CN116590168A - 一种包含奇异酵母菌体溶胞物的组合物的制备与应用 - Google Patents
一种包含奇异酵母菌体溶胞物的组合物的制备与应用 Download PDFInfo
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- CN116590168A CN116590168A CN202211628123.0A CN202211628123A CN116590168A CN 116590168 A CN116590168 A CN 116590168A CN 202211628123 A CN202211628123 A CN 202211628123A CN 116590168 A CN116590168 A CN 116590168A
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- 241001608472 Bifidobacterium longum Species 0.000 claims abstract description 66
- 229940009291 bifidobacterium longum Drugs 0.000 claims abstract description 66
- 240000001929 Lactobacillus brevis Species 0.000 claims abstract description 64
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- 241000218588 Lactobacillus rhamnosus Species 0.000 claims abstract description 64
- 238000000855 fermentation Methods 0.000 claims abstract description 37
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- PLXBWHJQWKZRKG-UHFFFAOYSA-N Resazurin Chemical compound C1=CC(=O)C=C2OC3=CC(O)=CC=C3[N+]([O-])=C21 PLXBWHJQWKZRKG-UHFFFAOYSA-N 0.000 description 2
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- GRVFOGOEDUUMBP-UHFFFAOYSA-N sodium sulfide (anhydrous) Chemical compound [Na+].[Na+].[S-2] GRVFOGOEDUUMBP-UHFFFAOYSA-N 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
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Abstract
本发明属于生物技术领域,具体涉及酵母菌和益生菌复合溶胞物的制备方法及其在化妆品行业领域的应用。本发明将奇异酵母用在短乳杆菌、鼠李糖乳杆菌及长双歧杆菌增殖中,并以四种菌体的溶胞物作为组合物,组合物中奇异酵母溶胞物的添加量为组合物的45%‑79%;短乳杆菌溶胞物的添加量为组合物的1%‑10%;鼠李糖乳杆菌溶胞物的添加量为组合物的5%‑20%;长双歧杆菌溶胞物的添加量为组合物的15%‑25%。本发明利用奇异酵母发酵产生的胞外多糖,促进短乳杆菌、鼠李糖乳杆菌及长双歧杆菌的增殖,节约发酵成本,减少资源浪费。溶胞物组合物的制备中以奇异酵母促进其他菌体的增殖,混合后的溶胞物功效更加全面。
Description
技术领域
本发明属于生物技术领域,具体涉及酵母菌和益生菌复合溶胞物的制备方法及其在化妆品行业领域的应用。
背景技术
皮肤是人体的主要屏障,是最直接与外界接触的器官。皮肤既能防止外界化学、物理、机械、生物诸多因素的侵入,又能防止水分,营养物质经表皮丢失而维持皮肤屏障功能的稳定。然而,过度的化学和物理刺激损伤皮肤的正常功能,诱发或加速其老化,导致皱纹的出现以及紧致度、柔韧度和弹性的损失。
益生菌是对人体有益的微生物的统称,在医药、食品、保健品及农业等领域有着广泛的应用。益生菌通常作为食物和药物用来维持胃肠道菌群平衡,近年来,随着对其研究的深入,发现益生菌还可以外用于皮肤,其在保护健康皮肤、防治皮肤疾病等方面,呈现巨大潜力。因此,益生菌逐渐被应用到化妆品领域中,为化妆品领域带来了新的发展方向。在现有益生菌溶胞物护肤品中,大多是添加单一的益生菌,其功效不够全面,组合添加的益生菌也存在配比不合理等问题。大多数益生菌培养采用单一的培养,或在培养基中加入益生菌营养剂(如:小核菌胶、α-葡聚糖寡糖),或加入益生元(如:低聚果糖、葡聚糖、抗性淀粉),来促进益生菌的增殖。这些额外添加的营养物质,在益生菌批量发酵的过程中会增加成本。
发明内容
本发明的目的在于,提供一种功效全面的复合菌体溶胞物,确定制备方法及其在护肤品中的应用,所述复合溶胞物可以作为原材料加入各种化妆品中。
为了实现上述效果,本发明采用如下技术方案:
本发明提供了奇异酵母在促进短乳杆菌、鼠李糖乳杆菌及长双歧杆菌增殖中的应用。
上述技术方案中,进一步地,所述所述奇异酵母菌(Saccharomyces paradoxus),其保藏编号为:CGMCC 2.5026;所述短乳杆菌(Lactobacillus brevis),其保藏编号CGMCC8581;鼠李糖乳杆菌(Lactobacillus rhamnosus),其保藏编号CICC 20257;长双歧杆菌(Bifidobacterium longum),其保藏编号GDMCC 1.263。
上述技术方案中,进一步地,在短乳杆菌、鼠李糖乳杆菌、长双歧杆菌的培养基中加入所述奇异酵母的发酵液;所述奇异酵母发酵液的添加量为培养基总量的3~30%,其中,奇异酵母胞发酵液中胞外多糖含量为25-35%。
上述技术方案中,进一步地,在短乳杆菌、鼠李糖乳杆菌、长双歧杆菌的培养基中加入所述奇异酵母的胞外多糖,奇异酵母胞外多糖的添加量为培养基总量的1%-8%。
本发明还提供了一种可用于皮肤的复合菌体溶胞物的组合物,包括菌体细胞溶胞物,所述菌体包含奇异酵母(Saccharomyces paradoxus),菌种保藏号CGMCC 2.5026;短乳杆菌(Lactobacillus brevis),菌种保藏号CGMCC 8581;鼠李糖乳杆菌(Lactobacillusrhamnosus),菌种保藏号CICC 20257;长双歧杆菌(Bifidobacterium longum)菌种保藏号GDMCC 1.263。
上述技术方案中,进一步地,奇异酵母(Saccharomyces paradoxus)溶胞物的添加量为组合物质量的45%-79%;短乳杆菌(Lactobacillus brevis)溶胞物的添加量为组合物质量的1%-10%;鼠李糖乳杆菌(Lactobacillus rhamnosus)溶胞物的添加量为组合物质量的5%-20%;长双歧杆菌(Bifidobacterium longum)溶胞物的添加量为组合物质量的15%-25%。
上述技术方案中,进一步地,所述组合物中各菌体溶胞物的制备方法包括以下步骤:(1)菌体的培养:各菌体经平板培养活化,挑取单菌落制得一级种子液,再进行扩大培养,得菌体发酵液;奇异酵母培养基为麦芽汁肉汤培养基,短乳杆菌和鼠李糖乳杆菌的培养基为MRS培养基,长双歧杆菌的培养基为BBL培养基;
(2)将步骤(1)得到的发酵混合物进行离心,得菌体沉淀,加入蒸馏水混合均匀,经超声破碎后,将混合物进行离心,得到细胞溶胞物,冷冻干燥备用;
优选地,短乳杆菌、鼠李糖乳杆菌和长双歧杆菌溶胞物的制备方法如下步骤:
(1)短乳杆菌和鼠李糖乳杆菌的MRS培养基成分及其添加比例为:蛋白胨1%、牛肉浸提物1%、酵母提取物0.5%、葡萄糖2%、醋酸钠0.5%、柠檬酸二胺0.2%、磷酸氢二钾0.2%、硫酸镁0.02%、硫酸锰0.005%、吐温0.1%;长双歧杆菌的BBL培养基成分及其添加比例为:蛋白胨1%、牛肉浸提物1%、酵母提取物0.5%、葡萄糖0.5%、磷酸氢二钾0.045%、磷酸二氢钾0.033%、氯化铵0.1%、硫酸镁0.01%、L-半胱氨酸0.05%、硫化钠0.05%、刃天青0.0001%;
(2)菌体培养方法为:菌体在37℃培养箱中,平板静置培养20-24h,注意长双歧杆菌是在厌氧培养箱中进行培养;挑取单菌落接至10mL液体培养基中,37℃培养20-24h,获得一级种子液;种子液按1%的接种量接至液体培养基中,并接入30-300ml奇异酵母发酵液,至1L体系,37℃培养24h,得到菌体发酵液;
(3)将得到的发酵混合物进行离心,8000rpm/10min,得菌体沉淀;
(4)将菌体沉淀加水混合均匀,进行超声破碎,超声破碎的条件为:功率300W,工作5s,停顿15s,工作时长10min,共超声两次;
(5)将制得细胞溶出物进行离心,8000rpm/10min,去掉细胞壁,得到细胞溶胞物;
(6)将得到的溶胞物进行冻干处理,冷冻温度:-40℃至-60℃,真空度1Pa-8Pa,冷冻干燥时间为24h-36h。
优选地,在短乳杆菌、鼠李糖乳杆菌及长双歧杆菌的扩大培养阶段加入奇异酵母发酵液或奇异酵母胞外多糖;
优选地,奇异酵母溶胞物的制备方法如下步骤:
(1)奇异酵母的麦芽汁肉汤培养基成分及其添加比例为:酵母膏0.3%,麦芽提取物0.3%,葡萄糖1%,蛋白胨0.5%;
(2)菌体培养方法为:菌体在28℃培养箱中,平板静置培养24-48h;挑取单菌落接至10mL液体培养基中,28℃培养24-48h,获得一级种子液;种子液按1%的接种量接至1L液体培养基中,28℃培养48h,得到菌体发酵液;
(3)将得到的发酵混合物进行离心,8000rpm/10min,得菌体沉淀和发酵液;
(4)将菌体沉淀加水混合均匀,进行超声破碎,超声破碎的条件为:功率300W,工作5s,停顿15s,工作时长10min,共超声两次;
(5)将制得细胞溶出物进行离心,8000rpm/10min,去掉细胞壁,得到细胞溶胞物;
(6)将得到的溶胞物进行冷冻干燥处理,冷冻温度:-40℃至-60℃,真空度1Pa-8Pa,冷冻干燥时间为24h-36h。
优选地,奇异酵母胞外多糖的制备方法如下步骤:
(1)将上述步骤(3)得到的奇异酵母发酵液进行浓缩,并透析,然后进行冷冻干燥处理。称取适当干粉,用蒸馏水溶解;
(2)采用苯酚-硫酸法测定奇异酵母胞外多糖含量。苯酚-硫酸法指浓硫酸将多糖或寡糖水解为单糖,并使得单糖迅速脱水为糠醛衍生物,在强酸条件下糠醛衍生物可与苯酚发生显色反应,生成的橙黄色物质在490nm处有最大吸收值。由于吸光值与多糖浓度在一定浓度范围内成线性关系,故可用比色法测定多糖含量。
(3)将浓缩并透析后的奇异酵母发酵液中加入4倍体积的100%乙醇,4℃静置过夜,然后进行离心,10000rpm/20min,收集沉淀,并进行冷冻干燥处理;
(4)将所得干粉加入蒸馏水复溶,与Sevage试剂按照体积比1:1进行混合,震荡反应20min后进行离心,4000rpm/10min,然后小心吸取上层溶液。将得到的溶液进行旋蒸除去有机溶剂,随后进行冷冻干燥处理,得到胞外多糖干粉。
本发明还提供了前述组合物在护肤品中的应用;优选地,所述应用于护肤品的功效物质为菌体细胞溶胞物。
上述技术方案中,进一步地,前述溶胞物在减少皮肤水分流失、增强皮肤弹性、增加皮肤厚度、缩小皮肤毛孔、减少皮肤皱纹、改善皮肤敏感方面的应用。
与现有技术相比,本发明的有益效果:
(1)本发明利用奇异酵母发酵产生的胞外多糖,促进短乳杆菌、鼠李糖乳杆菌及长双歧杆菌的增殖,不仅节约了发酵成本,还可以高效利用酵母发酵产物中的胞外多糖,减少资源浪费。(2)本发明将奇异酵母、短乳杆菌、鼠李糖乳杆菌及长双歧杆菌的溶胞物组合,制备中以奇异酵母促进其他菌体的增殖,混合后的溶胞物,功效更加全面,解决了现有益生菌护肤品的局限性,可以更好的提升肌肤紧致度,强化皮肤屏障。经实验结果可以得到,使用复合菌体溶胞物的化妆品后,皮肤经皮水分流失降低14.9-18.7%;皮肤弹性升高10.7-15.4%;皮肤厚度增加11.3-16.8%;皮肤毛孔缩小了12.4-17.1%;肌肤皱纹减少9.8-12.6%;皮肤敏感度降低13.8-17.9%。
附图说明
图1标准曲线图。
具体实施方式
以下结合具体实施例对本发明作进一步说明,但不以任何方式限制本发明。所用菌均已保藏或通过商业途径直接购买得到。
实施例1:奇异酵母胞外多糖对短乳杆菌、鼠李糖乳杆菌以及长歧双歧杆菌的促生长作用。
奇异酵母胞外多糖的制备,采用以下步骤:
A、菌体在28℃培养箱中,平板静置培养24-48h;挑取单菌落接至10mL液体培养基中,28℃培养24-48h,获得一级种子液;种子液按1%的接种量接至1L液体培养基中,28℃培养48h,离心8000rpm/10min,除去菌体沉淀,得到菌体发酵液;
B、将发酵液浓缩后进行透析,冷冻干燥处理后称取适量干粉,用蒸馏水溶解;
C、采用苯酚-硫酸法测定奇异酵母胞外多糖,具体做法:取葡萄糖,加蒸馏水溶解,配制1mg/mL葡萄糖标准溶液。葡萄糖标准溶液浓度0μg/mL、20μg/mL、40μg/mL、60μg/mL、80μg/mL、100μg/mL,取100μL葡萄糖标准品,依次加入100μL 5%苯酚溶液和500μL浓硫酸溶液,100℃煮沸10min,待其冷却后测定490nm处的吸光值,以蒸馏水作空白对照。每组实验三平行。通过标准曲线测得奇异酵母发酵液中胞外多糖含量为28%。(图1)
D、将浓缩并透析后的奇异酵母发酵液加入4倍体积的100%的乙醇,4℃静置过夜,然后进行离心,10000rpm/20min,收集沉淀,并进行冷冻干燥处理;
E、将所得干粉加入蒸馏水复溶,与Sevage试剂按照体积比1:1进行混合,震荡反应20min后进行离心,4000rpm/10min,然后小心吸取上层溶液。将得到的溶液进行旋蒸除去有机溶剂,随后进行冷冻干燥处理,得到奇异酵母胞外多糖干粉。
奇异酵母胞外多糖溶液对短乳杆菌、鼠李糖乳杆菌以及长歧双歧杆菌的促生长作用,具体实验如下:
A、在MRS培养基中分别添加培养基总量1.0%、2.0%、4.0%、8.0%的奇异酵母胞外多糖溶液,再分别接种短乳杆菌、鼠李糖乳杆菌和长歧双歧杆菌,37℃条件下培养24h,每组实验三平行;
B、紫外可见分光光度计在波长600nm处测定短乳杆菌、鼠李糖乳杆菌和长歧双歧杆菌的吸光度值,结果见表1。
表1:短乳杆菌、鼠李糖乳杆菌和长歧双歧杆菌在不同浓度的奇异酵母菌胞外多糖溶液培养基中的吸光度值(OD=600nm)
奇异酵母胞外多糖溶液质量分数 | 0% | 1% | 2% | 4% | 8% |
短乳杆菌 | 0.03 | 0.26 | 0.47 | 0.93 | 1.81 |
鼠李糖乳杆菌 | 0.05 | 0.31 | 0.52 | 1.02 | 1.96 |
长双歧杆菌 | 0.02 | 0.23 | 0.41 | 0.87 | 1.77 |
由表1可知,随着奇异酵母胞外多糖添加量的增加,皮肤益生菌短乳杆菌、鼠李糖乳杆菌和长歧双歧杆菌的生长均表现为增长的趋势。实验结果可以表明该多糖对益生菌的生长增殖起到很好的促进作用。
实施例2:一种奇异酵母和益生菌复合溶胞物组合物,在化妆品中的添加比例。
菌体溶胞物的制备方法如下:
短乳杆菌、鼠李糖乳杆菌和长双歧杆菌溶胞物的制备方法如下步骤:
(1)菌体培养方法为:菌体在37℃培养箱中,平板静置培养20-24h,注意长双歧杆菌是在厌氧培养箱中进行培养;挑取单菌落接至10mL液体培养基中,37℃培养20-24h,获得一级种子液;种子液按1%的接种量接至液体培养基中,并接入30-300ml奇异酵母发酵液,至1L体系,37℃培养24h,得到菌体发酵液;
短乳杆菌和鼠李糖乳杆菌的MRS培养基成分及其添加比例为:蛋白胨1%、牛肉浸提物1%、酵母提取物0.5%、葡萄糖2%、醋酸钠0.5%、柠檬酸二胺0.2%、磷酸氢二钾0.2%、硫酸镁0.02%、硫酸锰0.005%、吐温0.1%;长双歧杆菌的培养基成分及其添加比例为:(BBL培养基)蛋白胨1%、牛肉浸提物1%、酵母提取物0.5%、葡萄糖0.5%、磷酸氢二钾0.045%、磷酸二氢钾0.033%、氯化铵0.1%、硫酸镁0.01%、L-半胱氨酸0.05%、硫化钠0.05%、刃天青0.0001%;
(2)将得到的发酵混合物进行离心,8000rpm/10min,得菌体沉淀;
(3)将菌体沉淀加水混合均匀,进行超声破碎,超声破碎的条件为:功率300W,工作5s,停顿15s,工作时长10min,共超声两次;
(4)将制得细胞溶出物进行离心,8000rpm/10min,去掉细胞壁,得到细胞溶胞物;
(5)将得到的溶胞物进行冻干处理,冷冻温度:-40℃至-60℃,真空度1Pa-8Pa,冷冻干燥时间为24h-36h。
奇异酵母溶胞物的制备方法如下步骤:
(1)菌体培养方法为:菌体在28℃培养箱中,平板静置培养24-48h;挑取单菌落接至10mL液体培养基中,28℃培养24-48h,获得一级种子液;种子液按1%的接种量接至1L液体培养基中,28℃培养48h,得到菌体发酵液;奇异酵母的麦芽汁肉汤培养基成分及其添加比例为:酵母膏0.3%,麦芽提取物0.3%,葡萄糖1%,蛋白胨0.5%;
(2)将得到的发酵混合物进行离心,8000rpm/10min,得菌体沉淀和发酵液;
(3)将菌体沉淀加水混合均匀,进行超声破碎,超声破碎的条件为:功率300W,工作5s,停顿15s,工作时长10min,共超声两次;
(4)将制得细胞溶出物进行离心,8000rpm/10min,去掉细胞壁,得到细胞溶胞物;
(5)将得到的溶胞物进行冷冻干燥处理,冷冻温度:-40℃至-60℃,真空度1Pa-8Pa,冷冻干燥时间为24h-36h。
将得到的菌体溶胞物按照以下各表给出的比例,添加到以水为主要溶剂的化妆品中。
表2:四种菌体溶胞物的添加比例
组分 | 百分比(%) |
短乳杆菌溶胞物 | 1.0 |
鼠李糖乳杆菌溶胞物 | 5.0 |
长双歧杆菌溶胞物 | 15 |
奇异酵母溶胞物 | 79 |
实施例3:菌体溶胞物的制备方法如实施例2。
将得到的菌体溶胞物按照下表,添加到以水为主要溶剂的化妆品中。
表3:四种菌体溶胞物的添加比例
组分 | 百分比(%) |
短乳杆菌溶胞物 | 4.0 |
鼠李糖乳杆菌溶胞物 | 8.0 |
长双歧杆菌溶胞物 | 18 |
奇异酵母溶胞物 | 70 |
实施例4:菌体溶胞物的制备方法如实施例2。
将得到的菌体溶胞物按照下表,添加到以水为主要溶剂的化妆品中。
表4:四种菌体溶胞物的添加比例
组分 | 百分比(%) |
短乳杆菌溶胞物 | 6.0 |
鼠李糖乳杆菌溶胞物 | 12 |
长双歧杆菌溶胞物 | 20 |
奇异酵母溶胞物 | 62 |
实施例5:菌体溶胞物的制备方法如实施例2。
将得到的菌体溶胞物按照下表,添加到以水为主要溶剂的化妆品中。
表5:四种菌体溶胞物的添加比例
组分 | 百分比(%) |
短乳杆菌溶胞物 | 8.0 |
鼠李糖乳杆菌溶胞物 | 16 |
长双歧杆菌溶胞物 | 22 |
奇异酵母溶胞物 | 54 |
实施例6:菌体溶胞物的制备方法如实施例2。
将得到的菌体溶胞物按照下表,添加到以水为主要溶剂的化妆品中。
表6:四种菌体溶胞物的添加比例
组分 | 百分比(%) |
短乳杆菌溶胞物 | 10 |
鼠李糖乳杆菌溶胞物 | 20 |
长双歧杆菌溶胞物 | 25 |
奇异酵母溶胞物 | 45 |
选取对比例1-15,对实施例2-6添加了菌体复合溶胞物的化妆品的功效进行评估。其中,对比例中的添加量按照表6中的比例进行计算。
对比例1:并未添加本发明复合溶胞物的基础化妆品。
对比例2:添加了短乳杆菌溶胞物的化妆品。
对比例3:添加了鼠李糖乳杆菌溶胞物的化妆品。
对比例4:添加了长双歧杆菌溶胞物的化妆品。
对比例5:添加了奇异酵母溶胞物的化妆品。
对比例6:添加了短乳杆菌和鼠李糖乳杆菌溶胞物的化妆品,添加比例见下表7。
表7:短乳杆菌和鼠李糖乳杆菌溶胞物的添加比例
组分 | 百分比(%) |
短乳杆菌溶胞物 | 33.3 |
鼠李糖乳杆菌溶胞物 | 66.7 |
对比例7:添加了短乳杆菌和长双歧杆菌溶胞物的化妆品,添加比例见下表8。
表8:短乳杆菌和长双歧杆菌溶胞物的添加比例
组分 | 百分比(%) |
短乳杆菌溶胞物 | 28.6 |
长双歧杆菌溶胞物 | 71.4 |
对比例8:添加了短乳杆菌和奇异酵母溶胞物的化妆品,添加比例见下表9。
表9:短乳杆菌和奇异酵母溶胞物的添加比例
组分 | 百分比(%) |
短乳杆菌溶胞物 | 18 |
奇异酵母溶胞物 | 82 |
对比例9:添加了鼠李糖乳杆菌和长双歧杆菌溶胞物的化妆品,添加比例见下表10。
表10:鼠李糖乳杆菌和长双歧杆菌溶胞物的添加比例
组分 | 百分比(%) |
鼠李糖乳杆菌溶胞物 | 44.4 |
长双歧杆菌溶胞物 | 55.6 |
对比例10:添加了鼠李糖乳杆菌和奇异酵母溶胞物的化妆品,添加比例见下表11。
表11:鼠李糖乳杆菌和奇异酵母溶胞物的添加比例
组分 | 百分比(%) |
鼠李糖乳杆菌溶胞物 | 30.8 |
奇异酵母溶胞物 | 69.2 |
对比例11:添加了长双歧杆菌和奇异酵母溶胞物的化妆品,添加比例见下表12。
表12:长双歧杆菌和奇异酵母溶胞物的添加比例
组分 | 百分比(%) |
长双歧杆菌溶胞物 | 35.7 |
奇异酵母溶胞物 | 64.3 |
对比例12:添加了短乳杆菌、鼠李糖乳杆菌和长双歧杆菌溶胞物的化妆品,添加比例见下表13。
表13:短乳杆菌、鼠李糖乳杆菌和长双歧杆菌溶胞物的添加比例
组分 | 百分比(%) |
短乳杆菌溶胞物 | 18 |
鼠李糖乳杆菌溶胞物 | 36 |
长双歧杆菌 | 46 |
对比例13:添加了短乳杆菌、鼠李糖乳杆菌和奇异酵母溶胞物的化妆品,添加比例见下表14。
表14:短乳杆菌、鼠李糖乳杆菌和奇异酵母溶胞物的添加比例
组分 | 百分比(%) |
短乳杆菌溶胞物 | 13 |
鼠李糖乳杆菌溶胞物 | 26 |
奇异酵母 | 61 |
对比例14:添加了短乳杆菌、长双歧杆菌和奇异酵母溶胞物的化妆品,添加比例见下表15。
表15:短乳杆菌、长双歧杆菌和奇异酵母溶胞物的添加比例
组分 | 百分比(%) |
短乳杆菌溶胞物 | 12.5 |
长双歧杆菌溶胞物 | 31.25 |
奇异酵母 | 56.25 |
对比例15:添加了鼠李糖乳杆菌、长双歧杆菌和奇异酵母溶胞物的化妆品,添加比例见下表16。
表16:鼠李糖乳杆菌、长双歧杆菌和奇异酵母溶胞物的添加比例
组分 | 百分比(%) |
鼠李糖乳杆菌溶胞物 | 22.2 |
长双歧杆菌溶胞物 | 27.8 |
奇异酵母 | 50 |
检测试验:
1、受试人群:80名健康受试者,男女比例为1:1,年龄21-36岁。要求受试者皮肤的基础指标一致,无严重系统疾病者、无免疫缺陷或自身免疫性疾病者、无活动性过敏性疾病者、无体质高度敏感者、受试部位没有接受过皮肤治疗、近一个月未曾使用激素类药物及免疫抑制剂、近三个月内受试部位未参加其他临床试验者。
2、试验方法:将受试者分成八组,每组十人。分别涂抹对比例和实施例中的化妆品。产品涂抹至志愿者脸颊、额头和鼻翼,待化妆品吸收后,使用TMHEX仪器测量皮肤的水分流失值;使用/>MPA580皮肤弹性测试仪测量皮肤弹性;使用Ultrascan UC22皮肤超声诊断仪测量表皮皮肤厚度;使用VISIA-CR表面图像分析仪器,测量皮肤皱纹面积;通过反复的乳酸刺痛试验,测量皮肤敏感度。使用频率:每天早晚使用一次,连续使用4周。
3、检测结果:根据各志愿者的皮肤数据,统计出升降比例,试验结果见下表17。
表17:皮肤相关指标的变化值
经皮水分流失 | 皮肤弹性 | 皮肤厚度 | 皮肤毛孔面积 | 皮肤皱纹面积 | 皮肤敏感度 | |
对比例1 | 降低1.2% | 升高0.8% | 升高0.5% | 升高0.6% | 降低0.4% | 降低1.4% |
对比例2 | 降低2.9% | 升高1.8% | 升高1.9% | 降低1.3% | 降低1.4% | 降低2.1% |
对比例3 | 降低3.7% | 升高2.2% | 升高2.4% | 降低2.8% | 降低1.9% | 降低2.8% |
对比例4 | 降低3.9% | 升高2.6% | 升高2.9% | 降低3.1% | 降低2.2% | 降低3.2% |
对比例5 | 降低1.8% | 升高1.4% | 升高1.6% | 降低1.2% | 降低0.9% | 降低1.9% |
对比例6 | 降低6.8% | 升高4.9% | 升高4.6% | 降低4.8% | 降低3.9% | 降低5.7% |
对比例7 | 降低7.1% | 升高5.2% | 升高5.8% | 降低5.1% | 降低4.3% | 降低6.2% |
对比例8 | 降低4.2% | 升高2.8% | 升高3.2% | 降低2.1% | 降低1.9% | 降低3.3% |
对比例9 | 降低7.8% | 升高5.7% | 升高6.2% | 降低6.7% | 降低4.9% | 降低6.8% |
对比例10 | 降低5.1% | 升高3.4% | 升高3.6% | 降低3.5% | 降低2.2% | 降低3.8% |
对比例11 | 降低5.2% | 升高3.5% | 升高4.2% | 降低3.2% | 降低1.9% | 降低4.3% |
对比例12 | 降低11.2% | 升高7.2% | 升高7.8% | 降低8.6% | 降低7.3% | 降低9.7% |
对比例13 | 降低8.6% | 升高5.2% | 升高5.8% | 降低5.1% | 降低4.0% | 降低6.1% |
对比例14 | 降低8.8% | 升高5.6% | 升高6.6% | 降低5.3% | 降低4.2% | 降低6.9% |
对比例15 | 降低9.7% | 升高6.3% | 升高6.8% | 降低6.8% | 降低4.3% | 降低7.2% |
实施例2 | 降低14.9% | 升高10.7% | 升高11.3% | 降低12.4% | 降低9.8% | 降低13.8% |
实施例3 | 降低15.4% | 升高11.4% | 升高12.6% | 降低13.9% | 降低10.3% | 降低14.3% |
实施例4 | 降低16.8% | 升高13.1% | 升高13.9% | 降低15.1% | 降低11.1% | 降低15.9% |
实施例5 | 降低17.2% | 升高14.5% | 升高15.1% | 降低16.3% | 降低11.9% | 降低16.8% |
实施例6 | 降低18.7% | 升高15.4% | 升高16.8% | 降低17.1% | 降低12.6% | 降低17.9% |
由表17的试验数据可见,使用了实施例2-6的含有本发明的复合溶胞物化妆品后,与对比例1-15相比,受试者的皮肤状况得到显著的改善。使用复合菌体溶胞物的化妆品后,皮肤经皮水分流失降低14.9-18.7%;皮肤弹性升高10.7-15.4%;皮肤厚度增加11.3-16.8%;皮肤毛孔缩小了12.4-17.1%;肌肤皱纹减少9.8-12.6%;皮肤敏感度降低13.8-17.9%。
综上所述,本发明包含的奇异酵母胞外多糖可以促进短乳杆菌、鼠李糖乳杆菌及长双歧杆菌的生长增殖起到很好的促进作用。另外,奇异酵母、短乳杆菌、鼠李糖乳杆菌及长双歧杆菌的复合溶胞物,对皮肤具有良好保湿、收缩毛孔、抗皱等功效,并且可以增强皮肤弹性、增加皮肤厚度以及改善皮肤敏感的功效,可作为化妆品成分应用于化妆品领域。
对于任何熟悉本领域的技术人员而言,在不脱离本发明技术方案范围情况下,都可利用上述揭示的技术内容对本发明技术方案作出许多可能的变动和修饰,或修改为等同变化的等效实施例。因此,凡是未脱离本发明技术方案的内容,依据本发明的技术实质对以上实施例所做的任何简单修改、等同变化及修饰,均应仍属于本发明技术方案保护的范围内。
Claims (9)
1.奇异酵母在促进短乳杆菌、鼠李糖乳杆菌及长双歧杆菌增殖中的应用。
2.根据权利要求1所述的应用,其特征在于,所述奇异酵母菌(Saccharomycesparadoxus),其保藏编号为:CGMCC 2.5026;所述短乳杆菌(Lactobacillus brevis),其保藏编号CGMCC 8581;鼠李糖乳杆菌(Lactobacillus rhamnosus),其保藏编号CICC 20257;长双歧杆菌(Bifidobacterium longum),其保藏编号GDMCC 1.263。
3.根据权利要求1所述的应用,其特征在于,在短乳杆菌、鼠李糖乳杆菌、长双歧杆菌的培养基中加入所述奇异酵母的发酵液;所述奇异酵母发酵液的添加量为培养基总量的3~30%,其中,奇异酵母胞发酵液中胞外多糖含量为25-35%。
4.根据权利要求1所述的应用,其特征在于,在短乳杆菌、鼠李糖乳杆菌、长双歧杆菌的培养基中加入所述奇异酵母的胞外多糖,奇异酵母胞外多糖的添加量为培养基总量的1%-8%。
5.一种复合溶胞物组合物,其特征在于,包括菌体细胞溶胞物,所述菌体包括权利要求2所述奇异酵母(Saccharomyces paradoxus)、短乳杆菌(Lactobacillus brevis)、鼠李糖乳杆菌(Lactobacillus rhamnosus)、长双歧杆菌(Bifidobacterium longum)。
6.根据权利要求5所述的组合物,其特征在于,奇异酵母(Saccharomyces paradoxus)溶胞物的添加量为组合物的45%-79%;短乳杆菌(Lactobacillus brevis)溶胞物的添加量为组合物的1%-10%;鼠李糖乳杆菌(Lactobacillus rhamnosus)溶胞物的添加量为组合物的5%-20%;长双歧杆菌(Bifidobacterium longum)溶胞物的添加量为组合物的15%-25%。
7.根据权利要求5所述的组合物,其特征在于,所述组合物中各菌体溶胞物的制备方法包括以下步骤:
(1)菌体的培养:各菌体经平板培养活化,挑取单菌落制得一级种子液,再进行扩大培养,得菌体发酵液;奇异酵母培养基为麦芽汁肉汤培养基,短乳杆菌和鼠李糖乳杆菌的培养基为MRS培养基,长双歧杆菌的培养基为BBL培养基;
(2)将步骤(1)得到的发酵混合物进行离心,得菌体沉淀,加入蒸馏水混合均匀,经超声破碎后,将混合物进行离心,得到细胞溶胞物,冷冻干燥备用;
优选地,在短乳杆菌、鼠李糖乳杆菌及长双歧杆菌的扩大培养阶段加入奇异酵母发酵液或奇异酵母胞外多糖;
优选地,奇异酵母胞外多糖的制备方法包括以下步骤:
(1)奇异酵母发酵液经浓缩、透析处理后,加入4倍体积的100%的乙醇,4℃静置过夜,离心收集沉淀,并进行冷冻干燥处理;
(2)将所得干粉加入蒸馏水复溶,与Sevage试剂按照体积比1:1进行混合,震荡反应20min后进行离心,4000rpm/10min。然后小心吸取上层溶液,进行旋蒸除去有机溶剂,随后进行冷冻干燥处理,得到奇异酵母胞外多糖干粉。
8.权利要求6所述组合物在护肤品中的应用;优选地,所述应用于护肤品的功效物质为菌体细胞溶胞物。
9.根据权利要求8所述的应用,其特征在于,在减少皮肤水分流失、增强皮肤弹性、增加皮肤厚度、缩小皮肤毛孔、减少皮肤皱纹、改善皮肤敏感方面的应用。
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