Phylotype-Level Characterization of Complex Communities of Lactobacilli Using a High-Throughput, High-Resolution Phenylalanyl-tRNA Synthetase (pheS) Gene Amplicon Sequencing Approach.

The lactobacilli identified to date encompass more than 270 closely related species that were recently reclassified into 26 genera. Because of their relevance to industry, there is a need to distinguish between closely related and yet metabolically and regulatory distinct species, e.g., during monitoring of biotechnological processes or screening of samples of unknown composition. Current available methods, such as shotgun metagenomics or rRNA gene-based amplicon sequencing, have significant limitations (high cost, low resolution, etc.). Here, we generated a phylogeny of lactobacilli based on phenylalanyl-tRNA synthetase (pheS) genes and, from it, developed a high-resolution taxonomic framework which allows for comprehensive and confident characterization of the community diversity and structure of lactobacilli at the species level. This framework is based on a total of 445 pheS gene sequences, including sequences of 276 validly described species and subspecies (of a total of 282, including the proposed L. timonensis species and the reproposed L. zeae species; coverage of 98%), and allows differentiation between 265 species-level clades of lactobacilli and the subspecies of L. sakei The methodology was validated through next-generation sequencing of mock communities. At a sequencing depth of ∼30,000 sequences, the minimum level of detection was approximately 0.02 pg per µl DNA (equaling approximately 10 genome copies per µl template DNA). The pheS approach, along with parallel sequencing of partial 16S rRNA genes, revealed considerable diversity of lactobacilli and distinct community structures across a broad range of samples from different environmental niches. This novel complementary approach may be applicable to industry and academia alike.IMPORTANCE Species formerly classified within the genera Lactobacillus and Pediococcus have been studied extensively at the genomic level. To accommodate their exceptional functional diversity, the over 270 species were recently reclassified into 26 distinct genera. Despite their relevance to both academia and industry, methods that allow detailed exploration of their ecology are still limited by low resolution, high cost, or copy number variations. The approach described here makes use of a single-copy marker gene which outperforms other markers with regard to species-level resolution and availability of reference sequences (98% coverage). The tool was validated against a mock community and used to address diversity of lactobacilli and community structure in various environmental matrices. Such analyses can now be performed at a broader scale to assess and monitor the assembly, structure, and function of communities of lactobacilli at the species level (and, in some cases, even at the subspecies level) across a wide range of academic and commercial applications.

Rapid and accurate identification of species of the genus Pediococcus isolated from Korean fermented foods by matrix-assisted laser desorption/ionization time-of-flight MS with local database extension.

Pediococci are halophilic lactic acid bacteria, within the family Lactobacillaceae, which are involved in the fermentation of various salted and fermented foods, such as kimchi and jeotgal. In this study, a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS method was developed for the rapid identification of species of the genus Pediococcus. Of the 130 Pediococcus spectra aligned with the Biotyper taxonomy database, 122 isolates (93.9 %) yielded log scores <1.7, which means they were not identifiable. After registering the spectra of 11 reference strains of the genus Pediococcus, all of the isolates were correctly identified, of which 84 (64.6 %) and 46 (35.4 %) were identified at the species and genus level, respectively. In comparing food origins, no relationship was found between the bacterial characteristics and food environment. We were able to produce a Biotyper system for identification of members of the genus Pediococcus with locally extended Pediococcus reference strains. The MALDI-TOF MS method is fast, simple and reliable for discriminating between species in the genus Pediococcus and therefore will be useful for quality control in determining the spoilage of alcoholic beverages or in the production of fermented food.

Biodiversity and technological potential of lactic acid bacteria isolated from spontaneously fermented amaranth sourdough.

UNLABELLED: Spontaneous fermented sourdoughs prepared from amaranth flour were investigated for the presence of autochthonous lactic acid bacteria (LAB) predominating microbiota. The doughs were fermented with daily backslopping on a laboratory scale at 30°C for 10 days. LAB counts ranged from 2·60 to 8·54 log CFU g(-1) with a pH declined from 6·2 to 3·8 throughout fermentation. The combined use of randomly amplified polymorphic DNA (RAPD)-PCR analysis and sequence analysis of 16S rRNA was applied for LAB intraspecies differentiation and taxonomic identification, respectively. Enterococcus, Pediococcus and Lactobacillus species were present in amaranth sourdoughs (AS). After the first refreshment step, Lactobacillus plantarum dominated AS until the end of fermentation. In coincidence, when DGGE analysis was performed, the occurrence of a progressive change in bacterial communities allowed the selection of Lact. plantarum as a dominant species. Moreover, technological, functional and safety characteristics of representative RAPD-biotypes were investigated. Lact. plantarum CRL1898 was selected as a potential candidate for gluten-free amaranth sourdough starter. SIGNIFICANCE AND IMPACT OF THE STUDY: Nowadays, there is an increasing interest in ancient noncereal gluten-free (GF) crops such as amaranth, due to their reported nutritional and health benefits. However, the use of these grains is still limited to traditional foods and bread making processes that are not yet well standardized. Results on the dynamics of autochthonous lactic acid bacteria (LAB) microbiota during laboratory spontaneous amaranth sourdoughs (AS) fermentation will contribute to overcome challenges for GF-fermented products development. In addition, knowledge about LAB diversity involving Enterococcus, Pediococcus and Lactobacillus species, with Lactobacillus plantarum predominating during AS fermentation, and their technological and functional properties provides the basis for the selection of autochthonous strains as starters cultures for novel gluten-free bakery products with enhanced nutritional, sensory and/or safety quality.

A Genomic View of Lactobacilli and Pediococci Demonstrates that Phylogeny Matches Ecology and Physiology.

Lactobacilli are used widely in food, feed, and health applications. The taxonomy of the genus Lactobacillus, however, is confounded by the apparent lack of physiological markers for phylogenetic groups of lactobacilli and the unclear relationships between the diverse phylogenetic groups. This study used the core and pan-genomes of 174 type strains of Lactobacillus and Pediococcus to establish phylogenetic relationships and to identify metabolic properties differentiating phylogenetic groups. The core genome phylogenetic tree separated homofermentative lactobacilli and pediococci from heterofermentative lactobacilli. Aldolase and phosphofructokinase were generally present in homofermentative but not in heterofermentative lactobacilli; a two-domain alcohol dehydrogenase and mannitol dehydrogenase were present in most heterofermentative lactobacilli but absent in most homofermentative organisms. Other genes were predominantly present in homofermentative lactobacilli (pyruvate formate lyase) or heterofermentative lactobacilli (lactaldehyde dehydrogenase and glycerol dehydratase). Cluster analysis of the phylogenomic tree and the average nucleotide identity grouped the genus Lactobacillus sensu lato into 24 phylogenetic groups, including pediococci, with stable intra- and intergroup relationships. Individual groups may be differentiated by characteristic metabolic properties. The link between phylogeny and physiology that is proposed in this study facilitates future studies on the ecology, physiology, and industrial applications of lactobacilli.

Identification and characterization of wild lactobacilli and pediococci from spontaneously fermented Mountain cheese.

The Traditional Mountain Malga (TMM) cheese is made from raw cow's milk by spontaneously fermentation in small farms called "Malga" located in Trentino region. This study was designed to characterize the lactic acid bacteria (LAB) growing on MRS medium, of TMM-cheese at the end of the ripening. Ninety-five LAB were isolated and genotypically characterized by Randomly Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) with two primers, species-specific PCR and partial sequencing of 16S rRNA gene. The 95 LAB clustered in 70 biotypes. Pediococcus pentosaceus and Lactobacillus paracasei were the dominant species. Isolates were tested for their growth properties, carbohydrate metabolism, acidifying ability, proteolytic and lipolytic activities, acetoin production, amino-peptidase (AP) activity, biogenic amines production, bile salts hydrolysis, conjugated linoleic acid and γ-aminobutyric acid production. Lb. paracasei isolates resulted to be well adapted to Malga environment and to show the best AP activity and acetoin production. TMM-cheese related LAB showed also interesting health promoting properties and produced bioactive substances. In particular, one Lb. brevis biotype produced a GABA mean value of 129 mg/L that is considered a high concentration. The results confirmed that TMM-cheese resident LAB could be exploited for dairy production.

Characterisation of the bacterial microbiota of the vagina of dairy cows and isolation of pediocin-producing Pediococcus acidilactici.

BACKGROUND: Uterine infections in dairy cows lower profitability of dairy operations. Infections of the reproductive tract are related to the overgrowth of pathogenic bacteria during the first three weeks after parturition. However, alterations in the vaginal microbiota composition in the first weeks after parturition remain poorly documented. RESULTS: In this study, bacteria isolated from the vagina of healthy pregnant, and infected postpartum cows were characterised by random amplification of polymorphic DNA (RAPD) analysis and partial 16S ribosomal RNA (rDNA) gene sequencing. Populations of bacilli and lactic acid bacteria of the genera Enterococcus, Lactobacillus, and Pediococcus were present in both healthy and infected cows. Infected cows had a significant increase in the vaginal enteric bacteria population which consisted mainly of Escherichia coli. Three E. coli isolates harboured the gene coding for Shiga-like-toxin (SLT) I or II. Several isolates of the Pediococcus acidilactici were found to produce the bacteriocin pediocin AcH/PA-1. Quantitative PCR analyses of vaginal mucus samples collected from ten metritic cows before and after parturition confirmed the presence of the Lactobacillus group (Lactobacillus spp., Pediococcus spp., Leuconostoc spp., and Weissella spp.); Enterobacteriaceae, E. coli, and bacilli. The presence of the pediocin AcH/PA-1 structural gene and SLT genes were also confirmed with qPCR. CONCLUSIONS: In conclusion, overgrowth of pathogenic bacteria, particularly E. coli, after parturition likely contributes to the development of metritis. Our microbiota analysis extends the information related to the composition of commensal bacteria in the bovine female reproductive tract and may facilitate the development of novel intervention strategies for prevention of uterine infections in dairy cows.

Bacterial population dynamics during the ensiling of Medicago sativa (alfalfa) and subsequent exposure to air.

AIMS: To describe, at high resolution, the bacterial population dynamics and chemical transformations during the ensiling of alfalfa and subsequent exposure to air. METHODS AND RESULTS: Samples of alfalfa, ensiled alfalfa and silage exposed to air were collected and their bacterial population structures compared using 16S rRNA gene libraries containing approximately 1900 sequences each. Cultural and chemical analyses were also performed to complement the 16S gene sequence data. Sequence analysis revealed significant differences (P < 0·05) in the bacterial populations at each time point. The alfalfa-derived library contained mostly sequences associated with the Gammaproteobacteria (including the genera: Enterobacter, Erwinia and Pantoea); the ensiled material contained mostly sequences associated with the lactic acid bacteria (LAB) (including the genera: Lactobacillus, Pediococcus and Lactococcus). Exposure to air resulted in even greater percentages of LAB, especially among the genus Lactobacillus, and a significant drop in bacterial diversity. CONCLUSIONS: In-depth 16S rRNA gene sequence analysis revealed significant bacterial population structure changes during ensiling and again during exposure to air. SIGNIFICANCE AND IMPACT OF THE STUDY: This in-depth description of the bacterial population dynamics that occurred during ensiling and simulated feed out expands our knowledge of these processes.

Genotypic and phenotypic diversity of Pediococcus pentosaceus strains isolated from food matrices and characterisation of the penocin operon.

Lactic acid bacteria (LAB) are widely used in the food industry. Pediococcus spp. belong to the LAB group and include several species that are essential for the quality of fermented food. Pediococcus pentosaceus is the species that is most frequently isolated from fermented food and beverages but its uncontrolled growth during food fermentation processes can contribute to undesired flavours. Hence, the characterisation of these bacteria at the strain level is of great importance for the quality of fermented products. Despite their importance, misidentification at the species level is common for members of the genus Pediococcus. To clarify the taxonomic relationships among strains, a multilocus sequencing approach was developed for the characterisation of a collection of 29 field strains, 1 type strain and 1 reference strain of P. pentosaceus isolated from food. These strains were also tested for several phenotypic properties of technological interest and for the production of bacteriocins. The chromosomal operon involved in the synthesis of the bacteriocin penocin was also investigated. The present study enabled a good genomic characterisation, identifying 17 sequence types, with an overview of phenotypic characteristics related to different technological abilities, and also provides a thorough characterisation of the operon involved in penocin production.

Pediococcus acidilactici endocarditis successfully treated with daptomycin.

This report describes the first case of persistent bacteremia with endocarditis caused by Pediococcus acidilactici in a 32-year-old male with a history of short gut syndrome following a small bowel transplant. The results showed the utility of sequencing the intergenic spacer region for species identification and successful treatment using daptomycin.

Isolation of Pediococcus acidilactici Kp10 with ability to secrete bacteriocin-like inhibitory substance from milk products for applications in food industry.

BACKGROUND: Lactic acid bacteria (LAB) can be isolated from traditional milk products. LAB that secrete substances that inhibit pathogenic bacteria and are resistant to acid, bile, and pepsin but not vancomycin may have potential in food applications. RESULTS: LAB isolated from a range of traditional fermented products were screened for the production of bacteriocin-like inhibitory substances. A total of 222 LAB strains were isolated from fermented milk products in the form of fresh curds, dried curds, and ghara (a traditional flavor enhancer prepared from whey), and fermented cocoa bean. Eleven LAB isolates that produced antimicrobial substances were identified as Lactococcus lactis, Lactobacillus plantarum, and Pediococcus acidilactici strains by biochemical methods and 16S rDNA gene sequencing. Of these, the cell-free supernatant of Kp10 (P. acidilactici) most strongly inhibited Listeria monocytogenes. Further analysis identified the antimicrobial substance produced by Kp10 as proteinaceous in nature and active over a wide pH range. Kp10 (P. acidilactici) was found to be catalase-negative, able to produce ß-galactosidase, resistant to bile salts (0.3%) and acidic conditions (pH 3), and susceptible to most antibiotics. CONCLUSION: Traditionally prepared fermented milk products are good sources of LAB with characteristics suitable for industrial applications. The isolate Kp10 (P. acidilactici) shows potential for the production of probiotic and functional foods.

Pediococcus lolii DSM 19927T and JCM 15055T are strains of Pediococcus acidilactici.

Strain NGRI 0510Q(T), isolated from ryegrass silage, was recently classified as a representative of a novel Pediococcus species, Pediococcus lolii Doi et al. 2009. It was deposited in the DSMZ and JCM culture collections as DSM 19927(T) and JCM 15055(T), respectively. A polyphasic taxonomic study, including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, pheS and 16S rRNA gene sequence analysis, fluorescent amplified fragment length polymorphism and DNA-DNA hybridization, was used to prove that both subcultures of the type, and only, strain of this species are strains of Pediococcus acidilactici.

Lactobacillus and Pediococcus species richness and relative abundance in the vagina of rhesus monkeys (Macaca mulatta).

BACKGROUND: The rhesus monkey is an important animal model to study human vaginal health to which lactic acid bacteria play a significant role. However, the vaginal lactic acid bacterial species richness and relative abundance in rhesus monkeys is largely unknown. METHODS: Vaginal swab samples were aseptically obtained from 200 reproductive-aged female rhesus monkeys. Following Rogosa agar plating, single bacterial colonies representing different morphotypes were isolated and analyzed for whole-cell protein profile, species-specific polymerase chain reaction, and 16S rRNA gene sequence. RESULTS: A total of 510 Lactobacillus strains of 17 species and one Pediococcus acidilactici were identified. The most abundant species was Lactobacillus reuteri, which colonized the vaginas of 86% monkeys. Lactobacillus johnsonii was the second most abundant species, which colonized 36% of monkeys. The majority of monkeys were colonized by multiple Lactobacillus species. CONCLUSIONS: The vaginas of rhesus monkeys are frequently colonized by multiple Lactobacillus species, dominated by L. reuteri.

Genetic screening of functional properties of lactic acid bacteria in a fermented pearl millet slurry and in the metagenome of fermented starchy foods.

Lactic acid bacteria (LAB) (n = 152) in African pearl millet slurries and in the metagenomes of amylaceous fermented foods were investigated by screening 33 genes involved in probiotic and nutritional functions. All isolates belonged to six species of the genera Pediococcus and Lactobacillus, and Lactobacillus fermentum was the dominant species. We screened the isolates for the abilities to survive passage through the gastrointestinal tract and to synthesize folate and riboflavin. The isolates were also tested in vitro for their abilities to survive exposure to bile salts and to survive at pH 2. Because the ability to hydrolyze starch confers an ecological advantage on LAB that grow in starchy matrixes as well as improving the nutritional properties of the gruels, we screened for genes involved in starch metabolism. The results showed that genes with the potential ability to survive passage through the gastrointestinal tract were widely distributed among isolates and metagenomes, whereas in vitro tests showed that only a limited set of isolates, mainly those belonging to L. fermentum, could tolerate a low pH. In contrast, the wide distribution of genes associated with bile salt tolerance, in particular bsh, is consistent with the high frequency of tolerance to bile salts observed. Genetic screening revealed a potential for folate and riboflavin synthesis in both isolates and metagenomes, as well as high variability among genes related to starch metabolism. Genetic screening of isolates and metagenomes from fermented foods is thus a promising approach for assessing the functional potential of food microbiotas.

Evaluation of yogurt and various beverages as carriers of lactic acid bacteria producing 2-branched (1,3)-ß-D-glucan.

Probiotic cultures are increasingly being incorporated into a wide variety of food products. Although lactobacilli and bifidobacteria are the most frequently used, other lactic acid bacteria (LAB) have been reported to be potential probiotics. Of these, the cider isolates Pediococccus parvulus (strains 2.6 and CUPV22) and Lactobacillus suebicus CUPV221 produce a 2-branched (1,3)-ß-d-glucan exopolysaccharide that decreases serum cholesterol levels and affects the activation of human macrophages. For this reason, these 3 strains were incorporated into yogurt, orange juice, and 2 juice-milk beverages to evaluate the effect of the food matrix on the resistance of these strains to simulated gastrointestinal tract conditions. Our results showed that incorporation of the LAB did not significantly affect the physical and rheological properties of the food matrices tested. When incorporated in yogurt, LAB strains population decreased by 2 to 3 log orders of magnitude during the shelf life of the product (28 d). However, no significant decrease was observed in the juice and juice-milk beverages during the same storage period, except for Lb. suebicus, whose viability decreased by 3 log orders of magnitude. When strains were subjected to gastrointestinal tract conditions, a decrease in the survival was observed at the lower pH (1.8). However, incorporation of these LAB strains into orange juice increases their resistance to lower pH conditions, thus improving survival to gastrointestinal stress. Moreover, a protective effect was observed for P. parvulus CUPV22 and 2.6 to gastric stress in juice-milk beverages and to gastrointestinal stress in yogurt. Lactobacillus suebicus CUPV221 did not survive when incorporated into yogurt and juice-milk beverage.

Genotypic diversity of lactic acid bacteria isolated from African traditional alkaline-fermented foods.

AIM: To identify and compare lactic acid bacteria (LAB) isolated from alkaline fermentations of cassava (Manihot esculenta Crantz) leaves, roselle (Hibiscus sabdariffa) and African locust bean (Parkia biglobosa) seeds for production of, respectively, Ntoba Mbodi, Bikalga and Soumbala. METHODS AND RESULTS: A total of 121 LAB were isolated, identified and compared by phenotyping and genotyping using PCR amplification of 16S-23S rDNA intergenic transcribed spacer (ITS-PCR), repetitive sequence-based PCR (rep-PCR) and DNA sequencing. The results revealed a diversity of genera, species and subspecies of LAB in African alkaline fermentations. The isolates were characterized as nonmotile (in most cases) Gram-positive rods, cocci or coccobacilli, catalase and oxidase negative. ITS-PCR allowed typing mainly at species level, with differentiation of a few bacteria at subspecies level. Rep-PCR permitted typing at subspecies level and revealed significant genotypic differences between the same species of bacteria from different raw materials. DNA sequencing combined with use of API 50CHL and API 20Strep systems allowed identification of bacteria as Weissella confusa, Weissella cibaria, Lactobacillus plantarum, Pediococcus pentosaceus, Enterococcus casseliflavus, Enterococcus faecium, Enterococcus faecalis, Enterococcus avium and Enterococcus hirae from Ntoba Mbodi; Ent. faecium, Ent. hirae and Pediococcus acidilactici from Bikalga and Soumbala. CONCLUSION: LAB found in African alkaline-fermented foods belong to a range of genera, species and subspecies of bacteria and vary considerably according to raw material. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study confirms that LAB survive in alkaline fermentations, a first crucial stage in determining their significance and possible value as probiotic bacteria.

Purification of bacterial genomic DNA in less than 20 min using chelex-100 microwave: examples from strains of lactic acid bacteria isolated from soil samples.

We established a Chelex 100-Microwave method for the purification of bacterial genomic DNA (gDNA) in less than 20 min with high yield and good quality, useful for multiple purposes. It combines Chelex 100, proteinase K, RNase A and heating in a microwave oven. The resulting gDNA was used directly to identify bacterial species of the Order Lactobacillales by means of PCR amplification of their 16S rDNA gene, isolated from sediments on the Yucatan Peninsula, Mexico. This method produced gDNA free of phenolic and protein residual contaminants from 100 of these isolated bacteria. 16S rDNA amplification and sequencing showed Pediococcus acidilactici to prevail in inland lagoons, and Pediococcus pentosaceus, Lactobacillus plantarum, Lactobacillus sp., and Lactobacillus fermentum to be most abundant in the soils of livestock farms. The combination of Chelex 100, enzymes and microwave heating used in the Chelex 100-Microwave method produced large amounts of highly pure gDNA from Gram-positive and Gram-negative bacteria, in less than 20 min.

Culture-independent analysis of lactic acid bacteria diversity associated with mezcal fermentation.

Mezcal is an alcoholic beverage obtained from the distillation of fermented juices of cooked Agave spp. plant stalks (agave must), and each region in Mexico with denomination of origin uses defined Agave species to prepare mezcal with unique organoleptic characteristics. During fermentation to produce mezcal in the state of Tamaulipas, not only alcohol-producing yeasts are involved, but also a lactic acid bacterial community that has not been characterized yet. In order to address this lack of knowledge on this traditional Mexican beverage, we performed a DGGE-16S rRNA analysis of the lactic acid bacterial diversity and metabolite accumulation during the fermentation of a typical agave must that is rustically produced in San Carlos County (Tamaulipas, Mexico). The analysis of metabolite production indicated a short but important malolactic fermentation stage not previously described for mezcal. The denaturing gradient gel electrophoresis (DGGE) analysis of the 16S rRNA genes showed a distinctive lactic acid bacterial community composed mainly of Pediococcus parvulus, Lactobacillus brevis, Lactobacillus composti, Lactobacillus parabuchneri, and Lactobacillus plantarum. Some atypical genera such as Weissella and Bacillus were also found in the residual must. Our results suggest that the lactic acid bacteria could strongly be implicated in the organoleptic attributes of this traditional Mexican distilled beverage.

Diversity of lactic acid bacteria from Hussuwa, a traditional African fermented sorghum food.

The diversity of lactic acid bacteria associated with Hussuwa fermentation, a Sudanese fermented sorghum food, was studied using a polyphasic taxonomical approach. Predominant strains could be well characterised based on a combination of phenotypic tests and genotypic methods such as ARDRA, rep-PCR and RAPD-PCR, as well as 16S rRNA gene sequencing of representative strains. Thus, the majority (128 of 220, 58.3%) of strains exhibited phenotypic properties typical of heterofermentative lactobacilli and of these, 100 strains were characterised more closely using the genotyping methods. The majority (97/100) strains could be characterised as Lactobacillus fermentum strains. Seventy-two of 220 strains (32.7%) showed phenotypic properties that are characteristic of pediococci. Of 41 selected strains investigated by genotyping techniques, 38 (92.7%) could be characterised as Pediococcus acidilactici strains, while three (7.3%) could be characterised as Pediococcus pentosaceus strains. The Hussuwa fermentation thus appears to be dominated by L. fermentum strains and P. acidilactici strains. For this reason, we selected representative and predominant strains as potential starter cultures for Hussuwa fermentation. These strains, L. fermentum strains BFE 2442 and BFE 2282 and P. acidilactici strain BFE 2300, were shown on the basis of RAPD-PCR fingerprinting to predominate in a model fermentation when used as starter cultures inoculated at 1 x 10(6) CFU/g and to lower the pH of the fermentation to below pH 4.0 within 48 h. These cultures should be studied for further development as starter preparations in pilot scale studies in actual field fermentations.

Isolation and characterization of acid-sensitive mutants of Pediococcus acidilactici.

Acid-sensitive mutants of Pediococcus acidilactici BCC 9545, a starter culture of the Thai fermented pork sausage nham, were isolated as spontaneous neomycin resistant mutants. The mutants generally produced less acid and acidified the culture media less than the parent strain in a 72 h culturing period. Interestingly, the ATPase activities of the mutants did not differ considerably from that of the parent strain in acidic conditions. It was also found that the internal pH values of the mutant strains were somewhat lower in neutral environment, while at pH 5.0 their internal pHs were significantly lower compared to the parent's. Inhibiting the H(+)-ATPase activities in energized cells by N,N'-dicyclohexyl carbodiimide also revealed that protons were leaking from the mutants at neutral pH, which increased under acidic conditions. In contrast, the parent strain exhibited a smaller proton leak and only under acidic conditions. The membrane fatty acid analysis of the mutants indicated that under acidic conditions the mutants had a significantly smaller major unsaturated/saturated fatty acids ratio ((C(18:1)+C(18:3n6))/(C(16:0)+C(18:0))) compared to the parent strain's membrane. Taken together, these observations suggest there is a reasonable possibility that the membrane fatty acid profile differences in the mutants resulted in their acid-sensitivity.

[Lactic acid bacteria diversity in fermented cabbage estimated by culture-dependent and-independent methods].

OBJECTIVE: To study the diversity of lactic acid bacteria (LAB) and dominant LAB in fermented cabbage. METHODS: Culture-dependent and -independent (16S rRNA gene clone libraries were constructed) methods were used to determine the composition of LAB in fermented cabbage. RESULTS: Ninety LAB isolated from fermented cabbage were identified as species of Lactobacillus and Leuconostoc, whereas 115 clones of the 16S rRNA gene sequence from fermented cabbage DNA were identified as Lactobacillus, Weissella, Pediococcus and Leuconostoc. CONCLUSION: The significant difference of the LAB compositions by the two methods implies that some specialized nutrients may lead to a distinctive selection of the dominant organisms. Lactobacillus plantarum appeared as the dominant species in fermented cabbage by both methods.