A beer brewing method for increasing the content of terpenoid aroma substances

Abstract

The application provides a beer brewing method for improving the content of terpenoid aroma substances, and relates to the field of beer brewing. The brewing method comprises the following steps: inoculating Pichia kudriavzevii (P. Pichia kudriavzevii ) PK2 into a beer fermentation substrate to perform aroma fermentation, then inoculating Saccharomyces cerevisiae to perform main fermentation, and performing post-fermentation after the main fermentation is completed, so that beer is obtained. In the beer brewing process, the safe and efficient enzyme-producing non-Saccharomyces cerevisiae PK2 is added to perform synergistic fermentation with Saccharomyces cerevisiae, the hydrolysis of β-glucosidase to glycoside-bound terpenoid substances is strengthened, the complexity and richness of beer aroma are improved, the flavor balance of the beer body is optimized, the accumulation of adverse flavor substances is reduced, the flavor and quality of the beer are significantly improved, and the demand of the market for high-quality flavor beer is met.

Description

Technical Field

[0001] This invention relates to the field of beer brewing, and more specifically to a beer brewing method for increasing the content of terpenoid aroma substances. Background Technology

[0002] The unique aroma of beer mainly comes from malt, hops and yeast metabolites. Among them, terpenes in hops (such as linalool, nerol, geraniol, etc.) are the key components that give hops their characteristic fruity and herbal aromas.

[0003] However, most terpenoid aroma compounds in hops do not exist in a free state, but mainly in the form of glycosidic complexes of sugars and glycosidic aglycones. These bound substances are non-volatile and stable, and cannot directly contribute aroma to beer. They need to be hydrolyzed to release free aroma compounds in order to contribute flavor. β-glucosidase is the key enzyme catalyzing this hydrolysis reaction. It can specifically break glycosidic bonds, converting bound terpenoid precursors into free aroma compounds.

[0004] Traditional beer brewing often uses pure brewing yeast for fermentation. These yeasts produce β-glucosidase with extremely low activity, making it difficult to effectively hydrolyze glycoside-bound aroma compounds in hops, resulting in insufficient aroma complexity and a monotonous flavor profile in beer. To address this issue, existing technologies attempt to enhance hydrolysis by adding exogenous β-glucosidase preparations or screening for enzyme-producing microorganisms. However, several limitations remain: Firstly, exogenous enzyme preparations are expensive and exhibit poor stability in the complex environment of beer fermentation, characterized by low pH, ethanol, and iso-α-acids, leading to easy inactivation and poor hydrolysis efficiency. Secondly, most reported β-glucosidase-producing microorganisms are molds or bacteria. Mold-derived enzymes may introduce mycotoxins and pose safety risks, while bacterial-derived enzymes lack adaptability and flavor compatibility within the fermentation system.

[0005] In addition, the mixed-strain fermentation methods used in existing beer brewing processes mostly focus on enhancing single flavor components, resulting in insufficient release of aroma substances. At the same time, they may be accompanied by excessive accumulation of undesirable flavor substances such as higher alcohols and organic acids, ultimately leading to an unbalanced flavor in the beer, and even health risks due to the increased content of higher alcohols.

[0006] Therefore, how to effectively promote the release of terpenoid aroma substances during beer brewing, while regulating various flavor substances to achieve flavor balance in the beer and minimizing the health risks posed by higher alcohols, has become an urgent need in the beer brewing industry. Summary of the Invention

[0007] The present invention aims to overcome the shortcomings of existing beer brewing methods that make it difficult to simultaneously achieve the full release of terpenoid aroma substances, the balance of beer flavor, and the effective reduction of higher alcohol content, thereby providing a beer brewing method that increases the content of terpenoid aroma substances and beer brewed based on the method.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] In a first aspect, the present invention provides a beer brewing method for increasing the content of terpenoid aroma substances, comprising the following steps: inoculating a beer fermentation substrate with *Pichia pastoris* (Kudela spp.) Pichia kudriavzevii PK2 is used for aroma-enhancing fermentation, followed by the introduction of brewing yeast for primary fermentation. After the primary fermentation is completed, secondary fermentation is carried out to obtain the final product.

[0010] Kudria zwibichi yeast ( Pichia kudriavzevii PK2, with accession number CGMCC No. 29250, deposited at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on December 6, 2023. This strain has been published in Chinese patent document CN118291281A on July 5, 2024.

[0011] Furthermore, the aroma-enhancing fermentation temperature is 15~25 ℃, and the time is 30~40 h.

[0012] Furthermore, the aroma-enhancing fermentation temperature is 20~22 ℃, and the time is 36~42 h.

[0013] Furthermore, the aroma-enhancing fermentation temperature is 20 °C and the time is 36 h.

[0014] Furthermore, based on the volume of the beer fermentation substrate, the inoculation amount of the *Pichia kudrica* yeast is 5 × 10⁻⁶. 4 ~5×10 5 cfu / mL, preferably 1×10 5 ~3×10 5 cfu / mL, more preferably 1×10⁻⁶ 5 cfu / mL.

[0015] Furthermore, the brewing yeast specifically refers to brewing yeast US-05.

[0016] Furthermore, based on the volume of the beer fermentation substrate, the inoculation amount of the brewing yeast is 1 × 10⁻⁶. 3 ~5×10 4 cfu / mL, preferably 1×10 4 ~3×10 4 cfu / mL, more preferably 1×10⁻⁶ 4cfu / mL.

[0017] Furthermore, the primary fermentation temperature is 15~25 ℃, and the time is 7~14 days.

[0018] Furthermore, the primary fermentation temperature is 20~22 ℃, and the time is 11~12 days.

[0019] Furthermore, the primary fermentation temperature is 20 °C and the time is 11 days.

[0020] Furthermore, the post-fermentation temperature is 2-6 °C, and the time is 5-9 days.

[0021] Furthermore, the post-fermentation temperature is 4-5 °C, and the time is 7-8 days.

[0022] Furthermore, the post-fermentation temperature is 4 ℃ and the time is 7 days.

[0023] Furthermore, the method for preparing the beer fermentation substrate includes the following steps: (1) After crushing the malt, mix it with water and then saccharify the mixture. After saccharification, cool and filter to obtain the saccharified liquid. (2) Boil the saccharified liquid and add hops after boiling. After boiling, wort is obtained. (3) The wort is packaged and sterilized to obtain the beer fermentation substrate.

[0024] Further, in step (1), malt and water are mixed at a mass ratio of 1:3 to 5, preferably 1:4.

[0025] Further, in step (1), the saccharification process includes: heating the mixture to 50~55 ℃ and holding it at that temperature for 30~40 min; continuing to heat to 60~65 ℃ and holding it at that temperature for 1~2 h; continuing to heat to 66~72 ℃ and holding it at that temperature for 30~40 min; finally heating to 75~80 ℃ and holding it at that temperature for 10~20 min; preferably, heating the mixture to 52 ℃ and holding it at that temperature for 30 min; continuing to heat to 62 ℃ and holding it at that temperature for 1 h; continuing to heat to 68 ℃ and holding it at that temperature for 30 min; finally heating to 78 ℃ and holding it at that temperature for 10 min.

[0026] Further, in step (2), the boiling time is 1 to 1.5 hours. Hops are added 10 to 15 minutes after the start of boiling, with an addition amount of 0.2 to 0.4 g / L based on the volume of the saccharified liquid. Hops are added again 10 to 15 minutes before the end of boiling, with an addition amount of 0.6 to 0.8 g / L based on the volume of the saccharified liquid. Preferably, the boiling time is 1 hour. Hops are added 10 minutes after the start of boiling, with an addition amount of 0.3 g / L based on the volume of the saccharified liquid. Hops are added again 10 minutes before the end of boiling, with an addition amount of 0.7 g / L based on the volume of the saccharified liquid.

[0027] Furthermore, in step (3), the sterilization conditions are: high temperature and high pressure sterilization at 115 ℃ and 0.1 MPa for 20 min.

[0028] Secondly, the present invention provides beer obtained by the beer brewing method described above for increasing the content of terpenoid aroma substances.

[0029] The technical solution of this invention has the following advantages: 1. The beer brewing method provided by this invention involves adding *Pichia pastoris* PK2 before the primary fermentation of brewing yeast for aroma enhancement fermentation. This strain possesses excellent environmental tolerance and can stably adapt to the complex environment of beer fermentation. This strain has the outstanding characteristic of high β-glucosidase production (enzyme activity in fermentation broth reaches 308.93 U / mL). The β-glucosidase it produces has excellent tolerance to ethanol and iso-α-acids and can function stably without additional adjustments to the fermentation system. β-glucosidase PkBgl belongs to the GH1 family, with a protein size of 40 kDa. It can specifically hydrolyze glycoside-bound terpenes in hops, releasing free terpenes aroma compounds. After hydrolysis experiments of hop essential oils with the enzyme solution, the contents of linalool and nerol increased to 2.67 times and 3.06 times respectively, providing a new approach to improving the flavor and quality of beer.

[0030] 2. This invention introduces a safe and efficient enzyme-producing non-brewing yeast, PK2, into the beer brewing process for synergistic fermentation with brewing yeast. This enhances the hydrolysis of glycoside-bound terpenes by β-glucosidase, thereby improving the complexity and richness of beer aromas, optimizing the balance of beer flavors, reducing the accumulation of undesirable flavor substances, and significantly improving the flavor and quality of beer to meet the market demand for high-quality flavored beer.

[0031] 3. Compared with brewing yeast fermentation alone, co-fermentation with non-brewing yeast PK2 increases the content of terpenoid aroma compounds linalool, nerol, and geraniol in beer to 2.8 times, 11.4 times, and 93.6 times, respectively, compared to brewing yeast fermentation alone. The content of esters ethyl acetate, ethyl octanoate, and phenylethyl acetate increases to 5.6 times, 2.3 times, and 2.1 times, respectively, compared to fermentation alone. The content of higher alcohols isobutanol, isoamyl alcohol, and phenylethyl alcohol is effectively reduced, and the undesirable flavor compound decanoic acid is reduced from 0.87 mg / L to 0.26 mg / L. Phenylethyl acetate, linalool, and geraniol impart rich floral and fruity aromas to the beer, resulting in prominent floral and fruity notes. The reduction in organic acid content weakens the sourness of the beer, making the taste more refreshing. The reduction in higher alcohol content maintains a suitable body while avoiding the undesirable flavors and health risks caused by excessive higher alcohols, resulting in a smoother and more harmonious taste. The addition of non-brewing yeast PK2 not only increased the content of terpenoid aroma compounds in beer, but also significantly improved the sensory balance of beer in many ways, resulting in a sensory score of 96.76.

[0032] 4. The core strain of Pichia pastoris PK2 in the beer brewing process is derived from a natural fermentation system, is green and safe, has no risk of fungal toxin residue, and meets the safety requirements of the food fermentation field.

[0033] 5. The beer brewing method provided by this invention does not require the addition of exogenous enzyme preparations. Flavor enhancement is achieved through the enzyme production of the strain itself, which reduces the cost of production materials and simplifies the process, thus having a cost advantage for industrial application. Attached Figure Description

[0034] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0035] Figure 1 This is a graph showing the change in the content of terpenoids in hop oil before and after hydrolysis in Example 3 of the present invention. The ns group showed a significant difference between the two groups (P<0.05); the ns group showed no significant difference between the two groups (P≥0.05). Figure 2 This is a graph showing the effect of ethanol concentration on the activity of β-glucosidase PkBgl in Example 4 of the present invention; Figure 3This is a graph showing the effect of iso-α-acid concentration on the activity of β-glucosidase PkBgl in Example 4 of the present invention; Figure 4 This is a graph showing the effect of different fermentation methods on beer aroma components in Example 6 of the present invention; Figure 5 This is a radar chart showing the sensory evaluation of different beer samples in Embodiment 7 of the present invention. Detailed Implementation

[0036] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0037] Where specific experimental steps or conditions are not specified in the embodiments, they can be performed according to the conventional experimental steps or conditions described in the literature in this field. All raw materials or instruments used are commercially available conventional products, including but not limited to those used in the embodiments of this application.

[0038] Example 1: Isolation and Screening of Strains YPD solid culture medium composition: peptone 20 g / L, yeast extract 10 g / L, glucose 20 g / L, agar 20 g / L, with the remainder being water. Preparation method: Dissolve all the above components in 1 L of distilled water, then dispense into 100 mL Erlenmeyer flasks and sterilize at 115 ℃ for 20 min. To eliminate bacterial interference during the screening process, 60 μg / mL of chloramphenicol was added.

[0039] Take 10 g of soy sauce-flavored baijiu mash into a 250 mL sterile Erlenmeyer flask, add 90 mL of sterile physiological saline, seal the flask with a breathable sealing film, and activate at 30 ℃ for 30 min; take out the bacterial suspension and dilute it with sterile water to obtain a diluted solution, spread it on YPD solid medium, and incubate at 30 ℃ for 1-2 days. Isolate and streak-purify strains with typical yeast colony characteristics (purify 2-3 times depending on the isolation effect) until a single strain is obtained, and number the isolated strains.

[0040] The strain numbered KD4 was identified and classified as follows: Pichia kudriavzeviiThe strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on December 6, 2023, under the name PK2 and accession number CGMCC No. 29250. The address of the depository is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. This strain has been published in Chinese patent document CN118291281A on July 5, 2024.

[0041] Example 2: Preparation of crude β-glucosidase enzyme solution and determination of enzyme activity YPD liquid culture medium composition: peptone 20 g / L, yeast extract 10 g / L, glucose 20 g / L, the remainder being water. Preparation method: Dissolve the above components in 1 L of distilled water, adjust the pH to 5.0, then dispense into 100 mL Erlenmeyer flasks and sterilize at 115 ℃ for 20 min.

[0042] Preparation of crude β-glucosidase solution: Pichia kudrica PK2 was inoculated into YPD liquid medium and cultured at 30 ℃ and 220 rpm for 48 h for activation; the activated bacterial suspension was inoculated into a conical flask containing 100 mL of YPD liquid medium at a 6% inoculation rate and cultured at 30 ℃ and 220 rpm for 60 h; the cultured bacterial solution was aliquoted into centrifuge tubes and centrifuged at 6500 rpm for 13 min at 4 ℃ to obtain the supernatant, which was the crude enzyme solution for later use.

[0043] Construction of the p-nitrophenol (p-NP) standard curve: 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, and 6 mL of 0.1 mol / L p-NP standard solution were placed in 100 mL volumetric flasks, and then diluted to the mark with 1 mol / L sodium carbonate standard solution. The absorbance of each solution was measured at 400 nm. For the blank control group, 1 mL of ultrapure water was added, and the volume was diluted to 100 mL with sodium carbonate standard solution before measurement. A standard curve was plotted with p-NP concentration on the x-axis and absorbance on the y-axis, yielding the regression equation Y = 11.232x - 0.0098 (R²). 2 =0.9993).

[0044] Assay for β-glucosidase activity: In a 2 mL sterile EP tube, 0.2 mL of crude enzyme solution, 0.2 mL of 0.035 mol / L p-NPG solution, and 0.6 mL of citrate-phosphate buffer (pH 5.0) were added sequentially. The reaction system was placed in a 50 °C water bath for 10 min. The reaction was then terminated by rapidly adding 1 mL of 1 mol / L NaOH solution, and the absorbance was measured at 400 nm. The blank control group was prepared using inactivated crude enzyme solution and subjected to the same procedure. β-glucosidase activity is defined as the amount of enzyme required to catalyze the production of 1 nmol of p-nitrophenol per minute at 50 °C and pH 5.0; one unit of enzyme activity (U) is defined as the amount of enzyme required to catalyze the production of 1 nmol of p-nitrophenol per minute.

[0045] Enzyme activity is calculated using the following formula:

[0046] In the formula, U represents enzyme activity units (U / mL), C is p-NP concentration (mmol / L), V is the total volume of the reaction system (mL), N is the dilution factor of the crude enzyme solution, t is the reaction time (min), and 0.2 is the volume of the crude enzyme solution (mL).

[0047] The enzyme activity of the crude β-glucosidase solution prepared in this embodiment was finally measured to be 308.93 U / mL.

[0048] Example 3: Hydrolysis Experiment of Hop Oil Preparation of β-glucosidase lyophilized enzyme powder: Pichia pastoris PK2 was inoculated into YPD liquid medium and cultured at 30 ℃ and 220 rpm for 48 h for activation; the activated bacterial suspension was inoculated into an Erlenmeyer flask containing 100 mL of YPD liquid medium at a 6% inoculation rate and cultured at 30 ℃ and 220 rpm for 48 h; the cultured bacterial solution was aliquoted into centrifuge tubes and centrifuged at 6500 rpm for 13 min in a centrifuge at 4 ℃, and the supernatant was obtained as crude enzyme solution. The crude enzyme solution was freeze-dried to obtain lyophilized enzyme powder for later use.

[0049] Experimental groups: The blank group consisted of 5 mL of hop oil without added lyophilized enzyme powder; the experimental group consisted of 5 mL of hop oil with added 2 g of lyophilized enzyme powder.

[0050] Experimental method: Both groups of hop essential oils were reacted in a shaker at 30℃ and 220 rpm for 36 h. After the reaction was completed, GC-MS was performed for detection.

[0051] Chromatographic conditions: An HP-INNO Wax column (60 m × 320 μm × 0.5 μm) was used. The temperature program was as follows: the initial temperature was set at 40 ℃ and held for 3 min; then the temperature was increased to 120 ℃ at a rate of 5 ℃ / min and held for 3 min; next, the temperature was increased to 230 ℃ at a rate of 3 ℃ / min and held for 5 min. The injection port temperature was 250 ℃. Helium with a purity of 99.9% was used as the carrier gas, and its flow rate was controlled at 1.5 mL / min. The injection mode was splitless, and the solvent delay time was set to 3 min.

[0052] Mass spectrometry conditions: An electron impact (EI) ion source was used with an energy of 70 eV and a source temperature of 230 °C. The quadrupole temperature was set to 200 °C. Mass spectrometry detection was performed in full scan mode, with a scan range of 35–400 amu.

[0053] Qualitative and quantitative analysis of volatile substances in wine samples: Characteristic ions were extracted based on the mass spectrometry characteristics of volatile substances. The substances corresponding to each peak were identified by searching the GC-MS standard mass spectrometry database. The ratio of the peak area of ​​the flavor compound to the peak area of ​​the internal standard (2-octanol) was used as the result of quantitative analysis.

[0054] like Figure 1 As shown, after β-glucosidase enzymatic hydrolysis of hop essential oils, the linalool content increased to 2.67 times that of the control group, the nerol content increased to 3.06 times that of the control group, while the α-terpineol content did not change significantly. This indicates that β-glucosidase produced by the non-brewing yeast PK2 fermentation can effectively promote the release of glycosidic aroma substances in hop essential oils, providing a theoretical basis for PK2 to play a corresponding role in the beer brewing process.

[0055] Example 4: β-glucosidase tolerance analysis Preparation of β-glucosidase purified enzyme solution: Pichia kudrizii PK2 was inoculated into YPD liquid medium and cultured at 30 ℃ and 220 rpm for 48 h for activation. The activated bacterial suspension was inoculated into a conical flask containing 100 mL of YPD liquid medium at a 4% inoculation rate and cultured at 28 ℃ and 220 rpm for 48 h. The cultured bacterial solution was aliquoted into centrifuge tubes and centrifuged at 6500 rpm for 13 min at 4 ℃. The supernatant was collected, filtered through a 0.45 μm aqueous filter membrane, and added to a gravity column containing Ni-NTA agarose (GE Healthcare Biosciences, Sweden). The target protein was then eluted with 10 mL of ultrapure water containing 60 mmol / L imidazole. The eluent was collected to obtain the purified enzyme solution.

[0056] I. Ethanol Tolerance Test To investigate the ethanol tolerance of β-glucosidase, purified enzyme solution was mixed with pure ethanol to a total volume of 1 mL. Pure ethanol was added to achieve volume concentrations of 4%, 8%, 12%, 16%, and 20% in the mixture (the remainder was made up with purified enzyme solution). The mixture was incubated at 4 °C for 3 h. β-glucosidase activity was measured according to the method in Example 2 at the optimal temperature of 30 °C and the optimal pH of 5.0. The enzyme activity measured with the purified enzyme solution without added ethanol was considered 100%. Enzyme activities measured under other conditions were expressed as relative enzyme activities. Three parallel experiments were set up for each group.

[0057] like Figure 2 As shown, enzyme activity decreases with increasing ethanol concentration. At 4% ethanol concentration, the relative enzyme activity remains above 90%; at 8% ethanol concentration, it remains around 50%; however, when the ethanol concentration exceeds 12%, the enzyme activity drops to approximately 20%. Therefore, the β-glucosidase produced by fermentation using strain PK2 exhibits good stability at ethanol concentrations below 12%, but its activity is significantly affected at ethanol concentrations above 12%. Since the ethanol concentration in beer brewing is typically between 4% and 12%, the β-glucosidase produced by PK2 fermentation can tolerate the ethanol concentrations required for beer brewing.

[0058] II. Iso-α-acid tolerance test To investigate the tolerance of β-glucosidase to iso-α-acids, purified enzyme solution was mixed with pure iso-α-acid in a total volume of 1 mL. Pure iso-α-acid was added to achieve volume concentrations of 50 IBU, 100 IBU, 150 IBU, 200 IBU, and 250 IBU in the mixture (the remainder was made up with purified enzyme solution). After incubation at 4 °C for 3 h, β-glucosidase activity was measured at the optimal temperature of 30 °C and the optimal pH of 5.0, following the method in Example 2. The enzyme activity measured with the purified enzyme solution without added iso-α-acid was considered 100%. Enzyme activities measured under other conditions were expressed as relative activities. Three parallel experiments were conducted for each group.

[0059] like Figure 3 As shown, enzyme activity decreases with increasing isoalpha acid content. When the isoalpha acid content is between 50 and 150 IBU, its relative enzyme activity remains above 50%; when the isoalpha acid content is between 0 and 100 IBU, the relative enzyme activity remains above 80%. During beer brewing, the isoalpha acid content varies due to various factors, typically ranging from 10 mg / L to 100 mg / L (1 IBU equals 1 mg of isoalpha acid in 1 L of beer). Therefore, the β-glucosidase produced by PK2 fermentation can tolerate changes in isoalpha acid content during beer brewing.

[0060] In summary, the PK2 strain exhibited stable tolerance to multiple stress conditions that beer fermentation may encounter, such as high ethanol and high bitterness, and has the potential to adapt to the actual brewing environment.

[0061] Example 5: Beer Brewing Method I. Mixed fermentation samples (1) Brewing steps of UK sample 1 S1. Raw material preparation: a. Preparation of beer fermentation substrate (wort medium): Accurately weigh an appropriate amount of barley malt and lightly crush it; add an appropriate amount of water at a malt-to-water mass ratio of 1:4, and place the mixture in a water bath for staged saccharification: First, adjust the water bath temperature to 52 ℃ and hold for 30 min; then raise the temperature to 62 ℃ and hold for 1 h; raise the temperature again to 68 ℃ and hold for 30 min; finally, raise the temperature in a microwave oven to 78 ℃ and hold for 10 min, thus ending the saccharification stage; after saccharification, cool and filter the saccharified liquid, then boil for 1 h, adding hops twice during the boiling stage. The first addition is 10 min after the start of boiling (0.3 g / L hops), and the second addition is 10 min before the end of boiling (0.3 g / L hops); after boiling, dispense the wort into 250 mL Erlenmeyer flasks, 150 mL per flask, and then heat at 115 ℃ for 0.1... Sterilize under high temperature and high pressure at MPa for 20 min; b. Preparation of PK2 bacterial suspension: A single yeast colony was inoculated into 5 mL of YPD liquid medium and cultured at 28 ℃ and 220 rpm for 24 h for primary activation. 100 μL of the primary culture was then inoculated into 10 mL of YPD liquid medium and cultured at 28 ℃ and 220 rpm for 18 h for secondary activation. The secondary activated cells were then washed twice with 0.9% physiological saline. The cells were collected and a bacterial suspension was prepared using 0.9% physiological saline (adjusted to OD). 600 =1.0), resulting in PK2 bacterial culture with a bacterial activity of 1×10⁻⁶. 6 cfu / mL; c. Preparation of US-05 bacterial suspension: Dissolve Saccharomyces cerevisiae US-05 powder (SafAle US-05, Fermentis brand, provided by Lesaffre) in an appropriate amount of ultrapure water. Spread the bacterial suspension onto YPD solid medium and incubate upside down in a 30 ℃ constant temperature incubator for 24 h. After scattered single colonies appear on the plate, obtain US-05 bacterial suspension according to the preparation method of PK2 bacterial suspension described above, with a bacterial activity of 1×10⁻⁶. 6 cfu / mL; S2. Flavor-enhancing fermentation: Inoculate the beer fermentation substrate with PK2 bacterial solution for flavor-enhancing fermentation. The inoculation amount is 10% (based on the volume of the beer fermentation substrate). Fermentation conditions: Ferment at 20 ℃ for 36 h. S3. Primary fermentation: After the aroma-enhancing fermentation is completed, inoculate with US-05 bacterial solution for primary fermentation. The inoculation amount is 1% (based on the volume of the beer fermentation substrate). Fermentation conditions: ferment at 20 ℃ for 11 days. S4. Post-fermentation: After the main fermentation is completed, transfer to 4 ℃ for post-fermentation for 7 days to obtain the final product.

[0062] (2) Brewing steps of UK sample 2 The beer brewing steps were the same as those in UK Sample 1, except that the inoculum amount in S2 was 9% and the inoculum amount in S3 was 2%, with a fermentation time of 9 days.

[0063] (3) Brewing steps of UK sample 3 The beer brewing steps were the same as those in UK Sample 1, except that the inoculum amount in S2 was 8% and the inoculum amount in S3 was 3%, with a fermentation time of 7 days.

[0064] II. US-05 single-strain fermentation sample S1. Raw material preparation: Refer to brewing steps S1 of UK sample 1; S2. Primary fermentation: US-05 bacterial culture is inoculated into the beer fermentation substrate for primary fermentation. The inoculation amount is 1% (based on the volume of the beer fermentation substrate). Fermentation conditions: fermentation at 20 ℃ for 36 h + 11 d. S3, Post-fermentation: After the main fermentation is completed, transfer to 4 ℃ for post-fermentation for 7 days to obtain the final product.

[0065] III. PK2 single-strain fermentation samples S1. Raw material preparation: Refer to brewing steps S1 of UK sample 1; S2. Primary fermentation: Inoculate the beer fermentation substrate with PK2 bacterial solution for primary fermentation. The inoculation amount is 10% (based on the volume of the beer fermentation substrate). Fermentation conditions: Ferment at 20 ℃ for 36 h + 11 d. S3, Post-fermentation: After the main fermentation is completed, transfer to 4 ℃ for post-fermentation for 7 days to obtain the final product.

[0066] Example 6: Detection of flavor compounds in beer I. Experimental Samples The UK sample 1 (denoted as PK2+US-05), the US-05 single-strain fermentation sample (denoted as US-05), and the PK2 single-strain fermentation sample (denoted as PK2) obtained in Example 5.

[0067] II. Experimental Methods Volatile flavor compounds in beer samples were detected using gas chromatography-mass spectrometry (Agilent MSD-5977B) with a 50 / 30 μm DVB / CAR / PDMS extraction fiber. After ultrasonic degassing, 5 mL of the beer sample was added to a 20 mL headspace vial, along with 3 g NaCl and 10 μL of 1 g / L internal standard (2-octanol). The sample was equilibrated at 55 °C for 5 min, extracted for 40 min, and then desorbed for 5 min before injection. The chromatographic column was an HP-INNOWax (60 m × 320 μm × 0.5 μm), with the following temperature program: 40 ℃ for 3 min, ramped at 5 ℃ / min to 120 ℃ for 3 min, ramped at 3 ℃ / min to 230 ℃ for 5 min; the injection port temperature was 250 ℃, and helium was used as the carrier gas (flow rate 1.5 mL / min). Mass spectrometry employed an electron impact (EI) ion source with an energy of 70 eV and an ion source temperature of 230 ℃, covering a full scan range of 35–400 amu. Qualitative analysis was performed using the NIST11 library, and quantification was based on the ratio of the peak area of ​​flavor compounds to the peak area of ​​the internal standard.

[0068] III. Experimental Results The flavor compounds in beer mainly come from three sources: malt, hops, and aroma compounds produced by yeast metabolism. These flavor compounds mainly include: alcohols, esters, organic acids, terpenes, aldehydes, and phenols.

[0069] Testing revealed 48 volatile flavor compounds in the beer samples, including 18 esters, 8 alcohols, 9 organic acids, 7 terpenes, 4 aldehydes and ketones, and 2 phenols. Figure 4 As shown, the contributions of various compounds to beer flavor vary significantly.

[0070] The detection data of some major volatile flavor compounds are shown in Table 1.

[0071] Table 1. Some major volatile flavor compounds and their contents

[0072] Esters are the most abundant aroma compounds in beer. As shown in the table above, the PK2+US-05 co-fermentation significantly increased the content of ethyl acetate, ethyl octanoate, and phenylethyl acetate, by 5.6 times, 2.3 times, and 2.1 times, respectively, compared to the US-05 single-strain fermentation sample. These esters impart a rich ester aroma and a slight floral and fruity fragrance to beer, significantly enriching its aroma profile.

[0073] Higher alcohols, as metabolic byproducts of yeast, mainly include isobutanol, isoamyl alcohol, and phenylethyl alcohol, and their content directly affects the body and safety of beer. As shown in the table above, compared with single-strain fermentation of PK2 or US-05, mixed fermentation of PK2+US-05 can effectively reduce the content of isobutanol, isoamyl alcohol, and phenylethyl alcohol, maintaining an appropriate body while avoiding the unpleasant flavors and health risks caused by excessive higher alcohols, resulting in a smoother and more harmonious taste.

[0074] Terpenes have low thresholds and make a significant contribution to beer flavor. They mainly originate from hop and yeast metabolism and include linalool, nerol, and geraniol. As shown in the table above, the contents of linalool, nerol, and geraniol in the PK2+US-05 mixed fermentation sample were increased to 2.8 times, 11.4 times, and 93.6 times that of the US-05 single-strain fermentation sample, respectively, significantly enhancing the hop and fruit aroma characteristics and enriching the aroma complexity.

[0075] The increase in phenolic and aldehyde / ketone content was not significant and had little impact on the aroma characteristics of beer.

[0076] Example 7 Sensory Evaluation of Beer I. Experimental Samples The UK sample 1 (denoted as PK2+US-05), UK sample 2, UK sample 3, US-05 single-strain fermentation sample (denoted as US-05) and PK2 single-strain fermentation sample (denoted as PK2) obtained in Example 5.

[0077] II. Experimental Methods A sensory evaluation panel composed of 5 male and 5 female members from the laboratory research group evaluated the beer's appearance, foam, aroma, and taste. The detailed sensory scoring criteria for each item are shown in Table 2.

[0078] Table 2 Sensory Evaluation Details

[0079] III. Experimental Results Table 3 Sensory rating results

[0080] Sensory evaluation radar charts of PK2+US-05, US-05, and PK2 beer samples are shown below. Figure 5As shown, PK2 single-strain fermented beer exhibits prominent floral and fruity aromas, but its acidity is noticeable, and its appearance and taste are relatively poor. US-05 single-strain fermented beer demonstrates better taste, appearance, and acidity, but its floral and fruity aromas are weaker. The mixed-fermentation beer combines the advantages of both. Compared to US-05 single-strain fermentation, its floral and fruity aromas are significantly more prominent, its acidity is significantly reduced, and its taste and appearance are relatively more harmonious, resulting in superior overall sensory quality. These results indicate that adding a PK2 aroma-enhancing fermentation step to the US-05 fermentation process can significantly improve the floral and fruity aromas of beer while reducing acidity, resulting in a more harmonious taste.

[0081] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A beer brewing method for increasing the content of terpenoid aroma compounds, characterized in that, The steps include: first inoculating the beer fermentation substrate with Pichia pastoris (Kudela spp.). Pichia kudriavzevii PK2 is used for aroma-enhancing fermentation, followed by the inoculation of brewing yeast for primary fermentation. After the primary fermentation, secondary fermentation is carried out to obtain the product, which includes Kudriazwijki yeast (…). Pichia kudriavzevii The accession number for PK2 is CGMCC No. 29250.

2. The beer brewing method for increasing the content of terpenoid aroma substances according to claim 1, characterized in that, The aroma-enhancing fermentation is carried out at a temperature of 15-25°C for 24-48 hours; and / or Based on the volume of the beer fermentation substrate, the inoculum size of the *Pichia kudrica* is 5 × 10⁻⁶. 4 ~5×10 5 cfu / mL.

3. The beer brewing method for increasing the content of terpenoid aroma substances according to claim 1, characterized in that, The aroma-enhancing fermentation is carried out at a temperature of 20-22°C for a time of 36-42 hours; and / or The inoculation amount of *Pichia kudrica* is 1 × 10⁻⁶ based on the volume of the beer fermentation substrate. 5 ~3×10 5 cfu / mL.

4. The beer brewing method for increasing the content of terpenoid aroma substances according to claim 1, characterized in that, The brewing yeast specifically refers to brewing yeast US-05; and / or The inoculation amount of brewing yeast is 1 × 10⁻⁶ based on the volume of the beer fermentation substrate. 3 ~5×10 4 cfu / mL; and / or The primary fermentation temperature is 15~25 ℃, and the time is 7~14 days.

5. The beer brewing method for increasing the content of terpenoid aroma substances according to claim 1, characterized in that, The inoculation amount of brewing yeast is 1 × 10⁻⁶ based on the volume of the beer fermentation substrate. 4 ~3×10 4 cfu / mL; and / or The primary fermentation temperature is 20~22 ℃, and the time is 11~12 days.

6. The beer brewing method for increasing the content of terpenoid aroma substances according to claim 1, characterized in that, The post-fermentation temperature is 2~6 ℃, and the time is 5~9 days.

7. The beer brewing method for increasing the content of terpenoid aroma substances according to claim 1, characterized in that, The post-fermentation temperature is 4-5 ℃, and the time is 7-8 days.

8. The beer brewing method for increasing the content of terpenoid aroma substances according to claim 1, characterized in that, The method for preparing the beer fermentation substrate includes the following steps: (1) After crushing the malt, mix it with water and then saccharify the mixture. After saccharification, cool and filter to obtain the saccharified liquid. (2) Boil the saccharified liquid and add hops after boiling. After boiling, wort is obtained. (3) The wort is packaged and sterilized to obtain the beer fermentation substrate.

9. The beer brewing method for increasing the content of terpenoid aroma substances according to claim 8, characterized in that, In step (1): Mix malt and water in a mass ratio of 1:3 to 5; The saccharification process includes: heating the mixture to 50-55 °C and holding it at that temperature for 30-40 min; continuing to heat the mixture to 60-65 °C and holding it at that temperature for 1-2 h; continuing to heat the mixture to 66-72 °C and holding it at that temperature for 30-40 min; and finally heating the mixture to 75-80 °C and holding it at that temperature for 10-20 min. In step (2): The boiling time is 1 to 1.5 hours. Hops are added 10 to 15 minutes after the start of boiling, at a rate of 0.2 to 0.4 g / L based on the volume of the mashed liquid. Hops are added again 10 to 15 minutes before the end of boiling, at a rate of 0.6 to 0.8 g / L based on the volume of the mashed liquid. In step (3): The sterilization conditions were: high temperature and high pressure sterilization at 115 ℃ and 0.1 MPa for 20 min.

10. Beer obtained by the beer brewing method for increasing the content of terpenoid aroma substances according to any one of claims 1 to 9.

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