,, 李淼, 李永锋, 孙元,, 仇华吉,中国农业科学院哈尔滨兽医研究所/兽医生物技术国家重点实验室,哈尔滨150069Identification and Properties of Lactic Acid Bacteria Isolated from Wild Boar Feces
WANG Yi,, LI Miao, LI YongFeng, SUN Yuan,, QIU HuaJi,State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069通讯作者:
责任编辑: 林鉴非
收稿日期:2019-08-27接受日期:2020-02-19网络出版日期:2020-07-16
| 基金资助: |
Received:2019-08-27Accepted:2020-02-19Online:2020-07-16
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王翌, 李淼, 李永锋, 孙元, 仇华吉. 野猪粪便中乳酸菌的分离鉴定及特性研究[J]. 中国农业科学, 2020, 53(14): 2964-2973 doi:10.3864/j.issn.0578-1752.2020.14.019
WANG Yi, LI Miao, LI YongFeng, SUN Yuan, QIU HuaJi.
0 引言
【研究意义】当前,抗生素作为饲料添加剂广泛用于促进动物生长及疾病预防,而动物相关产品及制品中抗生素残留以及细菌耐药性问题日趋严峻并亟需解决[1]。因此,抗生素替代品的开发备受关注。益生菌是一类对宿主有益的活性微生物,不存在耐药性或残留的问题。在众多的益生菌产品中,最为常见的是乳酸菌,它能够利用碳水化合物发酵产生乳酸,是人体和哺乳动物肠道微生物的主要组成部分,对宿主的健康有着重要的促进作用,国家食品药品监督局将其评定为食品安全级的菌株[2]。【前人研究进展】乳酸菌具有广泛的益生作用,例如改善消化功能,增强机体免疫水平,缓解炎症性肠病和便秘,增强黏膜屏障作用等。乳酸菌作为益生菌不但有利于宿主的健康,一些乳酸菌分离株甚至具有抗癌或治疗糖尿病的效用[3,4,5,6,7]。为了发挥良好的益生效果,乳酸菌在胃肠道的转运过程中必须保持高水平的存活效率和定植能力[8]。因此,乳酸菌对酸性环境的抵抗力、对胆盐的适应浓度以及对肠上皮细胞的黏附特性等是评价其免疫调节功能的重要指标。【本研究切入点】目前,从乳制品、泡菜等食品和禽类、鱼等动物体内分离益生菌的研究较多,而对于从野猪中分离益生菌的研究鲜有报道。【拟解决的关键问题】本研究以大兴安岭野猪粪便中的乳酸菌株为研究对象,对其进行分离与鉴定,进一步评价了其对pH和胆盐的耐受能力、黏附能力、凝集能力和抑菌特性,同时评估了其安全性,以期筛选出安全且具有良好特性的乳酸菌,为进一步应用于饲料添加剂等动物相关产品奠定基础。1 材料与方法
1.1 主要材料
1.1.1 样品来源 2018年6月从黑龙江大兴安岭地区采集野猪粪便样品13份,用无菌棉签小心地拨开粪便,再用新的无菌棉签从粪便内部轻轻蘸取少许,收集于50 mL无菌离心管,编号后置于4℃保温箱迅速运回实验室。1.1.2 菌株、细胞和试验动物 标准致病菌株主要用于抑菌试验和共凝集试验,包括致病性埃希氏大肠杆菌（BNCC 337304）、金黄色葡萄球菌（ATCC 6538P）及鼠伤寒沙门氏菌（ATCC 14028）。模式菌株嗜酸乳杆菌（ATCC 4356）用于比较乳酸菌菌株的黏附能力和溶血性分析。结肠腺癌细胞Caco-2（BNCC 338148）使用含20%血清的MEM培养基,猪小肠上皮细胞IPEC-J2（BNCC 338252）使用含10%血清的DMEM培养基,在37℃ 5% CO2条件下培养,用于乳酸菌黏附试验。以上菌株及细胞均由兽医生物技术国家重点实验室保存。20只7周龄的雌性BALB/c小鼠购自辽宁长生生物技术有限公司,用于乳酸菌的体内安全性评价。
1.1.3 主要试剂 MRS培养基购自青岛高科园海博生物技术有限公司。细菌基因组DNA提取试剂盒购自天根生化科技（北京）有限公司。异硫氰酸荧光素（fluorescein isothiocyanate, FITC）购自哈尔滨宝士德生物科技有限公司。MEM培养基、DMEM培养基、胎牛血清（FBS）及0.25%胰酶购自Gibco公司。
1.2 乳酸菌的分离与鉴定
从大兴安岭采集野猪粪便,每管粪便样本中加入约5倍体积的MRS培养基,充分振荡混匀,分别取1 mL转接至8 mL MRS培养基中,于37℃温箱厌氧培养24 h,分别取微量过夜培养的菌液,在MRS固体培养基上进行划线,置于37℃温箱中厌氧培养24 h。挑取特征明显的菌落反复划线分离,直至分离出形态、大小一致的菌落,进行革兰氏染色。对菌体形态进行显微观察,确定为单一的革兰氏阳性菌后,使用细菌基因组DNA提取试剂盒提取分离菌株的基因组DNA。利用细菌16S rDNA通用引物进行扩增（表1）,PCR反应程序为：95℃ 5 min;95℃ 45 s,50℃ 1 min,72℃ 90 s,共10个循环;95℃ 45 s,58℃ 1 min和72℃ 90 s,共15个循环;95℃ 45 s,55℃ 1 min和72℃ 90 s,共10个循环;72℃延伸10 min。扩增片段测序后,使用BLAST与GenBank数据库中序列进行对比分析。Table 1
表1
表1PCR扩增引物
Table 1
| 基因名称 Gene | 引物序列 (5°-3°) Primer sequence (5°-3°) | 产物长度 Length (bp) |
|---|---|---|
| 16S rDNA | 27F: AGAGTTTGATCCATGGCTCAG | 1514 |
| 1541R: AAGGAGGTGATCCAGCC | ||
| ace | F: CAGGCCAACATCAAGCAACA | 125 |
| R: GCTTGCCTCGCCTTCTACAA | ||
| agg | F: AAGAAAAAGAAGTAGACCAAC | 1553 |
| R: AAACGGCAAGACAAGTAAATA | ||
| atpA | F: CCAGGTCGTGAAGCTTATCC | 110 |
| R: GGTAAGGCCGTCATTGAACC | ||
| cfa1 | F: ACGACCTGTTGTTCGACCTG | 150 |
| R: ACGACCTGTTGTTCGACCTG | ||
| ebp | F: AATGTGTTAAACCATCAAGGGAAT | 372 |
| R: ACTCCTTTTTGAACTTCACCAATC | ||
| espA | F: TTTGGGGCAACTGGAATAGT | 407 |
| R: CCCAGCAAATAGTCCATCAT | ||
| gelE | F: CGAAGTTGGAAAAGGAGGC | 372 |
| R: GGTGAAGAAGTTACTCTGA | ||
| groEL | F: GTTTGATCGCGGCTATCTGA | 150 |
| R: CCTTGTTGMACGATTTCTTG | ||
| hisD | F: TGAACCACTCGGTGACTACG | 150 |
| R: GGAGCTTCCTTAGCCAAAGC | ||
| mleS | F: ACAAGGTCTCAGCGTTCAGC | 140 |
| R: GACTGGGATTCCAGCTGATG | ||
| sprE | F: GGTAAACCAACCAAGTGAATC | 300 |
| R: R: TTCTTCCGATTGACGCAAAA |
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将分离的乳酸菌在酸性（pH 3.0）和胆盐（0.3%）条件下评价其耐受性[9],将过夜培养的菌液接种于5 mL pH 3.0的MRS和含0.3%胆盐的MRS培养基中,37℃孵育4 h,测定其OD600nm值,筛选出具备较强耐酸、耐胆盐特性的菌株用于后续试验。
1.3 乳酸菌的自凝集能力测定
按照COLLADO等[10]的方法稍作改进后进行乳酸菌的自凝集能力检测：将分离的乳酸菌转接于5 mL MRS培养基中,37℃温箱中静置培养18 h。以5 000×g离心10 min收集菌体,PBS洗涤2次后将其重悬于2 mL PBS中,调节OD600nm为0.25 ± 0.05,室温条件下静置,在不同时间点（0、2、4、6、10和24 h）分别取100 μL上层悬液测定其OD600nm。乳酸菌的自凝集率AA（%）计算公式如下：AA（%）= [(A0 - At)/A0]×100。其中,A0是0 h测定的乳酸菌悬液OD600nm值;At是静置后不同时间点测定的OD600nm值。1.4 乳酸菌与致病菌的共凝集能力测定
对乳酸菌进行共凝集能力检测,方法如下[11]：取乳酸菌悬液2 mL（OD600nm = 0.25 ± 0.05）分别与3株标准致病菌株等体积混合,室温静置,在不同时间点（0、2、4、6、10和24 h）分别取100 μL上层悬液测定其OD600nm。根据如下公式计算乳酸菌与致病菌的共凝集率AC（%）：AC（%）= [(Apro+Apat)-Amix]/ (Apro+Apat)×100。其中,Apro+Apat代表0 h混合悬液的OD600nm值;Amix是不同时间点测定的OD600nm值。1.5 乳酸菌的黏附能力测定
将Caco-2细胞和IPEC-J2细胞分别接种于24孔板,待其贴壁生长至80%—90%时进行黏附试验。将过夜培养的乳酸菌以5 000×g离心10 min收集菌体,PBS洗涤2次,将其以108 CFU/mL重悬于PBS（含100 μg·mL-1 FITC）中,37℃避光孵育1 h将细菌着色,随后PBS洗涤3次除去未结合的FITC,重悬于PBS中。用PBS将细胞洗涤3次,每孔加入500 μL FITC标记的菌悬液,使用多功能酶标仪测定其初始荧光强度（吸收光波长为485 nm,发射光波长为530 nm）,将24孔板置于37℃、5% CO2的培养箱中孵育1 h。PBS洗涤24孔板3次,去除未黏附的乳酸菌,加入100 μL 0.25%的胰酶作用10 min后,再加入400 μL含20% FBS的培养基终止反应,使用酶标仪测定其荧光强度 [12]。按照如下公式计算：黏附率（%）= C/C0×100。其中,C0代表乳酸菌黏附前的荧光强度,C代表乳酸菌黏附后的荧光强度。1.6 乳酸菌的抑菌能力测定
按照BHOLA等[13]的方法进行乳酸菌的抑菌能力测定。先将灭菌的牛津杯平放在底层MRS固体培养基上,将指示菌按1%接种于45—50℃的MRS固体培养基中,混匀后倒入放置好牛津杯的培养皿中,待其凝固后取出牛津杯,在杯孔中加入过夜培养的待测乳酸菌培养液,每孔100 μL,37℃温箱培养24 h后,使用游标卡尺测量抑菌环大小。1.7 乳酸菌的安全性评价
将过夜培养的模式菌株嗜酸乳杆菌、金黄色葡萄球菌和3株乳酸菌菌株在血平板上划线,37℃厌氧孵育18—24 h,观察细菌菌落周围是否形成溶菌环。根据文献中已报道的毒力因子基因在NCBI上查找相关序列送至南京金斯瑞生物科技有限公司进行基因合成,同时设计相应引物（表1）,以合成基因作为阳性对照,对分离菌株进行PCR扩增,检测是否存在毒力因子的编码基因,进行分离菌株的体外安全性评价[14,15,16,17,18]。过夜培养的M6-5、M2-38和M6-27菌株以5 000×g离心10 min收集菌体,将其以1010 CFU/mL重悬于PBS。7周龄的BALB/c小鼠随机分为4组,每组5只,分别将上述3种细菌悬液和PBS连续饲喂21 d,100 μL·d-1。测定小鼠的初始体重以及饲喂7、14和21 d的体重,计算体重变化情况;连续饲喂结束后,解剖获取小鼠的脾脏、肝脏和肾脏按照如下公式计算器官指数：器官指数=器官重量/小鼠体重,对乳酸菌菌株进行体内安全性评价[19]。1.8 统计学分析
采用SPSS 22统计软件分析试验数据,P<0.05表示差异显著,所有试验设置3个重复,数值以平均数±标准差表示。2 结果
2.1 乳酸菌分离株的分类
从野猪粪便样品中分离的细菌通过在MRS固体培养基反复纯化后,经革兰氏染色证明分离的菌株均为的革兰氏阳性菌（结果未显示）。耐酸、耐胆盐试验结果显示,其中有3株乳酸菌显示出一定程度的耐受性,在酸性和含胆盐的MRS培养基中存活率能达到50%—80%以上不等,其中M6-27菌株具备最为优良的耐酸、耐胆盐特性,在pH 3.0时存活率高达70.47%,胆盐浓度0.3%时存活率达80%以上。经16S rDNA鉴定,此3株乳菌分别为蒙氏肠球菌（M6-5）、耐久肠球菌（M2-38）和黏膜乳杆菌（M6-27）（表2）。Table 2
表2
表2三株乳酸菌的种属鉴定及耐酸、耐胆盐特性
Table 2
| 菌株 Isolate | 种属 Specie | pH 3.0 MRS中存活率 Survival rate at pH 3.0 (%) | 0.3%胆盐MRS中存活率 Survival rate at 0.3% bile salt (%) |
|---|---|---|---|
| M6-5 | 蒙氏肠球菌Enterococcus mundtii | 65.22 | 53.37 |
| M2-38 | 耐久肠球菌Enterococcus durans | 56.83 | 76.08 |
| M6-27 | 黏膜乳杆菌Lactobacillus mucosae | 70.47 | 82.99 |
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2.2 乳酸菌的自凝集能力与致病菌的共凝集能力
对分离的3株乳酸菌进行自凝集能力检测,结果如图1所示,随着时间的推移,3株乳酸菌的自凝集率稳步上升,其中,M6-27和M2-38菌株具有较强的自凝集能力,静置24 h后其自凝集率高达70%以上,明显高于M6-5菌株,而M6-5在静置24 h后凝集率也达到了50%（图1-A）。图1
新窗口打开|下载原图ZIP|生成PPT图13株乳酸菌自凝集能力及其与致病菌的共凝集能力
A：3株乳酸菌自凝集能力;B-D：3株乳酸菌与致病性埃希氏大肠杆菌、金黄色葡萄球菌及鼠伤寒沙门氏菌共凝集能力
Fig. 1The autoaggregation and coaggregation ability of three lactic acid bacteria with the pathogenic bacteria
A: Autoaggregation ability of the three strains of lactic acid bacteria; B-D: Coaggregation ability of the three strains of lactic acid bacteria with Escherichia coli, Staphylococcus aureus and Salmonella typhimurium
共凝集能力检测结果显示,分离的乳酸菌菌株均能凝集致病性埃希氏大肠杆菌、金黄色葡萄球菌和鼠伤寒沙门氏菌3种致病菌。其中M2-38凝集致病性埃希氏大肠杆菌能力最强,可达到70%以上（图1-B）。3株乳酸菌凝集金黄色葡萄球菌的能力接近,凝集率均在50%左右（图1-C）。3株乳酸菌凝集鼠伤寒沙门氏菌的能力有所不同,其中M6-27凝集能力最强,可达60%;M2-38次之,凝集率接近50%;M6-5凝集能力最弱,只有不到40%（图1-D）。
2.3 乳酸菌的黏附能力
黏附试验结果显示,3株乳酸菌菌株对Caco-2细胞的黏附率均达到50%以上,其中,M6-27菌株黏附率最高,可达72%,显著高于模式菌株ATCC 4356（P<0.05）;另外,2株M2-38和M6-5菌株黏附率分别为63%和56%,其中M2-38也显著高于ATCC 4356菌株（P<0.05）（图2-A）,结果表明,2株乳酸菌菌株M6-27和M2-38均具有较强的黏附能力。图2-B显示,3株乳酸菌菌株对IPEC-J2细胞的黏附能力极显著高于参考菌株（P<0.001）,其中乳酸菌菌株M6-27的黏附能力最高,可达77%。图2
新窗口打开|下载原图ZIP|生成PPT图23株乳酸菌黏附能力
A：3株乳酸菌对Caco-2的黏附能力;B：3株乳酸菌对IPEC-J2的黏附能力
Fig. 2The adhesion ability of three lactic acid bacteria
NS: not significant; *: P<0.05; ***: P<0.001 A: Adhesion ability of the three strains of lactic acid bacteria to Caco-2 cells;B: Adhesion ability of the three strains of lactic acid bacteria to IPEC-J2 cells
2.4 乳酸菌的抑菌能力
乳酸菌体外抑菌试验结果显示,3株乳酸菌均对鼠伤寒沙门氏菌有抑制作用,其中M6-27菌株抑菌能力最强,抑菌环直径为（13.61 ± 0.27）mm;M6-5和M6-27菌株对致病性埃希氏大肠杆菌有抑制作用,而只有M6-27菌株对金黄色葡萄球菌有抑制作用（表3）。以上结果表明,本研究筛选的3株乳酸菌抑菌能力不尽相同,M6-27菌株对3种致病菌均具有较强的抑菌活性。Table 3
表3
表33株乳酸菌对致病菌的抑制能力
Table 3
| 乳酸菌株 LAB strain | 抑菌环直径 Inhibition zone diameter (mm) | ||
|---|---|---|---|
| 埃希氏大肠杆菌Escherichia coli | 鼠伤寒沙门氏菌Salmonella typhimurium | 金黄色葡萄球菌Staphylococcus aureus | |
| M6-5 | 10.48 ± 0.19 | 9.91 ± 0.43 | — |
| M2-38 | — | 9.97 ± 0.21 | — |
| M6-27 | 13.63 ± 0.27 | 13.61 ± 0.24 | 13.48 ± 0.28 |
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2.5 乳酸菌的安全性
溶血性试验结果显示,金黄色葡萄球菌（ATCC 6538P）划线的血平板上菌落周围形成完全透明的溶血环（β型溶血）,模式菌株嗜酸乳杆菌（ATCC 4356）和3株乳酸菌菌株划线的血平板上菌落周围无溶血现象,表明3株乳酸菌均无溶血性（图3）。食物来源的肠球菌存在单一或多重毒力因子,尽管其在益生性肠球菌中的发生率较低,另外,也有关于乳杆菌中存在毒力因子的报导,因此仍需对本试验中分离的两株肠球菌和一株黏膜乳杆菌进行毒力因子的分析,以评估其安全性。在本研究中,以乳酸菌分离株基因组DNA为模板,使用多对毒力因子相应引物进行PCR扩增,均未检测到毒力因子（图4）。图3
新窗口打开|下载原图ZIP|生成PPT图3乳酸菌分离株的溶血性
A：M2-38;B：M6-5;C：M6-27;D：阴性对照：嗜酸乳杆菌（ATCC 4356）;E：阳性对照：金黄色葡萄球菌（ATCC 6538P）
Fig. 3The hemolytic activity of lactic acid bacteria
A: M2-38; B: M6-5; C: M6-27; D: Negative control: Lactobacillus acidophilus (ATCC 4356); E: Positive control: Staphylococcus aureus (ATCC 6538P)
图4
新窗口打开|下载原图ZIP|生成PPT图4乳酸菌分离株的部分毒力因子检测
M：DL2000 Marker;1-3、6-8、11-13和16-18：扩增gelE、ebp、espA、agg基因片段;4、9、14和19：阳性对照;5、10、15和20：阴性对照
Fig. 4Partial virulence factor detection results of lactic acid bacteria
M: DL2000 Marker; 1-3, 6-8, 11-13 and 16-18: Amplification fragment of gelE, ebp, espA and agg; 4, 9, 14 and 19: The positive control; 5, 10, 15 and 20: The negative control
与对照组相比,连续饲喂乳酸菌菌株的各组小鼠在第7、14天的体重增加比例无显著性差异（结果未显示）。在第21天,黏膜乳杆菌M6-27饲喂小鼠的体重增加百分率与对照组相比差异极显著（P<0.01）（图5-A）。此外,小鼠脾脏（图5-B）、肝脏（图5-C）和肾脏（图5-D）的器官指数在各小组间无显著差异。
图5
新窗口打开|下载原图ZIP|生成PPT图5乳酸菌在小鼠体内的安全性评价
A：3株乳酸菌对小鼠体重的影响;B-D：三株乳酸菌对小鼠脾脏、肝脏、肾脏指数的影响
Fig. 5Safety evaluation of lactic acid bacteria in mice
NS: not significant; **: P<0.01 A: Effect of probiotic supplementation on body weight gain of mice; B-D: The indices analysis of spleen, liver, and kidney in all the experimental groups
3 讨论
野猪,也称“山猪”,属于偶蹄目猪科猪属。与家猪相比,野猪体躯健硕、野性凶猛、抗逆性强。野猪为杂食性动物,主要以植物、种子、菌类、果实、昆虫及小型脊椎动物为食,其饮食习惯、活动模式等也与统一化饲喂的家猪不同,因此,长期的进化导致二者肠道正常菌群组成也不尽相同。目前,关于家猪体内益生菌分离鉴定的报导较为常见,而少有研究从野猪体内进行益生菌的分离,因此本研究选择对野猪粪便样本进行采集与益生菌的分离鉴定。大兴安岭地区位于黑龙江省西北部、内蒙古自治区东北部、大兴安岭山脉东北坡,是中国最北端的地级行政区,土地面积占全省51.8%,地广人稀,是黑龙江野生动物的主要活动基地[20]。大兴安岭地区野猪资源丰富,野猪生长活动于此,人为污染少,常见病原感染少,有望能够分离出天然、具备优良特性的益生菌株。益生菌在改善肠道功能和提高动物生产性能等方面发挥着重要作用。乳酸菌作为最为常见的益生菌,可以通过平衡肠道菌群维持宿主的肠道健康,甚至治疗人类和动物的肠道疾病[21]。同时,研究证实,益生菌作为饲料添加剂对不同生长期猪都能产生积极作用,比如改善动物健康和性能[22]。
为了获得具有优良特性的乳酸菌株,必须对其基本功能特性和安全性进行评价。因为胃中的酸性条件和十二指肠的胆盐是益生菌在胃肠道生存的最大障碍,所以在胃肠道定植的首要条件是拥有对低pH和胆盐的抵抗力[23,24]。不同菌株在酸性和胆盐条件下的存活率不同,可能是由于其对酸性和胆盐的耐受性机制不同所致[25]。在本研究中,我们筛选了3株不同的乳酸菌株,均具有较强的耐酸、耐胆盐能力。
黏附是乳酸菌有效地定植于肠上皮细胞表面、发挥益生作用的前提条件,因此黏附能力是评价益生菌的一个非常重要的指标[25,26]。益生菌的自凝集和共凝集能力在防止病原体表面定植方面发挥着重要作用。研究表明,聚集的益生菌能够形成覆盖肠道黏膜表面的物理屏障,防止病原体的定植,而益生菌与致病菌结合是宿主抵御感染的重要防御机制[27,28,29]。本研究中分离的乳酸菌株中,M6-27菌株具有较强的自凝集和共凝集致病菌的能力,同时可以抑制3种致病菌的活性。
通过比较分析3株乳酸菌的凝集能力、黏附特性和抗菌特性发现,每株益生菌各有特点,这可能与其生活环境、产生不同的副产物、生物特性以及其他因素等有关,但综合看来,M6-27各方面特性均较为优良。已有研究表明,部分益生菌株可能具有抗病毒和抗癌活性[30,31]。因此,深入探索3株益生菌的抗癌和抗病毒活性具有重要意义。
乳酸菌在乳制品、肉制品、泡菜和青贮饲料以及益生菌制品发酵中被广泛使用,具有悠久的历史,大部分乳酸菌被公认为安全级。但考虑到本研究中分离乳酸菌的目的是进一步作为饲料添加剂使用,因此,对其可能携带的毒力基因进行检测是十分必要的。在本研究中,分离得到的3株乳酸菌均无溶血性和毒力因子。同时,将乳酸菌连续饲喂小鼠后,对小鼠健康未产生任何不良影响,而饲喂黏膜乳杆菌（M6-27）的小鼠体重显著增加,表明这株乳酸菌在一定程度上能够提高生长性能,有望用于动物饲料添加剂的开发。
4 结论
从大兴安岭野猪粪便中分离得到3株乳酸菌,经16S rDNA鉴定,此3株乳酸菌分别为蒙氏肠球菌、耐久肠球菌和黏膜乳杆菌,在酸性和含胆盐培养基中存活能力较强。其中黏膜乳杆菌具备良好的黏附能力和凝集能力,并且能够抑制3种致病菌的生长,具有较强的抑菌活性,而连续饲喂21 d黏膜乳杆菌可显著增加小鼠体重,表明其具备良好的益生特性。后期将对黏膜乳杆菌的免疫调节活性、体内抑菌能力等进行进一步研究,使其能更好的作为益生制剂应用于饲料添加剂等动物相关产品中。参考文献 原文顺序
文献年度倒序
文中引用次数倒序
被引期刊影响因子
DOI:10.1007/s00253-018-9403-9URLPMID:30280241 [本文引用: 1]
Lactic acid bacteria (LAB), a heterogeneous group of bacteria that produce lactic acid as the main product of carbohydrate degradation, play an important role in the production and protection of fermented foods. Moreover, beside the technological use of these microorganisms added to control and steer food fermentations, their beneficial healthy properties are largely overt. Thus, numerous LAB strains have obtained the probiotic status, which entails the ability to maintain and promote a good health of consumers. In particular, increasing consideration is being focused on probiotic microorganisms that can improve the human immune response against dangerous viral and fungal enemies. For such beneficial microbes, the term
URLPMID:31335895 [本文引用: 1]
DOI:10.1007/s12602-017-9354-yURLPMID:29196920 [本文引用: 1]
Probiotics offer numerous health benefits, including digestive and immune health. Improved digestive health is linked to a more efficient absorption of important nutrients from our diet. This review focused on the rationale of using the probiotic Bacillus coagulans GBI-30, 6086 to aid protein absorption and utilization. B. coagulans GBI-30, 6086 can withstand the acidic environment of the stomach to reach the intestine where it germinates. Once active in the small intestine after germination, it has been shown to aid the digestion of carbohydrates and proteins. Co-administration of B. coagulans GBI-30, 6086 with protein has been shown to increase protein absorption and to maximize the health benefits associated with protein supplementation.
URLPMID:29118557 [本文引用: 1]
DOI:10.3920/BM2017.0097URLPMID:29488412 [本文引用: 1]
Alterations in the gut microbiota composition play a key role in the development of chronic diseases such as inflammatory bowel disease (IBD). The potential use of probiotics therefore gained attention, although outcomes were sometimes conflicting and results largely strain-dependent. The present study aimed to identify new probiotic strains that have a high potential for the management of this type of pathologies. Strains were selected from a large collection by combining different in vitro and in vivo approaches, addressing both anti-inflammatory potential and ability to improve the gut barrier function. We identified six strains with an interesting anti-inflammatory profile on peripheral blood mononuclear cells and with the ability to restore the gut barrier using a gut permeability model based on Caco-2 cells sensitized with hydrogen peroxide. The in vivo evaluation in two 2,4,6-trinitrobenzene sulfonic acid-induced murine models of colitis highlighted that some of the strains exhibited beneficial activities against acute colitis while others improved chronic colitis. Bifidobacterium bifidum PI22, the strain that exhibited the most protective capacities against acute colitis was only slightly efficacious against chronic colitis, while Bifidobacterium lactis LA804 which was less efficacious in the acute model was the most protective against chronic colitis. Lactobacillus helveticus PI5 was not anti-inflammatory in vitro but the best in strengthening the epithelial barrier and as such able to significantly dampen murine acute colitis. Interestingly, Lactobacillus salivarius LA307 protected mice significantly against both types of colitis. This work provides crucial clues for selecting the best strains for more efficacious therapeutic approaches in the management of chronic inflammatory diseases. The strategy employed allowed us to identify four strains with different characteristics and a high potential for the management of inflammatory diseases, such as IBD.
DOI:10.1002/ijc.28702URLPMID:24382758 [本文引用: 1]
Recent studies suggest health benefits including protection from cancer after eating fermented foods such as probiotic yogurt, though the mechanisms are not well understood. Here we tested mechanistic hypotheses using two different animal models: the first model studied development of mammary cancer when eating a Westernized diet, and the second studied animals with a genetic predilection to breast cancer. For the first model, outbred Swiss mice were fed a Westernized chow putting them at increased risk for development of mammary tumors. In this Westernized diet model, mammary carcinogenesis was inhibited by routine exposure to Lactobacillus reuteri ATCC-PTA-6475 in drinking water. The second model was FVB strain erbB2 (HER2) mutant mice, genetically susceptible to mammary tumors mimicking breast cancers in humans, being fed a regular (non-Westernized) chow diet. We found that oral supplement with these purified lactic acid bacteria alone was sufficient to inhibit features of mammary neoplasia in both models. The protective mechanism was determined to be microbially-triggered CD4+CD25+ lymphocytes. When isolated and transplanted into other subjects, these L. reuteri-stimulated lymphocytes were sufficient to convey transplantable anti-cancer protection in the cell recipient animals. These data demonstrate that host immune responses to environmental microbes significantly impact and inhibit cancer progression in distal tissues such as mammary glands, even in genetically susceptible mice. This leads us to conclude that consuming fermentative microbes such as L. reuteri may offer a tractable public health approach to help counteract the accumulated dietary and genetic carcinogenic events integral in the Westernized diet and lifestyle.
DOI:10.3168/jds.2018-15203URLPMID:30471910 [本文引用: 1]
The probiotic Lactobacillus gasseri SBT2055 (LG2055) has a protective effect against metabolic syndrome in rats and humans. Metabolic syndrome increases the risk of type 2 diabetes mellitus. In this study, Goto-Kakizaki rats were used as a diabetic model and fed diets containing LG2055-fermented or nonfermented skim milk for 4 wk. Indices of diabetes such as blood glucose levels, serum glucagon levels, plasma levels of insulin, C-peptide, and glucagon-like peptide-1, tissue glycogen contents, and pancreatic mRNA levels were measured. The plasma C-peptide levels and pancreatic mRNA levels of insulin genes (Ins1 and Ins2) and Pdx1 (a transcriptional factor of insulin genes) were increased in LG2055 diet-fed rats. The increase in insulin secretion corresponded to an improvement in serum and pancreatic inflammatory status, associated with decreases in serum levels of serum amyloid P and pancreatic levels of granulocyte colony-stimulating factor. Insulin resistance in Goto-Kakizaki rats was ameliorated by increased glycogen storage in the liver and quadriceps femoris muscles and decreased serum free fatty acid levels. This improvement may be related to the increased cecal production of short-chain fatty acids. In conclusion, dietary LG2055 improved insulin secretion in diabetic rats by improving the inflammatory status in the pancreas and serum.
DOI:10.3389/fmicb.2018.02220URLPMID:30323790 [本文引用: 1]
In order to eliminate the widespread use of antibiotics in livestock production, the research for alternatives has increased lately. This study examined the safety of 40 lactic acid bacteria (LAB) isolated from bovine feedlot environment and previously selected as potential probiotics. A high sensitivity prevalence to ampicillin (AMP, 100%), gentamicin (GEN, 96.3%), kanamycin (KAN, 96.3%), clindamycin (CLI, 85.2%), chloramphenicol (CHL, 92.6%) and streptomycin (STR, 88.9%) while moderate and high resistance against erythromycin (ERY, 48%) and tetracycline (TET, 79%) respectively, were determined. Feedlot enterococci and pediococci displayed high resistance to CLI, ERY, GEN and TET (73, 100, 54.5, and 73%, respectively). Among fifteen resistance genes investigated, seven were identified in lactobacilli; their presence not always was correlated with phenotypic resistance. STR resistance genes, aadA and ant(6) were observed in 7.4 and 3.7% of isolates, respectively; genes responsible for aminoglycosides resistance, such as bla (7.4%), and aph(3
URLPMID:29518093 [本文引用: 1]
URLPMID:17995806 [本文引用: 1]
[本文引用: 1]
URLPMID:30888623 [本文引用: 1]
URLPMID:31193267 [本文引用: 1]
DOI:10.3389/fmicb.2018.02220URLPMID:30323790 [本文引用: 1]
In order to eliminate the widespread use of antibiotics in livestock production, the research for alternatives has increased lately. This study examined the safety of 40 lactic acid bacteria (LAB) isolated from bovine feedlot environment and previously selected as potential probiotics. A high sensitivity prevalence to ampicillin (AMP, 100%), gentamicin (GEN, 96.3%), kanamycin (KAN, 96.3%), clindamycin (CLI, 85.2%), chloramphenicol (CHL, 92.6%) and streptomycin (STR, 88.9%) while moderate and high resistance against erythromycin (ERY, 48%) and tetracycline (TET, 79%) respectively, were determined. Feedlot enterococci and pediococci displayed high resistance to CLI, ERY, GEN and TET (73, 100, 54.5, and 73%, respectively). Among fifteen resistance genes investigated, seven were identified in lactobacilli; their presence not always was correlated with phenotypic resistance. STR resistance genes, aadA and ant(6) were observed in 7.4 and 3.7% of isolates, respectively; genes responsible for aminoglycosides resistance, such as bla (7.4%), and aph(3
DOI:10.3389/fmicb.2019.00300URLPMID:30863379 [本文引用: 1]
The present study focused on probiotic characterization and safety evaluation of Enterococcus isolates from different artisanal dairy products. All the isolates exhibited inhibitory activity against several food spoilage bacteria and food-borne pathogens, including Shigella flexneri, Staphylococcus aureus, Listeria monocytogenes, Yersinia enterocolitica, Klebsiella pneumoniae, Escherichia coli, and Bacillus subtilis. The PCR results indicated the presence of at least one enterocin structural gene in all the tested strains. The Enterococcus isolates were further evaluated regarding their safety properties and functional features. The isolates were susceptible to vancomycin, gentamycin, and chloramphenicol. The results of PCR amplification revealed that all the tested isolates harbored none of the tested virulence genes except E. faecalis (ES9), which showed the presence of esp gene. The Enterococcus isolates showed cholesterol lowering properties. The selected isolates showed a high tolerance to low pH, and toward bile salts. They also demonstrated hydrophobicity activity, auto-aggregation, and adhesion ability to the human intestinal Caco-2 cell line. These properties may contribute the bacteria colonizing the gut. This study revealed that the Enterococcus isolates, especially E. durans ES11, ES20 and ES32, might be excellent candidates for production of functional foods to promote health benefits.
DOI:10.17219/acem/38162URLPMID:25923096 [本文引用: 1]
BACKGROUND: The genus Enterococcus is of increasing significance as a cause of nosocomial infections, and this trend is exacerbated by the development of antibiotic resistance. OBJECTIVES: The aim of the present study was to estimate the potential virulence factors in enterococci and to ascertain their prevalence in Malaysian hospitals. MATERIAL AND METHODS: The study comprised 222 enterococcal strains isolated from blood, urine, exudates, sputum, stool and body fluid. These strains were collected from patients staying in three referral hospitals in Malaysia. All isolates were identified to the species level, and their MIC of vancomycin was determined using E test strips. Specific primers were designed for detection of the five potential virulence genes (gelE, PAI, esp, ace, and sprE) by PCR assay. RESULTS: Different patterns and frequency of virulence determinants were found for the E. faecalis and E. faecium isolates. E. faecalis isolates had more virulence determinants than E. faecium isolates. Clinical enterococcal isolates were found to possess more virulence determinants than enterococci isolated from faecal samples. The esp gene is significantly more common (p = 0.049) in vancomycin-resistant strains (85.7%) than in vancomycin-sensitive strains (44.2%). All of the vancomycin-resistant isolates were isolated from faecal samples. None of the classical virulence factors were found in 11% of enterococcal isolates, while all five virulence genes were found in 21% of enterococcal isolates. CONCLUSIONS: All the virulence genes considered in this study were important in the pathogenesis of enterococcal infections and further studies including more virulence genes and epidemiological data will be necessary in order to analyze the association and role of virulence genes in the pathogencity of enterococci.
URLPMID:25380802 [本文引用: 1]
URLPMID:25801971 [本文引用: 1]
DOI:10.1186/s12934-019-1161-6URLPMID:31217027 [本文引用: 1]
BACKGROUND: Yaks living in the high-altitude hypoxic environment of Tibetan plateau (3600 m) have special gut microbes. However, it is still little research on yak probiotics until now. Therefore, the purpose of our study was to evaluate the growth promoting effect, antioxidant capability, immune effect, and anti-inflammatory ability of Bacillus subtilis and Bacillus velezensis isolated from Tibetan yaks in mice model. RESULTS: The results showed that the isolated strains supplementation not only improve the growth performance but also increased the length of villus in the small intestine and intestinal digestive enzyme activity. Importantly, we observed that the T-AOC, SOD, and GSH-PX levels were increased and the MDA content was reduced in probiotic-treated mice, which implied that probiotics supplementation can ameliorate the antioxidative activity of mice. The levels of AST and ALT correlated with the hepatic injury were reduced and the levels of AKP, TP, GLB, ALB, Ca, and P were markedly higher than those in the control group. Additionally, mice treated with probiotics exhibited higher serum IgG, IgM and IgA, which can reflect the immune status to some extent. At the same time, the major pro-inflammatory factor TNF-alpha, IL-6, and IL-8 were down-regulated and the anti-inflammatory factor IL-10 was up-regulated compared with the control groups. CONCLUSIONS: In conclusion, these results demonstrated that Bacillus subtilis and Bacillus velezensis supplementation can increase overall growth performance and ameliorate the blood parameters related to inflammation and immunity of mice.
[本文引用: 1]
[本文引用: 1]
DOI:10.3389/fmicb.2015.00782URLPMID:26284059 [本文引用: 1]
Screening of lactic acid bacteria (LAB) isolated from ewe colostrum led to the identification and isolation of Enterococcus faecium CM33 with interesting features like high survival rates under acidic or bile salts condition, high tolerance for the simulated gastrointestinal condition, and high adhesive potential to Caco-2 cells. According the inhibition of pathogen adhesion test results, this strain can reduce more than 50% adhesion capacity of Escherichia coli, Shigella flexneri, Klebsiella pneumoniae, Listeria monocytogenes, and Staphylococcus aureus to Caco-2 cells. Based on the antibiotic sensitivity test findings, E. faecium CM33 was susceptible to gentamycin, vancomycin, erythromycin, ampicillin, penicillin, tetracycline, and rifampicin, but resistant to chloramphenicol, clindamycin, and kanamycin. Upon assessment of the virulence determinants for E. faecium CM33, this strain was negative for all tested virulence genes. Furthermore, the genome of this strain was evaluated for the incidence of the known enterocin genes by specific PCR amplification and discovered the genes encoding enterocins A, 31, X, and Q. Based on this study findings, the strain E. faecium CM33 can be considered as a valuable nutraceutical and can be introduced as a new potential probiotic.
DOI:10.1016/bs.aambs.2019.03.003URLPMID:31128749 [本文引用: 1]
An accurate understanding of properties of probiotics is a prerequisite for selecting probiotic organisms for use in swine production. This review aims to review selection criteria for probiotic organism in swine. The systematically investigated ecological history rather than the source of isolates should be regarded as the natural origin of probiotic strains, which helps to correct the inconsistencies arising from incorrect identification of the source. Moreover, in vivo studies are suggested as follow-up assessment to validate the characteristics of probiotic predicted by in vitro experiments. In addition, the intended probiotic effect depends on the age of the animal and disease prevention in young animals may require different probiotic strains when compared to growth promotion in older animals. With adequate selection criteria, the inclusion of probiotic in feed supplementation is a promising way to exert positive effects on sows, newborns, weanling animals and grower-finisher pigs. Both host-adapted probiotics and nomadic probiotics can be applied for pathogen inhibition but host adapted organisms appear to have a different mode of action. Host-adapted probiotic strains are likely to be associated with exclusive colonization while the nomadic or environmental strain exert better immune stimulating functions. Strains with potent enzymatic activity are fitter for grower pigs favoring feed digestion and enhancing growth performance.
DOI:10.1371/journal.pone.0194866URLPMID:29641608 [本文引用: 1]
Zearalenone (ZEN) is a mycotoxin produced by Fusarium species, which is one of the main animal feed contaminants causing reproductive disorders in livestock. The aim of this study was to evaluate the probiotic characteristics and ZEN removal ability of a Bacillus licheniformis strain CK1. The probiotic properties, including acidic tolerance, bile salt tolerance, adherence capability, and anti-pathogenic activities of CK1 were evaluated. CK1 survived after incubation at pH 2.0 or 3.0 for 3 h, grew well in LB broth containing 0.3% oxgall, possessed adherence capability to Caco-2 cells, and inhibited the growth of Escherichia coli O157:H7 and Listeria monocytogenes. The ZEN removal ability of CK1 was compared with a mineral mycotoxin-adsorbing agent, hydrated sodium calcium aluminosilicate (HSCAS), and a well-characterized biological mycotoxin-adsorbing agent, Lactobacillus rhamnosus GG (LGG). At 37 degrees C in phosphate-buffered saline (PBS, pH 7.0) containing 5 mug mL-1 of ZEN, the ZEN removal percentage of CK1 was 73.0%, which was significantly higher than that of HSCAS and LGG (45.9% and 48.4%, respectively). In the pH range of 2.5-8.0, CK1 removed up to 65% of ZEN. At temperatures between 4 and 42 degrees C, CK1 removed more than 75% of ZEN. In the adsorption stability analysis, the amounts of ZEN removed by CK1 was over 30% even after five consecutive rounds of washing procedures. These findings demonstrated that CK1 displayed probiotic characteristics and removed ZEN effectively. Therefore, CK1 has a great potential for the development of feed additive to remove ZEN.
URLPMID:30220016 [本文引用: 1]
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URLPMID:27682086 [本文引用: 1]
DOI:10.2527/jas.2010-2996URLPMID:21622872 [本文引用: 1]
Adherence to the gastrointestinal tract is a key element desirable for many of the proposed beneficial health effects of probiotic bacteria. The aims of this study were to determine the amounts of adhesion of 3 Lactobacillus salivarius strains (Lb6, Lb9, and Lb10) to porcine small intestinal mucins and to determine whether adhesion is a function of lectin-like activities. Dot and Western blot assays were performed to investigate bacterial adhesion. Several carbohydrates and glycoproteins were evaluated to determine whether they interfered with adhesion of the Lactobacillus strains to intestinal mucins and to determine whether they had lectin-like activities. The Lb9 and Lb10 strains had greater association with piglet mucins than did those from 22- to 24-wk-old finishing pigs (P = 0.021 and 0.037, respectively), whereas the Lb6 strain adhered to both (P = 0.138). Western blot assays showed that bacterial adhesion detected piglet mucosa from the duodenum, jejunum, and ileum. In finishing pigs, the adhesion was variable throughout the gastrointestinal tract. Galactose and mannose diminished the interaction of the Lb9 and Lb10 strains in intestinal mucosa (P = 0.028 and 0.026, respectively), whereas pig gastric mucin reduced the adhesion of the Lb6 strain (P = 0.013). Adhesion of the Lb9 and Lb10 strains to intestinal mucosa was less after protease treatment (P = 0.023 and 0.018, respectively), which indicates that proteins are needed for the Lb9 and Lb10 strains to recognize mucin. The Lb6 strain also demonstrated diminished adhesion after periodate treatment (P = 0.038). From these results, we suggest that the nature of the bacterial lectin-like substance is a surface protein that loosely binds to the bacterial cell surface. All the tested strains adhered to specific targets in the small intestinal mucosa of piglets, and the bacteria had lectin-like proteins involved in this adhesion.
URLPMID:25861634 [本文引用: 1]
DOI:10.1186/s13099-017-0162-4URLPMID:28286570 [本文引用: 1]
BACKGROUND: One of the working mechanisms of probiotic bacteria is their ability to compete with pathogens. To define the probiotic properties of seven Lactic Acid Bacteria (LAB) strains, we tested them for survival in simulated gastro-intestinal conditions, antimicrobial activities, co-aggregative abilities, and interferences studies against five human intestinal pathogens (Salmonella enteritidis ATCC 13076, Listeria monocytogenes ATCC 7644, Escherichia coli O157: H7 ATCC 35150, Cronobacter sakazakii ATCC 29544 and Campylobacter jejuni ATCC 33291). RESULTS: The LAB strains were able to survive the stomach simulated conditions, and varied in their abilities to survive the small intestinal-simulated conditions. The strains showed antibiotic susceptibility profiles with values equal or below the breakpoints set by the European Food and Safety Authority. The LAB cell-free cultures supernatants showed antimicrobial activities, with inhibition zones ranging from 10.0 to 17.2 mm. All the LAB strains showed moderate auto-aggregation abilities while the greatest co-aggregation abilities were observed for Bifidobacterium bifidum W23, Lactobacillus plantarum W21 and Lactobacillus rhamnosus W71. The individual LAB strains showed strain-specific abilities to reduce the invasion of intestinal pathogens in an interference model with Caco-2 cells. Increased invasion inhibition was found when different combinations of LAB strains were used in the interference tests. CONCLUSION: The LAB strains examined in this study may protect the intestinal epithelium through a series of barriers (antimicrobial activity, co-aggregation with pathogens, adherence) and interference mechanisms. Consequently, these LAB strains may be considered candidates for prophylactic use to prevent intestinal infections.
URLPMID:28879447 [本文引用: 1]
DOI:10.1007/s12602-016-9223-0URLPMID:27481236 [本文引用: 1]
This commentary was aimed at shedding light on the multifunction of bacteriocins mainly those produced by lactic acid bacteria. These antibacterial agents were first used to improve food safety and quality. With the increasing antibiotic resistance concern worldwide, they have been considered as viable agents to replace or potentiate the fading abilities of conventional antibiotics to control human pathogens. Bacteriocins were also shown to have potential as antiviral agents, plant protection agents, and anticancer agents. Bacteriocins were reported to be involved in shaping bacterial communities through inter- and intra-specific interactions, conferring therefore to producing strains a probiotic added value. Furthermore, bacteriocins recently were shown as molecules with a fundamental impact on the resilience and virulence of some pathogens.
