Study On The Enzymatic Preparation Of Bone Gelatin And Its Film-forming Properties

Obtained from partial hydrolysis of bone collagen,bone gelatin is widely used in food,medicine,cosmetic,photographic materials and sometimes irreplaceable for pharmaceutical uses?hard capsules,soft capsules?.However,conventional gelatin production based on liming is time-consuming and leads to significant land pollution and wastewater.Concerns over the water waste and the environmental pollution caused by the oldest industries lead to the appeal for new ways of production to reduce environmental impact.Thus,an efficient and ecologically compatible method of gelatin production from bone has to be developed.In this study,enzymatic process is used to replace the liming process to hydrolyze native collagen to gelatin from porcine bone.The advantages of this enzymatic process are short processing time,higher gelatin extraction,and less waste generation.One-step enzymatic method provides an alternative route to sustainable development for bone gelatin industry.In one-step enzymatic method,conventional pretreatment-demineralization,liming and deliming before gelatin extraction could be replaced by one step process-demineralization and enzyme hydrolysis at the same time.In this study,the novel one-step enzymatic method shows excellent results,with time reduction for ossein hydrolysis from 3?8 weeks to 3 h and without the need for liming and deliming,thereby reducing alkali usage and saving 60%water.In addition,4 steps with low yield involved in conventional gelatin extraction were replaced by 1step with relatively high yield in the new method.The gelatin obtained from the one-step enzymatic method possesses fine qualities?Bloom strength and viscosity?similar to that of commercial high-grade gelatin.All the physical and chemical indicators of gelatin obtained from one-step enzymatic method meet the requirements of the national standards for medical gelatin and gelatin for capsule.Moreover,the environmental benefit of one-step enzymatic method is also confirmed by a brief life cycle assessment?LCA?.There are some similarities between type E gelatin?obtained from one-step enzymatic method?and type B gelatin?obtained from conventional gelatin extraction?.Firstly,amino acid composition.The amino acids which account for 2/3 of the total amino acid residues are glycine,proline,hydroxyproline and alanine.The content of glycine,imino acid?proline&hydroproline?and alanine are 1/3,1/4 and 1/9,respectively.There are little aromatic amino acids and sulfur-containing amino acids,and no tryptophan in both types of gelatin.Secondly,molecular weight distribution.The main components in both types of gelatin are?₁ chain,?₂ chain and?chain.Thirdly,structural morphology.There are triple-helix structure and amorphous fraction in both types of gelatin,which are confirmed by CD,FTIR and XRD analysis.The fourth,dynamic rheology.Both types of gelatin exhibit similar dynamic viscoelastic profiles and thermal stability.The similarities between type E gelatin and type B gelatin are attributed to their same native collagen-porcine bone collagen.However,the isoelectric point?the isoelectric point of type E gelatin is 8.89,and that of type B gelatin is 4.84?,water holding capacity and fat binding capacity,foaming ability and foaming stability,emulsion activity and emulsion stability are different between these two types of gelatin.Because of the difference in the break site and extension degree of collagen chain during the process of gelatin production,the amino acid distribution and spatial conformation of type E gelatin and type B gelatin are different,which lead to the differences of their properties.The cleave site of enzyme during one-step enzymatic method has been studied.It is concluded through the primary,secondary and tertiary structure analysis of enzyme soluble collagen that enzyme hydrolysis doesn't damage the integrity of the original triple-helix structure.The results of fibrillogenesis test and peptide mass spectrometry identification exhibit the integrity of telopeptide,which means the cleave site is the crosslink bonds of native collagen.During one-step enzymatic method,the single chain of collagen was separated from collagen molecule by heating to form gelatin after enzymatic hydrolysis.The difference of the isoelectric point between type E gelatin and type B gelatin can be explained as follows.Firstly,the different spatial conformation of both types of gelatin may lead to the difference of the isoelectric point.Secondly,during prolonged exposure of the raw material to alkali,deamidation of glutamine?pI=5.65?and asparagine?p I=5.41?may occur,resulting in the formation of glutamic acid?pI=3.22?and aspartic acid?pI=2.97?,leading to a relatively low p I value?at pH 4.84?of type B gelatin.Thirdly,the degree of exposure of the high isoelectric point amino acid inside type E gelatin is higher than that of type B gelatin.Finally,the film-forming property of type E gelatin has been studied.Type E gelatin could form more covalent crosslinks when compared to that of conventional ones[type A gelatin?obtained from acid process?and type B gelatin]with the same amount of microbial transglutaminase?MTgase?,and the modified type E gelatin film exhibits high molecular weight,stable network structure,and high mechanical strength.This kind of gelatin film could retain its original shape even in boiling water.With the assistant of MTgase,type E gelatin could provide a strategy for fabricating water-insoluble gelatin film.Type E gelatin has narrow molecular weight distribution,which can recover the triple-helix structure of native collagen to a great extent.A collagen-inspired anisotropic gelatin film was successfully fabricated with the assistant of SO₄^2-?kosmotropic ion?and mechanical deformation using type E gelatin.SO₄^2-could make the gelatin film more stretchable,which is critical for the formation of hierarchical ordered structure under the mechanical deformation.The resulted anisotropic gelatin film shows excellent tensile strength and toughness,which are increased from 89.20 MPa and 3.13MJm^-3 to 220.71 MPa and 14.15 MJm^-3,respectively.Moreover,this anisotropic gelatin film has high transparency and birefringence,which can separate polarized light and tune the intensity of polarized light.