Effectiveness of a Commercial Lactic Acid Bacteria Intervention Applied to Inhibit Shiga Toxin-Producing Escherichia coli on Refrigerated Vacuum-Aged Beef

Because of their antagonistic activity towards pathogenic and spoilage bacteria, some members of the lactic acid bacteria (LAB) have been evaluated for use as food biopreservatives. The objectives of this study were to assess the antimicrobial utility of a commercial LAB intervention against O157 and non-O157 Shiga-toxigenic E. coli (STEC) on intact beef strip loins during refrigerated vacuum aging and determine intervention efficacy as a function of mode of intervention application. Prerigor strip loins were inoculated with a cocktail (8.9 ± 0.1 log10 CFU/ml) of rifampicin-resistant (100.0 μg/ml; RifR) O157 and non-O157 STEC. Inoculated loins were chilled to ≤4°C and treated with 8.7 ± 0.1 log10 CFU/ml LAB intervention using either a pressurized tank air sprayer (conventional application) or air-assisted electrostatic sprayer (ESS). Surviving STEC were enumerated on tryptic soy agar supplemented with 100.0 μg/ml rifampicin (TSAR) to determine STEC inhibition as a function of intervention application method (conventional, ESS) and refrigerated aging period (14, 28 days). Intervention application reduced STEC by 0.4 log10 CFU/cm2 (p < 0.05), although application method did not impact STEC reductions (p > 0.05). Data indicate that the LAB biopreservative may assist beef safety protection when utilized within a multi-intervention beef harvest, fabrication, and aging process.


Introduction
An estimated 175,905 Shiga-toxigenic Escherichia coli (STEC) foodborne disease cases occur in the United States each year, with non-O157 STEC being reportedly the causative agents in 64.1% of cases [1]. The US Department of Agriculture Food Safety and Inspection Service (USDA-FSIS) declared raw nonintact beef, as well as intact beef intended to be processed into nonintact beef, adulterated if found positive for E. coli belonging to serogroups O26, O45, O103, O111, O121, O145, and/or O157 [2]. Cattle serve as a reservoir of STEC [3][4][5]; eradication of these pathogens from the beef supply chain remains a challenge. Chemical food safety interventions such as lactic acid, peroxyacetic acid, and chlorine are commonly applied to reduce and/or eliminate spoilage and pathogenic organisms from beef surfaces [6][7][8]. However, consumer demand for natural or minimally processed foods [9,10] suggests a need for alternative beef safety interventions [11].
The lactic acid bacteria (LAB), as a type of biopreservative, are reported to be useful for preventing the growth of pathogenic microbes on meat products [12,13] and are in some instances classified as generally recognized as safe (GRAS) for use in nonintact, whole muscle cuts or carcasses, and ready-to-eat meats [14,15]. These organisms antagonize other bacteria, including human pathogens, through competition for nutrients and/or attachment sites, production of antimicrobial metabolites (e.g., reuterin, diacetyl, and fatty acids), bacteriocins (e.g., nisin, pediocin), and weak organic acids (e.g., lactic, acetic acid) [16,17]. Previous studies have explored the inhibitory mechanisms of specific protective 2 International Journal of Food Science  [26], who reported that STEC isolates identical to those used in the current study were able to achieve non-statistically differing counts following 24 h incubation at 35 ∘ C in a nutritious medium. Rif R organisms used in the current study were previously compared to antibiotic-sensitive parents for tolerance to common food safety interventions including heating, lactic acid exposure, and high pressure, with no statistically significant differences in survivors between parents and mutants [27]. The commercial biopreservative LactiGuard consisted of lyophilized powders containing a manufacturer-described set of Lactobacillus, Lactococcus, and Pediococcus spp. Immediately prior to use, a LactiGuard suspension was prepared by combining 7.5 g (11.0 log 10 CFU/g) of each organism (4 total) into 3.0 liters of sterile water to a concentration of 8.4 log 10 CFU/ml. LAB numbers in the antimicrobial suspension prior to application to beef surfaces were verified by enumerating on de Man, Rogosa, and Sharpe (MRS) lactobacilli agar (Becton, Dickinson and Co.) supplemented with streptomycin sulfate (40 g/ml, Amresco, Solon, OH, USA), sodium oxacillin (0.4 g/ml, Chem-Impex Int., Inc., Wood Dale, IL, USA), and gentamycin sulfate (5 g/ml, Amresco), as per intervention supplier instructions. Colonies were enumerated following anaerobic incubation for 48 h at 35 ∘ C.

Meat Preparation and Inoculation.
Prerigor beef strip loins were procured from a federally inspected establishment in Texas and harvested within 2 h of animal death. Once collected, each strip loin was transferred into a polyethylene bag and swathed in a thermal blanket (EverReady First Aid, Brooklyn, NY, USA) to minimize heat loss. Beef pieces were then immediately transported in insulated coolers containing activated instant hot packs (Dynarex, Orangeburg, NY, USA) to a Texas A&M AgriLife Research facility maintaining a BSL-2 Laboratory located within a 30 min drive of the beef slaughter establishment to inoculate strip loins as quickly as possible after collection (simulating prerigor carcass crosscontamination during beef harvest). At the facility, a prepared inoculum application spray bottle was primed and held 25 to 31 cm above the meat for application of three pumps of inoculum (1.0-1.5 ml per pump) onto the lean side of the strip loin surface. The bag containing the inoculated strip loin was closed with a zip tie and hand-tumbled for 1 min to distribute inoculum over beef surfaces. Beef pieces were held at 25 ∘ C for 30 min for inoculum attachment, loaded into a second polypropylene bag, and placed in insulated coolers containing frozen ice packs to initiate chilling. Coolers were transported to the Food Microbiology Laboratory (Texas A&M University) within 8 h of inoculation and bacterial attachment. Upon return, bagged strip loins were removed from coolers and placed on shelves in a single layer in a 4 ∘ C walk-in cooler and held until a total chilling period of 24 h had elapsed.
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Sampling and Microbiological Analysis.
In order to track changes in meat pH as a function of intervention application, external pH of individual strip loins was measured in triplicate using an ExStik5 pH and temperature meter (Extech Instruments Corp., Nashua, NH, USA) before and after STEC inoculation, before and after LAB intervention treatment, and again after 14 and 28 days of refrigerated vacuum aging. For microbiological sampling, three 10 cm 2 outlines were marked on the lean tissue surface using a flame-sterilized stainless steel borer, excised to a depth of 1-2 mm using flamesterilized scalpels and forceps, and composited into a sterile stomacher bag. Samples were then sealed and transported in insulated coolers packed with ice to the Food Microbiology Laboratory for analysis. Beef samples were assayed by adding 99 ml phosphate buffered saline (PBS; Sigma-Aldrich Co.) to each sample pouch, pummeling for 1 min in a stomacher, serially diluting in 0.1% peptone water, and spreading on an appropriate medium. LactiGuard LAB were enumerated on MRS agar supplemented with antibiotics described above and incubated anaerobically 48 h at 35 ∘ C prior to colony counting. Rif R STEC were spread on TSAR and colonies enumerated following incubation at 35 ∘ C for 24 h.

Statistical Analysis.
The experiment was completed via triplicate identical samples conducted over two replications ( = 6). All statistical analyses were performed using JMP Pro v11.0 (SAS Institute, Inc., Cary, NC, USA). Colony counts were transformed to log 10 CFU/cm 2 ; the limit of detection for the plating assays was 0.5 log 10 CFU/cm 2 . Differences between main effects (vacuum aging duration, and biopreservative intervention application method [control, conventional spray, ESS]) and their interaction were identified by analysis of variance (ANOVA) at a = 0.05. Statistical differences among means were separated with Tukey's Honestly Significant Differences (HSD) multiple comparisons test ( < 0.05).

Results and Discussion
To simulate cross-contamination during commercial animal slaughter prior to intervention application, beef strip loins were collected within 2 h of animal slaughter and inoculated as rapidly as possible. STEC inoculum fluid contained 8.9 ± 0.1 log 10 CFU/ml prior to application to beef surfaces; STEC populations on inoculated strip loins remained unchanged during chilled transportation to the food microbiology laboratory prior to treatment (Table 2). Although control strip loins (STEC-inoculated, untreated) were handled identically to those subjected to LAB intervention treatment, STEC numbers enumerated from nontreated controls after chilling (6.6 ± 0.1 log 10 CFU/cm 2 ) were statistically lower than those from intervention-treated samples (7.2 ± 0.1 log 10 CFU/cm 2 ) ( < 0.05) ( Table 2). This was unexpected; authors are uncertain as to the cause(s) behind this observed difference in STEC counts between controls and other samples eventually treated with LAB.

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International Journal of Food Science Table 2 presents surviving populations of STEC and LAB on strip loins immediately following treatment during vacuum aging at 4 ∘ C. Analysis of microbiological data determined method of intervention application (conventional spray, ESS) did not influence LAB numbers recovered from treated beef strip loins (6.5 ± 0.1 log 10 CFU/cm 2 ). Likewise, STEC numbers on treated strip loins did not differ as a function of the mode of LAB application method ( ≥ 0.05). Nevertheless, STEC numbers on biopreservative-treated strip loins differed by intervention application with respect to STEC counts before and after treatment on strip loins and by posttreatment aging period ( = 0.015) ( Table 2). Applying LAB onto beef strip loins resulted in a reduction in STEC of 0.4 ± 0.1 log 10 CFU/cm 2 ( < 0.05). Once treated, subprimals were individually vacuum packaged and aged at 4 ∘ C for 14 or 28 days. On day 14 posttreatment, STEC numbers increased to 7.1 log 10 CFU/cm 2 and were not different from STEC counts obtained immediately after intervention applications. Conversely, at 28 days of vacuum refrigerated aging, STEC counts (6.7 ± 0.1 log 10 CFU/cm 2 ) were significantly lower than pretreatment means ( < 0.05). At 14 days' vacuum refrigerated aging, LAB numbers on treated strip loin surfaces declined from posttreatment application counts to 6.2 log 10 CFU/cm 2 , though no further changes in LAB counts were detected at day 28 ( ≥ 0.05) ( Table 2). Overall, reductions in STEC counts following intervention application and refrigerated aging were modest and not likely of great antimicrobial significance.
Outcomes from previous studies evaluating the antimicrobial activity of LAB biopreservatives against pathogens on beef products are mixed with respect to pathogen reduction results. Smith et al. [22] inoculated ground beef with 5.0 log 10 CFU/g of E. coli O157:H7 and 7.0 log 10 CFU/g of LAB (Lactobacillus acidophilus strains NP 51, NP 25, NP 7, and NP 3) and then stored the product at 5 ∘ C under vacuum for up to 5 days prior to analysis of pathogen survival. Compared to untreated controls, E. coli O157:H7 in treated beef was reduced by >3.0 log 10 CFU/g following refrigeration for 5 days ( < 0.05). Echeverry et al. [28], conversely, reported that E. coli O157:H7 inoculated on refrigerated strip loins (5.0 log 10 CFU/cm 2 ) treated with a spray consisting of 7.7 log 10 CFU/ml LAB remained unchanged during refrigerated vacuum aging over 21 days, similar to findings reported herein. Finally, previous research has reported that reductions in E. coli O157:H7 on beef following treatment with LAB intervention were independent of LAB numbers applied, with no differences in E. coli O157:H7 numbers observed following incubation [29]. In the current study, numbers of LAB and STEC were nearly equivalent throughout the refrigerated aging period, yet LAB did not exert strong pathogen inhibition activity (Table 2). This may have resulted from insufficient levels of fermentable sugars for production of organic acids, lack of available oxygen for production of peroxides, or possibly other unanticipated factors. Finally, no effort to differentially count surviving STEC from intervention-treated or control strip loins during the experiment was attempted, disallowing authors from determining whether some members of the STEC inoculum were inhibited to greater or lesser extents than other inoculum members. While Kirsch et al. [26] reported differing morphological characteristics of STEC strains identical to those used herein on selective/differential media, it was noted that STEC isolates not bearing good isolation were frequently subject to misidentification on selective/differential plating medium surfaces. The enumeration of total surviving STEC from the inoculum would be expected to primarily yield counts of STEC isolates most tolerant to the antimicrobial intervention, though quantification of any differences in inhibition on individual STEC isolates was therefore precluded.
Mean surface pH of strip loins immediately prior to inoculation was 6.2 ± 0.1, while pH following meat inoculation and chilling of inoculated product declined to 5.8 ± 0.1 ( < 0.05) (data not shown). Whereas application of the LAB intervention by conventional pressurized sprayer versus ESS did not produce statistically significant differences in meat surface pH, surface pH significantly decreased from the point of treatment application (LAB, control) to 14 days of refrigerated vacuum storage (5.4 ± 0.1) ( < 0.05). A small, nonstatistically significant decline in meat surface pH was observed to occur between days 14 and 28 for refrigerated vacuum-aged strip loins (5.3 ± 0.1) ( > 0.05) (data not shown). These pH declines mirror those for similarly chilled and vacuum-aged refrigerated beef in other studies where researchers determined LAB-driven declines in beef meat pH during chilled aging [30][31][32]. The inhibition of STEC by LAB is thought to result largely from production of organic acids [33]. However, exposure to sublethal stress (cold storage, carbohydrate limitation) may have limited acid fermentation output of LAB [34,35]. Data indicate that acid fermentation of endogenous carbohydrate by applied LAB was insufficient to exert a greater degree of pathogen inhibition than that observed. This study investigated the efficacy of a commercial LAB food safety biopreservative intervention for inhibiting growth of eight O157 and non-O157 STEC isolates on beef strip loins during refrigerated vacuum aging. STEC numbers were reduced following treatment, and STEC populations on beef strip loins after 28 days of aging were lower than at pretreatment. However, pathogen reductions were small (<1.0 log 10 -cycle). While not likely effective as a sole antimicrobial intervention for beef safety protection, biopreservative food safety interventions such as LactiGuard can be utilized to gain useful reductions in STEC when integrated into a multi-intervention process for beef safety protection during beef harvest and fresh beef products manufacture.