| Microbial extraction of chitin is a method that uses crustacean waste as rawmaterials,removes proteins and minerals from raw materials and extracts chitin by using specific metabolites produced and accumulated in large quantities by microorganisms.The principle is to remove proteins in crustacean wastes by using protease-producing microorganisms,and to remove minerals in crustacean wastes by using acid-producing microorganisms.The principle is to remove protein in crustacean waste by using protease-producing microorganisms,and acid-producing microorganisms to remove minerals.Compared with the traditional chemical method,the microbiological method has the advantages of mild reaction conditions,low environmental pollution and low energy consumption.At the same time,it can avoid the hydrolysis of chitin due to the long-term acid-base action,and also makes it possible to recycle shrimp shells and other wastes.However,this method has the disadvantages of long time consumption,low efficiency and insufficient deproteinization and demineralization.Therefore,further improvement of the experiment is required before large-scale industrial production can be carried out.This article discusses the fermentation conditions and fermentation process of single-bacteria fermentation,successive two-step fermentation,ARTP mutagenesis fermentation and small fermentor fermentation to extract chitin.It provides a powerful reference value for the stepwise microbial fermentation process to treat shrimp shells and other wastes.The main research results of this paper are as follows:?1?After culturing,screening,and isolating bacteria from samples atdifferent stages of fermentation in traditional fermented shrimp paste,the lactic acid or protease-producing microorganisms were selected for isolation,purification,and identification,and their acid-producing ability or protease activity was identified.The strains determined for chitin extraction were Lactobacillus acidophilus?L.acidophilus?and Exiguobacterium profundum?E.profundum?.Two strains were used for the green extraction of chitin by microbial method.As a result,it was found that the fermentation of L.acidophilus removed 88.65%±2.20%of the minerals in the shrimp shell,and E.profundum fermentation removed 76.43%±0.92%of protein.The results showed that L.acidophilus was suitable for growth in a liquid medium with shrimp shells,and through low pH?about 3.5?and total acid production(about 2.0 g·mL-1),it was determined that L.acidophilus fermentation produced a large amount of lactic acid,which can be used in the process of shrimp shell demineralization.E.profundum can directly obtain carbon and nitrogen sources required for growth from protein hydrolysates.Shrimp shells could replace common medium components,reducing fermentation costs.?2?The results of successive two-step fermentation?L.acidophilusfermentation followed by E.profundum fermentation?showed that 85.94%±1.22%of the protein and 94.68%±3.25%of the minerals were removed from the sample,the chitin recovery was 47.82%,residual mineral content was 2.52%±0.25%,and residual protein content was 4.65%±0.58%.Characterization of chitin obtained by single-bacteria fermentation,successive two-step fermentation was performed by SEM,FT-IR and XRD.The deacetylation degree and crystallinity of the samples from the successive two-step fermentation were lower than single-fungus fermentation.?3?Through ARTP mutagenesis screening,the strain used for fermentationextraction of chitin was identified as MS 10017?Accession number was CCTCC M2019795?.Its protease activity is 36.39%higher than E.profundum.The fermented sample removed 91.48%±2.60%of protein and 99.84%±0.36%of minerals,and70.18%±2.68%of chitin was recovered.And the mutant strain can be used for multi-generation culture and fermentation without reducing protease activity.?4?The establishment of small-scale fermentor method under the optimalconditions of shake flask fermentation.After 72 h,the degree of deproteinization was about 91.60%,and the recovery of chitin was 83.22%±1.01%.After 72 h of fermentation,the degree of deproteinization could reach that of shake flask fermentation.Replacing the fresh water used in the fermentation process with sea water reduces the use of fresh water,while the use of chemical reagents is significantly reduced.The resulting protein-rich fermentation broth with high nutritional value has great potential for producing high-value proteins for human consumption.This study provides an effective way to make chitin from crustacean shell green. |
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