CN117683825A - 一种含丁酸乙酯的双菌发酵产物及其制备方法和护肤应用 - Google Patents
一种含丁酸乙酯的双菌发酵产物及其制备方法和护肤应用 Download PDFInfo
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Abstract
本发明属于微生物产品和化妆品领域，具体涉及一种含丁酸乙酯的双菌发酵产物及其制备方法和护肤应用。本发明在富含糖类的培养基中同时接种发酵糖类产生短链脂肪酸的产芽孢细胞和代谢糖类产生乙醇的酵母，在发酵过程中分别产生短链脂肪酸和乙醇，并在菌体产生的酯酶催化下形成短链脂肪酸酯。发酵结束后通过蒸馏工艺得到以短链脂肪酸乙酯为主要成分的双菌发酵产物，可将短链脂肪酸以酯类的形式施用在皮肤上会提高感官接受度，提高皮肤渗透性，更有利于发挥短链脂肪酸的有益于皮肤的生物学活性。
Description
技术领域
本发明属于微生物产品和化妆品领域，具体涉及一种含丁酸乙酯的双菌发酵产物及其制备方法和护肤应用。
背景技术
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解，而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
短链脂肪酸(SCFAs，short chain fatty acids)是碳链长度从2至6的脂肪酸，常见的SCFAs种类有乙酸、丙酸、乳酸、丁酸、异丁酸(2-甲基丙酸)、3-羟基丁酸、丁二酸(琥珀酸)、戊酸、异戊酸(3-甲基丁酸)、2-甲基丁酸、2-羟基-3-甲基丁酸、3-羟基-3-甲基丁酸、己酸等。在人体内，SCFAs一般由肠道菌群在消化道道后段发酵多糖类产生，对人体有多种有益功能，包括调节免疫、预防或改善结肠癌、维护葡萄糖稳态、降低血脂、改善骨质疏松等。与肠道和健康领域的研究相比，在皮肤护理方向上的SCFAs研究还处在初期。在来源上，包括肠道吸收并运输至皮肤、外用施加、皮肤菌群代谢产生。在作用机制上，SCFAs可通过细胞表面的游离脂肪酸受体FFAR2、FFAR3，G蛋白偶联受体GRP109A、嗅觉受体OR51E2启动信号途径，也可通过FFARs、单羧酸转运蛋白MCT、钠依赖性单羧酸转运蛋白SMCT运输至胞内发挥生理作用。研究最充分的是FFARs受体，FFAR2主要感受乙酸、丙酸、丁酸，FFAR3对丙酸至己酸的亲和性更高。
不同来源的SCFAs对皮肤的有益作用已有多项报道。肠道来源的SCFAs可通过调节免疫状态、减轻炎症、提高抗氧化能力等方式整体性/间接性改善皮肤状态，也有直接性的改善研究报道。在小鼠模型上喂食高纤维食物提高肠道SCFAs吸收或直接口服丁酸，可改善尘螨过敏原引起的皮肤屏障损坏，丁酸盐可改变角质形成细胞对过敏原反应并减轻下游炎症级联反应；在不同种类细胞共培养模型中证实丁酸可增强屏障功能并降低炎症反应。人体上口服瓜尔胶来源的多糖/膳食纤维通过提高肠道SCFAs吸收提高了皮肤完整性和皮肤水合度；小鼠模型中通过FFAR2/FFAR3加快皮肤伤口愈合、降低炎症反应、减少皮肤疤痕。
皮肤菌群也是皮肤接触到的SCFAs的重要来源，皮肤菌群产生的SCFAs也参与人体体味的构成，尤其是丙酸杆菌和葡萄球菌类。体外发酵检测中痤疮丙酸杆菌主要产生乙酸和丙酸，而丁酸、异丁酸、异戊酸产量很低，表皮葡萄球菌以乙酸为主，也产生丙酸、丁酸、异丁酸、异戊酸、琥珀酸，个别菌株的丁酸产量很高。Sunita(2019)报道了表皮葡萄球菌产生的丁酸通过FFAR2下调了小鼠皮肤在UVB刺激下的促炎性细胞因子IL-6，痤疮丙酸杆菌产生的丁酸或人工合成的丁酸衍生物可通过IL-6/p-ERK途径降低磷酸钙导致的瘙痒(2020)。Kao(2021)发现痤疮丙酸杆菌发酵产生的丙酸通过FFAR2抑制酪氨酸酶活性，降低UVB导致的黑色素合成。在体外细胞和小鼠皮肤上，痤疮丙酸杆菌产生的丙酸可增加皮脂合成，是通过PPARα/GPAT3途径起作用而非FFARs受体。表皮葡萄球菌发酵产生的丁酸通过FFAR2/p-ERK途径促进I型胶原的合成，具有潜在的抗衰老作用(Indira，2021)。SCFAs也被皮肤菌群作为相互竞争的工具，表皮葡萄球菌产生的琥珀酸可抑制痤疮丙酸杆菌及其毒力表达(Wang，2014、2016)，表皮葡萄球菌产生的丁酸可抑制金黄色葡萄球菌的生长，合成的丁酸衍生物可抑制金黄色葡萄球菌在皮肤上的定殖并降低炎症因子(Traisaeng，2019)，痤疮丙酸杆菌产生的丙酸、异丁酸、异戊酸可降低表皮葡萄球菌的生物膜形成(Nakamura，2021)。
随着人们对SCFAs对皮肤益处的认识，也开始在护肤品中添加SCFAs成分。CN114569536B公开了一种对衰老皮肤微生态和皮肤生理状态有改善作用的乳液，该乳液以褐藻酵母菌发酵溶胞物、乳酸杆菌/梨汁发酵滤液、短链脂肪酸盐为主要有效成分，每20g乳液中含有短链脂肪酸盐0.05～0.1g(0.25～0.5％)。CN116763703A公开了雌马酚、丁酸盐、暗绿龙舌兰提取物、羟基积雪草苷、依克多因和硫胺素组成的组中的一种以上作为有效成分的皮肤微生物组调节用组合物，但未说明各组分的比例。CN116650699A利用羟基丁酸可特异性显著抑制NLRP3炎症小体活性，减低巨噬细胞焦亡，以3-羟基丁酸或盐类为活性物、聚甲基丙烯酰胺化壳聚糖为基质制备了一种微针型伤口敷料。CN108392629B公开了一种基于C2～C5多元羧酸和400～460nm波长蓝光的组合，用于治疗痤疮和其他皮肤病症。
现有技术均中使用短链脂肪酸盐或短链脂肪酸盐和其他活性物的组合作为护肤品的活性成分，然而短链脂肪酸及其盐类往往有不易接受的气味。在气味上，乙酸为尖锐刺激性酸味、丙酸为酸臭或汗臭味、乳酸为酸臭味、丁酸至己酸均为腐败性酸臭味。其盐类也具有一定的不良气味，例如丙酸钠或丁酸钠的～0.2mg/ml水溶液涂抹在皮肤上，即可嗅到不良气味。短链脂肪酸及其盐类在皮肤上解离为带有负电荷的酸根形式，亲脂性降低而亲水性提高，透过角质层脂质屏障的能力降低，堆积在皮肤表层又会使不良气味更加明显，且无法深入皮肤组织产生作用。
发明内容
为了解决现有技术的不足，本发明的目的是提供一种含丁酸乙酯的双菌发酵产物及其制备方法和护肤应用。本发明在富含糖类的培养基中同时接种发酵糖类产生短链脂肪酸的产芽孢细胞和代谢糖类产生乙醇的酵母，在发酵过程中分别产生短链脂肪酸和乙醇，并在菌体产生的酯酶催化下形成短链脂肪酸酯。发酵结束后通过蒸馏工艺得到以短链脂肪酸乙酯为主要成分的双菌发酵产物，可将短链脂肪酸以酯类的形式施用在皮肤上会提高感官接受度，提高皮肤渗透性，更有利于发挥短链脂肪酸的有益于皮肤的生物学活性。
为了实现上述目的，本发明是通过如下的技术方案来实现：
第一方面，本发明提供了一种含丁酸乙酯的双菌发酵产物的制备方法，包括以下步骤：
将产芽孢细菌种子液和干酵母接种于灭菌的培养基后进行密闭发酵，发酵过程中控制发酵液pH为5.0～6.5，发酵结束后将发酵液的pH调节为7.0～8.0，进行减压提取，收集冷凝液并与水混合、分层、萃取除去水溶性杂质后得到含丁酸乙酯的双菌发酵产物。
发酵液中酸性越强，未形成酯类的短链脂肪酸越多以非解离形式存在(R-COOH)，与酸根形式(R-COO-)相比，非解离形式的短链脂肪酸的挥发性更强，在后续蒸馏提取时也随之进入产物中，导致所得双菌发酵产物具有不易接受的臭味，减低感官品质；pH过高为强碱性时，酯类分解增多，也会导致氨基酸等成分降解产生氨气或胺类，也会降低双菌发酵产物品质。
优选的，所述培养基中含有麦芽浸粉，所述麦芽浸粉的含量为5％～20％w/w。
优选的，所述产芽孢细菌为代谢糖类产生以丁酸为主要短链脂肪酸成分的细菌，包括枯草芽胞杆菌Bacillus subtilis subsp.subtilis CICC23592、热噬淀粉芽胞杆菌Bacillus thermoamylovorans CICC20802、巨大芽胞杆菌Bacillus megateriumCICC10024和丁酸梭菌Clostridium butyricum CICC23847中的至少一种。
优选的，酵母为具有良好酒精发酵能力的酵母菌株或商业化酵母产品，例如安琪耐高温酿酒高活性干酵母，或其他具有类似工艺特性的酵母产品。
优选的，产芽孢细菌种子液的接种量为2％～5％v/v，干酵母接种量为0.5～1g/L培养液。
优选的，发酵时间为3～10天。
优选的，减压提取时发酵液的温度为40～60℃，压力为-0.09～-0.06MPa，冷凝温度为10～20℃。
第二方面，本发明提供了一种含丁酸乙酯的双菌发酵产物，通过如第一方面所述的制备方法获得。
第三方面，本发明提供了一种组合物，包括如第二方面所述的含丁酸乙酯的双菌发酵产物和多元醇，所述双菌发酵产物和多元醇的质量比为5～90:95～10
优选的，所述多元醇包括1,3-丙二醇、1,2-丙二醇、1,3-丁二醇、1,2-丁二醇、甲基丙二醇、1,2-戊二醇和1,2-己二醇中的至少一种。
第四方面，本发明提供了如第二方面所述的含丁酸乙酯的双菌发酵产物和/或如第三方面所述的组合物在制备护肤产品中的应用。
第五方面，本发明提供了一种护肤产品，所述护肤产品中含有如第二方面所述的含丁酸乙酯的双菌发酵产物和/或如第三方面所述的组合物。
上述本发明的一种或多种技术方案取得的有益效果如下：
本发明通过产芽孢细胞和酵母进行双菌发酵获得以丁酸乙酯等短链脂肪酸酯为主要成分的双菌发酵产物，双菌发酵产物避免了短链脂肪酸及其盐带来的不良气味和溶解性问题，施用在皮肤上会提高感官接受度，提高皮肤渗透性，更有利于发挥短链脂肪酸的有益于皮肤的生物学活性。
本发明通过控制双菌发酵过程中发酵液pH等反应参数，防止双菌发酵产物中含有短链脂肪酸及其盐影响双菌发酵产物的品质。
本发明通过体外重建表皮模型的功效测试证明双菌发酵产物具有改善SLS导致的角质层结构损伤，增强兜甲蛋白、丝聚蛋白表达，降低炎症因子表达的效果，表明该双菌发酵产物及其混合物可作为一种活性成分用于护肤品中。
附图说明
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。
图1为实施例2中LOR、FLG、NF-κB的荧光免疫图像。
具体实施方式
如背景技术所述，短链脂肪酸及其盐类往往有不易接受的气味，其在皮肤上的解离会降低角质层屏障的透过性。人类的皮肤结构由外内向为表皮层和真皮层，表皮层表面还具有角质层，由已死亡的、扁平形状的角化细胞构成，角化细胞间填充有水-双层磷脂构成的多层结构，被称为“泥浆-砖块”模型，这种结构使角质层具有良好的屏蔽性，阻隔外界成分直接接触皮肤存活细胞，起到保护作用。但对于活性成分而言，角质层的屏蔽作用成为接触活细胞前需要跨越的障碍。由于角化细胞已经死亡，无法再通过主动转运蛋白等方式运输活性成分，活性成分只能依靠浓度差透过多层交替排列的水-磷脂双层结构。磷脂双层表面亲水内部疏水，亲脂性越高的成分越容易透过。短链脂肪酸及其盐类在皮肤上解离为带有负电荷的酸根形式，亲脂性降低而亲水性提高，透过角质层脂质屏障的能力降低，堆积在皮肤表层又会使不良气味更加明显。
本发明使用双菌发酵获得了含有丁酸乙酯的双菌发酵产物，双菌发酵产物中以短链脂肪酸酯为主要成分。在气味上，形成酯类后气味有明显改善，变为容易接受或乐于接受的香味。例如乳酸乙酯、丙酸乙酯、丁酸乙酯、异丁酸乙酯、戊酸乙酯、异戊酸乙酯、己酸乙酯均为不同类型的水果香气，3-羟基丁酸乙酯为果香和白酒样香气，丁二酸二乙酯为愉快的微弱香气。在皮肤渗透性上，对短链脂肪酸单体及其乙酯的LogP(辛醇-水分配系数的Log值，该值越大亲脂性越高，ACD/LogP为ACD/Labs Percepta Platform理论计算值)进行比较，如表1所示。从表1中数值可知，形成乙酯后亲脂性均有明显提高，更有利于透过皮肤表面的脂质屏障。本发明将短链脂肪酸以酯类的形式施用在皮肤上会提高感官接受度，提高皮肤渗透性，更有利于发挥短链脂肪酸的有益于皮肤的生物学活性。
表1部分短链脂肪酸单体及其乙酯的LogP值比较
为了使得本领域技术人员能够更加清楚地了解本发明的技术方案，以下将结合具体的实施例与对比例详细说明本发明的技术方案。
实施例1
选取B.subtilis CICC23592、B.thermoamylovorans CICC20802、B.megateriumCICC10024、C.butyricum CICC23847为产芽孢细菌，于补充2％麦芽糖的营养肉汤培养液培养，获得产芽孢细菌种子液。酵母菌使用高温型酒精活性干酵母(安琪酵母)，该产品为市售干酵母，每升发酵液加入0.5～1g。细菌培养物以冻干管形式购买获得，打开冻干管后加入1ml培养液使冻干物溶解后，吸取溶解后液体至20ml培养液，37℃下静置培养24至48小时至培养液明显浑浊，为一代种子液。一代种子液以2％v/v接种至新的培养液并在37℃下静置培养24小时为二代种子液。使用二代种子液进行发酵培养。
10％w/w麦芽浸粉培养基经调整pH为6.5～7.0、灭菌处理并冷却至35～40℃后，分别接种2％v/v产芽孢细菌种子液和1g/kg的酵母菌，摇匀后37℃±1℃培养箱内静置培养5天。发酵结束后，用NaOH溶液调节发酵液pH为7.0～7.5后，使用旋转蒸发器进行蒸馏提取，发酵液至于旋蒸瓶内，旋蒸瓶至于水浴锅中，水浴温度设为40.0℃，旋蒸瓶转速60～100rpm，使用可调真空阀使系统内真空度为-0.06～-0.07MPa，冷凝水温度约为10℃，收集冷凝液。冷凝液与等体积水混合后萃取上相，重复2次，得到提取物。提取物称重计算产率，不同菌株在0.57～0.82％w/w范围内。
对不同菌株所得提取物使用GC-MS做成分分析。仪器为7890B-5977A型GC-MS联用仪配DB-FFAP色谱柱(60mm×0.25mm，0.25μm)。GC条件为进样口温度：260℃；载气为高纯氮；恒流：柱流速1.0mL/min，不分流；进样量1μl；升温程序：初始温度35℃，以10℃/min升至50℃，保持20min；以1℃/min升至70℃，保持10min，再以3℃/min升至250℃，保持5min；传输管线温度260℃。MS条件为电子电离源；电子能量70eV；离子源230℃；四极杆温度150℃；无溶剂延迟；扫描模式fullscan；扫描质量范围m/z29～1000；调谐文件为标准调谐。如表2所示，分析结果显示所得提取物以短链脂肪酸的乙酯为主要成分，占总质量的85～95％范围内，鉴定出的其他成分包括长碳链酯或内酯类、缩醛类、醛酮类、芳香类、酸类、醇类、杂环类化合物等，不含有不宜于化妆品中使用的化学成分。
表2不同产芽孢细菌和酵母发酵后所得提取物的产率和成分分析(提取物中百分比％w/w)
实施例2
以CICC23592菌株在实施例1中所得双菌产物，按重量比取3份，与7份1,3-丁二醇混合均匀得到组合物，外观为均一的无色透明溶液，该组合物在2～10％w/w浓度下可溶于水中。
细胞毒性测试
1、材料：人角质形成细胞(广东博溪生物，Ep230407)、DMEM培养液(Gibco)、KcGrowth培养液(广东博溪生物)、PBS(索莱宝)、MTT(Sigma)、DMSO(Sigma)。
2、实验步骤
1)细胞接种：角质形成细胞按1×104个/孔的接种密度接种细胞至96孔板，培养箱(37℃、5％CO2)中孵育过夜。
2)试验分组：试验设置调零组、空白组(Control)、阳性对照组(PC，10％DMSO)与样品组，样品组为实施例2中制得组合物，样品组设置从0.008％～0.5％w/w共7个浓度梯度，每个浓度下设置3个重复孔。
3)给药：待96孔板中细胞铺板率达到40％～60％时进行给药。空白组每孔加入200μL培养液；阳性对照组每孔加入200μL含10％DMSO的培养液；样品组每孔加入200μL含有相应浓度样品的培养液；调零组无细胞接种，仅加入200μL细胞培养液。给药完成后将96孔板放置在培养箱(37℃、5％CO2)中培养24h。
4)检测：细胞孵育培养24h后弃掉上清，加入0.5mg/mL的MTT工作液，37℃避光孵育4h，孵育结束后，弃掉上清，每孔加150μLDMSO，在490nm处读取OD值。
5)细胞相对活力计算：细胞相对活力(％)＝(样品孔OD-调零孔OD)÷(空白孔OD-调零孔OD)×100％。
细胞活力数据见表3，测试结果显示实施例2所得组合物在0.125％w/w下细胞活力与空白对照无差别。
表3组合物在不同浓度下对角质形成细胞的毒性
组合物护肤性能测试
使用较大浓度的十二烷基硫酸钠(SLS)接触皮肤后能够损伤皮肤屏障，表现为炎症因子升高和屏障相关蛋白降低。在体外重建表皮模型上使用SLS刺激可作为检测皮肤屏障功能和炎症控制(舒缓功效)的模型。根据经验，一般情况下细胞安全浓度可放大10～20倍为皮肤使用的安全浓度，根据评价例1所得结果，选择2.0％w/w作为测试浓度。
1、材料：3D表皮皮肤模型，(广东博溪生物，批号：ES230504)、EpiGrowth培养液(广东博溪生物)、PBS(索莱宝)、地塞米松(中检所)、IL-1αELISA试剂盒(Abcam)、IL-8ELISA试剂盒(Abcam)、PGE2 ELISA试剂盒(ENZO)、NF-κB抗体(Abcam)、SLS(Sigma)、多聚甲醛(Biosharp)、WY14643(Sigma)、FLG抗体(Abcam)、LOR抗体(Abcam)。
2、实验步骤
1)工作液配置
0.1％SLS工作液：称取0.006g SLS溶于6mL PBS溶液中，0.22μm过滤，配制成0.1％SLS工作液备用。
地塞米松工作液配置：吸取2μL 10％母液溶于2mL培养液中，配制成0.01％地塞米松。
WY14643工作液配置：吸取10μL 30mM WY14643母液溶于6mL培养液中，配置成50μMWY14643。
样品工作液：实施例2中组合物0.2g与9.8g培养液混合，获得2.0％w/w样品工作液。
2)分组和检测指标
分为空白对照组(BC)、阴性对照组(NC)、阳性对照组(PC)、样品组，样品组使用0.1％SLS工作液处理后使用样品工作液处理，空白对照组不做处理，阴性对照组为使用0.1％SLS工作液处理，阳性对照组使用0.1％SLS工作液处理后根据不同测试指标使用不同的阳性对照工作液。
屏障功能指标：兜甲蛋白(LOR)、丝聚蛋白(FLG)，免疫组化后荧光定量检测，以50μM的WY 14643作为阳性对照。
炎症控制指标：IL-1α、IL-8、PGE2、NF-κB，NF-κB使用免疫组化后荧光定量检测，其余使用ELISA法定量检测，以0.01％地塞米松作为阳性对照。
3)操作步骤
6孔板内添加EpiGrowth培养液0.9mL后将表皮模型转移到6孔板中并对各孔根据分组进行编号。NC组、PC组和样品组表面添加25μL 0.1％的SLS溶液，孵育30min。孵育结束后，吸去SLS溶液，PC组液下添加相应浓度的阳性对照工作液，样品组取待测样品工作液12.5μL加于模型表面，涂抹均匀后置于CO2培养箱(37℃、5％CO2)中孵育24h。孵育结束后，用无菌PBS溶液清洗模型表面残留的受试物，用无菌棉签拭去模型表面残留液体。
ELISA检测：吸取模型组织下培养液于离心管中并置于-80℃冰箱冷冻暂存，根据ELISA试剂盒的操作说明进行检测分析。
免疫荧光检测：将用于检测的模型环切取下后用4％的多聚甲醛进行固定处理，固定24h后，进行免疫荧光检测，显微镜下拍照观察，采集图片并定量分析。各指标检测结果见表4，NC组与BC组相比各指标均有显著或极显著差异，表明SLS建立损伤和炎症模型的有效性。PC组和阳性组与NC组相比，LOR和FLG均有极显著提高，IL-1α等炎症因子均有极显著下降，表明本发明制备的以丁酸乙酯为特征性成分的组合物可提高皮肤屏障功能，降低刺激导致的炎症因子升高，具有舒缓作用。LOR、FLG和NF-κB的免疫荧光结果示例见图1。
表4、组合物对屏障功能相关蛋白和炎症因子水平的影响
实施例3
将实施例2的组合物以组合物以2％的w/w浓度溶于纯化水中，并加入适量化妆品用防腐剂，制备得到精华液。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (10)
1.一种含丁酸乙酯的双菌发酵产物的制备方法，其特征在于，包括以下步骤：
将产芽孢细菌种子液和干酵母接种于灭菌的培养基后进行密闭发酵，发酵过程中控制发酵液pH为5.0～6.5，发酵结束后将发酵液的pH调节为7.0～8.0，进行减压提取，收集冷凝液并与水混合、分层、萃取除去水溶性杂质后得到含丁酸乙酯的双菌发酵产物。
2.如权利要求1所述的制备方法，其特征在于，所述培养基中含有麦芽浸粉，所述麦芽浸粉的含量为5％～20％w/w。
3.如权利要求1所述的制备方法，其特征在于，所述产芽孢细菌为代谢糖类产生以丁酸为主要短链脂肪酸成分的细菌，包括枯草芽胞杆菌Bacillus subtilis subsp.subtilisCICC23592、热噬淀粉芽胞杆菌Bacillus thermoamylovorans CICC20802、巨大芽胞杆菌Bacillus megaterium CICC10024和丁酸梭菌Clostridium butyricum CICC23847中的至少一种。
4.如权利要求1所述的制备方法，其特征在于，产芽孢细菌种子液的接种量为2％～5％v/v，干酵母接种量为0.5～1g/L培养液。
5.如权利要求1所述的制备方法，其特征在于，发酵时间为3～10天。
6.如权利要求1所述的制备方法，其特征在于，减压提取时发酵液的温度为40～60℃，压力为-0.09～-0.06MPa，冷凝温度为10～20℃。
7.一种含丁酸乙酯的双菌发酵产物，其特征在于，通过如权利要求1-6任一项所述的制备方法获得。
8.一种组合物，其特征在于，包括如权利要求7所述的含丁酸乙酯的双菌发酵产物和多元醇，所述双菌发酵产物和多元醇的质量比为5～90:95～10；
优选的，所述多元醇包括1,3-丙二醇、1,2-丙二醇、1,3-丁二醇、1,2-丁二醇、甲基丙二醇、1,2-戊二醇和1,2-己二醇中的至少一种。
9.如权利要求7所述的含丁酸乙酯的双菌发酵产物和/或如权利要求8所述的组合物在制备护肤产品中的应用。
10.一种护肤产品，其特征在于，所述护肤产品中含有如权利要求7所述的含丁酸乙酯的双菌发酵产物和/或如权利要求8所述的组合物。
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