1 ANTIMICROBIAL EFFICACY OF FLOWER EXTRACT FROM ALPINIA GALANGA (LINN.) SWARTZ AGAINST LISTERIA MONOCYTOGENES AND STAPHYLOCOCCUS AUREUS IN A READY TO EAT TURKEY HAM PRODUCT By MELISSA E. CADET A THESIS PRESENTED TO THE GRAD UATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIR EMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2012
2 2012 Melissa Cadet
3 To mother and father; thank you for the enlightenment that allowed me to strive for the best. To my family and friends, thank you for your moral support. Above all, I am grateful to God for helping me throughout this journey.
4 ACKNOWLEDGEMENTS I would like to express my appreciation to the U niversity of Florida Graduate School Office of Graduate Minority Programs for providing me with a Florida Agricultural and Mechanical University Feeder Fellowship in order for me to complete my studies. I would like to thank Dr. Sally K. Williams for her d edicated support and generous guidance. I also extend appreciation towards my committee members, Dr. Amy Simonne and Dr. Keith Schneider, who offered valuable advice regarding my research. I also extend special gratitude to the Department of Animal Science s for their support and assistance during this research.
5 TABLE OF CONTENTS page ACKNOWLEDGEMENTS ................................ ................................ ................................ ............. 4 LIST OF TABLES ................................ ................................ ................................ ........................... 7 ABSTRACT ................................ ................................ ................................ ................................ ..... 8 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .................. 10 2 LITERATURE REVIEW ................................ ................................ ................................ ....... 12 Microbiology of Poultry ................................ ................................ ................................ ......... 12 Processing Plant Conditions ................................ ................................ ............................ 12 Pathogenic and Spoilage Microflora ................................ ................................ ............... 13 Spoilage Organisms in Poultry ................................ ................................ ........................ 13 Problems Associated with Pathogens in Poultry ................................ ............................. 13 Gram Ne gative Pathogens in Poultry ................................ ................................ .............. 14 Salmonella spp ................................ ................................ ................................ ........ 14 Campylobacter spp. ................................ ................................ ................................ .. 15 Gram Positive Pathogens in Poultry ................................ ................................ ................ 15 Listeria monocytoge nes ................................ ................................ ............................ 15 Listeria monocytogenes: Listeriosi s ................................ ................................ ......... 16 Listeria monocytogenes : Foods implicated ................................ .............................. 17 Listeria monocytogenes : Problems a ssociated with L. monocytogenes ................... 17 Staphylococcus aureus : Characteristics and growth conditions .............................. 19 Staphylococcus aureus : Staphylococcal food poisoning ................................ ......... 20 Staphylococcus aureus: Outbreaks ................................ ................................ .......... 21 Ingredients Currently Used to Control G rowth of Pathogens in Ready to Eat Meat and Poultry Products ................................ ................................ ................................ .................. 21 Natural Herbs as Antimicrobials ................................ ................................ ..................... 22 The use of galangal extract in food products ................................ ........................... 22 Antimicrobial properties of galangal extract ................................ ............................ 23 Other Nat ural Herbs as Antimicrobials in Food Products ................................ ............... 25 Further Research Based on Literature Review ................................ ................................ ....... 27 3 MATERIALS AND METHODS ................................ ................................ ........................... 28 Preparation, Cultivation, and Storage of Inoculum ................................ ................................ 28 Preparation of Galangal Extract for Turkey Ham Samples ................................ .................... 29 Sample Preparation, Inoculation and Treatment ................................ ................................ .... 29 Microbiology, pH, and L*a*b* Color Analyses ................................ ................................ ..... 30 Objective Color Analysis ................................ ................................ ................................ ........ 31 Data Anal ysis ................................ ................................ ................................ .......................... 31
6 4 RESULTS AND DISCUSSION ................................ ................................ ............................. 34 Microbiology ................................ ................................ ................................ .......................... 34 Total Plate Count ................................ ................................ ................................ ............. 34 Staphylococcus aureus Plate Count ................................ ................................ ................. 37 Listeria monocytogenes Plate Count ................................ ................................ ............... 39 pH and Objective Color Analyses ................................ ................................ .......................... 40 pH ................................ ................................ ................................ ................................ .... 40 Color L*a*b* ................................ ................................ ................................ ................... 40 5 SUM MARY AND CONCLUSION ................................ ................................ ....................... 46 LIST OF REFERENCES ................................ ................................ ................................ ............... 48 BIOGRAPHICAL SKETCH ................................ ................................ ................................ ......... 55
7 LIST OF TABLES Table page 3 1 Formulation for Turkey Hams ................................ ................................ ........................... 33 4 1 Least square means for the interactions of treatment combined storage time for to tal plate count in turkey hams in trial 1 ................................ ................................ ................... 41 4 2 Least square means for the interactions of treatment combined storage time for total plate count in turkey hams for trial 2 ................................ ................................ ................. 41 4 3 Least square means for the interactions of treatment combined storage time for total plate count in turkey hams for trial 3 ................................ ................................ ................. 42 4 4 Least squ are means for the interactions of treatment combined storage time for S. aureus plate count in turkey hams ................................ ................................ ..................... 42 4 5 Least square means for the interactions of treatment combined storage time for L. monocytogenes plate count in turkey hams ................................ ................................ ....... 43 4 6 Least square means for the interactions of treatment combined storage time for pH in turkey hams ................................ ................................ ................................ ........................ 43 4 7 color values in turkey hams ................................ ................................ ............................... 44 4 8 Least square means for the interactions of tr color values in turkey hams ................................ ................................ ............................... 44 4 9 color values in turkey hams ................................ ................................ ............................... 45
8 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science ANTIMICROBIAL EFFICACY OF FLOWER EXTRACT FROM A LPINIA GALANGA (LINN.) SWARTZ. AGAINST LISTERIA MONOCYTOGENES AND STAPHYLOCOCCUS AUREUS IN A READY TO EAT TURKEY HAM PRODUCT By Melissa Cadet August 2012 Chair: Sally K. Williams Major: Animal Sciences The increase in the consumption of ready to e at ( RTE) meats over the last decade has resulted in an increase in the number of illness outbreaks associated with these foods The objective s of this study were to determine the antimicrobial efficacy of galangal flower extract on a RTE turkey ham inoculated with Listeria monocytogenes and Staphylococcus aureus and stored at 4C, and ascertain the effects of the galangal extract (GE) on pH and objective color of the ham. Seven treatments were evaluated over a period of four w eeks. The treatments included 1 u ncooked sample, 2 cooked sample with no galangal extract (negative control), 3 inoculated cooked sample (positive control), 4 inoculated cooked sample with 0.5% galangal extract, 5 inoculated cooked sample with 1.0% galangal extract, 6 inoculated cooked sample with 0.5% galangal extract (post cooked), 7 inoculated cooked sample with 1.0% galangal extract (post cooked). In treatments 3 through 5, the galangal extract was incorporated into the raw mixture and cooked. In treatments 6 and 7, the gal angal extract was applied to the meat after it was cooked. After treatment all samples were vacuum packaged, s tored at 4 1C for 28 days and analyzed at 1 wk intervals. Samples were analyzed for pH, total plate count, L. monocytogenes, S. aureus, and c olor (L*A*B*).
9 The data revealed that t he GE treatments resulted in no significant (P > 0.05) log reductions against L. monocytogenes and S. aureus The counts for both organisms were similar (P > 0.05) for all samples treated with 0.5 and 1.0% GE (treatme nts 4 through 7) when compared to the positive control. However, turkey samples treated with 0.5% GE prior to cooking resulted in a one log cfu/gram reduction (P > 0.05) in S. aureus on day 0, and a one log cfu/g reduction (P > 0.05) in L. monocytogenes on days 0 when compared to the positive control. Although not significantly different (P > 0.05) S. aureus counts for treatments 4 and 5 were lower (P > 0.05) than the positive control (treatment 3) on day 0. Treatment 4 also had lower counts (P > 0.05) than treatment 6 of 0.40 or more log cfu/g for S. aureus on days 0, 7, 21, 28 and up to 1.87 log cfu/g in L. monocytogenes on all days. Treatment 5 had lower counts (P > 0.05) compared to treatment 7 of 0 .20 or more log cfu/g for S. aureus on days 0, 14. 21, 2 8 and 0.20 or more log cfu/g for L. monocytogenes on days 0, 14, 28. The raw samples (treatment 1) and cooked untreated samples (treatment 2) were lower (P > 0.05) than all treated samples through the 28 days of storage. This study was also used as a model to determine the best application method for the GE. Based on the bacterial counts for total plate count, S. aureus and L. monocytogenes the galangal extract would be more effective as an ingredient incorporated into the meat mixture prior to cooking co mpared to applying the GE on the surface of the cooked meat as a post processing treatment.
10 CHAPTER 1 INTRODUCTION Listeria monocytogenes and Staphylococcus aureus are two pathogens that are widely found in ready to eat (RTE) meat products. These orga nisms possess characteristics that will allow them to survive or even multiply. L. monocytogenes has high tolerance for salt and the ability to grow in foods that have low pH (Bell and Kyriakides, 2005). S. aureus is capable of producing exotoxins that are responsible for staphylococcal food poisoning (Dinges et al., 2000). The post contamination of S. aureus occurs solely during processing and packaging (Tassou et al., 2007). Increasing demand and consumption of RTE foods over the past two decades resulted in great economic importance for the RTE food industry (Cutler et al., 2003). However, the increase in the consumption of RTE foods has resulted in higher numbers of outbreaks involving these types of food products. The outbreaks have result ed from cross contamination of contaminated raw products, processing equipment or food handlers, or a combination of the three. Although, many of the RTE products are cooked to eliminate pathogens such as L. monocytogenes S. aureus and Salmonella, recontamination coul d possibly occur during post processing and packaging (Farber and Peterkin, 1999). These organisms have raised concerns in the food industry due to their mortality rates and implication in numerous cases. In 1999, it was reported by the Centers for Disease Control and Prevention (CDC) that L. monocytogenes had the second highest fatality rate of 20% and the highest hospitalization rate of 90% (Gallagher, 2003). Currently, RTE meat products are often being recalled due to the contamination of L. monocytogene s or S. aureus (Lindenberger, 2011; Cochran, 2011). To reduce the incidents of outbreaks and recalls in RTE meat products, researchers are investigating the use of natural herbs and spices that possess antimicrobial properties against
11 Gram positive organi sms such as L. monocytogenes and S. aureus Spices and herbs are common for their antimicrobial and antioxidant properties, as well as their flavors in foods. Some of the commonly used spices and herbs are clove, cinnamon, oregano, and rosemary, which are considered to have strong antimicrobial activity (Weerakkody et al., 2010). Other spices and herbs are being investigated for their antimicrobial characteristics, one of which is Alpinia galanga (Linn.) Swartz. Alpinia galanga flower is an herb that has be en commonly used i n Indian and Asian countries as medicine and as a flavorful spice in foods (Hsu et al., 2010). Parts of the distinctive herb are being viewed as an effective natural herb that will minimize Gram positive pathogens in RTE foods (Cheah and Gan, 2000 ; Hsu et al., 2010 ). The objective s of this project were to observe the antimicrobial efficacy of the galangal flower extract on a RTE turkey ham product inoculated with L. monocytogenes and S. aureus and stored at 4C, and ascertain the effects of the galangal extract on pH and objective color of the ham.
12 CHAPTER 2 LITERATURE REVIEW Microbiology of Poultry Processing Plant Conditions Over the past few decades the consumption of poultry products has doubled, which has led to a massive increase in animal produc tion ( USDA, 2011; Seo and Bohach, 2007). I ncreased poultry production has raised concerns of both contaminations with human and animal pathogens by consumers and government officials. One o f the most significant solution s used to solve this contamination problem was the rapid transition from handcraft operations (i.e., the use of manual tools) between the 1950s and 1960s to mechanical processing (Bolder, 1998). This change has influenced the outcome of the finished product. The guidelines of Hazard Analysis Critical Control Point (HACCP) were also another preventative measure used to control the contamination of microbiological and chemical hazards on the final product (Bolder, 1998). As a manufacturer, one of the factors that must be consid ered is the design and production of the machines bei ng used in the production of poultry products. Proper facility design will allow the production flow to avoid finished product contact with the raw materials. Placing cleaning stations throughout the pla nt also allows the employees and the production workers to maintain proper hygiene. This will also reduce or limit the transfer of pathogens such as S. aureus to the food prod ucts (Schmidt and Erickson, 2005 ). The same factors would apply to smaller poultr y producers, to ensure that their line workers are healthy. The use of convenient machines, such as moveable equipment for easy cleaning, also plays a major role in the production aspect. Producers must take into consideration that the machines should have the capability to be cleaned efficiently, even if it requires disassembling (Goddard, 2011). Both large and s mall producers share the desire to ensure safety for its consumers. Food producers would
13 consider food safety to be a significant fact or due to th e possible undesirable outcomes of a recall or outbreak, which would result in tremendous economic loss and bad media exposure for the company. Pathogenic and Spoilage Microflora Poultry is known to have a very complex microflora, which can be found in th e intestinal system based on the production methods being used. Live conventional poultry are raised in large flocks on litter floors, which can lead to contamination with pathogens such as Salmonella, Campylobacter, Listeria, E. coli, Clostridium, and S. aureus (Kotula and Pandya, 1995). Spoilage Organisms in Poultry Spoilage microorganisms such as Pseudomonads, lactic acid bacteria, and yeasts tend to be present in live animals and are potentially being transferred to their meat products. Additionally, s poilage organisms from the environment such as those in the water supply of the processing plant can also contribute to the added contamination of spoilage microorganisms. These spoilage organisms have resistant characteristics that can survive normal chlo rinated water treatments. Although during some processing steps such as scalding, these organisms can be destroyed, recontamination can occur during subsequent stages throughout the processing steps because these organisms favor wet surfaces (Mead, 1989). Problems A ssociated with P athogens in P oultry There are a number of factors that influence the contamination of poultry products. Some of these factors begin with hatching of the live bird to the final dressing of the carcass, or even further processing t steps that are critical in these production processes is the evisceration of the bird. A challenge that many producers face is trying to avoid tearing the intestines, which would caus e fecal contamination of the carcass. Another challenge would be to avoid cutting the esophagus to
14 prevent microbial contamination from leakage (Fanatico, 2003). These factors can lead to recalls or foodborne outbreaks in the poultry processing industry. T o control pathogens such as L. monocytogenes and S. aureus in the poultry industry, techniques such as good manufacturing practices, environmental sanitation, HACCP, pasteurization, and post processing treatments are being used. Current research focuses on taking a more natural approach with the use of natural herbs a nd spices that will help decrease or control the growth of these organisms. These spices and herbs have antimicrobial properties that are used in the product formula tion and post cooking marina des. Gram Negative Pathogens in Poultry Salmonella and Campylobacter are the two major pathogen species that remain of greatest concern in the poultry industry. Another concern in the poultry industry is the antimicrobial resistance of these poultry relat ed pathogens Salmonella spp Most of the Salmonella found in poultry products has the capability of causing hu man infection which can lead to acute diseases. An acute disease is a rapid onset infection. Two of the common types of Salmonella that are famil iar in poultry products are Salmonella Enteritidis and Salmonella Typhimurium. Salmonella has been found in different parts of the poultry Salmonella Enteritidis contamination usua lly takes place in the egg or during post production cross contamination (Simonsen, 1989). Salmonella spp. are facultative anaerobic, Gram negative, rod shaped bacteria that belong to the Enterobacteriaceae family. The species contains approximately 2,500 serotypes but only about 10 are responsible for human illness called salmonellosis. Salmonellosis is a disease that is transmitted from animals to man, but is primarily acquired through the
15 consumption of contaminated food products. Classic symptoms of th e disease include stomach pain, diarrhea, headache, and fever accompanied by chills. The bacteria can grow at 37C, but 2007). Salmonella can grow within a pH range of 3.6 to 9.5 with an optimum range of 6.5 to 7.5 (Marriott and Gravani, 2006). Campylobacter spp. Campylobacter is also a Gram negative, spirally curved, motile, rod shaped bacterium. It is a microaerophilic bacterium, which means it requires little to no oxygen to survive. The bacteria can grow in 3 to 5% oxygen and 2 to 10% carbon dioxide. Campylobacter can survive up to 4 weeks in 4C under moist, reduced oxygen conditions (Nachamkin, 2007). The two common Campylobacter spp. that is commonly found in the poultry industry are Campylobacter jejuni and Campylobacter coli. Campylobacter has been isolated primarily on the sur face of the carcasses, up to 10 6 cfu/carcass. Unlike S almonella that can survive in various foods such as seafood where as Campylobacter prefers the gastrointestinal tract of warm blooded animals. Campylobacter also t ends to multiply more in microbial cou nts than most other pathogens (Mbata, 2010). Gram Positive Pathogens in Poultry Listeria monocytogenes L. monocytogenes is a facultative anaerobic Gram positive, non spore forming, rod shaped bacterium that can be found in soil, water, and vegetation (R ocourt and Buchrieser, 2007; ICMSF 1996). Compared to Gram negative organisms, Gram positive organisms such as L. monocytogenes have a thicker peptidoglycan layer in their cell wall, which allows them to survive in an intracellular environment. The bacter ium was first isolated in the 1920s from rabbits and guinea pigs; however, it was not until the early 1980s that the significance of foods
16 was being observed as the primary route of transmission for human exposure to L. monocytogenes (ICMSF, 1996; WHO and FAO, 2004). Unlike other major foodborne microorganisms, L. monocytogenes can grow to significant numbers at refrigeration temperatures within hours (WHO and FAO, 2004). The organism can also survive for several weeks at 1 8C in various food substrates (G olden et al., 1988) but does not survive well under acidic environments (El Kest and Yousef, 2006). L. monocytogenes can sometimes survive without reproduction at 4C, however, it can grow well in sterile minced meat at 4C or naturally contaminated minced meat at 20C (ICMSF, 1996). Recent observations have shown that L. monocytogenes survives in f oods th at are stored at refrigeration for extended periods of time, having potential for contamination, and providing nutrients for the organism to survive (Wall s, 2005). The organism requires minimal oxygen in order to grow; therefore, it survives for long periods of time in the environment, foods, processing plants, and household refrigerators. Contamination of the organism in RTE foods normally occurs from cont act with contaminated equipment such as brine chill chambers, slicers, peelers, conveyors, and packaging machines (Tompkin, 2002). L. monocytogenes is considered to be an environmental contaminant that has been isolated from many natural surroundings such as water, soil, sewage, mud, and animals (Donnelly et al., 1992). Listeria monocytogenes: L isteriosi s L. monocytogenes is known to cause listeriosis especially in immuno compromised individuals, pregnant women, the elderly and infants, which are at a highe r risk. This disease can result in abortions, stillbirths, septicemia, meningitis, encephalitis, and death (Nelson et al., 2004). In the US, L. monocytogenes is responsible for approximately 2,500 cases of listeriosis each year, 91% of which are hospitaliz ation and 20% fatality (Mead et al., 1999; Scallan et al., 2011). Approximately one third of those cases involve pregnant women and their unborn or
17 newly born infants (ICMSF, 1996). Symptoms that are common in perinatal cases include mild fever in the moth er with or without gastroenteritis symptoms, but can result in major consequences such as meningitis or death for the fetus or newborns. Other symptoms that can occur in cases that are not perinatal are bacteremia, which is the presence of bacteria in the blood, and meningitis (ICMSF, 1996). Listeria monocytogenes : F oods i mplicated L. monocytogenes gained its name from its ability to infect the monocytes (white cells) in the blood (ICMSF, 1996). It is the main human pathogen of the Listeria genus that is re sponsible for a number of foodborne outbreaks of listeriosis that are related to (RTE) foods such as milk, soft ripened cheeses, coleslaw, and vacuum packaged meats (Ooi and Lorber, 2005). Listeria monocytogenes is a pathogen that is prevalent in the poul try industry. The organism is the leading cause of mortality and morbidity in humans. The pathogen can be found in a variety of foods especially in poultry products. A high dose (10 9 cfu) of this organism can cause human listeriosis, which is a rare diseas e (Mbata, 2010). The contamination of this pathogen normally occurs through the cross contamination of turkey or chicken in which the organism can spread to cooked and RTE foods. L. monocytogenes can be commonly found in natural environments and in the int estinal tract of animals. In the US, the organism has been isolated in approximately 6% of poultry carcass rinses and in 31% of poultry ground meat (USDA FSIS, 1997). Cooking can destroy the organism; however, recontamination can occur during post cooking techniques. Listeria monocytogenes : P roblems a ssociated with L monocytogenes Recalls. L. monocytogenes usually triggers class 1 type recalls, which are defined by the Food and Drug Administration (FDA) as a situation that has an exposure to a product tha t can cause adverse health consequences or death (FDA, 2012). RTE meat products are being recalled
18 due to possible contamination of L. monocytogenes In 2011, there was a total of 11 recalls issued by the USDA FSIS due to possible L. monocytogenes contamin ation, and six of which were RTE meat products. When the organism first emerged as a public health problem associated with deli meats and other processed foods, USDA L. monocytogenes in RTE food s. Any products that contain the organism at any level would be considered adulterated under the Federal Meat Inspection Act (FMIA) or the Poultry Products Inspection Act (PPIA) (USDA FSIS, 1998). One of the most recent meat recalls occurred in August 2011 for diced bacon products that was contaminated with the pathogen (Lindenberger, 2011). L. monocytogenes has also been associated with large outbreaks as well. Most of the large foodborne outbreaks of listeriosis have been associated with three serogroups of L. monocytogenes ; 1/2a, 1/2b, or 4b (Bell, 2000; Nelson et al., 2004). Outbreaks Over the last decade, there have been a number of outbreaks associated with L. monocytogenes which has resulted in chronic illnesses or even death in humans and more tha n 40 animal species (Nightingale et al., 2005). One of the earliest outbreaks associated with L. monocytogenes was in Germany in 1949, and was linked to the consumption of contaminated raw milk. A multi state outbreak that resulted in plant contamination with L. monocytogenes serotype 4b infected RTE hot dogs and deli meat that was produced by Sara Lee. This outbreak resulted in a total of 21 deaths, 15 of which were adults and the remaining 6 were miscarriages/stillbirths. The company issued a voluntary r ecall o n 35,000,000 lbs of meat, but was only able to recover 5,918,795 lbs (USDA FSIS, 1998; CDC, 1999). L. monocytogenes serotype 4b has been the most isolated serotype in many food outbreaks. This strain was involved in a multistate outbreak involving c ontaminated hot dogs and
19 deli meat in 1998 to 1999, which led to at least 50 illnesses where six adults died and two pregnant women had spontaneous abortions (CDC, 2001). Another case that involved L. monocytogenes serotype 4b was a multistate outbreak in November of 2002 involving RTE turkey deli meat. This outbreak resulted in 54 illnesses. Out of the 54 reported cases, eight people died and three pregnant women had spontaneous abortions (Gottlieb, 2006). Based on the number of outbreaks that have occurre d, it can be concluded that L. monocytogenes poses a serious threat to the RTE food supply that is produced throughout the world Staphylococcus aureus : C haracteristics and growth conditions The presence of S. aureus in poultry product s is an indication of improper handling techniques. This organism is normally found on the outer surfaces of the bird as well as the sinuses and lungs. Harry (1967) found that 49% of 276 chickens and turkeys from 162 farms were positive for S. aureus in the skin and nasal si nuses. This poses a threat to processed products such as deboned poultry meat that is used to produce chicken franks and hot dogs that are combined with other muscle meats. With the level of contamination that is found in live birds, this organism can be f ound in various parts of the processed carcass. Roberts (1972) isolated S. aureus from 65 of 172 frozen chicken carcasses that were processed from four different farms. Some of the S. aureus strains that are isolated from chickens or turkeys produce entero toxins, which are probably of human origin. The enterotoxins are initiated through the handling of cooked foods in the processing kitchen by an infected individual and subsequent temperature abuse. These toxic organisms have the ability to grow more rapidl y in cooked poultry products than in raw poultry products (Mead, 1989). Staphylococcus aureus is also a common microorganism that is associated with numerous foodborne outbreaks. S. aureus is a normal harmless resident flora of the human skin and nasal mem branes (John, 2004). It is carried by a third of the human population. However, the growth
20 of S. aureus in foods poses a threat since many of the strains produce enterotoxins that can cause food poisoning when ingested. Sir Alexander Ogston first identifie d the bacterium during a (Bell, 2000). S. aureus is a Gram positive, spherical bacterium. It can be transmitted into the food through many sources, including foo d handlers. The bacteria is unable to grow at refrigerated temperature, however, if abused it may multiply in foods such as irradiated cooked hams (Cabeza et al., 2010). S. aureus is a non spore forming bacteria that is resistant to radiation (Erdman et al ., 1961). S. aureus is also a facultative anaerobic organism that is the most common cause of staph intoxications The organism can grow in a temperature range between 7 and 48C and pr oduce enterotoxin from 10 to 48 C, with an optimum enterotoxin producti on at 40 to 45C. The pH for growth of the organism ranges between 4.0 and 9.8, with an optimum pH of 6.0 to 7.0 (Marriott and Gravani, 2006 ). S. aureus tends to grow in a non competitive environment that contains high concentrations of salt (Seo and Bohac h, 2007). Staphylococcus aureus : S taphylococcal f ood p oisoning Staphylococcal food poisoning (SFP), which is caused by S. aureus, is one of the most predominant causes of gastroenteritis in the world. This food poisoning results in the production of staph y lococcal enterotoxins (SEs). SFP is an intoxication that does not involve infection by, and growth of, the bacteria in the host (Seo and Bohach, 2007). SFP symptoms usually take place within a few hours of ingestion of toxin contaminated food, depending o n the toxic dose. These symptoms include nausea, abdominal cramps, diarrhea, and vomiting. The symptoms of SFP do not result in fatality, however, it has been reported that there is an average of two deaths per year due to SFP (Mead et al., 1999). Some str ains of the bacterium can produce exotoxin TSST 1, which is the causative agent of toxic syndrome. Other strains of S. aureus can produce enterotoxin, which can cause gastroenteritis in humans. Staphylococcal enterotoxins are resistant
21 to heat and cannot b e distinguished in infected foods (Seo and Bohach, 2007). S. aureus organisms can be destroyed through the use of heat, 66C for 12 min, however, the enterotoxins require heating for 30 min at 131C to inactivate (Marriott and Gravani, 2006). Staphylococc us aureus: O utbreaks Outbreaks that have been associated with this organism usually result in cross contamination of foods that are contaminated or food handlers that are infected with S. aureus One of the major multistate outbreaks occurred in 1998. This outbreak resulted in 225 ill persons who consumed a contaminated ham salad (CDC, 2010). Outbreaks that have been associated with S. aureus are not known to be as deadly or fatal as L. monocytogenes. CDC (2010) has reported a number of outbreaks that has taken place over the past decade but only a few have resulted in deaths. One of those outbreaks occurred in a nursing facility in South Dakota in July 2000. The source of the outbreak was consumption of chicken salad, which resulted in 95 ill persons. Out of the total ill persons, ther e were 13 people hospitalized, two of which resulted in death. Ingredients Currently U sed to C ontrol G rowth of Pathogens in Ready to Eat Meat and Poultry Products Poultry producers are currently using a number of antimicrob ial methods to reduce, retard, or eliminate the growth of L. monocytogenes and/or S. aureus Antimicrobials such as nisin have been used to remove or reduce bacterial populations in lean and fat pork (Nattress et al., 2001) and RTE turkey hams (Ruiz et al. 2010) through direct addition. Nisin has also been used to inhibit spoilage and pathogenic bacteria on RTE ham and bologna (Gill and Holley, 2000). Another form of antimicrobial that is commonly u sed in the poultry industry is chlorinated rinses. A chlori nated rinse usually contains 18 30 ppm of chlorine and must be maintained at 160F for approximately 2 hr (USDA FSIS, 2006). Marsden et al. (2000) used a
22 combination of 1,200 ppm sodium chlorite and 0.9% citric acid on Little Smokies sausages to reduce L. monocytogenes. This resulted in a 1.2 log reduction of L. monocytogenes. The proper u se of food handling and avoid ing cross contamination are good method s of prevention for S. aureus To destroy the organism, proper cooking techniques must be u sed (Marriot t and Gravani, 2006 ). Natural Herbs as Antimicrobials The use of galangal extract in food products Alpinia galanga (L.) is a member of the flowering plant family Zingiberaceae, which is comprised of approximately 1,200 species including ginger. The plant o riginated in tropical parts of Asia such as India, Malaysia, and Indonesia (Raina et al., 2002; Wong et al., 2009), but has been cultivated worldwide in places such as the United States (Hsu et al., 2010). Alpinia galanga, commonly known as the greater gal angal, is a species that is widely used in the foreign countries mentioned above in a variety of traditional cures and in foods as an ingredient. The rhizome of the plant is used as spice and food flavoring agent, whereas the leaves are consumed as vegetab les (Wong et al., 2009). The rhizome is also used for medicinal purposes to treat diseases such as fungal skin infections, intestinal infections, stomachaches, diarrhea, vomiting, and rheumatism (Wong et al., 2009; Hsu et al., 2010). The galangal plant is approximately 4 7 ft tall. It proliferates from underground rhizomes. The plant produces flowers in clusters of 2 5 and contains stems and leaves that are precisely hairy on the bottom (Staples and Kristiansen, 1999). In terms of cultivation, the plant re quires sunny or moderately shady locations to grow, which limits its availability to the summer months of the year. The plant also requires fertile and moist soil for best results. Once the plant has reached maximum size, it is harvested three months after planting for market purposes (Peter, 2004).
23 Antimicrobial properties of galangal e xtract The rhizomes of A. galanga consists of terpenoids with 1,8 cineol, which is an antimicrobial compound found in other natural herbs such as rosemary and lemon bark (We idner et al. 2003). Terpenoid compounds are created from acetate units that are originated from fatty acids (Cowan, 1999; Weidner et al., 2003). A number of studies have shown that the greater galangal plant has antimicrobial properties. Some of which were performed using the agar disc diffusion method for potential antimicrobial activity against L. monocytogenes and S. aureus (Hsu et al., 2010). Hsu et al. (2010) used the agar disc diffusion method to determine antimicrobial activities of oven dried or fre eze dried galangal flowers extracted in 190 proof ethanol or hexane against Salmonella E. coli O157:H7, L. monocytogenes S. aureus and Shigella. Pure bacterial cultures were swab inoculated on to surfaces of pre hardened Mueller Hinton agar Discs infu sed with the galangal extract solution were then placed on the surface of the incubated plates and incubated at 37C fo r 18 24 hr. Results showed that the galangal extracted in the ethanol showed a broad spectrum of antimicrobial activity against Gram posi tive bacteria, but showed little to no antimicrobial activity against the Gram negative bacteria. Amongst all of the bacteria that were being tested in this study, S. aureus and L. monocytogenes showed the most sensitivity against the test extracts. The ov en dried flowers extracted in ethanol showed an inhibition zone that ranged from 26 to 31 mm. However, a larger inhibition zone was illustrated in L. monocytogenes with the freeze dried samples extracted with hexane. This study also showed that there was a higher yield percentage of the extracts from the oven dried samples than the freeze dried samples. The oven dried samples produced in ethanol had a higher yield than those extracted using hexane. The rhizome of A. galanga has also been shown to be effect ive in certain meat products. In a study conducted by Cheah and Gan (2000) to observe the antioxidant properties of A.
24 galanga in minced beef, it was observed that there were antimicrobial activities in cooked beef instead of the raw beef. The study mentio ns that there was a 0.71 log cfu/g reduction in the total aerobic bacteria plate count The type of solvent used to extract the plant plays a major role in its antimicrobial activity. Extraction of the galangal plant using hexane showed greater results tha n the extraction using ethanol or water, which could be due to its non polar properties (Hsu et al., 2010; Weerakkody et al., 2010). Other studies have also shown that S. aureus a Gram positive organism, is more sensitive to the galangal extract than Gram negative organisms such as E. coli The results obtained by Weerakkody et al. (2011) are in agreement with other studies where the use of galangal extracts resulted in inhibition against S. aureus (Oonmetta aree et al., 2006). Oonmetta aree et al. (2006) used A. galanga extracted in 100% ethanol to observe its antimicrobial properties against S. aureus and E. coli With a larger inhibition zone measured for S. aureus using agar disc diffusion assay, S.aureus was shown to be more sensitive to the extract th an E. coli The minimum inhibitory concentration (MIC) of galangal extract that was used to determine the sensitivity of the bacteria was 0.325 mg/ml. The use of natural herbs in food products has been known for decades. These antimicrobials are used in f oods as food preservatives to control spoilage and to prevent and control the growth of microorganisms. Natural herbs have been well known for their antimicrobial and antioxidant properties. They also have the ability to add desirable intense flavors to fo ods (Uhl, 2000). Galangal extract has been used in a wide variety of food products. It is commonly used in Thai or Asian dishes. Meat and poultry producers are making the attempt to use the natural herbs in their products due to its effective antimicrobial properties against food borne pathogens such as L. monocytogenes and S. aureus Cheah and Gan (2000) conducted a
25 study that revealed the effects of galangal extracted in acetone on raw minced beef samples evalua ted for storage stability over seven days. T hree different levels of galangal treatment were employed : 0.02%, 0.05%, and 0.10% (wt/wt, fat basis). The total plate counts for the raw minced beef with the galangal extract were gradually lower than the control, which contained no galangal, over the sev en day period of this study. There was a log reduction of 0.1 cfu/g over a seven day period. This illustrates that the addition of galangal extend the shelf life of meat products. Cheah and Hasim (2000) stated that the galangal extract exhi bited antimicrob ial pr operties, which resulted in a 0.71 log reduction Other N atural H erbs as A ntimicrobials in F ood P roducts Due to the higher demands for foods that are produced with minimal chemical preservatives, natural herbs have become more appealing to food prod ucers and their consumers. This is a challenge that food producers have taken into consideration to use natural occurring food antimicrobials and antioxidants to reduce the use of chemical preservatives. Some of the most commonly used spices and herbs are rosemary, cinnamon, oregano, and cloves (Weerakkody et al., 2010). Like galangal, rosemary is a well known herb that has antimicrobial activities against foodborne pathogens due to its compounds. Studies have shown that combinations of galangal and rosemar y had synergistic antimicrobial activity against S. aureus and L. monocytogenes (Weerakkody et al., 2011) and antioxidant effects in food products. Weerakkody et al. (2010) conducted a study to compare the antimicrobial activities of four different spices and herbs including goraka ( Garcinia quaesita ), galangal ( Alpinia galanga ), lemon iron bark ( Eucalyptus staiger ana ) and mountain pepper ( Tasmannia lanceolata ) to three common spices including pepper ( Piper nigrum ), rosemary ( Rosmarinus officinalis ), and o regano ( Oreganum vulgare ). These spices were extracted in water, hexane, and ethanol and tested against four different foodborne pathogens: E. coli Salmonella Typhimurium, L.
26 monocytogenes and S. aureus using the agar disc diffusion and broth dilution me thod. Each 18 hr. The different solvent types showed great antimicrobial activity with the exception of P. nigrum which showed little to no activity. Each herb was applied to each bacterium individually at different usage levels, and then as four different combinations. The A. galanga extracted in hexane showed a larger inhibition zone (34.1 mm) against S. aureus compared to the other bacteria that were tested. L monocytogenes was inhibited by A. galanga hexane extract at a MIC of < 0.625 mg/ml after 24 hr, but the MIC increased to 1.25 mg/ml 48 hr later. The MIC levels of A. galanga against S. aureus were < 0.625 mg/ml and showed no difference after 24 hr. Ethan ol extracts of all the spices and herbs showed antimicrobial activity against the Gram positive bacteria, except for P. nigrum and T. lanceolata. L. monocytogenes was inhibited by A. galangal extracted in hexane and E. staigerana extracted in ethanol. The spices and herbs had a stronger effect on the Gram positive microorganisms than the Gram negatives, which used higher levels of the spices and herbs. As previously discussed, this is possibly due to the differences in the cell wall between Gram negative a nd Gram positive bacteria. Unlike the Gram negative bacteria, antimicrobial substances can penetrate through Gram positive bacterial cell wall and attack the cytoplasmic membrane, which causes leakage of the cytoplasm. One of the most effective herbs was t he A. galanga in the hexane and ethanol extracts against S. aureus and L. monocytogenes. This study showed that the extraction of these spices and herbs contained components that have different modes of antimicrobial actions. The study also showed that the phenolic compound had little to no effect on the antimicrobial activity of these spices and herbs. The spices and herbs that were
27 extracted in the ethanol and water seemed to have a higher total phenolic content than those extracted in hexane. Further Re search Based on Literature Review This literature review revealed that natural herbs and spices have potential as antimicrobials for use in the food industry, especially in meats. The studies in this review have revealed that the use of natural spices and herbs have the ability to reduce microbial activities i n certain RTE foods. Further research is needed to evaluate the antimicrobial properties of A. galangal in RTE meats such as turkey hams. Turkey ham is a popular meat product that is lower in fat, comp ared to the traditional pork ham (USDA FSIS, 2001). This product is fabricated from boneless, turkey thigh meat with all skin and surface fat removed. The objectives of this project were to determine the antimicrobial efficacy of galangal extract on a RTE turkey ham inoculated with L. monocytogenes and S. aureus, stored at 4C, and to ascertain the effects of the galangal extract on pH and objective color of the hams.
28 CHAPTER 3 MATERIALS AND METHOD S Preparation, Cultivation, and Storage of Inoculum Two ref erence strains of L. monocytogenes ATCC 51772 (Serotype 1/2a) and S. aureus ATCC 8095 were obtained from ABC Research Corporation in Gainesville, FL, and used as the inoculum in this stud y. Each strain was received on tryptic soy a gar (TSA, DF 0369 17 6, D ifco Laboratories, Detroit, MI) slants and transferred to four test tubes containing 10 mL of tryptic soy broth (TSB, DF 0370 17 3, Difco Laboratories, Detroit, MI) using a sterile disposable 3 mm inoculation loop. The tubes were incubated at 35C for 24 h r. After incubation, the cultures were poured into sterile 15 mL centrifuge tubes and centrifuged (RC 5 Super speed Centrifuge, Sorvall SS 34 Rotor, Dupont Instruments, Newton, CT) at 5000 rpm for 10 min. The supernatant was discarded and the pellets were resuspended in 10 mL of deionized/distilled water and recentrifuged. The supernatant was discarded and the pellets were resuspended in 1 mL of 3% TSB with 30% glycerol in a 2 mL cryovial (Cat. No. 03 374 2, Corning Incorporated, Corning, NY), stored at 45 C and used as the stock culture for the inoculation studies. A total of four vials were prepared. Twenty four hours prior to conducting the study, one tube of each of the individual strains was removed from the freezer and allowed to thaw for 10 min. A lo opful of the cultures from each strain was transferred and mixed into a test tube containing 10 mL of 3% TSB, vortexed, and incubated at 35C for 24 h. After incubation, the cultures were centrifuged at 5000 rpm for 10 min and washed with 0.1% sterile buff ered peptone water (BPW, Cat. No. DF O1897 17 4, Difco Laboratories, Detroit, MI) and serially diluted with BPW to concentrations of 10 1 to 10 8 and plated on TSA.
29 Pr eparation of Galangal Extract for Turkey Ham Samples Dried galangal flower powdered samp les, which were obtained from the galangal flowers from a local producer in Gainesville, FL, were extracted using 190 proof ethanol (moderately polar, spectrophotometric grade, Acros Organics, Fair Lawn, NJ, USA). Ten grams of the dried sample were extract ed using 150 mL of ethanol and shaken on an orbital shaker at room temperature for 24 h. After extraction, samples were filtered through Whatman No. 1 filters (Whatman International Ltd., Maidstone, UK) using B chner funnels to obtain clear filtrates. All filtrates were dried under reduced pressure at 40C using a rotary evaporator (B chi, Labortechnik AG, Flawil, Switzerland). The extraction yield (%) was calculated using the ratio of the final yield of dried extract (g)/10 g of original dried galangal sam ples and converted to a percentage (%). After the extract was completely dried, ethanol extract was reconstituted in ethanol solvent by adding 6mL of ethanol to the flask to obtain a stock solution of 300 mg/mL, yielding 6 mL of the extract. Galangal Extr disc filter (Millipore, Bilerica, MA, USA) and stored in sterile vial at 20C. A total of two vials were prepared for this experiment (Hsu et al. 2010). Sample Preparation, Inoculation and Trea tment Commercially available case ready ground turkey was purchased from a local supermarket as soon as the shipment arrived at the store and was used in this study. The ground turkey was labeled with a sell by date of at least 30 d ay The ground turkey pa cks were immediately transported to the research laboratory in transportable coolers and stored in a walk in cooler (4 1C) and used within 24 h r The ground turkey was divided into 7 different presterilized trays, 150 g each, to produce seven turkey ham treatments. The turkey ham treatments included the following:
30 1. Raw meat, no GE, no inoculum (negative control, raw meat only) 2. No GE + cook, no inoculum (effect of cooking only) 3. Cooked meat, no GE + inoculum (positive control) 4. 0.5% GE + cook, then inoculate (effect of cooking and 0.5% GE) 5. 1.0% GE + cook, then inoculate (effect of cooking and 1.0% GE) 6. Cook, then 0.5% GE, then inoculate (effect of adding 0.5% GE after cooking) 7. Cook, then 1.0% GE, then inoculate (effect of adding 1.0% GE after cooking) The L. monocytogenes and S. aureus cocktail was used as the inoc ulum for Treatments 3 through 7. Treatments 1 7 were formulated as outlined in Table 2 1 and mixed in pre sterilized sterile Ziploc bags. With the exception of Treatment 1, all samples were cooked in a water bath for approximately 30 min or until the internal temp erature of the meat reached 74 C (USDA recommended). Treatment 1 was not cooked and used as a control. Once cooked, the 6 treatments were allowed to cool for 8 10 min at room temperature. Treatments 3 7 were 8 cfu/mL inoculum. Inoculated samples were left for 20 min to allow bacterial attachment to ensure final concentration o f 10 4 cfu/g. Predetermined quantities of the cooked inoculated turkey ham were aseptically weighed and placed into prelabeled vacuum bags (FoodSaver bags, T150 00011 002, 164.232 cc/m2/24 hr @ 23C, 0.334 cc/m2/24 hr @ 23C). All samples for each treatment were then placed into individual Gallon sized Ziploc freezer bags and stored at 4 1C in a refrigerator for 28 d ay Duplicate samples per treatment were analyzed after 0, 7, 14, 21, and 28 d ay for aerobic plate count (APC), L monocytogenes, S. aureus p H, and color using a Hunter colorimeter. Aerobic plate counts were performed on d ay 0 to monitor sanitation and to ensure no cross contamination during sample preparation. Microbiology, pH, and L*a*b* C olor Analyses Eleven grams of turkey ham was transfer red aseptically from the vacuum bag to a sterile stomacher bag (01 002 44, Fischer Scientific) containing 99 mL of sterile 0.1% BPW (DF
31 O1897 17 4, BD Diagnostics, Sparks, MD) and was agitated for 60 s. The appropriate serial dilutions were prepared by tra nsferring 1.0 mL of the sample homogenate to 9 mL of sterile BPW. One microliter of the dilutions was pipetted onto prepoured modified Oxford agar plates (DF0225 17 0, BD Diagnostics) with Oxford media supplement (DF0214 60 9, BD Diagnostics) for L. monocy togenes, mannitol salt agar plates (MSA, R453902, Remel Inc., Lenexa, KS) for S. aureus and tryptic soy agar for total plate count. All plates were incubated for 48 h at 35 1C. After incubation, colony forming units from each plate were counted, record ed, averaged, and reported as log colony forming units per gram (cfu/g). Prior to microbiology analyses, pH was recorded for each sample using a pH meter (Accumet Basic, Model No. AB15). The pH probe was placed into the sample homogenate and allowed to eq uilibrate for 1 min before the reading was taken. All pH readings were performed in duplicate. Objective Color Analysis Vacuum packed samples were evaluated for color using the Miniscan XE plus Hunter Colorimeter (Cat. No. 4 320, Fischer Scientific) for d ay 0 28. Each treatment contained two vacuum packed samples that were scanned to yield L*, a*and b* values. The two L*, a*and b* values for each treatment were averaged using the colorimeter. The L* value measures from 100 (white) to 0 black. The a* and b* values have no numerical limits, but a positive a value is a measure of the redness in the sample and a negative a value is a mea sure of greenness. A positive b value is a measure of the yellowness in the sample and a negative b value is a measure of blueness (Hunter Lab, 2001). Data Analysis The data analysis of this experiment was designed using JMP Pro 9.0.2 (64 bit Edition, 70108654, 2010). A complete block design with seven treatments and two replications was used
32 to evaluate pH, microbiologic al analysis, and color analysis. A total of 140 samples were analyzed for pH, microbiology, and color over a course of five weeks (0, 7, 14, 21, 28 d). To obtain the standard errors of the mean (SEM), the General Linear Model and least squared means (LSM) were used. The SEM was used to analyze the differences between the treatment means The Multivariate Analysis of Variance (MANOVA) was used to determine the differences among treatments, storage days, and treatment by stor age day interaction. The T pairwise comparison was used to compare the treatment means.
33 Table 3 1. Formulation for Turkey Hams No GE (g) 1 0.5% GE (g) 2 1.0% GE (g) 3 Raw 4 Meat 150 150 150 150 Salt 2.25 2.25 2.25 -Sugar 1.13 1.13 1.13 -Sod ium Trypolyphosphate (STPP) 0.60 0.60 0.60 -Modern Cure 0.4 0.4 0.4 -Na Erythorbate 0.1 0.1 0.1 -Water 15 15 15 -Galangal Extract -0.75* 1.50* -1 Used for Treatments 2 and 3. 2 Used for Treatments 4 (GE added prior to cooking process) and 6 ( GE was added after cooking process)*. 3 Used for Treatments 5 (GE added prior to cooking process) and 7 (GE was added after cooking process)*. 4 Used for Treatment 1, which remained uncooked
34 CHAPTER 4 RESULTS AND DISCUSSI ON The main objective of this study wa s to evaluate the antimicrobial efficacy of the galangal extract on a RTE turkey ham product inoculated with L. monocytogenes and S. aureus stored at 4C, while observing the color and pH stability of the product. Microbiology Total Plate Count The dat a reveale d significant differences (P < 0.05) between trials 2 and 3, and no difference between trials 1 and 2, and 1 and 3 (P > 0.05). Significant treatment by time, and time and treatment differences were revealed. The total plate counts contained less t han 8 log cfu /g during the st orage period of 4 weeks (Table 4 1, 4 2, 4 3) for all trials. In some cases, the ham samples that were treated with the galangal extract illustrated bacteriostatic properties. Bacteriostatic properties are found in agent s that are used to prevent the growth of bacteria, keeping them in a stationary growth phase (Pankey and Sabath, 2004). The difference in trials 2 and 3 indicated that there was a time effect for each treatment, a treatment effect among the d ifferent treatments and a time by treatment interaction (P < 0.05). Except for day 7, i n trial 3, there was a significant (P < 0.05) decrease in bacterial counts in treatments 4 and 5 comp ared with the positive contro l on all storage days (Table 4 3) On day 7, only treatm ent 4 was lower (P < 0.05) than the positive control. A similar d ecrease in total plate counts was revealed for treatments 4 and 5 on d ays 14 and 28 when compared to positive control. The treatment difference can be largely attributed to the decrease in t otal plate count for treatment 4 and 5 when compared to the positive control on all storage days in trial 3 and days 14, 21, and 28 in trial 2. The time effect was due largely to the increa se in total plate counts after 2 8 days when compared to day 0 in al l trials. There appears to be a bacteriostatic effect. Trial 2
35 (Table 4 2) showed that there were no significant (P > 0.05) decreases on day 0. Although there was a difference of bacterial counts in the treatments, both trials illustrated that there was at least a 1 or less than 1 log difference of bacterial counts over time. The trials also showed that there was a higher decrease in bacterial counts in treatments 4 and 5 than in treatments 6 and 7, with the exception of day 14 in trial 2 and day 0 and 7 in trial 3. Trial 1 shows higher bacterial counts compared to trials 2 and 3. Trial 1 used turkey that was purchased as thigh meat and converted to ground turk ey, whereas, trials 2 and 3 used ground turkey that was already prepared prior to sample preparati on. However, the bacterial counts of the raw material (treatment 1) were higher in trials 2 and 3 compared to trial 1. According to the Nationwide Raw Ground Turkey Microbiological Survey (NRGTMS) ( USDA FSIS, 1996) the initial microflora that is present on the chilled carcasses of the animal primarily comprises of gram negative aerobic psychrot r ophic bacteria. Once the meat is grinded, the surface area bacteria are distributed throughout the product. This article also shows that meats that are processed at a larger scale produce more pathogens than those that are processed in smaller quantities. The thigh meat that was used in trial 1 was grounded in a smaller batch, whereas; trials 2 and 3 were commercially grounded on a larger scale. The NRGTMS (1996) show ed that out of the 296 samples that were observed, 100% of t he samples tested positive for aerobic plate c ounts (APC) Based on the results that were obtained from this observation, raw ground turkey contains 4.15 c fu /g of APC (USDA FSIS, 1996). Freshly pr ocessed carcasses can contain 10 3 to 10 4 cfu /cm 2 microorganisms (USDA FSIS, 1997). Trial 1 showed that the re was a time effect for each treatment over time a treatment effect between the different treatments, and a time*treatment interaction (P < 0.05) Table 4 1 shows that there was no significant difference in treatments 3 t h rough 7 on day 0, however, there
36 is a 2 and 1 log increase between treatments 3 and 4 and 3 and 5, respectively. This shows that the galangal extract induce microbial effect on th e treated samples compared to treatment 3. Day 7 shows that there was a significant decrease (P < 0.05) in treatments 6 and 7 compared to treatment 3. Treatment 3 shows bacteriostatic properties with the exception of days 7 and 28. Treatment 4 shows that there is a significant decrease (P < 0.05) over time where there is a log difference of more than 1 with the exception of day 7 and 28. The galangal extract has a bacteriostatic effect on Treatment 4 because there is a log decrease in the last two weeks c ompared to the first two weeks of the study, whereas, in treatment 3 no decrease was observed in the last two weeks compared to the first two. Treatment 5 shows that there is a significant difference (P < 0.05) in all of the days, with the exception of day s 7 and 28. Treatment 6 shows no significant difference (P > 0.05) amongst the days. Treatment 7 shows significant decrease (P < 0.05) between days 7 and 14. There is also a significant decrease (P < 0.05) on days 21 and 28 in treatment 7. Overa ll, table 4 1 shows that the extract had no effect on the treatments compared to the positive control, however, the extract seemed to stabilize growth over time. Trial 2 also showed that there was a time effect of each treatment, a treatment effect between the differ ent treatments, and a time*treatment interaction (P < 0.05). Table 4 2 shows that there was no significant difference in treatments 3 through 7 on day 0 as shown in trial 1. This again shows that the extract has no effect on the treated ham samples compare d to the positive control. Day 7 shows that treatments 4 and 5 were not significantly different (P > 0.05) to treatment 3; whereas, treatments 6 and 7 were significantly different (P < 0.05) to treatment 3. Treatments 6 and 7 have higher bacterial counts t han treatments 4 and 5 compared to the positive control. Over time, treatments 3, 4, and 5 showed a 1 or less log decrease from day 0 to day 7; whereas, treatments 6 and 7 showed a less than 1 log increase from day 0 to 7, which could be a
37 result of bacter iostatic. Treatments 3 through 7 ended up with higher counts on day 28 compared to day 0 and 7. There was no significant decrease (P < 0.05) in treatment 3 between day 0 and 7, but an increase on days 14, 21 and 28. Treatment 4 and 7 showed no significant difference (P > 0.05) with the exception of an increase on day 28 in treatment 7. Treatment 5 showed that there were no significant difference (P > 0.05) between day 0 and 21 and day 14 and 28. However, day 7 showed a significant (P < 0.05) decrease from d ay 0 in treatment 5. Treatment 6 was significantly increase (P < 0.05) on days 0, 7, 14 and 28; but showed a decrease on day 21. Trial 3 also showed that there was a time effect of each treatment, a treatment effect between the different treatments, and a time*treatment interaction (P < 0.05). Table 4 3 shows that there was a log or more reduction in treatments 4 through 7 compared to treatment 3 on day 0. Treatment 4 has a less (P < 0.05) bacterial count compared to treatment 6 on days 0, 7, 14, 21 and 28. This illustrated that the GE had an effect on treatment 4 compared to the positive control. Treatment 3 showed a significant decrease (P < 0.05) between day 0 and 7; but showed an increase on the bacterial counts on day 14. Treatment 5 showed a significan t decrease (P < 0.05) on day 14 compared to days 0, 7, 21 and 28. Treatment 6 showed a significant increase (P < 0.05) from day 0 compared to all of the other days and that there was at least a 1 or more log increase. Treatment 7 showed a significant incre ase (P < 0.05) from day 0. Both treatments 4 and 5 seemed to show stability in bacterial counts over time compared to treatments 3, 6, and 7, which showed a bacterial increase. The galangal treatment in treatments 4 and 5 showed bacteriostatic activity com pared to the other treatments. Staphylococcus aureus P late Count S. aureus counts were not significantly different (P > 0.05) among the treatments when compared to the positive control on all storage days (Table 4 4). The data revealed no significant tim e and time by treatment interaction (P > 0.05) (Table 4 4). A significant treatment difference
38 was revealed which was due to the significantly lower S aureus counts (P < 0.05) for the uncooked and cooked negative controls on all storage days when compared to all the GE treatments. In general there was treatment difference among the treatments when compared to the negative controls Day 0 showed no significant difference (P > 0.05) in S. aureus between the treatments with the exception of the negative cont rol and uncooked samp le (treatments 1 and 2) (Table 4 4). Treatment 4 and 5 was one l og lower (P > 0.05) than the positive control on day 0. Treatments 4 and 5 also had lower (P > 0.05) counts than treatments 6 and 7 on days 0, 21 and 28. This illustrates that the method of application of the galangal extract is a major factor. The galangal extract seems to have a more effective outcome as an ingredient in the formulation of the product than as a post treatment after cooking. Treatments 4 and 5 had a var iat ion of bacterial counts over t ime, which indicates that there was less than 0.5 log variation between the days with the exception of day 14 in treatment 4. The counts in treatment 5 through 7 were higher (P > 0.05) than the positive control on days 7. Days 7 through 28 showed that the extract had no significant difference (P > 0.05) or effect on the treatments 4 through 7. However, there was a decrease in bacterial counts in treatment 4 on day 7, 14 and 28 when compared to day 0. There is also a decrease in bacterial count s (P > 0.05) in treatment 5 on day 14 and 28. Comparing each treatment over the 4 week period, there was no significant difference (P > 0.05) in the bacterial counts, which shows that there were effects on each treatment over time. The nega tive controls had similar (P > 0.05) S. aureus counts, which remained less than 2 log cfu /gram, through 28 days storage. This also illustrates that the bacterial count remained less than 2 log cfu / gram and the survival phase of the bacteria has been extend ed over the storage time (Todar, 2012).
39 Studies have shown that S. aureus does not grow well with lactic acid bacteria present (Haines and Harmon, 1973). However, the organism has the ability to grow well in its preferred environment. S. aureus favors a te mperature of 7 47 C and optimum temperature of 37 C (Marriott and Gravani, 2006 ). Listeria m onocytogenes Plate Count The data revealed no significant difference (P > 0.05) in L. monocytogenes co unts among the trials (Table 4 5). The data also revealed significant time and treatment differences and significant time*treatment interaction In the time by treatment effect, the counts of L. monocytogenes increased over the storage days. The time difference was due to the increase in L. monocytogenes. For tr eatments 3 and 6, the counts increased on day 28 when compared to day 0. Table 4 5 reveals that there was no significant difference (P > 0.05) between the positive control and treatments 5 through 7 on day 0. However, treatment 4 had 1.87 log reductions co mpared to the positive control. Day 14, 21 and 28 also showed a 1 or more log reduction in treatments 4 and 5 compared to the positive control. Treatments 6 and 7 did not show a decrease in the bacterial counts compared to the positive control with the exc eption of treatment 7 on day 7, 14 and 21. Over time, treatments 3 increased less than 2 logs on day 28 compared to day 7. The other treatments also increased in bacterial counts, but not as much as the positive control. This indicates that bacterial count in treatment 3 has multiplied ( Whiting and Bagi, 200 2) compared to its beginning stages on day 0. The other treatments (4 through 7) that were treated with the galangal extract showed a lower (P > 0.05) increase due to the addition of the galangal extract compared to the positive control. Treatments 6 and 7 showed a higher (P > 0.05) bacterial count than treatments 4 and 5, which seems to be influenced by the method of application. The organism has the ability to survive or grow in vacuum packaged refriger ated meats. (Glass and
40 Doyle, 1989), which is what influenced the bacterial count increase in treatments 3 through 7 from day 0 to day 28. pH and Objective Color Analyses pH The pH values showed no significant difference (P > 0.05) among treat ments and ov er time f rom days 0 to 28 (Table 4 6). These findings revealed that the galangal extract had no effect on the pH of the RTE turkey hams. A study conducted by Glass and Doyle (1989) on the effects of pH in processed meats inoculated with 10 5 cfu/gram of L. monocytogenes showed that the microorganism grew well on meats near or above pH 6. Color L*a*b In general, the color values showed no significant difference (P > 0.05) amongst the treatments (Tables 4 7, 4 8, and 4 nd 50 in all of the treatments, which indicates the galangal extract had no effect on the black and whiteness of the o significant difference (P > 0.05) among the samples treated with the galangal extract when compared to the positive control. The no significant difference (P > 0.05) in all of the treatments, with the exc eption of treatment 3 and 4 on day 21 when compared to the raw sample (treatment 1) and treatm ents 1 and 2 on day 28 There was significant difference (P < 0.05) in the treatments 1, 2, 4, 5 and 7 on day 0 compared to day 7, 14, 21 and 28; but not a difference (P > 0.05) amo ng the positive control and treatment 6.
41 Table 4 1. Least square means f or the interactions of treat ment combined storage time for total plate count in turkey hams in trial 1 (Log cfu /g) Treatments* Day 0 Day 7 Day 14 Day 21 Day 28 1 3.85 b,w 4.36 c,w 5.52 d,v 6.16 b,uv 6.34 c,u 2 2.77 b,uv 3.34 d,u 1.00 e,v 1.00 c,v 1.00 d,v 3 6.6 6 a,w 7.89 a,u 6.94 a,vw 6.91 ab,vw 7.25 ab,v 4 8.16 a,u 8.25 a,u 6.68 b,vw 6.46 b,w 7.05 b,v 5 7.65 a,v 8.25 a,u 6.25 c,x 7.28 a,w 7.71 a,u 6 7.02 a,u 7.00 b,u 6.93 a,u 7.56 a,u 7.28 ab,u 7 6.90 a,uv 7.30 b,u 6.41 c,v 7.28 a,u 6.38 c,v SEM 0.31 0.09 0.04 0.14 0.11 a e means in same column with different superscripts are significantly different (p < 0.05) u x means in same row with different superscripts are significantly different (p < 0.05) *1=Raw meat (Negative Control), 2=No GE + cook, 3=Cooked meat, no GE + Inoculum (Pos itive Control), 4=0.5% GE + Inoculum, 5=1.0% GE + Inoculum, 6=0.5% GE (Post Treatment), 7=1.0% GE + Inoculum (Post Inoculum) SEM = Standard Error Mean Table 4 2. Least square means for the interactions of treatment combined storage time for total plate co unt in turkey hams for trial 2 (Log cfu /g) Treatment* Day 0 Day 7 Day 14 Day 21 Day 28 1 4.44 b,y 5.36 bc,x 7.29 a,u 6.93 a,v 6.62 bc,w 2 3.63 c,w 4.73 d,v 5.63 e,u 4.67 d,v 5.24 e,uv 3 5.86 a,v 4.86 d,w 6.83 b,u 6.96 a,u 7.12 ab,u 4 5.62 a,u 4.98 cd,u 5.77 de,u 5.59 c,u 5.59 de,u 5 5.57 a,v 4.85 d,w 6.30 c,u 5.46 c,v 6.22 cd,u 6 5.64 a,x 5.98 a,w 7.53 a,u 6.79 a,v 7.72 a,u 7 5.85 a,v 5.82 ab,v 6.17 cd,v 6.18 b,v 7.32 a,u SEM 0.09 0.08 0.08 0.10 0.12 a e means in same column with different superscripts are significantly different (p < 0.05) u y means in same row with different superscripts are significantly different (p < 0.05) *1=Raw meat (Negative Control), 2=No GE + cook, 3=Cooked meat, no GE + Inoculum (Positive Control), 4=0.5% GE + Inoculum, 5=1.0% GE + Inoculum, 6=0.5% GE ( Post Treatment), 7=1.0% GE + Inoculum (Post Inoculum) SEM = Standard Error Mean
42 Table 4 3. Least square means for the interactions of trea tment combined storage time for total plate count in turkey hams for trial 3 (Log cfu /g) Treatment* Day 0 Day 7 Da y 14 Day 21 Day 28 1 5.24 d,v 4.71 d,w 4.76 c,w 5.54 e,v 7.34 a,u 2 4.55 e,w 5.34 c,v 4.75 c,w 5.81 d,u 5.46 c,v 3 8.06 a,u 6.69 b,w 7.31 a,v 7.62 b,v 7.43 a,v 4 5.43 d,u 5.70 c,u 6.03 b,u 5.77 d,u 5.72 bc,u 5 6.64 b,u 6.52 b,u 5.47 bc,v 6.62 c,u 6.22 b,u 6 6.10 bc,v 7.22 a,u 7.45 a,u 7.97 a,u 7.24 a,u 7 5.60 cd,x 6.51 b,w 7.79 a,uv 8.00 a,u 7.31 a,v SEM 0.10 0.09 0.16 0.03 0.10 a e means in same column with different superscripts are significantly different (p < 0.05) u x means in same row with different superscripts are significan tly different (p < 0.05) *1=Raw meat (Negative Control), 2=No GE + cook, 3=Cooked meat, no GE + Inoculum (Positive Control), 4=0.5% GE + Inoculum, 5=1.0% GE + Inoculum, 6=0.5% GE (Post Treatment), 7=1.0% GE + Inoculum (Post Inoculum) SEM = Standard Error M ean Table 4 4. Least square means for the interactions of treatment combined storage time for S. aureus plate count in turkey hams (Log cfu /g) Treatment* Day 0 Day 7 Day 14 Day 21 Day 28 1 1.56 b,u 1.84 b,u 1.67 b,u 1.20 b,u 1.00 b,u 2 1.33 b,u 1.00 b,u 1.34 b,u 1.00 b,u 1.00 b,u 3 5.98 a,u 5.88 a,u 5.79 a,u 4.26 a,u 5.28 a,u 4 4.96 a,u 4.94 a,u 5.28 a,u 4.78 a,u 5.08 a,u 5 4.96 a,u 5.99 a,u 5.22 a,u 5.80 a,u 4.94 a,u 6 5.56 a,u 6.33 a,u 5.22 a,u 6.36 a,u 5.71 a,u 7 5.45 a,u 5.30 a,u 5.49 a,u 6.02 a,u 5.35 a,u SEM 0.39 0.41 0.38 0 .50 0.28 a b means in same column with different superscripts are significantly different (p < 0.05) u means in same row with different superscripts are significantly different (p < 0.05) *1=Raw meat (Negative Control), 2=No GE + cook, 3=Cooked meat, no G E + Inoculum (Positive Control), 4=0.5% GE + Inoculum, 5=1.0% GE + Inoculum, 6=0.5% GE (Post Treatment), 7=1.0% GE + Inoculum (Post Inoculum) SEM = Standard Error Mean
43 Table 4 5. Least square means for the interactions of treatment combined storage ti me for L. monocytogenes plate count in turkey hams (Log cfu /g) Treatment* Day 0 Day 7 Day 14 Day 21 Day 28 1 1.75 b,u 2.46 c,u 2.46 b,u 1.00 b,u 1.41 c,u 2 1.86 b,u 3.20 bc,u 1.77 b,u 1.67 b,u 1.47 c,u 3 5.53 a,w 5.82 a,vw 6.70 a,uv 6.98 a,u 7.23 a,u 4 3.66 ab,v 5.9 5 a,u 5.68 a,u 5.87 a,u 6.13 ab,u 5 5.37 a,u 5.72 a,u 5.23 a,u 5.80 a,u 5.90 b,u 6 5.59 a,v 5.72 a,v 6.88 a,uv 6.62 a,uv 7.06 ab,u 7 5.51 a,uv 4.93 ab,v 5.95 a,uv 5.91 a,uv 6.73 ab,u SEM 0.50 0.45 0.38 0.28 0.10 a c means in same column with different superscripts are s ignificantly different (p < 0.05) u w means in same row with different superscripts are significantly different (p < 0.05) *1=Raw meat (Negative Control), 2=No GE + cook, 3=Cooked meat, no GE + Inoculum (Positive Control), 4=0.5% GE + Inoculum, 5=1.0% GE + Inoculum, 6=0.5% GE (Post Treatment), 7=1.0% GE + Inoculum (Post Inoculum) SEM = Standard Error Mean Table 4 6. Least square means for the interactions of treatment combined storage time for pH in turkey hams (Log cfu /g) Treatment* Day 0 Day 7 Day 14 Day 21 Day 28 1 6.83 6.84 6.84 6.78 6.76 2 6.92 7.04 6.98 6.99 7.02 3 6.86 6.92 6.91 6.93 6.91 4 6.98 6.90 6.91 7.03 7.01 5 6.87 6.95 6.93 6.94 7.01 6 6.90 6.98 6.95 6.96 7.01 7 6.87 6.94 6.91 6.92 6.93 SEM 0.09 0.07 0.08 0.06 0.07 *1=Raw meat (Ne gative Control), 2=No GE + cook, 3=Cooked meat, no GE + Inoculum (Positive Control), 4=0.5% GE + Inoculum, 5=1.0% GE + Inoculum, 6=0.5% GE (Post Treatment), 7=1.0% GE + Inoculum (Post Inoculum) SEM = Standard Error Mean
44 Table 4 7. Least square mean s for the interactions of treatment combined storage time for color values in turkey hams (Log cfu /g) Treatment* Day 0 Day 7 Day 14 Day 21 Day 28 1 49.36 41.64 39.89 43.42 43.56 2 41.33 44.66 42.08 42.66 42.33 3 47.14 41.88 41.73 43.35 42.52 4 41.17 44.52 43.87 45.67 43.53 5 42.34 44.47 43.15 42.65 41.59 6 41.99 42.07 42.77 42.92 42.00 7 41.05 43.47 44.88 43.42 42.85 SEM 2.74 1.63 1.82 1.22 1.15 *1=Raw meat (Negative Control), 2=No GE + cook, 3=Cooked meat, no GE + Inoculum (Positive Contro l), 4=0.5% GE + Inoculum, 5=1.0% GE + Inoculum, 6=0.5% GE (Post Treatment), 7=1.0% GE + Inoculum (Post Inoculum) SEM = Standard Error Mean Table 4 color values in turke y hams (Log cfu /g) Treatment* Day 0 Day 7 Day 14 Day 21 Day 28 1 15.11 a,u 8.63 a,v 8.57 a,v 8.70 a,v 8.78 a,v 2 7.43 b,u 8.96 a,u 8.16 a,u 11.12 a,u 8.02 a,u 3 8.46 b,u 9.00 a,u 10.53 a,u 9.03 a,u 10.04 a,u 4 6.88 b,u 10.22 a,u 9.38 a,u 9.09 a,u 8.26 a,u 5 6.90 b,u 8.1 0 a,u 8.74 a,u 9.30 a,u 8.72 a,u 6 7.40 b,u 7.89 a,u 8.24 a,u 9.49 a,u 9.40 a,u 7 7.68 b,u 7.50 a,u 7.01 a,u 7.91 a,u 8.40 a,u SEM 1.06 0.77 1.37 1.07 0.95 a b means in same column with different superscripts are significantly different (p < 0.05) u v means in same column with different superscripts are significantly different (p < 0.05) *1=Raw meat (Negative Control), 2=No GE + cook, 3=Cooked meat, no GE + Inoculum (Positive Control), 4=0.5% GE + Inoculum, 5=1.0% GE + Inoculum, 6=0.5% GE (Post Treatment), 7=1.0% GE + Inoculum (Post Inoculum) SEM = Standard Error Mean
45 Table 4 color values in turkey hams (Log cfu /g) Treatment* Day 0 Day 7 Day 14 Day 21 Day 28 1 14.29 a,u 7.57 a,v 6.82 a,v 8.09 a,v 7.80 a,v 2 9.04 a 5.74 a,uv 4.24 a,v 6.42 ab,uv 4.16 b,v 3 11.52 a,u 5.06 a,u 5.72 a,u 5.34 b,u 5.60 ab,u 4 8.83 a,u 6.23 a,v 5.57 a,v 5.40 b,v 5.07 ab,v 5 9.80 a,u 6.01 a,v 6.57 a,v 6.43 ab,v 6.20 ab,v 6 9.39 a,u 5.21 a,u 5.05 a,u 5.70 ab,u 5.83 ab,u 7 9. 62 a,u 6.08 a,v 5.38 a,v 6.01 ab,v 6.84 ab,uv SEM 1.15 0.62 0.90 0.44 0.59 a b means in same column with different superscripts are significantly different (p < 0.05) u v means in same column with different superscripts are significantly different (p < 0.05) *1=Raw meat (Negative Control), 2=No GE + cook, 3=Cooked meat, no GE + Inoculum (Positive Control), 4=0.5% GE + Inoculum, 5=1.0% GE + Inoculum, 6=0.5% GE (Post Treatment), 7=1.0% GE + Inoculum (Post Inoculum) SEM = Standard Error Mean
46 CHAPTER 5 SUMMARY AND CONCLUSION The objective of this study was to observe the effects of galangal extract in RTE turkey hams against L. monocytogenes and S. aureus The study has shown that there was no significant (P > 0.05) effect against both organisms. However, treatm ent 4 (0.5% GE prior to cooking) showed stability in the bacterial counts over time. In general t urkey samples treated with GE prior to cooking resulted in 1 log cfu/gram reduction in (P > 0.05) S. aureus on day 0 and 1 log cfu/gram reductions (P > 0.05) in L. monocytogenes on days 0 (0.5% GE only), 14, 21 and 28 when comp ared to the positive control. Although the mechanisms of the GE have not yet been investigated, the antimicrobial effects of the extract seem to be more effective as an ingredient incorpo rated into the meat mixture when added prior to cooking as compared to applying the GE on the surface of the cooked meat (post processing) Other studies have shown that herbs that are extracted in ethanol have bactericidal activity against S. aureus and L monocytogenes (Hsu et al., 2010; Oonmetta aree et al 2006; Weerak k o dy et al., 2010, Weerakkody et al., 2011), however, these studies d id not factor in the composition of RTE meats seeing that th e disc diffusion method was used instead This study was u sed as a model to determine the best application method for the GE. The 1.0% maximum usage level of GE was insufficient to achieve at least 2 log reductions in L. monocytogenes and S. aureus in this study. In future studies the amount of GE should be incre ased from 1.0% to at least 5% (in 1 % increments) in order to ac hieve antimicrobial reductions in L. monocytogenes and S. aureus greater than 1.0 log. The goal is to achieve 5 log reductions while maintaining the inherent quality characteristics of the turk ey ham product. Cheah and Hasim (2000) used up to 10% galangal extract, produced from the galangal plant rhizome in cooked minced beef for antioxidant and antimicrobial properties. The
47 researchers achieved a reduction in total aerobic bacteria of 0.71 log cfu/gram. Galangal extract was produced from the galangal flower in our study. Future studies should also focus on bacterial count reductions during days 0, 7 and 14, which are the days when RTE meats are primarily consumed (MAFF, 2000).
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55 BIOGRAPHICAL SKETCH Melissa Cadet was born in Miami, Florida in 1986. In 2010, she was awarded a Bachelor of Science degree fr om the College of Engineering Sciences Technology and Agriculture at Florida Agricultural and Mechanical University in Tallahassee, Florida. She was awarded the Florida Agricultural and Mechanical University Feeder Fellowship at the University of Florida G raduate School Office of Graduate Minority Programs, to study for the Master of Science degree in the College of Agricultural and Life Sciences in the Department of Animal Sciences under the supervision of Dr. Sally K. Williams. Melissa will receive a M aster of Science degree in August 2012.