Study On Enzymatic Preparation Of Bone Gelatin And Its MTGase-mediated Crosslinking Properties And Application

Bone gelatin is a product purified from bone collagen by moderate hydrolysis.Because of its good ability for emulsification,foaming,gel,film formation,stability and thickening,bone gelatin is widely used in many fields such as food,medicine,cosmetics,and industry.The traditional bone gelatin is mainly produced by the alkaline method,which has some disadvantages,such as the long production cycle,waste of resources,and serious environmental pollution.Therefore,the production of bone gelatin urgently needs to realize the transformation from traditional production to green production of science and technology.In this paper,the enzymatic method was used to replace the traditional method to prepare bone gelatin.On the premise of ensuring the yield and quality of bone gelatin,the enzymatic preparation of bone gelatin shortened the production cycle of bone gelatin and reduced the use of fresh water,acid,and lye in the production process,which could provide a reference for the green transformation of bone gelatin production.First of all,taking the yield,viscosity,and gel strength of bone gelatin as indexes,the enzymatic hydrolysis process and gel extraction process were optimized by a single factor experiment.The optimum conditions of enzymatic hydrolysis of bone gelatin were as follows:the amount of trypsin was 150 U/g,the temperature was 30℃,the time was 2 h and the solid to liquid ratio was 1: 2.The optimum condition for extracting gelatin was p H = 5,at 60℃ for3 h,and the solid to liquid ratio was 1:2.Under these conditions,the crude glue can be obtained from bone powder by demineralization,enzymatic hydrolysis,and extraction,which takes only about 10 hours,compared with the alkaline liming process of 30 ～ 60 days,the production cycle was greatly shortened.The yield of bone gelatin was 10.75%,which was 3 times higher than that of bone gelatin prepared by the alkali method and 80% of the total yield of bone gelatin prepared by the alkali method.The gel strength of enzymatic bone gelatin was 370 Bloom g(converted to commercial gel strength is 255 Bloom g)and the viscosity was 3.34 m Pa·s.All of the quality indexes of enzymatic bone gelatin met the standards of edible gelatin and highgrade acid bone pharmaceutical gelatin(gel strength is 200 Bloom g).Secondly,under the optimal process,the structure and physicochemical properties of type E bone gelatin(gelatin prepared by enzymatic method)were studied and compared with type B bone gelatin(gelatin prepared by alkaline method)and type A bone gelatin(gelatin prepared by pickling method).It was found that the amino acid composition of type E bone gelatin was similar to that of type A and type B bone gelatin,and the higher contents were glycine and subamino acids(proline and hydroxyproline),accounting for about 44% of the total amino acids.Compared with type A and type B bone gelatin,the relative molecular weight distribution of type E bone gelatin was narrow,and the average relative molecular weight was higher.The secondary structure of type E bone gelatin is similar to that of type A and type B bone gelatin.The contents of the triple helix in the tertiary structure were typed A bone gelatin,type E bone gelatin,and type B bone gelatin from high to low.The rheological properties of the three types of bone gelatin were similar,the gel temperature(20.76℃)and melting temperature(31.27℃)of type E bone gelatin were higher than those of type A and type B bone gelatin.And the isoelectric point of type E bone gelatin was 9,which was higher than that of type A(p I = 7～8)and type B(p I = 5)bone gelatin.The foaming ability of type E bone gelatin was higher than that of type A and type B bone gelatin when deviating from the isoelectric point,the foaming stability was higher than that of type A and type B bone gelatin when nearing the isoelectric point,and the emulsion activity index and emulsion stability index of type E bone gelatin was the highest at p H = 2,which was higher than that of type A and type B bone gelatin.Then,the properties and characteristics of three types of bone gelatin modified by microbial transglutaminase(MTGase)were studied.It was found that the contents of lysine and glutamine in type E bone gelatin are higher than those in type A and type B bone gelatin,which provided abundant action sites for MTGase and made type E bone gelatin have higher MTGase crosslinking potential.When the MTGase amount was 1.44 U/g,the crosslinking degree of type E bone gelatin(79%)was about 4.9 times that of type A bone gelatin and 5.6 times that of type B bone gelatin.After MTGase modification,the α-helix in the secondary structure of the three types of bone gelatin disappeared,and the total content of β-folding and β-turning angles of type E and type A bone gelatin was the same(76%),which was higher than that of type B bone gelatin.The triple helix content of type E bone gelatin modified by MTGase decreased the most,which was 1.3times of that of type A bone gelatin and 3.1times of type B bone gelatin.After MTGase modification,the gel temperature,melting temperature,and apparent viscosity of the three types of bone gelatin increased with the increase of the concentration of MTGase.When the amount of MTGase was higher than 0.90 U/g,the type E bone gelatin was highly crosslinked with MTGase,and the apparent viscosity increased significantly,which could not be transformed into a liquid and gel state,and finally the thermal reversibility was lost.Finally,type E bone gelatin,type A bone gelatin,type B bone gelatin and MTGase were used in minced pork alone or cooperatively.It was found that the combined use of type E bone gelatin and MTGase could not only significantly improve the puncture strength of non-cooked minced pork gel but also effectively improve the texture properties of cooked minced pork gel,such as hardness,elasticity,stickiness and chewiness.The water-holding capacity of cooked minced pork gel increased and the cooking loss decreased when bone gelatin and MTGase were used together,especially the combined use of type E bone gelatin and MTGase was the most significant,but the use of bone gelatin alone could reduce the water-holding capacity of cooked minced pork gel and increase the cooking loss.The addition of bone gelatin could significantly change the color of cooked and non-cooked minced pork gels,and the combined use of type E bone gelatin and MTGase had the least effect on the color of cooked and non-cooked minced pork gels.