Saccharomyces cerevisiae with lactic acid resistance and high ethanol yield and application of saccharomyces cerevisiae in white spirit brewing

Abstract

The invention discloses a lactic acid-resistant high-ethanol-yield saccharomyces cerevisiae strain and application thereof in white spirit brewing, and belongs to the technical field of microorganisms. The Saccharomyces cerevisiae is Saccharomyces cerevisiae NCUF309.5-44, is preserved in the China Center for Type Culture Collection on December 14, 2023, has the preservation number of CCTCC M 20232557, is applied to solid-state brewing of white spirit, and is used for producing ethanol during solid-state fermentation of the white spirit. According to the invention, the saccharomyces cerevisiae NCUF309.5-44 obtained by screening through a strategy of combining mutagenesis and brewing lactic acid stress directional breeding is excellent in lactic acid resistance and can tolerate 46.15 g / L of lactic acid. After a seed solution containing the saccharomyces cerevisiae NCUF309.5-44 disclosed by the invention is added for the first time in a traditional white spirit solid-state brewing process, 48.59% of ethanol content and 160.43% of volatile component content in fermented grains can be increased, so that the brewing cost is reduced, the flavor quality of white spirit is improved, and the saccharomyces cerevisiae NCUF309.5-44 has a wide application prospect in the field of white spirit brewing industry.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a lactic acid-resistant and high-ethanol-producing brewer's yeast and its application in liquor brewing. Background Art

[0002] Chinese baijiu (white spirit) is a traditional fermented beverage in my country. Its unique flavor and brewing process have been developed over a long history, making it one of the world's most renowned distilled spirits. Made from high-starch grains like sorghum and rice, using koji (drinking yeast) as a saccharifying and fermenting agent, it utilizes traditional solid-state cooking, fermentation, and distillation processes, utilizing unique brewing techniques and fermentation equipment.

[0003] During the solid-state fermentation process of baijiu (white liquor), brewer's yeast (Saccharomyces cerevisiae) is a highly important and dominant fungus. It not only metabolizes grains to produce ethanol, but also generates a large number of flavor compounds such as higher alcohols, aldehydes, and esters through a series of complex biochemical reactions. These compounds play a vital role in the formation of baijiu's unique style. Among them, esters such as ethyl acetate, ethyl hexanoate, and ethyl lactate can impart typical aroma characteristics such as fruity, cellar-like, and honey-like aromas to baijiu. Alcohols such as isopentanol, isobutanol, and n-propanol not only provide a mellow and sweet taste, but also work together with esters to form baijiu's unique aroma and taste experience. However, due to the complex brewing environment in the mash, brewer's yeast is subject to stress from environmental factors including ethanol, temperature, organic acids, and high and osmotic pressure. Among them, lactic acid is the organic acid with the highest content in the solid-state liquor fermentation process, reaching 40g / kg in the liquor mash. It can freely diffuse through the yeast cell membrane into the cytoplasm, affecting the structure and function of key enzymes in the cell, thereby inhibiting the normal growth metabolism and fermentation of yeast to produce ethanol and the activity of flavor components, ultimately causing a negative impact on the liquor brewing efficiency and flavor. Therefore, improving the lactic acid tolerance of brewing yeast is crucial to the development of the solid-state liquor brewing industry.

[0004] Brewing lactic acid stress directed selection (BLASRB) is a new microbial cultivation and screening technology based on the liquor brewing system to carry out lactic acid stress adaptive evolution mutation of strains. It has the advantages of high lactic acid resistance mutation rate, strong selectivity, low reagent and labor costs, and good sample parallelism.

[0005] Therefore, the present invention selectively breeds lactic acid-tolerant and high-ethanol-yielding brewer's yeast that can be used in the field of food brewing through brewing lactic acid stress, and performs bio-fortification during the solid-state brewing process of liquor, which is a potential way to increase the ethanol content in liquor and improve the quality and characteristic aroma of liquor. Summary of the Invention

[0006] In view of the deficiencies and difficulties in the prior art, the present invention aims to provide a lactic acid-tolerant and high-ethanol-yielding brewer's yeast and its application in liquor brewing.

[0007] To achieve the above object, the present invention is implemented through the following technical solutions:

[0008] The first aspect of the present invention provides a lactic acid-tolerant and high-ethanol-producing brewer's yeast (Saccharomyces cerevisiae) NCUF309.5-44. The strain has strong lactic acid tolerance and ethanol production ability. It was deposited in the China Center for Type Culture Collection on December 14, 2023, with the deposit number: CCTCC M 20232557, and the deposit address is Wuhan University, No. 229, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0009] The above-mentioned Saccharomyces cerevisiae NCUF309.5-44 is obtained by the following steps:

[0010] (1) The brewer's yeast NCUF309.5 with strong lactic acid tolerance, which was isolated and screened from the naturally inoculated Daqu of a liquor brewing enterprise, was cultured at 28°C and 160 rpm / min for 12 h until the logarithmic growth phase.

[0011] (2) The dynamic culture conditions of liquor brewing from low to high concentrations of lactic acid (1.2%, 2.4%, 3.6% and 4% (v / v)) were simulated, and the induced or non-induced brewer's yeast strains were cultured according to the above concentration gradient. After culturing at 28°C for 12 hours, the strains were subcultured to conduct long-term brewing lactic acid stress directional selection to improve the lactic acid tolerance of brewer's yeast.

[0012] (3) The selected lactic acid-resistant Saccharomyces cerevisiae strain was inoculated into a slant culture medium, cultured at 28°C for 48 h, and then stored at 4°C.

[0013] (4) Through colony morphology characteristics, alcohol production content and esterification ability determination, and 26Sr RNA sequence identification experiments, a lactic acid-tolerant and high-ethanol-producing Saccharomyces cerevisiae strain was finally selected. The strain was sent to the China Center for Type Culture Collection on December 14, 2023, and its classification name is: Saccharomyces cerevisiae NCUF309.5-44, and the preservation number is: CCTCC M 20232557. It can tolerate 4% lactic acid.

[0014] The second aspect of the present invention provides the use of the above-mentioned lactic acid-tolerant and high-ethanol-producing Saccharomyces cerevisiae NCUF309.5-44 in liquor brewing. The Saccharomyces cerevisiae NCUF309.5-44 is used in solid-state liquor brewing, and produces ethanol during solid-state fermentation of liquor.

[0015] The specific steps are as follows: the brewer's yeast NCUF309.5-44 seed liquid is added to the liquor mash brewing system at a ratio of 20% to the Daqu. Specifically, in a conventional brewing process, the lactic acid-tolerant brewer's yeast NCUF309.5-44 seed liquid is added to the liquor mash at a ratio of 20% to the Daqu. In another group, no brewer's yeast NCUF309.5-44 seed liquid is added as a control group, and the ethanol content during the fermentation process and the content of volatile aroma components at the end of fermentation are detected.

[0016] The present invention has the following advantages:

[0017] (1) The present invention screened the brewer's yeast NCUF309.5-44 through a strategy combining mutagenesis and directed breeding under brewing lactic acid stress, which has excellent lactic acid resistance and can tolerate 46.15 g / L of lactic acid.

[0018] (2) When the seed liquid containing the brewer's yeast NCUF309.5-44 of the present invention is added for the first time in the traditional solid-state brewing process of liquor, the ethanol content in the mash can be increased by 48.59% and the volatile component content can be increased by 160.43%, thereby reducing the brewing cost and increasing the flavor quality of the liquor. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The adaptive evolution results of Saccharomyces cerevisiae NCUF309.5 under different lactic acid concentrations;

[0020] Figure 2 Ethanol production content of NCUF309.5 and lactic acid tolerant strains under 4% lactic acid stress;

[0021] Figure 3 The esterification capacity of NCUF309.5 and lactic acid tolerant strains under 4% lactic acid stress;

[0022] Figure 4 This is the plate colony morphology of Saccharomyces cerevisiae NCUF309.5-44 of the present invention;

[0023] Figure 5 This is the genetic stability result of NCUF309.5-44;

[0024] Figure 6 Growth curves of NCUF309.5-44 and NCUF309.5 under 4% lactic acid stress;

[0025] Figure 7 It is the ethanol content during the fermentation process of liquor mash;

[0026] Figure 8 It is the content of volatile components in the fermented mash of liquor at the end of fermentation. DETAILED DESCRIPTION

[0027] The invention provides a lactic acid-resistant and high-ethanol-producing brewer's yeast and application of the yeast in liquor brewing.

[0028] The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Without departing from the spirit or basic features of the present invention, the present invention will be further explained in conjunction with the embodiments.

[0029] The technical solution adopted in the present invention is as follows:

[0030] Example 1: Screening and identification of lactic acid-tolerant and high-ethanol-producing Saccharomyces cerevisiae NCUF309.5-44

[0031] (1) Raw material pretreatment

[0032] The starting strain Saccharomyces cerevisiae NCUF309.5 with strong lactic acid tolerance used in the present invention is separated from naturally inoculated Daqu of a liquor brewing enterprise and stored at 4°C.

[0033] YPD medium: 20 g glucose, 20 g peptone, 10 g yeast extract, 1000 mL distilled water, sterilized at 121°C for 20 min.

[0034] Solid-state fermentation medium: bran 100 g, glucose 0.5 g, distilled water 100 mL, sterilized at 121 °C for 40 min.

[0035] (2) Strain screening

[0036] Saccharomyces cerevisiae NCUF309.5 was cultured at 28°C and 160 rpm / min for 12 hours until the logarithmic growth phase, simulating the dynamic culture conditions of liquor brewing from low to high lactic acid stress. Mutagenized or non-mutagenized Saccharomyces cerevisiae strains were inoculated into YPD liquid medium containing 1-4% lactic acid for long-term stress gene mutagenesis and directed breeding to improve the lactic acid tolerance of Saccharomyces cerevisiae. The growth of the strains under different lactic acid concentrations was measured through 25 consecutive subcultures. The results of BLASRB adaptive evolution are shown in Figure 2. Figure 1 As shown, the three strains with the best growth in the mutant library samples were named NCUF309.5-21, NCUF309.5-41, and NCUF309.5-44. These three strains were plated and streaked 2-3 times. Single colonies were selected and inoculated onto slant culture media. The culture was then incubated at 30°C for 48 hours and stored at 4°C.

[0037] Single colonies of NCUF309.5 and the three yeast strains mentioned above were inoculated into 100 mL of YPD liquid medium and cultured at 28°C for 24 h. The bacterial solution was diluted to 108 CFU / mL and inoculated into fresh YPD liquid medium containing 4% lactic acid at a 1% inoculum. The ethanol content was determined by gas chromatography. The experimental results are as follows: Figure 2 As shown in the figure, NCUF309.5-44 had the highest ethanol production, which was 2.29 times higher than that of the starting strain at 24 hours. Another 5% inoculum was added to a solid fermentation medium containing 4% lactic acid and cultured at 28°C for 7 days. The esterification capacity was determined by reflux saponification. Figure 3 As shown, NCUF309.5-44 had the highest esterification capacity, which was 2.09 times higher than that of the starting strain.

[0038] (3) Strain identification

[0039] The three strains of potential lactic acid-tolerant Saccharomyces cerevisiae obtained above were subjected to molecular biological 26S rRNA characterization. PCR amplification was performed using the universal fungal primers ITS-1 and ITS-4 and yeast genomic DNA as the template. Amplification conditions included a 4-minute initial denaturation at 94°C, followed by 34 cycles of denaturation at 94°C for 1 minute, annealing at 58°C for 1 minute, and extension at 72°C for 1 minute, with a final extension cycle at 72°C for 10 minutes. Detection was performed by electrophoresis on a 1% agarose gel. The electrophoresis results were visualized using a gel imaging system, and samples with brighter bands were selected and sent to Shanghai Qingke Biotechnology Co., Ltd. for sequencing. The sequenced sequences were searched for homology in NCBI's GenBank using the BLAST tool, and a phylogenetic tree was constructed using multiple sequence alignment. The identification results showed that the strain with the highest ethanol production content and esterification capacity under lactic acid stress was Saccharomyces cerevisiae, which was named Saccharomyces cerevisiae NCUF309.5-44 and then deposited in the China Center for Type Culture Collection with the deposit number CCTCC M 20232557.

[0040] The colony morphology of Saccharomyces cerevisiae NCUF309.5-44 on the plate is as follows Figure 4 The genetic stability results are shown in Figure 5 As shown in the figure, there is no significant difference in its acid resistance after continuous passage, indicating that it has excellent genetic stability.

[0041] Example 2: Comparison of lactic acid tolerance between Saccharomyces cerevisiae NCUF309.5-44 and the original strain Saccharomyces cerevisiae NCUF309.5

[0042] The strains included in this example are: the original strain Saccharomyces cerevisiae NCUF309.5-44. NCUF309.5-44 is derived from the mutagenesis of Saccharomyces cerevisiae NCUF309.5, which is derived from naturally inoculated Daqu in a liquor brewing enterprise.

[0043] Pick a single yeast colony and inoculate it into 100 mL YPD liquid medium, culture it at 28℃ for 24 hours, dilute the bacterial solution to 108 CFU / mL, inoculate it into fresh YPD liquid medium containing 4% lactic acid at a 1% inoculum volume, culture it at 28℃ for 48 hours, and measure the OD value at 600 nm using a UV spectrophotometer. The results are as follows Figure 6 As shown, the OD value of Saccharomyces cerevisiae NCUF309.5-44 is significantly higher than that of the original strain Saccharomyces cerevisiae NCUF309.5, indicating that the lactic acid tolerance of the strain Saccharomyces cerevisiae NCUF309.5-44 of the present invention is significantly higher than that of the original yeast strain.

[0044] Example 3: Application of lactic acid-tolerant and high-ethanol-yielding Saccharomyces cerevisiae NCUF309.5-44 in solid-state liquor brewing

[0045] Pick a ring of lactic acid-tolerant brewer's yeast NCUF309.5-44 and put it into YPD liquid medium containing 4% lactic acid, and culture it at 28℃ for 48h. Count the bacterial suspension obtained by culture, inoculate it into YPD liquid medium containing 4% lactic acid at an inoculum size of 107CFU / mL, and culture it at 28℃ for 48h. According to the liquor brewing process, the lactic acid-tolerant brewer's yeast NCUF309.5-44 seed liquid is added to the liquor mash at a ratio of 20% of Daqu for brewing, and the mash with only traditional Daqu added is used as a control. The ethanol content of the mash during the fermentation process (0d, 3d, 6d, 10d, 15d, 20d, 25d, 30d) is tracked and measured, and the content of volatile components is measured after the fermentation is completed. The results are as follows: Figure 7 and Figure 8 As shown in the figure, with increasing amounts of lactic acid-tolerant Saccharomyces cerevisiae NCUF309.5-44 seed solution added, the ethanol and volatile component contents in the mash gradually increased after 30 days of fermentation. After adding 20% ​​lactic acid-tolerant Saccharomyces cerevisiae NCUF309.5-44 seed solution, compared with the control group, the ethanol content increased from 4.26% to 6.33%, the content of volatile flavor compounds increased by 160.43%, and the total amount of esters and alcohols increased significantly, by 146% and 182%, respectively, compared with the control group. This indicates that this strain can effectively accelerate fermentation and significantly enhance the flavor of baijiu in solid-state brewing.

[0046] The above detailed description is a specific description of one feasible embodiment of the present invention. This embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not depart from the present invention should be included in the scope of the technical solution of the present invention.

Claims

1. A lactic acid-tolerant, high-ethanol-producing yeast strain (Saccharomyces cerevisiae) NCUF309.5-44, characterized by: The brewer's yeast (Saccharomyces cerevisiae) NCUF309.5-44 was deposited in the China Center for Type Culture Collection on December 14, 2023, with the deposit number: CCTCC M 20232557, and the deposit address is Wuhan University, No. 229, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. The use of the lactic acid-tolerant and high-ethanol-producing Saccharomyces cerevisiae NCUF309.5-44 strain according to claim 1 in liquor brewing, characterized in that: The Saccharomyces cerevisiae NCUF309.5-4 is applied to the solid-state brewing of liquor and produces ethanol during the solid-state fermentation of liquor.

3. The use according to claim 2, characterized in that: The brewer's yeast NCUF309.5-44 seed liquid is added to the liquor mash brewing system at a Daqu ratio of 20%.

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