Reduction of Salmonella by two commercial egg white pasteurization methods.

The effect of pH, processing temperatures, and preheating steps in two commercial egg white pasteurization procedures (Armour and Standard Brands methods) were evaluated using a five-strain cocktail of Salmonella. We devised a benchtop pasteurization system that would more closely resemble the two commercial processes than could the traditional capillary tube method. The pasteurization methods both require hydrogen peroxide to be metered into the egg white stream between a required initial preheat step and the main heating regimen. Both processes were evaluated at three pH levels (pH 8.2, 8.6, 9.0), at four temperatures (51.7 degrees C/125 degrees F, 53.1 degrees C/127.5 degrees F, 54.4 degrees C/130 degrees F, 55.8 degrees C/132.5 degrees F), and over four residence times to allow calculation of D-values at each temperature. When compared at the minimum allowable time and temperatures for each process, our results showed at least a 1-log greater log reduction (P < 0.05) for the Standard Brands method than the Armour method in 10 of 12 of the pH and temperature combinations tested. Almost all runs at any given temperature showed more reduction at pH 9.0 than at pH 8.2 except for the Standard Brands method at 54.4 degrees C and 55.8 degrees C, which showed the most consistent reduction across all three pH levels tested. Analysis of the preheat portion of the two methods showed that there was no contribution (P > 0.05) toward Salmonella reduction when compared with the identical process without the preheating step. We generally observed a greater reduction of Salmonella with egg white at pH 9.0 that is typical of older, off-line processing than with low pH egg white (i.e., 8.2) that is typical of modern in-line processing facilities. This difference was as much as 3.5 log cycles depending on the processing conditions. The data has been used to make recommendations for minimum processing conditions for hydrogen peroxide-based egg white pasteurization.

Postpackage pasteurization of ready-to-eat deli meats by submersion heating for reduction of Listeria monocytogenes.

A mixed cocktail of four strains of Listeria monocytogenes was resuspended in product purge and added to a variety of ready-to-eat (RTE) meat products, including turkey, ham, and roast beef. All products were vacuum sealed in shrink-wrap packaging bags, massaged to ensure inoculum distribution, and processed by submersion heating in a precision-controlled steam-injected water bath. Products were run in pairs at various time-temperature combinations in either duplicate or triplicate replications. On various L. monocytogenes-inoculated RTE deli meats, we were able to achieve 2- to 4-log cycle reductions when processed at 195 degrees F (90.6 degrees C), 200 degrees F (93.3 degrees C), or 205 degrees F (96.1 degrees C) when heated from 2 to 10 min. High-level inoculation with L. monocytogenes (approximately 10(7) CFU/ml) ensured that cells infiltrated the least processed surface areas, such as surface cuts, folds, grooves, and skin. D- and z-value determinations were made for the Listeria cocktail resuspended in product purge of each of the three meat categories. However, reduction of L. monocytogenes in product challenge studies showed much less reduction than was observed during the decimal reduction assays and was attributed to a combination of surface phenomena, including surface imperfections, that may shield bacteria from the heat and the migration of chilled purge to the product surface. The current data indicate that minimal heating regimens of 2 min at 195 to 205 degrees F can readily provide 2-log reductions in most RTE deli meats we processed and suggest that this process may be an effective microbial intervention against L. monocytogenes on RTE deli-style meats.

Cloning, sequencing, and characterization of genomic subtracted sequences from Listeria monocytogenes.

Individual sequences of a genomic subtracted, PCR-amplified, mixed-sequence probe (GS probe) were cloned and sequenced. The GS probe differentiated restriction fragment length polymorphism patterns for Listeria monocytogenes but did not hybridize with members of other bacterial genera. Sequence analysis identified several L. monocytogenes sequences already present in the GenBank database; the putative identities of other sequences were inferred from homology data, and still other sequences did not exhibit significant levels of homology with any GenBank sequences.

Comparison of Conventional Plating Methods and Petrifilm for the Recovery of Microorganisms in a Ground Beef Processing Facility †.

The objective of this study was to compare recovery of microorganisms for various beef samples and beef contact surfaces using conventional pour plating techniques and Petrifilm methods. Comparisons for aerobic plate count (APC), coliform count (CC), and Escherichia coli count (ECC) were done for 104 fresh or frozen retail cuts and 56 food surface or food contact surfaces. Samples were taken at a midwestern retail ground beef processing plant during a 12-month project. APC comparisons were made for pour plating using Trypticase soy agar versus Aerobic Plate Count Petrifilm. CC and ECC were compared for pour plating using violet red bile + MUG agar versus E. coli Petrifilm. Overall, paired t tests revealed a significantly higher recovery for APC from fresh and frozen beef samples using the pour plating technique (P ≤ 0.05). No significant differences (P > 0.05) were observed for CC from fresh and frozen meat samples. Recovery of E. coli from many beef samples was better using Petrifilm. Significantly higher ECCs were observed from fresh and frozen meat samples using Petrifilm compared to the pour plating technique (P ≤ 0.05). For food surfaces and food contact surfaces, a comparison between pour plating and Petrifilm was done for aerobic plate count. No significant differences (P > 0.05) in recovery could be found between methods. A comparison between neutralizing buffer and letheen broth for recovery of surface microorganisms was done for both the APC pour plating method and APC Petrifilm. In both cases, recovery when using letheen broth was significantly (P ≤ 0.05) higher than neutralizing buffer. Because it is convenient and gave comparative results, Petrifilm offers a good alternative for environmental microbial testing and red meat product testing.

Pasteurization of eggs in the shell.

A small percentage of all eggs may be contaminated with Salmonella enteritidis (SE). To eliminate this hazard from the food supply, procedures for pasteurizing eggs in the shell have been developed. At least four research groups are attempting to devise a process to achieve a pasteurized shell egg. Only one of the groups has reported procedures and results. Sound shell eggs were washed to remove surface contaminants. The clean eggs were then inoculated with high levels of SE cells. The inoculated eggs were then heated by one of several means to a yolk temperature of about 55 C and held at that temperature for varying periods of time. The number of surviving cells was determined. It is possible to obtain a 7 log cycle reduction of SE in inoculated eggs without a significant change in functional or visual quality of the eggs.

Genomic subtraction in combination with PCR for enrichment of Listeria monocytogenes-specific sequences.

Genomic DNA from Listeria innocua and Listeria ivanovii was used in subtractive hybridization with DNA from Listeria monocytogenes involving two amplification strategies. Subtraction was accomplished by labelling the subtracting DNA with biotin and removal after liquid hybridization with tester DNA (L. monocytogenes) by reaction with streptavidin and phenol extraction. In one strategy, L. monocytogenes DNA was poly(A)-tailed with terminal transferase and amplified asymmetrically after subtraction. In another procedure, adapters ligated to the target DNA allowed symmetrical amplification after subtraction using an adapter-specific primer; in both amplifications, the amplified products were labelled with biotin-modified dUTP. Southern hybridization of the amplified/subtracted probes with tester- and subtractor-related strains demonstrated numerous L. monocytogenes-specific sequences. The genome-subtracted mixed probe identified 7 RFLP patterns among 13 strains of L. monocytogenes representing 11 L. monocytogenes serovars. Southern blot analysis demonstrated that the subtracted probe cross-hybridized to two bands among L. welshimeri strains but had little or no hybridization with five other species of Listeria including L. innocua, L. ivanovii, L. seeligeri, L. grayi, and L. murrayi. These data demonstrate that genomic subtraction via subtractive hybridization is a powerful method to enrich for specie-specific sequences in L. monocytogenes; the enriched sequences in the subtracted probe may be useful for typing L. monocytogenes strains by specific RFLP patterns or for cloning L. monocytogenes-specific sequences.

Plasmid profiles and resistance to antimicrobial agents among Salmonella enteritidis isolates from human beings and poultry in the midwestern United States.

In the study reported here, 121 Salmonella enteritidis isolates from human beings and 467 isolates from nonhuman sources were analyzed for plasmid pattern and susceptibility to a panel of antimicrobial agents commonly used as biologic markers. A significant (P < 0.05) number of isolates from nonhuman sources were resistant to beta-lactam antibiotics and tetracycline. Resistance to aminoglycosides, quinolones, and trimethoprim/sulfamethoxazole was uncommon. Of the 588 isolates, 445 (76%) were resistant to 2 or more antimicrobial agents. Sixty of 121 (50%) S enteritidis isolates from human beings were susceptible to all 12 antimicrobial agents, but 425 of 467 (91%) S enteritidis isolates from nonhuman sources expressed resistance to 1 or more of the antimicrobial agents used in the study. Analysis of plasmid profiles revealed that significantly (P < 0.05) more isolates from nonhuman sources had high molecular weight plasmids than did isolates from human beings. Isolates from ceca of chickens were associated with patterns of low molecular weight plasmids. Analysis of results of the study revealed similarities among S enteritidis from human beings and eggs, as determined on the basis of plasmid profiles and antibiotic susceptibility patterns, which may implicate eggs as one of the potential sources for infection of human beings. In addition, periodic monitoring of a substantial number of Salmonella isolates to detect drug resistance may be a prudent practice for use in revising the list of antimicrobial agents commonly used in human beings and other animals.

Purification and partial amino acid sequence of curvaticin FS47, a heat-stable bacteriocin produced by Lactobacillus curvatus FS47.

Curvaticin FS47, a bacteriocin produced by Lactobacillus curvatus FS47, is inhibitory to Listeria monocytogenes, as well as Lactobacillus, Pediococcus, Enterococcus, and Bacillus spp. The bacteriocin was purified by 40% ammonium sulfate precipitation, solid-phase extraction, and reversed-phase high-pressure liquid chromatography. Purified curvaticin FS47 was determined to be 4.07 kDa by mass spectrometry and was partially sequenced. Thirty-one N-terminal amino acids were identified; the curvaticin FS47 protein sequence did not show homology to the pediocin-like group of bacteriocins.

Detection, identification and characterization of bacteriocin-producing lactic acid bacteria from retail food products.

Forty bacteriocin-producing (Bac+) lactic acid bacteria (LAB) were isolated from food samples purchased from retail supermarkets and local farms. Of the 40 Bac+ isolates, 18 were isolated from 85 food samples by enrichment (21% isolation rate) whereas eight were obtained from 63 samples by direct plating (13% isolation rate). By direct plating, Bac+ LAB were detected at levels up to 2.4 x 10(5) cfu/g in ready-to-eat meats. The Bac+ isolates were identified by carbohydrate fermentation patterns, SDS-PAGE protein patterns, and other biochemical characteristics; SDS-PAGE proved invaluable in identifying strains that could not be identified by other means. Differential inhibitory spectra against indicator microorganisms assisted in the identification of 19 unique Bac+ isolates. Bac+ LAB included Enterococcus faecalis, Lactobacillus curvatus, Lb. delbrueckii, Lb. plantarum, Lactococcus lactis, and Pediococcus acidilactici. Lb. curvatus (four strains) and Lc. lactis (nine strains) were the only isolates inhibitory to foodborne pathogens including Listeria monocytogenes, Bacillus cereus, Clostridium perfringens and Staphylococcus aureus. Some Lc. lactis isolates inhibited as many as nine Gram-positive genera. Lb. curvatus FS47 and FS65 grew to high cell densities and produced bacteriocin at 6 degrees C; however, Lc. lactis FS56 produced greater levels of bacteriocin at lower cell densities. The high incidence of Bac+ LAB detected in retail foods indicates that the public is consuming a wide variety of Bac+ LAB that occur as natural contaminants. These data suggest a greater role for bacteriocins as biopreservatives in food.

Purification and Partial Characterization of Lacticin 481, a Lanthionine-Containing Bacteriocin Produced by Lactococcus lactis subsp. lactis CNRZ 481.

Lacticin 481, a bacteriocin produced during the growth of Lactococcus lactis subsp. lactis CNRZ 481, was purified sequentially by ammonium sulfate precipitation, gel filtration, and preparative and analytical reversed-phase high-pressure liquid chromatography. Ammonium sulfate precipitations resulted in a 455-fold increase in total lacticin 481 activity. The entire purification protocol led to a 107, 506-fold increase in the specific activity of lacticin 481. On the basis of its electrophoretic pattern in sodium dodecyl sulfate-polyacrylamide gels, lacticin 481 appeared as a single peptide band of 1.7 kDa. However, dimers of 3.4 kDa also exhibiting lacticin activity were detected. Derivatives of the lacticin-producing strain which did not produce lacticin 481 (Bac) were sensitive to this bacteriocin (Bac) and failed to produce the 1.7-kDa band. Amino acid composition analysis of purified lacticin 481 revealed the presence of lanthionine residues, suggesting that lacticin 481 is a member of the lantibiotic family of antimicrobial peptides. Seven residues (K G G S G V I) were sequenced from the N-terminal portion of lacticin 481, and these did not shown any homology with nisin or other known bacteriocin sequences.

Cloning, phenotypic expression, and DNA sequence of the gene for lactacin F, an antimicrobial peptide produced by Lactobacillus spp.

Lactacin F is a heat-stable bacteriocin produced by Lactobacillus acidophilus 11088. A 63-mer oligonucleotide probe deduced from the N-terminal lactacin F amino acid sequence was used to clone the putative laf structural gene from plasmid DNA of a lactacin F-producing transconjugant, L. acidophilus T143. One clone, NCK360, harbored a recombinant plasmid, pTRK160, which contained a 2.2-kb EcoRI fragment of the size expected from hybridization experiments. An Escherichia coli-L. acidophilus shuttle vector was constructed, and a subclone (pTRK162) containing the 2.2-kb EcoRI fragment was introduced by electroporation into two lactacin F-negative strains, L. acidophilus 89 and 88-C. Lactobacillus transformants containing pTRK162 expressed lactacin F activity and immunity. Bacteriocin produced by the transformants exhibited an inhibitory spectrum and heat stability identical to those of the wild-type bacteriocin. An 873-bp region of the 2.2-kb fragment was sequenced by using a 20-mer degenerate lactacin F-specific primer to initiate sequencing from within the lactacin F structural gene. Analysis of the resulting sequence identified an open reading frame which could encode a protein of 75 amino acids. The 25 N-terminal amino acids for lactacin F were identified within the open reading frame along with an N-terminal extension, possibly a signal sequence. The lactacin F N-terminal sequence, through the remainder of the open reading frame (57 amino acids; 6.3 kDa), correlated extremely well with composition analyses of purified lactacin F which also predicted a size of 51 to 56 amino acid residues. Molecular characterization of lactacin F identified a small hydrophobic peptide that may be representative of a common bacteriocin class in lactic acid bacteria.

Purification and partial characterization of lactacin F, a bacteriocin produced by Lactobacillus acidophilus 11088.

Lactacin F, a bacteriocin produced by Lactobacillus acidophilus 11088 (NCK88), was purified and characterized. Lactacin F is heat stable, proteinaceous, and inhibitory to other lactobacilli as well as Enterococcus faecalis. The bacteriocin was isolated as a floating pellet from culture supernatants brought to 35 to 40% saturation with ammonium sulfate. Native lactacin F was sized at approximately 180 kDa by gel filtration. Column fractions having lactacin F activity were examined by electron microscopy and contained micelle-like globular particles. Purification by ammonium sulfate precipitation, gel filtration, and high-performance liquid chromatography resulted in a 474-fold increase in specific activity of lactacin F. The purified bacteriocin was identified as a 2.5-kDa peptide by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The lactacin F peptide retained activity after extraction from SDS-PAGE gel slices, confirming the identity of the 2.5-kDa peptide. Variants of NCK88 that failed to exhibit lactacin F activity did not produce the 2.5-kDa band. Sequence analysis of purified lactacin F identified 25 N-terminal amino acids containing an arginine residue at the N terminus. Composition analysis indicates that lactacin F may contain as many as 56 amino acid residues.

Application of electroporation for transfer of plasmid DNA to Lactobacillus, Lactococcus, Leuconostoc, Listeria, Pediococcus, Bacillus, Staphylococcus, Enterococcus and Propionibacterium.

Plasmid DNA was introduced by electroporation into Bacillus, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Listeria, Pediococcus, Propionibacterium and Staphylococcus as an alternative to competent-cell or protoplast transformation. Plasmid-containing transformants were recovered in these recipients at frequencies ranging from 10(1) to 10(5) transformants micrograms-1 of pGK12. Several parameters of the protocol, including DNA concentration, voltage, plating regimen and electroporation buffers were evaluated to determine conditions that improved transformation frequencies for Lactobacillus acidophilus. Using optimized conditions, the following plasmids were introduced into L. acidophilus: pAMB1, pC194, pGB354, pGKV1, pSA3, pTRK13, pTV1 and pVA797. The ability to transfer plasmid DNA via eletroporation will greatly facilitate the application of recombinant DNA methodology and transposon technology to Gram-positive bacteria for cloning and analysis of significant genes.

Conjugal Transfer of Plasmid-Encoded Determinants for Bacteriocin Production and Immunity in Lactobacillus acidophilus 88.

Lactobacillus acidophilus 88 produced a bacteriocin, designated lactacin F, that demonstrated inhibitory activity toward L. acidophilus 6032, L. lactis 970, L. helveticus 87, L. bulgaricus 1489, L. leichmanii 4797, L. fermentum 1750, and Streptococcus faecalis 19433. Production of lactacin F was pH dependent and could be maximized in MRS broth cultures maintained at pH 7.0. Lactacin F was heat stable and sensitive to ficin, proteinase K, trypsin, and Bacillus subtilis protease. L. acidophilus 88 harbored plasmids of 4 and 27 megadaltons. Variants of L. acidophilus 88 which were deficient in lactacin F production (Laf) and lactacin F immunity (Laf) retained the two resident plasmids. A Laf Laf derivative, L. acidophilus 89, was used as a recipient in agar surface mating experiments with L. acidophilus 88 (Laf Laf). Two types of Laf Laf transconjugants were recovered. One type (T-E) had acquired two plasmids of 68 (pPM68) and 52 (pPM52) megadaltons that were not detected in either the conjugal donor or the other type of Laf Laf transconjugants (T-89). Laf and Laf were unstable in the plasmid-bearing transconjugant. Plasmid analysis of Laf Laf variants revealed that pPM52 and pPM68 were cured with loss of Laf and Laf. Bacteriocin production and immunity phenotypes were genetically stable in Laf Laf transconjugants not harboring pPM52 and pPM68, suggesting chromosomal integration of the transferred determinants. The data demonstrated intragenic conjugation in L. acidophilus and provided direct evidence for involvement of transient plasmid determinants in Laf and Laf.