多菌种酸奶中活性乳酸菌的计数方法研究

利用4种选择性培养基MRS、MRS-山梨醇、MRS-5．2、Elliker,采用平板涂布法和倾注接种法。在蜡烛缸法（缺氧）、封口膜法（微氧）和供氧条件下对4种市售多菌种酸奶中保加利亚乳杆菌、嗜热链球菌、嗜酸乳杆菌、双歧杆菌进行选择性计数方法研究。结果表明：蜡烛缸法较能反映乳酸菌活菌数量的实际情况,封口膜法次之;在不同培养条件下4种选择性培养基对于乳酸菌活菌的计数都是灵敏的;而涂布法和倾注法接种活菌数有显著差异,倾注法要优于涂布法。     

EZAL-MY96乳酸菌种菌株分离鉴定

目的：对引进法国罗地亚EZAL-MY96直投式酸奶发酵剂(DVS)进行菌株分离鉴定。方法：利用半选择性培养基进行发酵剂菌株分离,并采用糖发酵实验和RAPD指纹图谱进行鉴定。结果：EZAL-MY96中球菌的平板菌落与光镜显微观察、糖发酵实验和RAPD指纹图谱均只有1种情况,而杆菌均有2种情况。结论：从EZAL-MY96菌种中共分离到3株菌,其中1株鉴定为嗜热链球菌,1株为德氏乳杆菌保加利亚亚种,另1株为未知的乳杆菌。     

两株保加利亚乳杆菌产β-半乳糖苷酶条件的优化及酶活力测定

目的优化2株保加利亚乳杆菌产β-半乳糖苷酶的培养条件,测定其在最适于产酶条件下的酶活力,并初步观察酶活力稳定性。方法测定2株保加利亚乳杆菌——来源于酸奶的wch9901和标准菌株1．1480在不同培养时间、培养基碳源、摇床转速、气体环境下产生的β-半乳糖苷酶的活力,优化产酶条件。测定2株菌在其最适产酶条件下的酶活力。将制得的1．1480粗酶液分别置冰浴和20℃水浴,每隔1h测定一次粗酶液中β-半乳糖苷酶活力,观察酶活力稳定性。结果2株保加利亚乳杆菌的最适产酶条件分别为：1．1480于30℃有氧静止培养36h;wch9901于37℃厌氧静止培养18h,培养基含乳糖。在最适产酶条件下,1．1480的酶活力为0．321NLU／ml粗酶液,比酶活为2．469NLU／mg蛋白;wch9901的酶活力为0．401NLU／ml粗酶液,比酶活为6．169NLU／mg蛋白。1．1480粗酶液于冰浴可稳定保存6h,于20℃水浴可稳定保存5h。结论wch9901与1．1480的最适产酶条件有所差异,前者产酶迅速,产酶量多。     

发酵乳中动物双歧杆菌乳亚种分离及鉴定

采用改良MRS培养基从蒙牛冠益乳酸奶中选择性分离得到2种乳酸菌, 表型特征检测初步确定目标菌株BB-12, 该菌株符合双歧杆菌属特征描述。通过Biolog微生物自动鉴定系统鉴定、16S rDNA序列分析、atpD基因序列分析, 多相鉴定表明: 菌株BB-12为动物双歧杆菌乳亚种(Bifidobacterium animalis subsp. lactis)。系统发育学分析表明: atpD基因序列比16S rDNA序列在动物双歧杆菌亚种水平鉴定上有更好的分辨率。     

双歧杆菌凝固型无糖酸奶生产工艺研究

对双歧杆菌凝固型无糖酸奶的生产工艺进行了研究。采用接种双歧杆菌、保加利亚乳杆菌和嗜热链球菌3菌株进行发酵。确定最佳工艺条件为双歧杆菌发酵剂接种量5%、木糖醇6%于39℃条件下培养8h后,再接入接种比例为1:1、接种量为4%的保加利亚乳杆菌、嗜热链球菌混合发酵剂,于42℃条件下培养3 h。在此工艺条件下生产的酸奶风味纯正,凝乳状态良好,口感细腻,具有天然的奶香味。     

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。     

双歧杆菌与乳杆菌原生质体的融合及筛选

目的：通过双歧杆菌与乳杆菌的原生质体融合构建耐氧双岐杆菌,以解决双岐杆菌制剂开发中始终存在的“活菌数低”和存活时间短等问题。方法：采用不同浓度的Mutanolysin分别制备长岐杆菌和保加利亚乳杆菌的原生质体,在电场作用下诱导长双岐杆菌原生质体和经56℃热灭活30min的保加利亚乳杆菌原生质体融合,并在双层再生培养基上筛选融合菌株。结果：成功地构建出几种较为稳定的兼具长双岐杆菌和保加利亚乳杆菌生物学特性的融合菌株;其中融合株F2具有良好的耐氧性;且能在有氧、CO2条件下良好生长。结论：通过原生质体融合技术能改良双歧杆菌的严格厌氧特性,有利于对双岐杆菌的开发和应用。     

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.     

H.ylori尿素酶B亚单位在保加利亚乳杆菌的表达与鉴定

目的构建表达幽门螺杆菌（Helicobacter pylori,H、pylori）尿素酶B亚单位（UreB）的基因工程乳杆菌,并对其进行初步的安全性评估。方法采用高保真PCR从H.pylori标准菌株NCTC 11637中扩增ureB基因,插入乳酸菌表达质粒pMG36e,将重组质粒电转入保加利亚乳杆菌L6032中,获得表达ureB的基因工程乳杆菌。在含乳糖的MRS培养基诱导目的蛋白表达,Western blot鉴定其免疫原性。连续传代培养60代,检测基因工程乳杆菌的稳定性、形态学与生理生化特性以进行初步的安全性评估。结果特异PCR、酶切和测序鉴定均证实ureB基因克隆入表达载体pMG36e,SDS-PAGE结果显示,重组质粒pMG36e-ureB电转入保加利亚乳杆菌所构建的基因工程乳杆菌能表达约64KD的蛋白,Western blot证明该蛋白能与抗H.priori ureB的兔血清反应。稳定性、形态学与生理生化特性检测结果表明,基因工程乳杆菌与原始菌株保加利亚乳杆菌完全一致。结论成功构建能表达H.pylori UreB的保加利亚乳杆菌L6032-UreB,该基因工程菌在形态与生理生化特性上未发生任何变异,从而为探索幽门螺杆菌感染的益生菌制剂调理疗法奠定了坚实的基础。     

高活性乳酸菌发酵剂培养条件优化及活性测定

采用自行分离选育的乳酸菌进行混合培养,对浓缩培养条件进行正交试验,以德氏乳杆菌保加利亚亚种和嗜热链球菌的菌数及其比值作为考察指标。结果为以改良MRS为基础培养基,以6%番茄汁、0.3%玉米浆作为生长因素,42℃培养,过程采用流加氨水作中和试剂,使培养基酸度控制在pH 6.0,浓缩培养液中菌数可达9.15×109个/mL,球菌与杆菌比例为1.2∶1;最佳菌体收集时间为7.5 h;-30℃下冷冻干燥得到的乳酸菌发酵剂含活菌数达到8.2×1010个/g,球菌与杆菌比例为1.5∶1,发酵剂活性达0.435;0~4℃冷藏6个月,乳酸菌活性没有损失。产品可直接加入到灭菌奶中发酵,具有大众化特点,广泛用于各类乳品厂、饭店、食堂及家庭。     

Isolation and characterization of Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus from plants in Bulgaria

One of the traditional ways of preparation of yogurt starter in Bulgaria is placing a branch of a particular plant species into boiled sheep's milk maintained at about 45°C, which is further incubated until a dense coagulum is obtained. To investigate the possible origin of the yogurt starter bacteria, Lactobacillus delbrueckii ssp. bulgaricus ( L. bulgaricus ) and Streptococcus thermophilus ( S. thermophilus ), the traditional way of yogurt-starter preparation was followed. Hundreds of plant samples were collected from four regions in Bulgaria and incubated in sterile skim milk. The two target bacteria at low frequencies from the plant samples collected were successfully isolated. Phenotypic and genotypic characteristics of these bacterial isolates revealed that they were identified as L. bulgaricus and S. thermophilus . Twenty isolates of L. bulgaricus and S. thermophilus , respectively, were selected from the isolated strains and further characterized with regard to their performance in yogurt production. Organoleptic and physical properties of yogurt prepared using the isolated strains from plants were not significantly different from those prepared using commercial yogurt-starter strains.
It was therefore suggested that L. bulgaricus and S. thermophilus strains widely used for commercial yogurt production could have originated from plants in Bulgaria. To our knowledge, this is the first report on the isolation and characterization of L. bulgaricus and S. thermophilus strains from plants.     

ACCELERATION OF YOGURT FERMENTATION BY BACTERIAL MEMBRANE FRACTION BIOCATALYSTS

Bacterial membrane fraction biocatalysts obtained from Escherichia coli (E-8), Gluconobacter oxydans (Gox) and Acetobacter xylinum (Acx), as well as the commercial oxygen scavenger Oxyrase^TM, at concentrations of 0.1–2.0 U/ml effectively stimulated cell growth of Lactobacillus bulgaricus and Sreptococcus thermophilus, and lactic acid production during yogurt fermentation. The membrane fractions scavenged oxygen in the fermented milk to an optimal oxygen tension for growth of yogurt cultures. The yogurt culture populations with membrane fraction(s) increased faster than those without. Total counts in the presence of yogurts with Oxyrase, E-8, Gox, and Acx were 0.5–1, 0.5, 1.5, or 1.2 log cycles, respectively, greater than counts of the control after 3 h of fermentation. The commercial membrane fraction Oxyrase reduced the fermentation time by 1 h needed to reach pH 4.5 compared to the controls, while E-8, Gox, and Acx reduced time by about 0.5 h, 1.5 h and 1–1.5 h, respectively, depending on the membrane concentrations. The titratable acidity was corresponded well with the reduction in pH. Ratios of rods to cocci among the samples with and without membrane fraction supplementation were not different (P > .05). Each membrane fraction biocatalyst enhanced the depletion rate of dissolved oxygen in a yogurt mix differently.     

Detection of extracellular bound proteinase in EPS-producing lactic acid bacteria cultures on skim milk agar

AIMS: Skim milk agar was developed to investigate extracellular cell-bound proteinase in yogurt cultures, Streptococcus thermophilus and Lactobacillus bulgaricus. METHODS AND RESULTS: The Lact. bulgaricus cultures produced more extracellular cell-bound proteinase than did Strep. thermophilus cultures. Strong positive correlations between the size of the exopolysaccharide (EPS) layer and extracellular cell-bound proteinase were found for both Streptococcus and Lactobacillus cultures. CONCLUSION: Strong positive linear relationships existed between the EPS size and colony size and the diameter of clear zone and colony size for Streptococcus cultures, whereas weak positive linear relationships were observed for Lactobacillus cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: These data are useful to validate the relationship between extracellular proteinase and the EPS size of LAB. Also, a convenient medium to detect the presence of extracellular cell-bound proteinase of LAB is valuable for dairy industries.     

Production of natural folates by lactic acid bacteria starter cultures isolated from artisanal Argentinean yogurts

Folate is a B-group vitamin that cannot be synthesized by humans and must be obtained exogenously. Although some species of lactic acid bacteria (LAB) can produce folates, little is known about the production of this vitamin by yogurt starter cultures. Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains were isolated from artisanal Argentinean yogurts and were grown in folate-free culture medium (FACM) and nonfat milk after which intracellular and extracellular folate production were evaluated. From the initial 92 isolated LAB strains, 4 L. delbrueckii subsp. bulgaricus and 32 S. thermophilus were able to grow in the absence of folate. Lactobacillus delbrueckii subsp. bulgaricus CRL 863 and S.?thermophilus CRL 415 and CRL 803 produced the highest extracellular folate levels (from 22.3 to 135?μg/L) in FACM. In nonfat milk, these strains were able to increase the initial folate concentrations by almost 190%. This is the first report where native strains of L. delbrueckii subsp. bulgaricus were shown to produce natural folate. The LAB strains identified in this study could be used in developing novel fermented products bio-enriched in natural folates that could in turn be used as an alternative to fortification with the controversial synthetic chemical folic acid.     

Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus survive gastrointestinal transit of healthy volunteers consuming yogurt

To date, there is significant controversy as to the survival of yogurt bacteria (namely, Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus) after passage through the human gastrointestinal tract. Survival of both bacterial species in human feces was investigated by culture on selective media. Out of 39 samples recovered from 13 healthy subjects over a 12-day period of fresh yogurt intake, 32 and 37 samples contained viable S. thermophilus (median value of 6.3 x 10(4) CFU g(-1) of feces) and L. delbrueckii (median value of 7.2 x 10(4)CFU g(-1) of feces), respectively. The results of the present study indicate that substantial numbers of yogurt bacteria can survive human gastrointestinal transit.     

Growth and activity of Bulgarian yogurt starter culture in iron-fortified milk

Bulgarian yogurts were manufactured and fortified with 8, 15 and 27 mg of iron kg(-1) of yogurt. The growth and acidifying activity of the starter culture bacteria Streptococcus thermophilus 13a and Lactobacillus delbrueckii subsp. bulgaricus 2-11 were monitored during milk fermentation and over 15 days of yogurt storage at 4 degrees C. Fortifying milk with iron did not affect significantly the growth of the starter culture during manufacture and storage of yogurt. Counts of yogurt bacteria at the end of fermentation of iron-fortified milks were between 2.1 x 10(10) and 4.6 x 10(10) CFU ml(-1), which were not significantly different from numbers in unfortified yogurts. In all batches of yogurt, the viable cell counts of S. thermophilus 13a were approximately three times higher than those of L. delbrueckii subsp. bulgaricus 2-11. Greater decrease in viable cell count over 15 days of storage was observed for S. thermophilus 13a compared to L. delbrueckii subsp. bulgaricus 2-11. Intensive accumulation of lactic acid was observed during incubation of milk and all batches reached pH 4.5 +/- 0.1 after 3.0 h. At the end of fermentation process, lactic acid concentrations in iron-fortified yogurts were between 6.9 +/- 0.4 and 7.3 +/- 0.5 g l(-1). The acidifying activity of starter culture bacteria in the control and iron-fortified milks was similar. There was no increase in oxidized, metallic and bitter off-flavors in iron-fortified yogurts compared to the control. Iron-fortified yogurts did not differ significantly in their sensorial, chemical and microbiological characteristics with unfortified yogurt, suggesting that yogurt is a suitable vehicle for iron fortification and that the ferrous lactate is an appropriate iron source for yogurt fortification.     

Selective enumeration of Lactobacillus casei from yogurts and fermented milk drinks

A selective medium (LC agar) was developed for enumeration of Lactobacillus casei populations from commercial yogurts and fermented milk drinks that may contain strains of yogurt bacteria (Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus), probiotic bacteria (Lactobacillus acidophilus and bifidobacteria) and L. casei. Appropriate dilutions were pour-plated in specially formulated LC agar acidified to pH 5.1 and the plates incubated at 27°C for 72 to 96 h under anaerobic conditions. Growth of S. thermophilus was prevented by adjusting pH to 5.1. L. delbrueckii ssp. bulgaricus did not ferment ribose as the carbon source, as a result the organisms did not form colonies. L. acidophilus formed colonies on MRS-ribose agar; however, this organism did not grow in the specially formulated LC agar containing ribose. Similarly, Bifidobacterium spp. did not form colonies in LC agar. L. casei formed colonies on LC agar. © Rapid Science Ltd. 1998     

Improvement of carotenoid-synthesizing yeast Rhodotorula rubra by chemical mutagenesis

A mutant Rhodotorula rubra with enhanced carotenoid-synthesizing activity for synthesizing total carotenoids and beta-carotene was obtained by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. When co-cultivated with yogurt starter bacteria (Lactobacillus bulgaricus + Streptococcus thermophilus) in whey ultrafiltrate it produced 15.7 mg total carotenoids l(-1) culture fluid or 946 microg total carotenoids g(-1) dry cells of which 71% was beta-carotene. Grown as a monoculture in glucose substrate, the mutant shown 1.4 times lower carotenoid-synthesizing activity, and the relative share of beta-carotene in the total carotenoids was lower (63%). The individual pigments torulene and torularhodin were identified, whose mass fractions were (29% and 7%) and (24% and 4%), respectively, for the mutant grown as a monoculture and as a mixed culture with the yogurt bacteria.     

Exopolysaccharides produced by mixed culture of yeast Rhodotorula rubra GED10 and yogurt bacteria (Streptococcus thermophilus 13a + Lactobacillus bulgaricus 2-11)

AIMs: The studies of the production of exopolysaccharides by lactose-negative yeast and a yogurt starter co-cultivated in a natural substrate containing lactose may be considered of interest because they reveal the possibilities for high-efficiency synthesis of biopolymers by mixed cultivation. METHODS AND RESULTS: The mixed culture Rhodotorula rubra GED10 + (Streptococcus thermophilus 13a + Lactobacillus bulgaricus 2-11) was cultivated in cheese whey ultrafiltrate (WU) (44.0 g lactose l(-1)) at initial pH 6.0, 28 degrees C, under intensive aeration (air-flow rate 1.0 l l(-1) min(-1), agitation 220 rev min(-1)) in a MBR AG fermentor. The mixed culture manifested the highest activity for synthesis of exopolysaccharides (19.3 g l(-1)) and cell mass (21.0 g l(-1)) at the 84th hour. The yogurt starter synthesized neutral exopolysaccharides, while the mixed culture yeast + yogurt starter produced acidic exopolysaccharides containing uronic acid (6%). The neutral sugar composition was identified as mannose, glucose, galactose, xylose and arabinose. Mannose dominated in the polymer composition (83%) that was produced only by the yeast (97%). CONCLUSIONS: Lactose in the WU can be effectively utilized by a co-culture of lactose-negative yeast-yogurt starter for synthesis of exopolysaccharides. SIGNIFICANCE AND IMPACT OF THE STUDY: The present findings propose an alternative use of WU as a cost-effective carbohydrate substrate, and suggest that the lactose-negative yeast Rhodotorula rubra can have industrial application as producers of exopolysaccharides.     

Construction of a new shuttle vector for Lactobacillus.

To clone the malolactic enzyme gene from Lactobacillus sp. 89, construction of a shuttle vector able to express itself in Lactobacillus sp. 89 and Escherichia coli was undertaken. The shuttle plasmid pLE16 resulted from the union of pBR328 and of the pLB10 plasmid extracted from Lactobacillus bulgaricus 10. The bacterial transformation in Lactobacillus sp. 89 was performed by electroporation, and the clones were selected on MRS medium with 30 micrograms.mL-1 chloramphenicol added. Fifty percent of the clones from Lactobacillus sp. 89 lost their resistance to chloramphenicol following 28 generations when the selection pressure was not maintained. The restriction map of pLE16 (7600 bp) was established using several restriction enzymes.