CN114686415B - 凝结芽孢杆菌bc99在制备富硒药物或食品中的应用 - Google Patents
凝结芽孢杆菌bc99在制备富硒药物或食品中的应用 Download PDFInfo
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- CN114686415B CN114686415B CN202111586847.9A CN202111586847A CN114686415B CN 114686415 B CN114686415 B CN 114686415B CN 202111586847 A CN202111586847 A CN 202111586847A CN 114686415 B CN114686415 B CN 114686415B
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- bacillus coagulans
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Abstract
本发明属于微生物技术领域,尤其是涉及凝结芽孢杆菌BC99在制备富硒药物或食品中的应用。通过富硒驯化,凝结芽孢杆菌BC99菌体每克湿重中硒含量达101μg,而人体每天仅需几十微克,因此凝结芽孢杆菌BC99发酵菌体有用于人体有机硒补充食品药品的潜力。经富硒驯化后,其菌株DPPH清除率从67.01%提升到88.53%。本发明提供凝结芽孢杆菌BC99在制备富硒或抗氧化药品、食品中的应用。
Description
技术领域
本发明属于微生物技术领域,尤其是涉及凝结芽孢杆菌BC99在制备富硒药物或食品中的应用。
背景技术
硒是维持生命正常生长代谢的必须微量元素;已被联合国与农业组织(FAO)和世界卫生组织(WHO)共同确认为人体必须的一种重要微量矿质元素,与机体正常生理功能的维持以及多种疾病的发生发展密切相关,是哺乳动物体内多种酶(如谷胱甘肽过氧化物酶(GSH-Px)、磷脂氢谷胱甘肽过氧化物酶(GSH-Px2))活性中心的必需组分。适量的硒在清除体内过多的活性氧自由基,延缓衰老,抑癌抗癌,提高机体免疫力,解除重金属中毒,促进生殖能力方面具有显著效果。
硒在人体无法长期贮存,也无法合成,人体必须通过摄入谷物、水果、蔬菜、肉类等食物获得硒来满足机体需要。但这些食物不足以满足人体对硒的推荐摄入量(55ug/d)。据统计,我国2/3的人口居住在低硒或缺硒的地区,约有3亿人处于缺硒状态,因此,人体适量的补硒是非常必要的。补硒的来源主要有无机硒和有机硒;无机硒(如亚硒酸钠制成的口服制剂)不易被机体吸收,存在潜在毒性;而经无机硒生物转化后的有机硒安全性高,易吸收,环境污染小,受到广泛关注。
目前有机硒的获得主要有微生物转化法、植物天然转化法、种子发芽转化法、动物转化法等。微生物转化法中较多的是培养富硒酵母,目前在国内外已实现工业化生产。关于富硒酵母已进行了大量的研究,酵母菌主要将无机硒转化为硒代蛋氨酸,其插入蛋白的活性位点附近可能会影响蛋白的活性,因此硒代蛋氨酸有一定的不安全性。而乳酸菌能将无机硒转化为更安全的硒代半胱氨酸,它不会像硒代蛋氨酸那样随机插入机体蛋白,只能转化为H2Se,随后再转为HSePO3 2-合成硒蛋白,因此可安全的作为有机硒源用于膳食补充。近年来富硒乳酸菌已经成为乳酸菌领域一个的热点研究方向,有望取代富硒酵母成为首选的高效富硒微生态制剂,以应用于发酵食品来满足每日硒摄入量。
发明内容
鉴于现有技术存在的问题,本发明的目的在于针对现有技术中缺乏富硒及抗氧能力强且抗逆性好的乳酸菌的不足,提供具有富硒及抗氧化能力强的凝结芽孢杆菌BC99在制备富硒和抗氧化的药物或食品中的应用。
本发明提供具有富硒及抗氧化能力的凝结芽孢杆菌BC99,其微生物保藏号为CGMCC No.21801,分类命名为:凝结芽孢杆菌Bacillus coagulans;保藏时间:2021年02月01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;保藏地址:北京市朝阳区北辰西路1号院3号。该菌株首次公开在中国专利申请2021102858746中。
本发明所述凝结芽孢杆菌BC99耐热性能优异,具有较强的耐胃酸耐胆盐能力,具有较强的抑制病原菌的能力,对多种抗生素敏感。本发明所述的凝结芽孢杆菌BC99是通过30株凝结芽孢杆菌进行富硒能力筛选出来,表现出较强的富硒能力。进一步研究表明,富硒驯化后,能增强菌株BC99的抗氧化能力。
本发明所述的凝结芽孢杆菌BC99发酵后,菌泥硒含量能够达到101ug/g。其发酵液DPPH清除率从67.01%提高到88.53%。
本发明还提供富硒凝结芽孢杆菌BC99的制备工艺:
凝结芽孢杆菌菌种BC99从甘油管出发,接入试管斜面活化,经一级种子摇瓶(150ml/500mL)培养、二级种子摇瓶(150ml/500mL)培养后,进行5L发酵罐发酵,发酵罐中亚硒酸钠的加入浓度为20ug/mL,发酵时间20~24h,得到富硒发酵液,经12000rmp离心后收集富硒菌泥。如制备菌粉,可进行喷雾干燥喷粉后得到菌粉。
上述制备过程中,所用的种子培养基配方以重量百分比计,葡萄糖2.0%、酵母浸粉1.0%、蛋白胨1.0%、MgSO4·7H2O 0.03%、MnSO40.01%、NaCl 0.05%、K2HPO40.1%、FeSO4·7H2O 0.001%,CaCO30.05%,余量为水,调节初始pH值为7.0±0.1,115℃灭菌30min备用。
上述制备过程中,所用的发酵培养基配方以重量百分比计,葡萄糖3.0%、酵母浸粉3.5%、蛋白胨4.0%、MgSO4·7H2O 0.06%、MnSO40.01%、NaCl 0.1%、K2HPO40.2%、FeSO4·7H2O 0.002%,CaCO30.1%,余量为水,调节初始pH值为7.0±0.1,115℃灭菌30min备用。
本发明的另一目的提供凝结芽孢杆菌BC99在制备富硒或抗氧化药品或食品中的应用。所述富硒和或抗氧化药品或食品中含有本发明的富硒驯化后的凝结芽孢杆菌BC99的生物制剂。
所述食品包括固体饮料或液体饮料。
本发明的有益效果是:本发明凝结芽孢杆菌BC99具有较强的耐胃酸耐胆盐能力,对常见肠道致病菌具有较强的抑制作用,对多种抗生素敏感。通过富硒筛选,凝结芽孢杆菌BC99具有较强的富硒能力,凝结芽孢杆菌BC99菌体每克湿重中硒含量达101μg,而人体每天仅需几十微克,因此凝结芽孢杆菌BC99发酵菌体有用于人体有机硒补充食品药品的潜力。经富硒驯化后,其菌株DPPH清除率从67.01%提升到88.53%。本发明还提供凝结芽孢杆菌BC99在制备富硒或抗氧化药品、食品及饮料中的应用,具有非常广泛的应用前景。
附图说明
图1不同亚硒酸浓度下凝结芽孢杆菌的生长情况。
图2凝结芽孢杆菌BC99富硒浓度标准曲线。
图3凝结芽孢杆菌BC99的耐胃酸耐胆盐情况。
具体实施方式
下面结合实施例对本发明作进一步详细的描述,但本发明的实施方式不限于此。
下述实施例中涉及的培养基如下:
牛肉膏蛋白胨培养基:牛肉膏5g,蛋白胨10g,氯化钠5g,水1000ml,pH 7.0~7.2,118℃灭菌15min。
种子培养基:葡萄糖20g/L,蛋白胨10g/L,酵母浸粉10g/L,NaCl 0.5g/L,K2HPO41g/L,MgSO4·7H2O 0.3g/L,MnSO40.1g/L,FeSO4·7H2O 0.01g/L,CaCO3 0.5g/L,余量为水,pH7.0±0.1,115℃灭菌30min备用。
发酵培养基:葡萄糖3.0%、酵母浸粉3.5%、蛋白胨4.0%、MgSO4·7H2O 0.06%、MnSO40.01%、NaCl 0.1%、K2HPO40.2%、FeSO4·7H2O 0.002%,CaCO30.1%,余量为水,调节初始pH值为7.0±0.1,115℃灭菌30min备用。
固体培养基:在液体培养基的基础上添加1.5%的琼脂。
实施例1:耐硒凝结芽孢杆菌BC99的筛选
30株高效菌株来源于微康益生菌菌种资源库,通过公开的专利CN202110285880.1A中的筛选方法得到。
菌种活化:30株凝结芽孢杆菌菌种在牛肉膏蛋白胨固体培养基的平皿上划线,42℃恒温培养箱中培养48h,得到具有单菌落的平皿;挑取单菌落接入上述种子培养基中,42℃,250rpm摇床培养24h,得到种子液;
耐硒菌株筛选:上述种子液按照2%(V/V)的接种量接种到含有不同亚硒酸钠浓度(0、10、20、30、40μg/mL)的种子培养基中,42℃培养24h后,观察不同凝结芽孢杆菌菌体的颜色变化以及生长情况。结合菌体颜色变化和OD值筛选出耐硒能力强的凝结芽孢杆菌菌株。
菌体出现红色,说明菌体把一部分无机硒还原成单质硒,而不是转化成有机硒,随着亚硒酸钠质量浓度的升高,菌体转化出的单质硒会逐渐增多,颜色会越来越红。耐硒菌株初步定性判定,大部分菌株在10μg/mL亚硒酸钠浓度时,已经出现菌体颜色变红现象,而凝结芽孢杆菌BC99菌株在20μg/mL亚硒酸钠浓度时基本上未变色,在30μg/mL亚硒酸钠浓度时有略微变红。说明凝结芽孢杆菌BC99表现出较好的耐硒能力,耐亚硒酸钠浓度的范围是20-30μg/mL。
根据菌体的生长情况,如图1所示,随着亚硒酸钠浓度的升高,菌株的OD值逐渐降低,说明高浓度亚硒酸钠条件下会抑制菌体的正常生长代谢,但是不同菌株表现出不同耐受能力,不同浓度亚硒酸钠条件下,凝结芽孢杆菌BC99的生长OD值影响最小,在20μg/mL亚硒酸钠浓度时,基本上不影响。
结合菌体颜色变化和生长情况,凝结芽孢杆菌BC99表现出较强的耐硒能力。为避免过多无机硒转化为单质硒,确定20μg/mL的亚硒酸钠浓度为凝结芽孢杆菌BC99的最佳耐受浓度。
实施例2:凝结芽孢杆菌BC99富硒含量的测定
硒含量测定方法釆用国标“GB5009.93-2017食品中硒的测定”中的荧光分光光度法。
盐酸溶液(1%):量取5mL盐酸,用水稀释至500mL,混匀。
盐酸溶液(6mol/L):量取50mL盐酸,缓慢加入40mL水中,冷却后用水定容至100mL。
EDTA-氯化羟胺混合液:每250mL混合液中含EDTA二钠2.5g、氯化羟胺6.25g,用蒸馏水配置。
DAN试剂(1g/L):此试剂在暗室内配制。称取DAN0.2g于一带盖锥形瓶中,加入1%盐酸溶液200mL,振摇约15min使其全部溶解。加入约40mL环己烷,继续振荡5min。将此液倒入塞有玻璃棉(或脱脂棉)的分液漏斗中,待分层后滤去环己烷层,收集DAN溶液层,反复用环己烷纯化直至环己烷中荧光降至最低时为止(约纯化5次~6次)。将纯化后的DAN溶液储于棕色瓶中,加入约1cm厚的环己烷覆盖表层,于0℃~5℃保存。必要时在使用前再以环己烷纯化一次。
硒贮备液制备(1000mg/L):准确称量1g硒粉于50mL离心管,将离心管置于沸水浴中,逐步加入2mL浓硝酸、4mL高氯酸,迅速盖上离心管盖酸化50min,打开离心管盖10min后,加入6mol/L盐酸5mL,盖上管盖反应10min,将离心管取出,冷却至室温。转移至1L容量瓶,加入1%盐酸溶液定容。
硒标准溶液(5mg/L):准确吸取硒贮备液5mL至1L容量瓶中,加入1%盐酸溶液定容。
硒工作液(50μg//L):准确吸取硒标准溶液1mL至100mL容量瓶中,加入1%盐酸溶液定容。
标准曲线绘制:于暗室中分别准确量取0mL、1mL、2mL、4mL、8mL、10mL硒工作液100mL锥形瓶中,加水至30mL,用1%盐酸溶液调节pH至1.5~2.0;向锥形瓶中加入3mLEDTA-氯化羟胺混合液混匀;向锥形瓶中加入3mLDAN溶液,混匀;将锥形瓶转移至恒温水浴锅中,置沸水浴中加热5min,随后取出冷却至室温后,将样品转移至具塞试管中,准确加入10mL环己烷,剧烈震荡4min后,静置,待出现明显的分层后弃去水层;小心吸取2~3mL环己烷于比色皿中,置于酶标仪中,于激发光波长376nm、发射光波长520nm条件下测定荧光值。以硒质量为横坐标,荧光强度为纵坐标,制作硒标准曲线。计算出其线性回归方程为y=64.195x-11.976,相关系数为R2=0.9992,如图2所示。
(2)凝结芽孢杆菌BC99富硒含量测定
BC99从甘油管出发,接入试管斜面活化,经一级种子摇瓶(150ml/500mL)培养、二级种子摇瓶(150ml/500mL)培养后,进行5L发酵罐(发酵罐中亚硒酸钠的加入浓度为20ug/mL)发酵20~24h,发酵液离心,收集菌体,置于100ml的锥形瓶,加水至30mL,用1%盐酸溶液调节pH至1.5~2.0;后续步骤同硒标准曲线绘制过程,最终测得样品荧光值,代入硒标准曲线,换算可得硒含量。
实验结果表明:凝结芽孢杆菌BC99菌体每克湿重中硒含量达101μg,而人体每天仅需几十微克,因此凝结芽孢杆菌BC99发酵菌体有用于人体有机硒补充食品药品的潜力。
实施例3:BC99抗氧化能力的测定
(1)乳酸菌完整细胞IC的制备
将上述活化后的BC99及富硒后的BC99菌液经1000r/min离心收集菌体,PBS洗涤2次后重悬,调整菌数至109浓度,即为乳酸菌完整细胞(IC)悬浮液;
(2)DPPH清除实验
将0.8mL的样品(活菌数控制为109浓度)加入到1mL 0.2mmol/L新鲜配制的DPPH-无水乙醇溶液中,混匀,在黑暗中避光室温反应30min,10000r/min离心2min,取上清液在517nm波长处测定吸光值,记为As。空白组以等体积无水乙醇替换DPPH溶液测量吸光值,记为Ab;对照组以等体积蒸馏水(或PBS)替换样品溶液测量吸光值,记为Ac。
DPPH自由基清除率(%)=[1-(As-Ab)/Ac]×100
其中,As为样品组吸光值;Ac为对照组吸光值(蒸馏水替代样品);Ab为空白组吸光值(无水乙醇替代DPPH)。
实验结果表明:BC99菌株本身具有较强的抗氧化能力,其DPPH清除能力达到67.01%,经富硒驯化后,其抗氧化能力明显增强,其DPPH清除能力高达88.53%。
表1 BC99及其富硒后的DPPH清除能力实验结果
菌株编号 | BC99 | BC99-Se |
DPPH清除能力(%) | 67.01±1.05 | 88.53±1.17 |
实施例4:凝结芽孢杆菌BC99的模拟胃液和胆盐耐受性测定
A.模拟胃液试验
模拟胃液的配制:NaCl 0.5%、胃蛋白酶0.3%,用HCl溶液调整pH值为3.0,用0.22μm滤膜过滤除菌,4℃保存备用。
分别取凝结芽孢杆菌BC99的芽孢菌悬液1mL,离心、收集菌体,然后加入1mL的模拟胃液中,于37℃培养2.5h,用稀释平板法测定活菌总数(CFU/mL),并以0h测定值作为空白对照,计算存活率。
B.耐胆盐试验
耐胆盐培养基的配制:MRS培养基中添加0.2%(W/V)的巯基乙酸钠,并加入0.30%胆盐(W/V),118℃灭菌15min,冷却备用。
分别取凝结芽孢杆菌BC99的芽孢菌悬液1mL,离心、收集菌体,然后加入到1mL的0.3%胆盐培养基中,于37℃培养2.5h,用稀释平板法测定活菌总数(CFU/mL),并以0h测定值作为空白对照,计算存活率。
实验结果如图3所示,凝结芽孢杆菌BC99在体外模拟胃液及耐胆盐的耐受性测试中,其存活率均较高,基本上不受耐受压力的影响。
实施例5:凝结芽孢杆菌BC99抑制病原菌实验
指示菌株菌悬液准备:将大肠杆菌、金黄色葡萄球菌和沙门氏菌分别在LB固体平板培养基上活化,37℃培养24h,挑取菌苔至生理盐水制备菌悬液,调整菌悬液浓度至108CFU/ml
拮抗菌株菌液制备:将凝结芽孢杆菌按一定比例接种至液体MRS培养基中,42℃培养24h,得到培养液。
抑菌实验:将含有1.5%琼脂的肉汤培养基冷却至55℃左右,与指示菌株菌悬液按一定比例混匀,使得最终指示菌浓度在106CFU/mL,迅速倾注于预先放置牛津杯的平板中,待培养基冷却凝固后,取出牛津杯,在每孔中加入200μL拮抗菌液,以200μL无菌MRS液体培养基作为空白对照,静置数分钟后,放入37℃恒温培养箱中,培养适宜时间后观察,用游标卡尺测量抑菌圈直径,每个菌三个重复,结果见表2。
表2对病原菌的拮抗能力
表2结果表明,凝结芽孢杆菌BC99对大肠杆菌、金黄色葡萄球菌和沙门氏菌都有较强的抑制作用,其中对大肠杆菌的抑制作用最为显著。
实施例6:BC99的抗生素耐受性测定
凝结芽孢杆菌划线接种于MRS琼脂平板上,42℃培养48h后,得到单菌落。挑取单菌落至生理盐水中制备菌悬液,采用麦氏比浊法调整菌悬液浓度为108CFU/mL。取300ul菌悬液,用无菌棉签均匀涂布于整个MRS固体平板上,将抗生素药敏试纸片按照一定间距置于平板表面。37℃培养适宜时间后观察是否有抑菌圈。结果见表3。
表3凝结芽孢杆菌BC99对抗生素的敏感性
抗生素药敏片 | BC99 | 抗生素药敏片 | BC99 |
青霉素 | S | 链霉素 | S |
氨苄西林 | S | 庆大霉素 | S |
本唑西林 | S | 卡那霉素 | S |
阿莫西林 | S | 红霉素 | S |
头孢克洛 | S | 阿奇霉素 | S |
头孢曲松 | S | 四环素 | S |
头孢噻吩 | S | 万古霉素 | S |
新生霉素 | S | 甲氧嘧啶 | S |
新霉素 | S | 环丙沙星 | S |
克林霉素 | S | 利福平 | S |
供试菌株和20种常用抗生素进行药敏实验,表3结果表明,该菌株对20种抗生素都是敏感的,充分表明所筛选到的凝结芽孢杆菌BC99是具有富硒及抗氧化功能的安全益生菌株。
实施例7含有富硒及抗氧化功能的BC99制备胶囊制剂
凝结芽孢杆菌BC99菌种从甘油管出发,接入试管斜面活化,经一级种子摇瓶(150ml/500mL)培养、二级种子摇瓶(150ml/500mL)培养后,进行5L发酵罐发酵,发酵罐中亚硒酸钠的加入浓度为20ug/mL,发酵时间20-24h,得到富硒发酵液,经12000rmp离心后收集富硒菌体,菌体经喷雾干燥后,得到富硒及抗氧化功能的菌粉。
以重量份计,4-10份凝结芽孢杆菌BC99菌粉、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,采用通用方法配置成胶囊制剂。
本发明生产的胶囊制剂,硒载体是凝结芽孢杆菌BC99,由于具有富含硒及抗氧化,具有防癌抗癌功能。
实施例8:含有富硒及抗氧化功能的BC99制备固体饮料
称取菊粉40~50份、低聚果糖10~30份、脱脂乳粉5~10份、赤藓糖醇5~15份、麦芽糊精5~10份、蓝莓水果粉5~15份、按实施例7制备的富硒凝结芽孢杆菌BC99菌粉2~5份。
称取麦芽糊精按1:4~1:6重量比投入70℃温水中,充分搅拌至完全溶解后过80目筛,得到浆液待用;称取菊粉、低聚果糖、脱脂乳粉、赤藓糖醇、蓝莓水果粉,投入沸腾制粒机中,设置温度65℃,引风频率28Hz,混料20min至均匀,得到混合物料;更改设置沸腾制粒机温度至55℃,引风频率28Hz,将混合浆液在压力0.1MPa,流速80r/min条件下喷入沸腾制粒机中的混合物料中,喷浆时间50min至物料出现颗粒度均匀的状态,得到造粒体;更改设置沸腾制粒机温度65℃,引风频率25Hz,将造粒体干燥20min后停止加热,待料温降至50℃以下后收料;将所收料与富硒凝结芽孢杆菌BC99菌粉均匀混合后分装。
实施例9:含有富硒及抗氧化功能的BC99的面条制作
准备适量的面粉,食盐,富硒及抗氧化功能的凝结芽孢杆菌BC99菌粉,纯净水。将面粉、食盐、菌粉混合均匀。混合均匀的面粉加25-35wt%纯净水,进行揉搓和面,和面时间10-20分钟。将制成的初始面坯醒发10-15分钟,醒好的面坯放入压面机制成湿面条。将含有BC99的湿面条进行干燥,切断和包装。
实施例10:含有富硒及抗氧化功能的BC99果蔬饮料的制备
选用的新鲜蔬菜洗净后榨汁,接着进行高温瞬间灭菌,在温度140℃高温杀菌2秒后,立即降温至约37℃,再接入含有富硒及抗氧化功能的BC99菌粉,使其浓度达到106CFU/mL以上,在温度4℃冷藏保存,于是得到含本发明的富硒及抗氧化功能的BC99活菌果蔬饮料。
Claims (8)
1.一种富硒驯化的凝结芽孢杆菌BC99的制备方法,所述凝结芽孢杆菌BC99,其微生物保藏号为 CGMCC No .21801,其特征在于:
凝结芽孢杆菌菌种BC99从甘油管出发,接入试管斜面活化,经一级种子摇瓶培养、二级种子摇瓶培养后,进行5L发酵罐发酵,发酵罐中亚硒酸钠的加入浓度为20ug/mL,发酵时间20~24h,得到富硒发酵液,经12000rmp离心后收集富硒菌泥。
2.凝结芽孢杆菌BC99在制备富硒药品或食品中的应用,所述富硒药品或食品中含有按权利要求1所述方法富硒驯化后的凝结芽孢杆菌BC99的生物制剂。
3.根据权利要求2所述的应用,其特征在于,所述食品为固体饮料或液体饮料。
4.根据权利要求3所述的应用,其特征在于: 称取菊粉40~50份、低聚果糖10~30份、脱脂乳粉5~10份、赤藓糖醇5~15份、麦芽糊精5~10份、蓝莓水果粉5~15份、富硒凝结芽孢杆菌BC99菌粉2~5份;
称取麦芽糊精按1:4~1:6重量比投入70℃温水中,充分搅拌至完全溶解后过80目筛,得到浆液待用;称取菊粉、低聚果糖、脱脂乳粉、赤藓糖醇、蓝莓水果粉,投入沸腾制粒机中,设置温度65℃,引风频率28Hz,混料20min至均匀,得到混合物料;更改设置沸腾制粒机温度至55℃,引风频率28Hz,将混合浆液在压力0 .1MPa,流速80r/min条件下喷入沸腾制粒机中的混合物料中,喷浆时间50min至物料出现颗粒度均匀的状态,得到造粒体;更改设置沸腾制粒机温度65℃,引风频率25Hz,将造粒体干燥20min后停止加热,待料温降至50℃以下后收料;将所收料与富硒凝结芽孢杆菌BC99菌粉均匀混合后分装,得到富硒固体饮料。
5.根据权利要求2所述的应用,其特征在于:准备适量的面粉,食盐,富硒凝结芽孢杆菌BC99菌粉,纯净水;将面粉、食盐、菌粉混合均匀;混合均匀的面粉加25-35wt%纯净水,进行揉搓和面,和面时间10-20分钟;将制成的初始面坯醒发10-15分钟,醒好的面坯放入压面机制成湿面条,干燥,切断和包装,得到富硒面条。
6.根据权利要求2所述的应用,其特征在于:选用的新鲜蔬菜洗净后榨汁,接着进行高温瞬间灭菌,在温度140℃高温杀菌2秒后,立即降温至约37℃,再接入富硒BC99菌粉,使其浓度达到106 CFU/mL以上,在温度4℃冷藏保存,得到富硒果蔬饮料。
7.凝结芽孢杆菌BC99在制备抗氧化药品或食品中的应用,所述抗氧化药品或食品中含有按权利要求1所述方法富硒驯化后的凝结芽孢杆菌BC99的生物制剂。
8.根据权利要求7所述的应用,其特征在于,所述食品为固体饮料或液体饮料。
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