Arginine, citrulline and ornithine metabolism by lactic acid bacteria from wine

The catabolism of arginine, an amino acid found in grape juice and wine, citrulline and ornithine was investigated in four lactic acid bacteria. Only Lactobacillus hilgardii X1B catabolized arginine and excreted citrulline into the medium. The recovery of arginine as ornithine was lower than the expected theoretical value. The arginase-urease pathway was not detected indicating that the amino acid degradation was carried out only by the arginine dihydrolase pathway. Oenococcus oeni m, a strain not able to utilize arginine, degraded citrulline that was completely recovered as ornithine, ammonia and CO2. Lactobacillus hilgardii X1B catabolized citrulline but it was only 44% recovered as ornithine. The citrulline utilization by Oenococcus oeni m may be important for two reasons: it can gain extra energy for growth from citrulline metabolism, and the amino-acid diminution could avoid the possibility of ethyl carbamate formation from the citrulline naturally present in wine.     

Inhibitory substances produced by Lactobacilli isolated from sourdoughs--a review

Several sourdough lactic acid bacteria (LAB) produce inhibitory substances other than organic acids. Bacteriocins (bavaricin A, and plantaricin ST31), a bacteriocin-like inhibitory substance (BLIS C57), and a new antibiotic (reutericyclin) have been discovered. Maximum antimicrobial production was found in the pH range 4.0-6.0. Temperature optima vary strongly. The substances are resistant to heat and acidity, and inactivated by proteolytic enzymes, except for reutericyclin. Bavaricin A and plantaricin ST31 have been purified to homogeneity. Bavaricin A is classified as a class IIa bacteriocin. Reutericyclin is a new tetramic acid. The mode of action of bavaricin A, BLIS C57, and reutericyclin is bactericidal. Some of these substances are active towards some Bacilli, Staphylococci and Listeria strains. Up to now, only the application potential of purified bavaricin A has been examined. More research should be done to study the production, the activity, and the stability of these inhibitory substances in food systems as these often differ from the broths mostly used in this kind of studies. Furthermore, an extensive screening of the sourdough microflora must be performed, in particular towards Bacilli and fungi. This could lead to the discovery of additional inhibitory substances, although it seems that the frequency of isolating bacteriocin-producing sourdough LAB is low. However, potent antimicrobials towards Bacilli as well as antifungal substances will have to be found using rational screening strategies and novel purification and analytical techniques.     

Aerobic glycerol catabolism by Pediococcus pentosaceus isolated from wine

Glycerol catabolism was studied in Pediococcus pentosaceus N₅p isolated from wine, growing on different glycerol concentrations in aerobic conditions. Enzymatic activities of the glycerol kinase and glycerol dehydrogenase pathways were detected but the levels demonstrated allow to establish that glycerol was mainly degraded by glycerol kinase pathway. The highest levels of the activities of this pathway were expressed at the lowest concentration of glycerol, decreasing with the increase in glycerol concentration in the growth media. Glycerol was transformed in d-lactate, acetate, diacetyl and 2,3-butanediol. The enzymatic activities involved in the formation of these products were also modulated by glycerol. NAD-dependent lactate dehydrogenases were not affected by glycerol. Diacetyl reductase and acetoin reductase were inhibited by glycerol, whereas NAD-independent lactate dehydrogenases, NADH oxidase as CoA-independent acetaldehyde dehydrogenase, and acetate kinase were stimulated. The behavior of the enzymatic activities was in conformity with the fermentation balances; we propose a probable pathway for glycerol catabolism in P. pentosaceus N₅p.     

Transport of glycerol by Pediococcus pentosaceus isolated from wine

Pediococcus pentosaceus N(5)p is a strain isolated from wine that uses glycerol as its sole carbon source, mainly via the glycerol kinase pathway. The transport of glycerol was investigated in resting cells of this strain. Glycerol uptake followed a Michaelis-Menten relationship with an observed apparent K(m) of 33 microM and a V(max) of 2.5 nmol/min/mg of cell protein. The transport system was specific for glycerol, which was present in the cells grown either on glycerol or glucose suggesting its constitutive nature. The presence of uptake when resting cells were treated with HgCl(2) and the absence of counterflow indicate that facilitated diffusion is not involved in glycerol transport. On the other hand, glycerol uptake was inhibited by the metabolic poisons that affect ATP availability by acting on either electron transport or ATPase activity, and by the proton-conducting uncouplers without any effect on glycerol kinase activity. The restoration of glycerol uptake in de-energized cells by the addition of glucose and low concentration of cyanide-m-chlorophenyl hydrazone was achieved. These results, the first in the genus Pediococcus, provide evidence for an energy-dependent uptake of glycerol that involves the proton motive force directly or coupled with ATP synthesis.     

Technological characterisation of Lactobacilli isolated from Chinese artisanal fermented milks

Thirty‐six Lactobacilli, isolated from Chinese artisanal fermented milks, were evaluated for potential use as adjunct cultures. All isolates presented low acidification rate. Proteolytic activities of these Lactobacilli ranged from 17.7 to 48.5 mg Gly/L milk, and strain SB5 showed the highest activity. Aminopeptidase activities ranged from 15.1 to 71.0 U/mg. Strains M18L2, SB33 and SB29 were 71.0, 68.6 and 68.5 U/mg, respectively. Autolytic activities of isolates in simulated cheese‐like buffer were between 10% and 45%. Strains SB9 and SB11 showed the two highest values. In summary, the aforementioned six strains could be good candidates as adjunct cultures in cheese.     

Lactobacilli isolated from chicken intestines: potential use as probiotics

Lactobacillus strains were tested for their in vitro probiotic properties. Cell surface hydrophobicity was found to be very high for Lactobacillus fermentum subsp. cellobiosus and Salmonella Gallinarum; high values could indicate a greater ability to adhere to epithelial cells. Studies on Lactobacillus animalis indicated relative cell surface hydrophobicities smaller than those of L. fermentum subsp. cellobiosus and L. fermentum. L. animalis and Enterococcus faecalis were able to coaggregate with L. fermentum subsp. cellobiosus and L. fermentum, respectively, but not with Salmonella Gallinarum. After mixed-culture studies for determining suitable growth behavior, the pair of strains L. animalis plus L. fermentum subsp. cellobiosus was selected for an attempted challenge against Salmonella Gallinarum. Double and triple mixed-culture studies indicated that selected lactobacillus strains were able to retain their beneficial characteristics in the presence of Salmonella Gallinarum such as presence of lectins, production of antimicrobial compounds, and ability to grow and compete. The selected microorganisms can be considered as potential ingredients for a chicken probiotic feed formulation intended to control salmonellosis and also improve poultry sanitation.     

Genotyping by Amplified Fragment Length Polymorphism and malate metabolism performances of indigenous Oenococcus oeni strains isolated from Primitivo wine

The Amplified Fragment Length Polymorphism (AFLP) technique was applied for the first time to investigate the genotyping of Oenococcus oeni, the most important species involved in malolactic fermentation (MLF) in wine. A total of 87 out of 220 lactic acid bacteria, isolates from "Primitivo" wine (Apulia, Italy) undergoing MLF, identified as O. oeni by species-specific PCR and 16S rRNA sequence analysis, were studied by AFLP analysis. Four main clusters were distinguished and three of them showed intraspecific homology higher than 60%. A total of 28 strains, representative of AFLP clusters, were tested for malate metabolism in order to gain information on their malolactic performances. Significant differences were observed among strains for malic acid consumed, biomass produced and specific malic acid consumption rate. These findings indicated that AFLP technique is reliable for typing O. oeni strains and that, together with metabolism studies it may be used to individuate possible candidates as industrial malolactic starters.     

Evaluation of exopolysaccharide production by Leuconostoc mesenteroides strains isolated from wine

ABSTRACT:  Exopolysaccharide (EPS)-producing lactic acid bacteria are responsible for the alteration of wine and other fermented beverages. The potential to produce EPS was investigated for Leuconostoc mesenteroides strains isolated from Spanish grape must and wine. Most strains were able to produce EPS from sucrose containing media. Based on their EPS-producing phenotype and on their EPS monosaccharide composition, the L. mesenteroides strains analyzed could be arranged in 2 groups. One group comprises mucoid strains producing a glucan polymer, and the other group includes strains producing a fructan polymer. The presence of a glucosyltransferase encoding gene in the glucan producing L. mesenteroides strains was assayed by PCR. Two primer sets, PF1-PF8 and GTFF-GTFR, were used to amplify internal fragment of known glucosyltransferase genes. None of the glucan-producing strains gave a positive amplicon by the primer sets used. Therefore, new tools need to be developed to broaden the range of potentially spoiling agents detected by PCR in fermented beverages.     

中国传统乳制品中乳杆菌的益生菌潜力评估

目的：评估从中国传统乳制品中分离出的9株乳杆菌的功能及其益生菌潜力。 方法：从中国传统乳制品中分离纯化得到乳杆菌菌株,测试了它们的益生菌潜力,如溶血活性和抗氧化活性等。 结果：所有分离的乳杆菌均可耐受低pH值（pH 2.0、2.5和3.0）和高胆盐条件（0.3％,0.5％和1％）。 测试了分离的乳杆菌对10种抗生素的敏感性,结果表明,所有分离株对万古霉素均没有抗性,另一方面,大多数分离株对青霉素,氨苄青霉素,链霉素和四环素具有抗性。 所有乳杆菌对HT-29细胞具有优异的粘附能力,其值介于10.4％-39.0％之间,并且对DPPH自由基（清除率50.4％-80.4％）和超氧阴离子自由基具有出色的抗氧化活性（清除率17.5％-44.4％）。 另外,所有分离的乳杆菌均可以极大降低胆固醇含量（19.1-65.8 μg / mL）,且没有任何溶血性。结论：从中国乳制品中分离出的乳杆菌具有作为各种产品中益生菌的潜力。     

Tannase activity by lactic acid bacteria isolated from grape must and wine

We examined a range of oenological lactic acid bacteria species and reference strains for their potential to degrade tannins. Bacterial tannase activity was checked by a spectrophotometric and a visual reading method. None of the strains belonging to the oenological species of the genus Lactobacillus, Leuconostoc, Oenococcus or Pediococcus were tannase producers, with the exception of Lactobacillus plantarum. All the L. plantarum strains analyzed were positive for tannase activity and their identities were reconfirmed by L. plantarum PCR-specific assay or by sequencing the 16S rDNA. Tannase activity could be considered an important criterion for the selection of malolactic starter cultures since it might confer advantages in the winemaking process by reducing astringency and haze in wine.     

Phenotypic and genotypic characterisation of Lactobacilli isolated from camel cheese produced in India

Thirty‐two Lactobacilli strains were isolated from four samples of camel cheese collected from Bikaner, India. These isolates were identified based on phenotypic and genotypic characteristics. Sequencing of 16S rDNA was performed for species identification and diversity analysis. Lactobacillus delbrueckii and Lb. fermentum were found to be dominant species followed by Lb. plantarum and Lb. casei. On evaluation of technological properties of these isolates, 20 isolates were observed to be good acid producers, eight were found positive for citrate utilisation and 11 showed presence of Prtp gene. Isolates obtained can be potential for development of defined strain starter for camel cheese.     

Lactobacilli isolated from kefir grains: evidence of the presence of S-layer proteins

In the present study we report for the first time the presence of S-layer proteins in Lactobacillus kefir and Lactobacillus parakefir isolated from kefir grains. Soluble whole-cell protein profile obtained either by mechanical disruption (X-press) or by a combined treatment with lysozyme and SDS on whole cells, showed a significant band of apparent molecular mass of 66-71 kDa as measured by SDS-PAGE. The intensity of this band was considerably reduced when cells were treated with 5 M-LiCl. The above mentioned proteins were recovered in the LiCl extracts. After dialysis and concentration, the proteins extracted were able to reassemble in a regular array. Negative staining of these protein preparations were analysed by transmission electron microscopy and a paracrystalline arrangement was seen. Thin sections of bacteria analysed by transmission electron micrographs showed an outermost layer over the bacterial cell wall, that was lost after the LiCl treatment. The production of this surface structure under different culture conditions was also evaluated. Finally, the relationship between the presence of S-layer proteins and surface properties (e.g. adhesion to Caco-2 cells, autoaggregation, and hemagglutination) was investigated.     

Screening of biogenic amine production by lactic acid bacteria isolated from grape must and wine

The potential to produce the biogenic amines tyramine, histamine and putrescine, was investigated for lactic acid bacteria (LAB) of various origin, including commercial malolactic starter cultures, type strains and 78 strains isolated from Spanish grape must and wine. The presence of biogenic amines in a decarboxylase synthetic broth was determined by reverse-phase high performance liquid chromatography (RP-HPLC). Tyramine was the main amine formed by the LAB strains investigated. Leuconostoc strains were the most intensive tyramine formers. No potential to form biogenic amines was observed in Oenococcus oeni strains. Two strains of Latobacillus buchneri were associated with putrescine formation. None of the lactic acid bacteria produced histamine. According to these in vitro results, the commercial starter bacteria analyzed did not produce histamine, tyramine and putrescine.     

烧酒曲中扣囊复膜酵母的分离及鉴定

为了筛选1株可用于纯菌种酿造糯米酒的酵母菌,将烧酒曲稀释涂布在淀粉培养基(YPS)平板上培养,得到1株产淀粉酶酵母菌(YW12)。通过形态学、生理生化特征和18S rDNA、ITS区序列分析鉴定,YW12为扣囊复膜酵母(Saccharomycopsis fibuligera)。YW12在YPS液体培养基中28℃培养4 d,用Yoo改良法测其粗酶液的淀粉酶活力为49.8 U/mL。YW12在糯米糖化液(含175 g/L葡萄糖)中28℃发酵3 d的酒精度为5.63%(v/v)。YW12既能同化淀粉又能发酵产生酒精,具有用于纯菌种酿造糯米酒的潜力。     

Tyramine and phenylethylamine production among lactic acid bacteria isolated from wine

The ability of wine lactic acid bacteria to produce tyramine and phenylethylamine was investigated by biochemical and genetic methods. An easy and accurate plate medium was developed to detect tyramine-producer strains, and a specific PCR assay that detects the presence of tdc gene was employed. All strains possessing the tdc gene were shown to produce tyramine and phenylethylamine. Wines containing high quantities of tyramine and phenylethylamine were found to contain Lactobacillus brevis or Lactobacillus hilgardii. The main tyramine producer was L. brevis. The ability to produce tyramine was absent or infrequent in the rest of the analysed wine species.     

Structural analysis by 13C-nuclear magnetic resonance spectroscopy of glucans elaborated by gum-producing bacteria isolated from palm wine

The linkages of the glucans produced by palm wine bacteria in sterile palm sap and in sucrose broth were determined using ¹³C nmr spectroscopy. The glucose units appeared to be linked α(-1–6) in the main chain. Therefore, the glucans are likely to be dextrans. There were branch linkages, and these differed between the genera of lactic acid bacteria (LAB) and even within one genus. However, branching by α(-1–3) was a feature common to all the dextrans of the three organisms employed. The dextran of Leuconostoc dextranicum appeared to branch mainly by α(1–3) linkages with minor α(1–4) ones; that of Leconostoc mesenteroides, mainly by α(-1–2) and that of the Lactobacillus spp. by only α(1–3) linkages. The organisms were found to elaborate more highly branched dextrans in sucrose broth than in palm sap probably due to nutrient differences, but the branch linkage types remained the same. The degree of branching did not appear to affect the viscosity. It was concluded that gums produced by palm wine glucan-producers were dextrans and that these different dextran-producing bacteria, in palm wine, each produced its own peculiar type of dextran in the beverage.     

Effect of phenolic acids on glucose and organic acid metabolism by lactic acid bacteria from wine

The influence of phenolic (p-coumaric, caffeic, ferulic, gallic and protocatechuic) acids on glucose and organic acid metabolism by two strains of wine lactic acid bacteria (Oenococcus oeni VF and Lactobacillus hilgardii 5) was investigated. Cultures were grown in modified MRS medium supplemented with different phenolic acids. Cellular growth was monitored and metabolite concentrations were determined by HPLC-RI. Despite the strong inhibitory effect of most tested phenolic acids on the growth of O. oeni VF, the malolactic activity of this strain was not considerably affected by these compounds. While less affected in its growth, the capacity of L. hilgardii 5 to degrade malic acid was clearly diminished. Except for gallic acid, the addition of phenolic acids delayed the metabolism of glucose and citric acid in both strains tested. It was also found that the presence of hydroxycinnamic acids (p-coumaric, caffeic and ferulic) increased the yield of lactic and acetic acid production from glucose by O. oeni VF and not by L. hilgardii 5. The results show that important oenological characteristics of wine lactic acid bacteria, such as the malolactic activity and the production of volatile organic acids, may be differently affected by the presence of phenolic acids, depending on the bacterial species or strain.     

Assessment of antibiotic susceptibility within lactic acid bacteria strains isolated from wine

Susceptibility to 12 antibiotics was tested in 75 unrelated lactic acid bacteria strains of wine origin of the following species: 38 Lactobacillus plantarum, 3 Lactobacillus hilgardii, 2 Lactobacillus paracasei, 1 Lactobacillus sp, 21 Oenococcus oeni, 4 Pediococcus pentosaceus, 2 Pediococcus parvulus, 1 Pediococcus acidilactici, and 3 Leuconostoc mesenteroides. The Minimal Inhibitory Concentrations of the different antibiotics that inhibited 50% of the strains of the Lactobacillus, Leuconostoc and Pediococcus genera were, respectively, the following ones: penicillin (2, < or =0.5, and < or =0.5 microg/ml), erythromycin (< or =0.5 microg/ml), chloramphenicol (4 microg/ml), ciprofloxacin (64, 8, and 128 microg/ml), vancomycin (> or =128 microg/ml), tetracycline (8, 2, and 8 microg/ml), streptomycin (256, 32, and 512 microg/ml), gentamicin (64, 4, and 128 microg/ml), kanamycin (256, 64, and 512 microg/ml), sulfamethoxazole (> or =1024 microg/ml), and trimethoprim (16 microg/ml). All 21 O. oeni showed susceptibility to erythromycin, tetracycline, rifampicin and chloramphenicol, and exhibited resistance to aminoglycosides, vancomycin, sulfamethoxazole and trimethoprim, that could represent intrinsic resistance. Differences were observed among the O. oeni strains with respect to penicillin or ciprofloxacin susceptibility. Antibiotic resistance genes were studied by PCR and sequencing, and the following genes were detected: erm(B) (one P. acidilactici), tet(M) (one L. plantarum), tet(L) (one P. parvulus), aac(6')-aph(2") (four L. plantarum, one P. parvulus, one P. pentosaceus and two O. oeni), ant(6) (one L. plantarum, and two P. parvulus), and aph(3')-IIIa (one L. plantarum and one O. oeni). This is the first time, to our knowledge, that ant(6), aph(3')-IIIa and tet(L) genes are found in Lactobacillus and Pediococcus strains and antimicrobial resistance genes are reported in O. oeni strains.     

植物乳杆菌与费氏丙酸菌转化生成甘露醇的比较

对1株植物乳杆菌(Lactobacillus plantarum HSC 235)和1株费氏丙酸菌(Propionibacterium freudenreichiiHZP-35)生物转化果糖生成甘露醇能力进行了比较研究。在生长细胞、静息细胞以及无完整细胞粗酶提取物3种不同转化反应体系中,植物乳杆菌HSC 235和费氏丙酸菌HZP-35都表现出甘露醇脱氢酶活性,而生长细胞甘露醇产量和得率较高,分别为38 g/L和27%,静息细胞和粗酶提取物反应体系甘露醇的产量及得率则相关无几,表明甘露醇的转化过程与细胞生长紧密关联。在不同初始果糖浓度的试验中,较低果糖有利甘露醇得率的提高,50～150 g/L果糖浓度甘露醇得率稳定在27%左右,果糖浓度提高到200 g/L时则表现出对转化的抑制作用。植物乳杆菌可与费氏丙酸菌混合生长,但混菌发酵的实验结果表明,混合培养体系甘露醇得率比单菌种纯培养时甘露醇得率要低得多。植物乳杆菌甘露醇醇产量和得率要明显高于费氏丙酸菌。