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dissertation entitled

VOLATILE ANALYSIS OF WHOLE AND HINIHALLY PROCESS. FRUITS
AND VEGETABLES FOR PHYSIOLOGICAL AND SAFETY ANALYSIS

presented by

RUFINO PEREZ-BRENNAN

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1M WHO/WM“

VOLATILE ANALYSIS OF WHOLE AND NIINIMALLY PROCESSED FRUITS
AND VEGETABLES FOR PHYSIOLOGICAL AND SAFETY ANALYSIS

By

Rufmo Perez-Brennan

A DISSERTATION

Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Horticulture

1998

VOLATILE ANALYSIS OF WHOLE AND MINIMALLY PROCESSED FRUITS
AND VEGETABLES FOR PHYSIOLOGICAL AND SAFETY ANALYSIS

by Rufino Peru-Brennan

Solid phase micro extraction (SPME) sampling and rapid gas chromatography
(GC) in combination with time-of-ﬂight mass spectrometry (TOF-MS) was used for
volatile analysis of whole strawberry, cut carrot and broccoli ﬂorets to assess the
physiology and microbiology of these produce. Volatiles of strawberry stored under
refrigerated air (2°C) changed as the fruit senesced. Esters were found to be the dominant
volatile class. The production of methyl acetate was found to negatively correlate with
loss in ﬁrmness, suggesting that the production of this volatile may be related to cell wall
metabolism of the fruit. For cut carrot, the dominant volatile compounds were the
terpenes. Most terpenes detected decreased markedly during the ﬁrst 3 days of
evaluation. Alpha phellandrene was the only terpene to increase as the produce aged. In
broccoli, there was not a single group of volatiles which amply predominated during
storage. The types of volatiles found included terpenes, aldehydes, fatty acid derivatives,
sulﬁdes, ketones and benzoids. For E. coli and S. whimurium, 1-propanol may have the
potential to be used as a ﬁngerprint compound for E. coli in produce packages while
heavier alcohols such as dodecanol, octadecanol and nonadecanol appeared to be
characteristic for S. typhimurium. Today, with the availability of rapid, accurate and more
sensitive volatile analysis techniques, it is worthwhile to use volatiles produced by plants
and microorganisms as a research asset. Volatile analysis may be a useful tool to assess
some physiological events that take place in plants and microorganisms that need yet to be

ﬁilly understood.

Copyright by
RUFINO PEREZ-BRENNAN
1 998

DEDICATION

To Jenny, Laura and Nona: they own my inside.

iv

ACKNOWLEDGMENTS

Dr. Randolph M. Beaudry: I want to express my deepest thanks to my major
professor, Dr. Randolph M. Beaudry. He provided me
with the resources and guidance I needed. I feel
endebted to him forever.

Dr. Robert Hemer: Bob Hemer was one of the key people in helping me to
achieve my goals during my doctoral studies. His
moral support, wise advice and concern for me made
me build my strength and conﬁdence. He really made a
diﬁ‘erence in my studies both as a member of my
graduate committee and as a ﬁend.

Dr. John Linz: Besides being part of my graduate committee, Dr. Linz
was a rich source of knowledge for me when I was
conducting the study on bacterial ﬁngerprint
compounds. Thanks so much to him and his graduate

student Matt Rasieck.
Dr. Art Cameron, All of these professors were also part of my graduate
Dr. Jerry Cash, committee. I feel very thankful for their contribution to
Dr. Jack Giacin: my doctoral studies. Each of them contributed in his

unique way and made my research more challenging
and interesting.

Postharvest Technology I want to express my deepest appreciation to all

Laboratory Group: graduate students, post does, undergrads and
technicians in the Postharvest Technology and
Physiology Laboratory who in one way or the other
made my life more meaningful and made my experience
at MSU much more worthwhile. In particular, Dr.
Nazir N. Mir, Dr. Dina Kadirzhanova and Weimin

Deng.
Department of I want to thank the Department of Horticulture
Horticulture particularly its chairperson Dr. Wayne Loescher. Also

Lorri Busick, Joyce Jackson, Sherry Mulvaney and
Gabrielle Seyka, all of whom are beautiful people.

TABLE OF CONTENTS

DEDICATION ....................................................... i
ACKNOWLEDGMENTS ............................................... ii
TABLE OF CONTENTS .............................................. iiii
LIST OF TABLES .................................................... v
LIST OF FIGURES ................................................... vi
FOREWORD ........................................................ 1
INTRODUCTION .................................................... 2
CHAPTER I: LITERATURE REVIEW ................................... 7
Senescence Physiology of Fruit and Vegetables ......................... 8
Lipoxygenase .................................................. 10
Vegetable and Fruit Volatiles ...................................... 13
Modiﬁed Atmosphere Packaging of Fruits and Vegetables ................ 17
Modiﬁed Atmosphere Packaging and Temperature ...................... 19
Enzymatic Considerations in Modiﬁed Atmosphere Packaging ............. 20
Future of Modiﬁed Atmosphere Packaging ........................... 22
Food Microbiology ............................................. 22
Food-home Pathogens ........................................... 24
Spoilage of Fruit and Vegetable .................................... 29
Microbiology of Modiﬁed Atmosphere-Packaged Produce ................ 34
Detection and Identiﬁcation of Microorganisms in Food .................. 36
Food Safety and Control Systems ................................... 38
Hazard Analysis of Critical Control Points (HACCP) .................... 41
Food Quality and Food Quality Assessment ........................... 43
SPME Sampling System .......................................... 48
Cryo-cooling Technology ......................................... 50
Thesis Research ................................................ 50
CHAPTER II: SENESCENCE-RELATED VOLATILE PROFILES OF
STRAWBERRIES .............................................. 66
Abstract. ..................................................... 67
Material and Methods ........................................... 69
Results ....................................................... 74
Discussion .................................................... 75

CHAPTER HI: VOLATILE PROFILES OF BROCCOLI AND CARROT. ........ 90

Abstract ...................................................... 91
Material and Methods ........................................... 95
Results ....................................................... 96
Discussion .................................................... 97

CHAPTER IV: VOLATILE PROFILES OF MICROORGANISMS GROWN IN

LABORATORY MEDIA AND FOOD PRODUCTS .................. 118
Abstract ..................................................... 119
Material and Methods .......................................... 123
Results ...................................................... 127
Discussion ................................................... 129

CHAPTER V: STORAGE DURATION, LOW 02 AND ELEVATED CO2 ALTER

ESTER BIOSYNTHESIS IN STRAWBERRY ....................... 147
Abstract. .................................................... 148
Material and Methods .......................................... 150
Results ...................................................... 155
Discussion ................................................... 156

CHAPTER VI: AROMA PRODUCTION OF CARROT AND BROCCOLI AS A

FUNCTION OF 02 PARTIAL PRESSURE IN MAP .................. 168
Abstract. .................................................... 169
Material and Methods .......................................... 172
Results ...................................................... 173
Discussion ................................................... 174
SUMMARIES ...................................................... 185

vii

LIST OF TABLES

CHAPTER I

Table 1. Selected volatiles produced by strawberry over time . . . .

CHAPTER II

Table 1. Selected volatiles produced by carrot over time .......

Table 2. Selected volatiles produced by broccoli over time ......

CHAPTER V

Table 1. Changes of strawberry volatiles under MAP during d 3. .

viii

............... 82

.............. 105

.............. 106

............. 161

LIST OF FIGURES

CHAPTER H

Figure 1. Selected strawberry volatile on day 1 in a ﬂow-through system .......... 83
Figure 2. Ester production by strawberry during storage classiﬁed by group ........ 84
Figure 3. Changes of methyl esters produced by strawberry during storage. ........ 85
Figure 4. Ratios of methyl acetate to methyl hexanoate, methyl butanoate, ethyl

hexanoate and ethyl butanoate ...................................... 86
Figure 5. Softening of strawberry during storage, open circles, and correlation between

methyl acetate and softening, closed circles ............................ 87
Figure 6. Ratios of methyl acetate over chemical parameters ................... 88
Figure 7. Activity of strawberry lipoxygenase and peroxidases over time. ......... 89

CHAPTER IH

Figure l. volatiles proﬁle of carrot using time-of-ﬂight mass spectrometry ........ 107
Figure 2. Selected carrot volatile production over time. ..................... 108
Figure 3. Ratios of selected carrot volatiles over time ....................... 109
Figure 4. A representation of the general trend of selected carrot volatiles over time. 110
Figure 5. Volatiles proﬁle of broccoli using time-of-ﬂight mass spectrometry. ..... 111
Figure 6. Selected broccoli volatile production over time ..................... 112
Figure 7. Mechanism of formation of myrcene ............................. 113
Figure 8. Mechanism of formation of limonene ............................ 114
Figure 9. Mechanism of formation of linalool .............................. 115

ix

Figure 10. Mechanism of formation of B-phellandrene and alpha terpinolene ...... 116

Figure 7. Mechanism of formation of alpha pinene, and camphene .............. 117
CHAPTER IV
Figure 1. Spectrum of volatiles produced by E. coli in Super Broth media. ....... 137

Figure 2. Spectrum of volatiles produced by S. typhimurium in Super Broth media. . 138
Figure 3. E. coli grth over time as detected using spectrophotometer

measurements at 595 nm. ........................................ 139
Figure 4. Ratios of indole production by E. coli to population of the organism in the

media. . .................................................... 140
Figure 5. Indole production in Super Broth media with and without tryptophan. . . . 141
Figure 6. Indole (open symbols) and l-propanol (closed circles) production

over time. ................................................... 142
Figure 7. l-propanol production in broccoli packages at RT.. . ................ 143
Figure 8. 1-propanol production by E. coli in broccoli stored under modiﬁed

atmosphere packaging as a ﬁrnction of the bacterial initial population (IP).. . . 144
Figure 9. 1-Propanol accumulation in packages of carrot and lettuce at RT inoculated

with threonine solution contaminated with E. coli . . ................... 142
Figure 10. Schematic of the mode of action of E. coli on the breakdown of threonine

in packages of broccoli ........................................... 146

CHAPTER V

Figure l. GC-MS Spectrum of the total ion count (TIC) for strawberry enclosed in a

sealed jar for 40 min on day 0. ...................................... 162
Figure 2. Trend over time of four selected different strawberry volatiles in modiﬁed

atmosphere packages. . ......................................... 163
Figure 3. Trend of strawberry volatiles in modiﬁed atmosphere packaging and as

impacted by in-package O2 concentration ............................ 164
Figure 4. Ratios of methyl acetate/methyl butanoate and hexyl acetate/methyl hexanoate

in modiﬁed atmosphere-packed strawberry. ......................... 165
Figure 5. Range of O2 generated in a ﬂow through system (top) and changes in the

production of six different volatile esters at different 02 concentrations. . . . . 166
Figure 6. Proposed mode of action of O2 and CO2 on the biosynthesis of esters in

strawbeny packaged under modiﬁed atmospheres. ..................... 167

CHAPTER VI

Figure 1. Selected volatile compounds produced by MAP-stored broccoli as

affected by different levels of in-package O2 partial pressures. ............ 181
Figure 2. Effect of 02 on the accumulation of volatile sulfur compounds from

broccoli in modiﬁed atmosphere packaging ........................... 182
Figure 3. Selected volatile compounds produced by MAP-stored carrot as

affected by different levels of in—package O2 partial pressures. ............ 183
Figure 4. General trend of terpene volatile compounds as affected by 02 partial

pressure in MAP-stored broccoli ................................... 184

FOREWORD

In the United States and across the world, governments are calling for tougher
standards of sanitation and hygiene to be applied in the ﬂesh produce industry that
previously were found only in processed foods. The Food and Drug Administration
(FDA), for instance, is planning on putting food safety guidelines in place before the year
2000 for high risk fruits and vegetables. Federal authorities now have begun to
acknowledge that the microbial problems in the produce industry are not like other food
groups: they are more varied and complex because of the number of commodities and
growing practice and handling. Because of this higher awareness of the nature of the
problem of pathogens on produce, authorities are more willing to recommend produce
trace back systems, quicker approval of technologies such as irradiation, more research
and a comprehensive sanitation and hygiene program for the produce industry.

These concerns in conjunction with a rapidly changing consumer base and
exploding global trade present challenges for scientists, government and food industries at
the levels of fundamental and applied research. In this dissertation, volatile analysis of
whole and minimally processed hits and vegetables has been used to try to assess the
physiological condition of plant productss and tests for human pathogen presence. Volatile
emissions by fruits, vegetables, microorganisms and their combination were assessed for

their potential for the development of sensing technologies in the food industry.

INTRODUCTION

Quality control for millions of packages ﬁlm and vegetable products which pass
through the hands of farmers, shippers, fresh product storage operators, processors,
processed product storage operators, retailers and the consumers is limited at best. The
‘use by’ dates, printed on shipping boxes and packages, although helpful, cannot identify
packages with poor quality product. Sensing devices that are able to assess the
physiological status of the product and/or the presence of biological hazards at various
stages in product delivery are needed and should enhance reliability in product quality and
safety.

At present, enzyme-linked immuno—sorbent assays (ELISAs) are available to test
for the presence of a number of human pathogens (Samarajeewa et a1., 1991). However,
ELISA tend to be slow, subject to variable results and are not amenable to automation.
For a facility that may be shipping dozens of product lines to dozens of locations per day,
sampling under these conditions would not be prudent and would reveal nothing relative
to quality (e.g., ﬂavor, tissue integrity, state of senescence). A remote inspection
biological sensor is being developed to detect E. coli and Salmonella in contaminated
meat in real time (SatCon Technology Corp., Cambridge, MA). This technique uses a
laser spectrographic combination. It has a laser source that is reﬂected off and interacts
with pathogens on a carcass surface, then returns. Because pathogens have a very speciﬁc
signature, the system can make an identiﬁcation and give a general indication of the
number of organisms present in the packages. However, this technology while promising,

has yet to be implemented and its applicability and reliability out of the laboratory is yet to

be tested.

Recent developments in extremely rapid gas chromatography coupled with time-
of-ﬂight mass spectrometry (GC-MS), have made feasible real-time assessment of
biological materials. The emanation of speciﬁc volatiles, which can serve as ﬁngerprint or
signature-type compounds for some physiological processes such as senescence or the
presence of microorganisms and may oﬂ‘er signiﬁcant opportunities for quality assessment,
are poorly characterized at best.

The ﬁeld of gas chromatography and mass spectrophotometry will likely continue
to advance and become more accessible, which will make possible detection of volatiles
with sufﬁcient rapidity to permit the on-line monitoring of volatiles with the intention of
relating the changes to quality and safety of produce. Work is under way in the
development of sniffers for volatiles or classes of volatiles at the part-per-billion level
(Hetzroni et al., 1995). Volatile sensors for industrial process control systems are already
in use. In addition, the ﬁeld of biosensor applications continues to expand. With regard
to volatile emanations ﬁ'om plant products and microorganisms, there already exists a
biosensor for alcohols (Chematics, Inc., North Webster, IN.). Thus, volatile sensing in the
headspace of packaged products has the potential to be performed as an in-package
biosensor or via automated, rapid instrumentation. It would be of a great value to
develop a sensor to detect the presence of E. coli and/or other microorganisms in
headspace packaging, using ﬁngerprint signature-type volatiles.

A volatile detection system requires the development of a database relating the

physiological state of the product to changes in its volatiles proﬁle. This database does

4

not exist in any meaningful form for lightly-processed plant products. To a great extent,
senescence-related volatiles primarily refer to the biosynthesis of the gaseous hormones
ethylene and methyl-jasmonate and the generation of lipoxygenase pathway products,
which include aldehydes, alpha- and gamma-keto fatty acids, divinyl ether fatty acids and
others. While ethylene is a signal molecule for the induction of ripening and senescence
(Abeles et al., 1992) its presence may not be particularly closely tied to tissue degradation.
J asmonates offer more promise, perhaps, being more speciﬁcally tied to senescence and a
downstream product of lipoxygenase activity (Parthier and Sembdner, 1983; Reinboth et
el., 1994 ). Other lipoxygenase products such as aldehydes, offer much potential in that
they are produced in copious amounts and are directly linked to the loss of tissue integrity
(Galliard, T., 1978; Hildebrand et al., 1988; Hildebrand, 1989; Thompson, 1988). Further
characterization of volatile proﬁles is needed.

The effect of a polymer barrier on the retention of volatiles such as those noted
here is not well understood. Little information is available on the permeability of polymers
to organic volatiles. To a large extent, this is a ﬁrnction of the difﬁculty of making such
measurements. The permeability of LDPE to hexanal as well as a number of other plant
volatiles has been measured (Deng and Beaudry, unpublished data; Song et al., 1996).
Hexanal (a lipoxygenase product) and hexyl acetate (an apple ﬂavor compound)
permeability was found to be 50 to 100-fold higher than the permeability to 02. Despite
its high permeability, LDPE serves as a suﬂicient barrier to apple ﬂavor volatiles to permit
their detection in sealed packages using only a glass syringe with a volume of 50 uL.

Importantly, polymers of ever increasing selectivity and capacity are being developed that

5

may be able to preferentially retain classes of compounds such as those related to aroma.
The available information regarding volatiles emitted from plants and
microorganisms is sufﬁcient to suggest that one might be able to successﬁilly relate
speciﬁc volatiles to the physiological and quality status of whole or lightly processed
products. The development of detection systems for volatiles appears to be dynamic and
their ﬁiture promising. In addition, the need for additional monitoring by the product
industry is increasing. What remains to be done is to quantitatively and qualitatively
establish the relationship between package volatiles and speciﬁc measures of quality loss

and safety.

References

Abeles, F .B., P.W. Morgan and ME. Salveit, Jr. 1992. Ethylene in Plant Biology.
Academic Press. p. 120-181.

Hayes, PR. 1992. Food Microbiology and Hygiene. Elsevier. NY. 516 p.

Galliard T. 1978. Lypolitic and lipoxygenase enzymes in plants and their action in
wounded tissues. In: G. Kahl (ed.) Biochemistry of Wounded Plant Tissues.
Berlin. Walter de Gruyter, pp. 155-201.

Hildebrand, D. F., TR. Hamilton-Kemp, C.S. Leg and G. Bookjans. 1988. Plant
lipoxygenase: Occurrence, properties and possible ﬁinctions. Curr. Top. Plant
Bioch. Physiol. 7 :210-209.

Thompson, W.W. and Platt-Aloia. 1987. Ultra structure and senescence in plants. In:
W.W. Thompson, EA. Nothnagel, and RC. Huﬁ‘aker. (eds). Plant Senescence:
Its Biochemistry and Physiology. American Society of Plant Physiologists.
University of California. Riverside. pp. 20-42.

Sarnarajeewa, U. , C. I. Wei, T. S. Huang and M. R. Marshall. 1991. Application of
immunoassay in the food industry. Food Sci. Nutrition. 29:403-434.

Sembder, G. and B. Perthier. 1983. The biochemistry and the physiological signiﬁcance
of jasmonates. Ann. Rev. Plant Physiol. Plant Mol. Biol. 44:569-89.

Song, J ., B.D. Gardner, F.F. Holland, and RM. Beaudry. 1997. Rapid analysis of volatile
ﬂavor compound in apple fruit using SPME and GC/time of ﬂight mass
spectrometry. J. Agric. Food Chem. 45(5): 1801-1807.

CHAPTER I

LITERATURE REVIEW

8

Modiﬁed atmosphere packaging (MAP) effects include extension of shelf-life of
produce and enhancement of distribution and storage systems. The use of MAP, however,
has implications for changing microbiological activity, visual quality and product
taste/ﬂavor. GC-mass spectrometry (GC-MS) and solid phase micro extraction (SPME)
sampling technology are analytical techniques available that may allow us to better
monitor and evaluate these aspects of MAP. By doing so, we should be able to gain a
better undestanding of the physiology, physicochemistry and biochemistry of senescing
ﬁuits and vegetables. We may also be able assess produce safety and quality. Important
goals in this area include analysis and control of microbial development in produce
packages, design of hazard analysis critical control point (HACCP) programs, quality

modeling, and development of biosensors .

Senescence Physiology of Fruit and Vegetables

The idea that death of plant tissue or an organism might be actively induced by
endogenous factors that are a natural part of organism development has yet to be
contradicted. Nooden (1988) stated that senescence is related to certain internal changes
that increase the probability of death with increasing chronological age. Senescence could
also be deﬁned as the deteriorative processes that are natural causes of death of plants or
plant parts (Thimann, 1980). Thomson and Platt-Aloia (1987) suggest that the
ultrastructural changes during senescence include ﬁrst, a degradation of the contents of
the protein bodies followed by the fusion of the vacuolated protein bodies into large

vacuoles. Associated in time with these transitions, the cisternal endoplasmic reticulum

9

becomes tubular, resulting in the fragmentation of the tonoplast membrane of the vacuole
followed by a complete loss of cellular compartmentation.

Dalling (1987) suggests that the phenomenon of plant senescence, regardless of
whether a particular organ is evaluated or the plant as a whole, is invariably associated
with the progressive loss of soluble and insoluble protein. Within a cell, the amount of any
protein is determined by the balance between synthesis and degradation of that protein.
Because of its abundance and metabolic importance, there is more information on
ribulose-bisphosphate carboxylase (RuBPCase) than on other proteins. Brady (1988)
studied the relationship of senescence and some physiological changes in bean (Phaseolus
vulgaris L.) and reported that in P. vulgaris senescent tissue there is a decrease in
chlorophyll content, total protein, and rRNA biosynthesis.

Senescence of higher plants are genetically as well as environmentally regulated
processes intimately associated with hormonal interactions. Of the well-recognized plant
hormones known to date, ethylene is unique in that it is a simple, gaseous hydrocarbon
that is a principal feature of processes that enhance plant senescence (Mattoo and Aharoni,
1988). In addition, cytokinins play an important role in controlling many of the processes
that contribute to plant senescence (Nooden, 1980; Nooden and Leopold, 1978; Staden et
al., 1988; T himann, 1980). In fruits, chlorophyll loss and the conversion of chloroplast to
chromoplasts is frequently accompanied by a decrease in cytokinin (Staden et al., 1988).
Fruit of the nonripening rin tomato variety have a high cytokinin content at the full-green
stage, and this persists, whereas in the normal ripening ﬁuit, cytokinins decrease as the

hits change color (Davey and Staden, 1978) . Second to ethylene, abscisic acid is the

10

most well studied of the senescence promoters (Nooden, 1988). Poovaiah (1988) also
suggested that calcium distribution plays a critical role in cell ﬁrnction and that a high level
of free calcium in the cytoplasm is injurious to the cell. At such a level, calcium reacts
with inorganic phosphate to form an insoluble precipitate.

Kuo and Parking (1989) reported that lipid peroxidation plays a role in the
developmental process of senescence in fruits and vegetables. The peroxidation of
linolenic acid is the only known source of ethane, which is not metabolized ﬁrrther (Lyons
and Breidenbach, 1987). Thus, ethane production could be a good index for assessing
senescence if we take into consideration other physiological parameters such as changes in

ﬁrmness, electrolyte leakage, chlorophyll changes, and soluble solids.

Lipoxygenase (LOX)

Lipoxygenase (LOX) (linoleate : oxygen oxidoreductase, EC 1.13.11.12) is a
ubiquitous protein that catalyzes the degradation of fatty acids. Because many products of
the LOX reaction are aromatic, the presence of lipoxygenase activity in many foodstuffs
can affect their sensory properties, particularly during long-term storage, in both desirable
and undesirable ways. LOX is a member of a class of nonheme iron-containing
dioxigenases that catalyze the addition of molecular oxygen to fatty acids containing a cis,
cis-1,4-pentadiene system to give an unsaturated fatty acid hydroperoxide (Siedow, 1991).
LOX has been reported to be ubiquitous in plants, which means that failure to measure
LOX activity in a given species likely reﬂects the insensitivity of the detection method

used rather than absence of the enzyme activity .

11

In a resting LOX molecule, the single atom of nonheme iron at the active site
exists in a high-spin Fe (11) state. The active form of LOX can then be generated from the
resting enzyme by reaction with one equivalent of hydroperoxide product which oxidizes
the active site iron to a high-spin Fe (111) state.

Given the diversity of plants in which LOX activity has been reported, it is
somewhat surprising that the number of species from which the enzyme has been well
characterized is limited. However, where plant LOX has been characterized to some
extent, the enzyme consists of a protein whose molecular weight is in the vicinity of
95,000 with a range of 72,000 to 108,000 (Siedow, 1991). That LOX plays a role in
senescence is probably the oldest of the currently extant theories about this enzyme’s
function. The reason for this is obvious: senescence is essentially a degradative process,
which includes the loss of membrane integrity. The most prevalent source of potential
LOX substrate is found among the polyunsaturated fatty acid side chains present in
membrane lipids. In ﬁ'uit, a pool of free fatty acids released from the membrane by action
of hydrolysis of lipids via lipase during ﬁ'uit senescence (Lester, 1990). Likewise, the
products of LOX reaction are themselves relatively reactive and can result in further
membrane deterioration and increased permeability. Further evidence of the role of LOX
in senescence is the ability of the enzyme and its products to generate free radicals, a
factor that seems to be of critical importance in plant senescence.

Several studies have indicated that an increase in LOX activity is a common
feature of senescing plant tissues and that treatments believed to delay the onset of

senescence, such as the addition of cytokinins or antioxidants, reduce the levels of

12

exogenous LOX activity relative to untreated controls (Pearry and Prince, 1990).
However, the ﬁnding of high LOX activities in actively growing plant tissues contrasts
markedly with the literature suggesting a role for LOX in senescence (Siedow, 1991). A
ripening sequence has been proposed for ‘Golden Delicious’ which starts with the
maturity-controlled degradation of fatty acids by LOX into hexenal, followed by the
oxidation of the aldehyde into hexanoic acid and then into smaller carboxylic acids (Feys
et al., 1980).

Methods used for the determination of LOX activity include spectrophotometry,
oxygen depletion, immunology, and electrophoresis. The spectrophotometric method is
by far the most commonly used and is based on measurements of the increase in ultraviolet
(UV) light absorption at a wavelength of 234 run, when LOX acts upon essential fatty
acids (Surrey, 1963). The increase in absorption is related to the amount of peroxide
formed, which is proportional to time and enzyme concentration. The immunological
approach to measure LOX activity is based on the existence of antibodies against the
enzyme, providing a very sensitive probe for the detection of the enzyme. Probes for the
RNA messanger for LOX upon conserved regions within the nucleotide coding sequence
of the LOX gene and make possible the development of a very sensitive assay using
Northern blot techniques to ascertain the presence of the LOX transcripts (Siedow, 1991).
LOX can also be detected following the course of the reaction between LOX and the
unsaturated fatty acid by measuring the uptake of molecular oxygen with a Clarke-type
oxygen electrode (Grossman and Zakut, 1979).

Activation of the LOX pathway in plant tissue gives rise to a series of products

13

derived from fatty acids (Croft et al., 1993). Analysis by GC-MS of volatile products
produced through the LOX pathway indicates cis-3 -hexenol and trans-hexenal are

produced, which arise from the C-13-hydroperoxide of linoleic acid.

Vegetable and Fruit Volatiles

A large number of volatile compounds are common at some level of concentration
in most vegetables and fruits. They arise ﬁ'om unsaturated fatty acids, amino acids, and
other generally universal components of ﬁ'uits and vegetables (Buttery, 1971). Some
commonly occurring compounds in vegetables and fruits include aliphatic saturated
aldehydes, aliphatic monosaturated aldehydes, aliphatic diunsaturated aldehydes, aliphatic
saturated alcohols, aliphatic unsaturated alcohols, aromatic hydrocarbons, sulfur
containing compounds, esters, terpenes, and chemical of other classes. The main thing
that differentiates ﬁ'uits ﬁom vegetables is that ﬁ'uits have higher concentrations of sugars
and ﬁ'uit volatile oils (Buttery, 1971; Teranishi et al., 1971). Common components of
fruit volatile oils are aliphatic esters such as ethyl acetate, ethyl butyrate, ethyl hexanoate,
butyl hexanoate, butyl acetate, hexyl acetate and branched esters such as 2-methylbutyl
acetate, ethyl-2-methylbutarate and others. In many fruits, beside the background of
commonly occurring compounds, some unique compounds are frequently responsible for
the main aroma character of the food. Such compounds have sometimes been referred to
as character impact compounds (Nurten, 1977).

In both ﬁ'uits and vegetables, there are some aroma compounds present in the

intact product. These compounds apparently mostly produced in the normal metabolism

14

of the plant by the controlled use of enzymes (Nurten, 1977; Teranishi et al, 1971). Many
volatiles on the other hand, are produced only when the raw ﬁuit or vegetable is broken
open such as cutting, chewing, or blending. This is an uncontrolled effect, where enzymes
in one part of the fruit are mixed with chemical components in another part normally kept
separated (Schwimmer and Friedman, 1972). For example in bananas, oranges, pears,
carrot, and bell peppers, at least some part of the aroma can be perceived in the intact ﬁuit
or vegetable. However, in cucumbers, some melons, and tomatoes the intact material has
extremely little aroma (Buttery, 1971).

One of the most universal enzyme-driven occurrences in a wide variety of foods is
the enzyme-induced oxidative breakdown of a small percentage of the unsaturated fatty
acids in that food. The oxidation of unsaturated fatty acids by enzymes has been
investigated (Teranishi et al., 1971). There is evidence that indicates that a number of
different enzymes systems can be involved: (1) the lipoxygenase which, forms the initial
peroxides; (2) isomerases which can arrange the initially formed peroxides molecule to a
different peroxide; and (3) other enzymes, which can chemically change the initially
formed peroxide. Each plant has its own set of enzymes, pH, and other conditions that
can lead to different volatile products, starting with the same unsaturated fatty acids which
are principally linoleic and oleic acids. These fatty acids are usually in the form of
triglyceride, phospholipids, or lipoprotein in the plant material. Unsaturated fatty acids are
an essential part of every plant cell. Fatty acid degradation products, particularly a
homologous series of aliphatic saturated and unsaturated aldehydes, are some of the most

common volatiles found in foods. Fatty acid fragments (aldehydes, alcohols, and ketones)

15

of C6, C7, C8, C9, and C10 chain length are particularly common (Buttery, 1971). C6
compounds (Hex-3 -enol, hexanol, hex-2-enal, hexanal, and hex-3 -enal always seem to be
formed wherever green plant material is crushed. Apparently they are formed through
enzymatic oxidation of linoleic and linolenic acids. These compounds are usually
described as having “green” odors. C9 aldehydes and alcohols are produced in
cucumbers. Nona-E-Z-6, 6-dienal from breakdown of linolenic acid provides most of the
characteristic cucumber aroma. C7 compounds such as hept-4-en-2-ol are found in
tomato and are probably formed from linoleic acid. The corresponding ketone, hept-4-en-
2—one is found in banana. C8 and C10 types of compounds are both the result of
breakdown of linoleic acid and are found in citrus and beans. Some other compounds
such as sulﬁir-containing volatile compounds result from the amino acid metabolism
(Buttery et al., 1976). Pentina et al. (1995) reported a possible enzymatic pathway for the
production of some of the sulﬁrr-containing compounds found in broccoli. They
suggested that for a cell-free system of fresh broccoli, sulﬁir containing compounds are
formed from L-methionine and S-methionine. Simon et a1. (1980) and Kopas-Lane and
Warthensen (1995) reported that volatiles produced by carrot are mostly terpenes.
Hamilton-Kemp et a1. (1996) conducted studies on the metabolism of natural
volatile compounds by strawberry fruit. As part of these experiments to evaluate
naturally-occurring volatile compounds as ﬁrmigants to reduce microbial populations,
especially pathogenic ﬁingi on strawberry fruit, several test compounds were found to be
metabolized by fruit to yield additional volatile compounds. The three principal types of

volatile metabolites formed by strawberry ﬁ'uit as determined by GC and GC-MS analyze

16

of headspace vapor samples were esters produced from aliphatic alcohols (e. g., 3-hexenyl
acetate from 3-hexen-1-ol), alcohols formed by the reduction of aliphatic aldehydes (e.g.,
l-hexanol from hexanal), and saturated products from reduction of carbon-carbon double
bonds conjugated with an aldehyde or ketone carbonyl moiety (e. g., 2-nonanone from 3-
nonen-2-one). The pathways resulting in the above metabolites in strawberry appear to
involve three systems (Perez et al., 1993). The incorporation of alcohols into esters in
strawberry and other fruit is due to a developmentally regulated enzyme known as alcohol
acyl-CoA transferase. This enzyme is necessary for production of several esters that
generate characteristic aromas associated with the ﬁ'uit. The second group of metabolites,
alcohols, are produced following reduction of aliphatic aldehydes to their corresponding
alcohols is likely accomplished through the action of alcohol dehydrogenase (Bartley and
Hindley, 1980; Hamilton-Kemp et al., 1996). This enzyme system also produces ethanol
ﬁom acetaldehyde when plant tissue is placed under anaerobic conditions. A third system
resulting in the reduction of carbon-carbon double bonds adjacent to carbonyl groups is
not known; however, the closest analogy seems to be the reduction of unsaturated acyl
moieties during the chain elongation steps of fatty acid synthesis (Hamilton-Kemp et al.,
1996; Perez et al., 1993; Yamashita, 1976).

Much needs to be learned regarding the biosynthesis of volatile materials produced
by plant products. From a horticultural perspective, the impact of environmental factors
and cultural processes on aroma and other volatile production are at best poorly

understood.

17

Modified Atmosphere Packaging of Fruits and Vegetables

Modiﬁed atmosphere packaging could be deﬁned as the enclosure of food
products in gas-barrier materials, in which the gaseous environment has been changed in
order to inhibit spoilage processes and therefore either maintain a high quality within a
perishable food during its natural life or actually extend the shelf-life (Beaudry et al., 1992;
Cameron, 1989, 1994; Church and Parsons, 1994). The rationale for the application of
MAP to extend the shelf life of fresh produce is that by reducing O2 and increasing CO2
levels, respiration is inhibited, degreening and decay is prevented, and senescence is
subsequently postponed (Ben-Arie and Zutkhi, 1992; Kader et al., 1989; Vergano and
Pertruit, 1993).

With modiﬁed atmosphere packaging, the atmosphere inside a package generally is
low in 02 and high in C02, with typical values at steady-state of 2-3% and 5%,
respectively (Beaudry et al., 1992; Cameron, 1989; Church and Parsons, 1994; Gorris et
al., 1996). The composition of the atmosphere is the product of the initial gas
composition, the gas exchange through the packaging material and the respiratory activity
of the produce (Beaudry et al., 1992; Brackett, 1989; Cameron, 1989, 1994; Church and
Parsons, 1994; Gorris et al., 1996). One method of achieving modiﬁed atmosphere
packages is by ﬂushing pouches with a desired gas mixture before sealing the package.
MAP can also be done by shrinking ﬂexible ﬁlms tightly around an item, a method know
as shrink-wrapping or seal-packaging (Brackett, 1989; Exama et al., 1993).

Cameron et al. (1989) developed a model to determine O2 consumption as a

ﬁrnction of in-package 02 concentration by mathematical characterization of O2 depletion

18

by a tomato ﬁ'uit in a closed system. They were able to develop a mathematical model to
relate ﬁlm permeability characteristics as a ﬁinction of fruit weight to achieve desired
sealed package concentrations of O2 in modiﬁed atmosphere packages. Ben-Arie and
Zutkhi (1992) were able to extend the shelf-life of ‘Fuyu' persimmon from 6 to 18 weeks
at 1°C by modiﬁed atmosphere packaging in a low-density polyethylene (LDPE) ﬁlm by
retarding fruit softening and inhibiting development of peel and ﬂesh disorders.

MAP is often used with minimally processed produce. Wiley (1994) deﬁnes
minimally processed fruits and vegetable as products that contain live tissues or those that
have been only slightly modiﬁed from the ﬂesh-condition and are like-fresh in character
and quality. Minimal processing includes all of the unit operations (washing, sorting,
peeling, slicing, and packaging) that might be used prior to blanching in a conventional
processing line. Thus, minimally processed tissues have been exposed to substantial injury
due to cutting and handling (Rolle and Chism III, 1987). Unlike meat and ﬁsh, harvested
ﬁ'uits and vegetables are still living material and, although cut oﬂ‘ from their normal
nutrient supplies, are capable of a wide range of metabolic reactions, the most important
of which is respiration (Ballantyne, 1989). Even so, Wiley (1994) states that these tissues
do not exhibit the same physiological responses as normal or raw untreated intact live
plant tissues. Minimally processed foods, including cut vegetables and ﬁuits, represent
one of the fastest growing segments of the ﬂesh produce industry in the United States and
across the world (Stanley, 1997; Wiley, 1994). The international-cut produce association
claims that fresh-cut produce is the most promising segment of the market for fresh ﬁ'uits

and vegetables with a projected sale of $19 billion by 1999 relative to $5.8 billion in 1994

19

(Stanley, 1997).

Modiﬁed Atmosphere Packaging and Temperature

Fresh-cuts generally are much more perishable than intact products because they
have been subjected to severe physical stress, such as peeling, cutting, slicing, shredding,
trimming, and/or coring, and removal of protective epidermal cells. Consequently, ﬁesh-
cuts probably should be held at a lower temperature than that recommended for intact
commodities (W atada et al., 1996). Despite precautions, modiﬁed atmosphere packages
are often subject to a wide spectrum of external temperatures during handling, transport
and subsequent retailing. Although 0°C generally is the desirable temperature for most
fresh-cuts, many are prepared, shipped and stored at 5°C and sometimes as high as 10°C.
Storage at this elevated level can hasten deterioration substantially because Qlo of
biological reactions ranges from 3 to 4 and possibly as high as 7 within this temperature
region (Schlimme, 1995). Respiration rates of fresh-cuts increases with temperature, and
the degree of increase differs with commodities (W atada, 1996). In the 10 to 20°C
storage temperature range, the Q10 of most fresh-cuts is greater than in the 0 to 10°C
range. An increase in temperature causes a greater increase in the rate of 02 by harvested
produce than in polymer permeability creating the potential for injurious 02 levels as
temperature increases (Beaudry et al, 1992; Cameron and Talasila, 1994; Kader et al.,
1989)

Beaudry et a1. (1992) meassured the O2 partial pressure of blueberry modiﬁed

atmosphere packages of blueberry at which the fruit began to exhibit anaerobic CO2

20

production across a range of temperatures. They found that the package lower 02 limit
increased with increasing temperature, which implies that blueberry ﬁ'uit can be stored at

lower 02 partial pressure when stored at lower temperatures.

Enzymatic Considerations in Modified Atmosphere Packaging

As soon as vegetables and ﬁ'uits are harvested, physiological changes occur and
some of these lead to a loss in quality. Respiratory activity involving the breakdown of
carbohydrates by plant enzymes continues and the changes induced, whether advantageous
or deleterious, are oﬁen markedly inﬂuenced by the maturity of the plant when harvested;
thus some plants organs can be stored for lengthy periods with little change in quality if
harvested at the right time (Hayes, 1992).

Minimally processed fruits and vegetables with wounded tissue require special
preservation methods for the purpose of extending storage life and preventing spoilage
(W rley, 1994). The effects of wounding on plant metabolism have been studied
extensively in tissues prepared from bulky plant organs such as tubers and roots (Solomos,
1994). The vast literature on this subject has established that slicing or minimal processing
induces profound quantitative and qualitative changes in tissues metabolism including a
rise in respiration, DNA and RNA synthesis, induction of new enzymes, membrane
degradation, and the appearance of novel mRNA (King and Bolin, 1989; Laties, 1978;
Price and Floros, 1993; Rolle and Chism, 1987; Solomos, 1994).

Enzymes and substrates are normally located in different cellular compartments

and their transfer is actively regulated, thus, processing results in destruction of cells, loss

21

of compartmentation for cells at the cut surface and injury stress of underlying tissues
(Varoquax and Wiley, 1994). Quality loss due to lipid peroxidation and some browning
reactions of cut surface and ruptured cells are dependent upon loss of compartmentation.
Enzymatic peroxidation of unsaturated fatty acids results the modiﬁcation of natural
aromas. This peroxidation is catalyzed by LOX and leads to the formation of numerous
aldehydes and ketones (Hildebrand, 1989). With regard to browning, it is an enzymatic
process that occurs in bruised and cut plant tissues (Hildebrand, 1994; Mayer, 1997;
Solomos, 1994; Wiley, 1994). The enzymatic reactions involved in the brown
discoloration are still under investigation but are known to involve polyphenol oxidase
(PPO, tyrosinase, EC 1.14.181). The enzymatic activities markedly depend on pH. A pH
reduction of 0.5 in the natural pH of apple results in a 50% decrease in chloroplast
polyphenol oxidase (PPO) activity (Varoquax, 1994).

Luo and Barbosa-Canovas (1991) investigated enzymatic browning and its
inhibition in new apple cultivar slices using 4-hexylresorcinol in combination with ascorbic
acid. The quality of minimally processed apples of the cultivars ‘Braeburn’, ‘Criterion’,
‘Fuji’, ‘Gala’, and ‘Jonagold’ was assessed at 2.5, 12 and 20°C, and compared with
traditional cultivars ‘Golden Delicious’ and ‘McIntosh’. Among the new cultivars,

‘J onagold’ had the slowest browning rate and ‘Braebum’ had the fastest rate. Of the two
traditional cultivars, ‘McIntosh’ had the faster browning rate. The temperature sensitivity
of the browning varied among cultivars, where Qlo ranged from 1.3 to 4.4 and the
apparent energy of activation (Ea) ranged ﬁom 8.0 to 80.3 kcal/mol. The browning rate

dependence on temperature was as follows: Fuji < McIntosh < Criterion < Braeburn and

22

Gala < Golden Delicious < Jonagold.

Future of Modiﬁed Atmosphere Packaging

There remains a great need for research and innovation to determine the most
appropriate gas levels for extension of shelf-life and develop methods for consistently
obtaining these levels in MA packages (Cameron and Talasila, 1994). Cameron (1994)
stated that new developments to expand use of MA are necessary. To that end, a sense—
and-respond type of package was deemed to be the most appropriate. In their concept, a
sense-and-respond system would perceive environmental conditions and/or chemical cues
from the enclosed product, then change some factor such as 02 permeability, in response
to avoid the generation of unwanted conditions in the package atmosphere. An additional
case can be made for sense-and-respond packages that percieve the presence of unwanted

microorganisms and respond by the generation of an indicator.

Food Microbiology

Microbiology is the study of microscopic organisms and their activities. Within
this area is the study of the distribution of the organism, their beneﬁcial and harmﬁrl
effects, especially in relation to the changes they make in their environment of which food
is one particular example (Hayes, 1992; Jay, 1996). Six major groups of microorganisms
are generally recognized, namely bacteria, fungi, viruses, algae, protozoa and rickettsias.
The bacteria are the most important in relation to food safety but ﬁingi also have a

signiﬁcant role (Fields, 1979; Frazier and Westhoff, 1977; Hayes, 1992; Ramachandra,

23

1980)

Because food sources are of plant and animal origin, it is important to understand
the biological principles related to growth and control of the microbial ﬂora associated
with plants and animals in their natural habitats and respective roles. If one considers the
types of microorganism associated with plant and animal foods in their natural states, one
can then predict the general type of microorganisms to be expected on a particular food
product at some later stage in its history (Banwart, 1979; Bryant, 1983; Rose, 1983). As
our food are of plant and animal origin, it is worthwhile to consider those characteristics
of plant and animal tissues that affect the growth of microorganisms. The plants and
animals that serve as food sources have all evolved mechanisms of defense against the
invasion and proliferation of microorganisms, and some of these remain in effect in fresh
foods (Adams, 1898; Foster; 1989; Frank, 1922; Mossel and Ingran, 1955). By taking
these natural phenomena into account, one can make effective use of some or all in
preventing or retarding the microbial spoilage of the products that are derived from them.
The parameters of plant and animal tissue that are inherent in the tissues are referred to as
intrinsic parameters. Properties of the storage environment that aﬁ‘ect both the foods and
their microorganisms are regarded to as extrinsic parameters (Jay, 1996; Montville, 1987;
Mossel and Ingran, 1955; Mountney and Gould, 1988). Intrinsic parameters include
product pH, moisture content, oxidation-reduction potential, nutrient content,
antimicrobial constituents and biological structures. Extrinsic parameters include
temperature of storage, relative humidity of environment, presence and concentration of

gases and the presence and activity of other microorganism.

24

The examination of foods for the presence, types, and numbers of microorganisms
and/or their product is basic to food microbiology. In spite of the importance of precise
identiﬁcation and quantiﬁcation none of the methods in common use permits the
determination of the number of microorganisms in a food product (Jay, 1996; Roberts and
Skinner, 1983; Sharpe, 1985). Since 1960, many modern and sophisticated physical,
chemical, molecular and immunologic microbial detection methods have been developed.
Many of these methods can be used to estimate numbers of cells or quantity of cellular by-
products (Jay 1996; Montville, 1987; Roberts and Skinner 1983). Most of these detection
methods are based on metabolic activity of microorganisms on given substrates
measurements of growth response and the study of nucleic acids or combination of these

phenomena (Bryant, 1983; Frazier and Westhoff, 1977; Ramachandra, 1980; Rose, 1983).

Food-home Pathogens

Salmonellosis, staphylococcal food poisoning, and infectious hepatitis are only a
few of the illnesses that have been associated with the consumption of contaminated food
(Foster, 1989; Jay, 1996; Monk et al., 1995; Troller, 1983). Symptoms of nausea,
vomiting, diarrhea, abdominal cramps, and malaise are most common and generally last
from one to seven days. However, foodbome illness can also be fatal, particularly in very
young children, the elderly, and those with weakened immune systems. Bacteria that cause
foodbome illness do not necessarily make the food look, smell, or taste bad. There is no
deﬁnitive way to tell simply by looking at a food whether or not it is contaminated

(Montville, 1987). The best way to control pathogenic bacteria in food is to careﬁrlly

25

adhere to a few simple food handling principles (Hobbs, 1993; Klontz et al., 1995;
Montville, 1988; Pan et al., 1997).

The bacteria Escherichia coli 01571H7, also known just as EC 0157, is a
dangerous type of E. coli. Appearantly healthy fresh produce or ﬁuit juices carry the
bacteria. It can be transferred ﬁom animal to animal, animal to plant and plant to animals.
EC 0157 can survive reﬁigeration and freezer storage (Hobbs, 1993, Pan et al., 1997).
Thorough cooking to 70 °C, which kills EC 0157 bacteria, is recommended as a safeguard
against infection. Careful kitchen sanitation is also vital (Troller, 1993). Uncooked
hamburger and roast beef, raw milk, improperly processed cider, contaminated water and
mayonnaise and vegetables grown in cow manure have caused outbreaks. Contaminated
cantaloupe, strawberries, and hard, dry sausage (salami) have been contaminated and
caused illness upon consumption. Increasingly, cross-contamination at food service
outlets, grocery carryouts and salad bars is causing outbreaks (Klontz et al., 1995; Monk
et al., 1995; Pan et al., 1997). Transmission of the illness, particularly among children in
daycare, is another problem. Since January 1993, there have been nearly 50 outbreaks in
the United States . The National Center for Disease Control (CDC) estimates there may be
20,000 illnesses a year (Jay, 1996). It appears that just a few (1 to 12) of these bacteria
can make you sick. After an incubation period of 3-8 days, the disease normally lasts 4-10
days. Patients may suffer severe abdominal cramps, diarrhea and sometimes bloody
diarrhea. Young children, the elderly and inﬁrm may develop complications (Rajkowski
and Mariner, 1995; Ray, 1996). They can develop hemolytic uremic syndrome which can

cause kidney failure, brain damage, strokes and seizures (Eley, 1996).

26

Clostridium botulinum is another important food pathogen that has been involved
in fresh produce-related outbreaks (Doyle et al., 1997; Eley, 1996; Jay, 1996; Lilly et al.,
1996; ). C. botulinum is an anaerobic, Gram-positive, spore-forming rod that produces a
potent neurotoxin. The spores are heat—resistant and can survive in foods that are
incorrectly or minimally processed. Seven categories (A, B, C, D, E, F and G) of botulism
are recognized, based on the antigenic speciﬁcity of the toxin produced by each strain.
Types A, B, E and F cause human botulism (Jay, 1996; Ray, 1996). Types C and D cause
most cases of botulism in animals. Animals most commonly affected are wild fowl and
poultry, cattle, horses and some species of ﬁsh. Although type G has been isolated ﬁ'om
soil in Argentina, no outbreaks involving it have been recognized. Four types of botulism
are recognized: foodbome, infant, wound, and a form of botulism whose classiﬁcation is
as yet undetermined. Foodbome botulism (as distinct ﬁ'om wound botulism and infant
botulism) is a severe type of food poisoning caused by the ingestion of foods containing
the potent neurotoxin formed during growth of the organism under anaerobic conditions.
The toxin can be destroyed if heated at 80°C for 10 minutes or longer. The incidence of
the disease is low, but the disease is of considerable concern because of its high mortality
rate if not treated immediately and properly. Most of the 10 to 30 outbreaks that are
reported annually in the United States are associated with inadequately processed,
home-canned foods, but occasionally commercially produced foods have been involved in
outbreaks (Hobbs, 1993; Pan et al., 1997; Troller, 1983). Vegetables and seafood
products have been the most frequent vehicles for human botulism. The organism and its

spores are widely distributed in nature. They occur in both cultivated and forest soils,

27

bottom sediments of streams, lakes, and coastal waters, and in the intestinal tracts of ﬁsh
and mammals, and in the gills and viscera of crabs and other shellﬁsh. Certain foods have
been reported as sources of spores in cases of infant botulism and the undetermined
category; wound botulism is not related to foods (Eley, 1996; Jay, 1996; Montville, 1987).

The types of foods involved in botulism vary according to food preservation and
eating habits in different regions (Eley, 1996). Any food that is conducive to outgrth
and toxin production, that when processed allows spore survival, and is not subsequently
heated before consumption can be associated with botulism. Almost any type of food that
is not very acidic (pH above 4.6) can support growth and toxin production by C.
botulinum. Botulinal toxin has been demonstrated in a considerable variety of foods, such
as canned corn, peppers, green beans, soups, beets, asparagus, mushrooms, ripe olives,
spinach, tuna ﬁsh, chicken and chicken livers and liver pate, and luncheon meats, ham,
sausage, stuﬁ‘ed eggplant, lobster, and smoked and salted ﬁsh (Lilly et al., 1996; Reddy et
al., 1996).

Since botulism is foodbome and results ﬁ'om ingestion of thet toxin of C.
botulinum, determination of the source of an outbreak is based on detection and
identiﬁcation of toxin in the food involved (Doyle et al 1997; Ray, 1996; Reddy et al.,
1996). The most widely accepted method is the injection of extracts of the food into
passively immunized mice (mouse neutralization test). The test takes 48 hours. This
analysis is followed by culturing all suspect food in an enrichment medium for the
detection and isolation of the causative organism. This test takes 7 days (Eley, 1996;

Reddy et al., 1996).

28

Listeria monocytogenes has been associated with such foods as raw milk,
supposedly pasteurized ﬂuid milk, cheeses, ice cream, raw vegetables, fermented
raw-meat sausages, raw and cooked poultry, raw meats (all types), and raw and smoked
ﬁsh (Fenlon et al., 1996). Its ability to grow at temperatures as low as 3°C permits
multiplication in reﬁ'igerated foods. The methods for analysis of food are complex and
time consuming. The present FDA method, approved in September, 1990, requires 24
and 48 hours of enrichment, followed by a variety of other tests. Total time to
identiﬁcation is ﬁom 5 to 7 days. In the near future speciﬁc nonradiolabled DNA probes
should allow a simpler and faster conﬁrmation of suspect isolates. When the recombinant
DNA technology is ﬁilly applied it may even permit 2-3 day positive analysis (F enlon et
al., 1996; Jay, 1996).

Salmonella spp. is one of the most dangerous food borne bacteria. It is a
rod-shaped, motile bacterium. Nonmotile exceptions are S. gallinarum and S. pullorum.
Salmonella is nonspore-forming and Gram-negative. There is a widespread occurrence in
animals, especially in poultry and swine. Environmental sources of the organism include
water, soil, insects, factory surfaces, kitchen surfaces, animal feces, fresh produce, raw
meats, raw poultry, and raw sea foods, to name only a few (Doyle et al, 1997 ; Eley,
1996)

Salmonella typhi and the paratyphoid bacteria normally cause septicemic and
produce typhoid or typhoid-like fever in humans (Doyle et al., 1997; Jay, 1996). Other
forms of Salmonella generally produce milder symptoms. Fresh produce, raw meats,

poultry, eggs, milk and dairy products, ﬁsh, shrimp, frog legs, yeast, coconut, sauces and

29

salad dressing, cake mixes, cream-ﬁlled desserts and toppings, dried gelatin, peanut butter,
cocoa, and chocolate are sources or carrier of the bacteria. Various Salmonella species
have been isolated from the outside of egg shells (Bean et al., 1990; Doyle, 1990; Farber,
1991). The organism can also exist inside the egg, in the yolk. This and other information
strongly suggest vertical transmission, i.e., deposition of the organism in the yolk by an
infected layer hen prior to shell deposition. Foods other than eggs have also caused
outbreaks of S. enteritis disease (Bryant, 1983; Montville, 1987).

Methods for the determination of the presence of Salmonella sp. have been
developed for many foods having prior history of Salmonella sp. contamination. Although
conventional culture methods require 5 days for presumptive results, several rapid

methods are available which require only 2 days (F enlon et al., 1996; Jay, 1996).

Spoilage of Fruit and Vegetables

Although spoilage in ﬁ'uits and vegetables can be induced by autolytic enzymes, it
is caused usually by the activities of microorganism (Brackett, 1994; Charambous, 1986;
Hayes, 1992). Spoilage microorganisms are an important aspect that has to be considered
in minimally processed hits and vegetables. All food exposed to air at room
temperature will spoil unless preserved in some way. Eliminating microbiological causes
of spoilage are key targets for the maintaining of the quality of ﬂesh produce (Brackett,
1994; Gorris et al., 1996). There are three types of microorganisms that cause food
spoilage: yeasts, molds and bacteria (Jay, 1996; Lund, 1971). Bacteria, yeast, and molds

cause 15% of postharvest losses of fresh produce (Harvey, 197 8). There are two types of

30

yeasts that cause spoilage: true yeast and false yeast. True yeasts metabolize sugar in an
aerobic environment, producing alcohol and carbon dioxide gas in a process known as
fermentation (Pitt and Hocking, 1997). False yeast grows as a dry ﬁlm on a food surface,
such as on pickle brine. False yeast occurs in foods that have a high sugar or high acid
environment.

Molds grow in ﬁlaments forming a tough mass which is visible as ‘mold growth'.
Molds form spores which, when dry, ﬂoat through the air to ﬁnd suitable conditions where
they can start the grth cycle again (Jay, 1996 Pitt and Hocking, 1997). Both yeasts and
molds can thrive in high acid foods like ﬁ'uit, tomatoes, jams, jellies and pickles
(Splittstoesser, 1973). Both are easily destroyed by heat. Processing high acid foods at a
temperature of 100°C (212°F) in a boiling water canner for the appropriate length of time
destroys yeasts and molds (Cuppers et al., 1997; Jay 1996).

Bacteria may grow under a wide variety of conditions. There are many types of
bacteria that cause spoilage. They can be divided into: spore-forming and
nonspore-forming (Bean et al., 1990; Doyle, 1990; Farber et al., 1989; Jay 1996).
Bacteria generally prefer low acid foods like vegetables and meat (Bean et al., 1990;
Fowler et al., 1976; Garg et al., 1990). In order to destroy bacteria spores in a relatively
short period of time, low acid foods must be processed for the appropriate length of time
at 116°C (240°F) in a pressure canner (Giese, 1991; Harvey, 1989; Garg, 1990).
Minimally processed fresh fruits and vegetables are media in which microorganisms can
grow (Addy and Stuard, 1986; Nguyen and Carlin, 1994). Modiﬁed atmosphere

packaged-produce have been involved in outbreaks because of the consumption of

31

products contaminated by pathogens. The potential contamination points along the
produce chain include unclean irrigation sources, unsanitary conditions for harvesting and
ﬁeld packing, low chloride levels for packinghouse washes, repacking operation sanitation,
processing operation controls, and cross-contamination in restaurant kitchens, salad bars
or consumers kitchens (Waterﬁeld, 1996). Outbreaks of food-bome illness in produce
have grown in recent years, although no one knows for sure just how prevalent the
problem is.

The low pH (<45) of most ﬁuits means that spoilage is caused mainly by ﬁrngi
(Jay, 1996; Rose, 1983). On the other hand, the pH range of most vegetables varies
between 5.0 and 7.0 and thus spoilage can be caused by either fungi or bacteria although
the former are again the most important group. In terms of their spoilage characteristics,
ﬁrngi are often somewhat arbitrarily divided into two groups: the plant pathogen which
infect the plant before harvesting and the saprophytic ﬁingi which attack the commodity
after harvesting (Deﬁgueiredo, 1976). An important ﬁmgal spoilage organism is
Penicillium sp. many species of which are able to attack fruit. Perhaps as much as 30% of
all ﬁ'uit decay can be attributed to this genus (Montville, 1987). Many fruit-vegetables
such as tomatoes, cucumbers, apple, strawberry and vegetables such as potatoes, tomato
and beetroot are also susceptible. Another important disease is Rhizopus soft rot which
affect a wide range of ﬁ'uits and vegetables particularly during transit under poor
reﬁigeration (Fields, 1979; Rose, 1983). Harvested strawberries and potatoes are often
attacked by Rhizopus sexualis L. and spoilage is indicated by soft, mushy areas with

greyish mycelium evident in the affected areas (Hayes, 1992). Bottytis cinerea Pers. is

32

another very important fungi particularly in fruits (Snowdon, 1990). Botrytis can attack a
wide range of fruits including apples, appricots, stawberries, blueberries, cherries, citrus,
melons, nectarines, pears, and many others. The fungal survive as sclerotia in the soil and
on plant debries, and under favorable conditions asexual spores are formed. This fungus
can cause heavy losses and is particularly important in pears. It is found in all producer
countries, for example UK, Italy, Germany, Switzerland, Norway, Australia, South Aﬁica
and the USA. Another fungi able to cause spoilage in fresh produce is Colletotrichum
spp. This ﬁrngi causes serious losses of strawberries, tomatoes and avocado (Gupton and
Smith, 1987; Maas and Howard, 1985).

Although bacteria are of little importance in the spoilage of fruit some 35% of
microbial spoilage losses of ﬁuits and vegetables can be attributed to them (Brackett,
1994; Gorris et al, 1996). The bacteria responsible are chieﬂy members of the genera
Erwinia and Pseudomonas (Jay, 1996). The most common spoilage form is bacterial soft
rot which affect most vegetables and some ﬁuit-vegetables such as tomatoes and
cucumbers; soft rot bacteria can also cause pre-harvest infection (Fowler and Foster,
1976; Garg et al., 1990; Lund, 1971). The soft rot bacteria, of which Erwinia carotovora
L. is the most important, are present on the plant at harvest and they usually gain entry
through damaged tissue; even the injury created by cutting lettuce and cabbage ﬁom the
stern can provide an infection site for bacteria causing soft rots (Dennis, 1987; Garg et al.,
1990). Growth of such bacteria is so rapid that molds are unable to compete and are not
normally isolated ﬁ'om commodities infected with bacterial soft rot (Geeson, 1979).

Pectic enzymes are readily formed by the bacteria producing soft, mushy tissues and

33

within a few days there is major tissue breakdown (Rose, 1983). In the case of potatoes
the whole tuber may collapse, with tomatoes the outer skin may remain intact whilst the
entire contents have changed to turbid liquid and with leafy vegetables slimy masses are

produced (Bryant, 1983; Hayes, 1992; Montville, 1987; Ramachandra, 1980).

Many microorganisms gain access to or contaminate plant material during
harvesting or subsequent handling. Therefore, it is desirable to use equipment that is as
clean as possible and to minimize mechanical damage of plant material (Banwart, 1979;
Bryant, 1983; Fields, 1979; Jacob, 1989; Mountney, 1988). Many external organisms can
be removed ﬁom ﬁ'uits and vegetables by being washed in water although washing can
reduce the storage life of vegetables if they are inadequately drained afterward
(Deﬁgueiredo and Splittstoesser, 1976; Hayes, 1992). A suitable storage environment is
essential so that physiological and microbiological deterioration are minimized. Storage is
normally under chilled conditions (5°C) but certain commodities such as potatoes and
cucumbers are best stored at 7-10°C. Pseudomonads are usually responsible for bacterial
spoilage of vegetables stored at 2°C while Erwinia are more common at slightly higher
temperatures (Dennis, 1987; Garcia-Villanova et al., 1987; Jacob, 1989).

Changes to existing processes (eg. in source of raw materials) and the
development of new processes such as MAP and preservation methods may all create new
niches for emerging food spoilage and pathogenic microorganisms (F arber, 1991; Fowler
et al., 1976; Garcia-Villanoava et al., 1987; Hintlian et al, 1986). In the last ﬁve years,
substantial contribution has been made to understand the ways in which the structure of

complex foods inﬂuences microbial growth as well as the effect of local chemical

34

environments on the partition of antimicrobial compounds between the aqueous, oil and
micellar phases of food.

Mathematical approaches have been used in the development of food models to
predict the activity of food microorganisms. These are dynamic models that quantify the
physiological state of the cells and information is used for modeling growth of spoilage
organisms in food. For example, the relation to survival kinetics has also been assessed
for processed food by combining engineering equations for heat ﬂow with microbial
models to allow the extent of growth or inactivation to be predicted in three dimensional
foods when they are subject to thermal gradients (Geeson, 1979). Advanced mathematical
modeling is fundamental to research to study the effect of food structure on microbial
growth and ﬁrrther be used to predict both local difﬁcult-to-measure states and more
global effects. Rapid methods to assess food spoilage and microorganisms are badly

needed.

Microbiology of Modiﬁed Atmosphere-Packaged Produce

As processors move away ﬁ'om traditional preservation methods such as heat
treatment and brine to packaged fresh produce to meet customers' demands, it is not
always possible to rely on the accepted principles concerning microbial growth, survival
and death. Consumers have recently become more aware of the importance of fruits and
vegetables in maintaining health (Bracket, 1994). Consequently, consumers are
purchasing and eating more fresh produce and demanding a greater variety of these

products in the marketplace. This has forced food processors to take advantage of

35

modern technology and transportation to satisfy the consumer's demand (Bracket, 1994;
Wiley, 1994). Process innovation requires a complementary understanding of how
food-home organisms react to new processes or storage conditions (Pitt and Hocking,
1997). Key aspects for technological innovation in the ﬂesh produce industry are:
increased consumption of convenience foods particularly grth in the cook/chill market;
consumer demand for food that have undergone less processing; export potential for fresh
foods and increased consumer awareness of safety issues (Hao and Brackett, 1993). In
the recent past, conventional wisdom suggested that a reﬁigeration temperature from 2.2
to 44°C would generally control outgrowth of most pathogens including pathogenic
psychrotrophic types of microorganism (Wiley, 1994). However, there is considerable
evidence that the low temperature or reﬁigerator temperature treatment is not suﬁicient to
control some psychrotrophic organisms.

Microﬂora responsible for the spoilage of minimally processed hits and
vegetables include a large number of ﬁingi and bacteria species (Varoquax, 1994). MAP
has been known to limit the growth of certain microrganisms (Dennis, 1987). Different
gases have been assessed for their impact on the microﬂora of pre-cut produce (Clark and
Takacs, 1980). However, microorganisms differ in their sensitivity to gases normally
used in modiﬁed atmospheres. Nitrogen is often used in modiﬁed atmospheres but is
primarily used to displace O2 and has little other direct effect on microorganisms
(Brackett, 1994). On the other hand, CO2 has both direct and indirect effects on
microorganisms. The impact of CO2 will depend on the organism in question, the

concentration of gas, and temperature (Clark and Takacs, 1980). The general effects of

36

CO2 are related to displacement of 02, reduction of pH, and interference of cellular
metabolism. When the 02 concentration becomes low enough, modiﬁed atmospheres can
select for facultative or obligate anaerobes (Brackett, 1994).

Another issue in MAP system has been the alarm of possible hazards related to the
ﬁhn material itself (Hartman et al., 1990; Manura et al., 1990). There is presently a great
deal of concern for the potential health risks due to the consumption of food products
contaminated with packaging material residues. When using these packaging materials for
food preparation at high temperatures (the boiling point of water) such as those
encountered in microwave ovens, the problem may become exacerbated by the thermal
leaching of the packaging material residues from the packaging ﬁhn and eventual diffusion
into the food products (Hartman et al., 1990; Manura et al., 1990). This concern for the
contamination of food products by leaching of residual components ﬁ'om the packaging
materials will continue to be of major concern for both the scientiﬁc community and the
food industry especially with the increased use of recycled papers and plastics. There are
technologies available to detect volatiles emitted by wrapping ﬁlms which could
compromise the safety of the packaged product. Manura (1991) conducted a study to
detect and identify low levels of volatiles and semi-volatiles present in commercially
available microwave safe plastic food wraps, and to determine the viability of this
technique for the analysis of other packaging material analysis. He found that plastic food

wraps release detectable material residues, when food are exposed to higher temperatures.

37

Detection and Identiﬁcation of Microorganisms in Food

Analytical microbiology is a relatively new discipline that has arisen on the
interface between analytical chemistry and microbiology (Fox and Black, 1992). In the
1960's and 70's microbial chemistry established the composition of many major structural
components of bacteria (Fox and Black, 1992; Roboz, 1992). These structures include
peptidoglycans, liposaccharides and teichoic acids. Among physicochemical techniques,
mass spectrometry often offers the unique combination of great speed, high speciﬁcity,
and excellent sensitivity (Berkeley et al., 1992; Board et al., 1992). These features are
helpful for detection and characterization of microorganisms when the public health is
threatened by contaminated food (F enselau, 1992). Lipid biomarkers can be recovered
from isolates and environmental samples by single-phase chloroform/methanol extraction,
fractionation of the lipids on salicilic acid, and derivitazation prior to analysis by GC-MS
(White et al., 1992). Bacterial polysaccharides contain a diverse collection of unique
sugar monomers that can serve as markers to identify speciﬁc bacterial species or genera
(Fox and Black, 1992).

A multitude of species—speciﬁc biomolecules are needed by each microbial species
to perform the ﬁinctions to ﬁll, and survive in, its niche in nature (Cole and Enke, 1992).
The natural selective forces imposed on different microorganisms have required microbes
to adapt and optimize speciﬁc sets of biomolecules for survival in speciﬁc environment. A
few examples of these biomolecules include the lipids needed for membrane ﬁrnction and
integrity, the respiratory quinones needed for metabolism, and the polysaccharides needed

for protection from the environment (Cole and Enke, 1992; Fenselau, 1992). There are

38
technologies available, including chromatographic analysis, to identify and quantify a
number of these biomolecules. The detection of toxins by direct sensing of the speciﬁc
binding reaction of the toxin (antigen) with a speciﬁc antibody in ELISAs is a very
accurate method (Doyle et al., 1997; Hobbs, 1993; Troller, 1983). However, the
impossibility of doing real-time evaluation has limited its used at a wider scale (Bryant,

1983; Montville, 1987).

Food Safety and Control Systems

Reports of food-bom illness associated with foods in developed countries indicate
that contamination of raw products can be a health problem (Jacob, 1989). However, in a
worldwide basis, most such illness is caused by foods that have been mishandled or
mistreated during preparation (Klontz et al., 1995). Even though control at a single point
along the production chain may be effective in combating contamination, there are
considerable potential beneﬁts in approaches which eliminate or reduce microbial loads
throughout the process (Jay, 1996; Pan et al., 1997). Heavy levels of contamination could
be more difﬁcult to completely eradicate, or reduce to acceptable levels, ﬁrrther along the
chain. The risk of cross—contamination may also increase if contaminated surfaces,
equipment or food processors and handlers, come into contact with treated product (Bean
et al., 1990; Garg et al., 1990; Giese, 1991).

While it is ﬁ'equently not possible to entirely prevent the contamination of foods by
pathogenic organisms, the safety of such foods can be assured by systematic action to:

l) minimize the contamination of raw foods whether for direct consumption or for ﬁnther

39

processing (this includes animal husbandry and agricultural practices at the primary level),
2) destroy remaining organisms of concern in processed foods through processing
operations and 3) properly formulate and use correct practices (refrigeration, freezing,
hygiene etc.) in the packaging, storage and distribution of food, and in its preparation for
consumption (Banwart, 1979; Giese, 1991).

Control of food-bome pathogens at the production stage can be effected
systematically by: identifying the bacteria of concern, identifying the sources of
contamination, and taking action to effect control of contamination at source (Giese,
1991; Mountney and Gould, 1988). It also helps to identify appropriate control points and
develop processes for destroying the remaining pathogens of concern, preventing
subsequent re-contamination of product by the use of proper hygiene and handling
practices, and appropriate packaging materials, and/or creating an environment which is
unfavorable for the growth of the pathogen(s) of concern (Klontz et al., 1995; Monk et
al., 1995; Troller, 1983). Some of these practices include physical controls (such as
drying, lowering water activity, or storing the product at temperatures which do not favor
bacterial growth), or controlling storage times if appropriate storage temperatures cannot
be guaranteed ( Rajkowski et al., 1995; Ray, 1996).

Potential problems related to processed and minimally processed products are of
concern to the fresh produce industry. Microbial food safety issues to date have probably
not been addressed to the extent they should have, as activities have focused on pesticide
residue issues (Bean et al., 1990; Garbutt, 1997; Klontz et al., 1995). Presently there are

testing programs in place for coliforms, Salmonella and yeasts and molds in nuts. While

40

the issue of cross-contamination during the preparation of ready-to—eat salads is an area of
particular concern (Gorris et al., 1996; Hao and Brackett, 1993; Lund, 1971), there are no
similar testing programs in place.

Microorganisms, microbial metabolites and viruses of human health concern can
enter the food chain on the farm or growing environment, during transportation,
processing, as a result of poor hygienic practices, mishandling of food after processing and
during food preparation in homes and catering establishments and retail food outlets.
While a particular food may be safe at any given point, it can always be subsequently
contaminated by other foods, utensils, equipment and by food handlers (Charalambous,
1986; Montville, 1987; Troller, 1983).

In raw foods, improper handling, which favors bacterial growth normally, but not
always, causes spoilage organisms to grow to levels where visible signs of spoilage occur
and the food is clearly unﬁt for human consumption (Giese, 1991; Troller, 1983).
Pathogens that are present on such foods are unlikely to pose a signiﬁcant risk, as the
foods are normally discarded without consumption. Additionally, cooking destroys most
of the pathogens of concern. For this reason, raw foods, when they are cooked and
consumed immediately thereafter, usually present a low microbial risk to consumers (Jay,
1996). Processed foods are, however, different from raw foods. Processing, involving
high temperatures or bactericidal activity, usually destroys most of the natural spoilage
organisms that may be present on a raw food (Doyle, 1990; Garg et al., 1990; Hintlian and
Hotchkiss, 1986). Ifthe composition of the food or subsequent storage conditions (e. g.

poor temperature control of chilled foods), permit the growth of pathogens which have

41

survived processing, processed foods can pose a hazard to consumers before they show
obvious signs of spoilage.

Foods such as traditional vegetable dishes and stews which are cooked at
temperatures which kill any vegetative organisms and are then immediately consumed, are
arguably among the safest (Harvey, 1989; Rose, 1983). Even here there is a risk that some
heat-tolerant toxins may survive the cooking process. The fresh food industry needs rapid,
sensitive, selective and reliable method for the detection of the toxins or bacterial
ﬁngerprint compounds to ensure food safety (Doyle et al., 1997; Eley, 1996; Giese, 1991;

Roberts and Skinner, 1983; Sharpe, 1985).

Hazard Analysis of Critical Control Points (HACCP)

HACCP is a preventive measure inspection program that assesses hazards,
estimates risks, and establishes speciﬁc control measures that emphasize prevention rather
than reliance on end-point testing. HACCP is a system of science-based process controls
designed to identify and prevent physical, chemical and microbial hazards. The system is
designed to prevent problems before they occur, and to correct deviations as soon as they
are detected (Anderson, 1994; Kalish, 1991; Sarlett, 1991; Sperber, 1991 ). The HACCP
approach is a system of process controls that is widely recognized by scientiﬁc authorities
and international organizations and is used extensively in the food industry to produce
products in compliance with health and safety requirements. HACCP started in the 1960’s,
when National Aeronautic and Space Administration (NASA) was looking for a way to

guarantee that the food for astronauts on space ﬂights was totally safe (Sperber, 1991).

42

They gave the task of producing "Zero Defect" food to the Pilsbury Corporation who
responded by developing the system of Hazard Analysis Critical Control Points or
HACCP. From this original model has sprung most of our modern thinking about food
safety, whereby we look systematically for potential risks and then identify appropriate
control and monitoring systems, concentrating on those deemed critical to the safety of the
product. HACCP has today been the subject of an enormous amount of study (Sperber,
1991; Weilgold et al., 1994). It has been incorporated into the World Health
Organization/Food and Agriculture Organization standard, the Codex Alimentarius and is
now required of all food businesses in Europe under EC Directive (Dean, 1990; Kalish,
1991; Sperber, 1991; Weingold et al., 1994). By using a HACCP system, control is
transferred from end product testing (i.e. testing for failure) into the design and
manufacturing of foods (i.e., preventing failure). There will, however, always be a need for
some end product testing, particularly for on-going veriﬁcation of the HACCP process.
The HACCP system has seven principles:
1. Conduct a hazard analysis. Prepare a list of steps in the process where
signiﬁcant hazards can occur and describe the preventive measures.
2. Identify the critical control points (CCPs) in the process.
3. Establish critical limits for preventive measures associated with each identiﬁed
CCP.
4. Establish CCP monitoring requirements. Establish procedures for using the
results of monitoring to adjust the process and maintain control.

5. Establish corrective actions to be taken when monitoring indicates that there is

43

adeviation from an established critical limit.

6. Establish effective record-keeping procedures that document the HACCP

system.

7. Establish procedures for veriﬁcation that the HACCP system is working

correctly.

HACCP systems have to be accompanied by other preventive measures. Food
safety during transportation, storage, retail sale and preparation are the next important
links in the farm-to-table chain (Stevenson, 1990). Safe handling beyond the plant
becomes the responsibility of distributors, employees in retail stores and restaurants, and
consumers.

The FDA has worked very hard to develop standards governing the safety of foods
during transportation and storage and to support local and state authorities in their efforts
to improve food safety practices at the retail level (Tisler, 1991). As progress is made in
reducing contamination that occurs in the farm-to-retail process, increased awareness of
proper storage, handling and preparation practices in the home and in foodservice facilities
becomes even more critical in maintaining the safety of fresh produce. In addition to their
strong support for the government inspection service's transition to a more science-based
inspection program, fresh produce industries have invested millions of dollars US. to
develop HACCP plans and new technologies to reduce the risk of bacterial contamination.
For instance, in recent years, millions of dollars US. have been invested in research

designed to reduce the risk from E. coli 01572H7 (Rajkowski, 1995; Tisler, 1991).

44

Food Quality and Food Quality Assessment

The consumer expects a wide range of competitively priced food products of
consistently high quality. As the technology of agriculture and food preparation have
advanced and as populations have increased, the analytical problems concerning food have
become very complex. Problems faced include, among others, discerning of food products
of different quality, frauds, food preservation and development of novel food (Boelkel,
1996; Jay, 1996; Sims and Rodrick, 1995). Moreover, production processes for
preservation of nutritious food have become very important because of the need to store
food for long periods and to prevent chemical and microbiological deterioration, insect
infestation and bacterial contamination. Fierce competition in a saturated market has
forced food producers to introduce increasingly more new products to the market.
Modern food technology can lead to the production of components in food which possess
new signiﬁcant biological effects and result in the formation of unknown chemical
compounds, and microbiological development (Bianco, 1984; Diaz et al., 1997;
Charalambous, 1986; Murphy, 1983; Thorner and Manning, 1983; Troller, 1986). One of
the most common methods to assess quality of food is by sensory evaluation (SE). SE
uses selected consumers and/or trained panelists to assess the characteristics of product
under controlled conditions and using appropriate techniques (Cichy, 1983; Heintz and
Kader, 1983). SE provides feedback on eating quality, gives an estimation of shelf life,
allows analysis of product failure, indicates product development direction, provides a
quality assurance check and ingredient substitution (Chambers et al., 1981; Harries and

Smith, 1991). The major drawback of sensory analysis is the subjectivity of the results

45

and high variability. SE is different from other type of analysis that may be more difficult
to perform, but could offer greater precision and reproducibility (Gunasekaran and Ding,
1994; Martin, 1971; McIntire, 1983; Resurreccion, 1988; Tarrant, 1980).

Volatile and semi-volatile organic compounds are largely responsible for the ﬂavor
qualities of the foods we eat (Kader et a1, 1985) . Flavor is always considered one of the
most important factors of any of the quality categories when comparing various
horticultural products. Fresh produce, particularly fruits provide a great sense of eating
pleasure for their characteristic ﬂavor and taste as well as have an important role in a well
balanced diet.

Benady et a1. (1994) developed an electronic sniffer to measures ﬁ'uit ripeness
rapidly and nondestructively by measuring the aromatic volatiles that are naturally emitted
by ripening ﬁ'uit. In this study, the potential of using a ‘sniifer’ in the quality sorting of
blueberries was evaluated. Blueberries were ﬁrst visually classiﬁed into four distinct
ripeness classes: unripe; half-ripe; ripe; and over-ripe and quantitatively measured for
color, ﬁrrrmess, TSS, and sugar acid ratio. Ripeness classiﬁcation accuracy with the sniffer
matched or exceeded that of all other ripeness indices. The sniﬂ‘er diﬂ‘erentiated unripe,
ripe and over-ripe berries within one second, but could not distinguish between the unripe
and half-ripe class. Detection of 1 to 2 damaged or 1 to 2 soft ﬁuit spiked within a large
container of 24 to 37 high quality ripe fruit was also achieved, but required a response
time of 10 seconds. Electronic sensing of aromatic volatiles may be a useful new
technique in the grading and sorting. Simon et a1. (1980) reported a study on carrot

volatiles collected on porous polymer traps. In this study, an accurate and precise porous

46

polymer trapping method was adapted for the gas-liquid chromatography analysis of
volatiles from raw carrots. It was found that more volatiles are synthesized in the crown
of roots than in midsection or tip sections and that terpinolene and caryophyllene levels
were higher in the phloem than in the xylem of roots. Fragrance and odor characteristics
are critically important to many commercial products and applications (Gray and Robert,
1970; Lindsay, 1984; Kokini et al., 1977). Volatile analysis of hits and vegetables has
been gaining importance as a tool to assess the quality of produce. Consumers rely a great
deal on their perception of aroma of the food they buy to assess quality. This is
particularly true for produce.

Chromatography as a method of instrumental analysis is capable of
producing information which may describe the qualitative and quantitative composition of
mixtures of compounds (Schomburg, 1990). The two important parts of the
chromatographic system are the column, in which the separation takes place, and the
detector. The carrier gas supply unit, the main component of which is a controller for
pressures and ﬂows connected to a gas cylinder (for H2, He, N2, Ar) or to a liquid source
(for N2 or Ar) generates an optimized gas ﬂow through the column (Schamburg, 1990).

Analytical mass spectrometry (MS) was introduced commercially in 1941. For
two decades its main application was quantitative analysis of light hydrocarbon mixtures
and similar samples, often with accuracies of plus and minus 1% absolute ( McLaﬁ’erty
and Turecek, 1993; Turro, 1980). Gas chromatography (GC) is the most common
combination of chromatography interfaced directly to mass spectrometry (Anon, 1995).

For all GC/MS combinations, the data consist of a series of mass spectra that are acquired

47

sequentially. Instrument systems combining GC and MS have proven to be of high value
for analysis of complex mixtures, such as those ﬁom human biological ﬂuids, plant
extracts, pollutants, industrial processes, and samples for forensic study (Anon, 1995;
McLafferty and Turecek, 1993). A variety of detectors and column types can be used in
gas chromatography to assess the chemistry of food ﬂavor. The combination of gas
chromatography/mass spectrometry (GC/MS) analysis is becoming increasingly prevalent
due to the advantages it offers in terms of spectral information for peak identiﬁcation
along with high resolution and sensitivity (Hartman et al., 1990; Manura et al., 1990 ;
Schomburg, 1995; Yeung et al., 1995). The speciﬁc instrument conﬁguration will depend
on the requirements of the analysis, and different options can be used even for a particular
method, such as that used by the Environmental Protection Agency (EPA) low-level
volatiles in drinking water.

GC-MS technology has been utilized for the detection and identiﬁcation of volatile
organic, aromas and ﬂavor components in milk over time and how these differences in
volatile organic composition relate to the quality of milk (Careri et al., 1990). This
analytical technique can be utilized for the quality control during the production of milk as
well as a technique for the detection of potential contamination of milk. GC/MS systems
have successfully been used to characterize the aroma and ﬂavor of fruits and vegetables
(Song et al., 1997).

Volatile metabolites produced by microorganisms can be measured directly in
growth media by membrane inlet mass spectrometry (Lauritsen and Lloyd, 1992;

Schomburg, 1990). Membrane inlet mass spectrometry has become a popular technique

48

for the direct measurement of gases and volatile organic compounds in aqueous solutions.
The technique has been used especially for on-line measurements of dissolved gases in
bioreactors, kinetic studies of chemical and biological reactions, identiﬁcation of volatiles

produced by microorganisms and environmental monitoring (Lauritsen and Loyd, 1992).

SPME Sampling System

Solid phase extraction (SPE) has become a widely used technique in the analysis of
food. Since its introduction to the analytical community in the 1970’s (Hurst, 1991), it
has gained a wide acceptance. A relatively new SPE technique has been commercialized
and is referred to as solid phase micro extraction or SPME. SPME is designed to be used
with a gas chromatograph. A 1998 on-line search performed using the Yahoo and Alta
Vista web search engines to determine the number of citations on the use of SPME in food
analysis since 1991 garnered almost 300 citations.

Sample preparation techniques have been developed using SPME preconcentrate
trace compounds and/or separate analytes from sample matrix (Anon 1996; Arthur and
Pawliszyn, 1990; Arthur et al., 1990; Arthur et al., 1992; and Zhang et al., 1993). A
SPME unit consists of a length of ﬁised silica ﬁber coated with a polyacrylate or
polydimethylsiloxane phase (Anon, 1996). The ﬁber is attached to a stainless steel
plunger in a protective holder. When the SPME ﬁber is immersed in an aqueous sample, a
partitioning of the compounds in the sample between the aqueous phase and the ﬁber
surface occurs (Yang, 1994; Matich et al., 1996). SPME requires no solvents or

complicated apparati. It can be used to concentrate volatile and nonvolatile compounds in

49

both liquid and gaseous samples (Yang and Peppard, 1994).

Yang and Peppard (1994) conducted a study using SPME, headspace and liquid
sampling to test 25 common ﬂavor components in dilute aqueous solutions. They found
out that the addition of salt generally enhances SPME absorption and that larger sample
volume and a smaller volume of headspace over the liquid sample also increase the
sensitivity of SPME-based analysis. Matich et al. (1996) investigated SPME for use in the
quantiﬁcation of aroma volatile production by ‘Granny Smith’ apples during cool storage.
Matich et al. (1996) suggests that collection of high molecular weight volatiles by SPME
is hindered by the slow transport of analytes into the gaseous phase, which results in long
equilibration times and headspace depletion of analytes during sampling, and by adsorptive
losses onto walls of containers. Steﬂ’en and Pawliszyn (1996) investigated the ﬂavor
compounds of orange juice by using headspace SPME. They found that the addition of
salt to the matrix signiﬁcantly enhanced the amount of analytes extracted into the ﬁber
coating and that the partial removal of suspended solids ﬁ'om the juice was found to
enable a standard addition quantitation of the target analytes.

Song et al. (1997) examined SPME and time-of-ﬂight mass spectrometry for their
suitability and compatibility for rapid sampling, separation and detection of apple ﬂavor
volatiles. This study suggests that ﬂavor-contributing volatiles compounds are of
relatively high partition coefﬁcient on a 100 rim-thick SPME coating of
polydimethylsiloxane (PDMS). The time required to saturate the PDMS coating was
dependent on the absorbed material, varying from less than 2 minutes to greater than 30

minutes. Theyn also determined that GC response was linear across several orders of

50

magnitude. SPME has been used to compare various packaging materials to assess their
suitability for storing and shipping analytical materials (Penton, 1997) as well as in the
characterization of volatiles in wines (Penton, 1997 ; Yang and Peppard, 1994). In order
to make effective use of SPME, there needs to be an additional concentration step prior to
separation by chromatography. Generally, this is accomplished by cooling the GC inlet or

a portion of the GC column in a proces known as cryofocusing.

Cryo-cooling Technology

A cryofocusing trap consists of a small chamber cooled with liquid nitrogen
through which the volatiles pass. Cryofocusing permits the concentration of analytes in a
narrow band in the column due to the decrease of the kinetic energy of the gas exerted by
the abrupt decrease in temperature (Hartman et al., 1991; Manura et al., 1990).
Concentrating the analyte then rapidly heating improves instrument response, achieving a
higher resolution. Cryofocusing units are usually placed inside the GC oven just under the
GC injection port. Some cooling devise are directly installed into the GC oven.

Alternatively, the GC entire oven is cryofocussed using liquid C02.

Thesis Research

We are presenting the results of a research project in which we have used a
combination of SPME sampling method and time-of-ﬂight mass spectrometry for volatile
analysis of whole strawbeny (Fragaria ananasa Duch.), minimally processed carrot

(Carota daucus L.), and broccoli (Brassica oleracea L.) to assess the physiology and

51

microbiology of these products. We have evaluated the changes of volatiles produced by
strawberry during storage and studied the relationship of those volatiles with the process
of senescence (Chapter 11). Chapter III deals with the monitoring of senescence-related
volatiles of carrot and broccoli. In Chapter IV we present the results of the project
dealing with the identiﬁcation of ﬁngerprint volatiles produced by E. coli and S.
niphimurium grown in various substrates including ﬁuits and vegetables. We have also
investigated the impact of modiﬁed atmosphere packaging on aroma production by

strawberry (Chapter V) and by carrot and broccoli (Chapter VI).

52

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64

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66

CHAPTER H

SENESCENCE—RELATED VOLATILE PROFILES OF STRAWBERRIES
(Emzan'a mamas Duch.)

67

SENESCENCE—RELATED VOLATILE PROFILES OF STRAWBERRIES
(Engaria ananassa DuchJ

Additionaljndexmrds. methyl acetate, aroma, volatiles, ripening, fruit aging

Abstract. Volatiles of strawberry stored under reﬁigerated air (2°C) changed as ﬁuit
senesced. Esters were the dominant volatile class. Of ester types, those with a methyl
group on their alcohol portion comprised proportionally 74.64% of total , ethyl esters
comprised 11.48%, propyl esters comprised 8.28%, and hexyl esters made up 5.60%. All
ester classes decreased during storage, except methyl esters, which increased slightly over
time. The production of methyl acetate was found to negatively correlate with loss in
ﬁrmness, suggesting that the production of this volatile may be related to the cellular
breakdown of the hit. The ratios of methyl acetate production to the product of other
methyl esters (methyl butanoate, methyl hexanoate, ethyl butanoate and ethyl hexanoate)
increased continuously as the ﬁ'uit senesced. Methyl acetate production was also found to
have a good correlation with extractable lipoxygenase and peroxidase activities
suggesting thereby that methyl acetate may be linked to senescence-related enzymatic
changes of strawberry during storage. Methyl acetate is a volatile compound that appears

to have potential for use in assessing the physiological status of stored strawberry.

68

Aroma is an essential component of food ﬂavor, particularly in fruit, and
constitutes one of the key elements in food quality. The ﬂavor quality of food is a
combination of the sensory impressions of taste detected by the tongue and aroma sensed
by the nose (Manning, 1993). To assess quality, consumers rely a great deal on their
perception of the aroma of the food they buy. Understanding of the biochemistry of
naturally-occurring aroma of fruit is of great importance to the food industry for
determining optimal harvesting dates, storage conditions and shelf life. The relative
amount of particular volatiles and type of volatiles are like “ﬁngerprints” for different ﬁuit
species. Very little has been done to evaluate the volatile proﬁle of ﬁuit, including
strawberry, directly ﬁom the ﬁeld versus hit that have been stored for any length of
time. Therefore, little is known about changes of volatile proﬁles as the ﬁuit senesces.
Nevertheless, volatiles of strawberry have been thoroughly identiﬁed and characterized
(Douillard and Guichard, 1990; Pyysalo et al., 1979). The aroma biosynthetic process yet
to be fully understood. Esters, alcohols and carbonyls are important for strawberry ﬂavor
(Dirinck et al., 1981). It seems unreasonable to expect a different enzyme to catalyze the
formation of each compound as this would require a considerable expenditure of energy
for protein synthesis (Manning, 1993). At the same time, it seems logical to think that
more than one enzyme is responsible for aroma production in strawberry.

New advances in the ﬁeld of gas chromatography, mass spectrometry and
headspace sampling have made possible detection of volatiles with sumcient rapidity to
permit real-time monitoring (Arthur et al., 1992; Schomburg, 1990; Song et al., 1997).

Work is under way in the development of sensors for volatiles or classes of volatiles at the

69

part-per-billion level (Hetzroni et al., 1995). Volatile sensors for industrial process
control systems are already in use. In addition, the ﬁeld of biosensor applications
continues to expand. With regard to volatiles emanations from plant products, there
already exists a biosensor for alcohols (Chematics, Inc., North Webster, Inc.). Thus,
volatile sensing in the headspace of packaged products has the potential to be performed
as an in-package biosensor or via automated, rapid instrumentation

In this work, we have evaluated the changes of stored-strawberry volatiles over
time to identify those volatiles that could potentially be used as senescence indices. Physic
and biochemical analyses were performed to assess the physiological status of the fruit and
to thereby establish the relationship between the senescence state and the production of
particular volatiles. Physicochemical parameters include changes in ascorbic acid, citric
acid and pH. Biochemical parameters included changes in the senescence-related enzymes
lipoxygenase and peroxidase. We have also examined the pattern of volatile production
by strawberry during storage and tried to understand the biochemical nature of volatile

production by the fruit while it senesces.

Material and Methods
Plant material. Strawberry (Fragaria ananasa Duch. cv. Kent) were obtained as
fresh as possible ﬁom the Detroit Produce Terminal. Fruit were sorted for visible defects
and ﬁirther sanitized in a solution of 0.5 g'L'l Benlate (Du Pont de Nemours C0,,
Agricultural Product Department, Wilmington, Del.) and 0.5 g-L‘l Rovral ( Rhone-

poulenc Ag. Company, Research Triangle Park, NC.) and then rinsed with distilled water

70

and allowed to air-dry. Approximately 400 g of the hit were placed in ventilated glass
jars. A stream of air was passed through each jar at a ﬂow rate of 10 to 15 mL-min".
Three jars were used for continuous gas measurement (CO2 and Oz) and volatiles. One jar
containing about 20 berries was used for ﬁrmness measurements. Fruits used for ﬁrmness
were taken out every day for measurement. Firrniness was achieved by using the
F irmTechl Instrument (BioWork Co., Stillwater, Okla). FirmTechl Instrument measures
ﬁrmness by individual compression tests of each hit, i.e., each fruit is squeezed much in
the same manner as a consumer might do with their ﬁngers. As the fruit is squeezed in a
step manner, the force increases. The rate at which the force increases (g-mm") is deﬁned
as ﬁrmness. Berries in six other jars were used for destructive physicochemical and
biochemical analysis. These analysis included soluble solid, pH, ascorbic acid, citric acid
and enzymatic evaluations. Four or ﬁve berries were taken out every day for this purpose.
Analysis of volatiles. Volatile collection was accomplished using solid-phase
micro-extraction (SPME) device (Supelco, Inc., Bellefonte, Pa.) A ﬁber with a 100 pm-
thick, non-bonded, polydimethylsiloxane-based stationary phase was used. The absorption
time was 5 min. After absorption, volatiles were then thermally desorbed from the ﬁber
coating by directly inserting the ﬁber into the injection port (200°C) of a gas
chromatograph (HP-6890, Hewlett Packard C0,, Wilmington, Del.) for 90 s. Gases were
cryofocused on-column using liquid N2 and separated using a gas chromatograph ﬁtted
with a 10-meter, 0.1 mm ID, DB-5 capillary column (Hewlett Packard Co., Boise,
Idaho). The detector was a time-of-ﬂight (TOF) mass spectrometer (Pegasus H, LECO

Co., St. Joseph, Mich.) The TOF mass spectrometer permitted the resolution and

71

quantiﬁcation of compounds not separated chromatographically. Identiﬁcation of volatile
material was done by comparison of the spectra and GC retention times with authenticated
standard compounds. High purity (>95%) authenticated standards obtained from Aldrich
(Aldrich, Co., Milwaukee, WI). For the standard compounds, a mix of the targeted
compounds was prepared in a 4.85-L jar (standard jar), equipped with a self-sealing
rubber sampling port. The mix was prepared by ﬁrst placing 50 uL of each compound
into a 2.5-mL vial to generate a stock mix. Then, 10 pL were taken from the stock mix
and placed into the 4.85-L jar to be used as standard. Standard preparation was done at 5
°C and the standard jar was kept at the temperature of the experiment. Calculation of the
concentration of standard compounds was expressed in ppm. As sampling ﬁ'om the
packages was done, so was from the standard to estimate the actual production of the
targeted compounds regardless of the sensitivity of the detection system. Data were
reported in umol-kg'l-h".

Lipoxygenase activity. A modiﬁcation of the method of Lester (1990) was used as
extraction protocol for lipoxygenase. Fresh strawberry (10 g) were frozen by dipping fruit
slices into liquid N, Fruit were then kept at -80 °C until use. For extraction, samples
were grounded using liquid nitrogen in a mortar and by using a pestle. The powder was
placed in a beaker containing 40 mL 0.05 M potassium phosphate buffer adjusted to pH 7
containing 61.5 mL 0.5 M monoacid potassium phosphate (KZHPO4) and 38.5 mL of 0.5
M biacid potassium phosphate (M KH2P04). After shaking and blending for 30 s, the
homogenate was ﬁltered through miracloth and ﬁirther centrifuged (RCSC, Sorvall

Instrument Co, Duont, Colo.) at 4000x g for 15 min. The supernatant was used for the

72

enzyme assay. For the substrate preparation, 0.5 mL Tween 20 was dissolved in 10 mL
0.05 M potassium phosphate (pH 7.0) and 0.5 mL linoleic acid was added, as described by
Surrey (1964). The linoleic acid was added drop by drop to facilitate solubility. The
contents were throughly mixed so as to dispose the acid into a ﬁne emulsion. Following
this, 1.3 mL of 1 N NaOH was added and the mixture was again agitated until a clear
solution was obtained. To this solution, 90 mL of the potassium buﬂ‘er was added and the
ﬁnal volume was brought to 200 mL with distilled water. For the assay cocktail, 150 aL
of the enzyme extract was added to 2.85 mL of the linoleic acid substrate solution in a
quartz cuvette and mixed. The lipoxygenase activity was measured from the change in
absorbance at 234 nm over time using a spectrophotometer (Model 300 Hitachi
Instruments, Inc., Naperville, Ill.) The assay temperature was 25°C. One unit of enzyme
activity was deﬁned as the amount of protein that produced a change of 1 unit in
absorbance. Enzyme activity was expressed in umol'l-rnin-g'l in a fresh weight basis.
Peroxidase activity. A combination of the Prestamos and Manzano (1993) and
Rodriguez-Saona et al., (1995) methods was used for the peroxidases (s) enzyme
extraction from strawberry. Fresh strawberry (10 g) were frozen by dipping fruit slices
into liquid N, Frozen tissue was then kept at -80 °C until use. For extraction, samples
were grounded using liquid nitrogen, mortar and a pestle. Ten grams of strawberry
powder was placed into a beaker containing 40 mL 0.1 M phosphate buffer at pH 7.8.
The mixture was blended by using a Waring blender for 30 s and the homogenate was
ﬁltered through miracloth and ﬁrrther centriﬁiged (RCSC, Sorvall Instrument, Duont Co.)

at 17000x g for 30 min at a temperature of 4 °C. The supernatant was used as enzyme

73

extract. For the peroxidases substrate preparation, a modiﬁcation of Kar and Mishra
(1975) method was used. Catechol was used as substrate. 3.3 g of 99% catechol was
dissolved into 40 mL of distilled water to yield a 0.3 M catechol solution. One mL of
30% H202 was diluted into 30 mL of distilled water to create a 0.1 M solution of H202
which served as the electron donor. The assay cocktail for the spectrophotometric
determination of peroxidase activity consisted of 2 mL of 0.07 M phosphate buﬁ‘er (pH
5.9), 0.4 mL of catechol solution, 0.2 mL of the 1% H202 solution and 0.2 mL of freshly
prepared strawberry extract. The reference cuvette consisted of 2.5 mL of the buffer.
Measurements were made at 420 nm and activity was calculated based on the increase in
absorbance during the ﬁrst 30 s of the reaction. A unit of peroxidase activity is the
increase of absorbance of 0.001 per min. Results were expressed in pmol-'.rrrin"-g’l in a
fresh weight basis calculated by considering an extinction coefﬁcient of 25.5 cmz-jrmol'l at
wavelength of 420 nm and at a temperature of 25 °C.

Physicochemical Analysis. For pH measurements, 50 g mesocarp tissues from
strawberry fruit was homogenized in a blender for 2 min. The pH of the resulting puree
was measured with a pH meter (Accumet, Fisher Scientiﬁc Co., Liberty Lane, NH). For
the determination of citric acid, a modiﬁcation of the Lee and Lee (1992) method was
used. Strawberries were blended for 2 min and strained through 4 layers of cheesecloth.
Ten mL of juice was diluted with 90 mL of distilled water and titrated with 0.1 N NaOH
to pH 7. Citric acid was expressed in % on w/w basis. Ascorbic acid was determined by
the method described by Ruck (1963). A 30 g portion of homogenised sample was

blended with about 100 mL of 0.5% oxalic acid for 2 min in a Waring blender. The

74

blended misture was made to 500 mL with 0.4% oxalic acid and was ﬁltered; 20 mL of the
ﬁltrate were titrated with standard 2,6-dichorophenol inophenol. Results were expressed

as mg per 100 g fresh weight.

Results

Strawberry volatile proﬁle. Erighteen volatiles of strawberry stored under
reﬁigerated air (2 °C) could be detected (Fig. 1). Some strawberry volatiles increased
over time while others decreased (Table 1, Fig. 2). Volatiles that steadily increased as the
fruit senesced included methyl acetate, hexyl acetate, and 3-buten-2-one. Volatiles that
decreased included methyl butanoate, methyl pentanoate, methyl hexanoate, methyl
octanoate, ethyl butanoate, hexyl hexanoate and 2,5-dimethyl-4-hydroxy—3 (2H) ﬁrranone.
Esters were the dominant volatile class. Of ester types, those with a methyl group on their
alcohol portion comprised proportionally 74.64%, ethyl esters 11.48%, propyl esters
8.28%, and hexyl esters 5.60% when averaged over the duration of the study. The total
amount of esters declined as the fruit senesced (Fig. 2A). The proportion of methyl esters
slightly increased over time relative to the other classes of esters (Fig. 2B). However, the
only methyl ester that increased as the ﬁuit senesced was methyl acetate (Fig. 3). Both
methyl butanoate and methyl hexanoate decreased while methyl propanoate and methyl
octanoate were unchanged throughout storage. The ratios of methyl acetate to methyl
butanoate, methyl hexanoate, ethyl hexanoate and ethyl butanoate were evaluated. The
four ratios showed a continuous increasing trend during storage (Fig. 4). The ratios of

methyl acetate over most of the other strawbeny esters showed an increasing trend

75

throughout storage (data not shown).

Methyl acetate production as related to changes in ﬁrmness and some chemical
changes. The production of methyl acetate by strawbeny during storage correlated (r2
=0.86) with the rate of softening of the fruit (Fig. 5). Methyl acetate production was
negatively correlated with citric acid (r2 = 0.8), ascorbic acid (r2 = 0.96) and positively
correlated to soluble solid (r2 = 0.8) and pH (r2 = 0.92) (Fig. 6).

Volatiles and Senescence-related enzymes. Extractable lipoxygenase and
peroxidase activity increased as the hit senesced (Fig. 7). Methyl acetate correlated with

both lipoxygenase (0.9) and peroxidases activities (0.9).

Discussion

The increase of some aroma compounds of strawberry while others decrease as the
ﬁ'uit senesces gives way to several thoughts. First, the data indicate that there is not a
single enzymatic system or a single metabolic pathway responsible for the aroma
production in strawberry as suggested by Hamilton-Kemp (1996). Second, the data also
suggest that the activities of the enzymes that catalyzes the biosynthesis of volatiles in
strawberry are not uniformly aﬁected by the senescence process, resulting in the
suppression of some volatiles and in the enhancement of others. This ﬁnding is consistent
with results published by Yamashita et al. (1977). Third, the data may be taken to mean
that the degradation products of some volatiles are necessary for the formation of other
volatiles as reported by Yamashita et a1. (1976) and as shown in our results when

evaluating the ratios of methyl acetate to methyl butanoate, methyl hexanoate, ethyl

76

hexanoate and ethyl butanoate. As some substrates such as butanoate become limiting,
the ﬁuit necessarely favors the use of others substrates such as acetyl-CoA. Fourth,
perhaps, the genes responsible of expressing the enzyme (s) which catalyze the
biosynthesis of aroma in strawberry, are differentially activated by the phenomenon of
senescence, resulting in the increase of the expression of particular enzymes and a
decrease in the expression of others.

The increase in lipoxygenase may result in a higher peroxidative modiﬁcation of
polyunsaturated fatty acid and ultimately in the formation of various secondary lipid
oxidation products. The principal substrates for LOX in higher plants are linoleic (18
carbons long and two double bonds with one of the double bond being 6 carbons in from
the last methyl) or omega (w) end C1832, W6 and alpha linolenic (C1823, W3) (Hildebrand,
1989; Schewe, et al., 1986). The initial event in the lipoxygenase pathway is thought to be
the release of ﬁ'ee fatty acids from glycerolipids such as phospholipids. Lipoxygenase has
been implicated as playing a role in senescence (Hildebrand, 1989). Lipoxygenase and its
hydroperoxide products might directly participate in senescence by inactivating protein
synthesis, inhibition of photochemical activity in chloroplast and deterioration of cellular
membranes. Oxidation of polyunsaturated fatty acids in the membranes could lead to
increased membrane permeability which could then result in increase Ca2+ levels in cells,
stimulation of phospholipase and release of free fatty acids. The free fatty acids might
then be peroxidized by lipoxygenase, accelerating the cycle and increasing the
deterioration of membranes and metabolic ﬁmction.

Peroxidase activity also increased during storage of strawberry. An increase in

77

peroxidase activity suggests a possible role of this enzyme in degrading unsaturated fatty
acids and activating cell membrane and cell wall breakdown, yielding volatiles such as
methyl acetate thus linking them directly to quality deterioration of strawberry. Some of
the direct products of peroxidase activity in ﬁuits include the production of cis-3 -hexenol,
trans-2-hexenal, and other aldehydes and alcohols typical of some fruits and vegetables.
One ester of unsaturated aldehydes was detected in the volatile proﬁle of senescing
strawberry. Peroxidase catalyzed reactions may be envisioned as an integral aspect of the
biochemistry of aging cells since sub-cellular organelles undergo degeneration with
subsequent loss of compartmentation of enzymes and substrates (Dilley, 1970).
Peroxidase is considered to be a membrane bound or soluble enzyme.

Source of methyl acetate in senescent strawbeny. Methyl acetate is formed by the
condensation of methanol and acetyl CoA in a reaction castalyzed by alcohol acyl-CoA
transferase (AAT). Methanol has been reported to be produced from the degradation of
pectins (Siragusa et al., 1988). Pectins are a group of heterogenous polysaccharides with
a high molecular weight. They can be found in the intercellular regions and cell walls of
most ﬁuits and vegetables (Talmadge et al., 1973; Siragusa et al., 1988; Ahmed et al.,
1980). Pectins are composed of D-anhydrogalacturonic acid units linked through alpha
(1-> 4) glycocidic bonds, forming a polygalacturonic acid with some of the carboxyl
groups esteriﬁed with MeOH (Siragusa, 1988). When pectins are degraded, the methoxyl
groups are released as MeOH. MeOH has been reported as the major volatile produced
during seed growth and maturation in soybean (Obendorf et al., 1990) and peanut (Pattee

et al., 1970). Obendorf et a1. (1990) reported that pectin methyl esters are the most likely

78

source of methanol released in plant tissue. As a result, methanol has been used to
quantify pectin ester content and pectin methylesterase activity (Wood and Siddiqui, 1971).
The source of the acetate moiety of methyl acetate may be membrane lipids. Following
cellular breakdown as a result of the programmed physiological event of senescence, both
unsaturated and saturated free fatty acids are released by cellular membranes as reported
by previous researchers (Hildebrand, 1989; Vick and Zimmerman, 1976; Zimmerman,
1976; Brader, 1980). In order for most free fatty acids to be oxidized, they must be
primed or activated for reaction in an ATP-dependent acylation reaction to form fatty
acyl-CoA (Voet and Voet, 1990). The products of this reaction are are CoA derivatives
of fatty acids including acetyl-CoA. Acetyl-CoA, in the presence of methanol and acetyl-
CoA transferase would form methyl acetate. Nurten (1970) reported that co-enzyme A is
involved in the formation of esters and that therefore, it is associated with the fundamental
metabolism of the cell.

Given the high correlation between methyl acetate and an increase in softening of
strawberry during storage and considenring that methanol has been linked to cell wall
degradation, it may indicate that the cell wall degradation, membrane breakdown and the
resulting fatty acid release activate the mechanism of methyl acetate formation. It can also
be suggested that methyl acetate is the result of the esteriﬁcation of MeOH and acetic acid
(Brader, 1980; Hildebrand, 1989; Nurten , 1970; Vick and Zimmerman, 1976).

In conclusion, our results indicate that, in strawberry, ester volatiles as a whole
decrease as the ﬁuit senesces and thus a decrease of esters may be used as a general

indicator of the physiological age of the fruit during storage. Among esters, methyl

79

acetate seems to have unique-type relationships with other physiological parameters,
which may link this volatile with the senescence process of strawberry. The increase of
methyl acetate produced by strawberry during storage and the positive correlation of
methyl acetate with softening and changes of activity of the senescence-related enzymes
lipoxygenase and peroxidase and may give indication of a casual link between methyl
acetate production and senescence. Therefore, methyl acetate has the potential to be used

as a ﬁngerprint-type volatile to assess senescence in strawberry.

80

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Wood, P. J. and I. R. Siddiqui. 1971. Determination of methanol and its application to
meassurement of pectin ester content and pectin methylesterase activity. Anal.
Biochem. 39:418-428.

Yamashita, I. K., Y. Nemoto, S. Yoshikawa. 1976. Formation of volatile alcohols and
esters from aldehydes in strawberries. Pytochemistry 15: 1633-163 7.

 

82

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Retention Time (s)

Figure 1. Strawberry volatiles produced on day 1 of study in a ﬂow-through system: (1)
carbon dioxide, (2) methyl acetate, (3) methyl propanoate, (4) methyl butanoate,
(5) propyl acetate, (6) ethyl butanoate, (7) l-pentanol, 2,3-dimethyl, (8) 3-methyl
butyl acetate, (9) methyl hexanoate, (10) propyl butanoate, (11) hexyl acetate, (12)
3-hexen-l-ol, acetate, (13) ethy,2-methyl, 2-heptene, (14) 2,5-dimethyl-4-hydroxy-
3(2) Furanone, (15) ethyl hexanoate, (l6) methyl octanoate, (17) hexyl hexanoate,
' (18) octyl butanoate. Qauntitation of identiﬁed peaks is presented in Table 1.

84

 

 

 

 

 

 

 

 

 

A 0.3 L4 L1 1 r L l r 1 1 l l l 1 1_r 1 I l l
'7“ A —6— Methyl Esters
'1' . . -6— Ethyl Esters
5° 0-2‘ :_Y— Propyl Esters
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Figure 2. Ester production by strawbeny during storage classiﬁed by group (A) and the
proportion of each class of esters (B). Solid line represents the changes of total
esters during storage time. In B, open circles represent methyl esters, squares are
hexyl esters, diamonds are ethyl esters, and triangles are propyl esters.

85

 

 

 

 

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.1." : ° 3 —o— Methyl Acetate :
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. 0-051 —B— Methyl Hexanoate 7
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0

 

Figure 3. Changes of methyl esters produced by strawberry during storage. Closed circles
represent methyl acetate, open circles methyl butanoate, squares, methyl
hexanoate, and diamond methyl octanoate.

86

 

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Figure 4. Ratios of methyl acetate to methyl hexanoate (closed squares), methyl acetate
to methyl butanoate (open squares), methyl acetate to ethyl hexanoate (closed
circles), and methyl acetate to ethyl butanoate.

87

 

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Figure 5. Softening of strawberry during storage. Insert ﬁgures represents changes of
the ratio MA to ﬁrmness over time.

88

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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0.020 0.025 0.030 0.035 0.020 0.024 0.023 0.032
Methyl Acetate, nmollkg.s Methyl Acetate, nmollkg.s

Figure 6. Changes in strawbeny physicochemical parameters (top) and correlation of
methyl acetate produced by strawbeny and changes in physicochemical parameters
(bottom).

Enzyme Activity (pmollmin.g)

89

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Days

Figure 7. Activity of lipoxygenase (open circles, bottom) and peroxidases (closed
circles, bottom). Calculation were made in a fresh weigth basis.

90

CHAPTER III

VOLATILE PROFILES OF BROCCOLI (BESSIE m L.) AND CARROT
(Baum cam L-)-

91

VOLATILE PROFILES OF BROCCOLI (Dragging WELL.) AND CARROT

(Dams carnal...) DURING STORAGE

WW. terpenes, sulﬁde compounds, volatile ratios

Abstract. Carrots were cut into sticks and broccoli into ﬂorets for the monitoring of
volatile production during 9 days in a ﬂow through system. The most abundant class of
carrot volatiles were terpenes. Of the terpenes measured all but alpha phellandrene
declined as the commodity aged. This decline may give an indication of the physiological
status of carrot during storage and may provide information regarding shelf-life. For
broccoli there was no single class of volatiles that predominated. In broccoli, the types of
volatiles detected included terpenes (limonene and O-cymol), aldehydes (heptanal), fatty
acid derivatives (pentene-3 -methyl, hexene-2,5-dimethyl, and 1-heptene-4-methyl),
sulﬁdes (dimethyl disulﬁde), ketones (acetone), alcohol (linalool) and benzenoid
(ocimene). Dimethyl disulﬁde production increased as broccoli senesced. Dimethyl
disulﬁde, which has before been reported as anaerobic metabolism product is likely also a
senescence-related volatile compound. Dimethyl disulﬁde therefore has the potential to

serve as a senescence-type volatile in the designing of shelf-life prediction models.

92

To date, to our knowledge, research to assess the volatile organics in fresh
produce, such as broccoli and carrot as the commodity senesces has yet to be fully
explored (King and Morris, 1994). As tissues senesce they produce enzymes necessary to
recycle metabolically expensive materials and reroute the subunits to areas for use by
active systems elsewhere. Some of these materials are likely to be speciﬁc to the process
of senescence (King and Morris, 1994). Volatile production of produce during storage
could be a valuable asset to indirectly and non-destructively assess the physiology of the
produce and serve as a tool to make prediction regarding storage or shelf-life.

Broccoli is a compact, rapidly developing ﬂoral vegetable which is usually
harvested when the ﬂowering heads are immature and growing. When harvested, the
ﬂoral apices are separated from nutrients, hormones and energy supplied by the roots and
leaves. Consequently, broccoli ﬂorets senesce rapidly as is typical of commodities
harvested before physical growth has ceased (Huber, 1987). The most obvious feature of
broccoli postharvest senescence is sepal degreening due to degradation of chlorophyll
(King and Morris, 1994). To ﬁnd favor with consumers, however, broccoli must also be
mild in ﬂavor (Werner and Harris, 1974).

Tollsten and Bergstrom (1988) reported that terpenes were the major compounds
found in plants og the genus Brassica. Among terpenes, the most abundant compound
was the monoterpene trans-B-ocimene. Other prominent monoterpenes found were [3-
pinene, sabinene, myrcene, limonene, and B-phellandrene. Buttery et al. (1976) reported
some other groups of importance. These include sulﬁdes (dimethyl disulﬁde, dimethyl

sulﬁde, dimethyl trisulﬁde), isothiocyanate, cyanides, aliphatic aldehydes, aliphatic

93

alcohols and aromatic compounds. Pentina et al. (1995) reported a possible enzymatic
pathway for the production of some of the sulfur-containing compounds. They suggested
that for a cell-ﬁee system of ﬂesh broccoli, sulfur containing compounds are formed from
L-methionine and S-methionine. Terpene and benzenoid compounds are ﬂower-fragrance
components, being mainly released from undamaged inﬂorescences. They also reported
that alcohols and aldehydes are mainly degradation products from lipids while nitrogen-
containing compounds are formed through enzymatic degradation of different
glucosinolates (Tollsten and Bergstrom, 1988).

Carrot volatiles are mostly terpenes (Wagner and Warthesen, 1995). These
include alpha pinene, beta pinene, myrcene, alpha phellandrene, alpha terpinene, limonene,
gamma terpinene, terpinolene, terpinen-4-ol, methyl acetate, caryophyllene and gamma
bisabolene (Simon et al., 1980; Kopas—Lane and Warthesen, 1995). Terpenoid
biosynthesis begins with the condensation of acetyl-CoA and acetoacetyl-CoA to form 3-
hydroxy-3 -methylglutaryl-CoA (HMG-CoA). This reaction is catalyzed by the enzyme
HMG-CoA synthase (Dewick, 1997). Studies of plant-derived HMG-CoA have
suggested that in radish, both HMG—CoA synthase activity and that responsible for
acetoacetyl-CoA synthase (Acetoacetyl-CoA thiolase) are localized on a single
polypeptide. Thus, the soluble monomeric protein ﬁ'om radish membranes catalyzes the
two-step conversion of acetyl-CoA into HMG-CoA. Because of the critical role of HMG-
CoA on the terpenoid biosynthesis pathaway, this enzeme is considered the most
important control point in terpenoid biosynthesis. Whereas the mevalonate pathway to

terpenoids has been studied in considerable detail (Dewick, 1997; Grayson, 1997;

94

Cullmarm and Becker, 1998), it has been observed on several occasions that angiosperrns
incorporate little, if any, l4C-mevalonate into B-carotene and the phytol side of chlorophyll
as well as into other terpenoids (Arigoni et al., 1997). It has been suggested that diﬂ‘erent
pathways of terpenoid biosynthesis might be operative even in different compartments of
plant cells and carbohydrate (Arigoni et al., 1997). The conversion of geranyl
diphosphate (GPP) into simple cyclic monoterpenes involves initial isomerization to (3R)-
(-) or (3S)-(+)-lina]yl diphosphate (LDP) (Dewick, 1997). GPP is typically bound to the
enzyme as a complex with a divalent metal ion, and undergoes ionization to the allylic
linalyl cation, which allows formation of LPP, and the opportunity for cyclization via the
stereochemically favorable cation. A series of cation-diphosphate ion pairs participates in
the sequence, and both the isomerization and cyclization reactions are catalyzed by a
single enzyme. Interestingly, the biosynthesis of linalool in the ﬂowers of Clarkia breweri
does not proceed as far as LPP, but involves quenching of the linalyl cation with water.
The enzyme limonene synthase catalyzes the isomerization-cyclization of GPP to
limonene, the chirality in the product being determined by the enzyme and its source.
Thus, limonene synthase from spearmint (Mentha spicata) yields (4S)-(-)-limonene
(Dewick, 1997).

The Biosynthesis of (+)-3carene, a major component of turpentine, has been
studied by feeding experiments with labeled mavalonic acid precursors. Accordingly, the
formation of (+)-3-carene has been reinvestigated using cell-free extracts of Douglas ﬁr
(Psedotsuga menziesii) and partially puriﬁed carene synthase preparation from lodgepole

pine (Pinus contora). It has been established that label ﬁom (1-H)GPP was retained at C-

95
5 in carene, in keeping with the intennediacy of terpinyl cation, then cyclopropane ring
formation by loss of proton C-5 of the cation (Dewick, 1997)

There is a concern in the produce industry as to what effect time of storage has on
the quality and ﬂavor of produce to the consumer. In this project, we examined the
changes of volatile proﬁles of both broccoli and carrot during storage, with the objective
of identifying trends of volatile biosynthesis by these two commodities as they senesced.
We have also attempted to identify those particular senescence signature-type volatiles
with which we would be able to elaborate on the designing of prediction models to assess

shelf-life.

Material and Methods

Plant material. Broccoli and carrot were obtained as fresh as possible from the
Detroit Produce Terminal. Broccoli was cut into ﬂorets and carrot was cut into sticks
ranging from 2 to 5 inch length. The lightly processed products were then sanitized in a
smixture of 0.5 g-L‘1 of Benlate (Du Pont de Nemours Co., Agricultural Product
Department, Wilmington, Del.) and 0.5 g-L‘1 Rovral ( Rhone-poulenc Ag. Company,
Research Triangle Park, NC.) To prevent decay and then rinsed with distilled water and
allowed to air-dry. Approximately 450 g of carrot and 350 g of broccoli were placed in
glass jars for use in experiments. The storage temperature was 15 °C.

F low through sampling system. The jars were ﬁtted with T-shaped glass gas
sampling ports at the inlet and outlet. Connectors and gas lines were constructed of glass

and teﬂon, respectively, to minimize sorption and loss of volatiles ﬁom the system.

96

Temperature of the system was regulated by placing the apparatus in a controlled
temperature room. The jars were ventilated with air at ﬂow rates that ranged ﬁ'om 15-19
mL-min".

Headwace Analysis and Volatile Identiﬁcation. Volatile collection was
accomplished using solid-phase micro-extraction (SPME) device (Supelco, Inc.,
Bellefonte, Pa.) A ﬁber with a 100 um-thick, non-bonded, polydimethylsiloxane-based
stationary phase was used. The absorption time was 5 min. After absorption, volatiles
were then thermally desorbed from the ﬁber coating by directly inserting the ﬁber into the
injection port (200°C) of a gas chromatograph (HP-6890 Hewlett Packard Co.,
Wihnington, Del.) for 90 s. Gases were cryofocused on-column using liquid N2 and
separated using a gas chromatograph ﬁtted with a 10-meter, 0.1 mm ID, DB-S capillary
column (Hewlett Packard Co., Boise, Idaho). The detector was a time-of-ﬂight (TOF)
mass spectrometer (Pegasus H, LECO Co., St. Joseph, Mich.) The TOF mass
spectrometer permitted the resolution and quantiﬁcation of compounds not separated
chromatographically. Identiﬁcation of volatile material was done by comparison of the
spectra and GC retention times with authenticated standard compounds. High purity
(>95%) authenticated standards obtained from Aldrich (Aldrich, Co., Milwaukee, Wis).
For the standard compounds, a mix of the targeted compounds was prepared in a 4.85-L
jar (standard jar), equipped with a self-sealing rubber sampling port. The mix was
prepared by ﬁrst placing 50 uL of each compound into a 2.5-mL vial to generate a stock
mix. Then, 10 uL were taken from the stock mix and placed into the 4.85-L jar to be used

as standard. Standard preparation was done at 5 °C and the standard jar was kept at the

97

temperature of the experiment. Calculation of the concentration of standard compounds
was expressed in ppm. As sampling ﬁ'om the packages was done, so was ﬁom the
standard to estimate the actual production of the targeted compounds regardless of the

sensitivity of the detection system. Data were reported in umol-kg"°h“.

Results

Trend of carrot and broccoli volatile proﬁle. The most abundant chemical class
among carrot volatiles were the terpenoids (Table 1, Fig. 1). Fourteen terpenoids were
evaluated during the storage time and among these, only alpha phellandrene showed an
increase over time (Fig.2). Other terpenoids were found to decrease as the product aged
(Figs. 2 and 3). Most terpenoids decreased markedly during the ﬁrst 1-3 (1 of evaluation
and declined more slowly thereafter.

Ratios among carrot terpenoids were generated to assess the type of relationship
between the production of different carrot volatiles during storage (Fig. 4). Ratios
included camphene to alpha pinene, ocimene to camphene, sabinene to ocimene, alpha
phellandrene to cymene, myrcene to alpha phellandrene, and alpha pinene to myrcene.
Ratios of ocimene to camphene, myrcene to alpha phellandrene and alpha pinene to
myrcene decreased over time. The ratios of camphene to alpha pinene and alpha
phellandrene to cymene increased as the product aged.

In broccoli no single group of volatiles predominated. Volatiles detected included
terpenes (limonene and O-cymol), aldehydes (heptanal), fatty acid derivatives (pentene-3-

methyl, hexene-2,5-dimethyl, and l-heptene-4-methyl), sulﬁdes (dimethyl disulﬁde),

98

ketones (acetone), alcohol (linalool) and a benzenoid (ocimene) (Table 2, Fig. 5). Among
these groups, terpenes and fatty acid derivatives were the most abundant. Broccoli
volatiles which increased over time included heptanal and dimethyl disulﬁde. Volatiles
that decreased included acetone 3-methyl pentene, 4-methyl-1-heptene-, limonene, and
linalool. 2,5-dimethyl hexene, ocimene, and O-cymol stayed approximately at the same
levels throughout storage (Fig. 6). We have also provided a series of possible modes of
formation of particular terpenoid volatiles produced by carrot and broccoli that exist in the

literature (Dewick, 1997). (Figs. 7, 8, 9, 10 and 11).

Discussion

The carrot volatiles identiﬁed in this work including sabinene, ocimene, camphene,
alpha pinene, myrcene, alpha phellandrene, cymene, limonene, 3-carene, terpinolene, 1,2-
dimethyl-4-vinyl benzene, caryophyllene and bisabolene were consistent with previous
reports (Heatherbell et al., 1971a; Heatherbell et al., 1971b; Heatherbell and Wrolstad,
1971; Simon et al., 1980). Simon et a]. (1980) reported the volatile proﬁles of eight
carrot lines either blended, grated or sliced. They found that the volatile proﬁles for the
eight carrot lines were the same and that the difference was only in concentration.
Heatherbell and Wrolstad (1971b) reported the volatile composition of carrot as
inﬂuenced by variety, maturity and storage. They reported that differences were
quantitative rather than qualitative and that variation in concentration of individual
terpenes appeared to be consistent with descriptions of ﬂavor characteristics of diﬂ‘erent

varieties.

99

The formation of cyclic monoterpene such as alpha phellandrene in carrot is
usually via the intennediacy of alpha terpinyl cation which may be produced in either 4R
or 4S conﬁgurations depending on whether the LPP produced from GPP has 3R or 3S
conﬁguration respectively (Dewick, 1997). Biosynthesis of many other monoterpenes
then requires migration of the positive charge from the side-chain into the ring, and this
may be achieved by hydride shift. The precise nature of such shift in the formation of
alpha terpinene, for instance and alpha phellandrene has been investigated using cell-free
enzymes systems and speciﬁcally-labelled GPP substrates (Dewick, 1997). It has been
established that the a 1,2-hydride shift ﬁ'om C-4 to C-8 of the alpha terpinyl cation thus
generating the terpinen-4-yl cation is occurring during alpha terpinene formation (Dewick,
1997)

The decrease of terpene production by carrot as the product aged observed in our
experiment may help to understand the biochemical nature of the volatile biosynthesis
system of the product. These data indicate there may be a step in the terpenoid pathway
that is increasingly inhibited as the crop senesces. Most terpenes detected in carrot are
cyclic including sabinene, camphene, alpha pinene, alpha phellandrene, cymene, limonene,
3-carene, terpinolene, 2,2-dimethyl 1,4-vinyl benzene, caryophyllene and bisabolene.
George-Nascimento and Cori (1969) reported that the biosynthesis of cyclic
monoterpenes involves the transformation of mevalonic acid into 10-carbon
phosphorylated intermediates, and subsequent elimination of the pyrophosphate moiety.
The cyclization process leading to the formation of mono- or bicyclic-monoterpenes such

as limonene or the pinene apparently requires an adequate stereochemistry of the 10-

100

carbon precursor. The cis isomer neryl pyrophosphate has been postulated as the more
likely precursor of cyclic monoterpenes (George-Nascimento and Cori, 1969). The
process of senescence in carrot may have an impact in both the necessary stereochemistry
of the 10-carbon cyclic terpene precursor. However, the two straight-chain terpenes
ocimene and myrcene also behaved the same way as cyclic terpenes as the product aged,
implying that probably the effect of aging on terpene biosynthesis is somewhere in the
pathway before the formation of mevalonic acid. Ova et al. (1970) reported that both the
acetate and mevalonate used in terpene biosynthesis are produced through the formation
of pyruvate and acetyl—CoA in mitochondria.

A particular carrot volatile that increased over time while the product aged was
alpha phellandrene. Dewick (1997) reported that beta phellandrene is a derivative product
of geranyl pyrophosphate (GPP) (Fig. 10). It is then likely that isomerization occurs and
alpha phellandrene is produced. Biosynthesis of beta phellandrene requires migration of
the positive charge from the side-chain of the GPP molecule into a ring, and this may be
achieved by hydride shifts. The precise nature of such shift in the formation of beta
phellandrene in (Pinus cortorta) has been investigated using cell-ﬂee enzyme system and
speciﬁcally labeled GPP substrate (Fig. 10) (Dewick, 1997). A 1,3-hydride shift from 03
to C-8 of the alpha terpinyl cation giving the terpin-3 -yl cation and the interrnediacy of
(3 S)-LPP. It has been established that a 1,2-hydride shift from C-4 to C-8 of the alpha
terpinyl cation thus generating the terpinen-4-yl cation was occurring during landa-
terpinene formation. Alpha terpinene formation also involves the same 1,2-hydride shift,

and a similar proton loss, this time from C-3 of the terpinen-4-yl cation. An increase of

101

alpha phellandrene in carrot as the product ages as observed in our study, may indicate
that the 1,3-hydride shift, which results in the formation of (4S)-(+)-alpha terpinyl cation
into terpinen-3 -yl cation, is favored. This results in an increase in the ﬁnal product beta
and alpha phellandrene and a decrease in the production of landa and alpha terpinene. Our
results are consistent with this theory.

Broccoli volatiles identiﬁed in this study are typical of the genera Brassica as
reported by Tollsten and Bergstrom (1988). Both heptanal and dimethyl disulﬁde
increased as broccoli senesced and it may be worthwhile to evaluate their potential as
senescence-ﬁngerprint compounds. The diversity of types of volatiles during storage may
indicate that volatiles in broccoli come from different sources. Thus, the impact of
senescence on volatile production of broccoli may not be as easily targeted as in carrot.
Pentina et a1. (1995) and F omey et a1. (1991) have both reported sulfur compounds as off-
odor indicators in experiments conducted under anaerobic conditions. Dimethyl disulﬁde
was included in both studies. In our study, conducted under aerobic conditions, none of
the other sulfur containing compounds but dimethyl disulﬁde was detected. It may
indicate that dimethyl disulﬁde is rather a senescence-related volatile and not necessarily a
low-oxygen synthesized compound. It also suggests that dimethyl disulﬁde is the
responsible for the unpleasant odor of decaying broccoli under aerobic conditions.
Dimethyl disulﬁde may have the potential to serve as a senescence-type volatile.

The production of volatiles during storage merits further evaluation. Monitoring
of terpenes may provide a method to assess the physiological status of the product during

storage and should be judged for its value in prediction of shelf-life. Besides the general

102

trend of terpenes over time, ratios among terpene compounds may have potential to serve
as markers of aging and may have an application in the design of models to assess the
stage of development of the product as well. As for broccoli, dimethyl disulﬁde seemed

to be related to senescence and may have potential to be used as a senescence indication.

1 03
References

Arigoni, D., S. Sagner, C. Latzer, W. Eisenreich, A. Bacher and M. Zenk. 1997.
Terpenoid biosynthesis from 1-deoxy-D-xylulose in higher plants by intramolecular
skeletal rearrangement. Pro. Natl. Acad. Sci. 94:10600-10605.

Buttery, R. G., D. G. Guadagni, L. C. Ling, R.M. Seifert and W. Lipton. 1976.
Additional volatile components of cabbage, broccoli and cauliﬂower. J. Agric.
Food Chem. 24(4):829-832.

Cullrnann, F. and H. Becker. 1998. Terpenoid constituents of Pella epiphyla.
Phytochemistry. 47(2):237-245.

Dewick, P. M. 1997. The biosynthesis of C5-C2, terpenoid compounds. Nat. Pro. Rep.
14(2):111-144.

Fomey, C. F ., J. P. Mattheis and R. K. Austin. 1991. Volatile compounds produced by
broccoli under anaerobic conditions. J. Agric. Food Chem. 39(12):2257-2259.

George-Nascimento, C. and O. Cori. 1971. Terpene biosynthesis from geranyl and neryl
pyrophosphates by enzymes from orange ﬂavedo. Phytochemistry. 10:1803-1810.

Grayson, D. H. 1997. Monoterpenoids. Nat. Pro. Rep. 14(2):477-522.

Heatherbell, D. A. and R. E. Wrolstad. 1971. Carrot volatiles: Inﬂuence of variety,
maturity and storage. J. Food Science. 362225-227.

Heatherbell, D. A. and R. E. Wrolstad and M. Libbey. 1971a. Carrot volatiles:
Characterization and effects of canning and freeze drying. J. Food Science.
36:225-227.

Heatherbell, D. A. and R. E. Wrolstad and M. Libbey. 1971b. Isolation, concentration
and analysis of carrot volatiles using on-column trapping and gas-liquid
chromatography-mass spectrometry. J. Agr. Food Chem. 19:1009-1073.

Huber, D. J. 1987. Postharvest senescence. HortScience. 22:853-854.
King, A. G. and S. C. Morris. 1994. Physiological changes of broccoli during early
postharvest senescence and through the preharvest-postharvest continuum. J.

Amer. Soc. Hort. Sci. 119(2):270-275.

Kopas-Lane, L. M. And J. J. Warthesen. 1995. Carotenoid photostability in raw spinach
and carrot during cold storage. J. Food Sci. 60(3):773-776.

104

Lindsay, R. C. and J. F. Rippe. 1986. Enzymatic generation of methanethiol to assist in
the ﬂavor development of cheddar cheese and other foods. ACS Syrnp. Ser.
317:286-308.

Ova, K., H. Shibata, and I. Uritani. 1970. The mechanism supplying acetyl-CoA for
terpene biosynthesis in sweet potato with black rot: Incorporation of acetate-Z-“C,
pyruvate-3-“C and citrate-2,4-“C into ipomeamarone. Plant and Cell Physiol.

1 1 1507-510.

Pentima, J. H. D., J. J. Rios, A. Clemente, and J. M. Olias. 1995. Biogenesis of off-odor
in broccoli storage under low oxygen atmosphere. J. Agric. Food Chem.
43:1320-1313.

Simon, P. W., R. C. Linsay and C. E. Peterson. 1980. Analysis of carrot volatiles
collected on porous polymer traps. J. Agric. Food Chem. 28:549-552.

Tollsten, L. and G. Bergstrom. 1988. Headspace volatiles of whole plants and macerated
plant parts of Brassica and Sinapis. Phytochemistry. 27(7):2073-2077.

Wagner, L. A. and J. J. Warthesen. 1995. Stability of spray-dried encapsulated carrot
carotenes. 60(5):1048-1053.

105

 

 

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Figure 1. Volatiles proﬁle of carrot using time-of-ﬂight mass spectrometry: 1) sabinene,
2) ocimene, 3) camphene, 4) alpha pinene, 5) myrcene, 6) alpha phellandrene, 7)
cymene, 8) limonene, 9) 3-carene, 10) terpinolene, 11) 1,2-dimethy-4-vinyl
benzene, 12) caryophyllene, 13) bisabolene.

Volatile Production (pg/kgh)

108

 

 

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Figure 2. Production over time of selected carrot volatiles. a) camphene b) ocimene, c)
sabinene, (1) alpha phellandrene, e) myrcene, 0 alpha pinene, g) 3-carene, h)
limonene, i) cymene.

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Figure 3. Ratios of selected carrot volatiles over time: a) camphene to alpha pinene, b)
ocimene to camphene, c) sabinene to ocimene, (1) alpha phellandrene to cymene, e)
myrcene to alpha phellandrene, f) alpha pinene to myrcene.

110

 

 

 

 

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Figure 4. Effect of storage duration at 15 °C on the production of selected carrot
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(closed circles), bisabolene (closed squares), alpha mycerene (open circles).

111

 

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Figure 5. Volatiles proﬁle of broccoli using time-of-ﬂight mass spectrometry: 1) acetone,
2) heptanal, 3) pentene-B-methyl, 4) hexene, 2-Sdimethyl, S) 1-heptene-4-methyl,
6) dimethyl disulﬁde, 7) ocimene, 8) limonene, 9) o-cymol, 10) linalool.

Broccoli Volatiles (pg/ngt)

112

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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heptanal, c) acetone, d)dimethyl disulﬁde, e) l-heptene-4-methyl, i) hexene-2,5-
dimethyl, g) linalool, h) limonene, i) ocimene.

113

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OH
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Figure 7. Mechanism of formation of the monoterpene myrcene. From: Dewick (1997).

114

 

(4S)-(—)-limonene

Figure 8. Mechanism of formation of the monoterpene limonene. From: Dewick (1997)

 

   

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Figure 9. Mechanism of formation of the monoterpene linalool. From: Dewick (1997)

116

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Figure 10. Mechanism of formation of the monoterpenes b-phellandrene and alpha
terpinolene. From: Dewick (1997)

117

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(IQ-camphene

Figure 11. Mechanism of formation of the monoterpenes alpha pinene, beta pinene and
camphene. From: Dewick (1997)

118

CHAPTER IV:

VOLATILE PROFILES OF MICROORGANISMS GROWN IN LABORATORY
MEDIA AND FOOD PRODUCTS

119

VOLATILE PROFILES OF MICROORGANISMS GROWN IN LABORATORY
MEDIA AND FOOD PRODUCTS

WW5 Escherichia coli, S. typhimurium, indole, propanol, vegetables,

carrot, broccoli

Abstract. The volatile proﬁle of E. coli and S. typhimurium grown on both laboratory
media and selected horticultural products was studied to determine whether ﬁngerprint
volatiles could be identify for nondestructive assessment of human pathogen presence in
food. Mass spectrometrically-detectable volatile compounds produced by E. coli ATCC-
25922 in standard laboratory media in a closed system included carbon dioxide, ethanol,
l-propanol, cyclobutanol, l-butanol, 1-pentanol, disulﬁde dimethyl, cyclopropane-pentyl,
octanol and indole. Detectable volatiles produced by S. typhimurium grown under the
same conditions as E. coli included carbon dioxide, cyclobutanol, l-butanol, 2-methyl
cyclopentanol, 3-ethyl cyclopentanol, 2-hepten-1-ol, octane-3,3-dimethyl, 3-decanol,
2,2,3-trimethyl decane, 3,5-dimethyl dodecane, 3- methyl tridecane, 5,8-dimethyl
dodecane, pentadecane, 2,4,6-trimethyl dodecane, 2-heptadecanol, octadecanol,
nonadecanol, 2,5,6-trimethyl heptadecane. As a general assessment, it appears that
alcohols and some other compounds such as indole result from amino acid breakdown and
are typical volatile compounds of E. coli AT CC-25922 grown in standard laboratory
media. S. typhimurium produced a wider range of detectable alcohols. When E. coli was

present on broccoli, carrot and lettuce packages, the only reliably detected volatile was 1-

120

propanol. Indole, which is a compound produced in abundance by E. coli AT CC-25922 in
standard laboratory media was not detected in produce packages. Indole production
appeared to be inhibited by other sources of carbon as suggested by previous workers. 1-
propanol may have the potential to be used as a ﬁngerprint compound for E. coli in both
different growing media and in different produce packages. Heavier alcohols such
dodecanol, octadecanol and nonadecanol seemed to be characteristic for Salmonella, even
though we did not performe extensive monitoring nor investigate whether these
compounds are detectable in packages using the SPME/GC/MS system.

While the US. food supply is among the safest in the world, there are still millions
of US. residents stricken by illness every year from microbial pathogen contamination of
food they consume, and some 9,000 people die each year as a result (Jay, 1996). The
number of food-related human pathogens is large and their routes for ingestion varied
(Eley, 1996; Hobbs et al., 1993; Mott and Foster, 1995). E. coli 0157:H7 has been
detected in a wide array of food including meat, apple juice and lettuce (Gn'ﬂin and Tauxe,
1991; Moyer, 1996; Tarr et al., 1997). Salmonella has been found in eggs, vegetables and
poultry (Eley, 1996; McNab, 1997). Suzuki, 1994). Cyclospora has been encountered on
ﬁuit, Coptosporidium has infected people via drinking water and hepatitis A virus was
detected in frozen strawberries (Anonymous, 1997). As of May of 1997, it was estimated
that as many as 6.5 to 33 million illnesses in the United States each year are food-related
(Anonymous, 1997). The United State Department of Agriculture (USDA) estimates that
medical costs and productivity losses for 7 speciﬁc pathogens range between $6.5 billion

and $34.9 billion annually (Anonymous, 1997). Total costs for all foodbome illnesses are

121

likely to be much higher. Those estimates do not include the total burden placed on
society by the chronic illness caused by some foodbome pathogens. In comparison, the
total annual value of fresh produce grown in the U. S. is over $50 to 60 billion.

The foodbome outbreak paradigm has shiﬁed. In the past, an outbreak affected a
small local population, had a high attack rate, and involved locally prepared food products
with limited distribution (Eley, 1996). Nowadays, outbreaks involve larger populations
and may be multistate and/or international. In many cases, the pathogenic organism has a
low infective dose and it is unlikely that can be isolated from the food product. Delay in
identifying the causative agent can allow the outbreak to spread, increasing the number of
cases with time. Most pathogens cannot be easily detected in foods (Jay, 1996). Among
the needs for improved diagnostics include greater sensitivity, accuracy and speed for
detection of human pathogens in food (Briggs, 1987; Giese, 1993; Schaertel and
ﬁrstenberg-Eden, 1988).

At present, most of the microbial detection methods are time consuming and very
sophisticated, in many cases involving DNA ampliﬁcation. This does not allow real-time
monitoring (Jay, 196: Watanabe, 1987). The lack of technology for real-time pathogen
evaluation in food has encouraged the study of alternative detection technologies. Such
technologies may include volatile analysis for the detection of ﬁgerprint volatiles produced
by pathogenic organisms.

The development of new volatile analysis technologies in the last decade and the
increasing interest of designing automated quality control sensors in the food industry

make it worthwhile to explore the potential of possible ﬁngerprint or signature-type

122

volatiles emitted by microorganisms while present in food. The most proliﬁc methods of
detecting and classifying bacteria have been focused on the detection of speciﬁc fatty acids
for particular group of microorganisms (Phillips et al., 1976; Van den Bogaard et al.,
1986). Analytical instrumental methods have been used to identify and classify these
microorganisms (Fukuda et al., 1971). Identiﬁcation has been based on the determination
of the chemical composition of certain constituents in the microorganism such as proﬁling
of lipids, or the detection of a biomarker that signiﬁes a particular microorganism. A
variety of analytical techniques have been used in these analyses including gas
chromatography (GC), gas chromatography/mass spectrometry (GC-MS), pyrolysis mass
spectrometry (Py-MS), and pyrolysis gas chromatography/mass spectrometry (Py-
GC/MS). The most common approach in the classiﬁcation of bacteria through proﬁling is
the analysis of their fatty acid methyl esters.

Volatile emanations from human pathogenic microorganisms associated with fresh
or lightly-processed produce are, in many cases, unknown. However, volatile emanations
from some microorganisms have been measured. Many ﬁrngi are known to synthesize
ethylene (Fujii et al., 1985; Fukuda et al., 1971; Sembdner and Parthier, 1973).
Additionally, bacteria are known to emit C2 to C7 fatty acids which can be somewhat
volatile with enough speciﬁcity to permit identiﬁcation of the organism in clinical settings
(Phillips et al., 1976; Van den Bogaard et al., 1986). The potential to identify volatiles
associated with the presence of human pathogens has not received the same level of
scrutiny as, say, volatiles associated with aroma development by fruit during ripening.

Interesting or novel metabolites may lead to new avenues of investigation. Similarly,

123

volatile emanations from bacteria in relation to atmospheric composition and temperature
has not been previously described and may form the basis of future inquiries in the area of
microbial ecology.

In this work we have investigated the volatile proﬁles of E. coli A T CC-25922 and
S. typhimurium grown in or on various substrates including fresh produce. The major aim
was to identify those volatiles which could serve as ﬁngerprints for the identiﬁcation of the

microorganisms in food.

Material and Methods

Bacterial cultures and stocks. The medium for the growth of both E. coli and S.
typhimurium was LB (Luria-Bertani) broth (Maniatis et al., 1982). The broth was
prepared with 10 g of Bacto-tryptone, 5 g of Bacto-yeast, and 5 g of NaCl in a liter of
distilled water (Maniatis et al., 1982). The broth was autoclaved for 15 min. E. coli
ATCC-25922‘was added to the broth and incubated at 37 °C in a gyratory shaker at a
speed of 100 rpm (G-25 New Brunswick Scientiﬁc Corporation, New Brunswick, NJ).
Growth was monitored at 595nm using a spectrometric densitometer (model 601-G,
Milton Ray Co., Rochester, NY). The target concentration was 3.75x108 cells/mL
(OD=0.375). Measurements with the densitometer were made every half hour. A
growth curve was constructed.

When the target population was achieved, starting cultures were prepared. One
liter of solution containing E. coli AT CC-25922 was dispensed to 6-125mL rotor-GSA

centrifuge bottles with screw caps. The four bottles were then centrifuged (model Rc 5c

124

Superspeed Centrifuge, Sorvall Instruments, Dupon Co., Haﬂinan Estate, 111.) at 1600x g
for 10 min and the supernatant decanted. Cells were re-suspended in 3 mL of 20%
glycerol solution. The bacterial population was rechecked using spectrometry at a wave
length of 595 nm. Cultures were then diluted to 2.6x10lo cells/mL. One mL aliquots were
placed in 1.5-mL microcentrifuge tubes. Tubes were quickly frozen using ethanol and dry
ice (C02) and stored at -80 °C.

Viability Analysis. The number of viable cells per mL of solution was determined
by serial dilutions of the starter culture on agar plates. Agar-based medium was composed
of 10 g of Bacto-tryptone, 5 g of Bacto-yeast extract, 5 g of NaCl, and 12 g of agar.
Distilled water was added to the mix and brought to a volume of 1 L. Agar was added
after all other components were dissolved. Once the medium was prepared and
autoclaved for 30 min, 1 mL of starter culture was diluted serially and the solution was
dispersed evenly over the surface of the agar. Plates were incubated overnight at 37°C
and colony count was performed.

E. coli and S. whimurium in standard laboratory media in a closed system. E.
coli and S. typhimurium microorganisms were prepared in ﬁve different growing media to
monitor their volatile proﬁles over time. Media included NZY broth (5 g NaCl, 2 g
MgSO4.7H20, 5 g yeast extract, 10 g NZ amine or casein hydrolysate and adjusted to pH
7.5 with NaOI-I), 2X YT broth ( 10 g NaCl, 10 g yeast extract, and 16 g bacto-tryptone),
SM Buffer (5.8 g NaCl, 2 g MgSO4.7H20, 50ml 1M Tris-Cl, pH 7.5 and 5 mL of 2%
bacterial gelatin), LB broth (10 g NaCl, 10 g bacto-tryptone and 5 g yeast extract) and

Super Broth (5 g NaCl, 20 g bacto-yeast extract, 35 g tryptone and 1 mL 5 M NaOH).

125

About 50 mL of each media were placed in 120-mL glass jars equipped with sampling
ports. Media then were inoculated with 1 mL of overnight-enriched stock bacterial
sample. Jars were sealed and sampling of the headspace using SPME was initiated.
Alternatively, tryptophan or threonine was added to the Super Broth medium to evaluate
the capacity of E. coli to breakdown both amino acids and yield indole and l-propanol,
respectively. At this point we should mention that only the Super Broth medium was used
for time course monitoring and the other media were employed only in the preliminary and
exploratory stage. In Super Broth medium, the two compounds (indole and l-propanol)
that were evaluated more extensively were produced in copious amount relative to the
other media.

Headspace sampling of volatile. Volatile collection was accomplished using a
solid-phase micro-extraction(SPl\/1E) device (Supelco, Inc., Bellefonte, Pa.) as described
by Song et al. (1997). Using SPME, analytes are absorbed into a coating on a fused silica
ﬁber exposed to the jar headspace for 5 min. For this study, a ﬁber was coated with a 100
pm-thick polydimethylsiloxane-based non-bonded stationary phase. After absorption,
volatiles were then thermally desorbed from the ﬁber coating by directly inserting the ﬁber
into the injection port of a gas chromatograph (HP-6890, Hewlett-Packard Co.,
Wilmington, Del). For most fruit and vegetable volatiles, 5 min was sufﬁcient time to
achieve equilibrium, between the volatiles in the headspace and the ﬁber coating.
However, as much as 25 minutes were required for some volatiles. As long as absorption
time is held constant, however, detector response is linear with volatile partial pressure

(Song et al., 1997).

126

MS volatile analysis. Volatile analysis was conducted by using gas
chromatography/time-of-ﬂight mass spectrometry. Gases were cryofocused on-column
using liquid N2 and separated in the GC (HP-6890, Hewlett-Packard Co., Wilmington,
Del.) Using a 10-meter, 0.1 mm ID, DB-5 column (Hewlett-Packard Co., Wilmington,
Del). The detector was a benchtop time-of-ﬂight (TOF) mass spectrometer (Pegassus H,
LECO Co., St. Joseph, Mich). This mass spectrometer permitted the resolution and
quantiﬁcation of compounds not separated chromatographically. Identiﬁcation of volatile
material was done by comparison of the mass spectra with high purity authenticated
standard compounds (Aldrich, Co., Milwaukee, Wis). For the standard compounds, a
mix of the targeted compounds was prepared in a 4.85-L jar (standard jar), equipped with
a self-sealing rubber sampling port. The mix was prepared by ﬁrst placing 50 uL of each
compound into a 2.5-mL vial to generate a stock mix. Then, 10 “L were taken from the
stock mix and placed into the 4.85-L jar to be used as standard. Standard preparation was
done at 5 °C and the standard jar was kept at the temperature of the experiment.
Calculation of the concentration of standard compounds was expressed in ppm. As
sampling from the packages was done, so was from the standard to estimate the actual
production of the targeted compounds regardless of the sensitivity of the detection system.
Data were reported in nmol-kg'1-h".

Monitoring E. coli A TCC-25 922 volatiles in produce packages. Produce (brocoli,
carrot and lettuce) were obtained as fresh as possible from a local shipper (Steve Produce
Company, Lansing, Mich). Produce were sorted out so that defect-free material would

be used in experiments. Broccoli ﬂorets were used, lettuce was shredded and carrot was

127

cut in sticks. Produce was weighed and inoculated with E. coli. The number of cells
applied to the produce varied between 8 and 1x109 cells per 500 mL of dipping solution.
The inoculation was performed by dipping the product for 20 s in solutions containing E.
coli. The range of number of E. coli cells in packages was achieved by serially diluting 1
mL of stock solution containing E. coli in jars with 500 mL of tryptophan-based dipping
media. We were also interested in determining the effect of tryptophan availability on
indole production. The dipping solution was prepared in a way that different amounts of
tryptophan were available for the bacteria to evaluate the impact of substrate on the
production of targeted volatiles. Five different concentrations of tryptophan were
prepared (5, 4, 3, 2, and 1 g of tryptophan in 250 mL of distilled water). Alternatively,
threonine was used as the amino acid to evaluate the potential of E. coli to metabolize it
to 1-propanol. Produce were then placed in pouches of low density polyethylene (LDPE)
(LDPE, Dow chemical company, Midland, Mich.) equipped with a gas-sampling septum,
made of Dupont Silicone II tub/tiling glue on a short strip of electrical tape as described by
Beaudry et al. (1992). Pouches were heat-sealed and stored at 23 °C for evaluation. The
dimensions and thickness of the LDPE ﬂim used to construct the modiﬁed atmosphere
packaging was determined by using the model of Cameron et al. (1989) so that the

produce would generate an in-package 02 level of about 3-5 kPa.

Results
Volatile proﬁles of E. coli and S. typhimurium in standard laboratory media.

Volatile compounds produced by E. coli in LB media tentatively identiﬁed as carbon

128

dioxide, ethanol, l-propanol, cyclobutanol, 1-butanol, l-pentanol, dimethyl disulﬁde,
cyclopropane-pentyl, octanol and indole (Fig. 1). Volatiles produced by S. typhimurium
grown under the same conditions as E. coli were tentatively identiﬁed as carbon dioxide,
cyclobutanol, l-butanol, cyclopentanol-Z-methyl, cyclopentanol-3 -ethyl, 2-hepten-1-ol,
octane-3,3-dimethyl, 3-decanol, decane-2,2,3-trimethyl, dodecane-3,5-dimethyl, tridecane-
3-methyl, dodecane-5,8-dimethyl, pentadecane, dodecane-2,4,6-trimethyl, 2-heptadecanol,
octadecanol, nonadecanol, heptadecane-2,5,6-trimethyl (Fig. 2).

Impact of the type of media on the production of indole by E. coli. Indole was the
most abundant detectable volatile compound produced by E. coli. The highest indole
production by E. coli was observed in the Super Broth media (Fig. 3).

Indole production as a function of the number of E. coli cells. The biggest
diﬁ‘erence in the ratios of indole over E. coli population was between 10 and 25 h (Fig. 4).
The highest ratio of indole/E. coli population was achieved by using the medium Super
Broth. The ratios of indole to E. coli population had the order of Super Broth > 2xYT >
NZY. The amount of tryptophan available in the media had the same order. When free
tryptophan was added to the Super Broth media, indole production was 90-fold higher
than the control (Fig. 5).

Identifying microorganism-ﬁngerprint volatiles in modiﬁed atmosphere-packed
produce. l-propanol was detected in packages of broccoli, lettuce and carrot inoculated
with E. coli in a threonine-containing solution (Figs. 7, 8 and 9). Indole, however, was
not detected in the produce packages inoculated with an E. coli-containing solution. In

produce packages contaminated with E. coli, fewer E. coli-related alcohols were detected

129

as suppose in laboratory media.

Discussion

Sources of volatiles produced by bacteria. Carbon dioxide and l-butanol were the
only common compounds between E. coli and Salmonella volatile proﬁles, suggesting
therefore that each organism has a distinctive way of metabolizing the nutrients available
in the media. Microbiologists have long recognized that the enormous diversity of
microbial enzymes represent a valuable resource for the biosynthesis of desirable
molecules (Harper and Nelson, 1982; Jones and Turner, 1971; Stephenson, 1939; Webb,
1984). The fermentation of sugars to ethanol is one of the earliest applications dating back
to earliest recorded history, although its enzymatic basis was only revealed in 1897 using a
microbial cell-free system (Buchner, 1897). More recently, molecular biological methods
have extended the range of applications for microbial enzymes in biotechnology (Grund,
1995; Weast and Grasselli, 1992). For example, naphthalene dioxygenase genes, cloned
and expressed in E. coli, provide the last enzymatic step for a fermentation process to
make the blue jean dye indigo (Ensley et al., 1983).

In order to assess the mechanism and biochemistry of volatile production by E.
coli and S. typhimurium, we should start by evaluating the growing media and the possible
substrates for both organisms. The media used in this experiment for time course analysis
was Super Broth. Super Broth is rich in tryptone, a media component rich in tryptophan.
E. coli has been linked to the generation of indole in media containing the substrate

tryptophan (Happold, 1950; Gunsalus et al., 1955; Wood, 1947). E. coli synthesizes

130

tryptophanase which catalyzes the cleavage of tryptophan into indole, ammonia and
pyruvate. The indole nucleus of tryptophan undergoes many reactions which can be
utilized for analytical purposes. The indole detected in our experiment is likely to be the
result of the action of tryptophanase provided by E. coli (Gunsalus et al., 1955; Happold,
1950; Wood, 1947). S. typhimurium, to our knowledge, has not been reported to have
active tryptophanase and therefore does not cleave tryptophan to yield indole, ammonia
and pyruvate. Our results are in agreement with the literature since indole was not
detected in assays S. typhimurium,

Casein, one of the component of the Super Broth medium, has 20 amino acids in
addition to tryptophan. Amino acids may be broken down by microorganisms in a number
of ways in either anaerobic, semi-anaerobic or aerobic conditions (Stephenson, 193 9).
These include decarboxylation with the formation of some amines followed by hydrolytic
deamination with the formation of hydroxy acids, hydrolytic deamination and
decarboxylation with the formation of alcohol, reductive deamination with the formation
of saturated acids, reductive deamination and decarboxylation with the formation of
hydrocarbons, deamination and desaturation with the formation of unsaturated acid and
oxidative breakdown to compounds having fewer carbons. In our experiment, we suggest
that the series of alcohols detected in media containing E. coli (Fig. 1) and S. typhimurium
(Fig. 2) are likely to be the result of deamination of amino acids with subsequent
hydrolytic decarboxylation. In the case of E. coli, ethanol production can be attributed to
the action of the bacteria on the serine present in the tryptone of the Super Broth media.

Serine is ﬁrst hydrolytically attacked to remove the carbon dioxide and then deaminated,

131

thereby producing ethanol. The same mechanism is suggested for the microbial
biosynthesis of the other alcohols shown in Fig. 1. The amino acids attacked by E. coli to
produce other alcohols include threonine for l-propanol, proline for cyclobutanol,
glutamine for l-butanol, and lysine for l-pentanol. Octanol may be the result of the
condensation and reduction of pentanol and butanol.

Another alternative pathway through which E. coli could have biosynthesized
ethanol and butanol from Super Broth media is by using the metabolite pyruvate which
can be generated by E. coli via the action of tryptophanase. Moat and Foster (1995)
reported that some bacteria are able to produce at least three different alcohols in the
presence of pyruvate as is the case when tryptophan has been broken down into indole,
pyruvate and ammonia. Bacteria are able to generate acetyl-CoA ﬁ'om pyruvate by action
of the pyruvatezferrodoxin (F d) oxidoreductase. Acetyl-CoA can act as a hydrogen
acceptor giving rise to ethanol. Two moles of acetyl-CoA condense and form acetoacyl-
CoA which ﬁrrther is converted into acetone and butyrate via acetoacetate decarboxylase
and beta hydroxybutyryl-CoA dehydrogenase respectively. Acetone is ﬁrrther converted
into isopropanol and butyrate into butanol.

The dimethyl disulﬁde detected in the E. coli-containing media may form as a
result of the degradation of sulfur-containing amino acids such as methionine and cysteine
(Moat and Foster, 1995; Stephenson, 1939). Hydrogen sulﬁde, methyl mercaptan,
dimethyl disulﬁde, and ethyl sulﬁde have been reported as products of amino acid
degradation by microorganisms (Stutz, 1991). The production of methyl mercaptan, and

possibly dimethyl disulﬁde, depends on the presence of carbohydrate and therefore may

132

be due to the methylation of hydrogen sulﬁde. The lack of sulfur-containing volatiles from
S. typhimurium suggests that perhaps the enzymatic system that catalyzes the production
of sulﬁrr-containing compounds from sulfur-containing amino acids is missing in S.
typhimurium.

In a comparinson of E. coli volatiles versus S. typhimurium volatiles, it appears
that S. typhimurium synthesizes higher molecular weight alcohol compounds than E. coli.
S. typhimurium has also been reported to exhibit decarboxylase and deaminase activity for
amino acids including aspartate, glutamate, ornithine, lysine, arginine, tyrosine,
phenylalanine, cysteic acid, diaminopimelic acid, hydroxyphenyl serine, histidine,
tryptophan, 5-hydroxytryptophan, and possibly others (Moat and Foster, 1995). Most of
these amino acids were present in the growing media used in this experiment. This may
explain the production of various alcohols detected in the media containing S.
typhimurium (Fig. 2).

Another component of the media used in this experiment was yeast extract. Yeast
extracts are concentrates of the soluble components of yeast cells. Fresh yeast contains 6
to 8 % RNA. Special strains may contain 13 % or more (all ﬁgures referring to dry solids
contents). The yeast RNA is mainly degraded by bacteria into S prime nucleotides of
guanine (GMP), adenine (AMP), cytosine (CMP) and uracil (UMP) are formed
(Stephenson, 193 9). The decomposition of nucleotides can occur in three ways: a) by
only releasing the phosphate, b) by releasing all the phosphate, the ribose, and the base;
and c) by only releasing the base. However, no literature substantiating possible

production of alcohols, sulﬁdes, indole, esters, and fatty acids as nucleic acid breakdown

133

products and by action of E. coli or S. typhimurium was found

Potential of 1-propanol and indole as bacterial ﬁngerprint compounds. 1-
Propanol offers the best characteristics to be considered as a ﬁngerprint compound for E.
coli for the types of vegetables in packages (broccoli, lettuce, and carrot) that we used in
our experiments (Figs. 7, 8, 9). 1-Propanol increased in packages over time and the rate
of increase was dependent on the inoculum concentration (Fig. 8). Presumably the
biosynthesis mechanism of l-propanol by the action of the bacteria on threonine (Fig. 10),
is favored by a higher initial population of the bacteria.

Both indole and l-propanol have the potential to be used as ﬁngerprint compounds
for E. coli in both standard laboratory media and in different produce packages. 1-
propanol and indole are volatiles produced by E. coli but not by broccoli, carrot or lettuce
and this information may be exploited in a more practical way. Indole was not detected in
produce packages, however, due perhaps to the competition of other carbohydrate
sources. Nevertheless, indole may be promising in other types of food such as meat, dairy
products and egg. Fingerprint volatiles of microorganisms not only oﬂ‘er a new way of
identifying microbial development in food but also oﬁ‘er information on the biochemistry
and metabolism of the organisms. With the availability of rapid, accurate and more
sensitive volatile analysis techniques, it worthwhile to further investigate volatiles

produced by microorganisms as a research practical and research applications.

134

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GC-MS Relative response

137

 

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80 100 120 140 160 180 200 220 240 260 280 300 320 340

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Retention Time, s

Figure 1. Spectrum of volatiles produced by E. coli in Super Broth media. These
volatiles were generated in a closed system at room temperature. (1) carbon
dioxide, (2) ethanol, (3) l-propanol, (4) cyclopropanol, (5) butanol, (6) 1-
pentanol, (7) disulﬁde dimethyl, (8) cyclopropane-pentyl, (9) octanol, and (10)
indole. Identiﬁcation of compounds is tentative, based on comparison to spectra in

NIST.

138

 

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Retention Time, s

Figure 2. Spectrum of volatiles produced by S. typhimurium in Super Broth media.
These volatiles were generated in a closed systems at room temperature. (1)
carbon dioxide, (2) cyclobutanol, (3) 1-butanol, (4)2-methyl cyclopentanol, (5) 2-
ethyl cyclopentanol, (6) 2-hepten-l-ol, (7) 3,5-dimethyl octane, (8) decanol, (9)
2,2,3-trimethyl decane, (10) 3,5-dimethyl dodecane, (11) 3-methyl tridecane, (12)
5,8-dimethyl dodecane, (13) 2,4,6-trimethyl dodecane, (14) pentadecane, (15) 2-
heptadecanol, (16), Octadecanol, (17) nonadecanol, (18) 2,5,6-trimethyl
heptadecane. Identiﬁcation of compounds is tentative, based on comparison to

spectra in NIST.

E. coli population (log CFU/ml.)

GC-MS Response

139

 

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Figure 3. The top graph represents E. coli population over time as detected using
spectrophotometer measurements at 595 nm. The bottom graph represents indole
production by E. coli in three different standard laboratory media. Square symbols
represent indole in the Super Broth media, diamond symbols are for the 2xYT

media and circles are for the NZY media.

140

 

 

 

 

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Figure 4. Ratios of indole production by E. coli to population of the organism in the
media. Square symbols represent indole in the Super Broth media, diamond
symbols are indole from the 2xYT media and circles symbols are indole from the

NZY media.

GC-MS Response

141

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Figure 5. Indole production by E. coli in Super Broth media with (open circles) and

without (closed circles) tryptophan incubated at RT.

142

 

 

 

 

 

 

 

 

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Figure 6. Indole (open symbols) and l-propanol (closed circles) production. E. coli
grown in Super Broth medium and a closed system and at RT. Top graph

represents oxygen depletion in incubation chamber.

143

 

 

 

 

 

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Figure 7. Pproduction of l-propanol by E. coli in broccoli packages at RT. Closed
circles represent l-propanol detected in packages inoculated with E. coli, open
circles are l-propanol detected in packages without E. coli. Squares symbol

represent the depletion of in-package oxygen over time.

144

 

 

 

 

 

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Days of storage under MAP at RT

Figure 8. Accumulation of l-propanol produced by E. coli on broccoli in modiﬁed
atmosphere packages as a ﬁmction of the initial bacterial population (IP). Circles
represent 1x109 cells/mL in the dipping solution, squares represent IP = 2,000,000
cells/mL, triangles represent [P = 4,000 cells/mL, diamonds IP = 8 cells/mL

inverted triangles IP = 8 cells/500 mL and open circles IP = 0 cells/mL.

145

50000 4 1 1 4 ' 1 1 1 l . 4 1 I 1 1 1 I 1 1 1 I 1 1 1

 

400003 Carrot

   
  

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Figure 9. 1-Propanol accumulation in LDPE packages of carrot (top) and lettuce
(bottom) incubated at RT inoculated with threonine solution contaminated with

(closed circles) and without (open circles) E. coli.

146

 

 

 

 

clzo o H H20 co 0 H
“C — H— CH3 //’:) c= CH—CH3
l H l
N H3 N H2
Threonine Cl'hreonine Beaming )l/
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N H
J\;20
302 N H; / (f0 0 H
W ﬁ— CH-Z—C H3
HOCHz—CHTC H3 {1 O
n-propanol alpha-ketobutyrate

Figure 10. Schematic of the mode of action of E. coli on the breakdown of threonine in

packages of broccoli.

147

CHAPTER V:

STORAGE DURATION, LOW 0; AND ELEVATED CO; ALTER ESTER
BIOSYNTHESIS IN STRAWBERRY

148

STORAGE DURATION, LOW 02 AND ELEVATED COz ALTER ESTER
BIOSYNTHESIS IN STRAWBERRY
Addjﬂonaljndgzgmrds modiﬁed atmospheres, volatile, mass spectrometry, aroma,

F ragaria ananasa Dutch.

Abstract. A combination of solid phase micro extraction (SPME) as a sampling method
and gas chromatography/time-of-ﬂight mass spectrometry (GC-TOF-MS) for gas analysis
were used to investigate the volatiles produced by strawberry (Fragaria ananasa Dutch.)
stored for eight days in modiﬁed atmosphere packages. A range of steady-state
atmospheres (2 to 16 kPa 02, and 2 to 20 kPa CO,) was developed by enclosing the fruit
in LDPE pouches at 0 °C. There was a decrease in volatile production as the
concentration of O2 in the packages decreased and the concentration of CO2 increased.
Volatile esters, known to be very important in the ﬂavor of strawberry, declined with a
decrease in package 02 and a concomitant increase in CO, MAP methyl acetate, methyl
butanoate, methyl hexanoate and ethyl butanoate. Strawberry fruits were also exposed to
a range of 02 levels in a ﬂow-through system (1.3, 2.4, 3.6, 6.3, 9.5, and 16 kPa). Low
02 alone reduced the production of methyl butanoate, methyl hexanoate and hexyl acetate
but not methyl acetate and ethyl acetate. The data suggest that both low 02 and high CO2

alter ester production, each acting in speciﬁc ways.

149

The time devoted to domestic meal preparation has continuously decreased during
the last decade and the time spent in restaurants and eating places continue to rise
(Hobson and Tucker, 1996). In order to fulﬁll this consumer-led demand, new storage
handling and delivery techniques have had to be developed. One such technique is
modiﬁed atmosphere packaging (MAP) (Beaudry et al., 1993; Cameron et al., 1994;
Cameron et al., 1989; Church and Parsons, 1994). Modiﬁed atmosphere packaging could
be deﬁned as the enclosure of food products in gas-barrier materials, in which the gaseous
environment is manipulated in order to reduce unwanted metabolic events or inhibit
spoilage agents and therefore either maintain a higher quality within a perishable food
during its natural life (Beaudry et al., 1992; Ben-Arie and Zutkhi, 1992; Cameron et al.,
1989; Church and Parsons, 1994; Kader et al., 1989; Vergano and Pertruit, 1993). With
modiﬁed atmosphere packaging, the atmosphere inside a package generally is low in 02
and high in CO2 (Beaudry et al., 1992; Cameron, 1989; Church and Parsons, 1994; Gorris
et al., 1996). The composition of the atmosphere at any particular time is a ﬁinction of the
initial gas composition, package volume, gas transmission rate, and the respiratory activity
of the produce (Barmore and Rouse, 1976; Gorris et al., 1996; Li and Kader, 1989;
Sommer et al., 1973).

The eﬁ‘ect of O2 and CO2 on aroma compound production has not been well
characterized, although elevated CO2 and or reduced 02 levels have also been suggested
to be the causal factors in off-ﬂavors and off-odors in strawbeny stored in MAP. To
assess quality, consumers rely a great deal on their perception of the aroma of the food

they buy. Therefore, a better understanding of the relationship between the in-package

150

levels of O2 and CO2 in MAP-stored fruit and changes in both aroma-related and off-odor-
type volatiles may be of importance in the design of optimized packaging systems. Our
objective was to evaluate the impact of modiﬁed atmosphere packaging on aroma

production by strawberry and more speciﬁcally on ester production.

Material and Methods

Plant material. Strawberry (F ragaria ananasa Duch.) fruit of the cultivar ‘ Sweet
Darling’ not previously treated with CO2 were shipped overnight under reﬁigerated air
from Florida (BB1 Produce, Inc., Dover, Fla.) to the Detroit Produce Terminal in Detroit,
MI (Rocky Produce Co., Detroit, Mich). Fruit were sorted for visible defects and ﬁirther
sanitized in a solution of 0.5 g-L‘l Benlate (Du Pont de Nemours Co., Agricultural
Product Department, Wilmington, Del.) and 0.5 g-L‘l Rovral ( Rhone-poulenc Ag.
Company, Research Triangle Park, NC). Following application of the fungicide, ﬁ'uits
were air-dried at 0 °C for 5 h. Fruit were then placed in pouches of LDPE and stored at 0
°C for evaluation or in ﬂow-through chambers.

Modiﬁed atmosphere packaging of strawbeny. The design of modiﬁed
atmosphere packaging was achieved by using the approach of Beaudry et al. (1992) as
adapted from Cameron et al. (1989). Fruit were sealed in pouches made of 0.00762 cm (3
mil) thick low-density polyethylene (LDPE; LDF 550, Dow Chemical Company, Midland,
Mich). The initial step in the package design for strawberry was the determination of
respiration rates (r02 ) of whole strawberries stored at atmospheric O2 partial pressure

(20.7 kPa) at 2 °C. Using the estimated r02, the needed ﬁlm area and fruit weight were

151

calculated to generate 15 kPa O2 . From this high 02 setting, fruit weight was varied to
achieve a range of 02 levels. Fruit weights used were 15, 41, 120, and 360 g.
Determination of ﬁlm permeability. The O2 and CO2 permeability of the LDPE
ﬁlm (3 mil) was measured on three random ﬁlm samples at 0, 5, 10, 15, 20 and 25 °C
according to the method described by Beaudry et al. (1992). A specially-built stainless
steel permeability cell was submerged in a water bath (Lauda RC20; Brinkman Instrument
Co., West Bury, N.Y.). Temperature of the permeability cell was measured using
thermocouple and the temperature of the bath was veriﬁed with a mercury thermometer.
The permeability cell contained two circular 25-mL chambers separated by the ﬁlm sample
and sealed in place by an O-ring. The cell chambers were 8 cm in diameter and 0.5 in
depth and surface area 50 cm2. Copper coils tubing were attached to the inlet for the N2
carrier supply line. Exit lines were composed of stainless steel. The passage of the gas
through the coils before entering the cell allowed the system to be isothermic, i.e. the
entering gas had the same temperature as the cell. The permeant mixture of O2 and CO2
(65 and 35 kPa, respectively) was introduced to one chamber of the cell and N2 carrier gas
was introduced to the other chamber. The rate of O2 and CO2 permeation through the ﬁlm
was calculated from the steady-state partial pressure diﬂ‘erence between the two streams.
The partial pressure of O2 and CO2 in the carrier gas stream was determined from
concentration measurements obtained using a sequential combination of O2 and CO2
analyzers. To measure the 02 concentration, a calcia-zirconia analyzer (Ametek S3 A/II,
Ametek Co., Thermox Instrument Div., Pittsburgh, Pa.) Was used. CO2 was measured

with an infrared gas analyzer (ADC 225—MK3, Analytical Development Co.,

152

Hertfordshire, England). Concentrations were calculated relative to a certiﬁed standard
gas mixture (106 uL/L O2 and 100 uL/L CO2 in N2 gas). Flow rates were maintained
between 110 and 130 mL/min for all gases, and the chamber pressure were equalized and
maintained at about 0.4 kPa above atmosphere.

Concentration data were converted to partial pressure using the ideal gas law
equation. The partial pressure difference and the measured rate of permeant ﬂux ﬁom the
receiver cell were used to determine the permeability coefﬁcient using Fick’s Law. Values

of the permeability as a ﬁmction of the temperature were ﬁtted by the Arhenius equation:

where P, is permeability coeﬁicient at any temperature in Kelvin. Pc is permeability
constant. R is the gas constant (0.0083144kJ-mol'1-K"). Ep is activation energy in kJ.

mol". T is temperature in Kelvin. This equation was converted to:

1n(P,)=(I€—I;)+1n(Pc)

Package gas composition. Strawberries were placed into LDPE pouches and the
pouches were heat sealed. A gas-sampling septum, made of Dupont Silicone H tub/tiling
glue on a short strip of electrical tape was attached to the surface of the package as
described by Beaudry et a1. (1992). Gas samples were drawn from each package through

the self-sealing silicone septum using a 0.5 mL insulin syringe. Three gas samples were

153

analyzed from each package at each evaluation using an O2 analyzer (Sevomex
Paramagnetic O2 Transducer, Series 110, Servomex Co., Sussex, England) and CO2
analyzer (ADC analytical Development Co., Hoddesdon, England) connected in series,
with N2 as the carrier gas (ﬂow rate = 100 mL/min). Average of the three taken samples
was used in analysis. The gas composition of individual packages was calculated using a
certiﬁed standard (1.95% 02 and 4.85% C02). Gas partial pressure were monitored daily
for 8 days.

Analysis of volatiles. Volatile collection was accomplished using solid-phase
micro-extraction (SPME) device (Supelco, Inc., Bellefonte, Pa.). A ﬁber with a 100 pm-
thick, non-bonded, polydimethylsiloxane-based stationary phase was used. The absorption
time was 5 min. After absorption, volatiles were then thermally desorbed from the ﬁber
coating by directly inserting the ﬁber into the injection port (200°C) of a gas
chromatograph (HP-6890 Hewlett Packard Co., Wilmington, Del.) for 90 s. Gases were
cryofocused on-column using liquid N2 and separated using a gas chromatograph ﬁtted
with a 10-meter, 0.1 mm ID, DB-S capillary column (Hewlett Packard Co., Boise,
Idaho). The detector was a time-of-ﬂight (TOF) mass spectrometer (Pegasus H, LECO
Co., St. Joseph, Mich). The TOF mass spectrometer permitted the resolution and
quantiﬁcation of compounds not separated chromatographically. Identiﬁcation of volatile
material was done by comparison of the spectra and GC retention times with authenticated
standard compounds. High purity (>95%) authenticated standards were obtained from
Aldrich (Aldrich, Co., Milwaukee, Wis). For the standard compounds, a mix of the

targeted compounds was prepared in a 4.85-L jar (standard jar), equipped with a self-

154

sealing rubber sampling port. The mix was prepared by ﬁrst placing 50 11L of each
compound into a 2.5-mL via] to generate a stock mix. Then, 10 uL were taken from the
stock mix and placed into the 4.85-L jar to be used as standard. Standard preparation was
done at 5 °C and the standard jar was kept at the temperature of the experiment.
Calculation of the concentration of standard compounds was expressed in ppm. As
sampling from the packages was done, so was from the standard to estimate the actual
production of the targeted compounds regardless of the sensitivity of the detection system.

Data were reported in nmol-kg'I-h".

Results

Strawberry volatiles in packages. Strawberry volatiles detected include esters,
alcohols, hydrocarbons, and ketones (Fig. 1 and Table 1) although many of the
identiﬁcations are tentative in that appropriate standards are lacking. This is in agreement
with previous reports (Shamila et al., 1992; Shamaila et al., 1992a). Among these groups
of volatiles, esters represented the dominant class which is consistent with data reported
by Manning (1993). Identiﬁed esters included methyl, ethyl, propyl, butyl, pentyl, hexyl,
octyl, and nonyl esters. Sulfur compounds, which have been reported to be part of the
strawberry volatiles proﬁle (Dirinck et al. 1977; Dirinck et al., 1989; Lieten and Dirinck,
1991), were not detected in study. Furanones detected include, 2-(2-furanylmethyl)-5-
methyl furan and, 2,5-dimethyl butyl furan. Furanones have been referred as the character
impact compounds of strawberry (Manning, 1993).

Effect of modiﬁed atmosphere on strawberry volatiles over time. Aroma

155

production of most strawberry volatiles declined markedly during the 8 (1 package sealing,
independently of the O2 and CO2 concentration in the packages (Fig. 2). Volatiles which
declined over time such as methyl butanoate and acetone reached the lowest levels of
biosynthesis at d 8 and stayed low thereafter.

The degree of suppression of volatile production as a ﬁinction of in-package O2
and CO2 was evaluated for selected volatiles including methyl hexanoate, acetone, methyl
butanoate, ethyl butanoate, methyl acetate and hexyl acetate (Table 2). The rate of
suppression for the selected volatiles ranged from 10 fold to 26.2 fold. The rate of
suppression was estimated by dividing the GC-MS response in packages with 15 g of fruit
over the GC-MS response in packages with 360 g of fruit.

The ratios of methyl acetate to methyl butanoate and hexyl acetate to methyl
hexanoate did not seem to be affected by 02 and CO2 partial pressure combinations (Fig.
4). The ratio of methyl acetate to methyl butanoate was steady during the ﬁrst 6 d of
storage and increased markedly aﬁer d 6. The ratio of methyl acetate to methyl butanoate
may be used as indicator of shelf-life of MAP-stored strawberry. The break point was
estimated to be day 6. On the other hand, the ratio of hexyl acetate to methyl hexanoate
increased continuously over time. This ratio may also have potential to be used as a shelf-
life indicator.

Eﬂect of low 0, alone on the production of strawbeny volatiles. In order to
determine whether the impact of MA on volatile production by strawberry was a particular
effect of the combination of low 02 and high C02, we monitored volatile production by

strawberry exposed to low 02 alone. Low 02 alone has a different impact on volatile

156

production by strawberry than a combination of low 02 and high C02. Low 0,, for
instance, enhance the production of methyl acetate, acetone and ethyl butanoate (Fig. 5)

which appeard to be supressed by the combination of low 02 and high CO2 (Fig. 5).

Discussion

In contrast to what has been reported by Yahia et al. (1991) on the diﬁ‘erential
effect of low 02 and high CO2 levels on the production of speciﬁc volatiles by ‘McIntosh’
and ‘Cortland apples, in strawberry, there was a severe suppression of volatile production
by low 02 and high C02. It may indicate that atmosphere modiﬁcation in strawberry
affected the activity of key enzymes in the ester production system.

Williams and Knee (1977) reported that esterifying enzymes operate at similar
rates in fruits stored in air or in 2% 02. Thus, changes in the suppression of particular
volatiles such as methyl butanoate, acetone, and methyl hexanoate (Figs. 2, 3) in steady-
state atmospheres are suggested to be due to limited supply of volatile precursors, such as
alcohols and CoA fatty acids. Perhaps, at d 8, the production precursors to alcohols such
as amino acid derivatives necessary for the production of esters in strawberry stored in
MAP was limited. This theory, however, may not be likely in regard to alcohols in
strawberry since very few alcohols were identiﬁed. Nevertheless, the only alcohol of
importance we found, butanol, appeared to be markedly reduced by low 02 atmospheres
(Table. 1). A decrease in the in-package 02 from 17.5 kPa to 3 kPa induced a decrease in
butanol production in by 99.6%.

Isolation of the low-oxygen eﬂect on volatile production by strawbeny. The

157

information regarding ester production formation in modiﬁed atmosphere-packaged
strawberries with a combination of low 02 and high CO2 atmospheres and the response to
a range of O2 partial pressures with no CO2 permits the isolation of the effect of low 02
and high CO2 on ester biosynthesis (Fig. 5). Esters, except those of ferrnentative
metabolism (ethanol, ethyl acetate and ethyl butyrate), have been suggested to be
produced via B-oxidation to generate short chain fatty acids and via peroxidation to
generate a pool of alcohols (Ke et al., 1994). At least three controlling points can be
suggested in which low 02 and or high CO2 have effect on the production of esters in
strawbeny (Fig. 5). 1) Low 02 has a positive direct effect on the activity of both pyruvate
decarboxylase and acetaldehyde dehydrogenase. Low 02 activates these two enzymes and
the substrate ethanol is produced in abundance. High carbon dioxide may have less direct
effect, as reported by Ke et al. (1994) via induced changes in pH. 2) Low 02 can also
have some effect on one or more of the peroxidases responsible for peroxidation of
saturated fatty acids. Equally, high CO2 may also have some effect on these peroxidases
which may not be necessarily the same enzymes affected by low 02. The effects of either
or both low 02 and or high CO2 on these peroxidation enzymes would result in changes in
the production of alcohols which in turn will affect the total production of esters. This
concept is supported by our results which showed that the suppression of ester production
in strawberry is a combination of both low 02 and high C02. 3) Low 02 and or high CO2
might induce changes in ester production during B-oxidation of unsaturated fatty acids.
Possible enzymes that are affected include: a) the set of fatty acid dehydrogenase enzymes

and the B-ketoacyl-CoA thiolase responsible for the generation of acetyl-CoA out of fatty

158

acids, 3) The third and last point at which low 02 and/or CO2 may exert some effect is
during the formation of short chain fatty acids ﬁ'om elongating FA chains via an ACP
thioesterase that cleaves the FA from the acyl carrier protein. The synthesis of fatty acids
from acetyl-CoA and malonyl-CoA involves seven enzymatic reactions (Voet and Voet,
1990). These reactions include enzymes such as reductases and dehydrases as well as
acyl carrier proteins.

In conclusion, the impact of modiﬁed atmospheres on the production of esters by
strawberry is a combined inhibitory effect of low 02 and high CO2 . The mode of action of
O2 and C02, however, is not clear yet. It appears to be by interfering the biosynthesis of
substrates such as alcohols and acids since the enzyme alcohol acetyl CoA transferase has
been reported to have little sensitivity to either low 02 or high C02. However, the results
of this study suggest that developing the appropriate package design, as it affects O2 and
C02, is of paramount importance to achieve high quality product since the aroma at the

time the consumer opens a package is what is perceived and used to judge quality.

159

References

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Ben-Arie, R. and Y. Zutkhi. 1992. Extending the storage life of ‘ﬁiyu' persimmon by
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Cameron, A.C., W. Boylant-Pett, and J. Lee. 1989. Design of modiﬁed
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Church, 1.] and AL. Parsons. 1994. Modiﬁed atmosphere packaging
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Dirinck, P., H. Poorter, and N. de Champ. 1989. Aroma development in ripening ﬁ'uits.
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fruits and vegetables. CRC Crit. Rev. Food Sci. Nutr. 2821-30.

Ke, D., L. Zhou, and A. Kader. 1994. Mode of oxygen and carbon dioxide action on
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Lesschaeve, I., D. Langlois, and P. Etievant. 1991. Volatile compounds in strawberry
jam: Inﬂuence of cooking on volatiles. J. Food Sci. 5: 1393-1398.

Li, C. and AA. Kader. 1989. Residual effects of controlled atmospheres on postharvest
physiology and quality of strawberry. J. Am. Soc. Hort. Sci. 98:285-288.

Lieten, P. and P. Dirinck. 1991. Fruit quality of strawberries grown on substrates. Acta
Hortic. 294:117-123.

Manning, K. 1993. Soft Fruit. In: G. Seymour, J. Taylor and G. Tucker. (eds)
Biochemistry of Fruit Ripening. Chapman & Hall, London. pp 347-377.

Shamaila, M., W. D. Powire, and BI. Skura. 1992a. Sensory evaluation of strawbeny
fruit stored under modiﬁed atmosphere packaging (MAP) by quantitative
descriptive analysis. J. Food Sci. 521168-1172.

Shamaila, M., W. D. Powire, and BJ. Skura. 1992b. Analysis of volatiles compounds
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Food Sci. 5:1173-1176.

Sommer, N.F., Fortlage, R.J., Mitchell, PC, and EC. Maxie. 1973. Reduction of
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Williams, A. A. and M. Knee. 1977. The ﬂavor of Cox’s Orange pippin apples and its
variation with storage. Ann. Appl. Biol. 87:127-131.

Vergano, PI. and A]. Pertruit. 1993. Effect of modiﬁed atmosphere packaging on the
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24: 145-151.

 

161

strawberry volatile production after 3 d at 22 °C.

Table l. Eﬁ‘ect of fruit weight in 15 cm x 20 cm LDPE packages on 02, CO2 and

 

MS Response 3 d under MAP for each mass of strauberry in packages

 

No.

Mass

 

 

15 g 41g 120 g 360 g No. Mass 15 g 413 120 g 360 g
O/CO, levels O/CO, levels '
(kPa) 188/3 15/7.l 9/13 3/20 (kPa) 188/3 15l7.l 9/13 3/20
1 Ethanol .23 .28 .3 1.5 22 4-rnethyl l-heptane 83.2 9.7 1.82 1.25
2 Butane 6.9 1.98 .29 .23 23 Methyl Hexanoate 5.14 .85 7.16 11
3 Acetone 4.1 .75 .8 .56 24 3-rnethyl l-heptane 12.3 4.14 .47 .018
4 Butanol 28.8 7.3 .31 . 12 25 Ethyl hexanoate 12.6 4.06 .19 .29
5 Methyl 'aeetate 10.5 3.5 3.3 2.73 26 Hexyl acetate 11 7.8 1.49 .05
6 Ethyl acetate 0.35 0.60 1.5 1.9 27 2-methy1, 2 heptane 64 21.7 .24 4.4
Ethyl
7 Propyl acetate 26 15 2.8 .35 28 2,7-dimethyl octanol 62.24 5.64 10.67 3.7
8 Isopropyl acetate 1.4 1.7 1.6 1.99 29 4,6,8-trimethyl 1- 4.6 1.4 3.12 .13
nonene.
9 2.2-dimethyl 39.44 13 1.6 .04 30 Butyl hexanoate 13.3 4.1 .34 .3
Hexene
10 Methyl butanoate 1 16.5 24.3 5.3 4.57 31 S-butyl 4-nonene .62 .6 7 .11
1 1 2.5-dimethyl 2.72 1.1 8.55 1.57 32 2-(2-fru'anylmethyl)—5- 1.8 9 .22 .09
Hexene methyl flu-an
12 4-methy1 1-hepty1 26.6 7.16 .35 .095 33 2,5-ditert-buty1 furan 6.8 5.27 .33 .02
13 2,3,3-trimethyl 21.4 7.8 2.56 0 34 2,4,6-trimethy1 deeane 10.30 6.53 .045 .8
pentene
14 3-methy1heptane 17.7 3.2 1.38 .71 35 2.5,9-uimethy1 decane .92 ’ .45 5.93 .01
15 . Ethyl butanoate 48.7 1.36 1.82 .12 36 Pcntyl hexanoate 21.98 .69 .11 .26
16 Butyl acetate 86.7 19.8 .28 .19 37 Nonyl acetate 1.06 .59 .2 .06
17 Propyl butanoate 92.6 34 13 2.78 38 Tridecane 2.34 .41 .054 .08
18 Ethyl pentanoate 23.26 4.46 16.4 .23 39 Oetyl butanoate .72 .39 .21 .04
19 ' 3-methyl octene 2.54 .77 2.51 1.6 40 Oetanal dimethyl 2.32 1.06 .23 .03
20 Butyl butanoate 2.39 .78 .76 ‘ .22 .
21 Tridecene , 24 22 .055 .05

 

 

162

2.0

 

l

1.8“ 3 10 ten 23 282! _

'16; “ h
1.4‘ e —

'l p
1.2- ..
1.0- ..
0.8- _

J

0.6-1 s -

GC-MS Response 11 108

0.4—

 

5 12 1|

.2. A l ..
. 22
0 0 123 ‘ 7 . U i 1. ‘

100 120 140 160 180 200 220 240 260.' 280 300
Retention Time, s

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 1. GC-MS Spectrum of the total ion count (TIC) for strawberry enclosed in a sealed jar
for 40 min on day 0. Identiﬁed volatiles include: 1) carbon dioxide, 2) Ethanol, 3) butane,
4) acetone, 5) butanol, 6) methyl acetate, 7) ethyl acetate, 8) propyl acetate, 9) isopropyl
acetate, 10) 2,2-dimethyl hexene, 11) methyl butanoate, 12) 2,5-dimethyl hexene, 13) 4-
methyl-l-heptyl, l4) 2,3,3-trimethyl pentene, 15) 3-methyl heptane, l6) ethyl butanoate,
l7) butyl acetate, 18) propyl butanoate, l9) ethyl pentanoate, 20) 3-methyl octene, 21)
butyl butanoate, 22) tridecene, 23) 4-methyl-l-heptane, 24) ethyl hexanoate, 25) 3-methyl-
l-heptane, 26) ethyl hexanoate, 27) hexyl acetate, 28) 2-methyl-2-heptane ethyl, 29) 2,7-
dimethyl octanol, 30) 4,6,8-trimethyl-l-nonene, 31) butyl hexanoate, 32) 5-butyl-4-nonene,
33) 2(2)-furanyhnethyl)-5-methyl furan, 34) 2,5-dimethyl-butyl furan, 35) 2,4,6-trimethyl
decane, 36) 2,5,9-trimethyl decane, 37) pentyl hexanoate, 38) nonyl acetate, 39) tridecane,
40) octyl butanoate, 41) octanal 2,5-dimethyl acetate.

163

 

 

 

 

 

 

 

 

 

 

 

 

 

16" ' ' ' ' ' )_ 1.6; .
. a _ - i
”A 3 t 1-273 c) '
a ‘12-;1 :- 08:
5 0.8-3 :— ' E
a j E 0.4:
‘T 0.4“: - ‘
é” : : 034.
,5 0,0 , . , . , , , , ’ -. . - . - .
a ~+3609+1209+41g+159
5
$- 3 r t . t l t . 1.6 l . r . r , I I
9: 1.6 b) - 121 d)
g : '1
U ; 04-: '
E “1%
o 2 4. 15V 3 1o 0 2 j 4 ' 6 i 8 10
Days of Monitoring Days of Monttorlng

Figure 2. Trend over time of four selected different strawberry volatiles in modiﬁed
atmosphere packages. Figure (a) represents methyl butanoate, b) Methyl acetate,
c) Acetone, d) Methyl hexanoate. Each graph represents four different packages
with four different atmospheres as determined by different masses of the fruit in
the packages. Each dot is the average of three replicates.

Carbon Dioxide (kPa)

GC-MS Response x108

164

 

 

 

  

.4-

 

 

 

 

 

 

 

 

 

 

20"? A C C 93609 1
1s.-j
12:5
8 .1 : 2 3 120 g
‘ a. L‘- 1341 g
4.1 —
1 C C C 3 C C C ﬁ’> 15 g
0. 1 2‘
I "1‘7 1"Irj‘ ‘t'"‘17ﬁ‘71IT‘Tl“ﬁ‘l'I"T“"1""l
0 1 2 3 4 5 6 7 8 9 1O 11
Days
1.6 4 1 1 1 I 1 4 1 l - 1 - l . . . I . . 1 l 1 4 1 l - 1 1 I 1 1 1 1 1 #1 1 1 1 1 J 1
14.; O Methyl Hexanoate - L
1 25 A Acetone g E
O 3 V Methyl Butanoate ' - F
10- I Ethyl Butanoate :—
03“: 0 Methyl Acetate 7
0.5.3 as Hexyl Acetate L
0.4-j E-
i 1
0.21 "
0.0‘ 1 . Tej- w a .1
0 2 4 20

 

Oxygen Concentration in Packages '

Figure 3. Trend of strawberry volatiles in modiﬁed atmosphere packaging and as

impacted by in-package 02 concentration (lower graph). Top graph shows the
levels of CO2 in packages. The arrow shows the day when volatile measurements
were made. Amount of product in packages is shown for each level of CO2 in top

graph.

165

 

 

 

 

 

 

 

 

 

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Days of Monitoring

Figure 4. Ratios of methyl acetate/methyl butanoate and hexyl acetate/methyl hexanoate
in modiﬁed atmosphere-packed strawberry.

166

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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a
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Oxygen Partial Pressure, kPa

Figure 5. Range of O2 generated in a ﬂow through system (top) and changes in the
production of six different volatile esters at different 02 concentrations: a) acetone,
b) methyl acetate, c) methyl butanoate, d) ethyl butanoate, e) methyl hexanoate,
and t) hexyl acetate. Data were generated for day 3.

 

 

167

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Fatty Acid Carbohydrate
Oxrdation Metabolism
"‘ ““““ 8-0xidatio ' 1’
6 . n Peroxidatron Pyruvate
Ecetyl-COA (SE-’4‘: Peroxidasesj- ‘- 0
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- XI
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V

Figure 6. Proposed mode of action of O2 and CO2 on the biosynthesis of esters in
strawberry packaged under modiﬁed atmospheres. The arrows indicate the
possible points where low 02 and high CO2 may have inhibitory impact on the
biosynthesis of strawberry volatiles. The signs (-) or (+) indicate low or high

respectively.

168

CHAPTER VI

AROMA PRODUCTION OF CARROT (12111111; mm L.) AND BROCCOLI
(Brassica oleracea L.) As A FUNCTION OF 02 PARTIAL PRESSURE IN MAP

169

AROMA PRODUCTION OF CARROT (123119115 mm L.) AND BROCCOLI
(BESSIE cleracea L.) AS A FUNCTION OF O, PARTIAL PRESSURE IN MAP

Addjﬂgnaﬂndexms. modiﬁed atmospheres, volatile, mass spectrometry, sulfur
compounds, terpenes, quality
Abstract. A combination of solid phase micro extraction (SPME) as a sampling method
and gas chromatography/time-of-ﬂight mass spectrometry (GC-TOF-MS) for gas analysis
were used to investigate the volatiles produced by carrot sticks and broccoli ﬂorets stored
for eight days in modiﬁed atmosphere packages. Methanethiol, dimethyl sulﬁde and
dimethyl disulﬁde content in the headspace of packages containing broccoli increased as
package 02 decreased. Our results indicate that packages should be designed to maintain
02 levels > 5 to 6 pKa to prevent broccoli from producing sulﬁir volatile compounds
should. In carrot, most of the carrot volatiles detected were negatively impacted by MAP
when the in-package 02 levels were below 3-9 kPa. These results provide evidence that
the formation of terpenes, major component of the carrot volatile proﬁle, is dependent
upon the Oz/COZ-sensitive carbohydrate metabolism of higher plants. The impact of low
02 on the production of total terpenes suggests that most carrot volatiles are produced
through the same pathway and that thus their O,-dependent control points, or key
enzymatic steps, are common for all of them. Low 02 may act by restricting the

production of important precursors such as acetyl-CoA and mevalonate.

170

The technology of modiﬁed atmosphere packaging has been employed in extending
the shelf-life and preserving the fresh-like appearance of broccoli and carrot ( Barmore,
1987; Shewfelt, 1987; Riel, 1995). The 02 levels, however, in modiﬁed atmosphere
packages of fresh cut vegetables can be depleted to a low level by respiration. When the
oxygen drops below the extinction point, aerobic respiration diminishes and anaerobic
respiration becomes the dominate pathway. Some produce such as carrot can survive a
slight depletion of 02 below the extinction point (Hisashi and Watada, 1996). The
extension of shelf-life of vegetables including carrot and broccoli in modiﬁed atmosphere
packaging is due to a reduction in the rate of respiration and associated biochemical
processes delaying senescence (Ballantyne, 1989). Beneﬁts include delayed ripening,
improved color retention and reduction of microbial incidence. However, not much
attention has been paid to the impact of modiﬁed atmosphere packaging on the production
of volatiles by both broccoli and carrot during storage. Aroma is an essential component
of food ﬂavor. Flavor constitutes one of the key elements in food quality.

Fomey et al. (1991) reported that modiﬁed atmosphere-packed broccoli develop
strong off-ﬂavors. Fomey identiﬁed ethanol, methanethiol, hydrogen sulﬁde, ethyl
acetate, dimethyl disulﬁde, acetaldehyde, methyl acetate, and acetone in in the headspace
of packages broccoli stored under an atmosphere of 0.5% Oz. Hensen et al. (1992)
reported that B-ionone also contributes to the objectionable odor of modiﬁed atmosphere-
packed broccoli.

Heatherbell et al. (1971) reported that the volatiles acetaldehyde, sabinene,
myrcene, and terpinolene are considered to be important character-impact compounds in

raw carrot aroma. Heatherbell and Wrolstad (1971) conducted a study on the inﬂuence of

171

variety, maturity and storage duration on the type and concentration of volatiles in carrot.
Their result showed that the differences were quantitative rather than qualitative and that
the variation in concentration of individual terpenes and their summation appeared to be
consistent with description of the ﬂavor characteristics of the different varieties. In most
research done on modiﬁed atmosphere-packed carrot, the effect of MAP on aroma has not
been evaluated, even though aroma is on of the key components of minimally processed
carrot quality (Bolin and Huxoll, 1991; Bolin, 1992; Brackett, 1987; Bruemmer, 1987;
Carlin et al., 1990; Chervin et al., 1994; Heatherbell et al., 1971; Howard et al., 1995).
Simon et al. (1980) examined the volatiles of eight carrot lines by using a tenax GC
trapping technique. Among the volatiles reported by Simon et al. (1980) include alpha
pinine, beta pinene, myrcene, alpha phellandrene, alpha terpinene, limonene, gamma
terpinene, terpinolene, terpinen-4-ol, bomyl acetate, caryophellene and gamma bisabolene.
Simon et al. also found that slicing of carrot enhanced the production or release
caryophellene and alpha bisabolene, which is consistent with results published by
Heatherbell et al., ( 1970).

In this study, our major aim was to evaluate the volatile production of both
broccoli and carrot stored under modiﬁed atmosphere packaging to gain a better
understanding of the relationship between volatiles and levels of O2 and C02. as affected by
package design. We wished to identify those volatiles that could serve as ﬁngerprints to
assess the physiology of the packed produce and be able to assess shelf-life and storage

conditions.

172

Material and Methods

Plant material. Broccoli and carrot were obtained as fresh as possible from the
Detroit Produce Terminal. Broccoli was cut into ﬂorets and carrot was cut into sticks
ranging from 2 to 5 inch length. The lightly processed products were then sanitized in a
smixture of 0.5 g-L‘l of Benlate (Du Pont de Nemours Co., Agricultural Product
Department, Wilmington, Del.) and 0.5 g-L‘1 Rovral ( Rhone-poulenc Ag. Company,
Research Triangle Park, NO) TO prevent decay and then rinsed with distilled water and
allowed to air-dry. The storage temperature was 15 °C.

Modiﬁed atmosphere packaging of strawbeny. The design Of modiﬁed
atmosphere packaging was achieved by using the approach of Beaudry et al. (1992) as
adapted from Cameron et al. (1989). Fruit were sealed in pouches made of 0.00762 cm (3
mil) thick low-density polyethylene (LDPE; LDF 550, Dow Chemical Company, Midland,
Mich). The initial step in the package design for strawberry was the determination of
respiration rates (r02 ) of whole strawberries stored at atmospheric O2 partial pressure
(20.7 kPa) at 2 °C. Using the estimated r02, the needed ﬁlm area and fruit weight were
calculated to generate 15 kPa O2 . From this high 02 setting, ﬁ'uit weight was varied to
achieve a range of 02 levels.

Headspace Analysis and Volatile Identiﬁcation. Volatile collection was
accomplished using solid-phase micro-extraction (SPME) device (Supelco, Inc.,
Bellefonte, Pa). A ﬁber with a 100 um-thick, non-bonded, polydimethylsiloxane-based
stationary phase was used. The absorption time was 5 min. After absorption, volatiles
were then thermally desorbed from the ﬁber coating by directly inserting the ﬁber into the

injection port (200°C) of a gas chromatograph (HP-6890, Hewlett Packard CO.,

173

Wilmington, Del.) for 90 s. Gases were cryofocused on-column using liquid N2 and
separated using a gas chromatograph ﬁtted with a 10-meter, 0.1 mm ID, DB-S capillary
column (Hewlett Packard Co., Boise, Idaho). The detector was a time-of-ﬂight (TOF)
mass spectrometer (Pegasus 11, LECO Co., St. Joseph, Mich). The TOF mass
spectrometer permitted the resolution and quantiﬁcation of compounds not separated
chromatographically. Identiﬁcation of volatile material was done by comparison of the
spectra and GC retention times with authenticated standard compounds. High purity
(>95%) authenticated standards obtained from Aldrich (Aldrich, Co., Milwaukee, Wis).
For the standard compounds, a mix of the targeted compounds was prepared in a 4.85-L
jar (standard jar), equipped with a self-sealing rubber sampling port. The mix was
prepared by ﬁrst placing 50 11L of each compound into a 2.5-mL vial to generate a stock
mix. Then, 10 11L were taken from the stock mix and placed into the 4.85-L jar to be used
as standard. Standard preparation was done at 5 °C and the standard jar was kept at the
temperature of the experiment. Calculation of the concentration of standard compounds
was expressed in ppm. As sampling from the packages was done, so was from the
standard to estimate the actual production of the targeted compounds regardless of the
sensitivity of the detection system. Data were reported in umol-kg'1-h".
Results

Impact of IMAP on broccoli volatiles. Typical broccoli volatiles such as limonene
appeared to be negatively affect by 0, levels below 5-6 kPa. Limonene production was
markedly reduced at 02 levels less than 6 kPa (Fig. 1). The same effect is Observed for
acetone. Methanethiol, dimethyl sulﬁde and dimethyl disulﬁde were identiﬁed in low 0,

headspace (0.8-5 kPa) in a higher concentration than in headspace with higher levels of O,

174
(Fig. 1).
Impact of MAP on carrot volatiles. Most of the carrot terpene volatiles detected
using the SPME/GC-MS method were negatively impacted by MAP when the in-package
0, levels were below 3-9 kPa (Fig. 3). The reduction in headspace content was greater

thar 90% for all almost all volatiles as 02 level declined from 17 kPa to 1.3 kPa.

Discussion

A possible enzymatic pathway for the production of methanethiol, dimethyl sulﬁde
and dimethyl disulﬁde by broccoli was investigated by Pentima et al. (1995). In a cell-free
system of fresh broccoli, methanethiol formation occurred in response to L-methionine
and S-methylcysteine and dimethyl sulﬁde is formed ﬁ'om S-methylmethionine. In the
same study, Pentima et al. (1995) reported that an increase in free amino acids was
observed in broccoli under anaerobic conditions. Our results, are in agreement with
Pentima et al. (1995), and suggest that broccoli has the enzymatic capacity to produce
volatile sulﬁlr compounds when there are free amino acids available. Modiﬁed
atmosphere packaging, which usually consists of a range of O, of 1.5-6 kPa, enhance the
release of the amino acids that serve as precursors for the sulfur volatile compounds
detected. F omey et al. (1991) considered the methanethiol primarily responsible for the
off-odor produced by broccoli stored under low 0, atmospheres. Hensen et al. (1992)
found that the major compounds contributor to the offensive off-odor are methanethiol,
methyl trisulﬁde, and B-ionone. In our experiment, we did not detect either methyl
trisulﬁde nor B-ionone. Perhaps these compounds require lower oxygen levels or maybe

the concentrations of these compounds were below the detection limit of our method.

175

Pentina et al. (1995), reported that the three main factors involved in the formation
of volatile sulfur compounds, which are contributors to the objectable odor in broccoli
include: (1) the availability of substrates, free sulfur amino acids; (2) the presence of
enzyme with speciﬁcity to cleave these amino acids; and 3) the accessibility between
substrates and enzymes. Our results suggest that one or more of these factors is lacking in
modiﬁed atmosphere packages where the levels of methanethiol, dimethyl sulﬁde and
dimethyl disulﬁde are low. Perhaps the enzymatic system responsible for the cleavage of
amino acids is more active at low 0, levels (0.8-6 kPa). Or else, the amount of
precursors (amino acids) is higher at low 0, atmospheres. Our results are also in
agreement with Kasmire et al. (1974) who reported the effect of aeration rate on the
production of off-ﬂavor by broccoli heads during a simulated transit period. Kasmire et
al. (1974) reported that adequate ventilation is essential to prevent accumulation of off-
odors .

Makhlouf et al. (1990) reported that the combination Of moderately low 0, and
high CO, has a protective effect on membrane integrity of broccoli. In the same study
they suggested that protection may be achieved indirectly by delay of senescence, or
indirectly by the MAP action on the membranes. The later possibility would agree with
the hypothesis that states that the very ﬁrst event during senescence would be membrane
lipid degradation (Heemskerk and Witerrnans, 1987). Direct protection of membrane
lipids could be due to the high solubility of CO, in hydrophobic environment (I-Iayduck
and Buckley, 1971). Based on our results, we cannot elucidate whether the effect on
MAP on sulfur volatile compounds is due to the action of low 0,, high CO, or their

combination. However, our results suggest that the packaging design that would prevent

176

broccoli from producing sulfur volatile compounds should not induce 02 levels below 5-6
kPa in the packages. This information can be important in the formulation of
mathematical models for predicting shelf-life of broccoli.

Regarding the impact of modiﬁed atmospheres on the production of terpenes,
several studies have conducted to investigate the O,/CO,-sensitive carbohydrate
metabolism of higher plants. Using radiolabeled acetate-Z-"C and mevalonate-Z-“C, Oba
et al. (1970) found that the acetate and mevalonate incorporated into terpenes appear to
be produced ﬁ'om carbohydrate metabolism through the formation of pyruvate and acetyl-
COA in mitochondria Of sweet potato. Croteau et al. (1972) demonstrated that in vitro
biosynthesis of acetyl-CoA from sugars yields, concomitantly, ATP and reduced pyridine
nucleotides, both of which are required for utilization of acetyl-CoA in terpene
biosynthesis in peppermint. Croteau et al. (1972) also study the effect of CO, on the
assimilation of precursors in the formation of terpenes. This group of researchers found
that 5% CO, approximately doubled the incorporation of labeled mevalonate into
caryophellene.

Based on this worlg modiﬁed atmosphere-packed carrot, the inhibition of terpene
production is likely to be the result of the effect of low 0, (<5-6 kPa) on the metabolism
of carbohydrates. The overall pathway of terpinolene, for instance, include the formation
of acetyl-CoA, NADH, ATP and CO2 ﬁ'om sucrose. The acetyl-CoA generated along
with the energy produced as NADH and ATP are then used to produce the corresponding
terpenes. George-Nascimento and Cori (1971) conducted a study on terpene biosynthesis
from geranyl and neryl pyrophosphates by enzymes from orange ﬂavedo. In this study

they reported that the biosynthesis of monoterpenes such as limonene and alpha pinene

177

involves the transformation of mevalonic acid into 10-carbon pyrophosphorylated
intermediates, and subsequent elimination of the phosphate moiety. Terpinolene,
limonene, caryophellene and alpha pinene formation requires the supply of acetyl-CoA as
well as the generation of energy in the form of NADH obtained from the aerobic pathway
of carbohydrate metabolism. These results support our theory and result that the low 02
levels in MAP-packed carrot is the responsible for the marked reduction of terpene
production by carrot (Figs. 3, 4). The impact of low 0, on the production of total
terpenes also gives way to several observations: (1) it appears that most carrot volatiles
are produced through the same pathway and that thus their controlling points, or key
enzymatic steps, are common for all of them; (2) it seems likely that the low 02 is the
inhibition factor, perhaps by restricting the production of important precursors such as
acetyl-CoA and mevalonate; (3) the relationship between 02 levels in packages and the
production Of terpene volatiles in MAP-packed carrot allows us to have a better
understanding of the biochemistry of the produce during storage and could serve as asset
in the designing of automated quality control systems and mathematical models to predict

storage time and conditions.

178

References

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Bolin, H. R. and C. C. Huxoll. 1991. Control of minimally processed carrot surface
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atmospheres. ARS Report No. 73272. 7-73p

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Oba, K., H. Shibata and I. Uritani. 1970. The mechanism supplying acetyl-CoA for
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180

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181

 

 

   
   

 

 

 

 

 

 

 

    
 

 

 

 

 

 

24- r y 1 T 1 1 1 1 5° ‘ 1 1 ' l ' 1 ' 2
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16- 30— 3
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. 4g-
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Oxygen partial pressure in packages (kPa)

Figure 1. Selected volatile compounds produced by MA-stored broccoli as aﬁ'ected by
diﬁ‘erent levels of in-package 0, partial pressures.

182

 

 

 

 

 

Ll l l l l l l l I l l l l l l l I l l l l l l L J. L1 1 l 1
24.- ' . -
- C methanethiol
: I methyl sulﬁde
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Figure 2. Eﬂ‘ect of 02 on the accumulation of volatile sulfur compounds from broccoli in
modiﬁed atmosphere packaging on d 3 stored at 15 °C.

 

6.0-1

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12.0-

 

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Camphene

Ocimene

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183

 

 

 

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Oxygen partial pressure (kPa)

IIIII‘IIII

3-carene

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Figure 3. Selected volatile compounds produced by MA-stored carrot as aﬁ‘ected by
different levels of in-package O2 partial pressures.

184

 

    

 

 

 

70. ‘ ' ' ' 1 1 1 . l . l . l . l
‘ A sabenene -
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a . _

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O 2 4 6 8 10 12 14 16 18

Oxygen Partial Pressure in Packages (kPa)

Figure 4. General trend of terpene volatile compounds as affected by different 02 partial
pressure in MA-stored carrot.

185

SUMMARIES

Senescence-related volatile proﬁles of strawberry. Our results indicate that, in
strawberry, ester volatiles as a whole decrease as fruit senesce and thus a decrease of
esters may be used as a general indicator of the physiology of the fruit during storage.
Among esters, methyl acetate seems to have unique relationships with physiological
processes linked to the senescence of strawberry. The positive correlation of methyl
acetate with softening may be related to the release of methanol from the breakdown of
the cell wall pectins. Additionally, the acetate moiety of the ester is derived from acetyl
CoA, which may result from LOX activity and B-oxidation processes that are thought to
be involved in membrane degradation during senescence. Methyl acetate may be a good
candidate as a ﬁngerprint-type volatile to assess senescence in strawberry.

Volatile proﬁles of broccoli and carrot during storage. Volatiles of carrot sticks
decreased diring storage, possibly as a function of tissue aging. Besides the general trend
of terpenes over time, ratios among terpene compounds shifted markedly as well,
suggesting the ratios may be used as markers of carrot stick age. In broccoli ﬂorets,
dimethyl disulﬁde seemed to be related to senescence and may have potential to be used as
a senescence index.

Volatile proﬁles of E. coili and S. typhimurium grown in laboratory media and
food products. Both indole and l-propanol have the potential to be used as ﬁngerprint
compounds for E. coli in both standard laboratory media and in different produce
packages. 1-propanol and indole are volatiles produced by E. coli but not by broccoli,

carrot or lettuce and this information may be exploited in a more practical way. Indole

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was not detected in produce packages, however, due perhaps to the interference of some
other carbohydrate sources in produce. Nevertheless, indole may be promising in other
types of food such as meet, dairy products and egg. S. typhymurium, also produces
detectable volatiles which should be explored ﬁrrther. S. typhymurium appears to have the
enzymatic capacity to breakdown amino acids present in the standard laboratory media, to
produce alcohols.

Fingerprint volatiles of microorganisms not only offer a way of identifying
microbial development in food but also offer information on the biochemistry and
metabolism of the organisms. With the availability of rapid, accurate and more sensitive
volatile analysis techniques, it worthwhile to use volatiles produced by microorganisms as
a research asset in the development of bacterial detection systems in food packages.

Storage duration, low 0, and elevated C 02 alter ester biosynthesis in strawberry.
The impact of modiﬁed atmospheres on the production of esters by strawberry is a
combined inhibitory effect of low 02 and high CO2 . The mode of action of 02 and C02,
however, is not clear yet. 02 appears to interfere with the biosynthesis of substrates such
as alcohols and acids since the enzyme alcohol acyl transferase has been reported to have
little sensitivity to either low 02 or high C02. Nevertheless, the information regarding
changes of speciﬁc volatiles in packages may be used to assess aroma quality of packaged
hit and for the design of packages to optimize quality at the time of package opening by
the consumer.

Aroma production by carrot and broccoli as a function of 0, partial pressure in
modiﬁed atmosphere packages. Methanethiol, dimethyl sulﬁde and dimethyl disulﬁde

were identiﬁed in low 02 headspace of packages (0.8-5 kPa) in a higher concentration

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than in headspace with higher levels of 01 A possible enzymatic pathway for the
production of these compounds seems to be activated by low 02 levels in MAP-packed
broccoli that involves the breakdown of L-methionine, S-methylcysteine and S-
methylmethionine as suggested by previous workers. Our results indicate that packaging
design that would prevent broccoli from producing sulfur volatile compounds should not
induce 02 levels below 5-6 kPa in the packages. In carrot, most of the carrot volatiles
detected were negatively impacted by MAP when the in-package 02 levels were below 3-9
kPa. These results provide evidence that perhaps the formation of terpenes, major
component of the carrot volatile proﬁle, is sensitive to 02/C02 levels that limit sensitive
carbohydrate metabolism.

General remark. Today, with the availability of rapid, accurate and more sensitive
volatile analysis techniques, it worthwhile to use volatiles produced by plants and
microorganisms as a research asset. Volatile analysis may be a useﬁrl tool to assess some
physiological events that take place in plants and microorganisms which today are not fully
understood. Furthermore, signiﬁcant potential exists to exploit the volatiles produced by
packaged products and microbial contaminants to assist in developing technologies to

non-destructively assess product quality and safety.

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