Assessment of the genetic polymorphism and biogenic amine production of indigenous Oenococcus oeni strains isolated from Greek red wines

In the warm climate country of Greece malolactic fermentation (MLF) has received limited attention. Molecular techniques and High Performance Liquid Chromatography (HPLC) were used to study the genetic polymorphism of autochthonous lactic acid bacteria developing towards the end of spontaneous MLF of Greek red wines and for the assessment of their potential to produce harmful biogenic amines. This research revealed that native Oenococcus oeni isolates are very much adapted to specific winery conditions since the majority of spontaneous MLF were driven mostly or exclusively by a single strain of O. oeni. Native O. oeni strains showed only limited dispersion since cluster analysis uncovered only few common genotypes among indigenous isolates from different wineries. The genotype of a frequently used malolactic starter was more than often detected among autochthonous isolates without nevertheless compromising the biodiversity of natural microflora residing in wineries but rather becoming a part of it. For the majority of the wine samples studied, MLF implementation and storage in bottles resulted in negligible changes on the levels of the BA histamine, tyramine, phenylethylamine, cadaverine as well as of ethylamine, methylamine, isobutylamine. We provide evidence that autochthonous O. oeni isolates can only contribute to putrescine accumulation in Greek wines but still the specific trait behaves as strain-specific with a limited dispersion.     

Characterization and amino acid metabolism performances of indigenous Oenococcus oeni isolated from Chinese wines

Oenococcus oeni is a multiple physical stress-tolerant lactic acid bacterium that plays an important role in wine making. It is often added as a starter culture to carry out malolactic fermentation (MLF). In this study, a total of 22 out of 127 lactic acid bacteria, isolated from Chinese wines undergoing MLF, were identified as O. oeni by species-specific PCR and 16S rRNA sequencing. Single-enzyme amplified fragment length polymorphism (SE-AFLP) analysis showed that all strains could be typed under these conditions, and three main groups were determined by cluster analysis, which showed intraspecific homology higher than 69 %. Eight strains, representative of SE-AFLP clusters, were tested for malolactic activity. Significant differences were observed among strains with regard to the amount of malic acid consumed. Seventeen amino acids in different wines that were inoculated by 4 O. oeni strains, respectively, were analyzed before and after MLF. The results indicated that the amino acid metabolism of the 4 strains was significantly different between each strain.     

Inducing malolactic fermentation in Chardonnay musts and wines using different strains of Oenococcus oeni

Malolactic fermentations (MLF) were induced in a commercially prepared Washington State Chardonnay must to evaluate the influence of timing of inoculation and pre-culture conditions of Oenococcus oeni strains MCW, EQ-54, and WS-8. The must (pH 3.62, 21.5°Brix) was divided into lots and inoculated with Saccharomyces cerevisiae strain CY3079. Strains of O. oeni were pre-cultured by growing in diluted juice or by re-hydration of freeze-dried strains. Bacteria were inoculated into the musts before (Day 0) or after completion of the alcoholic fermentation (Day 22). Yeast populations exceeded 10⁷cfu/mL in all fermentations that proceeded to dryness. However, the viability of most strains of O. oeni quickly declined after inoculation regardless of the timing of inoculation or the strain used. MLF was induced in the wines inoculated with strains EQ-54 and WS-8 but not with MCW, and the rate depended on the time of inoculation. The method used to prepare bacterial starter cultures had no apparent influence on the completion of MLF. Values for volatile acidity were slightly higher (P< 0.05) in wines inoculated with O. oeni before alcoholic fermentation compared with those inoculated after alcoholic fermentation.     

Phenotypic and genotypic characterization of Pseudomonas aeruginosa strains isolated from mastitis outbreaks in dairy herds

During 1998-2002 outbreaks of Pseudomonas sp. mastitis among more than 15 Israeli sheep and goat dairy herds were observed. The animals presented a wide spectrum of clinical signs ranging from subclinical to gangrenous udder. Ninety-five isolates of Pseudomonas sp. were isolated from clinical and subclinical mastitis of 47 sheep, 17 goats and 31 cows from 34 different farms. Biochemical and genetic analyses revealed that the all-causative organism was Ps. aeruginosa. Selections of isolates were further analysed on the bases of colony morphology, biochemical traits and capacity to form biofilm. All the strains displayed a wide heterogeneity in all the tested traits. No association between bacterial isolates, farm of origin and type of animal was found. Pulsed-field gel electrophoresis and cluster analysis showed no clonality among the tested strains. The present study revealed that a large variety of Ps. aeruginosa strains may cause mastitis outbreaks in sheep, goat and cattle in Israel.     

宁夏酿酒产区酒酒球菌的快速鉴定及其PCR体系的优化

运用基于酒酒球菌16SrRNA高度保守序列设计的特异引物,通过对实验室保存优良酒酒球菌菌株的扩增验证,实现了对筛选自宁夏产区的未知苹果酸乳酸细菌的快速、可靠鉴定。通过对影响特异引物PCR反应体系的主要因素进行优化,建立了适合于实验室的特异引物扩增反应体系。结果表明:实验室保存酒酒球菌菌株均能扩增出唯一的、清晰的、大约995bp的条带,未知菌株通过扩增证明均为酒酒球菌;建立的特异引物扩增体系为:50μL的反应总体积,2.5UTaq酶、200μmol/LdNTP、0.2μmol/L上/下游引物、2.0mmol/LMg2+、DNA模板40ng。     

Phenotypic and genotypic characterization of lactobacilli isolated from Spanish goat cheeses

Lactic acid bacteria from 18 Spanish goat cheeses produced by seven dairies were isolated to evaluate the genetic diversity of this bacterial community. 136 representative isolates were characterized by phenotyping and Randomly Amplified Polymorphic DNA (RAPD-PCR) analysis. Ten species were identified with predominance of Lactobacillus paracasei subsp. paracasei. The presence of L. curvatus, L. pentosus, L. cellobiosus and L. rhamnosus has not hitherto been reported in Spanish goat cheeses. A high degree of genetic diversity was found for L. paracasei subsp. paracasei, L. curvatus and L. plantarum. Some of the identified strains displayed strong acidifying and proteolytic capacities.     

Phenotypic and genotypic aspects of Lactobacillus sanfranciscensis strains isolated from sourdoughs in Italy

Physiological properties and genetic characteristics of 44 strains of Lactobacillus sanfranciscensis, isolated in Italy from sourdough samples for salted and sweet baked products, were investigated. Data were compared with those obtained for reference strains belonging to the same and to related species. Phenotypic analysis showed the presence of two main groups, the first including strains able to ferment glucose and maltose, the second including strains fermenting only maltose. Other few isolates exhibited individual different fermentation patterns. Genetic diversity was evaluated by using Amplified Ribosomal 16S rDNA Restriction Analysis and Amplified 16S-23S rDNA Spacer Region Analysis. Electrophoretical patterns of fragments generated by Hinf I indicated a polymorphism within 16S rDNA gene that allowed the subdivision ofLb. sanfranciscensis strains in two groups. The reliability to use Dde I and Hinf I 16S rDNA restriction analysis for differentiation of Lb. sanfranciscensis strains from the other phylogenetically related Lactic Acid Bacteria was demonstrated. The amplification of the intergenic spacer region produced a unique electrophoretical profile of three bands (approximately 300, 390 and 580 bp) for all Lb. sanfranciscensis strains; this analysis proved to be helpful for the discrimination of sourdough lactobacilli at species level, but was unable to distinguish among the isolates belonging to the same species. Results of grouping, obtained by genetic analyses, did not agree with those attained with phenotypic analysis, and no relation with the geographic area of isolation or type of product was found.     

3种酒类酒球菌发酵性能研究及其对樱桃酒品质的影响

在樱桃酒的生产过程中,苹果酸-乳酸发酵(malolactic fermentation,MLF)是降低酒体酸度、改善口感、提升生物稳定性、提高风味复杂性的重要过程,因此,选择合适的乳酸菌株触发MLF对于保障樱桃酒的品质和安全性十分重要。以市售的3种酒类酒球菌(Enoferm ALPHA、VTT.D、Lalvin VP41)为研究对象,比较它们在苹果酸-乳酸发酵过程中的增殖情况,以及它们对樱桃酒基本理化指标、挥发性组分以及生物胺含量的影响,并据此选择适宜的发酵乳酸菌株。研究结果表明,3株酒类酒球菌均可在20天内完成降酸过程,生物胺含量均在可控范围,对酒体成分的影响也不存在显著性差异。Lalvin VP41能够增加樱桃酒多种挥发性物质的合成量,如苯乙醇、丁酸乙酯、乙酸异戊酯和α-松油醇等,从而增强了樱桃酒的果香和花香特征,因此,其更适宜作为樱桃酒发酵的降酸菌株。     

Phenotypic and genotypic characterization of foodborne bacteria isolated from Sinop Province,Turkey

In this study, genotypic and phenotypic characteristics of the strains, antibiotic susceptibility, and antibiotic resistance genes of the Escherichia coli, Bacillus cereus, Listeria spp., and Salmonella spp. isolated from various animal foods in Sinop province were determined. Resistance percentages of the isolated strains to the antibiotics penicillin, vancomycin, clindamycin, tetracycline, ampicillin/sulbactam, gentamicin, ciprofloxacin, and chloramphenicol were 87.8, 71.9, 69.7, 63.4, 27.5, 23.1, 20.9, and 13.4%, respectively. Antibiotic resistance genes tetA, bla_CMY-2, and cat1 were found in 45, 21.25, and 6.25% of all the strains, respectively. The virulence genes were also investigated in the isolated in E. coli strains; the eae gene was found in 13.1% of the strains, while the stx1 was not identified in any strains. As a result, we can say that SDS-PAGE method may provide discrimination at species level; however, surveys at subspecies level demand molecular approaches such as ERIC and (GTG)₅-PCR. In addition, we can suggest that molecular techniques may be especially helpful in the rapid identification of multidrug-resistant and isolates with virulence genes. This study has revealed that the investigated bacteria in animal foods is a significant reservoir of resistance genes.     

Phenotypic and genotypic characterization of Pseudomonas fluorescens isolated from bulk tank milk.

Pseudomonas fluorescens isolates (n = 55) isolated from farm bulk tank milk (n = 55) from dairy herds in eastern South Dakota and western Minnesota were examined for phenotypic (biotype, proteolytic, and lipolytic profiles) and genotypic (plasmid profiles and 16S-23S PCR ribotypes) characteristics. The observed phenotypic and genotypic characteristics were used to conduct phylogenetic analysis. Pseudomonas fluorescens belonged to 28 API 20 NE biotypes and 14 proteolytic and lipolytic profiles. It was observed that 80, 91, and 58% of the isolates were proteolytic at 7, 22, and 32 degrees C, respectively. Only 7, 44, and 7% of the isolates were lipolytic at the same three temperatures. Pseudomonas fluorescens was more likely to produce proteinases at 7 and 22 degrees C and lipases at 22 degrees C. Only 9 of 55 isolates of P. fluorescens harbored plasmids. This small percentage of plasmid-bearing isolates provided insufficient data for inferences related to the distribution of plasmid-bearing clonal types. Based on 16S-23S PCR ribotyping, P. fluorescens belonged to 14 subtypes. The 16S-23S PCR ribotyping technique allowed differentiation between strains; however, it did not concur with the biotypes and proteolytic and lipolytic profiles. Use of biotypes in conjunction with proteolytic and lipolytic profiles might have practical value for conducting trace-back studies related to P. fluorescens. Based on phylogenetic analysis, it was inferred that for the given geographical area and time period, P. fluorescens isolated from farm bulk tank milk consists of a large heterogeneous group of organisms.     

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.     

RNA fingerprinting analysis of Oenococcus oeni strains under wine conditions

Oenococcus oeni is a lactic acid bacterium of economic interest used in winemaking. This bacterium is the preferred species for malolactic fermentation (MLF) due its adaptability to the chemically harsh wine environment. MLF enhances the organoleptic properties and ensures deacidification of wines.     

酒酒球菌蛋白质提取方法研究

以裂解液为提取介质,分别采用超声法、反复冻融法和玻璃珠破碎3种方法提取细菌蛋白质,通过分析细菌破碎程度、蛋白质SDS-PAGE电泳及蛋白质质量浓度,比较各提取方法的效果。结果表明,超声法细胞破碎明显,提取蛋白质条带丰度最高,蛋白质质量浓度最大,有较好的稳定性和重复性,且操作简单。     

A putative glucan synthase gene dps detected in exopolysaccharide-producing Pediococcus damnosus and Oenococcus oeni strains isolated from wine and cider

Some lactic acid bacteria can induce viscosity in wine, beer and cider by production of exopolysaccharides (EPS). A polymerase chain reaction (PCR) assay was previously described for the detection of ropy Pediococcus damnosus strains in wine [J. Appl. Microbiol. 90 (2001) 535]. The primers used in that study, PF5 and PF6, are investigated in addition to new primers which broaden the range of spoiling agents detectable by PCR. Primers PF1 and PF8 allow the amplification of DNA from ropy P. damnosus strains isolated from wine, as was observed with PF5 and PF6. In addition, PF1 and PF8, unlike PF5 and PF6, are able to generate an amplicon using template DNA from a ropy P. damnosus strain isolated from cider and a ropy Oenococcus oeni strain isolated from champagne. Two different ropy Lactobacillus species were also isolated, but their DNA was not amplified using primers PF1 and PF8. The new primers PF1 and PF8 were chosen from the sequence of gene dps, a putative glucan synthase gene, found across all the ropy P. damnosus strains isolated, from both wine or cider, and also in a ropy O. oeni strain. To our knowledge, this is the first time that an EPS-producing O. oeni strain is described. Glucan biosynthesis was assessed by agglutination tests done with Streptococcus pneumoniae type 37-specific antibodies, which specifically detect glucan-producing cells. The results show that there is a direct correlation between glucan production and detection of gene dps. Therefore, Dps is considered a candidate for the glucan synthase enzyme responsible for EPS production by ropy strains of P. damnosus and O. oeni.     

Molecular cloning, heterologous expression, and characterization of Ornithine decarboxylase from Oenococcus oeni

Ornithine decarboxylase (ODC) is responsible for the production of putrescine, the major biogenic amine found in wine. Oenococcus oeni is the most important lactic acid bacterium in the winemaking process and is involved in malolactic fermentation. We report here the characterization of ODC from an O. oeni strain isolated from wine. Screening of 263 strains isolated from wine and cider from all over the world revealed that the presence of the odc gene appears to be strain specific in O. oeni. After cloning, heterologous expression in Escherichia coli, and characterization, the enzyme was found to have a molecular mass of 85 kDa and a pI of 6.2 and revealed maximal activity at pH 5.5 and an optimum temperature of 35°C. Kinetic studies showed that O. oeni ODC is specific for L-ornithine with a K(m) value of 1 mM and a V(max) of 0.57 U·mg(-1). The hypothesis that cadaverine, which results from lysine decarboxylation, may be linked to putrescine production is not valid since O. oeni ODC cannot decarboxylate L-lysine. As no lysine decarboxylase was detected in any of the O. oeni genomes sequenced, cadaverine synthesis may result from another metabolic pathway. This work is the first characterization of an ODC from a lactic acid bacterium isolated from a fermented product.     

葡萄酒中酒类酒球菌的分离——抑制剂对酵母菌生长的抑制效应

从葡萄酒中分离酒类酒球菌时 ,酵母菌是最主要的干扰菌。作者通过在ATB和改良MRS琼脂培养基中添加放线菌酮、山梨酸和制霉菌素 ,研究了不同抑制剂对酵母生长的抑制效应。结果表明 ,采用ATB + 5 0mg/L放线菌酮、改良MRS + 5 0mg/L放线菌酮、改良MRS + 10 0mg/L山梨酸、改良MRS + 5 0mg/L制霉菌素选择性培养基 ,能够完全抑制酿酒酵母的生长。     

Diversity of Candida zemplinina strains from grapes and Italian wines

The aim of this research was to genetically and technologically characterize Candida zemplinina strains isolated from different sources of enological interest. Phenotypic and genotypic subtyping, as well as enological characterization, were carried out on 36 C. zemplinina isolates collected from grapes, must and wines of different regions of Italy. RAPD-PCR fingerprinting of the isolates revealed a high genetic heterogeneity. At physiological level, yeasts were grouped into different clusters on the basis of sugar and ethanol tolerance. Common enological characteristics were examined and strains resulted to be highly fructophilic while presenting low ethanol and acetic acid production, high glycerol production, capacity to metabolize malic acid and slower fermentation kinetics when compared to Saccharomyces cerevisiae.The genetic and phenotypic intraspecies biodiversity of C. zemplinina gave useful data to understand its potential technological role in winemaking. This research represents a first step for the selection of C. zemplinina strains to be used as a starter in co-culture or in sequential inoculation with S. cerevisiae to improve the complexity and to enhance the particular characteristic of wines.     

Induction,purification and characterization of malolactic enzyme from Oenococcus oeni SD-2a

The aim of this study was to purify a malolactic enzyme (MLE) from Oenococcus oeni (O. oeni) strain and determine its properties in detail. O. oeni SD-2a was cultivated in the ATB broth supplemented with 7 g/L l-malic acid for harvesting the cells. After harvest, the cells were washed and disrupted for purification of MLE. MLE was purified from the supernatant of the disrupted cells through protamine sulfate precipitation, anion exchange chromatography and gel filtration chromatography. The purified MLE was identified using mass spectrometry. The MLE was purified by 43-fold with a yield of 0.42 % and possessed a specific activity of 419.2 U/mg. The purified enzyme with a nominal molecular mass of 59 kDa and a theoretical pI of 4.76 exhibited a maximum enzyme activity at 35 °C and pH 6.0, which retained over 50 % of its initial activity in the presence of 14 % (v/v) ethanol. Mn²⁺ was proven to be the most effective divalent cation to promote enzyme activity. Under the conditions of temperature 30 °C and pH 6.0, the K _m and V _max of MLE on l-malic acid were 12.5 × 10^?3 M and 43.86 μmol/(min × mg), respectively. Moreover, the purified enzyme exhibited a higher stability with 0.1 M NaCl in addition and had a half-life of 30 days at 4 °C.