Lactobacillus rhamnosus GS044, a freeze-dried powder thereof, and a probiotic in promoting growth and development and improving immunity

Abstract

The application provides a lactobacillus rhamnosus GS044 and a lyophilized powder and a probiotic of the lactobacillus rhamnosus GS044 in application to promoting growth and development and improving immunity, and belongs to the technical field of microorganisms.The application provides a lactobacillus rhamnosus GS044 with a preservation number of CGMCC No.33713.The application further provides a method for preparing gamma-aminobutyric acid by using the lactobacillus rhamnosus GS044, and a lyophilized powder prepared by using the lactobacillus rhamnosus GS044, a probiotic and application of the lyophilized powder and the probiotic.The lyophilized powder and the probiotic prepared by the application can effectively promote growth and development and improve immunity.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and specifically relates to application of Lactobacillus rhamnosus GS044, its freeze-dried powder, and postbiotics in promoting growth and development and improving immunity. Background Art

[0002] With the improvement of living standards and the strengthening of health awareness, promoting growth and development and enhancing immunity have become the focus of people's attention. Poor growth and development may lead to problems such as short stature and intellectual disability, while low immunity can easily lead to frequent infections and prolonged chronic diseases, seriously affecting the individual's quality of life and health level, and also placing enormous pressure on families and the medical system. At present, methods to promote growth and development and enhance immunity mainly include the intake of nutritional supplements, physical exercise, and medical intervention. However, nutritional supplements may have impure ingredients and cause adverse reactions due to excessive intake; physical exercise is difficult to maintain long-term due to venue, time and personal physical fitness limitations; medical interventions such as immunoglobulin injections are not only costly, but also may be accompanied by risks such as allergies. Therefore, exploring a new way to promote growth and development and enhance immunity that is safe, efficient and easy to implement has become an important issue that needs to be addressed urgently.

[0003] Probiotics are a type of active microorganisms that are beneficial to the host. They can colonize the human intestine, maintain the balance of intestinal flora, and play an important role in human health. Due to their relative safety and diverse efficacy, they are increasingly attracting the attention of researchers in this field and have been widely used in food, health products, and medicine. Lacticaseibacillus rhamnosus As a common probiotic, Lactobacillus belongs to the genus Lactobacillus. It is an anaerobic, acid-resistant, non-spore-forming, and Gram-positive bacterium that is widely present in the human and animal intestines. It exhibits multiple biological functions, such as acid resistance, bile salt resistance, and resistance to multiple antibiotics, enabling it to successfully colonize the human intestine and exert beneficial effects. Therefore, screening for Lactobacillus rhamnosus with significant efficacy in promoting growth and development and / or enhancing immunity, and developing related preparations, remains a key research topic in this field. Summary of the Invention

[0004] In order to solve the problems existing in the prior art, the first object of the present invention is to provide a rhamnosus lactobacillus ( Lacticaseibacillus rhamnosus ) GS044, depository: China General Microbiology Center of China Culture Collection Administration (CGMCC), deposit number: CGMCC No.33713, deposit date: March 4, 2025.

[0005] The second object of the present invention is to provide a method for preparing γ-aminobutyric acid, which can be used to prepare γ-aminobutyric acid.

[0006] The third object of the present invention is to provide a freeze-dried powder or postbiotic of Lactobacillus rhamnosus GS044, and a preparation method thereof.

[0007] The fourth object of the present invention is to provide the use of the above-mentioned freeze-dried powder or postbiotics in the preparation of drugs that promote growth and development, and the preparation of products that enhance immunity.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a rhamnosus lactobacillus GS044, wherein the rhamnosus lactobacillus ( Lacticaseibacillus rhamnosus ) The deposit number of GS044 is CGMCC No.33713.

[0010] The present invention also provides the use of the Lactobacillus rhamnosus GS044 in the preparation of gamma-aminobutyric acid.

[0011] The present invention also provides a method for preparing gamma-aminobutyric acid, comprising the following steps: fermenting and culturing the above-mentioned Lactobacillus rhamnosus GS044, and extracting gamma-aminobutyric acid from the fermentation product.

[0012] Preferably, the fermentation culture medium is based on Hami melon puree, and the Hami melon puree contains 15-25 g / L glucose and 25-35 g / L soybean peptide.

[0013] Preferably, the fermentation temperature is 36-38° C., and the fermentation time is 12-24 h.

[0014] The present invention also provides a method for preparing a freeze-dried powder, comprising the following steps: statically culturing the above-mentioned Lactobacillus rhamnosus GS044 until the residual sugar content is below 5 mmol / L; then, adding 4-6 g / L of sterile trehalose, 4-6 g / L of skim milk, 8-12 g / L of maltodextrin, and 0.3-0.7 g / L of vitamin E based on the volume of the culture product, thoroughly mixing, and freeze-drying.

[0015] The present invention also provides freeze-dried powder prepared by the above preparation method.

[0016] The present invention also provides a method for preparing a postbiotic, comprising the following steps: statically culturing the above-mentioned Lactobacillus rhamnosus GS044 until the residual sugar content is below 5 mmol / L; then, adding 4-6 g / L of sterile trehalose, 4-6 g / L of skim milk, 8-12 g / L of maltodextrin, and 0.3-0.7 g / L of vitamin E based on the volume of the culture product, thoroughly mixing, and spray drying.

[0017] The present invention also provides postbiotics prepared by the above preparation method.

[0018] The present invention also provides the use of the above-mentioned freeze-dried powder or postbiotics in any of the following:

[0019] (1) Preparation of drugs for promoting growth and development;

[0020] (2) Preparation of products that enhance immunity.

[0021] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0022] The present invention obtains a rhamnosus Lactobacillus GS044 for the first time, and provides a method for producing gamma-aminobutyric acid using the rhamnosus Lactobacillus GS044, as well as further preparing freeze-dried powder or postbiotics using the rhamnosus Lactobacillus GS044. The obtained freeze-dried powder or postbiotics can effectively promote growth and development or enhance immunity.

[0023] Biological Deposit Description

[0024] Lactobacillus rhamnosus GS044, classified as Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ), deposited at China General Microbiology Culture Collection Center (CGMCC), deposited at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, deposit number: CGMCCNo.33713, deposit date March 4, 2025. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] 图1 :The macroscopic morphology of the XW-9 colony in the plate;

[0026] 图2 : Microscopic morphology of strain XW-9;

[0027] 图3 : γ-aminobutyric acid mass spectrum;

[0028] 图4 : γ-aminobutyric acid high performance liquid chromatography. DETAILED DESCRIPTION

[0029] The present invention provides a rhamnosus lactobacillus GS044, wherein the rhamnosus lactobacillus ( Lacticaseibacillus rhamnosus ) GS044 is deposited with CGMCC No. 33713. The rhamnosus lactobacillus GS044 of the present invention is isolated and purified from breast milk during healthy lactation, and is identified as belonging to rhamnosus lactobacillus ( Lacticaseibacillus rhamnosus ), which was preserved in China General Microbiological Culture Collection Center on March 4, 2025, and the address of the preservation unit is No. 1, Beichen West Road, Yard 3, Beijing Chaoyang District, Institute of Microbiology, Chinese Academy of Sciences.

[0030] The application further provides application of the above-mentioned Lactobacillus rhamnosus GS044 in preparation of gamma-aminobutyric acid.

[0031] The application further provides a method for preparing gamma-aminobutyric acid, comprising the following steps: fermenting and culturing the above-mentioned Lactobacillus rhamnosus GS044, and extracting gamma-aminobutyric acid from a fermentation and culture product. The fermentation and culture temperature in the application is 36-38℃, preferably 37℃; the fermentation and culture time is 12-24h, preferably 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h or 23h; the fermentation and culture medium can be selected from modified MRS medium or a modified medium formula, the modified medium is based on Hami melon original plasma, the Hami melon original plasma contains 15-25g / L glucose and 25-35g / L soybean peptide, the content of the glucose is preferably 16g / L, 17g / L, 18g / L, 19g / L, 20g / L, 21g / L, 22g / L, 23g / L or 24g / L, and the content of the soybean peptide is preferably 26g / L, 27g / L, 28g / L, 29g / L, 30g / L, 31g / L, 32g / L, 33g / L or 34g / L; the inoculation amount of the fermentation and culture is 2-4%(v / v), preferably 3%(v / v). The extraction method of gamma-aminobutyric acid is not limited in the application, and a conventional extraction method in the field can be used for extraction.

[0032] The present invention also provides a method for preparing a freeze-dried powder, comprising the following steps: statically culturing the above-mentioned Lactobacillus rhamnosus GS044 until the residual sugar content is below 5 mmol / L; then, adding 4-6 g / L of sterile trehalose, 4-6 g / L of skim milk, 8-12 g / L of maltodextrin, and 0.3-0.7 g / L of vitamin E based on the volume of the culture product, thoroughly mixing, and freeze-drying. The temperature of the static culture of the present invention is 36-38° C., preferably 37° C.; the time of the static culture is 12-24 h, preferably 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h or 23 h; the culture medium of the static culture is based on Hami melon puree, and the Hami melon puree contains 15-25 g / L glucose and 25-35 g / L soybean peptide; the added amount of trehalose is preferably 5 g / L, the added amount of skim milk is preferably 5 g / L, the added amount of maltodextrin is preferably 9 g / L, 10 g / L or 11 g / L, and the added amount of vitamin E is preferably 0.4 g / L, 0.5 g / L or 0.6 g / L; the temperature of the freeze-drying is -30--40° C., and the time is 64-72 h.

[0033] The present invention also provides a freeze-dried powder prepared by the above preparation method, wherein the number of viable bacteria in the freeze-dried powder can reach 1.1×10 10 cfu / g, the extracellular polysaccharide content can reach 5392mg / 100g, and the γ-aminobutyric acid content can reach 55.79mg / g.

[0034] The present invention also provides a method for preparing a postbiotic, comprising the following steps: statically culturing the above-mentioned Lactobacillus rhamnosus GS044 until the residual sugar content is below 5 mmol / L; then, adding 4-6 g / L of sterile trehalose, 4-6 g / L of skim milk, 8-12 g / L of maltodextrin, and 0.3-0.7 g / L of vitamin E based on the volume of the culture product, thoroughly mixing, and spray drying. The temperature of the static culture of the present invention is 36-38° C., preferably 37° C.; the time of the static culture is 12-24 h, preferably 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h or 23 h; the culture medium of the static culture is based on Hami melon puree, and the Hami melon puree contains 15-25 g / L glucose and 25-35 g / L soybean peptide; the amount of trehalose added is preferably 5 g / L, the amount of skim milk added is preferably 5 g / L, the amount of maltodextrin added is preferably 9 g / L, 10 g / L or 11 g / L, and the amount of vitamin E added is preferably 0.4 g / L, 0.5 g / L or 0.6 g / L; the conditions for the spray drying are an inlet temperature of 185° C., an outlet temperature of 85° C., and an air volume flow rate of 2.5 m 3 / min, and the feed flow rate was 6.5 mL / min.

[0035] The present invention also provides a postbiotic prepared by the above preparation method, wherein the extracellular polysaccharide content in the postbiotic can reach 5417 mg / 100 g, and the γ-aminobutyric acid content can reach 56.04 mg / g.

[0036] The present invention also provides the use of the above-mentioned freeze-dried powder or postbiotic in any of the following: (1) preparing a drug for promoting growth and development; (2) preparing a product for enhancing immunity; the product is preferably a health product, food, or medicine. The freeze-dried powder or postbiotic of the present invention can promote the growth of young mice, increase the rate of weight gain, increase skeletal muscle growth, promote bone growth and development; promote the expression of IL-6, promote the development and maturation of the immune organs thymus and liver, promote the body's immune function, and enhance immunity.

[0037] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0038] In the following examples, unless otherwise specified, all methods are conventional.

[0039] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0040] Example 1

[0041] Isolation and identification of Lactobacillus rhamnosus GS044:

[0042] 1. Seven breast milk samples were collected from healthy lactating women and sent to the laboratory for strain isolation. Using commercially modified MRS solid isolation medium, the samples were diluted in multiple gradients and then anaerobically cultured at 37°C for 72 hours for strain isolation. Single colonies were picked and cultured until the strains were purified.

[0043] The purified colonies were subjected to Gram staining, microscopic examination and H2O2 catalase test, and a total of 5 spore-free, catalase-negative Gram-positive strains (named XW-1, XW-9, XW-26, XW-43, and XW-45) were picked out, placed in 15% glycerol, and stored at -80°C for future use.

[0044] The preserved strains were inoculated with 3% (v / v) MRS medium and incubated at 37°C for 18 hours. The strains were then cultured again using the same method. After two incubations, the strains were activated. The activated strains were inoculated at 0.1% (v / v) in MRS medium at pH 3.0 and incubated at 37°C for 24 hours. OD values ​​were measured to preliminarily screen for acid-resistant strains. The results are shown in the table below.

[0045] Table 1 OD values ​​of each strain

[0046]

[0047] Note: △OD 600 =24h(OD 600 )-0h(OD 600 ).

[0048] The results showed that after acid-resistant culture experiments, strains XW-9 and XW-43 were able to grow well in MRS medium at pH 3.0, and had a certain acid tolerance.

[0049] 2. Polysaccharide extraction of strains XW-9 and XW-43

[0050] Activation: Take the glycerol cryopreserved tubes of strains XW-9 and XW-43 stored in a -80°C refrigerator and thaw them at room temperature. Then, use an inoculation loop to pick the strains under a sterile environment, streak inoculate them on MRS solid culture medium plates, and incubate them anaerobically at 37°C for 36 hours. Observe the morphology of the colonies on the plates to confirm that they are the strain morphology and are free of contamination.

[0051] Seed culture: In a clean bench, pick a single colony from the activated MRS solid culture medium plate and inoculate it into the modified MRS medium. Incubate it at 37°C for 12 hours to obtain the first-level seed solution. Then, inoculate the first-level seed solution into fresh modified MRS medium at a 3.0% (v / v) inoculation volume. Incubate it at 37°C for 12 hours. When the OD value of strain XW-9 is 600 When the OD value of the seed solution and strain XW-43 reached 5.5, the OD value was obtained. 600 When it reaches 8, you get the seed liquid.

[0052] Fermentation: Inoculate XW-9 and XW-43 seed liquid into modified MRS medium at a 3% (v / v) inoculation rate and ferment at 37°C for 16 h to obtain fermentation broth of strains XW-9 and XW-43. At this time, the pH of the fermentation broth of the two strains reached 3.6-3.9.

[0053] Extraction of exopolysaccharides: Fermentation broths of strains XW-9 and XW-43 were centrifuged (8000 rpm, 20 min). 100 mL of the fermentation supernatant was collected. Three volumes of anhydrous ethanol were added to the supernatant to precipitate the polysaccharides in the fermentation broth. The mixture was mixed and precipitated overnight at 4°C for 14 h. The supernatant was then discarded and the precipitate was oven-dried at 50°C for 10 min. The remaining liquid was removed to obtain exopolysaccharides. The exopolysaccharides were weighed, as shown in the table below.

[0054] Table 2 Extracellular polysaccharide content of strains XW-9 and XW-43

[0055]

[0056] The results showed that strain XW-9 produced up to 1207 mg / 100 mL of exopolysaccharide. XW-43, despite its high OD, had a lower exopolysaccharide content. Strain XW-9, which is acid-resistant and produces exopolysaccharides, was selected as the subsequent experimental strain.

[0057] 3. Physiological and biochemical experiments of strain XW-9

[0058] The activated strain XW-9 was inoculated on solid MRS and cultured at 37°C for 48 hours. The morphology of the strain in the culture medium was observed. 图1 The results showed that milky white, smooth-surfaced, round colonies with neat edges could be seen on the culture medium.

[0059] After Gram staining, the strain was G + The microscopic morphology of bacteria was photographed using an electron microscope. 图2 The results showed that the bacteria had no whips and were short rod-shaped under microscope observation.

[0060] According to the direct inoculation method in the instruction manual for the complete set of biochemical tubes for identification of lactic acid bacteria (SHBG13): use an inoculation needle to pick up the same purified cultured colony from the plate and inoculate it into the biochemical tube to be tested. After inoculation, add sterilized liquid paraffin and seal the tube with a sterilized sealing film wiped with 75% alcohol. Incubate in a 37°C incubator for 36 hours and judge the physiological and biochemical tests of strain XW-9 by color reaction. The results are shown in the following table.

[0061] Table 3 Physiological and biochemical test results of strain XW-9

[0062]

[0063] Note: “+” indicates positive; “-” indicates negative.

[0064] Results showed that strain XW-9 could utilize glucose, lactose, sucrose, fructose, maltose, amygdalin, turanose, salicin, trehalose, and sorbitol, but could not utilize raffinose, melibiose, or arginine. Based on reference to the Bergey's Manual of Bacterial Identification (9th edition) and the Classification, Identification, and Test Methods of Lactic Acid Bacteria, this strain is likely Lactobacillus rhamnosus.

[0065] 4. Identification of strain XW-9

[0066]

[0067] The total 16SrDNA fragment of this strain was 1506bp, and the strain was identified as Lacticaseibacillus (Lactobacillus spp.), Lacticaseibacillus rhamnosus (Lactobacillus rhamnosus), named Lactobacillus rhamnosus GS044.

[0068] Lactobacillus rhamnosus GS044 was deposited in the General Microbiology Center of China Culture Collection Administration, the deposit address of which is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number: CGMCC No. 33713 and the deposit date: March 4, 2025.

[0069] Example 2

[0070] Determination of the adhesion ability of Lactobacillus rhamnosus GS044 to Caco-2 cells:

[0071] Lactobacillus rhamnosus GS044 and Lactobacillus rhamnosus LGG (purchased from Chr. Hansen) were cultured in MRS liquid medium at 37°C for 16 h and then used.

[0072] Adjust the Caco-2 cell concentration to 2 × 10 cells using a hemocytometer. 5 The cells were inoculated into 24-well culture plates with coverslips and cultured in a CO2 incubator at 37°C. After the cells grew into a monolayer, they were washed three times with sterile PBS to remove dead cells and antibiotics. Then, 1×10 suspensions of Lactobacillus rhamnosus GS044 and Lactobacillus rhamnosus LGG were added to the culture medium without antibiotics. 8 CFU / mL, cells were seeded into 24-well plates at 1 mL / well and incubated in a 37°C CO2 incubator for 2 hours. The supernatant was then carefully removed and the cells were rinsed five times with sterile PBS to remove unadhered bacteria. The cells were then digested with 0.25% trypsin (0.5 mL / well) and then 0.5 mL of PBS. The plates were serially diluted, mixed, and incubated at 37°C for counting. Simultaneously, cells in the uninoculated well were digested with 0.25% trypsin (0.5 mL / well) and then 0.5 mL of PBS was added. The cells were counted directly using a hemocytometer to calculate the cell concentration. The number of strains adhering to a single cell was calculated using the following formula, as shown in the table below.

[0073]

[0074] Table 4 Adhesion ability of each strain to Caco-2 cells

[0075]

[0076] Note: *: P<0.05.

[0077] As can be seen from the above table, the attachment ability of Lactobacillus rhamnosus GS044 is better than that of Lactobacillus rhamnosus LGG (P<0.05), further indicating that Lactobacillus rhamnosus GS044 has the potential to colonize the intestine.

[0078] Example 3

[0079] 1. Non-targeted metabolome analysis of Lactobacillus rhamnosus GS044:

[0080] Lactobacillus rhamnosus GS044 was inoculated into fresh modified MRS medium and cultured at 37°C for 24 h. The fermentation broth was collected, stored on dry ice, and mailed to Shanghai Biotechnology for non-targeted metabolomics analysis.

[0081] The detection method is as follows:

[0082] (1) Take the sample out of the -80°C freezer and thaw it on ice (all subsequent operations are performed on ice);

[0083] (2) Weigh 20 mg (± 1 mg) of sample into the corresponding numbered centrifuge tube;

[0084] (3) Add 400 μL of 70% methanol-water internal standard extract and vortex for 3 min;

[0085] (4) Ultrasonicate in an ice-water bath for 10 min, remove the sample and continue vortexing for 1 min, then place in a -20°C refrigerator for 30 min;

[0086] (5) Centrifuge at 12000 rpm for 10 min at 4°C and transfer 300 μL of the supernatant to another corresponding numbered centrifuge tube;

[0087] (6) Centrifuge at 12,000 rpm for 3 min at 4°C, and transfer 200 μL of the supernatant into the corresponding liner tube of the injection bottle for analysis.

[0088] T3 chromatographic conditions:

[0089] (1) Chromatographic column: Waters ACQUITY Premier HSS T3 Column 1.8µm, 2.1mm * 100mm;

[0090] (2) Mobile phase A: 0.1% formic acid / water; Mobile phase B: 0.1% formic acid / acetonitrile;

[0091] (3) Instrument column temperature: 40°C; flow rate: 0.4 mL / min; injection volume: 4 μL.

[0092] (4) Elution process:

[0093] Table 5 Elution process

[0094]

[0095] Mass spectrometry: Raw data from the mass spectrometer were converted to mzXML format using ProteoWizard. Peak extraction, alignment, and retention time correction were performed using the XCMS program. Peaks with a missingness rate >50% in each sample group were filtered, and blank values ​​were filled using KNN. Peak areas were corrected using the SVR method. Metabolites identified after correction were identified by searching the laboratory's own database, integrated public libraries, prediction libraries, and metDNA. Finally, substances with a comprehensive score of 0.5 or above and a QC sample CV value of less than 0.3 were extracted and identified. Positive and negative modes were then merged (retaining substances with the highest qualitative grade and the lowest CV value). This generated the file all_sample_data.xlsx.

[0096] Table 6 Mass spectrometry conditions

[0097]

[0098] The mass spectrum of γ-aminobutyric acid is shown in 图3 As shown in the figure, non-targeted metabolomics analysis revealed that the metabolites of Lactobacillus rhamnosus GS044 contained γ-aminobutyric acid (GABA) in positive ionization mode (POS) and negative ionization mode (NEG). GABA was further tested.

[0099] 2. The fermentation broth of Lactobacillus rhamnosus GS044 was tested by high performance liquid chromatography:

[0100] (1) Sample processing

[0101] Weigh 0.100 g (accurate to 0.001 g) of fermentation broth and dissolve it in a small amount of water. Add 200 μL of 0.2 mol / L dilute hydrochloric acid and dilute to 4 mL with water. Vortex for 1 minute and centrifuge at 8500 rpm for 10 minutes. Transfer 1 mL of the supernatant to a 10 mL brown bottle and add 1 mL of 0.5 mol / L sodium bicarbonate solution and shake thoroughly. Add 1 mL of 1% 2,4-dinitrofluorobenzene (DNFB) in acetonitrile and shake thoroughly. Derivatize in a 60°C water bath for exactly 1 hour. After derivatization, cool to room temperature and dilute to 10 mL with 0.2 mol / L ammonium acetate solution. Filter through a 0.22 μm organic filter membrane to prepare the test solution.

[0102] (2) Chromatographic conditions:

[0103] Analytical column: octadecyl bonded silica gel as filler (specifications: 4.6 mm × 250 mm, 5.0 μm);

[0104] Mobile phase A: 0.02 mol / L ammonium acetate solution; mobile phase B: 50% acetonitrile (acetonitrile-water mixed at a ratio of 1:1);

[0105] Perform linear gradient elution according to the following table:

[0106] Table 7 Linear gradient elution conditions

[0107]

[0108] Flow rate: 1.0 mL / min; injection volume: 20 μL; column temperature: 30°C; detector: UV (ultraviolet detector) 360 nm; the content of γ-aminobutyric acid in the sample is calculated according to the following formula:

[0109]

[0110] Where: X---the content of γ-aminobutyric acid in the sample, in mg / g; c---the concentration of the test solution calculated by checking the standard curve, in μg / mL; V---the total dilution volume of the sample, in mL (40mL); m---the amount of sample, in g.

[0111] Test results such as 图4 As shown in the detection spectrum, it can be seen that γ-aminobutyric acid is produced during the fermentation metabolism of Lactobacillus rhamnosus GS044, and the content is 6.21 mg / mL as determined by peak area.

[0112] Example 4

[0113] Improvement of exopolysaccharide and γ-aminobutyric acid metabolism in Lactobacillus rhamnosus GS044:

[0114] Activation: Take the glycerol cryopreserved tube of Lactobacillus rhamnosus GS044 stored in a -80℃ refrigerator, thaw it at room temperature, and then pick the bacteria with an inoculation loop under sterile conditions, streak inoculate on MRS solid medium plates, and culture anaerobically at 37℃ for 36 hours until the OD 600 is 12, and the activated Lactobacillus rhamnosus GS044 is obtained.

[0115] (1) After peeling and removing the seeds and cores of watermelon, Hami melon, and pear, use a pulper to prepare puree juice (without removing the fiber). The prepared watermelon puree, Hami melon puree, and pear puree are sterilized at 85°C for 30 minutes and set aside.

[0116] After activation, Lactobacillus rhamnosus GS044 was inoculated into prepared watermelon puree, cantaloupe puree, and pear puree at 3% (v / v). After incubation at 37°C for 18 hours, the number of viable fermenting bacteria was measured. The results are as follows:

[0117] Table 8 Effects of different fruit purees on viable bacterial counts

[0118]

[0119] The results showed that cantaloupe puree promoted the growth of Lactobacillus rhamnosus GS044, followed by pear puree, and watermelon puree had the worst effect on the growth of Lactobacillus rhamnosus GS044. Therefore, cantaloupe puree was used as a raw material for the growth of Lactobacillus rhamnosus GS044.

[0120] (2) Based on Hami melon puree, 20 g / L of glucose, lactose, sucrose, soybean peptide, corn oligopeptide, pea peptide, and wheat oligopeptide were added respectively to screen carbon and nitrogen sources suitable for the growth of rhamnosus Lactobacillus GS044.

[0121] The activated Lactobacillus rhamnosus GS044 was inoculated into the above culture medium at a 3% (v / v) inoculation rate. After static culture at 37°C for 16 hours, the number of viable bacteria was detected. The results are as follows:

[0122] Table 9 Effects of different carbon and nitrogen sources on viable bacterial counts

[0123]

[0124] The results showed that in terms of carbon source screening, glucose, lactose, and sucrose showed no significant differences, making glucose the preferred fermentation feedstock. In terms of nitrogen source screening, soybean peptides and corn oligopeptides performed well, with no significant differences, while pea peptides and wheat oligopeptides were less effective. Ultimately, the selected glucose, soybean peptides, and corn oligopeptides were used as the growth feedstock for Lactobacillus rhamnosus GS044.

[0125] (3) Effects of soybean peptides and corn oligopeptides on the metabolism of exopolysaccharides and γ-aminobutyric acid by Lactobacillus rhamnosus GS044:

[0126] After adding 20g / L glucose to cantaloupe puree, two portions were added with 20g / L soy peptide and 20g / L corn oligopeptide, respectively. After pasteurization at 85°C for 30 minutes, the mixture was cooled to 37°C and Lactobacillus rhamnosus GS044 was added at a 3% (v / v) inoculum. The mixture was incubated at 37°C until the residual sugar level dropped below 5mmol / L, at which point fermentation reached the endpoint. The viable cell count, exopolysaccharide, and γ-aminobutyric acid content after fermentation were measured. The results are as follows.

[0127] Table 10 The number of viable bacteria, extracellular polysaccharide and γ-aminobutyric acid content after fermentation

[0128]

[0129] The results showed that soybean peptides could promote the metabolism of Lactobacillus rhamnosus GS044 to produce a large amount of exopolysaccharides and γ-aminobutyric acid; corn oligopeptides promoted bacterial growth, but the metabolism of exopolysaccharides and γ-aminobutyric acid was relatively low. Soybean peptides were subsequently selected as fermentation raw materials.

[0130] (4) Screening of soybean peptide addition amount

[0131] The sugar content of Hami melon puree was 120 g / L as determined by Abbe refractometer, which contained fructose, glucose and sucrose. In this study, 20 g / L of glucose was added to promote the rapid growth and reproduction of Lactobacillus rhamnosus GS044, so glucose was not screened.

[0132] After adding 20g / L of glucose to cantaloupe puree, four portions were added with 10g / L, 20g / L, 30g / L, and 40g / L of soy peptide, respectively. After pasteurization at 85°C for 30 minutes, the mixture was cooled to 37°C and inoculated with Lactobacillus rhamnosus GS044 at a 3% (v / v) inoculum. The mixture was incubated at 37°C until the residual sugar level fell below 5mmol / L, and fermentation reached the endpoint. The viable cell count, exopolysaccharide content, and gamma-aminobutyric acid content were measured after fermentation. The results are shown below.

[0133] Table 11 The number of viable cells, extracellular polysaccharides and γ-aminobutyric acid contents after fermentation of soybean peptides at different concentrations

[0134]

[0135] The results showed that as the amount of soy peptide added increased, the number of viable bacteria of Lactobacillus rhamnosus GS044 increased, and the content of exopolysaccharides and γ-aminobutyric acid increased. When the soy peptide reached 30g / L, there was no significant difference in the number of viable bacteria, exopolysaccharides, and γ-aminobutyric acid compared with the fermentation with 40g / L of soy peptide. Therefore, the addition amount of soy peptide was 30g / L.

[0136] Example 5

[0137] Preparation of lyophilized powder of Lactobacillus rhamnosus GS044:

[0138] Hami melon was peeled, seeded and cored and prepared into puree juice (without removing fiber) using a pulper. 20 g / L glucose and 30 g / L soybean peptide were added to 1000 mL of Hami melon puree. The mixture was pasteurized at 85°C for 30 min, cooled to 37°C and added with 3% (v / v) inoculum of Lactobacillus rhamnosus GS044. The mixture was cultured at 37°C until the residual sugar level was below 5 mmol / L. After fermentation reached the end point, 5 g / L sterile trehalose, 5 g / L skim milk, 10 g / L maltodextrin and 0.5 g / L vitamin E were added. After thorough mixing, 500 mL of the fermentation broth was freeze-dried (-40°C for 72 h) to obtain 192.5 g of freeze-dried Lactobacillus rhamnosus GS044 powder.

[0139] Preparation of postbiotics from Lactobacillus rhamnosus GS044:

[0140] Hami melon was peeled, seeded and cored, and pulped into raw juice (without removing fiber). 20 g / L glucose and 30 g / L soybean peptide were added to 1000 mL of Hami melon pulp. After pasteurization at 85 °C for 30 min, the mixture was cooled to 37 °C and Lactobacillus rhamnosus GS044 was added at a 3% (v / v) inoculation rate. The mixture was cultured at 37 °C until the residual sugar content was below 5 mmol / L. After fermentation reached the end point, 5 g / L sterile trehalose, 5 g / L skim milk, 10 g / L maltodextrin and 0.5 g / L vitamin E were added. After thorough mixing, 500 mL of fermentation liquid was spray dried (inlet temperature 185 °C, outlet temperature 85 °C, air volume flow rate 2.5 m 3 / min, feed flow rate was 6.5 mL / min), and Lactobacillus rhamnosus GS044 postbiotics (187.3 g) were obtained.

[0141] The above results indicate that the solid content of Hami melon puree is approximately 15%. The freeze-dried powder and spray-dried powder (postbiotics) were tested for viable bacterial count, exopolysaccharides, and γ-aminobutyric acid, and the results are as follows:

[0142] Table 12 Contents of viable bacteria, exopolysaccharides and γ-aminobutyric acid in postbiotics and freeze-dried powder

[0143]

[0144] The results showed that the number of live bacteria in the freeze-dried powder reached more than 10 billion, while no live bacteria were detected in the spray-dried powder; the solid content of the fermentation broth reached 40% after adding trehalose, skim milk and maltodextrin, and there was no loss in the content of extracellular polysaccharides and γ-aminobutyric acid after freeze-drying and spray-drying.

[0145] Example 6

[0146] The freeze-dried powder and spray-dried powder (postbiotics) prepared in Example 5 were used to conduct a rat growth experiment:

[0147] 120 3-week-old SPF-grade SD rat pups (half male and half female) were fed a basal diet for one week and then randomly divided into 10 groups (12 rats per group, half male and half female): control group, low-dose γ-aminobutyric acid group, medium-dose γ-aminobutyric acid group, high-dose γ-aminobutyric acid group, low-dose lyophilized powder group, medium-dose lyophilized powder group, high-dose lyophilized powder group, low-dose spray-dried powder group, medium-dose spray-dried powder group, and high-dose spray-dried powder group. The animals had free access to food and water. Daily body weight and food intake were recorded. The ambient temperature was controlled at 20°C to 22°C, relative humidity at 35% to 75%, and the light cycle was 12 hours and the dark cycle was 12 hours. The experimental pups in each group were fed a basal diet. The gavage dosage and feeding days for each group are as follows:

[0148] Table 13 Oral administration dosage and feeding days of each group of experimental mice

[0149]

[0150] The body weight of the young mice was recorded before and after the experiment. After 28 days of gavage and 12 hours of fasting, blood was collected from the eye sockets, centrifuged, and the serum was stored at -80°C for later use. The right femur was removed, the muscle was removed, and the mice were rinsed with saline, dried with filter paper, and their length was measured with a vernier caliper. The stored serum was used to determine the IL-6 content in the serum according to the method of the kit (Xinbosheng Bio). The thymus and liver tissues were removed, the surface blood stains were rinsed with saline, dried with filter paper, weighed, and their organ indexes were calculated.

[0151] Calculation formula: Organ index = organ live weight (mg) / animal live weight (g) × 100%.

[0152] Table 14 Body weight of experimental mice in each group

[0153]

[0154] Note: Weight gain rate % = (final weight of experimental group - final weight of control group) / final weight of control group; Weight gain = final weight of each group - initial weight of each group; *: indicates P < 0.05; **: indicates P < 0.01

[0155] As can be seen from the table: there was a significant difference in weight gain between the high-dose freeze-dried powder group and the high-dose spray-dried powder group (P<0.01); there was a significant difference in weight gain between the medium- and high-dose γ-aminobutyric acid groups, the medium-dose freeze-dried powder group, and the medium-dose spray-dried powder group (P<0.05); compared with the freeze-dried powder group and the spray-dried powder group, the freeze-dried powder group and the spray-dried powder group had better growth effects on young mice. Freeze-dried powder and spray-dried powder contain extracellular polysaccharides. This experiment shows that extracellular polysaccharides play a certain role in the growth of young mice; compared with the freeze-dried powder group, the freeze-dried powder group had a slight advantage but the effect was not significant. Live bacteria of Lactobacillus rhamnosus GS044 highlighted its role here.

[0156] Table 15 Comparison of femurs of experimental mice in each group

[0157]

[0158] The results showed that compared with the control group, femoral length increased by 2.51% and 3.19% in the medium-dose freeze-dried powder group and the high-dose freeze-dried powder group, respectively. Femoral length increased by 2.13% and 2.91% in the medium-dose spray-dried powder group and the high-dose spray-dried powder group, respectively, but these differences were not statistically significant. The experiment demonstrated that Lactobacillus rhamnosus GS044 increased femoral growth in a dose-dependent manner. The increase in femoral length suggests that Lactobacillus rhamnosus GS044 promotes bone growth and development, benefiting the growth of young mice.

[0159] Table 16 IL-6 expression results of experimental mice in each group

[0160]

[0161] Compared with the control group, IL-6 levels increased in all nine experimental groups, with increases of 24.36% and 23.32% in the high-dose freeze-dried powder and spray-dried powder groups, respectively, but these differences were not statistically significant. The increase in IL-6 expression in the GABA, Lactobacillus rhamnosus GS044 freeze-dried powder, and spray-dried powder groups was as follows: GABA < Lactobacillus rhamnosus GS044 spray-dried powder < Lactobacillus rhamnosus GS044 freeze-dried powder. This suggests that GABA alone can increase IL-6 expression in young mice, but Lactobacillus rhamnosus GS044 produces a greater increase. Both GABA and exopolysaccharides produced by the strain promote IL-6 expression in young mice. IL-6 is primarily produced by multiple cell types, including macrophages, T cells, and B cells. It regulates the growth and differentiation of various cells, modulating immune responses, acute phase reactions, and hematopoiesis, and plays a crucial role in the body's anti-infective immune response. GABA and Lactobacillus rhamnosus GS044 freeze-dried and spray-dried powders can promote IL-6 expression, indicating that all three substances can enhance immunity.

[0162] Table 17 Organ index results of experimental mice in each group

[0163]

[0164] The results showed that the addition of γ-aminobutyric acid, Lactobacillus rhamnosus GS044 freeze-dried powder, and spray-dried powder had no significant effect on the liver and spleen indices of the experimental mice (P>0.05), but compared with the control group, the 9 experimental groups showed an upward trend. The results indicate that γ-aminobutyric acid, Lactobacillus rhamnosus GS044 freeze-dried powder, and spray-dried powder can promote the development and maturation of the immune organs thymus and liver, promote immune function, and enhance immunity. The Lactobacillus rhamnosus GS044 freeze-dried powder and spray-dried powder were more effective than γ-aminobutyric acid.

[0165] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A Lactobacillus rhamnosus GS044, characterized in that The Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) The deposit number of GS044 is CGMCC No.33713.

2. Use of the Lactobacillus rhamnosus GS044 according to claim 1 in the preparation of gamma-aminobutyric acid.

3. A method for preparing γ-aminobutyric acid, characterized in that: The method comprises the following steps: fermenting and culturing the Lactobacillus rhamnosus GS044 according to claim 1, and extracting gamma-aminobutyric acid from the fermentation product.

4. The method according to claim 3, characterized in that The culture medium for fermentation culture is based on Hami melon puree, to which 15-25 g / L of glucose and 25-35 g / L of soybean peptide are added.

5. The method according to claim 3, characterized in that The fermentation temperature is 36-38° C., and the fermentation time is 12-24 hours.

6. A method for preparing a freeze-dried powder, characterized in that: The method comprises the following steps: statically culturing the Lactobacillus rhamnosus GS044 described in claim 1 until the residual sugar content is below 5 mmol / L; then, adding 4-6 g / L of sterile trehalose, 4-6 g / L of skim milk, 8-12 g / L of maltodextrin and 0.3-0.7 g / L of vitamin E based on the volume of the culture product, thoroughly mixing the mixture and freeze-drying the mixture.

7. The freeze-dried powder prepared by the preparation method according to claim 6.

8. A method for preparing postbiotics, characterized in that: The method comprises the following steps: statically culturing the Lactobacillus rhamnosus GS044 described in claim 1 until the residual sugar content is below 5 mmol / L; then, adding 4-6 g / L of sterile trehalose, 4-6 g / L of skim milk, 8-12 g / L of maltodextrin and 0.3-0.7 g / L of vitamin E based on the volume of the culture product, thoroughly mixing the mixture and spray drying the mixture.

9. The postbiotics prepared by the preparation method according to claim 8.

10. Use of the freeze-dried powder according to claim 7 or the postbiotic according to claim 9 in any of the following: (1) Preparation of drugs for promoting growth and development; (2) Preparation of products that enhance immunity.

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