Composite functional bacterial agent for regulating nucleoside substances in Luzhou-flavor liquor as well as preparation method and application of composite functional bacterial agent

Abstract

The invention discloses a composite functional bacterial agent for regulating nucleoside substances in Luzhou-flavor liquor as well as a preparation method and application of the composite functional bacterial agent, and belongs to the technical field of liquor production. The compound functional bacterial agent is prepared from a liquid culture of pichia kudriavzevii GTY837 and a liquid culture of pichia kudriavzevii PKW194, or a liquid culture of pichia kudriavzevii GTY837 and a liquid culture of pichia kudriavzevii PKW194, or a liquid culture of pichia kudriavzevii PKW194, or a liquid culture of pichia kudriavzevii GTY837 and a liquid culture of pichia kudriavzevii PKW194. According to the composite functional bacterial agent provided by the invention, through the synergistic effect of the GTY837 and the PKW194, the content of seven nucleoside substances such as cytidine and hypoxanthine in the Luzhou-flavor liquor is effectively increased. The microbial agent solves the technical problem of unstable generation of nucleoside substances in traditional fermentation, and realizes synergistic improvement of the flavor and health attribute of Baijiu by enriching nucleoside substances with the functions of dispelling the effects of alcohol, protecting nerves and the like while enhancing the mellow feeling, delicate flavor and other sensory qualities of the Baijiu.

Description

Technical Field

[0001] This invention relates to the field of liquor production technology, and in particular to a compound functional microbial agent for regulating nucleoside substances in strong-aroma liquor, its preparation method, and its application. Background Technology

[0002] Baijiu (Chinese white liquor) brewing boasts a history of over a thousand years, renowned worldwide for its unique solid-state fermentation process and diverse styles, earning it the title of one of the world's six major distilled spirits. Strong-aroma baijiu, a typical representative of traditional Chinese distilled spirits, is beloved by consumers for its unique style characterized by "rich cellar aroma, mellow and sweet taste, harmonious flavors, and a clean, refreshing finish." Its complex flavor system originates from the synergistic effect of the microbial community during fermentation, resulting in a wide variety of trace flavor components. Typical production processes for strong-aroma baijiu include continuous fermentation, mixed steaming and mixing of mash, and continuous fermentation in old cellars.

[0003] In recent years, with the deepening of research on the flavor chemistry of baijiu (Chinese liquor), in addition to the traditionally focused esters, acids, and alcohols, nucleosides have been found to make significant contributions to the flavor and health attributes of baijiu. Studies have shown that nucleosides (such as cytidine, hypoxanthine, uridine, inosine, guanosine, thymidine, and adenosine) not only possess significant umami flavor, enhancing the body, fullness, and intensity of the liquor, forming the so-called "skeleton," but also participate as flavor precursors in complex chemical changes such as the Maillard reaction during storage and aging, further enriching the flavor layers of the liquor and positively impacting the persistence of the "lingering aroma in an empty glass." Furthermore, nucleosides contain various trace elements beneficial to the human body, stimulating the activity of alcohol dehydrogenase and aldehyde dehydrogenase, accelerating alcohol metabolism, and possessing rapid hangover relief and sobering functions; simultaneously, they can clear toxins from the blood and free radicals produced by ethanol metabolism, playing a positive role in protecting the liver, cardiovascular system, and other circulatory tissues. Among them, components such as adenosine and uridine also show potential value in improving central nervous system function, promoting sleep, and anti-epileptic effects.

[0004] However, in the natural fermentation process of traditional strong-aroma baijiu, the generation of nucleosides depends on a complex and unstable microbial ecosystem, and their content and proportion are difficult to control effectively, leading to challenges in the flavor stability and quality consistency of baijiu products. In existing technologies, in order to improve the quality of baijiu, the method of adding exogenous functional microbial agents is generally adopted, such as hexanoic acid bacteria agents and esterified red yeast rice, whose main functions are concentrated on increasing the content of main aroma substances (such as ethyl hexanoate) or adjusting the acid-ester ratio. For example, Xu Chunyan et al. pointed out in the paper "Targeted Regulation Application Research Based on Functional Core Microbial Community of Cellar Mud Microbiome" published in Food and Fermentation Industries (2021, 47(15):1-7) that by constructing a compound microbial agent based on the functional core microbial community of cellar mud microbiome, the content of ethyl hexanoate was increased and the composition of aroma substances was optimized; another technical solution uses compound microbial agents such as Bacillus subtilis and Saccharomyces mulliganum to improve the style of the liquor and enhance the characteristics of strong aroma. In addition, patent CN115838619A proposes to control the total acid and ethyl lactate content in Maotai-flavor liquor by using a segmented distillation method at the tail end of the liquor, so as to improve the grade of the liquor.

[0005] While the aforementioned methods have achieved some success in regulating ester metabolism, research on the metabolic regulation of various nucleosides in strong-aroma baijiu remains extremely lacking. Existing microbial agents have relatively limited functions, failing to cover the synthesis and accumulation of nucleosides, thus hindering the synergistic enhancement of baijiu's flavor and health attributes. Therefore, there is an urgent need in this field to develop a functional microbial agent capable of targeted regulation of the content of multiple nucleosides to enhance the richness and harmony of the baijiu's flavor, while simultaneously achieving the dual goals of rapid hangover relief and health protection, thereby promoting the baijiu industry towards a dual focus on flavor and health. Summary of the Invention

[0006] The purpose of this invention is to provide a compound functional microbial agent for regulating nucleoside compounds in strong-aroma baijiu, its preparation method, and its application, thereby solving the problems existing in the prior art. The compound functional microbial agent provided by this invention, through the synergistic effect of GTY837 and PKW194, effectively increases the content of seven nucleoside compounds, including cytidine and hypoxanthine, in strong-aroma baijiu. This microbial agent solves the technical problem of unstable nucleoside compound formation in traditional fermentation. While enhancing the mellowness and umami of the baijiu, it also achieves a synergistic improvement in the flavor and health attributes of baijiu by enriching nucleoside compounds with functions such as hangover relief and neuroprotection.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] This invention provides a compound functional microbial agent for regulating nucleoside substances in strong-aroma baijiu, which is composed of liquid cultures of Pichia kudriezweig GTY837 and Pichia kudriezweig PKW194, or Pichia kudriezweig GTY837 and Pichia kudriezweig PKW194.

[0009] The preservation number of Pichia pastoris GTY837 is CGMCC No. 26751;

[0010] The strain number of Pichia pastoris PKW194 is CICC 33194.

[0011] Optionally, the ratio of viable cells of Pichia kudriezweig GTY837 to Pichia kudriezweig PKW194 is 1:5 to 5:1.

[0012] This invention also provides a method for preparing the aforementioned composite functional microbial agent, comprising the following steps:

[0013] (1) Pichia kudriezwiyne GTY837 and Pichia kudriezwiyne PKW194 were activated;

[0014] (2) The activated strains were expanded to obtain bacterial cultures of Pichia kudriezweig GTY837 and Pichia kudriezweig PKW194, respectively.

[0015] (3) Mix the two bacterial solutions obtained in step (2) to obtain the composite functional bacterial agent.

[0016] Optionally, in step (2), the concentration of the bacterial culture of Pichia kudriezweig GTY837 and the bacterial culture of Pichia kudriezweig PKW194 are both (1-5)×10⁻⁶. 8 CFU / mL.

[0017] Optionally, in step (3), the bacterial culture of Pichia kudriezwiye GTY837 and the bacterial culture of Pichia kudriezwiye PKW194 are mixed in equal volume ratio.

[0018] Optionally, in step (2), the conditions for the expanded culture are 28-32℃, 150-200rpm shaking culture for 10-16h.

[0019] The present invention also provides an application of the aforementioned compound functional microbial agent in increasing the content of nucleoside substances in strong-aroma baijiu.

[0020] Optionally, the nucleoside analogues include cytidine, hypoxanthine, uridine, inosine, guanosine, thymidine, and adenosine.

[0021] The present invention also provides an application of the aforementioned compound functional microbial agent in enhancing the flavor of strong-aroma baijiu.

[0022] The present invention also provides an application of the aforementioned compound functional microbial agent in improving the sensory quality of strong-aroma baijiu.

[0023] The present invention discloses the following technical effects:

[0024] This invention successfully developed a compound functional microbial agent that synergistically enhances the content of various nucleoside compounds in strong-aroma baijiu by combining a specific strain, Pichia pastoris GTY837, with PKW194. Experimental results show that this compound microbial agent effectively promotes the balanced synthesis of seven nucleoside compounds—cytidine, hypoxanthine, uridine, inosine, guanosine, thymidine, and adenosine—during the fermentation process. Compared to the blank control group, the content of each nucleoside increased by 14.0% to 737.5%, with the most significant enhancement effect on the three key umami compounds: hypoxanthine, inosine, and guanosine.

[0025] Applying the compound microbial agent provided by this invention to the fermentation of strong-aroma baijiu effectively solves the technical problems of unstable nucleoside formation and difficult-to-control content in traditional fermentation processes, achieving a synergistic improvement in the flavor, quality, and health attributes of baijiu. On the one hand, it significantly enhances the mellowness, umami, and harmony of the liquor, improving its sensory quality; on the other hand, by enriching nucleoside substances with physiological activities such as hangover relief and neuroprotection, it endows baijiu with health benefits, promoting the development of the baijiu industry towards a dual focus on flavor and health. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 Figure showing the content of cytidine substances in enhanced fermentation of different treatment groups;

[0028] Figure 2 Figure showing the hypoxanthine content in different treatment groups after enhanced fermentation.

[0029] Figure 3 Figures showing the content of uridine substances in enhanced fermentation of different treatment groups;

[0030] Figure 4Figure showing the inosine content in different treatment groups during enhanced fermentation;

[0031] Figure 5 Figure showing the content of guanosine in enhanced fermentation in different treatment groups;

[0032] Figure 6 Figure showing the content of thymidine substances in different treatment groups during enhanced fermentation;

[0033] Figure 7 A graph showing the content of adenosine substances in different treatment groups for enhanced fermentation. Detailed Implementation

[0034] This invention provides a compound functional microbial agent for regulating nucleoside substances in strong-aroma baijiu, which is composed of Pichia kudriezweig GTY837 and another Pichia kudriezweig PKW194.

[0035] This invention investigates the practical effects of combining Pichia pastoris GTY837 with three other Pichia pastoris strains, both as single and double strains, on the production of strong-aroma baijiu. Through the determination and analysis of the contents of cytidine, hypoxanthine, uridine, inosine, guanosine, thymidine, and adenosine in the fermented strong-aroma baijiu, the aforementioned compound functional microbial agents that can effectively increase the content of various nucleoside substances in strong-aroma baijiu are screened.

[0036] The aforementioned *Pichia kudriavzevii* GTY837 strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) on March 6, 2023, with accession number CGMCC No. 26751 and classification name: *Pichia kudriavzevii*. This strain is disclosed in patent application number 202510529681.9.

[0037] The other three Pichia kudriezweig strains mentioned above were all purchased from the China Industrial Microbial Culture Collection Center, namely Pichia kudriezweig PKW194 with strain number CICC 33194, Pichia kudriezweig PKW293 with strain number CICC33293, and Pichia kudriezweig PKW335 with strain number CICC 33335.

[0038] In this invention, the preparation method of the aforementioned composite functional microbial agent includes the following steps:

[0039] (1) Pichia kudriezwiyne GTY837 and Pichia kudriezwiyne PKW194 were activated and expanded respectively;

[0040] (2) The cultures were cultured under shaking conditions to obtain two yeast cultures.

[0041] (3) Mix the two bacterial solutions obtained in step (2) according to the predetermined ratio of live bacteria to obtain the composite functional bacterial agent.

[0042] Preferably, in step (2), the rotation speed range of the oscillation culture is 150-200 rpm, the culture temperature range is 28-32℃, and the culture time range is 10-16h.

[0043] In one specific embodiment of the present invention, the rotation speed of the oscillation culture is 170 rpm, the culture temperature is 30°C, and the culture time is 12 h.

[0044] Preferably, in step (3), the predetermined live bacteria ratio ranges from 1:5 to 5:1.

[0045] In one specific embodiment of the present invention, the predetermined live bacteria ratio is 1:1.

[0046] The above-mentioned compound functional microbial agent is applied to the fermentation process of liquor mash to effectively increase the content of various nucleosides in strong-aroma liquor. The nucleosides include at least two of cytidine, hypoxanthine, uridine, inosine, guanosine, thymidine and adenosine, thereby synergistically improving the sensory quality and health characteristics of strong-aroma liquor and enhancing its flavor.

[0047] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0048] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0049] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0050] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0051] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0052] Example

[0053] This embodiment aims to screen and verify the synergistic effect of a compound functional microbial agent composed of Pichia kudriavzevii GTY837 and Pichia kudriavzevii PKW194 in increasing the content of various nucleosides in the mash during the actual brewing process of strong-aroma baijiu.

[0054] 1. Experimental Materials

[0055] Strains: Pichia kudriezweig GTY837, Pichia kudriezweig PKW194, Pichia kudriezweig PKW293 and Pichia kudriezweig PKW335.

[0056] Liquid culture medium: YPD liquid medium (1 wt% yeast extract, 2 wt% peptone and 2 wt% glucose).

[0057] Fermentation raw materials: Simulated strong-aroma baijiu mash, with the following dry weight ratio: 65% sorghum, 10% wheat, 10% glutinous rice, 10% corn, and 5% rice, mixed with 25% (based on total dry weight of grains) rice husks as filler, and 30% (based on total dry weight of grains) medium-high temperature strong-aroma baijiu starter.

[0058] 2. Experimental Methods

[0059] 2.1 Preparation of microbial agents

[0060] Four yeast strains, GTY837, PKW194, PKW293 and PKW335, were inoculated into Erlenmeyer flasks containing liquid culture medium, respectively.

[0061] Place the Erlenmeyer flask in a shaker and incubate at 30℃ and 170 rpm for 12 hours until the bacterial growth enters the late logarithmic phase, obtaining a viable count of not less than 1 × 10⁻⁶. 8 Seed culture solution with CFU / mL.

[0062] According to the experimental group design shown in Table 1, the corresponding seed bacterial solutions were directly prepared into bacterial agents, or mixed at a volume ratio of 1:1 to prepare compound bacterial agents for later use.

[0063] Table 1 Experimental group design

[0064]

[0065]

[0066] 2.2 Fermentation Experiment

[0067] Weigh an equal amount of mash raw material (150g) into a 500mL Erlenmeyer flask.

[0068] According to 1×10 5 The total inoculation amount was determined by adding the corresponding bacterial suspension to each Erlenmeyer flask (single bacterial group was inoculated with a single bacterial suspension, and compound group was inoculated with a compound bacterial suspension mixed in a 1:1 ratio). The blank control group was inoculated with an equal amount of sterile water.

[0069] The Erlenmeyer flasks were placed in a 30°C constant temperature incubator for solid-state fermentation, with a fermentation cycle of 30 days.

[0070] 2.3 Sample Testing

[0071] After fermentation, samples of the mash from each group were collected. The contents of seven nucleoside compounds (cytidine, hypoxanthine, uridine, inosine, guanosine, thymidine, and adenosine) in the mash were determined by liquid chromatography-quadrupole time-of-flight mass spectrometry (Q-TOF).

[0072] 3. Experimental Results

[0073] The results of the determination of the content of seven nucleoside compounds in the fermented mash are shown in Table 2 and... Figures 1-7 As shown in Table 2 (unit: μg / g, data are expressed as mean ± standard deviation).

[0074] Table 2. Nucleoside content (μg / g) of the compound functional microbial agent enhanced fermentation of the present invention.

[0075]

[0076] By analyzing the data in Table 1 and Figures 1-7 Systematic analysis revealed that the combination of GTY837 and PKW194 strains exhibited significant advantages, as detailed below:

[0077] 1. Synergistic effect of the compound (GTY837+PKW194) on nucleoside analogues

[0078] When GTY837 or PKW194 strains were inoculated alone, they not only failed to promote the production of most nucleoside compounds, but actually inhibited it. Table 1 shows that compared with the blank control group (CK), the contents of cytidine, uridine, guanosine, and adenosine in the GTY837 single-strain group decreased by 60.8%, 48.5%, 31.7%, and 24.7%, respectively. In the PKW194 single-strain group, the contents of cytidine, hypoxanthine, and adenosine decreased by 40.7%, 26.6%, and 62.3%, respectively.

[0079] However, when GTY837 and PKW194 were combined in a 1:1 ratio, a significant synergistic effect was achieved. Compared with the control group, the compound microbial agent group showed a significant increase in the content of all seven nucleoside compounds: cytidine increased from 16.18 μg / g to 23.36 μg / g, an increase of 44.4%; hypoxanthine increased from 16.50 μg / g to 19.23 μg / g, an increase of 16.5%; uridine increased from 5.40 μg / g to 7.38 μg / g, an increase of 36.7%; inosine increased from 1.05 μg / g to 1.58 μg / g, an increase of 50.5%; guanosine increased from 33.78 μg / g to 38.50 μg / g, an increase of 14.0%; thymidine increased from 0.16 μg / g to 1.34 μg / g, an increase of 737.5%; and adenosine increased from 0.77 μg / g to 1.34 μg / g, an increase of 74.0%.

[0080] An appropriate increase in uridine can accelerate alcohol metabolism, giving alcohol a unique flavor, promoting sleep and having anti-epileptic effects, improving memory function, and influencing neuronal plasticity. A moderate increase in adenosine can stimulate the activity of alcohol dehydrogenase and aldehyde dehydrogenase, rapidly detoxifying alcohol, significantly improving cell vitality, and playing an important role in the body's central nervous system, cardiovascular system, and coagulation system.

[0081] 2. Advantages compared to combinations with other strains

[0082] Figures 1-7 To compare the content of the seven nucleoside compounds in all experimental groups, in the systematic comparison of all 11 experimental groups, the combination of GTY837 and PKW194 showed a unique comprehensive advantage, as detailed below:

[0083] Figure 1The results showed that the highest cytidine content (45.51 μg / g) was achieved after simultaneous addition of Pichia pastoris kudrizii GTY837 and PKW293 for enhanced fermentation. The cytidine content of the GTY837+PKW293 combination was higher than that of the control group (CK), the single-strain GTY837 group, and the single-strain PKW293 group. In terms of cytidine content, the GTY837+PKW293 combination had the highest content, followed by the PKW194+PKW293 combination, then the PKW293+PKW335 combination, and finally the GTY837+PKW194 combination.

[0084] Figure 2 The results showed that the highest hypoxanthine content (19.23 μg / g) was achieved after simultaneous addition of Pichia pastoris GTY837 and PKW194 for enhanced fermentation. The hypoxanthine content of the GTY837+PKW194 combination was higher than that of the control group (CK), the single-strain GTY837 group, and the single-strain PKW194 group. Specifically, the hypoxanthine content of the GTY837+PKW194 combination was higher than that of the single-strain PKW293 group and the GTY837+PKW293 combination.

[0085] Figure 3 The results showed that the highest uridine content (10.86 μg / g) was achieved after enhancing fermentation with Pichia pastoris PKW293 alone. In terms of uridine content, the single-strain PKW293 group ranked first, followed by the GTY837+PKW335 combination, the PKW194+PKW293 combination, and the GTY837+PKW194 combination. The uridine content of the GTY837+PKW335, PKW194+PKW293, and GTY837+PKW194 combinations was very similar, with minimal differences.

[0086] Figure 4 The results showed that the highest inosine content (1.58 μg / g) was achieved after simultaneous addition of Pichia pastoris GTY837 and PKW194 for enhanced fermentation. The inosine content of the GTY837+PKW194 combination was higher than that of the control group (CK), the single-strain GTY837 group, and the single-strain PKW194 group. In terms of inosine content, the GTY837+PKW194 combination outperformed the single-strain PKW194 group, the single-strain PKW293 group, and the control group (CK) in that order.

[0087] Figure 5The results showed that the highest guanosine content (38.50 μg / g) was achieved after simultaneous addition of Pichia pastoris kudrizii GTY837 and PKW194 for enhanced fermentation. The guanosine content of the GTY837+PKW194 combination was higher than that of the control group (CK), the single-strain GTY837 group, and the single-strain PKW194 group. In terms of guanosine content, the GTY837+PKW194 combination outperformed the GTY837+PKW335 combination and the control group (CK).

[0088] Figure 6 The results showed that the highest thymidine content, reaching 1.63 μg / g, was achieved after enhancing fermentation with Pichia pastoris PKW293 alone. In terms of thymidine content, the single-strain PKW293 group performed best, followed by the GTY837+PKW194 and GTY837+PKW335 combinations, among others.

[0089] Figure 7 The results showed that the highest adenosine content, reaching 1.71 μg / g, was achieved after simultaneous addition of Pichia pastoris PKW194 and PKW335 for enhanced fermentation. In terms of adenosine content, the PKW194+PKW335 combination performed best, followed by the GTY837+PKW194 combination, then the GTY837+PKW293 combination, and finally the PKW293+PKW335 combination.

[0090] This demonstrates that the GTY837+PKW194 combination achieved the highest levels of hypoxanthine, inosine, and guanosine among all experimental groups. Inosine, a potent umami enhancer, saw a significant increase in its content (1.58 μg / g), playing a crucial role in enhancing the wine's rich umami and smooth sweetness. Guanosine, one of the strongest known umami enhancers, saw its content increase (38.50 μg / g), dramatically amplifying the wine's umami and richness. In terms of thymidine and adenosine content, this combination reached 1.34 μg / g and 1.34 μg / g respectively, ranking second among all experimental groups. Thymidine, a flavor precursor, contributes to enhancing the wine's aged aroma and mellowness; adenosine, an important endogenous neuromodulator, helps counteract alcohol-induced central nervous system excitation, fundamentally alleviating post-drinking discomfort. In terms of cytidine and uridine content, this combination reached 23.36 μg / g and 7.38 μg / g respectively. Although not the highest, the improvement effect was significant compared to the CK group and the corresponding single-strain group, and it ranked among the top in all experimental groups. Importantly, among all the strain combinations tested, only the compound inoculum of GTY837 and PKW194 achieved a stable and balanced increase in the content of all seven nucleoside substances, without any decrease in any indicator. This characteristic gives it the following unique value in achieving the dual goals of flavor and health in baijiu:

[0091] Flavor Enhancement: By synergistically enhancing umami substances such as inosine and guanosine, it effectively enhances the body's richness, fullness, and intensity, and improves the wine's harmony.

[0092] Health Promotion: By balancing and increasing all seven nucleoside compounds, a complete synergistic protective system is constructed to jointly exert the comprehensive effects of accelerating alcohol metabolism, protecting the nervous system, and alleviating drinking discomfort.

[0093] The results of this embodiment show that the combination of GTY837 and PKW194 produces a unique metabolic synergistic effect, which can effectively regulate the metabolic pathways of nucleosides during the fermentation of strong-aroma baijiu, providing a reliable technical solution for achieving synergistic improvement of baijiu flavor and health characteristics.

[0094] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A compound functional microbial agent for regulating nucleoside compounds in strong-aroma baijiu, characterized in that, It consists of liquid cultures of Pichia kudriezwiyne GTY837 and Pichia kudriezwiyne PKW194, or Pichia kudriezwiyne GTY837 and Pichia kudriezwiyne PKW194. The preservation number of Pichia pastoris GTY837 is CGMCC No. 26751; The strain number of Pichia pastoris PKW194 is CICC 33194.

2. The compound functional microbial agent according to claim 1, characterized in that, The ratio of viable cells of Pichia kudriezweig GTY837 to Pichia kudriezweig PKW194 is 1:5 to 5:

1.

3. A method for preparing a composite functional microbial agent as described in claim 1 or 2, characterized in that, Includes the following steps: (1) Pichia kudriezwiyne GTY837 and Pichia kudriezwiyne PKW194 were activated; (2) The activated strains were expanded to obtain bacterial cultures of Pichia kudriezweig GTY837 and Pichia kudriezweig PKW194, respectively. (3) Mix the two bacterial solutions obtained in step (2) to obtain the composite functional bacterial agent.

4. The preparation method according to claim 3, characterized in that, In step (2), the concentrations of the bacterial cultures of *Pichia kudriezweig* GTY837 and *Pichia kudriezweig* PKW194 are both (1-5)×10⁻⁶. 8 CFU / mL.

5. The preparation method according to claim 3, characterized in that, In step (3), the bacterial culture of Pichia kudriezwiye GTY837 and the bacterial culture of Pichia kudriezwiye PKW194 are mixed in equal volume ratio.

6. The preparation method according to claim 3, characterized in that, In step (2), the conditions for the expanded culture are 28-32℃ and 150-200rpm shaking culture for 10-16h.

7. The application of a compound functional microbial agent as described in claim 1 or 2 in increasing the content of nucleoside substances in strong-aroma baijiu.

8. The application according to claim 7, characterized in that, The nucleoside compounds include cytidine, hypoxanthine, uridine, inosine, guanosine, thymidine, and adenosine.

9. The application of a compound functional microbial agent as described in claim 1 or 2 in enhancing the flavor of strong-aroma baijiu.

10. The application of a compound functional microbial agent as described in claim 1 or 2 in improving the sensory quality of strong-aroma baijiu.

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