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thesis entitled

MICROBIAL LEVELS AND REDUCTION STRATEGIES FOR
MICHIGAN HIGHBUSH BLUEBERRIES

presented by

luliano Dumitru Pope

has been accepted towards fulﬁllment
of the requirements for the

MS degree in Food Science and Human
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MICROBIAL LEVELS AND REDUCTION STRATEGIES FOR MICHIGAN
HIGHBUSH BLUEBERRIES
By

luliano Dumitru Popa

A THESIS

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

MASTERS OF SCIENCE

Department of Food Science and Human Nutrition

2005

ABSTRACT

MICROBIAL LEVELS AND REDUCTION STRATEGIES FOR MICHIGAN
HIGHBUSH BLUEBERRIES

By

luliano Dumitru Popa

Highbush blueberries were collected from 18 Michigan ﬁelds during 2003-2004 and
assessed for mesophilic aerobic bacteria (MAB), coliforms, Escherichia coli, yeasts and
molds during harvesting and processing (pre-harvest, post-harvest, blower-exit, water
tank-exit, and pre-packaging for freezing) along with conveyor belt and chlorinated wash
water samples from six processing facilities. Microbial populations generally increased
~l .5 log between pre- and post-harvest and decreased < 1 log aﬁer exiting the water tank
(~10 to 200 ppm chlorine). Thus, microbial levels were higher aﬁer processing than
before harvesting with populations signiﬁcantly (P <0.05) higher on conveyor belts and
in chlorinated wash water during processing. Chlorine dioxide gas was also assessed as a
pre-processing microbial reduction strategy for blueberries. In preliminary work using
blueberries inoculated with several foodbome pathogens as well as spoilage yeasts and
molds, 12 h of gassing (0.16 mg C102 gas/g fruit) in a 20 L bucket decreased all microbial
populations >3 logs. When 600 lb pallets of naturally contaminated blueberries were
subsequently exposed to 0.13 mg C102 gas/g fruit for 12 h, signiﬁcant (P <0.05)
reductions of 2.12, 1.61, 0.72, 1.76, and 1.55 logs CFU/g were seen for MAB, yeasts,
molds, coliforms, and E. coli, respectively, compared to ungassed controls. Gassing of
additional blueberries with 0.19 mg C102 gas/g for 12 h did not affect the appearance,

aroma, texture, ﬂavor or overall fruit acceptability.

To my dearest son Bogdan and wife Darclee for all of your love and support

iii

ACKNOWLEDGMENTS

I would like to extend my sincere appreciation to my committee members Dr. Eric
Hansone, Dr. Kirk Dolan, and to my project collaborators Dr. Annemiek Schilder, Dr.
Ewen Todd.

I am grateful to my major professor Dr. Elliot Ryser for his excellent insight and
his scientiﬁc guidance in preparing this dissertation and my carrier.

I am thankful to Dr. Zhinog Yan for his unforgettable help in the laboratory as
well to all my laboratory colleagues.

Finally, I would like to thank my family for encouraging and supporting me. To
my mom, thank you for all of your love and encouragement. You have helped this whole

process to be a memorable experience that was worthwhile.

iv

TABLE OF CONTENTS

LIST OF FIGURES ................................................................................. vii
LIST OF TABLES ................................................................................... ix
1. INTRODUCTION ................................................................................. 1
2. LITERATURE REVIEW ......................................................................... 5
2.1 BLUEBERRY INDUSTRY ..................................................................... 5
2.1.1 CHARACTERIZATION OF BLUEBERRY .............................................. 5
2.1.2 MICHIGAN BLUEBERRY INDUSTRY .................................................. 6
A. Processed Market ........................................................................... 8
B. Processed Blueberry Standards .......................................................... 10
C. Fresh Market ............................................................................... 11
2.2 PROCUCE RELATED OUTBREAKS ...................................................... 11
2.2.1 SOURCES OF CONTAMINATION ...................................................... 12
2.2.2 BERRY- ASSOCIATED OUTBREAKS ................................................. 13
2.3 BLUEBERRY MICROBIAL LIMITS ...................................................... 14
2.4 HUMAN PATHOGENS ...................................................................... 16
2.4.1 ESCHERICHIA COL10157:H7 ............................................................ 16
A. Health signiﬁcance of E. coli 0157:H7 ................................................. 16
B. E. coli 0157: H7 outbreaks in produce .................................................. 17
2.4.2 SALMONELLA ............................................................................... 18
A. Health signiﬁcance of Salmonella ....................................................... 18
B. Salmonella outbreaks in produce ........................................................ 20
2.4.3 LIST ERIA MONOC YT OGENES .......................................................... 21
A. Health signiﬁcance of Listeria monocytogenes ....................................... 21
B. Listeria monocytogenes outbreaks in produce ......................................... 22

2.5 ECOLOGICAL FACTORS INFLUENCING HUMAN PATHOGENS

ON BLUEBERRY ............................................................................. 23
2.6. METHODS TO PREVENT AND ELIMINATE BLUEBERRY AND

HUMAN PATHOGENS ..................................................................... 24
2.7 SAN ITIZERS .................................................................................. 30

2.7.1 CLOROX TM/ SODIUM HYPOCHLORITE (NaClO) ................................. 32

A. General characteristics ................................................................... 32
B. Antimicrobial performance .............................................................. 33
2.7.2 CHLORINE DIOXIDE .................................................................... 34
A. General characteristics .................................................................... 34
B. Antimicrobial performance ............................................................... 35

3. MICROBIAL CONTAMINANTION IN HIGHBUSH BLUEBERIES BEFORE,

DURING, AND AFTER PROCESSING .................................................... 38
3.1 INTRODUCTION .............................................................................. 39
3.2 MATERIAL AND METHODS .............................................................. 40
3.3 RESULTS ....................................................................................... 44
3.4 DISCUSION .................................................................................. 48
4. EFFICACY OF CHLORINE DIOXIDE GAS SACHETS FOR ENHANCING THE

MICROBIOLOGICAL QUALITY AND SAFETY OF BLUEBERRIES .............. 51
4.1 INTRODUCTION ............................................................................. 52
4.2 MATERIAL AND METHODS .............................................................. 55

A. PILOT STUDY .............................................................................. 55

B. PALLET STUDY ........................................................................... 59
4.3 RESULTS AND DISCUSSION ............................................................. 63

A. PILOT STUDY .............................................................................. 64

B. PALLET STUDY ............................................................................ 66
4.4 CONCLUSIONS ............................................................................... 70
5.CONCLUSIONS ................................................................................. 72
APPENDIX A ....................................................................................... 75
APPENDIX B ....................................................................................... 86
APPENDIX C ....................................................................................... 97
APPENDIX D ....................................................................................... 102
APPENDIX E ......................................................................................... 138
REVERENCES ...................................................................................... 144

vi

FIGURE 3.1

FIGURE 3.2

FIGURE 3.3

FIGURE 3.4

FIGURE 3.5

FIGURE 4.1

FIGURE 4.2

FIGURE 4.3

FIGURE 4.4

LIST OF FIGURES

HIGHBUSH BLUEBERRY PROCESSING AND
SAMPLING DIAGRAM .............................................

POPULATIONS OF MAB, YEASTS, MOLDS,
COLIFORMS AND E. COLI ON BLUEBERRIES
SAMPLED AT PRE-HARVEST, POST-HARVEST,
BLOWER EXIT, WATER TANK EXIT AND PRE-
PACKAGING .........................................................

POPULATIONS OF MAB, YEASTS, MOLDS,
COLIFORMS AND E. COLI ON CONVEYER BELTS
ENTERING THE BLOWER (A) AND PRE-PACKAGING
AREAS (B), BEFORE AND AFTER FRUIT
PROCESSING .........................................................

POPULATIONS OF MAB, YEASTS, MOLDS,
COLIFORMS AND E. C OLI IN WATER SAMPLES
BEFORE AND AFTER FRUIT WASHING ......................

POPULATIONS OF MAB, YEASTS, MOLDS,
COLIFORMS AND E. COLI ON BLUEBERRIES BEFORE
AND AFTER 3 AND 6 MONTHS OF STORAGE AT - 20°
C .........................................................................

CHLORINE DIOXIDE GAS SYSTEMS USED TO TREAT
INOCULATED BLUEBERRIES IN PILOT STUDY ...........

PALLET STUDY WITH GASSED AND UN-GASSED
PALLETS .............................................................

SCHEMATIC DIAGRAMS OF A BLUEBERRY PALLET
AND LUG WITH SAMPLING LOCATIONS ...................

POPULATIONS OF L. MONOC YT OGENES,
SALMONELLA, E. COLI 01572H7, YEASTS AND MOLDS
ON INOCULATED BLUEBERRIES BEFORE AND AFTER
EXPOSURE TO C102 GAS IN A SEALED BUCKET ..........

vii

42

44

46

47

47

58

60

62

65

FIGURE 4.5

FIGURE 4.6

FIGURE 4.7

FIGURE A 1

FIGURE A 2

FIGURE A 3

FIGURE D 1

FIGURE E 1

FIGURE E 2

POPULATIONS OF MAB, YEASTS, MOLDS,
COLIFORMS, AND ECOLI RECOVERED FROM
BLUEBERRIES INITIALLY AND FROM GASSED AND
UN-GASSED FRUIT AFTER 12 H OF STORAGE ............

POPULATIONS 0F MAB, YEASTS, MOLDS,
COLIFORMS, AND E. COLI RECOVERED FROM
GASSED BLUEBERRY PALLETS AT DIFFERENT
PALLET LEVELS (A) AND BETWEEN THE BOTTOM
AND TOP SURFACE OF LUGS FROM THE SAME
PALLET LEVEL (B) .................................................

AVERAGE CONSUMER RATINGS FOR APPEARANCE,
AROME, TEXTURE, FLAVOR, AND OVERALL
ACCEPTABILITY OF TREATED AND UNTREATED
BLUEBERRIES ......................................................

REDUCTIONS OF MAB, YEASTS, AND MOLDS AFTER
A S-MINUTE EXPOSURE TO VARIOUS SANITIZERS IN
AN AQUEOUS MODEL SYSTEM ................................

REDUCTIONS OF MAB, YEASTS, AND MOLDS AFTER
A 5-MINUTE EXPOSURE OF INOCULATED FRUITS T0
VARIOUS SANITIZERS ............................................

AVERAGE CONSUMER ACCEPTABILITY FOR FRESH
BLUEBERRIES SUBJECTED FOR 5 MIN TO WASH
TREATMENT WITH SODIUM HYPOCHLORITE,
CHLORINE DIOXIDE, ORGANIC FATTY ACIDS AND
SDW AS CONTROL .................................................

POPULATIONS OF MAB, YEASTS, MOLDS,
COLIFORMS AND ECOLI ON BLUEBERRY SAMPLES
BEFORE AND AFTER FROZEN STORAGE IN
INDUSTRIAL CONDITIONS ......................................

GROWTH OF MAB, YEASTS, MOLDS, COLIFORMS,
AND E. COLI ON NATURALLY CONTAMINATED
BLUEBERRIES DURING STORAGE ............................

REDUCTIONS OF MAB, YEASTS, MOLDS,
COLIFORMS, AND E. C OLI ON INOCULATED AND
UNINOCULATED BLUEBERRIES AFTER 12-H
EXPOSURE TO C102 GAS IN A SEALED BUCKET .........

viii

67

69

70

81

83

84

102

141

142

TABLE 2.1

TABLE 3.1

TABLE 3.2

TABLE 3.3

TABLE 3.4

TABLE 4.1

TABLE 4.2

TABLE A.l

TABLE A.2

LIST OF TABLES

MICROBIAL LIMITS FOR MAB, YEASTS, MOLDS,
COLIFORMS AND HUMAN PATHOGENS SET BY
PURCHASERS OF BLUEBERRIES ....................................

POPULATIONS OF MAB, YEASTS, MOLDS, COLIFORMS
AND E. COLI ON BLUEBERRIES SAMPLED AT PRE-
HARVEST, POST-HARVEST, BLOWER EXIT, WATER TANK
EXIT AND PRE-PACKAGING ..........................................

POPULATIONS OF MAB, YEASTS, MOLDS, COLIFORMS
AND E. COL] ON CONVEYER BELTS ENTERING THE
BLOWER AND PRE-PACKAGING AREAS, BEFORE AND
AFTER FRUIT PROCESSING ...........................................

POPULATIONS 0F MAB, YEASTS, MOLDS, COLIFORMS
AND E. COLI IN WATER SAMPLES TAKEN BEFORE AND
AFTER FRUIT WASHING ................................................

POPULATIONS OF MAB, YEASTS, MOLDS, COLIFORMS
AND E. COL] ON BLUEBERRIES BEFORE AND AFTER 3 AND
6 MONTHS OF STORAGE AT - 20° C .........................

POPULATIONS OF L. MONOC YT OGENES, SALMONELLA, E.
COL] 01572H7, YEASTS AND MOLDS ON INOCULATED
BLUEBERRIES BEFORE, AFTER AND REDUCTIONS FROM
EXPOSURE TO C102 GAS IN A SEALED BUCKET ...............

POPULATIONS OF MAB, YEASTS, MOLDS, COLIFORMS,
AND ECOL] RECOVERED FROM BLUEBERRIES BEFORE
AND FROM GASSED AND UN-GASSED FRUIT AFTER 12 H
OF STORAGE AT 12 TO 14°C ..........................................

REDUCTIONS OF MAB, YEASTS, AND MOLDS AFTER A 5-
MINUTE EXPOSURE TO VARIOUS SANITIZERS IN AN
AQUEOUS MODEL SYSTEM ...........................................

REDUCTIONS OF MAB, YEASTS, AND MOLDS AFTER A 5-
MINUTE EXPOSURE OF INOCULATED FRUITS TO VARIOUS
SANITIZERS ..................................................

ix

15

86

87

88

89

90

91

93

94

TABLE E.l

TABLE E.2

GROWTH OF MAB, YEASTS, MOLDS, COLIFORMS, AND
E. COLI ON NATURALLY CONTAMINATED BLUEBERRIES
DURING STORAGE AT ROOM TEMPERATURE ......................

REDUCTIONS OF MAB, YEASTS, MOLDS, COLIFORMS, AND
E. COL] ON IN OCULATED AND UNINOCULATED
BLUEBERRIES AFTER 12-H EXPOSURE TO C102 GAS IN A
SEALED BUCKET .........................................................

95

96

1. INTRODUCTION

Consumption of fruits and vegetables in the United States has increased
substantially, with per capita consumption of produce increasing 24% (from 573 1b in
1970 to 711 pounds in 1997) (Putnam and Allshouse, 1997). Moreover, new production
and packaging technologies make possible year-around availability of numerous fresh
fruits and vegetables that also are increasing in consumption. Fresh fruits and vegetables
increased 3% in 2004 with a grth trend in consumption forecast by 2020 (Produce
Marketing Association, 2005). Changes in dietary habits, with a higher per capita
consumption of fresh and minimally processed fruits and vegetables, and increasing
demand for salad bars and meals eaten outside the home have also ampliﬁed the number
of foodbome outbreaks (Altekruse and Swerdlow, 1996). The median number of reported
produce-associated outbreaks increased from 2 outbreaks per year in the 19703 to 7 per
year in the 19805, and to 16 per year in the 19905. According to Sumathi et a1. (2004),
from 190 produce-associated outbreaks reported between 1973 through 1997, nine
involved berries - 4 raspberry, 4 strawberry, and 1 blackberry.

Food safety concerns surrounding blueberries include a possible linked to a 1984
outbreak of listeriosis in Connecticut after consumption of unwashed strawberries,
blueberries or nectarines (Ryser, 1999), a 2002 conﬁrmed outbreak of hepatitis A in New
Zealand with domestic blueberries traced back to a single orchard that revealed multiple
opportunities for contamination of blueberries by pickers (Calder et a1., 2003), and a 1998
recall involving an undetermined quantity of frozen blueberries from California, Illinois
and Australia that was contaminated with Listeria monocytogenes (FDA Enforcement

Report, 1998). These blueberry and other berry-types outbreaks in the past with their

economical implications highlights the need for food safety programs in the berry fruit
industry.

Although the domestic blueberry industry has not been negatively affected by any
widespread outbreak with foodbome pathogens, microbial safety remains a critical
concern for all segments of the blueberry industry that have indirectly affected producers
and marketers in the form of buyer demands for microbial testing and increasing
microbial speciﬁcation.

The United States is the world’s leading blueberry producer, and approximately
one-third of the total national highbush blueberry crop is produced in Michigan. Most of
Michigan blueberries are processed and frozen for later use (NASS/USDA, 2002). Some
buyers of frozen berries now demand microbial testing for levels of spoilage bacteria,
yeasts and molds in order to assure that the product does not exceed their Speciﬁcation
and also have a ‘zero tolerance’ policy for human pathogens. However, microbial
speciﬁcations for frozen blueberries vary considerably from buyer to buyer, in general
reﬂecting different uses, and becoming increasingly stringent. One example are the pie
manufacturers that need a low level of bacteria, yeast and molds in their product because
most of these microbial categories isolated from blueberries are producing the enzyme
amylase that reduce the viscosity of blueberry ﬁling (Schilder Annemieck — unpublished
data).

Blueberry producers have observed large variations in microbial levels that can
not be correlated just to ﬁeld conditions or harvesting procedures. A microbial
assessment study of blueberries from different locations in Michigan during 2002 and

2003, revealed that populations of bacteria, yeasts and ﬁlamentous fungi varied widely

among ﬁeld locations including those that were irrigated and non-irrigated although the
same populations increased considerably on blueberry surface in time from green stage to
the end of harvest (Schilder Annemieck — unpublished data).

A scientiﬁc understanding of the factors affecting variation in microbial levels,
the risk associated with various microbial levels, and the steps needed to attain particular
levels is needed in order to set microbial standards for blueberries. Understanding at
which points in production, harvesting, processing and packaging contamination is likely
to occur is clearly needed along with improved sanitizing and microbial reduction
strategies in order to obtain a science-based uniform standard for microbial levels that are
able to satisfy buyers and also be effective and affordable for growers and processors.

The emergence of new pathogens is a well-known fact. Pathogens that were
previously less virulent have increased their virulence, being now considered of public
health concern to certain categories of consumers. They acquired resistance to antibiotics,
and environment conditions (low temperature, low pH) in which they were unable to
survive before. The bacterial pathogens of greatest concern for the blueberry industry that
can use the less acidic surface of blueberries as vectors to produce outbreaks include
Listeria monocytogenes, Salmonella, and Escherichia coli 01571H7. All these pathogens
categories are included in this study in order to achieve the new microbial speciﬁcations
that include the ‘zero tolerance’ for the human pathogens; however, there is a general
lack of efficacy of aqueous chemical sanitizers in killing or removing these pathogens
from the surface of raw fruits and vegetables (Beuchat, 1998). This fact can be attributed
to difﬁculties in delivering aqueous chemical sanitizers to areas on the surface of fruits

with a hydrophobic surface such as blueberries, where these human and also fruit

pathogens that produce the spoilage may be lodged (Burnet and Beuchat, 2001). The
theory that hydrophobicity of microbial cells aids in their protection by inhibiting
penetration of the disinfectants has also been proposed (Buck, 2003).

Alternative sanitizers such as gaseous chlorine dioxide have been explored as
alternative to aqueous chemicals for sanitizing raw fruits and vegetables.

Studies have shown gaseous chlorine dioxide to be effective in microbial
reductions including enteric pathogens on several fruits and vegetables in laboratory
conditions. Gaseous chlorine dioxide has some advantages over chlorinated water that
consist in removing phenolic tastes and odor from water, does not react with organic
compounds, and has a greater oxidation and germicidal capacity including Spores, Viruses
and protozoa that are resistant to chlorine. Gaseous chlorine dioxide is increasing in
popularity by being used to effectively control the spread in molds in libraries (Weaver-
Meyers et a1., 1998), as a sanitizer for reducing yeast and mold populations in food
processing plants on stainless steel surfaces (Han et a1., 1999), and in the
decontamination of buildings in the US after the 2001 anthrax attacks.

The goal of this research is to (1) assess the levels of microbial contamination at
various points during blueberry harvesting, handling, cleaning, and packaging and (2)
assess the efﬁcacy of gaseous chlorine dioxide for inactivating Escherichia coli
0157:H7, Listeria monocytogenes, Salmonella, mesophilic aerobic bacteria, coliforms, E.
coli, yeasts and molds on the surface of blueberries in industrial conditions before
processing. These ﬁndings will eventually help to establish a science-based uniform
standard for microbial levels and improve the actual microbial reductions strategies in

blueberries that will satisfy the needs of buyers as well as growers and processors.

2. LITERATURE REVIEW

2.1 BLUEBERRY INDUSTRY
2.1.1 CHARACTERIZATION OF BLUEBERRY

The blueberry, which belongs to the genus Vaccinium, is native to North America.
Native Americans took advantage of these fruits to consume fresh or to preserve for
medicinal purposes and dyes (U.S. Highbush Blueberry Council, 2002). Although
Vaccinium includes over 450 species of blueberries, the highbush blueberry (V.
cotymbosum) is economically the most important. This specie is grown in the Mid-
Atlantic, Midwest and Paciﬁc Northwest regions of the United States, and along the
Paciﬁc Northwest of Canada (Bowling, 2000).

Blueberry fruits are round to slightly ﬂattened in shape with a diameter of about
0.5 inch. They have a crown-like structure termed the calyx on the bottom and a
depressed ring on the top where the stem is attached. The blue-to-blue dark epidermal
surface of the fruit is covered with a waxy bloom, giving the fruit a light blue appearance
(Pritts, 1992). During the fruit expansion the total B-diketone per fruit increase from 191
to 909 ug/ﬁ'uit. The hydrophobicity results from B-diketone arranged in a dense network
of interlocking branched rodlets or closed tubes within this wax layer (Freeman et a1.,
1979). Possingham et a1. (1976) suggested that the structural arrangement of wax on
blueberries, together with the hydrophobic nature of the surface, controls water
movement.

Blueberries have been important commercially in the northeastern United States

and Canada since the 18808. The Highbush blueberry industry, however, did not begin

until the early 19005 when Elizabeth White from New Jersey and Dr. Frederick Coville
from Maryland initiated research for domestication of the wild highbush blueberry. Their

work resulted in the development of blueberries that could be commercially grown by

farmers (U.S. Highbush Blueberry Council, 2002).

2.1.2 MICHIGAN BLUEBERRY INDUSTRY

Development of the blueberry industry in Michigan is attributed to Stanley
Johnston’s work at Michigan State University during the 19305 (Bowling, 2000).
Michigan’s climate and soil conditions are ideal for blueberries that require sandy soils
that are high in organic matter and very acidic (Hancock et a1., 2001). Hence, they can
thrive near the Lake Michigan shore, an area that was previously considered useless for
agriculture (Bowling, 2000).

The United States is the world’s leading blueberry producer with 55% of total
production followed by Canada at 28% (ESR/ USDA, 2003). Approximately one-third of
the total US. highbush blueberry crop comes from Michigan. The blueberry industry is
composed of two market categories: fresh and processed blueberries. In the US. about 45
percent of all highbush blueberries are sold to the fresh market, with the remainder being
further processed. Harvest begins in April in Florida, peaking in July when Michigan is
included, and ends in October in British Columbia, Canada (NASS/USDA, 2003)

In 2002, Michigan had 16,900 acres of highbush blueberries that yielded 64
million lb of fruit. Approximately 66 percent of these berries (42 million lb) were
processed and the remainder went to the fresh market (NASS/USDA, 2002). The

percentage of Michigan blueberries marketed as fresh fruit is much lower than the

national average because harvest begins in July. July - also known as National Blueberry
Month, is the peak harvest season for blueberries with the volume of fruit in the
marketplace exceeding demand. Given the drop in price during peak harvest, much of the
fruit is sold to the processed market. Allegan, Berrien, Muskegon, Ottawa and Van Buren
counties on the western side of Michigan’s Lower Peninsula are primary blueberry-
growing regions (Hancock et a1., 2001). The most popular varieties in Michigan are
Jersey, Bluecrop, Elliot, Duke and Rubel. Total Jersey acreage declined from about 55%
in 1970 to 40 % in 2000. Rubel, the other historically important variety, has also declined
and now representsless than 10% of total acreage. However, the remaining varieties have
increased over time with Bluecrop now comprising almost 30% of total production due to
its very high and dependable yields. The Elliot variety, almost nonexistent in the 1970’s,
has become increasingly popular due to its very late harvest and storability, with the
Duke variety also increasing signiﬁcantly after 2000 because of its large, ﬁrm fruit, late
bloom and early harvest (Hancock et a1., 2001). The best—suited varieties for fresh market
include Bluecrop, Duke, and Elliot; whereas the Jersey and Rubel varieties are small-
fruited and most popular for the processing market (Hancock et a1., 2001). Most growers
plant a range of varieties to extend the harvest season for both fresh and processed
markets.

The time of harvest depends on the blueberry variety, weather, and location. In
Michigan, harvesting typically begins in early July with handpicking for the fresh market,
and ends in September. Blueberries usually ripen over several weeks, and require two to
four pickings to harvest. After handpicking is complete, growers shift to mechanized

harvesting for the processed market. Machine-harvested bushes are usually picked when

60 to 70 % of the berries are blue and again 10 to 14 days later (Hanson and Hancock,
1998)

In 2000, Michigan had approximately 575 growers of which 6% had 100 or more
acres and accounted for 46% of the total acreage (Michigan Agricultural Statistic Service,
2002). From 1991 to 2000, the trend was for the number of farms to decrease and acreage
to increase. Although the numbers of acres planted has increased by almost 10 %, acreage
decreased for all size groups except the largest growers (200 or more acres) where
acreage increased by 93 %. Approximately half of all growers belong to the Michigan
Blueberry Growers Association (MBG) with the remainder market their fruit
independently. MBG members market their fresh fruit through Global Berry Farms
(MBG’s marketing company), and their processed fruits through Peterson Farms, Inc.
Some integrated operations also grow, pack and market their own fruit. Most growers
pack their own fresh fruit; however, a few growers have their own processing facility

(Bertelsen et a1., 1995).

A. Processed Market

The market structure for processed blueberries has changed in response to
demands by customers for more stringent food safety and quality standards. High costs
due to updating facilities, technologies and administration to meet these standards have
resulted in fewer individual growers operating their own processing facilities. Most
signiﬁcant was the opening of two new processing cooperatives in 1999 and 2001, in

addition to MBG: Northern Pride Processing (NPP) and West Michigan Processing

Cooperative (WMPC). Each processing cooperative has about 24 members (Michigan
Blueberry Growers Association, 2001).

The blueberry processing chain begins with mechanical harvesting by large over-
row harvesters that shake berries from the bushes onto conveyors. From conveyors, the
berries are loaded int020-lb lugs. The lugs are then stacked on pallets and transported to
the processing facility. Upon arrival at the processing facility, the ﬁ'uits are either
processed the same day or stored overnight on pallets until the next day. From lugs, fruits
are loaded onto the processing line (conveyer belts) that carries the fruit over a blower to
remove leaves, sticks and other debris. A tilt belt is then used to separate soft and cluster
berries. Next, the berries are passed through a water tank to remove any green unripened
fruit that ﬂoats to the surface. This water tank also contains a sanitizer (usually sodium
hypochlorite or chlorine dioxide) to decrease the microbial load on the berries.
Subsequently, the berries pass through a de-stemmer that consists of a series of rollers.
Finally, the fruit is inspected manually and/or automatically (color sorter/ optical sorter)
for quality characteristics before being boxed. The color sorter is most popular, but the
new optical inspection sorter can distinguish ripened berries from other berries (green,
red, multicolored) and also the Japanese beetle more accurately than hand sorting (Fruit
Growers News, 2002). Finally, the 30-pound boxes are coded, dated, passed through a
metal detector, and stacked on pallets for freezing (U.S. Highbush Blueberry Council,

2002)

B. Processed Blueberry Standards

Blueberry grades and standards have been set by USDA (USDA/AMS, 1995), and
MBG. Processors use MBG’s grading system because it is more demanding. In grading
fruit, considerations include stem counts, detritus, berry color, fruit damage and ripeness.
The most important standards for processors are those set by individual customers that
may include particular sizes (small and ﬁrm or larger), and/or particular characteristics
such as sweetness and color. Food safety standards are stringently implemented by
processors, who must now have in place a detailed and documented Hazard Analysis
Critical Control Point (HACCP) program, metal detection, multiple self-inspection
audits, and annual audits by outside parties including an independent, and third-party
food safety audit. All of these changes are necessary because of increasing food safety
concerns among customers (Gentry, 2002). In addition, some buyers require third-party
certiﬁcation (TPC). A cost-effective solution for meeting the high cost of TPC and
changing customer standards was the opening of the new processing co-ops including
NPP and WMPC.

Microbial standards are increasing because of customer demands. Particular
microbial standards set by customers reﬂect the need for safety (‘zero tolerance’ for
human pathogens) or a speciﬁc use. The industry needs to know how to meet these
increasing demands. A scientiﬁc understanding of the factors affecting variation in
microbial levels, the risk associated with various microbial levels, and the steps needed to
attain particular levels is needed in order to set microbial standards. Understanding at
which points in production, harvesting, processing and packaging contamination is likely

to occur is clearly needed along with improved sanitizing and microbial reduction

10

strategies. These efforts will eventually lead to a science-based uniform standard for
microbial levels that will satisfy buyers and also be effective and affordable for growers

and processors.

C. Fresh Market

Investments for fresh market are much lower than those for the processing
market. Most fresh blueberries are handpicked using seasonal migrant labor. Fresh
blueberries are not sanitized to decrease microbial levels, mainly because immersion in
water severely limits product shelf-life. Some large growers run berries through a blower
followed by a tilt belt (for soft berries), a color sorter, and an inspection belt before
marketing. Smaller growers simply inspect the fruit manually before packaging. In the
fresh market, adherence to good agricultural and hygienic practices is the basis for
minimizing microbial levels. Third-party certiﬁcation is also implemented in the fresh

sector because of requirements from some major retailers.

2.2 PRODUCE RELATED OUTBREAKS

During the past several decades the number of produce-related outbreaks reported
to Centers for Disease Control and Prevention (CDC) produced by foodbome pathogens
as greatly increased due to increased consumption of fresh produce, changes in food
production and distribution practices, and the emergence of new foodbome pathogens
(U .S.G.A.O., 2002). Changes in dietary habits, with a higher per capita consumption of
fresh and minimally processed fruits and vegetables, and increasing demand for salad

bars and meals eaten outside the home have further ampliﬁed the number of foodbome

ll

outbreaks (Altekruse and Swerdlow, 1996). Consumption of fruits and vegetables in the
United States has increased substantially, with per capita consumption of produce
increasing 24% (from 573 lb in 1970 to 711 pounds in 1997) (Putnam and Allshouse,
1997). The median number of reported produce-associated outbreaks increased from 2
outbreaks per year in the 19705 to 7 per year in the 19805, and to 16 per year in the
19905. According to Sumathi et a1. (2004), from 190 produce-associated outbreaks
reported between 1973 through 1997, nine involved berries - 4 raspberry, 4 strawberry,

and 1 blackberry.

2.2.1 SOURCES OF CONTAMINATION

Produce can be contaminated at any point during its growth, harvesting,
processing, distribution, and ﬁnal preparation (Beuchat et a1., 2003). Potential sources for
pre-harvest contamination of produce can include soil, feces, irrigation water, water used
to apply fungicides and insecticides, insects, dust, inadequately composed manure, wild
animals, and human handling (Beuchat, 1996). The ability of produce to internalize
pathogens, including Escherichia coli 0157:H7 (Solomon et a1., 2002), and Salmonella
(Guo et a1., 2002), from contaminated water was also recently reported. Thus, irrigation
wells and other sources for irrigation water should be monitored for human pathogens.
Manure should be adequately composted before being used as fertilizer. Domestic and
wild animals should also be also excluded from contact with produce ﬁelds. Post-harvest
sources of contamination include feces, human handling, harvesting equipment, transport

containers, insects, dust, rinse water, and processing equipment (Burnett and Beuchat,

2001).

12

Fruits and vegetables are major components of a healthy diet; however, eating
fresh uncooked produce is not risk free. Further efforts are needed to better understand
the complex interactions between microbes and produce and the mechanisms by which
contaminants are spread from ﬁeld or orchard to the table. Intensive investigations that
include traceback to the farm of origin are now being conducted to identify the root cause

of these outbreaks (Sumathi et a1., 2004).

2.2.2 BERRY-ASSOCIATED OUTBREAKS

Some berry-associated outbreaks have been traced back to single and/or multiple
growers that were located both in and outside of the United States. In 1990 one multistate
(Montana, Georgia) outbreak of hepatitis A, the source of contamination was traced to
frozen strawberries from one processing facility in California with the strawberries
coming from their own farm (N iu et a1., 1992). Another traceback of a multistate hepatitis
A virus outbreak in 1997 led to strawberries from four farms in Mexico that were
processed (frozen and packaged) at a single plant in California (Hutin et a1., 1999).
Several Cyclospora outbreaks were also traced to raspbem'es imported from Guatemala
(Herwaldt, 2000).

Regarding blueberries, in 1984 consumption of unwashed strawberries,
blueberries or nectarines may have been linked to an outbreak of listeriosis in
Connecticut (Ryser, 1999). More recently, blueberries were conﬁrmed as the source of
infection in a multi-district outbreak of hepatitis A in New Zealand with domestic
blueberries that were traced back to a single orchard. Fourteen tones of blueben'ies from

the orchard had been sold in New Zealand, 14 tones had been exported and 22 tones were

13

in cold storage (frozen). At the investigation was found a high contamination rate (3/6
samples) among frozen bluebenies (Calder et a1., 2003). In addition, in 1998 one
producer was forced to recall an undetermined quantity of frozen blueberries from
California, Illinois and Australia because of contamination with Listeria monocytogenes

(FDA Enforcement Report, 1998).

2.3 BLUEBERRY MICROBIAL LIMITS

Michigan blueberries have not yet been implicated in any outbreaks of illness.
However, since blueberries and other berry types have been linked to outbreaks in the
past, microbial safety remains a critical concern for all segments of the blueberry
industry. Large buyers now demand microbial testing and processors must meet
particular microbial speciﬁcations with a third-party certiﬁcation. These buyers have
‘zero tolerance’ policies for human pathogens such as L. monocytogenes, E. coli
0157:H7, Staphylococcus, and Salmonella. They also have limits on mesophilic aerobic
bacteria (MAB), coliforms, yeasts and molds that vary considerably from buyer to buyer

(Table 2.1).

14

Table 2.1 Microbial limits (CFU/g) for mesophilic aerobic bacteria (MAB), yeasts,
molds, coliforms, and human pathogens (Staphylococcus, Listeria and E. coli 0157:H7)
set by purchasers of blueberries (After MAFMA Research Proposal, 2003).

 

 

 

Test Company A Company B Company C Company D

M A B 150,000 50,000 50,000 10,000
Yeast 20,000 10,000 5,000 4 1,000
Mold 5,000 10,000 5,000 1,000
Colifonns 100 100 <1 ,000 10
E. coli Absent <10 Absent Absent
Staphylococcus Absent Absent Absent Absent
Listeria Absent Absent/25 g Absent/25 g Absent/25 g
E. coli 0157:H7 Absent Absent Absent/25g Absent

 

For example, Company A accepts levels of MAB, yeast, mold and colifonns on
fruit that are 15, 20, 5, and 10 times higher than Company D. Some limits appear
arbitrary, whereas others reﬂect speciﬁc uses. Pie manufacturers who use blueberries
with high yeast counts frequently ﬁnd that their pies do not “set up” because yeast grth
results in enzymatic breakdown of starch and other stiffening agents. This can, in turn,
lead to considerable ﬁnancial loss and frequently a change in blueberry suppliers. These
microbial standards are difﬁcult to meet since the level of MAB, yeasts and molds and
other contaminants can vary widely between ﬁelds, seasons, and the time of harvest
depending on factors such as moisture, temperature, insect level, plant health, and harvest

management practices (Schilder Annemieck — unpublished data).

15

2.4 HUMAN PATHOGENS
2.4.1 ESCHERICHIA COL] 0157:H7
A. Health signiﬁcance of E. coli 0157:H7

Enterohemorrhagic E. coli (EHEC) was ﬁrst isolated in 1975 and ﬁrst identiﬁed
as a human pathogen in 1982 during two outbreaks of hemorrhagic colitis in Oregon and
Michigan that were linked to undercooked ground beef hamburgers sold by one fast food
restaurant chain (Riley et a1., 1983). Individuals who are most susceptible population
include children age 2-10 and those over 65 years of age. The oral infective dose is very
low (0.3-15 CFU/g Of frozen ground beef) as evidenced from the largest multistate
outbreak to date. In this outbreak 731 cases were registered, 178 were hospitalized and 4
children died. The incubation period for E. coli 0157:H7 is 1-5 days after which
individuals develop nonbloody diarrhea that can be self-limiting or turn bloody and
persist for up to 2 weeks. Hemolytic uremic syndrome (HUS) - the most dangerous
complication in children, is characterized by acute renal failure and a high fatality rate.
Elderly patients can also develop thrombotic thrombocytopenic purpura (TTP), a rare
adult version of HUS that produces clots in the brain due to platelet aggregation with
neurological signs. These symptoms result from adherence of the organism to the
intestinal tract lining followed by production of one or more verotoxins. All EHEC
produce verotoxins (VT’s) that are cytotoxic to African green monkey kidney (V cm)
cells and similar to Shiga toxin produced by Shigella type 1 [Shiga-like toxins (SLT)]

(Doyle et a1., 1997).

16

B. E. coli 0157: H7 outbreaks in produce

Escherichia coli 0157: H7 has been traced to outbreaks involving lettuce, apple
juice, salad, cantaloupe, and alfalfa sprouts. In 1995, one outbreak was reported in
Montana in which 92 people become ill after consuming leaf lettuce consumption. In
1996, another outbreak in Connecticut, Illinois, and New York was traced to mesclun
lettuce (Hilbom et a1., 1999).

Traceback of lettuce in these outbreaks lead mainly to farms located in the United
States. Contamination on farms most likely occurred through contact with contaminated
irrigation water, wash water or animal feces (Ackers et a1., 1998; Hilbom et a1., 1999).
Fresh produce can easily become contaminated with fecal pathogens such as E. coli
0157:H7, which can remain viable in bovine feces for up to 70 days (Wang and Doyle,
1996). When grown adjacent to or downwind from a microbial source of pathogens such
as a dairy operation, tree fruits can also become contaminated with E. coli (George et al.,
2002)

A signiﬁcant number of outbreaks occurred with fresh apple cider and apple juice.
In 1991, 23 people in Massachusetts became ill and 3 developed HUS after consuming
unpasteurized cider. In 1996, 12 people in Connecticut were ill, 3 with HUS. In the same
year, in multiple states and Canada, another outbreak occurred in which56 people became
ill, 14 developed HUS and 1 died. All of these outbreaks were traced to consumption of
unpasteurized apple cider or juice. Practices that increased the risk of producing
contaminated juice included the use of dropped fruit or inadequate cleaning of the fruit
before juicing (Besser et a1., 1993; CDC, 1997; Cody et a1., 1999). These outbreaks

highlighted the unusual acid tolerance Of E. coli 0157:H7 in acidic foods such as apple

17

cider that typically has a pH of 3.5-4.0. This increased tolerance to acid is based on the
expression of acid shock proteins that provide protection to the organism from normally
lethal pH levels (Foster and Hall, 1990; Leyer and Johnson, 1995). Escherichia coli
0157: H7 can survive up to 7 days in unpasteurized apple cider at room temperature
(25°C), but when the cider is refrigerated (8°C) survival is increased to 20 days (Besser et
aL,1993)

Internalization has been shown to occur with Escherichia coli 0157: H7 in
sprouts (Itoh et a1., 1998) and apples (Buchanan et a1., 1999), and with Salmonella in
tomatoes (Zhuang et a1., 1995) and mangoes (Penteado et a1., 2004). When warm produce
is immersed in cold water, a pressure difference is formed between the produce core and
the surrounding water that facilitates entry of bacteria into the core, mainly through the

stem end (Bartz and Showalter, 1981).

2.4.2 SALMONELLA
A. Health signiﬁcance of Salmonella

Salmonellosis caused by the bacterium Salmonella is one of the most common
and widely distributed foodbome diseases. Millions of human cases are reported
worldwide every year and the disease results in thousands of deaths (WHO, 2005). The
contamination route is mostly fecal to oral with symptoms of infection usually appear 8-
72 h after initial exposure of incubation (Ryser, 1998). The clinical manifestations of
non-typhoid Salmonella infections can range from a self-limited gastroenteritis to
septicemia and death. In the mild cases symptoms of gastroenteritis, including nausea and

vomiting subside within a few hours. Fever, chills, prostration, myalgia and abdominal

18

pain that can resemble acute appendicitis with diarrhea can follow in the course of the
disease. Diarrhea, the predominant symptom can range from loose stools to bloody and
rice-water cholera-like stools in severe cases. The clinical condition can still be self-
lirniting with remission of signs without intervention within 5 days of onset (D’Aoust,
1997 and Ryser, 1998). In some cases, particularly in the very young and in the elderly,
the associated dehydration can become severe and life-threatening. In such cases, as well
as in cases where Salmonella causes bloodstream infections, effective antibiotics are
essential for treatment (WHO, 2005). Of public health concern is shedding of Salmonella
in infected patient’s stools at concentrations 10° to 109 CFU/g. Administration of
antibiotics for gastroenteritis can disturb and/or destroy the normal intestinal microﬂora
that competes with Salmonella for nutrients and binding sites, thereby increasing the
asymptomatic carrier state (D’Aoust, 1997 and Ryser, 1998).

Another public health concern is the emergence of multi-antibiotic resistant
serovars of Salmonella that have now become a serious worldwide problem (Glynn et a1.,
1998). Strains of Salmonella subsp. enterica serovar Typhimurium Deﬁnitive Type 104
isolated in the United States are commonly resistant to ampicillin, chloramphenicol,
streptomicyn, sulfonamides and tetracyclines with strains in England showing additional
resistance to trimethroprim, ﬂuoroquinolones and ciproﬂoxacin (Glynn et a1. 1998). This
resistance results from the use/misuse of antimicrobials in humans and animal husbandry
and national/intemational trade of infected animals is thought to play a major role in the
spread (WHO, 2005). A total of 2501 different Salmonella serotypes have been identiﬁed
up to 2004 and all can cause disease in humans with a total cost estimated at 3 billion

dollars annually in the United States. Salmonella Enteritidis and Salmonella

19

Typhimurium are the two most important serotypes for salmonellosis transmitted from

animals to humans (WHO, 2005).

B. Salmonella outbreaks in produce

Produce items frequently implicated in Salmonella outbreaks include sprouts,
melons, apple/orange juice, and tomatoes. Most of the sprout outbreaks involved alfalfa
sprouts, affected a high number of persons, and were multistate (Glynn et a1., 1998;
Mohle-Boetani et a1., 2001; Mouzin et a1., 1997) or international (Mahon et a1., 1997;
Van Beneden et a1., 1999). These outbreaks were associated with the use of fecally
contaminated seeds or contaminated water during sprouting. The melons implicated in
outbreaks (including watermelon and cantaloupe) were more likely surface contaminated
from the ground where they were grown (CDC, 1991). Between 2000 and 2002, several
salmonellosis outbreaks in United States and/or Canada were linked to Mexican-grown
cantaloupe. FDA conducted a survey of cantaloupe from Mexico and found that 5 percent
of samples were contaminated with Salmonella. Possible sources of contamination
included irrigation of ﬁelds with water containing sewage; processing (cleaning and
cooling) produce with Salmonella-contaminated water; poor hygienic practices of
workers who harvest and process the cantaloupe; pests in packing facilities, and
inadequate cleaning and sanitizing of equipment that comes in contact with cantaloupes.
FDA now detains whole and precut cantaloupes from Mexico until the producer can

demonstrate that the product can be exempt from detention (CDC, 2002).

20

In 1974 in New Jersey 298 people become ill after consumption of contaminated
apples. Twenty-one years later, 62 cases of salmonellosis were linked to consumption of
Florida orange juice. In both outbreaks, the unpasteurized juice was prepared from fruit
that dropped from the trees (CDC, 1975; Cook et a1., 1998). In 2000 another multistate S.
Enteritidis outbreak was associated with consumption of unpasteurized orange juice
prepare from oranges that received a sanitizer treatment before squeezing (Rangel, 2000).

Salmonella is another pathogen that develops tolerance to acidic environments
with serovars Hartford and Typhimurim able to survive up to 27 and 60 days in orange
juice at pH 3.5 and 4.1, respectively. Hence, consumer of unpasteurized juice can be

exposed potentially infectious levels of Salmonella (Parish et a1., 1997).

2.4.3 LISTERIA MONOCYTOGENES
A. Health signiﬁcance of Listeria monocytogenes
Listeria monocytogenes is a pathogenic bacterium that can be found in animal
feces. It is a natural inhabitant of soil and has been isolated from a wide variety of fresh
produce including bean sprouts, cabbage, chicory, cucumbers, eggplant, lettuce,
mushrooms, potatoes, radishes, and salad vegetables (Buck et a1., 2003). However, L.
monocytogenes was not Often found on broccoli, carrots, cauliﬂower and tomatoes, which
lead to speculation that less soil contact during growth of these vegetables or the presence
of some compounds that inhibits the pathogen (tomatoes and carrots) can be the reason.
The cause of the current “Listeria Hysteria” stems from the ubiquitous nature of
the pathogen, severity of the disease that has a fatality rate of 20-30%, and increased

susceptibility of pregnant women, the elderly, and immunocompromised adults.

21

Pregnant women may develop ﬂu-like symptoms, but the infection is much severe for the
unborn fetus with spontaneous abortion, fetal death or delivery of a stillborn infant.
Listeria monocytogenes is a psychrotrophic organism that is also acid and salt tolerant.
Although the oral infectious dose is thought to be as low as 1,000 cells, listeriosis
remains a rare disease with about 2500 cases and 500 fatalities reported annually in the
United States (Mead et a1., 1999). However, the gravity of this disease led to the current
“0 tolerance policy” for L. monocytogenes in ready-tO-eat (RTE) foods, and has made this
pathogen the leading cause of all microbiologically related Class I recalls.
B. Listeria monocytogenes outbreaks in produce

Although L. monocytogenes is widely distributed on plant vegetation (Beuchat,
1996), with produce contamination from soil, water, and manure, (Reina et a1., 1995),
fresh produce has largely remained an uncommon source of foodbome listeriosis. In
1979, raw celery, tomatoes and lettuce were epidemiologically linked as the possible
cause of a listeriosis outbreak involving 23 patients from the Boston area (Beuchat,
1996). Two years later, L. monocytogenes was ﬁrst conﬁrmed as a consumption of
Listeria-contaminated coleslaw was directly linked to 7 adult and 34 perinatal (17 death)
cases of listeriosis in the Maritime Provinces of Canada. Cabbage used in manufacturing
the coleslaw came from ﬁelds that were fertilized with fresh sheep manure from a ﬂock
of Listeria-infected sheep (Schlech, 1983).

Listeria monocytogenes is known as a “hardy” organism and is able to survive on
produce (Zhang and Farber, 1996). Furthermore, as a psychrotroph this pathogen can
grow on refrigerated foods (Lou and Yousef, 1999). Beuchat (1996) found that, L.

monocytogenes grew on endive and lettuce during storage at 10°C. In 1984, consumption

22

of unwashed strawberries, blueberries or nectarines may have been linked to an outbreak
of listeriosis in Connecticut (Ryser, 1999), and in 1998 one producer was forced to recall
an undetermined quantity of frozen blueberries from California, Illinois and Australia

because of contamination with Listeria monocytogenes (FDA Enforcement Report,

1998).

2.5 ECOLOGICAL FACTORS INFLUENCING HUMAN PATHOGENS 0N
BLUEBERRY

To generate disease, pathogens contaminating any food product need to survive at
levels sufﬁcient to cause illness. The minimum infectious dose is lower for Salmonella at
<100 cells (Blaser and Newman, 1982) and E. coli 0157:H7 at 2 to 45 cells (Tilden et a1.,
1996) than for La monocytogenes. Pathogens that contaminate the surface of fruit are
mainly inﬂuenced by pH, availability of nutrients, and the natural microﬂora of the
product. Fruits such as blueberries have a pH of 3.5 to 4.0 that is sufﬁciently low to
prevent or retard the growth of bacterial pathogens. However, the pH can be increased by
growth of post-harvest fungi that may permit the growth of pathogenic bacteria. In two
studies, populations of L. monocytogenes and E. coli 0157:H7 increased on the surface of
apples containing Glomerella cingulata with this attributed in part to the increase in pH
from 4.7 to around 7.0 (Conway et a1., 2000; Riordan et a1., 2000).

Produce with damaged tissue from spoilage fungi or bacteria may also be more
prone to pathogen growth due to the availability of nutrients in the exudates. In a study

with healthy and soft-rotted produce, the incidence of Salmonella on produce affected by

23

bacterial soft rot (Erwinia, and Pseudomonas) was twice that of the control samples
(Weissinger and Beuchat, 2000).

Growth of spoilage and non-spoilage microorganisms on fresh fruit and
vegetables can result in the formation of bioﬁlms that can protect bacteria from the
bactericidal action of sanitizers. On one study, L. monocytogenes was unaffected by
treatment with 500 ppm free chlorine when the pathogen was present in a multi-species

bioﬁlm with Pseudomonas fragi and Staphylococcus xylosus (Norwood and Gilmour,

2000).

2.6 METHODS TO PREVENT AND ELIMINATE BLUEBERRY AND HUMAN
PATHOGENS

Spoilage and illness prevention strategies available to consumers of blueberries
are primarily limited to washing. One method to improve the microbial quality of
bluebenies would involve to improvements in crop and harvest management. One
microbial assessment of blueberries from 9 and 12 locations in Michigan during 2002 and
2003, respectively, showed that populations of bacteria, yeasts and ﬁlamentous fungi
increased considerably from green to ripened fruit and from the 1St to the 2'1d harvest.
(Schilder Annemieck — unpublished data). Microbial populations varied widely among
ﬁeld locations including those that were irrigated and non-irrigated. Among ﬁlamentous
fungi were Colletotrichum (most common), Cladosporium, Epicoccum, Alternaria and
Phoma species. Most of these fungi are common in the environment and live on dead and

dying plant material. They attack ripe or ovenipe fruit and can spread from infected to

24

healthy berries upon contact and can result in serious spoilage and losses in stored
blueberries.

Colletotrichum acutatum, the causal fungus of anthracnose fruit rot, is a major
cause of pre- and post-harvest fruit rot in most blueberry-growing regions. In Michigan,
C. acutatum was found in nearly all ﬁelds and comprised 95-100% of fungal population
(Schilder Annemieck — unpublished data). Cane, twig, and leaf lesions are more sporadic.
Fruit rot manifests itself as sunken areas on ripe fruit with gelatinous, orange spore
masses. The fungus over-winters in remnants of old ﬁ'uiting twigs and infected canes. In
spring and summer, fruiting bodies release spores that are dispersed by rain to infect
ﬂowers, fruit and other tissues. Fruit infections remain latent until the fruit starts to ripen.
In Michigan, spore numbers peak around bloom. A second peak occurs when fruits are
ripening. Warm humid conditions favor the disease (Michigan blueberry facts, 2005).

Alternaria fruit rot occurs in most blueberry—growing regions. On ripe fruit,
sunken areas near the calyx are covered by a dark green, velvety growth. On stored fruit,
a grayish-green mold may appear on the stem scar or calyx end and spread over the entire
berry. Infected fruit becomes soft and shriveled. The fungus over-winters in old twigs and
in plant debris on the ground. Leaf infections occur in the spring during periods of cool,
wet weather. Fruit infections occur as the benies start to ripen. Disease development is
optimal at 68°F (20°C) (Michigan blueberry facts, 2005).

The fungus Phomopsis vaccinii over-winters in infected canes and twigs.
Phomopsis canker and twig blight occur in most blueberry-growing regions. In the
spring, spores are dispersed from fruiting bodies by rain. The fungus is active from bud

swell until after harvest. Plants that have been wounded mechanically or damaged by

25

freezing are more susceptible to infection than undamaged plants. Fruit infection leads to
white mold growth and soft fruit that splits when squeezed (Michigan blueberry facts,
2005).

Fungi management involves the use of plant resistant cultivars, pruning to remove
Old or infected wood, application of fungicides before harvest, harvesting in a timely
manner, handling berries dry, and rapidly cooling fruit after harvest (Michigan blueberry
facts, 2005).

Some of the common yeasts on blueberry fruit surfaces were identiﬁed as
Aureobasidium, and Cryptococcus (most common), Sporidiobolus, Bullera,
F ilobasidium, and Rhizosphaera species. These yeasts are also common on plant surfaces
and are not harmful to humans. Bacteria found on blueberries include Pseudomonas,
Bacillus, and Clavibacter spp (Schilder Annemieck - unpublished data).

Aureobasidium pullulans (yeast rot) produces a sporadic post-harvest rot
characterized by rapid collapse and a wet or slimy appearance of the fruit. Yeast growth
may appear as black, Shiny bumps with a white or pinkish slime (Michigan blueberry
facts, 2005). Representative bacteria, yeast and mold isolates were tested for production
of extracellular enzymes including amylase that reduce the viscosity of backed goods as
well as pectinase and cellulase that can lead to breakdown of the fruit itself. Most fruit
associated microbes were able to break down starch (Schilder Annemieck - unpublished
data). According to Janisiewicz and Korsten (2002) several yeast species including
Aureobasidium and Cryptococcus spp. possessed antimicrobial activity against these
spoilage organisms, thus suggesting their potential use as biocontrol agents for post-

harvest diseases of fruits.

26

Mechanical harvesting, storage and processing are the most important in
obtaining high quality and safe fruit for consumption. According to NeSmith et a1.
(2002) machine harvesting caused the greatest loss in fruit ﬁrmness (20-30%). This was
followed by a 10-15% loss in ﬁrmness due to grading and sorting. Keeping fruit at
ambient temperatures for 24 h after harvest resulted in only a 3-8% loss in ﬁrmness as
compared to cooling fruit immediately. Removal of harvester padding resulted in a 4-8%
loss in fruit ﬁrmness. The decay of machine-harvested bluebenies during post-harvest
holding is perhaps the industry’s biggest problem. Mechanical harvesters operate over-
the-row and result in loss of berries on the ground with the quality generally inferior to
handpicked fruit. However, one over-the-row harvester can cover up to 1 acre/h,
replacing over 100 hand pickers. Thus, economics may dictate mechanical harvesting.
Blueberry quality changes in response to pre-packing delays and holding temperatures.
Jackson et al. (1998) assessed blueberries for the following ten quality attributes during
storage: percentage of split benies, bloom, ﬁrmness, weight loss, moisture, soluble
solids, titratable acids, pH, microbial counts, and percentage of marketable berries at
different pre-packing temperatures up to 26°C, delayed processing times up to 45 h, and
subsequent storage times up to 21 d at 0°C. Increasing the pre-packing temperature and
delaying packing led to a ~1-log increase in microbial populations. Overall, minimizing
delays was the best means for maximizing blueberry quality with cooling before packing
being beneﬁcial when packing was delayed.

Freezing is one means of prolonging the shelf life of blueberries by preventing microbial
growth. The additional killing effect of freezing on microorganisms relates to temperature

shock, concentration of extracellular solutes, toxicity of intracellular solutes, and

27

dehydration and ice formation (Zaritzky, 2000). However, decades ago Schmidt-Lorenz
(1963) and Schmidt-Lorenz and Gutschmidt (1969) found that certain bacteria were still
able to grow to relatively high numbers at -7.5°C. The authors also found that the growth
limit for yeast was -10°C. Microbial grth or metabolic activity also has been reported
in permafrost bacteria at - 10°C (Gilichinsky et a1., 1995) with the temperature limit of
bacterial growth in frozen food now generally considered to be -8°C (Geiges, 1996).
Rivkina et a1. (2000) attempted to quantify metabolic activity at subzero temperatures in
the native bacterial population of Siberian permafrost by measuring the incorporation of
sodium acetate into lipids at seven temperatures from -5°C to -20°C for times up to 550
days. The minimum doubling times ranged from 1 day (5°C) to 20 days (-10°C) to ca.
160 days (~20°C). Thus, microbiological quality remains an issue until temperatures
below -10°C have been reached in the freezing process. Geiges (1996) reviewed the
available literature regarding the effect of slow and fast freezing on bacteria and
concluded that quick freezing and thawing would result in higher microbial survival rates
than those found for slow freezing and thawing. Today, cryogenic substances are
routinely used to maintain viability of microorganisms during under long-tenn storage.
Hence, consumers and manufacturers should not take the microbiological safety of
cryogenically frozen products for granted. Additional factors that affect the
microbiological quality of frozen foods relate to the physical and chemical characteristics
of the product, the pro-freezing microbiological quality of the product and the
temperature ﬂuctuations during storage that can lead to thawing and refreezing.

Methods to prevent consumption of fresh or frozen blueberries with foodbome

pathogens are limited to thorough washing, choosing unblemished product, avoidance Of

28

cross contamination in the kitchen, and eating or refrigerating the berries promptly after
purchase in order to prevent microbial growth. If contamination is limited to the surface,
various wash treatments have shown to be marginally effective in reducing levels of
surface microorganisms. Washing produce with water alone will typically reduce
microbial populations by only 1 or 2 logs (Beuchat et a1., 2003). Some products such as
cantaloupe and raspberries have difﬁcult-to-clean surfaces that support increased
adherence of pathogens (Ortega et a1., 1997). Moreover, some types of produce such as
apples are prone to internal contamination that results from ineffective surface washing
treatments (Zhuang et a1., 1995). When Crowe et al (2005) commercially washed
blueberries in Maine with a sterile spray of distilled water for 30 and 300 sec, populations
of bacteria, yeasts and molds decreased _<_ 0.43 log CFU/g.

Another prevention method is to avoid consumption Of high risk items such as
unpasteurized apple cider by persons at high-risk (young children, elderly). Laboratory
and ﬁeld studies on the behavior of microbes in produce during growing, harvesting, and
processing can Offer novel and important insights into potential control strategies with an
emphasis on bioprevention of pathogen contamination. New control measures that
prevent or reduce contamination of fresh produce are needed (Sumathi et a1., 2004).

Once fresh produce becomes contaminated with pathogens, the product remains a
potential risk until the time of consumption. Washing and rinsing some types of fruit and
vegetables will prolong the shelf life by reducing the number of microorganisms on the
surfaces. However, with this simple method only some of the pathogens are removed

from the surface. To be deemed effective, any antimicrobial treatment (sanitizer) that is

29

administrated to produce should reduce the populations of pathogens by more than 2 to 3

log CFU/g (Beuchat, 1998).

2.7 SANITIZERS

The use of chemical sanitizers to enhance the microbial safety and shelf-life of
fresh fruits and vegetables is of great interest to the food industry. The goal is to obtain a
5-log reduction as set by the Food and Drug Administration (FDA) for selected
commodities. Chlorine-based sanitizers are most common for fruits and vegetables
including Michigan blueberries; however, other sanitizers including fatty organic acids
(0A), aqueous chlorine dioxide and especially chlorine dioxide gas are of increasing
interest to blueberry processors. Sanitizers that can be used to wash fruits and vegetables
are regulated by the US. Food and Drug Administration in accordance with the Federal
Food, Drug and Cosmetic Act outlined in the Code of Federal Regulations, Title 21,
Ch], Section 173.315. The efﬁcacy of a sanitizer is a function of both time and
concentration. In general, greater sanitizer efﬁcacy is seen at increasing concentrations.
Increasing the contact time and the sanitizer concentration can also increase microbial
inactivation. Sodium hypochlorite at 200 ppm was found to be signiﬁcantly more
effective than 100 ppm in inactivating L. monocytogenes and E. coli 0157:H7 on
different types of produce after a 5-minute exposure (Rodgers, 2004). There is an
optimum sanitizer concentration below which the effectiveness is reduced and above
which there is no further improvement. Populations of fecal coliforms on green salad
leaves were reduced by 2-logs using 50 ppm chlorine with concentrations up to 200 ppm
being no more effective (Mazollier, 1988). Sanitizer antimicrobial action can be

inﬂuenced by temperature (chlorine compounds) with greater activity at 22°C than at 4°C

30

(Zhang and Farber, 1996). The pH of the solution can also inﬂuence effectiveness of
chlorine with a decrease in pH from 9 to 5 increasing the antimicrobial effect of chlorine
4-fold on lettuce (Adams, 1989). Pathogens also vary in their sensitivity to sanitizers with
L. monocytogenes generally being more resistant to chlorine than Salmonella and E. coli
0157:H7 (Beuchat, 1998).

The general lack of efﬁcacy of sanitizers on raw fruits and vegetables can be
attributed, in part, to their inaccessibility to locations within structures and tissues that
harbor pathogens. Pathogenic bacteria are able to inﬁltrate cracks, crevices, and
intercellular Spaces of seeds and produce. Inﬁltration is dependent on temperature, time,
and pressure, and only occurs when the water pressure on the produce surface overcomes
the internal gas pressure and the hydrophobic nature of the surface of the produce
(Beuchat, 2002; Burnet and Beuchat, 2001). Inﬁltration may also be enhanced by the
presence of surfactants and when the temperature of the fruit or vegetable is higher than
the temperature of the water. The protective mechanism of these sites is not well
understood, but the theory that hydrophobicity of microbial cells aids in their protection
by inhibiting penetration of the disinfectants has been proposed (Buck, 2003). Major
factors limiting sanitizer efﬁcacy include strength and rapidity of microbial attachment,
inaccessibility to attachment sites, attachment and growth in cuts and punctures,
internalization of microbial contaminants within plant tissues, and bioﬁlm formation

(Sapers, 2001).

31

2.7.1 CLOROX TM/ SODIUM HYPOCHLORITE (N aClO)
A. General characteristics

Chlorine was discovered several hundred years ago and has been since used to
noxious odors, and as calcium hypochlorite to sanitize morgues, sewers and hospitals.
Today most sanitizers used in the produce industry are chlorine-based (Beuchat, 1998
and Brackett, 1999). Chlorine is used at concentrations of 5 to 200 ppm with contact
times of few minutes for raw fruits and vegetables (Beuchat, 1996). Hypochlorites such
as calcium hypochlorite (CaCl2) and sodium hypochlorite (NaCl) are produced when the
chlorine compound is dissolved in water. Chlorine is released forming hypochlorous acid
(HOCl) and hydrochloric acid. The strong antimicrobial activity of HOCI results from a
high oxidation potential (Ong, 1996).
The dissociation rate of HOCI is pH dependent. At pH 5 2, and 2 10, chlorine is in the
elemental form and in the hypochlorite ion form, respectively. As the pH of a solution at
20°C is reduced, the concentration of HOCI increases t023 and 97% at pH of 8.0 and 6.0
respectively. Toxic chlorine (C12) gas is also formed at pH below 4 (Beuchat, 1996).
Temperature is another factor that inﬂuences the reaction with more HOCI produced at
lower temperatures and greater loss of chlorine gas at higher temperatures. Chlorine is
maximally soluble in aqueous solutions at 4°C (Burnett, 2001). Chlorine also rapidly
loses activity when in contact with organic matter, exposed to air, light, metals (copper,
cobalt, nickel, other catalysts) or ultraviolet light. The most common method for
measurement of “available chlorine content” (strength of hypochlorite solution) is the

iodometric method. This method is based on the principal that in the presence of

32

potassium iodine (KI), free chlorine in water liberates iodine that is titrated with a

standardized sodium thiosulfate solution as a quantitative indicator.

B. Antimicrobial performance

Chlorine has good antimicrobial activity in aqueous model systems. However, on
the surface Of fresh fruits and vegetables, chlorine is less effective in killing
microorganisms with microbial reductions typically not exceeding2 to 3 log CFU/ g
(Beuchat, 1998) due mainly to its inactivation by other organic materials that are present
on the product. According to Park and Beuchat (1999), treating cantaloupe with 2000
ppm chlorine reduced E. coli 0157:H7 populations less than 1 log due to interference
from organic matter. Treatment of blueberries with 100 ppm for 5 min reduced the
population of bacteria, yeast, and mold by 0.83, 0.77, and 0.61 log CFU/g, respectively
(Crowe et a1., 2005). Another factor that makes the effectiveness of chlorine compounds
unpredictable is insufﬁcient wetting of the hydrophobic surface (waxy cuticle) of ﬁ'uits
and vegetables (Adams, 1989). Although chlorine-based sanitizers are relatively
inexpensive, some concerns have been raised regarding corrosivity, instability and the
production of residual chlorine by-products such as chloroform, trihalomethane (THM),
bromodichloromethane, and MX [ 3-chloro-4-(dichloromethyl)-5-hydroxyl-2(5H)-
furanone] (Richardson, 1998). These organochloride compounds have been demonstrated
to cause cancer in laboratory animals (Wei, 1985). Because the organochloride
compounds can enter the environment through wastewater and gain access to drinking
water, the US Environmental Protection Agency established a maximal THM limit in

drinking water of 100 ug/L (Richardson, 1998). Because of these limitations in efﬁcacy

33

along with recent environmental and public health concerns, the produce industry is

stimulated to ﬁnd alternative treatments.

2.7.2 CHLORINE DIOXIDE
A. General characteristics

Chlorine dioxide is a yellow-green gas generated through oxidation when
concentrated hydrochloric acid is added to sodium chlorite. When done in water, the end
result is an aqueous chlorine dioxide solution. Chlorine dioxide is receiving increased
attention as a produce sanitizer due to the following advantages over chlorine: greater
effectiveness over a wider pH range, not affected by high levels of organic matter, no
rapid dissociation in water, an oxidation capacity 3 times greater than chlorine (White,
1972), does not react with organic compounds to produce carcinogenic by-products,
formation of by-products that are less toxic and 3-5 times lower compared with chlorine
(Richardson et a1., 1998), and a wide germicidal activity including spores (Richardson et
a1., 1994), viruses (Sobsey, 1988) and protozoa incuding Cryptosporidium and Giardia
oocysts ( Finch et a1., 1997) that are more resistant to chlorine. Because of these qualities
chlorine dioxide is used today in the disinfection of water, air, and was the principal

agent used in the decontamination of buildings in the US after the 2001 anthrax attacks.

The food and Drug Administration (FDA 2005) amended the food additive
regulation to allow the safe use of chlorine dioxide as an antimicrobial agent in water
used to wash fruits and vegetables that are not raw agricultural commodities in an
amount not to exceed 3 ppm residual chlorine dioxide. The treatment of the fruits and

vegetables with chlorine dioxide shall be followed by a potable water rinse or by

34

blanching, cooking, or canning. The US. Environmental Protection Agency (EPA)
approved use of chlorine dioxide as a disinfectant for potable water treatment limiting
the residual to 1 ppm (US. Federal Register, 2000). There is a lot of effort underway
to use chlorine dioxide gas for raw agricultural commodities such as tomato,
cantaloupe, onion, ﬂower bulb and vegetable seed trials (ICA TriNova, LLC Forest
Park, GA) but nothing is commercial yet.

The germicidal action of chlorine dioxide is based on a loss of membrane
permeability that results from oxidative damage to the outer cell membrane followed by
destruction of the trans-membrane ionic gradient and suspension of protein synthesis

(Berg et a1., 1986).

B. Antimicrobial performance

The efﬁcacy of chlorine dioxide is high when studied in model aqueous systems
with a > 5 log reduction for E. coli 0157:H7 and L. monocytogenes following a 5—min
exposure to 3 and 5 ppm chlorine dioxide (Rodgers et a1., 2004). On produce, the efficacy
of chlorine dioxide against pathogens is far lower with reductions ranging from 1 log on
shredded lettuce and cabbage (Zhang and Farber, 1996) to ~ 4 logs on whole apples
(Wisniewsky et a1., 2000). The surface seems to play a more important role for the
efficacy of sanitizers. A 3 mg/L aqueous C102 treatment achieved reductions of 3.7- and
0.4-log for L. monocytogenes on uninjured and injured surfaces of green pepper,
respectively, while populations decreased >6 and ~3.5 logs/ 5g on the same surfaces
using similar concentrations of chlorine dioxide gas (Han et a1., 2001). A C102 gas

treatment was the most effective in reducing L. monocytogenes on both uninjured and

35

injured green pepper surfaces, when compared with aqueous C102. The difference in log
reductions for uninjured and injured green pepper surfaces and results from confocal laser
scanning microscopy (CLSM) analysis suggested that injured surfaces protected more
bacteria from sanitation treatments than did uninjured surfaces or that the injured surfaces
could also support grth of the pathogen. Fruit treatment with aqueous chemical
solutions can promote yeast and mold growth. Growth of molds can increase the pH of
blueberries from ~ 3.35 (Jackson et a1., 1998) to over 4 and thus enhance the grth of
bacteria including foodbome pathogens and increasing safety risk.

Thus, alternatives to aqueous sanitizers such as gaseous chlorine dioxide have
been assessed for microbial reductions on fresh produce. Studies have shown gaseous
chlorine dioxide to be effective in microbial reductions including enteric pathogens on
apple (Du et a1., 2002; Du et a1., 2003), green peppers (Han et a1., 2000; Han et a1., 2001),
lettuce (Lee et a1., 2004), tomato, cabbage, carrots, peaches (Sy et al., 2005a) strawberries
(Han et a1., 2004; Sy et al., 2005b), raspberries and blueberries (Sy et al., 2005b) resulting
an increase in the popularity of using chlorine dioxide gas as a sanitizer. Chlorine dioxide
gas proved also to be effective as a sanitizer in food processing plants by reducing the > 4
log populations of yeast and molds on stainless steel surface of the tanks used for aseptic
juice storage below detectable limits (Han et a1., 1999) and in libraries by effectively
controlling the spread of molds (Weaver-Meyers et a1., 1998). Treatment of surface-
injured green peppers with 1.2 mg/L chlorine dioxide gas resulted also in a 6.4 log
reduction in E. coli 0157:H7 per spotted site (Han et a1., 2000). Treatment with 18 mg/L
for 10 min reduced numbers of E. coli 0157:H7 on calyx, stem, and skin surfaces of

apples by 3.8, 3.8, and >7 logs CFU per inoculated spot (Du et a1., 2003). Similarly, a

36

nonpathogenic strain of E. coli was reduced by 4.5 log CFU/g on whole apples using 0.3
mg/L chlorine dioxide gas (Sapers et a1., 2003). Gaseous chlorine dioxide was also tested
for reducing populations of Salmonella, yeasts and molds on blueberries surface (Sy et
a1., 2005). Treatment with 4.1 to 8 mg/L chlorine dioxide gas released in 30 to 120 min,
and 75 to 90% relative humidity (RH), reduced the populations of Salmonella, yeasts and
molds on spot-inoculated blueberries (skin, calyx and stem scar) by 1.9 to 3.7, and 1.4 to
2.5 log CFU/g, respectively. The reductions achieved using increased concentrations of
chlorine dioxide gas were similar, with higher reductions on skin than the stem scar and
calyx. Sensory attributes including appearance, color, aroma, and overall quality aﬁer
treatment with 4.1 mg/L chlorine dioxide gas were not signiﬁcantly different compared
with the ungassed control after 0 and 3 days of storage. However, after 7 and 10 days,
the treated samples were ranked signiﬁcantly higher for overall quality and aroma,

respectively.

37

CHAPTER3
MICROBIAL CONTAMINANTION IN HIGHBUSH BLUEBERIES BEFORE,

DURING, AND AFTER PROCESSING

ABSTRACT

Concerns regarding blueberry spoilage, safety, and development of microbiological
standards prompted a 2003-2004 survey in which highbush blueberries were collected
from 18 different Michigan ﬁelds before harvest and quantitatively examined for
mesophilic aerobic bacteria (MAB), coliforms, Escherichia coli, yeasts (Y) and molds
(M). Thereafter, blueberries from these same ﬁelds were harvested and similarly assessed
at different points during processing (after-harvest, blower exit, after washing, and before
packaging for freezing) at six facilities along with environmental samples (blower and
ﬁller conveyor belts, chlorinated wash water). Duplicate blueberry (100g), wash water
(50 ml) and environmental swab samples (~10 x 10 cm) were analyzed for MAB,
coliforms, E. coli, Y and M by plating on tryptic soy agar containing 0.6% yeast extract
and cyclohexamide, PetriﬁlmTM E. coli/coliform plates, and potato dextrose agar
containing streptomycin and ampicillin, respectively. Average MAB, Y and M counts on
blueberries were 3.49, 3.81 and 3.35 at pre-harvest, increasing to 4.95, 4.37, and 4.06 at
post-harvest, and decreasing to 4.21, 3.86 and 3.52 logs CFU/g after washing,
respectively. Coliform and E. coli counts increased 0.64 and 0.16 logs from pre-harvest
to after washing, respectively. Microbial populations were highest on the blower and
ﬁller belts and lowest in the chlorinated wash water. Overall, MAB populations

increased ~1.5 logs between harvest and processing (4 to 18 h) with chlorinated wash

38

water (~10 to 200 ppm chlorine) reducing populations <1 log. Thus, improved storage
strategies before processing and more effective microbial reduction strategies during

processing are needed to enhance the microbial quality of blueberries.

3.1 INTRODUCTION

The United States is the world’s leading blueberry producer with 55% of total
production and approximately one-third of the total US. highbush blueberry crop coming
from Michigan. In 2002, Michigan had 16,900 acres of highbush blueberries that yielded
64 million lb of fruit. Approximately 33 percent of these berries (42 million lb) were
marketed as fresh berries with the remainder processed and frozen for later use in jams
and baked goods (N ASS/U SDA, 2002).

Food safety concerns surrounding blueberries include a possible linked to a 1984
outbreak of listeriosis in Connecticut (Ryser, 1999), one conﬁrmed outbreak of hepatitis
A in New Zealand (Calder et a1., 2003), and a 1998 recall involving an undetermined
quantity of frozen blueberries from California, Illinois and Australia that was
contaminated with Listeria monocytogenes (FDA Enforcement Report, 1998). Michigan
blueberries have not yet been implicated in any outbreaks of illness. However, since
blueberries and other berry types have been linked to outbreaks in the past, microbial
safety remains a critical concern for all segments of the blueberry industry.

Besides ‘zero tolerance’ policies for L. monocytogenes, Escherichia coli
0157:H7 and Salmonella in blueberries, buyers of frozen berries now demand microbial
testing for levels of spoilage bacteria, yeasts and molds. Different purchasers have now

developed varying microbial standards for frozen blueberries with these standards

39

reﬂecting different uses. Pie manufacturers need a low level of amylase in their
blueberries in order to produce baked goods with a low viscosity blueberry ﬁlling.
Because most bacteria, yeasts and molds isolated from blueberries produce the enzyme
amylase (Schilder Annemieck — unpublished data), the microbial standards for frozen
berries are now becoming increasingly stringent. A better understanding of factors
contributing to high microbial levels in blueberries along with improved microbial
reduction strategies will allow blueberry growers and processors to better meet these
increasingly strict microbial standards.

The speciﬁc objective of this study was to assess the levels of microbial
contamination at various points during blueberry harvesting, handling, cleaning, and
packaging. These ﬁndings will eventually help to establish a science-based uniform
standard for microbial levels in blueberries that will satisfy the needs of buyers as well as

growers and processors.

3.2 MATERIAL AND METHODS
Blueberry samples

During the 2003 and 2004 harvest seasons, 18 500-g blueberry samples were
obtained by hand-picking speciﬁc rows or sections at 12 and 6 ﬁelds locations in
Michigan, respectively. These samples were than assessed for microbial levels at ﬁve
points during processing at six and two facilities in 2003 and 2004, respectively. Fruit
samples (500 g) were collected in 1- pint plastic clam shell containers at pre-harvest
(prior to mechanical harvesting), post-harvest (when loaded onto the conveyer belt 4 to

18 h after harvesting), blower-exit, water tank-exit, and pre-packaging for freezing

40

(Figure 2.1). All clam shells containing blueberries were placed in individual plastic
bags, stored on ice and analyzed within 24 h of collection. Portions of these samples
(450g) from the 2003 harvest season were placed in sterile 20 x 10 cm polyethylene bags
(Whirl-PackTM, Fisher Scientiﬁc, Pittsburgh, NJ.) and analyzed after 3 and 6 months of
storage at -20°C. Storage time between pre-harvest and post-harvest varied between 4 to

18h.

Environment samples

Environment samples were collected during processing (Figure 3.1) from 10 x 10
cm areas of the conveyer belt entering the blower and pre-packaging areas using Enviro-
sponges (Fisher Scientiﬁc, Pittsburgh, N.J.) that were hydrated in 30 ml of neutralizing
buffer (Difco, Becton Dickinson, Sparks, MD). Water tank samples were collected in 50-
ml sterile centrifuge tubes containing 1 ml of neutralizing buffer (Difco). All samples
were transported to the laboratory on ice and analyzed within 24 h of collection.

Microbial data and additional information obtained at the time of sampling
included the ﬁeld location, fruit variety, date and time of harvest, date and time of

processing, visual cleanliness of the fruit, and type of sanitizer (Appendix D).

41

 

Pre-harvest {—— Fruit sample

1

Mecllanical harvesting

Transportation and
storage

l

Loading onto «— Fruit sample
conveyer belt

l

Blower <——— Fruit and swab sample

l

Water tank ‘— Fruit and water sample

De-sthmer

l

Color/size sorter

Pre-Packaging 4— Fruit and swab sample
area for freezing
(End of processing)

 

 

 

Figure 3.1 Sample collection points for highbush blueberries. Swab and water samples
were taken before the start of processing and aﬁer the berries were processed.

42

Microbial Analysis

Blueberry samples (25 g) were placed in sterile 20 x 10 cm polyethylene bags
(Whirl-PackTM) containing 100 ml of neutralizing buffer (Difco). The sealed bags were
shaken horizontally at 100 rpm for 20 min on a G2 Gyratory Shaker platform (New
Brunswick Scientiﬁc Co., Inc., Edison, NJ.) and then pulsiﬁed for 1 min using a
Pulsiﬁer, (Filataflex Ltd., Ontario, Canada). Thereafter, l-ml samples were serially
diluted in sterile phosphate buffer solution (PBS) (Sigma-Aldrich Co., St. Louis, MO)
and spiral-plated (Autoplate 4000 —Spira1 Biotech, Exotech, Inc, Gaithersburg, MD) in
duplicate on tryptic soy agar (Difco) containing 0.6% yeast extract (Difco) and 100 ppm
cyclohexamide (Sigma) (TSAYE-C) for enumeration of mesophilic aerobic bacteria
(MAB), and Potato Dextrose Agar (Difco) containing 20 ppm streptomycin (Sigma) and
50 ppm ampicillin (Sigma) (PDA-SA) for enumeration of yeast and mold. Additional 1-
ml aliquots were plated on E. coli/Coliform PetriﬁlmTM plates (3M Corp., St. Paul, MN)
for quantiﬁcation of coliforms and E. coli. TSAYE-C and E. coli /Coliform plates were
counted after 48 h of incubation at 37°C. PDA-SA plates were counted after 72 to 96 h of
incubation at room temperature (22°C). Environmental sponge and water samples were

serially diluted in PBS and similarly examined for the same microorganisms.
Statistical analysis

Analysis of Variance (ANOVA) was done on all microbial count data obtained

from fresh / frozen fruit samples, environmental sponge, and water samples using the

43

Statistical Analysis System (SAS, Version 8, SAS© Institute Inc., Cary, NC) to assess
microbial changes during processing and frozen storage. Data in the graphs and tables
(Appendix B) are means from replicates and signiﬁcance between means were
determined using least signiﬁcant difference (LSD) test at the 95% conﬁdence level

(P=0.05).

3.3 RESULTS
Fruit samples

Numbers of MAB, yeasts, molds, coliforms and E. coli at pre-harvest varied from
1 to 3.41 logs for the different ﬁeld locations with highest populations seen near the end
of the harvest season (Appendix B, Table 3.1). Microbial populations increased 0.6 to
1.46 logs (Appendix B, Table 3.1) from pre-harvest to post-harvest with this difference

statistically signiﬁcant (P < 0.05) (Figure 3.2).

Pre-harvest

m Post-harvest
Exit blower

a Exrt water tank
I Pre-packaging

i
l
l
E

LogCFU/g
o-srowtsmoaxr;

     

Coliforms E.coli

Figure 3.2 Populations of MAB, yeasts, molds, coliforms and E. coli (mean i std. dev.,
n=l8) on blueberries sampled at pre-harvest, post-harvest, blower exit, water tank exit,
and pre-packaging. Values with different letters within the same microbial category are

signiﬁcantly different (P < 0.05).

44

Only washing of the fruit signiﬁcantly reduced (P < 0.05) the numbers of
microorganisms with no statistically signiﬁcant differences (P > 0.05) seen elsewhere
during processing. Microbial levels on fruit at pre-packaging and pro-harvest were similar
for yeasts, molds and E. coli, but were signiﬁcantly higher (P < 0.05) for bacteria and

coliforms (Figure 3.2).

Environmental samples

Populations of MAB, yeasts, molds, coliforms and E. coli in time from before to after
processing the fruits, increased on conveyer belts surface entering the blower area by
1.29, 1.00, 1.58, 1.39 and 0.97 log CFU/cmz, respectively (Fig. 3.3 A), and on conveyer
belts surface entering the pre-packaging area by 1.17, 1.15, 1.12, 1.06 and 0.66 log
CPU/cm2 (Fig. 3.3 B), respectively (Appendix B, Table 3.2), with all of these increases
being statistically signiﬁcant (P < 0.05).

(A)

l

~~ l

l .__ J- J- _-
T jIBeforelm Alter ; :

l

a l

a
”1101] 5
J __,'

Yeasts Molds Colifonns E.coli

    
 

.\“

 

Log CFU/cmz
o A N on .§ 01

   

45

was; Earner ;

a a
b
b a ‘
s11 all be
. j. --_,_.L[ll£lL_, I

M A B Yeasts Molds Colifonns E.coli

DANG-50'!

 

l
l
l
J 1

Figure 3.3 Populations of MAB, yeasts, molds, coliforms and E. coli (mean :1: std. dev.,
n=l8) in time from before to aﬁer processing the fruits, increased on conveyer belts
surface entering the blower (A) and pre-packaging areas (B). Values with different letters

within the same microbial category are signiﬁcantly different (P < 0.05).

Water tank samples

During fruit processing, populations of MAB, yeasts, molds, coliforms and E. coli in
water tanks increased in time from before to after processing the fruits by 0.56, 0.74,
0.86, 0.41 and 0.32 log CFU/ml (Table 3.3), respectively, with all these increases being

statistically signiﬁcant (P < 0.05) (Figure 3.4).

Frozen fruit storage studies
After 3 months at -20°C populations of yeasts, molds, and E. coli on blueberries
decreased by 0.44, 0.46, and 0.68 log CFU/g (Appendix B Table 3.4), respectively, with

all of these reductions being statistically signiﬁcant (P < 0.05) No change was seen for

46

MAB or coliforms with no reduction in numbers of MAB, yeasts, molds, and E. coli

evident after 6 months of frozen storage (Figure 3.5).

‘ 77 Before Ill. Alter

wwwm 2.2..

M A B Yeasts Molds Coliforms E. coli

Log CFUIml

o-‘NU-ho'l
##LJJJW'

r_. J,J. ‘— ,_, 7, .., 777-, 7* d. .. ,7 ., L, _. k ...~ . - . 7

Figure 3.4 Populations of MAB, yeasts, molds, coliforms and E. coli (mean :t std. dev.,
n=1 8) in water samples (log CF U/ml) collected at the time before and after fruit washing.
Values with different letters within the same microbial category are signiﬁcantly different
(P < 0.05).

       

Yeasts Molds Colifonns E. coli

1 _.._ __.. . J
I Fresh [1113 month 6 month

Figure 3.5 Populations of MAB, yeasts, molds, coliforms and E. coli (mean :i: std. dev.,
n=l8) on blueberries before and after 3 and 6 months of storage at -20°C. Values with

different letters within the same microbial category are signiﬁcantly different (P < 0.05).

47

3.4 DISCUSION S

Results showed some consistent trends. Populations of MAB, yeasts, molds, coliforms
and E. coli at pre-harvest varied widely among ﬁeld location, increased over the harvest
season for these same ﬁelds, and increased 1 log or more ﬁ'om pre-harvest to post-
harvest. These observations are similar to another microbial survey of Michigan
blueberries collected from irrigated and non-irrigated ﬁelds during 2002 and 2003
(Schilder Annemieck — unpublished data) with populations of bacteria, yeasts and
ﬁlamentous fungi increasing 1 to 2 logs between unripened green fruit and the 2"d
harvest.

Variations in microbial load between ﬁelds and increases within the same ﬁeld
over time suggest multiple pre-harvest sources of contamination including soil, feces,
irrigation water, water used to apply fungicides and insecticides, insects, dust,
inadequately composed manure, wild animals, and human handlers (Beuchat, 1996). The
ability of produce to internalize pathogens, including Escherichia coli 0157:H7
(Solomon et a1., 2002), and Salmonella (Guo et a1., 2002), from contaminated water was
also recently reported. Thus, sources for irrigation water, including wells, should be
monitored for microbial levels. Manure needs to be adequately composted before being
used as fertilizer. Domestic and wild animals also should be discouraged from entering
blueberry ﬁelds. Calder et al. (2003) conﬁrmed a multi-district outbreak of hepatitis A
(HAV) associated with consumption of fresh blueberries in New —Zealand with the
contaminant likely coming from infected ﬁeld workers or polluted groundwater.

During handpicking, blueberries can become contaminated with bacterial

pathogens that can readily survive on the non-acidic blueberry surface.

48

Aﬁer handpicking is complete, growers shift to mechanized harvesting for the
processed market. The increase in bacteria, yeast and mold during the harvest season is
due mainly to microbial spread and multiplication facilitated by mechanical harvesting.
According to NeSmith et a1. (2002) machine harvesting caused the greatest loss in
blueberry ﬁrmness (20-30%). Such damaged blueberries are more susceptible to attack
from spoilage fungi that are easily spread to non-infected by harvest equipment that can
result in serious spoilage and major ﬁnancial losses during extended storage (Schilder
Annemieck — unpublished data).

Pathogens that contaminate the surface of blueberries are mainly inﬂuenced by
pH, availability of nutrients, and the natural microﬂora. Blueberries have an internal pH
of 3.5 to 4.0 that is sufﬁciently low to prevent or retard the grth of bacterial pathogens.
However, pH can increase from growth of spoilage fungi that could lead to the grth of
bacterial pathogens. In two studies, growth of L. monocytogenes and E. coli 0157:H7 on
the surface of apples containing Glomerella cingulata was attributed to an increase in pH
from 4.7 to 7.0 (Conway et a1., 2000; Riordan et a1., 2000). In a study with healthy and
soft-rotted produce, the incidence of Salmonella on produce affected by bacterial soft rot
(Erwinia and Pseudomonas) was twice that of the control samples (Weissinger and
Beuchat, 2000).

In addition to contamination during mechanical harvesting, the higher microbial
load on blueberries at post-harvest also likely resulted from a delay in processing. Upon
arrival at the processing facility, most blueberries were held for 12 hours or more before
processing began. According to Jackson et al. (1998), microbial populations on fresh

bluebenies were ~l log higher after 45 h of storage at temperatures up to 26°C.

49

Microbial populations on blueberries decreased less than 1 log after exposure to ~
10 to 200 ppm chlorine in the water tank. The limited efﬁcacy of chlorinated water may
be due to short contact times, which were typically less than 1 minute. Increasing
amounts of organic matter in the wash water also decreased the effectiveness of chlorine
as evidenced by an increase in the levels of microorganisms in the water tank over time
with additional contaminants transferred from the conveyor belt to the berries after
washing. The fact that blueberries generally contain higher microbial levels after
processing than before harvest demonstrate the lack of efﬁcacy in reducing microbial
loads in industry settings.

Microbial populations on blueberries decreased less than 0.7 logs after either 3 or
6 months of storage at —20°C, demonstrating that maintaining freezing and maintaining
berries in the freezer for extended storage periods is not an effective microbial reduction
strategy. Decades ago, Schmidt-Lorenz (1963) and Schmidt-Lorenz and Gutschmidt
(1969) found that certain bacteria were still able to grow to relatively high numbers at -
75°C. The authors also found that the grth limit for yeast was -10°C. Microbial growth
or metabolic activity also has been reported in permafrost bacteria at - 10°C (Gilichinsky
et a1., 1995) with the temperature limit for bacterial grth in frozen food now generally
considered to be-8°C (Geiges, 1996). In a bacterial population study of Siberian
permafrost, Rivkina et a1. (2000) reported doubling times of l (5°C) to 20 days (~10°C)
and 160 days (-20°C). Thus, microbiological quality of blueberries remains an issue

during frozen storage.

50

CHAPTER4
EFFICACY OF CHLORINE DIOXIDE GAS SACHETS FOR ENHANCING THE

MICROBIOLOGICAL QUALITY AND SAFETY OF BLUEBERRIES

ABSTRACT

In response to increasingly stringent microbial speciﬁcations being imposed by
purchasers of blueberries, chlorine dioxide (C102) gas generated by a dry chemical sachet
was tested against three foodbome pathogens as well as ﬁve yeasts and molds known for
spoilage. Initially, ﬁve fresh blueberry samples (100 g) were separately inoculated with
Listeria monocytogenes, Salmonella, Escherichia coli 0157:H7 (3 strains each), and
yeasts and molds (5 genera each) to contain ~106 CFU/g and exposed to C102 (4 mg/L,
0.16 mg/g) for 12 h in a sealed 20 liter container (99.9% RH) at ~22°C (3 replicates).
After gassing, blueberries (25 g) were diluted 1:5 in neutralizing buffer, pulsiﬁed for l
min and plated using standard procedures to quantify survivors. This treatment yielded
reductions of 3.94, 3.62, 4.25, 3.10, and 3.17 log CFU/g for L. monocytogenes,
Salmonella, E. coli 0157:H7, yeasts and molds, respectively. Thereaﬁer, 30 lugs of
uninoculated blueberries (~9.1 kg/lug) were stacked on 4 x 4 ft pallets (5 lugs/level x 6
levels) (6 replicates), tarped, and exposed to C102 (18 mg/L, 0.13mg/g) for 12 h. After
gassing, signiﬁcant (P < 0.05) reductions of 2.33, 1.63, 0.48, 1.47, and 0.52 logs CFU/g
were seen for mesophilic aerobic bacteria (MAB), yeasts, molds, coliforms, and E. coli,
respectively, compared to ungassed controls. No signiﬁcant differences (P > 0.05) in

microbial inactivation were seen between lug levels and, with one exception (MAB),

51

between the bottom and top surface of individual lugs. Based on these ﬁndings, C102
sachets may provide a simple, economical and effective means of enhancing the

microbial shelf-life and safety of fresh blueberries.

4.1 INTRODUCTION

Microbial safety and quality are critical concerns to all segments of the blueberry
marketing chain. A single widely publicized outbreak of a blueberry-related illness would
negatively impact the entire industry. Although the blueberry industry in the US. has not
yet experienced such event, one recent multi-district outbreak of hepatitis A (HAV)
associated with the consumption of fresh blueberries was reported in New-Zealand with
these berries likely contaminated from infected food handlers or fecally polluted
groundwater (Calder et a1., 2003).

Most Michigan blueberries are processed and frozen rather than fresh marketed
for economic reasons (NASS/USDA, 2002). Some buyers of frozen blueberries now
demand microbial testing in order to ensure that the product does not exceed their
speciﬁcations for mesophilic aerobic bacteria (MAB), coliforms, yeasts and molds. These
buyers also have ‘zero tolerance’ policies for human pathogens such as L.
monocytogenes, E. coli 0157:H7, and Salmonella. Microbial speciﬁcations vary
considerably from buyer to buyer with many appearing arbitrary, whereas others reﬂect
speciﬁc uses. Pie manufacturers who use blueberries with high yeast populations
frequently ﬁnd that their pies do not “set up” because yeast growth results in enzymatic
breakdown of starch and other stiffening agents. In one Michigan study, most bacteria,

yeasts and molds isolated from fresh blueberries were found to produce amylase which

52

facilitates starch breakdown with some isolates also producing pectinase and cellulase
that can breakdown the fruit cell walls (Schilder Annemieck — unpublished data). These
enzymatic concerns can lead to considerable ﬁnancial loss and frequently a change in
blueberry suppliers.

For blueberry industry, these microbial standards are difﬁcult to meet since the
level of MAB, yeasts and molds and other contaminants can vary widely between ﬁelds,
seasons, and the time of harvest depending on factors such as moisture, temperature,
insect level, plant health, and harvest management practices with microbial populations
peaking at the end of the harvest season (Schilder Annemieck — unpublished data).

Given the relative ineffectiveness of chlorinated water in reducing microbial
levels on blueberries during processing, new microbial reduction strategies are needed in
order to meet the new microbial standards.

Chlorine dioxide is a yellow-green gas generated through oxidation when
concentrated hydrochloric acid is added to sodium chlorite. When done in water, the end
result is an aqueous chlorine dioxide solution. Chlorine dioxide is increasing in
popularity due to the numerous advantages over chlorine: greater effectiveness over a
wider pH range, not affected by high levels of organic matter, no rapid dissociation in
water, an oxidation capacity 3 times greater than chlorine (White, 1972), does not react
with organic compounds to produce carcinogenic by-products, formation of by-products
that are less toxic and 3-5 times lower compared with chlorine (Richardson et a1., 1998),
and a broader germicidal activity including spores (Richardson et a1., 1994 ), viruses (
Sobsey, 1988) and protozoa as Cryptosporidium and Giardia oocysts ( Finch et a1.,

1997) that are more resistant to chlorine. Thus, chlorine dioxide is used today in the

53

disinfection of water, air, and was the principal agent used in the decontamination of

buildings in the US after the 2001 anthrax attacks.

The Food and Drug Administration (FDA 2005) amended the food additive
regulation “to allow the safe use of chlorine dioxide as an antimicrobial agent to wash
ﬁ'uits and vegetables that are not raw agricultural commodities in an amount not to
exceed 3 ppm residual chlorine dioxide that shall be followed by a potable water rinse
or by blanching, cooking, or canning.” The US. Environmental Protection Agency
(EPA) approved use of chlorine dioxide as a disinfectant for potable water treatment
limiting the residual to 1 ppm (US. Federal Register, 2000).

Studies have shown gaseous chlorine dioxide to be effective in microbial
reductions including enteric pathogens on apple (Du et a1., 2002; Du et a1., 2003), green
peppers (Han et a1., 2000; Han et a1., 2001), lettuce (Lee et a1., 2004), tomato, cabbage,
carrots, peaches (Sy et al., 2005a) strawberries (Han et a1., 2004; Sy et al., 2005b),
raspberries and blueberries (Sy et al., 2005b) in laboratory conditions. Chlorine dioxide
gas proved also to be effective as a sanitizer in food processing plants by reducing the > 4
log populations of yeast and molds on stainless steel surface of the tanks used for aseptic
juice storage below detectable limits (Han et a1., 1999) and in libraries by effectively
controlling the spread of molds (Weaver-Meyers et a1., 1998).

This study assessed the efﬁcacy of gaseous chlorine dioxide for inactivating
Escherichia coli 0157:H7, Listeria monocytogenes, Salmonella, mesophilic aerobic
bacteria, coliforms, E. coli, yeasts and molds on the surface of blueberries in industrial

conditions before processing.

54

4.2 MATERIAL AND METHODS
EXPERIMENTAL DESIGN

Two different studies were conducted to assess the efﬁcacy of C10; gas for
microbial reductions on blueberries - (a) a pilot study in which fresh blueberries were
inoculated to contain various foodbome pathogens, spoilage yeasts or molds and then
exposed to C10; gas for 12 h in sealed 20 L buckets and -(b) a pallet study in which 30
lugs of bluebenies (~272 kg) were placed on a pallet and exposed to C102 gas for 12 h

under a tarp.

A. PILOT STUDY
Blueberries

Blueberries (Vaccinium corymbosum) variety ‘Bluecrop’ were obtained from a
local retailer and stored at 4°C for a maximum 2 days before inoculation. Before

inoculation the fruit was tempered for 1 to 2 h at room temperature (22i1°C).

Bacterial Strains

Three strains of Escherichia coli 0157:H7 (AR, AD305, AD3l7), and Listeria
monocytogenes (CWD 95,CWD102, CWD184) were previously obtained from Catherine
W. Donnely (Dept. of Nutrition and Food Sciences, University of Vermont, Burlington,
VT). Three additional Salmonella strains (S. Typhimurium H 3380, S. Heidelberg F5038
BGl, and S. Enteritidis H3502) were obtained from V.K. Juneja (USDA-ARS-ERRC,
Wyndmoor, PA). All strains were maintained at -80°C in trypticase soy broth (TSB)

(Difco, Becton Dickinson, Sparks, MD) containing 10% (v/v) glycerol. Individual strains

-55

 

ha

Sus
Wet

015

were separately activated by transferring a loop of frozen stock culture into 9 ml of sterile
TSB containing 0.6 % (w/v) yeast extract (TSBYE) (Difco) followed by 18-24 h of
incubation at 35°C and then subjected to an identical transfer in 20 ml of TSBYE before
use.
Yeasts and Molds

Five spoilage molds (Colletotrichum sp., Epicoccum sp., Cladosporium sp.,
Phoma sp., and Alternaria sp.) and yeasts (Aureobasidium sp., Bullera sp., Cryptococcus
sp., Sporidiobolus sp., and Filobasidiu sp.) originally isolated from blueberry ﬁelds in
Michigan were obtained from A.C. Schilder (Dept. of Plant Pathology, Michigan State
University, E. Lansing, MI). All yeasts and molds were maintained at -80°C in TSB
containing 10% (v/v) glycerol. Individual strains were separately activated by
transferring a loop of frozen stock culture onto duplicate plates of Potato Dextrose Agar
(PDA) (Difco) containing 20 ppm streptomycin (Sigma-Aldrich Co., St. Louis, MO) and
50 ppm ampicillin (Sigma) (PDA-SA) with the yeasts and molds incubated 3 - 4 and 10 -

12 days at 26°C, respectively, before use.

Preparation of the inoculum

The cultures of E. coli 0157:H7, L. monocytogenes and Salmonella were
harvested by centrifugation (Sorvall Super T21, Newtown, CT) at 7,000 rpm for 10 min
at 4°C and re-suspended in equal volumes of sterile phosphate buffer solution (PBS).
Suspensions of each strain containing approximately equal populations (109 CPU/ml)
were combined to generate three separate 3-strain cocktails (~60 ml each) of E. coli

0157:H7, L. monocytogenes and Salmonella. Populations in these cocktails were

56

determined by plating appropriate serial dilutions in PBS on Sorbitol McConkey Agar
(SMAC) (Difco), Modiﬁed Oxford Agar (MOX) (Difco), and McConkey Agar (MAC)
(Difco), respectively. Two separate 5-strain 100-ml cocktails of yeasts and molds
containing approximately equal populations (108 CF U/ml) were obtained by washing the
previous PDA-SA plates with 20 ml of PBS. Yeast and mold populations in these
cocktails were determined by surface plating appropriate serial dilutions on PDA-SA

followed by 3 - 4 and 10 - 12 days of incubation at 26°C, respectively.

Inoculation of blueberries

Five 125-g blueberry samples were placed in separate 25 x 20 cm sterile
polyethylene bags (Whirl-PackTM, Fisher Scientiﬁc, Pittsburgh, NJ.) containing 100 ml
of each of the ﬁve cocktails and then gently swirl-agitated at 100 rpm for 20 min on a G2
Gyratory Shaker platform (New Brunswick Scientiﬁc Co., Inc., Edison, NJ.) These
inoculated samples containing ~106 CFU/g of E. coli 0157:H7, L. monocytogenes,
Salmonella, yeasts or molds were then air-dried under laminar ﬂow in a biosafety cabinet

for 2 h, stored overnight at 4°C and ﬁnally re-dried under laminar ﬂow for 2 h before use.

Chlorine Dioxide Exposure

Five 100-g samples of inoculated blueberries were placed in separate half pint
plastic clamshell containers and in a 20 L bucket. The berries were exposed to C102 gas
(4 mg/L, 0.16mg/g fruit) in the sealed 20L bucket (Figure 4.1) for 12 h at ~ 22°C/99.9%
RH. Chlorine dioxide gas was generated inside the bucket using a 20-g commercial C102

sachet (ICA TriNova, LLC Forest Park, GA). The ends of the sachet were pulled to

57

remove the spine clip; granules from the two compartments of the sachet were fully
mixed and pooled at one end, aﬂer which the sachet was placed in the bottom of the
bucket. High humidity was maintained by placing a Petri dish containing 20 ml of SDW
on the bottom of the bucket next to the inoculated fruit and C102 sachet. Chlorine dioxide
gas was circulated inside the sealed bucket using a brushless 12 VDC cooling fan (5 by 5
by 1 cm) (Model DSOSM, RadioShack, Fort Worth, TX) that was attached to the
underside of the bucket lid. Temperature (22°C) and RH. were continuously monitored

using a Thermo-Hygrometer (Model Traceable®, Fischer Scientiﬁc) that was sealed in

the lid of the container.

 

Figure 4.1 Chlorine dioxide gas system used to treat inoculated blueberries in Pilot study.
This system consisted of a 20 L bucket, a Petri dish containing SDW (inside), a lid with a
fan and an attached Thenno-Hygrometer. Inoculated fruit was placed in the container and
exposed to C102 gas (4 mg/L, 0.16mg/g fruit) for 12 h at 22°C/99.9% RH.

58

Microbial Analysis

After treatment, fruit samples (25 g) were placed in sterile 20 x 10 cm
polyethylene bags (Whirl-PackTM) containing 100 ml of neutralizing buffer (Difco). The
sealed bags were shaken horizontally at 100 rpm for 20 min on a G2 Gyratory Shaker
platform and then pulsiﬁed for l min using a Pulsiﬁer (Filtaﬂex Ltd., Almonte, Ontario,
Canada). Samples of neutralizing buffer (1 ml) were serially diluted in PBS and spiral-
. plated (Autoplate 4000—Spiral Biotech, Exotech, Inc, Gaithersburg, MD) in duplicate on
SMAC, MOX, MAC, and PDA-SA to enumerate E. coli 0157:H7, L. monocytogenes,
Salmonella, and yeasts and molds, respectively. SMAC, MOX, and MAC plates were
counted after 48 h of incubation at 37°C, whereas PDA-SA plates were counted after 72

to 96 h of incubation at room temperature (22°C).

B. PALLET STUDY
Blueberries

Fresh mechanically harvested blueberries from different ﬁeld locations at the
same grower were obtained through the Michigan Blueberry Growers Association (Grand
Junction, MI). Thirty lugs of blueberries (~ 9.1 kg/lug) were stacked on 4 x 4 ft wooden
pallets (~ 272 kg fruit/pallet) at a blueberry processing facility with 5 lugs/level and 6

levels/pallet (Figure 4.2)

59

 

Figure 4.2 Pallet study with gassed (tarped) and un-gassed pallets (control).

Chlorine Dioxide Exposure

Each of the six replicated experiments included one gassed (tarped) and one un-
gassed pallet (control) that were held for 12 h at ~12-l4°C. Chlorine dioxide gas was
generated using three 3-kg sachet containers (ICA TriNova) according to the
manufacturer’s instructions: The sackets were removed from their containers and opened
after which their content were poured back into the container and thoroughly mixed by
shaking. Two containers per pallet were then placed on the top lugs with their lids
opened to allow C102 gas to escape. Two IO-cm diameter ﬂexible hoses ~2 meters in
length containing ventilating fans were run along two sides of each pallet from bottom to
top to circulate C102 through the pallet (Figure 4.2). The pallets were tarped with a

plastic sheet, sealed, and exposed to C102 gas for 12 h. According to the manufacturer 1 g

60

of this mixture generated 4 mg of C102 over a 12 h period with the three 3-kg sachets
generating a total of 36,000 mg C102 gas in each 2000 L pallet, giving a ﬁnal C102

estimated concentration of 18 mg/L or 0.13mg/g fruit.

Pallet samples

Before and after 12 h of gassing, blueberry samples (500-g) were collected from
the gassed and ungassed pallets. Each pallet contained 30 lugs (5 lugs x 6 levels) (Figure
4.3). Samples were taken from 25 surface and bottom collection points (5 surface and 5
bottom collection points/lug) and then composited to obtain one surface and one bottom
sample for each of the six levels. All samples were placed in individually bagged
clamshell containers, transported to the laboratory on ice and analyzed within 24 h of

collection.

Microbial analysis

All blueberry samples (100 g each) were placed in sterile 25 x 20 cm Whirl-
PackTM bags containing 200 ml of neutralizing buffer, shaken and pulsiﬁed as previously
described. Appropriate dilutions in PBS were spiral-plated in duplicate on trypticase soy
agar (Difco) containing 0.6% (w/w) yeast extract and 100 ppm cyclohexamide (Sigma-
Aldrich Co., St. Louis, MO) (TSAYE-C) for enumeration of mesophilic aerobic bacteria
(MAB), and PDA-SA for enumeration of yeasts and molds. Additional l-ml aliquots
were plated on E. coli /coliform count plates (PetriﬁlmTM, 3M Corp., St.Paul, MN) for

quantiﬁcation of coliforms and E. coli. TSAYE-C and E. coli / coliforrn count plates

61

were counted after 48 h of incubation at 37°C, whereas PDA-SA plates were counted

after 72 to 96 h of incubation at room temperature (22°C).

   

Surface

Bottom

QUI-BUJNI—

 

Figure 4.3 Blueberry pallet (leﬁ) and lug (right). Every lug has 5 sampling points from
the surface (shown) and 5 sampling points at the bottom (not shown). The pallet has 6
levels with 5 lugs per level. Surface and bottom samples were collected from each lug at
each level (25 surface and 25 bottom samples per level) and then composited to obtain 1

surface and 1 bottom sample for each of the 6 levels.

Statistical analysis

Analysis of Variance (ANOVA) was done on all microbial count data obtained
from the pilot and pallet studies using the Statistical Analysis System (SAS, Version 8,
SASO Institute Inc., Cary, NC). Data in the graphs and tables (Appendix B) are means
from replicates and signiﬁcance between means were determined using least signiﬁcant

difference (LSD) test at the 95% conﬁdence level (P=0.05).

62

Sensory Analysis

Uninoculated blueberries from Grand Junction, MI, variety ‘Bluecrop’ were
exposed to 0.19 mg C102 gas /g fruit under the same conditions as for inoculated ﬁ'uit (12
h at 22°C/99.9% R.H.). Gassed and un-gassed samples (control) were few seconds rinsed
in tap water, air-dried for 1 h, dispensed into individual sample cups with lids (15- 20
berries each) and stored aerobically overnight at 4°C before being evaluated by 110
panelists for various sensory attributes. Panelists were presented with a legal consent
form (Appendix C), individual trays containing coded samples, and a set of instructions
regarding evaluation. The samples were analyzed using a Hedonic scale with “like
extremely” (9) to “dislike extremely (1) for the following sensory attributes: appearance,
aroma, texture, ﬂavor, and overall acceptability. The data were analyzed using SIMS

2000 computer software program at a signiﬁcance level of P < 0.05.

4.3 RESULTS AND DISCUSSION

Blueberries were inoculated by dipping rather than spotting to more closely
mimic contamination during irrigation, mechanical harvesting, and processing including
direct contact with belts, blowers and water tanks. Surface washing was chosen as the
method to remove bacteria, yeasts and molds from the surface of blueberries rather than
homogenizing. Homogenizing blueberries in a diluent would decrease the pH to about
4.0, thereby hampering recovery of any injured cells. When Sy et al. (2005) compared
washing and stomaching for recovery of Salmonella from inoculated blueberries,
populations were 10-fold lower after stomaching. Han and Linton (2004) also reported

that stomaching was less effective than washing for recovery of L. monocytogenes from

63

inoculated strawberries. Although acid tolerant, Salmonella, E. coli 0157:H7, and L.
monocytogenes are unable to survive in foods at pH < 4.0 (D’Aoust, 2000). Pulsiﬁcation
by ultrasound aﬁer surface washing further improved release and subsequent recovery of
microorganisms from the surface without rupturing the fruit and decreasing the pH as

occurred after stomaching.

A. PILOT STUDY

Initial populations of L. monocytogenes, Salmonella, E. coli 0157:H7, yeasts and
molds on the inoculated berries before treatment were 6.46, 6.38, 6.35, 5.99, and 6.22
logs CFU/g, respectively. Signiﬁcant (P <0.05) microbial reductions of 3.94, 3.62, 4.25,
3.10, and 3.17 log CF U/g, respectively, were achieved after exposing the inoculated
blueberries to 4 mg/L, 0.16mg/g fruit C102 gas in a sealed bucket for 12 h at 22°C/99.9%
RH (Appendix B, Table 4.1). High relative humidity (99.9 %) was maintained to enhance
the lethality of chlorine dioxide gas against bacteria, yeasts and molds, with Han et al.
(1999) also reporting increased efﬁcacy at higher relative humidity. In later work by Han
et a1 (2001, 2000) L. monocytogenes and E. coli 0157:H7 populations decreased 3.5 and
6.4 logs on the surface of injured green peppers after exposure to 3 and 1.2 mg/L chlorine
dioxide gas for 30 min, respectively. When the skin, calyx and stem scar areas of apples
were surface-inoculated with L. monocytogenes and exposed to 4 mg/L chlorine dioxide
gas for 10 min at 21°C/90% RH, Du et al. (2002) obtained higher reductions of 5.5, 3.2,
and 3.6-logs, respectively. Exposing Salmonella-inoculated blueberries to 4.1 to 8 mg/L

chlorine dioxide gas / 75-95% RH for 30 to 120 min reduced Salmonella up to 3.7 logs

64

with populations of inherent yeasts and molds decreasing as much as 2.5-logs (Sy et al.,
2005).

Differences in the surface structure and microbial attachment sites on green
peppers, apples and blueberries are likely responsible for the different biocidal efﬁcacies
seen in these studies. Waxes on the surface of apples contain alcohols, morpholine and
surfactants that may enhance the penetration of sanitizers resulting in higher microbial
reductions (Kenney and Beuchat, 2002). The wax on the surface of blueberries is
composed mainly of B-diketone that produces a dense network of interlocking branched
rodlets or closed tube-like structures (Freeman et al., 1979). The hydrophobicity of [3-

diketone also hinders penetration of aqueous-based sanitizers leading to decreased

 

 

 

 

 

 

 

 

   

efficacy.
I I
, . - W . . _. _, ,
i . Before mAﬂer I l
a -—. m” -- HM» — I
‘ f a
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l o 4 , ‘33.}: b
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r \ 1 III 5
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Listeria Salmonella E. coli Yeast

 

Figure 4.4 Populations of L. monocytogenes, Salmonella, E. coli 0157:H7, yeasts and
molds (mean :t std. dev., n=3) on inoculated blueberries before and after exposure to 4
mg/L, 0.13mg/g C102 gas in a sealed 20 L bucket for 12 h at 22°C/99.9% RH. Values
with different letters within the same microbial category are signiﬁcantly different (P <

0.05).

65

 

10

Cl.
bl

de

B. PALLET STUDY

Initial populations of MAB, yeasts, molds, coliforms, and E. coli on naturally
contaminated untreated blueberries were 4.03, 4.32, 4.57, 1.12, and 0.51 logs CFU/g,
respectively (Appendix B, Table 4.2). Aﬁer 12 h of storage, signiﬁcant (P < 0.05)
microbial growth was observed in the ungassed pallets with populations of MAB, yeasts,
molds, coliforms, and E. coli increasing 0.69, 0.36, 0.11, 1.00 and 0.50 logs, respectively.
However, after 12 h of storage under the same conditions, signiﬁcant (P < 0.05)
reductions of 2.33, 1.63, 0.48, 1.47, and 0.52 log CF U/g, respectively, were seen between
the gassed and un-gassed pallets. Overall, reductions for bacteria, yeasts and molds were
1 to almost 3 logs higher on inoculated as compared to uninoculated blueberries. One
reason for this decrease in efﬁcacy is likely related to the lower chlorine dioxide
concentration in the pilot (0.16 mg/g) as opposed to the pallet study (0.13 mg/g).
Decreased efﬁcacy of chlorine dioxide gas in the pallet size study could also be related to
the use of uninoculated blueberries. Compared to bacteria, populations of yeasts and
molds naturally present on blueberries were less susceptible to chlorine dioxide gas.
These ﬁndings are in agreement with Rodgers et al. (2004) who reported reductions of up
to 5 log CFU/g for L. monocytogenes and E. coli 0157:H7 compared to 1.5 log CFU/g
for yeasts and molds when apples were treated with aqueous solutions of 3 and 5 ppm
chlorine dioxide. The lower reductions for coliforms and E. coli on gassed uninoculated
blueberries can be explained by the relatively low initial populations with numbers

decreasing below the limit of detection (0.48 log CFU/g) after gassing.

66

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Population recovered (Log CF U/g
N

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Figure 4.5 Populations of MAB, yeasts, molds, coliforms, and E. coli (mean i std. dev.,
n=6) recovered from blueberries initially (0 h) and from gassed and un-gassed fruit after
12 h of storage. Blueberry pallets containing fruit harvested from the same grower were
stored under the same conditions for 12 h at 12 to 14°C. Gassed pallets were exposed to
18 mg/L (0.13mg/g) C102 gas. The limits of detection were 1.78 (MAB, yeast, and mold)
and 0.48 log CF U/g (coliforms, and E. coli). Values with different letters within the same
microbial category are signiﬁcantly different (P < 0.05).

Uniformity in the reduction of MAB, yeasts, molds, coliforms, and E. coli on
palletized blueberries at different pallet levels (Figure 4.6 A) and sample locations within
the lugs (Figure 4.6 B) using ClOz gas was also evaluated. Aﬁer gassing, no signiﬁcant
differences (P > 0.05) in microbial counts were seen between pallet levels 1 to 6 and,
except for MAB, no signiﬁcant differences were evident between the bottom and top
surface of the lugs. Based on the analysis of variance, regarding gassed pallets levels (1
to 6) and positions (surface and bottom) no interactive effect resulted on the fruit
microbial load (Appendix B, Table 4.3). These ﬁndings demonstrate uniform dispersion

and penetration of chlorine dioxide gas throughout the pallets. Regarding quality of the

67

gassed blueberry pallets, no visible changes in the fruit were evident; however, some

bleaching of the blueberry leaves was observed.

 

 

 
 

      

      
            

  

  

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68

Figure 4.6 Populations of MAB, yeasts, molds, coliforms, and E. coli (mean :h std. dev.,
n=6) recovered from gassed blueberry pallets at different pallet levels (A) and between
the bottom and top surface of lugs from the same pallet level (B). Blueberry pallets were
stored under the same conditions at 12 to 14°C, exposed to 18 mg/L (0.13mg/g) C102
gas, tarped and sealed for 12 h. The limits of detection were 1.78 (MAB, yeast, mold) and
0.48 (coliforms, E. coli) logs CFU/g. Values with different letters within the same
microbial category are signiﬁcantly different (P < 0.05).

Sensory Analysis

No signiﬁcant (P < 0.05) differences regarding appearance, aroma, texture, ﬂavor,
or overall acceptability were seen between blueberries exposed to 0.19 mg/g C102 gas for
12 h and ungassed (control) blueberries (Figure 4.6). Similarly, Sy et al., 2005 reported
no signiﬁcant changes in sensory attributes including appearance, color, aroma, and
overall quality between blueberries exposed to 4.1 mg/L chlorine dioxide gas and
ungassed blueberries after 0 and 3 days of storage. However, in the same study the
treated samples were ranked signiﬁcantly higher for overall quality and aroma after 7 and

10 days of storage, respectively.

69

l
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appearance aroma texture ﬂavor overall

Figure 4.7 Average consumer (n=110) ratings for appearance, aroma, texture, ﬂavor, and

overall acceptability of treated (0.19 mg/g C102/12 h) and untreated (control) blueberries.

4.4 CONCLUSIONS

The results of these studies indicate that chlorine dioxide gas can be used by the
blueberry industry to reduce microbial populations before processing. This pre-
processing microbial reduction strategy will help blueberry processors to better meet
current the microbial standards that are being imposed by blueberry purchasers, without
changes in current processing technology. Gassing of fruit can be done when the fruit is
stored at the processing facility (up to 18 h). In most blueberry processing operations,
the sole microbial reduction step involves brief passage of the berries through a tank of
sanitizer solution. The water tanks were originally employed to separate buoyant green
unripened from mature blueberries but sanitizers are also used to decrease the microbial
load on the berries. In most processing facilities the fruit is typically exposed to a
chlorine-based sanitizer for less than one minute. These wash treatments are only

marginally effective in reducing the number of microbial contaminants on the surface of

70

 

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blueberries. Treatment of bluebenies with 100 ppm chlorine for 5 min reportedly reduced
the population of bacteria, yeast and mold by 0.83, 0.77, and 0.61 log CFU/g,
respectively (Crowe et al., 2005). Our preliminary work using chlorine, and aqueous
chlorine for 5 min on inoculated blueberries resulted in similar results (Appendix A).
Organic fatty acids used in the same preliminary work produced > 3 log reductions for
bacteria, yeasts and molds, but negatively (P <0 .05) impacted the sensory attributes
compared with SDW-washed ﬁ'uit used as the control (Appendix A).
To answer to the actual needs of the industry, this study evaluated gaseous chlorine
dioxide for its effectiveness in inactivation of the human pathogens E. coli 0157:H7, L.
monocytogenes, and Salmonella, as well as mesophilic aerobic bacteria, coliforms, E.
coli, yeasts and molds known for fruit spoilage. Small-scale experiments done on
inoculated blueberries yielded reductions of > 3 logs for known human pathogens and
spoilage yeasts and molds.
Pallet size studies using ~ 272 kg of uninoculated blueberries/pallet (industrial scale)
were done to determine the effectiveness of gaseous chlorine dioxide (18 mg/L,
0.13mg/g) on naturally occurring MAB, yeasts, molds, coliforms, and E. coli with
signiﬁcant (P<0.05) reductions achieved for all microbial categories compared to the
ungassed control pallets.
Overall, reductions of bacteria, yeasts and molds were higher on inoculated (Pilot study)
than on uninoculated blueberries (Pallet study). One reason would be the use of a lower
gas concentration in the pallet (0.13 mg/ g) as opposed to the pilot study (0.16 mg/ g).
Sensory analysis of blueberries exposed to chlorine dioxide (0.19 mg/g fruit)

revealed no signiﬁcant (P < 0.05) differences compared to ungassed (control) blueberries.

71

These results are in agreement with those of Sy et al. (2005) and suggest that chlorine
dioxide gas can be used as to both enhance the microbial quality of blueberries and meet

the present microbial standards being imposed by purchasers.

5. CONCLUSIONS

Microbial contamination on highbush blueberries before, during, and after
processing from the 2003-2004 survey demonstrated that populations of MAB, yeasts,
molds, coliforms and E. coli varied widely among ﬁeld location without a consistent
correlation to ﬁeld condition or handling procedure and increased over the harvest season
for the same ﬁelds, and from pre-harvest to post-harvest due mainly to microbial spread
and multiplication facilitated by mechanical harvesting, and from a 12-h or more delay in
processing, respectively. Microbial populations on the blueberries were not efficiently
reduced after exiting the chlorinated water tank (~ 10 to 200 ppm chlorine). Reduced
efﬁcacy of chlorinated water is due to the short contact times that were typically less than
1 minute. Increasing amounts of organic matter in the wash water also decreased the
effectiveness of chlorine as evidenced by an increase in the levels of microorganisms in
the water tank over time with additional contaminants transferred from the conveyor belt
to the berries after washing. The fact that blueberries generally contained higher
microbial levels after processing than before harvest demonstrates the lack of efﬁcacy in
reducing microbial loads in industry settings. Microbial reductions of less than 0.7 logs
after either 3 or 6 months of storage at -20°C conﬁrms that freezing is also not an

effective microbial reduction strategy for blueberries.

72

Treating blueberries with 100 ppm chlorine for 5 min reportedly reduced the
population of bacteria, yeast and mold by less than 1 log CFU/g (Crowe et al., 2005). Our
preliminary work using chlorine and aqueous chlorine dioxide for 5 min on inoculated
blueberries resulted in similar reductions (Appendix A). Organic fatty acids used in the
same preliminary work produced > 3 log reductions for bacteria, yeasts and molds, but
negatively (P <0 .05) impacted the sensory attributes compared with SDW-washed fruit
used as the control (Appendix A).

To answer to the actual needs of the industry, gaseous chlorine dioxide was evaluated for
its effectiveness in inactivation of the foodbome pathogens E. coli 0157:H7, L.
monocytogenes, and Salmonella, as well as mesophilic aerobic bacteria, coliforms, E.
coli, yeasts and molds known for fruit spoilage. Small-scale experiments on inoculated
blueberries (0.16 mg/g C102 gas) yielded reductions of > 3 logs for known human
pathogens and spoilage yeasts and molds. When 600 lb pallets of uninoculated
blueberries were exposed to 0.13 mg/g ClOz gas for 12 h, populations of naturally
occurring MAB, yeasts, molds, coliforms, and E. coli decreased signiﬁcantly (P < 0.05)
compared to the ungassed control pallets. Sensory analysis of blueberries exposed to
higher chlorine dioxide gas concentrations (0.19 mg/g fruit) revealed no signiﬁcant (P <
0.05) differences compared to ungassed (control) blueberries. Thus, chlorine dioxide
shows considerable promise to enhance the microbial quality of blueberries before
processing and perhaps of fresh fruit, and may help producers and processors meet the

present microbial standards demanded by purchasers.

73

Future research goals focusing on the microbial spoilage and safety of blueberry
would include de following:

l.Optimize chlorine dioxide gas treatment for sanitizing blueberries without
changing sensory attributes.

2.Scale up the gaseous chlorine dioxide treatment for industrial processing of
blueberries.

3.Assess the ability of this gaseous chlorine dioxide treatment followed by
washing of berries in water containing 200 ppm chlorine to meet the current microbial
standards demanded by buyers of frozen berries.

4.Assess the ability of gaseous chlorine dioxide to extend the shelf life of fresh

blueberries.

74

APPENDIX A
MICROBIAL REDUCTIONS ON BLUEBERRIES USING SODIUM

HYPOCHLORITE, CHLORINE DIOXIDE AND ORGANIC ACIDS

OBJECTIVE

The objective of this preliminary work study was to better characterize the
efficacy of chlorine, aqueous chlorine dioxide, organic fatty acid A and B sanitizers at
concentrations and exposure times deemed to appropriate for blueberries processors.
These sanitizers were tested at a contact time of 5 min in an aqueous model system and
on inoculated fruits against three strains each of Escherichia coli 0157:H7, Listeria
monocytogenes, and Salmonella as well as yeasts (Aureobasidium sp., Bullera sp.,
Cryptococcus sp., Sporidiobolus sp., and F ilobasidiu sp.) and molds (Colletotrichum sp.,

Epicoccum sp., Cladosporium sp., Phoma sp., and Alternaria sp.).

MATERIAL AND METHODS

All bacterial strains, yeasts and molds for this study were grown as described in
Chapter 4.
Sanitizers

The following sanitizer solutions were investigated for microbial reductions:
chlorine (100, 200 and 400 ppm), aqueous chlorine dioxide (C102) (3 and 5 ppm),
organic fatty acid A (OA) (caprylic acid, Emsorb 6915, and mineral oil) at concentrations

of 0.3%, 0.6%, and 0.9 % (v/v), and OA at 0.3% + organic fatty acid B (OB) (glycolic

75

acid, caprylic acid, and Emsorb 6915) at 0.15% (v/v), with sterile distilled water (SDW)
used as the control.

Sodium hypochlorite solutions containing 100, 200 and 400 ppm chlorine were
prepared by diluting CloroxTM (Clorox Company, Oakland, CA) with SDW and than
conﬁrming the concentration with a chlorine test kit (La Motte Chemical Products Co.,
Inc., Chestertown, MD). Aqueous chlorine dioxide solutions (3 and 5 ppm C102) were
prepared from commercial 20-g chlorine dioxide sachet (ICA TriNova, LLC Forest Park,
GA) by placing the mixed contents of the sachet in a sealed bottle containing 1150 ml of
SDW for 12 h. This stock solution containing 800 ppm ClOz was transferred in smaller
sterile bottles and stored at 4°C with the C102 concentration remaining stable up to 30
days. Working solutions containing 3 and 5 ppm C102 were obtained by diluting the stock
solution with SDW and then conﬁrming the C102 concentration with a chlorine test kit
(La Motte Chemical Products Co.). The organic fatty acid sanitizers were obtained from
Bob Coleman and are not yet commercially available. These fatty acid sanitizer solutions
were freshly prepared just before use by adding 1.5 ml OB, and 3, 6, or 9 ml OA stock
solution to 1 L SDW to obtain 0.15% OB, and 0.3, 0.6, or 0.9% CA work solutions,

respectively.

Aqueous Model System Study

Sterile centrifuge tubes containing 9-ml aliquots of each sanitizer solution at the
above concentrations were inoculated with 1ml of a l9-strain cocktail containing three
strains each of the mesophilic bacterial pathogens E. coli 0157:H7, L. monocytogenes,

and Salmonella as well as ﬁve yeasts (Aureobasidium sp., Bullera sp., Cryptococcus sp.,

76

Sporidiobolus sp., and F ilobasidium sp.) and ﬁve molds (Colletotrichum sp., Epicoccum
sp., Cladosporium sp., Phoma sp., and Alternaria sp.), with SDW used as the control.
Over a period of 5 minutes, 1 ml aliquots were removed and diluted 1:10 in neutralizing
buffer (Difco) for sodium hypochlorite, chlorine dioxide and. double strength neutralizing
buffer (Guthery, 2001) for organic fatty acids A and B, followed by serial dilution in
sterile phosphate buffer solution (PBS). Double strength neutralizing buffer solution
(Guthery, 2001) was prepared by adding 10 g peptone (Difco), 2 g sodium thiosulfate (JT
Baker, Phillipsburg, NJ), 0.5 g mono-potassium phosphate (JT Baker), 0.5 g catalase
(Sigma), 60 g Tween 80 (Sigma), and 10 g lecithin (Sigma) to 1 L of Letheen broth
(Difco). Appropriate dilutions in PBS were spiral-plated in duplicate on trypticase soy
agar (Difco) containing 0.6% yeast extract and 100 ppm cyclohexamide (Sigma-Aldrich
Co., St. Louis, MO) (TSAYE-C) for enumeration of mesophilic aerobic bacteria (MAB),
and potato dextrose agar (Difco) containing 20 ppm streptomycin (Sigma) and 50 ppm
ampicillin (Sigma) (PDA-SA) for enumeration of yeasts and molds. TSAYE-C plates
were counted after 48 h of incubation at 37°C. PDA-SA plates were counted after 72 to
96 h of incubation at room temperature (22°C).
Fruits

Fresh blueberries of uniform size and shape were obtained from the local market
and stored at 4°C. Before inoculation the fruits were held at room temperature to dry.
Inoculation

Blueberries (275g) were inoculated by immersion in a 25 x 20 cm sterile
polyethylene bag (Whirl-PackTM, Fisher Scientiﬁc, Pittsburgh, N.J.) containing ~370 ml

of the 19-strain cocktail of E. coli 0157:H7, L. monocytogenes, Salmonella, yeasts and

77

molds and gently swirl-agitated at 100 rpm for 20 min on a G2 Gyratory Shaker platform,
(New Brunswick Scientiﬁc Co., Inc., Edison, NJ). The fruits were air dried under
laminar ﬂow in a biosafety cabinet for 2 h, stored overnight at 4°C and then re-dried
under a laminar ﬂow hood for 2 h before use.
Sanitizer Exposure

Fruit samples (25g) placed in sterile 20x 10 cm polyethylene bags (Whirl-PackTM)
containing 100 ml of the various sanitizers were horizontally shaken at 100 rpm for 5
minutes on a G2 Gyratory Shaker platform (New Brunswick Scientiﬁc Co.).
Microbial Analysis

After treatment, the sanitizer was discarded and replaced by 100 ml of
neutralizing buffer (Difco) for sodium hypochlorite, chlorine dioxide, and 100 ml of
double strength neutralizing buffer (Guthery, 2001) for organic fatty acids A, and B. The
sealed bags were horizontally shaken at 100 rpm for 20 min on a G2 Gyratory Shaker
platform (New Brunswick Scientiﬁc Co.) and then pulsiﬁed for 1 min using a Pulsiﬁer,
(Filataﬂex Ltd., Ontario, Canada). Samples of neutralizing buffer (lml) were serially
diluted in sterile phosphate buffer solution (PBS) (9ml) and appropriate dilutions were
spiral-plated (Autoplate 4000 —Spiral Biotech, Exotech, Inc, Gaithersburg, MD) in
duplicate on TSAYE-C for entuneration of mesophilic aerobic bacteria (MAB), and
PDA-SA for enumeration of yeasts and molds. TSAYE-C plates were counted after 48 h
incubation at 37°C. PDA-SA plates were counted after 72 to 96 h incubation at room

temperature (22°C).

78

Statistical analysis

Analysis of Variance (ANOVA) was done on all microbial reduction data for each
sanitizer concentrations using the Statistical Analysis System (SAS, Version 8, SAS0
Institute Inc., Cary, NC). Data in the graphs and tables (Appendix B) are means from
replicates and signiﬁcance between means were determined using least signiﬁcant

difference (LSD) test at the 95% conﬁdence level (P=0.05).

Sensory Analysis

Uninoculated fruit samples were exposed to each of the following sanitizers for 5
min : 200 ppm chlorine, 5 ppm C102, 0.9% OA, , 0.3% OA + 0.15% OB. After
treatment, the samples were water rinsed for 1-2 sec., air dried for 1 h, dispensed into
individual sample cups with lids (15- 20 berries each) and stored aerobically overnight at
4°C before being given to 110 panelists comprising students, staff and faculty at
Michigan State University. The panelists were presented with a legal consent form
(Appendix C), individual trays containing the coded samples, and a set of computer
instructions regarding sample evaluation. The samples were analyzed using a Hedonic
scale with “like extremely” (9) to “dislike extremely” (1) for the following sensory
attributes: appearance, aroma, texture, ﬂavor, and overall acceptability. The data were

analyzed using SIMS 2000 computer software program at a signiﬁcance level of P<0.05.

79

RESULTS
Aqueous Model System Studies

Chlorine dioxide was the least effective sanitizer for all microbial categories.
Concentrations of 3 to 5 ppm C102 reduced MAB, yeast and mold populations by 1.61,
1.16 and 1.83 log CFU/ml (Appendix B, Table A.l), respectively, with these reductions
only statistically signiﬁcant (P< 0.05) for MAB, and molds. Chlorine and organic fatty
acids were more effective, with signiﬁcantly (P < 0.05) greater reductions than 5 ppm
C102 for MAB and molds, and signiﬁcantly (P < 0.05) greater reductions than 3 ppm
C102 for all microbial categories. These two sanitizers decreased populations >4 logs

with no greater reductions seen at higher concentrations (Figure A. 1 ).

 

 

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aﬁer a 5-minute exposure to various sanitizers in an aqueous model system: 3 ppm

chlorine dioxide (3 ppm C102), 5 ppm C102, 100 ppm sodium hypochlorite (100 ppm C1),

, 0.9% CA, and

0.3% CA + 0.15% OB (OA+OB). Microbial reductions with different letters are

signiﬁcantly different (P<0.05).

0.6% CA

200 ppm Cl, 400 ppm Cl, 0.3% organic fatty acid A (0.3% CA),

81

Blueberries Inoculation Studies

The two organic fatty acids were the most effective sanitizers. OA at a
concentration of 0.9% decreased MAB, yeast, and mold populations by 2.52, 3.77, and
3.72 logs CFU/g, respectively (Appendix B, Table A.2). Increasing the OA concentration
from 0.3 to 0.9% resulted in signiﬁcantly (P < 0.05) greater reductions for MAB and
yeast. Chlorine at 400 ppm was the next most effective sanitizer, reducing populations of
MAB, yeast, and mold by 0.91, 1.28, and 2.14 logs CFU/g, respectively. Similar
microbial reductions were seen when the Cl concentration was decreased from 400 to 100
ppm. Although 3 and 5 ppm 002 were least effective, these sanitizers were not
signiﬁcantly (P > 0.05) different from C1 for MAB and molds, and SDW for MAB and
yeast (Figure A.2). Treatment of inoculated blueberries with SDW for 5 minutes reduced

populations of MAB, yeast and mold by 0.24, 0.15 and, 0.49 log CFU/g, respectively.

 

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82

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after a 5-minute exposure of inoculated fruit to various sanitizers: 3 ppm chlorine dioxide
(3 ppm 002), 5 ppm C102, 100 ppm sodium hypochlorite (100 ppm Cl), 200 ppm Cl,
400 ppm Cl, 0.3% organic fatty acid A (0.3% OA), 0.6% OA, 0.9% OA, and 0.3% CA +
0.15% OB (OA+OB) and SDW. Microbial reductions with different letters are
signiﬁcantly different (P<0.05).

83

Sensory Analysis

The only statistically signiﬁcant differences between the sanitizer treatments were
observed for 0.9% OA and 0.3% OA +0.15% OB (OA+OB) for all sensory attributes
(Figure A 3). 0A 0.9% compared with OA+OB treated fruit was also signiﬁcantly (P

<0.05) lower in terms of texture, ﬂavor, and overall acceptability.

 

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Figure A 3 Average consumer acceptability for fresh blueberries subjected for 5 min to
wash treatment with 200 ppm sodium hypochlorite (200 ppm C1), 5 ppm chlorine dioxide
(5 ppm C102), 0.9% organic fatty acid A (0.9% OA), 0.3% CA + 0.15% OB (OA + OB),
and sterile distilled water (SDW) as control.

CONCLUSION

The most effective sanitizers were organic fatty acids A and B followed by
chlorine, and chlorine dioxide. On fruits organic fatty acid A and B negatively impacted
all sensory attributes (P < 0.05) compared to the control and other sanitizers. Aqueous

solutions of chlorine dioxide chlorine produced similar microbial reductions and sensory

84

attributes (P > 0.05) for fruit. SDW reduced microbial populations by < 0.5 logs
indicating that just simply washing of mu is not a solution for enhancing the safety and

quality of blueberries.

85

 

 

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96

APPENDIX C

Consumer Consent Form
“Microbial Reduction Strategies for Highbush Blueberries ”
Department of Food Science and Human Nutrition, Michigan State University

Sample:

Before you decide to sign this consent form and continue to participate in our study,
please read carefully and thoroughly the reverse side of this form for the sample
ingredients and preparation information, purpose and procedure of this study, potential
risks and beneﬁts from your participation, our assurance of your privacy, your rights as a
human subject in our study, etc. If you have any question during your reading this
consent form, or during or after your participation, please do not hesitate to contact the
on-site sensory evaluation leader and/or the principle investigator. Feel free to contact
Dr. Elliot Ryser, the principle investigator of this study, via phone at 517-355-7713
ext.185 (Department of Food Science and Human Nutrition, 2108 S. Anthony, Michigan
State University, East Lansing, MI 48823) You can also reach me via email at
ryser@msu.edu for any inquiry you might have due to your participation in our study. In
case you have questions or concerns about your role and rights as a research participant,
please feel free to contact Peter Vasilenko, Ph.D., Chair of University Committee on
Research Involving Human Subject (UCRIHS), 202 Olds Hall, Michigan State
University, East Lansing, MI 48824-1046, PHONE (517) 355-2180 FAX (517) 432-
4503, E-Mail - UCRIHS@msu.edu, WEB SITE -http://www.humanresearch.msu.edu

If you have read all the information given to you in this consent form and decide to
participate in this study and provide your valuable response to our questionnaire, you can
go ahead and sign this form now. Otherwise, you can stop here and feel free to
discontinue participation in our study without any penalty.

PLEASE NOTE UPON YOUR SIGNING THIS CONSENT FORM, YOU
VOLUNTARILY AGREE TO PARTICIPATE IN THIS STUDY. YOUR SIGNATURE
INDICATES YOU HAVE READ THE INFORMATION PROVIDED ABOVE AND
THAT YOU HAVE HAD AN ADEQUATE OPPORTUNITY TO DISCUSS THIS
STUDY WITH THE PRINCIPLE INVESTIGATOR AND HAVE HAD ALL YOUR
QUESTIONS ANSWERED TO YOUR SATISFACTION. YOU WILL BE GIVEN A
COPY OF THIS CONSENT FORM WITH YOUR SIGNATURE FOR YOUR
RECORDS UPON YOUR REQUEST.

SIGNED DATE

 

 

You indicate your voluntary agreement to participate by signing above.

97

Consent Form
“Microbial Reduction Strategies for Highbush Blueberries ”
Department of Food Science and Human Nutrition, Michigan State University

INVITATION TO PARTICIPATE
You are invited to participate in this study that assesses the quality attributes of blueberries.

PURPOSE OF THIS STUDY

This study is intended to study the quality of blueberries that have been washed in either water or
one or more FDA-approved sanitizer solutions that are widely used commercially. Texture,
appearance and ﬂavor characteristics of blueberries will be evaluated.

PROCEDURE OF THIS STUDY

Each participant will be presented with a series of three blueberry samples. They will be asked to
evaluate visually and after tasting, score the attributes as presented on the score sheet for each
sample. Samples will be presented using three digit random codes. Consumer marketing
questions will also be asked.

SAMPLE PREPARATION
All treatments given to our blueberries are FDA approved.

POTENTIAL RISKS

Because all ingredients we use in our study are food grade and FDA approved for food
applications, these samples pose no adverse health risk, provided the subject has not been
identiﬁed as being susceptible to an allergic reaction to the previously listed sample ingredients.
If you believe there is a potential of an allergic reaction upon snifﬁng and tasting, notify the
on-site sensory evaluation coordinator and/or principle investigator immediately. You will be
released from participating in this study. Please note if you are injured as a result of your
participation in this study, Michigan State University will provide emergency medical care, if
necessary, but this and any other medical expense must be paid from your own health insurance
program.

POTENTIAL BENEFITS

There are no beneﬁts gained directly from your participation in this study. However, your
participation and response will provide us valuable data, which can be used to identify optimum
microbial reduction strategies for blueberries that will help keep the Michigan blueberry industry
strong and proﬁtable.

ASSURANCE OF CONFIDENTIALTY

Any information obtained in connection with this study that could be identified with you will be
kept confidential by ensuring that all consent forms are securely stored. All data collected and
analyzed will be reported in an aggregate format that will not permit associating subjects with
speciﬁc responses or ﬁndings. Your privacy will be protected to the maximum extent
allowable by law.

WITHDRAWAL FROM THIS STUDY
Participation in this study is voluntary. Your decision to refuse participation or discontinue
participation during this study will be honored promptly and unconditionally.

98

Questionnaire
Product: Fresh blueberry

Instructions:

You will be provided with 6 ﬁ'uit samples and a questionnaire. Please look carefully at
the sample number and ﬁnd the corresponding blueberry cup with that number. Then
answer the following questions:

1. How do you like the APPEARANCE of the blueberries?
9 — Like extremely
8 - Like very much
7 — Like moderately
6 — Like slightly
5 — Neither like nor dislike
4 — Dislike slightly
3 - Dislike moderately
2 — Dislike very much
1 — Dislike extremely

Instruction: Please sniff the blueberries and answer to the following question.

2. How do you like the AROMA of the blueberries?
9 — Like extremely
8 — Like very much
7 — Like moderately
6 — Like slightly
5 — Neither like nor dislike
4 - Dislike slightly
3 — Dislike moderately
2 — Dislike very much
1 — Dislike extremely

99

Instruction:
Please taste the blueberries and answer the following questions.

3. How do you like the TEXTURE of the blueberries?
9 - Like extremely
8 - Like very much
7 — Like moderately
6 — Like slightly
5 — Neither like nor dislike
4 — Dislike slightly
3 — Dislike moderately
2 — Dislike very much
1 — Dislike extremely

4. How do you like the FLAVOR of the blueberries?
9 — Like extremely
8 - Like very much
7 — Like moderately
6 — Like slightly
5 — Neither like nor dislike
4 — Dislike slightly
3 — Dislike moderately
2 - Dislike very much
1 — Dislike extremely

5. How do you like the OVERALL ACCEPTABILITY of the blueberries?
9 — Like extremely
8 — Like very much
7 — Like moderately
6 — Like slightly
5 - Neither like nor dislike
4 — Dislike slightly
3 — Dislike moderately
2 — Dislike very much
1 - Dislike extremely

Instruction:
Please answer the following marketing questions.

6. Please check all that apply: Which of these factors are important to you as
reasons why you purchase blueberries.

[] Nutritious/healthy food
[] Good value
[] Taste

100

[] Little/no waste

[] Safe
[] Do not purchase blueberries

7. Thinking back over these past two weeks, did you purchase blueberries and if yes,
how did you purchase them?

5 - Did not purchase

4 — Other forms

3 - Fresh blueberries

2 — Frozen blueberries
1 — Canned blueberries

8. I prefer to eat fresh blueberries rather than eating frozen blueberries.

5 — Strongly agree

4 — Agree
3 — Neither agree nor disagree
2 — Disagree

1 — Strongly disagree
9. Are you Male or Female?

2 — Female

1 — Male

10. Of the following, which category best represents your 2004 household income
before taxes?

4 - Less than $20,000

3 - $20,000 to $39,999
2 — $40,000 to $59,999

1 — Greater than $60,000

11. How many people are in your household?

12. How many people in your household are under 18?

THANK YOU FOR YOUR PARTICIPATION!

101

APPENDIX D

Frozen Sampling Log 2005

Fruit samples of processed fruits before freezing were taken from Hartman on 8/25/2005
and microbial analysis within 4 h. Fruit were packed the same day in two-30 lb boxes.
Room temperature 66°F. Product temperature 60°F. Both boxes (A and B) were located
in the middle of the pallet. Product was frozen to 32 °F 8/27/05 6:45 AM @ freezer
according to RF ID Tag. Last read of RF ID Tag 9/19/05 @ 1:15 PM with 0° F recorded.

Fruit samples after freezing taken on 9/20/2005 and microbial analysis done within 4 h.

IBefore unAﬂer ?
.. 1

._..___I_._ _mj .

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\ N \\III ‘

Mold Coliform E. coli 1

 

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Figure D.1 Populations of MAB, yeasts, molds, coliforms and E. coli (mean d: std. dev.,
n= 6) on blueberry samples before and after frozen storage in industrial conditions.
Values with different letters within the same microbial category are signiﬁcantly different

(P<0.05).

102

Microbiological Survey of Blueberries 2003-2004

Sampling Log - 2004

Hartman 1 ‘Rancocas’ Type of blueberry.

Pre-harvest sample collection: 13 July 04 at 08:30 by Steven and luliano.
Weather Conditions: ~75°F, cloudy, calm, humidity 85 — 90%.
Machine—harvested sample collection: 11:00.

Weather Conditions: ~80°F, cloudy, calm, humidity 85 — 90%.
Processing began at 13:00 — 13:45 on the same day.

Wash water for processing: ‘Chlorox’ at 10 ppm Cl

HA 1- Populations of MAB, yeasts, molds, coliforms and E. coli on blueberries
(Log CFU/g)

 

 

Fruit sample MAB Yeast Mold Coliforms E. coli
Pre-harvest 1 4.78 4.9 4.3 1.6 0.7
Pre-harvest 2 5 5.04 4.9 0.7 0.7
Post-halvest 1 5.77 5.04 5.71 1.3 1.18
Post-harvest 2 5.9 5.73 5.9 1.74 1.4
Blower exit 1 4.85 5 5.62 3.18 2.23
Blower exit 2 5.96 5.18 5.23 3.18 2.48
Water tank exit 1 4.3 4.3 4.9 2.35 0.7
Water tank exit 2 5.04 4.7 4.7 1.48 0.7
PIC-packaging 1 5.23 4.8 4.7 1.81 0.7
Pre-packaging 2 4.8 4.6 4.7 2.08 0.7

 

103

HA l-Populations of MAB, yeasts, molds, coliforms and E. coli on conveyer belts (Log
CF U/cmz) entering the blower (BA) and pre-packaging areas (PA), before and after ﬁ'uit

processing.

 

Swab samples MAB Yeast Mold Coliforms E. coli

 

BA before 1.08 1.48 0.78 0 0
PA before 1.48 1.08 0.78 o 0
BA after 4.63 3.38 3.92 2.32 1.48
PA after 4.38 5.08 4.53 0 0

 

HA l-Populations of MAB, yeasts, molds, coliforms and E. coli in water tank samples
(log CFU/ml) taken before and after fruit washing.

 

Water sample MAB Yeast Mold Coliforms E. coli

 

Before 1.3 1.3 1.3 0 0
After 1.3 1.3 2.53 0 0

 

104

HA 2 ‘Rubel’ Type of blueberry.

Pre-harvest sample collection: harvested 19 July by Eric.

Weather conditions: unknown on 19 July, and sunny, still, ~78°F on July 20.

Processing began at 08:05- 08:15 on 20 July 04.

Wash water for processing: ‘Chlorox’. During processing time the free chlorine and the
pH of the water tank were: Water drops -10ppm and 8.51; Water tank 0 min-48 ppm and

8.90; 10 min (end) 44 ppm and 8.18.

HA 2- Populations of MAB, yeasts, molds, coliforms and E. coli on blueberries
(Log CFU/g)

 

 

Fruit sample MAB Yeast Mold Coliforms E. coli
Pre-harvest 1 3.04 3.34 2.9 0.7 0.7
Pre-harvest 2 3 3.62 2.3 0.7 0.7
Post-harvest 1 5.59 5.48 5.6 2.11 1
Post-harvest 2 5.75 5.36 5.58 1.95 2
Blower exit 1 5.79 5.77 5.54 2.78 1.7
Blower exit 2 5.91 5.67 5.9 2.18 1.7
Water tank exit 1 5.54 4.65 4.58 0.7 0.7
Water tank exit 2 5.94 4.57 5 2.3 1
Pre-packaging 1 5.58 4.48 4.96 2 1
Pre-packaging 2 5.63 4.48 4.59 2.54 0.7

 

105

HA 2-Populations of MAB, yeasts, molds, coliforms and E. coli on conveyer belts (Log
CFU/cmz) entering the blower (BA) and pre-packaging areas (PA), before and after fruit

processing.

 

Swab samples MAB Yeast Mold Coliforms E. coli

 

BA before 1.86 0.78 0.78 o 0
PA before 3.08 2.59 1.26 1.08 0
BA after 4.11 4.08 4.57 0.85 0.3
PA after 3.61 2.89 3.36 0.3 o

 

HA 2-Populations of MAB, yeasts, molds, coliforms and E. coli in water tank samples
(log CFU/ml) taken before and after fruit washing.

 

Water sample MAB Yeast Mold Coliforms E. coli

 

Before 1.3 1.3 1.3 o 0
After 1.3 1.9 1.3 0.3 0

 

106

HA 3 ‘Rancocas’ Type of blueberry.

Pre-harvest sample collection: 20 July 04 from 09:30 — 09:40 by Steven and luliano.
Weather Conditions: sunny, still, ~78°F.

Harvested (handpicked) for processing on 21 July from 12:20 — 13:20.

Weather Conditions: partly cloudy, still, ~83°F.

Processing began at 14:00, ﬁnished at 14:20.

Wash water for processing: ‘Chlorox’.

During processing time the free chlorine and the pH of the water tank were: 0 min-2 ppm
and 7.64; 10 min 20 ppm and 6.84; 20 min-18 ppm and 7.88.

HA 3- Populations of MAB, yeasts, molds, coliforms and E. coli on blueberries
(Log CFU/g)

 

 

Fruit sample MAB Yeast Mold Coliforms E. coli
Pre-harvest 1 5.41 4 5.08 0.7 0.7
Pre-harvest 2 4.9 4 4.7 0.7 0.7
Post-harvest 1 5.45 4.85 5.45 1 0.7
Post-harvest 2 5.2 4.9 5.52 1.85 1.4
Blower exit 1 5.08 4.78 5.38 2.65 1.7
Blower exit 2 5.53 4.7 5.36 2.48 1.7
Water tank exit 1 3.3 3.6 4 1.4 0.7
Water tank exit 2 3 3.6 4.34 1.7 0.7
Pre-packaging 1 3 3.7 4.52 1.18 0.7
Pre-packaging 2 3.48 4.1 1 4.58 1.95 0.7

 

107

HA 3-Populations of MAB, yeasts, molds, coliforms and E. coli on conveyer belts (Log
CF U/cmz) entering the blower (BA) and pre-packaging areas (PA), before and aﬁer fruit

processing.

 

Swab samples MAB Yeast Mold Coliforms E. coli

 

BA before 3.63 3.28 2.26 o 0
PA before 2.88 1.89 0.78 0 0
BA after 4.15 4.15 4.58 0.48 1.65
PA after 2.96 2.11 2.36 o o

 

HA 3-Populations of MAB, yeasts, molds, coliforms and E. coli in water tank samples
(log CFU/ml) taken before and after fruit washing.

 

Water sample MAB Yeast Mold Coliforms E. coli

 

Before 1.3 1.3 1.3 0 0
After 1.3 1.3 1.3 O 0

 

108

HA 4

Pre-harvest sample collection: July 26 taken by Eric and Steven from 12:00 — 12:15.
Weather Conditions: sunny, slight breeze, ~73F.

Harvested for processing on 26 July from 11:00 - ~13:00.

Processing began at 08:15 on 27 July.

Weather Conditions: cloudy, still, rain in the area, ~70F.

Wash water for processing: ‘Chlorox’.

During processing time the free chlorine and the pH of the water tank were: Water drops
-32 ppm and 8.62; 0 min-250 ppm and 9.08;5 min- 108ppm and 8.29;]0 min(end)

208ppm and 8.68, respectively.

HA 4- Populations of MAB, yeasts, molds, coliforms and E. coli on blueberries
(Log CFU/g)

 

 

Fruit sample MAB Yeast Mold Colifonns E. coli
Pre-harvest 1 2.48 3.67 3.4 0.7 0.7
Pre-harvest 2 2.85 3.23 3.28 0.7 0.7
Post-harvest 1 5.72 4.58 4.85 2 0.7
Post-harvest 2 5.18 4.81 4.08 2.81 0.7
Blower exit 1 6.23 4.48 4.58 1 0.7
Blower exit 2 5.83 4.52 4.56 2.18 1.18
Water tank exit 1 3.59 3.52 2.95 0.7 0.7
Water tank exit 2 3.46 3.6 2.78 1.4 1.4
Pre-packaging 1 4.15 3.3 3.94 1.7 0.7
Pre-packaging 2 4 3 3.96 0.7 0.7

 

109

HA 4-Populations of MAB, yeasts, molds, coliforms and E. coli on conveyer belts (Log
CF U/cmz) entering the blower (BA) and pre-packaging areas (PA), before and aﬁer fruit

processing.

 

Swab samples MAB Yeast Mold Coliforms E. coli

 

BA before 3.63 3.6 2.68 o 0
PA before 1.38 0.78 0.78 O 0
BA after 4.54 4.23 4.51 1.98 0.78
PA after 2.23 2.04 1.92 1.26 0.48

 

HA 4-Populations of MAB, yeasts, molds, coliforms and E. coli in water tank samples
(log CFU/ml) taken before and aﬁer fruit washing.

 

Water sample MAB Yeast Mold Coliforms E. coli

 

Before 1.3 1.3 1.3 0 0
After 1.3 1.3 1.3 o o

 

110

KA 1 ‘Bluecrop’ Type of blueberry.

Preharvest sample taken by Eric on 2 Aug 04.

Weather conditions: ~67F, sunny/foggy, still.

Harvested for processing on 2 Aug from 13:00 — 17:00.

Processing began next day at 11:45-12:00, ﬁnished at 12:30-12:45.

During processing time the free chlorine and the pH of the water tank were: 0min -12
ppm and 6.84; 5 min-18 ppm and 6.98; 15 min- 10ppm and 6.94; 20 min— 8ppm and 6.91;
rinse water-2ppm and 7.01.

KA 1- Populations of MAB, yeasts, molds, coliforms and E. coli on blueberries
(Log CFU/g)

 

 

Fruit sample MAB Yeast Mold Colifonns E. coli
Pre-harvest 1 2.7 3.78 2.3 0.7 0.7
Pre-harvest 2 3.2 4.66 2.48 0.7 0.7
Post-harvest 1 4.32 3.95 2 0.7 0.7
Post-harvest 2 4.08 3.48 2 0.7 0.7
Blower exit 1 4.61 4.23 2.3 0.7 0.7
Blower exit 2 4.26 3.78 2.3 0.7 0.7
Water tank exit 1 4.67 4.15 3.7 0.7 0.7
Water tank exit 2 4.36 4.11 3 0.7 0.7
Pre-packaging 1 4.28 4.2 3.6 0.7 . 0.7

Pre-packaging 2 3.95 4.4 3.9 1.4 o

 

111

KAI-Populations of MAB, yeasts, molds, coliforms and E. coli on conveyer belts (Log
CFU/cmz) entering the blower (BA) and pre-packaging areas (PA), before and aﬁer fruit

processing.

 

Swab samples MAB Yeast Mold Coliforms E. coli

 

BA before 3.46 4 3.91 0.48 0
PA before 3.3 2.62 1.82 o 0
BA aﬁer 3.86 3.38 4.15 1.78 0
PA after 2.38 2.08 2.08 0.9 o

 

KA l-Populations of MAB, yeasts, molds, coliforms and E. coli in water tank samples
(log CFU/ml) taken before and after fruit washing.

 

Water sample MAB Yeast Mold Coliforms E. coli

 

Before 1.3 1.3 1.3 0 0
After 1.3 1.3 1.3 o o

 

112

KA 2 ‘Bluecrop’ Type of blueberry.

Preharvest sample taken on 3 Aug 04from 13:25 — 13:35 by Steven and luliano.
Harvested for processing on 3 Aug from 13:00 — 17:00.

Weather conditions: raining, cloudy, cool, ~65F.

Processing began at 10:45, ﬁnished at 11:15 on 4 Aug.

During processing time the free chlorine and the pH of the water tank were: 0min -6 ppm

and 6.35; 5 min-l6 ppm and 6.9020 min- 22ppm and 7.00; rinse water-3ppm and 7.08.

KA 2- Populations of MAB, yeasts, molds, coliforms and E. coli on blueberries
(Log CFU/g)

 

 

Fruit sample MAB Yeast Mold Coliforms E. coli

Pre-harvest 1 3.85 3.7 2.7 0.7 0.7
Pre-hawest 2 4.26 3.6 2 0.7 0.7
Post-harvest 1 4.93 3.96 2.78 0.7 0.7
Post-harvest 2 4.79 3.99 3 0.7 0.7
Blower exit 1 5.04 4.61 3.7 2 0.7
Blower exit 2 4.99 4.65 4.34 2.3 0.7
Water tank exit 1 4.49 3.75 3.28 0.7 0.7
Water tank exit 2 4.08 3.18 2.3 1.48 0.7
Pre-packaging 1 3.51 4.08 2.6 1.3 0.7

Pre-packaging 2 3.61 4 2.3 0.7 0.7

 

113

KA 2-Populations of MAB, yeasts, molds, coliforms and E. coli on conveyer belts (Log
CFU/cmz) entering the blower (BA) and pre-packaging areas (PA), before and after fruit

processing.

 

Swab samples MAB Yeast Mold Colifonns E. coli

 

BA before 4.69 4.18 4.34 0.6 0.3
PA before 3.28 2.46 2.23 o 0
BA aﬁer 4.92 4.63 4.26 1.26 0
PA after 3.26 2.92 1.78 o o

 

KA 2-Populations of MAB, yeasts, molds, coliforms and E. coli in water tank samples
(log CF U/ml) taken before and after fruit washing.

 

Water sample MAB Yeast Mold Colifonns E. coli

 

Before 1.78 1.3 1.3 o 0
After 1.3 1.3 1.3 0 0

 

114

Sampling Log - 2003

EL 1 ‘Earliblue’ ﬁ'uit ﬁrst picking.

Preharvest sample taken on 7 July 03 by Hanson just NW of packing shed. Good shape,
little ﬁeld rot. About 70 F.

Processing samples taken on 8 July by Hanson and Berkheimer from Ellis processing line
in Grand Junction. First ﬁ'uit run of the season for them. Fruit had sat in shed @ 60-70F
all night. Ran ﬁrst thing in morning. Fruit collected over 2 hr period. Older shed and
equipment generally clean, but not an MBG-Marketing approved processor.

Some question about whether chlorine injector was working when pre-run water samples
were taken.

No swab samples taken —— all stainless steel mesh belts. Line included one blower, water

tank with bleach injector, sorting belt, and then back into lugs.

115

EL 1- Populations of MAB, yeasts, and molds on blueberries (Log CF U/g)

 

 

Fruit sample MAB Yeast Mold
Pre-harvest 1 2.17 3.47 1.69
Pre-harvest 2 3.86 4.72 2.69
Post-harvest 1 3.41 3.79 2.39
Post-harvest 2 2.87 3.79 3.07
Blower exit 1 3.68 4.04 2.54
Blower exit 2 3.79 4.41 3
Water tank exit 1 3.32 3.17 <1.69
Water tank exit 2 2.30 3.11 1.69

Pre-packaging 1
Pre-packaging 2

 

HA1 ‘Bluecrop’ Type of blueberry.

Preharvest sample take by Berkheimer on 28 July 03.

Weather conditions: sunny, still and warm; ~80F.

First mechanical harvest of ‘Bluecrop’on July 28. Field was hand-picked once.

Berries had sat in shed over night at 60-65F. Berries with 1-4% green plus some over-ripe
and rot. Fruit collected over 1 hr period.

Processing samples taken by Hanson and Montecino on July 29.

Line set up with two blowers, de-stemmer, custom water tank continuously ﬂushed with
bleach water (try to maintain 10-20 ppm), rinse spray as they leave water tank (2-4 ppm),
color sorter, hand sorting belts, and boxer. Berries in water for just short time, just to
treat, not to sort out the green.

Water chlorinated with Chlorox.

116

HA 1- Populations of MAB, yeasts, molds, coliforms and E. coli on blueberries

 

 

(Log CFU/g)

Fruit sample MAB Yeast Mold Coliforms E. coli
Pre-harvest 1 4.11 4.23 3.90 <0.69 <0.69
Pro-harvest 2 4.41 4.11 4.25 <0.69 <0.69
Post-harvest 1 4.75 3.59 3.30 1.30 0.69
Post-harvest 2 4.81 3.07 2.95 1.54 1
Blower exit 1 4.65 2.69 4.60 1.39 0.69
Blower exit 2 4.59 4.04 3.77 1.17 0.69
Water tank exit 1 4.27 3.65 3.04 1 <0.69
Water tank exit 2 4.54 3.23 3.49 0.69 <0.69
Pre-packaging 1 4.17 2.74 3.11 <0.69 <0.69
Pre-packaging 2 3.74 2.81 2.90 <0.69 <0.69

 

HA l-Populations of MAB, yeasts, molds, coliforms and E. coli on conveyer belts (Log

CFU/cmz) entering the blower (BA) and pro-packaging areas (PA), before and after fruit

 

 

processing.

Swab samples MAB Yeast Mold Coliforms E. coli
BA before 3.27 3.25 2.55 <-0.52 -0.52
PA before 1.77 1.07 0.47 <-0.52 <-0.52
BA after 3.94 3.74 3.77 2.05 1.25
PA after 2.66 2.50 1.07 1.50 0.90

 

117

HA 2-Populations of MAB, yeasts, molds, coliforms and E. coli in water tank samples
(log CFU/ml) taken before and after fruit washing. '

 

 

Water sample MAB Yeast Mold Coliforms E. coli
Before <1 <1 <1 <0 <0
Aﬁer 2.62 1.60 1.77 0.77 0.30

 

EL 2 ‘Bluecrop’ Type of blueberry.

 

Preharvest sample collected by Hanson and Montecino.

Third picking of fruit behind (west) of pond. Some fruit over-ripe and rot.

EL 2 - Populations of MAB, yeasts, molds, coliforms and E. coli on blueberries

 

 

(Log CFU/g)

Fruit sample MAB Yeast Mold Coliforms E. coli
Pre-harvest 1 2.30 2.92 1.69 <0.69 <0.69
Pre-harvest 2 2 2.87 1.69 <0.69 <0.69
Post-harvest 1 2.60 3.30 2.96 <0.69 <0.69
Post-harvest 2 3.04 3.27 3.33 <0.69 <0.69
Blower exit 1 2.30 3.38 3.38 <0.69 <0.69
Blower exit 2 2.87 3.14 3.14 <0.69 <0.69
Water tank exit 1 3.62 3.66 3.66 <0.69 <0.59
Water tank exit 2 2.83 3.79 3.79 0.69 <0.69
Pro-packaging 1 2.47 3.60 3.60 <0.69 <0.69
Pre-packaging 2 3.74 3.66 3.66 <0.69 <0.69

 

118

EL 2-Populations of MAB, yeasts, molds, coliforms and E. coli on conveyer belts (Log
CFU/cmz) entering the blower (BA) and pre-packaging areas (PA), before and after fruit

processing.

 

Swab samples MAB Yeast Mold Coliforms E. coli

 

BA before 2.75 2.47 1.79 <-0.52 <-0.52
PA before

BA after 2.67 1.91 2.41 <-0.52 -0.52
PA after

 

EL 2-Populations of MAB, yeasts, molds, coliforms and E. coli in water tank samples
(log CFU/ml) taken before and after fruit washing.

 

Water sample MAB Yeast Mold Colifonns E. coli

 

Before <1 <1 <1 <0 <0
After 3.54 3.46 2.23 0.60 0.30

 

119

 

HA 2 ‘Bluecrop’ Type of blueberry.
Pre-harvest sample collected on 4 Aug 03 by Hanson.
Harvest same day-3rd picking. Field placed on CR 380. Had rained 2 inches day before.

Fruits quite ripe. Bushes 15+ years-old.

HA 2- Populations of MAB, yeasts, molds, coliforms and E. coli on blueberries
(Log CFU/g)

 

 

Fruit sample MAB Yeast Mold Coliforms E. coli
Pre-harvest1 3.30 3.47 3 <1.69 <1.69
Pre-hawest 2 3.30 3.69 3.92 <1 .69 <1 .69
Post-harvest 1 5.44 5.17 3.60 3.20 3.04
Post-harvest 2 5.41 5.14 4.53 3.17 3.04
Blower exit 1 5.34 4.87 4.81 3.39 3
Blower exit 2 5.30 4.11 4.83 3.46 2.65
Water tank exit 1 4.88 4.14 3.65 2.69 2.17
Water tank exit 2 4.77 4.04 3 2.74 2.17
Pre-packaging 1 4.76 3.81 3 2.60 2
I’m-packaging 2 5.11 4.34 3.95 2.65 2.54

 

120

HA 2-Populations of MAB, yeasts, molds, coliforms and E. coli on conveyer belts (Log
CFU/cmz) entering the blower (BA) and pre-packaging areas (PA), before and after fruit

processing.

 

Swab samples MAB Yeast Mold Colifonns E. coli

 

BA before 3.17 3.41 <1.47 <0.47 <0.47
PA before 0.77 1.77 <1 .47 <0.47 <0.47
BA after 4.95 4.89 4.23 3.20 2.73
PA after 4.57 3.71 2.47 1.95 1.47

 

HA 2-Populations of MAB, yeasts, molds, coliforms and E. coli in water tank samples
(log CFU/ml) taken before and aﬁer fruit washing.

 

Water sample MAB Yeast Mold Coliforms E. coli

 

Before 2.65 1 <1 <0 0.30
After <2 <2 3.38 <0 <0

 

121

 

KAI ‘Bluecrop’ Type of blueberry.

Pre-harvest sample collected by Hanson.

First picking of Bluecrop ﬁeld at Riley x 152“. Field had overhead irrigation. Bushes 10-
15 years old. Well pruned, heavy crop. Soil dry - no recent rain.

Processing samples collected by Berkheimer, Montecino and Thombush.

KA l- Populations of MAB, yeasts, molds, coliforms and E. coli on blueberries
(Log CFU/g)

 

 

Fruit sample MAB Yeast Mold Colifonns E. coli
Pre-harvest 1 2.39 3.46 2.39 <0.69 <0.69
Pre-harvest 2 2.81 3.64 2.60 0.69 <0.69
Post-harvest 1 4.67 3.77 3 2.65 2.36
Post-harvest 2 4.47 4.11 3.30 2.30 2.17
Blower exit 1 3.77 3.81 3.17 1.25 1.11
Blower exit 2 4.14 4.07 3.47 1 1
Water tank exit 1 3.17 3.65 <2.69 <0.69 <0.69
Water tank exit 2 3.77 3.6 <2.69 <0.69 <0.69
Pre-packaging 1 3.60 3.65 3.17 <0.69 0.90
Pre-packaging 2 3.74 3.87 3 1.25 1.39

 

122

KA l-Populations of MAB, yeasts, molds, coliforms and E. coli on conveyer belts (Log
CFU/cmz) entering the blower (BA) and pre-packaging areas (PA), before and aﬁer fruit

processing.

 

Swab samples MAB Yeast Mold Coliforms E. coli

 

BA before 3.92 3.90 2.65 1.55 1.14
PA before 3.04 2.30 0.95 <-0.52 <-0.52
BA after 4.95 3.90 2.62 2.46 1.17
PA after 2.55 1.87 1.17 <0.47 <0.47

 

KA l-Populations of MAB, yeasts, molds, coliforms and E. coli in water tank samples
(log CPU/ml) taken before and after ﬁ'uit washing.

 

Water sample MAB Yeast Mold Coliforms E. coli

 

Before 1.47 <1 1.47 <0 <0
After <2 <2 <2 <1 <1

 

123

TH 1

Pre-harvest samples from this ﬁeld collected a few days earlier by Siva and luliano.
Processing samples taken on 10 Aug 03at Northern Pride facility in Hartford, by
Hanson and Berkheimer. Ran ﬁrst thing in morning. Berries delivered previous night,
left outside over-night. Sampled 2 pallets of fruit over a 10 min period.

Line set up with conveyor to single large blower, into water ﬁlled ﬂume to water tank
(all chlorinated), water tank quite large and retained fruit for unknown length of time on

bottom, Conveyed to color sorter, over de-stemmer, and to boxer.

TH 1- Populations of MAB, yeasts, molds, coliforms and E. coli on blueberries
(Log CFU/g)

 

 

Fruit sample MAB Yeast Mold Colifonns E. coli
Pre-harvest 1 3.99 4.34 3.23 0.69 0.69
Pre-harvest 2 3.99 4.34 3.28 0.69 0.69
Post-harvest 1 5.41 3.69 4.65 2.25 2.36
Post-harvest 2 5.50 5.20 4.39 2.47 2
Blower exit 1 6.04 4.65 5.04 3.25 3.20
Blower exit 2 5.75 4.30 4.54 3.04 3
Water tank exit 1 5.71 4 4.30 1.90 0.69
Water tank exit 2 5.59 3.36 4.59 1.65 <0.69
Pre-packaging 1 5.20 3.63 4.04 2.17 1.77
Pro-packaging 2 5.20 3.47 4.25 1.87 1.69

 

124

 

TH l-Populations of MAB, yeasts, molds, coliforms and E. coli on conveyer belts (Log
CFU/cmz) entering the blower (BA) and pre-packaging areas (PA), before and after fruit

processing.

 

Swab samples MAB Yeast Mold Coliforms E. coli

 

BA before 2.07 2.23 1.32 -0.52 -0.52
PA before 1.23 0.47 <0.47 -0.30 <.0.52
BA after 4.67 3.61 4.81 2.47 2.14
PA after 3.67 1.65 3.25 1.23 1.17

 

TH l-Populations of MAB, yeasts, molds, coliforms and E. coli in water tank samples
(log CPU/m1) taken before and after fruit washing.

 

Water sample MAB Yeast Mold Coliforms E. coli

 

Before 1 <1 <1 <0 <0
After 2 <2 2,17 <1 <1

 

125

KA 2 ‘Bluecrop’ Type of blueberry.

Pre-harvest sample collected on 11 Aug 03 by Hanson. First picking of 4-6 year-old
Bluecrop bushes on Brewer farm west of 148‘", just north of Kamphuis processing
facility. Fruit very nice, average crop load. Little rot.

Processing samples taken on 12 Aug by Berkheimer and Montecino (Denny Brewer).

Cloudy and warm. Run took 45 minutes.

Kamphuis 2- Populations of MAB, yeasts, molds, coliforms and E. coli on blueberries
(Log CFU/ g)

 

 

Fruit sample MAB Yeast Mold Colifonns E. coli
Pre-harvest 1 3.11 4.04 2.44 <0.69 <0.69
Pre-harvest 2 3.74 4.44 2.69 <0.69 1.17
Post-harvest 1 5.30 4.61 3 2.44 1.11
Post-harvest 2 5.61 4.53 2.39 2.59 1
Blower exit 1 5.14 4.44 3.11 2.07 1.17
Blower exit 2 5.17 4.46 3.30 1.94 0.90
Water tank exit 1 3.68 4.07 2.39 1.96 0.69
Water tank exit 2 4.30 3.90 3.86 2.64 <0.69
Pre-packaging 1 4.17 3.72 <2.69 0.69 <0.69
Pre-packaging 2 3.83 3.77 2.87 1 <0.69

 

126

KA 2-Populations of MAB, yeasts, molds, coliforms and E. coli on conveyer belts (Log
CFU/cmz) entering the blower (BA) and pre-packaging areas (PA), before and after fruit

processing.

 

Swab samples MAB Yeast Mold Colifonns E. coli

 

BA before 3.90 3.74 3.30 2.69 0.47
PA before 1.64 1.43 1.65 0.30 <-0.52
BA after 4.55 4.43 3.43 2.41 0.47
PA after 3.25 1.95 1.17 0.90 <0.47

 

KA 2-Populations of MAB, yeasts, molds, coliforms and E. coli in water tank samples
(log CFU/ml) taken before and after fruit washing.

 

Water sample MAB Yeast Mold Coliforms E. coli

 

Before <1 <1 <1 <0 <0
After <2 <2 2 <1 <1

 

127

HA 3 ‘Jersey’ Type of blueberry.

Pre-harvest sample taken on 19 Aug 03 by Berkheimer and Montecino.

Harvest same day. First picking (CPPU trial site). Fruit over-ripe with considerable rot.
30-50 year-old bushes. Weather conditions: sunny and hot. Processing samples taken on
20 Aug.

HA 3- Populations of MAB, yeasts, molds, coliforms and E. coli on blueberries
(Log CFU/g)

 

 

Fruit sample MAB Yeast Mold Coliforms E. coli
Pre-harvest 1 3.63 <2.69 4.79 <0.69 <0.69
Pre-hawest 2 3.94 3.11 4.79 0.69 <0.69
Post-harvest 1 5.87 4.60 5.43 3.99 3.23
Post-harvest 2 5.85 4.72 5.57 2.51 1.30
Blower exit 1 5.36 4 4.89 1.81 0.69
Blower exit 2 5.43 4.25 4.72 2.55 1
Water tank exit 1 5 3.69 4.57 <0.69 0.69
Water tank exit 2 4.63 3.69 4.86 <0.69 <0.69
Pre-packaging 1 5.34 4.74 4.36 1.25 <0.69
Pre-packaging 2 5.27 4.17 4.69 1.68 <0.69

 

128

 

HA 3-Populations of MAB, yeasts, molds, coliforms and E. coli on conveyer belts (Log
CFU/cmz) entering the blower (BA) and pre-packaging areas (PA), before and after fruit

processing.

 

Swab samples MAB Yeast Mold Coliforrns E. coli

 

BA before 3.92 3.79 3.25 0.30 <-0.52
PA before 1.92 2.49 0.47 <-0.52 <-0.52
BA after 4.61 4.60 4.11 2.25 1.36

PA after 3.50 3.38 3.72 1.90 <-0.52

 

HA 3-Populations of MAB, yeasts, molds, coliforms and E. coli in water tank samples
(log CFU/ml) taken before and after fruit washing.

 

Water sample MAB Yeast Mold Coliforms E. coli

 

Before 1 <1 <1 <0 <0
After 2 2 3.77 <1 <1

 

129

KL 1 ‘Elliott’ Type of blueberry.

Pre-harvest sample collected on 23 Aug 03 by Berkheimer and Montecino.

Weather conditions: dry day, little soil moisture, sunny and clear, ~80F.

Harvest with smaller harvester. First picking of mu.

Processing samples collected on 24 Aug by Hanson and Montecino, Hartmann processing
facility. Fruit delivered previous evening, held in shed over night. Ran very fast ﬁrst

thing in morning.

KL 1- Populations of MAB, yeasts, molds, coliforms and E. coli on blueberries

(Log CFU/g)

 

 

Fruit sample MAB Yeast Mold Coliforms E. coli
Pre-harvest 1 2.94 3.46 2.39 <0.69 <0.69
Pre-harvest 2 3.04 3.41 2.11 <0.69 <0.69
Post-harvest 1 4.49 3.54 4.11 <1 .69 <1 .69
Post-harvest 2 4.74 3.47 2.87 <1.69 <1.69
Blower exit 1 4.11 3.51 2.69 1.69 <1.59
Blower exit 2 4.39 4.04 <2.69 <1 .69 <1 .69
Water tank exit 1 2.69 3.57 <2.69 <1 .69 <1 .69
Water tank exit 2 2.69 3.63 <2.69 <1 .69 <1 .69
Pre-packaging 1 <2.69 3.81 2.69 <1 .69 <1 .69
Pre-packaging 2 <2.69 3.84 <2.69 <1.69 <1.69

 

130

KL l-Populations of MAB, yeasts, molds, coliforms and E. coli on conveyer belts (Log

CF U/cmz) entering the blower (BA) and pro-packaging areas (PA), before and after fruit

 

 

processing.

Swab samples MAB Yeast Mold Coliforms E. coli
BA before <1 .47 <1 .47 <1 .47 <-0.52 <-0.52
PA before <1 .47 <1 .47 <1 .47 <-0.52 <-0.52
BA after 3.53 3.62 2.89 0.77 <0.47

PA after 2.36 2.77 1.47 <0.47 <0.47

 

HA 3-Populations of MAB, yeasts, molds, coliforms and E. coli in water tank
samples (log CFU/ml) taken before and after fruit washing.

 

 

Water sample MAB Yeast Mold Coliforms E. coli
Before <1 <1 <1 <0 <0
After <2 <2 <2 <1 <1

 

131

HA 4 ‘Rubel’ and ‘Bluecrop’ Type of blueberry.

Pre-harvest sample collected on 24 Aug 03by Hanson and Montecino. Third picking of
mixed ﬁeld (very little left), 8th St west (N of Hartmann’s), cross RR tracks, ﬁrst 2-track
on the left. Bushes variable in size, 15-30 years-old.

Processing samples collected on 25 Aug by Hanson. Question about whether initial ﬁll
and sample from water tank was chlorinated. Run at 1:00 and took 20 min. Fruit sat in

shed overnight and through morning.

 

HA 4- Populations of MAB, yeasts, molds, coliforms and E. coli on blueberries
(Log CF U/g)

 

 

Fruit sample MAB Yeast Mold Coliforms E. coli
Pre-harvest 1 <2.69 4.17 3.30 <0.69 <0.69
Pre-harvest 2 <2.69 3.57 4.47 <0.69 <0.69
Post-harvest 1 5.43 5.07 4.76 3.79 2.17

Post-harvest 2 5.61 5.07 4.68 3.62 <1 .69
Blower exit 1 5.56 5.14 4.99 3.53 <1 .69
Blower exit 2 5.53 4.97 4.84 3.50 <1 .59
Water tank exit 1 4.59 3.92 4.23 3.55 <1 .69
Water tank exit 2 4.64 3.53 4.41 3.51 <1 .69
Pre-packaging 1 4.34 3.47 4.47 2.57 <1 .69
Pre-packaging 2 4.30 3.63 4.14 3.27 <1 .69

 

132

HA 4-Populations of MAB, yeasts, molds, coliforms and E. coli on conveyer belts (Log
CFU/cmz) entering the blower (BA) and pre-packaging areas (PA), before and aﬁer fruit

processing.

 

 

Swab samples MAB Yeast Mold Coliforms E. coli

 

BA before 2.96 1.85 1.25 1.23 <-0.52
PA before 0.95 0.69 <0.47 <-0.52 <-0.52 F
BA after 4.34 4.04 4.20 2.30 <0.47 2
PA after 3.46 3.72 2.77 2.17 <0.47

 

 

HA 4-Populations of MAB, yeasts, molds, coliforms and E. coli in water tank samples
(log CFU/ml) taken before and after ﬁ'uit washing.

 

Water sample MAB Yeast Mold Coliforms E. coli

 

Before <1 <1 <1 <0 <0
After 3.38 2.17 2 <1 <1

 

133

 

KA 3 First picking of older Burlington bushes.

Pre-harvest sample takenon Aug 27 by Hanson. Off North Holland Road, N of 160th.
Overhead irrigated, nice quality, average crop load.

Processing samples collected on 28 Aug by Hanson. Fruit sat in shed overnight at 65F.
Sampled over 25 min run. Line set up with blower, de-stemmer, water bath, rinse (3-5

ppm chlorine) blower-drier, color sorter, hand sorting belt, and boxer.

KA 3- Populations of MAB, yeasts, molds, coliforms and E. coli on blueberries
(Log CFU/g)

 

 

 

Fruit sample MAB Yeast Mold Coliforms E. coli
Pre-harvest 1 4.44 3.36 4.82 2.44 <1 .69
Pro-harvest 2 4.39 3.25 4.69 2.17 <1 .69
Post-harvest 1 5.53 4.77 5.20 2.96 1.69
Post-harvest 2 5.57 4.57 5.34 3.07 <1 .69
Blower exit 1 5.69 5.07 5.50 2.94 1.69
Blower exit 2 5.60 4.99 5.38 3.14 <1 .59
Water tank exit 1 5.77 4.87 4.89 3 1.69
Water tank exit 2 5.68 4.81 4.11 3.04 <1.69
Pre-packaging 1 5.23 4.63 4.57 2.74 2.09
Pre-packaging 2 5.55 4.89 4.96 2.89 <1 .69

 

134

KA 3-Populations of MAB, yeasts, molds, coliforms and E. coli on conveyer belts (Log
CFU/cmz) entering the blower (BA) and pre-packaging areas (PA), before and after fruit

processing.

 

Swab samples MAB Yeast Mold Coliforms E. coli

 

BA before 1.81 2.59 3.04 <-0.52 <-0.52
PA before 0.30 0.69 1.07 <-0.52 <-0.52
BA after 4.36 4.44 4.47 2.04 0.47

PA after 2.50 2.89 3.14 1.23 <0.47

 

KA 3-Populations of MAB, yeasts, molds, coliforms and E. coli in water tank samples
(log CFU/ml) taken before and after fruit washing.

 

Water sample MAB Yeast Mold Colifonns E. coli

 

Before <1 <1 1 <0 <0
After <1 1 1 .30 <0 <0

 

135

 

 

 

BO 1 ‘Jersey’ Type of blueberry.

Pre-harvest samples collected on 4 September by Hanson and Montecino. First thing in
morning From ﬁeld off Tyler west of U831. 2nd pick from old ﬁeld, light yield, nice
quality.

Weather conditions: dry, 65F and cloudy.

Processing samples run same morning at West Central Processing Facility. About 25 lugs
of 30 lb each, run in 15 min. Slowed down to accommodate us. Crew had cleaned the line
well before run, emptied and re-ﬁlled water tank. Tank maintained at 2 ppm with chlorine
dioxide generator. Setup blower, water tank, clean water rinse, de-stemmer, color sorter,

and then boxer.

BO 1- Populations of MAB, yeasts, molds, coliforms and E. coli on blueberries
(Log CF U/g)

 

 

Fruit sample MAB Yeast Mold Coliforms E. coli
Pre-harvest 1 3.83 4.39 4.97 2.57 <1 .69
Pre-harvest 2 3.18 4.34 4.26 2.11 <1 .69
Post-harvest 1 4.59 4.1 1 4.45 2.36 <1 .69
Post-harvest 2 4.30 4.26 4.23 2.30 <1 .69
Blower exit 1 4.55 4.18 4.32 2.58 <1 .69
Blower exit 2 4.49 4.28 4.34 2.63 <1 .69
Water tank exit 1 3.25 3.89 4.11 <1.69 <1.69
Water tank exit 2 3.94 3.94 3.68 <1 .69 <1 .69
Pre-packaging1 3.60 3.76 4.49 1.87 <1.69
Pre-packaging 2 3.60 3.65 4.28 1.87 <1.69

 

136

 

 

BO l-Populations of MAB, yeasts, molds, coliforms and E. coli on conveyer belts (Log
CFU/cmz) entering the blower (BA) and pre-packaging areas (PA), before and after fruit

processing.

 

Swab samples MAB Yeast Mold Coliforms E. coli

 

BA before 3.41 3.77 3.39 1.25 -0.52
PA before 2.97 2.27 1.91 0 <-0.52 "
BA aﬁer 4.14 3.74 4.11 1.47 0.47 E
PA after 2.77 2.84 2.79 0.95 <0.47

 

 

BO l-Populations of MAB, yeasts, molds, coliforms and E. coli in water tank samples
(log CFU/ml) taken before and after fruit washing.

 

Water sample MAB Yeast Mold Coliforms E. coli

 

 

Before 1.92 1.30 1.54 0.60 <0
After 1.47 1.77 2.02 <0 <0
137

APPENDIX E
EFFICACY COMPARISON OF CHLORINE DIOXIDE GAS SACHETS FOR
THE MICROBIOLOGICAL QUALITY ON SURFACE-INNOCULATED AND —

UNINOCULATED BLUEBERRIES

OBJECTIVE

The objective of this work study was to better characterize and compare the
efﬁcacy of chlorine dioxide gas generated by a commercial sachet in reducing the
populations of MAB, coliforms, E. coli, yeasts and molds on inoculated and uninoculated
blueberries. The sanitizer was tested at concentrations and exposure times similar to pilot

study (Chapter 4) that are appropriate for blueberries processors.

MATERIAL AND METHODS
Blueberries

During the 2005 harvest season, blueberry samples (500 g) were collected in 1-
pint plastic clamshell containers from a ﬁeld in Grand Junction, MI, placed in individual
plastic bags, stored on ice and analyzed within 4 h of collection.
Bacteria, yeasts and molds

For the inoculated fruit study, MAB, yeasts, molds, coliforms and E. coli strains
were isolated from these same blueberry ﬁeld fruits. Fruit samples (25 g) were placed in
sterile 20 x 10 cm polyethylene bags (Whirl-PackTM) containing 100 ml of neutralizing
buffer (Difco),shaken, pulsiﬁed, diluted, plated on TSAYE-C, E. coli / coliform count

plates and incubated as previously described for the isolation of MAB, E. coli and

138

coliforms, respectively. Five isolates each of MAB, E. coli and coliforms were
transferred from plates into 9 ml of TSBYE (Difco) followed by 18-24 h of incubation at
35°C and then subjected to an identical transfer in 30 ml of TSBYE before use. Five
representative yeasts and molds were isolated on PDA-SA from the same fruit 3 weeks
before using previously described procedure. Individual strains of yeasts and molds were
separately activated by transferring a loop culture from isolation plates onto culture PDA-
SA plates, surface-plated, and grown for 3 - 4 and 10 - 12 days at 26°C, respectively,
before use.

For the uninoculated fruit study, MAB, yeast, molds, coliforms and E. coli were
obtained by fruit storage in ventilated buckets at 22°C and 99.9 RH for 96 h. Fruit sample
(25g) were taken initially, at 24, 48, and 96 h and monitored for the microbial growth of

MAB, yeast, molds, coliforms and E. coli using similar procedure as described above.

Preparation of the inoculum

The ﬁve culture each of MAB, E. coli and coliforms were harvested by
centrifugation (Sorvall Super T21) at 7,000 rpm for 10 min at 4°C and re-suspended in
equal volumes (30ml) of sterile PBS. The ﬁve culture each of yeast and mold were
harvested by washing the previous yeast and molds culture plates with 30 ml of sterile
PBS. Suspensions (~3 0ml) of each bacterial strain containing approximately equal
populations (109CFU/ml) and each yeast and mold strain containing approximately equal
populations (108 CFU/ml) were combined to generate a single inoculum cocktail (~ 750

ml). Populations of MAB, yeast and molds, and E. coli, coliforms from inoculum cocktail

139

 

 

were determined by plating appropriate serial dilutions in PBS on TSAYE-C, PDA-SA,

and E. coli / coliform count plates, respectively.

Inoculation of blueberries
Blueberries (500g) were inoculated by immersion in a 25 x 20 cm sterile polyethylene

bag (Whirl-PackTM) containing ~750 ml of inoculum cocktail and gently swirl-agitated at
100 rpm for 20 min on a G2 Gyratory Shaker platform (New Brunswick Scientiﬁc Co.).
Inoculated fruit samples were then air-dried under laminar ﬂow in a biosafety cabinet for

2 h, stored overnight at 4°C and ﬁnally re-dried under laminar ﬂow for 2 h before use.

Sanitizer Exposure

Inoculated and uninoculated fruit samples (500 g) were placed in separate l-pint plastic
clam shell containers that were then placed in sealed 20 L buckets and were either gassed
(4 mg/L, 0.16mg/g fruit) or un-gassed (control) for 12 h at ~ 22°C/99.9 RH. Chlorine
dioxide gas was generated in the sealed bucket using a 20-g ClOz sachet (ICA TriNova).
After 12 h fruit samples (25g) were placed in sterile 20 x 10 cm polyethylene bags
(Whirl-PackTM) containing 100 ml of neutralizing buffer (Difco) and assessed for

popuations of MAB, yeasts, molds, E. coli and coliforms as previously described

Statistical analysis

Analysis of Variance (ANOVA) was done on all microbial count data obtained from
microbial growth, inoculated and un-inoculated fruit studies using the Statistical Analysis
System (Proc Anova, SAS, Version 8, SASO Institute Inc., Cary, NC). Data in the graphs

and tables (Appendix B) are means from replicates and signiﬁcance between means were

140

 

determined using least signiﬁcant difference (LSD) test at the 95% conﬁdence level
(P=0.05).

RESULTS AND DISCUSSIONS

Initial populations of MAB, yeasts, molds, coliforms, and E. coli on naturally
contaminated berries were 4.72, 4.83, 5.41, 0.85, and 0.69 logs CFU/g, respectively
(Appendix B, Table E.1). After 96 h of storage at 21i1°C/ 99% RH in a sealed and
ventilated bucket, signiﬁcant (P < 0.05) microbial grth was observed on the
blueberries with populations of MAB, yeast, mold, coliforms, and E. coli increasing 1.47,

1.36, 0.72, 2.73, and 2.62 logs, respectively.

1
1
1
1
1
1
I
1

    

Yeast Mold Coliform

7.011 1

g5 g; E96h 1
1 — 4 /E

v g; 1

1 E 3 Z: 1

E /E 1

1 '9 2 /§ 1
3 8 %E

1 51 g; 1

1 o ,_ 2g 1

 

Figure B 1. Growth (log CFU/g) of MAB, yeasts, molds, coliforms, and E. coli (mean :t
std. dev., n=3) on naturally contaminated blueberries during storage at 21i1°C/99 % RH.
Values with different letters within the same microbial category are signiﬁcantly different
(P < 0.05).

Populations of MAB, yeasts, molds, coliforms, and E. coli on inoculated blueberries

decreased 3.71, 2.78, 2.52, 3.39, and 3.44 logs CFU/g, respectively, after a 12-h exposure

141

to 4 mg/L (0.16 mg/g) C102 gas in a sealed bucket at 22°C 99.9% RH (Appendix B,
Table E.2). Signiﬁcantly (P < 0.05) greater microbial survival was seen after similarly
exposing naturally contaminated blueberries to chlorine dioxide. Numbers of MAB,
yeasts, molds, and E. coli decreased 1.05, 0.52, 0.65, and 0.26 logs CFU/g, respectively;
whereas the 0.05 log CFU/g reduction for coliforms was not signiﬁcant (P>0.05) lower.
Surface-inoculated microorganisms were likely attached to the waxy blueberry surface
and thus more susceptible to sanitizers than naturally occurring microbial contaminants
that were more likely embedded in the hydrophobic wax layer and thereby protected from

the action of chlorine dioxide (Freeman et al., 1979).

 

Figure E 2. Reduction (log CFU/g) of MAB, yeasts, molds, coliforms, and E. coli
(mean :t std.dev., n=4) on inoculated and uninoculated blueberries after a 12-h exposure
to 4 mg/L (0.16 mg/g) C102 gas in a sealed 20 L bucket 22°C/ 99.9% RH. Values with

different letters within the same microbial category are signiﬁcantly different (P < 0.05).

Higher efﬁcacy of the sanitizer on inoculated as opposed to uninoculated blueberries is

also likely due to less internalization of microorganisms on the surface-inoculated fruit

142

 

which results in greater exposure to chlorine dioxide gas as opposed to naturally
contaminated fruit with microorganisms embedded in the wax layer. Yeasts and molds
naturally present on blueberries were less susceptible to chlorine dioxide gas than
bacteria with these ﬁndings in agreement with those of Rodgers et al. (2004) and Sy et al.
(2005)

Work with inoculated vs. uninoculated blueberries revealed signiﬁcantly (P < 0.05) lower
microbial reductions after similar exposure of naturally contaminated blueberries
(uninoculated) to chlorine dioxide for MAB, yeasts, molds, and E. coli as well as
signiﬁcant (P > 0.05) differences for coliforms. Surface inoculation of the berries likely
led to microorganisms that were attached to the blueberry wax. These surface-inoculated
organisms were likely more exposed to chlorine dioxide gas than those on naturally
contaminated fruit which would tend to be embedded in the hydrophobic wax structure

and thus more protected ﬁ'om chlorine dioxide (Freeman et al., 1979).

I43

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