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LSU Historical Dissertations and Theses Graduate School

1994

Effects of Heat, Lactic Acid, and Modified Atmosphere Packaging Effects of Heat, Lactic Acid, and Modified Atmosphere Packaging

on Listeria Monocytogenes on Cooked Crawfish Tail Meat. on Listeria Monocytogenes on Cooked Crawfish Tail Meat.

Warren Joseph Dorsa Louisiana State University and Agricultural & Mechanical College

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Recommended Citation Recommended Citation Dorsa, Warren Joseph, "Effects of Heat, Lactic Acid, and Modified Atmosphere Packaging on Listeria Monocytogenes on Cooked Crawfish Tail Meat." (1994). LSU Historical Dissertations and Theses. 5692. https://digitalcommons.lsu.edu/gradschool_disstheses/5692

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Effects o f heat, lactic acid, and m odified atm osphere packaging on Listeria monocytogenes on cooked crawfish ta il m eat

Dorsa, Warren Joseph, Ph.D.

The Louisiana State University and Agricultural and Mechanical Col., 1994

UMI300 N. ZeebRd.Ann Arbor, MI 48106

EFFECTS OF HEAT, LACTIC ACID, AND MODIFIED ATMOSPHERE PACKAGING ON LISTERIA MONOCYTOGENES

ON COOKED CRAWFISH TAIL MEAT

A Dissertation

Submitted to the Graduate Faculty of the Louisiana State University and

Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of

Doctor of Philosophyin

The Department of Food Science

byWarren Joseph Dorsa

B.S., Nicholls State University, 1978 M.S., Mississippi State University, 1981

May 1994

ACKNOWLEDGEMENTS

The author would like to express gratitude to Dr. Douglas L. Marshall for his guidance while serving as chairman of his major professor. Dr. Marshall's constant thought provoking challenges provided him with the atmosphere he needed to conduct research in a manor conducive to doctorate work. He would also like to thank Dr. Robert M. Grodner, Dr. Michael W. Moody, Dr. John H. Wells, and Dr. Paul W. Wilson for their guidance while serving on his graduate committee.

Special thanks are extend to Harold Bennett and the Louisiana Crawfish Promotion and Research Board for aid in acquisition of product used in conducting my research.

He wishes to thank his friends and fiance Jessica, for their constant support, love, and encouragement during this undertaking. Finally, he wishes to dedicate this work to his parents Ted and Nancy, for without their unfaltering support, encouragement, and love throughout the years, his life would not be the joyous event it has become during the completion of this task.

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TABLE OP CONTENTS

pageACKNOWLEDGEMENTS..................................... i iLIST OF TABLES..... VLIST OF FIGURES..................................... viLIST OF ABBREVIATIONS AND SYMBOLS..................... viiiABSTRACT........................................... ixINTRODUCTION................... 1REVIEW OF LITERATURE..... 9

General Microbiology of L . monocytogenes....... 9Infection Characteristics....................... 10Foodborne Listeriosis .......................... 12Presence in Seafood............................ 13Thermal Tolerance............................. 15Affects of Organic Acids on L.monocytogens ..... 18Citric Acid.................................... 21Lactic Acid.................................... 2 3Sorbic Acid................................... 25Modified Atmosphere Packaging (MAP)............. 29

MATERIALS AND METHODS ............................... 33Sample Preparation ............................. 3 3Preparation of Inocula ......................... 3 3Thermal Inactivation Studies ................... 35

Three-neck Flask Method (TNF) ............... 35Sample preparation .................. 35Thermal treatment ......................... 3 6Enumeration of L. monocytogenes...... 36D value determination ..................... 37

Polyethylene Bag Method (PB) ................ 38Sample inoculation................ 38Preparation & application of lactic acid ... 39Introduction of modified atmospheres ....... 41Thermal treatment and enumeration ...... 41D value determination ..................... 44

Cellulose Casing Method (CC) ................ 46Sample inoculation............. 46

Sensory Evaluation ............................. 47

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pageStorage Studies............................... 48

Effect of Storage at 0, 6, & 12° C .......... 48Preparation of inoculated samples ........ 48Enumeration of bacteria ................... 49

Calculation of Generation Time................ 50Effect of Citric Acid & Potassium Sorbate onGrowth of L. monocytogenes................. 51Preparation of inoculated samples ......... 51

Effect of Lactic Acid and Atmospheres onGrowth of L. monocytogenes................. 52Preparation of inoculated samples ........ 52Enumeration of bacteria ................... 53Experimental protocol ..................... 53

RESULTS AND DISCUSSION ............................. 56Low Temperature Growth......................... 56Storage Studies Using Citric Acid & PotassiumSorbate............. 61

Thermal Inactivation .......................... 64Thermal Inactivation with Lactic Acid ......... 71Sensory Evaluation ............................ 75Heat and Modified Atmospheres.................. 784°C Storage Under Various Atmospheres & 1% LA . . . 83

SUMMARY AND CONCLUSIONS ........................... 91

REFERENCES......................................... 94VITA............................................... 108

iv

LIST OP TABLES

Table Page1. Treatment combinations for inoculated crawfish

samples ........................................ 402. Gas composition as determined immediately after

introduction to sample packs and after 60°C heat treatment..................................43

3. Gas content before and after 21 d storageat 4°C of package atmospheres and pH of crawfishtail meat treated with or without lacticacid (LA) .................................... 54

4. Generation times, least squares regression factorsand goodness of fit for Listeria monocytogenes in cooked crawfish tail meat treated with citric acid (CA) or potassium sorbate (PS) .......... 63

5. Thermal death times, least squares regressionfactors and goodness of fit for D value determinations using TNF methodology for L. monocytogenes in crawfish tail meat........... 65

6. Thermal death times, least squares regressionfactors and goodness of fit for D60 values determinations of L. monocytogenes using various methods in crawfish tail meat......... 68

7. Least squares regression factors and goodness offit for D-value determinations at 60°C for different levels of lactic acid or HCl adjusted crawfish.......................................72

8. Taste panel results of crawfish samples packedunder air and stored at 4°C. Values of 5.0 =standard quality, >5.0 = less favorable quality, and <5.0 = more favorable quality............. 77

9. Least squares regression factors and goodness offit for D-value determinations after 60°C thermal treatment of crawfish tail meat treated with or without 1% LA and packed in different modified atmospheres with head space .......... 80

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LIST OF FIGURESFigure Page

1. Inside dimensions of polybag used in both thermaland storage studies ................. 42

2. Center temperature for come-up and cool-down timesfor 10 g samples of crawfish tail meat in polybags (PB) and polybags with head space (PB-a) and cellulose casings (CC) ............. 45

3. Growth of L. monocytogenes on crawfish tail meatstored at 0, 4, 6, and 12 °C ................... 57

4. Generation times for L. monocytogenes indifferent food products at 4°C ............... 60

5. Growth of L. monocytogenes on crawfish tail meatat 4°C and treated with 0.03 g/kg Citric Acid (CA) or Potassium Sorbate (PS)............ 62

6. Thermal death curve for L. monocytogenes at 60°Cusing various levels of spray appliedlactic acid .............................. 73

7. Linear regression comparing the effect of 1%lactic acid and the equivalent HC1 adjusted pH, on lethality of 60°C toward L . monocytogenes on crawfish tail meat ...................... 74

8. Thermal death curve for L. monocytogenes oncrawfish tail meat at 60°C packed undervarious atmospheres .......................... 79

9. Thermal death curve for L. monocytogenes oncrawfish tail meat at 60°C spray treated with 1% lactic acid and packed under various atmospheres .......................... 82

10. Growth of L . monocytogenes stored under 4different atmospheres at 4°C for 21 d .... 84

11. Growth of L . monocytogenes on crawfish tail meatstored under Air and N2 alone and in combination with 1% LA at 4°C for 21 d .................... 87

12. Growth of L. monocytogenes stored under C02 and 02alone and in combination with 1% LA at 4°C for 21 d ......................................... 88

vi

Destruction of L. monocytogenes stored under Air, N2, C02 and 02 in combination with 1% LA at 4°C for 21 d ....................................

vii

LIST OF ABBREVIATIONS AND SYMBOLSAcH — pediocin (form of)ANOVA - analysis of variance

atm - atmosphere(s)BPB - buterfield phosphate

buffer°C - degrees CelsiusCA - citric acid

CC - cellulose casing method

CFU - colony forming units

co2 - carbon dioxideCAT -- catalased - day(s)6̂0 - D value at 60°Cop - degrees Fahrenheitg - gram(s)h - hour(s)

HCl - hydrochloric acidkg - kilograms

L - liter

LA - lactic acid

LOA - listeria oxoid agar

MAP - modified atmosphere packaging

min - minute(s)

mL - milliliter(s)MPa - millibar per

atmosphereP - probabilityPA-1 - pediocin (form of)

PB - polybag methodPB-a - polybag method

(with head space)PH - (-) log of hydrogen

ionsPKa - dissociation

constantPMF - proton motive forcePS - potassium sorbatesp - species (singular)spp - species (plural)SOD - superoxide dismutaseTDT - thermal death timeTNRF - 3 neck reaction

flask methodTSA - tryptic soy agarUSDA - U.S. Department

of AgricultureUSFDA- U.S. Food and Drug

AdministrationWt - weight

w/w - weight to weight

% percent

viii

ABSTRACT

The generation time of L. monocytogenes was calculated to be 72.2, 28.5, 17.0, and 6.9 h on crawfish tail meat held at 0, 4, 6, and 12°C, respectively. Potassium sorbate (PS) (0.3%) spray treatments on crawfish held at 4°C, extended the lag phase of the bacterium. However, once exponential growth began, a generation time not significantly different (P>0.05) than that of untreated crawfish was observed. Samples sprayed with 0.3% citric acid (CA) and held at 4°C supported growth of the bacterium equally well as that of untreated samples.

Thermal inactivation of L. monocytogenes using a three neck reactionary flask method yielded D55 6065 values of 10.23, 1.98, and 0.19 min, respectively. A z value of 5.5°C was calculated. Polybag and cellulose casing methodology yielded significantly higher (P> 0.05) D60 values of 4.68 and 3.84 respectively, for the bacterium. D60 values for L. monocytogenes decreased as the percent lactic acid (LA) spray treatments levels were increased. Increasing sensitivity to heat with LA was not solely due to a reduction of pH. D60 values for the bacterium on samples packed in air, 02, C02, or N2 were not significantlydifferent (P>0.05), however, D60 of samples packed under 02 were lowest.

ix

Generation times for heat damaged L . monocytogenes on crawfish meat stored at 4°C and packed under air, 02, C02/ or N2 was determined. Twenty percent of the cell population was damaged when thermally treated at 60°C for 12.5 min. The bacterium consistently grew slowest in C02 and 02. Samples receiving a treatment of 1% LA, packaging under either of the 4 different atmospheres, and storage at 4°C, were lethal to the bacterium.

These studies indicate that post-packaging heat treatments of 60°C or greater in the presence of C02 or 02, and the addition of 1% LA sprays, would be detrimental to the survival of L . monocytogenes on crawfish meat.

x

INTRODUCTION

Increased public awareness and perception of food quality and* safety has escalated government monitoring of seafood products. As a result, development of more safety focused processing techniques is critical. Employment of multi-barrier methodology through modification of presently used processes would be an economical way to accomplish this goal.

Outbreaks of foodborne listeriosis involving a variety of foods has established Listeria monocytogenes as an important foodborne pathogen (Fleming et al., 1985; Kvenberg, 1988; Linnan et al., 1988; Sclech et al., 1983). The association of L. monocytogenes with seafood products has also been established (McLauchlin, 1987). Frozen seafood products such as shrimp, crabmeat, lobster, langostinos, scallops, squid, and surimi have been shown to contain L. monocytogenes when samples were obtained from several different countries (Weagant et al., 1988). The study found that 2 6% of the products sampled contained L. monocytogenes. Farber (1991) found 13% of fish products at the wholesale level in the U.S.A., Britain, and Taiwan, to be positive for L. monocytogenes. He also found ready-to- eat shrimp, crab, and salmon to contain the bacterium. L. monocytogenes flourishes well in aquatic environments of

1

decaying vegetative matter (Gray and Killinger, 1966) , which is the typical environment in a crawfish pond. As a result L. monocytogenes can usually be found on live crawfish (crayfish) entering processing plants.

Crawfish tail meat is hand picked from parboiled whole crawfish and usually packed for retail sale in one pound, low oxygen permeable polybags, that are sometimes vacuum sealed. While boiling temperatures (100°C) are sufficient to kill L. monocytogenes (Fain et al., 1991), cross contamination from workers and food contact surfaces may occur. Postprocessing contamination with L . monocytogenes has been identified as a major source of contamination for many food products (Farber and Peterkin, 1991). Low numbers of L . monocytogenes have been observed in selected frozen crawfish tail meat (Dorsa and Marshall, 1993). One pound bags of crawfish tail meat are typically sold unfrozen on ice. Harrison et al. (1991) found that L. monocytogenes populations on vacuum packed iced shrimp did not increase. Despite this finding, it is important to consider that evenly distributed product in ice-packs or 0°C conditions are not always practical during product transportation or during retail display of packaged crawfish tail meat. As a result, it is possible for the product to be subjected to periods of warming, potentially resulting in proliferation of L . monocytogenes.

L. monocytogenes has been shown to grow well on many food products stored under refrigeration. The generation times for the bacterium has been determined for red meats, vegetables, and seafood products (Berrang et al., 1989; Marshall et al., 1991; Shelef, 1989). The generation time for the bacterium on crawfish tail meat has not been determined, however.

Most information available for the heat resistance of L. monocytogenes in foods has concentrated on milk and red muscle products. L. monocytogenes has been shown to survive improper heat treatments in red meats and chicken (Farber, 1989; Harrison and Carpenter, 1989). Little is known about the thermal resistance of L. monocytogenes in seafoods, although D-values for some products such as crab and lobster have been determined (Budu-Amoako et al., 1992; Harrison & Huang, 1990). Presently, there are few crawfish processors that subject crawfish tail meat to post-packaging heat treatments. Since post-packaging heat treatments might be beneficial in reducing post-processing contamination and insure product safety, there is a need to study the heat resistance of L. monocytogenes on this product.

L. monocytogenes has been detected in 53% of vacuum packed processed meats obtained from retail stores (Grau and Vanderlinde 1992). Several studies with modifiedatmospheres indicate that the bacterium can survive low

temperature conditions in reduced oxygen or anaerobic conditions. Marshall et al. (1991, 1992) found thatatmospheres lower in oxygen than air, reduced growth of the bacterium at low temperatures, but were not lethal. Manu- Tawiah et al. (1993) determined that atmospheres containing C02 concentrations normally used for commercial packaging of fresh red meats are unlikely to reduce the risk factor for listeriosis in ready-to-eat pork chops. Additional studies have determined that if L. monocytogenes is heat injured prior to incubation, exposure to oxygen will decrease its chances for recovery (Knabel et al., 1990). These findings agree with earlier researchers who theorized that severely heat injured cells acquire a strict anaerobic nature (McCord et al., 1971). Dallmier and Martin (1988) reported that catalase (CAT) and superoxide dismutase (SOD) are rapidly inactivated when the bacterium is heated. The subsequent buildup of toxic 02 radicals due to inactivation of these enzymes converts L. monocytogenes into an obligate anaerobe (Knabel et al., 1990). Consequently, the use of various modified package atmospheres in combination with heat treatments to control or eliminate L . monocytognes on crawfish tail meat warrants investigation.

Studies have shown that organic acids and their salts inhibit the growth of L. monocytogenes. Ahamad and Marth (1989) determined that citric and lactic acids, with large

dissociation constants, caused cell damage to L. monocytogenes. El-Shenawy and Marth (1988) observed that L. monocytogenes was susceptible to sorbic acid. Chung and Lee (1981) found that potassium sorbate on English sole delayed the onset of logarithmic growth of Pseudomonas sp. Organic acids are effective for controlling several bacteria in broth media and some food products. Continued investigation into the use of organic acids on other food products is warranted. The impact of combining organic acid treatments with other barriers, in an attempt to enhance their effectiveness in controlling L. monocytogenes on crawfish tail meat, remains unknown.

More specifically, Wei et al. (1991) determined that decreasing the pH of a Listeria spp. suspension for 2 h using 0.1 N HC1 was not effective in killing the bacterium below detectible levels. Ita and Hutkins (1991) suggested that inhibition of L. monocytogenes by acids may not be caused by a decrease in the intracellular pH, but rather by some specific effect of undissociated acid species on metabolic or other physiological activities. The inhibitory effects of LA and other organic acids can be correlated with their dissociation constants or pKa values. In general, weak acids having higher pKa values are thought to be more inhibitory to L. monocytogenes at a given pH than strong acids at the same pH (Ita and Hutckins, 1991) . These

studies allow the development of a working hypotheses where weak organic acids should be effective for controlling L. monocytogenes on crawfish tail meat.

The bacterial properties of lactic acid (LA) are well documented (Ingram et al., 1956; Van Netten and Mossel, 1980; Woolthuis and Smulders, 1985). Ockerman et al. (1974) found that spraying 12% LA on hot sheep carcasses reduced the surface aerobic colony counts by about 1 log10. Similar reductions with only 1.0% LA on hot beef carcasses were achieved by Snijders et al. (1979) . Woolthuis and Smulders (1985) determined that 1.25% LA solutions resulted in noticeable decontamination of calf carcasses. Ahamad and Marth (1989) observed that tryptose broth acidified with 0.3 or 0.5% LA was lethal to L . monocytogenes at 7, 13, 21, and 35°C. They also found the rate of inactivation to be much greater at 35°C than at lower temperatures. Considering the previous research relating to the use of LA to control L . monocytogenes on red meat carcasses, LA treatment on crawfish tail meat might prove effective for controlling the bacterium.

Roberts (1989) noted that except where one or two factors limit microbial growth, our understanding of the relative contributions of multiple factors to give safe and shelf-stable food products is surprisingly poor. As a result, there is an inadequate data base with which to

develop microbiologically stable and safe foods that have a long shelf life. Roberts (1989) stated that there is an urgent need for a better understanding of the effects that combinations of factors have on microbial growth and survival in foods. Chung et al. (1982) found that the presence of an organic acid magnified the effect of heat injury of several microorganisms isolated from seafood. Thus heat treatments in combination with an organic acid could form the basis of a multifactorial (multibarrier) processing technique to improve crawfish tail meat safety.

The overall objective of the present study was to determine the impact of heat, lactic acid, and various gasses when used independently and in combination against L. monocytogenes in cooked crawfish tail meat. A multibarrier approach was postulated to more aggressively destroy L. monocytogenes; however, such methodology could not be considered independent of desirable sensory qualities of crawfish tail meat. The specific objectives of this work were:

1. To determine the generation time for L. monocytogenes on crawfish tail meat at 0, 4, 6, and 12°C.

2. To develop and evaluate the effect of several thermal destruction methods used for D value determination of L. monocytogenes on crawfish tail meat.

To determine the lethal effects of heat, lactic acid and modified atmosphere packaging (MAP) applied both separately and in combination on L. monocytogenes on crawfish tail meat.To determine the effects resulting from process deviations of lactic acid in combination with MAP on survivability of heat injured L. monocytogenes at 4°C on crawfish tail meat.To measure the sensory effect of lactic acid during refrigerated storage of crawfish tail meat.

REVIEW OF LITERATURE

General Microbiology of L. monocytogenesL. monocytogenes is a foodborne pathogen that infects

humans, domesticated animals, wildlife and various aquatic species. It is considered an environmental contaminant widely present in plant, soil, and surface water samples (Moore, 1988; Weis and Seeliger, 1975). It also has been found in silage, sewage, slaughterhouse solid waste, effluent of abattoirs and poultry packing plants, milk of normal and mastitic cows, pond reared rainbow trout, and human and animal feces (Gray and Killinger, 1966; McCarthy, 1990; Watkins and Sleath, 1981) . Because of environmental preferences of the bacterium, both pond raised and wild stock crawfish (Crayfish, Procambarus clarkii) are natural hosts.

The bacterium was first described by Murray et al. (1926) . The pathogenic form of L. monocytogenes is a smooth, small gram-positive, nonsporeforming, facultatively anaerobic, non-acid fast, diphtheroid-like rod with rounded ends measuring 1.0 to 2.0 microns by 0.5 microns (Gray and Killinger, 1966). It is catalase positive and oxidase negative and expresses a beta-hemolysin which produces zones of clearing on blood agar (Farber and Peterkin, 1991) . The bacterium possesses peritrichous flagella, which give it a

9

10characteristic tumbling motility, occurring only in a narrow temperature range. When grown at temperatures between 20 and 25°C, flagellin is both produced and assembled at the cell surface, but at 37°C flagellin production is markedly reduced (Peel et al., 1988). Colonies of the bacterium demonstrate a characteristic blue-green sheen under obliquely transmitted light (Henry, 1933).

Infection CharacteristicsIn humans, the greatest impact of L. monocytogenes is

to the elderly, immunosuppressed, and to pregnant women and their fetuses. The symptoms of the resulting infection, listeriosis, include meningitis, central nervous system infection, stillbirths, abortions, premature labor and septicemia (Health and Welfare, 1990). There are, however, instances when apparently normal healthy individuals have become ill with listeriosis in both foodborne epidemics (Schultz, 1945) and sporadic cases (Azadian et al., 1989). Most cases of human listeriosis appear to be sporadic, although a portion of these cases may be previously unrecognized common-source clusters (Ciesielski et al., 1988) .

Serovar 4b of L. monocytogenes is the most virulent in human and animal disease. This serovar is only rarely isolated from foodstuffs, in contrast to the more common

11serovars l/2a and l/2c (Menudier et al., 1991). The existence of avirulent serovars l/2a strains may account for the infrequent involvement of this serovar in human disease. Serovar l/2c has been found to be less virulent than serovar 4b, which may help explain its relatively low involvement in human listeriosis (McLauchlin, 1990).

The infective route of orally ingested cells appears to be via the Peyer patches of the intestine and then into phagocytes, passing into the mesenteric lymph nodes by way of the lymphatic pathways (Farber and Peterkin, 1991). Mice infected orally have shown variable responses, with 50% infectious doses ranging from 1.7 X 103 to 9.9 X 106/g cells (Audurier et al., 1980; Golnazarian et al., 1989). Other studies on oral feeding of mice with L. monocytogenes have shown that very high (> 2.5 X 108/g cells) numbers are required to cause invasion of the Peyer's patches or death in normal mice (MacDonald and Carter, 1980). Miller and Burns (1970) found that a dose of >4 X 107/g cells caused fetal death in 6 of 10 pregnant mice. Feeding trials in which nonhuman primates were used showed that only animals receiving a dose of 109/g I#, monocytogenes cells became noticeably ill. Smaller numbers of cells did not result in noticeable symptoms of disease (Farber et al., 1990). The minimum number of pathogenic L. monocytogenes that must be ingested by humans to cause illness in either normal or

12susceptible individuals is not known. Farber & Peterkin (1991) compiled a list of cases in which the numbers causing illness were approximated for humans. In most of these cases the approximated number of cells that caused illness was 106/g of food.

Foodborne ListeriosisNot until the late 1970's was foodborne listeriosis

documented in North America and Europe. In 1986 the Council of State and Territorial Epidemiologists recommended that listeriosis become a reportable disease in the United States (Gellin and Broome, 1989). But as early as 1979, the first outbreak of listeriosis occurred in Boston. Lettuce, celery and tomatoes were implicated and 23 cases were reported with 5 fatalities (Ho et al., 1986). Coleslaw was the cause of a listeriosis outbreak in Nova Scotia in 1981 resulting in 18 deaths from 41 cases (Schlech et al., 1983). Pasteurized milk was implicated as the transmitter of a listeriosis outbreak in Massachusetts during 1983, where 49 cases were reported with 14 fatalities (Fleming et al., 1985). The largest outbreak occurred in southern California in 1985 where 142 cases were reported causing 48 deaths and was traced to Mexican style cheese (Linnan et al., 1988). Farber and Losos (1988) reported that all of these outbreaks were caused by L. monocytogenes serotype 4b.

In 1980 an epidemic of perinatal listeriosis in New Zealand suggested a possible link to the consumption of shellfish and raw fish, resulting in 22 reported cases with 5 deaths (Lennon et al., 1984) . To date, there have been no reported cases of listeriosis from the consumption of crawfish. However, since crawfish are crustaceans that are grown in static pond water, typically high in detritus loads, the potential for contamination of hand picked crawfish tail meat is omnipresent.

Presence in SeafoodListeria is considered an environmental contaminant and

can be found in waters of rivers, lakes, ponds and canals (Moore, 1988; Watkins & Sleath, 1981). In 1966, Gray and Killinger reported L. monocytogenes to be present in pond raised rainbow trout. Bracket (1988) hypothesized a cycle of infection for Listeria indicating that contaminated water infects fish and shellfish which in turn infect humans through consumption of the contaminated food product. Since crawfish are boiled as part of the peeling process, the packaged tail meat is considered cooked. Crosscontamination during peeling is a possible avenue of product inoculation with L. monocytogenes. It is conceivable that consumers might mistakenly consider crawfish tail meat

14ready-to-eat from the retail package, resulting in a possible infection with the bacterium in consumers.

A Class I recall was made by the U.S. Food and Drug Administration (USFDA) on vacuum and air packed smoked shad because of the presence of L. monocytogenes (Anonymous, 1992). Several surveys have detected various levels of Listeria spp. in seafood products from retail markets. Jemmi (1990) detected L. monocytogenes in 12% of smoked and marinated fish from 377 samples tested. Frozen seafood products such as shrimp, crabmeat, lobster, langostinos, scallops, squid, and surimi have been shown to contain L. monocytogenes in samples obtained from several different countries (Weagant et al., 1988). The study found that 26% of the products sampled contained L . monocytogenes. Fuchs and Sirvas (1991) surveyed the incidence of Listeria in ceviche products bought from markets in Lima and Callao, Peru. Approximately, 75% of the samples they tested contained Listeria innocua and 9% contained L. monocytogenes.

Noah et al. (1991) isolated Listeria from frozen lobster tails, shrimp, prawn, breaded shrimp, whole fish, and fish fillets. Of 211 composites, 28% were positive for Listeria spp. Wong et al. (1990) conducted a local market survey in Taiwan and found L. monocytogenes in 10.5% of seafoods containing fish and squid. Farber (1991) found 13%

15of ready-to-eat shrimp, crab, and salmon at the wholesale level in the U.S.A., Britain and Taiwan to be positive for L. monocytogenes. No surveys conducted to date have included peeled crawfish tail meat. Since live crawfish are raised in a potentially hazardous environment, it would warrant the inclusion of this product in future surveys for L . monocytogenes.

Thermal ToleranceFollowing the 1983 Massachusetts outbreak of

listeriosis from pasteurized milk, the thermal tolerance of L. monocytogenes was questioned (Fleming et al., 1985). Since this time, Bradshaw et al. (1985) determined the D633 value to range from 13.4 to 28.4 s. Donnelly et al. (1987) did similar studies and calculated D62 values to range between 6 and 24 s. The suggestion was made that an intracellular positioning of the bacterium provided a protective milieu (Bunning et al., 1988). However, after an extensive study involving sterile whole milk in which L. monocytogenes was either freely suspended or located within bovine milk phagocytes, Bunning et al. (1988) reported the intracellular position of the bacterium did not significantly increase thermal resistance.

Recent studies conducted on thermal resistance of xj . monocytogenes in red meats have resulted in several

interesting observations. The addition of beef fat does not enhance the heat resistance of the bacterium, but the presence of curing salt substantially increases it (Farber et al., 1989b; Mackey et al., 1990). However, work by Fain et al. (1991) with L. monocytogenes in lean and fatty turkey meat indicated that the bacterium was more resistant to heat when in fatty meat than in lean turkey meat. Crawfish tail meat is typically packaged with fat remaining. Since the present state of knowledge is inconclusive, the presence of fat in crawfish tail meat should be a consideration when conducting any thermal studies with L. monocytogenes on crawfish tail meat.

Additionally, the heat shock phenomenon must be taken into account when heating solids that heat much slower than liquids. Heat shock in this case would be defined as any sublethal exposure to heat that alters the ability of a bacterium to function or survive normally. Liquids, which heat rapidly, do not allow bacteria acclimation time to temperature induced stresses as do solids, in which some limited survival may result from a bacterial adjustment to environmental changes. This is especially true for meats, which heat slowly to a final internal temperature (Farber and Brown, 1990) as is the case for 1 lb crawfish tail meat packages.

Little research has been performed to determine the thermal resistance of L. monocytogenes in seafoods. Thermal death times for L. monocytogenes in crab meat and lobster meat have been evaluated (Budu-Amoako et al. , 1992; Harrison and Huang, 1990) . No research has been conducted to determine the effect of heat on the bacterium on crawfish tail meat. Harrison and Huang (1990) determined D values at 55 and 60°C to be 12.00 and 2.61 min, respectively for crab meat. Budu-Amoako et al. (1992) determined the D60 value on lobster for L. monocytogenes was 2.39 min. Interestingly, Harrison and Huang's (1990) D55.60 values ranged from 40.43 to 2.61 min when using non selective tryptic soy agar (TSA) as the plating medium, but when using a selective medium, modified Vogel-Johnson agar, the D55.60 values were reduced to 34.48 to 1.31 min. This implies that injured cells may have been inhibited on the selective medium.

McCarthy et al. (1990) tested the recovery of heat stressed L. monocytogenes from artificially and naturally contaminated shrimp. No L. monocytogenes were recovered from naturally contaminated shrimp boiled at 1, 3, or 5 min. However, L . monocytogenes were detected in experimentally internally-infected shrimp when boiled up to 5 min. This study also revealed the affects of freezing on heat stressed cells in frozen cooked shrimp. They found no L. monocytogenes cells survived the cook-freeze-thaw process.

This indicates that the combination effect of the multi­barriers, heat then cold, can be lethal to the bacterium. The increase in lethality observed by McCarthy et al. (1990) resulting from exposure of the bacterium to multi-barriers does not always hold for other combinations, however. Pickett and Murano (1993) determined that heat shock did not result in any significant difference in survival of L. monocytogenes when incubated at 37°C and exposed to the minimum inhibitory concentrations of chlorine-base, iodophor, phosphoric acid, or guaternary ammonium compounds or citric, lactic, or propionic acids. The addition of a third barrier, however, could possibly change the effect of any one barrier, a facet the present study will investigate.

Affects of Organic Acids on L. monocytogenesL. monocytogenes is more acid tolerant than most

foodborne pathogens and can grow or survive in broth media with pH values as low as 4.3-5.2 (Ahamad and Marth, 1989; Conner et al. 1986; El-Shenaway and Marth, 1988; Farber et al., 1989a; Parish & Higgins, 1989). Studies by Ahamad and Marth (1989) and Farber et al. (1989) indicated that the effects of various organic acids differ. These studies determined that acetic acid is more detrimental to Listeria spp. than either lactic, citric or hydrochloric acid. Acetic acid acts mainly by lowering the pH of the

environment to the point where microorganisms can no longer grow (Liewen and Marth, 1985). Although the undissociated form of acetic acid has antimicrobial action, it is primarily the hydrogen ion and the resulting decrease in pH that inhibits microorganisms (Cowles, 1941; Levine & Fellers, 1939). Subsequent studies by Buchanan et al. (1993) support these findings at low pH values. However, researchers determined that at higher pH values LA was more effective than acetic acid. The pH of crawfish tail meat was determined in the present study to be 7.6, supporting the use of LA rather than acetic acid, as a controlling agent for L. monocytogenes. Also the rate at which lactic and acetic acids inactivate L. monocytogenes is dependent on the identity of the acid, the concentration of the acid, and the pH of the system.

The inhibitory effects of citric, lactic and sorbic acids can be correlated with their dissociation constants or pKa values (Ita and Hutkins, 1991). In general, weak acids having higher pKa values are thought to be more inhibitory to L. monocytogenes, at a given pH, than strong acids, at the same pH (Ita and Hutkins, 1991) . The amount of the molecule in the undissociated form is determined by pH. As a result, this along with solubility properties, determines the foods in which these acids are most effective (Ray and Bullerman, 1982). For example the antimicrobial

20effectiveness of sorbic acid increases as the pH value approaches its pKa value of 4.75, the upper pH limit for activity being 6.0-6.5.

Many weak acids, in their undissociated or protonated form, have the ability to penetrate the cell membrane and accumulate within the cell cytoplasm (Ita and Hutkins,1991). If the interior of the cell is more alkaline than the pKa of the acid, more of the acid will dissociate, releasing a proton and acidifying the cytoplasm of the cell (Booth, 1985). These events are thought to result in a variety of detrimental effects (Kashket, 1987) . As a defense, many bacteria possess proton pumps or proton/cation exchange systems to deal with the influx of protons and to maintain the cytoplasm near neutral. However, if these pH regulatory systems are unable to function sufficiently (i.e., if the proton concentration is too great), then the pH gradient (the difference between the intracellular and the extracellular pH) will collapse. Intracellular acidification will then result in loss of cell viability or cell destruction. The acidified cell is forced to consume a great deal of energy in an attempt to maintain pH homeostasis. It has been further suggested that the internal or cytoplasmic pH is the relevant pH which ultimately affects cell metabolic activities (Booth, 1985).

It is also established that undissociated and uncharged weak lipophilic acids, such as acetic acid, are permeable through the cell membrane (Kashket, 1987). In contrast, lactic and citric acids are generally unable to passively diffuse across cell membranes; uptake of these acids by cells is instead thought to be carrier mediated (Kashket, 1987) . Ita and Hutkins (1991) determined that acetic acid (pKa = 4.76) accumulated in the cells of L. monocytogenes at a higher concentration than lactic (pKa = 3.86) or citric (pKa = 3.13) acid. Their work was consistent with other reports showing that L. monocytogenes was most inhibited by acetic acid, followed by lactic then citric acid (Ahamad & Marth, 1989; Sorrells et al., 1989; Young & Foegeding,1992). Since organic acids have been shown effective for inhibiting the growth of L. monocytogenes, studies that would evaluate their effectiveness as one part in a system of barriers to control the bacterium would be of interest.

Citric AcidCitric acid (CA)(molecular wt 192.12) is generally

recognized as safe (GRAS) for use in foods under FDA regulations (Food & Drug Admin., 1990). Thus no upper limits are imposed for foods not covered by Federal Standards of Identity. CA is presently used in the seafood

22industry primarily for flavor enhancement and to prevent discoloration.

Because CA is a weak acid having a higher dissociation constant (pKa = 3.13) than strong acids, it's ability to inhibit the growth of L. monocytogenes has been studied. Ahamad and Marth (1989) determined that citric and lactic acids, with smaller pK„' s, were less detrimental to L. monocytogenes than was acetic acid. In subsequent studies done by Ahamad and Marth (1990) it was found that even though acetic acid caused greater inactivation of L. monocytogenes, CA caused the greatest degree of injury followed by lactic and acetic acid. CA treated cells incurred more initial inactivation than the other two acids, however eventually acetic acid produced more total cell inactivation.

Ita and Hutkins (1991) observed that both citric and lactic acids were more effective in lowering intracellular pH of the pathogen than acetic acid. When citric and lactic acid-treated cells at pH values of 3.5, 4.0, and 4.5 were incubated for 24 h, both the pH gradient (intracellular pH- external pH) and the intracellular pH values decreased. They noted, however, that although the total amount of added LA reached 160 mM, the amount of undissociated acid was less than 50 mM (pKa of lactic acid = 3.86).

23Lactic Acid

LA (molecular wt 90.08) is listed as GRAS in the United States (Food and Drug Admin., 1990). Similarly, in Europe it is considered a harmless constituent of food (Lueck,1980). The bacterial properties of LA are well documented (Ingram et al., 1956; Van Netten and Mossel, 1980; Woolthuis and Smulders, 1985).

Ryser and Marth (1988) studied the effect of acetic and lactic acid on L . monocytogenes in cold-packed cheese. They observed the degree of dissociation related to the pKa of acidic (4.76) and lactic acid (3.86). They noted that at pH 5.0, 36.0 and 5.9% of acetic and lactic acid, respectively, will be undissociated. Therefore, the higher proportion of undissociated acetic as compared to LA in cheese food at pH 5.0 would most likely account for the increased capability of acetic acid to decrease a viable Listeria population. Buchanan, et al. (1993) determined that at pH levels <6.0 lactic acid was a more effective inactivator of the bacterium than acetic acid. Models produced from their studies also indicated that LA was more effective than acetic acid for inactivating the bacterium when expressed in terms of concentration of undissociated acid.

Ockerman et al. (1974) found that spraying 12% LA on hot sheep carcasses reduced the surface aerobic colony counts by about 1 log10 CFU/cm2 and did not bleach the meat.

Smijders et al. (1979) achieved similar reductions with only 1% LA on hot beef carcasses, however they reported that some bleaching of product did occur. Woolthuis and Smulders (1985) determined that LA solutions of 1.25% exhibited bactericidal qualities. When measured 24 h postmortem, aerobic colony counts (3 d, 3 0°C) were reduced as much as 1.3 log10 CFU/cm2 on calf carcasses. Additionally, 1.25% LA solutions did not produce commercially unacceptable calf carcass discoloration.

Ahamad and Marth (1989) observed that in tryptose broth (TB) acidified with 0.05% LA, L . monocytogenes proliferated under a wide range of temperatures (7, 13, 21, 35°C). This also was the case when 0.1% LA was employed except at 7°C where lag times were longer. They further determined that strain V7 of L. monocytogenes showed more resistance to LA than strain CA. In addition, the same study indicated that while no growth of the bacterium occurred when acid concentration was 0.2%, it did survive. At 0.3 and 0.5%, LA was lethal to the bacterium. The rate of inactivation was much greater at 3 5°C than at lower temperatures. The D- value of 0.3% citric acid (49.5 h) was also determined to be higher than that of 0.3% LA (29.53 h) at 35°C. At 0.5% acid the D-value for citric was 32.47 h and lactic was 14.6 h at the same temperature.

Bruno and Montville (19S3) determined the mechanisms by which bacteriocins from lactic acid bacteria inhibit L. monocytogenes. They found that the dissipation of the proton motive force (PMF) is accomplished by pediocin PA-1, AcH, leuconocin S and nisin. These antimicrobial proteins are classified as colicins and human defensins. They are characterized according to their energy-dependent or energy- independent mode of PMF depletion. They determined that pediocin PA-1, AcH and leuconocin S dissipated PMF in an energy-independent fashion, whereas action of nisin was energy-dependent. Morille et al. (1993) determined that nisin in a neutral-pH pudding product was limited as an antilisterial agent. However, in systems acidified to pH >5.0, nisin inhibited the growth of the bacterium at 6, 25, and 37°C.

Sorbic AcidSorbic acid (pKa = 4.75) is GRAS under regulations of

the U.S. FDA (Food & Drug Admin., 1990). As a result no upper limits are imposed for foods not covered by Federal Standards of Identity. In natural cheese that has a standard of identity for example, the maximum quantity of sorbic acid may not exceed 0.3% by weight. The upper limits are normally set at 0.1 and 0.2% in the U.S. in foods

26covered by Federal Standards of Identity (Liewen & Marth, 1985).

Sorbic acid is among the most thoroughly studied food preservatives. Since sorbic acid is only slightly soluble in water it is commonly solubilized with sodium or potassium hydroxide. The most widely used forms are sorbic acid and its potassium salt. These compounds are collectively known as sorbates (Liewen and Marth, 1985) . It has been found non-toxic in numerous acute, subchronic and chronic toxicity tests (Gaunt et al., 1975; Luck, 1976). Sorbic acid is metabolized in the body like other fatty acids and has a half-life of 40-110 min, depending on the dosage (Liewen & Marth, 1985). As a result, sorbic acid, which occurs naturally in mountain cish berries or can be chemically synthesized (Windholz et al., 1976), is used as an antimicrobial agent in a variety of foods (Sofos and Busta,1981). The acid serves to extend the storage life of such products as butter, cheese, meats, cereals and bakery items (Anonymous, 1979; Kaul et al., 1979), some fruits, berry products, vegetable products and other foods (Anonymous, 1978; Moline et al., 1963). The effectiveness of sorbic acid against various microorganisms has been reviewed (Liewen & Marth, 1985; Sofos & Busta, 1981) .

Work by Ryser and Marth (1988) showed that L. monocytogenes was inactivated more rapidly in cold-pack

27cheese food containing 0.3% sorbic acid than when it was absent. El-Shenawy and Marth (1988) observed the effect of sorbic acid on L. monocytogenes in tryptose broth supplemented with potassium sorbate ranging from 0 to 0.3%. They adjusted the pH of the broth to 5.6 or 5.0 and incubated it at several temperatures ranging from 4 to 35°C. The bacterium grew in potassium sorbate-free controls under all conditions except 4°C and pH 5.0. In all treatment combinations, as percent potassium sorbate increased, inhibition of the bacterium increased. This indicates an inhibitory effect of sorbic acid toward the bacterium. The study also indicates that the behavior of the bacterium in the presence of different concentrations of potassium sorbate is affected by incubation temperature and pH of the substrate.

Chung and Lee (1981) noted that potassium sorbate delayed the onset of microbial logarithmic growth in English sole but did not alter the rate of microbial growth nor ultimate predominance by Pseudomonas sp. in fish stored at 1.1°C. Other microorganisms such as Flavobacterium- Cytophaga sp., Arthrobacter, Acinetobacter, and Staphylococcus disappeared from samples treated with sorbic acid during 1.1°C storage. Chung and Lee (1982) determined the effect of potassium sorbate on sub-lethally injured bacteria found on English sole. They found that even though

28Pseudomonas sp. was relatively resistant to potassium sorbate and was not affected by 0.3% potassium sorbate after freeze-thaw treatment, it showed delayed onset of logarithmic growth for up to 20 h after heating at 50°C for 5 min.

The process by which sorbate inhibits microbial growth is not clear and a variety of mechanisms are probably involved in its antimicrobial action (Liewen and Marth, 1985). Melnick et al. (1954) proposed that sorbic acid inhibits certain dehydrogenase enzymes which are involved in the J3-oxidation of fatty acids. York and Vaughn (1964) proposed that sorbic acid uncouples oxidative phosphorylation by inhibiting enzymes within the cell. Troller (1965) proposed that the inhibitory action of sorbate resulted from inhibiting catalase, which would cause an increase of toxic hydrogen peroxide within the cell. Freese et al. (1973) have proposed that sorbate prevents microbial growth by inhibiting the transport of substrate molecules into cells. Yousef and Marth (1983) concluded that sorbate inhibited aflatoxin biosynthesis in the same manner. Inhibition of transport of carbohydrates has also been suggested as the inhibitory mechanism of sorbic acid (Deak and Novak, 1972).

Several investigators have shown that C02 and sorbate in combination will effectively inhibit microorganisms.

Elliot et al. (1981, 1982) showed that C02 and sorbateacting synergistically inhibited Salmonella enteriditis and Staphylococcus aureus. Carbon dioxide and sorbate will inhibit mold spoilage of grains (Danzinger et al., 1973). Sorbate will delay the growth of psychrotrophic bacteria in vacuum-packed pork loins (Myers et al., 1983). Since sorbates have proven successful for controlling many different bacteria in a variety of foods, its effectiveness in controlling L. monocytogenes in crawfish tail meat should be evaluated.

Modified Atmosphere Packaging (MAP)The use of MAP to extend shelf-life of foods and

suppress the growth of bacteria has been studied extensively (Gill and Tan, 1979; Hintlian and Hotchkiss, 1986). Killeffer (1930) conducted some of the earliest studies with C02 and seafood products. His work with cod was later corroborated by Coyne (1932, 1933) using fish held under high levels of C02 and by Stansby and Griffiths (193 5) who worked with haddock. Several subsequent studies have been conducted showing the beneficial effects of MAP to extend the shelf life of fresh fish (Gray et al., 1983; Mokhele et al. 1983; Molin et al., 1983). Przybylski et al. (1989) noted that while microbiological counts for catfish packaged in C02/air (80:20) atmosphere were significantly lower than

30those packaged in 100% air, growth did progress. Other researchers have observed reductions in microbiological levels of muscle products packaged in various C02 atmospheres (Banks et al., 1980; Mokhele et al,. 1983;Parkins et al., 1981; Wang & Ogrydziak, 1986).

Studies using C02 dissolved in refrigerated brines improved quality of rockfish and chum (Barnett et al., 1971) , silver hake (Hiltz et al. , 1976) , and pink shrimp(Bullard and Collins, 1978; Barnett et al., 1978). Brown et al. (1980) demonstrated that rockfish and silver salmon stored at 4.5°C, under high levels of C02, had lower microbial counts and less undesirable sensory changes than controls. They also determined that levels oftrimethylamine (TMA) and ammonia were markedly lower in samples held in atmospheres containing 20 or 40% C02.Gerdes et al. (1989) and Wang and Brown (1980) observed these same trends for TMA and ammonia in crawfish held under atmospheres of high C02.

Although MAP has been shown to suppress the growth of spoilage bacteria the subsequent increased potential for the growth of several pathogenic bacteria, such as L . monocytogenes, has been questioned (LaBell, 1986). Wei et al. (1991) determined that L. monocytogenes suspended in distilled water was completely killed after C02 treatments at 6.18 MPa (61.2 atm) and 35°C for 2 h. They observed a

31similar affect of C02 on L. monocytogenes in spiked shrimp. Garcia et al. (1987) and Garcia and Genigeorgis (1987) determined that when salmon fillets were stored at 4°C, under several modified atmospheres (vacuum, 100% C02/ and 70% C02+30% air), Clostridium botulinum did not produce detectable toxin for 60 d. Toxin detection coincided with spoilage at 30°C, but preceded spoilage at 8 and 12°C.

In studies done by Marshall et al. (1991, 1992), L.

monocytogenes on chicken was moderately inhibited under refrigeration temperatures by MAP (76 and 8 0% C02) compared with storage in air. They also observed that growth was not completely retarded nor was the organism killed by the applied storage environments.

Kallander et al. (1991) determined that spiked cabbage stored at 25°C under 70% C02 yielded a slightly lower count (>1 log/g) of L. monocytogenes after 24 h of storage than did cabbage stored in normal atmosphere (>2 logs/g). They also observed that during subsequent storage at 2 5°C, L. monocytogenes counts decreased to undetectable levels (<20 CFU/g) at 6 d of storage under both atmospheres. The decline, however, was more dramatic for the cabbage in modified than in normal atmosphere between days 3 and 4 of storage.

Ingham (1990) working with crawfish determined under MAP (80% C02/balanced propriety) and refrigeration, growth

of Aeromaonas hydrophila and Plesiomonas shigelloides was effectively inhibited. Since researchers have used MAP to successfully control L. monocytogenes in various muscle foods and MAP has proved inhibitory to pathogens found on crawfish, its application to control L. monocytogens on crawfish warrants investigation.

MATERIALS AMD METHODS

Sample PreparationFor all studies conducted, frozen crawfish tail meat

(Procambarus clarkii) was purchased from local suppliers, in one pound heat sealed retail packs. The frozen meat was stored in these retail packs at -10°C for less than 6 months until needed.

Before using, the frozen package was thawed under refrigeration for 24 h. After thawing, a mercury-in-glass thermometer was placed in the geometric center of the package through the top of the bag. The bag was thensubmerged into boiling water and heated to a coretemperature of 90°C (approximately 10 min) to reduce background microflora. The package of crawfish meat was immediately removed and placed into an ice bath until a core temperature of approximately 2 0°C was achieved.

Preparation of InoculaL . monocytogenes strains Scott A (Economics

Laboratories, St. Paul, MN), F5069, and S4b (C. Donnelly, Univ. of Vermont, Burlington, VT) were maintained as stockcultures through monthly transfers on trypticase soy 0.6%yeast extract agar slants (TSYEA) (BBL Microbiology Systems, Cockeysville, MD) and stored at 4°C. All L . monocytogenes

33

34cultures were verified to species using the API Listeria system (bioMerieux, La Balme-les-Grottes, France). This system consists of the following 10 tests to differentiate between L. innocua and L . monocytogenes: presence or absence of arylamidase (DIM test), hydrolysis of esculin, presence of a-mannosidase, and acid production from D-arabitol, D- xylose, L-rhamnose, a-methyl-D-glucoside, D-ribose, glucose- 1-phosphate, and D-tagatose. A study conducted by Bille et al. (1992) determined that with this system, 97.7% (252 of 258) of the L. monocytogenes strains tested were correctly identified and differentiated from 99.4% (175 of 176) of the L. innocua strains tested. Other gram-positive genera included by Bille et al. (1992) were Enterococcus spp., Lactobacillus spp., Carnobacterium spp., Erysipelothrix spp., Oerskovia spp., Corynebacterium spp., and Rhodococcus spp. The 3 biochemical characteristics that are positive for Listeria isolates include: hydrolysis of esculin and acid production from D-arabitol and a-methyl-D-glucoside. These tests easily eliminate the above listed non-Listeria cultures.

Individual strains of L. monocytogenes were subcultured by quiescent incubation for 20-22 h in trypticase soy 0.6% yeast extract broth at 30°C. Ten mL of each subcultured strain were mixed into a sterile centrifuge tube. The mixed strain cells were harvested by centrifugation (3000 X g) for

10 min at 4°C (RC5C, Sorval Instruments, Norwalk, CT) , washed twice in sterile Butterfield Phosphate Buffer (BPB), and resuspended to give desired target levels. Samples were spread plated on TSA to verify target levels. It is known that a particular species of bacterium grown strictly under the same conditions will always reach approximately the same maximum number of viable cells in a given time period (Nickerson and Sinskey, 1972). Using this established criterium, subsequent inoculation target levels for all experiments were successfully replicated under the same strict conditions.

Thermal Inactivation Studies Three-neck Flask Method (TNF)

Sample preparation

The first method employed to determine the lethal effects of heat on L . monocytogenes was TNF, as described by the Food Processors Institute (1988). For this method 25 g of heat treated crawfish tail meat was placed into a sterile stomacher bag with approximately 225 mL of sterile BPB to make a 1:10 dilution. The sample was blended for 2 min using a stomacher (Model STO, Tekmar, Cincinnati, OH) and aseptically transferred to a 3 neck 1000 mL sterile reaction flask.

36Thermal treatment

An initial uninoculated sample was taken from the flask to verify product sterility. The flask containing the sample was secured into a swirling water bath that was maintained at the targeted study temperature. Twenty-five mL of a mixed cell suspension of approximately 109 cells per mL, were aseptically pipetted into the sample slurry. The flask was swirled in the water bath, allowing the sample slurry and inoculum to mix. When the sample reached the study temperature a zero time sample was taken. Sample temperature was monitored using a high precision mercury-in- glass thermometer (Sargent-Welch, Skokie, IL) placed from the top of the flask into the middle of the sample slurry. The sample slurry remained in motion and completely immersed during the heat treatment. The swirling motion was stopped for several seconds during each sampling period. Five mL aliquots from all 3 test temperatures (55, 60, and 65°C)were removed at designated time intervals and placed into sterile tubes. Tubes were rapidly cooled by swirling in an ice slurry, then iced for up to 1 h until analyzed.

Enumeration of L. monocytogenes

After cooling, 1-mL portions of iced samples were transferred to 9 mL sterile BPB diluent and mixed thoroughly. The resulting 1:10 dilution was further

37serially diluted as required. Enumeration of the L . monocytogenes mix as conducted by spread plating 0.1 ml dilutions on Tryptic Soy Agar (TSA) modified with 0.6% yeast extract and 1.0% sodium pyruvate (Fain et al., 1991; Smith and Hunter, 1988). Plates were allowed to stand for 4 h at ambient temperature (approximately 2 5°C) after spread plating to enable recovery of injured cells (Smith, 1990). After this period, approximately 10 mL of Listeria Oxford Agar (LOA) (Unipath Limited, Hampshire, England) was used as an overlay. A study by Xavier and Ingham (1993) suggest that when determining the survival of facultatively anaerobic micro-organisms in foods that have been subjected to heat-shock, an anaerobic enumeration method produces higher counts. Their work showed that an overlay method could be used as an alternative to a total anaerobic enumeration method.

After being overlaid, plates were incubated at 27°C for 48 h before enumeration. L. monocytogenes were enumerated using standard microbial count methods (Messer et al., 1985).

D value determination

A thermal death time (TDT) plot was established by plotting logi0 number of survivors against time for each heating temperature (Nickerson and Sinskey, 1972). The time

38required to bring about a 90% reduction in the number of survivors (i.e. the D value for each treatment) was calculated from the slope of the best fit line representing data on the TDT plot. The log,0 of D value (min) was plotted versus temperature (°C) to determine the z value. Lotus Freelance 3.01 Software (Lotus Corp., Orem, UT) was used to determine slope, y intercept and goodness of fit values (e.g., R2) for the best fit line by least squares linear regression of the thermal death time (TDT) plot.

Polyethylene Bag Method (PB)

Sample inoculation

The second method employed 3-mil polyethylene bags (PB) (Koch, Kansas City, MO) that have a gas transmission rate of 9 cc/m2/24 h at 0°C. This type of PB is normally used to commercially pack crawfish meat. For this method, samples were inoculated by blending 1 mL of the mixed culture (109 cells per mL) with 100 g of crawfish tail meat, prepared as described previously, in a Waring blender to produce a homologous composite sample. Inoculation levels of approximately 107 CFU/g were consistently achieved. Two separate 100 g inoculated samples were aseptically transferred to a 4000 mL collection beaker then hand mixed under a laminar flow hood (Labconco, Kansas City, MO) for 5

39min, using a sterile spatula. At least 2 10-g subsamples of this homogeneous composite sample had L. monocytogenes enumerated to verify beginning inoculation levels. Inoculated samples were either left untreated or spray treated with LA as described in the following procedure. Treatment combinations are given in Table (1).

Preparation & application of lactic acid

A stock solution of 10% LA was prepared and filter sterilized. Appropriate amounts of the stock solution to produce desired concentrations of 0.5, 0.75, or 1.0% w/w were sprayed onto inoculated crawfish using an autoclavable, forced air, clinical atomizer. These concentrations were chosen because they cover the common range of usage for this preservative. Additionally, a preliminary sensory study indicated that the highest concentration did not significantly alter the sensorial quality of crawfish tail meat. The pH of the crawfish tail meat was measured using an Orion model 420A pH meter (Orion Research Inc., Boston, MA). To determine if the effect of LA was attributable to a reduction in pH caused by the addition of LA, the pH of additional crawfish samples was adjusted using 10% HC1 to values approximating that of 0.5 and 1% LA. Approximately 3.5 and 7.5 mL of HCl per 100 g of crawfish meat was required to achieve pH values of 6.5 and 5.4, respectively.

40

Table 1. Treatment combinations for inoculated crawfish samples

GAS TYPE TREATMENT CONCENTRATION°C % LA

AIR60 0.00 0.75

100% C02 0.50 1.00100% 02100% N2

AIR4 1.00

100% C02 100% 02100% N.2

41Treated crawfish were segregated into 10-g samples and

placed into polyethylene bags (Figure 1) . The bags were then heat sealed using a standard industrial heat sealer, model LS (Rennco, Homer, MI) after forcing as much air from the head space as possible without applying a vacuum.

Introduction of modified atmospheres

The polyethylene bags with samples were vacuum packed using a Multivac Model A300/22 vacuum-packing machine (Multivac SEPP Haggenmuller K.G., Germany) and held under vacuum for approximately 15 min. Bags were then opened and resealed using several methods. For the heating trials studying LA levels alone, bags were heat sealed with no head space using a standard industrial heat sealer, Model LS (Rennco, Homer, MI). If the sample bags were to be given a headspace of 100% air, 100% 02, 100% C02, or 100% N2 theywere backflushed with the appropriate gas using the Multivac vacuum-packing machine. Verification of 02 and C02 levels in the packs was done before and after heat treatments using a Servomex Food Package Analyzer, Series 1400 (Servomex, Inc., Croroborough, Sussex, England) (Table 2).

Thermal treatment and enumeration

For all thermal treatments the sample bags were clipped to a stainless steel test tube rack and completely submerged in a 60°C swirling water bath. Sample temperature was

4 2

HEAD SPACE

4.0 c m 16.5 c m

Figure 1. Inside dimensions of polybag used in both thermal and storage studies.

43

Table 2. Gas composition as determined immediately after introduction to sample packs and after 60°C heat treatment

Package Before heat After heatGas O2 CO2 O2 CO2

Air 21.6 0.6 21.1 0.302 9 9 . 0 0.1 99 . 2 0.1C02 0.0 99.6 0.4 99.5N2 0.0 0.1 0.2 0.1

44monitored and come up time recorded (Figure 2) using thermocouples placed in the geometric center of 2 uninoculated sample bags. Sample bags remained in motion and completely immersed during the entire heat treatment. Bags were removed at designated time intervals and agitated in an ice bath for rapid cooling until analysis.

After cooling, a zero time sample was immediately taken by aseptically removing it from the heat sealed bag and placing it into a stomacher bag to which a sufficient volume of sterile BPB was added to achieve an initial dilution of 1:10. The diluted sample was homogenized by stomaching (Tekmar) for 2 min and additional decimal platings were made by removing 0.1-mL from serially diluted samples. This procedure was repeated for all samples. Enumeration of L. monocytogenes was done as previously described.

D value determination

D values (min) for the polybag method were determined as described previously for the TNF method. D value means for thermal treatments of different LA concentrations and all atmospheres were subjected to ANOVA for determination of significant differences at the 5% probability level using Minitab Statistical Software (Minitab, Inc., PA). Student's t-test was used to quantify significant (P<0.05) differences between means of each individual analysis.

Tem

pera

ture

i°

C)

45

Time (min)

Figure 2. Center temperature for come-up and cool-downtimes for 10 g samples of crawfish tail meat in polybags (PB) and polybags with head space (PB-a) and cellulose casings (CC).

46Cellulose Casing Method (CC)

Sample inoculation

A third heat treating method was utilized to determine a D60 value for L. monocytogenes in crawfish tail meat. This method utilized 18 mm EZ Peel Nojax cellulose casings (CC) (Viskase Corp., Chicago, IL) . Cellophane and cellulose casings as a vessel for ground or emulsified food products inoculated with a variety of bacteria are used extensively for D value determinations (Zaika et al., 1990).

For this method crawfish tail meat was prepared and inoculated as described for the PB method. Ten g of inoculated product was aseptically stuffed, using a sterilized glass funnel, into individual casings. Casings were then sealed by tieing off before being secured to test tube racks and placed into the water bath for thermal treatment. Cell enumeration and D value determination was accomplished as described for the PB method. No additional treatments other than heat were applied to the crawfish tail meat using this method.

D60 value means for all three thermal treatment methods (TNF, PB, and CC) were subjected to ANOVA for determination of significant differences at the 5% probability level using Minitab Statistical Software (Minitab, Inc. PA).

47Sensory Evaluation

A trained sensory panel of 15 judges was used to evaluate all LA treatment levels. A single training session that involved group sampling of crawfish tail meat treated with known levels of LA was conducted. During the training session open discussion of any observed sensory alterations due to LA on crawfish meat was discussed among panelist. All levels of LA (0.5, 0.75 and 1.0%) that would be tested for by the taste panel were introduced to the panelist during the training session. Since these levels were relatively low and difficult to detect, samples treated with 10% LA also were used to sensitize the panel to the taste of LA in crawfish tail meat.

Uninoculated crawfish tail meat samples were spray treated with 0.0, 0.5, 0.75, and 1.0% LA then sealed inheatable polyethylene bags (Koch, Kansas City, MO). They were heat treated at 60°C for 10 min in a swirling water bath as described previously. Samples were tested immediately after treatment then held at 4°C and tested after 2 and 5 days of storage. Immediately prior to being given to the sensory panel, the samples were placed in boiling water for 10 min then cooled to ambient temperature to assure product safety.

Samples were presented to panelists at room temperature under white fluorescent lighting. All panelist were

48isolated from one another during sampling periods. For each LA treatment level, 2 segregated 20-g samples were used to evaluate four attributes. The samples were evaluated on a 10-point descriptive scale for odor (10=clean odor, l=strong odor), color (10=bright excellent look, l=graying dull look), taste (10=clean desirable flavor, l=strong acid/undesirable flavor), and texture (10=tender, l=tough).

Mean scores of each attribute for all LA treatment levels were subjected to ANOVA. Determination ofsignificant differences was done at the 5% probability level using Minitab Statistical Software (Minitab, Inc., PA).

Storage StudiesEffect of Storage at 0, 6, & 12°C Preparation of inoculated samples

One hundred g of heat treated crawfish tail meat was placed into a Waring blender (Waring Products, Co., Winsted, CT) prior to introduction of the inoculum. One mL of the diluted mixed L. monocytogenes culture (107 cells per mL) was added to the 100-g sample and blended to obtain a uniform distribution of cells. Each 100-g portion was aseptically transferred to a 4000 mL collection beaker after inoculation. The inoculation procedure was repeated 6 times in the same beaker until 600 g of crawfish tail meat was uniformly inoculated. The total inoculated 600 g of

49crawfish meat in the collection beaker was then hand mixed aseptically using a sterile spatula, under a laminar flow hood (Labconco, Kansas City, MO) for 5 min. This homogeneous composite sample contained approximately 10s CFU/g inoculum. Negative control samples were left uninoculated to test for sterility.

After inoculating the crawfish tail meat, 25-g samples were aseptically placed into sterile, 6-oz whirl pack bags and randomly assigned to 3 storage treatments of 0, 6, and 12°C for 20 d. Additionally, negative controls of 25-g portions were distributed to storage treatments.

Enumeration of bacteria

Two replicate experiments were conducted with each replicate consisting of 3 storage temperatures (0, 6, and 12°C). Samplings were made at 2-d intervals for 0 and 6°C, and at 1-d intervals for 12°C stored samples. For each sampling period, 2 25-g samples were analyzed to enumerate L. monocytogenes. In addition, a 25-g portion of uninoculated meat was used to verify the sterility of the initial uninoculated crawfish tail meat. Each sample was aseptically removed from a "whirl pack" and placed individually into a stomacher bag and stomached as previously described.

50Enumeration of L. monocytogenes was accomplished by

duplicate surface platings of serially diluted samples made on LOA (Unipath)• Counting of the bacterium was conducted using standard microbial count methods (Messer et al., 1985) Inoculated plates were incubated at 27°C for 48 h prior to counting. Mean values for each treatment were reported as the average of duplicate platings of all sampling points from a given time period. The number of bacteria present on the crawfish meat was determined for each sampling and expressed as log10 CFU/g.

Calculation of Generation TimeThe generation time of L. monocytogenes in the crawfish

tail meat was determined for each temperature using the following formula (Marshall and Schmidt, 1988) . Two points on the logarithmic growth phase of each curve were used in the calculation. Generation times were reported as the time required for L. monocytogenes populations to double in number.

Generation Time = (0.301(T2 - TJJ/flog P2 - logP,)

Where: Ti = time of Pi T2 = time of P2 P, = CFU/g at T!P2 = CFU/g at T2

51Effect of Citric Acid & Potassium Sorbate on Growth of L. monocytogenes

Preparation of inoculated samples

Initial storage studies were done with crawfish samples treated with potassium sorbate (PS) and citric acid (CA) . Crawfish tail meat samples were stored and prepared for inoculation as described previously in "Crawfish tail meat sample preparation". Working cultures of L. monocytogenes also were subcultured as described previously in "Preparation of inocula". Samples were inoculated as described previously for thermal inactivation studies using the polyethylene bag method in "Sample inoculation". Mixed cultures were serially diluted before using, allowing inoculation levels of approximately 105 CFU/g. Six separate inoculated 100 g samples were aseptically transferred to a 4000 mL collection beaker then hand mixed as described previously.

PS or CA were prepared as individual 10% stock solutions and sterilized by filtration. Three ml of stock solution were sprayed onto 100 g of inoculated crawfish to achieve a 0.03 g/kg (0.3% w/w) concentration. The spraying procedure employed the same method described for thermal studies with LA. Typical use levels in foods for PS and CA range from 0.002 to 0.3% (Liewen and Marth, 1985). Treated and untreated samples were then segregated aseptically into

5215-g sub-samples that were placed into the same type 3-mil polyethylene bags (Koch) used for thermal studies.

Effect of Lactic Acid & Atmospheres on Growth of L. monocytogenes

Preparation of inoculated samples

For storage studies done with LA and various gas atmospheres, 5 mL of each subcultured strain were transferred to a sterile centrifuge tube. The crawfish tail meat samples were then secured in a 60°C swirling water bath and heated for 12.5 min. This procedure yielded cell counts of approximately 106 CFU/g. Of this population,approximately 20% were heat damaged. The number of heat damaged cells was determined by the difference in number of CFU recovered on LOA divided by the number of CFU recovered on TSA.

Crawfish tail meat samples were inoculated as described previously for thermal inactivation studies using the polyethylene bag method in "Sample inoculation". Inoculation levels of approximately 104 CFU/g were consistently achieved. Nine separate 100 g inoculated samples were aseptically transferred to a 4 000 mL collection beaker then hand mixed as described previously for thermal inactivation studies. One percent LA treated samples and untreated controls were prepared for storage as described above. Treatment combinations are given in Table (1). The

53percentage of 02 and C02 for all package atmospheres was determined using the Servomex Food Package Analyzer (Servomex) both before and after a 21 d storage period at 4°C (Table 3).

Enumeration of bacteria

Bacteria were enumerated using standard microbial count methods as described previously (Messer et al., 1985). At 2-d intervals, 15-g crawfish tail meat samples treated with PS or CA were used for L. monocytogenes enumeration. For samples treated with LA and various gas atmospheres, counts were made at 3-d intervals. Samples were prepared for enumeration as described previously for thermal inactivation studies of the polyethylene bag method. Enumeration of L. monocytogenes was accomplished by duplicate surface platings made on LOA (Unipath). Inoculated plates were incubated at27°C for 48 h prior to counting.

Experimental protocol

Three replicate experiments were conducted at 4°C. At each sampling point, one randomly selected 15 or 2 5-gportion of crawfish tail meat was used to enumeratemicrobial populations for each replicate. In addition, a 15 or 25-g portion of uninoculated meat was used to verify the

Table 3. Gas content before and after 21 d storage at 4°C of package atmospheres and pH of crawfish tail meat treated with or without lactic acid (LA).

Package Before storage After storageGas pH 02 C02 pH 02 C02

Air 7.6 21.6 0.5 7.6 15.4 0.5+1% LA 5.4 21.4 0.4 5.5 15.9 0.3

02 7.6 99.1 0.1 7.6 98.0 0.5+1% LA 5.4 99. 3 0.1 5.4 97.8 0.9C02 7.6 0.0 99.7 6.8 ND ND

+1% LA 5.4 0.0 99.5 5.4 ND NDn2 7.6 0.1 0.0 7.6 0.2 0.5

+1% LA 5.4 0.0 0.0 5.4 0.0 0.0

ND - Gas content of packages back filled with 100% C02 lost all head space during storage resulting in non-readable head space gas content.

55sterility of the initial, uninoculated crawfish tail meat. Mean values for each treatment were reported as the average of duplicate platings of all sampling points of a given time period. The number of bacteria present on the crawfish meat was determined for each sampling and expressed as log10 CFU/g.

Generation time means for each treatment combination were subjected to ANOVA for determination of significant differences at the 5% probability level using Minitab Statistical Software (Minitab, Inc., PA). Lotus Freelance 3.01 software (Lotus Corp., Orem, UT) was used in determining slope, intercept, and goodness of fit values (e.g., R2) of log CFU/g versus temperature and best fit line by least squares linear regression, of the exponential growth phase of L. monocytogenes (Dogra and Schaffner, 1993). Slopes resulting from the exponential growth phase of the bacterium for each treatment were also compared using ANOVA.

RESULTS AND DISCUSSION

Low Temperature GrowthGrowth of L. monocytogenes on precooked crawfish tail

meat stored at 0, 6, and 12°C, either to stationary phase or 20 d, are shown in Figure 4. The initial population of L. monocytogenes on inoculated crawfish samples held at 0 and 6°C was approximately 104 CFU/g. At 0°C, less than 1 log10 CFU/g growth was observed for the entire storage time of 20 d (Figure 3). Similar results were reported for shrimp held on ice where no increase in L. monocytogenes populations occurred for 21 d (Harrison et al., 1991). An initial decrease in the population of L. monocytogenes stored at 0°C was observed (Figure 3) . Harrison et al. (1991) showed that shrimp samples held at -2 0°C had a decrease in Listeria spp. populations of less than 1 log when stored for 3 months. Any inhibitory environment that the bacterium is exposed to in cold storage seems to cause an initial decrease in population. If the bacterium can adjust to the 0°C environment, growth ensues.

Generation time of L. monocytogenes incubated at 0°C was approximately 72.2 h, dramatically longer than 17.0 or 6.9 h at 6 and 12°C, respectively. Consequently, as long as crawfish tail meat is constantly held at 0°C, L. monocytogenes will grow poorly.

56

Log

CFU

/g57

10

- o°c4°C 6°C

12°C

20 4 6 8 10 12 14 16 20Storage Time (days)

Figure 3. Growth of L. monocytogenes on crawfish tail meat stored at 0, 4, 6, and 12°C.

Studies conducted by Glass and Doyle (1989) indicated that at 4.4°C the rate of growth for L. monocytogenes depended largely upon product type and pH. In the present storage study, crawfish tail meat inoculated with approximately 104 CFU/g began exponential multiplication immediately with no observed lag phase at 6°C (Figure 3). A 1 log increase per 2-d period was observed until 10 d, at which time maximum population density for the bacterium on crawfish tail meat and stationary phase was reached. This temperature is important when determining the growth rates of L. monocytogenes in crawfish tail meat since 6°C (42.8°F) is close to the temperature of many retail display cases and home refrigerators. Because L. monocytogenes multiplies rapidly at this storage temperature, it may be beneficial to further heat process precooked crawfish tail meat prior to distribution.

At 12°C, the initial L. monocytogenes count of approximately 105 CFU/g underwent rapid exponential multiplication, with no observed lag phase, for only 3 d before maximum population density for the bacterium on crawfish tail meat and stationary phase was reached (Figure3). Even though 12°C is not considered properrefrigeration, it is possible to reach this abusive temperature if product mishandling occurs duringtransportation or storage. Crawfish tail meat, stored at

59abusive temperatures even for short periods of time, could support rapid growth of L. monocytogenes and should be regarded as unsafe for consumption unless it undergoes an additional heat treatment.

In the seafood industry, a 7 to 10 d shelf life is typical for fresh, iced, products. Czuprynski et al. (1989) observed that L. monocytogenes at reduced temperature (4°C) increased in virulence in intravenously inoculated mice, although it did not appear to affect mice that had been infected orally. Thus, an increase in the virulence of the bacterium in refrigerated foods may be possible. The present storage studies indicated that L. monocytogenes can flourish on precooked packaged crawfish tail meat when stored under refrigeration temperatures for several days.

The generation time for L. monocytogenes on crawfish tail meat appears shorter than for other non-seafood products. Generation times for the bacterium at 4°C on beef (74.1 h), beef extract (50.6 h) (Shelef, 1989), and chicken (43.3 h) (Marshall et al., 1991) have been determined. On asparagus, broccoli, and cauliflower, the bacterium experienced good multiplication when stored at 4°C (Berrang et al., 1989). The generation time for the bacterium on seafood products, especially crustaceans, appears to be lower than on non-seafood products (Figure 4) . Farber (1991) reported the generation time for the bacterium at 4°C

Figure

Beef S.Milk Lettuce B. Ext. Chick. Salm. CrawfishShrimp Crab Lobster

* Generation time for lettuce was determined at 5°C

4. Generation times for L. monocytogenes in different food products at 4°C.

61on crustaceans such as crab, lobster, and shrimp, to be 16.9, 15.5 and 25.3h, respectively. Further researchdirected at determining why the bacterium grows so successfully on shellfish products, may unlock a key to controlling the bacterium on all food products.

Storage Studies Using Citric Acid & Potassium SorbateChung and Lee (1981) observed that PS delayed onset of

exponential growth but did not alter rate of microbial growth nor ultimate predominance of Pseudomonas sp. in English sole stored at 1.1°C. The present study notes the same effect when 0.03 g/kg PS was sprayed onto crawfish inoculated with L. monocytogenes and held at 4°C (Figure 5) . The lag phase of the bacterium was extended by 2 d on treated crawfish. Once exponential growth began, however, the generation time was not significantly different (P>0.05) than the generation time for untreated crav/fish meat (Table4). Additional studies by Chung and Lee (1982) evaluating the effectiveness of PS for controlling growth of several seafood bacterial isolates (Arthrobacter sp., Moraxella sp, and Pseudomaonas sp.) found PS treatments ineffective unless amounts as high as 2.73% were used. Considering the findings of Chung and Lee (1981, 1982) and those of thepresent study, additional study of PS was not pursued.

Log

CFU

/g62

7.6

7.4

7.2Control

CA

PS6.8

6.6

6.4

6.2

6.8

6.6

6.4

6.2

4.6

4.60 2 4 6 8 10

STORAGE TIME (days)

Figure 5. Growth of L . monocytogenes on crawfish tail meat at 4°C and treated with 0.03 g/kg Citric Acid (CA) or Potassium Sorbate (PS).

63

Table 4. Generation times, least squares regression factors and goodness of fit for Listeria monocytogenes in cooked crawfish tail meat treated with citric acid (CA) or potassium sorbate (PS).

Treatment Generation Y-intercept Slope R2Time (h) (Log10 CFU)

none 28.5 5.45 0.239 + 0.028 0.958CA 29.5 5.47 0.229 ± 0.029 0.953PS 28.1 4.30 0.261 + 0.029 0.977

Samples sprayed with 0.03 g/kg CA supported growth of L. monocytogenes equally well as untreated samples (Figures) . The generation time of the bacterium on CA- treated samples was not significantly different (P>0.05) than on untreated samples (Table 4) . The present study indicated that 0.03 g/kg CA had no inhibitory effect against L. monocytogenes on cooked crawfish meat. These results indicate that post processing sprays of 0.03 g/kg PS or CA would have little or no benefit in adding any degree of safety against L. monocytogenes on crawfish tail meat. As a result, additional studies using mixed barriers and CA or PS were not perused. However, as discussed previously other researchers have had success controlling the growth of L. monocytogenes using CA at higher concentrations. Consequently, additional research directed at the use of CA as a barrier for controlling L. monocytogenes should not be overlooked in the future.

Thermal InactivationThe inoculation levels (log10 CFU/ml) at time zero for

each temperature tested were 9.6 (55°C) , 9.3 (60°C) , and 8.3 (65°C). The heat resistance of L. monocytogenes in crawfish tail meat, as designated by D values (min) determined using TNF methodology, is shown in Table 5. From these data, a TDT plot was constructed showing a z value of 5.5°C.

65

Table 5. Thermal death times, least squares regression factors and goodness of fit for D value determinations using TNF methodology for L. monocytogenes in crawfish tail meat.

Temperature D values Y intercept Slope W °C (min) ±s.d. (log10)

55 10.23 ±0.32 9.56 -0.783 0.92960 1.98 ±0.15 8.48 -0.550 0.95265 0.19 ±0.01 7.91 -0.080 0.987

66Regression analysis values for intercept, slope, and R2 of the log10 survivors versus temperature are shown in Table 5.

D value ranges for L. monocytogenes on crawfish tail meat determined using the TNF method were very similar to those determined previously on crab and lobster meat (Budu- Amoake et al. , 1992; Harrison and Huang, 1990). Harrison and Huang (1990) determined D values for the bacterium on crab meat at 55 and 60°C to be 12.00 and 2.61 min, respectively. Budu-Amoake et al. (1992) determined a D value for L. monocytogenes on lobster to be 2.39 min at 60°C. They calculated a z value for the bacterium of 5°C. D values for crawfish meat obtained using the TNF method were slightly lower than in both of those studies. Crawfish tail meat is packed with adherent hepatopancreas and fat having a total lipid content of 0.939 g/100 g(USDA, 1987). Crab meat has a total lipid content of 1.188 g/100 g (USDA, 1987). The higher D value observed for crab meat at 60°C might be attributable to the protective characteristics of fat (Mackey et al., 1990). However, this did not appear to be the case when comparing the D values of crawfish tail meat to that of lobster which has a total lipid content of 0.358 g/100 g (USDA, 1987).

Other factors may have contributed to the lower values observed in the TNF thermal study. The previous studies

67done with crab meat and lobster, both used non selective TSA to enumerate L. monocytogenes. The present study used TSA as a base medium and allowed a 4 h recovery time to resuscitate injured cells. An overlay of selective LOA was then added. The presence of the antimicrobial agent, acriflavine, in this selective medium may have inhibited the growth of some heat damaged cells that were unable to recover completely before being overlaid. Inhibition would cause the recovery of fewer cells than had actually survived thermal treatment, thus yielding a lower D value than the previous two studies. When Harrison and Huang (1990) used Modified Vogel-Johnson agar, a more selective medium than TSA, to enumerate L . monocytogenes from crab meat, fewer bacteria were recovered. The D values for 55 and 60°C using this medium were 9.18 and 1.31 min, respectively; both values are lower than those observed in the present study. Additionally, the use of a mixed strain inoculum in the present study, as opposed to strain Scott A singly, may have been a contributing factor producing the reported variable results.

D60 values for L . monocytogenes differed (P<0.05) when various methods of determination were employed (Table 6). The TNF method required the crawfish samples to be suspended in an aqueous environment, which leaves the bacterium less protected by constituents of the crawfish meat. Therefore,

68

Table 6. Thermal death times, least squares regression factors and goodness of fit for D60 values determinations of L. monocytogenes using various methods in crawfish tail meat.

Method D values (min) ±s.d.

Slope Std. Err. of slope

R

TNF 1.98 ±0.15a -0.550 0.078 0.952PB 4.68 ±0. 56b -0.215 0.012 0.979CC 3.84 ±0.23b -0.251 0.012 0.987

* D values with similar superscripts are not significantly different (P>0.05).

69a lower D value can be expected when using the TNF method over the PB or CC methods. The D60 valuesdetermined using the PB and CC methods did not differ (P>0.05) (Table 6).

Traditionally TDT tests to determine D values for bacteria in foods have utilized TDT tube, TDT can, or TNF methods. The use of CC method is one commonly used for meats. Since crawfish tail meat is typically packaged by the industry in polyethylene bags, the present study used this type of container and developed a new method (PB) for assessing thermal tolerance of bacteria. The CC and PB methods yielded similar D values (P>0.05) for L. monocytogenes. The D value determined using TNF method was significantly less than both the CC and PB method (P<0.05) . Other D value determinations discussed later, done using PB method with head space (PB-a) yielded significantly (P<0.05) higher D values (10.58 min) than PB or CC without head space. Variations in D values observed with different methods indicates that methods used for the determination of D values for L. monocytogenes on crawfish tail meat are critical. Since researchers have used many different methods to determine D values for the bacterium, it is very difficult to accurately compare behavior of the bacterium in different foods.

When head space was added to the PB samples (PB-a) , the D60 values (10.58 CFU/g) increased significantly (P<0.05).

Heating profile of PB-a samples was about 0.5 to 1.0 minslower to reach 60°C than the PB samples (Figure 3). Thismay have been caused by an air insulation effect due to the headspace. The head space in PB-a crawfish tail meat samples forces product away from some walls of thepolyethylene bags. This created a situation where less surface area of crawfish sample was in proximity to liquid heating medium (water), thus reducing the speed at which the core temperature reached 60°C. The reduction in come-up time would allow L. monocytogenes more time to acclimate to the rise in temperature and reduce the accumulation of lethality units (Nickerson and Sinskey, 1972). As a result, fewer bacteria were killed with the PB-a method than for CC and PB methods under similar time frames. It is alsopossible the head space may have allowed the thermocouples to shift slightly during agitation in the water bath. A slight shift from the geometric center of the samples may have caused the centers of the crawfish tail meat samples to remain at a slightly cooler temperature. This would happen because the thermocouple would have indicated that samples had reached the targeted treatment temperature premature to the actual occurrence. The resulting lower core temperature of the samples would reduce the effectiveness of the thermal treatment, yielding higher D values.

71Thermal Inactivation with Lactic Acid

A D60 value of 4.68 min was calculated for L.monocytogenes on untreated crawfish tail using the PBmethod. LA sprays were found to enhance the lethality of heat against L. monocytogenes (Table 7) . Although a stepwise increase of lethality of heat against the bacterium was observed between the control, 0.5, and 0.75% LA, no statistically significant improvement (P<0.05) in lethality was noted until treatments reached 1.0% LA (Table 7). A1.0% LA treatment increased lethality of heat during a 14-min thermal treatment by approximately 4 logs over the control (Figure 6).

D60 values calculated for L. monocytogenes on crawfish tail meat that was pH adjusted using HC1 to approximate the pH of 0.5 and 1.0% LA were 4.92 and 3.54 min, respectively. There was no significant difference between crawfish tail meat controls and samples adjusted with HC1 to approximate the pH of 0.5 or 1% LA treatments (Table 7). Although the D60 value (Table 7) and resulting linear regression of TDT slope (Figure 7) for L. monocytogenes on crawfish tail meat samples pH adjusted with HC1 to approximate that of 1.0% LA was smaller than the D60 for controls, it was significantly greater (P<0.05) than 1.0% LA treated samples. This indicates that while a reduction of pH may increase

72

Table 7. Least squares regression factors and goodness of fit for D60 values for L. monocytogenes on crawfish tail meat treated with different levels of lactic acid or HC1.

Acid %Acid

PH D-value Slope Std. Err. of Slope

R*

LA 0.00 7.6 4. 68“ -0.215 0.013 0.979LAHC1

0.500.35

6.46.5

4 .41a 4.92s

-0.237-0.216

0. 019 0. 026

0.9620.923

LA 0.75 6.3 3 .46a -0.289 0. 013 0.989LAHC1

1.000.75

5.55.4

2.49b 3. 54a

-0.417-0.282

0.0150.019

0.9940.973

a,b D values with similar superscripts are not significantly different when compared to the control (P>0.05).

Log

CFU

/g73

9

8

7

6

5

4

0.0%3

0 .5 %

0 .7 5 %21.0 %

1

0 2 4 6 8 1210 14TIME (min)

Figure 6. Thermal death curve for L. monocytogenes at 60°C using various levels of spray applied lactic acid.

Log

CFU

/g74

10

CONTROL {pH 7.6)

* - 0.215 ± .01

- - HCI (pH 5.5)* - 0.282 ± .02

- 1 .0% LA (pH 5.5) * -0.417 ± .02

0 2 4 6 8 10 12 14 16

Treatment Time (min)

* Slope + s.d. of least squared regressions

Figure 7. Linear regression comparing the effect of 1%lactic acid and the equivalent HCI adjusted pH, on lethality of 60°C toward L. monocytogenes on crawfish tail meat.

75lethality of heat towards L. monocytogenes, LA augments lethality independent of pH.

To date, the exact mechanism that makes environments containing weak acids like LA more lethal to L. monocytogenes than an equivalent low pH environment altered with strong acids has not been defined. It has, however, been established that weak acids in their undissociated or protonated form have the ability to penetrate the cell membrane and accumulate within the cell cytoplasm (Ita and Hutkins, 1991) . It may be that LA simply is able to penetrate the cell more effectively than other acids. Once the acid penetrates the interior of the cell, it releases a proton and acidifies the cytoplasm of the cell (Booth, 1985). The inability to maintain a proper pH gradient via a proton/cation exchange system, coupled with the exhaustion of energy needed to maintain normal metabolic processes, results in the cells inability to survive (Kashket, 1987). Continued research directed at establishing the exact mechanism is certainly warranted.

Sensory EvaluationSensory analysis was conducted to determine the effect

of study levels of LA on product acceptability. Although the sensory panel found no significant difference (P>0.05) for all characteristics tested between controls and any LA

76treated samples (Table 8) , the panel observed a slight toughening of the product when LA at any level was added. The panel also noted that the typical fishy odor and taste of crawfish was reduced with application of any level of LA. Additionally, LA treated crawfish appeared to be brighter in color. Many panelists found these characteristics to be a desirable change.

While crawfish tail meat was stored at 4°C under various gases, browning of the samples was visually observed in packages containing 02 after 6 d, when compared to other samples packed without 02. Paralleling this observation, Bentley et al. (1989) observed no surface discoloration on beef patties packaged under 100% N2, 100% C02, or Vacuum and stored at 0, 4, or 8°C for up to 21 d. Of atmospheresevaluated, they determined that beef patties stored in a C02environment produced the highest values for all sensory traits evaluated, including tenderness, juiciness, flavor, and overall desirability. When crawfish tail meat samples stored in 100% C02 were visually compared with samplespacked in all other atmospheres, an increase of colorbrightness was observed. This occurrence in crawfish has been previously quantified by Gerdes et al. (1989). Using a HunterLab color difference meter (HunterLab Assoc., Richmond, VA) , they documented an increase in redness color

77

Table 8. Taste panel results of crawfish samples packed under air and stored at 4°C. Values of 5.0 = standard quality, >5.0 = less favorable quality, and <5.0 = more favorable quality.

%LA DAY ODOR COLOR TASTE TEXTURE

0.00 0 4.3±1.3 4.311.2 4.411.4 4.311.42 4.7±1.4 4.411.2 4.811.5 4.511.45 5.Oil.5 4.511.2 4.511.3 4.811.3

0.50 0 4.811.7 4.411.5 4.611.8 4.211.52 4.611.4 4.311.2 4.511.4 4.711.55 4.711.1 4.410.9 4.411.1 4.811.2

0.75 0 4.611.0 4.411.0 5.011.2 4.711.32 4.211.1 4.211.2 4.011.4 4.311.55 5.111.5 4.611.0 5.411.4 5.311.2

1.00 0 4.611.5 4.511.0 4.811.4 4.511.12 4.611.1 4.411.4 4.711.2 5.011.35 5.111.1 4.611.2 5.011.5 4.911.1

78values when crawfish samples were stored on ice in high C02 atmospheres for 21 d. Consumers may find this change desirable, however, additional consumer surveys would be required to determine this.

In general it appears that LA treatment levels used for the present study are acceptable sensorially. As a result the use of these levels on crawfish tail meat could be recommended.

Heat and Modified AtmospheresDuring 60°C heat treatments of up to 20 min, gas

measurements of package headspace revealed no meaningful changes in C02 or 02 composition of any atmospheres studied (Table 2). The TDT curves for L. monocytogenes on crawfish tail meat are shown in Figure 8. There was no statistical difference in D60 values for L. monocytogenes on crawfish meat packed in any of the 4 atmospheres (Table 9) . D60values for L. monocytogenes on crawfish meat packed in atmospheres that contained 02 were lower, however than for atmospheres which were void or limited of 02 (Table 9) .

Recent research conducted on Escherichia coli as well as L. monocytogenes may offer an explanation for the decreased thermal resistance of L . monocytogenes in atmospheres rich in 02. Murano and Pierson (1993) determined D value of heat-shocked E. coli 0157:H7 to be

Log

CFU

/g79

9

8

7

6

5 Air

4

CO

3

2

0 4 8 12 16 20

TREATMENT TIME (min)

Figure 8. Thermal death curve for L. monocytogenes oncrawfish tail meat at 60°C packed under various atmospheres.

80

Table 9. Least squares regression factors and goodness of fit for D-value determinations after 60°C thermal treatment of crawfish tail meat treated with or without 1% LA and packed in different modified atmospheres with head space.

AtmosphereType

pH8 D-value Slope Std. Err. of Slope

R2

Air 7.6 10.58b -0.097 0.003 0. 995+1% LA 5.4 3.44c -0.360 0.031 0.986

o2 7.6 9.22b -0.110 0. 007 0.983+1% LA 5.4 3 .88° -0.274 0. 031 0.975C02 6.6 11. 68b -0.088 0.007 0.975

+1% LA 5.4 3 .48c -0.313 0.037 0.973

n2 7.6 12 .97b -0.081 0.005 0.983+1% LA 5.4 3.08° -0.340 0.014 0.996

a pH of samples are after heating at 60°Cb,c D values with similar superscripts are not

significantly different (P>0.05).

higher when cells were incubated anaerobically than when incubated aerobically. It has been theorized that severely heat-injured cells acquire a strict anaerobic nature (McCord et al., 1971). Thus, any cell damage due to oxidation reactions, which are normally enhanced by heat, are diminished in oxygen free environments. Additionally, Dallmier and Martin (1988) showed that after 10 min at 60°C, L. monocytogenes (Scott A) retained only 42% of its initial catalase (CAT) activity. It has been hypothesized that inactivation of enzymes like CAT and superoxide dismutase (SOD) during heating, would interrupt the cells ability to deactivate toxic oxygen radicals during subsequent recovery periods. The result would be a buildup of toxic oxygen radicals in an 02 rich environment. Consequently, the inactivation of these enzymes converts L. monocytogenes into an obligate anaerobe (Knabel et al., 1990).

The addition of 1% LA to crawfish tail meat caused a significant decrease (P<0.05) in D60 values for L. monocytogenes, regardless of packaging atmosphere (Table 9) . There was, however, no difference (P>0.05) between gas environments (Figure 9). Presence of LA masked the affect of an 02-free environment on thermal resistance. D60 values determined for L. monocytogenes on crawfish tail meat using 1% LA and head space containing molecular 02, were higher than D60 values determined from samples packed using 1% LA

Log

CFU

/g82

9

8

7

6

5X

control4

Air + 1 %LA

3 O , + 1 %LA

CO + 1 %LA2

1

00 4 8 12

TREATMENT TIME (min)

Figure 9. Thermal death curve for L . monocytogenes oncrawfish tail meat at 60°C spray treated with 1% lactic acid and packed under various atmospheres.

83and no head space (Tables 5 & 9) . This might beattributable to an inability of the bacterium to detoxify toxic products of molecular 02, the superoxide radical (02—) and hydrogen peroxide (H202) , after thermal destruction of the enzymes CA and SOD.

4 °C Storage Under Various Atmospheres & 1% LAGeneration times at 4°C for heat shocked L.

monocytogenes on crawfish tail meat packed under Air, 100% 02, 100% C02, and 100% N2 were 26.4, 33.6, 105.1, and 26.9 h, respectively. The most inhibitive package environment was 100% C02, exhibiting significantly (P<0.05) largergeneration times than all other gas treatments (Figure 10). Carbon dioxide penetrates cells very easily (Daniels et al., 1985) and this may facilitate its chemical effects on the internal metabolic processes of L . monocytogenes. It has been long established that increased inhibition of bacteria at lower temperatures is correlated to increased solubility of the gas in the water phase (Golding, 1945). Carbon dioxide was rapidly absorbed by crawfish tail meat in the present study. Approximately 30 min after packing crawfish tail meat samples in 100% C02 with 4:1 head space (gas to product) no head space remained in the packs. This rapid absorption enables conversion of C02 to carbonic acid. Carbonic acid produces a rapid acidification of bacterial

Log

CFU

/g84

10.00

8 .6 0

' CO

7 .2 0

V

5 .8 0

4 .4 0

3 .0 0

0 3 6 9 12 18 2115

TREATMENT TIME (days)

Figure 10. Growth of L. monocytogenes stored under 4different atmospheres at 4°C for 21 d.

85cell internal pH leading to altered metabolic activities (Daniels et al., 1985). During the present study, pH of crawfish tail meat dropped approximately unit during the 21 d storage period when packed under C02 (Table 3).

These findings contribute support to a hypothesis that as with heat, C02 appears to exert an effect on certain enzyme systems affecting growth of the bacterium (Foster and Davis, 1949; Fenestil et al., 1963; King and Nagel, 1975). Several mechanisms have been hypothesized as explanations for the reduction of growth of a bacterium in the presence of C02. Stimulated mitochondrial ATPase activity and the uncoupling effect on oxidative phosphorylation resulting in a decreased level of energy available to the bacterium for metabolism and growth is one (Fenestil et al., 1963). King and Nagel (1975) postulated that C02 concentrations above 50% inhibit the activity of isocitrate dehydrogenase, malate dehydrogenase and inhibits certain decarboxylation enzymes through a mass action effect.

The present study found that thermally treated L. monocytogenes tended to recover more effectively on crawfish tail meat packaged in N2 and air, which were free or limited in 02, than in an environment of 100% 02 (Figure 10) . Knabel et al. (1990) reported that after heat shock in milk, anaerobic recovery of L. monocytogenes resulted in higher numbers when compared with recovery under aerobic

conditions. Murano and Pierson (1993) found the same to be true for Escherichia coli 0157:H7 after heat shock. The present study determined that 20% of the cells were injured when thermally treated at 60°C for 12.5 min. Dallmier and Martin (1988) reported that CAT and SOD were rapidly inactivated when L. monocytogenes was heated at temperatures of 60°C. Consequently this inactivation of CAT and SODduring the 60°C thermal treatment and subsequent build up ofoxygen radicals in an oxygen rich environment may havecaused the bacterium to grow at a slower rate when comparedto air and N2. However, since a majority of surviving cellswere not injured by the thermal treatment, no significant difference (P>0.05) was observed in growth inhibition between 100% 02, air and N2 atmospheres.

One percent LA added to crawfish tail meat followed by packaging under air, 100% 02, 100% C02 and 100% N2atmospheres prevented growth of L . monocytogenes (Figures 11 & 12) . The most effective atmosphere plus LA combination appears to be 02 + 1% LA (Figure 13) . This combination was significantly (P<0.05) more lethal to the bacterium than the N2 + 1% LA combination and consistently more lethal than the C02 + 1% LA or Air + 1% LA combinations (Figure 13). The destructive element of this treatment combination is probably largely a result of build up of oxygen radicals by the bacterium. It appears that when this outcome is

CFU

/g87

10.00

Air

8.40 Air + 1 % LA

6.80

5.20

-=i—3.60

2.000 3 6 129 15 18 21

STORAGE TIME (days)

Figure 11. Growth of L . monocytogenes on crawfishtail meat stored under Air and N2 alone and incombination with 1% LA at 4°C for 21 d.

Log

CFU

/g88

10.00

8 .4 0CO

O + 1% LA

6 .8 0

5 .2 0

3 .6 0

2.000 3 6 9 12 18 2115

STORAGE TIME (days)

Figure 12. Growth of L. monocytogenes stored underC02 and 02 alone and in combination with 1% LA at4°C for 21 d.

Log

CFU

/g89

4.25

3.85

3.45

Air3.05

CO

2.65

2.25

0 3 6 9 12 2115 18STORAGE TIME (days)

Figure 13. Destruction of L. monocytogenes stored underAir, N2, C02, and 02 in combination with 1% LA at 4°C for 21 d.

90combined with the effects of LA, a package environment of 100% Oz + 1% LA becomes the most lethal of the treatment combinations studied.

As discussed earlier, when LA was absent, C02 was the most restrictive packaging atmosphere to growth of L. monocytogenes on crawfish tail meat. However, any effect of pH reduction resulting from carbonic acid production experienced with introduction of C02 was completely masked by the presence of 1% LA. This was evidenced by the significant difference (P<0.05) between C02 and the other 3 atmospheres without LA present (Figure 10) versus the lack of significant difference (P>0.05) between COz and other atmospheres when 1% LA was added as a treatment (Figure 13) .

SUMMARY AND CONCLUSIONS

Heat treatments of approximately 4 min at 60°C will reduce L. monocytogenes by 90% on packaged crawfish tail meat. The addition of LA spray treatments increases lethality of heat to the bacterium. The enhanced lethality of heat when LA was introduced was not strictly due to a simple pH effect. It appears incorporation of two barriers, heat and LA, in a process could contribute a significant degree of safety to the consumer in regards to crawfish tail meat.

When L. monocytogenes was heat treated in an 02-free environment D60 values were consistently higher than in treatments done in the presence of 02. This observation supports the hypothesis that absence of 02 in the environment used during heat treatments of L. monocytogenes acts to protect the bacterium from heat injury. To encourage greater lethal heat processes against the bacterium, inclusion of sufficient amounts of 02 in packaging environments should be considered.

Results from storage studies further strengthen the argument that packaging of crawfish tail meat and any subsequent heat treatment should be done in the presence of 02. As mentioned above, heat treatments administered under a 100% 02 packing environment were the most destructive to

91

92L. monocytogenes, compared with air, C02, and N2. Storage at 4°C of 1% LA treated crawfish tail meat packed under 100% 02 proved to be the most lethal barrier combination to heat injured L . monocytogenes.

It would appear that methodologies employed in the determination of thermal death times for L. monocytogenes in various food products need careful consideration. The present study indicates that methods which use no head space, thus allowing the bacterium little or no access to molecular 02, may yield higher D values for a given product than similar methods which include a head space containing some molecular 02. Therefore, application of heat treatments in a low-oxygen environment and subsequent storage of a product in a similar environment may increase, instead of decrease the probability of listeriosis being contracted from a given food product.

While maximum kill of the bacterium seems to occur when it is heated in the presence of 02, minimum recovery is realized. Tightly packed TDT tubes and cellulose casings that allow very little 02 to remain during heating trials, may yield higher D values for L . monocytogenes. Because of the threat of toxin production from Clostridium botulinum in an anaerobic environment, many industrial packaging methods allow inclusion of 02. Consequently, laboratory methods for determining D values of L. monocytogenes should be designed

93to allow the presence of 02. However, since complete recovery of heat injured L. monocytogenes in an aerobic environment appears impossible, media overlays which exclude 02 or pre-reduced media should be employed for enumeration of the bacterium.

Commercially processed crawfish are boiled for 5 to 10 min before hand peeling. Based on heat resistance of L. monocytogenes demonstrated in the present studies, this process would provide sufficient destruction of the bacterium before hand peeling and packaging. Thus, occurrence of the bacterium in packaged crawfish tail meat would be mainly due to cross contamination from plant workers or equipment. The adoption of strict in-plant sanitation programs to prevent post-boil cross contamination is paramount. Because of the possibility of process deviations, post-packaging heat treatments in the presence of 02 and addition of 1% LA sprays should be implemented. While more extensive investigations into the mechanisms which cause interactions of these barriers in crawfish tail meat and other food products is needed, the addition of these steps in a crawfish packing plant would increase safety of the product.

REFERENCES

Ahamad, N. and Marth, E.H. 1990. Behavior of Listeria monocytogenes at 7, 13, 21, and 35°C in trytose brothacidified with acetic, citric, or lactic acid. J. Food Prot. 52:688-695.Anonymous. 1978. Sorbic acid and potassium sorbate for preserving food freshness and market quality. Monsanto Industrial Chemicals Co., St. Louis. MO.Anonymous. 1979. Sorbic acid as a food preservative. Food Proc. Ind. 48:36-41.Anonymous. 1992. Class I recall made of shad because of Listeria. Food Chemical News 34(33):19.Audurier, A., Prdon, P., Marly, J., and Lantier, F. 1980. Experimental infection of mice with Listeria monocytogenes and L . innocua. Ann. Microbiol. (Paris) 131B:47-57.Azadian, B.S., Finnerty, G.T., and Pearson, A.D. 1989. Cheese-borne Listeria meningitis in immunocompetent patient. Lancet i:322-323.Banks, H., Nickalson II, R., and Finne, G. 1980. Shelf- life studies on carbon dioxide packaged finfish from the Gulf of Mexico. J.Food Sci. 45:157.Barnett, H.J., Nelson, R.W., Hunter, P.J., Bauer, S., and Groninger, H. 1971. Studies on the use of carbon dioxide dissolved in refrigerated brine for the preservation of whole fish. Fish Bull. 69:433.Barnett, H.J., Nelson, R.W., Hunter, P.J., and Groninger, H. 1978. Use of carbon dioxide dissolved in refrigerated brine for the preservation of pink shrimp (Panadalus spp.). Mar. Fish Rev. 40:24.Bentley, D.S., Reagan, J.O., and Miller, M.F. 1989. Effects of gas atmosphere, storage temperature and storage time on the shelflife and sensory attributes of vacuum packaged ground beef patties. J. Food Sci. 54:284-286.Berrang, M.E., Brackett, R.E., and Beuchat, L.R. 1989. Growth of Listeria monocytogenes on fresh vegetables stored under controlled atmosphere. J. Food Prot. 52:702-705.

94

95Bille, J., Catimel, B., Bannerman, E., Jacquet, C. , Yersin, M.-N., Caniaux, I., Monoget, D., and Rocourt, J. 1992. API Listeria, a new and promising one-day system to identify Listeria isolates. Appl. Environ. Microbiol. 58:1857-1860.Booth, I.R. 1985. Regulation of cytoplasmic pH inbacteria. Microbiol. Rev. 49:359-378.Brackett, R.E. 1988. Presence and persistence of Listeria monocytogenes in food and water. Food Technol. 42:162-164.Brown, W.D., Albright, M., Watts, D.A., Heyer, B., Spruce, B., and Price, R.J. 1980. Modified atmosphere storage of rockfish (Sebastes miniatus) and silver salmon (Oncorhynchus kisutch). J. Food Sci. 45:93-96.Bruno, M.E.C. and Montville, T.J. 1993. Commonantilisterial mechanism of bacteriocins from lactic acid bacteria. Abstract: IFT 1993 Annual Meeting. p42.Buchanan, R.L., Golden, M.H., and Whiting, R.C. 1993. Differentiation of the effects of pH and lactic or acetic acid concentration on the kinetics of Listeria monocytogenes inactivation. J. Food Protection 56:474-478,484.Budu-Amoako, E. , Toora, S., Walton C. , Ablett, R.F., and Smith, J. 1992. Thermal death times for Listeria monocytogenes in lobster meat. J. Food Prot. 55:211-213.Bullard, F.A. and Collins, J. 1978. Physical and chemical changes of pink shrimp, Pandalus borealis, held in carbondioxide modified refrigerated seawater compared with pinkshrimp held in ice. Fish Bull. 76:73.Bunning, V.K., Donnelly, C.W., Peeler, J.T., Briggs, E.H., Bradshaw, J.G., Crawford, R.G., Beliveau, C.M., and Tierney, J.T. 1988. Thermal inactivation of Listeria monocytogenes with bovine milk phagocytes. Appl. Environ. Microbiol. 54:364-370.Chung, Y.M. and Lee, J.S. 1981. Inhibition of microbial growth in English sole (Parophrys retulus). J. Food Prot. 44:66-68.Chung, Y.M. and Lee, J.S. 1982. Potassium sorbate inhibition of microorganisms isolated from seafood. J. Food Prot. 45:1310-1313.

96Ciesielski, C.A., Hightower, A.W., Parsons, S.K., and Broome, C.V. 1988. Listeriosis in the United States: 1980-1982. Arch. Intern. Med. 148:1416-1419.Conner, D.E., Brackett, R.E., and Beuchat, L.R. 1986. Effect of temperature, sodium chloride, and pH on growth of Listeria monocytogenes in cabbage juice. Appl. Environ. Microbiol. 52:59-63.Cowles, P.B. 1941. The germicidal action of the hydrogen ion and of the lower fatty acids. Yale J. Biol. Med. 13:571-578.Coyne, F.P. 1932. The effect of carbon dioxide on bacterial growth with special reference to the preservation of fish. Part 1. J. Soc. Chem. Ind. 51:1191.Coyne, F.P. 193 3. The effect of carbon dioxide on bacterial growth with special reference to the preservation of fish. Part 2. Gas storage of fresh fish. J. Soc. Chem. Ind. 52:19T.Czuprynski, C.J., Brown, J.F., and Roll, J.T. 1989. Growth at reduced temperatures increases the virulence of Listeria monocytogenes for intravenously but not intragastrically inoculated mice. Microb. Path. 7:213-223.Dallmier, A.W. and Martin, S.E. 1988. Catalase and superoxide dismutase activities after heat injury of Listeria monocytogenes. Appl. Environ. Microbiol. 54:581- 582.Deak, T. and E.K. Novak. 1972. Assimilation of sorbic acid by yeasts. Acta Aliment. 1:87-104.Daniels, J.A., Krishnamurthi, R., and Rizvi, S.S.H. 1985. A review of effects of carbon dioxide on microbial growth and food quality. J. Food Prot. 48:532-537.Danzinger, M.T., Steinberg, M.P. and Nelson, A.I. 1973. Effect of C02, moisture content and sorbate on safe storage for wet corn. Trans. Am. Soc. Agr. Eng. 16:679-682.Dogra, R. and Schaffner, D.W. 199 3. Determining differences in microbial growth rates using linear regression. Dairy Food Environ. Sani. 13:517-518.Dorsa, W.J. and Marshall, D.L. 1993. Unpublished data. Louisiana State Univ.

97Elliot, P.H., and Gray, R.J. 1931. Salmonella sensitivity in a sorbate-modified atmosphere combination system. J. Food Prot. 44:903-908.Elliot, P.H., R.I. Tomlins, and R.J.H. Gray. 1982. Inhibition and inactivation of Staphylococcus aureus in a sorbate-modified atmosphere combination system. J.Food Prot. 45:1112-1116.El-Shenawy M.A. and Marth, E.H. 1988. Inhibition and inactivation of Listeria monocytogenes by sorbic acid. J. Food Prot. 51(11):842-847.Fain, A.R., Jr., Line, J.E., Moran, A.B., Martin, L.M. , Feachowich, R.V., Carosella, J.M., and Brown, W.L. 1991. Lethality of heat to Listeria monocytogenes Scott A: D-Value and Z-Value determinations in ground beef and turkey. J. Food Prot. 54(10):756-761.Fanestil, D.D., Hastings, A.B., and Mahowald, T.A. 1963. Environmental carbon dioxide stimulation of mitochondrial adenosine triphosphatase activity. J. Bio. Chem. 238:836- 842.Farber, J.M. 1989. Thermal resistance of Listeriamonocytogenes in foods. Int. J. Food Microbiol. 8:285-291.Farber, J.M. 1991. Listeria monocytogenes in fishproducts. J. Food Protection. 54:922-924, 934.Farber, J.M. and Brown, B.E. 1990. Effect of prior heat shock on heat resistance of Listeria monocytogenes in meat. Appl. Environ. Microbiol. 56:1584-1587.Farber, J.M., Fournier, J., Daley, E., and Dodds, K. 1990. Abstr. Annu. Meet. Am. Soc. Microbiol. 1990, P-57, p.287.Farber, J.M. and Losos, J.Z. 1988. Listeria monocytogenes: a foodborne pathogen. CMAJ 138 (Mar):413.Farber, J.M. and Peterkin, P.I. 1991. Listeriamonocytogenes, a food-borne pathogen. Microbiol. Reviews. 55(1):476-511.Farber, J.M., Sander, G.W., Dunfield, S., and Prescott, R. 1989a. The effect of various acidulants on the growth of Listeria monocytogenes. Lett. Appl. Microbiol. 9:181-183.

98Farber, J.M., Hughes, A., Holly, R., and Brown, B. 1989b. Thermal resistance of Listeria monocytogenes in sausage meat. Acta Microbiol. Hung. 36:273-275.Fleming, D.W., Cochi, S.L., MacKonald, K.L., Brondum, J., Hayes, P.S., Plikaytis, B.D., Holmes, M.B., Audurier, A., Broome, C.V., and Reigold, A.L. 1985. Pasteurized milk as a vehicle of infection in an outbreak of listeriosis. N. Engl. J. Med. 312:404-407.Foster, J.W. and Davis, J.B. 1949. Carbon dioxide inhibition of anaerobic fumarate formation in molds Rhizopus nigricans. Arch. Biochem. 21:135-142.Food and Drug Administration. 1990. Substances generallyrecognized as safe. In Code of Federal Regulations, Title 21, Sec. 170. U.S. Government Printing Office, Washington, DC.Food Processors Institute. 1988. Canned Foods: Principles of thermal process control, acidification and container closure evaluation. Fifth Edition, 2nd Printing. U.S. Government Printing Office, Washington, DC.Freese, E., Wheu, C.W., and Galliers, E. 1973. Function of lipophilic acids as antimicrobial food additives. Nature 241:321-325.Fuchs, R.S. and Sirvas, S. 1991. Incidence of Listeria monocytogenes in an acidified fish product, ceviche. Letts. Appl. Microbiol. 12:88-90.Garcia, G.W., Genigeorgis, C. , and Lindroth, S. 1987. Risk of growth and toxin production by Clostridium botulinum nonproteolytic types B, E, and F in salmon fillets stored under modified atmospheres at low and abused temperatures. J. Food Prot. 50:330-336.Garcia, G.W. and Genigeorgis, C. 1987. Quantitative evaluation of Clostridium botulinum nonproteolytic types B, E, and F growth risk in fresh salmon tissue homogenated stored under modified atmospheres. J. Food Prot. 50:390- 397.Gaunt, I.F., Butterworth, K.R., Hardy, J., and Gangolli, S.D. 1975. Longterm toxicity of sorbic acid in the rat. Food Cosmet. Toxicol. 13:31-45.Gellin, B.G. and Broome, C.V. 1989 Listeriosis. JAMA 261(9):1313-1320.

99Gerdes, D.L., Hoffstein, J.J., Finerty, M.W., and Grodner, R.M. 1989. The effects of elevated C02 atmospheres on the shelf life of freshwater crawfish (Procambaris clarkii) tail meat. Lebensm.-Wiss. u.-Technol. 22:315-318.Gill, C.O and Tan, K.H. 1979. Effect of carbon dioxide on growth of Pseudomonas fluorescens. Appl. Environ.Microbiol. 38:237-240.Glass, K.A. and Doyle, M.P. 1989. Fate of Listeria monocytogenes and Listeria infections. Bacteriol. Rev. 30:309-382.Golding, N.S. 1945. The gas requirments of molds. IV. A preliminary interpretation of the growth rates of four common mold cultures on the basis of absorbed gases. J. Dairy Sci. 28:737-750.Golnazarian, C.A., Donnelly, C.W., Pintauro, S.J., and Howard, D.B. 1989. Comparison of infectious dose of Listeria monocytogenes F5817 as determined for normal versus compromised C57B1/6J mice. J. Food Prot. 52:696-701.Grau, F.H. and Vanderlinde, P.B. 1992. Occurrence, numbers, and growth of Listeria monocytogenes on some vacuum-packed processed meats. J. Food Prot. 55:4-7.Gray, M.L. and Killinger, A.H. 1966. Listeriamonocytogenes and Listeria infections. Bact. Review 30(2) :3 09-382.Gray, R.J.H., Hoover, D.G., and Muir, A.M. 1983. Attenuation of microbial growth on modified atmospheres- packaged fish. J. Food Prot. 46:610-613.Harrison, M.A. and Carpenter, S.L. 1989. Survival of large populations of Listeria monocytogenes on chicken breast processed using moist heat. J. Food Prot. 52:376-378.Harrison M.A. and Yao-Wen Huang. 1990. Thermal death times for Listeria monocytogenes (Scott A) in crabmeat. J. Food Prot. 53: 878-880.Harrison M.A., Yao-Wen Huang, Chao, C.H., and Shineman, T.1991. Fate of Listeria monocytogenes on packaged, refrigerated, and frozen seafood. J. Food Prot. 54:524- 527.

100Health and Welfare Canada. 1990. Listeria monocytogenes - An Information Handbook. Health and Welfare Canada,Agriculture Canada.Henry, B.S. 1933. Dissociation in the genus Brucella. J. Infect. Dis. 52:374-402.Hiltz, D.F., Lall, B.S., Lemon, D.W., and Dyer, W.J. 1976. Deteriorative changes during frozen storage in fillets and minced flesh of silver hake (Merluccius bilinearis) processed from round fish held in ice and refrigerated sea water. J. Fish. Res. Bd. Can. 33:2560.Hintlian, C.B and Hotchkiss, J.H. 1986. The safety ofmodified atmosphere packaging: a review. Food Technol.40(12):70-76Ho, J.L., Shands, K.N., Friedland, G. , Eckind, P., and Fraser, D.W. 1986. An outbreak of type 4b Listeria monocytogenes infection involving patients from eight Boston hospitals. Arch. Intern Med. 146:520-524.Ingham, S.C. 1990. Growth of Aeromons hydrophila and Plesiomonas shigelloides on cooked crayfish tails during cold storage under air, vacuum, and a modified atmosphere. J. Food Prot.Ingram, M. , Ottoway, F.J.M., and Loppock, J.B.M. 1956. The preservative action of acid substances in food. Chem. Ind. 42:1154-1158.Ita, P.S. and Hutkins, R.W. 1991. Intracellular pH and survival of Listeria monocytogenes Scott A in Tryptic soy broth containing acetic, lactic, citric and hydrochloric acids. J. Food Prot. 54:15-19.Jemmi, T. 1990. Actual knowledge of Listeria in meat and fish products. Mitt. Geb. Lebensmittelunters. Hyg. 31:144- 157.Kallander, K.D., Hitchins, A.D., Lancette, G.A., Schmieg, J.A„ , Garcia, G.R., Solomon, H.M. and Sofos, J.N. 1991. Fate of Listeria monocytogenes in shredded cabbage stored at 5 and 25°C under modified atmosphere. J. Food Prot. 54:302-304.Kashket, E.R. 1987. Bioenergetics of lactic acid bacteria: cytoplasmic pH and osmotolerance. FEMS Microbiol. Rev. 46:233-244.

101Kaul, A., Singh, J. , and Kuila, R.K. 1979. Effect of potassium sorbate on the microbiological quality of butter. J. Food Prot. 42:656-657.Killeffer, D. 1930. Carbon dioxide preservation of meat and fish. Ind. Eng. Chem. 22:140.King, A.D., Jr., and Nagel, C.W. 1975. The influence of carbon dioxide upon the metabolism of Pseudomonas aeruginosa. J. Food Sci. 40:362-366.Knabel, S.J., Walker, H.W., Hartman, P.A., and Mendonca, A.F. 1990. Effects of growth temperature and strictly anaerobic recovery on the survival of Listeria monocytogenes during pasteurization. Appl. Environ. Microbiol. 56:370- 376.Kvenberg, J.E. 1988. Outbreaks of listeriosis/Listeria- contaminated foods. Microbiol. Sci. 5:355-358.LaBell, F. 1986. Modified atmosphere packaging of meats, poultry, seafoods. Food Proc. Dec:135-136.Lennon, D., Lewis, B., Mantell, C., Becroft, D., Dove, B., Farmer, K., Tonkin, S., Yeates, N., Stamp, R., and Mickleson, K. 1984. Epidemic perinatal listeriosis. Pediatr. Infect. Dis. 3:30-34.Levine, A. S. and Fellers, C.R. 1939. Action of acetic acid on food spoilage microorganisms. J. Bacteriol. 39:499-515.Liewen, M.B. and Marth, E.H. 1985. Growth and inhibition of microorganisms in the presence of sorbic acid: A review. J. Food Prot. 48:364-375.Linnan, M.J., Nascola, L., Lou, X.D., Goulet, V., May, S., Salminen, C., Hird, D.W., Yonekura, M.L., Hays, P., Weaver, R., Audurier, A., Plikaytis, B.D., Fannin, S.L., Kleks, A., and Broome, C.V. 1988. Epidemic listeriosis associated with mexican-style cheese. N. Engl. J. Med. 319:823-828.Luck, E. 1976. Sorbic acid as a food preservative. Int. Flavors Food Additiv. 7:122-124, 127.Lueck, E. 1980. Antimicrobial food additives. Springer Verlag, New York.MacDonald, T.T., and Carter, P.B. 1980. Cell-mediated immunity to intestinal infection. Infect. Immun. 28:516- 523 .

102Mackey, B.M. , Pritchet, C. , Norris, A., and Mead, G.C.1990. Heat resistance of Listeriai strain differences and effects of meat type and curing salts. Lett. Appl. Microbiol. 10:251-255.Manu-Tawiah, W. , Myers, D.J., and Molins, R.A. 1993. Survival and growth of Listeria monocytogenes and Yersinia enterocolitica in pork chops packed under modified gas atmospheres. J. Food Sci. 58:475-479.Marshall, D.L. and Schmidt, R.H. 1988. Growth of Listeria monocytogenes at 10°C in milk preincubated with selected Pseudomonads. J. Food Prot. 51:277-282.Marshall, D.L., Wiese-Lehigh, P.L., Wells, J.H., and Farr, A.J. 1991. Comparative growth of Listeria monocytogenes and Pseudomonas fluorescens on precooked chicken nuggets stored under modified atmospheres. J. Food Prot. 54:841- 843,851.Marshall, D.L., Andrews, L.S., Wells, J.H. and Farr, A.J.1992. Influence of modified atmosphere packaging on the competitive growth of Listeria monocytogenes and Pseudomonas fluorescens on precooked chicken. Food Microbiol. 9:303- 309.McCarthy, S.A. 1990. Listeria in the environment, p.25-29. In A.J. Miller, J.L. Smith, and G.A. Somkuti (ed.), Foodborne listeriosis. Society for Industrial Microbiology. Elsevier Science Publishing, Inc. New York.McCarthy, S.A., Motes, M.L., and McPhearson, R.M. 1990. Recovery of heat-stressed Listeria monocytogenes from experimentally and naturally contaminated shrimp. J. Food Prot. 53:22-25.McCord, J.M., Keele, B.B, and Fridovich. 1971. An enzyme- based theory of obligate anaerobiosis: the physiological function of superoxide dismutase. Proc. Natl. Acad. Sci. USA. 68:1024-1027.McLauchlin, J. 1987. Listeria monocytogenes. Recent advances in the taxonomy and epidemiology of listeriosis in humans. J. Appl. Bacteriol. 63:1-11.McLauchlin, J. 1990. Distribution of serovars of Listeria monocytogenes isolated from different categories of patients with listeriosis. Eur. J. Clin. Microbiol. Infect. Dis. 9:210-213.

103Melnick, D. , Luckmann, F.H., and Gooding, C.M. 1954.Sorbic acid as a fungistatic agent for foods. VI. Metabolic degradation of sorbic acid in cheese by molds and the mechanism of mold inhibition. Food Res. 19:44-58.Menudier, A., Bosiraud, C., and Nicolas, J.A. 1991.Virulence of Listeria monocytogenes serovars and Listeria spp. in experimental infection of mice. J. Food Prot. 54:917-921.Messer, J.W., Behney, H.M., and Luedecke, L.O. 1985.Microbiological count methods. pl33. In G.H. Richardson (ed)Standard methods for the examination of dairy products, 15th ed. American Public Health Association, Washington, DC.Miller, J.K., and Burns, J. 1970. Histopathology ofListeria monocytogenes after oral feeding to mice. Appl. Microbiol. 19:772-775.Mokhele, K. , Johnson, A., Barret, E. , and Ogrydziak, D.1983. Microbiological analysis of rock cod (Sebastes sp.) stored under elevated carbon dioxide atmospheres. Appl. Environ. Microbiol. 45:878.Molin, G., Stenstrom, I.M., Ternstrom, A. 1983. The microbial flora of herring fillets after storage in carbondioxide, nitrogen or air at 2°C. J. Appl. Bacteriol.55:49-56.Moline, S.W., C.E. Colwell, andJ.E. Simeral. 1963. Sorbic acid. p. 589-599. In A.Standen (ed), Kirk-Othmer encyclopedia of chemical technology, 2nd ed. Vol. 18. John Wiley and Sons, NY.Moore, D. 1988. How to control Listeria in dairy plants. Modern Dairy 12:15.Morille, T.M., Young-Perkins, K., and Corrigan, J. 1993. The effect of nisin on the growth of Listeria monocytogenes in model ready-to-eat pudding products. Abstract: IFTAnnual Meeting. p4 3.Murano, E.A. and Pierson, M.D. 1993. Effect of heat shock and incubation atmosphere on injury and recovery of Escherichia coli 0157:H7. J. Food Prot. 56:568-572.Murray, E.G.D., R.A. Webb, and M.B.R. Swann. 1926. A disease of rabbits characterized by large mononuclear leucocytosis caused by a hitherto undescribed bacillus

104Bacterium monocytogenes (n. sp.) J. Pathol. Bacteriol. 29:407-439.Myers, B.R., J.E. Edmondson, M.E. Anderson, and R.T. Marshall. 1983. Potassium sorbate recovery of pectinolytic psychrotrophsfrom vacuum-packed pork. J. Food Prot. 46:499-502.Nickerson, J.T. and Sinskey, A.J. 1972. Microbiology of foods and food processing. American Elsevier Publishing Comp., Inc. New York, N.Y. pp 306.Noah, C.W., Perez, J.C., Ramos, N.C., McKee, C.R., and Gipson, M.V. 1991. Detection of Listeria spp. in naturally contaminated seafoods using four enrichment procedures. J. Food Prot. 54:174-177.Ockerman, H.W., Boton, R.J., Cahill, V.R., Parrt, N.A., and Hoffman, H.D. 1974. Use of acetic and lactic acid to control the quantity of microorganisms on lamb carcasses. J. Milk Food Technol. 37:203-204.Parish, M.E. and Higgins, D.P. 1989. Survival of Listeria monocytogenes in low pH model broth systems. J. Food Prot. 52:144-147.Parkin, K. , Wells, M. and Brown, W. 1981. Modified atmosphere storage of rockfish fillets. J. Food Sci. 47:181.Peel, M. , Donachie, W. and Shaw, A. 1988. Temperature dependent expression of flagella of Listeria monocytogenes studied by electron microscopy. SDS-PAGE and western blotting. J. Gen. Microbiol. 143:2171-2178.Pickett E.L. and Murano, E.A. 1993. Effect of heat and/or chemical shock on ability of Listeria monocytogenes to survive exposure to industrial sanitizers and organic acids. Abstract: IFT Annual Meeting. p43.Przybylski, L.A., Finerty, M.W., Grodner, R.M. and Gerdes,D.L. 1989. Extension of shelf-life of iced fresh channel catfish fillets using modified atmospheric packaging and low dose irradiation. J. Food Sci. 54:269-273.Ray, L.L. and Bullerman, L.B. 1982. Preventing growth of potentially toxic molds using antifungal agents. J. Food Prot. 45:953-963.

105Roberts, T.A. 1989. Combinations of antimicrobials and processing methods. Food Techn. Jan:156-163.Ryser, E.T and E.H. Marth. 1988. Survival of Listeria monocytogenes in cold-pack cheese food during refrigerated storage. J. Food Prot. 51:615-621.Schlech, W.F., P.M. Lavigne, R.A. Bortolussi, A.C. Allen,E.V. Haldane, A.J. Wort, A.W. Hightower, S.E. Johnson, S.H. King, E.S. Nicholls, and C.V. Broome. 1983. Epidemic listeriosis-evidence for transmission by food. N. Engl. J. Med. 308:203-206.Schultz, E.W. 1945. Listerella infections: a review.Stanford Me. Bull. 3:135-151.Shelef, L.A. 1989. Survival of listeria monocytogenes in ground beef or liver during storage at 4 and 25°C. J. Food Prot. 52:379-383.Smith, J.L. 1990. Stress-induced injury in Listeria monocytogenes, p.203-209. In A.J. Miller, J.L. Smith, and G.A. Somkuti (ed.), Foodborne listeriosis. Society for Industrial Microbiology. Elsevier Science Publishing, Inc. New York.Smith, J.L., and S.E. Hunter. 1988. Heat injury in Listeria monocytogenes-prevention by solutes. Lebensm.- Wiss. Technol. 21:307-311.Snijders, J.M.A., M.J.G. Schoenmakers, G.E. Gerats, and F.W. de Pijper. 1979. Dekontamination schlachtwarmerrinderkorper mit organischen sauren. Fleischwirtschaft 50:656-663.Sofos, J.N. and F.F. Busta. 1981. Antimicrobial activity of sorbate: A review. J. Food Prot. 44:614-647.Sorrells, K.M., D.C. Enigl, and J.R. Hatfield. 1989. Effect of pH, acidulant, time, and temperature on the growth and survival of Listeria monocytogenes. J. Food Prot. 52:571-573.Stansby, M.E. and Griffiths, F.P. 1935. Carbon dioxide in handling fresh fish. Ind. Eng. Chem. 27:1452.Troller, J.A. 1965. Catalase inhibition as a possible mechanism of the fungistatic action of sorbic acid. Can. J. Microbiol. 11:611-617.

106USDA (United States Department of Agriculture). 1987. Composition of foods: finfish and shellfish products. USDA Agri. Handbook #8-15.Van Netten, P., and Mossel, D.A.A. 1980. The ecological consequences of decontaminating raw meat surfaces with lactic acid. Arch. Lebensm. Hyg. 31:190-191.Watkins, J. and K.P. Sleath. 1981. Isolation and enumeration of Listeria monocytogenes from sewage, sewage sludge and river water. J. Appl. Bact. 50:1-9.Wang, M. and Brown, D. 1980. Effects of elevated C02 atmospheres on storage of freshwater crawfish (Pacifastacus bucusclus). J. Food Sci. 48:158-159.Wang, M. and Ogrydziak, D. 1986. Residual effect ofstorage in an elevated carbon dioxide atmosphere on the microbial flora of rock cod (Sebastes sp.). Appl. Environ. Microbiol. 52x727.

Weagant, S.D., P.N. Sado, K.G. Colburn, J.D. Torkelson, F.A. Stanley, M.H. Krane, S.C. Shields, and C.F. Thayer. 1988. The incidence of Listeria species in frozen seafoodproducts. J. Food Prot. 51:655-657.Wei, C.I., M.O. Balaban, S.Y. Fernando, and A.J. Peplow.1991. Bacterial effect of high pressure C02 treatment on foods spiked with Listeria or Salmonella. J. Food Prot. 54:189-193.Weis, J. , and H.P.R. Seeliger. 1975. Incidence of Listeriamonocytogenes in nature. Appl. Microbiol. 30:29-32.Windholz, M. , Budavari, S., Stroumtsos, L.Y., and Fertig, M.N. (eds.). 1976. The Merck index. 9th ed. Merck and Co., Inc., Rahway, NJ. pp.1095, 7614, 8492.Wong, H., Chao, W. and Lee, S. 1990. Incidence andcharacterization of Listeria monocytogenes in foods available in Taiwan. Appl. Environ. Microbiol. 56:3101- 3104.Woolthuis, C.H.J. and Smulders, F.J.M. 1985. Microbial decontamination of calf carcasses by lactic acid sprays. J. Food Prot. 48:832-837.Xavier, I.J. and Ingham, S. 1993. Increased D values for Salmonella enteritidis resulting from the use of anaerobic enumeration methods. Food Microbiol. 10:22 3-228.

107York, G.K. and Vaughn, R.H. 1964. Mechanisms in the inhibition of microorganisms by sorbic acid. J. Bacteriol. 88:411-417.Young, K.M. and Foegeding, P.M. 1992. Acetic, lactic and citric acids and pH inhibition of Listeria monocytogenes Scott A and the effect on intracellular pH. J. Appl. Bacteriol. 74:515-520.Yousef, A.E. and Marth, E.H. 1983. Incorporation of (14C) acetate by Aspergillus parasiticus in presence of antifungal agents. Eur. J. Appl. Microbiol. Biotechnol. 18:103-108.Zaika, L.L., Palumbo, S.A., Smith, J.L., Corral, F.D., Bhaduri, S., Jones, C.O., and Kim, A.H. 1990. Destruction of Listeria monocytogenes during frankfurter processing. J. Food Prot. 53:18-21.

VITA

The author was born on November 8, 1955, in New Orleans, Louisiana. He attended Archbishop Rummel High School, located in Metairie, Louisiana, and graduated in May, 1973. He entered Nicholls State University, located in Thibodaux, Louisiana and received his Bachelor of Science degree on May 13, 1978 in Marine Biology. He received his Master of Science degree from Mississippi State University located in Starkville, Mississippi, in the field of Fisheries Management on May 15, 1981.

Following completion of the M.S. degree he became employed in the seafood industry for ten years serving in both the production and processing aspects of the catfish industry in Mississippi, Arkansas, and Louisiana.

In January of 1990 he enrolled at the University of Louisiana and received the degree of Doctor of Philosophy in food science with emphasis in food microbiology in 1994.

108

DOCTORAL EXAMINATION AND DISSERTATION REPORT

Candidate: Warren Joseph Dorsa

Major Field: Food Science

Title of Dissertation: Effects of Heat, Lactic Acid, and Modified AtmospherePackaging on Listeria monocytogenes on Cooked Crawfish Tail Meat

Approved:

Major Professor and Chairman

Dean of the Graduate School

EXAMINING COMMITTEE:

fi

/ A.

Date of Examination:

December 16, 1993