Application of Lactobacillus brevis 146 in improving bioavailability of GABA (gamma-aminobutyric acid)

Abstract

The invention discloses an application of Lactobacillus brevis 146 (Lactobacillus brevis 146) in improving the bioavailability of GABA (gamma-aminobutyric acid) (gamma-aminobutyric acid). According to the present invention, the specific Lactobacillus brevis 146 is adopted, such that the component of GABA is enriched, the GABA is more diversified, and the GABA, the dead bacteria component of the postbiotics, and the other metabolic components such as the active components such as polysaccharide are provided, such that the bioavailability of the GABA is improved; therefore, the lactobacillus brevis 146 and the GABA can be combined for use, and the lactobacillus brevis 146 can be used for preparing the medicine for treating the nervous system and intestinal diseases, and can be used for preparing food and health care products.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and particularly relates to the use of Lactobacillus brevis 146 in improving the bioavailability of GABA. Background Art

[0002] GABA (γ-aminobutyric acid) is an inhibitory neurotransmitter widely present in the nervous system and gastrointestinal tract, and has important physiological functions and market demands. In the nervous system, GABA can inhibit the release of glutamate, thereby regulating the activities of neurons and the transmission of nerve signals, and participating in physiological and pathological processes such as pain, anxiety, and depression. In addition, GABA can also maintain the intestinal barrier function, inhibit the apoptosis of intestinal cells and the expression of inflammatory factors, and thus play an important role in the occurrence and development of intestinal diseases.

[0003] Due to the important physiological functions of GABA, the market demand is also very broad. In drug research and development, GABA can be used as a potential therapeutic drug for treating nervous system and intestinal diseases. For example, new antidepressant drugs and painkillers can be developed by regulating the expression level of GABA. In addition, GABA can also be used as an important bioactive substance for research and development in the fields of nutrition and food science. For example, foods added with GABA can improve intestinal health, promote nutrient absorption, enhance immunity, etc.

[0004] However, GABA encounters difficulties in absorption. Due to the relatively large molecular weight of GABA, it is difficult to diffuse through the semipermeable membrane and enter cells. Therefore, the bioavailability of orally administered GABA is relatively low, which limits the exertion of its drug efficacy and scope of action.

[0005] To solve this problem, scientific researchers have carried out a large number of studies to explore effective methods for improving the bioavailability of GABA. Among them, binding GABA with a suitable carrier can promote its absorption through intestinal cells. In addition, changing the molecular structure of GABA, such as synthesizing its analogs or modifying its structure, can also increase its absorption effect and biological activity.

[0006] These studies have important practical significance for the application of GABA in the fields of drug research and development, nutrition, and food science. By improving the bioavailability of GABA, its drug efficacy and scope of action can be enhanced, thereby providing new treatment strategies for treating nervous system and intestinal diseases. At the same time, the application of GABA in nutrition and food science can also be more widely promoted and applied, providing more choices and support for the health and nutritional needs of humans. Summary of the Invention

[0007] Objective of the Invention: The objective of the present invention is to provide a use of Lactobacillus brevis 146 in improving the bioavailability of GABA.

[0008] Technical Solution: The objective of the present invention is achieved by the following technical solutions:

[0009] The present invention provides a use of Lactobacillus brevis 146 in improving the bioavailability of GABA.

[0010] The present invention also provides a use of the combined use of Lactobacillus brevis 146 and GABA in the preparation of drugs for treating nervous system and intestinal diseases.

[0011] The present invention also provides a use of the combined use of Lactobacillus brevis 146 and GABA in the preparation of foods and health products.

[0012] The Lactobacillus brevis 146 described in the present invention is purchased from Synbiotic Biotechnology Co., Ltd. (Taiwan, China).

[0013] A preferred embodiment of the present invention is that the method for improving the bioavailability of GABA includes the following steps:

[0014] (1) Prepare a 2% high-concentration lactic acid bacteria powder with the initial lactic acid bacteria amount of about 1.0×10 10 CFU / mL of Lactobacillus brevis 146 strain;

[0015] (2) Add the high-concentration lactic acid bacteria powder prepared in step (1) to the sterilized 200 g / L sodium glutamate solution, and carry out fermentation at 37°C, pH 5.0, and 80 rpm for 24 hours to obtain a GABA fermentation broth with a concentration of 100 - 110 mg / mL;

[0016] (3) Heat-inactivate the GABA fermentation broth prepared in step (2);

[0017] (4) Dry the heat-inactivated GABA fermentation broth by the drum drying method and collect all the contents.

[0018] Postbiotics are a collective term for the metabolite components of probiotics after processing, including bacterial cells and metabolites. When lactic acid bacteria ferment to produce GABA, other postbiotics can also be produced, and GABA itself is also a type of postbiotic. Other postbiotics can better retain or increase the biological activity of GABA. Since postbiotics retain some of the physiological functions of microorganisms, they can better protect the biological activity of GABA and thus better exert its physiological effects. At the same time, GABA is an active ingredient of postbiotics and can better promote human health. Postbiotics can stimulate the immune system, improve intestinal health, etc., and these functions promote each other with the physiological functions of GABA, which can better promote human health and is beneficial to improving the bioavailability of GABA.

[0019] Further, in step (1), the number of viable cells of the Lactobacillus brevis 146 bacterial powder > 1.0×10 11 CFU / g of bacterial powder.

[0020] Further, in step (1), the directed activation of high-concentration lactic acid bacteria powder is prepared by the following steps: The Lactobacillus brevis 146 strain is activated twice with MRS + 1% sodium glutamate and then fermented;

[0021] The culture medium formula is as follows: 60 g / L glucose, 20 g / L yeast extract, 20 g / L peptone, 1 g / L dipotassium hydrogen phosphate, 1 g / L sodium citrate, 0.2 g / L manganese sulfate, 0.1 g / L magnesium sulfate, 20 g / L sodium glutamate;

[0022] Fermentation conditions: inoculum size 3%, temperature 37 °C, pH 5.0, 80 rpm. After culturing for 16 hours, the bacterial cells are collected by centrifugation, and then an equal weight of cryoprotectant is added for freeze-drying, and the viable cell count of the produced bacterial powder is > 5.0×10 11 CFU / g of bacterial powder;

[0023] The formula of the cryoprotectant: 100 g / L maltodextrin, 10 g / L glycerol.

[0024] Further, in step (2), the sterilization conditions of the sodium glutamate solution are: sterilization at 121 °C for 15 minutes.

[0025] Further, in step (3), the heat inactivation conditions of the GABA fermentation broth are: heat inactivation at 100 °C for 30 minutes.

[0026] Further, in step (4), the excipient used in the drum drying method is corn starch, the liquid-solid ratio is 3.5:1, and the drying temperature is 120 - 130 °C.

[0027] Furthermore, the collected content contains the bacterium Lactobacillus brevis 146, with a bacterial count of 3.3×10 9 cells / g and a GABA content of 22.7%.

[0028] The above method of the present invention enriches the components of GABA, making it more diverse. It not only contains GABA but also the components of the dead bacteria of postbiotics, as well as other active components such as polysaccharides in other metabolic components. This makes the final active effect of GABA more effective and diverse, thereby improving the bioavailability of GABA.

[0029] Beneficial effects:

[0030] The present invention uses a specific Lactobacillus brevis 146 to improve the bioavailability of GABA. Therefore, Lactobacillus brevis 146 can be used in combination with GABA for the preparation of drugs for treating nervous system and intestinal diseases, and for the preparation of foods and health products. Description of the drawings

[0031] Figure 1 Transport effect of Caco-2 cells on different GABA samples at 48 hours. Detailed implementation manners

[0032] The technical solutions of the present invention will be described in detail below through specific examples, but the protection scope of the present invention is not limited to the described examples.

[0033] For those not specifying specific techniques or conditions in the examples, the techniques or conditions described in the literature in this field or according to the product specifications are followed. For reagents or instruments not specifying the manufacturer, they are all conventional products that can be obtained through regular channels.

[0034] The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all commercially available products unless otherwise specified.

[0035] In the examples, Lactobacillus brevis 146 (LBR146) was purchased from Synbio Technologies Co., Ltd., and its bacterial count > 1.0×10 11 CFU / g bacterial powder.

[0036] Example 1 Preparation of 20% GABA solution

[0037] (1) Preparation of high-concentration lactic acid bacteria powder

[0038] Lactobacillus brevis 146 was activated twice from a glycerol tube with MRS (BD, catalog number 288130) + 1% sodium glutamate and then fermented.

[0039] The fermentation medium formula is as follows: 60 g / L glucose, 20 g / L yeast extract, 20 g / L peptone, 1 g / L dipotassium hydrogen phosphate, 1 g / L sodium citrate, 0.2 g / L manganese sulfate, 0.1 g / L magnesium sulfate, 20 g / L sodium glutamate.

[0040] The fermentation conditions are: inoculum amount 3%, temperature 37 °C, pH 5.0, 80 rpm. After culturing for 16 h, the cells were collected by centrifugation, and then an equal weight of cryoprotectant for freeze-drying (formula: 100 g / L maltodextrin, 10 g / L glycerol) was added, followed by freeze-drying, and the number of viable bacteria produced > 5.0×10 11 CFU / g bacterial powder.

[0041] (2) Preparation of GABA 20 (containing LBR146 cells)

[0042] Prepare a 200 g / L sodium glutamate solution and sterilize it at 121 °C for 15 min. Add 2% of the above bacterial powder (the initial amount of lactic acid bacteria is about 1.0×10 10 CFU / mL) for fermentation.

[0043] Fermentation conditions: 37 °C, pH 5.0, 80 rpm, culture for 24 h to obtain a fermentation broth with a GABA concentration of 110 mg / mL, then heat inactivation (100 °C, 30 min), and then dry by drum drying. The excipient used for drum drying is corn starch, the liquid-solid ratio is 3.5:1, and the drying temperature is 125 °C.

[0044] The number of viable bacteria in the prepared GABA 20 bacterial powder is 3.3×10 9 cells / g, and the GABA content is 22.7%. (Self-made, batch number: 221221)

[0045] (3) Dissolve the above bacterial powder in ultrapure water to a concentration of 25 mM and store it in a 4 °C refrigerator for later use.

[0046] Preparation of Syn GABA solution in Comparative Example 1

[0047] Syn GABA: Does not contain LBR146 cells, the GABA content is 23.5%, purchased from Biosyn Biotechnology, batch number 230309.

[0048] Preparation method: The GABA 20 solution prepared in Example 1 was centrifuged to remove LBR146 bacteria, and then the supernatant was dried using the drum drying method. The excipient used for drum drying was corn starch, the liquid-solid ratio was 3.5:1, and the drying temperature was 125°C. The above-mentioned bacterial powder was dissolved in ultrapure water to a concentration of 25 mM and stored in a 4°C refrigerator for later use.

[0049] Prepare the pharma GABA solution in Comparative Example 2

[0050] pharma GABA TM : Purchased from Shanghai Tongyuan, with a GABA content of 27.6%.

[0051] A raw material product of GABA fermented product made by Lactobacillus hilgardii K-3 strain by Japan's Pharma Foods International Co., Ltd.

[0052] The above-mentioned bacterial powder was dissolved in ultrapure water to a concentration of 25 mM and stored in a 4°C refrigerator for later use.

[0053] Determination of GABA transport across Caco-2 monolayers in Example 2

[0054] During the experiment, the GABA solutions of Example 1 and Comparative Examples 1-2 were diluted with DMEM culture medium (gibco) respectively, and the final GABA concentration was 10 mM.

[0055] For the experiment of determining GABA transport across Caco-2 monolayers, refer to the article published by Raveschota et al. (Cyril Raveschot et al., Probiotic Lactobacillus strains from Mongolia improve calcium transport and uptake by intestinal cells in vitro, Food Research International 133 (2020) 109201, https: / / doi.org / 10.1016 / j.foodres.2020.109201.

[0056] Take 4×10 4Caco-2 cells (Sigma-Aldrich) were seeded in the apical chamber of a transwell device placed in a 24-well culture plate and grown for 21 days. The Caco-2 cells were washed twice with PBS. The GABA solution was dissolved in DMEM and adjusted to the required experimental concentration of 10 mM, and then added to the apical chamber of the transwell device. It was cultured at 37 °C and 5% CO2. The basolateral culture medium was taken out at different time points and stored at -80 °C until analysis.

[0057] Meanwhile, the transepithelial electrical resistance (TEER) value of Caco-2 was measured at 48 hours using a transcellular membrane potential measurement system (Cica TEER Measuring, KANTO CHEMICAL C7902, Hongyi Technology). Samples of the basolateral culture medium of Caco-2 were determined using HPLC.

[0058] HPLC analysis method:

[0059] The HPLC system equipment (SHIMADZU LC-40 HPLC System) had a column of Ascentis C18 5 μm (15 cm × 4.6 mm). The column temperature was 30 °C, the flow rate was 1.0 mL / min, the detection wavelength was 436 nm, the injection volume was 20 μL, and the retention time was 45 minutes.

[0060] The mobile phase consisted of A: 40% methanol, 1% acetic acid, and B: 1% acetic acid dissolved in methanol.

[0061] Gradient elution program: From 0 - 1 minute, mobile phase B increased from 0% to 23%;

[0062] From 1 - 20 minutes, mobile phase B was maintained at 23%;

[0063] From 20 - 25 minutes, mobile phase B increased from 23% to 25%;

[0064] From 25 - 30 minutes, mobile phase B increased from 25% to 40%;

[0065] From 30 - 32 minutes, mobile phase B increased from 40% to 100%;

[0066] From 32 - 36 minutes, mobile phase B was maintained at 100%;

[0067] From 36 - 37 minutes, mobile phase B decreased from 100% to 0%;

[0068] From 37 - 45 minutes, mobile phase B was maintained at 0%.

[0069] The GABA sample to be measured is first derivatized: 100 μL of the GABA sample to be measured is added with 250 μL of 0.15 M NaHCO3 (pH 9.0) and 500 μL of 2 mg / mL DABS-Cl (Merck), vortexed for 30 seconds to mix evenly, dried in a 70 °C dry bath for 15 minutes, cooled to room temperature, filtered through a 0.22 μm filter membrane, and then analyzed by HPLC.

[0070] The Caco-2 monolayer cell layer cultured in a transwell device was treated with 10 mM concentrations of GABA20, SynGABA, and pharma GABA respectively. After 48 hours, the basal medium was collected, and the GABA content in the medium was measured using HPLC.

[0071] Figure 1 The transport effect of Caco-2 cells on different GABA samples at 48 hours (the numbers marked on the histogram are the experimental values minus the values of the blank group).

[0072] From Figure 1 The results showed that the GABA concentration in the GABA 20 experimental group was the highest at 48 h. The GABA concentration in the GABA 20 group was 5.3 times that of the syn GABA and Pharma GABA groups.

[0073] From the experimental results, it can be seen that using Lactobacillus brevis 146 and adopting the method of the present invention improved the bioavailability of GABA.

[0074] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as a limitation of the present invention itself. Various changes can be made in its form and details without departing from the spirit and scope of the present invention defined by the appended claims.

Claims

1. Use of Lactobacillus brevis 146 in improving the bioavailability of GABA.

2. Use of Lactobacillus brevis 146 in combination with GABA in the preparation of drugs for treating nervous system and intestinal diseases.

3. Use of Lactobacillus brevis 146 in combination with GABA in the preparation of foods and health products.

4. The use according to claim 1, characterized in that, The method for improving the bioavailability of GABA includes the following steps: (1) Prepare a 2% high-concentration lactic acid bacteria powder of Lactobacillus brevis strain 146 with an initial lactic acid bacteria amount of approximately 1.0×10 10 CFU / mL; (2) Add the high-concentration lactic acid bacteria powder prepared in step (1) to the sterilized 200 g / L sodium glutamate solution and ferment at 37 °C, pH 5.0, 80 rpm for 24 hours to obtain a GABA fermentation broth with a concentration of 100 - 110 mg / mL. (3) Heat-inactivate the GABA fermentation broth prepared in step (2). (4) Dry the heat-inactivated GABA fermentation broth by roller drying and collect all the contents.

5. The use according to claim 4, characterized in that, In step (1), the viable count of the Lactobacillus brevis 146 bacterial powder > 1.0×10 11 CFU / g of the bacterial powder.

6. The use according to claim 4, characterized in that, In step (1), the high-concentration lactic acid bacteria powder for directional activation is prepared by the following steps: Activate the Lactobacillus brevis 146 strain twice with MRS + 1% sodium glutamate and then ferment. The medium formula is as follows: 60 g / L glucose, 20 g / L yeast extract, 20 g / L peptone, 1 g / L dipotassium hydrogen phosphate, 1 g / L sodium citrate, 0.2 g / L manganese sulfate, 0.1 g / L magnesium sulfate, 20 g / L sodium glutamate. Fermentation conditions: inoculum size 3%, temperature 37 °C, pH 5.0, 80 rpm. After culturing for 16 hr, the cells were collected by centrifugation, and then a cryoprotectant of equal weight was added, followed by freeze-drying. The viable cell count of the resulting bacterial powder was >5.0×10 11 CFU / g bacterial powder; The formula of the lyophilization protectant: 100 g / L maltodextrin, 10 g / L glycerol.

7. The use according to claim 4, wherein In step (2), the sterilization conditions of the sodium glutamate solution are: sterilize at 121 °C for 15 min.

8. The use according to claim 4, characterized in that, In step (3), the heat-inactivation conditions of the GABA fermentation broth are: heat-inactivate at 100 °C for 30 min.

9. The use according to claim 4, characterized in that, In step (4), the excipient used in the roller drying method is corn starch, the liquid-solid ratio is 3.5:1, and the drying temperature is 120 - 130 °C.

10. The use according to claim 4, characterized in that, The collected contents contain the strain Lactobacillus brevis 146, with a cell count of 3.3×10 9 cells / g and a GABA content of 22.7%.

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