Rapid screening method of stress-resistant yeast suitable for ultrahigh-concentration brewing

Abstract

The invention provides a rapid screening method of stress-resistant yeast suitable for ultrahigh-concentration brewing, and belongs to the technical field of biology. According to the method, the ultrahigh-concentration brewed stress-resistant yeast is rapidly screened according to the content of trehalose in yeast measured under a stress condition after the yeast is activated and the measured residual sugar degree. According to the method, a fermentation test is not needed, the used time is short and can be shortened to 5 days, and all culture and detection can be completed in a 96-well plate.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a rapid screening method for stress-resistant yeasts suitable for ultra-high gravity brewing. Background Art

[0002] Trehalose is an isomer of maltose, a non-reducing sugar, which is composed of two glucose molecules connected by 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 demonstrated 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.

[0003] During the fermentation process, yeast is subjected to various stresses. When yeast resists dehydration, cold, heat, starvation, and osmotic pressure, alcohol, the trehalose content in yeast increases. Trehalose can enhance the tolerance of yeast to alcohol, osmotic pressure, cold, heat, and starvation. As the wort concentration increases, the stress on yeast during fermentation becomes greater, and a series of problems will occur in brewing, such as incomplete fermentation, slow or blocked fermentation, and a decrease in yeast activity and viability.

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

[0005] However, to determine whether a yeast strain can perform high-gravity brewing, fermentation tests usually need to be carried out. The entire fermentation process from yeast propagation to the end of fermentation takes about 1 month. If it is necessary to screen out yeast strains that can perform high-gravity brewing from multiple yeast strains, fermentation tests need to be carried out for each yeast strain, which is a large workload and time-consuming. Therefore, a method for rapid screening of yeast that is efficient, time-saving and does not require fermentation tests is needed. Summary of the Invention

[0006] The present invention provides a rapid screening method for stress-resistant yeasts suitable for ultra-high gravity brewing. This method does not require fermentation tests, takes a short time, can be shortened to 5 days, and all culturing and detections can be completed in a 96-well plate.

[0007] To achieve the above object, the present invention provides a rapid screening method for stress-resistant yeasts suitable for ultra-high gravity brewing, which rapidly screens out stress-resistant yeasts for ultra-high gravity brewing by measuring the trehalose content and the remaining sugar degree in yeast measured under stress conditions after yeast activation.

[0008] Preferably, the yeast is activated by culturing at 25 °C for 48 h.

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

[0010] Preferably, the stress medium used under the stress condition is YPD+(10%-30%) sorbitol.

[0011] Preferably, no alcohol is added to the stress medium.

[0012] Preferably, the trehalose content in the yeast is calculated as follows:

[0013] 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

[0014] Among them, 342.3 is the molecular weight of trehalose; 180.16 is the molecular weight of glucose; C standard is the concentration of the glucose standard, 1 mg / mL; V is the volume of the extraction solution added, 1 mL; V1 is the volume of the sample added, 0.01 mL; W is the fresh weight of the sample, g; 2 is that 1 molecule of trehalose is decomposed into 2 molecules of glucose; D is the dilution factor, which is 1 if not diluted.

[0015] Preferably, the trehalose content in the yeast should be ≥ 30 mg / g.

[0016] Preferably, the method for detecting the residual sugar degree is as follows:

[0017] Transfer the activated yeast to a deep well plate containing 28°P wort, with the yeast count of 36×10 6 cells / mL, and detect the residual sugar degree of the yeast after culturing at 25 °C for 96 h.

[0018] Preferably, the residual sugar degree of the yeast < 8°P.

[0019] Preferably, the yeast is Lager yeast.

[0020] Compared with the prior art, the advantages and positive effects of the present invention are as follows:

[0021] The rapid screening method for stress-resistant yeast suitable for ultra-high gravity brewing provided by the present invention does not require fermentation tests, takes a short time, can be shortened to 5 days, and all cultures and detections can be completed in a 96-well plate. Compared with the prior art, the method provided by the present invention takes a short time and is easy to operate, facilitating the rapid screening of yeast suitable for high-gravity brewing from a large number of yeasts, and can greatly reduce the screening workload. Description of the Drawings

[0022] Figure 1 Schematic diagram for detecting the trehalose content in yeast under alcohol stress provided by an embodiment of the present invention;

[0023] Figure 2 Schematic diagram for detecting the trehalose content in yeast under sorbitol stress provided by an embodiment of the present invention;

[0024] Figure 3 Schematic diagram for detecting the trehalose content in yeast under sorbitol stress provided by an embodiment of the present invention;

[0025] Figure 4 Schematic diagram for detecting the fermentation degree and the remaining sugar degree in a 96-well deep plate provided by an embodiment of the present invention. Detailed implementation manners

[0026] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0027] Example 1 Screening of stress-resistant yeasts suitable for high-gravity brewing

[0028] The yeasts A-D to be screened are all Lager yeasts, that is, four different Lager yeasts are selected.

[0029] The activation medium used is YPD medium, and the specific components are 1% yeast extract, 2% peptone, and 2% glucose; the stress media used are YPD + sorbitol (10% - 30%) and YPD + alcohol (5% - 9%).

[0030] 1.1 Preparation of stressed yeasts

[0031] The yeasts A-D to be screened are cultured at 25 °C for 48 h for activation;

[0032] The activated yeasts A-D are respectively transferred to media under different stress conditions and cultured at 25 °C for 48 h to obtain stressed yeasts.

[0033] 1.2 Screening of stress conditions

[0034] Stress conditions were created by adding different amounts of sorbitol (10%-30%) and alcohol (5%-9%) to the YPD medium. Yeast was cultured under different stress conditions, and the OD value and trehalose of the cultured yeast 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 trehalose content of yeast was relatively stable, and 30 mg / g of trehalose content was determined as the standard. That is, for the yeast cultured under this stress condition, when the detected trehalose content is greater than 30 mg / g, we consider its stress resistance ability to be strong and it has the potential for high-concentration fermentation.

[0035] 1.3 Detection of trehalose content

[0036] The trehalose content of the cultured stress yeast was detected, and the specific steps are as follows:

[0037] Yeast pretreatment:

[0038] Absorb 1 mL of the culture solution, centrifuge at 5000 rpm for 5 min to remove the supernatant, and add 4°C physiological saline to wash twice. Record the weight W of the yeast in the test tube after washing, and add 1 mL of distilled water at about 80°C; ultrasonically break the bacteria or cells in an ice bath (ice bath, power 20% or 200 W, ultrasonic for 3 s, interval 10 s, repeat 30 times), shake at room temperature for 30 min, centrifuge at 8000 rpm at room temperature (25°C) for 10 min, and take the supernatant.

[0039] Detection on the machine:

[0040] 1. Preheat the microplate reader for 30 min, set the temperature at 25°C, and set the wavelength to 510 nm.

[0041] 2. Before doing the experiment, several samples can be selected to find the dilution factor D suitable for the samples in this detection.

[0042] 3. Add the following in the 96-well plate in sequence:

[0043]

[0044]

Note

[0045] 2. If △A hovers around zero and the A measurement tube is less than 1, the sample volume V1 can be increased (such as increased to 40 μL, then the reagent three should be reduced accordingly), or the sample sampling mass W can be increased (such as 0.2 g or more), and the changed V1 and W need to be substituted into the calculation formula for recalculation.

[0046] Result calculation:

[0047] Content of trehalose (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

[0048] Among them, 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 extraction solution added, 1 mL; V1 is the volume of sample added, 0.01 mL; W is the fresh weight of the sample, g; 2 means that 1 molecule of trehalose decomposes into 2 molecules of glucose; D is the dilution factor, which is 1 if not diluted.

[0049] The detection data of yeast growth turbidity (OD600) and trehalose content under different stress conditions are shown in Tables 1 - 4.

[0050] Table 1 Yeast growth turbidity (OD600) under alcohol stress

[0051]

[0052] Table 2 Yeast trehalose (mg / g) under alcohol stress

[0053] Yeast strain YPD medium YPD + 5% alcohol YPD + 7% alcohol A 11.4 27.9 36.8 B 10.4 24.7 28.7 C 32.9 36.6 43.7 D 23.7 28.6 36.0

[0054] It can be seen from the data in Tables 1 - 2 that when alcohol is added to the YPD medium as an anti - stress condition, with the increase of alcohol addition amount, the growth rates of the 4 strains of yeast in the medium with different alcohol contents are significantly affected (since the growth of yeast in YPD + 9% alcohol is less, trehalose detection was not carried out). And, combined with Figure 1 it can be known that when alcohol is added to the YPD medium as an anti - stress condition, the content of trehalose in yeast shows an obvious upward trend, but there is no significant difference among different yeasts.

[0055] Table 3 Yeast growth turbidity (OD600) under sorbitol stress

[0056]

[0057] Table 4 Yeast trehalose (mg / g) under sorbitol stress

[0058]

[0059] It can be seen from the data in Tables 3 - 4 that when different contents of sorbitol are added to the YPD medium as an anti - stress condition, the growth of the 4 strains of yeast is also inhibited, but the amount of yeast does not affect the detection of trehalose content. And from Figure 2It can be seen from the data that the trehalose content is significantly divided into two groups. Among them, after culturing in YPD + 10% sorbitol, YPD + 20% sorbitol, and YPD + 30% sorbitol media, yeast A and B are in one group, and yeast C and D are in another group. The two groups are distinguished by a trehalose standard of 30 mg / g.

[0060] To further verify the stress caused by the addition of alcohol, and since the change in yeast trehalose is not conducive to the differentiation of yeast, a group of YPD + 30% sorbitol + 5% alcohol was also set up. Under this culture condition, yeast A, C, and D are in one group, and yeast B is in a separate group. The trehalose content of yeast A and B increased significantly and by different amounts compared to the previous groups. However, since a unified trehalose standard cannot be determined by this method, this stress method is not selected.

[0061] In summary, by comparing the stress effects of alcohol and sorbitol on yeast, due to the volatilization of alcohol, the concentration will volatilize during stress culture, resulting in changes in osmotic pressure, and the difference in trehalose content under alcohol stress conditions is small. Therefore, sorbitol addition is selected as the stress condition for the experiment. Moreover, in the stress condition experiment, YPD + 10% sorbitol, YPD + 20% sorbitol, and YPD + 30% sorbitol media can be selected, and no alcohol is added to the media.

[0062] Example 2 Screening of Yeast Glucose-Lowering Ability

[0063] The glucose-lowering ability of yeast has a high correlation with the fermentation degree after fermentation of the yeast. By screening for glucose-lowering ability, yeasts with strong glucose-lowering ability can be selected. Through two-step screening of stress resistance and glucose-lowering, yeasts capable of high-gravity fermentation can be screened out.

[0064] Specifically, the yeast to be tested is cultured at 25 °C for 48 h for activation;

[0065] The activated yeast is transferred to a deep-well plate containing 28°P wort, with a yeast count of 36×10 6 cells / mL, and cultured at 25 °C for 96 h. After the culture ends, the remaining sugar content is measured.

[0066] Table 5 Remaining Sugar Content after Deep-Well Plate Culture

[0067]

[0068] From Figure 3 the data, it can be seen that the increase in trehalose content of yeast A and B is significantly lower than that of yeast C and D when the osmotic pressure rises. We believe that yeast C and D are more stress-resistant. At the same time, combining the remaining sugar content data in Table 5, it can be seen that yeast C and D have stronger glucose-lowering ability. The combination of the two, that is, yeast C and D with strong stress resistance and glucose-lowering ability, is more suitable for high-gravity brewing.

[0069] Performance test

[0070] Test objective: To verify whether Yeast C and Yeast D screened by trehalose content and residual sugar content are more suitable for high-gravity brewing under stress conditions (10%-30% sorbitol).

[0071] Fermentation tests were carried out on 4 strains of yeast in 13°P and 28°P wort respectively.

[0072] 13°P wort, the number of yeast cells at pitching is 20×10 6 cells / mL, fermented at 12°C for 14 days;

[0073] 28°P wort, the number of yeast cells at pitching is 36×10 6 cells / mL, fermented at 14°C for 14 days;

[0074] Table 6 Data of cold-stored beer

[0075]

[0076] The data of cold-stored beer are shown in Table 6. It can be seen from the data in the table that after fermentation in 13°P wort, the fermentation degrees of the 4 strains of yeast are all about 66%, and the maturity index is good. After fermentation in 28°P wort, the fermentation degrees of Yeast A and Yeast B are about 62%, and the fermentation degrees of Yeast C and Yeast D are still above 66%. Moreover, the fermentation degree and the residual sugar content are highly negatively correlated, see Figure 4 . The fermentation results show that Yeast C and Yeast D are more suitable for high-gravity brewing. This also confirms the previous detection results of trehalose that Yeast C and Yeast D are more stress-resistant. Combining the residual sugar content, it is confirmed that Yeast C and Yeast D are more suitable for high-gravity brewing.

Claims

1. A rapid screening method for stress-resistant yeast suitable for ultra-high concentration brewing, characterized in that: The stress-resistant yeast for ultra-high concentration brewing was quickly screened by measuring the trehalose content in the yeast under stress conditions after yeast activation and the residual sugar content.

2. The rapid screening method according to claim 1, characterized in that The yeast was activated by culturing at 25°C for 48 h.

3. The rapid screening method according to claim 1, characterized in that 10%-30% sorbitol was used as stress condition.

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

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

6. The rapid screening method according to claim 1, characterized in that The trehalose content in yeast was calculated as follows: 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 Among them, 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 added extract, 1 mL; V1 is the volume of added sample, 0.01 mL; W is the fresh weight of the sample, g; 2 means that 1 molecule of trehalose is decomposed into 2 molecules of glucose; D is the dilution factor, which is 1 if not diluted.

7. The rapid screening method according to claim 6, characterized in that: The trehalose content in yeast should be ≥30 mg / g.

8. The rapid screening method according to claim 1, characterized in that The residual sugar content detection method is: The activated yeast was transferred to a deep-well plate containing 28°P wort, with a yeast count of 36×10 6 / mL, and the residual sugar content of the yeast was detected after culturing at 25℃ for 96h.

9. The rapid screening method according to claim 8, characterized in that The residual sugar content of the yeast is less than 8°P.

10. The rapid screening method according to any one of claims 1 to 9, characterized in that: The yeast is Lager yeast.

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