Investigation on the Effect of Packing Material, In-package Gas Composition, and Sanitizer on
the Safety and Quality of Fresh-cut Produce
By
Natalie Page

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Packaging - Master of Science
2014

ABSTRACT
Investigation on the Effect of Packaging Material, In-package Gas Composition, and Sanitizer on
the Safety and Quality of Fresh-cut Produce
By
Natalie Page
Due to safety concerns and increasing demand for high quality fresh-cut produce, there is a
communal need to better understand the implications of sanitizing and packaging on fresh-cut
produce. The effect of bio-based packaging and non-conventional atmospheres on the quality
and safety of chlorine-sanitized celery sticks (CS) inoculated with Listeria monocytogenes and
stored at 7 ºC for 21 days was investigated. New quantitative descriptive sensory analysis
methods were developed to evaluate the color and texture of CS. Additionally, the effect of inpackage atmospheres and sanitizers and their interactions on the growth of Salmonella
Typhimurium, mesophilic bacteria, and yeasts and molds as well as on the physico-chemical and
sensorial quality of diced onions (DO) was investigated throughout 14 days of storage at 7 °C.
The results show that that the initial gas composition and packaging material both impact the
quality and safety of CS. Overall, the combination PLA and 95 kPa O2 proved most beneficial in
maximizing both the safety and quality of CS during one week of storage at 7 ºC. The new
texture method involves bending CS until they break and comparing the breakage angle with a
reference scale and the new color method involves comparing CS to a Pantone® scale. 2-way
interactions between atmosphere and sanitizer that affected S. Typhimurium and pH in DO were
identified. 3-way interactions (atmosphere, sanitizer, and time) were only observed in the case of
headspace CO2. Elevated CO2/reduced O2 and sodium hypochlorite was the best in-package
atmosphere and sanitizer combination for enhancing the safety and quality of packaged DO.

I would like to dedicate my thesis to my parents, Larry and Angela Page.

iii

ACKNOWLEDGEMENTS

I would like to express my sincerest gratitude to my advisor, Dr. Eva Almenar for her
guidance, patience, unwavering support, and immeasurable time throughout my degree. I would
also like to thank my committee members, Dr. Elliot Ryser, Dr. Janice Harte, and Dr. Susan
Selke for their valuable insight, advice, and time throughout this process. I am also extremely
thankful to Dr. Jaime González-Buesa for teaching me so much and his continuous help
throughout my research. I would like to convey gratitude to my fellow graduate and
undergraduate students that made this research possible through their knowledge, time, and
onion peeling skills. I would like to show my appreciation for the Michigan State University
Food Safety Group that awarded me a graduate assistantship, helping to fund my continued
education.

iv

TABLE OF CONTENTS

LIST OF TABLES......................................................................................................................viii
LIST OF FIGURES........................................................................................................................ix
1. Introduction……..........................................................................................................................1
2. Literature Review…….................................................................................................................7
2.1 Means to reduce spoilage and pathogenic microorganisms in processed produce........7
2.1.1. Effect of gas compositions and sanitizers on spoilage microorganisms........8
2.1.2. Effect of gas compositions and sanitizers on pathogenic microorganisms..11
2.1.3. Non-traditional means to reduce microbial populations on minimally
processed produce..................................................................................................13
3. Effect of Non-conventional Atmospheres and Bio-based Packaging on the Quality and Safety
of Listeria monocytogenes-inoculated Fresh-cut Celery (Apium graveolens L.) During
Storage...........................................................................................................................................15
3.1. Materials and methods................................................................................................15
3.1.1. Celery...........................................................................................................15
3.1.2. Packaging.....................................................................................................15
3.1.2.1. Packaging and storage..................................................................16
3.1.2.2. Packaging material characterization.............................................16
3.1.3. Atmosphere composition.............................................................................17
3.1.4. Texture.........................................................................................................17
3.1.5. Color............................................................................................................18
3.1.6. Weight Loss.................................................................................................18
3.1.7. Preparation of inoculum, inoculation of samples, and microbiological
analysis...................................................................................................................18
3.1.8. Statistical analysis........................................................................................20
3.2 Results and discussion.................................................................................................20
3.2.1. Packaging material characterization............................................................20
3.2.2. Atmosphere composition.............................................................................21
3.2.3. Weight loss...................................................................................................24
3.2.4. Color............................................................................................................25
3.2.5. Texture.........................................................................................................30
3.2.6. Listeria monocytogenes...............................................................................32
4. Development of Novel Descriptive Sensory Methods to Evaluate the Quality of Fresh-cut
Celery.............................................................................................................................................36
4.1. Materials and Methods................................................................................................36
4.1.1. Sample preparation......................................................................................36
4.1.2. Panelist Selection.........................................................................................37
4.1.3. Development of novel descriptive sensory methods and training of
panelists..................................................................................................................37
v

4.1.3.1. Standardized color scale...............................................................37
4.1.3.2. Flexibility texture method.............................................................38
4.1.3.3. Hardness sensory method.............................................................39
4.1.4. Statistical analysis........................................................................................39
4.2. Results and Discussion...............................................................................................40
4.2.1. Texture.........................................................................................................40
4.2.2. Color............................................................................................................41
5. Interactions Between Sanitizers and Packaging Gas Compositions and their Effects on the
Growth of Spoilage Microorganisms and Salmonella on Fresh-cut Onions (Allium cepa
L.)…………...................................................................................................................................42
5.1. Materials and methods................................................................................................42
5.1.1. Onion dicing.................................................................................................42
5.1.2. Preparation of inoculum and inoculation of the diced onions.....................42
5.1.3. Sanitation of the diced onions......................................................................42
5.1.4. Packaging of the diced onions.....................................................................43
5.1.4.1. Packaging system characterization...............................................44
5.1.5. Analyses.......................................................................................................45
5.1.5.1. Microbiological analyses..............................................................45
5.1.5.2. Physico-chemical analyses............................................................46
5.1.5.2.1. In-package atmosphere..................................................46
5.1.5.2.2. Color..............................................................................46
5.1.5.2.3. Weight Loss...................................................................47
5.1.5.2.4. pH...................................................................................47
5.1.5.3. Sensory Analyses..........................................................................47
5.1.5.3.1. Descriptive sensory analysis..........................................47
5.1.5.3.2. Consumer acceptance sensory analysis.........................49
5.1.5.4. Statistical analysis.........................................................................49
5.1.6. Treatment labels...........................................................................................50
5.2. Results and discussion................................................................................................51
5.2.1. Packaging system characterization..............................................................51
5.2.2. Effect of atmosphere/sanitizer combinations on microbial growth in diced
onions.....................................................................................................................52
5.2.2.1. Salmonella Typhimurium.............................................................52
5.2.2.2. Mesophilic bacteria.......................................................................56
5.2.2.3. Yeasts and molds..........................................................................57
5.2.3. Effect of atmosphere/sanitizer combinations on physico-chemical changes
in diced onions.......................................................................................................58
5.2.3.1. In-package atmosphere.................................................................58
5.2.3.2. Color.............................................................................................63
5.2.3.3. Weight loss....................................................................................66
5.2.3.4. pH..................................................................................................66
5.2.4. Effect of atmosphere/sanitizer combinations on sensory attributes of diced
onions.....................................................................................................................67
5.2.4.1. Descriptive sensory analysis.........................................................68
5.2.4.2. Consumer acceptance sensory analysis........................................69
vi

CONCLUSIONS...........................................................................................................................70
FUTURE WORK..........................................................................................................................73
BIBLIOGRAPHY..........................................................................................................................75

vii

LIST OF TABLES

Table 1. Outbreaks and illnesses in the world associated with onions from 2004 to 2012...........4
Table 2. Carbon dioxide, oxygen, water and ethanol permeability coefficients for PLA and
PP/PE films....................................................................................................................................21
Table 3. Weight loss (%) and ethanol content (µL L-1) for fresh-cut celery in packages differing
in initial gas composition and packaging material during storage at 7 ºC.....................................25
Table 4. Lightness (L*) and hue (hº) values for surface and cut ends of the celery sticks packaged
in different materials and initial gas compositions during storage at 7 ºC...................................27
Table 5. Quantitative descriptive sensory analysis of the texture and color of fresh-cut celery
stored at 5°C for 21 days................................................................................................................41
Table 6. Packaging and sanitizer abbreviations that comprise the codes used in this paper.........51
Table 7. Transmission rates (TR) of gases and vapors in the two packaging systems used in this
study...............................................................................................................................................52
Table 8. P values resulting from Univariate ANOVA (SPSS) (P < 0.05 indicates effect of factor
(single or combined (interaction)) on each parameter)..................................................................55
Table 9. Effect of atmosphere/sanitizer combinations on the weight loss, in-package ethanol, and
pH of packaged diced onions during 14 days of storage at 7 oC...................................................61
Table 10. Mean sensory scores for freshly packaged diced onions (I) and diced onions packaged
in Cl CO2 (II) for 14 days at 7 °C..................................................................................................68
Table 11. Mean sensory scores for freshly packaged diced onions (I) and diced onions packaged
in Cl CO2 (II) for 14 days at 7 °C..................................................................................................69

viii

LIST OF FIGURES

Figure 1. Oxygen and carbon dioxide contents of celery stick packages for different materials
and initial gas compositions stored at 7 oC....................................................................................23
Figure 2. Celery stick pitting from O2-PLA bags after 21 days of storage at 7 oC.......................28
Figure 3. Effect of initial gas composition and packaging material on the color (a*, b* and hue)
evolution of the cut surfaces of celery sticks. Numbers (0, 3, 7, 14 and 21) indicate storage time
in days at 7 oC................................................................................................................................30
Figure 4. Firmness (a) and toughness (b) of celery sticks packaged in different materials and
initial gas compositions during storage at 7 oC..............................................................................32
Figure 5. Listeria monocytogenes populations on sanitized and unsanitized celery sticks
packaged in different materials and initial gas compositions during storage at 7o C ((a) compares
initial headspace gas compositions and (b) compares materials as well as sanitation).................34
Figure 6. Pantone® celery color scale............................................................................................38
Figure 7. a) Bending Scale used as a reference to compare with the ink impressions created from
bending celery sticks (1-very crisp to 9-very flexible) and b) angles (Ѳ1 and Ѳ2) of the ink
impressions created from bending celery sticks............................................................................39
Figure 8. Effect of sanitizer/atmosphere combinations on the growth of Salmonella
Typhimurium, Mesophilic bacteria, and yeasts and molds on diced onions during 14 days of
storage at 7 oC................................................................................................................................53
Figure 9. Effect of atmosphere/sanitizer combinations on the growth of Salmonella
Typhimurium, Mesophilic bacteria, and yeasts and molds on diced onions during 14 days of
storage at 7 oC................................................................................................................................56
Figure 10. Effect of atmosphere/sanitizer combinations on in-package gas composition of diced
onions during 14 days of storage at 7 oC.......................................................................................59
Figure 11. Effect of atmosphere/sanitizer combinations on the L* and b* values of diced onions
during 14 days of storage at 7 oC...................................................................................................64

ix

1. Introduction
Consumer demand for minimally processed fruits and vegetables has increased
dramatically over the last ten years due to convenience and consumers increased interest in
healthy foods (United Fresh Foundation, 2010). Onions (Allium cepa L.) are one of the most
widely utilized vegetables in the world, with an annual per capita consumption of nearly 6 kg
(The National Onion Association, 2011), and celery (Apium graveolens L.) is one of the most
desired fresh-cut products due to its taste, texture and low caloric content (United Fresh
Foundation, 2010).
As ready-to-use (RTU) produce items, celery and onions have short shelf lives damage
and stress during processing can lead to changes in color (Viña and Chaves, 2003; Gómez and
Artés, 2005; Viña et al., 2007; Rizzo and Muratore, 2009; Block et al., 1997; Howard et al.,
1994; Selman, 1993; Zaulia et al., 2013), pithiness development in celery (Saltveit and
Mangrich, 1996; Gómez and Artés, 2005), and loss of turgor (Prakash et al., 2000; Viña and
Chaves, 2003; Viña et al., 2007). Moreover, spoilage microorganisms rapidly proliferate due to
the easy accessibility to nutrients resulting from processing. In addition to spoilage
microorganisms, pathogenic microorganisms are a growing concern of the minimally processed
produce industry since they can also be transferred during cutting, washing, or drying stages.
As another analytical tool for assessing fresh produce quality, quantitative descriptive
sensory analysis (QDA) allows scientists to identify key sensory attributes of a product (Stone et
al., 1974). Evaluations using QDA are based on several types of scales. An anchored 15-point
scale method was developed by Poste et al. (1991) for sensory analysis of foods and adapted for
celery attributes, which include color, off-aroma, hardness, celery flavor, and off-flavor (Prakash
et al., 2000). A nine-point scale technique was developed by Kader et al. (1973) to assess quality
attributes of harvested lettuce. This scale has been adapted for analysis of celery sensory
1

attributes, which include aroma, flavor, texture, color, visual structural integrity and general
appearance (Gómez and Artés, 2004; Zhang et al., 2004). A nine-point scale for the assessment
of celery pithiness has also been developed (Saltveit and Mangrich, 1996).
Utilizing the Munsell color system is common in QDA (Meilgaard et al., 1999) to
identify the color of fresh produce on a given scale. The PANTONE® Company (Carlstadt, New
Jersey) has standardized colors for consistent use by applying the Munsell color theory.
PANTONE® is internationally recognized as a standard language for communicating colors in a
variety of industries (PANTONE, 2013). PANTONE® colors can be used to effectively assess
the color changes that occur during the shelf-life of various fresh-cut fruits and vegetables, but
have not yet been applied to celery.
Many attributes evaluated by QDA use Spectrum® methodology (Bett, 2002). Some
spectrum methods published in Meilgaard et al. (1999) include universal scales that assess
texture by crispness, hardness and juiciness. For fruits and vegetables, these attributes are
important because they can address the major issues associated with the texture quality.
However, these methods require that panelists compress products between their teeth and
evaluate it while it is in their mouths. For fresh-cut produce, flexibility is an alternative indicator
of freshness and texture changes can be determined by flexing or bending products, however, a
standardized scale has not yet been described for training and evaluations for flexibility.
Therefore, there is a need to develop a standardized method that allows researchers to assess
flexibility by training panelists to bend products.
Various strategies have been developed to better maintain celery freshness. Low
temperature storage combined with high CO2/low O2 controlled atmospheres can extend the shelf
life of celery by several days (Saltveit, 1997; Suslow and Cantwell, 2000; Gómez and Artés,

2

2004) as can passive modified atmosphere packaging (MAP) (Viña and Chaves, 2003; Gómez
and Artés, 2005; Viña and Chaves, 2006; Viña et al., 2007; Rizzo and Muratore, 2009).
However, no information has been published about the benefits from active MAP, in
which the package atmosphere is established at the time of sealing, over passive MAP on the
shelf life of fresh-cut celery. In addition, the effect of non-conventional active packaging
atmospheres, including noble gases, superatmospheric oxygen, and nitrogen, on shelf life
extension of fresh-cut produce has been explored for some products (Allende et al., 2004;
Rocculi et al., 2005; Escalona et al., 2006; Artés et al., 2009), but not for celery.
Furthermore, MAP with low O2 levels and/or high CO2 levels can extend the shelf life of
fresh-cut onions by reducing respiration and discoloration, maintaining sucrose content, and
preserving aroma (Blanchard et al., 1996; Forney et al., 2012; Liu and Li, 2006; Selman, 1993).
The beneficial effect of MAP on shelf life extension of RTU onions has also been proven by
sensory panels (Liu and Li, 2006; Blanchard et al., 1996).
Several studies have focused on the microbiological safety of fresh-cut celery (Prakash et
al., 2000; Lu et al., 2005; Kwak et al., 2011; Vandamm et al., 2013). However, very little
information has been published on Listeria monocytogenes in celery, most likely because fresh
produce has not been traditionally considered a special risk product (Aguado et al., 2004).
Recently, L. monocytogenes was identified in ready-to-eat salads containing raw celery (Cordano
and Jacquet, 2009) indicating that celery occasionally poses a risk to the consumers.
Furthermore, from January to October 2010, ten cases of hospital-acquired listeriosis in Texas
(USA), including five deaths, were traced to commercially diced celery that was used as an
ingredient in chicken salad (Gaul et al., 2013).

3

In addition, Salmonella spp in onions has been reported as responsible for 8 confirmed
outbreaks and 348 illnesses in the past decade (Table 1).
Table 1. Outbreaks and illnesses in the world associated with onions from 2004 to 2012.

Year

Country

Species /
Serotype

USA
Salmonella
2004 (Multistate) Berta

Total
Hospitalizations Deaths
Illnesses
155

16

N/R

Food
Vehicle

Contaminated
Source
ingredient

Egg, onion,
taco beef
beef

1

N/R

2

2004 Australia

Salmonella
Typhimurium
12a

28

3

0

gourmet
rolls/red
onion

USA
(New
2005 York)

Salmonella
Newport

27

2

0

onion,
tomato

root, vine
stalk

1

USA
Salmonella
2009 (Multistate) Javiana

9

N/R

N/R

green onion
/ scallion

N/R

1

Salmonella
Newport

33

7

0

sandwich,
salad

onion, lettuce

USA
Salmonella
2010 (Michigan) Javiana

41

5

0

potato salad yellow onion

1

25

N/R

N/R

green
onions

N/R

4

30

0

0

onion

N/R

5

USA
2009 (Kansas)

Canada
2010 (Ontario)
2011 Sweden

Salmonella
Oranienberg
Salmonella
Haifa

1, 3

N/R stands for Not Reported.
1

CDC's Foodborne Outbreak Online Database (http://wwwn.cdc.gov/foodborneoutbreaks/).

2

Reported foodborne illness and gastroenteritis in Australia: Annual report of the OzFoodNet
network, 2004. The OzFoodNet Working Group
(http://www.ncbi.nlm.nih.gov/pubmed/16119765).
3

Kansas Department of Health and Environment Investigative Reports
(http://www.kdheks.gov/epi/outbreaks.htm).
4

Archived News Release, Ministry of Health and Long-Term Care, Ontario (Canada)
(http://news.ontario.ca/mohltc/en/2010/08/salmonella-oranienberg-outbreak-in-ontario.html).
5

EFSA Panel on Biological Hazards Panel, 2013. Scientific Opinion on the risk posed by
pathogens in food of non-animal origin. EFSA Journal 2013; 11(1):3025
(http://www.efsa.europa.eu/en/efsajournal/pub/3025.htm).
4

Several technologies have been explored as means to reduce spoilage and pathogenic
microorganisms in RTU onions including controlled atmosphere (CA) (Blanchard et al., 1996),
sanitizing washes (Park et al., 1995), commercial fermentation products (Yang et al., 2011), UV
light (Hadjok et al., 2008), modified atmosphere packaging (MAP) (Zaulia et al., 2013), and
active packaging (Piercey et al., 2012). Among these technologies, gas compositions (CA and
MAP) and sanitizing washes combined, or not, have been most widely investigated. The growth
of psychrotrophic microorganisms on chlorine-sanitized diced yellow onions (Allium cepa L. cv.
Blitz) increased more than 4 log CFU/g in a CA of 2 kPa O2 + 0 kPa CO2 but only ~2.5 log
CFU/g in a CA of 2 kPa O2 + 10 kPa CO2 after 14 days at 4 °C, showing that the enrichment of a
low O2 atmosphere with CO2 can reduce psychrotrophic proliferation in diced yellow onions
(Blanchard et al., 1996).
Fresh-cut produce is commonly marketed in packages made from petroleum-based
materials. However, environmental concerns surrounding these packages have created a need for
more environmentally-friendly materials (Tharanathan, 2003). Polylactic acid (PLA), a
commercially produced bio-based plastic material, is one suitable alternative for packaging of
fresh produce (Almenar et al., 2008; Almenar et al., 2010; Joo et al., 2011; Forney et al., 2012).
PLA-based packages are clear and highly appealing to consumers (Koutsimanis et al., 2012).
Although this material has been previously evaluated for some types of whole and fresh-cut
produce, its suitability for fresh-cut celery and onion packaging has not yet been assessed.
The objectives of this study were: (1) to compare the effect of packaging material and
initial gas composition on the quality and safety of celery sticks stored at 7 ºC, (2) develop
reproducible methods for flexibility and color descriptive sensory assessments for fresh-cut
celery, (3) to compare the efficacy of different in-package gas compositions and of several

5

commercial sanitizing washes on the growth of S. Typhimurium, total aerobic bacteria, and
yeasts and molds on diced onions, (4) establish interactions between in-package gas
compositions and sanitizing washes and their effects on the microbiological safety and quality as
well as the physico-chemical quality of diced onions, and (5) to determine the best sanitizer and
in-package gas combination for enhancing the microbiological safety and quality of packaged
diced onions.

6

2. Literature Review
Due to changes in consumers’ lifestyles over the last decade, the demand for minimally
processed produce has notably increased (Fresh Cut, 2008). During this same period of increased
processed produce consumption, pathogenic microorganism contamination in RTU produce has
also increased. Salmonella spp. has been reported as responsible for 3 outbreaks and 184
illnesses (Erickson 2010) in fresh-cut onions. In addition to pathogenic microorganisms, spoilage
microorganisms are a growing concern of the minimally processed produce industry due to their
increased proliferation caused by the favorable environment resulting from processing. In
addition to traditional quality and microbial assays, sensorial analysis is crucial for
understanding the human aspect of produce freshness perception and purchasing behavior.
2.1 Means to reduce spoilage and pathogenic microorganisms in processed produce
Technologies such as controlled atmosphere (CA), sanitizing washes, commercial
fermentation products, UV light, modified atmosphere packaging (MAP), and active packaging
have been explored as means to reduce spoilage and pathogenic microorganisms in processed
onions (Blanchard et al., 1996; Hadjok et al., 2008; Yang, Fan, Jiang, Doucette, 2011; Zaulia et
al., 2013; Piercey et al., 2012). Additionally, these technologies can affect the sensory quality of
processed onions. Among these technologies, CA and MAP with or without a sanitizing wash
have most widely been investigated.
Various strategies have also been studied to better maintain celery freshness. Low
temperature storage combined with high CO2/low O2 controlled atmospheres can extend the shelf
life of celery by several days (Saltveit, 1997; Suslow and Cantwell, 2000; Gómez and Artés,
2004) as can passive modified atmosphere packaging (MAP) (Viña and Chaves, 2003; Gómez
and Artés, 2005; Viña and Chaves, 2006; Viña et al., 2007; Rizzo and Muratore, 2009).

7

However, no information has been published about the benefits from active MAP, in which the
package atmosphere is established at the time of sealing, over passive MAP on the shelf life of
fresh-cut celery. In addition, the effect of non-conventional active packaging atmospheres,
including noble gases, superatmospheric oxygen, and nitrogen, on shelf life extension of freshcut produce has been explored for some products (Allende et al., 2004; Rocculi et al., 2005;
Escalona et al., 2006; Artés et al., 2009), but not for celery.
2.1.1. Effect of gas compositions and sanitizers on spoilage microorganisms
Blanchard et al. (1996) carried out a CA study using diced yellow onions that had been
dipped in a 100 ppm chlorine solution for 30 seconds before storage in either: 2% O2 + 0% CO2,
2% O2 + 5% CO2, 2% O2 + 10% CO2, 2% O2 + 15% CO2, 20% O2 + 0% CO2, or air at 4 °C for 2
weeks. Throughout storage, 2% O2 + 10% CO2 had the lowest psychrotrophic growth (~2.5 log
CFU/g increase) while onions stored in 2% O2 + 0% CO2 or air had the most, with an increase of
more than 4 log CFU/g from day 0 to day 14. The storage atmosphere of 2% O2 + 10% CO2 also
had the best sensory scores, with a score of 8 out of 9 for general appearance, and 7.4 out of 9 for
aroma after 14 days. This atmosphere was significantly better than all others in terms of sensory
quality, while air was scored the worst (3.1/9 for appearance and 3.3/9 for aroma). This research
indicates that a storage atmosphere of 2% O2 + 10% CO2 can help to maintain the microbial
safety and quality, as well as consumer perceived quality.
Liu and Li (2006) studied the growth of total psychrotrophs and yeasts in sliced onions
packaged in LDPE bags containing 40% CO2 + 1 % O2 or air during storage at -2, 4, and 10 °C
for 2 weeks. The authors found that onions packaged in high CO2 at 4 °C maintained initial
psychrotroph counts (~3 log CFU/g) through day 5, and yeast growth was also maintained, with
both high CO2 and air stored onions at -2 and 4 °C containing ~4 log CFU/g of growth at day 7.

8

Thus calling attention to the fact that storage temperature is a more influential parameter than inpackage atmosphere for preventing yeast growth in sliced onions.
In diced red onions sanitized with peracetic acid and packaged in sealed polyethylene
(PE) bags, sealed polylactic acid (PLA) containers, or non-sealed PLA containers for 3 weeks at
4.5 °C, aerobic plate counts increased from 3.87±0.12 log CFU/g to 7.74, 6.82, and 8.21 log
CFU/g after 12 days in the sealed PE bags, sealed PLA containers, and non-sealed PLA
containers, respectively. The in-package CO2 accumulation, 15, >25, and 4% for the sealed PE
bags, sealed PLA containers, and non-sealed PLA containers, respectively, directly correlated
with the reduction of aerobic microorganisms, indicating that high in-package CO2
concentrations can help to improve microbial shelf life of red diced onions at 4.5 °C (Forney et
al., 2012).
Selmen (1993) reported that diced onions packaged in OPE/PE bags that were either
perforated or contained modified atmosphere showed varying shelf lives depending on
atmosphere and storage temperature. Diced onions experienced no discoloration when packaged
in air for 14 days at 5 °C and 11 days at 10 °C. Packages containing 10% O2 + 5% CO2 helped to
maintain onion quality for 1 week at 5 °C and 5 days at 10 °C, while perforated (control) bags
only allowed for 5 days at 5 °C and 2 days at 10 °C. In polyvinylchloride bags sealed with air,
diced onions experienced no discoloration for 7 days at 5 °C and 3 days at 10 °C indicating that
not only is temperature and in-package atmosphere important, but also packaging material for
maintaining typical onion color during storage.
A similar passive atmosphere packaging and chlorine sanitizing technique was studied by
Park and Lee (1995) with hand-diced onions in polystyrene trays wrapped with linear low
density polyethylene film for storage at 5 °C for 3 weeks. Diced onions were dipped into a 25 °C

9

sanitizing solution containing either 0 (control), 30, 50, or 100 ppm chlorine for 1 minute.
Mesophilic populations were reduced to approximately 0.5 log CFU/g at the initial sampling for
all samples treated with chlorine, while the control contained populations of nearly 2 log CFU/g.
There were no differences between sanitizer concentration and all treatments exceeded 8 log
CFU/g at the end of storage. The authors also observed that the surface color of the diced onions
did not vary between treatments throughout storage. Because there were no observed differences
between microbial populations or onion color over time, the minimum 10 ppm chlorine sanitizer
was determined to be sufficient for reducing initial microflora. This study helps to point out the
need for more innovative methods to reduce microbial populations in diced onions throughout
their storage to improve overall marketability.
Park and Lee also (1995) researched the effect of varying concentrations of chlorine
sanitizing wash on the initial total microbial populations, ascorbic acid content, and color of
diced onions. Similar to the previously mentioned packaging study, onions washed with 10, 30,
50, and 100 ppm chlorine at 25 °C showed little to no differences in the measured parameters.
The naturally occurring microbial populations were reduced from 1.7 log CFU/g to < 1 log
CFU/g for all concentrations, with only a slight destruction (0.3 mg/100g) of ascorbic acid in
onions washed in 10 ppm chlorine, again indicating that it is an adequate choice for an initial
reduction of microorganisms while maintaining quality.
The quality and bacterial populations of diced yellow onions with passive MAP and an
ethylene absorber were studied by Howard et al. (1994). Onions were first diced by hand with a
knife and then submerged in a 100 ppm chlorine solution before being packaged in PE bags and
stored at 2 °C for 10 days. Packages with and without an ethylene absorber reached a CO2
concentration of 7% after 10 days. Differences were observed, however, for mesophilic bacterial

10

populations, starting on day 3 of storage. Onions packaged with an ethylene absorber reached a
growth level of 7 log CFU/g, while those without the absorber were less than 6 log CFU/g on
day 10. These results indicate that the naturally accumulating ethylene inside the diced onion
package is beneficial to reduce the growth of bacterial microorganisms during storage, even at
low temperatures such as 2 °C.
Beerli et al. (2004) researched the effect of hydrogen peroxide (H2O2) and sodium
dichloroisocianurate (NaDCC) sanitizers on the microbial and physico-chemical quality of sliced
onions for 1 week at 4 °C. Onion slices treated with H2O2 (4 and 6%) contained lower aerobic
mesophiles and yeast and mold populations than NaDCC (50 and 100 ppm), however there were
not differences between the two sanitizers for aerobic psychrotrophs, total coliforms at 35 °C,
total soluble solids, and weight loss. While the NaDCC treated onions experienced an increase in
pH, H2O2 treated onions increased in firmness during storage, indicating that while some
sanitizers can help to reduce microbial populations, they can also damage sliced onions,
producing undesirable affects.
2.1.2. Effect of gas compositions and sanitizers on pathogenic microorganisms
Several studies have focused on the microbiological safety of fresh-cut celery (Prakash et
al., 2000; Lu et al., 2005; Kwak et al., 2011; Vandamm et al., 2013). However, very little
information has been published on Listeria monocytogenes in celery, most likely because fresh
produce has not been traditionally considered a special risk product (Aguado et al., 2004).
Recently, L. monocytogenes was identified in ready-to-eat salads containing raw celery (Cordano
and Jacquet, 2009) indicating that celery occasionally poses a risk to the consumers.
Furthermore, from January to October 2010, ten cases of hospital-acquired listeriosis in Texas

11

(USA), including five deaths, were traced to commercially diced celery that was used as an
ingredient in chicken salad (Gaul et al., 2013).
Farber et al. (1997) investigated the effect of passive MAP in combination with chlorine
sanitizer on the growth of Listeria monocytogenes on inoculated sliced onions. Whole peeled
onions were dipped in a 200 ppm sodium hypochlorite solution for 10 seconds before being
sliced. Onion slices were then inoculated with L. monocytogenes and stored in sealed OPE bags
at 4 and 10 °C for 9 days. In-package CO2 accumulation was 23% in bags stored at 4 °C and
52% in bags at 10 °C on day 9. There was no increase in L. monocytogenes growth in onion
slices stored at 4 °C, likely because Listeria does not grow well at low temperatures. There was a
slight (~1 log CFU/g) increase in L. monocytogenes populations from day 0 to day 9 in onions at
10 °C. The unexpectedly low increase of L. monocytogenes during the 9 day storage period can
be attributed to the large buildup of CO2 inside the bags, preventing Listeria proliferation.
Sy et al. (2005) applied gaseous chlorine dioxide (ClO2) to whole Vidalia onions to
determine the possible reductions in Salmonella and yeast/mold growth for the produce.
Salmonella enterica surface inoculated onions were treated with ClO2 in concentrations of 0
(control), 1.4, 2.7, and 4.1 mg/L, resulting in reductions of 0 (control), 0.83, 1.89, and 1.94 log
CFU/piece, respectively. Yeast and mold growth was also reduced on the onion surface after
ClO2 treatment, with reductions of 0.36 and 0.22 log CFU/piece for 1.4 and 2.7 mg/L treatments,
and an increase of 0.22 log CFU/piece for those treated with 4.1 mg/L ClO2. This study
illustrates that chlorine dioxide is a viable alternative sanitizing option for onions, but that the
proper concentration must be utilized to ensure that the onions are not damaged, allowing for
increased yeast and mold growth.

12

2.1.3. Non-traditional means to reduce microbial populations on minimally processed produce
As an alternative to immersion sanitizing techniques, Hadjok et al. (2008) applied
ultraviolet (UV) light combined with hydrogen peroxide (H2O2) to sliced Spanish onions to
reduce both surface and internal pathogenic microorganisms. The onion slices were submerged
in 500 mL of inoculum containing 7 log CFU/g of Salmonella Montevideo for 20 minutes before
being vacuumed cyclically to induce internalization of the bacteria. Each side of the onion slice
was treated with 50 °C 1.5% v/v H2O2 mist while exposed to a UV lamp (254 nm) for 1 minute
while control samples were treated with a 200 ppm calcium hypochlorite solution for 3 minutes.
Salmonella populations present on the surface of the slices were reduced 3.66 log CFU/g while
the calcium hypochlorite control was only reduced 0.34 log CFU/g. Internalized Salmonella were
reduced 0.97 log CFU/g when treated with UV/H2O2, and 0.05 log CFU/g for the control,
indicating that this experimental sanitizing technique is significantly more effective at reducing
surface populations of Salmonella Montevideo than calcium hypochlorite on sliced onions.
While the observed surface Salmonella reduction is notable, studying the additional effect of
storage time on microbial populations is necessary to deem UV/H2O2 as a viable alternative
sanitizing technique for sliced onions.
Purple Brazilian onions were sliced and diced in a study by Berno et al. (2014) to
determine the effect of storage temperature on the quality of fresh-cut onions. Hand-sliced or
diced onions were kept in polypropylene snap fit containers at 0, 5, 10, or 15 °C for up to 15
days. Sliced onions were darker (higher luminosity) than diced onions from day 0, and better
maintained their luminosity, Chroma, and Hue values throughout the storage study compared to
diced onions. Diced onions experienced greater surface color changes than sliced onions at all
temperatures, likely due to the increased surface area to volume ratio produced by the dicing

13

action. However, for all color attributes reported, onions (both sliced and diced) kept at 5 °C best
maintained their initial color compared to the other storage temperatures studied. This study
helps to stress the importance of finding better storage conditions that can help to mitigate the
loss of initial quality due to the processing conditions necessary for fresh-cut onions.
Although the effect of modified atmosphere packaging and assorted sanitizers on
spoilage and pathogenic microorganisms in minimally processed produce has been studied, there
is a lack of information on sanitizer and packaging material on pathogenic microorganism
growth in fresh-cut celery, lacking available methods for flexibility and color descriptive sensory
assessments for fresh-cut celery, and no information on the effect of interactions between inpackage gas compositions and sanitizers in regards to the microbiological safety and quality. In
addition, there is no information on the effect of either in-package gas compositions or sanitizing
washes on Salmonella Typhimurium growth on minimally processed onions.

14

3. Effect of non-conventional atmospheres and bio-based packaging on the quality and safety of
Listeria monocytogenes-inoculated fresh-cut celery (Apium graveolens L.) during storage
3.1. Materials and methods
3.1.1. Celery
Celery (Apium graveolens L.) was purchased from a local distributor (Stan Setas
Produce, Lansing, MI, USA), transported under refrigeration to the MSU School of Packaging,
maintained at 7 ºC and used within 2 d of delivery. Celery was visually inspected, selected, and
minimally processed with a stainless steel knife (Granton, Sheffield, UK) into 10 cm long sticks
(~ 20 g each). After processing, the celery sticks were rinsed with cold tap water and stored
inside a temperature controlled chamber at 7 ºC overnight. The following day, the celery sticks
were immersed in a commercial sanitizer containing 50 mg L-1 available chlorine at pH 7.5 (XY12, Ecolab, St. Paul, MN, USA) for 1 min at 7 ºC, centrifuged for 1 min using a batch spin dryer
(SD50 LT, Heinzen Manufacturing Intl., CA, USA) and packaged. The celery sticks were not
rinsed after sanitizing, following the current processing conditions of celery processors in the
United States, meaning some residual chlorine could be present after processing. The sticks
intended for L. monocytogenes inoculation were treated as described in Section 8.2.7.
3.1.2. Packaging
Two packaging films were used: A 51 µm-thick film composed of polypropylene (PP)
and low density polyethylene (LDPE) (commonly used in fresh-cut produce packaging in the
U.S.) and a 44-µm thick film made of polylactic acid (PLA) coated with high molecular weight
PLA (EVLON EV-HS1, BI-AX International Inc., Wingham, ON, Canada). Codes used for the
petroleum-based polyolefin and for the bio-based polyester from now on are PP/PE and PLA,
respectively.

15

3.1.2.1. Packaging and storage
Both packaging films were formed into 20 cm length × 15 cm width bags using an
impulse sealer (Ceratek, Sencorp Systems Inc., Hyannis, MA, USA). An amount of 10-12 celery
sticks were placed in each bag (≈190 grams per bag). Half of the PLA and PP/PE bags were
sealed without atmosphere replacement for passive modified atmosphere packaging (MAP) (APLA and A-PP/PE). The air in the remaining PLA bags was replaced with 95 kPa O2 + 5 kPa N2
(O2-PLA), 99 kPa N2 + 1 kPa O2 (N2-PLA), or 6 kPa O2 + 12 kPa CO2 + 82 kPa N2 (CO2-PLA)
using a Multivac flusher (Model A300/16, Sepp Haggenmüller KG, Wolfertschwenden,
Germany) for the active MAP treatments. As a control, celery was packaged under the same
conditions as in the passive MAP treatment, but the bottom of the bags was partially cut off,
creating a non-modified atmosphere package (coded as open bags). All packages were stored at 7
ºC until the day of testing.
3.1.2.2. Packaging material characterization
Oxygen, carbon dioxide, water vapor and ethanol transmission rates were assessed for
PLA and PP/PE. Oxygen transmission rate (OTR) was determined at 23 °C and 0% RH
according to the American Society for Testing Materials (ASTM) method D3985 (ASTM, 2005)
using an OX-TRAN® Model 2/21 (MOCON, Minneapolis, MN, USA). Carbon dioxide
transmission rate (CO2TR) was measured using a PERMATRAN-CTM Model 4/41 (MOCON)
under the same conditions. Water vapor transmission rate (WVTR) was determined at 23 °C and
100% RH according to ASTM method F1249 (ASTM, 2006) using a PERMATRAN-WTM
Model 3/33 (MOCON). Permeation cells made from stainless steel were used to gravimetrically
determine the ethanol transmission rate for PP/PE and PLA using a slightly modified ASTM
method E96-80 (ASTM, 1980). All testing was conducted in triplicate.

16

In all cases, the permeability coefficients (kg m m-2 s-1 Pa-1) were obtained as follows:
P = (TR × l)/∆p
where TR is the transmission rate value (kg m-2 s-1), l (m) is the film thickness, and ∆p is the
partial pressure differential across the film (Pa). The permselectivity coefficient (β; ratio of CO2
to O2 permeation) was also calculated.
3.1.3. Atmosphere composition
Progressive changes in CO2, O2, and N2 were monitored in all packages using a gas
chromatograph (Thermo Scientific Trace GC Ultra, Thermo Electron S.p.A., Rodano, Italy)
equipped with a thermal conductivity detector and a Carboxen 1010 Plot capillary column, 30 m
in length with a film thickness of 30 µm, and an internal diameter of 0.53 mm (Supelco,
Bellefonte, PA, USA). Helium was used as a carrier gas (3 mL min-1). Using a syringe (SGE,
Austin, TX, USA), a 100-µL headspace sample was collected through a silicone septum attached
to each package. Initial gas compositions were determined to verify correct flushing by the
Multivac machine. Subsequently, CO2, O2, and N2 levels were monitored after 1, 3, 5, 7, 10, 14,
and 21 d. New bags were used for each analysis day.
Ethanol concentration was determined after 3, 7, 14, and 21 d by injecting 1 mL
headspace samples into a gas chromatograph equipped with a flame ionization detector (Hach
Carle Series 100 AGC, Loveland, CO, USA) fitted with a 2 m long, 2 mm internal diameter
stainless steel column packed with Chromosorb OV-103, 60/80 mesh (Altech Associates Inc.,
Deerfield, IL, USA). Ethanol content was expressed as microliters of ethanol per liter (µL L-1).
3.1.4. Texture
A texture analyzer (TA-XT2i, Stable MicroSystem, Godalming, UK) equipped with a 50
kg-load cell was used to determine celery stick firmness and toughness. The assays were

17

performed using a 3-point bending probe (constant span length of 4 cm acting perpendicularly to
the celery), which was positioned horizontally with the outer convex surface upwards, centered
on the supports, and the pressing force was applied vertically to the middle of the celery. Each
stick was flexed at a constant speed of 2 mm s-1 until failure. Firmness (N) was calculated as the
measured force at 5 mm of the load-displacement curve. Toughness (J m-3) was defined as the
area below the stress-strain curve. For texture measurements, all sticks (≈10 per bag, ≈ 60 sticks
per treatment) were analyzed.
3.1.5. Color
Celery color was determined after 0, 3, 7, 14, and 21 d of storage using a Minolta CR300
colorimeter (Osaka, Japan) calibrated with a standard white plate (Y=93.84; x=0.3132;
y=0.3191) and set with a C illuminant, 2º observer. Readings taken from both the outer convex
surface and cut ends were expressed as L*, a* and b* parameters. Hue angle (hº) was calculated
using the a* and b* parameters (hº=tan-1(b*/a*)). For color measurements, all sticks (≈10 per
bag, ≈ 60 sticks per treatment) were measured at two points on the surface, and at the two cut
ends.
3.1.6. Weight Loss
After 21 d of storage, weight loss was determined for all celery stick treatments (6
bags/treatment) by subtracting the final weight from the initial weight with the results expressed
as % weight loss.
3.1.7. Preparation of inoculum, inoculation of samples, and microbiological analysis
Stock cultures of three avirulent strains of Listeria monocytogenes (J22F, M3, J29H)
were maintained at -80 °C and subcultured twice (24 h/37 °C) in 200 mL of trypticase soy broth
(Difco, Becton Dickinson, Sparks, MD, USA) containing 0.6% (w/v) yeast extract (Difco,

18

Becton Dickinson) (TSB-YE). Cultures were combined in equal volumes and diluted 1:100 in
tap water (~15 °C) to contain 7.19±0.13 log CFU/mL. The bacterial population in the final
cocktail culture was confirmed by appropriately diluting in sterile 0.1% phosphate buffer
solution and plating on Modified Oxford Agar (Neogen Corporation, Lansing, MI, USA) (MOX)
plates. These plates were then incubated at 37 ºC for 48 h prior to enumeration. The celery sticks
were then inoculated by immersion for 30 min to obtain 4.35±0.33 log CFU/g. After centrifugal
drying and 18–22 h of storage at 4 ºC, the celery sticks were immersed in 60 L of tap water
containing 50 mg L-1 available chlorine (XY-12, Ecolab, USA) for 1 min, centrifugally dried and
then packaged in both PLA and PP/PE bags in a glove chamber (Labconco 50004 Fiberglass
Glove Box, Kansas City, MO, USA) using the gas compositions described in section 2.2. The
chamber was used instead of the Multivac machine due to the special safety precautions required
for handling the L. monocytogenes. Additional PLA-packaged, passive modified atmosphere
samples were prepared using unsanitized inoculated fresh-cut celery (immersed in 60 L of tap
water).
Following 0, 1, 3, 5, 7, 10, 14, and 21 d of storage at 7 C, two celery stick samples (~50
g) from each package were diluted 1:4 in Difco Neutralizing Buffer (Becton Dickinson, Sparks,
MD, USA) and homogenized in a stomacher (Stomacher 400 Circulator, Seward, Worthington,
UK) at 260 rpm for 1 min. Samples were either appropriately diluted in sterile 0.1% phosphate
buffer solution and plated on MOX, or filtered using 0.45 μm membrane filters (Millipore,
Millipore Corporation, Billerica, MA) and placed on 60 mm diameter petri plates containing
MOX for quantification of L. monocytogenes after 48 h of incubation at 37C.

19

3.1.8. Statistical analysis
All of the experiments were conducted in triplicate and data was expressed as the means
± standard errors. Three replications consisting of two bags each (a total of 6 bags) were
analyzed per treatment on each day of testing for quality analysis. For Listeria analysis, one bag
for each of the three replicates was used (a total of 3 bags). Data were subjected to statistical
analysis using a statistical software package IBM SPSS version 19 (IBM Corporation Software
Group, Somers, NY, USA). One-way analysis of variance (ANOVA) and Duncan multiple range
tests at a significance level P < 0.05 were used, except for the microbiological and texture
analyses, where comparison of means was conducted with the least significant difference (LSD)
test (P < 0.05).
3.2. Results and discussion
3.2.1. Packaging material characterization
O2, CO2, water vapor and ethanol permeability coefficients of PLA and PP/PE are shown
in Table 2. The materials had different permeabilities to the gases and vapors tested. The O2 and
CO2 permeabilities of PLA were 4 and 5 times lower than those of the PP/PE, respectively.
Given that a low permeability is necessary to reduce gas exchange and maintain the flushed gas
inside package, PLA could be a viable bio-based material to maintain desired gas compositions
in active MAP. The CO2 and O2 permeability coefficients of PLA were very similar to those
reported for oriented PLA sheets (33±8 × 10-18 kg m m-2 s-1 Pa-1 and 4±0 × 10-18 kg m m-2 s-1 Pa1

, respectively) by Joo et al. (2011). The ethanol permeability of PLA was lower than that of

PP/PE. Information on the permeability of plastics to ethanol is very limited (Robertson, 2013),
and no data on the permeability of PLA to ethanol has been found in the literature. The ethanol
permeability coefficient of LDPE has been reported as 3.24 × 10-17 kg m m-2 s-1 Pa-1 (Robertson,

20

2013). This value is higher than our value for PP/PE, which indicates that PP is a better barrier to
ethanol than LDPE. In agreement, Scully (2009) reported that LDPE is not as good a barrier to
ethanol as PP. The water vapor permeability coefficient of PP/PE was 20 times lower than that of
PLA. Our PLA results are in agreement with those reported by Almenar and Auras (2010) under
the same analysis conditions (11.98 × 10-15 kg m m-2 s-1 Pa-1). The O2, CO2, and water
permeability values of PP/PE were between those reported for LDPE and PP (Almenar and
Auras, 2010).
Table 2. Carbon dioxide, oxygen, water and ethanol permeability coefficients for PLA and
PP/PE films.
Permeability coefficient
β
Material
CO2 a
O2 a
H2 O b
Ethanol a
(PCO2/PO2)
PLA

30.34±9.07

5.67±1.17

21.86±3.22

0.10±0.03

5.35

PP/PE

113.4±13.8

28.35±4.61

1.08±0.24

0.51±0.01

4.00

a
b

×10-18 kg m m-2 s-1 Pa-1
×10-15 kg m m-2 s-1 Pa-1

3.2.2. Atmosphere composition
The steady state atmospheres achieved in the A-PLA, N2-PLA, and CO2-PLA bags
ranged between 1-2 kPa O2 and 8-10 kPa CO2 (Figure 1) with these values close to the
atmospheres recommended by Gómez and Artés (2004) for shelf life extension of celery during
refrigerated storage. The steady state atmospheres reached in the O2-PLA bags was 2 kPa O2 and
22 kPa CO2 (Figure 1). The steady state atmosphere was achieved faster in CO2-PLA (3 d) than
A-PLA and N2-PLA bags (7 d). Levels of CO2 and O2 in the O2-PLA bags stabilized after 14 d
with the A-PP/PE bags reaching steady state after 10 d of storage (2.5 kPa O2 and 4 kPa CO2).
Bags made from PLA equilibrated faster than those made from PP/PE due to the greater

21

permselectivity of the PLA (5.35 and 4.00 for PLA and PP/PE, respectively) resulting from their
different permeability to O2 and CO2 (Table 2). Therefore, PLA would be preferred over PP/PE
for fresh-cut celery. Gas evolution in the A-PP/PE bags was very close to that obtained by
Gómez and Artés (2005) but non-optimal when celery sticks were packaged in 25 µm LDPE
bags and stored at 4 ºC. These same authors found that the higher CO2 (7 kPa) and lower O2 (5
kPa) levels achieved in 35 µm OPP bags worked better to maintain fresh-cut celery quality. The
relatively higher CO2 concentration reached in the bags with high O2 can be attributed to a more
stable respiration rate of the celery since O2 was not a limiting factor during the first 14 d of
storage. However, an increased respiration rate due to tissue damage (Cantwell and Suslow,
2002; Martínez et al., 2005; Iqbal et al., 2008), microbial growth (Jacxsens et al., 2003; ArtésHernandez et al., 2007; Silveira et al., 2008), or headspace volume reduction could be
contributing to CO2 accumulation for this treatment.

22

Figure 1. Oxygen and carbon dioxide contents of celery stick packages for different materials
and initial gas composition s stored at 7 oC.

Throughout storage, the headspace for PP/PE bags yielded far lower ethanol levels
compared to PLA bags age which is most likely due to the 5-fold greater ethanol permeability of
23

the PP/PE film which favored the escape of ethanol from the package headspace (Table 3).
Comparing the different in-package gas compositions, faster ethanol accumulation was observed
in the CO2-PLA and N2-PLA bags due to their low level of O2 (~ 1 kPa) (Table 3) which led to
earlier hypoxia. Many vegetables generate ethanol through fermentative metabolism at O2 levels
< 2 kPa. Since ethanol was not detected in the A-PLA and O2-PLA bags until day 14, the
packages remained sufficiently aerobic to minimize fermentation for at least a week (P < 0.05).
The ethanol content in all PLA bags increased from week 2 to 3 with the greatest increase seen in
O2-PLA bags, likely due to the higher CO2 content. High levels of CO2 alone (> 20 kPa CO2) or
in combination with low levels of O2 (> 20 kPa CO2 and < 2 kPa O2) have been related to the
accumulation of ethanol due to fermentation (Jacxsens et al., 2001). A sensory evaluation is
needed to determine the impact of ethanol concentration on consumer acceptance of packaged
celery sticks.
3.2.3. Weight loss
The weight of celery sticks in open bags decreased more than 30% after 21 d of storage at
7 ºC (Table 3), showing the need for hermetically sealed containers during the postharvest
period. Significantly greater (P < 0.05) weight loss was seen after 21 d of storage using PLA (~
4%) compared to PP/PE bags (< 1%) (Table 3). The higher weight loss of the celery sticks in the
PLA bags was due to the higher water vapor permeability coefficient of PLA (21.86±3.22 × 10-15
kg m m-2 s-1 Pa-1) compared to PP/PE (1.08±0.24 × 10-15 kg m m-2 s-1 Pa-1). The initial
atmosphere did not affect celery weight loss since similar weights were obtained for all PLApackaged celery at the end of the storage.

24

Table 3. Weight loss (%) and ethanol content (µL L-1) for fresh-cut celery in packages differing
in initial gas composition and packaging material during storage at 7 ºC.
Storage time (days)
Packaging
3
7
14
21
code
Ethanol
Ethanol
Ethanol
Ethanol
Weight loss
Open Bags 30.99±12.42
A-PP/PE
0.00±0.00 a*
0.06±0.11 a
0.32±0.25 a
0.39±0.28 a
0.73±0.77
a
ab
ab
ab
A-PLA
0.00±0.00
0.73±0.98
12.92±8.94
16.34±17.41
4.50±0.14
O2-PLA
0.00±0.00 a
0.13±0.16 a
12.99±6.80 ab
47.88±22.89 c
4.67±0.57
b
ab
b
bc
CO2-PLA
8.15±9.98
6.32±5.42
16.31±11.81
31.97±29.17
4.53±0.33
b
b
b
abc
N2-PLA
10.72±6.25
7.66±4.93
17.59±13.55
19.99±22.36
4.87±0.40
*
Each value represents the mean of three replicates ± standard deviation. Different letters
indicate significant differences (P < 0.05) between treatments for each time point.

Celery packaged in PLA bags exhibited a > 4% weight loss after 21 d, however, this was
less than the maximum 10% water loss considered as a limit of acceptance for celery (Robinson
et al., 1975). Therefore, celery sticks in PLA bags would likely remain acceptable for at least 21
d with PLA performing similarly to other plastics. Viña and Chaves (2003) reported a weight
loss of 6.2% for minimally processed celery packaged in polyvinylchloride film-covered
polystyrene trays after 28 d of storage at 10 ºC. Assuming a linear relationship between weight
loss and time, our results for celery sticks stored in PLA at 7 ºC are very similar to those of Viña
and Chaves (2003) after 21 d, although their water vapor transmission rate for polyvinyl chloride
(4.63 × 10-7 kg m-2 s-1) was 2.8 times lower than that of our PLA (13.08±1.92 × 10-7 kg m-2 s-1).
3.2.4. Color
The surface color (hue angle) of celery sticks was affected by both the packaging material
and gas composition (Table 4). At the end of storage, celery packaged in A-PLA was greener
(higher hº) than that packaged in A-PP/PE (P < 0.05) with the former maintaining the green
color of celery throughout storage. The difference was likely due to the higher CO2 level in APLA bags (Figure 1). In agreement, steady state CO2 and O2 concentrations of 5-7 kPa and 9-6
kPa, respectively, have been shown to preserve the external color of celery (Gómez and Artés,
25

2005). Among headspace gas compositions, the CO2-PLA bags maintained the green color of
celery better than the other treatments (P < 0.05) up to d 14, after which all samples were visibly
decayed. In contrast, celery sticks packaged in O2-PLA showed a more intense yellowing
compared to the other treatments (P < 0.05). In addition, celery sticks packaged in the O2-PLA
bags developed numerous small pits on the surface that became increasingly evident over time
(Figure 2). The pitting development could be due to the high oxygen, but CO2 accumulation
could be also affecting, so further research needs to be carried out to clarify the origin of this
disease. Surface lightness was affected by in-package gas composition, but not the packaging
material with celery sticks packaged in CO2-PLA bags exhibiting the lowest L* value during the
first week of storage (P < 0.05) (Table 4).

26

Table 4. Lightness (L*) and hue (hº) values for surface and cut ends of the celery sticks packaged in different materials and initial gas
compositions during storage at 7 ºC.
Storage time (days)
Analysis
location

Packaging
code

0

L*

3

hº

L*

7

hº

14

21

L*

hº

L*

hº

L*

hº

Open Bags

57.8±4.9

118.2±1.4

58.0±6.9 a* 117.5±1.9 ab

57.5±4.7 a

118.1±1.2 b

59.7±5.3 ns

117.4±2.3 ab

60.6±5.4 ns

116.9±2.4 b

A-PP/PE

57.8±4.9

118.2±1.4

60.6±5.1 b

117.0±2.6 a

59.4±6.2 ab

117.8±1.4 b

60.5±5.6 ns

117.4±3.2 ab

60.2±4.8 ns

116.1±3.2 a

A-PLA

57.8±4.9

118.2±1.4

59.0±6.1 ab

117.8±1.4 bc

59.3±5.0 ab

118.0±1.4 b

60.8±5.9 ns

117.7±2.2 b

60.5±5.0 ns

117.3±2.5 b

O2-PLA

57.8±4.9

118.2±1.4

59.1±5.3 ab

117.9±1.5 bc

60.9±5.7 b

117.3±1.7 a

60.6±5.7 ns

116.8±2.1 a

60.6±4.9 ns

115.7±2.9 a

CO2-PLA

57.8±4.9

118.2±1.4

57.8±5.9 a

118.9±1.9 d

58.8±5.7 a

118.8±1.4 c

59.9±6.3 ns

118.4±2.0 c

60.0±6.3 ns

117.3±2.1 b

N2-PLA

57.8±4.9

118.2±1.4

58.7±6.3 ab

118.3±1.4 c

59.0±5.9 a

118.3±1.7 b

60.8±5.7 ns

117.2±2.9 ab

59.2±5.3 ns

117.4±1.9 b

Open Bags

56.3±4.9

115.8±1.5

60.4±5.3 b

111.3±4.0 a

63.8±4.7 c

111.1±4.4 a

63.7±4.5 c

110.0±4.1 a

64.88±5.5 d

106.0±4.7 a

A-PP/PE

56.3±4.9

115.8±1.5

59.1±5.2 ab

113.0±2.6 b

59.4±5.6 a

111.9±3.8 ab

59.7±5.9 a

111.3±3.7 b

60.3±5.5 a

107.6±5.6 b

A-PLA

56.3±4.9

115.8±1.5

59.1±6.2 ab

113.7±3.0 b

61.9±5.9 b

113.4±3.0 c

62.4±5.8 b

112.9±3.2 c

61.9±6.2 abc

111.7±3.2 d

O2-PLA

56.3±4.9

115.8±1.5

60.6±5.8 b

113.4±2.6 b

61.8±5.1 b

112.4±3.1 bc

62.4±5.9 b

110.5±3.0 ab

61.3±7.0 ab

110.0±4.3 c

CO2-PLA

56.3±4.9

115.8±1.5

60.6±5.6 b

115.5±2.7 c

61.5±5.7 b

115.5±2.5 c

61.7±5.9 ab

114.3±2.9 d

63.5±5.5 cd

112.9±3.4 d

N2-PLA

56.3±4.9

115.8±1.5

57.8±7.5 a

115.3±2.5 c

61.6±5.6 b

114.8±2.8 c

61.9±6.2 ab

113.6±3.0 cd

62.5±5.0 bc

112.4±3.2 d

Surface

Cut
ends

*Each value represents the mean of three replicates ± standard deviation. Different letters indicate significant differences (P < 0.05)
between treatments for each time point and analysis location.

27

Figure 2. Celery stick pitting from O2-PLA bags after 21 days of storage at 7 oC.

The color of cut celery stick ends was also affected by packaging material and initial gas
composition (Table 4). Comparing the different materials, the cut celery stick ends in the A-PLA
bags maintained a higher hue value (greener color) than those in A-PP/PE bags throughout
storage. This was probably caused by the higher CO2 content in the A-PLA bags. The
effectiveness of low O2 and high CO2 levels in preserving the green color of the cut ends of
fresh-cut celery in OPP bags at 4 ºC (6 kPa O2 and 7 kPa CO2 at steady state) was reported by
Gómez and Artés (2005). However, initially high O2 levels caused the degradation of the green
color and promoted yellowing in the cut ends. In contrast, initially high O2 levels (95 kPa)
reduced discoloration of chicory endive (Jacxsens et al., 2001). CO2-PLA and N2-PLA
maintained a higher hue during storage, frequently associated to the maintenance of a greener
color. The L* value for the cut ends of celery rapidly increased during the first 7 d of storage and
then stabilized thereafter (Table 4). Celery sticks stored at 10 ºC in passive modified atmospheres
have reportedly exhibited similar behavior (Viña and Chaves, 2003) which is likely related to

28

increased hydration of the cut ends. Discoloration caused by reversible surface dehydration is
common in fresh-cut produce including carrots (Tatsumi et al., 1991; Cisneros-Zevallos et al.,
1995) and peaches (Gorny et al., 1998; González-Buesa et al., 2011). Although discoloration of
cut ends due to lignin accumulation has been reported in many fresh-cut products, this does not
seem to be the reason of the discoloration of the celery cut ends according to Viña and Chaves
(2003). Polymerized phenol could be the cause of discoloration. Headspace gas composition had
no effect on the L* value for celery cut ends, but packaging film did have an effect (Table 4).
PP/PE bags better maintained lightness of the celery cut ends after 7 and 14 d of storage. Cut
end lightness was associated with a lower level of dehydration compared to the PLA bags, with
this higher L* due to the greater water vapor permeability of PLA (Table 4).
Figure 3 shows the change in the a* and b* color coordinates and of the hue angle in the cut ends
of celery sticks during storage, providing additional information about the change in color. The
color evolution of the cut ends of the celery sticks in the A-PP/PE bags was close to that of the
cut ends of the celery sticks in the open bags, indicating that the CO2 accumulated inside the APP/PE was not enough to avoid color degradation. Comparing the different initial gas
compositions, the ends of the celery sticks in CO2-PLA bags showed more stable a* and b*
coordinates, especially during the first 14 d of storage, indicating a more well maintained color
to the initial one. However, O2-PLA and A-PLA showed a large change in the a* and b* values
during the first 7 d, indicating major changes in the color of the celery cut ends. The N2-PLA
bags after 3 and 7 d maintained intermediate values, indicating a less severe color evolution.

29

Figure 3. Effect of initial gas composition and packaging material on the color (a*, b* and hue)
evolution of the cut surfaces of celery sticks. Numbers (0, 3, 7, 14 and 21) indicate storage time
in days at 7 oC.

Transversal dotted lines indicate different guide hue angles.

These results show that a high initial concentration of CO2 in the headspace can help
maintain the initial green color of the surface and cut ends of celery sticks while a high O2
concentration promotes yellowing and other quality defects. While PLA can better maintain the
green color of both cut and uncut celery, dehydration can become an issue.
3.2.5. Texture
Figure 4a shows the evolution of firmness or loss of turgor of unpackaged and packaged
celery sticks during 21 days at 7 ºC, measured as the force at a displacement of 5 mm in the 3
bending point rig. Using open bags, celery stick firmness decreased from 56.2±10.6 N to
18.4±10.9 N during 21 d at 7 ºC. This change in texture is likely due to water loss and pectin
degradation as suggested by Viña and Chaves (2003). In contrast, both PLA and PP/PE bags
maintained celery firmness during 14 d of storage. At day 21, PLA-packaged celery softened due
to the relatively high water vapor permeability of PLA while celery sticks packaged in PP/PE

30

maintained their turgor (P < 0.05) (Table 3). The different initial gas compositions did not affect
the turgor of the celery sticks throughout the storage. This was due to the gas compositions
having no effect on the weight loss of the celery sticks. Unlike firmness, the celery toughness
was affected by the initial gas composition, but not the packaging material (Figure 4b). Celery
sticks maintained a slightly tougher texture in high O2 compared to other gas mixtures during
storage (P < 0.05). This was most likely due to maintenance of the celery respiration rate at high
O2 levels, which has the potential to increase lignin production and hardening of the celery
fibers. Viña and Chaves (2003) reported lignification of fibers and/or xylem vessels in fresh cut
celery during storage in passive modified atmospheres (> 10 kPa O2 and < 4 kPa CO2) with
similar results obtained for asparagus during exposure to 21 kPa O2 (Everson et al., 1992).
Another possible explanation is damage of tissues by the high O2 exposure during storage.

31

Figure 4. Firmness (a) and toughness (b) of celery sticks packaged in different materials and
initial gas compositions during storage at 7 oC.

3.2.6. Listeria monocytogenes
Growth of L. monocytogenes on celery during 21 d of storage at 7 ºC was impacted by
initial headspace gas composition (Figure 5a). While O2-PLA bags showed no increase in
Listeria populations during the first 10 d of storage (P < 0.05), CO2-PLA, N2-PLA, and A-PLA
supported growth. Amanatidou et al. (1999) reported only a slight reduction in Listeria growth
on agar plates that were exposed to 90 kPa O2 at 8 ºC. In other work, L. monocytogenes growth
32

in fresh, processed, mixed salads (endive, curly endive, radicchio, lollo rosso and lollo bionda)
was not affected by an initial oxygen level of 95 kPa during storage at 4 ºC (Allende et al.,
2002). Suppression of Listeria growth is most likely only visible above 4 ºC. In fact, Listeria
populations in fresh-cut celery stored at multiple temperatures in highly permeable bags and
containers (< 2 kPa CO2 and ≈ 18 kPa O2 at steady state) decreased at 4 ºC due to the low
temperature but not the gas composition (Vandamm et al., 2013).

33

Figure 5. Listeria monocytogenes populations on sanitized and unsanitized celery sticks
packaged in different materials and initial gas compositions during storage at 7o C ((a) compares
initial headspace gas compositions and (b) compares materials as well as sanitation).

After 10 and 14 d of storage at 7 ºC, Listeria populations in the CO2-PLA bags were
slightly higher than those in the N2-PLA and A-PLA bags, and significantly higher (P < 0.05)
than those in the O2-PLA bags (Figure 5). Enhanced growth of Listeria at higher CO2 levels has
been previously reported for other types of produce including cut chicory endive (Bennik et al.,
1996), shredded cabbage (Kallander et al., 1991), and shredded lettuce (Francis and O’Beirne,
34

2001). In contrast, an initial in-package gas composition of 5 kPa O2 + 5 kPa CO2 suppressed
Listeria growth in shredded iceberg lettuce (Carrasco et al., 2008). These observed differences in
Listeria behavior in packaged fresh-cut produce stored at low temperatures can be partially
explained by the type of produce, modified atmosphere, inherent microflora and Listeria strain
variations (Caleb et al., 2013).
At day 21, no differences (P > 0.05) were found between the Listeria populations on
celery for the different packaging methods, likely due to similarly low O2 levels in all packages.
The O2-PLA bags were the only packages able to maintain the initial Listeria populations during
the first 10 d of storage, but no longer, due to the shift to anaerobic conditions. This can be
attributed to the lack of competing aerobic microflora that occurred when O2 was depleted.
According to Farber et al. (1998), decreased growth of L. monocytogenes on cabbage packaged
using a highly-permeable film could be partially explained by the competing microflora such as
lactic acid bacteria.
No differences (P > 0.05) were found between the Listeria populations on celery sticks
packaged in different materials and between sanitized and unsanitized celery throughout storage
(Figure 5b), even though the CO2 level was higher and the O2 level lower for the PLA bags.

35

4. Development of Novel Descriptive Sensory Methods to Evaluate the Quality of Fresh-cut
Celery
4.1. Materials and methods
4.1.1. Sample preparation
Fresh celery bunches were purchased from a local distributor (Stan Setas Produce,
Lansing, MI, USA), stored under refrigeration, and used within 1 day of delivery. All bunches
were rinsed with ~4 °C tap water for 1 minute, before being manually cut into 10 cm sticks. The
cut sticks were sanitized in 70 ppm chlorine in ~4 °C tap water for one minute and drained in a
kitchen centrifuge. Before packaging, the celery sticks were carefully inspected and selected
based on similar size and visual appearance.
A polylactic acid (PLA) film (44-µm thick) coated with high molecular weight PLA
(EVLON EV-HS1, BI-AX International Inc., Wingham, ON, Canada) was used as the packaging
material in this study. The O2, CO2 and water vapor permeabilities were determined in previous
work (González et al., 2013) and are as follows: 30.34±9.07∙10-18 Kg m m-2 sec-1 Pa-1,
5.67±1.17∙10-18 Kg m m-2 sec-1 Pa-1, and 21.86±3.22 ∙ 10-15 Kg m m-2 sec-1 Pa-1, respectively.
Packaging film was formed into 20 x 15-cm bags using an impulse sealer (Ceratek, Sencorp
Systems Inc., Hyannis, MA, USA). Approximately 190 grams of selected fresh-cut celery was
packaged into each PLA pouch.
PLA pouches packaged with fresh-cut celery were stored at 5°C for 21 days. Samples
were evaluated by a trained descriptive sensory initially (Day 0) and after 7, 14, and 21 days as
explained below. Samples were prepared in triplicate (from 3 different bags) at each analysis
day.

36

4.1.2. Panelist Selection
Panelists to be trained for quantitative descriptive sensory method (QDA) were selected
based on their ability to discriminate. Twenty students and faculty members from Michigan State
University (East Lansing, Michigan) were contacted based on their involvement with fruit and
vegetable research or previous trained sensory panel experience. A preliminary screening
assessment using triangle tests was administered to potential panelists, to evaluate their ability to
discriminate between celery colors and textures. From the screening test results, nine panelists
were selected to be trained to evaluate fresh-cut celery throughout storage.
4.1.3. Development of novel descriptive sensory methods and training of panelists
4.1.3.1. Standardized color scale
A standardized color scale for the assessment of color changes in fresh-cut celery was
developed from PANTONE® colors. The scale was based on a 9-point scale (1-yellow/light
green to 9-dark green) using nine PANTONE® colors. The standard PANTONE® colors from
light to dark green used for the scale were 587, 586, 585, 584, 381, 382, 583, 378, and 581. The
complete color scale is featured in Figure 6 along with a typical range of celery sticks from one
bunch. Panelists were trained to use the scale under consistent fluorescent lighting conditions in
individual evaluation booths. The upper/outer side of the fresh-cut celery sticks were used for
color evaluation.

37

Figure 6. Pantone® celery color scale

4.1.3.2. Flexibility texture method
A new 9-point QDA reference scale (1-very crisp to 9-very flexible) was developed to
assess the flexibility of fresh-cut celery. The scale is diagramed in Figure 7a and represents
product breakage angles from 10° to 90°. The references were developed to show breakage angle
increments at every ten degrees. Panelists were trained to press celery sticks (with the outer edge
facing the panelist) along their entire length onto an ink pad, transfer them to an anchored piece
of paper, and bend them with constant pressure until they snap. The inked side of the celery stick
remained in contact with the paper while bending. The angle created between the initial ink
impression and the impression at the point where the celery stick broke was compared to the
reference scale. Figure 7b diagrams the two angles created by the ink impressions when bending
celery, signified as Ѳ1 and Ѳ2. The flexibility of the celery was evaluated by taking the average
of the two angles.

38

Figure 7 a) Bending Scale used as a reference to compare with the ink impressions created from
bending celery sticks (1-very crisp to 9-very flexible) and b) angles (Ѳ1 and Ѳ2) of the ink
impressions created from bending celery sticks.

Ѳ1

Ѳ2

4.1.3.3. Hardness sensory method
A universal 15-point spectrum scale was used to evaluate the hardness of fresh-cut celery
(Meilgaard, 1999). The scale was slightly modified to fit within a 9-point range for celery.
Within the 15-point scale, celery was determined to fit within the range of 7 to 12 (5-very soft
celery to 14-very hard celery). Standard references for hardness included one whole pitted green
olive (7), one whole roasted peanut (9), ½ inch cube of carrot (11), and one whole roasted
almond (12). Panelists were trained to compress reference and fresh-cut celery samples between
their molars and compare their hardness (the force it takes to completely bite through the sample)
throughout storage.
4.1.4. Statistical analysis
Analysis of variance and the Tukey-Kramer test were used to determine any significant
differences at a confidence level of 95% (P ≤ 0.05). Statistical analysis was performed with SAS
version 9.3 software (SAS Institute, Cary, N.C., U.S.A.)
39

4.2. Results and discussion
4.2.1. Texture
Firmness relates to the softening of the plant tissue and the strength and thickness of the
cell wall are major contributors (Toivonen and Brummell, 2008). Therefore, the less firm the
product is, the more flexible the product will become. As celery ages, celery firmness decreases
and subsequently, celery flexibility increases. A new QDA was developed to assess changes in
celery flexibility. For that, trained panelists were asked to bend celery sticks and compare their
results to a reference scale. The standard bending scale represents the firmness of the celery
sticks by relating their flexibility to the angle at which the product breaks when it has been
deformed by bending. The new QDA method was compared against a universal spectrum
method used for measuring hardness. Like a change in celery flexibility, a change in the hardness
of celery indicates that the firmness of the celery is changing. The results for bending and
hardness evaluations by 9 trained panelists over a 3 week storage period can be seen in Table 5.
Average scores by panelists are shown out of a 9 point scale for each attribute ± standard
deviation.
No significant differences were found for the flexibility of celery sticks between
evaluation dates by the trained panel (Table 5). Therefore, the PLA bag seems to be able to retain
enough moisture from leaving the bag that results in a maintained firmness of the celery sticks
over a storage period of 3 weeks at 5°C. No significant differences in the sensory assessment of
hardness were observed in celery sticks that had been stored in PLA bags either (Table 5). The
lack of observable changes in sensory hardness could be attributed to the stability of the celery
fibers. Therefore, results from hardness evaluated by the spectrum method and from flexibility

40

evaluated by the developed QDA method are in agreement and show no differences in the
firmness of the packaged celery sticks over a storage period of 3 weeks at 5°C.
4.2.2. Color
The Pantone® color scale (Figure 6) was used to indicate the light to dark green color
spectrum of celery and observe any sensory color changes. Sensory tests performed showed that
the celery studied did not significantly differ in green color over time (Table 5). The
effectiveness of low O2 and high CO2 levels in preserving the green color of fresh-cut celery at 4
ºC (6% O2 and 7% CO2 at steady state) was reported also by Gómez and Artés (2005).
Table 5. Quantitative descriptive sensory analysis of the texture and color of fresh cut celery
stored at 5 °C for 21 days.
Time (Days)
Sensory
0
7
14
21
Method
Bending

3.1 ± 0.8a*

3.1 ± 0.6a

3.2 ± 0.8a

3.8 ± 0.9a

Hardness

8.7 ± 0.7a

8.7 ± 0.6a

8.7 ± 0.6a

8.8 ± 0.8a

Surface Color
7.4 ± 0.4a
7.0 ± 0.9a
6.7 ± 0.7a
7.2 ± 0.9a
* Each value represents the mean of three replicates ± standard deviation. Means sharing the
same lowercase letter indicate no significant difference (P < 0.05) between time points for each
attribute.
These results indicate that for the storage conditions evaluated, celery sticks were able to
maintain their sensorial properties for 3 weeks. No differences were observed for flexibility
(bending method), hardness, or color, indicating that PLA is a viable packaging option for freshcut celery sticks stored at 5 °C for up to 3 weeks. It is worth noting that these results to not take
into consideration off-flavor development or consumer liking of the packaged celery sticks.

41

5. Interactions Between Sanitizers and Packaging Gas Compositions and their Effects on the
Growth of Spoilage Microorganisms and Salmonella on Fresh-cut Onions (Allium cepa L.)
5.1. Materials and methods
5.1.1. Onion dicing
Spanish yellow onions (Allium cepa L.) were purchased from a local distributor (Stan
Setas Produce, Lansing, MI, USA), stored under refrigeration, and used within 1 day of delivery.
All onions were visually inspected, had their ends and outer layers manually removed and finally
were diced using an Urschel mechanical dicer model HA (Valparaiso, IN, USA). The same
procedures were repeated using three different batches of onions to obtain three replicates.
5.1.2. Preparation of inoculum and inoculation of the diced onions
Stock cultures of one avirulent strain Salmonella enterica subspecies enterica serovar
Typhimurium (LT2 , obtained from D. Michelle Danyluk, University of Florida, Gainesville, FL,
USA) were maintained at -80 °C and subcultured twice (24 h/37 °C) in 9 mL of trypticase soy
broth (Difco, Becton Dickinson, Sparks, MD, USA) containing 6 g/L yeast extract (Difco,
Becton Dickinson, Sparks, MD, USA). The culture was diluted in tap water (~2 ºC) to contain 5
log CFU/ml and then the diced onions (~ 4 ºC) were immersed for 2 min. This led to diced
onions with 3.87 ± 0.05 log CFU/g.
5.1.3. Sanitation of the diced onions
Approximately 23 kg per replicate of both Salmonella-inoculated and un-inoculated diced
onions were sanitized by immersion for 2 minutes in 60 L at ~2 ºC of one of the following
commercial sanitizers: 0.08 g/L sodium hypochlorite at pH 6.5 (XY-12, Ecolab, St. Paul, MN,
USA), 0.08 g/L peroxyacetic acid at pH 5.9 (Tsunami 100, Ecolab, St. Paul, MN, USA), or 0.002
g/L liquid chlorine dioxide (CDG Environmental, Bethlehem, PA, USA). Liquid chlorine

42

dioxide was used as a control because it is currently used to sanitize packaged diced onions
according to industry contacts. Concentrations of active sodium hypochlorite and peroxyacetic
acid were measured using a chlorine or peracid/peroxide test kit #321 and #311 (Ecolab, St. Paul,
MN, USA), respectively, while chlorine dioxide concentration was measured using chlorine
dioxide test strips (InstaTest UX-99532-34, Cole-Parmer, Vernon Hills, IL, USA). The sodium
hypochlorite wash was acidified according to manufacturer's instructions by adding a 100 g/L
citric acid solution and monitoring the pH until stable. After sanitation, the excess sanitizing
wash was spun off of the diced onions using a SD50-LT spin drier (Heinzen Manufacturing Intl.,
Gilroy, CA, USA).
5.1.4. Packaging of the diced onions
The centrifugally dried Salmonella-inoculated and un-inoculated diced onions were
weighed out into 100 g batches for each individual package. Two different packaging systems
were used: snap-fit containers and bags. The bags with diced onions that were to be filled with an
active modified atmosphere were placed into a sterilized glovebox chamber (Labconco 50004
Fiberglass Glove Box, Kansas City, MO, USA). The chamber was closed and then flushed with
the appropriate atmosphere (99 kPa O2 + 1 kPa N2 or 15 kPa CO2 + 5 kPa O2 + 80 kPa N2).
Finally, the bags were sealed using an impulse sealer located inside the chamber to obtain active
modified atmosphere packages. Passive modified atmosphere packages were obtained by sealing
the remaining bags under air. Snap-fit containers were simply filled with 100 g batches and the
lids were fastened on before storage. Packaging was conducted under sterile conditions at ~4 ºC.
All packages were stored at 7 ºC until the appropriate analysis day.

43

5.1.4.1. Packaging system characterization
Snap-fit containers made of polyethylene terephthalate (PET) (Clear Lam, Elk Grove
Village, IL, USA) had approximate internal dimensions of 9.5 × 9.5 × 2.5-cm, with a total
volume of 210 mL, an external surface of 270 cm2, and an average thickness of 330 µm. These
containers were chosen to mimic what industry is currently using for diced packaged onions
based on information provided from industry contacts. Bags were made of polylactic acid (PLA)
film (EVLON EV-HS1, BI-AX International Inc., Wingham, ON, Canada) and were formed
using an impulse sealer (Ceratek, Sencorp Systems Inc., Hyannis, MA, USA). PLA was chosen
as the packaging material because of its consumer desired sustainability properties (Koutsimanis
et al., 2012), it has better thermal processibility than other biopolymers (Rasal et al., 2010), and
can be processed on standard converting equipment with minimal modifications (Lim et al.,
2008). The approximate internal bag dimensions were 11 × 12.5-cm, resulting in a total area of
275 cm2.
O2, CO2, water vapor, and ethanol transmission rates of the whole packages (snap-fit
container and sealed bag) were determined. For the snap-fit containers, due to the presence of a
non-hermetically sealed lid, the whole package transmission rate for O2 and CO2 was measured
using a static method, adapting the procedure described by González et al. (2008) to a whole
package. Water vapor and ethanol transmission rates in the snap-fit containers were measured
using a gravimetric method. 12 mL of water or ethanol was placed on the bottom of the
packages, the lids were then snapped and their weight losses were measured over time using a
precision balance (OHAUS, model Discovery DV314C, Florham Park, NJ, USA). All testing
was conducted in triplicate and the results were expressed as kg/s∙package.

44

O2, CO2, water vapor, and ethanol transmission rates of the PLA bags were estimated by
using the permeability values of the PLA film to these gases and vapors previously calculated by
this research team (30.34±9.07 and 5.67±1.17 × 10-18 kg∙m/m2∙s∙Pa for CO2 and O2, respectively,
21.86±3.22 × 10-15 kg∙m/m2∙s∙Pa for water vapor, and 20.01±5.76 × 10-18 kg∙m/m2∙s∙Pa for
ethanol (González-Buesa et al., 2014)), the average thickness of the PLA film, the total area of
the bag, and the pressure difference of gases and vapors. For O2 and CO2, the pressure difference
was 101,350 Pa, and for water vapor and ethanol, the pressure difference was 1,408 Pa and 7,821
Pa, respectively. The results were expressed as kg/s∙package.
5.1.5. Analyses
5.1.5.1. Microbiological analyses
After 0, 3, 7, 10, and 14 days of storage at 7 C, 25 g of Salmonella-inoculated or uninoculated diced onions from each package were diluted 1:1 in Difco Neutralizing Buffer (Difco,
Becton Dickinson, Sparks, MD, USA) and homogenized in a stomacher (Stomacher 400
Circulator, Seward, Worthington, UK) at 260 rpm for 1 min. Un-inoculated samples were
appropriately diluted in sterile 1 g/L phosphate buffer solution and plated on either trypticase soy
agar + yeast extract (Difco, Becton Dickinson, Sparks, MD, USA) for quantification of
mesophilic bacteria after 24 h of incubation at 37 C, or potato dextrose agar (PDA) (Neogen
Corporation, Lansing, MI, USA) acidified to pH 3.5 with a 100 g/L tartaric acid solution for
quantification of yeasts and molds after 48 h at 23 C (following the Neogen Accumedia method
for PDA #7149). Inoculated samples were also serially diluted but then plated on the selective
and differential media brilliant green agar (Neogen Corporation, Lansing, MI, USA) for
quantification of S. Typhimurium after 24 h of incubation at 37 C.

45

5.1.5.2. Physico-chemical analyses
5.1.5.2.1. In-package atmosphere
In-package O2, N2, and CO2 levels were determined using a gas chromatograph (Thermo
Scientific Trace GC Ultra, Thermo Electron S.p.A., Rodano, Italy) equipped with a thermal
conductivity detector and a Carboxen 1010 Plot capillary column, 30 m in length with a film
thickness of 30 µm, and an internal diameter of 0.53 mm (Supelco, Bellefonte, PA, USA).
Helium was used as a carrier gas (3 mL min-1). Using a syringe (SGE, Austin, TX, USA), a 100µL headspace sample was removed through a silicone septum that was attached to each package
on the appropriate headspace sampling day. The initial gas compositions of all the packages were
determined to verify correct flushing by the glovebox. Subsequently, O2, N2, and CO2 levels
were monitored on days 2, 6, 9, and 13 of storage at 7 °C. Snap-fit packages were also monitored
throughout storage for headspace gas concentrations. These results are not shown because the inpackage O2, N2, and CO2 levels were the same as those in atmospheric air.
In-package ethanol concentrations were determined after 7, 10, and 14 days of storage at
7 °C by injecting 1 mL of headspace gas into a gas chromatograph equipped with a flame
ionization detector (Hach Carle Series 100 AGC, Loveland, CO, USA) fitted with a 2 m long,
2 mm internal diameter stainless steel column packed with Chromosorb OV-103, 60/80 mesh
(Altech Associates Inc., Deerfield, IL, USA).
5.1.5.2.2. Color
Diced onion color was determined after 0, 3, 7, 10, and 14 days of storage at 7 °C using a
Minolta CR300 colorimeter (Osaka, Japan) calibrated with a standard white plate (Y=93.84;
x=0.3132; y=0.3191) and set with a C illuminant, 2º observer. Four samples of diced onions
were taken from each package and were then placed into glass sample cups (#04720900 Hunter

46

Lab, Reston, VA USA). Each sample cup was measured in three different places and L* and b*
values recorded. The average value of the twelve measurements was used to represent package
L* and b* values for each replicate.
5.1.5.2.3. Weight Loss
The weight loss of the diced onions was determined after 3, 7, 10, and 14 days of storage
at 7 °C for all treatments by weighing the entire package over time and using the formula shown
below, with the results expressed as % weight loss.
(𝑊𝑖 − 𝑊𝑓)
[
] ∗ 100
𝑊𝑖 − 𝑊𝑝
Where: Wi is the initial weight of the diced onions plus the weight of the package, Wf is the final
weight of the diced onions plus the weight of the package, and Wp is the weight of the package.
5.1.5.2.4. pH
The pH of the diced onions was measured on day 0, 3, 7, 10, and 14 of storage at 7 °C
using an Omega PHB-212 pH meter (OMEGA Engineering, Inc., Stamford, CT, USA),
calibrated everyday of testing with buffer solutions at pH 4 and 7. Approximately 10 g of diced
onions from each package were placed into sterile 50 mL centrifuge tubes (Corning, Tewksbury,
Massachusetts, USA), mixed with 10 mL of distilled water, and homogenized using a PT-2000
polytron (Kinematica, Lucerne, Switzerland) for 30 seconds. The pH of the resulting solution
was measured twice and the average value was used to represent package pH. Between each
sample the pH meter probe was rinsed with distilled water and patted dry.
5.1.5.3. Sensory Analyses
5.1.5.3.1. Descriptive sensory analysis
Descriptive sensory analysis was performed to determine the acceptable storage period
for diced onions packaged with the best atmosphere/sanitizer combination for maintenance of
47

physico-chemical and microbiological quality when compared with freshly packaged diced
onions. Panelists experienced with fresh produce quality and previous experience on a trained
sensory panel were recruited and screened based on their ability to discriminate between samples
of freshly diced onions and diced onions stored at 23 ºC for 12 hours using a triangle
discrimination test. The eight panelists selected were then trained to use 5-point intensity scales
prior to sample evaluation. Two samples of packaged diced onions were provided to the panelists
for evaluation on days 0, 3, 7, 10, and 14 of storage at 7 C . The two samples were 1) the best
packaging treatment as predetermined in the physico-chemical and microbiological results, and
2) packages containing freshly diced onions (less than 12 h). Panelists were also shown freshdiced onions to remind them of the desired characteristics for fresh onions each testing day.
Attributes of color, “off-odor”, and overall appearance were rated using 5-point intensity scales.
Color and overall appearance were evaluated from the outside of the package for both samples
first, followed by off-odor. To determine off-odor, panelists were given a small needle to poke a
hole in the bag so as to not overwhelm their olfactory sense by opening the entire bag. Finally,
the panelists were asked to determine if the packaged onions were acceptable for purchase at the
grocery store based on being trained as to what was an acceptable level of quality. Samples for
sensory evaluation were prepared under the same conditions used for the other portions
(microbiological and physico-chemical) of this study, including the use of the commercial dicer
to best mimic industry conditions. However, since the commercial dicer was previously used
with inoculated samples, it was not considered safe to ask panelists to consume the diced onions.
Because of this, panelists were restricted to the attributes and chosen methods (i.e. closed
packages) described above. The descriptive sensory training and analysis were held in the

48

sensory laboratory at Michigan State University, under controlled conditions (light, temperature,
aroma) with each panelist performing the test at their own booth to minimize distractions.
5.1.5.3.2. Consumer acceptance sensory analysis
A consumer acceptance sensory analysis was performed to determine the acceptability of
the packaged diced onions on day 14 by consumers. Eighty-five consumers of fresh onions were
recruited and asked to evaluate the same two sample packages as the descriptive sensory analysis
panelists evaluated. For both packages, panelists were provided a hedonic 9-point scale and
asked to evaluate the acceptability of the onion aroma from outside the package, since this is
how they would evaluate the package in the grocery store. They were also asked to rate their
acceptance of the onion color and overall appearance of the packaged diced onions. Finally, they
were asked if they would consider this product to be acceptable for purchase at the grocery store.
In addition, panelists were asked about their fresh-cut produce and onion purchasing behavior
and for feedback as to what would make them more likely to purchase fresh-cut onions if they
were not already. Panelists were asked to evaluate the aforementioned attributes and not to open
the packages for the same reasons as presented for the trained panel. The consumer acceptance
sensory test was held in the sensory laboratory at Michigan State University, under controlled
conditions (light, temperature, aroma) with each panelist performing the test at their own booth
to minimize distractions.
5.1.5.4. Statistical analysis
Both microbiological and physico-chemical analyses were conducted on three replicates
on each analysis day, and data were expressed as means ± standard deviation. Each of the three
replicates consisted of three packages: one was used for microbial analysis and the other two
were used for physico-chemical analyses. Three data points were obtained for Salmonella,

49

mesophilic bacteria, yeast/molds, and pH while six data points were obtained for O2, N2, CO2,
ethanol, and weight loss on each analysis day. A total of 36 data points were obtained for color
on each analysis day. Differences caused by time, atmosphere, and sanitizer and their
interactions for all the evaluated parameters were determined through two-way analysis of
variance (ANOVA) performed using the statistical software package IBM SPSS version 19 (IBM
Corporation Software Group, Somers, NY, USA) and confirmed with the GLIMMIX procedure
of SAS (SAS Inst. Cary, NY). Additionally, one-way ANOVA was performed using the
statistical software package IBM SPSS version 19 to show differences caused by time,
atmosphere, and sanitizer for weight loss, ethanol and pH at each analysis day. Tukey’s test at a
significance level P < 0.05 was used for both one-way and two-way ANOVA. The means of the
microbiological, color (L* and b*), and headspace analyses were also compared with the least
significant difference (LSD) test (P < 0.05).
5.1.6. Treatment labels
As previously described, all onions were sanitized with one of three sanitizers and
packaged in PLA bags containing either active or passive modified atmosphere or in PET snapfit containers which always contained atmospheric air. For simplicity, throughout this paper the
atmosphere/sanitizer combinations are presented as codes comprised of the abbreviation of the
initial in-package atmosphere, followed by the abbreviation of the sanitizer type used. Table 6
shows the abbreviations that comprise the codes (i.e., PA O2 for onions sanitized with
peroxyacetic acid and packaged in 99 kPa O2).

50

Table 6. Packaging and sanitizer abbreviations that comprise the codes used in this paper.

Sanitizer
Sodium hypochlorite
Peroxyacetic acid
Chlorine dioxide (liq.)

Sanitizer
Abbreviation
Cl
PA
ClO2

Packaging
Abbreviation
O2
CO2
Air
SF

Initial Atmosphere/ Packaging Design
99 kPa O2 + 1 kPa N2 / Bag
15 kPa CO2 + 5 kPa O2 + 1 kPa N2 / Bag
21 kPa O2 + 79 kPa N2 / Bag
21 kPa O2 + 79 kPa N2 / Snap-fit

5.2. Results and discussion
5.2.1. Packaging system characterization
O2, CO2, WV, and ethanol transmission rates of the PLA bag and the PET snap-fit container are
shown in Table 7. The O2, CO2, and ethanol transmission rates of the PET snap-fit container are
higher than those of the PLA bag. The greater gas exchange in the PET snap-fit container is due
to the significant diffusion of O2, CO2, and ethanol through air due to its non-hermetic closure. In
fact, the diffusion coefficient of O2 and CO2 through air was obtained to be approximately 450
and 62 million times, respectively, greater than through the PET sheet when using the approach
of Yasuda (1975) to make such comparisons possible, and the diffusion coefficient of O2 and
CO2 in air provided by Mannaperuma et al. (1999) and Massman (1998), respectively. In
contrast, the WV transmission rate of the PET container was lower than that of the PLA bag.
This resulted from the weaker barrier to WV of PLA compared to PET (Almenar and Auras,
2010), the reduced thickness of the PLA film compared to the PET sheet, and the lower water
vapor exchange through the PET container closure than through the PET container wall. Based
on these results, the PET snap-fit container behaves similar to a microperforated package, where
microperforations have a greater impact on O2 content than on WV content inside package
(Fishman et al., 1996).

51

Table 7. Transmission rates (TR) of gases and vapors in the two packaging systems used in this
study.
Packaging
Whole package TR (×10-10 kg/s∙package )
system
O2
CO2
WV
Ethanol
PET container1

1402 ± 91

1591 ± 117

3.6 ± 0.7
19 ± 6
PLA bag2
Whole package TR values calculated in this study.

45 ± 2

81 ± 29

192 ± 28

1.0 ± 0.3

1

2

Whole package TR values estimated from film permeability values in González-Buesa et al.
(2014).

5.2.2. Effect of atmosphere/sanitizer combinations on microbial growth in diced onions
5.2.2.1. Salmonella Typhimurium
Figures 1 and 2 show the growth of S. Typhimurium on diced onions under different
atmosphere/sanitizer combinations throughout two weeks of storage at 7 °C. Interactions
between atmosphere and time (P = 0.003), sanitizer and time (P = 0.040), and atmosphere and
sanitizer (P = 0.003) affected the growth of S. Typhimurium on packaged diced onions (Table 8).
None of the studied atmosphere and sanitizer combinations were able to completely stop the
growth of S. Typhimurium throughout time (P< 0.05), but some combinations were able to
reduce growth compared to others. The snap-fit packages had the diced onions with the most S.
Typhimurium growth (P < 0.05) throughout storage regardless of the type of sanitizer (Figure 8).
Diced onions sanitized with ClO2 contained significantly (P < 0.05) more S. Typhimurium than
those sanitized with Cl throughout storage regardless of the in-package atmosphere (Figure 9).

52

Figure 8. Effect of sanitizer/atmosphere combinations on the growth of Salmonella
Typhimurium, Total aerobic bacteria, and yeasts and molds on diced onions during 14 days of
storage at 7 oC.

This indicates that the commercially used package (snap-fit container) and sanitizer (liquid
chlorine dioxide) are not effective at preventing S. Typhimurium growth in the instance of
contamination. However, an in-package atmosphere other than air can reduce S. Typhimurium
growth on diced onions (P < 0.05). This is consistent with findings by other authors. Amanatidou
et al. (1999) reported that the combined application of high O2 and moderate CO2 had an
inhibitory effect on the growth of nonpathogenic S. Enteritidis. Martínez-Hernández et al. (2015)
showed a bacteriostatic effect on S. Enteritidis in fresh-cut broccoli sanitized with PA and stored
in either passive MAP or active MAP (90% O2) at 5 ºC. .
Interactions between atmospheres and sanitizers that affect S. Typhimurium growth on
diced onions were observed as well, with diced onions of the Cl CO2 treatment having the
53

lowest S. Typhimurium populations throughout storage (about 1.5 log CFU/g of growth from day
0, compared to 2.5 - 5 log CFU/g of growth for all other treatments). Therefore, the combination
of CO2 and Cl works to reduce S. Typhimurium growth in diced onions.

54

Table 8. P values resulting from Univariate ANOVA (SPSS) (P < 0.05 indicates effect of factor (single or combined (interaction)) on
each parameter).
Parameter
S. Typhimurium Bacteria Yeasts&Molds
O2
CO2
Factor
Atmosphere
0.000*
0.000*
0.000*
0.000* 0.000*
Sanitizer
0.019*
0.000*
0.000*
0.307 0.003*
Time
0.000*
0.000*
0.000*
0.000* 0.000*
Atmosphere*Sanitizer
0.003*
0.212
0.075
0.066 0.061
Atmosphere*Time
0.003*
0.021*
0.005*
0.000* 0.000*
Sanitizer*Time
0.040*
0.271
0.005*
0.551 0.197
Atmosphere*Sanitizer*Time
0.551
1.000
0.947
0.086 0.007*
* indicates significant (P < 0.05)

55

Ethanol
L*
b*
Weight Loss
0.000* 0.000* 0.000*
0.000*
0.530
0.804 0.462
0.000*
0.000* 0.000* 0.000*
0.000*
0.520
0.203 0.873
0.105
0.000* 0.000* 0.000*
0.000*
0.271
0.445 0.651
0.005*
0.996
0.778 0.949
0.199

pH
0.000*
0.001*
0.000*
0.011*
0.000*
0.247
0.476

Figure 9. Effect of atmosphere/sanitizer combinations on the growth of Salmonella
Typhimurium, Total aerobic bacteria, and yeasts and molds on diced onions during 14 days of
storage at 7 oC.

5.2.2.2. Mesophilic bacteria
The effect of different atmosphere/sanitizer combinations on the growth of mesophilic
bacteria on diced onions throughout two weeks of storage at 7 °C is illustrated in Figures 9 and
10. Mesophilic bacteria were affected by atmosphere and time (P = 0.021), sanitizer (P = 0.000),
and time (P = 0.000) (Table 8). Snap-fit packages had diced onions with significantly higher (P
< 0.05) mesophilic bacteria populations, 6 log CFU/g increase from day 0 to day 14, regardless
of the type of sanitizer (Figure 8). Therefore, it seems that an in-package atmosphere other than
air can reduce bacterial growth on diced onions. Howard et al. (1994) and Forney et al. (2012)
reported the positive effect of CO2 in reducing bacteria growth in packaged diced onions stored
at low temperature. Blanchard et al. (1996) found that reduced O2 accompanied with elevated
56

CO2 controlled atmospheres reduced mesophilic and psychrotrophic growth in diced onions
during 2 week storage at 4 °C. As for the effect of sanitizer on bacterial growth, diced onions
sanitized with Cl had significantly lower (P < 0.05) mesophilic bacterial populations than those
sanitized with PA or ClO2 regardless of the in-package atmosphere (Figure 9). Therefore, the
commercially used package (snap-fit container) and sanitizer (liquid chlorine dioxide) are not the
best choices for commercializing diced onions in terms of bacterial growth.
5.2.2.3. Yeasts and molds
Yeast and mold population changes over time as affected by atmosphere/sanitizer
combinations can be seen in Figures 1 and 2. Atmosphere, sanitizer, and time all had significant
effects on yeast and mold growth (P = 0.000 for all three; Table 8). Snap-fit and CO2 in-package
atmospheres had diced onions with the highest and the lowest yeast and mold populations (P <
0.05), respectively, compared to all other atmospheres regardless of sanitizer used (Figure 8). In
contrast, Liu and Li found no significant differences between yeast populations on sliced onions
packaged in 40 KPa CO2 + 1KPa O2 or air. Since the diced onions exposed initially to CO2 had
less yeast and mold populations than those exposed to the CO2 build-up during storage (O2 and
Air atmospheres; section 3.3.1), it can be concluded that the microbial quality in terms of yeasts
and molds on diced onions can be improved using active MAP with high CO2. Diced onions
sanitized with Cl had significantly lower (P < 0.05) mold and yeast counts than those sanitized
with either PA or ClO2 regardless of in-package atmosphere (Figure 9). These findings further
prove that the current commercially used atmosphere/sanitizer combination is not the best option
for packaging diced onions.

57

5.2.3. Effect of atmosphere/sanitizer combinations on physico-chemical changes in diced onions
5.2.3.1. In-package atmosphere
As expected, in-package atmosphere and time each had a significant effect (P = 0.000 for
both; Table 8) on the O2 levels inside the PLA bags, as seen in Figure 10. The bags with an inpackage atmosphere CO2 reached O2 equilibrium from the start (~1-2 kPa), while the bags with
an in-package atmosphere Air equilibrated (~1.5-3 kPa) around day 7 of storage and those with
an in-package atmosphere O2 did not reach O2 equilibrium (~1.5-3 kPa) until approximately day
9. Sanitizer or interactions between the investigated factors did not have a significant effect on
the O2 levels inside the PLA bags throughout storage (Table 8). In contrast, sanitizer (P = 0.003)
and 2-way (atmosphere and time; P = 0.000) and 3-way (atmosphere and sanitizer and time; P =
0.007) interactions affected the CO2 levels inside the PLA bags (Table 8). CO2 concentration
steadily increased from 0 to ~ 20 kPa in the Air and O2 packages while it was maintained quite
stable in the CO2 packages up to day 9 and then increased ~5 kPa. This lower accumulation of
CO2 in CO2 packages can be attributed to the faster reduction of O2 occurring in these packages
and/or the presence of high CO2 which reduced the respiration rate of the diced onions.
Similarly, Day (1989) observed a reduction in the respiration rate of cut onion under low O2.
Blanchard et al. (1996) suggested that both low O2 and high CO2 are necessary to reduce
respiration in diced onions. The increase in CO2 content of ~5 kPa after day 9 in the packages
CO2 was only observed for the diced onions sanitized with PA or ClO2 (3-way interaction P =
0.007; Table 8). This could be attributed to the lower S. Typhimurium populations for this
treatment (Figure 9), while the other treatments have greater microbial respiration attributing to
their in-package CO2 accumulation.

58

Figure 10. Effect of atmosphere/sanitizer combinations on in-package gas composition of diced
onions during 14 days of storage at 7 oC.

Packages containing onions sanitized with Cl had significantly less (P < 0.05) CO2
content than those sanitized with PA or ClO2, regardless of the in-package atmosphere. GómezLópez et al. (2008) observed an increased respiration rate in packaged RTU cabbage sanitized
with ClO2 compared to unsanitized RTU cabbage and Martínez-Sánchez et al. (2006) observed
an increase respiration rate in packaged rocket leaves sanitized with PA compared to lactic acid
or acidified sodium chloride. In addition, diced onions sanitized with Cl had significantly lower
(P < 0.05) mesophilic bacteria, mold and yeast counts than those sanitized with either PA or
ClO2, regardless of in-package atmosphere (Figure 9). The greater microbial respiration in the
packages with diced onions sanitized with either PA or ClO2 may have contributed to a higher
in-package CO2 buildup.
59

Headspace ethanol concentration (Table 9) was only significantly affected by in-package
atmosphere throughout storage (interaction atmosphere and time; P = 0.000; Table 8). The
packages containing diced onions exposed to the in-package atmosphere CO2 had the highest (P
< 0.05) ethanol concentrations (80-100 ppm) while those in snap-fit packages and O2 had the
lowest (P < 0.05) ethanol concentrations with no difference (P > 0.05) between them. Forney et
al. (2012) attributed a rapid increase of ethanol content in packaged diced onions to low O2
concentrations. This agrees with the presented results since the more rapid decrease of O2
occurred in the packages with the in-package atmosphere CO2 regardless of sanitizer, and these
were the packages containing the highest ethanol contents. The significantly greater (P < 0.05)
in-package ethanol content for the CO2 packaged diced onions compared to diced onions
packaged in Air, O2, and snap-fit containers could have also contributed to the lower yeast and
mold populations of the CO2 packaged diced onions. Ethanol has been reported to reduce mold,
yeast and bacteria growth and is most effective against mold (Almenar and Gartner, 2015). A
difference in ethanol concentration between snap-fit containers and PLA bags was expected due
to the faster diffusion of the ethanol through the non-sealed lid of the snap-fit container as shown
in Table 7. However, this difference was not observed between snap-fit containers and PLA bags
containing high O2 levels, which may indicate a lower production of ethanol in diced onions
when exposed to high O2 levels compared to air.

60

Parameter

Table 9. Effect of atmosphere/sanitizer combinations on the weight loss, in-package ethanol, and pH of packaged diced onions during
14 days of storage at 7 oC.
Stor
age
Tim
e
(Da
ys)
0

Cl O2

PA O2

ClO2 O2

Cl CO2

PA CO2

ClO2 CO2

Cl Air

PA Air

ClO2 Air

Cl SF

PA SF

ClO2 SF

n/a
0.26 ±
0.04 aB1
1.15 ±
0.06 bB1
1.82 ±
0.08 cB1
2.82 ±
0.17 dB1
n/a

n/a
0.23 ± 0.04
aB1*
1.10 ± 0.23
bB1
1.75 ± 0.09
cC1
2.77 ± 0.27
dB1
n/a

n/a
0.26 ± 0.05
aB1
1.18 ± 0.05
bB1
1.89 ± 0.12
cB1
2.92 ± 0.18
dB1
n/a

n/a
0.28 ±
0.06 aB1
1.17 ±
0.08 bB1
1.88 ±
0.08 cB1
2.91 ±
0.15 dB1
n/a

n/a
0.27 ± 0.03
aB1
1.17 ± 0.06
bB1
1.83 ± 0.04
cC1
2.69 ± 0.14
dB1
n/a

n/a
0.29 ±
0.06 aB1
1.27 ±
0.09 bB1
1.91 ±
0.13 cB1
2.95 ±
0.23 dB1
n/a

n/a
0.25 ±
0.05 aB1
1.17 ±
0.06 bB1
1.75 ±
0.08 cB2
2.82 ±
0.13 dB12
n/a

n/a
0.25 ± 0.05
aB1
1.19 ± 0.22
bB1
1.63 ± 0.04
cB1
2.65 ± 0.13
dB1
n/a

n/a
0.27 ± 0.04
aB1
1.15 ± 0.09
bB1
1.82 ± 0.05
cB2
2.94 ± 0.21
dB2
n/a

n/a
0.03 ±
0.02 aA1
0.30 ±
0.01 bA2
0.57 ±
0.07 cA1
0.91 ±
0.06 dA1
n/a

n/a
0.04 ±
0.01 aA1
0.23 ±
0.01 bA1
0.44 ±
0.04 cA1
0.83 ±
0.11 dA1
n/a

n/a
0.06 ±
0.01 aA1
0.27 ±
0.02 bA2
0.46 ±
0.06 bA1
0.77 ±
0.15 cA1
n/a

n/a
8.59 ±
4.94 aAB1

n/a
6.07 ± 1.75
aA1

n/a
7.39 ± 4.42
aAB1

n/a
23.85 ±
15.76 aB1

n/a
15.5 ±
15.57 aA1

n/a
14.48 ±
8.26 aAB1

n/a
15.43 ±
1.45 aAB1

n/a
18.85 ±
4.67 aB1

n/a
6.58 ±
3.71 aA1

n/a
5.47 ±
4.46 aA1

n/a
4.67 ±
3.93 aA1

10

10.42 ±
10.34 aA1

9.77 ± 7.06
aAB1

14.26 ±
4.18 aA1

60.67 ±
6.69 bC1

52.11 ±
15.09 abC1

46.32 ±
3.87 aC1

34.39 ±
0.16 abB1

n/a
16.17 ±
1.33 aA1
31.63 ±
15.85
abBC1

29.34 ±
10.11 abB1

4.09 ±
1.70 aA1

4.81 ±
2.94 aA1

4.77 ±
5.39 aA1

14

24.40 ±
8.53 aAB1

34.89 ±
10.82
bAB1
5.64 ± 0.03
cA1
5.55 ± 0.07
bcAB1
5.05 ± 0.42
abcAB1
4.92 ± 0.02
abAB1

82.04 ±
5.72 bC1

100.93 ±
42.54 bC1

83.63 ±
49.51 aB1

42.87 ±
15.84 bB1

66.6 ±
18.43 bBC1

79. 65 ±
39.50 bB1

7.52 ±
5.17 aA1

8.88 ±
6.75 aA1

10.08 ±
4.54 aA1

5.64 ±
0.03 aA1
5.51 ±
0.03 aA1
5.22 ±
0.62 aA1
4.77 ±
0.74 aA1

40.21 ±
12.25
bAB1
5.64 ± 0.03
aA1
5.51 ± 0.04
aA1
5.18 ± 0.54
aA1
5.19 ± 0.11
aB1

5.64 ±
0.03 aA1
5.73 ±
0.06 abB1
5.68 ±
0.04 abA1
5.76 ±
0.05 bA1

5.64 ± 0.03
aA1
5.67 ± 0.08
aA1
5.25 ± 0.48
aA1
5.18 ± 0.59
aAB1

5.64 ±
0.03 aA1
5.74 ±
0.11 aB1
5.50 ±
0.17 aB1
5.55 ±
0.17 aB1

5.64 ±
0.03 aA1
5.46 ±
0.04 aA1
5.53 ±
0.05 aA2
5.46 ±
0.25 aA1

5.64 ± 0.03
aA1
5.49 ± 0.10
aA1
5.37 ± 0.22
aA12
5.45 ± 0.10
aB1

5.64 ± 0.03
aA1
5.50 ± 0.06
aA1
5.01 ± 0.20
aAB1
5.23 ± 0.33
aB1

5.64 ±
0.03 bA1
5.56 ±
0.17 bA1
4.47 ±
0.38 aA1
4.38 ±
0.11 aA1

5.64 ±
0.03 bA1
5.49 ±
0.07 bA1
4.37 ±
0.24 aA1
4.53 ±
0.35 aA1

4.76 ±
0.63 aA1

5.15 ± 0.49
aA1

4.81 ± 0.41
aA1

5.91 ±
0.02 cB2

5.51 ± 0.06
aA1

5.43 ±
0.14 aA1

5.63 ±
0.18 aAB1

5.10 ± 0.99
aA1

5.44 ± 0.37
aA1

5.64 ±
0.03 bA1
5.53 ±
0.06 abA1
4.85 ±
0.24 aA1
5.35 ±
0.20 abA2
5.37 ±
0.48
abAB1

4.65 ±
0.09 aA1

4.73 ±
0.29 aA1

Wt Loss

3
7
10
14
0

Ethanol

3
7

0
3
pH

Treatment

7
10
14

61

Table 9 (cont’d)
* Means sharing the same lowercase letter in the same column show no significant (P < 0.05)
difference caused by time; means sharing the same uppercase letter in the same row for each
sanitizer show no significant (P < 0.05) difference caused by gas composition; means sharing the
same number in the same row for each gas composition show no significant (P < 0.05) difference
caused by sanitizer.

5.2.3.2. Color
As shown in Figure 11, regardless of sanitizer, the in-package atmosphere CO2 best
maintained the diced onions’ L* value throughout storage while the snap-fit containers promoted
the most (P < 0.05) darkening (interaction atmosphere and time; P = 0.000; Table 8). There was
no difference between the effect of in-package atmospheres Air and O2 on the luminosity of the
diced onions, which can be attributed to the same CO2 contents reached in these packages
throughout storage (Figure 10). Blanchard et al. (1996) reported that the higher the CO2 content
surrounding the diced onions, the lower the darkening since they observed that diced onions kept
in a controlled atmosphere of 2 KPa O2 + 15 KPa CO2 had the least browning after 14 days
compared to air and 2 KPa oxygen with 0, 5, and 10 kPa of CO2. The b* value for diced onions
(Figure 11) was also only significantly affected by in-package atmosphere throughout storage
(interaction atmosphere and time; P = 0.000; Table 8). Although a change in color was observed
in the diced onions of all packages, the diced onions in snap-fit containers were significantly (P <
0.05) more yellow over time. There was no difference in the yellowness of the diced onions
between in-package atmospheres CO2, Air, and O2. Blanchard et al. (1996) also observed less
yellowing in diced onions exposed to high CO2 contents. Therefore, it seems that an in-package
atmosphere rich in CO2 can minimize both enzymatic browning and tissue yellowing in diced
onions for all sanitizers studied. The sanitizers studied did not have an effect on either browning
or tissue yellowing in diced onions (P = 0.804). The color of the diced onions was the same

62

throughout storage for all the sanitizers investigated although the capability of ClO2 to promote
browning in some RTU products has been reported (Gómez-López et al., 2008).

63

Figure 11. Effect of atmosphere/sanitizer combinations on the L* and b* values of diced onions during 14 days of storage at 7 oC.

64

5.2.3.3. Weight loss
The weight loss of the diced onions increased as a function of time (P = 0.000; Table 8).
At the end of storage, diced onions in the bags had a weight loss of ~3 % while those in the snapfit containers had less than 1% weight loss, regardless of sanitizer (Table 9). The reason for this
difference was the different water vapor permeability of the two packaging designs. The PLA
bags had a water vapor transmission rate approximately 4 times greater than that of the PET
snap-fit containers as shown in Table 7 and discussed in section 3.1.
The sanitizer used also affected the weight loss of the onions (P = 0.000; Table 8), with
diced onions sanitized with PA having significantly lower (P < 0.05) weight loss than those
sanitized with Cl or ClO2. The potential of ClO2 to damage tissue of RTU products has
previously been reported in the literature (Gómez-López et al., 2008). An interaction between
sanitizer and time (P = 0.005) was observed for diced onion weight loss, with the diced onions in
CO2 or O2 sanitized with PA having the same weight loss at day 10. Regardless of packaging
system and sanitizer, the change in weight of the diced onions throughout storage was not big
enough to affect their marketability as shown in section 3.7. Moreover, Ohta et al. (2002)
reported that weight losses which exceed 5% of the initial weight led to limited marketability and
reduced retail value of fresh produce. Based on this criterion, the diced onions of all treatments
would be marketable throughout the 14 days of storage.
5.2.3.4. pH
Acidification of all the packaged diced onions occurred throughout storage (P = 0.000),
although differently depending on the in-package atmosphere and sanitizer (P = 0.000 and P =
0.001, respectively) (Table 8). Onions packaged in in-package atmospheres Air or CO2 had a
significantly higher (P < 0.05) pH than those packaged in in-package atmospheres O2 or snap-fit

65

containers (Table 9). Therefore, it seems that a combination high CO2 and low O2 is necessary to
reduce acidification in diced onions since the in-package atmospheres Air and O2 both had the
same high CO2 content throughout storage, but they differed in O2 content during most of the
storage period (Figure 10). Forney et al. (2012) also observed the least amount of acidification
for diced onions in packages with the highest/lowest amounts of CO2/O2.
Onions sanitized with Cl had a significantly higher (P < 0.05) pH than those sanitized
with PA or ClO2. The higher acidification of the diced onions sanitized with PA or ClO2 can be
attributed to a greater amount of bacterial growth since diced onions sanitized with Cl had
significantly lower (P < 0.05) mesophilic bacteria populations than those sanitized with PA or
ClO2, regardless of the in-package atmosphere (Figure 9). In agreement, Forney et al. (2012)
reported that increased microbial growth in diced onions caused a drop in their pH during
storage. In contrast, Beerli, Vilas Boas and Piccoli (2004) observed that hydrogen peroxide
decreased both counts of aerobic mesophiles and pH in packaged sliced onions compared to
sodium dichloroisocianurate. Interactions between atmosphere and sanitizer were also observed
with Cl SF and PA O2 resulting in less acidification of packaged diced onions (atmosphere and
sanitizer P = 0.011; Table 8).
5.2.4. Effect of atmosphere/sanitizer combinations on sensory attributes of diced onions
Cl CO2 was the best in-package gas and sanitizer combination for enhancing the safety
and quality of packaged diced onions as determined by the above microbiological and physicochemical results. In order to determine the consumer acceptability of diced onions packaged in
this gas composition/sanitizer combination, they were compared with freshly packaged diced
onions for two weeks using descriptive sensory analysis and after 14 days using consumer
acceptance sensory analysis. The results are presented below.

66

5.2.4.1. Descriptive sensory analysis
Trained panelists rated the off-odor, color, and overall appearance of diced onions
packaged in Cl CO2 with significantly (P < 0.05) higher scores (poorer quality) than freshly
packaged diced onions starting on day 7 of storage (Table 10). However, at day 14, panelists
scored the color and overall appearance of the two different packaged diced onions as not being
statistically different. The lack of differences detected at day 14 most likely was due to the use
of a different batch of onions to obtain the freshly packaged diced onions used on each analysis
day. However, a higher off-odor score was still obtained for the diced onions packaged in Cl
CO2 compared to the freshly packaged diced onions. Because the majority of panelists (6/8)
deemed diced onions packaged in Cl CO2 as acceptable for purchase after 14 days storage, this
storage period was determined to be the acceptable shelf life of the diced onions packaged in Cl
CO2 and chosen for the consumer panel to be performed in order to evaluate the consumer
acceptance of onions packaged in Cl CO2. Other authors have also reported that the combination
of chlorine sanitizer and elevated CO2/ reduced O2 atmosphere can lengthen the consumer
acceptability for diced onions. Blanchard et al. (1996) reported that a trained panel scored higher
(higher quality) chlorine-sanitized diced onions stored in 2 kPa O2 + 10 kPa CO2 and 2 kPa O2 +
15 kPa CO2 than those stored in air for both appearance and aroma after 14 days.

67

Table 10. Mean sensory scores for freshly packaged diced onions (I) and diced onions packaged
in Cl CO2 (II) for 14 days at 7 °C.
Off-odor
Color
Appearance
Acceptable for Purchase
I
II
I
II
I
II
I
II
Day
0
1 aA* 1.13 aA 1.06 aA
1 aA
1.13 aA 1.06 aA
100%
100%
3
1.38 aA 1.25 aA 1.56 aA 1.13 aA 1.56 aA 1.13 abA
100%
100%
7
1.25 aA 2.5 bB 1.31 aA 2.06 bB 1.38 aA 2.13 cB
100%
87.5%
10
1.31 aA 2.69 bB 1.25 aA 2.25 bB 1.25 aA 2.19 cB
100%
87.5%
14
1.44 aA 3.19 bB 1.38 aA 2.06 bA 1.44 aA
2 bcA
100%
75%
*Means sharing the same lowercase letter in the same column are not significantly (P < 0.05)
different. Means sharing the same uppercase letter in the same row are not significantly (P <
0.05) different. Number of trained panelists = 8. Three different 5-point scales were used (1 = no
off odor, 5 = extreme off odor, rancid; 1 = bright white, 5 = darkened/discolored; 1 = fresh
looking, typical onion appearance, 5 = mushy, dried out, wet on surface, spoiled).

5.2.4.2. Consumer acceptance sensory analysis
The results from the consumer panel (Table 11) show that panelists were not able to
differ (P < 0.05) between the aroma, color, and overall appearance of diced onions packaged in
Cl CO2 for 14 days and freshly packaged diced onions. In addition, the difference of purchase
acceptability between the diced onions in the two packages was minimal. Over 97% of panelists
said that the freshly packaged onions were acceptable for purchase versus 94% for diced onions
packaged in Cl CO2, indicating that the evaluated gas composition/sanitizer combination can
successfully maintain the accepted consumer quality of diced onions for 14 days. While the
diced onions were deemed acceptable for purchase after 14 days of storage, the test was not
carried out further because of the microbiological growth at that point in the experiment. Aerobic
bacterial populations reached approximately 6 log CFU/g after 14 days in Cl CO2 (Figure 9), at
which point the study was terminated.

68

Table 11. Mean sensory scores for freshly packaged diced onions (I) and diced onions packaged
in Cl CO2 (II) for 14 days at 7 °C.
Aroma
Color
Overall Appearance Acceptable for Purchase?
I
II
I
II
I
II
I
II
6.04 A*
5.94 A
7.57 A
7.27 A
7.39 A
7.09 A
97.6% A
94.1% A
*Means sharing the same letter in the same row are not significantly (P < 0.05) different.
Number of consumer panel = 85. Two 9-point hedonic score were used (1 = dislike extremely, 9
= like extremely).

69

CONCLUSIONS
The initial headspace gas composition and packaging material can significantly affect the
quality and safety of celery sticks during the marketable period. Active MAP (CO2-PLA) outperformed passive MAP (A-PLA) in maintaining celery stick quality but not safety.
Conventional active MAP (CO2-PLA) out-performed non-conventional active MAP (O2-PLA
and N2-PLA) in maintaining the quality of the celery sticks. Non-conventional active MAP with
95 kPa O2 suppressed the growth of L. monocytogenes, while a high concentration of CO2
promoted growth during the first 10 d of storage. When comparing PLA and PP/PE as a
packaging material, both impacted the quality of celery to varying degrees, but not Listeria
growth. PLA allowed quicker development of gas compositions that better maintained the green
color of the celery surface and cut ends, but increased cut end dehydration and weight loss.
However, this weight loss was well within tolerable limits with celery turgor also unaffected
during two weeks of storage. PLA retained ethanol that was released into the package headspace
while PP/PE favored its escape from the package. The combination PLA and an initial gas
composition of 95 kPa O2 would be a viable packaging option when storing celery for up to one
week due to the few quality changes and maintained Listeria populations. However, during
longer storage, celery exposed to this initial gas composition showed more visual and textural
defects along with high levels of ethanol.
Through descriptive sensory testing, the bio-based material was found to develop an
adequate atmosphere to preserve color, texture and sensory quality in fresh-cut celery. In
addition, the newly developed flexibility and color scales proved to be effective assessors for
descriptive sensory analysis of fresh-cut celery, and could be applied to other fresh produce.

70

Both in-package atmosphere and sanitizer had a significant (P < 0.05) effect on the
microbiological safety (S. Typhimurium) and quality (mesophilic bacteria, yeasts and molds) of
packaged diced onions. Snap-fit containers, regardless of sanitizer, had diced onions with the
most (P < 0.05) S. Typhimurium, mesophilic bacteria, yeasts and molds growth throughout
storage, showing that an in-package atmosphere other than air can improve the microbiological
safety and quality of diced onions. Regardless of in-package atmosphere, diced onions sanitized
with ClO2 contained the most (P < 0.05) S. Typhimurium, and those sanitized with Cl had the
least (P < 0.05) mesophilic bacteria, yeasts and molds, showing the different effectiveness of
sanitizers on S. Typhimurium, mesophilic bacteria, and yeast and mold growth. The Cl and CO2
combination had diced onions with the lowest (P < 0.05) S. Typhimurium populations
throughout storage, proving that in-package atmosphere and sanitizer interactions exist and can
reduce S. Typhimurium growth in diced onions.
In-package atmosphere had a greater effect on the physico-chemical quality of packaged
diced onions than sanitizer. While in-package atmosphere had a significant (P < 0.05) effect on
all parameters evaluated (weight loss, headspace ethanol, pH, L*, b*, headspace O2, and
headspace CO2), sanitizer, however, only had a significant effect on weight loss, pH, and
headspace CO2. Snap-fit containers, regardless of sanitizer, promoted more darkening,
yellowing, and acidification in diced onions than other in-package atmospheres, while CO2 best
maintained the L* value of diced onions and reduced yellowing and acidification. Snap-fit
containers had lower headspace ethanol and less weight loss compared to CO2, both of which
resulted from the different permeabilities of the packaging systems instead of their different
atmospheres. Regardless of the in-package atmosphere, diced onions sanitized with Cl had
significantly less (P < 0.05) CO2 content and acidification than those sanitized with PA or ClO2,

71

and diced onions sanitized with PA had significantly lower (P < 0.05) weight loss than those
sanitized with Cl or ClO2, indicating the greater damage to the onion tissue when ClO2 is used.
In-package atmosphere and sanitizer interactions that enhance the physico-chemical quality of
packaged onions were also identified, with the interactions Cl SF and PA O2 resulting in less
acidification of packaged diced onions.
Considering all of the parameters researched, the current commercially used in-package
atmosphere/sanitizer combination (ClO2 SF) was shown to not be the best choice for
maintaining the safety and quality of packaged diced onions during a 2 week storage at 7 °C.
Conversely, Cl CO2 was the best in-package atmosphere and sanitizer combination for
enhancing the safety and quality of packaged diced onions. Diced onions packaged in this inpackage atmosphere and sanitizer combination would be acceptable for purchase after 14 days as
verified by both a trained panel and a consumer panel. This period would also be acceptable in
terms of microbiological counts for mesophilic bacteria and yeasts and molds for diced onions.

72

FUTURE WORK
These studies show the importance of researching the impact of packaging material, inpackage atmosphere, sanitizer, and their combination on the quality and safety of fresh-cut
produce. More research on the topic would help aid in the understanding of how packaging
material, in-package atmosphere, sanitizer, and their combination affect the quality and safety of
fresh-cut produce, and could include the following:


A sensory evaluation would be needed to determine the impact of in-package ethanol
concentration on the consumer acceptance of packaged celery sticks.



Researching the effects of in-package atmosphere and sanitizer combinations on the
safety and quality of celery sticks at temperatures other than 7 °C.



Examination of the effects of alternative in-package atmospheres and sanitizers on the
safety and quality of fresh-cut celery.



Investigation of the effects of real life temperature storage (fluctuating temperatures) and
in-package atmospheres and sanitizers on the safety and quality of celery sticks.



Studying the impact of in-package atmosphere and sanitizer combinations on the growth
of other common pathogenic microorganisms on fresh-cut celery to determine the
effectiveness of those combinations at helping to mitigate the damage caused by
contamination.



Applying the newly developed celery sensory scales to other fresh produce items.



Studying the effects of other in-package atmospheres and sanitizers on the flexibility and
color change of fresh-cut celery



Qualify the newly developed flexibility and color scales with fresh-cut celery stored at
other or fluctuating temperatures

73



Develop more standardized color scales specific to fresh produce degradation



Researching the effects of in-package atmosphere and sanitizer combinations on the
safety and quality of diced onions at temperatures other than 7 °C.



Examination of the effects of alternative in-package atmospheres and sanitizers on the
safety and quality of diced onions.



Investigation of the effects of real life temperature storage (fluctuating temperatures) and
in-package atmospheres and sanitizers on the safety and quality of diced onions.



Studying the impact of in-package atmosphere and sanitizer combinations on the growth
of other common pathogenic microorganisms on diced onions to determine the
effectiveness of those combinations at helping to mitigate the damage caused by
contamination.



Determining the effect of in-package atmosphere and sanitizer combinations on the safety
and quality of other fresh-cut produce.

74

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