CN114574387B - 一株促进生长与生殖发育的高富集有机锌动物双歧杆菌 - Google Patents
一株促进生长与生殖发育的高富集有机锌动物双歧杆菌 Download PDFInfo
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- CN114574387B CN114574387B CN202210203177.6A CN202210203177A CN114574387B CN 114574387 B CN114574387 B CN 114574387B CN 202210203177 A CN202210203177 A CN 202210203177A CN 114574387 B CN114574387 B CN 114574387B
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- zinc
- bifidobacterium
- ccfm1230
- bifidobacterium animalis
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Classifications
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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- C—CHEMISTRY; METALLURGY
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- A—HUMAN NECESSITIES
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- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
本发明公开了一株促进生长与生殖发育的高富集有机锌动物双歧杆菌,属于微生物技术领域。本发明筛选得到的动物双歧杆菌(Bifidobacterium animals)CCFM1230能够将无机锌高效富集,转化为有机锌,被机体更好的吸收利用,且有效促进生长与生殖发育。经动物实验证明,富锌动物双歧杆菌能够促进雄性大鼠幼鼠的生长与生殖发育,发挥较无机锌更高的生物活性,达到补充锌的生理要求。使用本发明的动物双歧杆菌CCFM1230可对无机锌进行高效富集,用于制备促进生长与生殖发育的益生菌制剂,并在食品或医药领域具有巨大的应用前景。
Description
技术领域
本发明涉及一株促进生长与生殖发育的高富集有机锌动物双歧杆菌,属于微生物技术领域。
背景技术
锌是维持人体正常生长发育所必需的微量元素。人体含有2-3克锌,其中近90%存在于肌肉和骨骼中。健康成人每天从饮食中摄取约10-15mg锌,一般吸收率为20-30%。锌缺乏症被认为是世界范围内普遍存在的营养缺乏症,影响了全球约31%的人口,在发达国家和发展中国家都流行,尤其是在发展中国家。缺锌会导致生长迟缓和性腺机能减退。轻度至中度缺锌在世界范围内普遍存在,目前在我国一些贫困地区,由于动物性食物的摄入量低,对富锌的食物获取有限,出生后以植物性食物为主,植物性饮食中的植酸盐等会抑制锌的吸收,造成生长发育期长期锌缺乏,影响生长发育。
由于锌无法在人体内储存,为了维持机体所有功能的正常进行,需要每天补充锌,这就极易造成人体缺锌的状况发生。目前补锌主要是通过额外摄入含有机或无机锌的产品来实现的。市场上的锌补充剂分为三种:无机锌(ZnO、ZnSO4、ZnCl2等)、简单有机锌(葡萄糖酸锌、醋酸锌、丙酸锌等)和有机锌(氨基酸螯合锌、蛋白质络合锌等)。不同补锌剂的吸收效率不同。有研究表明,有机锌比无机锌更容易被人体吸收。无机锌吸收率低,副作用明显。单纯有机锌比无机锌吸收率高,但仍存在一定的刺激胃肠道等副作用。有机锌以人工合成为主,相对安全,但合成较为复杂。
微生物富集锌是近几十年来研究的热点,向培养基中添加无机盐使得微生物能够富集矿物离子,将微量元素富集在细胞表面或将它们转运到细胞内储存,它们以与氨基酸、蛋白质、脂类和多糖的复合物的形式存在,从而实现无机态微量元素向有机态微量元素的转化,为人体补充有机态微量元素提供了一个良好的膳食来源。目前利用微生物富集锌元素所用的菌株大多是酵母菌,较少用到双歧杆菌。相比之下,关于富锌双歧杆菌的报道很少,而双歧杆菌作为一类常见的并且对人体健康有益的微生物,同样具有富集金属离子的功能。而且双歧杆菌具有更多的益生特性,用双歧杆菌富集锌可能比用酵母菌富集锌具有更高的价值。富锌益生菌在补充锌的同时还可以调节肠道微生物。作为一种新型的含有活性益生菌的膳食锌源,富锌益生菌比益生菌或其他锌补充剂具有更多的优势,值得探索。与单独补充无机锌相比,有必要对富锌益生菌高效富集无机锌,转化为有机锌进行研究,开发出一种价格低廉、制备方法简单、安全可靠且补锌更高效的锌补充剂。
通过在培养过程中添加无机锌(非吸附),最终筛选出一株富锌量高且有机生物态含量高的动物双歧杆菌CCFM1230,其干菌粉锌含量能够达到3.8mg/g且有机锌转化率可达到95.3%。其余菌株的锌含量均不超过2.5mg/g。张青松等人对6株两歧双歧杆菌、青春双歧杆菌、动物双歧杆菌、短双歧杆菌进行了筛选,对锌的富集量在0.064mg/g~0.50mg/g之间不等。Leonardi等人测定了5株双歧杆菌(短双歧杆菌、婴儿双歧杆菌、假小链双歧杆菌)对锌的富集,发现双歧杆菌富集锌的量在0.975mg/g~2.08mg/g之间。在公开号为CN101971921B的专利申请文本中,胡文锋等人所述的双歧杆菌锌含量为0.35mg/g。在公开号为CN108220208B的专利申请文本中,王宝维等人所述的枯草芽孢杆菌锌含量为2.51mg/g。以上所述文献中双歧杆菌对锌的富集含量均较低,无法达到本专利菌株高富锌量、高有机锌转化率的理想效果。
发明内容
本发明提供了一株高富集有机锌的动物双歧杆菌CCFM1230,分类学命名为:动物双歧杆菌(Bifidobacterium animals),已于2022年2月11日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No:62248,保藏地址为广州市先烈中路100号大院59号楼5楼。
所述动物双歧杆菌CCFM1230具有如下特征:
(1)该菌株在MRS培养基上培养48h后呈小的、白色、不透明菌落;
(2)可将无机锌高效富集,转化为有机锌,能够被机体更好的吸收利用;
(3)菌株结果富锌发酵后,每克菌粉锌含量能够达到3.8mg以上,有机锌转化率可达到95.3%,活菌数能够达到4.73×109CFU/g以上。
本发明还提供了含有所述动物双歧杆菌CCFM1230,或含有动物双歧杆菌CCFM1230经富锌培养后所获得的细胞,或含有机锌的细胞裂解物的益生菌制剂。
在一种实施方式中,每g或每mL所述益生菌制剂中的有机锌含量≥3605.45μg。
在一种实施方式中,每g或每mL益生菌制剂中含有≥1×1010CFU/g或≥1×1010CFU/mL动物双歧杆菌CCFM1230或所述经富锌培养后所获得的细胞。
在一种实施方式中,所述细胞包括但不限于活细胞或死细胞;所述死细胞包括但不限于自然失去活性的细胞或经灭活处理后的细胞。
在一种实施方式中,所述富锌培养是将所述动物双歧杆菌CCFM1230在富锌培养基中培养至菌体数量≥1×108CFU/mL。
在一种实施方式中,所述富锌培养是将所述动物双歧杆菌CCFM1230在富锌培养基中培养一段时间;所述富锌培养基中的锌离子浓度为200~500mg/L。
在一种实施方式中,所述动物双歧杆菌CCFM1230经富锌培养后还经过干燥处理;干燥处理的方式包括但不限于:真空冷冻干燥、喷雾干燥、真空干燥、流化床干燥。
本发明还提供富锌动物双歧杆菌CCFM1230的制备方法,所述方法包括如下步骤:
(1)将所述的动物双歧杆菌在改良MRS固体培养基上划线,平板于37℃倒置培养36~48h,挑取单菌落接入到改良MRS液体培养基中于37℃培养24h。再以2%(v/v)的接种量接入到改良MRS液体培养基中37℃培养12~18h作为后续培养的种子菌液;
(2)将动物双歧杆菌的种子菌液以2%(v/v)的接种至富锌液体培养基中培养12~18h;
(3)发酵完成后将菌液在4℃条件下8000g/min离心20min,取湿菌体用纯水漂洗2次,得到富锌长双歧杆菌菌泥。
在一种实施方式中,所述步骤(2)中所述的富锌液体培养基中锌离子浓度为200~500mg/L。
在一种实施方式中,所述富锌液体培养基含有:葡萄糖20-30g/L,氮源15-25g/L(酵母浸粉、蛋白胨的质量比为1:2)、无水乙酸钠2g/L、柠檬酸氢二胺2g/L、K2HPO4·3H2O2.6g/L、MgSO4·7H2O 0.1g/L、MnSO4·7H2O 0.05g/L、吐温-80 1g/L、半胱氨酸0.5g/L、硫酸锌(按锌离子浓度为200~500mg/L换算添加)。
在一种实施方式中,所述富锌动物双歧杆菌菌泥还经过干燥处理,得到高富集有机锌的动物双歧杆菌菌粉。
在一种实施方式中,所述富锌动物双歧杆菌菌泥还经过任意干燥处理;所述干燥包括但不限于喷雾干燥、真空干燥、流化床干燥或真空冷冻干燥。
在一种实施方式中,所述富锌动物双歧杆菌菌泥经过灭活后再经任意干燥方式处理,得到无细胞活性的高富集有机锌的动物双歧杆菌CCFM1230菌粉;所述干燥使用蛋白或糊精作填充剂,或不使用任何填充剂。
本发明还提供应用所述方法制备的富锌菌粉。
本发明还提供所述动物双歧杆菌CCFM1230或所述益生菌制剂在制备食品、药品或保健品中的应用。
本发明还提供所述动物双歧杆菌CCFM1230或所述益生菌制剂在促进幼年哺乳动物生长和生殖发育方面的应用。
有益效果:
本发明提供了一株可高富集有机锌的动物双歧杆菌,该菌株能够将无机锌富集吸收并在菌体内转化为生物态锌,该菌株经过富锌培养后,每克菌粉锌含量能够达到3.8mg以上,有机锌含量可达到95.3%;菌粉中活菌数能够达到4.73×109CFU/g以上。无论有无活性,经该菌株富集生产的有机锌能够被机体更好的吸收利用,可有效促进哺乳动物的生长与生殖发育。
生物材料保藏
动物双歧杆菌(Bifidobacterium animals)CCFM1230,分类命名为Bifidobacterium animals,已于2022年2月11日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No:62248,保藏地址为广州市先烈中路100号大院59号楼5楼。
附图说明
图1为补锌对大鼠血清中ALP活性的影响;注:不同字母代表组别间具有显著性差异(*p<0.05,**p<0.01);
图2为补锌对大鼠血清中胰岛素样生长因子和睾酮浓度的影响;注:不同字母代表组别间具有显著性差异(*p<0.05,**p<0.01)。
具体实施方式
下面结合具体实施例对本发明进行进一步的阐述。
下述实施例中涉及的硫酸锌(产品货号:10024018、CAS:7446-20-0)购自国药集团化学试剂有限公司;硝酸(产品货号:yb2-308、CAS:7697-37-2)购于国药集团化学试剂有限公司。
下述实施例中涉及的培养基如下:
改良MRS液体培养基(g/L):蛋白胨10g/L、牛肉膏10g/L、酵母提取物5g/L、葡萄糖20g/L、无水乙酸钠2g/L、柠檬酸氢二胺2g/L、K2HPO4·3H2O 2.6g/L、MgSO4·7H2O 0.1g/L、MnSO4·7H2O 0.05g/L、吐温-80 1g/L、半胱氨酸0.5g/L、蒸馏水1000g/L。
改良MRS固体培养基(g/L):蛋白胨10g/L、牛肉膏10g/L、酵母提取物5g/L、葡萄糖20g/L、无水乙酸钠2g/L、柠檬酸氢二胺2g/L、K2HPO4·3H2O 2.6g/L、MgSO4·7H2O 0.1g/L、MnSO4·7H2O 0.05g/L、吐温-80 1g/L、半胱氨酸0.5g/L、琼脂20g/L、蒸馏水1000g/L。
富锌液体培养基(g/L):葡萄糖20-30g/L,氮源15-25g/L(酵母浸粉、蛋白胨的质量比为1:2)、无水乙酸钠2g/L、柠檬酸氢二胺2g/L、K2HPO4·3H2O 2.6g/L、MgSO4·7H2O 0.1g/L、MnSO4·7H2O 0.05g/L、吐温-80 1g/L、半胱氨酸0.5g/L、蒸馏水1000g/L、硫酸锌(按锌离子浓度为200~500mg/L换算添加)。
实施例1:动物双歧杆菌的筛选、菌种鉴定及保存
1、筛选
以来源于上海市的婴儿粪便为样本,用无菌生理盐水进行10倍梯度稀释至10-6,然后分别取100μL稀释倍数为10-4、10-5、10-6的稀释液于改良MRS固体培养基上进行平板涂布,37℃培养48h,观察并记录菌落形态;挑取改良MRS固体培养基上不同形态的菌落进行划线分离,经37℃培养48h后,再次挑取改良MRS固体培养基上不同形态的单菌落进行划线分离,直至得到形态一致的纯的单菌落;挑取改良MRS固体培养基上的纯菌落接种于含有硫酸锌的富锌液体培养基中,37℃培养18h;将菌液转移至无菌离心管中,8000g/min离心10min后弃去上层培养基,得到的菌泥漂洗2遍后冷冻干燥,得到富锌菌粉。使用原子吸收分光光度计检测菌粉中的锌含量,选出富锌能力较强的菌株。
2、鉴定
将分离得到的富集锌能力较强的菌株进行PCR扩增16S rDNA,PCR产物送至英潍捷基(上海)贸易有限公司进行测序,将测序得到的结果在NCBI中进行核酸序列比对,最终得到1株动物双歧杆菌,命名为动物双歧杆菌(Bifidobacterium animals)CCFM1230。
3、保存
将动物双歧杆菌(Bifidobacterium animals)CCFM1230接种于改良MRS液体培养基中,37℃培养18h;取1mL菌液于无菌离心管中,8000g/min离心10min后弃去上层培养基,菌泥重悬于30%甘油溶液中置于-80℃中保藏。
实施例2:富锌动物双歧杆菌的制备方法
(1)将实施例1筛选获得的动物双歧杆菌CCFM1230在改良MRS固体培养基上划线,平板于37℃倒置培养48h;挑取单菌落接入MRS液体培养基中于37℃培养24h;以2%(v/v)的接种量接入到改良MRS液体培养基中37℃培养12-18h作为后续培养的种子菌液。
(2)将步骤(1)获得的种子菌液以2%(v/v)的接种量接入到含富锌液体培养基的发酵瓶中培养12-18h。
(3)将步骤(2)发酵完成后的菌液在4℃条件下8000g/min离心20min,取湿菌体用纯水漂洗2次,以质量分数13%的脱脂乳作为冻干保护剂,将洗涤后的湿菌体与冻干保护剂以质量比1:1混匀,冷冻干燥,得到高富集锌的动物双歧杆菌菌粉,活菌数为4,73×109CFU/g菌粉,菌粉中的有机锌含量可达3605.45μg以上。
可选地,还可将富锌动物双歧杆菌灭活、经干燥处理制备菌粉,干燥方式可选用喷雾干燥或真空干燥或流化床干燥或真空冷冻干燥。
可选地,灭活高富集锌动物双歧杆菌菌粉可按如下方法制备:将前述步骤(2)发酵完成后将菌液在4℃条件下8000g/min离心20min,取湿菌体用纯水漂洗2次,将洗涤后的湿菌体进行喷雾干燥或真空干燥或流化床或真空冷冻干燥,使用蛋白或糊精作填充剂(菌泥与填充剂溶液质量比为1:1,填充剂溶液为质量分数13%的乳清蛋白或胶原蛋白或大豆蛋白或糊精溶液),得到无活性的高富集锌动物双歧杆菌菌粉,菌粉中的有机锌含量可达3605.45μg以上。
实施例3:富锌动物双歧杆菌锌含量及有机锌检测
1、富锌动物双歧杆菌锌含量检测
(1)微波消解
称取实施例2制备的动物双歧杆菌(Bifidobacterium animals)CCFM1230菌粉样品0.1g~0.15g于微波消解罐中,加入5mL硝酸后,进行微波消解。冷却后取出消解罐,在电热板上于140℃~160℃赶酸至1mL左右。消解罐放冷后,将消化液转移至25mL容量瓶中,用少量水洗涤消解罐2次~3次,合并洗涤液于容量瓶中,用水定容至刻度,混匀备用。同时做试剂空白试验。
(2)标准溶液配制
①锌标准储备液(1000mg/L):准确称取1.2447g(精确至0.0001g)氧化锌,加少量体积分数50%的硝酸溶液,加热溶解,冷却后移入1000mL容量瓶,加水至刻度,混匀。
②锌标准中间液(10mg/L)准确吸取锌标准储备液(1000mg/L)0.5mL于50mL容量瓶中,加体积分数为5%的硝酸溶液至刻度,混匀。
③锌标准系列溶液:分别准确吸取锌标准中间液0mL、0.5mL、1mL、2mL、4mL、和5mL于50mL容量瓶中,加硝酸溶液(5+95)至刻度,混匀。此锌标准系列溶液的质量浓度分别为0mg/L、0.1mg/L、0.2mg/L、0.4mg/L、0.8mg/L、1mg/L。
参照中华人民共和国国家标准GB 5009.14-2017中的第一种方法火焰原子吸收光谱法测量样品中的锌含量,其检测结果为3.8mg/g。
2、富锌动物双歧杆菌有机锌分析
准确称取0.5g的富锌动物双歧杆菌CCFM1230菌粉于烧杯中,加入45ml的蒸馏水,然后用稀酸或稀碱调节蒸馏水pH值至6.5,调好后用50ml容量瓶定容,定容蒸馏水的pH值也调至6.5,定容毕,将富锌动物双歧杆菌溶液转移至烧杯中,用玻璃棒缓慢匀速搅拌5~10min,搅拌毕,用8000r/min于室温离心15min。收集离心获得的上清液用于测量富锌动物双歧杆菌细胞表面水溶性锌(即无机锌)含量。沉淀中加入45ml的10mmol/L EDTA溶液,然后用稀酸或稀碱调节EDTA溶液pH值至6.5,调好后用50ml容量瓶定容,定容的EDTA溶液pH值也调至6.5,定容毕,将富锌动物双歧杆菌溶液转移至烧杯中,用玻璃棒缓慢匀速搅拌5~10min,搅拌毕,于8000r/min室温离心15min。收集离心获得的上清液用于测量富锌动物双歧杆菌细胞壁多糖及蛋白质络合的锌含量。第二次离心沉淀用于测量富锌动物双歧杆菌细胞内有机大分子或小分子结合锌含量。
有机化程度=(细胞壁多糖及蛋白质络合的锌含量+细胞内有机大分子或小分子结合锌含量)/总锌含量
参照中华人民共和国国家标准GB 5009.14-2017中的第一种方法火焰原子吸收光谱法测量各组分中的锌含量,其检测结果如下:
表1富锌动物双歧杆菌CCFM1230菌粉锌含量分析
由此可见,富锌动物双歧杆菌CCFM1230菌粉总锌含量达3.8mg/g,表明该动物双歧杆菌对锌的富集能力较强。无机态锌含量为4.7%,说明该动物双歧杆菌对无机锌的同化效果较好。有8.0%的锌以有机体的形式结合在细胞壁的多糖和蛋白质等大分子上;87.2%的锌与动物双歧杆菌细胞内有机大分子或小分子结合。采用相同方法检测无活性的高富集锌动物双歧杆菌菌粉中有机锌含量,结果显示灭活后的菌泥制备的菌粉中总锌含量为3.8mg/g,无机态锌含量为4.7%,有机态锌含量为95.3%。
对比例1:不同富锌双歧杆菌的富锌量和有机化程度
检索并收集不同来源的益生菌富锌培养后的锌含量。其中,两歧双歧杆菌O4、青春双歧杆菌W5、青春双歧杆菌HuNan-2016 MRS 11-2、动物双歧杆菌HuNan-2016 22-3、短双歧杆菌HuNan-2016 49-7、短双歧杆菌GuXi-2016 6-7、罗伊氏乳杆菌138-1、保加利亚乳杆菌MJ-1公开于论文《乳酸菌对锌的富集特性及富锌乳酸菌对小鼠结肠炎的缓解作用》;短双歧杆菌WC 0421、短双歧杆菌WC 0480、短双歧杆菌WC 0481、婴儿双歧杆菌WC 0460、假小链双歧杆菌WC 0455公开于论文《Zinc Uptake by Lactic Acid Bacteria》中;枯草芽孢杆菌NZ56公开于公开号为CN 108220208 B的专利中。
表2不同富锌双歧杆菌的锌含量对比
按照实施例2相同的方法培养表3所示的菌株,并检测培养18h后的锌含量。其中,短双歧杆菌F-JS-ZJ-1-M5、鼠李糖乳杆菌DG11-1、植物乳杆菌NFM11、干酪乳杆菌RS-2-1、发酵乳杆菌NT65-2为自行筛选获得的富锌菌株。
表3不同富锌双歧杆菌有机化程度对比
以上为已有文献或专利中所述的菌株,其对锌的富集含量和有机锌转化率均相对较低,无法达到本发明菌株的高富锌量、高有机锌含量的理想效果。
实施例4:富锌长双歧杆菌对雄性大鼠幼鼠生长及生殖发育的影响
1、造模:选取3周龄雄性SD大鼠幼鼠40只,随机分为正常组、缺锌组、无机锌组、富锌动物双歧组4组,每组5只。缺锌组、无机锌组、富锌动物双歧组用缺锌饲料TP0690-01G(1ppm)(订购于南通特洛菲饲料科技有限公司)缺锌喂养一周进行缺锌造模,正常组喂食对照饲料。
2、干预:从第2周开始按照1.0mL的灌胃量进行灌胃。
无机锌组在饲喂缺锌饲料的同时按照每日0.7mg Zn/只剂量的氧化锌溶液进行灌胃;
富锌动物双歧组在饲喂缺锌饲料的同时按照每日0.7mg Zn/只剂量的菌悬液(将按照锌含量计的实施例2制备的菌粉溶于生理盐水中)进行灌胃;
缺锌对照组饲喂缺锌饲料并灌胃等体积的生理盐水;
正常对照组饲喂对照饲料并灌胃等体积的生理盐水,灌胃两周。
3、实验结果:饲养期间定期观察大鼠反应,活动情况,精神状况,毛发变化,拍照记录大鼠形态变化,最后一次灌胃后24小时收集粪便,然后夜间禁食。次日对大鼠实施安乐死,采集血液、肝脏、睾丸、肾、胰。肝脏、睾丸、肾、胰组织在液氮中速冻并保存在-80℃。将血样吸入促凝管,离心后收集血清。样品在-20℃冷冻保存直至分析。我们的结果表明缺锌对幼鼠的生长和生殖发育有负面影响。不同的锌补充剂由于吸收利用的差异,对身体各项指标的恢复能力也不同。富锌动物双歧杆菌CCFM1230的生物利用度要高于无机锌,能够被机体更好的吸收利用。
表4大鼠身长、身宽
注:不同字母代表组别间具有显著性差异(p<0.05)
表5大鼠睾丸重量
注:不同字母代表组别间具有显著性差异(p<0.05)
表6大鼠组织中的锌含量
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
Claims (8)
1.一株高富集有机锌的动物双歧杆菌(Bifidobacterium animals)CCFM1230,已于2022年2月11日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No:62248。
2.一种益生菌制剂,其特征在于,含有(a)或(b):
(a)权利要求1所述的动物双歧杆菌CCFM1230;
(b)权利要求1所述动物双歧杆菌CCFM1230经富锌培养后的细胞。
3.根据权利要求2所述的益生菌制剂,其特征在于,所述细胞是活细胞或死细胞;所述死细胞是自然失去活性的细胞或经灭活处理后的细胞。
4.根据权利要求2或3所述的益生菌制剂,其特征在于,每g或每mL益生菌制剂中含有≥1×1010 CFU/g或≥1×1010CFU/mL动物双歧杆菌CCFM1230或所述经富锌培养后所获得的细胞。
5.根据权利要求2所述的益生菌制剂,其特征在于,所述富锌培养是将所述动物双歧杆菌CCFM1230在富锌培养基中培养至菌体数量≥1×108 CFU/mL。
6.制备权利要求1所述的动物双歧杆菌CCFM1230的方法,其特征在于,将权利要求1所述动物双歧杆菌CCFM1230在富锌培养基中培养至菌体数量≥1×108 CFU/mL;所述富锌培养基中锌离子浓度为200~500 mg/L。
7.根据权利要求6所述的方法,其特征在于,所述富锌培养基含有:葡萄糖 20-30g/L,氮源15-25g/L、无水乙酸钠2 g/L、柠檬酸氢二胺2 g/L、K2HPO4·3H2O 2.6 g/L、MgSO4·7H2O0.1 g/L、MnSO4·7H2O 0.05 g/L、吐温-80 1 g/L、半胱氨酸0.5g/L、硫酸锌;所述氮源包括质量比为1:2的酵母浸粉和蛋白胨。
8.权利要求1所述的动物双歧杆菌CCFM1230或权利要求2~5任一所述的益生菌制剂在制备改善生长发育功能的药品中的应用。
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