Compound bacteria for promoting synthesis of hexanoic acid as well as preparation method and application of compound bacteria

Abstract

The invention belongs to the technical field of microbial fermentation, and particularly relates to compound bacteria for promoting synthesis of hexanoic acid as well as a preparation method and application of the compound bacteria. In order to solve the problem of unstable batch quality of wine products in the wine brewing fermentation process, the invention provides the compound bacterium for promoting the synthesis of hexanoic acid, and the compound bacterium contains Caproicillium sp. BJN0012 with the preservation number of CGMCC 1.18097 and Clostridium sp. BJN0013 with the preservation number of CGMCC No.46148. The invention also provides a preparation method of the compound bacterium for promoting the synthesis of hexanoic acid. Through the synergistic effect of the two bacteria, the yield of caproic acid is increased to 15 times or more of that of single-bacterium culture. The co-culture technology provided by the invention solves the problems of insufficient synthesis amount and unstable batch of caproic acid in the Baijiu fermentation process, and has important meanings for constructing the Baijiu fermentation minimum core flora and directionally regulating and controlling the generation of caproic acid.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to a complex bacterium that promotes hexanoic acid synthesis, its preparation method, and its application. Background Technology

[0002] Baijiu, along with brandy, whiskey, vodka, gin, and rum, is considered one of the world's six major distilled spirits. Over 120 kinds of acids have been detected in baijiu, such as acetic acid, propionic acid, butyric acid, hexanoic acid, valeric acid, heptanoic acid, phenylpropionic acid, 2-methylpropionic acid, 4-methylvaleric acid, and 2-methylbutyric acid. These are widely present in various types of baijiu, forming the basis of its flavor, with strong-aroma baijiu having the highest acid content. Acids act as "regulators" in the baijiu fermentation process, effectively reducing its spiciness and bitterness, increasing its sweetness, and significantly impacting its taste. Adding an appropriate proportion of organic acids can make the baijiu fuller, more harmonious, and with a longer finish, helping to eliminate bitterness and promote the aging of new baijiu. Hexanoic acid has the most significant impact on product quality among known acids, directly affecting the grade of strong-aroma baijiu. However, due to the relatively extensive production process, strong-aroma baijiu is still unable to control the synthesis of flavor substances that are important for fermentation during the production process, resulting in large fluctuations in fermentation batches. This is particularly evident in the unstable metabolic synthesis of hexanoic acid, an important acid, which is a production bottleneck problem that urgently needs to be solved.

[0003] The synthesis of acids is directly related to acid-producing bacteria in the fermentation system of strong-aroma baijiu. These bacteria, such as *Clostridium coccidioides*, are concentrated in the fermentation pit mud and exhibit a high hexanoic acid synthesis capacity, demonstrating good effects in enhanced fermentation. However, due to the wide geographical distribution and complex fermentation system of strong-aroma baijiu production, the application of single acid-producing bacteria has limitations, such as poor performance in cold winter regions. Existing research shows that synergistic compound microbial agents are significantly more effective than single-species agents and have better adaptability to fermentation environments. Therefore, in-depth research into the acid-producing bacteria in the fermentation pit system and screening for compound microbial agents that promote hexanoic acid synthesis are key to effectively solving current production bottlenecks; however, related research is extremely scarce. Summary of the Invention

[0004] To enrich the biological resources of compound microbial agents that promote hexanoic acid synthesis in the brewing industry and effectively solve the problem of inconsistent batch quality of wine products during brewing fermentation, this invention provides a compound microorganism that promotes hexanoic acid synthesis, its preparation method, and its application.

[0005] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows: In a first aspect, the present invention provides a compound bacteria that promotes hexanoic acid synthesis, which contains... Caproicibacter sp.BJN0012 and Clostridiumsp. BJN0013; the aforementioned Caproicibacter sp. BJN0012, accession number CGMCC1.18097; the Clostridium sp. BJN0013, accession number CGMCC No. 46148.

[0006] Furthermore, the aforementioned Caproicibacter sp. BJN0012 and Clostridium The cell number ratio of sp. BJN0013 was 1–10:1–10.

[0007] The Caproicibacter sp. BJN0012 (accession number CGMCC 1.18097) is described in the literature *Caproicibacter sp. BJN0012, a potential new species isolated from cellar mud for caproic acid production from glucose*. Journal of Biotechnology Volume 388, published on 10 June 2024, pages 11-23; Clostridium sp. BJN0013 (accession number CGMCC No. 46148) is published in patent CN 119391590 A.

[0008] Secondly, the present invention provides a method for preparing the aforementioned compound bacteria that promote hexanoic acid synthesis, which involves... Caproicibacter sp. BJN0012 and Clostridium Sp. BJN0013 was mixed at a cell ratio of 1-10:1-10 and then cultured anaerobically in a fermentation medium.

[0009] Further, the fermentation medium comprises: KH2PO4 0.05–0.1 g / L, K2HPO4 0.05–0.1 g / L, CaCl2 0.001–0.003 g / L, Fe2(SO4)3·3H2O 0.0001–0.0004 g / L, (NH4)2SO4 0.1–0.2 g / L, MgSO4·7H2O 0.02–0.04 g / L, glucose 10–15 g / L, sodium acetate 20–25 g / L, yeast extract 5–10 g / L, calcium carbonate 10–12 g / L, sodium resazurin 0.1–0.5 mg / L, L-cysteine ​​0.0025–0.004 g / L, and ethanol 2–5% (V / V).

[0010] Furthermore, the fermentation culture temperature is 35±5℃.

[0011] Thirdly, the present invention also provides an application of the above-mentioned compound bacteria that promote hexanoic acid synthesis in the field of distilled spirits brewing.

[0012] Furthermore, the distilled spirit is selected from at least one of the following: spirits, brandy, whiskey, vodka, rum, gin, juniper berry spirit, tequila, or fruit spirits.

[0013] Preferably, the liquor is a strong-aroma type of liquor.

[0014] Fourthly, the present invention provides a method for promoting the synthesis of hexanoic acid, comprising the following steps: in Caproicibacter During the fermentation process of sp. BJN0012, add butyric acid and continue fermentation.

[0015] Furthermore, the butyric acid concentration is 1–10 g / L.

[0016] Beneficial effects: This invention will produce hexanoic acid. Caproicibacter sp. BJN0012 (accession number CGMCC1.18097) and capable of producing butyric acid Clostridium When sp. BJN0013 (CGMCC No. 46148) was co-cultured as a compound inoculum, under the same culture conditions, due to the promoting effect of butyric acid, the co-culture fermentation system showed increased activity. Caproicibacter Compared to its single-culture fermentation system, sp. BJN0012 utilizes more glucose and produces more hexanoic acid, increasing hexanoic acid production by more than 15 times compared to single-strain culture. Furthermore, the fermentation system is simple and controllable. Both strains used in this invention originate from the cellar mud in the baijiu brewing system and can be directly applied to the baijiu brewing process, avoiding the unsuitability of exogenous strains in this environment and the risk of strain contamination. Moreover, the co-culture interaction mechanism described in this invention can provide a reference for elucidating the interaction mechanisms of other baijiu fermentation microorganisms, and is of great significance for constructing a minimal core microbial community for baijiu fermentation and for the targeted regulation of hexanoic acid production. Attached Figure Description

[0017] Figure 1 This is a comparison of the acid production curves of co-culture and single culture of the two bacteria with different inoculation ratios in Example 1; Figure 2 Example 2 shows the effect of whether the two bacteria come into contact or not on the co-culture fermentation of the strains: (A) Experimental culture group settings, (B) Acid production and metabolism results after 14 days of fermentation; Figure 3 The original liquid chromatography chromatograms for the detection of glucose, acetic acid, ethanol, butyric acid and hexanoic acid in the co-culture system of Group III in Example 2 are shown. Figure 4This is a verification of the high hexanoic acid production from the co-culture of two bacteria in Example 3, along with an acid production curve. Detailed Implementation

[0018] To make the technical problems, solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with the embodiments. Unless otherwise defined, all technical terms used herein have the same meaning as understood by one of ordinary skill in the art.

[0019] In one embodiment of the present invention, strains capable of producing hexanoic acid are isolated, screened, and identified from the fermentation pit mud of baijiu (Chinese liquor) brewing. Caproicibacter sp. BJN0012 (accession number CGMCC 1.18097) and butyric acid-producing strains Clostridium sp. BJN0013 (CGMCC No. 46148) was mixed at a cell ratio of 1-10:1-10 and co-cultured in a fermentation medium for anaerobic fermentation to increase the yield of hexanoic acid.

[0020] This invention discovers Caproicibacter sp. BJN0012 has the ability to produce hexanoic acid, and Clostridium sp. BJN0013 exhibits the characteristic of metabolizing acetic acid and ethanol to produce high levels of butyric acid. Co-culturing the two strains under anaerobic conditions with glucose, acetic acid, and ethanol as carbon sources at pH 7.0 and 35±5℃ utilized more glucose and produced more hexanoic acid compared to fermentation systems where the strains were cultured alone. Hexanoic acid production increased by more than 15 times, and the fermentation system was simple and controllable.

[0021] To investigate the mechanism by which co-culturing two bacteria promotes hexanoic acid synthesis, another embodiment of the present invention provides a method for promoting hexanoic acid synthesis. Caproicibacter During the fermentation process of sp. BJN0012, the addition of butyric acid and continued fermentation showed that it also significantly increased the yield of hexanoic acid. This indicates that... Clostridium The function of sp. BJN0013 is to convert carbon sources (acetic acid, ethanol) in the fermentation system into butyric acid (a key intermediate). Caproicibacte r sp. BJN0012 utilizes Clostridium Butyric acid produced by sp. BJN0013 can be further extended to synthesize hexanoic acid.

[0022] The following specific embodiments will be provided to explain the solution of the present invention. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0023] Caproicibacter The isolation, screening, and identification process of sp. BJN0012 (accession number CGMCC 1.18097) has been documented in the literature: Caproicibacter sp. BJN0012, a potential new species isolated from cellar mud for caproic acid production from glucose. Journal of Biotechnology Volume 388, 10 June 2024, Pages 11-23 (Published); Clostridium sp. BJN0013 (accession number CGMCC No. 46148) has been published in patent CN 119391590 A.

[0024] The Clostridium fortified culture medium consisted of: 10 g / L peptone, 5 g / L beef meal, 5 g / L glucose, 5 g / L sodium chloride, 3 g / L yeast extract, 3 g / L sodium acetate, 1 g / L soluble starch, 0.5 g / L cysteine ​​hydrochloride, 0.5 g / L agar, at natural pH, and sterilized at 121℃ for 15 min.

[0025] The fermentation medium consisted of: 0.05 g / L KH2PO4, 0.05 g / L K2HPO4, 0.001 g / L CaCl2, 0.0001 g / L Fe2(SO4)3·3H2O, 0.1 g / L (NH4)2SO4, 0.02 g / L MgSO4·7H2O, 10 g / L glucose, 20 g / L sodium acetate, 5 g / L yeast extract, 10 g / L calcium carbonate, 0.1 mg / L sodium resazurin, 0.0025 g / L L-cysteine, and 2% ethanol (v / v). The pH was natural, and the medium was sterilized at 115℃ for 20 min.

[0026] The detection method for metabolites uses liquid chromatography: Column: Bio Rad Aminex HPX-87H.

[0027] Detection conditions: mobile phase was 0.006 M H2SO4 solution, flow rate was 0.9 mL / min, column temperature was 65℃, injection volume was 10 μL, detector was a differential refractive index detector, detector internal temperature was 35℃, and detection time was 30 min.

[0028] Example 1: Strain Culture Microbial activation: Under aseptic conditions, the microbial strain is activated by... Caproicibactersp. BJN0012 and Clostridium sp. BJN0013 was inoculated at 1-10% (V / V), preferably 5%, into 30 mL test tubes containing 5 mL of Clostridium tumefaciens reinforcement medium and incubated anaerobically for 5-7 days at 35°C.

[0029] Co-culture seeding: according to cell number ratio Caproicibacter sp. BJN0012: Clostridium sp. BJN0013 was inoculated into the fermentation medium at ratios of 10:1, 5:1, 1:1, 1:5, and 1:10 (initial OD600 = 0.1), and static anaerobic fermentation was carried out for 14 days at 35℃. After 14 days of co-cultivation with different inoculation ratios, the hexanoic acid yields reached 3.17±0.36 g / L, 3.17±0.19 g / L, 3.10±0.31 g / L, 3.63±0.28 g / L, and 2.88±0.19 g / L, respectively. Caproicibacter After 14 days of fermentation alone, sp. BJN0012 produced 12.00–15.13 times the yield of hexanoic acid. Figure 1 ).

[0030] Example 2: The effect of contact on co-culture fermentation of strains The contact interaction was analyzed using 0.4 μm 6-well Transwell cell culture chambers. Caproicibacter sp.BJN0012 and Clostridium The effect of interaction with sp. BJN0013. An equal volume of fermentation medium was added to the culture chambers. This example includes a total of 5 groups (…). Figure 2 A), Group I, Upper Indoor Vaccination Clostridium Fermentation of sp. BJN0013, inoculated in the lower chamber. Caproicibacter Fermentation of sp. BJN0012; Group II, upper chamber inoculation Caproicibacter Fermentation of sp. BJN0012, lower chamber culture Clostridium Fermentation with sp. BJN0013; Groups III, IV, and V do not embed nested support membranes in the culture wells, and Group III is inoculated. Caproicibacter sp. BJN0012 and Clostridium sp. BJN0013 co-culture fermentation, group IV inoculation Caproicibacter sp. BJN0012 was cultured and fermented separately, group V was inoculated. Clostridium sp. BJN0013 was cultured and fermented alone. After static culture in an anaerobic environment at 35℃ for 14 days, the contents of glucose, acetic acid, ethanol, butyric acid and hexanoic acid were determined.

[0031] Figure 2 B illustrates the results of this embodiment, showing that the upper and lower chambers of Combination I exhibit similar acid-producing metabolic characteristics. The lower chamber of Combination II is...Clostridium The septal culture of sp. BJN0013, with the upper chamber Caproicibacter Compared to the spaced culture of sp. BJN0012, less glucose and more acetic acid were consumed, resulting in a higher final ethanol concentration, consistent with the acid-producing metabolic characteristics of either cultured alone. The combined III chamber consumed more glucose. Figure 3 The image shows the chromatograms of metabolites in compartment III, but overall, the images from compartment II are more representative. Caproicibacter The hexanoic acid production in the growth chamber of sp. BJN0012 was not significantly different from that in the combined chamber III. Furthermore, regardless of physical contact, co-culture exhibited better carbon source utilization and hexanoic acid metabolic activity compared to monoculture. Therefore, this example demonstrates… Clostridium sp.BJN0013 promotes Caproicibacter Hexanoic acid production, sp. BJN0012, is primarily driven by non-contact metabolite exchange.

[0032] Example 3: Interaction Mode of Microbial Co-culture and Optimization of Acid-Producing Fermentation Medium To further verify Clostridium Butyric acid produced by sp. BJN0013 Caproicibacter The promoting effect of sp. BJN0012 on hexanoic acid production was investigated by adding exogenous butyric acid to the fermentation medium and observing its effect on... Caproicibacter The effects of sp. BJN0012 on growth and metabolism. Caproicibacter sp. BJN0012 was inoculated into the fermentation medium, when Caproicibacter When sp. BJN0012 reached the logarithmic growth phase, butyric acid was added exogenously to the fermentation medium, increasing the butyric acid concentration in the fermentation medium by 1 g / L, 5 g / L, and 10 g / L, respectively. Caproicibacter sp.BJN0012 was continued to undergo anaerobic fermentation at 35°C for 14 days in fermentation media containing different concentrations of butyric acid. Samples were taken every 2 days to determine the yield of hexanoic acid. Fermentation under conditions without exogenous butyric acid addition was used as a control group.

[0033] Caproicibacter After 14 days of fermentation in fermentation media supplemented with 1 g / L, 5 g / L, and 10 g / L butyric acid, sp. BJN0012 produced hexanoic acid yields of 1.71±0.06 g / L, 1.80±0.03 g / L, and 1.49±0.05 g / L, respectively. The addition of butyric acid to the fermentation medium significantly promoted the production of hexanoic acid. Caproicibacter The production of hexanoic acid (sp. BJN0012) Figure 4 ).illustrate Clostridium Butyric acid, produced by the metabolism of sp. BJN0013, is a promoter of... CaproicibacterOne of the key reasons for the production of hexanoic acid by sp. BJN0012 is that the hexanoic acid yield in monoculture after optimization of the culture medium was not as high as that in the co-culture system, indicating that... Caproicibacter sp. BJN0012 and Clostridium The microbial co-culture system of sp. BJN0013 is a more stable and effective means to promote the production of hexanoic acid.

Claims

1. A complex bacterium that promotes hexanoic acid synthesis, characterized in that: contain Caproicibacter sp. BJN0012 and Clostridium sp. BJN0013; the aforementioned Caproicibacter sp. BJN0012 has the accession number CGMCC 1.18097; the Clostridium sp. BJN0013 has the accession number CGMCC No. 46148.

2. The compound bacteria for promoting hexanoic acid synthesis according to claim 1, characterized in that: The Caproicibacter sp. BJN0012 and Clostridium The cell number ratio of sp. BJN0013 was 1–10:1–10.

3. The method for preparing the complex bacteria that promote hexanoic acid synthesis according to claim 1 or 2, characterized in that, Includes the following steps: Will Caproicibacter sp. BJN0012 and Clostridium Sp. BJN0013 was mixed at a cell ratio of 1-10:1-10 and then cultured anaerobically in a fermentation medium.

4. The method for preparing the compound bacteria that promote hexanoic acid synthesis according to claim 3, characterized in that, The fermentation medium comprises: KH2PO4 0.05–0.1 g / L, K2HPO4 0.05–0.1 g / L, CaCl2 0.001–0.003 g / L, Fe2(SO4)3·3H2O 0.0001–0.0004 g / L, (NH4)2SO4 0.1–0.2 g / L, MgSO4·7H2O 0.02–0.04 g / L, glucose 10–15 g / L, sodium acetate 20–25 g / L, yeast extract 5–10 g / L, calcium carbonate 10–12 g / L, sodium resazurin 0.1–0.5 mg / L, L-cysteine ​​0.0025–0.004 g / L, and ethanol 2–5% (V / V).

5. The method for preparing the compound bacteria that promote hexanoic acid synthesis according to claim 3 or 4, characterized in that: The fermentation culture temperature is 35±5℃.

6. The application of the compound bacteria that promote hexanoic acid synthesis as described in claim 1 or 2 in the field of distilled spirits brewing.

7. The application according to claim 6, characterized in that: The distilled spirit is selected from at least one of the following: spirits, brandy, whiskey, vodka, rum, gin, gin, tequila, or fruit spirits.

8. A method for promoting the synthesis of hexanoic acid, characterized in that, Includes the following steps: exist Caproicibacter During the fermentation process of sp.BJN0012, butyric acid is added, and fermentation continues; Caproicibacter sp. BJN0012 has the accession number CGMCC 1.18097.

9. The method for promoting hexanoic acid synthesis according to claim 8, characterized in that: The butyric acid concentration is 1–10 g / L.

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