The High Expression Of Umami Peptide BMP And Its High Density Fermentation

Umami peptide,as an important base material for condiment,is a typical green and safe peptide flavor enhancer,endowing a unique flavor in food.It is an issue to obtain new umami peptides for the further development of industry of protein flavor enhancer.However,the related researches about umami peptides have been focusing on the enzymatic abstraction and chemical synthesis.To solve the problems of low purity and potential toxicity of chemical reagents caused by the traditional methods,new method based on a biological aspect was expected.Beefy Meaty Peptide（BMP）was an octopeptide with umami taste isolated from beef hydrolysate.It has been proved that BMP has a good synergistic effect with salt and monosodium glutamate,which could enhance the beef flavor.Furthermore,it has good thermal stability which could meet the heat treatment requirements in food industry.In the light of the above problems,this paper chosen the identified BMP and its multiple copies sequence as the candidates from the perspective of food biotechnology.It combined the molecular docking,cloning expression and high density fermentation,systematically carries out the molecular docking studies of the umami peptide and umami taste receptor（hT1R1/T1R3）to research the interaction between 8 BMP and hT1R1/T1R3.Based on the molecular docking results,we chosen eight copies sequence of BMP（8 BMP）as the research object,using genetic engineering method to construct a food safety grade recombinant strain Bacillus subtilis168/pMA0911-8BMP,and compared the taste difference of the 8 BMP purification solution and the 8 BMP Maillard reaction solution though the sensory evaluation.Finally,the exponential flow rate was used to obtain umami peptide 8 BMP though the high density fermentation technology.In this paper,the structure of single BMP and eight copy number BMP were optimized by Discovery Studio software based on the molecular simulation.Then SWISS-MODEL software was used to construct the model of the human umami taste receptor hT1R1/T1R3（hT1R1,Human taste receptor type 1 member 1）,and found the Venus Fly Trap Domain（VFTD）,which was the active pocket of BMP and its umami taste receptor hT1R1/T1R3.Finally,the interaction between BMP and hT1R1/T1R3 receptor was studied though molecular docking.The results showed that both the single BMP and 8 BMP could interact with the key amino acids on VFTD,and 8 BMP has more H-bond numbers with the umami receptor,leading to more effective umami taste.In order to express and purify the umami peptide BMP effectively,eight copies BMP was selected as the research object in this paper.To avoid the potential safety problem of genetic engineering bacteria in food process,the food safety grade bacteria B.subtilis 168 was used as the host bacteria of expression vector.The gene sequence of the 8 BMP was first optimized in Bacillus subtilis（B.subtilis）and the optimized gene fragment was inserted into the plasmid vector pMA0911 by double enzyme cutting method,followed by the expression vector pMA0911-8BMP by agarose gel electrophoresis.Then,pMA0911-8BMP vector was transformed into the food safety grade engineering bacteria B.subtilis 168.The results showed that the recombinant strain B.subtilis168/pMA0911-8BMP was successfully constructed though the resistance screening and protein electrophoresis.Finally,the culture conditions of the recombinant bacteria in the shaking bottle were optimized though the single factor tests.The results showed that the highest expression of umami peptide is 0.16 g/L on the condition of 37℃,7.5 pH and 2:1 C/N for 30 h.Finally,the sensory evaluation of the 8 BMP purification solution and the 8 BMP Maillard reaction solution were compared.The results showed that both of them had a good umami taste,and the umami taste of the product of 8 BMP mallard reaction could be significantly improved,after the mallard reaction of 8 BMP and the substances,such as salt and xylose.The fermentation liquid was inoculated into the 5 L fermenting tank for high density fermentation which was obtained from the shaking bottle.The formula of the exponential flow rate of the recombinant bacteria at the feeding stage was obtained from the balance equation of the restrictive substrate and Monod dynamic equation.The results showed that the bacterial community and 8 BMP expression products by the exponential feeding method could obtain with OD₆₀₀ 90,45 g/L（by dry cell weight,DCW）and 2.56 g/L 8 BMP.