First Line of Title

ENHANCED REMOVAL OF SALMONELLA TYPHIMURIUM AND E. COLI

O157:H7 FROM BLUEBERRIES AND STRAWBERRIES BY SOLUTIONS

CONTAINING SODIUM DODECYL SULFATE AND ORGANIC ACIDS OR

HYDROGEN PEROXIDE

Yingying Li

A thesis submitted to the Faculty of the University of Delaware in partial fulfillment of the requirements for the degree of Master of Science in Food Science

Fall 2013

ENHANCED REMOVAL OF SALMONELLA TYPHIMURIUM AND E. COLI

O157:H7 FROM BLUEBERRIES AND STRAWBERRIES BY SOLUTIONS

CONTAINING SODIUM DODECYL SULFATE AND ORGANIC ACIDS OR

HYDROGEN PEROXIDE

Yingying Li

Approved: ______Changqing Wu, Ph.D. Professor in charge of thesis on behalf of the Advisory Committee

Approved: ______Jack Gelb, Ph.D. Chair of the Department of Animal and Food Sciences

Approved: ______Mark W. Rieger, Ph.D. Dean of the College of Agriculture and Natural Resources

Approved: ______James G. Richards, Ph.D. Vice Provost for Graduate and Professional Education ACKNOWLEDGMENTS

My first and sincere appreciation goes to my advisor Dr. Changqing Wu for her continuous help and support in all stages of this thesis. I would like to thank my committee members Dr. Haiqiang Chen and Dr. Rolf Joerger for their interest in my work. In addition, I would like to thank my labmate, Wenqing Xu, who was always a great support in all my struggles and frustrations in my work and life in this country. I am also thankful to Melissa Ehrich, Kyle LeStrange, and Patrick Spanninger; they are wonderful labmates and friends. Finally, I would like to thank my parents for always believing in me, for their continuous love and their supports in my decisions. Without whom I could not have made it here.

iii TABLE OF CONTENTS

LIST OF TABLES ...... viii LIST OF FIGURES ...... ix ABSTRACT ...... xi

Chapter

1 INTRODUCTION ...... 1

REFERENCES ...... 3

2 LITERATURE REVIEW ...... 5

2.1 U.S. Disease Outbreaks Associated with Blueberries and Strawberries ... 5 2.2 Microorganisms of Concern ...... 6

2.2.1 Salmonella ...... 6 2.2.2 Escherichia coli ...... 7

2.3 Washing Treatment ...... 8

2.3.1 Chlorine ...... 9 2.3.2 Organic acids ...... 9 2.3.3 Hydrogen peroxide ...... 11 2.3.4 Trisodium phosphate ...... 11 2.3.5 Sodium dodecyl sulfate ...... 12

2.4 Frozen Fruit Safety ...... 13

REFERENCES ...... 15

3 ENHANCED INACTIVATION OF SALMONELLA TYPHIMURIUM FROM BLUEBERRIES BY SOLUSIONS CONTAINING SODIUM DODECYL SULFATE AND ORGANIC ACIDS OR HYDROGEN PEROXIDE ...... 21

iv Abstract ...... 21

3.1 Introduction ...... 22 3.2 Materials and Methods ...... 24

3.2.1 Bacterial strains and inoculum preparation ...... 24 3.2.2 Contamination of blueberry surfaces and washing procedures ... 24 3.2.3 Enumeration of Salmonella ...... 25 3.2.4 Effect of washing treatments on the physical properties of blueberries ...... 26 3.2.5 Total phenolic and anthocyanins content ...... 26 3.2.6 Impact of the two most effective treatments coupled with low temperature storage on Salmonella, yeasts and molds counts ..... 27 3.2.7 Statistical analysis ...... 28

3.3 Results and Discussion ...... 28

3.3.1 Effect of the combination of chlorine and SDS solutions on inactivation of Salmonella ...... 28 3.3.2 Effect of combinations of organic acids and SDS solutions on inactivation of Salmonella ...... 31 3.3.3 Effect of combinations of hydrogen peroxide and SDS solutions on inactivation of Salmonella ...... 33 3.3.4 Effect of combinations of trisodium phosphate and SDS solutions on inactivation of Salmonella ...... 35 3.3.5 Effect of washing treatments on the physical properties of blueberries ...... 36 3.3.6 Impact of treatments coupled with low temperature storage on Salmonella, yeasts and molds counts ...... 38 3.3.7 Comparison of Salmonella inactivation after washing for 1 and 5 min ...... 41

REFERENCES ...... 43

4 SURVIVAL OF SALMONELLA TYPHIMURIUM AND E. COLI O157:H7 ON BLUEBERRIES DURING FROZEN STROAGE AFTER WASHING WITH COMBINATIONS OF SODIUM DODECYL SULFATE AND ORGANIC ACIDS OR HYDROGEN PEROXIDE ...... 46

Abstract ...... 46

4.1 Introduction ...... 47 4.2 Materials and Methods ...... 48

v 4.2.1 Bacterial strains and inoculum preparation ...... 48 4.2.2 Inoculation of blueberry surfaces ...... 49 4.2.3 Washing procedures and frozen storage ...... 49 4.2.4 Microbial analysis ...... 50 4.2.5 Sensory evaluation after 12-weeks of frozen storage ...... 51 4.2.6 Physical properties and total bacteria, yeasts and molds counts of blueberries during frozen storage ...... 51 4.2.7 Total phenolic and anthocyanins content ...... 52 4.2.8 Statistical analysis ...... 52

4.3 Results and Discussion ...... 52

4.3.1 Effect of the combination of peroxyacetic acid and SDS on inactivation of Salmonella ...... 52 4.3.2 Combination of SDS and organic acids or hydrogen peroxide on inactivation of Salmonella and E. coli during frozen storage 54 4.3.3 Sensory analysis ...... 57 4.3.4 Physical properties and total bacteria and yeasts and molds counts of blueberries during frozen storage ...... 58

REFERENCES ...... 63

5 COMPARISON OF PROMISING TREATMENTS EFFICACY ON SPOT OR DIP INOCULATED BLUEBERRIES AND STRAWBERRIES ...... 66

Abstract ...... 66

5.1 Introduction ...... 67 5.2 Materials and Methods ...... 69

5.2.1 Bacterial strains and inoculum preparation ...... 69 5.2.2 Contamination of blueberries and strawberries ...... 69 5.2.3 Washing procedures ...... 70 5.2.4 Microbial analysis ...... 70 5.2.5 Statistical analysis ...... 71

5.3 Results and Discussion ...... 71

5.3.1 Effect of combinations of SDS and organic acids or hydrogen peroxide on inactivation of Salmonella and E. coli on spot-, calyx- or dip-inoculated blueberries ...... 71 5.3.2 Effect of a combination of SDS and organic acids or hydrogen peroxide on inactivation of Salmonella and E. coli on spot- or dip-inoculated strawberries ...... 75

vi REFERENCES ...... 81

6 FUTURE WORK ...... 83

REFERENCES ...... 85

vii LIST OF TABLES

Table 3.1 The test values for color, pH, texture, total phenolic and anthocyanins content of treated and untreated berries ...... 37

Table 4.1 Sensory test results for fresh blueberries and blueberries washed with antimicrobial solutions and stored for 12 weeks ...... 57

viii LIST OF FIGURES

Figure 3.1 Recovery of Salmonella from spot-inoculated blueberries after washing with combinations of chlorine and SDS at different concentrations...... 30

Figure 3.2 Recovery of Salmonella from surface-inoculated blueberries after washing with combinations of organic acid and SDS at different concentrations. .... 33

Figure 3.3 Recovery of Salmonella from spot-inoculated blueberries after washing with combinations of hydrogen peroxide and SDS at different concentrations...... 34

Figure 3.4 Recovery of Salmonella from spot-inoculated blueberries after washing with combinations of trisodium phosphate and SDS at different concentrations...... 35

Figure 3.5 Impact of two promising treatments on inactivation of Salmonella (A) and molds and yeasts (B) coupled with low temperature storage on the microbiological quality...... 40

Figure 3.6 Comparison of two promising alternatives on inactivation of Salmonella between 1 min and 5 min washing...... 42

Figure 4.1 Recovery of Salmonella from surface-inoculated blueberries after washing with combinations of peroxyacetic acid and SDS at different concentrations...... 53

Figure 4.2 Log10 reductions by washing blueberries contaminated with Salmonella (A) and E. coli O157:H7 (B) with SDS and organic acids or hydrogen peroxide followed by frozen storage...... 56

Figure 4.3 Physical properties of blueberries during frozen storage ...... 60

Figure 4.4 Chemical properties of blueberries during frozen storage...... 61

Figure 4.5 Inactivation of total bacteria (A) and molds and yeasts (B) during frozen storage after treatments with a combination of SDS and organic acids or hydrogen peroxide...... 62

Figure 5.1 Inactivation of Salmonella on spot-, calyx- and dip-inoculated blueberries by combinations of SDS and organic acids or hydrogen peroxide...... 73

ix Figure 5.2 Inactivation of E. coli on spot-, calyx- and dip-inoculated blueberries by combinations of SDS and organic acids or hydrogen peroxide...... 74

Figure 5.3 Inactivation of Salmonella on spot- inoculated (A) and dip- inoculated (B) strawberries by combinations of SDS and organic acids or hydrogen peroxide...... 79

Figure 5.4 Inactivation of E. coli on spot- inoculated (A) and dip- inoculated (B) strawberries by combinations of SDS and organic acids or hydrogen peroxide...... 80

x ABSTRACT

Increasing consumption of raw blueberries has led to the need for improved food safety in the berry fruit industry. This study was undertaken to evaluate enhanced Salmonella and E. coli inactivation on blueberries by washing with sodium dodecyl sulfate (SDS) in combination with other common antimicrobial agents, such as organic acids and hydrogen peroxide. The addition of 5000 ppm SDS in 500 ppm acetic acid, 200 ppm hydrogen peroxide, and 20 ppm peroxyacetic acid resulted in more than 4.0 log10 CFU/g reductions of Salmonella and E. coli. Low-temperature frozen storage had a significant impact (P<0.05) on microbial counts of both treated and untreated blueberries. None of the washings decreased the total phenolic, anthocyanins content and apparent quality, but frozen storage caused significant damage to the texture of both treated and untreated blueberries. A solution containing

500 ppm acetic acid plus 5000 ppm SDS, 200 ppm hydrogen peroxide in combination with 5000 ppm SDS, and 20 ppm peroxyacetic acid coupled with 5000 ppm SDS holds promise in enhancing the safety of blueberries and frozen storage has the potential to enhance their effectiveness. However, these three washing solutions did not inactivate Salmonella and E. coli on strawberries presumably due to the rough surface of strawberries and the presence of numerous surface-borne achenes (seeds), which provide hidden areas for the bacteria to attach and are less accessible to sanitizing solutions. Thus, these experiments indicate that once strawberries were contaminated with Salmonella Typhimurium and E. coli O157:H7, there is a potential health hazard to cause illness for consumers.

xi Chapter 1

INTRODUCTION

Small fruits such as blueberries, strawberries, and raspberries are important agricultural commodities, which are generally not treated or processed before sale, in order to increase the shelf life of the product. Consequently, microbial contaminants that were introduced by irrigation water, soil, equipment, pickers, and food handlers persist and can reach the consumer (Han et al., 2004).

Most fruits and vegetables are placed in flume tanks containing 150 to 200 ppm of free chlorine, typically at pH 6.5 to 7, at 10 ℃ for a short period of time before packing (Bartz et al., 2001). For example, processed blueberries are usually washed or sprayed with chlorinated water containing 50-200 ppm active chlorine to reduce microorganisms (Good Agricultural and

Manufacturing (Handling) Safety and Food Defense Practices, 2010). Strawberries are not washed because of their high susceptibility to fungal deterioration when getting wet (Gulati et al., 2001). Despite their common usage, chlorine treatments are not very effective, resulting in less than a 2-log reduction in the numbers of bacterial organisms on fresh fruits and vegetables (Beuchat, 1992, 1999; Brackett, 1999). The effectiveness of chlorine is reduced by organic matter surrounding the bacterial organisms, such as carbohydrates on the surface of fresh produce. The chlorine-based washing solutions also bring safety risks such as the formation of by-products such as chloroform (CHCl3), suspected to have carcinogenic or mutagenic effects (Artes et al., 2009; Nieuwenhuijsen et al., 2000). For these reasons, alternative sanitizers must be developed to increase decontamination efficiency and to overcome the safety issues associated with chlorinated water.

1 Recent investigations have shown that organic acids, hydrogen peroxide, and trisodium phosphate may have potential as alternatives to the use of chlorine-based washing solutions in eliminating foodborne pathogens on raw fruits and vegetables (Crowe, Bushway, and Bushway, 2005; Sapers and Sites, 2003). Organic acids, hydrogen peroxide, and trisodium phosphate are generally recognized as safe (GRAS) according to the U.S. Food and Drug Administration (FDA) classification. Sodium dodecyl sulfate (SDS) is a surface-active compound that also has GRAS status as a multipurpose additive (21 CFR 172.822). This compound makes the liquid spread more easily and lowers the interfacial tension between two liquids or between a liquid and a solid. The additions of SDS to levulinic-acid enhanced removal of Salmonella on chicken breast meats (Zhao et al., 2009), and combinations of SDS with chlorinated water also improved inactivation of human norovirus surrogates on produce (Predmore and Li, 2011). The objective of the present study was to develop an antimicrobial washing solution using a common sanitizer (chlorine, organic acid, hydrogen peroxide and trisodium phosphate) and SDS to enhance the Salmonella and E. coli O157:H7 inactivation on blueberries and strawberries. In addition, the impacts of the effective treatments on sensory quality, total phenolic, anthocyanins content, molds and yeasts during the storage were determined.

2 REFERENCES

Artes, F., Gomez, P., Aguayo, E., Escalona, V., Artes-Hernundez, F., 2009. Sustainable sanitation techniques for keeping quality and safety of fresh-cut plant commodities. Postharvest Biology and Technology 51, 287-296.

Bartz, J.A., Eayre, C.G., Mahovic, M.J., Concelmo, D.E., Brecht, J.K., Sargent, S.A., 2001. Chlorine concentration and the inoculation of tomato fruit in packinghouse dump tanks. Plant Disease 85, 885-889.

Beuchat, L.R., 1992. Surface disinfection of raw produce. Dairy, Food and Environmental Sanitation 12, 6-9.

Beuchat, L.R., 1999. Survival of enterohemorrhagic Escherichia coli O157:H7 in bovine faeces applied to lettuce and the effectiveness of chlorinated water as a disinfectant. Journal of Food Protection 62, 845-849.

Brackett, R.E., 1999. Incidence, contributing factors, and control of bacterial pathogens in produce. Postharvest Biology and Technology 15, 305-311.

Crowe, K.M., Bushway, A.A., Bushway, R.J., 2005. Effects of alternative postharvest treatments on the microbiological quality of lowbush blueberries. Small Fruits Review 4, 29-39.

Good Agricultural and Manufacturing (Handling) Safety and Food Defense Practices as Part of Fresh Blueberry Farming, Packing and Distribution, March 2010.

Gulati, B.R., Allwood, P.B., Hedberg, C.W., Goyal, S.M., 2001. Efficacy of commonly used disinfectants for the inactivation of Calicivirus on strawberry, lettuce, and a food-contact surface. Journal of Food Protection 64, 1430-1434.

Han, Y., Selby, T.L., Schultze, K.K., Nelson, P.E., Linton, R.H., 2004. Decontamination of strawberries using batch and continuous chlorine dioxide gas treatments. Journal of Food Protection 67, 2450-2455.

Nieuwenhuijsen, M.J., Toledano, M.B., Elliott, P., 2000. Uptake of chlorination disinfection by- products; a review and a discussion of its implications for exposure assessment in epidemiological studies. Journal of Exposure Analysis and Environmental Epidemiology 10, 586-599.

Predmore, A., Li, J., 2011. Enhanced removal of a human norovirus surrogate from fresh vegetables and fruits by a combination of surfactants and sanitizers. Applied Environmental Microbiology 77, 4829-4838.

3 Sapers, G.M., Sites, J.E., 2003. Efficacy of 1% hydrogen peroxide wash in decontaminating apples and cantaloupe melons. Journal of Food Science 68, 1793-1797.

Zhao, T., Zhao, P., Doyle, M.P., 2009. Inactivation of Salmonella and Escherichia coli O157:H7 on lettuce and poultry skin by combinations of levulinic acid and sodium dodecyl sulfate. Journal of Food Protection 72, 928-936.

4 Chapter 2

LITERATURE REVIEW

2.1 U.S. Disease Outbreaks Associated with Blueberries and Strawberries In recent years, outbreaks associated with the consumption of fresh produce have led to an increased need for food safety programs. Factors that contribute to the contamination of pathogenic microorganisms on fresh fruits include preharvest operations (soil, irrigation water, water used to apply pesticides, green or inadequately composted manure, air, wild and domestic animals, insects and human handling) and various postharvest procedures, , such as handling by workers and consumers, contact with harvesting equipment, wash and rinse water, and equipment used for sorting, packing, cutting and other processing steps (Wiley, 1994; Beuchat and Ryu, 1997).

Since blueberries are generally consumed raw or only minimally processed without a pasteurization step, microbial contaminants can easily reach the consumer. For example, in 1984, an outbreak of listeriosis in Connecticut was possibly associated with fresh blueberries (Ryser, 1999). A more recent outbreak of hepatitis A in New Zealand was linked to the consumption of raw blueberries, which were likely contaminated by infected food handlers or fecally contaminated groundwater (Calder et al, 2003). Additionally, according to the Foodborne Outbreak Online Database, in June 2009 a confirmed outbreak of Salmonella Muenchen on blueberries caused 14 people to become ill.

A multistate outbreak of hepatitis A was traced to frozen strawberries processed at a single plant in 1990 (Niu et al, 1992). Fifteen people in a school in Georgia developed hepatitis A, as well as 13 people in the institution for the developmentally disabled in Montana.

5 Contamination occurred before distribution, most likely from an infected picker. In 1997, a total of 153 cases of hepatitis A related to the consumption of strawberries were reported in Calhoun County, Michigan (CDC, 1997). E. coli O157:H7 or Salmonella infection have also been associated with the consumption of raw and minimally processed strawberries. Salmonella and E. coli O157:H7 are capable of surviving on fresh strawberries for over 7 d (Knudsen et al., 2001). A FDA survey found that 1 out of 143 imported strawberry samples tested positive for Salmonella (FDA, 1999). In 2011, fresh strawberries were implicated in an E. coli O157 outbreak (Christine, 2011). Oregon Public Health officials determined that fresh strawberries from a Newberg farm were likely contaminated with E. coli O157:H7. The outbreak caused at least 15 people to become sick, and led to the death of one person. These outbreaks highlight the need for more effective control measures in the berry industry.

2.2 Microorganisms of Concern

2.2.1 Salmonella Salmonella are gram-negative, rod-shaped bacilli, consisting of more than 2300 serotypes (Sudarsan et al., 2012). Salmonella is one of the most frequent causes of foodborne illness worldwide (Rabsch et al., 2001).Salmonella Enteritidis and Salmonella Typhimurium are the most common serotypes causing salmonellosis (Tauxe, 1991; Todd, 1996). Any person is susceptible to infection with Salmonella, but especially those with weakened immune systems (El-GazzarandMarth, 1992).The infectious dose depends on several factors, but it is especially low for immunocompromised individuals and the very young and the old (Jay, 2000). Acute symptoms of salmonellosis include nausea, vomiting, abdominal cramps, minimal diarrhea, fever, and headache. Typically the onset of symptoms occurs 5 to 72 hours after ingesting contaminated food and the symptoms will last for 1 to 4 days (Doyle and Cliver, 1990).

6 According to a summary of foodborne disease from 1996-2010, Salmonella infection has not declined in 15 years (CDC, 2011). Outbreaks of salmonellosis have been linked to a wide variety of fresh fruits and vegetables including apple, cantaloupe, alfalfa sprout, mango, lettuce, cilantro, unpasteurized orange juice, tomato, melon, celery and parsley (Pui et al., 2011). In 2012, there were two Salmonella outbreaks associated with the consumption of fresh mango and cantaloupe. A total of 127 persons were infected with Salmonella Braenderup from mangoes from Agricola Daniella. Thirty-three persons were hospitalized (CDC, 2012).The FDA blocked importation of Agricola Daniella mangoes into the United States unless the importer showed they were free of Salmonella (FDA, 2012). Salmonella Typhimurium and Salmonella Newport- contaminated cantaloupes originating from Chamberlain Farms caused 94 individuals to become ill, and resulted in 3 deaths (CDC, 2012). In August of 2012, a recall was issued for all cantaloupes from Chamberlain Farms (FDA, 2012).

2.2.2 Escherichia coli Escherichia coli are gram-negative, nonsporeforming bacilli, which include a broad variety of types (Kaper et al., 2004). There are E. coli strains that are a virulent and others that are highly pathogenic to humans and animals. Most E. coli strains can grow at a temperature range from 15 to 48 ℃, with a maximal growth rate in the range of 37-42 ℃; at a pH range of approximately 5.5-8.0 with best growth at neutrality. Since pathogens are easily ingested with food, the human gastro-intestinal tract is susceptible to diarrhoeagenic E. coli infections (Sousa, 2006).

E. coli O157:H7 has emerged as a widespread foodborne pathogen that may cause hemorrhagic colitis and hemolytic uremic syndrome (Padhye and Doyle, 1991). The possible vehicles of E. coli O157:H7 are a wide variety of foods, including meat, milk, fruit juices, and vegetables (Singh et al., 2002). The largest reported E. coli O157:H7 outbreak resulted from

7 contaminated radish sprouts, which affected about 6000 people in Japan (Yoh et al., 1997). In 2012, the CDC collaborated with the FDA to investigate a multistate outbreak of E. coli O157:H7 infections linked to romaine lettuce. Fifty-eight persons infected with the outbreak strain of E. coli O157:H7 were reported. Contamination of the lettuce likely occurred before the product reached grocery stores. (CDC, 2012)

Recent studies have shown that E. coli O157:H7 can survive on freshly peeled Hamlin orange, watermelon, and cantaloupe for prolonged periods (Del Rosario, 1995). Tichert (2000) also found that E. coli O157:H7 could survive on broccoli, cucumber, and green pepper at 4 ℃ and maintain initial levels or grow at 15 ℃. Studies on radish sprouts suggest that E. coli O157:H7 contamination could result from contaminated hydroponic water (Hara et al., 2000; Itoh et al., 1998). Knudsen et al. (2001) indicated that E. coli O157:H7 is capable of surviving on fresh strawberries for over 7 d.

2.3 Washing Treatment Traditionally, the produce industry utilizes water washes with and without chemical sanitizers in an effort to decontaminate fresh produce prior to further processing. However, the efficacy of common chemical sanitizers to bring about significant microbial reductions on the surface of fruits and vegetables may be limited and unpredictable (Nguyen and Carlin, 1994). A battery of chemical disinfectants has been tested to determine their effectiveness, including sodium hypochlorite; chlorine dioxide; hydrogen peroxide; combinations of above chemicals with acids such as citric, lactic, acetic, phosphoric or other organic acids; trisodium phosphate; sodium hydroxide; peroxyacetic acid; ozone (Sapers et al., 1999; Sapers, 2000; Wisniewsky et al., 2000; Venkitanarayanan et al., 2002; Zhuang and Beuchat, 1996; Beuchat et al.,1998; Venkitanarayanan et al., 1999; Sapers et al., 2000; Liao et al., 2003; Wright et al., 2000; Kenney et al., 2002). All disinfectants must be used at concentrations no more than the levels

8 recommended as safe by the Canadian Food Inspection Agency (CFIA) or the Food and Drug Administration (FDA) of the United States.

2.3.1 Chlorine Fresh produce is generally washed or sprayed with chlorinated water containing 50-200 ppm total active chlorine to reduce microbial contamination (GAMP, 2010); however, this chlorine-based washing solution has shown limited efficacy. For example, a five-minute- exposure to 100 ppm chlorine reduced populations of bacteria, yeast, and mold on blueberries by only 0.83, 0.77, and 0.61 log10 CFU/g, respectively (Crowe et al., 2005). The reduced effectiveness of chlorine at inactivating surface microorganisms may be a result of organic matter surrounding the target cells (Beuchat et al., 2001). If organic materials, like plant tissues, interact with chlorine before it makes contact with target cells, the free chlorine in solution becomes neutralized on contact. In addition, the pH of the chlorinated wash water also influences the effectiveness of chlorine. The antimicrobial activity of chlorine is dependent upon the concentration of hypochlorous acid (HOCl) in solution; therefore, the pH of the solution should remain at or below pH 7, a point at which hypochlorous acid remains undissociated. Chlorine- based washing solutions also bring safety risks such as the formation of by-products such as chloroform (CHCl3), suspected to have carcinogenic or mutagenic effects (Artes et al., 2009; Nieuwenhuijsen et al., 2000).

2.3.2 Organic acids Some organic acids have lethal or inhibitory effects on microorganisms and have GRAS status. Shapiro and Holder (1960) indicated that 150 ppm and 500 ppm citric acid solutions were capable of inactivating bacterial growth on salad greens for 96 hours. The 0.5% citric acid solution was effective in reducing aerobic mesophillic bacteria (APC), yeast, and mold on blueberries (Crowe et al., 2005). The effects of lactic and acetic acid, either alone or in

9 combination with chlorine, on survival of L. monocytogenes inoculated onto shredded lettuce were studied by Zhang and Farber (1996). Only 1% lactic acid and combinations of 0.5% or 1% lactic acid and 100 ppm chlorine reduced numbers of L. monocytogenes.

Álvarez-Ordóñez and others (2010) studied the ability of Salmonella Typhimurium cultivated in Brain Heart Infusion (BHI) to grow in the presence of different acidic conditions at different temperatures. Acetic acid was the best antimicrobial against Salmonella Typhimurium, and the effectiveness of the acids decreasing order of acetic>lactic>citric>hydrochloric. The antimicrobial activity of organic acids on microorganisms primarily rests on their undissociated forms. Since undissociated weak acids can penetrate bacterial cell membranes through permeases or porins, the cytoplasmatic pH is reduced by their intracellular dissociation, which affects the metabolic activity of the cell (Cherrington et al., 1991; Davidson, 2001). Acetic acid can easily penetrate bacterial membranes due to its low molecular weight and its liposolubility, whereas lactic and citric acids diffuse slowly (Fernándezet al., 2009). However, acetic acid at concentrations of 2 and 5% reduced E. coli O157:H7 on strawberries by approximately 1.6 log units, whereas 5% acetic acid caused a 3-log reduction in pathogens on apples compared to unrinsed controls. The reduced effectiveness on strawberries is likely due to the relatively uneven surface of the fruit (Yu et al., 2001).

In 1986, the FDA approved the use of peroxyacetic acid (PPA) as a food-grade sanitizer at concentrations not exceeding 100 ppm (21 CFR 172.1010). PPA disinfects by oxidizing the outer cell membrane of vegetative bacterial cells, endospores, yeast, and mold spores. PPA has a higher oxidation potential than chlorine but less than ozone. Gulati et al. (2001) found 1- and 2- log reduction of cultivable feline calicivirus (FCV) on respectively strawberries and lettuce treated with a 150 ppm PPA solution. Treatment of ready-to-use salads with 90 ppm PPA reduced total counts and faecal coliforms nearly 100-fold, similar to reductions with 100 ppm chlorine (Beuchat, 1998). Populations of E. coli O157:H7 and L. monocytogenes decreased to

10 nondetectable levels on strawberries after 5 min of exposure to 80 ppm peroxyacetic acid (Rodgers et al., 2004). Additionally, PPA has been used extensively as a sanitizer for food processing equipment where it is particularly effective against biofilms. However, a possible disadvantage of organic acids is that these treatments may change the flavor and aroma of treated products.

2.3.3 Hydrogen peroxide

H2O2 acts on the microorganisms through its release of nascent oxygen. H2O2 produces free hydroxyl radicals that damage proteins and DNA. Dipping strawberries in 1% and 3% H2O2 for 1 minute resulted in 1.4 and 2.2 log10 reductions of E. coli O157:H7 (Yu et al., 2001). Blueberries treated with 1% hydrogen peroxide for 120 seconds resulted in population reductions two times greater than reductions observed on samples treated with 100 ppm chlorine (Crowe et al., 2005). H2O2 at a concentration of 1% is an effective alternative to chlorine for improving the microbiological quality and safety of lowbush blueberries during processing without compromising blueberry color. However, 5% H2O2 resulted in a mildly bleached appearance on alfalfa sprouts and cantaloupe cubes. Hence, some fruits and vegetables (e.g. mushrooms, some types of berries and lettuce) may not be amenable to disinfecting with H2O2 because of adverse changes in surface color.

2.3.4 Trisodium phosphate In 1992, the use of trisodium phosphate (TSP) was approved by the USDA as a processing aid to reduce Salmonella on raw poultry carcasses (Giese, 1993). TSP is a GRAS food additive according to the FDA, and has been approved by the USDA for use during poultry processing. Zhuang and Beuchat (1996) investigated the effectiveness of TSP in wash-water in killing Salmonella Montevideo on the surface and in core tissue of inoculated mature green

11 tomatoes. Dipping tomatoes in 15% TSP solution for 15 seconds resulted in complete inactivation of Salmonella (5.18 log10 CFU/cm2) on the tomato surface. However, the use of TSP to remove L. monocytogenes from shredded lettuce was less promising. Treatment of lettuce with 2% TSP had almost no effect on reducing the population of L. monocytogenes (Zhang and Farber, 1996). Solutions containing more than 10% TSP damaged the sensory quality of lettuce. Other investigators have reported that L. monocytogenes was resistant to TSP (Somers et al., 1994). Escherichia coli O157:H7, on the contrary, was sensitive to 1% TSP, as 106 CFU/ ml or

105 CFU/cm2 of biofilm were killed within 30 seconds at room temperature. TSP at 2 and 5% concentrations in aqueous solutions led to a reduction of E. coli O157:H7 on strawberries of approximately 1.6 to 1.9 log10 CFU/g. (Yu et al., 2001). It should be noted that the pH of TSP solutions is in the 11 to 12 range, thus perhaps limiting their application as a disinfectant of fruits and vegetables to commercial use.

2.3.5 Sodium dodecyl sulfate Sodium dodecyl sulfate (SDS) is a surface-active compound that can be added to the water used to wash fresh produce. This compound makes the liquid spread more easily and lowers the interfacial tension between two liquids or between a liquid and a solid. SDS also is GRAS as a multipurpose additive (21 CFR 172.822). This compound has been widely studied as a surfactant and is approved for use in a variety of foods, including egg whites, fruit juices, vegetable oils, and gelatin as a whipping or wetting agent (FDA, 2007). The additions of SDS to levulinic-acid enhanced removal of Salmonella from chicken breast meats (Zhao et al., 2009), and combinations of SDS with chlorinated water also improved inactivation of human norovirus surrogates on produce (Predmore and Li, 2011). SDS could have significant bactericidal effect when pH was between 1.5 and 3.0, which might be related with the ability of SDS to denature protein surfaces and damage cell membranes (Tamblyn and Conner, 1997; Williams and Payne, 1964). SDS has virucidal activity against papillomaviruses, herpes simplex virus 2 (HSV- 2), and

12 human immunodeficiency virus type 1 (HIV-1) (Howett and Kuhl, 2005; Howett et al., 1999; Urdaneta et al., 2005). Incubation of a human norovirus surrogate (MNV-1) in a solution containing 200 ppm of SDS at 37 ℃ for 4 h resulted in a 3 log10 reduction, as well as a 5 log10 reduction of vesicular stomatitis virus (VSV) under the same incubation conditions (Predmore and Li, 2011).

2.4 Frozen Fruit Safety Freezing is the most traditional method of preserving berries as it provides for a long shelf life and has minimal impact on quality and nutrition. Overall, frozen foods have an excellent safety record. Although illnesses associated with frozen berries are rare, freezing also preserves the viability of some pathogens. An outbreak of hepatitis A in UK was linked to the consumption of frozen raspberries, which were likely contaminated at the picking stage or when the plastic tubs were being filled and their weight adjusted prior to freezing (Reid and Robinson, 1987). In March 1997, a total of 153 cases of hepatitis A were suspected to be associated with the consumption of frozen strawberries (CDC, 1997). A more recent outbreak of hepatitis A in September 2013 was attributed to the consumption of “Townsend Farms Organic Antioxidant Blend”, which is a frozen berry mix, including cherries, blueberries, pomegranate seeds, raspberries and strawberries. A total of 161 people became ill (CDC, 2013).

Salmonella spp. are known for their tolerance to freezing (Archer, 2004). Salmonella

Typhimurium remained viable on frozen fish stored at -17.9 ℃ for over 1 year with only 1 log10 reduction in numbers (Raj and Liston, 1961). Additionally, it was demonstrated that a portion of Salmonella Typhimurium cells on frozen sausage and minced beef were only sublethally damaged after storage at -18 ℃ for up to 10 weeks (Barrell, 1988). Moreover, Doyle and Schoeni (1984) reported little change in the number of surviving E. coli O157:H7 on ground beef maintained at -80 ℃ for 30 min and then stored at -20 ℃ for up to 9 months.

Berries picked for frozen processing are usually full-flavored, ripe berries with uniform size and tender skins. They are hulled in the field, transported to the processing facility, and washed prior to freezing (Zhao, 2007). Because of the limited effectiveness of chlorine on the inactivation of pathogens on fruit, there is a need for alternative methods to improve the safety of frozen berries.

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20 Chapter 3

ENHANCED INACTIVATION OF SALMONELLA TYPHIMURIUM FROM

BLUEBERRIES BY SOLUSIONS CONTAINING SODIUM DODECYL SULFATE AND

ORGANIC ACIDS OR HYDROGEN PEROXIDE

Abstract

Like other fruits and vegetables that are consumed raw, blueberries are also potential vehicle for the transmission of pathogens introduce pre- or post-harvest. The present study was undertaken to evaluate Salmonella inactivation on blueberries by washing with a solution containing sodium dodecyl sulfate (SDS) in combination with the antimicrobial agents, chlorine, lactic acid, acetic acid, citric acid and hydrogen peroxide. The impacts of the most effective treatments on sensory quality, total phenolic and anthocyanin content, mold and yeast counts, and Salmonella survival during the storage of blueberries were also determined. Maximum reductions of Salmonella after washing with 500 ppm acetic acid plus 5000 ppm SDS and 200 ppm hydrogen peroxide containing 5000 ppm SDS were 4.0 and 4.2 log10 CFU/g, respectively. Without the addition of SDS to the acetic acid or hydrogen peroxide solutions,

Salmonella reductions were less than 2 log10 CFU/g. The 500 ppm acetic acid plus 5000 ppm SDS and 200 ppm hydrogen peroxide containing 5000 ppm SDS showed similar Salmonella reductions as those using 200 ppm chlorine (P>0.05). None of these washings decreased the total phenolic, anthocyanins content and apparent quality. The two antimicrobial washes that resulted in maximum Salmonella inactivation also suppressed further growth of Salmonella on the blueberries during three days of storage at 4 ℃. Both treatments also decreased counts of yeasts and molds throughout the storage. Therefore, the use of 500 ppm acetic acid plus 5000 ppm

21 SDS, 200 ppm hydrogen peroxide in combination with 5000 ppm SDS may be an alternative to the use of chlorine-based washing solutions for blueberries.

3.1 Introduction Salmonella is one of the leading causative microorganisms of foodborne illness in the United States and many other countries. Scallan et al. (2011) estimated that 31 pathogens caused 228,744 hospitalizations annually, 35% of which resulted from Salmonella. Outbreaks of salmonellosis have been linked to a wide variety of fresh fruits and vegetables including apple, cantaloupe, alfalfa sprout, mango, lettuce, cilantro, unpasteurized orange juice, tomato, melon, celery and parsley (Pui et al., 2011). For example, 127 persons were infected with Salmonella Braenderup by mangoes and 33 of them were hospitalized (CDC, 2012). Cantaloupes contaminated with Salmonella Typhimurium and Salmonella Newport contaminated caused 94 individuals to become ill and resulted in three deaths (CDC, 2012).

Since blueberries are generally sold raw or in an only minimally processed state in order to increase the shelf life of the product, any pathogens that contaminated the fruits during production, e. g. from contaminated irrigation water, soil, and equipment, or from infected pickers and food handlers, will likely reach the consumer. In 1984, an outbreak of listeriosis was associated with fresh blueberries in Connecticut (Ryser, 1999). A more recent outbreak of hepatitis A in New Zealand was linked to the consumption of raw blueberries, which were likely contaminated by infected food handlers or fecally contaminated groundwater (Calder et al.,

2003). According to the Foodborne Outbreak Online Database (2013), a confirmed outbreak caused by Salmonella Muenchen residing on blueberries caused 14 people to become ill in June 2009, which implicates Salmonella as an important potential contaminant of blueberries.

22 In commercial settings, harvested blueberries are usually washed or sprayed with chlorinated water containing 50-200 ppm active chlorine to reduce microorganisms (Good Agricultural and Manufacturing (Handling) Safety and Food Defense Practices, 2010). However, this chlorine-based washing solution has shown limited efficacy in deactivating foodborne pathogens. Five-minute exposure to 100 ppm chlorine reduced populations of bacteria, yeasts, and molds on blueberries by only 0.83, 0.77, and 0.61 log10 CFU/g, respectively (Crowe et al., 2005). The chlorine-based washing solutions also bring safety risks such as the undesirable chlorine by-products, some of which are suspected carcinogens or mutagens (Artes et al., 2009; Nieuwenhuijsen et al., 2000). Treatments with citric acid or hydrogen peroxide to protect blueberries from decaying, to prolong shelf life, and to secure product safety have been explored (Shapiro and Holder, 1960; Crowe et al., 2005; Sapers and Sites, 2003; Sapers and Simmons, 1998; Crowe et al., 2007), but effective antimicrobial treatments can also lead to adverse changes in flavor and surface color (Abee and Wouters, 1999; Wesche et al., 2009; Beuchat, 1998).

Sodium dodecyl sulfate (SDS) is a surface-active compound that lowers the interfacial tension between two liquids or between a liquid and a solid. The additions of SDS to levulinic- acid enhanced removal of Salmonella from chicken breast meats (Zhao et al., 2009), and combinations of SDS with chlorinated water improved inactivation of human norovirus surrogates on produce (Predmore and Li, 2011). Therefore, the objective of present study was to develop an antimicrobial washing solution using a common sanitizer (chlorine, lactic acid, acetic acid, citric acid and hydrogen peroxide) and SDS to enhance the Salmonella inactivation on blueberries. In addition, the impacts of the most effective treatments on sensory quality, total phenolic, anthocyanins content, molds and yeasts, and Salmonella survivors during the storage of blueberries were also determined.

23 3.2 Materials and Methods

3.2.1 Bacterial strains and inoculum preparation Salmonella enterica serovar Typhimurium DT 104 was obtained from the culture collection of the Department of Animal and Food Sciences at the University of Delaware (Newark, DE). Stock cultures on tryptic soy agar (TSA; Difco Laboratories, Becton Dickinson,

Spark, Md) were stored at 4 ℃. A single colony from a TSA plate was cultured into tryptic soy broth (TSB, Difco Laboratories, Becton Dickinson, Spark, Md) and grown at 37 ℃ for 24 h before inoculation of blueberries. Viable cell counts were determined by serially diluting suspensions in sterile 0.1% peptone water (Difco) and spread plating 0.1 ml on the xylose lysine deoxycholate (XLD; Difco) plate.

3.2.2 Contamination of blueberry surfaces and washing procedures Fresh blueberries were obtained from a local market on the day of experimentation. Intact surfaces were selected for inoculation. Inoculation was achieved by applying 25 μl of inoculum

(6 to 10 small droplets) onto the surface of each blueberry. After inoculation, blueberries were left in a laminar flow hood at room temperature (21±1 ℃) for about 2 h to allow for attachment of the microorganisms. Inoculation resulted in approximately 106 CFU of Salmonella per g of fresh blueberries.

The washing solutions included: chlorine (4, 12.5, 25, 50 and 100 ppm, prepared from sodium hypochlorite, Sigma–Aldrich, Inc., St. Louis, MO, USA), organic acids (lactic acid, acetic acid, and citric acid at 50 ppm or 500 ppm, Sigma–Aldrich, Inc., St. Louis, MO, USA), trisodium phosphate (50, 200 and 500 ppm, Sigma–Aldrich, Inc., St. Louis, MO, USA) and hydrogen peroxide (50, 100 and 200 ppm, prepared from 30% hydrogen peroxide, Sigma– Aldrich, Inc., St. Louis, MO, USA), as well as these antimicrobial solutions combined with SDS (50, 500, 5000 ppm, Sigma–Aldrich, Inc., St. Louis, MO, USA). Distilled water (DI) washing

24 served as negative control. Four g of blueberries was submerged in 80 ml of the rinse solution with continuous agitation provided by starring bar in a beaker for 1 or 5 min. The 5-min washing was used in initial tests to determine the best treatments, based on previous research (Crowe et al., 2005). After identifying the most effective treatments, 80 g of blueberries were used for further evaluation of the antimicrobial efficacy of these treatments and their impacts on blueberry qualities, as described in sections 2.4, 2.5, and 2.6. In addition, a 1-min washing time was studied for the best effective treatments to determine if a shorter time was effective in

Salmonella inactivation.

3.2.3 Enumeration of Salmonella After treatments, blueberries at 4 g were placed in a sterile stomacher sample bag containing 10 ml of elution buffer (phosphate-buffered saline [PBS]) and pummeled in a stomacher (Colworth Stomacher 400, A. J. Seward and Co., Ltd., London, UK) for 2 min at medium speed. For the quantification of surviving bacteria, 1 ml aliquots from 10 ml of homogenate were plated on three XLD agar plates. Plates were incubated at 37 ℃ for 24 h before presumptive-positive black colonies were counted (Andrews, Jacobson, and Hammack, 2011).

The detection limit was 0.39 log10 CFU/g.

For Salmonella enrichment, 1 ml of the blueberry homogenate in PBS and 9 ml of preenrichment broth (TSB) were incubated at 37 ℃ for 24 h. After incubation, 1 ml of TSB was plated on three XLD agar plates. Plates were incubated at 37 ℃ for 24 h and examined for the presence of Salmonella colonies (Lu and Wu, 2010).

25 3.2.4 Effect of washing treatments on the physical properties of blueberries Fresh blueberry color was determined by a color reader (MINOLTA model CR-10, Minolta Camera Co., Ltd., Osaka, Japan) to determine the following color values: L* (brightness/darkness), a* (redness/ greenness), and b* (yellowness/blueness). Measurements were taken at three different parts of treated and untreated un-inoculated blueberries. Texture was measured by a TA.XT2 texture analyzer (Texture Technology Corp., Scarsdale, N.Y., U.S.A.) using a TA-91 Kramer shear probe with a rounded end. For pH measurements, 5 g of blueberries were placed in sterile stomacher sample bags and homogenized in a stomacher for 2 min at medium speed. The pH values of homogenates were measured by a pH meter (FiveEasy FE20, Mettler-Toledo AG, Greifensee, Switzerland).

3.2.5 Total phenolic and anthocyanins content The total phenolic contents of untreated and treated blueberries were determined as described by Tsai et al. (2008). Gallic acid was used as a standard. Twenty μl of supernatants from centrifuged homogenates of treated and untreated blueberries, 180 μl of distilled water, 100 μl of the Folin-Ciocalteau reagent(Sigma–Aldrich, Inc., St. Louis, MO, USA), and 0.5 ml of a 20% sodium carbonate (Sigma–Aldrich, Inc., St. Louis, MO, USA) solution made up the reaction mixtures. The reaction mixtures were covered, vortexed vigorously, and allowed to react at room temperature for 2 h in microcentrifuge tubes. Two-hundred μl of these mixtures was added into each blank well of a clear 96-well plate. Absorbance was measured at 765 nm by a Synergy 2 multimode microplate reader (BioTek Instruments, Inc., Winooski, VT) and the total phenolic content was calculated as mg gallic acid (Sigma–Aldrich, Inc., St. Louis, MO, USA) equivalent/100 g of berries.

The method for determination of total anthocynins content was performed according to Qi et al. (2011) with modifications. Fresh blueberry samples at 5 g and 30 ml of 80:20 (v/v)

26 methanol–water solution containing 0.1 ml/L acetic acid were added to a tube and homogenized by an ultra-turrax instrument for 2 min. The mixture (total volume of 30 ml) was placed in the dark for 1 h and then sonicated for 15 min. After centrifugation at 5000 rpm for 30 min at room temperature, the volume of the supernatants was recorded. The total anthocyanins content was measured by the pH differential method with some modification. The anthocyanins extract was dissolved in a 0.025 M potassium chloride buffer (pH 1.0) and 0.4 M sodium acetate buffer (pH 4.5) with a dilution factor at 6.0. The absorbance of each dilution was measured at 510 nm and

700 nm. The absorbance (A) of the diluted sample was calculated by the following formula:

A = (A510- A700) pH 1.0-(A510 - A700) pH 4.5 The monomeric anthocyanin pigment concentration in the original sample was expressed in equivalence of cyaniding-3-glucoside according to the following formula: anthocyanin content (mg/l)=(A ×MW× DF×1000)／ε×1

where MW (449.2) is the molecular weight of cyanidin-3-glucoside in; DF is the dilution factor; and ε is the molar absorptivity, which is equal to 26, 900 for cyanidin-3-glucoside.

3.2.6 Impact of the two most effective treatments coupled with low temperature storage on Salmonella, yeasts and molds counts Inoculated blueberries (80 g) were washed for 5 min in 20 ml of washing solutions per 1 g of blueberries with continuous agitation. The two washing solutions were a combination of

500 ppm acetic acid and 5000 ppm SDS, and a combination of 200 ppm H2O2 with 5000 ppm SDS. Following treatment, samples were removed for microbial analysis at time 0h and transferred to a plastic storage package prior to storage at 4 ℃ for 3 days. Untreated blueberries served as the control for the study. Distilled water and 200 ppm chlorine was also applied as comparisons. Viable cells of Salmonella after 0, 5, 12, 24, 48 and 72 h post washing were determined. Counts of yeasts and molds were determined on Potato Dextrose Agar (Difco)

27 acidified to a final pH of 3.5 with tartaric acid. The plates were incubated at 25 ℃ for 3-5 days (Tournas et al., 2001).

3.2.7 Statistical analysis All experiments were conducted in three independent trials, and triplicate samples were analyzed in each trial. The data are represented as mean values ± standard deviation. Microbial survivors after treatments, color, pH measurements, and nutrient contents were analyzed for significant treatment differences by one-way analysis of variance and the fit model test of JMP (v. 10.0, SAS Institute Inc., Cary, NC). The different effect in washing experiments was assessed by the fit model. Significance was determined at P values of 0.05. Log reductions were calculated as difference between mean log Salmonella population of unwashed samples and log survivor counts of each treated sample.

3.3 Results and Discussion

3.3.1 Effect of the combination of chlorine and SDS solutions on inactivation of Salmonella The reductions of Salmonella after washing with 4, 12.5, 25, 50, and 100 ppm chlorine plus SDS at 50, 500 and 5000 ppm for 5 mins are shown in Fig. 3.1. The microbial load on untreated control samples was 4.6 log10 CFU/g. All treatments resulted in survivor counts significantly lower than those from unwashed blueberries (P<0.05). Chlorinated water at 4 ppm alone was able to achieve 3.2 log10 CFU/g reductions of Salmonella, however, the addition of 50,

500, and 5000 ppm SDS in chlorine treatment did not show any significant improvement in log10 reduction. A similar pattern was also observed for chlorinated water at other concentrations (12.5, 25, 50, and 100 ppm). It is interesting to notice that combinations of SDS at 10~50 ppm with chlorinated water (200 ppm) improved inactivation of human norovirus surrogates on

28 blueberries (Predmore and Li, 2011), but not Salmonella on the blueberries. Even though adjusted pH combinations of chlorine with SDS washing solutions resulted in slightly higher log reduction than the unadjusted pH combinations (Fig. 3.1), no statistical significant difference was observed.

Figure 3.1 Recovery of Salmonella from spot-inoculated blueberries after washing with combinations of chlorine and SDS at different concentrations.

Note: A: chlorine unadjusted pH to 6.25; B: chlorine adjusted pH to 6.25. Microbial counts represent the mean of three independent trials, and triplicate samples were in each trail. Values represent means±SD.

3.3.2 Effect of combinations of organic acids and SDS solutions on inactivation of Salmonella Commercial blueberry processing mostly involves a brief passage of the berries through a tank of chlorinated sanitizer solution. According to Sapers and Simmons (1998), a 1- to 2-log population reduction is the most that can be expected when chlorine is used at permitted concentrations. Hence, given the limited efficacy of chlorinated water, efficiency of washing blueberries with organic acids (lactic acid, acetic acid, and citric acid) and SDS to inactivate surface inoculated Salmonella were evaluated individually or in combination. When an organic acid was combined with SDS, treatments resulted in an increase in log10 reductions for Salmonella. When washed with lactic acid solution at 50 ppm in combination with SDS, reductions of Salmonella ranged from 2.6 to 3.2 log10 CFU/g for solutions containing 50 ppm SDS and 5000 ppm SDS, respectively. These reductions were significantly higher than those for lactic acid alone (1.8 log10 CFU/g reduction) (P<0.05). Additionally, combinations of SDS with higher concentration of acid (lactic acid at 500 ppm with 50 ppm SDS) resulted in a significantly higher log10 reduction of Salmonella compared to that achieved with a lower level of acid (lactic acid at 50 ppm with 50 ppm SDS) (P<0.05). Lactic acid at 500 ppm plus 50 ppm SDS, or citric acid at 500 ppm plus 50 ppm SDS showed similar Salmonella reductions to those obtained with 100 ppm chlorine (P>0.05). Furthermore, 500 ppm acetic acid plus 5000 ppm SDS achieved the greatest reductions, as they reduced Salmonella populations from 4.64 log10 CFU/g to 0.63 log10 CFU/g (Fig. 3.2). As this treatment resulted in zero colony counts on six plates out of total of nine plates, enrichment was conducted and three out of six enrichments were negative for

Salmonella. The efficacy of this treatment was significantly higher (P<0.05) than that of the treatment consisting of 500 ppm acetic acid and 500 ppm SDS.

31 Several organic acids have lethal or inhibitory effects on microorganisms and have generally recognized as safe (GRAS) status. Shapiro and Holder (1960) indicated that 150 ppm and 500 ppm citric acid solutions were capable of inhibiting bacterial growth in salad greens for 96 hours. At a concentration of 0.5% citric acid was effective in reducing aerobic mesophillic bacteria (0.24 log), yeast (0.14 log), and mold (0.40 log) on blueberries (Crowe et al., 2005). Álvarez-Ordóñez et al. (2010) studied the effect of different types of acids on growth of Salmonella Typhimuriumin Brain Heart Infusion (BHI) under acidic conditions at different temperatures. The authors showed that acetic acid was the best antimicrobial against Salmonella Typhimurium, and that the order of effectiveness was: acetic>lactic>citric>hydrochloric acid. The results obtained with the current studies agree with those of Álvarez-Ordóñez et al. (2010). The primary mechanism of organic acids to affect microbial activity is their undissociated form. Since undissociated weak acids penetrate bacterial cell membranes through permeases or porins, cytoplasmatic pH is reduced by their intracellular dissociation, which affects the metabolic activity of the cell (Cherrington et al., 1991; Davidson, 2001). Acetic acid can easily penetrate bacterial membranes due to its low molecular weight and its great liposolubility, whereas lactic and citric acids diffuse slowly (Fernández et al., 2009).

SDS is GRAS multipurpose additive (21 CFR 172.822). This compound has been widely studied as a surfactant and is approved for use in a variety of foods, including egg whites, fruit juices, vegetable oils, and gelatin as a whipping or wetting agent (U.S. FDA, 2007). In the present study, SDS at 50, 500, 5000 ppm alone caused Salmonella reductions of only 1.6, 1.8 and

1.8 log10 CFU/g, respectively. The bactericidal effect of 500 ppm acetic acid or 5000 ppm SDS alone is not sufficient to kill more than 2 log10 CFU/g Salmonella within 5 min, but bactericidal activity was increased to 4.0 log10 CFU/g when 5000 ppm SDS was combined with the acetic acid. Our results indicate that addition of 5000 ppm SDS into 500 ppm acetic acid can significantly enhance inactivation of Salmonella on blueberries after a relatively short time washing. SDS could have significant bactericidal effect when pH was between 1.5 and 3.0,

32 which might be related with the ability of SDS to denature protein surfaces and damage cell membranes (Tamblyn and Conner, 1997; Williams and Payne, 1964). Our finding is in good agreement with previous research.

Figure 3.2 Recovery of Salmonella from surface-inoculated blueberries after washing with combinations of organic acid and SDS at different concentrations.

Note: Microbial counts represent the mean of three independent trials, and triplicate samples were in each trail. Values represent means±SD.

3.3.3 Effect of combinations of hydrogen peroxide and SDS solutions on inactivation of Salmonella

Combinations of H2O2 with higher concentrations of SDS resulted in a significantly higher reduction of Salmonella than those containing the lower SDS (Fig. 3.3). Reductions of

Salmonella were 1.8 and 3.0 log10 CFU/g for 50 ppm hydrogen peroxide solution containing 50 ppm SDS and 5000 ppm SDS, respectively. Despite nonstatistical differences among treatment

33 effectiveness at 50, 100 ppm H2O2 plus 50, 500 ppm SDS, addition of 5000 ppm SDS in 50 and

100 ppm H2O2 result in higher log10 reduction than those with 50 and 500 ppm SDS (P<0.05).

Furthermore, 200 ppm H2O2 containing 5000 ppm SDS resulted in the highest removal of Salmonella on inoculated berry surfaces, as the population of Salmonella was reduced from 4.93 log10 CFU/g to 0.73 log10 CFU/g. In addition, 200 ppm H2O2 containing 5000 ppm SDS showed similar Salmonella reductions as treatments with 200 ppm chlorine (P>0.05). Therefore, increased microbial reductions with 5000 ppm SDS added to the H2O2 solutions (50, 100 and

200 ppm) was observed in blueberries. That data showed H2O2 concentrations of less than 1% (200 ppm) can effectively inactivate Salmonella when SDS is present.

Figure 3.3 Recovery of Salmonella from spot-inoculated blueberries after washing with combinations of hydrogen peroxide and SDS at different concentrations.

Note: Microbial counts represent the mean of three independent trials, and triplicate samples were in each trail. Values represent means±SD.

34 3.3.4 Effect of combinations of trisodium phosphate and SDS solutions on inactivation of Salmonella Washing blueberries for 1 min with a combination of trisodium phosphate and SDS resulted in survivor counts significantly lower than those from unwashed blueberries (P<0.05)

(Fig. 3.4). Trisodium phosphate at 500 ppm alone was able to achieve a 2.8 log10 CFU/g reduction in Salmonella counts. However, additional SDS did not show any significant improvement in log10 reduction. A similar pattern was observed for trisodium phosphate solution at concentrations of 50 and 200 ppm.

Figure 3.4 Recovery of Salmonella from spot-inoculated blueberries after washing with combinations of trisodium phosphate and SDS at different concentrations.

Note: Microbial counts represent the mean of three independent trials, and triplicate samples were in each trail. Values represent means±SD.

35 3.3.5 Effect of washing treatments on the physical properties of blueberries The effects of the treatments that resulted in the highest reductions in Salmonella counts, namely 500 ppm acetic acid plus 5000 ppm SDS and 200 ppm H2O2 containing 5000 ppm SDS on the quality of treated berries were determined. For each of the treatments, visual quality analysis was performed (Table 3.1). None of the color parameters (L*, a*, and b*) changed significantly after washing by treatments (P>0.05). It has been documented previously that 5%

H2O2 resulted in a mildly bleached appearance on produce, although it resulted in more than 2 log10 reductions of Salmonella (Beuchat, 1997). In the current study, berries washed with 200 ppm H2O2 combined with 5000 ppm SDS did not show adverse changes in surface color. No significant difference was detected with respect to the pH and total anthocyanins values of untreated and treated blueberries. The pH and total anthocyanins content of untreated blueberries was 3.13 and 0.344 mg/g, while the pH of treated blueberries was around 3.2, and the total anthocyanins content of treated blueberries ranged from 0.308 to 0.393 mg/g, respectively. The treated berries had similar values for visual quality, pH, and anthocyanins contents as berries treated with 200 ppm chlorine.

The texture value of untreated blueberries was 2.6 N, while that of treated blueberries was around 2.3 N. All of the washings, including the 200 ppm chlorine or DI washings, decreased texture values as compared with no washings applied; however, there was no difference between the values after washing with the two chosen solutions and washing with 200 ppm chlorine. The total phenolic content of untreated blueberries was 74.73 mg/100g while the total phenolic content of treated blueberries was around 76 mg/100g. When compared with 200 ppm chlorine treatment, both effective treatments resulted in higher total phenolic contents in blue berries (P<0.05), the underlying mechanism remainsto be determined.

Blueberry quality and safety are greatly impacted by the methods of harvesting, storage, and processing as populations of bacteria, yeasts, and filamentous fungi can increase

36 considerably. One of the industry’s biggest concerns is the loss in fruit firmness caused by machine harvesting (NeSmith, Prussia, Tetteh, andKrewer, 2002). Since loss of firmness leads to blueberry decay during postharvest procedures as fungi are capable to invade the berries and increase the pH allowing growth of bacterial pathogens and spoilage organisms. In thepresent study, the use of 500 ppm acetic acid plus 5000 ppm SDS, 200 ppm hydrogen peroxide in combination with 5000 ppm SDS did not have any negative effect on pH, but a slight loss of firmness was determined in washed berries.

Table 3.1 The test values for color, pH, texture, total phenolic and anthocyanins content of treated and untreated berries

Total Total Color Texture anthocyanins phenolic Treatments PH (N) content content (mg/g) (mg/100g) L Chroma

200 ppmH O + 2 2 29.6±0.8 a 5.0±0.7 a 3.12±0.15 a 2.3±0.4 b 0.308±0.031 a 80.68±4.41 b 5000 ppm SDS

500 ppm acetic acid+ 5000 29.0±0.7 a 5.3±0.6 a 3.06±0.19 a 2.3±0.3 b 0.324±0.030 a 78.34±0.85 b ppm SDS

200 ppm Cl 29.0±0.6 a 5.3±0.5 a 3.22±0.27 a 2.3±0.3 b 0.347±0.085 a 73.77±0.77 a

DI 28.4±0.6 a 6.5±0.7 a 3.26±0.23 a 2.2±0.3 b 0.314±0.098 a 77.89±1.81b

No wash 29.4±0.9a 5.8±1.1 a 3.13±0.04 a 2.6±0.5 a 0.344±0.063 a 74.73±4.68 a

Note: Data are shown as means ± standard deviation. Data followed by the same superscript was insignificantly different to that of no wash berries in the same column (P>0.05). DI: distilled water washing.

37 3.3.6 Impact of treatments coupled with low temperature storage on Salmonella, yeasts and molds counts

Berries were washed with in 500 ppm acetic acid plus 5000 ppm SDS or 200 ppm H2O2 containing 5000 ppm SDS for 5 min, and stored at 4 ℃ for 3 days. There was no statistical difference between the outcomes of the two treatments at 0 h (Fig. 3.5); however, both treatments resulted in significantly lower Salmonella counts when compared to those found on unwashed blueberries and berries washed with DI water (P<0.05). Initial Salmonella counts on the unwashed and DI water washed berries were 4.5 log10 CFU/g and 3.5 log10 CFU/g, respectively, while the average survivors count on the two treated berries was 0.9 log10 CFU/g. Although low temperature storage decreased Salmonella counts in both treated and control samples, the acetic acid plus SDS, or hydrogen peroxide plus SDS treated berries still had significantly lower Salmonella counts when compared to those in non-treated controls or DI water-washed berries after 3 days storage (P<0.05). In fact, populations of Salmonella remained relatively constant on treated blueberries throughout storage. The berries washed with 500 ppm acetic acid plus 5000 ppm SDS or 200 ppm H2O2 containing 5000 ppm SDS had similarly low Salmonella counts as berries treated with 200 ppm chlorine.

The initial level of yeasts and molds on unwashed blueberries was about 3.6 log10 CFU/g (Fig. 3.5). Treatments with 500 ppm acetic acid plus 5000 ppm SDS or 200 ppm hydrogen peroxide in combination with 5000 ppm SDS resulted in yeasts and molds counts that were significantly lower than those of unwashed blueberries. The mean reductions of 500 ppm acetic acid plus 5000 ppm SDS and 200 ppm hydrogen peroxide containing 5000 ppm SDS were 2.4 and 2.0 log10 CFU/g, respectively, and these values are not different at time 0 h. Both treated berries had similar log reduction of yeasts and molds as observed in 200 ppm chlorine treated berries. Crowe et al. (2005) reported that a 5-min spray of 100 ppm chlorine reduced populations of bacteria, yeast, and mold on blueberries by only 0.83, 0.77, and 0.61 log10 CFU/g,

38 respectively. The lower values may have been caused by shorted exposure times due to spraying instead of submerging the berries.

After 3 days storage at 4 ℃, both treatments had significantly lower yeasts and molds counts than those of unwashed or DI water-washed blueberries (P<0.05). Additionally, the numbers of viable yeasts and molds decreased during the storage.

Figure 3.5 Impact of two promising treatments on inactivation of Salmonella (A) and molds and yeasts (B) coupled with low temperature storage on the microbiological quality.

Note: Microbial counts represent the mean of three independent trials, and triplicate samples were in each trail. Values represent means±SD.

40 3.3.7 Comparison of Salmonella inactivation after washing for 1 and 5 min Two washing time (1 and 5 min) were compared to determine if a shorter time was feasible to obtain effective Salmonella inactivation. Washing in 200 ppm H2O2 containing 5000 ppm SDS resulted in significantly more efficacy in killing Salmonella after 5 min washing (4.2 log10 CFU/g reduction) compared with 1 min washing (3.2 log CFU/g reduction), but no significant difference was detected when the berries were washed in 500 ppm acetic acid plus 5000 ppm SDS for 5 or 1 min (Fig. 3.6, P>0.05). Crowe et al. (2007) reported that increased treatment time from 60s to 120s when using 1% H2O2 did not significantly improve log reductions of Pseudomonas fluorescenson wild blueberries. The difference in outcomes may have been due to different H2O2 concentrations, treatment times and pathogens used. In the current study, all 4 single treatments including 200 ppm chlorine washing showed no difference inefficacy between the 1-and 5-min washings. Both treatment combinations had similar efficacy as that of a 200-ppm chlorine wash. When compared with single treatments (5000 ppm SDS, 200 ppm H2O2 or 500 ppm acetic acid), the combinations with SDS, 200 ppm H2O2 containing 5000 ppm SDS or 500 ppm acetic acid plus 5000 ppm SDS, still had better efficacy to inactivate

Salmonella (Fig. 4, P<0.05) in either 1- or 5-min washes.

Figure 3.6 Comparison of two promising alternatives on inactivation of Salmonella between 1 min and 5 min washing.

Note: Microbial counts represent the mean of three independent trials, and triplicate samples were in each trail. Values represent means±SD.

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45 Chapter 4

SURVIVAL OF SALMONELLA TYPHIMURIUM AND E. COLI O157:H7 ON

BLUEBERRIES DURING FROZEN STROAGE AFTER WASHING WITH

COMBINATIONS OF SODIUM DODECYL SULFATE AND ORGANIC ACIDS OR

HYDROGEN PEROXIDE

Abstract

Salmonella spp. and E. coli are known for their tolerance of freezing. The objective of this study was to investigate survival of the two organisms during storage at -18 ±2 ℃ for 12 weeks on blueberries after washing with: 500 ppm acetic acid plus 5000 ppm sodium dodecyl sulfate (SDS) (AA/SDS), 20 ppm peroxyacetic acid plus 5000 ppm SDS (PPA/SDS), or 200 ppm hydrogen peroxide plus 5000 ppm SDS (H2O2/SDS). Following a 60 s contact time with one of the three solutions, the treatments showed 3.3 to 3.9 log10 CFU/g reductions in Salmonella Typhimurim and E. coli O157:H7 counts. After 2-weeks of frozen storage, significant reductions of Salmonella and E. coli were observed (3.9 to 4.2 log10 CFU/g reductions). After 12-weeks of frozen storage, no Salmonella and E. coli survivors were detected by direct plating. Results suggest that low-temperature frozen storage had a significant impact (P<0.05) on microbial counts of both treated and non-treated blueberries. Although none of these washings decreased the total phenolic and anthocyanins contents and apparent quality at time 0, frozen storage caused significant damage on the texture of both treated and non-treated blueberries. No significant decrease in the total phenolic, anthocyanins content and apparent quality was observed during the frozen storage. The counts of total bacteria, yeasts and molds decreased throughout storage for treated and untreated berries. This study demonstrates that the three wash solutions can enhance the safety of frozen berries.

4.1 Introduction Freezing is the most traditional method of preserving berries since it provides for a long shelf life and minimal impacts on quality and nutrition. Although illnesses associated with frozen berries are rare, freezing also preserves the viability of some pathogenic microorganisms. An outbreak of hepatitis A in UK was linked to the consumption of frozen raspberries, which were likely contaminated at the picking stage or when the plastic tubs were filled and their weight adjusted prior to freezing (Reid and Robinson, 1987). In March 1997, a total of 153 cases of hepatitis A were suspected to be associated with the consumption of frozen strawberries (CDC, 1997). A hepatitis A in September 2013 involving 161 cases of illness was attributed to the consumption of a frozen berry mix, including cherries, blueberries, pomegranate seeds, raspberries and strawberries (CDC, 2013).

Berries picked for frozen processing are usually full-flavored, ripe berries with uniform size and tender skins. The berries are usually washed or sprayed with chlorinated water containing 50-200 ppm active chlorine to reduce microorganisms (Good Agricultural and Manufacturing (Handling) Safety and Food Defense Practices, 2010); however, this chlorine- based washing solution has shown limited efficacy to deactivate foodborne pathogens on blueberries, with 0.83, 0.77, and 0.61 log10 CFU/g reductions of bacteria, yeast, and mold after washing with 100 ppm chlorine for 5 minutes (Crowe et al., 2005). The chlorine-based washing solutions also bring increased scrutiny from regulatory agencies due to the undesirable chlorine by-products (Artes et al., 2009; Nieuwenhuijsen et al., 2000). Hence, because of limited effectiveness and issues surrounding public health and environmental safety, there is a need for more effective and practical methods for removal of pathogens from fresh blueberries prior to freezing.

47 Sodium dodecyl sulfate (SDS) is a surface-active compound that lowers the interfacial tension between two liquids or between a liquid and a solid. Additions of SDS to a levulinic-acid solution enhanced removal of Salmonella on chicken breast meats (Zhao et al., 2009), and combinations of SDS with chlorinated water improved inactivation of human norovirus surrogates on produce (Predmore and Li, 2011). Enhanced inactivation of Salmonella Typhimurim on blueberries was determined after washing with solutions combining SDS (50, 500, and 5000 ppm) with other antimicrobials (lactic acid, acetic acid, citric acid or hydrogen peroxide) (Li and Wu, 2013). Three washing solutions, 500 ppm acetic acid plus 5000 ppm SDS (AA/SDS), 20 ppm peroxyacetic acid combined with 5000 ppm SDS (PPA/SDS), or 200 ppm hydrogen peroxide containing 5000 ppm SDS (H2O2/SDS) resulted in more than 3 log10 CFU/g reductions of Salmonella Typhimurium. To further evaluate the antimicrobial efficacy of the three effective washing treatments, their impacts on E. coli O157:H7 surface inoculated on blueberries were determined. In addition, treated blueberries were stored in -18 ±2 ℃ freezer for 12 weeks, and the survival of Salmonella and E. coli was determined. The impacts of these treatments on sensory quality, total phenolic, anthocyanins content, molds and yeasts, and total bacteria account were also tested during the frozen storage of the blueberries.

4.2 Materials and Methods

4.2.1 Bacterial strains and inoculum preparation Salmonella enterica serovar Typhimurium (DT 104) and E. coli O157:H7 strain 250 (sprout outbreak isolate) obtained from the culture collection in the Department of Animal and

Food Sciences at the University of Delaware (Newark, DE). A mutant of the E. coli strain was isolated that is able to grow in the presence 100 μg/ml of nalidixic acid (Fisher Scientific, Hampton, NH, USA) and 100 μg/ml of streptomycin (Sigma, St. Louis, MO, USA). Stock cultures of Salmonella on tryptic soy agar (TSA; Difco Laboratories, Becton Dickinson, Spark,

48 Md) and E. coli on TSA plus 0.6% yeast extract (Difco) supplemented with 100 μg/mL of nalidixic acid and 100 μg/mL of streptomycin (TSAYE-NS) were stored at 4 ℃. A single colony of Salmonella and E. coli from TSA plate was cultured into tryptic soy broth (TSB, Difco Laboratories, Becton Dickinson, Spark, Md) and TSB plus 0.6% yeast extract (Fisher) supplemented with same antibiotics (TSBYE-NS), respectively, and then grown at 37 ℃ for 24 h The culture was transferred to fresh TSB and TSBYE-NS and incubated at 37 ℃ for another 24 h. Before inoculation on the blueberries, viable counts were determined by serially diluting suspensions in sterile 0.1% peptone water (Difco) and spread plating 0.1 ml on the xylose lysine deoxycholate (XLD; Difco) plate for Salmonella and TSAYE-NS for E. coli.

4.2.2 Inoculation of blueberry surfaces Fresh blueberries were obtained from a local market on the day of experimentation. Intact surfaces were selected for inoculation. Inoculation was achieved by applying 25 μl of inoculum (6 to 10 small droplets) onto the surface of each blueberry. After inoculation, blueberries were left in a laminar flow hood at room temperature (21±1 ℃) for about 2 h to allow for attachment of the microorganisms. Inoculated blueberries had approximately 106 CFU/g of Salmonella and E. coli.

4.2.3 Washing procedures and frozen storage The washing solutions included: 200 ppm chlorine (prepared from sodium hypochlorite, Sigma–Aldrich, Inc., St. Louis, MO, USA), 80 ppm peroxyacetic acid (prepared from 32% peroxyacetic acid, Sigma–Aldrich, Inc., St. Louis, MO, USA), combinations of acetic acid (500 ppm, Sigma–Aldrich, Inc., St. Louis, MO, USA), peroxyacetic acid (5, 10 and 20 ppm, prepared from 32% peroxyacetic acid, Sigma–Aldrich, Inc., St. Louis, MO, USA), hydrogen peroxide (200 ppm, prepared from 30% hydrogen peroxide, Sigma–Aldrich, Inc., St. Louis, MO, USA)

49 and 5000 ppm SDS (Sigma–Aldrich, Inc., St. Louis, MO, USA). Distilled water (DI) washing served as negative control. Blueberries at 50 g were submerged in 1000 ml of the washing solutions with continuous agitation provided by starring bar in a beaker for 1 min. For further evaluation of the antimicrobial efficacy of these effective treatments and their impacts on blueberry qualities, as described in 2.6 and 2.7, blueberries of 80 g were used.

Following each treatment, samples were removed for microbial analysis at time 0 and transferred to commercially plastic storage bags prior to storage at -18 ±2 ℃ for 12 weeks. Viable cells of Salmonella and E. coli after each treatment were determined at 1, 2, 4, 8, and 12 weeks.

4.2.4 Microbial analysis After washing, two randomly picked blueberries (4 g) from each treatment were placed in a sterile stomacher sample bag containing 10 ml of elution buffer (phosphate-buffered saline

[PBS]) and pummeled in a stomacher (Colworth Stomacher 400, A. J. Seward and Co., Ltd., London, UK) for 2 min at medium speed. For the quantification of surviving bacteria, 1 ml aliquots from 10 ml of homogenate were plated on three XLD agar plates for Salmonella and three TSAYE-NS plates for E. coli. Plates were incubated at 37 ℃ for 24 h before presumptive- positive colonies were counted (Andrews, Jacobson, and Hammack, 2011). The microbial population detection limit was 0.39 log10 CFU/g.

Salmonella enrichment was carried out by adding 10 ml of TSB to the PBS homogenate and incubating at 37 ℃ for 24 h. After incubation, a loopful of enrichment solution was streaked onto XLD plate for Salmonella and TSAYE-NS for E. coli and incubated for 24 h at 37 ℃. Additionally, 0.1 ml of enrichment solution was transferred to 10 ml universal preenrichment broth (Difco) for Salmonella and MacConkey broth (Difco) for E. coli, respectively, incubated at

50 37 ℃ for 24 h, and a loopful of the cultures was streaked onto XLD agar for Salmonella and MacConkey agar for E. coli.

4.2.5 Sensory evaluation after 12-weeks of frozen storage Sensory evaluation (overall appearance, color, texture, and aroma) was conducted using a 5-point subjective scale. For overall appearance, the following scale was used: 5, excellent quality, fresh appearing; 4, good quality, minor defects; 3, fair quality; 2, poor quality, excessive defects; 1, extremely poor quality, not usable. For color, texture and aroma, similar 5-point scales were used. Ten untrained panelists performed subjective assessments for all samples. Frozen blueberry samples for each panelist were enclosed in a 20-ml plastic container, allowed to thaw for 15 min at ambient temperature before being presented to the panelists. Each panelist evaluated six blueberries from each treatment.

4.2.6 Physical properties and total bacteria, yeasts and molds counts of blueberries during frozen storage Color, texture, and pH of un-inoculated blueberries were determined at 0, 1, 2, 4, 8, and 12 weeks. Color was determined by a color reader (MINOLTA model CR-10, Minolta Camera Co., Ltd., Osaka, Japan) to determine the following color values: L* (brightness/darkness), a*

(redness/ greenness), and b* (yellowness/blueness). Measurements were taken at three different parts of treated and untreated un-inoculated blueberries. Texture was measured by a TA.XT2 texture analyzer (Texture Technology Corp., Scarsdale, N.Y., U.S.A.) using a TA-91 Kramer shear probe with a rounded end. For the pH measurement, 5 g blueberries were placed in sterile stomacher sample bags and homogenized in a stomacher for 2 min at medium speed. The pH values of homogenates were measured by a pH meter (FiveEasy FE20, Mettler-Toledo AG, Greifensee, Switzerland).

51 The survival of total bacteria and yeasts and molds on uninoculated blueberries during 12-week frozen storage was also determined. Total bacteria account was enumerated on TSA plates and incubated at 37 ℃ for 24 h. Yeasts and molds were enumerated on Potato Dextrose Agar (Difco) acidified to a final pH of 3.5 with tartaric acid. The plates were incubated at 25 ℃ for 3-5 days (Tournas et al., 2001).

4.2.7 Total phenolic and anthocyanins content The total phenolic and anthocyanins contents of untreated and treated blueberries were determined as described by Li and Wu (2013) using the gallic acid as standard curve method and the pH differential method, respectively.

4.2.8 Statistical analysis All experiments were conducted in three independent trials. The data are represented as mean values ± standard deviation. Microbial survivors after treatments, color, pH measurements, and nutrient contents were analyzed for significant treatment differences by one-way analysis of variance, fit model test of JMP (v. 10.0, SAS Institute Inc., Cary, NC). The different effect in washing experiments was assessed by fit model. Significance was determined at P values of 0.05. Log reductions were calculated as difference between mean log Salmonella or E. coli population of unwashed samples and log survivor population of each treated sample.

4.3 Results and Discussion

4.3.1 Effect of the combination of peroxyacetic acid and SDS on inactivation of Salmonella It is well known that peroxyacetic acid has a higher oxidation potential than chlorine sanitizers but a lower potential than ozone. The effect of peroxyacetic acid and SDS on the

52 inactivation of Salmonella on blueberries was evaluated individually or in combination. The initial microbial load on untreated control samples was 5.0 log10 CFU/g (Fig. 4.1). Addition of SDS to the peroxyacetic acid solution at 5 and 10 ppm did not show any significant improvement in log10 reduction. A combination of 20 ppm peroxyacetic acid combined and5000 ppm SDS yielded a maximum reduction of 3.9 log after 5 min of washing, a reduction similar to that achieved 200ppm of chlorine (P>0.05). Reduced microbial growth can be partially attributed to the residual effect of acetic acid released by the degradation of peroxyacetic acid. Park and

Beuchat (1999) found that 200ppm chlorine was as effective as 80 ppm peroxyacetic acid for treating cantaloupes. In the present study, 5000 ppm SDS improved the antimicrobial effect of peroxyacetic acid at 20 ppm, which also brought the benefit of reducing the amount of peroxyactic acid in the washing solutions.

Figure 4.1 Recovery of Salmonella from surface-inoculated blueberries after washing with combinations of peroxyacetic acid and SDS at different concentrations.

53 Note: Microbial counts represent the mean of three independent trails. Values represent means±SD.

4.3.2 Combination of SDS and organic acids or hydrogen peroxide on inactivation of Salmonella and E. coli during frozen storage Figure 4.2 shows the effect of washing and 12 week of frozen storage on survival of

Salmonella on blueberries. The initial microbial load on untreated control samples was 4.67 log10 CFU/g. Washing the inoculated blueberries with the combinations of antimicrobials reduced

Salmonella counts by 3.3 to 3.7 log10 CFU/g. However, no significant differences were detected among the three combinations (P>0.05).The solution containing 80 ppm peroxyacetic acid reduced the population of Salmonella from 4.67 log10 CFU/g to 0.57 log10 CFU/g, a reduction similar to that with a200-ppm chlorine wash (P>0.05).Our results indicate that addition of 5000 ppm SDS into 500 ppm acetic acid resulted in a 3.7-log reduction of Salmonella, which is in good agreement with previous research (Li and Wu, 2013).

Further reduction in Salmonella number with three combination washing was noticed after frozen storage for 2 weeks as AA/SDS, PPA/SDS, and H2O2/SDS reduced viable counts of

Salmonella by 4.1, 4.0 and 3.9 log10 CFU/g, respectively, which was a significant reduction compared to the control (0.5 log10 CFU/g reduction) (P<0.0001). There was no significant difference in the effectiveness of the three combination washing solutions and treatment with

200 ppm chlorine after 2-week frozen storage (P>0.05). After 8 weeks of frozen storage, the

Salmonella counts with three combination washing treatments decreased to about 0.4 log10 CFU/g. After 12weeks of frozen storage, no survivors among three combination washing treatments were detected by direct plating. However, Salmonella was detected in the enrichment cultures. Salmonella spp. are known for their tolerance to freezing (Archer, 2004). Salmonella

Typhimurium survived on frozen fish and stored at -17.9 ℃ for over 1 year with only 1 log10 reduction in numbers (Raj and Liston, 1961). Salmonella Typhimurium cells on frozen sausage and minced beef were only sublethally damaged after frozen storage at -18 ℃ for up to 10 weeks

54 (Barrell, 1988). Therefore, our results are consistent with previous studies that Salmonella cannot be eliminated during frozen storage.

When organic acids or hydrogen peroxide plus SDS combination treatments were evaluated for killing E. coli O157:H7 on blueberries during frozen storage (Figure 4.2), washes with AA/SDS for 60s reduced E. coli cell numbers by 3.9 log10 CFU/g, whereas PPA/SDS treatment reduced E. coli by 3.4 log10 CFU/g in comparison to the no-wash control. Similarly, E. coli was reduced by 3.5 log10 CFU/g when treated with H2O2/SDS for 60s. Treatment with AA/SDS showed similar reductions to those obtained with 200 ppm chlorine (P>0.05). After 1- week frozen storage, no significant differences were detected not only among three combination wash solutions tested (P>0.05) but also between each combination wash solution and 200 ppm chlorine (P>0.05). Further slightly reductions in E. coli numbers were observed during frozen storage for up to 12 weeks. For example, AA/SDS reduced E. coli numbers from 4.61 log10

CFU/g to 0.57 log10 CFU/g with 1-week frozen storage, whereas AA/SDS was able to reduce E. coli numbers from 0.57 log10 CFU/g to 0.40 log10 CFU/g during frozen storage from 1-week to 8-week. After 12-week frozen storage, E. coli on blueberries treated with the combination wash solutions was detectable by enrichment culture but not by directly plating. Our results are in good agreement with Doyle and Schoeni’s finding (Doyle and Schoeni, 1984) that E. coli O157:H7 survive well on food products during frozen storage.

Figure 4.2 Log10 reductions by washing blueberries contaminated with Salmonella (A) and E. coli O157:H7 (B) with SDS and organic acids or hydrogen peroxide followed by frozen storage.

Note: Microbial counts represent the mean of three independent trails. AA/SDS: 500 ppm acetic acid plus 5000 ppm SDS; H2O2/SDS: 200 ppm hydrogen peroxide containing 5000 ppm SDS; PPA/SDS: 20 ppm peroxyacetic acid combined with 5000 ppm SDS.

4.3.3 Sensory analysis Freezing significantly affected the overall appearance of blueberries when compared with fresh ones (P<0.05) (Table 4.1). No significant difference in the overall appearances were observed among the berries that underwent combination treatments, 200 ppm chlorine or DI washes prior to frozen storage (P>0.05).Freezing did not have a noticeable effect on color and aroma, but caused significant damage to the texture of both treated and non-treated blueberries.

Table 4.1 Sensory test results for fresh blueberries and blueberries washed with antimicrobial solutions and stored for 12 weeks Overall appearance Color Texture Aroma Washing treatment score score score score

AA/SDS (frozen) 2.3±0.67B 4.3±1.06A 2±1.05B 4.3±0.82A

B A B A H2O2/SDS(frozen) 2.4±0.84 4.1±0.99 1.5±0.53 3.5±1.08

PPA/SDS(frozen) 2.3±0.82B 4.2±1.14A 1.4±0.52B 3.7±0.95A

80ppm peroxyacetic acid (frozen) 1.9±0.57B 4.3±0.82A 1.6±0.84B 3.6±1.07A

200ppm Cl (frozen) 1.9±0.99B 3.8±1.4A 1.6±0.84B 3.1±1.29A

DI (frozen) 2.3±1.06B 3.8±1.03A 1.7±0.95B 3.6±0.84A

No wash (frozen) 2.7±0.95B 3.9±0.57A 2.9±0.88B 4.1±0.74A

Fresh blueberries 4.9±0.32A 3.4±1.35A 4.6±0.52A 3.3±1.16A

Note: Values are means ± standard deviations (scale of 5 to 1) (n~10). Data followed by the same superscripts indicate was insignificantly different to that of no wash berries in the same column (P>0.05). DI: distilled water washing; AA/SDS: 500 ppm acetic acid plus 5000 ppm SDS; H2O2/SDS: 200 ppm hydrogen peroxide containing 5000 ppm SDS; PPA/SDS: 20 ppm peroxyacetic acid combined with 5000 ppm SDS.

57 4.3.4 Physical properties and total bacteria and yeasts and molds counts of blueberries during frozen storage The effect of washing treatment on the physical properties (color and texture) during cold storage is shown in Figure 4.3. Color parameters (L*, a*, and b*) were not significantly affected by washing treatments, whereas the value of L* slightly decreased during frozen storage. The texture value of untreated blueberries was 3.6 N, while the texture values of treated blueberries were around 3.2 N at time 0. However, 1-week frozen storage decreased texture value of treated and non-treated blueberries to 1.2 N. Apparently, freezing caused severe damage on the texture of treated and non-treated blueberries, which was related to the skin (epidermal and subepidermal) damage, flesh (parenchymal) damage, and leakage from the vascular tissue during frozen storage (Allan-Wojtas et al., 1999). There was no significant difference in pH value between untreated and treated blueberries after washing or during the frozen storage (P>0.05). For example, the pH value of untreated blueberries before frozen storage was 3.36, whereas the pH value of treated blueberries before frozen storage was around 3.38. With 12-week frozen storage, the pH value of untreated blueberries was 3.45, while the pH value of treated blueberries was around 3.46. No significant difference was detected in total anthocyanins and total phenolic content between untreated and treated blueberries at time 0 (P>0.05) as the total anthocyanins and phenolic contents of untreated blueberries were 36.9 and 72.9 mg/100g while the total anthocyanins content of treated blueberries were around 37.7 mg/100g, and the total phenolic content of treated blueberries ranged from 72.3 to 80.4 mg/100g. After 12-week frozen storage, only small changes in the total anthocyanins and phenolic contents were observed (Figure 4.4). The total anthocyanins and total phenolic content of untreated blueberries was respectively increased to 38.2 mg/100g and decreased to 66.2 mg/100g, whereas the total anthocyanins and the total phenolic contents of treated blueberries were around 38.1 and 67.2 mg/100g, respectively. Furthermore, no significant difference was observed in total anthocyanins and phenolic contents between each combination treatment and 200 ppm chlorine or DI wash. Therefore, in our present study, the use of 500 ppm acetic acid plus 5000 ppm SDS, 20 ppm

58 peroxyacetic acid combined with 5000 ppm SDS, and 200 ppm hydrogen peroxide in combination with 5000 ppm SDS did not have any negative effect on the physical and chemical properties of blueberries before frozen storage.

The initial total bacteria and yeast and mold counts on unwashed blueberries were about

1.8 and 3.4 log10 CFU/g, respectively (Figure 4.5). The combination treatments (AA/SDS,

PPA/SDS, and H2O2/SDS) resulted in total bacteria and yeasts and molds counts that were significantly lower than those of unwashed blueberries. The mean reductions by combination treatments from total bacteria and yeasts and molds counts were 0.4 and 1.5 log10 CFU/g, respectively, but these values were not statistically different at time 0. However, 200 ppm chlorine showed reduced effectiveness at inactivation molds and yeasts at time 0. Crowe et al. (2005) reported a 5 min spray of 100 ppm chlorine reduced populations of bacteria, yeast, and mold on blueberries by only 0.83, 0.77, and 0.61 log10 CFU/g, respectively. The reduced effectiveness of chlorine at inactivating surface microorganisms in previous research may be a result of organic matter surrounding the target cells. Further reduction in total bacteria and yeast and mold counts with the combination washes was noticed after frozen storage for 2 weeks. Additionally, all combination treatments had significantly lower total bacteria and yeast and mold counts than those of unwashed or DI-washed blueberries (P<0.05). After 12-week frozen storage, the counts for total bacteria and molds and yeasts with the combination washing treatments decreased to about 0.5 and 0.8 log10 CFU/g, respectively. Moreover, there was no significant difference in the effectiveness of these three combination washing solutions and 200 ppm chlorine on the inactivation of total bacteria and molds and yeasts during frozen storage

(P>0.05). Therefore, AA/SDS, PPA/SDS, and H2O2/SDS showed effective removal and inactivation of total bacteria and molds and yeasts on blueberries.

Figure 4.3 Physical properties of blueberries during frozen storage

Note: A: color (L); B: color (chroma); C: texture. Data were expressed in the average of three independent trails. Values represent means±SD. AA/SDS: 500 ppm acetic acid plus 5000 ppm SDS; H2O2/SDS: 200 ppm hydrogen peroxide containing 5000 ppm SDS; PPA/SDS: 20 ppm peroxyacetic acid combined with 5000 ppm SDS.

Figure 4.4 Chemical properties of blueberries during frozen storage.

Note: A: pH; B: total anthocynins content, C: total phenolic content. Data were expressed in the average of three independent trails. Values represent means±SD. AA/SDS: 500 ppm acetic acid plus 5000 ppm SDS; H2O2/SDS: 200 ppm hydrogen peroxide containing 5000 ppm SDS; PPA/SDS: 20 ppm peroxyacetic acid combined with 5000 ppm SDS.

Figure 4.5 Inactivation of total bacteria (A) and molds and yeasts (B) during frozen storage after treatments with a combination of SDS and organic acids or hydrogen peroxide.

Note: Microbial counts represent the mean of three independent trails. Values represent means±SD. AA/SDS: 500 ppm acetic acid plus 5000 ppm SDS; H2O2/SDS: 200 ppm hydrogen peroxide containing 5000 ppm SDS; PPA/SDS: 20 ppm peroxyacetic acid combined with 5000 ppm SDS.

62 REFERENCES

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63 Crowe, K.M., Bushway, A.A., Bushway, R.J., Davis-Dentici, K., Hazen, R.A., 2007. A comparison of single oxidants versus advanced oxidation processes as chlorine-alternatives for wild blueberry processing (Vaccinium angustifolium). International Journal of Food Microbiology 116, 25-31.

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Nieuwenhuijsen, M.J., Toledano, M.B., Elliot, P., 2000. Uptake of chlorination disinfection by-products; a review and a discussion of its implications for exposure assessment in epidemiological studies. Journal of Exposure Analysis and Environmental Epidemiology 10, 586-599.

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64 Tamblyn, K.C., Conner, D.E., 1997. Bactericidal activity of organic acids in combination with transdermal compounds against Salmonella typhimurium attached to broiler skin. Food Microbiology 14, 477-484.

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65 Chapter 5

COMPARISON OF PROMISING TREATMENTS EFFICACY ON SPOT OR

DIP INOCULATED BLUEBERRIES AND STRAWBERRIES

Abstract

Salmonella Typhimurium and E. coli O157:H7 are able to survive on blueberries and strawberries. In the current study, three different washing solutions were examined for inactivation of Salmonella Typhimurium and E. coli O157:H7 dip or spot inoculated onto blueberries and strawberries. The washes were carried out with

500 ppm acetic acid plus 5000 ppm SDS (AA/SDS), 200 ppm H2O2 containing 5000 ppm SDS (H2O2/SDS), and 20 ppm peroxyacetic acid combined with 5000 ppm SDS (PPA/SDS). Surface or calyx of blueberries was inoculated with approximately 106 colony-forming units (CFU/g) of Salmonella and E. coli. Additionally, surface of strawberries were dip or spot inoculated with Salmonella or E. coli (106 CFU/g). For tests involving surface spot inoculation, 1 min washing with AA/SDS resulted in the greatest reduction of Salmonella and E. coli by approximately 4 log10 CFU/g on blueberries. For calyx and dip contaminated blueberries, the treatments reduced

Salmonella and E. coli from 2.5 to 3.0 log10 CFU/g; however, washing inoculated strawberries with the solutions for 1 min only reduced Salmonella and E. coli by no more than 2.2 log10 CFU/g. Increasing the wash times of 2 and 5 min did not further enhance microbial inactivation. These findings show that dip inoculated berries were more difficult to decontaminate by the 1 min washing treatments than spot inoculated

66 berries. If strawberry fruit were contaminated with Salmonella Typhimurium or E. coli O157:H7, they could present a health hazard due to the relatively low effectiveness of common sanitizing agents.

5.1 Introduction Multistate outbreaks associated with consumption of blueberries and strawberries have been reportedin recent years. A multistate outbreak of hepatitis A was traced to frozen strawberries processed at a single plant in 1990 (Niu et al, 1992). Additionally, an outbreak of hepatitis A in New Zealand was linked to the consumption of raw blueberries, which were likely contaminated from infected food handlers or fecally contaminated groundwater (Calder et al., 2003). A more recent outbreak of hepatitis A in September 2013 was attributed to the consumption of a frozen berry mix that included cherries, blueberries, pomegranate seeds, raspberries and strawberries and caused confirmed illness in 161 people (CDC, 2013). E. coli

O157:H7 and Salmonella infection have been associated with the consumption of raw and minimally processed strawberries. A U.S. Food and Drug Administration (FDA) survey found that 1 out of 143 imported strawberry samples tested positive for Salmonella (FDA, 1999). In 2011, fresh strawberries were implicated in an E. coli O157 outbreak (Stone, 2011). Oregon Public Health officials have identified fresh strawberries as possible source of an E. coli O157:H7 outbreak that made at least 15 people sick, and caused the death of one person. These outbreaks highlight the need for effective control measures in the berry industry.

67 After harvesting, blueberries are usually washed or sprayed with chlorinated water containing 50-200 ppm active chlorine to remove dirt and agricultural chemicals and to reduce microorganisms (Crowe et al., 2005). However, commercial strawberries are not washed due to their high susceptibility to fungal deterioration. Knudsen et al. (2001) reported that both Salmonella and E. coli O157:H7 are capable of surviving on fresh strawberries for over 7 d. For these reasons, procedures and sanitizers must be developed that allow decontamination of these types of fruit and to overcome the safety issues associated with chlorinated water.

While spot inoculation represents contamination from a point source such as contact with soil, workers’ hands, or surfaces of equipment and has been recommended for testing the efficacy of sanitizers in killing foodborne pathogens on tomatoes, lettuce, and parsley, dipping inoculation simulates contamination from contaminated irrigation, run-off, or flume water. Therefore, the objective of this study was to further investigate the antimicrobial effects of three previously studied wash solutions (500 ppm acetic acid plus 5000 ppm SDS (AA/SDS), 200 ppm H2O2 containing 5000 ppm SDS (H2O2/SDS), and 20 ppm peroxyacetic acid combined with 5000 ppm SDS (PPA/SDS)) on decontamination of Salmonella Typhimurium and E. coli O157:H7 spot or dip inoculated onto surface or calyx of blueberries and the surface of strawberries.

68 5.2 Materials and Methods

5.2.1 Bacterial strains and inoculum preparation Salmonella enterica serovar Typhimurium (DT 104) and E. coli O157:H7 strain 250 (sprout outbreak isolate) used for this study was obtained from the culture collection in the Department of Animal and Food Sciences at the University of Delaware (Newark, DE). A mutant of the E. coli strain was isolated that is able to grow in the presence 100 μg/ml of nalidixic acid (Fisher Scientific, Hampton, NH,

USA) and 100 μg/ml of streptomycin (Sigma, St. Louis, MO, USA). Stock cultures of Salmonella on tryptic soy agar (TSA; Difco Laboratories, Becton Dickinson, Spark, Md) and E. coli on TSA plus 0.6% yeast extract (Difco) supplemented with 100 μg/mL of nalidixic acid and 100 μg/mL of streptomycin (TSAYE-NS) were stored at

4 ℃. A single colony of Salmonella and E. coli from TSA plate was cultured into tryptic soy broth (TSB, Difco Laboratories, Becton Dickinson, Spark, Md) and TSB plus 0.6% yeast extract (Fisher) supplemented with same antibiotics (TSBYE-NS), respectively, and then grown at 37 ℃ for 24 h The culture was then transferred to fresh TSB and TSBYE-NS and incubated at 37 ℃ for another 24 h. Before inoculation on the blueberries, populations of the cell culture were determined by serially diluting suspensions in sterile 0.1% peptone water (Difco) and spread plating 0.1 ml on the xylose lysine deoxycholate (XLD; Difco) plate for Salmonella and TSAYE-NS for E. coli.

5.2.2 Contamination of blueberries and strawberries Fresh blueberries and strawberries were obtained from a local market on the day of experimentation. Intact surfaces were selected for inoculation. Spot inoculation

69 was achieved by applying 25 μl of inoculum (6 to 10 small droplets) onto the surface of each blueberry and strawberry. For calyx inoculation, 25 μl of inoculum was placed on the calyx of blueberry. To dip inoculate blueberries and strawberries, blueberries and strawberries were fully submersed in 107 CFU/ml inoculums in a sterile stomacher bag. Bags were heat-sealed immediately and slight shaking was applied for 1 min. Blueberries and strawberries were taken out with sterile tweezers. After inoculation, berries were left in a laminar flow hood at room temperature (21±1 ℃) for about 2 h to allow for attachment of the microorganisms. Inoculated blueberries and strawberries had approximately 106 CFU/g of Salmonella and E. coli.

5.2.3 Washing procedures The washing procedures were the same as described in Chapter 4.2.3. Following treatment, samples were removed for microbial analysis.

5.2.4 Microbial analysis After washing, two blueberries (approximately 4 g) from each treatment were placed in a sterile stomacher sample bag containing 10 ml of elution buffer

(phosphate-buffered saline [PBS]) and pummeled in a stomacher (Colworth Stomacher 400, A. J. Seward and Co., Ltd., London, UK) for 2 min at medium speed. Strawberry at 20 g was placed in a stomacher bag with 50 ml PBS buffer for stomaching. For the quantification of surviving bacteria, , 1 ml aliquots from of the blueberry homogenate were plated on three XLD agar plates for Salmonella and three TSAYE-NS plates for E. coli, whereas 100 μl of strawberry homogenate was plated on

70 XLD agar for Salmonella and TSAYE-NS agar for E. coli. Plates were incubated at

37 ℃ for 24 h before presumptive-positive colonies were counted (Andrews, Jacobson, and Hammack, 2011).

5.2.5 Statistical analysis All experiments were conducted in three independent trials. The data are represented as mean values ± standard deviation. Microbial survivor counts after treatments were analyzed for significant treatment differences by one-way analysis of variance, fit model test of JMP (v. 10.0, SAS Institute Inc., Cary, NC). The different effect in washing experiments was assessed by the fit model. Significance was determined at P values of 0.05. Log reductions were calculated as difference between mean log Salmonella or E. coli counts of unwashed samples and log survivor counts of each treated sample.

5.3 Results and Discussion

5.3.1 Effect of combinations of SDS and organic acids or hydrogen peroxide on inactivation of Salmonella and E. coli on spot-, calyx- or dip-inoculated blueberries The efficacy of combination of SDS and organic acids or hydrogen peroxide in reducing populations of Salmonella on blueberries is presented in Figure 5.1. For spot inoculated blueberries, 80 ppm peroxyacetic acid achieved the highest log reduction of

Salmonella, which was significantly higher than those obtained with 200 ppm chlorine (P<0.05). Yuk et al (2006) reported 75 ppm peroxyacetic acid treatment on bell peppers for 60 or 120 s reduced Salmonella populations on smooth surfaces to

71 undetectable levels (about 4.0-log reduction). Our finding is in good agreement with those from Yuk’s research. AA/SDS and PPA/SDS showed similar Salmonella reductions as 200 ppm chlorine (P>0.05). Moreover, the results indicate that addition of 5000 ppm SDS into 500 ppm acetic acid, 200 ppm H2O2 or 20 ppm peroxyacetic acid can significantly enhance inactivation of Salmonella on spot inoculated blueberries. For example, the 500 ppm acetic acid or 5000 ppm SDS alone is not sufficient to kill more than 2 log10 CFU/g Salmonella within 1 min washing, but its activity was increased to 4.3 log10 CFU/g when 5000 ppm SDS was combined with the acetic acid. More than 3 log reductions were observed with the treated blueberries that had been spot inoculated on smooth surfaces.

When blueberries were spot inoculated onto the calyx, 80 ppm peroxyacetic acid, 200 ppm chlorine and AA/SDS resulted in 3.4, 3.2 and 2.8 log reductions, respectively, compared with the unsanitized control. These combination solutions represented a 0.3 to 0.9 log10 CFU/g decrease in the reduction of Salmonella numbers on the calyx of blueberries compared with spot surface inoculated blueberries, and no significant difference was observed between these treatments and 200 ppm chlorine (P>0.05). For dip inoculated blueberries, approximately 2.2-log reductions relative to unsanitized controls were observed with all treatments, whereas 80 ppm peroxyacetic acid and 200 ppm chlorine resulted in approximately 2.8 log reductions of Salmonella. In contrast to the effective inactivation on smooth surfaces, the aqueous sanitizers showed a lower efficacy to reduce Salmonella on calyx- and dip- inoculated blueberries. The dip inoculation of blueberries likely facilitated the infiltration of Salmonella through the stem scar, calyx, and other open areas (Durak et al., 2012).

72 Therefore, washing treatments may have no or little effect on the removal of pathogens. The current results agree with previous findings that dip inoculated produce was more difficult to decontaminate than spot inoculated samples.

Figure 5.1 Inactivation of Salmonella on spot-, calyx- and dip-inoculated blueberries by combinations of SDS and organic acids or hydrogen peroxide.

The reductions of E. coli after washing with combination of SDS and organic acids or hydrogen peroxide for 1 min are shown in Figure 5.2. For spot-inoculated blueberries, no significant difference in effectiveness on inactivation of E. coli was observed between the combination treatments and 200 ppm chlorine as well as between 200 ppm chlorine and 80 ppm peroxyacetic acid (P>0.05). Washing spot-

73 inoculated blueberries with the combination solutions reduced E. coli by approximately 4.0 log10 CFU/g, but when blueberries were calyx- or dip- inoculated, no more than 2.7 log reductions were observed. Results in Figure 5.2 indicate that 80 ppm peroxyacetic acid and 200 ppm chlorine had no significant (P > 0.05) effects on killing of the bacteria on both calyx- (2.7 log10 CFU/g reductions) and dip-inoculated blueberries (2.8 log10CFU/g reductions).

Figure 5.2 Inactivation of E. coli on spot-, calyx- and dip-inoculated blueberries by combinations of SDS and organic acids or hydrogen peroxide.

74 5.3.2 Effect of a combination of SDS and organic acids or hydrogen peroxide on inactivation of Salmonella and E. coli on spot- or dip-inoculated strawberries

Previous research found that AA/SDSS, H2O2/SDS, and PPA/SDS can achieve promising Salmonella inactivation efficacy on blueberries (approximate 4 log10 CFU/g reductions) (Li and Wu, 2013). It was thus interesting to test the effect of these solutions on inactivation of Salmonella on spot-inoculated strawberries (Figure 5.3A). Considering that a short wash time is preferred by the industry, 1-min washes were first investigated. Initial Salmonella counts on the unwashed and DI water washed spot-inoculated strawberries were 5.5 log10 CFU/g and 4.7 log10 CFU/g, respectively.

No significant difference was observed between 200 ppm chlorine (2.3 log10 CFU/g reductions) and 80 ppm peroxyacetic acid (2.4 log10 CFU/g reductions) treatments (P>0.05). Solutions containing 500 ppm acetic acid, 200 ppm hydrogen peroxide, and

20 ppm peroxyacetic acid reduced Salmonella populations from 1.4 to 1.8 log10 CFU/g, whereas 5000 ppm SDS resulted in 1.6 log10 CFU/g reductions. When combined with 5000 ppm SDS, Salmonella log reduction increased significantly, ranging from 2.0 to

2.2 log10 CFU/g. Additionally, there was no significant difference among all combinations and 200 ppm chlorine (P>0.05). To further increase the degree of Salmonella inactivation on spot inoculated strawberries, washing times of 2 and 5 min were applied. While a wash time of 2 min did not significantly improve the reduction observed with a 1-min exposure (P>0.05), 5-min exposures increased reductions to between 2.8 and 3.0 log10 CFU/g (this is a 0.8-log higher reduction, similar to what was achieved with 200 ppm chlorine and 80 ppm peroxyacetic acid treatments).

Similarly, 200 ppm chlorine and 80 ppm peroxyacetic acid treatments caused an additional 0.8 log10 CFU/g reduction as the washing time was increased from 1 min to 5 min. Whereas for blueberries, a 1-min washing time was sufficient for each

75 treatment to obtain effective Salmonella inactivation (about 4.0 log10 CFU/g reductions) (Li and Wu, 2013), even a washing time of 5 min did not achieve a promising Salmonella inactivation on strawberries (no more than 3.0 log10 CFU/g).

For dip inoculated strawberries, an even lower Salmonella inactivation efficiency was observed compared to that obtained for spot-inoculated strawberries (Figure 5.3B). Washing dip-inoculated strawberries with the combination solutions for

1 min reduced Salmonella by about 1.8 log10 CFU/g, and no significant differences were detected among the solutions (P>0.05). Solutions of 500 ppm acetic acid, 200 ppm hydrogen peroxide, 20 ppm peroxyacetic acid, and 5000 ppm SDS applied separately only resulted in approximately 0.9 log10 CFU/g reductions of Salmonella.

Exposure to 200 ppm chlorine reduced the population of Salmonella from 5.03 log10

CFU/g to 3.17 log10 CFU/g, a reduction similar to that observed with 80 ppm peroxyacetic acid (P>0.05). As observed with spot-inoculated strawberries, an increase in the washing time to 2 min did not improve inactivation of Salmonella. When the washing time was increased to 5 min, a similar inactivation was observed for each combination treatment. For example, AA/SDS was able to reduce Salmonella numbers by 2.9 log10 CFU/g, a result which was significantly higher than that obtained with1-min washing (1.8 log10 CFU/g reductions).

Figure 5.4A shows the effect of washing with combinations of SDS and organic acids or hydrogen peroxide on survival populations of E. coli on spot- inoculated strawberries. Reductions of E. coli after 1 min washing were approximate

1.2 log10 CFU/g for 500 ppm acetic acid, 200 ppm hydrogen peroxide, and 20 ppm

76 peroxyacetic acid. Although the differences among treatments were not significant, additional SDS significant enhanced inactivation of E. coli. For example, PPA/SDS resulted in 2.0 log10 CFU/g reductions of E. coli and H2O2/SDS reduced the surviving numbers of E. coli by 1.7 log10 CFU/g. Moreover, as washing time increased from 1 min to 5 min, E. coli reductions of each combination treatment significantly increased from 1.8 to 3.0 log10 CFU/g, while less than 1 log enhancement was observed in single solution treatments. Treatment with 200 ppm chlorine and 80 ppm peroxyacetic acid for 5 min caused approximately 2.6 log10 CFU/g reductions of E. coli. When strawberries were dip inoculated with E. coli, less than 2 log10 CFU/g reductions were observed when the combination treatments were applied for 1 min (Figure 5.4B).

When the wash time was increased to 5 min, no more than 3 log10 CFU/g reductions were achieved when dip-inoculated strawberries were treated with each combination solution. A similar pattern was observed for E. coli on dip-inoculated strawberries as that obtained with Salmonella.

Yu et al. (2001) used acetic acid at 2 and 5% and hydrogen peroxide at 1 and 3% to decontaminate E. coli O157: H7 on strawberries. The results from treating contaminated strawberries with acetic acid at 2 and 5% and hydrogen peroxide at 1 and 3% for 1 min revealed less than 2 log reductions in E. coli. Gurtler et al. (2012) evaluated the effectiveness of 85 ppm peroxyacetic acid, 200 ppm chlorine and 1% acetic acid on inactivation of a cocktail of Escherichia coli O157:H7 and Salmonella enterica on strawberries. Reductions were 2.84, 1.73, and 1.34 log10 CFU/g, respectively, when 85 ppm peroxyacetic acid, 200 ppm chlorine and 1% acetic acid

77 were applied for 2 min. Apparently, our finds are in good agreement with previous research. It is believed that the relative ineffectiveness of sanitizers on the inactivation of pathogens on strawberries is due to the rough surface of strawberries and the presence of numerous surface-borne achenes (seeds), which provide hidden areas for the bacteria to attach and are less accessible to sanitizing solutions. Therefore, these experiments indicate that once strawberries are contaminated with Salmonella Typhimurium and E. coli O157:H7, there is a potential health hazard to cause illness due to the survivability of the pathogen and the low effectiveness of common sanitizing agents.

Figure 5.3 Inactivation of Salmonella on spot- inoculated (A) and dip- inoculated (B) strawberries by combinations of SDS and organic acids or hydrogen peroxide.

Figure 5.4 Inactivation of E. coli on spot- inoculated (A) and dip- inoculated (B) strawberries by combinations of SDS and organic acids or hydrogen peroxide.

80 REFERENCES

Andrews, W.H., Jacobson, A., Hammack, T., 2011. Bacteriological Analytical Manual. Chapter 5: Salmonella. Available from: http://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm0701 49.htm

Calder, L., Simmons, G., Thornley, C., Taylor, P., Pritchard, K., et al. 2003. An outbreak of hepatitis A associated with consumption of raw blueberries. Epidemiology and Infection 131, 745-751.

CDC, 2013. Multistate outbreak of hepatitis A virus infections linked to pomegranate seeds from Turkey. Available from: http://www.cdc.gov/hepatitis/Outbreaks/2013/A1b-03-31/index.html

Crowe, K.M., Bushway, A.A., Bushway, R.J., 2005. Effects of alternative postharvest treatments on the microbiological quality of lowbush blueberries. Small Fruits Review 4, 29-39.

Durak, M.Z., Churey, J.J., Gates, M., Sacks, G.L., Worobo, R.W., 2012. Decontamination of green onions and baby spinach by vaporized ethyl pyruvate. Journal of Food Protection 75, 1012-1022.

FDA, Food and Drug Administration. 1999. FDA survey of imported fresh produce. Available from: http://www.cfsan.fda.gov/∼dms/prodsir6/html. Accessed September 13, 2004.

Gurtler, J., Bailey, R., Jin T., 2012, Inactivation of Escherichia coli O157:H7 and Salmonella enterica on strawberries by sanitizing solutions, International Association for Food Protection Meeting. https://iafp.confex.com/iafp/2012/webprogram/Paper2734.html

Knudsen, D.M., Yamamoto, S.A., Harris, L.J., 2001. Survival of Salmonella spp. and Escherichia coli O157:H7 on fresh and frozen strawberries. Journal of Food Protection 64, 1438-1488.

81 Niu, M.T., Polish, L.B., Robertson, B.H., et al. 1992. Multistate outbreak of hepatitis A associated with frozen strawberries. Journal of Infectious Diseases 166, 518- 524.

Stone, C., 2011. Fresh Strawberries implicated in E. coli O157 outbreak in NW Oregon, New York berry news 10 (7).

Yuk, H.G., Bartz, J.A., Schneider, K.R., 2006. The effectiveness of sanitizer treatments in inactivation of Salmonella spp. from bell pepper, cucumber, and strawberry. Journal of Food Science 71, 95-99.

Yu, K., Newman, M.C., Archbold, D.D., Hamilton-Kemp, T.R., 2001.Survival of Escherichia coli O157:H7 on strawberry fruit and reduction of the pathogen population by chemical agents. Journal of Food Protection 64, 1334-1340.

82 Chapter 6

FUTURE WORK

Although 500 ppm acetic acid plus 5000 ppm SDS (AA/SDS), 200 ppm hydrogen peroxide in combination with 5000 ppm SDS (H2O2/SDS), and 20 ppm peroxyacetic acid coupled with 5000 ppm SDS (PPA/SDS) holds promise in enhancing the safety of blueberries, these three washing solutions were ineffective in inactivating Salmonella and E. coli on strawberries. In addition, based on the hepatitis A outbreak associated with a frozen berry mix, including cherries, blueberries, pomegranate seeds, raspberries and strawberries in 2013 and the low efficacy of chemical sanitizers, there is a need to develop more effective treatment to control the safety of strawberry.

It is conceivable that edible coating creates a micro-modified atmosphere around produce to extend shelf-life of fresh fruits (Baldwin, 1994). Several edible coating formulations, including chitosan- or hydroxypropyl methylcellulose (HPMC)- based coatings and polysaccharide- or protein-based polymers containing chemical fungicides have been studied (Park et al., 2005; Garcia et al., 2001). Chitosan was approved by the FDA as a feed additive in 1983 (Knorr, 1986) and also has demonstrated antifungal activity against several post-harvest pathogens (Ghaouth et al.,

1991; Zhang and Quantick, 1998; Jiang and Li, 2001). Although FDA has not approved the use of chitosan in food, significant research has been conducted to understand its safety and potential applications in foods. In addition, a limited number

83 of coating studies on fresh strawberries have been published. The effectiveness of chitosan, chitosan containing 5% Gluconal® CAL, and chitosan containing 0.2% DL- α-tocopheryl acetate to prolong the shelf-life of fresh strawberries was studied (Han et al., 2004). Park et al. (2005) showed that chitosan containing 3% potassium sorbate not only inhibited fungal growth but also reduced total aerobic and coliform counts on strawberries. Based on the antifungal effectiveness of edible coating treatment, it is possible to hypothesize that edible coatings can reduce Salmonella and E. coli when combined with antimicrobial substances, such as organic acids, ethyl pyruvate, and essential oils.

In the future, more work is needed to explore an efficient and effective edible coating formulation for removal of pathogens from fresh strawberries. Meanwhile, sensory studies to assess consumer acceptance of coated strawberries are necessitated.

84 REFERENCES

Baldwin, E.A., 1994. Edible coatings for fresh fruits and vegetables: past, present and future. In: Krochta J, Baldwin E, Nisperos-Carriedo M, editors. Edible coatings and films to improve food quality. Basel: Technomic Publ. Co. p 25.

Garcia, M.A., Martino, M.N., Zaritzky, N.E., 2001. Composite starch-based coatings applied to strawberries (Fragaria x ananassa). Nahrung/Food 45, 267-272.

Ghaouth, A.E., Arul, J., Ponnampalam, R., Boulet, M., 1991. Chitosan coating effect on storability and quality of fresh strawberries. Journal of Food Science 56, 1618-1620.

Han, C., Zhao, Y., Leonard, S.W., Traber, M.G., 2004. Edible coatings to improve storability and enhance nutritional value of fresh and frozen strawberries (Fragaria × ananassa) and raspberries (Rubus ideaus). Postharvest Biology and Technology 33, 67-78.

Jiang, Y., Li, Y., 2001. Effects of chitosan coating on postharvest life and quality of longan fruit. Food Chemistry 73, 139-143.

Knorr, D., 1986. Nutritional quality, food processing, and biotechnology aspects of chitin and chitosan: a review. Process Biochemistry 6, 90-92.

Park, S., Stan, S. D., Daeschel, M.A., Zhao, Y.Y., 2005. Antifungal coatings on fresh strawberries (Fragaria × ananassa) to control mold growth during cold storage, Journal of Food Science 70, 202-207.

Zhang, D., Quantick, P., 1998. Antifungal effects of chitosan coating on fresh strawberries and raspberries during storage. Journal of Horticultural Science and Biotechnology 73, 763-767.