CN115029284A - 一种活性益生菌及其在食品中的应用 - Google Patents
一种活性益生菌及其在食品中的应用 Download PDFInfo
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- CN115029284A CN115029284A CN202210831505.7A CN202210831505A CN115029284A CN 115029284 A CN115029284 A CN 115029284A CN 202210831505 A CN202210831505 A CN 202210831505A CN 115029284 A CN115029284 A CN 115029284A
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Abstract
本发明公开了一种活性益生菌,由特殊的发酵培养基发酵制备而成,该特殊的发酵培养基能显著的促进益生菌的增殖,本发明制备的活性益生菌具有药食同源、降血糖效果,将该活性益生菌应用于制备红树莓酵素中,能有效地提高红树莓酵素清除DPPH自由基的作用。
Description
技术领域
本发明涉及功能性食品技术领域,尤其涉及一种活性益生菌及其在食品中的应用。
背景技术
随着人民生活水平的不断提高,人们对均衡全面的营养摄入有了越来越充分的认识,对食品的品质、功能需求不断提高。其中具有一定营养保健功能、能够帮助预防和辅助治疗某些疾病并提高免疫力的食品,受到越来越多的关注。
益生菌是能够以活菌形式进入消化道并能够耐受胃中的酸性环境和肠道中的胆盐而定植下来从而对人体健康发挥有益作用的活性有益微生物的总称。其益生作用主要是通过直接或者间接的调整宿肠道微生物的组成,激活宿主内源性微生物群或者免疫系统的活性来实现。目前证实益生菌可以有效的治疗和缓解便秘、腹泻、炎症性肠病等疾病,缓解乳糖不耐受,促进基体营养吸收,降低血清胆固醇,还可预防癌症和抑制肿瘤生长等。此外,益生菌还可以增加生物体和人类健康所必需的重要化合物,如丁酸等短链脂肪酸。
糖尿病是一种由多种病因引起的以高血糖为特征的慢性代谢紊乱性疾病,病因涉及遗传、环境、免疫炎症等各方面,胰岛素耐受导致机体炎症应激的增加,肠道菌群与环境、遗传因素的相互影响导致了糖尿病的发生。目前治疗糖尿病多为口服降糖药物,如磺酰脲类、双胍类药物或注射促胰岛素分泌剂、胰岛素类似物或者胰岛血糖素抑制剂等。尽管其治疗效果明显,但是降血糖药物大多对肝脏、肾和胃肠道有一定的损害,因此,急切需要一种安全没有副作用或副作用较轻的治疗糖尿病的方案,尤其是从益生菌或其代谢产物中选择具有降糖功效的保健食品,目前成为治疗糖尿病发展的重要方向。
CN 111374256 A公开了益生菌组合物及其用途、食品和药品,所述益生菌组合物包括:短双歧杆菌;鼠李糖乳杆菌;以及乳双歧杆菌。本发明的益生菌组合物中三种菌可以协同共生,相互之间促进生长代谢,使其在肠道内发挥益生作用,该发明主要解决的技术问题是减轻体内炎症,促进脂肪代谢,从而达到减肥降脂功效。但是该发明的活均数并不高,活菌数的保质期也大大受到限制,因此也限制了该益生菌组合物在其它方面的应用。
发明内容
有鉴于现有技术的上述缺陷,本发明所要解决的技术问题是解决现有技术中降糖药物在治疗糖尿病过程中副作用较大的问题。
为实现上述目的,本发明提供了一种具有药食同源的、降血糖功效的活性益生菌,该活性益生菌具有较好的降糖效果,并将该活性益生菌应用于制备红树莓酵素,还提高了红树莓酵素的DPPH清除自由基效果。
实现上述发明目的,本发明采用了如下的技术方案:
一种活性益生菌的制备方法,其特征在于,包括如下步骤:
(1)菌株活化:固体培养基灭菌后接种罗伊氏乳杆菌,接种量为2-4wt%,于30-40℃下培养20-30h,得到活化菌株;
(2)扩大培养:扩大培养基灭菌后接种步骤(1)制得的活化菌株,接种量为2-5wt%,于温度为35-42℃、震荡速度为200-400rpm条件下震荡培养10-18h,调节溶液pH为6-7得到种子液;
(3)发酵:特种发酵培养基灭菌后接种种子液,接种量为2-5wt%,于30-35℃下无氧发酵25-30h,调节溶液pH为6-7,得到发酵物;
(4)菌泥收集及乳化:将步骤(3)得到的发酵物离心、弃上清液、收集菌泥;按质量比1:(2-3)将菌泥加入到冻干保护剂1中乳化,得到乳化液;
(5)制备冻干粉:将步骤(4)得到的乳化液冷冻干燥、粉碎,得到所述活性益生菌。
优选的,所述活性益生菌的制备方法,包括如下步骤:
(1)菌株活化:将2-7wt%葡萄糖、0.5-1wt%NaCl、2-3wt%琼脂粉、余量为水混合均匀制成的固体活化培养基加热至110-130℃灭菌10-30min、冷却至20-40℃,然后将罗伊氏乳杆菌在上述培养基中于30-40℃下培养20-30h,接种量为2-4wt%,得到活化菌株;
(2)扩大培养:将5-15wt%脱脂奶粉、2-5wt%菊粉、0.5-1.5wt%磷酸二氢钠、0.1-1wt%硫酸镁、余量为水混合均匀制成的扩大培养基加热至110-130℃灭菌10-30min、冷却至20-40℃,然后将步骤(1)制得的活化菌株接种于上述扩大培养基中,接种量为2-5wt%,于温度为35-42℃、震荡速度为200-400rpm条件下震荡培养10-18h,然后用2-3mol/L的碳酸氢钠水溶液调节溶液pH为6-7得到种子液;
(3)发酵:将特种发酵培养基加热至110-130℃灭菌10-30min、冷却至20-40℃,然后将种子液接种于特种发酵培养基中于30-35℃下无氧发酵25-30h,接种量为2-5wt%,然后用2-3mol/L的碳酸氢钠水溶液调节溶液pH为6-7,得到发酵物;
(4)菌泥收集及乳化:将上述发酵物置于转速为4000-8000rpm离心机中离心20-30min,弃掉上清液,收集菌泥,按质量比1:2-3将菌泥加入到冻干保护剂1中,在转速为200-300rpm下乳化20-40min,得到乳化液;所述冻干保护剂1由3-10wt%亚麻籽、1-3wt%食用明胶、0.5-2wt%益生元、2-6wt%维生素C、余量为水混合均匀制备而成。
(5)制备冻干粉:将乳化液置于-20~-60℃下冷冻2-5h,再置于真空温度为-40~-60℃、真空度为0.1-0.5mbar条件下干燥20-30h,粉碎过20-50目筛,得到所述活性益生菌。
所述特种发酵培养基由0.5-2wt%酪蛋白胨、0.05-1wt%硫酸锰、0.01-0.5wt%亚硒酸钠、余量为水混合均匀制成。
优选的,所述特种发酵培养基由1-5wt%绿豆肽、0.5-2wt%酪蛋白胨、0.05-1wt%硫酸锰、0.01-0.5wt%亚硒酸钠、余量为水混合均匀制成。
进一步优选的,所述特种发酵培养基由质量百分比为1-5wt%绿豆肽、2-5wt%胡柚提取物、0.5-2wt%酪蛋白胨、0.05-1wt%硫酸锰、0.01-0.5wt%亚硒酸钠、余量为水混合均匀制成。
最优选的,所述特种发酵培养基由质量百分比为1-5wt%绿豆肽、2-5wt%胡柚提取物、1-4wt%海带提取物、0.5-2wt%酪蛋白胨、0.05-1wt%硫酸锰、0.01-0.5wt%亚硒酸钠、余量为水混合均匀制成。
所述胡柚提取物的制备方法如下:
1)将胡柚果肉洗净干燥、粉碎后,取100-200重量份的胡柚果肉粉末,加入质量比为1:2-3的水,再加入果胶酶进行酶解,酶解温度为50-70℃,酶解时间为1-2h,酶解完成后用1-3mol/L的碳酸氢钠水溶液调节溶液的pH值为5-7得到混合液,果胶酶的用量为柚子果肉粉末质量的0.001-0.01倍;
2)按体积比1:0.2-0.4向上述混合液中加入的70-90wt%乙醇水溶液,在70-78℃条件下反应1-2h后冷却至20-40℃,离心收集上清液,将上清液减压蒸除溶剂后得到胡柚提取物。
本发明采用特种发酵培养基及发酵方法培育的益生菌活菌数较普通发酵方式明显提高。绿豆肽,富含各种氨基酸,尤其是赖氨酸、苯丙酸含量较高,还富含各种维生素(硫胺素、核、尼克酸、维生素E等)和各种微量元素(钾、钠、钙、镁、铁、猛、硒等),还含有许多生物活性物质,包括鞣酸、香豆素、生物碱,植物甾醇和黄酮类物质;胡柚是柚和酸橙的杂交后代,其香气独特、浓郁,呈鲜黄色,富有光泽,果肉呈金黄色、汁液丰富,具有清凉祛火、镇咳化痰、润喉醒酒等诸多药理功效;海带,是一种重要的海生资源,含有酸性聚糖类物质、岩藻半乳多糖硫酸酯、大叶藻素、半乳糖醛酸、昆布氨酸、牛磺酸、双歧因子等多种活性成分,还含有丰富的褐藻酸、纤维素、甘露醇及多种微量元素和大量的膳食纤维,能加速胃排空,降低肠腔pH值,促进胆汁酸代谢,降低血中胆固醇,提高胰岛细胞外周敏感性以降低血糖等作用。发明人通过大量的实验发现绿豆肽、胡柚提取物、海带提取物三者的协同能有效促进罗伊氏乳杆菌的增殖,还能够抑制杂菌的生长,提高益生菌的存活率。发明人采用特殊的冻干保护剂能包围在益生菌蛋白质分子周围,形成玻璃态,阻止了大分子物质的运动,阻碍了益生菌蛋白质三维空间结构的变化,同时又可保持蛋白质活性,从而保护细胞免受损伤,能进一步提高活性益生菌的存活率。
另外发明人发现用特殊培养基培养的罗伊氏乳杆菌益生菌有着优秀的单糖和混合单糖的利用率,能够抑制转运蛋白SGLT1和GLUT5基因的表达,从而减少葡萄糖、半乳糖、果糖从肠道中被肠黏膜细胞吸收进入血液,降低血糖浓度。发明人通过大量的实验发现本发明用特殊培养基培养的罗伊氏乳杆菌益生菌菌株可明显改善糖尿病小鼠的葡萄糖代谢,改善糖尿病小鼠的血糖水平和含量。
本发明制备的活性益生菌可以应用于食品中,更具体的应用为治疗糖尿病的食品中。
一种含活性益生菌的食品,其制备方法,包括如下步骤:
M1红树莓果浆预处理:将红树莓冻果解冻,用水清洗干净沥干,用榨汁机破碎打浆,充分搅拌均匀后置于在80-100℃下杀菌10-20min得到预处理后的红树莓果浆;
M2发酵:将预处理后的红树莓果浆加入到发酵罐中进行无氧发酵,接种上述制得的活性益生菌冻干粉,接种量为2-3wt%,发酵温度为25-35℃,发酵时间为20-24h,然后加热至110-130℃下灭菌10-30min,得到发酵的红树莓果浆;
M3制备红树莓酵素冻干粉:将发酵的红树莓果浆与冻干保护剂2按质量比为1:2-3混合均匀后置于-20~-60℃下冷冻2-5h,再置于真空温度为-40~-60℃、真空度为0.05-0.1Mpa条件下干燥40-48h,粉碎过10-40目筛,得到含活性益生菌的食品;所述冻干保护剂2由5-8wt%海藻糖、1-3wt%山梨醇、0.5-2wt%玉米面、余量为水混合均匀制备而成。
酵素是用一种或几种蔬菜、水果等材料经破碎榨汁后,添加单一或多种益生菌定向或非定向发酵而来,富含多种维生素、生物酶、矿物质和次生代谢产物等生物活性物质的发酵产品。益生菌在自身代谢的过程中,原材料发生复杂的中间代谢或交叉代谢,实现物质转化,产生高生理活性的物质和生物酶,即保留果实中原有的营养,又获得了新的活性物质,红树莓具有极高的抗氧化活性,加入本发明制备的活性益生菌发酵后,其生物活性物质与活性益生菌共同作用,对肠道菌群多样性产生积极影响。
与现有技术相比,本发明的有益效果:
1、本发明采用特定培养基能有效促进罗伊氏乳杆菌培育的益生菌菌株的增殖,且能提高益生菌菌株的存活率。
2、本发明采用特殊的冻干保护剂能防止益生菌在冷冻干燥中造成损伤,进一步提高活性益生菌的存活率。
3、本发明制备的活性益生菌能有效调节小鼠的血糖含量,达到降血糖的效果。
4、用本发明制备的活性益生菌制备的红树莓酵素提高了红树莓果浆的DPPH自由基清除效果。
具体实施方式
本发明中所用部分原料来源如下:
罗伊氏乳杆菌,购买自中国工业微生物菌种保藏管理中心,菌株保藏编号为:CICC6126。
绿豆肽,购买自西安汇林生物科技有限公司,总蛋白质含量≥99%,肽含量≥80%,分子量为130-750D,外观为类白色粉末。
胡柚,产地浙江常山。
海带提取物,购买自扶风慈缘生物科技有限公司,外观为棕色粉末,为水提物。
果胶酶,购买自惠州源乐生物科技有限公司,酶活力:10万U/g,CAS号为9032-75-1。
对比例1
一种含活性益生菌的食品,其制备方法,包括如下步骤:
M1红树莓果浆预处理:将红树莓冻果解冻,用水清洗干净沥干,用榨汁机破碎打浆,充分搅拌均匀后置于在80℃下杀菌15min得到预处理后的红树莓果浆;
M2发酵:将预处理后的红树莓果浆加入到发酵罐中进行无氧发酵,接种活性益生菌冻干粉,接种量为2wt%,发酵温度为28℃,发酵时间为24h,然后加热至110℃下灭菌10min,得到发酵的红树莓果浆;
M3制备红树莓酵素冻干粉:按质量比为1:2将发酵的红树莓果浆与冻干保护剂2混合均匀后置于-40℃下冷冻4h,再置于真空温度为-40℃、真空度为0.08Mpa条件下干燥48h,粉碎过30目筛,得到含活性益生菌的食品;所述冻干保护剂2由6wt%海藻糖、2wt%山梨醇、1wt%玉米面、余量为水混合均匀制备而成。
所述活性益生菌的制备方法,包括如下步骤:
(1)菌株活化:将5wt%葡萄糖、0.8wt%NaCl、3wt%琼脂粉、余量为水混合均匀制成的固体活化培养基加热至110℃灭菌20min、冷却至35℃,然后将罗伊氏乳杆菌在上述培养基中于38℃下培养30h,接种量为3wt%,得到活化菌株;
(2)扩大培养:将10wt%脱脂奶粉、4wt%菊粉、1.0wt%磷酸二氢钠、0.5wt%硫酸镁、余量为水混合均匀制成的扩大培养基加热至110℃灭菌20min、冷却至35℃,然后将步骤(1)制得的活化菌株接种于上述扩大培养基中,接种量为4wt%,于温度为35℃、震荡速度为300rpm条件下震荡培养18h,然后用3mol/L的碳酸氢钠水溶液调节溶液pH为7得到种子液;
(3)发酵:将3wt%绿豆肽、2wt%胡柚提取物、1.5wt%酪蛋白胨、0.2wt%硫酸锰、0.2wt%亚硒酸钠、余量为水混合均匀制成的特种培养基加热至110℃灭菌20min、冷却至35℃,然后将种子液接种于特种发酵培养基中于35℃下无氧发酵30h,接种量为4wt%,然后用3mol/L的碳酸氢钠水溶液调节溶液pH为7,得到发酵物;
(4)菌泥收集及乳化:将上述发酵物置于转速为6000rpm离心机中离心30min,弃掉上清液,收集菌泥,按质量比1:2将菌泥加入到冻干保护剂1中,在转速为200rpm下乳化30min,得到乳化液;所述冻干保护剂1由8wt%亚麻籽、2wt%食用明胶、1.5wt%益生元、5wt%维生素C、余量为水混合均匀制备而成。
(5)制备冻干粉:将乳化液置于-40℃下冷冻4h,再置于真空温度为-40℃、真空度为0.3mbar条件下干燥30h,粉碎过40目筛,得到活性益生菌。
所述胡柚提取物的制备方法如下:
1)将胡柚果肉洗净干燥、粉碎后,取200重量份的胡柚果肉粉末,加入600重量份的水,再加入果胶酶进行酶解,酶解温度为60℃,酶解时间为2h,酶解完成后用3mol/L的碳酸氢钠水溶液调节溶液的pH值为6得到混合液,果胶酶的用量为柚子果肉粉末质量的0.005倍;
2)按体积比1:0.3向步骤1)的混合液中加入90wt%乙醇水溶液,在78℃条件下反应2h后冷却至30℃,离心收集上清液,将上清液减压蒸除溶剂后得到胡柚提取物。
对比例2
一种含活性益生菌的食品,其制备方法,包括如下步骤:
M1红树莓果浆预处理:将红树莓冻果解冻,用水清洗干净沥干,用榨汁机破碎打浆,充分搅拌均匀后置于在80℃下杀菌15min得到预处理后的红树莓果浆;
M2发酵:将预处理后的红树莓果浆加入到发酵罐中进行无氧发酵,接种活性益生菌冻干粉,接种量为2wt%,发酵温度为28℃,发酵时间为24h,然后加热至110℃下灭菌10min,得到发酵的红树莓果浆;
M3制备红树莓酵素冻干粉:按质量比为1:2将发酵的红树莓果浆与冻干保护剂2混合均匀后置于-40℃下冷冻4h,再置于真空温度为-40℃、真空度为0.08Mpa条件下干燥48h,粉碎过30目筛,得到含活性益生菌的食品;所述冻干保护剂2由6wt%海藻糖、2wt%山梨醇、1wt%玉米面、余量为水混合均匀制备而成。
所述活性益生菌的制备方法,包括如下步骤:
(1)菌株活化:将5wt%葡萄糖、0.8wt%NaCl、3wt%琼脂粉、余量为水混合均匀制成的固体活化培养基加热至110℃灭菌20min、冷却至35℃,然后将罗伊氏乳杆菌在上述培养基中于38℃下培养30h,接种量为3wt%,得到活化菌株;
(2)扩大培养:将10wt%脱脂奶粉、4wt%菊粉、1.0wt%磷酸二氢钠、0.5wt%硫酸镁、余量为水混合均匀制成的扩大培养基加热至110℃灭菌20min、冷却至35℃,然后将步骤(1)制得的活化菌株接种于上述扩大培养基中,接种量为4wt%,于温度为35℃、震荡速度为300rpm条件下震荡培养18h,然后用3mol/L的碳酸氢钠水溶液调节溶液pH为7得到种子液;
(3)发酵:将3wt%绿豆肽、3wt%海带提取物、1.5wt%酪蛋白胨、0.2wt%硫酸锰、0.2wt%亚硒酸钠、余量为水混合均匀制成的特种培养基加热至110℃灭菌20min、冷却至35℃,然后将种子液接种于特种发酵培养基中于35℃下无氧发酵30h,接种量为4wt%,然后用3mol/L的碳酸氢钠水溶液调节溶液pH为7,得到发酵物;
(4)菌泥收集及乳化:将上述发酵物置于转速为6000rpm离心机中离心30min,弃掉上清液,收集菌泥,按质量比为1:2将菌泥加入到冻干保护剂1中,在转速为200rpm下乳化30min,得到乳化液;所述冻干保护剂1由8wt%亚麻籽、2wt%食用明胶、1.5wt%益生元、5wt%维生素C、余量为水混合均匀制备而成。
(5)制备冻干粉:将乳化液置于-40℃下冷冻4h,再置于真空温度为-40℃、真空度为0.3mbar条件下干燥30h,粉碎过40目筛,得到活性益生菌。
对比例3
一种含活性益生菌的食品,其制备方法,包括如下步骤:
M1红树莓果浆预处理:将红树莓冻果解冻,用水清洗干净沥干,用榨汁机破碎打浆,充分搅拌均匀后置于在80℃下杀菌15min得到预处理后的红树莓果浆;
M2发酵:将预处理后的红树莓果浆加入到发酵罐中进行无氧发酵,接种活性益生菌冻干粉,接种量为2wt%,发酵温度为28℃,发酵时间为24h,然后加热至110℃下灭菌10min,得到发酵的红树莓果浆;
M3制备红树莓酵素冻干粉:按质量比为1:2将发酵的红树莓果浆与冻干保护剂2混合均匀后置于-40℃下冷冻4h,再置于真空温度为-40℃、真空度为0.08Mpa条件下干燥48h,粉碎过30目筛,得到含活性益生菌的食品;所述冻干保护剂2由6wt%海藻糖、2wt%山梨醇、1wt%玉米面、余量为水混合均匀制备而成。
所述活性益生菌的制备方法,包括如下步骤:
1)菌株活化:将5wt%葡萄糖、0.8wt%NaCl、3wt%琼脂粉、余量为水混合均匀制成的固体活化培养基加热至110℃灭菌20min、冷却至35℃,然后将罗伊氏乳杆菌在上述培养基中于38℃下培养30h,接种量为3wt%,得到活化菌株;
(2)扩大培养:将10wt%脱脂奶粉、4wt%菊粉、1.0wt%磷酸二氢钠、0.5wt%硫酸镁、余量为水混合均匀制成的扩大培养基加热至110℃灭菌20min、冷却至35℃,然后将步骤(1)制得的活化菌株接种于上述扩大培养基中,接种量为4wt%,于温度为35℃、震荡速度为300rpm条件下震荡培养18h,然后用3mol/L的碳酸氢钠水溶液调节溶液pH为7得到种子液;
(3)发酵:将2wt%胡柚提取物、3wt%海带提取物、1.5wt%酪蛋白胨、0.2wt%硫酸锰、0.2wt%亚硒酸钠、余量为水混合均匀制成的特种培养基加热至110℃灭菌20min、冷却至35℃,然后将种子液接种于特种发酵培养基中于35℃下无氧发酵30h,接种量为4wt%,然后用3mol/L的碳酸氢钠水溶液调节溶液pH为7,得到发酵物;
(4)菌泥收集及乳化:将上述发酵物置于转速为6000rpm离心机中离心30min,弃掉上清液,收集菌泥,按质量比为1:2将菌泥加入到冻干保护剂1中,在转速为200rpm下乳化30min,得到乳化液;所述冻干保护剂1由8wt%亚麻籽、2wt%食用明胶、1.5wt%益生元、5wt%维生素C、余量为水混合均匀制备而成。
(5)制备冻干粉:将乳化液置于-40℃下冷冻4h,再置于真空温度为-40℃、真空度为0.3mbar条件下干燥30h,粉碎过40目筛,得到活性益生菌。
所述胡柚提取物的制备方法如下:
1)将胡柚果肉洗净干燥、粉碎后,取200重量份的胡柚果肉粉末,加入600份的水,再加入果胶酶进行酶解,酶解温度为60℃,酶解时间为2h,酶解完成后用3mol/L的碳酸氢钠水溶液调节溶液的pH值为6得到混合液,果胶酶的用量为柚子果肉粉末质量的0.005倍;
2)按体积比为1:0.3向步骤1)的混合液中加入90wt%乙醇水溶液,在78℃条件下反应2h后冷却至30℃,离心收集上清液,将上清液减压蒸除溶剂后得到胡柚提取物。
实施例1
一种含活性益生菌的食品,其制备方法,包括如下步骤:
M1红树莓果浆预处理:将红树莓冻果解冻,用水清洗干净沥干,用榨汁机破碎打浆,充分搅拌均匀后置于在80℃下杀菌15min得到预处理后的红树莓果浆;
M2发酵:将预处理后的红树莓果浆加入到发酵罐中进行无氧发酵,接种活性益生菌冻干粉,接种量为2wt%,发酵温度为28℃,发酵时间为24h,然后加热至110℃下灭菌10min,得到发酵的红树莓果浆;
M3制备红树莓酵素冻干粉:按质量比为1:2将发酵的红树莓果浆与冻干保护剂2混合均匀后置于-40℃下冷冻4h,再置于真空温度为-40℃、真空度为0.08Mpa条件下干燥48h,粉碎过30目筛,得到含活性益生菌的食品;所述冻干保护剂2由6wt%海藻糖、2wt%山梨醇、1wt%玉米面、余量为水混合均匀制备而成。
所述活性益生菌的制备方法,包括如下步骤:
1)菌株活化:将5wt%葡萄糖、0.8wt%NaCl、3wt%琼脂粉、余量为水混合均匀制成的固体活化培养基加热至110℃灭菌20min、冷却至35℃,然后将罗伊氏乳杆菌在上述培养基中于38℃下培养30h,接种量为3wt%,得到活化菌株;
(2)扩大培养:将10wt%脱脂奶粉、4wt%菊粉、1.0wt%磷酸二氢钠、0.5wt%硫酸镁、余量为水混合均匀制成的扩大培养基加热至110℃灭菌20min、冷却至35℃,然后将步骤(1)制得的活化菌株接种于上述扩大培养基中,接种量为4wt%,于温度为35℃、震荡速度为300rpm条件下震荡培养18h,然后用3mol/L的碳酸氢钠水溶液调节溶液pH为7得到种子液;
(3)发酵:将3wt%绿豆肽、2wt%胡柚提取物、3wt%海带提取物、1.5wt%酪蛋白胨、0.2wt%硫酸锰、0.2wt%亚硒酸钠、余量为水混合均匀制成的特种培养基加热至110℃灭菌20min、冷却至35℃,然后将种子液接种于特种发酵培养基中于35℃下无氧发酵30h,接种量为4wt%,然后用3mol/L的碳酸氢钠水溶液调节溶液pH为7,得到发酵物;
(4)菌泥收集及乳化:将上述发酵物置于转速为6000rpm离心机中离心30min,弃掉上清液,收集菌泥,按质量比为1:2将菌泥加入到冻干保护剂1中,在转速为200rpm下乳化30min,得到乳化液;所述冻干保护剂1由8wt%亚麻籽、2wt%食用明胶、1.5wt%益生元、5wt%维生素C、余量为水混合均匀制备而成。
(5)制备冻干粉:将乳化液置于-40℃下冷冻4h,再置于真空温度为-40℃、真空度为0.3mbar条件下干燥30h,粉碎过40目筛,得到活性益生菌。
所述胡柚提取物的制备方法如下:
1)将胡柚果肉洗净干燥、粉碎后,取200重量份的胡柚果肉粉末,加入600份的水,再加入果胶酶进行酶解,酶解温度为60℃,酶解时间为2h,酶解完成后用3mol/L的碳酸氢钠水溶液调节溶液的pH值为6得到混合液,果胶酶的用量为柚子果肉粉末质量的0.005倍;
2)按体积比为1:0.3向步骤1)的混合液中加入90wt%乙醇水溶液,在78℃条件下反应2h后冷却至30℃,离心收集上清液,将上清液减压蒸除溶剂后得到胡柚提取物。
测试例1
益生菌增殖测试:
活菌数的测定:各取1g罗伊氏乳杆菌冻干粉、对比例1-3和实施例1收集到的菌泥于离心管中,向离心管中加入10mL质量体积浓度为0.85%生理盐水,充分震荡摇匀后,通过梯度稀释法,取1mL均匀接种于已灭菌冷却至45℃左右的琼脂培养基中,振荡摇匀后置于37℃条件下培养48h,采用平板计数法测定活菌数,每个样品测3次,测试结果取平均值,测试结果如表1所示:
表1:益生菌增殖测试结果
通过表1中益生菌的活菌数可以知道本发明通过添加绿豆肽、胡柚提取物、海带提取物制备的培养基对罗伊氏乳杆菌有很好的增殖效果,可能的原因是三种物质中的有效活性物质起了协同,促进了罗伊氏乳杆菌的发酵增殖。
测试例2
降血糖功效测试:检测对象为对比例1-3、实施例1制备的活性益生菌冻干粉,试验动物为SPF级健康ICR成年小鼠50只,雌雄各半,体重30.4±1.2g。将50只试验小鼠随机平均分为5组,每组10只,分别为实验组和空白对照组。试验小鼠由湖南斯莱克景达实验动物有限公司提供(生产许可证号SCXK(湘)2016-0002)
对实验组制作高血糖动物模型,空白对照组不进行高血糖动物模型的处理。高血糖动物模型制作方法为:小鼠禁食24小时后,尾静脉注射四氧嘧啶(46mg/kg.bw),5天后禁食5小时,测血糖值,血糖值在10-25mmol/L之间为高血糖模型成功动物,实验组所有小鼠均成功制作了高血糖动物模型。将对比例1-3、实施例1制备的活性益生菌冻干粉用37℃水配成浓度为100mg/kg的益生菌水溶液,对实验组小鼠给予灌胃3次,每次用量为0.5mL,空白组每天给予37℃水灌胃3次,连续喂养30天后,禁食5小时测空腹血糖值,比较各组动物血糖值及血糖下降百分率,血糖下降百分率的计算公式如下:
血糖下降百分率=(实验前血糖值-实验后血糖值)/实验前血糖值×100%
测试结果如表2所示:
表2:活性益生菌降血糖功效的测试结果
通过表2的实验数据可知,本发明制备的活性益生菌具有很好的降血糖的功能,实施例1制备的活性益生菌的降血糖效果最好,相较于其他对比例,实施例1添加了绿豆肽、胡柚提取物、海带提取物,三者协同,能使培养的罗伊氏乳杆菌益生菌抑制转运蛋白SGLT1和GLUT5基因的表达,改善了肠道中的菌群种类,从而减少了葡萄糖、半乳糖、果糖从肠道中被肠黏膜细胞吸收进入血液,从而降低了血糖浓度。
测试例3
DPPH自由基清除试验:分别用水将红树莓果浆、对比例1-3、实施例1制备的含活性益生菌的食品稀释成1wt%的溶液,加入400μL的DPPH甲醇溶液(1×10-4M),于37℃下遮光反应30min,测定样品在紫外波长为517nm处的吸光值,将对照溶液、样品溶液、样液本底的测定值分别记为A0、A1、A2,由如下公式计算自由基清除率,代入Vc标准曲线,用Vc当量含量(mgVc/L)表示:
表3:含活性益生菌的食品的自由基清除率测试结果
V<sub>C</sub>/L | |
红树莓果浆 | 20.53 |
对比例1 | 26.46 |
对比例2 | 27.53 |
对比例3 | 28.61 |
实施例1 | 35.68 |
从表3的数据可知,含本发明制备的活性益生菌的食品还具有很好的自由基清除效果,通过对比可知,红树莓果浆经过本发明制备的活性益生菌发酵后对DPPH自由基的清除能力有显著的提高。可能的原因是本发明制备的活性益生菌促进了红树莓果浆发酵后产生了更多的有机酸、多酚等生物活性物质,这些物质能作为氢离子供体起到抗氧化作用,另外本发明制备的活性益生菌在发酵后期,会有部分菌体自溶,胞内物质溶出,协同起到了清除自由基的效果。而绿豆肽、胡柚提取物、海带提取物三者的协同促进了罗伊氏乳杆菌益生菌的增殖,也促进了含活性益生菌食品的清除自由基的效果。
Claims (10)
1.一种活性益生菌的制备方法,其特征在于,包括如下步骤:
(1)菌株活化:固体培养基灭菌后接种罗伊氏乳杆菌,接种量为2-4wt%,于30-40℃下培养20-30h,得到活化菌株;
(2)扩大培养:扩大培养基灭菌后接种步骤(1)制得的活化菌株,接种量为2-5wt%,于温度为35-42℃、震荡速度为200-400rpm条件下震荡培养10-18h,调节溶液pH为6-7得到种子液;
(3)发酵:特种发酵培养基灭菌后接种种子液,接种量为2-5wt%,于30-35℃下无氧发酵25-30h,调节溶液pH为6-7,得到发酵物;
(4)菌泥收集及乳化:将步骤(3)得到的发酵物离心、弃上清液、收集菌泥;按质量比1:(2-3)将菌泥加入到冻干保护剂1中乳化,得到乳化液;
(5)制备冻干粉:将步骤(4)得到的乳化液冷冻干燥、粉碎,得到所述活性益生菌。
2.如权利要求1所述的活性益生菌的制备方法,其特征在于:所述步骤(3)中特种发酵培养基由0.5-2wt%酪蛋白胨、0.05-1wt%硫酸锰、0.01-0.5wt%亚硒酸钠、余量为水混合均匀制成。
3.如权利要求1所述的活性益生菌的制备方法,其特征在于:所述步骤(3)中特种发酵培养基由1-5wt%绿豆肽、0.5-2wt%酪蛋白胨、0.05-1wt%硫酸锰、0.01-0.5wt%亚硒酸钠、余量为水混合均匀制成。
4.如权利要求1所述的活性益生菌的制备方法,其特征在于:所述步骤(3)中特种发酵培养基由1-5wt%绿豆肽、2-5wt%胡柚提取物、0.5-2wt%酪蛋白胨、0.05-1wt%硫酸锰、0.01-0.5wt%亚硒酸钠、余量为水混合均匀制成。
5.如权利要求1所述的活性益生菌的制备方法,其特征在于:所述步骤(3)中特种发酵培养基由1-5wt%绿豆肽、2-5wt%胡柚提取物、1-4wt%海带提取物、0.5-2wt%酪蛋白胨、0.05-1wt%硫酸锰、0.01-0.5wt%亚硒酸钠、余量为水混合均匀制成。
6.如权利要求4所述的活性益生菌的制备方法,其特征在于,所述胡柚提取物的制备方法如下:
1)将胡柚果肉洗净干燥、粉碎后,取100-200重量份的胡柚果肉粉末,加入200~600重量份的水,再加入果胶酶进行酶解,酶解温度为50-70℃,酶解时间为1-2h,酶解完成后用1-3mol/L的碳酸氢钠水溶液调节溶液的pH值为5-7得到混合液,果胶酶的用量为柚子果肉粉末质量的0.001-0.01倍;
2)按体积比1:(0.2-0.4)向步骤1)的混合液中加入70-90wt%乙醇水溶液,在70-78℃条件下反应1-2h后冷却至20-40℃,离心收集上清液,将上清液减压蒸除溶剂后得到胡柚提取物。
7.如权利要求1所述的活性益生菌的制备方法,其特征在于:所述步骤(4)中冻干保护剂1由3-10wt%亚麻籽、1-3wt%食用明胶、0.5-2wt%益生元、2-6wt%维生素C、余量为水混合均匀制备而成。
8.一种活性益生菌,其特征在于:由权利要求1-7任一项所述的活性益生菌的制备方法制备而成。
9.如权利要求8所述的活性益生菌在食品中的应用。
10.一种含活性益生菌的食品的制备方法,其特征在于,包括如下步骤:
M1红树莓果浆预处理:将红树莓冻果解冻,用水清洗干净沥干,用榨汁机破碎打浆,充分搅拌均匀后置于在80-100℃下杀菌10-20min得到预处理后的红树莓果浆;
M2发酵:将预处理后的红树莓果浆加入到发酵罐中进行无氧发酵,接种权利要求8所述的活性益生菌,接种量为2-3wt%,发酵温度为25-35℃,发酵时间为20-24h,然后加热至110-130℃下灭菌10-30min,得到发酵的红树莓果浆;
M3制备红树莓酵素冻干粉:将发酵的红树莓果浆与冻干保护剂2按质量比为1:2-3混合均匀后置于-20~-60℃下冷冻2-5h,再置于真空温度为-40~-60℃、真空度为0.05-0.1Mpa条件下干燥40-48h,粉碎过10-40目筛,得到含活性益生菌的食品。
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