Study On The Dynamic Changes Of Metabolites During Neutral Protease Fermentation Process

Neutral proteinase produced by microbial fermentation is widely used in food, brewing, textile, chemical industry and so on. A comprehensive and in-depth study of the fermentation process is of great significance to reduce the production cost and increase the production of neutral proteinase. At present, the neutral protease for commercial use is manly fermented by Bacillus, of which Bacillus subtilis AS 1.398 is widely used in the industry.Selection of an appropriate sample pretreatment method is crucial to the quality of metabolomic analysis. Great Variability exists in terms of sample pretreatment techniques for microbial metabolomics in the current literature and thus it is uncertain of a suitable sample preparation procedure for metabolomic analysis of Bacillus subtilis. In this study, the dynamic changes of intracellular and extracellular metabolites has been analyzed and some useful conclusions had been provided. The main work are as follows:(1) To improve the quality of metabolomic analysis of B. subtilis based on GC-MS, the leakage of intracellular metabolites with five different quenching solvents was compared in this study, and 60% methanol/0.9% (NH4)2CO3 were found to have the least leakage; effects of different cell disruption methods including bead-milling, liquid nitrogen grounding and ultrasonication on the extraction of metabolites were compared. The bead-milling method was shown to have the best extraction efficiency. The effects of derivatization time (0.5-2.5 h) on relative abundance of metabolite distribution were also compared and derivatization for 2 and 2.5 h was shown to be adequate. The optimal sample pretreatment method for metabolomic analysis of B. subtilis was determined as follows:60% methanol/0.9% (NH4)2CO3 as quencher, glass bead breaker used for cell disruption and the derivatization time 2 h. With this method,223 metabolites were detected, including a large number of amino acids, organic acids and carbohydrates in B. subtilis.(2) Samples were gained during the fermentation process of B. subtilis AS 1.398 in LB medium every 2 hours and they were analyzed by GC-MS.123 and 104 kind of compounds had been analyzed for intracellular and extracellular metabolites respectively. The results of GC-MS were used for Principal Component Analysis (PCA). The PCA results showed that Alanine, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Proline, Tryptophan, Tyrosine, Valine, Threonine and Phenylalanine had a significant influence on both intracellular and extracellular metabolites.(3) Based on the previous results, samples were gained during the fermentation process of Bacillus subtilis AS 1.398 in fermentation medium every 6 hours and they were analyzed by GC-MS. The results of GC-MS were used for Principal Component Analysis (PCA). The PCA results also showed that Alanine, Glycine, Isoleucine, Leucine, Lysine, Methionine, Proline, Tryptophan, Threonine and Phenylalanine had a significant influence on both intracellular and extracellular metabolites.(4) According to the amino acid composition of soybean protein it was inferred that the amino acid provide in the medium is not equal to the demand of neutral proteinase synthesis. The neutral protease synthesis was limited by the inadequate supply of Methionine, Lysine, Glutamic acid, Histidine. Neutral protease was produced in shake flask scale based on the results of the study on the dynamic changes of metabolites during the neutral protease fermentation process by Bacillus subtilis AS1.398. The results showed that adding 2% Methionine the enzyme activity increased 67.3%. Adding 0.5% Lysine, Glutamic acid and Histidine, the enzyme activity increased 68.46%,27.8% and 16.18% respectively. It showed that the level of neutral protease synthesis is limited by the inadequate supply of amino acid.This study should facilitate the comprehensive metabolic analysis of this important industrial microbe. It contributed to the understanding of the neutral protease fermentation process, and provided an important theoretical basis for the rational improvement of the fermentation techonology.