CN113262181B - 一种柳叶蜡梅花发酵物及其制备方法与应用 - Google Patents
一种柳叶蜡梅花发酵物及其制备方法与应用 Download PDFInfo
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- CN113262181B CN113262181B CN202110465817.6A CN202110465817A CN113262181B CN 113262181 B CN113262181 B CN 113262181B CN 202110465817 A CN202110465817 A CN 202110465817A CN 113262181 B CN113262181 B CN 113262181B
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- salicifolia
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Abstract
本发明提供一种柳叶蜡梅花发酵物及其制备方法与应用，涉及化妆品技术领域，本发明包括如下制作项目：鲜花采摘、分段烘干、粉碎、发酵、过滤、菌体的破壁与提取、冷冻干燥以及高压蒸馏，采用发酵工程等现代生物技术，添加有益菌种进行分段发酵，运用膜过滤，结合绿色溶剂甘油联合超声波提取技术，并经高压蒸馏制得柳叶蜡梅花发酵物，改良了柳叶蜡梅花香气太冲的难题，并赋予了柳叶蜡梅花发酵物自然水果香气的味道。
Description
技术领域
本发明涉及化妆品技术领域，尤其涉及一种柳叶蜡梅花发酵物及其制备方法与应用。
背景技术
柳叶蜡梅为蜡梅科蜡梅属半常绿灌木，为中国特有植物，主产于江浙皖南一带，《中国药典》记载柳叶蜡梅叶又名食凉茶、伤风草、茅山茶、香风茶、黄金茶等，主要治疗因感受风寒而引起的肚痛、肚胀、腹泻，或伤食所致的消化不良、腹部胀痛等症，还用于防治感冒和流行性感冒，现代药理学表明，柳叶蜡梅叶中含有挥发油、生物碱、香豆素类、黄酮类、蒽醌类、黄酮等活性成分，具有抗菌、消炎、抗病毒、抗氧化及增强机体免疫等功效。
柳叶蜡梅花是柳叶蜡梅的花朵，属完全花，花瓣一般淡黄色，开花时间为当年11月至次年2月，柳叶蜡梅是以采收叶片为主的经济作物，柳叶蜡梅开花结果属于柳叶蜡梅的生殖生长，会争夺柳叶蜡梅养分，致使柳叶蜡梅叶产量下降、质量降低，柳叶蜡梅花被认为是柳叶蜡梅叶生产中的“废料”，在柳叶蜡梅叶生产过程中会使用开花抑制剂或者通过施肥调控等措施来抑制柳叶蜡梅花的生长，市场上有少量柳叶蜡梅花产品，但普遍存在的问题是，由于柳叶蜡梅花的香味太冲，气味难以被普通消费人群接受，目前，国内外学者对柳叶蜡梅的研究多集中在叶，对其花的研究相对较少，柳叶蜡梅花资源有效利用率较低。
目前未见有柳叶蜡梅花加工与应用的技术公开，更未查询到有关改良柳叶蜡梅花香味的相关文献，且利用传统工艺所制备的柳叶蜡梅花香气太冲、刺激较大，气味难以被普通消费人群接受，不能满足柳叶蜡梅花功能性化妆品的工业化开发需求。
发明内容
本发明的目的在于提供一种柳叶蜡梅花发酵物及其制备方法与应用，以解决上述技术问题。
本发明为解决上述技术问题，采用以下技术方案来实现：一种柳叶蜡梅花发酵物的制备方法，其特征在于，包括如下制作项目：
鲜花采摘、分段烘干、粉碎、发酵、过滤、菌体的破壁与提取、冷冻干燥以及高压蒸馏；
1)鲜花采摘，选用当年十一月份新鲜柳叶蜡梅花朵；
2)分段烘干，将新鲜柳叶蜡梅花于烘干设备内进行分段式温度递增烘烤，其中首次烘烤温度为38℃-45℃，时间1-2小时，下次烘烤温度为40℃-55℃，时间为8-12小时，末次烘烤温度50℃-65℃，时间1-3小时；
3)粉碎，将干燥后的柳叶蜡梅花转移至破壁机中进行气流粉碎；
4)发酵，将柳叶蜡梅花粉末与去离子水按照重量比1:15-1:30混合均匀，在以清酒乳杆菌、乳酸乳球菌依序进行二段式发酵获得发酵液；
5)过滤，将发酵液经过石英砂过滤器进行过滤后，再送入平均孔径30-50微米的陶瓷膜过滤器中过滤去除菌体，得到发酵上清液；
6)菌体的破壁与提取，在菌体中加入10-70％的甘油，以150-300瓦数的功率超声，超声温度为25℃-35℃，超声时间为5-10分钟，在转速2500-3500rpm条件下离心15-30分钟，收集上清液进行旋转蒸发回收甘油，剩余的即为菌体活性液；
7)冷冻干燥，将发酵上清液和菌体活性液按照质量比(3-8)：1比例混合，以50-100rpm的转速搅拌10-15分钟，混匀，在进行-45℃至-55℃下的真空干燥；
8)高压蒸馏，真空干燥处理后，设定加压压力数值为100-350MPa，在温度45℃-60℃下，蒸馏10-40分钟，将蒸馏产生的精油和蒸汽的混合气体进行冷凝处理，得到馏出液，对馏出液进行油水分离处理，收集浮于上层的精油，即为柳叶蜡梅花发酵物。
优选的，步骤4)中清酒乳杆菌的添加量为0.01-0.5％(w/w)，发酵温度为20℃-28℃，发酵时间为1-2天，振荡转速为100-200rpm。
优选的，步骤4)乳酸乳球菌的添加量为1.5-7.5％(w/w)，发酵温度为25℃-35℃，发酵时间为5-12天，振荡转速为100-200rpm。
优选的，步骤4)中发酵液达到糖度范围1.5°-3°、pH<4.2等，则判定发酵完成。
优选的，步骤5)中陶瓷膜过滤过程中压力0.15-0.35MPa。
一种柳叶蜡梅花发酵物的应用，其特征在于，包括面膜纸和精华液，其中精华液重量百分比计其组分如下：
柳叶蜡梅花发酵物0.3-3.5％
红豆杉叶提取物0.5-2％
黄精提取物1.5-4.5％
金黄洋甘菊提取物1.5-3％
白及提取物1.2-2.7％
保湿剂2.75-8％
助剂1.68-7％和剩余量为去离子水。
优选的，保湿剂包括透明质酸钠0.01-0.2％、羟乙基纤维素0.04-0.05％、海藻糖0.2-2.5％、甘油1-3％、丁二醇1.5-3％。
优选的，助剂包括螯合剂0.01-0.03％、增稠剂0.15-1.2％、防腐剂1.5-6.2％、pH调节剂0.01-0.025％、增溶剂0.01-0.15％。
优选的，螯合剂为辛酰羟肟酸和植酸钠，增稠剂为卡波姆和长角豆胶、防腐剂为对羟基苯乙酮、1,2己二醇和丁二醇，pH调节剂为10％NaOH溶液，增溶剂为PEG20氢化蓖麻油和PEG60氢化蓖麻油。
本发明的有益效果是：
1、本发明的目的是提供一种柳叶蜡梅花发酵物的制备方法，采用发酵工程等现代生物技术，添加有益菌种进行分段发酵，运用膜过滤，结合绿色溶剂甘油联合超声波提取技术，并经高压蒸馏制得柳叶蜡梅花发酵物，改良了柳叶蜡梅花香气太冲的难题，并赋予了柳叶蜡梅花发酵物自然水果香气的味道。
2、本发明工艺制得的柳叶蜡梅花发酵物的活性成分含量保留较高、提取效率更高，获得的发酵物不仅包含了柳叶蜡梅花本身的活性物质，而且还含有微生物的次级代谢产物，增强了发酵产物的抑菌和抗炎症功效。
3、本发明的另一个目的是充分利用柳叶蜡梅花废弃资源，提高柳叶蜡梅花资源的有效利用率，提供的一种柳叶蜡梅花发酵物面膜，其功效成分均来源于天然植物，安全可靠，且通过各功效组分间的系统配伍，能有效抑制肌肤炎症，促进了炎症肌肤的修复，进而减少毛孔堵塞引发的痤疮。
附图说明
图1为本发明制备的柳叶蜡梅花发酵物的GC-MS总离子色谱图；
图2为以水蒸气蒸馏法制备的柳叶蜡梅花精油的GC-MS总离子色谱图。
具体实施方式
基于实施方式中的实施例，本领域技术人员在没有做出创造性劳动的前提下所获得其它实施例，都属于本发明的保护范围。
1、柳叶蜡梅花发酵物的制备及其应用
实施例1
一种柳叶蜡梅花发酵物的制备方法，包括如下制作项目：
鲜花采摘、分段烘干、粉碎、发酵、过滤、菌体的破壁与提取、冷冻干燥以及高压蒸馏；
1)鲜花采摘，选用当年十一月份新鲜柳叶蜡梅花朵；
2)分段烘干，将新鲜柳叶蜡梅花于烘干设备内进行分段式温度递增烘烤，其中首次烘烤温度为38℃-45℃，时间1-2小时，下次烘烤温度为40℃-55℃，时间为8-12小时，末次烘烤温度50℃-65℃，时间1-3小时；
3)粉碎，将干燥后的柳叶蜡梅花转移至破壁机中进行气流粉碎；
4)发酵，将柳叶蜡梅花粉末与去离子水按照重量比1:15-1:30混合均匀，在以清酒乳杆菌、乳酸乳球菌依序进行二段式发酵获得发酵液；
5)过滤，将发酵液经过石英砂过滤器进行过滤后，再送入平均孔径30-50微米的陶瓷膜过滤器中过滤去除菌体，得到发酵上清液；
6)菌体的破壁与提取，在菌体中加入10-70％的甘油，以150-300瓦数的功率超声，超声温度为25℃-35℃，超声时间为5-10分钟，在转速2500-3500rpm条件下离心15-30分钟，收集上清液进行旋转蒸发回收甘油，剩余的即为菌体活性液；
7)冷冻干燥，将发酵上清液和菌体活性液按照质量比(3-8)：1比例混合，以50-100rpm的转速搅拌10-15分钟，混匀，在进行-45℃至-55℃下的真空干燥；
8)高压蒸馏，真空干燥处理后，设定加压压力数值为100-350MPa，在温度45℃-60℃下，蒸馏10-40分钟，将蒸馏产生的精油和蒸汽的混合气体进行冷凝处理，得到馏出液，对馏出液进行油水分离处理，收集浮于上层的精油，即为柳叶蜡梅花发酵物。
实施例2
以水蒸气蒸馏法制备的柳叶蜡梅花精油为对比组。
实施例3
一种含有柳叶蜡梅花发酵物的抑菌消炎面膜，包括面膜纸和精华液，
精华液按重量百分比计其组分如下，柳叶蜡梅花发酵物1.5％、红豆杉叶提取物0.5％、黄精提取物3％、金黄洋甘菊提取物2％、白及提取物1.5％，保湿剂6％，助剂4.5％和剩余量为去离子水。
保湿剂包括透明质酸钠0.1％、羟乙基纤维素0.05％、海藻糖1.35％、甘油2％、丁二醇2.5％。
助剂包括螯合剂0.01％、增稠剂0.2％、防腐剂4.27％、pH调节剂0.01％、增溶剂0.01％。
螯合剂为辛酰羟肟酸和植酸钠，增稠剂为卡波姆和长角豆胶，防腐剂为对羟基苯乙酮、1,2己二醇和丁二醇，pH调节剂为10％NaOH溶液，增溶剂为PEG20氢化蓖麻油和PEG60氢化蓖麻油。
2、柳叶蜡梅花发酵物的化学组成
色谱条件：选用HP-5MS弹性石英毛细光柱(0.25μm，30m×0.25mm)，载气为高纯氦气，载气流速为1.0mL/min，分流比为40:1，进样量为1.0μL，进样口温度为280℃，色谱柱初始温度为60℃，保持3min，以5℃/min升至280℃，保持10min。
质谱条件：离子源温度230℃，电离方式为EI，电子能量为70eV，四级杆温度为150℃；质量扫描范围为35-450u，溶剂延迟时间3min，采用NIST08标准谱库进行检索。
图1为本发明制备的柳叶蜡梅花发酵物的GC-MS总离子色谱图，经过NIST08谱库检索和质谱图核对，从柳叶蜡梅花发酵物中鉴定出21个化合物，占总峰面积92.15％。
图2为以水蒸气蒸馏法制备的柳叶蜡梅花精油的GC-MS总离子色谱图，从对比实施例2鉴定出14个化合物，占总峰面积92.55％，由表1可知，对比实施例2的主要化学成分为桉叶油醇、α-蒎烯，其含量分别高达60.51％和13.6％，而高含量的桉叶油醇和α-蒎烯导致新鲜柳叶蜡梅花香气太冲、刺激较大。
表1实施例1和对比实施例2的化学成分及其相对含量
本发明采用发酵工艺制备的柳叶蜡梅花发酵物，其桉叶油醇和α-蒎烯含量下降至9.02％和1.57％，且通过微生物发酵作用产生了新的化学成分，如柠檬烯、α-香柑油烯、双环大牦牛儿烯、麝子油烯、γ-毕澄茄烯和1-去氢白菖烯等，改良了柳叶蜡梅花香气太冲的难题，并赋予了柳叶蜡梅花发酵物自然水果香气的味道，而且采用本发明提取方法制得的柳叶蜡梅花发酵物的活性成分含量保留较高、不混有其它杂质、提取效率更高，并由于采用了高压萃取技术，对柳叶蜡梅花发酵物还起到了很好的杀菌作用，更易于保藏。
3、柳叶蜡梅花发酵物的抑菌作用
菌种活化及菌悬液制备：将大肠杆菌、金黄葡萄球菌、痤疮丙酸杆菌和绿脓杆菌接种到试管培养基斜面进行活化，于37℃恒温培养箱中培养24h，长出菌落后，在无菌操作台内各挑取已活化好的菌种接种到无菌生理盐水中，配置各菌悬液浓度约为106～107CFU/mL。
抑菌圈法测定抑菌活性：在无菌操作台中将熔化并灭菌的培养基倒入已灭菌的培养皿中，冷却凝固后，加入菌悬液100μL,用涂布棒涂布均匀，用移液枪于无菌直径6mm的滤纸片中央加入10μL样品，以灭菌蒸馏水为空白对照，以对羟基苯甲酸丙酯为阳性对照，37℃倒置培养24h后测量抑菌圈直径，结果重复3次，取平均值。
表2为本发明制备的柳叶蜡梅花发酵物的抑菌作用结果，相比于对比实施例2，实施例1对大肠杆菌、金黄葡萄球菌、绿脓杆菌和痤疮丙酸杆菌具有更为显著的抑菌效果，在同样浓度条件下，柳叶蜡梅花发酵物与阳性对照对羟基苯甲酸丙酯的抑菌作用相当，研究结果首次揭示了柳叶蜡梅花发酵物对4种供试的革兰氏阳性细菌和革兰氏阴性细菌都有明显的抑制作用，说明采用本发明制得的发酵物增强了抑菌功效，为柳叶蜡梅花发酵物的功能性化妆品开发奠定了科学依据。
表2实施例1和对比实施例2对4种菌的抑菌圈直径
4、柳叶蜡梅花发酵物的抗炎症作用
RAW264.7细胞培养及分组：小鼠RAW264.7细胞置于10％胎牛血清的DMEM高糖培养液中培养，培养条件为37℃、5％CO2且相对饱和湿度，实验设置空白对照组、LPS模型组、实施例1组、对比实施例2组。
细胞毒性的检测：取对数生长期的RAW264.7细胞，以1x105个/mL接种于96孔板中，每孔100μL，贴壁培养24h，样品处理后，培养24h，每孔加入10μL的MTT，继续培养3h后，吸弃上清液，每孔中加入200μL DMSO溶液，避光震荡，使紫色结晶充分溶解，于570nm波长处测各孔的吸光值。
NO含量的检测：RAW264.7细胞以1x106个/mL接种于96孔板中，每孔200μL，培养24h，在实验组分别加入样品，孵育1h后加入20μL LPS(1μg/mL)作用18h，收集上清，用Griess法检测细胞上清液中NO水平。
表3为本发明制备的柳叶蜡梅花发酵物的抗炎症作用结果，由表3可知，在0.05mg/mL浓度下，柳叶蜡梅花发酵物对RAW264.7细胞无毒性作用，空白对照组RAW264.7细胞液中NO含量为(15.47±1.20)％，细胞经LPS刺激后，NO含量明显升高，表明体外RAW264.7细胞炎症模型建立成功，与对比实施例2相比，实施例1的抗炎症作用效果愈加显著，与上述的抑菌作用结果相一致，研究结果首次揭示了采用本发明制得的柳叶蜡梅花发酵物具有更为显著的抗炎症作用，说明采用本发明制得的发酵物增强了抗炎症功效，为柳叶蜡梅花发酵物的功能性化妆品开发奠定了理论基础。
表3实施例1和对比实施例2对RAW264.7细胞活力及LPS诱导的细胞NO含量的影响
5、柳叶蜡梅花发酵物面膜的消炎作用评价
选择具有60名脸部痤疮肌肤，伴有油脂分泌旺盛、炎症的肌肤问题的志愿者(18-25岁)，随机平均分为3组，第1、第2组志愿者使用实施例3制得的柳叶蜡梅花发酵物面膜产品，第3组志愿者使用市售一般面膜作为对比例，治疗标准如下：
显效：炎症完全消失，不再复发；
有效：炎症部分消失；
无效：炎症没有改善甚至加重。
每一样品使用二周，由表4可知，有97.5％以上的试验者认为添加了柳叶蜡梅花发酵物、红豆杉叶提取物、黄精提取物、金黄洋甘菊提取物、白及提取物等所制得的柳叶蜡梅花发酵物面膜能有效抑制肌肤炎症，促进了炎症肌肤的修复，进而减少毛孔堵塞引发的痤疮，维持肌肤的健康状态，综上所述，本发明的面膜完全符合要求，相比于市售产品的安神效果更好。
表4柳叶蜡梅花发酵物面膜的消炎作用评价(试验期2周)
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的仅为本发明的优选例，并不用来限制本发明，在不脱离本发明精神和范围的前提下，本发明还会有各种变化和改进，这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
Claims (6)
1.一种柳叶蜡梅花发酵物的制备方法，其特征在于，包括如下制作项目：
鲜花采摘、分段烘干、粉碎、发酵、过滤、菌体的破壁与提取、冷冻干燥以及高压蒸馏；
1）鲜花采摘，选用当年十一月份新鲜柳叶蜡梅花朵；
2）分段烘干，将新鲜柳叶蜡梅花于烘干设备内进行分段式温度递增烘烤，其中首次烘烤温度为38℃-45℃，时间1-2小时，下次烘烤温度为40℃-55℃，时间为8-12小时，末次烘烤温度50℃-65℃，时间1-3小时；
3）粉碎，将干燥后的柳叶蜡梅花转移至破壁机中进行气流粉碎；
4）发酵，将柳叶蜡梅花粉末与去离子水按照重量比1:15-1:30混合均匀，在以清酒乳杆菌、乳酸乳球菌依序进行二段式发酵获得发酵液；
步骤4）中清酒乳杆菌的添加量为0.01-0.5％(w/w)，发酵温度为20℃-28℃，发酵时间为1-2天，振荡转速为100-200rpm；
步骤4）乳酸乳球菌的添加量为1.5-7.5％ (w/w)，发酵温度为25℃-35℃，发酵时间为5-12天，振荡转速为100-200rpm；
步骤4）中发酵液达到糖度范围1.5°-3°和pH<4.2，则判定发酵完成；
5）过滤，将发酵液经过石英砂过滤器进行过滤后，再送入平均孔径30-50微米的陶瓷膜过滤器中过滤去除菌体，得到发酵上清液；
6）菌体的破壁与提取，在菌体中加入浓度为10-70%的甘油，以150-300瓦数的功率超声，超声温度为25℃-35℃，超声时间为5-10分钟，在转速2500-3500rpm条件下离心15-30分钟，收集上清液进行旋转蒸发回收甘油，剩余的即为菌体活性液；
7）冷冻干燥，将发酵上清液和菌体活性液按照质量比（3-8）：1比例混合，以50-100rpm的转速搅拌10-15分钟，混匀，在进行-45℃至-55℃下的真空干燥；
8）高压蒸馏，真空干燥处理后，设定加压压力数值为100-350MPa，在温度45℃-60℃下，蒸馏10-40分钟，将蒸馏产生的精油和蒸汽的混合气体进行冷凝处理，得到馏出液，对馏出液进行油水分离处理，收集浮于上层的精油，即为柳叶蜡梅花发酵物。
2.根据权利要求1所述的一种柳叶蜡梅花发酵物的制备方法，其特征在于，步骤5）中陶瓷膜过滤过程中压力0.15-0.35MPa。
3.一种含有柳叶蜡梅花发酵物的面膜，其特征在于，所述面膜包括面膜纸和精华液，柳叶蜡梅花发酵物由权利要求1中所述柳叶蜡梅花发酵物的制备方法得到，其中精华液重量百分比计其组分如下：
柳叶蜡梅花发酵物0.3-3.5%，
红豆杉叶提取物0.5-2%
黄精提取物1.5-4.5%
金黄洋甘菊提取物1.5-3%
白及提取物1.2-2.7%
保湿剂2.75-8%
助剂1.68-7%和剩余量为去离子水。
4.根据权利要求3所述的一种含有柳叶蜡梅花发酵物的面膜，其特征在于，保湿剂包括透明质酸钠0.01-0.2%、羟乙基纤维素0.04-0.05%、海藻糖0.2-2.5%、甘油1-3%、丁二醇1.5-3%。
5.根据权利要求3所述的一种含有柳叶蜡梅花发酵物的面膜，其特征在于，助剂包括螯合剂0.01-0.03%、增稠剂0.15-1.2%、防腐剂1.5-6.2%、pH调节剂 0.01-0.025%、增溶剂0.01-0.15%。
6.根据权利要求5所述的一种含有柳叶蜡梅花发酵物的面膜，其特征在于，螯合剂为辛酰羟肟酸和植酸钠，增稠剂为卡波姆和长角豆胶、防腐剂为对羟基苯乙酮、1,2 己二醇和丁二醇，pH调节剂为10％NaOH溶液，增溶剂为PEG20氢化蓖麻油和PEG60氢化蓖麻油。
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