【作者】 李瑜；

【导师】 余晓斌；

【作者基本信息】 江南大学 ， 发酵工程， 2018， 硕士

【摘要】 生物美白剂富含抗氧化活性成分及多种氨基酸,具有易于被皮肤吸收、安全性高等优点,抗氧化及美白功效较好,已经被广泛应用于美白祛斑等护肤品中。目前,已有微生物发酵单一中药制备化妆品的报道,复方中药美白效果较好,但关于微生物发酵复方中药的报道很少。因此,开发复方中药发酵原浆并研究其应用显得尤为重要。本文对选定的28种中药进行研究,对4种美白能力较强的中药进行复配及效果评价,制备复方中药发酵液并研究其活性成分及功效性,采用HPLC法对没食子酸的抗氧化活性进行评价,进而研究绿茶对五倍子发酵品的影响,以提高五倍子发酵品中没食子酸含量,通过一系列的手段,为生物美白剂的开发提供了新思路。具体研究内容及结果如下:（1）对28种中药进行研究,五倍子60%乙醇提取物抗氧化效果最好,酪氨酸酶抑制作用最强,为96.73%,总酚及黄酮含量均最高,分别为729.49 mg GAE·g^-1、3.14%,红景天、绿茶叶及丁香60%乙醇提取物的酪氨酸酶抑制率也较高,分别为73.47%、57.23%、67.39%。Pearson相关性分析表明,总酚含量与总还原力、酪氨酸酶抑制率极显著相关（P<0.01）,R值分别为0.990、0.853。（2）选取红景天、五倍子、绿茶叶和丁香进行中药的复配,正交试验得出最佳复配含量为红景天0.50%、五倍子0.10%、绿茶叶0.25%、丁香1.00%,该复配组合的酪氨酸酶抑制率为74.53%。采用金氏公式对中药复配效果进行评价,五倍子与低质量浓度中药的复配均表现为协同增效作用。（3）评价15株酵母菌与12株乳酸菌的体外抗氧化效果,扣囊复膜酵母和粪肠球菌的抗氧化效果最好,用这两株菌分别发酵复方中药后酪氨酸酶抑制率、总还原力、总酚含量显著提高,酵母菌发酵后分别提高37.34%、28.64 mmol·L^-1、39.54 mg GAE·g^-1,乳酸菌发酵后分别提高35.24%、28.75 mmol·L^-1、35.90 mg GAE·g^-1。采用单因素试验得出发酵复方中药的最佳条件为酵母菌比乳酸菌1:1,总接种量10%,培养方式为30℃摇床培养48 h。（4）复方中药发酵液中多酚、多糖、黄酮、蛋白质含量明显升高,分别达到147.80 mg GAE·g^-1、4.36 mg·g^-1、0.12%、2.22 mg·g^-1,氨基酸含量增加显著,其中谷氨酸含量最高,为0.078 mg·g^-1,5%（w/v）复方中药发酵液的酪氨酸酶活性抑制率为82.41%。（5）没食子酸抗氧化活性的评价。复方中药发酵液中没食子酸与DPPH自由基有较强的相对结合强度,达到55.65%,说明没食子酸抗氧化效果较好。（6）黑曲霉SXG-Z-6所产单宁酶在显色平板中形成的降解圈面积最大,所产单宁酶活力最高。添加1.25 g绿茶叶的五倍子加茶渣茶汁体系,发酵效果最佳,没食子酸含量为42.31 mg·m L^-1。更多还原

【Abstract】 Biological whitening agent is rich in antioxidant active ingredients and amino acids,and it has the advantages of easy absorption by skin and high safety.Biological whitening agent has good anti-oxidation and whitening efficacy,and it has been widely applied in skin care products such as whitening and freckle-removing.At present,there are reports on the preparation of single Chinese herbs cosmetic fermented by microorganisims.The whitening efficacy of compound Chinese herbs is better.However,there are few reports on the compound Chinese herbs fermented by microorganisms.For this reason,it is particularly important to develop and apply fermented compound Chinese herbs puree.In this paper,the selected 28 kinds of Chinese herbs were studied,4 kinds of Chinese herbs with better whitening ability among them were selected for compounding,and the compound effectiveness was evaluated.Fermentation broth of compound Chinese herbs was prepared,and its active ingredients and effectiveness of compound Chinese herbs fermentation broth were investigated.The antioxidant effect of gallic acid was estimated by HPLC.In order to improve gallic acid content in fermented Galla chinensis products,the effect of green tea on different fermented Galla chinensis products was investigated.Through a series of means,it provides new ideas of developing biological whitening agent.The specific researches and results are listed as follows:（1）Research on the selected 28 kinds of Chinese herbs.The antioxidant effect of Galla chinensis 60%ethanol extract was the best,and tyrosinase inhibitory effect was the strongest,which was 96.73%.The content of total phenols and flavonoids was the highest,which was729.49 mg GAE·g^-1 and 3.14%,respectively.The 60%ethanol extract of Rhodiola rosea,Green tea and Eugenia caryophyllata also had high tyrosinase inhibition rate,which was 73.47%,57.23%and 67.39%,separately.Pearson analysis showed that total phenols content was significantly correlated with total reducing power and tyrosinase inhibition rate（P<0.01）,and the R value was 0.990 and 0.853.（2）The Rhodiola rosea,Galla chinensis,Green tea and Eugenia caryophyllata were selected for compounding.The best compound composition was 0.50%Rhodiola rosea,0.10%Galla chinensis,0.25%Green tea,and 1.00%Eugenia caryophyllata,which was obtained by orthogonal designed experiment.The tyrosinase inhibition rate of the best compound combination was 74.53%.The compound efficacy of Chinese herbs was assessed by Jin’s formula.The complex of Galla chinensis and low-quality concentration of Chinese herbs showed synergistic effect.（3）In vitro antioxidant effect of 15 yeasts and 12 lactic acid bacterias was assessed.The best antioxidant strains were Saccharomycopsis fibuligera and Enterococcus faecalis.The compound Chinese herbs was fermented by the two strains.The tyrosinase inhibition rate,the total reducing power and total phenols content increased significantly.The increased amount of fermented compound Chinese herbs by yeast was 37.34%,28.64 mmol·L^-1,and 39.54 mg GAE·g^-1,respectively.The increased amount of fermented compound Chinese herbs by lactic acid bacteria was 35.24%,28.75 mmol·L^-1,and 35.90 mg GAE·g^-1,respectively.The single factor test showed that the best conditions were:yeast:lactic acid bacteria（1:1）,total inoculation amount 10%,and then cultivated in a rotary shaker at 30℃for 48 h.（4）The content of phenolics,polysaccharides,flavonoids,protein in compound Chinese herbs fermentation broth increased obviously,which reached to 147.80 mg GAE·g^-1,4.36 mg·g^-1,0.12%,and 2.22 mg·g^-1,separately.The content of amino acid increased significantly,and the fermentation broth contained the highest glutamate content,which was 0.078 mg·g^-1.Tyrosinase inhibition rate of 5%（w/v）fermentation broth reached to 82.41%.（5）Evaluation of the anti-oxidant activity of gallic acid.There was a strong relative binding strength beween gallic acid and DPPH radical,which reached to 55.65%in the compound Chinese herbs fermentation broth.The result showed that antioxidant effect of gallic acid was better.（6）The tannase produced by Aspergillus niger SXG-Z-6 had the largest area of degrating circle formed in the color plate,and the activity of tannase was the highest.The fermentation effect of Green tea with 1.25 g addition was the best,the content of gallic acid reached to 42.31mg·m L^-1.更多还原