ABSTRACT

MICROBIOLOGICAL QUALITY OF RETAIL “RAW” ALMONDS IN THE UNITED STATES

Almonds are an important crop to California’s economy and make up 83% of the world’s almond supply. Due to outbreaks of Salmonella in retail almond products in the early 2000s, a new rule established under the California almond marketing order was issued in 2007 requiring almonds be pasteurized before they are packaged and sold to consumers. These almonds are labeled “raw” or “natural” as it is thought that the changes in nutritional and sensory characteristics are negligible. Since the 2007 rule, no reported study has tested retail almonds for the presence of Salmonella, nor has any reported study established a microbiological profile of retail almonds. A total of 116 raw retail almond samples were collected and tested for the presence of Salmonella spp., total mesophilic aerobic plate count, coliform count, and generic

Escherichia coli count. The presence of Salmonella was not detected in any of the samples. The total aerobic mesophilic plate count was 5.14 log CFU/g, yeast count was 3.32 log CFU/g, mold count was 3.44 log

CFU/g, and coliform count was 2.83 log CFU/g. No generic E. coli was detected. These results corroborate prior laboratory findings that the required pasteurization of almonds is effective in protecting consumers from foodborne illness. Pasteurization is a valid food safety intervention method for nut processors, especially in the wake of several recent recalls for Salmonella in other nuts (walnuts, macadamia nuts, pecans, cashews, pistachios, hazelnuts, and pine nuts).

Brittany Blanco August 2015

MICROBIOLOGICAL QUALITY OF RETAIL “RAW” ALMONDS IN THE UNITED STATES

by Brittany Blanco

A thesis submitted in partial fulfillment of the requirements for the degree of

Master of Science in Food and Nutritional Sciences in the Jordan College of Agricultural Sciences and Technology California State University, Fresno August 2015 APPROVED

For the Department of Food Science and Nutrition:

We, the undersigned, certify that the thesis of the following student meets the required standards of scholarship, format, and style of the university and the student's graduate degree program for the awarding of the master's degree.

Brittany Blanco Thesis Author

Erin Dormedy (Chair) Food Science and Nutrition

Steven Pao Food Science and Nutrition

Dennis Ferris Food Science and Nutrition

For the University Graduate Committee:

Dean, Division of Graduate Studies AUTHORIZATION FOR REPRODUCTION

OF MASTER’S THESIS

I grant permission for the reproduction of this thesis in part or in its entirety without further authorization from me, on the condition that the person or agency requesting reproduction absorbs the cost and provides proper acknowledgment of authorship.

X Permission to reproduce this thesis in part or in its entirety must be obtained from me.

Signature of thesis author: ACKNOWLEDGMENTS

I would like to thank Dr. Erin Dormedy for making it possible for me to attend graduate school at Fresno State and introducing me to the local IFT section. If it were not for her teaching, belief in me, and support, I would not have fallen in love with the food industry as I have. I would also like to thank my husband who has always believed in me and supported me through everything. If it were not for his support and willingness to do whatever it took to help me while I was going to school I would not have been able to be as successful as I have. I would not be the person I am today and would not have accomplished the things I have without him. TABLE OF CONTENTS Page

LIST OF TABLES ...... vi

CHAPTER 1: INTRODUCTION ...... 1

Research Statement...... 2

CHAPTER 2: LITERATURE REVIEW ...... 3

Food Safety ...... 3

Almonds ...... 5

Microbiological Testing ...... 11

CHAPTER 3: METHODOLOGY ...... 14

Sampling Plan ...... 14

Microbiological Testing ...... 14

Statistical Analysis ...... 19

CHAPTER 4: RESULTS AND DISCUSSION ...... 20

CHAPTER 5: CONCLUSION ...... 24

REFERENCES ...... 27

APPENDICES ...... 33

APPENDIX A: DILUTION SCHEME ...... 34

APPENDIX B: ALMOND SAMPLE CLASSIFICATION ...... 36

LIST OF TABLES

Page

Table 1. Summary of studies conducted on the incidence of Salmonella in almonds...... 9

Table 2. E. coli, Coliform, Yeast, Mold, and APC Test Results (n=115) .. 21

1

CHAPTER 1: INTRODUCTION

Almonds are the top valued crop grown in California and have had the largest export value for the past 3 years (California Department of Food and Agriculture 2014). California almonds make up 83% of the world’s supply, and 100% of the United States’ supply (Almond Board of California 2015a). Raw retail almond products were recalled in 2001 and

2004 in Canada and the US due to the presence of Salmonella. After these 2 outbreaks the Almond Board of California worked with the USDA to put in place a pasteurization rule under the California almond marketing order for almond products to prevent future recalls due to the presence of Salmonella. Salmonella is the second leading cause of foodborne illness with about 1.2 million cases every year (Scallan and others 2011). The pasteurization rule requires a 4 log reduction of

Salmonella and multiple methods have been validated for use (Agricultural Marketing Service 2007). To date only studies conducted in the United Kingdom have tested retail almond products for the presence of Salmonella (Little and others 2009, 2010). The only reported study performed in the United States, after the pasteurization rule, tested for the risk of Salmonella contamination in almond samples from processors in California (Lambertini and others 2012). The detection and enumeration of other microorganisms is also important to the food industry. It is important to have an understanding of the microbial profile of a food item as an indication of process control and sanitation.

A processor can target areas of improvement for quality as well as food safety standards during processing. The total mesophilic aerobic plate 2 2 count of a food product provides an overview of the amount of microorganisms in the product. Generic Escherichia coli and coliform bacteria are important to test for as they are an indication of fecal contamination. Yeast and mold are also of importance as they can cause quality defects in food products and may produce metabolites that are hazardous to human health. No reported study, after the pasteurization mandate, has established the microbiological profile of retail almonds.

Research Statement The purpose of this study is to provide data regarding the presence of Salmonella in retail “raw” almonds and aid in the confirmation of the effectiveness of the required pasteurization of almonds. This study will also provide data establishing the general microbiological profile of retail almonds. 3

CHAPTER 2: LITERATURE REVIEW

Food Safety Food safety is a large concern of the food industry. It is important to ensure the food supply is safe for consumers. Part of the U.S. Food and Drug Administration’s (FDA) mission statement includes they are “responsible for protecting the public health by assuring the safety …of our nation’s food supply” (United States Food and Drug Administration 2014a). Food safety is also included in the Institute of Food Technologists’ (IFT) mission statement which includes they “exist to advance the science of food. Our long-range mission is to ensure a safe and abundant food supply” (Institute of Food Technologists). Detection of foodborne pathogens and the prevention of foodborne illness are important to ensure a safe food supply. According to a 2011 U.S. Centers for Disease Control and Prevention (CDC) study, there are an estimated

9.4 million cases of foodborne illness every year, which includes 55,961 hospitalizations and 1,351 deaths (Scallan and others 2011). These values are only those that have been reported, as most cases of foodborne illness go unreported and undocumented. These hospitalizations and deaths are caused by the 31 major pathogens present in the United States. Foodborne illness is estimated to cost $14.1 to $152 billion every year due to hospitalizations (Hoffmann and others 2012; Scharff 2012). This does not include the cost of food recalls and the damage to a company’s reputation and sales. Government agencies such as the United States Department of Agriculture (USDA) are 4 currently funding research to find and reduce pathogens in the food supply.

Salmonella Salmonella is the second leading cause of foodborne illness. It causes 1.2 million illnesses, 23,000 hospitalizations, and 450 deaths every year (Scallan and others 2011). Salmonella causes the most deaths due to foodborne illness (Centers for Disease Control and Prevention

2014). People that contract foodborne illness due to Salmonella may experience a variety of symptoms including fever, diarrhea, abdominal cramps, headache, nausea, and vomiting (National Institute of Allergy and Infectious Disease 2014). Symptoms typically appear around 12 to 72 hours after consumption of an infected food item and last between 4 to 7 days (Centers for Disease Control and Prevention 2015). Antibiotics are used to treat the infection. If left untreated, Salmonella can enter the bloodstream and spread to other locations in the body and cause death. According to the CDC estimates of foodborne illness in the United States,

28% of deaths caused by foodborne illness are due to Salmonella (Centers for Disease Control and Prevention 2014). Salmonella is most commonly associated with raw chicken and eggs. However, outbreaks have also occurred in unpasteurized milk, raw fruits and vegetables, spices, and nuts (United States Food and Drug Administration 2015b). 5 Almonds

Production The United States produces 83% of the world’s almond supply, with the other 17% being produced in Australia, the European Union, Iran, Turkey, Tunisia, and others (Almond Board of California 2015b). In the United States, almonds, which are only grown and produced in California, have surpassed grapes as the top valued California agricultural export, and have the largest production and per capita consumption compared to other nuts. According to the USDA the United States’ 2014 nut production was valued at $10.5 billion, with over half ($6.46 billion) attributed to almonds (United States Department of Agriculture National Agricultural Statistics Service 2015). About 30% of the California almond crop is sold in the US, while the additional 70% is exported and sold internationally (Almond Board of California 2015a).

Almonds are harvested from mid-August through October using mechanical harvesters. The harvesters shake the almond trees to release the almonds on the ground where they are dried for 8-10 days. Once the almonds are dry they are swept into rows and picked up by a mechanical harvester. It is during harvesting where almonds have the largest potential to become contaminated with microorganisms, most notably

Salmonella (Harris and others 2013).

Outbreaks and Legislation In 2001 there was an outbreak of a rare type of Salmonella enteritidis in Canada due to raw whole almonds (Isaacs and others 2005). The bacterium affected 157 people in Canada, 11 people in the 6

United States, and was found on the recalled almond products, processing equipment, and associated farmers’ orchards. In May 2004 about 15 million pounds of almonds were recalled due to the presence of

Salmonella enteritidis (Agricultural Marketing Service 2007). In June 2004 the CDC reported the outbreak affected 29 people with 7 hospitalized. The infected almond products were found in 12 states and throughout Canada (Centers for Disease Control and Prevention 2004). The USDA Agricultural Marketing Service works with the food industry to enact marketing orders which are, “initiated by the industry to help provide stable markets for dairy products, fruits, vegetables and specialty crops” (United States Department of Agriculture Agricultural Marketing Service 2015). Due to the 2001 and 2004 outbreaks, public safety concerns, and the industry reputation, an almond marketing order was enacted in 2007. The marketing order was designed to maintain the quality of the almonds, produced in California, by requiring a pasteurization step. The method of pasteurization used can be steam, water, oil roasting or gas, and is required to achieve a minimum 4-log reduction. Pasteurization must be performed by handlers or any person handling almonds during any crop year (Agricultural Marketing Service 2007). Pasteurizing almonds has been found to have no significant effect on the nutrient profile compared to unpasteurized almonds (Almond Board of California 2015d). This makes it possible for retail almonds to be labeled as “raw” or “natural”. The rule does not apply to growers who sell their almonds at a roadside stand they operate which includes selling at farmers markets. The exemption also applies to people who sell less than 100 pounds of almonds to one customer each day (Agricultural 7

Marketing Service 2007). Hazelnuts, pistachios, and walnuts also have marketing orders but they do not include a pasteurization requirement.

Since the 2007 pasteurization requirement there have not been any outbreaks or recalls of almonds. There have and continue to be recalls and outbreaks due to walnuts, macadamia nuts, pecans, cashews, pistachios, hazelnuts, and pine nuts (Palumbo and others 2015).

Conducted Studies A study was conducted in Australia that tested for Salmonella in multiple types of nut samples that were processed in Australian facilities.

No presence of Salmonella was detected (Eglezos 2010). Retail nut samples were studied, but further information regarding the sample type was not provided. It is unknown if the products tested were roasted, salted, or further processed in a way that would inhibit Salmonella growth. A similar study was conducted in the United Kingdom where 83 samples of roasted nut kernels were tested, and one pistachio sample was found to contain Salmonella (Little and others 2009). Another study was conducted on 359 almond samples, among other types of nuts, purchased at retail stores, that were either heat processed or chemically treated, and tested for the presence of Salmonella (Little and others 2010). Salmonella was detected in a sample of Brazil nuts and a sample of mixed nuts. In the United States, a study was conducted at the University of California at Davis (U.C. Davis) that tested for the presence of Salmonella in almond samples from processing facilities (Lambertini and others 2012). Salmonella was found in less than 1% of the samples tested. No study found has tested for the presence of Salmonella in retail 8 almond products sold in the United States to aid in showing the effectiveness of the pasteurization requirements for almonds. Table 1 contains a summary of the studies conducted on the presence of

Salmonella in almonds.

Microbiological Profile Microorganisms other than Salmonella are also of concern in the food industry. It is important for processors to have an understanding of what microorganisms may be in their food products to know how to control them. During harvesting and processing, in addition to

Salmonella, almonds may also become contaminated with yeast, mold,

Escherichia coli, coliforms, and other bacteria. An aerobic mesophilic plate count, also known as aerobic plate count (APC) and standard plate count, provide a general count of the bacteria in a food product. APC tests for bacteria that require oxygen and mesophilic temperatures for growth.

This includes Streptococcus bacteria which can also be used as an indication of the effectiveness of sanitary processing (Jay and others

2005). Escherichia coli and coliform bacteria are important to test for, as they are indicators of fecal contamination (Feng and others 2002). This provides an indication as to the effectiveness of sanitary processing conditions and prevention of cross contamination. Lastly, yeast and mold can indicate a quality defect in food products. The metabolites of some molds that may infect food products are mycotoxins. Mycotoxins can survive processing even if the organism that produces it is killed during processing (Tournas and others 2001). The toxins are a known carcinogen and almonds are one of the primary nut types affected by

Table 1. Summary of studies conducted on the incidence of Salmonella in almonds. Author No. & Type of Microorganism & Confirmation Country Sampling Results Comparison and Year Samples Method Method Lambertini United 13,972 almond Samples taken upon receipt at Salmonella - Danyluk Not retail samples, and others 137 samples positive MPN States samples 7 processing facilities 2007 found Salmonella 2012 XLD & BS agar Retail packs immediately taken Detected in all colonies streaked after packing and taken to lab; sample types tested APC - 3M Petrifilm on cystine lactose 42 retail almonds 10 g sample homogenized in Almonds = avg. 2.4 Different country, Eglezos electrolyte Australia 43 retail mixed 90 mL 0.1% peptone soln. in CFU/g same methods as 2010 deficient media, packs stomacher for 1 min., my research confirmed on Salmonella samples in buffered Salmonella - BAX Not detected Medvet Microbact peptone water E. coli - 3M petrifilm Not detected 24E strips 1 almond sample Little and 359 retail almonds E. coli - MPN Different country, United 2 mixed nut samples others 329 retail mixed different method = Kingdom Salmonella - if 2010 nuts 1 mixed nut sample MPN out of date present, then MPN Little and 1:10 dil. in buffered peptone Salmonella - RVS and MacKonkey & United 83 retail almonds ND Different country, others water, stomacher for 30 sec - 3 XLD agar Nutrient agar, Kingdom 63 retail mixed nuts different method 2009 min. E. coli - MPN ND PCR optional 60 almond samples APC - 3M Petrifilm 4.4 CFU/g Different country, Eglezos from processing 10 g into 90 mL peptone processing and others Australia E. coli - 3M petrifilm ND XLD & BS agar facilities, prior to solution samples, same 2008 roasting Salmonella - BAX present in 1 sample methods Aerobic mesophilic 15 Almond samples Different country, Vural and bacteria - Oxoid plate 1.1 x 10³ - 1.4 x 10⁵ 16 mixed nut Analyzed immediately, 10g into different methods, Kergan Turkey count agar XLD agar samples in several 90mL buffered peptone water use results as basis 2008 Coliform - violet red markets ND for dilutions bile agar

9

Yeast & Mold - potato 2.0 x 101 - 5.8 x 10³ Table 1 (cont.) dextrose agar Salmonella - RVS 46.54% of samples Author No. & Type of Microorganism & Confirmation Country Sampling Results Comparison and Year Samples Method Method 9,274 almonds Salmonella - RV broth 81 samples Before samples 2001-2005 APC avg. = 1,800 CFU/g Danyluk pasteurization, United Canada almond Samples stored at 4°C before BS, XLD agar, and others E. coli - MPN 125 samples processor samples, States samples associated and after testing MPN 2007 older methods, with 2000, 2001 avg. mold = 2,900 Yeast & Mold found Salmonella recall CFU/g Total Viable Count - 0.55 x 10³ - 3.3 x 10³ oxoid plate count agar Coliform - violet red ND bile agar Different country, BS agar and Candlish United Almond retail Yeast & Mold - rose different method, BioMerieux API 2001 Kingdom samples bengal agar, use levels for 20E kits oxytetracycline- ND dilutions glucose-yeast extract agar Salmonella - RV broth ND Bacteria - plate count agar with avg. 2,828-6,955 172 almond cycloheximide Before United samples 1966 Yeast & Mold - BBL trypticase pasteurization, use King 1970 Stored at 2°C until testing States 99 almond samples acidified potato avg, 1,494-47,159 soy agar levels as basis for 1967 dextrose starch dilutions Coliform - violet red avg. 8-11 CFU/g bile agar Conclusion: No one in the US, since the pasteurization requirement, has tested retail almond samples. Only the 2007 study provides a microbiological quality overview of almonds.

10

11 contamination (Campbell and others 2003, National Toxicology Program 2014). There are no set industry standards or recommendations for the presence and levels of these microorganisms in almonds. However, according to the Almond Board of California, common industry levels of the microorganisms of concern are: APC <4.70 log CFU/g, coliform <3.00 log CFU/g, E. coli <1 log CFU/g, yeast and mold <3.70 log CFU/g and Salmonella negative/25g (Almond Board of California 2015e). A study performed with the 1966 California almond crop tested the microbial quality and found the almonds tested had an average APC count of 3.45 log CFU/g, yeast and mold count of 3.17 log CFU/g, and coliform count of 0.90 log CFU/g (King and others 1970). The only other study conducted in the United Sates tested almond samples from processors that were involved in the 2000-2001 recall. The study tested for the level of APC, E. coli, yeast, and mold counts in Salmonella positive almond samples and found an average of 3.26 log CFU/g, 0.3 mpn/g, <1 log CFU/g, and 3.46 log CFU/g respectively (Danyluk and others 2007). No other reported studies in the United States have tested the microbiological quality of almonds.

Microbiological Testing Numerous methods are available to test for the presence of microorganisms in almond samples. Whether testing samples for research purposes or industry use, it is important to follow standard test methods that have been outlined and validated by governing authorities.

The FDA’s Bacteriological Analytical Manual (BAM) is considered the 12 standard when testing for the presence of microorganisms in food products (United States Food and Drug Administration 2015a).

Salmonella To test for the presence of Salmonella the Bacteriological Analytical Manual (BAM) provides instructions for the use of various types of media. These media selectively grow Salmonella and can take upwards of 4 days to receive results (Andrews and others 2014). Most food processors are unable to place their products on hold to wait for test results that take 4 days to confirm. Rapid test methods have been developed to reduce the amount of time needed to wait for confirmation of the presence of Salmonella. These rapid test methods utilize Polymerase Chain Reaction (PCR) testing.

PCR detects the presence of Salmonella based on a DNA sequence that is unique to Salmonella. The method separates the DNA in the almond sample and uses a DNA template to find the target sequence that is unique to Salmonella. When the template finds the target sequence it will replicate the sequence until there are millions of copies of the target sequence. The equipment used will then detect the presence of the target sequence and provide a positive test results in less than one day. The Dupont™ BAX® System was chosen to be used to test for the presence of Salmonella. The BAX® has been validated for Salmonella detection and is AOAC Official Method 2013.02 (DuPont Nutrition and Health 2013). The BAX® is used by the United States Department of

Agriculture-Food Safety Inspection Service (USDA-FSIS) and located in their Microbiology Laboratory Guidebook for the “Screening Salmonella in 13

Meat, Poultry, Pasteurized Egg, and Catfish Products and Carcass and Environmental Sponges” (United States Department of Agriculture Food

Safety and Inspection Service 2014). The BAX® has been found to be

“equivalent to Chapter 5 (Salmonella) of FDA’s Bacteriological Analytical Manual in accuracy, precision, and sensitivity in detecting Salmonella enteritidis” (United States Food and Drug Administration 2014c).

Microbiological Quality As with Salmonella testing, rapid methods have been created for the detection and enumeration of yeast, mold, E. coli, coliform, and APC, for use by the food industry. 3M™ Petrifilm™ is one of the rapid methods available for use by food processors. As with Salmonella testing, this rapid method provides results in 24-48 hours instead of several days using more traditional methods. 3M™ Petrifilm™ use can be found in the FDA BAM and USDA-FSIS Microbiology Laboratory Guidebook (United

States Food and Drug Administration 2015a, United States Department of Agriculture Food Safety and Inspection Service 2015). Its use is recommended by the Almond Board of California in their Pathogen Environmental Monitoring Program (Almond Board of California 2015c), and is an AOAC International Official Method of Analysis (3M™ 2014). 11

CHAPTER 3: METHODOLOGY

Sampling Plan Almond products that were only pasteurized, and were considered raw, were tested. Sugar and salt, in any form, added to almond products during processing have an inhibitory effect on potential microorganism contamination, and such products were not tested. Raw almond samples were purchased from numerous retail stores under various brand labels. When samples from the same brand and style were purchased, they were required to be from different lot or production batches. A total of 116 almond samples were collected from retail stores, farmer markets, and purchased online.

Microbiological Testing

Salmonella Lactose broth was prepared by adding 500 mL of distilled water into a 1 L Erlenmeyer flask. Thirteen g of lactose broth (Oxoid item CM0137, lot 1342712) were weighed out and added to the flask. The flask was placed on a stir plate (Dataplate, PMC 720 series) and the solution mixed until the broth was completely dissolved. An additional 500 mL of distilled water was added to the flask and mixed. Aliquots of dilution bottles were filled with 235.13 mL (225 mL plus the autoclave factor of 0.045) of lactose broth, capped, and placed in the autoclave (Market Forge Sterilmatic) and processed at 121°C for 30 minutes. Brain Heart Infusion (BHI) broth was prepared by adding 50 mL distilled water to a 100 mL Erlenmeyer flask. The flask was placed on a stir plate and 3.7 g of BHI was added and mixed until completely dissolved. An 15 additional 50 mL of distilled water was added to the flask and mixed. A dilution bottle was filled with the BHI broth and autoclaved following the same procedure as the lactose broth. Snap-cap microcentrifuge tubes (Fisherbrand, item number 02-681-10) were placed in an autoclavable container, closed with aluminum foil, and autoclaved. Once the materials were done being autoclaved, the lactose broth and BHI was placed in a refrigerator (Fisher Scientific Low Temp Incubator). The cooling block was placed in a refrigerator. Samples were prepared and enriched as recommended in the DuPont BAX® System Q7 Instrument User Guide (DuPont 2013). A sterile Whirl-Pak filter bag (Nasco item B01318, lot 04267) was placed in a beaker and on a scale (Fisher Science Education, item ALF1202). 25 g from one of the retail almond samples was added and the weight recorded. Sterile lactose broth (225 mL) was added to the bag. The bag was sealed and mixed at medium speed for 120 seconds using a lab blender (Seward Stomacher 400 type BA 7021). This process was repeated for all almond samples tested. A negative control was made for each run of samples by adding only lactose broth to a Whirl-Pak bag. A positive control as made for each run of samples by adding a microtubule of Salmonella typhimurium (ATCC 2514, lot 253-23-4), warmed to room temperature, to lactose broth in a Whirl-Pak bag. The samples were incubated (Fisher Scientific Isotemp) at 35°C for 22-26 hours. The BHI was placed in an incubator (Lab-line Instruments Inc., model 120) at 37°C. Microtubules were arranged in a holder according to the almond samples to be tested. After 22-26 hours the almond samples were removed from the incubator. The warmed BHI was removed from the 16 incubator and 490 µL were pipetted into each of the microtubules. 10 µL of the first almond sample was added to the first microtubule. The microtubule was capped and inverted twice to mix the contents. Microtubules were filled one for each almond sample and placed in an incubator at 37°C for 3 hours. The BAX® was turned on and a rack file was created with the information for each sample tested. The heating block (DuPont Thermal Block, model DUP-1000) was turned on and the “Gram Negative” program was selected. The appropriate number of cluster tubes was arranged according to the created rack file. The protease and lysis buffer was removed from the refrigerator. 150 µL of protease was added to a 12 mL bottle of lysis buffer and mixed. 200 µL of the mixed lysis buffer was added to each of the cluster tubes. 5 µL of the first almond sample in BHI was added to the first cluster tube. This was repeated for each almond sample that was tested. Caps were placed on the cluster tubes and secured. The cluster tubes were placed on the heating block and the “Gram Negative” program started. Once the program was completed, the heating block read “Sample PCR Ready”. The cooling block was removed from the refrigerator and the appropriate amount of PCR tubes were placed in the cooling block according to the rack file. The cluster tube caps and PCR caps were removed. 50 µL of each almond sample solution was pipetted into the appropriate PCR tube. Optical caps were placed on the PCR tubes and placed in the BAX®. The BAX® program was run to completion. The results were reviewed and presented as a green negative sign to represent negative for Salmonella, or a red positive sign to represent positive for Salmonella (Dupont 2013). When samples tested 17 positive for Salmonella, the lactose broth enriched almond samples were retained for confirmation testing based on the FDA’s Bacteriological

Analytical Manual (United States Food and Drug Administration, 2015a). Rapaport-Vassiliadis Soya Peptone Broth (RV) tubes were prepared by adding 13.38 g RV media (Oxoid product CM0866, lot 1533097) and 125 mL of DI water in a 1 L Erlenmeyer flask. The flask was placed on a hot plate and boiled. Once the media dissolved completely it was removed from the hot plate and 375 mL cold DI water was added. 9.4 mL (includes autoclave factor) of media was dispensed into test tubes which were then capped and autoclaved. 1.045 L of DI water, an empty flask, stir bar, and spatula were autoclaved. 50 mL of autoclaved DI water as added to the flask. 55 g of XLD agar (Remel product R459902, lot 620956) was weighed out using the autoclaved material. The agar was transferred to the flask and mixed thoroughly using the stir bar. The remaining DI water was added to the flask and mixed until completely suspended. The media was then heated to boiling until completely dissolved. Once dissolved the media was immediately cooled to about 47- 50°C in a water bath and poured into petri plates. 1 mL of presumptive positive lactose broth enriched almond sample was aseptically pipetted into a 9 mL RV tube. RV tubes were created for each presumptive positive almond sample. The RV tubes were incubated for 18-20 hours at 42°C. For each RV tube, 1mL was aseptically transferred to an XLD plate and BD Biplate (product 292765, lot 4308668). A second plate of each type for each RV tube was streaked. The plates were incubated at 37°C for 18-24 hours. The plates were then incubated at 25°C for 18-24 hours and read. 18 Yeast & Mold, E. coli & Coliform, and Aerobic Plate Count A Whirl-Pak filter bag was placed in a beaker on the scale and the scale was tared. 11 g of the first almond sample to be tested was added to the bag. 99mL of a pre-sterilized flip-top dilution bottle with Butterfield’s buffer (3M™, product FTBFD9966, lot2015-11AN) was added to the bag. The contents of the bag were mixed using a lab blender on medium speed for 120 seconds. Serial dilutions were made using Butterfield’s buffer (3M™, product FTBFD9966, lot2015-11AN). The dilutions were lined up in order on the lab countertop and plated in duplicate on E. coli and Coliform Petrifilm™ plates (3M™, product 6404, lot 2016-10KE), APC Petrifilm™ plates (3M™, product 6400, lot 2016- 10TB), Rapid Yeast and Mold Petrifilm™ plates (3M™, product 6475, lot 2016-10KB) by lifting the top film and adding 1 mL of the appropriate dilution. The top film was gently rolled down onto the plate to prevent air bubbles. The film was sealed by placing the spreader on the plate and gently pressing down to evenly distribute the sample. The Petrifilm™ was allowed to set for 1 minute before placing in an incubator. This was repeated for all Petrifilm™ and almond samples to be tested. The APC Petrifilm™ plates were placed in stacks, no more than 20 high, in an incubator at 35°C for 48 hours. The E. coli and Coliform Petrifilm™ plates were placed in stacks, no more than 20 high, in an incubator at 35°C for 24 hours. The Yeast and Mold Petrifilm™ plates were placed in stacks, no more than 40 high, in an incubator at 25-28°C for 48 hours. After the designated amount of time, the Petrifilm™ plates were removed and the colonies counted and calculated according to the 3M™ interpretation guides. 19 Statistical Analysis An Analysis of Variance (ANOVA) was conducted using Excel 2010 to determine if there was a significant difference between samples purchased at retail stores, at locations considered road-side stands, or from locations whose classification is unknown. The test was also used to determine if there was a significant difference between samples which were known to be pasteurized, unpasteurized, or those whose pasteurization status was unknown. These comparisons were performed on the APC, coliform, yeast and mold results. 21

CHAPTER 4: RESULTS AND DISCUSSION

A total of 116 almond samples were tested with 74 from retail stores and 22 from locations that can be considered farmers markets, which are exempt from the new mandated pasteurization rule. Twenty of the samples tested were purchased online and it is unknown if the company they came from is considered a retail store or can be classified as a ‘roadside stand’. Of the samples tested 65 were known to be pasteurized, 4 were known to be unpasteurized, and for 47 samples it was unknown if they were pasteurized or unpasteurized.

During the first set of Salmonella testing 4 out of 40 of the tested samples were randomly selected and the pH was tested using the lactose broth enriched samples. The average sample pH was 6.60 ±0.08. This pH level is within the required pH for the test (6.6-6.8) method so a pH adjustment step was not used. Five samples tested presumptive positive for Salmonella. These samples were retested for the presence of Salmonella using the BAX®, subjected to confirmation testing, and were found to be negative.

Several recalls due to Salmonella have recently occurred in nut types other than almonds. The recalls included raw macadamia nuts, raw pine nuts, walnuts, and mixed nut products from multiple manufacturers and sold at multiple retail stores from March 20, 2015 to May 15, 2015 (United States Food and Drug Administration 2015b). A sample of almonds that was found negative for Salmonella was purchased from one of the retailers involved in one of the recalls involving unpasteurized nut products. 21 21

One hundred fifteen of the almond samples tested for the presence of Salmonella were also subjected to E. coli, coliform, yeast, mold, and APC testing (Table 2).

Table 2. E. coli, Coliform, Yeast, Mold, and APC Test Results (n=115) Coliform Yeast Mold APC log CFU/g log CFU/g log CFU/g log CFU/g Industry Specification <3 <3.70 <4.70 Mean 2.83 3.23 3.44 5.14 Standard Error 2.06 2.85 2.76 4.79 Median 2.31 2.85 2.89 3.21 Range 1.48-3.76 2.00-4.80 1.70-4.42 1.00-6.83

The average APC count was 5.14 log CFU/g, which is larger than the common industry specification of less than 4.70 log CFU/g, according to the Almond Board of California. This is higher than what was found in Australia by Eglezos in 2010 (0.38 log CFU/g), in Turkey by Vural and others in 2008 (3.04-5.15 log CFU/g), in the United Kingdom by Candlish and others in 2001 (2.74-3.52 log CFU/g), and in The United States by King in 1970 (3.45-3.84 log CFU/g) and Danyluk and others in 2007 (3.26 log CFU/g). The high APC count may indicate an issue with the equipment sanitation or cross contamination of the almonds between pasteurization and packaging. There was no significant (p≥ 0.05) difference in APC counts between the samples from retail stores, locations classified at road-side stands, and those whose classification is unknown. There was also no significant (p≥ 0.05) difference in APC counts for samples that were known to be pasteurized 22 22 and unpasteurized, and for those whose pasteurization status was unknown.

The average yeast count was 3.32 log CFU/g and the average mold count was 3.44 log CFU/g. Combined, the values are within the common industry specification of less than 3.70 log CFU/g for yeast and mold. This value is comparable to what was found in Turkey by Vural and others in 2008 (1.30-3.76 log CFU/g), and in The United States by King in 1970 (3.45-3.84 log CFU/g) and Danyluk and others in 2007 (3.46 log CFU/g). The amount of yeast and mold present in almonds is comparable to the levels found before the 2007 rule. There was no significant (p≥ 0.05) difference in yeast and mold counts between the samples from retail stores, locations classified at road-side stands, and those whose classification is unknown. There was also no significant difference (p≥ 0.05) in yeast and mold counts for samples that were known to be pasteurized and unpasteurized, and for those whose pasteurization status was unknown.

No generic E. coli was found which is comparable to the results found in Australia by Eglezos in 2010, and in the United Kingdom by Little and others in 2009. The average coliform count was 2.83 log CFU/g. This is within the industry standard of less than 3.00 log CFU/g and is higher than the level found in Turkey by Vural in 2008 (not detected), in the United Kingdom by Candlish and others in 2001 (not detected) and in The United States by King in 1970 (0.90-1.04 log

CFU/g). Based on the studies that tested the level of E. coli and coliform in almonds, it appears that the amount of coliforms in almonds has increased since the 2007 rule. The amount of coliforms present may 23 23 indicate cross contamination after pasteurization and before packaging. There was no significant difference (p≥ 0.05) in coliform counts between the samples from retail stores, locations classified at road-side stands, and those whose classification is unknown. There was also no significant difference (p≥ 0.05) in coliform counts for samples that were known to be pasteurized and unpasteurized, and for those whose pasteurization status was unknown. 25

CHAPTER 5: CONCLUSION

Almonds are very important to the California economy as they are the largest valued export crop contributing over $3.3 billion annually (California Department of Food and Agriculture 2014). Ensuring a safe food supply is extremely important as foodborne illness affects about 9.4 million people every year (Scallan and others 2011). Food safety is especially important in terms of controlling Salmonella as it causes about 23,000 hospitalizations and 450 deaths every year and it is estimated that for every one confirmed case, about 23 cases are unreported. Since 2001 there have been 69 recalls of nut products, with 41 of the recalls due to Salmonella, and 7 outbreaks due to Salmonella in pistachio, pine nut, hazelnut, cashew, and almond products (Palumbo and others 2015). Pasteurization is a food safety intervention method that reduces the amount of microorganisms, including Salmonella, in food products. In this study the presence of Salmonella in retail almonds was not found and there is no evidence that the pasteurization is not working. In 2000 it was reported that the USDA, FDA, and industry associations do not collect information on the economic impact of recalls due to foodborne illness (United States General Accounting Office 2000). It can be assumed that recalls can have a large economic impact on food companies due to lost profits of the recalled products, damage caused to a brand’s reputation, and lost business as consumers avoid recalled brands and switch to purchasing a competitor’s product. It is known that during the 2004 outbreak of Salmonella in almonds, approximately 13 million pounds of almonds were recalled. It is likely that recalls of other 25 25 nuts may have experienced a similar economic loss that could have been avoided if the products underwent a pasteurization step. Although this study, with its limited amount of samples, does not establish the incidence of Salmonella it is recommended that studies be conducted to further investigate and ensure the safety of nuts including almonds. According to the results of this study, pasteurization has not affected the level of yeast and mold on the almond samples tested, and the APC and coliform counts are higher compared to previously performed studies. These levels indicate the possibility of cross contamination between the pasteurization step and before packaging of the almonds. Cross contamination can be caused by a lack of sanitary design of a processing facility, a lack of good manufacturing practices (GMPs), poor product handling, and an insufficient pest control program (Podolak and others 2010). In a study performed in the United Kingdom,

57% of outbreaks, when the cause of the outbreak was known, were due to cross contamination (Powell and others 1998). To prevent foodborne illness and reduce cross contamination the Food Safety and Modernization Act (FSMA) of 2011 calls for “science-based preventative controls” to be implemented by food processors (United States Food and Drug Administration 2014b). These preventative controls are similar to the Hazard Analysis Critical Control Points (HACCP) system that is mandatory in some industries such as the seafood and juice industry. To assist almond processors with their food safety systems, the Almond Board of California has created resources including guidance for developing a HACCP plan, Good Manufacturing Practices (GMPs) guidance, model forms for Sanitation Standard Operating Procedures 26 26

(SSOPs), and a Pathogen Environmental Monitoring (PEM) program (Almond Board of California 2015c). Efforts and resources should be spent to assist processors and growers with the implementation of a HACCP plan, or other “science-based preventative controls,” GMPs, and

SSOPs. The current PEM program focuses on Salmonella which deemphasizes other potential microorganism contaminants. It is recommended a revised PEM be created that also includes cross contamination prevention and testing for other microorganisms such as yeast, mold, coliforms, and total aerobic mesophilic plate count. Pasteurization has aided the food industry in ensuring a safe supply of almonds. The same could be said of other nut types if they were pasteurized before being sold to consumers. 28

REFERENCES 29

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APPENDICES 37

APPENDIX A: DILUTION SCHEME 35 35

APC: target = 25-250 CFU/petrifilm, lit. = 2,800-6,900 CFU/g

Yeast & Mold: target = <150 CFU/petrifilm, lit. = 1,400-47,000 CFU/g

E. coli & Coliform: target = <150 CFU/petrifilm, lit. = 8-11 CFU/g

37

APPENDIX B: ALMOND SAMPLE CLASSIFICATION 37

Retailer (R) or Pasteurized (P), Unpasteurized Sample ID Farmers Market (F) (UP), Unknown (U) 2013.10 1a, 1b R P 2013.10 2a, 2b R P 2013.10 3 F U 2013.11.22 1 F U 2013.11.23 1 R P 2013.11.27 1a, 1b F U 2013.11.27 2a, 2b R U 2013.12.18 1a, 1b R P 2013.12.18 2a, 2b R P 2013.12.18 3a, 3b R P 2014.1.9 1a, 1b R P 2014.2.19 1 F U 2014.2.19 2 F U 2014.2.19 3 F U 2014.2.19 4 F U 2014.3.1 1a, 1b F U 2014.3.21 1 F U 2014.3.31 1a, 1b R P 2014.4.11 1 F U 2014.4.11 2 F U 2014.4.11 3 F U 2014.4.26 1a-1c F U 2014.7.14 1 F U 2014.8.31 1a, 1b R P 2014.9.2 1a, 1b R P 2014.12.6 1a, 1b R P 2014.12.6 2a, 2b R P 2014.12.6 3 F U 2014.12.6 4 F U 2014.12.6 5 F U 2014.12.6 6a, 6b R P 2014.12.6 7a, 7b R P 2014.12.6 8 R P 2014.12.6 9 R P 2014.12.6 10 R P 2014.12.6 11 R P 38 38

Retailer (R) or Pasteurized (P), Unpasteurized Sample ID Farmers Market (F) (UP), Unknown (U) 2014.12.6 12a, 12b R P 2014.12.6 13a, 13b R P 2014.12.6 14a, 14b R P 2014.12.6 15a, 15b R P 2014.12.6 16a, 16b R P 2014.12.6 17a, 17b R P 2014.12.6 18a, 18b R P 2014.12.6 19a, 19b R P 2014.12.19 1a, 1b R P 2014.12.20 1a, 1b R P 2014.12.20 2a, 2b R P 2014.12.20 3a, 3b R P 2014.12.20 4a, 4b R P 2014.12.20 5 R P 2014.12.20 6 R P 2014.12.20 7 R P 2014.12.20 8a, 8b R P 2014.12.20 9a, 9b R P 2014.12.27 1a, 1b R P 2015.1.4 1a, 1b R P 2015.1.4 2a, 2b R P 2015.1.4 3a, 3b R P 2015.2 1 F U 2015.2 2 F U 2015.3.4 1a, 1b R P 2015.3.4 2a, 2b R P 2015.3.14 1a, 1b R P 2015.3.14 2a, 2b R P 2015.3.14 3a, 3b R P 2015.3.14 4a, 4b R P 2015.3.31 1a, 1b U U 2015.3.31 2a, 2b R UP 2015.3.31 3a, 3b R U 2015.3.31 4a, 4b R U 2015.3.31 5a, 5b R U 2015.3.31 6a, 6b R U 39 39 Retailer (R) or Pasteurized (P), Unpasteurized Sample ID Farmers Market (F) (UP), Unknown (U) 2015.3.31 7a, 7b R P 2015.3.31 8a, 8b R U 2015.3.31 9a, 9b R P 2015.3.31 10 R U 2015.4.1 1a, 1b R P 2015.4.1 2a, 2b R U 2015.4.2 1a, 1b R P 2015.4.2 2 U UP 2015.4.2 3a, 3b U UP 2015.4.2 4 U U 2015.4.2 5 U P 2015.4.2 6 U U 2015.4.2 7 U U 2015.4.2 8 U P 2015.4.2 9 U P 2015.4.3 1a, 1b R P 2015.4.3 2 U P 2015.4.3 3 U P 2015.4.6 1 R U 2015.4.7 1a, 1b R U 2015.4.7 2a, 2b R U 2015.4.7 3 U UP 2015.4.7 11a, 11b R U 2015.4.9 1 U U 2015.4.9 2 U U 2015.4.15 1 U U 2015.4.15 2 U U 2015.4.15 3 U U 2015.4.15 4 U U 2015.4.15 5a, 5b R P 2015.4.15 6a, 6b U P 2015.4.15 7 U U 2015.4.15 8a, 8b R P 2015.4.15 9a, 9b R U 2015.4.15 10a, 10b R U 2015.4.17 1 F U 2015.4.17 2 F U 40 40 Retailer (R) or Pasteurized (P), Unpasteurized Sample ID Farmers Market (F) (UP), Unknown (U) 2015.4.17 3 F U 2015.5.1 1a, 1b R P Unknown 1a, 1b R P Unknown 2a, 2b R P Unknown 3a-3c R P Unknown 4a, 4b R P Unknown 5a, 5b R P

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