CN117016789A - 一种具有拮抗汞的富硒益生菌微生态制剂配方及其制备方法与应用 - Google Patents
一种具有拮抗汞的富硒益生菌微生态制剂配方及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种具有拮抗汞的富硒益生菌微生态制剂配方及其制备方法与应用。具有拮抗汞功能的益生菌微生态制剂,由复合益生菌冻干粉、活性多糖、菌粉膳食益生元组成;益生菌冻干粉以富硒副干酪乳杆菌,复配以植物乳杆菌、凝结芽孢杆菌、短双歧杆菌中的一种或几种。本发明中的益生菌微生态制剂能够显著降低人体内的汞水平,缓解由于汞富集导致的人体炎症刺激,并提升人体的硒水平,在拮抗汞方面具有广泛的前景。
Description
技术领域
本发明属于微生物制剂领域,尤其是指一种具有拮抗汞的富硒益生菌微生态制剂配方及其制备方法与应用。
背景技术
汞是一种剧毒的非必需元素,广泛存在于各种环境介质(水、土壤、大气)和生物圈(尤其是鱼类)中,形成了“汞循环”。汞是一种具有强烈神经毒性的元素,其元素(Hg)和汞化合物[HgCl2、(CH3)2Hg、CH3HgCl等]都含有毒性。长期摄入会对人体造成慢性毒性。自2017年8月16日起,《水俣公约》在中国正式生效。同年10月27日,世界卫生组织国际癌症研究机构公布了一份初步致癌物清单供参考。汞和无机汞化合物被列入三类致癌物清单。2019年7月23日,汞及其化合物被列入《有毒有害水污染物名录(第一批)》。
“汞循环”是生态系统中重金属元素的典型循环之一,汞在水、土壤、大气和生物圈中以离子状态不断迁移和转化。作为生态系统中唯一可以改善循环的重金属。汞排放到水体中后,就形成了汞污染。通过食物链,鱼类中甲基汞的浓度可能是水中甲基汞浓度的一万倍。在微生物的作用下,金属汞、二价离子汞等无机汞会转化为甲基汞和二甲基汞,称为汞的生物甲基化。甲基汞易被人体吸收,具有毒性大、放电周期长的特点。原因是:甲基汞易溶于脂质,不易在体内分解,而且由于其分子结构中的C-Hg键能高,不易断裂。
一般来说,摄入少量液态汞不会引起严重的毒性反应。一些研究表明,它会在生物体内形成有机化合物,但汞蒸气和溶解的汞盐(溶解度极低的汞盐除外,如硫化汞)具有剧毒,暴露、吸入和口服都会导致大脑干燥损伤。最危险的汞有机化合物是二甲基汞[(CH3)2Hg]。几微升的皮肤接触便会导致死亡。汞可以在生物体内积累,很容易被皮肤、呼吸道和消化道吸收。水俣病是汞中毒的一种。汞会损害中枢神经系统,并对口腔、粘膜和牙齿产生不利影响。长期暴露在高汞环境中会导致大脑损伤和死亡。尽管汞的沸点很高,但在室内温度下饱和的汞蒸气已达到毒性剂量的数倍。
硒是动物的必需营养素,也是植物的有益营养素。硒在自然界中以两种方式存在:无机硒和植物活性硒。无机硒一般指亚硒酸钠、硒酸钠等,可从金属矿的副产物中获得;后者是硒通过生物转化捕获含硫氨基酸(蛋氨酸、胱氨酸和半胱氨酸)的硫位置而形成的。
硒是人体必需的微量元素。它是心脏的重要组成部分,具有谷胱甘肽过氧化物酶(GPx)、碘甲(状)腺原氨酸脱碘酶(IDD)、硫氧还蛋白还原酶(TR)等多种酶活性,并具有抗氧化、保护心血管和心肌健康、抗肿瘤活性、调节免疫等多种生理功能,近年来备受关注。
硒是人体、动物和植物内循环中的一种关键营养元素。硒在拮抗重金属毒性方面的重要作用已被广泛研究和证明。重金属对生物体的毒性具有很长的积累周期,很难去除。硒拮抗汞,特别是甲基汞,缓解其神经毒性的机制受到了人们的特别关注。硒是抗氧化系统的重要组成部分,可以改善重金属引起的脂质过氧化,降低对生物体的毒性。同时,其他金属具有较强的亲和力,与重金属结合后可形成硒蛋白复合物,降低重金属的毒性。
研究表明,硒酵母对汞中毒引起的GPx活性下降具有显著的拮抗作用。由此可见,硒提高了抗氧化酶的活性,提高了机体的抗氧化能力。此外,硒拮抗Hg毒性的另一部分原因是它增强了机体的抗氧化能力,而主要原因是Se-Hg大分子复合物在体内形成,大大降低了游离Hg的毒性。也有专家从生物化学角度阐述了硒的解毒机制:硒蛋白P可以通过形成复合物来消除血液中Hg的毒性作用。例如:亚硒酸钠被红细胞选择性摄入后,代谢为还原形式的Se并进入血浆。离子Hg与还原形式的Se相互作用,形成(Hg-Se)n,然后与硒蛋白P上的阴离子中心和阳离子中心相互作用,形成无活性的[(Hg-Se)n]m-P复合物,再排出体外,达成解毒效果。
益生菌具有调节人体肠道微生物群的平衡、增强人体免疫力、预防疾病等生理功能,益生菌在人体中的作用具有广阔的发展前景。肠道微生物群是人类第二大基因组,由人类胃肠道细菌和其他微生物组成,在调节代谢和维持肠上皮细胞稳态方面发挥着重要作用。益生菌作为一类调节宿主微生物群组成并对宿主有益的活性微生物,在缓解通过食物来源暴露的重金属污染方面具有潜在价值。然而,益生菌作为肠道微生物群的组成部分,如何协调宿主与肠道微生物群之间的相互作用、维持宿主的健康、减轻重金属的毒性的机理仍然在研究之中。
研究表明:植物乳杆菌、凝结芽孢杆菌等具有吸附汞的能力。不仅能够在环境中对汞进行吸附,且在肠道内也能够发挥吸附作用,但不排除益生菌对汞的耐受性问题,因而需要即时补充外源性益生菌以发挥其解毒作用。因此通过筛选能够分离出耐汞乳酸菌成为潜在的汞吸附剂和解毒剂。而乳酸菌作为食品安全级别的益生菌,在食品中应用广泛,使用食品安全级的乳酸菌作为汞吸附剂添加到食品中或作为膳食补充剂,最终通过粪便将汞排出体外,防止汞在体内进行积累。此外,乳酸菌能否在人体肠道内环境中存活和定植是益生菌发挥其保健作用的重要前提之一。因此急需研发一种具有拮抗汞功能的高活性复合益生菌微生态制剂。
发明内容
为解决上述技术问题,本发明提供了一种具有拮抗汞的富硒益生菌微生态制剂配方及其制备方法与应用。本发明所得富硒益生菌微生态制剂配方具有高活性,在拮抗人体富集的汞方面具有潜在的应用价值,对汞元素的拮抗产生有益效果。
本发明的第一个目的在于提供一种具有拮抗汞的富硒益生菌微生态制剂配方,所述富硒益生菌微生态制剂的原料组成包括复合益生菌冻干粉、低聚半乳糖、低聚木糖、菊粉;所述复合益生菌冻干粉的复合益生菌选自富硒副干酪乳杆菌和/或其他菌株;所述其他菌株选自凝结芽孢杆菌、植物乳杆菌、短双歧杆菌、副干酪乳酪杆菌中的一种或多种。
进一步的,所述复合益生菌冻干粉的复合益生菌优选选富硒副干酪乳杆菌和其他菌株。
在本发明的一个实施例中,所述的富硒益生菌微生态制剂,各原料的含量:按质量百分数计,复合益生菌冻干粉含量不超过15wt%,菊粉含量不超过50wt%。
进一步的,各原料的组成含量优选为冻干菌粉10wt%、低聚半乳糖15wt%、低聚木糖35wt%、菊粉40wt%。
在本发明的一个实施例中,所述复合益生菌冻干粉通过以下方法制备得到:收集益生菌的稳定期的菌泥并清洗干净,将清洗过的菌泥与保护剂混合,得到菌体悬浮液,冻干,得到复合益生菌冻干粉。
进一步的,所述冻干的条件为:-45℃预冻2h,-30℃保持4h,-3℃保持6h,25℃保持4h,30℃保持8h。
进一步的,冻干后,在无菌环境下进行研磨,得到复合益生菌冻干粉,复合益生菌冻干粉采用无菌样品袋收集,置于-20℃以下的环境保存。
进一步的,所述复合益生菌冻干粉中富硒副干酪乳杆菌冻干菌粉含量大于50wt%。
进一步,所述复合益生菌冻干粉优选为富硒副干酪乳杆菌冻干菌粉、凝结芽孢杆菌冻干菌粉、植物乳杆菌冻干菌粉。其中,所述富硒副干酪乳杆菌冻干菌粉、凝结芽孢杆菌冻干菌粉、植物乳杆菌冻干菌粉的质量比为6:3:1。
在本发明的一个实施例中,所述保护剂通过以下方法制备得到:将脱脂乳粉溶解于水中,灭菌后,得到第一混合液;将蔗糖、海藻糖、水苏糖溶解于水中,灭菌,得到第二混合液;将混合液1和混合液2混匀,得到所述保护剂。
在本发明的一个实施例中,所述第一混合液和第二混合液的体积比为(1:1)-(4:1)。
在本发明的一个实施例中,所述蔗糖、海藻糖、水苏糖的质量比为(2-8):(1-6):(1-6)。
在本发明的一个实施例中,所述菌泥与保护剂的质量比为(1:2)-(1:5)。
本发明的第二个目的在于提供所述的富硒益生菌微生态制剂配方的制备方法,包括以下步骤:将低聚半乳糖、低聚木糖、菊粉混合,随后加入复合益生菌冻干粉,混合均匀,得到所述富硒益生菌微生态制剂。
本发明的第三个目的在于提供所述的富硒益生菌微生态制剂配方在制备拮抗汞制剂中的应用。
本发明的上述技术方案相比现有技术具有以下优点:
本发明基于富硒副干酪乳杆菌,由于其含有氨基酸硒成分,协同其他益生菌自身具有相应的吸附汞的效果,因此益生菌制剂具备良好的拮抗人体重金属汞有良好效果;通过对植物乳杆菌等益生菌的筛选及组合设计,结合多糖有益生菌的增殖因子,可以帮助更快的增殖,菊粉等益生元具有稳定肠道的功能,最终使得制备得到的益生菌制剂在拮抗人体重金属-汞方面有着极高的应用前景。
附图说明
为了使本发明的内容更容易被清楚的理解,下面根据本发明的具体实施例并结合附图,对本发明作进一步详细的说明,其中,
图1是本发明实施例1中益生菌的汞吸附含量对比。
图2是本发明实施例中不同时长摄入组毛发中汞含量的下降率。
图3是本发明实施例中人群毛发中硒与汞的摩尔质量浓度(μM/kg)的比值及其与阈值对比图。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好地理解本发明并能予以实施,但所举实施例不作为对本发明的限定。
下述实施例中涉及的材料如下:
副干酪乳杆菌CCFM 1089由苏州硒泰克生物科技有限公司授权使用。详细信息如菌株的形态、核苷酸序列已记载于专利CN111004753B中。
本发明中,涉及MRS培养基配方,如下:
MRS液体培养基的组成为:蛋白胨10g/L、牛肉浸膏10g/L、酵母提取物4g/L、葡萄糖20g/L、三水·乙酸钠2g/L、柠檬酸氢二胺2g/L、磷酸氢二钾2g/L、七水·硫酸镁0.1g/L、硫酸锰0.05g/L、吐温80 1g/L、L-半胱氨酸0.5g/L。
MRS固体培养基的组成为:蛋白胨10g/L、牛肉浸膏10g/L、酵母提取物4g/L、葡萄糖20g/L、三水·乙酸钠2g/L、柠檬酸氢二胺2g/L、磷酸氢二钾2g/L、七水·硫酸镁0.1g/L、硫酸锰0.05g/L、吐温80 1g/L、L-半胱氨酸0.5g/L、琼脂15g/L。
下述实施例中涉及的检测样品和检测方法如下:
1、测试指标的选择及要求
毛发作为人体内重金属排泄出体外的主要途径,毛发具有采集方便、运输便捷、前处理容易的优点。相对于生物液体能够更准确地反映人群长期接触的环境汞水平,较生物液体,毛发中的元素含量相对更高,而对于检测设备的要求不高。因此选用毛发能真实反映人群真正的汞接触水平及环境的蓄积程度。
样品:新生寒毛,长度不超过2cm或者头发,发根处向上2cm的新生发。
2、硒元素的测定
参照GB5009.93—2017食品安全国家标准食品中硒的测定中的第一法氢化物原子荧光光谱法测定。
3、汞元素的测定
参照GB5009.17—2021食品安全国家标准食品中总汞及有机汞的测定中的第二法直接进样测汞法。
4、冻干菌粉的制备方法为:乳酸菌活化、扩大培养、低温离心、低温洗涤,保护剂重悬,最终得到菌体悬浮液;菌体悬浮液经过预冻后冷冻干燥,得到冻干菌粉。
实施例1益生菌的筛选及汞吸附效果的对比
益生菌对于汞的拮抗效果体现在培养过程中对发酵液中汞元素的吸附。在上述液体MRS培养基中,加入汞标准溶液2mg/L;按照重量计2%接种量将富硒的副干酪乳酪杆菌(保藏编号为GDMCC No.60880)、长双歧杆菌WCFM1001、长双歧杆菌CCFM729、长双歧杆菌BL21、副干酪乳酪杆菌CCFM1089、干酪乳杆菌LC89、鼠李糖乳杆菌LRa05、鼠李糖乳杆菌CCFM1028、植物乳杆菌Lp05、植物乳杆菌CW006、植物乳杆菌CCFM639、嗜酸乳杆菌LA85、凝结芽孢杆菌BC99、分别接种于蓝盖瓶中,于37℃的恒温培养箱中培养24h,得到稳定期的菌液,对菌液进行离心、干燥测定其纯菌粉中汞元素的含量,其汞吸附效果的对比结果如图1所示。结果表明:富硒的副干酪乳酪杆菌较普通副干酪乳杆菌具有明显的汞拮抗优势,其原因应与其细胞内转化的硒蛋白相关;而在普通菌株中,凝结芽孢杆菌属、植物乳杆菌属也展现出较为良好的汞元素吸附效果,因此可用于配方的组合。
实施例2:一种具有拮抗汞的富硒益生菌微生态制剂配方及制备方法
本实施例提供了一种制剂配方及其配制方法,具体如下:
步骤1、冻干菌粉的制备,具体如下
(1)、稳定期菌液的培养方法
配方中的富硒副干酪乳杆菌按照专利CN111004753B所述培养基和方法进行培养;
并将配方中的富硒的副干酪乳杆菌(保藏编号为GDMCC No.60880)、凝结芽孢杆菌BC99、植物乳杆菌CW006的组合益生菌按照以MRS液体培养基的重量计2%接种量分别接种,在37℃的恒温培养箱中培养24h,可得到稳定期的菌液;
其中,培养基的制备方法为:
将蛋白胨10g/L、牛肉浸粉5g/L、酵母提取物4g/L、葡萄糖20g/L、三水·乙酸钠5g/L、柠檬酸氢二胺2g/L、三水·磷酸氢二钾2g/L、七水·硫酸镁0.5g/L、一水·硫酸锰0.2g/L、吐温80 1mL/L混合,加入去离子水混匀后,调节pH至6.2-6.4,115℃下灭菌15min,得到培养基。
步骤2、保护剂及其制备方法
将脱脂乳粉16wt%,溶解于500mL去离子水中,105℃灭菌10min后,得到脱脂乳粉混合液,置于4℃冰箱保存备用;同时,将蔗糖4%、海藻糖2%、水苏糖1%均溶解于500mL去离子水中,115℃灭菌20min,得到糖混合液。将脱脂乳粉混合液和糖混合液以体积比为1:1混匀,得到所述保护剂;
步骤3、菌体收集及冻干方法
从稳定期菌液中收集(低温离心的方法收集)的菌泥用已灭菌的去离子水反复清洗3次,按保护剂:菌泥的质量比为2:1的比例重新调配,即得菌体悬浮液;设置冻干机程序:-45℃预冻2h,-30℃保持4h,-3℃保持6h,25℃保持4h,30℃保持8h,得到冻干菌粉,于无菌环境下研磨,得到冻干纯益生菌菌粉,纯益生菌菌粉采用无菌样品袋收集,置于-20℃以下的环境保存。
步骤4、益生微生态制剂的制备
按质量百分数,将步骤3中所得三种冻干纯益生菌菌粉、低聚半乳糖、低聚木糖、菊粉混合,得到基于富硒益生菌的微生态制剂;其中,冻干菌粉、低聚半乳糖、低聚木糖、菊粉分别为10wt%、15wt%、35wt%、40wt%,其中,冻干纯益生菌菌粉由富硒副干酪乳杆菌冻干菌粉、凝结芽孢杆菌冻干菌粉、植物乳杆菌冻干菌粉三者组成,其质量比为6:3:1。复配时,先将低聚半乳糖、低聚木糖、菊粉三者以100rpm混合10min,冻干纯益生菌菌粉加入后,再次混合5min。得到复配后的富硒益生菌微生态制剂,活菌含量达5×1010CFU/g。
实施例3益生微生态制剂的经口急性毒性实验
取5周龄健康雄、雌性小鼠各10只,分为空白对照组和配方实验组。期间每间隔24小时进行一次灌胃[0.2g的膳食补充剂溶液(系指用无菌去离子水配制的益生微生态制剂溶液)稀释成的溶液0.2mL,以下实施例都采用此溶液作为配方组],连续喂养14日,期间记录小鼠的体重、摄食量及死亡状况,空白组不喂食膳食补充剂。
结果如表1和表2所示,膳食补充剂的喂养未对小鼠造成任何负面影响,小鼠正常生长,无死亡现象产生,且无任何病理症状。
表1喂食膳食补充剂对小鼠体重(g)的影响
表2喂食膳食补充剂对小鼠摄食量的影响
实施例4不同摄入时长对人群汞水平的影响及评价
为了验证该富硒益生菌微生态制剂在长周期条件下的拮抗效果。先后募集了2000名志愿者,其中有效样本1530例(其中:空白组284例,不同时长摄入组1246例;空白组期间不摄入任何益生菌或硒补充制剂,其他人群按照2g/天补充该富硒益生菌微生态制剂,下述组别中补充量均为:2g/天),各组别的测定结果如表3所示。
表3空白组与不同时长摄入组的毛发汞含量(mg/kg)
通过Mann-Whitney U检验,空白组与各个不同时长摄入组的毛发的汞含量存在显著差异(P<0.05),具有统计学意义;而空白组与摄入超过两年的组别其毛发的汞含量存在极显著差异(P<0.01)。
通过计算不同补充时间下的平均下降率,在上述的五个时间周期中,依次为0~2、3~6、7~12、13~24、>24~,其周期内的汞浓度的下降率依次为:19.0%、15.1%、19.8%、15.5%、23.4%。该结果表明,该复合富硒益生菌微生态制剂具有良好的拮抗汞的效果。
实施例5长时间(大于24个月)摄入膳食补充剂(富硒益生菌微生态制剂)对人群汞水平的影响及评价
通过连续稳态的跟踪摄入该产品24个月以上的人群(初始跟踪例:284人,24个月保持跟踪例:173人,数据获取率:60.9%),通过对毛发的硒检测、汞检测及可视化的数据分析,其结果如图所示。由图知,该富硒益生菌微生态制剂的拮抗汞的效果是显著的。
显然,上述实施例仅仅是为清楚地说明所作的举例,并非对实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
Claims (10)
1.一种具有拮抗汞的富硒益生菌微生态制剂配方,其特征在于,所述富硒益生菌微生态制剂配方的原料组成包括复合益生菌冻干粉、低聚半乳糖、低聚木糖、菊粉;所述复合益生菌冻干粉的复合益生菌选自富硒副干酪乳杆菌和/或其他菌株;所述其他菌株选自凝结芽孢杆菌、植物乳杆菌、短双歧杆菌、副干酪乳酪杆菌中的一种或多种。
2.根据权利要求1所述的富硒益生菌微生态制剂配方,其特征在于,所述复合益生菌冻干粉的复合益生菌选自富硒副干酪乳杆菌和其他菌株。
3.根据权利要求1所述的富硒益生菌微生态制剂配方,其特征在于,所述复合益生菌冻干粉含量不超过15wt%,菊粉含量不超过50wt%。
4.根据权利要求1所述的富硒益生菌微生态制剂配方,其特征在于,所述复合益生菌冻干粉通过以下方法制备得到:收集益生菌的稳定期的菌泥并清洗干净,将清洗过的菌泥与保护剂混合,得到菌体悬浮液,冻干,得到复合益生菌冻干粉。
5.根据权利要求3所述的富硒益生菌微生态制剂配方,其特征在于,所述保护剂通过以下方法制备得到:将脱脂乳粉溶解于水中,灭菌后,得到第一混合液;将蔗糖、海藻糖、水苏糖溶解于水中,灭菌,得到第二混合液;将混合液1和混合液2混匀,得到所述保护剂。
6.根据权利要求4所述的富硒益生菌微生态制剂配方,其特征在于,所述第一混合液和第二混合液的体积比为(1:1)-(4:1)。
7.根据权利要求4所述的富硒益生菌微生态制剂配方,其特征在于,所述蔗糖、海藻糖、水苏糖的质量比为(2-8):(1-6):(1-6)。
8.根据权利要求3所述的富硒益生菌微生态制剂配方,其特征在于,所述菌泥与保护剂的质量比为(1:2)-(1:5)。
9.权利要求1-8中任一项所述的富硒益生菌微生态制剂配方的制备方法,其特征在于,包括以下步骤:将低聚半乳糖、低聚木糖、菊粉混合,随后加入复合益生菌冻干粉,混合均匀,得到所述富硒益生菌微生态制剂。
10.权利要求1-8中任一项所述的富硒益生菌微生态制剂配方在制备拮抗汞制剂中的应用。
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