Screening and application of strain with high-yield gamma-aminobutyric acid and xanthine degradation characteristics

Abstract

The invention discloses screening and application of a strain with the characteristics of high yield of gamma-aminobutyric acid and degradation of xanthine, the strain is Heynickia coagulans LPB-13, has been preserved in China General Microbiological Culture Collection Center (CGMCC) on December 29, 2025, the preservation number is CGMCC No: 37230, and the classification number is Heynickia coagulans. The fermentation liquor of the strain LPB-13 can rapidly degrade xanthine within 4 hours, the degradation rate is 68.16%, and 9.9 g / L gamma-aminobutyric acid can be generated after 72 hours of fermentation. Therefore, the hahndersella coagulans LPB-13 disclosed by the invention breaks through the application limitation of a single-function microbial agent, and has important practical application value and industrialization prospect in research and development of a compound xanthine degradation microbial agent.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to the screening and application of a strain that exhibits both high production of γ-aminobutyric acid (GABA) and xanthine degradation characteristics. Background Technology

[0002] Hyperuricemia ( Hyperuricemia, HUA Hyperuricemia is a chronic clinical syndrome caused by disordered purine metabolism and abnormal uric acid excretion, resulting in a significantly higher than normal blood uric acid level. With changes in dietary levels, the prevalence of hyperuricemia is increasing year by year, and it often leads to chronic diseases such as diabetes, hypertension, and urate nephropathy. Long-term high uric acid levels can cause a series of adverse consequences for the blood vessels, heart, and kidneys.

[0003] Currently, clinical treatment for hyperuricemia involves two approaches: exogenous medication and restriction of high-purine food intake. Most uric acid-lowering drugs rely on xanthine oxidase (…). Xanthine Oxidase, XO Uric acid-lowering drugs include purine inhibitors (allopurinol) and uricosuric drugs (benzbromarone). However, these drugs are prone to causing allergic reactions, gastrointestinal reactions, liver damage, and other toxic side effects, and are poorly tolerated by patients. Long-term use can lead to serious adverse reactions. Furthermore, due to the abundance of high-purine food sources, it is difficult to control hyperuricemia through dietary restriction alone. At the same time, flavor enhancers in food additives are also purine substances, and patients with hyperuricemia must also control their intake of flavor enhancers, which significantly impacts their dietary habits of pursuing good flavor. Therefore, it is urgent to find new ways to lower uric acid levels in the blood of people with hyperuricemia.

[0004] Gamma-aminobutyric acid (GABA), also known as amino acid, is a naturally occurring amino acid widely found in plants, animals, and microorganisms. It is derived from glutamate through the catalytic conversion of glutamate by glutamate decarboxylase (GAD). In the central nervous system of human mammals, this non-protein-based natural amino acid is a key inhibitory neurotransmitter, inhibiting excessive excitation of the central nervous system, thereby promoting relaxation and relieving nervous tension, and improving symptoms such as neurasthenia and insomnia. Therefore, GABA is an important ingredient in sleep aids. Currently, there are two main methods for synthesizing GABA: chemical and biological methods. Biological methods include plant enrichment and microbial fermentation. Microorganisms, due to their short growth cycle and rapid reproduction, have been widely used in GABA production in recent years. Compared to existing GABA products, GABA-rich foods prepared through microbial fermentation are naturally produced and do not require the addition of chemically purified GABA, making them greener, healthier, and pollution-free, with promising application prospects.

[0005] Therefore, screening for Heinrich's coagulans strains with multiple excellent functions is of great practical significance for the food and related health industries. Summary of the Invention

[0006] The purpose of this invention is to provide a strain with both high production of γ-aminobutyric acid (GABA) and xanthine degradation characteristics for screening and application.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of this invention provides a strain of *Hydrilla verticillata* LPB-13, which was deposited on December 29, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No:37230 and classification number *Hydrilla verticillata* (…). Heyndrickxia coagulans ).

[0008] A second aspect of the present invention provides a microbial agent containing the aforementioned Heinrichia coagulans LPB-13.

[0009] Further, the specific steps are as follows: the seed culture of *Hydroxypyrella coagulans* LPB-13 is inoculated into liquid culture medium at an inoculation rate of 2-6% v / v and cultured at 35-40℃ and 120-200 rpm for 10-72 h to prepare the culture.

[0010] Furthermore, the liquid culture medium is formulated as follows: 12.0 g / L beef extract, 12.0 g / L yeast extract, 22.0 g / L glucose, 2.0 g / L dipotassium hydrogen phosphate, 2.0 g / L triammonium citrate, 5.0 g / L sodium acetate, 1.0 mL / L Tween 80, 0.4 g / L MgSO4·7H2O, and 0.1 g / L MnSO4·H2O.

[0011] A third aspect of the present invention provides the use of the above-mentioned Heinrich's coagulating bacteria LPB-13 or the above-mentioned microbial agent in the production of γ-aminobutyric acid.

[0012] Furthermore, it was inoculated into a liquid culture medium containing 1-5% monosodium glutamate and fermented for 48-96 hours; the liquid culture medium was formulated as follows: beef extract powder 12.0 g / L, yeast extract powder 12.0 g / L, glucose 22.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, triammonium citrate 2.0 g / L, sodium acetate 5.0 g / L, Tween 80 1.0 mL / L, 0.4 g / L MgSO4·7H2O, and 0.1 g / L MnSO4·H2O.

[0013] The fourth aspect of the present invention provides the use of the above-mentioned Heinrich's coagulating bacteria LPB-13 or the above-mentioned microbial agents in the degradation of xanthine.

[0014] Furthermore, the microbial inoculum was centrifuged at 6000-8000 rpm for 2 min, the supernatant was discarded, an equal volume of xanthine solution was added, and the mixture was incubated at 35-40℃ and 120-200 rpm for 3-5 h.

[0015] The fifth aspect of the present invention provides the use of the above-mentioned Heinrich's coagulating bacteria LPB-13 or the above-mentioned microbial agents in the preparation of uric acid-lowering products.

[0016] The beneficial effects of this invention are as follows: This invention screened and obtained a bifunctional strain of Heinrich's coagulation bacterium (Hyndrix bacillus). Heyndrickxia coagulans LPB-13 strain can degrade xanthine within 4 hours, with a degradation rate of 68.16%. This has important practical significance for developing highly efficient microbial agents for degrading xanthine.

[0017] Furthermore, this strain LPB-13 also possesses the function of high-yield γ-aminobutyric acid (GABA), producing 9.9 g / L of GABA after 72 hours of fermentation, which can be used for the efficient preparation of GABA. GABA, as a functional active ingredient, has physiological effects such as regulating metabolism, soothing nerves, improving sleep, and assisting in blood pressure regulation, making it a high-quality additive widely used in the food industry. The *Hydrilla verruca coagulans* LPB-13 strain of this invention combines the dual functions of efficient xanthine degradation and high-yield GABA synthesis, overcoming the application limitations of single-function microbial agents. It has significant practical application value and industrialization prospects in the development of compound, multifunctional xanthine-degrading microbial agents and their application in the food and other fields. Attached Figure Description

[0018] Figure 1 This is a diagram showing the purified colony morphology of the strain of this invention.

[0019] Figure 2 This is a Gram staining image of the strain of this invention.

[0020] Figure 3 This is a graph showing the acid production capacity of the strain of this invention after 48 hours.

[0021] Figure 4 This is the phylogenetic tree of the strains of this invention.

[0022] Figure 5 This is a diagram showing the inoculation amount of the strain of this invention.

[0023] Figure 6 This is a diagram showing the nitrogen source optimization for the strain of this invention.

[0024] Figure 7 This is a diagram showing the optimized nitrogen source combination for the strain of this invention.

[0025] Figure 8 This is a diagram showing the optimized carbon source for the strain of this invention.

[0026] Figure 9 This is a diagram showing the optimized carbon source content of the strain of this invention.

[0027] Figure 10 This is an optimized diagram of inorganic salts for the strain of this invention.

[0028] Figure 11 This is the standard curve for γ-aminobutyric acid in Example 3.

[0029] Figure 12 This is the xanthine standard curve in Example 4. Detailed Implementation

[0030] The present invention will be further described in detail below through specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0031] Unless otherwise specified, all raw materials involved in the following embodiments of the present invention are commercially available.

[0032] Example 1 Isolation and Identification of Strains (1) Bacterial liquid enrichment Take 10 mL of beer sample (from fermented beer by local residents in Huai'an) and add an equal volume of MRS liquid medium. Shake thoroughly at 120 rpm for 8 h, centrifuge at 6000~8000 rpm for 15 min, discard the supernatant, add 10 mL of sterile PBS buffer solution (pH=7.0) to the precipitate and wash twice. Finally, resuspend the bacterial cells in 1 mL of 0.85% sterile physiological saline to obtain the enriched bacterial solution.

[0033] (2) Initial screening The MRS solid culture medium formulation used in this step is as follows: peptone 10.0 g / L, beef extract 8.0 g / L, yeast extract 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, triammonium citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate heptahydrate 0.2 g / L, manganese sulfate monohydrate 0.05 g / L, Tween 80 1.0 mL / L, and agar 15.0 g / L.

[0034] The formulation of MRS liquid medium containing xanthine is as follows: peptone 10.0 g / L, beef extract 8.0 g / L, yeast extract 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, triammonium citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate heptahydrate 0.2 g / L, manganese sulfate monohydrate 0.05 g / L, Tween 80 1.0 mL / L, xanthine 0.1 g / L; The formula for the MRS-bromocresol purple primary screening medium is as follows: peptone 10.0 g / L, beef extract 8.0 g / L, yeast extract 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, triammonium citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate heptahydrate 0.2 g / L, manganese sulfate monohydrate 0.05 g / L, Tween 80 1.0 mL / L, and bromocresol purple 0.02 g / L; The enriched bacterial solution obtained in step (1) above was serially diluted with 0.85% physiological saline, and 100 μL of each solution was taken. -5 ~10 -7The diluted solution was spread on MRS solid medium and placed in a constant temperature incubator. It was incubated upright at 37°C for 10 min, the plate was dried, and then inverted for further incubation. The growth of colonies in the plate was observed and recorded every 48 h. After the colonies grew, typical single colonies (round or irregularly round, milky white, opaque, with a dense texture, smooth edges, rough, dry and dull surface, thin and small colonies) were picked from the plate and inoculated into MRS liquid medium containing xanthine. The medium was incubated overnight at 37°C and 120 rpm. One loopful of bacterial solution was streaked on MRS-bromocresol purple primary screening medium to obtain the primary screening plate.

[0035] (3) Secondary screening The formulation of the MRS bromocresol purple-xanthine secondary screening plate involved in this step is as follows: 100 μL of xanthine aqueous solution (300 mg / L) is spread on the MRS-bromocresol purple primary screening medium to obtain the secondary screening medium.

[0036] Single colonies obtained from the initial screening were inoculated into a secondary screening culture. Based on colony size, color, shape, edge expansion, and surface smoothness, the single colonies on the secondary screening plates were preliminarily classified, and healthy single colonies from each category were selected. The secondary screening medium was then streaked until a single colony appeared, yielding the secondary screening strain. Figure 1 The final selected strain was named LPB-13 and preserved in glycerol tubes.

[0037] (4) Identification of strains The selected strain LPB-13 was subjected to Gram staining for secondary screening, and preliminary identification was performed based on microscopic examination. Results are as follows: Figure 2 As shown, the Gram staining result was positive, and the colony morphology in the microscopic field was uniform, with each cell appearing as a rod shape.

[0038] Further, the growth curve of strain LPB-13 in MRS liquid medium at 37℃ and 120 rpm for 48 h was plotted and its acid production capacity was recorded. This strain exhibited strong acid production capacity within 48 h. Figure 3 ).

[0039] Genomic DNA was extracted from strain LPB-13 for fragment amplification (reaction system and procedure are shown in Tables 1 and 2). Universal primers for the 16S rDNA gene were used: 27F: 5-AGAGTTTGATCCTGGCTCAG-3, 1492R: 5-CTACGGCTACCTTGTTACGA-3. The PCR products were subjected to 1.0% agarose gel electrophoresis and then sent to Sangon Biotech for sequencing analysis. The sequencing results are shown in SEQ ID NO.1. Sequence alignment was performed using BLAST from NCBI GenBank, and the phylogenetic tree is shown below. Figure 4Based on this, the strain was identified as *Hydroxypyrella coagulans*. Heyndrickxia coagulans The strain LPB-13 was deposited at the China General Microbiological Culture Collection Center on December 29, 2025, with accession number CGMCCNo:37230.

[0040] Table 1 PCR amplification reaction system

[0041] Table 2 PCR amplification reaction procedure Example

[0042] The MRS liquid culture medium used in this step has the following formulation: peptone 10.0 g / L, beef extract 8.0 g / L, yeast extract 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, triammonium citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate heptahydrate 0.2 g / L, manganese sulfate monohydrate 0.05 g / L, and Tween 80 1.0 mL / L. Subsequent fermentation conditions were optimized based on this formulation.

[0043] Preparation of seed culture: 100 µL of bacterial culture LPB-13 was taken from a -80°C glycerol storage tube and inoculated into MRS liquid medium. The culture was then incubated overnight at 37°C and 180 rpm to obtain the activated seed culture.

[0044] Optimization of fermentation conditions for strain LPB-13: (1) Optimization of strain inoculum size: To determine the optimal inoculum size, experiments were conducted at five gradients: 2%, 3%, 4%, 5%, and 6% (v / v). The activated seed culture (OD) was then... 600 =1.0±0.1) were inoculated into 50 mL of MRS liquid culture medium according to the above ratio, and incubated at 37°C and 180 rpm for 24 hours before the final OD was measured. 600 The results are shown below. Figure 5 The optimal inoculation rate is 4.0%.

[0045] (2) Optimization of nitrogen source types and their compounding ratio: In the culture medium, peptone, beef extract, soybean peptone, acid-hydrolyzed protein, yeast extract, yeast extract, ammonium chloride, and urea were used as the main nitrogen sources to replace peptone, beef extract, and yeast extract in the MRS liquid medium, respectively. The carbon source and other conditions remained constant. Seed culture was inoculated at a 4.0% inoculum and cultured at 37°C and 180 rpm for 24 hours. OD was then measured. 600The most suitable nitrogen sources were beef extract and yeast extract. Figure 6 ).

[0046] Two optimal nitrogen sources (beef extract and yeast extract) were selected, with a fixed total addition of 24 g / L, and different mass ratios were set (1:0, 1:2, 1:3, 3:1, 2:1). Seed culture was inoculated at a 4.0% inoculum and cultured at 37°C and 180 rpm for 24 hours, after which the OD was measured. 600 The optimal compound ratio was determined to be 1:1 (by weight of beef extract powder and yeast extract powder). Figure 7 ).

[0047] Optimization of carbon source type and concentration: Fermentation media were prepared using equal masses (20 g / L) of glucose, D-fructose, α-lactose, maltose, soluble starch, and sucrose as the sole carbon source. Seed culture was inoculated at a 4% inoculum, and after culturing at 37°C and 180 rpm for 24 hours, the OD was measured. 600 Compare the differences in bacterial cell growth ( Figure 8 The results showed that glucose was the optimal carbon source.

[0048] Glucose was selected as the optimal carbon source, and experiments were conducted at five concentration gradients: 1.8%, 2.0%, 2.2%, 2.4%, and 2.6% (w / v). Keeping other conditions constant, the OD values ​​at each concentration were measured after 24 hours of inoculation and culture. 600 The optimal concentration was determined to be 2.2% (w / v), which is 22 g / L. Figure 9 ).

[0049] Optimization of the impact of the amount added: The optimized basal culture medium obtained according to steps (1) to (3) has the following formula: 12.0 g / L beef extract, 12.0 g / L yeast extract, 22.0 g / L glucose, 2.0 g / L dipotassium hydrogen phosphate, 2.0 g / L triammonium citrate, 5.0 g / L sodium acetate, 0.2 g / L magnesium sulfate heptahydrate, 0.05 g / L manganese sulfate monohydrate, and 1.0 mL / L Tween 80.

[0050] The inorganic salts were further optimized in the already optimized basal culture medium, and four experimental groups were set up: Group A (blank control) without the addition of MgSO4·7H2O and MnSO4·H2O, Group B with 0.4 g / L MgSO4·7H2O alone, Group C with 0.1 g / L MnSO4·H2O alone, and Group D with a combination of 0.4 g / L MgSO4·7H2O and 0.1 g / L MnSO4·H2O. Seed culture was inoculated at a 2% inoculum, and the cultures were incubated at 37°C and 180 rpm for 24 hours. The OD of the fermentation broth in each group was then measured.600 Values ​​were used to evaluate the effects of different inorganic salt combinations on bacterial cell growth. Figure 10 The results showed that the combined addition of 0.4 g / L MgSO4·7H2O and 0.1 g / L MnSO4·H2O to the culture medium yielded the best results.

[0051] In summary, the final optimized culture medium formula is as follows: beef extract 12.0 g / L, yeast extract 12.0 g / L, glucose 22.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, triammonium citrate 2.0 g / L, sodium acetate 5.0 g / L, Tween 80 1.0 mL / L, 0.4 g / L MgSO4·7H2O, and 0.1 g / L MnSO4·H2O.

[0052] Example 3 The ability to produce γ-aminobutyric acid was determined by the Berthelot colorimetric method, the specific method being as follows: Accurately weigh 0.1 g of GABA sample, add ultrapure water to make up to 10 mL, prepare a stock solution with a GABA concentration of 10 mg / mL, and then serially dilute it to prepare standard solutions of 2 mg / mL, 4 mg / mL, 6 mg / mL, 8 mg / mL, and 10 mg / mL.

[0053] Take 0.5 mL of γ-aminobutyric acid standard solution and add 0.4 mL of 0.1 mol / L sodium tetraborate solution, 2 mL of 6% redistilled phenol solution, and 1 mL of 7% sodium hypochlorite solution sequentially. After thorough shaking, heat in a boiling water bath for 10 min, then place in ice water and cool for 20 min until the solution turns a stable blue-green color. Add 4 mL of 60% ethanol and shake thoroughly again to mix well. Measure the absorbance (OD) of the solution at a wavelength of 635 nm. 635 (), with γ-aminobutyric acid concentration as the x-axis and corresponding standard sample OD, 635 Plot a standard curve with the values ​​on the ordinate to establish the γ-aminobutyric acid (GABA) standard curve, and calculate the standard regression equation. Figure 11 ).

[0054] 100 µL of bacterial culture was drawn from a -80°C glycerol storage tube and inoculated into MRS liquid medium. The culture was incubated overnight at 37°C and 180 rpm. The activated strain was then inoculated into the fermentation liquid medium at a 4% inoculum size and incubated at 37°C and 180 rpm for 72 h to obtain the fermentation broth. The fermentation liquid medium was prepared by adding 2% monosodium glutamate to the final optimized medium from Example 2.

[0055] (4) After centrifuging the fermentation broth at 8000 rpm for 10 min, collect the supernatant and perform the same treatment as in step (2). The yield of γ-aminobutyric acid was calculated to be 9.9 g / L.

[0056] Example 4 (1) Prepare xanthine standard aqueous solutions of 0.001~0.005 mg / L respectively, filter them through an aqueous filter membrane with a pore size of 0.22 µm, and measure the absorbance (OD) of each solution at a wavelength of 267 nm. 267 ), and then plot the standard curves in sequence ( Figure 12 ).

[0057] (2) The seed culture of strain LPB-13 was inoculated into 100 mL of liquid culture medium at an inoculation rate of 4%, and cultured overnight at 37°C and 180 rpm. An appropriate amount of the overnight culture was centrifuged at 6000-8000 rpm for 2 min, the supernatant was discarded, and an equal volume of xanthine solution of known concentration was added. The culture was then incubated at 37°C and 160 rpm for 4 h. The liquid culture medium was the final optimized culture medium of Example 2.

[0058] (3) Centrifuge the solution obtained in step (2) at 10000~12000 rpm for 5 min, collect the supernatant, and detect OD. 267 The xanthine concentration was calculated based on the standard curve, and the degradation rate of xanthine by strain LPB-13 was then determined. After 4 hours of incubation, the degradation rate of xanthine by strain LPB-13 reached 68.16%, which is of significant practical importance for developing highly efficient xanthine-degrading microbial agents and applying them in the food and other fields.

[0059] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A strain of Heinrich's coagulans LPB-13, characterized in that: The *Hydrilla verticillata* LPB-13 strain was deposited on December 29, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No:37230 and classification number *Hydrilla verticillata* (…). Heyndrickxia coagulans ).

2. A microbial inoculant, characterized in that: The microbial agent contains *Hydrilla verruca coagulans* LPB-13 as described in claim 1.

3. The method for preparing the microbial inoculant as described in claim 2, characterized in that: The specific steps are as follows: the seed culture of *Hydrilla verticillata* LPB-13 is inoculated into liquid culture medium at an inoculation rate of 2-6% v / v, and cultured at 35-40℃ and 120-200 rpm for 10-72 h.

4. The preparation method according to claim 3, characterized in that: The liquid culture medium is formulated as follows: 12.0 g / L beef extract, 12.0 g / L yeast extract, 22.0 g / L glucose, 2.0 g / L dipotassium hydrogen phosphate, 2.0 g / L triammonium citrate, 5.0 g / L sodium acetate, 1.0 mL / L Tween 80, 0.4 g / L MgSO4·7H2O, and 0.1 g / L MnSO4·H2O.

5. The use of *Hydrilla verticillata* LPB-13 as described in claim 1 or the microbial agent as described in claim 2 in the production of γ-aminobutyric acid.

6. The application according to claim 5, characterized in that: The product was obtained by inoculating it into a liquid culture medium containing 1-5% monosodium glutamate and fermenting for 48-96 hours. The liquid culture medium was formulated as follows: 12.0 g / L beef extract, 12.0 g / L yeast extract, 22.0 g / L glucose, 2.0 g / L dipotassium hydrogen phosphate, 2.0 g / L triammonium citrate, 5.0 g / L sodium acetate, 1.0 mL / L Tween 80, 0.4 g / L MgSO4·7H2O, and 0.1 g / L MnSO4·H2O.

7. The application of *Hydrilla verruca coagulans* LPB-13 as described in claim 1 or the microbial agent as described in claim 2 in the degradation of xanthine.

8. The application according to claim 7, characterized in that: Centrifuge the microbial inoculum at 6000-8000 rpm for 2 min, discard the supernatant, add an equal volume of xanthine solution, and incubate at 35-40℃ and 120-200 rpm for 3-5 h.

9. The use of the *Hydrilla verticillata* LPB-13 as described in claim 1 or the microbial agent as described in claim 2 in the preparation of uric acid-lowering products.

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