Preparation Of Chicken Flavor Base By Enzymatic Hydrolysis And Its Flavor Formation Mechanism

With the economic development and the improvement of living standards,condiments as the main substance to improve the flavor of food,show the trend toward high grade and nutrition.Chicken skeleton is the main by-product of chicken meat processing.However,for a long time,due to the limitations of processing technology,a large number of chicken skeletons have been directly processed into low value-added products such as bone sludge and feed,and some have even been discarded.Therefore,it results in a great waste of resources,and has a certain impact on the environment.Chicken and its bones contain a variety of umami components with a strong flavor.They can be used as raw materials for food or flavor precursor substances for the production of seasonings,to meet people’s consumption needs and promote the development of food industry.In this paper,the optimum enzymatic hydrolysis conditions for the preparation of chicken flavor base were determined by singlefactor experiment and orthogonal test.The change rules of peptides,free amino acids,nucleotides,organic acids and volatile flavor substances in the hydrolysates at different enzymatic hydrolysis times were analyzed.The best umami components were screened by isolation and purification techniques,and the umami peptides were identified and synthesized to investigated their taste characteristics.The interaction between the umami peptides and the umami receptor T1R1/T1R3 was explored by molecular docking means to elucidate the umami presentation mechanism.Firstly,the composite substrate of chicken skeleton and chicken breast was hydrolyzed using enzymatic hydrolysis technology.The umami and overall flavor of the product obtained by the compound enzyme including Protamex and Flavourzyme were found to be outstanding by the index of hydrolysis degree and sensory evaluation.The enzymatic hydrolysis process was optimized by single factor experiment and orthogonal experiment.The results pointed out that the taste of hydrolysate was better using simultaneous hydrolysis with Protamex and Flavourzyme by the ratio of 3:1.The optimal enzymolysis conditions were as follows: ratio of chicken skeleton to chicken breast 9:1,solid-liquid ratio 1:2,temperature 50℃,p H 7.5,enzyme dosage 0.6%,and hydrolysis time 3 h.Under these conditions,the hydrolysate had a prominent umami taste,obvious meat flavor and good taste,with hydrolysis degree and sensory score of 13.60% and 4.7,respectively.Then,the peptides,free amino acids,nucleotides,organic acids and volatile flavor substances in the products of different enzymatic hydrolysis times were analyzed.The content of peptides with low molecular weight and the total amount of free amino acids in the products increased with the increase of enzymatic hydrolysis time.At 3 h of enzymatic hydrolysis,peptides with molecular weight <3 k Da accounted for 93.26%,including 69.36% of peptides with molecular weight <1 k Da.The content of umami and sweet amino acids in the products increased to 15.09 mg/g at 3 h,and the content of bitter amino acids was 52.91 mg/g,accounting for 76.33% of the total free amino acids.Histidine and glutamic acid were the main amino acids contributing to taste.The content of 5’-guanosine monophosphate(5’-GMP)increased to 1.17 and 1.22 mg/g at 3 h and 5 h of enzymatic hydrolysis,respectively.The content of 5’-adenosine monophosphate(5’-AMP)and 5’-inosine monophosphate(5’-IMP)decreased with the extension of enzymatic hydrolysis time and did not change significantly after 1 h.The content of lactic acid showed an increasing trend with the extension of enzymatic hydrolysis time,and it did not change significantly after 2 h,which was 78.98 mg/g at 3 h.The content of succinic acid decreased and then increased during enzymatic hydrolysis,and it was 71.93 mg/g at 3 h.When the time was extended to 5 h,the content of succinic acid reached77.40 mg/g.After enzymatic hydrolysis,the equivalent umami concentration(EUC)increased significantly and the product had stronger umami.The kinds of volatile flavor substances increased after enzymatic hydrolysis,from 21 at 0 h to 35 at 3 h.The content of each kind of volatile substances firstly increased and then decreased in general.Flavor substances mainly included esters,alcohols,aldehydes and ketones.Moreover,flavor substances such as pyrazine and thiazole were detected after enzymatic hydrolysis,which contributed to the formation of characteristic flavor of chicken meat.Finally,the complex hydrolysate obtained under the optimal conditions was firstly ultrafiltered to obtain the strong flavor fraction F4(<1 k Da),and then further separated and purified by dextran gel filtration chromatography to obtain the outstanding flavor fraction F41.Seven peptides including GCDKYGCGY,FAGDDAPRA,AHDGGRYY,TERGYSF,GNSNLPCS,TNPYDYHY and GDPHVGH were identified from the F41 fraction using liquid chromatography tandem mass spectrometry(LC-MS/MS).The bioinformatics prediction results revealed that the six peptides except GNSNLPCS were potential umami peptides.The six small molecule peptides were synthesized,and their taste characteristics were analyzed by sensory evaluation and electronic tongue.The results showed that the umami taste thresholds of the six synthetic peptides ranged from 0.16 to 0.54 mg/m L,and the umami taste enhancement thresholds were in the range of 0.02-0.11 mg/m L.Peptides GCDKYGCGY and AHDGGRYY had prominent umami taste,with the umami taste thresholds of 0.16 and 0.20mg/m L,respectively,which were lower than that of monosodium glutamate(MSG)(0.30mg/m L).Moreover,the two peptides had significant umami and salty taste enhancement effects.The T1R1/T1R3 model was constructed by homology simulation,and could be used as a umami receptor.The docking results of umami peptides with T1R1/T1R3 molecules showed that GCDKYGCGY preferred to bind to the T1R3 subunit,and Ser306 was the key binding site for the receptor to interact with the umami peptide.While AHDGGRYY preferred to bind to the T1R1 subunit,and His71 was the important amino acid binding site.The main forces of peptides binding to the receptor were hydrogen bonds and hydrophobic interactions.