Method for producing GABA and low-molecular-weight collagen through fermentation by using lactobacillus brevis BJ20 (KCTC11377BP) and lactobacillus plantarum BJ21 (KCTC18911p), and use thereof

Abstract

The present invention relates to a method for simultaneously producing GABA and low-molecular-weight collagen with a molecular weight of less than 300 Da by using Lactobacillus brevis BJ-20 (KCTC11377BP) and L. plantarum BJ21 (KCTC18911P). A culture medium composition containing GABA and low-molecular-weight collagen, which are prepared according to the preparation method, can exhibit the effects of improving joint function, improving sleep activity, and improving skin condition.

Description

A manufacturing method for producing GABA and low-molecular-weight collagen through fermentation using Lactobacillus brevis BJ20 (KCTC11377BP) and Lactobacillus plantarum BJ21 (KCT18911P), and its use

[0001] The present invention relates to a manufacturing method for producing GABA and low-molecular-weight collagen through fermentation using Lactobacillus brevis and Lactobacillus Plantarum, and to a use thereof.

[0002] GABA (gamma-aminobutyric acid) is a neurotransmitter found in the brain and spinal cord of vertebrates. Its physiological role is to increase cerebral blood flow and oxygen supply, thereby enhancing brain cell metabolic function. GABA is known to be effective in stabilizing nerves, relieving stress, enhancing memory, regulating blood pressure, alleviating depression, preventing stroke and dementia, alleviating insomnia, improving obesity, alleviating menopausal symptoms, and improving diabetes.

[0003] As the diverse functional properties of GABA become known, active research is underway to utilize it not only as a pharmaceutical but also as a functional ingredient. In particular, numerous studies are being conducted to increase GABA content in foods that naturally contain small amounts of GABA, such as brown rice, green tea, malt, and cabbage. However, GABA production from natural plant sources is limited, and research using microorganisms is necessary for mass production.

[0004] Collagen, particularly low-molecular-weight collagen, is known to improve skin elasticity and moisturizing properties. As skin ages, it undergoes various changes. The epidermis, dermis, and subcutaneous tissue thin, and the composition of the extracellular matrix (ECM) changes, leading to a loss of elasticity. Collagen production, which accounts for 70-80% of the ECM, decreases rapidly, and collagen, elastin, glycosaminoglycans, and fibronectin, which form the skin's connective tissue, become oxidized and lose their function. These changes lead to a loss of elasticity, wrinkles, and an aged appearance. While active research is being conducted on skin moisturizing and wrinkle improvement using existing collagen and elastin, high-molecular-weight collagen suffers from difficulty in percutaneous absorption. It has been reported that smaller collagen molecules increase their absorption rate in the body.

[0005] Accordingly, as modern people's interest in a healthy life increases, there is a demand for the development of functional materials such as GABA and low-molecular collagen that have the effect of improving sleep and skin barrier damage.

[0006] The purpose of the present invention is to provide a method for simultaneously producing GABA and low-molecular-weight collagen of less than 300 Da through fermentation using Lactobacillus brevis and Lactobacillus Plantarum.

[0007] Another object of the present invention is to provide a culture composition containing GABA and low molecular collagen that exhibits effects of improving skin condition, improving sleep activity, and improving joint health.

[0008] The present disclosure provides a manufacturing method for simultaneously producing GABA and low-molecular-weight collagen of less than 300 Da using Lactobacillus brevis BJ-20 (KCTC11377BP) and Lactobacillus Plantarum BJ21 (KCT18911P).

[0009] The above manufacturing method may include a) a step of adding an enzyme to polymer collagen to hydrolyze it to produce a collagen hydrolysate, b) a step of producing a medium including the collagen hydrolysate and one or more selected from monosodium glutamate (MSG), L-glutamic acid, or a mixture thereof, c) a step of inoculating the medium with Lactobacillus brevis BJ-20 (KCTC11377BP) and performing a primary fermentation, and d) a step of inoculating the medium with Lactobacillus Plantarum BJ21 (KCT18911P) and performing a secondary fermentation.

[0010] In the above step a), the enzyme may include at least one selected from collagenase, protease, peptidase, trypsin, chymotrypsin, papain, bromelain, protease K, and serine protease.

[0011] The above step a) may be to add an enzyme to high molecular collagen and hydrolyze it at 40 to 80°C for 8 to 18 hours.

[0012] In the above step b), the medium may contain 1 to 30 wt% of MSG and L-glutamic acid based on the total weight.

[0013] It may further include a step of autoclaving and cooling the medium manufactured in step b).

[0014] The above steps c) and d) may be performed independently of each other at a temperature of 30 to 80°C and for 6 to 48 hours.

[0015] The above manufacturing method may further include a step of e) heat-treating the secondary fermented mixture at high temperature to extract fermentation metabolites.

[0016] The above heat treatment may be performed at a temperature of 100 to 150°C for 5 to 30 minutes.

[0017] In the above step a), the polymer collagen may be extracted from fish skin, cow skin, pig skin or livestock bones.

[0018] In the step b), the medium may further include a carbon source selected from the group consisting of glucose, sucrose, maltose, fructose, lactose, xylose, galactose, arabinose, and mixtures thereof; a nitrogen source selected from the group consisting of yeast extract, soytone, peptone, beef extract, tryptone, casitone, and mixtures thereof; and an inorganic component selected from the group consisting of magnesium sulfate, sodium acetate, manganese sulfate, ferric sulfate, calcium chloride, polysorbate, and mixtures thereof.

[0019] The weight ratio of the above-mentioned generated GABA and low molecular collagen may be 1:1.5 to 1:10.

[0020] The present disclosure provides a culture medium composition containing GABA and low molecular collagen, prepared according to one embodiment of the present disclosure.

[0021] The above culture composition may contain glycine and GABA in a weight ratio of 1:2 to 1:20.

[0022] The above culture composition may be for improving joint function.

[0023] The above culture composition may be for improving sleep activity.

[0024] The above culture composition may be for improving skin condition.

[0025] The manufacturing method of the present disclosure can simultaneously produce GABA, low-molecular-weight collagen, and postbiotics, and the culture composition manufactured thereby can have the effects of improving skin condition, improving sleep activity, and improving joint health.

[0026] Figure 1 shows the results of observing chondrocytes in the experimental and control groups using H&E staining (A) and the results of quantifying the thickness of chondrocytes (B).

[0027] Figure 2 shows the results of observing chondrocytes in the experimental and control groups using Safranin-O staining (A) and the results of quantifying the staining area (B).

[0028] Figure 3 shows the results of measuring the expression of MMP-3 (A), MMP-13 (B), and ADAMTS5 (C) enzymes in the experimental and control groups, respectively.

[0029] Figure 4 shows the results of evaluating anti-inflammatory efficacy such as cytotoxicity (A), cell viability (B), and NO production (C) in the experimental and control groups, respectively.

[0030] Figure 5 shows the results of analyzing the production of inflammatory cytokines IL-6(A) and IL-1β in the experimental and control groups, respectively.

[0031] Figure 6 shows the results of measuring wake time (A), total sleep time (B), REM time (C), Non-REM time (D), and Orexin expression (E) in the experimental and control groups, respectively, as measured by SleepSign Ver. 3 software (Kissei Comtec, Nagano, Japan).

[0032] Figure 7 shows the results of observing pigmentation in the experimental and control groups (A) and the numerical results thereof (B), respectively.

[0033] Figure 8 shows the mRNA expression levels of AP-1 and MMPs (MMP1, 3, 9) in the experimental and control groups, respectively.

[0034] Figure 9 shows the results of observing the epidermal thickness in the stratum corneum in the experimental and control groups (A) and the results of quantifying the thickness (B).

[0035] Figure 10 shows the expression levels of antioxidant enzymes SOD and 9-OHdG in the experimental and control groups, respectively.

[0036] Figure 11 shows the expression levels of inflammatory cytokines (IL-6, IL-1α, IL-10, TNF-α) in the experimental and control groups, respectively.

[0037] Figure 12 shows skin elasticity (A) and changes in skin elasticity (B) in the experimental and control groups, respectively.

[0038] The present invention will be described in detail below. Terms used herein, unless specifically defined, should be interpreted as generally understood by those skilled in the art. The drawings and examples in this specification are intended to facilitate the understanding and practice of the present invention by those skilled in the art. Content that may obscure the gist of the invention may be omitted from the drawings and examples, and the present invention is not limited to the drawings and examples.

[0039] The singular forms used in this specification are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0040] Additionally, the numerical range used in the present invention includes lower and upper limits and all values ​​within that range, increments logically derived from the shape and width of the defined range, all doubly defined values, and all possible combinations of upper and lower limits of numerical ranges defined in different shapes. Unless otherwise specifically defined in the specification of the present invention, values ​​outside the numerical range that may arise due to experimental error or rounding of values ​​are also included in the defined numerical range.

[0041] In addition, units used in the specification of the present invention without special mention are based on weight, and for example, units of % or ratio mean weight% or weight ratio.

[0042] In this specification, terms such as include, have, and have mean that a feature or component described in the specification exists, and unless specifically limited, do not preclude the possibility that one or more other features or components may be added.

[0043] The present disclosure provides a method for producing a culture composition containing GABA and low-molecular-weight collagen, which simultaneously produces GABA and low-molecular-weight collagen of less than 300 Da using Lactobacillus brevis BJ-20 (KCTC11377BP) and Lactobacillus Plantarum BJ21 (KCT18911P).

[0044] The above GABA is an abbreviation for gamma-aminobutyric acid (γacid; GABA), a non-protein amino acid well known as the main inhibitory neurotransmitter in the central nervous system of animals. GABA is involved in many physiological mechanisms, and in animals, it activates cerebral blood flow, increases oxygen supply, improves the metabolic function of brain cells, and is also effective in regulating the secretion of prolactin and growth hormones, lowering blood pressure, relieving pain, and alleviating insomnia.

[0045] In a manufacturing method according to one embodiment of the present disclosure, GABA produced by a mixed strain of Lactobacillus brevis BJ-20 (KCTC11377BP) and Lactobacillus Plantarum BJ21 (KCT18911P) has a higher GABA production rate than other lactic acid bacteria, so that production can be improved, and the mixed strain can simultaneously produce GABA and low-molecular-weight collagen of less than 300 Da.

[0046] Specifically, the Lactobacillus brevis BJ20 is a Gram-positive rod-shaped bacterium isolated from salted seafood and is optimized for GABA (γ-aminobutyric acid) production. The lactic acid bacteria exhibit optimal growth conditions at 37°C, pH 7, and a NaCl concentration of 1%.

[0047] Additionally, the Lactobacillus plantarum BJ21 strain is a Gram-positive bacillus isolated from kimchi and possesses anti-cholesterol and antioxidant properties. The optimal growth conditions for this lactic acid bacterium are 37°C and pH 6.5.

[0048] According to one embodiment, the manufacturing method of the present disclosure may include a) a step of adding an enzyme to polymer collagen to hydrolyze it to produce a collagen hydrolysate, b) a step of preparing a medium including the collagen hydrolysate and one or more selected from monosodium glutamate (MSG), L-glutamic acid, or a mixture thereof, c) a step of inoculating the medium with Lactobacillus brevis BJ-20 (KCTC11377BP) and performing a primary fermentation, and d) a step of inoculating the medium with Lactobacillus Plantarum and performing a secondary fermentation.

[0049] According to one embodiment, in the step a), the enzyme may include at least one selected from amylase, lipase, cellulase, collagenase, protease, peptidase, trypsin, chymotrypsin, papain, bromelain, protease K, and serine protease, and specifically, may include at least one selected from α-amylase, lipase, cellulase, and protease, and more specifically, may be a prozyme including α-amylase, lipase, cellulase, and protease.

[0050] The above prozyme may include alpha-lamylase having an enzyme activity of 2000 SKBU / gram, lipase having an enzyme activity of 200 U / gram, cellulase having an enzyme activity of 50 CU / gram, and protease having an enzyme activity of 8 GDU / gram, respectively.

[0051] According to one embodiment, the step a) may be to add an enzyme to high molecular collagen and hydrolyze it at 40 to 80°C for 8 to 18 hours, and specifically, to hydrolyze it at 50 to 60°C for 10 to 14 hours.

[0052] According to one embodiment, in step b), the medium may contain 1 to 30 wt% of MSG and L-glutamic acid based on the total weight, and specifically, 1 to 10 wt%. If it is lower than the above range, it may be difficult to produce sufficient GABA, and if it is higher than the above range, the fermentation time by Lactobacillus brevis BJ-20 (KCTC11377BP) may be prolonged, resulting in a decrease in hourly production efficiency. In addition, the L-glutamic acid may be in the form of L-glutamate, and can maintain the pH at which the enzyme maximally activates glutamate (MSG) and L-glutamic acid are converted into GABA by glutamate decarboxylase, an enzyme of fermentation lactic acid bacteria, thereby improving the production speed and yield of GABA. According to one embodiment, the manufacturing method of the present disclosure may further include a step of autoclaving and cooling the medium prepared in step b).

[0053] GABA exists in general grains, but the content of GABA in grains is extremely small, 1-40 mg / 100 g in general rice, 4-8 mg / 100 g in brown rice, and 10-100 mg / 100 g in germinated brown rice. On the other hand, when GABA is produced through a manufacturing method using Lactobacillus brevis BJ-20 (KCTC11377BP) and Lactobacillus Plantarum BJ21 (KCT18911P) according to an embodiment of the present invention, it can be produced in a very high concentration, thereby maximizing the efficacy of GABA.

[0054] Specifically, by sequentially culturing Lactobacillus brevis BJ-20 (KCTC11377BP) and Lactobacillus Plantarum BJ21 ((KCT18911P), which are lactic acid bacteria for fermentation, on the above-mentioned MSG (mono sodium glutamate) and L-glutamic acid, GABA is produced, and MSG (mono sodium glutamate) and L-glutamic acid can be converted into GABA by glutamate decarboxylase, which is an enzyme of the above-mentioned lactic acid bacteria for fermentation. Lactobacillus Sakei, Lactobacillus hilgardii, Lactobacillus Although Lactobacillus helveticus and Lactococcus lactis can also be used, the present invention sequentially ferments Lactobacillus brevis BJ-20 (KCTC11377BP) and Lactobacillus Plantarum BJ21 ((KCT18911P) to have a superior GABA conversion rate compared to other lactic acid bacteria, resulting in a greater production of GABA, and can simultaneously produce lower molecular collagen, so a composition using the same can exhibit improved effects.

[0055] According to one embodiment, steps c) and d) may be performed independently of each other at a temperature of 30 to 80°C and for 6 to 48 hours, and specifically, independently of each other at a temperature of 30 to 40°C and for 20 to 28 hours.

[0056] According to one embodiment, the manufacturing method of the present disclosure may further include the step of e) heat-treating the secondary fermented mixture at high temperature to extract fermentation metabolites.

[0057] Specifically, the heat treatment may be performed at a temperature of 100 to 150°C for 5 to 30 minutes, and more specifically, at a temperature of 120 to 140°C for 15 to 30 minutes.

[0058] According to one embodiment, in step a), the polymer collagen may be extracted from fish skin, cow skin, pig skin or livestock bones.

[0059] Collagen is a protein with a triple helix structure composed of three intertwined polypeptide chains. It is particularly high in hydroxyproline, and is known for its anti-aging and moisturizing effects. Collagen is derived from animal bone, cartilage, skin, tendon, and ligament tissues. Its molecular weight is 100,000 Da, and its three-phase structure of polypeptide bonds gives it a total molecular weight of approximately 300,000 Da.

[0060] The above hydroxyproline is rarely found in proteins other than collagen, and the amount of hydroxyproline is used as an indicator of the amount of synthesis and decomposition of the entire collagen protein.

[0061] Gelatin is extracted by hydrolyzing the above collagen and has a molecular weight of approximately 100,000 Da, and collagen peptides are produced by hydrolyzing collagen or gelatin and have a total molecular weight of 5,000 Da or less.

[0062] The collagen used in the manufacturing method according to one embodiment of the present invention may be selected from the group consisting of collagen, gelatin, and collagen peptides, and may be collagen peptides, but is not limited thereto.

[0063] According to one embodiment, in step b), the medium may further include a carbon source selected from the group consisting of glucose, sucrose, maltose, fructose, lactose, xylose, galactose, arabinose, and mixtures thereof; a nitrogen source selected from the group consisting of yeast extract, soytone, peptone, beef extract, tryptone, casitone, and mixtures thereof; and an inorganic component selected from the group consisting of magnesium sulfate, sodium acetate, manganese sulfate, ferric sulfate, calcium chloride, polysorbate, and mixtures thereof.

[0064] Specifically, the carbon source may be included in an amount of 0.1 to 3 wt%, the nitrogen source in an amount of 0.1 to 3 wt%, and the inorganic component in an amount of 0.01 to 0.1 wt%, based on the total weight of the medium, but is not limited thereto.

[0065] If the content of the above nitrogen source is less than 0.1 wt%, MSG (Mono Sodium Glutamate) is used as a nitrogen source due to a lack of nitrogen source, so the concentration of GABA produced decreases, and if it exceeds 3 wt%, spray drying may not proceed well due to the Maillard reaction of the remaining nitrogen source and carbon source, which is not desirable.

[0066] According to one embodiment, the weight ratio of GABA and low molecular collagen produced in the manufacturing method of the present disclosure may be 1:1.5 to 1:10, and specifically 1:2.5 to 1:8.

[0067] The present disclosure provides a culture medium composition containing GABA and low-molecular-weight collagen manufactured according to a manufacturing method according to one embodiment of the present disclosure.

[0068] The above low-molecular collagen-containing culture composition may include postbiotics derived from Lactobacillus brevis and Lactobacillus Plantarum, and the postbiotics may exhibit improved effects for the purposes described below through synergistic action with GABA and low-molecular collagen included in the above composition.

[0069] According to one embodiment, the culture composition may contain glycine and GABA in a weight ratio of 1:2 to 1:20, specifically, in a weight ratio of 1:5 to 1:15, and more specifically, in a weight ratio of 1:8 to 1:12. The culture composition of the present disclosure may exhibit improved effects for the purposes described below by containing glycine and GABA in the above-described weight ratio as active ingredients.

[0070] A culture composition according to one embodiment of the present disclosure may be for improving joint function.

[0071] A culture composition according to one embodiment of the present disclosure may be for improving sleep activity.

[0072] A culture composition according to one embodiment of the present disclosure may be for improving skin condition.

[0073] Hereinafter, a method for manufacturing a culture medium composition containing GABA and low-molecular-weight collagen according to the present invention will be described in more detail through specific examples. However, the following examples are merely references for explaining the present invention in detail, and the present invention is not limited thereto, and can be implemented in various forms. Furthermore, the terminology used in the description of the present invention is merely for the purpose of effectively describing specific examples and is not intended to limit the present invention.

[0074] [Example 1]

[0075] 58 g of prozyme enzymes including alpha-amylase, lipase, and cellulase were added to 1160 g of high-molecular-weight collagen (Hainan Pure Peptide Technology Co,ltd., Fish Collagen Peptide (1000 Da)), and the high-molecular-weight collagen was hydrolyzed at 55°C±2°C for 12 hours to prepare a collagen hydrolysate.

[0076] (Specifically, the prozyme comprises alpha-lamylase having an enzyme activity of 2000 SKBU / gram, lipase having an enzyme activity of 200 U / gram, cellulase having an enzyme activity of 50 CU / gram, and protease having an enzyme activity of 8 GDU / gram.)

[0077] Afterwards, 1160 g of the collagen hydrolyzate, 12 g of glucose (Qwon, hydrous crystalline glucose), 12 g of yeast extract (LALLEMAND, powdered yeast extract-2), 220 g of L-glutamic acid (Samin Chemical, L-glutamic acid), and 2576 ml of water were added, and sterilized at 121°C for 15 minutes.

[0078] After inoculating 10 ml of Lactobacillus brevis BJ-20 (KCTC11377BP) culture solution cultured for 24 hours in a sterilized medium, fermentation was performed by culturing at 37°C for 24 hours to produce a fermented solution (A).

[0079] Afterwards, 10 ml of Lactobacillus Plantarum BJ21 ((KCT18911P) culture solution cultured for 24 hours was inoculated into the fermentation solution (A), and fermentation was performed by culturing at 37°C for 24 hours to prepare a fermentation solution (B).

[0080] In order to extract postbiotic from the above fermentation solution (B), it was cooled at 121°C for 15 minutes, and then sterilized and spray-dried to obtain 1300 g of powdered fermentation product, which was named Gabalagen.

[0081]

[0082] [Experimental Example 1] Analysis of GABA content

[0083] Each 4g of Gabalagen manufactured through Example 1 was dissolved in a 0.02N hydrochloric acid solution, and then 200μL of OPA solution prepared by adding 1mL MeOH to 50mg of refrigerated OPA (OPhthaldialdehyde, Sigma79760), 800μL of borate, and 20μL of MPA (Mercaptopropionic acid, Sigma 63768) were added to prepare a measurement sample.

[0084] A SUPERSIL column (4.6 mm 250 mm) was used as the HPLC column, and GABA was detected with a 338 nm UV detector. The results of the GABA content analysis are shown in Table 1 below.

[0085] GABA wt% (GABA weight produced in g)Glutamate wt%Example 114.6% (207.9g)0.8% (11.39g)

[0086] <Generated GABA weight = GABA weight% x product weight / 100%>

[0087]

[0088] [Experimental Example 2] Collagen Molecular Weight Analysis

[0089] The molecular weight of the low-molecular collagen contained in the collagen raw material, Gabalagen, manufactured through Example 1, was analyzed. The molecular weight was measured using GPC (gel permeation chromatography) equipped with a TOSOH G3000PWXL column. The solvent used was DW:Acetic acid (30:70), and the detector used was an RI detector. The raw collagen had a molecular weight of 1028 Da, and the collagen of Example 1 had a molecular weight of 275 Da, respectively.

[0090] In Comparative Examples 1 and 2, it can be confirmed that collagen was not depolymerized, whereas in Example 1 of the present invention, the raw collagen was depolymerized.

[0091]

[0092] [Experimental Example 3] Analysis of the effect of improving joint function (1)

[0093] Ten-week-old C57BL / 6J mice were used, and the mice were raised under conditions of temperature 20-22℃, humidity 55%, and photoperiod 12 hours light / 12 hours dark, with free access to water and food. The gabaragen manufactured in Example 1 was administered to the mice by oral administration, and the concentrations of the fermentation product were set to low concentration (20 mg / kg), medium concentration (150 mg / kg), and high concentration (300 mg / kg). The experimental period was set to 8 weeks, and a 1-week acclimation period was conducted before the experiment.

[0094] The experimental group was composed as follows.

[0095] Sham group (n=10): control group that did not undergo surgery

[0096] DMM group (n=10): group that underwent medial meniscus tear (DMM) surgery

[0097] DMM + L-GABA (300 mg / kg) group (n=10): A group administered high-dose GABA after DMM surgery.

[0098] DMM + M-GABA (75 mg / kg) group (n=10): group administered low-dose GABA after DMM surgery

[0099] DMM + H-GABA (150 mg / kg) group (n=10): Group administered intermediate concentration of GABA after DMM surgery

[0100] DMM + IND (Indometacin, 3 mg / kg) group (n=10): Group administered indomethacin after DMM surgery

[0101] Some of the above experimental groups underwent destabilized medial meniscus (DMM) surgery to induce osteoarthritis, and gabaragen or indomethacin at the same concentration as described above was administered orally daily for 8 weeks, starting the week following the surgery. After 8 weeks, each mouse was sacrificed and the results of the joint function improvement evaluation are shown in Figures 1 to 7.

[0102]

[0103] Analysis of changes in cartilage thickness

[0104] The results of observing chondrocytes through H&E staining and Safranin-O staining of the experimental and control groups, respectively, are shown in Fig. 1A and Fig. 2A, and the results of quantifying the thickness of cartilage are shown in Fig. 1B and Fig. 2B.

[0105] According to Figures 1 and 2, it can be confirmed that the thickness of cartilage significantly increased and degenerative arthritis was recovered in the group administered with gabaragen compared to the DMM group.

[0106] Analysis of inflammatory enzyme expression

[0107] The results of measuring the mRNA expression levels of the three enzymes MMP-3, MMP-13, and ADAMTS5 in each experimental and control group are shown in Figure 5.

[0108] According to Figure 3, compared to the Sham group, the DMM group showed increased expression of MMP-3, MMP-13, and ADAMTS5, and it was confirmed that the expression of inflammatory enzymes was significantly reduced in the group administered with gabaragen.

[0109]

[0110] [Experimental Example 4] Analysis of the effect of improving joint function (2)

[0111] RAW 264.7 macrophages were cultured and inflammatory responses were induced using LPS. Subsequently, they were treated with low (50 μg / ml), medium (100 μg / ml), and high (200 μg / ml) concentrations of gabalate or Dexa (Dexamethasone). Anti-inflammatory efficacy evaluation results, including cytotoxicity, cell viability, and NO production, are shown in Figure 4, and the results of analyzing the production of inflammatory cytokines IL-6 and IL-1β are shown in Figure 5.

[0112] Cell viability

[0113] According to Figure 4A, cell viability was 100% in all experimental and control groups. Meanwhile, according to Figure 4B, cell viability decreased in both the experimental and control groups when inflammation occurred due to LPS treatment, and the experimental group treated with gabaragen showed a mild decrease in viability.

[0114] NO (Nitric Oxide) production

[0115] According to Figure 4C, the amount of NO produced rapidly increased with LPS treatment, which actively triggered an inflammatory response, but it can be confirmed that NO production was significantly reduced in the experimental group treated with gabaragen.

[0116] Proinflammatory cytokine production

[0117] According to Figure 5, the production of IL-6 and IL-1β increased with LPS treatment, but it can be confirmed that the production of the cytokines was significantly suppressed in the experimental group treated with gabaragen.

[0118]

[0119] [Experimental Example 5] Analysis of the effect of improving sleep activity

[0120] Seven-week-old adult male Sprague Dawley rats were used, and were raised under conditions of temperature 23-25℃, humidity 45-60%, light intensity 200-300 LUX 12 h / day, and noise level 40 dB or less, with free access to food and water. Gabaragen was administered orally to each experimental group for 5 days as follows. Afterwards, pentobarbital (40 mg / kg) was injected intraperitoneally into each experimental group to induce sleep, and the effect of gabaragen on sleep was analyzed through EEG and EMG measurements.

[0121]

[0122] Normal group (n=6): No sleep induction, oral saline solution administration

[0123] Negative control group (n=6): Pentobarbital 40 mg / kg intraperitoneally administered, saline solution orally administered

[0124] L-GABA group (n=6): Pentobarbital 40 mg / kg intraperitoneally, gabagen 100 mg / kg orally

[0125] H-GABA group (n=6): Pentobarbital 40 mg / kg intraperitoneally, gabapentin 250 mg / kg orally

[0126] Positive control group (n=6): Pentobarbital 40 mg / kg intraperitoneally, Diazepam (DZP) 10 mg / kg orally

[0127]

[0128] Electroencephalography (EEG) & Electromyography (EMG) Surgery

[0129] Each experimental group was anesthetized with intraperitoneal pentobarbital (50 mg / kg). The skin was shaved and an incision was made from the front of the eyes to behind the ears. Two holes were drilled just below the bregma (AP: -1.5, ML: ±1.5) and one above the lambda (AP: 0, ML: -2). The membrane was pierced with a 1 ml syringe needle. Screws with soldered wires were secured into the two holes below the bregma using a screwdriver. The two upper parts of the connector were soldered to wire #3, and the middle screw was secured to the hole above the lambda using a screwdriver. The two lower parts (with the wires connected) were sewn to the neck muscles. The wires on the skull were bent, and the wires for the neck muscles were placed under the skin. Rebase powder and liquid were mixed to an appropriate concentration and applied generously to ensure the device was securely in place. The incisions were sutured with black silk and Rebase was reapplied to secure the device in place. The patients were allowed to rest for one week after surgery.

[0130]

[0131] EEG measurement and analysis

[0132] After surgical recovery, the rats were acclimated to experimental conditions and then sleep measurements were performed. EEG recordings were performed (for 12 hours, from 8:00 PM to 8:00 AM). The measured EEG and recording results were analyzed using SleepSign Ver. 3 software (Kissei Comtec, Nagano, Japan), a specialized animal sleep analysis program. The ratios of Wake, REM, and Non-REM sleep were recorded for 9 hours after the measurement, and the results are shown in Figure 6.

[0133] Figure 6A shows the ratio of wakefulness times for each experimental and control group. The normal group had the highest wakefulness time, while the drug-administered groups (L-GABA, H-GABA), the control group, and the positive control group all showed decreased wakefulness times. This suggests that GABAgen has the effect of reducing wakefulness time and promoting sleep induction.

[0134] Figure 6B shows the percentage of total sleep time in each experimental group. Compared to the normal group, the total sleep time increased in the control and drug administration groups, and in particular, the total sleep time increased the most in the H-GABA administration group. This indicates that GABAgen is effective in increasing total sleep time.

[0135] Figure 8C shows the ratio of REM sleep time in each experimental group. Compared to the normal group, the ratio of total REM sleep period increased in the control group and drug administration group, and in particular, the REM sleep time increased significantly in the L-GABA and H-GABA administration groups.

[0136] Figure 6D shows the ratio of NON-REM sleep time in each experimental group. In the control group, NON-REM sleep time tended to increase slightly, and in the L-GABA and H-GABA administration groups, NON-REM sleep time increased significantly in a concentration-dependent manner.

[0137] Figure 6E shows the expression level of Orexin in the lateral hypothalamus (LH) region of each experimental group. Compared to the normal group, the expression of Orexin tended to decrease in the negative control group and the positive control group, but no significance was found. Compared to the control group, the expression of Orexin tended to decrease in H-GABA, but no significance was found.

[0138]

[0139] [Experimental Example 6] Analysis of skin condition improvement effects

[0140] In order to evaluate the efficacy of GABALAGEN prepared in Example 1 on skin damage caused by UV rays, female mice (7 weeks old) of the HRM-2 strain were irradiated with UVB after a 7-day acclimation period to produce a freckle model. Out of the total 13-day UVB irradiation schedule of 8 times, irradiation was performed once every other day for 5 minutes each, 6 times, until the 11th day. To promote melanin production, irradiation was performed twice a day for 5 minutes each after the 11th day. After the freckle model was produced, GABALAGEN at various concentrations and indicator substances GABA, GPH, and Collagen were orally administered to each group for 28 days. Specifically, 150, 250, and 350 mg / kg of GABALAGEN prepared in Example 1 were orally administered to the freckle model mice every day for 28 days, and the mice were then sacrificed to evaluate the skin condition improvement effect as follows. The results are shown in FIGS. 7 to 12.

[0141] Analysis of pigmentation changes

[0142] The results of analyzing the degree of pigmentation by sacrificing mice from each of the above experimental groups are shown in Figure 7.

[0143] According to Figure 7, after UVB exposure, the experimental group not treated with Gabaragen showed increased pigmentation, whereas the group administered with Gabaragen showed a significant decrease in pigmentation.

[0144] Analysis of skin wrinkle formation efficacy

[0145] The changes in the mRNA expression levels of AP-1, Mmp1, Mmp3, and Mmp9, which are skin wrinkle-generating factors, were shown in Figure 8 by sacrificing mice from each of the above experimental groups.

[0146] According to Figure 8, the mRNA expression of AP-1 increased in the UVB exposure group but decreased in the gabaragen administration group, and the decrease was maintained at doses above 250 mg / kg. In addition, the mRNA expression of MMPs (MMP1, 3, 9) increased in the UVB irradiation group but decreased in a concentration-dependent manner in the gabaragen administration group.

[0147] Analysis of the efficacy of controlling epidermal thickness

[0148] The results of analyzing the effect of regulating epidermal thickness by sacrificing mice from each of the above experimental groups are shown in Figure 9.

[0149] According to Figure 9, the epidermal layer thickened due to keratinocyte proliferation caused by UVB exposure, and it can be seen that the thickness of the epidermis was significantly reduced when Gabaragen was administered.

[0150] Skin antioxidant efficacy analysis

[0151] The results of confirming the expression of antioxidant enzymes SOD (Superoxide Dismutase) and 9-OHdG (Superoxide Dismutase) by sacrificing mice from each of the above experimental groups are shown in Figure 10.

[0152] According to Figure 10, it can be seen that the expression level of antioxidant enzymes decreased in the UVB exposure group, but increased significantly in the gabagen administration group.

[0153] Analysis of inflammation control efficacy

[0154] The results of confirming the expression of inflammatory cytokines (IL-6, IL-1α, IL-10, TNF-α) by sacrificing mice from each of the above experimental groups are shown in Figure 11.

[0155] According to Figure 11, it can be seen that the expression level of each cytokine increased in the UVB irradiation group, but decreased significantly in the gabaragen treatment group.

[0156] Analysis of skin elasticity control efficacy

[0157] The results of sacrificing mice from each of the above experimental groups to check changes in skin elasticity are shown in Figure 12.

[0158] According to Figure 12, it can be confirmed that skin elasticity decreased in the UVB irradiation group, but significantly increased in the Gabaragen treatment group.

[0159]

[0160] As described above, the present invention has been described through specific matters and limited examples and comparative examples, but these are provided only to help a more general understanding of the present invention, and the present invention is not limited to the above examples, and those skilled in the art to which the present invention pertains can make various modifications and variations based on this description.

[0161] Therefore, the idea of ​​the present invention should not be limited to the described embodiments, and all things that are equivalent or equivalent to the following claims as well as the claims are considered to fall within the scope of the idea of ​​the present invention.

Claims

1. A manufacturing method for simultaneously producing GABA and low-molecular-weight collagen of less than 300 Da using Lactobacillus brevis BJ-20 (KCTC11377BP) and Lactobacillus Plantarum BJ21 (KCT18911P).

2. In paragraph 1, a) A step of adding enzymes to high molecular collagen to hydrolyze it and produce collagen hydrolysate. b) a step of preparing a medium including the collagen hydrolyzate; and one or more selected from monosodium glutamate (MSG), L-glutamic acid, or a mixture thereof; c) Inoculating Lactobacillus brevis BJ-20 (KCTC11377BP) into the above medium and performing primary fermentation; and d) A manufacturing method comprising a step of inoculating Lactobacillus Plantarum BJ21 (KCT18911P) into the above medium and performing secondary fermentation.

3. In paragraph 2, A manufacturing method, wherein in the step a), the enzyme comprises at least one selected from collagenase, protease, peptidase, trypsin, chymotrypsin, papain, bromelain, protease K, and serine protease.

4. In paragraph 2, The above step a) is a manufacturing method in which an enzyme is added to high molecular collagen and hydrolyzed at 40 to 80°C for 8 to 18 hours.

5. In paragraph 2, A manufacturing method, wherein in the step b), the medium contains 1 to 30 wt% of MSG and L-glutamic acid based on the total weight.

6. In paragraph 2, A manufacturing method further comprising a step of autoclaving and cooling the medium manufactured in step b).

7. In paragraph 2, A manufacturing method wherein the above steps c) and d) are performed independently at a temperature of 30 to 80°C and for 6 to 48 hours.

8. In paragraph 2, e) A manufacturing method further comprising a step of heat-treating the secondary fermented mixture at high temperature to extract fermentation metabolites.

9. In paragraph 8, A manufacturing method wherein the above heat treatment is performed at a temperature of 100 to 150°C for 5 to 30 minutes.

10. In paragraph 2, A manufacturing method wherein, in the above step a), the polymer collagen is extracted from fish skin, cow skin, pig skin or livestock bones.

11. In paragraph 2, In the step b), the medium comprises a carbon source selected from the group consisting of glucose, sucrose, maltose, fructose, lactose, xylose, galactose, arabinose, and mixtures thereof; A nitrogen source selected from the group consisting of yeast extract, soytone, peptone, beef extract, tryptone, casitone and mixtures thereof; and Magnesium sulfate, sodium acetate, manganese sulfate, ferric sulfate, calcium chloride, polysorbate and A manufacturing method further comprising an inorganic component selected from the group consisting of mixtures thereof.

12. In paragraph 1, A manufacturing method wherein the weight ratio of the above-mentioned GABA and low molecular collagen is 1:1.5 to 1:

10.

13. A culture medium composition containing GABA and low-molecular-weight collagen, manufactured according to the manufacturing method of any one of claims 1 to 12.

14. In paragraph 13, A culture medium composition containing GABA and low-molecular-weight collagen, wherein the culture medium composition comprises glycine and GABA in a weight ratio of 1:2 to 1:

20.

15. In paragraph 13, The above culture composition is a culture composition containing GABA and low molecular collagen for improving joint function.

16. In paragraph 13, The above culture medium composition is a culture medium composition containing GABA and low molecular collagen for improving sleep activity.

17. In paragraph 13, The above culture solution composition is a culture solution composition containing GABA and low molecular collagen for improving skin condition.

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