CN110100954A - 一种赭曲霉毒素a解毒剂的制备方法 - Google Patents
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Abstract
本发明公开一种赭曲霉毒素A解毒剂的制备方法。本发明涉及的微生态制剂由植物乳杆菌、两歧双歧杆菌菌液与陈皮粗提物混合而成。本发明可安全高效的解除粮食中的赭曲霉毒素A,且将含量严格控制在1 μg/kg以下,脱除率达到82%~95%。相比于各组分单独作用,微生态制剂能将脱除效率提升30%以上,还能起到保护人体和动物肝肾健康、缓解肠道炎症的重要作用。在本发明微生态制剂中,益生菌与益生元分别按照15%~85%的比例进行混合,之和为1。其中,植物乳杆菌和两歧双歧杆菌能够高效脱除赭曲霉毒素A,陈皮粗提物起到缓解肠道炎症、修复损伤的作用,本发明在对粮食中赭曲霉毒素A的脱毒具有广阔的应用前景。
Description
技术领域
本发明涉及复合微生态制剂领域,特别涉及一种赭曲霉毒素A解毒剂的制备方法。
技术背景
研究表明,动物摄入过量的赭曲霉毒素A(OTA)会引起肠黏膜炎症和坏死。而人体在食用体内含有OTA毒素的动物后,OTA进入人体也会对人体造成持续性损伤,增强肝脏、肾脏等器官对毒素代谢的负担。赭曲霉毒素(Ochratoxin)主要由多种曲霉及青霉菌产生,其中,毒性最大、与人类健康关系最为密切的为OTA。因此,缓解并解除OTA对人体和动物肠道的损伤是当前研究的重点。前期的研究发现,植物乳杆菌能够有效吸附OTA,并且在发酵过程中产生的酸可保护本发明中的微生态制剂耐受胃酸的侵蚀。本发明微生态制剂中,两歧双歧杆菌具备一定的OTA解毒功能,其分泌的抗菌肽能增加肠道黏液层厚度。陈皮作为一种药材,具有抗炎、抗氧化的功效,研究表明,适量的服用陈皮可起到降肝火、润肾燥的作用。
有报道指出,乳酸菌可能是通过物理作用将毒素吸附在细胞壁上,因此,益生菌的作用越来越多的得到了科研工作者们的关注。《湖南农业大学学报》,41(03):286-293指出植物乳杆菌BS22对黄曲霉毒素B1(AFB1)进行吸附后,可使得动物排出体外的AFB1含量增加。申请号201810554175.5公开了一种含有植物乳杆菌的降解毒素的制剂,该制剂能减少霉菌毒素侵染原料而造成的浪费。申请号201410564991.6公开了一种由枯草芽孢杆菌、植物乳杆菌等组成的霉菌毒素降解吸附剂,能够对饲料中的几种常见霉菌毒素有降解、吸附等多重作用。申请号201810842975.7公开了一种降解AFB1的发酵剂的制备方法,所制得的发酵剂由嗜热链球菌和双歧杆菌等菌剂混合,其降解产物安全并对动物体无毒害。上述的研究证实了植物乳杆菌、两歧双歧杆菌在对毒素的吸附及降解方面具有广阔的应用前景。
然而,仅仅依靠益生菌的作用来解除毒素是不够的,肠道中的微环境对益生菌的生长及活性均会造成不同程度的影响,使得益生菌在作用的过程中效率降低,因此,通常加入益生元来保护益生菌抵达肠道并促进益生菌的增殖。《食品工业》,39(09):232-236提出陈皮可促进乳酸杆菌的增殖,并且对出血性大肠杆菌有抑制作用。申请号201910000153.9公开了一种益生茶,陈皮、甘草等药材的配合能够清体内湿毒郁火,调养身体阴阳平衡。多种益生元的配伍不仅促进了益生菌的增殖,还起到了稳定肠道微生态环境,提高肠黏膜免疫力功能等作用。本发明中微生态制剂的制备对解除OTA,保持人和动物身体健康具有重要意义。
现有技术和手段用于OTA的吸附及降解已经日益展现出优势,但是仍有不够完善之处。因此本发明提出一种高效脱除OTA的方法,以益生元、益生菌为材料制成合生元。本发明的研究重点在于将植物乳杆菌、两歧双歧杆菌菌液与陈皮粗提物而制备一种高效脱除OTA的合生元。本发明中植物乳杆菌主要起到降解OTA的作用,其所代谢的天然防腐剂,如:乳酸、细菌素等能有效的保持微生态制剂的作用活性;两歧双歧杆菌可以抑制病原菌侵入肠道,避免毒素对人体造成二次伤害,在改善肝脏功能的基础上,两歧双歧杆菌还能起到一定的脱毒效果;陈皮粗提物能缓解肠道炎症,还能起到调节摄入毒素后食欲不振的现象。
发明内容
本发明提供一种赭曲霉毒素A解毒剂的制备方法。为实现上述目的,本发明所采用的技术方案是:
本发明中植物乳杆菌(Lactobacillus plantarum)和两歧双歧杆菌(Bifidobacterium bifidum)购于菌种保藏中心,对应编号为ATCC8014,ATCC29521。陈皮保存时间为1~3年。粮食来源于农场。
植物乳杆菌、两歧双歧杆菌菌液与陈皮粗提物三种物质按照一定的百分比混合制成复合制剂。
进一步,所述制备方案中,植物乳杆菌、两歧双歧杆菌菌液与陈皮粗提物分别按照15%~85%的体积比混合而成,之和为1。其中,植物乳杆菌、两歧双歧杆菌对OTA起到吸附并降解的作用,陈皮粗提物起到缓解肝脏、肾脏负担,调节肠道炎症的作用。
本发明一种赭曲霉毒素A解毒剂的制备方法包括以下步骤:
将植物乳杆菌、两歧双歧杆菌在适宜条件下活化并进行发酵扩大培养。在无菌条件下收集发酵液,获得两种菌株的混合发酵液,并与陈皮粗提物按照一定比例混合得到能够高效脱除OTA的合生元。
进一步,在降解粮食中的OTA时,微生态制剂与粮食的比例为1/80 L/kg~1/15 L/kg。
进一步,微生态制剂的作用温度为30℃~37℃,pH为4.5~7.5,吸附时间为12~26 h,在上述条件下,该微生态制剂对赭曲霉毒素A的脱除率可达到82%~95%,相比于各组分单独作用,该微生态制剂可将脱除率提升30%以上。
在解除粮食中的OTA时,OTA在发霉粮食中的初始含量均超过安全限2~5倍,利用国标法(GB 5009.96-2016)检测粮食中OTA的含量。本发明的微生态制剂能够将发霉粮食中的OTA含量降解到1 μg/kg以内。
本发明通过将植物乳杆菌、两歧双歧杆菌菌液与陈皮粗提物混合制备一种高效脱除赭曲霉毒素A的合生元,具备安全、高效降解粮食中OTA并减轻人和动物肾脏、肝脏负担,缓解肠道炎症等作用。首先植物乳杆菌对OTA起到吸附作用,其产生的酸可使微生态制剂耐受胃酸的腐蚀,并且生物防腐剂的合成能够有效维持微生态制剂的作用活性。其次人或动物的肠道中存在一定量的两歧双歧杆菌,在代谢毒素的过程中,菌的数量会有所减少,通过微生态制剂的服用,一方面可以良好的维持肠道内的平衡,另一方面,两歧双歧杆菌对OTA起到降解作用,其分泌的抗菌肽还可抑制产生内毒素的有害菌。作为微生态制剂的重要组成部分,陈皮粗提物不仅可以促进益生菌的生长,还具有缓解肠道炎症的效果。
本发明中的微生态制剂能够在多种条件下取得较好的使用效果,在高效脱除OTA的过程中免受肠道微环境及有害微生物的腐蚀和侵染,且具备极好的保鲜功能。
具体实施方式
下面结合实施例,对本发明的具体的实施方式进一步详细描述。以下实施例是用来进一步说明本发明的内容,而不限制本发明的保护范围。对于本发明所涉及的相关内容及所做的修改,均属于本发明保护范围。
实施例1:
将植物乳杆菌和两歧双歧杆菌分别接种于固体营养培养基进行活化,并在适宜条件下于液体发酵培养基中恒温发酵一定时间,发酵结束后在无菌条件下,将两种菌液按照25%、25%的比例混合均匀,离心获取菌体并重悬于无菌水中。将植物乳杆菌、两歧双歧杆菌菌液与陈皮水分别以25%、25%、50%的体积比混合均匀,从而得到一种高效脱除OTA的微生态制剂,备用。
用适量的陈皮水对微生态制剂进行稀释,并以喷洒的方式加入到OTA含量超出安全限2倍的发霉粮食中,搅拌混匀,使微生态制剂与粮食的添加量的比例为1/55 L/kg。将添加过微生态制剂的粮食在30℃,pH7.5条件下处理12 h后,检测粮食中OTA含量。实验结果显示,经过微生态制剂的高效解毒之后,OTA的含量为0.6 μg/kg,脱除率达到87%,相比于各组分单独作用,该复合制剂将脱除率提升了30%。
实施例2:
将植物乳杆菌和两歧双歧杆菌分别接种于固体营养培养基进行活化,并在适宜条件下于液体发酵培养基中恒温发酵一定时间,发酵结束后在无菌条件下,将两种菌液按照30%、40%的混合均匀,过滤收集菌体并重悬于无菌水中。将植物乳杆菌、两歧双歧杆菌菌液和陈皮煎煮液分别以30%、40%、30%的组分比混合均匀,从而得到一种高效脱除OTA的微生态制剂,备用。
用适量的陈皮煎煮液对微生态制剂进行稀释,逐滴滴加到OTA含量超出安全限2.5倍的发霉粮食中,搅拌混匀,使微生态制剂与粮食的添加量的比例为1/43 L/kg。将微生态制剂分2次每隔12 h加入到粮食中,在37℃,pH5.5下恒温处理24 h后,检测粮食中OTA含量。实验结果表明,经第一次处理后,OTA含量降低至1.5 μg/kg,第二次处理后,OTA含量控制在0.8 μg/kg,相比于各组分单独作用,OTA总脱除率提升了32%。
实施例3:
将植物乳杆菌和两歧双歧杆菌分别接种于固体营养培养基进行活化,并在适宜条件下于液体发酵培养基中恒温发酵一定时间,发酵结束后在无菌条件下,将两种菌液按照20%、25%的比例搅拌均匀,离心收集菌体并重悬于无菌水中。将陈皮打磨成粉,过100~120目筛,溶于陈皮煎煮液中。随后,将植物乳杆菌、两歧双歧杆菌菌液和陈皮溶液分别以20%、25%、45%的组分比混合均匀,从而得到一种高效脱除OTA的微生态制剂,备用。
用适量的陈皮煎煮液对微生态制剂进行稀释,分次流加至OTA含量超出安全限2倍的发霉粮食中,搅拌混匀,使微生态制剂与粮食的添加量的比例为1/72 L/kg。将微生态制剂分3次每个6 h添加到粮食中,于35℃,pH5.0下处理18 h后,检测粮食中OTA含量。实验结果表明,三次的OTA含量分别为2.2 μg/kg,1.5 μg/kg和0.46 μg/kg,相比于各组分单独作用,OTA总脱除率提升了36%。
实施例4:
将植物乳杆菌和两歧双歧杆菌分别接种于固体营养培养基进行活化,并在适宜条件下于液体发酵培养基中恒温发酵一定时间,发酵结束后在无菌条件下,将两种菌液按照30%、30%的比例充分混匀,过滤收集菌体并重悬于无菌水中。将植物乳杆菌、两歧双歧杆菌菌液和陈皮浸提液分别以30%、30%、40%的体积比混合均匀,从而得到一种高效脱除OTA的微生态制剂,备用。
用适量的陈皮浸提液对微生态制剂进行稀释,并将OTA含量超出安全限4倍的发霉粮食与微生态制剂搅拌混匀,使微生态制剂与粮食的添加量的比例为1/80 L/kg。将添加过微生态制剂的粮食在33℃,pH6.0条件下处理26 h后,检测粮食中OTA含量。实验结果表明,经过微生态制剂的高效解毒之后,OTA的含量降低为0.69 μg/kg,脱除率可达90%,相比于各组分单独作用,该复合制剂将脱除率提升了31%。
Claims (6)
1.一种赭曲霉毒素A解毒剂的制备方法,主要由植物乳杆菌、两歧双歧杆菌菌液与陈皮粗提物混合而成。
2.如权利要求1所述的高效脱除赭曲霉毒素A的微生态制剂,其特征在于由植物乳杆菌、两歧双歧杆菌菌液与陈皮粗提物分别按照15%~85%的体积比混合而成,之和为1。
3.如权利要求1和权利要求2所述的一种赭曲霉毒素A解毒剂的制备方法,在解除粮食中的赭曲霉毒素A时,微生态制剂与粮食的比例为1/80 L/kg~1/15 L/kg。
4.权利要求1~3所述方法制备的微生态制剂在温度为30℃~37℃,pH为4.5~7.5,吸附时间为12~26 h条件下对粮食进行作用。
5.权利要求1~4所述的微生态制剂,其特征在于该微生态制剂能将赭曲霉毒素A含量控制在1μg/kg以内,脱除率达到82%~95%,相比于各组分单独作用,该微生态制剂可将脱除率提升30%以上。
6.根据权利要求1~5所述的微生态制剂的应用,其特征在于该微生态制剂在粮食中解除赭曲霉毒素A中的应用。
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