侗族传统发酵肉的微生物特性

目的：对侗族传统发酵肉的微生物生态系的构成进行分析。方法：稀释平板法,结果：乳酸细菌在发酵30d达到最大值,4个处理的logCFU/g值均在8.2以上;280个MRS平板分离物中,米酒乳杆菌占37.91%,片球菌占20.71%。MSA平板培养物在发酵全过程中,均呈上升趋势;60d后,4个处理的logCFU/g值平均为5.5,增加了2.0;腐生葡萄球菌、肉葡萄球菌和木糖葡萄球菌是其优势菌群。酵母菌的数量在发酵过程中未见明显增加,发酵完毕,菌数的logCFU/g值在5.7左右,鉴定结果表明主要是德巴利酵母和球拟酵母。革兰阴性的肠细菌群发酵50d后,其logCFU/g值在2.0以下,结论：对发酵肉品微生态系的不断了解和肉品发酵技术不断完善,将为人们提供新颖、营养、安全的食品。     

藏灵菇微生物种群结构的分子特性研究*

用PCR-DGGE指纹技术,研究了藏灵菇中微生物多样性及藏灵菇发酵奶发酵过程微生物种群动力学。结果表明,藏灵菇中细菌的种群结构较酵母菌的复杂,不同来源的藏灵菇中细菌种群结构的相似性为78%~84%,酵母菌种群结构的相似性为80%~92%。发酵过程中细菌种群结构变化图谱中的条带B和条带E,以及酵母菌种群结构变化图谱中的条带N贯穿于整个发酵过程,是发酵过程的优势菌。序列分析表明,细菌种群结构的DGGE图谱中的绝大多数条带与乳酸菌相对应,其中最亮条带(条带E)的序列与乳酸乳球菌的相似性为100%。     

藏灵菇微生物种群结构的分子特性研究

用PCR—DGGE指纹技术,研究了藏灵菇中微生物多样性及藏灵菇发酵奶发酵过程微生物种群动力学。结果表明,藏灵菇中细菌的种群结构较酵母菌的复杂,不同来源的藏灵菇中细菌种群结构的相似性为78％-84％,酵母菌种群结构的相似性为80％-92％。发酵过程中细菌种群结构变化图谱中的条带B和条带E,以及酵母菌种群结构变化图谱中的条带N贯穿于整个发酵过程,是发酵过程的优势菌。序列分析表明,细菌种群结构的DGGE图谱中的绝大多数条带与乳酸菌相对应,其中最亮条带（条带E）的序列与乳酸乳球菌的相似性为100％。     

锡林郭勒地区鲜马奶中细菌多样性分析

【背景】传统的酸马奶是由鲜马奶自然发酵而成,鲜马奶中的微生物在很大程度上决定了酸马奶的品质。然而,鲜马奶中的微生物组成却鲜有报道。【目的】解析鲜马奶中细菌多样性,比较分析鲜马奶和酸马奶中微生物的差异,探究马奶发酵过程中微生物的变化。【方法】采用PacBio SMRT测序技术,对6份采集自内蒙古锡林浩特牧区的鲜马奶中细菌16SrRNA基因序列全长进行测序,在种水平解析鲜马奶样品的细菌组成,并与前期同一地区的酸马奶菌群组成作对比。【结果】鲜马奶中包含大量的乳酸菌,主要为乳酸乳球菌(12.43%)、格氏乳球菌(3.53%)、肠膜明串珠菌(1.77%)等。此外还有部分板崎肠杆菌等条件致病菌的存在。马奶发酵前后菌群变化较大,鲜马奶中微生物多样性显著高于酸马奶,其中乳酸乳球菌和瑞士乳杆菌为鲜马奶和酸马奶的共有细菌。鲜马奶中的肠膜明串珠菌与乳酸乳球菌、格氏乳球菌呈正相关关系,与嗜水气单胞菌等有害菌为负相关关系,表明在马奶发酵过程中乳酸菌可能会抑制有害菌的生长。【结论】在马奶的发酵过程中,乳酸菌的大量生长抑制了马奶中有害菌生长,降低了马奶中微生物的多样性,提升了酸马奶的品质。为酸马奶的工业化生产提供了参考。     

卡氏德巴利酵母植酸酶在毕赤酵母中的异源表达及优化

【背景】植酸是一种能螯合金属离子和蛋白质的有机磷类化合物,广泛存在于植物组织中,影响动物对营养元素的吸收。在饲料中加入植酸酶可有效降解植酸。【目的】构建毕赤酵母异源表达卡氏德巴利酵母(Debaryomyces castellii,D. castellii)植酸酶的菌株,促进卡氏德巴利酵母植酸酶的研究及工业应用。【方法】将卡氏德巴利酵母植酸酶基因进行密码子优化后转入毕赤酵母GS115中,通过筛选多拷贝、敲除蛋白酶、过表达分子伴侣及转运蛋白的方法获取优势菌株。【结果】所得重组菌株GS115/DCphy(ΔPep4)(BFR2)的产酶酶活是低拷贝菌株的7倍。【结论】研究结果为卡氏德巴利酵母植酸酶的异源表达及潜在工业应用提供了一定的指导。     

苹果酸降解相关基因在酿酒酵母中的表达

微生物降酸是现代葡萄酒酿造重要工艺。将裂殖酵母苹果酸通透酶基因(mae1)和苹果酸酶基因(mae2)克隆到酿酒酵母中,构建了苹果酸酒精酵母;将mae1基因和乳酸乳球菌的苹果酸乳酸酶基因(mleS)克隆到酿酒酵母中,构建了苹果酸乳酸酵母。构建的酵母重组子能够有效地分解发酵基质中的苹果酸。     

糊米汁对烟叶发酵过程中微生物的影响

研究了糊米汁对烟叶发酵过程中微生物种群及种群数量的变化,初步了解了糊米汁对烟叶微生物的影响。通过添加糊米汁的培养基对烟叶微生物进行液态培养和固态培养。结果表明:在糊米汁堆积式发酵与糊米汁培养基中,糊米汁对烟叶的好氧细菌、厌氧细菌都有非常明显的抑制作用,糊米汁用量越多,抑制作用越明显;但是在普通培养基中施加糊米汁,却对霉菌和酵母没有明显的抑制作用。糊米汁只在烟叶堆积式固态发酵过程中表现出抑制霉菌和酵母的作用,说明糊米汁抑制微生物生长的机制主要是它具有黏性,施加到烟叶中会使烟叶相互粘结,阻碍了O2的传递进而抑制了霉菌和酵母的生长。     

基于高通量测序的传统甜面酱自然发酵过程中的微生物群落结构及其动态演替

【背景】传统自然发酵的甜面酱滋味鲜美、风味独特,但目前尚没有采用高通量测序技术研究其发酵过程中微生物多样性的报道。【目的】解析传统甜面酱自然发酵过程中微生物的群落结构及其动态演替规律。【方法】采用高通量测序技术对传统甜面酱自然发酵过程中的微生物多样性进行研究。【结果】共检出100个真菌菌属和432个细菌菌属,前发酵阶段和后发酵0-90 d的优势真菌为曲霉属(Aspergillus,≥76.96%);后发酵120–180 d的优势真菌为接合酵母属(Zygosaccharomyces,≥85.42%)。芽胞杆菌属(Bacillus)在整个发酵阶段一直是最主要的优势细菌(其中前发酵43 h时占98.07%)。曲样和酱醪中的优势细菌菌属还有葡萄球菌属(Staphylococcus)、雷尔氏菌属(Ralstonia)、泛菌属(Pantoea)、伯克霍尔德氏菌属(Burkholderia)、片球菌属(Pediococcus)、鞘氨醇单胞菌属(Sphingomonas)、双歧杆菌属(Bifidobacterium)、霍氏真杆菌属(Faecalibacterium)、考克式菌属(Kocuria)和乳酸菌属(Lactobacillus)等。【结论】确定了传统甜面酱自然发酵过程中不同时期的优势菌群,为研究微生物对传统甜面酱风味形成的影响提供了理论依据。     

高盐稀醪酱油发酵原油中微生物区系研究

对传统的高盐稀醪酱油发酵过程中的正常原油和异常原油中的微生物区系进行了分析,并对主要的细菌和酵母菌进行了菌种鉴定。同时,初步探讨了温度对原油中微生物区系的影响。实验结果表明,正常原油和异常原油中细菌总数、芽孢菌数、肠道杆菌、乳酸菌数和厌氧菌数没有明显差异,而酵母菌数和耐盐菌数有明显的差异。正常原油中优势酵母为球拟酵母属(Torulopsis)和酵母属(Saccharomyces),分别占酵母总数的55.9%和35.3%。异常原油中优势酵母为毕赤酵母属(Pichia)、假丝酵母属(candida)和酵母属(Saccharomyces),分别占酵母总数的62.8%、17.9%和9.0%。     

芝麻香型白酒微生物多样性及其与胁迫因子相关性

【背景】近年来芝麻香型白酒的生产工艺日臻成熟,然而相应的科学研究却没有同步发展起来。高通量测序技术越来越多地应用于物种多样性的研究,但偏重于研究物种的相对丰度,没有关注物种的生物数量。【目的】深度解析芝麻香型白酒发酵过程微生物群落结构变化及其与胁迫因子相关性,并研究主要酵母菌与细菌的相关性,为揭示芝麻香型白酒发酵机理和控制发酵质量提供理论支撑。【方法】使用Thermofisher的Ion S5~(TM)XL测序平台进行16S rDNA和ITS rDNA扩增子高通量测序,结合微生物传统的定量方法,测定芝麻香型白酒发酵过程微生物群落结构的变化,同时监测发酵过程乳酸、乙酸、乙醇的含量变化,通过样品复杂度分析、多样品比较分析、环境因子关联性分析探究发酵过程微生物群落及其与胁迫因子的关系。通过Pearson相关性分析酵母菌与细菌的相关性。【结果】发酵前期纤维素菌、魏斯氏菌和芽孢杆菌占主要优势,发酵中后期乳杆菌占绝对优势,其次是纤维素菌、魏斯氏菌和芽孢杆菌。整个发酵过程伊萨酵母占绝对优势,其次是维克霉菌、酿酒酵母、假丝酵母。大部分微生物与胁迫因子呈负相关,只有乳杆菌与乙酸呈极显著性正相关。酵母菌与部分细菌呈正相关性。【结论】白酒发酵过程胁迫因子和微生物间的相互作用促进了群落演替过程,发酵后期乳杆菌和芽孢杆菌发酵产酸抑制了大部分不耐酸菌,有机酸是影响群落结构变化的主要胁迫因子。微生物数量结合相对丰度揭示了发酵过程群落结构演替及其与环境因子相关性的更多信息。     

Antagonism Between Osmophilic Lactic Acid Bacteria and Yeasts in Brine Fermentation of Soy Sauce

Brine fermentation by osmophilic lactic acid bacteria and yeasts for long periods of time is essential to produce a good quality of shoyu (Japanese fermented soy sauce). It is well known that lactic acid fermentation by osmophilic lactic acid bacteria results in the depression of alcoholic fermentation by osmophilic yeasts, but the nature of the interaction between osmophilic lactic acid bacteria and yeasts in brine fermentation of shoyu has not been revealed. The inhibitory effect of osmophilic lactic acid bacteria on the growth of osmophilic yeasts was investigated. It was recognized that osmophilic shoyu yeasts such as Saccharomyces rouxii and Torulopsis versatilis were inhibited by a metabolite produced by osmophilic lactic acid bacteria (belonging to Pediococcus halophilus) in brine fermentation of shoyu. The primary inhibitor was considered to be acetic acid, although lactic acid was slightly inhibitory.     

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。     

Molecular diversity of lactic acid bacteria from cassava sour starch (Colombia)

Lactic acid bacteria and more particularly lactobacilli and Leuconostoc, are widely found in a wide variety of traditional fermented foods of tropical countries, made with cereals, tubers, meat or fish. These products represent a source of bacterial diversity that cannot be accurately analysed using classical phenotypic and biochemical tests. In the present work, the identification and the molecular diversity of lactic acid bacteria isolated from cassava sour starch fermentation were assessed by using a combination of complementary molecular methods: Randomly Amplified Polymorphic DNA fingerprinting (RAPD), plasmid profiling, hybridization using rRNA phylogenetic probes and partial 16S rDNA sequencing. The results revealed a large diversity of bacterial species (Lb. manihotivorans, Lb. plantarum, Lb. casei, Lb. hilgardii, Lb. buchneri, Lb. fermentum, Ln. mesenteroides and Pediococcus sp.). However, the most frequently isolated species were Lb. plantarum and Lb. manihotivorans. The RAPD analysis revealed a large molecular diversity between Lb. manihotivorans or Lb. plantarum strains. These results, observed on a rather limited number of samples, reveal that significant bacterial diversity is generated in traditional cassava sour starch fermentations. We propose that the presence of the amylolytic Lb. manihotivorans strains could have a role in sour starch processing.     

Microbiology of Lebanon Bologna

Various aspects of the microbiology of the Lebanon bologna process were studied. Manufacture of Lebanon bologna appeared to be similar to that of summer sausage and other fermented sausages and consisted of a lactic acid fermentation by lactobacilli accompanied by the production of cured meat color from the reduction of nitrate by micrococci. The traditional process consists of aging coarse ground beef at 5 C for several days. Aging the beef for about 10 days was necessary to allow development of lactic acid bacteria; for successful fermentation, the concentration of lactic acid producers must be 10(4)/g or more. At least 3% NaCl was necessary to suppress the development of pseudomonads during the aging period; higher concentrations of salt suppress the development of the lactic acid-producing flora. During aging, in the presence of salt, the predominant flora developing on the meat consisted of catalase-positive, gram-positive rods and cocci; during fermentation at 35 C, the predominant flora became catalase-negative, gram-positive rods with characteristics of lactobacilli. Lebanon bologna could be made from frozen beef if the meat was thawed, salted, and aged. However, bolognas could not be made from unaged beef unless a lactic acid starter culture was used. The microflora of several commercial bolognas is reported also.     

Evolution of Yeasts and Lactic Acid Bacteria During Fermentation and Storage of Bordeaux Wines

The levels of yeasts and lactic acid bacteria that naturally developed during the vinification of two red and two white Bordeaux wines were quantitatively examined. Yeasts of the genera Rhodotorula, Pichia, Candida, and Metschnikowia occurred at low levels in freshly extracted grape musts but died off as soon as fermentation commenced. Kloeckera apiculata (Hanseniaspora uvarum), Torulopsis stellata, and Saccharomyces cerevisiae, the dominant yeasts in musts, proliferated to conduct alcoholic fermentation. K. apiculata and eventually T. stellata died off as fermentation progressed, leaving S. cerevisiae as the dominant yeast until the termination of fermentation by the addition of sulfur dioxide. At least two different strains of S. cerevisiae were involved in the fermentation of one of the red wines. Low levels of lactic acid bacteria (Pediococcus cerevisiae, Leuconostoc mesenteroides, and Lactobacillus spp.) were present in grape musts but died off during alcoholic fermentation. The malolactic fermentation developed in both red wines soon after alcoholic fermentation and correlated with the vigorous growth of at least three different strains of Leuconostoc oenos.     

Lactic fermentation during the storage of 'Alorena' cultivar untreated green table olives

The development of lactic fermentation processes for the storage of directly brined olives (Aloreña cultivar) was investigated by three procedures: (1) a modification of the traditional method with an initial brine containing 9% (w/v) NaCl and 0.2% (w/v) acetic acid; (2) induced lactic fermentation with 6% NaCl and 0.2% acetic acid; and (3) conservation in acidified brine containing 6% NaCl and 0.6% acetic acid. In all cases, strains of Lactobacillus plantarum and Pediococcus spp. were present in each, indicating the great tolerance of these micro-organisms to high levels of lactic and acetic acids. They also appeared in an altered sequence. Counts of Pediococcus remained moderate (higher than Lact. plantarum ) throughout the last part of the preservation period. A commercial starter improved colonization by Lact. plantarum. Yeasts coexisted with the lactic bacteria throughout the preservation period although their importance in the fermentation process was very limited. The brine characteristics obtained after fermentation were suitable for assured product preservation. There was no spoilage. These results encourage research on the mechanism of lactic acid bacteria inhibition in brines and the development of lactic fermentation processes for directly brined olives from other olive cultivars.     

Production of sour maize bread using starter-cultures

Eight lactic acid bacteria were isolated from fermenting maize meal. They were identified as Lactobacillus brevis, L. casei, L. fermentum, Pediococcus acidilacti, P. pentosaceus, Lactobacillus spp. I and Pediococcus spp. I and II. L. brevis and Lactobacillus spp. I isolated from the spontaneously fermented maize meal together with L. brevis isolated from rye sour dough and L. plantarum from ogi, a fermented maize gruel, were selected as starter organisms. There was a decrease in the final pH from 4.9 to 3.8 and an increase in the acid equivalent and temperature of the spontaneously-generated sour maize meal at the end of 24h fermentation. There was a decrease in the pH and moisture content of the sour maize breads relative to the conventional wheat bread. An improvement in the shelf-life of the bread samples was also obtained. Crude protein values of the sour maize breads were between 4.36% and 8.87%, while crude fat contents ranged between 3.66% to 7.67%. The ash contents increased from 2.29% to 2.54% while total carbohydrate values were between 46.31% and 65.3%. Calcium, phosphorus and potassium contents ranged from 0.015, 0.26 and 0.018% to 0.036, 0.47 and 0.036% respectively. Physical examination of the bread samples showed that all were cracked and relatively hard. Weight, height and volume ranged from 316 to 380g; 4.2 to 5.2cm and 200 to 320cm3 respectively. Statistical analysis of the sensory attributes revealed a consumer acceptance of the sour maize breads, although ranking test showed preference for the baker's yeast leavened bread that served as a control.     

Design and evaluation of a Lactobacillus manihotivorans species-specific rRNA-targeted hybridization probe and its application to the study of sour cassava fermentation

Based on 16S rRNA sequence comparison, we have designed a 20-mer oligonucleotide that targets a region specific to the species Lactobacillus manihotivorans recently isolated from sour cassava fermentation. The probe recognized the rRNA obtained from all the L. manihotivorans strains tested but did not recognize 56 strains of microorganisms from culture collections or directly isolated from sour cassava, including 29 species of lactic acid bacteria. This probe was then successfully used in quantitative RNA blots and demonstrated the importance of L. manihotivorans in the fermentation of sour cassava starch, which could represent up to 20% of total lactic acid bacteria.