阅读说明：本技术 一种2’-岩藻糖基乳糖的分批发酵方法 (Batch fermentation method of 2' -fucosyllactose ) 是由 王硕 王远一飞 于 2021-01-27 设计创作，主要内容包括：本发明公开了一种2’-岩藻糖基乳糖的分批发酵方法,该方法在原有基础上更适用于大规模发酵,生产效率和最终产量更高,更易于工业扩大培养,具有较高的应用前景和经济价值。本发明使用的发酵培养基组分简单,无有毒有害成分,经济廉价且易于获得,并且能得到较优的发酵效果和较高的2’-岩藻糖基乳糖产量。(The invention discloses a batch fermentation method of 2' -fucosyllactose, which is more suitable for large-scale fermentation on the original basis, has higher production efficiency and final yield, is easier for industrial expanded culture, and has higher application prospect and economic value. The fermentation medium used in the invention has simple components, no toxic and harmful components, economy, low price and easy acquisition, and can obtain better fermentation effect and higher 2' -fucosyllactose yield.)

1. A batch fermentation method of 2' -fucosyllactose, which is characterized in that: the method comprises the following steps:

1) preparing first-stage seed fermentation liquor: inoculating-80% glycerol strain Escherichia coli frozen in refrigerator into LB culture medium at volume percentage of 1-5%, and shake culturing at 37 deg.C and 220rpm for 4-8 hr;

2) preparing secondary seed fermentation liquor: inoculating the first-stage seed fermentation liquid into LB culture medium according to the inoculum size of 5-10%, and performing shake cultivation at 37 ℃ and 220rpm for 3-6 h;

3) batch fermentation: after the culture medium is prepared and sterilized, the secondary seed fermentation liquid is measured according to 10-20 percent of inoculation amount and inoculated, the mixture is stirred at the temperature of 37 ℃ and the speed of 400rpm, the aeration is 0.8-1.5vvm, the pH is constant and 7.0, 0.2mM of inducer is added after the mixture is fermented for 4-6h, then the temperature is set to be 30 ℃, the stirring speed is reduced to 400rpm at the temperature of 300 ℃, the aeration is reduced to 0.5-1vvm, the substrate lactose is added to 10-15g/L after the inducer is added for 2-4h, the antifoaming agent is added to 0.05mL/L, and the fermentation is finished after the continuous fermentation for 96 h.

2. A batch fermentation process of 2' -fucosyllactose according to claim 1, wherein: the batch fermentation is done in shake flasks or fermentors.

3. A batch fermentation process of 2' -fucosyllactose according to claim 1, wherein: the inducer is IPTG.

4. A batch fermentation process of 2' -fucosyllactose according to claim 1, wherein: the Escherichia coli is Escherichia coli E.

5. A batch fermentation process of 2' -fucosyllactose according to claim 1, wherein: the defoaming agent is polydimethylsiloxane.

6. A batch fermentation process of 2' -fucosyllactose according to claim 1, wherein: the specific formula of the culture medium prepared in the step (3) in batch fermentation is as follows:

wherein the yeast extract powder, peptone, glucose, MgSO₄、CaCl₂Methionine needs to be separately consumed and then fed into the fermentation tank.

7. A batch fermentation process of 2' -fucosyllactose as claimed in claim 6, wherein: the trace element solution was as follows:

8. a batch fermentation process of 2' -fucosyllactose as claimed in claim 6, wherein: the metal solutions were as follows:

9. the batch fermentation method of 2 '-fucosyllactose according to claim 1, wherein 4-10g/L of 2' -fucosyllactose is obtained.

Technical Field

The invention belongs to the field of microbial fermentation, and particularly relates to a fermentation tank batch fermentation method of 2' -fucosyllactose.

Background

2' -Fucosyllactose (2 ' -Fucosyllactose, 2' -FL) is the most abundant one of breast milk oligosaccharides, and has a molecular weight of 488.44 Da. It is a very special natural trisaccharide with fucose residue and galactose end of lactose connected by alpha 1-2 glycosidic bond. 2 '-FL is a neutral oligosaccharide which can resist the digestion of enzymes in the gastrointestinal tract of a human body, so that the 2' -FL can directly reach the colon part of the human body, is digested and decomposed by microorganisms in the intestinal tract of the human body, promotes the proliferation of related beneficial bacteria and plays a role of prebiotics.

In recent years, a great deal of research shows that oral administration of 2' -FL can regulate the immune function of suckling mice, reduce inflammation, enhance the intestinal barrier function, reduce anaphylactic reaction and even improve the cognitive ability of the mice. In addition, studies have shown that 2' -FL has a protective effect against pathogen infection. Furthermore, in 2015 and 2016, the FDA and the european union also approved the addition of 2' -FL to infant formula, even as a dietary supplement and a new food product. Commercially, there are also many companies that have added 2' -FL to infant formulas in an attempt to close the gap between the formula and breast milk. However, since 2' -FL is found only in large amounts in breast milk and in trace or no amounts in other mammals, it can only be obtained by artificial synthesis, it is expensive and safety-critical. Therefore, secure mass acquisition of 2' -FL is a hot spot that has been explored throughout the industry. Currently, there are three main synthetic routes for 2' -FL, chemical synthesis, enzymatic synthesis and microbial synthesis. Craft et al chemically synthesized a variety of breast milk oligosaccharides including 2' -FL in 2017, but the steps were tedious, byproducts were complex, and the yield was low; alberman et al have first synthesized 2 '-FL by enzyme method in 2001, Choi et al have synthesized 2' -FL from mannose by two-enzyme method, but the production conditions are harsh, and the production of tool enzyme itself needs complicated production process and is expensive; compared with other production methods, the 2' -fucosyllactose produced by microbial synthesis has high yield, is easy to expand and is safer, 2' -FL is produced by Lee and other groups at home and abroad by using escherichia coli in 2012 and has the yield as high as 1.2g/L, and in recent years, the yield is reported to be higher than that by other groups at home and abroad, but the yield of 2' -FL needs a large rise space compared with the yield of hundreds of grams per liter of other functional polysaccharides.

For the biological synthesis of 2' -FL, there are a large number of other engineered bacterial systems in addition to the E.coli system. Yu equals 2018 and 2' -FL is also produced by Saccharomyces cerevisiae with a yield of 0.5 g/L. In addition, there is also the production of 2' -FL by constructing Bacillus subtilis and Corynebacterium glutamicum, but the production is far inferior to that of E.coli because of its complex and unstable system. Therefore, in terms of the current technology, the exploration of an Escherichia coli fermentation process for efficiently producing 2 '-FL is extremely necessary for the production application and scientific research of 2' -FL.

Disclosure of Invention

In brief, the present invention generally discloses a fermenter batch fermentation method for producing 2' -fucosyllactose using e.coli-XYY1, which is simple and easy to operate, has stable yield, and has higher and more stable yield than the existing methods. In order to achieve the purpose, the invention provides the technical scheme that:

a batch fermentation method of 2' -fucosyllactose comprises the following steps:

1) preparing first-stage seed fermentation liquor: inoculating-80% glycerol strain Escherichia coli frozen in refrigerator into LB culture medium at volume percentage of 1-5%, and shake culturing at 37 deg.C and 220rpm for 4-8 hr;

2) preparing secondary seed fermentation liquor: inoculating the first-stage seed fermentation liquor into an LB culture medium according to the inoculation amount of 5-10% of the volume percentage, and performing shake culture at 37 ℃ and 220rpm for 3-6 h;

3) batch fermentation: after the culture medium is prepared and sterilized, the secondary seed fermentation liquid is measured according to the inoculation amount with the volume percentage of 10-20% and inoculated, the mixture is stirred at the temperature of 37 ℃ and the speed of 400 plus rpm, the aeration is 0.8-1.5vvm, the constant pH is 7.0, (the acid alkali liquid is 25% ammonia water and the acid liquid is 10% phosphoric acid) are automatically added through the constant pH mode of a fermentation tank), the inducer is added after the mixture is fermented for 4-6h, the concentration of 0.2mM, the temperature is set to 30 ℃, the stirring speed is reduced to the speed of 300 plus rpm and the aeration is reduced to 0.5-1vvm, the substrate lactose is added to 10-15g/L after the inducer is added for 2-4h, the antifoaming agent is added to 0.05mL/L, and the fermentation.

Preferably, the batch fermentation is performed in a shake flask or a fermentor.

Preferably, the inducer is IPTG.

Preferably, the Escherichia coli is E.coli-XYY 1.

Preferably, the defoamer is polydimethylsiloxane.

A batch fermentation method of 2' -fucosyllactose comprises the following specific formula of a culture medium prepared in batch fermentation:

wherein the yeast extract powder, peptone, glucose, MgSO₄、CaCl₂Methionine needs to be separately consumed and then fed into the fermentation tank.

Preferably, the solution of trace elements is as follows:

preferably, the metal solution is as follows:

an object of the present invention is to provide a medium composition for de novo production of 2' -fucosyllactose using e.coli-XYY 1. coli-XYY1 in a fermenter through a de novo route to produce 2' -fucosyllactose, but not limited to a specific species or a specific fermenter. This method can be used for the production of 2' -fucosyllactose by fermentation of all E.coli in a fermenter via the de novo route, with expansion. The method can be used for producing 2' -fucosyllactose by fermentation from the de novo route by using Escherichia coli. Glucose is one of the only carbon source and substrate for this strain in the medium, NH₄Cl is the only nitrogen source, and the change of the content of Cl has great influence on the growth of thalli and the yield of products. Both too much and too little of the resulting product will be reduced in amount.

Another two objects of the present invention are to provide a fermenter batch fermentation method for producing 2' -fucosyllactose de novo using e.coli-XYY 1. The medium used in the fermentation process is the above-described medium components of the present invention, but is not limited to specific medium components or specifications of a specific fermentor. On the other hand, this method is suitable for all batch fermentations using E.coli-XYY1 for de novo production of 2' -fucosyllactose. On the other hand, this method is suitable for all batch fermentations in which 2' -fucosyllactose is first produced using E.coli. The method is suitable for all fermentations which utilize Escherichia coli to produce 2' -fucosyllactose from the head.

Compared with the prior art, the invention has the advantages that:

1) the fermentation medium used in the invention has simple components, no toxic and harmful components, economy, low price and easy acquisition, and can obtain better fermentation effect and higher 2' -fucosyllactose yield.

2) The fermentation conditions used by the invention are simple and easy to implement and easy to expand, and the 2 '-fucosyllactose with 4-10g/L can be obtained by matching with the culture medium formula of the invention, wherein the fermentation effect is better and the yield of the 2' -fucosyllactose is higher. The concentration difference obtained by different strains and different fermentation methods is huge, the concentration of the product can be ensured to be 4-10g/L, the concentration is stable, and the method has high economic value.

3) The methionine, the triton and the tween-80 added in the invention are not seen in the original fermentation method, the inhibition effect of acetic acid generated by a strain in the fermentation process on the growth of the strain can be reduced by adding the methionine, and the transport of a product from the inside to the outside of a cell can be accelerated by adding the triton and the tween-80. And slowing the fermentation agitation rate during the induction fermentation stage, all of which improve the yield of the fermented product.

Description of the drawings:

FIG. 1 is a liquid chromatogram of a fermentation liquid obtained by an original fermentation method and a fermentation liquid obtained by the fermentation method of the present invention under a differential detector

FIG. 2 is a graph of different NH₄Effect of Cl concentration on 2' -fucosyllactose production

FIG. 3 is a graph of the effect of different glucose concentrations on the production of 2' -fucosyllactose

FIG. 4 is a graph of the effect of different inoculum sizes on the production of 2' -fucosyllactose

FIG. 5 is a graph of the effect of different agitation rates on the production of 2' -fucosyllactose

FIG. 6 is a graph of the effect of different aeration volumes on 2' -fucosyllactose production

FIG. 7 is the effect of different IPTG addition times on 2' -fucosyllactose production

FIG. 8 is the effect of different induction temperatures on the production of 2' -fucosyllactose

FIG. 9 is a graph of the effect of different methionine concentrations on the production of 2' -fucosyllactose

FIG. 10 is a graph of the effect of different aeration on 2' -fucosyllactose production during the induction fermentation stage.

Detailed Description

The present invention will be further described with reference to the following specific examples, but the present invention is not limited to these examples. All those skilled in the art can adapt and apply the invention to the needs of their own process.

In the examples, the 2 '-fucosyllactose was quantified by external standard liquid chromatography using a Waters Binary HPLC Pump 1525-Detector 2414/2489 column chromatography using Phenomenex Rezex ROA-Organic Acid H + (8%), and the 2' -fucosyllactose standard was 98% of Japanese Glycaro standard.

Example 1:

the fermentation operation was carried out using a displacement fermenter of 5L volume, and the liquid loading was 3L.

1) Preparing first-stage seed fermentation liquor: the glycerol strain E.coli-XYY1 frozen in a-80 refrigerator was inoculated in 50mL LB medium at 2% inoculum size and shake-cultured at 37 ℃ and 220rpm for 8 h.

2) Preparing secondary seed fermentation liquor: inoculating 30mL of the first-order seed fermentation liquid into 300mL of LB culture medium, and performing shake culture at 37 ℃ and 220rpm for 4 hours.

3) Preparing a fermentation tank culture medium: na was added to a 5L pot₂HPO₄·12H₂O 51.3g，NaCl 1.5g，KH₂PO₄9 g，NH₄Cl 12g, triton 0.3mL, tween-800.3 mL, diluting to 2L with deionized water, and standing at 121 deg.CThe mixture was sterilized in a sterilizer for 15min and then cooled to 37 ℃. In addition, MgSO is prepared₄72mg in 10mL deionized water, CaCl₂13.5mg in 10mL deionized water, 48g glucose in 500mL deionized water, 0.75g yeast extract powder in 100mL deionized water, and 1.5g peptone in 100mL deionized water, respectively sterilizing in a high temperature sterilization pot at 121 ℃ for 15min, cooling to 37 ℃, and aseptically adding into a fermentation tank. In addition, 150mg of methionine is taken in 10mL of deionized water, 3mL of vitamin solution and 3mL of metal solution are taken, and the vitamin solution and the metal solution are respectively filtered by a 0.22 mu m water system filter membrane for sterilization and then are added into a fermentation tank.

The vitamin solution needs to be prepared in advance, and the formula is as follows: weighing VB-x 120mg and VB₃ 8mg，VB₆ 1.5mg，VB₁₂ 5mg，VB₇ 1mg，VB₅ 5mg，VB₁ 10mg，VB₂ 2.5mg，V_B91mg of lipoic acid and 2.5mg of lipoic acid are dissolved in 100mL of deionized water and stored at 4 ℃ in the dark. The metal solution needs to be prepared in advance, and the formula is as follows: weighing FeCl₃·6H₂O 250mg，ZnCl₂ 20mg，H₃BO₃ 5mg，Na₂MoO₄·2H₂O 20mg，CuSO₄·5H₂O 20mg，CoSO₄·7H₂O10 mg was dissolved in 100mL of deionized water and stored at 4 ℃ in the dark.

4) Batch fermentation: inoculating 300mL of secondary seed fermentation broth into a fermentation tank, stirring at 37 ℃ and 500rpm, ventilating for 1vvm, keeping the pH constant at 7.0 (the alkali solution is 25% ammonia water, and the acid solution is 10% phosphoric acid), fermenting for 4h, adding 6mL of sterile 0.1M IPTG, setting the temperature at 30 ℃, reducing the stirring speed to 300rpm, and reducing the ventilation amount to 0.6 vvm. Adding 300mL of 10% sterile lactose solution after 2h, adding 0.2mL of defoaming agent polydimethylsiloxane, continuously fermenting for 96h, unloading the tank, and detecting the components by liquid chromatography, wherein the concentration of the 2' -fucosyllactose is 4.17 g/L.

Example 2:

the fermentation operation was carried out using an in situ fermentation tank having a capacity of 30L, and the liquid loading was 20L.

1) Preparing first-stage seed fermentation liquor: the glycerol strain E.coli-XYY1 frozen in a-80 refrigerator was inoculated in 100mL LB medium at 5% inoculum size and shake-cultured at 37 ℃ and 220rpm for 6.5 h.

2) Preparing secondary seed fermentation liquor: taking 100mL of first-order seed fermentation liquid, inoculating the first-order seed fermentation liquid into 2L of LB culture medium, and carrying out shake cultivation at 37 ℃ and 220rpm for 6 h.

3) Preparing a fermentation tank culture medium: adding Na into a 30L tank₂HPO₄·12H₂O 342g，NaCl 10g，KH₂PO₄60g，NH₄80g of Cl, 2mL of triton and 802mL of Tween, diluting to 13L with deionized water, sterilizing with high-temperature steam at 121 ℃ for 15min, and cooling to 37 ℃. In addition, MgSO is prepared₄480mg in 10mL deionized water, CaCl₂90mg in 10mL of deionized water, 320g of glucose in 1.6L of deionized water, 5g of yeast extract powder in 500mL of deionized water, and 10g of peptone in 500mL of deionized water, sterilizing in a high-temperature sterilization pot at 121 ℃ for 15min, cooling to 37 ℃, and then adding into a fermentation tank in an aseptic operation. In addition, 1g of methionine was added to 100mL of deionized water, 20mL of vitamin solution and 20mL of metal solution were added to the fermentor after they were sterilized by 0.22 μm aqueous membrane, respectively. The vitamin solution and the metal solution were prepared in advance, and the formulation was the same as in example 1.

4) Batch fermentation: inoculating 2L of secondary seed fermentation broth into a fermentation tank, stirring at 37 ℃ and 600rpm, ventilating for 1.5vvm, keeping pH constant at 7.0 (alkaline solution is 25% ammonia water, and acid solution is 10% phosphoric acid), fermenting for 6h, adding 20mL of sterile 0.1M IPTG, setting the temperature at 30 ℃, reducing the stirring speed to 400rpm, and reducing the ventilation to 0.6 vvm. And 2L of 10% sterile lactose solution is added after 2h, 1mL of defoaming agent polydimethylsiloxane is added, the fermentation is continued for 96h, then the tank is unloaded, and the components are detected by liquid chromatography, wherein the concentration of the 2' -fucosyllactose is 6.22 g/L.

Example 3

The fermentation operation was carried out using an in situ fermentation tank having a capacity of 30L, and the liquid loading was 20L.

(1) Preparing first-stage seed fermentation liquor: the glycerol strain E.coli-XYY1 frozen in a-80 refrigerator was inoculated in 100mL LB medium at 3% inoculum size and shake-cultured at 37 ℃ and 220rpm for 6 h.

(2) Preparing secondary seed fermentation liquor: inoculating 100mL of first-stage seed fermentation liquor into 2L of LB culture medium, and performing shake cultivation at 37 ℃ and 220rpm for 4 h;

(3) preparing a fermentation tank culture medium: adding Na into a 30L tank₂HPO₄·12H₂O 342g，NaCl 10g，KH₂PO₄60g，NH₄80g of Cl, 2mL of triton and 802mL of Tween, diluting to 13L with deionized water, sterilizing with high-temperature steam at 121 ℃ for 15min, and cooling to 37 ℃. In addition, MgSO is prepared₄480mg in 10mL deionized water, CaCl₂90mg in 10mL of deionized water, 320g of glucose in 1.6L of deionized water, 5g of yeast extract powder in 500mL of deionized water, and 10g of peptone in 500mL of deionized water, sterilizing in a high-temperature sterilization pot at 121 ℃ for 15min, cooling to 37 ℃, and then adding into a fermentation tank in an aseptic operation. In addition, 1g of methionine was added to 100mL of deionized water, 20mL of vitamin solution and 20mL of metal solution were added to the fermentor after they were sterilized by 0.22 μm aqueous membrane, respectively. The vitamin solution and the metal solution were prepared in advance, and the formulation was the same as in example 1.

(4) Batch fermentation: inoculating 2L of secondary seed fermentation broth into a fermentation tank, stirring at 37 ℃ and 500rpm, ventilating for 1.2vvm, keeping the pH constant at 7.0 (the alkali solution is 25% ammonia water, and the acid solution is 10% phosphoric acid), fermenting for 5h, adding 20mL of sterile 0.1M IPTG, setting the temperature at 30 ℃, reducing the stirring speed to 350rpm, and reducing the ventilation amount to 0.6 vvm. Adding 2L of 10% sterile lactose solution after 3h, adding 1mL of defoaming agent polydimethylsiloxane, continuously fermenting for 96h, unloading the tank, and detecting the components by liquid chromatography, wherein the concentration of 2' -fucosyllactose is 6.22 g/L.