CN117417856A - 一种具有多种水解酶分泌能力的贝莱斯芽孢杆菌及其培养方法和应用 - Google Patents
一种具有多种水解酶分泌能力的贝莱斯芽孢杆菌及其培养方法和应用 Download PDFInfo
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Abstract
本发明提供了一种具有多种水解酶分泌能力的贝莱斯芽孢杆菌及其培养方法和应用,属于微生物技术领域,所述贝莱斯芽孢杆菌为贝莱斯芽孢杆菌Bacillus sp.QJS2.1,已于2023年5月29日保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:M 2023838。本发明提供的菌株具有很好的益生性能,对胆盐、强酸、人工胃肠液均具有较好的耐受性能,不会产生溶血现象;该菌株能够有效定殖在肠道内,起到整肠及调节肠道菌群的作用;无抗生素耐药性,可用于制备动物饲料和发酵食品,应用于动物饲料时可延长饲料的保存期限,并能提高饲料利用率,促进禽畜生长;且具有提高免疫力的作用。
Description
技术领域
本发明属于微生物技术领域,尤其涉及一种具有多种水解酶分泌能力的贝莱斯芽孢杆菌及其培养方法和应用。
背景技术
随着畜牧养殖业的迅速发展,“人畜共粮”的矛盾日益严重,引起了世界各国不仅是科技界还有工业界的关注。在努力探寻新的饲料来源的过程中,蛋白质饲料尤其受到重视。从20世纪中期开始,在动物饲粮中添加抗生素等物质显著促进了动物的生产,并在畜牧养殖业取得了很大成果。但是随着时间的推进,人们发现在日粮中添加抗生素会带来非常显著的危害,因此,受到了行业各界人士的广泛关注。
随着全面禁抗时代的到来,发展安全、健康的养殖业环境是当务之急,其中利用微生物发酵饲料正在受到人们的格外青睐。微生物发酵饲料分为固态发酵饲料和液体发酵饲料,因固态发酵干物质损失少,条件易于控制,发酵效果较好,被广泛应用于畜牧养殖业中。贝莱斯芽孢杆菌是一种新型细菌,对该菌的研究主要集中在生防、医疗和工业酶制剂等领域,而对禽畜养殖方面的研究较少。
发明内容
有鉴于此,本发明的目的在于提供一种具有多种水解酶分泌能力的贝莱斯芽孢杆菌及其培养方法和应用,该菌株能够产生芽孢和多种水解酶,具有延长饲料保存期限、提高饲料利用率、促进禽畜生长的作用;且该菌株能够有效定殖在肠道内,起到整肠及调节肠道菌群的作用;无抗生素耐药性。
为实现上述目的,本发明提供了以下技术方案:
一种贝莱斯芽孢杆菌,所述贝莱斯芽孢杆菌为贝莱斯芽孢杆菌Bacillussp.QJS2.1,于2023年5月29日保藏于中国典型培养物保藏中心,保藏编号为CCTCCNO:M 2023838。
本发明还提供了所述贝莱斯芽孢杆菌的培养方法,将贝莱斯芽孢杆菌Bacillussp.QJS2.1接种于LB培养基上进行培养。
优选的,所述培养的温度为30~40℃,所述培养的时间为20~28h。
本发明还提供了所述贝莱斯芽孢杆菌在制备调节肠道菌群的药物、食品和保健品中的应用。
本发明还提供了所述贝莱斯芽孢杆菌在制备提高免疫力的药物、食品和保健品中的应用。
本发明还提供了所述贝莱斯芽孢杆菌在制备发酵食品中的应用。
优选的,所述发酵食品包括酒精饮料、乳制品、豆制品、发酵蔬菜和调味品。
本发明还提供了所述贝莱斯芽孢杆菌在制备动物饲料中的应用。
优选的,所述动物饲料包括青贮饲料、生物发酵饲料和饲料添加剂。
与现有技术相比,本发明具有如下有益效果:
(1)本发明中的贝莱斯芽孢杆菌QJS2.1具有很好的益生性能,不仅对胆盐、强酸具有良好的耐受性,对人体的胃肠液也具有较好的耐受性能,不会产生溶血现象;且通过了小鼠体内安全试验,因此,可用于制备发酵饲料。
(2)本发明提供的贝莱斯芽孢杆菌QJS2.1在进入人体胃肠道后,仍然有较多的活菌存在,可有效定殖在肠道内,起到整肠及调节肠道菌群的作用;该菌株无抗生素耐药性,可在肠道内长期发挥作用;且其具有一定的抑菌能力,能够维护畜禽肠道健康。
(3)经小鼠血清免疫指标检测,可知该菌株能够在一定程度上提高小鼠的免疫功能。
(4)该菌株能够分泌多种水解酶,如,淀粉酶、纤维素酶和蛋白酶,能够通过发酵提高饲料预消化率,进而提高畜禽生产性能。
(5)本发明提供的贝莱斯芽孢杆菌QJS2.1的上述特性使其能够应用于制备发酵食品和动物饲料。
附图说明
图1为菌株QJS2.1的生理生化试验图;
图2为菌株QJS2.1的16S rDNA扩增产物图;
图3为菌株QJS2.1的系统发育树;
图4为菌株革兰氏染色的结果;
图5为菌株在平板上的菌落形态;
图6为菌株耐酸性试验中测得的菌株的存活率;
图7为菌株耐胆盐试验中测得的菌株的存活率;
图8为菌株耐人工胃液试验中测得的菌株的存活率;
图9为菌株耐人工肠液试验中测得的菌株的存活率;
图10为菌株产酶试验的结果,其中,从左至右依次为产蛋白酶、淀粉酶和纤维素酶的结果图;
图11为菌株耐药性试验的结果;
图12为菌株溶血性试验的结果;
图13为菌株易位试验的结果。
生物保藏说明
贝莱斯芽孢杆菌Bacillus sp.QJS2.1,保藏于中国典型培养物保藏中心(CCTCC),保藏地址为中国.武汉.武汉大学,保藏日期为2023年5月29日,保藏编号为CCTCC NO:M2023838。
具体实施方式
一种贝莱斯芽孢杆菌,所述贝莱斯芽孢杆菌为贝莱斯芽孢杆菌Bacillussp.QJS2.1,于2023年5月29日保藏于中国典型培养物保藏中心,保藏编号为CCTCCNO:M 2023838。
本发明还提供了所述贝莱斯芽孢杆菌的培养方法,将贝莱斯芽孢杆菌Bacillussp.QJS2.1接种于LB培养基上进行培养。
在本发明中,所述培养的温度优选为30~40℃,进一步优选为32~38℃,更进一步优选为34~37℃,再进一步优选为35~36℃;所述培养的时间优选为20~28h,进一步优选为22~27h,更进一步优选为24~26h,再进一步优选为25h。
本发明还提供了所述贝莱斯芽孢杆菌在制备调节肠道菌群的药物、食品和保健品中的应用。
本发明还提供了所述贝莱斯芽孢杆菌在制备提高免疫力的药物、食品和保健品中的应用。
本发明还提供了所述贝莱斯芽孢杆菌在制备发酵食品中的应用。
在本发明中,所述发酵食品包括酒精饮料、乳制品、豆制品、发酵蔬菜和调味品。
本发明还提供了所述贝莱斯芽孢杆菌在制备动物饲料中的应用。
在本发明中,所述动物饲料包括青贮饲料、生物发酵饲料和饲料添加剂;所述青贮饲料优选的包括玉米青贮、玉米秸青贮、牧草青贮、秧蔓青贮、叶菜类青贮和混合青贮等;所述生物发酵饲料优选的包括全混合日料发酵饲料、浓缩饲料发酵饲料、单一原料发酵饲料和地源性饲料资源发酵饲料;所述饲料添加剂优选的包括微生物活菌添加剂类、保健和促生长剂类、生理代谢调节剂类、增进食欲助消化类、饲料加工及保存剂类等。
下面结合实施例对本发明提供的技术方案进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
实施例1贝莱斯芽孢杆菌Bacillus sp.QJS2.1的筛选和鉴定
1.1菌种的分离、纯化
取甘肃省定西市安定区凤翔镇张家庄村的清浆水1mL,装入9mL无菌生理盐水中,混匀,采用梯度稀释法分别获得10-2、10-4、10-6稀释度的菌液,分别取100μL三个稀释度的菌液并分别涂布于LB固体培养基上,置于37℃恒温培养箱中培养24h,从出现单菌落的培养基上挑取菌株,通过平板反复划线纯化菌株,经革兰氏染色处理后,观察芽孢杆菌的形态,并置于4℃冰箱中进行斜面传代保藏。
其中,LB固体培养基的配方为:蛋白胨1g,酵母粉0.5g,氯化钠0.5g,琼脂18g,蒸馏水1000mL,pH7~7.2,配制完成后经121℃灭菌处理20min。
观察结果显示,分离、纯化后的菌株的菌落呈乳白色,直径2-3mm,表面粗糙、边缘整齐、中央隆起;镜检形态为短杆状,中央空芽孢,单个、成对排列;如图4所示,革兰氏染色的结果为蓝紫色,即该菌株属于革兰氏阳性菌;且是一种好氧菌;将分离纯化获得的菌株命名为QJS2.1。
1.2菌株的生理生化鉴定试验
采用细菌微量生化反应管对分离、纯化获得的菌株QJS2.1进行生理生化鉴定试验。鉴定结果如表1所示。
表1QJS2.1的生理生化试验结果
注:“+”代表阳性;“-”代表阴性。
由表1可知,菌株QJS2.1能够利用阿拉伯糖、甘露糖、葡萄糖等,不能利用蕈糖、半乳糖、棉子糖等,甲基红、MR-VP、硝酸盐还原等试验为阳性,硫化氢、胆汁七叶苷和明胶试验为阴性,且该菌株能够在6.5%的高盐肉汤上生长。
1.316S rDNA分子鉴定
采用16S rDNA法对分离纯化获得的菌株进行分子鉴定。16S rDNA扩增及序列测定由上海生物工程股份有限公司完成。
结果显示,菌株QJS2.1的16S rDNA的核苷酸序列为:AGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGTGAAGTCGTA(SEQ ID No.1)。
将测得的16S rDNA序列置于NCBI(National Center forBiotechnologyInformation,美国国立生物技术信息中心)数据库中进行比对,结果发现,菌株QJS2.1的基因序列,在所有相似的序列中,与贝莱斯芽孢杆菌(Bacillussp.QJS2.1)菌株19742-2、19742-1和HAB-2在一簇上;结合其生理生化特征,鉴定菌株QJS2.1是贝莱斯芽孢杆菌。
该分离自清浆水的贝莱斯芽孢杆菌(Bacillus sp.QJS2.1),菌株QJS2.1已于2023年05月29日保藏于中国典型培养物保藏中心,其保藏编号为CCTCC NO:M 2023838。
实施例2菌株QJS2.1的耐酸性试验
将活化后的菌株制成菌体浓度为108CFU/mL的菌悬液,按4%的接种量接入pH值分别为1.0、2.0、3.0的LB液体培养基内,37℃静置培养,分别在培养至0h和5h时吸取菌悬液,进行梯度稀释,涂布至LB固体培养基上,置于37℃的恒温培养箱中倒置培养48h,选取菌落数在30~300之间的平板进行计数,计算同一培养时间下的平均值,获得菌株在不同酸性溶液中的耐受力(存活率)。其中,对同一培养时间获得的菌悬液设定3个平行处理组。结果如表2和图6所示。
其中,LB液体培养基的配方为:蛋白胨1g,酵母粉0.5g,氯化钠0.5g,蒸馏水1000mL,pH7~7.2,配制完成后经121℃灭菌处理20min。LB固体培养基为实施例1中的LB固体培养基。
表2菌株QJS2.1的耐酸性结果
由表2和图6可知,菌株QJS2.1具有较好的耐酸性;相较于在pH2.0环境中的菌株,在pH3.0环境中的菌株出现了生长的现象;在pH1.0和pH2.0的环境中,随着培养时间的延长,菌株的存活数均有所下降。
实施例3菌株QJS2.1的耐胆盐试验
将活化后的菌株制成菌体浓度为108CFU/mL的菌悬液,按4%的接种量接入胆盐浓度分别为0.3、1.5、3g/L的LB液体培养基(同实施例2)中,37℃静置培养,分别在培养至0h和5h时吸取菌悬液,进行梯度稀释,涂布至LB固体培养基(同实施例1)上,置于37℃的恒温培养箱中倒置培养48h,选取菌落数在30~300之间的平板进行计数,计算同一培养时间下的平均值,并根据公式(1)计算菌株对胆盐溶液的耐受力(存活率)。其中,对同一培养时间获得的菌悬液设定3个平行处理组。结果如表3和图7所示。
胆盐耐受力(%)=(5h活菌数Nt/0h活菌数N0)×100%公式(1)
表3菌株QJS2.1的耐胆盐能力
由于人体小肠中胆盐含量在0.3-3.0g/L范围内波动,结合试验测得的菌株QJS2.1在胆盐浓度为0.3g/L的环境中的耐受力为94.28%,在胆盐浓度为1.5g/L的环境中的耐受能力为78.50%;可知,该菌株具有较好的耐胆盐能力,能够长期定殖于人体肠道,并发挥相应的益生作用。
实施例4菌株QJS2.1对人工胃液的耐受力试验
人工胃液的制备:取浓度为20%盐酸16.4mL,调节其pH值至3.0,加入胃蛋白酶(颗粒),使溶液的终浓度达到0.01g/mL,待胃蛋白酶充分溶解后,用0.22μm规格的微孔膜过滤除菌。
将活化后的菌株制成菌体浓度为108CFU/mL的菌悬液,按4%的接种量接入人工胃液中,分别在0、2、4h时吸取菌悬液,进行10倍梯度稀释,并涂布至LB固体培养基上(同实施例1),37℃倒置培养,选取菌落数在30~300之间的平板进行计数,计算同一培养时间下的平均值,并根据公式(2)计算菌株对人工胃液的耐受力(存活率)。其中,对同一培养时间获得的菌悬液设定3个平行处理组。结果如表4和图8所示。
胃液耐受力(%)=(4h/2h活菌数Nt/0h活菌数N0)×100%公式(2)
表4菌株QJS2.1对人工胃液的耐受力
试验结果表明:培养2h后菌株耐受力达到50%以上,随着培养时间的延长,菌株的数量略有下降,但耐受力仍达到46.98%,表明菌株具有优秀的耐胃液能力。
实施例5菌株QJS2.1对人工肠液的耐受力试验
人工肠液的配置方法为:向100mL无菌蒸馏水中加入1g胰蛋白酶(颗粒),使溶液的终浓度达到0.01g/mL,待胰蛋白酶充分溶解后,用0.22μm的微孔膜过滤除菌,即得。
将活化后的菌株制成菌体浓度为108CFU/mL的菌悬液,按4%的接种量接入人工肠液中,分别于0、2、4h从中吸取菌悬液,进行10倍梯度稀释,涂布至LB固体培养基上(同实施例1),37℃倒置培养,选取菌落数30~300之间的平板进行计数,计算同一培养时间下的平均值,并根据公式(3)计算菌株对人工肠液的耐受力(存活率)。其中,对同一培养时间获得的菌悬液设定3个平行处理组。结果如表5和图9所示。
所述菌株对人工肠液的耐受力根据公式(3)进行计算:
肠液耐受力(%)=(4h/2h活菌数Nt/0h活菌数N0)×100%公式(3)
表5菌株QJS2.1对人工肠液的耐受力
试验结果表明:菌株在人工肠液中的耐受力较高,随着培养时间的延长,菌株的耐受力略有降低,但培养4h后菌株耐受力仍达到44.35%,可以在动物肠道中存活。
实施例6菌株QJS2.1产酶能力的测定
从实施例1中保藏的斜面中用接种环挑取一环菌体接种于10mL的LB液体培养基(同实施例2)中培养24h,制备浓度为108CFU/mL的菌悬液。将菌种液分别点种在酪蛋白选择培养基、纤维素选择培养基、淀粉选择培养基上,37℃培养24h,测定不同平板上水解圈的直径D(mm),每组处理3个重复。同时,通过判断对应的培养基上是否有透明圈判定菌株能否产生蛋白酶、纤维素酶和淀粉酶。结果如表6和图10所示。
其中,酪蛋白选择培养基的配方为:牛肉净粉3g/L,氯化钠5g/L,蛋白胨10g/L,脱脂奶粉15g/L,琼脂18g/L,pH值7.0±0.2,121℃灭菌20min;
纤维素选择培养基的配方为:磷酸二氢钠1g/L,硫酸镁0.3g/L,氯化镁0.1g/L,硝酸钠2.5g/L,六水氯化钙0.1g/L,氯化铁0.01g/L,稻草粉20g/L,琼脂18g/L,pH值7.0±0.2,121℃灭菌20min;
淀粉选择培养基的配方为:蛋白胨10g/L,氯化钠5g/L,可溶性淀粉2g/L,牛肉膏5g/L,琼脂18g/L,pH值7.0±0.2,121℃灭菌20min。
表6菌株QJS2.1的产酶能力
结果显示,菌株QJS2.1可产生淀粉酶、纤维素酶和蛋白酶。
实施例7菌株QJS2.1对抗生素的敏感实验
采用药敏纸片琼脂扩散法(K-B)进行药敏试验,评价菌株对抗生素的耐药性。具体步骤如下:
取1mL菌体浓度为108CFU/mL菌种液接种于100mL LB固体培养基内,摇匀到平皿,待培养基冷却凝固后,将纸片分别均匀平贴在LB固体培养基(与实施例1相同)表面。
所用的药敏纸片分别为:万古霉素(30μg)、羧苄西林(30μg)、青霉素(30μg)、四环素(75μg)、庆大霉素(30μg)、复方新诺敏(30μg)、庆大霉素(10μg)、头孢哌酮(5μg)、头孢呋辛(15μg)、哌拉西林(75μg)、N新霉素(30μg)、E红霉素(15μg)、疾特灵(30μg),抗生素剂量参照美国临床实验室标准化研究所(Clinicaland Laboratory Standards Institute,CLSI/NCCLS)2012版。
将贴好药物纸片的平板分别放置于37℃培养箱中培养48h,用游标卡尺测量各药敏纸片的抑菌圈直径(D)大小,通过引用《刘玉庆,李璐璐,骆延波,等.EUCAST欧盟药敏试验标准[J].中国标准导报,2016(5):60》文中提供的抑菌圈直径评判标准判定菌株对抗生素的敏感性,测定的药敏结果如表7所示。
敏感率(中等敏感率)=菌株敏感性(中等敏感)药物个数/测的总药物个数×100%
表7菌株QJS2.1对抗生素的敏感实验结果
结果表明:QJS2.1只对四环素表现出相对耐药,对其他抗生素均表现出敏感,敏感率和中等敏感率分别为91.22%、8.33%。表明菌株作为功能菌加入动物饲料是安全的,其无耐药基因,不会将其携带的耐药基因水平传达给肠道中的致病类菌导致此类菌获得耐药性。
实施例8菌株QJS2.1的溶血实验
对分离、纯化后的菌株进行活化、增菌处理,在血平板上划线,37℃培养24h,观察菌落周围是否有溶血性环。结果如图12所示。
由于一些细菌能够产生细菌素,导致红细胞裂解破碎,在血琼脂平板上产生透明或半透明的溶血环。结合图12可知,菌株QJS2.1不产生细菌素,因此,可以将其用于生产实践中。
实施例9小鼠试验
选取SPF级昆明小鼠40只,分为实验组(低、中、高剂量,即菌液中菌体细胞浓度分别为108、109、1010CFU/mL)和对照组(生理盐水),每组10个重复,每只小鼠灌胃剂量为0.2mL。在环境为20℃,湿度为50%,光照12h的条件下,给予足够的食物和纯净水喂养3~5天。灌胃开始前16h禁食,但不限制饮水。每天灌胃前记录小鼠的体重和采食量变化,并观察记录小鼠的毛色、整体精神状态及分泌物、排泄物状况。灌胃结束后,第8d将全部的小鼠用颈椎脱臼法处死,观察记录主要器官的变化。
在正式试验期的第1d和试验的最后1d,对试验小鼠进行空腹称重,计算平均日增重。称重记录小鼠的投喂料量和剩料量,按照公式(4)计算平均日采食量。
平均日采食量=(投料量-剩料量)/试验天数 公式(4)
将小鼠处死后取肝脏、脾脏、肾脏称重,根据公式(5)计算脏器指数。
脏器指数(%)=(脏器质量(g)/体质量(g))×100% 公式(5)
结果如表8~表10所示。
表8灌胃菌株QJS2.1对小鼠体质量的影响
表9灌胃菌株QJS2.1对小鼠采食量(g)的影响
从表8、表9可以看出,灌胃芽孢杆菌QJS2.1后24h小鼠采食量有所下降可能是由于灌胃时刺激到小鼠的食管使小鼠采食量有不同程度的下降,但灌胃7d内平均采食量呈上升趋势,对小鼠体质量、平均日采食量无显著差异(P>0.05)。
表10灌胃菌株QJS2.1对小鼠脏器指数(%)的影响
从表10可以看出,与对照组相比,灌胃芽孢杆菌QJS2.1的处理组对小鼠的心、肝、脾、肺、肾均无显著影响(P>0.05)。
此外,对小鼠的主要器官进行易位试验,即在灌胃的最后一天对小鼠的心、肝、脾、肺、肾进行研磨破碎后涂布在LB固体培养基上(同实施例1),37℃培养24h,观察。结果如图13所示。
由图13可知,在平板上均未发现与QJS2.1形态相似的菌落。说明通过灌胃方式给予小鼠菌液后细菌不会穿过粘膜进入无菌的组织或器官。
实施例10免疫功能试验
在上述实施例9中小鼠试验的最后1d,采集试验小鼠的血液10.0mL,在4℃冰箱中冷却2h后3000r/min离心20分钟,得到血清样品。
使用AST测试盒、ALT测试盒测定血清中谷草转氨酶(AST)(比色法)、谷丙转氨酶(ALT)(微板法)活性。
使用T-AOC、SOD、GSH测试盒测定血清总抗体能力(T-AOC)(Fe3+还原法)、超氧化物歧化酶(SOD)(羟胺法)和谷胱甘肽过氧化物酶(GSH-Px比色法)。
使用IgA、IgM、IL-2、TNF-α测试盒测定血清样品中免疫球蛋白A(IgA)、免疫球蛋白M(IgM)、白细胞介素-2(IL-2)、肿瘤坏死因子-α(TNF-α)含量。
上述测试盒均购自南京建成生物工程研究所。
测定结果如表11所示。
表11灌胃菌株QJS2.1对小鼠免疫功能的影响
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注:同行数据不同字母表示差异显著(P<0.05),相同字母或无字母表示差异不显著(P>0.05)
由表11可知,与对照组相比,高、中、低剂量组小鼠血清中的超氧化物歧化酶、谷胱甘肽、免疫球蛋白M和白细胞介素含量显著提升(P<0.05)。各剂量组小鼠血清谷草转氨酶、谷丙转氨酶、肿瘤坏死因子α和免疫球蛋白A含量差异均不明显(P>0.05),无统计学意义。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (9)
1.一种贝莱斯芽孢杆菌,其特征在于,所述贝莱斯芽孢杆菌为贝莱斯芽孢杆菌Bacillus sp.QJS2.1,于2023年5月29日保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:M 2023838。
2.如权利要求1所述的贝莱斯芽孢杆菌的培养方法,其特征在于,将贝莱斯芽孢杆菌Bacillus sp.QJS2.1接种于LB培养基上进行培养。
3.根据权利要求2所述的培养方法,其特征在于,所述培养的温度为30~40℃,所述培养的时间为20~28h。
4.如权利要求1所述的贝莱斯芽孢杆菌在制备调节肠道菌群的药物、食品和保健品中的应用。
5.如权利要求1所述的贝莱斯芽孢杆菌在制备提高免疫力的药物、食品和保健品中的应用。
6.如权利要求1所述的贝莱斯芽孢杆菌在制备发酵食品中的应用。
7.根据权利要求6所述的应用,其特征在于,所述发酵食品包括酒精饮料、乳制品、豆制品、发酵蔬菜和调味品。
8.如权利要求1所述的贝莱斯芽孢杆菌在制备动物饲料中的应用。
9.根据权利要求8所述的应用,其特征在于,所述动物饲料包括青贮饲料、生物发酵饲料和饲料添加剂。
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