抑制黑色素合成的乳酸菌胞外多糖的筛选和性质研究
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摘要
【目的】筛选可抑制黑色素合成的乳酸菌胞外多糖。【方法】通过观察凝乳拉丝外观筛选产胞外多糖的乳酸菌菌株,测量胞外多糖对B16黑色素瘤细胞黑色素合成和细胞活力的影响。对胞外多糖进行纯化,并通过PMP衍生-HPLC、红外光谱、抑制酪氨酸酶活性、抗氧化能力对其单糖组成和结构、作用机制进行研究。【结果】筛选到一株乳酸菌Lactobacillus rhamnosus HLAB122,发酵产生的胞外多糖在5 g/L浓度下可使B16细胞黑色素产量下降至空白对照的32.7%,且在96 h内对细胞活力无影响。纯化后的多糖由鼠李糖、葡萄糖、半乳糖构成,各单糖摩尔比为1?5.44?5.37。该胞外多糖不抑制酪氨酸酶活力且抗氧化性微弱。【结论】L.rhamnosus HLAB122产生的胞外多糖在个人护理产品中有潜在应用价值。
[Objective] To screen exopolysaccharide from lactic acid bacteria for inhibiting melanin synthesis. [Methods] Exopolysaccharide-producing lactic acid bacteria were screened by observing the ropy appearance in skim milk media. Effects of exopolysaccharides on melanin synthesis and cell viability were measured on B16 melanoma cell. Exopolysaccharide was purified by chromatography columns of DEAE-52 cellulose and Sepahdex G100. Monosaccharide composition and basic structural information were studied by PMP-HPLC and FT-IR method. Tyrosinase inhibiting activity and antioxidant activity were studied. [Results] Exopolysaccharide form Lactobacillus rhamnosus HLAB122(LR122EPS) could inhibit melanin synthesis of B16 melanoma cell. Compared with the control, melanin production decreased to 32.7% at 5 g/L of LR122 EPS, cell viability was not changed within 96 hours. Purified LR122 EPS was composed of rhamnose, glucose, and galactose in a molar ratio of 1:5.44:5.37. LR122 EPS could not inhibit tyrosinase activity and its antioxidation was weak. [Conclusion] Exopolysaccharide LR122 EPS had the potential in products toinhibit melanin synthesis.
[Objective] To screen exopolysaccharide from lactic acid bacteria for inhibiting melanin synthesis. [Methods] Exopolysaccharide-producing lactic acid bacteria were screened by observing the ropy appearance in skim milk media. Effects of exopolysaccharides on melanin synthesis and cell viability were measured on B16 melanoma cell. Exopolysaccharide was purified by chromatography columns of DEAE-52 cellulose and Sepahdex G100. Monosaccharide composition and basic structural information were studied by PMP-HPLC and FT-IR method. Tyrosinase inhibiting activity and antioxidant activity were studied. [Results] Exopolysaccharide form Lactobacillus rhamnosus HLAB122(LR122EPS) could inhibit melanin synthesis of B16 melanoma cell. Compared with the control, melanin production decreased to 32.7% at 5 g/L of LR122 EPS, cell viability was not changed within 96 hours. Purified LR122 EPS was composed of rhamnose, glucose, and galactose in a molar ratio of 1:5.44:5.37. LR122 EPS could not inhibit tyrosinase activity and its antioxidation was weak. [Conclusion] Exopolysaccharide LR122 EPS had the potential in products toinhibit melanin synthesis.
引文
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[17]Wang W,Wang XQ,Ye H,et al.Optimization of extraction,characterization and antioxidant activity of polysaccharides from Brassica rapa L.[J].International Journal of Biological Macromolecules,2016,82:979-988
[18]Yuan JF,Cai H,Shan XY,et al.Isolation and purification of exopolysaccharide from the fermentation broth of Bacillus sp.And its antioxidant effect[J].Microbiology China,2009,36(10):1466-1470(in Chinese)袁建锋,蔡恒,单咸旸,等.一株芽孢杆菌胞外多糖的分离纯化及其抗氧化性测定[J].微生物学通报,2009,36(10):1466-1470
[19]Daniel W.Latest skin care development analysed[A]//Caroline J.Cosmetic Science Technology 2012[M].Hertfordshire:T4International,2013:151-154
[20]Verschoore M,Liu W,Zhen YX.Basic Science for Modern Cosmetic Dermatology[M].Beijing:People’s Medical Publishing House,2011(in Chinese)Verschoore M,刘玮,甄雅贤.现代美容皮肤科学基础[M].北京:人民卫生出版社,2011
[21]Tian FW,Ding HS,Ding N,et al.Fast screening and identification of exopolysaccharide-producing lactic acid bacteria[J].Food and Fermentation Industries,2008,34(3):15-19(in Chinese)田丰伟,丁虎生,丁纳,等.产胞外多糖的乳酸菌的简便筛选与鉴定[J].食品与发酵工业,2008,34(3):15-19
[22]Stingele F,Neeser JR,Mollet B.Identification and characterization of the eps(Exopolysaccharide)gene cluster from Streptococcus thermophilus Sfi6[J].Journal of Bacteriology,1996,178(6):1680-1690
[23]Ruas-Madiedo P,de los Reyes-Gavilán CG.Invited review:methods for the screening,isolation,and characterization of exopolysaccharides produced by lactic acid bacteria[J].Journal of Dairy Science,2005,88(3):843-856
[24]Hassan AN,Frank JF,Qvist KB.Direct observation of bacterial exopolysaccharides in dairy products using confocal scanning laser microscopy[J].Journal of Dairy Science,2002,85(7):1705-1708
[25]Hassan AN,Ipsen R,Janzen T,et al.Microstructure and rheology of yogurt made with cultures differing only in their ability to produce exopolysaccharides[J].Journal of Dairy Science,2003,86(5):1632-1638
[2]Broadbent JR,Mc Mahon DJ,Welker DL,et al.Biochemistry,genetics,and applications of exopolysaccharide production in Streptococcus thermophilus:a review[J].Journal of Dairy Science,2003,86(2):407-423
[3]de Vuyst L,Degeest B.Heteropolysaccharides from lactic acid bacteria[J].FEMS Microbiology Reviews,1999,23(2):153-177
[4]Gu XM,Kong J,Wang FS,et al.Exopolysaccharide I(EPS I)purified from a strain of lactic acid bacterium Z222 and studying on the effect of EPS I on the immunofunction of mice planted with S180[J].Acta Microbiologica Sinica,2003,43(2):251-256(in Chinese)顾笑梅,孔健,王富生,等.一株乳酸菌所产胞外多糖对荷瘤小鼠机体免疫功能影响的研究[J].微生物学报,2003,43(2):251-256
[5]Li WX,Chen Q,Li PL,et al.Study on immunoregulation effect of exopolysaccharide produced by Bifidobacterium spp.[J].Microbiology China,2009,36(6):931-935(in Chinese)李伟欣,陈倩,李平兰,等.一种双歧杆菌胞外多糖免疫调节功能研究[J].微生物学通报,2009,36(6):931-935
[6]Chen X,Jiang HR,Yang YM,et al.Effect of exopolysaccharide from Bifidobacterium bifidum on cell of gastric cancer and human telomerase reverse transcriptase[J].Acta Microbiologica Sinica,2009,49(1):117-122(in Chinese)陈旭,江虹锐,杨艳梅,等.两歧双歧杆菌胞外多糖对胃癌细胞及端粒酶逆转录酶的影响[J].微生物学报,2009,49(1):117-122
[7]Li C,Wang CF,Yang GL.Progress in intestinal adhension and immunoregulatory effect of extracellular polysaccharides of lactic acid bacteria[J].Food Science,2014,35(11):314-318(in Chinese)李超,王春凤,杨桂连.乳酸菌胞外多糖肠道黏附及免疫调节作用研究进展[J].食品科学,2014,35(11):314-318
[8]Welman AD,Maddox IS.Exopolysaccharides from lactic acid bacteria:perspectives and challenges[J].Trends in Biotechnology,2003,21(6):269-274
[9]Yand SH,Liu HI,Tsai SJ.Edible tremella polysaccharide for skin care:U.S.,11/095,525[P].2005-4-1.https://www.google.com/patents/US20060222608
[10]Kwon SS,Yeom MH,Kim DH,et al.Method for preparing polysaccharide of green tea and cosmetic composition for skin whitening,moisturization and anti-wrinkle effects comprising the polysaccharide:U.S.,14/450,349[P].2014-8-4.https://www.google.com/patents/US20140341827
[11]Yeom MH,Park WS,Kim DH,et al.Composition containing Chamaecyparis obtusa polysaccharides to be externally applied to the skin:U.S.,13/319,765[P].2010-5-27.https://www.google.com/patents/US20120065159
[12]Chen JB.Isolation,purification,structure analysis and activity study of water solube polysaccharide from mussel cooking liquor[D].Hangzhou:Master’s Thesis of Zhejiang Gongshang University,2008(in Chinese)陈静波.贻贝蒸煮液水溶性多糖的分离纯化,结构分析和活性研究[D].杭州:浙江工商大学硕士学位论文,2008
[13]Kang YY,Zhang MY,Xing SJ,et al.Toxicity and lightening effects of several natural active products on melanocytes[J].China Surfactant Detergent&Cosmetics,2005,35(6):361-363,379(in Chinese)康琰琰,张美英,邢少璟,等.几种天然活性物对黑色素细胞毒性及美白功效的比较[J].日用化学工业,2005,35(6):361-363,379
[14]Koji I,Morimasa T,Seiji K,et al.Skin-beautifying agent:Japan,09-249524[P].1997-9-22.https://www19.j-platpat.inpit.go.jp/PA1/html/input_e.html
[15]Ouyang QB,Li PL,Li WX,et al.Isolation and purification of exopolysaccharides produced by Bifidobacterium sp.22-5[J].Food and Fermentation Industries,2005,31(6):127-130(in Chinese)欧阳清波,李平兰,李伟欣,等.双歧杆菌22-5胞外多糖(EPS)的分离、纯化及纯度鉴定[J].食品与发酵工业,2005,31(6):127-130
[16]Zhang WJ.Biochemical Research Technology of Glycoconjugate[M].2nd Edition.Hangzhou:Zhejiang University Press,1999(in Chinese)张惟杰.糖复合物生化研究技术[M].第2版.杭州:浙江大学出版社,1999
[17]Wang W,Wang XQ,Ye H,et al.Optimization of extraction,characterization and antioxidant activity of polysaccharides from Brassica rapa L.[J].International Journal of Biological Macromolecules,2016,82:979-988
[18]Yuan JF,Cai H,Shan XY,et al.Isolation and purification of exopolysaccharide from the fermentation broth of Bacillus sp.And its antioxidant effect[J].Microbiology China,2009,36(10):1466-1470(in Chinese)袁建锋,蔡恒,单咸旸,等.一株芽孢杆菌胞外多糖的分离纯化及其抗氧化性测定[J].微生物学通报,2009,36(10):1466-1470
[19]Daniel W.Latest skin care development analysed[A]//Caroline J.Cosmetic Science Technology 2012[M].Hertfordshire:T4International,2013:151-154
[20]Verschoore M,Liu W,Zhen YX.Basic Science for Modern Cosmetic Dermatology[M].Beijing:People’s Medical Publishing House,2011(in Chinese)Verschoore M,刘玮,甄雅贤.现代美容皮肤科学基础[M].北京:人民卫生出版社,2011
[21]Tian FW,Ding HS,Ding N,et al.Fast screening and identification of exopolysaccharide-producing lactic acid bacteria[J].Food and Fermentation Industries,2008,34(3):15-19(in Chinese)田丰伟,丁虎生,丁纳,等.产胞外多糖的乳酸菌的简便筛选与鉴定[J].食品与发酵工业,2008,34(3):15-19
[22]Stingele F,Neeser JR,Mollet B.Identification and characterization of the eps(Exopolysaccharide)gene cluster from Streptococcus thermophilus Sfi6[J].Journal of Bacteriology,1996,178(6):1680-1690
[23]Ruas-Madiedo P,de los Reyes-Gavilán CG.Invited review:methods for the screening,isolation,and characterization of exopolysaccharides produced by lactic acid bacteria[J].Journal of Dairy Science,2005,88(3):843-856
[24]Hassan AN,Frank JF,Qvist KB.Direct observation of bacterial exopolysaccharides in dairy products using confocal scanning laser microscopy[J].Journal of Dairy Science,2002,85(7):1705-1708
[25]Hassan AN,Ipsen R,Janzen T,et al.Microstructure and rheology of yogurt made with cultures differing only in their ability to produce exopolysaccharides[J].Journal of Dairy Science,2003,86(5):1632-1638