Study On Preparation Of Chitin By Biological Fermentation

Chitin is the second largest biological resource in nature.Due to its excellent properties,such as biocompatibility and biodegradability,it has great potential in the fields of food,medicine and biomaterials.The current methods for extracting chitin are mainly chemical method and biological fermentation method.Although the extraction efficiency of chemical method is much faster than biological method,it consumes more energy and easily causes environmental pollution.Comparing to the chitin obtained by chemical method,the degree of acetylation of chitin prepared by biological method is higher than that of chemical method,and it has less environmental pollution.Although biological fermentation has the disadvantages of long fermentation cycle and low product purity,it has gradually become a research hotspot.This experiment mainly studied the extraction of chitin from shrimp shells by biological fermentation.The main contents were as follows: firstly,the proteolytic enzymes and acid-producing activity were used as the standard to screen bacteria with higher proteolytic and acid-producing activities.Secondly,using deproteinization rate(DP%)and demineralization rate(DM%)as the main measurement standards of the fermentation methods,the single-bacteria fermentation,the continuous two-step fermentation and the mixed-bacteria simultaneous fermentation on the preparation of chitin were explored.The specific contents were as follows:(1)The 9 bacteria with proteolytic enzymes activity and the 3 bacteria with acidproducing activity were screened from soil and yogurt powder.Then,the 3 acidproducing strains and the 3 strains with the highest protease activity selected from the 9protease strains were identified by 16 S r RNA.Based on the results of 16 S r RNA,the 3strains with the highest protease activity could be categorized as Bacillus mobilis strain,Bacillus zanthoxyli strain,Bacillus proteolytic strain respectively,and the 3 high acidproducing strains belonged to the same strain,which was Streptococcus thermophilus strain.(2)According to the DP% and DM%,the Bacillus zanthoxyli strain(named B2)and Streptococcus thermophilus strain(named L)were screened as the dominant strains.The B2 and L strains were be used in the single-strain fermentation.In the continuous two-step fermentation,the strain B2 was used in the first fermentation stage and the strain L was used in the second fermentation stage,and the fermentation process above was named B2 → L-C(C inferred the replacement of fresh broth in the second fermentation stage).In the mixed-bacteria simultaneous fermentation process,the B2 and L strain were inoculated in the same time,and the process above was named B2-L.Compared experiments results above,under the initial fermentation conditons,the B2single-strain fermentation(the DP% and DM% were 39.95% and 58.46%,respectively)and the continuous two-step fermentation of B2→L-C(the DP% and DM% were 31.53%and 68.00%,respectively)had more balanced results,the L single-strain fermentation and the mixed-bacteria simultaneous fermentation of B2-L had the highest DM%(75.59%and 79.89%,respectively).(3)Optimized the three fermentation processes.The optimized conditions of the single-bacterium fermentation method included: shrimp shell size(2.00-<0.2mm),carbon source(glucose,sucrose,maltose),nitrogen source(tryptone,yeast extract powder),inoculation amount(2%,4%,6%,8%,10%),temperature(25℃,30℃,35℃,40℃,45℃),p H(6,6.5,7,7.5,8)and carbon source content(2.5%,5%,7.5%,10%,12.5%).Based on the optimization of single-bacteria fermentation conditions,this study also carried out the optimization of the continuous two-step fermentation and the mixedbacteria simultaneous fermentation.In the continuous two-step fermentation,the fermentation conditions of B2 strain with the highest DP% was selected,and the fermentation conditions of L strain with highest DM% was used.Besides the singlestrain fermentation conditions,the optimization of the inoculation ratio and the carbon sources ratio were considered in the mixed-bacteria simultaneous fermentation.The final fermentation conditions were as follows.B2 strain: 7.5% sucrose,30℃,2%inoculum,p H 7.5,2% trypsin.L strain: 5% glucose,30℃,4% inoculum,p H 6.0,2%yeast extract powder.The group of B2-L: inoculation ratio 1:1(B2: L),carbon sources ratio 1:1(glucose: sucrose),4% inoculum,p H 7,7.5% carbon source content,30℃.The group of B2→L-C: the first stage(B2 strain: 2.5% sucrose,30℃,2% inoculum,p H 7.5),the second stage(L strain: 10% glucose,30℃,4% inoculum,p H 6.0,2% yeast extract powder).(4)Using the optimal fermentation conditions above,the final fermentation results were as follows.B2 strain: the DP% and DM% were 61.78% and 87.41%;L strain: the DP% and DM% were 19.55% and 89.19%;B2→L-C: the DP% and DM% were 76.64%and 57.57%;B2-L: the DP% and DM% were 68.89% and 83.80%.We believe that the the mixed-bacteria simultaneous fermentation(B2-L)has more balanced results,which has the potential to extract high-quality chitin.(5)In the simultaneous fermentation of mixed bacteria(B2-L),using the optimal size of shrimp shells,the final results of DP% and DM% were 83.76% and 91.48%,respectively.And the final degree of acetylation could be reached to 93.47%.The infrared spectra of the products prepared in this experiment was consistent with the characteristic functional groups of commercial chitin.These data indicated that chitin was successfully produced from shrimp shells using the method of the mixed-bacteria simultaneous fermentation.Comparing to single-bacteria fermentation and continuous two-step fermentation,the simultaneous fermentation of mixed bacteria can be used to extract chitin,which provides a feasible fermentation method for the large-scale production of chitin in the future.