嗜酸乳杆菌和双歧杆菌益生特性的研究

以两株嗜酸乳杆菌和三株动物双歧杆菌为研究对象,详细研究它们的耐胃酸性、耐胆汁盐性、抑菌性和抗药性。结果表明:五株益生菌具有良好的耐酸耐胆汁盐性,双歧杆菌的耐酸性较强于嗜酸乳杆菌;抑菌性和抗药性结果表明:五株益生菌对致病菌大肠杆菌、金黄色葡萄球菌和沙门氏菌均有抑制作用,嗜酸乳杆菌的抑菌性较强于双歧杆菌;五株益生菌对三种抗生素乙酰螺旋霉素、链霉素和青霉素均敏感,敏感程度各异。     

Lactic acid starter and probiotic bacteria: a comparative “in vitro” study of probiotic characteristics and biological barrier resistance

Probiotic characteristics (deconjugation of bile salts, hydrophobicity and β-galactosidase activity) and the resistance to biological barriers (gastric juice and bile salts) of 24 strains of lactic acid starter bacteria (Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus and Lactococcus lactis) and 24 strains of probiotic bacteria (Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus rhamnosus and bifidobacteria) were compared. Among the probiotic bacteria tested, Lactobacillus acidophilus was the most interesting species since it showed high values of resistance to gastric juice and bile, hydrophobicity and β-galactosidase and bile salts deconjugation activities. Bifidobacterium bifidum strains showed the same behavior, although the values of the parameters investigated were slightly lower than those obtained for Lactobacillus acidophilus. On the other hand, it was demonstrated that Lactobacillus delbrueckii subsp. bulgaricus was the lactic acid starter species with the best probiotic characteristics among the starter species assessed. It was resistant to gastric juice and bile, and showed high values for β-galactosidase activity. On the other hand, lactic acid starter bacteria showed hydrophobicity values similar to or higher than those obtained for the strains of the Lactobacillus casei . According to the results found, the total probiotic value of a fermented dairy product should take into account not only the intestinal probiotic cultures used in the formulation but also the probiotic contribution of the lactic acid starter microflora.     

Acid, bile, and heat tolerance of free and microencapsulated probiotic bacteria

ABSTRACT:  Eight strains of probiotic bacteria, including Lactobacillus rhamnosus , Bifidobacterium longum, L. salivarius, L. plantarum , L. acidophilus , L. paracasei , B. lactis type Bl-O4, and B. lactis type Bi-07, were studied for their acid, bile, and heat tolerance. Microencapsulation in alginate matrix was used to enhance survival of the bacteria in acid and bile as well as a brief exposure to heat. Free probiotic organisms were used as a control. The acid tolerance of probiotic organisms was tested using HCl in MRS broth over a 2-h incubation period. Bile tolerance was tested using 2 types of bile salts, oxgall and taurocholic acid, over an 8-h incubation period. Heat tolerance was tested by exposing the probiotic organisms to 65 °C for up to 1 h. Results indicated microencapsulated probiotic bacteria survived better ( P < 0.05) than free probiotic bacteria in MRS containing HCl. When free probiotic bacteria were exposed to oxgall, viability was reduced by 6.51-log CFU/mL, whereas only 3.36-log CFU/mL was lost in microencapsulated strains. At 30 min of heat treatment, microencapsulated probiotic bacteria survived with an average loss of only 4.17-log CFU/mL, compared to 6.74-log CFU/mL loss with free probiotic bacteria. However, after 1 h of heating both free and microencapsulated probiotic strains showed similar losses in viability. Overall microencapsulation improved the survival of probiotic bacteria when exposed to acidic conditions, bile salts, and mild heat treatment.     

新疆发酵驼乳中分解植酸盐酵母菌的筛选及鉴定

为发掘和保护新疆牧区传统发酵驼乳中潜在益生酵母菌资源,从新疆伊犁地区不同牧区采集12份传统发酵驼乳样品,通过酵母浸出粉胨葡萄糖（YPD）培养基分离酵母菌,通过富含植酸二钾的培养基初筛及降解植酸盐能力的测定从中筛选可降解植酸盐的酵母菌,通过形态观察、生理生化试验及5.8S rDNA ITS1/ITS4区域序列同源性分析进行菌种鉴定,并测定其耐酸及耐胆盐性能。结果表明,筛选出11株可分解植酸盐的酵母菌,鉴定8株为库德毕赤酵母（Pichia kudriavzevii）,2株为白地霉（Geotrichum candidum）,1株为乳酸克鲁维酵母（Kluyveromyces lactis）,其中库德毕赤酵母菌BJ9-12在模拟胃液和模拟肠液中的存活率分别为87.79%和81.50%,耐酸及耐胆盐性能较好,且培养48 h时分解植酸盐的透明圈直径达到3.45 cm,在发酵食品中具有良好的应用潜力。     

鼠李糖乳杆菌耐酸及胆盐能力研究

本实验对来源于婴儿粪便鼠李糖乳杆菌进行耐酸、耐胆盐能力表征及模拟胃液和模拟肠液实验。结果获得鼠李糖乳杆菌菌株B在pH1.8条件下,维持2h,活菌数达到107CFU/ml以上;在pH值为2.2以上的培养基中生长良好,而且随着时间的延长菌株的数量增加。在胆盐浓度在0.5%以下的MRS-broth培养基中鼠李糖乳杆菌B菌株维持4h,活菌数均达到108CFU/ml以上,而且随着时间的延长活菌数均有所增加。模拟胃液和模拟肠液实验结果表明该菌株能够有效通过胃环境,并能在肠道中繁殖。结果可见,鼠李糖乳杆菌B具有较强的酸的胆盐的耐受能力,符合微生态制剂和乳酸菌发酵功能食品菌种的要求。     

Viability,Acid and Bile Tolerance of Spray Dried Probiotic Bacteria and Some Commercial Probiotic Supplement Products Kept at Room Temperature

Production of probiotic food supplements that are shelf‐stable at room temperature has been developed for consumer's convenience, but information on the stability in acid and bile environment is still scarce. Viability and acid and bile tolerance of microencapsulated Bifidobacterium spp. and Lactobacillus acidophilus and 4 commercial probiotic supplements were evaluated. Bifidobacterium and L. acidophilus were encapsulated with casein‐based emulsion using spray drying. Water activity (a_w) of the microspheres containing Bifidobacterium or L. acidophilus (SD GM product) was adjusted to 0.07 followed by storage at 25 °C for 10 wk. Encapsulated Bifidobacterium spp. and Lactobacillus acidophilus and 4 commercial probiotic supplement products (AL, GH, RE, and BM) were tested. Since commercial probiotic products contained mixed bacteria, selective media MRS‐LP (containing L‐cysteine and Na‐propionate) and MRS‐clindamycin agar were used to grow Bifidobacterium spp. or L. acidophilus, respectively, and to inhibit the growth of other strains. The results showed that a_w had a strong negative correlation with the viability of dehydrated probiotics of the 6 products. Viable counts of Bifidobacterium spp. and L. acidophilus of SD GM, AL, and GH were between 8.3 and 9.2 log CFU/g, whereas that of BM and RE were between 6.7 and 7.3 log CFU/g. Bifidobacterium in SD GM, in AL, and in GH products and L. acidophilus in SD GM, in AL, and in BM products demonstrated high tolerance to acid. Most of dehydrated probiotic bacteria were able to survive in bile environment except L. acidophilus in RE product. Exposure to gastric juice influenced bacterial survivability in subsequent bile environment.     

Fermentation characteristics and transit tolerance of Lactobacillus casei Zhang in reconstituted mare milk during storage

The fermentation characteristics of Lactobacillus casei Zhang in mare milk and transit tolerance during refrigerated storage for 28 days were evaluated. There were no dramatic changes in the viable counts of L. casei Zhang in the fermented mare milk samples during storage at various inoculation levels. L. casei Zhang showed good tolerance to the simulated transit juice and maintained high viability in mare milk (above 10⁸cfu/g) during storage. Our results showed that L. casei Zhang had good probiotic properties, and suggest that mare milk could serve as the vehicle for the delivery of L. casei Zhang.     

产乳酸芽孢杆菌DU-106益生特性的研究

以产乳酸芽孢杆菌（Bacillus sp.）DU-106为研究对象,对其益生特性进行研究。测定菌株DU-106发酵过程中的生长曲线、产酸曲线、高胆盐耐受性、模拟胃肠液耐受性、酸性pH耐受性和抗生素敏感性。结果表明,菌株DU-106在发酵36 h后达到稳定的生长期和较高的产酸量,产酸约1.7 g/L;经过0.3%和0.6%浓度的胆盐处理4 h后,存活率分别为91.8%和73.9%;经过4 h模拟胃液和24 h模拟肠液处理后存活率分别为66.5%和71.7%;在pH值为2的环境中经过1 h和3 h培养后存活率分别为58.8%和83.3%;对庆大霉素、四环素、红霉素、克林霉素、万古霉素、诺氟沙星、环丙沙星、加替沙星、磷霉素、亚胺培南等11种常用的抗生素敏感。菌株DU-106具有良好的体外益生性能,可作为益生菌进一步开发。     

In vitro tolerance to digestive stresses of propionibacteria: influence of food matrices

This study investigated Propionibacterium freudenreichii tolerance to stresses encountered during food technology or in the digestive tract. The impact of food matrices on stress survival was evaluated. The ability of P. freudenreichii strains SI 41 and CNRZ 81 to survive both acid and bile salts stresses was studied in vitro. Stress tolerance was examined after inclusion of bacteria in different food matrices (alginate beads, xanthan–gellan beads, fermented milk) and compared to the stress tolerance of the same strains in pure cultures (either in exponential growth phase, in stationary phase, or after freeze-drying). Fermented milk was determined as the best probiotic vector to protect propionibacteria from this trial. This in vitro investigation gave promising results. Indeed, some food matrices can significantly improve protection of bacterial cells from stress injury. These data, which should be confirmed in an in vivo study, will be taken into consideration for the improvement of technological processes and for the choice of an adequate probiotic vector.     

In Vitro Assessment of Probiotic Properties of Lactobacillus Strains from Infant Gut Microflora

Lactobacillus isolates from infant feces were identified and investigated in vitro for their probiotic properties. The isolates belonged to the species L. acidophilus, L. rhamnosus, L. paracasei subsp. paracasei, L. fermentum, L. delbrueckii subsp. delbrueckii, L. cellobiosus, L. plantarum, L. brevis and L. crispatus. The strains were examined for acid and bile tolerance, adhesion to Caco-2 cells, antibiotic susceptibility and antimicrobial activity against selected enteric pathogens. According to the obtained results, a L. deblueckii subsp. delbrueckii strain was found to exhibit probiotic properties. Other strains from L. paracasei subsp. paracasei and L. fermentum group also could be further investigated as potential probiotic candidates.     

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。     

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。     

Acid tolerant mutants of Bifidobacterium animalis subsp. lactis with improved stability in fruit juice

The potential of UV mutagenesis combined with a specific selection step to generate more acid-resistant Bifidobacterium animalis subsp. lactis Bb-12 strains with improved viability in low pH food matrices was investigated in this study. A total of 144 Bb-12 UV-mutants were initially characterised. After prolonged storage in apple juice (pH 3.5) and various pheno- and genotypic tests (acid and bile tolerance, substrate utilisation, antibiotic susceptibility, aerotolerance, RAPD) two mutants (2.20 and 2.56) were chosen for further studies including cell surface morphology, stability of various traits after repeated inoculations, and performance in a fermenter and during down-stream processing. 2.20 and 2.56 showed over two Log-values better viability in pH 3.5 juice compared to Bb-12. Alterations in cell surface structures of 2.20 and 2.56 were detected with AFM, whereas other studied traits remained unchanged.In conclusion, UV mutagenesis and subsequent incubation in acidic medium enabled improving the stability of B. animalis subsp. lactis in low pH juice. Acid tolerance testing (HCl, pH 2.5, 2 h) results did not predict long-term stability of the strains in acidic food matrix (apple juice pH 3.5).     

产Reuterin罗伊氏乳杆菌的定向筛选及其生物学特性

依据菌株宿主适应性特点，从健康家禽新鲜粪便中定向筛选具有开发潜力的罗伊氏乳杆菌。经16S rDNA序列分析和pduC基因筛查，获得3 株产抗菌物质Reuterin的罗伊氏乳杆菌，对其耐受模拟胃肠道环境、抗生素敏感性、甘油发酵上清液抑菌活性及肠上皮细胞黏附能力等生物学特性进行系统评价。结果表明，罗伊氏乳杆菌WLRE01、WLRE03、WLRE04均具有较强耐受极端酸（pH 2.5和pH 3.5）、胆盐（1.5 g/L）及模拟胃肠液的能力；经1 mmol/L过氧化氢处理6 h，WLRE03、WLRE04的活菌数能基本维持初始浓度；甘油发酵上清液对金黄色葡萄球菌具有极强的抑制作用，抑菌圈最高达（28.1±1.2）mm；3 株菌对HT-29细胞均具有良好的黏附特性，其中WLRE01黏附能力最强。综上所述，健康家禽肠道源的3 株产Reuterin的罗伊氏乳杆菌可作为益生菌候选菌株进行深入研究。     

小麦提取物对乳酸杆菌胆盐耐受力的影响

模拟人十二指肠高胆盐环境,研究小麦提取物对乳酸杆菌胆盐耐受力的影响,探讨作为生产益生菌食品的小麦基质对乳酸杆菌的保护作用。将约氏乳酸杆菌JJB3、SNZ10,罗伊氏乳酸杆菌MNZ8、KNZ4 株菌,在含有小麦提取物的10g/L 胆盐溶液,pH7.0 的磷酸盐缓冲液中作用4h,检测其活菌数。未添加小麦提取物的对照4 株乳酸杆菌活菌数显著下降(P ＜ 0.05),其中MNZ8 对胆盐最为敏感,活菌数下降1.74 lg(CFU/ml),KNZ4 的耐受力最强,活菌数下降1.16 lg(CFU/ml);添加小麦提取物后,4 株乳酸杆菌的胆盐耐受力均显著提高(P ＜ 0.05),但对不同菌株提高程度有差异,其中,JJB3 胆盐耐受力提高最明显,其活菌仅减少了0.11 lg(CFU/ml)。葡萄糖和游离氨基氮(FAN)的浓度与乳酸杆菌耐胆盐能力的提高呈正相关关系,但葡萄糖的保护作用显著高于游离氨基氮。小麦提取物对4 株乳酸杆菌耐受模拟十二指胆盐环境具有较好的保护作用,对约氏乳酸杆菌JJB3 保护作用最好。     

酵素源乳酸菌的益生功能及抑菌特性评价

为筛选到发酵性能稳定且高效的功能性菌株,以韶关紫背桃源生产的酵素中分离出的12株乳酸菌为试验菌株,测定其益生特性。分别对12株乳酸菌进行了自聚集性、耐酸性,以及对胆盐、胃蛋白酶、胰蛋白酶与抗生素的耐受性和对肠道致病菌的拮抗作用实验,结果表明：12株受试乳酸菌均表现出良好的自聚集性;除XZJ07外,其他11株菌株在pH值为2.5的模拟胃液中培养3 h存活率仍达75%以上;在人工模拟胃液以及肠液中对胃蛋白酶和胰蛋白酶孵育2、3 h存活率仍超过75%;在含有胆盐浓度为3 g/L MRS培养基中培养3 h后,活菌数仍达106 CFU/mL,另外,受试菌株对金黄色葡萄球菌、大肠杆菌均有一定的抑制作用,对大肠杆菌抑菌圈直径均大于15 mm,对金黄色葡萄球菌抑菌圈直径均大于17 mm;受试菌株对氨苄青霉素敏感,对四环素、红霉素、土霉素较为耐受。研究结果预示着受试菌株具有作为优质益生性发酵剂的潜在能力。     

Identification and functional traits of lactic acid bacteria isolated from Ciauscolo salami produced in Central Italy

Lactic acid bacteria (LAB) from Ciauscolo salami produced in Marche Region of Central Italy, and LAB strains belonging to our laboratory collection were examined for their capability to survive at low pH and bile, to adhere to Caco-2 cells, and for antibiotic resistance. LAB from Ciauscolo were identified by ARDRA and RAPD-PCR. Our study showed that all LAB strains had good adaptation to gastric juice and moderate tolerance to bile. The adhesiveness was variable among strains but significantly lower in LAB from food. Antibiotic resistance was broadly spread among food strains, with level of resistance exceeding 15% for all the antibiotics tested. The resistance determinants erm(B) and tet(M) were found in nine strains of food origin (21.4%) while tet(L) in one strain of our collection (5%). Our work suggests that fermented foods are valuable sources of bacterial strains with functional traits of intestinal lactobacilli. These bacteria may be further studied for their use in probiotic applications.     

发酵酸菜来源乳酸菌的益生特性及其在发酵乳中的应用

通过pH值、胆盐耐受性实验从发酵酸菜中筛选性能优良的益生乳酸菌株,经16S rRNA序列分析鉴定得4 株植物乳杆菌A44、B51、B54、C53和2 株戊糖乳杆菌A16、B72。经疏水、黏附、自凝聚和溶血能力实验评价6 株乳酸菌的益生特性,其中植物乳杆菌A44对氯仿和二甲苯的疏水性均大于80%,对Caco-2细胞的黏附率为13.57%,放置5 h的自凝聚率超过60%,与其他菌株相比具有更好的益生特性且无溶血活性。因此选用植物乳杆菌A44进一步研究其在发酵乳中的功能特性,结果表明：植物乳杆菌A44作为辅助发酵剂添加后对4 ℃贮藏7 d期间发酵乳pH值、滴定酸度和持水性均无显著影响（P＞0.05）,但是可以显著提高发酵乳的活菌数和黏度（P＜0.05）,活菌数达到8.45（lg（CFU/mL））。本研究筛选得到的植物乳杆菌A44是一株性能优良的益生乳酸菌,具有作为发酵乳益生菌辅助发酵剂的潜在应用前景。     

双歧杆菌冻干菌粉制备工艺的研究

优化双歧杆菌冻干菌粉的制备工艺及检测冻干菌粉在模拟人体胃肠环境中的耐受性和贮存稳定性,采用3 种方法制备双歧杆菌冻干菌粉。方法1：双歧杆菌在牛奶还原培养基中的扩大培养液加入甘露醇作为保护剂制备冻干菌粉;方法2：双歧杆菌在PTYG 培养基中的扩大培养液经低温离心得到菌泥,向菌泥中加入保护剂甘露醇、增量赋形剂脱脂乳粉制备冻干菌粉;方法3：同方法2 得到菌泥,向其中加入保护剂甘露醇、包埋剂明胶及增量赋形剂脱脂乳粉制备冻干菌粉。由方法2 得到菌粉活菌数较高,为1.31 × 1010CFU/g;经模拟胃肠环境处理后,活菌数仍保持在4.19 × 108CFU/g;对胃酸、胆汁酸具有很高的耐受力。经典加速实验证明由方法2 制备的菌粉室温保存1 年以上活菌数仍保持在107 数量级,具有较好的贮存稳定性。     

乳杆菌在胆盐MRS培养基中的传代稳定性

该实验探究提高乳杆菌传代稳定性的方法以提高乳杆菌在胃肠道的定植能力,为工业生产提供依据。该研究以植物乳杆菌M547、M621、M748、戊型乳杆菌M750为研究对象,研究胆盐环境对菌株生长的影响,利用生长曲线仪确定合适胆盐培养菌株的浓度,对比胆盐培养和MRS培养的菌株在耐胆盐能力、产酸能力、耐酸能力的表现。0.04%的胆盐培养后,菌株在传代过程中对于0.075%胆盐的耐受存活率提升11%~18%左右,0.1%胆盐的耐受存活率提升20%~34%。胆盐培养对于产酸能力,耐酸能力及稳定性的提高没有明显作用,但对菌株的耐胆盐能力及传代稳定性有提高作用,可为研究改善益生菌的益生特性的方法提供理论支持。