假单胞菌F12中氨对产酶及L-半胱氨酸合成的影响

目的:探索假单胞菌F12氨的代谢及积累对产酶和转化合成L-半胱氨酸的影响。方法:本研究考察了氨对菌体生长、对DL-2-氨基-△2-噻唑啉-4-羧酸(DL-ATC)的代谢及产酶的影响,以及转化过程中氨的积累对L-半胱氨酸合成的影响。结果:氨浓度大于60mmol/L时对菌体生长有明显抑制作用,并没有影响DL-ATC的代谢和产酶,但同时加入葡萄糖后产酶显著下降;转化过程中有氨的释放,但DL-ATC消耗引起的氨的积累不影响L-半胱氨酸的合成。结论:氨的积累在产酶阶段和转化阶段对L-半胱氨酸的合成影响不显著。     

溶氧对假单胞菌TS1138生产L-半胱氨酸合成酶系的影响

假单胞菌(Pseudomonas sp.)TS1138在含有DL-2-氨基-Δ2-噻唑啉-4-羧酸(DL-2-amino-Δ2-thiazoline-4-carboxylic acid,DL-ATC)的环境中能够转化DL-ATC生成L-半胱氨酸的酶系。通过固定不同溶氧浓度,探索溶氧对假单胞菌TS1138发酵产酶的影响。摇瓶实验结果表明：溶氧浓度过低,菌体生长缓慢,细胞产酶能力较低;溶氧浓度过高,菌体生长速度快,但不利于细胞产酶。7L罐发酵实验结果表明：在产酶中后期,应根据溶氧浓度变化来调节搅拌转速或空气流量,使溶氧浓度控制在30%以上。     

自选酿酒酵母（KDLYS9-3）产β-D-葡萄糖苷酶动力学研究

自选高产β-D-葡萄糖苷酶的酿酒酵母(KDLYS9-3)在葡萄酒酿造过程中具有增强香气的效果。依据菌体生长和产酶试验,利用Logistic方程、Dose Resp方程和Nelder方程建立了菌体生长和产酶,以及菌体生长速率与酶生成速率之间关系的动力学模型,通过Origin8.0软件进行非线性拟合,并利用Lineweaver-Burk法作图测定了该菌所产β-D-葡萄糖苷酶的动力学参数K_m值和V_(max)值。结果显示:自选酿酒酵母KDLYS9-3的菌体生长与产酶的相关性为部分偶联型,动力学模型与试验值吻合度好,方程能够反映菌体生长与产酶的变化规律。菌体生长8 h后开始产酶,菌体进入对数生长期时酶大量生成,到39 h菌体生长进入稳定期,随着菌体生长进入衰亡期后酶也随之停止产生;利用Lineweaver-Burk法求得酶动力学参数K_m=8.492 579 mmol/L,V_(max)=1.030 715(μmol/L)/min。研究结果为该菌株的理论研究和实际应用奠定了基础。     

氯化胆碱对L-缬氨酸发酵的影响

为探究氯化胆碱在微生物菌体培养方面的作用,采用微生物发酵法生产L-缬氨酸,以谷氨酸棒状杆菌XV0505(Leu~-+Ile~-+2-TA~r+α-AB~r+SG~r)为供试菌株,探究了其在不同氯化胆碱添加量以及在底物添加与联合随糖流加2种添加方式下生长、耗糖、产酸的情况。结果表明,发酵过程中氯化胆碱的添加方式为底物添加联合随糖流加,底物中氯化胆碱最适添加浓度为0. 5 g/L。此方式下,发酵周期内产酸量增加了18 g/L,较未添加氯化胆碱批次提升了26. 4%。氯化胆碱的外源添加能够有效增强菌体的活力,加快菌体生长,提高菌体产酸速率,为L-缬氨酸的生产提供了参考。     

酶转化三七茎叶总皂苷制备稀有人参皂苷C-K

为了提高三七茎叶总皂苷的利用率和C-K酶转化得率,采用Aspergillus niger sp. G8菌所产酶与Aspergillus niger sp. G4菌所产酶的等体积混合酶(以下简称G8-G4混合酶),研究了酶转化三七茎叶总皂苷进而制备C-K的方法。结果表明,市销的三七茎叶总皂苷中,主要成分是人参二醇类皂苷,其中含量最多的皂苷是Rb3、C-Mx1、Rc和Fc,质量分数分别为26. 38%、14. 28%、12. 73%、10. 25%。G8-G4混合酶的最佳反应条件为45℃、p H 5. 0、底物质量浓度25 g/L、反应24 h。三七茎叶总皂苷经G8-G4混合酶转化后得到4种产物为C-K、C-Mx、Fc、R7,其中C-K的转化得率为32. 7%。     

L-乳酸细菌培养条件的研究

对突变株干酪乳杆菌(Lactobacillus casei)FH2-8-9菌体培养条件、液体培养基成分进行了研究.结果证明,发酵过程中pH的调节及H 和游离乳酸的添加可以促进菌体生长,并提高了L-乳酸的产量;培养基中添加乳糖和果糖、酵母膏和蛋白胨、CaCO3对菌体生长和产酸均有重要影响;调节葡萄糖与丙酮酸钠的比例为9:3,并在发酵42 h时添加丙酮酸钠,L-乳酸产量可达75 g/L.     

大肠杆菌高密度培养发酵L-色氨酸

为实现高水平的大肠杆菌高密度培养,提升发酵法产L-色氨酸的生产效率,以大肠杆菌TRTH为供试菌株,在初始发酵工艺的基础上,先后对发酵接种量和底物KH_2PO_4添加量各设置4个梯度进行发酵对比试验,并着重考察了底物KH_2PO_4添加量对菌体生长、产酸、磷酸盐消耗、糖酸转化率及副产物积累的影响。试验结果表明,在接种量为20%(体积分数),底物KH_2PO_4添加量为10 g/L时,最高菌体密度达到65. 39 g/L,最终L-色氨酸产量为59. 55 g/L,分别较优化前提高了45. 99%和31. 17%,发酵延滞期明显缩短,菌体生长迅速,原料利用率大幅提高,主要副产物积累量较低,发酵总体水平达到最优。为大肠杆菌高密度培养在L-色氨酸发酵中的应用提供了一个成本低、可行性高的新策略,同时为L-色氨酸发酵生产中底物磷酸盐的调控提供了参考。     

VC混菌发酵中大菌不同胞外组分对小菌的影响

在维生素C(VC)二步混菌发酵中,巨大芽孢杆菌可显著促进氧化葡萄糖酸杆菌产VC前体2-酮基-L-古龙酸(2-KLG)。采用硫酸铵分级盐析沉淀、柱层析及电泳技术对巨大芽孢杆菌胞外液中的活性物质进行了分离纯化。通过测定氧化葡萄糖酸杆菌生长细胞转化L-山梨糖为2-KLG的生成量、产酸菌关键酶L-山梨糖脱氢酶(SDH)酶活性及产酸菌的活菌数,研究了VC两步发酵中大菌不同胞外组分对小菌的作用。结果表明:大菌胞外36 000和44 300两个组分蛋白质对小菌产酸、SDH酶活具有促进作用。     

产天冬氨酸酶菌株大肠杆菌HY-05C发酵培养基及培养条件的优化

为提高大肠杆菌HY-05C在L-天冬氨酸生产过程中的菌体浓度,进而提高单位体积培养液中L-天冬氨酸酶酶活,本文对该菌株的培养基和培养条件进行了优化。首先通过正交实验对大肠杆菌HY-05C培养基组成进行优化,确定了最佳培养基配方(g/L):富马酸铵10,玉米浆干粉8,酵母粉2,蛋白胨7,氯化钠5,磷酸二氢钾1,硫酸镁0.2。又进一步考察了对菌体浓度和酶活影响较大的因素初始p H和接种量,得到了最适p H=6.0,最佳接种量为1.0‰,在上述最佳培养基及培养条件下对大肠杆菌HY-05C的酶转化进程进行测定,优化后培养液的转化效率较初始提高150%,同时也验证了通过发酵条件优化提高单位体积的菌体浓度,提高L-天冬氨酸转化效率方法的可行性。     

产碱性脂肪酶热带假丝酵母LYSC-3的筛选、鉴定及发酵条件的优化

以橄榄油为唯一碳源,以溴钾酚紫为显色剂,采用琼脂平板法从富含油脂的土壤中筛选到一株产碱性脂肪酶野生型菌株LYSC-3。经形态学、生理生化和分子生物学的鉴定,确定菌株LYSC-3为热带假丝酵母(Candida tropicalis)。经发酵条件优化,菌株LYSC-3的最佳产碱性脂肪酶的发酵条件为:橄榄油10g·L-1、蛋白胨5g·L-1、蔗糖20g·L-1、(NH4)2SO40.5g·L-1、MgSO4·7H2O0.2g·L-1,K2HPO40.2g·L-1,pH8.0,35℃,150r·min-1摇床振荡培养3d,获得碱性脂肪酶最高酶活力达38.6U·mL-1。     

沙门氏菌选择性化学限定培养基的研制

目的 拟用氨基酸、维生素、无机盐替代牛肉膏和蛋白胨,开发质量可控的化学限定沙门氏菌选择性培养基。方法 分别研究20种氨基酸、11种维生素或4种无机盐对大肠杆菌ATCC 25922 生长特性的影响,并确定生长最适浓度,替换亚硫酸铋(BS)琼脂或Hektoen Enteric(HE)琼脂中的牛肉膏和蛋白胨,研制出化学限定BS琼脂或HE琼脂培养基。结果 在36 ℃下培养48 h,基础培养基中添加天冬酰胺、天冬氨酸、缬氨酸、盐酸硫胺或核黄素有利于大肠杆菌ATCC 25922生长,OD₅₉₅值均高于0.1,而添加丝氨酸、赖氨酸盐酸盐、异亮氨酸、尼克酸、生物素或磷酸氢二钠对细菌增殖没有促进作用,OD₅₉₅值几乎为0。将35种营养物质按照最佳浓度替代牛肉膏、蛋白胨制成化学限定BS琼脂或HE琼脂,经过质控菌株验证,化学限定BS琼脂或HE琼脂在菌体生长指数、菌落形态和特殊显色反应等方面均与商业培养基相似。结论 化学限定BS琼脂和HE琼脂均符合国标GB 4789.28—2013对 培养基和试剂的质量要求中关于参考培养基的质控要求。     

阿魏菇原生质体制备及再生条件的建立

以新疆阿魏菇为材料,研究菌龄、酶解时间、酶解液对阿魏菇菌丝原生质体产量的影响,同时还研究了不同渗透压稳定剂对原生质体再生的影响。结果显示,在0.6 mol/L KCl稳渗剂条件下,菌龄为8 d阿魏菇菌丝体(0.1 g)在0.8 mL混合酶解液中(1.5%离析酶+0.5%纤维素酶+2%溶菌酶)制备原生质体,35℃酶解3 h获得原生质体达5×106个/mL。原生质体再生结果显示,在0.8 mol/L蔗糖稳渗剂下,原生质体再生时间为16 d,其再生率可达0.6%。该文通过对阿魏菇原生质体分离和再生条件的研究,旨在从细胞水平上探索一条食用菌育种新途径,为发展和完善食用菌新菌株选育提供理论依据和技术借鉴。     

A new chemically defined medium for wine lactic acid bacteria

Growth and activity of lactic acid bacteria is crucial for the production and quality of grape wines but studying their metabolism is difficult in wines or complex laboratory media because of the undefined substrate pools. This work presents a new chemically defined medium that meets the fastidious nutritional requirements of wine lactic acid bacteria and yields rapid and strong growth. The new medium is composed of 44 constituents and a precise protocol is provided for its preparation. Maximum specific growth rates and growth yields of the wine strains studied were comparable to those obtained in common laboratory media, and the new medium allows for various modifications, such as changing the medium pH to the wine range, addition of L-malic acid or utilization of different carbon sources while maintaining growth of wine lactic acid bacteria. The medium was successfully tested with 22 wine strains of the genera Oenococcus, Lactobacillus and Pediococcus. It is suggested that this chemically defined medium be considered for the investigation of the nutritional requirements and metabolism of wine lactic acid bacteria.     

大豆蛋白水解物促酸奶乳酸菌增殖及生长动力学

由于乳酸菌缺乏一些生物代谢途径,不能合成生长必需的氨基酸、维生素等物质,营养要求十分苛刻,必需依赖生长环境提供必需氨基酸、维生素等生长因子才能获得高密度生长。应用化学限定培养基进行大豆蛋白水解物、全价氨基酸和生长基本氨基酸3种氮源条件下乳酸菌的生长动力学研究,结果表明:保加利亚乳杆菌和嗜热链球菌在分子量小于5 ku的大豆蛋白水解物培养基中的最大生长速率μmax分别为0.215/h和0.262/h,约为全价氨基酸的2倍,细胞密度增加到5倍。20种全价氨基酸氮源培养基对两种乳酸菌的生长效能很低,保加利亚乳杆菌和嗜热链球菌的最大生长速率分别为0.10/h和0.12/h,pH值只达到pH4.5～5.0。基本氨基酸氮源只能满足乳酸菌生长的最低要求。分子量小于5 ku的大豆蛋白水解物比全价氨基酸对乳酸菌生长具有显著的促进作用,表明乳酸菌生长的最佳氮源是寡肽而不是氨基酸,大豆蛋白水解物可以用作乳酸菌高密度培养添加物。     

地衣芽孢杆菌固体发酵培养基优化研究

研究了麦麸含量、加水量、碳源、氮源等对地衣芽孢杆菌固体发酵产碱性蛋白酶的影响。采用正交试验法优化发酵培养基配方,确定最佳发酵条件为:麦麸20 g,大豆分离蛋白30%,加水量160%,麦芽糖5%,MgSO40.6%,(NH4)2SO40.5%,K2HPO40.5%,吐温-80 0.05%,发酵时间72 h,在此发酵条件下,酶活力最高达到16 08 U/mL。     

A natural strategy to improve the shelf life of the loaf bread against toxigenic fungi: The employment of fermented whey powder

Whey powders are used as food ingredients in many applications, from bakery goods, soups and sauces to baby food. The objective of the study was to evaluate the antifungal property of a whey‐based medium (WM) fermented by lactic acid bacteria. The antifungal activity of the WM was evaluated using antifungal tests on solid and liquid media. MIC and MFC ranged from 15.6 to 250 mg/mL and 62.5 to 250 mg/mL, respectively. Using fermented WM for dough preparation produced a reduction of Penicillium expansum growth of 0.5–0.6 log CFU/g and an improvement in shelf life of 1–2 days in relation to control bread.     

与酵母菌具有共生作用乳酸菌LC5培养基的优化

用对酵母菌(YE4)有促进作用的乳酸菌(LC5)为研究对象,以OD值和活菌数为测定指标筛选合适的培养基。通过对5种乳酸菌常用培养基的筛选,确定M17培养基中LC5生长良好。以M17培养基作为基础培养基,进一步利用单因素试验和正交试验的设计方法对其进行优化,得出改良M17培养基的最佳成分为:大豆蛋白胨为0.6%,胰蛋白胨为0.6%,牛肉膏为0.4%,酵母浸提粉为0.5%,K2HPO4为0.7%,盐液A为0.4%,甘油为0.8%,乳糖为0.6%。优化后的培养基乳酸菌LC5的活菌数可达到1.89×109cfu/mL,是基础培养基的5.9倍,OD值是M17基础培养基的1.22倍。     

Growth responses of coliform bacteria to purified immunoglobulin G from cows immunized with ferric enterobactin receptor FepA

The ability of purified bovine immunoglobulin (Ig) G from cows immunized with ferric enterobactin receptor FepA to inhibit the growth of coliform bacteria derived from bovine intramammary infection was investigated in iron-restricted media. All isolates of Escherichia coli (n = 21) and Klebsiella pneumoniae (n = 21) were tested for growth in a chemically defined medium containing 0.5 mg/ml of apolactoferrin and in a pooled source of dry cow secretion. The addition of 4 mg/ml of purified bovine IgG directed against FepA in the synthetic medium resulted in significant growth inhibition for both E. coli and K. pneumoniae isolates. Growth reduction of E. coli was greater than that of K. pneumoniae. In dry cow secretions, the growth of each E. coli isolate but of less than half of K. pneumoniae isolates (43%) was inhibited by IgG from cows immunized with FepA. Purified bovine IgG from cows immunized with E. coli J5 had a minimal inhibitory effect on the growth of both E. coli and K. pneumoniae isolates in the synthetic medium. In dry cow secretions, IgG from cows immunized with E. coli and K. pneumoniae isolates. Supplementation with 50 microM of ferric chloride to the medium completely reversed the inhibitory effects of the antibodies and lactoferrin. Bovine IgG directed against FepA apparently inhibited the growth of coliform bacteria by interfering with the binding of the ferric enterobactin complex to the cell surface receptor FepA.     

Tolerance to high osmolality of the lactic acid bacterium Oenococcus oeni and identification of potential osmoprotectants

Growth of the lactic acid bacterium Oenococcus oeni under hyperosmotic constraint was investigated in a chemically defined medium. The bacterium could grow on media with an elevated osmolality, preferably below 1.5 Osm kg(-)(1) H(2)O. At osmolalities comprised between 0.6 and 1.5 Osm kg(-)(1) H(2)O, the growth deficit elicited by the sugars glucose and fructose was slightly more severe than with salts (NaCl or KCl). In contrast to what was observed in other lactic acid bacteria, proline, glycine betaine and related molecules were unable to relieve inhibition of growth of O. oeni under osmotic constraint. This was correlated to the absence of sequences homologous to the genes coding for glycine betaine and/or proline transporters described in Lactococcus lactis and Lactobacillus plantarum. The amino acid aspartate proved to be osmoprotective under electrolyte and non-electrolyte stress. Examination of the role of peptides during osmoregulation showed that proline- and glutamate-containing peptides were protective under salt-induced stress, and not under sugar-induced stress. Under high salt, PepQ a cytoplasmic prolidase that specifically liberated proline from di-peptides increased activity, while PepX (X-prolyl-dipeptidyl aminopeptidase) and PepI (iminopeptidase) activities were unaffected. Our data suggest that proline- and glutamate-containing peptides may contribute to the adaptation of O. oeni to high salt through their intracellular hydrolysis and/or direct accumulation.     

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。