Growth of yeasts, lactic and acetic acid bacteria in palm wine during tapping and fermentation from felled oil palm (Elaeis guineensis) in Ghana

AIM: To investigate the microbiological and biochemical changes which occur in palm wine during the tapping of felled oil palm trees. METHODS AND RESUlts: Microbiological and biochemical contents of palm wine were determined during the tapping of felled oil palm trees for 5 weeks and also during the storage. Saccharomyces cerevisiae dominated the yeast biota and was the only species isolated in the mature samples. Lactobacillus plantarum and Leuconostoc mesenteroides were the dominated lactic acid bacteria, whilst acetic acid bacteria were isolated only after the third day when levels of alcohol had become substantial. The pH, lactic and acetic acid concentrations during the tapping were among 3.5-4.0%, 0.1-0.3% and 0.2-0.4% respectively, whilst the alcohol contents of samples collected within the day were between 1.4% and 2.82%; palm wine which had accumulated over night, 3.24% to 4.75%; and palm wine held for 24 h, over 7.0%. CONCLUSION: Accumulation of alcohol in palm wine occurs in three stages during the tapping and marketing with the concurrent lactic and acetic acid fermentation taking place as well. SIGNIFICANCE AND IMPACT OF THE STUDY: Yeasts, lactic and acetic acid bacteria are all important in the fermentation of palm wine and influence the composition of the product.     

The sourdough microflora. Interactions between lactic acid bacteria and yeasts: metabolism of amino acids

Co-culture of Lactobacillus brevis subsp. lindneri or L. plantarum with Saccharomyces cerevisiae or S. exiguus from sourdough did not modify the yield of the yeasts but gave higher growth rates and final yields of both lactic acid bacteria (LAB) than in their respective mono-cultures. Co-cultures of L. brevis subsp. lindneri with S. cerevisiae or S. exiguus in a medium without valine or leucine, which are essential for growth of the LAB, led to growth of the LAB due to excretion of these amino acids by the yeasts.The authors are with the Institute of Dairy Microbiology, Faculty of Agriculture, University of Perugia, Via S. Costanzo, 06100 Perugia, Italy     

16S-ARDRA, a tool for identification of lactic acid bacteria isolated from grape must and wine

Lactic acid bacteria (LAB) are found in a great variety of habitats, including grape must and wines. There is a close relationship between the species of LAB which develop during fermentation and the eventual quality of the wine. For these reasons analytical techniques allowing fast and reliable identification of wine LAB are needed. In this work a simple and accurate protocol for identifying species of LAB isolated from grape must and wine is presented. This protocol is based on the amplification, directly from colony, of 16S rDNA and later digestion with one of the following restriction enzymes BfaI, MseI and AluI. A sequential use of the three enzymes is proposed to simplify LAB wine identification, first MseI, then BfaI and finally, if necessary, AluI digestion. The technique was able to discriminate 32 of the 36 LAB reference species tested and allowed the identification of 342 isolates from musts and wines. The isolates belonged to the species: Lactobacillus brevis, L. collinoides, L. coryniformis, L. bilgardii, L. mali, L. paracasei, Leuconostoc mesenteroides, Oenococcus oeni, Pediococcus parvulus and P. pentosaceus.     

Spoilage of bottled red wine by acetic acid bacteria

AIMS: To determine the bacterial species associated with an outbreak of spoilage in commercially bottled red wine where the bottles had been stored in an upright vertical compared with horizontal position. METHODS AND RESULTS: Bottled wines comprising Cabernet Sauvignon, Pinot Noir, Shiraz, Merlot and blended red varieties were examined for visible spoilage. Analysis of visibly affected and non-affected wines revealed a spectrum of aroma and flavour defects, ranging from loss of fruity aroma, staleness, oxidized character to overt volatile acidity. Only acetic acid bacteria, and not yeast or lactic acid bacteria, could be isolated from both spoiled and unspoiled wines and were found to grow only on Wallerstein Nutrient (WL) medium supplemented with 10% red wine or 1-2% ethanol. Analysis of the 16S rRNA region and RAPD-PCR analysis showed the isolates to be a closely related group of Acetobacter pasteurianus, but this group was differentiated from the group comprising beer, vinegar and cider strains. CONCLUSIONS: A. pasteurianus was the species considered responsible for the spoilage but the isolates obtained had atypical properties for this species. In particular, they failed to grow on WL nutrient medium without ethanol or wine supplementation. Storage of the bottles of wine containing A. pasteurianus in an upright vertical position specifically induced growth and spoilage in a proportion of the bottles under conditions that were inhibitory for horizontally stored bottles. We hypothesize that the upright position created a heterogeneous environment that allowed the growth of bacteria in only those bottles sealed with cork closures that had upper limit for the natural permeability to oxygen. Such a heterogeneous environment would not exist in horizontally stored bottles as the larger volume of wine adjacent to the cork would strongly compete with the bacteria for the oxygen as it diffuses through the cork closure. SIGNIFICANCE AND IMPACT OF THE STUDY: A low level of bacteria (acetic acid bacteria) in wine can proliferate and cause wine spoilage in bottles stored in an upright vertical as opposed to an horizontal position under conditions that would normally limit bacterial development.     

泡菜中优良乳酸菌的分离、鉴定及发酵特性

从几种泡菜中分离出86株菌,对其在适温和低温下产酸速率及硝酸盐降解能力进行测定,筛选出5株产酸速率快、硝酸盐降解能力强的菌株。经形态学鉴定及生理生化反应试验,初步鉴定为：植物乳杆菌2株,短乳杆菌1株,戊糖乳杆菌1株,肠膜明串珠菌葡聚糖亚种1株,并对5株菌的发酵性能进行了测定。     

Degradation of free and sulfur-dioxide-bound acetaldehyde by malolactic lactic acid bacteria in white wine

AIMS: Acetaldehyde is the major carbonyl compound formed during winemaking and has implications for sensory and colour qualities of wines as well as for the use of the wine preservative SO(2). The current work investigated the degradation of acetaldehyde and SO(2)-bound acetaldehyde by two commercial Oenococcus oeni starters in white wine. METHODS AND RESULTS: Wines were produced by alcoholic fermentation with commercial yeast and adjusted to pH 3.3 and 3.6. While acetaldehyde was degraded rapidly and concurrently with malic acid at both pH values, SO(2)-bound acetaldehyde caused sluggish bacterial growth. Strain differences were small. CONCLUSIONS: Efficient degradation of acetaldehyde can be achieved by commercial starters of O. oeni. According to the results, the degradation of acetaldehyde could not be separated from malolactic conversion by oenococci. While this may be desirable in white winemaking, it may be necessary to delay malolactic fermentation (MLF) in order to allow for colour development in red wines. SO(2)-bound acetaldehyde itself maybe responsible for the sluggish or stuck MLF, and thus bound SO(2) should be considered next to free SO(2) in order to evaluate malolactic fermentability. SIGNIFICANCE AND IMPACT OF THE STUDY: The current study provides new results regarding the metabolism of acetaldehyde and SO(2)-bound acetaldehyde during the MLF in white wine. The information is of significance to the wine industry and may contribute to reducing the concentration of wine preservative SO(2).     

Quantification of glycosidase activities in selected yeasts and lactic acid bacteria

Using a model system, the activities of α-L-arabinofuranosidase, β-glucosidase, and α-L-rhamonopyranosidase were determined in 32 strains of yeasts belonging to the genera Aureobasidium, Candida, Cryptococcus, Hanseniaspora, Hansenula, Kloeckera, Metschnikowia, Pichia, Saccharomyces, Torulaspora and Brettanomyces (10 strains); and seven strains of the bacterium Leuconostoc oenos. Only one Saccharomyces strain exhibited β-glucosidase activity, but several non-Saccharomyces yeast species showed activity of this enzyme. Aureobasidium pullulans hydrolyzed α-L-arabinofuranoside, β-glucoside, and α-L-rhamnopyranoside. Eight Brettanomyces strains had β-glucosidase activity. Location of enzyme activity was determined for those species with enzymatic activity. The majority of β-glucosidase activity was located in the whole cell fraction, with smaller amounts found in permeabilized cells and released into the growth medium. Aureobasidium pullulans hydrolyzed glycosides found in grapes. Received 02 February 1999/ Accepted in revised form 26 June 1999     

猪源乳酸菌的分离、鉴定及其生物学特性

【目的】将分离自猪肠道粘膜、食糜和粪便的乳酸菌,通过产乳酸能力、生长性能、耐酸和耐胆盐性能及抑菌能力评价,筛选适应养猪生产的潜在益生特性的菌株。【方法】共分离获得155株乳酸菌纯菌株,从中筛选出4株产酸能力较强的乳酸菌,结合生理生化试验及细菌16S rRNA测序鉴定其种属,评价候选乳酸菌的生长情况、耐酸、耐胆盐及抑菌特性。【结果】综合变色时间(8 h)、pH值(3.9)和乳酸含量(100 mmol/L),筛选出4株(L45、L47、L63和L79)候选菌株,经鉴定依次为罗伊氏乳杆菌、植物乳杆菌、约氏乳杆菌和粪肠球菌。该4株乳酸菌均可在体外快速生长;L47和L79能够耐受pH 2.5的酸性环境,L47能够耐受0.5%胆盐环境;各乳酸菌上清液与指示菌共培养,发现对E coli K88和沙门氏菌均产生了抑制作用,其中L47上清液对指示菌的抑制作用较强。【结论】L47具有较好的产酸性能与生长性能、可耐受猪胃酸和肠道胆盐环境,对E.coli K88和沙门氏菌具有较好的抑制作用,说明该乳酸菌具有潜在的益生特性。     

水质净化乳酸菌的分离鉴定及发酵参数优化

【目的】从水产养殖环境和养殖生物体中选育具有水质净化功能的乳酸菌,以期为水产养殖提供专用高效的菌种资源。【方法】在低温和常温条件下从皮皮虾、南美白对虾肠道及养殖池底质活性污泥中分离具有水质净化功能的乳酸菌,对筛选的优良菌株采用形态、生理生化实验及16S r RNA序列分析进行鉴定,并对菌株的发酵参数进行了研究。【结果】低温和常温条件下从3种介质中共分离到乳酸菌136株,经水质净化能力筛选,发现常温分离的r13对模拟水体中亚硝态氮去除效果较强,72 h能将11.5 mg/L的亚硝态氮彻底去除,且对13.0 mg/L氨氮的去除率达到29.1%。经形态特征、生理生化特性及16S r RNA基因序列分析,鉴定菌株r13为植物乳杆菌Lactobacillus plantarum。发酵参数研究结果表明,该菌最适培养基组成为:酵母膏6.0 g/L,葡萄糖20.0 g/L,乙酸钠4.0 g/L,柠檬酸氢二铵2.0 g/L,K_2HPO_4 2.0 g/L,番茄汁50 m L/L;培养条件为:初始p H 6.0,接种量5%,装液量45/50,培养温度为34°C;在上述优化培养条件下发酵72 h,菌液的生物量达28.4 g/L湿重,有效活菌浓度达4.4×10~9 CFU/m L。     

Isolation, characterization and identification of lactic acid bacteria and yeasts from sour Mifen, a traditional fermented rice noodle from China

Aims:  Considering the effect of natural fermentation on the textural improvement of fermented rice noodles in China and South Asia, and given the lack of reports concerning microbial populations and structure in the fermentation process, this study aims to determine the number of viable micro-organisms and identify the species isolated from the local factories, and to assess their potential use as a starter culture from their enzymatic profiles.
Methods and Results:  Fourteen samples from three local factories were analysed for the presence of micro-organisms. A total of 170 lactic acid bacteria (LAB) and 96 yeasts were isolated from the factories. The isolates were phenotypically characterized by using API 50 CHL kits, API 20 Strep kits, API ID 32 C kits and by performing additional biochemical tests. The enzymatic profiles of isolates were assessed by using API ZYM kits. Lactobacillus plantarum and Saccharomyces cerevisiae were identified as predominant species in the fermented supernatants. A majority of the isolates of LAB and yeasts displayed activities of α-glucosidase, β-glucosidase, lipase and trypsin.
Conclusions:  The microbial composition and strain characteristics present in the fermentation supernatant demonstrate that a majority of micro-organisms have the ability to digest starch, sugar, protein or lipid. It supports our previous work in which the rice starch was modified and purified by fermentation and thus improves the texture of rice noodles.
Significance and Impact of the Study:  The dominant strains would be important in developing a starter culture. The results can form the basis for the improvement of product quality and consistency.     

Determination of lactic acid bacteria producing biogenic amines in wine by quantitative PCR methods

Aims: To develop rapid methods allowing enumeration of lactic acid bacteria producing biogenic amines in wines and to analyse wine samples by the methods. Methods and Results: Methods based on quantitative PCR targeting bacterial genes involved in histamine, tyramine and putrescine production were developed and applied to detect and quantify the bacteria producing these biogenic amines in wine. Analysis of 102 samples revealed low populations of the targeted bacteria in grape must samples, an increased bacteria biomass in wine samples after alcoholic fermentation, reaching the highest population levels (above 10⁶ cells ml^?1) during spontaneous malolactic fermentation. A minimum of 10³ ml^?1 producing cells was required for production of more than 1 mg l^?1 of biogenic amines. Accumulation of putrescine in wine was correlated with the presence of bacteria carrying an ornithine decarboxylation pathway. Trials of winemaking showed that the use of selected bacteria for inducing malolactic fermentation was efficient to limit the proliferation of undesirable bacteria and the production of biogenic amines. Conclusion: Methods using quantitative PCR are efficient to enumerate biogenic amines‐producing cells in wine. Significance and Impact of the Study: The methods can help to better control and to improve winemaking conditions in order to avoid biogenic amine production.     

Lactic acid bacteria and yeasts in kefir grains and kefir made from them

In an investigation of the changes in the microflora along the pathway: kefir grains (A)→kefir made from kefir grains (B)→kefir made from kefir as inoculum (C), the following species of lactic acid bacteria (83–90%) of the microbial count in the grains) were identified: Lactococcus lactis subsp. lactis, Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus helveticus, Lactobacillus casei subsp. pseudoplantarum and Lactobacillus brevis. Yeasts (10–17%) identified were Kluyveromyces marxianus var. lactis, Saccharomyces cerevisiae, Candida inconspicua and Candida maris. In the microbial population of kefir grains and kefir made from them the homofermentative lactic streptococci (52–65% and 79–86%, respectively) predominated. Within the group of lactobacilli, the homofermentative thermophilic species L. delbrueckii subsp. bulgaricus and L. helveticus (70–87% of the isolated bacilli) predominated. Along the pathway A→B→C, the streptococcal proportion in the total kefir microflora increased by 26–30% whereas the lactobacilli decreased by 13–23%. K. marxianus var. lactis was permanently present in kefir grains and kefirs, whereas the dominant lactose-negative yeast in the total yeast flora of the kefir grains dramatically decreased in kefir C. Journal of Industrial Microbiology & Biotechnology (2002) 28, 1–6 DOI: 10.1038/sj/jim/7000186 Received 02 August 2000/ Accepted in revised form 15 July 2001     

Isolation and characteristics of lactic acid bacteria from koshu vineyards in Japan

Aims:  To isolate, characterize and identify lactic acid bacteria (LAB) in the vineyards where koshu grapes, a primary wine grape cultivar in Japan, are grown.
Methods and Results:  Sixty samples, including leaves, undamaged grape berries and soil under damaged berries, were collected at four koshu vineyards in Yamanashi Prefecture, Japan. One hundred and 15 acid-producing cultures were isolated from these samples, and the isolates were divided into classes by phenotype and then into groups by restriction fragment length polymorphism analysis and sequencing of 16S ribosomal DNA (rDNA). Phenotypic and biochemical characteristics identified seven different bacterial groups (A to G). Lactococcus lactis ssp. lactis was the most abundant type of LAB distributed in three koshu vineyards, and Leuconostoc pseudomesenteroides was the most abundant LAB found in the remaining vineyard. Forty-six isolates produced bacteriocin-like inhibitory substances (BLIS) against the indicator strain Lactobacillus sakei JCM 1157^T.
Conclusions:  These results suggest that various LAB are distributed in koshu vineyards, of which a large number produce BLIS.
Significance and Impact of the Study:  This is the first report describing the distribution and varieties of LAB that exist in koshu vineyards.