Preparation Of Oligopeptide Chelated Cu/Zn By Solid-state Fermentation Of Soybean Meal And Characterization Of Their Structural Properties

As a by-product of soybean processing,soybean meal（SBM）is rich in protein and is a high-quality and inexpensive vegetable protein resource.Copper and zinc are one of the indispensable nutrients for animals.Deficiency of copper/zinc can lead to loss of appetite,economic disorders and reduced reproductive capacity,thus breeders usually add a certain amount of copper/zinc supplements to the feed.Copper sulfate and zinc sulfate have a low absorption rate in the intestinal tract of animals and are prone to heavy metal contamination of the soil,while oligopeptide/amino acid-based copper/zinc supplements have a high bioavailability and can achieve the purpose of supplementing dual nutrition.However,amino acid-based copper/zinc supplements（copper/zinc glycinate,copper/zinc glutamate,etc.）are costly to prepare and monotonous in variety.Therefore,in this study,soybean meal oligopeptides（SBMOP）that could be used to chelate metal ions were prepared at low cost by fungal solid-state fermentation（SSF）of SBM,and the chelation mechanism and in vitro stability of SBMOP with Cu/Zn ions were investigated.The main results of the study are as follows:（1）Three fungi（Aspergillus oryzae NCUA 101,Aspergillus niger NCUA 102,and Mucor NCUA 103）were used for SSF of SBM.Comprehensive consideration of protease species and activity,Aspergillus oryzae was an optimal strain for the production of protease in SSF of SBM.Single-factor and response surface experiments showed that the optimal conditions for alkaline protease production by Aspergillus oryzae SSF of SBM were fermentation time of 86 h,inoculation ratio of 43%,and liquid-substrate ratio of 0.8.The alkaline protease activity for the optimum conditions above was 574.43 U/g.The degree of hydrolysis（DH）and total Kjeldahl nitrogen（TKN）content of SBM protein reached 38.97%and 45.36 mg/g,respectively,after 6 h of hydrolysis at 55°C.Antioxidant experiments showed that the soybean meal peptides（SBMP）prepared by SSF of Aspergillus oryzae had high antioxidant properties.The IC₅₀ values of ABTS⁺radical scavenging ability,hydroxyl radical scavenging ability and DPPH radical scavenging ability of SBMP were 2.56 mg/m L,3.35 mg/m L and 1.71mg/m L,respectively,which could be used as an effective source of antioxidant peptides with promising applications in functional products.（2）The preparation process of SBMP-Cu/Zn chelate was optimized using single factor and orthogonal.The optimal chelation conditions were 50°C,60 min,p H 3.0and mass ratio 1:2,under which the Cu/Zn chelating capacities of the SBMP were 94.26mg/g and 99.46 mg/g,respectively.The membrane separation experiments showed that the peptide fraction of 1-5 k Da presented the highest chelating capacity for Cu and Zn After further purification by weak anion exchange chromatography and gel filtration chromatography,the Cu/Zn chelating capacity of SBMP reached 288.47 mg/g and325.75 mg/g.The chelated peptides were analyzed by LC-MS/MS,and a total of 27oligopeptides were identified and screened,all with molecular weights less than 1500Da,and each oligopeptide contained at least two acidic amino acids（aspartic acid and glutamic acid）,25 oligopeptides contained basic amino acids（arginine,histidine,lysine）,and 4 oligopeptides contained serine.The results showed that acidic and basic amino acids played a key role in the chelation of the oligopeptide with Cu/Zn ions.In addition,phosphorylation of serine residues created suitable binding sites for copper/zinc ions.（3）UV,FTIR,SEM and TG techniques were used to characterize the structural stability of SBMOP and their Cu/Zn chelates.The main active groups for Cu/Zn chelation by SBMOP were–COOH and–NH₂.The crystallinity of SBMOP-Cu/Zn chelate was increased,and rough spherical agglomerates were formed on its surface.The negative charge on the surface of the oligopeptide after Cu/Zn chelation was neutralized,and the zeta potential increased from-31.3 m V to-27.0 m V and-24.9 m V,respectively.The maximum tolerable temperature of SBMOP should not exceed 133°C,and the thermal stability of chelated Cu/Zn was significantly increased to 244°C and380°C,respectively.The crucial amino acids for chelating Cu/Zn in SBMOP were acidic amino acids（aspartic acid and glutamic acid）and basic amino acids（lysine,arginine,and histidine）.The acidic and basic amino acid contents of SBMOP-Cu chelate reached 36.22%and 30.59%,respectively.SBMOP-Zn chelate contained 30.54%and 45.24%of acidic and basic amino acids,respectively.In addition,SBMOP-Cu/Zn chelate showed higher acid and base resistance,heat resistance,salt resistance,and in vitro digestive stability than SBMOP,indicating the potential of SBMOP-Cu/Zn chelate to improve Cu/Zn bioavailability.The results of this study provide theoretical basis and technical support for the low-cost and efficient preparation of oligopeptide-Cu/Zn chelate supplements and the high-value resource utilization of SBM.