产酱香风味芽孢杆菌类菌株发酵代谢产物及其酶分析研究

酱香白酒生产过程中的制曲、堆积等工艺环节可富集大量芽孢杆菌,这些芽孢杆菌是酱香风味成分形成的基础和关键.对从酱香型白酒生产制曲、堆积酒醅和发酵期酒醅中分离的地衣芽孢杆菌、解淀粉芽孢杆菌、枯草芽孢杆菌3类芽孢杆菌的分离、鉴定、产香、产酶等进行总结,详述了3类产香芽孢杆菌发酵代谢产香的特征性成分、特征性酶、产香过程风味的特征性变化和群体微生物发酵机制.     

沉香型酒醅中产香芽孢杆菌的分离鉴定及代谢产物分析

从沉香型酒醅中分离产香芽孢杆菌并进行发酵风味分析。通过平板分离得到4株产香较好的芽孢杆菌,并进行16S rDNA序列分析和构建系统发育树,4株菌分别为阿式芽孢杆菌（Bacillus aryabhattai）、地衣芽孢杆菌（Bacillus licheniform）、枯草芽孢杆菌（Bacillus subtilis）、解淀粉芽孢杆菌（Bacillus amyloliquefaciens）。其中地衣芽孢杆菌产蛋白酶和淀粉酶能力均为最强且最适生长温度为50 ℃。以玉米粉为底物进行单菌液态发酵,采用固相萃取和气相色谱-质谱法(SPME-GC-MS)分析发酵液中的风味物质,一共检测到24种风味物质,其优势产物主要为3-羟基-2-丁酮、2,3-丁二醇和一些酸类等风味物质。由此而见,阿式芽孢杆菌、地衣芽孢杆菌、枯草芽孢杆菌、解淀粉芽孢杆菌这4类菌对沉香型白酒风味的形成均具有重要作用。     

牛栏山二锅头酒醅中芽孢杆菌分离鉴定及发酵风味分析

从牛栏山二锅头酒醅中分离筛选出5株产风味物质能力较好的芽孢杆菌,通过16SrDNA序列分析和构建系统发育树,5株细菌分别为地衣芽孢杆菌(Bacillus licheniformis)、蜡样芽孢杆菌(Bacillus cereus)、短小芽孢杆菌(Bacillus pumilus)和枯草芽孢杆菌(Bacillus subtilis)。分别对它们进行发酵风味分析,其发酵液经固相微萃取和GC-MS分析,并除去空白培养基中物质,地衣芽孢杆菌BL-1发酵液共检测得到14种风味物质,蜡样芽孢杆菌BC-1和短小芽孢杆菌BP-1发酵都得到12种风味物质,枯草芽孢杆菌BS-1好氧发酵共得到16种风味物质,枯草芽孢杆菌BS-2厌氧发酵共得到19种风味物质。除短小芽孢杆菌外,其他4株芽孢杆菌都含有较多数量的酯类化合物,且主要代谢风味物质都是3-羟基-2-丁酮,而短小芽孢杆菌BP-1则含有数量较多的烃类化合物,其主要风味物质是苯乙醇。     

牛栏山二锅头酒醅中芽孢杆菌分离鉴定及发酵风味分析

从牛栏山二锅头酒醅中分离筛选出5株产风味物质能力较好的芽孢杆菌,通过16SrDNA序列分析和构建系统发育树,5株细菌分别为地衣芽孢杆菌（Bacillus licheniformis）、蜡样芽孢杆菌（Bacillus cereus）、短小芽孢杆菌（Bacillus pumilus）和枯草芽孢杆菌（Bacillus subtilis）。分别对它们进行发酵风味分析,其发酵液经固相微萃取和GC-MS分析,并除去空白培养基中物质,地衣芽孢杆菌BL-1发酵液共检测得到14种风味物质,蜡样芽孢杆菌BC-1和短小芽孢杆菌BP-1发酵都得到12种风味物质,枯草芽孢杆菌BS-1好氧发酵共得到16种风味物质,枯草芽孢杆菌BS-2厌氧发酵共得到19种风味物质。除短小芽孢杆菌外,其他4株芽孢杆菌都含有较多数量的酯类化合物,且主要代谢风味物质都是3-羟基-2-丁酮,而短小芽孢杆菌BP-1则含有数量较多的烃类化合物,其主要风味物质是苯乙醇。      

基于酶学特性筛选大曲来源芽孢杆菌用于强化酿酒

通过分析来源于清香型、浓香型和酱香型大曲的105 株芽孢杆菌的产淀粉酶和蛋白酶的能力,优选出1 株地衣芽孢杆菌（Bacillus licheniformis）B.L-1和1 株枯草芽孢杆菌（B. subtilis）B.S-1,将其分别应用于清香型白酒酿造中,探究强化接种微生物对发酵的影响。发酵结束后,在酒醅中共检测到挥发性代谢产物38 种,通过多元统计分析,在强化B.L-1组和B.S-1组中分别筛选出差异代谢物21 个和11 个（VIP＞1;P＜0.05）,其中4-乙基-2-甲氧基苯酚、辛酸乙酯、3-甲基丁酸乙酯和四甲基吡嗪在2 组强化中均为差异代谢物,含量显著升高。结果表明,用酶学特性进行菌株筛选能够快速有效地获得特定功能菌株,由此获得的菌株进行强化发酵可显著提高酒醅中挥发性风味物质含量。该方法可以有效地为大曲微生物合成群落重塑提供菌种资源。     

高温大曲中产吡嗪芽孢杆菌的分离鉴定及发酵产物分析

该试验从高温大曲中筛选产吡嗪芽孢杆菌（Bacillus）,通过形态观察及磷脂脂肪酸（PLFA）分析对其进行鉴定,并采用发酵试验分析其代谢产物。共筛选出3株芽孢杆菌,即菌株B1、B2、B3。经分析鉴定后得出：菌株B1为蜡样芽孢杆菌（Bacillus cereus）;菌株B2和B3为萎缩芽孢杆菌（Bacillus atrophaeus）。3株菌发酵产物分析结果表明,在液态发酵条件下3株菌发酵产吡嗪,其中菌株B2和B3发酵产物中吡嗪相对含量较高,均＞60%;固态发酵条件下生成吡嗪前体物质3-羟基-2-丁酮,且发酵产物中相对含量均＞75%。表明这3株芽孢杆菌对酱香型白酒风味物质的产生有着一定的影响。     

陶融型酒醅中产香芽孢杆菌的筛选、鉴定及发酵产物分析

通过传统微生物分离法和纯种固态发酵产香试验,从陶融型酒醅微生物资源中分离、筛选产香能力优良的芽孢杆菌,采用形态观察、生理生化试验及分子生物技术对其进行鉴定,并对其耐受性进行研究。结果表明,从陶融型酒醅中共分离得到11株芽孢杆菌,其中,菌株YSB11产香较好,固态发酵产物中含有浓郁的焦香、糊香和酱香味,包含酚类、吡嗪类、呋喃类、芳香类、酯类等多种白酒中重要的呈香物质,为优良产香芽孢杆菌。菌株YSB11被鉴定为贝莱斯芽孢杆菌（Bacillus velezensiss）,可耐高温53 ℃、酒精度6%vol、NaCl含量8%及低pH值6.0。     

高温大曲曲房空气中可培养细菌的分离鉴定及产酶特性

以高温大曲曲房空气中可培养细菌为研究对象,对其进行分离鉴定及产酶特性研究;将得到的优势功能细菌进行高粱汁液态发酵,利用气相色谱—质谱联用法分析其代谢产物。结果表明：从高温大曲曲房空气中共分离出8种菌株,分别为解淀粉芽孢杆菌（Bacillus amyloliquefaciens）、枯草芽孢杆菌（Bacillus subtilis）、蜡样芽孢杆菌（Bacillus cereus）、Pedobacter suwonensis、血红鞘氨醇单孢菌（Sphingomonas sanguinis）、Massilia sp.、Delftia sp.和 Sphingobacterium sp.;经产酶试验筛选得到3株优势功能菌：枯草芽孢杆菌（Bacillus subtilis）、解淀粉芽孢杆菌（Bacillus amyloliquefaciens）和蜡样芽孢杆菌（Bacillus cereus）,均具有纤维素分解能力、淀粉和蛋白水解能力;3株菌的生长曲线表明液体培养4 h达到对数生长期,在高粱汁液态发酵48 h后,优势代谢产物为3-羟基-2-丁酮,且四甲基吡嗪含量也较高,并代谢产生少量高级醇、酚类、酯类等芳香物质。     

婴幼儿配方奶粉中的菌相分析

从市场上随机抽取不同生产厂家的婴幼儿奶粉,通过菌落总数测定,单菌落的分离,利用细菌通用引物27f/1492r对不同菌株进行16S rDNA片段扩增、测序,建立菌株的系统发育树进行分析。结果表明,婴幼儿配方奶粉中主要有地衣芽孢杆菌(Bacillus licheniformis),短小芽孢杆菌(Bacillus pumilus)、蜡样芽孢杆菌(Bacillus cereus)、枯草芽孢杆菌(Bacillus subtilis)和屎肠球菌(Enterococcus faecium),其中,地衣芽孢杆菌(Bacillus licheniformis)、短小芽孢杆菌(Bacillus pumilus)、蜡样芽孢杆菌(Bacillus cereus)为优势菌种。说明婴幼儿配方奶粉中存在的微生物主要为芽孢杆菌。     

酱香型白酒酿造酒醅中产香芽孢杆菌的分离鉴定

为了改善并增强酱香型白酒香气,使其具有独特的风味特征,本研究从七轮次混合酒醅中分离到的7株菌中筛选到一株产香性能较优的菌株DAI bj-1。通过观察该菌株在肉汤琼脂培养基上的菌落形态及细胞形态特征（革兰氏染色为阳性）,初步判断该菌为革兰氏阳性菌。通过革兰氏阳性菌鉴定卡（GP）、芽孢菌鉴定卡（BCL）等生理生化实验结果,PCR扩增、上机测序以及系统发育树的构建分析,该菌株与标准菌株序列的相似性为99.93%,即酒醅中分离筛选到的菌株DAI bj-1为解淀粉芽孢杆菌（Bacillus amyloliquefaciens）。     

浓香型白酒酒醅中乳酸菌分离及其对模拟固态发酵的影响

从发酵酒醅中分离得到21株产乳酸菌株,其中16株为芽孢杆菌。利用梅里埃微生物鉴定仪鉴定,得到干燥棒杆菌（Corynebacterium xerosis）、耳葡萄球菌（Staphylococcus auricularis）、枯草芽孢杆菌（Bacillussubtilis）、巨大芽孢杆菌（Bacillus megaterium）、腊状芽孢杆菌（Bacillus cereus group）、浸麻芽孢杆菌（Paenibacillusmacerans）6种产乳酸菌。将6株产酸菌进行模拟固态发酵,结果显示所有乳酸菌都不同程度的抑制了己酸、己酸乙酯以及乳酸乙酯的产量,其中浸麻芽孢杆菌、巨大芽孢杆菌属的2株产乳酸菌严重抑制了乙醇和己酸乙酯的生成量,巨大芽孢杆菌的一株还严重抑制了己酸的产量。     

清香型白酒酒醅发酵菌株分离鉴定及细菌群落结构分析

采用纯培养技术对清香型白酒酒醅中的乳酸菌和芽孢杆菌进行分离鉴定,同时通过Illumina MiSeq高通量测序技术解析清香型白酒酒醅细菌群落结构。结果表明,所有酒醅样品共分离得到13株乳酸菌,分别鉴定为乳酸片球菌（Pediococcus acidilactici）4株、副干酪乳杆菌（Lactobacillus paracasei）3株、短乳杆菌（Lactobacillus brevis）3株、粪肠球菌（Enterococcus faecium）2株以及希尔氏乳杆菌（Lactobacillus hilgardii）1株。从酒醅中分离得到的17株芽孢杆菌菌株分别鉴定为地衣芽孢杆菌（Bacillus licheniformis）9株、枯草芽孢杆菌（Bacillus subtilis）2株、短小芽胞杆菌（Bacillus pumilus）2株、澳洲芽孢杆菌（Bacillus australimaris）2株、解淀粉芽孢杆菌（Bacillus amyloliquefaciens）1株以及蜡样芽孢杆菌（Bacillus cereus）1株。Illumina MiSeq高通量测序技术结果显示,酒醅中优势菌门分别为厚壁菌门（Firmicutes）（97.96%）、放线菌门（Actinobacteria）（1.24%）和变形菌门（Proteobacteria）（0.76%）;优势细菌属为乳酸杆菌属（Lactobacillus）（97.42%）、栖热嗜油菌属（Thermoleophilum）（1.22%）和假单胞菌属（Pseudomonas）（0.72%）。     

Enterotoxins and emetic toxins production by Bacillus cereus and other species of Bacillus isolated from Soumbala and Bikalga, African alkaline fermented food condiments

The ability of various species of Bacillus from fermented seeds of Parkia biglobosa known as African locust bean (Soumbala) and fermented seeds of Hibiscus sabdariffa (Bikalga) was investigated. The study included screening of the isolates by haemolysis on blood agar, detection of toxins in broth and during the fermentation of African locust bean using the Bacillus cereus Enterotoxin Reverse Passive Latex Agglutination test kit (BCET-RPLA) and the Bacillus Diarrhoeal Enterotoxin Visual Immunoassay (BDEVIA). Detection of genes encoding cytotoxin K (CytK), haemolysin BL (Hbl A, Hbl C, Hbl D), non-hemolytic enterotoxin (NheA, NheB, NheC) and EM1 specific of emetic toxin producers was also investigated using PCR with single pair and multiplex primers. Of 41 isolates, 29 Bacillus belonging to the species of B. cereus, Bacillus subtilis, Bacillus licheniformis and Bacillus pumilus showed haemolysis on blood agar. Using RPLA, enterotoxin production was detected for three isolates of B. cereus in broth and all B. cereus (9) in fermented seeds. Using BDEVIA, enterotoxin production was detected in broth as well as in fermented seeds for all B. cereus isolates. None of the isolates belonging to the other Bacillus species was able to produce enterotoxins either by RPLA or BDEVIA. Nhe genes were detected in all B. cereus while Hbl and CytK genes were detected respectively in five and six B. cereus strains. A weak presence of Hbl (A, D) and CytK genes was detected in two isolates of B. subtilis and one of B. licheniformis but results were inconsistent, especially for Hbl genes. The emetic specific gene fragment EM1 was not detected in any of the isolates studied.     

CYTOTOXICITY ASSESSMENT OF BACILLUS STRAINS ISOLATED FROM STREET-VENDED FOODS IN JOHANNESBURG, SOUTH AFRICA

Twenty-one isolates each of Bacillus (B.) cereus, B. licheniformis and B. subtilis from street foods, collected in central Johannesburg, were randomly selected to test for cytotoxicity against McCoy 5A Mouse cells using a 3-(4,5-dimethylthizol-2-yl)-2,5-diphenyltetrazolium bromide assay, and observation by confocal scanning laser microscopy (CSLM) and scanning electron microscopy (SEM). Forty-eight percent of B. cereus, 33% of B. licheniformis and 19% of B. subtilis strains produced cytotoxic compounds. For B. cereus strains, all supernatants exhibiting cytotoxic effects were inactivated by heat treatment at 121C for 15 min. By contrast, 24% of B. licheniformis and 10% of B. subtilis supernatants exhibited cytotoxic effects following heat treatment. CSLM and SEM showed that McCoy cells treated with cytotoxic supernatants exhibited leakage and necrosis. Presence of B. cereus, B. licheniformis and B. subtilis in street foods in high numbers may pose potetnial safety risks due to production of cytotoxic compounds.     

麦麸阿魏酸糖酯微生物发酵工艺优化及体外抗氧化和益生活性评价

以麦麸为原料,对固态发酵制备麦麸阿魏酸糖酯(Feruloylated glycosides,FGs)的工艺进行优化,并对其体外抗氧化及益生活性进行评价.以植物乳杆菌、枯草芽孢杆菌、地衣芽孢杆菌、酿酒酵母为发酵菌种,采取单菌发酵和混菌发酵筛选最优菌种组合,考察接种量、发酵温度、发酵时间、料水比对麦麸FGs产量的影响,通过...     

In vitro fermentation studies for selection and evaluation of Bacillus strains as starter cultures for the production of okpehe, a traditional African fermented condiment

Selected Bacillus and Enterococcus strains, isolated from traditional okpehe fermentations, were studied for their suitability as starter cultures in laboratory-scale fermentations of Prosopis africana seeds for the production of okpehe, a traditional fermented vegetable product of Nigeria. The strains were selected on the basis of highest proteolytic activity, as determined with the APIZYM (BioMerieux) test. The choice of starter strains was narrowed to Bacillus subtilis strains BFE 5301 and BFE 5372. These were determined as the best starter combination because of rapid growth, high amylolytic and proteolytic activities, high levels of polyglutamic acid production by strain BFE 5372, as well as bacteriocin production by strain BFE 5301. Other mixed culture fermentations did not yield sensorically acceptable products. Although a monoculture fermentation, using only B. subtilis strain BFE 5372, produced okpehe with very good sensory characteristics, the growth of B. cereus could be detected after 48 h fermentation, indicating that this starter did not sufficiently contribute to product safety. Mixed culture fermentation with the combination of bacteriocin-producing starter B. subtilis BFE 5301 and the non-bacteriocin-producing B. subtilis BFE 5372, produced a product with good sensory characteristics, in which growth of B. cereus was delayed. The bacteriocin produced by B. subtilis strain BFE 5301 was identified as subtilisin, using subtilisin-specific primers and PCR amplification of the subtilisin gene. The bacteriocin was heat-stable at 100 degrees C for 10 min and exhibited highest activity at pH values lower or equal to pH 6.0. The bacteriocin was sensitive to the proteolytic enzymes trypsin and alpha-chymotrypsin at concentrations of 10 mg/ml.     

Diversity and functionality of Bacillus and related genera isolated from spontaneously fermented soybeans (Indian Kinema) and locust beans (African Soumbala)

A total of 126 isolates of Bacillus and related genera from indigenous, spontaneously fermented soybeans (Kinema) and locust beans (Soumbala) were characterized with the purpose of defining interspecific, as well as intraspecific relationships among the components of their microflora. B. subtilis was the dominant species, and species diversity was more pronounced in Soumbala than in Kinema. While from Kinema, six species were isolated (B. subtilis, B. licheniformis, B. cereus, B. circulans, B. thuringiensis and B. sphaericus), in Soumbala, the species found were B. subtilis, B. thuringiensis, B. licheniformis, B. cereu, B. badius, Paenibacillus alvei, B. firmus, P. larvae, Brevibacillus laterosporus, B. megaterium, B. mycoides and B. sphaericus. Genomic diversity in the isolates of B. subtilis was investigated by random amplified polymorphic DNA (RAPD) analysis using the polymerase chain reaction (PCR). The RAPD-PCR fingerprint analysis showed a high level of diversity. With more than 90% similarity, all 52 RAPD subdivisions were source and continent-wise homogeneous. Profiles of carbon source fermentation also showed a wide but corresponding phenotypic diversity, largely corresponding with RAPD subdivisions. The various strains were tested for several criteria for functionality in soybean fermentation, viz. protein degradation, pH increase, and development of desirable stickiness caused by viscous exopolymers. Profiles of functionality, based upon estimations of pH, free amino nitrogen and stickiness were associated with genotypic and phenotypic profiles. Notwithstanding the heterogenous fermentation results for some genotypic profiles, a ranking of RAPD groups is possible and can be useful in the further selection and study of B. subtilis strains.