International Journal of Food Microbiology, 18 (1993) 223-232 223 © 1993 Elsevier Science Publishers B.V. All rights reserved 0168-1605/93/$06.00

FOOD 00584

Fate of Listeria monocytogenes in raw and cooked ground beef with meat processing additives

E. Harmayani, J.N. Solos and G.R. Schmidt Department o f Animal Sciences and Department of Food Science and Human Nutrition, Colorado State

University, Fort Collins, CO, USA

(Received 15 July 1992; revision received 5 November 1992; accepted 4 December 1992)

The effect of sodium lactate (1.8% w/w), sodium erythorbate (0.1% w/w), kappa-carrageenan (1% w/w), and the alginate meat binder (0.4% w/w, sodium alginate; 0.6% w/w lactic acid; and 0. 075% w/w calcium carbonate) on Listeria monocytogenes survival and growth was determined in raw and cooked ground beef stored aerobically at 4°C. There was no significant (P > 0.05) increase in numbers of L. monocytogenes during storage of raw ground beef. However, L. monocytogenes numbers were generally lower in treatments with sodium lactate, and higher in sodium erythorbate compared to controls and meat with other additives. Increases in total aerobic plate counts were less pronounced in raw meat formulated with sodium lactate and alginate meat binder than with other additives. Cooking meat with initial inoculum levels of 6.52 to 7.03 L. monocytogenes log CFU/g to 65°C resulted in lower destruction (0.56 and 1.18 log CFU/g) in samples with added alginate meat binder and kappa-carra- geenan, respectively, compared to the control. Survivors (2.11-3.73 log CFU/g) decreased initially and then increased slightly, but not significantly (P > 0.05), during storage (4°C, 6 days) of the cooked products.

Key words: Listeria monocytogenes; Meat; Food additive

Introduction

The frequent incidence of Listeria monocytogenes (McClain and Lee, 1988; Truscott and McNab, 1988; Farber, 1989; Farber and Peterkin, 1991; Johnson et al., 1990) emphasizes the need for prevention or elimination of the pathogen from meat and meat products in order to protect human health. Among meat products, the pathogen was isolated from 20 of 41 ground beef samples (McClain and Lee, 1988), and 29 of 50 lean ground beef samples (Truscott and McNab, 1988). In addition to its frequent occurrence, L. monocytogenes has also been shown to survive during the manufacture and thermal processing of meat products (Johnson

Correspondence address: J.N. Solos, Department of Animal Sciences and Department of Food Sciences and Human Nutrition, Colorado State University, Fort Collins, CO 80523, USA.

224

et al., 1988a,b; Karaioannoglou and Xenos, 1980; Farber et al., 1988). Boyle et al. (1990) found that heating ground beef to 50, 60 or 65°C resulted in 0.2-0.9, 1.6-3.4 and 4.4-6.1 log reductions per gram product. Fain et al. (1991) reported D-values of 0.5 and 1.1 min. for L. monocytogenes strain Scott A in lean and fatty ground beef at 145°F (62.8°C).

In recent years, additives have been used in meat not only as preservatives but also to reduce fat content, enhance water retention, meat particle cohesion and fat binding, and development and preservation of color. Additives that are commonly used or have commercial potential in meat products include sodium erythorbate, kappa-carrageenan, sodium lactate and the algin/calcium meat binder. There is a need to determine the effects of these additives on L. monocytogenes. Sodium erythorbate is widely used with nitrite in meat products as a curing accelerator at the level of 0.0547% (Schmidt, 1986). The use of kappa-carrageenan (0.5-1.0%) is increasing in the meat industry, especially for the formulation of low-fat meat products, because it can effectively increase rigidity and water-holding capacity of meat (Foegeding and Ramsey, 1987). Rosenow and Marth (1987) reported that addition of carrageenan alone did not have a significant effect on the growth of L. monocytogenes in milk, but in the presence of cocoa and sugar it enhanced the growth of the pathogen. Sodium lactate is approved to be used in meat products as a flavoring agent at a level of 2% based on its solid weight. The salt showed inhibitory effects against growth and aminopeptidase activity of Pseudomonas fragi, but not against the growth of Salmonella typhimurium (Harmayani et al., 1990). The compound was also found to delay Clostridium botulinum toxin produc- tion (Maas et al., 1989) and to decrease the number of survivors of L. monocyto- genes and Clostridium sporogenes (Unda et al., 1990). Use of the algin/calcium binder in restructured meat has been patented by Schmidt and Means (1986), and is approved by the United States Department of Agr icu l tu re /Food Safety and Inspection Service (1986) for use in meat and poultry products. Ensor et al. (1990) indicated that a formulation of 0.4% sodium alginate, 0.075% calcium carbonate and 0.6% lactic acid was optimal for restructuring beef. Information on the effect of this binder on growth of L. monocytogenes in meat is nonexistent. Studies by Farber (1989) and Yen et al. (1991) have indicated a protective effect of meat curing ingredients on thermal destruction of L. monocytogenes in ground beef and pork. Siragusa and Dickson (1992) found that lactic acid and acetic acid immobi- lized with calcium alginate gels reduced the population of L. monocytogenes inoculated on raw lean beef tissue more than did acid treatment alone. Alginate coatings, however, did not enhance acid inhibition on fat tissue.

This study was conducted to determine: (1) the effect of sodium lactate, kappa-carrageenan, sodium erythorbate, and a combination of sodium alginate / lactic ac id/calc ium carbonate, used as a meat binder, on growth of L. monocyto- genes in raw ground beef stored at refrigeration temperature (4°C) for 15 days; (2) the effect of these additives on survival of L. monocytogenes during cooking of the ground beef treatments to an internal temperature of 65°C over a 20 min period; and (3) the fate of surviving cells during storage of these cooked products at refrigeration temperature (4°C) for 6 days.

225

Materials and Methods

Culture preparation A mixture of nine strains of L. monocytogenes was used in this study, which

included Scott A (serotype 4b, human isolate), Brie-1 (4b, Brie cheese isolate), V7 (la, raw milk isolate) and V37CE (4b, raw milk isolate) provided by Dr Wesley (United States Department of Agriculture, Agriculture Research Service, National Animal Disease Center); LM101M (4b, sausage isolate) and LM103M (la, sausage isolate) provided by Dr Doyle (University of Wisconsin-Madison, Madison); F5027 (la, raw milk isolate), F5069 (4b, raw milk isolate) provided by Dr Donnelly (University of Vermont, Burlington, Vermont); and LCDC 81-861 (4b, coleslaw isolate) provided by Dr Farber (Bureau of Microbial Hazards, Ottawa, Ontario, Canada). Stock cultures were maintained on tryptic soy agar slants (TSA) (Difco Laboratories, Detroit, MI). The inoculum was prepared by growing individual strains in 10 ml of tryptic soy broth (Difco) at 37°C for 24 h. Equal portions of each culture were then mixed into a single sterile flask prior to inoculation of meat. A mixed culture was used in this study in order to challenge the products with strains of potentially variable sensitivities. Serial dilutions in sterile phosphate buffer solution (pH 7.2) were made to reach initial target inoculum levels of 103-104 and 106-107 colony forming units (CFU) per gram of ground beef for the first (raw) and the second (cooked) experiments, respectively. The initial inoculum levels of the second experiment were higher than the first in order to quantitate destruction during cooking.

Sample preparation Top round beef purchased from a local grocery store was ground through a 0.32

cm plate and split into 500 g batches for different treatments. The history of the beef was unknown. The initial aerobic bacterial count of the beef before inocula- tion was 5.5 log CFU/g. For the first experiment, the ground beef was mixed with the L. monocytogenes inoculum (0.2 ml) in a sterile Kitchen Aid R mixer for 2 min (speed two) and then mixed with the additives for 2 min. Control ground beef received 0.2 ml of phosphate buffer. Treatments tested (Table I) included a mixture of 0.4% (w/w) sodium alginate (Manugel DMB, Kelco, San Diego, CA), 0.075% (w/w) calcium carbonate (Gamma Sperse 80, Georgia Marble, Tate, GA) and 0.6% (w/w) encapsulated lactic acid (Balchem Corp., Slate Hill, NY); 1.8% (w/w) sodium lactate (3% of 60% commercial solution) (Purac Inc., Arlington Heights, IL); 1% (w/w) kappa-carrageenan (FMC Corporation, Philadelphia, PA) and 0.1% (w/w) sodium erythorbate (Griffith Laboratories, Alsip, IL). Approxi- mately 35-g portions from each treatment were put into plastic cups (96.2 ml) which were closed loosely, stored and analysed at day 0, 3, 6, 9, 12 and 15 during 4°C storage. The procedures of the second experiment were the same as those of the first except that the meat (500 g) was inoculated with 2 ml of inoculum, or phosphate buffer for control, before mixing with the additives. The samples (35 g) were put in tubes (50 ml) then cooked in a waterbath (69°C) until their internal temperature reached 65°C. Sterile thermocouples were inserted in the center of

226

T A B L E I

E x p e r i m e n t a l d e s i g n o f g r o u n d b e e f t r e a t m e n t s w i t h v a r i o u s a d d i t i v e s to s t u d y t h e i r e f f e c t s o n t h e f a t e

o f Listeria m o n o c y t o g e n e s a

I n g r e d i e n t s T r e a t m e n t s ( % , w / w o f g r o u n d b e e f )

1 2 3 4 c o n t r o l

S o d i u m a l g i n a t e 0 . 4 % . . . .

E n c a p s u l a t e d l ac t i c a c i d 0 . 6 % . . . .

C a l c i u m c a r b o n a t e 0 . 0 7 5 % . . . .

S o d i u m l a c t a t e ( 6 0 % ) - 3 % - - -

K a p p a - c a r r a g e e n a n - - 1 % - -

S o d i u m e r y t h o r b a t e - - - 0 . 1 % -

a D u p l i c a t e s a m p l e s w e r e d o n e f o r e a c h r e p l i c a t i o n .

tubes to monitor temperature increase, and the tubes were immersed in water below the level of the meat. After cooking, the samples were stored at 4°C for 6 days and analysed at day 0, 2, 4 and 6.

The samples were analysed for L. monocytogenes counts using 25-g portions taken aseptically, diluted (1 : 10) in sterile phosphate buffer solution (pH 7.2), and macerated for 2 min in a stomacher (model 400, Tekmar Co., Cincinati, OH). Serial dilutions were made and 0.1 ml portions were spread plated in duplicate onto LiCl-phenylethanol-moxalactam-tellurite (LPMT) agar (Shelef, 1989) for L. monocytogenes counts and onto tryptic soy agar for total aerobic bacterial counts. The plates were incubated at 37°C for 48 h under aerobic conditions. Black with blue tint, smooth raised, umbrella top-shaped colonies, 1-2 mm in diameter, were counted as L. monocytogenes on LPMT agar. An Orion pH meter (Model 901, Orion Research Inc, Cambridge, MA) equipped with a Corning flat combination electrode (Corning Glass Work, Medfield, MA) was used to measure the pH values of the meat blends used for microbial analyses.

Statistical analysis Both experiments were replicated twice on different occassions with different

meat. Analysis of variance (ANOVA) was used to analyse the differences in bacterial numbers and pH values among treatments. Analyses were run at least in duplicate for each replicate.

Results and Discussion

Raw ground beef Colony counts of L. monocytogenes did not change significantly (P > 0.05)

during the 15 days of storage of raw ground meat, with or without the various ingredients (Fig. 1). Of all the additives tested, however, sodium lactate resulted in the lowest number of L. monocytogenes colonies after 15 days at 4°C, but they

227

c

L)

o ~

o

E

ep %

(D

RAW GROUND BEEF 0 Na alginote/Lactic ocid/CaCO 5 (0.4%:0.6%:0.075%) • Na-lactate (1.8%) Z~ Kappa ca r rogeenan (1%) • N a - e r y t h o r b a t e (0.1%) [ ] Contro l 1

0 5 6 9 12 15

DAYS OF STORACE AT 4°C

Fig. 1. Effect of meat processing additives on growth of Listeria monocytogenes in raw ground beef during aerobic storage at 4°C. Points represent mean values of four samples :t: s tandard error (n = 2 per

replication). The concentration of each ingredient is a % (w/w) of ground beef.

were not significantly (P > 0.05) different from counts of other treatments. The initial numbers of L. monocytogenes colonies from samples with sodium alginate/ lactic acid/calcium carbonate, sodium lactate, kappa-carrageenan, sodium ery- thorbate, and control samples were 4.38, 3.59, 3.62, 3.46 and 3.89 log CFU/g, respectively. After 15 days of storage at 4°C the respective counts were 3.75, 3.51, 3.83, 4.85 and 4.22 log CFU/g. It appears that only in the formulation with sodium erythorbate, were colony counts more than one log higher after 15 days at 4°C, compared to day 0.

Johnson et al. (1988b) reported that the viable cell population of L. monocyto- genes in raw ground beef stored at 4°C for 14 days was not substantially changed. Buchanan and Klawitter (1991) also observed lack of growth of L. monocytogenes in either untreated or irradiation-sterilized raw ground beef. They found that temperature history (5-42°C) of the inoculum did not account for the lack of growth in raw meat, suggesting that there was an inhibitory condition or compo- nent present in raw ground beef.

The antimicrobial activity of sodium lactate has been shown against various microorganisms, including lactic acid bacteria, Staphylococcus aureus and S. ty- phimurium in culture media incubated at 30°C (de Wit and Rombouts, 1990). A previous study in our laboratory (Harmayani et al., 1991) found sodium lactate inhibitory against growth and aminopeptidase activity of P. fragi in autoclaved ground beef. Maas et al. (1989) demonstrated the antibotulinal effect of sodium lactate in turkey products and reported that sodium lactate concentration rather than sodium concentration, ionic strength or brine content was the principal antibotulinal factor. Shelef and Yang (1991) reported that 4% sodium lactate suppressed the growth of L. monocytogenes in sterile comminuted beef and chicken stored at 35, 20 and 5°C for 80 h, 10 and 30 days, respectively. Since there was no extensive multiplication of L. monocytogenes in control raw ground beef in this study, no major inhibitory activity by lactate or other additives could be observed. However, during storage for 15 days at 4°C, samples with added sodium

228

RAW GROUND BEEF

O No alginate/Lactic acid/CoCO 3 (04%:0.6%:0.075%) u-; 1 2 F Z • No-lactate (1.8%) . ~ - - , i , ~ l l

o . ~ 8

a (~ i r) O 4 & Kappa carrageenon (1%) OS • No erythorbote (0.1%) 0 2 [U Control £L N]

0 .5 6 9 12 15 DA"S OF STORAGE AT 4°C

Fig. 2. Effect of meat processing additives on total bacterial counts in raw ground beef during aerobic storage at 4°C. Points represent mean values of four samples + s tandard error (n = 2 per replication).

The concentration of each ingredients is a % (w/w) of ground beef.

lactate had lower total aerobic bacterial counts than other treatments (Fig. 2). Papadopoulos et al. (1991) also found that sodium lactate resulted in reduced aerobic plate counts in cooked, vacuum-packaged beef top rounds stored for up to 84 days at 0°C. In our study, no spoilage signs were visually detected in samples with added sodium lactate, while samples with other ingredients spoiled after 15 d of storage. Samples with the meat binder of sodium alginate/lactic acid/calcium carbonate also produced significantly ( P < 0.05) lower total aerobic bacterial counts compared to control samples (Fig. 2). Products with sodium erythorbate and kappa-carrageenan had similar total aerobic bacterial counts as compared to control samples (Fig. 2).

The initial pH values (5.38-5.56) of all products were not significantly (P > 0.05) different (Fig. 3), but during storage, samples with added sodium lactate or sodium alginate/lactic acid/calcium carbonate had lower pH values compared to the control. Addition of kappa-carrageenan and sodium erythorbate did not change

RAW GROUND BEEF 8.0

O Na-alginate/Ioctic acid/CoCO 5 (04%:0.6%:0.075%) No-lactate (1.8%) ~

A Kappa- . . . . . g . . . . . (1%) | / / f [ ~ 7 . 0 A Na-ery[horbote (0.1%) . ~ / .~-- '~

-Z ,~ cont~o, i/¢~,Z 1

5.0 . . . . . 0 5 6 9 1 2 1 5

DAYS OF STORAGE AT 4 ° C

Fig. 3. Effect of meat processing additives on pH values of raw ground beef during aerobic storage at 4°C. Points represent mean values of four samples_+ standard error ( n = 2 per replication). The

concentration of each ingredients is a % (w/w) of ground beef.

229

I 8 3 uJ I ;I. 6

k/ ( 3

8 t 2 GI

® 0

COOKED GROUND BEEF 0 Na-alginate/Lactic acid/CaCO 3 (0,4~;:0 6%:0 075%)

• No lactate (1,8%) Kappa-carrageenan (1%)

• NG erythorbate (0.1%) Q Control

RAW 0 2 4 6 ! . . . . . . . COOKED I

DAYS OF STORAOE AT 4°C

Fig. 4. Effect of meat processing additives on thermal destruction and survival of Listeria monocyto- genes after cooking to 650C and during aerobic storage at 4°C. Points represent mean values of four samples + s tandard error (n = 2 per replication). The concentration of each ingredients is a % (w/w) of

ground beef.

the initial pH values of the samples, but pH of samples with these treatments increased significantly (P < 0.05) during storage, similar to the control. In sum- mary, the results of this study indicated that addition of sodium lactate and the meat binder of sodium alginate/lactic acid/calcium carbonate resulted in lower aerobic plate counts in raw ground beef, but had little effect on L. rnonocytogenes counts, which did not show significant increases, even in control samples. Of the other ingredients, kappa-carrageenan had no major inhibitory effect on total counts or fate of L. monocytogenes, while sodium erythorbate allowed growth of L. monocytogenes by more than one log after 15 days of aerobic storage at 4°C. It is not known whether the pH (5.38-5.56) or other factors did not allow major increases in L. monocytogenes counts in raw ground beef with or without additives.

Cooked ground beef Cooking to 65°C was achieved after 20 min of heating in a waterbath of 69°C.

The initial L. monocytogenes counts of 7.03, 6.52, 6.79, 6.77 and 6.68 log CFU/g were reduced by 3.68, 4.41, 3.06, 4.22 and 4.24 log CFU/g after cooking ground beef treatments with sodium alginate/lactic acid/calcium carbonate, sodium lactate, kappa-carrageenan, sodium erythorbate and control, respectively (Fig. 4). Heating rates were similar among treatments with different additives (data not shown). Thus, destruction of L. monocytogenes was 0.56 and 1.18 log CFU/g less in samples with added sodium alginate/lactic acid/calcium carbonate and kappa- carrageenan than in control samples, respectively. Thermal destruction of L. monocytogenes was similar to the control in ground beef with sodium erythorbate and sodium lactate.

Surviving L. monocytogenes were detected in all treatments by direct plating immediately (less than 1 h) after cooking. In general, the numbers of surviving L. monocytogenes decreased initially during storage of cooked ground beef at 4°C (2 days) and then remained constant or increased slightly. Sodium lactate significantly

230

E/] F 12 z m 10 0 ( ) or, 8

t~ ZD L 6 <~ ( )

CL~ ~ 4

0 2

~ 0 <c

COOKED GROUND BEEF

0 Na alginate/lactic acid/CaCO 5 (04%:06%0.075%) • Nu-tactate (1.8%) /~ Koppa cqrrageenan (1%)

\ , • Na erythorbate (0.1%) FJ Control

/ ~ / . . . . . . . . . .

RAW 0 2 4 6 i COOKED I

DAYS OF STORAGE AT 4°C

Fig. 5. Effect of meat processing additives on total aerobic bacterial counts of ground beef before and after cooking at 65°C and during aerobic storage at 4°C. Points represent mean values of four samples + s tandard error (n = 2 per replication). The concentration of each ingredients is a % (w/w) ot

ground beef.

(P < 0.05) decreased the numbers of the surviving L. monocytogenes cells during the first 2 days of storage of the cooked product. Unda et al. (1990) reported that sodium lactate decreased numbers of L. monocytogenes and C. sporogenes in microwave-ready roast beef after they survived heat processing. Although counts of L. monocytogenes were higher in cooked meat containing added sodium alginate/ lactic acid/calcium carbonate and kappa-carrageenan than controls and samples with other treatments, there were no major increase in cell numbers during aerobic storage at 4°C for 6 days. No major increases in total aerobic bacterial counts of any of the treatments tested were detected during the 6 days of storage at 4°C (Fig. 5). The initial pH values after cooking (5.46-5.58) were similar (P > 0.05) for all treatments of ground beef and remained constant during 6 days of storage at 4°C (data not shown).

In summary, the results of this study suggested that the various additives tested had no effect on survival or growth of L. monocytogenes in raw ground beef of pH 5.50 stored aerobically at 4°C for 15 days. Furthermore, minimal effects were observed in the cooked products stored for 6 days at 4°C. It is also concluded that under the conditions of this study, L. monocytogenes is capable of surviving refrigeration storage of raw or cooked ground beef of pH 5.50, even in the presence of the additives tested, provided the initial inoculum levels permit survival during cooking, however, little or no growth was detected during storage.

Acknowledgements

This work was supported in part by the Agricultural Experiment Station of Colorado State University, by the National Live Stock and Meat Board, and by the Food and Nutrition Development and Research Center (FANDARC) of Gadjah Mada University, Indonesia.

231

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