CN112842952A - 酵母菌发酵的桦树汁及其在防脱生发组合物中的应用 - Google Patents
酵母菌发酵的桦树汁及其在防脱生发组合物中的应用 Download PDFInfo
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- CN112842952A CN112842952A CN201911189622.2A CN201911189622A CN112842952A CN 112842952 A CN112842952 A CN 112842952A CN 201911189622 A CN201911189622 A CN 201911189622A CN 112842952 A CN112842952 A CN 112842952A
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- yeast
- birch juice
- birch
- fermented
- juice
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Abstract
酵母菌发酵的桦树汁及其在防脱生发组合物中的应用。本发明涉及一种酵母菌发酵的桦树汁,其通过采用酵母菌作为菌种在包含桦树汁和任选的生姜粉的培养基中进行发酵得到的。本发明还涉及一种生产酵母菌发酵的桦树汁的方法,其包括采用酵母菌作为菌种在包含桦树汁和任选的生姜粉的培养基中进行发酵的步骤。
Description
技术领域
本发明涉及酵母菌发酵的桦树汁及其在防脱生发组合物中的应用,其中所述酵母菌发酵的桦树汁是通过采用酵母菌作为菌种在包含桦树汁和任选的生姜粉的培养基中进行发酵而得到的。
背景技术
脱发是常见的皮肤科疾病,以雄激素性脱发最为常见,脱发影响美观,给患者带来心理压力。目前防脱生发问题的主要解决方法是生发药物和手术。手术治疗费用较高,有创伤,需要一定的恢复期;药物治疗主要是非那雄胺和米诺地尔,往往有较严重的副作用。
桦树汁(也称桦树液)是桦树树皮被划开或树干钻孔流出的新鲜汁液,无色或浅黄色,无沉淀及杂质,具有淡淡的桦树清香。桦树汁是白桦树的生命之源,富含人体需要的多种果糖、氨基酸、维生素、生物素、矿物质等,具有抗疲劳、抗衰老、治疗烧烫伤、促进细胞生长的功效。北欧有传统使用桦树汁洗头和滋养头皮的记载。
酵母菌发酵能够富集营养成分,降低毒副作用,在化妆品领域的应用具有广阔前景。目前,使用酵母菌发酵的桦树汁来防止脱发的应用还没有报道。
为探索发酵的桦树汁的应用,本发明人采用酵母菌,以桦树汁为主要底物,任选地向其中添加促生长成分,例如生姜粉,进行发酵工艺,所得酵母菌发酵的桦树汁具有良好的防脱生发功效。
发明内容
本发明人经研究发现,采用酵母菌作为菌种,桦树汁作为主要底物,任选地向其中添加生姜粉进行发酵,可以得到一种具有改进性能的酵母菌发酵的桦树汁产物,其在具有桦树汁本身活性成分的同时还富含发酵工艺产生的活性成分,可作为防脱生发组合物的活性原料。
一方面,本发明涉及一种生产酵母菌发酵的桦树汁的方法,其包括采用酵母菌作为菌种在包含桦树汁和任选的生姜粉的培养基中进行发酵的步骤。
所述方法还包括过滤发酵中得到的发酵液,得到作为上清液的发酵的桦树汁滤液(即作为产物的“酵母菌发酵的桦树汁”),和得到酵母菌菌体副产物。
进一步地,所述方法还包括破碎所得到的酵母菌菌体,然后进行过滤,得到作为上清液的可溶性酵母菌溶胞物,和将所得酵母菌溶胞物与所述发酵的桦树汁滤液混合,过滤,得到发酵的桦树汁滤液产物(即作为优选产物的“酵母菌发酵的桦树汁”)。
在一个优选的实施方案中,所述方法包括下述步骤:
(1)采用酵母菌作为菌种,在包含桦树汁和任选的生姜粉的培养基中进行发酵,得到发酵液产物;
(2)过滤所述发酵液产物,分别得到酵母菌菌体和发酵的桦树汁滤液;
(3)破碎所述酵母菌菌体,然后进行过滤,得到作为上清液的可溶性酵母菌溶胞物,和破碎的菌体;和
(4)将所得酵母菌溶胞物与所述发酵的桦树汁滤液混合,过滤,得到发酵的桦树汁滤液产物(即作为优选产物的“酵母菌发酵的桦树汁”)。
本发明中可采用的酵母菌包括酿酒酵母菌、布拉迪酵母菌、解脂耶氏酵母菌等,其中优选酿酒酵母菌。所述酵母菌可以干酵母菌形式商购,例如商购于杜邦丹尼斯克有限公司、安琪酵母菌有限公司等;或者,所述酵母菌也可由野生酵母菌种扩大培养得到。
本发明中所涉及的桦树汁得自桦木科桦树属,其可来自白桦(Betula alba)、柔毛桦(Betula pubescens)、垂枝桦(Betula Pendula)和亚洲白桦(Betula platyphylla)等品种。所述桦树汁为在解冻至早春发叶之间,人工在桦树的树干基部钻孔收集而得的无色透明、无沉淀、无杂物,具有桦树清香营养丰富的汁液。所述桦树汁可商购得到并原样采用,例如可购自大兴安岭超越野生浆果开发有限责任公司。
本发明中可采用的桦树汁为桦树汁原液或浓缩的桦树汁,其中浓缩的桦树汁的浓缩倍数为约1.2-12倍,优选约1.5-6倍,更优选约2-4倍。
所述浓缩的桦树汁是通过将商购得到的桦树汁产品进行浓缩得到的。浓缩方法是本领域已知的,例如加热浓缩、低温真空浓缩、膜浓缩等。在本发明中,优选通过低温冷冻浓缩或膜浓缩工艺进行浓缩,例如,将商购的桦树汁原液输入低温干燥设备,降温至约-40℃至-70℃,抽真空至约0.1-30Pa而进行低温真空浓缩,从而得到不同浓缩倍数的浓缩桦树汁。
上述步骤(1)发酵是本领域已知的。例如,按照约60-70%(v/v)的装液量,将桦树汁培养基加入约100-12000L发酵罐中,在约105℃下灭菌约20-30分钟;以发酵培养基体积为准,将酵母菌种子液在无菌条件下接入发酵罐中,在约25-30℃温度和约180-350rmp的转速下进行搅拌,在约1.2-2.0vvm的通气量下,连续发酵约8-48小时,停罐,即完成发酵过程,得到发酵产物。
其中,上述酵母菌种子液可通过使活性干酵母菌复水得到。活性干酵母菌复水是本领域已知的,例如,按照约0.1-0.8g/L,优选约0.3-0.5g/L桦树汁底物的用量称取活性干酵母菌,将其加入到约5-15倍体积无菌水中,在约28-35℃下复水约15-20分钟,即得到酵母菌种子液。或者,酵母菌种子液也可以通过将野生酵母菌在培养基中扩大培养得到,这种培养是本领域已知的。
所述桦树汁培养基可以采用桦树汁、尤其是浓缩的桦树汁,例如浓缩1.5-6倍的桦树汁作为底物来制备。所述桦树汁在所述桦树汁培养基中的含量在约93%以上,优选约95%以上,基于所述桦树汁培养基的总重量。
优选地,可将生姜粉加入桦树汁培养基中。所述生姜粉可商购得到,例如可购自南京松冠生物科技有限公司。所述生姜粉在所述桦树汁培养基中的含量通常为约0-5%,优选约0.5-2%,基于所述桦树汁培养基的总重量。
进一步地,可将pH调节剂加入到所述桦树汁培养基中,以将桦树汁培养基的pH调节到约4.5-5.0。所述pH调节剂是本领域已知的,例如,包括但不限于乳酸、柠檬酸、乳酸钠、柠檬酸钠和氢氧化钠,优选柠檬酸钠。
此外,还可以将促进酵母菌生长的无机盐加入到培养基中,其实例包括但不限于磷酸二氢钾、无水硫酸镁、磷酸二氢钠等。所述无机盐的用量是本领域已知的,例如约0.05-0.2%,基于所述桦树汁培养基的总重量。
在包含生姜粉、pH调节剂和/或无机盐的情况下,将这些物质加入桦树汁中即可制备桦树汁培养基。
上述步骤(2)离心和过滤所得发酵产物是本领域已知的。通常,在例如高速离心机上,在约6000-10000rpm下,进行约15-30分钟。所述离心和过滤步骤将发酵产物中的酵母菌菌体和发酵的桦树汁滤液(上清液)分开。
上述步骤(3)高压破碎所得酵母菌菌体是本领域已知的。例如,使用3-5倍质量的发酵桦树汁将所得酵母菌菌体重悬,得到酵母菌菌体的悬浮液,然后采用高压细胞破碎机,在1-2升/分钟的流速和1000-1200巴压力下均匀处理所述悬浮液,直至酵母菌菌体的破碎率达到95%以上。然后,将经破碎的酵母菌菌体液离心过滤,通常在6000-10000rpm下进行15-30分钟,所得上清液即为可溶性酵母菌溶胞物。
上述步骤(4)中,将步骤(3)所得可溶性酵母菌溶胞物与步骤(2)所得发酵的桦树汁滤液混合均匀,然后进一步过滤,所得滤液即为酵母菌发酵的桦树汁滤液产物。
所述方法可进一步包括将所得产物经超高温瞬时杀菌,其中杀菌温度为约115±5℃,时间为约10-30秒;然后,将杀菌过的产物转至存储罐存储。
通过上述方法得到的酵母菌发酵的桦树汁色浅、透明,其通常包含100-1000mg/L的多酚,2-10g/L的多糖,0.05-1.6%的蛋白质和300-1100mg/L的核酸。因此,所得酵母菌发酵的桦树汁含有丰富的活性营养物,可以作为原料营养物用于防脱生发化妆品组合物中,具有显著的防脱生发功效。
另一方面,本发明涉及一种酵母菌发酵的桦树汁,其是通过采用酵母菌作为菌种在包含桦树汁和任选的生姜粉的培养基中进行发酵得到的。
通常,所述酵母菌发酵的桦树汁包含100-1000mg/L的多酚,2-10g/L的多糖,0.05-1.6%的蛋白质和300-1100mg/L的核酸。
又一方面,本发明涉及酵母菌发酵的桦树汁在防脱生发组合物中的用途。
再一方面,本发明涉及一种防脱生发组合物,其包含(A)酵母菌发酵的桦树汁。所述防脱生发组合物显示了显著的防脱生发功效。
所述酵母菌发酵的桦树汁在所述防脱生发组合物中的含量为大于0至小于100%,优选为约20-95%,基于所述防脱生发组合物的总重量。
防脱生发涉及两个非常关键的方面:其一是5α-还原酶的活力,抑制5α-还原酶的活力,可以有效减少二氢睾酮的生成,避免毛囊萎缩;其二是毛乳头细胞的活力,毛乳头细胞增殖活力强可以影响整个毛囊的状态,使其保持生长期的状态,促进毛发新生,延长毛发生长时间。桦树汁能够抑制5α-还原酶的活力,调节毛乳头细胞的增殖与分化,以及防脱生发相关基因的转录和蛋白质的表达。生姜粉能够抑制5α-还原酶的活力。与未发酵的桦树汁原液相比,发酵的桦树汁和/或生姜粉具有显著更好的防脱生发功效,表现为能够抑制5α-还原酶的活力,调节毛乳头与防脱生发相关基因的转录和蛋白质的表达,促进毛乳头细胞的增殖与分化。意想不到的是,本发明的所述发酵的桦树汁表现出比桦树汁和生姜粉显著更好的防脱生发效果。
除了所述酵母菌发酵的桦树汁外,本发明的防脱生发组合物还任选地包含(B)毛发化妆品中常用的成分,其中包括媒介物、活性成分和辅料等。组分(B)是本领域已知的,本领域技术人员可根据需要选择其类型和用量,例如,组分(B)的含量为约4-88%,基于所述防脱生发组合物的总重量。
所述媒介物包括例如稀释剂、分散剂或载体等,其实例包括但不限于乙醇、双丙甘醇、丁二醇等。所述媒介物在所述防脱生发组合物中的含量是本领域已知的,例如,其通常占组分(B)总重量的约0.02-15%。
所述活性成分包括例如防脱生发剂、保湿剂、发质调理剂等。
所述防脱生发剂的实例包括但不限于苍术提取物、白术提取物、雪莲花提取物、土木香提取物、洋甘菊提取物、千里光提取物、高良姜提取物、生姜提取物、莪术提取物、姜黄提取物、郁金提取物、肉豆蔻提取物、白芍提取物、牡丹皮提取物、升麻提取物、秦皮提取物、白鲜皮提取物、陈皮提取物、化橘红提取物、佛手提取物、山楂叶提取物、仙鹤草提取物、细辛提取物、八角茴香提取物、肉桂提取物、乌药提取物、荜茇提取物、胡椒提取物、秦艽提取物、龙胆提取物、甘松提取物、没药提取物、乳香提取物、辣椒提取物、独一味提取物、沙棘提取物、人参提取物、日本獐牙菜提取物、益母草提取物、茶叶提取物等中的一种或多种。所述防脱生发剂在所述防脱生发组合物中的含量是本领域已知的,例如其通常占组分(B)总重量的约0.01-40%。
所述保湿剂的实例包括但不限于甘油、双甘油、丁二醇、丙二醇、1,3-丙二醇、双丙甘醇、1,2-戊二醇、聚乙二醇-8、聚乙二醇-32、甲基葡糖醇聚醚-10、甲基葡糖醇聚醚-20、PEG/PPG-17/6共聚物、甘油聚醚-7、甘油聚醚-26、甘油葡糖苷、PPG-10甲基葡糖醚、PPG-20甲基葡糖醚、PEG/PPG/聚丁二醇-8/5/3甘油、蔗糖、海藻糖、鼠李糖、甘露糖、棉子糖、甜菜碱、赤藓醇、木糖醇、尿素、甘油聚醚-5乳酸酯、透明质酸钠、水解透明质酸钠、乙酰化透明质酸钠、聚谷氨酸钠、水解小核菌胶、出芽短梗酶多糖、银耳多糖、酸豆籽多糖等中的一种或多种。所述保湿剂在所述防脱生发组合物中的含量是本领域已知的,例如,其通常占组分(B)总重量的约0.01-40%。
所述发质调理剂的实例包括但不限于脂肪醇、甾醇、脂肪酸、单甘油酯、三甘油脂、羊毛脂、肌氨酸酯、脂肪酸酯、白油、角鲨烷、聚季铵盐-10、聚季铵盐-7、聚季铵盐-39、聚季铵盐-52、聚季铵盐-73、瓜尔胶羟丙基三甲基氯化铵、聚乙烯吡咯烷酮、明胶、果胶、皂荚提取物、丹参提取物、当归根提取物、丁二醇、何首乌提取物、扁柏叶提取物、姜根提取物、黄芩根提取物、苦参根提取物、胀果甘草根提取物、尿囊素、三七根提取物、木瓜果提取物、等中的一种或多种。所述发质调理剂成分在所述防脱生发组合物中的含量是本领域已知的,例如,其通常占组分(B)总重量的约0.01-50%。
所述辅料包括例如发泡剂、表面活性剂、乳化剂、增稠剂、防腐剂、香料等。
所述发泡剂的实例包括但不限于月桂醇聚醚硫酸酯钠、十三烷醇聚醚硫酸钠、癸基葡糖苷、月桂酰肌氨酸钠、C14-16烯烃磺酸钠、椰子油酸单乙醇酰胺、椰子油酸二乙醇酰胺、椰子油酰基丙基甜菜碱、月桂醇硫酸酯铵、椰油酰两性基乙酸钠等中的一种或多种。所述发泡剂在所述防脱生发组合物中的含量是本领域已知的,例如,其通常占组分(B)总重量的约0.01-50%。
所述表面活性剂的实例包括但不限于椰油酰胺丙基甜菜碱、月桂醇聚醚硫酸酯钠、PEG-150二硬脂酸酯、乙二醇二硬脂酸酯、月桂基聚氧乙烯醚硫酸铵、十二烷基醇醚硫酸钠、棕榈酰胺丙基甜菜碱、椰油酰胺二乙醇胺、十二烷基醚硫酸盐、十二烷基硫酸铵(K12A)、脂肪醇聚乙烯醚硫酸钠(AES)、脂肪醇聚乙烯醚硫酸铵(AESA)、月桂醇醚硫酸铵、月桂醇硫酸铵、月桂醇硫酸钠、十二烷基聚醚硫酸铵、十二烷基醇硫酸铵、椰油基单乙醇酰胺、椰油基两性醋酸钠、聚季銨盐-7、聚季铵盐-10、聚季铵盐-37、羟丙基瓜尔胶羟丙基三甲基氯化銨、羟丙基三甲基氯化铵瓜尔胶、菩提子提取物等中的一种或多种。所述表面活性剂在所述防脱生发组合物中的含量是本领域已知的,例如,其通常占组分(B)总重量的约0.01-50%。
所述乳化剂的实例包括但不限于鲸蜡硬脂醇橄榄油酸酯、山梨坦橄榄油酸酯、聚山梨醇酯-60、聚山梨醇酯-80、甲基葡糖倍半硬脂酸酯、PEG-20甲基葡糖倍半硬脂酸酯、PEG-40氢化蓖麻油、PPG-26-丁醇聚醚-26、PEG-4聚甘油-2硬脂酸酯、PEG-60氢化蓖麻油、硬脂醇聚醚-2、硬脂醇聚醚-21、PPG-13-癸基十四醇聚醚-24、鲸蜡硬脂基葡糖苷、PEG-100硬脂酸酯、甘油硬脂酸酯、甘油硬脂酸酯SE、椰油基葡糖苷、鲸蜡硬脂醇聚醚-25、PEG-40硬脂酸酯、聚甘油-3甲基葡糖二硬脂酸酯、甘油硬脂酸酯柠檬酸酯、聚甘油-10硬脂酸酯、聚甘油-10肉豆蔻酸酯、聚甘油-10二油酸酯、聚甘油-10月桂酸酯、聚甘油-10异硬脂酸酯、聚甘油-10油酸酯、聚甘油-10二异硬脂酸酯、聚甘油-6月桂酸酯、聚甘油-6肉豆蔻酸酯、蔗糖硬脂酸酯、蔗糖多硬脂酸酯等中的一种或多种。所述乳化剂在所述防脱生发组合物中的含量是本领域已知的,例如,其通常占组分(B)总重量的约0.01-30%。
所述增稠剂的实例包括但不限于卡波姆类、丙烯酸(酯)类及其衍生物、黄原胶、阿拉伯胶、聚乙二醇-14M、聚乙二醇-90M、琥珀酰聚糖、羟乙基纤维素、羟丙基纤维素、羟丙基甲基纤维素等高分子聚合物中的一种或多种。所述增稠剂在所述防脱生发组合物中的含量是本领域已知的,例如,其通常占组分(B)总重量的约0.1-20%。
所述防腐剂的实例包括但不限于羟苯甲酯、羟苯乙酯、羟苯丙酯、苯氧乙醇、苯甲醇、苯乙醇、双(羟甲基)咪唑烷基脲、山梨酸钾、苯甲酸钠、氯苯甘醚、脱氢乙酸钠、辛酰羟肟酸、1,2-己二醇、1,2-戊二醇、对羟基苯乙酮、辛甘醇、甘油辛酸酯、十一碳烯酸甘油酯、山梨坦辛酸酯、乙基己基甘油、牡丹根提取物等中的一种或多种。所述防腐剂在所述防脱生发组合物中的含量是本领域已知的,例如,其通常占组分(B)总重量的约0.01-10%。
本发明的防脱生发组合物可以通过本领域已知的任何合适的方法制备。例如,使用化妆品领域中常用的溶解槽、乳化锅、分散器、输送泵等设备制备。制备时先将水溶性物质投入水相溶解釜,油溶性物质投入油相溶解釜,将两个釜的温度加热至约80℃,其中对于易结块的原料,可先用分散器将其预分散。待溶解完成后将油相和水相输送至乳化锅中,均质乳化约5-15分钟。乳化完成后将料体温度降至常温,加入任选的香精、防腐剂等,并视需要调节产物的pH。相关检测指标都合格后方可灌装出货。以上制备方法可根据剂型要求进行删减或调整。
本发明的防脱生发组合物可以是洗去型的防脱生发洗发香波形式,也可以是留存型的防脱生发液形式。本发明的防脱生发组合物可根据需要制备成各种剂型,例如,膏、霜、乳液、液体等。
实施例
以下结合实施例,对本发明进行进一步详细说明。但是,应当理解为,这些实施例、对比例仅仅是用于更详细地说明本发明,而不应理解为用于以任何形式限制本发明所附权利要求书的范围。
实施例1
(1)使活性干酵母菌复水
将干酵母菌按1:10的比例投放于30℃的温水中复水15分钟,得到酵母菌种子液。
(2)桦树汁培养基的制备
以东北大兴安岭采集的桦树汁原液(brix 1.2)为底物,向其中添加1%生姜粉作为补充碳源,添加0.08%磷酸二氢钾和0.04%无水硫酸镁,使用1M柠檬酸钠水溶液调整桦树汁培养基的pH为4.5。
(3)接种发酵
按照65%(v/v)体积装液量,将步骤(2)所配制的桦树汁培养基加入到100L发酵罐中,105℃灭菌20分钟;将适量酵母菌种子液投入发酵培养基中,于30℃、搅拌转速300rmp、通气量1.6vvm的条件下,连续发酵24小时,停罐,得到发酵液产物。
(4)离心分离,得到发酵滤液和菌体
使用蝶式离心得机处理发酵液产物,其中离心转速为6000rpm,离心时间30分钟,得到作为上清液的发酵的桦树汁滤液产物,并将其打入储存罐;和得到作为菌泥的酵母菌菌体。
取发酵的桦树汁滤液产物,测定其中的多糖、蛋白质、核酸、总酚含量,结果如表1所示。
实施例2
(1)使活性干酵母菌复水
将干酵母菌按1:10的比例投放于30℃的温水中复水15分钟,得到酵母菌种子液。
(2)桦树汁培养基的制备
以东北大兴安岭采集的桦树汁的浓缩液(浓缩3倍,brix 3.58)为底物,向其中添加1%生姜粉作为补充碳源,添加0.08%磷酸二氢钾和0.04%无水硫酸镁,使用1M柠檬酸钠水溶液调整桦树汁培养基的pH为4.5。
(3)接种发酵
按照65%(v/v)体积装液量,将步骤(2)所配制的桦树汁培养基加入到100L发酵罐中,105℃灭菌20分钟;将适量酵母菌种子液投入发酵培养基中,于30℃、搅拌转速300rmp、通气量1.6vvm的条件下,连续发酵24小时,停罐,得到发酵液产物。
(4)离心分离,得到发酵滤液和菌体
使用蝶式离心得机处理发酵液产物,其中离心转速为6000rpm,离心时间30分钟,得到作为上清液的发酵的桦树汁滤液产物,并将其打入储存罐;和得到作为菌泥的酵母菌菌体。
取发酵的桦树汁滤液产物,测定其中的多糖、蛋白质、核酸、总酚含量,结果如表1所示。
实施例3
(1)使活性干酵母菌复水
将干酵母菌按1:10的比例投放于30℃的温水中复水15分钟,得到酵母菌种子液。
(2)桦树汁培养基的制备
以东北大兴安岭采集的桦树汁原液(brix 1.12)为底物,添加0.08%磷酸二氢钾和0.04%无水硫酸镁,使用1M柠檬酸钠水溶液调整桦树汁培养基的pH为4.5。
(3)接种发酵
按照65%(v/v)体积装液量,将步骤(2)所配制的桦树汁培养基加入到100L发酵罐中,105℃灭菌20分钟;将适量酵母菌种子液投入发酵培养基中,于30℃、搅拌转速300rmp、通气量1.6vvm的条件下,连续发酵24小时,停罐,得到发酵液产物。
(4)离心分离,得到发酵滤液和菌体
使用蝶式离心得机处理发酵液产物,其中离心转速为6000rpm,离心时间30分钟,得到作为上清液的发酵的桦树汁滤液产物,并将其打入储存罐;和得到作为菌泥的酵母菌菌体。
取发酵的桦树汁滤液产物,测定其中的多糖、蛋白质、核酸、总酚含量,结果如表1所示。
实施例4
(1)使活性干酵母菌复水
将干酵母菌按1:10的比例投放于35℃的温水中复水15分钟,得到酵母菌种子液。
(2)桦树汁培养基的制备
以东北大兴安岭采集的桦树汁的浓缩液(浓缩3倍,brix 3.58)为底物,添加0.08%磷酸二氢钾和0.04%无水硫酸镁,使用1M柠檬酸钠水溶液调整桦树汁培养基的pH为4.5。
(3)接种发酵
按照65%(v/v)体积装液量,将步骤(2)所配制的桦树汁培养基加入到100L发酵罐中,105℃灭菌20分钟;将适量酵母菌种子液投入发酵培养基中,于30℃、搅拌转速300rmp、通气量1.6vvm的条件下,连续发酵24小时,停罐,得到发酵液产物。
(4)离心分离,得到发酵滤液和菌体
使用蝶式离心得机处理发酵液产物,其中离心转速为6000rpm,离心时间30分钟,得到作为上清液的发酵的桦树汁滤液产物,并将其打入储存罐;和得到作为菌泥的酵母菌菌体。
取发酵的桦树汁滤液产物,测定其中的多糖、蛋白质、核酸、总酚含量,结果如表1所示。
实施例5
与实施例1类似,包括其中所述的步骤(1)-(4),但进一步包括如下步骤:
(5)将作为菌泥的酵母菌菌体用5倍体积上清液重悬,得到酵母菌悬液;利用高压破碎设备,在用1000巴、1.2升/分钟的条件下,将所得酵母菌悬液进行高压均质破碎;然后使用硅藻土板框过滤,即得到可溶性的酵母菌溶胞物。
(6)将步骤(5)所得酵母菌溶胞物与步骤(4)所得发酵的桦树汁滤液混合,然后进一步使用硅藻土板框过滤,得到最终的酵母菌发酵的桦树汁产物。
取最终的酵母菌发酵的桦树汁产物,测定其中的多糖、蛋白质、核酸、总酚含量,结果如表1所示。
实施例6
与实施例2类似,包括其中所述的步骤(1)-(4),但进一步包括如下步骤:
(5)将所得上清液打入储存罐,和将作为菌泥的酵母菌菌体用5倍体积上清液重悬,得到酵母菌悬液;利用高压破碎设备,在用1000巴、1.2升/分钟的条件下,将所得酵母菌悬液进行高压均质破碎;然后使用硅藻土板框过滤,即得到可溶性的酵母菌溶胞物。
(6)将步骤(5)所得酵母菌溶胞物与步骤(4)所得发酵的桦树汁滤液混合,然后进一步使用硅藻土板框过滤,得到最终的酵母菌发酵的桦树汁产物。
取最终的酵母菌发酵的桦树汁产物,测定其中的多糖、蛋白质、核酸、总酚含量,结果如表1所示。
实施例7
与实施例3类似,包括其中所述的步骤(1)-(4),但进一步包括如下步骤:
(5)将作为菌泥的酵母菌菌体用5倍体积上清液重悬,得到酵母菌悬液;利用高压破碎设备,在用1000巴、1.2升/分钟的条件下,将所得酵母菌悬液进行高压均质破碎;然后使用硅藻土板框过滤,即得到可溶性的酵母菌溶胞物。
(6)将步骤(5)所得酵母菌溶胞物与步骤(4)所得发酵的桦树汁滤液混合,然后进一步使用硅藻土板框过滤,得到最终的酵母菌发酵的桦树汁产物。
取最终的酵母菌发酵的桦树汁产物,测定其中的多糖、蛋白质、核酸、总酚含量,结果如表1所示。
实施例8
与实施例4类似,包括其中所述的步骤(1)-(4),但进一步包括如下步骤:
(5)将作为菌泥的酵母菌菌体用5倍体积上清液重悬,得到酵母菌悬液;利用高压破碎设备,在用1000巴、1.2升/分钟的条件下,将所得酵母菌悬液进行高压均质破碎;然后使用硅藻土板框过滤,即得到可溶性的酵母菌溶胞物。
(6)将步骤(5)所得酵母菌溶胞物与步骤(4)所得发酵的桦树汁滤液混合,然后进一步使用硅藻土板框过滤,得到最终的酵母菌发酵的桦树汁产物。
取最终的酵母菌发酵的桦树汁产物,测定其中的多糖、蛋白质、核酸、总酚含量,结果如表1所示。
表1:发酵的桦树汁滤液产物的总酚、多糖、蛋白质、核酸含量
实施例9:发酵的桦树汁滤液产物的防脱发功效测试
在该实施例中,根据5α-还原酶活性抑制率和防脱生发相关基因和蛋白质表达的影响,评价实施例1-8中发酵的桦树汁滤液产物的防脱发功效,具体如下。
(1)5α-还原酶活性抑制率评价
1)粗酶制备:取正常健康小鼠5只,颈部脱臼致死,打开腹腔,取其睾丸,等分为数份,置于2mL EP管中,按1:4比例加入适量粗酶提取液,于4℃下破壁机破碎制成匀浆液,4℃高速冷冻离心(10000rpm/分钟,10分钟),取其上清液4℃保存。BCA法测定蛋白质浓度,1mg/mL以上即可进行后续实验;
2)空白对照的测定:分别取2组200μL EP管(每组4个),均加入5α-还原酶粗酶、PBS溶液(pH=7.4)、还原型辅酶Ⅱ,一组混合后立即放入沸水中煮沸5分钟,离心后吸取50μL液体进行后续El isa检测,为空白对照组起始睾酮含量;另一组混合后放置恒温摇床中混匀60分钟,放入沸水中煮沸5分钟,离心后吸取50μL液体进行后续El isa检测,其与起始含量的差值为空白对照组转化后睾酮含量;
3)样品组的测定:分别取2组200μL EP管(每组4个),均加入5α-还原酶粗酶、PBS溶液(pH=7.4)、还原型辅酶Ⅱ和测试原料,一组混合后立即放入沸水中煮沸5分钟,离心后吸取50μL液体进行后续Elisa检测,为抑制剂组起始睾酮含量;另一组混合后放置恒温摇床中混匀60分钟,放入沸水中煮沸5分钟,离心后吸取50μL液体进行后续Elisa检测,其与起始含量的差值为抑制剂组转化后睾酮含量。实验每次4个平行样。
5α-还原酶抑制率按如下公式计算:
I%=(ΔA0-ΔAn)/ΔA0*100%
其中:ΔA0--对照组睾酮减少量;ΔAn--抑制剂组睾酮减少量。
测试结果如表2所示。
(2)防脱生发相关基因和蛋白质表达情况
基于毛乳头细胞的基因和蛋白质表达分析步骤如下:
以适宜的毛乳头细胞接种量接种至6孔板中,37℃,5%CO2培养箱孵育过夜;待6孔板中细胞铺板率达到40-50%时,进行分组给药,每孔给药量为2mL,每组设3个复孔。37℃,5%CO2培养箱继续培养24小时。
a.培养24小时后,收集细胞培养上清液于EP管中,收集完后将用于VEGF含量检测的样品置于-80℃冰箱冷冻保存;根据VEGF ELISA试剂盒的操作说明书进行检测分析。
b.收集细胞:培养24小时后,收集细胞上清后,1mL/孔PBS清洗两次,每孔加入1mLRNAiso Plus,吹打裂解细胞后,收样。提取RNA,反转录至cDNA后,进行荧光定量PCR检测,采用2-△△CT方法进行结果计算,检测TGF-β2、β-catenin、AR基因表达
测试结果如表2所示。
表2:发酵的桦树汁滤液5α-还原酶活性抑制率和防脱发相关基因表达
实施例10:防脱生发洗发水
采用实施例1中制备的发酵的桦树汁产物来制备防脱生发洗发水,其配方如下:
上述防脱生发洗发水的制备方法如下:
按照上述表中的比例加入发酵的桦树汁滤液,将其加热到80℃,并持续半小时。将十二烷基醇醚硫酸钠、椰油酰谷氨酸钠、瓜尔胶按照顺序依次加入水中,持续搅拌,保持10分钟。对上述混合物进行均质20-30分钟。
预先将1,2-戊二醇、1,3-BG、D-泛醇、薄荷醇、桉树叶油混合好,待上述均质混合物温度降到室温时,加入体系中,搅拌10分钟。加入的柠檬酸和氯化钠。调节pH=5-7,最后加入去离子水到100%处方重量。经1200目过滤后,灌装,得到洗发水。
临床人体试验:选取150名脱发患者(脱发数大于100根/天),随机分成两组,分别使用上述实验组和对照组洗发水,每组男女各半。受试者每隔一天洗一次头,早晚2次梳理头发,收集脱落头发并计数。每15天记录一次,持续使用和记录3个月。
以上结果表明,含本发明的发酵的桦树汁的洗发水在抑制脱发方面明显优于空白组,随着使用时间的延长,防脱效果也逐渐增加。
实施例11:防脱生发液
采用实施例5中制备的发酵的桦树汁产物来制备防脱生发液,其配方如下:
上述防脱生发液制备方法如下:
1.首先将A相各组分按照质量配比加入主锅,并加热至80℃,混合;
2.将B相各组分按照质量配比加入边锅,加热融化并搅拌均匀,并将其投入到A相中,与A相搅拌混合均匀;
3.将C相中的氨基酸、营养增补剂、控油剂和烟酰胺依次加入主锅中,并搅拌均匀;
4.将D相中的二氨基嘧啶氧化物、D相中的溶剂依次加入主锅中,并搅拌均匀;
5.将主锅降温至50℃,依次加入E相的酒精和E相中的溶剂,并搅拌均匀;
6.再次将主锅降温至45℃,依次加入F相的何首乌提取物和生姜提取物,并搅拌均匀;
7.将G相中的吐温-20和香精混合后预溶,并搅拌至透明后加入到主锅中,再次搅拌均匀;
8.最后向主锅中加入H相,搅拌均匀后,冷却至室温,检测合格后出料,得到防脱生发液。
临床人体试验:选取120名脱发患者(脱发数大于100根/天),随机分成四组,分别使用上述四种生发液,每组男女各半。受试者每天涂于头皮部,并按摩吸收,早晚2次梳理头发,收集脱落头发并计数。每周记录一次,持续使用和记录4周。
以上结果表明,含本发明的酵母菌发酵的桦树汁的防脱生发液在抑制脱发方面明显优于空白组,随着使用时间的延长,防脱生发效果也逐渐增加。
以上所述实施例的技术方案是本发明优选实施方式,在不脱离本发明原理的前提下还可以进行若干改进和变换,这些改进和变化也应视为在本发明的保护范围内。
Claims (18)
1.一种生产酵母菌发酵的桦树汁的方法,其包括采用酵母菌作为菌种在包含桦树汁和任选的生姜粉的培养基中进行发酵的步骤。
2.权利要求1所述的方法,其包括下述步骤:
(1)采用酵母菌作为菌种,在包含桦树汁和任选的生姜粉的培养基中进行发酵,得到发酵液产物;
(2)过滤所述发酵液产物,分别得到酵母菌菌体和发酵的桦树汁滤液;
(3)破碎所述酵母菌菌体,然后进行过滤,得到作为上清液的可溶性酵母菌溶胞物;和
(4)将所得酵母菌溶胞物与所述发酵的桦树汁滤液混合,过滤,得到发酵的桦树汁滤液产物,即作为产物的酵母菌发酵的桦树汁。
3.权利要求1或2所述的方法,其中所述桦树汁是浓缩倍数为1.2-12倍,优选约1.5-6倍,更优选约2-4倍的浓缩的桦树汁。
4.权利要求1-3任一项所述的方法,其中所述酵母菌选自酿酒酵母菌、布拉迪酵母菌和解脂耶氏酵母菌。
5.权利要求4所述的方法,其中所述酵母菌为酿酒酵母菌。
6.权利要求1-5任一项所述的方法,其中所述桦树汁在所述桦树汁培养基中的含量在93%以上,优选95%以上,基于所述桦树汁培养基的总重量。
7.权利要求1-6任一项所述的方法,所述生姜粉在所述桦树汁培养基中的含量为0-5%,优选0.5-2%,基于所述桦树汁培养基的总重量。
8.可通过权利要求1-7任一项所述的方法得到的酵母菌发酵的桦树汁。
9.一种酵母菌发酵的桦树汁,其是通过采用酵母菌作为菌种在包含桦树汁和任选的生姜粉的培养基中进行发酵而得到的。
10.权利要求8或9所述的酵母菌发酵的桦树汁,其包含100-1000mg/L的多酚,2-10g/L的多糖,0.05-1.6%的蛋白质和300-1100mg/L的核酸。
11.权利要求9或10所述的酵母菌发酵的桦树汁,其中所述桦树汁是浓缩倍数为1.2-12倍,优选约1.5-6倍,更优选约2-4倍的浓缩的桦树汁。
12.权利要求9-11任一项所述的酵母菌发酵的桦树汁,其中所述酵母菌选自酿酒酵母菌、布拉迪酵母菌和解脂耶氏酵母菌。
13.权利要求12所述的酵母菌发酵的桦树汁,其中所述酵母菌为酿酒酵母菌。
14.权利要求9-13任一项所述的酵母菌发酵的桦树汁,其中所述桦树汁在所述桦树汁培养基中的含量在93%以上,优选95%以上,基于所述桦树汁培养基的总重量。
15.权利要求9-14任一项所述的酵母菌发酵的桦树汁,所述生姜粉在所述桦树汁培养基中的含量为0-5%,优选0.5-2%,基于所述桦树汁培养基的总重量。
16.权利要求8-15任一项所述的酵母菌发酵的桦树汁在防脱生发组合物中的用途。
17.一种防脱生发组合物,其包含权利要求8-15任一项所述的酵母菌发酵的桦树汁。
18.权利要求17所述的防脱生发组合物,其中酵母菌发酵的桦树汁在所述防脱生发组合物中的含量为大于0至小于100%,优选为20-95%,基于所述防脱生发组合物的总重量。
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