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Determination of the effect of sodium lactate on the survival and heat resistance of Escherichia coli O157:H7 in two commercial beef patty formulations
Food Microbiology, 2002, 19, 211^219 Available online at http://www.idealibrary.com on
doi:10.1006/fmic.2001.0462
ORIGINAL ARTICLE
Determination of the e¡ect of sodium lactate on the survival and heat resistance of Escherichia coli O157:H7 in two commercial beef patty formulations C. M. Byrne1; *, D. J. Bolton1, J. J. Sheridan1, I. S. Blair2 and D. A. McDowell2 The e¡ect of 4% sodium lactate (NaL) in beefburger patty formulations on the survival and heat resistance of Escherichia coli O157:H7 was investigated. Fresh beef trimmings were inoculated with E. coli O157:H7 to a concentration of 6?0^7?0 log10 cfu g 1 and subjected to the processing stages of beefburger patty production.Two commercial beefburger patty formulations were produced: a‘quality’patty (100% beef) and an ‘economy’ patty (70% beef, 30% other ingredients, including onion, water, salt, seasoning, rusk and soya concentrate). Sodium lactate (4% w/v) was added to the beefburger patties during mincing and the formed patties were frozen and stored for 1month. Beefburger patties without added NaL were used as controls. After frozen storage for 1 month, patties were examined for E. coli O157:H7 counts.There was a synergistic e¡ect between freezing and NaL, which resulted in a small but signi¢cant reduction (P o 0?05) (approximately 0?5 log10 cfu g 1) in E. coli O157:H7 numbers. The frozen beefburger patties were also heat-treated at 50, 55 and 601C and the data analysed to derive D-values for E. coli O157:H7 cells. At each temperature treatment, the D-values of the quality and economy beefburger patties with 4% NaL were signi¢cantly lower (P o 0?001) than the D-values of the patty formulations without NaL. The study demonstrates that the presence of 4% NaL in beefburger patty formulations can reduce the overall risks posed to consumers by the presence of E. coli O157:H7 by, ¢rst; reducing pathogen survival during freezing and frozen storage of the uncooked product; and, second, by increasing the susceptibility of the pathogen to heat during normal cooking processes. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction *Corresponding author. Present address: Microbial Food Safety Unit, Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 600 East Mermaid Lane, Wyndmoor, Pennsylvania 19038, USA.Tel: 001 215 836 3773; Fax: 001 215 233 6581. E-mail: [email protected] 0740 -0020/02/2^30211+09 $35.00/0
Since Escherichia coli O157:H7 ¢rst came to prominence in 1982, a range of foods have been associated with outbreaks worldwide. Ground (minced) beef and beefburger patties, however, are the foods most often implicated in such disease (Willshaw et al. 1994, Meng and Doyle 1998). r 2002 Elsevier Science Ltd. All rights reserved.
Received: 16 April 2001 1 Teagasc,The National Food Centre, Dunsinea, Castleknock, Dublin15, Eire 2 Food Microbiology Research Group, Faculty of Science, The University of Ulster, Jordanstown, Newtownabbey, Co. Antrim BT37 OQB, UK
212 C. M. Byrne et al.
Beefburger patty production processes do not signi¢cantly reduce the risks posed to consumers by beef trimmings that become contaminated by low infective dose pathogens, such as E. coli O157:H7, during slaughter and carcass dressing (Line et al. 1991, Ahmed et al. 1995, Juneja et al. 1997). Current inability to prevent contamination highlights the importance of adequate cooking of patties to eliminate E. coli O157:H7 prior to consumption. However, the frequent association of E. coli O157:H7 foodborne illness with beefburger patties suggests that such products are not always adequately cooked, possibly as a result of consumer demand for rare patties, or errors in the cooking process (Riley et al. 1983, CDC 1993, Jordan et al. 1999). As it is not possible to guarantee adequate cooking of patties consistently, it is important to try to limit the numbers and incidence of E. coli O157:H7 in the uncooked product. Successful achievement of this depends on the development and application of strategies which focus on reducing or eliminating E. coli O157:H7 during each step of the beefburger patty production process (Buchanan and Doyle 1997). The inclusion of antimicrobial agents in the uncooked beefburger patty may be a possible strategy to reduce or inactivate such pathogenic bacteria. Antimicrobial agents provide additional hurdles to control pathogens, thereby providing increased margins of safety to the products. Sodium lactate (NaL) is one such antimicrobial agent. It is a⁄rmed as a ‘Generally Regarded as Safe’ (GRAS) substance by the Food and Drugs Administration in the US for use as a human food ingredient, and is added to meat and poultry products to extend shelf life and inhibit growth of foodborne pathogens (Duxbury 1990, Miller and Acu¡ 1994, Nnanna et al. 1994, Houtsma et al. 1996, Venkitanarayanan et al. 1999). Some compounds, including organic acids and their salts, can have signi¢cant antimicrobial e¡ects when combined with a heat treatment (Shelef and Potluri 1995, McMahon et al. 1999). Thus, McMahon et al. (1999) reported that the presence of 2?4 and 4?8% NaL decreased the heat resistance of Yersinia enterocolitica and Listeria monocytogenes in ground beef. For this reason, the inclusion of NaL in
beefburger patties could provide an additional margin of product safety in relation to the presence of E. coli O157:H7, by increasing the susceptibility of the pathogen to heat. Most studies have concentrated on the e¡ect of NaL on growth and survival of pathogens at refrigeration (41C) temperatures or conditions of low temperature abuse (101C)(Maas et al. 1989, Brewer et al. 1992, Weaver and Shelef 1993, Miller and Acu¡ 1994). There is, however, limited information available on the e¡ect of NaL on E. coli O157:H7 during beefburger patty production and frozen storage, and during subsequent heating (i.e. cooking). Accordingly, the aim of the current study was to investigate the e¡ect of 4% NaL on: ¢rst the survival of the pathogen in two beefburger patty formulations during commercial processing and frozen storage; and, second, the thermal resistance of E. coli O157:H7 during heat treatment of beefburger patties.
Materials and Methods Organism A non-toxigenic strain of E. coli O157:H7 (NCTC 12900), resistant to streptomycin sulphate (Sigma Chemical Co., St. Louis, Missouri, USA) and nalidixic acid (Sigma) was prepared as described by Park (1978). Such antibiotic-resistant mutants can be enumerated in the presence of other contaminant or resident micro£ora by plating on a selective agar, Sorbitol MacConkey agar (SMAC; Oxoid, Basingstoke, UK), supplemented with nalidixic acid (NAS; 50 mg ml 1 ) and streptomycin sulphate (1000 mg ml21 ) (SMAC+NAS). Stock cultures for the double antibiotic-resistant mutant of E. coli O157:H7 were maintained on a frozen bead storage system (Protect Technical Consultant Services Ltd, Lancashire, UK) at 201C.
Preparation of bacterial inoculum One Protect bead of E. coli O157:H7 was aseptically transferred to 30 ml brain^heart infusion broth (BHI; Oxoid) and incubated at 371C for 24 h. A 3?0 -ml sample from the resultant culture was transferred to 300 ml fresh BHI broth
The e¡ect of sodium lactate on E. coli O157:H7 in beef burger patties 213
and incubated for a further 18 h at 371C to produce a stationary phase culture containing 9?0^10?0 log10 E. coli O157:H7 ml 1 . Stationary phase cultures have been shown to have greater heat resistance than log phase cultures (Tomlins and Ordal 1976). The cells were recovered from the stationary phase culture by centrifugation at 3000 g for 10 min at 41C (Eppendorf Centrifuge 5403, Germany); they were then washed three times in 9 -ml volumes of maximum recovery diluent (MRD, Oxoid) and the resultant pellet was resuspended in 9 ml of MRD.The numbers of E. coli O157:H7 cells ml 1 of this cell suspension were estimated using the membrane ¢ltration epi£uorescent staining technique of Wall et al. (1989). The cell suspension was diluted in MRD to form 1 l volumes of inoculum suspension containing 7?0 log10 E. coli O157:H7 ml 1 .
Preparation and inoculation of beef trimmings Beef trimmings (plate, £ank, forequarter and fat trim) were obtained from the on-site abattoir at The National Food Centre. Fat content of the beef trimmings was determined by the Food Analysis Department at The National Food Centre, using an automated, integrated microwave moisture and methylene chloride extraction method (JVA Analytical Ltd, Dublin, Eure) (Bostian et al. 1985). The results were used to calculate the exact quantity of additional beef fat to add to give the required fat:lean ratio to the formed beefburgers. The beef trimmings were cut into approximately 4^5 cm3 pieces (25^30 g) and inoculated with E. coli O157:H7 by immersion in an 1l-inoculum suspension for 5 s. The meat was allowed to drain for 5 s, transferred to sterile Stomacher bags (Seward Ltd, London, UK) and massaged thoroughly to obtain an even distribution of micro-organisms. This process yielded beef trimmings with an initial E. coli O157:H7 count of approximately 6?0^7?0 log10 cfu g 1. Inoculated trimmings were weighed into 500-g batches and sealed into Cryovac BB4L bags (approximately 15 20 cm: W.R. Grace Ltd, Dublin, Eure) using a portable heat sealer (Salton Bag Sealer, Pifco Ltd, Manchester, UK).
Beef trimmings inoculated with E. coli O157:H7 ( 7.0 log 10 cfu g-1 )
Frozen to –18˚C over a 37 h period and stored at –18˚C for 1 month
Tempered using microwave method (–18 to –5˚C (±2˚C) in 6 min)
Tempered beef trimmings (-3˚C ± 2.0˚C)
Formation of 10 g beefburger patty samples (quality or economy formulations)
Frozen to –18˚C and stored at –18˚C for 1 month
Figure 1. Commercial beefburger patty production from beef trimmings to frozen storage of the formed patties. Beef burger patty production process The speci¢cations and methods of routine commercial beefburger patty production, including the various physical stages and ingredient/ product temperature pro¢les, were identi¢ed and modelled within a laboratory-based system to mimic normal industrial processes (Fig. 1). Details of commercial beefburger patty production were carried out according to the methods of Byrne (2001).
Formation of ‘quality’ and ‘economy’ beef burger patties Two beefburger patty formulations, an ‘economy’ patty, and a ‘quality’ patty, were prepared according to the commercial speci¢cations.
214 C. M. Byrne et al.
Quality patty meat mixture was prepared in 1-kg batches, to contain 56% frozen ( 31C 7 2?01C) and 44% fresh (01C 7 11C) beef trimmings. The ¢nal formulation had a total fat content of approximately 24%. This process yielded samples (10 g) containing E. coli O157:H7 concentrations of approximately 5?0 log10 cfu g 1. Economy patty meat mixture was prepared in 1-kg batches to contain 69?9% frozen ( 31C 7 2?01C) beef trimmings and 30?1% other ingredients.The ingredients included tap water (13?5%), pinhead rusk (5?27%), frozen onion (7?59%), seasoning/spices (1?54%), salt (0?76%) and soya concentrate (1?73%)? The ¢nal formulation had a total fat content of approximately 30%.This process yielded samples (10 g) with an initial E. coli O157:H7 count of approximately 5?0 log10 cfu g 1.
ture, i.e. 50, 55 or 601C (7 1?01C). Processing times were measured from the time the core temperature of the test substrate reached the target temperature (o 3 min). Once the desired temperature was reached, duplicate samples were removed and cooled to 01C within 10 min in iced water. Duplicate samples were withdrawn at regular intervals during the heat treatment, cooled and subjected to microbiological analysis within 10 min. The temperature of the test substrate, the water at the thermal challenge temperature and the iced water were continuously monitored throughout the course of the experiment using thermocouples (1?2 -mm diameter 100 mm) attached to a temperature microprocessor (Code SSA12080G700TS, Ellab A/S, Copenhagen, Denmark).
Preparation of beefburger patties containing 4% sodium lactate
Chemical analyses
Economy and quality beefburger patty formulations were prepared as described above, except that 6?7 ml of NaL solution (sodium salt of DL-Lactic Acid, 60% w/w syrup)(Sigma) per 100 g of minced ingredients was added immediately after the ¢rst mincing step. The ¢nal ingredient mixes, including NaL, were doubleminced and formed into patties as previously described. The concentration of NaL, present was determined spectrophotometrically (Spectromics Kino MT Genesyst 5, Milton Roy Co. Analytical Products Division, Rochester, New York, USA) at 340 nm using a D-lactic acid/L lactic acid combination test kit (Boehringer Mannheim). The process yielded beefburger patty samples (10 g) with an initial E. coli O157:H7 count of approximately 5?0 log10 cfu g 1.
Thermal inactivation studies of E. coli O157:H7 in beefburger patties Thermal inactivation studies were carried out on both economy and quality beefburger patties (10 g), with and without added NaL, over a range of heating temperatures (501C, 551C and 601C). Bags containing the frozen inoculated beef patty samples were completely submerged in a water bath (Techne, AGB Scienti¢c, Dublin, Eure) with water at the desired tempera-
Estimations of pH and water activity of the beefburger patties were carried out according to the methods of Bendall (1973) and Richard and Labuza (1990), respectively.
Microbiological analyses Recovery and enumeration of E. coli O157: H7. Beefburger patties (10 g) were stomached in 90 ml MRD for 1 min in a Colworth Stomacher (Seward). Samples (1 ml) were serially diluted (1:10) in 9 ml volumes of MRD and spread-plated in duplicate (0?1 ml and 1?0 ml aliquots) onto SMAC+NAS and tryptone soya agar (TSA, Oxoid). Samples plated on SMAC+NAS were incubated at 371C for 48 h to obtain an estimate of uninjured E. coli O157:H7 numbers. Samples plated on TSA were incubated at 371C for 2 h, overpoured with SMAC+NAS (10 ml) and incubated at 371C for a further 48 h. The latter process allows the recovery of any injured cells that may be present (Ray and Speck 1978, Doyle and Schoeni 1984, Du¡y et al.1999) and, therefore, provided an estimate of the total E. coli O157:H7 numbers (injured plus uninjured).
Statistical analyses Frozen storage studies were repeated three times with each beefburger patty formulation.
The e¡ect of sodium lactate on E. coli O157:H7 in beef burger patties 215
The data was subjected to analysis of variance (ANOVA) and signi¢cant di¡erences between mean D-values were identi¢ed using t-tests. The heating trials were repeated three times with each beefburger patty formulation at each heating temperature. Estimates of heat resistance were performed by plotting the log10 of the number of survivors against time for each heating temperature to give a survival curve. The decimal reduction time or D-value (the time required, at a particular temperature, to reduce the number of bacteria by 90%, i.e. 1 log10 ) was derived by regression analysis of the linear portion of the log curve (Miles and Mackey 1994) using the Statistical Analysis Systems (SAS) procedure for the General Linear Model (SAS Institute Inc., Cary, North Carolina, USA). The data was subjected to analysis of variance (ANOVA) and signi¢cant di¡erences between mean D-values were identi¢ed using t-tests.
Results Data from proximate analyses (pH and aw ) of the beefburger patties are presented in Table 1. There were no signi¢cant di¡erences
between the mean pH values of the economy formulations and those of the quality formulations. This pattern was observed for formulations with and without added NaL. The addition of NaL resulted in a signi¢cant reduction in the mean pH values of both the economy (P o 0?001) and quality (P o0?05) beefburger patty formulations. The mean aw of the economy samples was signi¢cantly lower (P o 0?05) than the mean aw of the quality samples. This pattern was observed for formulations with and without added NaL. The addition of NaL (4%) resulted in a signi¢cant reduction (P o0?001) in the mean aw of both the quality and economy beefburger formulations. Table 2 presents the numbers of E. coli O157:H7 in quality, economy, quality with NaL and economy with NaL beefburger patties, immediately after production, and after freezing and storage at 181C for 1 month. Analysis of variance (ANOVA) determined that there was a signi¢cant interaction between freezing, the presence of NaL and the agar used for recovery. There was no signi¢cant di¡erence in E. coli O157:H7 numbers recovered from fresh burger samples without NaL and fresh samples with NaL on either SMAC (uninjured cells) or
Table 1. Proximate analyses of quality and economy beef burger patty formulations, with and without sodium lactate (0% NaL and 4% NaL) Proximate Analyses
Quality beefburger patty 0% NaL
pH (d.f. 19) aw (d.f. 23)
5?85 (0?03) 0?993 (0?002)
4% NaL 5?68 (0?10) 0?967 (0?007)
Economy beef burger patty 0% NaL 5?99 (0?03) 0?978 (0?003)
4% NaL 5?7 (0?14) 0?949 (0?005)
d.f. Degrees of freedom; aw ,Water activity. Standard deviation in parentheses.
Table 2. The numbers of E. coli O157:H7 (log10 cfu g 1 ) in quality and economy beefburger patties immediately after production (fresh), and after freezing and frozen storage for 1 month (frozen), detected using SMAC+NAS and TSA/SMAC+NAS Burger type
Quality (0% NaL) Quality (4% NaL) Economy (0% NaL) Economy (4% NaL)
Fresh beefburger patties
Frozen beefburger patties
SMAC+NAS
TSA/SMAC+NAS
SMAC+NAS
TSA/SMAC+NAS
4?79 (0?14) 4?72 (0?12) 4?71 (0?11) 4?67 (0?14)
5?38 (0?17) 5?37 (0?20) 5?18 (0?11) 5?09 (0?16)
4?71 (0?32) 4?60 (0?22) 4?64 (0?30) 4?47 (0?26)
5?42 (0?43) 5?03 (0?37) 5?10 (0?22) 4?62 (0?46)
Standard deviation in parentheses; d.f., degrees of freedom = 80. For details of media see text.
216 C. M. Byrne et al.
Table 3. Comparison of D-values (min) for E. coli O157:H7 in quality and economy beefburger patties, with and without sodium lactate (4% and 0% NaL), heated at three temperatures (50, 55 and 601C) and plated on TSA/SMAC+NAS Temperature (1C)
501C 551C 601C
Quality beefburger patty
Economy beefburger patty
Control (0% NaL)
NaL Added (4% NaL)
Control (0% NaL)
NaL Added (4% NaL)
152?8 (22?3) 14?8 (1?8) 2?7 (0?5)
76?0 (17?4) 9?8 (1?3) 1?5 (0?1)
170?5 (7?4) 12?4 (2?9) 3?5 (0?3)
61?8 (6?8) 12?4 (0?6) 2?2 (0?3)
Standard deviation in parentheses; d.f., degrees of freedom = 24.
TSA/SMAC (total cell count). However, with recovery on TSA/SMAC, E. coli O157:H7 counts in frozen burger samples containing NaL were signi¢cantly lower (P o 0?05) than counts of the pathogen in frozen samples without NaL. This pattern was observed in both the quality and economy formulations. In most cases there were no signi¢cant differences between numbers of E. coli O157:H7 recovered on TSA/SMAC+NAS (total counts) and those on SMAC+NAS (uninjured counts). Hence, there were no signi¢cant di¡erences between D-values derived from TSA/SMAC counts and those derived from SMAC+NAS counts. Data and D-values presented are those derived using TSA/SMAC only. Table 3 presents the derived D-values for E. coli O157:H7 in quality and economy formulations, with and without NaL, during heating at 50, 55 and 601C, as derived from plate counts on TSA/SMAC+NAS. Under most temperature treatments, the Dvalues in NaL-supplemented patties were signi¢cantly lower (P o 0?001) than the D-values in unsupplemented patties. There was one exception to this pattern. In samples heat-treated at 551C, there was no signi¢cant di¡erence between the D-values of economy samples with or without NaL. In most cases, the D-values for economy beefburger patties were signi¢cantly higher (P o 0?05) than the D-values for quality beefburger patties at the same temperature. This pattern was observed in both the NaL-supplemented and unsupplemented samples.
Discussion In the current study, the addition of 4% NaL resulted in signi¢cant reductions in both the pH
and the aw of the quality and economy beefburger patty formulations. The changes of the magnitudes demonstrated in the present study are in agreement with the ¢ndings of previous studies (Debevere 1989, Shelef and Yang 1991, Chen and Shelef 1992, Weaver and Shelef 1993, Bloukas et al. 1997, McMahon et al. 1999). Such changes have been reported to have little or no deleterious e¡ects on the sensory characteristics or acceptability of the product (Brewer et al. 1992; Papadopoulos et al. 1991). For a substance to be successful as a preservative, it must demonstrate antimicrobial activity against the target organisms without negatively a¡ecting the quality attributes of the food product, e.g. colour, odour or £avour. Sodium lactate meets these criteria and therefore has potential as an antimicrobial in such products as beefburger patties. In this study, the synergistic e¡ect of freezing and 4% NaL resulted in a small but signi¢cant reduction (approximately 0?5 log10 cfu g 1 ) in the numbers of total E. coli O157:H7 in frozen stored products. This result is smaller than, but generally in agreement with, the ¢ndings of Conner and Hall (1994), who demonstrated a signi¢cant reduction of approximately 1?0 log10 cfu g 1 in the numbers of E. coli O157:H7 in chicken meat supplemented with 4% NaL and stored in a frozen state for 18 months. Any reduction, even such relatively small reductions, in E. coli O157:H7 numbers, are to be welcomed. However, considering the levels of E. coli O157:H7 contamination that beef trimmings may carry, and the very low infective dose of this pathogen, such limited reductions in E. coli O157:H7 numbers in beefburger patties are, in isolation, unlikely to make a major contribution to the reduction of the risks posed to consumers by contaminated patties.
The e¡ect of sodium lactate on E. coli O157:H7 in beef burger patties 217
The current study noted signi¢cantly lower D-values in NaL-supplemented samples than in unsupplemented samples, demonstrating that the addition of 4% NaL reduced the heat resistance of E. coli O157:H7 in both the economy and quality burger patty formulations. In some instances the addition of 4% NaL reduces the D-value by 50%, i.e. when heated at 501C the D-value for quality samples with NaL is 76 min, compared to 153 min for quality samples alone. Such synergism between heat and NaL demonstrated in the present study has been reported previously (Shelef and Potluri 1995, McMahon et al. 1999). For example, McMahon et al. (1999) demonstrated that the addition of 4?8% NaL to minced beef signi¢cantly reduced the heat resistance of Yersinia enterocolitica (P o 0?001) and Listeria monocytogenes (P o 0?01) at 551C. Although the e¡ects of NaL, alone or in combination with other inhibitory agents, have been noted in a number of studies, the speci¢c mode of action of this additive has not been fully elucidated. However, numerous theories regarding its antimicrobial activity have been presented. Several investigators have suggested that the antimicrobial e¡ect of NaL may be caused by the e¡ect of pH on the bacterial cell, or speci¢cally the ability of the undissociated molecule to cross the cell membrane, dissociate, release protons and acidify the cell interior (Maas et al. 1989, Shelef 1994). Consequently, cell growth rate is reduced as more of the cell’s energy is expended in maintaining a constant internal pH. Therefore, in the present study, the small but signi¢cant reduction in product pH caused by the addition of NaL is likely to have rendered conditions less favourable to the survival of the pathogen, resulting in lower heat resistance. The reduction in the aw values of the beef patties caused by the addition of NaL in the current study may also account for the lower heat resistance in NaLsupplemented samples. Bacterial cells are generally more heat-resistant in food systems with low aw (Kaur et al. 1998). However, it has been reported that if the aw is decreased below a certain threshold value, the protective e¡ect is lost and the lower aw values act as a component of a more adverse microbial environment, leading to a reduction in bacterial heat resistance
(Kaur et al. 1998). Thus, the addition of NaL to the beefburger formulations may have reduced the water activity (aw ) to a point where the protective e¡ect of aw was lost, leading to a reduction in bacterial heat resistance. In the current study, the E. coli O157:H7 strain used was made resistant to two antibiotics, streptomycin sulphate and nalidixic acid, to facilitate the recovery and enumeration of the strain in the presence of other contaminant or resident meat micro£ora. However, previous reports have noted that the presence of antibiotics in the growth and/or enumerating media may a¡ect the D-values obtained (Doherty et al. 1998, Dombroski et al. 1999). For example, Dombroski et al. (1999) reported that the D-values for an antibiotic-resistant (AR) strain (nalidixic acid) of Vibrio vulni¢cus were signi¢cantly higher at 471C, lower at 501C and not signi¢cantly di¡erent at 521C than those of the wild-type (WT) strain. Therefore, the D-values obtained for the AR strain in the present study may be di¡erent from those of the WT strain. However, the D-values for the AR strain in this study are similar to those in previous reports of D-values for WT E. coli O157:H7 strains in minced beef (Ahmed et al. 1995, Juneja et al. 1997) and are within the range speci¢ed within a recent review of the published data on the heat resistance of E. coli O157:H7 (Stringer et al. 2000). In addition, the use of the AR strain does not detract from the major ¢nding of the current study, that the addition of NaL to the beef patty formulations signi¢cantly decreased the heat resistance of E. coli O157:H7 and increased the margin of safety of such products. This study has demonstrated that the addition of NaL to beefburgers may reduce the risks posed by E. coli O157:H7 in beefburgers by: ¢rst, reducing the numbers of E. coli O157:H7 during freezing and frozen storage of the uncooked product; and, second, decreasing the heat resistance of the pathogen during heat treatment. In isolation, the reductions in E. coli O157:H7 numbers in NaL-supplemented burger samples during frozen storage (approximately 0?5 log10 cfu g 1 ), are unlikely to have much practical impact in reducing the risks posed by contaminated beefburgers. Perhaps of greater signi¢cance in this regard is the e¡ect of
218 C. M. Byrne et al.
NaL on the heat resistance of E. coli O157:H7 in beefburgers noted in this study, i.e. the addition of NaL to the beefburger formulations signi¢cantly reduced the heat resistance of the pathogen. Thus, the addition of NaL should be considered as a means of increasing the margins of safety of such products, by making the E. coli O157:H7 cells more susceptible to heat treatment. It is clear that E. coli O157:H7 is an all too frequent and undesirable contaminant in beef products. Until robust strategies are available to ensure the consistent exclusion of this and other pathogens from such high-risk products, at the earliest possible stage of the human food chain, it will be necessary to explore and exploit all possible opportunities to reduce the persistence of these organisms. This study has demonstrated that the addition of NaL to beefburger patties o¡ers the potential of reducing pathogen survival during frozen storage of the uncooked product and may be helpful by rendering pathogens less resistant to commercially applied heat treatments.
Acknowledgements The authors gratefully acknowledge the assistance of Dr. Dermot Harrington and Ms. Paula Reid, of the Statistics Department, Teagasc Headquarters, Sandymount, Dublin, in performing the statistical analysis on these results. This project is supported by the Irish Department of Agriculture, Food and Rural Development through EU and national funds.
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The e¡ect of sodium lactate on E. coli O157:H7 in beef burger patties 219
spoilage organisms and pathogens at di¡erent pH values and temperatures. J. Food Prot. 59, 1300^1304. Jordan, D., McEwen, S. A., Lammerding, A. M., McNab, W. B., and Wilson, J. B. (1999) Preslaughter control of Escherichia coli O157:H7 in beef cattle: a simulation study. Preventive Veterinary Medicine. 41, 55^74. Juneja,V. K., Snyder, O. P., and Marmer, B. S. (1997) Thermal destruction of Escherichia coli O157:H7 in beef and chicken: determination of D- and z-values. Int. J. Food Microbiol. 35, 231^237. Kaur, J., Ledward, D. A., Park, R.W. A., and Robson, R. L. (1998) Factors a¡ecting the heat resistance of Escherichia coli O157:H7. Lett. Appl. Microbiol. 26, 325^330. Line, J. E., Fain, A. R., Moran, A. B., Martin, L. M., Lechowich, R. V., Carosella, J. M., and Brown, W. L. (1991) Lethality of heat to Escherichia coli O157:H7: D-value and z-value determinations in ground beef. J. Food Prot. 54, 762^766. Maas, M. R., Glass, K. A., and Doyle, M. P. (1989) Sodium lactate delays toxin production by Clostridium botulinum in cook-in-bag turkey products. Appl. Environ. Microbiol. 55, 2226^2229. McMahon, C. M. M., Doherty, A. M., Sheridan, J. J., Blair, I. S., McDowell, D. A., and Hegarty,T. (1999) Synergistic e¡ect of heat and sodium lactate on the thermal resistance of Yersinia enterocolitica and Listeria monocytogenes in minced beef. Lett. Appl. Microbiol. 28, 340^344. Meng, J. and Doyle, M. P. (1998) Escherichia coli O157:H7 and other shiga toxin-producing E. coli strains. In Microbiology of Shiga Toxin-producing Escherichia coli in Foods (Eds J. B. Kaper and A. D. O’Brien) pp. 92^108.Washington DC, American Society for Microbiology. Miles, C. A. and Mackey, B. M. (1994) A mathematical analysis of microbial inactivation at linearly rising temperatures: calculation of the temperature rise needed to kill Listeria monocytogenes in di¡erent foods and methods for dynamic measurements of D and z values. J. Appl. Bacteriol. 77, 14^20. Miller, A. J. and Acu¡, G. R. (1994) Sodium lactate affects pathogens in cooked beef. J. Food Sci. 59, 15^19. Nnanna, I. A., Ukuku, D. O., McVann, K. B., and Shelef, L. A. (1994) Antioxidant activity of sodium lactate in meat and model systems. Lebessm.-Wiss. 27, 78^85. Papadopoulos, L. S., Miller, R. K., Ringer, L. J., and Cross, H. R. (1991) Sodium lactate e¡ect on sensory characteristics, cooked meat color and chemical composition. J. Food Sci. 56, 621^626.
Park, R.W. A. (1978) The isolation and use of streptomycin-resistance mutants for following development of bacteria in mixed cultures. In Techniques for the Study of Mixed Populations (Eds D. Lovelock and R. Davies) pp. 107^112. London, Academic Press. Ray, B. and Speck, M. L. (1978) Plating procedure for the enumeration of coliforms from dairy products. Appl. Environ. Microbiol. 35, 820^822. Richard, J. and Labuza,T. P. (1990) Rapid determination of the water activity of some reference solutions, culture media, and cheese using a new instrument based on the dew-point method. Science Des Aliments. 10, 57^64. Riley, L.W., Remis, R. S., Helgerson, S. D., McGee, H. B.,Wells, J. G., Davis, B. R., Hebert, R. J., Olcott, E. S., Johnson, L. M., Hargrett, N. T., Blake, P. A., and Cohen, M. L. (1983) Hemorrhagic colitis associated with a rare Escherichia coli serotype. New Eng. J. Med. 308, 681^685. Shelef, L. A. (1994) Antimicrobial e¡ects of lactates: a review. J. Food Prot. 57, 445^450. Shelef, L. A. and Yang, Q. (1991) Growth suppression of Listeria monocytogenes by lactates in broth, chicken, and beef. J. Food Prot. 54, 283^287. Shelef, L. A. and Potluri,V. (1995) Behaviour of foodborne pathogens in cooked liver sausage containing lactates. Food Microbiol. 12, 221^227. Stringer, S. C., George, S. M., and Peck, M. W. (2000) Thermal inactivation of Escherichia coli O157:H7. J. Appl. Microbiol. 88, 79S^89S. Tomlins, R. I. and Ordal, Z. J. (1976) Thermal injury and inactivation in vegetative bacteria. Soc. Appl. Bacteriol. Symposium Series No. 5, 153^190. Venkitanarayanan, K. S., Zhao, T., and Doyle, M. P. (1999) Inactivation of Escherichia coli O157:H7 by combinations of GRAS chemicals and temperature. Food Microbiol. 16, 75^82. Wall, I., Sheridan, J. J., and Levett, P. N. (1989) A rapid method of enumeration of microorganisms from beef, using an acridine orange directcount technique. Irish J. Food Sci. and Technol. 13, 23^31. Weaver, R. A. and Shelef, L. A. (1993) Antilisterial activity of sodium, potassium or calcium lactate in pork liver sausage. J. Food Safety. 13, 133^146. Willshaw, G. A., Thirlwell, J., Jones, A. P., Parry, S., Salmon, R. L., and Hickey, M. (1994) Vero cytotoxin-producing Escherichia coli O157 in beefburgers linked to an outbreak of diarrhoea, haemorrhagic colitis and haemolytic uraemic syndrome in Britain. Lett. Appl. Microbiol. 19, 304^307.
doi:10.1006/fmic.2001.0462
ORIGINAL ARTICLE
Determination of the e¡ect of sodium lactate on the survival and heat resistance of Escherichia coli O157:H7 in two commercial beef patty formulations C. M. Byrne1; *, D. J. Bolton1, J. J. Sheridan1, I. S. Blair2 and D. A. McDowell2 The e¡ect of 4% sodium lactate (NaL) in beefburger patty formulations on the survival and heat resistance of Escherichia coli O157:H7 was investigated. Fresh beef trimmings were inoculated with E. coli O157:H7 to a concentration of 6?0^7?0 log10 cfu g 1 and subjected to the processing stages of beefburger patty production.Two commercial beefburger patty formulations were produced: a‘quality’patty (100% beef) and an ‘economy’ patty (70% beef, 30% other ingredients, including onion, water, salt, seasoning, rusk and soya concentrate). Sodium lactate (4% w/v) was added to the beefburger patties during mincing and the formed patties were frozen and stored for 1month. Beefburger patties without added NaL were used as controls. After frozen storage for 1 month, patties were examined for E. coli O157:H7 counts.There was a synergistic e¡ect between freezing and NaL, which resulted in a small but signi¢cant reduction (P o 0?05) (approximately 0?5 log10 cfu g 1) in E. coli O157:H7 numbers. The frozen beefburger patties were also heat-treated at 50, 55 and 601C and the data analysed to derive D-values for E. coli O157:H7 cells. At each temperature treatment, the D-values of the quality and economy beefburger patties with 4% NaL were signi¢cantly lower (P o 0?001) than the D-values of the patty formulations without NaL. The study demonstrates that the presence of 4% NaL in beefburger patty formulations can reduce the overall risks posed to consumers by the presence of E. coli O157:H7 by, ¢rst; reducing pathogen survival during freezing and frozen storage of the uncooked product; and, second, by increasing the susceptibility of the pathogen to heat during normal cooking processes. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction *Corresponding author. Present address: Microbial Food Safety Unit, Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 600 East Mermaid Lane, Wyndmoor, Pennsylvania 19038, USA.Tel: 001 215 836 3773; Fax: 001 215 233 6581. E-mail: [email protected] 0740 -0020/02/2^30211+09 $35.00/0
Since Escherichia coli O157:H7 ¢rst came to prominence in 1982, a range of foods have been associated with outbreaks worldwide. Ground (minced) beef and beefburger patties, however, are the foods most often implicated in such disease (Willshaw et al. 1994, Meng and Doyle 1998). r 2002 Elsevier Science Ltd. All rights reserved.
Received: 16 April 2001 1 Teagasc,The National Food Centre, Dunsinea, Castleknock, Dublin15, Eire 2 Food Microbiology Research Group, Faculty of Science, The University of Ulster, Jordanstown, Newtownabbey, Co. Antrim BT37 OQB, UK
212 C. M. Byrne et al.
Beefburger patty production processes do not signi¢cantly reduce the risks posed to consumers by beef trimmings that become contaminated by low infective dose pathogens, such as E. coli O157:H7, during slaughter and carcass dressing (Line et al. 1991, Ahmed et al. 1995, Juneja et al. 1997). Current inability to prevent contamination highlights the importance of adequate cooking of patties to eliminate E. coli O157:H7 prior to consumption. However, the frequent association of E. coli O157:H7 foodborne illness with beefburger patties suggests that such products are not always adequately cooked, possibly as a result of consumer demand for rare patties, or errors in the cooking process (Riley et al. 1983, CDC 1993, Jordan et al. 1999). As it is not possible to guarantee adequate cooking of patties consistently, it is important to try to limit the numbers and incidence of E. coli O157:H7 in the uncooked product. Successful achievement of this depends on the development and application of strategies which focus on reducing or eliminating E. coli O157:H7 during each step of the beefburger patty production process (Buchanan and Doyle 1997). The inclusion of antimicrobial agents in the uncooked beefburger patty may be a possible strategy to reduce or inactivate such pathogenic bacteria. Antimicrobial agents provide additional hurdles to control pathogens, thereby providing increased margins of safety to the products. Sodium lactate (NaL) is one such antimicrobial agent. It is a⁄rmed as a ‘Generally Regarded as Safe’ (GRAS) substance by the Food and Drugs Administration in the US for use as a human food ingredient, and is added to meat and poultry products to extend shelf life and inhibit growth of foodborne pathogens (Duxbury 1990, Miller and Acu¡ 1994, Nnanna et al. 1994, Houtsma et al. 1996, Venkitanarayanan et al. 1999). Some compounds, including organic acids and their salts, can have signi¢cant antimicrobial e¡ects when combined with a heat treatment (Shelef and Potluri 1995, McMahon et al. 1999). Thus, McMahon et al. (1999) reported that the presence of 2?4 and 4?8% NaL decreased the heat resistance of Yersinia enterocolitica and Listeria monocytogenes in ground beef. For this reason, the inclusion of NaL in
beefburger patties could provide an additional margin of product safety in relation to the presence of E. coli O157:H7, by increasing the susceptibility of the pathogen to heat. Most studies have concentrated on the e¡ect of NaL on growth and survival of pathogens at refrigeration (41C) temperatures or conditions of low temperature abuse (101C)(Maas et al. 1989, Brewer et al. 1992, Weaver and Shelef 1993, Miller and Acu¡ 1994). There is, however, limited information available on the e¡ect of NaL on E. coli O157:H7 during beefburger patty production and frozen storage, and during subsequent heating (i.e. cooking). Accordingly, the aim of the current study was to investigate the e¡ect of 4% NaL on: ¢rst the survival of the pathogen in two beefburger patty formulations during commercial processing and frozen storage; and, second, the thermal resistance of E. coli O157:H7 during heat treatment of beefburger patties.
Materials and Methods Organism A non-toxigenic strain of E. coli O157:H7 (NCTC 12900), resistant to streptomycin sulphate (Sigma Chemical Co., St. Louis, Missouri, USA) and nalidixic acid (Sigma) was prepared as described by Park (1978). Such antibiotic-resistant mutants can be enumerated in the presence of other contaminant or resident micro£ora by plating on a selective agar, Sorbitol MacConkey agar (SMAC; Oxoid, Basingstoke, UK), supplemented with nalidixic acid (NAS; 50 mg ml 1 ) and streptomycin sulphate (1000 mg ml21 ) (SMAC+NAS). Stock cultures for the double antibiotic-resistant mutant of E. coli O157:H7 were maintained on a frozen bead storage system (Protect Technical Consultant Services Ltd, Lancashire, UK) at 201C.
Preparation of bacterial inoculum One Protect bead of E. coli O157:H7 was aseptically transferred to 30 ml brain^heart infusion broth (BHI; Oxoid) and incubated at 371C for 24 h. A 3?0 -ml sample from the resultant culture was transferred to 300 ml fresh BHI broth
The e¡ect of sodium lactate on E. coli O157:H7 in beef burger patties 213
and incubated for a further 18 h at 371C to produce a stationary phase culture containing 9?0^10?0 log10 E. coli O157:H7 ml 1 . Stationary phase cultures have been shown to have greater heat resistance than log phase cultures (Tomlins and Ordal 1976). The cells were recovered from the stationary phase culture by centrifugation at 3000 g for 10 min at 41C (Eppendorf Centrifuge 5403, Germany); they were then washed three times in 9 -ml volumes of maximum recovery diluent (MRD, Oxoid) and the resultant pellet was resuspended in 9 ml of MRD.The numbers of E. coli O157:H7 cells ml 1 of this cell suspension were estimated using the membrane ¢ltration epi£uorescent staining technique of Wall et al. (1989). The cell suspension was diluted in MRD to form 1 l volumes of inoculum suspension containing 7?0 log10 E. coli O157:H7 ml 1 .
Preparation and inoculation of beef trimmings Beef trimmings (plate, £ank, forequarter and fat trim) were obtained from the on-site abattoir at The National Food Centre. Fat content of the beef trimmings was determined by the Food Analysis Department at The National Food Centre, using an automated, integrated microwave moisture and methylene chloride extraction method (JVA Analytical Ltd, Dublin, Eure) (Bostian et al. 1985). The results were used to calculate the exact quantity of additional beef fat to add to give the required fat:lean ratio to the formed beefburgers. The beef trimmings were cut into approximately 4^5 cm3 pieces (25^30 g) and inoculated with E. coli O157:H7 by immersion in an 1l-inoculum suspension for 5 s. The meat was allowed to drain for 5 s, transferred to sterile Stomacher bags (Seward Ltd, London, UK) and massaged thoroughly to obtain an even distribution of micro-organisms. This process yielded beef trimmings with an initial E. coli O157:H7 count of approximately 6?0^7?0 log10 cfu g 1. Inoculated trimmings were weighed into 500-g batches and sealed into Cryovac BB4L bags (approximately 15 20 cm: W.R. Grace Ltd, Dublin, Eure) using a portable heat sealer (Salton Bag Sealer, Pifco Ltd, Manchester, UK).
Beef trimmings inoculated with E. coli O157:H7 ( 7.0 log 10 cfu g-1 )
Frozen to –18˚C over a 37 h period and stored at –18˚C for 1 month
Tempered using microwave method (–18 to –5˚C (±2˚C) in 6 min)
Tempered beef trimmings (-3˚C ± 2.0˚C)
Formation of 10 g beefburger patty samples (quality or economy formulations)
Frozen to –18˚C and stored at –18˚C for 1 month
Figure 1. Commercial beefburger patty production from beef trimmings to frozen storage of the formed patties. Beef burger patty production process The speci¢cations and methods of routine commercial beefburger patty production, including the various physical stages and ingredient/ product temperature pro¢les, were identi¢ed and modelled within a laboratory-based system to mimic normal industrial processes (Fig. 1). Details of commercial beefburger patty production were carried out according to the methods of Byrne (2001).
Formation of ‘quality’ and ‘economy’ beef burger patties Two beefburger patty formulations, an ‘economy’ patty, and a ‘quality’ patty, were prepared according to the commercial speci¢cations.
214 C. M. Byrne et al.
Quality patty meat mixture was prepared in 1-kg batches, to contain 56% frozen ( 31C 7 2?01C) and 44% fresh (01C 7 11C) beef trimmings. The ¢nal formulation had a total fat content of approximately 24%. This process yielded samples (10 g) containing E. coli O157:H7 concentrations of approximately 5?0 log10 cfu g 1. Economy patty meat mixture was prepared in 1-kg batches to contain 69?9% frozen ( 31C 7 2?01C) beef trimmings and 30?1% other ingredients.The ingredients included tap water (13?5%), pinhead rusk (5?27%), frozen onion (7?59%), seasoning/spices (1?54%), salt (0?76%) and soya concentrate (1?73%)? The ¢nal formulation had a total fat content of approximately 30%.This process yielded samples (10 g) with an initial E. coli O157:H7 count of approximately 5?0 log10 cfu g 1.
ture, i.e. 50, 55 or 601C (7 1?01C). Processing times were measured from the time the core temperature of the test substrate reached the target temperature (o 3 min). Once the desired temperature was reached, duplicate samples were removed and cooled to 01C within 10 min in iced water. Duplicate samples were withdrawn at regular intervals during the heat treatment, cooled and subjected to microbiological analysis within 10 min. The temperature of the test substrate, the water at the thermal challenge temperature and the iced water were continuously monitored throughout the course of the experiment using thermocouples (1?2 -mm diameter 100 mm) attached to a temperature microprocessor (Code SSA12080G700TS, Ellab A/S, Copenhagen, Denmark).
Preparation of beefburger patties containing 4% sodium lactate
Chemical analyses
Economy and quality beefburger patty formulations were prepared as described above, except that 6?7 ml of NaL solution (sodium salt of DL-Lactic Acid, 60% w/w syrup)(Sigma) per 100 g of minced ingredients was added immediately after the ¢rst mincing step. The ¢nal ingredient mixes, including NaL, were doubleminced and formed into patties as previously described. The concentration of NaL, present was determined spectrophotometrically (Spectromics Kino MT Genesyst 5, Milton Roy Co. Analytical Products Division, Rochester, New York, USA) at 340 nm using a D-lactic acid/L lactic acid combination test kit (Boehringer Mannheim). The process yielded beefburger patty samples (10 g) with an initial E. coli O157:H7 count of approximately 5?0 log10 cfu g 1.
Thermal inactivation studies of E. coli O157:H7 in beefburger patties Thermal inactivation studies were carried out on both economy and quality beefburger patties (10 g), with and without added NaL, over a range of heating temperatures (501C, 551C and 601C). Bags containing the frozen inoculated beef patty samples were completely submerged in a water bath (Techne, AGB Scienti¢c, Dublin, Eure) with water at the desired tempera-
Estimations of pH and water activity of the beefburger patties were carried out according to the methods of Bendall (1973) and Richard and Labuza (1990), respectively.
Microbiological analyses Recovery and enumeration of E. coli O157: H7. Beefburger patties (10 g) were stomached in 90 ml MRD for 1 min in a Colworth Stomacher (Seward). Samples (1 ml) were serially diluted (1:10) in 9 ml volumes of MRD and spread-plated in duplicate (0?1 ml and 1?0 ml aliquots) onto SMAC+NAS and tryptone soya agar (TSA, Oxoid). Samples plated on SMAC+NAS were incubated at 371C for 48 h to obtain an estimate of uninjured E. coli O157:H7 numbers. Samples plated on TSA were incubated at 371C for 2 h, overpoured with SMAC+NAS (10 ml) and incubated at 371C for a further 48 h. The latter process allows the recovery of any injured cells that may be present (Ray and Speck 1978, Doyle and Schoeni 1984, Du¡y et al.1999) and, therefore, provided an estimate of the total E. coli O157:H7 numbers (injured plus uninjured).
Statistical analyses Frozen storage studies were repeated three times with each beefburger patty formulation.
The e¡ect of sodium lactate on E. coli O157:H7 in beef burger patties 215
The data was subjected to analysis of variance (ANOVA) and signi¢cant di¡erences between mean D-values were identi¢ed using t-tests. The heating trials were repeated three times with each beefburger patty formulation at each heating temperature. Estimates of heat resistance were performed by plotting the log10 of the number of survivors against time for each heating temperature to give a survival curve. The decimal reduction time or D-value (the time required, at a particular temperature, to reduce the number of bacteria by 90%, i.e. 1 log10 ) was derived by regression analysis of the linear portion of the log curve (Miles and Mackey 1994) using the Statistical Analysis Systems (SAS) procedure for the General Linear Model (SAS Institute Inc., Cary, North Carolina, USA). The data was subjected to analysis of variance (ANOVA) and signi¢cant di¡erences between mean D-values were identi¢ed using t-tests.
Results Data from proximate analyses (pH and aw ) of the beefburger patties are presented in Table 1. There were no signi¢cant di¡erences
between the mean pH values of the economy formulations and those of the quality formulations. This pattern was observed for formulations with and without added NaL. The addition of NaL resulted in a signi¢cant reduction in the mean pH values of both the economy (P o 0?001) and quality (P o0?05) beefburger patty formulations. The mean aw of the economy samples was signi¢cantly lower (P o 0?05) than the mean aw of the quality samples. This pattern was observed for formulations with and without added NaL. The addition of NaL (4%) resulted in a signi¢cant reduction (P o0?001) in the mean aw of both the quality and economy beefburger formulations. Table 2 presents the numbers of E. coli O157:H7 in quality, economy, quality with NaL and economy with NaL beefburger patties, immediately after production, and after freezing and storage at 181C for 1 month. Analysis of variance (ANOVA) determined that there was a signi¢cant interaction between freezing, the presence of NaL and the agar used for recovery. There was no signi¢cant di¡erence in E. coli O157:H7 numbers recovered from fresh burger samples without NaL and fresh samples with NaL on either SMAC (uninjured cells) or
Table 1. Proximate analyses of quality and economy beef burger patty formulations, with and without sodium lactate (0% NaL and 4% NaL) Proximate Analyses
Quality beefburger patty 0% NaL
pH (d.f. 19) aw (d.f. 23)
5?85 (0?03) 0?993 (0?002)
4% NaL 5?68 (0?10) 0?967 (0?007)
Economy beef burger patty 0% NaL 5?99 (0?03) 0?978 (0?003)
4% NaL 5?7 (0?14) 0?949 (0?005)
d.f. Degrees of freedom; aw ,Water activity. Standard deviation in parentheses.
Table 2. The numbers of E. coli O157:H7 (log10 cfu g 1 ) in quality and economy beefburger patties immediately after production (fresh), and after freezing and frozen storage for 1 month (frozen), detected using SMAC+NAS and TSA/SMAC+NAS Burger type
Quality (0% NaL) Quality (4% NaL) Economy (0% NaL) Economy (4% NaL)
Fresh beefburger patties
Frozen beefburger patties
SMAC+NAS
TSA/SMAC+NAS
SMAC+NAS
TSA/SMAC+NAS
4?79 (0?14) 4?72 (0?12) 4?71 (0?11) 4?67 (0?14)
5?38 (0?17) 5?37 (0?20) 5?18 (0?11) 5?09 (0?16)
4?71 (0?32) 4?60 (0?22) 4?64 (0?30) 4?47 (0?26)
5?42 (0?43) 5?03 (0?37) 5?10 (0?22) 4?62 (0?46)
Standard deviation in parentheses; d.f., degrees of freedom = 80. For details of media see text.
216 C. M. Byrne et al.
Table 3. Comparison of D-values (min) for E. coli O157:H7 in quality and economy beefburger patties, with and without sodium lactate (4% and 0% NaL), heated at three temperatures (50, 55 and 601C) and plated on TSA/SMAC+NAS Temperature (1C)
501C 551C 601C
Quality beefburger patty
Economy beefburger patty
Control (0% NaL)
NaL Added (4% NaL)
Control (0% NaL)
NaL Added (4% NaL)
152?8 (22?3) 14?8 (1?8) 2?7 (0?5)
76?0 (17?4) 9?8 (1?3) 1?5 (0?1)
170?5 (7?4) 12?4 (2?9) 3?5 (0?3)
61?8 (6?8) 12?4 (0?6) 2?2 (0?3)
Standard deviation in parentheses; d.f., degrees of freedom = 24.
TSA/SMAC (total cell count). However, with recovery on TSA/SMAC, E. coli O157:H7 counts in frozen burger samples containing NaL were signi¢cantly lower (P o 0?05) than counts of the pathogen in frozen samples without NaL. This pattern was observed in both the quality and economy formulations. In most cases there were no signi¢cant differences between numbers of E. coli O157:H7 recovered on TSA/SMAC+NAS (total counts) and those on SMAC+NAS (uninjured counts). Hence, there were no signi¢cant di¡erences between D-values derived from TSA/SMAC counts and those derived from SMAC+NAS counts. Data and D-values presented are those derived using TSA/SMAC only. Table 3 presents the derived D-values for E. coli O157:H7 in quality and economy formulations, with and without NaL, during heating at 50, 55 and 601C, as derived from plate counts on TSA/SMAC+NAS. Under most temperature treatments, the Dvalues in NaL-supplemented patties were signi¢cantly lower (P o 0?001) than the D-values in unsupplemented patties. There was one exception to this pattern. In samples heat-treated at 551C, there was no signi¢cant di¡erence between the D-values of economy samples with or without NaL. In most cases, the D-values for economy beefburger patties were signi¢cantly higher (P o 0?05) than the D-values for quality beefburger patties at the same temperature. This pattern was observed in both the NaL-supplemented and unsupplemented samples.
Discussion In the current study, the addition of 4% NaL resulted in signi¢cant reductions in both the pH
and the aw of the quality and economy beefburger patty formulations. The changes of the magnitudes demonstrated in the present study are in agreement with the ¢ndings of previous studies (Debevere 1989, Shelef and Yang 1991, Chen and Shelef 1992, Weaver and Shelef 1993, Bloukas et al. 1997, McMahon et al. 1999). Such changes have been reported to have little or no deleterious e¡ects on the sensory characteristics or acceptability of the product (Brewer et al. 1992; Papadopoulos et al. 1991). For a substance to be successful as a preservative, it must demonstrate antimicrobial activity against the target organisms without negatively a¡ecting the quality attributes of the food product, e.g. colour, odour or £avour. Sodium lactate meets these criteria and therefore has potential as an antimicrobial in such products as beefburger patties. In this study, the synergistic e¡ect of freezing and 4% NaL resulted in a small but signi¢cant reduction (approximately 0?5 log10 cfu g 1 ) in the numbers of total E. coli O157:H7 in frozen stored products. This result is smaller than, but generally in agreement with, the ¢ndings of Conner and Hall (1994), who demonstrated a signi¢cant reduction of approximately 1?0 log10 cfu g 1 in the numbers of E. coli O157:H7 in chicken meat supplemented with 4% NaL and stored in a frozen state for 18 months. Any reduction, even such relatively small reductions, in E. coli O157:H7 numbers, are to be welcomed. However, considering the levels of E. coli O157:H7 contamination that beef trimmings may carry, and the very low infective dose of this pathogen, such limited reductions in E. coli O157:H7 numbers in beefburger patties are, in isolation, unlikely to make a major contribution to the reduction of the risks posed to consumers by contaminated patties.
The e¡ect of sodium lactate on E. coli O157:H7 in beef burger patties 217
The current study noted signi¢cantly lower D-values in NaL-supplemented samples than in unsupplemented samples, demonstrating that the addition of 4% NaL reduced the heat resistance of E. coli O157:H7 in both the economy and quality burger patty formulations. In some instances the addition of 4% NaL reduces the D-value by 50%, i.e. when heated at 501C the D-value for quality samples with NaL is 76 min, compared to 153 min for quality samples alone. Such synergism between heat and NaL demonstrated in the present study has been reported previously (Shelef and Potluri 1995, McMahon et al. 1999). For example, McMahon et al. (1999) demonstrated that the addition of 4?8% NaL to minced beef signi¢cantly reduced the heat resistance of Yersinia enterocolitica (P o 0?001) and Listeria monocytogenes (P o 0?01) at 551C. Although the e¡ects of NaL, alone or in combination with other inhibitory agents, have been noted in a number of studies, the speci¢c mode of action of this additive has not been fully elucidated. However, numerous theories regarding its antimicrobial activity have been presented. Several investigators have suggested that the antimicrobial e¡ect of NaL may be caused by the e¡ect of pH on the bacterial cell, or speci¢cally the ability of the undissociated molecule to cross the cell membrane, dissociate, release protons and acidify the cell interior (Maas et al. 1989, Shelef 1994). Consequently, cell growth rate is reduced as more of the cell’s energy is expended in maintaining a constant internal pH. Therefore, in the present study, the small but signi¢cant reduction in product pH caused by the addition of NaL is likely to have rendered conditions less favourable to the survival of the pathogen, resulting in lower heat resistance. The reduction in the aw values of the beef patties caused by the addition of NaL in the current study may also account for the lower heat resistance in NaLsupplemented samples. Bacterial cells are generally more heat-resistant in food systems with low aw (Kaur et al. 1998). However, it has been reported that if the aw is decreased below a certain threshold value, the protective e¡ect is lost and the lower aw values act as a component of a more adverse microbial environment, leading to a reduction in bacterial heat resistance
(Kaur et al. 1998). Thus, the addition of NaL to the beefburger formulations may have reduced the water activity (aw ) to a point where the protective e¡ect of aw was lost, leading to a reduction in bacterial heat resistance. In the current study, the E. coli O157:H7 strain used was made resistant to two antibiotics, streptomycin sulphate and nalidixic acid, to facilitate the recovery and enumeration of the strain in the presence of other contaminant or resident meat micro£ora. However, previous reports have noted that the presence of antibiotics in the growth and/or enumerating media may a¡ect the D-values obtained (Doherty et al. 1998, Dombroski et al. 1999). For example, Dombroski et al. (1999) reported that the D-values for an antibiotic-resistant (AR) strain (nalidixic acid) of Vibrio vulni¢cus were signi¢cantly higher at 471C, lower at 501C and not signi¢cantly di¡erent at 521C than those of the wild-type (WT) strain. Therefore, the D-values obtained for the AR strain in the present study may be di¡erent from those of the WT strain. However, the D-values for the AR strain in this study are similar to those in previous reports of D-values for WT E. coli O157:H7 strains in minced beef (Ahmed et al. 1995, Juneja et al. 1997) and are within the range speci¢ed within a recent review of the published data on the heat resistance of E. coli O157:H7 (Stringer et al. 2000). In addition, the use of the AR strain does not detract from the major ¢nding of the current study, that the addition of NaL to the beef patty formulations signi¢cantly decreased the heat resistance of E. coli O157:H7 and increased the margin of safety of such products. This study has demonstrated that the addition of NaL to beefburgers may reduce the risks posed by E. coli O157:H7 in beefburgers by: ¢rst, reducing the numbers of E. coli O157:H7 during freezing and frozen storage of the uncooked product; and, second, decreasing the heat resistance of the pathogen during heat treatment. In isolation, the reductions in E. coli O157:H7 numbers in NaL-supplemented burger samples during frozen storage (approximately 0?5 log10 cfu g 1 ), are unlikely to have much practical impact in reducing the risks posed by contaminated beefburgers. Perhaps of greater signi¢cance in this regard is the e¡ect of
218 C. M. Byrne et al.
NaL on the heat resistance of E. coli O157:H7 in beefburgers noted in this study, i.e. the addition of NaL to the beefburger formulations signi¢cantly reduced the heat resistance of the pathogen. Thus, the addition of NaL should be considered as a means of increasing the margins of safety of such products, by making the E. coli O157:H7 cells more susceptible to heat treatment. It is clear that E. coli O157:H7 is an all too frequent and undesirable contaminant in beef products. Until robust strategies are available to ensure the consistent exclusion of this and other pathogens from such high-risk products, at the earliest possible stage of the human food chain, it will be necessary to explore and exploit all possible opportunities to reduce the persistence of these organisms. This study has demonstrated that the addition of NaL to beefburger patties o¡ers the potential of reducing pathogen survival during frozen storage of the uncooked product and may be helpful by rendering pathogens less resistant to commercially applied heat treatments.
Acknowledgements The authors gratefully acknowledge the assistance of Dr. Dermot Harrington and Ms. Paula Reid, of the Statistics Department, Teagasc Headquarters, Sandymount, Dublin, in performing the statistical analysis on these results. This project is supported by the Irish Department of Agriculture, Food and Rural Development through EU and national funds.
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