Discrimination of probiotic Lactobacillus strains for poultry by repetitive sequenced‐based PCR fingerprinting

BACKGROUND: Four repetitive element sequence‐based polymerase chain reaction (rep‐PCR) methods, namely repetitive extragenic palindromic PCR (REP‐PCR), enterobacterial repetitive intergenic consensus PCR (ERIC‐PCR), polytrinucleotide (GTG)₅‐PCR and BOX‐PCR, were evaluated for the molecular differentiation of 12 probiotic Lactobacillus strains previously isolated from the gastrointestinal tract of chickens and used as a multistrain probiotic. This study represents the first analysis of the comparative efficacy of these four rep‐PCR methods and their combination (composite rep‐PCR) in the molecular typing of Lactobacillus strains based on a discriminatory index (D). RESULTS: Species‐specific and strain‐specific profiles were observed from rep‐PCR. From the numerical analysis of composite rep‐PCR, BOX‐PCR, (GTG)₅‐PCR, REP‐PCR and ERIC‐PCR, D values of 0.9118, 0.9044, 0.8897, 0.8750 and 0.8529 respectively were obtained. Composite rep‐PCR analysis was the most discriminative method, with eight Lactobacillus strains, namely L. brevis ATCC 14869^T, L. reuteri C 10, L. reuteri ATCC 23272^T, L. gallinarum ATCC 33199^T, L. salivarius ATCC 11741^T, L. salivarius I 24, L. panis JCM 11053^T and L. panis C 17, being differentiated at the strain level. CONCLUSION: Composite rep‐PCR analysis is potentially a useful fingerprinting method to discriminate probiotic Lactobacillus strains isolated from the gastrointestinal tract of chickens. Copyright © 2011 Society of Chemical Industry     

罗非鱼源肠道益生菌的分离鉴定及其体外益生特性分析

为了筛选出优良的鱼源益生菌，作者以吉富罗非鱼(Oreochromis niloticus)为实验材料，进行肠道芽孢杆菌和乳酸菌的分离、鉴定，并对分离得到的菌株进行抑菌活性、耐酸性、耐胆盐、人工胃肠液耐受、溶血活性和抗生素敏感性等体外益生特性分析。结果表明，从罗非鱼肠道中共分离得到109株菌，其中包括79株乳酸菌和30株芽孢杆菌，经过一系列的实验最终筛选出3株乳酸乳球菌（Lactococcus lactis）。生物学特性研究表明，3株菌均无溶血活性，对抗生素具有一定的敏感性，对强酸、胆盐和人工胃肠液具有一定的耐受性，对水产病原菌具有不同的抑菌活性。筛选得到的3株菌丰富了鱼源益生菌资源，它们均可作为优良的鱼源益生菌候选菌株。其中，菌株R1对强酸和人工胃肠液耐受性最强，菌株R29耐胆盐能力最强，可进行进一步的益生特性研究，挖掘其作为食品源益生菌的益生效果。     

Encapsulation in an alginate–goats’ milk–inulin matrix improves survival of probiotic Bifidobacterium in simulated gastrointestinal conditions and goats’ milk yoghurt

In this work, a new encapsulating matrix, alginate–goats’ milk–inulin, was used to encapsulate Bifidobacterium animalis subsp. lactis BB‐12. The addition of inulin resulted in capsules with a compact structure, and a higher probiotic cell count under simulated gastrointestinal conditions and in probiotic goats’ milk yoghurt during refrigerated storage. Encapsulation of the probiotic bacteria led to slower post‐acidification yoghurts. The results of this study showed that the alginate–goats’ milk–inulin matrix has potential to be used as a new encapsulation material to encapsulate probiotics for use in goats’ milk‐based probiotic fermented dairy products, avoiding the cross‐contamination caused by using capsules based on cows’ milk.     

The gyrase B gene as a molecular marker to resolve interspecific relationships within the Acetobacter pasteurianus group and a novel target for species-specific PCR

Members of the Acetobacter pasteurianus are popular acetic acid bacteria (AAB) for the production of vinegar. Neither phenotypic nor the most frequently applied genotypic marker (16S ribosomal DNA) provides sufficient resolution for accurate identification of the AAB strains. In this study, the gyrB gene was used for species discrimination by direct DNA sequencing and as marker in a species-specific PCR assay. All examined A. pasteurianus strains were clearly distinguished from the closely related species by comparative sequence analysis of the gyrB gene. The average sequence similarity for the gyrB gene (82.2 %) among type strains was significantly lower than that of the 16S rRNA sequence (98.2 %). Therefore, the gyrB gene can be proposed as an additional molecular marker for A. pasteurianus and related taxa that provides higher resolution than 16S rRNA. In addition, the species-specific primers were also developed based on the gyrB and 16S rRNA gene sequences, which were then employed for PCR using the template DNA of Acetobacter strains. The PCR primer pairs were shown to be specific for A. pasteurianus, A. peroxydans and papayae. Our data indicate that the phylogenetic relationships in the A. pasteurianus group are easily resolved by direct sequencing of the gyrB gene and combined with species-specific PCR assays.     

AN ACETOBACTER LETHAL TO YEASTS IN BOTTLED BEER

A strain of Acetobacter killed yeasts in bottled beer and so prevented natural conditioning from taking place. This ability to kill yeasts, which was not shared by other strains of Acetobacter, could not be divorced from the simultaneous presence of Acetobacter and yeasts. The active principle could pass through a dialysis membrane but was very quickly destroyed and was not isolated. The activity of the Acetobacter was lost if the bacteria were killed by heat or chemicals; it was maintained if they were killed by streptomycin. The lethal activity against yeasts was increased with rising temperature, it was counteracted by oxygen, it was enhanced by alcohol, and it was strongest at pH 3·5–4·5 and absent above pH 5. The active strain closely resembled Acetobacter rancens and was similar to an authentic culture of A. rancens although the latter had no anti-yeast activity. Many species of yeasts were killed by this strain of Acetobacter; lactobacilli were also killed but pediococci were not.     

Usefulness of a set of simple in vitro tests for the screening and identification of probiotic candidate strains for dairy use

A set of simple in vitro tests (identification by species-specific PCR, genetic diversity, phage sensitivity, growth and viability in milk, resistance to salts and flavor compounds, bacterial interactions, tolerance to simulated gastric juice and bile, bile salts deconjugation, hydrophobicity and β-galactosidase and antibacterial activities), that can be carried out in almost every laboratory of microbiology, mainly in developing countries where there is often limited access to sophisticated techniques, allowed us to identify, among 19 intestinal human isolates, a potential candidate for new probiotic dairy foods for the local market. Lactobacillus gasseri LgF_37/1 performed well in the culture media used for the enumeration of probiotic bacteria in argentinian dairy products. This strain showed also high tolerance to the technological challenges assessed, bile salts resistance, the capacity to produce bacteriocin-like metabolites, to inhibit pathogenic bacteria, to deconjugate bile salts and high hydrophobicity. Further in vivo research should be carried out with this strain before claiming probiotic properties for it. However, the use of a set of simple in vitro techniques proved to be important to determine which strains should undergo future and more complex studies.     

Binding of aflatoxin B1 by probiotic bacteria

The ability of six probiotic bacteria to bind a common food carcinogen, aflatoxin B₁, was assessed. The studied strains included Lactobacillus strains and one Bifidobacterium strain. The strains were incubated in vitro with alfatoxin B₁ and the toxin residue in the supernatant was measured using high‐performance liquid chromatography. The aflatoxin‐binding capacity of the strains was found to range from 5.8 to 31.3%. The results further support the observation that a number of probiotic bacteria are able to bind specific dietary contaminants. Although the extent of binding varies depending on the bacterial strain used, the data may explain some of the antimutagenic and anticarcinogenic effects of probiotic micro‐organisms. © 2000 Society of Chemical Industry     

The isolation and identification of microbes from a fermented tea beverage, Haipao, and their interactions during Haipao fermentation

Samples of Haipao from three cities of Taiwan were analyzed for their microbial population. Two species of acetic acid bacteria and three species of yeast were isolated from tea fungus Haipao using appropriate isolation media. The isolated bacteria were identified asAcetobacter acetiandAcetobacter pasteurianus, based on their biochemical properties, and compared with those of the type strains of the genusAcetobacter. The yeasts wereSaccharomyces cerevisiae, Zygosaccharomyces bailii, andBrettanomyces bruxellensisaccording to conventional phenotypic characterization combined with the Yeast Identification Program. The brew broth analyzed by high−performance liquid chromatography (HPLC) was shown to contain glycerol, acetic acid and ethanol. The symbiosis phenomenon between the yeast andAcetobacterwas studied. It was found that the autoclaved yeast cells and ethanol produced by yeasts were helpful forAcetobacterto grow or produce acetic acid. The acetic acid produced byAcetobactercould stimulate the yeast to produce ethanol. The ethanol and acetic acid produced by yeasts andAcetobactermight prevent the competition from other micro-organisms.     

In vitro characterisation of probiotic properties of Enterococcus faecium and Enterococcus durans strains isolated from raw milk and traditional dairy products

In this study, some probiotic characteristics such as antimicrobial activity, vancomycin resistance, growth ability at pH, resistance to bile salts, bile salt deconjugation and hydrophobicity of 30 Enterococcus faecium and Enterococcus durans strains isolated and identified from raw milk and various dairy products were investigated. According to the study results, antimicrobial activity profiling, pH and bile salt resistance and bile salt deconjugation ability of Enterococcus strains varied depending on the species and strains and all the strains showed resistance to the tested bile salt concentrations. It was concluded that the E. faecium and E. durans strains tested showed probiotic characteristics and have the potential to be used in food production.     

Identification of Acetobacter strains from Thai fermented rice products based on the 16S rRNA gene sequence and 16S-23S rRNA gene internal transcribed spacer restriction analyses

BACKGROUND: Fermented rice flour (khao‐khab, a non‐glutinous rice) and related products are Thai traditional products. The types of acetic acid bacteria (AAB) microflora in khao‐khab have not been reported. In this study, Acetobacter strains were isolated and identified based on the phenotypic and chemotaxonomic characteristics and molecular aspects. RESULTS: Twenty‐five acetic acid bacteria isolated from fermented rice products and a starter for sweetened rice in Thailand by an enrichment culture approach, were assigned to the genus Acetobacter by phenotypic and chemotaxonomic characterisations. On the basis of the 16S rRNA gene sequence and 16S–23S rRNA gene ITS restriction analyses, 25 isolates were divided into six groups and identified at the specific level: (1) Group 1 included five isolates, which were identified as A. indonesiensis; (2) Group 2 included two isolates, which were identified as A. lovaniensis; (3) Group 3 included one isolate, which was identified as A. orientalis; (4) Group 4 included eleven isolates, which were identified as A. pasteurianus; (5) Group 5 included three isolates, which were identified as A. syzygii and (6) Group 6 included three isolates, which were unidentified and considered to constitute a new species. CONCLUSION: Results revealed that various Acetobacter species were distributed in Thai fermented rice flour and related products. A novel Acetobacter species was isolated from the product. Copyright © 2011 Society of Chemical Industry     

Performance in Nondairy Drinks of Probiotic L. casei Strains Usually Employed in Dairy Products

The increase in vegetarianism as dietary habit and the increased allergy episodes against dairy proteins fuel the demand for probiotics in nondairy products. Lactose intolerance and the cholesterol content of dairy products can also be considered two additional reasons why some consumers are looking for probiotics in other foods. We aimed at determining cell viability in nondairy drinks and resistance to simulated gastric digestion of commercial probiotic lactobacilli commonly used in dairy products. Lactobacillus casei LC‐01 and L. casei BGP 93 were added to different commercial nondairy drinks and viability and resistance to simulated gastric digestion (pH 2.5, 90 min, 37 °C) were monitored along storage (5 and 20 °C). For both strains, at least one nondairy drink was found to offer cell counts around 7 log orders until the end of the storage period. Changes in resistance to simulated gastric digestion were observed as well. Commercial probiotic cultures of L. casei can be added to commercial fruit juices after a carefull selection of the product that warrants cell viability. The resistance to simulated gastric digestion is an easy‐to‐apply in vitro tool that may contribute to product characterization and may help in the choice of the food matrix when no changes in cell viability are observed along storage. Sensorial evaluation is mandatory before marketing since the product type and storage conditions might influence the sensorial properties of the product due to the possibility of growth and lactic acid production by probiotic bacteria.     

Identification of yeast and acetic acid bacteria isolated from the fermentation and acetification of persimmon (Diospyros kaki)

Persimmon (Diospyros kaki) is a seasonal fruit with important health benefits. In this study, persimmon use in wine and condiment production was investigated using molecular methods to identify the yeast and acetic acid bacteria (AAB) isolated from the alcoholic fermentation and acetification of the fruit. Alcoholic fermentation was allowed to occur either spontaneously, or by inoculation with a commercial Saccharomyces cerevisiae wine strain, while acetification was always spontaneous; all these processes were performed in triplicates. Non-Saccharomyces yeast species were particularly abundant during the initial and mid-alcoholic fermentation stages, but S. cerevisiae became dominant toward the end of these processes. During spontaneous fermentation, S. cerevisiae Sc1 was the predominant strain isolated throughout, while the commercial strain of S. cerevisiae was the most common strain isolated from the inoculated fermentations. The main non-Saccharomyces strains isolated included Pichia guilliermondii, Hanseniaspora uvarum, Zygosaccharomyces florentinus and Cryptococcus sp. A distinct succession of AAB was observed during the acetification process. Acetobacter malorun was abundant during the initial and mid-stages, while Gluconacetobacter saccharivorans was the main species during the final stages of these acetifications. Four additional AAB species, Acetobacter pasteurianus, Acetobacter syzygii, Gluconacetobacter intermedius and Gluconacetobacter europaeus, were also detected. We observed 28 different AAB genotypes, though only 6 of these were present in high numbers (between 25%–60%), resulting in a high biodiversity index.     

Dynamics and species diversity of communities of lactic acid bacteria and acetic acid bacteria during spontaneous cocoa bean fermentation in vessels

To speed up research on the usefulness and selection of bacterial starter cultures for cocoa bean fermentation, a benchmark cocoa bean fermentation process under natural fermentation conditions was developed successfully. Therefore, spontaneous fermentations of cocoa pulp-bean mass in vessels on a 20 kg scale were tried out in triplicate. The community dynamics and kinetics of these fermentations were studied through a multiphasic approach. Microbiological analysis revealed a limited bacterial species diversity and targeted community dynamics of both lactic acid bacteria (LAB) and acetic acid bacteria (AAB) during fermentation, as was the case during cocoa bean fermentations processes carried out in the field. LAB isolates belonged to two main (GTG)₅-PCR clusters, namely Lactobacillus plantarum and Lactobacillus fermentum, with Fructobacillus pseudofilculneus occurring occasionally; one main (GTG)₅-PCR cluster, composed of Acetobacter pasteurianus, was found among the AAB isolates, besides minor clusters of Acetobacter ghanensis and Acetobacter senegalensis. 16S rRNA-PCR-DGGE revealed that L. plantarum and L. fermentum dominated the fermentations from day two until the end and Acetobacter was the only AAB species present at the end of the fermentations. Also, species of Tatumella and Pantoea were detected culture-independently at the beginning of the fermentations. Further, it was shown through metabolite target analyses that similar substrate consumption and metabolite production kinetics occurred in the vessels compared to spontaneous cocoa bean fermentation processes. Current drawbacks of the vessel fermentations encompassed an insufficient mixing of the cocoa pulp-bean mass and retarded yeast growth.     

Anti‐obesity effect of feeding probiotic dahi containing Lactobacillus casei NCDC 19 in high fat diet‐induced obese mice

Nowadays, studies about the anti‐obesity potential of probiotics are of growing interest. Lactobacilli are one of the well‐studied probiotics owing to their preventing effect on metabolic disorders. This study was undertaken to access the anti‐obesity effect of probiotic dahi containing Lactobacillus casei NCDC 19 on C57BL/6 mice. Feeding of probiotic dahi showed reduce body weight gain and epididymal fat weights. Moreover, blood glucose, plasma lipids and expression level of leptin were reduced and caecal bifidobacteria counts and adiponectin expression levels were significantly increased. It can be concluded that feeding of probiotic dahi containing L. casei NCDC 19 showed potential anti‐obesity effects.     

Molecular discrimination of new isolates of Bifidobacterium animalis subsp. lactis from reference strains and commercial probiotic strains

Fifteen new isolates from pig faeces were identified as Bifidobacterium animalis subsp. lactis by polymerase chain reaction (PCR) using primers specific for this subspecies. Ten of the isolates could be differentiated at strain level by randomly amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE). These new strains were different from the type strain and other reference strains of this subspecies, and from all commercial dairy strains with probiotic functionality. Thus, possibly some of the isolates are potential candidates for new probiotic Bifidobacterium strains that can be unambiguously identified by RAPD-PCR and PFGE, which could be of interest for the food industry. In contrast, reference strains and commercial dairy strains revealed great genetic homogeneity, showing almost identical DNA fingerprints using RAPD-PCR and PFGE. Some reference strains and all commercial probiotic strains that had been originally designated as B. animalis or B. lactis have to be assigned to B. animalis subsp. lactis to be correctly labelled.     

An Influence of Fructan Containing Concentrate from Jerusalem Artichoke Tubers on the Development of Probiotic Dairy Starters on Milk and Oat-based Substrates

Supplementation of milk and oat hydrolysate containing medium with Jerusalem artichoke concentrate (JAC) and subsequent fermentation with probiotic dairy starters resulted in substantial stimulation of probiotics Bifidobacterium lactis and Lactobacillus acidophilus as well as yogurt starter culture Lactobacillus bulgaricus development and acidification rate. The strain-specific responses of the general yogurt cultures, as well as probiotics to the addition of JAC, should be considered to achieve optimal composition of probiotic strains and conformable fermentation conditions. JAC is suggested to be perspective prebiotic additive for fermented synbiotic milks or oat-hydrolysate-based products.     

Evaluation of in vitro probiotic potential of phytase‐producing bacterial strain as a new probiotic candidate

Sixty‐three phytase‐producing bacterial strains were isolated from natural sources, and their probiotic potential was evaluated. Of these, only fifteen strains were selected on the basis of confirmatory plate assay. Among these, five phytase‐producing strains exhibiting potent probiotic properties were identified as Bacillus cereus P1, Bacillus subtilis P6, B. subtilis P7, Pseudomonas aeruginosa P12 and P. aeruginosa P15 on the basis of morphological, biochemical and molecular characteristics. Maximum phytase activity (2.74 EU mL^?1) with potential probiotic properties, that is more than 70% survivability at pH 2.0, up to 2.0% bile salt tolerance, sporulation efficiency of more than 80% and survival in anaerobic condition (94.31%), was revealed by B. subtilis P6 as compared to the well‐established commercial probiotic strains Lactobacillus sporogenes and Lactobacillus casei. Thus, phytase‐producing B. subtilis P6 with promising probiotic features can be used in food and animal feed applications for betterment of mankind after further validation.     

Determination of the relationship between oxalate degradation and exopolysaccharide production by different Lactobacillus probiotic strains

The aim of this study was to evaluate the protective effects of exopolysaccharide (EPS) production on resistance to gastrointestinal digestive conditions and oxalate by probiotic strains of the species Lactobacillus rhamnosus, Lactobacillus fermentum and Lactobacillus brevis. The oxalate‐degrading ability of the strains was determined using an enzymatic assay. The correlation between oxalate degradation rate and EPS production was not significant (P > 0.05); however, the high‐EPS‐producing L. fermentumIP5 and L. brevisYG7 strains showed high oxalate‐degrading activity, whereas the low‐EPS‐producing strain L. fermentumBP5 demonstrated low oxalate‐degrading activity. The present findings suggest that dietary supplementation with the probiotic strain L. fermentumIP5 could be a promising strategy for the prevention of oxalate stone disease.     

Identification and quantification of acetic acid bacteria in wine and vinegar by TaqMan–MGB probes

A Real-Time PCR (RT-PCR) assay was developed using TaqMan minor groove binder (MGB) probes for the specific detection and quantification of five acetic acid bacteria (AAB) species (Acetobacter pasteurianus, Acetobacter aceti, Gluconacetobacter hansenii, Gluconacetobacter europaeus and Gluconobacter oxydans) in wine and vinegar. The primers and probes, designed from the 16S rRNA gene, showed good specificity with the target AAB species. The technique was tested on AAB grown in glucose medium (GY) and inoculated samples of red wine and wine vinegar. Standard curves were constructed with the five target species in all these matrices. Quantification was linear over at least 5 log units using both serial dilution of purified DNA and cells. When this technique was tested in GY medium and inoculated matrices, at least 10²–10³ cells/ml were detected. To quantify low populations of AAB in microbiologically complex samples, a PCR enrichment including part of the 16S–23S rRNA gene ITS region was needed to increase the amount of target DNA compared to non-target DNA.     

Identification of Microbiota Present on the Surface of Taleggio Cheese Using PCR–DGGE and RAPD–PCR

Abstract: Microbial DNA from 9 batches of Taleggio PDO cheese sampled at various times during ripening, brines, swabs of wooden shelves used for cheese dry‐salting, and 13 commercial cheeses were analyzed by denaturing gradient gel electrophoresis (PCR‐DGGE) and/or random amplification of polymorphic DNA (RAPD‐PCR). Sequencing allowed the detection of 12 genera, 27 species, and 2 unclassified bacteria. Molecular analysis allowed for the detection of microorganisms not previously associated with Taleggio such as Lactobacillus paracasei, Carnobacterium maltaromaticum, Bacillus licheniformis, Corynebacterium variabile, Psychrobacter cibarius, and Staphylococcus carnosus. For the first time Massilia spp. was detected in a dairy ecosystem. Practical Application: Indigenous species and strains of bacteria identified by this study could be used for the selection of dairy cultures to be employed routinely by manufacturers to control the Taleggio cheese production. The new cultures may give the bases for driving dairy processes and, consequently, control the typical flavor resulting from metabolic actions of environmental microorganisms.