Study On The Preparation Of Substitute Nitrogen Source And Flavor Peptide By Hydrolysis Of Mulberry Seed Protein

The defatted mulberry seed contains 30-50%protein,which is an ideal raw material for the preparation of alternative nitrogen source and flavor peptides.However,the low solubility of mulberry seed protein limits its extraction and utilization.Therefore,this article pretreated defatted mulberry seeds by ultrasound and microwave to improve the solubility and extraction rate of mulberry seed protein;on the basis of single-frequency ultrasound,the molecular diffusion dynamic model was used to describe the leaching process of mulberry seed protein by multi-frequency countercurrent ultrasound treatment,and the mechanism and law of multi-frequency ultrasound extraction of mulberry seed protein were thoroughly explored;the microfluidic immobilized protease-ionic liquid catalytic system wasconstructed to improve the hydrolysis rate of mulberry seed protein,and separate,analyze,identify characteristic flavor peptides from mulberry seed protein hydrolysate.The specific research results are as follows:?1?The feasibility of using protein hydrolysate of defatted mulberry seed as a substitute for nitrogen source to produce lipid was investigated for the first time.The results show that the ultrasound pretreatment at the solid-liquid ratio of 1:40,treatment time of 9min,treatment temperature of 60?,output power of 300 W,the extraction rate was up to71.22%,which was 3.7 times of untreated;the microwave pretreatment at the solid-liquid ratio of 1:30,treatment time of 3 min,treatment temperature of 60?,output power of 300W,the extraction rate was up to 33.19%andonly 13%morethanuntreated.On this basis,the effects of ultrasound and microwave on the physicochemical properties of mulberry seed protein were predicted and evaluated from the secondary structure of mulberry seed protein and antioxidant activity of hydrolysates.After ultrasound and microwave treatment,the?-helix of mulberry seed protein decreased from 41.36%to 31.61%and 39.92%,respectively.The solubility of mulberry seed protein increased from 25.2%to 30.89%and28.33%,and DPPH scavenging activity increased from 35.67%to 74.15%and 44.96%,respectively.Using defatted mulberry seed protein hydrolysate as a nitrogen source to culture Schizochytriumsp.SR21 fermentation to production lipid,the growth curve during the fermentation process was measured.The result shows that the amino acid composition of mulberry seed protein was not significantly different from the amino acid composition of yeast extract.The 17.5%DHA in the lipid of Schizochytrium sp.SR21 production?17.45%culturedby yeast extract?,indicated that mulberry seed protein hydrolysate could replace yeast extract to produce oil the fitting model R²was 0.99.The amino acid composition of mulberry seed proteinhydrolysate.In summary,the mulberry seed protein hydrolysate can replace yeast extract as an alternative nitrogen source to cultivate microorganisms produce lipid.?2?Themicrofluidized immobilized protease-ionic liquid catalyzed protein hydrolysis system for mulberry seed was constructed The molecular diffusion dynamic model was used to comprehensively simulate the extraction process of multi-frequency countercurrent ultrasound pretreatment of mulberry seed protein.The results show that the apparent rate parameterskandk_dweredetermined;thechange law of y?k_d?k?D_swith the change of ultrasound power and treat temperature was obtained.Whensolid-liquid ratio set as 1:30,temperature of 50?,powerdensity of 300 W,ultrasoundfrequency of triple frequency combination28K/52K/40K,the protein extraction rate was 86.42%;compared with single-frequency ultrasound treatment,it was increased by 16.2%,and which was 4.47times of untreatedsimple.The R²of the fitted model was 0.9783,and the conditions of model calculation processing were consistent with the above results.In summary:the multi-frequency countercurrent ultrasound can further enhance the extraction efficiency of mulberry seed protein under specific processing conditions;the model based on the Fick second law was suitable for predicting the pretreatment conditions of multi-frequency ultrasonic extractionproceing of mulberry seed protein;the DPPH scavenging activity,SRASA,reducing power test and chelating Fe²⁺activity of mulberry seed protein hydrolysate treated with multi-frequency ultrasound were significantly higher than the anti-oxidation activity of hydrolysate after single-frequency ultrasound treatment.The above results indicate that multi-frequency ultrasound can further improve the physicochemical properties of mulberry seed protein on single frequency ultrasound?3?The catalytic system of microfluidic immobilized proteases-ionic liquid was constructed.The experimental results show thatthe combination of complex protease-choline chloride?CC?makes the protease activity increase by 1.8 times.The micro-reactor hydrolysis process was determined with flow rate of 2.5?L/min,temperature of 50?,and the protein dilution concentration of 1:50.The proteolysis rate reached 78.33%,which was twice of no ionic liquid added.The stability test results of the immobilized enzyme showed that the complex protease still had 80%relative activity after being reused for 12 times.After adding the ionic liquid,the K_mvalue of the protease decreased by 1.25 mg/m L,and the V_maxvalue increased by 178.13 U;the thermal stability of the protease was evaluated based on the kinetic parameters(k_d,T_1/2,and D).The T_1/2of the complex protease was increased by 90 minutes,it shows that after the addition of ionic liquid,the affinity of the substrate and the enzyme was increase,and the thermal stability of the complex protease wasimproved,it shows that the addition of ionic liquid makes the enzyme play a better catalytic role.?4?The electronic tongue technology was used to detect the taste characteristics of mulberry seed proteolysate,and to separate and screen and identify sweet peptides.The results showed that the mulberry seed proteolysis contained flavor peptides or amino acids with various flavors.The taste characteristics of the mulberry seed proteolysis were mainly sweet,and the sweetness intensity was slightly different due to the choice of protease.Among them,the compound protease can hydrolyze mulberry seed protein more fully,and the sweet taste intensity of the hydrolysed hydrolysate was 3108.13.Therefore,sweet peptides were screened from the product of catalyzed mulberry proteolysis by complex protease.The three sweet peptides derived from mulberry seed protein were separated and identified by ultrafiltration,gel chromatography,reversed-phase chromatography separation,mass spectrometry and other techniques,namely FEGGSIE,KDFPEAHSQAT,and GSQPAEGAK.In these three peptides,the proportion of sweet amino acids in total amino acids was 44.45%,63.64%,44.40%.The amino acid composition of the three sweet taste peptides contains very few hydrophobic amino acids,so there are no other odor effects.