Molecular Verification of Two Potent Bacteria Isolated from Darfiyeh Cheese: <i>Lactococcus lactis</i>Subsp. <i>Lactis</i>and <i>Lactobacillus plantarum</i>

Raw goat milk cheeses are known for their natural microflora linked to many biodiversity factors such as the use of raw milk. That microflora serves probiotic attribution conferring a beneficial health impact on the consumer. Darfiyeh is an artisanal raw goat milk cheese manufactured traditionally in Northern Lebanese Mountains. To emphasize its clinical significance in both digestive and immune system, and to provide health remunerations to the consumer, the cheese microbiota will be investigated. To serve that purpose, the presence of the two potent probiotic Lactic Acid Bacteria (LAB): Lactococcus lactis subsp. lactis and Lactobacillus plantarum will be investigated. For bacterial identification, selection and isolation: culture-dependent techniques that imply the use of laboratory media will be implemented, and culture independent techniques: Polymerase Chain Reaction (PCR) will be applied for further validation. Both bacteria were further verified as Lactococcus lactis subsp. lactis and Lactobacillus plantarum by implementing specie-specific primers for the qualitative PCR.

嗜酸乳杆菌(Lactobacillus acidophilus)合成纳米硒特性研究

乳酸菌具有还原亚硒酸钠为纳米硒的能力。为了采用更加环境友好的方式获得纳米硒颗粒,本研究首先探索了培养基中加硒浓度、加硒时间,以及培养时长对嗜酸乳杆菌(Lactobacillus acidophilus)合成纳米硒的影响,随后对所产的纳米硒颗粒的粒径、zeta电位及抑菌活性进行研究。结果表明,当培养基中硒含量为0~600μg/mL时,硒浓度越高,转化得到的纳米硒越多;当培养基中硒浓度高于600μg/mL时,发酵液中的纳米硒含量反而减少。在嗜酸乳杆菌生长的对数中前期,即第6 h时添加亚硒酸钠,更有利于纳米硒的合成。而培养至32 h后,纳米硒几乎不再继续合成。通过扫描电镜及粒径和电位仪分析发现,嗜酸乳杆菌所产的纳米硒颗粒呈球形,且超声破碎有助于其释放;纳米硒颗粒带负电荷,电位绝对值为40~50 mV,粒径大小较多分布于170~210 nm,具有良好的稳定性。抑菌实验结果表明,嗜酸乳杆菌还原亚硒酸钠所产的纳米硒对大肠杆菌、沙门氏菌、金黄色葡萄球菌和枯草芽孢杆菌的生长均具有抑制作用。综上,嗜酸乳杆菌可以将亚硒酸钠还原合成纳米硒,且所产的纳米硒颗粒具有良好的抑菌活性。

没食子酸对植物乳杆菌Lactobacillus plantarum的抑制和促进生长

该文以微生物生长曲线、多酚浓度变化和培养液pH值变化为指标,研究了没食子酸(GallicAcid,GA)和表没食子儿茶素没食子酸酯(Epigallocatechin Gallate,EGCG)对植物乳杆菌CICC 6253生长的影响。结果表明GA和EGCG对植物乳杆菌生长的影响具有浓度依赖性,可表现出抑制和促进的双向调节作用,且二者的作用存在构效差异性。在GA和EGCG分别在5 mg/mL和7 mg/mL以上时,两者都抑制了植物乳杆菌的生长,而当GA和EGCG分别低于4 mg/mL和6 mg/mL时二者都促进了细胞的增殖,且随质量浓度增加其促进作用增大。GA可作为碳源被植物乳杆菌迅速利用,而EGCG(4 mg/mL)因其良好的细胞亲和性表现出显著的促进作用,高效地促进了植物乳杆菌细胞的增殖,菌落数在28h内由7.42lgCFU/mL增加到12.54lgCFU/mL,与空白对照组相比增加了2个数量级以上。本研究表明,GA和EGCG均表现出对植物乳杆菌的双向调节作用,其中EGCG表现出更佳的益生特性。本文研究结果可为GA和EGCG及类似食源性多酚在益生菌生长调节和相关食品研发中提供参考。

Identification,Characterization,and Probiotic Potentials of Lactobacillus pentosus SF-1

In recent years probiotics have been considered as a potential substitution of antibiotics to control pathogens and treat infectious diseases in aquaculture.In the present study a strain of Lactobacillus pentosus,named as L.pentosus SF-1,was isolated from waters in aquaculture.The species identification of this strain was conducted by 16S rRNA sequence,and the physiological and biochemical characteristics of this strain were assessed.Furthermore,the virulence,antibiotic sensitivity,cell surface characteristics and acid/base-resistance of L.pentosus SF-1 were determined to evaluate the probiotic potentials of this strain.Specifically,L.pentosus SF-1 is sensitive to most common antibiotics,and no hemolysin was generated from it,indicating the safety of this strain to hosts.In addition,L.pentosus SF-1 was able to tolerate the artificial gastric juice at pH 3 for 4 h and the artificial intestinal fluid at pH 6.8 or 8.0 for 6 h.Moreover,the analysis of self-aggregation and the adhesion of L.pentosus SF-1 to organic solvents suggested a high potential of L.pentosus SF-1 to inhabit the hosts,which was confirmed by testing the colonization of L.pentosus SF-1 in germ-free zebrafish.Interestingly,L.pentosus SF-1 displayed a high bactericidal activity against several bacterial pathogens.Consistently,the incubation of L.pentosus SF-1 significantly promoted the expression of antimicrobial components in zebrafish,contributing to the protection of the fish from E.tarda infection in vivo.Taken together,the probiotic strain L.pentosus SF-1 could be applied as anti-infection reagent in aquaculture.

Lactobacillus from fermented bamboo shoots prevents inflammation in DSS-induced colitis mice via modulating gut microbiome and serum metabolites

Fermented bamboo shoots(FBS)is a region-specific food widely consumed in Southwestern China,with Lactobacillus as the predominant fermenting bacteria.However,the probiotic potential of Lactobacillus derived from FBS reminds largely unexplored,especially for diseases with a low prevalence in areas consuming FBS,namely,inflammatory bowel disease.In this study,Lactiplantibacillus pentosus YQ001 and Lentilactobacillus senioris YQ005 were screening by in vitro probiotic tests to further investigate the probioticlike bioactivity in dextran sulfate sodium(DSS)-induced ulcerative colitis(UC)mouse.They exhibited more positive probiotic effects than Lactobacillus rhamnosus GG in preventing intestinal inflammatory response.The results revealed that both strains improved the abundance of deficient intestinal microbiota in UC mice,including Muribaculaceae and Akkermansia.In the serum metabolome,they modulated the DSS-disturbed levels of metabolites,with significant increment of cinnamic acid.Meanwhile,they reduced the expression levels of interleukin-1β(IL-1β),interleukin-6(IL-6)inflammatory factors and increased zonula occludens-1(ZO-1),Occludin,and cathelicidin-related antimicrobial peptide(CRAMP)in the colon.Consequently,these results demonstrated that Lactobacillus spp.isolates derived from FBS showed promising probiotic activity based on the gut microbiome homeostasis modulation,anti-inflammation and intestinal barrier protection in UC mice.

干酪乳杆菌干酪亚种Lactobacillus casei subsp.casei SY_(13)对衰老模型小鼠的抗氧化作用

【目的】利用一株经体外筛选具有较强抗氧化活性的干酪乳杆菌干酪亚种(Lactobacillus casei subsp.casei)SY13活菌制剂和灭活制剂研究D-半乳糖所致衰老模型小鼠体内抗氧化能力。【方法】采用SY13活菌制剂和灭活制剂饲喂由D-半乳糖连续皮下注射诱导的亚急性衰老模型小鼠,以各组小鼠血清和肝组织匀浆中超氧化物岐化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)的活力、总抗氧化能力(T-AOC)及脂质过氧化产物丙二醛(MDA)的含量为指标,全面评价其在小鼠体内的抗氧化能力。【结果】Lactobacillus casei subsp.casei SY13活菌制剂高、中剂量组(20、10mL·kg-1)能降低连续6周注射D-半乳糖诱发的衰老小鼠血清和肝脏组织中的MDA含量,同时提高血清和肝脏组织中GSH-Px活力以及总抗氧化能力(T-AOC),SOD活力变化不明显。【结论】SY13活菌制剂能有效提高亚急性衰老小鼠的抗氧化能力,具有一定延缓衰老的作用。

Lactobacillus菌株的益生功能评价

本研究评价了分离自传统发酵乳制品中4株L.paracasei supsp.paracasei菌株在模拟胃、肠液中的存活性,在HT-29细胞上的粘附性,对致病菌的抑制能力,以及刺激外周血单核细胞(PBMCs)增殖的能力。其中菌株Lactobacillus paracasei supsp.paracaseiM5AL、J23AL在模拟胃液存活率较高,而菌株Lactobacillus paracasei supsp.paracasei G15AL、T3AL在胃液中暴露3 h即失去活性;4株菌在模拟肠液中存活率较高。菌株L.paracasei supsp.paracasei M5AL和G15AL在HT-29细胞上有较强粘附能力(>40 bacteria cellsper 100 HT-29 cells),并抑制病原微生物E.coli ATCC25922、S.typhimurium ATCC 14028、S.sonnei ATCC 25931生长。4株菌活菌体刺激PBMCs增殖,具有潜在免疫调节功能。

益生菌Lactobacillus helveticus 130B4在牛乳中的生长特性及其促长因子研究

将具有ACE抑制活性的乳酸菌Lactobacillus helveticus 130B4用于发酵乳制品中,研究其在还原脱脂乳中的生长特性,并探讨通过添加生长促进物质对Lactobacillus helveticus 130B4生长特性以及对ACE抑制活性的影响。结果表明,Lb.helveticus 130B4菌株在还原脱脂乳培养基中生长较缓慢,产酸能力较弱,37℃培养36 h后的pH值为4.31,酸度达0.8%,活菌数为8.93 g-1;72 h后的pH值为3.45,酸度可以达到1.85%,活菌数达9.1g-1;酵母浸膏、大豆肽、核酸关联物质对该菌株的产酸能力有一定的促进作用。

Lactobacillus delbrueckii QS306对牛乳蛋白质水解能力与ACE抑制活性的研究

通过研究Lactobacillus delbrueckii QS306在10%的还原脱脂牛乳中的生长特性,探讨培养基中添加核酸关联物质时对蛋白质水解能力及其水解产物对血管紧张素转化酶(ACE)抑制活性的影响。结果表明,菌株QS306在10%的还原脱脂牛乳中生长缓慢,产酸量和生物量逐渐上升,在37℃培养第4 d时,滴定酸度和生物量分别达到68.24(°T)和6.91(OD410 nm×10),蛋白质水解能力为623.21μg·m L-1Tyr。在培养基中添加核酸关联物质鸟嘌呤(Guanine)时蛋白质水解能力提高了43.7%,ACE抑制活性可达到61.99%。

益生菌Lactobacillus helveticus H9在脱脂乳中复配发酵特性研究

将益生菌Lactobacillus helveticusH9分别与Lactococcus lactis、Streptococcus thermophilus及Lactobacillus delbrueckiisubsp.bulgaricus和Streptococcus thermophilus三类不同的菌种复配发酵脱脂乳,对其复配发酵乳的ACE抑制率、Val-Pro-Pro浓度、Ile-Pro-Pro浓度、游离氨基氮浓度、滴定酸度、p H值、活菌数、黏度、脱水收缩性和凝乳时间进行测定。结果表明,L.helveticusH9与Streptococcus thermophilus IMAU40115,Lactococcus lactis subsp.cremoris IMAU60065,Lactococcus lactissubsp.cremoris IMAU60064,L.delbrueckiisubsp.bulgaricus IMAU20094和Streptococcus thermophilus IMAU40051的复配发酵乳的ACE抑制率大于60%,而且和IMAU40115、IMAU20094和IMAU40051的复配发酵乳具有较高的Val-Pro-Pro和Ile-Pro-Pro浓度。从复配菌种的种类分析发现,Lactococcus lactis较Streptococcus thermophilus和酸奶发酵剂更适宜与L.helveticus H9复配发酵。本文为进一步研究L.helveticus H9发酵乳的开发提供了借鉴。

面包乳杆菌(Lactobacillus panis)C-M_2细菌素的分离纯化及特性分析

本实验对面包乳杆菌C-M2所产的新型细菌素进行分离纯化和特性研究。通过乙酸乙酯萃取、阳离子交换层析和半制备液相三步分离纯化该细菌素。最终细菌素的比活力达到5 044.96 AU/mg,纯化倍数为79.8倍,但回收率仅为0.35%。通过液相色谱-串联质谱法分析,该细菌素的分子质量为863.52 D,氨基酸序列为MVKKTSAV,它是一种新型的Ⅱ类细菌素。该细菌素具有广泛的抑菌谱,可以抑制革兰氏阳性和阴性的食品腐败菌。该细菌素对热和p H值稳定,即使在121℃灭菌15 min,仍保留82.1%的抑菌活性,在p H 6条件下保留85.6%的抑菌活性。它能被多种蛋白酶失活,不能被脂肪酶和淀粉酶失活,这些结果表明该细菌素具有作为食品生物防腐剂的潜在应用价值。

Lactobacillus helveticus 9的酪蛋白水解作用研究

基于酪蛋白水解度和多肽含量数据,研究了两株瑞士乳杆菌(L.helveticus 9和L.helveticus 6)与酪蛋白共培养后的蛋白水解效果,菌株L.helveticus 9蛋白水解活性显著高于菌株L.helveticus 6。在不同发酵时间下,菌株L.helveticus9对酪蛋白的水解作用随时间延长而逐渐递增,培养48h时酪蛋白的水解度可达17.3%,多肽含量达到0.62mg/ml;SDS-PAGE分析和Sephadex G-25层析鉴定表明,酪蛋白水解产物多肽含量随培养时间延长而增加。显然,具有较高蛋白水解活力的菌株L.helveticus 9可以用于从酪蛋白中来制备潜在的生物活性肽。

益生菌Lactobacillus casei Zhang和Bifidobacterium lactis V9在活性乳酸菌饮料中的应用

以L.casei Zhang和B.lactis V9为发酵菌株制备活性乳酸菌饮料,评价2株益生菌对活性乳酸菌饮料感官品质的影响及在货架期内的存活稳定性,为L.casei Zhang和B.lactis V9在产品中的应用提供基础数据。研究结果显示,利用L.casei Zhang单独发酵制备活性乳酸菌饮料的最适添加量为1.0×107CFU/g,4℃28 d贮藏期内保持高于3.0×108CFU/g水平;最佳复配发酵组合为L.casei Zhang+B.animalis V9(1∶1,2.0×107CFU/g),4℃28 d贮藏期内样品中总的益生菌活菌数保持5.0×108CFU/g以上,且在口感和滋气味方面感官评价分值最高;B.animalis V9与L.casei Zhang共同发酵发生协同效应,B.animalis V9的添加可促进L.casei Zhang的增殖,且有利于改善L.casei Zhang单株发酵样品的感官品质。

植物乳杆菌(Lactobacillus plantarum)对过敏原原肌球蛋白免疫活性的消减作用

为探究植物乳杆菌(Lactobacillus plantarum)对过敏原原肌球蛋白(tropomyosin,TM)免疫活性的影响,以富集纯化的TM为研究对象,利用植物乳杆菌的菌体、破碎内容物、菌体碎片、胞内酶提取液,以及去除蛋白、脂肪和脂磷壁酸、羧基酯化、氨基甲基化后的菌体分别水解TM 12~48 h,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、蛋白免疫印迹和竞争性酶联免疫吸附剂测定(enzyme linked immunosorbent assay,ELISA)等方法测定免疫活性,利用表位多克隆抗体结合红外光谱技术研究植物乳杆菌对TM二级结构及其致敏表位的影响,以解析植物乳杆菌消减TM免疫活性的作用位点。免疫学分析结果发现,不同提取物水解TM 48 h后,菌体对TM免疫活性的消减率最高(76.9%),而菌体碎片和氨基甲基化菌体的消减率最低,分别为60.7%、61.7%,表明植物乳杆菌对原肌球蛋白免疫活性有一定消减作用。红外光谱及ELISA结果显示植物乳杆菌各成分提取物会较大程度改变TM的二级结构并破坏其致敏表位,而氨基甲基化菌体和菌体碎片对TM的α-螺旋结构和致敏表位的破坏最小,且TM的折叠化结构较少,对TM免疫活性消减效果最差。此外,研究发现氨基可能是植物乳杆菌消减TM免疫活性的一个重要作用位点。本研究可为食物过敏原的活性控制及低致敏性水产加工制品的开发提供理论参考。

Lactobacillus plantarum M616生长及代谢特性研究

植物乳杆菌(Lactobacillus plantarum)是酸面团中优势菌群之一,为提供植物乳杆菌M616在酸面团发酵中应用的理论依据,采用比浊方法测定该菌株的生长曲线、产酸能力以及耐酸、耐盐、耐胆盐特性,并对其抑菌性能进行分析。结果表明:植物乳杆菌M616为革兰氏阳性菌,细胞呈直或弯短杆状;在37℃时MRS摇瓶中发酵30 h,p H 3.89,具有良好的产酸能力;在pH值为2.0、3.0的条件下培养6 h,OD600 nm分别为0.03、0.05;胆盐质量浓度为0.3 g/100 m L时培养4 h,OD600 nm为0.03;当NaCl质量分数达到8%以上时,菌株几乎不能生长。在抑菌方面,该菌株对大肠杆菌、金黄色葡萄球菌以及单增李斯特菌均有抑制效果,抑菌圈直径分别达14、15、17 mm。因此,植物乳杆菌M616具备较强的产酸能力,代谢性能良好,可作为酸面团发酵用菌株。

Effects of Yogurt Containing <i>Lactobacillus gasseri</i>OLL2716 on Autonomic Nerve Activities and Physiological Functions

The purpose of this study was to investigate the effects of yogurt containing Lactobacillus gasseri OLL2716 (LG21) on autonomic nerve activities, peripheral blood flow, skin condition (skin pig-mentations and moisture), saliva s-IgA and examination of quality of life (QOL). 20 healthy female volunteers (yogurt containing LG21 group: 10 people, yogurt containing Bifidobacterium (Bif) group: 10 people) were examined. The subjects ingested 100 g of yogurt twice daily for 4 weeks. Analysis was before and after 4 weeks dosage. By the effects for the autonomic nervous activity, parasympathetic increase was observed in the LG21 yogurt group, but was not significant increase. The LG21 yogurt was significantly increased on the peripheral blood flow. The LG21 yogurt was significantly increased on saliva s-IgA. The LG21 yogurt and Bif yogurt were significantly decrease on skin pigmentation. Also, LG21 yogurt was significantly increased on skin moisture. As a result of QOL questionnaire, incomplete evacuation, lower abdominal fullness, cold extremities and pimply or rough skin improved in LG21 yogurt and Bif yogurt after the administration period. These results suggest that the improvement effects of LG21 yogurt may be related to the activity of the parasympathetic nervous system.

Fermented Black Turmeric Designed by Lactobacillus Rearranged Leukocyte Subsets and Anti-Oxidative Activity

A plant fermentation was carried out by Yeast and Lactobaccilli against fermented black turmeric, Kaempferia parviflora (FBT). These materials were proved by as safe in animal safety experiment. We tried to investigate changes of immune-competent cells that commonly utilized FBT, including after administration of immno-suppressed animals, the effects by FBT on the regulated effect on the cells were evaluated. Our results showed that FBT augmented the level of lymphocytes in number, while FBT regulated the level of granulocytes in both number and function. In our clinical study with 20 healthy volunteers, granulocyte and lymphocyte ratio suggesting their constitution as neutral in peripheral blood were increased significantly 30 days after the administration of FBT in rodents, and compromised host was prepared with cancer chemotherapeutic agent (Mytomycin-C). Our observations showed against intracellular parasite, and that FBT augmented intercellular pathogen through humoral immunity. We discussed the significance and mechanism of FBT on the level of leukocyte subsets in number and function that were considered to be potential indicators for the activation of the compromised host. We also proposed an idea that FBT exhibited tonic effects via activating complement components. The evidences were shown by immune-electrophoretic method. Moreover, we tried to access further to the anti-oxidative activities of this FBT. This modification brought to the significant lift up for antibody producing cells and anti-oxidative activity for phagocytic cells.

Improving the Antioxidant Properties of Fermented Camel Milk Using Some Strains of <i>Lactobacillus</i>

This study aimed at improving the antioxidant capacity of fermented camel milk using some single strains of Lactobacillus (Lb. helveticus B-734, Lb. casei subsp. casei B-1922, Lb. paracasei subsp. paracasei B-4560, Lb. rhamnosus B-1445 or Lb. rhamnosus B-442), as well as evaluating the acceptability of the final products. The acidity, proteolysis degree, antioxidant activity, viscosity and organoleptic properties of fermented milk were assessed during 14 days of storage at 4°C. Total phenolic content (TPC), DPPH radical scavenging activity, Ferrous ion chelating ability (FCA) and Ferric reducing antioxidant power (FRAP) assays were used to determine the antioxidant activity of fermented milks. The results indicated that fermented milks differed significantly (P < 0.05) in all studied parameters due to the type of starter culture used. During storage period, samples containing Lb. helveticus had the highest proteolysis degree, while samples with commercial starter culture (control) showed the lowest degree of proteolysis. Fermented milks containing Lactobacillus strains showed higher DPPH radical scavenging activity compared to those samples containing commercial starter culture. By the end of storage, there was a significant improvement (P < 0.05) in scavenging activity for all fermented milk samples. Regarding FCA, at the beginning of storage fermented milks containing Lb. paracasei, Lb. rhamnosus B-442 or commercial starter culture had the highest FCA values, while Lb. rhamnosus B-1445 samples recorded the highest value at the end of storage. Lb. helveticus samples had the highest TPC and FRAP values (P < 0.05) throughout the storage. There was a high significant correlation (P < 0.0001) between the proteolysis degree and the values of FRAP and TPC. Samples containing Lb. rhamnosus B-442, Lb. rhamnosus B-1445 or commercial starter culture received the highest taste and overall acceptability scores while Lb. helveticus samples were the lowest. It is recommended to use Lb. rhamnosus B-442 and Lb. rhamnosus B-1445 for producing fermented camel milk with high antioxidant activity and acceptability.

Effect of Active Dried Yeast on Intestine Development,Intestinal Flora and Serum Cholesterol Mass Concentration of Quails

[ Objective] The experiments aimed to investigate the effect of dietary active dried yeast on intestine development, intestinal flora and serum cholesterol mass concentration of quails. [ Method] One-day-old 180 quails were randomly divided into 4 groups, 3 repetitions, each with 15 quails. Control group was fed with basic dietary, while experimental groups were added with 0.6, 1,2 g/kg active dried yeast respectively in basic dietary, [ Result] Addition of active dried yeast had little effect on quails＇ intestine development（ P 〉0.05）. With the increasing adding amount active dried yeast, the number of colibacillus decreased significantly （P 〈0.05） while lactobacillus content increasing sharply （P 〈 0.05）. Dietary with 1 and 2 g/kg active dried yeast can significantly decrease the mass concentration of cholesterol in serum （ P 〈 0.05）. [ Conclusion ] Adding active dried yeast in feed can decrease the number of colibacillus while increasing lactobacillus content in intestinal tract of quails, also can low serum cholesterol mass concentration.

Use of <i>Lactococcus lactis</i>Subsp. <i>Lactis</i>Strains to Inhibit the Development of Pathogens

Bovine mastitis affects the udder health and thus causing significant economic losses. Probiotic products based on the use of lactic acid bacteria (LAB) to limit pathogens multiplication and pre-infection risks can be an interesting alternative to post infection allopathic treatment with antibiotics. Lactococcus lactis is one of the most important bacteria used in dairy technology. In this work, a total of 21 Lactococcus lactis subsp. Lactis strains, 20 from goat milk whey and one strain from cow milk were used to evaluate their antibacterial activity against four pathogenic germs responsible for mastitis: Escherichia coli, Staphylococcus aureus, Streptococcus uberis and Streptococcus agalactiae. The nisin-producing cow milk strain was active against St. uberis and Str. Agalactiae using the well diffusion method. For the strains isolated from goat milk whey, no antimicrobial effect was observed against these pathogens. However, a different approach based on the growth of pathogenic bacteria interacting with the Lactococcus lactis strains in a minimum medium was used to study the barrier effect of LAB. The Lactococcus lactis strains S1 and S2 from goat milk whey depleted the growth of Sa. aureus, St. uberis and E. coli during 8 h and stopped the development of St. agalactiae.