Purification Of Enzymatic Hydrolysate From Oyster Crassostrea Gigas And Their Effects On Proliferation And Testosterone Secretion In TM3 Leydig Cells

Oyster meat is delicious and has high nutritional value.It is known as the “root source” and “sea milk”.According to the classics,oyster meat has the potential medicinal value to enhance male sexual function.Oyster peptides also have good biological activity and have great potential for improving the development of sexually functional foods.With the increasing emergence of contemporary social trend of population aging,life pressures,environmental pollution,weak awareness of public movement and other issues,testosterone deficiency disease incidence gradually increased.As the main androgen,testosterone plays a key role in maintaining muscle mass and strength,libido and erections.Testosterone deficiency has a serious impact on the development of various organ systems in men.In this paper,active peptides from Crassostrea gigas oyster was prepared by enzymatic hydrolysis,isolation and purification.And its activity in promoting testosterone secretion of mice in vitro was evaluated.Below are key research findings:(1)The 16 s rRNA gene detection method was used to identify species of raw oysters.Using the Basic Local Alignment Search Tool(BLAST)method,the resulting gene sequence was 100% coincident with the 16 s rRNA gene sequence of the Crassostrea gigas in the NCBI database,which proved that the oyster used in the experiment was Crassostrea gigas.(2)Through the single factor experiment,taking peptid yield as an indicator,the effect of the different enzyme amount,pH value,enzymatic hydrolysis temperature and enzymatic hydrolysis time on it was compared.The orthogonal test analysis was carried out according to the results of single factor experiments.The optimal enzymatic conditions for papain were as follows: enzyme dosage 3000U/g,pH 6.5,enzymatic hydrolysis temperature 62.5 ?,enzymatic hydrolysis time 4.5h,and its peptide concentration was 14.95mg/mL.(3)Compared with the blank control group,the oyster hydrolysate and its ultrafiltration fraction in the range of 0.25-1mg/mL,especially <5 ku and 5~10 ku,can significantly increase the proliferation and secretion of testosterone in TM3 cells.Considering the protein recovery rate and molecular weight distribution,it is decided to further separate and purify the component OP-I(<5 ku)and detect its activity in vitro.(4)The purified enzymatic ultrafiltration fraction OP-I(<5 ku)was separated and purified by Sephadex G-25 gel chromatography,and the three components(OP-Ia,OP Ib and OP Ic)were separated and collected.They were evaluated to promote testosterone secretion activity of mice in vitro.The OP-Ib fraction significantly enhanced the cell proliferation activity of TM3 cells at different concentrations(0.1,0.2,0.5 mg/mL).The optimal concentration of OP-Ib on TM3 cell proliferation during the 24 h duration was 100?g/mL.In this concentration,OP-Ib has a significant concentration-dependent effect on the proliferation activity of TM3 cells.Compared with the blank control group and the other two isolated components OP-Ia,OP Ic,the OP-Ib component significantly promoted the secretion of testosterone in TM3 cells at three concentrations(50,100 and 200?g/mL).(5)Further separation and purification,OP-Ib was separated by Sephadex G-15 gel chromatography to obtain two peaks.These two components were evaluated to promote the testosterone secretion activity of mice in vitro.The activity of F2 component was significantly better than that of F1,and it showed a concentrationdependent effect on promoting testosterone secretion of mice in vitro.