Preparation Of Collagen By Microbial Fermentation And Its Properties And Applications

China's aquatic resources are extremely rich,and tilapia,as the main breeding economic variety,produces a large amount of scrap during processing.Such as fish skin,fish scales and other parts of the mass accounted for about 40% to 55% of the total mass.In the past,these wastes were discarded,which not only caused environmental pollution,but also caused huge waste of resources.Collagen has received much attention in recent years due to its good biological properties and a wide range of biological sources.Therefore,the experiment used tilapia skin as the main raw material to prepare collagen.It helps to solve the above problems and also helps to reduce dependence on mammals.It will help increase fish utilization,increase added value,and create greater economic and social benefits.In this experiment,high-yield collagenase strains were screened from the soil,and strain species were identified.The tilapia skin was fermented by this strain,and the optimal collagen preparation process was obtained by orthogonal test.The physicochemical properties of the collagen prepared by the method and the conventional method are compared.Finally,the collagen obtained by fermentation was added to the yogurt to explore its effect on the quality of the yogurt.The details are as follows:(1)Screening and identification of high-yield collagenase strainsFirstly,the collagenase-producing microbial strain was screened from the soil,and the enzyme activity was determined by the Folin-phenol method to obtain a high-yield collagenase strain.Then,the culture was carried out for morphological observation.After that,the strain genome was extracted,and the 16 S rDNA gene was amplified and sent for detection.The sequencing results were compared with known sequences to obtain strain species,and a phylogenetic tree was constructed.After screening,the six strains of high-yield collagenase were finally obtained from Bacillus,Acinetobacter,Enterobacter,Enterobacter,Aeromonas,and Achromobacter.Among them,Bacillus sp.H3 had the highest activity,which was(8.89±0.62)U/mL,so it was selected for subsequent tilapia skin fermentation test.(2)Process optimization of collagen preparation by microbial fermentationUsing tilapia skin as raw material,the seed liquid was prepared by using Bacillus sp.H3 strain with the highest enzyme activity,and collagen was prepared by fermentation.The effects of inoculum size,fermentation temperature,extraction time and feed water ratio on the extraction rate of collagen were investigated.Combined with the single factor test results,the four-factor three-level orthogonal test was designed to optimize the process conditions.The results showed that the optimum conditions for the preparation of collagen by microbial fermentation of tilapia skin were as follows: seed inoculum was 8%,fermentation temperature was 35 ?,extraction time was 24 h,and the volume of liquid was 45 mL.(3)Comparison of extraction rate and physicochemical properties of four collagensUsing tilapia skin as an experimental material,enzyme-soluble collagen,acid-soluble collagen,alkali-soluble collagen and water-soluble collagen were prepared by microbial fermentation,acid method,alkali method and hot water method.The extraction rate of collagen prepared by the four methods and various physicochemical properties were compared,including infrared spectrum scanning,ultraviolet spectrum scanning,amino acid analysis,solubility,water absorption,oil absorption,and water holding capacity.The results showed that the collagen extracted by the four methods had a complete triple helix structure,high purity and certain water absorption and oil absorption.The extraction rate of microbial fermentation is higher than other methods,and the amino acid composition is similar to the acid method and the hot water method.Moreover,the collagen extracted by the method has high solubility,water holding capacity and emulsifying property.(4)Effect of fish skin collagen on the viscosity and sensory evaluation of yogurtIn the experiment,collagen yoghurt was prepared by adding tilapia skin collagen to the traditional yoghurt making process.The effects of the amount of collagen added,the amount of white sugar added and the inoculum of the starter on the viscosity and sensory evaluation of collagen yoghurt were studied by single factor test and orthogonal test.The results showed that the amount of collagen had a significant effect on the viscosity and sensory evaluation of yogurt.Combined with the results of the orthogonal test,the optimal formula of collagen yoghurt was 1.5% collagen,6% white sugar,1fermentation inoculum,and the most yoghurt at 8 h at 43?..The collagen yoghurt prepared under this condition is fine and uniform,suitable for sweet and sour,and has a rich mouthfeel.