Factors affecting growth and lipase production by meat lactobacilli strains and Brochothrix thermosphacta

Lipolytic activity of lactobacilli strains and Brochothrix thermosphacta was cell-related; no significant activity was found in the supernatant fluids. Most lipase was produced during the logarithmic phase of growth and was greatly affected by growth conditions. The optimal temperatures for growth and lipase production were respectively 24 degrees C for B. thermosphacta and 30 degrees C for lactobacilli. For all strains, an initial pH of around 7.0 for the medium and low glucose concentration stimulated lipase production. Tributyrin inhibited both growth and lipase production at a concentration of 0.1% for B. thermosphacta or 1% for lactobacilli. Butyric acid (0.1%) and anaerobic culture inhibited lipase production by B. thermosphacta while these two factors had no effect on enzyme production by lactobacilli.     

Cell location and partial characterization of Brochothrix thermosphacta and Lactobacillus curvatus lipases

The lipases of Brochothrix thermosphacta and Lactobacillus curvatus were in the soluble fraction of the cell and not membrane- or wall-bound. Their optimal activities were around pH 6mD5 and at 37°C. Enzymes were stable between 4 and 30°C and at freezing temperatures (— 20°C and —60°C). The lipase of Lact. curvatus was stable in the pH range of 4mD0 to 11mD0, whereas that of B. thermosphacta was denatured at pH lower than 5mD0 and greater than 9mD0. Among triglycerides tested, maximal activity was found with tributyrin and hydrolysis of other triglycerides decreased as the length of fatty acid increased.     

Evaluation of the lipolytic activity of starter cultures for meat fermentation purposes

A modified pork fat based agar method was specially developed to determine the lipolytic activity of starter strains used for meat fermentations. The lipolytic ability of strains of lactobacilli, pediococci, staphylococci and micrococci, was examined on agar plates using different substrates and in a lipid broth model system. The screening results indicate that lipase activity was confined to strains of Staphylococcus and Micrococcus . None of the lactic acid bacteria tested showed lipolytic activity. A strain of Staphylococcus xylosus , which displayed high lipolytic activity during the screening process was examined to find its optimum conditions for lipase activity regarding pH, temperature and NaCl concentration. The lipolytic activity of seven bacterial strains was determined under optimum conditions (0% salt, pH 7 and 30 °C) and sausage conditions (3·5% salt, pH 5 and 20 °C) using fat buffer model systems. High lipolytic activity was determined for all seven strains under optimal conditions, whereas no activity was detected under fermented sausage conditions.     

Antimicrobial activity of lactobacilli: preliminary characterization and optimization of production of acidocin B, a novel bacteriocin produced by Lactobacillus acidophilus M46

Approximately 1000 lactobacillus strains were isolated and screened for the production of antimicrobial activity, using a target panel of spoilage organisms and pathogens. Only eight positive strains were found; two of these were studied in more detail. Lactobacillus salivarius M7 produces the new broad spectrum bacteriocin salivaricin B which inhibits the growth of Listeria monocytogenes, Bacillus cereus, Brochothrix thermosphacta, Enterococcus faecalis and many lactobacilli. A new atypical bacteriocin produced by Lact. acidophilus M46, acidocin B, combines the inhibition of Clostridium sporogenes with a very narrow activity spectrum within the genus Lactobacillus and was selected for further characterization. Acidocin B is sensitive to trypsin, heat-stable (80°C for 20 min) and can be extracted from the culture supernatant fluid with butanol. Native acidocin B occurs as a large molecular weight complex (100 kDa), while with SDS-PAGE the partly purified activity migrates as a peptide of 2·4 kDa. Optimization of the cultivation conditions resulted in an eightfold increase of the amount of acidocin B produced during growth. Growth is not necessary for acidocin B production; washed producer cells can synthesize the bacteriocin in a chemically defined production medium. The application potential of acidocin B is discussed.     

Minimum inhibitory concentration of smoke wood extracts against spoilage and pathogenic micro-organisms associated with foods

Antimicrobial activity of seven commercial smoke preparations (four liquid and three solid) was studied. The minimum inhibitory concentration (MIC) was determined against a selection of food spoilage and pathogenic micro-organisms. The main smoke components were identified and quantified by gas chromatography/mass spectrometry. The most effective condensate was S2. All strains except Salmonella enteritidis were inhibited by S2 with an MIC <0·5–1·5%. Smoke extract L2 inhibited growth of Vibrio vulnificus, Yersinia enterocolitica, Bacillus subtilis, Staphylococcus aureus, Listeria monocytogenes, L. inocua, Brochothrix thermosphacta and Lactococcus lactis ssp. lactis with an MIC of <0·2–0·8%. The condensate L3 inhibited effectively V. vulnificus, B. subtilis, L. innocua and Staph. aureus. L1, L4, S1 and S3 had no inhibitory effects at levels tested against most micro-organisms. Vibrio vulnificus was the most susceptible micro-organism to test compounds. The antimicrobial activity of smoke preparations was related to the concentration of phenols.     

Amines in fresh beef of normal pH and the role of bacteria in changes in concentration observed during storage in vacuum packs at chill temperatures

E dwards , R.A., D ainty , R.H., H ibbard , C.M. & R amantanis , S.V. 1987. Amines in fresh beef of normal pH and the role of bacteria in changes in concentration observed during storage in vacuum packs at chill temperatures.
The amine content of fresh and vacuum-packaged beef of normal pH stored at 1°C was evaluated by high performance liquid chromatography of dansyl derivatives. Fresh samples contained five amines, viz. putrescine, cadaverine, histamine, sperm-ine and spermidine. Development of a natural spoilage flora during storage led to increases in concentration of putrescine and cadaverine and the production of a sixth amine, tyramine. Pure culture meat inoculation experiments showed tyramine formation to be restricted to lactobacilli and to strains of Lactobacillus divergens and Lact. carnis in particular; strains of leuconostocs, Enterobacteriaceae, Pseudomonas spp. and Brochothrix thermosphacta were negative. Production of tyramine at cell densities >log₁₀ 6/cm² indicated its potential as an objective measure of acceptability/spoilage.     

Spoilage microflora in fresh chicken breast stored at 4 °C : influence of packaging methods

Chicken breasts with skin were packaged either in air, under vacuum or in modified atmospheres of (i) 30% CO₂/70% N₂ and (ii) 70% CO₂/30% N₂. After 3, 7, 14 and 21 days of storage at 4 °C, the samples were evaluated for spoilage microbial growth, odour and overall aspect. As expected, pseudomonads grew well in air or under vacuum, but growth was suppressed in both types of modified atmosphere packaging (MAP). However, growth of lactobacilli, Enterobacteriaceae and Brochothrix thermosphacta was not inhibited in MAPs. Modified atmosphere packaging (ii) extended shelf-life up to 21 days compared to 5 days for air-packed samples.     

Research of quality indices for cold-smoked salmon using a stepwise multiple regression of microbiological counts and physico-chemical parameters

AIMS: The aim of the study was to assess the relationships between the remaining shelf-life (RSL) of cold-smoked salmon and various microbiological and physico-chemical parameters, using a multivariate data analysis in the form of stepwise forward multiple regression. METHODS AND RESULTS: Thirteen batches of French cold-smoked salmon were analysed weekly during vacuum-packed storage at 5 degrees C for their lipid, water, salt, phenol, pH, total volatile basic nitrogen (TVBN) and trimethylamine contents, total psychrotrophic count, lactic acid bacteria, lactobacilli, B. thermosphacta, Enterobacteriaceae and yeast counts. At the sensory rejection time, the flora was dominated by lactobacilli, lactobacilli/Enterobacteriaceae or Carnobacteria/B. thermosphacta. Shelf-life was very variable (1->6 weeks) and was related to the initial Enterobacteriaceae load (P < 0.05), depending on hygienic conditions in the smokehouse. High correlations existed between the RSL and lactobacilli count (P < 0.01), yeast count (P < 0.05) and TVBN concentration (P < 0.01). A polynomial fitting the RSL as a function of those three factors was proposed (R(2) = 0.80). Assuming that lactobacilli count could not exceed 109 cfu g-1, a minimum of 36 mg-N 100 g-1 was necessary for a product to be rejected, with a yeast count of 104 cfu g-1. CONCLUSION: Estimation of cold-smoked salmon quality is possible by measuring three parameters: lactobacilli and yeast counts and TVBN concentration. SIGNIFICANCE AND IMPACT OF THE STUDY: The technical content is important for the smoked salmon industry and for development of quality standards for cold-smoked salmon.     

Incorporation of nisin into a meat binding system to inhibit bacteria on beef surfaces

In two separate experiments, the bacteriocin, nisin, was incorporated into a commercially available meat binding system (Fibrimex^®) and applied to meat surfaces as a way of inhibiting the meat spoilage organism, Brochothrix thermosphacta during extended refrigerated storage. In experiment 1, pre-rigor lean beef carcass tissue (BCT) was inoculated with B. thermosphacta , left untreated (U), treated with 10 μg ml⁻¹ nisin (N), Fibrimex^® (F) or Fibrimex^® containing 10 μg ml⁻¹ nisin (FN), held aerobically at 4 °C for up to 7 d, and populations of B. thermosphacta and nisin activity determined. Experiment 2 determined the effects of the same treatments but on post-rigor, frozen and thawed lean BCT that was inoculated, vacuum-packaged, and stored at 4 °C for up to 14 d. In both experiments, N- and FN-treated tissues exhibited significantly lower populations of B. thermosphacta compared to U- and F-treated tissues, for the duration of refrigerated storage. Nisin activity was detected up to 7 d in N- and FN-treated samples from experiment 1. However, activity was detected only to days 0 and 2 in FN- and N-treated samples, respectively, from experiment 2. These studies indicate that the addition of a bacteriocin to a meat binding system and application to meat surfaces may be useful in reducing undesirable bacteria in restructured meat products.     

Influence of growth temperature on the production of extracellular virulence factors and pathogenicity of environmental and human strains of Aeromonas hydrophila

The biochemical properties, virulence for mice and trout, and the extracellular virulence factors at 28° and 37°C of 11 environmental and nine human strains of Aeromonas hydrophila were compared. All the environmental isolates and four of the human group were virulent for trout at 3 x 10⁷ cfu, but only human strains were able to cause death or lesions in mice by the intramuscular route. Extracellular virulence factors such as haemolysins, cytotoxins and proteases were also investigated in supernatant fluids of cultures grown at 28°C and 37°C. The production of haemolysins, caseinases, elastases and growth yields of environmental strains decreased sharply during cultivation at 37°C but cytotoxins were produced to the same extent, or slightly less, than at 28°C. The human strains differed from the environmental strains in response to growth temperatures: protease activity decreased at 37°C, although growth yield was not affected, but more haemolysins and cytotoxins were produced by the virulent strains at this temperature than at 28°C. Sodium caseinate SDS-PAGE of culture supernatant fluids of selected human strains revealed that temperature selectively inhibited the production of certain proteases.     

Effect of pH and NaCl on growth from spores of non-proteolytic Clostridium botulinum at chill temperature

The effect of combinations of temperature (2°, 3°, 4°, 5°, 8° and 10°C), pH (5·0–7·2) and NaCl (0·1–5·0% w/w) on growth from spores of non-proteolytic Clostridium botulinum types B, E and F was determined using a strictly anaerobic medium. Inoculated media were observed weekly for turbidity, and tests were made for the presence of toxin in conditions that approached the limits of growth. Growth and toxin production were detected at 3°C in 5 weeks, at 4°C in 3/4 weeks and at 5°C in 2/3 weeks. The resulting data define growth/no growth boundaries with respect to low temperature, pH, NaCl and incubation time. This is important in assessment of the risk of growth and toxin production by non-proteolytic Cl. botulinum in minimally processed chilled foods.     

Hemicellulases of Bacillus species: preliminary comparative studies on production and properties of mannanases and galactanases

A range of Bacillus subtilis strains and other Bacillus species were screened for mannanase, β-mannosidase and galactanase activities. Maximum mannanase activity, 106.2 units/ml, was produced by B. subtilis NRRL 356. β-Mannosidase and galactanase activities from all strains were relatively low. The effect of carbon and nitrogen source on mannanase and galactanase production by B. brevis ATCC 8186, B. licheniformis ATCC 27811, B. polymyxa NRRL 842 and B. subtilis NRRL 356 was investigated. Highest mannanase production was observed in the four strains tested when the mannan substrate, locust bean gum, was used as carbon source. Induction was most dramatic in the case of B. subtilis NRRL 356 where only basal enzyme levels were produced in the presence of other carbon sources. β-Mannosidase was induced in the four Bacillus cultures by locust bean gum. Results indicated that galactose acted as an inducer for production of galactanase. Organic and inorganic nitrogen sources resulted in induction of high mannanase titres in B. subtilis. Highest galactanase activity was produced by each organism in media containing sodium nitrate as nitrogen source. Mannanases from B. brevis, B. licheniformis, B. polymyxa and B. subtilis retained 100% residual activity after a 3 h incubation at 65°C, 65°C, 60°C and 55°C respectively. Galactanases retained more than 95% activity at 55°C after 3 h. The pH optima of mannanases ranged from 6.5–6.8 whereas galactanases ranged from 5.1 in the case of B. brevis to 7.0 for B. polymyxa.     

Bacterial synergism or antagonism in a gel cassette system

The growth and the metabolic activity of Shewanella putrfaciens, Brochothrix thermosphacta, and Pseudomonas sp., when cultured individually or in all possible combinations in gel cassettes system supplemented with 0.1% glucose at 5 degrees C, were investigated. The overall outcome was that the coexistence of the above-mentioned microorganisms affected not only each growth rate but also their type of metabolic end products compared to the control cultures. These effects were varied and depended on the selection of the combination of the tested bacteria. For example, the growth of Pseudomonas sp. strains cocultured with either B. thermosphacta or S. putrefaciens strains resulted in different effects: a promoting one for the first and an inhibitory one for the second. Moreover, the production of formic acid and two unidentified organic acids (peaks a and b) was characteristic in all cases in which S. putrefaciens was cultured.     

Lactobacilli and enterococci — Potential probiotics for dogs

Forty strains of enterococci and forty strains of lactobacilli isolated from feces of 10 healthy dogs were tested for the antimicrobial activity, tolerance to bile and adhesion activity. The total count of fecal enterococci reached 5.5 log CFU/g and of lactobacilli 7.6 log CFU/g. Screening for production of bacteriocin-like substances showed an to partly inhibit the growth of Enterobacter sp. (hazy zones of inhibition). Ten strains of Enterococcus sp. and nine strains of Lactobacillus sp. were found without any inhibitory activity against all indicators used. Seven enterococcal strains and six lactobacilli with the broadest antimicrobial spectrum were selected for further probiotic assays. In the presence of 1% bile, the survival rate of selected enterococci (71.7-97.5%) was higher than that of lactobacilli (66.7-75.4%). The adhesion of strains to human intestinal mucus (5.1-8.2% by enterococci, 2.7-8.3% by lactobacilli) was found to be similar as adhesion to canine intestinal mucus (3.7-10.6% by enterococci, 2.1-6.0% by lactobacilli). Strain AD1, one lactobacillus isolate, reduced the higher level of serum cholesterol and alanine aminotransferase after oral administration to dogs suffering from diseases of the gastrointestinal tract.     

Effect of culture medium composition on inhibition of growth ofNeisseria gonorrhoeae by lactobacilli

The role of genital microorganisms in resistance to gonococcal infection is usually based on their in vitro inhibition of gonococcal growth. Three different culture media (GC, DSA, and MRS) were evaluated for their ability to support the growth of 23 lactobacilli strains and the detection of the antigonococcal activity of these bacteria. The MRS medium was the most suitable medium for the growth of lactobacilli since it favored a good growth of all the lactobacilli strains tested, but it was inhibitory toNeisseria gonorrhoeae. Decreasing the concentration of Tween 80, ammonium citrate, and sodium acetate to one-tenth of their original concentrations yielded a modified MRS medium which still supported good growth of the lactobacilli and was no longer inhibitory to the gonococci. While GC medium did not allow any detection of the production of antigonococcal activity by the lactobacilli, both modified MRS and DSA media allowed the detection of this activity by the agar overlay technique. The use of modified MRS medium is recommended since it is less selective than DSA medium for the growth of lactobacilli.     

Amines in fresh beef of normal pH and the role of bacteria in changes in concentration observed during storage in vacuum packs at chill temperatures

The amine content of fresh and vacuum-packaged beef of normal pH stored at 1 degree C was evaluated by high performance liquid chromatography of dansyl derivatives. Fresh samples contained five amines, viz. putrescine, cadaverine, histamine, spermine and spermidine. Development of a natural spoilage flora during storage led to increases in concentration of putrescine and cadaverine and the production of a sixth amine, tyramine. Pure culture meat inoculation experiments showed tyramine formation to be restricted to lactobacilli and to strains of Lactobacillus divergens and Lact. carnis in particular; strains of leuconostocs, Enterobacteriaceae, Pseudomonas spp. and Brochothrix thermosphacta were negative. Production of tyramine at cell densities less than log10 6/cm2 indicated its potential as an objective measure of acceptability/spoilage.     

Antifungal activity of two Lactobacillus strains with potential probiotic properties

Aflatoxin (highly toxic and carcinogenic secondary metabolites produced by fungi) contamination is a serious problem worldwide. Modern agriculture and animal production systems need to use high-quality and mycotoxin-free feedstuffs. The use of microorganisms to preserve food has gained importance in recent years due to the demand for reduced use of chemical preservatives by consumers. Lactic acid bacteria are known to produce various antimicrobial compounds that are considered to be important in the biopreservation of food and feed. Lactobacillus rhamnosus L60 and Lactobacillus fermentum L23 are producers of secondary metabolites, such as organic acids, bacteriocins and, in the case of L60, hydrogen peroxide. The antifungal activity of lactobacilli strains was determined by coculture with Aspergillus section Flavi strains by two qualitative and one quantitative methods. Both L23 and L60 completely inhibited the fungal growth of all aflatoxicogenic strains assayed. Aflatoxin B (1) production was reduced 95.7-99.8% with L60 and 27.5-100% with L23. Statistical analysis of the data revealed the influence of L60 and L23 on growth parameters and aflatoxin B (1) production. These results are important given that these aflatoxicogenic fungi are natural contaminants of feed used for animal production, and could be effectively controlled by Lactobacillus L60 and L23 strains with probiotic properties.     

THE SELECTIVE ACTION OF THALLOUS ACETATE FOR LACTOBACILLI

SUMMARY: The effect of thallous acetate has been examined on the growth of 23 strains of lactobacilli representing 13 different species. All strains grew satisfactorily in both broth and agar media containing 0.1% of thallous acetate. Of the 18 other organisms also examined the growth of the streptococci was unsuppressed, staphylococci, a micrococcus, B. licheniformis and Pr. mirabilis were partially inhibited and strains of Bact. coli, B. cereus, Chr. prodigiosum and Ps. fluorescens were almost completely inhibited by that concentration. By its use a valuable selective medium for isolating lactobacilli from natural habitats of mixed flora was obtained, other organisms, with the exception of streptococci, being almost completely eliminated.     

Identification and characterization of Lactobacillus species isolated from fillets of vacuum-packed smoked and salted herring (Clupea harengus)

Seventy-eight strains were isolated from fillets of vacuum-packed smoked and salted herring ( Clupea harengus ) on APT glucose medium at 5°C or 20°C . All the isolates belonged to the genus Lactobacillus . CO₂ production and arginine degradation were characteristic of the isolates. A dichotomous key is proposed, using only four characters (growth on Rogosa broth, fermentation of ribose, presence of meso -diaminopimelic acid, isomer of lactate produced) for presumptive identification of lactobacilli from fish. The isolates could be allocated to three groups related to Lactobacillus groups II or III of Kandler and Weiss. Most of the strains possess important technological capacities : tolerance of high levels of NaCl, liquid smoke and bile salts. No lipolytic activity was found but some proteolytic activity was found on casein. Inhibitory activity against pathogenic or spoilage organisms was frequent and probably due to the production of hydrogen peroxide, or organic acids or both. No Carnobacterium strains were isolated despite the use of suitable temperature and isolation medium, probably because of the smoking of the fish.     

The Acid-Dependent and Independent Effects of Lactobacillus acidophilus CL1285, Lacticaseibacillus casei LBC80R,and Lacticaseibacillus rhamnosus CLR2 on Clostridioides difficile R20291

Clostridioides difficile infections (CDI) result from antibiotic use and cause severe diarrhea which is life threatening and costly. A specific probiotic containing Lactobacillus acidophilus CL1285, Lacticaseibacillus casei LBC80R, and Lacticaseibacillus rhamnosus CLR2 has demonstrated a strong inhibitory effect on the growth of several nosocomial C. difficile strains by production of antimicrobial metabolites during fermentation. Though there are several lactobacilli shown to inhibit C. difficile growth by processes relying on acidification, this probiotic has demonstrated potency for CDI prevention among hospitalized patients. Here, we describe the acid-dependent and independent mechanisms by which these strains impair the cytotoxicity of a hypervirulent strain, C. difficile R20291 (CD). These bacteria were co-cultured in a series of experiments under anaerobic conditions in glucose-rich and no-sugar medium to inhibit or stimulate CD toxin production, respectively. In glucose-rich medium, there was low CD toxin production, but sufficient amounts to cause cytotoxic damage to human fibroblast cells. In co-culture, there was acidification by the lactobacilli resulting in growth inhibition as well as ≥ 99% reduced toxin A and B production and no observable cytotoxicity. In the absence of glucose, CD produced much more toxin. In co-culture, the lactobacilli did not acidify the medium and CD growth was unaffected; yet, the amount of detected toxin A and B was decreased by 20% and 41%, respectively. Despite the high concentration of toxin, cells exposed to the supernatant from the co-culture were able to survive. These results suggest that in addition to known acid-dependent effects, the combination of L. acidophilus CL1285, L. casei LBC80R, and L. rhamnosus CLR2 can interfere with CD pathogenesis without acidification: (1) reduced toxin A and B production and (2) toxin neutralization. This might explain the strain specificity of this probiotic in potently preventing C. difficile-associated diarrhea in antibiotic-treated patients compared with other probiotic formulae.