Добірка наукової літератури з теми "Lactobacillus hilgardii"

The production of mead holds great value for the Polish liquor industry, which is why the bacterium that spoils mead has become an object of concern and scientific interest. This article describes, for the first time, Lactobacillus hilgardii FLUB newly isolated from mead, as a mead spoilage bacteria. Whole genome sequencing of L. hilgardii FLUB revealed a 3 Mbp chromosome and five plasmids, which is the largest reported genome of this species. An extensive phylogenetic analysis and digital DNA-DNA hybridization confirmed the membership of the strain in the L. hilgardii species. The genome of L. hilgardii FLUB encodes 3043 genes, 2871 of which are protein coding sequences, 79 code for RNA, and 93 are pseudogenes. L. hilgardii FLUB possesses three clustered regularly interspaced short palindromic repeats (CRISPR), eight genomic islands (44,155 bp to 6345 bp), and three (two intact and one incomplete) prophage regions. For the first time, the characteristics of the genome of this species were described and a pangenomic analysis was performed. The concept of the pangenome was used not only to establish the genetic repertoire of this species, but primarily to highlight the unique characteristics of L. hilgardii FLUB. The core of the genome of L. hilgardii is centered around genes related to the storage and processing of genetic information, as well as to carbohydrate and amino acid metabolism. Strains with such a genetic constitution can effectively adapt to environmental changes. L. hilgardii FLUB is distinguished by an extensive cluster of metabolic genes, arsenic detoxification genes, and unique surface layer proteins. Variants of MRS broth with ethanol (10–20%), glucose (2–25%), and fructose (2–24%) were prepared to test the strain’s growth preferences using Bioscreen C and the PYTHON script. L. hilgardii FLUB was found to be more resistant than a reference strain to high concentrations of alcohol (18%) and sugars (25%). It exhibited greater preference for fructose than glucose, which suggests it has a fructophilic nature. Comparative genomic analysis supported by experimental research imitating the conditions of alcoholic beverages confirmed the niche specialization of L. hilgardii FLUB to the mead environment.

After 6 days of Lactobacillus hilgardii 5w incubation at 4°C, the viable cell counts diminish 31.9, 45.6, and 89.0% when suspended in control wine (2,600 mg/liter gallic acid equivalents [GAE]), three-fold concentrated wine (6,150 mg/liter GAE), and six-fold concentrated wine (13,000 mg/liter GAE), respectively. At 20°C in the same conditions, the cell viabilities decrease 74.2, 80.5, and 100.0%, respectively. In decolorized wines, which result in tannin losses, the viable cell counts increase. There is a relationship between L. hilgardii 5w tannin binding and its viability loss.

Of the 40 strains isolated from several spoiled ciders where glycerol was degraded, 36 were identified as Lactobacillus collinoides, three were Lactobacillus hilgardii, and one was Lactobacillus mali. However, only 30 L. collinoides and two L. hilgardii could degrade glycerol. The glycerol dehydratase activity was shown. The main product of the transformation was 1,3 propanediol. Two DNA primers GD1 and GD2 were chosen in the region encoding one of the subunits of glycerol dehydratase of Citrobacter freundii, Klebsiella pneumoniae, Klebsiella oxytoca, Salmonella Typhimurium, and Clostridium pasteurianum. A 279-bp amplicon in polymerase chain reaction amplification was obtained with the genomic L. collinoides IOEB 9527 DNA as template. The amino acid sequence deduced from the amplicon DNA sequence showed a very high similarity and identity with the gene of gram-negative and C. pasteurianum species. After labeling, the amplicon was used as DNA probe in dot-blot hybridization with the genomic DNA of all the tested strains. Only strains that could degrade glycerol hybridized. Moreover, polymerase chain reactions using GD1 and GD2 revealed only glycerol dehydratase genes of positive L. collinoides and L. hilgardii strains. The primers and the amplicon proved to be suitable and reliable tools to detect the lactic acid bacteria involved in the deterioration of cider.

Lactobacillus ferintoshensis has recently been described as a novel species, distinct from its close phylogenetic neighbours Lactobacillus buchneri, Lactobacillus kefiri and Lactobacillus hilgardii. Two highly related species with validly published names, Lactobacillus parakefiri and Lactobacillus parabuchneri, were not considered in the study due to the lack of 16S rRNA gene sequence data at that time. Since the publication of the study, the sequences have become available and have revealed that L. ferintoshensis and L. parabuchneri share 99·7 % 16S rRNA gene sequence similarity. Further genomic and phenotypic data, derived from fluorescent amplified fragment length polymorphism, DNA–DNA hybridization and API 50 CHL analyses, have demonstrated that the species are synonymous.

Five strains of lactic acid bacteria were isolated from a compost of distilled shochu residue in Japan. The isolates were separated into two groups on the basis of 16S rRNA gene sequence similarity, and two subclusters were formed that comprised micro-organisms closely related to Lactobacillus buchneri, L. diolivorans, L. hilgardii, L. kefiri, L. parabuchneri and L. parakefiri. DNA–DNA relatedness results revealed that the isolates could be separated into two groups, and these groups correlated well with the subclusters generated using the phylogenetic analysis. Moreover, the levels of DNA–DNA relatedness showed clear separation of the two groups from their phylogenetic relatives. Therefore, the two groups represent two novel species, for which the names Lactobacillus farraginis sp. nov. (type strain NRIC 0676T=JCM 14108T=DSM 18382T) and Lactobacillus parafarraginis sp. nov. (type strain NRIC 0677T=JCM 14109T=DSM 18390T) are proposed.

En vinification, les bactéries lactiques conduisent la fermentation malolactique, processus de désacidification du vin. Cependant, certaines d’entre elles peuvent être à l’origine d’altérations. Le contrôle de la flore microbienne est donc souhaitable à toutes les étapes de la vinification, grâce à la mise en œuvre de méthodes d’identification de plus en plus efficaces. L’identification moléculaire des bactéries lactiques est désormais réalisée par une méthode d’hybridation ADN/ADN. Mais, des hybridations croisées sont observées entre plusieurs souches L. Hilgardii et L. Brevis, différenciées au préalable d’après leur profils API. Les souches fermentant ce pentose sont classées au sein de l’espèce L. Brevis, les autres au sein de l’espèce L. Hilgardii. La caractérisation moléculaire des espèces L. Hilgardii et L. Brevis constitue donc l’essentiel de ce travail. Des sondes et tests PCR spécifiques de l’espèce L. Hilgardii ont été élaborés. Par contre, dans le cas de L. Brevis, les polymorphismes génomiques observés laissent supposer que le regroupement de souches effectué sur la base de profil fermentaire ne correspond pas véritablement à une espèce. Ainsi, trois souches fermentant l’arabinose, mais possédant toutes les caractéristiques génomiques requises, ont été reclassés au sein de l’espèce L. Hilgardii, remettant ainsi en cause la validité du test biochimique. Une étude métabolique et génétique de l’assimilation de l’arabinose a donc été abordée chez différentes souches L. Hilgardii et L. Brevis. Les différences phénotyques observées entre les souches L. Hilgardii ne porteraient pas forcément sur la capacité ou non à fermenter l’arabinose. Enfin, dans le but de réaliser un contrôle de qualité efficace des vins, une méthode d’hybridation in situ a été adaptée au contrôle de la flore lactique avec les sondes d’ores et déjà utilisées pour des méthodes d’hybridation en tache ou sur colonie.

This study examined the complex array of ester synthesis and hydrolysis activities in whole cells by cloning, heterologous expression, partial purification, and biochemical characterisation of EstA2, EstB28 and EstCOo8 esterase proteins from O. oeni alongside EstC34, an esterase from Lactobacillus hillgardii. With a view to applying these esterases to wine, enzyme function under frequently encountered harsh physicochemical conditions in wine was examined. EstB28, EstCOo8 and EstC34 showed highest activity with pNPacetate (C₂) and pNP-butanoate (C₄). The highest activity of EstA2 was observed with pNP-hexanoate (C₆). All enzymes retained at least some activity under conditions relevant to winemaking. However, only EstB28 and EstA2 showed an increase in activity above 10% ethanol. EstB28 and EstA2 also had a higher relative activity to EstCOo8 and EstC34. Once characterisation of these esterases showed that they should retain at least partial activity under wine-like conditions, EstA2 and EstB28 were assayed for activity in two separate wine samples by SPME-GCMS. Studies were also conducted to determine activity on natural substrates. Synthesis and hydrolysis of ethyl esters with acyl chain lengths of between C₂-C₈ was measured by SPME-GCMS. All four esterases where found to have the ability to synthesise and hydrolyse ethyl esters under optimal conditions (pH 5.0). Observed strain-specific differences in whole cell ester hydrolysis were also investigated. Firstly wine MLF trials were conducted in Chardonnay and Cabernet Sauvignon wines to determine if these strain specific differences could be translated into hydrolysis ability in wine. Eight strains of LAB were initially used in this study and strains with the lowest activity against pNP-linked ester substrates (Ooeni2, Ooeni3, Ooeni6, Lac34 and Lac40) were compared to the strains with the highest activity (Ooeni8, Ooeni12 and Ooeni28). In an effort to better understand the O. oeni strain specific differences in esterase activity real-time qPCR was carried out on an independent set of samples (n = 30) using strain Ooeni28 (‘high’ activity against pNP-linked ester substrates). Expression of esterase genes was measured in the presence of known esterase substrates. Both estA2 and estA7 showed an increase in expression in the presence of ethanol and butyric acid, whereas estB28 and estC expression increased in the presence of ethyl butyrate. However, when the same experiment was repeated with Ooeni2 (‘low’ activity) there was little change in the expression of the characterised esterase genes. Further investigation is required to determine if this is response is related to the phenotype of ‘low’ activity. Finally esterase genes were also sequenced from strains demonstrating ‘low’ and ‘high’ esterase hydrolysis activity to establish if there are any differences in predicted protein sequences amongst these strains. All strains sequenced had a homologue of EstA2, EstA7 and EstB28. Based on EstA2 and EstB28 sequences the strains with ‘high’ whole cell activity can be separated from strains with „low‟ whole cell activity through single nucleotide polymorphisms (SNP). This study will improve the understanding of the functioning of LAB esterases in wine conditions and the reason for strain specific differences in activity, with a view towards modulating and controlling the impact of LAB in ester profile modifications during the MLF.
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2013