Effect of a bacteriocin produced by Pediococcus acidilactici against Listeria monocytogenes and Clostridium perfringens on Spanish raw meat

The inhibitory effect of a bacteriocin, produced by Pediococcus acidilactici, against Listeria monocytogenes and Clostridium perfringens on Spanish raw meat surface, was evaluated by in situ assays. Samples were incubated with the bacteriocin and then with a culture of the pathogenic bacteria. The treatment with 500, 1000 or 5000 bacteriocin units/ml (BU/ml) reduced the counts of L. monocytogenes after storage at 15°C during 72h by 1, 2 or 3 log cycles and with 1000 or 5000 BU/ml after storage at 4°C during 21 days by 2.5 or 3.5 log cycles, respectively, compared to the control. With C. perfringens a bacteriostatic effect could be observed.     

Production of salami from beef, horse, mutton, Blesbok (Damaliscus dorcas phillipsi) and Springbok (Antidorcas marsupialis) with bacteriocinogenic strains of Lactobacillus plantarum and Lactobacillus curvatus

Lactobacillus plantarum 423, producer of bacteriocin 423, Lactobacillus curvatus DF38, producer of curvacin DF38, and a bacteriocin-negative mutant of L. plantarum 423 (423m) were evaluated as starter cultures in the production of salami from beef, horse, mutton, Blesbok (Damaliscus dorcas phillipsi) and Springbok (Antidorcas marsupialis). Growth of L. plantarum 423 and L. curvatus DF38 was best supported in Blesbok salami, as revealed by the highest growth rate during sweating, cold smoking and maturation, and final cell numbers after 70 days (1 × 10⁸ and 5 × 10⁷ cfu/g, respectively). Growth of Listeria innocua was the best suppressed in Blesbok salami fermented with L. plantarum 423 and L. curvatus DF38. Growth of L. innocua in horse salami was best suppressed when fermented with L. curvatus DF38. The final pH of salami fermented with L. plantarum 423 and L. plantarum 423m was slightly lower (4.4) compared to the pH of salami produced with L. curvatus DF38 (pH 4.7). No significant differences (P > 0.05) were recorded by a trained sensory taste panel amongst the three starter cultures regarding colour and venison like aroma. Horse, Blesbok and Springbok salami were rated significantly higher (P ? 0.05) in salami flavour than mutton salami, which was rated the lowest for this attribute. Blesbok salami was rated the highest for sour meat aroma, while beef salami was rated the lowest. Springbok salami was rated the highest in terms of oily mouth feel. Beef salami had the most compact structure and horse salami the softest structure of all meat types fermented. In general, salami produced with L. plantarum 423 yielded the best sour meat aroma, colour, texture, venison like flavour, sour meat flavour and oily mouthfeel and is considered superior to the L. plantarum mutant (strain 423m) and L. curvatus DF38.     

Biochemical,antimicrobial and molecular characterization of a noncytotoxic bacteriocin produced by Lactobacillus plantarum ST71KS

Lactobacillus (Lb.) plantarum ST71KS was isolated from homemade goat feta cheese and identified using biochemical and molecular biology techniques. As shown by Tricine-SDS-PAGE, this lactic acid bacterium produces a bacteriocin (ST71KS) with an estimated molecular weight of 5.0 kDa. Bacteriocin ST71KS was not affected by the presence of α-amylase, catalase and remained stable in a wide range of pH and after treatment with Triton X-100, Triton X-114, Tween 20, Tween 80, NaCl, SDS, urea and EDTA. This bacteriocin also remained active after being heated at 100 °C for 2 h and even after 20 min at 121 °C; however, it was inactivated by proteolitic enzymes. Production of bacteriocin ST71KS reached 6400 AU/mL during stationary growth phase of Lb. plantarum cultivated in MRS at 30 °C and 37 °C. Bacteriocin ST71KS displayed a bactericidal effect against Listeria monocytogenes strains 603 and 607 and did not adsorb to the producer cells. Lb. plantarum ST71KS harbors two bacteriocin genes with homology to plantaricin S and pediocin PA-1. These characteristics indicate that bacteriocin ST71KS is a class IIa bacteriocin. The peptide presented no toxic effect when tested in vitro with kidney Vero cells, indicating safe technological application to control L. monocytogenes in foods.     

Effects of curing sodium nitrite additive and natural meat fat on growth control of Listeria monocytogenes by the bacteriocin-producing Lactobacillus curvatus strain CWBI-B28

In realistic model meat systems, the separate and combined effects of fat content and sodium nitrite on the antilisterial activity of the bacteriocin of Lactobacillus curvatus CWBI-B28 were studied. In laboratory fermentations where Listeria monocytogenes was co-cultured at 4 °C with bacteriocin-producing CWBI-B28 in lean pork meat (fat content: 13%) without added nitrite, a strong antilisterial effect was observed after one week. The effect was maintained for an additional week, after which a slight and very gradual rebound was observed. Both added nitrite (20 ppm) and a high-fat content (43%) were found to antagonise this antilisterial effect, the Listeria cfu count reached after six weeks being 200 times as high in high-fat meat with added nitrite than in lean meat without nitrite. This antagonism could not be attributed to slower growth of the bacteriocin-producing strain, since CWBI-B28 grew optimally in fat-rich meat with 20 ppm sodium nitrite. Bacteriocin activity was also measured in the samples. The observed activity levels are discussed in relation to the degree of antilisterial protection conferred.     

Coculture-inducible bacteriocin activity of Lactobacillus plantarum strain J23 isolated from grape must

Detection and characterization of bacteriocin production by Lactobacillus plantarum strain J23, recovered from a grape must sample in Spain, have been carried out. Bacteriocin activity was degraded by proteolytic enzymes (trypsin, alfa-chymotrypsin, papaine, protease, proteinase K and acid proteases), and it was stable at high temperatures (121 degrees C, 20min), in a wide range of pH (1-12), and after treatment with organic solvents. L. plantarum J23 showed antimicrobial activity against Oenococcus oeni, and a range of Lactobacillus and Pediococcus species. Bacteriocin production was detected in liquid media only when J23 was cocultivated with some inducing bacteria, and induction took place when intact cells or 55 degrees C heated cells of the inducer were cocultivated with J23, but not with their autoclaved cells. Bacteriocin activity of J23 was not induced by high initial J23 inocula, and it was detected in cocultures during the exponential phase. The presence of ethanol or acidic pH in the media reduced bacteriocin production in the cocultures of J23 with the inducing bacteria. The presence of plantaricin-related plnEF and plnJ genes was detected by PCR and sequencing. Nevertheless, negative results were obtained for plnA, plnK, plNC8, plS and plW genes.     

Combined use of bacteriocin‐producing strains to control Listeria monocytogenes regrowth in raw pork meat

Avoiding the presence of Listeria in meat and dairy products is a major challenge for the food industry. In this work, a Lactobacillus curvatus strain producing the bacteriocin sakacin P and a Pediococcus acidilactici strain producing another bacteriocin, pediocin AcH, were used as starter cultures under laboratory conditions in a Listeria‐seeded raw‐pork‐meat matrix, which was then stored for 6 weeks at 4 °C. At 1 week intervals during the storage period, the antilisterial activity was evaluated. When either strain was added alone, the Listeria monocytogenes cfu count initially dropped from 10² cfu g^?1 to an undetectable level by the end of week 1 or 2, but this was followed by a rebound (regrowth) 1 week later. When both strains were added together to the meat matrix, rebound was delayed, Listeria remaining undetected from the end of week 1 to the end of week 5. A rebound was observed 6 weeks post‐inoculation, but fewer than 10 cfu g^?1 were counted. The use of more than one bacteriocin‐producing strain may thus overcome some of the problems limiting the effectiveness of bacteriocins in food systems.     

Satureja horvatii essential oil: In vitro antimicrobial and antiradical properties and in situ control of Listeria monocytogenes in pork meat

The dominant compounds in Satureja horvatii oil were p-cymene (33.14%), thymol (26.11%) and thymol methyl ether (15.08%). The minimum inhibitory concentration (MIC) varied from 0.03 to 0.57 mg/mL for bacteria, and from 0.56 to 2.23 mg/mL for yeast strains, while minimum bactericidal/yeast-cidal concentration (MBC/MYC) varied from 0.07 to 1.15 mg/mL and 1.11 to 5.57 mg/mL for bacteria and yeasts, respectively. The antiradical potential of the essential oil was evaluated using hydroxyl radical (•OH) generated in Fenton reaction. The meat preserving potential of essential oil from Satureja horvatii was investigated against L. monocytogenes. Essential oil successfully inhibited development of L. monocytogenes in pork meat. Sensorial evaluation on flavor and color of meat was performed. The color and flavor of meat treated with essential oil improved after 4 days of storage. S. horvatii essential oil can act as a potent inhibitor of food spoiling microorganisms, in meat products and also can be a useful source of natural antioxidants.     

Mixed species biofilms of Listeria monocytogenes and Lactobacillus plantarum show enhanced resistance to benzalkonium chloride and peracetic acid

We investigated the formation of single and mixed species biofilms of Listeria monocytogenes strains EGD-e and LR-991, with Lactobacillus plantarum WCFS1 as secondary species, and their resistance to the disinfectants benzalkonium chloride and peracetic acid. Modulation of growth, biofilm formation, and biofilm composition was achieved by addition of manganese sulfate and/or glucose to the BHI medium. Composition analyses of the mixed species biofilms using plate counts and fluorescence microscopy with dual fluorophores showed that mixed species biofilms were formed in BHI (total count, 8-9 log₁₀ cfu/well) and that they contained 1-2 log₁₀ cfu/well more L. monocytogenes than L. plantarum cells. Addition of manganese sulfate resulted in equal numbers of both species (total count, 8 log₁₀ cfu/well) in the mixed species biofilm, while manganese sulfate in combination with glucose, resulted in 1-2 log₁₀ more L. plantarum than L. monocytogenes cells (total count, 9 log₁₀ cfu/well). Corresponding single species biofilms of L. monocytogenes and L. plantarum contained up to 9 log₁₀ cfu/well. Subsequent disinfection treatments showed mixed species biofilms to be more resistant to treatments with the selected disinfectants. In BHI with additional manganese sulfate, both L. monocytogenes strains and L. plantarum grown in the mixed species biofilm showed less than 2 log₁₀ cfu/well inactivation after exposure for 15 min to 100 μg/ml benzalkonium chloride, while single species biofilms of both L. monocytogenes strains showed 4.5 log₁₀ cfu/well inactivation and single species biofilms of L. plantarum showed 3.3 log₁₀ cfu/well inactivation. Our results indicate that L. monocytogenes and L. plantarum mixed species biofilms can be more resistant to disinfection treatments than single species biofilms.     

Characterization of Lactobacillus isolates from fermented olives and their bacteriocin gene profiles

Near one hundred isolates of Lactobacillus paraplantarum, Lactobacillus pentosus and Lactobacillus plantarum from table olives were studied. Strains were genotyped by rep-PCR. Although the technique failed to differentiate some isolates at the species level, it proved a robust and easy procedure that could be useful for distinguishing between related strains of L. paraplantarum, L. pentosus and L. plantarum from a large pool of unrelated strains of these species. A PCR-based screening revealed the presence of the plantaricin encoding genes plnA, plnB, plnC, plnD, plnE/F, plnF, plnI, plnJ, plnK, plnG and plnN in most isolates of the three species. Sequences of bacteriocin genes present in L. paraplantarum and L. pentosus were homologous to L. plantarum genes. Through a discriminating analysis of the bacteriocin gene profiles, it was possible to establish a relationship between the origin of isolation and the LAB isolates, regardless of species.     

Purification and characterization of a new bacteriocin active against Campylobacter produced by Lactobacillus salivarius SMXD51

Strain SMXD51, isolated from chicken ceca and identified as Lactobacillus salivarius, produced a component that inhibits the growth of Gram-positive and Gram-negative bacteria and especially Campylobacter jejuni. The active peptide from the cell-free supernatant of Lb. salivarius SMXD51 was purified in three steps: (i) precipitation with 80% saturated ammonium sulfate, (ii) elution on a reversed phase SPE UPTI-CLEAN cartridge using different concentrations of acetonitrile, (iii) final purification by reversed phase HPLC on a C₁₈ column. The mode of action of this peptide of 5383.2 Da was identified as bactericidal, and its amino acid composition was established. This new bacteriocin SMXD51 appears potentially very useful to reduce Campylobacter in poultry prior to processing.     

High prevalence,low counts and uncommon serotypes of Listeria monocytogenes in linguiça, a Brazilian fresh pork sausage

Linguiça is a highly popular and appreciated pork product in Brazil, frequently consumed undercooked. Aiming at collection of data for a future risk assessment, this study evaluated the prevalence and counts of Listeria monocytogenes in linguiça samples collected at retail level in Sao Paulo, SP, Brazil. ISO methods were used for detection and enumeration of the pathogen (11290-1 and 11290-2, respectively). Isolates were submitted to Simplex-PCR for hlyA gene and those with biochemical features of L. monocytogenes and hlyA positive were serotyped using a Multiplex PCR. Ninety percent of the samples were positive for Listeria spp., and L. monocytogenes was detected in 42% of the samples, with counts below 10² CFU/g in all samples. A prevalence of uncommon serotypes 4a and 4c was observed.     

Microbial heat resistance of Listeria monocytogenes and the impact on ready-to-eat meat quality after post-package pasteurization

Several methods using bactericides, hydrostatic pressure, and post-package pasteurization technologies to control Listeria monocytogenes (LM) in ready-to-eat meats have been attempted. In addition to controlling LM contamination, any newly developed technology must have minimal effects on organoleptic properties. The objectives of this study were to: (1) determine the heat resistance of LM in two brands (A and B) of bologna differing in formulations, and, (2) evaluate the effects of post-package pasteurization on product quality. Fat content did not affect LM heat resistance in bologna at 55, 60, and 65 °C; however, Brand B bologna had a numerically lower inactivation rate. Microbial heat resistance differed (P < 0.05) with changes in pasteurization temperature. Time and temperature affected (P < 0.05) cook-loss and L^∗ Hunter color value for both bologna brands. These data show that post-package pasteurization is effective but suggest that meat formulations may need modification to prevent development of negative quality characteristics.     

Expression of Lactobacillus pentosus B96 bacteriocin genes under saline stress

This research studied the influence of sodium chloride on bacteriocin activity of table olives’ strain Lactobacillus pentosus B96. The strain was cultured in MRS under different NaCl concentrations (0, 4, 6 and 8%, in w/v). In MRS, maximum bacteriocin activity was achieved 9 h later. A medium containing 4 or 6% NaCl (w/v) increased the total bioactivity of the strain and an 8% NaCl reduced it. Real-time PCR was used to monitor the genetic expression of the bacteriocin genes plnA, plnB, plnC, plnE/F, plnJ, plnK, plnN and plantaricin S. Cultured in MRS, plantaricin S reached its maximum expression during the lag phase while plnE/F expresses during the exponential phase. The presence of sodium chloride in the medium moved the maximum expression of plantaricin S to the stationary phase, independently of the concentration. 4% (w/v) of NaCl didn’t affect the expression pattern of plnE/F while promotes the expression of plnN during both the lag and the exponential phases. More sodium chloride, 6% (w/v) maintained the expression of plnN in the pag phase but not in the exponential and moved plnE/F expression to the stationary phase. Plantaricin S, plnE/F and plnN over-expressed during the stationary phase in the higher sodium chloride concentration assayed, 8% (w/v). The relative expression level of plsA was 1000-fold higher than that of the plnE/F and plnN genes and even the ldhD constitutive gene used. Under our conditions, expression of plnA, plnB, plnC, plnJ and plnK genes was not observed.     

Quantifying Listeria monocytogenes prevalence and concentration in minced pork meat and estimating performance of three culture media from presence/absence microbiological testing using a deterministic and stochastic approach

Listeria monocytogenes poses a serious threat to public health, and the majority of cases of human listeriosis are associated with contaminated food. Reliable microbiological testing is needed for effective pathogen control by food industry and competent authorities. The aims of this work were to estimate the prevalence and concentration of L. monocytogenes in minced pork meat by the application of a Bayesian modeling approach, and also to determine the performance of three culture media commonly used for detecting L. monocytogenes in foods from a deterministic and stochastic perspective. Samples (n = 100) collected from local markets were tested for L. monocytogenes using in parallel the PALCAM, ALOA and RAPID'L.mono selective media according to ISO 11290-1:1996 and 11290-2:1998 methods. Presence of the pathogen was confirmed by conducting biochemical and molecular tests. Independent experiments (n = 10) for model validation purposes were performed. Performance attributes were calculated from the presence–absence microbiological test results by combining the results obtained from the culture media and confirmative tests. Dirichlet distribution, the multivariate expression of a Beta distribution, was used to analyze the performance data from a stochastic perspective. No L. monocytogenes was enumerated by direct-plating (<10 CFU/g), though the pathogen was detected in 22% of the samples. L. monocytogenes concentration was estimated at 14–17 CFU/kg. Validation showed good agreement between observed and predicted prevalence (error = −2.17%). The results showed that all media were best at ruling in L. monocytogenes presence than ruling it out. Sensitivity and specificity varied depending on the culture-dependent method. None of the culture media was perfect in detecting L. monocytogenes in minced pork meat alone. The use of at least two culture media in parallel enhanced the efficiency of L. monocytogenes detection. Bayesian modeling may reduce the time needed to draw conclusions regarding L. monocytogenes presence and the uncertainty of the results obtained. Furthermore, the problem of observing zero counts may be overcome by applying Bayesian analysis, making the determination of a test performance feasible.     

Factors affecting the adsorption of Lactobacillus plantarum bacteriocin bacST8KF to Enterococcus faecalis and Listeria innocua

Bacteriocin ST8KF (bacST8KF), produced by Lactobacillus plantarum ST8KF isolated from kefir, inhibits the growth of Enterococcus faecalis E88, Lactobacillus casei LHS, Lactobacillus curvatus DF38, Lactobacillus sakei DSM 20017^T, Lactobacillus salivarius 241 and Listeria innocua LMG 13568. Eighty per cent of bacST8KF adsorbed to La. casei LHS and La. sakei DSM 20017^T. However, the same level of adsorption was recorded to bacST8KF-resistant Lactobacillus paraplantarum ATCC 700211^T and Streptococcus caprinus ATCC 700066, suggesting that adherence to the cell surface occurs at random and not always at specific receptor sites. Adsorption of bacST8KF is affected by inorganic salts, acids, surfactants, alcohols, temperature and pH, as observed for E. faecalis E88 and Li. innocua LMG 13568. The target of bacST8KF is the cytoplasmic membrane, as indicated by changes in cell morphology and leakage of DNA and β-galactosidase from cells of E. faecalis E88 and Li. innocua LMG 13568 treated with bacST8KF. However, the mode of activity against E. faecalis E88 and Li. innocua LMG 13568 is bacteriostatic, as concluded from slight increases in optical density levels after a few hours in the presence of bacST8KF. Activity of bacST8KF doubled (from 12 800 AU mL⁻¹ to 25 600 AU mL⁻¹) when strain ST8KF was grown in the presence of Li. innocua LMG 13568, which may indicate some sort of quorum sensing.     

Bacteriocinogenic Lactobacillus plantarum ST16Pa isolated from papaya (Carica papaya) — From isolation to application: Characterization of a bacteriocin

Strain ST16PA, isolated from papaya was identified as Lactobacillus plantarum based on biochemical tests, PCR with species-specific primers and 16S rDNA sequencing. L. plantarum ST16PA produces a 6.5 kDa bacteriocin, active against different species from genera Enterobacter, Enterococcus, Lactobacillus, Pseudomonas, Streptococcus and Staphylococcus and different serotypes of Listeria spp. The peptide is inactivated by proteolytic enzymes, but not when treated with ??-amylase, catalase, lipase, Triton X-100, SDS, Tween 20, Tween 80, urea, NaCl and EDTA. However, presence of 1% Triton X-114 deactivates the bacteriocin. No change in activity was recorded after 2 h at pH values between 2.0 and 12.0, and after treatment at 100 °C for 120 min or 121 °C for 20 min. The mode of activity against Lactobacillus sakei ATCC 15521, Enterococcus faecalis ATCC 19443 and Listeria innocua 2030C was bactericidal, resulting in cell lysis and enzyme-leakage. No significant differences in cell growth and bacteriocin production were observed when strain ST16Pa was cultured in MRS broth at 26 °C and 30 °C for 24 h (25 600 AU/ml). However, even though strain ST16PA grows well in MRS broth at 15 °C and 37 °C, a reduction of bacteriocin production was observed (400 AU/ml and 1600 AU/ml, respectively). In addition, effect of MRS medium components, different initial pH and additions of glycerol or vitamins to the media on bacteriocin ST16Pa production was studied.Peptide ST16PA adsorbs (400 AU/ml) to producer cells. However, bacteriocin ST16Pa was adsorbed at 50% to cells of L. innocua 2030C and at 75% to L. sakei ATCC 15521 and E. faecalis ATCC 19433 when experiments were conducted at 30 °C and pH 6.5. Adsorption of bacteriocin ST16Pa to target cells at different temperatures, pH and in presence of potassium sorbate, sodium nitrate, sodium chloride, ascorbic acid, Tween 80 and Tween 20 were also studied. To the best of our knowledge, this is the first report on detection of L. plantarum in papaya.     

Diversity of stress tolerance in Lactobacillus plantarum, Lactobacillus pentosus and Lactobacillus paraplantarum: A multivariate screening study

Sixty-three strains of the taxonomically related species Lactobacillus plantarum subsp. plantarum, L. plantarum subsp. argentoratensis, L. paraplantarum and L. pentosus isolated from sourdoughs and other food and non-food sources and 14 strains of other members of the genus Lactobacillus were screened for their tolerance of acid, alkaline, heat, oxidative, osmotic, detergent and starvation stresses in order to evaluate the diversity of stress response. Most strains of the L. plantarum group were highly tolerant of acid, alkaline and osmotic stress and highly sensitive to detergent stress, while a larger diversity was found for other stress. Multivariate analysis allowed grouping the strains in clusters with similar response patterns. Stress response patterns in the L. plantarum group were similar to those of species of the L. casei/L. paracasei group but clearly different from those of other mesophilic Lactobacillus. No relationship was found between grouping obtained on the basis of stress response patterns and by genotypic fingerprinting (rep-PCR), nor with the taxonomic position or isolation source of the strains. Further experiments with selected strains showed that exponential phase cells were generally but not always more sensitive than stationary phase cells. The ability to grow under stressful conditions showed a slightly better correlation with the ecological conditions prevailing in the isolation niches of the strains.This study will be the basis for further investigations to identify and exploit the basis of diversity in the stress response of lactic acid bacteria.     

Strain dependent expression of stress response and virulence genes of Listeria monocytogenes in meat juices as determined by microarray

A subgenomic array, encompassing 54 probes targeting genes responsible for virulence, adhesion and stress response in Listeria monocytogenes, was used in order to study their expression in food systems. RNA extracted from L. monocytogenes inoculated in BHI and in situ (i.e. in minced meat and fermented sausage juices) and incubated at 4 °C, was hybridized on the array and the results obtained were compared in order to understand the effect that the food juice has on the expression. Three different strains of L. monocytogenes were tested, in order to determine the effect of the strain provenience. As determined by cluster analysis, each strain behaved in a different way when inoculated in food juices. The goal was to respond to acidic and osmotic stresses encountered in the food, particularly in the fermented sausage juice. No differences in the expression profile between the three strains were observed, when they were inoculated in BHI. On the other hand, in the meat and sausage juices, the iap, gadC and gadE genes, together with different internalin encoding genes, were significantly differentially expressed in the three strains.     

Selective enumeration of Lactobacillus delbrueckii ssp. bulgaricus,Streptococcus thermophilus,Lactobacillus acidophilus,bifidobacteria, Lactobacillus casei,Lactobacillus rhamnosus,and propionibacteria

Nineteen bacteriological media were evaluated to assess their suitability to selectively enumerate Lactobacillus delbrueckii ssp. bulgaricus, Streptococcus thermophilus, Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus acidophilus, bifidobacteria, and propionibacteria. Bacteriological media evaluated included Streptococcus thermophilus agar, pH modified MRS agar, MRS-vancomycine agar, MRS-bile agar, MRS-NaCl agar, MRS-lithium chloride agar, MRS-NNLP (nalidixic acid, neomycin sulfate, lithium chloride and paramomycine sulfate) agar, reinforced clostridial agar, sugar-based (such as maltose, galactose, sorbitol, manitol, esculin) media, sodium lactate agar, arabinose agar, raffinose agar, xylose agar, and L. casei agar. Incubations were carried out under aerobic and anaerobic conditions at 27, 30, 37, 43, and 45 degrees C for 24, 72 h, and 7 to 9 d. S. thermophilus agar and aerobic incubation at 37 degrees C for 24 h were suitable for S. thermophilus. L. delbrueckii ssp. bulgaricus could be enumerated using MRS agar (pH 4.58 or pH 5.20) and under anaerobic incubation at 45 degrees C for 72 h. MRS-vancomycine agar and anaerobic incubation at 43 degrees C for 72 h were suitable to enumerate L. rhamnosus. MRS-vancomycine agar and anaerobic incubation at 37 degrees C for 72 h were selective for L. casei. To estimate the counts of L. casei by subtraction method, counts of L. rhamnosus on MRS-vancomycine agar at 43 degrees C for 72 h under anaerobic incubation could be subtracted from total counts of L. casei and L. rhamnosus enumerated on MRS-vancomycine agar at 37 degrees C for 72 h under anaerobic incubation. L. acidophilus could be enumerated using MRS-agar at 43 degrees C for 72 h or Basal agar-maltose agar at 43 degrees C for 72 h or BA-sorbitol agar at 37 degrees C for 72 h, under anaerobic incubation. Bifidobacteria could be enumerated on MRS-NNLP agar under anaerobic incubation at 37 degrees C for 72 h. Propionibacteria could be enumerated on sodium lactate agar under anaerobic incubation at 30 degrees C for 7 to 9 d. A subtraction method was most suitable for counting propionibacteria in the presence of other lactic acid bacteria from a product. For this method, counts of lactic bacteria at d 3 on sodium lactate agar under anaerobic incubation at 30 degrees C were subtracted from counts at d 7 of lactic bacteria and propionibacteria.