汉氏葡糖醋杆菌Komagataeibacter hansenii HDM1-3不同发酵时间的转录组测序分析

为了探究汉氏葡糖醋杆菌HDM1-3(Komagataeibacter hansenii HDM1-3)在不同发酵时间的差异基因表达水平,挖掘差异表达基因在细菌纤维素(Bacterial cellulose,BC)生物合成代谢相关基因中的功能,利用Illumina HiSeq平台对发酵前期(18 h)和发酵后期(36 h)的Komagataeibacter hansenii HDM1-3菌株进行转录组测序分析,通过荧光定量PCR验证转录组测序结果的准确性,通过GO富集和KEGG富集分析差异表达基因。转录组测序共获得3154个CDS序列,其中57个未被注释,注释率达98.22%。差异表达基因分析表明,菌株HDM1-3发酵36 h相比于发酵18 h,共有684个显著性差异表达基因,其中上调基因369个(53.9%),下调基因315个(46.1%)。GO功能富集分析结果表明,发酵后期碳水化合物代谢相关功能基因下调,而细胞膜相关功能基因上调。KEGG富集分析结果表明,主要的能量代谢途径,包括糖酵解途径、磷酸戊糖途径和柠檬酸循环都显著下调,两个BC合成酶基因显著上调。这些结果为进一步分析HDM1-3菌株BC合成相关基因、提高BC产量的研究提供理论参考。

Debaryomyces hansenii supplementation in low fish meal diets promotes growth,modulates microbiota and enhances intestinal condition in juvenile marine fish

Background The development of a sustainable business model with social acceptance,makes necessary to develop new strategies to guarantee the growth,health,and well-being of farmed animals.Debaryomyces hansenii is a yeast species that can be used as a probiotic in aquaculture due to its capacity to i)promote cell proliferation and differen-tiation,ii)have immunostimulatory effects,iii)modulate gut microbiota,and/or iv)enhance the digestive function.To provide inside into the effects of D.hansenii on juveniles of gilthead seabream(Sparus aurata)condition,we inte-grated the evaluation of the main key performance indicators coupled with the integrative analysis of the intestine condition,through histological and microbiota state,and its transcriptomic profiling.Results After 70 days of a nutritional trial in which a diet with low levels of fishmeal(7%)was supplemented with 1.1%of D.hansenii(17.2×10^(5) CFU),an increase of ca.12%in somatic growth was observed together with an improve-ment in feed conversion in fish fed a yeast-supplemented diet.In terms of intestinal condition,this probiotic modu-lated gut microbiota without affecting the intestine cell organization,whereas an increase in the staining intensity of mucins rich in carboxylated and weakly sulphated glycoconjugates coupled with changes in the affinity for certain lectins were noted in goblet cells.Changes in microbiota were characterized by the reduction in abundance of several groups of Proteobacteria,especially those characterized as opportunistic groups.The microarrays-based transcrip-tomic analysis found 232 differential expressed genes in the anterior-mid intestine of S.aurata,that were mostly related to metabolic,antioxidant,immune,and symbiotic processes.Conclusions Dietary administration of D.hansenii enhanced somatic growth and improved feed efficiency param-eters,results that were coupled to an improvement of intestinal condition as histochemical and transcriptomic tools indicated.This probiotic yeast stimulated host-microbiota interactions without altering the intestinal cell organization nor generating dysbiosis,which demonstrated its safety as a feed additive.At the transcriptomic level,D.hansenii pro-moted metabolic pathways,mainly protein-related,sphingolipid,and thymidylate pathways,in addition to enhance antioxidant-related intestinal mechanisms,and to regulate sentinel immune processes,potentiating the defensive capacity meanwhile maintaining the homeostatic status of the intestine.

Co-inoculation of Debaryomyces hansenii and lactic acid bacteria: a strategy to improve the taste and odour profiles of dry sausages

The effects of the co-inoculation of Debaryomyces hansenii separately with 3 lactic acid bacteria(LAB),Lactobacillus sakei,Lactobacillus plantarum and Lactobacillus curvatus,on the taste and odour profi les of dry sausages were investigated.The co-inoculated sausages showed higher free amino acid and organic acid contents than the non-inoculated control and sausages inoculated with D.hansenii alone.Meanwhile,the sausages inoculated with D.hansenii+L.plantarum,D.hansenii+L.sakei and D.hansenii+L.curvatus had the highest contents of aldehydes,esters and alcohols,respectively.The results of electronic tongue,electronic nose and sensory evaluation demonstrated that compared with the sausage inoculated with D.hansenii,the sour taste and fl oral odour increased and the fatty odour decreased in the sausage inoculated with D.hansenii+L.sakei;this was more favourable for the development of a desirable fl avour in sausages.Moreover,the partial least squares regression analysis indicated that 10 taste and 33 odour compounds were mainly responsible for the differences in the flavour profiles among the sausages.Overall,these findings contributed to a more comprehensive understanding of the formation of sensory characteristics in dry sausages co-inoculated with yeast and LAB.

化风丹药母中3种优势微生物的实时荧光定量PCR方法的建立

目的:建立化风丹药母中3种优势微生物米曲霉(Aspergillus oryzae)、汉逊德巴利酵母菌(Debaryomyces hansenii)、阿氏芽孢杆菌(Bacillus aryabhattai)快速、有效的实时荧光定量PCR检测方法。方法:以米曲霉、汉逊德巴利酵母菌、阿氏芽孢杆菌的重组质粒为标准质粒,设计特异性引物并进行特异性、灵敏性及重复性实验。结果:3种优势微生物的熔解曲线不在同一峰值。米曲霉、汉逊德巴利酵母菌、阿氏芽孢杆菌的荧光定量PCR最低检测限度分别为0.256、514、5.08 copies/μL。不同质量浓度的标准质粒变异系数(CV)均小于5%。结论:研究表明以荧光定量PCR技术建立的米曲霉、汉逊德巴利酵母菌、阿氏芽孢杆菌的检测方法具有重复性好、特异性强、灵敏度高等优点,可用于化风丹药母发酵过程中微生物的检测及定量。

Optimization of Production and Preliminary Characterization of New Exopolysaccharides from <i>Gluconacetobacter hansenii</i>LMG1524

The influence of different carbon and nitrogen sources, of ethanol concentration, the optimal pH, temperature and medium composition were evaluated on extracellular polysaccharides (EPS) synthesis and bacterial growth of Gluconacetobacter hansenii LMG1524, and preliminary characterization of EPS was investigated. The highest EPS yields were obtained using glycerol and ammonium sulphate as carbon and nitrogen sources, respectively. The increase of ethanol concentration in the medium did not influence the EPS synthesis but reduced the bacterial growth. The optimum temperature and pH for polysaccharides production were respectively 25℃ and 5;whereas for cell growth were respectively 30℃ and 4. The optimal culture medium composition was determined as follows: 10 g/L sucrose, 0.892 g/L (NH4)2SO4, 0.34 g/L NaNO3, 3 mL acetic acid, 1.5 g/L KH2PO4, 1.5 g/L K2HPO4 and 0.6 g/L MgSO4. The polysaccharides produced were of 14 and 10 polymerization degrees (DP) and constituted mainly of glucose, galactose and mannose, in relative percent of 36.36, 33.94 and 22.42, respectively.

Improving Effect of Acetic Acid Bacteria (Gluconacetobacter hansenii GK-1) on sIgA and Physical Conditions in Healthy People: Double-Blinded Placebo-Controlled Study

It is known that the consumption of bacteria such as lactobacilli and bifidobacteria has beneficial effects on human immune function. Most of them are Gram-positive bacteria, and there are few reports on Gram-negative bacteria. In this study, we evaluated the effects of intake of Gluconacetobacter hansenii GK-1 (GK-1), Gram-negative acetic acid bacteria, for 12 weeks on physical condition and immune indices. We conducted a randomized, double-blind, placebo-controlled, parallel-group study in 100 healthy adults. The subjects were randomized into the GK-1 and the placebo groups. The diary-administered physical condition survey was conducted during the study period. The evaluation of salivary sIgA levels, NK-cell activity, and serum IFN-γ levels and quality of life survey was conducted before, in 6 weeks, and 12 weeks after the start of ingestion. Based on the physical condition survey, the cumulative onset-days of symptoms were significantly suppressed in the GK-1 group compared to the placebo group regarding the evaluation of 13 symptoms related to immunity, every 3 weeks. Additionally, salivary sIgA levels per hour were significantly increased in the GK-1 group compared with the placebo group at 6 and 12 weeks. Despite no significant differences in the NK-cell activity, serum IFN-γ levels or quality of life survey between the groups. Serum IFN-γ levels in the GK-1 group were significantly elevated at 12 weeks after the start of ingestion compared with those before ingestion. In conclusion, intake of GK-1 was shown to increase salivary sIgA levels and improve physical condition. This suggested that oral intake of GK-1 may help maintain the immune system.

不同发酵剂对发酵香肠品质的影响

微生物群落对发酵香肠的品质特征的形成具有重要作用,目前很多研究应用单一菌种作为发酵剂来改善发酵香肠品质,但将酵母菌与腐生葡萄球菌混合作为发酵香肠的发酵剂的相关研究还未见报道。该文研究了自然发酵(空白组)、接种腐生葡萄球菌、德巴汉逊氏酵母菌以及同时接种两种菌(1∶1)对发酵香肠的微生物、理化性质和风味的影响。结果表明,发酵结束后,空白组的菌落总数和pH值显著高于其他3组;3个接菌组香肠的亮度值、红度值和黄度值显著高于空白组;接种单一和混合发酵剂显著抑制了发酵香肠的脂肪和蛋白质氧化;接种不同发酵菌株显著增加了发酵香肠挥发性风味物质的丰度,减少了醛类物质的产生。综上,德巴汉逊氏酵母与腐生葡萄球菌单一和混合接入发酵香肠后,均可在不同程度上抑制杂菌生长,促进发色,促进酯类等风味物质产生,其中,混菌发酵组效果最佳。

纤维素产生菌汉氏葡糖醋杆菌HDM1-3全基因组测序分析

为全面了解汉氏葡糖醋杆菌(Komagataeibacter hansenii,K.hansenii)HDM1-3的发酵特性,为提高纤维素产量提供基因组信息,对其基因组数据进行测序分析。采用PacBio RSⅡ平台对该菌株进行全基因组测序,基因组由1个3659612 bp染色体和2个质粒组成,编码3820个蛋白质,含有7个纤维素合成酶基因。基于16S rRNA的系统发育分析表明了K.hansenii HDM1-3相对于醋酸杆菌科菌株的进化地位。在基因组中,共注释到碳水化合物活性酶88个。通过KEGG注释到代谢通路相关基因共3132个,其中碳水化合物代谢相关基因287个。通过基因组测序获得了K.hansenii HDM1-3完整的基因组信息,为改造该菌株提供了基因组学基础。

Evaluation and selection of yeasts as potential aroma enhancers for the production of dry-cured ham

Yeasts play a critical role in the flavor formation of dry-cured ham.In this study,41 yeast isolates from the dry-cured ham at different processing stages were evaluated for their technological properties.Debaryomyces hansenii was the most dominant yeast and has been detected at each phase of dry-cured ham,followed by Candida zeylanoides which was mainly detected in salting phase.Yarrowia bubula and Yarrowia alimentaria were found at the first two-phase of dry-cured ham.All isolates of yeast showed enzymatic activities against milk protein and tributyrin,while only 4 strains displayed proteolytic activity on meat protein.Yeast strains were grown in a meat model medium and volatile compounds were identified.The result showed that inoculated yeast strains could promote the production of volatiles and there were significant differences among strains.D.hansenii S25 showed the highest production of volatile compounds,followed by the strain C.zeylanoides C4.D.hansenii S25 was the highest producer of alcohols showing the highest production of benzeneethanol and 3-(methylthio)-1-propanol.Based on OAV and PLS analysis,D.hansenii S25 was strongly correlated with overall flavor and key volatile compounds of dry-cured ham,which could be selected as potential starter cultures.

化风丹药母发酵过程中优势菌种分离鉴定及不同温度和pH值下菌株生长情况

目的对化风丹药母发酵过程中优势菌种进行分离鉴定,考察不同温度和pH值对优势菌株生长的影响。方法用选择性培养基对化风丹药母发酵过程中0周至6周样品中的细菌、霉菌、酵母菌分别进行培养、菌落计数、分离纯化优势菌种;采用16S rDNA,26S rDNA序列对优势细菌和真菌进行鉴定;分别考察阿氏芽孢杆菌、汉逊德巴利酵母菌、米曲霉的最适宜生长温度、pH值。结果化风丹药母发酵过程中细菌0周至4周数量增加,4周至6周数量趋于稳定;酵母菌在发酵0周至3周时数量增加,4周至6周数量不断减少;霉菌在0周时没有生长,1周至4周数量平稳增加,5周至6周霉菌数量呈现下降趋势。通过构建系统发育树,鉴定筛选出化风丹药母发酵过程中的优势菌种,以阿氏芽孢杆菌、汉逊德巴利酵母菌、米曲霉为研究对象,其生长的最适温度分别为35、25~30、35℃;最适pH值分别为8、6、7。结论化风丹药母发酵过程中优势微生物分别为阿氏芽孢杆菌、汉逊德巴利酵母菌、米曲霉。

汉逊德巴利酵母对采后柑橘青霉病的防治及贮藏品质的影响

利用汉逊德巴利酵母不同浓度梯度的处理液对指状青霉体外和柑橘上进行生物防治试验。体外实验表明,108CFU/mL酵母溶液牛津杯产生的抑菌圈为20.6 mm,在PDB培养基中青霉孢子24 h后的萌发率和芽管长度分别为5.2%,11.4μm。柑橘体上实验表明,108CFU/mL酵母溶液处理组8 d后的病斑直径和发病率分别为3.6 mm和7.4%,病变指数仅为7.8%,防治效率达到了92.3%。拮抗酵母可以防止柑橘总糖和Vc的损失,防止柑橘霉变引起的总酸增长。25℃左右的条件下,108CFU/mL的酵母溶液能够较好地抑制柑橘青霉病的发生也保证柑橘的贮藏品质。

一株酵母菌的鉴定及其挥发性物质防病测定

为进一步研究用于防治草莓灰霉病的生防酵母菌,针对一株经平板测定和离体接种筛选得到的酵母菌菌株W4682,进行分类鉴定,并对其产生的挥发性物质防治草莓灰霉病的能力进行测定。结果发现,菌株W4682应属于汉逊德巴利酵母(Debaryomyces hansenii)。菌株W4682产生的挥发性物质不但可以抑制灰霉菌的生长,对草莓灰霉病的发生也具有显著的防治效果。同时菌株W4682接种草莓后不仅可以在被接种果实上定殖,控制其病害发生,其产生挥发性物质还可以控制同一密闭空间内未接种的草莓上病害的发生。因而,汉逊德巴利酵母菌株W4682具有较好的应用潜力。

如式香肠用发酵剂多菌种混培养技术的研究

以耳式葡萄球菌C131、德巴利汉逊酵母Y163、米酒乳杆菌L04为受试菌株构建发酵剂混培养技术体系,当L04、C131和Y163的起始菌构成比分别为1:2:1、1:4:1,使用加有0.1g/L NaNO2的模拟肉汤培养基,于30℃混培养24、12h,混培养液pH值为4.8和5.6时,总菌数达到最高值,L04对C131弱拮抗作用得到有效控制。将这两种混培养液以3%加入如式香肠结果酸度适中,品质优良。

表面活性剂对D-阿拉伯糖醇发酵的影响

为提高D-阿拉伯糖醇的产量,研究不同类型表面活性剂对德巴利汉逊酵母(Debaryomyces hansenii)发酵生产D-阿拉伯糖醇的影响。结果表明:阳离子和阴离子表面活性剂对D-阿拉伯糖醇的生成几乎没有影响,部分非离子表面活性剂对D-阿拉伯糖醇的生产有促进作用,其中Trition X-100的影响最为显著。在不同发酵时间加入不同浓度的Trition X-100均对D-阿拉伯糖醇的生产有促进作用,当发酵24 h添加30 g/LTrition X-100时,D-阿拉伯糖醇的产量达到最高(92.9 g/L),相比于对照增加了27.2%。

传统凉果盐胚中有害和有益微生物的分离与鉴定

为从凉果盐胚中分离到有害和有益微生物菌株并进行菌种鉴定,评价凉果盐胚食用安全性。通过采取菌株分离,采用稀释平板法从不同食盐质量分数的8种凉果盐胚中分离不同类型菌株,菌种鉴定分别采用显微观察、生理生化指标检测和16 S rDNA基因序列分析,建立菌株的系统发育树。结果表明:分离到细菌32株,真菌7株,并分别对其中的条件致病菌株A鉴定为浅绿气球菌和有益酵母菌株B鉴定为汉逊酵母,供试的凉果盐胚食用存在着安全隐患。

利用氨基酸废水发酵生产饲料用汉逊德巴利酵母的研究

以毛发水解法提取胱氨酸后的废液为氮源,发酵生产饲料酵母。从高盐环境中筛选获得一株高耐盐酵母。通过分子生物学鉴定,证实该菌株为汉逊德巴利酵母,其可用于饲料添加剂。经人工驯化进一步提高其耐盐能力后,通过正交及单因子实验探索其最优生长条件。结果显示,其最佳发酵条件为:氨基酸废水33.3%(v/v),玉米淀粉7%(w/v),K2HPO4 1.5%(w/v),酵母粉0.3%(w/v),FeSO40.6%(w/v);最佳发酵条件如下:培养时间5 d,装液量50 ml/250 ml,初始pH值5.0,种龄4 d,接种量10%(v/v)。此条件下,菌体干重达4.9%(w/v),较优化前提高了48.5%。本研究中分离得到的饲用汉逊德巴利酵母可有效将毛发水解法提取胱氨酸后废水中的营养成分转化为优质饲料用菌体蛋白,具有较高的应用价值。

果汁发酵生产细菌纤维素

为提高果汁发酵生产细菌纤维素的产量,开发特色纤维素功能性食品,以葡糖醋杆菌CGMCC 3917为实验菌种,以苹果汁和梨汁为发酵培养基生产细菌纤维素(BC),研究果汁用量和酵母膏添加量对细菌纤维素产量的影响,比较分析两种果汁生产的细菌纤维素在产量、结构和性质方面的差别。结果表明:梨汁发酵生产的细菌纤维素产量明显高于苹果汁,可达46.343g/100mL;其BC干膜复水率显著高于苹果汁,BC干膜的总糖含量稍高于苹果汁。两种果汁发酵生产的细菌纤维素在湿膜持水量及干膜的纤维素含量、蛋白质含量、脂肪含量以及微观结构上没有明显差异。

氯化锂诱变选育3-羟基丙酸高产菌株

本文以实验室筛选得到的翰逊德巴利酵母菌(Debaryomyces hansenii)DH01为出发菌株,利用氯化锂诱变选育3-羟基丙酸(3HP)产量高并且可以用于实际生产的突变株。经过两轮氯化锂诱变,筛选得到一株产酸量较高的菌株wt06。在培养温度为28℃,120 r/min的条件下,经过48 h培养后,该菌株3HP的产量最高达到23.70 g/L,是原始菌株产量的5.44倍,且该菌株具有良好的遗传稳定性。

基于氮阻遏效应分析汉逊德巴利酵母组胺调控

研究添加L-组氨酸和不添加L-组氨酸两种不同氮源水平情况下,汉逊德巴利酵母(Debaryomyces hansenii)氮代谢阻遏效应调控机制。以添加L-组氨酸为实验组,不添加组为对照,利用Illumina高通量测序平台对两组进行转录组测序,通过多种生物信息学方法对数据进行分析处理。对照组测序结果共得到42.921 6 M clean reads;实验组测序结果共得到41.415 4 M clean reads。两组样品分析显示共有133个显著差异表达基因,78个基因表达上调,50个基因表达下调,基因本体论(Gene Ontology,GO)和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)富集分析表明,差异表达基因参与了组胺代谢相关酶系代谢。本研究为汉逊德巴利酵母组胺代谢进一步研究提供了参考。

聚乙烯醇和海藻酸钠联合固定化汉逊德巴利酵母产3-羟基丙酸的研究

采用微胶囊固定化技术,利用聚乙烯醇、海藻酸钠和氯化钙制备汉逊德巴利酵母微胶囊,探讨了聚乙烯醇、海藻酸钠、氯化钙的浓度对汉逊德巴利酵母微胶囊的制备工艺及其产3-羟基丙酸量的影响。结果表明,随着聚乙烯醇、海藻酸钠和氯化钙浓度的分别增加,3-羟基丙酸含量先增加后减少。聚乙烯醇最佳浓度为10.0%,此时3-羟基丙酸含量为(20.01±0.66)g/L;海藻酸钠为最佳浓度为1.0%,此时3-羟基丙酸含量为(18.71±0.54)g/L;氯化钙最佳浓度为2.1%,此时3-羟基丙酸含量为(18.37±0.45)g/L。聚乙烯醇和海藻酸钠联合固定汉逊德巴利酵母制备微胶囊最佳工艺为10.0%聚乙烯醇,1.0%海藻酸钠和2.1%氯化钙,在此条件下3-羟基丙酸含量为(21.53±1.12)g/L。