Growth medium for ruminococcus buchneri fermentation and preparation method thereof

Abstract

The invention discloses a ruminococcus buchneri fermentation growth medium and a preparation method thereof. The growth medium is prepared from the following raw materials: a carbon source, a nitrogen source, inorganic salt, a reducing agent, a redox indicator, cooked beef granules and bovine rumen fluid, wherein the carbon source is resistant starch, and the concentration of the carbon source is 3.0-7.0 g / L; the concentration of the nitrogen source is 16.0-67.0 g / L; the reducing agent is L-cysteine hydrochloride, and the concentration of the reducing agent is 0.1-0.7 g / L; the redox indicator is resazurin, and the concentration of the redox indicator is 0.001-0.007 g / L; the volume ratio of the bovine rumen fluid is 5%-25%; the mass volume ratio content of the cooked beef granules is 10.0-40.0 g / L. By optimizing the formula of the culture medium, the growth efficiency and metabolism of the ruminococcus buchneri are remarkably improved, and the growth activity is prolonged, which is specifically reflected in multiple aspects such as thallus growth density, physiological functions and economy.

Description

Technical Field

[0001] This invention relates to a culture medium for Ruminococcus brucelli, and more particularly to a growth culture medium for Ruminococcus brucelli fermentation and its preparation method; it belongs to the field of microbial culture medium technology. Background Technology

[0002] Based on the rate and extent of starch digestion, starch can be classified into rapidly digestible starch (RDS, hydrolyzed within the first 20 minutes under in vitro digestion conditions), slowly digestible starch (SDS, hydrolyzed within 20–120 minutes), and resistant starch (RS, partially undigested starch after 120 minutes, continuing into the large intestine where it is completely or partially degraded into short-chain fatty acids and other metabolites by the colonizing microbiota). Resistant starch provides a growth substrate for gut microbiota, which obtain the energy needed for growth by degrading resistant starch. The degradation of resistant starch by gut microbiota produces short-chain fatty acid metabolites containing acetic acid, propionic acid, butyric acid, etc. For a long time, resistant starch, as a poorly digestible dietary fiber, has had its dense molecular structure constitute a barrier to utilization by ordinary microorganisms, making it difficult for most microorganisms to ferment and utilize in the gut. Therefore, resistant starch has not been used as a carbon source in culture media. Related research indicates that *Ruminococcus brucelli* is a key species in the human gut for degrading resistant starch.

[0003] Ruminococcus bromii, a core component of the gut microbiota in humans and ruminants, plays an irreplaceable role in degrading resistant starch, and its metabolically produced short-chain fatty acids have a profound impact on host health. Short-chain fatty acids help improve inflammation and oxidative stress, enhance visceral sensitivity, and promote other functions. Achieving efficient in vitro culture of this strain is crucial for probiotic development and microbial ecology research. From an application perspective, Ruminococcus bromii is considered a candidate for next-generation probiotics, with its strong resistant starch degradation ability being the core of its probiotic function. Achieving high-density, low-cost, and standardized industrial-scale culture using optimized media will directly promote the development of live bacteria or prebiotic formulations targeting intestinal metabolic disorders, energy imbalances, and related diseases. However, the difficulty in culturing this bacterium in vitro and its slow growth severely limit its application in probiotic, synthetic formulation, and enzyme preparation development.

[0004] Chinese invention patent application CN120478418A discloses the application of *Ruminococcus bromii* and its in vitro pure culture metabolic supernatant in promoting intestinal development in piglets. The *Ruminococcus bromii* used was purchased from Guangzhou Kangruis Biotechnology Co., Ltd., with accession number ATCC51896. CMC medium is commonly used. Chopped Meat Carbohydrate Broth (CMC) is a liquid culture medium specifically designed for anaerobic bacteria (especially obligate anaerobes) and requires the addition of ground beef granules and specific additives. Its typical formulation includes casein peptone, beef extract, yeast extract, buffer salts, glucose, maltose, cellobiose, starch, and bovine rumen fluid at a volume ratio of up to 30%. Ruminococcus brucelli cannot utilize reducing sugars such as glucose and maltose as carbon sources. However, the carbon source used in this CMC medium results in slow growth, low biomass (OD value), long lag phase, and low production of beneficial metabolites, making high-density culture impossible and only achieving a certain OD value. 600nm With a concentration of 0.8-1.0, the amount of acetic acid produced is only 7.0 mM. Summary of the Invention

[0005] The purpose of this invention is to provide a growth culture medium for Ruminococcus brucelli that increases the number of viable bacteria, enhances physiological functions, reduces cultivation costs, and improves cultivation efficiency, as well as a method for its preparation.

[0006] To achieve the purpose of the invention, the present invention adopts the following technical solution:

[0007] A growth medium for Ruminococcus brucelli: the raw materials include carbon source, nitrogen source, inorganic salt, reducing agent, redox indicator, beef granules and bovine rumen juice;

[0008] The carbon source is resistant starch with a concentration of 3.0–7.0 g / L; the nitrogen source has a concentration of 16.0–67.0 g / L; the reducing agent is L-cysteine ​​hydrochloride with a concentration of 0.1–0.7 g / L; and the redox indicator is resazurin with a concentration of 0.001–0.007 g / L.

[0009] The volume percentage of the bovine rumen fluid is 5% to 25%; the mass-volume ratio of the cooked beef granules is 10.0 to 40.0 g / L.

[0010] To further achieve the objectives of this invention, preferably, the resistant starch is selected from at least one of potato resistant starch, banana resistant starch, Hylon VIII, and Ylon VII. Hylon VIII and Ylon VII are both high-amylose corn resistant starches.

[0011] Preferably, the nitrogen source comprises one or more of beef extract, tryptone, and yeast extract.

[0012] Preferably, the nitrogen source further includes fish peptone, which together with beef extract and / or casein peptone constitutes the nitrogen source.

[0013] Preferably, the concentrations of the components in the nitrogen source are: beef extract 5.0–30.0 g / L, tryptone 10.0–40.0 g / L, yeast extract 1.0–30.0 g / L, and fish peptone 10.0–20.0 g / L.

[0014] Preferably, the inorganic salt is selected from K2HPO4, KH2PO4, (NH4)2SO4 or MgSO4·7H2O.

[0015] A method for preparing a growth medium for Ruminococcus brucelli: dissolve all the raw materials of the above-mentioned Ruminococcus brucelli growth medium in water to obtain a culture medium solution; subject the culture medium solution to anaerobic treatment to obtain a growth medium for Ruminococcus brucelli.

[0016] Preferably, the anaerobic treatment involves heating the basal culture medium solution to boiling and replacing the oxygen in the culture medium solution with nitrogen for 10-20 minutes. The growth medium for *Ruminococcus brucelli* must meet the anaerobic growth environment requirements of *Ruminococcus brucelli*.

[0017] Preferably, the growth medium for *Ruminococcus brucelli* further includes sterilization.

[0018] Preferably, the sterilization process involves heating the anaerobic culture medium solution to 117-121°C for 20-40 minutes using a high-pressure steam method.

[0019] A method for culturing Ruminococcus brucelli involves inoculating Ruminococcus brucelli bacterial suspension at a rate of 3% into the aforementioned basal culture medium and then anaerobically incubating it at 30-40℃ for at least 20 hours.

[0020] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0021] 1) The growth medium for *Ruminococcus brucelli* of this invention significantly improves the culture efficiency and physiological efficacy of *Ruminococcus brucelli*, prolonging its growth activity, specifically in terms of cell density, enhanced metabolism, and economic efficiency. Compared to existing CMC culture media (cell density OD...), 600nm With only 0.8-1.0, acetic acid production and 7mM), this invention optimizes the carbon source structure by adding resistant starch (optimal concentration 5g / L), thus increasing OD. 600nmThe value was increased to above 1.4, the cell density increased by 52%, and the acetic acid production reached above 10.0 mM, an increase of 42.9%. As a characteristic beneficial metabolite of this bacterium, the significant increase in the production of acetic acid directly confirms the effectiveness of this invention in enhancing the core metabolic function of the strain.

[0022] 2) The growth medium for *Ruminococcus brucelli* of this invention supports high-density growth. In CMC medium, the bacteria typically enter the death phase after about 36 hours of culture; however, using the growth medium of this invention, the exponential growth phase and stationary phase of the bacteria are extended until about 48 hours before entering the death phase, thus extending the effective activity of the strain by about 33%. This provides a more ample and flexible time window for controlling the termination of the fermentation process, harvesting the bacteria, and subsequent process operations.

[0023] 3) Within the formulation framework of resistant starch as the core carbon source, the culture medium of this invention exhibits good adaptability to the specific composition and ratio of nitrogen sources, as well as the type and concentration of buffer salts. Using the growth medium of this invention, key indicators such as cell growth density and acetic acid yield remain stable and excellent, demonstrating the robustness and reproducibility of the culture medium formulation, which is beneficial for the stable implementation of large-scale production. Attached Figure Description

[0024] Figure 1 Growth curves of Ruminococcus brevicornu ATCC51896 in Examples 1-4 using different resistant starch substrates.

[0025] Figure 2 The utilization rate of different resistant starch by *Ruminococcus brevicornu* ATCC51896 in Examples 1-4 is shown.

[0026] Figure 3 The effects of different resistant starches on the pH of Ruminococcus brucelli ATCC51896 in Examples 1-4.

[0027] Figure 4 The effect of different resistant starches on the acetic acid production of Ruminococcus brevicornu ATCC51896 in Examples 1-4.

[0028] Figure 5 The effects of different amounts of resistant starch added in Examples 5-7 on the growth of Ruminococcus brucelli ATCC51896.

[0029] Figure 6 The effect of different amounts of resistant starch added in Examples 5-7 on the acetic acid production of Ruminococcus brevicornu ATCC51896.

[0030] Figure 7 The effect of different nitrogen source conditions on the growth of Ruminococcus brevicornus ATCC51896 in Example 8.

[0031] Figure 8 The effect of different nitrogen source conditions on the growth of Ruminococcus brevicornus ATCC51896 in Example 8.

[0032] Figure 9 This describes the effect of different nitrogen source conditions on the acetic acid production of *Ruminococcus brevicornus* ATCC51896 in Example 8.

[0033] Figure 10 The effect of different buffer salt conditions on the growth of Ruminococcus brevicornus ATCC51896 in Example 9.

[0034] Figure 11 The effect of different buffer salt conditions on the growth of Ruminococcus brevicornus ATCC51896 in Example 9.

[0035] Figure 12 The effect of different buffer salt conditions on the acetic acid production of Ruminococcus brevicornus ATCC51896 in Example 9.

[0036] Figure 13 The effects of different growth factors on the growth of Ruminococcus brevicornus ATCC51896.

[0037] Figure 14 The effects of different growth factors on the acetic acid production of Ruminococcus brevicornu ATCC51896 in Examples 10-13.

[0038] Figure 15 The effects of different amounts of bovine rumen fluid added in Examples 14-17 on the growth of Rumenococcus brevicornus ATCC51896 were investigated.

[0039] Figure 16 The effect of different amounts of bovine rumen fluid added in Examples 14-17 on the acetic acid production of Ruminococcus brevicornus ATCC51896.

[0040] Figure 17 The growth curves of *Ruminococcus brevicornus* ATCC51896 in Comparative Example 1 are shown in CMC medium and optimized medium.

[0041] Figure 18 The pH change of Ruminococcus brevicornus ATCC51896 in CMC medium was compared with that of Comparative Example 1.

[0042] Figure 19 The amount of acetic acid produced by *Ruminococcus brevicornus* ATCC51896 in CMC medium in Comparative Example 1 is given.

[0043] Figure 20 The growth curves of *Ruminococcus brucelli* in Comparative Example 2 on different concentrations of soluble starch are shown.

[0044] Figure 21 The amount of acetic acid produced by the growth of Ruminococcus brucelli on different concentrations of soluble starch in Comparative Example 2 is shown.

[0045] Figure 22 The growth curves of *Ruminococcus brucelli* in Comparative Example 3 on different concentrations of potato resistant starch are shown.

[0046] Figure 23 The amount of acetic acid produced by the growth of Ruminococcus brucelli on potato resistant starch at different concentrations in Comparative Example 3. Detailed Implementation

[0047] To better understand the present invention, it will be further described below with reference to the accompanying drawings and specific embodiments. However, the implementation of the present invention is not limited thereto. The described embodiments are some, but not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0048] The Ruminococcus bromii involved in this invention is Ruminococcus bromii with accession number ATCC 51896, deposited at the U.S. Culture Collection Center.

[0049] In existing technologies, universal culture media for culturing Ruminococcus brucelli (such as CMC medium) typically use glucose or maltose as carbon sources. However, these carbon sources result in insufficient activation of the natural substrate utilization pathways of the strains during in vitro culture, preventing them from exhibiting their unique degradation functions and metabolic activities in vitro in a truly efficient manner, thus limiting the efficiency of obtaining highly active and functionalized bacterial cells.

[0050] This invention discovers that using resistant starch to replace glucose and maltose in existing technologies, along with a nitrogen source, inorganic salts, reducing agents, redox indicators, beef granules, and bovine rumen fluid to obtain a growth medium for *Ruminococcus brucelli*, can significantly optimize the growth kinetics of *Ruminococcus brucelli*, such as its growth efficiency and metabolism, and prolong its growth activity. Compared to the existing CMC medium, this method significantly improves the cell density (OD) of *Ruminococcus brucelli* culture. 600nm With an OD of only 0.8-1.0 and an acetic acid production of only 7.0 mM, the growth medium for *Ruminococcus brucelli* of this invention produces an OD of only 0.8-1.0 mM. 600nm The value can be increased to over 1.4, and the acetic acid production reaches 10.0 mM; the cell density increases by 52%, the acetic acid production increases by 42.9%, and the strain activity retention rate increases by 33%, achieving efficient and continuous culture, improving growth rate and efficiency, extending the growth cycle, promoting vigorous metabolism, and fully activating and strengthening physiological functions.

[0051] The present invention discloses a growth culture medium for *Ruminococcus brucelli*: the raw materials include a carbon source, a nitrogen source, inorganic salts, a reducing agent, a redox indicator, beef granules, and bovine rumen fluid; wherein the carbon source is resistant starch with a concentration of 3.0–7.0 g / L; the nitrogen source has a concentration of 16.0–67.0 g / L; the reducing agent is L-cysteine ​​hydrochloride with a concentration of 0.1–0.7 g / L; the redox indicator is resazurin with a concentration of 0.001–0.007 g / L; the volume percentage of bovine rumen fluid is 5%–25%; and the mass-volume percentage of beef granules is 10.0–40.0 g / L.

[0052] It should be noted that, in this invention, the mass-to-volume ratio of beef granules refers to the ratio of the mass of beef granules to the total volume of the growth medium; the volume percentage of bovine rumen fluid refers to the ratio of the volume of bovine rumen fluid to the total volume of the growth medium. In the examples, the percentage of bovine rumen fluid is a volume percentage (V / V), meaning the added volume of bovine rumen fluid is a percentage of the total volume of the culture medium. The total volume of the culture medium refers to the sum of the volumes of the bovine rumen fluid and the aqueous solutions of all other culture medium components besides the bovine rumen fluid.

[0053] Resistant starch, due to its difficulty in being enzymatically broken down in the small intestine, is primarily used in existing technologies as a functional food or feed additive to regulate postprandial blood glucose, increase satiety, or serve as a non-specific prebiotic. Its core application lies in utilizing its "anti-digestion" physicochemical properties. This invention, however, leverages the unique probiotic properties of resistant starch for *Ruminococcus brucelli*, using it as a specific substrate and key carbon source for the efficient cultivation of this strain. By matching the unique enzyme system and metabolic pathways of *Ruminococcus brucelli*, the strain can fully exert its evolutionarily acquired core physiological function in vitro—efficiently degrading resistant starch and converting it into energy and acetic acid. Simultaneously, the slow-release carbon source properties of resistant starch effectively maintain the stability of the metabolic flux in the culture system, avoiding metabolic imbalances caused by rapid fermentation. In traditional culture media, strains often experience rapid growth followed by a rapid decline, with a short metabolic activity window. This invention, by adding resistant starch, provides the strain with a stable environment characterized by moderate metabolic stress and continuous nutrient supply.

[0054] In this invention, OD is an abbreviation for Optical Density. 600nm The OD value refers to the absorbance of the tested solution at a wavelength of 600 nm. Within a certain range, the absorbance value is directly proportional to the concentration of the light-absorbing substance in the solution; that is, the higher the bacterial content, the higher the OD value. 600nm The larger the value, the better. In this invention, a 0.5cm cuvette is used to test the OD. 600nm value.

[0055] The following are the descriptions of the activation and testing of the bacterial strains in this invention:

[0056] Strain activation (primary seed culture): Ruminococcus bromii strain ATCC 51896 or ATCC 27255, preserved in glycerol tubes at -80℃, was aseptically transferred to a container holding sterilized primary seed culture medium (CMC medium) at an inoculation rate of 10% (v / v). The culture was then incubated statically in a 37℃ anaerobic glove box for 10–20 hours to obtain activated primary seed culture.

[0057] Seed amplification (secondary seed culture): The above primary seed culture was transferred at an inoculum rate of 3% (v / v) to a container containing sterilized CMC medium. The mixture was then incubated statically in an anaerobic glove box at 37°C for 10–20 hours to obtain a highly viable secondary seed culture, which is the seed culture used for subsequent fermentation.

[0058] The method for culturing Ruminococcus brucelli using the growth medium of the present invention is as follows: Ruminococcus brucelli bacterial suspension is inoculated into the growth medium of the present invention at a volume of 3% (v / v) and anaerobic static culture at 30-40°C for no less than 20 hours.

[0059] Cell density determination: During fermentation, samples were taken every 6 hours to measure OD. 600nm The growth curves of Ruminococcus brevicornu ATCC51896 in different resistant starch media were plotted.

[0060] Starch utilization rate was determined after 48 hours of fermentation: the starch utilization rate was calculated by measuring the total sugar content of different starch samples before and after fermentation. The total sugar content in the culture was determined by the phenol-sulfuric acid assay.

[0061] pH determination after 48 hours of fermentation: During the fermentation process, pH values ​​were measured every 12 hours, and pH curves of Ruminococcus brucelli ATCC51896 in optimized culture media with different resistant starches were plotted.

[0062] Determination of acetic acid after 48 h of fermentation: The growth of Ruminococcus brucelli mainly produces acetic acid, but not propionic or butyric acid. The amount of acetic acid produced was measured by sampling after 48 h of fermentation. The acetic acid production of Ruminococcus brucelli ATCC51896 in optimized media with different resistant starches was plotted.

[0063] Example 1:

[0064] A culture medium formula for *Ruminococcus brucelli* is as follows: 5 g / L potato resistant starch; 30.0 g / L casein peptone; 10 g / L beef extract; 5.0 g / L yeast extract; 5.0 g / L K₂HPO₄; 0.5 g / L L-cysteine ​​hydrochloride; 0.001 g / L resazurin; 10.0 g / L beef granules; and 30% (v / v) bovine rumen fluid. Here, 30% (v / v) refers to the addition of bovine rumen fluid at 30% (v / v) of the total culture medium volume.

[0065] In preparation, all materials in the culture medium formulation were dissolved in water, and the pH was adjusted to 6.5 with NaOH aqueous solution to obtain the growth medium for Ruminococcus brucelli. The solution was heated to boiling, and the oxygen in the solution was replaced with nitrogen for 10 minutes to obtain the anaerobic culture medium, which was then sterilized at 121°C for 20 minutes before use.

[0066] Example 2:

[0067] A culture medium formula for *Ruminococcus brucelli* is as follows: 5 g / L banana resistant starch; 30.0 g / L casein peptone; 10 g / L beef extract; 5.0 g / L yeast extract; 5.0 g / L K2HPO4; 0.5 g / L L-cysteine ​​hydrochloride; 0.001 g / L resazurin; 30% (V / V) bovine rumen fluid; and 10.0 g / L beef granules.

[0068] The specific preparation method is the same as in Example 1, except that the oxygen in the solution is replaced with nitrogen for 20 minutes.

[0069] Example 3:

[0070] A culture medium formula for *Ruminococcus brucelli* is as follows: Hylon VIII 5 g / L; tryptone 30.0 g / L; beef extract 10 g / L; yeast extract 5.0 g / L; K2HPO4 5.0 g / L; L-cysteine ​​hydrochloride 0.5 g / L; resazurin 0.001 g / L; bovine rumen fluid 30% (V / V); and beef granules 10.0 g / L.

[0071] The specific preparation method is the same as in Example 1, except that the oxygen in the solution is replaced with nitrogen for 15 minutes.

[0072] Example 4:

[0073] A culture medium formula for *Ruminococcus brucelli* is as follows: Hylon VII 5 g / L; tryptone 30.0 g / L; beef extract 10 g / L; yeast extract 5.0 g / L; K2HPO4 5.0 g / L; L-cysteine ​​hydrochloride 0.5 g / L; resazurin 0.001 g / L; bovine rumen fluid 30% (V / V); and beef granules 10.0 g / L.

[0074] The specific preparation method is the same as in Example 1, except that the oxygen in the solution is replaced with nitrogen for 10 minutes.

[0075] Measurements in Examples 1-4:

[0076] Cell density determination: During fermentation, samples were taken every 6 hours to measure OD. 600nm The growth curves of *Ruminococcus brevicornu* ATCC51896 in different resistant starch media were plotted. Figure 1 .Depend on Figure 1 It was found that *Ruminococcus brucelli* entered the stationary phase after 24 hours of cultivation in optimized culture media using different resistant starches as substrates. At a cultivation time of 24 hours, the OD value measured using banana resistant starch as the substrate was... 600nm The value was 1.4, and the OD was measured when potato starch was used as the substrate. 600nm The value is 1.5, and the OD measured with Hylon VIII as the substrate is... 600nm The value is 1.4, and the OD is measured with Hylon VII as the substrate. 600nm The value was 1.3, demonstrating good growth effects in different resistant starches. Furthermore, using different resistant starches as substrates, the stationary phase was significantly prolonged, entering the decline phase at 48 hours, indicating that the addition of resistant starch to the culture medium can maintain the activity of the strain for a long time. This suggests that resistant starch, as a slow-release carbon source, can provide a continuous and stable energy supply to the bacteria, effectively delaying the onset of the decline phase.

[0077] Starch utilization rate determination after 48 hours of fermentation: Starch utilization rate was calculated by measuring the total sugar content before and after fermentation of different starch samples. The total sugar content in the culture was determined by the phenol-sulfuric acid assay method. Figure 2 .Depend on Figure 2 It can be seen that Ruminococcus brucelli degraded different resistant starches in the culture medium, with a degradation rate of 80%, achieving efficient degradation of resistant starches.

[0078] pH determination after 48 hours of fermentation: pH values ​​were measured every 12 hours during fermentation. pH curves of *Ruminococcus brucelli* ATCC51896 in optimized media with different resistant starches were plotted. (See attached image) Figure 3 .Depend on Figure 3 It can be seen that the pH value of the optimized culture medium with different resistant starch substrates all showed a significant decreasing trend. Furthermore, the pH value gradually decreased with the extension of the culture time and gradually stabilized after 36 hours, indicating that the addition of resistant starch promoted the growth rate of Ruminococcus brucelli ATCC51896 and produced a larger amount of acidic metabolites (such as short-chain fatty acids), which directly reflects the significant enhancement of its metabolic activity.

[0079] Determination of acetic acid after 48 hours of fermentation: *Ruminococcus brucelli* primarily produces acetic acid, with no propionic or butyric acid. After 48 hours of fermentation, samples were taken to determine the amount of acetic acid produced. The acetic acid production of *Ruminococcus brucelli* ATCC51896 in optimized media with different resistant starches was plotted. Figure 4 At 48°C, acetic acid production reached 10.0 mM. This further verified that the addition of resistant starch to the culture medium, replacing the original carbon source, promoted the growth rate of *Ruminococcus brucelli* and the production of beneficial metabolites. Acetic acid fermentation is the main energy acquisition pathway for *Ruminococcus brucelli*. High acetic acid production signifies the efficient operation of its carbon and energy flows, providing direct energy support for high-density cell growth and maintenance of high activity.

[0080] The test results of Examples 1-4 show that different resistant starches have no significant effect on the growth of Ruminococcus brucelli.

[0081] Example 5

[0082] A culture medium formula for *Ruminococcus brucelli* is as follows: 3 g / L potato resistant starch; 30.0 g / L casein peptone; 10 g / L beef extract; 5.0 g / L yeast extract; 5.0 g / L K2HPO4; 0.5 g / L L-cysteine ​​hydrochloride; 0.001 g / L resazurin; 30% (V / V) bovine rumen fluid; and 10.0 g / L beef granules.

[0083] Example 6

[0084] A culture medium formula for *Ruminococcus brucelli* is as follows: 5 g / L potato resistant starch; 30.0 g / L casein peptone; 10 g / L beef extract; 5.0 g / L yeast extract; 5.0 g / L K2HPO4; 0.5 g / L L-cysteine ​​hydrochloride; 0.001 g / L resazurin; 30% (V / V) bovine rumen fluid; and 10.0 g / L beef granules.

[0085] Example 7

[0086] A culture medium formula for *Ruminococcus brucelli* is as follows: 7 g / L potato resistant starch; 30.0 g / L casein peptone; 10 g / L beef extract; 5.0 g / L yeast extract; 5.0 g / L K2HPO4; 0.5 g / L L-cysteine ​​hydrochloride; 0.001 g / L resazurin; 30% (V / V) bovine rumen fluid; and 10.0 g / L beef granules.

[0087] The preparation methods for Examples 5-7 are the same as in Example 1. *Ruminococcus brucellosis* culture was inoculated at a rate of 3% (V / V) into 30 mL anaerobic tubes containing different concentrations of potato resistant starch (Examples 5-7). The tubes were then incubated statically in a 37°C anaerobic glove box for 48 h, and the cell density was measured. The test results for Examples 5-7 are as follows: Figure 5 and Figure 6 As shown.

[0088] Growth curves of Ruminococcus brucelli under different resistant starch concentrations are as follows: Figure 5 As shown, within the test range of 3.0–7.0 g / L, when resistant starch was used as the carbon source, the bacterial cells entered the stationary phase within 24 hours, and the activity could be stably maintained until 48 hours. The final cell density (OD) was [data missing]. 600 The OD values ​​remained above 1.3, confirming that resistant starch effectively supports the vigorous growth and sustained metabolic activity of *Ruminococcus brucelli*. The bacterial cell density reached its peak (OD) when the potato resistant starch concentration was 5 g / L. 600 =1.4). After the concentration exceeded this value, the cell density did not increase significantly, indicating that in this optimized culture medium system, 5 g / L is close to the carbon source saturation point, and other nutrients or culture environment become new limiting factors. Continuing to increase the starch concentration alone cannot break through this new growth bottleneck.

[0089] The amount of acetic acid produced after 48 hours of fermentation was determined (see...). Figure 6 It was found that at all effective concentrations, the yield of acetic acid remained stable above 10 mM after 48 h of culture, significantly higher than that in traditional CMC medium (Comparative Example 1). This reveals a deep match between resistant starch and the metabolic characteristics of *Ruminococcus brucelli*. The dense structure of resistant starch is slowly degraded by the enzyme system unique to *Ruminococcus brucelli*, avoiding the rapid consumption and waste of carbon sources commonly found in fast carbon sources, and ensuring a continuous and directional flow of carbon to the acetic acid fermentation pathway. The culture medium, with resistant starch as its core, precisely matches the unique nutritional requirements of *Ruminococcus brucelli* as a "professional resistant starch-degrading bacterium," allowing it to reach its maximum growth potential in vitro.

[0090] Example 8

[0091] A culture medium formula for *Ruminococcus brucelli* is as follows: 5 g / L potato resistant starch; 5.0 g / L K₂HPO₄; 0.5 g / L L-cysteine ​​hydrochloride; 0.001 g / L resazurin; 30% bovine rumen fluid (v / v); 10.0 g / L beef granules; and a nitrogen source. The nitrogen source is one of the nitrogen sources listed in conditions 1-9 below.

[0092] Nitrogen source condition 1: 30.0 g / L of beef extract powder;

[0093] Nitrogen source condition 2: tryptone 30.0 g / L;

[0094] Nitrogen source condition 3: Yeast extract 30.0 g / L;

[0095] Nitrogen source condition 4: 15.0 g / L beef extract powder, 15.0 g / L fish peptone;

[0096] Nitrogen source condition 5: tryptone 15.0 g / L, fish peptone 15.0 g / L;

[0097] Nitrogen source condition 6: 10.0 g / L tryptone, 5.0 g / L beef extract, and 1.0 g / L yeast extract;

[0098] Nitrogen source condition 7: tryptone 20.0 g / L, beef extract 10.0 g / L, yeast extract 3.0 g / L;

[0099] Nitrogen source condition 8: tryptone 30.0 g / L, beef extract 15.0 g / L, yeast extract 5.0 g / L;

[0100] Nitrogen source condition 9: 40.0 g / L casein peptone, 20.0 g / L beef extract, and 7.0 g / L yeast extract.

[0101] The growth curve and acetic acid production of *Ruminococcus brucelli* after culture are shown below. Figure 7 , Figure 8 and Figure 9 As shown, under all the above nitrogen source conditions, *Ruminococcus brevicornu* ATCC 51896 exhibited a growth and metabolic pattern highly consistent with the aforementioned examples: the cells entered the stationary phase after approximately 24 hours of culture, maintained activity until 48 hours, and the final cell density (OD) reached a certain level. 600 The nitrogen content consistently remained above 1.3. After 48 hours of cultivation, the yield of acetic acid, a characteristic beneficial metabolite, in the fermentation broth consistently remained above 10 mM, significantly superior to the control group cultured in traditional CMC medium. Despite significant changes in the type (single or combined) of nitrogen sources, the proportion of nitrogen source combinations, and even some nitrogen source components (such as replacement with fish peptone), the high cell density and high acetic acid yield of *Ruminococcus brucelli* remained consistently high and excellent. This demonstrates that in the culture medium system of this invention, resistant starch is an irreplaceable and decisive key carbon source and functional inducing factor driving the high-level growth and functional metabolism of *Ruminococcus brucelli*. The nitrogen source in this system primarily provides basic growth support, and changes in its specific composition did not significantly affect the core functional advantages conferred by resistant starch. The culture medium framework centered on resistant starch has good tolerance for nitrogen source conditions. This provides flexibility for the practical application of this invention; while ensuring the core effect, the nitrogen source can be appropriately adjusted according to cost, raw material availability, and other factors, enhancing the industrial application potential of the technology.

[0102] Example 9:

[0103] A culture medium formulation for *Ruminococcus brucelli* is as follows: 5 g / L potato resistant starch; 30.0 g / L tryptone; 15.0 g / L beef extract; 5.0 g / L yeast extract; buffer salt; 0.5 g / L L-cysteine ​​hydrochloride; 0.001 g / L resazurin; 30% (v / v) bovine rumen fluid; and 10.0 g / L beef granules. The buffer salt is selected from the buffer salt conditions 1-8 below.

[0104] Buffer salt condition 1: K2HPO4 1.0 g / L;

[0105] Buffer salt condition 2: K2HPO4 3.0 g / L;

[0106] Buffer salt condition 3: K2HPO4 5.0 g / L;

[0107] Buffer salt condition 4: K2HPO4 7.0 g / L;

[0108] Buffer salt condition 5: KH2PO4 5.0 g / L;

[0109] Buffer salt condition 6: (NH4)2SO4 5.0 g / L;

[0110] Buffer salt condition 7: MgSO4·7H2O 5.0 g / L;

[0111] Buffer salt conditions 8: K₂HPO₄ 0.50 g / L; KH₂PO₄ 0.45 g / L; (NH₄)₂SO₄ 0.9 g / L; MgSO₄·7H₂O 0.09 g / L;

[0112] The growth curve and acetic acid production of *Ruminococcus brucelli* after culture are shown below. Figure 10 , Figure 11 and Figure 12 As shown, in all tests under buffered salt conditions 1 to 8, *Ruminococcus brucelli* exhibited the same superior growth and metabolic pattern as in the above examples, namely, a high cell density (OD) after 24 hours of culture. 600 The activity remained stable above 1.3 for up to 48 hours, and the acetic acid yield remained at a high level of over 10 mM. This indicates that within the culture medium framework centered on resistant starch, the culture effect showed significant tolerance to the specific types and combinations of buffer salts.

[0113] Example 10:

[0114] A culture medium formula for *Ruminococcus brucelli* is as follows: 5 g / L potato resistant starch; 30.0 g / L casein peptone; 15.0 g / L beef extract; 5.0 g / L yeast extract; 5.0 g / L K2HPO4; 0.1 g / L L-cysteine ​​hydrochloride; 0.001 g / L resazurin; 30% (V / V) bovine rumen fluid; and 10.0 g / L beef granules.

[0115] Example 11:

[0116] A culture medium formula for *Ruminococcus brucelli* is as follows: 5 g / L potato resistant starch; 30.0 g / L casein peptone; 15 g / L beef extract; 5.0 g / L yeast extract; 5.0 g / L K2HPO4; 0.3 g / L L-cysteine ​​hydrochloride; 0.003 g / L resazurin; 30% (V / V) bovine rumen fluid; and 20.0 g / L beef granules.

[0117] Example 12:

[0118] A culture medium formula for *Ruminococcus brucelli* is as follows: 5 g / L potato resistant starch; 30.0 g / L casein peptone; 15 g / L beef extract; 5.0 g / L yeast extract; 5.0 g / L K2HPO4; 0.5 g / L L-cysteine ​​hydrochloride; 0.005 g / L resazurin; 30% (V / V) bovine rumen fluid; and 30.0 g / L beef granules.

[0119] Example 13:

[0120] A culture medium formula for *Ruminococcus brucelli* is as follows: 5 g / L potato resistant starch; 30.0 g / L casein peptone; 15 g / L beef extract; 5.0 g / L yeast extract; 5.0 g / L K2HPO4; 0.7 g / L L-cysteine ​​hydrochloride; 0.007 g / L resazurin; 30% (V / V) bovine rumen fluid; and 40.0 g / L beef granules.

[0121] The effects of different growth factor concentrations on the growth and acetic acid production of *Ruminococcus brucelli* ATCC51896 in Examples 10-13 are shown in the figures below. Figure 13 and Figure 14 Within the tested concentration range (L-cysteine ​​hydrochloride: 0.1-0.7 g / L; resazurin: 0.001-0.007 g / L; beef granules: 10.0-40.0 g / L), *Ruminococcus brucelli* showed good growth, and the bacterial density (OD) after 24 h of culture was [data missing]. 600 All reached high levels. *Ruminococcus brucelli* exhibited the same excellent growth and metabolic pattern as in the above examples, i.e., high cell density (OD) after 24 hours of culture. 600The concentration remained stable above 1.3, and the activity was maintained for up to 48 hours, with acetic acid production remaining at a high level of over 10 mM. In the culture medium system of this invention, which uses resistant starch as the core, the above-mentioned growth factors can effectively support cell growth over a wide concentration range, demonstrating the robustness of the culture medium formulation.

[0122] Example 14:

[0123] A culture medium formula for *Ruminococcus brucelli* is as follows: 5 g / L potato resistant starch; 30.0 g / L casein peptone; 15 g / L beef extract; 5.0 g / L yeast extract; 5.0 g / L K2HPO4; 0.5 g / L L-cysteine ​​hydrochloride; 0.005 g / L resazurin; 10.0% (V / V) bovine rumen fluid; and 30.0 g / L beef granules.

[0124] Example 15:

[0125] A culture medium formula for *Ruminococcus brucelli* is as follows: 5 g / L potato resistant starch; 30.0 g / L casein peptone; 15 g / L beef extract; 5.0 g / L yeast extract; 5.0 g / L K2HPO4; 0.5 g / L L-cysteine ​​hydrochloride; 0.005 g / L resazurin; 15.0% (V / V) bovine rumen fluid; and 30.0 g / L beef granules.

[0126] Example 16:

[0127] A culture medium formula for *Ruminococcus brucelli* is as follows: 5 g / L potato resistant starch; 30.0 g / L casein peptone; 15 g / L beef extract; 5.0 g / L yeast extract; 5.0 g / L K2HPO4; 0.5 g / L L-cysteine ​​hydrochloride; 0.005 g / L resazurin; 20.0% (V / V) bovine rumen fluid; and 30.0 g / L beef granules.

[0128] Example 17:

[0129] A culture medium formula for *Ruminococcus brucelli* is as follows: 5 g / L potato resistant starch; 30.0 g / L casein peptone; 15 g / L beef extract; 5.0 g / L yeast extract; 5.0 g / L K2HPO4; 0.5 g / L L-cysteine ​​hydrochloride; 0.005 g / L resazurin; 25.0% (V / V) bovine rumen fluid; and 30.0 g / L beef granules.

[0130] The effects of different amounts of bovine rumen fluid added in Examples 14-17 on the growth of *Ruminococcus brevicornu* ATCC51896 and the amount of acetic acid produced are shown in the following examples. Figure 15 and Figure 16The total volume of the culture medium refers to the sum of the volumes of bovine rumen fluid and the aqueous solutions of all other culture medium components besides bovine rumen fluid. Inoculation was performed at a rate of 3% (V / V) into 30 mL anaerobic tubes containing different concentrations of bovine rumen fluid. The tubes were incubated statically in a 37°C anaerobic glove box for 24 h, and then the bacterial cell density was measured. With different amounts of bovine rumen fluid added, *Ruminococcus brucelli* exhibited the same excellent growth and metabolic pattern as in the above examples. When the amount of bovine rumen fluid added was 15%, the bacterial cell density of *Ruminococcus brucelli* reached its peak, with an OD value of [missing value]. 600nm The value was 1.4, and the bacterial concentration did not increase significantly as the amount of bovine rumen fluid added gradually increased. This is because the OD value was 1.4 at 20% and 15% bovine rumen fluid concentrations. 600nm The values ​​are not significantly different.

[0131] Comparative Example 1

[0132] Replace the optimized culture medium in the anaerobic tubes with CMC culture medium.

[0133] Cell density determination: During fermentation, samples were taken every 6 hours to measure OD. 600nm The growth curves of *Ruminococcus brevicornus* ATCC51896 in optimized and CMC media were plotted. Figure 17 .

[0134] Depend on Figure 17 It can be seen that *Ruminococcus brucelli* ATCC51896 entered the stationary phase after 24 hours of culture in both the media of Examples 1-4 and CMC medium. During this phase, the cell number essentially reached its maximum and remained stable. The OD value was measured after 24 hours of culture in CMC medium. 600nm The value was 0.8-1.0. After culturing in the media of Examples 1-4 for 24 hours, the OD600nm value was measured to be 1.3-1.5. The cell density of the culture media of Examples 1-4 (OD600 = 1.3-1.5) was higher than that of the CMC medium (OD600 = 1.3-1.5). 600 =0.8-1.0) significantly increased by more than 50% (up to 52%). Furthermore, in CMC medium, the cells entered the death phase within 36 hours; while in the optimized medium, cell growth activity was maintained for a longer period, not entering the death phase until 48 hours. This indicates that the medium of the present invention not only increases cell density but also significantly prolongs the exponential growth phase and stationary phase of the cells, extending the effective activity retention time of the strain by approximately 33%. This provides a wider process window and higher time-yield efficiency for large-scale fermentation culture.

[0135] After 48 hours of incubation in both culture media: the pH value of the culture medium was measured. Figure 18 ) and acetic acid production ( Figure 19 ),Depend on Figure 18It can be seen that the pH value stabilized in CMC medium after 24 hours of incubation, while it remained stable in the mediums of Examples 1-4. Figure 3 The pH value in the medium stabilized after 36 hours of incubation, and was significantly lower than that in CMC medium. This lower stable pH is a typical characteristic of the cells continuing vigorous carbohydrate fermentation metabolism. Figure 19 It can be seen that acetic acid is produced rapidly in CMC medium after 12 hours of cultivation, and then gradually levels off after 24 hours, with a production amount of only 7.0 mM. However, in the medium of this invention... Figure 4 As the culture time increased, the production of acetic acid gradually increased, reaching 10.0 mM, an increase of 42.9%. This increase in acetic acid production not only verifies the enhanced metabolic activity of the bacteria but also demonstrates the outstanding effect of the culture medium of this invention in promoting the accumulation of beneficial metabolites in a targeted manner. The increase in acetic acid production, along with improvements in cell density and activity period, is not an isolated phenomenon but rather a comprehensive reflection of the synergistic effects achieved by optimizing the carbon source structure (using resistant starch), balancing nutrient supply, and simulating key ecological factors in the culture medium of this invention.

[0136] The culture medium of this invention uses resistant starch as the core carbon source. This medium can efficiently activate and support the function of the resistant starch-degrading enzyme system unique to *Ruminococcus brucelli*, directing its metabolic flux fully towards energy acquisition and product synthesis pathways centered on acetic acid fermentation. Compared to traditional culture media using easily degradable carbon sources (such as glucose and soluble starch), this invention avoids rapid depletion of carbon sources and metabolic leakage, thereby enabling *Ruminococcus brucelli* to achieve continuous and efficient energy metabolism and biosynthesis.

[0137] The culture medium of this invention is beneficial for obtaining high biomass, high metabolic activity, and functionally enhanced (high acetic acid production) Ruminococcus brevicornus.

[0138] The *Ruminococcus brucelli* ATCC51896 exhibited high cell concentration, good growth, and significantly increased yield of metabolic beneficiaries. The culture medium of this invention provides more suitable nutrients and a more optimal growth environment for *Ruminococcus brucelli* ATCC51896, thereby promoting its growth and metabolism.

[0139] Comparative Example 2

[0140] The carbon source in the culture medium of the present invention in the anaerobic tube of Example 15 was replaced with soluble potato starch, and the concentration of soluble potato starch was set (1 g / L, 3 g / L, 5 g / L, 7 g / L), while other conditions or parameters were the same as in Example 15.

[0141] Cell density determination: During fermentation, samples were taken every 6–12 hours to measure OD. 600nmThe growth curves of *Ruminococcus brevicornu* ATCC51896 under different soluble starch conditions were plotted. Figure 20 .

[0142] Determination of acetic acid after 48 hours of fermentation: Samples were taken after 48 hours of fermentation to detect the amount of acetic acid produced. The acetic acid production of *Ruminococcus brucelli* ATCC51896 in different soluble starch media was plotted. Figure 21 .

[0143] In soluble starch medium, even at its optimal concentration (5 g / L), the highest cell density (OD) reached during the stationary phase. 600 The OD200 of the culture medium of this invention is only 1.1. However, the optimized culture medium of this invention achieves a stable cell density of over 1.3 (peak OD200) within the same culture time. 600 =1.4), biomass increased by more than 27%. In the medium using soluble starch as the carbon source, the cell growth cycle was shortened, entering the death phase after 36 hours of culture. In contrast, in the medium of this invention, cell growth activity was maintained for a longer period, entering the death phase only after 48 hours, extending the effective active growth period by approximately 33%. In the soluble starch medium, the final yield of the characteristic beneficial metabolite acetic acid was only 7.0 mM. However, in the medium of this invention, the acetic acid yield remained stable at over 10.0 mM, and the metabolite yield increased by more than 42%.

[0144] Unlike soluble starch, resistant starch, as a slow-release carbon source, has a dense structure that can be slowly and continuously degraded by the specific enzyme system of *Ruminococcus brucelli*. This avoids the carbon waste and metabolic imbalance caused by the instantaneous oversupply of rapid carbon sources, allowing energy and carbon to be used more persistently and specifically for cell growth and acetic acid synthesis. Therefore, the resistant starch of this invention overcomes the technical problems of low *Ruminococcus brucelli* biomass and low culture efficiency in existing technologies, providing an effective solution for large-scale production of this cell.

[0145] Comparative Example 3

[0146] The concentration of potato resistant starch in the culture medium of the anaerobic tube in Example 15 was replaced with 0.3 g / L, 0.5 g / L, 0.7 g / L, 9 g / L, 12 g / L, and 14 g / L, while other conditions or parameters remained the same as in Example 15. Specifically, the concentration of potato resistant starch in the culture medium of this invention was set to six gradients, both below the effective range (0.3, 0.5, 0.7 g / L) and above the effective range (9, 12, 14 g / L), and compared with the effective concentration (5 g / L) in the example.

[0147] Growth curves of *Ruminococcus brucelli* ATCC51896 grown under different concentrations of resistant starch are shown below. Figure 22As shown, at the concentrations of this invention (3.0-7.0 g / L), the cell density rapidly increased and reached a plateau (OD) within 24 hours. 600 Compared to the concentration group (≈1.4), the low concentration group (0.3-0.7 g / L) showed significantly slower cell growth, with insufficient biomass accumulation throughout the culture period, resulting in a final cell density far below 1.0. This indicates that when the resistant starch concentration is below 1.0 g / L, the carbon source becomes a key limiting factor for cell proliferation. The high concentration group (9-14 g / L) also did not show increased cell growth with increasing concentration, with a final density reaching only about 1.2, still significantly lower than the peak value at the protected concentration of this invention. This suggests that concentrations exceeding 7.0 g / L may, due to changes in osmotic pressure, substrate inhibition, or accumulation of metabolic byproducts, actually inhibit cell growth.

[0148] Acetic acid production of *Ruminococcus brevicornu* ATCC51896 grown under different concentrations of resistant starch, as follows: Figure 23 As shown in Comparative Example 3, along with growth restriction, acetic acid production also decreased simultaneously, failing to reach a high level of production above 10 mM. This indicates that insufficient or unbalanced carbon source supply directly weakens the core metabolic function of Ruminococcus brucelli.

[0149] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A growth culture medium for Ruminococcus brucelli, characterized in that, The raw materials include carbon source, nitrogen source, inorganic salt, reducing agent, redox indicator, beef granules and bovine rumen juice; The carbon source is resistant starch with a concentration of 3.0–7.0 g / L; the nitrogen source has a concentration of 16.0–67.0 g / L; the reducing agent is L-cysteine ​​hydrochloride with a concentration of 0.1–0.7 g / L; and the redox indicator is resazurin with a concentration of 0.001–0.007 g / L. The volume percentage of the bovine rumen fluid is 5% to 25%; the mass-volume ratio of the cooked beef granules is 10.0 to 40.0 g / L.

2. The growth medium for *Ruminococcus brucelli* according to claim 1, characterized in that, The resistant starch is selected from at least one of potato resistant starch, banana resistant starch, Hylon VIII, and Hylon VII.

3. The growth culture medium for *Ruminococcus brucelli* according to claim 1, characterized in that, The nitrogen source comprises one or more of beef extract, tryptone, and yeast extract.

4. The growth medium for *Ruminococcus brucelli* according to claim 3, characterized in that, The nitrogen source also includes fish peptone, which together with beef extract and / or casein peptone constitutes the nitrogen source.

5. The growth medium for *Ruminococcus brucelli* according to claim 3 or 4, characterized in that, The concentrations of the components in the nitrogen source are as follows: beef extract 5.0–30.0 g / L, tryptone 10.0–40.0 g / L, yeast extract 1.0–30.0 g / L; fish peptone 10.0–20.0 g / L.

6. The growth medium for *Ruminococcus brucelli* according to claim 1, characterized in that, The inorganic salt is selected from K2HPO4, KH2PO4, (NH4)2SO4 or MgSO4·7H2O.

7. A method for preparing a growth culture medium for Ruminococcus brucelli, characterized in that, All raw materials of the growth medium of Ruminococcus brucelli according to any one of claims 1 to 4 are dissolved in water to obtain a culture medium solution; the culture medium solution is subjected to anaerobic treatment to obtain the growth medium of Ruminococcus brucelli.

8. The method for preparing the growth culture medium for *Ruminococcus brucelli* according to claim 7, characterized in that, The anaerobic treatment involves heating the culture medium solution to boiling and replacing the oxygen in the culture medium solution with nitrogen for 10-20 minutes.

9. The method for preparing the growth culture medium for *Ruminococcus brucelli* according to claim 7, characterized in that, The growth medium for *Ruminococcus brucelli* also includes sterilization.

10. The method for preparing the growth culture medium for *Ruminococcus brucelli* according to claim 7, characterized in that, The sterilization process involves heating the anaerobic culture medium solution to 117-121°C for 20-40 minutes using a high-pressure steam method.

Images