CN113332354B - 一种抗糖化抗衰老发酵组合物及其制备方法 - Google Patents
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Abstract
本发明提供了一种抗糖化抗衰老发酵组合物,按质量份计,包括洋甘菊花40~120份,鱼腥草40~120份,余甘子20~100份,山楂10~80份,发酵菌粉0.1~3份,酶制剂6~40份和水1500~3000份;所述发酵菌粉由植物乳杆菌、嗜酸乳杆菌、干酪乳杆菌、两歧双歧杆菌、乳酸乳球菌乳酸亚种和肠膜明串珠菌肠膜亚种组成。上述药食同源中药为主要成分,在酶制剂和发酵菌粉作用下,可提高中药中有效活性成分槲皮素、绿原酸和没食子酸的有效含量;而发酵将中药大分子物质分解经微生物分分解小分子,更利于物质吸收;且经实验证明发酵后产物具有抗糖化和抗衰老的功效。
Description
技术领域
本发明属于中药发酵技术领域,具体涉及一种抗糖化抗衰老的发酵组合物及其制备方法。
背景技术
非酶糖基化是一系列复杂的非酶促反应,蛋白质和葡萄糖在体内发生非酶促反应形成席夫碱和Amadori产物等早期糖基化产物,进而经过氧化、重排、交联等过程,形成不可逆的非酶糖基化终产物(AGEs)。AGEs在体内长期积累,可引发一系列病理变化,最终导致肾脏疾病,阿尔兹海默症,II型糖尿病,心血管疾病等疾病的发生和发展。就皮肤而言,AGEs的形成导致皮肤中的胶原蛋白发生变质,使肌肤失去弹性,变的暗黄松弛,造成皮肤衰老。
衰老是人类生命过程中的必然规律,也是不可抗拒的自然现象。而随着生活水平的提高,人们对延缓皮肤衰老的迫切愿望,出现了各式各样的抗糖基化、抗衰老的产品。市面上抗糖基化防衰老的外敷化妆品中,不可避免的要添加一些对人体有害的化学物质,虽然这些化学物质对皮肤有短期的美白、抗衰老作用,但如果长期使用,反而会使皮肤受到损害,出现长痘、色斑和油脂分泌失调等。因而研发逐渐趋向于内服天然功能性食品。
例如中国专利CN109007819A公开了一种抗糖化产品及其制备方法,其包括生姜0.5-2份、石榴3-10份、鱼腥草2-8份、富锌酵母0.5-2份、针叶樱桃2-8份、胶原蛋白肽3-10份、绿茶2-10份、葡萄籽1-5份、白藜芦醇1-5份、红酒多酚1-5份、神经酰胺1-5份、绿豆2-10份、异麦芽酮糖醇 20-30份、麦芽糊精1-5份、羧甲基纤维素钠1-5份。上述组合物优选多糖、多酚、黄酮、萜类、甾醇、皂苷进行组合搭配,抑制非酶糖基化终产物AGEs 的生成。然而,上述产品的制备仅为混合压片,存在鱼腥草、生姜等生物利用率低,不利于人体肠道吸收及抗糖效果仍有待提高的问题。
发明内容
因此,本发明要解决的技术问题在于克服现有技术中的抗糖化产品中药利用率低,不利于人体肠道吸收及抗糖效果有待提高的问题,从而提供了一种抗糖化抗衰老发酵组合物及其制备方法。
本发明的技术方案为:
一种抗糖化抗衰老发酵组合物,按质量份计,包括洋甘菊花40~120份,鱼腥草40~120份,余甘子20~100份,山楂10~80份,发酵菌粉0.1~3份,酶制剂6~40份和水1500~3000份;所述发酵菌粉由植物乳杆菌、嗜酸乳杆菌、干酪乳杆菌、两歧双歧杆菌、乳酸乳球菌乳酸亚种和肠膜明串珠菌肠膜亚种组成。
优选的,所述发酵菌粉由植物乳杆菌0.1~0.5份、嗜酸乳杆菌0.1~0.5 份、干酪乳杆菌0.1~0.5份、两歧双歧杆菌0.1~0.5份、乳酸乳球菌乳酸亚种0.1~0.5份和肠膜明串珠菌肠膜亚种0.1~0.5份组成。
优选的,所述植物乳杆菌活菌数为1000~3000亿CFU/g,所述嗜酸乳杆菌1000~5000亿CFU/g,所述干酪乳杆菌1000~3000亿CFU/g,所述两歧双歧杆菌500~2000亿CFU/g,所述乳酸乳球菌乳酸亚种800~3000 亿CFU/g,所述肠膜明串珠菌肠膜亚种1000~3000亿CFU/g。
优选的,所述酶制剂包括纤维素酶1~5份和果胶酶1~5份,所述纤维素酶活力为10000~30000U,果胶酶活力为10000~50000U。
进一步优选的,还包括酸碱调节剂1~10份。
更进一步优选的,按质量份计,包括洋甘菊花40~120份,鱼腥草40~120 份,余甘子20~100份,山楂10~80份,植物乳杆菌0.1~0.5份、嗜酸乳杆菌0.1~0.5份、干酪乳杆菌0.1~0.5份、两歧双歧杆菌0.1~0.5份、乳酸乳球菌乳酸亚种0.1~0.5份、肠膜明串珠菌肠膜亚种0.1~0.5份,纤维素酶3~20 重量份,果胶酶3~20重量份,酸碱调节剂1~10份和水1500~3000份。
更进一步优选的,按质量份计,包括洋甘菊花100份,鱼腥草80份,余甘子80份,山楂60份,植物乳杆菌0.1份、嗜酸乳杆菌0.1份、干酪乳杆菌0.3份、两歧双歧杆菌0.4份、乳酸乳球菌乳酸亚种0.2份、肠膜明串珠菌肠膜亚种0.5份,纤维素酶4份,果胶酶4份,酸碱调节剂7份和水 2200份;
或者包括洋甘菊花120份,鱼腥草100份,余甘子60份,山楂50份,植物乳杆菌0.3份、嗜酸乳杆菌0.2份、干酪乳杆菌0.2份、两歧双歧杆菌 0.5份、乳酸乳球菌乳酸亚种0.1份、肠膜明串珠菌肠膜亚种0.2份,纤维素酶3份,果胶酶3份,酸碱调节剂7份和水2000份。
一种制备上述抗糖化抗衰老发酵组合物的制备方法,包括如下步骤:
(1)将洋甘菊花、鱼腥草、余甘子和山楂粉碎过筛至目数为100~600 目,加水混合均匀后添加酸碱调节剂至pH为4.0~5.0,得混合液;
(2)向所述混合液中加入酶制剂酶解,酶解温度40~70℃,酶解时间90~240min,酶解结束后添加酸碱调节剂至pH为5.0~6.0,得酶解液;
(3)将所述酶解液在温度80~102℃下提取10~90min,得提取液;
(4)向所述提取液中加入所述发酵菌粉发酵,发酵温度为15~45℃,时间为36~96h,得发酵液,将所述发酵液固液分离得到发酵清液。
优选的,所述发酵清液可采用真空低温浓缩工艺,浓缩温度45~75℃,真空度-0.03~-0.09Mpa下制备成发酵浸膏。
进一步优选的,向所述发酵浸膏加入辅料后采用喷雾干燥工艺,进风温度88~125℃,出风温度75~115℃,泵速8~50%下喷粉干燥得发酵粉末。
本发明技术方案,具有如下优点:
1.洋甘菊花:洋甘菊花含有绿原酸、木樨草甘、槲皮素等多种有效成份。其中,绿原酸等苯丙素类物质可有效阻碍蛋白质糖化反应。洋甘菊花还具有抗氧化、消炎、抗变应性和抗病毒的功效,具有很好的舒敏、修护敏感肌肤、调整肤色不均等美容作用。
鱼腥草:辛,微寒。归肺经。清热解毒,消痈排脓,利尿通淋。鱼腥草可以抑制中性脂肪吸收,防止其蓄积。在抗糖化方面,鱼腥草含有挥发油、多糖、槲皮素和芸香甙等组分,其中,槲皮素等组分能够与含氨基物质结合,从而抑制该含氨基物质与糖类进行糖基化作用。槲皮素能对抗自由基,络合或捕获自由基防止机体脂质过氧化反应,对癌症、衰老、心血管疾病的治疗和预防有重要意义。
余甘子:甘、酸、涩,凉。归肺、胃经。清热凉血,消食健胃,生津止咳。余甘子含有大量的多酚物质(没食子酸)和维生素C,具有抑制AGEs 形成的作用,作用随剂量的增加而加强,可以改善因老化带来的高脂血症和肾脏功能障碍。
山楂:酸、甘,微温。归脾、胃、肝经。消食健胃,行气散瘀,化浊降脂。山楂含有的槲皮素、绿原酸等丙苯素类物质具有较强的抗氧化性活性,可以阻碍3-脱氧葡萄糖醛酮和羧甲基赖氨酸的生成,可阻止蛋白质架桥形成,从而阻碍糖化反应。
本发明选用上述药食同源中药为主要成分,在酶制剂和由植物乳杆菌、嗜酸乳杆菌、干酪乳杆菌、两歧双歧杆菌、乳酸乳球菌乳酸亚种和肠膜明串珠菌肠膜亚种6种菌株组成的发酵菌粉作用下,可提高中药中有效活性成分槲皮素、绿原酸和没食子酸的有效含量;而发酵将中药大分子物质分解经微生物分分解小分子,更利于物质吸收;且经实验证明发酵后产物具有抗糖化和抗衰老的功效。
2.本发明提供的抗糖化抗衰老发酵组合物,选用合适比例和活菌数的发酵菌粉,可进一步提高槲皮素、绿原酸和没食子酸的有效含量,进一步降低羧甲基赖氨酸的生成,提高抗糖化和抗衰老的功效。
3.本发明提供的抗糖化抗衰老发酵组合物,酸碱调节剂可调节发酵过程中pH值,更适合菌株的生产,提高发酵产率。
4.本发明提供的抗糖化抗衰老组合物的发酵清液,可作为基础原料,应用在普通食品,保健食品,药品或化妆品中,也可制备成膏剂,粉剂等不同的剂型,应用在普通食品,保健食品,药品和化妆品中。
具体实施方式
为使本发明的目的,技术方案和优点更加清楚,下面将对本发明实施方式做进一步的详细描述。基于本发明公开中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本公开保护的范围。
实施例中未注明具体实验步骤或条件者,按照本领域内文献所描述的常规步骤的操作或条件即可进行。所有试剂未注明生产厂商者,均为可以通过市购获得的常规试剂产品。
洋甘菊的别称又叫罗马洋甘菊、德国洋甘菊,罗马洋甘菊又称为“春黄菊”。本发明中选用的洋甘菊花为洋甘菊完全开放的花序。
实施例1
本实施例提供了一种抗糖化抗衰老发酵组合物,包括洋甘菊花100g,鱼腥草80g,余甘子80g,山楂60g,植物乳杆菌0.1g、嗜酸乳杆菌0.1g,干酪乳杆菌0.3g,两歧双歧杆菌0.4g,乳酸乳球菌乳酸亚种0.2g,肠膜明串珠菌肠膜亚种0.5g,纤维素酶4g,果胶酶4g,碳酸氢钠7g和水2200g。其制备方法为:
(1)洋甘菊花100g、鱼腥草80g、余甘子80g和山楂60g分别粉碎过300目筛网,并与2200g水混合均匀后加入4g碳酸氢钠调节至pH为 4.8,得到混合液;
(2)向所述混合液中加入纤维素酶4g和果胶酶4g酶解,在温度 58℃下酶解150min,酶解结束后加入3g碳酸氢钠调节pH至5.6,得酶解液。其中纤维素酶活力为30000U,果胶酶酶活力为30000U;
(3)将酶解液升温至95℃,搅拌提取30min,得提取液;
(4)待温度降温至35℃向提取液中加入植物乳杆菌0.1g、嗜酸乳杆菌0.1g,干酪乳杆菌0.3g,两歧双歧杆菌0.4g,乳酸乳球菌乳酸亚种0.2g 和肠膜明串珠菌肠膜亚种0.5g,在温度为35℃下发酵72h,得发酵液;其中植物乳杆菌活菌数为1200亿CFU/g、嗜酸乳杆菌1800亿CFU/g、干酪乳杆菌2000亿CFU/g、两歧双歧杆菌1000亿CFU/g、乳酸乳球菌乳酸亚种2200亿CFU/g、肠膜明串珠菌肠膜亚种2800亿CFU/g。
(5)将上述发酵液固液分离,得发酵清液,发酵清液pH为3.2,可溶性固形物含量9.6%,总酸1.53%(以乳酸计);
(6)将上述发酵清液采用真空低温浓缩工艺浓缩,浓缩温度62℃,真空度-0.08MPa,得发酵浸膏;发酵膏密度为1.16g/cm3,可溶性固形物含量 35.6%;
(7)将发酵浸膏和15g脱脂奶粉混合后,在进风温度为118℃,出风温度为85℃,泵速为20%的条件下喷粉,得发酵粉。
实施例2
本实施例提供了一种抗糖化抗衰老发酵组合物,包括洋甘菊花120g,鱼腥草100g,余甘子60g,山楂50g,植物乳杆菌0.3g、嗜酸乳杆菌0.2g,干酪乳杆菌0.2g,两歧双歧杆菌0.5g,乳酸乳球菌乳酸亚种0.1g,肠膜明串珠菌肠膜亚种0.2g,纤维素酶3g,果胶酶3g和水2000g。
其制备方法为:
(1)将洋甘菊花120g、鱼腥草100g、余甘子60g和山楂50g分别粉碎过300目筛网,并与2000g水混合均匀后加入5g碳酸氢钠调节至 pH为5.0,得到混合液;
(2)向所述混合液中加入纤维素酶3g和果胶酶3g酶解,在温度 55℃下酶解120min,结束后加入2g碳酸氢钠调节pH至5.5,得酶解液。其中纤维素酶活力为20000U,果胶酶酶活力为40000U;
(3)将酶解液升温至92℃,搅拌提取45min,得提取液;
(4)待温度降温至32℃向提取液中加入植物乳杆菌0.3g、嗜酸乳杆菌0.2g,干酪乳杆菌0.2g,两歧双歧杆菌0.5g,乳酸乳球菌乳酸亚种0.1g 和肠膜明串珠菌肠膜亚种0.2g,在温度为32℃下发酵48h,得发酵液;其中植物乳杆菌活菌数为1000亿CFU/g、嗜酸乳杆菌2000亿CFU/g、干酪乳杆菌1000亿CFU/g、两歧双歧杆菌500亿CFU/g、乳酸乳球菌乳酸亚种800亿CFU/g、肠膜明串珠菌肠膜亚种3000亿CFU/g;
(5)将上述发酵液固液分离,得发酵清液,发酵清液pH为3.8,可溶性固形五含量11.4%,总酸1.08%(以乳酸计)。
(6)将上述发酵清液采用真空低温浓缩工艺浓缩,浓缩温度70℃,真空度-0.09MPa,得发酵浸膏;发酵膏密度为1.12g/cm3,可溶性固形物含量 36.6%;
(7)将发酵浸膏和20g麦芽糊精混合后,在进风温度为120℃,出风温度为88℃,泵速为30%的条件下喷粉,得发酵粉。
实施例3
本实施例提供了一种抗糖化抗衰老发酵组合物,包括洋甘菊花40g,鱼腥草120g,余甘子20g,山楂80g,植物乳杆菌0.5g、嗜酸乳杆菌0.5g,干酪乳杆菌0.1g,两歧双歧杆菌0.1g,乳酸乳球菌乳酸亚种0.1g,肠膜明串珠菌肠膜亚种0.5g,纤维素酶4g,果胶酶4g,碳酸氢钠7g和水1500g。其制备方法为:
(1)春黄菊花40g、鱼腥草120g、余甘子20g和山楂80g分别粉碎过300目筛网,并与1500g水混合均匀后加入4g碳酸氢钠调节至pH为 4.7,得到混合液;
(2)向所述混合液中加入纤维素酶4g和果胶酶4g酶解,在温度 60℃下酶解200min,酶解结束后加入4g碳酸氢钠调节pH至5.6,得酶解液。其中纤维素酶活力为20000U,果胶酶酶活力为40000U;
(3)将酶解液升温至98℃,搅拌提取40min,得提取液;
(4)待温度降温至40℃向提取液中加入植物乳杆菌0.5g、嗜酸乳杆菌0.5g,干酪乳杆菌0.1g,两歧双歧杆菌0.1g,乳酸乳球菌乳酸亚种0.1g 和肠膜明串珠菌肠膜亚种0.5g,在温度为40℃下发酵84h,得发酵液;其中植物乳杆菌活菌数为1200亿CFU/g、嗜酸乳杆菌1800亿CFU/g、干酪乳杆菌2000亿CFU/g、两歧双歧杆菌1000亿CFU/g、乳酸乳球菌乳酸亚种2200亿CFU/g、肠膜明串珠菌肠膜亚种2800亿CFU/g;
(5)将上述发酵液固液分离,得发酵清液,发酵清液pH为3.3,可溶性固形物含量9.5%,总酸1.51%(以乳酸计)。
(6)将上述发酵清液采用真空低温浓缩工艺浓缩,浓缩温度52℃,真空度-0.08MPa,得发酵浸膏;发酵膏密度为1.19g/cm3,可溶性固形物含量 36.5%;
(7)将发酵浸膏和20g脱脂奶粉混合后,在进风温度为108℃,出风温度为80℃,泵速为30%的条件下喷粉,得发酵粉。
实施例4
本实施例提供了一种抗糖化抗衰老发酵组合物,包括洋甘菊花90g,鱼腥草40g,余甘子100g,山楂10g,植物乳杆菌0.2g、嗜酸乳杆菌0.3g,干酪乳杆菌0.5g,两歧双歧杆菌0.1g,乳酸乳球菌乳酸亚种0.3g,肠膜明串珠菌肠膜亚种0.1g,纤维素酶4g,果胶酶4g,碳酸氢钠7g和水3000g。其制备方法为:
(1)洋甘菊花90g、鱼腥草40g、余甘子100g和山楂10g分别粉碎过300目筛网,并与3000g水混合均匀后加入4g碳酸氢钠调节至pH为 4.8,得到混合液;
(2)向所述混合液中加入纤维素酶4g和果胶酶4g酶解,在温度 60℃下酶解220min,酶解结束后加入3g碳酸氢钠调节pH至5.6,得酶解液。其中纤维素酶活力为30000U,果胶酶酶活力为30000U;
(3)将酶解液升温至85℃,搅拌提取60min,得提取液;
(4)待温度降温至40℃向提取液中加入植物乳杆菌0.2g、嗜酸乳杆菌0.3g,干酪乳杆菌0.5g,两歧双歧杆菌0.1g,乳酸乳球菌乳酸亚种0.3g 和肠膜明串珠菌肠膜亚种0.1g,在温度为40℃下发酵72h,得发酵液;其中植物乳杆菌活菌数为1200亿CFU/g、嗜酸乳杆菌1800亿CFU/g、干酪乳杆菌2000亿CFU/g、两歧双歧杆菌1000亿CFU/g、乳酸乳球菌乳酸亚种2200亿CFU/g、肠膜明串珠菌肠膜亚种2800亿CFU/g;
(5)将上述发酵液固液分离,得发酵清液,发酵清液pH为3.1,可溶性固形物含量9.4%,总酸1.49%(以乳酸计)。
(6)将上述发酵清液采用真空低温浓缩工艺浓缩,浓缩温度55℃,真空度-0.05MPa,得发酵浸膏;发酵膏密度为1.16g/cm3,可溶性固形物含量 36.2%;
(7)将发酵浸膏和15g脱脂奶粉混合后,在进风温度为118℃,出风温度为85℃,泵速为20%的条件下喷粉,得发酵粉。
对比例1
本对比例提供了一种抗糖化抗衰老组合物,包括洋甘菊花100g,鱼腥草80g,余甘子80g,山楂60g,碳酸氢钠7g和水2200g。
其制备方法为:
(1)洋甘菊花100g、鱼腥草80g、余甘子80g和山楂60g分别粉碎过300目筛网,并与2200g水混合均匀后加入4g碳酸氢钠调节至pH为 4.8,得到混合液;
(2)将上述混合液在温度58℃下保温150min后加入3g碳酸氢钠调节pH至5.6;
(3)将上步骤混合液升温至95℃,搅拌提取30min,得提取液;
(4)将提取液降温至35℃后,保温72h;随后固液分离,得清液。
(6)将上述清液采用真空低温浓缩工艺浓缩,浓缩温度62℃,真空度 -0.08MPa,得浸膏;
(7)将浸膏和15g脱脂奶粉混合后,在进风温度为118℃,出风温度为85℃,泵速为20%的条件下喷粉,得固体粉。
该对比例与实施例1的区别在于,未经过酶解和发酵。
对比例2
该对比例同实施例1的区别在于,将洋甘菊花替换为滁菊,其余组分含量及制备方法与实施例1相同。
对比例3
该对比例同实施例1的区别在于,发酵菌粉中不包括肠膜明串珠菌肠膜亚种、嗜酸乳杆菌,干酪乳杆菌,两歧双歧杆菌和乳酸乳球菌乳酸亚种,即本对比例中发酵菌粉由植物乳杆菌1.6g组成,其余组分含量及制备方法与实施例1相同。
对比例4
该对比例同实施例1的区别在于,发酵菌粉中各菌株比例不在优选范围内,即本对比例中发酵菌粉由植物乳杆菌0.05g,嗜酸乳杆菌0.05g,干酪乳杆菌0.3g,两歧双歧杆菌0.4g,乳酸乳球菌乳酸亚种0.5g和肠膜明串珠菌肠膜亚种0.5g组成,其余组分含量及制备方法与实施例1相同。
实验例1活性成分检测
鱼腥草、山楂中含有槲皮素活性成分,洋甘菊花、山楂中含有的绿原酸等苯丙素类物质等活性物质,余甘子中含有多酚类物质没食子酸等活性成分,上述活性均具有较强的抗氧化活性,本实验目的在于验证各实施例和对比例中得到发酵清液或清液中槲皮素、绿原酸和没食子酸的含量。
槲皮素含量检测
色谱条件:色谱柱:C18色谱柱(250mm×4.6mm,5μm);流动相:0.4%磷酸缓冲液-甲醇(40:60,V/V);流速:0.8mL/min;检测波长:373nm。
对照品溶液的制备:
取槲皮素对照品0.001g,于100mL量瓶中加适量甲醇溶解,定容配置成10μg/mL的溶液100mL,备用。
标准曲线制作:
取槲皮素对照品溶液5mL置50mL量瓶中,加甲醇(色谱纯),超声处理20min后过滤,加甲醇(色谱纯)定容至刻度。精密量取上述对照品溶液0.20,0.25,1.2,2.0mL,分别置于4个10mL量瓶中,加甲醇(色谱纯)稀释至刻度,摇匀,分别量取20μL注入色谱仪,记录色谱图,以浓度 (Cμg/mL)为横坐标,峰面积为纵坐标进行线性回归,得线性方程 Y=117.05X+7598.9。相关系数r=0.9994。
测试样品的制备及测定:
取实施例1和对比例2-4对应得到的发酵粉和对比例1得到的固体粉末,过100目筛,各称取粉末0.12g,加甲醇-25%盐酸(4:1)25mL,水浴回流30min,放冷,置于50mL的量瓶中,加甲醇使其稀释至刻度,摇匀。精密称取1mL置于25mL量瓶中,加甲醇至刻度线,称定重量,超声处理20min,放冷,称定重量,用20%甲醇补足使完全溶解并稀释至刻度,摇匀,过滤,精密量取20μL精密量取20μL,注入液相色谱仪,记录色谱图。每个样品测量4次。实验结果如表1所示。
没食子酸含量检测
色谱条件:
色谱柱:C18色谱柱(250mm×4.6mm,5μm);流动相:0.5%磷酸缓冲液-甲醇(5:95,V/V);流速:1.0mL/min;检测波长:270nm。
对照品溶液的制备:
取没食子酸对照品适量加甲醇配置成每1mL含47.8μg/mL的溶液备用。
标准曲线制作:
精密称取对照品溶液(47.8μg/mL)1,2,4,6,8,10,12μL,依次注入高校液相色谱仪,测定峰面积,以峰面积为纵坐标,没食子酸进样量为横坐标,绘制标准曲线,得回归方程为:Y=332.65+297.34,r=0.9994。
测试样品的制备及测定:
取实施例1和对比例2-4对应得到的发酵粉和对比例1得到的固体粉末,过100目筛,各称取粉末0.5g,置于50mL的量瓶中,加20%甲醇使其稀释至刻度,称定重量,超声处理20min,放冷,称定重量,用 20%甲醇补足使完全溶解并稀释至刻度,摇匀,过滤,精密量取20μL,注入液相色谱仪,记录色谱图。每个样品测量4次。实验结果如表1所示。
绿原酸含量测定
色谱条件:
色谱柱:C18色谱柱(300mm×4.6mm,10μm);流动相:甲醇-乙腈 -0.01mol/L磷酸二氢钠溶液(用冰醋酸调节pH为3.0)(10:13:77,V/V);流速:1.0mL/min;检测波长:326nm。
对照品溶液的制备:
取绿原酸对照品10mg,置10mL棕色量瓶中,加50%甲醇溶液并稀释至刻度,摇匀;精密称取1mL,置25mL棕色瓶中,加50%甲醇稀释至刻度,摇匀即得(每1mL中含有绿原酸40μg)。
标准曲线绘制:
精密称取对照品溶液2,4,8,12,16,20μL分别注入液相色谱柱,以峰面积为纵坐标,绿原酸进样量为横坐标,绘制标准曲线。
测试样品的制备及测定:
取实施例1和对比例2-4对应得到的发酵粉和对比例1得到的固体粉末,过100目筛,各称取粉末0.05g,置于50mL的量瓶中,加50%甲醇使其稀释至刻度,称定重量,超声处理60min,放冷,称定重量,用 20%甲醇补足使完全溶解并稀释至刻度,摇匀,过滤,精密量取20μL,注入液相色谱仪,记录色谱图。每个样品测量4次。实验结果如表1所示。
表1
槲皮素/mg·g | 绿原酸/mg·g | 没食子酸/mg·g | |
实施例1 | 15.06±0.12 | 36.27±0.18 | 52.48±0.21 |
对比例1 | 6.39±0.26 | 13.17±0.19 | 26.52±0.26 |
对比例2 | 7.39±0.13 | 15.24±0.17 | 31.29±0.43 |
对比例3 | 10.48±0.35 | 17.44±0.36 | 39.56±0.39 |
对比例4 | 12.82±0.28 | 20.33±0.20 | 43.01±0.48 |
从三组样品的有效物质指标来看,实施例1中3项指标均为最高,其中,具有抗糖化作用的槲皮素含量是对比例1的2.36倍,具有抗氧化作用的绿原酸含量是对比例1的2.75倍,没食子酸含量是对比例1的1.98 倍。结果证明,实施例1制备工艺的效果明显优于对比例1。
对比样品实施例1和对比例2组,同样可以发现,实施例1中槲皮素、绿原酸、没食子酸含量分别是对比例2组的2.04倍、2.38倍、1.68倍。证明采用洋甘菊花制备的组合物各项功能物质指标含量优于采用滁菊组。
对比样品实施例1组和对比例3组发现,实施例1中的槲皮素、绿原酸和没食子酸含量分分别是对比例3组的1.44倍,2.07倍和1.33倍,表明本发明选用的菌株组合为优选的菌株组合。
对比样品实施例1和对比例4组发现,实施例1中的槲皮素、绿原酸和没食子酸含量分分别是对比例3组的1.17倍,1.78倍和1.22倍,表明本发明选用的菌株比例为优选菌株比例。
实验例2体外抑制抗糖化终产物的测定实验
测试样品:实施例1的发酵粉命为M1样品,对比例1得到的固体粉命为M2样品,对比例2得到的发酵粉命为M3样品,对比例3得到的发酵粉为命M4样品,对比例4得到的发酵粉为命M5样品。
实验步骤:以pH7.4磷酸盐缓冲液为基质液,配制终浓度含体积分数为5%牛血清白蛋白溶液及1mol/L葡萄糖的溶液(蛋白质高糖溶液);用此溶液分别配制不同浓度的M1、M2、M3、M4、M5样品,样品M1设置低 (0.5g·L-1)、中(2g·L-1)、高(4g·L-1)三个剂量组,M2、M3、M4和M5 采用高剂量组(4g·L-1),同时配制阳性对照组和阴性对照组,其中采用氨基胍(4g·L-1)为阳性对照组,不含待测物的蛋白质高糖溶液为阴性对照组。以上液体均用0.2μm滤膜除菌过滤,分装于1.5mL尖底离心管内。37℃恒温有氧避光条件下连续孵育28d,每间隔7d取不同实验组样品一次,其中每次连续取样10次,所有样品取出后-20℃冰箱冷冻保存。
蛋白糖化终末产物由日本岛津RF-510荧光分光光度计测定,激发波长 370nm,发射波长440nm。
抑制率=(阴性对照组荧光强度-实验组荧光强度)/阴性对照组荧光强度。抑制率结果如表2所示,
表2
注:表2中抑制率为每次取样后根据公式的计算得到的抑制率的平均值和方差。
AGEs含量与荧光强度呈正相关,抑制率数值越大,代表产品抑制AGEs 效果更好。由表2可知,随着孵育时间的延长,各实验组AGEs生成抑制率都有一定程度的提高。尤其是样品M1高剂量组生成抑制率同时间下要优于氨基胍阳性对照组,表明本发明的产品具有体外抗糖化的功效。
与此同时可以看出,高剂量下,样品M1 AGEs生成抑制率要优于M3, M4和M5,要显著优于M2。表明采用实施例1工艺制备的样品M1抗糖化效果明显优于对比例1制备得到的样品M2,即酶解+发酵的工艺能明显增强组合物的抗糖化效果。
另一方面,采用实施例1制备的样品M1抗糖化效果也要优于对比例2 得到的样品M3,即,采用洋甘菊花制备的发酵组合物抗糖化效果优于采用滁菊制备出来的样品。
另一方面,采用实施例1制备的样品M1抗糖化效果也要优于对比例3 得到的样品M4,即,采用本发明优选的植物乳杆菌、嗜酸乳杆菌、干酪乳杆菌、两歧双歧杆菌、乳酸乳球菌乳酸亚种和肠膜明串珠菌肠膜亚种的菌株组合方式,要高于植物乳杆菌、嗜酸乳杆菌、干酪乳杆菌、两歧双歧杆菌和乳酸乳球菌乳酸亚种的组合方式,本发明的菌株组合方式为优选菌株组合。
另一方面,采用实施例1制备的样品M1抗糖化效果也要优于对比例4 得到的样品M5,即,本发明选用的菌株比例为优选比例。
实验例3利用小鼠衰老模型验证抗糖化能力实验
测试样品:实施例1的发酵粉命为M1样品,对比例1得到的固体粉命为M2样品,对比例2得到的发酵粉命为M3样品,对比例3得到的发酵粉为命M4样品,对比例4得到的发酵粉为命M5样品。
实验设计:
ICR小鼠,体质量(18±2)g,雌雄兼用。随机共分为9组,分别是正常对照组,模型对照组,样品M1低剂量组,样品M1中剂量组,样品M1高剂量组,样品M2高剂量组,样品M3高剂量组,样品M4高剂量组,样品 M5高剂量组,每组12只小鼠。
采用皮下注射D-半乳糖(1g/kg)造小鼠衰老模型,D-半乳糖需现配现用。模型对照组,样品M1低剂量组,样品M1中剂量组,样品M1高剂量组,样品M2高剂量组,样品M3高剂量组,样品M4高剂量组,样品M5高剂量组每日进行皮下注射给药D-半乳糖,给药体积为0.1ml/10g,连续每日背部皮下注射42天,正常对照组注射同等体积的生理盐水。42天后,小鼠毛色逐渐枯槁,无光泽,易脱落,皮肤弹性差,精神萎靡,嗜睡倦怠,进食量减少,食欲不振,呈现出明显衰老体征,表明造模成功。
对上述样品M1低剂量组,样品M1中剂量组,样品M1高剂量组,样品M2高剂量组,样品M3高剂量组,样品M4高剂量组,样品M5高剂量组每日灌胃给药,其中低剂量组的给药剂量为2.5g/kg,中剂量组的给药剂量为5.0g/kg,高剂量组的给药剂量为10.0g/kg。正常对照组、模型对照组每日给予10.0g/kg的生理盐水。42天后,摘眼球放血处死小鼠,取血进行检测超氧化物歧化酶(SOD酶)、谷胱甘肽过氧化物酶(GSH-Px)活力。并检测皮肤中丙二醛(MDA)、羟脯氨酸含量。
超氧化物歧化酶(SOD酶)检测参照南京森贝伽生物科技有限公司的小鼠超氧化物歧化酶(SOD酶)检测试剂盒说明书方法检测SOD活力;谷胱甘肽过氧化物酶(GSH-Px)检测参照南京森贝伽生物科技有限公司的小鼠谷胱甘肽过氧化物酶(GSH-Px)检测试剂盒说明书方法检测GSH-Px活力。
10%皮肤匀浆的制备:取脱毛背部皮肤组织0.5g,经预冷生理盐水漂洗,除去皮下脂肪和其它结缔组织,滤纸拭干,称重,量取该组织块9倍重量预冷生理盐水,用内切式组织匀浆机制成10%组织匀浆,反复冻融3次,使细胞完全破碎,内容物全部游离在液相中,用此检测丙二醛(MDA)、羟脯氨酸
羟脯氨酸检测参照南京森贝伽生物科技有限公司的小鼠羟脯氨酸检测试剂盒说明书方法检测;丙二醛(MDA)检测参照南京森贝伽生物科技有限公司的小鼠丙二醛(MDA)检测试剂盒说明书方法检测,实验结果如表 3所示
表3各实验组小鼠皮肤及血液生化指标检测结果(n=12)
注:*,与模型对照组相比,P<0.05;**,与模型对照组相比,P<0.01。+,与样品M2高剂量组相比,P<0.05。
SOD酶、GSH-Px酶活力提高,表明机体清除自由基能力增强,抗氧化能力提高。丙二醛含量降低,则表示皮肤中堆积的脂质过氧化产物得到清除,羟脯氨酸含量高,则代表皮肤中胶原蛋白含量增加,皮肤趋于年轻化,有抗衰老效果。
从上表可以看出,与模型对照组相比,样品M1高剂量组能十分显著提高血液中SOD酶、GSH-Px酶活力(P<0.01),显著提高抗氧化能力,皮肤自由基防护功能增强。同时,能十分显著提高皮肤中羟脯氨酸含量(P< 0.01),降低丙二醛含量(P<0.01)。
相比而言,样品M2高剂量组和样品M3虽能一定程度提高SOD酶、GSH-Px 酶活力(P<0.05),降低丙二醛含量(P<0.05),但效果明显不如样品M1 高剂量组(P<0.05)。结果表明,采用实施例1发酵法制备的组合物效果要明显优于对比例1未发酵制备方法,且优于对比例2成分替换后的组合物。
相比之下,样品M4高剂量组和样品M5高剂量组也能显著提高SOD酶、 GSH-Px酶活力(P<0.01),显著提高皮肤中羟脯氨酸含量(P<0.5),降低丙二醛含量(P<0.5),但生化指标整体低于样品M1高剂量组。
综上所述,通过本发明的特定配伍及特定制备工艺,制备出来的洋甘菊花、鱼腥草、余甘子、山楂发酵组合物,能够协同增效,显著增加SOD 酶、GSH-Px酶抗氧化活力表达、提高皮肤羟脯氨酸及降低丙二醛,具有实现皮肤的抗糖化与抗衰老能力的提升。
实验例4人群试服实验
测试样品:实施例1的发酵粉命为M1样品,称重,20g/袋;对比例1 得到的固体粉命为M2样品,称重,20g/袋;对比例2得到的发酵粉命为M3 样品,称重,20g/袋。
人群选择
选取90名年龄在35~60岁的女性,皮肤有雀斑、黄褐斑或老年斑,面色暗沉或有痤疮,毛孔粗大,面部有明显皱纹,肤质较差者。受试者在试验期间不得服用其他影响皮肤的功能食品或药物及停用影响结果判定的化妆品。试服期间保持原来的饮食习惯,正常饮食。
实验设计
实验人员随机分为3组,每组30人,分别服用对应的测试样品。每天服用2次,每次20g,用适量温水冲服,连续服用30天。记录皮肤试服前和试服后变化,包括皮肤肤色、皱纹测试和皮肤整体状态。测试前需清洁面部。
分析方法
皮肤肤色测试:皮肤光泽度使用德国CK多功能皮肤测试仪MPA10测定,数值越高,则皮肤光泽度越好。测试部位:脸颊,测量3次取平均值。
皮肤皱纹测试:使用FPCANF-PRIMOS高分辨率皮肤老化成像仪评价皮肤皱纹状况,测量3次取平均值。
皮肤整体状态评分:采用5分制评分标准,根据个人对试服前后的皮肤整体状态的变化程度打分,打分标准如下:
5分:皮肤完全好转,皮肤白皙,有光泽,有弹性,色斑、痤疮或皱纹全部消失;
4分:皮肤较试服前有明显改善,色斑明显变浅,痤疮或皱纹有明显减少:
3分:皮肤状况稍有改善,色斑变浅,痤疮或皱纹稍有减少;
2分:皮肤前后无明显变化,无改善效果;
1分:试服后皮肤变差。
表4试服前后皮肤指标及皮肤状态整体打分结果
从上表可以看出,三组试服后皮肤光泽度都有改善,皱纹数量也有降低,试服者普遍认为试服后皮肤状态比试服前有所改善。其中,样品M1组各项指标均最优,其改善皮肤的效果要明显优于M2,也优于M3组。
显然,上述实施例仅仅是为清楚地说明所作的举例,而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的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸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 (4)
1.一种抗糖化抗衰老发酵组合物,其特征在于,按质量份计,包括洋甘菊花100份,鱼腥草80份,余甘子80份,山楂60份,植物乳杆菌0.1份、嗜酸乳杆菌0.1份、干酪乳杆菌0.3份、两歧双歧杆菌0.4份、乳酸乳球菌乳酸亚种0.2份、肠膜明串珠菌肠膜亚种0.5份,纤维素酶4份,果胶酶4份,酸碱调节剂7份和水2200份;或者包括洋甘菊花120份,鱼腥草100份,余甘子60份,山楂50份,植物乳杆菌0.3份、嗜酸乳杆菌0.2份、干酪乳杆菌0.2份、两歧双歧杆菌0.5份、乳酸乳球菌乳酸亚种0.1份、肠膜明串珠菌肠膜亚种0.2份,纤维素酶3份,果胶酶3份,酸碱调节剂7份和水2000份;上述组合物按照下述制备方法制得,制备方法包括步骤:(1)将洋甘菊花、鱼腥草、余甘子和山楂粉碎过筛至目数为100-600目,加水混合均匀后添加酸碱调节剂至pH为4.0-5.0,得混合液;(2)向所述混合液中加入酶制剂酶解,酶解温度40-70℃,酶解时间90-240min,酶解结束后添加酸碱调节剂至pH为5.0-6.0,得酶解液;(3)将所述酶解液在温度80-102℃下提取10-90min,得提取液;(4)向所述提取液中加入发酵菌粉发酵,发酵温度为15-45℃,时间为36-96h,得发酵液,将所述发酵液固液分离得到发酵清液。
2.根据权利要求1所述的抗糖化抗衰老发酵组合物,其特征在于,所述植物乳杆菌活菌数为1000-3000亿CFU/g,所述嗜酸乳杆菌1000-5000亿CFU/g,所述干酪乳杆菌1000-3000亿CFU/g,所述两歧双歧杆菌500-2000亿CFU/g,所述乳酸乳球菌乳酸亚种800-3000亿CFU/g,所述肠膜明串珠菌肠膜亚种1000-3000亿CFU/g。
3.根据权利要求1所述的抗糖化抗衰老发酵组合物,其特征在于,所述纤维素酶活力为10000-30000U,果胶酶活力为10000-50000U。
4.根据权利要求1-3任一所述的抗糖化抗衰老发酵组合物的制备方法,其特征在于,包括如下步骤:
(1)将洋甘菊花、鱼腥草、余甘子和山楂粉碎过筛至目数为100-600目,加水混合均匀后添加酸碱调节剂至pH为4.0-5.0,得混合液;
(2)向所述混合液中加入酶制剂酶解,酶解温度40-70℃,酶解时间90-240min,酶解结束后添加酸碱调节剂至pH为5.0-6.0,得酶解液;
(3)将所述酶解液在温度80-102℃下提取10-90min,得提取液;
(4)向所述提取液中加入发酵菌粉发酵,发酵温度为15-45℃,时间为36-96h,得发酵液,将所述发酵液固液分离得到发酵清液。
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