火龙果酵素在发酵过程中功能成分变化规律及其与抗氧化相关性

对火龙果酵素发酵487 d过程中总酸、有机酸、总多酚等功能成分的变化及其与抗氧化的相关性进行研究,其中抗氧化能力通过ABTS自由基清除能力和还原力来检测;此外,利用主成分分析法、因子分析法对不同发酵时间的火龙果酵素总酸含量、pH、总多酚含量、ABTS自由基清除能力和还原力5个评价指标进行分析.结果表明:随着发酵时间的延长,火龙果酵素发酵液中总酸、总多酚含量总体上呈逐步增加的趋势,发酵液的pH呈缓慢降低的趋势,从3.75降到3.40;利用高效液相色谱(HPLC)法检测有机酸,从火龙果原料和火龙果酵素发酵液中分别检测到9种和8种有机酸,原料中苹果酸的含量较多,发酵液中乳酸和醋酸的含量较多.与原料相比,酵素发酵液中新检测到了莽草酸;富马酸和抗坏血酸在原料中含量极少,酵素发酵液中则未检测到;火龙果经发酵后,检测到的8种有机酸的含量均有所增加.抗氧化分析结果表明:火龙果酵素发酵过程中ABTS自由基清除能力、还原力总体上呈逐步增加趋势;火龙果酵素发酵过程中总多酚含量、总酸含量、莽草酸和醋酸与ABTS自由基清除能力之间呈中度相关,总多酚含量还原力之间呈中度相关.主成分分析和因子分析结果显示:从检测指标中提取出2个因子,代表了抗氧化因子和环境因子;发酵至359d时,综合评价指标达到最高值且趋于稳定.     

无花果酵素自然发酵过程中代谢产物与抗氧化活性的相关性研究CSCD

为了解无花果自然发酵过程中代谢产物与抗氧化活性之间的相关性,测定其发酵过程中pH、总酸、总糖、总酚、总黄酮、有机酸等理化指标和活性物质,以DPPH自由基、羟基自由基和超氧自由基清除能力及还原力这4个指标考察其体外抗氧化能力,利用主成分分析方法,对代谢产物和抗氧化活性进行相关性分析,并对无花果酵素发酵的各个过程进行综合性评价,确定自然发酵最优的发酵时间。结果表明:随着发酵时间的延长,pH持续降低,总黄酮呈现波动变化,总酸、总酚、DPPH自由基、羟基自由基、超氧自由基清除能力和还原力均呈现先升高后降低的趋势。无花果酵素中检测出了13种主要有机酸,其中含量比较丰富的是乳酸、琥珀酸、苹果酸、柠檬酸、葡萄糖醛酸和泛酸。在发酵第60天时,综合评价值(CEI)最高,发酵80天之后持续下降,以此可以作为无花果酵素前发酵结束的依据,为无花果酵素发酵阶段精准调控奠定相关理论基础。     

龙胆酵素的发酵工艺优化及其生化功效研究

以龙胆草为发酵底物,龙胆苦苷为评价指标,采用正交实验优化龙胆酵素的发酵工艺,用高效液相色谱(HPLC)分析龙胆酵素中龙胆苦苷含量,并评估其生化功效。结果表明,最优龙胆酵素发酵工艺为菌株组合酿酒酵母加植物乳杆菌1∶1、接种量10%、发酵温度30℃、发酵时间72 h。在此优化工艺条件下,发酵液龙胆苦苷含量达到(1 823±21)μg/mL,其透明质酸酶抑制率、DPPH、·OH和O2-·自由基的清除能力分别达到89.2%、77.5%、70.2%和74.1%,表明该工艺的发酵液具有很好的抗敏和自由基清除功效。     

蛹虫草固体发酵大豆基质的成分及抗氧化活性变化研究

以大豆为培养基质,对蛹虫草固体发酵过程中的pH值、淀粉酶、蛋白酶、总糖、还原糖、总酚、ABTS自由基清除率和FRAP进行测定,分析各种物质含量及抗氧化活性变化。结果表明,发酵过程中pH值、淀粉酶和蛋白酶先增加后减少,还原糖含量随着发酵时间延长不断下降,可溶性总糖先减少后增加。总酚含量、ABTS自由基清除率和FRAP值随着发酵时间延长先减少后增加,22d达最大值。发酵成品有望开发成为食品基料及抗氧化食品。     

红曲霉-金钗石斛双向发酵工艺优化

红曲霉(Monascus sp.)代谢产物具有很强的抗氧化性,是天然的抗氧化剂重要来源.金钗石斛是中国传统中药材,具有很多生物活性,结合红曲霉与中草药材双向发酵有望增强其抗氧化能力和改变代谢通路,为探究筛选最佳的金钗石斛-红曲霉发酵条件,提高双向发酵代谢产物的抗氧化活性,采用ABTS法、DPPH法和抗脂质过氧化法三种体外抗氧化模型,通过单因素实验确定影响抗氧化活性的发酵条件,然后设计了四因素三水平正交实验,确定了最优发酵条件为:接种量10％、料水比1∶0.7、培养时间16d和温度(28±1)℃.在此条件下,石斛红曲的脂质过氧化抑制率为69.32％,DPPH自由基清除率为45.65％,ABTS+自由基清除率90.63％,比单独红曲霉发酵代谢产物的抗氧化性分别提高了13.04％,14.10％,29.07％,比金钗石斛的抗氧化性分别提高了42.48％,44.99％,74.63％.可为新型双向发酵的抗氧化产品的开发提供一定的依据.     

混菌发酵芝麻粕制备芝麻多肽及其体外抗氧化活性研究

活性氧自由基(ROS)是细胞有氧代谢过程中产生的一类活性物,利用食源蛋白制备具有抗氧化能力的天然活性肽是当前的研究热点。本研究以机械冷榨芝麻粕为原料,比较了解淀粉芽孢杆菌、植物乳杆菌单菌及其混菌发酵芝麻粕制备芝麻多肽的过程,以DPPH·与·OH清除能力为评价指标,研究其体外抗氧化活性。数据表明,解淀粉芽孢杆菌、植物乳杆菌单菌及其混菌发酵芝麻粕36 h,芝麻多肽提取液中芝麻多肽的浓度分别达到9.841 mg/m L、11.620 mg/m L、14.412 mg/m L,DPPH·清除率分别是71.378%、77.013%、87.622%,·OH清除率分别是68.427%、82.507%、92.460%。因此,芝麻多肽具有较强的抗氧化活性,芝麻粕是制备抗氧化活性肽的良好原料;微生物发酵制备芝麻多肽时,解淀粉芽孢杆菌与植物乳杆菌混菌发酵显著优于单菌发酵,其多肽产量及DPPH·与·OH清除能力均优于相应单菌发酵。     

接种发酵和自然发酵中酿酒酵母菌株多样性比较

【目的】探究自然发酵和接种发酵两种发酵方式,对霞多丽葡萄发酵中酵母菌种多样性和酿酒酵母菌株遗传多样性的影响。【方法】以霞多丽葡萄为原料,分别进行自然发酵和接种不同酿酒酵母菌株(NXU17-26、UCD522和UCD2610)的发酵,利用26S rDNA D1/D2区序列分析和Interdelta指纹图谱技术分别进行酵母菌的种间及种内水平的区分,通过聚类分析及多样性指数对不同发酵方式下酿酒酵母菌株的多样性进行分析和比较。【结果】自然发酵的发酵曲线较平缓,接种发酵的发酵速度显著快于自然发酵。26S rDNA D1/D2区序列分析将4个发酵中分离到的酵母菌鉴定为6属11种,自然发酵中分离的酵母有5属6种,均为非酿酒酵母(non-Saccharomyces);而接种发酵中的酵母多样性远低于自然发酵,均由酿酒酵母和两种非酿酒酵母组成。Interdelta指纹图谱分析表明,接种UCD2610的发酵中,发酵后UCD2610是优势菌株,其基因型占比为48.78%;接种NXU17-26和UCD522的发酵中,未发现与NXU17-26和UCD522相同的基因型。聚类分析表明,分离自接种UCD522发酵中的酿酒酵母菌株间的遗传差异性较小;而分离自NXU17-26和UCD2610发酵中的酿酒酵母菌株间遗传差异性较大。多样性指数结果表明,接种UCD2610发酵中的优势菌株(UCD2610)在发酵过程中占据更加突出的地位;接种UCD522发酵中分离的酿酒酵母具有更高的多样性,影响其菌株多样性的未知因素较多,且不同基因型酿酒酵母的集中度较高。【结论】发酵方式对霞多丽葡萄发酵中酵母菌种多样性、以及酿酒酵母菌株遗传多样性的影响显著,研究结果对葡萄酒发酵中的微生物控制具有指导意义。     

固态发酵苦荞制备多肽菌种的筛选

【目的】筛选固态发酵苦荞高产多肽及发酵产物液具有抗菌、抗氧化活性的菌株。【方法】采用米曲霉、酱油曲霉、雅致放射毛霉和少孢根霉分别对苦荞进行固态发酵,以蛋白酶活力、水解度、可溶性肽得率、抑菌率和体外自由基清除率作为筛菌指标。【结果】米曲霉固态发酵苦荞的可溶性肽得率最高达38.83%±1.18%,发酵产物液对大肠杆菌和金黄色葡萄球菌的抑菌率分别为96.62%±1.66%和97.54%±0.54%,同时羟自由基(·OH)清除率和二苯基苦味酰基苯肼自由基(DPPH·)清除率分别为55.65%±1.25%和10.84%±1.03%。对米曲霉发酵2 d发酵产物液的不同分子量分布及活性分析表明,分子量大小对抗菌及抗氧化活性有一定的影响。【结论】米曲霉可作为固态发酵苦荞制备多肽且发酵产物液具有抗菌及抗氧化活性的最佳菌株,并在多肽产量提升及抗菌、抗氧化活性的研究上具有巨大空间。     

霍山铁皮石斛糖蛋白的制备及抗氧化活性

以霍山铁皮石斛枫斗为试验材料,经脱脂脱色、0.3 mol/L盐溶液浸提、Sevage法脱蛋白、硫酸铵分级沉淀、透析、聚乙二醇10 000浓缩、冷冻干燥得到粗品;经SDS-PAGE电泳、DEAE 52离子柱和Sephadex G-100凝胶柱分离纯化得到糖蛋白并测定其抗氧化活性。SDS-PAGE电泳显示硫酸铵饱和度越大,盐析作用越强,糖蛋白条带越明显,糖蛋白分子量在6.62×10~4附近;DEAE 52离子柱和Sephadex G-100凝胶柱逐步分离纯化得到2个组分的糖蛋白GP1和GP2;分光光度法测定GP1和GP2中糖蛋白含量分别约为95%和87%;抗氧化结果表明,霍山铁皮石斛糖蛋白具有较强的还原能力且可有效地清除自由基·DPPH、·OH和·O~-_2。     

混菌发酵中不同分子量代谢产物对非酿酒酵母胞内蛋白及酒体有机酸的影响

本研究采用透析袋发酵法研究了酿酒酵母产生的不同分子量代谢产物对非酿酒酵母胞内蛋白和酒体有机酸的影响。当截留分子量为3.5 kD和10.0 kD时非酿酒酵母的存活时间分别延长至18 d和22 d,表明通过改变菌种之间物质的交换可以调节非酿酒酵母的存活时间。蛋白质解析发现共有65个蛋白质斑点表达出现差异,占蛋白质总数的13%,经质谱鉴定表明与这些蛋白同类固醇、赖氨酸、有机酸和ATP的生物合成有关。与截留分子量为10 kD的透析袋发酵相比,在截留分子量为3.5 kD的混菌发酵中,酒石酸含量升高5.1%、醋酸含量下降44.3%,说明通过限定菌体间代谢产物的沟通可以调整酒的滴定酸度和挥发酸度。     

Pilot scale vinification process using immobilized Candida stellata cells and Saccharomyces cerevisiae

Sequential grape juice fermentation first with immobilized Candida stellata and then with an inoculum of Saccharomyces cerevisiae was carried out at pilot scale and under non-sterile conditions in order to evaluate the dynamics of yeast microflora and their influence on the analytical profile of wine. Non-Saccharomyces yeast were adequately controlled while S. cerevisiae wild strains were consistently present after 3 days of fermentation and could compete with the inoculated S. cerevisiae strain. However, the metabolism of immobilized C. stellata cells strongly influenced the analytical profile of wines with a consistent increase in glycerol (70%) and succinic acid content in comparison with values for a S. cerevisiae fermentation control.     

表达透明颤菌血红蛋白基因对酿酒酵母生长及细胞内氧化状态的影响*

透明颤菌血红蛋白基因vgb在多种研究和工业发酵菌中异源表达很好的解决了高密度发酵中的溶氧率问题。酿酒酵母是经典的真核模型,且在发酵工业中具有重要的应用价值,但vgb在酿酒酵母中异源表达对细胞生长的影响并不清楚。以ADH1为启动子构建了含透明颤菌(Vistreoscilla)血红蛋白基因vgb的异源表达质粒YEplac195-ADH1pr-vgb,并转化至酿酒酵母BY4741。通过生长敏感性实验,发现在发酵碳源和非发酵碳源中,vgb的异源表达均抑制了菌株生长。接着,通过2',7'-二氯荧光黄双乙酸盐和PI染色和脂质过氧化产物检测分析,发现过表达vgb的酿酒酵母细胞中活性氧(ROS)的积累、细胞膜通透性改变以及脂质过氧化。结果表明,酿酒酵母中过表达vgb改变细胞的氧化状态促进活性氧的累积,氧化应激导致菌株的生长抑制。     

Utilization of amino acids to enhance glutathione production in Saccharomyces cerevisiae

The effects of amino acids on glutathione (GSH) production by Saccharomyces cerevisiae T65 were investigated in this paper. Cysteine was the most important amino acids, which increased intracellular GSH content greatly but inhibited cell growth at the same time. The suitable amino acids addition strategy was two-step addition: in the first step, cysteine was added after two hours culture to 2 mM and then, the three amino acids (glutamic acid, glycine, and serine) were added after seven hours culture. The optimum concentration of those three key amino acids (10 mM glutamic acid, 10 mM glycine, and 10 mM serine) was obtained by orthogonal matrix method. With the optimum amino acids addition strategy a 1.63% intracellular GSH content was obtained in shake flask culture. Intracellular GSH content was 55.2% higher than the experiments without three amino acids addition. The cell biomass and GSH yield were 9.4 g/L and 153.2 mg/L, respectively. Using this amino acids addition strategy in the fed-batch culture of S. cerevisiae T65, GSH content, the biomass, and GSH yield reached 1.41%, 133 g/L, and 1875 mg/L, respectively, after 44 hours fermentation. GSH yield was about 2.67 times as that of amino acids free.     

Respirofermentative metabolism in Kluyveromyces lactis: Ethanol production and the Crabtree effect

Kluyveromyces lactis is a yeast widely used in processes related to milk whey use and lactose fermentation. However, contradictory information about some aspects related to the respirofermentative metabolism of this yeast is found in the literature. We have studied ethanol production and oxygen use in discontinuous and continuous cultures of K. lactis under hypoxic and aerobic conditions. Growth in nonfermentable carbon sources reflects a more efficient respiratory capacity of K. lactis in relation to Saccharomyces cerevisiae; however, in both species, similar glucose fermentation levels under aerobic oxygen-limited conditions are found. Continuous K. lactis cultures in fully oxidative conditions show the oxygen and substrate uptake rates typical of a respiration-unlimited Crabtree-negative yeast; however, a small residual fermentation is present even when respiration is not limited. Some aspects of the Crabtree effect in K. lactis are discussed. The impossibility of including K. lactis in any group of the metabolism-based classification from Alexander and Jeffries (1990) has led us to the formulation of a new group which incorporates the peculiarities of this and other related yeasts.     

A Dictyostelium discoideum DNA fragment complements a Saccharomyces cerevisiae ura3 mutant

Dictyostelium discoideum DNA fragments have been inserted into the chimeric bacterium-yeast plasmid YEp13. Recombinant plasmids were used to transform yeast using a strain of Saccharomyces cerevisiae deficient in OMP decarboxylase activity. Several clones were selected for growth in uracil-free medium. One clone was further analysed and contains a plasmid with a segment of D. discoideum DNA which complements a yeast ura3 mutation.     

Fermentative capability and aroma compound production by yeast strains isolated from Agave tequilana Weber juice

Five yeast strains isolated from agave juice were studied for their fermentative and aromatic capacity. The experiments were performed using agave juice supplemented with ammonium sulphate, as is commonly done in tequila distilleries. Three strains classified as Saccharomyces cerevisiae showed high biomass and ethanol production, as well as higher ethanol tolerance than those classified as Kloeckera africana and Kloeckera apiculata, which showed scarce growth. The results suggest that Kloeckera strains were affected by nutritional limitation and/or toxic compounds present in agave juice. Agave juice analyses showed a lower amino acid content than those reported in grape juice. S. cerevisiae strains produced predominantly amyl and isoamyl alcohols, n-propanol, 2-phenyl ethanol, succinic acid, glycerol, methanol, isoamyl acetate, ethyl hexanoate, acetaldehyde and isobutanol, whereas Kloeckera strains showed a high production of acetic acid, 2-phenyl ethyl acetate and ethyl acetate. The methanol concentration was significantly different among the yeasts studied. The diversity between three S. cerevisiae strains were higher for the aromatic profile than for genetic level and kinetic parameter. On the other hand, the diversity of Kloeckera yeasts were lower than Saccharomyces yeasts even when belonging to two different species.     

Subunit composition of RNA polymerase II from the fission yeast Schizosaccharomyces pombe

The subunit composition of RNA polymerase II (polII) was compared between the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe. For this purpose, we partially purified the enzyme from S. pombe. Judging from the co-elution profiles in column chromatographies of both the RNA polymerase activity and the two large subunit polypeptides (subunit 1 (prokaryotic β' homologue) and subunit 2 (β homologue)), the minimum number of S. pombe polII-associated polypeptides was estimated to be ten, less than the proposed subunit number of the S. cerevisiae enzyme. These ten putative subunits of S. pombe polII correspond to subunits 1, 2, 3, 5, 6, 7, 8, 10, 11 and 12 of the S. cerevisiae counterparts     

Yield improvement of heterologous peptides expressed in yps1-disrupted Saccharomyces cerevisiae strains

Heterologous protein expression levels in Saccharomyces cerevisiae fermentations are highly dependent on the susceptibility to endogenous yeast proteases. Small peptides, such as glucagon and glucagon-like-peptides (GLP-1 and GLP-2), featuring an open structure are particularly accessible for proteolytic degradation during fermentation. Therefore, homogeneous products cannot be obtained. The most sensitive residues are found at basic amino acid residues in the peptide sequence. These heterologous peptides are degraded mainly by the YPS1-encoded aspartic protease, yapsin1, when produced in the yeast. In this article, distinct degradation products were analyzed by HPLC and mass spectrometry, and high yield of the heterologous peptide production has been achieved by the disruption of the YPS1 gene (previously called YAP3). By this technique, high yield continuous fermentation of glucagon in S. cerevisiae is now possible.     

Maximizing production of glutathione by amino acid modulation and high-cell-density fed-batch culture of Saccharomyces cerevisiae

In this paper the high-cell-density fed-batch culture and optimal amino acid modulation were combined together to enhance glutathione production in Saccharomyces cerevisiae T65. Ethanol concentration in the broth was an important parameter for feedback control in fed-batch culture. Low ethanol concentration was propitious to both the cell growth and glutathione synthesis. The feedback control of a low ethanol concentration was an efficient way to realize high-cell-density culture and the biomass reached 140 g/L after 57 h fermentation. With optimal amino acid addition to elevate the glutathione content continually, the maximum glutathione yield achieved 2190 mg/L.     

Interactions between Saccharomyces cerevisiae and lactic acid bacteria in sourdough

The aim of this study was to assess the interactions between Saccharomyces cerevisiae and lactic acid bacteria that either form a stable consortium in Greek wheat sourdoughs (i.e. Lactobacillus sanfranciscensis and L. brevis) or occasionally constitute the secondary microbiota (i.e. Weissella cibaria, L. paralimentarius, Pediococcus pentosaceus and Enterococcus faecium). For this purpose, wheat dough was prepared by using strains of the above mentioned species either as single starters, or in combination of the yeast with each of the lactic acid bacteria strains. The determination of the metabolic products in sourdough samples was performed by HPLC analysis. Presence of lactic acid bacteria had no effect on S. cerevisiae final cell yield but affected negatively the maximum specific growth rate. Ethanol production was primarily affected negatively while the co-culture had a variable effect on glycerol production. On the other hand, the presence of S. cerevisiae favoured mannitol and acetic acid production, had a species-dependent effect on maximum specific growth rate and had no effect on final cfu/g sourdough and lactic acid production by the lactic acid bacteria and at the same time caused the depletion of glucose, fructose and maltose.