Study On Umami Properties And Flavor Formation Mechanism Of The Maillard Reaction Obtained From Enzymatic Hydrolysate Of Pleurotus Citrinopileatus

Pleurotus citrinopileatus is deeply loved by consumers because of its rich nutrition and delicious taste.In addition,P.citrinopileatus was identified to be rich in glutamic acid（Glu）,aspartic acid（Asp）,and 5’-nucleotides,making it a suitable material to prepare high quality flavor condiments.However,in recent years,most studies of P.citrinopileatus have concentrated on its biological activity evaluation and genetic diversity,whereas few reports focusing on the utilization of flavor compounds in P.citrinopileatus have been conducted.The efficient hydrolysis of P.citrinopileatus by protease obtained from Actinomucor elegans to prepare taste peptides-rich seasoning.On this base,umami peptides were identified from P.citrinopileatus enzymatic hydrolysate after multi-stage separation and purification techniques.The taste characteristics of umami peptide were analyzed to explore its flavor formation mechanism.In order to obtain good flavor precursor,the influence of different sulfur-containing compound and peptide fractions on the properties of Maillard reaction product were evaluated and compared,which provided theoretical support for the development of mushroom seasoning base material by Maillard reaction.Firstly,mucor protease was prepared by the solid-state fermentation of P.citrinopileatus with Actionmucor elegans,and its enzymatic properties were evaluated.Taking protease activity as the evaluation index,the medium composition and culture conditions were optimized through uniform design experiment and response surface methodology,respectively.The results suggested that the best composition for mucor protease production included P.citrinopileatus powder 5%,sucrose 6.14%,Mg SO₄ 0.05%,Ca Cl₂ 0.03%,and the protease activity got 99.72U/g.The optimal parameters were fermentation temperature of 27°C,the ratio of substrate to water 1:1.4,and the culture time of 104h,and the protease activity was 132.45U/g.The optimum temperature and pH of mucor protease were 50°C and pH7.0 respectively,and its pH-stability range from 6.0 to8.0.Mg²⁺,K⁺,Ga²⁺could significantly increase the protease activity.Secondly,the feasibility of a productive protease system from Actinomucor elegans to prepare P.citrinopileatus hydrolysate（PCH）was investigated.The Actinomucor elegans crude protease（AECP）was prepared from the solid-state fermentation product of P.citrinopileatus by A.elegans.AECP and four commercial proteases（alcalase,neutrase,papain,and protamex）were applied to acquire five kinds of PCHs.The physical-chemical properties of PCHs as well as its concentration and composition of nonvolatile compounds were comparatively analyzed.Sensory evaluation and electronic tongue analysis were utilized to evaluate sensory characteristics.AECP was found to be the most effective protease,with the highest hydrolysis degree（35.91%）and protein recovery（81.46%）.The result of molecular weight distribution indicated that peptides below 500 Da were the main fraction of AECP hydrolysates,while AECP hydrolysates showed the highest content of monosodium glutamate-like（20.23±0.16 mg/g）and flavor 5’-nucleotide（4.30±0.07 mg/g）peptides.In summary,the AECP hydrolysate had superior sensory profiles compared with otherhydrolysates.In addition,AECP hydrolysates exhibited favorable kokumi taste in which peptides below 500 Da showed the highest correlation with kokumi by the results of partial least-squares regression.Thirdly,P.citrinopileatus hydrolysates were isolated and purified from using ultrafiltration,nanofiltration,Sephadex G-15 gel filtration chromatography and reversed-phase high-performance liquid chromatography.The peptide fraction with the strongest umami taste was selected by sensory evaluation.The amino acid composition of the peptides was determined by ultra-performance liquid chromatography-mass spectrometry.The synthesis of the required peptides was carried out using solid-phase method,and their taste proerties were investigated by sensory evaluation and electronic tongue.The molecular docking technique was used to explore the interaction mechanisms between umami peptides and their taste receptors T1R1/T1R3.The results showed that four novel umami peptides,including VEDL,LELE,LDAL and FEEL,had obvious umami taste characteristics,and the recognition thresholds of umami peptides were in the range0.125-0.50 mg/m L.The interaction between molecular were mainly linked by van der Waals interactions and hydrogen bonds.Glu128,His125,Asp196 and Ser127 may play major roles in the binding and interactions between umami peptides and T1R1/T1R3receptors.Fourthly,the influence of L-cysteine（Cys）,L-methionine（Met）and thiamine（Thi）on the structure,sensory profiles and antioxidant activities of Maillard Reaction products（MRPs）derived from P.citrinopileatus was evaluated and compared.MRPs were prepared from P.citrinopileatus hydrolysate（PCH）,D-xylose and Cys/Met/Thi by heating at 100°C for duration of 2h at pH 7.4.The results illustrated that physicochemical properties,sensory attributes and antioxidant activities of MRPs were largely improved by sulfur-containing substances variety.Cys-MRPs showed stronger fluorescence intensity and lower particle size in comparison with other MRPs.Cys-MRPs exhibited stronger meat-like flavor and kokumi taste with better overall acceptability,while Met-MRPs and Thi-MRPs showed higher caramelic flavor and bitter taste.In addition,the antioxidant activity of Cys-MRPs was relatively high.Finally,three peptide fractions F0（5000-200Da）,F1（5000-500Da）,F2（500-200Da）were separated from P.citrinopileatus hydrolysate by ultrafiltration and nanofiltration.The Maillard Reaction products were prepared using different peptide fractions/D-xylose/L-Cys model system heated at 100°C for 2h.The effect of different peptide fractions on the Maillard reaction rate,aroma profile and sensory characteristics of MRPs was evaluated.The results indicated that F2 exhibited relatively high Maillard reaction rate,manifested as elevated UV-absorbance and browning intensity,decreased pH value and free amino acid content,and altered polypeptide structure.Moreover,the types and contents of volatile compounds were significantly increased by the Maillard reaction.Sensory evaluation results showed that the flavor enhancement effect of MRPs was superior to those of EHPs and TDPs.F2-MRPs showed good overall acceptability with stronger taste,while F0-MRPs had the strongest caramel-like flavor.The low molecular peptides had a great contribution to the generation of meat-like flavor and kokumi taste during the Maillard reaction.