CN112195122A - 一种抑制肿瘤生长的富硒动物双歧杆菌及应用 - Google Patents
一种抑制肿瘤生长的富硒动物双歧杆菌及应用 Download PDFInfo
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- CN112195122A CN112195122A CN202011077654.6A CN202011077654A CN112195122A CN 112195122 A CN112195122 A CN 112195122A CN 202011077654 A CN202011077654 A CN 202011077654A CN 112195122 A CN112195122 A CN 112195122A
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- bifidobacterium animalis
- bifidobacterium
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- selenium
- freeze
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Abstract
本发明涉及一种抑制肿瘤生长的富硒动物双歧杆菌及应用,所述动物双歧杆菌的菌株编号为V9,分类命名为动物双歧杆菌Bifidobacterium animalis,保藏号为:CGMCC No 4473,保藏日期为2010年12月14日,保藏单位:中国微生物菌种保藏管理委员会普通微生物中心。本发明提供的动物双歧杆菌V9可进一‑步用于富硒培养,且具有良好的肠道定殖能力以及肠道保护和肝损伤保护功能,可以用于制备预防或治疗胃肠道疾病或肝类疾病产品,比如提取其活性成分制成冻干制剂用于制药,或利用其发酵功能制成食品。
Description
技术领域
本发明涉及一种抑制肿瘤生长的富硒动物双歧杆菌及应用,属于微生物技术应用领域。
背景技术
硒是人体必需的微量元素,是一种多功能的生命营养元素,其性质介于金属和非金属之间。适量的硒具有清除体内自由基、抗氧化、抑制过氧化脂质产生、增强免疫的功能,还有保护细胞膜、增强***调节、防止血凝块、清除胆固醇、促进细胞摄取糖、促进血液循环、防止皮肤老化、降低致癌物质的诱癌性、清除体内突变的癌细胞、防止癌细胞的***生长等功能。世界上有40多个国家的土壤中缺硒,我国有3/4的面积属缺硒地区,呈东北一-西南走向。调查研究结果显示,在低硒地区生活的人群,癌症发病率明显高于其他地区。
益生菌是一类能够对宿主产生有益作用的微生物,与人体的健康密切相关。益生菌一般定植于人体的肠道和生殖***内,对于维持人体内微生态平衡具有重要作用。已有的研究结果表明,人体微生物群的稳定对于维持人体健康非常重要,衰老、高血压、糖尿病及肠道疾病等都与人体微生物群有关。人体内的微生物群失衡,会直接或间接导致这些疾病的产生。双歧杆菌是人体中常见的一种生理性益生菌,定植于人体的胃肠道内,对于维持和调节人体内的肠道菌群十分重要。许多研究表明,以乳酸菌、双歧杆菌等益生菌制备得到的活菌制剂,可以代替抗生素,起到保护肠道,维持肠道平衡的作用, 从而防治腹泻、便秘等消化疾病,并具有许多明显的益生功效,如降糖降胆固醇、保护肝脏、增强免疫、抗癌等。根据最新的(2004年)伯杰细菌鉴定手册,基于16S rDNA测序技术得到的双歧杆菌共分成34个不同的种,包括青春双歧杆菌、角双歧杆菌、动物双歧杆菌、星状双歧杆菌、两歧双歧杆菌、牛双歧杆菌、短双歧杆菌、齿双歧杆菌、婴儿双歧杆菌(即,长双歧杆菌婴儿亚种)、乳双歧杆菌(即,动物双歧乳脂亚种)、长双歧杆菌、假小链双歧杆菌以及嗜热双歧杆菌和嗜酸热双歧杆菌,等等。
动物双歧杆菌(Bifidobacterium animalis),是人和许多哺乳动物肠道中的优势菌之一。在微生态学上属于菌群。1899年法国巴斯德研究院的蒂瑟尔(Tissier)首次从母乳喂养的婴儿大便中分离到了该菌,并指出它对乳儿的营养和预防肠道疾病具有重要作用。该菌是人和动物肠道中重要的生理性细菌。参与免疫、营养、消化和保护等一系列的生理过程,发挥着重要的功能。
大量动物实验表明,摄入动物双歧杆菌活菌或死菌均可提高机体的抗体水平,提高巨噬细胞的吞噬活性,这对提高机体的抗感染能力、预防、抑制和杀死肿瘤细胞的产生具有重要作用。肠道腐生菌在代谢中会产生许多致癌产物,如吲哚、胺、酚等,有的还能将一些致癌前体物转化为致癌物。例如,将偶氮化合物和芳香化合物还原成有致癌作用的N,N-二苯亚硝基化合物。动物双歧杆菌可通过抑制腐生菌的生长和分解致癌物起到防癌作用。另外,动物双歧杆菌能吸收和消除致癌物的毒害作用,抑制突变的致突变作用,有效减少潜在的癌症的发生,动物双歧杆菌体细胞壁的肽聚糖、脂磷壁酸和多糖都有抗肿瘤作用。
发明内容
本发明所要解决的技术问题是提供一种抑制肿瘤生长的富硒动物双歧杆菌及应用,可以制备预防或治疗胃肠道疾病或肝类疾病产品,解决上述技术问题。
本发明解决上述技术问题的技术方案如下:一种抑制肿瘤生长的富硒动物双歧杆菌,所述动物双歧杆菌的菌株编号为V9,分类命名为动物双歧杆菌Bifidobacteriumanimalis,保藏号为:CGMCC No 4473,保藏日期为2010 年12月14日,保藏单位:中国微生物菌种保藏管理委员会普通微生物中心,保藏单位地址为:北京市朝阳区北辰西路1号院3。
本发明还涉及一种所述抑制肿瘤生长的富硒动物双歧杆菌在制备预防或治疗胃肠道疾病或肝类疾病产品中的应用。
在上述技术方案的基础上,本发明还可以做如下改进。
进一步,所述产品为胶囊制剂或益生菌酸奶。
进一步,所述胶囊制剂由所述动物双歧杆菌V9的冻干制剂、硬脂酸镁、乳糖、淀粉和低聚异麦芽糖配置而成。
进一步,所述动物双歧杆菌V9的冻干制剂的制备方法为:将动物双歧杆菌V9在TPV培养基中37℃厌氧培养24h,生理盐水离心洗涤3次,得到动物双歧杆菌V9的湿菌体,将其与冻干保护剂按1:1混合后进行冷冻干燥,所述冻干保护剂包括海藻糖、脱脂奶粉和Vc-Na,其质量比为10:20:2,冷冻干燥参数为:压强10-50Pa,加热板温度为-40~-35℃,样品厚度为8-12mm;干燥时间在40-72小时,制备活菌的冻干制剂。
进一步,以重量份计,所述胶囊制剂由4-10份所述动物双歧杆菌V9的冻干制剂、0.3-0.6份硬脂酸镁、25-35份乳糖、40-50份淀粉和20-30份低聚异麦芽糖配置而成。
进一步,所述益生菌酸奶由所述动物双歧杆菌V9的菌种液与保加利亚乳酸杆菌、嗜酸乳杆菌和嗜热链球菌的混合菌种液在乳制品中发酵而成。
进一步,所述动物双歧杆菌V9的菌种液的制备方法为:取牛乳在 108-115℃下灭菌25-30分钟,冷却至40℃以下,按牛乳体积的1-5%的接种动物双歧杆菌V9种子液,在37-38℃下发酵培养24-30h,制得动物双歧杆菌V9的菌种液;所述动物双歧杆菌V9种子液由动物双歧杆菌V9在TPV培养基分离培养而得。
进一步,以重量份计,所述益生菌酸奶由1000份牛乳、40-60份白砂糖、 10-15份动物双歧杆菌V9的菌种液和8-12份混合菌种液混合发酵而成,其中混合菌种液中保加利亚乳酸杆菌、嗜酸乳杆菌和嗜热链球菌的体积比为 1:1:2。
进一步,以每克产品为基准,所述动物双歧杆菌的含量为106-1011CFU。
本发明的有益效果是:本发明提供的动物双歧杆菌V9可进一-步用于富硒培养,且具有良好的肠道定殖能力以及肠道保护和肝损伤保护功能,可以用于制备预防或治疗胃肠道疾病或肝类疾病产品,比如提取其活性成分制成冻干制剂用于制药,或利用其发酵功能制成食品。
具体实施方式
以下对本发明的原理和特征进行描述,所举实例只用于解释本发明,并非用于限定本发明的范围。
本发明所用的试剂、培养基组成以及实验学方法等在没有特别说明的情况下,均为本领域常规使用的试剂、培养基组成以及实验学方法。在没有特别说明的情况下,以下所测得的数值均是在实验条件下测定至少8个平行样品所得的平均值。
实施例1动物双歧杆菌V9的分离筛选
本发明动物双歧杆菌V9是从健康老人粪便中筛选出来的最优菌株,其具体筛选方法如下:
(一)乳酸菌的分离筛选
取采样对象新鲜中段粪便1g,加入10mL生理盐水充分混匀,室温静置5min,收集上层悬液。取上层悬液1mL,接种于50mL的TPV培养基中,37℃厌氧培养24h,得到培养液。
取培养液,用生理盐水梯度稀释10-1,10-2,10-3,10-4,10-5倍,得到菌悬液, 吸取0.1ml菌悬液涂布于BBL培养基上,37℃厌氧培养24h,得到单菌落。
挑取单菌落,将菌落培养物划线接种于BBL培养基上,37℃厌氧培养24h;再将长出的单菌落继续划线接种于BBL培养基上,37℃厌氧培养24h,连续分离纯化三次。将最后挑取的单菌落进行革兰氏染色,镜检为革兰氏阳性菌,长杆或顶部分叉状的纯化菌株,即为分离获得的纯培养物。
(二)筛选富硒动物双歧杆菌
将纯培养物分装至含有5μg/mL***钠培养基的96深孔板中,厌氧培养24小时,96深孔板每孔取200μL到96酶标板中进行0D600检测,0D值最高的3个孔稀释至105-106倍涂布到5μg/mL***钠平皿上。平皿涂布后放进厌氧培养箱培养18小时,待单菌落长出后,挑取点大且颜色浅的单菌落(说明该菌落在对应浓度的***钠培养基.上生长良好,且不易将***钠转化为红色的单质硒)于装有5μg/mL***钠培养基的96深孔板中培养。厌氧培养18小时后,每孔取培养液到96酶标板中进行0D600检测,0D 值最大的3个孔稀释105-106倍涂布到10μg/mL***钠平皿上。平皿涂布后放进厌氧培养箱培养18小时,待单菌落长出后,挑取点大且颜色浅的单菌落于装有10μg/mL***钠培养基的96深孔板中,厌氧培养18小时后,每孔取培养液到96酶标板中进行0D600检测,OD值最大的3个孔稀释105-106倍涂布到15μg/mL***钠平皿上,如此反复循环,直到800μg/mL***钠压力为止。最后获得硒压力耐受能力最强,生长情况最好的单菌落,划线获得纯培养,并进行菌种鉴定。
利用形态特征、培养性状和生理生化特征等微生物学特性以及16SrDNA 等分子生物学手段对该菌株鉴定为动物双歧杆菌,将其进行菌种保藏,编号为V9,分类命名为动物双歧杆菌Bifidobacterium animalis,保藏号为: CGMCC No 4473,保藏日期为2010年12月14日,保藏单位:中国微生物菌种保藏管理委员会普通微生物中心,保藏单位地址为:北京市朝阳区北辰西路1号院3号。
实施例2:利用动物双歧杆菌V9制备胶囊制剂
方案1:(1)制备动物双歧杆菌V9的冻干制剂,将动物双歧杆菌V9 在TPV培养基中37℃厌氧培养24h,生理盐水离心洗涤3次,得到动物双歧杆菌V9的湿菌体,将其与冻干保护剂按1:1混合后进行冷冻干燥,所述冻干保护剂包括海藻糖、脱脂奶粉和Vc-Na,其质量比为10:20:2,冷冻干燥参数为:压强10Pa,加热板温度为-40℃,样品厚度为8mm;干燥时间在40小时,制备活菌的冻干制剂,其活菌数达到5×1010CFU/g。
(2)制备胶囊制剂,以重量份计,取4份所述动物双歧杆菌V9的冻干制剂、0.3份硬脂酸镁、25份乳糖、50份淀粉和20份低聚异麦芽糖,采用通用方法配置成胶囊制剂。
方案2:(1)制备动物双歧杆菌V9的冻干制剂,将动物双歧杆菌V9 在TPV培养基中37℃厌氧培养24h,生理盐水离心洗涤3次,得到动物双歧杆菌V9的湿菌体,将其与冻干保护剂按1:1混合后进行冷冻干燥,所述冻干保护剂包括海藻糖、脱脂奶粉和Vc-Na,其质量比为10:20:2,冷冻干燥参数为:压强50Pa,加热板温度为-35℃,样品厚度为12mm;干燥时间在72小时, 制备活菌的冻干制剂,其活菌数为2×1010CFU/g。
(2)制备胶囊制剂,以重量份计,取10份所述动物双歧杆菌V9的冻干制剂、0.6份硬脂酸镁、35份乳糖、40份淀粉和30份低聚异麦芽糖,采用通用方法配置成胶囊制剂。
方案3:(1)制备动物双歧杆菌V9的冻干制剂,将动物双歧杆菌V9 在TPV培养基中37℃厌氧培养24h,生理盐水离心洗涤3次,得到动物双歧杆菌V9的湿菌体,将其与冻干保护剂按1:1混合后进行冷冻干燥,所述冻干保护剂包括海藻糖、脱脂奶粉和Vc-Na,其质量比为10:20:2,冷冻干燥参数为:压强30Pa,加热板温度为-38℃,样品厚度为10mm;干燥时间在60小时, 制备活菌的冻干制剂,其活菌数达到6×1010CFU/g。
(2)制备胶囊制剂,以重量份计,取6份所述动物双歧杆菌V9的冻干制剂、0.5份硬脂酸镁、30份乳糖、45份淀粉和25份低聚异麦芽糖,采用通用方法配置成胶囊制剂。
本发明生产的胶囊制剂,不但具有防癌抗癌功能,而且该制剂的硒载体选择动物双歧杆菌,此菌为人体正常生理细菌,在增进机体免疫力方面具有重要的功能。
实施例4:利用动物双歧杆菌V9制备胶囊制剂制备益生菌酸奶
方案1:(1)制备动物双歧杆菌V9种子液,将动物双歧杆菌V9在TPV 培养基中37℃厌氧培养24h,得到动物双歧杆菌V9种子液。
(2)制备动物双歧杆菌V9的菌种液,取牛乳在108℃下灭菌25分钟,冷却至40℃以下,按牛乳体积的1的接种动物双歧杆菌V9种子液,在37℃下发酵培养24h,得到动物双歧杆菌V9的菌种液。
(3)制备混合菌种液:取牛乳在108℃下灭菌25分钟,冷却至50℃以下,按牛乳体积的1接种保加利亚乳酸杆菌、嗜酸乳杆菌和嗜热链球菌的混合菌种子液(市售直接购买),其中嗜热链球菌、嗜酸乳杆菌及保加利亚乳杆菌体积之比为2:1:1,在37-43℃下发酵培养2h,制得含嗜热链球菌、嗜酸乳杆菌及保加利亚乳杆菌的混合菌种液;
(4)制备益生菌酸奶,将1000kg牛乳冷却至4℃后贮存,配制时在其中加入白砂糖40kg,在15Mpa下均质,于120℃下杀菌5秒或85℃下杀菌5 分钟,冷却至40℃后,加入含硒量为1.0g/L的***钠溶液0.1L和硒氨基酸转化与益生菌生长促进剂2L(按每升水中含有20g寡糖、2g蛋氨酸、2g 丝氨酸、2g半胱氨酸配制混合溶液),充分搅拌均匀,然后接入动物双歧杆菌V9的菌种液10L和混合菌种液10L,在40℃下发酵3.5h,控制酸度60T,最后在2℃下冷藏8h,即得到益生菌酸奶。
方案2:(1)制备动物双歧杆菌V9种子液,将动物双歧杆菌V9在TPV 培养基中37℃厌氧培养24h,得到动物双歧杆菌V9种子液。
(2)制备动物双歧杆菌V9的菌种液,取牛乳在115℃下灭菌30分钟,冷却至40℃以下,按牛乳体积的5%的接种动物双歧杆菌V9种子液,在38℃下发酵培养30h,得到动物双歧杆菌V9的菌种液。
(3)制备混合菌种液:取牛乳在115℃下灭菌30分钟,冷却至50℃以下,按牛乳体积的5%接种保加利亚乳酸杆菌、嗜酸乳杆菌和嗜热链球菌的混合菌种子液(市售直接购买),其中嗜热链球菌、嗜酸乳杆菌及保加利亚乳杆菌体积之比为2:1:1,在43℃下发酵培养6h,制得含嗜热链球菌、嗜酸乳杆菌及保加利亚乳杆菌的混合菌种液;
(4)制备益生菌酸奶,将1000kg牛乳冷却至4℃后贮存,配制时在其中加入白砂糖60kg,在25Mpa下均质,于140℃下杀菌15秒或95℃下杀菌 20分钟,冷却至50℃后,加入含硒量为1.0g/L的***钠溶液0.3L和硒氨基酸转化与益生菌生长促进剂10L(按每升水中含有100g寡糖、10g蛋氨酸、 10g丝氨酸、10g半胱氨酸配制混合溶液),充分搅拌均匀,然后接入动物双歧杆菌V9的菌种液20L和混合菌种液20L,在42℃下发酵6h,控制酸度 80T,最后在8℃下冷藏24h,即得到益生菌酸奶。
方案3:(1)制备动物双歧杆菌V9种子液,将动物双歧杆菌V9在TPV 培养基中37℃厌氧培养24h,得到动物双歧杆菌V9种子液。
(2)制备动物双歧杆菌V9的菌种液,取牛乳在110℃下灭菌28分钟,冷却至40℃以下,按牛乳体积的3%的接种动物双歧杆菌V9种子液,在37℃下发酵培养26h,得到动物双歧杆菌V9的菌种液。
(3)制备混合菌种液:取牛乳在110℃下灭菌28分钟,冷却至50℃以下,按牛乳体积的3%接种保加利亚乳酸杆菌、嗜酸乳杆菌和嗜热链球菌的混合菌种子液(市售直接购买),其中嗜热链球菌、嗜酸乳杆菌及保加利亚乳杆菌体积之比为2:1:1,在40℃下发酵培养4h,制得含嗜热链球菌、嗜酸乳杆菌及保加利亚乳杆菌的混合菌种液;
(4)制备益生菌酸奶,将1000kg牛乳冷却至4℃后贮存,配制时在其中加入白砂糖50kg,在20Mpa下均质,于130℃下杀菌10秒或90℃下杀菌 10分钟,冷却至45℃后,加入含硒量为1.0g/L的***钠溶液0.2L和硒氨基酸转化与益生菌生长促进剂5L(按每升水中含有60g寡糖、7g蛋氨酸、 6g丝氨酸、8g半胱氨酸配制混合溶液),充分搅拌均匀,然后接入动物双歧杆菌V9的菌种液15L和混合菌种液15L,在41℃下发酵5h,控制酸度70T,最后在5℃下冷藏16h,即得到益生菌酸奶。
每升所述益生菌酸奶中,所述动物双歧杆菌的含量为107-109CFU,含硒量为0.1-0.32mg,其中硒氨基酸含量在50%以上,主要组成为硒代蛋氨酸和硒代半胱氨酸。
本发明由于在牛乳中接入对人体有益的动物双歧杆菌、保加利亚乳酸杆菌、嗜酸乳杆菌和嗜热链球菌,通过合适的制备工艺,使本发明的酸奶中的动物双歧杆菌活菌数保持较高的水平,可达107cfu/g以上。
本发明利用动物双歧杆菌、保加利亚乳酸杆菌、嗜酸乳杆菌和嗜热链球菌,在酸奶发酵过程中,将添加于牛乳的无机硒转化为硒氨基酸,其转化率大于50%,且无机硒可显著促进动物双歧杆菌、保加利亚乳酸杆菌、嗜酸乳杆菌和嗜热链球菌的增殖。
本发明所制成的富含硒氨基酸的益生菌酸奶,可同时发挥酸奶、益生菌以及有机硒三者的综合保健功能;这显然能够更好的改善机体内生态环境、拮抗病原微生物、增强机体免疫功能、抵抗肿瘤、预防疾病。
尽管上面已经示出和描述了本发明的实施例,可以理解的是,上述实施例是示例性的,不能理解为对本发明的限制,本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
Claims (10)
1.一种抑制肿瘤生长的富硒动物双歧杆菌,其特征在于,所述动物双歧杆菌的菌株编号为V9,分类命名为动物双歧杆菌Bifidobacterium animalis,保藏号为:CGMCC No 4473,保藏日期为2010年12月14日,保藏单位:中国微生物菌种保藏管理委员会普通微生物中心。
2.一种根据权利要求1所述抑制肿瘤生长的富硒动物双歧杆菌在制备预防或治疗胃肠道疾病或肝类疾病产品中的应用。
3.根据权利要求2所述的应用,其特征在于,所述产品为胶囊制剂或益生菌酸奶。
4.根据权利要求3所述的应用,其特征在于,所述胶囊制剂由所述动物双歧杆菌V9的冻干制剂、硬脂酸镁、乳糖、淀粉和低聚异麦芽糖配置而成。
5.根据权利要求4所述的应用,其特征在于,所述动物双歧杆菌V9的冻干制剂的制备方法为:将动物双歧杆菌V9在TPV培养基中37℃厌氧培养24h,生理盐水离心洗涤3次,得到动物双歧杆菌V9的湿菌体,将其与冻干保护剂按1:1混合后进行冷冻干燥,所述冻干保护剂包括海藻糖、脱脂奶粉和Vc-Na,其质量比为10:20:2,冷冻干燥参数为:压强10-50Pa,加热板温度为-40~-35℃,样品厚度为8-12mm;干燥时间在40-72小时,制备活菌的冻干制剂。
6.根据权利要求4所述的应用,其特征在于,以重量份计,所述胶囊制剂由4-10份所述动物双歧杆菌V9的冻干制剂、0.3-0.6份硬脂酸镁、25-35份乳糖、40-50份淀粉和20-30份低聚异麦芽糖配置而成。
7.根据权利要求3所述的应用,其特征在于,所述益生菌酸奶由所述动物双歧杆菌V9的菌种液与保加利亚乳酸杆菌、嗜酸乳杆菌和嗜热链球菌的混合菌种液在乳制品中发酵而成。
8.根据权利要求7所述的应用,其特征在于,所述动物双歧杆菌V9的菌种液的制备方法为:取牛乳在108-115℃下灭菌25-30分钟,冷却至40℃以下,按牛乳体积的1-5%的接种动物双歧杆菌V9种子液,在37-38℃下发酵培养24-30h,制得动物双歧杆菌V9的菌种液;所述动物双歧杆菌V9种子液由动物双歧杆菌V9在TPV培养基分离培养而得。
9.根据权利要求7所述的应用,其特征在于,以重量份计,所述益生菌酸奶由1000份牛乳、40-60份白砂糖、10-15份动物双歧杆菌V9的菌种液和8-12份混合菌种液混合发酵而成,其中混合菌种液中保加利亚乳酸杆菌、嗜酸乳杆菌和嗜热链球菌的体积比为1:1:2。
10.根据权利要求3-9任一项所述的应用,其特征在于,以每克产品为基准,所述动物双歧杆菌的含量为106-1011CFU。
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