Lactic acid bacteria and yeasts associated with gowé production from sorghum in Bénin

AIMS: To identify the dominant micro-organisms involved in the production of gowé, a fermented beverage, and to select the most appropriate species for starter culture development. METHODS AND RESULTS: Samples of sorghum gowé produced twice at three different production sites were taken at different fermentation times. DNA amplification by internal transcribed spacer-polymerase chain reaction of 288 lactic acid bacteria (LAB) isolates and 16S rRNA gene sequencing of selected strains revealed that the dominant LAB responsible for gowé fermentation were Lactobacillus fermentum, Weissella confusa, Lactobacillus mucosae, Pediococcus acidilactici, Pediococcus pentosaceus and Weissella kimchii. DNA from 200 strains of yeasts was amplified and the D1/D2 domain of the 26S rRNA gene was sequenced for selected isolates, revealing that the yeasts species were Kluyveromyces marxianus, Pichia anomala, Candida krusei and Candida tropicalis. CONCLUSIONS: Gowé processing is characterized by a mixed fermentation dominated by Lact. fermentum, W. confusa and Ped. acidilactici for the LAB and by K. marxianus, P. anomala and C. krusei for the yeasts. SIGNIFICANCE AND IMPACT OF THE STUDY: The diversity of the LAB and yeasts identified offers new opportunities for technology upgrading and products development in gowé production. The identified species can be used as possible starter for a controlled fermentation of gowé.     

Effect of starter culture inoculation on feed hygiene and microbial population development in fermented pig feed composed of a cereal grain mix with wet wheat distillers' grain

Aims:  Investigating the influence of an added starter culture on the properties of fermented liquid pig feed.
Methods and Results:  Diets of cereal grain blended with wet wheat distillers' grain that were either not inoculated (WWDG), inoculated with a silage starter culture at start (WWDGsc1) or at start and at each backslopping (replacement of 80% the content with fresh mixture, simulating feed outtake, WWDGsc5) were fermented for 5 days, followed by 5 days of daily backslopping. Numbers of undesirable micro-organisms (enterobacteria, moulds) were reduced in all fermentations; particularly enterobacteria in the starter culture inoculated diets. Lactobacillus plantarum present in the starter culture became dominant in diets WWDGsc1 and WWDGsc5. However, Lactobacillus panis that was dominating WWDG was also abundant in WWDGsc1 and WWDGsc5. Yeast populations were not influenced by the starter culture, with Pichia fermentans dominating all fermentations. All diets had similar chemical characteristics with the exception of a significant increase of all tested organic acids in WWDGsc5.
Conclusions:  The addition of a starter culture influences the bacterial population in fermented liquid feed, but there is also a strong impact of the flora already present in the feed ingredients. The yeast population is not influenced by adding a lactic acid bacteria (LAB) starter culture. A consortium of LAB and yeast strains adapted to the fermentation should be used as starter culture.
Significance and Impact of the Study:  The results suggest that it is possible to influence the current unpredictable and spontaneous process of feed fermentation when appropriate starter cultures are used. For this purpose, LAB and yeasts with desirable characteristics should be isolated.     

Genome sequence of the ethanol-producing Zymomonas mobilis subsp. mobilis lectotype strain ATCC 10988

Zymomonas mobilis ATCC 10988 is the type strain of the Z. mobilis subsp. mobilis taxon, members of which are some of the most rigorous ethanol-producing bacteria. Isolated from Agave cactus fermentations in Mexico, ATCC 10988 is one of the first Z. mobilis strains to be described and studied. Its robustness in sucrose-substrate fermentations, physiological characteristics, large number of plasmids, and overall genomic plasticity render this strain important to the study of the species. Here we report the finishing and annotation of the ATCC 10988 chromosomal and plasmid genome.     

Analyses of microbial consortia in the starter of Fen Liquor

Aims:  This study aimed to determine the microbial diversity in the starter of Fen Liquor.
Methods and Results:  The plate method was used to enumerate the micro-organisms; meanwhile, the 16S rDNA of bacteria and the internal transcribed spacer of fungi were used to determine microbial diversity. Several genera were accordingly identified. Among the bacteria, Lactobacillales and Actinomycetales were detected only on the surface of the starter, whereas Bacillales was dominant within the starter. Among the fungi, Saccharomycopsis and Issatchenkia were the main genera in surface and interior starter, respectively; in addition, Thermomyces was found in interior starter, while other species of fungi were detected on the surface.
Conclusions:  The culture-dependent and polymerase chain reaction-based methods revealed the significant microbial diversity in different locations in the starter of Fen Liquor.
Significance and Impact of the Study:  This study is the first to identify the bacterial and fungal communities associated with the starter of Fen Liquor using both traditional and molecular methods; it is also the first to compare the microbial diversity on the surface of starter with that in the interior. The results enrich our knowledge on liquor-related micro-organisms, and can be used to promote the development of the traditional fermentation technology.     

Yeast communities associated with artisanal mezcal fermentations from <Emphasis Type="Italic">Agave salmiana</Emphasis>

The aims of this work were to characterize the fermentation process of mezcal from San Luis Potosi, México and identify the yeasts present in the fermentation using molecular culture-dependent methods (RFLP of the 5.8S-ITS and sequencing of the D1/D2 domain) and also by using a culture-independent method (DGGE). The alcoholic fermentations of two separate musts obtained from Agave salmiana were analyzed. Sugar, ethanol and major volatile compounds concentrations were higher in the first fermentation, which shows the importance of having a quality standard for raw materials, particularly in the concentration of fructans, in order to produce fermented Agave salmiana must with similar characteristics. One hundred ninety-two (192) different yeast colonies were identified, from those present on WL agar plates, by RFLP analysis of the ITS1-5.8S- ITS2 from the rRNA gene, with restriction endonucleases, HhaI, HaeIII and HinfI. The identified yeasts were: Saccharomyces cerevisiae, Kluyveromyces marxianus, Pichia kluyveri, Zygosaccharomyces bailii, Clavispora lusitaniae, Torulaspora delbrueckii, Candida ethanolica and Saccharomyces exiguus. These identifications were confirmed by sequencing the D1-D2 region of the 26S rRNA gene. With the PCR-DGGE method, bands corresponding to S. cerevisiae, K. marxianus and T. delbrueckii were clearly detected, confirming the results obtained with classic techniques.     

Selected nondigestible carbohydrates and prebiotics support the growth of probiotic fish bacteria mono-cultures in vitro

Aims:  To search for nondigestible but fermentable (NDF) carbohydrates and prebiotics with a potency to promote the growth of selected bacteria in vitro .
Methods and Results:  The growth of three reference bacteria strains Bacillus subtilis LMG 7135^T, Carnobacterium piscicola LMG 9839, Lactobacillus plantarum LMG 9211 and one candidate probiotic bacteria Lactobacillus delbrueckii subsp. lactis was investigated over a minimum period of 48 h in the presence of β -glucan, xylo-oligosaccharide, arabinoxylo-oligosaccharide, inulin, oligofructose and glucose. Besides the capability to grow on inulin and oligofructose containing media, a distinct high growth in β -glucan based substrates and a low growth in (arabino)xylooligosaccharide containing media were evident for most bacteria tested. With the exception of B. subtilis and L. plantarum , other bacteria grew equally well or even better on different substrates than on glucose. The fermentation of studied carbohydrates by these micro-organisms was dominated by the production of acetic acid as the main short chain fatty acid.
Conclusions:  Selected bacteria are able to ferment and grow on NDF and prebiotic carbohydrates but in a substrate dependent manner.
Significance and Impact of the Study:  This study delivers a first screening of which NDF or prebiotic carbohydrates are the most promising for aquaculture feed supplementations.     

应用16S rDNA克隆文库法分析有机物料腐熟菌剂细菌组成

应用16SrDNA克隆文库法对有机物料腐熟菌剂A和B样品中的细菌组成进行分析研究。结果表明,样品A有14个OTU,主要是融合乳杆菌(Weissella confusa)、枯草芽孢杆菌(Bacillus subtilis)和短小芽孢杆菌(Bacillus pumilus),其比例分别占总克隆文库的28.6%、30.4%和23.2%;样品B有43个OTU,主要是布氏乳杆菌(Lactobacillus buchneri)、香肠乳杆菌(Lactobacillus farciminis)和耐酸乳杆菌(Lactobacillus acetotolerans),占总克隆文库的比例分别为18.03%、18.86%和13.12%;所得出的结果均与产品标注存在差异,样品A未提及细菌的种类,而样品B只标注短小芽孢杆菌。研究表明这一方法在微生物菌剂细菌组成分析及其质量检测中具有良好的应用前景。     

The characterization of lactic acid producing bacteria from the rumen of dairy cattle grazing on improved pasture supplemented with wheat and barley grain

Aims:  To identify and characterize the major lactic acid bacteria in the rumen of dairy cattle grazing improved pasture of rye grass and white clover and receiving a maize silage and grain supplement with and without virginiamycin.
Methods and Results:  Eighty-five bacterial isolates were obtained from the rumen of 16 Holstein-Friesian dairy cows. The isolates were initially grouped on the basis of their Gram morphology and by restriction fragment length polymorphism analysis of the PCR amplified 16S rDNA. A more definitive analysis was undertaken by comparing the 16S rDNA sequences. Many of the isolates were closely related to other previously characterized rumen bacteria, including Streptococcus bovis, Lactobacillus vitulinus , Butyrivibrio fibrisolvens , Prevotella bryantii and Selenomonas ruminantium . The in vitro production of l - and/or d -lactate was seen with all but five of the isolates examined, many of which were also resistant to virginiamycin.
Conclusion:  Supplementation of grain with virginiamycin may reduce the risk of acidosis but does not prevent its occurrence in dairy cattle grazing improved pasture.
Significance and Impact of the Study:  This study shows that lactic acid production is caused, not only by various thoroughly researched types of bacteria, but also by others previously identified in the rumen but not further characterized.     

Effects of the high pressure of homogenization on some spoiling micro-organisms, representative of fruit juice microflora, inoculated in saline solution

Aims:  This study was aimed to investigate the effects of a high-pressure homogenization (HPH) treatment on some micro-organisms, involved in the spoilage of fruit juices.
Methods and Results:  Lactobacillus plantarum , Lactobacillus brevis , Bacillus coagulans cells, Saccharomyces bayanus , Pichia membranaefaciens and Rhodotorula bacarum were separately inoculated in a saline solution (0·9% NaCl); the initial inoculum was ca. 5 log CFU ml⁻¹. Then, the samples were processed through a homogenizer at 10–150 MPa for 1, 2 or 3 times. Yeasts were completely inactivated at 50–110 MPa with a single pass treatment, while lactic acid bacteria counts were reduced to approximately 1 log CFU ml⁻¹ after a three-steps HPH processing.
Conclusions:  Yeasts were the most sensitive micro-organisms, followed by B. coagulans . On the other hand, lactic acid bacteria appeared resistant to HPH.
Significance and Impact of the Study:  The results of this study provided some useful information on the susceptibility of microflora of juices to homogenization; moreover, they suggested that HPH could be used successfully to inactivate yeasts.     

新疆地区酸马奶中酵母菌的鉴定及其生物多样性分析

从新疆少数民族牧民家庭采集的28份传统工艺酿造酸马奶样品中分离出87株酵母菌,并对其进行了生理生化鉴定、分子生物学鉴定和生物多样性分析。生化试验结果表明,新疆地区酸马奶中的酵母菌为Saccharomyces unisporus(占总分离株的48.3%),Kluyveromyces marxianus(27.6%),Pichia membranaefaciens(15.0%)和Saccharomyces cerevisiae(9.2%)。选取其中的6株酵母菌和1株参考菌株,进行大亚基(26S)rDNA D1/D2区域(600bp左右)碱基序列分析,并通过GenBank进行同源序列搜索以确定各菌株的归属,进一步验证生理生化方法的正确性。从得到的结果中可以看出,S.unisporus和K.marxianus为新疆地区酸马奶中的优势菌。     

Rapid detection of lactobacillus and yeast concentrations using a particle size distribution analyser

Aims:  To see the possibility of particle size distribution analyser (PSDA) in detecting concentration of lactobacillus contaminants in yeast fermentation.
Methods and Results:  A PSDA was used to rapidly determine the size and concentration of lactobacillus and Saccharomyces cerevisiae . Data showed that the aerodynamic diameters of Lactobacillus casei and S. cerevisiae cells were around 0·63 and 2·9 μm, respectively, with both cultures showing a linear relationship between cell density and particle count on a size distribution curve of PSDA. In addition, Lactobacillus fermentum showed high similarity in bacterial size distribution and particle count numbers with L. casei . The PSDA also rapidly detected (within 1 min) the cell concentrations of S. cerevisiae and L. casei in a mixed sample with different concentration ratios with 10⁷–10⁹ cells ml⁻¹ of detection range.
Conclusions:  PSDA was demonstrated to be useful for the rapid detection of lactobacillus and S. cerevisiae concentrations.
Significance and Impact of the Study:  This is the first report concerning PSDA to detect the concentration of bacteria and yeast. This method can be useful in the actual field during ethanol fermentation because of relatively easy handling and rapid detection.     

Quality of silages from Italian farms as attested by number and identity of microbial indicators

AIMS: This study evaluated the quality and possible hygiene risks related to farm-made silages by analysing the presence and number of micro-organisms that influence the preservation and safety in samples from four Italian regions. METHODS AND RESULTS: Lactic acid bacteria, clostridia, lactate-fermenting yeasts and propionibacteria (PAB) were isolated and identified by random amplified polymorphic DNA PCR, sequencing of the V2-V3 16S rRNA gene region, 5.8S-ITS rDNA RFLP and species-specific PCR. The Lactobacillus plantarum cluster was the most numerous and comprised strains mostly isolated from alfalfa silage. The Lactobacillus buchneri cluster, second in number, comprised isolates from both alfalfa and maize silage. Anaerobic spore formers were assigned to the species Clostridium baratii, Clostridium beijerinkii, Clostridium butyricum, Clostridium perfringens, Clostridium saccharolyticum, Clostridium tyrobutyricum and Paenibacillus macerans. Yeast isolates were identified as Candida apicola, Candida mesenterica and Pichia fermentans. PAB strains, detected only in unifeed, were all identified as Propionibacterium acidipropionici. CONCLUSIONS: The occurrence of spoiling micro-organisms was frequent and the possibility of contamination by potentially pathogenic clostridia was demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest the need for improved ensiling practices and appropriate control measures to safeguard the hygienic and nutritional quality of silages produced in farms.     

Preservation of probiotic strains isolated from kefir by spray drying

Aims:  This work aims to investigate the survival of Lactobacillus kefir CIDCA 8348, Lactobacillus plantarum CIDCA 83114 and Saccharomyces lipolytica CIDCA 812, all isolated from kefir, during spray drying and subsequent storage.
Methods and Results:  Micro-organisms were grown in De Man, Rogosa, Sharpe (MRS) or yeast medium (YM) medium and harvested in the stationary phase of growth. The thermotolerance in skim milk ( D and Z values), the survival of spray drying at different outlet air temperatures and subsequent storage in different conditions during 150 days were studied. The resistance to the heat treatments was higher in Lact. plantarum compared to Lact. kefir and S. lipolytica . The three micro-organisms studied varied considerably in their ability to survive to spray drying processes . Lactobacillus plantarum showed the highest survival rate for all the tested outlet air temperatures and also to the further storage in the dried state. The survival rates of Lact. kefir and S. lipolytica through drying and subsequent storage in the dried state decreased when the drying outlet air temperatures increased.
Conclusions:  Spray drying is a suitable method to preserve micro-organisms isolated from kefir grains. A high proportion of cells were still viable after 80 days of storage at refrigerated temperatures
Significance and Impact of Study:  It is the first report about spray-dried probiotic strains isolated from kefir grain and contributes to the knowledge about these micro-organisms for their future application in novel dehydrated products.     

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.     

Distribution of the phosphoenolpyruvate:glucose phosphotransferase system in fermentative bacteria.

A number of selected fermentative bacteria were surveyed for the presence of the phosphoenolpyruvate:glucose phosphotransferase system, with particular attention to those organisms which ferment glucose by pathways other than the Embden-Meyerhof-Parnas pathway. The phosphoenolpyruvate:glusoe phosphotransferase system was found in all homofermentative lactic acid bacteria tested that ferment glucose via the Embden-Meyerhof-Parnas pathway, but in none of a group of heterofermentative species of Lactobacillus or Leuconostoc, which ferment glucose via the phosphoketolase pathway. A phosphoenolpyruvate:glucose phosphotransferase system was also absent in Zymomonas mobilis, which ferments glucose via an anaerobic Entner-Doudoroff pathway. It thus appears that the phosphotransferase mode of glucose transport is limited to bacteria with the Embden-Meyerhof-Parnas mode of glucose fermentation.     

西藏曲拉和云南乳饼中酵母菌的鉴定及其生物多样性

【目的】探讨西藏曲拉和云南乳饼中酵母菌的生物多样性及其分布特征,为我国传统乳制品中酵母菌资源的利用提供基础数据。【方法】从西藏和云南分别采集的5份曲拉样品和8份乳饼样品中分离出41株酵母菌,利用26SrDNAD1/D2区域序列分析对这些菌株进行了分类鉴定。【结果】曲拉和乳饼样品中酵母菌的总数分别在106-107cfu/g和102-106cfu/g之间,曲拉样品的酵母菌平均数比乳饼样品中的高34倍。共鉴定出10属12种,其中西藏曲拉的优势菌株为发酵毕赤氏酵母(Pichia fermentans)和酿酒酵母(Saccharomyces cerevisiae);云南乳饼的优势菌株为类筒假丝酵母(Candida zeylanoides)和喜仙人掌毕赤氏酵母(Pichia cactophila)。毕赤氏酵母属(Pichia)是曲拉和乳饼的共同优势属。【结论】西藏曲拉和云南乳饼中的酵母菌都具有丰富的生物多样性,但其差异性很大。     

秦岭地区子囊菌酵母物种多样性研究

从陕西秦岭地区的果实和叶等不同基物上分离得到子囊菌酵母262株,利用26SrDNA的D1/D2区域序列分析并结合形态学特征对这些菌株进行了分类学研究,探讨了该地区子囊菌酵母的物种多样性及其分布。共鉴定出10属31种,其中优势属为假丝酵母属Candida,12种,(其中新种2个,另文发表)、酿酒酵母属Saccharomyces,5种、毕赤酵母属Pichia,5种和有孢汉逊酵母属Hanseniaspora,3种。对分离自陕西秦岭不同地区与海拔的同一个种的不同菌株进行了D1/D2序列分析比较,以探讨子囊菌酵母种内序列稳定性和变异幅度。     

芝麻香型白酒发酵过程中乳酸菌多样性及其演替规律

【背景】乳酸菌是白酒发酵过程中一类非常重要的微生物,其种类及动态变化对于白酒品质具有重要影响。然而,目前对于芝麻香型白酒发酵过程中乳酸菌群落结构及其演替规律的认识并不全面。【目的】揭示芝麻香型白酒发酵过程中乳酸菌的多样性及菌群的演替规律,为更好地探索白酒酿造机理和控制白酒品质提供生物学依据。【方法】利用高通量测序技术对芝麻香型白酒发酵过程中乳酸菌菌群演替进行跟踪分析,同时采用实时荧光定量PCR对发酵过程中乳酸菌的生物量进行定量分析。【结果】高通量测序结果显示,芝麻香型白酒发酵过程涉及5个属的乳酸菌:魏斯氏菌属(Weissella)、片球菌属(Pediococcus)、乳杆菌属(Lactobacillus)、明串珠菌属(Leuconostoc)和乳球菌属(Lactococcus),共计43种乳酸菌。其中,在发酵过程中平均相对丰度大于0.5%的乳酸菌有10种,分别是类肠膜魏斯氏菌(Weissella paramesenteroides)、食窦魏斯氏菌(Weissella cibaria)、融合魏斯氏菌(Weissella confusa)、戊糖片球菌(Pediococcus pentosaceus)、假肠膜明串珠菌(Leuconostoc pseudomesenteroides)、发酵乳杆菌(Lactobacillus fermentum)、植物乳杆菌(Lactobacillus plantarum)、副干酪乳杆菌(Lactobacillus paracasei)、耐酸乳杆菌(Lactobacillus acetotolerans)和Lactobacillus sp.。在堆积发酵过程中,Weissella属占细菌总量的50%以上,其次是Pediococcus属和Lactobacillus属,而Leuconostoc属和Lactococcus属相对较少。在窖池发酵过程中Lactobacillus属的乳酸菌逐渐成为优势细菌,尤其是Lactobacillus sp.在窖池发酵中后期相对丰度达到80%以上。实时荧光定量PCR结果显示,在堆积发酵和窖池发酵前期乳酸菌总量变化不大;从窖池发酵5 d开始,乳酸菌总量迅速上升,30 d时达到最大值。【结论】对白酒发酵过程中乳酸菌种类及动态变化的研究有助于探究白酒酿造过程中乳酸菌功能,进而解析白酒酿造机理,最终达到控制白酒品质的目的。     

PCR-DGGE analysis of lactic acid bacteria and yeast dynamics during the production processes of three varieties of Panettone

Aims:  To study lactic acid bacteria (LAB) and yeast dynamics during the production processes of sweet-leavened goods manufactured with type I sourdoughs.
Methods and Results:  Fourteen sourdough and dough samples were taken from a baking company in central Italy during the production lines of three varieties of Panettone. The samples underwent pH measurements and plating analysis on three solid media. The microbial DNA was extracted from both the (sour)doughs and the viable LAB and yeast cells collected in bulk, and subjected to PCR-denaturing gradient gel electrophoresis (DGGE) analysis. The molecular fingerprinting of the cultivable plus noncultivable microbial populations provide evidence of the dominance of Lactobacillus sanfranciscensis , Lactobacillus brevis and Candida humilis in the three fermentation processes. The DGGE profiles of the cultivable communities reveal a bacterial shift in the final stages of two of the production processes, suggesting an effect of technological parameters on the selection of the dough microflora.
Conclusions:  Our findings confirm the importance of using a combined analytical approach to explore microbial communities that develop during the leavening process of sweet-leavened goods.
Significance and Impact of the Study:  In-depth studies of sourdough biodiversity and population dynamics occurring during sourdough fermentation are fundamental for the control of the leavening process and the manufacture of standardized, high-quality products.