Evaluation of Lactic Acid and Sodium Metasilicate against Pathogens of Concern on Fresh and Deli Meats by Staci Lynn DeGeer A dissertation submitted to the Graduate Faculty of Auburn University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Auburn, Alabama May 4, 2014 Keywords: beef, pork, deli meats, lactic acid, sodium metasilicate, food safety Copyright 2014 by Staci Lynn DeGeer Approved by Christy Bratcher, Chair, Associate Professor of Animal Sciences Luxin Wang, Assistant Professor of Animal Sciences Manpreet Singh, Associate Professor of Food Science, Purdue University S. F. Bilgili, Professor and Extension Poultry Scientist, Poultry Science *Prepared per Meat Science guidelines ii Abstract Lactic acid and sodium metasilicate have been used in meat processing facilities as antimicrobial compounds. Their uses vary from carcasses rinses to ready to eat product applications at a variety of concentrations and temperatures. Utilizing these antimicrobials in different stages during meat processing may assist in the reduction of the risk of pathogenic microorganisms. The purpose of this study was to determine optimum concentrations and temperatures of application of lactic acid and sodium metasilicate for pathogen reduction on beef bottom round muscles. Using this information, optimal concentrations and temperatures were then applied to fresh pork and processed deli meats in addition to fresh beef. Four consecutive studies were conducted. In the first study, lactic acid (LA) was applied at 1, 2, 3, and 4% (LA1, LA2, LA3, and LA4) and sodium metasilicate (SM) was applied to fresh beef bottom rounds at 2, 3, 4, and 5% (SM2, SM3, SM4, and SM5) levels. In the next study, LA4 (v/v), SM4 (w/v), the combination of the two solutions (LASM), and distilled water (control) were applied to fresh beef bottom rounds at 4, 25, and 60 ?C. During the third study, LA4, SM4, and distilled water were applied to fresh beef and pork lean muscle and roast beef, ham, and turkey deli meats that were manufactured at the Lambert Powell Meat Laboratory without the use of antimicrobial solutions. All data were analyzed using the PROC MIXED procedure of SAS and Tukey pairwise comparisons, where appropriate (P > 0.05). LA and SM reduced (P < 0.05) the bacterial load of all the meat samples. Temperature of iii application had no effect (P > 0.05) on bacterial counts in any of the treatments. LA or SM alone were more effective (P < 0.05) in reduction of microbes than when used together (LASM). The control treatment resulted in higher microbial counts regardless of inoculum or species than either the LA or SM treatments (P < 0.01). Treatments including a hot water dip decreased the bacterial load of samples in comparison to those that did not receive the post packaging lethality treatment (P < 0.01). Regardless of hot water dip treatments, there were no differences among treatment groups in regards to microbial counts (P > 0.73). SM4 and LA4 were determined to be the lowest concentrations most effective against all microorganisms. Meat processors can apply LA or SM at refrigeration temperatures with the same benefits as applying them at a higher temperature. Both lactic acid and sodium metasilicate can be applied to fresh beef and pork as an effective hurdle technology in the fight for food safety. Treating deli meats with lactic acid or sodium metasilicate did not reduce L. monocytogenes loads. However, adding a post-packaging lethality treatment was able to minimize overall microbial contamination. iv Acknowledgments My journey in completing my PhD has been full of helpful individuals along the way who have been my support system, my motivation, and more importantly my inspiration. I would like to first thank the meat lab staff. Pete, TJ and the student workers did a phenomenal job ensuring that I had just what I needed whenever I needed it. They also provided a safe place to decompress and laugh during long days and short nights. Barney was forever keeping me sane and reminding me that I was not alone in the journey, all I had to do was ask for help. My fellow students, both graduate and undergraduate, and Heidi were vital in keeping my research moving in a forward direction. Having a great group of people to work with, play with, and just do life with makes everything better at the end of the day. The friendships formed will last a lifetime. My experience at Auburn wouldn?t have been the same without my advisor. Dr. Christy Bratcher provided me with all of the opportunities I asked for (and even some I didn?t). I was able to develop as a researcher, a manager, and a communicator. My committee was extremely supportive of my goals and timetables, even when they only made since to me. It is bittersweet to be ready to start the next chapter of my life. Thank you to everyone for making Auburn home. v Table of Contents Abstract ............................................................................................................................... ii Acknowledgements ............................................................................................................ iv List of Tables ................................................................................................................... viii Chapter I. Review of Literature ...........................................................................................1 Food Safety .............................................................................................................1 Intrinsic Factors Affecting Microorganisms ............................................................2 Water Activity ..............................................................................................2 pH .................................................................................................................3 Nutrient Availability ....................................................................................3 Biological Structures ....................................................................................4 Oxidative-Reduction Potential .....................................................................4 Naturally Occurring Antimicrobials ............................................................5 Extrinsic Factors Affecting Microorganisms ...........................................................5 Storage Temperatures ..................................................................................5 Atmosphere Compisition .............................................................................6 Microorganisms .......................................................................................................6 Escherichia coli ...........................................................................................6 Salmonella spp. ...........................................................................................8 Listeria monocytogenes ...............................................................................9 vi Topical Treatment ..................................................................................................10 Lactic Acid .................................................................................................10 Other Organic Acids ..................................................................................12 Sodium Metasilicate...................................................................................13 Other Alkaline Solutions............................................................................15 Research Objectives ...............................................................................................15 References ..............................................................................................................19 Chapter II. Evaluation of Multiple Concentrations and Temperatures of Lactic Acid and Sodium Metasilicate against Pathogens of Concern on Fresh Beef ..................................24 Abstract ..................................................................................................................24 Introduction ............................................................................................................25 Materials and Methods ...........................................................................................28 Culture Strains ...........................................................................................28 Treatment Preparation ................................................................................29 Sample Preparation ....................................................................................30 Statistical Analysis .....................................................................................31 Results and Discussion ..........................................................................................31 Concentration .............................................................................................31 Temperature ...............................................................................................34 Conclusion .............................................................................................................36 References ..............................................................................................................37 Chapter III. Evaluation of Lactic Acid and Sodium Metasilicate against Pathogens of Concern on Fresh Beef, Pork, and Deli Meats ..................................................................48 Abstract ..................................................................................................................48 vii Introduction ............................................................................................................50 Materials and Methods ...........................................................................................53 Culture Strains ...........................................................................................53 Treatment Preparation ................................................................................54 Sample Preparation ....................................................................................55 Statistical Analysis .....................................................................................56 Results and Discussion ..........................................................................................56 Conclusion .............................................................................................................58 References ..............................................................................................................60 Chapter VII. Implications and Conclusions .......................................................................69 viii List of Tables Table 1. Selected intrinsic factors affecting chosen pathogen growth...............................17 Table 2. Examples of gas mixtures used for selected MAP products.. ..............................18 Table 3. Strains of microorganisms used. ..........................................................................40 Table 4. pH values of lactic acid at 1, 2, 3, 4% (LA1, LA2, LA3, LA4), sodium metasilicate at 2, 3, 4, 5% (SM2, SM3, SM4, SM5), and distilled water. ..........41 Table 5. Concentration effects of lactic acid at 1, 2, 3, 4% (LA1, LA2, LA3, LA4) on fresh beef bottom round steaks after pathogen inoculation and a 30min contact time. ....................................................................................................................42 Table 6. Concentration effects of sodium metasilicate at 2, 3, 4, 5% (SM2, SM3, SM4, SM5) on fresh beef bottom round steaks after pathogen inoculation and a 30min contact time. ........................................................................................................43 Table 7. Temperature effects of 4% lactic acid (LA), 4% sodium metasilicate (SM), and 4% lactic acid + 4% sodium metasilicate (LASM) on Escherichia coli O157:H7 at 4, 25, and 60 ?C on fresh beef bottom round steaks after pathogen inoculation and a 30min contact time. ...................................................................................44 Table 8. Temperature effects of 4% lactic acid (LA), 4% sodium metasilicate (SM), and 4% lactic acid + 4% sodium metasilicate (LASM) on non-O157 STEC at 4, 25, and 60 ?C on fresh beef bottom round steaks after pathogen inoculation and a 30min contact time. .............................................................................................45 Table 9. Temperature effects of 4% lactic acid (LA), 4% sodium metasilicate (SM), and 4% lactic acid + 4% sodium metasilicate (LASM) on Salmonella spp. at 4, 25, and 60 ?C on fresh beef bottom round steaks after pathogen inoculation and a 30min contact time. .............................................................................................46 Table 10. Temperature effects of 4% lactic acid (LA), 4% sodium metasilicate (SM), and 4% lactic acid + 4% sodium metasilicate (LASM) on Listeria monocytogenes at 4, 25, and 60 ?C on fresh beef bottom round steaks after pathogen inoculation and a 30min contact time. ...................................................................................47 Table 11. Strains of microorganisms used. ........................................................................63 ix Table 12. pH values of lactic acid at 4% (LA4), sodium metasilicate at 4% (SM4), and distilled water. .....................................................................................................64 Table 13. Microbial effects of 4% lactic acid (LA) and 4% sodium metasilicate (SM) on Escherichia coli O157:H7 at 4?C on fresh beef bottom round steaks and fresh pork ham steaks after pathogen inoculation and a 30min contact time.. ............65 Table 14. Microbial effects of 4% lactic acid (LA) and 4% sodium metasilicate (SM) on non-O157 STEC at 4?C on fresh beef bottom round steaks and fresh pork ham steaks after pathogen inoculation and a 30min contact time.. ............................66 Table 15. Microbial effects of 4% lactic acid (LA) and 4% sodium metasilicate (SM) on Salmonella spp. at 4?C on fresh beef bottom round steaks and fresh pork ham steaks after pathogen inoculation and a 30min contact time.. ............................67 Table 16. Microbial effects of 4% lactic acid (LA) and 4% sodium metasilicate (SM) on Listeria monocytogenes at 4?C on deli roast beef, ham, and turkey with and without a post packaging lethality treatment.. ....................................................68 1 Chapter I Review of Literature Food Safety Food safety is a constant concern in the meat industry and consideration is given to methods of ensuring a safe food supply by reduction of pathogens. The Centers for Disease Control and Prevention (CDC) estimates that 1 in 6 Americans become ill each year due to foodborne illness. That is equivalent to approximately 48 million people. Of these, 128,000 are hospitalized and about 3,000 die of foodborne diseases (Weber, O?Brien, & Bender, 2004). Regulatory agencies, researchers, and employees in the meat industry mainly focus on Escherichia coli O157:H7 and non-O157 shiga-toxin producing E. coli (STEC) serotypes and Salmonella spp. in fresh meat. In ready to eat meats Listeria monocytogenes is the common pathogen of concern. While Salmonella spp., E. coli, and L. monocytogenes are common pathogens on the ?top five foodborne pathogens? lists compiled by the CDC (Weber, O?Brien, & Bender, 2004). The United States Department of Agriculture Food Safety and Inspection Service (USDA FSIS) has a zero tolerance policy for L. monocytogenes in ready-to-eat meat and poultry products. This rule requires meat processors to control L. monocytogenes by using one of three alternatives. Alternative 1 includes both a post-lethality treatment and a process or antimicrobial agent, Alternative 2 requires either a post-lethality treatment or a process or antimicrobial agent, and Alternative 3 requires the implementation of sanitation procedures and frequent USDA FSIS environmental testing (USDA, 2003). More recently, the USDA 2 FSIS expanded its ruling on E. coli O157 in raw, non-intact beef to include six non-O157 serotypes including: O26, O45, O103, O111, O121, and O145 (USDA 2011). Intrinsic Factors Affecting Microorganisms Water Activity Microorganisms need water to grow and thrive in food products. The water activity (aw) of foods is normally how this water described (FDA, 2013). The ratio of water vapor pressure of the food substrate to the vapor pressure of pure water at the same temperature is defined as water activity (Jay, Loessner, & Golden, 2006). Thus, aw describes how much water is ?unbound? and available for chemical/biochemical reactions and microbial growth facilitation (FDA, 2013). Pure water has an aw of 1.0 and most fresh foods have an aw of > 0.98 (Nester, Anderson, Roberts, Pearsall, & Nester, 2001). The aw in foods can be lowered with the addition of salts and sugars, binding the unbound water, or by physically removing the unbound water through drying, baking, or cooking the food (FDA, 2013). Most microorganisms require an aw > 0.90 (Nester, Anderson, Roberts, Pearsall, & Nester, 2001); however, the taxonomic classification of the microorganism can indicate how sensitive the bacteria will be to aw changes (FDA, 2013). Gram negative bacteria are usually more sensitive to low aw than Gram positive microorganisms (FDA, 2013). Selected pathogen aw requirements are listed in Table 1. Small changes in aw can have differential effects on bacterial growth (Gill & Newton, 1976). Most fresh meats have an aw of 0.99 ? 1.00, while cured meats generally have an aw of 0.87 ? 0.95 (FDA, 2013). 3 pH The pH of food can affect what microorganisms can survive on the food surface or within the food matrix (Nester, Anderson, Roberts, Pearsall, & Nester, 2001). Pathogens generally do not grow at pH levels below 4.6; however, there are some exceptions (FDA, 2013). Ground beef has a typical pH of 5.1 ? 6.1 and ham generally has a pH of 5.9 ? 6.1 (FDA, 2013). Normal meat pH is between 5.5 and 5.7; however, there can be differences in pH between carcasses and between different muscles from the same carcass (Gill & Newton, 1978). Table 1 includes approximate pH values allowing the growth of selected pathogens in food. Increasing the acidity of foods either through fermentation or the use of weak acids has been used as a food preservation method since ancient times (FDA, 2013). Meats can resist pH changes better than other foods such as vegetables because of the buffering ability of meat (Jay, Loessner, & Golden, 2006). The protein in meat contributes to the buffering capacity of the meat (Jay, Loessner, & Golden, 2006). Nutrient Availability Bacteria need five nutrients in order to grown and function normally: water, a source of energy, a source of nitrogen, vitamins, and minerals (Jay, Loessner, & Golden, 2006). An organism that requires a vitamin that the organism cannot synthesize will not grow (Nester, Anderson, Roberts, Pearsall, & Nester, 2001). Nutrients vary among foods. For example, meats have high levels of proteins, lipids, minerals, and vitamins with low levels of carbohydrates; most vegetables are high in carbohydrates, but have varying levels of proteins, vitamins, and minerals (FDA, 2013). In general, Gram positive 4 microorganism have more stringent nutrient requirements than Gram negative microorganism as Gram positive microbes are less likely to be able to synthesize required nutrients not in the environment (Jay, Loessner, & Golden, 2006). Gram negative bacteria are more likely to receive the nutrients they need from the food environment they are in (Jay, Loessner, & Golden, 2006). The most predominate microorganisms found in food are those that can use the nutrients available in the food source (FDA, 2013). Biological Structures Some foods have biological structures that may prevent the entry and growth of pathogens (FDA, 2013). Rinds, shells, and other coverings provide protection from some bacteria (Nester, Anderson, Roberts, Pearsall, & Nester, 2001). However, if the protective covering becomes damaged the covering will no longer be protective (Jay, Loessner, & Golden, 2006). In the case of meats, the hide of the animal protects the muscle and then the outside surface of an intact piece of meat protects the inner meat (Jay, Loessner, & Golden, 2006). These barriers will be destroyed once the meat is cut, chopped or ground; thus, allowing for bacteria to gain access to the interior of the meat (FDA, 2013). Oxidation-Reduction Potential Oxidation-reduction potential (Eh) is defined as the ratio of the total oxidizing (electron accepting) power to the total reducing (electron donating) power of the substance (FDA, 2013). Aerobic microorganisms require positive (oxidized) Eh values, anaerobic microorganisms require negative (reduced) Eh values, and facultative anaerobic bacteria can survive and grow in either condition (Jay, Loessner, & Golden, 5 2006). Raw, post-rigor muscle has an Eh of -60 to -150 mV and cooked sausages and canned meat have an Eh of -20 to -150 mV (FDA, 2013). Naturally Occurring Antimicrobials Some foods naturally contain antimicrobials such as lysozyme in egg whites (Nester, Anderson, Roberts, Pearsall, & Nester, 2001). Food processing techniques, such as smoking, can form antimicrobial compounds on the surface of the meat (Mossel, Corry, Struijk, & Baird, 1996). Phenol is found in smoke condensate and is not only an antimicrobial, but also lowers the pH (FDA, 2013). Extrinsic Factors Affecting Microorganisms Storage Temperatures All microorganisms have a defined temperature range in which they grow (FDA, 2013). The range of select pathogens can be found in Table 1. Microbes can be divided into four groups depending on the optimum temperature in which they grow and thrive: psychrotrophs, psychrophiles, mesophiles, and thermophiles (FDA, 2013). Almost all human pathogens are included in the mesophile group (FDA, 2013). When determining storage temperatures, the quality of the food must be kept in mind (Jay, Loessner, & Golden, 2006). Mesophilic bacteria can be inhibited by the use of cold storage; however, those same cold temperatures will facilitate the growth of psychrotrophic organisms. Small temperature changes can change the microbial profile of meat (Sun & Holley, 2012). 6 Atmosphere Composition Some gasses, such as carbon dioxide (CO2), ozone (O3), and oxygen (O2), are toxic to certain pathogens (FDA, 2013). Incorporating these gases into the packaging of the food can provide an antimicrobial affect (Nester, Anderson, Roberts, Pearsall, & Nester, 2001; Gill & Newton, 1978). Technologies commonly used to control the atmosphere of the food storage environment include modified atmosphere packaging (MAP), controlled atmosphere packaging (CAP), controlled atmosphere storage (CAS), direct addition of carbon dioxide (DAC), and hypobaric storage (Loss & Hotchkiss, 2001). MAP is most commonly used in the meat processing industry (FDA, 2013). Common compositions of gasses used in MAP can be found in Table 2 for select food types. Another common packaging type is vacuum packaging which restricts the O2 levels and allows for CO2 levels of about 20%, largely inhibiting the growth of Gram negative aerobes (Gill & Newton, 1978). Microorganisms Escherichia coli Escherichia coli is a Gram-negative non-spore forming short rod. There are both non-pathogenic and pathogenic E. coli groups. There are six pathogenic groups enterotoxigenic (ETEC), enteropathogenic (EPEC), enterohemorrhagic (EHEC), enteroinvasive (EIEC), enteroaggregative (EAEC), and diffusely adherent (DAEC). While all six strains are pathogenic, EHEC organisms are most commonly associated with foodborne outbreaks (FDA, 2012). 7 While about 75% of all E. coli foodborne infections world-wide are caused by O157:H7 there is another group of non-O157 E. coli serotypes that is currently being tested for in the United States. The ?big 6? non-O157 are O26, O45, O103, O111, O121, and O145. The ?big 6? account for 71% of the non-O157:H7 infections; however, there are other infectious serotypes that can result in illness (Brooks, et al., 2005). EHEC has a very low infectious dose of 10 to 100 cells (FDA, 2012). Symptoms of EHEC infection, developing after an incubation period of 2 hours to 6 days, can include nausea, abdominal cramps, vomiting, and watery or bloody diarrhea (Nester, Anderson, Roberts, Pearsall, & Nester, 2001). In some extreme cases, hemorrhagic colitis can progress to hemolytic uremic syndrome (HUS) 3%-7% of the time. Patients with HUS have a mortality rate of 4%-5% (FDA, 2012). E. coli O157:H7 results in 4% of domestically acquired foodborne illnesses that result in hospitalization (CDC, 2011). While most infections continue to be a result of ground beef or beef products, there is an increasing amount of produce products (FDA, 2012). There are about 63,153 cases of O157:H7 EHEC and about 112,752 cases of non- O157 EHEC infections annually (Scallan, et al., 2011). The largest meat recall associated with EHEC to date was in 1997 when Hudson Foods recalled six lots of frozen ground beef patties and burgers (CDC, 1997). Fifteen individual patients were determined to be affected by contaminate meat from Hudson Foods; five were hospitalized, but none developed HUS (CDC, 1997). The most recent meat related EHEC outbreak was attributed to Lebanon bologna in 2011 (CDC, 2011b). Fourteen persons from five states 8 were infected with the outbreak strain; none developed HUS, but three were hospitalized (CDC, 2011b). The last reported case of HUS associated with a meat product was in 2010 when beef non-intact steaks were recalled (CDC, 2010). Salmonella spp. Salmonella is a Gram-negative, non-spore forming, motile rod. S. enterica and S. bongori are the two species that cause illness in humans. There are six subspecies of S. enterica. The most common subspecies is S. enterica subsp. enterica. Serotypes within the subspecies include Enteritidis and Typhimurium ? two of the most common serotypes in the United States out of the 2,579 identified (FDA, 2012). Nontyphoidal salmonellosis and typhoid fever (only caused by S. Typhi or S. Paratyphi A.) are the two types of illnesses that can be caused by Salmonella infection. Nontyphoidal salmonellosis can have an infectious dose of one cell while typhoid fever?s infectious dose is fewer than 1,000 cells. Mortality rates of nontyphoidal salmonellosis range from less than 1% to about 3.6%; however, untreated typhoid fever can have a mortality rate of up to 10% (FDA, 2012). Salmonella is found widely in the environment and in the intestines of many animals. However, S. Typhi and S. Paratyphi A. are only found in human hosts. Salmonella illnesses have been linked to meats, poultry, and poultry products; as well as, peanut butter, cocoa, and produce (FDA, 2012). There are an estimated 1,027,561 9 nontyphoidal (foodborne) salmonellosis cases and 1,821 typhoid fever cases annually (Scallan, et al., 2011). In foodborne cases, the incubation period before symptoms begin is 6 to 72 hours. Symptoms of salmonellosis most often include diarrhea and vomiting. Less common symptoms are a prolonged fever, headache, abdominal pain, abscesses, and shock (Nester, Anderson, Roberts, Pearsall, & Nester, 2001). In 2007, 401 reported cases of salmonellosis with 338 hospitalizations were linked to frozen pot pies (CDC, 2008). ConAgra Foods, Inc. recalled 9 brands of pot pies and labeling issues were addressed highlighting the need to thoroughly cook not-ready-to-eat frozen foods (CDC, 2008). Salmonella spp. contamination in beef products is becoming an important issue. Cargill Meat Solutions recalled about 29,000 pounds of fresh ground beef in 2012 due to Salmonella Enteritidis infections (CDC, 2012). In total, 46 cases were reported in 9 states resulting in 12 hospitalizations (CDC, 2012). The most recent salmonellosis outbreak associated with poultry was in January 2014 when 9 reported cases in Tennessee were linked to Tyson brand mechanically separated chicken (CDC, 2014). Listeria monocytogenes Listeria monocytogenes is a Gram-positive, motile rod. There are thirteen serotypes, of which, three have been associated with the largest amount of foodborne illnesses. L. monocytogenes is both salt and cold tolerant and is widely found within the environment (FDA, 2012). Across organisms, L. monocytogenes is not a leading cause of illness, but it is a leading cause of death from foodborne illness (Scallan, et al., 2011). 10 The CDC estimates 1,591 cases of foodborne illness resulting from L. monocytogenes with about 255 resulting in death (Scallan, et al., 2011). The infectious dose is unknown, but estimated to be less than 1,000 cells. L. monocytogenes infection occurs more often in pregnant women than other populations. While the women generally have mild flu-like symptoms the infection results in spontaneous abortions and stillbirths one third of the time (FDA, 2012). Many foods have been associated with L. monocytogenes outbreaks including raw and ready-to-eat meats, dairy, dairy products, and produce. The ability to grow and thrive at refrigeration temperatures creates a unique problem for the food industry (FDA, 2012). The most recent listeriosis outbreak associated with meat products was in 2002 when Pilgram?s Pride recalled 27.4 million pounds of fresh and frozen ready-to-eat turkey and chicken products. Forty-six confirmed cases (including 7 deaths and 3 stillbirths or miscarriages) were linked to the contaminated products (CDC, 2002). Preventing listeriosis requires interventions in all stages of the food chain from the processing facility to the home (Lianou, et al., 2007). Topical Treatments Lactic Acid Lactic acid is a ?Generally Recognized As Safe? (GRAS) food additive commonly used in the meat industry. Lactic acid is an organic acid that has been used on abattoirs as a hot carcass rinse (Huffman, 2002). Lactic acid has been used at 1 to 2% to 11 decontaminate red meat carcasses without affecting meat quality (Theron & Lues, 2007). When used at high temperatures (>60 ?C) lactic acid has been proven to control pathogenic bacteria on carcasses (Theron & Lues, 2007). Organic acids, such as lactic acid, cause bactericidal and bacteriostatic results by reducing the pH of the substrate to lower the internal cellular pH that then disrupts the cell membrane (Chung & Murdock, 1991). Spraying or dipping cured meats post-processing in organic acids has been proven to reduce L. monocytogenes (Theron & Lues, 2007). Lactic acid at 2% has been found to reduce E. coli O157:H7 and Salmonella Typhimurium 3.54 and 4.68 CFU/cm2, respectively (Yoder, et al., 2012). Gill and Badoni (2004) found that 4% lactic acid reduced bacteria by >1 log unit compared to a water treatment. Lactic acid?s acceptable limit for use in products is the highest level that will not negatively impact sensory characteristics; however, higher acid concentrations can affect the buffering ability of the meat (Smulders, Barendsen, Vanlogtestijn, Mossel, & Vanermarel, 1986). The use of lactic acid use in other areas of meat processing has not been widely explored. Lactic acid (2%) has been showed to reduce the amount of surface bacteria on turkey rolls (L. monocytogenes), pork bellies (Salmonella), and chicken skins (Salmonella), but on not beef plates (E. coli O157:H7) (Carpenter, Smith, & Broadbent, 2011). Relatively high levels of E. coli can be reduced on cuts or trimmings with 5% lactic acid at ? 0.1 ml/cm2 by between 0.5 and 1 log units (Youssef, Yang, Badoni, & Gill, 2012). Lactic acid (4%) applied as a spray effectively reduced both non-O157 and O157:H7 STEC when beef flanks were inoculated at approximately 104 CFU/cm2 (Kalchayanand, et al., 2012). 12 When utilizing meat brines, adding 3.3% lactic acid has been shown to decrease E. coli O157:H7 during storage (Adler, et al., 2011). Stivarius et al. (2002) found that 5% lactic acid applied to beef trimmings before grinding reduced E. coli, coliforms, and aerobic plate count, but not Salmonella Typhimurium. Concentration and temperature of lactic acid have an affect on L. monocytogenes when applied to frankfurters (Byelashov, et al., 2010). Other Organic Acids Aside from lactic acid, there are many other organic acids currently being used in the food industry. Acetic acid, acetates, diacetates, and dehyroacetic acid are primarily used in dairy products and meats to target yeasts and bacteria; sodium propionate is commonly used in meat products to target molds (Mani-Lopez, Garcia, & Lopez-Malo, 2011). Other products utilized in meat and meat products as food preservatives are propionic, citric, and benzoic acids (Theron & Lues, 2007). Acetic acid, the principal ingredient of vinegar, has a pungent odor and flavor that limits its use in foods; many pickled products include acetic acid, acetates, or diacetates (Mani-Lopez, Garcia, & Lopez-Malo, 2011). As a beef carcass wash, fumaric acid has been shown to be effective against L. monocytogenes alone and in combination with lactic and acetic acids (Podolak, Zayas, Kastner, & Fung, 1995a). Citric acid and citrates are commonly used in the poultry industry in chill tanks to control Salmonella spp. (Mani-Lopez, Garcia, & Lopez- Malo, 2011). Other organic acids with similar uses that are not as commonly utilized include malic, propionic, and tartaric acids (Mani-Lopez, Garcia, & Lopez-Malo, 2011). Fumaric, acetic, and lactic acids have all proved to reduce E. coli O157:H7, Salmonella 13 Typhimurium, and L. monocytogenes counts on beef lean muscle over time (Podolak, Zayas, Kastner, & Fung, 1995a; Podolak, Zayas, Kastner, & Fung, 1995b). Spray application of peroxyacetic acid did not effectively reduce E. coli O111, but it did reduce other STEC strains when inoculated at about 104 CFU/cm2 (Kalchayanand, et al., 2012). The same study found that under the same inoculation conditions acidified sodium chlorite did not reduce E. coli O26, O111, or O145 serogroups (Kalchayanand, et al., 2012). In general, organic acids are more effective at reducing bacteria than hot water, but the discoloration and off-odor properties that organic acid use can lead to are main concerns when determining which acids and concentrations should be utilized (Sun & Holley, 2012) Sodium Metasilicate Very little research has been performed on the use of sodium metasilicate on meat and meat products. This area has previously been unexplored especially in regards to pork and processed meats. Sodium metasilicate is approved for antimicrobial use in ready-to-eat meat and poultry products up to 6% (USDA, 2013). It is an alkaline solution that has proven to be effective in reducing Gram-negative bacteria from the surface of meat and meat products (Carlson, et al., 2008; Pohlman, Dias-Morse, & Rajaratnam, 2005; Pohlman, et al., 2009; Weber, et al., 2004). Sodium metasilicate acts on the cytoplasmic membrane and causes the cells to lyse (Sharma, Williams, Schneider, Scmidt, & Rodrick, 2013b). Little research has been conducted on the effectiveness of sodium metasilicate on Gram-positive bacteria, but one in vitro study found that sodium metasilicate at 1, 2, or 3% reduced L. monocytogenes by >5 logs after a 30 min exposure 14 time (Sharma, Williams, Schneider, Schmidt, & Rodrick, 2012a). Sharma et al. (2012a) also discovered that applying 4% sodium metasilicate to a culture of L. monocytogenes with a 30 min exposure time resulted in an undetectable amount of L. monocytogenes. Sodium metasilicate has been explored as a treatment for fresh beef trimmings before grinding. Geornaras et al. (2012b) found that 4% sodium metasilicate reduced E. coli O157:H7 and multidrug-resistant and antibiotic susceptible Salmonella by 1.3-1.5 log CFU/cm2 when applied as a dip to beef trimmings. Sodium metasilicate (4%) applied to beef trimmings before grind has been shown to reduce coliforms, E. coli, aerobic plate counts, and Salmonella over 7 days of storage (Pohlman, et al., 2009). One study explored the ability of sodium metasilicate to reduce E. coli and Salmonella typhimurium when used as a dip on beef trimmings before grinding and discovered that both species were decreased; however, the bacteria were inoculated in the same solution and some reduction could be the result of organisms outcompeting each other (Pohlman, F. W., Dias-Morse, P. N., & Rajaratnam, G., 2005). When used as part of a multi-hurdle program, 4% sodium metasilicate in combination with 3% potassium lactate or 200-ppm peroxyacetic acid did not reduce E. coli or Salmonella typhimurium when applied to beef trimming before grinding (Quilo, et al., 2010). However, 4% sodium metasilicate in combination with 3% potassium lactate or 200-ppm peroxyacetic acid improved or maintained ground beef odor when applied to beef trimmings before grinding, and 4% sodium metasilicate in combination with 200-ppm peroxyacetic acid enhanced beef color when applied to beef trimming before grinding (Quilo, et al., 2010). Sodium metasilicate at 2.2% has shown the ability to reduce E. coli O157:H7 in brines both immediately and throughout storage (Adler, et al., 2011). While being effective against microorganisms 15 sodium metasilicate doesn?t affect meat quality (Quilo, et al., 2009). Other Alkaline Solutions Several alkaline solutions are used to reduce the risk of pathogens in the meat industry. Potassium hydroxide, sodium hypochlorite, and sodium hydroxide have been used as food surface cleaners (Sharma & Beuchat, 2004). Sharma & Beuchat (2004) discovered that high pH and chlorine combined with sodium hydroxide or potassium hydroxide add to the bactericidal effectiveness of alkaline cleaners. Sodium hypochlorite has been used to reduce E. coli, Campylobacter, and Salmonella from broiler carcasses after spray washing (Northcutt, Smith, Ingram, Hinton, & Musgrove, 2006). Using 0.1% ammonium hydroxide in a brine solution was less effective on total aerobic and anaerobic bacteria than 4.5% sodium tripolyphosphate (Parsons, VanOverbeke, Goad, & Mireles DeWitt, 2010); however, in a similar study the opposite was reported (Cerruto-Noya, VanOverbeke, & Mireles DeWitt, 2009). Research Objectives There are many different antimicrobial solutions that are currently being used in food applications. Expanding the use of these solutions may decrease foodborne pathogenic bacteria. Identifying cross-functional solutions that can be used in the meat industry may increase the safety of the meat supply. The effectiveness of lactic acid and sodium metasilicate will depend on the concentration, temperature, and whether the solutions are applied separately or in combination. While other researchers have investigated similar topics, those studies did not investigate individual inoculation of 16 microorganisms, fresh beef or pork steaks, or deli meats. Therefore, the objectives of this research addressed those concerns. The objective of the first study was to determine the optimal application concentration and temperature of lactic acid and sodium metasilicate for bactericidal reduction of inoculated pathogens. Each of these antimicrobials has been used at a variety of concentrations. Lactic acid was analyzed at 1, 2, 3, and 4% (LA1, LA2, LA3, and LA4) and sodium metasilicate at 2, 3, 4, and 5% (SM2, SM3, SM4, and SM5). Concentrations were determined based on current industry use and United States Department of Agriculture Food Safety and Inspection Service Directive 7120.1 revision 15 (USDA, 2013). Temperature of application may change the effectiveness. Temperatures of 4, 25, and 60 ?C were utilized. Temperatures were selected to represent meat processing (4 ?C), room (25 ?C), and warm (60 ?C) temperatures. The optimal concentration of LA, SM, and their combination (LASM) were utilized. The objective of the third study was to determine the bactericidal reduction of inoculated pathogens of the optimal concentration and temperature of LA, SM, and LASM on fresh beef, pork, and deli meats (roast beef, ham, and turkey). 17 Table 1. Selected intrinsic factors affecting chosen pathogen growth. Microorganism Approximate aw values for growth of selected pathogens in food Approximate pH values allowing the growth of selected pathogens in food Approximate temperatures allowing growth of selected pathogens (?C) Minimum Optimum Maximum Minimum Optimum Maximum Minimum Optimum Maximum Enterohemorrhagic Escherichia coli 0.95 0.99 4.4 6.0 ? 7.0 9.0 7 35 ? 40 46 Salmonella spp. 0.94 0.99 > 0.99 4.2a 7.0 ? 7.5 9.5 5 35 ? 37 45 ? 47 Listeria monocytogenes 0.92 4.39 7.0 9.4 0 30 ? 37 45 Sources: Tables 3-2, 3-5, 3-10; FDA 2013 a pH minimum as low as 3.8 has been reported when acidulants other than acetic acid or equivalent are used. 18 Table 2. Examples of gas mixtures used for selected MAP products. Product % Carbon Dioxide % Oxygen % Nitrogen Fresh Meat 30 30 30 15 ? 40 60 ? 85 0 Cured Meat 20 ? 50 0 50 ? 80 Sliced Cooked Roast Beef 75 10 15 Processed Meats 0 0 100 Source: Table 3-8; FDA, 2013 19 References Adler, J., Geornaras, I., Byelashov, O., Belk, K., Smith, G., & Sofos, J. (2011). Survival of Escherichia coli O157:H7 in meat product brines containing antimicrobials. Journal of Food Science, 76(7), M478-485. Brooks, J. T., Sowers, E. G., Wells, J. G., Greene, K. D., Griffin, P. M., Hoekstra, R. M., Strockbine, N. A. (2005). Non-O157 shiga-toxin producing Escherichia coli infections in the United States, 1983-2002. Journal of Infectious Disease, 192, 1422-1429. Byelashov, O., Daskalov, H., Geornaras, I., Kendall, P., Belk, K., Scanga, J., Smith, G., & Sofos, J. (2010). Reduction of Listeria monocytogenes on frankfurters treated with lactic acid solutions of various temperatures. Food Microbiology, 27(6), 783- 790. Carlson, B., Ruby, J., Smith, G., Sofos, J., Bellinger, G., Warren Serna, W., Centrella, B., Bowling, R., & Belk, K. (2008). Comparison of antimicrobial efficacy of multiple beef hide decontamination strategies to reduce levels of Escherichia coli O157:H7 and Salmonella. Journal of Food Protection, 71(11), 2223-2227. Carpenter, C. E., Smith, J. V., & Broadbent, J. R. (2011). Efficacy of washing meat surfaces with 2% levulinic, acetic, or lactic acid for pathogen decontamination and residual growth inhibition. Meat Science, 88(2), 256-260. CDC. (2008). Escherichia coli O157:H7 infections associated with eating a nationally distributed commercial brand of frozen ground beef patties and burgers ? Colorado, 1997. Morbidity and Mortality Weekly Reports. 46(33): 777-778. CDC. (2002). Public health dispatch: outbreak of listeriosis ? Northeastern United States, 2002. Morbidity and Mortality Weekly Reports. 51(42): 950-951. CDC. (2008). Multistate outbreak of Salmonella infections associated with frozen pot pies ? United States, 2007. Morbidity and Mortality Weekly Reports. 57(47): 1277-1280. CDC. (2010). Multistate outbreak of E. coli O157:H7 infections associated with beef from National Steak and Poultry (final update). Access Date March 19, 2014. http://www.cdc.gov/ecoli/2010/0105.html. CDC. (2011a). CDC Estimates of foodborne illness in the United States. Access Date January 3, 2014. 20 http://www.cdc.gov/foodborneburden/PDFs/FACTSHEET_A_FINDINGS_updat ed4-13.pdf. CDC. (2011b). Investigation announcement: multistate outbreak of E. coli O157:H7 infections associated with Lebanon bologna. Access Date March 19, 2014. http://www.cdc.gov/ecoli/2011/O157_0311/index.html. CDC. (2012). Multistate outbreak of Salmonella Enteritidis infections linked to ground beef (final update). Access Date March 19, 2014. http://www.cdc.gov/salmonella/enteritidis-07-12/index.html. CDC. (2014). Outbreak of Salmonella Heidelberg infections linked to Tyson brand mechanically separated chicken at a correctional facility (final update). Access Date March 19, 2014. http://www.cdc.gov/salmonella/heidelberg-01- 14/index.html. Cerruto-Noya, C. A., VanOverbeke, D. L., & DeWitt, Mireles DeWitt, C. A. (2009). Evaluation of 0.1% ammonium hydroxide to replace sodium tripolyphosphate in fresh meat injection brines. Journal of Food Science, 74(7), C519-C525. Chung, K-T. & Murdock, C. A. (1991). Natural systems for preventing contamination and growth of microorganisms in foods. Food Structure, 10, 361-374. FDA. (2012). Bad bug book, foodborne pathogenic microorganisms and natural toxins. (Second ed.). Access Date January 3, 2014. http://www.fda.gov/downloads/food/foodborneillnesscontaminants/ucm297627.p df. FDA. (2013). Evaluation and definition of potentially hazardous foods ? chapter 3. Factors that influence microbial growth. Access Date March 19, 2014. http://www.fda.gov/Food/FoodScienceResearch/SafePracticesforFoodProcesses/u cm094145.htm. Geornaras, I., Yang, H., Moschonas, G., Nunnelly, M. C., Belk, K. E., Nightingale, K. K., Woerner, D. R., Smith, G. C., & Sofos, J. N. (2012b). Efficacy of chemical interventions against Escherichia coli O157:H7 and multi-drug resistant and antibiotic-susceptible Salmonella on inoculated beef trimmings. Journal of Food Protection, 75(11), 1960-1967. Gill, C. O., & Badoni, M. (2004). Effects of peroxyacetic acid, acidified sodium chlorite or lactic acid solutions on the microflora of chilled beef carcasses. International Journal of Food Microbiology, 91(1), 43-50. Gill, C. O. & Newton, K. G. (1978). The ecology of bacterial spoilage of fresh meat at chill temperatures. Meat Science, 2, 207-217. Huffman, R. D. (2002). Current and future technologies for the decontamination of carcasses and fresh meat. Meat Science, 62(3), 285-294. 21 Jay, J. M., Loessner, M. J., & Golden, D. A. (2006). Modern food microbiology. (7th ed.) New York. Springer, (Chapter 3). Kalchayanand, N., Arthur, T. M., Bosilevac, J. M., Schmidt, J. W., Wang, R., Shackelford, S. D., & Wheeler, T. L. (2012). Evaluation of commonly used antimicrobial interventions for fresh beef inoculated with shiga toxin-producing Escherichia coli serotypes O26, O45, O103, O111, O121, O145, and O157:H7. Journal of Food Protection, 75(7), 1207-1212. Loss, C. R. & Hotchkiss, J. H. (2002). Inhibition of microbial growth by low-pressure and ambient pressure gasses. In V. K. Juneja & J. N. Sofos (Eds.), Control of foodborne microorganisms (pp. 245-279). New York: Marcel Dekker. Lianou, A., Geornaras, I., Kendall, P. A., Belk, K. E., Scanga, J. A., Smith, G. C., & Sofos, J. N. (2007). Fate of Listeria monocytogenes in commercial ham, formulated with or wihout antimicrobials, under conditions simulating contamination in the processing or retail environment and during home storage. Journal of Food Protection, 70(2), 378-385. Mani-Lopez, E., Garcia, H. S., & Lopez-Malo, A. L. (2012). Organic acids as antimicrobials to control Salmonella in meat and poultry products. Food Research International, (45, 713-721. Mossel, D. A. A., Corry, J. E. L., Struijk, C. B., & Baird, R. M. (1996). Essentials of the microbiology of foods: a textbook for advanced studies. Chichester (England): John Wiley and Sons. Nester, E. W., Anderson, D. G., Roberts Jr., C. E., Pearsall, N. N., & Nester, M. T. (2001). Microbiology a human perspective. New York: McGraw-Hill. Northcutt, J., Smith, D., Ingram, K. D., Hinton Jr., A., & Musgrove, M. (2007). Recovery of bacteria from broiler carcasses after spray washing with acidified electrolyzed water of sodium hypochlorite solutions. Poultry Science, 86, 2239-2244. Parsons, A. N., VanOverbeke, D. L., & Mireles DeWitt, C. A. (2011). Retail display evaluation of steaks from select beef strip loins injected with a brine containing 1% ammonium hydroxide. part 1: fluid Loss, oxidation, color, and microbial plate counts. Journal of Food Science, 76(1), S63-S71. Podolak, R. K., Zayas, J. F., Kastner, C. L., Fung, D. Y. C. (1995a). Inhibition of Listeria monocytogenes and Escherichia coli O157:H7 on beef by application of organic acids. Journal of Food Protection, 59(4), 370-373. Podolak, R. K., Zayas, J. F., Kastner, C. L., Fung, D. Y. C. (1995b). Reduction of Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella Typhimurium during storage on beef sanitized with fumaric, acetic, and lactic acids. Journal of Food Safety, 15, 283-290. 22 Pohlman, F. W., Dias-Morse, P. N., & Rajaratnam, G. (2005). Microbial characteristics of ground beef treated with potassium lactate, sodium metasilicate, peroxyacetic acid, and acidified sodium chlorite. Arkansas Animal Science Department Report, 2005 (AAES Research Series 535), 142-144. Pohlman, F. W., Dias-Morse, P. N., Quilo, S. A., Brown, A. H., Crandall, P. G., Baublits, R. T., Story, R. P., Bokina, C., & Rajaratnam, G. (2009). Microbial, instrumental color and sensory characteristics of ground beef processed from beef trimmings treated with potassium lactate, sodium metasilicate, peroxyacetic acid or acidified sodium chlorite as single antimicrobial interventions. Journal of Muscle Foods, 20(1), 54-69. Quilo, S. A., Pohlman, F. W., Dias-Morse, P. N., Brown, A. H., Crandall, P. G., Dias Morse, P. N., & Story, R. P. (2010). Microbial, instrumental color and sensory characteristics of inoculated ground beef produced using potassium lactate, sodium metasilicate or peroxyacetic acid as multiple antimicrobial interventions. Meat Science, 84(3), 470-476. Scallan, E., Hoekstra, R. M., Angulo, F. J., Tauxe, R. V., Widdowson, M. A., Roy, S. L., Jones, J. L., & Griffin, P. M. (2011). Foodborne illness acquired in the United States?major pathogens. Emerging Infectious Diseases, 17(1), 7-15. Sharma, C., Williams, S., Schneider, K., Schmidt, R., & Rodrick, G. (2012a). Antimicrobial effects of sodium metasilicate against Listeria monocytogenes. Foodborne Pathogens and Disease, 9(9), 822-828. Sharma, C. S., Williams, S. K., Schneider, K. R., Schmidt, R. H., & Rodrick, G. E. (2013). Mechanism of antimicrobial action of sodium metasilicate against Salmonella enterica serovar Typhimurium. Foodborne Pathogens and Disease, 10, 995-1001. Sharma, M. & Beuchat, L. R. (2004). Sensitivity of Escherichia coli O157:H7 to commercially available alkaline cleaners and subsequent resistance to heat and sanitizers. Applied and Environmental Microbiology, 70(3), 1795-1803. Smulders, F. J. M., Barendsen, P., Vanlogtestijn, J. G., Mossel, D. A. A., & Vandermarel, G. M. (1986). Review ? lactic acid - considerations in favor of its acceptance as a meat decontaminant. Journal of Food Technology, 21(4), 419-436. Stivarius, M. R., Pohlman, F. W., McElyea, K. S., & Waldroup, A. L. (2002). Effects of hot water and lactic acid treatment of beef trimmings prior to grinding on microbial, instrumental color and sensory properties of ground beef during display. Meat Science, 60(4), 327-334. Sun, X. D. & Holley, R. A. (2012). Antimicrobial and antioxidative strategies to reduce pathogens and extend the shelf life of fresh red meats. Comprehensive Reviews in Food Science and Food Safety, 11, 340-354. 23 Theron, M. M., & Lues, J. F. R. (2007). Organic acids and meat preservation: a review. Food Reviews International, 23(2), 141-158. USDA, F. S. I. S. (2003). Control of Listeria monocytogenes in ready-to-eat meat and poultry products; final rule. Federal Register, 68, 34208-34254. USDA, F. S. I. S. (2011). Shiga toxin-producing Escherichia coli in certain raw beef products. Federal Register, 76(182), 58157-58165. USDA, F. S. I. S. (2013). Safe and suitable ingredients used in the production of meat, poultry, and egg products. Access Date January 3, 2014. http://www.fsis.usda.gov/OPPDE/rdad/FSISDirectives/7120.1.pdf. Weber, G., O'Brien, J., & Bender, F. (2004). Control of Escherichia coli O157:H7 with sodium metasilicate. Journal of Food Protection, 67(7), 1501-1506. Yoder, S., Henning, W., Mills, E., Doores, S., Ostiguy, N., & Cutter, C. (2012). Investigation of chemical rinses suitable for very small meat plants to reduce pathogens on beef surfaces. Journal of Food Protection, 75(1), 14-21. Youssef, M. K., Yang, X., Badoni, M., & Gill, C. O. (2012). Effects of spray volume, type of surface tissue and inoculum level on the survival of Escherichia coli on beef sprayed with 5% lactic acid. Food Control, 25(2), 717-722. Zhou, G. H., Xu, X. L., & Liu, Y. (2010). Preservation technologies for fresh meat - a review. Meat Science, 86(1), 119-128. 24 Chapter II Evaluation of Multiple Concentrations and Temperatures of Lactic Acid and Sodium Metasilicate against Pathogens of Concern on Fresh Beef Abstract Lactic acid (LA) has been widely used in abattoirs as an antimicrobial spray for carcass intervention. Sodium metasilicate (SM) has been approved for use on carcasses, trimmings, and ready to eat products. Each of these antimicrobials has been used at a variety of concentrations and temperatures. Utilizing these antimicrobials in different stages during meat processing may assist in the reduction of the risk of pathogenic microorganisms. The first purpose of this study was to determine optimum concentrations of usage of lactic acid and sodium metasilicate for pathogen reduction on beef bottom round muscles. Lactic acid was applied at 1, 2, 3, and 4% (LA1, LA2, LA3, and LA4) and sodium metasilicate was applied at 2, 3, 4, and 5% (SM2, SM3, SM4, and SM5). The second purpose of this study was to determine optimum temperatures of usage of lactic acid and sodium metasilicate for pathogen reduction on beef bottom round muscles. Lactic acid 4% (LA, v/v), sodium metasilicate 4% (SM, w/v), the combination of the two solutions (LASM), and a distilled water control were applied at 4, 25, and 60 ?C. Antimicrobials were mixed into solution with distilled water. Beef bottom round was cut into 100 cm2 pieces. Pieces were then inoculated with Escherichia coli O157:H7 (5 strains), non-O157 shiga-toxin producing Escherichia coli (STEC, 1 strain each of the 25 ?Big 6?), Salmonella spp. (5 strains), or Listeria monocytogenes (5 strains). After 30 min of contact time, samples were treated with the antimicrobial solution or control and then allowed 30 min of contact time. Samples were serially diluted and plated on MacConkey Agar with Sorbitol (E. coli), XLT4 (Salmonella spp.), or Modified Oxford Medium (L. monocytogenes). Data were analyzed using the PROC MIXED procedure of SAS and Tukey pairwise comparisons. For all microorganisms, increasing the concentration of lactic acid or sodium metasilicate increased the effectiveness of the treatment. SM4 and LA4 were determined to be the lowest concentrations most effective against all microorganisms tested in this study. By utilizing the lowest concentration of antimicrobial solution necessary to achieve effective pathogen reduction, meat processors can provide a safe and wholesome meat supply. LA and SM reduced (P<0.05) the microbial contamination of the meat samples. Temperature of application had no effect (P>0.05) on bacterial counts in any of the treatments. LA or SM alone were more effective (P<0.05) in reduction of microbes than when used together (LASM). Meat processors can apply LA or SM at refrigeration temperatures and gain the same benefits of applying them at a higher temperature. Both solutions can serve as a hurdle technology in meat processing facilities. Introduction Food safety is a constant concern in the meat industry and consideration is given to methods of ensuring a safe food supply by reduction of pathogens. The Centers for Disease Control and Prevention (CDC) estimates that 48 million (1 in 6) Americans become ill each year due to foodborne illness. Of these, 128,000 are hospitalized and 26 about 3,000 die of foodborne diseases (Weber, O?Brien, & Bender, 2004). Salmonella spp., E. coli, and L. monocytogenes are common pathogens of concern in fresh and processed meats on the ?top five pathogens? lists compiled by the CDC (Weber, O?Brien, & Bender, 2004). Escherichia coli is a Gram-negative non-spore forming short rod. While about 75% of all E. coli foodborne infections world-wide are caused by O157:H7 there is another group of non-O157 E. coli serotypes that is currently being tested for in the United States. The ?big 6? non-O157 are O26, O45, O103, O111, O121, and O145. The ?big 6? account for most of the non-O157:H7 foodborne infections (FDA, 2012). E. coli O157:H7 results in 4% of domestically acquired foodborne illnesses that result in hospitalization (CDC, 2011a). While most infections continue to be a result of ground beef or beef products, there is an increasing amount of produce that has been implicated sporeforming, motile rod. Salmonella illnesses have been linked to meats, poultry, and poultry products; as well as, peanut butter, cocoa, and produce (FDA, 2012). There are an estimated 1,027,561 nontyphoidal salmonellosis cases and 1,821 typhoid fever cases annually (Scallan, et al., 2011). Nontyphoidal salmonellosis and typhoid fever (only caused by S. Typhi or S. Paratyphi A.) are the two types of illnesses that can be caused by Salmonella infection. Salmonella illnesses have been linked to meats, poultry, and poultry products; as well as, peanut butter, cocoa, and produce (FDA, 2012). Listeria monocytogenes is a Gram-positive, motile rod. Across organisms, L. monocytogenes is not a leading cause of illness, but it is a leading cause of death from foodborne illness (Scallan, et al., 2011). L. monocytogenes is both salt and cold tolerant and is widely found 27 within the environment (FDA, 2012). Many foods have been associated with L. monocytogenes outbreaks including raw and ready-to-eat meats, dairy, and dairy products. The ability of L. monocytogenes to grow and thrive at refrigeration temperatures creates a unique problem for the food industry (FDA, 2012). Lactic acid is a ?Generally Recognized As Safe? (GRAS) food additive commonly used in the meat industry. Lactic acid is an organic acid that has been used in abattoirs as a hot carcass rinse (Huffman, 2002). Lactic acid has been used at 1 to 2% to decontaminate red meat carcasses without affecting meat quality (Theron & Lues, 2007). Lactic acid at 2% has been found to reduce E. coli O157:H7 and Salmonella Typhimurium by 3.54 and 4.68 CFU/cm2, respectively (Yoder, et al., 2012). When used at high temperatures (>60 ?C) lactic acid has controlled pathogenic bacteria on carcasses (Theron & Lues, 2007). Gill and Badoni (2004) found that 4% lactic acid reduced bacteria by >1 log unit compared to a water treatment. Lactic acid effectiveness is impacted by concentration and lactic acid temperature on L. monocytogenes when applied to frankfurters (Byelashov, et al., 2010). There is very little research on the use of sodium metasilicate on meat and meat products as an antimicrobial. This is an area that has previously been unexplored especially in regards to pork and processed meats. Sodium metasilicate is approved for antimicrobial use in ready-to-eat meat and poultry products at 6% (USDA, 2013). It is an alkaline solution that has proven to be effective in reducing Gram-negative bacteria from the surface of meat and meat products (Carlson, et al., 2008; Pohlman, et al., 2009; 28 Weber, O?Brien, & Bender, 2004). Sodium metasilicate has been explored as a treatment for fresh beef trimmings before grinding. When used as part of a multi-intervention program, 4% sodium metasilicate in combination with 3% potassium lactate or 200-ppm peroxyacetic acid does not reduce E. coli or Salmonella Typhimurium when applied to beef trimming before grinding (Quilo, et al., 2010). However, 4% sodium metasilicate in combination with 3% potassium lactate or 200-ppm peroxyacetic acid does improve or maintain ground beef odor when applied to beef trimmings before grinding, and 4% sodium metasilicate in combination with 200-ppm peroxyacetic acid enhances beef color when applied to beef trimming before grinding (Quilo, et al., 2010). Each of these antimicrobials has been used at a variety of concentrations. Utilizing these antimicrobials in different stages during meat processing may assist in the reduction of the risk of pathogenic microorganisms. The purpose of this study was to determine optimum concentrations of usage of lactic acid and sodium metasilicate for pathogen reduction on beef bottom round muscles. Materials and Methods Culture Strains Five strains of Escherichia coli O157:H7, 1 strain of each of the big 6 STECs, 5 strains of Salmonella spp., and 5 strains of Listeria monocytogenes (Table 3) were used for this study. All media was purchased from Neogen Corporation, Lansing, Michigan unless otherwise stated. Cultured microorganisms were individually transferred to 9 ml sterile tryptic soy broth, vortexed (Labnet International, Inc., Edison, New Jersey), and 29 incubated at 35 ?C for 24h (Jeio Tech, Inc., Des Plaines, Illinois). The overnight culture produced approximately 9 log CFU/ml culture suspensions which were used for inoculation. Cultures were centrifuged at 3650 rpm for 20 min at 37 ?C (5810R Eppendorf, Hauppauge, New York). Using the same method as Wang & Harris (2011), the supernatant was discarded and the precipitate was re-suspended in 0.85% sodium chloride (Fisher Scientific, Fair Lawn, New Jersey) solution until a spectrometer (Amersham Biosciences Corporation, Piscataway, New Jersey) absorbance reading of 0.60 was determined yielding about 8 log CFU/ml cultures. To create the culture cocktail used for inoculation, equal parts of each strain of microorganism were combined and vortexed to result in cocktails of E. coli O157:H7, non-O157 STECs, Salmonella spp., and L. monocytogenes. The culture cocktails were serially diluted using 9ml peptone (Becton Dickinson and Company, Sparks, Maryland) and plated on MacConkey Sorbitol Agar (E. coli), XLT4 (Salmonella spp.) or Modified Oxford Medium (L. monocytogenes) to determine cell density. All plates were enumerated after incubation at 35 ?C for 24h. Treatment Preparation Lactic acid and sodium metasilicate antimicrobial treatments were utilized at various concentrations and temperatures of application. Lactic acid (analytical grade, Sigma Aldrich, St. Louis, Missouri) concentrations were 1, 2, 3, and 4% (v/v) while sodium metasilicate (analytical grade, Sigma Aldrich, St. Louis, Missouri) concentrations were 2, 3, 4, and 5% (w/v). A control treatment of distilled water was also tested. Lactic acid (analytical grade, Sigma Aldrich, St. Louis, Missouri), 4% (v/v), and sodium metasilicate (analytical grade, Sigma Aldrich, St. Louis, Missouri) 4% (w/v), were 30 applied at 4, 25, and 60 ?C. Antimicrobials were mixed into solution with distilled water (Podolak, Zayas, Kastner, & Fung, 1995a; Podolak, Zayas, Kastner, & Fung, 1995b). A control treatment of distilled water at 25 ?C was also tested. Table 4 contains the pH values of all treatments. Tap water in the research facility is of poor quality and consistency; thus, distilled water was used to maintain better control over the process. Sample Preparation Fresh beef bottom round steaks were cut at the Lambert Powell Meat Laboratory without the use of antimicrobial solutions. Lean meat samples were cut to 100 cm2 pieces. Each piece was individually inoculated and treated with the antimicrobial treatment assigned. Fresh beef steaks were inoculated with the culture cocktails of E. coli O157:H7, non-O157 STECs, Salmonella spp., or L. monocytogenes. The surface of the meat was inoculated with 1mL of a cocktail culture and then evenly spread using a disposable L- shaped culture spreader (VWR International, LLC, Radnor, Pennsylvania). Samples were allowed 30 min to allow the bacteria to adhere to the surface of the meat before antimicrobial solutions were applied. Antimicrobial treatments were randomly assigned. Ten ml of the assigned treatment were evenly applied over the surface of the meat. After treatment application, the samples were allowed an additional 30 min contact time. A modified plating method from Podolak, Zayas, Kastner, & Fung (1995a) was utilized. Since samples were not stored after dilution, a buffered solution was not utilized 31 and a simple diluent of 0.1% peptone was used instead. One hundred ml of 0.1% peptone was added to each of the meat samples in sterile stomacher bags (Nasco Whirl-Pak, Fort Atkinson, Wisconsin) and then samples were stomached for 2 min at 300 rpm (400 Circular Seward Medical, London, England). Serial dilutions with 9ml 0.1% peptone were created and dilutions were plated on MacConkey Sorbitol Agar (E. coli), XLT4 (Salmonella spp.), or Modified Oxford Medium (L. monocytogenes) to determine cell density. All plates were enumerated after incubation at 35 ?C for 24h. Results are reported in CFU/cm2. Statistical Analysis A completely random design was used to conduct these experiments. Each experiment was conducted in triplicate with 3 replications (on separate days) resulting in 9 samples per treatment. No more than 1 data outlier was removed as a sample possibly contaminated with pathogens before inoculation in each treatment group. All data were converted to log10 CFU/cm2 before statistical analysis. Statistics were completed using PROC MIXED in SAS 9.2 (SAS Institute, Inc., Cary, North Carolina). The fixed effect was the treatment. There were no differences in replications, and no treatment by replication interactions were included as no practical differences were observed. Tukey pairwise comparisons were utilized due to the potential unequal sample sizes that resulted when data points were removed. Results and Discussion Concentration 32 All LA treatments except LA1 reduced (P < 0.01) microbial counts of E. coli O157:H7 compared to the control sample. There were no differences in non-O157 STEC counts among the control, LA1, and LA2 (P > 0.37, Table 5). All LA treatments reduced L. monocytogenes counts compared to the control treatment (P < 0.01, Table 5). LA4 was the only LA treatment that reduced Salmonella spp. counts in comparison to the control sample (P < 0.01, Table 5). Lactic acid at a 2% concentration has been shown to reduce both E. coli O157:H7 and Salmonella as a beef carcass wash (Hardin, Acuff, Lucia, Oman, & Savell, 1995). In contrast, a study conducted using 5% lactic acid did not show a decrease in Salmonella Typhimurium in ground beef samples (Stivarius, Pohlman, McElyea, & Waldroup, 2002). It is possible that the increased fat in the Stivarius et al. (2002) study (15%) protected the bacteria more than the current study?s surface inoculation and treatment method allowed. However, the same study found that 5% lactic acid reduced E. coli and coliform counts. Lactic acid (2%) has also been shown to reduce the amount of surface bacteria on turkey rolls (L. monocytogenes), pork bellies (Salmonella), and chicken skins (Salmonella), but not beef plates (E. coli O157:H7) (Carpenter, Smith, & Broadbent, 2011). LA4 was most effective in combating L. monocytogenes (P < 0.03, Table 4). Gill and Badoni (2004) found that 4% lactic acid reduced total aerobic bacteria by >2 log units compared to a water treatment on chilled, raw beef. In the present study, Salmonella spp. and L. monocytogenes decreased; however, E. coli O157:H7 and non-O157 STEC did not. One study reported that treating cut muscle surfaces with 5% lactic acid resulted in greater effectiveness when the cut muscle surface was inoculated at a higher rather than lower level of E. coli (Youssef, Yang, Badoni, & Gill, 2012). While lower inoculation levels of E. coli were reduced 33 there was a greater overall reduction when inoculated at a higher level. This outcome is logical as there is overall less bacteria on the surface of the meat. Yoder et al. (2012) reported that 2% lactic acid reduced Salmonella Typhimurium and E. coli O157:H7 by 4.68 and 3.54 log CFU/cm2, respectively on inoculated beef plates. It is possible that these results differ from the current study because of lactic acid application methods. Yoder et. al (2012) utilized a pressurized spray system which could account for the increased bacterial reduction. Lactic acid is one of the most common organic acids used as a whole carcass decontamination step (Huffman, 2002). This data suggests that its effectiveness can be expanded past the abattoir and into fabrication. As the concentration of sodium metasilicate increased, the pathogen counts decreased. This was also reported by Huang, Williams, Sims, & Simmone (2011) when testing total psychotropic bacteria of fresh chicken breasts marinated in sodium metasilicate at 1, 2, 3, and 4% during cold storage. All SM treatments reduced E. coli O157:H7 counts compared to the control sample (P < 0.04, Table 6). SM5 was more effective than SM2 (P < 0.01) in reducing E. coli O157:H7 counts. SM3 and SM4 were equally as effective compared to SM2 and SM5 (P > 0.16, Table 6) on E. coli O157:H7. No differences were discovered in non-O157 STEC counts among the control, SM2, and SM3 (P > 0.15, Table 6). SM4 and SM5 were both more effective than the control sample (P < 0.01, Table 6) in controlling non-O157 STEC contamination. Compared to the control treatment, SM4 and SM5 reduced the Salmonella spp. counts of samples (P = 0.02 and P < 0.01, respectively, Table 6). Several studies have utilized 4% sodium metasilicate that have shown favorable bacteria reduction of E. coli O157:H7 or 34 Salmonella in ground beef (Geornaras, et al., 2012a; Geornaras, et al., 2012b; Pohlman, et al., 2009; Pohlman, Dias Morse, & Rajaratnam, 2005; Quilo, et al., 2009; Quilo, et al., 2010). Each of these studies utilized a shorter contact time (30s or 3 min) than the current procedure, but the results were still similar. There were no differences in L. monocytogenes counts among the control, SM2, and SM3 (P > 0.94, Table 6). SM4 and SM5 were most effective in reducing L. monocytogenes compared to the control (P < 0.03, Table 6). Listeria monocytogenes decreased as sodium metasilicate concentration increased as was observed in an in vitro study with a 30 min contact time conducted by Sharma et. al (2012a). It is possible that more favorable results were shown in the in vitro study than in the current study due to the ability of the meat to buffer the antimicrobial solutions and L. monocytogenes ability to form biofilms on the meat. Temperature E. coli O157:H7 and non-O157 STEC counts were reduced by all treatments compared to the inoculated control (P < 0.01, Tables 5 and 6, respectively). The control treatment was ineffective in reducing microbial Salmonella spp. contamination compared to all other treatments except LASM at 25 and 60 ?C (P < 0.01 and P > 0.11, respectively, Table 9). Compared to the control treatment, the SM and LA treatments effectively lessened L. monocytogenes contamination (P < 0.01 and P > 0.85, respectively); however, the combination treatment (LASM) did not (P > 0.05, Table 10). There were no differences in bacteria prevalence among the application temperatures within a treatment group (P > 0.33, Tables 5-8) for any microorganisms 35 tested. Lactic acid and sodium metasilicate have been used with positive results at a variety of temperatures. In a brine solution, sodium metasilicate has reduced E. coli O157:H7 at both 4 and 15 ?C (Adler, et al., 2011). At 25 ?C sodium metasilicate has been shown to reduce E. coli O157:H7, non-O157 STECs, and Salmonella Typhimurium on beef trimmings (Geornaras, et al., 2012a; Geornaras, et al., 2012b). Lactic acid (4%) at 55 ?C and > 60 ?C reduced microbial counts on beef carcasses (Castillo, Lucia, Mercado, & Acuff, 2001). While there was no difference in effectiveness of reducing E. coli O157:H7 between LA and LASM (P > 0.15, Table 7), there was an improvement between SM and LASM (P < 0.01, Table 7). All LA and SM treatments were equally effective (P > 0.57) against non-O157 STEC, but LA and LASM were only equally effective at 4 ?C (P = 0.12, Table 8). In all other comparisons, LA and SM provided a greater advantage in reducing non-O157 STEC than the combination, LASM (P < 0.02, Table 8). The greatest effect on Salmonella spp. reduction was observed by the LA treatment (P < 0.01, Table 9). The combination treatment (LASM) did not improve effectiveness when compared with LA or SM used alone in Salmonella spp. contamination. The only difference among LASM and SM treatments was SM at 25 ?C improving Salmonella spp. counts compared to LASM at 60 ?C (P = 0.05, Table 9). The LA treatment was the most effective in reducing the L. monocytogenes load (P < 0.01, Table 10). While utilizing multiple solutions as part of a multistep intervention program has proven positive (Quilo, et al., 2010), the lactic acid and sodium metasilicate combination did not enhance the individual antimicrobial effectiveness of either solution. Quilo, et al. (2010) combined 4% sodium 36 metasilicate with 3% potassium lactate or 200-ppm peroxyacetic acid and dipped inoculated beef trimmings in the treatment solution and then ground the meat before sampling. This research found that neither combination reduced E. coli or Salmonella Typhimurium contamination (Quilo, et al., 2010). Conclusion Sodium metasilicate, 4% and lactic acid, 4% were determined to be the lowest concentrations most effective against all microorganisms. While lower concentrations of lactic acid were equally effective against non-O157 STEC (LA3) and Salmonella spp. (LA2 and LA3); LA4 was more effective against E. coli O157:H7 and L. monocytogenes. Similarly, SM3 was consistently as effective as SM4 against E. coli O157:H7, non-O157 STEC, and L. monocytogenes, but not against Salmonella spp. Therefore, to have the greatest impact on all bacteria the greater concentration of 4% is recommended for both lactic acid and sodium metasilicate. By utilizing the lowest concentration of antimicrobial solution necessary to achieve effective pathogen reduction, meat processors can provide a safe and wholesome meat supply. Meat processors can apply 4% lactic acid or 4% sodium metasilicate at refrigeration temperatures (4 ?C) and obtain the same microbial benefits of decreased counts of E. coli O157:H7, non-O157 STEC, Salmonella spp., and L. monocytogenes of applying them at a greater temperatures (25 or 60?C). Combining the two antimicrobial solutions (LASM) is not recommended. The high pH of sodium metasilicate (12.82) combined with the low pH of lactic acid (1.84) creates a solution with a pH of 12.53 which is ineffective against pathogenic bacteria. However, individually both solutions can serve as a hurdle technology in meat processing facilities. 37 References Adler, J., Geornaras, I., Byelashov, O., Belk, K., Smith, G., & Sofos, J. (2011). Survival of Escherichia coli O157:H7 in meat product brines containing antimicrobials. Journal of Food Science, 76(7), M478-485. Byelashov, O., Daskalov, H., Geornaras, I., Kendall, P., Belk, K., Scanga, J., Smith, G., & Sofos, J. (2010). Reduction of Listeria monocytogenes on frankfurters treated with lactic acid solutions of various temperatures. Food Microbiology, 27(6), 783- 790. Carlson, B., Ruby, J., Smith, G., Sofos, J., Bellinger, G., Warren Serna, W., Centrella, B., Bowling, R., & Belk, K. (2008). Comparison of antimicrobial efficacy of multiple beef hide decontamination strategies to reduce levels of Escherichia coli O157:H7 and Salmonella. Journal of Food Protection, 71(11), 2223-2227. Carpenter, C. E., Smith, J. V., & Broadbent, J. R. (2011). Efficacy of washing meat surfaces with 2% levulinic, acetic, or lactic acid for pathogen decontamination and residual growth inhibition. Meat Science, 88(2), 256-260. Castillo, A., Lucia, L. M., Mercado, I., & Acuff, G. R. (2001). In-plant evaluation of a lactic acid treatment for reduction of bacteria on chilled beef carcasses. Journal of Food Protection, 64(5), 738-740. CDC. (2011). CDC estimates of foodborne illness in the United States. Access Date January 3, 2014. http://www.cdc.gov/foodborneburden/PDFs/FACTSHEET_A_ FINDINGS_updated4-13.pdf. FDA. (2012). Bad bug book, foodborne pathogenic microorganisms and natural toxins. (Second ed.). Access Date January 3, 2014. Geornaras, I., Yang, H., Manios, S., Andritsos, N., Belk, K. E., Nightingale, K. K., Woerner, D. R., Smith, G. C., & Sofos, J. N. (2012a). Comparison of decontamination efficacy of antimicrobial treatments for beef trimmings against Escherichia coli O157:H7 and 6 Non-O157 shiga toxin-producing E. coli serogroups. Journal of Food Science, 77(9), M539-M544. Geornaras, I., Yang, H., Moschonas, G., Nunnelly, M. C., Belk, K. E., Nightingale, K. K., Woerner, D. R., Smith, G. C., & Sofos, J. N. (2012b). Efficacy of chemical interventions against Escherichia coli O157:H7 and multi-drug resistant and 38 antibiotic-susceptible Salmonella on inoculated beef trimmings. Journal of Food Protection, 75(11), 1960-1967. Gill, C. O., & Badoni, M. (2004). Effects of peroxyacetic acid, acidified sodium chlorite or lactic acid solutions on the microflora of chilled beef carcasses. International Journal of Food Microbiology, 91(1), 43-50. Hardin, M. D., Acuff, G. R., Lucia, L. M., Oman, J. S., & Savell, J. W. (1995). Comparison of methods for decontamination from beef carcass surfaces. Journal of Food Protection, 58(4), 368-374. Huang, H., Williams, S. K., Sims, C. A., & Simmone, A. (2011). Sodium metasilicate affects antimicrobial, sensory, physical, and chemical characteristics of fresh commercial chicken breast meat stored at 4 degrees C for 9 days. Poultry Science, 90(5), 1124-1133. Huffman, R. D. (2002). Current and future technologies for the decontamination of carcasses and fresh meat. Meat Science, 62(3), 285-294. Podolak, R. K., Zayas, J. F., Kastner, C. L., Fung, D. Y. C. (1995a). Inhibition of Listeria monocytogenes and Escherichia coli O157:H7 on beef by application of organic acids. Journal of Food Protection, 59(4), 370-373. Podolak, R. K., Zayas, J. F., Kastner, C. L., Fung, D. Y. C. (1995b). Reduction of Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella Typhimurium during storage on beef sanitized with fumaric, acetic, and lactic acids. Journal of Food Safety, 15, 283-290. Pohlman, F. W., Dias Morse, P. N., & Rajaratnam, G. (2005). Microbial characteristics of ground beef treated with potassium lactate, sodium metasilicate, peroxyacetic acid, and acidified sodium chlorite. Arkansas Animal Science Department Report, AAES Research Series 535, 142-144. Pohlman, F. W., Dias-Morse, P. N., Quilo, S. A., Brown, A. H., Crandall, P. G., Dias Morse, P. N., Baublits, R. T., Story, R. P., Bokina, C., & Rajaratnam, G. (2009). Microbial, instrumental color and sensory characteristics of ground beef processed from beef trimmings treated with potassium lactate, sodium metasilicate, peroxyacetic acid or acidified sodium chlorite as single antimicrobial interventions. Journal of Muscle Foods, 20(1), 54-69. Quilo, S. A., Pohlman, F. W., Dias-Morse, P. N., Brown, A. H., Crandall, P. G., Dias Morse, P. N., & Story, R. P. (2010). Microbial, instrumental color and sensory characteristics of inoculated ground beef produced using potassium lactate, sodium metasilicate or peroxyacetic acid as multiple antimicrobial interventions. Meat Science, 84(3), 470-476. 39 Quilo, S. A., Pohlman, F. W., Dias-Morse, P. N., Brown, A. H., Crandall, P. G., Baublits, R. T., & Aparicio, J. L. (2009). The impact of single antimicrobial intervention treatment with potassium lactate, sodium metasilicate, peroxyacetic acid, and acidified sodium chlorite on non-inoculated ground beef lipid, instrumental color, and sensory characteristics. Meat Science, 83(3), 345-350. Scallan, E., Hoekstra, R. M., Angulo, F. J., Tauxe, R. V., Widdowson, M. A., Roy, S. L., Jones, J. L., & Griffin, P. M. (2011). Foodborne illness acquired in the United States?major pathogens. Emerging Infectious Diseases, 17(1), 7-15. Sharma, C., Williams, S., Schneider, K., Schmidt, R., & Rodrick, G. (2012a). Antimicrobial effects of sodium metasilicate against Listeria monocytogenes. Foodborne Pathogens and Disease, 9(9), 822-828. Stivarius, M. R., Pohlman, F. W., McElyea, K. S., & Waldroup, A. L. (2002). Effects of hot water and lactic acid treatment of beef trimmings prior to grinding on microbial, instrumental color and sensory properties of ground beef during display. Meat Science, 60(4), 327-334. Theron, M. M., & Lues, J. F. R. (2007). Organic acids and meat preservation: a review. Food Reviews International, 23(2), 141-158. USDA, F. S. I. S. (2013). Safe and suitable ingredients used in the production of meat, poultry, and egg products. Access Date January 3, 2014. http://www.fsis.usda.gov/OPPDE/rdad/FSISDirectives/7120.1.pdf. Wang, Luxin, and Harris, L.J. 2011. Rdar morphotype and its relationship to desiccation tolerance in Salmonella spp. International Association for Food Protection, Milwaukee, Wisconsin, USA, July 31-August 3. Weber, G., O'Brien, J., & Bender, F. (2004). Control of Escherichia coli O157:H7 with sodium metasilicate. Journal of Food Protection, 67(7), 1501-1506. Yoder, S., Henning, W., Mills, E., Doores, S., Ostiguy, N., & Cutter, C. (2012). Investigation of chemical rinses suitable for very small meat plants to reduce pathogens on beef surfaces. Journal of Food Protection, 75(1), 14-21. Youssef, M. K., Yang, X., Badoni, M., & Gill, C. O. (2012). Effects of spray volume, type of surface tissue and inoculum level on the survival of Escherichia coli on beef sprayed with 5% lactic acid. Food Control, 25(2), 717-722. 40 Table 3. Strains of microorganisms used Microorganism ATCC number or ID Code Source Escherichia coli O157:H7 ATCC 35150 Human ? HC Escherichia coli O157:H7 ATCC 43894 Human ? HC Escherichia coli O157:H7 AU ? 1 Laboratory strain (301) Escherichia coli O157:H7 AU ? 2 Laboratory strain (505B) Escherichia coli O157:H7 AU ? 3 Laboratory strain Non-O157 STEC (O145) TWO9356 Human ? HUS Non-O157 STEC (O26) TWO7814 Human ? HUS Non-O157 STEC (O121) TWO8039 Human Non-O157 STEC (O45) TWO14003 Human Non-O157 STEC (O111) TWO7926 Human ? HC Non-O157 STEC (O103) TWO8101 Human Salmonella AU ? Enteritidis Laboratory strain Salmonella AU ? Kentucky Laboratory strain Salmonella AU ? Montevideo Laboratory strain Salmonella AU ? Thompson Laboratory strain Salmonella AU ? Stanley Laboratory strain Listeria monocytogenes ATCC 49594 Petite Scott A Listeria monocytogenes ATCC 19115 Human ? Serotype 4b Listeria monocytogenes ATCC 7644 Human Listeria monocytogenes AU ? 4 Laboratory strain (101M serotype 4b) Listeria monocytogenes AU ? 5 Laboratory strain (108M serotype 1/2b) 41 Table 4. pH values of lactic acid at 1, 2, 3, 4% (LA1, LA2, LA3, LA4), sodium metasilicate at 2, 3, 4, 5% (SM2, SM3, SM4, SM5), and distilled water. Solution pH LA1 1.92 LA2 1.89 LA3 1.89 LA4 1.84 SM2 12.82 SM3 12.83 SM4 12.82 SM5 12.82 Distilled Water 4.90 LA4 + SM4 12.53 42 Table 5. Concentration effects of lactic acid at 1, 2, 3, 4% (LA1, LA2, LA3, LA4) on fresh beef bottom round steaks after pathogen inoculation and a 30min contact time. Microorganism Treatment Mean log10 CFU/cm2 SEMz E. coli O157:H7 Control 7.04a 0.19 LA1 6.74a 0.11 LA2 6.20b 0.12 LA3 5.90b 0.11 LA4 5.76b 0.11 Non-O157 STEC Control 7.00a 0.25 LA1 6.71a 0.15 LA2 6.33ab 0.15 LA3 6.04bc 0.15 LA4 5.43c 0.15 Salmonella spp. Control 6.71a 0.43 LA1 6.41a 0.25 LA2 5.20ab 0.25 LA3 5.26ab 0.25 LA4 4.76b 0.25 L. monocytogenes Control 7.24a 0.15 LA1 6.45b 0.09 LA2 6.37b 0.09 LA3 6.36b 0.09 LA4 5.93c 0.09 a, b, c Means within a bacteria group lacking a common superscript differ (P < 0.05). z Standard Error of the Mean 43 Table 6. Concentration effects of sodium metasilicate at 2, 3, 4, 5% (SM2, SM3, SM4, SM5) on fresh beef bottom round steaks after pathogen inoculation and a 30min contact time. Microorganism Treatment Mean log10 CFU/cm2 SEMz E. coli O157:H7 Control 7.04a 0.19 SM2 6.32b 0.11 SM3 6.12bc 0.11 SM4 5.87bc 0.11 SM5 5.70c 0.11 Non-O157 STEC Control 7.00a 0.25 SM2 6.20ab 0.15 SM3 6.20ab 0.15 SM4 5.88bc 0.15 SM5 5.52c 0.15 Salmonella spp. Control 6.71a 0.43 SM2 5.92ab 0.25 SM3 6.16a 0.25 SM4 4.93bc 0.25 SM5 4.70c 0.25 L. monocytogenes Control 7.24a 0.15 SM2 7.20a 0.09 SM3 7.03ab 0.09 SM4 6.64b 0.09 SM5 6.65b 0.09 a, b, c Means within a bacteria group lacking a common superscript differ (P < 0.05). z Standard Error of the Mean 44 Table 7. Temperature effects of 4% lactic acid (LA), 4% sodium metasilicate (SM), and 4% lactic acid + 4% sodium metasilicate (LASM) on Escherichia coli O157:H7 at 4, 25, and 60 ?C on fresh beef bottom round steaks after pathogen inoculation and a 30min contact time. Treatment Temperature (?C) Mean log10 CFU/cm2 SEMz Control 25 7.15a 0.10 LA 4 6.15bc 0.09 25 6.19bc 0.09 60 6.00bcd 0.09 SM 4 5.71d 0.09 25 5.59d 0.09 60 5.83cd 0.09 LASM 4 6.39b 0.09 25 6.47b 0.09 60 6.37b 0.09 a, b, c, d Means lacking a common superscript differ (P < 0.05). z Standard Error of the Mean 45 Table 8. Temperature effects of 4% lactic acid (LA), 4% sodium metasilicate (SM), and 4% lactic acid + 4% sodium metasilicate (LASM) on non-O157 STEC at 4, 25, and 60 ?C on fresh beef bottom round steaks after pathogen inoculation and a 30min contact time. Treatment Temperature (?C) Mean log10 CFU/cm2 SEMz Control 25 6.97a 0.08 LA 4 5.77cd 0.08 25 5.80cd 0.08 60 5.87cd 0.08 SM 4 5.70d 0.08 25 5.65d 0.08 60 5.64d 0.08 LASM 4 6.21bc 0.08 25 6.38b 0.08 60 6.32b 0.08 a, b, c, d Means lacking a common superscript differ (P < 0.05). z Standard Error of the Mean 46 Table 9. Temperature effects of 4% lactic acid (LA), 4% sodium metasilicate (SM), and 4% lactic acid + 4% sodium metasilicate (LASM) on Salmonella spp. at 4, 25, and 60 ?C on fresh beef bottom round steaks after pathogen inoculation and a 30min contact time. Treatment Temperature (?C) Mean log10 CFU/cm2 SEMz Control 25 6.60a 0.18 LA 4 3.87d 0.18 25 3.62d 0.18 60 3.57d 0.18 SM 4 5.28bc 0.18 25 5.13c 0.18 60 5.44bc 0.18 LASM 4 5.63bc 0.18 25 5.85abc 0.18 60 5.96ab 0.18 a, b, c, d Means lacking a common superscript differ (P < 0.05). z Standard Error of the Mean 47 Table 10. Temperature effects of 4% lactic acid (LA), 4% sodium metasilicate (SM), and 4% lactic acid + 4% sodium metasilicate (LASM) on Listeria monocytogenes at 4, 25, and 60 ?C on fresh beef bottom round steaks after pathogen inoculation and a 30min contact time. Treatment Temperature (?C) Mean log10 CFU/cm2 SEMz Control 25 7.19a 0.06 LA 4 6.24e 0.06 25 6.16e 0.06 60 6.03e 0.06 SM 4 7.10d 0.06 25 7.05bcd 0.06 60 7.06cd 0.06 LASM 4 6.72ab 0.06 25 6.85abc 0.06 60 6.82abc 0.06 a, b, c, d, e Means lacking a common superscript differ (P < 0.05). z Standard Error of the Mean 48 Chapter III Evaluation of Lactic Acid and Sodium Metasilicate against Pathogens of Concern on Fresh Beef, Pork, and Deli Meats Abstract An important aspect of the meat industry is food safety. Lactic acid is a ?Generally Recognized As Safe? (GRAS) food additive commonly used in the meat industry. Lactic acid is most commonly used as a hot carcass rinse. Sodium metasilicate is approved for antimicrobial use in ready-to-eat meat and poultry products. There is very little research about sodium metasilicate use on meat and meat products especially in regards to pork. The United States Department of Agriculture Food Safety and Inspection Service (USDA FSIS) has a zero tolerance policy for L. monocytogenes in ready-to-eat meat and poultry products. Spraying or dipping cured meats post-processing in organic acids has been proven to reduce L. monocytogenes. Lactic acid concentration and temperature impacts the effectiveness as an antimicrobial agent on L. monocytogenes when applied to frankfurters. There is very little research concerning sodium metasilicate as an antimicrobial agent on meat and meat products. This is an area that has previously been unexplored especially in regards to processed meats. The purpose of this study was to determine if lactic acid and sodium metasilicate could effectively lower pathogenic bacteria on fresh beef and pork and deli meats. Lactic acid 4% (LA, v/v), sodium metasilicate 4% (SM, w/v), and a distilled water control were applied to fresh beef and 49 pork lean muscle and deli meats. Antimicrobials were mixed in solution with distilled water. Fresh meat of beef bottom round and pork ham steaks were cut into 100 cm2 pieces. Roast beef, ham, and turkey deli meats were manufactured at the Lambert Powell Meat Laboratory without the use of antimicrobial solutions. Meat samples were cut to 100 cm2 pieces. Each piece was individually inoculated and treated with the antimicrobial treatment assigned. Fresh meat pieces were inoculated with Escherichia coli O157:H7 (5 strains), non-O157 shiga-toxin producing Escherichia coli (STEC, 1 strain each of the ?Big 6?), or Salmonella spp. (5 strains). Deli meats were inoculated with Listeria monocytogenes (5 strains). After 30 min of contact time samples were treated with the antimicrobial solution or control and then allowed 30 min of contact time. Half of the deli meat samples were vacuum packaged and treated in a hot water bath for 2 min at 90.6 ?C. Samples were serially diluted and plated on MacConkey Agar with Sorbitol (E. coli), XLT4 (Salmonella spp.), or Modified Oxford Medium (MOX, L. monocytogenes). Data were analyzed using the PROC MIXED procedure of SAS and Tukey pairwise comparisons. In fresh meat samples, the control treatment resulted in greater microbial counts regardless of inoculum or species than either the LA or SM treatments (P < 0.01). Within species, the SM treatment was more effective at reducing E. coli O157:H7 contamination than the LA treatment (P < 0.01). Beef treated with LA had less Salmonella spp. than pork treated with SM (P = 0.03). Both lactic acid and sodium metasilicate can be applied to fresh beef and pork as an effective hurdle technology in the fight for food safety. The deli meat treatments including post-packaging lethality decreased the bacterial load of samples in comparison to those that did not receive the post packaging lethality treatment (P < 0.01). Regardless of post-packaging lethality 50 treatments, there were no differences in microbial counts among treatment groups (P > 0.73). Treating deli meats with lactic acid or sodium metasilicate did not reduce L. monocytogenes loads. However, adding a post-packaging lethality treatment was able to minimize microbial contamination. Introduction Food safety is a major concern in the meat industry. The Centers for Disease Control and Prevention (CDC) estimates that 1 in 6 Americans become ill each year due to foodborne illness. Of these 48 million, 128,000 are hospitalized and about 3,000 die of foodborne diseases (Weber, O?Brien, & Bender, 2004). Fresh meat pathogens of concern include: Escherichia coli O157:H7, non-O157 shiga toxin producing E. coli (STEC) serotypes, and Salmonella spp. These pathogens, Salmonella spp. and E. coli are pathogens on the ?top five pathogens? lists compiled by the CDC (Weber, et al., 2004). The USDA FSIS has expanded its ruling on E. coli O157 in raw, non-intact beef to include six non-O157 serotypes including: O26, O45, O103, O111, O121, and O145 ? the ?big 6? (USDA, 2011). In ready to eat meats Listeria monocytogenes is the pathogen of concern. L. monocytogenes is on the ?top five pathogens? lists compiled by the CDC (Weber, O?Brien, & Bender, 2004). The United States Department of Agriculture Food Safety and Inspection Service (USDA FSIS) has a zero tolerance policy for L. monocytogenes in ready-to-eat meat and poultry products. This rule requires meat processors to control L. monocytogenes by using one of three alternatives. Alternative 1 includes both a post-lethality treatment and a process or antimicrobial agent, Alternative 2 requires either a post-lethality treatment or a process or antimicrobial agent, and 51 Alternative 3 requires the implantation of sanitation procedures and frequent USDA FSIS environmental testing (USDA, 2003). Lactic acid is a ?Generally Recognized As Safe? (GRAS) food additive commonly used in the meat industry. Lactic acid is an organic acid that has been used in abattoirs as a hot carcass rinse (Huffman, 2002). Gill and Badoni (2004) found that 4% lactic acid on chilled beef carcasses reduced bacteria by > 1 log unit compared to a water treatment. Similarly, lactic acid at 2% has been found to reduce E. coli O157:H7 and Salmonella Typhimurium by 3.54 and 4.68 CFU/cm2, respectively on beef surfaces (Yoder, et al., 2012). Lactic acid (2%) has been showed to reduce the amount of surface bacteria on pork bellies (Salmonella), and chicken skins (Salmonella), but not beef plates (E. coli O157:H7) (Carpenter, Smith, & Broadbent, 2011). Its use in other areas of meat processing has not been widely explored. Spraying or dipping cured meats post- processing in organic acids has proven to reduce L. monocytogenes (Theron & Lues, 2007). Lactic acid (2%) has been showed to reduce the amount of surface bacteria on turkey rolls (L. monocytogenes) (Carpenter, Smith, & Broadbent, 2011). Lactic acid concentration and temperature impacts the effectiveness as an antimicrobial agent on L. monocytogenes when applied to frankfurters (Byelashov, et al., 2010). Very little research has been performed on the use of sodium metasilicate on meat and meat products. This is an area that has previously been unexplored especially in regards to pork and processed meats. Sodium metasilicate is approved for antimicrobial use in ready-to-eat meat and poultry products at 6% (USDA, 2013). It is an alkaline 52 solution that has proven to be effective in reducing Gram-negative bacteria from the surface of meat and meat products (Carlson, et al., 2008; Pohlman, et al., 2009; Weber, O?Brien, & Bender, 2004). Sodium metasilicate has been explored as a treatment for fresh beef trimmings before grinding. Geornaras, et al. (2012b) found that 4% sodium metasilicate reduced E. coli O157:H7 and multidrug-resistant and antibiotic susceptible Salmonella by 1.3-1.5 log CFU/cm2 when applied as a treatment to beef trimmings. Sodium metasilicate (4%) applied to beef trimmings before grind has been shown to reduce coliforms, E. coli, aerobic plate counts, and Salmonella over 7 days of storage (Pohlman, et al., 2009). Little research has been conducted on the effectiveness of sodium metasilicate on Gram-positive bacteria, but one in vitro study found that sodium metasilicate at 1, 2, or 3% reduced L. monocytogenes by >5 logs after a 30 min exposure time (Sharma, Williams, Schneider, Schmidt, & Rodrick, 2012a). Sharma et al. (2012a) also discovered that applying 4% sodium metasilicate with a 30 min exposure time resulted in an undetectable amount of L. monocytogenes. Escherichia coli is a Gram-negative non-spore forming short rod. About 75% of all E. coli foodborne infections world-wide are caused by O157:H7. The ?big 6? account for 71% of the non-O157:H7 infections; however, there are other infectious serotypes that can result in illness (Brooks, et al., 2005). There are some other infectious serotypes that can result in illness (FDA, 2012). While most infections continue to be a result of ground beef or beef products, there is an increasing amount of produce products (FDA, 2012). There are about 63,153 cases of O157:H7 EHEC and about 112,752 cases of non- O157:H7 EHEC infections annually (Scallan, et al., 2011). 53 Salmonella is a Gram-negative, non-sporeforming, motile rod. Salmonella is found widely in the environment and in the intestines of many animals. Salmonella illnesses have been linked to meats, poultry, and poultry products; as well as, peanut butter, cocoa, and produce (FDA, 2012). There are an estimated 1,027,561 nontyphoidal salmonellosis cases annually (Scallan, et al., 2011). Listeria monocytogenes is a Gram-positive, motile rod. It is not a leading cause of illness, but it is a leading cause of death from foodborne illness (Scallan, et al., 2011). The CDC estimates 1,591 cases of foodborne illness resulting from L. monocytogenes with about 255 resulting in death (Scallan, et al., 2011). Many foods have been associated with L. monocytogenes outbreaks including raw and ready-to-eat meats, dairy, and dairy products. The ability of L. monocytogenes to grow and thrive at refrigeration temperatures creates a unique problem for the food industry (FDA, 2012). Materials and Methods Culture Strains Five strains of Escherichia coli O157:H7, 1 strain of each of the big 6 STECs, 5 strains of Salmonella spp., and 5 strains of Listeria monocytogenes (Table 11) were used for this study. All media was purchased from Neogen Corporation, Lansing, Michigan unless otherwise stated. Cultured microorganisms were individually transferred to 9 ml sterile tryptic soy broth, vortexed (Labnet International, Inc., Edison, New Jersey), and incubated at 35 ?C for 24h (Jeio Tech, Inc., Des Plaines, Illinois). These approximately 9 54 log CFU/ml culture suspensions were used for inoculation. Cultures were centrifuged at 3650 rpm for 20 min at 37 ?C (5810R Eppendorf, Hauppauge, New York). Using the same method as Wang & Harris (2011), the supernatant was discarded and the precipitate was re-suspended in 0.85% sodium chloride (Fisher Scientific, Fair Lawn, New Jersey) solution until a spectrometer (Amersham Biosciences Corporation, Piscataway, New Jersey) absorbance reading of 0.60 was determined yielding about 8 log CFU/ml cultures. To create the culture cocktail used for inoculation, equal parts of each strain of microorganism were combined and vortexed to result in cocktails of E. coli O157:H7, non-O157 STECs, Salmonella spp., and L. monocytogenes. The culture cocktails were serially diluted using 9ml peptone (Becton Dickinson and Company, Sparks, Maryland) and plated on MacConkey Sorbitol Agar (E. coli), XLT4 (Salmonella spp.) or Modified Oxford Medium (L. monocytogenes) to determine cell density. All plates were enumerated after incubation at 35 ?C for 24h. Treatment Preparation Lactic acid (analytical grade, Sigma Aldrich, St. Louis, Missouri), 4% (v/v), and sodium metasilicate (analytical grade, Sigma Aldrich, St. Louis, Missouri) 4% (w/v), were applied at 4 ?C. Antimicrobials were mixed in solution with distilled water (Podolak, Zayas, Kastner, & Fung, 1995a; Podolak, Zayas, Kastner, & Fung, 1995b). A control treatment of distilled water at 4 ?C was also tested. Table 12 contains the pH of all treatments. Tap water in the research facility is of poor quality and consistency; thus, distilled water was used to maintain better control over the process. 55 Sample Preparation Fresh beef bottom round steaks, fresh pork ham steaks, roast beef, ham, and turkey deli meats were manufactured at the Lambert Powell Meat Laboratory without the use of antimicrobial solutions. Lean meat samples were cut to 100 cm2 pieces. Each piece was individually inoculated and treated with the antimicrobial treatment assigned. Fresh beef steaks were inoculated with the culture cocktails of E. coli O157:H7, non-O157 STECs, or Salmonella spp.. The surface of the deli meats was inoculated with L. monocytogenes cocktail culture. The surface of the meat was inoculated with 1mL of a cocktail culture and then evenly spread using a disposable L-shaped culture spreader (VWR International, LLC, Radnor, Pennsylvania). Samples were allowed 30 min to allow the bacteria to adhere to the surface of the meat before antimicrobial solutions were applied. Antimicrobial treatments were randomly assigned. Ten ml of the assigned treatment were evenly applied over the surface of the meat. After treatment application, the samples were allowed an additional 30 min contact time. Half of the deli meat sample were then vacuum packaged (Promax Packaging Solutions, Claremont, California) and treated in a hot water bath (Thermo Scientific, Marietta, Ohio) for 2 min at 90.6 ?C (Murinana, Qumby, Davidson, & Grooms, 2002). A modified plating method from Podolak, Zayas, Kastner, & Fung (1995a) was utilized. Since samples were not stored after dilution, a buffered solution was not utilized and a simple diluent of 0.1% peptone was used instead. One hundred ml of 0.1% peptone was added to each of the meat samples in sterile stomacher bags (Nasco Whirl-Pak, Fort 56 Atkinson, Wisconsin) and then samples were stomached for 2 min at 300 rpm (400 Circular Seward Medical, London, England). Serial dilutions with 9ml 0.1% peptone were created and dilutions were plated on MacConkey Sorbitol Agar (E. coli), XLT4 (Salmonella spp.), or Modified Oxford Medium (L. monocytogenes) to determine cell density. All plates were enumerated after incubation at 35 ?C for 24h. Results are reported in CFU/cm2. Statistical Analysis A completely random design was used to conduct these experiments. Each experiment was conducted in triplicate with 3 replications (on separate days) resulting in 9 samples per treatment. No more than 1 data outlier was removed as a sample possibly contaminated with pathogens before inoculation in each treatment group. All data were converted to log10 CFU/cm2 before statistical analysis. Statistics were completed using PROC MIXED in SAS 9.2 (SAS Institute, Inc., Cary, North Carolina). The fixed effect was the treatment. There were no differences in replications, and no treatment by replication interactions were included as no practical differences were observed. Tukey pairwise comparisons were utilized due to the potential unequal sample sizes that resulted when data points were removed. Results and Discussion Both LA and SM treatments decreased E. coli O157:H7 and non-O157 STEC counts regardless of species than the control treatment (P < 0.01, Tables 11 and 12, respectively). Previous studies showed that lactic acid at a lower concentration (2%) does 57 not improve E. coli O157:H7 on beef plates (Carpenter, Smith, & Broadbent, 2011; Yoder, et al., 2012). The increased concentration of lactic acid in study 1 showed that 4% lactic acid was better at controlling E. coli O157:H7 than 2% lactic acid. In contrast, lactic acid at a greater concentration (5%) was more effective at reducing E. coli contamination when beef products were heavily contaminated than when they were lightly contaminated (Youssef, Yang, Badoni, & Gill, 2012). Similar to the current research, pork loins inoculated with E. coli O157:H7 showed decreased bacterial counts after exposure to 3% lactic acid (Choi, Kim, Kim, Kim, & Rhee, 2009). Within species, the SM treatment was more effective at reducing E. coli O157:H7 contamination than the LA treatment (P < 0.01, Table 13). There were no differences in E. coli O157:H7 between the LA treatment when applied to beef and pork or the SM treatment when applied to both species (P = 0.56 and P = 0.41, respectively, Table 13). The LA and SM treatments were equal in non-O157 STEC counts regardless of species (P > 0.31, Table 14). Like the current research, Geornaras, et al. (2012a, 2012b), found that beef trimmings treated with 4% sodium metasilicate showed a reduction of pathogens when inoculated with both E. coli O157:H7 and non-O157 STEC or Salmonella. The control treatment resulted in higher Salmonella spp. counts than that of either the LA or SM treatments (P < 0.01, Table 15). At a lower concentration (2%), lactic acid reduced Salmonella on pork bellies and beef plates (Carpenter, Smith, & Broadbent, 2011; Yoder, et al., 2013); results that coincide with the current research. Within a treatment group, Salmonella spp. counts remained equal regardless of the species (P > 0.81, Table 15). Beef treated with LA had less Salmonella spp. than pork treated with SM (P = 0.03, 58 Table 15). Similarly, Salmonella Typhimurium was reduced with 3% lactic acid on pork loins (Choi, Kim, Kim, Kim, & Rhee, 2009). Treatments including the hot water dip decreased the bacterial load of samples in comparison to those that did not receive the hot water dip (P < 0.01, Table 16). Similarly, L. monocytogenes counts were reduced in inoculated ready-to-eat meat products utilizing a post-package pasteurization process defined as 90.6 ?C for 2 min (Muriana, Quimby, Davidson, & Grooms, 2002). Regardless of post-packaging hot water dips, there were no differences among treatment groups in regards to L. monocytogenes counts (P > 0.73, Table 16). In a previous study, lactic acid at a lower concentration (2%) effectively reduced L. monocytogenes on turkey rolls when compared with a no wash control; however, it did not reduce microbial contamination any more than a water wash (Carpenter, Smith, & Broadbent, 2011). The current study also found no differences in pathogenic contamination among the treatments and the water control (P > 0.73, Table 16). In contrast, when applied as a dip to frankfurters, 3% lactic acid at 4 ?C reduced L. monocytogenes contamination compared to a distilled water control (Byelashov, et al., 2010). An in vitro study found 4% sodium metasilicate reduced a high load of L. monocytogenes to an undetectable level after 30 min of exposure (Sharma, Williams, Schneider, Schmidt, & Rodrick, 2012a). This may suggest that L. monocytogenes has the ability to form biofilms on meat surfaces to protect it from the sodium metasilicate. Conclusion 59 Both 4% lactic acid and 4% sodium metasilicate are effective against E. coli O157H7, non-O157 STEC, and Salmonella spp. on beef bottom rounds and pork ham steaks. Overall, neither antimicrobial solution outperformed the other within species. Sodium metasilicate (4%) on beef was more effective against E. coli O157:H7 than 4% lactic acid. In regards to Salmonella spp., 4% lactic acid was more effective on beef than 4% sodium metasilicate was on pork. However, both lactic acid and sodium metasilicate can be applied to fresh beef and pork as an effective hurdle technology in the fight for food safety. Treating deli meats with 4% lactic acid or 4% sodium metasilicate did not reduce L. monocytogenes loads compared to the control. However, adding a hot water dip was able to minimize microbial contamination. Therefore, by using either sodium metasilicate or lactic acid at 4% in combination with a hot water post-packaging dip could be an effective hurdle technology for deli meat processors. 60 References Brooks, J. T., Sowers, E. G., Wells, J. G., Greene, K. D., Griffin, P. M., Hoekstra, R. M., Strockbine, N. A. (2005). Non-O157 shiga-toxin producing Escherichia coli infections in the United States, 1983-2002. Journal of Infectious Disease, 192, 1422-1429. Byelashov, O., Daskalov, H., Geornaras, I., Kendall, P., Belk, K., Scanga, J., Smith, G., & Sofos, J. (2010). Reduction of Listeria monocytogenes on frankfurters treated with lactic acid solutions of various temperatures. Food Microbiology, 27(6), 783- 790. Carlson, B., Ruby, J., Smith, G., Sofos, J., Bellinger, G., Warren Serna, W., Centrella, B., Bowling, R., & Belk, K. (2008). Comparison of antimicrobial efficacy of multiple beef hide decontamination strategies to reduce levels of Escherichia coli O157:H7 and Salmonella. Journal of Food Protection, 71(11), 2223-2227. Carpenter, C. E., Smith, J. V., & Broadbent, J. R. (2011). Efficacy of washing meat surfaces with 2% levulinic, acetic, or lactic acid for pathogen decontamination and residual growth inhibition. Meat Science, 88(2), 256-260. Choi, Y. M., Kim, O. Y., Kim, K. H., Kim, B. C., & Rhee, M. S. (2009). Combined effect of organic acids and supercritical carbon dioxide treatments against nonpathogenic Escherichia coli, Listeria monocytogenes, Salmonella typhimurium and E. coli O157:H7 in fresh pork. Letters in Applied Microbiology, 49(4), 510-515. FDA. (2012). Bad bug book, foodborne pathogenic microorganisms and natural toxins. (Second ed.). Access Date January 3, 2014. http://www.fda.gov/downloads/food/foodborneillnesscontaminants/ucm297627.p df. Geornaras, I., Yang, H., Manios, S., Andritsos, N., Belk, K. E., Nightingale, K. K., Woerner, D. R., Smith, G. C., & Sofos, J. N. (2012a). Comparison of decontamination efficacy of antimicrobial treatments for beef trimmings against Escherichia coli O157:H7 and 6 Non-O157 shiga toxin-producing E. coli serogroups. Journal of Food Science, 77(9), M539-M544. Geornaras, I., Yang, H., Moschonas, G., Nunnelly, M. C., Belk, K. E., Nightingale, K. K., Woerner, D. R., Smith, G. C., & Sofos, J. N. (2012b). Efficacy of chemical interventions against Escherichia coli O157:H7 and multi-drug resistant and 61 antibiotic-susceptible Salmonella on inoculated beef trimmings. Journal of Food Protection, 75(11), 1960-1967. Gill, C. O., & Badoni, M. (2004). Effects of peroxyacetic acid, acidified sodium chlorite or lactic acid solutions on the microflora of chilled beef carcasses. International Journal of Food Microbiology, 91(1), 43-50. Huffman, R. D. (2002). Current and future technologies for the decontamination of carcasses and fresh meat. Meat Science, 62(3), 285-294. Muriana, P. M., Quimby, W., Davidson, C. A., & Grooms, J. (2002). Postpackage pasteurization of ready-to-eat deli meats by submersion heating for reduction of Listeria monocytogenes. Journal of Food Protection, 65(6), 963-969. Podolak, R. K., Zayas, J. F., Kastner, C. L., Fung, D. Y. C. (1995a). Inhibition of Listeria monocytogenes and Escherichia coli O157:H7 on beef by application of organic acids. Journal of Food Protection, 59(4), 370-373. Podolak, R. K., Zayas, J. F., Kastner, C. L., Fung, D. Y. C. (1995b). Reduction of Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella Typhimurium during storage on beef sanitized with fumaric, acetic, and lactic acids. Journal of Food Safety, 15, 283-290. Pohlman, F. W., Dias-Morse, P. N., Quilo, S. A., Brown, A. H., Crandall, P. G., Dias Morse, P. N., Baublits, R. T., Story, R. P., Bokina, C., & Rajaratnam, G. (2009). Microbial, instrumental color and sensory characteristics of ground beef processed from beef trimmings treated with potassium lactate, sodium metasilicate, peroxyacetic acid or acidified sodium chlorite as single antimicrobial interventions. Journal of Muscle Foods, 20(1), 54-69. Scallan, E., Hoekstra, R. M., Angulo, F. J., Tauxe, R. V., Widdowson, M. A., Roy, S. L., Jones, J. L., & Griffin, P. M. (2011). Foodborne illness acquired in the United States?major pathogens. Emerging Infectious Diseases, 17(1), 7-15. Sharma, C., Williams, S., Schneider, K., Schmidt, R., & Rodrick, G. (2012a). Antimicrobial effects of sodium metasilicate against Listeria monocytogenes. Foodborne Pathogens and Disease, 9(9), 822-828. Theron, M. M., & Lues, J. F. R. (2007). Organic acids and meat preservation: a review. Food Reviews International, 23(2), 141-158. USDA, F. S. I. S. (2003). Control of Listeria monocytogenes in ready-to-eat meat and poultry products; final rule. Federal Register, 68, 34208-34254. USDA, F. S. I. S. (2011). Shiga toxin-producing Escherichia coli in certain raw beef products. Federal Register, 76(182), 58157-58165. 62 USDA, F. S. I. S. (2013). Safe and suitable ingredients used in the production of meat, poultry, and egg products. Access Date January 3, 2014. http://www.fsis.usda.gov/OPPDE/rdad/FSISDirectives/7120.1.pdf. Wang, Luxin, and Harris, L.J. 2011. Rdar morphotype and its relationship to desiccation tolerance in Salmonella spp. International Association for Food Protection, Milwaukee, Wisconsin, USA, July 31-August 3. Weber, G., O'Brien, J., & Bender, F. (2004). Control of Escherichia coli O157:H7 with sodium metasilicate. Journal of Food Protection, 67(7), 1501-1506. Yoder, S., Henning, W., Mills, E., Doores, S., Ostiguy, N., & Cutter, C. (2012). Investigation of chemical rinses suitable for very small meat plants to reduce pathogens on beef surfaces. Journal of Food Protection, 75(1), 14-21. Youssef, M. K., Yang, X., Badoni, M., & Gill, C. O. (2012). Effects of spray volume, type of surface tissue and inoculum level on the survival of Escherichia coli on beef sprayed with 5% lactic acid. Food Control, 25(2), 717-722. 63 Table 11. Strains of microorganisms used Microorganism ATCC number or ID Code Source Escherichia coli O157:H7 ATCC 35150 Human ? HC Escherichia coli O157:H7 ATCC 43894 Human ? HC Escherichia coli O157:H7 AU ? 1 Laboratory strain (301) Escherichia coli O157:H7 AU ? 2 Laboratory strain (505B) Escherichia coli O157:H7 AU ? 3 Laboratory strain Non-O157 STEC (O145) TWO9356 Human ? HUS Non-O157 STEC (O26) TWO7814 Human ? HUS Non-O157 STEC (O121) TWO8039 Human Non-O157 STEC (O45) TWO14003 Human Non-O157 STEC (O111) TWO7926 Human ? HC Non-O157 STEC (O103) TWO8101 Human Salmonella AU ? Enteritidis Laboratory strain Salmonella AU ? Kentucky Laboratory strain Salmonella AU ? Montevideo Laboratory strain Salmonella AU ? Thompson Laboratory strain Salmonella AU ? Stanley Laboratory strain Listeria monocytogenes ATCC 49594 Petite Scott A Listeria monocytogenes ATCC 19115 Human ? Serotype 4b Listeria monocytogenes ATCC 7644 Human Listeria monocytogenes AU ? 4 Laboratory strain (101M serotype 4b) Listeria monocytogenes AU ? 5 Laboratory strain (108M serotype 1/2b) 64 Table 12. pH values of lactic acid at 4% (LA4), sodium metasilicate at 4% (SM4), and distilled water. Solution pH LA4 1.84 SM4 12.82 Distilled Water 4.90 65 Table 13. Microbial effects of 4% lactic acid (LA) and 4% sodium metasilicate (SM) on Escherichia coli O157:H7 at 4?C on fresh beef bottom round steaks and fresh pork ham steaks after pathogen inoculation and a 30min contact time. Treatment Meat Mean log10 CFU/cm2 SEMz Control Beef 6.98 a 0.08 Pork 6.91a 0.08 LA Beef 5.93 bc 0.08 Pork 6.12b 0.08 SM Beef 5.51 d 0.08 Pork 5.71cd 0.08 a, b, c, d Means lacking a common superscript differ (P < 0.05). z Standard Error of the Mean 66 Table 14. Microbial effects of 4% lactic acid (LA) and 4% sodium metasilicate (SM) on non-O157 STEC at 4?C on fresh beef bottom round steaks and fresh pork ham steaks after pathogen inoculation and a 30min contact time. Treatment Meat Mean log10 CFU/cm2 SEMz Control Beef 6.97 a 0.08 Pork 6.93a 0.08 LA Beef 5.91 b 0.08 Pork 5.86b 0.08 SM Beef 5.66 b 0.08 Pork 5.86b 0.08 a, b Means lacking a common superscript differ (P < 0.05). z Standard Error of the Mean 67 Table 15. Microbial effects of 4% lactic acid (LA) and 4% sodium metasilicate (SM) on Salmonella spp. at 4?C on fresh beef bottom round steaks and fresh pork ham steaks after pathogen inoculation and a 30min contact time. Treatment Meat Mean log10 CFU/cm2 SEMz Control Beef 6.67 a 0.16 Pork 6.39a 0.16 LA Beef 4.48 c 0.16 Pork 4.52bc 0.16 SM Beef 5.13 bc 0.16 Pork 5.17b 0.16 a, b, c Means lacking a common superscript differ (P < 0.05). z Standard Error of the Mean 68 Table 16. Microbial effects of 4% lactic acid (LA) and 4% sodium metasilicate (SM) on Listeria monocytogenes at 4?C on deli roast beef, ham, and turkey with and without a post packaging lethality treatment. Treatment Meat Mean log10 CFU/cm2 SEMz Control Without Hot Water Dip Roast Beef 7.41a 0.48 Ham 7.33a 0.48 Turkey 7.26a 0.48 LA Without Hot Water Dip Roast Beef 6.84a 0.48 Ham 6.86a 0.48 Turkey 7.03a 0.48 SM Without Hot Water Dip Roast Beef 7.01a 0.48 Ham 7.05a 0.48 Turkey 6.91a 0.48 Control With Hot Water Dip Roast Beef 1.50b 0.48 Ham 1.89b 0.48 Turkey 2.01b 0.48 LA With Hot Water Dip Roast Beef 0.91b 0.48 Ham 1.29b 0.48 Turkey 2.46b 0.51 SM With Hot Water Dip Roast Beef 1.16b 0.48 Ham 1.76b 0.48 Turkey 1.65b 0.48 a, b Means lacking a common superscript differ (P < 0.05). z Standard Error of the Mean 69 Chapter VII Implications and Conclusions Sodium metasilicate, 4% and lactic acid, 4% were determined to be the lowest concentrations most effective against all the tested microorganisms. While in some cases such as Salmonella spp. lower concentrations were equally effective; in order to have the greatest impact on all tested bacteria the higher concentration of 4% was used. By utilizing the lowest concentration of antimicrobial solution necessary to achieve effective pathogen reduction, meat processors can provide a safe and wholesome meat supply. Meat processors can apply lactic acid or sodium metasilicate at refrigeration temperatures and obtain the same benefits of applying them at a higher temperature. Combining the two antimicrobial solutions is not recommended as it is ineffective against pathogens. However, individually both solutions can serve as a hurdle technology in meat processing facilities. Both lactic acid and sodium metasilicate can be applied to fresh beef and pork as an effective hurdle technology in the fight for food safety. Treating deli meats with lactic acid or sodium metasilicate did not reduce L. monocytogenes loads. However, adding a hot water treatment was able to minimize microbial contamination. Results of the first three studies indicate that lactic acid and sodium metasilicate are viable options for hurdle technologies beyond the abattoirs. The final study shows that neither antimicrobial is a good option for controlling L. monocytogenes in processed meats; 70 however, by applying a post-packaging hot water dip in combination with either treatment they could provide an effective hurdle technology for deli meat processors.