Study On The Antioxidant Activity Of Soybean Fermentation Broth

There are many polyphenols in soybean,and the high content of protein in soybean is a potential source of active peptides.Both polyphenols and soybean peptides have excellent antioxidant activities.Fermentation can improve the yield of active compounds in soybean or enhance their activity through the biochemical reaction.Keratinocytes and melanocytes are located on the surface of the skin.Keratinocytes gradually differentiate outward to form the stratum corneum,thereby providing barrier protection for the skin.Excessive ROS will cause oxidative damage to cells.Therefore,improving the antioxidant capacity of keratinocytes is beneficial to maintain skin health.Ultraviolet radiation will induce melanogenesis in melanocytes to prevent oxidative damage,but excessive production of melanin can cause pigmentation.The work prepared soybean fermentation broth?S-FB?with excellent antioxidant properties,meanwhile,studied its antioxidant effect on skin cells of HaCa T?keratinocytes?and B16?melanocytes?to clarify the protective effect on skin care,which was significant for the application of S-FB in cosmetics.The main research contents were as follows:Firstly,preparation of S-FB was carried out.ABTS assay and DPPH assay were applied to test the antioxidant activity of fermented products.Bacillus subtilis?BS-136?was screened out to be the best strain for fermentation,followed by the single factor test and the orthogonal test to determine the optimal fermentation conditions,namely 37??fermentation temperature?,60h?fermentation time?and 10 g/L?glucose concentration?.Compared with the unfermented soybean medium?SM?,the optimized S-FB had 284%and 145%higher rates of scavenging ABTS⁺radical and DPPH radical,respectively,and its total polyphenol content and total peptide content increased by 187%and 229%,respectively.Further experiments investigated the antioxidation of S-FB in Ha CaT cells.With analyzing the effect of S-FB on intracellular ROS,S-FB?5%,10%,and 20%?increased the ROS level;after cells were treated with 5%S-FB for 12 or 24 h and without S-FB for12 h,ROS decreased below the normal level;under the sustaining treatment with 5%S-FB,the intracellular ROS level stabilized in 36 and 48 h.H₂O₂+S-FB?5%,10%and 20%S-FB;treated with H₂O₂and S-FB successively?induced less ROS than H₂O₂or 5%S-FB did.Thus,S-FB improved the antioxidant capacity of Ha CaT cells when induced ROS generation,additionally,S-FB could eliminate ROS in cells with a high ROS level.Research was developed to explore the effect of S-FB on the intracellular antioxidant system.Under the sustaining treatment with 5%S-FB,the cells got an improved Nrf2 level,meanwhile,comparing to 0 h,SOD activity increased by32.48%and 32.11%in 36 and 48 h,respectively,CAT activity increased by 19.62%and17.50%,respectively,and GSH content increased by 9.38%in 48 h.After treatment with 5%S-FB for 36 h and 48 h,respectively,the survival rates in UVB damage were both 1.3 times higher than that of the normal Ha CaT cells.Therefore,S-FB increased the Nrf2 level in HaCaT cells and increased the downstream SOD,CAT and GSH of Nrf2,moreover,cells'antioxidant capacity was improved.Then,research focused on the antioxidant activity of S-FB in B16 cells.With testing the inhibition of S-FB on tyrosinase,its activity was close to 150?mol/L kojic acid.The intracellular ROS was tested in cells pretreated with S-FB,and the result manifested that S-FB?5%,10%and 20%?reduced the intracellular ROS.Through comparing the effects of the primary and secondary incubation with S-FB on ROS removal,the ROS gap between 24 h+3 h?5%S-FB treated cells for 24 and 3 h successively;the below followed?and 24 h was significantly greater than that between 12 h+3 h and 12 h as well as 0 h+3 h and 0 h,respectively.S-FB's effect on intracellular SOD,CAT and GSH was investigated,and it suggested that the improvement of treatment with S-FB for 24 h on SOD and CAT activities and GSH content was better than for 12 h.From the test of damaging cells with H₂O₂,pretreatment with S-FB for 24h increased their survival?20%S-FB improved that by about 27%while 5%and 10%S-FB by about 22%?.Besides,UVB was applied to cause oxidative stress in cells,and the result indicated that UVB induced less ROS and melanin in cells pretreated with S-FB?5%,10%and 20%?.Therefore,S-FB could remove ROS directly or indirectly by improving the antioxidant capacity of B16 cells;moreover,S-FB could inhibit tyrosinase activity,and in B16 cells,it was able to suppress UVB-induced melanin production.