| ?-poly-L-lysine(?-PL),which usually consists of 25~30 residues of an essential amino acid L-lysine,is a homo-poly-amino acid characterized by the peptide bond between the carboxyl and ?-amino groups of L-lysine,and its molecular weight is approximately 3500~4500.?-PL has a wide range of antimicrobial activity,strong stability,safety and efficiency,and has been widely concerned at home and abroad.In 2014,?-PL was recognized as a new type of food additive in China.Although a small number of companies have realized small-scale ?-PL industrial production,with the expansion of application field and the rapid increase of demand,it is urgent to further promote the industrial production of ?-PL,so it is of profound significance to study ?-PL systematically.In this paper,starting from the screening of strains producing ?-PL,the ?-PL production performance of the screening strains was investigated through the preliminary fermentation of shake flasks and fermenters after identification of strains.Through the preliminary exploration of the separation and extraction process,the key constraints of extraction and separation are determined.It lays the foundation for the later breeding of bacteria and the extraction process optimization.The specific research content of this paper is as follows:(1)In order to obtain ?-PL producing strains,soil samples were collected to make the bacteria liquid,and the ?-PL resistant strains were obtained by using 2 g/L ?-PL medium plate and methylene blue transparent circle to screen the ?-PL resistant strains.Actinomycetes were enriched with complex antibiotic bacteriostatic agents,and actinomycetes producing ?-PL were screened with methylene blue transparent circle.Based on the special precipitation reaction of ?-PL and Dragendorff reagent,five strains producing ?-poly-L-lysine were obtained,and they were One strain of Bacillus amyloliquefaciens,one Bacillus cereus,one Streptomyces sp.,one Saccharopolyspora sp.and one Streptomyces albulus.(2)Five strains were fermented in flask,and the yield was 0.08 g/L,0.06 g/L,0.70 g/L,0.82 g/L,1.56 g/L,respectively.Fermentation was performed in 5 L fermentor with a yield of 6.99 g/L.The fermentation product was extracted,and analyzed by thin layer chromatography.The results of TLC analysis showed that hydrolysis products of ?-PL standards and ?-PL samples crudely extracted,and lysine standards,all have the same Rf value on the chromatographic plate.This proves that the fermentation product of Streptomyces albulus wzj5 is the polymer of lysine.(3)Three important steps in the extraction of ?-PL were optimized by single factor test,which were heated deproteinization,ion exchange and activated carbon decoloring.The optimal conditions for removal of miscellaneous proteins were pH 3.0,90 min,95 ?,and 80.18% of the hybrid proteins were removed by using the optimized conditions;The optimal conditions for ion exchange were as follows: initial pH 9.0,resin dosage 125 g/L,sample flow rate and elution flow rate is 3 ml/min.The average recovery of ?-PL was 77.19%;The optimum conditions of pigment removal were as follows: pH 4.0,75 ?,120 min,and the amount of activated carbon added to the solution was 2%(w/v),according to which the average yield of ?-PL was 92.18% and the average decolorization rate was 83.06%.The total recovery of ?-PL was 31.36%.Finally,the purified ?-PL solid powder was used to test the bacteriostasis of Escherichia coli,and the minimum inhibitory concentration is 0.25 mg/ml. |
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