不同凝结芽孢杆菌在单一及混合碳源下的发酵特性

本文研究了凝结芽孢杆菌BH1、HL5和36D1在木糖、海藻糖、葡萄糖、低聚异麦芽糖、蔗糖和麦芽糖等单一碳源和葡萄糖分别加其他五种碳源(即混合碳源)下的底物利用和代谢情况,对三株菌以葡萄糖和低聚异麦芽糖作为混合碳源进行5 L罐发酵,进一步考察三株菌发酵特性,以期确定一株菌株作为后续高效乳酸发酵菌株筛选的出发菌株。结果显示:单一碳源发酵下,凝结芽孢杆菌36D1可以完全利用海藻糖、木糖和葡萄糖,而BH1和HL5对木糖和海藻糖几乎无利用,同时三株菌对蔗糖均无明显利用;混合碳源发酵下,凝结芽孢杆菌36D1发酵过程中表现出明显的葡萄糖阻遏效应,BH1和HL5对其他糖(尤其是麦芽糖和异麦芽糖)的利用几乎不受影响;5 L罐发酵结果表明,HL5对异麦芽糖的利用能力较优。凝结芽孢杆菌HL5对麦芽糖、异麦芽糖以及葡萄糖的利用能力较高,最终确定HL5作为后续菌株筛选的出发菌株。     

凝结芽孢杆菌13002高密度培养

本实验旨在通过优化培养条件提高凝结芽孢杆菌13002发酵培养液中的菌体浓度,为生产高光学纯度L-乳酸奠定基础。通过单因素实验,最陡爬坡实验,Box-Behnken响应面实验确定凝结芽孢杆菌13002的最优培养基配方和最佳培养条件,并于自动发酵罐中进行高密度分批补料培养。结果表明,最优培养基配方为:葡萄糖20 g/L、细菌学蛋白胨10 g/L、酵母提取粉25 g/L、Na Cl 10 g/L,初始p H6.5。在此优化条件下,以6%的接种量,培养温度45℃,培养至16 h,以0.5 g/(L·h)速率添加补料至结束,最大菌体浓度达5.399 g/L,活菌总数为3.095×10~9CFU/m L,最终冻干后达0.730×10~(11)CFU/g。     

基于pH下降速率的反馈补料策略高密度培养保加利亚乳杆菌NQ2508

保加利亚乳杆菌是酸奶发酵剂和益生菌制剂的重要菌种,其高密度培养对于提高菌种在应用产品生产中的效率具有重要的现实意义。葡萄糖浓度和发酵pH是影响保加利亚乳杆菌生长的关键因子,底物葡萄糖被转化为细胞菌体和主要代谢产物乳酸,探究葡萄糖消耗速率与表征乳酸生成的发酵pH下降速率的关系,是研究保加利亚乳杆菌代谢流方向的重要方法。该实验通过培养基组成和培养条件对保加利亚乳杆菌生长的影响进行研究,同时对葡萄糖消耗速率-pH降低速率相关模型研究,并基于底物质量守恒定律建立了一条基于pH下降速率的保加利亚乳杆菌高密度培养的反馈补料发酵策略。通过化学中和法控制发酵pH为6.0～5.0,基于pH下降速率进行反馈补料将葡萄糖质量浓度控制在8～2 g/L,发酵液的活菌数达到3.6×10⁹ CFU/mL,是批次分批培养的2.4倍。该策略的应用结果为提高保加利亚乳杆菌的工业化生产效率提供参考。     

不同温度对β-葡聚糖酶分批发酵动力学的影响

在5 L发酵罐中研究不同温度对枯草芽孢杆菌(Bacillus subtilis)ZJF-15菌体生长、底物消耗和产酶量的影响。分批发酵的工艺条件为:搅拌转速400 r/min、通气量1.0 L/(L·min)、p H 7.0、接种量5%、装液系数0.6、发酵温度35~37℃。结果表明:温度升高会导致反应速率加大、生长代谢加快、生产期提前、酶失活速率加快、发酵周期缩短、最终产酶量减少。实验建立了B.subtilis ZJF-15分批发酵的菌体生长、底物消耗和β-葡聚糖酶产酶的动力学方程。     

培养基成分对酸土脂环酸芽孢杆菌生长及芽孢形成的影响

本研究通过单因素和双因素交互实验,研究多种营养因子对酸土脂环酸芽孢杆菌生长和芽孢形成的影响。结果表明,葡萄糖、酵母浸粉、Mn2+、Mg2+对酸土脂环酸芽孢杆菌的菌体生长和芽孢形成影响显著(p<0.05)。其中高浓度的Mn2+对酸土脂环酸芽孢杆菌的菌体生长有抑制作用,但能促进其芽孢形成。而Ca2+、NH₄+和K+单一作用时,对其菌体生长和芽孢形成影响不显著(p>0.05),但Ca2+与K+或者NH₄+协同作用时,可以促进酸土脂环酸芽孢杆菌的菌体生长和芽孢形成。研究结果揭示了培养基中的营养成分对该菌菌体生长和芽孢形成的影响,为控制该菌芽孢污染及酸性果蔬产品保藏提供实验和理论依据。     

植物乳杆菌发酵动力学及高密度培养研究

为提高具有益生特性植物乳杆菌的产量,基于发酵动力学模拟,对其培养基组成、发酵条件和补料方式进行了优化。在该研究中,通过建立人工神经网络(artificial neural network, ANN)和遗传算法(genetic algorithm, GA)结合的智能模型对培养基成分进行优化,结果为碳源36.64 g/L(葡萄糖-麦芽糖质量比2∶3)、氮源47.83 g/L(酵母粉-蛋白胨质量比1∶1)、柠檬酸二铵33.27 g/L。发酵动力学采用Logistic和Luedeking-Piret模型,对植物乳杆菌的发酵过程进行拟合,菌体生长、底物消耗和产物生成动力学模型相关系数R²分别为0.995,0.998,0.993,表明这些模型能够精确的模拟植物乳杆菌的发酵过程。此外,对培养条件和分批补料进行了优化,最佳培养条件为温度35℃,初始pH值5.0,接种量4%。在分批添加中和剂NH₃·H₂O和葡萄糖30 g/L的情况下,菌体产量可达12.64 g/L。     

蜡样芽孢杆菌高密度发酵条件与过程的优化

蜡样芽孢杆菌的生物量对其生物功能具有重要影响。为了提高蜡样芽孢杆菌的产量与芽孢率,通过单因素实验筛选出最适宜其生长的碳源与氮源,分别为质量比1∶1复配的葡萄糖与水溶性淀粉和质量比1∶1复配的大豆蛋白胨与酵母提取物。在此基础上,于7 L发酵罐中探究流加方式、p H、搅拌转速和通气量等因素对其生物量及芽孢率的影响。最优发酵条件为：在0～9 h之间,转速250 r/min,通气量3 L/min,pH恒定6.5;9～18 h时,转速升高至350 r/min,通气量升高至4.5 L/min,并以0.45 mL/min流量补加培养基浓缩液;18 h时,转速降低至150 r/min,通气量降低至2.25 L/min,pH调高至7.5,停止补料至发酵结束。基于以上发酵策略,在18 h时,蜡样芽孢杆菌活菌数达2.2×10¹⁰CFU/mL,是未优化前的4.88倍;发酵结束时,芽孢率超过90%。本研究结果为蜡样芽孢杆菌的工业化应用提供了一定基础。     

石斛汁乳酸发酵饮料的研制

研究了凝结芽孢杆菌、植物乳杆菌和鼠李糖乳杆菌在石斛汁中的生长规律,及发酵过程中的总酸、还原糖、p H值、活菌数、氨基态氮含量变化和之间的内在联系。从活菌数、产酸速度、产品的感官、风味等方面进行了评价,筛选出适用于发酵石斛汁的乳酸菌种。研究表明,发酵48 h后,凝结芽孢杆菌、植物乳杆菌、鼠李糖乳杆菌发酵石斛汁的酸度(以乳酸计)分别达到7.85、6.93和7.66 mg/m L;还原糖含量分别减少了78.64%、67.28%和76.85%;从产酸能力和产酸速度来说,凝结芽孢杆菌、鼠李糖乳杆菌要优于植物乳杆菌。     

凝结芽孢杆菌抗葡萄糖分解代谢物阻遏突变株合成培养基的筛选和发酵特性研究

以实验室前期选育的耐高温菌凝结芽孢杆菌Bacillus coagulans HL5-C以及其突变株5E-1为研究对象,筛选适合两菌株生长和代谢的合成培养基并对其发酵特性进行研究。单因素添加实验结果表明,合成培养基中尿嘧啶是影响HL5-C生长的关键物质,而赖氨酸、脯氨酸和苯丙氨酸则是影响5E-1生长的关键因素,由此分别得到了适合于HL5-C和5E-1生长的合成培养基MCDM4和MCDM5。在此基础上,考察了HL5-C和5E-1在单一碳源和混合碳源条件下的发酵情况,结果表明当非速效碳源与葡萄糖共存时,出发菌株HL5-C优先利用葡萄糖,其他糖的利用被延滞,即出现了碳源分解代谢物阻遏效应(carbon catabolite repression,CCR);突变株5E-1在利用葡萄糖的同时还利用非速效碳源生产乳酸,说明5E-1能消除碳源利用顺序的差异,即5E-1能有效解除或缓解葡萄糖引起的分解代谢物阻遏。本文研究结果为后续凝结芽孢杆菌葡萄糖分解代谢物阻遏效应机制研究奠定了基础。     

不同发酵条件对模拟葡萄酒中酒酒球菌柠檬酸代谢的影响

以酒酒球菌31-DH进行模拟酒苹果酸-乳酸发酵,使用离子排斥色谱法测定相关代谢物质的含量,研究苹果酸-乳酸发酵期间及发酵后p H、乙醇和葡萄糖对柠檬酸代谢的影响。结果表明:该方法耗时短、前处理简单、分离度良好、回收率为87%~108%、精密度为0.77%~4.33%,满足分析要求。当发酵液处于低p H,高酒精度,低葡萄糖浓度时,柠檬酸代谢缓慢,乳酸、乙酸及2,3-丁二醇的产量较低,双乙酰含量峰值较高。当发酵液中不含有乙醇时,苹果酸、柠檬酸、葡萄糖代谢迅速,伴随菌体的大量生长,乳酸、乙酸、2,3-丁二醇含量较高,但无双乙酰的产生。当发酵液中不含有葡萄糖时,柠檬酸代谢速率加快,乳酸产量较低,2,3-丁二醇及双乙酰产量较高。p H、乙醇、葡萄糖均显著影响酒酒球菌31-DH对柠檬酸的代谢。     

Open L-lactic acid fermentation of food refuse using thermophilic Bacillus coagulans and fluorescence in situ hybridization analysis of microflora

In the production of commercially useful poly-L-lactic acid plastic from biomass wastes, a feasible fermentation process to produce optically active L-lactic acid would be required. Here, model kitchen refuse (MKR) was inoculated with Bacillus coagulans NBRC12583 under nonsterilized openculture conditions. At temperatures below 45 degrees C, a racemic mixture of D- and L-lactic acids was accumulated, whereas only L-lactic acid was selectively accumulated by incubation at 50-65 degrees C. At 45 degrees C, the results of fermentation could not be consistently reproduced. To analyze microflora in this type of mixed culture system, whole-cell fluorescence in situ hybridization (FISH) using 16S rRNA-targeted oligonucleotide probes for B. coagulans, Bcoa191, and LAC722(L), a group-specific probe for a wide range of mesophilic lactic acid bacteria was applied. The dominancy of mesophilic lactic acid bacteria at lower temperatures, and that of B. coagulans at higher temperatures were confirmed. By using a saccharified liquid of collected kitchen refuse, 86 g/l of L-lactic acid was accumulated under nonsterile conditions by a 5-d incubation at 55 degrees C, pH 6.5, with 53% carbon yield and 97% optical purity. To conclude, high temperature open lactic acid fermentation is a simple and promising method for producing high-grade L-lactic acid from biomass waste, and FISH analysis of such mixed-culture systems is helpful for monitoring the microflora in these cultures.     

利用一株凝结芽孢杆菌发酵酸解玉米秸秆生产乳酸

该研究拟考察凝结芽孢杆菌（Bacillus coagulans）利用玉米秸秆生物炼制乳酸的效果。以凝结芽孢杆菌CGMCC No.7635为菌种,利用2%硫酸预处理后的玉米秸秆为碳源、20 g/L酵母粉为氮源,添加20 FPU/g纤维素酶后开展糖化发酵生产乳酸实验。结果表明,发酵65 h后可获得乳酸含量为（38.38±1.03） g/L,其中L-乳酸光学纯度为（99.23±0.22）%。进一步使用补料发酵工艺,添加经预处理的玉米秸秆,可最终获得乳酸含量为（82.56±1.28） g/L。建立的玉米秸秆生物炼制乳酸工艺操作简单、产物浓度高,具有工业应用潜力。     

凝结芽孢杆菌中与乳酸生产相关的乳酸脱氢酶基因的研究

对5株凝结芽孢杆菌乳酸生产情况进行研究,并以凝结芽孢杆菌ATCC7050作为乳酸脱氢酶基因研究的对象,确定得出其乳酸生产关键的乳酸脱氢酶基因。结果表明:凝结芽孢杆菌生产的乳酸分两种类型:L型乳酸和D型乳酸,但主要以L型乳酸为主,其光学纯度达到97%以上。在凝结芽孢杆菌ATCC7050基因组中存在的ldhL1、ldhL2和ldhD三种乳酸脱氢酶基因中,ldhL2基因没有检测到转录信号,而ldhD转录水平很低,在生长对数期ldhL1基因的转录水平是ldhD基因转录水平的68倍,这说明ldhL1基因是凝结芽孢杆菌ATCC7050乳酸生产的关键性基因。本研究确定了凝结芽孢杆菌ATCC7050乳酸合成中最主要的乳酸脱氢酶基因,对凝结芽孢杆菌代谢研究和基因改造奠定了基础。     

pH and dose-dependent effects of quercetin on the fermentation capacity of Lactobacillus plantarum

This article reports a study of how quercetin affects the capacity of Lactobacillus plantarum RM71 to ferment different media, including a chemically defined medium (CDM) and media relevant for practical fermentation processes. It is shown for the first time that quercetin exerts pH- and dose-dependent effects on the fermentation performance of L. plantarum. At an initial pH of 5.5, quercetin promoted quicker growth upon inoculation at increased quercetin concentrations, while a detrimental dose-dependent lengthening of the lag phase was observed at an initial pH of 6.5. The time course of sugar consumption and lactic acid production data tracking in pH 5.5 CDM showed that quercetin promoted quicker sugar consumption as a result of earlier sugar uptake and lactic acid production than in the control. A model wine and a similar medium with modified sugar composition were fermented with L. plantarum RM71 on quercetin. Quercetin improved several key fermentation traits for the performance of L. plantarum in food production, including accelerated fermentation of various sugars, and accelerated malolactic fermentation and lactic acid production. Quercetin was not catabolized by L. plantarum in the fermentations, so the antioxidant properties of the flavonol were protected against degradation while the bacterium improved its growth performance.     

pH值对乳酸菌生长和乳酸产量的影响

研究了分别用HCl和乳酸调节培养基初始pH以及在发酵过程中加入不同的酸中和剂对乳酸菌生长和乳酸产量的影响。结果表明 ,终产物乳酸的积累是抑制乳酸菌生长和导致乳酸产量下降的直接原因 ,该情况随发酵液中H+浓度的升高 (即pH的降低 )而增强 ,H+的积累对菌体生长和产酸的影响是间接的 ;用CaCO3 调节发酵液pH优于用NaOH和NH4OH。     

乳酸菌混菌发酵制备豆清液多肽动力学模型的建立

为探究乳酸菌混菌发酵豆清液制备多肽的动态过程及钙螯合活性变化，建立描述菌体生长、多肽生成及蛋白质消耗动力学模型，测定了发酵过程中的pH值、蛋白酶酶活和钙螯合活力。结果表明，动力学模型具有良好可靠性；乳酸菌混菌发酵过程中多肽含量在对数期达到5.31 mg/mL，pH值下降至3.5左右后稳定；酸性蛋白酶酶活在4～8 h较高，最大酶活为3.6 U/mL；钙螯合活力总趋势为先升高后降低，在14 h达到最高值33.35%。     

Production of antimicrobial metabolites by strains of Lactobacillus or Lactococcus co-cultured with Bacillus cereus in milk

During co-culture of Lactobacillus (five strains) or Lactococcus (two strains) with Bacillus cereus, organic acids and other potentially antimicrobial metabolites are produced. Lactic acid was produced at very different rates by the lactic acid bacteria (LAB) and the final concentrations varied much, however, the crucial point of rapid pH reduction during the initial hours of fermentation coincides with lactic acid production. Moderate amounts of acetic acid were produced during fermentation and the final concentrations were much smaller compared to lactic acid. According to these experiments, production of diacetyl, carbon dioxide and ethanol was considered too small to contribute to inhibition of B. cereus. The inhibitory substance produced by the LAB strains was not sensitive to proteinase K, trypsin or pepsin, so it was not likely that the LAB strains produced bacteriocins antagonistic against B. cereus. The strains that produced lactic acid fastest inhibited B. cereus best. Increased concentrations of lactic and acetic acid and carbon dioxide were also observed after co-culture with B. cereus compared to growth of the LAB strains alone, which indicates that B. cereus stimulates the biosynthetic capacities of the LAB strains.     

Effect of different cultivation conditions on Lactobacillus manihotivorans OND32T, an amylolytic lactobacillus isolated from sour starch cassava fermentation

Study of the cassava sour starch fermentation has led to the isolation of a new homofermentative amylolytic lactic acid bacterium, Lactobacillus manihotivorans OND32T, whose nutritional requirements have been investigated in this work. The main effect of deleting one of the substrate components of the MRS-starch medium was to reduce the amylase production. When starch fermentation with nitrogen as a gas phase was compared to fermentation under aerobic conditions, both growth and amylase production were reduced whereas lactic acid formation was not affected. Addition of carbon dioxide (> or = 20% v/v) to the nitrogen gas phase restored growth and amylase production. The amylase production was high with starch, maltose or cellobiose contrary to glucose, fructose and sucrose. During mixed fermentation of glucose and maltose, a diauxic growth was observed. The maltose consumption and the amylase production started after the glucose depletion. The presence of maltose altered the carbon assimilation from glucose, whereas the energetic pathway was not affected. It is concluded that the elimination of soluble sugars by the wet extraction of starch during the processing of cassava, together with the expected in situ CO2 production, are conditions favouring the growth and the amylase synthesis. However, these are likely to be limited by the low nitrogen content in cassava.