Tortuaspora delbrueckii BYA-1, screening method thereof and application of Tortuaspora delbrueckii BYA-1 in special beer brewing
By screening the Torulaspora delbrueckii BYA-1 strain, the problem of producing distinctive beers with high alcohol content, refreshing sourness, and tropical fruit aromas in existing technologies has been solved, achieving efficient fermentation and excellent flavor in beer production.
Patent Information
- Application Number
- CN202511790119.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-01
- Publication Date
- 2026-01-13
AI Technical Summary
Currently, there is no suitable Dale Kelvin spore-forming yeast available for producing specialty beers with high alcohol content, thorough fermentation, a refreshing sourness, and tropical fruit aromas.
Torulaspora delbrueckii BYA-1 strain was screened using enrichment and separation media. The screening method included enrichment media containing wort, ethanol, and chloramphenicol, combined with separation media containing YPD, sodium propionate, and chloramphenicol. The strains with excellent performance were screened through initial screening of maltose utilization ability, alcohol tolerance, and thiol production ability.
It achieves high-alcohol fermentation of specialty beer, with a refreshing sour taste and obvious tropical fruit aroma. The fermentation degree is 55.41%, the alcohol content is 4.65% vol, the acetic acid yield is 304 mg/L, the thiol 3MH yield is 178.5 ng/L, the 3MHA yield is 9.3 ng/L, and the 4MMP yield is 32.7 ng/L.
Smart Images
Figure CN121320119A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of specialty beer brewing technology, and particularly relates to a... Torulaspora delbrueckii BYA-1 The screening methods and their application in specialty beer brewing. Background Technology
[0002] The ancestors of modern brewing yeasts are wild yeasts, naturally occurring yeasts found in nature. Currently, consumer demand for new flavors and non-alcoholic beers, the popularity of craft and specialty beers, and a greater focus on health are collectively defining new trends in the beer industry. New flavors and varieties, as well as low-calorie and non-alcoholic beers, are the main drivers of product diversification. Brewers are seeking products with new flavors and characteristics, and wild yeasts have become a new approach to producing these products. The research community is also interested in this topic, reflected in the increasing number of scientific articles published over the past 15 years.
[0003] Because non-yeast yeasts are undomesticated strains, their fermentation characteristics are more variable than those of yeasts, affecting the consistency and quality of the beer produced. To date, the number of products using these unconventional species as alternative yeasts in brewing remains limited. Generally, non-yeast yeasts are considered to play a negative role in the brewing process due to problems associated with changes in beer turbidity, filterability, viscosity, phenolic off-flavors (POF), acidity, and other flavor characteristics. Successful application of unconventional yeasts in brewing involves handling these new yeasts in a controlled manner to obtain the desired beer characteristics. Because unconventional yeast species typically have lower ethanol production performance compared to brewer's yeast, they are rarely used as pure starter cultures for alcoholic beverages; instead, they are often used in co-fermentation or sequential fermentation with brewer's yeast.
[0004] Currently, commercially available yeast strains exist, such as WLP618 "maltose-negative" yeast strain from WHITELAB. Saccharomycodes ludwigii WLP603 Torulaspora delbrueckii WLP604 Pichia kudriavzevii Raman's WildBrew™ Philly Sour Lachancea (Philly Sour lactic acid yeast). Meanwhile, European brewers have also been using certain wild yeasts to brew commercial beers, such as Heineken. Saccharomyces eubayanus H41 Wild Beer, brewed by Finnish brewers Saccharomyces paradoxus The resulting beer is "pure Finnish." Therefore, it is of great significance to use non-yeast yeasts to produce new flavored beers. Summary of the Invention
[0005] To address the shortcomings of existing technologies, the technical problem this invention aims to solve is overcoming the inability to select suitable *DellKip* spore-forming yeast for the production of specialty flavored beers. The invention proposes a method for producing beers with high alcohol content, thorough fermentation, a refreshing sourness, and pronounced tropical fruit aromas. Torulaspora delbrueckii BYA- 1 The screening methods and their application in specialty beer brewing.
[0006] To solve the aforementioned technical problem, the technical solution adopted by the present invention is as follows: This invention provides one aspect Torulaspora delbrueckii BYA-1 It was deposited at the China General Microbiological Culture Collection Center on November 24, 2025, with accession number CGMCC No. 39053.
[0007] In some embodiments, the nucleotide sequence is as shown in SEQ ID NO:1.
[0008] The present invention provides the above-mentioned aspects. Torulaspora delbrueckii BYA-1 The screening method Torulaspora delbrueckii BYA-1 It was obtained by enrichment, separation and screening using enrichment culture media, which included wort, ethanol and chloramphenicol.
[0009] In some embodiments, the enrichment medium contains 5-10% ethanol by mass, 50-150 ug / mL of chloramphenicol, and a pH of 3.8-4.1.
[0010] In some embodiments, the enrichment medium contains 8% ethanol by mass, 100 μg / mL chloramphenicol, and pH 4.0.
[0011] In some embodiments, the enriched strains are isolated using an isolation medium comprising YPD, sodium propionate, and chloramphenicol. The sodium propionate content in the isolation medium is 0.5-1.6%, and the concentration of chloramphenicol is 50-150 ug / mL.
[0012] In some embodiments, the sodium propionate content in the separation medium is 1%, and the concentration of chloramphenicol is 100 ug / mL.
[0013] In some embodiments, during enrichment, the substrate and enrichment medium are added to the enrichment tube, left for a period of time to allow the medium to soak the substrate, the tube wall is tapped to expel all air bubbles from the substrate, the enrichment medium missing after the air bubbles are expelled is replenished, the tube is cultured, and the enrichment is continuously observed. When a large number of air bubbles are generated in the enrichment tube or even overflows, or white bacterial cells are visible to the naked eye at the bottom of the tube, it can be taken out for plate dilution and plating. Dilute and coat the enriched solution using YSC plates, diluting it to 10. -3 -10 -5Spread the plating, incubate the spread plates, and observe the results. Plates with few or no colonies can be re-spread by adjusting the dilution factor. For plates with mold growth, promptly remove colonies that morphologically resemble Saccharomyces cerevisiae. Other plates can be left to stand until the colonies are slightly larger and their morphology is clear. Then, visually inspect the plates and streak colonies that morphologically resemble yeast strains onto YPD plates.
[0014] In some embodiments, the isolated strains are initially screened for maltose utilization ability in a multi-well plate with maltose as the sole carbon source, and strains with OD600 not less than 1.26 after 3 days of culture are selected.
[0015] In some embodiments, the isolated strains are initially screened for alcohol tolerance in YPD and multi-well plates containing alcohol-stressed medium, and strains with an OD600 of not less than 1.32 after 3 days of culture are selected.
[0016] In some embodiments, the isolated strains are initially screened for thiol production capacity in a multi-well plate containing YNB, cysteine, and glucose, and strains with an OD600 of not less than 0.689 after 3 days of culture are selected.
[0017] The present invention also provides the above. Torulaspora delbrueckii BYA-1 Its application in specialty beer brewing Torulaspora delbrueckii BYA-1 Wheat fermentation is carried out. Torulaspora delbrueckii BYA-1 The 13°P wort has a fermentation degree of 50-60%, an alcohol content of 4.05-5.15% vol, an acetic acid yield of 200-400 mg / L, a thiol 3MH yield of 160-350 ng / L, a 3MHA yield of 6-15 ng / L, and a 4MMP yield of 30-45 ng / L.
[0018] In some embodiments, for Torulaspora delbrueckii BYA-1 Wheat fermentation is carried out. Torulaspora delbrueckii BYA-1 The 13°P wort had a fermentation degree of 55.41%, an alcohol content of 4.65% vol, an acetic acid yield of 304 mg / L, a thiol 3MH yield of 178.5 ng / L, a 3MHA yield of 9.3 ng / L, and a 4MMP yield of 32.7 ng / L.
[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides Torulaspora delbrueckii BYA-1 It is a wild strain, and the strain will be... Torulaspora delbrueckii BYA-1When used in the brewing of specialty beers, its 13°P wort has a fermentation degree of 55.41%, an alcohol content of 4.65% vol, an acetic acid yield of 304 mg / L, a thiol 3MH yield of 178.5 ng / L, a 3MHA yield of 9.3 ng / L, and a 4MMP yield of 32.7 ng / L. The fermented product has a high alcohol content, is thoroughly fermented, has a refreshing sour taste, and a distinct tropical fruit aroma. Attached Figure Description
[0020] Figure 1 Provided by the embodiments of the present invention Torulaspora delbrueckii BYA-1 A schematic diagram of its shape; Figure 2 Provided by the embodiments of the present invention Torulaspora delbrueckii BYA-1 A comparative illustration of the evaluation of fermented wort. Detailed Implementation
[0021] The technical solutions in specific embodiments of the present invention will be described in detail and completely below. Obviously, the described embodiments are only some specific implementations of the overall technical solution of the present invention, and not all implementations. Based on the overall concept of the present invention, all other embodiments obtained by those skilled in the art fall within the protection scope of the present invention.
[0022] This invention provides one aspect Torulaspora delbrueckii BYA-1 It was deposited at the China General Microbiological Culture Collection Center on November 24, 2025, with accession number CGMCC No. 39053.
[0023] As one of the evolutionary centers of the yeast genus, China possesses abundant wild yeast resources with immense potential for development and utilization. Dajishan Mountain in Laizhou is located in the hilly and low-mountain region of Jiaodong Peninsula, belonging to the warm-temperate deciduous broad-leaved forest zone, with zonal vegetation dominated by oak forests. This invention provides... Torulaspora delbrueckii BYA-1 It originates from Dajishan, Laizhou. In some embodiments, its nucleotide sequence is shown in SEQ ID NO:1.
[0024] About this invention Torulaspora delbrueckii BYA-1 The nucleotide sequence of the strain is required. It should be noted that the sequence is unique for each strain and requires sequencing. Then, a BLAST comparison is performed with the ITS sequence from NCBI. The strain with the highest sequence similarity (over 99%) is considered the correct strain. This invention... Torulaspora delbrueckii BYA-1 The nucleotide sequence was obtained through sequencing, and then a BLAST comparison of the ITS sequence was performed. Torulaspora delbrueckii The homology of a strain of (Delkirk's spore-forming yeast) reached 99.46%.
[0025] The present invention provides the above-mentioned aspects.Torulaspora delbrueckii BYA-1 The screening method Torulaspora delbrueckii BYA-1 The bacteria were obtained by enrichment, isolation, and screening using an enrichment medium consisting of wort, ethanol, and chloramphenicol. The wort provided maltose as a carbon source, which was beneficial for enriching strains suitable for beer fermentation; the addition of ethanol facilitated the enrichment of alcohol-tolerant strains; and chloramphenicol inhibited bacterial growth.
[0026] In some embodiments, the enrichment medium contains 8% ethanol by mass, 100 μg / mL chloramphenicol, and a pH of 4.0. This technical solution limits the ethanol and chloramphenicol content in the enrichment medium because too low an ethanol concentration will not allow for the enrichment of ethanol-tolerant strains, while too high an ethanol concentration will inhibit the growth of most suitable yeasts. Similarly, too low a chloramphenicol concentration will not inhibit bacterial growth during the enrichment process, while too high a chloramphenicol concentration will inhibit yeast growth.
[0027] In some embodiments, an isolation medium is used to isolate the enriched bacterial strains. The isolation medium includes YPD, sodium propionate, and chloramphenicol, with sodium propionate comprising 1% by mass and chloramphenicol at a concentration of 100 ug / mL. This technical solution limits the composition and content of the isolation medium because too low a concentration of sodium propionate will not inhibit the growth of mold mycelia during the isolation process, while too high a concentration will inhibit the growth of all yeasts; too low a concentration of chloramphenicol will not inhibit the growth of bacteria during the isolation process, while too high a concentration will inhibit the growth of yeasts.
[0028] In some embodiments, during enrichment, the substrate and enrichment medium are added to the enrichment tube, left for a period of time to allow the medium to soak the substrate, the tube wall is tapped to expel all air bubbles from the substrate, the enrichment medium missing after the air bubbles are expelled is replenished, the tube is cultured, and the enrichment is continuously observed. When a large number of air bubbles are generated in the enrichment tube or even overflows, or white bacterial cells are visible to the naked eye at the bottom of the tube, it can be taken out for plate dilution and plating. Dilute and coat the enriched solution using YSC plates, diluting it to 10. -3 -10 -5 Spread the plating, then incubate the spread plates and observe the results. Plates with few or no colonies can be re-spread by adjusting the dilution ratio. Remove any plates showing mold growth, focusing on colonies that morphologically resemble *Saccharomyces* strains. Figure 1 As shown, other plates can be placed until the colonies are slightly larger and the colony morphology is obvious. Then, by visual observation, colonies that look more like yeast strains can be streaked onto YPD plates.
[0029] In some embodiments, the isolated strains are initially screened for maltose utilization ability in a multi-well plate with maltose as the sole carbon source, and strains with OD600 not less than 1.26 after 3 days of culture are selected.
[0030] In some embodiments, the isolated strains are initially screened for alcohol tolerance in YPD and multi-well plates containing alcohol-stressed medium, and strains with an OD600 of not less than 1.32 after 3 days of culture are selected.
[0031] In some embodiments, the isolated strains are initially screened for thiol production capacity in a multi-well plate containing YNB, cysteine, and glucose, and strains with an OD600 of not less than 0.689 after 3 days of culture are selected.
[0032] The present invention relates to *Cyclophorus deltaea*, whose nucleotide sequence is SEQ ID NO:1. Torulaspora delbrueckii BYA-1 , Torulaspora delbrueckii Commercial strains, strains not screened using the screening method of this invention Torulaspora delbrueckii Preliminary screening of wild-type strains, including their sugar utilization capacity, alcohol tolerance, and β-lyase (thiol production capacity), revealed that the present invention is *Cyclophorus deltaeniae*. Torulaspora delbrueckii BYA-1 It exhibits absolute advantages in sugar utilization, alcohol tolerance, and β-lyase (thiol production capacity). Furthermore, combined with subsequent wort test results, it can be seen that the *DellKip* spore-forming yeast of this invention... Torulaspora delbrueckii BYA-1 The degree of fermentation of 13°P wort was 55.41%, the alcohol content was 4.65% vol, the acetic acid yield was 304 mg / L, the thiol 3MH yield was 178.5 ng / L, the 3MHA yield was 9.3 ng / L, and the 4MMP yield was 32.7 ng / L, which were significantly higher than those of commercial strains and similar strains.
[0033] The present invention also provides the above. Torulaspora delbrueckii BYA-1 Its application in specialty beer brewing Torulaspora delbrueckii BYA-1 Wheat fermentation is carried out. Torulaspora delbrueckii BYA-1 The 13°P wort had a fermentation degree of 55.41%, an alcohol content of 4.65% vol, an acetic acid yield of 304 mg / L, a thiol (3MMH) yield of 178.5 ng / L, a 3MHA yield of 9.3 ng / L, and a 4MMP yield of 32.7 ng / L. Tasting notes described the flavor as green apple, apple cider vinegar, and tropical fruit, with the thiol flavor (tropical fruit) being the most prominent and representing the desired characteristic of the product.
[0034] To more clearly and in detail describe the embodiments provided by the present invention Torulaspora delbrueckii BYA-1 The screening method and its application in specialty beer brewing will be described below with reference to specific embodiments.
[0035] Example 1 Enrichment Screening Torulaspora delbrueckiiThe sugar utilization ability varies greatly among strains, with a large proportion of strains unable to utilize maltose. To screen strains from the natural environment that have strong wort fermentation capabilities, high alcohol production capacity, and high thiol production, targeted enrichment and screening methods need to be designed.
[0036] 1) Sample collection: This project collected samples of Fagaceae plants from Dajishan Mountain in Laizhou. 2) Enrichment and Separation Strategy: The enrichment medium consisted of wort + ethanol (8%) + 100 μg / mL chloramphenicol, with the pH adjusted to 4.0. The wort provided maltose as a carbon source, which is beneficial for enriching strains suitable for beer fermentation. The addition of ethanol facilitated the enrichment of alcohol-tolerant strains, while chloramphenicol inhibited bacterial growth. During enrichment, a 50 ml screw-cap centrifuge tube was used, with approximately 2 g of substrate added and about 20 mL of enrichment medium added. The tube was left to allow the medium to soak the substrate, and the tube walls were tapped to remove all air bubbles to avoid affecting subsequent observation and inducing mold growth. Any missing enrichment medium was replenished after removing the air bubbles, and the tube was incubated at 18°C (the temperature for beer fermentation). The enrichment process was continuously observed; when a large number of air bubbles were produced in the enrichment tube, or even overflow, or when white bacterial deposits were visible at the bottom of the tube, the tube was removed and plated.
[0037] 3) Strain isolation: The isolation medium was YSC medium: YPD + 1% sodium propionate + 100 ug / mL chloramphenicol (sodium propionate inhibits mold growth). The strain was diluted and spread on YSC plates, and the enriched solution was diluted to 10... -3 -10 -5 The coating process is carried out using a disposable coating stick, with 200 μL of diluted solution applied. After spreading the agar plates, incubate them at 25°C for about 3 days and observe the results. Plates with few or no colonies can be re-spread by adjusting the dilution ratio. For plates showing mold growth, promptly remove colonies that morphologically resemble *Saccharomyces cerevisiae* strains to prevent mold contamination and inoculation. Other plates can be left to stand until the colonies are larger and their morphology is clear. Visually inspect the plates and then streak colonies that morphologically resemble yeast strains onto YPD plates.
[0038] Example 2 Identification and preliminary screening of isolated yeast (1) Identification of the isolated strains using ITS sequence analysis; The PCR primers were ITS1: TCCGTAGGTGAACCTGCGG; ITS4: TCCTCGCTTATTGATATGC.
[0039] The PCR results were cut and the target band was purified and recovered. The recovered product was then subjected to Sanger sequencing, and the obtained sequence is shown in SEQ ID NO:1.
[0040] The sequences were compared with the ITS sequence data in NCBI to obtain the accession number of the homologous sequence. The latest version of the nt library was selected as the nucleic acid database, and the accession number was obtained by performing a BLAST comparison with the nt library.
[0041] The comparison results are Torulaspora delbrueckii It is deposited at the China General Microbiological Culture Collection Center, accession number CGMCC No. 39053.
[0042] (2) Preliminary screening of sugar utilization capacity, alcohol tolerance and β-lyase (thiol production capacity) was carried out, and preliminary screening of wort plate flavor was carried out; among them, the method for determining OD600 was as follows: the multi-well plate method of the microplate reader was used, the instrument used was a microplate reader with a 600nm filter or grating, and the multi-well plate used was a 96-well transparent flat-bottom microplate; the steps were: turn on the machine and preheat for 10-15 minutes, set the wavelength to 600nm, select "endpoint method"; blank culture medium should be included in the multi-well plate as a blank control, each sample needs 3 parallel wells, put the multi-well plate into the measurement chamber of the microplate reader, select 20s of shaking before measurement; after measurement, the average reading of the sample is subtracted from the average reading of the blank control to obtain the OD600 data of the strain.
[0043] 1) Initial screening of maltose utilization capacity: The growth rate of yeast strains in multi-well plates with maltose as the sole carbon source was used. Single colonies from YPD plates were picked and grown in YPD multi-well plates for 5 days. Basically, all strains had the same OD600. 20 μL of each strain was inoculated into the above multi-well plates, and the OD600 was measured at regular intervals. The growth rate of yeast can be obtained as shown in Table 1, thus reflecting the yeast's maltose utilization capacity.
[0044] Table 1. Preliminary screening results of maltose utilization capacity
[0045] 2) Initial screening of alcohol tolerance: The growth rate of yeast strains in YPD and multi-well plates containing 6% alcohol stress was used. After single colonies from YPD plates were picked and grown in YPD multi-well plates for 5 days, the OD600 of almost all strains were the same. 20 μL of each strain was inoculated into the above multi-well plates, and the OD600 was measured at regular intervals. The growth rate of yeast can be obtained as shown in Table 2, thus reflecting the alcohol tolerance of yeast.
[0046] Table 2 Initial screening results of alcohol tolerance
[0047] 3) High-throughput screening of β-lyase activity based on cysteine utilization: The culture medium was YNB (0.17%) + 15 mM cysteine + 1% glucose; aliquoting: 140 μL of culture medium was added to each well of a 96-well plate; inoculation: 10 μL of the previously cultured YPD culture was taken, sealed with a film, and cultured; incubation: 25°C for 7 days. The OD600 was measured to obtain the yeast growth rate, as shown in Table 3, thus reflecting the β-lyase activity of the yeast. Yeast with higher activity had a stronger ability to produce thiols.
[0048]
[0049] The results in Tables 1, 2, and 3 show that, based on the initial screening of capabilities, the yeast strains obtained in this invention... Torulaspora delbrueckii BYA-1 ( T. delbrueckii (BYA-1) exhibits excellent alcohol tolerance and maltose utilization, and high β-lyase activity.
[0050] In Tables 1, 2, and 3, Torulaspora delbrueckii Strain 1 was purchased from outside; the specific strain is CBS 1146. Torulaspora delbrueckii The type strain; Torulaspora delbrueckii Isolation strain 2 and Torulaspora delbrueckii Strain 3 was isolated from the wild. The isolation method was as follows: 2 g of a Fagaceae plant sample was added to a 50 mL centrifuge tube, followed by 25 mL of sterile water. The mixture was vortexed for 2 min, and the supernatant was then plated onto a YSC agar plate. This method differs from the method used for the yeast strain specified in this invention, as it lacks the steps of enrichment with wort and alcohol. The isolated strain exhibited weak maltose fermentation ability and low alcohol tolerance.
[0051] Example 3 Evaluation of brewing performance of early-stage isolated yeast A fermentation experiment was conducted at 13°P wort and 18°C, and the yeast fermentation performance was obtained, as shown in Tables 4, 5, and 6. In the tables, T. delbrueckii (BYA-1) is Torulaspora delbrueckii BYA-1, T. delbrueckii Commercial strain 1 is Torulaspora delbrueckii Commercial strain 1, T. delbrueckii Collect strain 1 is Torulaspora delbrueckii Collect strain 1.
[0052] The flavor compounds were detected by headspace-solid phase microextraction (SPME) GC-MS: after filtering to remove yeast and degassing, saturated NaCl was added to the beer, and 2-octanol was used as an internal standard. After equilibration, SPME extraction was performed at 50℃ for 45 min. After extraction, the sample was desorbed at 250℃ and entered into a DB-WAX gas chromatography column with a programmed temperature increase of 40℃ (3 min) → 5℃ / min → 230℃ (5 min). The alcohols and esters were quantified by GC-MS internal standard method.
[0053] The organic acid detection method is ion chromatography: beer samples are filtered through 0.22 μm or 0.45 μm filter membranes, and the processed samples are injected into a Metrosep Organic Acids analytical column using an autosampler. The eluent is a mixture of 0.3 mM sulfuric acid and 15% acetone. The organic acids are separated in the chromatographic column according to their differences in migration rate, and finally detected by a suppressed conductivity detector. The retention time of the organic acids in the sample is compared with the standard for qualitative analysis; quantitative analysis is performed by external standard method using peak area by plotting a standard curve.
[0054] Thiol detection method: Headspace derivatization solid-phase microextraction-GC-MS / MS: After filtering out yeast and degassing the beer sample, borate buffer (pH 8.5) was added, followed by saturated NaCl, and then an internal standard. Immediately afterward, PFBBr was added for derivatization (final concentration 0.1 mmol / L). -1 After equilibration, SPME extraction was performed at 50℃ for 45 min using a 65µm DVB / CAR / PDMS triple extraction head. The sample was then desorbed at 250℃ and inserted into a DB-5MS 30m×0.25mm×0.25µm column with a programmed temperature increase of 40℃ (2 min) → 10℃ (min). -1 →160℃→25℃min -1 →280℃ (5 min), GC-MS / MS MRM detection, quantification using internal standard method. Table 4: Detection results of flavor compounds.
[0055] Table 5 Results of Organic Acid Detection
[0056] Table 6. Results of thiol detection
[0057] 13°P wort T. delbrueckii (BYA-1) had a fermentation degree of 55.41% and an alcohol content of 4.65% vol. The acetic acid yield reached 304 mg / L, giving the wine a sour taste similar to apple cider vinegar. At the same time, its 3MH yield was 178.5 ng / L, 3MHA yield was 9.3 ng / L, and 4MMP yield was 32.7 ng / L, which were significantly higher than those of commercial strains and similar strains.
[0058] After evaluation, such as Figure 2 As shown, the flavor profile is described as green apple, apple cider vinegar, and tropical fruit. Among these, the thiol flavor (tropical fruit) is the most prominent and is the desired characteristic of the product.
Claims
1. A kind Torulaspora delbrueckii BYA-1 Its characteristics are, It was deposited at the China General Microbiological Culture Collection Center on November 24, 2025, with accession number CGMCC No. 39053.
2. As described in claim 1 Torulaspora delbrueckii BYA-1 Its characteristics are, Its nucleotide sequence is shown in SEQ ID NO:
1.
3. As described in claim 1 or 2 Torulaspora delbrueckii BYA-1 The screening method is characterized by, The Torulaspora delbrueckii BYA-1 The sample was obtained by enrichment, separation, and screening using an enrichment medium, which included wort, ethanol, and chloramphenicol.
4. The method according to claim 3 Torulaspora delbrueckii BYA-1 The screening method is characterized by, The enrichment medium contains 5-10% ethanol by mass, 50-150 ug / mL chloramphenicol, and a pH of 3.8-4.
1.
5. The method according to claim 3 Torulaspora delbrueckii BYA-1 The screening method is characterized by, The enriched strains were isolated using an isolation medium comprising YPD, sodium propionate, and chloramphenicol. The sodium propionate content in the isolation medium was 0.5-1.6%, and the concentration of chloramphenicol was 50-150 ug / mL.
6. The method according to claim 3 Torulaspora delbrueckii BYA-1 The screening method is characterized by, During enrichment, the substrate and the enrichment medium are added to the enrichment tube and left for a period of time to allow the medium to soak the substrate. The tube wall is tapped to remove all air bubbles from the substrate. The enrichment medium that was missing after the air bubbles were removed is replenished. The tube is then incubated and the enrichment is continuously observed. When a large number of air bubbles are generated in the enrichment tube or even overflows, or when white bacterial cells are visible to the naked eye at the bottom of the tube, the tube can be removed and plated for dilution. Dilute and coat the enriched solution using YSC plates, diluting it to 10. -3 -10 -5 Spread the plating, incubate the spread plates, and observe the results. Plates with few or no colonies can be re-spread by adjusting the dilution factor. For plates with mold growth, promptly remove colonies that morphologically resemble Saccharomyces cerevisiae. Other plates can be left to stand until the colonies are slightly larger and their morphology is clear. Then, visually inspect the plates and streak colonies that morphologically resemble yeast strains onto YPD plates.
7. The method according to claim 3 Torulaspora delbrueckii BYA-1 The screening method is characterized by, The isolated strains were initially screened for maltose utilization ability in multi-well plates with maltose as the sole carbon source. Strains with OD600 not lower than 1.26 after 3 days of culture were selected.
8. The method according to claim 3 Torulaspora delbrueckii BYA-1 The screening method is characterized by, The isolated strains were initially screened for alcohol tolerance in YPD and multi-well plates containing alcohol-stressed medium. Strains with an OD600 of not less than 1.32 after 3 days of culture were selected.
9. The method according to claim 3 Torulaspora delbrueckii BYA-1 The screening method is characterized by, The isolated strains were initially screened for their thiol production capacity in multi-well plates containing YNB, cysteine, and glucose. Strains with an OD600 of not less than 0.689 after 3 days of culture were selected.
10. The method according to claim 1 or 2 Torulaspora delbrueckii BYA-1 Its application in specialty beer brewing is characterized by... Regarding the Torulaspora delbrueckii BYA-1 During wort fermentation, the Torulaspora delbrueckii BYA-1 The 13°P wort has a fermentation degree of 50-60%, an alcohol content of 4.05-5.15% vol, an acetic acid yield of 200-400 mg / L, a thiol 3MH yield of 160-350 ng / L, a 3MHA yield of 6-15 ng / L, and a 4MMP yield of 30-45 ng / L.