Characterization,Identification and Application of Lactic Acid Bacteria Isolated from Forage Paddy Rice Silage

There has been growing interest to develop forage rice as a new feed resource for livestock. This study was to characterize the natural population of lactic acid bacteria (LAB) and select potentially excellent strains for paddy rice silage preparation in China. One hundred and twenty-six strains were isolated and screened from paddy rice silage prepared using a small-scale fermentation system, and ninety-nine of these isolates were considered to be LAB based on their Gram-positive and catalase-negative morphology and the production of most of their metabolic products as lactic acid. These isolates were divided into eight groups (A-H) on the basis of their morphological and biochemical characteristics. The Group A to H strains were identified as Lactobacillus (L.) plantarum subsp. plantarum (species ratio: 8.1%), L. casei (5.1%), Leuconostoc (Ln.) pseudomesenteroides (11.1%), Pediococcus (P.) pentosaceus (24.2%), Enterococcus (E.) mundtii (12.1%), Lactococcus (Lc.) garvieae (15.2%), E. faecium (9.1%) and Lc. lactis subsp. lactis (15.2%) based on sequence analyses of their 16S rRNA and recA genes. P. pentosaceus was the most abundant member of the LAB population in the paddy rice silage. A selected strain, namely L. casei R 465, was found to be able to grow under low pH conditions and to improve the silage quality with low pH and a relatively high content of lactic acid. This study demonstrated that forage paddy rice silage contains abundant LAB species and its silage can be well preserved by inoculation with LAB, and that strain R 465 can be a potentially excellent inoculant for paddy rice silage.     

Presence of erythromycin and tetracycline resistance genes in lactic acid bacteria from fermented foods of Indian origin

Lactic acid bacteria (LAB) resistant to erythromycin were isolated from different food samples on selective media. The isolates were identified as Enterococcus durans, Enterococcus faecium, Enterococcus lactis, Enterococcus casseliflavus, Lactobacillus salivarius, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus fermentum, Pediococcus pentosaceus and Leuconostoc mesenteroides. Of the total 60 isolates, 88 % harbored the ermB gene. The efflux gene msrA was identified in E. faecium, E. durans, E. lactis, E. casseliflavus, P. pentosaceus and L. fermentum. Further analysis of the msrA gene by sequencing suggested its homology to msrC. Resistance to tetracycline due to the genes tetM, tetW, tetO, tetK and tetL, alone or in combination, were identified in Lactobacillus species. The tetracycline efflux genes tetK and tetL occurred in P. pentosaceus and Enterococcus species. Since it appeared that LAB had acquired these genes, fermented foods may be a source of antibiotic resistance.     

Natural populations of lactic acid bacteria associated with silage fermentation as determined by phenotype, 16S ribosomal RNA and recA gene analysis

One hundred and fifty-six strains isolated from corn (Zea mays L.), forage paddy rice (Oryza sativa L.), sorghum (Sorghum bicolor L.) and alfalfa (Medicago sativa L.) silages prepared on dairy farms were screened, of which 110 isolates were considered to be lactic acid bacteria (LAB) according to their Gram-positive and catalase-negative characteristics and, mainly, the lactic acid metabolic products. These isolates were divided into eight groups (A-H) based on the following properties: morphological and biochemical characteristics, γ-aminobutyric acid production capacity, and 16S rRNA gene sequences. They were identified as Weissella cibaria (36.4%), Weissella confusa (9.1%), Leuconostoc citreum (5.3%), Leuconostoc lactis (4.9%), Leuconostoc pseudomesenteroides (8.0%), Lactococcus lactis subsp. lactis (4.5%), Lactobacillus paraplantarum (4.5%) and Lactobacillus plantarum (27.3%). W. cibaria and W. confusa were mainly present in corn silages, and L. plantarum was dominant on sorghum and forage paddy rice silages, while L. pseudomesenteroides, L. plantarum and L. paraplantarum were the dominant species in alfalfa silage. The corn, sorghum and forage paddy rice silages were well preserved with lower pH values and ammonia-N concentrations, but had higher lactic acid content, while the alfalfa silage had relatively poor quality with higher pH values and ammonia-N concentrations, and lower lactic acid content. The present study confirmed the diversity of LAB species inhabiting silages. It showed that the differing natural populations of LAB on these silages might influence fermentation quality. These results will enable future research on the relationship between LAB species and silage fermentation quality, and will enhance the screening of appropriate inoculants aimed at improving such quality.     

Lactic acid bacteria against post-harvest moulds and ochratoxin A isolated from stored wheat

A total of 54 lactic acid bacteria (LAB) were isolated from stored wheat samples sourced from grain silos in North Tunisia. Fifteen representative isolates were identified by 16S rDNA sequencing as Pediococcus pentosaceus, Lactobacillus plantarum, Lactobacillus graminis, Lactobacillus coryniformis and Weissella cibaria. These isolates were screened for antifungal activity in dual culture agar plate assay against eight post-harvest moulds (Penicillium expansum, Penicillium chrysogenum, Penicillium glabrum, Aspergillus flavus, Aspergillus niger, Aspergillus carbonarius, Fusarium graminearum and Alternaria alternata). All LAB showed inhibitory activity against moulds, especially strains of L. plantarum which exhibited a large antifungal spectrum. Moreover, LAB species such as L. plantarum LabN10, L. graminis LabN11 and P. pentosaceus LabN12 showed high inhibitory effects against the ochratoxigenic strain A. carbonarius ANC89. These LAB were also investigated for their ability to reduce A. carbonarius ANC89 biomass and its ochratoxin A (OTA) production on liquid medium at 28 and 37 °C and varied pH conditions. The results indicated that factors such as temperature, pH and bacterial biomass on mixed cultures, has a significant effect on fungal inhibition and OTA production. High percentage of OTA reduction was obtained by L. plantarum and L. graminis (>97%) followed by P. pentosaceus (>81.5%). These findings suggest that in addition to L. plantarum, L. graminis and P. pentosaceus strains may be exploited as a potential OTA detoxifying agent to protect humans and animals health against this toxic metabolite.     

Phenotypic and genotypic analyses of lactic acid bacteria in local fermented food, breast milk and faeces of mothers and their babies

Lactic acid bacteria (LAB) are generally accepted as beneficial to the host and their presence is directly influenced by ingestion of fermented food or probiotics. While the intestinal lactic microbiota is well-described knowledge on its routes of inoculation and competitiveness towards selective pressure shaping the intestinal microbiota is limited. In this study, LAB were isolated from faecal samples of breast feeding mothers living in Syria, from faeces of their infants, from breast milk as well as from fermented food, typically consumed in Syria. A total of 700 isolates were characterized by genetic fingerprinting with random amplified polymorphic DNA (RAPD) and identified by comparative 16S rDNA sequencing and Matrix Assisted Laser Desorption Ionization-Time-Of-Flight Mass Spectrometry (MALDI-TOF-MS) analyses. Thirty six different species of Lactobacillus, Enterococcus, Streptococcus, Weissella and Pediococcus were identified. RAPD and MALDI-TOF-MS patterns allowed comparison of the lactic microbiota on species and strain level. Whereas some species were unique for one source, Lactobacillus plantarum, Lactobacillus fermentum, Pediococcus pentosaceus and Lactobacillus brevis were found in all sources. Interestingly, identical RAPD genotypes of L. plantarum, L. fermentum, L. brevis, Enterococcus faecium, Enterococcus faecalis and P. pentosaceus were found in the faeces of mothers, her milk and in faeces of her babies. Diversity of RAPD types found in food versus human samples suggests the importance of host factors in colonization and individual host specificity, and support the hypothesis that there is a vertical transfer of intestinal LAB from the mother's gut to her milk and through the milk to the infant's gut.     

Polyphasic taxonomic studies of lactic acid bacteria associated with Tunisian fermented meat based on the heterogeneity of the 16S–23S rRNA gene intergenic spacer region

The objective of this work was to investigate the structure and diversity of lactic acid bacteria (LAB) communities in traditionally fermented meat collected from different areas of Tunisia. A polyphasic study, which involves phenotypic tests and ribosomal DNA-based techniques, was used to identify Gram-positive and catalase-negative isolates. PCR amplification of the 16S–23S rDNA ISR of 102 isolates and other reference LAB strains gave (1) one type of rrn operon (M-ISR) for lactococci, (2) two types of rrn operon (S-ISR and M-ISR) for enterococci, (3) two types of rrn operon (S-ISR and L-ISR) for Lactobacilli, and (4) three PCR amplicons (S-ISR, M-ISR, and L-ISR) obtained for Pediococcus spp. and Weissella genus. The clustering and comparison of ISR–RFLP profiles given by the isolates with those given by reference LAB strains, allowed their identification as Lactococcus lactis, Enterococcus faecium, Enterococcus faecalis, Enterococcus sanguinicola, Enterococcus hawaiiensis, Lactobacillus sakei, Lactobacillus curvatus, Lactobacillus plantarum, Lactobacillus alimentarius, Pediococcus pentosaceus, and Weissella confusa. Combined 16S–23S rDNA ISR and RFLP patterns can be considered as a good potential target for a rapid and reliable differentiation between isolates of LAB and provided further information on the organization of their rrn operons.     

Identification of a Cold Shock Gene in Lactic Acid Bacteria and the Effect of Cold Shock on Cryotolerance

When Lactic Acid Bacterial cultures were frozen at −20°C for 24 h, the cell viability decreased drastically, but when they were cold shocked at 10°C for 2 h prior to freezing, viability improved significantly for the Lactococcus lactis subsp. lactis strains (25–37%) and Pediococcus pentosaceus PO2 (18%), but not for the Lactococcus lactis subsp. cremoris strains tested or for one strain of Lactobacillus helveticus LB1 and Streptococcus thermophilus TS2. When the period for cold shock was extended to 5 h, the viability increased even further for those strains that displayed cold shock cryotolerance. Use of degenerate PCR primers based on the major cold shock protein (csp) of both Escherichia coli and Bacillus subtilis resulted in PCR products from all strains tested. The PCR product from Lactococcus lactis ssp. lactis M474 was cloned and sequenced, and the deduced amino acid sequence displayed a high sequence similarity to other csp's. Use of PCR primers based on the M474 sequence resulted in PCR products being produced only from the lactococcal strains studied and not from the Lactobacillus helveticus, Streptococcus thermophilus, or Pediococcus pentosaceus strains tested. Received: 18 October 1996 / Accepted: 28 January 1997     

Taxonomic Structure and Monitoring of the Dominant Population of Lactic Acid Bacteria during Wheat Flour Sourdough Type I Propagation Using Lactobacillus sanfranciscensis Starters

The structure and stability of the dominant lactic acid bacterium population were assessed during wheat flour sourdough type I propagation by using singly nine strains of Lactobacillus sanfranciscensis. Under back-slopping propagation with wheat flour type 0 F114, cell numbers of presumptive lactic acid bacteria varied slightly between and within starters. As determined by randomly amplified polymorphic DNA-PCR and restriction endonuclease analysis-pulsed-field gel electrophoresis analyses, only three (LS8, LS14, and LS44) starters dominated throughout 10 days of propagation. The others progressively decreased to less than 3 log CFU g⁻¹. Partial sequence analysis of the 16S rRNA and recA genes and PCR-denaturating gradient gel electrophoresis analysis using the rpoB gene allowed identification of Weissella confusa, Lactobacillus sanfranciscensis, Lactobacillus plantarum, Lactobacillus rossiae, Lactobacillus brevis, Lactococcus lactis subsp. lactis, Pediococcus pentosaceus, and Lactobacillus spp. as the dominant species of the raw wheat flour. At the end of propagation, one autochthonous strain of L. sanfranciscensis was found in all the sourdoughs. Except for L. brevis, strains of the above species were variously found in the mature sourdoughs. Persistent starters were found in association with other biotypes of L. sanfranciscensis and with W. confusa or L. plantarum. Sourdoughs were characterized for acidification, quotient of fermentation, free amino acids, and community-level catabolic profiles by USING Biolog 96-well Eco microplates. In particular, catabolic profiles of sourdoughs containing persistent starters behaved similarly and were clearly differentiated from the others. The three persistent starters were further used for the production of sourdoughs and propagated by using another wheat flour whose lactic acid bacterium population in part differed from the previous one. Also, in this case all three starter strains persisted during propagation.     

Microbial Diversity in Ngari,Hentak and Tungtap,Fermented Fish Products of North-East India

Ngari, hentak and tungtap are traditional fermented fish products of North-East India. Eighteen samples of ngari, hentak and tungtap were collected and were analysed for microbial load. Lactic acid bacteria, endospore-forming rods, yeasts and aerobic mesophilic counts ranged from 4.0 to 7.2, 3.3–4.6, <1–3.5 and 4.3–7.3 log c.f.u./g, respectively. Lactic acid bacteria were identified as Lactococcus lactis subsp. cremoris, Lactococcus plantarum, Enterococcus faecium, Lactobacillus fructosus, Lactobacillus amylophilus, Lactobacillus coryniformis subsp. torquens and Lactobacillus plantarum. Endospore-forming rods were identified as Bacillus subtilis and Bacillus pumilus, aerobic coccal strains were identified as Micrococcus. Yeasts were identified as species of Candida and Saccharomycopsis. Pathogenic contaminants were detected in all samples, however, none of the sample contained more than 10² c.f.u./g of Bacillus cereus, 10³ c.f.u./g of Staphylococcus aureus and enterobacteriaceae population, respectively. Enzymatic and antimicrobial activities of the isolates were tested. None of the strains produced biogenic amines in the method applied. Most strains of LAB had a high degree of hydrophobicity, indicating their ‘probiotic’ characters. This study has demonstrated the microbial diversity within the species of lactic acid bacteria, Bacillus and yeasts. This revised version was published online in July 2006 with corrections to the Cover Date.     

Synthesis of γ-Aminobutyric Acid by Lactic Acid Bacteria Isolated from a Variety of Italian Cheeses

The concentrations of γ-aminobutyric acid (GABA) in 22 Italian cheese varieties that differ in several technological traits markedly varied from 0.26 to 391 mg kg⁻¹. Presumptive lactic acid bacteria were isolated from each cheese variety (total of 440 isolates) and screened for the capacity to synthesize GABA. Only 61 isolates showed this activity and were identified by partial sequencing of the 16S rRNA gene. Twelve species were found. Lactobacillus paracasei PF6, Lactobacillus delbrueckii subsp. bulgaricus PR1, Lactococcus lactis PU1, Lactobacillus plantarum C48, and Lactobacillus brevis PM17 were the best GABA-producing strains during fermentation of reconstituted skimmed milk. Except for L. plantarum C48, all these strains were isolated from cheeses with the highest concentrations of GABA. A core fragment of glutamate decarboxylase (GAD) DNA was isolated from L. paracasei PF6, L. delbrueckii subsp. bulgaricus PR1, L. lactis PU1, and L. plantarum C48 by using primers based on two highly conserved regions of GAD. A PCR product of ca. 540 bp was found for all the strains. The amino acid sequences deduced from nucleotide sequence analysis showed 98, 99, 90, and 85% identity to GadB of L. plantarum WCFS1 for L. paracasei PF6, L. delbrueckii subsp. bulgaricus PR1, L. lactis PU1, and L. plantarum C48, respectively. Except for L. lactis PU1, the three lactobacillus strains survived and synthesized GABA under simulated gastrointestinal conditions. The findings of this study provide a potential basis for exploiting selected cheese-related lactobacilli to develop health-promoting dairy products enriched in GABA.     

Diversity of Lactic Acid Bacteria Associated with Fresh Coffee Cherries in Taiwan

A total of 102 lactic acid bacteria (LAB) were isolated from three different coffee farms in Taiwan. These isolates were classified and identified by the restriction fragment length polymorphism analysis and sequencing of 16S ribosomal DNA. Heterofermentative Leuconostoc, and Weissella species were the most common LAB found in two farms located at an approximate altitude of 800 m. Lactococcus lactis subsp. lactis was the most common LAB found in the remaining farm was located at an approximate altitude of 1,200 m. It is therefore suggested that the altitude and climate may affect the distribution of LAB. On the basis of phylogenetic analysis, two strains included in the genera Enterococcus were considered as two potential novel species or subspecies. In addition, a total of 34 isolates showed the antifungal activity against Aspergillus flavus. Moreover, seven Lactococcus lactis subsp. lactis strains and one Enterococcus faecalis strain were found to have bacteriocin-like inhibitory substance-producing capability. These results suggest that various LAB are associated with fresh coffee cherries in Taiwan. Some of the isolates found in this study showed potential as antifungal agents.     

Screening and molecular identification of potential probiotic lactic acid bacteria in effluents generated during ogi production

Screening and molecular identification of probiotic lactic acid bacteria (LAB) in effluents generated during the production of ogi, a fermented cereal (maize, millet, and sorghum) were done. LAB were isolated from effluents generated during the first and second fermentation stages in ogi production. Bacterial strains isolated were identified microscopically and phenotypically using standard methods. Probiotic potential properties of the isolated LAB were investigated in terms of their resistance to pH 1.5 and 0.3% bile salt concentration for 4 h. The potential LAB isolates ability to inhibit the growth of pathogenic organisms (Escherichia coli, Staphylococcus aureus, and Salmonella typhimurium) was evaluated in vitro. The pH and LAB count in the effluents ranged from 3.31 to 4.49 and 3.67 to 4.72 log cfu/ml, respectively. A total of 88 LAB isolates were obtained from the effluents and only 10 LAB isolates remained viable at pH 1.5 and 0.3% bile salt. The zones of inhibition of the LAB isolates with probiotic potential ranged from 7.00 to 24.70 mm against test organsisms. Probiotic potential LAB isolates were molecularly identified as Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus reuteri, Enterococcus faecium, Pediococcus acidilactici, Pediococcus pentosaceus, Enterococcus faecalis, and Lactobacillus brevis. Survival and proliferation of LAB isolates at low pH, 0.3% bile salt condition, and their inhibition against some test pathogens showed that these LAB isolates could be a potential probiotics for research and commercial purposes.     

Diversity and Probiotic Potential of Lactic Acid Bacteria Isolated from Horreh,a Traditional Iranian Fermented Food

The aim of this study was to evaluate the probiotic potential of lactic acid bacteria (LAB) strains isolated from Horreh. Some probiotic properties, e.g., resistance to acid, bile tolerance, antibacterial activity, and antibiotic susceptibility, were investigated. A total of 140 Gram-positive and catalase-negative isolates from Horreh were subjected to identification and grouping by cultural methods and the 16S rRNA sequencing. The new isolates were identified to be Lactobacillus (fermentum, plantarum, and brevis) Weissella cibaria, Enterococcus (faecium and faecalis), Leuconostoc (citreum and mesenteroides subsp. mesenteroides) and Pediococcus pentosaceus. Probiotic potential study of LAB isolates showed that Lb. plantarum and Leu. mesenteroides subsp. mesenteroides isolates were able to grow at pH 2.5 and 3.5. Lactobacillus plantarum (isolate A44) showed the highest cell hydrophobicity (84.5%). According to antibacterial activity tests, Listeria innocua and Staphylococcus aureus were the most sensitive indicators against the selected LAB strains, while Escherichia coli and Bacillus cereus were the most resistant. In addition, all the isolated LAB species were resistant to vancomycin. The results of the present study suggested that the Lactobacillus fermentum and plantarum isolated from Horreh, characterized in this study, have potential use for industrial purposes as probiotics.     

内蒙古呼伦贝尔地区传统发酵乳中乳酸菌的多样性分析

【目的】对内蒙古呼伦贝尔地区传统发酵乳制品中乳酸菌资源的生物多样性进行研究。【方法】采用纯培养和16S rRNA基因序列分析法对内蒙古呼伦贝尔地区传统发酵乳中的乳酸菌进行多样性分析。【结果】从8份传统发酵乳制品(6份酸牛奶和2份酸马奶)样品中分离到24株乳酸菌,通过16S rRNA基因序列分析和系统进化关系分析将24株乳酸菌鉴定为2株Lactobacillus kefiranofaciens、2株Lactobacillus kefiri、5株Lactobacillus paracasei、3株Lactobacillus plantarum、1株Lactobacillus rhamnosus、6株Lactococcus lactis subsp.lactis、2株Leuconostoc mesenteroides subsp.dextranicum、2株Streptococcus thermophilus和1株Enterococcus faecium。【结论】Lactococcus lactis subsp.lactis为内蒙古呼伦贝尔地区传统发酵乳制品的优势菌种,占总分离株的25%,其次为Lactobacillus paracasei,占总分离株的20.83%。     

Production of a Heterologous Nonheme Catalase by Lactobacillus casei: an Efficient Tool for Removal of H2O2 and Protection of Lactobacillus bulgaricus from Oxidative Stress in Milk

Lactic acid bacteria (LAB) are generally sensitive to H₂O₂, a compound that they can paradoxically produce themselves, as is the case for Lactobacillus bulgaricus. Lactobacillus plantarum ATCC 14431 is one of the very few LAB strains able to degrade H₂O₂ through the action of a nonheme, manganese-dependent catalase (hereafter called MnKat). The MnKat gene was expressed in three catalase-deficient LAB species: L. bulgaricus ATCC 11842, Lactobacillus casei BL23, and Lactococcus lactis MG1363. While the protein could be detected in all heterologous hosts, enzyme activity was observed only in L. casei. This is probably due to the differences in the Mn contents of the cells, which are reportedly similar in L. plantarum and L. casei but at least 10- and 100-fold lower in Lactococcus lactis and L. bulgaricus, respectively. The expression of the MnKat gene in L. casei conferred enhanced oxidative stress resistance, as measured by an increase in the survival rate after exposure to H₂O₂, and improved long-term survival in aerated cultures. In mixtures of L. casei producing MnKat and L. bulgaricus, L. casei can eliminate H₂O₂ from the culture medium, thereby protecting both L. casei and L. bulgaricus from its deleterious effects.     

Use of real time PCR to determine population profiles of individual species of lactic acid bacteria in alfalfa silage and stored corn stover

Real-time polymerase chain reaction (RT-PCR) was used to quantify seven species of lactic acid bacteria (LAB) in alfalfa silage prepared in the presence or absence of four commercial inoculants and in uninoculated corn stover harvested and stored under a variety of field conditions. Species-specific PCR primers were designed based on recA gene sequences. Commercial inoculants improved the quality of alfalfa silage, but species corresponding to those in the inoculants displayed variations in persistence over the next 96 h. Lactobacillus brevis was the most abundant LAB (12 to 32% of total sample DNA) in all of the alfalfa silages by 96 h. Modest populations (up to 10%) of Lactobacillus plantarum were also observed in inoculated silages. Pediococcus pentosaceus populations increased over time but did not exceed 2% of the total. Small populations (0.1 to 1%) of Lactobacillus buchneri and Lactococcus lactis were observed in all silages, while Lactobacillus pentosus and Enterococcus faecium were near or below detection limits. Corn stover generally displayed higher populations of L. plantarum and L. brevis and lower populations of other LAB species. The data illustrate the utility of RT-PCR for quantifying individual species of LAB in conserved forages prepared under a wide variety of conditions.Disclaimer: Mention of products is for informational purposes only and does not imply a recommendation or warranty by USDA over other products that may also be suitable     

Antimicrobial susceptibility of microflora from ovine cheese

Strains identified in ovine cheese and bryndza by matrix-assisted laser desorption/ionization time-of-flight analysis belonged to ten species of non-enterococcal lactic acid bacteria and included Lactobacillus casei/Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus helveticus, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus brevis, Lactococcus lactis, Pediococcus pentosaceus and Pediococcus acidilactici. The susceptibility toward antibiotics was determined in lactobacilli, lactococci and pediococci and also in Escherichia coli for comparison. Analysis of L. fermentum and pediococci revealed the presence of non-wild-type epidemiological cut-offs in streptomycin, clindamycin or gentamicin. E. coli were resistant to ampicillin, tetracycline, enrofloxacin and florfenicol. No extended spectrum β-lactamases were detected.