酸乳中混合乳酸菌的分离及鉴定

利用MRS琼脂培养基和Elliker琼脂培养基分别对保加利亚乳杆菌和嗜热链球茵进行分离.然后,分别挑取疑似茵落进行革兰氏染色,对镜检结果为革兰氏阳性茵,且细茵形状为杆状和球状的菌种进行生物化学鉴定.采用糖发酵试验进行生化鉴定.对保加利亚乳杆茵进行麦芽糖、七叶苷、乳糖、蔗糖和葡萄糖5种糖的发酵试验,而对于嗜热链球菌进行葡萄糖、麦芽糖和蔗糖的发酵试验.而后,对葡萄糖、蔗糖发酵阳性,麦芽糖发酵阴性的菌种进行45℃培养.结论表明:在MRS琼脂培养基上,茵落较大、透明、灰白色,镜检细菌形状为杆状,麦芽糖和七叶苷发酵试验均为阴性,乳糖发酵阳性的茵种,即为保加利亚乳杆菌.在Elliker琼脂培养基上分离出的菌种,镜检细菌形状为球状.葡萄糖、蔗糖发酵呈阳性,麦芽糖发酵阴性且能在45℃条件下生长的茵种即为嗜热链球茵.     

新疆传统发酵酸驼乳中乳酸菌的分离和鉴定

利用MRS琼脂培养基对新疆传统发酵酸驼乳中的乳酸菌进行分离,分别挑取可疑菌落进行革兰氏染色,对镜检结果为革兰氏阳性菌,且细菌形状为球状和杆状的两种菌种进行糖发酵实验与生物化学鉴定.结论是:在两种不同pH值的MRS琼脂培养基上,菌落为灰白色、有透明圈,分别呈球状和杆状的两种菌种为乳球菌和乳杆菌.     

酸奶中嗜热链球菌的分离和鉴定

利用Elliker琼脂培养基对嗜热链球菌进行分离。然后,分别挑取疑似菌落进行革兰氏染色,对镜检结果为革兰氏阳性菌,且细菌形状球状的菌种进行生物化学鉴定。采用糖发酵试验进行生化鉴定。对嗜热链球菌进行葡萄糖、麦芽糖和蔗糖的发酵试验。而后,对葡萄糖、蔗糖发酵阳性,麦芽糖发酵阴性的菌种进行45℃培养。结论:在Elliker琼脂培养基上分离出的菌种,镜检细菌形状为球状,葡萄糖、蔗糖发酵呈阳性,麦芽糖发酵阴性且能在45℃条件下生长的菌种即为嗜热链球菌。     

保加利亚乳杆菌冻干菌种的研究

对酸奶发酵菌种之一--保加利亚乳杆菌的增菌培养基进行了筛选,得到了高活菌数的保加利亚乳杆菌增菌培养基,并进行冷冻升华干燥得到保加利亚乳杆菌的冻干菌种。结果表明:在添加有10%麦芽汁、10%番茄汁、0.05%CaCO3、1.0%乳糖的MRS基础培养基中培养保加利亚乳杆菌18h后活菌数可达到3.96×1011cfu/g。其冻干菌种的活菌数为2.53×1011cfu/g。     

保加利亚乳杆菌浓缩培养的研究

对培养保加利亚乳杆菌的基础培养基进行筛选,确定为MRS培养基。然后优化培养条件:发酵时间为12h,起始pH值为5.80,发酵温度为40℃。最佳培养基:MRS培养基 7.5%番茄汁 12%麦芽汁 0.009mol/LCaCl2 2%乳清。通过中和试验,保加利亚乳杆菌菌体浓度可达到9.55×109mL-1。     

保加利亚乳杆菌高密度培养的初步研究

对培养保加利亚乳杆菌的基础培养基进行优选,确定为基础MRS培养基.然后优化培养条件发酵时间为12h,起始pH为6.4,发酵温度为37℃.培养基MRS培养基 7.5%番茄汁 12%麦芽汁 5%海带汁 2%乳清.通过正交试验证明,保加利亚乳杆菌菌体浓度可达到1.0×1012 cfu/mL.     

乳酸菌培养基-菠萝皮汁与MRS的对比试验

以植物乳杆菌为发酵菌种,对菠萝皮汁和MRS两种培养基进行了对比试验,结果表明:植物乳杆菌在两种培养基中的生长速度、产酸能力和最大活茵数在方面均无显著性差异,菠萝皮汁替代MRS培养基作为乳酸菌发酵培养基具有可行性.     

冻干保加利亚乳杆菌增菌培养基的筛选

本文采用正交实验对冻干保加利亚乳杆菌的增菌培养基进行了筛选,研究了保加利亚乳杆菌在增菌培养基中的活菌数及菌活力,以寻找冻干保加利亚乳杆菌发酵的最适收获期。结果表明:冻干保加利亚乳杆菌的增菌培养基为:MRS基础培养基、麦芽汁体积分数为10%、番茄汁体积分数10%、CaCO3的质量分数为0.05%、乳糖的质量分数为1.0%。保加利亚乳杆菌的最适收获期为发酵18h,此时为对数生长期后期,活菌数为3.96×1011cfu/g,OD值为0.922,菌活力为500T、pH4.46。增菌培养基增菌效果明显,经增菌培养基培养所得活菌数是基础培养基的202倍,且菌活力也较基础培养基强,OD值和滴定酸度分别是基础培养基的5.6倍和1.3倍。     

马铃薯乳酸菌饮料发酵菌种研究

为生产出发酵时间短及活菌数高的马铃薯发酵饮料,以保加利亚乳杆菌、嗜热链球菌、干酪乳杆菌、双歧杆菌为试验菌种,以发酵时间和发酵活菌数为评价指标,进行单菌种和复合菌种发酵试验,筛选出最佳的菌种组合。试验结果表明:在接种量5%、各菌种间接种比例均为1:1、发酵温度为40℃的条件下,保加利亚乳杆菌、干酪乳杆菌和双歧杆菌3菌种复合发酵效果最佳,发酵时间为9.5 h,发酵结束时滴定酸度为44.33°T,活菌数达到9.30×10~8 cfu/mL。     

乳酸菌基础培养基比较研究

研究了脱脂乳中性蛋白酶的最适酶解条件，并以保加利亚乳杆菌和嗜热链球菌为混合发酵菌种，对MRS、脱脂乳和脱脂乳酶解液3种基础培养基进行了比较研究。结果表明：（1）脱脂乳中性蛋白酶的适宜酶解条件为：加酶量5000U／g蛋白质，在pH=7．0，50℃下酶解2h；（2）脱脂乳酶解液是保加利亚乳杆菌和嗜热链球菌混合菌种较好的基础培养基，其冻干发酵剂的质量和数量均优于脱脂乳培养基。以脱脂乳酶解液为基础培养基制备的冻干发酵剂的活菌数达到3．5×10^11cfu／g，冻干发酵剂的重量是脱脂乳的1．93倍。     

发酵香肠的研制

利用自行筛选的保加利亚乳杆菌和嗜热链球菌作为发酵剂,以猪肉为原料进行发酵制备乳酸发酵香肠。确定了最佳培养基:MRS培养基上的菌落相对多,且菌落较大,较为适合菌种生长,PYG培养基次之,LAB培养基菌落数目相对少。确定了最适工艺条件:乳糖添加量0.9%、接种量为4%、发酵温度36℃、发酵时间10h。制品色泽均匀,瘦肉切面呈玫瑰红色,香味浓郁纯正,有发酵型乳酸香肠持有的酸味,润滑的质构,口感舒适。     

Antifungal activity of sodium acetate and Lactobacillus rhamnosus

The inhibition of molds by sodium acetate in deMan Rogosa Sharpe (MRS) medium, along with the antifungal activity of Lactobacillus rhamnosus VT1, was studied by the slope agar plate method. MRS agar prepared with and without sodium acetate was used as the agar substrate. A total of 42 strains of Aspergillus, Penicillium, Fusarium, Alternaria, Cladosporium, and Rhizopus were used to compare sensitivities to the inhibitory activity of sodium acetate and L. rhamnosus VT1. It was found that sodium acetate in MRS medium affected the growth of 33 of the 42 mold strains tested to various degrees. The highest sensitivity to sodium acetate was shown by strains of Fusarium, followed by strains of Penicillium, Aspergillus, and Rhizopus. L. rhamnosus VT1 also inhibited mold growth. A significant finding was that sodium acetate and L. rhamnosus VT1 in combination exhibited a possible synergistic action. Thirty-nine of the 42 mold strains tested were completely inhibited by the presence of both antifungal agents. This finding confirms that sodium acetate, a basic component of commercial MRS medium, has strong antifungal properties, and this must be taken into consideration when evaluating the antifungal activity of Lactobacillus cultures grown in MRS broth.     

四川泡菜中植物乳杆菌的筛选与鉴定

该研究旨在从四川泡菜中筛选植物乳杆菌,为益生菌筛选提供来源。经MRS琼脂培养基筛选,采用革兰氏染色法,生化鉴定法,结合分子生物学方法,对筛选的菌株进行鉴定。VITEK 2棒状杆菌鉴定卡（CBC）共涉及到41个生化反应,能够鉴定到种属水平,该方法能够很好的筛选植物乳杆菌。经CBC卡鉴定,20个菌株中筛选到12个菌株,可鉴定为植物乳杆菌。这些菌株经16S rRNA PCR扩增测序后,在NCBI网站上进行同源性比对,确定为植物乳杆菌。由邻接法构建的系统进化树可知,该12个菌株与已知植物乳杆菌序列聚为一簇,6号菌株与Lactobacillus plantarum 3356聚为一支,亲缘关系更近;4号、5号、14号、15号、23号和38号菌株与Lactobacillus plantarum NCU116,Lactobacillus plantarum MBEL2169,Lactobacillus plantarum AN7等序列相似性极高,无遗传距离。综合所有的鉴定结果,这12个菌株都被鉴定为植物乳杆菌。CBC鉴定卡是初筛植物乳杆菌较好的方法。     

A collaborative study of a method for the enumeration of probiotic bifidobacteria in animal feed

An enumeration method to be used as an official control method in the framework of Council Directive 70/524/EEC for probiotic bifidobacteria used as feed additives was validated. Seventeen laboratories in 11 European Countries carried out a collaborative study. A spread plate method following BS ISO 15214:1998 using four different agars, Man Rogosa Sharpe (MRS), acidified MRS, MRS with triphenyl tetrazolium chloride (TTC) and a selective bifidobacteria medium, was validated. Precision data in terms of repeatability (r) and reproducibility (R) of the method for each medium using different feeding stuffs with a high and a low inoculation level were determined. Bifidobacteria were present in the samples as a single component or in mixtures with other probiotics. The enumeration of bifidobacteria on all agars showed a relative standard deviation of repeatability (RSD(r)) between 1.2% and 6.3% and a relative standard deviation of reproducibility (RSD(R)) between 2.6% and 8.7%. MRS agar was preferred, followed by acidified MRS and MRS+TTC agar. The selective bifidobacteria medium gave similar counts as the MRS media. For routine analysis, the use of MRS agar with supplementation of cysteine hydrochloride (the selective bifidobacteria medium without antibiotics) is recommended. Depending on the presence and concentration of other probiotics such as enterococci, lactobacilli and pediococci, acidified MRS or MRS+TTC agar is recommended. The selective bifidobacteria medium was selective for bifidobacteria.An official control method for enumeration of probiotic bifidobacteria as a single component and in mixtures with other probiotic microorganisms in feeding stuffs was validated. The methodology is not applicable to mineral feed. The results are intended for consideration for adaptation as CEN and ISO standards.     

培养基pH调控法高密度发酵唾液乳杆菌XH4B研究

本研究以MRS培养基为基础,通过优化碳、氮源及无机盐的配方和用量,再结合补料发酵,最终实现唾液乳杆菌XH4B高密度培养的目的。以乳酸菌的生物量为指标,同时考查发酵液pH值、乳酸含量等,最终确定酵母粉和蔗糖为最佳氮、碳源,同时增加乙酸钠用量至2%、磷酸二氢钠0.6%,可以对发酵液酸化时提供一定的缓冲作用。采用优化的PY-Suc培养基,唾液乳杆菌XH4B的生物量最高能达到6.91 g/L,明显高于MRS培养基的5.01~6.30 g/L(P0.05)。等量补料培养并且采用NaOH中和发酵液pH值时,乳酸最高积累速度可以达到5.958 g/(L·h),但是随着培养时间延长,积累速度迅速下降。发酵酸化较严重时(乳酸含量9~10 g/L),唾液乳杆菌XH4B的生物量积累变缓。结论:优化MRS培养基,并加大乙酸钠、磷酸二氢钠等能够缓冲发酵液的无机盐用量,结合补料发酵,可以实现唾液乳杆菌XH4B的高密度培养。     

酸乳菌种分离纯化方法

本文研究了在脱脂乳中保藏的发酵酸乳菌种的分离纯化办法。确定了德氏乳杆菌保加利亚亚种的分离纯化培养基为改良MRS，以10^-2稀释度划线分离法在烛缸中厌氧培养可以达到很好的分离效果，添加2％的玉米浆后有利于纯菌株的生长；采用涂布分离法和真空袋培养，在链球菌基础培养基上可很好地分离出唾液链球菌嗜热业种的纯菌株。经鉴定所分离出菌株为纯种后，采用斜面保藏在不含乳基质的培养基上，在15d内可以保持其活力。     

Evaluation of culture media for enumeration of Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium animalis in the presence of Lactobacillus delbrueckii subsp bulgaricus and Streptococcus thermophilus

The study compared the growth capability of probiotic (Lactobacillus acidophilus La05, Lactobacillus casei Lc01 and Bifidobacterium animalis Bb12) and non-probiotic (Lactobacillus delbrueckii subsp bulgaricus and Streptococcus thermophilus) cultures on twenty-one culture media grouped according to selectivity: non-selective agars, selective agars without antibiotics and MRS agars containing different combinations of lithium chloride, cystein, bile salts and antibiotics. Four of these media were selected for quantitative enumeration of L. acidophilus La05, L. casei Lc01, and B. animalis Bb12. The best culture media and incubation conditions for enumeration of the probiotic cultures were: B. animalis: MRS agar with dicloxacillin, 37 °C or 42 °C, anaerobiosis; L. acidophilus: MRS agar with bile salts, 37 °C or 42 °C, aerobiosis; L. casei: MRS agar with lithium chloride and sodium propionate, 37 °C or 42 °C, aerobiosis or anaerobiosis. Plating on MRS with glucose replaced by maltose, 37 °C or 42 °C, anaerobiosis, will distinguish probiotic from non-probiotic cultures. For enumeration of each probiotic in a mixed culture, the following media and incubation conditions were recommended: B. animalis: 4ABC-MRS, 42 °C, anaerobiosis, L. acidophilus: LC medium, 42 °C, aerobiosis or anaerobiosis and L. casei: LP-MRS, 42 °C, aerobiosis or anaerobiosis. In all experiments, differences in counts using pour plating or surface plating were not significant (P ≤ 0.05).     

啤酒有害菌检测培养基的比较

比较了乳酸短杆菌、四联球菌、醋化醋酸杆菌在NBB、MRS、Raka -Ray、VLB -S7、番茄汁培养基、自配培养基A和自配培养基UBA上的菌落生量。结果表明 ,NBB对乳酸杆菌的检测效果最好 ,乳酸菌在自制配养基UBA上不生长。 7种培养基对四联球菌的检测效果相当。醋酸是绝对好氧菌 ,在厌氧条件下不生长。     

导致黄酒酸败的食果糖乳杆菌的分离、鉴定及其检测条件优化

为提高黄酒中腐败微生物的检测效率,通过传统培养,结合感官评价和总酸变化对导致黄酒酸败的微生物进行分析,同时选择性筛选出目标腐败菌ZH-1和ZH-2,经形态学、生化实验和16S rRNA基因鉴定,并进一步采用单因素和Box-Behnken响应面试验设计方法对目标腐败菌的检测条件进行优化。结果表明:黄酒腐败呈浑浊、异味和总酸升高等特点;分离得到的菌ZH-1和菌ZH-2均鉴定为食果糖乳杆菌,该菌是导致黄酒此类酸败的目标腐败菌,表现为在固体培养基上生长缓慢等特点,采用GB 4789.35—2016《食品微生物学检验乳酸菌检验》的方法,需要9～10 d出结果;对食果糖乳杆菌检测方法优化得到最优的培养条件为:在MRS固体培养基基础上,添加质量分数为0.08%的L-Cys,调节pH值为5.4,临用时加入体积分数为9%的无水乙醇,培养温度为30℃。采用优化后的方法,定量检出时间缩短为3～4 d。     

嗜酸乳杆菌、双歧杆菌和干酪乳杆菌的选择性计数

介绍了干酪乳杆菌、嗜酸乳杆菌和双歧杆菌的选择性计数方法。LC培养基只能计数干酪乳杆菌，MRS-水杨素（或山梨醇）培养基可以计数嗜酸乳杆菌和干酪乳杆菌；而MRS培养基可以计数这3种益生菌，通过减法原则从嗜酸乳杆菌、干酪乳杆菌和双歧杆菌混合物中单独计数。另外，MRS-NNLP培养基也可用于选择性计数双歧杆菌。