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PubMed1998

Isolation, purification and partial characterization of plantaricin 423, a bacteriocin produced by Lactobacillus plantarum.

Lactobacillus plantarum 423, isolated from sorghum beer, produces a bacteriocin (plantaricin 423) which is inhibitory to several food spoilage bacteria and food-borne pathogens, including Bacillus cereus, Clostridium sporogenes, Enterococcus faecalis, Listeria spp. and Staphylococcus spp. Plantaricin 423 is resistant to treatment at 80 degrees C, but loses 50% of its activity after 60 min at 100 degrees C and 75% of its activity after autoclaving (121 degrees C, 15 min). Plantaricin 423 remains active after incubation at pH 1-10 and is inactivated when treated with pepsin, papain, alpha-chymotrypsin, trypsin and Proteinase K. Plantaricin 423 was partially purified and its size estimated at 3.5 kDa, as determined by tricine-SDS-PAGE. The mechanism of activity of plantaricin 423 is weakly bactericidal, as determined against Oenococcus oeni (previously Leuconostoc oenos). High DNA homology was obtained between the plasmid DNA of strain 423 and the pediocin PA-1 operon of Pediococcus acidilactici PAC 1.0, suggesting that plantaricin 423 is plasmid-encoded and related to the pediocin gene cluster.

Bacteria↗
PubMed1998

Coagulin, a bacteriocin-like inhibitory substance produced by Bacillus coagulans I4.

A protease-sensitive antibacterial substance produced by Bacillus coagulans I4 strain, isolated from cattle faeces, was classified as a bacteriocin-like inhibitory substance and named coagulin. The inhibitory spectrum included B. coagulans and unrelated bacteria such as Enterococcus, Leuconostoc, Oenococcus, Listeria and Pediococcus. Coagulin was stable at 60 degrees C for 90 min, at a pH ranging from 4 to 8 and appeared to be unaffected by alpha-amylase, lipase or organic solvents (10% v/v). Coagulin exhibited a bactericidal and a bacteriolytic mode of action against indicator cells. The apparent molecular mass was estimated to be about 3-4 kDa by SDS-PAGE. The B. coagulans I4 strain harbours a plasmid, pI4, approximately 14 kb in size. Novobiocin curing experiments yielded two derivatives that no longer produced the bacteriocin-like inhibitory substance. Plasmid content of these two derivatives showed that one had lost pI4, whereas the second harboured a deleted form of this plasmid, thus suggesting a plasmid location for the genes for coagulin production.

Animals↗
PubMed1999

Study of the micro-organisms associated with the fermented bread (khamir) produced from sorghum in Gizan region, Saudi Arabia.

Traditional bread (khamir) was made from sorghum flour of two local varieties, Bayadh and Hamra. The bread was prepared by mixing the sorghum flour with water and spices (onion, garlic, lemon juice and fenugreek) in a 1:0.8 (w/w) ratio and fermented for 24 h at 30 degrees C. Two other fermentations were carried out using an inoculum from the previous fermentation. The micro-organisms were isolated from different plates and identified using different characterization systems. Both total bacterial populations and lactic acid bacteria increased with fermentation time and reached the highest number at 16 h (first fermentation) and at 8 h (second and third fermentation). The content of lactic acid was increased with time to reach 1.2%, but the increase was higher for the second and third fermentations (1.6% each). The pH dropped with time from 6.77 to 4.35 in the first fermentation and from 6.65 to 4.18, and 6.57-3.93, in the second and third fermentations, respectively. The microorganisms, which were isolated and characterized during the 24 h fermentation, included: bacteria (Pediococcus pentosaceus, Lactobacillus brevis, Lact. lactis subsp. lactis, Lact. cellobiosus, Klebsiella oxytoca, Kl. pneumoniae, Enterobacter aerogenes, Ent. sakazakii, Serratia marcescens and Ser. odourifera), moulds (Penicillium sp., Rhizopus sp., Aspergillus niger, Alternaria sp., Fusarium sp. and Mucor sp.) and yeasts (Candida parapsilosis, C. orvegnsis and Rhodotorula glutinis).

Bread↗
PubMed2000

Solvent extraction of bacteriocins from liquid cultures.

A solvent extraction method was developed to concentrate lacidin from the culture of Lactobacillus acidophilus OSU133. The new method concentrates the bacteriocin at the interface between chloroform and the aqueous culture of the producing bacterium. Compared with other extraction procedures, the new method effectively recovers higher bacteriocin yield and results in relatively clean preparations. Recovery of lacidin by the chloroform extraction procedure, compared with ammonium sulphate precipitation and cell acidification methods, was >10-fold and about 100-fold greater, respectively. The new extraction procedure saves time and is easy to perform. This method is also effective in recovering subtilin, bacillicin, pediocin and nisin from cultures of Bacillus subtilis ATCC 6633, B. subtilis OSY1115/C, Pediococcus acidilactici PO2 and Lactococcus lactis ATCC 11454, respectively.

Ammonium Sulfate↗
PubMed2000

Identification and enumeration of oleic acid and linoleic acid hydrating bacteria in the rumen of sheep and cows.

The diversity and population densities of facultative anaerobic bacteria with the capacity to hydrate oleic acid and linoleic acid in the rumen of sheep and dairy cows were determined. The screening of representative colonies, from rumen fluid plated aerobically on a range of agar media, revealed that sheep rumen fluid contained hydration-positive strains of Streptococcus, Staphylococcus, Enterococcus, Lactobacillus and Pediococcus, whereas cow rumen fluid contained hydration-positive strains of Streptococcus, Lactobacillus and Staphylococcus. Mean counts of facultative anaerobic bacteria in sheep and cattle rumen were log10 7.29 and log10 6.40, respectively, and were independent of diet. Approximately 56% of facultative anaerobic bacteria were able to hydrate oleic and/or linoleic acid in anaerobic broth culture. For both sheep and cows, the most numerous hydration-positive isolates were strains of Strep. bovis. The results, which are the first to show that pediococci have the capacity to hydrate unsaturated fatty acids, suggest that lactic acid bacteria are the major unsaturated fatty acid hydrating bacteria in the rumen.

Animals↗
PubMed2000

Enterocin 012, a bacteriocin produced by Enterococcus gallinarum isolated from the intestinal tract of ostrich.

Enterococcus gallinarum strain 012, isolated from the duodenum of ostrich, produced enterocin 012 which is active against Ent. faecalis, Lactobacillus acidophilus, Lact. sake, Listeria innocua, Propionibacterium acidipropionici, Propionibacterium sp., Clostridium perfringens, Pseudomonas aeruginosa and Salmonella typhimurium. One of the four pathogenic strains of Escherichia coli isolated from the intestinal tract of ostrich was inhibited by enterocin 012. No antimicrobial activity was recorded against Bacillus cereus, Cl. sporogenes, Cl. tyrobutyricum, Leuconostoc cremoris, Pediococcus pentosaceus, Staphylococcus carnosus and Streptococcus thermophilus. Enterocin 012 was resistant to treatment with lysozyme, catalase, lipase and papain, but sensitive to Proteinase K, alpha-chymotrypsin, trypsin and pepsin. Treatment of enterocin 012 with gastric juice from the duodenum resulted in a 50% loss of antibacterial activity. Half of the activity was lost when incubated at 80 degrees C for 30 min, or when kept overnight at a pH of 1.0-5.0 and pH 11.0 and 12.0, respectively. Enterocin 012 production started in mid-logarithmic growth and reached a maximum of 800 AU ml-1, but increased further to 1600 AU ml-1 in the stationary growth phase. The peptide is approximately 3.4 kDa in size, as determined after partial purification with Amberlite XAD-1180 and ammonium sulphate precipitation, followed by tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The mechanism of antimicrobial activity against Lact. sake LMG 13558 is bactericidal and caused cell lysis of active growing cells.

Animals↗
PubMed2001

Comparison of pattern recognition techniques for the identification of lactic acid bacteria.

AIMS: The goal of this study was to evaluate three pattern recognition methods for use in the identification of lactic acid bacteria. METHODS AND RESULTS: Lactic acid bacteria (21 unknown isolates and 30 well-characterized strains), including the Lactobacillus, Lactococcus, Streptococcus, Pediococcus and Oenococcus genera, were tested for 49 phenotypic responses (acid production on carbon sources). The results were scored in several ways. Three procedures, k-nearest neighbour analysis (KNN), k-means clustering and fuzzy c-means clustering (FCM), were applied to the data. CONCLUSION: k-Nearest neighbour analysis performed better with five-point-scaled than with binary data, indicating that intermediate values are helpful to classification. k-Means clustering performed slightly better than KNN and was best with fuzzified data. The best overall results were obtained with FCM. Genus level classification was best with FCM using an exponent of 1.25. SIGNIFICANCE AND IMPACT OF THE STUDY: The three pattern recognition methods offer some advantages over other approaches to organism classification.

Bacterial Typing Techniques↗
PubMed2001

The gram-positive tonsillar and nasal flora of piglets before and after weaning.

AIMS: To investigate gram-positive nasal and tonsillar microbial flora of piglets before and after weaning. METHODS AND RESULTS: The nasal and tonsillar gram-positive bacterial flora of 20 non-weaned piglets (2 weeks of age) and 20 weaned piglets (6 weeks of age), obtained from four different piggeries, was quantified by culture and identified by tDNA-PCR. The most widely occurring species from nasal conchae before as well as after weaning in the different piglets investigated were Streptococcus suis and Rothia nasimurium. After weaning a wide variety of Lactobacillus species appeared but in low numbers. In the tonsils, Strep. suis, Strep. dysgalactiae, S. hyicus, S. aureus, Arcanobacterium pyogenes and Actinomyces hyovaginalis were the species isolated from the largest number of pigs before and after weaning. S. aureus and most lactobacilli became more prevalent after weaning. Bacteria not known to be associated with pigs found in the present study included R. nasimurium, Strep. gallolyticus, Pediococcus pentosaceus and some Lactobacillus species. CONCLUSIONS: Over 30 different gram-positive bacterial species may occur in nasal conchae and tonsils of unweaned piglets at 2 weeks of age and of 6-week-old weaned piglets. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated that weaning is associated with changes in prevalence of only a small minority of the highly diversified bacterial flora of the nares and tonsils of pigs.

Animals↗
PubMed2002

Persistence of Escherichia coli O157:H7 in barley silage: effect of a bacterial inoculant.

AIMS: The effect of a lactic acid producing bacterial (LAB) inoculant on the elimination of Escherichia coli O157:H7 from barley forage was assessed. METHODS AND RESULTS: Triplicate mini-silos were prepared for four treatments and six sampling times (1, 3, 7, 15, 30 and 42 d post-ensiling). The treatments were (i) 10(5) cfu g(-1) Pediococcus pentosaceus and Propionibacterium jenzenii (P2); (ii) 10(5) cfu g(-1) E. coli O157:H7 strain 3081 and 10(5) cfu g(-1) E. coli Biotype 1 strains 719IE10, 719IE14 and 614ME49 (EC); (iii) P2 + EC; and (iv) the control (sterile distilled water). Triplicate mini-silos were opened at each sampling time for pH, volatile fatty acid (VFA) and lactate determinations and E. coli, E. coli O157:H7 and LAB were enumerated. On d 3 and 7, numbers of E. coli O157:H7 in P2 + EC were significantly lower than in EC (P < 0;05). Escherichia coli O157:H7 was not detected in P2 + EC and EC at 7 and 15 d post-ensiling, respectively. On d 15 through 42, E. coli Biotype 1 was not detected in P2 + EC or EC. Populations of LAB were higher in P2 and P2 + EC than in the control and EC on d 3 and 7 (P < 0.05). After 3 d of ensiling, lactate levels were higher (P < 0.05) and pH was lower (P < 0.05) in P2 and P2 + EC as compared to the control and EC. Bacteriocins of P2 were not found to be inhibitory to E. coli O157:H7 using the agar-spot procedure. Escherichia coli O157:H7 inoculated into the control silage at a level of 10(3) cfu g(-1) and exposed to aerobic conditions at 22 degrees C was not detected after 1 d and remained undetectable for the 28 d exposure period. CONCLUSIONS: Silage inoculant P2 increased lactate levels and decreased pH more rapidly during ensiling, which appeared to hasten the elimination of E. coli O157:H7 from the silage. SIGNIFICANCE AND IMPACT OF THE STUDY: Results emphasize the importance of adequate ensiling since E. coli O157:H7 may be maintained and spread among cattle through feed.

Aerobiosis↗
PubMed1998

Detection and characterization of a bacteriocin produced by Lactococcus lactis subsp. cremoris R isolated from radish.

Bacteria isolated from radish were identified as Lactococcus lactis subsp. cremoris R and their bacteriocin was designated lactococcin R. Lactococcin R was sensitive to some proteolytic enzymes (proteinase-K, pronase-E, proteases, pepsin, alpha-chymotrypsin) but was resistant to trypsin, papain, catalase, lysozyme and lipase, organic solvents, or heating at 90 degrees C for 15, 30 and 60 min, or 121 degrees C for 15 min. Lactococcin R remained active after storage at -20 and -70 degrees C for 3 months and after exposure to a pH of 2-9. The molecular weight of lactococcin R was about 2.5 kDa. Lactococcin R was active against many food-borne pathogenic and food spoilage bacteria such as Clostridium, Staphylococcus, Listeria, Bacillus, Micrococcus, Enterococcus, Lactobacillus, Leuconostoc, Streptococcus and Pediococcus spp., but was not active against any Gram-negative bacteria. Lactococcin R was produced during log phase and reached a maximum activity (1600 AU ml-1) at early stationary phase. The highest lactococcin R production was obtained in MRS broth with 0.5% glucose, at 6.5-7.0 initial pH values, 30 degrees C temperature and 18-24-h incubation times. Lactococcin R adsorbed maximally to its heat-killed producing cells at pH 6-7 (95%). Crude lactococcin R at 1280 AU ml-1 was bactericidal, reducing colony counts of Listeria monocytogenes by 99.98% in 3 h. Lactococcin R should be useful as a biopreservative to prevent growth of food-borne pathogenic and food spoilage bacteria in ready-to-eat, dairy, meat, poultry and other food products. Lactococcin R differs from nisin in having a lower molecular weight, 2.5 kDa vs 3.4 kDa, and in being sensitive to pepsin and alpha-chymotrypsin to which nisin is resistant.

Bacteriocins↗
PubMed1999

Intrinsically vancomycin-resistant gram-positive organisms: clinical relevance and implications for infection control.

Intrinsic resistance to vancomycin in gram-positive bacteria presumably predates acquired vancomycin resistance in enterococci but it has only recently generated interest. Intrinsically resistant enterococci possessing the vanC gene and the non-enterococcal genera Leuconostoc, Lactobacillus, Pediococcus and Erysipelothrix are known to cause human infection. This review examines the available data on their identification, resistance mechanisms, epidemiology, clinical infections and antimicrobial susceptibility. Intrinsically vancomycin-resistant gram-positives are usually opportunistic pathogens. Although serious infections may occur, treatment options remain available. No additional infection control measures for the intrinsically resistant genera appear justified with currently available evidence, although vigilance should be maintained to detect future changes in susceptibility patterns.

Anti-Bacterial Agents↗
PubMed2001

Characterisation of IS153, an IS3-family insertion sequence isolated from Lactobacillus sanfranciscensis and its use for strain differentiation.

An insertion sequence has been identified in the genome of Lactobacillus sanfranciscensis DSM 20451T as segment of 1351 nucleotides containing 37-bp imperfect terminal inverted repeats. The sequence of this element encodes two out of phase, overlapping open reading frames, orfA and orfB, from which three putative proteins are produced. OrfAB is a transframe protein produced by -1 translational frame shifting between orf A and orf B that is presumed to be the transposase. The large orfAB of this element encodes a 342 amino acid protein that displays similarities with transposases encoded by bacterial insertion sequences belonging to the IS3 family. In L. sanfranciscensis type strain DSM 20451T multiple truncated IS elements were identified. Inverse PCR was used to analyze target sites of four of these elements, but except of their highly AT rich character not any sequence specificity was identified so far. Moreover, no flanking direct repeats were identified. Multiple copies of IS153 were detected by hybridization in other strains of L. sanfranciscensis. Resulting hybridization patterns were shown to differentiate between organisms at strain level rather than a probe targeted against the 16S rDNA. With a PCR based approach IS153 or highly similar sequences were detected in L. acidophilus, L. casei, L. malefermentans, L. plantarum, L. hilgardii, L. collinoides L. farciminis L. sakei and L. salivarius, L. reuteri as well as in Enterococcus faecium, Pediococcus acidilactici and P. pentosaceus.

Amino Acid Sequence↗
PubMed2004

Genotypic analyses of lactobacilli with a range of tannase activities isolated from human feces and fermented foods.

A total of 77 tannase producing lactobacilli strains isolated from human feces or fermented foods were examined for their genotypic profiles and intensities of tannase production. With a PCR-based assay targeting recA gene, all strains except one isolate were assigned to either Lactobacillus plantarum, L. paraplantarum, or L. pentosus whereas a 16/23S rDNA targeted PCR-based assay identified all except 6 isolates (inclusive of the above one isolate) as one of the closely related species. Subsequent DNA/DNA hybridization assays revealed that these 6 exceptional isolates showed low homology (between 1.2% and 55.8% relative DNA binding) against type strains of the three species. Supplemental carbohydrate fermentation profiles on the 6 isolates indicated that two of them were identified as L. acidophilus, one as Pediococcus acidilactici, one as P. pentosaceus, and two remained unidentifiable. The evidence suggests that the 16/23S rDNA targeted PCR assay can be used as a reliable identification tool for the closely related lactobacilli, and that the tannase gene is widely distributed within members of the Lactobacillaceae family. Meanwhile, a randomly amplified polymorphism DNA (RAPD) analysis revealed that all except 8 isolates were well allocated in 4 major RAPD clusters, though not species specific, consisting of two L. plantarum predominant clusters, one L. paraplantarum predominant, and one L. pentosus predominant. The RAPD patterns of the 8 non-clustered isolates, which consisted of the 6 unidentifiable isolates and 2 isolates identified as L. pentosus, were <40% similarity to those belonging to the 4 clusters. A quantitative assay of the tannase activities showed that there was a marked variation in the activities among the strains, which did not correlate with either species identification or clustering by RAPD.

Carbohydrates↗
PubMed2002

Factors affecting the adsorption of buchnericin LB, a bacteriocin produced by Lactobacillus [correction of Lactocobacillus] buchneri.

Buchnericin-LB adsorbs to gram-positive but not to gram-negative bacteria. The tested gram-positive bacteria were species of Lactobacillus, Pediococcus, Leuconostoc, Enterococcus, Lactococcus, Listeria, Bacillus, Staphylococcus; gram-negative bacteria belonged to the genera Salmonella, Escherichia, Yersinia and Pseudomonas. Buchnericin-LB adsorption depended on pH but not on time and temperature. Also some anions of salts and lipoteichoic acid reduced or inhibited its adsorption. Treatment of cells and cell walls of sensitive bacteria with detergents, organic solvents or enzymes did not affect subsequent binding of buchnericin-LB. Treatment with buchnericin-LB caused sensitive cells to lose high amounts of intracellular K+ ions and UV-absorbing materials and became more permeable to o-nitrophenol-beta-D-galactopyranoside. Buchnericin-LB (640-2560 AU/ml) decreased the colony forming units (99%) and absorbance values of Listeria monocytogenes and Bacillus cereus. These results indicate that the mode of action of buchnericin-LB is bactericidal and its lethal effect is very rapid.

Adsorption↗
PubMed1987

Tissue folates in fruit bats (Rousettus aegyptiacus) with nitrous oxide-induced vitamin B12 deficiency and neurological impairment.

1. Long-term exposure of the fruit bat Rousettus aegyptiacus to nitrous oxide, which inactivates methylcobalamin, leads to neurological impairment and ataxia. 2. In N2O-exposed animals, liver concentrations of total folates and methyl folates decreased to less than one-fifth that of control animals. Pediococcus cerevisiae-active folates were also reduced. 3. In brain, there were no changes in total or methyl folates, but P. cerevisiae-active folates were lower in N2O-exposed animals. 4. Supplementation with methionine retarded the development of neurological impairment and the fall in liver total and methyl folates, but not that in P. cerevisiae-active folates. 5. Supplementation with serine failed to retard the development of neurological impairment or fall in hepatic folates. 6. The present results suggest that the N2O-induced neurological impairment in the bat is not related to depletion of cerebral folates, but do not exclude changes in the subcellular distribution of folates.

Animals↗
PubMed2003

The effects of using lactic acid bacteria inoculants in maize silage on the formation of biogenic amines.

Silages from five ripened varieties of silage maize with dry matter contents ranging between 275 and 410 g/kg were prepared in five laboratory experiments. Whole-plant maize was fermented at 22 degrees C and silages were then stored at the same temperature for 4 months. Spontaneously fermented silages were prepared as control variants and compared with silages inoculated with commercial strains of Lactobacillus plantarum, Lactobzcillus buchneri and a mixed preparation Microsil containing L. plantarum, Lactobacillus casei, Enterococcus faecium and Pediococcus pentosaceus. The starter cultures were applied at doses 5 x 10(5) and 5 x 10(6) CFU/g of chopped maize. Seven biogenic amines and polyamines were extracted from silages with perchloric acid and determined as N-benzamides by micellar electrokinetic capillary chromatography. Common chemical criteria of silage quality were also determined. All three inoculants, mainly at the higher dose, decreased significantly contents of tyramine, putrescine and cadaverine, three undesirable amines occurring at the highest levels. L. plantarum was the most effective. Contents of histamine and tryptamine were low in all experimental silages. Also relatively low were levels of polyamines spermidine and mainly of spermine.

Animals↗
PubMed2004

Cloning vectors based on cryptic plasmids isolated from lactic acid bacteria: their characteristics and potential applications in biotechnology.

Lactic acid bacteria (LAB) are Gram positive bacteria, widely distributed in nature, and industrially important as they are used in a variety of industrial food fermentations. The use of genetic engineering techniques is an effective means of enhancing the industrial applicability of LAB. However, when using genetic engineering technology, safety becomes an essential factor for the application of improved LAB to the food industry. Cloning and expression systems should be derived preferably from LAB cryptic plasmids that generally encode genes for which functions can be proposed, but no phenotypes can be observed. However, some plasmid-encoded functions have been discovered in cryptic plasmids originating from Lactobacillus, Streptococcus thermophilus, and Pediococcus spp. and can be used as selective marker systems in vector construction. This article presents information concerning LAB cryptic plasmids, and their structures, functions, and applications. A total of 134 cryptic plasmids collated are discussed.

Biotechnology↗
PubMed2002

The lactic acid bacteria: a literature survey.

The purpose of this review article on the lactic acid bacteria grew from an early curiosity and a desire to convey and impart the broad scope of literary information on their functions as starter cultures, in the manufacture of fermentation products such as dairy products and alcoholic beverages, as well as their contribution to better health. This review article is an attempt to empower the reader and to circumvent the difficult task in acquiring and elucidating a large body of information. The intent is to familiarize the reader with the various lactic species, their habitat or source, associated food, physiological characteristics, colonial morphology, biochemical characteristics, culture media (enrichment, nonselective, and selective), classic description, and taxonomy. This review provides information on Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Carnobacterium, and Enterococcus. Trends are presented, such as the use of nisin to extend food shelf-life and the current research premise that Probiotic strains may alter the intestinal flora and thus prevent intestinal wall penetration by pathogens.

Culture Media↗