CN113980848B - 一种戊糖片球菌sbc5及其应用 - Google Patents
一种戊糖片球菌sbc5及其应用 Download PDFInfo
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- CN113980848B CN113980848B CN202111284009.6A CN202111284009A CN113980848B CN 113980848 B CN113980848 B CN 113980848B CN 202111284009 A CN202111284009 A CN 202111284009A CN 113980848 B CN113980848 B CN 113980848B
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- sbc5
- pediococcus pentosaceus
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- fermentation
- pediococcus
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Abstract
本发明公开了一种戊糖片球菌SBC5及其应用,该菌已于2021年1月5日保藏于北京市朝阳区北辰西路1号院3号中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC NO.21595。本发明的戊糖片球菌SBC5具有多种功能特性,同时还可以通过发酵生产有机酸组合物,生产出的多功能代谢物质可进一步应用于生物制剂的制备。
Description
技术领域
本发明属于微生物技术领域,涉及一种菌株,具体地说是一种戊糖片球菌SBC5及其应用。
背景技术
乳酸菌(Lacticacid bacteria)是发酵一系列糖类物质的主要终产物为乳酸的细菌总称,主要以革兰氏阳性菌为主,表现为不运动、不产生芽孢,不具有过氧化氢酶,是兼性气性微生物,也是国际公认的食品安全级微生物(GRAS);乳酸菌能够将各种多羟基的醛类和酮类化合物分解成为乳酸。作为已经被认可和证实的益生菌,乳酸菌的益生作用已经应用于各个领域中,例如,乳酸菌通过与致病菌竞争肠道结合位点、营养成分和生存空间,形成乳酸菌保护层,阻断致病菌对机体侵害,可以用于预防呼吸道感染、结肠直肠癌、急性胃肠炎等症状的发生,同时,乳酸菌还可以产生短链脂肪酸、乳酸、甲酸、细菌素、过氧化氢及小分子肽类等一系列代谢产物来拮抗肠道致病菌和抑制腐败及病原微生物的生长;乳酸菌作为定居肠道中的有益菌群,能够进营养成分的分解和吸收、维持微生态平衡和肠道机能、降低血清胆固醇水平、增强免疫功能、预防肿瘤等;对腹泻、肠易激综合征、机体过敏、乳糖不耐受、刺激反应等均有一定的治疗功效;利用乳酸菌制备的饲料,品质稳定、适口性好和饲料营养成分破坏较小、营养价值高,饲喂动物后能够改善机体营养状况,提高饲料转化率和动物生长性能;同时其无残留、无毒害,不产生耐药性,既保证人类健康又确保生态环境安全;利用乳酸菌生产的肉制品、乳制品、发酵蔬菜等食品,其营养价值更好、品质更好、更安全。
随着科技和社会的不断发展,对于优良乳酸菌的筛选和研究也越来越广泛和深入,不同乳酸菌甚至同一种乳酸菌的不同菌株,对环境的耐受能力,抗菌、黏附、降胆固醇、免疫调节等生物活性差异巨大,筛选具有优良特性的乳酸菌一直是国内外研发的重点。
戊糖片球菌(Pediococcus pentosaceus)是我国批准的可用于普通食品的菌种之一,于中华人民共和国国家卫生和计划生育委员会2014年第6号公告。其隶属链球菌科片球菌属,兼性厌氧,革兰氏阳性菌,因为能发酵葡萄糖产生乳酸,所以属于乳酸菌的一种,戊糖片球菌具有以下益生功能:抑制致病菌的生长繁殖、增强免疫力、减少体脂沉积、调节肠道免疫、降低胆固醇、抗肿瘤等。目前,对于戊糖片球菌益生功能的研究越来越受到重视,在科研和生活中得到各界人士的青睐。然而国内外的研究多集中于筛选高效的单功能菌株上,对多功能菌株的关注度较低。因此,筛选出具有多种益生功能、发酵性能良好的戊糖片球菌将拥有十分广泛的应用前景和较高的工业应用价值。
发明内容
本发明的目的,是要提供一种戊糖片球菌SBC5,以达到降血糖、抗氧化、抑菌等有益效果;
本发明的另一个目的,是要提供上述戊糖片球菌SBC5的应用。
为了实现上述目的,本发明采用的技术方案是:
一种戊糖片球菌SBC5,它保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏地址是北京市朝阳区北辰西路1号院3号,保藏日期为2021年1月5日,保藏编号为CGMCC NO.21595,拉丁文名称为Pediococcus pentosaceus。
作为一种限定,所述戊糖片球菌SBC5的16SrRNA基因序列如下:
AGGCTCAGGATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGAACTTCCGTTAATTGATTATGACGTACTTGTACTGATTGAGATTTTAACACGAAGTGAGTGGCGAACGGGTGAGTAACACGTGGGTAACCTGCCCAGAAGTAGGGGATAACACCTGGAAACAGATGCTAATACCGTATAACAGAGAAAACCGCATGGTTTTCTTTTAAAAGATGGCTCTGCTATCACTTCTGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCAGTGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTAAAGAAGAACGTGGGTAAGAGTAACTGTTTACCCAGTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTCTTTTAAGTCTAATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGATTACTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGTAATCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAAGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTCTGACAGTCTAAGAGATTAGAGGTTCCCTTCGGGGACAGAATGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTACTAGTTGCCAGCATTAAGTTGGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTCGCGAGACCGCGAGGTTAAGCTAATCTCTTAAAACCATTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGCCGGTGGGGTAACCTTTTAGGAGCTAGCCGTCTAAGGTGGGACAGATGATTAGGGTGAAGTCT。
作为另一种限定,所述戊糖片球菌SBC5是从发酵食品中分离筛选出来的。
本发明还提供了上述戊糖片球菌SBC5的一种应用,所述戊糖片球菌SBC5通过发酵生产有机酸组合物。
作为一种限定,所述发酵,所述发酵,培养基的碳源为蔗糖或葡萄糖;
所述有机酸组合物,包括乳酸、醋酸、柠檬酸、苹果酸、草酸、羟基苯乳酸、焦谷氨酸、甲酸、丙酸;
所述有机酸组合物,用于制备具有降血糖、抗氧化和抑菌性能的食品、药品、食品添加剂或饲料添加剂。
作为另一种限定,温度为33-37℃,pH值为6.0-7.0,时间为12-24h。
本发明还提供了上述戊糖片球菌SBC5的另一种应用,所述戊糖片球菌SBC5用于制备生物菌剂。
作为一种限定,所述生物菌剂,是将戊糖片球菌SBC5进行发酵,离心,收集菌泥,加入保护剂,真空冷冻干燥后制备而成的。
作为进一步限定,所述离心,转速为4000-6000r/mi n,时间为8-15mi n;
所述保护剂,为脱脂乳,用量为菌泥体积的10-15%。
作为另一种限定,所述菌剂,用于制备食品、药品、保健品或饲料添加剂。
由于采用了上述技术方案,本发明与现有技术相比,所取得的技术进步在于:
①本发明获得的戊糖片球菌SBC5,是从传统发酵食品中筛选获得,安全可靠;
②本发明获得的戊糖片球菌SBC5,具有降血糖、抗氧化和抑菌等多种益生功能;
③本发明获得的戊糖片球菌SBC5,通过发酵所得的代谢物质,具有降血糖、抗氧化和抑菌等多种性能。
本发明适用于分离戊糖片球菌SBC5,分离得到的戊糖片球菌SBC5,可以通过发酵生产多功能代谢物质,生产出的多功能代谢物质,具有降血糖、抗氧化和抑菌等多种性能。
附图说明
下面结合附图及具体实施例对本发明作更进一步详细说明:
图1为本发明实施例1中戊糖片球菌SBC5的菌落形态图;
图2为本发明实施例1中戊糖片球菌SBC5系统发育树;
图3为本发明实施例2中戊糖片球菌SBC5在不同碳源中的生长曲线;
图4为本发明实施例2中戊糖片球菌SBC5在不同温度下的生长情况;
图5为本发明实施例2中戊糖片球菌SBC5在不同pH下的生长情况;
图6为本发明实施例2中戊糖片球菌SBC5的生长曲线;
图7为本发明实施例16中小鼠空腹血糖值;
图8为本发明实施例16中小鼠餐后血糖值;
图9为本发明实施例16中小鼠糖耐量变化;
图10为本发明实施例16中小鼠AUC变化。
具体实施方式
下面通过具体实施例对本发明做进一步详细说明,应当理解所描述的实施例仅用于解释本发明,并不限定本发明。
实施例1一种戊糖片球菌SBC5的筛选与鉴定
一、菌种信息
戊糖片球菌SBC5是从发酵食品中分离筛选出来的,其菌株于2021年1月5日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏地址是北京市朝阳区北辰西路1号院3号,保藏编号为CGMCC NO.21595,拉丁文名称是Pediococcus pentosaceus;
二、戊糖片球菌SBC5的分离筛选方法
该分离纯化方法按照以下步骤依次进行:
S1.分别从河北、四川、广州、广西地区采集泡菜、母水、酒糟、醋糟、乳制品样品40个,在MRS培养基上,采用溶钙圈法,在含0.5%CaCO3的固体MRS上,以产生透明圈作为筛选依据,进行菌株初步筛选;经过筛选,获得86株能够形成明显透明圈的菌株,分别编号为X1-X86;
1.菌株的分离筛选
S1.收集液体发酵食品:母水、乳制品;
收集固体发酵食品:泡菜、酒糟、醋糟;
S2.直接吸取液体发酵食品,进行梯度稀释,分别吸取10-4、10-5、10-6三个稀释度的样品,得样品Ⅰ-Ⅲ;
将固体发酵食品采用打浆机进行粉碎,用0.85%的灭菌生理盐水提取5min,取上层液体,得样品Ⅳ-Ⅵ;
S3.用溶钙圈法,于37℃条件下在含0.5%CaCO3的固体MRS培养基上培养40h,挑选86个透明圈明显的菌落,作为初筛菌株X1-X86;
S4.对初筛菌株X1-X86进行耐受性的复筛,筛选出耐酸性和耐胆盐性优良的菌株,获得一株透明圈大、耐受性好的乳酸菌,编号为乳酸菌SBC5;
其中,乳酸菌SBC5的耐受性结果见表1和表2;
表1菌株SBC5在模拟人工胃液中的耐受性
表2菌株SBC5在模拟人工肠液中的耐受性
由表1和表2可知,菌株SBC5在模拟的人工胃液和肠液中均具有良好的耐受性;
三、乳酸菌SBC5菌株的鉴定
a1.形态观察
将乳酸菌SBC5在固体CaCO3-MRS平板上划线观察,菌落形态如图1a所示,挑取单菌落进行革兰氏染色并在显微镜下观察其形态结构如图1b所示,测定菌株的H2O2酶特性为阴性,与乳酸菌的特性相吻合;
由图1a可知,其菌落为乳白色,圆形凸起,边缘整齐,半透明,表面湿润光滑;由图1b可知,结果显示紫色,确定为革兰氏阳性菌,无芽孢形成,为球状;测定菌株的H2O2酶特性为阴性,与乳酸菌的特性相吻合。
a2.分子生物学鉴定
将乳酸菌SBC5进行16S rDNA序列测定,其16S rDNA序列全长为1513bp,具体序列如下:
AGGCTCAGGATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGAACTTCCGTTAATTGATTATGACGTACTTGTACTGATTGAGATTTTAACACGAAGTGAGTGGCGAACGGGTGAGTAACACGTGGGTAACCTGCCCAGAAGTAGGGGATAACACCTGGAAACAGATGCTAATACCGTATAACAGAGAAAACCGCATGGTTTTCTTTTAAAAGATGGCTCTGCTATCACTTCTGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCAGTGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTAAAGAAGAACGTGGGTAAGAGTAACTGTTTACCCAGTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTCTTTTAAGTCTAATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGATTACTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGTAATCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAAGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTCTGACAGTCTAAGAGATTAGAGGTTCCCTTCGGGGACAGAATGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTACTAGTTGCCAGCATTAAGTTGGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTCGCGAGACCGCGAGGTTAAGCTAATCTCTTAAAACCATTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGCCGGTGGGGTAACCTTTTAGGAGCTAGCCGTCTAAGGTGGGACAGATGATTAGGGTGAAGTCT
将16SrDNA序列提交到NCBI数据库(登录号MT313180),利用BLAST比对基因序列,利用软件MEGA7构建系统发育树进行同源性比较,如图2所示;
由图2可知,该菌株鉴定为戊糖片球菌,命名为Pediococcus pentosaceus SBC5。
实施例2戊糖片球菌SBC5的发酵条件优化
本实施例为戊糖片球菌SBC5的发酵条件优化实验,具体的方法为:
S1.挑取戊糖片球菌SBC5,加入MRS培养基δ1中,37℃条件下静置培养24h,连续活化2代,得待测菌株α;
S2.碳源的优化
用蔗糖、果糖、乳糖分别替代MRS培养基中的葡萄糖作为碳源,得培养基δ2-δ4,将戊糖片球菌SBC5分别加入培养基δ2-δ4中,37℃条件下静置培养24h,结果如图3所示;
由图3可知,戊糖片球菌SBC5在添加葡萄糖或蔗糖的培养基,菌体生长较快,菌体量高;
S3.培养温度的优化
将戊糖片球菌SBC5接种于改良MRS培养基δ2中,分别于30℃、33℃、36℃、39℃条件下恒温培养24h,结果如图4所示;
由图4可知,戊糖片球菌SBC5在不同温度下的生长情况不同,适宜温度为33-37℃;
S4.初始pH的优化
调节改良MRS培养基δ2的pH值为2.0-8.0,接种戊糖片球菌SBC5,37℃条件下恒温培养24h,结果如图5所示;
由图5可知,戊糖片球菌SBC5在pH值为6.0-7.0的条件下,表现出较好的生长特性,可以维持其最佳生长状态;
S5.培养时间的优化
将戊糖片球菌SBC5在改良MRS培养基δ2中培养,结果如图6所示;
由图6可知,12h后基本进入稳定期,活菌数在108CFU/mL以上。
实施例3-8戊糖片球菌SBC5的发酵方法
本实施例3-8分别为一种戊糖片球菌SBC5的发酵方法,具体为:将戊糖片球菌SBC5接种于改良MRS培养基δ2中,在温度为33-37℃,pH值为6.0-7.0,接种量为2%-4%的条件下发酵12-24h;它们的不同之处仅在于原料、用量、初始pH值及工艺参数的不同,具体详见表3。
表3戊糖片球菌SBC5的发酵方法参数一览表
各实施例在发酵结束后,得含戊糖片球菌SBC5的发酵液θ1-θ6,分别将发酵液θ1-θ6于转速为6000r/min的条件下离心10min,收集上清液,得发酵上清液ε1-ε6,收集菌体,用0.85%的生理盐水清洗2-3次后进行重悬,调整菌悬液为1×109CFU/ml得菌体悬液ψ1-ψ6。
实施例9戊糖片球菌SBC5及其发酵上清液的功能特性
本实施例为戊糖片球菌SBC5及其发酵上清液的功能特性检测实验,具体方法如下:
S1.体外降糖特性
S11.对α-淀粉酶的抑制率
将200μl戊糖片球菌SBC5菌体悬液ψ1和200μl发酵上清液ε1分别与100μl浓度为0.05mg/ml的α-淀粉酶混合,分别于37℃条件下保温10min、加入0.1%淀粉溶液625μl,37℃条件下反应15min后,终止反应,加入碘液显色,加蒸馏水定容至5ml,于波长为660nm下分别测吸光值,计算对α-淀粉酶的抑制率,结果如表4所示;
表4戊糖片球菌SBC5及其发酵液的体外降糖特性
由表4可知,戊糖片球菌SBC5发酵上清液ε1对α-淀粉酶的抑制效果高于菌体悬液ψ1,其抑制率可达46.93%;
S12.对α-葡萄糖苷酶的抑制率
将100μl戊糖片球菌SBC5菌体悬液ψ4和100μl发酵上清液ε4分别与α-葡萄糖苷酶混合,分别于37℃条件下孵育10min,加入500μl浓度为1mmol/L的PNG,培养20min,终止反应,于波长为405nm下分别测吸光值,计算对α-葡萄糖苷酶的抑制率,结果如表4所示;
由表4可知,戊糖片球菌SBC5发酵上清液ε4对α-葡萄糖苷酶具有一定的抑制作用,抑制率为10.25%。
综上,戊糖片球菌SBC5及其发酵上清液在体外均能够抑制降糖相关酶的活性,且其代谢物质的抑制率更优;
S2.体外抗氧化特性
S21.羟自由基清除能力
向容器中依次加入0.5ml水杨酸-乙醇溶液、0.5g硫酸亚铁、0.5mlH2O2和1ml戊糖片球菌SBC5上清液ε6或戊糖片球菌SBC5菌体悬液ψ6,混合均匀,于37℃条件下恒温孵育15min,于转速为6000r/min的条件下离心15min,以抗坏血酸作为阳性对照,测定溶液在510nm处吸光值,计算对羟自由基的清除能力,结果如表5所示;
S22.超氧阴离子清除能力
取0.1ml戊糖片球菌SBC5发酵上清液ε2或戊糖片球菌SBC5菌体悬液ψ2,加入4.5ml的Tris-HCl,25℃水浴20min,加入0.4ml邻苯三酚,25℃水浴5min,以HCl终止反应,测定溶液在325nm处吸光值,计算对超氧阴离子自由基的清除能力,结果如表5所示;
S23.DPPH清除能力
取1ml戊糖片球菌SBC5发酵上清液ε5或戊糖片球菌SBC5菌体悬液ψ5与1mlDPPH(0.2mmol/L,无水乙醇溶解)溶液混匀,25℃避光反应30min,于转速为6000r/min的条件下离心10min,取上清液在517nm处测吸光度,用1ml无水乙醇代替DPPH溶液为空白组,等体积蒸馏水代替样品溶液为对照组,计算对DPPH自由基的清除能力,结果如表5所示;
表5戊糖片球菌SBC5的体外抗氧化能力
由表5可知,戊糖片球菌SBC5及其发酵液对羟基自由基、DPPH自由基和超氧阴离子自由基均具有一定的清除能力,且其发酵代谢物的清除率高于菌悬液,总体来说戊糖片球菌SBC5具有抗氧化作用;
S3.体外抑菌特性
以金黄色葡萄球菌ATCC 25923、单核细胞增生李斯特菌10403s、大肠杆菌ATCC44752和鼠伤寒沙门氏菌ATCC 14028作为指示菌;
S31.戊糖片球菌SBC5菌体对指示菌的拮抗作用
采用双层平板法测定戊糖片球菌SBC5菌体的抑菌活性,底层为MRS固体培养基,将戊糖片球菌SBC5菌体悬液ψ6点种于MRS培养基中,于37℃静置培养4h后,分别将含有106CFU/mL金黄色葡萄球菌ATCC 25923、单核细胞增生李斯特菌10403s、大肠杆菌ATCC44752和鼠伤寒沙门氏菌ATCC 14028的营养琼脂培养基加至MRS培养基上,继续培养20h,观察抑菌效果,结果如表6所示;
表6戊糖片球菌SBC5菌体对指示菌的抑菌作用
对大肠杆菌 | 对鼠伤寒沙门氏菌 | 对金黄色葡萄球菌 | 对单核细胞增生李斯特菌 |
+ | + | + | + |
注:“+”表示有拮抗性,“-”表示无拮抗性;
由表6可知,戊糖片球菌SBC5菌体对选择的指示菌均具有一定的拮抗作用;
S32.戊糖片球菌SBC5发酵上清液对指示菌的抑制作用
采用牛津杯琼脂扩散法测量戊糖片球菌SBC5发酵上清液ε6的抑菌性,分别制备金黄色葡萄球菌ATCC 25923、单核细胞增生李斯特菌10403s、大肠杆菌ATCC 44752和鼠伤寒沙门氏菌ATCC 14028的抑菌测定平板,将戊糖片球菌SBC5发酵上清液ε6点于牛津杯中,4℃扩散10h后,37℃条件下恒温培养10h,测量抑菌圈大小,结果如表7所示;
表7戊糖片球菌SBC5发酵上清液对指示菌的抑菌作用
由表7可知,戊糖片球菌SBC5发酵上清液对四种指示菌均具有抑制作用,且抑制效果较好,抑菌圈直径在20mm以上。
实施例10一种戊糖片球菌SBC5菌剂的制备方法
本实施例为一种戊糖片球菌SBC5菌剂的制备方法,具体为:
取100ml含戊糖片球菌SBC5的发酵液θ1,于4℃、6000r/min的条件下离心10min,收集菌泥,得菌体A,将菌体用12%的脱脂乳作为保护剂重悬,真空冷冻干燥36h,即得戊糖片球菌SBC5菌剂ω1。
实施例11-15戊糖片球菌SBC5菌剂的制备方法
实施例11-15分别为一种戊糖片球菌SBC5菌剂的制备方法,它们的步骤与实施例10基本相同,不同之处仅在于离心的转速和时间不同,具体详见表8:
表8实施例11-15中离心参数一览表
最终制得戊糖片球菌SBC5菌剂ω2-ω6。
实施例16戊糖片球菌SBC5在糖尿病小鼠降糖中的应用
本实施例为验证戊糖片球菌SBC5在糖尿病小鼠降糖中的应用的实验,具体操作如下:
S1.取500ml实施例8中制备的含戊糖片球菌SBC5的发酵上清液ε6,冷冻干燥,制备为发酵上清液的冻干剂Ω;
S2.取2g实施例10中制备的戊糖片球菌SBC5菌剂ω1,分为两等份,用浓度为0.85%的生理盐水稀释,分别调整至为108CFU/mL和109CFU/mL;
S3.按照表9进行小鼠分组,从第3周开始分组进行灌胃,至第12周;
表9实验动物分组
S4.从第一周开始至实验结束前,每周称量各组小鼠的体重和血糖测量,包括其空腹血糖和餐后血糖,实验结束前一周通过灌胃葡萄糖溶液,对小鼠的口服葡萄糖耐量进行检测,实验结束时,检测肝脏、肾脏、脾脏和糖基化血红蛋白含量,结果如图7、图8和图9所示,具体数据如表10所示;
表10戊糖片球菌SBC5对各组小鼠脏器系数和糖基化蛋白的影响
组别 | 心(%) | 肝(%) | 脾脏(%) | 肾脏(%) | 肺(%) | HbA1c/(mmol/L) |
N | 0.56±0.09a | 6.29±0.15a | 0.20±0.07a | 1.94±0.12a | 0.65±0.05a | 7.56±0.18a |
M | 0.54±0.21a | 6.28±0.14a | 0.19±0.14a | 1.95±0.07a | 0.67±0.13a | 12.36±0.20d |
P | 0.55±0.07a | 6.28±0.25a | 0.21±0.05a | 1.99±0.02a | 0.67±0.07a | 7.54±0.19a |
SBC5(1) | 0.55±0.17a | 6.27±0.28a | 0.18±0.12a | 1.96±0.08a | 0.66±0.16a | 10.31±0.18b |
SBC5(2) | 0.56±0.26a | 6.29±0.32a | 0.20±0.03a | 1.95±0.13a | 0.67±0.03a | 10.34±0.19c |
SBC5(3) | 0.55±0.12a | 6.28±0.18a | 0.20±0.04a | 1.97±0.04a | 0.65±0.11a | 10.29±0.19b |
S5.构建Ⅱ型糖尿病小鼠模型
通过注射STZ诱导构建Ⅱ型糖尿病小鼠模型,根据小鼠的空腹血糖和餐后血糖值(空腹血糖>7.0mmol/L,餐后2h血糖>11.0mmol/L)可以判断Ⅱ型糖尿病小鼠模型造模成功,此时,正常组N组小鼠活动表现正常,精力充沛,毛发充足,白色顺滑,形态正常,小鼠饮食与饮水均正常,粪便为黑色;高血糖组小鼠均表现异常,精神萎靡,体态瘦小、毛色发黄易脱落,饮水与排尿量明显增多,粪便为灰色;
S6.小鼠空腹及餐后血糖
由图7和图8可知,空白对照M组的血糖增长速率远远大于其他各组;经过给药治疗和灌胃,阳性对照P组和SBC5实验组的空腹血糖和餐后血糖水平均显著低于M组,随着戊糖片球菌SBC5灌胃时间的增长,高血糖小鼠的血糖水平可以得到一定同程度降低和改善,且戊糖片球菌SBC5菌剂的缓解和改善效果优于发酵上清液;
S7.小鼠口服葡萄糖糖耐量
实验结束前一周各组小鼠灌胃葡萄糖溶液(2g/Kg),于15、30、60、90和120min时测定小鼠血糖的含量,通过图9和图10的各组糖耐受的结果可知,正常饲喂N组的糖耐受性好,血糖可以恢复到正常水平;高血糖M组血糖值高,耐受性差,阳性对照P组的耐受性接近于N组,说明治疗效果显著;SBC5实验组的糖耐受AUC值与M组相比差异性明显,血糖上升的速率和幅度也显著低于M组,下降速率比M组明显;说明戊糖片球菌SBC5或其发酵上清液对Ⅱ型糖尿病小鼠的糖耐量具有显著改善作用,戊糖片球菌SBC5菌剂的改善程度高于其发酵上清液;
由表10可知,喂食戊糖片球菌SBC5后可改善小鼠血清中糖基化血红蛋白HbA1c水平且不影响小鼠的脏器比。
SEQUENCE LISTING
<110> 河北科技大学
<120> 一种戊糖片球菌SBC5及其应用
<130> 20211101
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1513
<212> DNA
<213> 戊糖片球菌SBC5(Pediococcus pentosaceus)
<400> 1
aggctcagga tgaacgctgg cggcgtgcct aatacatgca agtcgaacga acttccgtta 60
attgattatg acgtacttgt actgattgag attttaacac gaagtgagtg gcgaacgggt 120
gagtaacacg tgggtaacct gcccagaagt aggggataac acctggaaac agatgctaat 180
accgtataac agagaaaacc gcatggtttt cttttaaaag atggctctgc tatcacttct 240
ggatggaccc gcggcgtatt agctagttgg tgaggtaaag gctcaccaag gcagtgatac 300
gtagccgacc tgagagggta atcggccaca ttgggactga gacacggccc agactcctac 360
gggaggcagc agtagggaat cttccacaat ggacgcaagt ctgatggagc aacgccgcgt 420
gagtgaagaa gggtttcggc tcgtaaagct ctgttgttaa agaagaacgt gggtaagagt 480
aactgtttac ccagtgacgg tatttaacca gaaagccacg gctaactacg tgccagcagc 540
cgcggtaata cgtaggtggc aagcgttatc cggatttatt gggcgtaaag cgagcgcagg 600
cggtctttta agtctaatgt gaaagccttc ggctcaaccg aagaagtgca ttggaaactg 660
ggagacttga gtgcagaaga ggacagtgga actccatgtg tagcggtgaa atgcgtagat 720
atatggaaga acaccagtgg cgaaggcggc tgtctggtct gtaactgacg ctgaggctcg 780
aaagcatggg tagcgaacag gattagatac cctggtagtc catgccgtaa acgatgatta 840
ctaagtgttg gagggtttcc gcccttcagt gctgcagcta acgcattaag taatccgcct 900
ggggagtacg accgcaaggt tgaaactcaa aagaattgac gggggcccgc acaagcggtg 960
gagcatgtgg tttaattcga agctacgcga agaaccttac caggtcttga catcttctga 1020
cagtctaaga gattagaggt tcccttcggg gacagaatga caggtggtgc atggttgtcg 1080
tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc ttattactag 1140
ttgccagcat taagttgggc actctagtga gactgccggt gacaaaccgg aggaaggtgg 1200
ggacgacgtc aaatcatcat gccccttatg acctgggcta cacacgtgct acaatggatg 1260
gtacaacgag tcgcgagacc gcgaggttaa gctaatctct taaaaccatt ctcagttcgg 1320
actgtaggct gcaactcgcc tacacgaagt cggaatcgct agtaatcgcg gatcagcatg 1380
ccgcggtgaa tacgttcccg ggccttgtac acaccgcccg tcacaccatg agagtttgta 1440
acacccaaag ccggtggggt aaccttttag gagctagccg tctaaggtgg gacagatgat 1500
tagggtgaag tct 1513
Claims (7)
1.一种戊糖片球菌SBC5,其特征在于:它保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏地址是北京市朝阳区北辰西路1号院3号,保藏日期为2021年1月5日,保藏编号为CGMCC NO.21595,拉丁文名称为Pediococcus pentosaceus。
2.根据权利要求1所述的戊糖片球菌SBC5的一种应用,其特征在于:所述戊糖片球菌SBC5通过发酵生产有机酸组合物;
所述发酵,培养基的碳源为蔗糖或葡萄糖;
所述有机酸组合物,包括乳酸、醋酸、柠檬酸、苹果酸、草酸、羟基苯乳酸、焦谷氨酸、甲酸、丙酸。
3.根据权利要求2所述的一种戊糖片球菌SBC5的应用,其特征在于:所述发酵,温度为33-37℃,pH值为6.0-7.0,时间为12-24h。
4.根据权利要求1所述的戊糖片球菌SBC5的一种应用,其特征在于:所述戊糖片球菌SBC5用于制备生物菌剂。
5.根据权利要求4所述的戊糖片球菌SBC5的一种应用,其特征在于:所述生物菌剂,是将戊糖片球菌SBC5进行发酵,离心,收集菌泥,加入保护剂,真空冷冻干燥后制备而成的。
6.根据权利要求5所述的戊糖片球菌SBC5的一种应用,其特征在于:所述离心,转速为4000-6000r/min,时间为8-15min;
所述保护剂,为脱脂乳,用量为菌泥体积的10-15%。
7.根据权利要求4-6中任一项所述的戊糖片球菌SBC5的一种应用,其特征在于:所述菌剂,用于制备食品、药品或饲料添加剂。
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