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Lactic acid bacteria isolated from wine fermentations, particularly from the malolactic fermentation, and belonging to Lactobacillus plantarum, Lactobacillus hilgardii, Lactobacillus paracasei and Lactococcus lactis species were tested for their effectiveness in inhibiting the development of different microorganisms. The different strains showed, to varying degrees, an antagonistic effect against bacteria of the genera Bacillus and Staphylococcus. The specificity of the species L. hilgardii that inhibits only strains of the genus Bacillus is remarkable, on the other hand, L. plantarum was more effective against the strains of the genus Staphylococcus. The greatest effectiveness, considering both the degree of inhibition and the number of inhibited species, was presented by strains of L. lactis and L. paracasei. Seven strains belonging to the species that showed a more broad-spectrum activity, L. paracasei and L. plantarum, were also tested for their ability to inhibit the growth of fungi. All of them showed, in different degree (55&ndash, 76%), activity against Fusarium oxysporum. Finally, the ability of the L. paracasei LPAUV12 and L. plantarum LPLUV10 strains was evaluated to protect Lycopersicon esculentum plants against the fungus F. oxysporum and promote its growth. Strain LPLUV10, showed capacity to significantly inhibit the harmful effect of F. oxysporum in tomato plants as well as to significantly stimulate their growth.

Acetic and some lactic acid bacteria are the main reason for the loss of quality of musts and wines, giving rise to defects such as “vinegary”, “chopped” or preventing alcoholic and / or malolactic fermentation. The solution to this problem consists in the application of authorized bactericidal agents, such as sulfurous. The aim of this work is to isolate bacteriophages from musts and wines of different grape varieties, able to eliminate lactic and acetic acid bacteria spoilages. Musts obtained from grape-berries of Vitis vinifera cv. Chardonnay and Moscatel and a red wine made with the Tintilla de Rota variety were used to isolate bacteriophages. It were isolated by classical virology methods and identified by electron microscopy. Host bacteria used in the study were the lactic acid bacteria of the species Lactobacillus hilgardii, L. plantarum and Oenococcus oeni and the acetic bacteria Acetobacter aceti. A comparative study was performed on musts and wines, previously inoculated with bacteria, by phage titration and SO2 addition to study the effectiveness of bacteriophages against bacteria. Bacteriophages were obtained from all musts and wine, belonging to the order of Caudovirals and Tectivirals. The comparative study showed that a cocktail of bacteriophages at low concentration is as effective as sulfur.

While lactic acid bacteria are widespread in various ecological niches with the ex cess of nutrients and extensive use in food industry, many aspects of the nitrogen metabolism of remain unexplored. The most preferred nitrogen sources for most bacteria are glutamine and ammonium ions. In bacterial cells an enzyme glutamine synthetase synthetizes glutamine from glutamate and ammonium ion. While L. hilgardii LMG 7934 genome carries two genes encoding glutamine synthetases with 55% of mutual identity of amino acid sequences, which protein plays the major role is still unclear. One gene is located within classical glnRA operon with the gene of GlnR-like transcriptional regulator, while the second is monocistronic. In this study we report the cloning of both genes encoding glutamine synthetases from L. hilgardii LMG 7934.

Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed was asked to deliver a scientific opinion on the safety and efficacy of a strain of Lactobacillus hilgardii and of Lactobacillus buchneri when used as a technological additive intended to improve ensiling at a proposed application rate of 3.0 x 108 colony forming units (CFU)/kg fresh material. The two bacterial species are considered by EFSA to be suitable for the qualified presumption of safety approach to safety assessment. In previous opinions the identity of the strains has been clearly established and no antibiotic resistance of concern was detected. Therefore, the use of the strains as a silage additive is considered safe for livestock species, for consumers of products from animals fed the treated silage and for the environment. In the absence of data, no conclusion can be drawn on the skin and eye irritancy or skin sensitisation of the additive. The additive should be considered a potential respiratory sensitiser. In a previous application, three studies involving both strains used in combination at the same concentration were made with samples of whole crop maize with varied dry matter content. The same studies have been submitted in this application. The Panel reiterates its previous conclusions that Lactobacillus hilgardii CNCM I‐4785 and Lactobacillus buchneri NCIMB 40788/CNCM I‐4323 at 1.5 × 108 CFU of each active agent per kg of fresh forage, equivalent to 3 x 108 CFU total lactobacilli/kg of forage significantly improve the aerobic stability of the silage.

Chinese Tianshan tibico grains were collected from the rural area of Tianshan in Xinjiang province, China. Typical tibico grains are known to consist of polysaccharide matrix that embeds a variety of bacteria and yeasts. These grains are widely used in some rural regions to produce a beneficial sugary beverage that is slightly acidic and contains low level of alcohol. This work aimed to characterize the microbiota composition of Chinese Tianshan tibicos using the single molecule, real-time sequencing technology, which is advantageous in generating long reads. Our results revealed that the microbiota mainly comprised of the bacterial species of Lactobacillus hilgardii, Lactococcus raffinolactis, Leuconostoc mesenteroides, Zymomonas mobilis, together with a Guehomyces pullulans-dominating fungal community. The data generated in this work helps identify beneficial microbes in Chinese Tianshan tibico grains.

This work evaluated the effect of lysozyme on lactobacilli isolated from Port wine. Bacterial growth experiments were conducted in MRS/TJ medium and inactivation studies were performed in phosphate buffer (KH2PO4), distilled water and wine supplemented with different concentrations of lysozyme. The response of bacteria to lysozyme was found to be highly strain dependent. Some strains of Lactobacillus hilgardii together with Lactobacillus collinoides and Lactobacillus fructivorans were found to be resistant to concentrations of lysozyme as high as 2000 mg/L. It was observed that among the L. hilgardii taxon the resistant strains possess an S-layer coat. Apparently, the strains of L. collinoides and L. fructivorans studied are also S-layer producers as suggested by the total protein profile obtained by SDS-PAGE. Thus, the hypothetical protective role of the S-layer against the action of lysozyme was investigated. From the various treatments used to remove the protein from the surface of the cells, the one employing LiCl (5 M) was the most effective. LiCl pre-treated cells exposed to lysozyme (2000 mg/L) in KH2PO4 buffer maintained its resistance. However, when cells were suspended in distilled water an increased sensitivity to lysozyme was observed. Moreover, it was found that the addition of ethanol (20% v/v) to the suspension medium (distilled water) triggered a strong inactivation effect especially on cells previously treated with LiCl (reduction of >6 CFU log cycles). The results suggest that the S-layer exerts a protective effect against lysozyme and that the cell suspension medium influences the bacteriolysis efficiency. It was also noted that ethanol enhances the inactivation effect of lysozyme.

The microbial basis of acidification process during spontaneous cheese whey wastewater fermentation was decrypted by implementing both culture-dependent and culture-independent techniques. Lac tobacillus and Bifidobacterium were the predominant taxa among the microbiota growing on MRS (deMan, Rogosa, and Sharpe), while Kazachstania unispora and Dekkera anomala yeast species were also isolated. Almost all Lactobacillus isolates were heterofermentative that could ferment glucose and lactose, with most of them being related to Lactobacillus hilgardii (99.0–100 % similarity). By employing fluorescence techniques, the dominance of long crescent-shaped bacteria in the acidogenic sludge was observed. Temperature gradient gel electrophoresis (TGGE), clone library, and next-generation sequencing techniques revealed the dominance of Selenomonas lacticifex. Based on Illumina data, Selenomonas in the continuous stirred-tank reactor (CSTR) represented 70.13 ± 4.64 % of the bacterial reads, while other Veillonellaceae taxa (Megasphaera and Pectinatus) represented a notable proportion (6.54 %). Prevotella was only detected by Illumina sequencing as an important constituent of the microbial population (14.97 ± 1.71 %). Budding yeasts represented 97 % of the fungal population in the CSTR, with Yarrowia strains representing 88.85 ± 5.52 % of the fungal reads. Spontaneous cheese whey acidification can favor the dominance of rumen bacteria and here was driven by the rarely reported S. lacticifex-type fermentation, which should be taken into consideration during evaluation of acidogenesis in process simulation and modelling. Moreover, the important nervonic acid content detected indicates that acidogenic sludge can be used as a source for the production of high value-added biomedical substrates.

Water kefir is a beverage fermented by a microbial consortium captured in kefir grains. The kefir grains matrix is composed of polysaccharide, primarily dextran, which is produced by members of the microbial consortium. In this study, we have isolated lactic acid bacteria (LAB) from non-commercial water kefir grains (from Belgrade, Serbia) and screened for dextran production. Among twelve LAB isolates three produced slime colonies on modified MRS (mMRS) agar containing sucrose instead of glucose and were presumed to produce dextran. Three LAB were identified, based on morphological, physiological and biochemical characteristics and 16S rRNA sequencing, as Leuconostoc mesenteroides (strains T1 and T3) and Lactobacillus hilgardii (strain T5). The isolated strains were able to synthesize a substantial amount of dextran in mMRS broth containing 5% sucrose. Maximal yields (11.56, 18.00 and 18.46 g/l) were obtained after 16, 20 and 32 h for T1, T3 and T5, respectively. Optimal temperature for dextran production was 23oC for two Leuconostoc mesenteroides strains and 30 °C for Lactobacillus hilgardii strain. The produced dextrans were identified based on paper chromatography, while the main structure characteristics of purified dextran were observed by FT-IR spectroscopy. Our study shows that water kefir grains are a natural source of potent dextran producing LAB. Vodeni kefir je napitak koji nastaje fermentacionom aktivnošću bakterija mlečno-kiselinskog vrenja, kvasca i sirćetnih bakterija. Ova kompleksna smeša mikroorganizama smeštena je unutar polisaharidnog matriksa - kefirnog zrna. Glavnu šećernu komponentu matriksa čini dekstran koji sintetišu bakterije mlečno-kiselinskog vrenja. Dekstran je homo polisaharid velike molekulske mase čija je monomerna jedinica glukoza. Zahvaljujući dobroj rastvorljivosti i stabilnosti, dekstran ima široku primenu u različitim granama industrije, kao što su medicina, farmacija, prehrambena, tekstilna i hemijska industrija. Nerastvorni dekstran može da posluži kao nosač za imobilizaciju biomolekula. Imajući u vidu veliku primenjivost dekstrana, cilj ovog rada je bio izolovati bakterije mlečno-kiselinskog vrenja iz zrna vodenog kefira koje produkuju ovaj egzopolisaharid sa visokim prinosima. U radu je prikazana izolacija i karakterizacija tri selektovana soja i optimizacija uslova za produkciju dekstrana. Na osnovu morfoloških, fizioloških i biohemijskih osobina i 16S rRNK sekvenciranja utvrđeno je da su izolati T1 i T3 sojevi vrste Leuconostoc mesenteroides, dok je izolat T5 identifikovan kao Lactobacillus hilgardii. Za produkciju dekstrana korišćena je modifikovana MRS podloga sa 5% saharoze, kao jedinim izvorom ugljenika. Maksimalni prinosi dekstrana (11,56, 18,00 i 18,46 g/l) su dobijeni nakon 16, 20 i 32 h fermentacije, za T1, T3 i T5, redom. Optimalne temperature za produkciju dekstrana su 23 °C za dva Leuconostoc mesenteroides soja, a za Lactobacillus hilgardii 30 °C. Uzorci sintetisanih polisaharida su identifikovani kao dekstrani na osnovu papirne hromatografije, dok su glavne strukturne karakteristike prečišćenih dekstrana utvrđene na osnovu FTIR spektroskopije. .

Vineyard- and winery-associated lactic acid bacteria (LAB) from two major PDO regions in Greece, Peza and Nemea, were surveyed. LAB were isolated from grapes, fermenting musts, and winery tanks performing spontaneous malolactic fermentations (MLF). Higher population density and species richness were detected in Nemea than in Peza vineyards and on grapes than in fermenting musts.Pediococcus pentosaceusandLactobacillus graminiswere the most abundant LAB on grapes, whileLactobacillus plantarumdominated in fermenting musts from both regions. No particular structure ofLactobacillus plantarumpopulations according to the region of origin was observed, and strain distribution seems random. LAB species diversity in winery tanks differed significantly from that in vineyard samples, consisting principally ofOenococcus oeni. Different strains were analysed as per their enological characteristics and the ability to produce biogenic amines (BAs). Winery-associated species showed higher resistance to low pH, ethanol, SO2, and CuSO4than vineyard-associated isolates. The frequency of BA-producing strains was relatively low but not negligible, considering that certain winery-associatedLactobacillus hilgardiistrains were able to produce BAs. Present results show the necessity of controlling the MLF by selected starters in order to avoid BA accumulation in wine.

Eight water kefir fermentation series differing in the presence of oxygen, the nutrient concentration, and the nutrient source were studied during eight consecutive backslopping steps. The presence of oxygen allowed the proliferation of acetic acid bacteria, resulting in high concentrations of acetic acid, and decreased the relative abundance of Bifidobacterium aquikefiri. Low nutrient concentrations resulted in slow water kefir fermentation and high pH values, which allowed the growth of Comamonas testosteroni/thiooxydans. Further, low nutrient concentrations favored the growth of Lactobacillus hilgardii and Dekkera bruxellensis, whereas high nutrient concentrations favored the growth of Lactobacillus nagelii and Saccharomyces cerevisiae. Dried figs, dried apricots, and raisins resulted in stable water kefir fermentation. Water kefir fermentation with dried apricots resulted in the highest pH and water kefir grain growth, whereas that with raisins resulted in the lowest pH and water kefir grain growth. Further, water kefir fermentation with raisins resembled fermentations with low nutrient concentrations, that with dried apricots resembled fermentations with normal nutrient concentrations, and that with fresh figs or a mixture of yeast extract and peptone resembled fermentations with high nutrient concentrations.

Fortified wines contain a high level of unfermented sugars and are prone to spoilage by alcohol-tolerant lactic acid bacteria. A total of 62 strains were isolated from various production stages of one of the more popular fortified wines produced in South Africa. The strains were identified by using numerical analysis of total soluble cell protein patterns and 16S rRNA sequence analyses. The species most frequently isolated were Lactobacillus vermiforme (24 strains) and Lactobacillus casei subsp. casei (32 strains). Twenty-four of the strains of L. vermiforme, three strains of Lactobacillus buchneri, one strain of Lactobacillus plantarum and two strains of L. casei subsp. casei were isolated from spoiled fortified wine which contained 22% (vol/vol) ethanol. The majority of strains of L. casei subsp. casei (25 of the 32) and two strains of Lactobacillus zeae were isolated from wine before submerged fermentation. Five strains of L. casei subsp. casei were isolated from wine undergoing submerged fermentation, with an alcohol content of 11.92% (vol/vol). No strain was isolated from unbottled wine which underwent the complete fermentation process and with an alcohol content of 17.20% (vol/vol). Three distinct phenotypic groups of L. vermiforme were identified at r ≥ 0. 70, separate from Lactobacillus brevis,' L. buchneri and Lactobacillus hilgardii. Three phenotypic clusters have been identified for L. casei subsp. casei. This is the first report of the presence of L. vermiforme, L. zeae, L. casei subsp. casei and L. plantarum in fortified wines.

Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed was asked to deliver a scientific opinion on the safety and efficacy of a strain of Lactobacillus hilgardii when used as a technological additive intended to improve ensiling at a proposed application rate of 1.5 × 108 colony‐forming units (CFU)/kg fresh material. The bacterial species L. hilgardii is considered by EFSA to be suitable for the qualified presumption of safety approach to safety assessment. As the identity of the strain has been clearly established and as no antibiotic resistance of concern was detected, the use of the strain as a silage additive is considered safe for livestock species, for consumers of products from animals fed the treated silage and for the environment. In the absence of data, no conclusion can be drawn on the skin and eye irritancy or skin sensitisation of the additive. The additive should be considered as a potential respiratory sensitiser. Three studies with laboratory‐scale silos were made using samples of easy and moderately difficult to ensile forage. In each case, replicate silos containing untreated forage were compared with identical silos containing the same forage treated with the combination of Lactobacillus hilgardii CNCM I‐4785 and Lactobacillus buchneri NCIMB 40788 each at an intended concentration of 1.5 × 108 CFU/kg fresh matter each. The results showed that the addition of Lactobacillus hilgardii CNCM I‐4785 at the proposed dose, when used in combination with an equal concentration of Lactobacillus buchneri NCIMB 40788, improves significantly the aerobic stability of the silage tested. This was shown in maize forage with a dry matter content ranging from 30% to 40%.

The present study aimed to explore a novel attribute of the bacterium Lactobacillus hilgardii IITRKH159, isolated from Sapodilla, which was found to produce Bacterial cellulose. The compositional analysis of BC was carried out by HPLC system using pre-column derivatization with 1-phenyl-3-methyl-5-pyrazolone showed that it consisted of glucan. The Physiochemical study of BC showed that it had bacterial nano-cellulose like structure, immensely pure, highly crystalline (90.25%) and significantly enriched with Iα form of cellulose. Additionally, FE-SEM and TEM of the BC revealed the morphological similarities with bacterial nano-cellulose due to the presence of a reticulated structure consisting of nano-sized fibrils with an average width of 45 nm. The effect of different carbon sources was analyzed to evaluate BC production and demonstrated the production of 7.23 ± 0.59 g L−1 BC with conversions yield (0.30 g BC/g sugar) and productivity (0.45 ± 0.15 g L−1 d−1) in a modified Y-medium after 16 days of cultivation.

Silage making has become a significant method of forage conservation worldwide. To determine how tomato pomace (TP) may be used effectively as animal feed, it was ensilaged for 90 days and microbiology counts, fermentation characteristics and chemical composition of tomato pomace silage (TPS) were evaluated at the 30th, 60th, and 90th days, respectively. In addition, 103 lactic acid bacteria were isolated from TPS. Based on the phenotypic and chemotaxonomic characteristics, 16S rDNA sequence and carbohydrate fermentation tests, the isolates were identified as 17 species namely: Lactobacillus coryniformis subsp. torquens (0.97%), Lactobacillus pontis (0.97%), Lactobacillus hilgardii (0.97%), Lactobacillus pantheris (0.97%), Lactobacillus amylovorus (1.9%), Lactobacillus panis (1.9%), Lactobacillus vaginalis (1.9%), Lactobacillus rapi (1.9%), Lactobacillus buchneri (2.9%), Lactobacillus parafarraginis (2.9%), Lactobacillus helveticus (3.9%), Lactobacillus camelliae (3.9%), Lactobacillus fermentum (5.8%), Lactobacillus manihotivorans (6.8%), Lactobacillus plantarum (10.7%), Lactobacillus harbinensis (16.5%) and Lactobacillus paracasei subsp. paracasei (35.0%). This study has shown that TP can be well preserved for 90 days by ensilaging and that TPS is not only rich in essential nutrients, but that physiological and biochemical properties of the isolates could provide a platform for future design of lactic acid bacteria (LAB) inoculants aimed at improving the fermentation quality of silage.

Molecular identification of microorganisms associated with submerged cassava fermentation was carried out and isolates of lactic acid bacteria (LAB) were examined for antimicrobial activity, including ability to produce antimicrobial peptides as a first step to define starter cultures for controlled cassava fermentations. A total of 75 isolates, including 41 LAB, 31 aerobic bacteria (AB) and three anaerobic bacteria were isolated from unfermented and fermenting cassava roots, cassava leaves and fermented cassava dough and identified by a combination of phenotypic tests and sequencing of 16S rRNA, rpoA, rpoB and pheS genes. Microbial diversity at interspecies and intraspecies level was screened by, respectively, PCR amplification of the 16S-23S rDNA intergenic transcribed spacer (ITS-PCR) and repetitive sequence based PCR (rep-PCR). Antimicrobial activity of LAB cultures and supernatants against indicator bacteria; Escherichia coli, Salmonella enterica serotype Typhimurium (S. Typhimurium), Bacillus cereus and Staphylococcus aureus was studied using agar diffusion tests. Furthermore, inactivation of indicator bacteria was investigated in both liquid medium and during controlled cassava fermentation. Results revealed a diversity of bacterial genera, species and subspecies associated with submerged cassava fermentation. DNA sequencing enabled identification of LAB isolates as Lactobacillus plantarum, Weissella confusa, Weissella paramesenteroides, Lactobacillus rhamnosus, Lactobacillus hilgardii, Lactobacillus paracasei, Leuconostoc mesenteroides, Enterococcus faecium, Enterococcus casseliflavus, and Pediococcus acidilactici. Lactobacillus spp. were the predominant LAB and were present in all cassava samples studied. Aerobic bacteria were predominantly Bacillus spp., including Bacillus subtilis, Bacillus amyloliquefaciens and B. cereus. Other species identified included Staphylococcus pasteuri and Clostridium beijerinkii. Cells, supernatants and cell free supernatants (CFS) of selected LAB isolates were able to inhibit both Gram positive and Gram negative pathogenic bacteria. LAB isolates inactivated all indicator organisms during controlled cassava fermentations, with a 4–6 log reduction after 48 h fermentation. The antimicrobial effect of the LAB was attributed to acid production.

Evolution of lactic acid bacteria (LAB) from two wineries located in Rioja Alavesa region has been analyzed during the whole winemaking process. Special attention was focused on determining their biogenic amine (BA) forming potential. From 295 isolates, 36 different genotypic profiles were identified, from which 17 belonged to Oenococccus oeni. Although LAB species evolution differed in both wineries during winemaking process, O. oeni became the predominant species in both wineries once malolactic fermentation (MLF) began. Each winery showed an exclusive ecosystem with own microbiota, as low coincident O. oeni strains were identified. Both phenotypical and molecular methods revealed the low incidence of BA producing LAB strains, as exclusively Lactobacillus hilgardii strains were positive for putrescine production. Other bacteria genus rarely found in wine environment such as Staphylococcus and Paenibacillus were also identified. Indeed, this is the first work in which those species have been reported as BA producers in wine. In addition, BA concentration was also monitored. Both wineries showed low concentrations for each BA, far from limits recommended for safety and quality principles. These results emphasize the potential application of these autochthonous strains in order to minimize BA formation during the whole winemaking process.

-Aminobutyric acid(GABA) is a four carbon non-protein amino acid that has several well-known phys-iological functions, such as a postsynaptic inhibitory neurotransmitter in the brain and induction of hypotensive and tran-quilizer effects. A lactic acid bacterium was isolated from button mushroom bed, which is showing high GABAproductivity by TLC or HPLC analysis. The strain was identified as Lactobacillus hilgardii by analysis of 16S rDNAgene sequence. When the maximum production of GABA by L. hilgardii was investigated with various concentration ofmonosodium glutamate, the yield of GABA reached to be 53.65 mM at 1% mono sodium glutamate (MSG) in flask cul-tivation. A Glutamate decarboxylase (GAD) enzyme, which was known to convert MSG to GABA, was purified froma cell-free extract of L. hilgardii and the molecular weights of purified GAD was estimated to 60,000 by SDS-PAGE.The optimum pH and temperature of GAD were at pH4.6 and at 37 o C, respectively. The GAD activity was increasedby the addition of sulfate ions such as ammonium sulfate, sodium sulfate and magnesium sulfate, indicating that theincrease of hydrophobic interaction causes the increase of GAD activity.

Oenococcus oeni is the best malolactic bacterium adapted to low pH and the high SO2 and ethanol concentrations in wine. Leuconostoc mesenteroides and Leuconostoc paramesenteroides (now classified as Weissella paramesenteroides) have also been isolated from wine. Pediococcus damnosus is not often found in wine and is considered a contaminant of high pH wines. Pediococcus inopinatus, Pediococcus parvulus and Pediococcus pentosaceus have occasionally been isolated from wines. Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus buchneri, Lactobacillus hilgardii (previously Lactobacillus vermiforme), Lactobacillus fructivorans (previously Lactobacillus trichoides and Lactobacillus heterohiochii) and Lactobacillus fermentum have been isolated from most wines. Lactobacillus hilgardii and L. fructivorans are resistant to high acid and alcohol and have been isolated from spoiled fortified wines. Lactobacillus vini, Lactobacillus lindneri, Lactobacillus nagelii and Lactobacillus kunkeei have been described more recently. The latter two species are known to cause sluggish or stuck alcoholic fermentations in wine. Although Lactobacillus collinoides and Lactobacillus mali (previously Lactobacillus yamanashiensis) decarboxylate L-malic acid, they are more often found in cider and fruit juices. Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus diolivorans, Lactobacillus jensenii and Lactobacillus paracasei are seldomly isolated from wines. Some strains of Lactobacillus casei may be closer related to Lactobacillus paracasei or a distant relative, Lactobacillus zeae. Oenococcus kitaharae, isolated from compost is genetically closely related to Oenococcus oeni, but does not decarboxylate malate, prefers higher growth pH and is phenotypically well distinguished from O. oeni. This review summarises the current taxonomic status of malolactic bacteria and lists key phenotypic characteristics that may be used to identify the species.

Lactobacillus buchneri belongs to the group of heterofermentative lactic acid bacteria and is a common member of the silage microbiome. Here we report the completely annotated genomic sequence of L. buchneri CD034, a strain isolated from stable grass silage. The whole genome of L. buchneri CD034 was sequenced on the Roche Genome Sequencer FLX platform. It was found to consist of four replicons, a circular chromosome, and three plasmids. The circular chromosome was predicted to encode 2319 proteins and contains a genomic island and two prophages which significantly differ in G+C-content from the remaining chromosome. It possesses all genes for enzymes of a complete phosphoketolase pathway, whereas two enzymes necessary for glycolysis are lacking. This confirms the classification of L. buchneri CD034 as an obligate heterofermentative lactic acid bacterium. A set of genes considered to be involved in the lactate degradation pathway and genes putatively involved in the breakdown of plant cell wall polymers were identified. Moreover, several genes encoding putative S-layer proteins and two CRISPR systems, belonging to the subclasses I-E and II-A, are located on the chromosome. The largest plasmid pCD034-3 was predicted to encode 57 genes, including a putative polysaccharide synthesis gene cluster, whereas the functions of the two smaller plasmids, pCD034-1 and pCD034-2, remain cryptic. Phylogenetic analysis based on sequence comparison of the conserved marker gene rpoA reveals that L. buchneri CD034 is more closely related to Lactobacillus hilgardii strains than to Lactobacillus brevis and Lactobacillus plantarum strains. Comparison of the L. buchneri CD034 core genome to other fully sequenced and closely related members of the genus Lactobacillus disclosed a high degree of conservation between L. buchneri CD034 and the recently sequenced L. buchneri strain NRRL B-30929 and a more distant relationship to L. buchneri ATCC 11577 and L. brevis ssp. gravesensis ATCC 27305, which cluster together with L. hilgardii type strain ATCC 8290. L. buchneri CD034 genome information will certainly provide the basis for further postgenome studies with the objective to optimize application of the strain in silage production.

BACKGROUND: Lactobacillus hilgardii, a wine lactic acid bacterium, is able to use arginine, through the arginine deiminase pathway with the formation of citrulline, a precursor of the carcinogen ethyl carbamate. The influence of different Argentine wine varieties (Merlot, Cabernet Sauvignon and Malbec), on bacterial growth and arginine metabolism was examined. Furthermore, the effect of different components normally present in wines on the enzyme activities of the arginine deiminase system was determined. RESULTS: Malbec wine under all conditions assayed (33, 50 and 100% supplemented wine:basal media) showed higher arginine consumption and citrulline production than the other wines, as well as the highest bacterial growth and survival of Lactobacillus hilgardii X1B. Glucose and L-malic inhibited both arginine deiminase enzymes while fructose and citric acid only inhibited arginine deiminase. The red wines assayed in this study had different composition, and this is an explanation for the different behavior of the bacterium. CONCLUSION: The highest citrulline production in Malbec wine could be correlated with its lower concentrations of glucose, fructose, citric and phenolic acid than the other wines. Therefore, a wine with lower concentration of these sugars and acids could be dangerous due to the formation of ethyl carbamate precursors. Copyright © 2012 Society of Chemical Industry

In order to analyze the genes related to histamine production in halophilic lactic acid bacteria, 16 strains of histamine-producing bacteria were isolated from three fermented seafoods produced in the Hokuriku region of Japan. Phenotypic and 16S rRNA gene sequence analyses identified all of the strains as those of Tetragenococcus muriaticus. Pyruvoyl-dependent histidine decarboxylase gene (hdcA) was determined from all strains using the PCR method with an hdcA-specific detection primer set. Genetic analyses (Southern blot and restriction fragment length polymorphism analysis) of hdcA and genes related to histamine production (the hdc cluster) confirmed that all of the strains harbored 21–23 kbp plasmids encoding a single copy of hdcA. The four representative strains were selected based on isolation source and genetic analysis, and subsequently full sequences of plasmids harbored in these strains were determined. hdc cluster sequences from the plasmids showed very high similarity (>99 %) to known hdc clusters of T. halophilus, Lactobacillus hilgardii 0006, and other lactic acid bacteria. The structures of the plasmids, the replication region, the hdc cluster, and the plasmid maintenance system were conserved between the plasmids present in new isolates and the T. halophilus strains. These results indicate that plasmids encoding hdcA are widely distributed among T. halophilus and T. muriaticus and function in both species.

The influence of seven phenolic compounds, normally present in wine, on the growth and arginine deiminase system (ADI) of Lactobacillus hilgardii X1B, a wine lactic acid bacterium, was established. This system provides energy for bacterial growth and produces citrulline that reacts with ethanol forming the carcinogen ethyl carbamate (EC), found in some wines. The influence of phenolic compounds on bacterial growth was compound dependent. Growth and final pH values increased in presence of arginine. Arginine consumption decreased in presence of protocatechuic and gallic acids (31 and 17%, respectively) and increased in presence of quercetin, rutin, catechin and the caffeic and vanillic phenolic acids (between 10 and 13%, respectively). ADI enzyme activities varied in presence of phenolic compounds. Rutin, quercetin and caffeic and vanillic acids stimulated the enzyme arginine deiminase about 37-40%. Amounts of 200 mg/L gallic and protocatechuic acids inhibited the arginine deiminase enzyme between 53 and 100%, respectively. Ornithine transcarbamylase activity was not modified at all concentrations of phenolic compounds. As gallic and protocatechuic acids inhibited the arginine deiminase enzyme that produces citrulline, precursor of EC, these results are important considering the formation of toxic compounds.

Aims To investigate the impact of acetaldehyde- and pyruvic acid-bound sulphur dioxide on wine lactic acid bacteria (LAB). Methods and results Growth studies were performed where Oenococcus oeni, Pediococcus parvulus, Ped. damnosus and Lactobacillus hilgardii were inoculated into media containing various concentrations of acetaldehyde or pyruvic acid and an equimolar concentration of SO(2) at pH 3.50 and 3.70. Low concentrations of acetaldehyde- and pyruvic acid-bound SO(2) were inhibitory to the growth of all bacteria although acetaldehyde-bound SO(2) was generally more inhibitory than pyruvic acid-bound SO(2). Inhibition was greater at pH 3.50 than 3.70, and Lact. hilgardii was the most sensitive to acetaldehyde-bound SO(2), while O. oeni was the most sensitive to pyruvic acid-bound SO(2). Degradation of SO(2)-bound acetaldehyde was observed for all LAB, and aside from O. oeni, there was also complete degradation of SO(2)-bound pyruvic acid at both pH values. O. oeni only degraded pyruvic acid at pH 3.70. Degradation of SO(2)-bound acetaldehyde or pyruvic acid did not correlate with bacterial growth as inhibition was always observed in media containing bound SO(2). Conclusions Acetaldehyde- and pyruvic acid-bound SO(2) were inhibitory to wine LAB growth at concentrations as low as 5 mg l(-1). Despite this inhibition, all wine LAB degraded SO(2)-bound acetaldehyde and pyruvic acid suggesting that bound SO(2) may have a bacteriostatic rather than bacteriocidal action. Significance and impact of the study Sulphur dioxide bound to acetaldehyde or pyruvic acid is inhibitory to growth of wine LAB and must be considered when conducting the malolactic fermentation or controlling the growth of spoilage bacteria such as Pediococcus and Lactobacillus.

The application of high hydrostatic pressure (HHP) to inactivate microorganisms in a grape must (crushed grapes), coupled with autoclavable microscale fermentors, was investigated as a potential tool for studying the impact of yeast on red wine aroma and flavor. A Pinot noir grape must was inoculated with Saccharomyces cerevisiae, Brettanomyces bruxellensis, Kluveromyces thermotolerans, Lactobacillus hilgardii, Oenococcus oeni, and Acetobacter aceti at ~1 × 105 cfu/mL and subjected to HHP treatment for 10 min at 551 MPa (5510 bar). After HHP treatment no viable cells were detected in the grape must. Autoclavable microscale fermentors were then used to conduct replicate fermentations of HHP-treated and untreated Pinot noir grapes producing sufficient wine for chemical and sensory analysis. No differences were observed in fermentation rate between treatments, and variability between replicates was very low. No significant differences in color or hue were observed, but wine produced from HHP-treated grapes contained higher total phenolics. Sensory analysis of the wines by a trained panel revealed that other than a slight increase in overall fruit aroma there were no significant differences between any aroma and flavor descriptors. Results suggest that HHP processing of grapes, in conjunction with autoclavable microscale fermentors, could be used to conduct experimental red wine fermentations without the influence of native yeast and bacteria present on grapes and fermentation equipment.

Five strains (Ryu1-2T, Gon2-9, Ryu4-3, Nog8-1 and Aza1-1) of lactic acid bacteria were isolated from flowers in mountainous areas in Japan, Oze National Park, Iizuna mountain and the Nikko area. The five isolates were found to share almost identical (99.6–100 % similar) 16S rRNA gene sequences and were therefore deemed to belong to the same species. These isolates exhibited low levels of 16S rRNA gene sequence similarity to known lactic acid bacteria; the closest recognized relatives to strain Ryu1-2T were the type strains of Lactobacillus hilgardii (92.8 % similarity), Lactobacillus kefiri (92.7 %), Lactobacillus composti (92.6 %) and Lactobacillus buchneri (92.4 %). Comparative analyses of rpoA and pheS gene sequences demonstrated that the novel isolates did not show significant relationships to other Lactobacillus species. The strains were Gram-stain-positive, catalase-negative and homofermentative. The isolates utilized a narrow range of carbohydrates as sources of carbon and energy, including glucose and fructose. On the basis of phenotypic characteristics and phylogenetic data, these isolates represent a novel species of the genus Lactobacillus, for which the name Lactobacillus floricola sp. nov. is proposed. The type strain is Ryu1-2T ( = NRIC 0774T = JCM 16512T = DSM 23037T).

International audience; A collection of 810 lactic acid bacteria (LAB) strains isolated from wine and cider was screened for potential biogenic amine (BA) producers by combining molecular and phenotypic approaches. A newly developed multiplex PCR method allowed for the simultaneous detection of four genes involved in the production of histamine (histidine decarboxylase, hdc), tyramine (tyrosine decarboxylase, tyrdc) and putrescine (via either ornithine decarboxylase, odc, or agmatine deiminase, agdi) while TLC and HPLC analysis allowed for BA-production determination. One hundred and fifty-eight LAB strains were monitored by the molecular/phenotypic double approach and revealed a good correlation between genotypic and phenotypic data. Eighteen per cent of the tested strains were positive for at least one BA target gene with up to three detected simultaneously, in particular amongst Lactobacillus brevis and Lactobacillus hilgardii isolates for the tyrdc and agdi genes. The most frequent gene corresponded to the agdi gene detected in 112 strains (14% of all LAB strains) of 10 different LAB species. The tyrdc gene was detected in 67 strains represented by 7 different LAB species (8% overall), especially those isolated from wine. Lower levels of hdc+ (2% of strains) and especially odc+ (0.5% of strains) strains were observed. Interestingly, species that have never been described to carry BA-producing pathway genes were identified in this study. Furthermore, only one cadaverine-producer was detected and corresponded to Lactobacillus 30a, a collection strain not found in fermented beverages, although cadaverine is commonly detected in wines.

The aim of this work was to determine the ability of six yeast and two bacterial species associated with wine spoilage to form biofilms in mono- or co-culture using the Calgary Biofilm Device (CBD). Moreover, the efficacy of several disinfectants was evaluated against these spoilage microorganisms, both in the planktonic and the biofilm states. Results showed that Dekkera bruxellensis, Saccharomyces cerevisiae, Saccharomycodes ludwigii, Schizosaccharomyces pombe and Acetobacter aceti formed biofilms both in wine and in synthetic medium. Zygosaccharomyces bailii formed biofilm only in wine and Pichia guilliermondii and Lactobacillus hilgardii formed biofilms only in synthetic medium. In wine, D. bruxellensis presented the same biofilm population when grown in pure culture or in mixed culture with acetic acid bacteria. There was a 3-log increase in biofilm formed by A. aceti in mixed culture with L. hilgardii. Alkaline chlorine-based disinfectant was the most effective in decontaminating spoilage yeast and bacteria both in planktonic and biofilm tests. Sodium hydroxide-based detergents and peracetic-based disinfectant were also efficient against suspended cells, but at least 10-fold more concentrated solutions were needed to remove the biofilms. Furthermore, the results showed that, except for the neutral detergent VK10, the tested agents were actually effective when used under the conditions recommended by manufacturers. In any case, biofilms showed greater tolerance to biocides when compared to the same microorganisms in the planktonic state. To our knowledge, this is the first study in which the CBD is used to assess the ability of wine spoilage microorganisms to form biofilms and their susceptibilities to disinfectant agents.

The aim of this study was to develop species-specific primer sets for kimchi Lactobacillus. Known gene sequences of Lactobacillus 16S rRNA were collected from the NCBI Gene bank, and 69 primer sets were designed using the homologous gene sequence. Six species of kimchi Lactobacilli were used as reference strains: Lactobacillus brevis KCTC3102, Lactobacillus farciminis KCTC3681, Lactobacillus fermentum KCTC3112, Lactobacillus hilgardii KCTC3500, Lactobacillus plantarum KCTC3099, and Lactobacillus sanfranciscensis KCTC3205. PCR amplification and gel electrophoresis were performed to identify the accuracy and specificity of the developed primer set. The results show that the primer set of 5'-aagcctgcgaaggcaag-3' & 5'-aggccaccggctttg-3', 5'-acatactatgcaaatctaagagattagacg-3' & 5'-actgagaatggctttaagagattagcttac-3' resulted in a specific PCR band on L. hilgardii, and primer set of 5'-ctaataccgcataacaactactttcacat-3' & 5'-aacttaataaaccgcctacattctctttac-3' on L. farciminis. The results indicate that the developed primer sets can provide a useful tool for the identification and differentiation of L. hilgardii and L. farciminis from other Lactobacillus species of kimchi.

The objective of this study was to design and develop an analytical protocol using chemical and molecular techniques to detect microorganisms able to produce biogenic amines (BAs). The protocol consists of: an initial screening by TLC (Thin Layer Chromatography) to detect BA production by microorganisms; multiplex PCR to identify decarboxylase genes in the BA-positive samples; and HPLC to quantify BA production. The protocol was used to verify the ability of a Lactobacillus hilgardii strain isolated from wine to produce histamine during malolactic fermentation. The results showed that this analytical approach is an useful tool to determine the production of BAs by microorganisms. Commercial dry yeast starters routinely used in wine-making were examined by this protocol and the results showed that the presence of bacterial contamination in these preparations is a possible origin of BAs in wine.

The bacteriocins produced by Lactococcus lactis subsp. lactis C101910 (C101910) and NBRC 12007 (NBRC 12007) were used to prevent the growth of sake spoiling hiochi bacteria (Lactobacillus hilgardii, Lactobacillus fructivorans, and Lactobacillus paracasei) in namazake, which is raw (unpasteurized) sake. The bacteriocin concentrations required for decreasing the viable cell concentrations of L. hilgardii and L. fructivorans below the detection limit (1.0 x 10(2) cells/ml) in 24 h from the initial concentration of 4.0-9.5 x 10(5) cells/ml in the namazake at pH 4.5 and at 4 degrees C, were 18-35 U/ml and 5.6 U/ml for the bacteriocin from C101910 and NBRC 12007, respectively. To decrease the viable cell concentration of L. paracasei from the initial concentration of 7.5 x 10(5) cells/ml to below the detection limit (1.0 x 10(2) cells/ml) in 24 h, 350 U/ml bacteriocin from C101910 and 140 U/ml bacteriocin from NBRC 12007 were required. In experiments using McIlvaine buffer (pH 4.5) with 15% ethanol instead of namazake as the medium, the viable cell concentrations of L. hilgardii and L. paracasei decreased to less than 1.0 x 10(2) cells/ml, whereas those of L. fructivorans decreased to less than 1.0 x 10(3) cells/ml, when bacteriocins were added at the concentrations that had proven effective in namazake. The membrane depolarization assay using a fluorescent probe showed that the presence of ethanol stimulated the collapse of the membrane potential induced by bacteriocins. The ethanol induced collapse of the membrane potential suggests that the application of bacteriocins at the storage stage of namazake is more beneficial than when used in other stages of the sake brewing process.

Aims: The objective of this study was to investigate the presence of genes coding for enzymes of oenological relevance in wine Lactobacillus strains isolated from South African grape and wine samples during the 2001 and 2002 harvest seasons. Methods and Results: A total of 120 wine lactobacilli isolates belonging to Lactobacillus plantarum, Lactobacillus hilgardii, Lactobacillus brevis, Lactobacillus pentosus, Lactobacillus paracasei, Lactobacillus sakei and Lactobacillus paraplantarum were genetically screened for enzyme-encoding genes using PCR with primers specific for β-glucosidase, protease, esterase, citrate lyase and phenolic acid decarboxylase. The results of PCR screening showed that the Lactobacillus strains possessed different combinations of enzymes and that some strains did not possess any of the enzymes tested. Confirmation analysis with gene sequencing also showed high similarity of genes with those available in GenBank database. Conclusion: In this study, we have demonstrated the existence of genes coding for wine-related enzymes in wine lactobacilli that could potentially hydrolyse wine precursors to positively influence wine aroma. Significance and Impact of the Study: An expansion of knowledge on the genetic diversity of wine-associated lactic acid bacteria will enable the selection of novel malolactic fermentation starter cultures with desired oenological traits for the improvement of the organoleptic quality of the wine, and hence wine aroma.

The comparative effects of the fibrinolytic, and tyrosinase inhibition activities and electrophoretical protein patterns with freeze-drying silkworm powder (FDSW), heating-drying silkworm powder (HDSW) and fermented silkworm powder by Bacillus subtilis or Lactobacillus hilgardii were investigated. When total protein patterns of FDSW, HDSW, both fermented SW, were analyzed by native- and SDS-polyacrylamide gel electrophoresis (PAGE), there were slightly varietal differences in electrophoretical protein patterns. Major minerals of FDSW and HDSW were K, Ca, Mg, and Zn. Major compositional amino acids of FDSW and HDSW were glycine, alanine, glutamic acid, aspartic acid, and serine. Major fatty acids of FDSW and HDSW were linolenic acid, oleic acid, and palmitic acid. Fibriolytic activity was the highest in the fermented FDSW by 5% B. subtilis among the various samples. Tyrosinase inhibition activity was higher in the water and 70% methanolic extract of FDSW than in HDSW. DPPH radical scavenging activity was slightly stronger in HDSW than in FDSW. In addition, DPPH radical scavenging activity was higher in FDSW or HDSW fermented by L. hilgardii than that fermented by B. subtilis, however, all samples exhibited a relatively low activity compared to the butylated hydroxytoluene (BHT). These results may provide the basic data to understand the biological activities of fermented SW.

Aims: To investigate the inactivation properties of different classes of phenolic compounds present in wine against two wine isolates of Lactobacillus hilgardii and Pediococcus pentosaceus, and to explore their inactivation mechanism. Methods and Results: After a first screening of the inactivation potency of 21 phenolic compounds (hydroxybenzoic and hydroxycinnamic acids, phenolic alcohols, stilbenes, flavan-3-ols and flavonols) at specific concentrations, the survival parameters (MIC and MBC) of the most active compounds were determined. For the L. hilgardii strain, the flavonols morin and kaempferol showed the strongest inactivation (MIC values of one and 5 mg l−1, and MBC values of 7·5 and 50 mg l−1, respectively). For the P. pentosaceus strain, flavonols also showed the strongest inactivation effects, with MIC values between one and 10 mg l−1 and MBC values between 7·5 and 300 mg l−1. Observations by epifluorescence and scanning electron microscopy revealed that the phenolics damaged the cell membrane and promoted the subsequent release of the cytoplasm material into the medium. Conclusions: The antibacterial activity of wine phenolics against L. hilgardii and P. pentosaceus was dependent on the phenolic compound tested, and led not only to bacteria inactivation, but also to the cell death. Significance and Impact of the Study: New information about the inactivation properties of wine lactic acid bacteria by phenolic compounds is presented. It opens up a new area of study for selecting/obtaining wine phenolic preparations with potential applications as a natural alternative to SO2 in winemaking.

In this study we isolated over 250 lactic acid bacteria (LAB) candidates from fruit, flowers, vegetables and a fermented food to generate an LAB library. One strain, designated 925A, isolated from kimchi (a traditional Korean fermented dish made from Chinese cabbage) produced a novel type of bacteriocin, brevicin 925A, which is effective against certain LAB, including strains ofLactobacillus,Enterococcus,Streptococcus,BacillusandListeria. Strain 925A, identified asLactobacillus brevis, harboured at least four plasmids and we determined the entire nucleotide sequence of each one. The four plasmids were designated pLB925A01–04, and have molecular sizes of 1815, 3524, 8881 and 65 037 bp, respectively. We obtained bacteriocin non-producing derivatives by treatment of strain 925A with novobiocin. All of these derivatives, which were susceptible to their own antibacterial product, lost the largest plasmid, pLB925A04, suggesting that the genes for bacteriocin biosynthesis (breBandbreC) and immunity (breE) are located on pLB925A04. The partial amino acid sequence of purified brevicin 925A and sequence analysis of pLB925A04 showed thatbreBis the structural gene for brevicin 925A. We constructed a shuttle vector (pLES003, 6134 bp) that can replicate in bothEscherichia coliand LAB such asLactobacillus plantarum,Lb. brevis,Lactobacillus helveticus,Lactobacillus hilgardiiandEnterococcus hirae. To determine the function of genebreE, which displays no significant similarity to any other sequences in theblastsearch database, the gene was inserted into pLES003. A pLB925A04-cured derivative transformed with pLES003 carryingbreEacquired immunity to brevicin 925A, suggesting thatbreEencodes an immunity protein.

Thirty six Gram-positive, rod-shaped, non-spore-forming, non-motile bacterial strains were isolated from the non-salted pickle solution used in producing sunki products, a traditional Japanese pickle. The novel strains were discriminated and separated into four groups by amplified fragment length polymorphism profiling, and by analysis based on recA gene sequences. The strains were classified into four species groups belonging to the Lactobacillus buchneri species group, which consists of L. buchneri, Lactobacillus diolivorans, Lactobacillus hilgardii, Lactobacillus kefiri, Lactobacillus parabuchneri and Lactobacillus parakefiri. The phenotypic and genotypic features of the four groups demonstrated that they represented four novel species, for which the names Lactobacillus kisonensis sp. nov. (type strain YIT 11168(T)=NRIC 0741(T)=JCM 15041(T)=DSM 19906(T)), Lactobacillus otakiensis sp. nov. (type strain YIT 11163(T)=NRIC 0742(T)=JCM 15040(T)=DSM 19908(T)), Lactobacillus rapi sp. nov. (type strain YIT 11204(T)=NRIC 0743(T)=JCM 15042(T)=DSM 19907(T)) and Lactobacillus sunkii sp. nov. (type strain YIT 11161(T)=NRIC 0744(T)=JCM 15039(T)=DSM 19904(T)) are proposed.

Glycerol catabolism was studied in Lactobacillus hilgardii X1B from wine, growing on glycerol and limiting glucose or fructose concentrations in anaerobiosis and microaerophilia. Glycerol consumption occurred simultaneously with sugar use, and it was higher with fructose as a cofermenting sugar in microaerophilia. Enzymatic activities of the glycerol kinase and glycerol dehydratase pathways were detected in both incubation conditions. In anaerobiosis, the main products were lactate, acetate, ethanol, and the intermediary product of the glycerol dehydratase pathway, 3- hydroxypropionaldehyde. However, in microaerophilia, 1,3-propanediol was also detected. In anaerobic glucose + glycerol and fructose + glycerol cultures as in microaerophilic glucose + glycerol cultures, glycerol was degraded mainly through the reductive pathway. However, when L. hilgardii X1B was grown on fructose + glycerol cultures in microaerophilia, glycerol dissimilation occurred mainly via the glycerol kinase way. According to these results, L. hilgardii X1B can degrade glycerol by producing 3-hydroxypropionaldehyde and acetic acid, both undesirable products for wine sensorial quality. © 2009 American Chemical Society. Fil: Pasteris, Sergio Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; Argentina Fil: Strasser de Saad, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina

The bacteriocins produced by Lactococcus lactis subsp. lactis C101910 (C101910) and NBRC 12007 (NBRC 12007) were used to prevent the growth of Lactobacillus hilgardii, a hiochi bacterium, at the mizu-koji stage in the sake brewing process. The bacteriocins produced by C101910 and NBRC 12007 were slowly inactivated in the koji extract by proteases, depending on the pH and temperature of the koji extract. However, the activities of bacteriocins from C101910 and NBRC 12007 remained about 50% and 70% of the initial values, respectively, even after incubation at pH 3 and 10℃ for 12 h. By adding the bacteriocin solutions, prepared according to the method reported previously, from C101910 and NBRC 12007 to koji extract (pH 3 and 10℃) at a volume ratio of 5% (v/v) and 1% (v/v), respectively, the number of viable L. hilgardii cells decreased to below the detection limit (1.0×10 2 cells/ml) from the initial concentration (3.2×10 5 cells/ml) within 12 h. In mizu-koji consisting of rice koji and lactic acid solution at pH 3 and 10℃, the growth-inhibitory effect of bacteriocins from C101910 and NBRC 12007 on L. hilgardii decreased as compared with that in koji extract. However, the number of viable L. hilgardii cells decreased by more than two orders of magnitude as compared with the initial value (1.0×10 5 cells/ml) by adding the bacteriocin solution at a volume ratio of 10% (v/v).

We investigated the antimicrobial activity of several bacteriocins, which were produced by newly isolated lactic acid bacteria in koji extract medium supplemented with rice protein hydrolyzate (RPH), against strains of NBRC type culture and spoilage bacteria isolated from deteriorated sake. The partial sequences of the 16S rRNA gene of the spoilage bacteria isolated from sake showed a high similarity to those of Lactobacillus fructivorans, Lactobacillus hilgardii, and Lactobacillus paracasei, with 99.6-100% identity. Enterococcus durans C102901 (C102901), Lactococcus lactis subsp. lactis C101910 (C101910), and Lactococcus lactis subsp. lactis NBRC 12007 (NBRC 12007) grew well and produced bacteriocins with high activity in koji extract medium supplemented with RPH. When culture supernatants containing bacteriocins from C101910 and NBRC 12007 were added to the medium at a volume ratio of 10% (v/v), the growth of L. fructivorans NBRC 13954 T was significantly inhibited and the viable cell concentration decreased below the detection limit (1.0×10 2 cells/ml) at 4 h. Further, by the addition of bacteriocin solutions from C102901, C101910, and NBRC 12007 to the medium at a volume ratio of 1% (v/v), the growth of L. hilgardii NBRC 15886 T and H130 (closely related to L. hilgardii) isolated from putrid sake was bactericidally inhibited and the colony-forming units fell by more than three orders of magnitude within 4-12 h as compared with the initial cell concentration.

A total of 330 lactic acid bacteria isolated from South African red wines during alcoholic and malolactic fermentations and 9 commercial malolactic bacteria starter cultures were screened for antimicrobial activity. Of the entire screened isolates, 26 strains, belonging to the species Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus hilgardii and Oenococcus oeni, showed activity towards various wine-related and non-wine-related indicator strains. A PCR-based screening revealed the presence of the plantaricin encoding genes plnA, plnEF, plnJ and plnK in five selected Lb. plantarum strains. Furthermore, a co-culture experiment with Lb. plantarum and Enterococcus faecalis was performed. A complete inhibition of cell growth of Ent. faecalis was observed within 72 h. Four putative bacteriocin-encoding genes in the genome of O. oeni were identified and sequenced.

In bioprocesses, the industrial use of lactic acid bacteria as starter cultures is directly related to the effectiveness of preservation technologies which must guarantee high cell survival and functionality rates through the processing stages and after storage and rehydration. In this work, flow cytometry (FC) technique in combination with fluorescent probes allowed to monitor, almost in real-time conditions, the physiological states of cider malolactic starter cultures (Lactobacillus hilgardii) throughout preservation processes (freezing or freeze-drying) and their further progress during the first stages of culture implantation in the fermentation medium. A subpopulation of non-culturable but metabolically active cells, which could contribute to further fermentation, was determined when comparing FC results and viable counts. Metabolically active and dead cell counts determined by FC and colony forming units determined by the standard plate counting method showed that reactivation of frozen L. hilgardii cells during 24 h, using apple must supplemented with YE as cultivation and rehydration medium, resulted in the more suitable procedure for obtaining malolactic starter cultures.

This article aims to study putrescine production in Lactobacillus hilgardii strain X1B, an agmatine degrader isolated from wine, and to compare it with three other different species, previously reported as putrescine producers from agmatine: Pseudomonas aeruginosa PAO1, Enterococcus faecalis ATCC11700 and Bacillus cereus CECT 148T. The effect of different biogenic amines, organic acids, cofactors, amino acids and sugars on putrescine production was evaluated. In some cases, a similar effect was found in all the strains studied but the magnitude differed. Arginine, glucose and fructose showed an inhibitory effect, whereas the presence of agmatine induced the production of putrescine in all microorganisms. In other cases, the effect differed between P. aeruginosa PAO1 and the other microorganisms. Histamine and tyramine poorly influenced the utilization of agmatine, although a small increase in putrescine production was observed in P. aeruginosa PAO1. Succinate, spermidine and spermine also led to an increase in putrescine production in P. aeruginosa PAO1, whereas the succinate had no effect in the other microorganisms. Spermine and spermidine always produced a diminution in agmatine deamination. In this work, we have also demonstrated that pyridoxal 5-phosphate, Mg2+ and Mn2+ had no effect on putrescine production from agmatine. Results presented in this paper indicate differences in regulation mechanisms of agmatine deiminase pathway among P. aeruginosa PAO1 and L. hilgardii X1B, E. faecalis ATCC11700 and B. cereus CECT 148T. These results are significant from two points of view, first food quality, and second the toxicological and microbiological aspects. It should be taken into account that putrescine, whose origin is still controversial, is quantitatively the main biogenic amine found in food. Fil: Landete, J. M.. Universidad de Valencia; España Fil: Arena, Mario Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto de Química del Noroeste. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química del Noroeste; Argentina Fil: Pardo, I.. Universidad de Valencia; España Fil: Manca, Maria Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Ferrer, S.. Universidad de Valencia; España

Enhanced production of bacteriocin by Staphylococcus sp. NPSI 38 (NPSI 38) using koji extract medium was investigated. The bacteriocin produced by NPSI 38 in MRS medium was found to be effective for inhibiting the growth of Lactobacillus hilgardii NBRC 15886T, one of the representative hiochi-bacteria. In cultivations using test tubes without pH control, meat extract and Polypepton were effective for bacteriocin production by NPSI 38 with 10%(v/v) koji extract medium. When the koji extract medium supplemented with hydrolyzate of a rice protein preparation was used instead of meat extract and Polypepton, NPSI 38 produced 160 U/ml of bacteriocin in the cultivation with pH control, which was almost as high as that (156 U/ml) observed in cultivation using MRS medium with pH control. When the cells of L. hilgardii NBRC 15886T collected at logarithmic growth and stationary phases were inoculated into fresh modified MRS medium with 11 U/ml of the bacteriocin, negligible cell growth was observed, irrespective of different growth phases of cells inoculated. Little or no increase in cell concentrations in the media containing bacteriocin showed that the action of the bacteriocin produced by NPSI 38 was bacteriostatic against the hiochi-bacterium.

The aim of this work was to investigate the effect of wine phenolic aldehydes, flavonoids and tannins on growth and viability of strains of Oenococcus oeni and Lactobacillus hilgardii. Cultures were grown in ethanol-containing MRS/TJ medium supplemented with different concentrations of phenolic aldehydes or flavonoids and monitored spectrophotometrically. The effect of tannins was evaluated by monitoring the progressive inactivation of cells in ethanol-containing phosphate buffer supplemented with grape seed extracts with different molecular weight tannins. Of the phenolic aldehydes tested, sinapaldehyde, coniferaldehyde, p-hydroxybenzaldehyde, 3,4-dihydroxybenzaldehyde and 3,4,5-trihydroxybenzaldehyde significantly inhibited the growth of O. oeni VF, while vanillin and syringaldehyde had no effect at the concentrations tested. Lact. hilgardii 5 was only inhibited by sinapaldehyde and coniferaldehyde. Among the flavonoids, quercetin and kaempferol exerted an inhibitory effect especially on O. oeni VF. Myricetin and the flavan-3-ols studied (catechin and epicatechin) did not affect considerably the growth of both strains. Condensed tannins (particularly tetramers and pentamers) were found to strongly affect cell viability, especially in the case of O. oeni VF. In general, this strain was found to be more sensitive than Lact. hilgardii 5 to the phenolic compounds studied. This work contributes to the knowledge of the effect of different phenolic compounds on the activity of wine lactic acid bacteria, which, especially in the case of aldehydes and of different molecular weight fractions of tannins, is very scarce.

Aims: Ethyl carbamate (EC) is a toxic compound of major concern for the hygiene quality of wines. Its natural synthesis in wine is connected to the ethanolysis of molecules such as urea (alcoholic fermentation metabolite) and citrulline and carbamyl phosphate (malolactic fermentation metabolites) during ageing. This study reports the effect of adenine, arginine, urea and ammonium phosphate of Vitis vinifera L. cv. Tempranillo must, on EC synthesis in fermented wine using two lactic acid bacteria strains under different microbiological and physicochemical conditions.Methods and results: EC was quantified by gas chromatography-mass spectrometry detection. The Lactobacillus hilgardii strain evaluated was associated with particularly high EC production. No significant effect on EC formation was observed for nitrogen enrichment of the starting must.Conclusion: Residual arginine levels at the end of alcoholic fermentation from arginine- and ammonium-enriched musts showed no direct relationship to EC levels found after incubation with either of the LAB strains. Overall results suggest that the different nitrogenous substrates added to V. vinifera L. cv. Tempranillo must do not have any influence on EC formation due to LAB growth in wines under the experimental conditions used.Significance and impact of study: The composition of the nitrogen status of must has frequently been said to be a factor of concern on the final urethane concentration of wines. High contents of arginine coming from over fertilised vineyards are known to render significant levels of urea after alcoholic fermentation if conducted by arginase (+) yeast strains. This urea is always likely to undergo ethanolysis. No significant correlations were found between any of the nitrogenous compounds tested and final EC. High levels of arginine in the starting must did not lead to greater EC concentrations in the resulting wines.

Ethnic people of the Himalayan regions of India, Nepal, Bhutan and China consume a variety of indigenous fermented milk products made from cows milk as well as yaks milk. These lesser-known ethnic fermented foods are dahi, mohi, chhurpi, somar, philu and shyow. The population of lactic acid bacteria (LAB) ranged from 10(7) to 10(8) cfu/g in these Himalayan milk products. A total of 128 isolates of LAB were isolated from 58 samples of ethnic fermented milk products collected from different places of India, Nepal and Bhutan. Based on phenotypic characterization including API sugar test, the dominant lactic acid bacteria were identified as Lactobacillus bifermentans, Lactobacillus paracasei subsp. pseudoplantarum, Lactobacillus kefir, Lactobacillus hilgardii, Lactobacillus alimentarius, Lactobacillus paracasei subsp. paracasei, Lactobacillus plantarum, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris and Enterococcus faecium. LAB produced a wide spectrum of enzymes and showed high galactosidase, leucine-arylamidase and phosphatase activities. They showed antagonistic properties against selected Gram-negative bacteria. None of the strains produced bacteriocin and biogenic amines under the test conditions used. Most strains of LAB coagulated skim milk with a moderate drop in pH. Some strains of LAB showed a high degree of hydrophobicity, suggesting these strains may have useful adhesive potential. This paper is the first report on functional lactic acid bacterial composition in some lesser-known ethnic fermented milk products of the Himalayas.

Different lactic acid bacteria strains have been shown to cause wine spoilage, including the generation of substances undesirable for the health of wine consumers. The aim of this study was to investigate the occurrence of selected species of heterofermentative lactobacilli, specifically Lactobacillus brevis, Lactobacillus hilgardii, and Lactobacillus plantarum in six different Slovak red wines following the fermentation process. In order to identify the dominant Lactobacillus strain using quantitative (real time) polymerized chain reaction (qPCR) method, pure lyophilized bacterial cultures from the Czech Collection of Microorganisms were used. Six different red wine samples following malolactic fermentation were obtained from selected wineries. After collection, the samples were subjected to a classic plate dilution method for enumeration of lactobacilli cells. Real-time PCR was performed after DNA extraction from pure bacterial strains and wine samples. We used SYBR® Green master mix reagents for measuring the fluorescence in qPCR. The number of lactobacilli ranged from 3.60 to 5.02 log CFU mL(-1). Specific lactobacilli strains were confirmed by qPCR in all wine samples. The number of lactobacilli ranged from 10(3) to 10(6) CFU mL(-1). A melting curve with different melting temperatures (T(m)) of DNA amplicons was obtained after PCR for the comparison of T(m) of control and experimental portions, revealing that the most common species in wine samples was Lactobacillus plantarum with a T(m) of 84.64°C.