Method for screening stress-resistant yeast suitable for very high gravity brewing

Abstract

Provided in the present application is a method for screening stress-resistant yeast suitable for very high gravity brewing. In the method, the stress-resistant yeast suitable for very high gravity brewing is obtained by performing screening on the basis of the trehalose content in the yeast and the residual sugar concentration under stress conditions after yeast activation.

Description

Screening method of stress-resistant yeast suitable for ultra-high gravity brewing

[0001] The present application claims priority to the Chinese patent application No. 202510381552.X filed on March 28, 2025 and entitled "A rapid screening method of stress-resistant yeast suitable for ultra-high gravity brewing", the whole content of which is incorporated herein by reference. TECHNICAL FIELD

[0002] The present application belongs to the field of biotechnology, and in particular relates to a screening method of stress-resistant yeast suitable for ultra-high gravity brewing. BACKGROUND

[0003] Yeast produces alcohol and flavor substances by metabolizing wort sugar in the beer brewing process, which has an important influence on the taste of beer. With the increase of wort concentration, the stress on yeast during fermentation is increasing, and a series of problems may occur in brewing, such as incomplete fermentation, slow or blocked fermentation, and decreased yeast activity and viability.

[0004] In the process of ultra-high gravity brewing (fermentation process with wort concentration greater than 18 °P), the increase of alcohol concentration and osmotic pressure has a certain inhibitory effect on the growth and metabolism of yeast, which leads to the decrease of yeast viability in the middle and late stages of fermentation, resulting in slow or blocked fermentation. Therefore, beer yeast for ultra-high gravity brewing requires good tolerance to high alcohol concentration and high osmotic pressure, and high stress resistance.

[0005] However, whether a strain of yeast can be used for ultra-high gravity brewing usually needs to be determined by fermentation test, and the whole fermentation process from yeast expansion to the end of fermentation takes about one month. If you want to select yeast that can be used for ultra-high gravity brewing from multiple strains of yeast, you need to conduct fermentation test for each strain of yeast, which is a lot of work and time-consuming. Therefore, a method for rapid screening of yeast is needed, which is efficient, time-saving and does not require fermentation test. SUMMARY

[0006] In view of at least one deficiency in the prior art, the present application provides a screening method of stress-resistant yeast suitable for ultra-high gravity brewing.

[0007] In order to achieve the above-mentioned purpose, the present application provides a screening method of stress-resistant yeast suitable for ultra-high gravity brewing, which selects stress-resistant yeast suitable for ultra-high gravity brewing by measuring the trehalose content in yeast and the residual sugar degree under stress conditions after yeast activation.

[0008] In some embodiments, the yeast is activated by culturing at 25℃ for 48h.

[0009] In some embodiments, 10%-30% sorbitol is used as the stress condition.

[0010] In some embodiments, the stress medium used in the stress condition is YPD+(10%-30%) sorbitol.

[0011] In some embodiments, no alcohol is added in the stress medium.

[0012] In some embodiments, the trehalose content in the stress-resistant yeast suitable for ultra-high gravity brewing should be ≥30 mg / g.

[0013] In some embodiments, the residual gravity of the stress-resistant yeast suitable for ultra-high gravity brewing should be <8 °P.

[0014] In some embodiments, the trehalose content in the yeast is calculated by the following formula: trehalose content (mg / g fresh weight) = (Cstandard x V1) x ΔAtrehalose ÷ (Astandard-Ablank) ÷ 2 x 342.3 ÷ 180.16 ÷ (W x V1 ÷ V) x D = 0.95 x ΔAtrehalose ÷ (Astandard-Ablank) ÷ W x D;

[0015] wherein 342.3 is the molecular weight of trehalose; 180.16 is the molecular weight of glucose; Cstandard is the concentration of the glucose standard, 1 mg / mL; V is the volume of the yeast extract, 1 mL; V1 is the volume of the added yeast extract sample, 0.01 mL; W is the fresh weight of the yeast in the yeast extract, g; 2 is the decomposition of 1 molecule of trehalose into 2 molecules of glucose; and D is the dilution factor, 1 for no dilution.

[0016] In some embodiments, the residual gravity detection method is as follows:

[0017] The activated yeast is transferred to a deep-well plate containing 28 °P wort, with a yeast number of 36 x 10 6 6 / mL, and the residual gravity of the yeast is detected after 96 h of culture at 25 °C.

[0018] In some embodiments, the yeast is Lager yeast.

[0019] Compared with the prior art, the advantages and positive effects of the present application are as follows:

[0020] The screening method for stress-resistant yeast suitable for ultra-high gravity brewing provided by the present application does not need to perform fermentation test, the time used is short, can be shortened to 5-6 days, and all the culture and detection can be completed in a 96-well plate. Compared with the prior art, the method provided by the present application is short in time and simple in operation, and is convenient for quickly screening the yeast suitable for ultra-high gravity brewing from a large number of yeasts, and can greatly reduce the screening workload. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 is a schematic diagram of the results of detecting trehalose content in yeast under alcohol stress in the present application;

[0022] Figure 2 is a schematic diagram of the results of detecting trehalose content in yeast under different stress conditions according to the embodiments of the present application;

[0023] Figure 3 is a schematic diagram of the results of detecting trehalose content in yeast under sorbitol stress according to the embodiments of the present application;

[0024] Figure 4 is a schematic diagram of fermentation degree and residual sugar degree detection according to the embodiments of the present application. DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0026] Trehalose is an isomer of maltose, which is a non-reducing sugar and is formed by the connection of two glucose molecules through a non-reducing end. Trehalose exists not only in the cytoplasm of yeast, but also in the cell wall and cell membrane. It has been proved that trehalose can be transported from the cytoplasm to the cell membrane by a transport factor. Trehalose is mainly used to stabilize the cell membrane, especially when the cell is under stress.

[0027] During fermentation, yeast is subjected to various stresses. When yeast resists dehydration, cold, heat, hunger and osmotic pressure, alcohol, the trehalose content in yeast increases. Trehalose can enhance the tolerance of yeast to alcohol, osmotic pressure, cold, heat and hunger.

[0028] In view of the inhibitory effect of high alcohol concentration and high osmotic pressure stress on yeast growth and metabolism in ultra-high concentration brewing process, the trehalose content is positively correlated with the survival and fermentation capacity of yeast in this environment, which can directly reflect the stress resistance level. At the same time, combined with the detection of residual sugar degree, the sugar reduction capacity of yeast can be comprehensively evaluated, which is highly related to fermentation degree. The two are used together to effectively predict the adaptability of yeast under ultra-high concentration brewing conditions.

[0029] Based on the above research, the present application provides a screening method for stress-resistant yeast suitable for ultra-high concentration brewing. The stress-resistant yeast suitable for ultra-high concentration brewing is screened by the measured trehalose content in yeast and the measured residual sugar degree under stress conditions after yeast activation.

[0030] The screening method for stress-resistant yeast suitable for ultra-high concentration brewing provided by the present application comprises the following steps:

[0031] The yeast to be screened is activated and cultured;

[0032] The activated yeast is cultured under stress conditions, and the trehalose content in the yeast is determined;

[0033] The activated yeast is cultured in wort, and the residual sugar content of the yeast is determined;

[0034] According to the determination results of the trehalose content and the residual sugar content in the yeast, the stress-resistant yeast suitable for ultra-high concentration brewing is screened.

[0035] The screening method in the present application uses the trehalose content and the residual sugar content in the yeast as screening indexes, without the need for complete fermentation test, and can shorten the screening process to 5-6 days. Compared with the prior art, the screening period is significantly shortened, the operation steps are simplified, the stress-resistant yeast with the required performance can be quickly screened from a large number of yeasts, the screening workload is greatly reduced, and the screening efficiency is improved.

[0036] In some embodiments, the activation culture is to activate the yeast by culturing at 25°C for 24-48h.

[0037] In some embodiments, the stress condition is 10%-30% sorbitol. Among them, 10%-30% is the mass-volume ratio, that is, the mass of sorbitol to the volume of the culture medium.

[0038] In some embodiments, the stress culture medium used under the stress condition is YPD+(10%-30%) sorbitol. 10-30g of sorbitol is added per 100mL of YPD.

[0039] In some embodiments, no alcohol is added to the stress culture medium.

[0040] In some embodiments, the trehalose content in the stress-resistant yeast suitable for ultra-high concentration brewing should be ≥30mg / g.

[0041] In some embodiments, the trehalose content in the yeast is calculated by the following method: trehalose content (mg / g fresh weight) = (Cstandard x V1) x ΔAtrehalose ÷ (Astandard-Ablank) ÷ 2 x 342.3 ÷ 180.16 ÷ (W x V1 ÷ V) x D = 0.95 x ΔAtrehalose ÷ (Astandard-Ablank) ÷ W x D;

[0042] Wherein, 342.3 is the molecular weight of trehalose; 180.16 is the molecular weight of glucose; Cstandard is the concentration of glucose standard, 1mg / mL; V is the volume of yeast extract, 1mL; V1 is the volume of added yeast extract sample, mL; W is the fresh weight of yeast in the yeast extract, g; 2 is that 1 molecule of trehalose is decomposed into 2 molecules of glucose; D is the dilution multiple, which is 1 without dilution;

[0043] △Atrehalose = Ameasured - Acontrol, Ameasured is the absorbance value of the detection reagent added with the yeast extract sample after hydrolysis by trehalase, Acontrol is the absorbance value of the detection reagent added with the yeast extract sample without hydrolysis by trehalase, and △A trehalose represents the absorbance value change of the glucose produced by the hydrolysis of trehalose in the yeast extract sample. In this way, the background value of free glucose can be corrected to obtain the trehalose content, and meanwhile, the interference of other disaccharides (such as maltose and lactose, etc.) on the detection result can be avoided.

[0044] Astandard is the absorbance value of the detection reagent added with the glucose standard, and Ablank is the absorbance value of the detection reagent without adding the glucose standard and the yeast extract sample. The absorbance value of the glucose standard is calculated by Astandard - Ablank, and then the glucose content obtained by the hydrolysis of trehalose in the yeast extract sample is multiplied by the glucose content in the glucose standard, i.e. △A trehalose ÷ (Astandard - Ablank).

[0045] The above detection reagent is a conventional reagent for detecting the absorbance value of glucose, which is not limited in the present application.

[0046] The above absorbance value can be detected at a wavelength of 510 nm.

[0047] In some embodiments, the volume V1 of the yeast extract sample added during detection is 0.01 mL, but it can be understood that V1 can also be adjusted according to the detection result of the absorbance value: if △A trehalose is near zero and Ameasured is lower than 1, the sample addition amount can be increased, i.e. V1 is increased, or the yeast mass W in the yeast extract is increased (such as 0.2 g or more). The adjusted V1 or W is re-substituted into the above calculation formula for calculation.

[0048] It can be understood that the detection and calculation method of the trehalose content in the yeast is not limited to the above embodiments, and other methods in the prior art can also be used to achieve the same.

[0049] In some embodiments, the residual sugar degree of the stress-resistant yeast suitable for ultra-high concentration brewing is < 8 °P.

[0050] In some embodiments, the residual sugar degree detection method is as follows:

[0051] The activated yeast is transferred to 28 °P wort, and the yeast number is 36 × 10 6 After 96 h of culture at 25 °C, the residual sugar degree of the yeast is detected.

[0052] In the above embodiments, a deep well plate can be used as the culture container, and the activated yeast is transferred to the deep well plate containing 28 °P wort. The deep well plate is a 96-well plate, each well has a volume of 1.6 mL, the bottom of the well is circular, and the cross-sectional shape of the well is square.

[0053] In some embodiments, the ultra-high concentration brewing refers to a fermentation process with wort concentration greater than 18 °P.

[0054] In some embodiments, the yeast is Lager yeast.

[0055] Yeast stress resistance screening

[0056] The present application determines and analyzes the stress resistance of yeast under different stress conditions, and screens suitable stress conditions through the following tests.

[0057] The yeast A-D to be screened are all Lager yeast, i.e., four different Lager yeasts are selected.

[0058] The activation medium used is YPD (Yeast Extract Peptone Dextrose) medium, and the specific components are 1% yeast powder, 2% peptone, and 2% glucose; the stress medium used is YPD + sorbitol (10%-30% by mass / volume), YPD + alcohol (5%-9% by volume / volume).

[0059] 1.1 Preparation of stress-resistant yeast

[0060] The yeast A-D to be screened is cultured at 25°C for 48 h for activation;

[0061] The activated yeast A-D is transferred to the culture medium under different stress conditions and cultured at 25°C for 48 h to obtain stress-resistant yeast.

[0062] 1.2 Detection of trehalose content

[0063] The trehalose content of the cultured stress-resistant yeast is detected, and the specific steps are as follows:

[0064] Yeast pretreatment:

[0065] 1 mL of culture solution is centrifuged at 5000 rpm for 5 min to remove the supernatant, and washed twice with 4°C physiological saline. Record the weight W of the yeast in the test tube after washing, add 1 mL of about 80°C distilled water; ice bath ultrasonic bacterial or cell crushing (ice bath, power 20% or 200W, ultrasonic 3s, interval 10s, repeat 30 times), room temperature shaking extraction for 30 min, 8000 rpm room temperature (25°C) centrifugation for 10 min, take the supernatant, and obtain the yeast extract.

[0066] Machine detection:

[0067] 1, enzyme label instrument preheating 30 min, set temperature at 25℃, set wavelength to 510 nm.

[0068] 2, can select several samples before doing the experiment, find out the dilution multiple D suitable for the sample detection this time.

[0069] 3, in 96 well plate in turn add:

[0070]

Note

[0071] 2. if △A is around zero, and A determination tube is less than 1, can increase the sample amount V1 (such as increased to 40 μL, then the reagent three phase should be reduced), or increase the sample sampling quality W (such as 0.2 g or more), then the changed V1 and W need to be substituted into the calculation formula to recalculate.

[0072] In the above table, reagent one is trehalase; reagent two is glucose detection enzyme reagent, for example, peroxidase, glucose oxidase, 4-aminoantipyrine, sodium azide; reagent three is phosphate buffer with PH = 7; reagent four is color developing agent, for example, phenol solution.

[0073] Result calculation: trehalose content (mg / g fresh weight) = (C standard × V1) × △A trehalose ÷ (A standard-A blank) ÷ 2 × 342.3 ÷ 180.16 ÷ (W × V1 ÷ V) × D = 0.95 × △A trehalose ÷ (A standard-A blank) ÷ W × D

[0074] Wherein, 342.3 is the molecular weight of trehalose; 180.16 is the molecular weight of glucose; C standard is the concentration of glucose standard, 1 mg / mL; V is the volume of yeast extract, 1 mL; V1 is the volume of added yeast extract sample, 0.01 mL; W is the fresh weight of yeast in yeast extract, g; 2 is that 1 molecule of trehalose is decomposed into 2 molecules of glucose; D is the dilution multiple, not diluted is 1.

[0075] The yeast growth turbidity (OD600) and trehalose content detection data under different stress conditions are shown in Tables 1-4.

[0076] Table 1 Yeast growth turbidity (OD600) under alcohol stress

[0077] Table 2 Trehalose content (mg / g) in yeast under alcohol stress

[0078] As can be seen from the data in Table 1 and Table 2, the growth rate of the four yeasts in the medium with different alcohol contents was obviously affected as the alcohol content in the YPD medium increased. As can be seen from Figure 1, the content of trehalose in the yeasts increased obviously under the stress condition of adding alcohol to the YPD medium, but the yeasts could not be distinguished from each other.

[0079] Table 3 Yeast growth turbidity (OD600) under sorbitol stress

[0080] Table 4 Trehalose content (mg / g) in yeasts under sorbitol stress

[0081] As can be seen from the data in Table 3 and Table 4, the growth of the four yeasts was inhibited when different contents of sorbitol were added to the YPD medium as the stress condition, but the content of trehalose in the yeasts could not be affected. As can be seen from the data in Figure 3, the content of trehalose in the four yeasts was obviously divided into two groups. Among them, after being cultured in the YPD + 10% sorbitol, YPD + 20% sorbitol and YPD + 30% sorbitol medium, the yeasts A and B were divided into one group, and the yeasts C and D were divided into another group, and the yeasts in the two groups were distinguished by taking 30 mg / g of trehalose as the standard.

[0082] In order to further verify the stress caused by adding alcohol, which is not conducive to the distinction of the content of trehalose in the yeasts, YPD + 30% sorbitol + 5% alcohol group was also set. As shown in the rightmost column of data in Table 4 and Figure 2, under this culture condition, the yeasts A, C and D were divided into one group, and the yeast B was alone in a group, and the content of trehalose in the yeasts A and B increased obviously and the increasing amplitude was different from the previous groups, but since this method could not determine a unified screening standard for trehalose, alcohol was not selected as the stress condition.

[0083] 1.3 Screening of stress conditions

[0084] Different stress conditions were made by adding different contents of sorbitol (10%-30%) and alcohol (5%-9%) to the YPD medium. The yeasts were cultured under different stress conditions, and the OD value and trehalose of the yeasts after culture were detected. Through the detection results, it was finally determined that sorbitol (10-30%) was used as the stress condition for yeast screening. Under this condition, the content of trehalose in the yeasts was relatively stable, and 30 mg / g of trehalose content could be determined as the standard. That is, when the content of trehalose detected in the yeasts cultured under this stress condition is greater than 30 mg / g, it is considered that the yeasts have strong stress resistance and have the potential for ultra-high concentration fermentation.

[0085] Based on the above analysis, by comparing the effects of alcohol and sorbitol on yeast stress, alcohol is volatile, and the concentration of alcohol will change during stress culture, resulting in changes in osmotic pressure. In addition, the trehalose content is not significantly different under alcohol stress conditions. Therefore, sorbitol is selected as the stress condition for the test. In addition, YPD + 10% sorbitol, YPD + 20% sorbitol, and YPD + 30% sorbitol media can be selected for stress condition tests, and no alcohol is added to the media.

[0086] Yeast sugar reduction ability screening

[0087] The sugar reduction ability of yeast is highly correlated with the fermentation degree after fermentation of the yeast. By screening the sugar reduction ability, yeast with strong sugar reduction ability is selected. By two-step screening of stress resistance and sugar reduction, yeast suitable for ultra-high concentration brewing process can be screened.

[0088] Specifically, the yeast to be screened is cultured at 25°C for 48h for activation;

[0089] The activated yeast is transferred to a deep well plate containing 28°P wort, and the yeast number is 36x10 6 ML, 25°C for 96h, and the residual sugar degree is detected at the end of the culture.

[0090] Table 5 Residual sugar degree at the end of deep well plate culture

[0091] As can be seen from the data in Figure 3, the increase in trehalose content of A and B yeast is significantly lower than that of C and D yeast when the osmotic pressure rises. Therefore, it is believed that C and D yeast are more resistant to stress. In combination with the residual sugar degree data in Table 5, it can be seen that C and D yeast have stronger sugar reduction ability. Both C and D yeast have strong stress resistance and sugar reduction ability, and are more suitable for ultra-high concentration brewing.

[0092] Verification of screening results

[0093] Test purpose: Verify whether C and D yeast obtained by screening trehalose content and residual sugar degree under stress conditions (10%-30% sorbitol) are more suitable for ultra-high concentration brewing.

[0094] Four strains of yeast were fermented in 13°P and 28°P wort, respectively.

[0095] 13°P wort, full tank yeast number 20x10 6 ML, 12°C for 14 days;

[0096] 28°P wort, full tank yeast number 36x10 6 ML, 14°C for 14 days;

[0097] Table 6 Cold storage wine data

[0098] As can be seen from the data in Table 6, the fermentation degrees of the cold storage wine after fermentation of the four strains of yeast in 13 °P wort are all about 66%, and the maturity indexes are good. After fermentation in 28 °P wort, the fermentation degrees of A and B strains of yeast are about 62%, and the fermentation degrees of C and D strains of yeast are still above 66%, and the fermentation degrees are highly negatively correlated with the residual sugar degrees, as shown in Figure 4. The fermentation results show that C and D yeasts are more suitable for ultra-high concentration brewing. This is consistent with the result that C and D yeasts obtained through screening by trehalose content and residual sugar degree indexes are suitable for ultra-high concentration brewing, thereby verifying the reliability and accuracy of the screening method.

Claims

1. A method for screening of stress resistant yeast suitable for ultra-high gravity brewing, characterized in that, Screening of stress-resistant yeast suitable for ultra-high gravity brewing by measuring trehalose content in yeast and residual sugar degree after yeast activation under stress conditions.

2. The screening method according to claim 1, characterized in that, The yeast is activated by culturing at 25℃ for 48h.

3. The screening method according to claim 1, characterized by, 10%-30% sorbitol is used as stress condition.

4. The screening method according to claim 3, characterized in that, YPD+(10%-30%) sorbitol is used as stress medium under stress conditions.

5. The screening method according to claim 4, characterized in that, No alcohol is added in the stress medium.

6. The screening method according to claim 1, wherein, The trehalose content in stress-resistant yeast suitable for ultra-high gravity brewing should be ≥30mg / g.

7. The screening method according to claim 1, wherein, The residual sugar degree of stress-resistant yeast suitable for ultra-high gravity brewing should be <8°P.

8. The screening method according to any one of claims 1 to 7, characterized in that, The ultra-high gravity brewing refers to fermentation process with wort concentration greater than 18°P.

9. The screening method according to claim 1, wherein, The trehalose content in yeast is calculated by the following method: trehalose content (mg / g fresh weight) = (Cstandard x V1) x △Atrehalose ÷ (Astandard-Ablank) ÷ 2 x 342.3 ÷ 180.16 ÷ (W x V1 ÷ V) x D = 0.95 x △Atrehalose ÷ (Astandard-Ablank) ÷ W x D Wherein, 342.3 is the molecular weight of trehalose; 180.16 is the molecular weight of glucose; Cstandard is the concentration of glucose standard, 1mg / mL; V is the volume of yeast extract, 1mL; V1 is the volume of added yeast extract sample, 0.01mL; W is the fresh weight of yeast in yeast extract, g; 2 is 1 molecule of trehalose decomposed into 2 molecules of glucose; D is the dilution factor, 1 for no dilution. The method for detecting residual sugar degree is as follows:

10. The screening method according to claim 1, wherein, The yeast is Lager yeast. Activated yeast was transferred to 28°P wort, yeast number 36 x 10 6 After 96h incubation at 25°C, the residual sugar of the yeast was measured.

11. The screening method according to claim 1, wherein, ​

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