A Saccharomyces cerevisiae strain tolerant to caproic acid and its application

Abstract

The present invention discloses a Saccharomyces cerevisiae strain tolerant to caproic acid and its application. The Saccharomyces cerevisiae is taxonomically named Saccharomyces cerevisiae( Saccharomyces cerevisiae ) M1, and the preservation number is CCTCC NO: M 20191005. This Saccharomyces cerevisiae strain was isolated from fermented grains. After multiple rounds of mutagenesis and screening, it can tolerate 100 g / L of caproic acid, and when the caproic acid concentration reaches 70 g / L, it has no significant effect on ethanol fermentation.

Description

Technical Field

[0001] The present invention belongs to the technical field of food brewing, and specifically relates to a Saccharomyces cerevisiae strain tolerant to caproic acid and its application. Background Art

[0002] Yeast is one of the important microorganisms in the production of Luzhou-flavor liquor and also one of the main functional microorganisms in the liquor brewing process. During the fermentation of fermented grains, the daqu saccharifying enzyme converts polysaccharides in raw materials into monosaccharides, and then yeast converts monosaccharides into ethanol. The generated ethanol will undergo an esterification reaction with various organic acids produced during the fermentation of fermented grains, generating ester flavor substances in Luzhou-flavor daqu liquor. Therefore, the growth of yeast during the fermentation of fermented grains will directly affect the fermentation quality of fermented grains and the flavor of liquor products. Nevertheless, the growth of yeast in fermented grains is restricted by the conditions of entering the pit and the ratio of raw materials. In production practice, if only apparent data such as the acidity of entering the pit are concerned, it is easy to cause obvious differences in the flavor of liquor products due to problems such as the acidity of entering the pit, which indicates that organic acids in fermented grains have an important impact on the fermentation efficiency of fermented grains.

[0003] Low-concentration organic acids are beneficial to the growth of yeast, while high-concentration organic acids will inhibit the growth of yeast, making it difficult for yeast to normally carry out ethanol fermentation under high-concentration organic acid conditions, restricting its application in some liquor brewing processes. Caproic acid is an important precursor substance of ester aroma compounds in the liquor brewing process, but the accumulated caproic acid during fermentation will inhibit the synthesis of ethanol by Saccharomyces cerevisiae, thereby reducing the accumulation of important index substances such as ethyl caproate. Screening Saccharomyces cerevisiae strains tolerant to caproic acid is not only beneficial to improving the liquor yield during the liquor brewing process but also can promote the synthesis of main aroma substances. Summary of the Invention

[0004] The first object of the present invention is to provide a Saccharomyces cerevisiae strain tolerant to caproic acid. This Saccharomyces cerevisiae is a highly efficient strain with high resistance to caproic acid, capable of tolerating high concentrations of caproic acid and simultaneously carrying out normal ethanol fermentation in the presence of high concentrations of caproic acid, and can be an effective candidate strain for liquor brewing.

[0005] To achieve the above technical object, the present invention adopts the following technical scheme:

[0006] A Saccharomyces cerevisiae strain tolerant to caproic acid, classified and named as Saccharomyces cerevisiae( Saccharomyces cerevisiae )M1, with the preservation number of CCTCC NO: M 20191005.

[0007] Another object of the present invention is to provide the application of the above Saccharomyces cerevisiae in the production of ethanol.

[0008] The present invention provides a specific application method, which includes subjecting Saccharomyces cerevisiae to plate culture and seed culture, and then inoculating the seed liquid into a fermentation medium containing caproic acid for fermentation culture to produce ethanol.

[0009] As a preferred embodiment, the media for plate culture and seed culture are YPD solid medium and YPD liquid medium respectively, and 40 - 60 g / L of caproic acid is added to the medium for seed culture.

[0010] As a preferred embodiment, the concentration of caproic acid in the fermentation medium is 0.1 - 100 g / L.

[0011] As a preferred embodiment, the temperatures for plate culture, seed culture and fermentation culture are 25 - 36 °C.

[0012] As a preferred embodiment, the temperatures for plate culture, seed culture and fermentation culture are 34 - 36 °C.

[0013] As a preferred embodiment, the carbon source of the fermentation medium is one or more of glucose, glycerol, molasses, and starch; the nitrogen source of the fermentation medium is one or more of yeast powder, peptone, and soybean meal.

[0014] As a preferred embodiment, the inoculation amount of the seed liquid is 5 - 10%.

[0015] As a preferred embodiment, the time for fermentation culture is 48 - 72 h.

[0016] The Saccharomyces cerevisiae of the present invention Saccharomyces cerevisiae M1 has resistance to caproic acid with a concentration of 100 g / L, and 70 g / L of caproic acid does not affect the ethanol production capacity of the yeast itself. As a microbial strain for ethanol synthesis in the process of Baijiu brewing, this strain has good application prospects both in developing new brewing processes and improving production efficiency, and has great economic benefits. Specific Embodiments

[0017] The following examples illustrate the method of the present invention in detail, but do not limit the present invention.

[0018] The biological material involved in the present invention is classified and named as: Saccharomyces cerevisiae( Saccharomyces cerevisiae )M1, which has been deposited in the China Center for Type Culture Collection (CCTCC), with the deposit number CCTCC NO: M 20191005, the deposit date being December 4, 2019, and the deposit address: Wuhan, China.

[0019] The original strain used in the examples was a wild-type Saccharomyces cerevisiae screened from the fermented grains of Tanggou Distillery Saccharomyces cerevisiaeA (He Xun, Jiang Xuejian, Hua Jiawei, Chen Kequan, Bai Jianxin. Effect of in-situ pretreatment of sugarcane molasses on ethanol production by a thermotolerant Saccharomyces cerevisiae mutant strain Saccharomyces cerevisiae AQ [J]. Guangxi Sciences, 23(1): 1-6.) was obtained by mutagenesis and screening. The Saccharomyces cerevisiae Saccharomyces cerevisiae M1 morphology: It is oval in shape and the strain color is milky white.

[0020] The culture temperature range is 25-36 °C, the optimum is 34 °C, the pH range is 4.0-6.0, and the optimum is 5.0.

[0021] The culture media used in the examples are as follows:

[0022] Plate culture medium: That is, YPD solid medium, and the composition of the components is 1% Yeast Extract (yeast extract), 2% Peptone (peptone), 2% Dextrose (glucose) (glucose), 2% agar powder.

[0023] Seed culture medium: That is, YPD liquid medium, and the composition of the components is 1% Yeast Extract (yeast extract), 2% Peptone (peptone), 2% Dextrose (glucose) (glucose), 20-60 g / L caproic acid.

[0024] Fermentation culture medium: That is, YPD liquid medium containing caproic acid, and the composition of the components is 1% Yeast Extract (yeast extract), 2% Peptone (peptone), 2% Dextrose (glucose) (glucose), 0.1-100 g / L caproic acid.

[0025] The carbon source in the fermentation culture medium can be equivalently replaced with glucose, glycerol, molasses and / or starch; the nitrogen source can be equivalently replaced with yeast powder, peptone and / or soybean meal.

[0026] Example 1

[0027] This example specifically illustrates the screening method of Saccharomyces cerevisiae Saccharomyces cerevisiae M1.

[0028] The specific steps to obtain Saccharomyces cerevisiae Saccharomyces cerevisiae M1 are as follows:

[0029] 1) Preparation of bacterial suspension

[0030] Take the fresh slant of the original strain cultured at 30 °C for 1 day, add 15 mL of sterile water, scrape and wash the strain and transfer it into a 250 mL Erlenmeyer flask with glass beads. Add sterile water containing 2% lithium chloride to the Erlenmeyer flask to make a bacterial suspension, and adjust the concentration of yeast to 10 3 cells / mL;

[0031] 2) Ultraviolet - lithium chloride compound mutagenesis

[0032] Take 15 mL of the bacterial suspension in step 1) and transfer it into a sterile empty petri dish containing a magnetic rotor. Under a stirring speed of 50 rpm, place it 25 cm away from a 30 W ultraviolet lamp for ultraviolet irradiation mutagenesis for 60 s. After the mutagenesis, take the bacterial suspension and spread it on a plate medium containing 2% lithium chloride, and incubate it in the dark and upside - down at 30 °C for 3 d;

[0033] 3) Low - energy ion implantation mutagenesis

[0034] Wash the yeast after cultivation in step 2) twice with sterile water and make it into a bacterial suspension. Take 0.1 mL of the bacterial suspension and evenly spread it on a sterile empty petri dish, dry it with sterile air. Under the conditions of 3 kV / cm × 2 min and an implantation dose of 20×10 14 ions / cm 2 carry out nitrogen ion implantation on the yeast. After the ion implantation is completed, take out the petri dish, wash it off with 1 mL of sterile water under sterile conditions, spread it on the plate medium, and incubate it in the dark and upside - down at 30 °C for 3 d;

[0035] 4) Primary screening

[0036] Inoculate several single colonies obtained by mutagenesis in step 3) onto a slant medium for activation. After incubating it upside - down at 3° C for 2 d, use an inoculation loop to pick up one loop of the activated strain into its seed medium respectively, with a stirring speed of 150 rpm. After culturing for 20 h, then inoculate it into the fermentation medium in a 1 L fermenter at an inoculation amount of 1% (v / v), with a stirring speed of 200 rpm, and ferment for 60 h. Detect the ethanol content in the fermentation broth, and primarily screen out the single colonies with an ethanol content greater than 5% in the fermentation broth.

[0037] 5) Secondary screening

[0038] Inoculate the single colonies obtained by primary screening in step 4) onto a slant medium for activation. After incubating it upside - down at 30 °C for 2 d, use an inoculation loop to pick up one loop of the activated strain into the seed medium, with a stirring speed of 200 rpm. After culturing for 20 h, inoculate it into the fermentation medium in a 5 L fermenter at an inoculation amount of 5% (v / v), with a stirring speed of 500 rpm, and ferment for 62 h; Detect the ethanol content in the fermentation broth, and secondary - screen out the single colonies with a high ethanol content in the fermentation broth.

[0039] Then prepare the single colonies obtained by secondary screening in step 5) into a bacterial suspension, and repeat steps 1) to 5). When the ethanol content in the fermentation broth measured by the SBA - 40E biosensor is 6% and above, the corresponding strain is the target strain Saccharomyces cerevisiae Saccharomyces cerevisiae M1.

[0040] Example 2

[0041] This example specifically describes a method for fermentatively synthesizing ethanol using Saccharomyces cerevisiae Saccharomyces cerevisiae M1.

[0042] This example investigated the growth of Saccharomyces cerevisiae Saccharomyces cerevisiae M1 in a fermentation medium containing 70 g / L of caproic acid. Saccharomyces cerevisiae Saccharomyces cerevisiae M1 was inoculated into a plate medium and cultured at 30 °C for 24 h to obtain single colonies on the plate; a single colony was picked from the solid plate and inoculated into a seed medium, and cultured at 30 °C and 200 rpm for 30 h to obtain a seed solution; ethanol fermentation: the seed solution was drawn from the seed medium at an inoculation amount of 10% into a YPD medium containing 70 g / L of caproic acid and cultured at 30 °C for 72 h, and the ethanol concentration in the fermentation broth was 6.42%. As a control, the seed solution was drawn from the seed medium at an inoculation amount of 10% into a YPD medium without caproic acid and cultured at 30 °C for 72 h, and the ethanol concentration in the fermentation broth was 6.41%.

[0043] Example 3

[0044] This example investigated the effect of different culture temperatures on ethanol synthesis by Saccharomyces cerevisiae Saccharomyces cerevisiae M1

[0045] The difference between this example and Example 2 is only that the temperatures of plate culture, seed culture, and fermentation culture are adjusted to 25 °C, 34 °C, and 36 °C, respectively.

[0046] When the temperatures of plate culture, seed culture, and fermentation culture are 25 °C, the ethanol concentration in the fermentation broth after 72 h of fermentation culture is 5.89%. Under the same conditions, the ethanol concentration in the fermentation broth after 72 h of fermentation of the M1 strain in a YPD medium without caproic acid is 5.88%.

[0047] When the temperatures of plate culture, seed culture, and fermentation culture are 34 °C, the ethanol concentration in the fermentation broth after 72 h of fermentation culture is 6.83%. Under the same conditions, the ethanol concentration in the fermentation broth after 72 h of fermentation of the M1 strain in a YPD medium without caproic acid is 6.80%.

[0048] When the temperatures of plate culture, seed culture, and fermentation culture are 36 °C, the ethanol concentration in the fermentation broth after 72 h of fermentation culture is 6.36%. Under the same conditions, the ethanol concentration in the fermentation broth after 72 h of fermentation of the M1 strain in a YPD medium without caproic acid is 6.35%.

[0049] Example 4

[0050] This example investigated the effect of the selection of carbon source / nitrogen source in the fermentation medium on Saccharomyces cerevisiae Saccharomyces cerevisiaeInfluence of M1 on ethanol synthesis.

[0051] According to the experimental results of Examples 2 to 3, 34 °C was selected as the culture temperature. Except for the temperature and the culture medium components listed below, other experimental conditions were the same as those in Example 2.

[0052] Experimental group 1: The carbon source of the fermentation medium was 2% molasses, and the nitrogen source was 3% soybean meal. After 72 h of fermentation culture, the ethanol concentration in the fermentation broth was 6.64%. Under the same conditions, when the M1 strain was fermented in a medium without caproic acid for 72 h, the ethanol concentration in the fermentation broth was 6.62%.

[0053] Experimental group 2: The carbon source of the fermentation medium was 2% starch, and the nitrogen source was 3% soybean meal. After 72 h of fermentation culture, the ethanol concentration in the fermentation broth was 6.78%. Under the same conditions, when the M1 strain was fermented in a medium without caproic acid for 72 h, the ethanol concentration in the fermentation broth was 6.79%.

[0054] Experimental group 3: The carbon source in the fermentation medium was 2% glycerol, and the nitrogen source was 3% soybean meal. After 72 h of fermentation culture, the ethanol concentration in the fermentation broth was 6.72%. Under the same conditions, when the M1 strain was fermented in a medium without caproic acid for 72 h, the ethanol concentration in the fermentation broth was 6.73%.

[0055] Example 5

[0056] The difference between this example and Example 2 was only that the caproic acid concentrations in the fermentation medium were adjusted to 0.1 g / L and 100 g / L, respectively.

[0057] The M1 strain was fermented and cultured in YPD medium containing 0.1 g / L caproic acid for 72 h, and the ethanol concentration in the fermentation broth was 6.41%.

[0058] The M1 strain was fermented and cultured in YPD medium containing 100 g / L caproic acid for 72 h, and the ethanol concentration in the fermentation broth was 5.97%. This indicated that the M1 strain could produce ethanol under the condition of 100 g / L caproic acid, but compared with the condition of 70 g / L caproic acid, 100 g / L caproic acid had a certain inhibitory effect.

[0059] Example 6

[0060] Saccharomyces cerevisiae Saccharomyces cerevisiaeM1 was inoculated into a solid medium and cultured at 25 °C for 24 h to obtain single colonies on the plate; a single colony was picked from the solid plate and inoculated into a seed medium, and cultured at 25 °C and 200 rpm for 30 h to obtain a seed solution; ethanol fermentation: the seed solution was sucked from the seed medium into a YPD medium containing 0.1 g / L caproic acid at an inoculation amount of 5%, and cultured at 25 °C for 48 h. The ethanol concentration in the fermentation broth was 5.77%. As a control, the seed solution was sucked from the seed medium into a YPD medium without caproic acid at an inoculation amount of 5%, and cultured at 25 °C for 24 h. The ethanol concentration in the fermentation broth was 5.79%.

[0061] Example 7

[0062] This example investigated Saccharomyces cerevisiae Saccharomyces cerevisiae M1 and the original strain Saccharomyces cerevisiae A, mutant strain Saccharomyces cerevisiae Comparison of the caproic acid tolerance effects of AQ.

[0063] The original strain Saccharomyces cerevisiae A and the mutant strain Saccharomyces cerevisiae AQ were fermented to synthesize ethanol under the conditions of Example 2. The results showed that when there was 20 g / L caproic acid in the medium for the original strain Saccharomyces cerevisiae A, the ethanol concentration in the fermentation broth was 2.11%. When there was 20 g / L caproic acid in the medium for the mutant strain Saccharomyces cerevisiae AQ, the obtained ethanol concentration was 3.28%. When there was 60 g / L caproic acid in the medium, no ethanol was detected in the fermentation broth of the original strain Saccharomyces cerevisiae A and the mutant strain Saccharomyces cerevisiae AQ.

Claims

1. A Saccharomyces cerevisiae strain tolerant to caproic acid, characterized in that, The classification name is Saccharomyces cerevisiae( Saccharomyces cerevisiae ) M1, and the deposit number is CCTCC NO: M 20191005.

2. Use of the Saccharomyces cerevisiae according to claim 1 in the production of ethanol.

3. The application according to claim 2, wherein After the Saccharomyces cerevisiae is subjected to plate culture and seed culture, the seed liquid is inoculated into a fermentation medium containing caproic acid for fermentation culture to produce ethanol.

4. The application according to claim 3, wherein The media for plate culture and seed culture are YPD solid medium and YPD liquid medium respectively, and 40 - 60 g / L of caproic acid is added to the medium for seed culture.

5. The application according to claim 3, characterized in that, The concentration of caproic acid in the fermentation medium is 0.1 - 100 g / L.

6. The application according to claim 3, characterized in that, The temperatures for plate culture, seed culture and fermentation culture are 25 - 36 °C.

7. The application according to claim 6, characterized in that, The temperatures for plate culture, seed culture and fermentation culture are 34 - 36 °C.

8. The application according to claim 6, characterized in that, The carbon source of the fermentation medium is one or more of glucose, glycerol, molasses, starch; the nitrogen source of the fermentation medium is one or more of yeast powder, peptone, soybean meal.

9. The application according to claim 6, wherein The inoculation amount of the seed liquid is 5 - 10%.

10. The application according to claim 6, characterized in that, The fermentation culture time is 48 - 72 h.