Clostridium high-efficiency culture medium based on yellow water, culture method, preparation and application

Abstract

The application provides a Clostridium efficient culture medium based on yellow water, a culture method, a preparation and an application. The yellow water culture medium comprises 20%-40% of yellow water, a byproduct of liquor brewing, and the rest is deionized water, and the pH value is adjusted to 6.5-7.5. The method for culturing Clostridium from pit mud source comprises the following steps: after the culture medium is sterilized, Clostridium from pit mud source is inoculated at an inoculation amount of 5%-15%, and then the mixture is placed and cultured anaerobically at 35-37 DEG C for 24-100 hours. The yellow water is converted into an efficient special culture medium for Clostridium, which can significantly promote the growth and proliferation and metabolic activity of Clostridium from pit mud source at the same time. Not only high-biomass Clostridium culture can be obtained, but also a breakthrough can be made to activate and support various Clostridium from pit mud source to synthesize organic acids such as hexanoic acid and butyric acid. The Clostridium culture rich in organic acids and the functional microbial preparation comprising the Clostridium culture can be used for strengthening liquor fermentation, maintaining pit mud or constructing artificial pit mud, and the high-value utilization of the byproduct of liquor brewing is realized. The cost is low, and the effect is remarkable.

Description

Technical Field

[0001] This invention relates to the field of microbial culture technology, specifically to a high-efficiency Clostridium culture medium based on yellow water, a culture method, a preparation, and its application. Background Technology

[0002] Clostridium is one of the core functional microorganisms in the cellar mud of strong-aroma and sauce-aroma baijiu. The short-chain fatty acids produced by its metabolism are precursors of key flavor substances in baijiu, directly affecting the aroma and taste of the liquor. Therefore, the efficient cultivation and rejuvenation of high-performance Clostridium strains are of great significance for baijiu microbiology research, process optimization, and product quality improvement.

[0003] Currently, anaerobic Clostridium is mostly cultured in the laboratory using commercial media such as reinforced Clostridium medium (RCM). Although these media have a certain degree of selectivity, they also have obvious shortcomings: First, they are expensive, which is not conducive to large-scale or long-term culture; second, their nutrient composition is mainly designed for common Clostridium and has not been fully optimized for the physiological characteristics and metabolic needs of Clostridium from pit mud, which often results in the bacterial growth rate, biomass and target acid synthesis capacity not reaching the optimal level.

[0004] On the other hand, the brewing process of baijiu (Chinese liquor) generates a large amount of "yellow water," a byproduct rich in organic acids, residual starch, sugars, amino acids, vitamins, microbial cells and their metabolites, representing a potentially high-value nutritional resource. However, currently, yellow water is mostly treated as waste liquid and has not yet been systematically and standardizedly utilized in functional microbial culture systems, resulting not only in resource waste but also increasing the environmental burden on enterprises.

[0005] Despite its rich nutritional content, the direct use of yellow water as a culture medium faces numerous technical obstacles. Firstly, the composition of yellow water is complex and unstable, significantly influenced by brewing processes, fermentation pits, and seasonal factors, potentially leading to poor reproducibility of the culture medium structure. Secondly, yellow water may contain ethanol, salts, metal ions, or microbial metabolic inhibitors, requiring appropriate treatment before it can be used for pure Clostridium culture. Furthermore, there is no mature solution for scientifically combining other nutrient components to achieve high-density Clostridium culture and targeted metabolism while maintaining the advantages of yellow water resources. Therefore, developing a standardized, high-efficiency Clostridium culture medium based on yellow water, combining low cost, resource recycling, and performance optimization, has significant scientific research value and promising industrial application prospects. Summary of the Invention

[0006] The purpose of this invention is to provide a high-efficiency culture medium for Clostridium based on yellow water, a culture method, a preparation and its application. The yellow water culture medium of this invention is low in cost, can be recycled, and can significantly promote the growth and metabolic acid production of Clostridium from pit mud.

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

[0008] A yellow water culture medium for culturing Clostridium perfringens from pit sludge, the culture medium comprising, by volume percentage:

[0009] Yellow water: 20%-40%;

[0010] Deionized water: 60%-80%;

[0011] The yellow water is a by-product of strong-aroma baijiu brewing, and the yellow water has a pH value of 3.0-4.0, a total acid content of 5.0-15.0 g / L, a reducing sugar content of 0.2-2.0 g / L, and a total nitrogen content of 2000-8000 mg / L.

[0012] The yellow water culture medium enables the inoculated Clostridium difficile from the pit mud to metabolize and synthesize organic acids during the culture process; and / or

[0013] It can support the high-density reproduction of Clostridium perfringens from the pit mud during the cultivation process.

[0014] As a preferred embodiment of the present invention, the yellow water contains a precursor substance that supports the synthesis of hexanoic acid, and the organic acid is at least one of acetic acid and butyric acid.

[0015] The present invention also provides a method for culturing Clostridium difficile from pit sludge using yellow water culture medium, wherein the method includes the following steps:

[0016] (1) The filtered yellow water is mixed with deionized water in a certain proportion, and the pH is adjusted to 6.5-7.5 to obtain yellow water culture medium;

[0017] (2) The yellow water culture medium is sterilized and then inoculated with Clostridium difficile from the pit mud;

[0018] (3) Under anaerobic conditions, the Clostridium culture was obtained by static incubation at 35-37℃ for 24-100h;

[0019] Among them, after culturing for 30-54 hours, the biomass OD of the Clostridium perfringens from the pit mud was... 600 The value is not less than 1.0;

[0020] and / or

[0021] After 24 hours of cultivation, the Clostridium culture contained organic acids synthesized by the Clostridium from the pit mud source.

[0022] As a preferred embodiment of the present invention, the Clostridium perfringens sourced from the pit mud is selected from at least one of Clostridium butyricum, Clostridium butyricum, Clostridium tumefaciens, Clostridium cochlearii, and Clostridium krylloides.

[0023] As a preferred embodiment of the present invention, the Clostridium perfringens from the pit mud is inoculated into the sterilized yellow water culture medium at an inoculation rate of 5%-15% by volume.

[0024] The present invention also provides a Clostridium preparation derived from pit mud, comprising the aforementioned Clostridium culture.

[0025] The present invention also provides a functional microbial preparation for baijiu brewing, comprising the aforementioned cellar mud-derived Clostridium preparation.

[0026] This invention also provides an application of a functional microbial preparation in the brewing of baijiu (Chinese liquor), the application including at least one of: enhancing baijiu fermentation, maintaining cellar mud, and constructing artificial cellar mud.

[0027] The present invention also provides the application of yellow water culture medium in the preparation of microbial culture agents for promoting the growth of Clostridium perfringens from pit mud.

[0028] The present invention also provides the application of yellow water culture medium in the preparation of microbial culture agents for promoting the metabolic synthesis of organic acids by Clostridium perfringens from pit mud.

[0029] As can be seen from the above technical solutions, the technical solutions of the present invention provide a high-efficiency Clostridium culture medium based on yellow water, a culture method preparation, and its application, which have the following beneficial effects:

[0030] 1. This invention achieves high-value resource utilization of brewing byproducts, combining economic and environmental benefits. It successfully develops the wastewater from baijiu brewing—yellow water—as the main raw material for a specialized culture medium for cultivating core functional microorganisms (clostridium from cellar mud). This not only significantly reduces the cost of the culture medium and eliminates dependence on expensive commercial media, but also enables large-scale, resource-based recycling of brewing waste, effectively alleviating environmental pressure and aligning with the green and sustainable development philosophy of production and research.

[0031] 2. Significantly promoted the high-density and rapid growth of Clostridium difficile from pit mud. Experiments have shown that, compared with traditional RCM medium, the nutrient composition and ratio of the yellow water medium of this invention are more suitable for the physiological needs of Clostridium difficile from pit mud, which can significantly shorten the growth lag phase of Clostridium difficile, accelerate its entry into the logarithmic growth phase, and obtain a higher final cell concentration (OD). 600 (Value). For example, the OD values ​​of Clostridium butyricum and Clostridium caseinate after 30 hours of culture in yellow water medium. 600 The value can reach above 1.0, which is much higher than the level in the same period in RCM medium (about 0.42-0.52), achieving efficient enrichment and culture of Clostridium.

[0032] 3. Significantly improved the efficiency of Clostridium metabolites in synthesizing organic acids and achieved precursor conversion of specific flavor acids. In conventional culture media with well-defined compositions (such as RCM), the tested Clostridium from pit mud had limited ability to synthesize organic acids such as butyric acid. However, the yellow water culture medium of this invention not only provides Clostridium with abundant initial substrates (such as lactic acid and acetic acid), but more importantly, its unique nutrient composition greatly promotes the further conversion of these substrates into high-value organic acids. Experiments show that after culturing in the yellow water medium, the concentration of key organic acids such as butyric acid achieved a net increase, with a significantly higher increase than in RCM medium. This proves that the yellow water medium can effectively drive the metabolic flux of Clostridium and support its efficient synthesis of target products. This indicates that the specific component combination in the yellow water effectively provides the precursors and reducing power required for the organic acid synthesis pathway, solving the technical problem that traditional culture media cannot support the efficient synthesis of organic acids by Clostridium.

[0033] 4. It provides a nutrient and microenvironment highly compatible with the habitat of the pit mud. The yellow water itself originates from the pit, and its complex organic acid spectrum, trace elements, and anaerobic metabolites constitute a chemical environment highly similar to that of natural pit mud. Using yellow water as a culture medium provides a more suitable and "familiar" growth and metabolic environment for Clostridium difficile from the pit mud, which is one of the fundamental reasons why it can simultaneously achieve rapid, high-density growth and reproduction and efficient acid production. This is also an advantage that is difficult to replicate with synthetic culture media.

[0034] 5. The method exhibits excellent versatility in strain selection and stability of culture results. It demonstrates broad applicability to various common functional Clostridium species found in pit mud (such as Clostridium butyricum, Clostridium butyricum, Clostridium krillii, and Clostridium cochlearii), significantly improving growth and acid production. By standardizing the concentration and pH of the yellow water, batch-to-batch fluctuations in the yellow water raw material are effectively balanced, ensuring the reproducibility of the culture process and the stability of the results, laying the foundation for industrial application.

[0035] 6. Broad application prospects and outstanding industrial value. The highly active, high-product-concentration Clostridium cultures obtained using the method of this invention can be directly used as functional microbial agents in areas such as strengthening the fermentation process of baijiu (Chinese liquor), repairing and maintaining aging cellar mud, and the targeted construction of artificial cellar mud. This provides an efficient microbial technology for improving baijiu quality, maintaining the health of the cellar mud microecology, and shortening the cellar cultivation cycle, and has broad prospects for industrial application. It should be understood that all combinations of the foregoing concepts and the additional concepts described in more detail below can be considered part of the inventive subject matter of this disclosure, provided that such concepts do not contradict each other.

[0036] The foregoing and other aspects, embodiments, and features of the teachings of the present invention will be more fully understood from the following description in conjunction with the accompanying drawings. Other additional aspects of the invention, such as features and / or beneficial effects of exemplary embodiments, will become apparent from the following description or may be learned through practice of specific embodiments according to the teachings of the present invention. Attached Figure Description

[0037] The accompanying drawings are not drawn to scale. In the drawings, each identical or nearly identical component shown in the various figures may be denoted by the same reference numeral. For clarity, not every component is labeled in each figure. Embodiments of various aspects of the invention will now be described by way of example and with reference to the accompanying drawings, wherein:

[0038] Figure 1 This is an example of the organic acid production of Clostridium in yellow water culture medium in Example 2 of the present invention. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the described embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical or scientific terms used herein should have the ordinary meaning understood by those skilled in the art.

[0040] The terms "first," "second," and similar words used in the specification and claims of this patent application do not indicate any order, quantity, or importance, but are merely used to distinguish different components. Similarly, unless the context clearly indicates otherwise, the singular forms of "an," "a," or "the," etc., do not indicate a quantity limitation, but rather indicate the presence of at least one. Terms such as "comprising" or "including" mean that the element or object preceding "comprising" encompasses the features, wholes, steps, operations, elements, and / or components listed following "comprising" or "including," and do not exclude the presence or addition of one or more other features, wholes, steps, operations, elements, components, and / or collections thereof. Terms such as "upper," "lower," "left," and "right" are used only to indicate relative positional relationships; when the absolute position of the described object changes, the relative positional relationship may also change accordingly.

[0041] On one hand, this invention provides a yellow water culture medium for cultivating Clostridium difficile from cellar mud, wherein the yellow water used is a byproduct generated during the brewing of strong-aroma baijiu and is collected in the middle and late stages of fermentation. Before use, the collected yellow water is allowed to settle to remove large particulate impurities, the supernatant is taken, and then diluted, pH adjusted and sterilized before being stored at room temperature for later use.

[0042] The yellow water collected in this embodiment of the invention has the following physicochemical characteristics: pH value of 3.0-4.0, preferably 3.2-3.6; total acid content of 7.0-10.0 g / L; total nitrogen content of 2000-8000 mg / L; and reducing sugar content of 0.5-1.0 g / L, wherein the reducing sugar includes glucose and fructose, and glucose is used as the unit of measurement in this embodiment. When preparing the culture medium from the pretreated yellow water, the yellow water is the main component, accounting for 20%-40% by volume, with the remainder being deionized water. Therefore, the yellow water culture medium of this invention is a liquid culture medium. Optionally, an appropriate amount of agar or other substances can be added to adjust it to a semi-solid or solid culture medium.

[0043] Another aspect of the present invention provides a method for culturing Clostridium difficile from pit mud using the above-mentioned yellow water culture medium, specifically comprising the following steps:

[0044] (1) Adjust the pretreated yellow water to a suitable volume concentration according to the volume ratio, preferably 20%-40%, and adjust its pH to 7.0±0.2 with 40% NaOH to obtain yellow water culture medium.

[0045] While undiluted yellow water has a rich impact, its ethanol, high salt concentration, metal ions, and microbial metabolic byproducts may inhibit the pure culture of Clostridium. Adjusting its concentration to 20%-40% effectively dilutes these potential inhibitors while retaining sufficient core carbon and nitrogen sources and growth factors to support high-density Clostridium growth and target product synthesis. This maximizes the metabolic potential of the cells while mitigating growth stress that may be caused by high-concentration substrates. As a brewing byproduct, the composition and concentration of yellow water naturally fluctuate due to factors such as fermentation pits, season, and processing techniques. Using a fixed concentration range, rather than a variable stock solution, and combining it with standardized dilution with deionized water, is a crucial step in transforming this naturally variable resource into a relatively stable culture medium raw material. This significantly reduces the fluctuations in culture effects caused by batch-to-batch variations in raw materials, providing a predictable and reproducible process basis for laboratory research and industrial applications. This concentration range is an optimized range that has been experimentally validated, enabling efficient utilization of yellow water resources while ensuring excellent culture results. Compared to using the stock solution directly, this method significantly expands the amount of culture medium prepared per batch of yellow liquid, improves the scale and economy of resource recycling, and better meets the cost control requirements for industrialization.

[0046] Furthermore, from a microbiological perspective of Clostridium culture, this embodiment of the invention adjusts the pH of the yellow water from the initial 3.0-4.0 to 6.5-7.5. As an anaerobic bacterium, Clostridium's intracellular enzyme system and membrane transport function are most active in a neutral to slightly alkaline environment. An initial pH below 4.0, a strongly acidic environment, directly leads to proton stress, inhibits the activity of key metabolic enzymes, hinders nutrient absorption, and severely limits or even completely inhibits Clostridium germination and early growth. Adjusting the pH to neutral is a necessary physiological initiation condition for Clostridium to rapidly initiate logarithmic growth after inoculation. During fermentation, Clostridium produces a large amount of short-chain fatty acids, which are themselves acidic and continuously lower the pH of the culture system. If the initial pH is too low, the acid produced in the early stages of fermentation will rapidly lower the environmental pH below the cell's tolerance limit, leading to premature termination of growth and inhibition of metabolic pathways. Adjusting the initial pH to a neutral range provides a buffer zone throughout the fermentation cycle, ensuring that Clostridium maintains metabolic activity for a longer period during acid production, thereby facilitating higher biomass and the accumulation of target products. Many metal ions are essential components of Clostridium coenzymes or key enzymes, and their bioavailable forms in solution are significantly affected by pH. At excessively low pH levels, these ions may precipitate or exist in forms that are difficult for the organism to utilize. Adjusting the pH to near neutral helps maintain the solubility and bioavailability of these trace elements, thereby supporting vigorous growth and metabolism in Clostridium.

[0047] (2) After the yellow water culture medium is treated with high-pressure high-temperature sterilization at 110-130℃ for 20-30 minutes, it is inoculated with Clostridium difficile from the pit mud at an inoculation rate of 5%-15%. The preferred Clostridium difficile from the pit mud is at least one of Clostridium butyricum, Clostridium butyricum, Clostridium tumefaciens, Clostridium cochlearii, Clostridium krylloides, and Clostridium tumefaciens.

[0048] High-pressure, high-temperature sterilization completely inactivates various wild microorganisms (such as contaminating bacteria, yeasts, molds, and their spores) carried by the yellow water itself or introduced during preparation, thus creating a sterile starting environment for the pure culture of Clostridium perfringens from the target pit mud. This temperature range ensures reliable sterilization while avoiding excessive heat treatment that could significantly damage heat-sensitive nutrients in the culture medium, achieving a balance between eliminating contaminating bacteria and preserving nutrient activity.

[0049] The inoculum size used in this embodiment of the invention is based on the following considerations: Too low an inoculum size will result in an excessively long lag phase, making it difficult for the bacteria to quickly establish a growth advantage, prolonging the culture period, and making them susceptible to trace residues or accidental contamination during subsequent operations; while too high an inoculum size may lead to premature depletion of the nutrient substrate or rapid accumulation of metabolic products due to an excessively high initial bacterial concentration, thereby inhibiting the growth and metabolic activity of the bacteria themselves. Within this inoculum size, it is possible to ensure that Clostridium adapts quickly to the growth environment, shortens the lag phase, enters a stable and efficient logarithmic growth phase, and ultimately achieves a balance between high biomass and high target synthesis rate.

[0050] (3) Under anaerobic conditions, the Clostridium species were cultured at a temperature of 35-37℃ for 24-100 h to obtain Clostridium cultures. In the Clostridium cultures, Clostridium from the pit mud existed in a high biomass and metabolized and accumulated at least one organic acid, including acetic acid and butyric acid.

[0051] The yellow water culture medium provided by this invention can significantly promote the proliferation of Clostridium difficile from pit mud, enabling its high-density cultivation; and / or, can effectively promote the metabolic synthesis of at least one organic acid by Clostridium difficile from pit mud, wherein the organic acid preferably includes one or more of butyric acid and acetic acid. Compared with conventional commercial culture media, the yellow water culture medium of this invention not only enables Clostridium difficile from pit mud to obtain a higher final biomass and a faster growth rate, but also specifically activates or enhances its ability to synthesize key flavor acids such as organic acids, solving the problem of insufficient acid production or no acid production of Clostridium difficile under traditional culture media.

[0052] Thirdly, the present invention provides the application of yellow water culture medium in the preparation of microbial culture agents for promoting the metabolic synthesis of organic acids by Clostridium difficile from pit mud.

[0053] Fourthly, the present invention provides the application of yellow water culture medium in the preparation of microbial culture agents for promoting the growth of Clostridium perfringens from pit mud.

[0054] Fifthly, the present invention provides a Clostridium difficile preparation derived from pit sludge, which comprises the aforementioned Clostridium difficile culture. The dosage form of this Clostridium difficile preparation can be liquid, concentrated solution, or solid form adsorbed onto a carrier, facilitating storage, transportation, and use.

[0055] In a sixth aspect, the present invention provides a functional microbial preparation for the brewing of baijiu (Chinese liquor), the functional microbial preparation comprising the above-mentioned Clostridium difficile preparation derived from cellar mud.

[0056] Seventhly, the present invention provides an application of a functional microbial preparation in the brewing of baijiu, including but not limited to: inoculating it into the fermentation system to enhance the baijiu fermentation process and promote the generation of flavor precursor substances; using it to maintain old cellar mud, supplement and revitalize beneficial functional microbial communities; and using it as a core functional strain to construct artificial cellar mud, shorten the cellar mud maturation cycle and improve its quality.

[0057] Example 1

[0058] The yellow water used in this embodiment came from the old cellar of a liquor production workshop of a liquor production enterprise in Jiangsu Province. The main physicochemical indicators of the collected yellow water were tested according to standards and the results are shown in Table 1 below.

[0059] Table 1. Main Physicochemical Indicators of Huangshui

[0060]

[0061] Table 1 clearly shows from the chemical oxygen demand item that the yellow water is rich in a large amount of oxidizable organic matter, such as residual starch, sugars, organic acids, alcohols, esters, etc. These organic substances are the valuable carbon source and energy required for the growth and metabolism of microorganisms (such as Clostridium perfringens from the pit mud).

[0062] The strains cultured in this invention are Clostridium species derived from cellar mud, preferably *Clostridium butyricum*, *Clostridium tyrobutyricum*, *Clostridium vini*, *Clostridium cochlearium*, *Clostridium kluyveri*, and *Clostridium peptidivorans*. These Clostridium species were isolated and purified from old cellar mud and identified by 16S rRNA gene sequencing. The genus *Clostridium* encompasses a wide variety of species with diverse physiological and metabolic characteristics, making accurate identification difficult based solely on morphological observation or traditional physiological and biochemical experiments. 16S rRNA gene sequencing, by comparing the sequences with known standard strains, can objectively and reliably determine the taxonomic position of the strains, avoiding misclassification.

[0063] Comparison of Clostridium growth on yellow water medium and RCM (reinforced Clostridium medium).

[0064] (1) Culture medium preparation:

[0065] Group A (control): Commercially available RCM medium, prepared according to the instructions.

[0066] The commercially available RCM culture medium in this embodiment of the invention has the following typical composition (per liter): 10.0g peptone, 10.0g beef extract, 3.0g yeast extract, 5.0g glucose, 1.0g soluble starch, 5.0g sodium chloride, 3.0g sodium acetate, 0.5g L-cysteine ​​hydrochloride, and distilled water to a final volume of 1.0L; if preparing a solid culture medium, 15.0g agar can be added.

[0067] Group B (Yellow Water Culture Medium): Take the pretreated yellow water, dilute it with deionized water to a volume concentration of 30%, adjust the pH to 7.0±0.2 with 40% NaOH, and autoclave at 118℃ for 20 minutes. This is to kill all microbial vegetative cells and spores introduced into the yellow water and during the preparation process, especially wild bacteria carried by the yellow water itself, such as aerobic bacteria, facultative anaerobic bacteria, yeast, mold, potential bacteriophages, and other microorganisms that may contaminate during the preparation process. This provides a sterile starting culture environment for the subsequent culturing of the target Clostridium, avoiding competition or interference from other microorganisms in the experimental results.

[0068] (2) Inoculation: Common Clostridium butyricum, Clostridium butyricum, Clostridium tumefaciens, Clostridium cochlearii, Clostridium krylloides, and Clostridium tumefaciens, which are commonly found in pit mud, were selected and activated. Each strain was inoculated into 6 groups of A culture medium and 6 groups of B culture medium at an inoculation rate of 10%, with each strain inoculated into the culture medium separately to facilitate subsequent performance analysis.

[0069] Optionally, in practical applications, the Clostridium to be cultured can be subjected to pure culture of a single strain or mixed culture of multiple strains as needed. For example, when preparing compound functional microbial preparations, a mixed culture of multiple strains in a specific ratio can be carried out.

[0070] (3) Culture conditions: static culture in an anaerobic workstation at 36℃.

[0071] (4) Detection: Samples were taken at 0h, 4h, 6h, 24h, 30h, 48h, 54h, 76h and 100h of culture and the OD was measured. 600 The value (representing the optical density of the culture medium measured at a wavelength of 600 nm, used to characterize cell concentration or biomass) is used to characterize the amount of cell growth.

[0072] Results: Table 2 shows the OD values ​​of Clostridium in RCM medium. 600 Table 3 shows the OD values ​​of Clostridium perfringens in yellow water culture medium. 600 value.

[0073] Table 2. OD of Clostridium in RCM medium 600 value

[0074]

[0075] Table 3. OD of Clostridium in yellow water culture medium 600 value

[0076]

[0077] As shown in Tables 2 and 3, all Clostridium species exhibited faster growth rates and higher maximum biomass in Group B (30% yellow water medium). Particularly at 30 hours, the OD values ​​of most species in Group B (Clostridium butyricum, Clostridium butyricum, and Clostridium cochlearii) were significantly higher. 600 The value reached or exceeded 1.0. In comparison, the OD value of Group A in the same period was... 600 The highest value was around 0.5, and the maximum value during the entire culture cycle was only around 0.7, not reaching 1.0, indicating that the yellow water culture medium of the present invention can greatly promote the rapid enrichment of Clostridium and achieve high-density culture compared with the existing Clostridium culture medium.

[0078] Example 2

[0079] This embodiment is mainly used to compare the amount of organic acids produced by Clostridium tumefaciens cultured in yellow water medium with the amount of organic acids produced by Clostridium tumefaciens cultured in RCM medium, so as to demonstrate the promoting effect of the yellow water medium of the present invention on the acid production of Clostridium tumefaciens.

[0080] (1) Culture method: Same as in Example 1, using two culture media A and B to culture six Clostridium species respectively.

[0081] (2) Detection: Samples were taken at 0h, 24h, 48h and 76h of culture, and the supernatant was centrifuged. The concentrations (g / L) of acetic acid, propionic acid, butyric acid and hexanoic acid were determined by gas chromatography. The results are shown in Tables 4 and 5.

[0082] (3) Blank group: The sterilized yellow water culture medium was placed in the same anaerobic environment without inoculation of the strain. The concentration of organic acids (g / L) in the yellow water during the standing period of 0-76h was also detected by gas chromatography.

[0083] Results: Table 4 shows the organic acid content of Clostridium in RCM medium, Table 5 shows the organic acid content of Clostridium in yellow water medium, and Table 6 shows the organic acid content of uninoculated yellow water medium. This example assesses the acid-producing capacity of Clostridium by monitoring changes in the concentration of organic acids in the culture system.

[0084] Table 4. Organic acid content of Clostridium in RCM medium (unit: g / L)

[0085]

[0086] Table 5. Organic acid content of Clostridium in yellow water culture medium (unit: g / L)

[0087]

[0088] It should be noted that in Tables 4 and 5, "-" indicates that the organic acid was not detected, or that the amount of the organic acid was so small that it could not be effectively detected.

[0089] As can be seen from Table 4, the acetic acid concentration in RCM medium (Group A) changed little or decreased. For example, the concentration of Clostridium butyricum decreased from 2.2 g / L to 0.9 g / L. Except for Clostridium butyricum (76 h) and Clostridium krusei, which produced very little propionic acid, the other Clostridium species did not produce any. Except for Clostridium tumefaciens, the other Clostridium species showed a slight increase in butyric acid. None of the Clostridium species produced hexanoic acid.

[0090] Based on the analysis in Table 5, the specific yields of acetic acid, propionic acid, butyric acid, and hexanoic acid synthesized by various Clostridium species in the yellow water culture medium are as follows:

[0091] Acetic acid status of Clostridium butyricum: Acetic acid concentration first increases and then decreases, because the carbon flow is diverted to butyric acid synthesis and acetic acid is reused.

[0092] Acetic acid status of Clostridium butyricum: Acetic acid concentration first increases and then slightly decreases. This is due to the carbon metabolism flowing to butyric acid synthesis and the reuse of acetic acid.

[0093] Acetic acid status of Clostridium tumefaciens: The overall acetic acid concentration showed a gradual upward trend, mainly because the main metabolic pathway of Clostridium tumefaciens uses acetic acid as the final product.

[0094] Acetic acid content in *Clostridium cochlearii*: The acetic acid concentration is initially high, then decreases or remains essentially unchanged, exhibiting no regularity. This is because during its growth, the bacterium continuously switches between utilizing different types of sugars or amino acids and other complex substrates in the yellow water, resulting in alternating metabolic pathways of acetic acid production and consumption. Therefore, the acetic acid concentration fluctuates irregularly. For example, as the concentration of a certain substrate decreases due to its utilization, the bacterium switches to utilizing other types of sugars or amino acids, leading to a dynamic alternation between the metabolic pathways of acetic acid synthesis and consumption.

[0095] Acetic acid status in Clostridium kuristeum: Acetic acid concentration initially increases and then decreases. This is because in the early stage, rapid acid production from sugar consumption leads to acetic acid accumulation. In the later stage, when substrate is depleted, reverse metabolism is initiated to maintain redox balance, replenishing acetic acid and causing its concentration to decrease.

[0096] Acetic acid content in peptide-digested Clostridium: Overall, there was an upward trend. This is mainly because, in this yellow water medium, the dominant metabolic pathway of this strain stably directs substrates towards acetic acid synthesis, thereby achieving a continuous net accumulation of acetic acid.

[0097] In summary, in the yellow water medium, the acetic acid concentration of some Clostridium species initially increased and then decreased. This may be because Clostridium utilizes acetic acid in the yellow water as a carbon source for growth in the early stages, but later switches to synthesizing other acids (such as butyric acid). Simultaneously, acetic acid is consumed for energy metabolism. This pattern of synthesis followed by consumption demonstrates that the yellow water medium successfully activates the complete metabolic pathway of Clostridium, achieving efficient and targeted conversion of carbon to the target flavor acid (such as butyric acid). In the RCM medium, the acetic acid concentration decreased even earlier, indicating nutrient deficiency.

[0098] In the yellow water culture medium of the present invention, all Clostridium species produce a small amount of propionic acid. Specifically, after culturing in the yellow water culture medium for 24 hours, the propionic acid concentration in the fermentation broths of Clostridium butyricum, Clostridium butyricum, Clostridium sacchariformis, Clostridium cochlearii, Clostridium krusei, and Clostridium peptide-digesting remains essentially unchanged. This is mainly because the yellow water culture medium lacks key precursors for propionic acid synthesis, and the main metabolic pathway of these bacteria is not the propionic acid pathway.

[0099] Propionic acid is not a major product of Clostridium. Clostridium typically produces propionic acid via the succinate pathway, but this pathway requires specific enzyme systems (e.g., the methylmalonyl-CoA pathway). The small variation in propionic acid concentration in yellow water medium may be due to Clostridium's lack of efficient propionic acid synthesis capacity, or the propionic acid being further metabolized. Propionic acid was almost undetectable in RCM medium, indicating that yellow water may contain trace amounts of precursors, but insufficient to drive significant synthesis.

[0100] In yellow water culture medium, the butyric acid synthesis of various Clostridium species can be divided into two categories: strains with significantly increased butyric acid production and strains with basically unchanged butyric acid production.

[0101] Among them, *Clostridium butyricum*, *Clostridium caseosa*, *Clostridium cochlearii*, *Clostridium krusei*, and *Clostridium peptide-digesting* all showed a stable upward trend in butyric acid concentration in their fermentation broths during cultivation. This indicates that butyric acid synthesis is the dominant metabolic pathway for these strains in the yellow water medium. The cumulative butyric acid production is mainly attributed to: a complete metabolic pathway, with the strains expressing and actively exercising the complete set of synthases from acetyl-CoA to butyric acid; sufficient carbon flux guidance, where carbon sources in the yellow water medium are effectively directed to the butyric acid synthesis pathway, and the metabolic flux of this pathway is higher than that of other competing pathways.

[0102] The butyrate concentration in *Clostridium tumefaciens* remained essentially constant throughout the culture. This is consistent with its inherent metabolic characteristics, as butyrate synthesis is not the primary metabolic pathway for this strain. The main reason is that *Clostridium tumefaciens* primarily directs its metabolic flux toward other products such as acetic acid or ethanol, resulting in weak or absent activity of its butyrate synthesis pathway (thiolases and butyrate kinase system), thus hindering the efficient conversion of carbon flux into butyrate.

[0103] In summary, such as Figure 1As shown, butyric acid is the main metabolic product of most Clostridium species. Lactic acid and acetic acid in the yellow water can serve as substrates and be converted into butyric acid through the butyric acid synthesis pathways of Clostridium (e.g., phosphotransacetase and butyrate kinase pathways). Yellow water is rich in lactic acid, which Clostridium can utilize to generate pyruvate, and subsequently, butyric acid. In RCM medium, the limited substrate hinders butyric acid synthesis. Table 5 shows that the butyric acid concentration of most Clostridium species increases with culture time, indicating that yellow water provides an abundant carbon source and reducing power. This also demonstrates that Clostridium has a very strong butyric acid synthesis capacity in the yellow water medium of this invention, far exceeding its performance in RCM medium.

[0104] In the yellow water culture medium, the concentration of hexanoic acid in all Clostridium species did not increase significantly with culture time, indicating that none of them produced hexanoic acid.

[0105] Analysis of the overall acid production profiles in the two culture media, based on Tables 4 and 5, revealed that Clostridium in group B culture medium exhibited more active metabolism, a broader acid production profile, and higher total acid yield, with a particularly significant advantage in the production of target flavor acids. This is because the yellow water provided a diverse range of substrates, such as various organic acids and sugars, activating multiple metabolic pathways in Clostridium.

[0106] Therefore, the culture medium and its cultivation method with yellow water as the main component provided by this invention can simultaneously and efficiently promote the biomass accumulation of Clostridium perfringens from cellar mud and the synthesis of flavor-related organic acids, with effects far superior to traditional RCM culture medium. This provides an efficient and economical new strategy for the development and utilization of baijiu microbial resources.

[0107] Table 6. Changes in organic acid concentration in yellow water culture medium (unit: g / L)

[0108]

[0109] In Table 6, "-" indicates that the organic acid was not detected, or that the amount of the organic acid was so small that it could not be effectively detected.

[0110] Table 6 shows that the concentration (g / L) of organic acids (including acetic acid, propionic acid, butyric acid and hexanoic acid) in the blank control group did not change significantly during this period, while the concentration (g / L) of organic acids in the experimental group inoculated with Clostridium showed a clear increase compared with the baseline value of the blank group, and this increase was positively correlated with the bacterial growth curve, proving that the net synthesis of organic acids comes from Clostridium metabolism.

[0111] While the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the invention. Those skilled in the art can make various modifications and refinements without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention shall be determined by the claims.

Claims

1. A yellow water culture medium for culturing Clostridium perfringens from pit sludge, characterized in that, The culture medium comprises, by volume percentage: Yellow water: 20%-40%; Deionized water: 60%-80%; The yellow water is a by-product of strong-aroma baijiu brewing, and the yellow water has a pH value of 3.0-4.0, a total acid content of 5.0-15.0 g / L, a reducing sugar content of 0.2-2.0 g / L, and a total nitrogen content of 2000-8000 mg / L. The yellow water culture medium enables the inoculated Clostridium difficile from the pit mud to metabolize and synthesize organic acids during the culture process; and / or It can support the high-density reproduction of Clostridium perfringens from the pit mud during the cultivation process.

2. The yellow water culture medium for culturing Clostridium perfringens from pit sludge according to claim 1, characterized in that, The organic acid is at least one of acetic acid and butyric acid.

3. A method for culturing Clostridium difficile from pit sludge using yellow water culture medium, characterized in that, Using the yellow water culture medium as described in claim 1 or 2, the method includes the following steps: (1) The filtered yellow water is mixed with deionized water in a certain proportion, and the pH is adjusted to 6.5-7.5 to obtain yellow water culture medium; (2) The yellow water culture medium is sterilized and then inoculated with Clostridium difficile from the pit mud; (3) Under anaerobic conditions, the Clostridium culture was obtained by static incubation at 35-37℃ for 24-100h; Among them, after culturing for 30-54 hours, the biomass OD of the Clostridium perfringens from the pit mud was... 600 The value is not less than 1.0; and / or After 24 hours of cultivation, the Clostridium culture contained organic acids synthesized by the Clostridium from the pit mud source.

4. The method for culturing Clostridium difficile from pit mud using yellow water culture medium according to claim 3, characterized in that, The Clostridium sourced from the pit mud is selected from at least one of Clostridium butyricum, Clostridium butyricum, Clostridium tumefaciens, Clostridium cochlearii, Clostridium krylloides, and Clostridium tumefaciens.

5. The method for culturing Clostridium difficile from pit mud using yellow water culture medium according to claim 3, characterized in that, The Clostridium perfringens from the pit mud was inoculated into the sterilized yellow water culture medium at an inoculation rate of 5%-15% by volume.

6. A Clostridium preparation derived from pit mud, characterized in that, It includes the Clostridium culture as described in claim 3.

7. A functional microbial preparation for use in Baijiu (Chinese liquor) brewing, characterized in that, It contains the Clostridium difficile preparation derived from pit mud as described in claim 6.

8. The application of a functional microbial preparation in Baijiu brewing, characterized in that, The applications include at least one of the following: enhancing the fermentation of baijiu (Chinese liquor), maintaining cellar mud, and constructing artificial cellar mud.

9. The application of a yellow water culture medium in the preparation of a microbial culture agent for promoting the growth of Clostridium perfringens from pit mud.

10. The application of a yellow water culture medium in the preparation of a microbial culture preparation for promoting the metabolic synthesis of organic acids by Clostridium difficile from pit mud.

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