CN117085079A - 植物乳杆菌sf-l38的发酵制品及其制备方法和应用 - Google Patents
植物乳杆菌sf-l38的发酵制品及其制备方法和应用 Download PDFInfo
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- CN117085079A CN117085079A CN202311362112.7A CN202311362112A CN117085079A CN 117085079 A CN117085079 A CN 117085079A CN 202311362112 A CN202311362112 A CN 202311362112A CN 117085079 A CN117085079 A CN 117085079A
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- lactobacillus plantarum
- fermented product
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Abstract
本发明涉及微生物发酵制品技术领域，具体涉及一种植物乳杆菌SF‑L38的发酵制品及其制备方法和应用，被发酵底物包括如下重量份数的原料：净化水30~50份，红景天提取物2~4份，樱花提取物2~5份，玫瑰提取物2~5份，丁香花提取物1~3份，洋甘菊提取物3~5份，番红花提取物1~2份，金盏花提取物2~3份；植物乳杆菌（Lactobacillus plantarum）SF‑L38已于2021年9月23日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏地址为北京市朝阳区北辰西路1号院3号，保藏编号为CGMCC No.23475。该发酵制品能够直接涂抹于皮肤表面，有效清除自由基、抑制活性氧的形成、减少细胞外基质降解，具有缓解紫外损伤、修复皮肤光老化的显著功能，方便快捷，效果快速持久。
Description
技术领域
本发明涉及微生物发酵制品技术领域，具体涉及一种植物乳杆菌SF-L38的发酵制品及其制备方法和应用。
背景技术
皮肤老化是人体衰老过程中的一个伴随症状，也是皮肤众多健康问题中最受关注的一个现象。老化的皮肤除美观性下降外，其防御损伤的功能也明显受损。皮肤老化的因素有很多，其中主要的一大因素是紫外辐射。紫外辐射导致的皮肤老化又称为光老化（photoaging）。光老化是导致皮肤老化的主要外因，80%以上的面部老化均由光老化引起。
光老化形成与细胞内的活性氧自由基（ROS）有密切联系。紫外辐射中的UV-B作用于皮肤中的光敏物质或色基，可诱导产生ROS，如果ROS积累过量，超出了机体的清除能力，过剩的ROS破坏皮肤自身的抗氧化体系，使抗氧化酶活性水平下降，造成脂质、蛋白质、核酸等生命物质的氧化变性，破坏细胞膜的流动性和通透性，导致细胞结构和功能的改变，最终引起皮肤系统紊乱，皮肤出现变薄、干燥、皱纹细小、出汗不足以及敏感性增加等老化症状。因此，清除自由基、增强抗氧化能力是减少紫外损伤、延缓皮肤衰老的重要途径之一。
据报道，植物乳杆菌的菌体及其代谢产物有清除自由基、抗氧化的能力。中国专利CN104127443B公开了一种植物乳杆菌C88与人参多糖组合物、制备方法及应用，该组合物具有良好的抗氧化性能，体外有效清除DPPH自由基和ABTS自由基，体内试验中可增强自然衰老小鼠血清及肝脏中抗氧化酶的活力，降低自然衰老小鼠血清及肝脏中MDA的含量。但是该类产品主要通过口服调节、改善机体机能，逐渐达到延缓衰老的效果，改善皮肤状况的周期长、见效慢。
发明内容
针对抗衰老产品改善皮肤状况周期长、见效慢等技术问题，本发明提供一种植物乳杆菌SF-L38的发酵制品及其制备方法和应用。
第一方面，本发明提供一种植物乳杆菌SF-L38的发酵制品，发酵制品的原料包括被发酵底物、植物乳杆菌（Lactobacillus plantarum）SF-L38和辅料；被发酵底物包括如下重量份数的组分：净化水30~50份，红景天提取物2~4份，樱花提取物2~5份，玫瑰提取物2~5份，丁香花提取物1~3份，洋甘菊提取物3~5份，番红花提取物1~2份，金盏花提取物2~3份；植物乳杆菌SF-L38已于2021年9月23日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏地址为北京市朝阳区北辰西路1号院3号，保藏编号为CGMCC No.23475。
进一步的，辅料包括如下重量份数的组分：油脂3~6份、乳化剂3~5份。
进一步的，油脂为橄榄油，乳化剂为甘油硬脂酸酯。
本发明使用原料的各种组分具有以下功效：
红景天提取物：减少皮肤黑色素的生成，改善皮肤色素沉着状态，达到皮肤美白功效；
樱花提取物：美白祛斑，抗氧化，防止衰老；
玫瑰提取物：滋润养颜，祛斑，清除自由基，保湿、锁水；
丁香花提取物：抗氧化，抗炎，抗皱，皮肤调理；
洋甘菊：补水保湿，修复敏感肌，改善浮肿、黑眼圈，预防面部衰老；
番红花：抗炎，抗氧化，美白，防晒，抗衰老；
金盏花提取物：舒缓抗敏，保湿，抗菌，改善毛孔粗大；
橄榄油：抗衰老，保湿，去角质；
甘油硬脂酸酯：润滑，乳化。
第二方面，本发明还提供一种上述发酵制品的制备方法，包括如下步骤：
（1）将被发酵底物各组分混合，得混合浆料；
（2）将混合浆料高温灭菌，冷却得灭菌液；
（3）将植物乳杆菌SF-L38接种到MRS液体培养基中，37℃下培养20~30h，得接种剂；
（4）将接种剂接种到灭菌液中，37℃下发酵24~48h，得发酵液；
（5）将发酵液破壁，与辅料混合后得发酵制品。
进一步的，步骤（3）中，植物乳杆菌SF-L38的接种量为1.5%。
进一步的，步骤（4）中，接种剂的接种量为1.5%。
进一步的，步骤（5）中，破壁方法为：将发酵液输送至高压喷雾破壁机中，压力设置为22Mpa，进行破壁。
第三方面，本申请还提供一种上述发酵制品在制备具有抗紫外损伤、延缓皮肤光老化功能的产品方面的应用。
本发明的有益效果在于：
经试验验证，本发明提供的植物乳杆菌SF-L38的发酵制品能够直接涂抹于皮肤表面，有效清除自由基、抑制活性氧的形成、减少细胞外基质降解，具有缓解紫外损伤、修复皮肤光老化的显著功能，方便快捷，效果快速持久。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，对于本领域普通技术人员而言，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1是本发明实施例6中表皮厚度测试结果图。
图2是本发明实施例6中经皮水分丢失量（TEWL）测试结果图。
图3是本发明实施例6中含水量测试结果图。
具体实施方式
为了使本技术领域的人员更好地理解本发明中的技术方案，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，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，都应当属于本发明保护的范围。
下述实施例使用的MRS液体培养基包括以下组分：
蛋白胨10g、牛肉粉5g、酵母粉4g、K2HPO4·7H2O 2g、柠檬酸三铵2g、葡萄糖20g、CH3COONa·3H2O 5g、MgSO4·7H2O 0.2g、MnSO4·4H2O 0.05g、吐温80 1mL、蒸馏水1000mL。
下述实施例使用的MRS固体平板培养基包括以下组分：
蛋白胨10g、牛肉粉5g、酵母粉4g、K2HPO4·7H2O2g、柠檬酸三铵2g、葡萄糖20g、CH3COONa·3H2O 5g、MgSO4·7H2O 0.2g、MnSO4·4H2O 0.05g、吐温-80 1mL、琼脂15.0g、蒸馏水1000mL；
下述实施例中的被发酵底物各组分的制备方法为：
洋甘菊提取物、番红花提取物：取洋甘菊或番红花干燥花朵5.0kg，加12倍蒸馏水，加热回流提取3次，每次1.5h，过滤，合并提取液，旋蒸至1mL溶液含1g生药，加95%乙醇，醇沉，使溶液含醇量达60%，静置48h，过滤，旋蒸，60℃恒温干燥，粉碎，得洋甘菊提取物或番红花提取物；
红景天提取物：红景天加水煎煮，浓缩后70%乙醇沉淀、红景天加水超声提取、红景天加水煎煮、红景天加70%乙醇超声提取、红景天加70%乙醇回流提取、红景天加水煎煮，浓缩后加60%乙醇沉淀，50℃下真空干燥，得到红景天提取物；
樱花提取物：取樱花花瓣经过干燥处理，粉碎至60目，与提取剂（1,2-己二醇和甲基丙二醇1:1混合）以质量比1:60混合，80℃条件下浸提2.5h，得到樱花提取液，浓缩后加70%乙醇沉淀，60℃下真空干燥，得到樱花提取物；
丁香花提取物：取丁香花花瓣经干燥、粉碎得丁香花粉，取丁香花粉20 g，按乙醇浓度80%，料液比1∶11，提取温度60℃，提取时间100 min，水浴加热，回流提取3次，合并每次提取液，减压浓缩后，真空干燥，得丁香花提取物；
玫瑰提取物：玫瑰花称重，干燥，粉碎，用90%~95%乙醇渗滤，乙醇用精制盐酸调pH至4~5，渗滤3~4小时，取乙醇滤液，滤液减压回收乙醇至无醇味，得浓缩液，将浓缩液喷雾干燥，得玫瑰花提取物；
金盏花提取物：将金盏花与浓度为60%~70%的酒精混合，以12000r/min的转速进行组织破碎3次，每次20分钟左右，静置4小时得混合液，用膜过滤，得到料液，并放置蒸馏器中蒸馏，得到精料，将精料放置在油水分离器中分离，得到金盏花提取物。
植物乳杆菌（Lactobacillus plantarum）SF-L38为申请人分离鉴定所得，已于2021年9月23日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏地址为北京市朝阳区北辰西路1号院3号，保藏编号为CGMCC No.23475，相关信息已公开于中国专利CN114437997B。
实施例1 制备植物乳杆菌SF-L38发酵制品
（1）首先将净化水40份，红景天提取物3份，樱花提取物3.5份，玫瑰提取物3.5份，丁香花提取物2份，洋甘菊提取物4份，番红花提取物1.5份，金盏花提取物2.5份混合，得混合浆料。
（2）将混合浆料输送至灭菌容器中，经过120℃高压灭菌30min，然后冷却至40℃，得到灭菌液；
（3）将植物乳杆菌SF-L38接种到MRS液体培养基中，接种量为1.5%，在37℃下培养24h制备接种剂；
（4）将接种剂接种于灭菌液中，接种量为1.5%，在37℃下培养40h，制备发酵液；
（5）将发酵液输送至高压喷雾破壁机中，压力设置22MPa，进行破壁处理；
（6）将破壁后的发酵液与橄榄油4份、甘油硬脂酸酯4份混合调配后，得发酵制品。
实施例2 制备植物乳杆菌SF-L38发酵制品
（1）首先将净化水50份，红景天提取物4份，樱花提取物5份，玫瑰提取物5份，丁香花提取物3份，洋甘菊提取物5份，番红花提取物2份，金盏花提取物3份混合，得混合浆料。
（2）将混合浆料输送至灭菌容器中，经过120℃高压灭菌30min，然后冷却至40℃，得到灭菌液；
（3）将植物乳杆菌SF-L38接种到MRS液体培养基中，接种量为1.5%，在37℃下培养20h制备接种剂；
（4）将接种剂接种于灭菌液中，接种量为1.5%，在37℃下培养24h，制备发酵液；
（5）将发酵液输送至高压喷雾破壁机中，压力设置22MPa，进行破壁处理；
（6）将破壁后的发酵液与橄榄油6份、甘油硬脂酸酯5份混合调配后，得发酵制品。
实施例3 制备植物乳杆菌SF-L38发酵制品
（1）首先将净化水30份，红景天提取物2份，樱花提取物2份，玫瑰提取物2份，丁香花提取物1份，洋甘菊提取物3份，番红花提取物1份，金盏花提取物2份混合，得混合浆料。
（2）将混合浆料输送至灭菌容器中，经过120℃高压灭菌30min，然后冷却至40℃，得到灭菌液；
（3）将植物乳杆菌SF-L38接种到MRS液体培养基中，接种量为1.5%，在37℃下培养30h制备接种剂；
（4）将接种剂接种于灭菌液中，接种量为1.5%，在37℃下培养48h，制备发酵液；
（5）将发酵液输送至高压喷雾破壁机中，压力设置22MPa，进行破壁处理；
（6）将破壁后的发酵液与橄榄油3份、甘油硬脂酸酯3份混合调配后，得发酵制品。
对比例1 制备植物乳杆菌JYLP-326发酵制品
对比例1与实施例1制备方法不同的是，采用植物乳杆菌JYLP-326进行发酵，其它处理均同实施例1。
对比例2 制备植物乳杆菌JYLP-375发酵制品
对比例2与实施例1制备方法不同的是，采用植物乳杆菌JYLP-375进行发酵，其它处理均同实施例1。
实施例4 菌株抗氧化特性测试
对比菌株为：植物乳杆菌JYLP-375、植物乳杆菌JYLP-326和发酵乳杆菌XJC60，其中发酵乳杆菌XJC60为行业内公认的具有抗氧化能力的乳杆菌菌种标品，上述菌种均来源于开放的菌种保藏库，能够在市面上购买得到。
对植物乳杆菌SF-L38、植物乳杆菌JYLP-375、植物乳杆菌JYLP-326和发酵乳杆菌XJC60进行如下抗氧化特性测试：
（1）DPPH自由基清除能力检测
将四种菌株分别按1.5%的接种量接种于MRS液体培养基中，37℃培养18~24h，发酵菌液8000r/min离心15分钟，收集乳杆菌发酵上清液。
在96孔板中每孔加入100µL 0.2mmol/L DPPH乙醇溶液和100µL乳杆菌发酵上清液，震荡混匀后避光反应30min，于517nm处测吸光值（Ai），用100µL MRS液体培养基代替乳杆菌发酵上清液，同操作得到吸光值（Aj），同时设置100µL无水乙醇+100µL蒸馏水的空白孔（Ab）和100µL 0.2mmol/L DPPH乙醇溶液+100µL蒸馏水的对照孔（Ac），每个处理3个重复。计算DPPH自由基清除率：
发酵制品的DPPH自由基清除率越高，其抗氧化特性越强。如表1结果所示，植物乳杆菌SF-L38发酵制品的DPPH自由基清除能力为42%，具有很强的抗氧化特性。
表1 植物乳杆菌SF-L38 DPPH自由基清除率测试结果
（2）羟基自由基清除能力检测
在96孔板中每孔加入30µL 0.75mmol/L邻二氮菲溶液后，加入60µL 0.2mol/L磷酸盐缓冲溶液（PBS，pH=7.40）和30µL 0.75mmol/L FeS04，制成工作液。在该工作液中分别加入30µL的乳杆菌发酵上清液，最后加入30µL 0.01%(v/v) H2O2，37℃水浴60min，在536nm处测定各样品吸光度（Ai），用30µL MRS液体培养基代替乳杆菌发酵上清液，同操作得到吸光值（Aj），同时设置在工作液中加入60µL蒸馏水的空白孔（Ab）和30µL的0.01%(v/v) H2O2+30µL蒸馏水的对照孔(Ac），每个处理3个重复。羟基自由基清除率按下式计算：
样品的羟基自由基清除率越高，样品的抗氧化特性越强。SF-L38的羟基自由基清除能力为70%，抗氧化特性显著，如表2结果所示。
表2植物乳杆菌SF-L38羟基自由基清除率测试结果
实施例5 发酵制品抗皮肤细胞紫外损伤能力的评估
（1）建立紫外辐射的人角质细胞光损伤细胞模型
采用人角质细胞（中山大学附属第一医院皮肤科馈赠）作为研究对象，采用完全培养基对人角质细胞进行培养。将传代人角质细胞种于孔板中，当细胞增殖覆盖孔板80%面积时，进行紫外辐射。
紫外辐照的条件为：采用UVB-313EL灯管（ANTOINE，波峰值313nm，40W），调整辐射强度为0.02mW/cm2，辐射剂量为18mJ/cm2，照射时间为15min。
（2）实施例1和对比例1、对比例2制备的发酵制品对紫外损伤的人角质细胞存活率的影响
人角质细胞培养至对数期，收集细胞。96孔板内设空白对照组和三个处理组，调整细胞悬液浓度，以1×105个/mL每孔接种100µL细胞悬液，每组均设3个实验孔。边缘孔用无菌PBS填充，细胞培养板置于5%CO2，37℃培养箱培养24h。培养24h细胞长至单层后，吸走培养基，用无菌PBS清洗3次，清除原有培养基后，每孔加入100µL无菌PBS。三个处理组和空白对照组给予15min的紫外照射，辐射剂量为18mJ/cm2。
照射后，第一个处理组每孔加入100µL含有 10%实施例1发酵制品的完全培养基，第二个处理组每孔加入100µL含有 10%对比例1发酵制品的完全培养基，第三个处理组每孔加入100µL含有10%对比例2发酵制品的完全培养基，空白对照组只加入完全培养基。培养24h后，用CCK-8法测定细胞存活率。
如表3结果所示，植物乳杆菌SF-L38发酵上清液对紫外损伤的人角质细胞存活率有显著提高作用。
表3 植物乳杆菌SF-L38对紫外损伤的人角质细胞存活率测试结果
实施例6 植物乳杆菌SF-L38发酵制品抗皮肤细胞紫外损伤能力的评估
选用30只成年雌小鼠 （体重为250~350g）进行正式试验，试验前约24h，将实验动物背部脊柱两侧毛剪掉，不可损伤表皮，备4块去毛区，每块去毛面积约为2cm×2cm。
首先随机选取10只小鼠，进行MED测试。根据小鼠身材大小，制作固定装置，只辐照小鼠背部去毛区域，每只照射不同剂量的UVB，剂量以25％递增，照射时间10min，24小时根据肉眼判断出皮肤对紫外照射的红斑反应情况，以出现可见红斑反应剂量确定本次实验的MED值。紫外辐照计测量出固定高度下紫外线到达小鼠背部皮肤的辐照强度为83.4μW/cm2，MED=83.4μW/cm2ｘ10minｘ60＝50mJ/cm2。因此本次实验的MED值为50mJ/cm2。
将30只小鼠随机分为3组，对照组、药物干预组和紫外照射组，每组10只。药物干预组和紫外照射组，每两天进行一次紫外照射，照射时间为5min，一次照射总剂量为75mJ/cm2，共照射3次。药物干预组每天都给小鼠背部涂抹实施例1的发酵制品，紫外照射组每天涂抹MRS液体培养基。
照射完成24小时后，借用多功能皮肤检测仪对小鼠背部皮肤进行检测，检测的参数有经皮水分丢失量（TEWL）、表皮厚度和含水量。结果如图1-3所示，可以看出，紫外照射组中表皮厚度显著厚于对照组，表皮组织增生、变厚是皮肤衰老的明显信号，从TEWL和含水量上也可以得出，紫外照射组小鼠光衰老表型显著。药物干预组的表皮厚度明显低于紫外照射组，TEWL和含水量明显高于紫外照射组。因此，实施例1的发酵制品可以有效抵抗紫外损伤、延缓皮肤光老化。
尽管通过参考附图并结合优选实施例的方式对本发明进行了详细描述，但本发明并不限于此。在不脱离本发明的精神和实质的前提下，本领域普通技术人员可以对本发明的实施例进行各种等效的修改或替换，而这些修改或替换都应在本发明的涵盖范围内/任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到变化或替换，都应涵盖在本发明的保护范围之内。
Claims (8)
1. 一种植物乳杆菌SF-L38的发酵制品，其特征在于，发酵制品的原料包括被发酵底物、植物乳杆菌（Lactobacillus plantarum）SF-L38和辅料；
被发酵底物包括如下重量份数的组分：净化水30~50份，红景天提取物2~4份，樱花提取物2~5份，玫瑰提取物2~5份，丁香花提取物1~3份，洋甘菊提取物3~5份，番红花提取物1~2份，金盏花提取物2~3份；
植物乳杆菌SF-L38已于2021年9月23日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏地址为北京市朝阳区北辰西路1号院3号，保藏编号为CGMCC No.23475。
2.如权利要求1所述的发酵制品，其特征在于，辅料包括如下重量份数的组分：油脂3~6份、乳化剂3~5份。
3.如权利要求2所述的发酵制品，其特征在于，油脂为橄榄油，乳化剂为甘油硬脂酸酯。
4.一种如权利要求1所述发酵制品的制备方法，其特征在于，包括如下步骤：
（1）将被发酵底物各组分混合，得混合浆料；
（2）将混合浆料高温灭菌，冷却得灭菌液；
（3）将植物乳杆菌SF-L38接种到MRS液体培养基中，37℃下培养20~30h，得接种剂；
（4）将接种剂接种到灭菌液中，37℃下发酵24~48h，得发酵液；
（5）将发酵液破壁，与辅料混合后得发酵制品。
5.如权利要求4所述的制备方法，其特征在于，步骤（3）中，植物乳杆菌SF-L38的接种量为1.5%。
6.如权利要求4所述的制备方法，其特征在于，步骤（4）中，接种剂的接种量为1.5%。
7.如权利要求4所述的制备方法，其特征在于，步骤（5）中，破壁方法为：将发酵液输送至高压喷雾破壁机中，压力设置为22Mpa，进行破壁。
8.一种如权利要求1所述发酵制品在制备具有抗紫外损伤、延缓皮肤光老化功能的产品方面的应用。
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