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A new bacteriophage (phage ggg) and its host, Leuconostoc gelidum LRC-BD, were isolated from vacuum-packaged pork loins. Homogenates of pork loin tissue were enriched with L. gelidum LRC-BD to isolate phages. Cultural, biochemical and genetic methods were used to compare L. gelidum LRC-BD and the type strain, L. gelidum ATCC 49366. The phages were characterized by host range, morphology and phage-bacterial interaction in All Purpose Tween (APT) broth and on pork adipose tissue. With the exception of its inability to produce dextran from sucrose and the fermentation of l-arabinose, L. gelidum LRC-BD was culturally and biochemically similar to L. gelidum ATCC 49366. DNA-relatedness of the strains was confirmed by sequencing of the 16s rRNA gene. Electron microscopic observation revealed that phage ggg was a member of the Siphoviridae. The host range was limited to L. gelidum isolates from meats. Phages were able to replicate and limit the growth of L. gelidum LRC-BD in APT broth incubated aerobically and anaerobically at 4 degrees C, with a multiplicity of infection (MOI) of 0.001. When inoculated pork adipose tissue was stored at 4 degrees C in air or vacuum, phages could multiply but a higher MOI (0.01 to 1000) was necessary to limit the growth of L. gelidum LRC-BD. Naturally occurring phages may affect the numbers of L. gelidum and other lactic acid bacteria residing in meats and thereby alter the storage quality or the preservative potential of competitive strains.

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The effect of a bacteriocinogenic Brochothrix campestris ATCC 43754 upon the growth of Brochothrix thermosphacta and a 4 strain mixture of Listeria monocytogenes was determined in All Purpose Tween (APT) broth and on pork adipose tissue discs at 4 degrees C. Inocula were prepared to give initial numbers of B. campestris of 6-7 log cfu/ml or cm(2) and 3-4 log cfu/ml or cm(2) of B. thermosphacta and L. monocytogenes. Adipose tissue discs were evaluated by a sensory panel to determine the intensity and acceptability of any off-odours produced during the growth of B. campestris. During co-culture in APT broth with B. campestris the growth of B. thermosphacta or L. monocytogenes was 4 log cycles less than growth in its absence. B. campestris showed limited growth on inoculated pork adipose tissue, increasing from initial numbers of about 6 log cfu/cm(2) to a maximum of 7 log cfu/cm(2) within 7d. B. campestris at numbers of 7 log cfu/cm(2) produced slight off-odours but these were not perceived by the panel as unacceptable. When co-inoculated on adipose tissue discs with B. campestris the numbers of B. thermosphacta or L. monocytogenes was limited to about 2-3 log units less than the numbers attained in its absence. B. campestris ATCC 43754 may be useful for meat preservation because it can inhibit B. thermosphacta and L. monocytogenes in situ while producing little change in the sensory properties of the product.

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Bacteriophages are measurable components of the natural microflora in the food production continuum from the farm to the retail outlet. Phages are remarkably stable in these environments and are readily recovered from soil, sewage, water, farm and processing plant effluents, feces, and retail foods. Purified high-titer phage lysates have been used for the species-specific control of bacteria during the pre- and postharvest phases of food production and storage. For example, the inhibition of the phytopathogens Erwinia amylovara and Xanthomonas campestris has reduced the incidence of diseases such as fire blight in apples and bacterial spot of tomato and peaches. Research on preslaughter treatment of food animals has demonstrated phage control of salmonellosis in chickens, enteropathogenic Escherichia coli infections in calves, piglets, and lambs, and E. coli O157:H7 shedding by beef cattle. Phages have also been applied to control the growth of pathogens such as Listeria monocytogenes, Salmonella, and Campylobacter jejuni in a variety of refrigerated foods such as fruit, dairy products, poultry, and red meats. Phage control of spoilage bacteria (e.g., Pseudomonas spp. and Brochothrix thermosphacta) in raw chilled meats can result in a significant extension of storage life. Phage biocontrol strategies for food preservation have the advantages of being self-perpetuating, highly discriminatory, natural, and cost-effective. Some of the drawbacks of biopreservation with phages are a limited host range, the requirement for threshold numbers of the bacterial targets, phage-resistant mutants, and the potential for the transduction of undesirable characteristics from one bacterial strain to another. Most research to date has involved experimentally infected plants and animals or artificially inoculated foods. This technology must be transferred to the field and to commercial environments to assess the possibility of controlling natural contaminants under more realistic production and processing conditions.

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In a commercial process for the production of moisture-enhanced pork, boneless pork loins were conveyed through a recirculating injection apparatus, and brine (sodium phosphate, sodium chloride, and lemon juice solids) was pumped into the meat through banks of needles inserted automatically into the upper surfaces of cuts. Brine samples were collected at intervals during the production process and analyzed to determine the total plate count and the numbers of lactic acid bacteria, pseudomonads, Brochothrix thermosphacta, and Enterobacteriaceae. Listeria monocytogenes numbers in the brine were determined using a PCR with primers for the hemolysin gene in combination with a most probable numbers determination. Maximum numbers of bacteria (log CFU/ml) recovered from the brine after 2.5 h of recirculation were as follows: total plate count, 4.50; lactic acid bacteria, 2.99; pseudomonads, 3.95; B. thermosphacta, 2.79; and enterics, 3.01. There was an increase in the number of L. monocytogenes in the recirculating brine with time, reaching a maximum of 2.34 log CFU/100 ml after 2.5 h of moisture-enhanced pork production. Thus, recirculating brines can harbor large populations of spoilage bacteria and L. monocytogenes and are an important source of contamination for moisture-enhanced pork.

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This study was undertaken to determine the impact of the moisture enhancement process on the bacterial contamination and storage life of vacuum-packaged pork loins. Bone-in and boneless pork loins injected with brine (sodium chloride, sodium phosphate, lemon juice) were obtained from a commercial processing facility and stored for 5 weeks in vacuum packaging at 2 and 5 degrees C. At weekly intervals, samples were excised to determine numbers of spoilage bacteria and pathogens. The loins were subjectively evaluated by a sensory panel to quantify appearance and odor acceptability. Moisture-enhanced loins were initially contaminated with a population of psychrotrophic bacteria that was approximately 2 log units higher than that for noninjected boneless loins. This difference was largely due to contamination by larger numbers of pseudomonads in the brine-injected loins. There were no significant differences in the initial numbers of lactic acid bacteria, Enterobacteriaceae, or Brochothrix thermosphacta. Similar trends in spoilage bacterial populations were observed for moisture-enhanced loins with bones, but Enterobacteriaceae counts were also found to be approximately 1 log unit higher for the injected product. Brine-injected loins generally had larger bacterial numbers at each storage time, but there were no consistent injection treatment effects on bacterial growth. Brine injection did not affect color or odor deterioration, and the storage life for vacuum-packaged loins was the same as that for noninjected controls. The incidence of Listeria monocytogenes was 21% for control loins and 27% for moisture-enhanced loins. Although the brine injection process resulted in an increase in bacterial contamination, there was no evidence that this contamination would affect the storage life of vacuum-packaged loins, and further research is necessary to determine the significance of the increased incidence of L. monocytogenes.

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Adipose tissue discs were coinoculated with Brochothrix thermosphacta and homologous bacteriophages (phages) to determine the effects these had on phage multiplication, bacterial growth, and off-odor development during storage at 2 degrees C or under simulated retail display at 6 degrees C. In the presence of about 10(5) bacteria/cm2 and an equivalent number of phages, there was a 3-log increase in phage numbers and a 2-log decrease in bacterial numbers, and objectionable off-odors were suppressed during refrigerated storage. Up to 68% of the surviving bacterial population were resistant to phages. The storage life of adipose tissue could be increased from 4 days in controls to 8 days in phage-treated samples by preventing the development of off-odors associated with the growth of B. thermosphacta. Phages may provide a novel approach to extending the storage quality of chilled meats.

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Hydrophobic grid membrane filtration (HGMF) was investigated as an alternative to conventional plate counts for enumerating spoilage bacteria recovered from raw beef. The HGMF method was compared to conventional procedures for the selective enumeration of total psychrotrophic bacteria, pseudomonads, total Enterobacteriaceae . Brochothrix thermosphacta , and lactic acid bacteria. Bacteria were recovered both from beef which had been artificially inoculated with identified strains and from naturally contaminated beef from a commercial abattoir. There were no significant differences (P > 0.05) in numbers of any bacterial group recovered from naturally contaminated beef using HGMF procedures when compared to conventional plating on selective media. The recoveries of the total psychrotrophic population, Escherichia coli . B. thermosphacta , and Lactobacillus sake inoculated onto meat were unaffected by the enumeration procedure (P > 0.05). However, the populations of Pseudomonas sp. recovered from inoculated beef by a HGMF procedure were 0.8 log cycles lower (P < 0.05) when compared to the conventional spread plate procedure.

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The effects of homologous bacteriophages upon growth of a beef spoilage pseudomonad and the retail case life of beef were examined under conditions of simulated retail display. Initial studies with an aqueous extract of beef muscle showed Pseudomonas growth was significantly limited by phages for up to 3 d at 7°C. Subsequently, it was shown that the treatment of Pseudomonas -inoculated steaks with high titer phage lysates (108 PFU/ml) resulted in a 1- to 2-log reduction in the level of bacterial contamination and a 2-log increase in phage numbers after 4 d of retail display. These changes were accompanied by a marked decrease in steak surface discoloration and a concurrent improvement in retail acceptance. Steak case life was positively correlated with phage concentration within the range of 10 to 108 PFU/ml. At the highest concentration of phages tested (108 PFU/ml) steak case life was significantly increased from 1.6 to 2.9 d. It was concluded that phages could multiply on the steak surface and have the potential for the biological control of beef spoilage.

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To determine effects of wholesale and retail contamination on steak retail case life, rib steaks were fabricated from wholesale beef ribs using laboratory-simulated extremes of retail processing sanitation. Steak retail case life was more highly correlated with the psychrotrophic bacterial load on the surface of wholesale ribs and retail steaks than with the level of retail processing sanitation. Although steak case life could be predicted as a function of both steak and rib bacterial loads or rib and retail equipment bacterial loads the, following is recommended as the most useful predictor of case life: Steak Case Life (days) = 3.97 - 0.19 (log bacteria/cm2 on wholesale ribs) -0.14 (log bacteria/cm2 on retail processing equipment). This equation could also be used by the retailer to assess the quality of wholesale product received from different suppliers.

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In both beef extract medium and on the surface of rib-eye steaks, potassium sorbate inhibited growth of psychrotrophic beef-spoilage bacteria by prolonging the lag phase of growth without affecting rate of growth. As a result, steak retail shelf life was extended by 2 days following a 10% potassium sorbate dip.

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A total of 40 beef spoilage pseudomonads was used as bacterial hosts for the isolation of psychrotrophic bacteriophages (phages) from spoiled rib steaks. Thirty-eight homologous phages, lytic for 25 of these hosts, were isolated and purified. An additional 12 bacterial isolates were susceptible to heterologous phage lysis and only three of the bacteria examined were resistant to lysis by any of the phage tested. On the basis of heterologous cross-sensitivity to phages, the meatborne Pseudomonas strains and 4 identified ATCC Pseudomonas hosts were differentiated into 37 distinct phage lysotypes. In addition, Pseudomonas phages were found to inhibit bacterial growth in tryptic soy broth by significantly extending the lag phase under psychrotrophic conditions (7°C). However, the incubation of bacteria with phages resulted in the selection of phage resistant bacterial mutants.

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Using laboratory-simulated retail conditions, a variety of retail case blower temperatures were selected to determine their influence upon the surface temperature of displayed rib eye steaks, bacterial growth and steak shelf life. Steak surface temperature was found to be significantly correlated with blower temperature and exceeded the temperature of the incoming blower air by 9 C. Furthermore, bacterial generation time and steak shelf life were significantly and inversely related to blower temperature. Shelf life was also dependent upon the initial psychrotrophic bacterial load. Retail blower temperature could be easily adjusted to reduce steak surface temperature to 2 C, and thereby extend the visual shelf life from 3.8 to 8.2 days. From a practical standpoint, these results indicate that a relatively simple temperature adjustment by the retailer could improve the microbial quality and more than double the shelf life of meats on display.

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The influence of retail sanitation on the psychrotrophic bacterial load and subsequent shelf life of rib steaks was investigated under laboratory-simulated retail processing conditions. Steaks were fabricated employing extensively sanitized processing equipment (< 10 bacteria/cm2 of surface) or with highly contaminated equipment where bacterial counts approached 106/cm2. No significant differences were observed in the initial psychrotrophic bacterial load, bacterial growth rate or in the organoleptic deterioration of steaks processed under these extremes of retail sanitation. These results were confirmed with ground beef fabricated under similar extremes of grinding sanitation. Although the psychrotrophic bacterial load on steaks could not be related to the degree of retail processing sanitation, it was significantly correlated with the level of surface contamination on wholesale ribs. In addition, the extent of steak surface discoloration and overall retail appearance were significantly correlated to the psychrotrophic bacterial load.

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Laboratory-simulated retail conditions were employed to examine the effects of retail display temperature upon growth of psychrotrophic bacteria and the retail case-life of rib-eye steaks. The temperature of continuously displayed steaks ranged from 7.5 to 14.2 C and under these conditions an average case-life of 2.4 days was determined. However, when steaks were limited in their time of retail display, by overnight refrigeration in a walk-in cooler (1 C), steak temperatures were reduced to 1 C and retail case-life substantially extended to 4.9 days. Increased retail case-life resulted from a reduction in psychrotrophic bacterial growth which was related to the rate of deterioration of steak retail appearance. These findings provide an alternative to the continuous retail display of beef which, if applied, could greatly improve beef case-life and reduce spoilage losses.