CN111363687A - 鲁氏结合酵母菌株及含其的玫瑰发酵液与应用 - Google Patents
鲁氏结合酵母菌株及含其的玫瑰发酵液与应用 Download PDFInfo
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- CN111363687A CN111363687A CN202010247861.5A CN202010247861A CN111363687A CN 111363687 A CN111363687 A CN 111363687A CN 202010247861 A CN202010247861 A CN 202010247861A CN 111363687 A CN111363687 A CN 111363687A
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Abstract
本发明提供一种鲁氏结合酵母菌株及含其的玫瑰发酵液,其制备方法与应用。该菌株的保藏号为CGMCC No.19335。其源于大理白族地区传统玫瑰糖,通过PDA培养基、麦芽汁培养基、察氏培养基、YPD培养基和沙氏培养基中的一种或多种培养不同发酵年限的大理白族地区传统玫瑰糖稀释液进行纯化后获得。该鲁氏结合酵母菌株与含其的玫瑰发酵液能够显著增加玫瑰液的总多酚和总黄酮含量,进而提高玫瑰液的抗氧化活性和酪氨酸酶抑制活性。其可以作为功能性食品直接食用或作为抗衰老和美白等功能性化妆品的主要原料或辅料用于化妆品应用领域。
Description
技术领域:
本发明属于功能性食品发酵及功能性化妆品应用领域,具体涉及一株能够增强功能性食品或化妆品的鲁氏结合酵母菌株 (Zygosaccharomycesrouxii),以及含有鲁氏结合酵母菌株的玫瑰发酵液及其应用。
背景技术:
玫瑰糖是云南地区的特色药食同源食品,主要集中在云南昆明、文山、大理和丽江等地,其中以大理白族地区久负盛名,玫瑰糖属于自然发酵,发酵过程中主要受发酵环境和植物资源微生物的影响。因此,微生物在发酵食品的风味、质地和活性方面扮演着极其重要的角色。而且,发酵促进了复杂的风味成分,这些化合物共同影响了被发酵物的理化性质和生理活性。
鲁氏结合酵母菌(Zygosaccharomycesrouxii)在分类学上属于真核生物原界,酵母亚门,酵母纲,酵母目,酵母科,鲁氏接合酵母属。菌落呈圆形,有光泽,平坦,边缘整齐。鲁氏结合酵母菌 (Zygosaccharomycesrouxii)是常见的耐高渗透酵母菌,传统上,主要用于发酵生产酱油、甜酱、味增剂等制品。工业生产中可发酵产***醇糖。在食品技术领域,鲁氏结合酵母菌(Zygosaccharomycesrouxii) 还可用于培养吸附重金属镉。然而,在功能食品和功能化妆品中的应用几乎未见相关报道。
多酚和黄酮类化合物广泛存在于植物中,是天然的抗氧化剂,其能够清除机体内的活性氧和自由基,防止氧化应激作用。氧化应激是导致衰老和疾病的一个重要因素,而多酚和黄酮类化合物含有多羟基结构,能够与金属离子螯合形成络合物,不仅能够抑制金属引发的脂质氧化等导致的衰老和相关疾病,而且能螯合酪氨酸酶活性部位的铜离子形成酪氨酸酶活性抑制,而且,近年来多酚和黄酮类化合物受到化妆品行业的重视,在功能性食品和化妆品研发中具有重要前景。
本发明通过对大理白族地区的传统玫瑰糖进行微生物分离,一株鲁氏结合酵母菌株(Zygosaccharomycesrouxii)被分离鉴定并被加入到玫瑰液中进行发酵,发酵后玫瑰液总多酚和总黄酮含量显著提高,增加了玫瑰液的抗氧化活性和酪氨酸酶抑制活性。因此,提供一株能够增强功能性食品或化妆品活性的鲁氏结合酵母菌株(Zygosaccharomycesrouxii),对于功能性食品和化妆品的研发具有重要的应用价值。
发明内容:
本发明的第一个目的是提供一种鲁氏结合酵母菌株,保藏号为 CGMCC No.19335。该鲁氏结合酵母菌株(Zygosaccharomycesrouxii),由北京擎科生物科技有限公司鉴定,现保存于中国科学院昆明植物研究所,编号为TFR-1。并于2020年01月13日在中国微生物菌种保藏管理委员会普通微生物中心进行保藏,保藏地址:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所菌种保藏中心,保藏号为CGMCC No.19335。
本发明的第二个目的是提供上述鲁氏结合酵母菌的来源,该酵母菌通过PDA培养基、麦芽汁培养基、察氏培养基、YPD培养基和沙氏培养基中的一种或多种培养不同发酵年限的大理白族地区传统玫瑰糖稀释液进行纯化后获得。
本发明的第三个目的是要解决提供一种能够增强功能性食品或化妆品活性的制备方法的关键技术问题。
为解决上述技术问题,本发明提供了如下的技术方案:
一种鲁氏结合酵母菌株(Zygosaccharomycesrouxii),保藏号为 CGMCCNo.19335。
如所述的一种鲁氏结合酵母菌株(Zygosaccharomycesrouxii),其源于大理白族地区传统玫瑰糖,其通过PDA培养基、麦芽汁培养基、察氏培养基、YPD培养基和沙氏培养基中的一种或多种培养不同发酵年限的大理白族地区传统玫瑰糖稀释液进行纯化后获得。
如所述的一种鲁氏结合酵母菌株(Zygosaccharomycesrouxii),所述玫瑰糖发酵年限是指传统玫瑰糖发酵15天-20年中的一个时间点或多个时间点;大理白族地区是指大理古城、大理喜洲、大理鹤庆、大理巍山、大理剑川、喜洲古镇和大理洱源的一个或多个地区;传统玫瑰糖稀释液是指传统玫瑰糖经无菌水稀释10-106倍后的玫瑰糖液。
所述的鲁氏结合酵母菌株(Zygosaccharomycesrouxii)在功能性食品发酵中的应用。
所述的鲁氏结合酵母菌株(Zygosaccharomycesrouxii)在生产功能性化妆品原料中的应用。
所述的鲁氏结合酵母菌株(Zygosaccharomycesrouxii)在制备抗氧化活性和酪氨酸酶抑制活性剂中的应用。
本发明同时提供了一种包含有鲁氏结合酵母菌株 (Zygosaccharomycesrouxii)的玫瑰发酵液,该玫瑰发酵液是按以下组分重量比进行配料:玫瑰花瓣20-30%,白沙糖10-30%,红糖10-30%,蜂蜜1-10%,食用白酒1-5%,无菌水40-60%,4.15*106CFU/ml的鲁氏结合酵母菌株菌液1-5%。
一种玫瑰发酵液,其是将所述的鲁氏结合酵母菌株 (Zygosaccharomycesrouxii)添加到玫瑰液中进行发酵所得,其是由下述步骤制备而得:
(1)原料:按以下组分重量比进行配料:玫瑰花瓣20-30%,白沙糖10-30%,红糖10-30%,蜂蜜1-10%,食用白酒1-5%,无菌水 40-60%,4.15*106CFU/ml的鲁氏结合酵母菌株菌液1-5%;
(2)灭菌:对发酵罐灭菌进行高压灭菌,原料灌装前和接种前进行酒精消毒,灌装后、接种前进行紫外灭菌,灭菌时间45-90min;
(3)接种:将步骤(2)灭菌后的发酵罐和原料按重量比添加 4.15*106CFU/ml的鲁氏结合酵母菌株(Zygosaccharomycesrouxii)菌液 1-5%中的一种,混合均匀;
(4)发酵:将步骤(3)所得发酵罐密封,控制发酵温度20-37℃,发酵时间24h-720h;
(5)样品保存:将步骤(4)发酵所得样品在4℃下,4000rmp/min 离心15min,收集上清液,保存于-20℃。
根据所述的玫瑰发酵液,该玫瑰发酵液在0.1-10%浓度下时, DPPH自由基清除率为78-100%;酪氨酸酶抑制率为2-87%。
本发明另外还提供了所述的玫瑰发酵液的制备方法,该方法包括下述步骤:
(1)原料:按以下组分重量比进行配料:玫瑰花瓣20-30%,白沙糖10-30%,红糖10-30%,蜂蜜1-10%,食用白酒1-5%,无菌水 40-60%,4.15*106CFU/ml的鲁氏结合酵母菌菌液1-5%;
(2)灭菌:对发酵罐灭菌进行高压灭菌,原料灌装前和接种前进行酒精消毒,灌装后、接种前进行紫外灭菌,灭菌时间45-90min;
(3)接种:将步骤(2)灭菌后的发酵罐和原料按重量比添加 4.15*106CFU/ml的鲁氏结合酵母菌株(Zygosaccharomycesrouxii)菌液 1-5%中的一种,混合均匀;
(4)发酵:将步骤(3)所得发酵罐密封,控制发酵温度20-37℃,发酵时间24h-720h;
(5)样品保存:将步骤(4)发酵所得样品在4℃下,4000rmp/min 离心15min,收集上清液,保存于-20℃。
此外提供了所述的玫瑰发酵液在制备功能性食品或抗衰老和美白功能性化妆品中的应用。
与现有技术相比,本发明具备如下的优益性:
本发明提供了一株能够增强功能性食品或化妆品活性的鲁氏结合酵母菌株(Zygosaccharomycesrouxii)及含其的玫瑰发酵液,该菌株来源于大理白族地区不同发酵年限的传统玫瑰糖,其可添加到玫瑰液等食品中进行发酵,增加玫瑰液的总多酚和总黄酮含量,进而提高玫瑰液的抗氧化活性和酪氨酸酶抑制活性。该菌株及含其的玫瑰发酵液可以作为丰富总多酚和总黄酮的策略应用于增加功能性食品和化妆品中。鲁氏结合酵母菌株(Zygosaccharomycesrouxii)的玫瑰液发酵产品可以作为功能性食品直接食用或作为抗衰老和美白等功能性化妆品的主要原料或辅料用于化妆品应用领域。
本发明的鲁氏结合酵母菌株(Zygosaccharomycesrouxii),由北京擎科生物科技有限公司鉴定,现保存于中国科学院昆明植物研究所,编号为TFR-1。并于2020年01月13日在中国微生物菌种保藏管理委员会普通微生物中心进行保藏,保藏地址:北京市朝阳区北辰西路1 号院3号,中国科学院微生物研究所菌种保藏中心,保藏号为CGMCC No.19335。
附图说明:
图1为本发明鲁氏结合酵母(Zygosaccharomycesrouxii)菌株的培养形态。
具体实施方式:
下面结合附图,用本发明的实施例来进一步说明本发明的实质性内容,但并不以此来限定本发明。
实施例1
鲁氏结合酵母(Zygosaccharomycesrouxii)菌株的分离与鉴定。
1.菌株的分离与纯化
将购买自大理白族地区不同发酵年限的传统玫瑰糖用无菌水稀释102倍,并用涂布器涂布到PDA培养基平板中,置于28℃的恒温培养箱中培养,待有菌落长出后,采用Z型平板划线法进行菌落纯化。所述的玫瑰糖不同发酵年限是指传统玫瑰糖发酵15天-20年中的一个时间点或多个时间点;大理白族地区是指大理古城、大理喜洲、大理鹤庆、大理巍山、大理剑川和大理洱源的一个或多个地区;传统玫瑰糖稀释是指传统玫瑰糖经无菌水稀释10-106倍后的玫瑰液。
2.菌落特征及菌体形态
纯化后PDA培养基平板上的菌均为乳白色,表面光滑,湿润,菌落大小适中。在PDA培养基平板中的菌落形态如图1所示。
3.菌株的分子生物学鉴定
挑取纯化后的菌落,使用TSINGKE植物DNA提取试剂盒(通用型)提取基因组DNA,用真菌通用性引物对正向引物ITS1、反向引物ITS4按以下规格:模板:1-3ul,正向引物:1ul(10P),反向引物: 1ul(10P),酶mix:15ul(2X),ddH2O补充至30ul后扩增基因组DNA。 PCR扩增条件为:94℃预变性5min后进入以下循环:94℃变性30s, 56℃退火30s,72℃延伸30s,35个循环,72℃延伸5min。PCR产物用1.0%琼脂糖凝胶电泳检测并切胶回收,得产物鲁氏结合酵母菌株 (Zygosaccharomyces Rouxii),将其保藏于中国科学院昆明植物研究所,保藏编号TFR-1,分类命名为鲁氏结合酵母 (Zygosaccharomycesrouxii)。同时于2020年01月13日在中国微生物菌种保藏管理委员会普通微生物中心保藏,保藏号为CGMCC No. 19335。
实施例2
鲁氏结合酵母菌株(Zygosaccharomycesrouxii)在玫瑰液发酵中增加总多酚和总黄酮的试验。
含鲁氏结合酵母菌株的玫瑰发酵液的制备:
(1)备料:按以下组分(重量比)进行配料:玫瑰花瓣25%,白沙糖0%,红糖25%,蜂蜜0%,食用白酒0%,无菌水45%, 4.15*106CFU/ml的鲁氏结合酵母菌变株(Zygosaccharomyces rouxii) 菌液5%。
(2)灭菌:高压灭菌,酒精消毒,紫外灭菌。
(3)接种:将步骤(2)灭菌后的发酵罐和原料按重量比添加 4.15*106CFU/ml的鲁氏结合酵母菌变株(Zygosaccharomyces rouxii) 菌液5%,混合均匀。
(4)发酵:将步骤(3)所得发酵罐密封,控制发酵温度28℃,发酵时间24h-720h。
(5)样品保存:将步骤(4)发酵所得样品在4℃下,4000rmp/min 离心15min,收集上清液,保存于-20℃。
表1原料组分重量比重
表2发酵过程中总多酚和总黄酮含量变化
实施例3
玫瑰液发酵液在功能性食品和化妆品中的评价。
1、参照实施例2制备玫瑰发酵液。
2、抗氧化活性评价:
将待测样品玫瑰液发酵液与DPPH(终浓度为100μM)混合反应,设定3个重复孔,同时设置不同样品的空白对照孔和Trolox阳性对照孔,30℃,1h,酶标仪测定OD值,检测波长为515nm,计算得到抗氧化率。
抗氧化率(%)=(1-实验孔OD515nm/空白孔OD515nm)×100%
3、酪氨酸酶抑制活性评价:
将待测样品玫瑰液发酵液与L-Dopa混合,加入酪氨酸酶(终浓度25U/mL)开始反应,设定3个重复孔,同时设置不含样品的空白对照和Kojic Acid阳性对照,室温,5min,酶标仪测定OD值,检测波长为490nm。计算得到酪氨酸酶活性抑制率。
酪氨酸酶活性抑制率(%)=(1–样品OD490nm/实验对照孔 OD490nm)×100
表3玫瑰发酵液样品体外抗氧化作用
表4玫瑰发酵液样品对酪氨酸酶的抑制作用
实施例4:
本发明的鲁氏结合酵母菌株(Zygosaccharomycesrouxii)和含其的玫瑰液发酵液可用于制作食品、甜味剂,用作食品时,可直接食用,或配以食品常规辅料制作而成。
颗粒剂的制备工艺采用:按实施例1和2所得的鲁氏结合酵母菌株(Zygosaccharomycesrouxii)和玫瑰液发酵液3份,糊精2份,适量 50%乙醇。将上述原辅料按照常规制备颗粒剂的工艺制备,即先对原辅料进行检验称重—制软材—制粒—干燥—整粒—成品分装。颗粒剂规格:20g/袋。为便携式制剂,配料简单,最大限度保持了营养成分,可增强免疫力,抗氧化、抑制酪胺酶等,老少皆宜,适合各类人群使用。
实施例5:
鼻喷雾剂的应用
鼻喷雾剂:鲁氏结合酵母菌株或玫瑰液发酵液 0.2%
氯化钠 8mg
EDTA 1mg
磷酸钠缓冲液(pH6.5) 10mg
多乙氧基醚 10mg
重蒸馏水 2ml
制备方法:搅拌下于适当体积的重蒸馏水中每次加入一种成分,直至完全深解,然后再加入另一种成分。加重蒸馏水至2ml后,将该溶液在无菌过滤器上过滤,装入瓶中并按照适当的剂量分隔。
实施例6:
美白类化妆品的应用
美白霜配方(W%):
鲁氏结合酵母菌株或玫瑰液发酵液 0.5%
硬脂酸 8.0
C16醇 2.0
自乳化单甘酯 2.0
氢化羊毛脂 2.0
液体石蜡 12.0
甘油 7.0
乳化剂 1.5
防腐剂 0.2
香精 0.2
去离子水加至100
按常规制作化妆品的方法制得本发明上述配方的化妆品。
实施例7:
抗衰老类化妆品的应用
乳剂配方(W%):
去离子水加至100ml
按常规制作化妆品的方法制得本发明上述配方的化妆品。
实施例8:
化妆水应用
化妆水配方
水相:聚丙烯酸钠0.35克,甘油4克,1,3-丁二醇2.5克,金缕梅提取液2.5克,维生素B5 0.5克,熊果甘0.5克,EDTA-Na2 50mg;去离子水,适量,加至100ml。制备方法:将聚丙烯酸钠0.3克溶入 70ml水中,搅拌,充分溶胀。缓缓加入处方中其余成分,不停搅拌;然后加入0.2%的鲁氏结合酵母菌株或玫瑰液发酵液,搅拌使其成匀质。去离子水,加至100ml,包装即可。
实施例9:
修复霜类应用
修复霜处方:甘油6克,卡波姆1.5克,三乙醇胺1.5克,丙二醇6克,尼伯金乙酯0.2克,丹参酮提取物1.2克,香榧精油,去离子水适量,加至总重量为100克。制备方法:取卡波姆于适量去离子水中,搅匀,静置过夜,使其充分溶胀后,加入甘油后,用三乙胺调节pH值,使凝胶基质粘稠性增加。另取处方量的丹参酮提取物、丙二醇及去离子水,混合均匀。再将卡波姆凝胶例入,加入0.5%鲁氏结合酵母菌株或玫瑰液发酵液,尼伯金乙酯,合并搅拌至均匀细腻,加蒸镏水至足量,研磨均匀,即可。
本发明的能够增强功能性食品或化妆品活性的鲁氏结合酵母菌株(Zygosaccharomycesrouxii)源于大理白族地区的传统玫瑰糖,该鲁氏结合酵母菌株按以下组分(重量比)进行配料:玫瑰花瓣20-30%,白糖10-30%,红糖10-30%,蜂蜜1-10%,食用白酒1-5%,无菌水 40-60%,4.15*106CFU/ml的鲁氏结合酵母菌株(Zygosaccharomycesrouxii)菌液1-5%,在发酵温度20-37℃,发酵时间24h-720h的条件下能够显著增加玫瑰液的总多酚和总黄酮含量,进而提高玫瑰液的抗氧化活性和酪氨酸酶抑制活性。其可以作为功能性食品直接食用或作为抗衰老和美白等功能性化妆品的主要原料或辅料用于化妆品应用领域。
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围不局限于申请文件所界定的内容。
Claims (11)
1.一种鲁氏结合酵母菌株(Zygosaccharomycesrouxii),保藏号为CGMCC No.19335。
2.如权利要求1所述的一种鲁氏结合酵母菌株,其特征在于其源于大理白族地区传统玫瑰糖,其是通过PDA培养基、麦芽汁培养基、察氏培养基、YPD培养基和沙氏培养基中的一种或多种培养不同发酵年限的大理白族地区传统玫瑰糖稀释液进行纯化后获得。
3.如权利要求2所述的一种鲁氏结合酵母菌株,其特征在于,所述玫瑰糖发酵年限是指传统玫瑰糖发酵15天-20年中的一个时间点或多个时间点;大理白族地区是指大理古城、大理喜洲、大理鹤庆、大理巍山、大理剑川,喜洲古镇和大理洱源的一个或多个地区;传统玫瑰糖稀释液是指传统玫瑰糖经无菌水稀释10-106倍后的玫瑰糖液。
4.权利要求1或2或3所述的鲁氏结合酵母菌株在功能性食品发酵中的应用。
5.权利要求1或2或3所述的鲁氏结合酵母菌株在生产功能性化妆品中的应用。
6.权利要求1或2或3所述的鲁氏结合酵母菌株在制备抗氧化活性剂和酪氨酸酶抑制活性剂中的应用。
7.一种玫瑰发酵液,其特征在于,该玫瑰发酵液是按以下组分重量比进行配料:玫瑰花瓣20-30%,白沙糖10-30%,红糖10-30%,蜂蜜1-10%,食用白酒1-5%,无菌水40-60%,4.15*106CFU/ml的鲁氏结合酵母菌株菌液1-5%。
8.一种玫瑰发酵液,其特征在于,其是将权利要求1所述的鲁氏结合酵母菌株添加到玫瑰液中进行发酵所得,其是由下述步骤制备而得:
(1)原料:按以下组分重量比进行配料:玫瑰花瓣20-30%,白沙糖10-30%,红糖10-30%,蜂蜜1-10%,食用白酒1-5%,无菌水40-60%,4.15*106CFU/ml的鲁氏结合酵母菌株菌液1-5%;
(2)灭菌:对发酵罐灭菌进行高压灭菌,原料灌装前和接种前进行酒精消毒,灌装后、接种前进行紫外灭菌,灭菌时间45-90min;
(3)接种:将步骤(2)灭菌后的发酵罐和原料按重量比添加4.15*106CFU/ml的鲁氏结合酵母菌株菌液1-5%中的一种,混合均匀;
(4)发酵:将步骤(3)所得发酵罐密封,控制发酵温度20-37℃,发酵时间24h-720h;
(5)样品保存:将步骤(4)发酵所得样品在4℃下,4000rmp/min离心15min,收集上清液,保存于-20℃。
9.根据权利要求7或8所述的玫瑰发酵液,其特征在于,该玫瑰发酵液在0.1-10%浓度下时,DPPH自由基清除率为78-100%;酪氨酸酶抑制率为2-87%。
10.权利要求7或8所述的玫瑰发酵液的制备方法,其物征在于该方法包括下述步骤:
(1)原料:按以下组分重量比进行配料:玫瑰花瓣20-30%,白沙糖10-30%,红糖10-30%,蜂蜜1-10%,食用白酒1-5%,无菌水40-60%,4.15*106CFU/ml的鲁氏结合酵母菌菌液1-5%;
(2)灭菌:对发酵罐灭菌进行高压灭菌,原料灌装前和接种前进行酒精消毒,灌装后、接种前进行紫外灭菌,灭菌时间45-90min;
(3)接种:将步骤(2)灭菌后的发酵罐和原料按重量比添加4.15*106CFU/ml的鲁氏结合酵母菌株菌液1-5%中的一种,混合均匀;
(4)发酵:将步骤(3)所得发酵罐密封,控制发酵温度20-37℃,发酵时间24h-720h;
(5)样品保存:将步骤(4)发酵所得样品在4℃下,4000rmp/min离心15min,收集上清液,保存于-20℃。
11.权利要求7或8所述的玫瑰发酵液在制备功能性食品或抗衰老和美白功能性化妆品中的应用。
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