藏灵菇源克鲁维酵母M3菌株胆盐水解酶的分离纯化研究

为获得藏灵菇马克斯克鲁维酵母M3菌株胆盐水解酶(BSH)的纯品,探讨了BSH粗品的分离纯化方法.粗酶液采用硫酸铵分级沉淀,再以DEAE-Sepharose Fast Flow离子交换介质,分别考察缓冲液pH值、流速和洗脱方式等对BSH分离纯化的影响.结果表明,在40%～70%饱和度的硫酸铵条件下BSH提取效率最高.最佳层析条件为采用pH值为6.5、50mmol/L磷酸钠缓冲体系、1.5mL/min的流速,进行分步洗脱(100mmol/L、350mmol/L、500mmol/L～600mmol/L NaCl及50mmol/L磷酸盐缓冲液3步洗脱).在优化纯化条件下BSH的比活力可达479.55AU/mg,是原粗酶液的23.66倍.     

藏灵菇源克鲁维酵母K1菌株胆盐水解酶的特性研究

探讨藏灵菇马克斯克鲁维酵母K1菌株胆盐水解酶作用底物的反应条件与不同化学试剂对酶活性的影响及其发酵动力学类型。采用单因素多水平试验方法,在pH值4～8、温度31℃～43℃、底物浓度4mmol/L～8mmol/L及不同化学试剂(SDS、EDTA、尿素、Cu2+、Mg2+、Ca2+、Fe3+、Al3+、Mn2+)的条件下,胆盐水解酶与底物反应30min,检测酶活力;平板活菌计数法分析细胞生长与产酶的关系;双倒数作图法求得酶促反应动力学常数Km。结果表明,胆盐水解酶最适反应条件为:pH值为6.0、底物浓度7mmol/L、温度37℃,Fe3+、Ca2+、Mn2+及尿素对酶活性有较大提高作用,Mg2+、Al3+及SDS对酶的激活作用次之,Cu2+及EDTA对酶活性影响不大。K1菌株在18h～21h进入稳定期,于21h对数生长期的末期时酶活性达到最高,表明其胆盐水解酶发酵动力学类型为生长偶联合成型。其酶促反应动力学常数Km为2.10mmol/L,说明该酶与最适底物的亲和力较大。     

藏灵菇源酸奶复合菌发酵剂对肠道菌群平衡的作用

目的:探讨藏灵菇源酸奶复合菌发酵剂对实验性高胆固醇血症大鼠调节肠道菌群平衡的作用.方法:根据大鼠血清总胆固醇水平分组.高脂模型组和试验Ⅰ、Ⅱ组饲喂高脂饲料,对照组饲喂基础饲料;试验Ⅰ组灌胃脱脂酸奶复合菌发酵剂,试验Ⅱ组灌胃全脂酸奶复合菌发酵剂,对照组和模型组给予蒸馏水,连续8周.结果:对照组大鼠的粪便中乳杆菌、肠杆菌和产气荚膜梭菌的活菌数量处于平稳状态;4周后高脂模型组大鼠的粪便中乳杆菌的活菌数量极显著低于对照组(p＜0.01),而6周后肠杆菌和产气荚膜梭菌的活菌数量极显著高于对照组(p＜0.01);试验Ⅰ组和试验Ⅱ组大鼠粪便中乳杆菌的活菌数量分别在4周和6周开始极显著高于高脂模型组(p＜0.01),而肠杆菌和产气荚膜梭菌的活菌数量极显著低于高脂模型组(p＜0.01);试验Ⅰ组调节肠道菌群平衡效果优于试验Ⅱ组.结论:藏灵菇源酸奶复合菌发酵剂能够促进高胆固醇血症大鼠肠道有益菌的生长,并抑制有害菌的繁殖,具有明显调节肠道菌群平衡作用.     

藏灵菇及其优势菌群的发酵性能研究

以藏灵菇为发酵剂生产酸乳的工艺条件为:发酵温度37℃,发酵时间10h。获得的酸乳产品pH较低。用MRS培养基、Elliker培养基、TYC培养基和SL培养基从藏灵菇中分离出乳球菌、乳杆菌、链球菌和明串珠菌。将优选的分离菌组合发酵鲜牛乳,产酸速度更快,黏度较高。     

克鲁维酵母菌产胆盐水解酶发酵条件的优化研究

利用从藏灵菇中筛选的产胆盐水解酶(bile salt hydrolase,BSH)的马克斯克鲁维酵母菌K1(Kluyveromyces marxianus K1)研究提高酶活力的环境因素。针对主要影响胆盐水解酶合成的4个因素,采用4因素3水平L9(34)正交试验确定了高产胆盐水解酶优化发酵条件:发酵温度为32℃,发酵时间为24h,培养基的初始pH值为6.0,接种量为2%;在优化发酵条件下,K1菌株产BSH活力比优化前提高了52.4%,为开发研制降胆固醇功能食品及微生态制剂提供了试验基础。     

藏灵菇耐胃肠道环境酵母菌和乳酸菌的筛选

以藏灵菇发酵乳液为原料,分别利用改良的马铃薯乳糖培养基和改良MRS培养基分离出8株酵母菌和10株乳酸菌。以pH值为3.0,质量分数为0.3%牛胆盐的培养液模拟胃肠道环境作为初筛条件,以不同pH值和牛胆盐质量分数作为耐酸和耐胆盐的复筛条件,筛选得到耐受性好活力高的两株酵母菌C3和C8,两株乳酸菌M1和M3。     

藏灵菇牦牛酸乳发酵过程中乳酸菌和酵母菌对品质的作用

藏灵菇酸乳是由乳酸菌、酵母菌、醋杆菌和霉菌共同发酵形成的一种发酵乳制品,其品质特征与普通酸乳有显著差异。为研究藏灵菇发酵酸乳中乳酸菌和酵母菌分别发挥的作用,利用放线菌酮和青链霉素分别抑制发酵过程中的酵母菌和乳酸菌,测定并比较正常发酵酸乳、抑制乳酸菌发酵酸乳和抑制酵母菌发酵酸乳的风味、口感和质构等相关指标。实验结果显示抑制酵母菌发酵的酸乳中乙醇含量由1.85g/kg上升为2.77g/kg,VB2含量由168μg/100g下降为157μg/100g,与正常发酵组差异较大;抑制乳酸菌发酵的酸乳中酸度由18°T上升为28°T,氨基酸态氮由65mg/100g下降为53mg/100g,乳酸含量持续2mg/g,乙酸由1mg/g下降为0.7mg/g,VB1含量由20μg/100g下降为18μg/100g,与正常发酵组差异较大。乳酸菌在酸乳发酵过程中,对酸度、氨基酸态氮、质构、乳酸、乙酸与VB1的生成贡献较大;酵母菌对乙醇、VB2的生成贡献较大。     

藏灵菇中醋酸菌和酵母菌的分离及初步鉴定

结合藏灵菇扫描电镜结果,从藏灵菇叶中分离出1株醋酸菌和4株酵母菌依据其彤态、培养特征和部分生理牛化研究,初步鉴定醋酸菌为巴氏醋杆菌（Acetobacter pasteurianus）,4株酵母菌分别为酒香酵母属（Brettanomyces）、类酵母属（Saccharomycodes）和酵母属（Saccharomycs）的菌株。     

藏灵菇源酵母菌和乳酸菌降胆固醇蛋乳发酵饮料的研制

利用藏灵菇中筛选的马克斯克鲁维酵母菌(Kluyveromyces marxianus) M3、干酪乳杆菌(Lactobacillus casei) KL1、嗜热链球菌(Streptococcus thermoptilus) Tx、乳酸乳球菌乳酸亚种(Lactococcus lactis sub sp.lactis) KS4混合发酵,以鸡蛋和牛奶为原料,研制降胆固醇蛋乳发酵饮料.利用KL1、Tx、KS4这3株乳酸菌进行一次发酵,再利用酵母菌进行二次发酵.采用3因素3水平正交试验优化发酵条件为发酵温度34℃,接种量1％,发酵时间24h.在优化条件下,蛋乳发酵饮料的降低胆固醇率为74.99％,显示藏灵菇源酵母菌M3菌株具有良好的降胆固醇作用.     

藏灵菇酵母菌K1耐胃肠道逆环境特性及降胆固醇的实验研究

利用从藏灵菇中筛选的马克斯克鲁维酵母K1菌株探讨胃肠道耐受性及体外降胆固醇功效。分别在pH 1.5~4.5、胆盐浓度0.3%~3.0%、NaCl浓度3.0%~6.0%的模拟胃肠道环境下,采用活菌计数法检测K1菌株的耐胃肠道逆环境特性,以邻本二甲醛法检测其降胆固醇效果。结果表明,在pH2.5~4.5作用4h活菌数超过106cfu/mL,0.3%~3.0%胆盐及3.0%~6.0%NaCl作用8h活菌数为104cfu/mL~106cfu/mL,在pH1.5~3.0(4h)及胆盐浓度0.3%~3.0%的模拟人体胃肠道环境下降胆固醇率40.0%~52.8%。显示K1菌株具有较强的耐胃肠道逆环境特性及良好的降胆固醇能力。     

Bioinformatic characterization of the extracellular lipases from Kluyveromyces marxianus

Lipases are hydrolytic enzymes that break the ester bonds of triglycerides, generating free fatty acids and glycerol. Extracellular lipase activity has been reported for the nonconventional yeast Kluyveromyces marxianus, grown in olive oil as a substrate, and the presence of at least eight putative lipases has been detected in its genome. However, to date, there is no experimental evidence on the physiological role of the putative lipases nor their structural and catalytic properties. In this study, a bioinformatic analysis of the genes of the putative lipases from K. marxianus L-2029 was performed, particularly identifying and characterizing the extracellular expected enzymes, due to their biotechnological relevance. The amino acid sequence of 10 putative lipases, obtained by in silico translation, ranged between 389 and 773 amino acids. Two of the analysed putative proteins showed a signal peptide, 25 and 33 amino acids long for KmYJR107Wp and KmLIP3p, and a molecular weight of 44.53 and 58.23 kDa, respectively. The amino acid alignment of KmLIP3p and KmYJR107Wp with the crystallized lipases from a patatin and the YlLip2 lipase from Yarrowia lipolytica, respectively, revealed the presence of the hydrolase characteristic motifs. From the 3D models of putative extracellular K. marxianus L-2029 lipases, the conserved pentapeptide of each was determined, being GTSMG for KmLIP3p and GHSLG for KmYJR107Wp; besides, the genes of these two enzymes (LIP3 and YJR107W) are apparently regulated by oleate response elements. The phylogenetic analysis of all K. marxianus lipases revealed evolutionary affinities with lipases from abH15.03, abH23.01, and abH23.02 families.     

马克斯克鲁维酵母对发酵乳中糖代谢的影响

采用高效液相色谱等方法,对乳酸菌单独发酵和向其中添加马克斯克鲁维酵母这两种发酵乳中乳糖代谢主要产物及关键酶活力的变化情况进行对比分析,以确定添加酵母菌对乳糖无氧代谢产生乳酸途径的影响。结果表明：添加酵母菌后乳糖降解速率明显加快（P＜0.05）,贮藏期间马克斯克鲁维酵母可以对积累的半乳糖进行利用;发酵过程中,由于酵母菌的添加使β-半乳糖苷酶及乳酸脱氢酶活力有显著提高（P＜0.05）,糖酵解途径关键限速酶--己糖激酶和丙酮酸激酶活力增加（P＜0.05）;含有酵母菌的发酵乳pH值下降（滴定酸度上升）较乳酸菌单菌发酵快（P＜0.05）,这与添加酵母菌后发酵乳中乳酸含量显著增加（P＜0.05）有关;丙酮酸含量变化不显著（P＞0.05）。该研究揭示了马克斯克鲁维酵母的添加对乳糖酵解具有一定促进作用。     

Enzymatic Production of Ribonucleotides from Autolysates of Kluyveromyces marxianus Grown on Whey

After 20h fermentation of medium containing 5% (w/v) dehydrated whey, at 30°C, pH 4.5, yeast cells were harvested, diluted in 0.1M KH₂PO₄, and autolyzed at different pHs (6.5–7.5) and temperatures (45–55°C). Phosphodiesterase (0.2–1.0% w/v, 65°C, pH 6.5, 6h) and adenyl deaminase (0.5-1.0% w/v, 60°C, pH 5.5, 4h) were added to the autolysates. After heat treatment (100°C, 15 min), samples were analyzed by RP-HPLC and LC/MS. Production of 5′-ribonucleotides was maximized at 50°C, pH 6.5. Yields of 5′-AMP (800 μg/g of biomass) and 5′-GMP (2000 μg/g) increased considerably after addition of 1.0% phosphodiesterase. 5′-IMP increased only after addition of 1.0% adenyl deaminase.     

雪莲菌中乳酸菌的益生特性

对分离自雪莲菌的乳酸菌进行益生特性评价。耐酸性和模拟胃肠液耐受性初筛结果表明,20株乳酸菌中有11株乳酸菌在pH 3.0和pH 2.5的培养基中具有耐受性,经过模拟胃肠液培养后有8株乳酸菌的活菌数大于10⁶ CFU/mL;疏水性、自凝聚性、抗氧化能力和抑菌实验结果表明,副干酪乳杆菌(Lactobacillus paracasei)KF2-5和鼠李糖乳杆菌(Lactobacillus rhamnosus)RG疏水性较高、抑菌性最强,菌株RG的自凝聚能力和抗氧化能力较强;耐药性实验结果显示,菌株KF2-5和RG对红霉素、卡那霉素、氯霉素和四环素敏感,对头孢噻肟和万古霉素具有耐药性;溶血性实验表明,2株菌均无溶血。本研究筛选得到2株具有益生特性的乳酸菌KF2-5和RG,在益生产品开发中具有潜在的应用前景。     

一株来自河南的雪莲菌TK3优势菌相分析

对一株来自河南省的雪莲菌TK3进行初步菌相分析,扫描电镜结果表明：TK3是由乳酸菌及酵母菌构成的混菌共生体系,菌体附着在基质上,形成紧密不易分离的颗粒。对其菌相进行分离纯化得到6种优势菌,经镜检分析、生理生化分析及16S rDNA/26S rDNA鉴定,鉴定结果分别为：乳明串珠菌(Leuconostoc lactis)、乳酸乳球菌(Lactococcus lactis)、植物乳杆菌(Lactobacillus plantarum)、单孢酿酒酵母(Kazachstania unispora)、马克斯克鲁维酵母(Kluyveromyces marxianus)以及exigua酿酒酵母(Kazachstania exigua),在发酵乳中存在的数量级分别为107、107、107、106、106、107CFU/mL,并且在4℃存放7d后各个菌群数量仍保持稳定。     

Induction and characterization of a novel amine oxidase from the yeast Kluyveromyces marxianus

An amine oxidase from the yeast Kluyveromyces marxianus was induced, purified and completely characterized; it was shown to belong to the class of copper-containing amine oxidases (E.C. 1.4.3.6). The enzyme was induced by putrescine and, very strongly, by copper(II); structural-functional characterization of the enzyme was performed, including determination of molecular weight, glycosylation, copper and TPQ content, isoelectric point, K(M) and k(CAT) (with benzylamine as substrate), pH, temperature and ionic strength effect on catalysis, substrate and inhibitor specificity. A 700 bp clone was isolated containing the cDNA that encodes for the C-terminus of the enzyme; the amino acid sequence deduced (the first available for a benzylamine oxidase from yeast) was compared to that of other copper amine oxidases from microorganisms and higher organisms. From the results obtained, the putrescine/benzylamine oxidase from Kluyveromyces marxianus was found to have a good homology with other enzymes of this class from microorganisms, and particularly with AO I from Aspergillus niger. Nonetheless, some features resulted closer to those of animal amine oxidases and histaminases. Some potential biotechnological applications are proposed. The cDNA Accession No. is AJ320485.     

‘平阳黄汤’对高脂饮食大鼠肠道屏障和肠道菌群的影响

为探讨‘平阳黄汤’对高脂饮食大鼠的肥胖调控作用及其与肠道屏障和菌群的关系,将SD雄性大鼠分为正常饮食组、高脂饮食组以及平阳黄汤低、中、高剂量组（75、150、300 mg/kg mb）,建立预防肥胖模型。通过分析大鼠体质量变化、摄食量、脂肪指数、血清指标、肝脏指标和肝脏、结肠组织切片评估‘平阳黄汤’对高脂饮食大鼠的肥胖调控作用;通过分析肠道的病理形态、杯状细胞数量和紧密连接蛋白的表达评估肠道屏障作用;通过分析微生物多样性和菌群结构评估对肠道菌群的影响。结果：高剂量‘平阳黄汤’能极显著降低高脂饮食大鼠的体质量增长量、附睾和肾周脂肪质量（P＜0.01）,降低血清血脂、脂肪因子和氧化应激水平（P＜0.01）,降低肝脏甘油三酯和总胆固醇水平（P＜0.01）,有效调节肝脏的脂质堆积和炎症损伤。同时,高剂量平阳黄汤还显著改善了高脂饮食大鼠结肠的形态,减少绒毛脱落和炎症浸润现象,显著恢复肠道杯状细胞数量,提高闭锁小带蛋白和闭锁蛋白的表达（P＜0.01）,有效降低了血清中脂多糖、肿瘤坏死因子α和白细胞介素6水平（P＜0.01）,有效预防代谢性内毒素和全身慢性炎症,提高了大鼠肠道菌群的丰度和多样性,促进了与肥胖发生相关有益菌的增殖,抑制有害菌的生长。结论：‘平阳黄汤’能通过维护肠道屏障和调节肠道菌群紊乱,有效调控高脂饮食引起的肥胖。     

Influence of carbon and nitrogen source on production of volatile fragrance and flavour metabolites by the yeast Kluyveromyces marxianus

The yeast Kluyveromyces marxianus produces a range of volatile molecules with applications as fragrances or flavours. The purpose of this study was to establish how nutritional conditions influence the production of these metabolites. Four strains were grown on synthetic media, using a variety of carbon and nitrogen sources and volatile metabolites analysed using gas chromatography–mass spectrometry (GC–MS). The nitrogen source had pronounced effects on metabolite production: levels of the fusel alcohols 2‐phenylethanol and isoamyl alcohol were highest when yeast extract was the nitrogen source, and ammonium had a strong repressing effect on production of 2‐phenylethyl acetate. In contrast, the nitrogen source did not affect production of isoamyl acetate or ethyl acetate, indicating that more than one alcohol acetyl transferase activity is present in K. marxianus. Production of all acetate esters was low when cells were growing on lactose (as opposed to glucose or fructose), with a lower intracellular pool of acetyl CoA being one explanation for this observation. Bioinformatic and phylogenetic analysis of the known yeast alcohol acetyl transferases ATF1 and ATF2 suggests that the ancestral protein Atf2p may not be involved in synthesis of volatile acetate esters in K. marxianus, and raises interesting questions as to what other genes encode this activity in non‐Saccharomyces yeasts. Identification of all the genes involved in ester synthesis will be important for development of the K. marxianus platform for flavour and fragrance production. Copyright © 2014 John Wiley & Sons, Ltd.