CN111254169B

CN111254169B - Application of escherichia coli CF16 in glutamic acid or sodium glutamate production

Info

Publication number: CN111254169B
Application number: CN201911397692.7A
Authority: CN
Inventors: 张国立; 郭永泽; 张国芝; 马洪梅
Original assignee: Guangzhou Shuguo Bio Co ltd
Current assignee: Guangzhou Shuguo Bio Co ltd
Priority date: 2019-12-30
Filing date: 2019-12-30
Publication date: 2023-05-12
Anticipated expiration: 2039-12-30
Also published as: CN111254169A

Abstract

The invention discloses application of escherichia coli CF16 in glutamic acid or sodium glutamate production. The invention discovers that Escherichia coli (CF 16) can efficiently produce glutamic acid, and can be applied to the production of glutamic acid and sodium glutamate. And contributes a basic resource for popularization of the sodium glutamate additive industry.

Description

Application of escherichia coli CF16 in glutamic acid or sodium glutamate production

Technical field:

the invention belongs to the field of microorganism application, and particularly relates to application of escherichia coli CF16 in glutamic acid or sodium glutamate production.

The background technology is as follows:

sodium glutamate is widely used as a natural additive in the fields of food, biological medicine, chemical synthesis and the like, and is very interesting in recent years. Sodium glutamate is known in life as monosodium glutamate. White needle-like crystal powder, an organic compound with carbon chain as skeleton. In the past, the production of monosodium glutamate is mainly carried out by a wheat gluten (glutelin) hydrolysis method, and the mass production is carried out by a microbial fermentation method instead, so that the screening of a proper strain is a precondition for producing sodium glutamate with over-hard quality.

The invention comprises the following steps:

the invention aims to provide an application of Escherichia coli (Escherichia coli) CF16 in efficiently producing glutamic acid.

Experiments show that the escherichia coli CF16 can ferment and produce glutamic acid. And glutamic acid reacts with sodium salt to produce sodium glutamate.

It is therefore a first object of the present invention to provide the use of E.coli CF16 for the production of glutamic acid or sodium glutamate.

A second object of the present invention is to provide a method for producing glutamic acid by fermentation culture using Escherichia coli CF16 as a fermentation strain, and isolating glutamic acid from the fermentation culture.

Preferably, escherichia coli CF16 is inoculated in a fermentation culture medium for culture, 15ml of molasses wastewater, 5g of peptone, 7g of sodium chloride, 1g of citric acid, 2g of ammonium chloride and 15g of agar are added into each liter of water of the fermentation culture medium, and the mixture is uniformly mixed, and the pH is adjusted to 7.6-8.2.

The culture condition is that the culture is carried out at 28 ℃.

The invention provides efficient and convenient strain resources from the perspective of resource screening under the background of lack of microbial resources for sodium glutamate product production under the research hot spot trend of microbial fermentation product utilization, and contributes basic resources for popularization of additive industry.

The invention discovers that Escherichia coli (CF 16) can efficiently produce glutamic acid, and can be applied to the production of glutamic acid and sodium glutamate. And contributes a basic resource for popularization of the sodium glutamate additive industry.

Coli CF16 of the present invention was deposited at the cantonese institute of microbiological bacterial strain (GDMCC), address: the collection number of the building 5 of the Guangzhou city first-violent Zhonglu No. 100 college No. 59 is: GDMCC 1.1478.

The specific embodiment is as follows:

the following examples are further illustrative of the invention and are not intended to be limiting thereof.

Example 1: isolation and identification of E.coli (Escherichia coli) CF16 Strain

1. Isolation of E.coli (Escherichia coli) CF16 Strain

Screening the culture medium:

TSA medium: 15g of tryptone, 5g of soybean peptone, 5g of sodium chloride, 15g of agar and 1000mL of distilled water, and uniformly mixing the above components, and adjusting the pH to 7.1-7.5;

the Escherichia coli (CF 16) strain is obtained by separating and purifying feces of physical examination people in southern Guangzhou hospital. The separation and purification method comprises the following steps:

1g of fecal sample was isolated and enriched in 100mL of TSA medium. The turbidity of the fermentation broth was checked during the enrichment process by different times. The enrichment culture is carried out at 37 ℃ for 1 day, and after obvious turbidity is observed, the culture is transferred to a newly prepared screening culture medium for continuous culture for 1 day under the same condition. Then, the mixed flora is taken from the culture solution of the enrichment culture in the previous step, is coated into a solid screening culture medium containing agar for culture in anaerobic working at 37 ℃, and the fast-growing colony is selected for purification and oil stain removal test, and finally, the strain CF16 with fast and efficient degradation capability on oil stains is obtained.

2. The physicochemical parameters of strain CF16 are as follows:

1. strain characterization

a. The strain CF16 is gram negative, rod-shaped, oxidase negative and can not move by adopting a conventional bacterial physiological and biochemical identification method and observation by an electron microscope.

b. After 20h of culture on TSA medium plates, the colony surface was beige, round and free of protrusions.

2. Main physicochemical properties of Escherichia coli CF 16:

the 16S rRNA gene sequence of the strain CF16 is sequenced, and BLAST comparison shows that the strain CF1616S rRNA gene sequence has higher similarity with the Escherichia coli existing model strain. Wherein the sequence similarity to Escherichia coli DSM30083T was 99%.

In view of the above physiological and biochemical characteristics and the 16S rRNA gene sequence results, the strain CF16 of the present invention should be assigned to Escherichia coli, named as Escherichia coli (Escherichia coli) CF16, deposited at the Guangdong province microorganism culture Collection (GDMCC) on month 07 of 2018, address: the collection number of the building 5 of the Guangzhou city first-violent Zhonglu No. 100 college No. 59 is: GDMCC 1.1478, a publicly shared strain, which anyone can purchase with the Guangdong province microorganism collection.

Example 2:

1. preparation of fermentation liquor:

inoculating Escherichia coli (Escherichia coli) CF16 into a 1L glutamic acid seed liquid culture medium, and culturing for 24 hours to obtain fermentation seed liquid. And then inoculating the fermentation seed liquid into a 5L fermentation medium for fermentation at the volume fraction of 8%, wherein the fermentation temperature is controlled at 28 ℃ and the fermentation time is 24 hours, so as to obtain the glutamic acid fermentation liquid.

The glutamic acid seed liquid culture medium and the fermentation culture medium are prepared from the following components: 15mL of molasses wastewater, 5g of peptone, 7g of sodium chloride, 1g of citric acid, 2g of ammonium chloride and 1000mL of distilled water, uniformly mixing the components, adjusting the pH to 7.6-8.2, and sterilizing to obtain the product.

2. Determination of glutamic acid

2.1 preparation of solutions

Preparing a glutamic acid standard solution: accurately weighing 0.2000g of dry glutamic acid, and fixing the volume to 100mL to obtain mother liquor.

Accurately sucking 10mL of mother solution, adding water to fix volume to 100mL to obtain 200 mug/mL of glutamic acid standard solution. Preparation of 2% ninhydrin solution: 1g of ninhydrin was dissolved in 35mL of hot water, 40mg of stannous chloride was added, the mixture was stirred well, placed in the dark, placed overnight, filtered, and then added with water to a volume of 50mL. Phosphate buffer solution: phosphate buffer solutions with pH of 5.0, 6.0, 7.0, 8.0 and 9.0 were prepared respectively.

2.2 sample treatment: centrifuging the liquid sample at 5000r/min for 15min, and removing impurities such as thalli. 5mL of supernatant is taken, an equal amount of 6mol/L hydrochloric acid solution is added into a colorimetric tube, and the colorimetric tube is put into a blast drying oven at 110 ℃ for hydrolysis for 24 hours. After hydrolysis, neutralization reaction is carried out by using 6mol/L NaOH solution, the pH is adjusted to about 7, and then the solution is poured into a 200mL volumetric flask for quantitative dilution.

2.3 Experimental methods

1mL of diluted sample is sucked into a 25mL colorimetric tube, a certain amount of phosphate buffer solution and ninhydrin solution are added, a proper amount of deionized water is added, and the mixture is placed into a water bath shaking table for heating. After cooling, the volume was set to 25mL and the absorbance was measured at 570 nm.

2.4 Standard Curve

Glutamic acid standard solution (200 mug/mL) 0.0, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0mL are placed in a 25mL volumetric flask, water is added to supplement the solution to the volume of 4mL, ninhydrin (20 g/L) and buffer solution with pH value of 8.04 are added into each 1mL, and the mixture is placed in a water bath shaker at 100 ℃ for heating for 20min. After cooling for 10min 25mL was filled with deionized water and the values were measured at 570nm in a spectrophotometer. And drawing a standard curve by taking the standard glutamic acid concentration as an abscissa and the absorbance A as an ordinate. As can be seen from table 1, the concentration of glutamic acid solution is linearly related to absorbance, and the regression equation thereof is y=0.0022x-0.0388, and the correlation coefficient r2= 0.9977.

TABLE 1

2.5 detection of the concentration of glutamic acid in fermentation broth

1mL of a fermentation medium without bacteria is taken as a control, placed in a 25mL volumetric flask, added with water to be supplemented to a volume of 4mL, added with ninhydrin (20 g/L) and 1mL of a pH 8.04 buffer solution respectively, and placed in a water bath shaker at 100 ℃ for heating for 20min. After cooling for 10min 25mL was filled with deionized water and a value of 0.0203 was measured at 570nm in a spectrophotometer. According to regression equation, 26.86. Mu.g of glutamic acid was contained in 1ml of the medium.

1mL of glutamic acid fermentation broth is taken, placed in a 25mL volumetric flask, added with water to be added to a volume of 4mL, added with ninhydrin (20 g/L) and 1mL of buffer solution with pH 8.04 respectively, and placed in a water bath shaker at 100 ℃ for heating for 20min. After cooling for 10min 25mL was filled with deionized water, and a value of 0.279 was measured at 570nm in the spectrophotometer. According to regression equation, 144.45 mug of glutamic acid was contained in 1ml of fermentation broth.

2mL of glutamic acid fermentation broth is taken, placed in a 25mL volumetric flask, added with water to be added to a volume of 4mL, added with ninhydrin (20 g/L) and 1mL of buffer solution with pH 8.04 respectively, and placed in a water bath shaker at 100 ℃ for heating for 20min. After cooling for 10min 25mL was filled with deionized water, and the value was 0.512 at 570nm in the spectrophotometer. According to regression equation, 125.18 mug of glutamic acid was contained in 1ml of fermentation broth.

3mL of glutamic acid fermentation broth is taken, placed in a 25mL volumetric flask, added with water to be added to a volume of 4mL, added with ninhydrin (20 g/L) and 1mL of buffer solution with pH 8.04 respectively, and placed in a water bath shaker at 100 ℃ for heating for 20min. After cooling for 10min 25mL was filled with deionized water and a value of 0.786 was measured at 570nm in the spectrophotometer. According to regression equation, 124.97 mug of glutamic acid was contained in 1ml of fermentation broth.

Claims

1. The application of the escherichia coli CF16 in the production of glutamic acid or sodium glutamate is that the escherichia coli CF16 is inoculated into a fermentation culture medium for culture, 15ml of molasses wastewater, 5g of peptone, 7g of sodium chloride, 1g of citric acid, 2g of ammonium chloride and 15g of agar are added into each liter of water of the fermentation culture medium, the mixture is uniformly mixed, the pH value is regulated to 7.6-8.2, and the escherichia coli CF16 is preserved in the microorganism strain preservation center of Guangdong province in the following preservation number in 2018 and 07 month 13: GDMCC 1.1478.

2. A method for producing glutamic acid, characterized in that Escherichia coli CF16 is used as a fermentation strain for fermentation culture, and glutamic acid is separated from the fermentation culture; inoculating escherichia coli CF16 into a fermentation culture medium for culture, adding 15ml of molasses wastewater, 5g of peptone, 7g of sodium chloride, 1g of citric acid, 2g of ammonium chloride and 15g of agar into each liter of water of the fermentation culture medium, uniformly mixing, adjusting pH to 7.6-8.2, and preserving the escherichia coli CF16 in a microorganism strain preservation center of Guangdong province in 2018 and 13 days, wherein the preservation number is: GDMCC 1.1478.

3. The method according to claim 2, wherein the culturing is performed at 28 ℃.