阅读说明：本技术 一种提高辅酶i产量的发酵方法 (Fermentation method for increasing output of coenzyme I ) 是由 王伟 金永红 潘丽英 普坤 周多玲 王磊 于 2020-07-24 设计创作，主要内容包括：本发明公开了一种提高辅酶I产量的发酵方法,包括如下步骤：S1、将酿酒酵母接种到培养基中,然后进行发酵,当发酵液的OD<Sub>600</Sub>值≥30时,补加糖液保持发酵液中的残糖浓度为2g/L,当酿酒酵母生长进入停止期后,停止补加糖液；S2、向停止期的发酵液中加入生物素和烟酸,继续发酵至辅酶I含量停止增长时,收集酿酒酵母,破壁得到含有辅酶I的破壁液即可。本发明选用合适的酿酒酵母菌种并配合合适的发酵工艺,大幅提高了辅酶I的产量。(The invention discloses a fermentation method for improving the output of coenzyme I, which comprises the following steps: s1, inoculating the saccharomyces cerevisiae into the culture medium, and then fermenting when the OD of the fermentation liquid is 600 When the value is more than or equal to 30, adding sugar liquor to keep the concentration of residual sugar in the fermentation liquor at 2g/L, and stopping adding sugar liquor when the growth of saccharomyces cerevisiae enters a stop period; and S2, adding biotin and nicotinic acid into the fermentation liquor in the stop period, continuing fermenting until the content of coenzyme I stops increasing, collecting saccharomyces cerevisiae, and breaking the wall to obtain the wall-broken liquid containing the coenzyme I. The invention selects proper saccharomyces cerevisiae strains and is matched with a proper fermentation process, thereby greatly improving the output of the coenzyme I.)

1. A fermentation method for improving the output of coenzyme I is characterized by comprising the following steps:

s1, inoculating the saccharomyces cerevisiae into the culture medium, and then fermenting when the OD of the fermentation liquid is₆₀₀When the value is more than or equal to 30, adding sugar liquor to keep the concentration of residual sugar in the fermentation liquor at 2g/L, and stopping adding sugar liquor when the growth of saccharomyces cerevisiae enters a stop period;

and S2, adding biotin and nicotinic acid into the fermentation liquor in the stop period, continuing fermenting until the content of coenzyme I stops increasing, collecting saccharomyces cerevisiae, and breaking the wall to obtain the wall-broken liquid containing the coenzyme I.

2. The coenzyme I production-improving fermentation method according to claim 1, wherein in S1, the Saccharomyces cerevisiae is a high-sugar Saccharomyces cerevisiae.

3. The fermentation method for improving the production of coenzyme I according to claim 1 or 2, wherein the inoculation amount in S1 is 5%.

4. The fermentation method for improving the production of coenzyme I according to any one of claims 1 to 3, wherein in S1, the medium comprises: glucose 60 g/L; casein hydrolysate 15 g/L; KH (Perkin Elmer)₂PO₄11.17g/L；MgSO₄·7H₂O 5g/L；ZnCl₂0.01 g/L; calcium pantothenate 0.01 g/L; vitamin B10.1g/L; inositol 0.1 g/L; 0.01g/L ferric citrate; 5g/L of urea; 2g/L of copper sulfate; 1g/L of manganese sulfate; 0.2mL/L defoaming agent and water.

5. The fermentation method for improving the production of coenzyme I according to any one of claims 1 to 4, wherein in S1, the inoculation parameters are: inoculating at 29.5-30.5 deg.C, culture medium pH of 5.2-5.6, and ventilation amount of 1m³The stirring speed is 175rpm, the DO value of the culture medium dissolved oxygen calibration is 100 percent, the tank pressure is 0.05Mpa, and CO is added₂The amount was 50%.

6. The fermentation method for increasing the production of coenzyme I according to any one of claims 1 to 5, wherein in S1, the fermentation temperature is 29.5 to 30.5 ℃, the pH of the culture medium is 5.2 to 5.6, the tank pressure is 0.03 to 0.07MPa, and CO is added in the whole fermentation process₂The amount is 49.95-50.05%, and the DO value of the fermentation liquor dissolved oxygen is more than 30%.

7. The fermentation method for increasing the production of coenzyme I according to any one of claims 1 to 6, wherein in S1, the sugar solution comprises: 1Kg of an aqueous glucose solution having a content of 60% by weight and 9.6mL of a mixed aqueous solution of potassium iodide having a concentration of 0.2g/L and cobalt chloride having a concentration of 1 g/L.

8. The coenzyme I production improving fermentation method according to any one of claims 1 to 7, wherein the OD of the fermentation broth is determined as S1₆₀₀When the value is 100, firstly adjusting the pH value of the fermentation liquor to be more than 5.43, and then adding sugar liquor.

9. The coenzyme I production improving fermentation method according to any one of claims 1 to 8, wherein the OD of the fermentation broth is determined as S1₆₀₀When the value is more than 200, the OD is detected every 2h₆₀₀Value, when OD₆₀₀When the value stops increasing, the growth of the saccharomyces cerevisiae enters a stop period, and the sugar liquid is stopped being supplemented.

10. The fermentation method for improving the production of coenzyme I according to any one of claims 1 to 9, wherein 0.5g of biotin and 6g of nicotinic acid are added per 1L of the fermentation broth at S2; preferably, in S2, the fermentation temperature is 29.5-30.5 deg.C, culture medium pH is 5.2-5.6, tank pressure is 0.03-0.07Mpa, and CO is added into the whole fermentation process₂The amount is 49.95-50.05%, and the ventilation amount is 0.8m³The DO value of dissolved oxygen calibration is more than 30 percent; preferably, in S1, S2, the pH of the fermentation broth is adjusted with an aqueous solution of phosphoric acid having a concentration of 85g/L or 12.5% by volume of aqueous ammonia.

Technical Field

The invention relates to the technical field of coenzyme I, in particular to a fermentation method for improving the output of coenzyme I.

Background

Coenzyme I (NAD), which is chemically named as nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide diphosphate or nicotinamide adenine dinucleotide, exists in mammals in two states of oxidation type (NAD +) and reduction type (NADH), is important coenzyme in human body redox reaction, participates in various physiological activities such as metabolism of cell substances, energy synthesis, cell DNA repair and the like, and plays an important role in the immunity of organisms. Under the healthy state, the concentration of nicotinamide adenine dinucleotide in the human body is stable, and the normal functions of various cells are maintained. The concentration of nicotinamide adenine dinucleotide in the body determines the process and extent of cellular senescence, and a decrease in concentration accelerates the process of cellular senescence.

Coenzyme I is mainly present in the mitochondria of yeast cells and belongs to intracellular enzymes. Coenzyme I is usually obtained by a method of extracting coenzyme I from yeast cells, but the content of coenzyme I in yeast is limited, the yield of the extracted coenzyme I is not high, and a new method for improving the yield of the coenzyme I is needed.

Disclosure of Invention

Based on the technical problems in the background art, the invention provides a fermentation method for improving the output of coenzyme I.

The invention provides a fermentation method for improving the output of coenzyme I, which comprises the following steps:

s1, inoculating the saccharomyces cerevisiae into the culture medium, and then fermenting when the OD of the fermentation liquid is₆₀₀When the value is more than or equal to 30, adding sugar liquor to keep the concentration of residual sugar in the fermentation liquor at 2g/L, and stopping adding sugar liquor when the growth of saccharomyces cerevisiae enters a stop period;

and S2, adding biotin and nicotinic acid into the fermentation liquor in the stop period, continuing fermenting until the content of coenzyme I stops increasing, collecting saccharomyces cerevisiae, and breaking the wall to obtain the wall-broken liquid containing the coenzyme I.

Preferably, in S1, the saccharomyces cerevisiae is a high-sugar saccharomyces cerevisiae.

The above-mentioned high-sugar type Saccharomyces cerevisiae (Saccharomyces cerevisiae) is commercially available, such as Mary's yeast, Inc., Angel Yeast, Inc.; preferred is Mary's yeast, Zintenna, Inc.

The saccharomyces cerevisiae can be directly inoculated into a culture medium.

Preferably, in S1, the inoculation amount is 5%.

The inoculation amount is the ratio of the volume of the seed solution to the volume of the culture medium after inoculation.

Preferably, in S1, the medium comprises: glucose 60 g/L; casein hydrolysate 15 g/L; KH (Perkin Elmer)₂PO₄11.17g/L；MgSO₄·7H₂O 5g/L；ZnCl₂0.01 g/L; calcium pantothenate 0.01 g/L; vitamin B10.1g/L; inositol 0.1 g/L; 0.01g/L ferric citrate; 5g/L of urea; 2g/L of copper sulfate; 1g/L of manganese sulfate; 0.2mL/L defoaming agent and water.

Preferably, in S1, the inoculation parameters are: inoculating at 29.5-30.5 deg.C, culture medium pH of 5.2-5.6, and ventilation amount of 1m³The stirring speed is 175rpm, the DO value of the culture medium dissolved oxygen calibration is 100 percent, the tank pressure is 0.05Mpa, and CO is added₂The amount was 50%.

Preferably, in S1, the fermentation temperature is 29.5-30.5 deg.C, culture medium pH is 5.2-5.6, tank pressure is 0.03-0.07Mpa, and CO is added into the whole fermentation process₂The amount is 49.95-50.05%, and the DO value of the fermentation liquor dissolved oxygen is more than 30%.

When the DO value of the fermentation liquor dissolved oxygen calibration is less than or equal to 30%, the dissolved oxygen can be controlled by adjusting the stirring rotating speed and the ventilation quantity, the DO value of the dissolved oxygen calibration is ensured to be more than 30%, and when the stirring rotating speed reaches the upper limit, the ventilation quantity is adjusted to control the dissolved oxygen.

Preferably, in S1, the sugar solution includes: 1Kg of an aqueous glucose solution having a content of 60% by weight and 9.6mL of a mixed aqueous solution of potassium iodide having a concentration of 0.2g/L and cobalt chloride having a concentration of 1 g/L.

In the above S1, OD of fermentation broth₆₀₀When the value is more than or equal to 30, detecting the concentration of residual sugar in the fermentation liquor, and when the concentration of the residual sugar is less than 2g/L, adding sugar liquor into the fermentation liquor in a flowing manner, and keeping fermentationThe residual sugar concentration in the fermentation liquid is 2g/L, and during the process of adding sugar liquid in flowing manner, when the ethanol content in the fermentation liquid is too high and the ethanol taste in the fermentation chamber is strong, or the residual sugar concentration is more than 2g/L, or CO₂The amount is not equal to 49.95-50.05%, and when the DO value of the fermentation liquor dissolved oxygen calibration is less than or equal to 30%, the adding speed of the sugar solution is reduced or the adding amount of the sugar solution is reduced.

Preferably, in S1, when the OD of the fermentation broth is₆₀₀When the value is 100, firstly adjusting the pH value of the fermentation liquor to be more than 5.43, and then adding sugar liquor.

Preferably, in S1, when the OD of the fermentation broth is₆₀₀When the value is more than 200, the OD is detected every 2h₆₀₀Value, when OD₆₀₀When the value stops increasing, the growth of the saccharomyces cerevisiae enters a stop period, and the sugar liquid is stopped being supplemented.

Preferably, 0.5g biotin and 6g nicotinic acid per 1L fermentation broth is added in S2.

Preferably, in S2, the fermentation temperature is 29.5-30.5 deg.C, culture medium pH is 5.2-5.6, tank pressure is 0.03-0.07Mpa, and CO is added into the whole fermentation process₂The amount is 49.95-50.05%, and the ventilation amount is 0.8m³The DO value of dissolved oxygen calibration is more than 30 percent.

Preferably, in S1, S2, the pH of the fermentation broth is adjusted with an aqueous solution of phosphoric acid having a concentration of 85g/L or 12.5% by volume of aqueous ammonia.

CO as described above₂The amount of CO in the tail gas of the fermentation tank₂The content of (b) can be detected on line in real time by a fermentation tail gas analysis technology.

In the fermentation process, a proper amount of defoaming agent aqueous solution with the volume fraction of 20 percent can be added to eliminate foams generated by fermentation.

The sugar solution, phosphoric acid aqueous solution and defoaming agent aqueous solution can be used after moist heat sterilization.

The culture medium can be used after being sterilized at 121 deg.C for 20 min.

Has the advantages that:

the invention does not directly extract coenzyme I from yeast, but firstly provides a method for improving the content of coenzyme I in the saccharomyces cerevisiae by fermentation, selects a proper saccharomyces cerevisiae strain and a proper fermentation process, screens the whole process parameters and culture medium, selects a proper amount of glucose aqueous solution and potassium iodide and cobalt chloride mixed aqueous solution to form sugar solution, selects biotin and nicotinic acid as substrates and controls the addition amount of the substrates, and finally obtains a proper fermentation process, so that the fermented saccharomyces cerevisiae is subjected to wall breaking treatment to obtain wall breaking liquid containing high-concentration coenzyme I; compared with the saccharomyces cerevisiae before fermentation, the coenzyme I yield of the invention is about 2 times of that of the saccharomyces cerevisiae before fermentation.

Drawings

FIG. 1 is a high performance liquid chromatogram of the coenzyme I-containing wall-broken liquid obtained in comparative example 1.

FIG. 2 is a high performance liquid chromatogram of the coenzyme I-containing wall-broken liquid obtained in example 1.

Detailed Description

The technical solution of the present invention will be described in detail below with reference to specific examples.

The Saccharomyces cerevisiae of example 1 and comparative example 1 below were all high-sugar Saccharomyces cerevisiae (Saccharomyces cerevisiae) available from Mary's Tokyo Maries, Inc.