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。     

乳酸菌对果蔬中霉菌拮抗作用的研究

应用保加利亚乳杆菌(Lactobacillus bulgaricus)、嗜热链球菌(Streptococcus thermophilus)、乳酸乳球菌(Lactococcus lactis)和嗜酸乳杆菌(Lactobacillus acidophilus)4种乳酸菌对从梨、黄瓜、葡萄等果蔬中分离的6种霉菌进行拮抗作用研究.短横线软琼脂拮抗孢子法结果表明,保加利亚乳杆菌、嗜热链球菌、嗜酸乳杆菌对6种霉菌生长平均抑制率分别为67.59％、69.37％和70.18％.菌饼法研究表明,保加利亚乳杆菌、嗜热链球菌、嗜酸乳杆菌对根霉A和D以及曲霉E都具有较强的拮抗作用,其中嗜热链球菌和嗜酸乳杆菌对霉菌D的抑制率分别为63.16％和57.17％,保加利亚乳杆菌对霉菌E抑制率为72.20％.保加利亚乳杆菌对霉菌E拮抗作用最强,嗜热链球菌和嗜酸乳杆菌对霉菌D的生长也具有较强的抑制作用,且在7d内拮抗作用稳定,乳酸乳球菌对所有霉菌没有拮抗作用.     

酸奶用乳酸菌的人工消化耐受性研究

对酸奶尤其益生菌酸奶加工常用的嗜热链球菌、保加利亚乳杆菌、嗜酸乳杆菌和双歧杆菌4支菌种,在人工模拟胃肠消化条件下进行了耐受性研究.结果表明,嗜热链球菌和保加利亚乳杆菌在经过胃肠消化后活菌数几乎消失殆尽,嗜酸乳杆菌和双歧杆菌耐受消化的能力较强.本研究初步证实了嗜酸乳杆菌和双歧杆菌的益生菌功效.     

影响乳酸菌产率因素的初步研究

本文对影响嗜热链球菌，保加利亚乳杆菌生长的多种因素进行了初步研究。结果表明；胰胨、蛋白胨、大豆蛋白胨、西人、ＰＢＳ体系对嗜热链球菌生长有明显促进作用，而肝浸液，ＰＢＳ体系对保加利亚乳杆菌生长有显著的促进作用。     

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。     

分段发酵提高酸乳中活菌总数的研究

为提高酸乳中乳酸菌活菌数量,在测定不同温度下嗜酸乳杆菌生长曲线及保加利亚乳杆菌与嗜热链球菌1∶1混菌发酵生长曲线的基础上,采用分段发酵的方式,发酵初期按1%接种量接入嗜酸乳杆菌,待发酵进行4h,嗜酸乳杆菌的数量达到107时,再接入保加利亚乳杆菌和嗜热链球菌,接种量均为1%。发酵结束后,测得最终活菌数可达109,相比3种菌一次性接种发酵,活菌数量提高了1个数量级。     

蜂蜜酸奶发酵过程中乳酸菌生长动力学的初步研究

本文研究了蜂蜜酸奶发酵过程中乳酸菌双菌的生长规律,同时研究了活菌数、pH、乳酸含量和还原糖转化率与时间变化的相关性。研究表明:在乳酸发酵过程中,保加利亚乳杆菌和嗜热链球菌的生长动力学性质满足典型对数生长曲线规律,且证实了两者之间存在共生作用;并建立了发酵过程中保加利亚乳杆菌和嗜热链球菌的数学模型,确定了世代时间大约为9.3-29.1min;随着发酵的进行,乳酸含量不断增大,体系的pH逐渐下降,还原糖转化率持续升高,在4h达到最高后下降。     

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。     

保加利亚乳杆菌和嗜热链球菌相互作用的研究

探讨了嗜热链球菌和保加利亚乳杆菌之间的相互作用(拮抗和共生效果)，指出这两个菌种的许多菌株在代谢过程中会产生过氧化氢、细菌素等抑菌性物质，导致嗜热链球菌与保加利亚乳杆菌间表现出非协同生长的现象。在此基础上，建立了评价发酵剂之间相互作用的新的指标体系，旨在方便优良酸奶发酵剂菌种的筛选工作。     

提高嗜酸乳杆菌在豆乳中发酵性能的研究

测定了嗜酸乳杆菌、保加利亚乳杆菌和嗜热链球菌发酵豆乳的单菌产酸曲线与混菌产酸曲线,并采用正交实验分析了它们对发酵豆乳的品质影响及与单菌发酵豆乳贮存期质量变化的比较。结果表明,嗜酸乳杆菌、保加利亚乳杆菌是影响酸豆乳质量的主要因素,嗜热链球菌次之。按体积分数为2.2%嗜酸乳杆菌、体积分数为3.0%保加利亚乳杆菌、体积分数为1.8%嗜热链球菌配比共同发酵,可得到组织状态、口感风味较好的酸豆乳,克服了单一嗜酸乳杆菌发酵豆乳产酸慢、凝乳时间长、活菌数不高且贮存期活菌数下降快的缺陷。     

Antibiotic resistance of lactic acid bacteria isolated from Chinese yogurts

The aim of this study was to evaluate the susceptibility of 43 strains of lactic acid bacteria, isolated from Chinese yogurts made in different geographical areas, to 11 antibiotics (ampicillin, penicillin G, roxithromycin, chloramphenicol, tetracycline, chlortetracycline, lincomycin, kanamycin, streptomycin, neomycin, and gentamycin). The 43 isolates (18 Lactobacillus bulgaricus and 25 Streptococcus thermophilus) were identified at species level and were typed by random amplified polymorphic DNA analysis. Thirty-five genotypically different strains were detected and their antimicrobial resistance to 11 antibiotics was determined using the agar dilution method. Widespread resistance to ampicillin, chloramphenicol, chlortetracycline, tetracyclines, lincomycin, streptomycin, neomycin, and gentamycin was found among the 35 strains tested. All of the Strep. thermophilus strains tested were susceptible to penicillin G and roxithromycin, whereas 23.5 and 64.7% of Lb. bulgaricus strains, respectively, were resistant. All of the Strep. thermophilus and Lb. bulgaricus strains were found to be resistant to kanamycin. The presence of the corresponding resistance genes in the resistant isolates was investigated through PCR, with the following genes detected: tet(M) in 1 Lb. bulgaricus and 2 Strep. thermophilus isolates, ant(6) in 2 Lb. bulgaricus and 2 Strep. thermophilus isolates, and aph(3')-IIIa in 5 Lb. bulgaricus and 2 Strep. thermophilus isolates. The main threat associated with these bacteria is that they may transfer resistance genes to pathogenic bacteria, which has been a major cause of concern to human and animal health. To our knowledge, the aph(3')-IIIa and ant(6) genes were found in Lb. bulgaricus and Strep. thermophilus for the first time. Further investigations are required to analyze whether the genes identified in Lb. bulgaricus and Strep. thermophilus isolates might be horizontally transferred to other species.     

市售酸奶中乳酸菌耐药性及耐药基因的检测

为探讨酸奶中乳酸菌所携带耐药基因对人类健康的潜在影响,对市售酸奶中的乳酸菌进行分离和鉴定,并通过药物敏感性实验确定菌株的耐药谱,同时利用聚合酶链式反应（polymerase chain reaction,PCR）扩增技术调查链霉素、庆大霉素、磺胺类和四环素等耐药基因的分布情况。结果表明：25 份市售酸奶样品中共分离得到56 株乳酸菌,包括26 株德氏乳杆菌保加利亚亚种、3 株植物乳杆菌、2 株嗜酸乳杆菌以及25 株嗜热链球菌。药敏结果显示,31 株乳杆菌对链霉素（87.1%）、庆大霉素（80.6%）、环丙沙星（74.2%）和四环素（61.3%）的耐药率较高,对头孢菌素类则较为敏感;而25 株嗜热链球菌同样对链霉素的耐药率最高,达76.0%;其次分别为万古霉素（32.0%）、环丙沙星（32.0%）和四环素（20.0%）。56 株乳酸菌中共检出5 种不同的耐药基因,分别为链霉素耐药基因ant(6)（检出率1.8%）、庆大霉素耐药基因aac(6')-aph(2')（检出率7.1%）、四环素耐药基因tetM（检出率5.4%）以及磺胺类耐药基因sulⅠ（检出率14.3%）和sulⅡ（检出率1.8%）。受试的乳酸菌中共有13 株检出耐药基因,其中有4 株携带两种不同的耐药基因。长期以来被认为安全并广泛应用于发酵食品领域的乳酸菌可能成为潜在的耐药基因贮存库。     

实时荧光定量PCR法鉴定发酵乳中嗜热链球菌

该研究通过系统发育树分析确定目标基因,根据目的基因设计特异性引物和探针,建立一种能够快速准确鉴定发酵乳中嗜热链球菌的实时荧光定量聚合酶链式反应(RT-fqPCR)法,通过特异性、灵敏性和抗干扰实验对所建立方法进行验证,并使用该方法对市售的60份标识含有嗜热链球菌的发酵乳样品进行检测。结果表明,rec A基因具有种间特异性,种间差异率>10%,以其为目的基因建立的RT-fqPCR方法能够特异性的检测嗜热链球菌;绝对灵敏度达1 pg/μL,相对灵敏度达10³CFU/m L;在培养物水平和基因组水平抗干扰能力良好。采用该方法从60份标识含有嗜热链球菌的发酵乳样品中均能检测出嗜热链球菌,说明实时荧光定量PCR方法能够快速、准确的对发酵乳中嗜热链球菌进行检测。     

EFFECT OF A BACTERIOCIN-LIKE SUBSTANCE (BLS) PRODUCED BY STREPTOCOCCUS THERMOPHILUS STRAINS ON LISTERIA SPP. STRAINS

In this study, fifty-two strains of Streptococcus, isolated from Turkish yogurt were tested for their ability to inhibit Listeria strains. Only two isolates exhibited antimicrobial activity (CFF; cell-free filtrate) against eight strains of Listeria innocua, four strains of Listeria monocytogenes and one strain of Listeria welshimeri by agar diffusion. However, bacteriocin-like substance (BLS) producing strains were determined under conditions with the effects of organic acids and hydrogen peroxide eliminated. Inhibitory activities of Streptococcus thermophilus ET5 and S. thermophilus H22 were due to BLS. BLS of S. thermophilus ET5 was observed to exhibit more significant inhibition to the Listeria strains than BLS of S. thermophilus H22.
Inhibitory effect of nisin in 50, 100, 150, 200, 250 and 300 ppm concentrations was compared with BLS producing S. thermophilus strains against Listeria spp. strains. While all Listeria strains were resistant to 50 and 100 ppm concentrations of nisin, some strains of Listeria spp. were only sensitive to 150, 200, 250 and 300 ppm concentrations. The inhibitory activity of S. thermophilus ET5 was more effective against Listeria strains than the inhibitory activity of nisin. It appears that BLS of S. thermophilus ET5 could provide greater potential and usefulness as bacteriocin than nisin. These results indicate that the potential application of antimicrobial-production of S. thermophilus ET5 and H22 as bio-preservatives against Listeria contamination on dairy products could be suggested.     

环丙沙星对嗜热链球菌grx02生理特性的影响

研究环丙沙星对嗜热链球菌(S. thermophilus)grx02生理特性的影响。测定环丙沙星对S. thermophilus grx02的最低抑菌质量浓度(MIC),然后将S. thermophilus grx02分别接种于加入0、1/2 MIC、1/10 MIC和1/50 MIC环丙沙星的MRS液体培养基中,测定其生长曲线和pH值变化,分别以光镜和电镜进行形态学观察,并测定对胞内乳糖代谢关键酶活性的影响。结果显示：嗜热链球菌grx02对环丙沙星的最低抑菌质量浓度为3.89μg/mL,加入1/2 MIC环丙沙星后,嗜热链球菌grx02生长速度和产酸速度均明显降低(P＜0.01); 加入不同亚抑菌质量浓度(低于MIC)环丙沙星后,菌体形态呈半椭圆形,且荚膜变得薄而致密;加入1/2 MIC环丙沙星后胞内的β-半乳糖苷酶和乳酸脱氢酶酶活力均明显下降,加入1/50 MIC环丙沙星后β-半乳糖苷酶活力下降,而乳酸脱氢酶酶活力反而增大。上述结果表明,在环丙沙星胁迫下,嗜热链球菌启动了相应的应激系统,细胞分裂被抑制,嗜热链球菌生理机制发生了复杂的变化。     

Oxygen uptake activity and aerobic metabolism of Streptococcus thermophilus STH450

Streptococcus thermophilus STH450 had a very high oxygen uptake. This strain was then compared with aerobic metabolism to S. thermophilus ATCC 19258, a reference strain for aerobic metabolism. Molecular oxygen, which was absorbed by S. thermophilus STH450 during aerobic glycolytic metabolism, was involved in the oxidation of NADH by the catalytic activity of NADH oxidase. The portion of pyruvate that corresponded to the oxidized NADH was committed to form alpha-acetolactate, acetoin, and diacetyl. Both strains were deficient in peroxidase and pyruvate oxidase activities; therefore, NADH oxidase was probably the terminal oxidase in aerobic glycolytic metabolism. Oxygen uptake and NADH oxidase activities were significantly higher in S. thermophilus STH450 than in S. thermophilus ATCC 19258. alpha-Acetolactate, acetoin, and diacetyl also accumulated during aerobic glycolytic metabolism of S. thermophilus STH450. However, when both strains were grown in the presence of pyruvate, these metabolites were equivalent. Hence, less oxygen might be needed for pyruvate metabolism.     

The effect of heteropolysaccharide‐producing strains of Streptococcus thermophilus on the texture and organoleptic properties of low‐fat yoghurt

Heteropolysaccharide‐producing Streptococcus thermophilus strains (LY03, ST 111, or 5077) were compared with nonropy S. thermophilus strains (NR or 5137), in respect of texture development of low‐fat yoghurt produced at 42°C. Whether milk solids were added or not, lower syneresis and improved texture and sensory properties were obtained in yoghurts produced with S. thermophilus LY03 or S. thermophilus 5077 compared with nonropy strains. Texture effects, lower syneresis and improved sensory properties because of the use S. thermophilus ST 111 were only noticeable in the presence of milk solids. Milk solids had no significant effect on the textural parameters of nonropy strains.     

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.     

基于基因组学分析嗜热链球菌KLDS SM的蛋白质水解系统和氨基酸合成途径

为从遗传水平上探究嗜热链球菌KLDS SM蛋白质水解和氨基酸合成的能力,首先基于Illumina Hiseq 2500与Pacbio RSII测序平台对嗜热链球菌KLDS SM进行全基因组测序并绘制基因组图谱;随后从胞外蛋白酶、转运系统、胞内肽酶以及氨基酸合成等方面所涉及的基因进行生物信息学分析;最后对15?株已完成全基因组测序的嗜热链球菌的氨基酸合成能力进行比较基因组学研究。结果表明：菌株KLDS?SM的基因组由一个1?856?787?bp环状染色体组成,GC含量为39.08%,含有1?732?个蛋白质编码基因;菌株KLDS?SM具有完整的蛋白水解系统和8?种氨基酸的合成能力;15?株嗜热链球菌在氨基酸合成方面上相对保守,仅在组氨酸合成途径存在较大的差异。本研究为该菌株氮代谢能力的挖掘提供了理论依据,并在将其开发为发酵剂方面上具有一定指导意义。     

嗜热链球菌培养过程中的细胞凋亡研究

对直投型酸奶发酵剂中的嗜热链球菌进行了培养，测定了培养过程中菌体的细胞凋亡、菌体的生长曲线、LDH活力、LD含量。结果表明，嗜热链球菌培养过程中存在细胞凋亡现象，与菌体增殖和产酸规律有关。