CN116333940A - 一株动物双歧杆菌的富硒培养方法与应用 - Google Patents
一株动物双歧杆菌的富硒培养方法与应用 Download PDFInfo
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Abstract
本发明涉及一种动物双歧杆菌的富硒培养方法与应用,所述富硒培养方法为:将所述动物双歧杆菌复活;再通过CM0231固体培养基活化;活化后将动物双歧杆菌接种至pH 7.5的CM0231液体培养基中,振荡培养得种子液;将种子液接种于pH 9的CM0231液体培养基中,振荡培养得到对数期菌液;将亚硒酸钠加入菌液中使菌液硒含量为100mg·L‑1,振荡培养,观察菌液颜色呈红色,即得到富硒动物双歧杆菌。本发明动物双歧杆菌可以应用于制备补硒食品、药物或者饲料,保证人体和动物对硒元素的需求。
Description
技术领域
本发明涉及微生物技术领域,尤其涉及可以将亚硒酸钠转化为生物利用度高的红色纳米硒的动物双歧杆菌的高效富硒培养方法与应用。
背景技术
硒已被联合国FAO/IAEA/WHO共同确认为人体必需的重要微量元素之一,对动物和人类的正常发育至关重要,具有抗氧化、提高机体免疫力等生理功能。硒(Se)被瑞典化学家Jakob Berzelius发现,最初被认为是一种有毒元素。随着对硒参与的各种生理反应的不断研究,发现硒是机体必不可少的膳食补充剂。世界卫生组织建议硒的摄入量为40~400μg/d。研究表明,在安全阈值范围内,硒不仅具有抗氧化、抗衰老、保护和修复细胞的活性,还可以增强机体的免疫系统和解毒功能,而在安全阈值外,缺硒可导致克山病和大骨节病,过量摄入硒则可能导致硒中毒。目前,补硒的方法大多仍是采用无机硒化合物即亚硒酸钠制成口服制剂,但亚硒酸钠具有较强的毒副作用。研究表明,无机硒经生物转化后形成有机硒化物,有机硒及纳米硒生物毒性更小、更有利于动植物吸收及安全利用,在激发免疫反应上较无机硒效果显著,而且吸收率更高,这对于我国推广并改善硒缺乏的现状具有重要意义。获得有机硒的途径中,有机硒及纳米硒可以通过物理和化学方法合成,但其生产成本高,产量低,在应用上受到了限制,所以不少学者在生物的吸收、转化上进行了大量探索。利用微生物实现无机硒转化为高营养价值的有机及纳米形式的硒,逐渐成为人们关注的焦点。
将益生菌等微生物作为补硒载体,同时起到保健和补硒两大功效。大多数补硒都是以无机硒(亚硒酸钠等)为原料,无机硒对微生物的生长有一定的抑制作用,同时未完全利用的无机盐被人体过量食用也会造成一定的危害。通过益生菌的发酵,将无机硒转化成有机硒,然后再被机体吸收利用,可以保证安全补硒。
益生菌是富硒微生物的主要研究对象,包括富硒酵母、富硒乳酸菌和富硒双歧杆菌。富硒益生菌具有多种益处,如抗氧化、抗致病、抗诱变、抗癌和抗炎活性。应用于食物中的富硒益生菌不仅能提供胃肠道保护屏障,而且能改变肠道环境pH提供非致病菌的增殖,从而增强宿主的免疫应答能力,提升抗氧化能力,产生抗菌物质与其在受体肠道中进行竞争,还能直接提供丰富的有机硒源(主要是硒蛋白)。首先,益生菌对硒的生物转化能力提供了人类和动物营养所需的廉价有机硒来源。其次,益生菌在肠道中长期存活后,可以从饮食中积累硒,从而影响宿主的硒摄入量,进而改变宿主中几种硒蛋白的表达,因此,可以从养生和营养元素补充两个角度满足人体对硒的需求。但现有的富硒酵母、富硒乳酸菌和富硒双歧杆菌富硒转化率均不高。
发明内容
要解决的技术问题:
本发明提出一种动物双歧杆菌高效富硒培养方法与应用,目的在于提供一种富硒转化率高的动物双歧杆菌的富硒培养方法,培养后的动物双歧杆菌能够高效的对亚硒酸钠转化为纳米硒,该菌能将无机亚硒酸钠转化为低毒、生物利用度高的红色纳米硒。
技术方案:
本发明的技术方案如下:
本发明第一方面提出动物双歧杆菌的富硒培养方法,该方法的具体步骤为:
a.将所述动物双歧杆菌复活,复活后转接至CM0231固体培养基上,37℃静置培养48h;
b.将步骤a所得的菌液通过CM0231固体培养基转接至2-3代活化;
c.将步骤b所得的动物双歧杆菌接种至pH 7.5的CM0231液体培养基中,在37℃、160rpm振荡培养12h,得到种子液;
d.将步骤c所得的种子液以3%(V/V)接种量接种于pH 9的CM0231液体培养基中,35℃摇床160rpm振荡培养9h,得到对数期菌液;
e.将亚硒酸钠加入至步骤d所得的菌液中使菌液硒含量为100mg·L-1,在35℃、160rpm条件下振荡培养24h,观察菌液颜色呈红色,即得到富硒动物双歧杆菌。
可选的,CM0231固体培养基成分每升为:蛋白胨10.0g、牛肉膏10.0g、酵母粉5.0g、葡萄糖5.0g、K2HPO4 0.45g、KH2PO40.33g、NH4Cl 1.0g、MgSO4 7H2O 0.1g、L-半胱氨酸0.5g、刃天青0.001g、Na2S 9H2O 0.5g、琼脂15g。
可选的,CM0231液体培养基成分每升为:蛋白胨10.0g、牛肉膏10.0g、酵母粉5.0g、葡萄糖5.0g、K2HPO4 0.45g、KH2PO40.33g、NH4Cl 1.0g、MgSO4 7H2O 0.1g、L-半胱氨酸0.5g、刃天青0.001g、Na2S 9H2O 0.5g。
本发明第二方面提出所述的培养方法培养的动物双歧杆菌在制备补硒食品、药品或者饲料中的应用。
可选地,所述补硒食品、药品或者饲料中包括动物双歧杆菌活菌株或干菌株。
可选地,所述补硒食品、药品包括药学上可接受的载体,所述载体为片剂、胶囊剂、口服液或冻干粉。
有益效果:
本发明开发一种动物双歧杆菌富硒方法,利用动物双歧杆菌作为补硒载体,提高其富硒能力,最终得到一株健康高营养的富硒动物双歧杆菌,在35℃培养箱160rpm振荡培养9h后加入亚硒酸钠,继续在该条件下培养24h使得动物双歧杆菌对亚硒酸钠转化率可达95.59%。本发明的动物双歧杆菌可应用于人体补硒的食品、药品中或动物饲料中,保证人体和动物对硒元素的需求。
附图说明
图1是富硒动物双歧杆菌培养工艺流程;
图2是硒标准曲线;
图3是动物双歧杆菌在不同初始pH的CM0231培养基转化Na2SeO324h后上清液未转化硒的浓度和转化效率;
图4是动物双歧杆菌在不同温度培养条件下转化Na2SeO324h后上清液未转化硒的浓度和转化效率。
具体实施方式
下面结合具体实施例及附图对本发明进行进一步说明。以下实施例用于说明本发明,但不用来限制本发明的范围。
实施例1:如图1所示,明确一株动物双歧杆菌(Bifidobacterium animalis)为优势富硒益生菌
一株可以转化亚硒酸钠的动物双歧杆菌(Bifidobacterium animalis),所述动物双歧杆菌Bifidobacterium animalis购买于中国工业微生物菌种保藏管理中心,保藏编号为CICC 6165,平台资源号:1511C0005000004655。所述动物双歧杆菌为优势富硒益生菌。所述动物双歧杆菌可进一步经富硒驯化培养,制备富硒动物双歧杆菌。
所述动物双歧杆菌的富硒培养方具体步骤为:
(1)菌种复活:取-20℃保存的动物双歧杆菌西林瓶,用75%酒精棉擦拭西林瓶表面进行消毒,用无菌吸管吸取0.5ml左右CM0231液体培养基于西林瓶中将冻干粉全部溶解,将溶解后的菌悬液转移至盛有4-5ml CM0231液体培养基的试管中混匀,残留在吸管中的1-2滴菌悬液转接至CM0231固体培养基上,37℃静置培养48h。
(2)菌种活化:将所得的菌液和平板转接至2-3代恢复活力。再将固体培养基平板上的动物双歧杆菌接种至pH 7.5的CM0231液体培养基中,37℃摇床160rpm振荡培养12h,得到种子液。
(3)配制亚硒酸钠储备液:首先称取2.19g亚硒酸钠溶于100mL去离子水中,配制10g·L-1的含硒储备液。
(4)转化验证:将种子液以3%(V/V)的接种量接种至装有pH9的CM0231液体培养基的厌氧瓶中,37℃摇床160rpm振荡培养9h,之后加入无菌处理的亚硒酸钠储备液(用无菌注射器将亚硒酸钠储备液经过无菌0.22μm的尼龙膜处理),37℃摇床160rpm振荡培养24h。由于亚硒酸钠溶液为无色而经生物转化后的纳米硒为红色,因此可以观察菌悬液培养前后颜色由无色转变为红色。
实施例2:动物双歧杆菌转化亚硒酸钠为纳米硒的富硒培养
(1)菌种复活:取-20℃保存的动物双歧杆菌西林瓶,用75%酒精棉擦拭西林瓶表面进行消毒,用无菌吸管吸取0.5ml左右CM0231液体培养基于西林瓶中将冻干粉全部溶解,将溶解后的菌悬液转移至盛有4-5ml CM0231液体培养基的试管中混匀,残留在吸管中的1-2滴菌悬液转接至CM0231固体培养基上,37℃静置培养48h。
(2)菌种活化:将所得的菌液和平板转接至2-3代恢复活力。再将固体培养基平板上的动物双歧杆菌接种至pH 7.5的CM0231液体培养基中,37℃摇床160rpm振荡培养12h,得到种子液。
(3)配制亚硒酸钠储备液:首先称取2.19g亚硒酸钠溶于100mL去离子水中,配制10g·L-1的含硒储备液。
(4)制备富硒动物双歧杆菌:将种子液以3%(V/V)的接种量接种至装有pH 9的CM0231液体培养基的厌氧瓶中,35℃摇床160rpm振荡培养9h,之后加入无菌处理的亚硒酸钠储备液(用无菌注射器将亚硒酸钠储备液经过无菌0.22μm的尼龙膜处理),35℃摇床160rpm振荡培养24h。由于亚硒酸钠溶液为无色而经生物转化后的纳米硒为红色,因此可以观察菌悬液培养前后颜色由无色转变为红色。
(5)动物双歧杆菌对亚硒酸钠的转化率
绘制硒标准曲线:取硒1000mg·L-1的标准溶液1mL用10%的稀盐酸定容至100mL,作为10mg·L-1硒标准中间液,硒标准中间液用10%的稀盐酸进一步稀释至1、2、5、10、20μg·L-1不同浓度梯度。用氢化法原子荧光光谱仪进行检测,仪器的还原液为15%的硼氢化钾水溶液。以浓度梯度为横坐标,荧光强度作为纵坐标绘制标准曲线,如图2所示。
对经过生物转化后的红色菌悬液进行离心,取上清液稀释到一定倍数经原子荧光光谱仪检测其中未转化硒的浓度Ct,仪器的还原液为15%的硼氢化钾水溶液。
上式中:
C0——动物双歧杆菌转化亚硒酸钠的初始硒浓度;
Ct——经过t时间后上清液中未转化硒的浓度。
实施例3:动物双歧杆菌在CM0231液体培养基初始pH不同的条件下对亚硒酸钠的生物转化情况
将动物双歧杆菌接种至CM0231液体培养基培养12h,作为种子液,动物双歧杆菌在CM0231液体培养基初始pH不同的条件下对亚硒酸钠的生物转化情况。将CM0231培养基分成7组,每组3个平行,在使用前用0.1mol·L-1HCl和0.1mol·L-1NaOH溶液校正CM0231培养基初始pH值,使7组CM0231培养基初始pH值分别为3、4、5、6、7、8、9。将3%的种子液接种至不同初始pH值的CM0231液体培养基中,置于温度为37℃,转速为160rpm的培养箱中连续发酵9h,随后加入无菌亚硒酸钠储备液使菌悬液中硒浓度为100mg·L-1,并继续发酵24小时,得到终点动物双歧杆菌菌液。对终点菌液进行离心分离上清液,并通过原子荧光分析仪检测上清液中未转化硒的浓度,进而确定动物双歧杆菌在不同pH情况下对硒浓度为100mg·L-1的亚硒酸钠的生物转化情况。
表1动物双歧杆菌在不同初始pH的CM0231培养基条件下对Na2SeO3转化率
如图3所示,在CM0231初始培养基pH为3~6时,亚硒酸钠经过动物双歧杆菌24h的转化上清液中未转化硒的浓度随pH的增大而减小,即亚硒酸钠的生物转化效果越好。当pH为6~9时,上清液中未转化硒的浓度随pH的上升而增大。因此在CM0231初始pH为9时动物双歧杆菌对亚硒酸钠的转化率最高,为94.84%。
实施例4:动物双歧杆菌在不同温度条件下对亚硒酸钠生物转化情况
将动物双歧杆菌接种于CM0231液体培养基培养12h,作为种子液,研究动物双歧杆菌在不同温度条件下对亚硒酸钠转化情况。将CM0231培养基分成5组,每组3个平行,将种子液以3%的接种量加入各组CM0231液体培养基中,各组分别在20、25、30、35、40℃培养箱中160rpm振荡培养9h,随后加入无菌亚硒酸钠储备液使菌悬液中硒浓度为100mg·L-1,使其继续生长24小时,得到终点动物双歧杆菌菌液。取终点菌液进行离心分离上清液,并通过原子荧光分析仪检测上清液中未转化硒的浓度,进而确定动物双歧杆菌在不同培养温度条件下对亚硒酸钠的生物转化情况。
表2动物双歧杆菌在不同温度培养条件下对Na2SeO3转化率
如图4所示,动物双歧杆菌在不同温度培养条件下转化Na2SeO324h,在温度为20~35℃时,亚硒酸钠经过动物双歧杆菌24h的转化后上清液中未转化硒的浓度随温度增大而减小,当温度为35~40℃时,上清液中未转化硒的浓度随温度增大而增大,35℃上清液中未转化硒的浓度最小,为4.41mg·L-1。因此在温度为35℃时动物双歧杆菌对亚硒酸钠的转化率最高,为95.59%。
综上,选择在35℃培养箱160rpm振荡培养9h后加入亚硒酸钠,继续在该条件下培养24h使得动物双歧杆菌对亚硒酸钠转化率为95.59%。
对比例1
徐洲等人(徐洲等,2017)通过对红发夫酵母的硒添加量、培养时间、装液量的研究,确定了培养的最适条件,在PDA培养基中红发夫酵母生长旺盛,分2次添加硒溶液将更利于有机硒的转化,最佳培养条件为:装液量80m L/500m L,硒添加浓度20mg/L,培养时间30h。在上述条件下,培养有机硒转化率达63.2%。本发明实施例4中动物双歧杆菌对亚硒酸钠转化率高达95.59%,高于对比例51.25%。
本发明的动物双歧杆菌菌株来源明确,安全健康,经富硒培养后可应用于制备补硒食品、药物或者饲料,保证人体和动物对硒元素的需求。具体可以应用于乳制品发酵、食品饮料,保健产品生产及动物饲料等行业。
以上对本发明的实例进行了详细说明,但所述内容不认为限定本发明的实施范围,对于本领域技术人员凡在本发明基础上依托本发明精神的进行修改和改进,均属于本发明的保护范围。
Claims (6)
1.一种动物双歧杆菌的富硒培养方法,其特征在于:该方法的具体步骤为:
a.将所述动物双歧杆菌复活,复活后转接至CM0231固体培养基上,37℃静置培养48h;
b.将步骤a所得的菌液通过CM0231固体培养基转接至2-3代活化;
c.将步骤b所得的动物双歧杆菌接种至pH 7.5的CM0231液体培养基中,在37℃、160rpm振荡培养12h,得到种子液;
d.将步骤c所得的种子液以3%(V/V)接种量接种于pH 9的CM0231液体培养基中,35℃摇床160rpm振荡培养9h,得到对数期菌液;
e.将亚硒酸钠加入至步骤d所得的菌液中使菌液硒含量为100mg·L-1,在35℃、160rpm条件下振荡培养24h,观察菌液颜色呈红色,即得到富硒动物双歧杆菌。
2.根据权利要求1所述动物双歧杆菌的富硒培养方法,其特征在于:CM0231固体培养基成分每升为:蛋白胨10.0g、牛肉膏10.0g、酵母粉5.0g、葡萄糖5.0g、K2HPO4 0.45g、KH2PO40.33g、NH4Cl 1.0g、MgSO4 7H2O 0.1g、L-半胱氨酸0.5g、刃天青0.001g、Na2S 9H2O 0.5g、琼脂15g。
3.根据权利要求1所述动物双歧杆菌的富硒培养方法,其特征在于:CM0231液体培养基成分每升为:蛋白胨10.0g、牛肉膏10.0g、酵母粉5.0g、葡萄糖5.0g、K2HPO4 0.45g、KH2PO40.33g、NH4Cl 1.0g、MgSO4 7H2O 0.1g、L-半胱氨酸0.5g、刃天青0.001g、Na2S 9H2O 0.5g。
4.如权利要求1所述的培养方法培养的动物双歧杆菌在制备补硒食品、药品或者饲料中的应用。
5.根据权利要求4所述的应用,其特征在于:所述补硒食品、药品或者饲料中包括动物双歧杆菌活菌株或干菌株。
6.根据权利要求4所述的应用,其特征在于:所述补硒食品、药品包括药学上可接受的载体,所述载体为片剂、胶囊剂、口服液或冻干粉。
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