Application of a high-GABA-producing Lactobacillus brevis strain with the ability to regulate intestinal flora in the production of plant-based fermented milk

Abstract

The present invention discloses an application of a high-yield GABA Lactobacillus brevis strain with the ability to regulate intestinal flora in the production of plant-based fermented milk, and belongs to the field of food biotechnology. The present invention provides an application of Lactobacillus brevis CGMCCNO.14385 in the preparation of antioxidant health products, medicines or health products for improving gastrointestinal flora, and medicines for alleviating and / or treating obesity caused by a high-fat diet. The strain can adapt to the production of various types of plant-based fermented milk. Without adding any other culture medium components, the strain ferments and enzymatically hydrolyzes oat milk to produce 499 mg / L of GABA. The strain is suitable for freeze-drying to prepare direct-injection bacterial agents and direct-injection fermentation. Lactobacillus brevis D17 has no potential virulence factors and drug resistance risks, is non-toxic to mice, and has excellent obesity-delaying effects. It can significantly reduce the weight gain caused by a high-fat diet, and has important application value in plant-based fermentation and other food production.

Description

Technical Field

[0001] The invention relates to an application of a high-GABA-yielding Lactobacillus brevis strain capable of regulating intestinal flora in the production of plant-based fermented milk, belonging to the field of food biotechnology. Background Art

[0002] In recent years, people's living standards have been improving, and animal-derived foods such as meat, eggs, and milk have accounted for an increasing proportion of the dietary structure. As a result, the incidence of many chronic diseases such as hypertension and obesity has also increased year by year. However, plant-derived foods do not contain substances such as saturated fatty acids and cholesterol that promote the above chronic diseases, and are rich in many bioactive substances with antioxidant activity such as polyphenols and flavonoids, which are also in line with the current global vegetarian trend. Therefore, plant-derived foods are gradually becoming an important choice for people to adjust to a healthy dietary structure.

[0003] Since plant-based matrices usually have some unpleasant flavors such as bitterness and beany smell, and the nutrition is relatively simple. However, plant-based matrices can not only eliminate unpleasant odor substances after being fermented by lactic acid bacteria, but also further enrich the taste and nutrition. Therefore, fermenting plant-based beverages with lactic acid bacteria has gradually become a research hotspot in plant-based beverages. At present, there are not many fully plant-based lactic acid bacteria fermented beverages on the market, most of which are mainly oat products. The sales market is mainly in developed regions such as Europe, the United States, and Japan. The domestic industry is still in its infancy. Lactic acid bacteria used in fully plant-based fermentation include dairy-derived lactic acid bacteria, such as Lactobacillus bulgaricus, Streptococcus thermophilus, Lactobacillus paracasei, Lactobacillus acidophilus, and plant-derived lactic acid bacteria such as Lactobacillus plantarum.

[0004] Since lactic acid bacteria are deficient in most amino acids, they require a higher nutritional environment for growth. Plant-based matrices are mostly simple in nutrition and cannot directly provide certain amino acids, vitamins and other growth factors required for the growth and reproduction of lactic acid bacteria. Therefore, lactic acid bacteria, especially small-genome lactic acid bacteria such as Lactobacillus brevis, may lack many genes encoding enzymes that hydrolyze proteins in plant-based matrices, and have higher nutritional requirements, which makes fermenting plant-based matrices full of difficulties and challenges.

[0005] In the previous study, the research group has screened a strain of Lactobacillus brevis D17 (preservation number: CGMCC NO.14385) with high γ-aminobutyric acid (GABA) production from the acid fermentation mash of liquor. It can produce 26.1g / L of GABA under uncontrolled pH fermentation conditions, which is 2.3 times higher than the typical strain Lactobacillus brevis ATCC 367; under controlled pH conditions, the GABA production is as high as 177.74g / L. GABA is an important inhibitory neurotransmitter in the central nervous system, with multiple physiological functions such as anti-obesity, anticonvulsant, lowering blood pressure, improving brain function and calming the mind. Moreover, the lactic acid bacteria in the liquor brewing system have been domesticated for a long time in a high-starch, high-acid environment, making them have high starch matrix growth adaptability and high acid resistance. Based on the above characteristics, the GABA-producing Lactobacillus brevis from the liquor brewing system has good plant-based fermentation and probiotic properties.

[0006] Lactobacillus brevis is a common lactic acid bacteria existing in traditional fermented foods. For example, Lactobacillus brevis is the dominant lactobacillus in the early stage of liquor brewing (Qiao Meiling et al., Bacterial community succession and self-assembly mechanism during the fermentation of traditional light-flavor liquor [J]. Food Science, 2023, 44(22): 139-148), and is also the dominant lactobacillus in the late stage of pickling kimchi (Liu, et al. Isolation and characterization of bacteria that produce quorum sensing molecules during the fermentation and deterioration of pickles [J]. International Journal of Food Microbiology, 2022, 379: 109869). In addition, Lactobacillus brevis has also been included in the GRAS certification list of the US Food and Drug Administration and the QPS certification list of the European Food Safety Authority. In the field of food fermentation, Lactobacillus brevis is often used to ferment dairy products (Seo, et al. γ-aminobutyric acid production in skim milk co-fermented with Lactobacillus brevis 877G and Lactobacillus sakei 795 [J]. Food Science and Biotechnology, 2013, 22 (3): 751-755), dough (Guo Xiaoyu et al., Processing and rheological properties of chickpea sourdough enriched with γ-aminobutyric acid [J]. Food and Fermentation Industries, 2023, 49 (15): 215-220), and fermented bean curd (Bao, et al. Influence of Lactobacillus brevis on metabolite changes in bacteria-fermented sufu [J]. Journal of Food Science and Biotechnology, 2013, 22 (3): 751-755). Science, 2020, 85(1):165-172), vegetables and fruits (Yang Rude, Modern Industrial Microbiology [M]. Guangzhou: South China University of Technology Press, 2001:337-340.), etc., in order to achieve the purpose of enhancing nutrition and improving flavor. In summary, these studies show that Lactobacillus brevis is a fermentation strain that has long existed in traditional fermented foods and has been used, and can be used in food production.

[0007] However, in the field of plant-based fermentation, Lactobacillus brevis is a rare lactic acid bacteria for plant-based fermentation. Currently, only a few literatures involve Lactobacillus brevis fermentation of plant-based, specifically: Lactobacillus brevis JCM 1059 fermentation of date residue (HASEGAWA M, et al. Gamma-aminobutyric acid fermentation with date residue by a lactic acid bacterium, Lactobacillus brevis [J]. Journal of Bioscience and Bioengineering, 2018, 125 (3): 316-319), Lactobacillus brevis FPA3709 fermentation of black soybean (Ko CY, et al. Gamma-aminobutyric acid production in black soybean milk by Lactobacillus brevis FPA3709 and the antidepressant effect of the fermented product on a forced swimming rat model [J]. Process Biochemistry, 2013, 48 (4): 559-568), Lactobacillus brevis grx-SOS04 fermentation of soybean (CN116536186 A), fermentation of bran and legumes by Lactobacillus brevis ATCC 14869 (XIE C, et al. Fermentation of cereal, pseudo-cereal and legume materials with Propionibacteriumfreudenreichii and Levilactobacillus brevis for vitamin B12 fortification[J]. Lwt-Food Science and Technology, 2021, 137: 9), fermentation of quinoa by Lactobacillus brevis TR055 (JESKE S, et al. Polyol-producing lactic acid bacteria isolated from sourdough and their application to reduce sugar in a quinoa-based milk substitute[J]. International Journal of Food Microbiology, 2018, 286: 31-36).Among them, only the plant-based fermented milk of Lactobacillus brevis JCM 1059 and Lactobacillus brevis FPA3709 involved the detection of GABA content. The GABA yield of Lactobacillus brevis JCM 1059 in fermentation of enzymatically hydrolyzed date palm residue was only 103mg / L; the GABA yield of Lactobacillus brevis FPA3709 in fermentation of black beans was higher, reaching more than 1g / L, but this high GABA yield was achieved under the premise of exogenous addition of culture medium components (monosodium glutamate). Therefore, it is of great application value to develop an oat fermented milk or other plant-based fermented milk with high GABA content produced by excellent strains of Lactobacillus brevis without exogenous addition of auxiliary growth agents and glutamic acid and glutamate. Summary of the invention

[0008] The invention provides a fermentation method of a high-yield GABA Lactobacillus brevis D17 strain (with a deposit number of CGMCC NO.14385, recorded in a Chinese invention patent with a publication number of CN108034599B) from fermented grains based on different plant-based matrix characteristics. The method uses a model strain Lactobacillus brevis ATCC 367 as a reference and includes a method for preparing a plant-based matrix and a strain fermentation method.

[0009] The invention provides an application of Lactobacillus brevis CGMCC NO.14385 in preparing antioxidant health products, medicines or health products for improving gastrointestinal flora. The antioxidant activity is capable of significantly increasing the content of serum superoxide dismutase (SOD); the improvement of gastrointestinal flora comprises significantly restoring the relative abundance of beneficial intestinal bacteria Lactobacillus, Muribaculaceae, Faecalibaculum, Bifidobacterium and Akkermansia, and significantly reducing the relative abundance of potentially harmful intestinal bacteria Flintibacter, Leptogranulimonas and Alistipes.

[0010] The invention provides application of Lactobacillus brevis CGMCC NO.14385 in preparing medicine for alleviating and / or treating obesity caused by high-fat diet.

[0011] In one embodiment of the present invention, the amount of Lactobacillus brevis CGMCC NO.14385 added to the drug is not less than: 1×10 9 CFU / mL.

[0012] The present invention provides a plant-based fermented milk, which is prepared according to the following steps:

[0013] (1) adding protease to the plant-based matrix for enzymatic hydrolysis to obtain an enzymatic hydrolyzate;

[0014] (2) Adding Lactobacillus brevis D17 bacterial agent to the enzymatic hydrolyzate obtained in step (1) for fermentation to prepare plant-based fermented milk.

[0015] In one embodiment of the present invention, the plant-based matrix is ​​pre-hydrolyzed oat milk, pre-hydrolyzed caramel malt milk, pre-hydrolyzed rice milk or pre-hydrolyzed corn milk; the preparation method of the plant-based matrix comprises the following steps:

[0016] Preparation of pre-hydrolyzed oat flour:

[0017] The oatmeal powder is mixed with water, α-amylase is added for enzymolysis, saccharifying enzyme is added to the enzymolysis solution for saccharification, and the enzyme is inactivated after the saccharification is completed; neutral protease is added to the enzyme-inactivated saccharification solution for enzymolysis, and the enzyme is inactivated to obtain an enzymolysis solution; the obtained enzymolysis solution is concentrated and dried to obtain pre-hydrolyzed oatmeal powder;

[0018] The prepared pre-hydrolyzed oatmeal powder is mixed with water in a material-water ratio of 1-2:5-10, and sterilized to prepare oatmeal milk; protease is added to the obtained oatmeal milk for enzymolysis to obtain pre-hydrolyzed oatmeal milk.

[0019] Preparation of other pre-hydrolyzed cereal raw milk:

[0020] The caramel malt powder, rice flour and corn flour are respectively mixed with water and gelatinized, and after cooling, α-amylase is added to the gelatinized solution for enzymolysis, and then the enzyme is inactivated, and neutral protease is added to the enzyme-inactivated solution for enzymolysis, and then the enzyme is inactivated to obtain an enzymolysis solution; the obtained enzymolysis solution is concentrated and dried to obtain pre-hydrolyzed caramel malt powder, pre-hydrolyzed rice flour and pre-hydrolyzed corn flour respectively;

[0021] The prepared prehydrolyzed caramel malt powder, prehydrolyzed rice powder and prehydrolyzed corn powder are mixed in a material-water ratio of 1-2:5-10, and sterilized to prepare prehydrolyzed caramel malt milk, prehydrolyzed rice milk or prehydrolyzed corn milk.

[0022] In one embodiment of the present invention, the preparation of pre-hydrolyzed oat flour: oat flour (purchased from Xuzhou Fangde Food Co., Ltd.) and water are mixed in a mass ratio of 1:4, and then 2% (m / v) of α-amylase (named BAN480 L, purchased from Novozymes) is added and enzymolyzed at 60°C for 60min. Subsequently, 2% (m / v) of amylase (named AmylaseAG 300L, purchased from Novozymes) is added to the enzymolysis solution and enzymolyzed at 60°C for 60min. After the enzymolysis is completed, the enzyme is inactivated at 90°C for 10min. Subsequently, 0.2% (m / v) of neutral protease (named Neutrase 0.8L, purchased from Novozymes) is added to the enzyme-inactivated enzymolysis solution and enzymolyzed at 50°C for 60min. After the enzymolysis is completed, the enzyme is inactivated again at 90°C for 10min. It is then filtered with a 60-mesh sieve. The enzymatic hydrolysate is concentrated, then drum-dried, and finally ground into powder for later use.

[0023] In one embodiment of the present invention, caramel malt powder (purchased from Jinan Shuangmai Beer Materials Co., Ltd.), rice flour (purchased from Jiangxi Guyifeng Food Technology Co., Ltd.), and corn flour (purchased from Jiangxi Guyifeng Food Technology Co., Ltd.) are mixed at a material-liquid ratio of 1:5 (m / v). After cooking and gelatinization for 30 minutes and cooling to room temperature, 0.05% (m / v) of α-amylase (named BAN480 L) is added to the gelatinized solution, and enzymolysis is carried out at 95°C for 80 minutes. Subsequently, the enzyme is inactivated at 90°C for 10 minutes. Subsequently, 0.05% (m / v) of neutral protease (named Neutrase0.8L) is added to the enzyme-inactivated enzymolysis solution, and enzymolysis is carried out at 50°C for 60 minutes. After the enzymolysis is completed, the enzyme is inactivated at 90°C for another 10 minutes. Subsequently, it is filtered with four layers of gauze, and the filtered enzymolysis solution is concentrated, then drum-dried, and finally ground into powder for standby use.

[0024] In one embodiment of the present invention, the protease includes but is not limited to: cereal hydrolase ZB3-1P, lytic enzyme Viscozyme L, neutral protease Neutrase 0.8L.

[0025] In one embodiment of the present invention, the Lactobacillus brevis CGMCC NO.14385 bacterial agent is Lactobacillus brevis CGMCC NO.14385 fermentation liquid, Lactobacillus brevis CGMCC NO.14385 wet bacterial body or Lactobacillus brevis CGMCC NO.14385 freeze-dried powder.

[0026] In one embodiment of the present invention, the fermentation broth is prepared by inoculating the bacterial solution stored in a glycerol tube into a liquid MRS medium, culturing at 37°C for 24 hours, and activating the culture broth three times. The fully activated bacterial solution is inoculated into 100 mL of liquid MRS medium at a 10% inoculation amount, and cultured to the mid-logarithmic phase (10-12 hours). The culture broth is diluted to 1×109 CFU / mL as fermentation broth.

[0027] In one embodiment of the present invention, in the bacterial agent, the added amount of Lactobacillus brevis CGMCC NO.14385 is not less than 5-10%.

[0028] In one embodiment of the present invention, the wet cells are obtained by centrifuging the expanded culture of Lactobacillus brevis D17 cultured to the mid-logarithmic phase in 100 mL of MRS liquid medium to collect the cells, washing the cells three times with sterile saline, and centrifuging again to collect clean cells, i.e., wet cells.

[0029] In one embodiment of the present invention, the lyophilized powder is, the enlarged culture fluid of the short lactobacillus D17 cultured to the mid-logarithmic period in 100mL MRS liquid medium is centrifuged to collect thalline, and thalline is washed 3 times with sterile physiological saline. Centrifuged again to collect clean thalline, then in thalline, add 10 (bacterial liquid volume before centrifugation): 18% skim milk (protective agent) of 1 (skim milk volume) (v / v). 10 times of concentrated milk bacteria suspensions are precooled 6h at 4 ℃, precooled 12h at -20 ℃ and precooled 24h at -80 ℃ successively, after being precooled completely, be placed in vacuum drying machine and carry out freeze drying, the freeze-dried sample is ground into powder for standby use.

[0030] In one embodiment of the present invention, the preparation method of the direct-injection bacterial agent is:

[0031] The activated bacterial liquid is inoculated into the seed culture medium to obtain the seed culture liquid; the prepared seed culture liquid is centrifuged to collect the bacterial bodies, and skim milk is added to the collected bacterial bodies as a protective agent at a volume ratio of 10-20:1-2 to obtain a bacterial suspension, and the prepared bacterial suspension is precooled at 4-8°C for 6-8h, -18--20°C for 10-12h and -80--100°C for 24-48h in sequence, and after being completely precooled, it is placed in a vacuum dryer for freeze-drying, and the freeze-dried sample is ground into powder for later use.

[0032] In one embodiment of the present invention, the bacterial solution stored in the glycerol tube was connected to the liquid MRS medium, cultured at 37°C for 24 hours, and activated three times. The fully activated bacterial solution was inoculated into 100 mL of liquid MRS medium at an inoculum amount of 10% (v / v), and cultured to the mid-logarithmic phase (fermentation time was 10-12 hours) to obtain a culture solution, at which time the bacterial concentration of the culture solution was 2.8×10 10CFU / mL as the fermentation seed liquid; the fermentation seed liquid is centrifuged to collect the bacteria, and the bacteria are washed 3 times with sterile saline. Centrifuge again to collect the clean bacteria, and then add 18% skim milk (protective agent) at a ratio of 10 (volume of bacterial liquid before centrifugation): 1 (volume of skim milk) (v / v) to the bacteria to obtain a 10-fold concentrated milk bacteria suspension. The 10-fold concentrated milk bacteria suspension is precooled at 4°C for 6h, -20°C for 12h, and -80°C for 24h in turn, and after being completely precooled, it is placed in a vacuum dryer for freeze-drying, and the freeze-dried sample is ground into powder for standby use.

[0033] In one embodiment of the present invention, in the bacterial agent, the added amount of Lactobacillus brevis CGMCC NO.14385 is not less than 5-10%.

[0034] In one embodiment of the present invention, a direct-injection fermentation method is used, and the method is as follows: using wet cells as a control, the wet cells are resuspended with an equal amount of sterile saline before centrifugation, and inoculated into a plant-based matrix at an inoculum size of 10% (m / v). Weigh the freeze-dried bacterial powder with an equal amount of CFU and resuspend it with sterile saline, inoculate it into a plant-based matrix, and ferment it at 37°C and 200rpm for 24h, taking samples every 4h. The contents of maltose, glucose, lactic acid, acetic acid, ethanol, glutamic acid and GABA in the fermentation broth are detected by high performance liquid chromatography (HPLC).

[0035] In one embodiment of the present invention, the fermentation conditions are: 35-40° C., 100-200 rpm, and fermentation for 12-36 hours.

[0036] In one embodiment of the present invention, prehydrolyzed cereal raw materials (prehydrolyzed oatmeal, prehydrolyzed malt powder, prehydrolyzed rice powder, prehydrolyzed corn powder) are used to prepare prehydrolyzed oat milk, prehydrolyzed malt milk, prehydrolyzed rice milk, and prehydrolyzed corn milk at a material-water ratio of 1:5 (m / v), and the prehydrolyzed oat milk, prehydrolyzed malt milk, prehydrolyzed rice milk, and prehydrolyzed corn milk are sterilized at 115°C for 30 minutes.

[0037] In one embodiment of the present invention, the fermentation broth was inoculated with the above four different pre-hydrolyzed cereal emulsions at an inoculum amount of 10%, and fermented at 37° C. and 200 rpm for 24 hours, with samples taken every 4 hours. The contents of maltose, glucose, lactic acid, acetic acid, ethanol, glutamic acid and GABA in the fermentation broth were detected by high performance liquid chromatography (HPLC).

[0038] The present invention also provides an oat milk rich in GABA prepared by fermentation using Lactobacillus brevis D17 strain without exogenous addition of auxiliary growth factors, glutamic acid and glutamate, wherein the oat milk is prepared according to the following steps:

[0039] (1) mixing oat flour with water, adding α-amylase for enzymolysis, adding saccharifying enzyme to the enzymolysis solution for saccharification, and performing enzyme inactivation after saccharification; adding neutral protease to the enzyme-inactivated saccharification solution for enzymolysis, inactivating the enzyme, and obtaining an enzymolysis solution; concentrating and drying the obtained enzymolysis solution to obtain pre-hydrolyzed oat flour; mixing the obtained pre-hydrolyzed oat flour according to a material-water ratio of 1-2:5-10, sterilizing, and preparing pre-hydrolyzed oat milk;

[0040] (2) Adding Lactobacillus brevis CGMCC No. 14385 bacterial agent to the pre-hydrolyzed oat milk obtained in step (1) and fermenting it to prepare oat milk.

[0041] In one embodiment of the present invention, the preparation method of oat milk is:

[0042] (1) Preparation of pre-hydrolyzed oat milk:

[0043] Oatmeal powder (purchased from Xuzhou Fangde Food Co., Ltd.) was mixed with water at a mass ratio of 1:4, and then 2% (m / v) α-amylase (named BAN480 L) was added and enzymolyzed at 60°C for 60 minutes. Subsequently, 2% (m / v) amylase (named Amylase AG 300L) was added to the enzymolysis solution and enzymolyzed at 60°C for 60 minutes. After the enzymolysis was completed, the enzyme was inactivated at 90°C for 10 minutes. Subsequently, 0.2% (m / v) neutral protease (named Neutrase 0.8L) was added to the inactivated enzymolysis solution and enzymolyzed at 50°C for 60 minutes. After the enzymolysis was completed, the enzyme was inactivated at 90°C for another 10 minutes. Then it was filtered with a 60-mesh sieve. The enzymolysis solution was concentrated, then drum-dried, and finally ground into powder to obtain pre-hydrolyzed oatmeal powder. The prepared prehydrolyzed oatmeal powder is mixed with water in a material-water ratio of 1-2:5-10 and then sterilized to prepare prehydrolyzed oatmeal milk.

[0044] (2) 1% (m / v) protease ZB3-1P (purchased from Shandong Longkote Enzyme Preparation Co., Ltd., license number: SC20137132300142) was exogenously added to the pre-hydrolyzed oat milk obtained in step (1), and the mixture was placed in a 45°C water bath for enzymatic hydrolysis for 8 hours, and then sterilized and inactivated at 115°C for 30 minutes to obtain enzymatic oat milk for fermentation.

[0045] The proteases include but are not limited to: cereal hydrolase ZB3-1P, lytic enzyme Viscozyme L, neutral protease Neutrase0.8L.

[0046] In one embodiment of the present invention, the Lactobacillus brevis CGMCC NO.14385 bacterial agent is Lactobacillus brevis CGMCC NO.14385 fermentation liquid, Lactobacillus brevis CGMCC NO.14385 wet bacteria or Lactobacillus brevis CGMCC NO.14385 freeze-dried powder; preferably, in the bacterial agent, the addition amount of Lactobacillus brevis CGMCC NO.14385 is not less than: 5-10%.

[0047] In one embodiment of the present invention, in step (1), the enzymatic hydrolysis conditions are: 40-50° C., 7-10 h; and in step (2), the fermentation conditions are: 35-40° C., 100-200 rpm, and fermentation for 12-36 h.

[0048] The present invention also provides a method for increasing the GABA content in oat milk prepared by fermentation of Lactobacillus brevis, wherein the method does not require exogenous addition of auxiliary growth factors, glutamic acid and glutamate, and specifically comprises the following steps:

[0049] (1) After adding protease to the pretreated oats for enzymolysis, an enzymolysis solution is obtained; the pretreatment method is: oatmeal powder (purchased from Xuzhou Fangde Food Co., Ltd.) and water are mixed at a mass ratio of 1:4, and then 2% (m / v) α-amylase (named BAN480 L) is added to the enzymolysis solution, and the enzymolysis is carried out at 60°C for 60 minutes. Subsequently, 2% (m / v) amylase (named Amylase AG 300L) is added to the enzymolysis solution, and the enzymolysis is carried out at 60°C for 60 minutes. After the enzymolysis is completed, the enzyme is inactivated at 90°C for 10 minutes. Subsequently, 0.2% (m / v) neutral protease (named Neutrase 0.8L) is added to the inactivated enzymolysis solution and the enzymolysis is carried out at 50°C for 60 minutes. After the enzymolysis is completed, the enzyme is inactivated at 90°C for another 10 minutes. Subsequently, it is filtered with a 60-mesh sieve. The enzymatic hydrolysate is concentrated, then drum-dried, and finally ground into powder to obtain pre-hydrolyzed oatmeal powder. The obtained pre-hydrolyzed oatmeal powder is mixed with water in a material-water ratio of 1-2:5-10 and then sterilized to obtain pre-hydrolyzed oatmeal milk.

[0050] (2) adding Lactobacillus brevis CGMCC No. 14385 to the enzymatic hydrolyzate obtained in step (1) for fermentation to prepare oat milk;

[0051] In one embodiment of the present invention, the protease includes but is not limited to: cereal hydrolase ZB3-1P, lytic enzyme Viscozyme L, neutral protease Neutrase 0.8L.

[0052] In one embodiment of the present invention, the Lactobacillus brevis CGMCC NO.14385 bacterial agent is Lactobacillus brevis CGMCC NO.14385 fermentation liquid, Lactobacillus brevis CGMCC NO.14385 wet bacterial body or Lactobacillus brevis CGMCC NO.14385 lyophilized powder;

[0053] In one embodiment of the present invention, in the bacterial agent, the added amount of Lactobacillus brevis CGMCC NO.14385 is not less than 5-10%.

[0054] In one embodiment of the present invention, in step (1), the enzymatic hydrolysis conditions are: 40-50° C., 7-10 h; and in step (2), the fermentation conditions are: 35-40° C., 100-200 rpm, and fermentation for 12-36 h.

[0055] The present invention also provides the use of the Lactobacillus brevis CGMCC NO.14385 in the preparation of antioxidant health products or medicines, and health products or medicines for improving gastrointestinal flora.

[0056] The present invention also provides the use of the Lactobacillus brevis CGMCC NO.14385 in preparing medicines for alleviating and / or treating obesity caused by a high-fat diet.

[0057] In one embodiment of the present invention, the amount of Lactobacillus brevis CGMCC NO.14385 added to the drug is not less than: 1×10 9 CFU / mL.

[0058] The present invention also provides a direct-injection bacterial agent of the Lactobacillus brevis D17 strain. By evaluating the survival ability and fermentation performance of the direct-injection bacterial agent, it is found that the strain is suitable for freeze-drying, and the direct-injection bacterial agent will not have a significant impact on its survival rate when refrigerated for a certain period of time, and can be used for direct-injection fermentation. The preparation method of the direct-injection bacterial agent comprises the following steps:

[0059] (1) Preparation of direct-injection microbial agent

[0060] The enlarged culture fluid of the short lactobacillus D17 that will be cultivated to the mid-logarithmic period in 100mL MRS liquid nutrient medium is centrifugal to collect thalline, wash thalline 3 times with sterile physiological saline.Centrifugal again to collect clean thalline, then in thalline, add 18% skim milk (protective agent) of 10 (bacterial liquid volume before centrifugation): 1 (skim milk volume) (v / v).10 times of concentrated milk bacteria suspension precooling 6h at 4 ℃, precooling 12h at-20 ℃ and precooling 24h at-80 ℃, treat to be placed in vacuum drier and carry out freeze drying after complete precooling, the freeze-dried sample is ground into powder for standby use.

[0061] (2) Determination of freeze-dried survival rate

[0062] Add an equal amount of sterile saline to the freeze-dried bacterial preparation for rehydration, mix well, dilute and plate count, and calculate the number of viable bacteria in the bacterial powder. The number of viable bacteria in the freeze-dried bacterial powder divided by the number of viable bacteria in the bacterial solution before freeze-drying is the survival rate of the strain after freeze-drying.

[0063] (3) Determination of the effect of refrigeration time on the survival rate of freeze-dried bacterial preparations

[0064] The freeze-dried bacterial agents stored in a refrigerator at 4 degrees for 0 days, 7 days and 28 days were taken out and rehydrated with an equal amount of sterile saline. After mixing, they were diluted and plated for counting to monitor the effect of refrigeration time on the survival rate of the freeze-dried bacterial agents.

[0065] (4) Direct fermentation assay

[0066] The wet bacteria (mud) obtained by centrifugation before freeze-drying was used as a control. The mud was resuspended with an equal amount of sterile saline before centrifugation and inoculated into enzymatic oat milk at an inoculum of 10% (m / v). A certain amount of freeze-dried bacterial powder (ensuring that the CFU number is consistent with that of the 10% mud resuspension) was weighed and resuspended with an equal amount of sterile saline, inoculated into enzymatic oat milk, and fermented at 37°C and 200rpm for 24h, with samples taken every 4h. The contents of maltose, glucose, lactic acid, acetic acid, ethanol, glutamic acid and GABA in the fermentation broth were detected by high performance liquid chromatography (HPLC).

[0067] The present invention also provides a method for safety assessment of Lactobacillus brevis D17 strain, the evaluation method comprising strain genome-wide analysis, antibiotic resistance test and in vivo toxicity test in mice. The method comprises the following steps:

[0068] (1) Whole genome analysis of strains

[0069] The whole genome sequence of Lactobacillus brevis D17 strain was compared with the Virulence Factor Database (VFDB) and the Antimicrobial Resistance Gene Database (CARD). With reference to the relevant guidelines of the European Food Safety Authority (EFSA), the virulence factors and antimicrobial resistance genes were predicted with identity greater than 80%, coverage greater than 70% and E value less than 1×e-10 as the screening criteria.

[0070] (2) Antibiotic resistance test

[0071] The KB disk diffusion method was used to determine the resistance of Lactobacillus brevis D17 to 12 commonly used antibiotics. The seed solution of Lactobacillus brevis D17 was diluted to a bacterial concentration of 1×10 8CFU / mL, draw 1mL of bacterial solution into 15mL MRS liquid medium containing agar on a plate and mix well. After solidification, use sterile tweezers to evenly stick the drug-sensitive paper containing a certain concentration of antibiotics on the plate. Place it in a 37℃ incubator and culture it for 48h before measuring the diameter of the inhibition zone on the plate. Determine according to the antibiotic sensitivity test execution standards established by the Clinical and Laboratory Standards Institutes.

[0072] (3) In vivo toxicity test in mice

[0073] Twenty 6-8 week old SPF BALB / c mice (10 males and 10 females, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were selected and lived in a 12 h light-dark cycle, constant ambient temperature (22 ± 2 °C), humidity (55 ± 2%), free access to food and water. After one week of adaptation, the mice were randomly divided into two groups, 10 mice in each group (5 females and 5 males, and housed separately), and the groups were as follows: (1) SD group (control group): mice were fed with standard diet (Co60 irradiated experimental mouse maintenance diet, purchased from Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd.) and 200 μL sterile saline; (2) D17 group: mice were fed with standard diet supplemented with 200 μL (concentration of 1×10 9 CFU / mL) of Lactobacillus brevis D17 suspension. Daily gavage lasted for 28 days. During the gavage period, the mice were weighed daily, and the food intake, drinking water, activity status and mental state of the mice were observed and recorded. On the last day of the gavage period, all mice were fasted overnight, and then orbital blood was collected and the mice were euthanized by the carbon dioxide method. The liver, spleen, kidney and colon were then immediately removed for subsequent analysis. This experiment strictly followed the relevant ethical guidelines for experimental animals and was approved by the Experimental Animal Ethics Committee of Jiangnan University (Ethics Approval Number: JN.No20220315b0300601).

[0074] The present invention also evaluates the in vivo beneficial function of the Lactobacillus brevis D17 strain and finds that the strain has excellent obesity-delaying effect. This is achieved by the following technical solutions:

[0075] Twenty-four 8-week-old SPF-grade C57BL / 6J male mice were selected and fed with normal diet for 1 week. After 1 week of adaptation, the mice were divided into 3 groups according to their body weight, as follows: (1) Normal diet group (NFD): normal diet + 0.2 mL sterile saline; (2) High-fat diet group (HFD, 60% kcal fat, purchased from Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd.): high-fat diet + 0.2 mL sterile saline; (3) Lactobacillus brevis D17 group (HFD+D17): high-fat diet + 0.2 mL 10 9CFU / mL of D17 bacterial suspension. 8 mice in each group were gavaged daily for 8 weeks. After 8 weeks of gavage, the mice were fasted overnight, and after orbital blood collection, they were euthanized by carbon dioxide method. The liver, kidney, spleen, epididymal white adipose tissue (eWAT) and inguinal subcutaneous white adipose tissue (iWAT) of each mouse were precisely dissected, weighed and collected for further analysis. This experiment strictly followed the relevant ethical guidelines for experimental animals and was approved by the Experimental Animal Ethics Committee of Jiangnan University (Ethics Approval Number: JN.No20220930c0321220).

[0076] The present invention provides a method for preparing GABA using oatmeal as a substrate, wherein the method does not require exogenous addition of auxiliary growth factors, glutamic acid and glutamate, and specifically comprises the following steps:

[0077] (1) mixing oat flour with water, adding α-amylase for enzymolysis, adding saccharifying enzyme to the enzymolysis solution for saccharification, and performing enzyme inactivation after saccharification; adding neutral protease to the enzyme-inactivated saccharification solution for enzymolysis, inactivating the enzyme, and obtaining an enzymolysis solution; concentrating and drying the obtained enzymolysis solution to obtain pre-hydrolyzed oat flour; mixing the obtained pre-hydrolyzed oat flour according to a material-water ratio of 1-2:5-10, sterilizing, and preparing an oat hydrolyzate; adding protease ZB3-1P to the obtained oat hydrolyzate for enzymolysis, and obtaining a pre-hydrolyzed oat substrate;

[0078] (2) Adding Lactobacillus brevis CGMCC No. 14385 bacterial agent to the pre-hydrolyzed oat substrate obtained in step (1) for fermentation to prepare a fermentation liquid containing GABA, and extracting to prepare GABA.

[0079] In one embodiment of the present invention, the protease includes but is not limited to: cereal hydrolase ZB3-1P, lytic enzyme Viscozyme L, neutral protease Neutrase 0.8L.

[0080] In one embodiment of the present invention, the Lactobacillus brevis CGMCC NO.14385 bacterial agent is Lactobacillus brevis CGMCC NO.14385 fermentation liquid, Lactobacillus brevis CGMCC NO.14385 wet bacteria or Lactobacillus brevis CGMCC NO.14385 freeze-dried powder; preferably, in the bacterial agent, the addition amount of Lactobacillus brevis CGMCC NO.14385 is not less than: 5-10%.

[0081] In one embodiment of the present invention, in step (1), the enzymatic hydrolysis conditions are: 40-50° C., 7-10 h; and in step (2), the fermentation conditions are: 35-40° C., 100-200 rpm, and fermentation for 12-36 h.

[0082] In one embodiment of the present invention, the conditions for adding α-amylase for enzymolysis are: enzymolysis at 55-65°C for 60-70 min; the conditions for saccharifying by saccharifying by saccharifying by saccharifying by 55-65°C for 60-70 min; the conditions for adding neutral protease for enzymolysis are: enzymolysis at 50-60°C for 60-70 min; the oatmeal powder and water are mixed in a ratio of 1-2:4-8, the addition amount of the α-amylase is: 2-3% (m / v); the addition amount of the saccharifying enzyme is: 2-3% (m / v); the addition amount of the neutral protease is: 0.2-0.3% (m / v).

[0083] Beneficial Effects

[0084] (1) The Lactobacillus brevis D17 of the present invention can directly ferment oat milk and other various types of hydrolyzed plant-based matrices.

[0085] (2) Without adding any other culture medium components and any form of glutamate or glutamate salt, the Lactobacillus brevis D17 of the present invention can ferment and enzymatically hydrolyze oat milk to achieve high GABA production.

[0086] (3) The Lactobacillus brevis D17 of the present invention can be made into a direct-injection fermentation agent for direct-injection fermentation.

[0087] (4) The Lactobacillus brevis D17 of the present invention has been proven to be a safe and usable strain through in vitro and in vivo tests.

[0088] (5) The Lactobacillus brevis D17 of the present invention has excellent effects of reducing fat and delaying obesity. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] Figure 1 The substrate utilization and main metabolite production of pre-hydrolyzed oat milk fermented by Lactobacillus brevis ATCC 367 and Lactobacillus brevis D17.

[0090] Figure 2 To investigate the substrate utilization and main metabolite production of pre-hydrolyzed caramel malt milk fermented by Lactobacillus brevis ATCC 367 and Lactobacillus brevis D17.

[0091] Figure 3 To investigate the substrate utilization and major metabolite production of pre-hydrolyzed rice milk fermented by Lactobacillus brevis ATCC 367 and Lactobacillus brevis D17.

[0092] Figure 4 To investigate the substrate utilization and major metabolite production of pre-hydrolyzed corn milk fermented by Lactobacillus brevis ATCC 367 and Lactobacillus brevis D17.

[0093] Figure 5 The glutamate utilization and GABA production of oat milk fermented with enzymatic hydrolysis of Lactobacillus brevis D17 strain.

[0094] Figure 6 Glutamate utilization and GABA production in oat milk fermented by Lactobacillus brevis D17 with added peptide powder.

[0095] Figure 7 The substrate utilization and main metabolite production of oat milk enzymatically hydrolyzed by direct fermentation of Lactobacillus brevis D17 strain.

[0096] Figure 8 This is the effect of Lactobacillus brevis D17 on the body weight, food intake and organ indexes of mice.

[0097] Fig. 9 This is the effect of Lactobacillus brevis D17 on the microstructure of representative organ tissues of mice.

[0098] Fig.10 This is the effect of Lactobacillus brevis D17 on mouse serum indicators.

[0099] Fig.11 To investigate the effects of Lactobacillus brevis D17 on body weight and organ indexes under high-fat diet intervention.

[0100] Fig.12 This is the effect of Lactobacillus brevis D17 on tissue morphology under high-fat diet feeding.

[0101] Fig.13 To investigate the effects of Lactobacillus brevis D17 on serum indicators of high-fat dietary disorders. DETAILED DESCRIPTION

[0102] The Lactobacillus brevis ATCC 367 strain involved in the following examples was purchased from the American Type Culture Collection (ATCC).

[0103] The culture medium involved in the following embodiments is:

[0104] Liquid MRS medium: glucose 20g / L, peptone 10g / L, beef extract 10g / L, yeast extract 5g / L, ammonium citrate 2g / L, dipotassium hydrogen citrate 2g / L, Tween 80 1ml / L, sodium acetate 5g / L, magnesium sulfate 0.58g / L, manganese sulfate 0.25g / L.

[0105] The Lactobacillus brevis CGMCC NO.14385 strain described in the following examples is the same strain as Lactobacillus brevis D17. The deposit number of high-yielding GABA Lactobacillus brevis D17 is CGMCC NO.14385, which is recorded in the Chinese invention patent text with publication number CN108034599B.

[0106] The technical solution of the present invention is described in detail below in conjunction with the embodiments of the accompanying drawings.

[0107] Example 1: Safety evaluation of Lactobacillus brevis D17 strain

[0108] This test example evaluated the safety of Lactobacillus brevis D17 and ensured the application safety of Examples 1-3.

[0109] The specific test method steps are:

[0110] (1) Whole genome analysis of strains

[0111] The whole genome sequence of Lactobacillus brevis D17 strain was compared with the Virulence Factor Database (VFDB) and the Antimicrobial Resistance Gene Database (CARD), and the identity (identity) greater than 80%, coverage (coverage) greater than 70% and E value less than l×e were used as reference. -10 As screening criteria, virulence factors and drug resistance genes were predicted.

[0112] Comparison with the Virulence Factor Database (VFDB) and the Drug Resistance Gene Database (CARD) showed that Lactobacillus brevis D17 had no potential virulence factors and drug resistance genes.

[0113] (2) Antibiotic resistance test

[0114] The KB disk diffusion method was used to determine the resistance of Lactobacillus brevis D17 strain to 12 commonly used antibiotics (penicillin G, ampicillin, erythromycin, norfloxacin, ciprofloxacin, chloramphenicol, cotrimoxazole, cefazolin, gentamicin, amikacin, tetracycline, and vancomycin). The seed solution of Lactobacillus brevis D17 strain was diluted to a bacterial concentration of 1×10 8 CFU / mL, pipette 1mL of bacterial solution into 15mL of MRS liquid medium containing agar on a plate and mix well. After solidification, use sterile tweezers to evenly stick antibiotic sensitive paper on the plate. Place in a 37℃ incubator and incubate for 48h before measuring the diameter of the inhibition zone on the plate. Determine according to the antibiotic sensitivity test execution standards established by the Clinical and Laboratory Standards Institutes.

[0115] The results are shown in Table 1 below. Lactobacillus brevis D17 is sensitive to penicillin G, erythromycin, chloramphenicol, ampicillin, cefazolin and gentamicin; moderately sensitive to tetracycline and cotrimoxazole; and resistant to amikacin, norfloxacin, ciprofloxacin and vancomycin. Lactobacillus such as Lactobacillus plantarum and Lactobacillus brevis have intrinsic resistance to fluoroquinolones (norfloxacin, ciprofloxacin), aminoglycoside antibiotics (gentamicin, amikacin) and vancomycin, while the intrinsic resistance of lactobacillus generally does not cause safety problems. This shows that Lactobacillus brevis D17 only has intrinsic resistance that does not cause safety problems, and there is no risk of transferable resistance with potential safety hazards.

[0116] Table 1 Drug sensitivity results of Lactobacillus brevis D17

[0117]

[0118] Note: S means sensitive; I means moderately sensitive; R means resistant

[0119] (3) In vivo toxicity test in mice

[0120] Twenty 6-8 week old SPF BALB / c mice (10 males and 10 females, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were selected and lived in a 12 h light-dark cycle, constant ambient temperature (22 ± 2 °C), humidity (55 ± 2%), free access to food and water. After one week of adaptation, the mice were randomly divided into two groups, 10 mice in each group (5 females and 5 males, and housed separately), and the groups were as follows: (1) SD group (control group): mice were fed with standard diet (Co60 irradiated experimental mouse maintenance diet, purchased from Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd.) and 200 μL sterile saline; (2) D17 group: mice were fed with standard diet supplemented with 200 μL (concentration of 1×10 9 CFU / mL) of Lactobacillus brevis D17 suspension. Daily gavage lasted for 28 days. During the gavage period, the mice were weighed daily, and the food intake, drinking water, activity status and mental state of the mice were observed and recorded. On the last day of the gavage period, all mice were fasted overnight, and then orbital blood was collected and the mice were euthanized by the carbon dioxide method. The liver, spleen, kidney and colon were then immediately removed for subsequent analysis. This experiment strictly followed the relevant ethical guidelines for experimental animals and was approved by the Experimental Animal Ethics Committee of Jiangnan University (Ethics Approval Number: JN.No20220315b0300601).

[0121] Organ index determination: The liver, spleen and kidneys were rinsed with physiological saline, dried with filter paper and weighed. The ratio of the weight to the final weight of the animal was the organ index.

[0122] Serum biochemical indexes determination: Commercial detection kits (purchased from Nanjing Jiancheng Bioengineering Institute) were used to detect serum alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), malondialdehyde (MDA), superoxide dismutase (SOD), triglyceride (TG), cholesterol (CHO), low-density lipoprotein cholesterol (LDL) and high-density lipoprotein cholesterol (HDL) levels.

[0123] Histopathological examination: Tissue sections of the liver, spleen and colon were obtained, fixed with 4% paraformaldehyde, dehydrated, embedded, sectioned and stained with hematoxylin-eosin, and finally the tissue morphology was observed under an optical microscope.

[0124] The results are as follows Figure 8 , Fig. 9 , Fig.10 As shown in the 28-day mouse toxicology experiment, no mouse died, nor did it show other adverse health symptoms such as mental depression. The appetite, mental state, mouse skin, and fur were all normal. Lactobacillus brevis D17 had no effect on the weight and food intake of mice. The weight showed a gradual increase, and the weight gain and food intake of male mice were higher than those of female mice, which was in line with normal rules ( Figure 8 AB in the figure). Lactobacillus brevis D17 had no significant effect on the liver index, kidney index and spleen index, indicating that neither strain would cause abnormal weight changes in the liver, kidney and spleen of mice ( Figure 8 CE in the experiment). By observing the tissue morphology of the liver, spleen and colon, it was found that the liver, spleen and colon tissue morphology of the mice in the Lactobacillus brevis D17 strain experimental group were normal, similar to the control group (SD), and there was no histopathological abnormality in the liver, spleen cells and colon tissues ( Fig. 9 ), further indicating that feeding Lactobacillus brevis D17 strain will not have a negative impact on the visceral and intestinal tissues of mice. In addition, Lactobacillus brevis D17 has no significant effect on serum alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), malondialdehyde (MDA), total protein (TP), triglycerides (TG), cholesterol (CHO) and low-density lipoprotein cholesterol (LDL), but can significantly increase the serum superoxide dismutase (SOD) content of male mice (increased by 10.2%). SOD is an important antioxidant enzyme that removes superoxide anion free radicals in the body and protects cells from damage by oxygen free radicals ( Fig.10 ).

[0125] In summary, whole genome analysis and antibiotic resistance tests showed that Lactobacillus brevis D17 had no virulence factors or antibiotic resistance genes that could pose a safety hazard, and its drug resistance safety met multiple assessment criteria. In vivo safety assessments showed that Lactobacillus brevis D17 was non-toxic to mice, indicating that Lactobacillus brevis D17 was a safe and usable strain.

[0126] Example 2: Evaluation of the probiotic function of Lactobacillus brevis D17 strain and its ability to regulate intestinal flora

[0127] This test example evaluated the probiotic function of Lactobacillus brevis D17 and provided a theoretical basis for the probiotic effects of Examples 1-3.

[0128] The specific test method steps are:

[0129] Twenty-four 8-week-old SPF C57BL / 6J male mice were selected and fed with normal feed for 1 week. After 1 week of adaptation, the mice were divided into 3 groups according to their body weight. The specific groups are as follows:

[0130] (1) Normal feed group (NFD): normal feed + 0.2 mL sterile saline;

[0131] (2) High-fat diet group (HFD, 60% kcal fat, purchased from Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd.): high-fat diet + 0.2 mL sterile saline;

[0132] (3) Lactobacillus brevis D17 group (HFD+D17): high-fat diet + 0.2 mL 10 9 CFU / mL D17 bacterial suspension; the preparation method of D17 bacterial suspension is: prepare fermentation seed liquid according to the method of step (3) of Example 1, centrifuge the fermentation seed liquid at 8000 rpm for 10 min at 4°C to collect bacteria, wash the bacteria with sterile saline for 3 times, resuspend with sterile saline and dilute to a bacterial concentration of 1×10 9 CFU / mL, and fresh bacterial suspension was prepared every day.

[0133] There were 8 mice in each group, and they were gavaged daily for 8 weeks. After 8 weeks of gavage, the mice were fasted overnight, and after orbital blood collection, they were euthanized by carbon dioxide method. The liver, kidney, spleen, epididymal white adipose tissue (eWAT) and inguinal subcutaneous white adipose tissue (iWAT) of each mouse were precisely dissected, weighed and collected for further analysis. This experiment strictly followed the relevant ethical guidelines for experimental animals and was approved by the Experimental Animal Ethics Committee of Jiangnan University (Ethics Approval Number: JN.No20220930c0321220).

[0134] Organ index determination: epididymal white adipose tissue (eWAT) and inguinal subcutaneous white adipose tissue (iWAT) were rinsed with physiological saline, dried with filter paper and weighed. The ratio of the eWAT to the final body weight of the animal was the organ index.

[0135] Serum biochemical index determination: Commercial detection kits (purchased from Nanjing Jiancheng Bioengineering Institute) were used to detect serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), malondialdehyde (MDA), superoxide dismutase (SOD), total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL) and high-density lipoprotein cholesterol (HDL) levels.

[0136] Histopathological examination: Tissue sections of the liver and adipose tissue (eWAT, iWAT) were obtained, fixed with 4% paraformaldehyde, dehydrated, embedded, sectioned, and stained with hematoxylin-eosin. Finally, the tissue morphology was observed using an optical microscope.

[0137] Intestinal flora analysis: Mouse fecal DNA was extracted according to the kit QIAamp fast DNA stool mini kit. The V3-V4 region of the 16S rRNA gene was amplified with primers: 338Fmod F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806Rmod R (5'-GGACTACNVGGGTWTCTAAT-3'). The amplified product was sent to Guangdong Meige Gene Technology Co., Ltd. for library construction and sequencing. The original fastq file was quality filtered using Trimmomatic software, and the obtained double-end sequences were denoised, merged and clustered into amplicon sequence variants (ASVs) using the DADA2 program in QIIME2 (v2021.11). Based on the Silva138 99% OUTs database, the naive Bayes classifier trained by QIIME2 was used to classify and annotate the obtained ASV sequences, and a standardized ASV table that reflects species abundance was generated. Based on the ASV table, QIIME2 was used to calculate the alpha diversity index and analyze the composition of the intestinal flora.

[0138] The results are as follows Fig.11 , Fig.12 , Fig.13 As shown, the results show:

[0139] (1) Weight changes:

[0140] Table 2 Changes in body weight over time in different experimental groups

[0141]

[0142] After 28 days of high-fat diet intervention, the mice in the HFD group gained 23.7% weight, which was 20% higher than that in the NFD group, meaning that an obesity model was successfully created after 4 weeks.

[0143] The weight gain caused by the high-fat diet continued until the end of the intervention period. At the end of the intervention period (day 56), the weight of mice in the HFD group increased by 46.5% compared with the NFD group. Lactobacillus brevis D17 supplementation significantly alleviated the weight gain caused by the high-fat diet, reducing the weight gain by 28.4% ( Fig.11 A).

[0144] (2) Epididymal fat index and inguinal subcutaneous fat index

[0145] A high-fat diet can lead to a significant increase in the epididymal fat index (0.056) and the inguinal subcutaneous fat index (0.031), that is, the obese mice in the HFD group have a higher body fat rate, while the supplementation of Lactobacillus brevis D17 can significantly reduce the epididymal fat index (0.04) and the inguinal subcutaneous fat index (0.022), which are reduced by 28.6% and 27.8% respectively compared with the model group. ( Fig.11 BC in.

[0146] (3) Observation of tissue morphology

[0147] From the observation of tissue morphology, it can be seen that the liver of obese mice induced by high-fat diet showed obvious pathological changes. This includes an increase in the number of fat vesicles, the presence of macrovesicular fatty degeneration, and the accumulation of lipid droplets. These changes mean that the mice are at a higher risk of non-alcoholic fatty liver disease (NAFLD). At the same time, obvious adipocyte growth was also shown in the epididymal adipose tissue and inguinal adipose tissue. However, the introduction of Lactobacillus brevis D17 as a dietary supplement can significantly reduce the hepatic fatty degeneration and adipocyte growth induced by the high-fat diet, making its tissue morphology closer to that of the NFD group mice ( Fig.12 ).

[0148] (4) Serum indicators

[0149] Table 3 Serum index contents in different experimental groups

[0150]

[0151] Serum biochemical analysis shows that intervention with a high-fat diet will significantly increase the level of leptin in serum, which is a hormone secreted by adipose tissue that can regulate fat production. Obese mice usually show leptin-resistant hyperleptinemia, which is also called type 2 obesity. Supplementation with Lactobacillus brevis D17 can reduce leptin levels by 73.9%. In addition, intervention with a high-fat diet will also significantly increase the levels of serum triglycerides (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL), and reduce the level of serum high-density lipoprotein cholesterol (HDL). However, with the supplementation of Lactobacillus brevis D17, the TC content decreased by 17%, the TG content decreased by 26%, the LDL content decreased by 22%, and the HDL content increased by 13% ( Fig.13 ).

[0152] (5) Intestinal flora analysis

[0153] Table 4 α diversity results of different experimental groups

[0154]

[0155]

[0156] From the evaluation of α diversity, it can be seen that the Chao1 index (reflecting species richness) of the intestinal microorganisms of mice in the HFD group was significantly lower than that in the NFD group and the HFD+D17 group, indicating that a high-fat diet significantly reduces the species richness of the mouse intestine, while the supplementation of Lactobacillus brevis D17 significantly restores the species richness of the intestinal flora. The Shannon index (reflecting diversity) of the intestinal microorganisms of mice in the HFD group was not significantly different from that in the NFD group, but was significantly lower than that in the D17 supplementation group, indicating that the supplementation of Lactobacillus brevis D17 is beneficial to improving the species diversity of the intestinal flora. In summary, these results reflect that Lactobacillus brevis D17 has an improving effect on the intestinal flora. Tables 5 and 6 show the specific intestinal flora that can be improved after intervention with Lactobacillus brevis D17.

[0157] Table 5 Relative abundance of beneficial intestinal bacteria in different experimental groups (%)

[0158]

[0159] Table 6 Relative abundance of potentially harmful intestinal bacteria in different experimental groups (%)

[0160]

[0161] It can be seen that high-fat dietary intervention will lead to a significant decrease in the abundance of beneficial intestinal genera Lactobacillus, unclassified Muribaculaceae, Faecalibaculum, Bifidobacterium and Akkermansia in obese mice, while the supplementation of Lactobacillus brevis D17 will significantly restore the relative abundance of these beneficial intestinal genera. In addition, high-fat dietary intervention will lead to the enrichment of some potentially harmful genera such as Flintibacter, Leptogranulimonas and Alistipes in the intestines of mice, while the supplementation of Lactobacillus brevis D17 will significantly reduce the relative abundance of these potentially harmful intestinal genera, making the intestinal flora abundance closer to that of normal group (NFD) mice.

[0162] In summary, Lactobacillus brevis D17 has a significant effect in delaying obesity, can effectively control a series of adverse changes caused by a high-fat diet, and has an excellent prebiotic effect.

[0163] Example 3: Fermentation capacity of Lactobacillus brevis D17 on different plant-based substrates

[0164] The specific steps are as follows:

[0165] (1) Preparation of prehydrolyzed cereal raw materials

[0166] Preparation of pre-hydrolyzed oat flour: Oat flour (purchased from Xuzhou Fangde Food Co., Ltd.) was mixed with water at a mass ratio of 1:4, and then 2% (m / v) α-amylase (named BAN480 L, purchased from Novozymes) was added and enzymolyzed at 60°C for 60 minutes. Subsequently, 2% (m / v) amylase (named Amylase AG 300L, purchased from Novozymes) was added to the enzymolysis solution and enzymolyzed at 60°C for 60 minutes. After the enzymolysis was completed, the enzyme was inactivated at 90°C for 10 minutes. Subsequently, 0.2% (m / v) neutral protease (named Neutrase 0.8L, purchased from Novozymes) was added to the inactivated enzymolysis solution and enzymolyzed at 50°C for 60 minutes. After the enzymolysis was completed, the enzyme was inactivated at 90°C for another 10 minutes. Then it was filtered with a 60-mesh sieve. The enzymolysis solution was concentrated, then drum-dried, and finally ground into powder.

[0167] Preparation of pre-hydrolyzed caramel malt powder: Caramel malt powder (purchased from Jinan Shuangmai Beer Materials Co., Ltd.) was mixed at a material-liquid ratio of 1:5 (m / v). After cooking and gelatinization at 65°C for 30 minutes, 0.05% (m / v) α-amylase (named BAN480 L) was added to the gelatinization solution and enzymolyzed at 95°C for 80 minutes. Subsequently, the enzyme was inactivated at 90°C for 10 minutes. Subsequently, 0.05% (m / v) neutral protease (named Neutrase 0.8L) was added to the inactivated enzymolysis solution and enzymolyzed at 50°C for 60 minutes. After the enzymolysis was completed, the enzyme was inactivated at 90°C for another 10 minutes. Subsequently, it was filtered with four layers of gauze, and the filtered enzymolysis solution was concentrated, then drum dried, and finally ground into powder.

[0168] Preparation of pre-hydrolyzed rice flour: Rice flour (purchased from Jiangxi Guyifeng Food Technology Co., Ltd.) was mixed at a material-liquid ratio of 1:5 (m / v). Cook and gelatinize at 65°C for 30 minutes. After cooling to room temperature, 0.05% (m / v) α-amylase (named BAN480 L) was added to the gelatinized liquid and hydrolyzed at 95°C for 80 minutes. Subsequently, the enzyme was inactivated at 90°C for 10 minutes. Subsequently, 0.05% (m / v) neutral protease (named Neutrase 0.8L) was added to the inactivated enzymolysis solution and enzymolyzed at 50°C for 60 minutes. After the enzymolysis was completed, the enzyme was inactivated at 90°C for another 10 minutes. Then, it was filtered with four layers of gauze, and the filtered enzymolysis solution was concentrated, then drum dried, and finally ground into powder.

[0169] Preparation of pre-hydrolyzed corn flour: Corn flour (purchased from Jiangxi Guyifeng Food Technology Co., Ltd.) was mixed at a material-liquid ratio of 1:5 (m / v). Cook and gelatinize at 65°C for 30 minutes. After cooling to room temperature, 0.05% (m / v) α-amylase (named BAN480 L) was added to the gelatinized liquid and hydrolyzed at 95°C for 80 minutes. Subsequently, the enzyme was inactivated at 90°C for 10 minutes. Subsequently, 0.05% (m / v) neutral protease (named Neutrase 0.8L) was added to the inactivated enzymolysis solution and hydrolyzed at 50°C for 60 minutes. After the enzymolysis was completed, the enzyme was inactivated at 90°C for another 10 minutes. Then, it was filtered with four layers of gauze, and the filtered enzymolysis solution was concentrated, then drum dried, and finally ground into powder.

[0170] (2) Preparation of pre-hydrolyzed vegetable milk

[0171] The prehydrolyzed cereal raw materials (prehydrolyzed oatmeal powder, prehydrolyzed malt powder, prehydrolyzed rice powder, and prehydrolyzed corn powder) obtained in step (1) are mixed uniformly at a material-water ratio of 1:5 (m / v) to prepare prehydrolyzed oat milk, prehydrolyzed malt milk, prehydrolyzed rice milk, and prehydrolyzed corn milk, and the mixture is sterilized at 115° C. for 30 min.

[0172] (3) The bacterial solution stored in the glycerol tube was added to liquid MRS medium and cultured at 37°C for 24 h. After activation three times according to the above method, the bacterial solution was obtained.

[0173] The fully activated bacterial solution was inoculated into 100 mL of liquid MRS medium at a 10% (v / v) inoculation volume and cultured at 37°C until the mid-logarithmic phase (fermentation time was 10-12 h) to obtain the culture solution. The culture solution was diluted to 1×10 9 CFU / mL was used as the fermentation seed solution.

[0174] According to the above method, ATCC 367 seed solution and D17 seed solution were prepared respectively;

[0175] (4) The prepared fermentation seed liquid was inoculated into the four different pre-hydrolyzed grain emulsions obtained in the above step (2) at an inoculum amount of 10% (v / v), and fermented at 37° C. and 200 rpm for 24 hours, with samples taken every 4 hours. The contents of maltose, glucose, lactic acid, acetic acid, ethanol, glutamic acid and GABA in the fermentation liquid were detected by high performance liquid chromatography (HPLC).

[0176] The results show:

[0177] (1) Figure 1 As shown in the figure, the prehydrolyzed oat milk has a high concentration of maltose, with a maltose content of 37g / L and a glucose content of 7g / L. Both ATCC 367 and D17 strains can ferment prehydrolyzed oat milk well, and both show obvious heterotypic fermentation characteristics. During the 24h fermentation cycle, the ATCC 367 strain consumed 4g / L maltose and 6.58g / L glucose, producing 8.53g / L lactic acid, 1.15g / L acetic acid, and 1.62g / L ethanol. The D17 strain consumed 5.4g / L maltose and 5.53g / L glucose, producing 10.1g / L lactic acid, 1.01g / L acetic acid, and 1g / L ethanol. In addition, the D17 strain can also use the glutamate in pre-hydrolyzed oat milk to produce GABA. The glutamate content dropped from 26.3 mg / L (pre-hydrolyzed oat milk) to 14.3 mg / L (after fermentation), and GABA 27 mg / L was detected (after fermentation). The ATCC 367 strain's ability to produce GABA (9.3 mg / L) by fermenting pre-hydrolyzed oat milk was second. Figure 1 FG in ).

[0178] (2) By Figure 2It can be seen that no maltose was detected in the pre-hydrolyzed caramel malt milk, and the glucose content was 4.5g / L. Both ATCC 367 and D17 strains can successfully ferment pre-hydrolyzed caramel malt milk, and both show obvious heterotypic fermentation characteristics. In the 24h fermentation cycle, the ATCC 367 strain consumed 4g / L glucose and produced 0.9g / L lactic acid, 1.33g / L acetic acid, and 1.8g / L ethanol. The D17 strain consumed 3.37g / L glucose and produced 0.89g / L lactic acid, 1.68g / L acetic acid, and 1.24g / L ethanol.

[0179] In addition, no glutamate was detected in the pre-hydrolyzed caramel malt milk, and no GABA was detected in the fermentation broth.

[0180] (3) By Figure 3 It can be seen that 6.8g / L of maltose and 3.14g / L of glucose were detected in the prehydrolyzed rice milk. Both ATCC 367 and D17 strains can successfully ferment prehydrolyzed rice milk, and both show obvious heterotypic fermentation characteristics. In the 24h fermentation cycle, the ATCC 367 strain consumes 0.85g / L maltose and 2.13g / L glucose, and can produce 1.99g / L of lactic acid, 0.44g / L of acetic acid, and 0.95g / L of ethanol. The D17 strain consumes 1.48g / L maltose and 2.41g / L glucose, producing 2.19g / L of lactic acid, 0.35g / L of acetic acid, and 0.89g / L of ethanol.

[0181] In addition, no glutamate was detected in pre-hydrolyzed rice milk, and no GABA was detected in the fermentation broth.

[0182] (4) By Figure 4 It can be seen that 7.81g / L of maltose and 4.3g / L of glucose were detected in the pre-hydrolyzed corn milk. Both ATCC 367 and D17 strains can successfully ferment pre-hydrolyzed corn milk, and both show obvious heterotypic fermentation characteristics. During the 24h fermentation cycle, the ATCC 367 strain consumed 0.28g / L maltose and 3.56g / L glucose, and produced 3.33g / L of lactic acid, 0.66g / L of acetic acid, and 0.31g / L of ethanol. The D17 strain consumed 0.44g / L maltose and 3.69g / L glucose, and produced 3.34g / L of lactic acid, 0.54g / L of acetic acid, and 0.24g / L of ethanol.

[0183] In addition, no glutamate was detected in pre-hydrolyzed corn milk, and no GABA was detected in the fermentation broth.

[0184] It can be seen that the reference strains of Lactobacillus brevis ATCC 367 and Lactobacillus brevis D17 can adapt to various types of plant-based matrices for fermentation. From the perspective of sugar utilization and lactic acid production, the fermentation ability of the strain in prehydrolyzed oat milk is significantly stronger than that in prehydrolyzed caramel malt milk, prehydrolyzed rice milk and prehydrolyzed corn milk. This may be because the degree of hydrolysis of the other three plant-based matrices is insufficient, resulting in less available sugar in the matrix. It is worth noting that the heterotypic fermentation characteristics of the reference strain of Lactobacillus brevis ATCC 367 are more prominent than those of the D17 strain, that is, ATCC 367 has a stronger ability to produce acetic acid and ethanol than D17. Obviously, excessive heterotypic fermentation will lead to a prominent irritating sour taste and excessive ethanol production, which is not suitable for beverage production.

[0185] In addition, GABA was only detected in pre-hydrolyzed oat milk, and the ability of D17 strain to produce GABA by fermenting pre-hydrolyzed oat milk was significantly stronger than that of ATCC 367 strain, indicating that pre-hydrolyzed oat milk is a potential plant-based matrix for fermentation to produce GABA.

[0186] Example 4: Fermentation and enzymatic hydrolysis of oat milk by Lactobacillus brevis D17 to achieve high GABA production

[0187] The specific steps are as follows:

[0188] 1. Preparation of GABA-enriched oat milk

[0189] (1) Preparation of prehydrolyzed oat flour

[0190] Oatmeal powder (purchased from Xuzhou Fangde Food Co., Ltd.) was mixed with water at a mass ratio of 1:4, and then 2% (m / v) α-amylase (named BAN480 L) was added and enzymolyzed at 60°C for 60 minutes. Subsequently, 2% (m / v) amylase (named Amylase AG 300L) was added to the enzymolysis solution and enzymolyzed at 60°C for 60 minutes. After the enzymolysis was completed, the enzyme was inactivated at 90°C for 10 minutes. Subsequently, 0.2% (m / v) neutral protease (named Neutrase 0.8L) was added to the inactivated enzymolysis solution and enzymolyzed at 50°C for 60 minutes. After the enzymolysis was completed, the enzyme was inactivated at 90°C for another 10 minutes. Then it was filtered with a 60-mesh sieve. The enzymolysis solution was concentrated, then drum-dried, and finally ground into powder to obtain pre-hydrolyzed oatmeal powder.

[0191] (2) Preparation of prehydrolyzed oat milk

[0192] The pre-hydrolyzed oatmeal powder in (1) is mixed with water in a material-water ratio of 1:5 (m / v) and then sterilized to prepare the product.

[0193] (3) Enzymatic hydrolysis

[0194] 1% (m / v) protease ZB3-1P (purchased from Shandong Longkote Enzyme Preparation Co., Ltd., license number: SC20137132300142) was exogenously added to the pre-hydrolyzed oat milk obtained in step (2), placed in a 45°C water bath for enzymatic hydrolysis for 8 hours, and then sterilized and inactivated at 115°C for 30 minutes to obtain enzymatic oat milk for fermentation.

[0195] (4) Strain fermentation:

[0196] The bacterial solution stored in the glycerol tube was inoculated into liquid MRS culture medium and cultured at 37°C for 24 hours. The bacterial solution was obtained after activation three times according to the above method.

[0197] The fully activated bacterial solution was inoculated into 100 mL of liquid MRS medium at a 10% (v / v) inoculation volume and cultured at 37°C until the mid-logarithmic phase (fermentation time was 10-12 h) to obtain the culture solution. The culture solution was diluted to 1×10 9 CFU / mL was used as the fermentation seed liquid to obtain the Lactobacillus brevis D17 seed liquid.

[0198] The fermented seed liquid of the Lactobacillus brevis D17 strain was inoculated into the enzymatic oat milk at an inoculation rate of 10% (v / v), and fermented at 37° C. and 200 rpm for 24 hours, with samples taken every 4 hours. The contents of glutamate and GABA in the fermented liquid were detected by high performance liquid chromatography (HPLC).

[0199] The results are as follows Figure 5 As shown, after adding 1% protease exogenously to the pre-hydrolyzed oat milk, the endogenous glutamate content in the oat milk can be increased to 294 mg / L, and 499 mg / L of GABA can be produced after fermentation with D17 strain.

[0200] Therefore, oat milk hydrolyzed by protease (referred to as enzymatic oat milk) is an ideal substrate for strain fermentation to produce GABA.

[0201] 2. Control Example: Preparation of wheat oligopeptide powder with added oat milk

[0202] The specific steps are the same as step 1, except that (3) in step 1 is adjusted to:

[0203] 1% (m / v) wheat glutamine peptide (purchased from Guangdong Zhongshiyingke Biotechnology Co., Ltd., patent number: ZL201110439352.3, prepared according to the method described in paragraphs 21 to 22 of the specification) is added exogenously to the pre-hydrolyzed oat milk obtained in step (2), and then sterilized and enzyme-inactivated at 115°C for 30 minutes to obtain enzymatic oat milk for fermentation.

[0204] Perform the test according to the method in step 1;

[0205] The results show: Figure 6 As shown, when 1% (m / v) wheat glutamine peptide was added exogenously to oat milk, the endogenous glutamate content in the oat milk was only 19.35 mg / L, and 29.1 mg / L of GABA could be produced after fermentation by D17 strain.

[0206] Example 5: Preparation method of direct-injection bacterial agent of Lactobacillus brevis D17 strain, and determination of survival ability and direct-injection fermentation ability of direct-injection bacterial agent

[0207] (1) Preparation of direct-injection microbial agent

[0208] The bacterial solution stored in the glycerol tube was connected to the liquid MRS medium and cultured at 37°C for 24 hours. The bacterial solution was activated three times according to the above method to obtain the bacterial solution. The fully activated bacterial solution was inoculated into 100 mL of liquid MRS medium at an inoculum of 10% (v / v) and cultured to the mid-logarithmic phase (fermentation time was 10-12 hours) to obtain the culture solution. At this time, the bacterial concentration of the culture solution was 2.8×10 10 CFU / mL, as fermentation seed liquid;

[0209] The fermented seed solution was centrifuged to collect the bacterial cells, and the bacterial cells were washed three times with sterile saline.

[0210] Centrifuge again to collect clean bacterial cells, and then add 18% skim milk (protective agent) in a ratio of 10 (volume of bacterial solution before centrifugation): 1 (volume of skim milk) (v / v) to the bacterial cells to obtain a 10-fold concentrated milk bacterial suspension. The 10-fold concentrated milk bacterial suspension is precooled at 4°C for 6h, -20°C for 12h, and -80°C for 24h in sequence, and after being completely precooled, placed in a vacuum dryer for freeze drying, and the freeze-dried sample is ground into powder for standby use.

[0211] (2) Determination of freeze-dried survival rate

[0212] Add equal amounts of sterile saline to the above freeze-dried bacterial preparations for rehydration, mix well, dilute and plate count, and calculate the number of viable bacteria in the bacterial powder. The number of viable bacteria in the freeze-dried bacterial powder divided by the number of viable bacteria in the bacterial solution before freeze-drying is the survival rate of the strain after freeze-drying.

[0213] The results are shown in Table 7 below. The viable counts of Lactobacillus brevis D17 freeze-dried powder after rehydration with an equal amount of saline were 2.2×10 10 CFU / mL. Compared with the viable bacterial count of D17 bacterial solution before freeze-drying, it was 2.8×10 10 CFU / mL, after vacuum freeze-drying, the survival rate of D17 strain was still 78.6%.

[0214] Table 7 Freeze-dried survival rate of Lactobacillus brevis D17

[0215]

[0216] (3) Determination of the effect of refrigeration time on the survival rate of freeze-dried bacterial preparations

[0217] The freeze-dried bacterial agents stored in a refrigerator at 4 degrees for 0 days, 7 days and 28 days were taken out and rehydrated with an equal amount of sterile saline. After mixing, they were diluted and plated for counting to monitor the effect of refrigeration time on the survival rate of the freeze-dried bacterial agents.

[0218] The results are shown in Table 8 below. The freeze-dried powder of D17 strain was placed in a 4°C refrigerator, and the survival rate after 7 days of refrigeration was 96.8%, and the survival rate after 28 days of refrigeration was still 77.7%. This shows that Lactobacillus brevis D17 is suitable for vacuum freeze drying, and refrigeration storage for a period of time will not have a significant impact on its survival rate.

[0219] Table 8: Survival rate of freeze-dried bacterial preparation of Lactobacillus brevis D17 under 4°C refrigeration conditions

[0220]

[0221] (4) Direct Fermentation

[0222] The wet bacterial cells (bacterial mud) obtained by centrifugation before freeze-drying were used as a control. The bacterial mud was resuspended with an equal amount of sterile physiological saline before centrifugation and inoculated into the enzymatic oat milk (prepared according to the method of step (3) of Example 2) at an inoculum amount of 10% (m / v);

[0223] Weigh a certain amount of freeze-dried bacterial powder (ensuring that the CFU number is consistent with that of the 10% bacterial sludge resuspension), resuspend it in an equal amount of sterile saline, and inoculate it into enzymatic oat milk (prepared according to the method of step (3) of Example 2);

[0224] The above systems were fermented at 37°C and 200 rpm for 24 hours, and samples were taken every 4 hours. The contents of maltose, glucose, lactic acid, acetic acid, ethanol, glutamic acid and GABA in the fermentation broth were detected by high performance liquid chromatography (HPLC).

[0225] The results are as follows Figure 7As shown in the figure, the D17 freeze-dried bacterial powder was directly fermented in a direct-throwing manner, and the enzymatic oat milk could be successfully fermented, indicating that the D17 strain is suitable for the preparation of direct-throwing starter. During the 24-hour fermentation cycle, the D17 bacterial mud group consumed 6.8 g / L maltose, 8 g / L glucose and 365 mg / L glutamic acid, and produced 12.3 g / L lactic acid, 2.73 g / L acetic acid, 1.52 g / L ethanol and 492 mg / L GABA. The D17 freeze-dried bacterial agent group consumed 6.7 g / L maltose, 8 g / L glucose and 402 mg / L glutamic acid, and produced 12.3 g / L lactic acid, 2.98 g / L acetic acid, 0.86 g / L ethanol and 507 mg / L GABA. It can be seen that the direct-throwing fermentation performance of Lactobacillus brevis D17 is not inferior to that of bacterial mud fermentation, and direct-throwing fermentation may be an effective measure to further improve the fermentation performance.

[0226] Example 6: Preparation of postbiotic preparation containing GABA by Lactobacillus brevis D17 using oats as substrate

[0227] The specific steps are as follows:

[0228] 1. Preparation of GABA-containing postbiotic preparations by Lactobacillus brevis D17 using cereal protein as substrate

[0229] 1. Lactobacillus brevis D17 produces GABA by fermentation with oats as substrate

[0230] (1) Preparation of prehydrolyzed oat flour:

[0231] Oatmeal powder (purchased from Xuzhou Fangde Food Co., Ltd.) was mixed with water at a mass ratio of 1:4, and then 2% (m / v) α-amylase (named BAN480 L, purchased from Novozymes) was added and enzymolyzed at 60°C for 60 minutes. Subsequently, 2% (m / v) amylase (named Amylase AG 300L, purchased from Novozymes) was added to the enzymolysis solution and enzymolyzed at 60°C for 60 minutes. After the enzymolysis was completed, the enzyme was inactivated at 90°C for 10 minutes. Subsequently, 0.2% (m / v) of neutral protease (named Neutrase 0.8L, purchased from Novozymes) was added to the inactivated enzymolysis solution and enzymolyzed at 50°C for 60 minutes. After the enzymolysis was completed, the enzyme was inactivated at 90°C for another 10 minutes. Then it was filtered with a 60-mesh sieve. The enzymolysis solution was concentrated, then drum-dried, and finally ground into powder.

[0232] (2) Preparation of prehydrolyzed oat mixture

[0233] The prehydrolyzed oatmeal powder obtained in step (1) was mixed uniformly with water at a material-water ratio of 1:5 (m / v) to prepare a prehydrolyzed oatmeal mixture, which was sterilized at 115° C. for 30 min.

[0234] (3) The bacterial solution stored in the glycerol tube was added to liquid MRS medium and cultured at 37°C for 24 h. After activation three times according to the above method, the bacterial solution was obtained.

[0235] The fully activated bacterial solution was inoculated into 100 mL of liquid MRS medium at a 10% (v / v) inoculation volume and cultured at 37°C until the mid-logarithmic phase (fermentation time was 10-12 h) to obtain the culture solution. The culture solution was diluted to 1×10 9 CFU / mL was used as the fermentation seed solution.

[0236] According to the above method, ATCC 367 seed solution and D17 seed solution were prepared respectively;

[0237] (4) The prepared fermentation seed liquid was inoculated into the pre-hydrolyzed oat mixture obtained in the above step (2) at an inoculum amount of 10% (v / v), and fermented at 37° C. and 200 rpm for 24 hours, with samples taken every 4 hours. The content of glutamate and GABA in the fermentation liquid was detected by high performance liquid chromatography (HPLC).

[0238] (5) Contrast:

[0239] The specific implementation method is the same as steps (1) to (4), except that the pre-hydrolyzed oat flour is adjusted to be pre-hydrolyzed caramel malt flour, pre-hydrolyzed rice flour, or pre-hydrolyzed corn flour;

[0240] Preparation of pre-hydrolyzed caramel malt powder: Caramel malt powder (purchased from Jinan Shuangmai Beer Materials Co., Ltd.) was mixed at a material-liquid ratio of 1:5 (m / v). After cooking and gelatinization at 65°C for 30 minutes, 0.05% (m / v) α-amylase (named BAN480 L) was added to the gelatinization solution and enzymolyzed at 95°C for 80 minutes. Subsequently, the enzyme was inactivated at 90°C for 10 minutes. Subsequently, 0.05% (m / v) neutral protease (named Neutrase 0.8L) was added to the inactivated enzymolysis solution and enzymolyzed at 50°C for 60 minutes. After the enzymolysis was completed, the enzyme was inactivated at 90°C for another 10 minutes. Subsequently, it was filtered with four layers of gauze, and the filtered enzymolysis solution was concentrated, then drum dried, and finally ground into powder.

[0241] Preparation of pre-hydrolyzed rice flour: Rice flour (purchased from Jiangxi Guyifeng Food Technology Co., Ltd.) was mixed at a material-liquid ratio of 1:5 (m / v). Cook and gelatinize at 65°C for 30 minutes. After cooling to room temperature, 0.05% (m / v) α-amylase (named BAN480 L) was added to the gelatinized liquid and hydrolyzed at 95°C for 80 minutes. Subsequently, the enzyme was inactivated at 90°C for 10 minutes. Subsequently, 0.05% (m / v) neutral protease (named Neutrase 0.8L) was added to the inactivated enzymolysis solution and enzymolyzed at 50°C for 60 minutes. After the enzymolysis was completed, the enzyme was inactivated at 90°C for another 10 minutes. Then, it was filtered with four layers of gauze, and the filtered enzymolysis solution was concentrated, then drum dried, and finally ground into powder.

[0242] Preparation of pre-hydrolyzed corn flour: Corn flour (purchased from Jiangxi Guyifeng Food Technology Co., Ltd.) was mixed at a material-liquid ratio of 1:5 (m / v). Cook and gelatinize at 65°C for 30 minutes. After cooling to room temperature, 0.05% (m / v) α-amylase (named BAN480 L) was added to the gelatinized liquid and hydrolyzed at 95°C for 80 minutes. Subsequently, the enzyme was inactivated at 90°C for 10 minutes. Subsequently, 0.05% (m / v) neutral protease (named Neutrase 0.8L) was added to the inactivated enzymolysis solution and hydrolyzed at 50°C for 60 minutes. After the enzymolysis was completed, the enzyme was inactivated at 90°C for another 10 minutes. Then, it was filtered with four layers of gauze, and the filtered enzymolysis solution was concentrated, then drum dried, and finally ground into powder.

[0243] The contents of glutamate and GABA in the fermentation broth were detected by high performance liquid chromatography (HPLC).

[0244] (6) Preparing a postbiotic preparation containing GABA from the fermentation end product obtained in step (4)

[0245] The fermentation end product containing the bacteria is concentrated at 60-65°C for 1-2h, and after the total solid content reaches 20-40%, it is spray-dried to obtain the postbiotic powder containing GABA and inactivated bacteria. The spray drying conditions are: feed temperature 30-40°C, inlet temperature 130-150°C, outlet temperature 50-60°C, inlet air pressure 0.25-0.3MPa, centrifugal turntable speed 19000-20000r / min, and drying time 5-15s.

[0246] 2. Experimental results:

[0247] The results show:

[0248] (1) D17 strain can also use glutamate in pre-hydrolyzed oats to produce GABA. The glutamate content dropped from 26.3 mg / L (pre-hydrolyzed oat milk) to 14.3 mg / L (after fermentation), and GABA 27 mg / L was detected (after fermentation). ATCC 367 strain had the second highest GABA production capacity (9.3 mg / L) when fermenting pre-hydrolyzed oats.

[0249] (2) Comparative data:

[0250] No glutamate was detected in the prehydrolyzed caramel malt, and no GABA was detected in the fermentation broth.

[0251] No glutamate was detected in prehydrolyzed rice, and no GABA was detected in the fermentation broth.

[0252] Glutamic acid was not detected in prehydrolyzed corn, and GABA was not detected in the fermentation broth.

[0253] In summary, GABA was only detected in prehydrolyzed oats, and the ability of D17 strain to produce GABA by fermenting prehydrolyzed oats was significantly stronger than that of ATCC 367 strain, indicating that prehydrolyzed oats is a potential plant-based matrix for preparing GABA-containing postbiotics by fermentation without exogenous glutamate / sodium glutamate.

[0254] 2. Improving GABA production in postbiotic preparations containing GABA prepared by Lactobacillus brevis D17 using oats as substrate

[0255] 1. Preparation of GABA-enriched oat milk

[0256] (1) Preparation of prehydrolyzed oat flour

[0257] Oatmeal powder (purchased from Xuzhou Fangde Food Co., Ltd.) was mixed with water at a mass ratio of 1:4, and then 2% (m / v) α-amylase (named BAN480 L) was added and enzymolyzed at 60°C for 60 minutes. Subsequently, 2% (m / v) amylase (named Amylase AG 300L) was added to the enzymolysis solution and enzymolyzed at 60°C for 60 minutes. After the enzymolysis was completed, the enzyme was inactivated at 90°C for 10 minutes. Subsequently, 0.2% (m / v) neutral protease (named Neutrase 0.8L) was added to the inactivated enzymolysis solution and enzymolyzed at 50°C for 60 minutes. After the enzymolysis was completed, the enzyme was inactivated at 90°C for another 10 minutes. Then it was filtered with a 60-mesh sieve. The enzymolysis solution was concentrated, then drum-dried, and finally ground into powder to obtain pre-hydrolyzed oatmeal powder.

[0258] (2) Preparation of prehydrolyzed oat mixture

[0259] The pre-hydrolyzed oatmeal powder in (1) is mixed with water in a material-water ratio of 1:5 (m / v) and then sterilized to prepare the product.

[0260] (3) Enzymatic hydrolysis

[0261] 1% (m / v) protease ZB3-1P (purchased from Shandong Longkote Enzyme Preparation Co., Ltd., license number: SC20137132300142) was exogenously added to the pre-hydrolyzed oat mixture obtained in step (2), placed in a 45°C water bath for enzymolysis for 8 hours, and then sterilized and inactivated at 115°C for 30 minutes to obtain an enzymolyzed oat mixture for fermentation.

[0262] (4) Strain fermentation:

[0263] The bacterial solution stored in the glycerol tube was inoculated into liquid MRS culture medium and cultured at 37°C for 24 hours. The bacterial solution was obtained after activation three times according to the above method.

[0264] The fully activated bacterial solution was inoculated into 100 mL of liquid MRS medium at a 10% (v / v) inoculation volume and cultured at 37°C until the mid-logarithmic phase (fermentation time was 10-12 h) to obtain the culture solution. The culture solution was diluted to 1×10 9 CFU / mL was used as the fermentation seed liquid to obtain the Lactobacillus brevis D17 seed liquid.

[0265] The fermented seed liquid of the Lactobacillus brevis D17 strain was inoculated into the enzymatic oat mixture at an inoculation rate of 10% (v / v), and fermented at 37° C. and 200 rpm for 24 hours, with samples taken every 4 hours. The content of glutamate and GABA in the fermented liquid was detected by high performance liquid chromatography (HPLC).

[0266] The results are as follows Figure 5 As shown, after adding 1% protease exogenously to the pre-hydrolyzed oat milk, the endogenous glutamate content in the oat milk can be increased to 294 mg / L, and 499 mg / L of GABA can be produced after fermentation with D17 strain.

[0267] (5) Preparing a postbiotic preparation containing GABA from the fermentation end product obtained in step (4)

[0268] The fermentation end product containing the bacteria is concentrated at 60-65°C for 1-2h, and after the total solid content reaches 20-40%, it is spray-dried to obtain the postbiotic powder containing GABA and inactivated bacteria. The spray drying conditions are: feed temperature 30-40°C, inlet temperature 130-150°C, outlet temperature 50-60°C, inlet air pressure 0.25-0.3MPa, centrifugal turntable speed 19000-20000r / min, and drying time 5-15s.

[0269] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A plant-based fermented milk, characterized in that: The plant-based fermented milk is prepared according to the following steps without adding exogenous glutamic acid and glutamate: Lactobacillus brevis ( Lactobacillus brevis ) CGMCC NO.14385 is added to a plant-based matrix for fermentation to prepare a plant-based fermented milk; the plant-based matrix is ​​pre-hydrolyzed oat milk; the medium for activating and culturing Lactobacillus brevis CGMCC NO.14385 is MRS medium; The preparation method of the pre-hydrolyzed oat milk is as follows: oat flour is mixed with water, 2-3% (m / v) α-amylase is added, and after enzymolysis at 55-65° C. for 60-70 min, 2-3% (m / v) saccharifying enzyme is added to the enzymolysis solution for saccharification, and the saccharification conditions are as follows: saccharification at 55-65° C. for 60-70 min, and enzyme inactivation is performed after the saccharification is completed; 0.2-0.3% (m / v) neutral protease is added to the enzyme-inactivated saccharification solution, and after enzymolysis at 50-60° C. for 60-70 min, the enzyme is inactivated to obtain an enzymolysis solution; the obtained enzymolysis solution is concentrated and dried to obtain pre-hydrolyzed oat flour; the prepared pre-hydrolyzed oat flour is mixed according to a material-water ratio of 1-2:5-10, and sterilized to prepare oat milk; protease ZB3-1P is added to the obtained oat milk, and enzymolysis is performed at 40-50° C. for 7-10 min. After h, prehydrolyzed oat milk was obtained.

2. The plant-based fermented milk according to claim 1, characterized in that: The fermentation conditions are: 35-40°C, 100-200 rpm for 12-36 h.

3. The plant-based fermented milk according to claim 1 or 2, characterized in that The oatmeal powder and water are mixed in a ratio of 1-2:4-8.

4. A method for preparing oat milk rich in GABA without exogenous addition of glutamate and glutamate, characterized in that: The oat milk is prepared according to the following steps: (1) Oatmeal powder is mixed with water, 2-3% (m / v) α-amylase is added, and after enzymatic hydrolysis at 55-65°C for 60-70 min, 2-3% (m / v) saccharifying enzyme is added to the enzymatic hydrolysis solution for saccharification, and the saccharification conditions are as follows: saccharification at 55-65°C for 60-70 min, and enzyme inactivation is performed after saccharification is completed; 0.2-0.3% (m / v) neutral protease is added to the enzyme-inactivated saccharification solution, and after enzymatic hydrolysis at 50-60°C for 60-70 min, the enzyme is inactivated to obtain an enzymatic hydrolysis solution; the obtained enzymatic hydrolysis solution is concentrated and dried to obtain pre-hydrolyzed oatmeal powder; the obtained pre-hydrolyzed oatmeal powder is mixed according to a material-water ratio of 1-2:5-10, and sterilized to prepare oat milk; protease ZB3-1P is added to the obtained oat milk, and after enzymatic hydrolysis at 40-50°C for 7-10 h, pre-hydrolyzed oat milk is obtained; (2) Adding Lactobacillus brevis CGMCC NO.14385 to the pre-hydrolyzed oat milk obtained in step (1) and fermenting the mixture to obtain oat milk; the medium for activating and culturing Lactobacillus brevis CGMCC NO.14385 is MRS medium.

5. The method according to claim 4, characterized in that The fermentation conditions in step (2) are: 35-40°C, 100-200 rpm, and fermentation for 12-36 h.

6. A method for preparing GABA using oatmeal as a substrate, characterized in that: The method does not require exogenous addition of glutamate and glutamate, and specifically comprises the following steps: (1) Oatmeal powder was mixed with water, 2-3% (m / v) α-amylase was added, and the mixture was enzymatically hydrolyzed at 55-65°C for 60-70 min. Then, 2-3% (m / v) saccharifying enzyme was added to the enzymatic hydrolyzate for saccharification. The saccharification conditions were as follows: saccharification at 55-65°C for 60-70 min, and enzyme inactivation was performed after the saccharification was completed. 0.2-0.3% (m / v) neutral protease was added to the enzyme-inactivating saccharification solution, and the mixture was enzymatically hydrolyzed at 50-60°C for 60-70 min, and enzyme inactivation was performed to obtain enzymatic hydrolyzate. The obtained enzymatic hydrolyzate was concentrated and dried to obtain pre-hydrolyzed oatmeal powder. The obtained pre-hydrolyzed oatmeal powder was mixed according to a material-water ratio of 1-2:5-10, and sterilized to prepare oat hydrolyzate. Protease ZB3-1P was added to the obtained oat hydrolyzate, and enzymatic hydrolysis was performed at 40-50°C for 7-10 min. h later, the prehydrolyzed oat substrate was obtained; (2) Adding Lactobacillus brevis CGMCC No. 14385 to the pre-hydrolyzed oat substrate obtained in step (1) for fermentation to prepare a fermentation liquid containing GABA, and extracting GABA to prepare GABA; the medium for activating and culturing Lactobacillus brevis CGMCC No. 14385 is MRS medium.

7. The method according to claim 6, characterized in that The fermentation conditions in step (2) are: 35-40°C, 100-200 rpm, and fermentation for 12-36 h.

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