一株高效降解胆固醇乳酸菌的筛选鉴定及其益生潜能初探

从新鲜牛粪中分离出的5株乳酸菌中筛选得到一株具有高效降胆固醇能力的乳酸菌,其体外胆固醇脱出率达到50.83%。经API50CHL系统及16S rRNA序列比对鉴定为发酵乳杆菌Lactobacillus fermentum,命名为发酵乳杆菌DF-4。模拟胃液中的低pH和小肠中的胆汁盐浓度进行了耐受性试验,结果表明该菌株具有较强的耐酸和耐胆盐的性能,对致病大肠杆菌和金黄色葡萄球菌均有显著抑制作用,是一株具有开发前景的高效降胆固醇的益生菌。     

Antibacterial activity of Lactobacillus sake isolated from dry fermented sausages

Lactic acid bacteria isolated from Spanish dry fermented sausages were screened for antagonistic activities under conditions that eliminated the effects of low pH and hydrogen peroxide. From 720 isolates tested 119 were inhibitory to Lactobacillus fermentum CECT285. The isolates showing the largest inhibitory activity exhibited an antagonistic effect against several other lactobacilli and the selected foodborne pathogens Staphylococcus aureus and Listeria monocytogenes. Comparison of the antimicrobial spectra of the supernatants suggested that the inhibitory compounds were not identical. The isolates were tentatively characterized as Lactobacillus sake. One of the isolates, L. sake 148 was chosen for further study. The compound excreted by L. sake 148 was active against various lactobacilli and several Gram-positive foodborne bacteria, but not against the Gram-negative bacteria tested. The antagonistic effects were almost eliminated by treatment with proteases, whereas they were heat resistant and bacteriostatic rather than bacteriocidal.     

Lactobacillus fermentum ACA-DC 179 displays probiotic potential in vitro and protects against trinitrobenzene sulfonic acid (TNBS)-induced colitis and Salmonella infection in murine models

Lactobacillus fermentum ACA-DC 179, Lactobacillus plantarum ACA-DC 287 and Streptococcus macedonicus ACA-DC 198 were studied for their probiotic potential. Firstly, strains were screened for antimicrobial activity towards a broad range of target strains, including lactic acid bacteria, food spoilage and pathogenic bacteria. L. fermentum ACA-DC 179 was active against five streptococci, including the two pathogenic strains Streptococcus oralis LMG 14532T and Streptococcus pneumoniae LMG 14545T. S. macedonicus ACA-DC 198 was active against the majority of the strains tested, including not only lactic acid bacteria but also many food spoilage or pathogenic species. The three potential probiotic strains were found to survive variably at pH 2.5 and were unaffected by bile salts. Only S. macedonicus ACA-DC 198 exhibited bile salt hydrolase activity, while none of the strains was haemolytic. Moreover, strains exhibited variable susceptibility towards commonly used antibiotics. L. plantarum ACA-DC 287 and S. macedonicus ACA-DC 198 induced the secretion of the pro-inflammatory cytokines IL-12, IFN-gamma and TNF-alpha by human peripheral blood mononuclear cells. Also elevated levels of the anti-inflammatory IL-10 were observed with L. fermentum ACA-DC 179. This strain consequently was found to significantly reduce colitis in a TNBS-induced colitis mouse model. Furthermore, L. fermentum ACA-DC 179 was successfully applied in an experimental Salmonella-infection mouse model. To conclude, strain L. fermentum ACA-DC 179 possesses desirable probiotic properties, such as antimicrobial activity and immunomodulation in vitro, which were confirmed in vivo by the use of animal models.     

西藏地区发酵牛乳中产细菌素乳酸菌的筛选及鉴定

从西藏地区藏族传统发酵乳中分离乳酸菌,采用生理生化特性和16S基因序列同源性分析对其进行鉴定,通过双层琼脂平板扩散法筛选具有抑菌活性的菌株。结果表明,共分离37株乳酸菌,其中,乳杆菌属(Lactobacillus)35株、明串珠菌属(Leuconostoc)2株;35株乳酸杆菌为Lactobacillus casei 16株、Lactobacillus paracasei 7株、Lactobacillus plantarum 4株、Lactobacillus fermentum 2株、L.delbrueckii subsp.bulgaricus 2株、Lactobacillus helveti-cus 1株、Lactobacillus diolivorans 3株;7株L.casei和L.paracasei的发酵上清液对3株细菌指示菌表现出明显抑制作用,所有菌株对真菌无抑菌活性;在排除有机酸、H2O2等的干扰和经蛋白酶K处理后,初步确定7株乳酸菌发酵上清液中的抑菌物质为细菌素。     

A new probiotic cheese with antioxidative and antimicrobial activity

The aim of our study was to develop an original probiotic cheese based on the Estonian open-texture, smear-ripened, semisoft cheese "Pikantne." Cheese was produced by two methods using cheese starter cultures (Probat 505) in combination with 0.04% of probiotic Lactobacillus fermentum strain ME-3 (10(9) cfu/mL) with high antimicrobial activity and antioxidative properties. The probiotic Lactobacillus was added into milk simultaneously with starter cultures (cheese A) and into drained curd (cheese B). After addition of probiotic L. fermentum ME-3, the cheese composition, flavor, and aroma were comparable to the control cheese (score values = 4.5, 4.2, and 3.7 for control cheese, cheese A, and cheese B, respectively). Cheese A, which had good sensory properties, was chosen for further testing of viability and probiotic properties. The probiotic strain was found to withstand the technological processing of cheese, surviving and sustaining moderate antimicrobial and high antioxidative activity throughout ripening and storage (the ripened cheese contained approximately 5 x 10(7) cfu/g viable ME-3 cells), although the viability of the ME-3 strain incorporated into the cheese showed a slight decrease between d 24 and 54 after cheese preparation. Semisoft cheese "Pikantne" serves as a suitable carrier of antimicrobial and antioxidative L. fermentum ME-3.     

Lactobacilli isolated from chicken intestines: potential use as probiotics

Lactobacillus strains were tested for their in vitro probiotic properties. Cell surface hydrophobicity was found to be very high for Lactobacillus fermentum subsp. cellobiosus and Salmonella Gallinarum; high values could indicate a greater ability to adhere to epithelial cells. Studies on Lactobacillus animalis indicated relative cell surface hydrophobicities smaller than those of L. fermentum subsp. cellobiosus and L. fermentum. L. animalis and Enterococcus faecalis were able to coaggregate with L. fermentum subsp. cellobiosus and L. fermentum, respectively, but not with Salmonella Gallinarum. After mixed-culture studies for determining suitable growth behavior, the pair of strains L. animalis plus L. fermentum subsp. cellobiosus was selected for an attempted challenge against Salmonella Gallinarum. Double and triple mixed-culture studies indicated that selected lactobacillus strains were able to retain their beneficial characteristics in the presence of Salmonella Gallinarum such as presence of lectins, production of antimicrobial compounds, and ability to grow and compete. The selected microorganisms can be considered as potential ingredients for a chicken probiotic feed formulation intended to control salmonellosis and also improve poultry sanitation.     

ε-聚赖氨酸抑菌性能研究

分别采用分光光度法、菌体量法以及孔扩散法研究了ε-聚赖氨酸对细菌和真菌的抑菌活性及ε-聚赖氨酸浓度、pH值和温度对其抑菌活性的影响。结果表明,ε-聚赖氨酸对金黄色葡萄球菌、大肠杆菌、黄曲霉、枯草芽孢杆菌、酿酒酵母、保加利亚乳杆菌等6种供试微生物均有一定的抑制作用,对大肠杆菌、金黄色葡萄球菌、保加利亚乳杆菌抑制效果较好,对黄曲霉较差;对各种菌的最小抑菌浓度(MIC)为大肠杆菌、金黄色葡萄球菌、保加利亚乳杆菌12.5 mg/L;枯草芽孢杆菌25 mg/L;酿酒酵母800 mg/L;黄曲霉1 600 mg/L。ε-聚赖氨酸的抑菌活性随其浓度的增加而增强,热稳定性非常好,能耐100℃的高温,在pH5～8抑菌活性最强,与甘氨酸、Nisin都有协同增效的作用。     

乳杆菌耐消化应激能力及消化应激对其肠道黏附能力的影响

首先通过体外模拟唾液-胃液-肠液应激实验研究乳杆菌的耐消化应激能力,然后研究其对肠道黏蛋白和Caco-2细胞的黏附及抑制肠道病原菌黏附的能力,最后探讨消化应激对乳杆菌黏附能力的影响。结果表明,副干酪乳杆菌W125、m111和发酵乳杆菌146在依次经过模拟唾液-胃液-肠液应激后存活率分别为2.70%、3.53%及11.15%,活菌数分别为7.46、7.24(lg(CFU/mL))及8.35(lg(CFU/mL)),且对黏蛋白和Caco-2细胞的黏附率显著高于其他菌株（P<0.05）,分别为15.67%、8.75%、8.38%和11.47%、21.34%、10.44%;3株菌株均可通过排除、竞争和替代的方式抑制大肠杆菌CICC10899和沙门菌WX29对肠道的黏附,黏附抑制率均大于13.51%;消化应激显著降低了副干酪乳杆菌W125和发酵乳杆菌146对肠道的黏附能力（P<0.05）,但显著增加了副干酪乳杆菌m111的黏附能力（P<0.05）,黏附率由17.60%增加到30.45%,且主要黏附素由消化应激前的表层蛋白变为应激后的蛋白和多糖;消化应激前后副干酪乳杆菌m111均...     

New efficient amylase-producing strains of Lactobacillus plantarum and L. fermentum isolated from different Nigerian traditional fermented foods

Amylolytic lactic acid bacteria (ALAB) were isolated from Nigerian traditional fermented foods (fufu, burukutu, ogi-baba and kunu-zakki) with the aim of selecting efficient amylase-producing strains. Nine isolates were characterized on the basis of their phenotypic and taxo-molecular characteristics. Three groups could be distinguished by their fermentation profiles and this was confirmed by DNA restriction analysis. Though fermentation profiles gave good identification of strain K9 (unique representative of group III) as Lactobacillus fermentum, they could not be used to ascertain the taxonomic position of strains of groups I and II. Analysis of partial 16S rRNA sequences led to the identification of these groups as L. plantarum strains and confirmed the species of strain K9 as L. fermentum. The two distinct phenotypic groups of L. plantarum differed in their use of D-xylose, L-arabinose, melibiose and were different from the previously described amylolytic L. plantarum A6 isolated from retted cassava in Congo. L. fermentum K9 was different from L. fermentum OgiE1 and Mw2 isolated from Benin maize sourdough and it is the first amylolytic L. fermentum described from Nigerian fermented products. Enzymatic profiles showed some differences between the strains of a similar fermentation group. One of the most relevant characteristics of the isolates was a higher yield of amylase production than those reported for previously described ALAB grown under the same conditions. Furthermore, all isolates were tolerant to an exposure at pH 2 and to bile salts.     

Acid adaptation to improve viability and X‐prolyl dipeptidyl aminopeptidase activity of the probiotic bacterium Lactobacillus fermentum HA6 exposed to simulated gastrointestinal tract conditions

In this work, the viability of the probiotic bacterium Lactobacillus fermentum HA6 isolated from naturally fermented vegetables in Vietnam was improved by growing the bacterium into a mild acid condition (pH 4.0). Viability and probiotic functionality [X‐prolyl dipeptidyl aminopeptidase (PepX) activity] of the acid‐adapted bacterium exposed to simulated gastrointestinal conditions were investigated. After 180 min in the simulated gastric juice (0.3 g/L pepsin, pH 2.0), the viability of acid‐adapted L. fermentum HA6 (11.5%) was higher than that of control L. fermentum HA6 (2.2%). Specific PepX activity of acid‐adapted cells (24.5 U/mg) was higher than that of control cells (17.8 U/mg). After 180‐min exposure to the simulated small intestinal medium (0.3 g/L bile salts, 0.1 g/L pancreatin, pH 8.0), the viability of acid‐adapted L. fermentum HA6 (13.5%) was twofold as high as that of control L  fermentum HA6 (8.0%). Our results suggested that acid adaptation has a key role in acquiring cross‐protection mechanism, which in this study resulted in higher survival of L. fermentum HA6 after simulated gastrointestinal stresses. The strategy of acid adaptation could be valuable for the production of robust probiotics.     

发酵乳杆菌的生长限制性因素分析及高密度培养工艺优化

为提高发酵乳杆菌的增殖浓度,对其高密度发酵培养基成分及培养工艺进行优化以提高其活菌数。结果表明,酵母粉复合大分子肽的蛋白胨是发酵乳杆菌的最适氮源,缓冲盐在恒pH培养时对菌株生长无促进作用,Mn²⁺和Mg²⁺均是发酵乳杆菌的限制性微量元素。另外,中性条件下酸根的积累不会对发酵乳杆菌有特异性毒害作用,其生长主要是受到渗透压的抑制。以菌株生长速率被抑制时的碳氮消耗比作为培养基中的碳氮源比例,基于菌株生长速率被抑制时的渗透压确定碳氮源的添加量。进一步优化恒pH分批培养和恒pH自动反馈补糖培养工艺,得到各菌株的最优培养策略:发酵乳杆菌FXJCJ6-1、发酵乳杆菌FGDLZR161、发酵乳杆菌CCFM422分别在恒pH 6.0、5.5、5.5分批培养时,活菌数分别达到(1.3±0.1)×10¹⁰、(1.1±0.1)×10¹⁰、(9.5±0.5)×10^(9 )CFU/mL,较在MRS培养基静置培养时的活菌数提高了3.1、3.8和4.6倍。该研究结果的应用将显著提高发酵乳杆菌的工业化生产效率。     

不同乳酸菌产谷氨酸脱羧酶特性研究

通过对发酵乳杆菌、植物乳杆菌、短乳杆菌所产谷氨酸脱羧酶(GAD)的酶学性质进行比较,分析酶反应温度、pH值、磷酸吡哆醛(PLP)浓度、酶浓度与酶活性的关系,并对高活性乳酸菌GAD酶的米氏常数进行测定。结果表明,3株乳酸菌GAD酶的最适温度为40℃,最适pH值为4.5,在PLP添加量为0.1mol/L时,对酶的促进作用达到最高,发酵乳杆菌Km值为0.05215mmol/L,最大反应速度Vmax=2.08μmol/min;短乳杆菌Km值为0.0243mmol/L,最大反应速度Vmax=1.01μmol/min。     

一株发酵乳杆菌的筛选鉴定及其高密度培养的研究

自黑龙江鹤岗自制酸菜汁中分离得到两株乳杆菌,经16SrDNA序列分析鉴定均为发酵乳杆菌并命名为发酵乳杆菌L1和L3,其中L1具有良好的耐酸和耐胆盐能力。用改良MRS培养基对发酵乳杆菌L17L发酵罐的高密度培养进行了研究,通过补碱和补糖有效克服了发酵过程中产酸和碳源短缺对菌体生长的抑制,最高菌浓可达10.19lgCFU/mL,较分批培养有显著提高,为发酵乳杆菌的工业化生产奠定了一定的基础。     

发酵乳杆菌细胞壁蛋白酶的提取

确定提取发酵乳杆菌细胞壁蛋白酶的最佳方法和最佳提取条件。在单因素试验的基础上,采用多指标正交试验设计和综合平衡分析法研究溶菌酶结合非离子表面活性剂法提取发酵乳杆菌细胞壁蛋白酶的裂解液成分和提取条件。结果表明：裂解液的成分为50mmol/L Tris-HCl、150mmol/L NaCl、体积分数3% 吐温-20、2mg/mL 溶菌酶、pH8.9;提取条件为裂解液与菌体的比例10:1(V/m)、保温时间5h、保温温度37℃时酶比活力达38.957U/mg,此时可最大效率的获得发酵乳杆菌的细胞壁蛋白酶。     

泡菜源乳酸菌的分离筛选及其对小鼠氧化应激水平的改善作用

为筛选具有较好体外抗性以及体内抗氧化效果的乳酸菌,本研究从重庆农家自然发酵泡菜中分离得到12株菌株,经16S rDNA种属分析表明,其中植物乳杆菌（Lactobacillus plantarum） 4株,发酵乳杆菌（Lactobacillus fermentum） 7株,短乳杆菌（Lactobacillus brevis） 1株。采用pH 3.0人工胃液和0.3%胆盐对乳酸菌进行体外抗性筛选,发现12株乳酸杆菌具有较好的体外抗性,其中10株乳酸杆菌在pH 3.0人工胃液中处理3 h后的存活率超过80%;2株乳酸杆菌在0.3%胆盐中的存活率超过60%。选取体外抗性好且为食品可用菌种的Lactobacillus fermentum CQPC-11（L. fermentum CQPC-11）为实验菌株,评价其对活性炭冰水诱导氧化应激损伤小鼠的改善作用,结果表明H-LF CQPC11组小鼠血清中总超氧化物歧化酶（T-SOD）、谷胱甘肽过氧化物酶（GSH-Px）和过氧化氢酶（CAT）的酶活力分别为67.73 U/mL、2742.35酶活力单位、136.59 U/mL,显著高于模型组（p<0.05）,而丙二醛（MDA）的含量为1.99 nmol/L,显著低于模型组（p<0.05）。综上所述,L. fermentum CQPC-11不仅具备良好的耐胃酸和耐胆盐能力,还能降低氧化应激模型小鼠的氧化应激反应,可通过后续进一步的实验探讨该菌株抗氧化作用的具体机理,为新型微生态制剂以及功能性乳酸菌食品的开发提供理论依据和可用菌株。     

蔬菜发酵菌种的筛选及发酵特性

为了选择适合蔬菜发酵的发酵菌种,本研究从四川泡菜老汤中分离的6 种乳酸菌Lactobacillus plantarum、Lactobacillus fermentum、Lactobacillus brevi、Lactobacillus pentosus、Leuconostoc mesenteroides、Lactobacillus lactics筛选发酵菌种。6 种乳酸菌接入亚硝酸盐的MRS培养液中,6 种乳酸菌还原亚硝酸盐的大小顺序为Lactobacilluspentosus＞Lactobacillus plantarum＞Leuconostoc mesenteroides＞Lactobacillus fermentum＞Lactobacillus lactics＞Lactobacillus brevis。以pH值降低的速率为发酵速率,Lactobacillus plantarum、Lactobacillus pentosus、Leuconostocmesenteroides这3 种发酵剂的发酵速率较其他3 种快,并且通过乳酸菌的全细胞蛋白电泳实验得出此3 种乳酸菌在甘蓝发酵过程中能够成为优势菌。从乳酸菌还原亚硝酸盐的能力、发酵速率、发酵菌种的生存能力（是否能成为优势菌）的实验结果表明Lactobacillus plantarum、Lactobacillus pentosus、Leuconostoc mesenteroides这3 种乳酸菌作为蔬菜发酵的发酵剂。通过对3 种发酵菌种发酵特性的研究可知,Leuconostoc mesenteroides较另两种生长周期短,稳定期维持时间短,很快进入衰退期。Lactobacillus plantarum菌较Lactobacillus pentosus、Leuconostoc mesenteroides耐酸,Leuconostoc mesenteroides对酸敏感。Leuconostoc mesenteroides最适生长温度为30 ℃,Lactobacillus plantarum、Lactobacillus plantarum两种菌的最适生长温度是37 ℃。15 ℃条件下Leuconostoc mesenteroides的光密度（OD600 nm）值很低,说明Leuconostoc mesenteroides较Lactobacillus plantarum、Lactobacillus pentosus对低温敏感。     

Identification of lactic acid bacteria in traditional fermented yak milk and evaluation of their application in fermented milk products

In this study, 53 strains of lactic acid bacteria (LAB) isolated from Xueo, a traditional fermented yak milk in the western Sichuan Plateau of China, were identified and their use in fermented milk was evaluated. All gram-positive and catalase-negative strains were divided into 6 groups at the level of 87% similarity using amplified ribosomal DNA restriction analysis. These groups were identified as 6 species using 16S rDNA sequence analysis and atpA gene analysis. The dominant LAB strains in Xueo were Enterococcus durans, Lactobacillus fermentum, and Lactobacillus paracasei, accounting for 45.3, 22.6, and 17.0% of isolates, respectively. Milk fermented with most of the representative strains was high in quality, exhibiting relatively high viscosity, moderate acidity, good sensory quality, and high counts of viable LAB. Fermented milk of E. durans SCA16 and L. fermentum SCA52 achieved the highest scores for overall sensory quality. Most strains displayed antimicrobial activity against at least 1 of 9 spoilage microorganisms. Lactic acid was the main factor inhibiting the growth of spoilage bacteria, and H(2)O(2) was also inhibitory to some extent. Excluding the influence of acid and H(2)O(2), strains SCA52 (L. fermentum) and SCA7 (Lactobacillus plantarum) were antagonistic against some of the indicators, suggesting that the 2 strains may produce a bacteriocin-like substance. Therefore, the development of superior strains isolated from Xueo to ferment milk with similar flavor and texture to Xueo is expected.     

西藏牦牛发酵乳中乳酸菌及酵母菌的特性与相互作用

研究了西藏牦牛发酵乳中的优势菌株:发酵乳杆菌166A、乳酒假丝酵母37、酿酒酵母100、郎比克假丝酵母58在牛乳中的生长特性。探讨了发酵乳杆菌与酵母菌间的相互作用,在发酵过程中3株酵母菌均对发酵乳杆菌166A的生长有促进作用,发酵乳杆菌166A能抑制乳酒假丝酵母37的生长。酵母菌与发酵乳杆菌166A共同接种有利于保持产品冷藏期间活菌数的稳定,菌株之间可能存在共生作用。     

六株人源益生菌的表面性质与黏附性能研究

通过测定六株人源益生菌的自凝集及疏水性能力,并以人结肠腺癌细胞系Caco-2和HT-29作为体外细胞黏附模型评价其黏附能力,最后探究生长阶段、菌体浓度和孵育时间对发酵乳杆菌CECT5716黏附能力的影响。结果表明嗜酸乳杆菌F-1的自凝集率最高,为69.92%;疏水性最强的是鼠李糖乳杆菌MP-108,达到90.10%;乳双歧杆菌AD011对Caco-2和HT-29的黏附性均为最强,分别为(42.80±0.68)、(47.01±0.20) CFU/cell;不同益生菌的自凝集能力、疏水性及黏附能力均存在菌株特异性,其自凝集能力、疏水性及黏附能力间无明显相关性。此外,发酵乳杆菌CECT5716黏附能力随菌体浓度增大而显著(p<0.05)增加,当菌体浓度达108 CFU/mL以上时黏附能力不再显著提高;随着共孵育的时间延长,其黏附能力也增强,在共孵育2 h后不再提升;且菌体生长稳定期时表现出的黏附能力最强,达到(3.61±0.20) CFU/cell。所考察的益生菌大部分都具有较强的潜在黏附能力,其中以乳双歧杆菌AD011最强。这些益生菌具有可在人体肠道定殖生长的潜在益生功能,在食品和药品等行业中具有良好的应用前景。     

Characterization of lactic acid bacteria isolated from Italian Bella di Cerignola table olives: selection of potential multifunctional starter cultures

Lactic acid bacteria (19 isolates) from Bella di Cerignola Italian table olives were investigated for their technological and probiotic properties for the selection of multifunctional starter cultures for table olives. The bacteria were first identified by phenotyping and genotyping, then characterized for the production of biogenic amines, growth at different pH, NaCl concentrations, and temperatures. The potentiality of the bacteria to have some probiotic properties (antimicrobial activity against foodborne pathogens, survival in low pH and in the presence of bile salts, ability to adhere to the mammalian cells model IPEC-J2) was also investigated. Eighteen of the studied isolates were identified as Lactobacillus plantarum and one as Enterococcus faecalis. All bacteria were able to grow at a range of pH between 4.0 and 10.0 as well as in media supplemented with 2.5 to 7.5% of NaCl and 0.3% bile salts and survived in MRS broth acidified at pH 2.5; moreover, they inhibited significantly Escherichia coli O157:H7. The adhesion to IPEC-J2 cells was in general low to moderate (5.3 to 8.3%); however, 2 isolates of L. plantarum (c16 and c19) showed interesting higher adhesion values (up to 16%). Our results suggest that at least 3 isolates could be possible multifunctional starters for Bella di Cerignola olives: L. plantarum 16 and 19 for mainly their probiotic properties and L. plantarum 10 for mainly its technological characteristics. Practical Application: A functional starter is a microorganism exerting benefits on human health (probiotic) and able to guide a fermentation (starter). The main goal of this article was to select a functional starter for table olives.