具有抑制肠道致病菌和黏附Caco-2细胞作用的益生性乳酸菌的筛选及鉴定

为了筛选具有肠道益生特性的乳酸菌,进一步开发益生菌资源,本文研究采用牛津杯、人工模拟胃肠液及体外黏附Caco-2细胞等方法,以鼠李糖乳杆菌GG(Lactobacillus rhamnosus GG,LGG)为对照菌株,对实验室保藏15株乳酸菌的益生特性进行筛选与评估。实验结果表明:15株乳酸菌对两株沙门氏菌都具有一定抑制效果,抑菌圈直径为10.95~22.06 mm;乳酸菌C174、D24、D599、C37、D512具有较好的耐酸耐胆盐能力,在人工胃液3 h内存活率达到60%以上,人工肠液4 h内存活率达到80%以上;在黏附试验中,C174对Caco-2细胞具有较强黏附能力,黏附数量为740 CFU/100细胞,略高于对照菌株鼠李糖乳杆菌(543 CFU/100细胞);通过对具有高黏附性的菌株C174进行生理生化和16S rRNA分子测序鉴定,结果表明菌株C174为植物乳杆菌(Lactobacillus plantarum),命名为Lactobacillus plantarum ZJ-174。Lactobacillus plantarum ZJ-174的抑菌能力强、能够耐酸耐胆盐,并具有很强的黏附能力,是一株潜在的益生菌,具有治疗或预防人和动物肠道疾病等应用价值。     

乳源乳杆菌的益生特性及抗氧化活性研究

以5株乳源乳杆菌为研究对象,鼠李糖乳杆菌（Lactobacillus rhamnosus）LGG为阳性对照菌株,通过pH和胆盐耐受性试验、胃肠液耐受性试验、抗生素敏感性试验、抑菌活性试验、黏附Caco-2细胞试验、抗氧化活性试验考察5株菌的益生特性。结果表明,5株乳杆菌对环境胁迫均具有一定的抗逆性;对大肠杆菌、金黄色葡萄球菌和单增李斯特菌具有一定的抑制能力,抑菌圈直径均分别＞10 mm、＞20 mm、＞13 mm;其中鼠李糖乳杆菌260益生特性较优,黏附能力较强,黏附率为4.16%,DPPH自由基清除能力56.36%、羟自由基的清除能力23.09%、总抗氧化能力（T-AOC）为5.26 μmol/mL,具有潜在的应用价值。     

鼠李糖乳杆菌(LGG)的功能特性及其应用前景

鼠李糖乳杆菌LGG(Lactobacillus rhamnosus GG,ATCC 53103)是目前最受全球关注的益生菌之一。该菌能够耐受动物消化道环境,具有能够在人和动物肠道内定植,起到调节肠道菌群、预防和治疗腹泻、排除毒素、预防龋齿和提高机体免疫力等功能特性。     

鼠李糖乳酪杆菌对乳鼠肠道发育和阪崎克罗诺杆菌致坏死性肠炎的影响

为研究鼠李糖乳酪杆菌（Lacticaseibacillus rhamnosus）SW-02 对乳鼠肠道发育的影响,将新生C57BL/6 小鼠以SW-02（1×107 CFU/d）灌胃,饲养2 周后,检测体质量、脏器指数、肠道组织苏木精-伊红染色、细胞增殖标志基因和紧密连接蛋白的mRNA 水平及免疫球蛋白A（immunoglobulin A,IgA）水平;将新生小鼠分为对照组、阪崎克罗诺杆菌组（CS 组）、SW-02 干预组（CS+SW-02 组）,饲养至第10 天,检测体质量、肠道组织苏木精-伊红染色、细胞增殖标志基因和紧密连接蛋白的mRNA 水平及炎症因子水平,研究SW-02 对阪崎克罗诺杆菌致坏死性小肠结肠炎的影响。结果显示,鼠李糖乳酪杆菌SW-02 能够促进乳鼠早期的生长,促进机体肠道发育和免疫功能,还可以有效改善阪崎克罗诺杆菌对乳鼠肠道造成的损伤,降低炎症。     

新疆哈萨克酸马奶中功能性乳酸菌株的筛选、鉴定及功能评价

摘 要: 目的 从新疆哈萨克传统发酵酸马奶中筛选和鉴定益生性性乳酸菌,并探究其胃肠道消化耐受性和抗氧化潜力。方法 通过稀释涂布平板法和生理生化鉴定来分离纯化乳酸菌属,利用耐酸性和耐胆盐性筛选出潜在的胃肠道消化耐受性强的候选菌株,采用体外模拟消化、硫酸铁铵比色法和TG试剂盒评估候选菌株的胃肠道存活率、降胆固醇能力和降甘油三酯能力。同时,通过DPPH自由基清除能力、ABTS阳离子自由基清除能力和铁还原能力测试候选菌株的抗氧化潜力,并与德式乳杆菌保加利亚亚种(Lactobacillus delbrueckii subsp. bulgaricus)进行比较分析。最后,采用16S rRNA高通量测序对候选菌株进行精确鉴定。结果 从新疆哈萨克传统发酵酸马奶中分离纯化出96株菌株,40株被鉴定为乳酸菌属。10株候选菌株表现出较高的耐酸性和耐胆盐性,并被鉴定为鼠李糖乳杆菌(Lactobacillus rhamnosus)、植物乳杆菌(Lactobacillus plantarum)、戊糖乳杆菌(Lactobacillus pentosus)和副干酪乳杆菌(Lactobacillus paracasei)。其中,M2菌株（L. rhamnosus）表现出最佳的益生特性,胆固醇和甘油三酯降解率高,模拟胃肠液中存活率高,对DPPH和ABTS自由基的清除能力显著高于德式乳杆菌保加利亚亚种。结论 本研究成功从新疆哈萨克传统发酵酸马奶中筛选鉴定出高耐受性、高抗氧化性的L. rhamnosus M2,为发掘和利用酸马奶中的功能性益生菌提供了理论依据。     

益生性植物乳杆菌的体外抗氧化活性研究

对从内蒙古奶豆腐及新生儿肠道中分离的5株植物乳杆菌的抗氧化活性进行分析.将保存的5株植物乳杆菌用MRS液体培养基活化培养后,对其耐酸特性、耐胆盐特性、疏水性和抗氧化能力进行分析.结果表明,5株植物乳杆菌均具有一定的耐酸和耐胆盐特性,其中植物乳杆菌MGSHC4302能够耐受pH=2.0的酸性环境,植物乳杆菌CRFA4608、MGSHC4301和MGSHC4302能够耐受1.0％的胆盐:5株植物乳杆对二甲苯和三氯甲烷的疏水性明显好于LGG,表明这些菌株具有较好的黏附性.实验结果表明,植物乳杆菌CRFA4608和MGSHC4302对羟自由基的清除能力高于对照菌株鼠李糖乳杆菌LGG,除植物乳杆菌MGSHC4303外其他4株茵对DPPH的清除能力均高于鼠李糖乳杆菌LGG或与之相近,而植物乳杆菌MGSHC4302、MGSHC4303具有较高的超氧阴离子清除能力.综上所述,植物乳杆菌MGSHC4302可以作为今后开发具有益生性产品的潜在菌株.     

鼠李糖乳杆菌B6抗氧化活性和细胞保护作用研究

以鼠李糖乳杆菌LGG(Lactobacillus rhamnosus GG)菌株为对照,将几株乳杆菌进行体外抗氧化能力的比较,以DPPH自由基、·OH及·O₂^-清除率为指标综合筛选出抗氧化能力较全面的鼠李糖乳杆菌B6,并比较不同培养基条件下其代谢产物的抗氧化能力;然后通过建立PC12细胞氧化损伤模型,测定益生菌代谢产物处理后丙二醛、活性氧的含量,探究鼠李糖乳杆菌B6代谢产物对细胞氧化应激的影响。结果表明,鼠李糖乳杆菌B6发酵MRS上清液的DPPH自由基、·OH以及·O₂^-清除率分别达到63.23%、99.38%和26.35%,都显著高于LGG。B6发酵脱脂乳得到的代谢产物与发酵MRS得到的代谢产物相比较,DPPH自由基清除率和·OH清除率都有显著升高,两者·O₂^-清除率相当。在PC12细胞氧化损伤实验中B6发酵脱脂乳的代谢产物降低了PC12细胞的氧化应激损伤,提高了氧化应激状态下的抗氧化能力。     

具有降血压功能乳酸菌的筛选及其益生作用

为高血压治疗方式提供更多选择,筛选1株具有较强降血压功能的益生菌菌株,探究其降血压能力及其益生特性。通过ACEKitWST试剂盒对12株益生菌菌株进行血管紧张素转化酶（ACE）活性抑制的检测,选出ACE抑制率最强的菌株,测定其在胃肠液中的存活率及对酸和胆盐的耐受性。结果表明,鼠李糖乳杆菌（Lactobacillus rhamnosus）HCS01-013的ACE抑制率最高（61.54%）,其在人工胃液中处理3 h、人工肠液中处理4 h后,存活率分别为99.9%、99.7%;分别在pH 2.0、1.5%胆盐的环境中培养17 h,存活率为71.95%、96.79%。说明鼠李糖乳杆菌HCS01-013是一株具有较强降血压功能且耐逆性能较强的益生菌菌株。     

基于主成分分析法的降血脂乳酸菌筛选

采用体外实验方法研究了12株乳酸菌对模拟胃肠道环境的耐受力、抗氧化能力及降胆固醇和降甘油三酯能力,并使用主成分分析法做综合评价,以期获得一株具有辅助降脂作用的乳酸菌。结果表明:鼠李糖乳杆菌LV108具有较强的耐胃/肠液能力、·OH清除力、DPPH·清除力及还原能力,胆盐耐受性为47.99%、胆固醇降解率为28.76%,甘油三酯降解率为21.11%,均居于所测定菌株的最高值。主成分分析显示Lactobacillus rhamnosus LV108的综合得分Y为0.909,高于其他菌株。L.rhamnosus LV108具备辅助降脂能力。     

臭味发酵食品中益生菌分离鉴定及功能性研究

目的分析臭味天然发酵食品中常见益生菌种类。方法本实验从臭味天然发酵食品中选取了4种常见益生菌株,并对耐酸耐胆盐能力、抗药性进行了研究。结果 4株益生菌株经16S rDNA分子鉴定为鼠李糖乳杆菌(Lactobacillus rhamnosus)、植物乳杆菌(Lactobacillus plantarum)、嗜酸乳杆菌(Lactobacillus acidophilus)和粪肠球菌(Enterococcus faecium)。嗜酸乳杆菌在pH 4的培养基培养16 h后,其相对OD_(600 nm)值为51.13%,具有较强的耐酸能力。植物乳杆菌在0.3 g/L和0.6 g/L胆盐质量浓度下培养16 h后,其相对OD_(600 nm)值分别为98.88%、66.22%,具有较强的耐胆盐能力。实验结果表明这2株菌的耐药性都低于鼠李糖乳杆菌和粪肠球菌。结论植物乳杆菌、嗜酸乳杆菌为耐酸耐胆盐低耐药性特点的益生菌株。     

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均为长梭状、圆润的杆状,表层物质的厚度也没有发生显著变化（P＞0.05）。副干酪乳杆菌m111具有较强的耐消化应激以及对肠道黏附的能力,且消化应激可提高其黏附肠道的能力,是1 株潜在的具有良好应用价值的益生乳杆菌。     

In vitro testing of commercial and potential probiotic lactic acid bacteria

Probiotics are defined as live microorganisms which when administered in adequate amounts confer a health benefit on the host. The objective of this study was to investigate the diversity of selected commercial and potential probiotic lactic acid bacteria using common in vitro screening assays such as transit tolerance in the upper human gastrointestinal tract, adhesion capacity to human intestinal cell lines and effect on epithelial barrier function. The selected bacteria include strains of Lactobacillus plantarum, Lactobacillus pentosus, Lactobacillus farciminis, Lactobacillus sakei, Lactobacillus gasseri, Lactobacillus rhamnosus, Lactobacillus reuteri and Pediococcus pentosaceus. Viable counts after simulated gastric transit tolerance showed that L. reuteri strains and P. pentosaceus tolerate gastric juice well, with no reduction of viability, whereas L. pentosus, L. farciminis and L. sakei strains lost viability over 180min. All strains tested tolerate the simulated small intestinal juice well. The bacterial adhesion capacity to human intestinal cells revealed major species and strain differences. Overall, L. plantarum MF1298 and three L. reuteri strains had a significant higher adhesion capacity compared to the other strains tested. All strains, both living and UV-inactivated, had little effect on the epithelial barrier function. However, living L. reuteri strains revealed a tendency to increase the transepithelial electrical resistance (TER) from 6 to 24h. This work demonstrates the diversity of 18 potential probiotic bacteria, with major species and strain specific effects in the in vitro screening assays applied. Overall, L. reuteri strains reveal some interesting characteristics compared to the other strains investigated.     

传统发酵豆腐酸浆中高产酸乳酸菌的分离鉴定及特性分析

该研究从采集于全国具有代表性的6大豆腐产地的7种豆腐酸浆老汤中分离筛选各样品中高产酸乳酸菌,通过形态观察及分子生物学技术对其进行鉴定,并对其产酸特性、耐酸特性及耐盐特性进行研究。结果表明,分离筛选到7株产酸性能优良的乳酸菌,并鉴定菌株L2-03、L3-01、L6-07与L7-01为玉米乳杆菌（Lactobacillus zeae）,菌株L1-02、L8-03与L10-01为副干酪乳杆菌（Lactobacillus paracasei）。除菌株L6-07外,6株乳酸菌均能产草酸、酒石酸、丙酮酸、乳酸、乙酸、富马酸及琥珀酸共7种有机酸,其中乙酸与乳酸为主要有机酸。产酸能力方面,菌株L6-07产酸量最高,达25.69 g/L,产酸能力最强;耐酸性方面,菌株L6-07在pH 2.5的酸性条件下仍能保持生长,耐酸性最好;耐盐性方面,菌株L1-02在5%NaCl条件下表现出较强的耐受性,耐盐性最强,菌株L6-07次之。     

Manufacture of Fior di Latte cheese by incorporation of probiotic lactobacilli

This work aimed to select heat-resistant probiotic lactobacilli to be added to Fior di Latte (high-moisture cow milk Mozzarella) cheese. First, 18 probiotic strains belonging to Lactobacillus casei, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus reuteri were screened. Resistance to heating (65 or 55°C for 10 min) varied markedly between strains. Adaptation at 42°C for 10 min increased the heat resistance at 55°C for 10 min of all probiotic lactobacilli. Heat-adapted L. delbrueckii ssp. bulgaricus SP5 (decimal reduction time at 55°C of 227.4 min) and L. paracasei BGP1 (decimal reduction time at 55°C of 40.8 min) showed the highest survival under heat conditions that mimicked the stretching of the curd and were used for the manufacture of Fior di Latte cheese. Two technology options were chosen: chemical (addition of lactic acid to milk) or biological (Streptococcus thermophilus as starter culture) acidification with or without addition of probiotics. As determined by random amplified polymorphic DNA-PCR and 16S rRNA gene analyses, the cell density of L. delbrueckii ssp. bulgaricus SP5 and L. paracasei BGP1 in chemically or biologically acidified Fior di Latte cheese was approximately 8.0 log(10)cfu/g. Microbiological, compositional, biochemical, and sensory analyses (panel test by 30 untrained judges) showed that the use of L. delbrueckii ssp. bulgaricus SP5 and L. paracasei BGP1 enhanced flavor formation and shelf-life of Fior di Latte cheeses.     

Assessment of adhesion properties of novel probiotic strains to human intestinal mucus

Potential new probiotic strains Lactobacillus brevis PELI, L. reuteri ING1, L. rhamnosus VTT E-800 and L. rhamnosus LC-705 were assessed for their adhesion properties using the human intestinal mucus model. The effect on the adhesion of exposure to acid and pepsin and to milk were tested to simulate gastric and food processing conditions, and the effect of different growth media on adhesion was tested. The properties of the four strains were compared to the well-investigated probiotic L. rhamnosus strain GG. Three of the tested strains showed significant adhesion properties in the mucus model, while L. brevis PELI had intermediate adhesion and L. rhamnosus LC-705 adhered poorly. Pretreatment with different milks decreased the adhesion and low pH and pepsin treatment reduced the adhesion of all tested strains except L. rhamnosus LC-705. No competitive exclusion of pathogenic Salmonella typhimurium or Escherichia coli SfaII was observed. The results indicate that major differences exist between tested proposed probiotic strains. The growth media and the food matrix significantly affect the adhesive ability of the tested strains. This has previously not been taken into account when selecting novel probiotic strains.     

Characterization of certain bacterial strains for potential use as starter or probiotic cultures in dairy products

The present work was aimed at characterizing 12 strains of lactic acid bacteria (LAB) to obtain improved potential starter or probiotic cultures that could be used for making dairy products from ewe's milk and cow's milk. Eight strains with antimicrobial properties, isolated from ewe's milk and from cheese made from ewe's and/or cow's milk, were studied. They were identified as Enterococcus faecalis (five strains), Lactococcus lactis subsp. cremoris, Leuconostoc mesenteroides, and Lactobacillus paracasei subsp. paracasei (one strain of each species). Additionally, four strains were obtained from the American Type Culture Collection: Lactobacillus casei 393 (isolated from cheese), L. lactis subsp. lactis 11454 (origin nonspecified and a producer of nisin), and two strains isolated from human feces (L. paracasei subsp. paracasei 27092 and Lactobacillus rhamnosus 53103, antibacterial agent producer). All E. faecalis strains showed at least one virulence factor (either hemolysin or gelatinase), which emphasizes the importance of these studies in this species. Both L. lactis strains and most Lactobacillus spp. were good acidifiers in ewe's milk and cow's milk at 30°C. High β-galactosidase activity, as well as aminopeptidase activities that favor the development of desirable flavors in cheese, were detected in all Lactobacillus spp. strains. Furthermore, L. rhamnosus ATCC 53103 showed α-fucosidase activity (thought to help colonization of the intestine) and lack of α-glucosidase activity (a trait considered positive for diabetic and obese humans). This last enzymatic activity was also lacking in L. lactis ATCC 11454. L. mesenteroides was the only strain D(2)-lactic acid producer. The selection of any particular strain for probiotic or dairy cultures should be performed according to the technological and/or functional abilities needed.     

Isolation and identification of lactobacilli from novel-type probiotic and mild yoghurts and their stability during refrigerated storage.

A total of 26 Lactobacillus strains were isolated from various mild yoghurts and novel-type probiotic dairy products and from a starter culture preparation and were identified by using DNA-DNA hybridization technique. The species present in those products were found to be Lactobacillus acidophilus, L. johnsonii, L. crispatus, L. casei, L. paracasei and L. rhamnosus. DNA homology analysis revealed that some strains had been misclassified by their investigators. Three strains designated as L. acidophilus (L. acidophilus LA-1, L. acidophilus ATCC 43121 and the Lactobacillus strain from Biogarde culture) were found to belong to L. johnsonii and L. acidophilus L1 to be L. crispatus. Strains designated as L. casei were found to be members of three separate species: L. casei, L. paracasei and L. rhamnosus. Viable numbers of lactobacilli in mild and probiotic yoghurts varied greatly including some products with very low Lactobacillus counts. The majority of the probiotic yoghurts, however, contained viable counts above 10(5) per g even at the end of the best before use period.     

乳杆菌发酵对黑米多酚含量及组成的影响

以植物乳杆菌（Lactobacillus plantarum 115,LP）、嗜酸乳杆菌（Lactobacillus acidophilus 14,LA）、干酪乳杆菌（Lactobacillus casei GB,LC）、保加利亚乳杆菌（Lactobacillus bulgaricus 134,LB）、副干酪乳杆菌（Lactobacillus paracasei 37,LPC）和鼠李糖乳杆菌（Lactobacillus rhamnosus GG,LGG）为发酵菌种,研究乳杆菌发酵对黑米游离酚和结合酚的含量及组成的影响。结果表明,6株乳杆菌发酵均显著提高了黑米游离酚的含量,但不同乳杆菌发酵后提高的程度具有显著差异,其中提高最显著的是LB。不同乳杆菌发酵后黑米中9种游离态和结合态单体酚的含量变化存在显著差异,其中,6株乳杆菌发酵后黑米中游离态的香草酸和对羟基苯甲酸的含量均显著降低（P<0.05）,LP菌株除外游离态咖啡酸的含量均显著提高（P<0.05）,而6株乳杆菌发酵后结合态阿魏酸含量均显著降低（P<0.05）。该研究为黑米的高效利用和天然活性产品的开发提供参考。