阅读说明：本技术 一种产2’-岩藻基乳糖菌株最佳发酵条件的构建方法 (Construction method of optimal fermentation condition of strain for producing 2' -fucosyllactose ) 是由 胡学超 史静 于 2019-08-15 设计创作，主要内容包括：本发明涉及大肠杆菌菌株技术领域,本发明首先采用单因素水平实验,从发酵的转速,接种量,装液量,时间,温度,氮源,碳源,pH值以及乳糖浓度因素进行实验分析,得到最佳的单因素水平,然后选择有代表性的水平组合进行正交实验,分析,确定最佳发酵条件,发酵后,获得的2’-岩藻基乳糖的含量提高了2.5％,产量为3.5g/L,所得的2’-岩藻基乳糖与人乳中低聚寡糖2’-岩藻基乳糖结构以及性质相同,它作为营养强化剂,作为益生元类物质的来源之一,常用于婴儿配方食品,较大婴儿和幼儿配方食品,特殊医学用途婴儿配方食品。(The invention relates to the technical field of escherichia coli strains, firstly, a single-factor level experiment is adopted, the experiment analysis is carried out on the factors of the fermentation rotating speed, the inoculation amount, the liquid loading amount, the time, the temperature, the nitrogen source, the carbon source, the pH value and the lactose concentration to obtain the optimal single-factor level, then, a representative level combination is selected to carry out an orthogonal experiment and analysis to determine the optimal fermentation condition, the content of the obtained 2 '-fucosyllactose is improved by 2.5 percent after the fermentation, the yield is 3.5g/L, the structure and the property of the obtained 2' -fucosyllactose are the same as those of the oligosaccharide 2 '-fucosyllactose in human milk, and the 2' -fucosyllactose is used as a nutrition enhancer and is one of sources of prebiotic substances and is commonly used for infant formula foods, infant formula foods for larger infants and children and infant formula foods.)

1. A construction method of the optimal fermentation condition of a strain for producing 2' -fucosyllactose is characterized by comprising the following steps: (1) initial fermentation medium: 10g/L glycerol, 12g/L K2HPO4, 3g/L KH2PO4, 5g/L (NH4)2SO4, 0.1g/L NaCl, MgSO4 & 7H2O0.3g/L CaCl 2.2 H2O0.015g/L, FeSO4 & 7H 2O/sodium citrate, 15mL/L (7.5g/LFeSO4 & 7H2O plus 100g/L sodium citrate), 7.5 μ g/L thiamine, 1g/L yeast powder, 1mL/L metallic element (10g/L MnSO4 & nH2O, 4g/L CoCl2, 2g/LNa 2MoO4 & 2H2O, 2g/L pHO 4 & 7H2O, 1g/L LCuCl2 & 2H2O, 670.5 g/L BO 3. 3.);

(2) activating strains: collecting a preservation strain E.coli K-12 ZK-2 in a refrigerator at-80 ℃, inoculating the preservation strain into an LB culture medium, and activating overnight;

(3) fermentation: inoculating Escherichia coli E.coli K-12 ZK-2 bacterial liquid into a culture medium, fermenting in a fermentation shake flask, adding 0.1mM isopropyl thiogalactoside and lactose when OD600 value reaches 0.6, and inducing expression;

(4) single factor, multi-level experiment: A. rotational speed

5 rotation speed levels of 140rpm, 160rpm, 180rpm, 200rpm and 220rpm are respectively selected, an initial fermentation culture medium is adopted for carrying out a fermentation experiment, other factors are unchanged, and the yield of the 2' -FL is measured after the fermentation is carried out for 24 hours;

B. amount of inoculation

Respectively selecting 5 inoculation quantity levels of 0.5%, 1%, 1.5%, 2% and 2.5%, performing fermentation experiment by using an initial fermentation culture medium, measuring the OD600 of the initial fermentation culture medium every other hour to measure the growth condition of the initial fermentation culture medium, and measuring the yield of 2' -FL of the initial fermentation culture medium after 24 hours of fermentation;

C. liquid loading amount

Respectively selecting 10%, 20%, 30%, 40% and 50% of 5 liquid loading levels, performing fermentation experiments by adopting an initial fermentation culture medium, selecting the optimal rotation speed condition at the rotation speed, selecting the optimal inoculation quantity, keeping other factors unchanged, measuring the OD600 of the strain every other hour to measure the growth condition of the strain, and measuring the yield of 2' -FL of the strain after fermenting for 24 hours;

D. pH value

Adjusting the pH value of the culture medium to 3.0, 4.5, 6.0, 7.5 and 9.05 pH values by NaOH and HCI respectively, performing fermentation experiments by adopting an initial fermentation culture medium, selecting the optimal rotating speed condition by rotating speed, selecting the optimal inoculation amount by the inoculation amount, selecting the optimal liquid loading amount by the liquid loading amount, and keeping other factors unchanged. Measuring the OD600 of the strain at intervals of one hour in an initial growth stage to measure the growth condition of the strain, and then measuring the yield of 2' -FL of the strain after fermenting for 24 hours in a product generation stage;

E. time of fermentation

Selecting the optimal rotation speed condition, selecting the optimal inoculation amount, selecting the optimal liquid loading amount, selecting the optimal pH value as the pH value, and sampling every 10, 20, 30, 40 and 50 hours to measure the 2' -FL yield, wherein other factors are unchanged;

F. carbon source

Selecting three carbon sources of glucose, sucrose and glycerol, fermenting respectively according to the addition amount of 10g/L, selecting the medium-optimal rotating speed condition at the rotating speed, selecting the medium-optimal inoculation amount, selecting the medium-optimal liquid loading amount, selecting the medium-optimal pH value at the pH value, keeping other factors unchanged, measuring the OD600 of the medium-optimal liquid loading amount every other hour to measure the growth condition of the medium-optimal liquid loading amount, and measuring the yield of 2' -FL of the medium-optimal liquid loading amount after the optimal fermentation time;

G. nitrogen source

NH4NO3, (NH4)2SO4, NH4Cl, urea, peptone and yeast powder 6 nitrogen sources are fermented respectively according to the addition amount of 5g/L, the rotation speed selects the medium optimal rotation speed condition, the inoculation amount selects the medium optimal inoculation amount, the liquid loading amount selects the medium optimal liquid loading amount, the pH value selects the medium optimal pH value, the carbon source selects the medium optimal carbon source, other factors are unchanged, the OD600 is measured every one hour to measure the growth condition, and the 2' -FL yield is measured after the optimal fermentation time;

H. temperature of

Selecting 5 temperature gradients of 16 ℃, 22 ℃, 28 ℃, 34 ℃ and 40 ℃, selecting the medium-optimal rotating speed condition at the rotating speed, selecting the medium-optimal inoculation amount at the inoculation amount, selecting the medium-optimal liquid loading amount at the liquid loading amount, selecting the medium-optimal pH value at the pH value, selecting the medium-optimal carbon source at the carbon source, selecting the medium-optimal nitrogen source at the nitrogen source, keeping other factors unchanged, sampling after the optimal fermentation time, and measuring the yield of 2' -FL;

I. lactose concentration

Respectively selecting 5 temperature gradients of 1g/L, 2g/L, 3g/L, 4g/L and 5g/L, selecting the medium-optimal rotating speed condition by rotating speed, selecting the medium-optimal inoculation amount by inoculation amount, selecting the medium-optimal liquid filling amount by liquid filling amount, selecting the medium-optimal pH value by pH value, selecting the medium-optimal carbon source by carbon source, selecting the medium-optimal nitrogen source by nitrogen source, selecting the optimal temperature by temperature, and keeping other factors unchanged. After the optimal fermentation time, samples were taken and the 2' -FL yield was determined.

(5) Multifactor, multilevel experiments: selecting four factors with larger influence factors, namely a nitrogen source, time B, temperature C and lactose concentration D, designing three levels 1, 2 and 3, performing a group of orthogonal tests, and finally determining the optimal component.

2. The method for constructing the optimal fermentation condition of the strain for producing 2' -fucosyllactose as claimed in claim 1, wherein the liquid content of the culture medium in the fermentation of the E.coli K-12 ZK-2 strain of step (1) is controlled to 10%.

3. The method for constructing the optimal fermentation condition of the strain for producing 2' -fucosyllactose as claimed in claim 1, wherein the pH value of the culture medium of the E.coli K-12 ZK-2 strain of step (1) is controlled to be 7.2 during fermentation.

4. The method for constructing the optimal fermentation condition of the strain for producing 2' -fucosyllactose as claimed in claim 1, wherein the temperature for activating the E.coli K-12 ZK-2 strain in step (2) is controlled within 37 ℃.

5. The method for constructing the optimal fermentation condition of the strain for producing 2' -fucosyllactose as claimed in claim 1, wherein the temperature of the E.coli K-12 ZK-2 strain in the step (3) is controlled within 37 ℃ before the DO600 reaches 0.6.

6. The method for constructing the optimal fermentation condition of the strain for producing 2' -fucosyllactose as claimed in claim 1, wherein the fermentation broth of step (3) is inoculated into the culture medium in an amount of 1%.

7. The method for constructing the optimal fermentation conditions of the strain for producing 2 '-fucosyllactose as claimed in claim 1, wherein the final concentration of lactose to be added is set at 2g/L during the fermentation of the E.coli strain E.coli K-12 ZK-2 in step (3), so as to realize the production of 2' -fucosyllactose.

8. The method for constructing the optimal fermentation conditions of the strain for producing 2' -fucosyllactose as claimed in claim 1, wherein the fermentation time of the E.coli strain E.coli K-12 ZK-2 in step (3) is controlled within 24 hours.

9. The method for constructing the optimal fermentation conditions for the strain producing 2' -fucosyllactose as claimed in claim 1, wherein the rotation speed is adjusted to 120rpm when the E.coli strain E.coli K-12 ZK-2 in step (3) is fermented.

10. The method for constructing the optimal fermentation conditions of the strain for producing 2' -fucosyllactose as claimed in claim 1, wherein the initial fermentation medium and the culture conditions of the strain E.coli K-12 ZK-2 in step (4) are the same as those in step (3).

Technical Field

The invention relates to the technical field of escherichia coli strains, in particular to a construction method of an optimal fermentation condition of a strain for producing 2' -fucosyllactose.

Background

Breast milk is an important source of nutrition for infants, and among infant nutrition, milk powder is the third most abundant solid nutrient, in which Human Milk Oligosaccharides (HMO) are contained, which play a key role in infant health, and fucosylated HMO, including 2 '-fucosyllactose (2' -FL), 3-fucose (3-FL), lactoneon-tetraose (LNnT), etc., which selectively stimulate the growth of bifidobacteria, and can form analogues of pathogen receptors, thereby protecting infants against infection by intestinal pathogens, such as enteropathogenic escherichia coli, vibrio cholerae, and salmonella. Furthermore, fucosylated HMOs play a key role in inflammation and tumor metastasis like the Lewis antigen sugar chain structure. The ability to recognize and metastasize the structure of tumor cells has attracted considerable pharmaceutical interest in fucosylated HMOs, 2' -FL, where they inhibit bacterial or viral adhesion to epithelial cells due to their potential use in nutrition and health care.

Companies have obtained a method capable of producing 2' -fucosyllactose by genetic engineering methods, but the yield thereof is low, the production thereof is limited, and the cost is increased.

The development of metabolic engineering and fermentation microbiology provides possibility for improving the microorganism to synthesize 2 '-fucose lactose, and researches on the content of 2' -fucose lactose are improved by controlling the fermentation conditions and processes and preventing the contamination of the fermentation process.

Disclosure of Invention

In order to solve the problems in the background art, the invention aims to provide a construction method of the optimal fermentation condition of the strain for producing the 2' -fucosyllactose, which provides possibility for being used in infant formula milk powder in the future and can greatly reduce the production cost.

In order to solve the technical problems, the technical scheme provided by the invention is as follows: a construction method of the optimal fermentation condition of a strain for producing 2' -fucosyllactose is prepared by the following steps: (1) initial fermentation medium: 10g/L glycerol, 12g/L LK2HPO4, 3g/L KH2PO4, 5g/L (NH4)2SO4, 0.1g/L NaCl, MgSO 4.7H 2O.0.3 g/L CaCl 2.2H 2O.015 g/L, FeSO 4.7H 2O/sodium citrate, 15mL/L (7.5g/LFeSO 4.7H 2O plus 100g/L sodium citrate), 7.5 μ g/L thiamine, 1g/L yeast powder, 1mL/L metallic elements (10g/L MnSO 4. nH2O, 4g/L CoCl2, 2g/L Na2MoO 4. multidot.2H 2 BO O, 2g/L ZnSO 4.7H 2O, 1g/L CuCl 2. multidot.2H 2 BO, 0.5g/L Na2MoO 4. multidot. O, pH 593.593 g/L592 BO, pH 9);

(2) activating strains: collecting a preservation strain E.coli K-12 ZK-2 in a refrigerator at-80 ℃, inoculating the preservation strain into an LB culture medium, and activating overnight;

(3) fermentation: inoculating Escherichia coli E.coli K-12 ZK-2 bacterial liquid into a culture medium, fermenting in a fermentation shake flask, adding 0.1mM isopropyl thiogalactoside and lactose when OD600 value reaches 0.6, and inducing expression;

(4) single factor, multi-level experiment: A. rotational speed

5 rotation speed levels of 140rpm, 160rpm, 180rpm, 200rpm and 220rpm are respectively selected, an initial fermentation culture medium is adopted for carrying out a fermentation experiment, other factors are unchanged, and the yield of the 2' -FL is measured after the fermentation is carried out for 24 hours;

B. amount of inoculation

Respectively selecting 5 inoculation quantity levels of 0.5%, 1%, 1.5%, 2% and 2.5%, performing fermentation experiment by using an initial fermentation culture medium, measuring the OD600 of the initial fermentation culture medium every other hour to measure the growth condition of the initial fermentation culture medium, and measuring the yield of 2' -FL of the initial fermentation culture medium after 24 hours of fermentation;

C. liquid loading amount

Respectively selecting 10%, 20%, 30%, 40% and 50% of 5 liquid loading levels, performing fermentation experiments by adopting an initial fermentation culture medium, selecting the optimal rotation speed condition at the rotation speed, selecting the optimal inoculation quantity, and keeping other factors unchanged. Measuring the OD600 of the fermented soybean every other hour to measure the growth condition of the fermented soybean, and measuring the 2' -FL yield of the fermented soybean after 24 hours of fermentation;

D. pH value

Adjusting the pH value of the culture medium to 3.0, 4.5, 6.0, 7.5 and 9.05 pH values by NaOH and HCI respectively, performing fermentation experiments by adopting an initial fermentation culture medium, selecting the optimal rotating speed condition by rotating speed, selecting the optimal inoculation amount by the inoculation amount, selecting the optimal liquid loading amount by the liquid loading amount, and keeping other factors unchanged. Measuring the OD600 of the strain at intervals of one hour in an initial growth stage to measure the growth condition of the strain, and then measuring the yield of 2' -FL of the strain after fermenting for 24 hours in a product generation stage;

E. time of fermentation

Selecting the optimal rotation speed condition, selecting the optimal inoculation amount, selecting the optimal liquid loading amount, selecting the optimal pH value as the pH value, and sampling every 10, 20, 30, 40 and 50 hours to measure the 2' -FL yield, wherein other factors are unchanged;

F. carbon source

Selecting three carbon sources of glucose, sucrose and glycerol, fermenting respectively according to the addition amount of 10g/L, selecting the medium-optimal rotation speed condition at the rotation speed, selecting the medium-optimal inoculation amount at the inoculation amount, selecting the medium-optimal liquid loading amount at the liquid loading amount, selecting the medium-optimal pH value at the pH value, and keeping other factors unchanged. Measuring the OD600 of the fermented soybean every other hour to measure the growth condition of the fermented soybean, and measuring the yield of 2' -FL after the optimal fermentation time;

G. nitrogen source

NH4NO3, (NH4)2SO4, NH4Cl, urea, peptone and yeast powder 6 nitrogen sources are fermented respectively according to the addition amount of 5g/L, the rotation speed is selected from the medium optimal rotation speed condition, the inoculation amount is selected from the medium optimal inoculation amount, the liquid loading amount is selected from the medium optimal liquid loading amount, the pH value is selected from the medium optimal pH value, the carbon source is selected from the medium optimal carbon source, other factors are unchanged, the OD600 is measured every one hour to measure the growth condition, and the 2' -FL yield is measured after the optimal fermentation time;

H. temperature of

Selecting 5 temperature gradients of 16 ℃, 22 ℃, 28 ℃, 34 ℃ and 40 ℃, selecting the medium-optimal rotating speed condition at the rotating speed, selecting the medium-optimal inoculation amount at the inoculation amount, selecting the medium-optimal liquid loading amount at the liquid loading amount, selecting the medium-optimal pH value at the pH value, selecting the medium-optimal carbon source at the carbon source, selecting the medium-optimal nitrogen source at the nitrogen source, keeping other factors unchanged, sampling after the optimal fermentation time, and measuring the yield of 2' -FL;

I. lactose concentration

Respectively selecting 5 temperature gradients of 1g/L, 2g/L, 3g/L, 4g/L and 5g/L, selecting the condition of the rotation speed to be the optimal rotation speed, selecting the inoculation amount to be the optimal inoculation amount, selecting the liquid loading amount to be the optimal liquid loading amount, selecting the pH value to be the optimal pH value, selecting the carbon source to be the optimal carbon source, selecting the nitrogen source to be the optimal nitrogen source, selecting the optimal temperature to be the temperature, keeping other factors unchanged, sampling after the optimal fermentation time, and measuring the yield of 2' -FL.

(5) Multifactor, multilevel experiments: selecting four factors with larger influence factors, namely a nitrogen source, time B, temperature C and lactose concentration D, designing three levels 1, 2 and 3, performing a group of orthogonal tests, and finally determining the optimal component.

As a modification, the liquid content of the culture medium in the fermentation of the E.coli K-12 ZK-2 strain in the step (1) is controlled to be 10%.

As an improvement, the pH value of the culture medium of the E.coli K-12 ZK-2 strain in the step (1) is controlled to be 7.2 during fermentation.

As an improvement, the temperature for activating the E.coli K-12 ZK-2 strain in the step (2) is controlled within 37 ℃.

As an improvement, the temperature of the E.coli K-12 ZK-2 strain in the step (3) is controlled within 37 ℃ before the DO600 reaches 0.6.

As an improvement, the zymophyte liquid in the step (3) is inoculated into a culture medium according to the inoculation amount of 1%.

As an improvement, when the escherichia coli strain e.coli K-12 ZK-2 in the step (3) is fermented, the final concentration of lactose to be added is set at 2g/L, and the production of 2' -fucosyllactose can be realized.

As an improvement, the fermentation time of the Escherichia coli strain E.coli K-12 ZK-2 in the step (3) is controlled within 24 hours.

As an improvement, when the Escherichia coli strain E.coli K-12 ZK-2 in the step (3) is fermented, the rotation speed is adjusted to 120 rpm.

As an improvement, when the escherichia coli strain E.coli K-12 ZK-2 in the step (4) is fermented, the initial fermentation medium and the culture conditions are consistent with those in the step (3).

The invention has the beneficial effects that: the 2' -fucosyllactose is prepared by adopting the escherichia coli strain E.coli K-12 ZK-2, and the shake flask yield is increased from 1g/L to 3.5g/L through the optimization of fermentation conditions, so that the possibility is provided for being used in infant formula milk powder in the future, and the production cost can be greatly reduced.

Drawings

FIG. 1 is an experimental grouping of the multifactor, multi-level experiment of the present invention.

Detailed Description

The invention is illustrated below by means of specific examples, without being restricted thereto.