Study On Preparation Of Potato Protein Hydrolysate And Its Promotion On The Growth Of MDCK Cells

Potato protein is a by-product of the potato starch processing.At present,the potato starch process generate a large amount of potato processing waster(PPW)which contains high concentration of ammonia nitrogen,biochemical oxygen demand(BOD)and protein,not only wastes of protein resource,but also causes several pollution to the environment.The protein in PPW was decomposed through biochemical digester,it has not been effectively recovered as a resource.In order to achieve the high-value utilization,the potato protein was hydrolyzed by protease,the components of the potato protein hydrolysate(PPH)containing small molecular weight fractions were collected and characterized in terms of functional properties,molecular weight distribution,free amino acid content and peptide sequences.Finally,PPH was applied to MDCK cell culture in low-serum in vitro.The effects of the PPH on cell growth promotion,growth metabolism and cell freeze storage were studied.The main research contents and results are as follows:(1)The potato protein concentrates was used as raw material.The five kinds of proteases(alkaline protease,neutral protease,papain,flavor protease,compound protease)were screened through the potato protein hydrolysis process to select the best performing protease,which was flavor protease.The single factor experiment and the response surface methodology experiment were carried out to study the optimal hydrolysis conditions.The results is:temperature 50?,p H 6.2,E/S 24000:1U/g,the hydrolysis degree was 43.49%.The PPH was subjected to 5 KD ultrafiltration to obtain potato proteolytic concentrate,then potato proteolytic concentrate was freeze-dried and stored.(2)The results of free amino acid detection showed that there were 24 kinds of free amino acids in PPH,and the total amount of amino acids was up to 38.44%(w/w%),among which the essential amino acids accounted for 13.22%,and the branching amino acids(leucine,valine and hetero bright amino acids)accounted for8.54%.The results of HPLC and mass spectrometry showed that the average molecular weight of 997 D was the highest,accounting for about 64%.The molecular weight of PPH peptides ranged from 573 D to 3867 D,and contained 101kinds of polypeptide sequences.(3)MDCK adherent cell culture was used in vitro.The results showed that the optimal addition amount of potato protein hydrolysate in MDCK adherent cell culture was 1 g/L.When the cells were fully adapted to the growth environment,the maximum proliferation concentration of cells was 9.11×10⁴ cells·m L^-1,which was higher than that of the control group at the 9^thgeneration.The cell growth rate was0.0163±0.0002 h^-1 and the doubling time was 15.71 h.The cell growth effect was significantly higher than that of 5%NBS control group(P<0.05),but there was no significant difference between 10%NBS control group and 5%NBS control group(P>0.05).This study the preparation of potato protein hydrolysate added to low serum culture medium,serum alternative part for cell culture,to promote the MDCK adherent cell growth,cell biological metabolic changes are different.In the preliminary evaluation of the effect of PPH on cell cryopreservation,the 10th generation cells cultured with PPH were cultured to the logarithmic growth phase and stored at the density of 2×10⁶cell·m L^-1 for one week.After recovery,the cells were found to be able to grow and passage normally with good cell morphology.The evaluation results of cell cryopreservation using PPH instead of serum showed that PPH could protect MDCK adherant cells in the cryopreservation process and could replace 5?10%serum.The specific mechanism needs to be further explored in the future.In conclusion,the research results prove that the PPH research route and process are effective and feasible in the development and application of low-serum culture media,,which provides a new idea for the high-value application of potato protein,a reference for the development of potato protein hydrolysates,and a new approach for the recovery and purification of potato protein,which is often used as industrial waste,as a substrate and biological material for cell culture.