CN101818183A - Method for improving trehalose content of yeast cell - Google Patents

Abstract

The invention relates to a method for increasing the trehalose content of yeast cells. The yeast fermentation culture liquid is centrifuged to obtain yeast milk with a cell concentration of 13-17%, and an aqueous solution of maltose, sodium chloride and calcium chloride is added to the yeast milk. Cultivate for 2-3 hours at 35°C, pH 5.2, and 1.0-1.2vvm ventilation conditions, and centrifuge to collect the yeast sludge. The trehalose content in the obtained yeast is more than 16%. The present invention can make yeast cells in a short time The trehalose is increased from 8% to more than 16%, which shortens the fermentation time and saves fermentation equipment and power consumption compared with the existing technology.

Description

A method for increasing the trehalose content of yeast cells

technical field

The invention relates to a method for increasing the trehalose content of yeast cells, in particular to a method for increasing the trehalose content of dried bread yeast cells, which belongs to the technical fields of fermentation engineering and microorganisms.

Background technique

Active dry yeast is made from fresh yeast or pressed yeast through granulation, drying and dehydration. The activity and storage time of active dry yeast are important indicators for evaluating the quality of yeast. These indicators are closely related to the content of trehalose in yeast cells.

Trehalose (Trehalose), also known as mushroom sugar, Qin sugar, its molecular formula is C ₁₂ H ₂₂ O ₁₁ 2H ₂ O, chemical name α-D-glucopyranosyl α-D-glucopyranoside (α- D-glycopyranosyl-α-D-glycopyranoside), is a non-reducing disaccharide formed by condensation of two glucose molecules through hemiacetal hydroxyl. Trehalose widely exists in lower plants, algae, bacteria, filamentous fungi, yeast, and insects.

The stress tolerance of certain species to harsh external environments, such as drought, high temperature, dehydration, freezing, high osmotic pressure and toxic substances, etc., is closely related to the trehalose present in these species, and has unique differences. The biological characteristics of other carbohydrates can protect the components of biological cells such as proteins, fats, sugars, and nucleic acids from damage in the above-mentioned environment, and can protect DNA from damage caused by radiation. Exogenous trehalose can also protect Organisms and biomacromolecules have good non-specific protective effects, so this makes them have many uses, such as cell antifreeze in medicine and microbiology, diagnostic reagents and stabilizers for biological products, active ingredients in cosmetics, food Preservatives, cultivation of drought-resistant crops in agriculture, etc.

As a stress metabolite of yeast, trehalose can endow microorganisms such as yeast with the ability to resist harsh environments such as nutrient deficiency, high temperature, low temperature, dryness, high osmotic pressure, and toxic substances. It has the ability to protect organisms in harsh environments. Biofilm , liposome, protein, nucleic acid and other special effects of structure and function, it keeps the cells moist and prevents the loss of various nutrients in the cells, so that these organisms live in an anhydrous state, such as in the production of active dry yeast, The dry yeast whose trehalose content is below 11% (calculated as solid matter) has a short active storage period and is not easy to store; when the trehalose content is about 12% to 14%, its storage period can be extended for 4-6 months; and When the trehalose content is more than 16%, its shelf life can reach more than 24 months.

Active dry yeast is made from fresh yeast (or squeezed yeast) through granulation, drying and dehydration. The activity of dry yeast is usually expressed by fermentation power. In general, 3.5g of fresh yeast is needed to produce 1g of active dry yeast, but in terms of activity (fermentation power), 1g of active dry yeast is only equivalent to 2-3g of fresh yeast: after rehydration, the live cells are generally 80- 90%. Fresh yeast is made into active dry yeast, and after a certain period of storage and rehydration before use, there is a certain loss of activity.

The content of trehalose in yeast has a great influence on the activity and storage of active dry yeast. To a certain extent, it determines the activity and storage resistance of high-activity dry yeast, and the amount of trehalose increases, which is beneficial to the stability of cells during drying and storage. Higher trehalose content is beneficial to extend the storage period of active dry yeast. The level of phosphorus content has a certain impact on the accumulation of trehalose, the level of nitrogen content in cells and the activity of enzymes. The content of trehalose in general active dry yeast is about 14% of the dry matter. Therefore, the degree of trehalose accumulation must be considered in the yeast cultivation process. The synthesis of trehalose by yeast needs to go through two stages, one is the stage of mass reproduction of yeast cells, the content of trehalose in the yeast is low at this stage, but it can provide a sufficient amount of bacteria; the other is the accumulation stage of trehalose, mainly through the control External conditions allow it to accumulate.

Contents of the invention

Aiming at the deficiencies of the prior art, the present invention proposes a method for increasing the trehalose content of yeast cells, which can achieve the trehalose content in yeast cells reaching more than 16% in a relatively short period of time.

Terminology Explanation:

The unit of ventilation volume, vvm, refers to the volume ratio of the amount of air introduced per minute to the amount of fermentation broth.

Cell concentration: refers to the mass concentration of Saccharomyces cerevisiae in the fermentation broth or yeast milk.

The "baker's yeast" mentioned in the present invention is a trade name of a product produced by the Saccharomyces cerevisiae species.

Technical scheme of the present invention is as follows:

A method for increasing the trehalose content of yeast cells. The yeast fermentation culture liquid is centrifuged to obtain yeast milk with a cell concentration of 13-17%, and an aqueous solution of maltose, sodium chloride and calcium chloride is added to the yeast milk, and the yeast milk and The mass volume ratio of water is 1: 1, and the consumption of maltose, sodium chloride and calcium chloride accounts for the total mass percent of yeast milk to be respectively 2.4-3.0% maltose, 3.2-4.0% sodium chloride and 1.0% calcium chloride, in Cultivate for 2-3 hours at 35°C, pH 5.2, and 1.0-1.2vvm ventilation conditions, collect the yeast sludge by centrifugation, and measure the trehalose content in the yeast to be 16-17%, based on the dry weight of yeast cells. The mass-to-volume ratio is in g/mL or kg/L. The yeast is Saccharomyces cerevisiae.

The addition of maltose in the method of the present invention can increase the accumulation of trehalose, because the yeast cells contain maltose isomerase, which can convert maltose into trehalose in a short time.

In the above-mentioned method of the present invention, the yeast fermentation broth is used as the starting material, and the preparation of the yeast fermentation broth can be according to the prior art. The present invention provides the preparation method of following yeast fermentation culture liquid, as one of preferred of the present invention:

1. Medium

(1) Seed medium: glucose 20g, KH ₂ PO ₄ 1g, (NH ₄ ) ₂ SO ₄ 5g, MgSO ₄ ·7H ₂ O 0.2g, ZnSO ₄ ·7H ₂ O 0.05g, pH 5.4, distilled water 1000mL.

(2) Fermentation medium

① Fermentation base medium

Corn steep liquor (containing 28wt% of solids) 20%, glucose 1%, nutrient salt solution 5%, water 74%, are volume ratio. Among them, nutrient salt solution: KH ₂ PO ₄ 13.0g, (NH ₄ ) ₂ SO ₄ 94.16g, MgSO ₄ ·7H ₂ O 1.4g, ZnSO ₄ ·7H ₂ O 0.28g, add water to make 1000mL.

②Fed-in feed components during fermentation

Sugar solution: make a 250g/L solution of glucose.

Nutrient salt solution: KH ₂ PO ₄ 13.0g, (NH ₄ ) ₂ SO ₄ 94.16g, MgSO ₄ ·7H ₂ O 1.4g, ZnSO ₄ ·7H ₂ O 0.28g, add water to make 1000mL.

Alkaline solution: 100g/L Na ₂ CO ₃ solution.

2. Seed culture

Yeast strains were purchased from the Institute of Microbiology, Chinese Academy of Sciences, name: Saccharomyces cerevisiae, code: AS 2.146, AS 2.502, AS 2.503 or AS 2.504. Put the yeast slant strain into a 250mL Erlenmeyer flask containing 20mL seed medium, and culture it in a shake flask at 200r/min at 30°C for 24h, then transfer it to a 500mL Erlenmeyer flask with 80mL seed medium, shake the flask at 200r/min at 30°C Cultivate for 16 hours to obtain seed culture solution, which is set aside.

3. Fermentation culture

Sterilize the real tank in a 50L fermenter with 25L of fermentation basal medium, and the sterilization condition is 120°C for 30min. Then when the temperature is lowered to 32°C, insert 3L of seed culture solution, adjust the pH to 5.2, control the culture temperature at 30°C, add sugar solution and nutrient salt solution, and control the total fermentation time to 18-21h. - After adding in 5 hours, the amount of sugar solution to be added is based on the growth of the yeast, so that the glucose content in the fermentation system is always controlled below 0.5%. During the fermentation process, the pH was controlled at 5.2, and the stirring speed was controlled at 200 r/min. Other process conditions are preferably as follows:

Culture temperature control: 30°C for 1-8 hours of fermentation, 32°C for 9-15 hours, and 35°C for 16 hours to the end of fermentation.

Ventilation: Control the ratio of the volume of sterile air to the volume of fermentation broth at 2.5vvm per minute during the 1-15h period of fermentation (early and mid-stage), and adjust the valve during the trehalose accumulation stage from 16h to the end of fermentation (late stage). Ventilation drops to 1.2vvm.

Preferably, in the fermentation and cultivation step of the above method, the amount of sugar solution added is controlled so that the glucose content in the fermentation system is 0.30-0.50% by volume; the pH regulator is 10wt% Na ₂ CO ₃ solution.

The yeast fermentation culture liquid prepared by the above method has a cell concentration of 4.5-5.5%, and the content of trehalose is 8-10% through measurement. Further used as raw material for the method of the present invention.

Determination method of trehalose content in yeast

In yeast cells, it contains mannan and insoluble dextran, which will react with anthrone reagent. Therefore, selecting a suitable extractant to control the interference of these substances on the determination of trehalose is the key to the determination of trehalose in yeast. Yeast was extracted with 0.5mol/L cold trichloroacetic acid, and only trehalose existed in the obtained solution. The trichloroacetic acid extract and 0.2% anthrone sulfate solution were mixed at a ratio of 1:4 in a boiling water bath for 2 minutes, and the absorbance of the reaction solution was measured at 590 nm. Calculate the content of trehalose in the extract, that is, the amount of trehalose in the yeast.

Using paper chromatography separation and elution method: when using paper chromatography to quantitatively analyze trehalose, the two samples on the filter paper strips are the same sample, and the sample volume is controlled at 40-100 μg, one of which is the reference point, and the other is the reference point. One is a quantitative measurement point. The filter paper strip after sample application was developed in the developer n-butanol: acetone: water: acetic acid = 10:3:6:2 (v/v), air-dried, and cut from the middle. Spray color on half of the reference point to determine the exact position of the trehalose spot, cut off the part corresponding to the spot on the other half of the filter paper strip and cut it into pieces, put it in the eluent 0.1mol/L HCl and wash at room temperature After stripping for 16 hours, the supernatant after elution was determined by the sulfuric acid-anthrone colorimetric method to determine the trehalose content therein.

The technical characteristics and excellent effects of the method for accumulating trehalose through yeast culture are as follows:

①Temperature: In the early stage of fermentation (1-8h), the culture temperature is 30°C, in the middle stage (9-15h) the culture temperature is 32°C, and in the later stage (16h to the end of fermentation) the culture temperature is 35°C.

②pH: During the yeast culture process, add 10% Na ₂ CO ₃ solution to adjust the pH to 5.2.

③Dissolved oxygen: During the yeast culture process, under the condition of constant ventilation, increasing the stirring rate can effectively increase the oxygen transfer rate of the fermenter, so as to ensure the dissolved oxygen supply required for cell growth and metabolism. In the method of the present invention, the stirring speed is controlled at 200r/min, the aeration volume is controlled at 2.5vvm for 1-15h, and the aeration volume is reduced to 1.2vvm during the trehalose accumulation stage from the fermentation time of 16h to the end of fermentation.

④ After a large number of cells are obtained from yeast culture, yeast milk with a concentration of 13-17% is obtained through centrifugation, maltose, sodium chloride and calcium chloride are added to the yeast milk, and 2- 3 hours to achieve a high concentration of trehalose accumulation in the yeast. The present invention can increase the trehalose in yeast cells from 8% to more than 16% in a relatively short period of time. Compared with the existing general process, the fermentation time is shortened.

Since the raw material of the present invention is yeast milk, the concentration of yeast cells is 13-17%, and the concentration of yeast in the yeast fermentation liquid is 4.5-5.5%, thereby saving fermentation equipment and power consumption.

Description of drawings

Fig. 1 Yeast milk of method embodiment 1 of the present invention accumulates trehalose under the condition of adding salt and maltose;

Figure 2 Determination of trehalose content in the yeast sludge of Example 1 by sulfuric acid-anthrone colorimetry: when using paper chromatography to quantitatively analyze trehalose, the two samples on the filter paper strips are the same sample, and the sample volume is controlled at 40～100μg, one of which is the reference point and the other is the quantitative measurement point.

Detailed ways

The technical solution of the present invention will be further elaborated below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto. Bacterial classification and substratum used in the embodiment are:

Saccharomyces cerevisiae strains were purchased from the Institute of Microbiology, Chinese Academy of Sciences, and the strain collection center of the Institute of Microbiology, Chinese Academy of Sciences. The strain catalog code: AS 2.146; AS 2.502; AS 2.503; AS 2.504.

Seed medium: glucose 20g, KH ₂ PO ₄ 1g, (NH ₄ ) ₂ SO ₄ 5g, MgSO ₄ ·7H ₂ O 0.2g, ZnSO ₄ ·7H ₂ O 0.05g, pH 5.4, distilled water 1000mL.

Fermentation medium: including fermentation basal medium and fermentation process feeding components, as mentioned above.

Example 1

Yeast species: Saccharomyces cerevisiae AS 2.146. Put the yeast slant strain into a 250mL Erlenmeyer flask containing 20mL seed medium, and culture it in a shake flask at 200r/min at 30°C for 24h, then transfer it to a 500mL Erlenmeyer flask with 80mL seed medium, shake the flask at 200r/min at 30°C Cultivate for 16 hours to obtain seed culture solution, which is set aside.

Add 3 L of the above-mentioned seed culture solution into 25 L of sterilized fermentation base medium (50 L of fermenter volume), and the culture conditions are as follows. ①Temperature: During the 1-8h period of fermentation, the cultivation temperature is 30°C; during the 9-15h fermentation period, the cultivation temperature is 32°C; during the 16-20h fermentation period, the cultivation temperature is 35°C. ②pH: During the yeast culture process, add 10% Na ₂ CO ₃ solution to adjust the pH to 5.2. ③ Dissolved oxygen: The stirring speed is controlled at 200r/min, the ventilation rate is controlled at 2.5vvm for 1-16 hours, and the ventilation rate is reduced to 1.2vvm through the valve during 16-20 hours of fermentation. Add sugar solution and nutrient salt solution, the total amount of nutrient salt solution is 2.5L, and the fermentation time is 16h, and the sugar solution is added according to the feedback of ethanol and the growth of yeast, so that the glucose content in the fermentation broth is always controlled at 0.40 -0.45% (volume ratio), after 20 hours of fermentation, the yeast cells in the obtained fermentation broth are 4.78% (dry weight), and the trehalose content is 8.2%.

The above-mentioned fermented liquid was centrifuged to collect yeast milk with a large-capacity centrifuge under the condition of 3500 r/min for 10 minutes. Discard the supernatant from the centrifuge tube and remove the yeast milk. In 1000mL of distilled water, add 40g of sodium chloride, 10g of calcium chloride and 30g of maltose, dissolve completely, sterilize at 100°C, control the temperature to 30°C in a water bath, add to 1000g of yeast milk, and place in a 10L fermenter. The culture temperature was controlled at 35°C, and the pH was adjusted to 5.2 with 10wt% Na ₂ CO ₃ solution; under the condition of 1.20vvm ventilation, the culture time was 3 hours, the yeast sludge was collected by centrifugation, and the trehalose content in the yeast cell dry weight was determined to be 16.88%.

Example 2

Yeast strain: Saccharomyces cerevisiae AS 2.502. The preparation of the seed culture solution was the same as in Example 1.

Put the yeast slant strain into a 250mL Erlenmeyer flask containing 20mL seed medium, and culture it in a shake flask at 200r/min at 30°C for 24h, then transfer it to a 500mL Erlenmeyer flask with 80mL seed medium, shake the flask at 200r/min at 30°C Cultivate for 16 hours to obtain seed culture solution, which is set aside.

Add 3 L of the above-mentioned seed culture solution into 25 L of sterilized fermentation base medium (50 L of fermenter volume), and the culture conditions are as follows. ①Temperature: During 1-8 hours of fermentation, the culture temperature is 30°C; during 9-15 hours of fermentation, the culture temperature is 32°C; during 16-19 hours of fermentation, the culture temperature is 35°C. ②pH: During the yeast culture process, add 10% Na ₂ CO ₃ solution to adjust the pH to 5.2. ③ Dissolved oxygen: The stirring speed is controlled at 200r/min, the ventilation rate is controlled at 2.5vvm for 1-16 hours, and the ventilation rate is reduced to 1.2vvm through the valve during 16-20 hours of fermentation. Add sugar solution and nutrient salt solution, the total amount of nutrient salt solution is 2.5L, and the fermentation time is 16h, and the sugar solution is added according to the feedback of ethanol and the growth of yeast, so that the glucose content in the fermentation broth is always controlled at 0.40 -0.48% (volume ratio), after 19 hours of fermentation, the yeast cells in the obtained fermentation liquid were 4.56% (dry weight), and the trehalose content was 9.0%.

The above fermented liquid was centrifuged at 3500r/min for 10min to collect the yeast milk. Discard the supernatant from the centrifuge tube and remove the yeast milk. In 1000mL of distilled water, add 32g of sodium chloride, 10g of calcium chloride, and 24g of maltose, dissolve completely, sterilize at 100°C, control the temperature to 30°C in a water bath, add to 1000g of yeast milk, and place in a 10L fermenter. The culture temperature was controlled at 35°C, and the pH was adjusted to 5.2 with 10% Na ₂ CO ₃ solution; under the condition of 1.20vvm ventilation, the culture time was 3 hours, the yeast sludge was collected by centrifugation, and the trehalose content in the yeast cell dry weight was determined to be 16.00%.

Example 3

Yeast species: Saccharomyces cerevisiae AS 2.503. The preparation of the seed culture solution was the same as in Example 1.

Put the yeast slant strain into a 250mL Erlenmeyer flask containing 20mL seed medium, and culture it in a shake flask at 200r/min at 30°C for 24h, then transfer it to a 500mL Erlenmeyer flask with 80mL seed medium, shake the flask at 200r/min at 30°C Cultivate for 16 hours to obtain seed culture solution, which is set aside.

Add 3 L of the above-mentioned seed culture solution into 25 L of sterilized fermentation base medium (50 L of fermenter volume), and the culture conditions are as follows. ①Temperature: During the 1-8h period of fermentation, the cultivation temperature is 30°C; during the 9-15h fermentation period, the cultivation temperature is 32°C; during the 16-20h fermentation period, the cultivation temperature is 35°C. ②pH: During the yeast culture process, add 10% Na ₂ CO ₃ solution to adjust the pH to 5.2. ③ Dissolved oxygen: The stirring speed is controlled at 200r/min, the ventilation rate is controlled at 2.5vvm for 1-16 hours, and the ventilation rate is reduced to 1.2vvm through the valve during 16-20 hours of fermentation. Add sugar solution and nutrient salt solution, the total amount of nutrient salt solution is 2.5L, and the fermentation time is 16h, and the sugar solution is added according to the feedback of ethanol and the growth of yeast, so that the glucose content in the fermentation broth is always controlled at 0.40 -0.45% (volume ratio), after 20 hours of fermentation, the yeast cells in the obtained fermentation liquid are 5.72% (dry weight), and the trehalose content is 7.72%.

The above fermented liquid was centrifuged at 3500r/min for 10min, and the yeast milk was collected by centrifugation. Discard the supernatant from the centrifuge tube and remove the yeast milk. In 1000mL of distilled water, add 36g of sodium chloride, 10g of calcium chloride and 26g of maltose, dissolve completely, sterilize at 100°C, control the temperature to 30°C in a water bath, add to 1000g of yeast milk, and place in a 10L fermenter. The culture temperature was controlled at 35°C, and the pH was adjusted to 5.2 with 10% Na ₂ CO ₃ solution; under the condition of 1.20vvm ventilation, the culture time was 3 hours, the yeast sludge was collected by centrifugation, and the trehalose content in the yeast cell dry weight was determined to be 16.01%.

Example 4

Yeast strain: Saccharomyces cerevisiae AS 2.504. The preparation of the seed culture solution was the same as in Example 1.

Put the yeast slant strain into a 250mL Erlenmeyer flask containing 20mL seed medium, and culture it in a shake flask at 200r/min at 30°C for 24h, then transfer it to a 500mL Erlenmeyer flask with 80mL seed medium, shake the flask at 200r/min at 30°C Cultivate for 16 hours to obtain seed culture solution, which is set aside.

Insert 3L of the above-mentioned seed culture solution into the sterilized 25L fermentation basal medium (fermentation tank volume 50L), the culture conditions are as in Example 1, after 20h of fermentation, the yeast cells in the obtained fermentation solution are 5.11% (dry weight), seaweed Sugar content 6.2%.

The above fermentation broth was centrifuged at 3500r/min for 10min to collect the yeast milk. Discard the supernatant from the centrifuge tube and remove the yeast milk. In 1000mL of distilled water, add 38g of sodium chloride, 10g of calcium chloride, and 28g of maltose, dissolve completely, sterilize at 100°C, control the temperature to 30°C in a water bath, add to 1000g of yeast milk, and place in a 10L fermenter. The culture temperature was controlled at 35°C, and the pH was adjusted to 5.2 with 10% Na ₂ CO ₃ solution; under the condition of 1.20vvm ventilation, the culture time was 3 hours, the yeast sludge was collected by centrifugation, and the trehalose content in the yeast cell dry weight was determined to be 16.22%.

Claims (4)

1. A method for improving the trehalose content of yeast cells, centrifuging the yeast fermentation culture liquid to obtain yeast milk with a cell concentration of 13-17%, adding an aqueous solution of maltose, sodium chloride and calcium chloride to the yeast milk, and yeast The mass volume ratio of milk and water is 1:1, the amount of maltose, sodium chloride and calcium chloride accounted for the total mass percentage of yeast milk is 2.4-3.0% maltose, 3.2-4.0% sodium chloride and 1.0% calcium chloride respectively , cultured at 35°C, pH 5.2, and 1.0-1.2vvm ventilation conditions for 2-3 hours, centrifuged to collect the yeast sludge, and determined the trehalose content in the yeast to be 16-17%, based on the dry weight of yeast cells; The yeast is Saccharomyces cerevisiae; The mass-to-volume ratio is in g/mL or kg/L. 2. the method for improving yeast cell trehalose content as claimed in claim 1, is characterized in that the preparation of described yeast fermentation medium, the steps are as follows: (1) culture medium Seed medium: glucose 20g, KH ₂ PO ₄ 1g, (NH ₄ ) ₂ SO ₄ 5g, MgSO ₄ 7H ₂ O 0.2g, ZnSO ₄ 7H ₂ O 0.05g, pH 5.4, distilled water 1000mL; Fermentation medium: ① Fermentation base medium Corn steep liquor (containing 28wt% solids) 20%, glucose 1%, nutrient salt solution 5%, water 74%, all in volume ratio; wherein, the formula of nutrient salt solution is KH ₂ PO ₄ 13.0g, (NH ₄ ) ₂ SO ₄ 94.16g, MgSO ₄ 7H ₂ O 1.4g, ZnSO ₄ 7H ₂ O 0.28g, add water to make 1000mL; ②Fed-in feed components during fermentation Sugar solution: Glucose made into 250g/L solution; Nutrient salt solution: KH ₂ PO ₄ 13.0g, (NH ₄ ) ₂ SO ₄ 94.16g, MgSO ₄ 7H ₂ O 1.4g, ZnSO ₄ 7H ₂ O 0.28g , add water to make 1000mL; lye: 100g/L Na ₂ CO ₃ solution; (2) Seed cultivation The yeast strain is Saccharomyces cerevisiae, code name: AS 2.146, AS 2.502, AS 2.503 or AS 2.504; put the yeast slant strain into a 250mL Erlenmeyer flask containing 20mL seed medium, shake at 30°C and 200r/min Cultivate in the bottle for 24 hours, transfer to a 500mL Erlenmeyer flask containing 80mL of seed medium, and culture in a shaker flask at 30°C and 200r/min for 16h to obtain a seed culture solution for later use; (3) Fermentation culture Sterilize the real tank in a 50L fermenter with 25L of fermentation basic medium. The sterilization condition is 120°C for 30 minutes; then when the temperature is lowered to 32°C, add 3L of seed culture solution, adjust the pH to 5.2, and control the culture temperature 30°C, add sugar solution and nutrient salt solution, control the total fermentation time to 18-21h, add nutrient salt solution 3-5 hours before the end of fermentation, add sugar solution according to the growth of yeast, make the fermentation system The glucose content is always controlled below 0.5%; during the fermentation process, the pH is controlled at 5.2, and the stirring speed is controlled at 200r/min. 3. The method for increasing the trehalose content of yeast cells as claimed in claim 2, characterized in that the culture temperature in the step (3) fermentation culture is controlled as follows: 1-8 hours of fermentation time, the culture temperature is 30 ° C, and the culture time is 9-15 hours The temperature is 32°C, and the culture temperature is 35°C from 16h to the end of fermentation. 4. the method for improving yeast cell trehalose content as claimed in claim 2 is characterized in that step (3) ventilation in fermentation culture: control sterile air volume and fermented liquid volume ratio per minute at 1-15h in fermentation time 2.5vvm, during the trehalose accumulation stage from 16h to the end of fermentation, adjust the valve to reduce the ventilation to 1.2vvm.

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