两株乳酸菌浓缩培养过程中的细胞凋亡研究

采用优化培养基和二次培养对保加利亚乳杆菌(L.b)和嗜热链球菌(S.t)进行了超浓缩培养,研究了超浓缩培养过程中菌体的细胞凋亡、生长曲线和乳酸浓度变化曲线。结果表明,保加利亚乳杆菌和嗜热链球菌超浓缩培养过程中存在细胞凋亡现象,与产酸规律密切相关。本研究为揭示超浓缩培养过程中保加利亚乳杆菌和嗜热链球菌的细胞凋亡机制提供了实验依据。     

全脂乳中2种乳酸菌的HPLC研究

研究了可应用于高效液相离子交换色谱分析的嗜热链球菌及保加利亚乳杆菌的培养方法,同时探讨了全脂乳中培养的乳酸菌HPLC上样菌悬液的制备方法。对全脂乳中培养不同时间的嗜热链球菌及保加利亚乳杆菌进行色谱表征,并首次发现保加利亚乳杆菌在全脂乳培养过程中菌体形态变化与色谱表征的一致性。     

嗜热链球菌MN002冻干菌粉的制备工艺

为制备高活性嗜热链球菌MN002冻干菌粉,对富集培养后不同离心条件下嗜热链球菌MN002的存活率进行了研究。同时,以菌体浓缩倍数为指标,对蛋白溶解剂进行了组合筛选。进而通过单因素实验和二次正交旋转组合设计实验,考查了11种不同冻干保护剂对嗜热链球菌MN002的冻干保护效果,以获得嗜热链球菌MN002真空冷冻干燥的最佳保护剂配方。结果表明,嗜热链球菌MN002在离心转速为8 000 r/min,离心温度4℃条件下,菌体离心存活率最高。同时,以0.15%六偏磷酸钠和0.5%柠檬酸钠作为组合蛋白洗脱液,可大大提高MN002菌体浓缩倍数。试验确定嗜热链球菌MN002的最佳保护剂配方为:5%蔗糖、2%甘油、0.8%谷氨酸钠、1%抗坏血酸、5%海藻糖,在此条件下,嗜热链球菌MN002菌体存活率可达84.8%。     

不同pH值培养乳酸球菌对其发酵活力的影响

为了确定嗜热链球菌St-1最适生长pH值,研究了在不同pH值条件下培养嗜热链球菌St-1,对其活菌数、发酵活力及菌体形态学特征的影响.结果表明,当培养体系的pH值恒定在6.0时,其活菌数和发酵活力最高;对比发酵活力曲线和生长曲线表明,发酵活力最好时的活菌数并不是最高的;对比不同恒定pH值条件下培养活力最高时的嗜热链球菌St-1菌体形态特征发现,嗜热链球菌St-1的链越长,其发酵活力越低,链越短发酵活力越高.     

嗜热链球菌对生物活性肽QEPV吸收和利用的研究

以嗜热链球菌和生物活性肽Gln-Glu-Pro-Val(QEPV)为研究对象,探索了乳酸菌对生物活性肽的吸收和利用。首先,用添加荧光标记QEPV的M17培养基培养嗜热链球菌,采用流式细胞术检测QEPV能否被嗜热链球菌吸收。其次,测定不同剂量生物活性肽QEPV对嗜热链球菌活菌数、发酵液p H值、菌体浓度的影响,以探索嗜热链球菌对生物活性肽的利用情况。流式细胞术检测结果表明,FAM-QEPV与FITC-QEPV能够穿过嗜热链球菌细胞膜,推测QEPV能够被嗜热链球菌吸收、利用。并且0.5 mg/m L的QEPV能有效促进嗜热链球菌的生长繁殖,提高嗜热链球菌的发酵能力。     

嗜热链球菌M5-5冻干保护剂配方的优化

为了研究不同冻干保护剂在真空冷冻干燥过程中对嗜热链球菌M5-5菌体细胞的保护效果,采用单因素试验和正交试验,以冻干后的菌体存活率为评价指标,考察9种冻干保护剂在冷冻干燥过程中对M5-5菌体的保护效果,以期获得最优保护剂配方。结果表明,海藻糖、甘油和脱脂乳作为单一冻干保护剂时,菌体细胞存活率分别为43.85%、47.51%和40.86%,显著高于其他6种保护剂（P＜0.05）;通过正交试验确定冻干保护剂的最优配方为海藻糖60 g/L、谷氨酸钠20 g/L、甘油20 g/L、脱脂乳150 g/L,此时嗜热链球菌M5-5的冻干存活率可达88.41%。扫描电镜结果显示,添加保护剂配方组菌体细胞完整,表面光滑,说明该保护剂配方能有效减少冷冻干燥过程对菌体细胞的损伤。     

嗜热链球菌分批发酵工艺放大研究

近年来,随着国内消费者对酸奶制品需求量的激增,使得乳酸菌浓缩培养成为亟待解决的难题。针对这种情况,在确定嗜热链球菌适宜的碳、氮源分别为乳糖和蛋白胨、适宜的碳氮比例为2∶1的基础上,将嗜热链球菌从小试培养放大到10L发酵罐。试验结果初步证实,三颈瓶比三角瓶更适于将实验室成果向小规模工业发酵过渡,只要保证关键参数与发酵罐相同或接近,嗜热链球菌经三颈瓶培养6h的最大活细胞数可达到3.81×109cfu/mL,与10L发酵罐的3.78×109cfu/mL菌体产量相似。     

抗冻多肽对嗜热链球菌冷冻干燥的保护作用

目的:研究抗冻多肽对嗜热链球菌冷冻干燥的保护作用并探讨其机理。方法:应用OD快速检测法检测冷冻干燥后的嗜热链球菌存活率,将不同浓度的抗冻多肽溶液添加到嗜热链球菌菌泥中,冷冻干燥后检测其存活率。结果:抗冻多肽能够降低嗜热链球菌冷冻干燥损伤,通过比较冻干菌粉中乳酸菌存活率得到抗冻多肽最佳添加质量浓度为1 mg/mL,最适pH 8.0,此时添加抗冻多肽的样品组存活率显著高于空白组和阳性对照组。在扫描电镜下观察嗜热链球菌冻干菌粉,添加抗冻多肽的菌体细胞饱满、完整,空白组则出现皱缩和塌陷,表明抗冻多肽对菌体细胞有保护作用。     

乳酸菌冷冻保护剂配方设计

乳酸菌浓缩发酵剂的制备过程中,冷冻对菌体的损伤比较明显。通过在保加利亚乳杆菌和嗜热链球菌混菌菌悬液中添加不同的保护剂并测定冷冻前后乳酸菌菌数,筛选出适用的保加利亚乳杆菌和嗜热链球菌混菌的保护剂。最终确定的保加利亚乳杆菌和嗜热链球菌混菌保护剂配方为:蔗糖浓度为10%,脱脂乳浓度为10%,谷氨酸钠浓度为5%。菌体经冷冻处理后,冷冻存活率可达到95.0%,比不添加保护剂的菌体存活率大大提高。此配方也简单易得,完全可以满足生产需要。     

红曲酸奶中乳酸菌变化情况研究

利用超声波浸提法提取红曲中的有效成分,将红曲水提取液与全脂奶粉混合发酵制成新型红曲酸奶。通过荧光原位杂交（FISH）技术跟踪发酵过程中乳酸菌含量的变化,结果表明：红曲酸奶中保加利亚乳杆菌和嗜热链球菌数量显著高于普通酸奶。发酵2 h时,红曲酸奶中保加利亚乳杆菌与嗜热链球菌菌体数量分别为普通酸奶的2.63和2.24倍;发酵5 h时,发酵接近终点,红曲酸奶中保加利亚乳杆菌与嗜热链球菌菌体数量分别为普通酸奶的2.29和2.75倍。红曲酸奶的保藏期可达15 d,乳酸菌数符合国家标准的要求。     

酶解法制备草鱼皮蛋白水解物增殖嗜热链球菌

嗜热链球菌被认为是一种多功能益生菌,将Neutrase 0.8L、Protease P “Amano”6、ProteAX、Alcalase 2.4LFG和风味中性蛋白酶5 种草鱼皮蛋白水解物作为培养嗜热链球菌的氮源,以水解物对嗜热链球菌的增殖效果为评价指标,选择最佳水解用酶,并利用正交试验优化酶解条件。结果表明,5 种微生物蛋白酶中,Neutrase 0.8L是水解草鱼皮蛋白的最佳用酶,最佳的酶解条件为：加酶量1%、酶解时间80 min、起始pH 6.5、料水比1∶2（m/V）。利用Neutrase 0.8L酶解法制备得到的富含胶原蛋白肽的草鱼皮蛋白水解物,是一种培养嗜热链球菌的理想氮源。     

Effect of chemico-physical parameters on the histidine decarboxylase (HdcA) enzymatic activity in Streptococcus thermophilus PRI60

In this study the activity of the histidine decarboxylase (HdcA) of Streptococcus thermophilus PRI60 was determined during growth and in crude enzyme preparations to evaluate its hazardousness in dairy products. The effect of different pH values, lactose availability, NaCl concentration, and growth temperature on histamine production was evaluated in M17 medium during 168 h incubation. In each case, the production of histamine increased concomitantly with the cell number with a relatively small further rise during the stationary phase. In all cultures the maximum histamine levels were reached at the end of active growth. Histamine was detectable (10 to 55 mg/L) even when growth was strongly inhibited. The HdcA enzyme in crude cell-free extracts was mostly active at acidic pH values common in dairy products. NaCl concentrations lower than 5% did not affect its activity. The enzyme was quite resistant to heat treatments resembling low pasteurization, but was inactivated at 75 °C for 2 min. Given the features of the enzyme studied, efforts must be dedicated to a thorough risk analysis and development of strategies to contrast the presence of histaminogenic S. thermophilus strains in products from raw or mildly heat-treated milk. PRACTICAL APPLICATION: During its growth Streptococcus thermophilus can produce histamine over a wide range of conditions encountered in cheesemaking and cheese ripening. The histidine-decarboxylase is even more active in cell-free extract and histamine can be accumulated independently of cell viability.     

三种乳酸菌混合培养的研究

在5L罐中对保加利亚乳杆菌、嗜酸乳杆菌和嗜热链球菌进行了单独培养及混合培养研究。实验结果表明,嗜酸乳杆菌及嗜热链球菌会抑制保加利亚乳杆菌的生长,保加亚利乳杆菌与嗜酸乳杆菌可能对嗜热链球菌的生长具有促进作用,嗜热链球菌对嗜酸乳杆菌具有促进作用,保加利亚乳杆菌对嗜酸乳杆菌的生长没有促进作用。这种实验结果为混合培养益生菌提供的实验参考。     

Contribution of Streptococcus thermophilus to growth-stimulating effect of yogurt on rats

The origin of the growth-stimulating factor in yogurt was studied in rats fed liquid or freeze-dried diets of milk, yogurt, milks fermented individually by Streptococcus thermophilus and Lactobacillus bulgaricus, milks to which cells of Streptococcus thermophilus and Lactobacillus bulgaricus were added. Diets containing sonicated cells, cell supernatant, and cell fractions also were fed. Milk fermented by Streptococcus thermophilus and milk plus Streptococcus thermophilus cells stimulated growth as effectively as did yogurt. That finding and the absence of stimulation in rats fed Lactobacillus bulgaricus showed that Streptococcus thermophilus is responsible for stimulation of growth by yogurt. Growth was stimulated by an intracellular factor and not by fermentative changes in the milk.     

Influence of temperature on associative growth of Streptococcus thermophilus and Lactobacillus bulgaricus

Compatibility of Streptococcus thermophilus and Lactobacillus bulgaricus during associative growth as dependent on optimum growth temperature was determined. Optimum growth temperatures for 9 strains of S. thermophilus and 10 strains of L. bulgaricus ranged from 35 to 42 degrees C for S. thermophilus and 43 to 46 degrees C for L. bulgaricus. Streptococcus thermophilus and L. bulgaricus strains exhibiting similar to divergent optimum growth temperatures were combined (1:1 vol/vol) and incubated in milk at 37, 42, and 45 degrees C until pH 4.2 was reached. Initial and postincubation cell numbers were determined by plate count method. Streptococcus thermophilus strains reached greater cell numbers than L. bulgaricus at 37, 42, and 45 degrees C in 93.3% of the mixed culture trials. Average rod-coccus ratios obtained at 37, 42, and 45 degrees C were 1:2.2, 1:8, and 1:2.4, respectively. Optimum growth temperatures had no influence on growth of L. bulgaricus and S. thermophilus in mixed culture. Rather, temperature appeared to influence compatibility by determining the concentration or type of stimulatory factor(s) produced by L. bulgaricus. All strains of S. thermophilus exhibited an uncoupling of growth from acid production. Optimum temperature for acid production ranged from 2 to 8 degrees C above optimum growth temperature. These findings warrant consideration in the manufacture of yogurt and other fermented milk products.     

THE SEPARATION FROM YOGHURT CULTURES OF LAGTOBACILLI AND STREPTOCOCCI USING REINFORCED CLOSTRIDIAL AGAR AT pH 5·5 AND PLATE COUNT AGAR INCORPORATING MILK

Reinforced clostridial agar, best known for its use in the isolation of Clostridia species, has been shown to be selective for Lactobacillus bulgaricus found in yoghurt by suppressing the growth of Streptococcus thermophilus when the pH of the agar is 5·5. Str. thermophilus was isolated on plate count agar (PCA) incorporating 10 per cent milk by incubating at 37°C for a maximum of 24 h, since L. bulgaricus colony development was only sparse after this period of incubation.     

三聚氰胺对嗜热链球菌生长影响的研究

通过在MRS液体培养基里分别添加0‰、0.25‰、0.5‰、1‰、2‰、3‰及饱和的三聚氰胺,接种嗜热链球菌,在42℃下培养48h,通过测定培养液的pH变化和计数细菌总数来考察三聚氰胺对嗜热链球菌生长的影响。结果表明:嗜热链球菌不能以三聚氰胺为氮源物质而加以降解利用,三聚氰胺在水溶解度范围内对嗜热链球菌的生长没有促进作用,只有微弱的抑制效果。     

Effect of thermophilic lactic acid bacteria on the viability of Salmonella serovar Typhimurium PT8 during milk fermentation and preparation of buffalo's yogurt

Viability of dairy-borne Salmonella enterica ssp. enterica serovar Typhimurium PT8 was studied during the fermentation of skim milk by thermophilic lactic acid bacteria (LAB). Longer generation times of Salmonella were found in mixed cultures of skim milk containing Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus or a mixture of them (1:1), as compared with single cultures of the pathogen. Salmonella was less able to survive in mixed cultures with these LAB during prolonged incubation at 41°C and also during cold storage of the fermented milk. L actobacillus ssp. bulgaricus and its mixture with S. thermophilus were more inhibitory to the growth and survival of Salmonella than was S. thermophilus . This was associated with higher ability of L . ssp. bulgaricus and the mixture to develop acidity in milk than S. thermophilus . Examining the antibacterial activity of these LAB towards Salmonella showed that other factors including heat-resistant and heat-labile compounds were involved in inhibiting the pathogen by these cultures. The viability of the same Salmonella strain during the preparation and cold storage of buffalo's yogurt was also examined. Salmonella was found to survive longer in yogurt made with starter containing probiotic bacteria than in that prepared with the traditional starter. This was ascribed to the development of lower pH by the traditional starter.     

唾液链球菌嗜热亚种荚膜多糖生理功能特性研究

通过对唾液链球菌嗜热亚种的研究发现,细胞固有发酵活性,抵抗外界不良环境能力英膜无关。破坏唾液链球菌嗜热亚种ＬＣＸ２００１的英膜,会使发酵速度加快,但同时对外界不良环境的抵抗力下降。这些研究结果表明,有无英膜,或英膜大小对同一亚种所表现出来的不同生理特性没有决定作用;破不英膜,会影响细胞体与外界物质交换能力,对细胞体许多生理功能有影响,英膜起到阻止外界物质的进入细胞和细胞内产生的乳酸向外界释放的作用     

二株嗜热链球菌益生特性的体外研究

对嗜热链球菌ST1和嗜热链球菌KS1的部分益生性及功能特性进行了研究。考察了ST1和KS1对酸和胆盐的耐受性,结果表明,ST1和KS1在pH值为3.0条件下培养3h的存活率分别为67.17%和67.49%,在pH值为4.0或质量分数为0.3%胆盐环境中仍可以生长,表现出较高的存活力。通过抑菌实验表明ST1和KS1可以抑制常见的致病菌金黄色葡萄球菌、大肠杆菌、鼠伤寒沙门氏菌和福氏志贺氏菌。在对抗生素敏感实验中,ST1对其中的3种抗生素有耐药性,而KS1对6种抗生素有耐药性。研究还发现ST1和KS1还具有较高的降胆固醇的能力,胆固醇的去除率分别为65.14%和74.49%。