Experimental Study On Preparation And Whitening Activity Of Poly γ-glutamic Acid With Different Molecular Weight

Objective Poly-γ-glutamate(γ-PGA)is a kind of water-soluble,anionic,biodegradable and non-toxic biopolymer.Our previous study found that γ-PGA can reduce the production of melanin and achieve the effect of whitening by inhibiting the related enzymes and factors in the process of melanin synthesis.In this experiment,γ-PGA with different molecular weight was prepared on the basis of previous research,and the whitening activity of γ-PGA with different molecular weight was further studied with tyrosinase activity inhibition rate and melanin formation inhibition rate as indicators.Methods Using Bacillus subtilis as the producing strain,the crude γ-PGA was prepared by microbial fermentation and water extraction and alcohol precipitation.Uniform γ-PGA was obtained by adsorption and decolorization of diatomite and activated carbon.The content,molecular weight and structure ofγ-PGA were characterized by high performance liquid chromatography(HPLC),gel permeation chromatography(GPC),thin layer chromatography(TLC),infrared absorption spectrophotometry(IR)and ultraviolet absorption spectrometry(UV).γ-PGA with different molecular weight was prepared by chemical degradation.The whitening activity of γ-PGA with different molecular weight was studied by MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)detection method,scavenging rate of hydroxyl radical,inhibition rate of tyrosinase activity and the inhibition rate of melanin production.Results Five groups of γ-PGA with different molecular weight were prepared by microbial fermentation combined with chemical degradation.Their weight-average molecular weight(Mw)were 21,117,282,432 and 501 k Da.IR showed that the fermentation product contained amide group,carboxyl group,methylene group and other characteristic absorption;UV showed that the maximum absorption wavelength of the fermentation product was at 216 nm.TLC comparison showed that there was only one chromatographic spot on the same horizontal line between the hydrolysate after complete hydrolysis and the standard solution of glutamic acid.The structure,characteristic absorption and polymerization monomer of the product were characterized by the above three methods.It was proved that the product was γ-PGA;in vitro tyrosinase activity inhibition experiment and hydroxyl radical scavenging experiment in vitro showed that five groups of γ-PGA with different molecular weight could inhibit the formation of tyrosinase,diphenolase and hydroxyl radical in different degrees,and the inhibitory effect increased with the increase of molecular weight of γ-PGA.Through MTT experiment,it was determined that different molecular weight γ-PGA had almost no toxicity to B16 mouse melanoma cells when the concentration was lower than or equal to 1 mg/ml,and the results of intracellular tyrosinase activity inhibition and melanogenesis inhibition experiments showed that different molecular weight γ-PGA could inhibit B16 cell tyrosinase activity and melanin production at 1 mg/ml or lower concentration.Conclusions In this study,a group of γ-PGA was prepared by fermentation of Bacillus subtilis,and then five groups of γ-PGA of different molecular weights were obtained by chemical degradation.Different molecular weight γ-PGA exert whitening activity by inhibiting the rate-limiting enzyme tyrosinase in the process of melanin formation,and the whitening activity increases with the increase of molecular weight,which provides a theoretical basis for the follow-up application of γ-PGA with different molecular weights in the field of skin whitening.