Efficacy of vacuum steam pasteurization for inactivation of Salmonella PT 30, Escherichia coli O157:H7 and Enterococcus faecium on low moisture foods

Accepted Manuscript Efficacy of vacuum steam pasteurization for inactivation of Salmonella PT 30, Escherichia coli O157:H7 and Enterococcus faecium on low moisture foods

Manoj K. Shah, Gladys Asa, Julie Sherwood, Kari Graber, Teresa M. Bergholz PII: DOI: Reference:

S0168-1605(17)30003-X doi: 10.1016/j.ijfoodmicro.2017.01.003 FOOD 7494

To appear in:

International Journal of Food Microbiology

Received date: Revised date: Accepted date:

2 May 2016 21 December 2016 6 January 2017

Please cite this article as: Manoj K. Shah, Gladys Asa, Julie Sherwood, Kari Graber, Teresa M. Bergholz , Efficacy of vacuum steam pasteurization for inactivation of Salmonella PT 30, Escherichia coli O157:H7 and Enterococcus faecium on low moisture foods. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Food(2017), doi: 10.1016/j.ijfoodmicro.2017.01.003

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ACCEPTED MANUSCRIPT Efficacy of vacuum steam pasteurization for inactivation of Salmonella PT 30, Escherichia coli O157:H7 and Enterococcus faecium on low moisture foods Manoj K. Shaha, Gladys Asab, Julie Sherwooda, Kari Graberb, Teresa M. Bergholza Department of Microbiological Sciences, North Dakota State University, Fargo, North Dakota,

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a

Napasol North America, Fargo, North Dakota, 58102

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b

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58108

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Email:

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Manoj K. Shah: [email protected]

Julie Sherwood: [email protected]

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Gladys Asa: [email protected]

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Kari Graber: [email protected]

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Teresa M. Bergholz: [email protected]

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Corresponding author: Teresa M. Bergholz

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PO 6050 Dept 7690, North Dakota State University Fargo, ND 58108-6050, United States Phone: 701-231-7692

Keywords: vacuum steam pasteurization, low moisture foods, inactivation, Salmonella, E. coli O157:H7, E. faecium

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ACCEPTED MANUSCRIPT Abstract Low moisture foods such as nuts, spices, and seeds have been implicated in several outbreaks due to Salmonella or E. coli O157:H7 contamination. Such foods may be consumed raw, and can be used as ingredients in other food products. While numerous thermal inactivation

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studies have been conducted for Salmonella on nuts, studies on other seeds and grains are

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minimal. Product water activity can influence the thermal resistance of pathogens, where thermal

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resistance increases as water activity decreases, leading to a requirement for higher temperatures and longer exposure times to achieve significant reduction of pathogen numbers. Vacuum steam

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pasteurization uses steam under vacuum, which can be operated at temperatures above and below

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100C. The objective of this study was to determine the efficacy of vacuum steam pasteurization for inactivation of pathogens on whole flaxseed, quinoa, sunflower kernels, milled flaxseed and

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whole black peppercorns. The use of E. faecium as a potential surrogate for Salmonella and E.

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coli O157:H7 in vacuum steam pasteurization was also evaluated. Pasteurization for 1 minute at

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75C yielded average log reductions of 5.48±1.22, 5.71±0.40 and 5.23±0.61 on flaxseed, 4.29±0.92, 5.89±0.26 and 2.39±0.83 on quinoa, and 4.01±0.74, 5.40±0.83 and 2.99±0.92 on

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sunflower kernels for Salmonella PT 30, E. coli O157:H7 and E. faecium, respectively.

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Similarly, on milled flaxseed and black peppercorns average log reductions of 3.02±0.79 and 6.10±0.64 CFU/g were observed for Salmonella PT 30 after 1 minute of treatment at 75C but, on average, >6.0 log reductions were observed after pasteurization at 85C. Our data demonstrate that vacuum steam pasteurization can be effectively used to reduce pathogens on these low moisture foods at temperature as low as 75 and 85C, and that E. faecium may be used as a potential surrogate for Salmonella PT 30 and E. coli O157:H7.

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ACCEPTED MANUSCRIPT 1.

Introduction Low moisture foods such as seeds, grains, and spices have been demonstrated to have

beneficial nutrients such as antioxidants, dietary fiber, proteins, lignans, omega-3 fatty acids, and other bioactive compounds (Carraro et al., 2012; Okarter and Liu, 2010; Su et al., 2007). While

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foods such as flaxseed, quinoa, sunflower kernels and peppercorns have been consumed since

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ancient times, the presence of foodborne pathogens in low moisture foods is an increasing

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concern (Beuchat et al., 2013; Young et al., 2015). These foods may be assumed to be safe for consumption in their raw form, though recent outbreaks of Salmonella and E. coli O157:H7

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associated with low moisture foods would suggest otherwise.

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Salmonella is a major pathogen of concern in low moisture foods with a number of outbreaks and recalls in recent years. In the U.S., E. coli O157:H7 has also been attributed to a

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few outbreaks in low moisture foods. Of the total Salmonella enterica illnesses from 1998-2008,

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13.0 % were attributed to fruits and nuts and 2.9 % were attributed to grains and beans (Painter et al., 2013). Similarly, 21.1% of total illnesses due to E. coli O157 were attributed to fruits and

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nuts, and 0.7% were attributed to grains and beans. From 2010-2015, there were four outbreaks

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due to Salmonella in low moisture foods (nut butter, tahini sesame sauce, Turkish pine nuts, and red and black pepper in salami) causing 310 illnesses, 65 hospitalizations, and one death (CDC,

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2015). In addition, there were 46 recalls in 2015 due to possible Salmonella contamination in low moisture products including milled flaxseed, whole flaxseeds, sunflower kernels, and organic milled coriander (FDA, 2015). Little data exist on the prevalence of Salmonella on edible seeds, grains, and flour. A survey of edible seeds found that 1/284 and 1/976 samples were positive for Salmonella on flaxseed and sunflower, respectively (Willis et al., 2009). The incidence of pathogens in edible seeds is estimated to be 2.07 log CFU/g (FAO/WHO, 2014).

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ACCEPTED MANUSCRIPT Contamination of low moisture foods with pathogens can occur at any stage of production, including during cultivation, processing, and/or packaging (Podolak et al., 2010). The estimated annual risks due to pathogen contamination of nuts and dried fruits rank from moderate to high for number of illnesses, hospitalizations, and deaths (Painter et al., 2013).

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Thermal processes such as dry heat are widely used to inactivate pathogens on nuts and edible

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seeds (Abd et al., 2012; Beuchat and Mann, 2011; Nascimento Mda et al., 2012). However, they

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are most effective at inactivation of pathogens at high treatment temperatures after long pasteurization times. Some of the drawbacks of high processing temperatures include loss of

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quality and/or functionality of the product, as well as reduction of some nutrients (Gerstenmeyer

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et al., 2013; Kiralan, 2012). Other methods such as irradiation, fumigation, and high pressure processing have been evaluated for inactivation of pathogens in low moisture foods. However,

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most of these technologies have been evaluated for reduction of pathogens on almonds and

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walnuts. Implementation of the Food Safety Modernization Act control measures would require the food industry to show adequate reduction of pathogens of concern in all types of foods (FDA,

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2016).

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The application of steam under a vacuum allows for pasteurization temperatures within the range of 70-100°C, which allows processors to balance pathogen reduction while maintaining

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desirable quality attributes of low moisture foods. Our goal was to quantify pathogen reduction in a variety of low moisture foods including seeds, grains, and spices (whole/milled flaxseed, quinoa, sunflower kernels, and whole black peppercorns) using vacuum steam pasteurization at a range of processing temperatures. The non-pathogenic bacterium, Enterococcus faecium, is commonly used as a surrogate for Salmonella in thermal processing (Bianchini et al., 2014; Ceylan and Bautista, 2015; Jeong et al., 2011). We also wanted to determine if E. faecium would

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ACCEPTED MANUSCRIPT serve as an effective surrogate for inactivation of pathogens on vacuum steam pasteurized foods. The data presented here demonstrate that vacuum steam pasteurization can be an effective method to significantly reduce pathogens on low moisture foods at relatively low temperatures.

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2. Materials and methods

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2.1 Inoculation of foods

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Raw whole and milled flaxseed (Linnum usitatissimum), quinoa (Chenopodium quinoa), sunflower kernels (Helianthus annuus), and whole black peppercorns (Piper nigrum) were

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obtained from local commodity suppliers. Upon receipt, these products were tested for

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Salmonella and E. coli and were found to be negative. Each product was separately mixed, packaged into Mylar bags, (Uline Inc., Pleasant Prairie, WI) and sealed. Bags were stored at

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room temperature until the product was needed for inoculation.

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Salmonella enterica subsp. enterica serovar Enteritidis PT 30 (ATCC BAA-1045) and Enterococcus faecium NRRL B-2354 (ATCC 8459) were obtained from the American Type

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Culture Collection (Manassas, VA) and Escherichia coli O157:H7 Sakai (RMID 0509952) was

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obtained from the Thomas S. Whittam STEC Center (Michigan State University, East Lansing, MI). The cultures were stored at -80C in Brain Heart Infusion Broth (BHI) (Difco, Becton

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Dickinson, Sparks, MD) with 10% glycerol. The cultures were streaked onto BHI agar plates and incubated overnight at 35C. An isolated colony was transferred to 5 ml BHI broth and incubated at 35C for 20 h. For each culture, 250 µl was spread onto BHI agar plates (100 mm x 15 mm) and plates were incubated at 35C for 24 h. An inoculation procedure similar to those described by using agar-grown cultures harvested in a minimal amount of water was used to inoculate the food products directly (Fudge

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ACCEPTED MANUSCRIPT et al., 2016; Smith and Marks, 2015). 2.2 kg of each product was required for each replicate. As preliminary experiments showed that a lawn of culture from a single BHI plate was sufficient to provide a target inoculum level of ~7-8 log CFU/g when added to 100 g of product, cultures recovered from 22 BHI agar plates were used for each replicate.

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Lawn cultures from 11 BHI agar plates were collected with a sterile cell spreader (Fisher

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Scientific, Waltham, MA) into a beaker containing 2.5 ml of sterile water and were stirred to

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suspend the cells. The bacterial suspension was poured into 1.1 kg product in a Whirl pak bag (Nasco, Fort Atkinson, WI). The culture and food were mixed by hand massaging for 5 minutes.

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The same procedure was followed for the remaining 1.1 kg of the product. After mixing, the

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bags were transferred to a sterile stainless steel tray (12" X 9") for water activity (aw) equilibration. Salmonella PT 30, Escherichia coli O157:H7, and E. faecium were inoculated onto

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whole flaxseed, quinoa, and sunflower kernels. Pasteurization was conducted only for

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Salmonella PT 30 on milled flaxseed and peppercorns due to the high number of native microbes on the raw product, typically yeasts and molds, which interfered with colony counts on non-

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selective media. All inoculation procedures were conducted in a class II biosafety cabinet.

2.2 aw equilibration

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aw was measured using an Aqualab 4TE aw meter (Aqualab, Pullman, WA). The aw of the raw products increased upon inoculation. Following inoculation, 2.2 kg of the inoculated product in the stainless steel tray was placed in a closed chamber (Coleman cooler 24" X 16", Coleman Company, Inc., Kingfisher, OK). Approximately 25-50 g of magnesium chloride 6Hydrate crystal (Avantor Performance materials, Inc., Center Valley, PA) or lithium chloride anhydrous 99% -20 Mesh (Alfa Aesar, Ward Hill, MA) were weighed in plastic trays (Fisher

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ACCEPTED MANUSCRIPT Scientific) and saturated with water. These trays were placed in the closed chamber with the inoculated product to equilibrate the aw to the initial level, as described by Jeong et al (2012). The aw was equilibrated to the original level within 24 h. Inactivation experiments were

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conducted at the equilibrated aw.

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2.3 Assessing homogeneity of inoculation

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Multiple samples of the inoculated and aw equilibrated foods were enumerated to ensure homogenous distribution of inoculum in the food products. Immediately after inoculation, two 25

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g samples of food were weighed in a sterile plastic bag (Nasco) and Butterfield’s dilution buffer

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was added in appropriate amounts. Bags were homogenized for 90 s and appropriate serial dilutions were spread plated onto Luria Bertani (LB) agar plates (Difco) in duplicate and

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incubated at 37±2C for 48 hours. Then, eight 25 g samples were plated in duplicates 24, 48, and

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72 hours post-inoculation for each product type. The assessment of homogeneity was performed

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for each food type.

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for two replicates of Salmonella PT 30 and 1 replicate each for E. coli O157:H7 and E. faecium

2.4 Heat resistance test

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Heat resistance of the inoculum on each product was verified using the guidelines provided by the Almond Board of California (Almond Board of California, 2004). After aw equilibration, 100g of each inoculated product with each inoculum type were held at 280°F for 15 minutes in an Isotemp oven (Fisher Scientific, model no. 6903). Three 25g samples of each inoculum were diluted in Butterfield’s buffer and spread plated onto LB agar in duplicate, and plates were incubated at 37±2C for 48 hours to quantify bacteria survival.

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2.5 Vacuum steam pasteurization The vacuum steam pasteurization system at the pilot plant facility of Napasol North America (Fargo, ND) was used to conduct the pasteurization studies. Vacuum steam

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pasteurization (SMC, Statisol 50, model no. NA 07, Via Lazzaris, 17, Spresiano TV, Italy) uses

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steam under vacuum to obtain treatment temperatures below 100C. The pasteurization system

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works in three steps: pre-heating, steam treatment and cooling. Pre-heating is used to elevate the

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temperature of the raw product to a pre-determined level to facilitate homogenous distribution of steam during the steam treatment, which is the inactivation step. Products are placed in a metal

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bin and pre-heated to a set temperature using forced hot air and then loaded into the Statisol for steam treatment. The steam treatment process works in four steps: pre-vacuum, vacuum,

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pasteurization, and post vacuum, which all occur in a closed system. The amount of time and

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pressure at the pre-vacuum and vacuum phases can be adjusted as needed dependent upon matrix

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type. During the pasteurization stage, the pressure is dependent upon the set temperature. Cooling of the post-pasteurized products is done with forced air. For our experiments, the

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inoculated samples were subjected only to the steam treatment process.

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2.6 Pasteurization conditions

After aw equilibration, the inoculated foods were divided into 25 g portions and were placed into cotton bags (Uline). Three bags of each bacterial strain were treated at each pasteurization condition and represent one biological replicate for that bacteria and product combination. The sterilizer bin was filled with product specific to the inoculated samples (45.0 kg for whole flaxseed, quinoa, and sunflower kernels, and 22.5 kg for milled flaxseed and black

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ACCEPTED MANUSCRIPT peppercorns) and was pre-heated. Whole flaxseed, quinoa, and whole black peppercorns were pre-heated to 40±3C, and sunflower seeds and milled flaxseed were pre-heated to 60±3C, and 50±3C, respectively, using forced dry heat (Table S1). After pre-heating, the nine inoculated bags and three temperature data loggers (Madge Tech, Inc., Warner, NH) were placed at an even

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depth halfway among the product in the sterilizer bin, which was placed into the Statisol.

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Pasteurization temperatures of 75, 85, 95, and 105C were used for all products. Data loggers

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measured the temperature during the processing cycles (Table S2). Upon initial injection of

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steam, the set pasteurization temperature was obtained within 0.11 – 2.97 min, following pasteurization at a given temperature, it took 10.8 – 15.16 min to lower to the pre-heat

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temperature (Table S2). During pasteurization, the observed pasteurization temperatures varied

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by approximately ±3C from the set pasteurization temperatures (Table S2). The treatment condition, which includes the measured pre-pasteurization, pasteurization and post-pasteurization

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temperatures, will be referred to as the set pasteurization temperature (Table S2). The pre- and

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post-pasteurization times with their corresponding temperatures increased with higher set pasteurization temperatures (Table S2).

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At 75 and 85C, pasteurization was conducted for 0.5, 1, 2, 3, 4, and 5 minutes and at 95

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and 105C, pasteurization was conducted for 1, 2, 3, and 4 minutes. After pasteurization, the inoculated bags were retrieved and left to cool for ~15 minutes at room temperature. The product (fill volume) used in the sterilizer bin was cooled to the pre-heat temperature before loading for another pasteurization cycle. The pasteurization cycles are conducted in batches; to obtain one replicate of data at all temperatures and pasteurization times for a product, 20 pasteurization cycles were conducted. For each replicate, three inoculated samples that were unpasteurized were used as the controls.

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2.7 Enumeration of surviving bacteria Following pasteurization, the samples were weighed into sterile plastic bags (Nasco) and Butterfield’s dilution buffer was added in appropriate amounts. Bags were homogenized or

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massaged for 90 seconds and appropriate serial dilutions were spread plated in duplicate

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manually or using an Autoplate 4000 (Spiral Biotech, Norwood, MA). LB agar plates were used

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to enumerate Salmonella PT 30, E. coli O157:H7 and Enterococcus faecium after pasteurization of whole flaxseed, quinoa, and sunflower kernels. Modified Tryptic soy yeast extract agar

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(TSAYE) supplemented with ammonium ferric citrate and sodium thiosulfate (Difco) (Smith and

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Marks, 2015) was used to enumerate Salmonella PT 30 on milled flaxseed and whole black peppercorns as this non-selective but differential agar allowed the differentiation of Salmonella

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from the numerous surviving native yeasts and molds at lower dilutions. The respective agar

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plates were incubated at 37±2C for 48 hours. Following the incubation, the colonies were

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counted using a Q-Count model 350 (Spiral Biotech).

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2.8 Parameter estimates for microbial survival curves The Geeraerd-tail model (Geeraerd et al., 2000) was used to estimate survival parameters

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for Salmonella PT 30, E. coli O157:H7 and E. faecium on each food type after pasteurization at 75C and 85C using GInaFiT Version 1.6 (Geeraerd et al., 2005). The goodness of fit of data generated by the model was tested by comparing the f value against F table value at 95% Confidence (den Besten et al., 2006). The equation with its parameters are listed and defined below. N= (No- Nres) * e(-kmax * t) + Nres

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ACCEPTED MANUSCRIPT Where N is the population at time t (CFU/g), No is the population at time 0 (CFU/g), Nres is the heat resistant population, t is the time in minutes and Kmax is the maximum specific inactivation rate.

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2.9 Statistical Analysis

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For each bacterial strain, three inoculated 25 g samples were pasteurized at each set

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temperature and time combination, and the experiment was repeated three times providing nine data points at each pasteurization condition. The duplicate counts obtained for each sample in

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CFU/g were averaged and log transformed. The limit of detection (1.0 – 1.38 log CFU/g) was

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used as the count for samples where no colonies were detected. Log reductions were calculated by subtracting the log CFU/g of the pasteurized samples from the log CFU/g of the

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unpasteurized control samples. Analysis of Co-variance (ANCOVA) was conducted using Proc

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GLM in SAS V.9.4 (SAS Institute, Cary, NC). For ANCOVA, log reduction was considered as the dependent variable. Temperature and time measurements during pre-pasteurization,

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pasteurization, and post-pasteurization were included as dependent variables. Based on LS

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means adjusted for significant interactions, comparisons where the Tukey adjusted p value <

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0.05 were considered significantly different.

3. Results

3.1 Inoculation and aw equilibration The inoculum levels of each strain on each product were quantified and their heat resistance verified prior to conducting the pasteurization experiments. Inoculation of Salmonella PT 30, Escherichia coli O157:H7, and Enterococcus faecium resulted in ~7 to 8 log CFU/g for

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ACCEPTED MANUSCRIPT each product (Table S3). When stored for three days after inoculation, <0.5 log CFU/g decline in bacterial numbers were observed (Table S3). Heat resistance tests performed after aw equilibration on the inoculated foods following the Almond Board of California protocol showed, on average, <2.5 log CFU/g reduction (Table S4). As inoculations were conducted for

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each strain separately, the aw of the products inoculated with each strain were measured to be

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within ±0.01 of each other (Table 1). Following inoculation, the equilibrated aw were

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approximately 0.36, 0.54, 0.49, 0.44 and 0.42 for whole flaxseed, milled flaxseed, quinoa,

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sunflower kernels and whole black peppercorns, respectively (Table 1).

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3.2 Pasteurization of whole flaxseed

Inoculated Salmonella PT 30, E. coli O157:H7 and E. faecium on whole flaxseed ranged

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from 7.410.22 to 8.110.10 log CFU/g (Table 1). At 75°C, pasteurization for 30 s yielded an

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average log reduction of 2.241.17, 2.501.36 and 1.810.58 for Salmonella PT 30, E. coli

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O157:H7 and E. faecium, respectively, and significantly greater log reductions of 5.481.22, 5.710.40 and 5.230.61 were achieved for each strain after 1 min (Fig. 1), (adj.p < 0.05). On

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average, greater than 6.0 log CFU/g reduction was observed after pasteurization for longer than 1

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minute at 75C for Salmonella and E. coli O157:H7. For each strain, similar log reductions were observed over pasteurization time from 1 through 5 min (adj.p > 0.05). At each pasteurization time, the average log reductions for all bacteria were not significantly different from each other (adj.p > 0.05), except for Salmonella PT 30 and E. faecium after 4 min of pasteurization at 75C (adj.p < 0.05). Pasteurization at 85C for 30 s significantly increased the average log reduction to 5.590.14, 5.250.02 and 4.970.21 for Salmonella PT 30, E. coli O157:H7 and E. faecium,

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ACCEPTED MANUSCRIPT respectively, when compared to the log reduction observed after 30 s at 75C (Fig. 1), (adj.p < 0.05). Among the three strains of bacteria, there were no significant differences in log reduction after 30 s at 85C (adj.p > 0.05). The maximum log reduction was achieved at 3 minutes of pasteurization at 85C for all three strains, as no further significant increases in inactivation were

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observed with increasing pasteurization times (adj.p > 0.05). Pasteurization at 95C and 105C

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for all strains resulted in on average ≥ 6 log CFU/g reductions irrespective of time (Fig. 1),

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(adj.p > 0.05). The limit of detection for this set of experiments was 1.25 log CFU/g, which was

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reached only at 4 time-temperature combinations (Fig. 1). When comparing log reduction across pasteurization temperatures, similar average log reductions were achieved by pasteurization at

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75C for 1 min, 85C for 30 s, 95C for 1 min, and 105C for 1 min for all bacteria (adj.p >

3.3 Pasteurization of quinoa

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0.05).

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Inoculated Salmonella PT 30, E. coli O157:H7 and E. faecium on quinoa ranged from 7.390.17 to 8.220.18 log CFU/g (Table 1). Pasteurization at 75C for 30 s yielded, on average,

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4.090.67, 5.830.31, and 2.450.74 log CFU/g reduction for Salmonella PT 30, E. coli

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O157:H7 and E. faecium, respectively (Fig. 2), (adj.p < 0.05). For each strain, similar log reductions were observed for pasteurization times of 1 through 5 min (adj.p > 0.05). For Salmonella PT 30 and E. coli O157:H7, pasteurization at 85C for 30 s resulted in greater than 6.0 log CFU/g reduction (Fig. 2). For E. faecium, a 6.0 log CFU/g reduction was observed only after pasteurization for 3 min. Comparing across strains, log reductions were similar for Salmonella PT 30 and E. coli O157:H7 at all pasteurization times at 85C, which were similar to log reductions of E. faecium after pasteurization for 3-5 min (adj.p > 0.05). At

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ACCEPTED MANUSCRIPT 95C, log reductions observed for Salmonella PT 30, E. coli O157:H7 and E. faecium were not different from those observed at 105C (adj.p > 0.05). Comparing across pasteurization temperatures, log reductions achieved after pasteurization for 30 s at 85C for each strain were similar to their respective log reductions obtained after 1 min of pasteurization at 95 and 105C

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(adj.p > 0.05) due to reaching the limit of detection of 1.25 log CFU/g at most time points at

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these temperatures (Fig. 2).

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3.4 Pasteurization of sunflower kernels

For sunflower kernels, the inoculum levels ranged from 7.850.18 to 8.310.11 log

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CFU/g for Salmonella PT 30, E. coli O157:H7 and E. faecium, respectively (Table 1).

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Pasteurization at 75C for 30 s resulted in average log reductions of 3.921.18, 4.851.33 and 3.140.58 CFU/g for Salmonella PT 30, E. coli O157:H7 and E. faecium, respectively, that were

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not significantly different (adj.p > 0.05) (Fig. 3). For each strain, similar average log reductions

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were observed after pasteurization at 75C irrespective of pasteurization times (adj.p > 0.05).

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Pasteurization at 85C resulted in similar log reduction of >6.4 log CFU/g for E. coli O157:H7 (adj.p > 0.05) at all pasteurization times. For Salmonella PT 30, the minimum and

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maximum log reductions of 4.831.01 and 6.750.28 CFU/g were observed after pasteurization for 0.5 and 4 minutes, respectively (adj.p > 0.05) (Fig. 3). Similarly, average minimum and maximum log reductions of 3.910.67 and 6.380.41 CFU/g were observed after pasteurization for 0.5 and 3 minutes, respectively, at 85C for E. faecium (adj.p > 0.05) (Fig. 3). Pasteurization at 95 and 105C yielded on average log reductions of greater than 6.24 CFU/g at all treatment time for all bacteria with the highest reduction of 7.050.25 observed for Salmonella PT 30 at 95C after pasteurization for 2 min (Fig. 3). Log reductions observed at 95 and 105C for 14

ACCEPTED MANUSCRIPT Salmonella PT 30, E. coli O157:H7 and E. faecium were not significantly different from each other at any of the pasteurization times, often reaching the limit of detection (1.38 log CFU/g) (adj.p > 0.05) (Fig. 3).

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3.5 Pasteurization of milled flaxseed

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Inoculated Salmonella PT 30 on milled flaxseed ranged from 7.810.15 to 7.920.42 log

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CFU/g (Table 1). Pasteurization at 75C for 30 s led to an average log reduction of 2.211.06

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CFU/g, which was significantly lower than the average log reduction of 3.020.79 CFU/g observed after pasteurization for 1 min (Fig. 4), (adj.p < 0.05). Vacuum steam pasteurization at

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75C did not result in a 4.0 CFU/g log reduction on milled flaxseed. Pasteurization for 30 s at

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85C yielded an average log reduction of 5.561.31 CFU/g that was significantly lower than log reductions observed after 1-5 minutes of pasteurization (Fig. 4), (adj.p < 0.05). At 95 and 105C

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no colonies were detected (LOD = 1.0 log CFU/g) at any of the pasteurization times. The log

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(Fig. 4), (adj.p > 0.05).

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reduction observed at 85C after 1 min was similar to log reductions observed at 95 and 105C

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3.6 Pasteurization of whole black peppercorns Inoculated Salmonella PT 30 on black peppercorns ranged from 7.760.08 to 7.800.16 log CFU/g (Table 1). Pasteurization of peppercorns at 75C and 85°C resulted in similar log reductions of Salmonella PT 30 across all pasteurization times (Fig. 5), (adj.p > 0.05). Pasteurization at 95C for 1 min yielded an average log reduction of 6.350.54 CFU/g, which were similar to log reductions at all pasteurization times at 95 and 105C (adj.p > 0.05). There were no significant differences in log reduction observed after 30 s of pasteurization at 75C and

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ACCEPTED MANUSCRIPT any other pasteurization times at 85, 95 and 105C (adj.p > 0.05), though the LOD (1.08 log CFU/g) was not reached for any of the samples (Fig. 5).

3.7

Survival curves were non-linear and showed high inactivation rates

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As bacterial survival was non-log linear, the Geeraerd-tail model was used to generate

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survival curves (Table 2, Fig. S1-S4). In most instances, pasteurization for just 0.5 or 1 minute at

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95 and 105°C yielded a rapid decline in bacterial counts at or near the limit of detection, not

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allowing survival curves to be fit to these data. Therefore, only data obtained after pasteurization at 75 and 85°C were used to generate survival curves. The Kmax values (inactivation rate per min)

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for pasteurization of whole flaxseed at 75°C were 12.71, 13.62 and 12.16 for Salmonella PT 30, E. coli O157H7 and E. faecium, respectively (Table 2). Pasteurization of whole flaxseed at 85°C

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yielded an increase in inactivation rates, to 27.71, 27.48 and 25.03 for Salmonella PT 30, E. coli

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O157H7 and E. faecium, respectively (Table 2). A similar increase in Kmax values between 75°C

PT

and 85°C was observed for all strains on quinoa and sunflower kernels. At both pasteurization temperatures, Kmax values for E. faecium were observed to be the lowest in all instances

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compared to Salmonella PT 30 and E. coli O157:H7 (Table 2). Pasteurization of milled flaxseed provided Kmax values of 9.42 and 25.84 for Salmonella PT 30 after pasteurization at 75 and 85°C,

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respectively, whereas similar Kmax values of 26.38 and 24.98 was observed for Salmonella PT 30 on whole black peppercorns at both pasteurization temperatures (Table 2). The estimated 4D values (4 log reduction) from this model were predicted to be less than 1 minute for all food types at both 75 and 85°C (Table 2). Also, the 4D values are lower at 85°C than at 75°C for all bacteria and all food types except for black peppercorns. There were four instances at the pasteurization temperature of 75°C for E. coli O157:H7 on quinoa, E. faecium on

16

ACCEPTED MANUSCRIPT quinoa and sunflower kernels and Salmonella PT 30 on milled flaxseed where 4D values could not be estimated (Table 2).

4. Discussion

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4.1 Vacuum steam pasteurization at 75 and 85C is effective for inactivation of pathogens

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Salmonellae are known to have exceptional heat resistance when present in food matrices

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with low water activity. For example, D49°C-values for Salmonella in corn flour ranged from 18

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to 594 minutes (VanCauwenberge et al., 1981). In wheat flour at aw=0.57, D75°C-values for Salmonella were ~30 minutes, and increased to 150 minutes in wheat flour at aw=0.26 (Archer et

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al., 1998). This high level of heat resistance on low-moisture foods can pose significant challenges when developing a process expected to achieve a 5 log reduction in pathogen

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numbers. Heat treatments ranging from 104°C for infrared heating (Brandl et al., 2008), 149°C

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for dry heating (Beuchat and Mann, 2011), and 127°C for oil roasting (Du et al., 2010), are

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required to achieve a 5 log reduction of Salmonella on almonds or walnuts. Many of these processing methods and temperatures would not be feasible, or lead to significant losses of

and grains.

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quality and/or product functionality for low-moisture foods other than nuts, such as seeds, spices,

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When inoculating low moisture foods to assess the efficacy of processing for inactivation of pathogens, it is important to achieve a homogeneous distribution of inoculum on the product and only minimally alter aw. A high density of inoculum is also required in order to demonstrate a 5 log reduction in pathogen numbers. Here, we used agar-grown cells harvested with a minimal amount of water as inoculum for the 5 different food matrices, whole flaxseed, quinoa, sunflower kernels, milled flaxseed and whole black peppercorns, similar to methods described by others

17

ACCEPTED MANUSCRIPT (Fudge et al., 2016; Smith and Marks, 2015), resulting in ~7-8 log CFU/g for Salmonella PT 30, E. coli O157:H7 and E. faecium on each of the products. Researchers have used other methods to inoculate products using a minimal amount of moisture, such as dry transfer from inoculated sand or chalk, though these methods led to inoculum levels of just 5 log CFU/g (Blessington et

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al., 2013). Smith and Marks demonstrated that product aw at the time of processing determines

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the thermal resistance of Salmonella (Smith and Marks, 2015), highlighting the importance of

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measuring aw before and after inoculation, and equilibrating the inoculated product to the original aw. Here, we used magnesium chloride or lithium chloride salts to equilibrate aw of

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inoculated products to the initial level, as described by Jeong et al. (Jeong et al., 2012).

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Data presented here show that vacuum steam pasteurization at 75°C results in >5.0 log CFU/g reduction of Salmonella PT 30 and E. coli O157:H7 in 5 min or less for whole flaxseed

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and sunflower kernels, and for Salmonella PT 30 on peppercorns. Variability in survival between

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replicates was observed at 75C, with large standard deviations in log reduction for the shorter

PT

pasteurization times. Increasing the pasteurization temperature to 85°C led to reduced variability and > 5 log reductions for both Salmonella PT 30 and E. coli O157:H7 on all products tested.

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Steam-based processing methods have been effective at reducing pathogens on almonds, with

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~8.5 log CFU/g reduction for Salmonella PT 30 after treatment for 65 s, at 95C (Chang et al., 2010), and 5.760.38 and 4.100.62 log CFU/g reduction of Salmonella Enteritidis on nonpareil and mission almonds after 65 s at 931C (Lee et al., 2006). The use of superheated steam at 200°C led to > 6.0 log reductions of Salmonella Typhimurium, Salmonella PT 30, and E. coli O157:H7 on both almonds and pistachios after just 15 s (Ban and Kang, 2016). Both the superheated steam and vacuum steam pasteurization use dry saturated steam and are applied in a closed system, as compared to the other steam based methods described, which likely contributes

18

ACCEPTED MANUSCRIPT to the relatively rapid and large reduction in pathogen numbers. While data on inactivation of foodborne pathogens on nuts are abundant, less is known about the efficacy of different inactivation methods for pathogens on seeds, grains, or spices. Dry roasting sesame seeds for 30 min at 130°C reduced Salmonella numbers by ~ 5 log CFU/g

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(Torlak et al., 2013). Exposure to aerated steam for 90 s led to > 5 log reduction of E. coli

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O157:H7 and Salmonella Weltevreden on alfalfa and mung bean seeds (Studer et al., 2013).

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Black peppercorns treated with atmospheric pressure plasma resulted in a log reduction of ~5 CFU/g Salmonella after 80 s (Sun et al., 2014). Here we present the first sets of data on

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inactivation of Salmonella and E. coli O157:H7 on flaxseed, sunflower kernels, and quinoa.

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These data highlight that inactivation parameters need to be assessed for different types of low moisture products, as some may require higher pasteurization temperatures than others to

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achieve the targeted reduction in pathogen numbers. Here we found the greatest log reduction in

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Salmonella was on peppercorns, where counts were below the limit of detection after

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pasteurization for 1 minute at 75C. In contrast, increasing the pasteurization time and temperature was necessary to achieve a similar log reduction on quinoa and milled flaxseed.

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Application of steam has been demonstrated to impact sensory properties or shelf life of some products. Black peppercorns exposed to steam at 100°C for 16 min were stored for 6

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months and were significantly darker in color than peppercorns that were irradiated and had a lower piperine content (Waje et al., 2008). Almonds treated with vacuum steam pasteurization, similar to the process used here, at 88C for 4 min had lower peroxide values compared to unpasteurized almonds during an accelerated shelf life study (Ivarsson, 2011). One of the limitations of our study is that changes in sensory properties and shelf life were not examined, though the pathogen inactivation data provide a starting point to begin assessing which vacuum

19

ACCEPTED MANUSCRIPT steam pasteurization conditions could be effective at reducing pathogens and maintaining sensory and functional properties.

4.2 E. faecium can be utilized as a conservative surrogate for Salmonella PT 30 and E. coli

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O157:H7 when using vacuum steam pasteurization

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E. faecium has been widely used as a surrogate for its similar thermal sensitivity as

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Salmonella (Bianchini et al., 2014; Ceylan and Bautista, 2015; Jeong et al., 2011), and its safety for use in processing facilities has been evaluated (Kopit et al., 2014). However, it is important to

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assess the utility of E. faecium as a surrogate for specific strains of pathogens in different product

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types, as it may not be appropriate for all Salmonella strains, as evidenced by a D-value for E. faecium to be 1/10th to that of Salmonella PT 42 in flour (Fudge et al., 2016). However, studies

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show that similar or greater log reductions are observed for E. coli O157:H7 when compared to

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log reductions obtained for Salmonella (He et al., 2011; Kim et al., 2012) and that the log reduction in E. faecium is often lower than that of Salmonella at the same thermal processing

PT

conditions (Jeong et al., 2011; Song et al., 2014). Therefore, in this study, E. faecium has been

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assessed as a potential surrogate for Salmonella PT 30 and E. coli O157:H7 exposed to vacuum steam pasteurization.

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Our data demonstrate that, overall, E faecium could serve as an effective surrogate for both Salmonella PT 30 and E. coli O157:H7 for vacuum steam pasteurization of whole flaxseed, quinoa, and sunflower kernels. At 75C, E. faecium had the greatest survival on quinoa and sunflower kernels, with significantly lower log reductions compared to E. coli O157:H7. At 85C, the log reduction in E. coli O157:H7 was observed to be greater than that of Salmonella PT 30 and E. faecium, with E. faecium being the most resistant bacteria on all three products. At

20

ACCEPTED MANUSCRIPT 95C and 105C, similar log reductions were observed for all bacteria.

4.3 Survival curves showed non log-linear kinetics after vacuum steam pasteurization For the three bacterial strains examined here, inactivation due to vacuum steam

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pasteurization was not of first order kinetics, rather they were non log-linear. We found that the

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Geeraerd-tail model provided better goodness of fit with low RMSE (Root Mean Square Error).

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The predicted survival curves generated at 75 and 85C yielded greater inactivation rates at 85°C

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than at 75°C as expected. The inactivation rates of E. faecium were observed to be the lowest, showing lower log reductions of E. faecium during this study when compared to Salmonella PT

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30 and E. coli O157:H7. In addition, the 4D (4 log reduction) values that were estimated using the model were similar to the log reductions observed in that amount of time during the

M

pasteurization study. The survival curves obtained in our experiment were also upward concave.

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It is also important to note that the tailing effect observed in our data may have been impacted to

PT

an extent due to the fact that lowest limit of detection was used when no colonies were detected,

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5. Conclusions

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which occurred mainly after pasteurization for 3-5 minutes at 85C.

Data presented here demonstrate that vacuum steam pasteurization can be used to effectively reduce pathogens on low moisture foods. The pilot scale equipment used here led to > 5 log reduction of pathogens on whole flaxseed, sunflower kernels, and peppercorns at 75°C and on milled flaxseed and quinoa at 85°C. Inactivation of E. faecium was similar to Salmonella PT 30 and E. coli O157:H7, indicating it can be used as a surrogate for these strains when evaluating or validating vacuum steam pasteurization. While scale up experiments and sensory evaluations

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ACCEPTED MANUSCRIPT will need to be conducted, the data presented here provide a starting point for further evaluation of the pasteurization parameters that can lead to effective reduction of pathogens.

6. Acknowledgements

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This work was funded by project #BDAPUC15-18 from the North Dakota Department of

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Commerce Agricultural Products Utilization Commission. We thank Katharine Sanders, Autumn

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Lea Kraft, Luiz Gustavo Conde Lima and Ryan Callahan for their technical assistance with this

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PT

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AN

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project.

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Food Prot 78, 2264-2278.

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Table 1. Inoculum levels and aw of food products used in this study

0.325 ± 0.05

Salmonella PT 30

0.438 ± 0.02

E. coli O157:H7

Controls CFU/g

0.421 ± 0.02

0.358 ± 0.02

7.90 ± 0.29

7.94 ± 0.11

E. faecium

0.432 ± 0.01

0.361 ± 0.02

Salmonella PT 30

0.550 ± 0.17

0.494 ± 0.12

E. coli O157:H7

0.554 ± 0.02

0.489 ± 0.07

E. faecium

0.551 ± 0.02

Salmonella PT 30

0.505 ± 0.03

E. coli O157:H7

0.501 ± 0.04

E. faecium

0.505 ± 0.05

0.567 ± 0.05

Salmonella PT 30

0.417 ± 0.04

Salmonella PT 30

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0.349 ± 0.02

for 95&105°Cab 8.11 ± 0.10

0.445 ± 0.03

milled flaxseed black peppercorns

aw After Equilibrationa

for 75&85°Cab 8.08 ±0.17 7.41 ± 0.22

7.44 ± 0.14

8.22 ± 0.18

8.01 ± 0.29

7.92 ± 0.31

7.85 ± 0.29

0.488 ± 0.02

7.39 ± 0.17

8.02 ± 0.30

0.436 ± 0.04

8.17 ± 0.22

8.31± 0.11

0.438 ± 0.05

8.03 ± 0.18

7.89 ± 0.44

0.446 ± 0.03

7.85 ± 0.18

7.91 ± 0.23

0.605 ± 0.01

0.543 ± 0.05

7.81 ± 0.15

7.92 ± 0.42

0.553 ± 0.06

0.419 ± 0.04

7.80 ± 0.16

7.76 ± 0.08

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0.513 ± 0.16

sunflower kernels

aw After Inoculationa

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quinoa

Strain

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whole flaxseed

aw Before Inoculation

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Product

averages and standard deviations for 3 replicates are reported

b

pasteurization at 75 & 85°C and 95 & 105°C were conducted on two separate days

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PT

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a

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ACCEPTED MANUSCRIPT Table 2. Parameter estimates obtained from the Geeraerd-tail model

quinoa

75°C

85°C

sunflower kernels

75°C

RMSE 0.7598

4D ±0.75

E. coli O157:H7

13.62

0.637

±0.7

E. faecium

12.16

0.3974

±0.8

Salmonella PT 30

27.71

0.3584

±0.35

E. coli O157:H7

27.48

0.2679

±0.35

E. faecium

25.03

0.4

±0.4

Salmonella PT 30

19.17

0.9372

±0.5

E. coli O157:H7

-

-

-

E. faecium

11.73

1.0452

-

Salmonella PT 30

30.82

0.4146

±0.3

E. coli O157:H7

29.87

E. faecium

27.58

Salmonella PT 30

18.34

E. coli O157:H7

22.94

E. faecium

±0.35

1.0731

±0.55

0.9088

±0.45

0.746

-

22.3

0.8889

±0.45

31.37

0.3386

±0.3

E. faecium

10.63

0.8285

±0.9

Salmonella PT 30

9.42

0.9689

-

85°C

Salmonella PT 30

25.84

0.9276

±0.4

75°C

Salmonella PT 30

26.36

0.5523

±0.35

85°C

Salmonella PT 30

24.98

0.9625

±0.4

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75°C

denotes values that were not estimated/predicted from the models

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-

±0.35

15.43

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black peppercorn

0.487

0.5112

E. coli O157:H7

PT

milled flaxseed

Salmonella PT 30

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85°C

29

T

kmax 12.71

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85°C

Strain Salmonella PT 30

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Temp 75°C

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Product whole flaxseed

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Geeraerd-tail

ACCEPTED MANUSCRIPT Figure legends Fig. 1. Average log reduction in CFU/g after vacuum steam pasteurization of whole flaxseed for

Salmonella PT 30 (white), E. coli O157:H7 (grey) and E. faecium (black) at pasteurization temperatures of 75, 85, 95 and 105C. Log reductions are average of three independent replicates

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and three technical replicates for each strain. Error bars denote standard deviation and ‘*’ shows

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pasteurization condition where the limit of detection (1.25 log CFU/g) was reached for all replicates.

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Fig. 2. Average log reduction in CFU/g after vacuum steam pasteurization of quinoa for

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Salmonella PT 30 (white), E. coli O157:H7 (grey) and E. faecium (black) at pasteurization temperatures of 75, 85, 95 and 105C. Log reductions are average of three independent replicates

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and three technical replicates for each strain. Error bars denote standard deviation and ‘*’ shows

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pasteurization condition where the limit of detection (1.25 log CFU/g) was reached for all replicates.

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Fig. 3. Average log reduction in CFU/g after vacuum steam pasteurization of sunflower kernels

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for Salmonella PT 30 (white), E. coli O157:H7 (grey) and E. faecium (black) at pasteurization temperatures of 75, 85, 95 and 105C. Log reductions are average of three independent replicates

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and three technical replicates for each strain. Error bars denote standard deviation and ‘*’ shows pasteurization condition where the limit of detection (1.38 log CFU/g) was reached for all replicates. Fig. 4. Average log reduction in CFU/g after vacuum steam pasteurization of milled flaxseed for

Salmonella PT 30 at pasteurization temperatures of 75, 85, 95 and 105C. Log reductions are average of three independent replicates and three technical replicates for each strain. Error bars

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peppercorns for Salmonella PT 30 at pasteurization temperatures of 75, 85, 95 and 105C. Log

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strain. Error bars denote standard deviations.

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Figure 2

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Figure 4

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Figure 5

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Highlights

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Low aw foods were inoculated with Salmonella PT 30, E. coli O157:H7 and E. faecium Inoculated foods were exposed to vacuum steam pasteurization >5 log reduction of pathogens on flaxseed and sunflower seeds at 75°C >5 log reduction of Salmonella on peppercorns at 75°C and milled flaxseed at 85°C E. faecium can be used as a surrogate for both Salmonella PT 30 and E. coli O157:H7

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