Lactic acid bacterium producing γ-aminobutyric acid and use thereof

Abstract

Provided is a Lactobacillus brevis ATIT-054 producing γ-aminobutyric acid (GABA). Further provided are a dry powder made from the Lactobacillus brevis and a use thereof in preparing a composition for scavenging free radicals, inhibiting angiotensin converting enzyme I, inhibiting α-glucosidase, promoting collagen protein production, and for anti-inflammatory and anti-allergic uses.

Description

GABA-producing lactic acid bacteria and uses thereof TECHNICAL FIELD

[0001] The present invention relates to GABA-producing lactic acid bacteria and uses thereof. In particular, it relates to the production of GABA using a novel Lactobacillus brevis ATIT-054, a bacterial powder made therefrom, and uses thereof. BACKGROUND

[0002] GABA (γ-aminobutyric acid) is a non-proteinogenic natural amino acid with functions such as mood relaxation, obesity prevention, blood pressure reduction, and blood sugar reduction, and is currently used in various fields such as food, cosmetics, and feed additives. GABA can be obtained using chemical synthesis, natural product extraction, and microbial fermentation. Chemical synthesis uses potassium phthalimide, chloroacetonitrile, or pyrrolidinone to produce GABA through chemical reactions. This method has the advantages of rapid reaction and high production efficiency, but has high production costs and generates toxic substances during production, so it is not suitable for use as a food or feed additive. Natural product extraction involves isolating and purifying GABA from plants containing GABA, such as mulberry leaves or germinated brown rice. Microbial fermentation involves using microorganisms with GABA production ability, such as Lactoccocus lactis, Lactobacillus paracasei, or Lactobacillus plantarum, for fermentation culture to obtain GABA. Although there are previous examples of using microorganisms to produce GABA, there is still room for improvement in GABA yield. For example, Taiwan patent "Method for preparing high-yield γ-aminobutyric acid" (I689593) discloses that Lactobacillus brevis CPC202 produces 29.82 g / L of GABA in MRS medium. Taiwan patent "Lactobacillus brevis LB919 strain and method for producing γ-aminobutyric acid using the same" (I754936) discloses that Lactobacillus brevis LB919 produces 953.16 mg / L of GABA in a designed medium. Taiwan patent "Lactic acid bacteria with γ-aminobutyric acid production ability" (I379003) discloses that Lactobacillus rennini KG34 produces 19.5 g / L of GABA in a designed medium.

[0003] The present invention aims to provide a novel GABA-producing bacterial strain, a method for producing GABA, and uses of the product thereof.

[0004] SUMMARY

[0005] To achieve the above object, the present application provides a Lactobacillus brevis ATIT-054, which is deposited in the NITE Patent Microorganisms Depositary National Institute of Technology and Evaluation (NPMD) (Address: 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba 292-0818, Japan) on May 13, 2024, with the accession number NITE BP-04112, and the classification name: Lactobacillus brevis.

[0006] According to another object of the present application, a method for preparing a GABA-containing powder is provided, wherein: the GABA-containing powder is made from Lactobacillus brevis ATIT-054 (the strain is also deposited in the Biological Resource Center of the Food Industry Development Institute, Taiwan, China, with the accession number BCRC 911213); and the method comprises the following steps: (a) inoculating the bacterial cells of the lactic acid bacteria into a plate culture medium for solid-state culture to form colonies; (b) inoculating the colonies cultured in step (a) into a pre-culture medium for liquid culture to obtain a liquid culture medium containing bacterial cells; (c) inoculating the liquid culture medium containing bacterial cells in step (b) into a fermentation tank containing a complex culture medium for liquid culture, and then adding a feed medium to obtain a bacterial liquid; (d) sterilizing the bacterial liquid in step (c); (e) mixing the sterilized bacterial liquid in step (d) with excipients and drying to obtain a bacterial powder.

[0007] Preferably, the pre-culture medium comprises pre-culture medium I, pre-culture medium II, or a combination thereof.

[0008] Preferably, the complex culture medium comprises complex culture medium I, complex culture medium II, or a combination thereof.

[0009] Preferably, the feed medium comprises feed medium I, feed medium II, feed medium III, or a combination thereof.

[0010] Preferably, the medium combination comprises the pre-culture medium I, the complex culture medium I, the feed medium I, and / or the feed medium II.

[0011] Preferably, the medium combination comprises the pre-culture medium II, the complex medium II, and the feed medium III.

[0012] Preferably, the pre-culture medium comprises 25-50 g / L of a carbon source, 10-20 g / L of a yeast extract, 10-20 g / L of a nitrogen source, 1-3 mL / L of Tween 80, 20-35 g / L of monosodium glutamate (MSG), and 0.005-0.01 g / L of inorganic salts, and has a pH of 5.5-6.5.

[0013] Preferably, the complex medium comprises 25-50 g / L of a carbon source, 10-50 g / L of a yeast extract, 0-2 g / L of a nitrogen source, 0.5-1.5 mL / L of Tween 80, 30-50 g / L of monosodium glutamate (MSG), and 3-6 g / L of inorganic salts, and has a pH of 5.5-6.5.

[0014] Preferably, the feed medium comprises 50-170 g / L of a carbon source, 30-130 g / L of a yeast extract, 0-5 g / L of a nitrogen source, 1-3 mL / L of Tween 80, 200-430 g / L of monosodium glutamate (MSG), and 4-16 g / L of inorganic salts, and has a pH of 5.5-6.5.

[0015] Preferably, the drying of step (d) comprises spray drying, freeze drying, drum drying, vacuum drying, foam drying, or fluidized bed drying.

[0016] According to yet another object of the present application, there is provided a use of Lactobacillus brevis ATIT-054 and / or an active substance thereof for the preparation of a food composition, a cosmetic composition, or a pharmaceutical composition for scavenging free radicals, inhibiting angiotensin converting enzyme I, inhibiting alpha-glucosidase, promoting collagen production, anti-inflammation, and anti-allergy. Preferably, the scavenging of free radicals can refer to scavenging DPPH free radicals, ABTS·+ free radicals, or superoxide anion free radicals. The promotion of collagen production can refer to the promotion of collagen production by human skin fibroblast CCD966SK.

[0017] Preferably, the Lactobacillus brevis ATIT-054 and / or the active substance thereof are prepared in the form of a bacterial powder, a liquid, a tablet, a capsule, or a jelly.

[0018] Through the above technical features, the Lactobacillus brevis ATIT-054 provided by the present application can produce GABA, and the bacterial powder and the composition prepared therefrom have the effects of scavenging free radicals, inhibiting angiotensin converting enzyme I, inhibiting alpha-glucosidase, promoting collagen production, anti-inflammation, and anti-allergy. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is a flow chart of the fermentation culture of the lactic acid bacterial strain according to an embodiment of the present application.

[0020] Figure 2 is a flow chart of the fermentation culture of the lactic acid bacterial strain according to another embodiment of the present application.

[0021] Figure 3 is a flow chart of the method for preparing the GABA-containing bacterial powder according to an embodiment of the present application.

[0022] Figure 4 is a comparison of the results of the analysis of the GABA production ability of 113 lactic acid bacterial strains using thin layer chromatography.

[0023] Figure 5 is an analysis of the GABA production of 10 lactic acid bacterial strains using colorimetry.

[0024] Figure 6 is the growth of the bacterial cells and the GABA production of Lactobacillus brevis ATIT-054 in different fermentation media. (A) Fermentation culture using complex medium I with feed medium I and II; (B) Fermentation culture using complex medium II with feed medium III.

[0025] Figure 7 is the ability of the GABA-containing bacterial powder (A) and the commercially available high-purity GABA (B) to scavenge DPPH free radicals.

[0026] Figure 8 is the ability of the GABA-containing bacterial powder (A) and the commercially available high-purity GABA (B) to scavenge ABTS+free radicals.

[0027] Figure 9 is the ability of the GABA-containing bacterial powder (A) and the commercially available high-purity GABA (B) to scavenge superoxide anion free radicals.

[0028] Figure 10 is the ability of the GABA-containing bacterial powder (A) and the commercially available high-purity GABA (B) to inhibit α-glucosidase.

[0029] Figure 11 is the ability of the GABA-containing bacterial powder (A) and the commercially available high-purity GABA (B) to inhibit angiotensin converting enzyme I.

[0030] Figure 12 is the effect of the GABA-containing bacterial powder and the commercially available high-purity GABA on the survival rate (A) and the collagen content (B) of human skin fibroblast CCD966SK cells.

[0031] Figure 13 is the effect of the GABA-containing bacterial powder and the commercially available high-purity GABA on the survival rate (A) and the ability to inhibit the production of nitric oxide (B) of mouse macrophage RAW264.7 cells.

[0032] Figure 14 is a graph showing the survival rate of RBL-2H3 cells (A) and the inhibition of degranulation reaction (B) after DNP-BSA treatment of RBL-2H3 cells sensitized with IgE.

[0033] Figure 15 is a graph showing the survival rate of RBL-2H3 cells (A) and the inhibition of degranulation reaction (B) of RBL-2H3 cells treated with DNP-BSA.

[0034] Main reference numeral explanation:

[0035] 100, 200: fermentation culture method

[0036] 300: dry powder preparation method

[0037] S101-S304: steps

[0038] Preservation of microorganism material for patent procedure:

[0039] Lactobacillus brevis ATIT-054 strain:

[0040] Date of preservation: May 13, 2024;

[0041] Preservation organization: NITE Patent Microorganisms Depositary National Institute of Technology and Evaluation (NPMD);

[0042] Address of preservation organization: Chiba, Japan (#122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba 292-0818, Japan);

[0043] Preservation number: NITE BP-04112;

[0044] Taxonomic designation: Lactobacillus brevis. DETAILED DESCRIPTION

[0045] In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the disclosed implementations. However, it will be apparent to one skilled in the art that one or more implementations can be practiced without these specific details. In other instances, well-known structures and processes are not shown in detail in order to simplify the drawings.

[0046] The details of various specific examples of the present application are set forth in the description below. Other features of the present application will become apparent from the following detailed description, which, when taken in conjunction with the drawings, discloses various embodiments of the present application. Those skilled in the art will realize that the application can be practiced with the less or different components, or with other components, and with various modifications, as apparent from the disclosure, without departing from the spirit and scope thereof. The following description is, therefore, not to be taken in a limited sense, and the scope of the present application is defined by the appended claims.

[0047] As used herein, the term "cell" refers to the structure of the lactic acid bacteria used in the present disclosure at various stages of cultivation. In one embodiment, the term "cell" refers to the structure of the lactic acid bacteria in whole or in part. In one embodiment, the term "cell" refers to the colony of the lactic acid bacteria formed by the division and reproduction of a single bacterium. In one embodiment, the term "cultivation stage" refers to solid-state cultivation, liquid-state cultivation, or liquid-scale-up in a fermentation tank.

[0048] As used herein, the term "active substance" refers to the substance selected from the lactic acid bacteria cell used in the present disclosure after a specific experimental procedure, or the mixture of the cell and the culture medium or broth. In one embodiment, the term "active substance" refers to the culture broth, sterilized broth, and dried cell powder. The method of drying the broth can include spray drying, freeze drying, drum drying, vacuum drying, foam drying, fluidized bed drying, etc., and the present disclosure is not limited thereto. In one embodiment, the term "active substance" refers to the solid-state culture medium containing the cell, the liquid-state culture medium containing the cell, or the broth after liquid-scale-up in a fermentation tank.

[0049] The pre-culture medium used in the embodiments of the present disclosure comprises 25-50 g / L of a carbon source, 10-20 g / L of a yeast extract, 10-20 g / L of a nitrogen source, 1-3 mL / L of Tween 80, 20-35 g / L of monosodium glutamate (MSG), and 0.005-0.01 g / L of inorganic salts, with a pH of 5.5-6.5.

[0050] The complex culture medium used in the embodiments of the present disclosure comprises 25-50 g / L of a carbon source, 10-50 g / L of a yeast extract, 0-2 g / L of a nitrogen source, 0.5-1.5 mL / L of Tween 80, 30-50 g / L of monosodium glutamate (MSG), and 3-6 g / L of inorganic salts, with a pH of 5.5-6.5.

[0051] The feed medium used in the embodiments of the present disclosure comprises 50-170 g / L of a carbon source, 30-130 g / L of a yeast extract, 0-5 g / L of a nitrogen source, 1-3 mL / L of Tween 80, 200-430 g / L of monosodium glutamate (MSG), and 4-16 g / L of inorganic salts, with a pH of 5.5-6.5.

[0052] In the present embodiment, 113 strains of lactic acid bacteria are screened, and strains with potential for GABA production are selected for a second screening. The strain with the highest GABA yield is selected for culture and preparation of dried powder for subsequent tests for free radical scavenging, angiotensin converting enzyme I inhibition, alpha-glucosidase inhibition, collagen production promotion, anti-inflammatory, and anti-allergic effects.

[0053] The materials and methods used in the present application, and the detailed procedures of each experiment, are described below.

[0054] Materials and Methods

[0055] 1. Screening of GABA-producing lactic acid bacteria

[0056] The lactic acid bacteria isolated from different sources were streaked onto deMan Rogosa Sharpe (MRS) plate medium (Merck, USA) and incubated at 30°C in a facultative anaerobic environment for 48 hours. Subsequently, single colonies were selected and inoculated into FGM medium (20 g / L glucose, 9 g / L yeast extract, 1 mL / L tween 80, 2 g / L K2HPO4, 3.014 g / L sodium acetate, 2 g / L ammonium citrate, 0.2 g / L MgSO4·7H2O, pH 5.7) containing 3% sodium glutamate. The culture was incubated at 30°C for 24 hours. After centrifugation (20,630 x g, 10 minutes, room temperature), the supernatant was collected and analyzed for GABA production by different lactic acid bacteria using thin layer chromatography (TLC). The procedure for TLC is described as follows. 1 μL of lactic acid bacteria fermentation supernatant, 1 μL of GABA standard solution (10 mg / mL), or 1 μL of sodium glutamate standard solution (10 mg / mL) was spotted onto a silica gel 60 TLC plate (Merck, USA). After the sample was air-dried, the plate was inclined and placed in a developing tank containing developing solution (50% n-butanol, 25% acetic acid, 25% deionized water). After development was complete, the TLC plate was removed and air-dried, and then 0.3% ninhydrin solution was uniformly sprayed onto the plate. After air-drying, color development was performed using heating, and the Rf value of the sample and standard was calculated. The type of lactic acid bacteria metabolite was determined by the Rf value.

[0057] 2. Quantitative analysis of GABA using colorimetry

[0058] After diluting the sample to the appropriate concentration with deionized water, 200 μL of the diluted sample was pipetted into a 1.75 mL microcentrifuge tube. 200 μL of 0.1 M sodium tetraborate, 120 μL of 7% sodium hypochlorite, and 240 μL of 5% phenol solution were added in sequence, mixed well by shaking, and then heated in a 100°C dry bath for 10 minutes. After the reaction was completed, the reaction solution was removed and placed on ice for 5 minutes. Finally, 400 μL of 60% ethanol was added and allowed to stand at room temperature for 20 minutes. 100 μL of the reaction solution was pipetted into a 96-well plate, and the OD 640 absorbance values. GABA standards were also tested at concentrations of 0.4, 0.3, 0.2, 0.1, and 0 mg / mL, and a standard curve of GABA concentration versus absorbance value was prepared. The absorbance value of the sample was entered into the standard curve, and the GABA concentration of the sample was calculated. The actual GABA concentration of the sample was then calculated by multiplying the GABA concentration by the dilution factor.

[0059] 3. Fermentation of GABA-producing lactic acid bacteria

[0060] (1) Fermentation using complex medium I with feed media I and II

[0061] Referring to the flow chart of the fermentation culture method 100 of FIG. 1, it includes strain thawing culture S101, pre-culture medium I liquid culture S102, complex culture medium I liquid culture S103, addition of feed medium I S104, and addition of feed medium II S105. According to the embodiment of the present application, the L. brevis ATIT-054 storage tube is taken out from -80°C, and after thawing at room temperature, the bacterial liquid is picked up using a loop and streaked onto FGM flat plate medium. After 48 hours of culture at 30°C in a facultative anaerobic environment, a single colony is selected and inoculated into a 50 mL centrifuge tube containing 32 mL of pre-culture medium I (50 g / L glucose, 12.5 g / L yeast extract, 12.5 g / L soytone, 2 mL / L Tween 80, 28 g / L MSG, 0.0076 g / L MnSO4H2O, pH 6.0) and incubated at 30°C for 24 hours. The pre-culture bacterial liquid is added into a 500 mL serum bottle containing 320 mL of pre-culture medium, and incubated at 30°C for 16 hours. 150 mL of the pre-culture bacterial liquid is added into a 5 L fermenter containing 1.5 L of complex culture medium I (16.5 g / L black sugar, 17.05 g / L maltose, 12.8 g / L yeast extract, 2 g / L K2HPO4, 1 mL / L Tween 80, 3.014 g / L sodium acetate, 30 g / L MSG, 0.2 g / L MgSO4·7H2O, 0.04 g / L MnSO4H2O, pH 6.0), and cultured at 30°C, pH 6.0, 1 VVM aeration, and 100 rpm stirring speed. Feed medium I (165.3 g / L maltose, 128.55 g / L yeast extract, 6 g / L K2HPO4, 3 mL / L Tween 80, 9.042 g / L sodium acetate, 240 g / L MSG, 0.2 g / L MgSO4·7H2O, 0.04 g / L MnSO4H2O, pH 6.0) is added at 8-10 hours of culture, with a total feed volume of 300 mL. Feed medium II (100 g / L black sugar, 5 g / L phytone, 15 g / L yeast extract, 2 g / L K2HPO4, 1 mL / L Tween 80, 3.014 g / L sodium acetate, 424.8 g / L MSG, 0.2 g / L MgSO4·7H2O, 0.04 g / L MnSO4H2O, pH 6.0) is added at 22-26 hours of culture, with a total feed volume of 2,500 mL. Before the addition of feed medium II, the pH value is adjusted to 5.0 and aeration is stopped. The fermentation is collected after 144 hours of culture. The OD 600 The supernatant is collected and subjected to GABA concentration analysis.

[0062] (2) Fermentation culture with complex medium II and feeding medium III

[0063] Please refer to the flow chart of fermentation culture method 200 of FIG. 2, which includes strain thawing culture S201, pre-medium II liquid culture S202, complex medium II liquid culture S203, and feeding medium III S204. According to the embodiment of the present application, the Lactobacillus brevis ATIT-054 storage tube is taken out from -80°C, thawed at room temperature, and then the bacterial liquid is taken with a loop and streaked on FGM flat plate medium. After 48 hours of culture at 30°C in a facultative anaerobic environment, a single colony is selected and inoculated into a 50 mL centrifuge tube containing 32 mL of pre-medium II (25 g / L maltose, 12.5 g / L yeast extract, 12.5 g / L soybean protein, 2 mL / L Tween 80, 28 g / L MSG, 0.0076 g / L MnSO4·H2O, pH 6.0) and incubated at 30°C for 24 hours. The pre-culture bacterial liquid is added to a 500 mL serum bottle containing 320 mL of pre-culture medium, and incubated at 30°C for 16 hours. 150 mL of pre-culture bacterial liquid is added to a 5 L fermenter containing complex medium II (50 g / L maltose, 42.85 g / L yeast extract, 1.82 g / L K2HPO4, 1 mL / L Tween 80, 2.74 g / L sodium acetate, 1.475 g / L (NH4)2SO4, 50 g / L MSG, 0.018 g / L CaCO3, 0.18 g / L MgSO4·7H2O, 0.01 g / L MnSO4·H2O, pH 6.0), and incubated at 30°C, pH 6.0, 1 vvm aeration, and 100 rpm stirring speed. Feeding medium III (55 g / L maltose, 33 g / L yeast extract, 1.82 g / L K2HPO4, 1 mL / L Tween 80, 2.74 g / L sodium acetate, 1.475 g / L (NH4)2SO4, 400 g / L MSG, 0.018 g / L CaCO3, 0.18 g / L MgSO4·7H2O, 0.01 g / L MnSO4·H2O, pH 6.0) is added at 15-20 hours of culture, with a total feeding volume of 1,500 mL. The pH value is adjusted to 5.0 and aeration is stopped before feeding the feeding medium III. The fermentation is collected after 120 hours of culture. The OD 600 and the supernatant is collected for GABA concentration analysis.

[0064] 4. Spray drying of lactic acid bacteria fermentation broth

[0065] Referring to the flow chart of the preparation method 300 of the GABA-containing bacterial powder of FIG. 3, the method comprises a high-temperature sterilization process S301, addition of malt dextrin and uniform mixing S302, water dilution S303, and spray drying S304. Specifically, after the fermentation broth after the fermentation culture is treated at 121°C for 15 minutes, 10 mL of the sterilized broth is taken for solid content analysis. An appropriate amount of malt dextrin is added to the sterilized broth (the ratio of the solid content of the sterilized broth to the malt dextrin is 1:0.5-1.5), and after uniform stirring and mixing, sterile deionized water is added to dilute the total solid content to 18-23%. Spray drying is performed at an inlet air temperature of 160°C, an outlet air temperature of 90°C, and a feed flow rate of 1.8-2.0 L / min to produce the bacterial powder.

[0066] 5. Analysis of the antioxidant activity of the GABA-containing bacterial powder

[0067] (1) Analysis of the DPPH radical scavenging ability

[0068] The antioxidant capacity of the GABA-containing bacterial powder was determined by the DPPH (α, α-Dipenyl-β-picrylhydrazyl) radical scavenging ability experiment, and the experimental steps were modified according to the method of Shimamura et al. (2014). The GABA-containing bacterial powder was re-dissolved in deionized water to prepare solutions with concentrations of 60 mg / mL, 30 mg / mL, 15 mg / mL, 7.5 mg / mL, and 3.75 mg / mL, and the actual GABA concentrations in the solutions were 12.5 mg / mL, 6.25 mg / mL, 3.125 mg / mL, 1.563 mg / mL, and 0.781 mg / mL, respectively. Commercially available high-purity GABA (Sigma, USA) was used as a control group, and solutions with concentrations of 12.5 mg / mL, 6.25 mg / mL, 3.125 mg / mL, 1.563 mg / mL, and 0.781 mg / mL were prepared. After 100 μL of the sample to be tested, 400 μL of 0.1M Tris-HCl (pH 7.4), and 500 μL of DPPH (0.2 mM; dissolved in anhydrous ethanol) were mixed uniformly, they were placed in the dark at 25°C for 30 minutes. Centrifugation was performed at room temperature at 20,640 x g for 5 minutes. After the supernatant was removed and shaken uniformly, 200 μL of the supernatant was taken to measure the absorbance value at 517 nm using an infinite200Pro enzyme labeler. The lower the absorbance value, the stronger the sample's ability to scavenge DPPH. Scavenging effect % = [(AC-(AS-Asample blank)] / AC x 100 (Formula 1);

[0069] where AC = control group without sample addition; AS = sample; Asample blank = group without DPPH solution addition. The half maximal scavenging concentration (IC50) of the sample was further calculated using the scavenging rate. 50 ).

[0070] (2) ABTS·+ radical cation scavenging ability analysis

[0071] ABTS·+ radical cation scavenging ability analysis of the GABA-containing bacterial powder was performed, and the experimental procedure was as follows. The GABA-containing bacterial powder was dissolved in deionized water to prepare solutions with concentrations of 60 mg / mL, 30 mg / mL, 15 mg / mL, 7.5 mg / mL, and 3.75 mg / mL, and the actual GABA concentrations in the solutions were 12.5 mg / mL, 6.25 mg / mL, 3.125 mg / mL, 1.563 mg / mL, and 0.781 mg / mL, respectively. Commercially available high-purity GABA was used as a control group, and solutions with concentrations of 12.5 mg / mL, 6.25 mg / mL, 3.125 mg / mL, 1.563 mg / mL, and 0.781 mg / mL were prepared. 0.25 mL of catalase (44 U / mL), 0.25 mL of 1 mM ABTS solution, 1.5 mL of deionized water, and 0.25 mL of 500 μM H2O2 were mixed uniformly and placed in the dark. After the stable blue-green ABTS·+ radical cation was generated, 0.25 mL of the sample was added and allowed to react for 10 minutes. After centrifugation at 20,630 x g for 5 minutes at 25°C, 100 μL of the supernatant was taken to a 96-well plate, and the absorbance value was measured at 734 nm using an infinite 200 Pro microplate reader. The lower the absorbance value, the stronger the ABTS·+ radical cation scavenging ability of the sample. Scavenging effect (%) = [1 - (absorbance value of the sample at 734 nm) / (absorbance value of the control group without sample addition at 734 nm)] x 100 (Equation 2).

[0072] The half maximal scavenging concentration (IC50) of the sample was further calculated using the scavenging rate.

[0073] (3) Superoxide anion radical scavenging ability analysis

[0074] The SOD assay kit-WST (Dojindo, Japan) was used to analyze the superoxide anion radical scavenging ability of the GABA-containing bacterial powder. The experimental procedure was as follows. The GABA-containing bacterial powder was dissolved in deionized water to prepare solutions with concentrations of 60 mg / mL, 30 mg / mL, 15 mg / mL, 7.5 mg / mL, and 3.75 mg / mL. The actual GABA concentrations in the solutions were 12.5 mg / mL, 6.25 mg / mL, 3.125 mg / mL, 1.563 mg / mL, and 0.781 mg / mL, respectively. Commercially available high-purity GABA was used as a control group and was prepared into solutions with concentrations of 100 mg / mL, 50 mg / mL, 25 mg / mL, 12.5 mg / mL, and 6.25 mg / mL. Twenty μL of each solution was added to a 96-well plate, followed by the addition of 200 μL of water-soluble tetrazolium salt solution (WST working solution) and 20 μL of xanthine oxidase solution (enzyme working solution). The mixture was incubated at 37°C for 20 minutes. The absorbance value of the sample at 450 nm was measured using an infinite 200Pro microplate reader. The clearance rate was calculated using the following formula. Clearance rate (%) = [(A blank1 -A blank3 )-(A sample -A blank2 )] / (A blank1 -A blank3 ) x 100 (Equation 3),

[0075] where blank 1 represents the group without the sample, blank 2 represents the group without the addition of xanthine oxidase solution, and blank 3 represents the group without the sample and xanthine oxidase solution. The clearance rate was used to further calculate the half-clearance concentration of the sample.

[0076] 6. Analysis of the angiotensin I-converting enzyme inhibitory activity of the GABA-containing bacterial powder

[0077] The renin-angiotensin system (RAS) is an important humoral regulatory system composed of a series of peptide hormones and enzymes, and its main function is to regulate and maintain the balance of blood pressure and electrolytes in the human body. Angiotensin I converting enzyme (ACE) in the RAS is a key enzyme that triggers blood pressure elevation. ACE-inhibitor (ACEI) can inhibit the activity of ACE enzyme, which helps to prevent the conversion of angiotensin I to angiotensin II in the body, thereby slowing down the vasoconstriction effect, thus delaying the increase in blood pressure. In this experiment, the ACE Kit-WST kit (Dojindo, Japan) was used to analyze the ability of GABA-containing bacterial powder to inhibit ACE. The experimental operation steps are as follows. The GABA-containing bacterial powder was dissolved in deionized water to prepare solutions with concentrations of 3.750 mg / mL, 1.875 mg / mL, 0.938 mg / mL, 0.469 mg / mL, and 0.234 mg / mL. The actual GABA concentrations in the solutions were 0.783 mg / mL, 0.392 mg / mL, 0.196 mg / mL, 0.098 mg / mL, and 0.049 mg / mL, respectively. High-purity GABA available on the market was used as a control group, and solutions with concentrations of 100 mg / mL, 50 mg / mL, 25 mg / mL, 12.5 mg / mL, and 6.25 mg / mL were prepared. 20 μL of the above solutions were taken into a 96-well plate, and 20 μL of substrate buffer, 20 μL of enzyme working solution, and 200 μL of indicator working solution were added in sequence. After reacting at room temperature for 10 minutes, the absorbance value of the sample at 450 nm was measured using an infinite 200 Pro microplate reader. The inhibition rate was calculated using the following formula. Inhibition rate (%) = [(A blank1 -A sample ) / (A balnk1 -A blank2 )] × 100 (Formula 4),

[0078] where blank 1 represents the group without inhibitor; blank 2 represents the group without enzyme. The half-inhibitory concentration of the sample was further calculated using the inhibition rate.

[0079] 7. Analysis of the α-glucosidase inhibitory activity of GABA-containing bacterial powder

[0080] Alpha-glucosidase inhibitors can delay the digestion and absorption of carbohydrates, thus lowering postprandial blood glucose levels. This experiment used an α-glucosidase inhibitor screening kit (Abcam, UK) to analyze the inhibitory ability of GABA-containing bacterial powder on α-glucosidase. The experimental procedures are as follows: GABA-containing bacterial powder was reconstituted in deionized water to prepare solutions with concentrations of 550 mg / mL, 275 mg / mL, 137.5 mg / mL, 68.75 mg / mL, and 34.375 mg / mL, respectively. The actual GABA concentrations in these solutions were 115 mg / mL, 57.5 mg / mL, 28.75 mg / mL, 14.375 mg / mL, and 7.188 mg / mL, respectively. Commercially available high-purity GABA was used as a control group, with solutions prepared at concentrations of 100 mg / mL, 50 mg / mL, 25 mg / mL, 12.5 mg / mL, and 6.25 mg / mL. Pipette 10 μL of the above solution into each well of a 96-well plate, and sequentially add 10 μL of α-glucosidase enzyme solution and 80 μL of α-glucosidase assay buffer. Incubate at 25°C for 20 minutes. Then, add 20 μL of α-glucosidase substrate mix solution and immediately measure the absorbance of the sample at 410 nm using an Infinite 200Pro microplate reader; this is the absorbance value after 0 hours of reaction. Afterward, incubate at 25°C in the dark for 60 minutes, and measure the absorbance of the sample at 410 nm; this is the absorbance value after 60 minutes of reaction. Calculate the inhibition rate using the following formula: Inhibition rate (%) = [(Slope...] EC -Slope BC )-(Slope S -Slope BC ) / (Slope EC -Slope BC )]×100 (Equation 5),

[0081] Slope EC The absorbance difference between the enzyme control group and the control group over 0 to 60 minutes; Slope BC The difference in absorbance values ​​between the background control group and 60 minutes; Slope S The absorbance difference of the sample group from 0 to 60 minutes is represented. The half-maximal inhibitory concentration (MCC) of the sample is further calculated using the inhibition rate.

[0082] 8. Analysis of the ability of GABA-containing bacterial powder to promote collagen production by human skin fibroblast CCD966SK cells

[0083] The ability of GABA-containing bacterial powder to promote collagen production by human fibroblast CCD966SK cells was analyzed using a procollagen type I c-peptide (PIP) EIA kit (Takara, Japan) according to the following procedure. CCD966SK cells were purchased from the Bioresource Collection and Research Center (BCRC) of the Food Industry Development Institute, Taiwan. The medium used for culturing the cells contained 88.2% minimum essential media (MEM), 10% fetal bovine serum (FBS), 0.9% 100 mM sodium pyruvate, and 0.9% 100x MEM non-essential amino acids solution. The cell freezing tube was placed in a 37°C water bath. After complete thawing, the cell solution was added to a container containing an appropriate amount of medium and placed in an incubator for culturing under the following conditions: 37°C, 5% carbon dioxide, and 90% humidity. When the cell density reached more than 80%, the cells were subcultured. During subculture, the original medium was removed, the plate was rinsed with phosphate buffered saline (PBS), and trypsin was added to suspend the cells. Next, an appropriate amount of cells was added to a container containing fresh medium for culturing. After at least two generations of culturing the thawed cells, the CCD966SK cells were subcultured at a density of 7 x 10 3The CCD966SK cells were seeded in 96-well cell culture plates at a density of 1 cell / well. After overnight incubation, the culture medium in each well was replaced with 90 μL of fresh medium. The GABA-containing bacterial powder was dissolved in sterile ultrapure water to prepare solutions with concentrations of 239.4 mg / mL, 47.9 mg / mL, and 4.79 mg / mL, and the actual GABA concentrations in the solutions were 50 mg / mL, 10 mg / mL, and 1 mg / mL, respectively. Commercially available high-purity GABA was used as a control group, and solutions with concentrations of 50 mg / mL, 10 mg / mL, and 1 mg / mL were prepared. In each well of the 96-well cell culture plate, 10 μL of the sample was added, and 10 μL of sterile ultrapure water was added to the control group. After 48 hours of incubation, the culture medium was removed and stored, and the cells were used for survival rate detection, and the culture medium was used for PIP content determination. The cell survival rate was detected as follows. Fresh medium was mixed with a 5 mg / mL 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) PBS solution at a ratio of 5:1, and 120 μL was added to each well of the 96-well plate containing the CCD966SK cells. After 2 hours of incubation, the culture medium was removed, and 100 μL of dimethyl sulfoxide (DMSO) was added and incubated at 37°C for 15 minutes. The absorbance value at 570 nm was measured using an infinite 200Pro microplate reader. The cell survival rate was calculated using the following formula. Cell survival rate (%) = [(A sample -A blank ) / (A control -A blank )] x 100 (Formula 6),

[0084] where A sample is the absorbance value measured after different sample treatments; A blank is the absorbance value without cells; and A control is the absorbance value measured after treatment with sterile ultrapure water.

[0085] The PIP content determination method is performed according to the original manufacturer's instructions, which is briefly described as follows. Add 100 μL of Solution I (peroxidase-labeled anti-PIP monoclonal antibody) to each well of a 96-well plate, and then add 20 μL of sample or standard. Incubate at 37°C for 3 hours. After removing the liquid, rinse 4 times with Wash solution, and then add 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate solution. Incubate at 25°C for 15 minutes, and then add 100 μL of 1% HC1. Measure the absorbance value at 450 nm using an infinite 200 Pro microplate reader. Plot the standard curve of the absorbance values of the standard against the concentration, and calculate the PIP concentration in different samples. The PIP content in cells is calculated according to the following formula. PIP concentration (%) = [(PIP concentration sample / PIP concentration control )] x 100 (Formula 7),

[0086] wherein PIP concentration sample is the PIP concentration measured after treatment of different samples; and PIP concentration control is the PIP concentration measured after treatment with sterile ultrapure water.

[0087] 9. Anti-inflammatory ability analysis of GABA-containing bacterial powder

[0088] The effect of the sample to be tested on the secretion of nitric oxide (NO) is analyzed using a mouse macrophage cell line RAW264.7 to evaluate the anti-inflammatory ability, and the experimental method is described as follows. RAW264.7 is purchased from BCRC. The medium used for culturing cells contains 90% Dulbecco's modified Eagle's medium (DMEM) and 10% fetal bovine serum (FBS). The cell freezing tube is placed in a 37°C water bath. After complete thawing, the cell solution is added to a container containing an appropriate amount of medium and placed in an incubator for culture. The culture conditions are 37°C, 5% carbon dioxide, and 90% humidity. When the cell density reaches more than 80%, the cells are subcultured. During subculture, the cells are scraped with a cell scraper. Then, an appropriate amount of cells is added to a container containing fresh medium for culture. After at least 2 passages of thawed cells, RAW264.7 cells are subcultured to a density of 7 x 10 4The cells were seeded in 96-well cell culture plates at a density of 1 x 104cells / well. After overnight incubation, the culture medium was replaced with 88 μL of fresh medium. The GABA-containing bacterial powder was dissolved in sterile ultrapure water to prepare solutions with concentrations of 383 mg / mL, 359.1 mg / mL, 335.1 mg / mL, 311.2 mg / mL, and 287.3 mg / mL, and the actual GABA concentrations in the solutions were 80 mg / mL, 75 mg / mL, 70 mg / mL, 65 mg / mL, and 60 mg / mL, respectively. Commercially available high-purity GABA was used as a control group, and solutions with concentrations of 80 mg / mL, 75 mg / mL, 70 mg / mL, 65 mg / mL, and 60 mg / mL were prepared. Ten microliters of the sample or 10 μL of sterile ultrapure water were added to the 96-well plate, and the treatment was performed for 1 hour. Subsequently, 2 μL of 50 μg / mL lipopolysaccharide (LPS) was added to each well, and 2 μL of PBS was added to the negative control group. After 24 hours of incubation, the culture medium was removed, and the cells were used for survival rate detection, and the culture medium was used for NO concentration determination. The detection method of cell survival rate is as follows. Fresh medium and PBS solution containing 5 mg / mL MTT were mixed at a ratio of 5:1, and 120 μL was added to each well of the 96-well plate containing RAW264.7 cells. After 1 hour of incubation, the culture medium was removed, 100 μL of DMSO was added, and the plate was incubated at 37°C for 15 minutes. The absorbance value at 570 nm was measured using an infinite 200 Pro microplate reader. The calculation formula of cell survival rate is as follows.

[0089] The NO content determination method was performed according to the manufacturer's instructions, and is briefly described as follows. The A reagent and the B reagent in the Griess Reagent Nitrite Measurement Kit (Cell Signaling, US) were mixed at a ratio of 1:1. Twenty microliters of the experimental culture medium were added to each well of a 96-well transparent plate, and 80 μL of the sample or the standard was added. Subsequently, 100 μL of the mixed solution of the A reagent and the B reagent was added, and the absorbance value at 550 nm was measured using an infinite 200 Pro microplate reader. The standard curve was plotted by corresponding the absorbance values of the standard to the concentration, and the NO concentration in the different samples was calculated. The inhibition rate was calculated according to the following formula. Inhibition rate (%) = 1-[(NO concentration sample - NO concentration negative control) / (NO concentration positive control- NO concentration negative control)] x 100 (Formula 8),

[0090] wherein NO concentration sampleNO concentration of different samples after treatment and LPS-induced inflammatory reaction; NO concentration of positive control is the NO concentration of samples after treatment with sterile ultrapure water and LPS-induced inflammatory reaction; NO concentration of negative control is the NO concentration of samples after treatment with sterile ultrapure water but without LPS-induced inflammatory reaction.

[0091] 10. Analysis of anti-allergic effect of GABA-containing bacterial powder

[0092] (1) Immunoglobulin E (IgE) against 2,4-dinitrophenol (DNP) and dinitrophenol-bovine serum albumin (DNP-BSA) induced allergy model

[0093] RBL-2H3, a rat basophilic cell line, was treated with anti-DNP IgE and DNP-BSA to induce degranulation, and the degree of inhibition of β-hexosaminidase release by the test sample was analyzed to evaluate the anti-allergic ability. The RBL-2H3 was purchased from BCRC. The culture medium used for the cells contained 83.2% MEM, 15% FBS, 0.9% 100 mM sodium pyruvate, and 0.9% 100x MEM non-essential amino acids solution. The cell freezing tube was placed in a 37°C water bath. After complete thawing, the cell solution was added to a container containing an appropriate amount of culture medium and placed in an incubator for culture under the following conditions: 37°C, 5% carbon dioxide, and 90% humidity. When the cell density reached more than 80%, the cells were subcultured. During subculture, the original culture medium was removed, the plate surface was rinsed with PBS, and trypsin was added to suspend the cells. Then, an appropriate amount of cells was added to a container containing fresh culture medium for culture. After at least 2 passages of thawed cells, the RBL-2H3 cells were seeded in a 24-well or 96-well cell culture plate at a density of 5x10 5 cells or 1x10 4 cells, respectively, for degranulation inhibition and cell survival rate tests. The culture medium containing 0.3 μg / mL of anti-DNP IgE was used for sensitization.

[0094] After the 24-well cell culture plates for the degranulation inhibition reaction test were incubated overnight, the culture solution was removed and the cells were rinsed with 400 μL of degranulation buffer (200 mM sodium chloride, 5 mM potassium chloride, 5.6 mM glucose, 0.4 mM magnesium chloride, 1 mM calcium chloride, 25 mM PIPES, 40 mM sodium hydroxide, and 0.1% bovine serum albumin, pH 7.2), and 180 μL of degranulation buffer was added to each well. The bacterial powder containing GABA was dissolved in sterile ultrapure water to prepare solutions with concentrations of 143.6 mg / mL, 95.8 mg / mL, and 47.9 mg / mL, and the actual GABA concentrations in the solutions were 30 mg / mL, 20 mg / mL, and 10 mg / mL, respectively. Commercially available high-purity GABA was used as a control group, and solutions with concentrations of 30 mg / mL, 20 mg / mL, and 10 mg / mL were prepared. Each well was added with 20 μL of the sample, and pretreated for 0.5 hours; the negative control group and the control group were added with 20 μL of sterile ultrapure water; and the positive control group was added with 20 μL of 200 μM quercetin. Then, 2 μL of 30 μg / mL DNP-BSA was added to each well, and the negative control group was added with 2 μL of degranulation buffer, and the degranulation reaction was performed for 1 hour. After the 24-well cell culture plates were left to stand on ice for 5 minutes, 50 μL of the supernatant was taken to a 96-well transparent plate, and 50 μL of p-nitrophenyl-N-acetyl-β-D-glucosaminide (PNAG) solution (46 mM sodium citrate, 54 mM citric acid, and 1 mM PNAG, pH 4.5) was added to each well, and the reaction was performed at 37°C for 1 hour. Then, 100 μL of stop solution (54 mM sodium carbonate, 46 mM sodium bicarbonate, pH 10) was added to each well, the absorbance value at 405 nm was measured by an infinite 200 Pro microplate reader, and the inhibition rate was calculated according to the following formula. Inhibition rate (%) = 1 - [(A sample -A sample blank )-(A negative control -A control blank )] / [(A control -A control blank )-(A negative control -Anegative control blank)] x 100 (Formula 9),

[0095] wherein A sample is the absorbance value measured after different samples or quercetin treatment and then DNP-BSA treatment; A control is the absorbance value measured after sterile ultrapure water treatment and then DNP-BSA treatment; and A negative controlA is the absorbance value measured after the sterile ultrapure water treatment but without DPN-BSA treatment; A sample blank A is the absorbance value of different samples or quercetin; A control blank A is the absorbance value of the degranulation buffer.

[0096] After the 96-well cell culture plate for cell survival test was incubated overnight, 90 μL of fresh culture medium was added to each well. The bacterial powder containing GABA was dissolved in sterile ultrapure water to prepare solutions with concentrations of 143.6 mg / mL, 95.8 mg / mL and 47.9 mg / mL, and the actual GABA concentrations in the solutions were 30 mg / mL, 20 mg / mL and 10 mg / mL, respectively. Commercially available high-purity GABA was used as a control group, and solutions with concentrations of 30 mg / mL, 20 mg / mL and 10 mg / mL were prepared. 10 μL of the sample was added to each well. After 24 hours of incubation, 120 μL of culture medium was added to each well, which was mixed with 5:1 of PBS solution containing 5 mg / mL MTT. After 2 hours of incubation, the culture medium was removed, 100 μL of DMSO was added to each well, and the absorbance value at 570 nm was measured by an infinite 200Pro microplate reader after 15 minutes of incubation at 37°C. The cell survival rate was calculated using the cell survival calculation formula (Formula 6).

[0097] (2) Calcium ion carrier A23187-induced anaphylaxis model

[0098] The rat basophilic cell line RBL-2H3 was treated with the calcium ion carrier A23187 to induce degranulation of the cells, and the degree of inhibition of the release of β-hexosaminidase by the degranulation reaction by the test sample was analyzed to evaluate the anti-allergic ability, and the experimental method is described as follows. The RBL-2H3 culture method is as described above. After the thawed cells were cultured for at least 2 generations, RBL-2H3 cells were seeded in 24-well or 96-well cell culture plates at a density of 5×10 5 cells or 1×10 4 cells, respectively, and the degranulation inhibition and cell survival tests were performed.

[0099] The 24-well cell culture plates for the degranulation inhibition reaction test were incubated overnight, and then the culture medium was removed and the cells were rinsed with 400 μL of degranulation buffer (200 mM sodium chloride, 5 mM potassium chloride, 5.6 mM glucose, 0.4 mM magnesium chloride, 1 mM calcium chloride, 25 mM PIPES, 40 mM sodium hydroxide, and 0.1% bovine serum albumin, pH 7.2). Then, 180 μL of degranulation buffer was added to each well. The bacterial powder containing GABA was dissolved in sterile ultrapure water to prepare solutions with concentrations of 143.6 mg / mL, 95.8 mg / mL, 47.9 mg / mL, and 23.9 mg / mL. The actual GABA concentrations in the solutions were 30 mg / mL, 20 mg / mL, 10 mg / mL, and 5 mg / mL, respectively. Commercially available high-purity GABA was used as a control group, and solutions with concentrations of 30 mg / mL, 20 mg / mL, 10 mg / mL, and 5 mg / mL were prepared. Each well was added with 20 μL of the sample for pretreatment for 0.5 hours. The negative control group and the control group were added with 20 μL of sterile ultrapure water, and the positive control group was added with 20 μL of 200 μM quercetin. Then, 2 μL of 200 μM A23187 was added to each well, and the negative control group was added with 2 μL of degranulation buffer for degranulation reaction for 1 hour. The 24-well cell culture plates were placed on ice for 5 minutes, and then 50 μL of supernatant was taken to a 96-well transparent plate. Then, 50 μL of p-nitrophenyl-N-acetyl-β-D-glucosaminide (PNAG) solution (46 mM sodium citrate, 54 mM citric acid, and 1 mM PNAG, pH 4.5) was added to each well, and the reaction was carried out at 37°C for 1 hour. Then, 100 μL of stop solution (54 mM sodium carbonate, 46 mM sodium bicarbonate, pH 10) was added to each well, and the absorbance value at 405 nm was measured using an infinite 200Pro microplate reader. The inhibition rate was calculated according to the following formula. Inhibition rate (%) = 1 - [(A sample -A sample blank )-(A negative control -A control blank )] / [(A control -A control blank )-(A negative control -Anegative control blank)] × 100 (Formula 10),

[0100] wherein A sample is the absorbance value measured after different samples or quercetin treatment and then A23187 treatment; A control is the absorbance value measured after sterile ultrapure water treatment and then A23187 treatment; Anegative control Absorbance value measured after treatment with sterile ultrapure water but without A23187 treatment; A sample blank Absorbance value of different samples or quercetin solutions; A control blank Absorbance value of degranulation buffer.

[0101] After the 96-well cell culture plate for cell survival test was incubated overnight, each well was replaced with 90 μL of fresh culture medium. The GABA-containing bacterial powder was dissolved with sterile ultrapure water and high-purity GABA on the market to prepare solutions with GABA concentrations of 10 mg / mL, 20 mg / mL, and 30 mg / mL, and 10 μL of the sample was added to each well. Fresh culture medium and PBS solution containing 5 mg / mL MTT were mixed at a ratio of 5:1, and 120 μL of the mixture was added to each well to replace the culture solution in the 96-well plate. After 2 hours of incubation, the culture solution was removed, 100 μL of DMSO was added to each well, and the plate was incubated at 37°C for 15 minutes. The absorbance value at 570 nm was measured using an infinite 200Pro microplate reader. The cell survival rate was calculated using the cell survival calculation formula (Formula 6).

[0102] Results

[0103] 1. Screening of GABA-producing lactic acid bacteria

[0104] One hundred and thirteen lactic acid bacteria were streaked onto MRS plate medium and incubated at 30°C in a facultative anaerobic environment for 48 hours. After the colonies grew, a single colony was inoculated into FGM screening medium containing 3% MSG. After incubation at 30°C for 24 hours, the supernatant was collected for TLC analysis. As shown in FIG. 4, ten of the 113 lactic acid bacteria had better GABA-producing ability, namely ATIT-013, ATIT-016, ATIT-017, ATIT-053, ATIT-054, ATIT-066, ATIT-067, ATIT-068, ATIT-069, and ATIT-070. Among them, ATIT-016 was Lactobacillus plantrum, and the rest were Lactobacillus brevis. Colorimetric analysis showed that ATIT-054 had a higher GABA yield (FIG. 5), and this strain was used for subsequent fermentation culture tests.

[0105] 2. Fermentation culture of GABA-producing lactic acid bacteria

[0106] (1) Fermentation culture using complex medium I with feed medium I and II

[0107] After the secondary pre-culturing of ATIT-054 in the pre-culture medium I, 10% of the pre-cultured bacteria was inoculated into a 5L fermenter containing 1.5L of the complex medium I. The fermentation was carried out at 30°C, pH 6.0, 1vvm aeration and 100rpm agitation speed for 8-10 hours. At this time, the pH signal began to rise, indicating that the carbon source in the medium had been depleted, and the feeding of the feeding medium I could be carried out. After 22-26 hours of culture, when the feeding of the feeding medium I was completed, the pH value was adjusted to 5.0, and the aeration was stopped. The feeding of the feeding medium II was then carried out. The fermentation was harvested at 144 hours. The GABA concentration in the culture broth was detected, and the results showed that the GABA concentration in the harvested broth was 159.9g / L [Figure 6(A)].

[0108] (2) Fermentation culture using complex medium II and feeding medium III

[0109] After the secondary pre-culturing of ATIT-054 in the pre-culture medium II, 10% of the pre-cultured bacteria was inoculated into a 5L fermenter containing 1.5L of the complex medium II. The fermentation was carried out at 30°C, pH 6.0, 1vvm aeration and 100rpm agitation speed for 15-20 hours, and the feeding of the feeding medium III was carried out. The fermentation was harvested at 120 hours. The GABA concentration in the culture broth was detected, and the results showed that the GABA concentration in the harvested broth was 139.9g / L [Figure 6(B)].

[0110] 3. Spray drying of GABA-containing lactic acid bacteria fermentation broth

[0111] After sterilizing the fermentation broth of ATIT-054 at 121°C for 15 minutes, samples were taken for solid content analysis. An appropriate amount of malt dextrin was added to the sterilized broth and stirred until evenly mixed. Sterile deionized water was then added to dilute the total solid content to 18-23%. Spray drying was carried out under specific conditions, and GABA content analysis was carried out on the spray-dried powder. Colorimetric quantitative analysis showed that the powder obtained by spray drying the fermentation broth obtained by fermentation culture using complex medium I and feeding medium I and II contained 21.49% GABA; the powder obtained by spray drying the fermentation broth obtained by fermentation culture using complex medium II and feeding medium III contained 20.89% GABA. Subsequently, the powder obtained by sterilization and spray drying of the fermentation broth obtained by fermentation culture using complex medium II and feeding medium III was subjected to functional analysis.

[0112] 4. Antioxidant activity analysis of GABA-containing bacterial powder

[0113] The GABA-containing bacterial powder was diluted to an appropriate concentration, and then subjected to DPPH radical scavenging ability test, ABTS+radical scavenging ability test, and superoxide anion radical scavenging ability test. The results showed that the GABA-containing bacterial powder had antioxidant ability, and the antioxidant effect increased with the increase of the concentration of the spray-dried powder [Fig. 7(A), Fig. 8(A), and Fig. 9(A)]. The half scavenging concentration of the GABA-containing bacterial powder for DPPH radical, ABTS+radical, and superoxide anion radical was 8.61 mg / mL, 4.75 mg / mL, and 6.40 mg / mL, respectively (here, the calculation was based on the concentration of GABA, and if the calculation was based on the weight of the bacterial powder, the values would be 41.52 mg / mL, 22.73 mg / mL, and 30.70 mg / mL, respectively); the commercially available high-purity GABA did not have obvious antioxidant ability [Fig. 7(B), Fig. 8(B), and Fig. 9(B)]. The above results showed that the antioxidant ability of the GABA-containing bacterial powder of the present application was better than that of high-purity GABA.

[0114] 5. Inhibition of α-glucosidase by GABA-containing bacterial powder

[0115] The GABA-containing bacterial powder was diluted to an appropriate concentration, and then subjected to α-glucosidase inhibition ability test. The results showed that the GABA-containing bacterial powder had α-glucosidase inhibition ability, and the half inhibition concentration was 78.92 mg / mL (here, the calculation was based on the concentration of GABA, and if the calculation was based on the weight of the bacterial powder, the value would be 377.88 mg / mL) [Fig. 10(A)]; the commercially available high-purity GABA did not have obvious α-glucosidase inhibition ability [Fig. 10(B)]. The above results showed that the α-glucosidase inhibition ability of the GABA-containing bacterial powder of the present application was better than that of high-purity GABA.

[0116] 6. Inhibition of angiotensin converting enzyme I by GABA-containing bacterial powder

[0117] The GABA-containing bacterial powder was diluted to an appropriate concentration, and then subjected to angiotensin converting enzyme I inhibition ability test. The results showed that the GABA-containing bacterial powder had angiotensin converting enzyme I inhibition ability, and the half inhibition concentration was 0.667 mg / mL (here, the calculation was based on the concentration of GABA, and if the calculation was based on the weight of the bacterial powder, the value would be 3.20 mg / mL) [Fig. 11(A)]; the commercially available high-purity GABA also had angiotensin converting enzyme I inhibition ability, and the half inhibition concentration was 17.672 mg / mL [Fig. 11(B)]. The above results showed that the angiotensin converting enzyme I inhibition ability of the GABA-containing bacterial powder of the present application was better than that of high-purity GABA.

[0118] 7. GABA-containing bacterial powder promotes the ability of human skin fibroblast CCD966SK to produce collagen protein

[0119] After diluting the GABA-containing bacterial powder to an appropriate concentration, the ability of the GABA-containing bacterial powder to promote the production of collagen protein by human skin fibroblast CCD966SK was tested. The results showed that the GABA-containing bacterial powder and commercially available high-purity GABA did not have significant cytotoxicity at a GABA concentration of 0.1 to 5 mg / mL [Fig. 12(A)], and both could promote the production of collagen protein by human skin fibroblast CCD966SK. The GABA-containing bacterial powder increased the collagen protein content by 1.69 times at a GABA concentration of 5 mg / mL (here, the GABA concentration was calculated; if the weight of the bacterial powder was calculated, it would be 23.94 mg / mL); commercially available high-purity GABA could only increase the collagen protein content by 1.17 times at the same concentration [Fig. 12(B)]. The above results showed that the GABA-containing bacterial powder of the present application had a better effect than high-purity GABA on promoting the production of collagen protein by human skin fibroblast CCD966SK.

[0120] 8. Anti-inflammatory ability of GABA-containing bacterial powder

[0121] After diluting the GABA-containing bacterial powder to an appropriate concentration, the ability of the GABA-containing bacterial powder to inhibit the production of nitric oxide by mouse macrophage RAW264.7 was tested as an analysis of anti-inflammatory ability. The results showed that the GABA-containing bacterial powder and commercially available high-purity GABA did not have significant cytotoxicity at 6 to 8 mg / mL [Fig. 13(A)]. The GABA-containing bacterial powder could inhibit the production of nitric oxide; when the GABA concentration in the powder was 8 mg / mL (here, the GABA concentration was calculated; if the weight of the bacterial powder was calculated, it would be 38.30 mg / mL), the NO production inhibition rate was 76.8%; commercially available high-purity GABA products did not have any effect on inhibiting the production of NO [Fig. 13(B)]. The above results showed that the GABA-containing bacterial powder of the present application had an anti-inflammatory effect, while commercially available high-purity GABA did not.

[0122] 9. Anti-allergic ability of GABA-containing bacterial powder

[0123] The GABA-containing bacterial powder was diluted to an appropriate concentration, and the degree of inhibition of the release of β-hexosaminidase from the degranulation reaction of rat basophilic cell RBL-2H3 was analyzed. The RBL-2H3 cells sensitized by IgE and the RBL-2H3 cells sensitized by A23187 did not show significant cytotoxicity after being treated with the GABA-containing bacterial powder and commercially available high-purity GABA for 24 hours [Fig. 14(A) and Fig. 15(A)]. After the IgE-sensitized RBL-2H3 cells were treated with DNP-HSA antigen, the GABA-containing bacterial powder inhibited the degranulation reaction, and at a concentration of 2 mg / mL (calculated based on the concentration of GABA; if calculated based on the weight of the bacterial powder, the concentration was 9.57 mg / mL), the degranulation reaction was inhibited by 58.1%; the commercially available high-purity GABA at a concentration of 2 mg / mL only had an inhibitory effect of 8.3% [Fig. 14(B)]. After the RBL-2H3 cells were treated with A23187, the GABA-containing bacterial powder inhibited the degranulation reaction, and at a concentration of 2 mg / mL (calculated based on the concentration of GABA; if calculated based on the weight of the bacterial powder, the concentration was 9.57 mg / mL), the degranulation reaction was inhibited by 103.4%; the commercially available high-purity GABA had no effect [Fig. 15(B)]. The above results show that the GABA-containing bacterial powder has a better anti-allergic effect.

[0124] In summary, the Lactobacillus brevis ATIT-054 of the present application can produce a high content of GABA after being cultured according to the culture method of the present application. The bacterial culture liquid is then made into a powder, which has the effects of removing free radicals, inhibiting angiotensin-converting enzyme I, inhibiting α-glucosidase, promoting collagen production, anti-inflammation, and anti-allergy. Furthermore, the bacterial powder can be formulated into different dosage forms, such as liquid, tablet, capsule, and jelly, and applied in the fields of food, cosmetics, and medicine.

Claims

1. Lactobacillus brevis ATIT-054, which is deposited with the NITE Patent Microorganisms Depositary National Institute of Technology and Evaluation (NPMD) under accession number NITE BP-04112.

2. A method for preparing a γ-aminobutyric acid-containing powder, wherein: the γ-aminobutyric acid-containing powder comprises Lactobacillus brevis ATIT-054, which is deposited with the NITE Patent Microorganisms Depositary National Institute of Technology and Evaluation (NPMD) under accession number NITE BP-04112; and the method comprises the steps of: (a) inoculating the bacterial cells of the lactic acid bacteria into a plate medium to perform solid-state culture to form colonies; (b) inoculating the colonies cultured in step (a) into a pre-culture medium to perform liquid-state culture to obtain a bacterial cell-containing liquid-state culture solution; (c) inoculating the bacterial cell-containing liquid-state culture solution of step (b) into a fermentation tank containing a complex culture medium to perform liquid-state culture, and then adding a feed medium to obtain a bacterial solution; (d) sterilizing the bacterial solution of step (c); and (e) mixing the sterilized bacterial solution of step (d) with excipients and drying to obtain a bacterial powder.

3. The method of claim 2, wherein the pre-culture medium comprises pre-culture medium I, pre-culture medium II, or a combination thereof.

4. The method of claim 2, wherein the complex culture medium comprises complex culture medium I, complex culture medium II, or a combination thereof.

5. The method of claim 2, wherein the feed medium comprises feed medium I, feed medium II, feed medium III, or a combination thereof.

6. The method of claim 2, wherein the combination of culture media comprises the pre-culture medium I, the complex culture medium I, the feed medium I, and / or the feed medium II.

7. The method of claim 2, wherein the combination of culture media comprises the pre-culture medium II, the complex culture medium II, and the feed medium III.

8. The method of claim 2, wherein the pre-culture medium comprises 25-50 g / L of a carbon source, 10-20 g / L of a yeast extract, 10-20 g / L of a nitrogen source, 1-3 mL / L of Tween 80, 20-35 g / L of sodium glutamate, and 0.005-0.01 g / L of inorganic salts, and has a pH of 5.5-6.

5. ​ ​ ​ ​ ​ ​ ​ 9. The method according to claim 2, wherein the complex medium comprises 25-50 g / L of a carbon source, 10-50 g / L of a yeast extract, 0-2 g / L of a nitrogen source, 0.5-1.5 mL / L of Tween 80, 30-50 g / L of sodium glutamate, and 3-6 g / L of inorganic salts, and has a pH of 5.5-6.

5.

10. The method according to claim 2, wherein the feed medium comprises 50-170 g / L of a carbon source, 30-130 g / L of a yeast extract, 0-5 g / L of a nitrogen source, 1-3 mL / L of Tween 80, 200-430 g / L of sodium glutamate, and 4-16 g / L of inorganic salts, and has a pH of 5.5-6.

5.

11. The method according to claim 2, wherein the drying of step (d) comprises spray drying, freeze drying, drum drying, vacuum drying, foam drying, or fluidized bed drying.

12. Use of Lactobacillus brevis ATIT-054 and / or an active substance thereof for the preparation of a composition for scavenging free radicals, wherein the Lactobacillus brevis ATIT-054 is deposited with the NITE Patent Microorganisms Depositary National Institute of Technology and Evaluation (NPMD) under accession number NITE BP-04112.

13. The use according to claim 12, wherein the scavenging of free radicals refers to scavenging of DPPH free radicals, ABTS+ free radicals, or superoxide anion free radicals.

14. Use of Lactobacillus brevis ATIT-054 and / or an active substance thereof for the preparation of a composition for inhibiting angiotensin converting enzyme I, wherein the Lactobacillus brevis ATIT-054 is deposited with the NITE Patent Microorganisms Depositary National Institute of Technology and Evaluation (NPMD) under accession number NITE BP-04112.

15. Use of Lactobacillus brevis ATIT-054 and / or an active substance thereof for the preparation of a composition for inhibiting alpha-glucosidase, wherein the Lactobacillus brevis ATIT-054 is deposited with the NITE Patent Microorganisms Depositary National Institute of Technology and Evaluation (NPMD) under accession number NITE BP-04112.

16. Use of Lactobacillus brevis ATIT-054 and / or an active substance thereof for the production of a composition for promoting collagen production, wherein the Lactobacillus brevis ATIT-054 is deposited with the NITE Patent Microorganisms Depositary National Institute of Technology and Evaluation (NPMD) under accession number NITE BP-04112.

17. The use according to claim 16, wherein the promoting collagen production refers to promoting collagen production by human skin fibroblast CCD966SK.

18. Use of Lactobacillus brevis ATIT-054 and / or an active substance thereof for the production of a composition for anti-inflammation, wherein the Lactobacillus brevis ATIT-054 is deposited with the NITE Patent Microorganisms Depositary National Institute of Technology and Evaluation (NPMD) under accession number NITE BP-04112.

19. Use of Lactobacillus brevis ATIT-054 and / or an active substance thereof for the production of a composition for anti-allergy, wherein the Lactobacillus brevis ATIT-054 is deposited with the NITE Patent Microorganisms Depositary National Institute of Technology and Evaluation (NPMD) under accession number NITE BP-04112.

20. The use according to any one of claims 12 to 19, wherein the Lactobacillus brevis ATIT-054 and / or an active substance thereof is in the form of a bacterial powder, a liquid, a tablet, a capsule or a jelly.

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