丙酸高产菌株的复合诱变选育

以费氏丙酸菌IFFI.10019为出发菌株,经2次紫外线、2次亚硝基胍、1次亚硝基胍-氯化锂多重复合诱变处理,选育获得丙酸高产菌株NL—3,丙酸产量由原来的0.20g/L,提高到1.23g/L,提高率达到515%。实验证明采用多因子复合诱变,特别是NTG-LiCl复合诱变,能有效改变菌株对诱变因素的敏感性,提高变异率,逐步提高突变株的产丙酸水平。     

丙酸菌HZ-P-35的分离鉴定及其生成共轭亚油酸的研究

从奶牛青贮饲料中分离筛选出1株丙酸菌菌株HZ-P-35,液相色谱检测证明该微生物能从葡萄糖生产丙酸、乙酸和虎珀酸。通过平板菌落形态、液体培养、透射电镜等形态学手段观察、生理生化实验鉴定以及16SrRNA序列分析,初步确定所得菌株HZ-P-35为1株费氏丙酸杆菌变种,命名为Propionibacterium frudenreichiivar LL。细胞发酵转化实验结果表明,菌株HZ-P-35在MRS培养基中具有转化亚油酸生成共轭亚油酸的能力,气相色谱测定表明共轭亚油酸的质量浓度达24.37μg/mL。     

清香型白酒大曲中高产四甲基吡嗪菌株的筛选、鉴定及发酵培养基优化

为通过生物法改善并提高清香型白酒酿造中功能性成分四甲基吡嗪的含量,该研究采用传统分离方法从清香型白酒大曲中分离纯化芽孢杆菌（Bacillus sp.）,通过气相色谱（GC）法筛选高产四甲基吡嗪的菌株,通过形态观察及分子生物学技术对其进行菌种鉴定,并对其产四甲基吡嗪发酵培养基配方进行研究。结果表明,共分离得到31株芽孢杆菌,从中筛选得到7株产四甲基吡嗪的芽孢杆菌,其中菌株q94的四甲基吡嗪产量最高,为（0.36±0.01）g/L,其被鉴定为枯草芽孢杆菌（Bacillus subtilis）。菌株q94产四甲基吡嗪的最优培养基组成为：蔗糖80 g/L,蛋白胨10 g/L、酵母浸粉20 g/L、磷酸氢二铵30 g/L、磷酸二氢钾15 g/L,在此优化条件下,菌株q94的四甲基吡嗪产量可达（3.32±0.18）g/L。     

响应面法优化P.acidipropionici产酸发酵培养基

利用响应面分析法优化产酸丙酸杆菌发酵产酸培养基.在单因素试验的基础上,采用Box-Behnken试验设计,选定甘油、混合氮源(酵母提取物:胰酶大豆肉汤=2∶1)和磷酸氢二钾3个关键因子为响应因子,以丙酸产量为响应值建立多元二次回归方程,在分析各个因素的显著性和交互作用后,确定了产酸丙酸杆菌发酵产酸的最优培养基为:甘油44.4g/L、混合氮源(酵母提取物:胰酶大豆肉汤=2∶1)27.0g/L、K2HPO4 2.0g/L,丙酸产量最大预测值为20.75g/L.经过优化,丙酸产量提高了151％,实验值与预测值基本相符.     

地衣芽孢杆菌MB01产抗菌性脂肽的发酵条件优化

该课题组分离得到一株对副溶血性弧菌有良好抑制作用的地衣芽孢杆菌,对其产抗菌脂肽的发酵培养基进行优化。结果表明,3种基础培养基中,Landy改进培养基为最佳培养基;Plackett-Burman试验结果表明葡萄糖(P0.05)、L-谷氨酸钠(P0.01)、MgSO_4(P0.01)和KH_2PO_4(P0.01)是影响脂肽产量的关键因素。在此基础上,采用响应面试验确定了培养基关键因素最佳水平,得到最优组合为:葡萄糖31.06 g/L、L-谷氨酸钠6.01 g/L、MgSO_4 0.77 g/L、KH_2PO_4 1.35 g/L,在此条件下,脂肽产量为476.23 mg/L,相较优化前产率提升15.32%。研究结果为产抑制副溶血性弧菌的抗菌脂肽大规模生产奠定基础。     

产壳聚糖酶菌株的筛选、鉴定及发酵条件优化

以壳聚糖为唯一碳源利用透明圈法,从养殖虾的池塘污泥中分离出8株产壳聚糖酶酶活较高的菌种。经过进一步的复筛,最终得到一株产非诱导型壳聚糖酶的菌株,确定该菌株TCCC150018为试验菌株,其酶活为2.48U/m L。经过形态特征观察以及16S r DNA序列分析对菌株进行鉴定,确定TCCC150018为蜡状芽孢杆菌(Bacillus cereus)。在单因素试验的基础上,通过响应面分析法确定在酵母浸粉16g/L,葡萄糖11.5g/L,吐温-80 1.2g/L条件下酶活力可达到4.92 U/m L,是优化前的1.98倍,在工业生产方面具有良好的应用前景。     

抗食品腐败真菌的乳酸菌筛选、复配及混合发酵工艺优化

应用化学防腐剂控制食品中腐败微生物,防腐剂残留对人体具有一定的潜在性危害。本文筛选抗食品腐败真菌的乳酸菌,并对其进行复配及混合发酵以提高其发酵液抗真菌活性。通过24孔板双层琼脂法及分级抑菌浓度法从多株乳酸菌和丙酸杆菌中筛选抗真菌较强的菌株并确定混合菌最优组合。通过Plackett-Burman试验,最陡爬坡试验,对其发酵工艺进行优化,用96孔板酶标仪法测其发酵上清液的抗真菌活性。结果表明:最优混合菌组合为植物乳杆菌L9和费氏丙酸杆菌D5;发酵培养基优化配方:葡萄糖55 g/L,碳酸钙6.7 g/L,酵母浸粉14.8 g/L,磷酸氢二钾 0.25 g/L,硫酸锰 0.1 g/L,乙酸钠 5.0 g/L,柠檬酸铵 2.0 g/L,接种比例为5:1（D5:L9）,发酵温度为37 ℃。对筛选出的L9和D5在优化后的发酵培养基进行混合发酵验证,其发酵液抗真菌活性可高达47.07 AU。     

布氏乳杆菌产γ-氨基丁酸发酵条件的优化

从中国传统发酵蔬菜中分离获得2株高产γ-氨基丁酸的布氏乳杆菌S37和布氏乳杆菌J68,为进一步提高其产γ-氨基丁酸的能力,对菌株的发酵条件进行优化。结果表明,2株菌产γ-氨基丁酸的最优条件为:发酵时间72 h、发酵温度35℃、底物L-谷氨酸钠浓度400 mmol/L、初始pH 5.0。在此条件下,菌株的γ-氨基丁酸产量分别为233.9 mmol/L和159.3 mmol/L,对应的L-谷氨酸钠转化率分别为58.5%和39.8%。单因素试验发现叶酸、L-半胱氨酸和氯化锰的添加能显著提高菌株的γ-氨基丁酸产量,在此基础上进一步通过响应面试验对其进行优化,发现对于菌株S37最优添加水平分别为8.37 mg/L、0.94 g/L和0.60 g/L,对于菌株J68其最优添加水平分别为10.16 mg/L、0.97 g/L和0.60 g/L。在该最优条件下,2株菌的γ-氨基丁酸产量分别达到312.6 mmol/L和251.2 mmol/L,对应的L-谷氨酸钠转化率分别为78.2%和62.8%。     

产γ-氨基丁酸乳酸菌的分离鉴定及其发酵条件优化

以浆水作为菌株分离源,分离筛选产γ-氨基丁酸（GABA）乳酸菌,采用薄层层析法和高效液相色谱法定性定量分析GABA,并对筛选菌株进行形态学观察、生理生化试验及分子生物学种属鉴定、发酵特性分析及发酵条件优化。结果表明,共分离筛选出21株乳酸菌,具有产GABA能力的有6株,经鉴定其中5株为植物乳杆菌（Lactobacillus plantarum）,1株为发酵乳杆菌（Lactobacillus fermentans）。选取GABA产量最高的一株植物乳杆菌,编号为2,通过单因素试验及响应面试验确定其最适发酵条件为：初始 pH值5.8,发酵温度36 ℃,发酵时间60 h。在此优化条件下,GABA产量可达0.78 g/L,比优化前产量（0.22 g/L）提高约3.5倍。     

植物乳杆菌与费氏丙酸菌转化生成甘露醇的比较

对1株植物乳杆菌(Lactobacillus plantarum HSC 235)和1株费氏丙酸菌(Propionibacterium freudenreichiiHZP-35)生物转化果糖生成甘露醇能力进行了比较研究。在生长细胞、静息细胞以及无完整细胞粗酶提取物3种不同转化反应体系中,植物乳杆菌HSC 235和费氏丙酸菌HZP-35都表现出甘露醇脱氢酶活性,而生长细胞甘露醇产量和得率较高,分别为38 g/L和27%,静息细胞和粗酶提取物反应体系甘露醇的产量及得率则相关无几,表明甘露醇的转化过程与细胞生长紧密关联。在不同初始果糖浓度的试验中,较低果糖有利甘露醇得率的提高,50～150 g/L果糖浓度甘露醇得率稳定在27%左右,果糖浓度提高到200 g/L时则表现出对转化的抑制作用。植物乳杆菌可与费氏丙酸菌混合生长,但混菌发酵的实验结果表明,混合培养体系甘露醇得率比单菌种纯培养时甘露醇得率要低得多。植物乳杆菌甘露醇醇产量和得率要明显高于费氏丙酸菌。     

丙酸对费氏丙酸杆菌生长及脱氧腺苷钴胺素合成的影响

通过添加控制丙酸浓度考查了费氏丙酸杆菌发酵生产VB12的过程中丙酸抑制细胞生长的浓度范围,在此基础上利用树脂对发酵过程中丙酸进行了选择性吸附后细胞的生长和脱氧腺苷钴胺素合成的变化。研究结果表明,丙酸的浓度在10.0g/L时,对菌体细胞生长产生了明显的抑制。在丙酸浓度积累到10.0g/L之前,利用树脂对其吸附分离出2.5g/L的丙酸后,生物量提高了37.5%,脱氧腺苷钴胺素产量提高了50.0%。该实验为实现丙酸的在线分离和丙酸/VB12高效联产的新型耦合发酵工艺提供了基础。     

Research on some factors influencing acid and exopolysaccharide produced by dairy propionibacterium strains isolated from traditional homemade Turkish cheeses

In this study, a total of 32 isolated strains and 5 reference strains of dairy propionibacteria were analyzed for acid and exopolysaccharide (EPS) production in skim milk and yeast extract-lactate broth (YEL) media in order to investigate the physiological background and preservative role of acid and EPS. The effects of final culture pH and optical density on acid and EPS production were also determined. On average, all strains produced more acid and reached lower final pH values in skim milk than in YEL medium. While the correlations obtained between the acid produced by propionibacterium strains and their final culture pH in skim milk medium were significant (P < 0.01), no correlations were found between optical density, final pH, and produced acid in YEL medium. Sixteen isolated and five reference strains of propionibacteria were tested further for the ability to produce propionic and acetic acids. On average, Propionibacterium freudenreichii subsp. shermanii and P. freudenreichii subsp. freudenreichii strains produced higher amounts of propionic and acetic acids than did Propionibacterium jensenii in YEL medium. The acid produced by these strains may be used as a preservative in the food industry for replacement or reduction of the increasing use of chemical additives. The EPS production by propionibacterium strains during growth in YEL medium was 72 to 168 mg/liter, while in skim milk it was 94 to 359 mg/liter. The monomer compositions of the EPSs formed by the six selected dairy propionibacteria strains were analyzed. The EPSs may have applications as food grade additives and viscosity-stabilizing agents.     

Production of extracellular bifidogenic growth stimulator by anaerobic and aerobic cultivations of several propionibacterial strains

Production of a bifidogenic growth stimulator (BGS) by propionic acid bacteria was investigated under anaerobic and aerobic culture conditions. To measure the concentration of extracellular BGS produced by propionic acid bacteria, we evaluated the effects of bioassay conditions using Bifidobacterium longum as a test microorganism on the formation of a growth-stimulation zone. The diameter of the growth-stimulation zone was significantly affected by both the component concentrations and the pH of a bioassay medium. The optimum component concentrations and pH of a bioassay medium were one-half of the normal values and 8.5, respectively. Using the bioassay method, we can measure the concentration of BGS produced by propionic acid bacteria ranging in concentrations from 0.1 microg/l to 1 mg/l using 1,4-dihydroxy-2-naphthoic acid (DHNA) and 2-amino-3-carboxy-1,4-naphthoquinone (ACNQ) as standards. Of six dairy propionic acid bacterial strains tested, the four strains (Propionibacterium freudenreichii ET-3, P. shermanii PZ-3, P. acidipropionici JCM 6432, and P. jensenii JCM 6433) produced BGS at a concentration range of 4-23 mg/l under the anaerobic culture conditions. Analysis of high performance liquid chromatography (HPLC) showed that more than 70% of total BGS produced in supernatant samples was DHNA and no ACNQ was produced by the strains. The effect of oxygen supply on BGS production was investigated for the four BGS-producing strains. The aerobic conditions exerted in positive effects on BGS production by only P. acidipropionici JCM 6432. The concentration of BGS obtained in the aerobic cultivation of P. acidipropionici JCM 6432 was 1.3-fold than that in anaerobic cultivation. Different properties (BGS production as well as cell growth and glucose metabolism) occurring in response to the aerobic conditions were observed, depending on the propionic acid bacterial strain used. This paper is the first report on BGS production by propionibacterial strains except for P. freudenreichii.     

高产酸扣囊复膜酵母的筛选与培养基配方优化

该研究对10种不同的米酒曲和黄酒曲中的高产酸酵母菌进行了分离、筛选及鉴定,并以酵母菌的生物量为评价指标,采用单因素试验和响应面法,对菌株的液态发酵培养基进行优化。结果表明,经过分子生物学鉴定,共分离获得6株扣囊复膜酵母（Saccharomycopsis fibuligera）。通过菌株产酸能力和耐受性的比较,筛选到一株产酸率高、耐高温和乙醇能力强的扣囊复膜酵母菌株3-1,其总酸（以乳酸计）产量达5.4 g/L。最佳培养基配方为：糖蜜7.5 g/L,葡萄糖7.7 g/L,大豆蛋白胨1.7 g/L,酵母浸粉1.7 g/L。在此优化条件下,菌株3-1的生物量达2.45×108个/mL,总菌数比对照培养基提高了63.3%。     

产GABA乳酸菌的筛选鉴定及其发酵条件研究

为了筛选得到一株高产γ-氨基丁酸(γ-aminobutyric acid,GABA)的乳酸菌,本研究选取传统发酵食品作为菌株来源,采用薄层层析法和高效液相色谱法对分离出的20株乳酸菌的GABA生产能力进行定性和定量分析,并对高产菌株进行种属鉴定及产GABA影响因素研究。结果表明:从传统发酵食品中分离得到一株GABA高产菌株14#,其发酵液中GABA含量为2.30 g/L,经菌落形态、生理生化特性以及16S rDNA基因序列分析鉴定该菌株为植物乳杆菌(Lactobacillus plantarum),研究其产GABA的影响因素并确定发酵优化条件为:MRS基础发酵培养基中以15 g/L的葡萄糖作为碳源,以10 g/L的酵母粉和15 g/L的牛肉膏作为复合氮源,添加1.5% L-谷氨酸钠,接种量为4%,初始pH为6.0,37 ℃静置培养48 h。优化后GABA含量可达9.12 g/L,比优化前产量(2.30 g/L)提高了近3倍。     

Genetic manipulation system in propionibacteria

Members of the genus Propionibacterium are widely used in the production of vitamin B12, tetrapyrrole compounds, and propionic acid as well as in probiotic and cheese industries. Shuttle vectors were developed in propionibacteria using replicons from endogenous plasmids in Propionibacterium and Escherichia coli and an appropriate selection marker. The efficient transformation was achieved using the shuttle vector prepared from Propionibacterium freudenreichii to overcome the high restriction modification system in propionibacteria. Expression vectors with native promoters for use in propionibacteria were also developed. Using this system, cholesterol oxidase, which is used as a diagnostic enzyme, was produced in P. freudenreichii. Genes involved in 5-aminolevulinic acid (ALA) and vitamin B12 biosynthesis in propionibacteria were isolated. ALA in propionibacteria could be synthesized via both the C4 pathway (condensation of glycine and succinyl CoA) and the C5 pathway (from glutamate). The hemA gene encoding ALA synthase from Rhodobacter spheroides, was overexpressed and ALA accumulated in P. freudenreichii. Thus, the genetic manipulation systems in propionibacteria will facilitate genetic studies of probiotics and the vitamin B12 biosynthetic pathway.     

Production of acetic and propionic acids from labneh whey by fermentation with propionibacteria

Whey produced during the manufacture of labneh was supplemented with yeast extract (10 g/1), and then fortified with lactose, treated with β-galactosidase or fermented with Lactobacillus helveticus, prior to inoculation with free living cells of Propionibacterium freudenreichii ssp shermanii or Propionibacterium acidipropionici or cells immobilized in aliginate beads. Under anaerobic batch conditions, fermentation of the whey with Lb helveticus followed by P acidipropionici (free cell system) for 2.5 days at 32°C gave a broth with 5.9 g/l of propionic acid and 2.4 gll of acetic acid, while immobilized cells of the same organisms gave a broth with 11.0 gll propionic acid and 3.2 g/l acetic acid over 4 days. These latter values were the maximum levels recorded with any of the treatments, and it is suggested that such yields might make recovery economically feasible in certain countries.     

外源有机酸对于Propionibacterium freudenreichii CCTCC M207015发酵生产丙酸的影响

考察了外源有机酸(丙酸、乙酸、琥珀酸)对于游离细胞与纤维床反应器生产丙酸的影响。结果表明,以Propionibacterium freudenreichii CCTCC M207015为生产菌株时,纤维床反应器具有比游离细胞更强的丙酸、乙酸耐受性,其中丙酸的抑制现象比乙酸的更加严重。此外,外源琥珀酸可以作为丙酸合成的前体物质对P.freudenreichii CCTCC M207015发酵生产丙酸产生促进作用。     

产赤藓糖醇菌株的筛选与鉴定

利用含有300g/L 葡萄糖的高渗培养基从蜂蜜、花粉、土壤等样品中筛选耐高渗酵母菌,经薄层层析和高效液相色谱分析得到两株产赤藓糖醇且不产甘油的酵母菌,通过高碘酸氧化法筛选出其中赤藓糖醇产量较高的一株菌株E54。菌株E54 在含葡萄糖200g/L、酵母膏5g/L 的发酵培养基中发酵90h,赤藓糖醇产量为41.1g/L,转化率为22.8%。通过形态观察、生理生化实验、5.8S rDNA 序列分析并构建系统进化树,初步鉴定E54 为Moniliellaacetoabutans(丛梗孢酵母)。