保加利亚乳杆菌微胶囊化工艺研究

目的制备保加利亚乳杆菌微胶囊,提高菌株的酸、热耐受性及降低菌体的分离成本。方法以保加利亚乳杆菌(Lactobacillus bulgaricus)为研究对象,海藻酸钠(SA)为壳材、CaCl2为固化剂,制备保加利亚乳杆菌微胶囊;包埋率、颗粒平均化程度、机械强度等为考核指标,研究保加利亚乳杆菌微胶囊化的工艺。结果当海藻酸钠浓度为0.75%、CaCl2浓度为3%、电压为600V、泵速为1.96mL/min、震动频率为80Hz时,微胶囊化包埋效果最佳,经固定化后的菌微胶囊保持了良好的保加利亚乳杆菌的活性,微囊化保加利亚乳杆菌经过2次连续发酵后的产酸量分别达到59.4g/L和55.8g/L。结论本研究为工业化生产乳酸提供了一条具有经济价值的途径。     

双歧杆菌NQ1501双层微胶囊制备及其特性研究

目的提高双歧杆菌NQ1501对环境的耐受性。方法采用流化床底喷工艺,以蔗糖、麦芽糊精、海藻糖和改性淀粉为内层壁材,聚丙烯酸树脂为外层壁材制备双歧杆菌NQ1501微胶囊,并对制得微囊进行评价。结果制得4种双歧杆菌NQ1501微胶囊均具有较好的耐酸和肠溶能力,25℃贮存6个月后对照菌粉存活率为4.8%,采用4种不同内壁材的微胶囊存活率分别为蔗糖28.6%,麦芽糊精45.7%,海藻糖38.9%,改性淀粉55.2%。结论采用该制备工艺制得双歧杆菌NQ1501微胶囊具备较好的耐酸性、肠溶性和常温保存稳定性。     

保加利亚乳杆菌冻干保护剂的优化

目的 研究4种常用冻干保护剂的添加量.方法 以保加利亚乳杆菌为出发菌株,以其冻干菌粉活菌数为指标,通过正交试验优化4种常用冻干保护剂的添加量.结果 保加利亚乳杆菌的最佳冻干保护剂配比为乳糖∶谷氨酸钠∶抗坏血酸∶脱脂奶粉=3∶1∶2∶7,其中脱脂奶粉对菌体保护效果极显著(P＜0.01),乳糖对菌体保护效果显著(P＜0.05).结论 通过对保加利亚乳杆菌的4种常用冻干保护剂的添加量进行优化,为保加利亚乳杆菌的冻干工艺提供基础.     

酸奶中保加利亚乳杆菌分离条件的优化

为优化从酸奶中分离保加利亚乳杆菌的培养条件,初步建立一种较简单有效的分离方法。以成都市市售的主要酸奶－华西牌瓶装酸牛奶为来源,采用MRS、SL、LS、蕃茄酵母4种经典的乳酸菌选择性培养基,并分别进行有氧及烛缸厌氧分离培养测定其得率。采用X^2检验对不同组的分离得率进行统计学分析。结果表明MRS培养基为酸奶中保加利亚乳杆菌分离首选培养基,分离得率达66．67％;微氧环境优于有氧环境（PO＜0．05）。     

保加利亚乳杆菌原生质体的制备与回复研究

目的:通过对保加利亚乳杆菌的原生质体的制备和回复的方法学探讨,为乳杆菌的基因操作和其相关研究提供技术思路和实验条件.方法:用酶浓度分别为1 μg/ml、4 μg/ml、10μg/ml Mutanolysin(变溶菌素)对保加利亚乳杆菌进行处理,脱去细胞壁以探讨其原生质体形成与时间和酶浓度的关系;并选用较为适宜的酶浓度制备其原生质体,在自制的双层再生培养基上观察其原生质体在普通培养、CO2培养、厌氧培养时的回复生长情况.结果:保加利亚乳杆菌对Mutanolysin较敏感,酶浓度为1 μg/ml时只需40min大部分菌体形成原生质体.经1μg/ml Mutanolysin处理后制得的保加利亚乳杆菌原生质体倾入自制的双层再生培养基中,置于CO2和厌氧环境条件下培养能很好的回复生长.结论:本文的研究为乳杆菌的基因工程方面的研究提供了相关的技术条件和实验基础.     

酸奶中保加利亚乳杆菌的抗药性实验研究

测定了10种常用抗菌药物对市售酸奶中分离的57株保加利亚乳杆菌的MIC,结果显示泰能、克林霉素、林可霉素、头孢唑啉和红霉素对乳杆菌的抗菌活性最强,MIC90值为0.25～2.0mg/L,敏感率为96.5%～86.0%。其次是阿莫西林、复方新诺明、青霉素,其MIC90值为2.0～4.0mg/L,敏感率为84.3%～78.9%。庆大霉素、氯霉素其MIC90值为4.0～16.0mg/L,敏感率为63.2%～59.7%。可见,酸奶中的保加利亚乳杆菌存在抗药性,其抗药性基因是否具转移性有待于进一步研究。     

保加利亚乳杆菌H+-ATPase缺陷型菌株的筛选

【目的】从传统乳制品中筛选具有新霉素抗性的H+-ATPase缺陷的德氏乳杆菌保加利亚亚种自发突变株,为最终开发弱后酸化的酸奶发酵剂奠定基础。【方法】利用API 50 CH细菌鉴定系统和16s rRNA基因序列分析对菌株进行鉴定。新霉素作为筛选压力,筛选具有新霉素抗性自发突变菌株,比较亲本和突变菌株的H+-ATPase活力及其代谢情况。【结果】从内蒙古地区的传统发酵酸奶中分离鉴定出一株德氏乳杆菌保加利亚亚种（Lactobacillus delbrueckii subsp. bulgaricus）,并命名为KLDS 1.9201。以此为出发菌株,筛选出两株H+-ATPase缺陷的自发突变株,分别命名为KLDS 1.9201-1、KLDS 1.9201-4,它们的H+-ATPase活力分别比亲本KLDS 1.9201降低了46%和60%。在MRS培养基中生长24 h后,KLDS 1.9201、KLDS 1.9201-1和KLDS 1.9201-4对初始葡萄糖的代谢率分别为65%、41%和31%,终产物中乳酸的浓度分别为26g/L、18g/L和15g/L,突变菌株的生物量均低于亲本。【结论】H+-ATPase活力降低的德氏乳杆菌保加利亚亚种的自发突变株具有较低的生长速率和弱产酸能力,它们可被用于制作弱后酸化的酸奶发酵剂。     

发酵中含氮类原料对保加利亚乳杆菌活菌数的影响

目的研究生产发酵过程中培养基含氮原料与发酵的相关性。方法实验通过对不同生产厂家三种原料进行理化指标检测,并通过正交试验,比较不同原料发酵后保加利亚乳杆菌发酵液活菌数。结果最佳条件为酪蛋白胨氨基氮含量高,酵母浸粉的炽灼残渣低,牛肉粉总氮含量高。结论经过相同原料不同生产厂家的配方发酵后的发酵液活菌数存在差异,其理化指标对发酵有一定影响。     

德氏乳杆菌保加利亚亚种电转化平台的构建和优化

德氏乳杆菌保加利亚亚种(Lactobacillus delbrueckiisub sp.bulgaricus)是最具经济价值的乳酸菌之一,在世界上广泛应用于酸奶和其它发酵乳生产。当前对该菌的代谢机制研究甚少。外源基因的转化效率是制约其分子代谢机制研究的重要因素。本研究以pMG36c为材料,对L.delbrueckiisub sp.bulgaricus CH3进行电转化条件研究。结果表明,在电转化过程中,电场强度、质粒的浓度、细胞生长状态均对转化效率有明显影响,得到了该菌株的最适电转化条件为:对数初期的细胞,质粒浓度为100 ng/50μl菌液,在10 kV/cm电场强度下电转化,转化后细胞在复壮培养液中培养3 h后涂布选择性培养基,转化率可达2.6×103CFU/μg DNA。以甘氨酸、醋酸锂、二硫苏糖醇处理细胞壁,发现醋酸锂和二硫苏糖醇共同处理对转化效率有明显改善,可提高转化效率。     

ldhL-ldb0094基因敲除对保加利亚乳杆菌产L-乳酸的影响

以保加利亚乳杆菌Lactobacillus delbrueckii subsp. bulgaricus CICC21101为出发菌株,利用PCR扩增L-乳酸脱氢酶(ldhL)基因上下游序列ldhL1、ldhL2,获得ldhL基因缺失且包含上下游序列的片段,连接到乳酸菌专用温敏性基因敲除质粒pGhost4,将构建好的敲除载体电转入保加利亚乳杆菌CICC21101,低温筛选。结果表明,成功获得敲除ldhL基因的敲除突变株,敲除后的工程菌D-乳酸产量由30. 5g/L降为4. 8g/L,L-乳酸的产量由25. 4g/L增至58. 3g/L,光学纯度由54. 56%增至90%。同时发现ldhL-ldb0094基因的敲除致使ldhL-ldb1020表达的上调,D-乳酸脱氢酶(ldbD)基因表达量没有变化,ldhL基因敲除株的成功构建将为进一步研究该基因在保加利亚乳杆菌中的功能及后续高光学活性D-乳酸工程菌构建奠定基础。     

基于正交实验法优化从栀子黄色素中制备藏红花酸的工艺

基于正交实验法,优化从栀子黄色素中提取制备藏红花酸的碱水解工艺,以期可以简单高效地获得高纯度藏红花酸。建立高效液相色谱法(HPLC)测定藏红花酸含量,以藏红花酸的含量和得率为考察指标,采用正交实验法考察碱水解工艺中的料液比、NaOH浓度、水解温度和水解时间对产品中藏红花酸含量和得率的影响。确定栀子黄色素碱水解的最佳条件:料液比1∶6 g/mL、NaOH浓度3 mol/L、水解温度55℃、水解时间60 min。在该条件下制备获得的藏红花酸得率可达15.33%±1.25%;含量可达到97.24%±0.78%。优化后方法步骤简单易行,绿色无污染,一步制得高纯度藏红花酸,适用于工业化生产。     

Response Surface Optimization of Lyoprotectant for Lactobacillus bulgaricus During Vacuum Freeze-Drying

The individual and interactive effects of skimmed milk powder, lactose, and sodium ascorbate on the number of viable cells and freeze-drying survival for vacuum freeze-dried powder formulation of Lactobacillus bulgaricus were studied by response surface methodology, and the optimal compound lyoprotectant formulations were gained. It is shown that skim milk powder, lactose, and sodium ascorbate had a significant impact on variables and survival of cultures after freeze-drying. Also, their protective abilities could be enhanced significantly when using them as a mixture of 28% w/v skim milk, 24% w/v lactose, and 4.8% w/v sodium ascorbate. The optimal freeze-drying survival rate and the number of viable cells of Lactobacillus bulgaricus were observed to be (64.41 ± 0.02)% and (3.22 ± 0.02) × 10¹¹ colony-forming units (CFU)/g using the optimal compound protectants, which were very close to the expected values 64.47% and 3.28 × 10¹¹ CFU/g.     

Spray drying as a method for preparing concentrated cultures of Lactobacillus bulgaricus

P. TEIXEIRA, H. CASTRO AND R. KIRBY. 1995. Spray drying and freeze drying as methods for concentration of Lactobacillus bulgaricus starter cultures were compared in terms of viability, lag phase until onset of pH decrease and total acid production. For the experimental conditions used, no significant differences were detected between the methods.
The effect of spray drying on the cell membrane of Lactobacillus bulgaricus was studied. Five separate methods were used to study the theory that spray drying causes cell membrane damage; three relating to leakage of intracellular components from the cell into the surrounding environment (260 and 280 nm absorbing materials, potassium ions and proteins); and two relating to increased cell permeability (increased sensitivity to NaCl and increased permeability to o -nitrophenyl-β-D-galactopyranoside (ONPG). Partial loss of some cytoplasmic material from the damaged cells was observed. The dried cells also became sensitive to NaCl and permeable to ONPG. Heat shock increased the survival of exponential cells as compared to controls but did not result in normal levels found with unshocked stationary phase cells. Heat shock had no effect on stationary phase cells. Different rehydration methods and media were investigated: slow rehydration increased survival.     

Transfection of Lactobacillus bulgaricus protoplasts by bacteriophage DNA

A protoplast transfection system has been developed for Lactobacillus bulgaricus. The procedure involves a polyethylene glycol-mediated fusion of bacteriophage DNA encapsulated in liposomes into mutanolysin-treated cells. With L. bulgaricus B004 and DNA isolated from the phage phi c5004, transfection reached a maximum when at least 95% of the cells were osmotically fragile. The incorporation of phage DNA into liposomes was essential; no transfectants were detected in the absence of liposomes. The largest number of transfectants was observed after longer periods (20 min) of fusion of mutanolysin-treated cells and liposomes with polyethylene glycol. The maximum efficiency of 5 x 10(7) PFU/microgram of DNA was reached after a 24-h incubation in growth media prior to plating transfected cells in an agar overlay to detect the appearance of plaques. A minimum of 4 h of incubation in growth medium after fusion was required to detect the production and release of virions. The possibility that the high frequencies observed were due to bursting of transfected cells and subsequent infection of additional cells was found not to be a factor. The number of transfectants observed was directly proportional to the quantity of DNA added. These results define conditions appropriate for the introduction of DNA into L. bulgaricus.     

Semi-continuous lactic fermentation of whey by Lactobacillus bulgaricus

Semi-continuous tests of lactic fermentation of whey by Lactobacillus buigaricus were carried out using a mixture of hydrolysed milk and vitamins. The volume of the Inoculum varied from 20% to 50% of the reactor working volume. A Monod-like equation correlates the lactic acid productivity and the volume fraction of inoculum.The authors are with the Centro de Desenvolvimento Biotecnológico de Santa Catarina (Biotechnological Development Centre of Santa Catarina), R. Princesa isabel, 114, 89200, Joinville, SC, Brazil. CDEB. L.A. Kulay was an undergraduate student, in 1989, of the Escola de Engenharia Mauá (Mauá Engineering School), São Caetano do Sul, SP, Brazil. W. Borzani is the corresponding author.     

Transfection of Lactobacillus bulgaricus protoplasts by bacteriophage DNA.

A protoplast transfection system has been developed for Lactobacillus bulgaricus. The procedure involves a polyethylene glycol-mediated fusion of bacteriophage DNA encapsulated in liposomes into mutanolysin-treated cells. With L. bulgaricus B004 and DNA isolated from the phage phi c5004, transfection reached a maximum when at least 95% of the cells were osmotically fragile. The incorporation of phage DNA into liposomes was essential; no transfectants were detected in the absence of liposomes. The largest number of transfectants was observed after longer periods (20 min) of fusion of mutanolysin-treated cells and liposomes with polyethylene glycol. The maximum efficiency of 5 x 10(7) PFU/microgram of DNA was reached after a 24-h incubation in growth media prior to plating transfected cells in an agar overlay to detect the appearance of plaques. A minimum of 4 h of incubation in growth medium after fusion was required to detect the production and release of virions. The possibility that the high frequencies observed were due to bursting of transfected cells and subsequent infection of additional cells was found not to be a factor. The number of transfectants observed was directly proportional to the quantity of DNA added. These results define conditions appropriate for the introduction of DNA into L. bulgaricus.     

Optimization of Exopolysaccharide Production by Lactobacillus delbrueckii subsp. bulgaricus RR Grown in a Semidefined Medium

The optimal fermentation temperature, pH, and Bacto-casitone (Difco Laboratories, Detroit, Mich.) concentration for production of exopolysaccharide by Lactobacillus delbrueckii subsp. bulgaricus RR in a semidefined medium were determined by using response surface methods. The design consisted of 20 experiments, 15 unique combinations, and five replications. All fermentations were conducted in a fermentor with a 2.5-liter working volume and were terminated when 90% of the glucose in the medium had been consumed. The population of L. delbrueckii subsp. bulgaricus RR and exopolysaccharide content were measured at the end of each fermentation. The optimum temperature, pH, and Bacto-casitone concentration for exopolysaccharide production were 38°C, 5, and 30 g/liter, respectively, with a predicted yield of 295 mg of exopolysaccharide/liter. The actual yield under these conditions was 354 mg of exopolysaccharide/liter, which was within the 95% confidence interval (217 to 374 mg of exopolysaccharide/liter). An additional experiment conducted under optimum conditions showed that exopolysaccharide production was growth associated, with a specific production at the endpoint of 101.4 mg/g of dry cells. Finally, to obtain material for further characterization, a 100-liter fermentation was conducted under optimum conditions. Twenty-nine grams of exopolysaccharide was isolated from centrifuged, ultrafiltered fermentation broth by ethanol precipitation.     

Inhibition of Growth of Lactobacillus bulgaricus by Purine Deoxyribonucleotides.

Morris, George K. (University of Georgia, Athens), and William L. Williams. Inhibition of growth of Lactobacillus bulgaricus by purine deoxyribonucleotides. J. Bacteriol. 90:715-719. 1965.-An inhibition of growth of Lactobacillus bulgaricus GS was observed with deoxyadenylic acid and deoxyguanylic acid. Deoxynucleotides of cytosine, thymine, and uracil, and the deoxynucleosides of adenine, guanine, cytosine, and thymine were inactive as inhibitors. The inhibition was reversed by liver extract (a crude source of two unidentified growth factors for this organism). With suboptimal concentrations of liver extract, the inhibition was reversed by nucleotides of adenine, guanine, uracil, cytosine, and thymine. When the medium contained partially purified sources of the two growth factors rather than crude liver extract, fewer compounds reversed the inhibition. Adenylic acid and guanylic acid reversed the action of either inhibitor. Inosinic acid reversed inhibition caused by deoxyguanylic acid, but not that caused by deoxyadenylic acid. Thymidylic acid reversed inhibition caused by deoxyadenylic acid better than that caused by deoxyguanylic acid. Uridylic acid and cytidylic acid were no longer effective in reversing the inhibitions. This organism preferentially responded to monophosphorylated compounds as inhibitors and as reversers of inhibitions. Studies on the acid-soluble nucleotide pool revealed an accumulation of adenosine triphosphate, guanosine triphosphate, and an unidentified compound which resembled a nucleotide in its physical properties. These data cannot be explained by known metabolic pathways of nucleic acid biosynthesis. This organism responds differently from other related organisms to nucleic acid derivatives; therefore, it may be a new useful tool for studying nucleic acid metabolism and biosynthesis.     

Identification of Stimulants for Lactobacillus bulgaricus in Tomato Juice

Acid production in milk by Lactobacillus bulgaricus was stimulated by tomato juice or its serum. Preliminary purification of the stimulants involved adsorption and elution on a cation-exchange resin. Unidimensional paper chromatography in two solvent systems was employed in further isolation and purification. Identification of the stimulatory components was based on ultraviolet spectral analysis and thin-layer chromatography. The stimulants were identified as adenine and adenosine.