Breeding of a high β-glucanase-producing pichia kluyveri strain and its application in beer fermentation

Abstract

The application discloses a high-yield beta-lyase Pichia kluyveri strain breeding and application in beer fermentation, belonging to the field of bioengineering technology. Pichia kluyveri The Pichia kluyveri (Pichia kluyveri) B21 is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 34028. The strain can produce high-yield beta-lyase, the enzyme catalyzes the production of volatile mercaptans, and can impart beer fruity aroma. The Pichia kluyveri B21 produces high content of ethyl acetate, isoamyl acetate, isobutyl acetate, ethyl nonanoate, ethyl decanoate, phenethyl acetate, linalool and nerol in hop simulation culture medium, and these volatile substances can increase beer fruity aroma, floral aroma and rich beer flavor. The Pichia kluyveri B21 is expected to be added to the production of other food and beverage to enrich flavor substance components.

Description

Technical Field

[0001] This invention relates to the selection and application of a high-yield β-lyase-producing Pichia pastoris strain in beer fermentation, belonging to the field of bioengineering technology. Background Technology

[0002] In recent years, with the improvement of people's economic level and the younger generation's consumption, traditional ales and lagers are no longer able to meet consumer demands, and the beer industry is moving towards high quality and diversification. Furthermore, consumers are no longer satisfied with the single flavor of traditional fermented beverages and are placing higher demands on the diversity of flavors. Yeast strains are the fermenting agents in beer fermentation and are closely related to the formation of flavor compounds; excellent yeast strains can bring unique taste and flavor to beer. Therefore, the selection and breeding of flavor compound-producing yeast strains and the study of their brewing performance have become current research hotspots.

[0003] Yeast strains secrete various extracellular enzymes that act on precursors in hops, influencing beer flavor. In studies of volatile thiols, β-lyases have received considerable attention. Under the action of β-lyases, volatile thiols are released from their non-volatile precursors—cysteine ​​conjugates. Among these volatile thiols, 4-mercapto-4-methylpentan-2-one (4MMP) has been extensively studied, exhibiting aromas of boxwood, currant, and blackcurrant. Next is 3-mercaptohexan-1-ol (3MH), exhibiting aromas of grapefruit and guava, while 3-mercaptohexyl acetate (3MHA), derived from the third thiol 3MH, exhibits aromas of passion fruit. These three thiols have extremely low thresholds; even trace amounts can significantly impact beer flavor.

[0004] Hops impart unique aromas to beer and influence its quality. Hops contain a large amount of volatile thiol precursors (bound thiols) and free thiols. Some studies suggest that hops rich in thiol precursors (bound thiols) should be added during whirlpool settling or after the settling process, as this helps the β-lyase produced by yeast during fermentation to catalyze these precursors. On the other hand, varieties with high free thiol content can be dry-hopped, as the yeast's β-lyase is not needed to release the thiols. For example, hops rich in 3MH are suitable for whirlpool settling because over 99% of 3MH exists in the hops in a bound form.

[0005] Kluyveromycin ( Pichia kluyveriKluwer Pichia pastoris is a non-brewing yeast that can be found in the natural environment and can be used to produce fermented foods such as alcoholic beverages and bread. K Hong et al. used a mixture of Kluwer Pichia pastoris and brewer's yeast for fermentation to enhance the aroma of beer; Q Wu et al. used a mixture of Kluwer Pichia pastoris and brewer's yeast for fermentation of high-alcohol kiwi wine. Studies have found that Kluwer Pichia pastoris (… Pichia kluyveri While yeasts with low acetic acid and high glycerol production exhibit faster fermentation rates compared to brewer's yeasts, research on β-lyase and its catalyzed thiols is lacking. Consequently, beer products with characteristic fruity flavors resulting from high β-lyase and thiols content are scarce. Therefore, there is an urgent need to breed yeasts that produce high levels of β-lyase to compensate for the shortage of fruity flavors in beer brewing, enrich beer flavor profiles, and improve beer quality. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a strain of Kluyveromyces yeast ( Pichia kluyveri B21 is used in beer fermentation to increase the content of volatile thiols 4-mercapto-4-methyl-2-pentanone, 3-mercapto-1-hexanol, and 3-mercaptoacetate flavor compounds, thereby enhancing the tropical fruit aroma of beer and enriching its flavor.

[0007] The first objective of this invention is to provide a strain of Kluyveki yeast ( Pichia kluyveri B21, the Kluyveromyces yeast ( Pichia kluyveri B21 was deposited on March 31, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34028. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0008] A second object of the present invention is to provide a product containing the aforementioned Kluyveromycin yeast ( Pichia kluyveri B21 microbial inoculants.

[0009] In one embodiment, the Kluyveromyces yeast in the microbial agent ( Pichia kluyveri The concentration of B21 cells should not be less than 3 × 10⁻⁶. 8 CFU / mL or 3×10 8 CFU / g.

[0010] A third object of the present invention is to provide a product containing the aforementioned Kluyveromyces B21 or the aforementioned microbial agent.

[0011] In one embodiment, the product includes food, medicine, or health products.

[0012] In one embodiment, the food includes a beverage; preferably, the beverage includes alcohol or soft drinks.

[0013] The fourth object of the present invention is to provide a beer fermentation method, wherein the Kluwer Pichia pastoris (Kluwer Pichia pastoris) is used to ferment beer. Pichia kluyveri B21, or the microbial agent described herein, is inoculated into the fermentation medium for fermentation.

[0014] In one embodiment, the fermentation medium comprises wort.

[0015] In one embodiment, the wort concentration is 11-12°P.

[0016] In one embodiment, the fermentation temperature is 20-22°C.

[0017] In one embodiment, the fermentation time is 5-7 days.

[0018] The fifth objective of this invention is to provide the above-mentioned Kluyveromyces yeast ( Pichia kluyveri B21, or the application of the above-mentioned microbial agents in improving the fruity flavor of beer.

[0019] In one embodiment, the enhancement of beer fruit flavor is achieved by increasing the content of at least one of 4-mercapto-4-methyl-2-pentanone, 3-mercapto-1-hexanol, and 3-mercaptoacetate.

[0020] The sixth object of the present invention is to provide the above-mentioned Kluyveromyces yeast ( Pichia kluyveri B21, or the above-mentioned microbial agents, in the preparation of 4-mercapto-4-methyl-2-pentanone, 3-mercapto-1-hexanol and / or 3-mercaptoacetate hexyl ester.

[0021] Beneficial effects: The strain Kluyveromyces yeast obtained by screening in this invention ( Pichia kluyveri The β-lyase activity of B21 was 157.86 ± 0.17 U / g.

[0022] The Kluyveromyces yeast described in this invention ( Pichia kluyveri B21 can be used to ferment wort and prepare beer. Under the action of β-lyase, volatile thiols are released from their non-volatile precursors—cysteine ​​conjugates. After fermentation, the contents of 4-mercapto-4-methyl-2-pentanone, 3-mercapto-1-hexanol, and 3-mercaptoacetate reach 4.61±0.42 ng / L, 271.02±9.20 ng / L, and 9.75±0.89 ng / L, respectively, which can impart blackcurrant, grapefruit, and passion fruit flavors to beer.

[0023] Therefore, Kluyvekistrodon (Kluyvekistrodon) Pichia kluyveri B21 has broad application prospects in beer brewing.

[0024] Preservation of biological materials: Kluyveromycin ( Pichia kluyveri B21, classified as Kluyveromyces bacillus. Pichia kluyveri It was deposited on March 31, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34028. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. Attached Figure Description Picture 1 The results are from spotting YCB-SMC selective medium for β-lyase.

[0025] Picture 2 The results of TTC plate staining for the initial screening strains are shown.

[0026] Picture 3 Kluyveromyces (Kluyveromyces) Pichia kluyveri Colony morphology of 13.

[0027] Picture 4 Kluyveromyces (Kluyveromyces) Pichia kluyveri Phylogenetic tree of 13.

[0028] Picture 5 Kluyveromyces (Kluyveromyces) Pichia kluyveri The growth curve of 13.

[0029] Picture 6 Kluyveromyces yeast under different mutagenesis times ( Pichia kluyveri The mortality rate was 13%.

[0030] Picture 7 Different pH values ​​were used to test the effects of different pH values ​​on the mutant strain Kluyveromyces (Kluyveromyces) Pichia kluyveri The effect of B21 growth.

[0031] Picture 8 To investigate the effects of different temperatures on the mutant strain Kluyveromyces ( Pichia kluyveri The effect of B21 growth.

[0032] Picture 9 To investigate the effects of different ethanol concentrations on the mutant strain Kluyveromyces ( Pichia kluyveri The effect of B21 growth.

[0033] Picture 10 Kluyveromyces (Kluyveromyces) Pichia kluyveri Changes in B21 β-lyase activity and biomass.

[0034] Picture 11 Heatmap of flavor compounds in Idaho No. 7 hops simulated culture medium.

[0035] Picture 12 Heatmap of flavor compounds in mosaic hops simulated culture medium. Picture 13 The production of volatile thiols by different strains. Detailed Implementation

[0036] The culture media involved in the following examples are as follows: YPD liquid medium (g / L): glucose 20.0, peptone 20.0, yeast extract 10.0.

[0037] YPD medium plates (g / L): glucose 20.0, peptone 20.0, yeast extract 10.0, agar powder 20.0.

[0038] YCB-SMC medium: 1.2% (w / v) yeast carbon source basal medium, 0.1% (w / v) S-methyl-L-cysteine, 0.01% (w / v) 5 ， - Pyridoxal phosphate and 2% agar. Autoclave the agar solution, adjust all other components to pH 3.5 with 1 mol / L HCl and filter (0.22 µm), then mix the two components in the agar solution at approximately 60°C.

[0039] Quantitative assay of β-lyase activity: Culture medium: 0.17% yeast natriuretic carbonate (YNB) free of (NH4)2SO4 and amino acids, 1% glucose, 0.01% 5 ， -Pyridoxal phosphate and 15 mM L-cysteine.

[0040] Mosaic and Idahua No. 7 hop simulation culture formula: 90mL sterile water, 1g hop granules.

[0041] Glucose-simulated culture medium formula (g / L): glucose 120.0, peptone 20.0, yeast extract 10.0.

[0042] The wort preparation method described in the following examples is as follows: Barley malt is pulverized and then saccharified. The barley is added at 45°C with a feed-to-water ratio of 1:5, and the temperature is raised to 48°C and held for 30 minutes. The temperature is then raised to 63°C and held for 1 hour, raised to 72°C and held for 20 minutes, and finally raised to 78°C and held for 10 minutes. The wort is filtered and then boiled for 1 hour. Hops (Cascade, Arkema) are added in three portions, totaling 3 g / L. After boiling, the wort is cooled to room temperature and allowed to swirl for 20 minutes, during which 10 g / L of dry hops is added. The hops are filtered, and the original wort concentration is adjusted to 12°P to serve as the wort culture medium.

[0043] Example 1: Screening for yeasts that produce high levels of β-lysin (1) YCB-SMC selective medium was used for initial screening of yeasts that produce high levels of β-lyase. Preparation of seed culture: First, banana and grape samples were enriched and cultured at 28℃ for 48 h, and then serial dilutions of different concentrations were performed, with the dilution gradient set at 10. -1 -10 -7 Triple replicates were performed for each gradient. An appropriate amount of the diluted solution was spread onto YPD plates supplemented with ampicillin and incubated at 28°C for 2-3 days. Based on morphological characteristics, strains exhibiting good growth viability, spherical, oval, or elliptical colonies with a milky-white color, rough or smooth edges, and typical yeast morphology were selected and added to YPD liquid medium for overnight incubation at 28°C.

[0044] YCB-SMC selective culture medium plate spotting: Adjust the concentration of the activated bacterial solution to 2×10⁻⁶. 8 CFU / mL, and according to 10 -1 -10 -4 Serial dilutions were performed, with 2.5 μL of each dilution spotted onto YCB-SMC agar plates. After incubation at 28°C for 72 h, colony growth was observed. Based on colony growth on the plates, a preliminary assessment of β-lyase activity was made. Good growth of the strain on YCB-SMC agar indicated high β-lyase activity. Strains exhibiting good growth on the plates were selected for further enzyme activity assays. Results of β-lyase spotting on YCB-SMC selective agar are shown below. Picture 1 As shown.

[0045] (2) Quantitative determination of β-lyase activity: β-lyase activity was estimated based on its ability to consume 15 mM L-cysteine ​​as the sole nitrogen source. The enzyme activity assay medium contained 0.17% yeast nitrogen base (YCB) without (NH4)2SO4 and amino acids, 1% glucose, 0.01% pyridoxal 5-phosphate, and 15 mM cysteine. The activated bacterial culture was taken and the cell concentration was adjusted to 2 × 10⁻⁶ cells / day. 8 CFU / mL. 1 mL of culture medium and 100 µL of bacterial suspension were placed in each well of a 96-well plate and sealed with a BreatheEasy membrane. The reaction was carried out at 25°C for 5 days. The L-cysteine ​​content of the culture medium was determined using the DTNB method.

[0046] Enzyme activity is defined as follows: The amount of enzyme that catalyzes 1 mmol of the substrate L-cysteine ​​at 25°C for 5 days is defined as one unit of enzyme activity (U). The enzyme activity corresponding to one unit of dry bacterial cells is calculated and denoted as β-lyase activity (U / g). The dry weight of the bacterial cells is determined by taking 1 mL of bacterial solution, centrifuging and discarding the supernatant, and drying the bacterial sludge at 105°C. The unit is g. The cell concentration is determined by taking 1 mL of bacterial solution and measuring the cell count using a Counterstar instrument. The unit is CFU. Dry weight and cell concentration can be converted between the same volume of bacterial solution. 0.5 mg of dry bacterial cells corresponds to 5 × 10⁵ cells / g. 8 CFU cells.

[0047] The quantitative determination results of β-lyase activity of 30 strains obtained from the initial screening are shown in Table 1.

[0048] Table 1. Quantitative determination results of β-lyase activity

[0049] Ten yeast strains with high β-lyase activity were screened using YCB-SMC selective medium plates and quantitative β-lyase assays: strains 1, 5, 7, 9, 10, 11, 12, 13, 15, and P7. These 10 strains were then used for further experiments.

[0050] Example 2: Physiological and biochemical analysis of yeast producing high levels of β-lysin. (1) Analysis of alcohol production capacity TTC is a chromogenic agent that reacts with dehydrogenases in yeast to produce a red color. The intensity of the red color is closely related to the activity of respiratory enzymes in the yeast; therefore, strains with higher alcohol production capacity will produce a deeper color upon reaction with TTC. The yeast suspension obtained from activation was diluted and spread onto a TTC lower plate, and incubated upside down for 2 days. The TTC upper medium was then poured in to cover the existing colonies, and the plate was incubated in the dark for 2 hours. The TTC plate color development results are shown below. Picture 2 As shown in Table 2, the TTC display intensity classification of the strains is presented. Based on the TTC plate colorimetric analysis, 10 strains showed weak alcohol production capacity.

[0051] Table 2 Classification of TTC staining intensity of strains

[0052] (2) Gas production capacity analysis The strength of a yeast strain's gas production capacity can be used to measure the speed of yeast fermentation. Yeast strains with strong gas production capacity ferment quickly and have strong fermentation ability in anaerobic environments.

[0053] Ten yeast strains with high β-lyase activity obtained from the initial screening were used for Durham tube fermentation. The fermentation capacity of the strains was represented by the gas production of the yeast during fermentation. The gas production of the 10 yeast strains obtained after the initial screening is shown in Table 3.

[0054] Table 3 Gas production of the strain

[0055] Note: The rate of gas production in yeast is observed through the Durham tubules. "+" indicates that the gas production volume is less than 2 / 3 of the Durham tubule volume, "++" indicates that the gas production volume is more than 2 / 3 of the Durham tubule volume, and "+++" indicates that the gas production volume reaches the Durham tubule volume.

[0056] As shown in the table, all 10 yeast strains have gas-producing capabilities, with 7 strains releasing more gas and exhibiting extremely strong gas-producing abilities. These strains are 1, 5, 7, 9, 11, 12, and P7, and these strains have stronger fermentation capabilities than the other strains.

[0057] (3) Analysis of carbon and nitrogen source assimilation capacity The assimilation capacity of carbon and nitrogen sources is closely related to the fermentation capacity of microbial strains. Understanding the carbon and nitrogen assimilation capacity of different strains helps to understand their physiological performance. A spot plate experiment was used to evaluate the carbon and nitrogen source utilization capacity of the strains.

[0058] Table 4. Nitrogen and carbon source utilization capacity of strains

[0059] As shown in the table above, the 10 yeast strains were able to utilize various carbon sources for growth, but the utilization efficiency varied considerably. The 10 strains showed the best utilization of glucose and fructose, but weaker utilization of maltose and sucrose. Nitrogen source is crucial for yeast growth and metabolism; the 10 strains showed the best utilization of peptone, but weaker growth on ammonium chloride, ammonium sulfate, and potassium nitrate. Furthermore, none of the strains could utilize urea as a nitrogen source.

[0060] (4) Analysis of β-lyase activity and evaluation of fermentation aroma during fermentation Ten strains with high β-lyase activity were screened and inoculated onto YPD medium, and cultured at 28°C for 2 days. The yeast was then transferred to 12°P wort medium, with an initial inoculum size of 5 × 10⁶. 6 CFU / mL, fermented at 22℃ for 5 days. β-lyase activity was measured and the aroma of the fermentation broth was evaluated by olfaction to screen strains with high enzyme activity and tropical fruit aromas during fermentation.

[0061] Table 5. β-Lysase Activity of Strains During Fermentation

[0062] Table 6 Evaluation of fermentation aroma of different strains

[0063] Table 7 Determination of Basic Fermentation Indicators

[0064] Preliminary bromine analysis revealed that strain 1 produced a rose aroma, strain 9 developed a banana aroma after fermentation, strain 12 exhibited banana and citrus aromas during fermentation, and strain P7 produced a cantaloupe aroma. Based on β-cleavage activity, strain 13 was selected as the starting strain for subsequent experiments. This strain effectively brought out the fruity aromas of the hops, producing a pleasant fragrance. Fermentation index measurements showed that strains 10 had low alcohol production capacity, with alcohol contents ranging from 0.48% to 0.71% v / v, and a true degree of fermentation of 7.64% to 8.84%.

[0065] Example 3: Biological identification of yeast producing high levels of β-lysin (1) Morphology of high-yield β-lysin-producing yeast The strain obtained in Example 2 was activated, streaked onto YPD plates, and colony morphology was observed. For example... Picture 3 The colonies are milky white with wrinkled edges.

[0066] (2) Molecular biological identification of yeasts that produce high levels of β-lysin Step 1: The starting strain was inoculated into YPD liquid medium at a 2% (v / v) inoculum for activation and cultured at 28℃ and 180 rpm for 24 h to obtain seed culture. An appropriate amount of culture was centrifuged at 12000 rpm and 4℃ for 1 min, the supernatant was discarded, and an appropriate amount of bacterial cells were obtained. Genomic DNA was extracted using the Shanghai Sangon Yeast Genomic DNA Extraction Kit.

[0067] Step 2: Extract DNA from the strain according to the yeast DNA extraction kit instructions for PCR amplification of 18S rDNA. Primer ITS1: 5 ， -TCCGTAGGTGAACCTGCGG- 3 ， And ITS4:5 ， -TCCTCCGCTTATTGATATGC- 3 ， 25µL reaction system: Taq DNA polymerase 12.5µL, ITS1 1µL, ITS4 1µL, template DNA 0.5µL, ddH2O 10µL. PCR reaction system: 95℃ 3min; 95℃ 30s; 56℃ 15s, 72℃ 1min, 35 cycles; 72℃ 5min. PCR products were detected by 1.0% agarose gel electrophoresis. Products were sent to Genewiz Biotechnology Co., Ltd. for sequencing.

[0068] Step 3: Perform 18S rDNA sequencing analysis on the starting strain, and compare the sequencing results with yeast sequences in GenBank using BLAST. The phylogenetic tree results of the BLAST analysis of the tested strain are shown below. Picture 4 As shown, species of the same kind are grouped into a single main branch, indicating close phylogenetic relationship, while different species are grouped into different subbranches, showing clear differences in the D1 / D2 region sequences. Strain 13 is related to *Kluyveromyces* (…). Pichia kluyveri The gene sequence similarity was >99%, confirming the strain as Kluyveromyces oryzae (Kluyveromyces oryzae). Pichia kluyveri ).

[0069] Example 4: ARTP mutagenesis and selection of high-yield β-lysin yeast (1) Growth curve of the starting strain The starting strain from Example 3 was activated, and after 24 hours, 1 mL was transferred to a 100 mL YPD liquid Erlenmeyer flask and cultured with shaking at 28℃ and 180 rpm. Samples were taken every 2 hours to determine the cell concentration, and a growth curve was plotted. Picture 5 The results showed that the strain gradually adapted to the new environment within 0-6 hours and was in the delayed growth phase; from 6-16 hours, the strain grew rapidly and was in the logarithmic growth phase; after 16 hours, the growth slowed down and entered the stationary growth phase.

[0070] When bacteria are in the logarithmic growth phase, their metabolic activity becomes particularly vigorous, and their enzyme production capacity is significantly enhanced. During this period, the bacteria's sensitivity to mutagens increases significantly, making them more susceptible to the effects of mutagens compared to other growth stages, thus leading to gene mutations. Therefore, based on the growth curve of the starting strain, 16 hours was selected as the culture time for starting strain 13.

[0071] (2) Determination of the optimal mutagenesis time 1. Preparation of bacterial suspension One loopful of bacterial culture was picked from the slant and activated. After 24 hours, it was transferred to the culture. 2 mL of the culture after 16 hours was centrifuged at 10000 rpm for 1 min, and the supernatant was discarded. The wet bacterial cells were washed with sterile water and centrifuged at 7000 rpm for 5 min, repeated three times. Finally, the cells were resuspended in 2 mL of sterile physiological saline, and the bacterial concentration was measured. Further dilution was performed to a concentration of 1 × 10⁻⁶. 7 CFU / mL. 2. ARTP mutagenesis Spread 10 µL of bacterial culture evenly onto a pre-sterilized ARTP metal slide, then place it in an ARTP mutagenesis instrument for treatment times of 20 s, 40 s, 60 s, 80 s, 100 s, and 120 s. After treatment, place the metal slide into a centrifuge tube containing 990 µL of sterile physiological saline and vortex for 1 min to elute. Serially dilute the bacterial culture 10-1 -1-10 -4 Take 100 µL and spread it evenly on a YPD solid plate, then incubate at 28 °C for 2 days. Calculate the lethality rate.

[0072] Cultivating the starting strain for 16 hours, with an ARTP mutagenesis lethality of 80%-95%, is more beneficial for subsequent screening. If the lethality rate exceeds 95%, the overall growth performance of the strain will be impaired, negatively impacting the flavor of the finished wine. Picture 6 As can be seen, plasma has a strong killing effect on Pichia pastoris; even a short ARTP treatment results in a high mortality rate. The mortality rate increases with prolonged mutagenesis time. At 30 seconds, the mortality rate of strain 13 was 89%, and at 40 seconds, it was 98.5%. At 80 seconds, the mortality rate of strain 13 reached 100%, with no viable cells found. When the mortality rate is between 80% and 95%, the mutation probability and magnitude of the strain are relatively high. Therefore, 30 seconds was selected as the optimal time for ARTP mutagenesis.

[0073] Starting strain 13 was mutagenized with ARTP to obtain 110 mutant strains. The enzyme activities of the mutant strains are shown in Table 8 below.

[0074] Table 8. β-Lysase Activity of Mutant Strains

[0075] After ARTP mutagenesis, strain B21 was selected as having the highest enzyme activity, at 157.86±0.17 U / g. Compared with the starting strain 13 (125.21±0.12 U / g), the enzyme activity increased by 26.07%.

[0076] The Kluyvechi yeast ( Pichia kluyveri B21 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34028.

[0077] Example 5: Environmental tolerance of Kluyveromyces b21 (1) Effects of different pH values ​​on the growth of Kluyveromyces B21 Yeast can carry out normal physiological and metabolic processes at a pH between 4.5 and 5.5. During fermentation, a pH that is too low will inhibit yeast metabolism and growth, and may even cause yeast death. The Kluyveromyces b21 from Example 4 was fermented at 5 × 10⁻⁶... 6 CFU / mL inoculum was inoculated into YPD liquid medium at pH 3, 4, 5, 6, 7, and 8, and cultured at 28℃ for 48 h. The bacterial concentration was then determined.

[0078] like Picture 7As shown, Kluyveromyces b21 hardly grows at pH=7 and pH=8, grows slowly at pH=3, and has the optimal growth pH between 4 and 6.

[0079] (2) Effects of different temperatures on the growth of Kluyveromyces b21 Temperature is one of the important factors affecting yeast growth. Suitable temperatures can promote yeast growth and reproduction, while excessively high or low temperatures will affect yeast growth activity. The Kluyveromyces b21 yeast from Example 4 was grown at 5 × 10⁻⁶... 6 CFU / mL inoculum was added to YPD liquid medium, and the culture was carried out at temperatures of 4℃, 10℃, 15℃, 20℃, 25℃, 28℃, and 30℃ for 48 hours. The cell concentration was then measured. Picture 8 As shown, Kluyveromyces b21 grows slowly at temperatures of 4℃, 10℃, 20℃ and 30℃, with the optimal growth temperature being 22℃-28℃.

[0080] (3) Effect of different ethanol concentrations on the growth of Kluyveromyces b21 As fermentation progresses, nutrients are continuously consumed, and the ethanol concentration gradually increases, putting stress on the cells. The ethanol content in the fermentation broth is a significant cause of decreased cell viability. Therefore, the yeast's tolerance to ethanol directly affects the completeness of fermentation and the utilization of nutrients. Non-Saccharomyces cerevisiae have lower ethanol tolerance and shorter fermentation cycles; therefore, screening for non-Saccharomyces cerevisiae with high alcohol tolerance can further improve their survival rate and increase flavor compounds in subsequent fermentation processes. The Kluyveromyces b21 from Example 4 was fermented at 5 × 10⁻⁶... 6 Inoculate with CFU / mL into YPD liquid medium containing different ethanol concentrations of 0%, 3%, 6%, 9%, 12%, and 15% (v / v), and incubate at 28°C for 48 hours. Then determine the cell concentration. Picture 9 As shown, Kluyveromyces B21 hardly grows at ethanol concentrations of 8%-12%, grows slowly at ethanol concentrations of 7%, and grows well at ethanol concentrations of 0%-6%. Therefore, Kluyveromyces B21 can tolerate ethanol concentrations of 7%.

[0081] Example 6: Analysis of β-lyase activity during the fermentation process of Pichia pastoris B21 The Kluyveromyces b21 strain with high β-lyase activity obtained in Example 4 was subjected to wort fermentation. The fermentation performance of the strain was evaluated by measuring the degree of fermentation and alcohol content of the fermentation broth. Enzyme activity was measured during fermentation to assess changes in enzyme activity in the mutant strain. Kluyveromyces b21 was fermented at 5 × 10⁻⁶... 6 CFU / mL was inoculated into the wort and fermented at 22℃ for 6 days. Biomass, CO2 weight loss, and β-lyase activity were monitored and measured during fermentation. After fermentation, alcohol content and true degree of fermentation were measured.

[0082] like Picture 10 As shown, during fermentation, the β-lyase activity exhibited a trend of first decreasing, then increasing, and then decreasing again. It reached its highest value on the first day, at 149.6 ± 0.01 U / g, at which point yeast growth and reproduction were vigorous, suggesting that this phenomenon is closely related to yeast growth. The fermentation indicators of Kluyveromyces B21 are shown in Table 9. The results indicate that the alcohol content of Kluyveromyces B21 was 0.91 ± 0.02 (%v / v), and the degree of fermentation was 12.15 ± 0.05 (%), both superior to the starting strain.

[0083] Table 9 Fermentation parameters of the starting strain and mutant strain

[0084] Example 7: Analysis of the ability of mutant strain B21 to convert hop and glucose flavor compounds (1) Kluyveromycin ( Pichia kluyveri B21, Abnormal Wickham Yeast ( Wickerhamomyces anomalus M3 (self-screened strain), LA01 (alcohol-free yeast), and US-05 (Ale brewer's yeast) were activated, and after 24 hours, 1 mL was transferred to a 100 mL YPD liquid Erlenmeyer flask and cultured with shaking at 28℃ and 180 rpm. The bacterial concentration of the amplification solution was adjusted to 2 × 10⁻⁶. 8 CFU / mL was inoculated into 10 mL of a 1% (w / v) hop simulant medium, using either Idahua 7 or Mosaic hop granules. After 5 days of static incubation at 22°C, the flavor compounds in the simulant medium were determined. Uninoculated hop simulant medium served as a control. Volatile flavor compounds were detected using GC-MS, and the results are shown below. Picture 11 , 12 The quantitative results of the compounds are shown in Tables 10 and 11.

[0085] In Idahua No. 7 hop-simulated medium, the fermentation products of Kluwer Pichia pastoris B21 showed a 60% increase in ethyl acetate content (25.81 mg / L), a 68% increase in isoamyl acetate content (0.45 mg / L), and a 3% increase in ethyl hexanoate content (0.03 mg / L). Furthermore, B21 produced new compounds including isobutyl acetate, ethyl nonanoate, ethyl decanoate, and phenylethyl acetate. Isobutyl acetate exhibits pear and raspberry aromas. Ethyl nonanoate has fruity, rose, and waxy aromas. Ethyl decanoate has rose and coconut aromas. Phenethyl acetate has a rose aroma.

[0086] Table 10. Analysis of flavor compounds in Idahua No. 7 hop simulated culture medium.

[0087] In a mosaic hop-simulated medium, the fermentation products of strain B21 showed the following improvements compared to the control without added bacterial culture: ethyl acetate content increased by 51% to 24.33 mg / L; isobutyl acetate content increased by 14-fold to 0.13 mg / L; isoamyl acetate content increased by 3-fold to 1.10 mg / L; linalool content increased by 19% to 0.25 mg / L; and nerol content increased by 22% to 0.018 mg / L. Furthermore, strain B21 newly produced phenethyl acetate. Phenethyl acetate has a rose aroma, isobutyl acetate has a banana aroma, and isoamyl acetate has banana and pear aromas. Using strain B21 in beer fermentation can produce more fruity and floral aromas, enriching the aroma profile of beer and improving its quality.

[0088] Table 11 Analysis of flavor compounds in mosaic hop simulated culture medium

[0089] (2) Activate strains B21, US05, and LA01, and after 24 hours, transfer 1 mL to a 100 mL YPD liquid Erlenmeyer flask and incubate with shaking at 28℃ and 180 r / min. Adjust the bacterial concentration of the amplification solution to 2 × 10⁻⁶. 8CFU / mL was inoculated into 10 mL to 90 mL of glucose-simulated medium. After static incubation at 22℃ for 5 days, the flavor compounds in the simulated medium were measured. The results are shown in Table 12. In the glucose-simulated system, strains B21, US05, and LA01 synthesized 24, 18, and 17 volatile flavor compounds, respectively. Among them, after yeast biotransformation, strain B21 produced the most new volatile compounds, with 18 volatile compounds, including 11 esters, 3 higher alcohols, and 4 terpenoids. Strain US05 and strain LA01 both produced 13 new volatile compounds. In the glucose-simulated medium supplemented with strain B21, three esters had an OAV > 1: isoamyl acetate, isoamyl propionate, and phenethyl acetate. Isoamyl acetate had the highest OAV at 16.52, contributing most significantly to the fruity flavor. The total ester compounds produced by B21, US05, and LA01 from glucose conversion were 1885.98, 152.01, and 108.36 μg / L, respectively, with B21 producing the highest content of ester compounds. In glucose-simulated medium supplemented with strain B21, isoamyl propionate was the main ester compound, with a concentration of 741.94 μg / L, primarily contributing to the aromas of tropical fruits and stone fruits. Isoamyl propionate is produced by the esterification reaction of propionic acid and isoamyl alcohol catalyzed by yeast esterase. The nitrogen sources in the glucose-simulated medium were peptone and yeast extract, and yeast can synthesize higher alcohols through amino acid catabolism. The total concentrations of higher alcohols synthesized by B21, US05, and LA01 were 3069.35, 1928.06, and 1779.95 μg / L, respectively, with the ability of the three yeast strains to synthesize higher alcohols from highest to lowest being B21, US05, and LA01. Strain B21 produced the highest content of phenylethanol, at 1721.72 μg / L. In the glucose simulation system, the core pathway for yeast synthesis of phenylethanol is the Ehrlich pathway: phenylalanine first undergoes transamination to produce phenylpyruvic acid, which is then decarboxylated to form phenylacetaldehyde, and finally reduced to phenylethanol. All three yeast strains can metabolize and produce various flavor compounds such as isoamyl acetate, phenylethanol, isobutanol, and isoamyl alcohol, and their metabolic characteristics show significant differences between strains. Strain B21 contributes most significantly to fruit aroma; strains US05 and LA01 have more balanced flavor compound synthesis profiles and higher overall flavor complexity.

[0090] Table 12 Analysis of flavor compounds after fermentation in glucose-simulated culture medium

[0091] Example 8: Analysis of the ability of Pichia pastoris B21 to convert hop thiols Pichia kluyveri B21, Pichia kluyveri 13, Wickerhamomyces anomalus M3, and Saccharomyces cerevisiae US-05 were activated and inoculated at 7 × 10⁶ cells / year. 6 CFU / mL was added to 12°P wort medium and fermented at 22°C for 5 days. The volatile thiol content of the fermented samples was determined. Strains included strain 13 (starting strain), B21 (mutant strain), and M3 (a non-Saccharomyces cerevisiae strain with good flavor selected in the laboratory). Picture 13 According to Table 13, LC-MS analysis showed that the concentrations of volatile thiols 4MMP, 3MH, and 3MHA in the B21 fermented sample were 4.61±0.42 ng / L, 271.02±9.20 ng / L, and 9.75±0.89 ng / L, respectively. Their OAV values ​​were 1.15, 4.92, and 6.50, all >1. These three substances contribute to the passion fruit, guava, and grapefruit flavors of beer.

[0092] Table 13 Thiol production by different strains

[0093] Example 9: Effect of co-fermentation of Pichia kluwer B21 and Saccharomyces cerevisiae US05 on beer flavor Kluyveromyces b21 and Saccharomyces cerevisiae US-05 were inoculated at the optimal ratio of 5:1 (total inoculation amount was 3×10⁻⁶). 7 The inoculation method was simultaneous inoculation, and the wort was fermented at 22℃ for 8 days. The content of flavor compounds such as thiols was measured at the end of fermentation. The control experiment used single-strain fermentation with US05 at an inoculation amount of 5 × 10⁻⁶. 6 CFU / mL.

[0094] As shown in Table 14, compared with single-strain fermentation, mixed-strain fermentation resulted in increased concentrations of esters, terpenes, and thiols in beer. Esters mainly included ethyl acetate, isobutyl acetate, and isoamyl acetate, which impart fruity, sweet, and ester-like aromas to beer. Terpenols are more hydrophilic than terpenes and are considered important substances for hop flavor. The main terpenol compounds were linalool and α-terpineol. Notably, α-terpineol was not produced during US-05 single-strain fermentation, indicating that this was due to biotransformation by B21. Furthermore, mixed-strain fermentation significantly increased the concentration of phenylethanol to 4.97 mg / L, enhancing the rosé aroma of beer. The concentrations of volatile thiols 4MMP, 3MH, and 3MHA, which impart tropical fruit aromas to beer, also significantly increased. Studies have shown that 10-20% of thiols in hops exist in bound form and require β-lyase catalysis for release. This indicates that the β-lyase produced by B21 plays a positive role in the release of thiols from hops. Compared with beer fermented by a single strain, mixed-strain fermentation increases the concentration of esters, terpenoids, and thiols, and this strain contributes more to the flavor profile.

[0095] Table 14. Concentration of flavor compounds in mixed-culture fermented beer

[0096] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A strain of Kluyveromyces yeast ( Pichia kluyveri B21, characterized in that, The Kluyveromyces b21 was deposited at the China General Microbiological Culture Collection Center on March 31, 2025, with accession number CGMCC No. 34028.

2. A microbial inoculum containing the Kluyveromyces b21 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The concentration of Kluyveromyces b21 cells in the microbial agent is not less than 3 × 10⁻⁶. 8 CFU / mL or 3×10 8 CFU / g.

4. A product containing the Kluyveromyces b21 of claim 1 or the microbial agent of claim 2 or 3.

5. The product according to claim 4, characterized in that, The products include food, medicine, or health products; the food includes beverages; preferably, the beverages include alcohol or soft drinks.

6. A method for preparing beer, characterized in that, The Kluyveromyces b21 of claim 1, or the microbial agent of claim 2 or 3, is inoculated into the fermentation medium for fermentation.

7. The method according to claim 6, characterized in that, The fermentation medium comprises wort; preferably, the concentration of the wort is 11-12°P.

8. The method according to claim 7, characterized in that, Ferment at 20-22℃ for 5-7 days.

9. The application of Kluyveromyces B21 as described in claim 1 or the microbial agent as described in claim 2 or 3 in improving the fruity flavor of beer.

10. The use of Kluyveromyces b21 of claim 1 or the microbial agent of claim 2 or 3 in the preparation of 4-mercapto-4-methyl-2-pentanone, 3-mercapto-1-hexanol and / or 3-mercaptoacetate hexyl ester.

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