流式细胞术分析与评价酸奶菌种对模拟胃酸和胆汁盐的耐受力

研究传统酸奶菌种对人体胃酸和胆汁盐的耐受性,对评价酸奶的益生性具有重要意义。实验选取普通酸奶中的嗜热链球菌和保加利亚乳杆菌作为研究对象,模拟胃肠环境以探讨所选两种菌株在不同pH 值和不同质量浓度脱胆盐下的耐受力,用流式细胞仪(FCM)结合羧基荧光素二醋酸酯(5-(6)-carboxyfluorescein diacetate,5(6)-cFDA)和碘化丙锭(propidium iodide,PI)两种荧光染料评价其活力。结果表明：pH2.0 时,菌体细胞活性较低,其活细胞标记率仅为41.8%,在pH3.0、4.0、5.0、6.0、7.0 时,活细胞标记率越来越高,对其抑制性的影响逐渐减弱;在1g/100mL 的脱胆盐(DBS)中,活细胞标记率为48.7%,而在0.5、0.25、0.1、0.05g/100mL 的DBS 中时,活细胞的标记率越来越高;同时比较FCM 与平板菌落计数法检测菌体细胞的耐受水平。实验证实酸奶菌种能耐受pH 值为5.0～7.0 的胃酸和0.05～0.5g/100mL 的DBS;FCM 检测的菌体细胞的状况更能恰当地反映发酵酸奶品质的优劣。     

发酵乳制品中乳酸菌的流式检测方案探索与研究

目的 建立基于流式细胞术的发酵乳制品中乳酸菌快速计数方法, 实现发酵乳制品中乳酸菌的快速定量检测。方法 使用5(6)-羧基荧光素二乙酸酯（5-(and 6)-Carboxyfluorescein diacetate,cFDA） 和碘化丙啶（Propidium iodid,PI）2种荧光染料对乳酸菌进行染色,经流式细胞仪检测乳酸菌的荧光信号,从而实现细菌定量计数。在分析过程中对染色剂浓度、荧光通道阈值进行优化,并将流式计数结果与现行国标方法进行对比研究。结果 在样品浓度为104 CFU/mL(g)时,通过0.05 μg/mL的PI和10 μmol/L的cFDA染色,在绿色荧光通道增益为100,红色荧光通道增益达到1000时,能够达到最佳的流式检测条件,且样品检测结果与现行国标检测方法存在显著的正相关关系（P<0.01）,2种方法测试结果相关系数值为0.949。结论 流式细胞术具有灵敏度高、检测时间短、稳定性高、重现性好等优点,对于货架期普遍较短,放行压力大的乳制品生产企业来说,能够缩短检测时间,实现产品快速放行上架。     

直投式发酵剂制备工艺对乳酸菌存活率的影响

为明确直投式发酵剂制备过程中菌体细胞存活率的变化,选择植物乳杆菌R23为实验菌,采用扫描电镜、透射电镜和正交试验等手段对收集条件、预冻条件和冻干时间等进行研究。结果表明,植物乳杆菌R23优化收集时间为15 h,此时菌量达到最大且活力较高;在优化的离心条件下菌体细胞存活率达92.4%,其中离心力起关键作用;采用梯度预冻即-20,-40,-80℃各1,2,3 h,可使物料达到较优冻结效果;发酵剂物料冻干至水质量分数为1.20%时最有利于贮藏。研究在明确菌剂制备关键因素对菌体细胞干预机制的基础上,还获得了适宜的制备条件,对直投式发酵剂的规模化生产具有重要的指导意义。     

番茄汁酸奶的研制

用保加利亚乳杆菌（L.bulgaricus）与嗜热链球菌（S.thermophilus）按1：1比例混合作发酵剂，用新鲜优质牛乳为原料，加入0.2%-0.3%的稳定剂，经90-95℃加热处理5min，冷却至45℃左右，加入经杀菌后的新鲜蕃茄汁8%，按2%-3%比例加入发酵剂，装瓶于39-43℃培养2-2.5h，冷藏可制得风味纯正，营养价值高的酸奶。     

5L立瓶发酵制备益生菌直投式发酵剂及应用试验

对长双歧杆菌、保加利亚乳杆菌、嗜热乳酸链球菌在5 L立瓶中分别进行培养,确定了各自的发酵最适收获期,菌体收集后经制备所获得冷冻浓缩直投式发酵剂活菌数大于1010mL-1,冷冻干燥直投式发酵剂活菌数大于1011g-1。以制备的冷冻浓缩、冷冻干燥直投式发酵剂,进行牛乳发酵应用试验,生产的益生菌酸奶风味纯正、品质良好。     

5(6)-cFDA 标记乳酸菌细胞的影响因素

选用常规发酵乳菌种,探讨不同溶剂、不同浓度的羧基荧光素二醋酸酯(5-(6)-Carboxyfluorescein diacetate,5(6)-cFDA)标记乳酸菌细胞的影响因素。结果显示：水、PBS 和二甲基亚砜(DMSO)配制的5(6)-cFDA 标记初期,细胞标记率分别为94.8%、95.2% 和99.77%,放置30d 后细胞标记效果分别下降到51.53%、55.6% 和96.7%;每毫升菌悬液加60μL 的5mg/mL 5(6)-cFDA(相当于终浓度为6.52 × 10-7mol/L),其荧光强度与5(6)-cFDA 浓度成正比,且标记效果最理想。实验还证实了1 × 106～1 × 107cells/mL 是流式细胞仪检测细胞活性较适合的浓度,且标记率较高;在pH7.0 的条件下对细胞活性和发光强度影响最小,其标记率达到98.3%。     

直投式纳豆菌发酵剂制备工艺优化

为探索出一种工艺简便、成本低且活菌数高的直投式纳豆菌（Bacillus natto）发酵剂的制备方法，该研究以大米粉为基质替代传统的种子液培养基培养纳豆菌后直接将其烘干制成直投式纳豆菌发酵剂，通过单因素及正交试验优化直投式纳豆菌发酵剂的制备工艺。结果表明，用含1.5%蛋白胨的大米粉作为培养基，在接种量4.0%、加水量40%、培养温度43 ℃条件下培养纳豆菌28 h，菌体生长良好，将培养物烘干后制成的直投式纳豆菌发酵剂中的活菌数含量达4.71×109 CFU/g。此技术操作简便，可推广至工业生产中，简化生产过程。     

乳酸菌溶源性的检测及紫外诱变后溶源菌蛋白表达分析

用紫外诱变的方法对本实验室保藏的嗜热链球菌（Streptococcus thermophilus）和保加利亚乳杆菌（Lactobacillus bulgaricus）进行溶源性的检测；并采用单向聚丙稀酰氨凝胶电泳，对不同诱变剂量下溶源菌菌体蛋白质组分的变化做了进一步分析，发现诱变前后菌体的蛋白组成发生了明显的变化，即随着诱变剂量的增加，菌体内蛋白种类，尤其是小分子蛋白有上升的趋势。     

流式细胞术检测单增李斯特菌与酿酒酵母

为探讨流式细胞术对单增李斯特菌和酿酒酵母的活菌与热灭活菌的检出效果,本文采用荧光染色试剂SYTO-9和碘化乙锭(PI)对单增李斯特菌和酿酒酵母的活菌与热灭活菌的细胞悬液进行染色,采用流式细胞仪同时测量红色荧光与绿色荧光从而得出细胞悬液中的细菌和酵母的含量。结果表明经核酸荧光染料染色后,再结合流式细胞术对细菌与酵母菌进行检测,步骤简单、耗时短。该法不仅简化了测量步骤且分辨率高,对单增李斯特菌和酿酒酵母均具有良好的检出结果,能分辨同一体系中同一菌种的活细胞与热灭活细胞和同一体系中的细菌与酵母活细胞;该法检出限低,将单增李斯特菌稀释后,最低检出限可达1.2×104 cells/mL,将酿酒酵母稀释后,最低检出限可达6×103 cells/mL,因此能大大缩短增菌时间或者避免繁复的增菌步骤。     

菌株性能及球杆菌比例对酸奶品质的影响

分析测定了从某直投式发酵剂中分离的两株嗜热链球菌（Streptococcus thermophilus）S.t S1、S.t R1和一株保加利亚乳杆菌（Lactobacillus delbrueckii subsp.bulgaricus）L.bL1的发酵性能,并将球菌与杆菌以不同比例组合发酵,比较酸奶发酵时间、粘度、乳清析出、后酸化和感官评价差异。结果表明,嗜热链球菌S.tS1生长速度略慢于S.tR1,酸化速度相当,但S.tS1的胞外多糖产量和发酵乳粘度明显高于S.tR1,保加利亚乳杆菌L.bL1的上述性能均比两株球菌差。S.tS1与L.bL1菌株以100：1的比例进行组合发酵时,粘度达0.549Pa·s;乳清析出仅2.5mL/10g;4℃冷藏保存15d后,酸度上升12°T,酸奶产品各项指标优于其他菌株组合。菌株性能和组合方式对酸奶产品的品质具有显著的影响。     

嗜热链球菌噬菌体对酸奶质量的影响

研究嗜热链球菌噬菌体对酸奶直投式发酵剂(direct vat set,DVS)发酵产酸、嗜热链球菌与保加利亚乳杆菌比例、酸奶黏度、口感、后酸化、乳清析出等方面的影响。结果表明：嗜热链球菌噬菌体可影响酸奶发酵剂发酵产酸、明显改变酸奶嗜热链球菌与保加利亚乳杆菌比例,导致产品黏度降低、口感变差、乳清析出及后酸化严重,嗜热链球菌噬菌体对DVS 生产酸奶有着严重的危害。     

保加利亚乳杆菌与嗜热链球菌抗噬菌体菌株的原生质体制备及融合

为获得具有抗噬菌体功能且发酵性能优良的乳酸菌融合子,采用单亲灭活及正交分析方法,研究了保加利亚乳杆菌与嗜热链球菌抗噬菌体菌株的原生质体制备及融合条件。结果表明：保加利亚乳杆菌原生质体制备的最适条件是以磷酸盐缓冲液和甘露醇制作的高渗溶液为原生质体稳定剂,1.0 mg/mL的溶菌酶36 ℃处理30 min,原生质体的形成率为（89.02±2.31）%,再生率为（4.62±0.22）%。嗜热链球菌抗噬菌体菌株原生质体制备的最适条件是以Tris-HCl和蔗糖制作的高渗溶液为原生质体稳定剂,0.1 mg/mL的溶菌酶42 ℃处理30 min,原生质体的形成率为（99.15±0.23）%,再生率为（5.79±0.17）%。单亲灭活保加利亚乳杆菌与嗜热链球菌抗噬菌体菌株原生质体融合的最适条件为聚乙二醇6000（质量浓度为400 g/L,添加0.01 mol/L CaCl2、0.02 mol/L MgCl2）40 ℃促融2 min,融合率可达（1.85±0.12）×10－6。所得融合子各项性能优良,适合于酸奶生产。     

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.     

Sensory Acceptability and Stability of Probiotic Microorganisms and Vitamin C in Fermented Acerola (Malpighia emarginata DC.) Ice Cream

ABSTRACT:  Six fermented acerola ice creams were produced, containing different starter cultures ( Bifidobacterium longum , Bi.lactis , and traditional yogurt starter culture— Streptococcus thermophilus and Lactobacillus delbrueckii spp. bulgaricus ) and final pH (5 and 4.5). The ice creams were evaluated for probiotic culture viability, vitamin C stability, and sensory acceptance. Mix fermentations were stopped when pH 5.0 and 5.5 were attained. However, after the addition of acerola pulp the determined pH were 4.5 and 5, respectively. Mixes were frozen and stored for 15 wk at −18 °C. The viable counts for probiotic cultures remained above the recommended minimum limit of 10⁶ cfu/g during 15 wk storage even in products with pH 4.5. Vitamin C concentration remained around 140 mg/100 g of product. The attributes of aroma, taste, texture, and overall acceptance obtained scores in the range of 5.15 to 7.22. The fermented acerola ice cream was a suitable food for the delivery of vitamin C and Bifidobacterium strains with excellent viability and acceptable sensory characteristics.     

Effect of carbon dioxide under high pressure on the survival of cheese starter cultures

A new processing method that rapidly forms curds and whey from milk has the potential to improve cheesemaking procedures if cheese starter cultures can tolerate the processing conditions. The survival of Lactobacillus delbrueckii ssp. bulgaricus, Lactococcus lactis ssp. lactis, or Streptococcus thermophilus through this new process was evaluated. Inoculated milk containing 0, 1, or 3.25% fat or Lactobacillus MRS broth or tryptone yeast lactose broth (depending on microorganism used) was sparged with CO2 to a pressure of 5.52 MPa and held for 5 min at 38 degrees C. Broth contained 7.93 to 8.78 log CFU/ ml before processing and 7.84 to 8.66 log CFU/ml afterward. Before processing, milk inoculated with L bulgaricus, L. lactis, or S. thermophilus contained 6.81, 7.35, or 6.75 log CFU/ml, respectively. After processing, the curds contained 5.68, 7.32, or 6.50 log CFU/g, and the whey had 5.05, 6.43, or 6.14 log CFU/ml, respectively. After processing, the pHs of control samples were lower by 0.41 units in broth, 0.53 units in whey, and 0.89 units in curd. The pH of the processed inoculated samples decreased by 0.3 to 0.53 units in broth, 0.32 to 0.37 units in whey, and 0.93 to 0.98 units in the curd. Storing curds containing L. lactis at 30 degrees C or control curds and curds with L. bulgaricus or S. thermophilus at 37 degrees C for an additional 48 h resulted in pHs of 5.22, 5.41, 4.53, or 4.99, respectively. This study showed that milk inoculated with cheese starter cultures and treated with CO2 under high pressure to precipitate casein-produced curds that contained sufficient numbers of viable starter culture to produce lactic acid, thereby decreasing the pH.     

Effects of ingestion of yogurts containing Bifidobacterium and Lactobacillus acidophilus on spleen and Peyer's patch lymphocyte populations in the mouse

Certain probiotic lactic acid bacteria have been reported to improve immune system function. Here, the effects of ingesting yogurts on lymphocyte populations in the spleens and Peyer's patches were determined in mice. Three probiotic-supplemented yogurts containing Streptococcus thermophilus, Lactobacillus bulgaricus, Bifidobacterium, and Lactobacillus acidophilus and one conventional yogurt containing only S. thermophilus and L. bulgaricus were prepared from commercial starter cultures and used in the study. B6C3F1 female mice were fed the four different types of yogurts mixed with an AIN-93G diet in a 50:50 (wt/wt) ratio. Nonfat dry milk mixed at a 50:50 (wt/wt) ratio with AIN-93G diet was used as the control. After a 14-day feeding period, spleen and Peyer's patches were removed and lymphocytes subjected to phenotype analysis by flow cytometry. Ingestion of the four yogurts had no effect on percentages of CD8+ (cytotoxic T cells), B220+ (B cells), IgA+, or IgM+ cells in spleen or Peyer's patches. The percentage of CD4+ (T helper) cells was significantly increased in the spleens from one group of mice fed a yogurt containing Bifidobacterium and L. acidophilus, and a similar trend was found in the remaining two probiotic-supplemented yogurts. Effects on CD4+ populations were not observed in spleens of mice fed conventional yogurt or in the Peyer's patches of any of the four yogurt groups. In total, the results suggested that ingestion of conventional or probiotic-supplemented yogurts for 2 weeks had very little effect on lymphocyte distribution in the systemic or mucosal immune compartments.     

Effect of addition of Versagel on microbial, chemical, and physical properties of low-fat yogurt

The objective of this study was to examine the effect of Versagel on the growth and proteolytic activity of Streptococcus thermophilus 1275 and Lactobacillus delbrueckii ssp. bulgaricus 1368 and angiotensin-I converting enzyme inhibitory activity of the peptides generated thereby as well as on the physical properties of low-fat yogurt during a storage period of 28 d at 4 degrees C. Three different types of low-fat yogurts, YV0, YV1, and YV2, were prepared using Versagel as a fat replacer. The fermentation time of the low-fat yogurts containing Versagel was less than that of the control yogurt (YV0). The starter cultures maintained their viability (8.68 to 8.81 log CFU/g of S. thermophilus and 8.51 to 8.81 log CFU/g of L. delbrueckii ssp. bulgaricus) in all the yogurts throughout the storage period. There was some decrease in the pH of the yogurts during storage and an increase in the concentration of lactic acid. However, the proteolytic and ACE-inhibitory potential of the starter cultures was suppressed in the presence of Versagel. On the other hand, the addition of Versagel had a positive impact on the physical properties of the low-fat yogurt, namely, spontaneous whey separation, firmness, and pseudoplastic properties.     

Short communication: effect of oxygen on symbiosis between Lactobacillus bulgaricus and Streptococcus thermophilus

Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) and Streptococcus thermophilus are traditionally used for the manufacture of yogurt. It is said that a symbiotic relationship exists between Strep. thermophilus and L. bulgaricus and this decreases fermentation time. It is well known that L. bulgaricus is stimulated by the formate produced by Strep. thermophilus, and Strep. thermophilus is stimulated by free amino acids and peptides liberated from milk proteins by L. bulgaricus in symbiotic fermentation. We found that acid production by starter culture LB81 composed of L. bulgaricus 2038 and Strep. thermophilus 1131 was greatly accelerated by decreasing dissolved oxygen (DO) to almost 0 mg/kg in the yogurt mix (reduced dissolved oxygen fermentation) and that DO interferes with the symbiotic relationship between L. bulgaricus 2038 and Strep. thermophilus 1131. We attributed the acceleration of acid production of LB81 by reduced dissolved oxygen fermentation mainly to the acceleration of formate production and the suppression of acid production of LB81 by DO mainly to the suppression of formate production.