Isolation and identification of lactic acid bacteria from sediments of a coastal marsh using a differential selective medium

Aims:  To identify lactic acid bacteria (LAB) colonies isolated from sediments of a coastal marsh by the reduction of 2,3,5‐triphenyltetrazolium chloride (TTC) in MRS medium. Methods and Results:  Single colonies isolated from sediments of a coastal marsh by enrichment in MRS broth were selected from MRS‐TTC plates and classified according to colony phenotype based on TTC reduction. A total of 37 colonies grouped in seven different phenotypes were identified by analysis of its 16S ribosomal gene sequence. Most isolates belonged to the Firmicutes phylum, mainly to orders Bacillales and Lactobacillales. LAB were represented by 20 isolates, 15 of which belong to the genus Weissella. Conclusions:  Enrichment in MRS was highly selective for the isolation of bacteria belonging to phylum Firmicutes. Several different phenotypes were developed by LAB and must be considered during LAB isolation based on TTC reduction. Significance and Impact of the Study:  To our knowledge, this is the first study aimed at determining a relationship between colony phenotype from TTC reduction and a partial identification of isolates based on 16S ribosomal gene sequence similarities. Besides, this is the first report of isolation of W. cibaria from environmental samples.     

Isolation and characteristics of lactic acid bacteria from koshu vineyards in Japan

Aims:  To isolate, characterize and identify lactic acid bacteria (LAB) in the vineyards where koshu grapes, a primary wine grape cultivar in Japan, are grown.
Methods and Results:  Sixty samples, including leaves, undamaged grape berries and soil under damaged berries, were collected at four koshu vineyards in Yamanashi Prefecture, Japan. One hundred and 15 acid-producing cultures were isolated from these samples, and the isolates were divided into classes by phenotype and then into groups by restriction fragment length polymorphism analysis and sequencing of 16S ribosomal DNA (rDNA). Phenotypic and biochemical characteristics identified seven different bacterial groups (A to G). Lactococcus lactis ssp. lactis was the most abundant type of LAB distributed in three koshu vineyards, and Leuconostoc pseudomesenteroides was the most abundant LAB found in the remaining vineyard. Forty-six isolates produced bacteriocin-like inhibitory substances (BLIS) against the indicator strain Lactobacillus sakei JCM 1157^T.
Conclusions:  These results suggest that various LAB are distributed in koshu vineyards, of which a large number produce BLIS.
Significance and Impact of the Study:  This is the first report describing the distribution and varieties of LAB that exist in koshu vineyards.     

泡菜中优良乳酸菌的分离、鉴定及发酵特性

从几种泡菜中分离出86株菌,对其在适温和低温下产酸速率及硝酸盐降解能力进行测定,筛选出5株产酸速率快、硝酸盐降解能力强的菌株。经形态学鉴定及生理生化反应试验,初步鉴定为：植物乳杆菌2株,短乳杆菌1株,戊糖乳杆菌1株,肠膜明串珠菌葡聚糖亚种1株,并对5株菌的发酵性能进行了测定。     

Influence of carbohydrates on the isolation of lactic acid bacteria

Aims: To determine the influence of carbohydrates on enrichment isolation of lactic acid bacteria from different niches. Methods and Results: Lactic acid bacteria in three traditional fermented products in southern Africa (amasi, mahewu and tshwala) and in three fresh samples (two flowers and a fruit) were enrichment cultured in media supplemented with 13 different carbohydrates. Diversity of lactic acid bacteria was determined by PCR‐denaturing‐gradient gel electrophoresis. Carbohydrates used in enrichment media had a big impact on the isolation of lactic acid bacteria from fermented products. Depending on the carbohydrates tested, the number of species detected ranged from one to four in amasi, one to five in mahewu and one to three in tshwala. Fructose and mannitol selected for relatively higher numbers of lactic acid bacteria in fermented products. Specific relationships between substrates and lactic acid bacteria have been noted. On the other hand, small influences were found among carbohydrates tested in flowers and fruit. Conclusion: Carbohydrates have a big impact on the isolation of a variety of lactic acid bacteria in fermented food. Significance and Impact of the Study: This is the first study that reports the influence of carbohydrates on the enrichment of lactic acid bacteria.     

Removal of patulin from apple juice using inactivated lactic acid bacteria

Aims: Apples and apple products are the most notably commodities contaminated with patulin (PAT), which cause detrimental effects on human health and economic problems. The primary objective of this study was to investigate the removal of PAT contamination from apple juice using 10 different inactivated lactic acid bacteria (LAB) strains. Methods and Results: Significant quantities of PAT ranging from 47 to 80% were bound to all tested bacterial strains, whereas Lactobacillus rhamnosus 6224 and Enterococcus faecium 21605 caused a decrease of PAT by 80·4 and 64·5%, respectively. The results showed that the binding of PAT depends on the initial concentration of toxin and the adsorption temperature, also the differences in biomass existed among the 10 bacterial strains. IR analysis was performed to identify potential functional groups and the possible binding sites related to PAT adsorption. Conclusions: The removal of PAT was observed to be strain specific. The results indicated that the biosorption process did not affect the quality of juice. FTIR analysis showed that the cell wall plays a key role in PAT adsorption. Significance and Impact of the Study: Our results proof that inactivated LAB have the potential as a novel and promising adsorbent to bind PAT effectively.     

Isolation and characterization of lactic acid bacteria from suan-tsai (fermented mustard), a traditional fermented food in Taiwan

AIMS: To isolate, characterize, and identify lactic acid bacteria (LAB) in suan-tsai (fermented mustard), a traditional fermented food in Taiwan. METHODS AND RESULTS: Suan-tsai samples were collected at five time points from a fixed fermenting bucket. Fifty cultures were isolated from suan-tsai samples, and isolates were divided into classes by phenotype and then into groups by restriction-fragment length polymorphism analysis and sequencing of 16S ribosomal DNA. Phenotypic and biochemical characteristics identified two different bacterial groups (A and B), and the results showed that Pediococcus pentosaceus was the most abundant LAB during the initial fermentation time. However, the more NaCl-tolerant species Tetragenococcus halophilus took the place of P. pentosaceus and became the most abundant LAB later. All isolates were grown in de Man, Rogosa, and Sharpe (MRS) broth containing 6% NaCl, but T. halophilus could grow only in MRS broth containing 10% NaCl. CONCLUSIONS: These results suggest that the LAB P. pentosaceus and T. halophilus play roles in the fermentation of suan-tsai. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report describing the distribution and varieties of LAB that exist in the suan-tsai fermentation process.     

Evaluation of lactic acid bacteria autolysate for the supplementation of lactic acid bacteria fermentation

At the end of culture in a carbon-limited medium, i.e. the best conditions for subsequent autolysis, lactic acid bacteria were harvested and autolysed at 50 °C for 24 h. The resulting supernatant was then successfully tested as a substitute for industrial yeast extract for the supplementation of whey permeate and its conversion into lactic acid: for almost equivalent total nitrogen amounts of both supplements, the same growth and production rates were recorded.     

Isolation, characterization and identification of lactic acid bacteria and yeasts from sour Mifen, a traditional fermented rice noodle from China

Aims:  Considering the effect of natural fermentation on the textural improvement of fermented rice noodles in China and South Asia, and given the lack of reports concerning microbial populations and structure in the fermentation process, this study aims to determine the number of viable micro-organisms and identify the species isolated from the local factories, and to assess their potential use as a starter culture from their enzymatic profiles.
Methods and Results:  Fourteen samples from three local factories were analysed for the presence of micro-organisms. A total of 170 lactic acid bacteria (LAB) and 96 yeasts were isolated from the factories. The isolates were phenotypically characterized by using API 50 CHL kits, API 20 Strep kits, API ID 32 C kits and by performing additional biochemical tests. The enzymatic profiles of isolates were assessed by using API ZYM kits. Lactobacillus plantarum and Saccharomyces cerevisiae were identified as predominant species in the fermented supernatants. A majority of the isolates of LAB and yeasts displayed activities of α-glucosidase, β-glucosidase, lipase and trypsin.
Conclusions:  The microbial composition and strain characteristics present in the fermentation supernatant demonstrate that a majority of micro-organisms have the ability to digest starch, sugar, protein or lipid. It supports our previous work in which the rice starch was modified and purified by fermentation and thus improves the texture of rice noodles.
Significance and Impact of the Study:  The dominant strains would be important in developing a starter culture. The results can form the basis for the improvement of product quality and consistency.     

Genomics of lactic acid bacteria

Lactic acid bacteria (LAB) are found to occupy a variety of ecological niches including fermented foods as well as mucosal surfaces of humans and other vertebrates. This review is based on the genomic content of LAB that is responsible for the functional and ecological diversity of these bacteria. These genomes reveal an ongoing process of reductive evolution as the LAB have specialized to different nutritionally rich environments. Species-to-species variation in the number of pseudogenes as well as genes directing nutrient uptake and metabolism reflects the adaptation of LAB to food matrices and the gastrointestinal tract. Although a general trend of genome reduction was observed, certain niche-specific genes appear to be recently acquired and appear on plasmids or adjacent to prophages. Recent work has improved our understanding of the genomic content responsible for various phenotypes that continue to be discovered, as well as those that have been exploited by man for thousands of years.     

Exploiting expolysaccharides from lactic acid bacteria

Microbial exopolysaccharides (EPSs) synthesized by lactic acid bacteria (LAB) play a major role in the manufacturing of fermented dairy products. EPS production is characterized by a large variety in terms of quantity, chemical composition, molecular size, charge, type of sidechains and rigidity of the molecules. Monosaccharide unit's composition, linkages, charge and size determine the EPS' intrinsic properties and their interactions with other milk constituents. EPSs contribute to texture, mouthfeel, taste perception and stability of the final product. Furthermore, it was reported that EPS from food grade organisms, particularly LAB, have potential as food additives and as functional food ingredients with both health and economic benefits. A better understanding of structure-function relationships of EPS in a dairy food matrix and of EPS biosynthesis remain two major challenges for further applications of EPS and the engineering of functional polysaccharides.     

Peptidases and amino acid catabolism in lactic acid bacteria

The conversion of peptides to free amino acids and their subsequent utilization is a central metabolic activity in prokaryotes. At least 16 peptidases from lactic acid bacteria (LAB) have been characterized biochemically and/or genetically. Among LAB, the peptidase systems of Lactobacillus helveticus and Lactococcus lactis have been examined in greatest detail. While there are homologous enzymes common to both systems, significant differences exist in the peptidase complement of these organisms. The characterization of single and multiple peptidase mutants indicate that these strains generally exhibit reduced specific growth rates in milk compared to the parental strains. LAB can also catabolize amino acids produced by peptide hydrolysis. While the catabolism of amino acids such as Arg, Thr, and His is well understood, few other amino acid catabolic pathways from lactic acid bacteria have been characterized in significant detail. Increasing research attention is being directed toward elucidating these pathways as well as characterizing their physiological and industrial significance.     

Exocellular polysaccharides produced by lactic acid bacteria

Abstract The production of homopolysaccharides (dextrans, mutans) and heteropolysaccharides by lactic acid bacteria, their chemical composition, their structure and their synthesis are outlined. Mutans streptococci, which include Streptococcus mutans and S. sobrinus produce soluble and insoluble α-glucans. The latter may contain as much as 90%α-1–3 linkages and possess a marked ability to promote adherence to the smooth tooth surface causing dental plaque. Dextrans produced by Leuconostoc mesenteroides are high molecular weight α-glucans having 1–6, 1–4 and 1–3 linkages, varying from slightly to highly branched; 1–6 linkages are predominant. Emphasis is put on exopolysaccharide producing thermophilic and mesophilic lactic acid bacteria, which are important in the dairy industry. The produced polymers play a key role in the rheological behaviour and the texture of fermented milks. One of the main problems in this field is the transitory nature of the thickening trait. This instability is not yet completely understood. Controversial results exist on the sugar composition of the slime produced, but galactose and glucose have always been identified with galactose predominating in most cases.     

Biogenic amine production by lactic acid bacteria isolated from cider

AIMS: To study the occurrence of histidine, tyrosine and ornithine decarboxylase activity in lactic acid bacteria (LAB) isolated from natural ciders and to examine their potential to produce detrimental levels of biogenic amines. METHODS AND RESULTS: The presence of biogenic amines in a decarboxylase synthetic broth and in cider was determined by reversed-phase high-performance liquid chromatography (RP-HPLC). Among the 54 LAB strains tested, six (five lactobacilli and one oenococci) were biogenic amine producers in both media. Histamine and tyramine were the amines formed by the LAB strains investigated. Lactobacillus diolivorans were the most intensive histamine producers. This species together with Lactobacillus collinoides and Oenococcus oeni also seemed to produce tyramine. No ability to form histamine, tyramine or putrescine by Pediococus parvulus was observed, although it is a known biogenic amine producer in wines and beers. CONCLUSIONS: This study demonstrated that LAB microbiota growing in ciders had the ability to produce biogenic amines, particularly histamine and tyramine, and suggests that this capability might be strain-dependent rather than being related to a particular bacterial species. SIGNIFICANCE AND IMPACT OF THE STUDY: Production of biogenic amines by food micro-organisms has continued to be the focus of intensive study because of their potential toxicity. The main goal was to identify the microbial species capable of producing these compounds in order to control their presence and metabolic activity in foods.     

Vanillin production from simple phenols by wine-associated lactic acid bacteria

AIMS: The ability of lactic acid bacteria (LAB) to metabolize certain phenolic precursors to vanillin was investigated. METHODS AND RESULTS: Gas chromatography-mass spectrometry (GC-MS) or HPLC was used to evaluate the biosynthesis of vanillin from simple phenolic precursors. LAB were not able to form vanillin from eugenol, isoeugenol or vanillic acid. However Oenococcus oeni or Lactobacillus sp. could convert ferulic acid to vanillin, but in low yield. Only Lactobacillus sp. or Pediococcus sp. strains were able to produce significant quantities of 4-vinylguaiacol from ferulic acid. Moreover, LAB reduced vanillin to the corresponding vanillyl alcohol. CONCLUSIONS: The transformation of phenolic compounds tested by LAB could not explain the concentrations of vanillin observed during LAB growth in contact with wood. SIGNIFICANCE AND IMPACT OF THE STUDY: Important details of the role of LAB in the conversion of phenolic compounds to vanillin have been elucidated. These findings contribute to the understanding of malolactic fermentation in the production of aroma compounds.     

Isolation and characterization of fructophilic lactic acid bacteria from fructose-rich niches

Fourteen strains of fructophilic lactic acid bacteria were isolated from fructose-rich niches, flowers, and fruits. Phylogenetic analysis and BLAST analysis of 16S rDNA sequences identified six strains as Lactobacillus kunkeei, four as Fructobacillus pseudoficulneus, and one as Fructobacillus fructosus. The remaining three strains grouped within the Lactobacillus buchneri phylogenetic subcluster, but shared low sequence similarities to other known Lactobacillus spp. The fructophilic strains fermented only a few carbohydrates and fermented d-fructose faster than d-glucose. Based on the growth characteristics, the 14 isolates were divided into two groups. Strains in the first group containing L. kunkeei, F. fructosus, and F. pseudoficulneus grew well on d-fructose and on d-glucose with pyruvate or oxygen as external electron acceptors, but poorly on d-glucose without the electron acceptors. Strains in this group were classified as “obligately” fructophilic lactic acid bacteria. The second group contained three unidentified strains of Lactobacillus that grew well on d-fructose and on d-glucose with the electron acceptors. These strains grew on d-glucose without the electron acceptors, but at a delayed rate. Strains in this group were classified as facultatively fructophilic lactic acid bacteria. All fructophilic isolates were heterofermentative lactic acid bacteria, but “obligately” fructophilic lactic acid bacteria mainly produced lactic acid and acetic acid and very little ethanol from d-glucose. Facultatively fructophilic strains produced lactic acid, acetic acid and ethanol, but at a ratio different from that recorded for heterofermentative lactic acid bacteria. These unique characteristics may have been obtained through adaptation to the habitat.     

一株能降解胆固醇的乳酸菌的选育

利用选择性培养基从成年人的粪便中分离出1株能降解胆固醇的乳酸菌,该菌能以胆固醇作为生长的唯一能源。在10％牛奶的发酵试验中,该菌能使牛奶发酵并凝固。在液体发酵中,胆固醇的降解率为34．6％。经初步鉴定,该菌为双歧杆菌（Bifidoacterium sp.)。     

Boza, a natural source of probiotic lactic acid bacteria

Aims: To evaluate the probiotic properties of strains isolated from boza, a traditional beverage produced from cereals. Methods and Results: The strains survived low pH conditions (pH 3·0), grew well at pH 9·0 and were not inhibited by the presence of 0·3% (w/v) oxbile. Cytotoxicity levels of the bacteriocins, expressed as CC₅₀, ranged from 38 to 3776 μg ml^?1. Bacteriocin bacST284BZ revealed high activity (EC₅₀ = 735 μg ml^?1) against herpes simplex virus type 1. Growth of Mycobacterium tuberculosis was 69% repressed after 5 days in the presence of bacST194BZ. Various levels of auto‐cell aggregation and co‐aggregation with Listeria innocua LMG 13568 were observed. Adhesion of the probiotic strains to HT‐29 cells ranged from 18 to 22%. Conclusions: Boza is a rich source of probiotic lactic acid bacteria. All strains survived conditions simulating the gastrointestinal tract and produced bacteriocins active against a number of pathogens. Adherence to HT‐29 and Caco‐2 cells was within the range reported for Lactobacillus rhamnosus GG, a well‐known probiotic. In addition, the high hydrophobicity readings recorded define the strains as good probiotics. Significance and Impact of the Study: Boza contains a number of different probiotic lactic acid bacteria and could be marketed as a functional food product.     

A method for the identification of proteins secreted by lactic acid bacteria grown in complex media

Lactic acid bacteria (LAB) are known for their special nutritional requirements, being usually cultured in complex media to achieve optimal growth. In this paper, a protocol based on trichloroacetic acid precipitation of peptides and proteins is presented. The method has been tested on four probiotic LAB strains grown in De Man Rogosa Sharpe (MRS) broth, a complex medium that is often used for the culture of such bacteria. This protocol allowed the detection of 19 proteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 10 of them being successfully identified by tandem MS. Thereafter, the 10 were found to be secreted or surface associated by bioinformatic means. In conclusion, this work supplies a method for the identification of proteins secreted by LAB, allowing discrimination between the proteins present in the MRS and those produced by probiotic LAB.     

一株产共轭亚油酸乳酸菌的鉴定及其特性

从酸菜汁中分离筛选到一株产共轭亚油酸(CLA)能力较高的乳酸菌。经鉴定,确定为植物乳杆菌Lactobacliius plantarum。微氧条件可提高CLA的产量,催化亚油酸(LA)生成CLA的酶受着LA的诱导。37℃对细胞生长和CLA生成最为有利。对数生长期为6～12h,18h后进入稳定期。在14～22h,CLA生成量快速增加,24h时达到最高值。该菌的培养物经萃取、甲酯化后,进行了气相色谱分离,生成的CLA产物为c9/t9,c11-CLA和t10,c12-CLA异构体的混合物。