阅读说明：本技术 一种提高腺苷发酵产量的方法 (Method for improving adenosine fermentation yield ) 是由 曹华杰 刘小都 孙超超 于 2020-12-31 设计创作，主要内容包括：本发明涉及一种提高腺苷发酵产量的方法,属于发酵工程技术领域。本发明方法以枯草芽孢杆菌为生产菌株,发酵培养基通过添加一定量的酵母浸粉和异亮氨酸,使发酵后期腺苷产量持续增长,从而使腺苷的产量提高至60.7g/L,与未使用该方法相比产量提高43.5%。该方法不仅可以提高腺苷的产量,发酵过程简单无需补料,减少染菌几率,非常适合工业化生产。(The invention relates to a method for improving adenosine fermentation yield, and belongs to the technical field of fermentation engineering. According to the method, bacillus subtilis is taken as a production strain, and a certain amount of yeast extract powder and isoleucine are added into a fermentation medium, so that the adenosine yield is continuously increased in the later fermentation period, the adenosine yield is increased to 60.7g/L, and the adenosine yield is increased by 43.5% compared with that in the case of not using the method. The method can improve the yield of adenosine, has simple fermentation process without material supplement, reduces the probability of bacterial contamination, and is very suitable for industrial production.)

1. A method for improving the fermentation yield of adenosine comprises the following steps:

step one, slant culture: inoculating bacillus subtilis serving as an original strain into a slant solid culture medium for activation, and culturing for 15-20 h at 28-32 ℃;

step two, seed culture: scraping strains on the slant solid culture by using an inoculating ring, inoculating the strains to a seed culture medium, and culturing at 30-36 ℃ and 200-500 rpm for 8-10 h to obtain a fermented seed solution;

step three, fermentation culture: inoculating 12-15% of the fermented seed liquid obtained in the step twoTransferring the mixture to a fermentation tank filled with a fermentation culture medium, maintaining the dissolved oxygen at 20-30% at 33-36 ℃, and using NH₃·H₂Regulating the pH value to 6.6-7.0 by O, and culturing for 40-50 h;

the liquid fermentation medium comprises the following components in g/L: 80-120 parts of glucose, 30-50 mL/L of corn steep liquor, 3-5 parts of yeast extract powder, 5-10 parts of polysaccharide antler essence, 3-5 parts of monosodium glutamate and K₂HPO₄1 to 3, isoleucine 0.1 to 0.2, xanthine 0.1 to 0.2, hypoxanthine 0.1 to 0.2, MgSO₄·7H₂O 1~5，MnSO₄0.03 to 0.06 and FeSO₄·7H₂O 0.03~0.06。

2. The method of claim 1, wherein: the slant solid culture medium in the first step comprises the following components in g/L: 1-3 parts of glucose, 5-10 parts of peptone, 2-5 parts of yeast powder, 0.1-0.15 part of xanthine and 20-30 parts of agar, and the pH value is 7.0-7.2.

3. The method of claim 1, wherein: the seed culture medium of the second step comprises the following components in g/L: 15-20% of glucose, 15-25 mL/L of corn steep liquor, 5-10% of protein fine powder, 5-10% of yeast powder and KH₂PO₄0.5~1.5，MgSO₄·7H₂0.2 to 0.8O, 0.1 to 0.15 xanthine and 0.01 to 0.05 histidine, pH6.5 to 7.0.

Technical Field

The invention belongs to the technical field of fermentation engineering, and particularly relates to a method for improving adenosine fermentation yield.

Background

Adenosine, also known as adenosine, is a pure white crystalline powder, odorless, bitter in taste. Adenosine formula C₁₀H₁₃N₅O₄And the molecular weight is 267.24. Adenosine is an important nucleotide derivative and is a dephosphorylated product of adenine nucleotide. As an endogenous nucleoside distributed throughout human cells, the nucleoside can directly enter cardiac muscle to generate adenylic acid through phosphorylation and participate in cardiac muscle energy metabolism. Adenosine plays a biochemical important role, including the transfer of energy in the form of Adenosine Triphosphate (ATP) or Adenosine Diphosphate (ADP), or the signaling of cyclic adenosine monophosphate (cAMP), among others. In addition, adenosine is an inhibitory neurotransmitter, and plays an important role in neurotransmission.

Along with the continuous expansion of the action range of adenosine, the market demand is also continuously increased, the production method of adenosine mainly comprises a chemical synthesis method, an enzymatic method and a fermentation method, and the production of adenosine at home at present mainly comprises the chemical method and the enzymatic method, so that the cost is high, the pollution is serious, and the popularization and the application are severely limited. The method for producing adenosine by fermentation has the advantages of mild reaction conditions, low cost, greenness, cleanness, environmental protection and the like. Therefore, under the condition of low carbon and environmental protection, the adenosine is produced by using a fermentation method, the fermentation process of the adenosine is optimized, the yield of the adenosine is further improved, and the method has important significance.

Disclosure of Invention

In view of the above problems, the present invention aims to provide a method for increasing adenosine fermentation yield, which is characterized in that yeast extract powder and isoleucine are added into a culture medium to provide sufficient nutrients for the growth and fermentation of bacteria, so as to prolong the stable growth period of the bacteria, facilitate the efficient synthesis of adenosine at the later stage, and achieve the purpose of increasing adenosine fermentation yield.

In order to achieve the purpose, the invention adopts the specific scheme that:

a method for improving the fermentation yield of adenosine comprises the following steps:

step one, slant culture: inoculating bacillus subtilis serving as an original strain into a slant solid culture medium for activation, and culturing for 15-20 h at 28-32 ℃;

step two, seed culture: scraping strains on the slant solid culture by using an inoculating ring, inoculating the strains to a seed culture medium, and culturing at 30-36 ℃ and 200-500 rpm for 8-10 h to obtain a fermented seed solution;

step three, fermentation culture: transferring the fermentation seed liquid obtained in the step two into a fermentation tank filled with a fermentation culture medium according to the inoculation amount of 12-15%, maintaining the dissolved oxygen at 20-30% at 33-36 ℃, and using NH₃·H₂Regulating the pH value to 6.6-7.0 by O, and culturing for 40-50 h;

the liquid fermentation medium comprises the following components in g/L: 80-120 parts of glucose, 30-50 mL/L of corn steep liquor, 3-5 parts of yeast extract powder, 5-10 parts of polysaccharide antler essence, 3-5 parts of monosodium glutamate and K₂HPO_4,1 to 3, isoleucine 0.1 to 0.2, xanthine 0.1 to 0.2, hypoxanthine 0.1 to 0.2, MgSO₄·7H₂O1~5，MnSO₄0.03 to 0.06 and FeSO₄·7H₂O 0.03~0.06。

Further, the slant solid culture medium in the first step comprises the following components in g/L: 1-3 parts of glucose, 5-10 parts of peptone, 2-5 parts of yeast powder, 0.1-0.15 part of xanthine and 20-30 parts of agar, and the pH value is 7.0-7.2.

Further, the seed culture medium of the second step comprises the following components in g/L: 15-20% of glucose, 15-25 mL/L of corn steep liquor, 5-10% of protein fine powder, 5-10% of yeast powder and KH₂PO₄0.5~1.5，MgSO₄·7H₂0.2 to 0.8O, 0.1 to 0.15 xanthine and 0.01 to 0.05 histidine, pH6.5 to 7.0.

Has the advantages that:

according to the invention, bacillus subtilis XGL is used as a fermentation strain, a fermentation medium is optimized, yeast extract powder and isoleucine are added on the basis of a 50L fermentation tank pilot scale, the production time of metabolites is prolonged, and the yield of adenosine is improved.

(1) Compared with the traditional chemical method and enzyme method, the method has the advantages of simple production equipment, little pollution, accordance with the current low-carbon environment, low cost and suitability for large-scale production.

(2) The microbial strains used in the invention have stable genetic markers and are not easy to lose, and the microbial strains are transferred after more than ten generations, so that the yield is basically kept stable.

(3) According to the invention, the yeast extract powder and the amino acid are added into the fermentation tank bottom material, so that the production strength of adenosine is improved to 60.7g/L, and compared with the method without adding the yeast extract powder and isoleucine, the yield is improved by 43.5%.

Detailed Description

A method for improving adenosine fermentation yield is characterized in that yeast extract powder and isoleucine are added to improve the adenosine fermentation yield, the method adopts a fed-batch mode to ferment and produce adenosine, and a fermentation strain adopts Bacillus subtilis XGL (which is published in China bioengineering journal in 2011, 12 and 31 days, and the article is 'influence of overexpression purA gene on adenosine accumulation', and is now preserved in the strain collection center of institute of bioengineering, university of Tianjin technology).

The specific operation of the method comprises the following processes:

step 1, slant culture: inoculating bacillus subtilis XGL as an original strain to an activated inclined plane, and culturing for 15-20 h at 28-32 ℃;

step 2, seed culture: scraping slant strains by using an inoculating ring, inoculating the slant strains to a seed culture medium, and culturing at 30-36 ℃ and 200-500 rpm for 8-10 h to serve as a fermentation seed solution;

step 3, fermentation culture: transferring the strain into a fermentation tank filled with a fermentation culture medium according to the inoculation amount of 12-15%, maintaining the dissolved oxygen at 20-30% at 33-36 ℃, and using NH₃·H₂Adjusting the pH value to 6.6-7.0 by O, and culturing for 40-50 h.

Wherein the slant solid culture medium comprises the following components in g/L: 1-3 parts of glucose, 5-10 parts of peptone, 2-5 parts of yeast powder, 0.1-0.15 part of xanthine, 20-30 parts of agar and 7.0-7.2 parts of pHs;

the seed culture medium comprises the following components in g/L: 15-20% of glucose, 15-25 mL/L of corn steep liquor, 5-10% of protein fine powder, 5-10% of yeast powder and KH₂PO₄0.5~1.5，MgSO₄·7H₂0.2 to 0.8O, 0.1 to 0.15 xanthine, 0.01 to 0.05 histidine, pH6.5 to 7.0;

the liquid fermentation medium comprises the following components in g/L: 80-120 parts of glucose, 30-50 mL/L of corn steep liquor, 3-5 parts of yeast extract powder, 5-10 parts of polysaccharide antler essence, 3-5 parts of monosodium glutamate and K₂HPO_4,1 to 3, isoleucine 0.1 to 0.2, xanthine 0.1 to 0.2, hypoxanthine 0.1 to 0.2, MgSO₄·7H₂O1~5，MnSO₄0.03~0.06，FeSO₄·7H₂O0.03~0.06。

The invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that the specific material ratios, process conditions and results described in these specific examples are merely illustrative of the invention and do not limit the scope of the invention in any way.

The analysis method adopted for the adenosine content of the fermentation product in each of the following examples is as follows:

the product adenosine in the fermentation broth is measured by an Agilent1100 High Performance Liquid Chromatograph (HPLC). The chromatographic column was a kromasil c18 column (250mm × 416mm i.d., 5 μm), the column temperature was 30 ℃, the detector was an ultraviolet detector (259 nm), the mobile phase was water: acetonitrile = 90: 10; the flow rate is 1.0 ml/min; the amount of the sample was 20. mu.L. Accurately weighing an adenosine standard substance, and preparing a standard substance solution with the mass concentration of 1g/L by using deionized water; diluting the pretreated fermentation liquid sample to be detected to a proper concentration by using deionized water, filtering by using a 0.22 mu m microporous filter membrane to be used as a fermentation sample solution to be detected, and calculating the adenosine yield of the fermentation sample according to the peak area.

Example 1:

the formula of the slant culture medium is calculated by g/L: glucose 2, peptone 5, yeast powder 5, xanthine 0.1, agar 20, pH 7.0;

the formula of the seed culture medium is calculated in g/L: 20 parts of glucose, 20mL/L of corn steep liquor, 10 parts of protein fine powder, 5 parts of yeast powder and KH₂PO₄1，MgSO₄·7H₂O0.5, xanthine 0.15, histidine 0.05, pH 7.0;

the fermentation medium is measured by g/L: 80 parts of glucose, 40mL/L of corn steep liquor, 6 parts of polysaccharide antler essence, 5 parts of yeast extract powder, 4 parts of monosodium glutamate and K₂HPO₄3, xanthine 0.15, hypoxanthine 0.2, MgSO₄·7H₂O1，MnSO₄0.05，FeSO₄·7H₂O0.05。

Inoculating Bacillus subtilis XGL as original strain to activated slant, culturing at 30 deg.C for 18 hr, scraping slant strain with inoculating loop, inoculating to seed culture medium, culturing at 36 deg.C and 200rpm for 9 hr, inoculating to 50L fermentation tank containing fermentation culture medium at an inoculum size of 15% (v/v), maintaining dissolved oxygen at 36 deg.C to 30%, and culturing with NH₃·H₂Adjusting pH, maintaining the pH at 6.4, monitoring the residual glucose amount in the fermentation broth on line, and feeding glucose until the residual glucose amount in the fermentation broth is 10g/L when the residual glucose amount in the fermentation broth is reduced to 10g/L, fermenting for 40h, wherein the adenosine yield reaches 54.3 g/L.

Example 2:

the formula of the slant culture medium is calculated by g/L: glucose 2, peptone 5, yeast powder 5, xanthine 0.1, agar 20, pH 7.0;

the formula of the seed culture medium is calculated in g/L: 20 parts of glucose, 20mL/L of corn steep liquor, 10 parts of protein fine powder, 5 parts of yeast powder and KH₂PO₄1，MgSO₄·7H₂O0.5, xanthine 0.15, histidine 0.05, pH 7.0;

the fermentation medium is measured by g/L: 80 percent of glucose, 40mL/L of corn steep liquor, 6 percent of polysaccharide antler essence, 4 percent of monosodium glutamate and K₂HPO₄3, isoleucine 0.1-0.2, xanthine 0.15, hypoxanthine 0.2, MgSO 0₄·7H₂O1，MnSO₄0.05，FeSO₄·7H₂O0.05。

Inoculating bacillus subtilis XGL as an original strain to an activated inclined plane, culturing at 30 ℃ for 18h, scraping the inclined plane strain by using an inoculating ring, inoculating to a seed culture medium, and culturing at 36 ℃ and 200rpm for 9h; transferring the mixture into a 50L fermentation tank filled with a fermentation medium according to the inoculation amount of 15% (v/v), maintaining the dissolved oxygen at 20-30% at 36 ℃, and using NH₃·H₂Adjusting pH, maintaining the pH at 6.4, monitoring the residual glucose amount in the fermentation broth on line, and feeding glucose until the residual glucose amount in the fermentation broth is 10g/L when the residual glucose amount in the fermentation broth is reduced to 10g/L, fermenting for 40h, wherein the adenosine yield reaches 50.7 g/L.

Example 3:

the formula of the slant culture medium is calculated by g/L: glucose 2, peptone 5, yeast powder 5, xanthine 0.1, agar 20, pH 7.0;

the formula of the seed culture medium is calculated in g/L: 20 parts of glucose, 20mL/L of corn steep liquor, 10 parts of protein fine powder, 5 parts of yeast powder and KH₂PO₄1，MgSO₄·7H₂O0.5, xanthine 0.15, histidine 0.05, pH 7.0;

the fermentation medium is measured by g/L: 80 parts of glucose, 40mL/L of corn steep liquor, 6 parts of polysaccharide antler essence, 5 parts of yeast extract powder, 4 parts of monosodium glutamate and K₂HPO₄3, isoleucine 0.1-0.2, xanthine 0.15, hypoxanthine 0.2, MgSO 0₄·7H₂O1，MnSO₄0.05，FeSO₄·7H₂O0.05。

Inoculating Bacillus subtilis XGL as original strain to activated slant, culturing at 30 deg.C for 18 hr, scraping slant strain with inoculating loop, inoculating to seed culture medium, culturing at 36 deg.C and 200rpm for 9 hr, inoculating to 50L fermentation tank containing fermentation culture medium at an inoculum size of 15% (v/v), maintaining dissolved oxygen at 36 deg.C to 30%, and culturing with NH₃·H₂Adjusting pH with O, maintaining pH at 6.4, and online monitoring glucose in fermentation brothWhen the glucose residual quantity is reduced to 10g/L, glucose is fed in until the residual sugar quantity of the fermentation liquor is maintained to be 10g/L, and the adenosine yield reaches 60.7g/L after fermentation for 40 hours.

Comparative example 1:

the fermentation was carried out in a fermentor without adding yeast extract powder and isoleucine under the same conditions as in example 1. The final adenosine content was determined to be 42.3 g/L.

As can be seen from example 1 and comparative example 1, the addition of 5g/L yeast extract powder in the fermenter can increase the adenosine yield by 28.4%, which indicates that the addition of organic nitrogen source yeast extract powder can provide sufficient nitrogen source for the growth of the thallus and prolong the stable growth period of the thallus. As can be seen from the example 2 and the comparative example 1, the effect of adding isoleucine into the culture medium on the yield of adenosine is large, the yield of adenosine is increased by 20%, and isoleucine can improve the activity of thalli and the yield of adenosine in a tank. As can be seen from example 3 and comparative example 1, the addition of yeast extract and isoleucine at the same time can significantly improve the adenosine yield by 43.5%, and the effect is relatively obvious.

It should be noted that the above-mentioned embodiments illustrate rather than limit the scope of the invention, which is defined by the appended claims. It will be apparent to those skilled in the art that certain insubstantial modifications and adaptations of the present invention can be made without departing from the spirit and scope of the invention.