Prevalent lactic acid bacteria in cider cellars and efficiency of Oenococcus oeni strains

Malolactic fermentation (MLF) is an important step in cider production in order to allowing for improvement of microbiological stability and organoleptic characteristics of cider. Induction of this fermentation by using starter cultures enables a better control over this bioprocess, but although it is a common practice in winemaking, starters specifically focussed for cider MLF are not yet commercially available. Proper starter cultures need to present the ability to degrade l-malic acid conferring pleasing sensory characteristics while avoiding toxicological risks. In this work, lactic acid bacteria (LAB) were first isolated from MLF industrial cider samples, obtained in a cellar in the main cider-producing region of Spain, Asturias. Isolates, identified by molecular tools, belonged to the Lactobacillus brevis and Oenococcus oeni species. After a phylogenetic analysis, representative strains of both identified species were evaluated in order to determine their fermentation capacity, showing O. oeni the best behaviour in this cider fermentation, as previously demonstrated for wine in the literature. Consequently, and with the aim to test the influence at strain level, selection of O. oeni isolates as starters for cider fermentation has been undergone. In order to check the influence of geography over biodiversity, O. oeni strains from six different industrial cellars representing the distinct producing areas in the region (located in a ratio of 30 km) were analyzed by using a specific RAPD method. In this way, isolates were typed in five distinct groups, mainly corresponding to each producing area. All strains isolated from the same cellar showed the same RAPD profile revealing the significance of geographical origin in the indigenous cider LAB. Molecular tools were applied to reject those isolates exhibiting presence of genes related to organoleptic spoilage (exopolysaccharides and acrolein production) or food safety (biogenic amine production), as key selection criteria. Representative strains of each of the five O. oeni RAPD groups were tested as pure cultures to evaluate their technological utility for cider production. Experimental data of malic acid degradation and cell concentration obtained were fitted to previously selected kinetic models aimed to optimization and prediction of bioprocess performance. Four strains revealed as suitable potential starter cultures for conducting MLF in cider production.     

酒类酒球菌快速特异性PCR鉴定体系的优化

研究采用目前应用较多的快速鉴定方法.种属特异性PCR技术(species-specific PCR)对酒类酒球菌进行鉴定.比较了不同提取方法所提DNA模板对species-specific PCR扩增结果的影响.尝试通过优化反应体系和反应条件,直接使用菌液和菌落作为模板进行扩增.结果表明,使用优化后的方法提取的DNA不需要纯化就能得到稳定的扩增结果,在优化后的反应体系下,可直接使用菌液或菌落作为模板进行扩增,扩增结果稳定,条带清晰.     

Acidity reduction in northern region berry juices by the malolactic bacterium Oenococcus oeni

Acidity of juices from northern berries was reduced by inoculating with a malolactic microorganism, Oenococcus oeni. The berries were white and black currant, bilberry, lingonberry, and, for comparison, an apple cultivar. Malic acid was first converted to lactic acid in all fermentations, while soluble sugars and citric acid remained unattacked. Upon exhaustion of malic acid, sugar and citric acid degradations were initiated simultaneously. Sequential utilization of substrates by O. oeni offers a basis for multitude compositional changes in acidic juices. Elimination of malic acid alone led to a noticeable reduction of acidic taste in these berry juices.     

分子生物学技术在酒类酒球菌分类鉴定方面的应用

酒类酒球菌(Oenococcus oeni)是葡萄酒进行苹果酸乳酸发酵(malolactic fermentation,MLF)的主要菌群,对其进行快速、高效的分离鉴定非常重要,该文针对不同分子生物学技术在筛选优良酒球菌的原理及应用进行了分析,同时对其前景进行了展望。     

酒酒球菌β-葡萄糖苷酶活性与耐酸胁迫能力的相关性分析

目的:β-葡萄糖苷酶是葡萄酒中结合态香气物质释放的关键酶,但其活性受诸多因素的影响。本研究旨在从酒酒球菌自身耐酸能力的视角去分析评估菌株糖苷酶活性,探索酸胁迫下不同耐酸表型酒酒球菌与其β-葡萄糖苷酶活性之间存在的相关性关系。方法:结合利用离子注入诱变与胁迫环境筛选的方式,获得耐酸(p H 3.0)、酸敏(p H 9.0)突变酒酒球菌菌株,对其β-葡萄糖苷酶的活力进行测定,并筛选3组酸胁迫下表型差异大的β-葡萄糖苷酶基因送样测序。结果:耐酸突变酒酒球菌的β-葡萄糖苷酶酶活力是出发菌株的2~4倍,是相应酸敏突变株的2~7倍。测序结果显示,除菌株a3的β-葡萄糖苷酶基因在108位(G置换成C)和1 232(A置换成T)位处发生了突变外,其余菌株β-葡萄糖苷酶的基因均未发生改变。结论:酒酒球菌β-葡萄糖苷酶活性与菌株酸胁迫能力显著相关(P0.05)。耐酸胁迫能力越强的菌株,β-葡萄糖苷酶活性越高。     

高盐辣椒坯中一株耐盐乳杆菌的分离及鉴定

目的采用选择性的MRS培养基,从湖南地区特色的盐坯辣椒中分离筛选出耐盐优势菌。方法得到一株能在18%NaCl培养基中生长的耐盐菌进行了生理生化实验,通过菌落PCR扩增其16S rDNA基因序列并测序,经过序列比对构建系统发育树。结果这株菌有较好的产乳酸、耐盐的能力,经过形态观察和生理生化试验验证,属于乳酸杆菌。结论经过16S rDNA进一步鉴定为植物乳杆菌。     

Potential osmoprotectants for the lactic acid bacteria Pediococcus pentosaceus and Tetragenococcus halophila

The physiological responses of the lactic acid bacteria Pediococcus pentosaceus and Tetragenococcus halophila (formely known as P. halophila), subjected to osmotic stress in the presence of molecules known to act as osmoprotectants for other bacteria were studied. In a defined medium, glycine betaine, dimethylsulfonioacetate, choline, proline and L-carnitine were able to relieve inhibition of growth at 0.8 M NaCl. The five compounds were shown to efficiently compete with glycine betaine transport, suggesting the existence of common transporter(s) for these molecules. T. halophila, the most tolerant strain, exhibited a larger spectrum of compatible solutes including dimethylsulfonioacetate, dimethylsulfoniopropionate and ectoine. Preliminary data suggest that restoration of growth by ectoine under osmotic constraint seems specific to the genus Tetragenococcus.     

Exploration of phenomena contributing to the diversity of Oenococcus oeni exopolysaccharides

Many food-grade bacteria produce exopolysaccharides (EPS) that may modify the food texture or affect their survival rate during food processing. This is the case of O. oeni, a bacterial species who drives malolactic fermentation in wine. The five strains analyzed in the present study all display both isolated genes dedicated to homopolysaccharide synthesis and gene clusters potentially associated with heteropolysaccharide synthesis. The number of isolated glycosyltransferase gene present and the gene composition of one of the operons change from one strain to the other. The soluble EPS yields and the EPS monomer composition vary depending on the strain and or the medium composition. O. oeni appears as a bacterium able to synthesize both homo and heteropolysaccharides. This unique property has rarely been described. Moreover, the abundance of the genetic determinants associated with EPS metabolism suggests that it is very important for the adaptation of the bacteria to wine.     

朝鲜辣白菜中抗扩展青霉乳酸菌的筛选与鉴定

该研究采用菌饼法从朝鲜辣白菜中筛选抗扩展青霉（Penicillium expansum）的乳酸菌,采用形态观察、生理生化试验及分子生物学技术进行菌种鉴定,并对乳酸菌抗扩展青霉的机理进行初步研究。结果表明,经过筛选和鉴定,获得1株对扩展青霉抑制效果最佳的清酒乳杆菌（Lactobacillus sakei）PC-3,抑菌率为68.62%。菌株PC-3无细胞上清液（CFS）的抗真菌活性随pH的升高呈现先升高后降低的趋势;随温度的升高逐渐下降。初步判定菌株PC-3 CFS中抗扩展青霉的有效成分是乳酸和乙酸,其对扩展青霉抑菌率分别为67.58%和66.73%。经扫描电镜观察发现,菌株PC-3 CFS抗扩展青霉的机制为破坏其细胞结构,导致胞内物质泄漏。     

产广谱细菌素口乳杆菌菌株的筛选和鉴定

从冷藏鲜肉制品中分离筛选到的36株乳酸菌中筛选出1株对指示菌具有明显抑菌作用的菌株,排除有机酸、过氧化氢等干扰因素后,该菌株发酵液仍有很强的抑菌作用.胃蛋白酶处理导致其抑菌活性急剧下降,表明该抑菌物质具有蛋白质性质,是一种细菌素.抑菌谱实验显示,这是一类广谱细菌素.通过形态学、生理生化特性分析,该菌株被初步鉴定为口乳杆菌.     

DNA标记技术在乳酸菌分类鉴定中的应用

通过DNA标记技术研究乳酸菌的基因型特征，阐明它们与类似属种在系统发育上的亲缘关系成为乳酸菌分类和鉴定的研究热点。综述了DNA标记技术在乳酸菌分类鉴定中的研究进展。     

高抗氧化乳酸菌的筛选鉴定

为了寻找天然的抗氧化剂,对18株乳酸菌的菌体细胞和无细胞提取物的DPPH自由基清除能力、羟自由基的清除能力、超氧阴离子自由基的清除能力、还原能力、抗脂质过氧化能力等5项抗氧化指标进行比较,并对高抗氧化活性的乳酸菌进行抗性筛选,鉴定出符合要求的菌株。结果表明:18株乳酸菌具有不同的抗氧化能力,编号为L4、L5、L8、L14、L18的菌株具有较高的抗氧化活性,其中,菌株L14抗人工胃液和胆盐的能力较强,有作为天然抗氧化剂的应用前景。结合生化特性鉴定和16S r DNA鉴定,确定其为类干酪乳杆菌类干酪亚种(Lactobacillus paracasei subsp.paracasei)。     

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.     

一株安徽本地泡菜中产细菌素乳酸菌的筛选与鉴定

从安徽本地泡菜中筛选出一株具有抑菌活性的乳酸菌(编号为PC-3)。通过菌落形态观察、染色镜检、生理生化和分子生物学实验鉴定分析,鉴定为植物乳杆菌(Lactobacillus plantarum),并命名为Lactobacillus plantarum PC-3。通过实验排除乳酸、乙酸和过氧化氢干扰后,L. plantarum PC-3的无细胞发酵上清液(cell-free fermentation supernatant,CFS)对大肠杆菌(ATCC 8099)和金黄色葡萄球菌(ATCC 6538)仍具有抑制作用,且经胰蛋白酶、胃蛋白酶和胃蛋白酶K处理后抑菌作用下降明显,说明L. plantarum PC-3所产主要抑菌物质为蛋白类物质,初步分析该抑菌活性物质为细菌素类。L. plantarum PC-3可作为食品运输和贮藏过程中控制大肠杆菌和金黄色葡萄球菌的拮抗制剂出发菌株,能有效地防止杂菌污染,延长食品保质期。     

乳酸菌制剂的免疫赋活作用

对乳酸菌制剂免疫赋活作用的类型、全身性免疫赋活作用及肠道免疫赋活的作用分别作了叙述。对乳酸菌制剂的免疫赋活作用机制进行了探讨。结果表明，乳酸菌的免疫赋活机制，首先是菌体刺激活化非特异性免疫赋活作用的巨噬细胞；NK细胞对肿瘤细胞的杀伤活性，其次为异性免疫反应的增强作用，产生肿瘤伤害性TC细胞。对乳酸菌制剂免疫赋活的意义作了论述。     

酒酒球菌抗酸相关基因RAPD标记的筛选

对影响RAPD 反应体系的主要因素进行优化,建立适合本实验室的RAPD 反应体系。在此基础上以33 株抗酸性酒酒球菌和9 株酸敏性菌株为试材,采用BSA 法和RAPD 技术,通过对45 个随机引物的筛选以及在单菌株中的筛选验证,最终获得与酒酒球菌抗酸性状相关的3 个RAPD 标记：S40-1400、S333-2500、S333-650。     

新疆葡萄酒产区优良酒类酒球菌的分离、鉴定

为了分离筛选出适合葡萄酒酿造的优良酒类酒球菌(Oenococcus oeni)菌株,从我国新疆葡萄酒产区分离纯化出30株酒类酒球菌,依据形态特征、生理生化特性,它们均被鉴定为酒类酒球菌。分别对其进行单因子(pH值、酒精、SO2)耐受性实验,选出单因子抗性较好的9株菌进行复合因子(pH值×酒精×SO2)耐受性实验,结果表明,有3株酒类酒球菌具有较好的发酵适应性。最后通过Species-specific PCR以及16S rRNA序列同源性分析对其进行验证,并构建相应的系统发育树,系统发育分析表明所筛菌株与酒类酒球菌的同源性均达到了99%以上,说明本实验所筛选的9株性能较好的菌株均为酒类酒球菌。     

Characterisation of malolactic conversion by Oenococcus oeni to reduce the acidity of apple juice

The high concentration of malic acid is responsible for the acidity and sourness in apple juice. Bio‐conversion of malic acid to lactic acid through malolactic conversion (MC) in apple juice using Oenococcus oeni was investigated. When apple juice was inoculated with O. oeni (1 × 10⁶ CFU mL^?1), over 90% of malic acid was converted into lactic acid within 96 h at room temperature. When pH of apple juice was adjusted to 4.1 prior to inoculation, MC was completed within 60 h. MC was enhanced at a higher temperature (30°C) when compared with room temperature. The rate of MC was directly proportional to the number of bacteria added and MC was completed within 24 h at 1 × 10⁹ CFU mL^?1 initial cell density. MC occurred equally under aerobic and anaerobic conditions. The sensory analysis of partial MC‐applied juice when compared against control revealed potential for use of MC for manufacture of low‐acid apple juice.     

一株安徽传统酸奶中产抑菌物质的乳酸菌的筛选与鉴定

目的从安徽传统发酵酸奶中筛选出一株具有抑菌活性的乳酸菌(编号为SN-5)。方法通过菌落形态观察、染色镜检、生理生化和分子生物学实验进行鉴定分析,利用牛津杯法进行抑菌实验。结果通过实验排除乳酸、乙酸和过氧化氢干扰后,L.delbrueckii ssp.bulgaricus SN-5的无细胞发酵上清液(cell-free fermentation supernatant,CFS)对大肠杆菌(ATCC8099)和金黄色葡萄球菌(ATCC6538)仍具有抑制作用,且经胰蛋白酶、胃蛋白酶和胃蛋白酶K处理后抑菌作用下降明显,说明L. delbrueckii ssp. bulgaricus SN-5所产主要抑菌物质为蛋白类物质。结论 L. delbrueckii ssp. bulgaricus SN-5可作为食品运输和贮藏过程中控制大肠杆菌和金黄色葡萄球菌的拮抗制剂出发菌株,为新型生物防腐剂的开发奠定了一定的理论基础。     

潜在益生乳酸菌分离和鉴定研究进展

利用益生菌提高人类健康水平是当前营养健康领域最具前景的方向之一。乳酸菌是食品中常用的益生菌,迄今为止,联合国粮食及农业组织（FAO）和世界卫生组织（WHO）推荐仅从人的胃肠道分离的乳酸菌可在食品中使用。而越来越多的研究从食品来源中分离出具有益生功能的菌株以扩大益生菌的选择范围。随着分子生物学和检测分析等技术的发展,基质辅助激光解吸电离-飞行时间质谱（MALDI-TOF MS）、全基因组测序、实时荧光定量聚合酶链式反应（RT-FQ-PCR）等技术可以鉴定到亚种及菌株水平上的益生菌。鉴于分离鉴定技术是益生菌安全规范应用的先决条件,该文综述了近年来针对潜在益生乳酸菌有效分离和鉴定技术的研究进展,以期为我国益生乳酸菌自主开发利用提供借鉴。