Preparation And Bioactivity Of Immunopeptide In Vitro From Oviductus Ranae

Oviductus Ranae is the essence of frog.It is a traditional nourishing good product in China.It is rich in nutritional value,and has many effects such as promoting development,delaying aging,nourishing kidney and essence,moisturizing lung and yin,beauty and beauty,strengthening body and so on.At present,the method of consuming Oviductus Ranae is mainly by soaking and cooking.Although the traditional edible method retains the nutrients of the raw materials to the greatest extent,it cannot be fully absorbed and utilized in the human body,causing Great waste.In this study,the immunoreactive peptides were obtained by enzymatic hydrolysis,ultrafiltration and gel chromatography,and the structure identification and in vitro immunoreactivity of the prepared highly active peptides were studied.The results obtained by this research are as follows:(1)Use five kinds of proteases such as neutral protease,papain,alkaline protease,trypsin and flavor protease to enzymatically digest defatted Oviductus Ranae,using amino nitrogen content as an indicator,and comprehensively consider related issues such as production costs Finally,the best protease for the preparation of Oviductus Ranae hydrolysate was selected as the neutral protease.The effects of four factors including hydrolysis time,temperature,p H,and amount of enzyme added on the hydrolysis of Oviductus Ranael were investigated.The L9(34)orthogonal test was used to optimize the conditions of Oviductus Ranae digestion.The orthogonal test results showed that the optimal process parameters for preparing the Oviductus Ranae hydrolysate with neutral protease were: enzymolysis time 5h,enzymolysis temperature 55?,p H value 7.0,and the amount of enzyme added 5000 U/g.(2)The effects of Oviductus Ranae immunoactive peptides on the immunoregulatory effects of mouse spleen cells and RAW264.7 cells: F1,F2 and F3 can induce the proliferation of mouse spleen cells and RAW264.7 cells and increase the phagocytic activity of RAW264.7 cells.Among them,F2 works better.When F2 concentration was 320 ?g / m L,the proliferation rate of RAW264.7 cells was increased by 26.5%,and the phagocytic activity was increased by 58.5% under theLPS cooperation and the proliferation rate of spleen cells was increased by 51.9%under Con A cooperation.(3)The structure of F2 was identified by high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS),and the identification results were screened to obtain three oviductus ranae immunoreactive peptides with amino acid sequences of AAGFLMK,SDLEWIR and HYDQSYR respectively.(4)F2 has the effect of alleviating CTX immunosuppression,promoting the proliferation of mouse spleen cells and RAW264.7,increasing the concentrations of cytokines IL-1? and TNF-? in the cell supernatant,and increasing the concentration of NO.