Lactobacillus plantarum CX1-3-2 with effects of producing gamma-aminobutyric acid and improving sleep and application of lactobacillus plantarum CX1-3-2

Abstract

The invention belongs to the technical field of microorganisms, and particularly discloses lactobacillus plantarum CX1-3-2 capable of producing gamma-aminobutyric acid and improving sleep and application of the lactobacillus plantarum CX1-3-2. The preservation number of the lactobacillus plantarum CX1-3-2 strain is CGMCC (China General Microbiological Culture Collection Center) No. 34340. The lactobacillus plantarum CX1-3-2 can achieve the effect of improving sleep by improving the levels of GABA and 5-HT in brain tissues and reducing the levels of glutamic acid Glu, dopamine DA and noradrenaline NE; the scavenging rate of DPPH free radicals of the strain reaches 96.4%, the scavenging rate of ABTS free radicals of the strain reaches 78.3%, and the strain has an excellent anti-oxidation effect; the diameters of inhibition zones of the strain on staphylococcus aureus, bacillus cereus, escherichia coli and pseudomonas fluorescens are all larger than 17 mm, and the strain has an excellent antibacterial effect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a plant lactobacillus CX1-3-2 with the functions of producing gamma-aminobutyric acid and improving sleep and application thereof. BACKGROUND

[0002] Probiotics are a class of active microorganisms that are beneficial to the host, which widely exist in the human intestinal tract and reproductive system, can produce definite health effects to improve the microecological balance of the host and play a probiotic role. In addition, more and more evidence shows that the change in the composition of intestinal microorganisms is related to the change in mood, pain and cognitive-related behaviors, and the bidirectional communication pathway between the microbiome and the brain plays a crucial role in health and disease. Based on this, the use of probiotics to assist in improving sleep has become a research hotspot in recent years. Lactobacillus plantarum is a species in the genus of lactobacillus, which occupies a key position in the fermentation industry, has the functions of regulating intestinal flora balance, enhancing intestinal barrier, immune regulation and improving nutrient absorption, and has great significance for meeting the increasing demand of the food, health care and pharmaceutical industries and continuously contributing to human health and food innovation.

[0003] In view of the various probiotic functions of probiotics, advanced fermentation technology and drying process are currently used in the industry to produce probiotic preparations with high activity and high stability on a large scale, such as bacterial powder, capsules and the like. Probiotic powder, as a popular nutritional supplement, takes carefully selected probiotic strains as the core ingredient, such as lactobacillus and bifidobacterium, and thus has various probiotic functions. However, most of the products on the market use a single strain, and the probiotic function of lactobacillus plantarum has not been fully explored and developed. In addition, these products are mostly directly fermented using ordinary MRS medium, resulting in poor smell and taste of the product, and even other additives need to be added to adjust, which not only increases the health risk, but also is not conducive to the probiotic function in the intestinal tract. Therefore, it is necessary to further explore more lactobacillus plantarum with probiotic function, and to explore the use of natural medium as fermentation medium, so as to make the probiotic powder have a broader application prospect and development space. SUMMARY

[0004] In view of this, the present application provides a Lactobacillus plantarum CX1-3-2 strain with the functions of producing gamma-aminobutyric acid and improving sleep, and its application. The strain has the effects of improving sleep, antioxidation and bacteriostasis. The Lactobacillus plantarum CX1-3-2 is inoculated in a fruit and vegetable juice culture medium or a milk powder culture medium for fermentation. The specific culture medium is conducive to improving the amount of gamma-aminobutyric acid produced by the Lactobacillus plantarum CX1-3-2 and the number of viable Lactobacillus plantarum CX1-3-2 in the fermentation product. The fermentation product is applied in the bacterial powder, which not only can improve the effects of the bacterial powder in improving sleep, antioxidation and bacteriostasis, but also can improve the taste of the bacterial powder, and has high application value.

[0005] To achieve the above-mentioned application purposes, the present application adopts the following technical solutions:

[0006] A Lactobacillus plantarum CX1-3-2 strain with the functions of producing gamma-aminobutyric acid and improving sleep, and its application. The classification name of the strain is Lactobacillus plantarum, which was preserved in the China General Microbiological Culture Collection Center on April 25, 2025, and the preservation number is CGMCC No.34340. The preservation address is No.3, Beichen West Road, Chaoyang District, Beijing.

[0007] Compared with the prior art, the Lactobacillus plantarum CX1-3-2 with the preservation number of CGMCC No.34340 provided by the present application has the following advantages:

[0008] (1) The Lactobacillus plantarum CX1-3-2 provided by the present application has good acid and bile salt resistance, and also has certain hydrophobicity and self-aggregation, which can ensure that the Lactobacillus plantarum CX1-3-2 reaches and colonizes in the intestinal tract, so as to fully exert its functions;

[0009] (2) The Lactobacillus plantarum CX1-3-2 provided by the present application can improve the levels of brain tissue GABA and 5-hydroxytryptamine (5-HT), and reduce the levels of glutamic acid (Glu), dopamine (DA) and norepinephrine (NE), thereby achieving the effect of improving sleep;

[0010] (3) The Lactobacillus plantarum CX1-3-2 provided by the present application can scavenge ABTS free radicals and DPPH free radicals, thereby achieving the effect of antioxidation;

[0011] (4) The Lactobacillus plantarum CX1-3-2 provided by the present application can inhibit or kill Staphylococcus aureus, Bacillus cereus, Escherichia coli and Pseudomonas fluorescens, and has excellent bacteriostatic effect.

[0012]

[0013] The application provides application of the Lactobacillus plantarum CX1-3-2 strain in production of gamma-aminobutyric acid.

[0014] The application provides application of the Lactobacillus plantarum CX1-3-2 strain in preparation of sleep-improving, antioxidant and bacteriostatic products.

[0015] Exemplarily, the bacteriostasis is represented by inhibition of at least one of Staphylococcus aureus, Bacillus cereus, Escherichia coli or Pseudomonas fluorescens.

[0016] The application provides a fermentation product, which is prepared by fermentation of the Lactobacillus plantarum CX1-3-2 strain.

[0017] Exemplarily, the fermentation metabolite in the fermentation product comprises at least one of gamma-aminobutyric acid, hydroxybenzyl lactic acid, citric acid, malic acid, succinic acid, lactic acid, formic acid, acetic acid, propionic acid, isobutyric acid or benzyl lactic acid.

[0018] Preferably, the fermentation specifically comprises the following operation: inoculating seed liquid of the Lactobacillus plantarum CX1-3-2 strain into a culture medium for fermentation culture.

[0019] The culture medium is a fruit and vegetable juice culture medium or a milk powder culture medium.

[0020] The Lactobacillus plantarum CX1-3-2 is fermented and cultured by using the fruit and vegetable juice culture medium or the milk powder culture medium, and the specific culture medium can significantly improve the yield of gamma-aminobutyric acid and the number of viable Lactobacillus plantarum CX1-3-2 in the fermentation product, and when the fermentation product is applied to a bacterial powder, the sleep-improving ability of the bacterial powder can be obviously improved.

[0021] The natural nutritional factors in the fruit and vegetable juice not only promote the growth of the strain, but also help to improve the yield of GABA and promote the production of more abundant secondary metabolites, and when the fruit and vegetable juice is applied to the bacterial powder, the function of the bacterial powder can be further improved; in addition, the fruit and vegetable juice is used as the fermentation culture medium, the production cost is further reduced, the fermentation product has a rich fruit and vegetable fragrance, also has the nutritional ingredients of fruits and vegetables, the taste of the bacterial powder is improved, and the fermentation product has high application value.

[0022] The milk powder is rich in lactose, whey protein, calcium, phosphorus and other substances, can provide balanced and easily utilized nutrition for the Lactobacillus plantarum CX1-3-2, and promote efficient multiplication of the bacterial body; meanwhile, the milk powder has wide sources and controllable cost, and when the milk powder is made into a culture medium and used for fermentation of the Lactobacillus plantarum CX1-3-2, the culture medium not only adapts to the needs of the Lactobacillus plantarum CX1-3-2, but also meets the natural adaptability of the Lactobacillus plantarum CX1-3-2 to the milk source nutrition, is beneficial to large-scale fermentation production, can promote the bacterial body to synthesize more functional metabolites, and improve the application value of the fermentation product.

[0023] Preferably, the fermentation culture is carried out at a temperature of 27-47℃ for 24-72 hours.

[0024] Preferably, the inoculation amount of the seed liquid of the Lactobacillus plantarum CX1-3-2 strain is 1-5%, and the viable cell count of the seed liquid of the Lactobacillus plantarum CX1-3-2 strain is 10 8 CFU / mL-10 9 CFU / mL.

[0025] Preferably, the fruit and vegetable juice culture medium comprises water, fruit and vegetable juice, carbon source, nitrogen source and L-glutamate sodium.

[0026] The present application further limits the components of the fruit and vegetable juice culture medium, which can further promote the production of γ-aminobutyric acid and the viable cell count of Lactobacillus plantarum CX1-3-2, and thus improve the performance of the fermentation product and the bacterial powder.

[0027] Preferably, the carbon source is at least one of glucose, maltose, sucrose or lactose.

[0028] Preferably, the nitrogen source is at least one of peptone, yeast extract powder, corn syrup powder or beef extract.

[0029] Preferably, the fruit and vegetable juice is at least one of tomato juice or grape juice.

[0030] Preferably, the addition amount of fruit and vegetable juice is 20-100% based on the sum of the volumes of water and fruit and vegetable juice, and the balance is water.

[0031] Preferably, the concentration of the carbon source in the fruit and vegetable juice culture medium is 0-5g / 100mL.

[0032] Preferably, the concentration of the nitrogen source in the fruit and vegetable juice culture medium is 0.5-2.5g / 100mL.

[0033] Preferably, the concentration of L-glutamate sodium in the fruit and vegetable juice culture medium is 0-4g / 100mL.

[0034] Preferably, the pH value of the fruit and vegetable juice culture medium is 5-7.

[0035] Preferably, the milk powder culture medium comprises water, whole milk powder or skim milk powder, carbon source, nitrogen source and L-glutamate sodium.

[0036] The present application further limits the components of the fruit and vegetable juice culture medium, which can further promote the production of γ-aminobutyric acid and the viable cell count of Lactobacillus plantarum CX1-3-2, and thus improve the performance of the fermentation product and the bacterial powder.

[0037] Preferably, the milk powder is added in an amount of 4% to 20% based on the total mass of the water and the milk powder, and the remainder is water.

[0038] Preferably, the concentration of the carbon source in the milk powder medium is 0g / 100g to 5g / 100g.

[0039] Preferably, the concentration of the nitrogen source in the milk powder medium is 0.5g / 100g to 2.5g / 100g.

[0040] Preferably, the concentration of the sodium L-glutamate in the milk powder medium is 0g / 100g to 4g / 100g.

[0041] Preferably, the pH value of the milk powder medium is 5 to 7.

[0042] Preferably, the carbon source is at least one of glucose, maltose, sucrose or lactose.

[0043] Preferably, the nitrogen source is at least one of peptone, yeast extract, corn steep liquor or beef extract.

[0044] Preferably, the milk powder is at least one of whole milk powder or skim milk powder.

[0045] The present application provides a bacterial powder, which comprises at least one of the above-mentioned Lactobacillus plantarum CX1-3-2 strain or the above-mentioned fermentation product.

[0046] Preferably, the bacterial powder is prepared by mixing the above-mentioned fermentation product and a protective agent and then drying.

[0047] Preferably, the content of gamma-aminobutyric acid in the bacterial powder is 7mg / g to 10mg / g, and the viable bacterial count of Lactobacillus plantarum CX1-3-2 is 10 7 CFU / g to 10 8 CFU / g.

[0048] Preferably, the mass-to-volume ratio of the protective agent to the fermentation product is (4 to 12)g:100mL.

[0049] Preferably, the protective agent is at least one of skim milk powder, whole milk powder, trehalose, lactose or maltodextrin.

[0050] Preferably, the drying comprises vacuum freeze-drying or spray drying.

[0051] The present application also provides the use of the above-mentioned Lactobacillus plantarum CX1-3-2 strain, the above-mentioned fermentation product or the above-mentioned bacterial powder in the preparation of food, medicine, health products, feed, food additives or feed additives.

[0052] The Lactobacillus plantarum CX1-3-2 provided by the present application has multiple functions such as improving sleep, resisting oxidation and inhibiting bacteria, and has the advantages of synergistic effect, efficiency improvement and diversified health demand satisfaction compared with single-function strains, and can provide more comprehensive health benefits by regulating multiple physiological processes, and has higher practical value in health product development and actual application. BRIEF DESCRIPTION OF DRAWINGS

[0053] Figure 1 Figure 1 is a bacterial colony morphology diagram of the Lactobacillus plantarum CX1-3-2 strain in the embodiment 1 of the present application;

[0054] Figure 2 Figure 1 is a bacterial colony morphology diagram of the Lactobacillus plantarum CX1-3-2 strain in the embodiment 1 of the present application;

[0055] Figure 3 Figure 3 is a phylogenetic tree of the Lactobacillus plantarum CX1-3-2 strain in the embodiment 1 of the present application;

[0056] Figure 4 Figure 8 is a statistical diagram of the influence of different treatment groups on the pentobarbital sodium sleep time prolongation in the embodiment 88 of the present application;

[0057] Figure 5 Figure 9 is a statistical diagram of the influence of different treatment groups on the pentobarbital sodium sleep latency in the embodiment 88 of the present application;

[0058] Figure 6 Figure 10 is a change diagram of the influence of different treatment groups on the body weight of mice in the embodiment 88 of the present application. DETAILED DESCRIPTION

[0059] In order to make the purpose, technical scheme and advantages of the present application clearer and more apparent, the present application will be further described in detail below in combination with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and do not limit the present application.

[0060] The strains used in the following embodiments of the present application are as follows:

[0061] The Lactobacillus plantarum CX1-3-2, whose classification name is Lactobacillus plantarum, was preserved in the China General Microbiological Culture Collection Center on April 25, 2025, and its preservation number is CGMCC No. 34340; the preservation address is No. 3, Beichen West Road, Chaoyang District, Beijing.

[0062] MRS liquid medium: glucose 20 g, peptone 10 g, beef extract 10 g, yeast extract 5 g, sodium acetate 5 g, potassium phosphate dibasic 2 g, triammonium citrate 2 g, manganese sulfate 0.25 g, magnesium sulfate 0.58 g, Tween 80 1 mL, supplemented with water to 1000 mL, pH adjusted to 6.2-6.4, sterilized at 121°C for 15 min.

[0063] MRS solid medium: glucose 20 g, peptone 10 g, beef extract 10 g, yeast extract 5 g, sodium acetate 5 g, potassium phosphate dibasic 2 g, triammonium citrate 2 g, manganese sulfate 0.25 g, magnesium sulfate 0.58 g, Tween 80 1 mL, agar 15-20 g, supplemented with water to 1000 mL, pH adjusted to 6.2-6.4, sterilized at 121°C for 15 min.

[0064] Other experimental materials and instruments, if not specified, can be purchased through commercial channels.

[0065] Example 1

[0066] This example provides a Lactobacillus plantarum CX1-3-2 strain, which is screened by the following steps:

[0067] 1. Preliminary screening of lactic acid bacteria

[0068] Samples of homemade pickles from Cangzhou, Hebei were collected, and after enrichment culture in MRS liquid medium, they were diluted and plated on MRS solid medium containing 3% CaCO3, colonies producing transparent circles were isolated and further purified, and preserved in a -80°C refrigerator with 25% glycerol.

[0069] 2. Rescreening of GABA-producing strains

[0070] (1) High performance liquid chromatography conditions:

[0071] In this study, the content of GABA in the fermentation supernatant was determined using a Waters E2695 high-performance liquid chromatograph (HPLC). The chromatographic conditions were as follows: chromatographic column: Waters Symmetry C18 chromatographic column (4.6 mm x 250 mm); column temperature: 40°C; injection volume: 20 μL; mobile phase A: 20 mmol / L anhydrous sodium acetate aqueous solution, mobile phase B: pure acetonitrile; flow rate: 0.8 mL / min; detection wavelength: 334 nm. The elution gradient was as follows: 0-15 min, B increased from 20% to 50%; 15 min-18 min, B remained at 50%; 18 min-20 min, B decreased from 50% to 20%; 20 min-25 min, B remained at 20%.

[0072] (2) Preparation of γ-aminobutyric acid (GABA) standard curve:

[0073] Accurately weigh 10 mg of GABA standard into a 10 mL volumetric flask, dissolve with water, dilute to the mark, obtain a stock solution with a mass concentration of 1 mg / mL, and sequentially dilute to 0.8 mg / mL, 0.6 mg / mL, 0.4 mg / mL, 0.2 mg / mL, and 0 mg / mL.

[0074] Respectively, 400 μL of boric acid buffer (2.47 g of boric acid is dissolved in 85 mL of water, and the pH is adjusted to 10.4 with sodium hydroxide, and diluted to 100 mL), 80 μL of o-phthaldehyde (OPA) derivative solution (5 mg of OPA is ultrasonically dissolved in 2.5 mL of acetonitrile, and 10 μL of β-mercaptoethanol is added), and 80 μL of GABA standard solution are measured, mixed, derivatized for 5 min, filtered through a 0.22 μm organic filter membrane, immediately injected for analysis and detection, and the whole derivatization operation time is strictly controlled. The standard curve is plotted with GABA concentration as the abscissa and peak area as the ordinate.

[0075] (3) Strain culture treatment:

[0076] Take the strain preserved at -80℃, inoculate 100 μL into 50 mL of MRS liquid medium, and incubate at 37℃ for 24 h, then inoculate at 2% into the fermentation medium, and incubate at 37℃ for 48 h, centrifuge at 8000 r for 10 min, take the supernatant, and measure the γ-aminobutyric acid yield after derivatization according to the standard product derivatization method. The Lactobacillus plantarum CX1-3-2 with high γ-aminobutyric acid yield is screened, and the γ-aminobutyric acid yield is 212.79 mg / L.

[0077] 3. Strain CX1-3-2 identification

[0078] (1) Strain morphological identification

[0079] The strain CX1-3-2 screened is re-streaked and cultured on MRS solid medium, and gram staining is performed, and the morphology and color of the bacteria are observed under the oil lens of a microscope. The strain is gram-positive, short rod-shaped, and the colony is milky white, smooth, and fine. The colony morphology and staining pictures are shown in Figure 1 and 2 .

[0080] (2) 16S rDNA sequence identification

[0081] The strain CX1-3-2 is sent to Shenzhen Huada Gene Co., Ltd. for sequencing, and the 16S rDNA of the strain CX1-3-2 is uploaded to the NCBI database for BLAST homology search, and the MEGA11 software is used to construct a phylogenetic tree, as shown in Figure 3As shown, the strain CX1-3-2 has the highest homology with Lactobacillus plantarum, which is 100.00%, and therefore the strain CX1-3-2 is identified as Lactobacillus plantarum, named as Lactobacillus plantarum CX1-3-2.

[0082] Example 2

[0083] The present example provides a fermentation product of a Lactobacillus plantarum CX1-3-2 strain, which is prepared by fermentation of the Lactobacillus plantarum CX1-3-2 strain, and the fermentation specifically comprises the following steps:

[0084] (1) After the grape stems are removed and washed, the grape is put into a juicer to squeeze juice, and then filtered with gauze for standby;

[0085] (2) 20 mL of grape juice and 80 mL of water are mixed, then 1 g of peptone and 1 g of L-glutamic acid sodium are added, mixed uniformly, the pH value is adjusted to 6.5, and sterilized at 115°C for 15 min as a fermentation medium;

[0086] 10 9 mL of Lactobacillus plantarum CX1-3-2 seed liquid is inoculated into the fermentation medium at an inoculation amount of 2% for fermentation culture, and the fermentation culture temperature is 37°C, and the fermentation culture time is 24 h.

[0087] Example 3

[0088] The present example provides a fermentation product of a Lactobacillus plantarum CX1-3-2 strain, which is different from example 2 in that the addition amount of grape juice is 40 mL, and the addition amount of water is 60 mL;

[0089] The other ingredients and operation methods are the same as those of example 2.

[0090] Example 4

[0091] The present example provides a fermentation product of a Lactobacillus plantarum CX1-3-2 strain, which is different from example 2 in that the addition amount of grape juice is 60 mL, and the addition amount of water is 40 mL;

[0092] The other ingredients and operation methods are the same as those of example 2.

[0093] Example 5

[0094] The present example provides a fermentation product of a Lactobacillus plantarum CX1-3-2 strain, which is different from example 2 in that the addition amount of grape juice is 80 mL, and the addition amount of water is 20 mL;

[0095] The other ingredients and operation methods are the same as those of example 2.

[0096] Example 6

[0097] This example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which is different from Example 2 in that the amount of grape juice added is 100 mL;

[0098] The other ingredients and the method of operation are the same as in Example 2.

[0099] Example 7

[0100] This example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which is different from Example 2 in that the amount of grape juice added is 80 mL, the amount of water added is 20 mL, and the peptone is replaced with an equal amount of corn steep powder;

[0101] The other ingredients and the method of operation are the same as in Example 2.

[0102] Example 8

[0103] This example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which is different from Example 7 in that the corn steep powder is replaced with an equal amount of yeast extract powder;

[0104] The other ingredients and the method of operation are the same as in Example 7.

[0105] Example 9

[0106] This example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which is different from Example 7 in that the corn steep powder is replaced with an equal amount of beef extract;

[0107] The other ingredients and the method of operation are the same as in Example 7.

[0108] Example 10

[0109] This example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which is different from Example 7 in that the amount of corn steep powder added is 0.5 g;

[0110] The other ingredients and the method of operation are the same as in Example 7.

[0111] Example 11

[0112] This example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which is different from Example 7 in that the amount of corn steep powder added is 1.5 g;

[0113] The other ingredients and the method of operation are the same as in Example 7.

[0114] Example 12

[0115] The present example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which is different from Example 7 in that the amount of corn steep powder added is 2 g.

[0116] The other ingredients and the method of operation are the same as in Example 7.

[0117] Example 13

[0118] The present example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which is different from Example 7 in that the amount of corn steep powder added is 2.5 g.

[0119] The other ingredients and the method of operation are the same as in Example 7.

[0120] Example 14

[0121] The present example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which is different from Example 7 in that no L-glutamic acid sodium is added.

[0122] The other ingredients and the method of operation are the same as in Example 7.

[0123] Example 15

[0124] The present example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which is different from Example 7 in that the amount of L-glutamic acid sodium added is 2 g.

[0125] The other ingredients and the method of operation are the same as in Example 7.

[0126] Example 16

[0127] The present example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which is different from Example 7 in that the amount of L-glutamic acid sodium added is 3 g.

[0128] The other ingredients and the method of operation are the same as in Example 7.

[0129] Example 17

[0130] The present example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which is different from Example 7 in that the amount of L-glutamic acid sodium added is 4 g.

[0131] The other ingredients and the method of operation are the same as in Example 7.

[0132] Example 18

[0133] The present example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which is different from Example 7 in that the pH is adjusted to 5.

[0134] The other ingredients and the method of operation are the same as in Example 7.

[0135] Example 19

[0136] This example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which is different from Example 7 in that the pH value is adjusted to 5.5;

[0137] Other ingredients and operating methods are the same as Example 7.

[0138] Example 20

[0139] This example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which is different from Example 7 in that the pH value is adjusted to 6;

[0140] Other ingredients and operating methods are the same as Example 7.

[0141] Example 21

[0142] This example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which is different from Example 7 in that the pH value is adjusted to 7;

[0143] Other ingredients and operating methods are the same as Example 7.

[0144] The viable cell count of Lactobacillus plantarum CX1-3-2 in the fermentation products provided in Examples 2-21 was determined, and the results are shown in Table 1. When grape juice was used as the fermentation medium, the optimal formulation was as follows: fruit and vegetable juice was grape juice, and the addition amount was 60% to 100%; the nitrogen source was corn syrup powder, and the addition amount was 1.5 g / 100 mL to 2.5 g / 100 mL; the addition amount of L-glutamic acid sodium was 0 to 2 g / 100 mL; and the pH value was 5.5 to 6.5. Under these conditions, the viable cell count of Lactobacillus plantarum CX1-3-2 in the fermentation product reached 10 7 CFU / mL to 10 9 CFU / mL.

[0145] Table 1 Viable cell count of Lactobacillus plantarum CX1-3-2 fermentation product

[0146]

[0147]

[0148] Example 22

[0149] This example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which is different from Example 7 in that the pH value is adjusted to 5.5;

[0150] (1) The grapes were cleaned and then put into a juicer to extract juice, which was then filtered with gauze and prepared for use;

[0151] (2) 80 mL of grape juice and 20 mL of water were mixed, then 2 g of corn syrup powder and 1 g of L-glutamic acid sodium were added, mixed uniformly, the pH value was adjusted to 6, and sterilized at 115°C for 15 min as a fermentation medium;

[0152] 10 9 CFU / mL of Lactobacillus plantarum CX1-3-2 seed liquid was inoculated into the fermentation medium at an inoculation amount of 2% for fermentation culture, the fermentation culture temperature was 37°C, and the fermentation culture time was 48 h.

[0153] Example 23

[0154] This example provides a fermentation product of Lactobacillus plantarum CX1-3-2 strain, which is different from example 22 in that the inoculation amount is 1%;

[0155] The other ingredients and operation methods are the same as those of example 22.

[0156] Example 24

[0157] This example provides a fermentation product of Lactobacillus plantarum CX1-3-2 strain, which is different from example 22 in that the inoculation amount is 3%;

[0158] The other ingredients and operation methods are the same as those of example 22.

[0159] Example 25

[0160] This example provides a fermentation product of Lactobacillus plantarum CX1-3-2 strain, which is different from example 22 in that the inoculation amount is 4%;

[0161] The other ingredients and operation methods are the same as those of example 22.

[0162] Example 26

[0163] This example provides a fermentation product of Lactobacillus plantarum CX1-3-2 strain, which is different from example 22 in that the inoculation amount is 5%;

[0164] The other ingredients and operation methods are the same as those of example 22.

[0165] Example 27

[0166] This example provides a fermentation product of Lactobacillus plantarum CX1-3-2 strain, which is different from example 22 in that the fermentation culture temperature is 27°C;

[0167] The other ingredients and operation methods are the same as those of example 22.

[0168] Example 28

[0169] This example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which differs from Example 22 in that the temperature of the fermentation culture is 32°C;

[0170] The other ingredients and methods of operation are the same as in Example 22.

[0171] Example 29

[0172] This example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which differs from Example 22 in that the temperature of the fermentation culture is 42°C;

[0173] The other ingredients and methods of operation are the same as in Example 22.

[0174] Example 30

[0175] This example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which differs from Example 22 in that the temperature of the fermentation culture is 47°C;

[0176] The other ingredients and methods of operation are the same as in Example 22.

[0177] Example 31

[0178] This example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which differs from Example 22 in that the time of the fermentation culture is 24h;

[0179] The other ingredients and methods of operation are the same as in Example 22.

[0180] Example 32

[0181] This example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which differs from Example 22 in that the time of the fermentation culture is 36h;

[0182] The other ingredients and methods of operation are the same as in Example 22.

[0183] Example 33

[0184] This example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which differs from Example 22 in that the time of the fermentation culture is 60h;

[0185] The other ingredients and methods of operation are the same as in Example 22.

[0186] Example 34

[0187] This example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which differs from Example 22 in that the time of the fermentation culture is 72h;

[0188] Other ingredients and operating methods and Example 22 are the same.

[0189] The determination of the GABA content in the fermentation products of Examples 22-34 is shown in Table 2. The optimal fermentation conditions for Lactobacillus plantarum CX1-3-2 using grape juice as the fermentation medium are as follows: inoculation amount of 2%-4%; fermentation temperature of 32-42°C; and fermentation time of 48-72h. Under these conditions, the GABA yield in the fermentation product reached 553.21mg / L.

[0190] Table 2 GABA content in the fermentation product of Lactobacillus plantarum CX1-3-2

[0191] Group GABA content in fermentation product (mg / L) pH value of fermentation product Example 22 489.25 3.91 Example 23 440.60 3.85 Example 24 508.34 3.98 Example 25 501.57 4.03 Example 26 476.31 4.01 Example 27 397.56 3.91 Example 28 466.43 3.88 Example 29 305.32 3.90 Example 30 190.36 4.89 Example 31 346.59 4.03 Example 32 396.45 3.95 Example 33 511.63 3.90 Example 34 553.21 3.88

[0192] Example 35

[0193] This example provides a fermentation product of Lactobacillus plantarum CX1-3-2 strain, which is prepared by fermentation of Lactobacillus plantarum CX1-3-2 strain. The fermentation specifically includes the following steps:

[0194] (1) Wash the tomatoes, cut them into small pieces, and juice them in a juicer. Then centrifuge the juice at 4000r for 10min at 4°C to obtain the supernatant, which is used as needed;

[0195] (2) Add 2g of glucose, 1g of corn syrup powder, and 1g of L-glutamate sodium to 100mL of tomato juice, mix well, adjust the pH to 6.5, and sterilize at 115°C for 15min to obtain the fermentation medium;

[0196] Take 10 9 CFU / mL of Lactobacillus plantarum CX1-3-2 seed liquid, inoculate it into the fermentation medium at an inoculation amount of 2% for fermentation culture. The fermentation culture temperature is 37°C, and the fermentation culture time is 24h.

[0197] Example 36

[0198] This example provides a fermentation product of Lactobacillus plantarum CX1-3-2 strain, which is different from Example 35 in that the glucose is replaced with an equal amount of sucrose;

[0199] Other ingredients and operating methods are the same as in Example 35.

[0200] Example 37

[0201] This example provides a fermentation product of Lactobacillus plantarum CX1-3-2 strain, which is different from Example 35 in that the glucose is replaced with an equal amount of maltose;

[0202] Other ingredients and operating methods are the same as in Example 35.

[0203] Example 38

[0204] This example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which differs from Example 35 in that the glucose is replaced with an equal amount of lactose;

[0205] The other ingredients and the method of operation are the same as in Example 35.

[0206] Example 39

[0207] This example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which differs from Example 35 in that the amount of glucose added is 1 g;

[0208] The other ingredients and the method of operation are the same as in Example 35.

[0209] Example 40

[0210] This example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which differs from Example 35 in that the amount of glucose added is 3 g;

[0211] The other ingredients and the method of operation are the same as in Example 35.

[0212] Example 41

[0213] This example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which differs from Example 35 in that the amount of glucose added is 4 g;

[0214] The other ingredients and the method of operation are the same as in Example 35.

[0215] Example 42

[0216] This example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which differs from Example 35 in that the amount of glucose added is 5 g;

[0217] The other ingredients and the method of operation are the same as in Example 35.

[0218] Example 43

[0219] This example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which differs from Example 35 in that the corn steep powder is replaced with an equal amount of yeast extract powder;

[0220] The other ingredients and the method of operation are the same as in Example 35.

[0221] Example 44

[0222] This example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which is different from example 35 in that the corn steep powder is replaced with an equal amount of peptone;

[0223] The other ingredients and the method of operation are the same as in example 35.

[0224] Example 45

[0225] This example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which is different from example 35 in that the corn steep powder is replaced with an equal amount of beef extract;

[0226] The other ingredients and the method of operation are the same as in example 35.

[0227] Example 46

[0228] This example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which is different from example 35 in that the amount of corn steep powder added is 0.5 g;

[0229] The other ingredients and the method of operation are the same as in example 35.

[0230] Example 47

[0231] This example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which is different from example 35 in that the amount of corn steep powder added is 1.5 g;

[0232] The other ingredients and the method of operation are the same as in example 35.

[0233] Example 48

[0234] This example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which is different from example 35 in that the amount of corn steep powder added is 2 g;

[0235] The other ingredients and the method of operation are the same as in example 35.

[0236] Example 49

[0237] This example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which is different from example 35 in that the amount of corn steep powder added is 2.5 g;

[0238] The other ingredients and the method of operation are the same as in example 35.

[0239] Example 50

[0240] This example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which is different from example 35 in that no L-sodium glutamate is added;

[0241] The other ingredients and the method of operation are the same as in Example 35.

[0242] Example 51

[0243] This example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which differs from Example 35 in that the amount of L-glutamic acid sodium added is 2 g;

[0244] The other ingredients and the method of operation are the same as in Example 35.

[0245] Example 52

[0246] This example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which differs from Example 35 in that the amount of L-glutamic acid sodium added is 3 g;

[0247] The other ingredients and the method of operation are the same as in Example 35.

[0248] Example 53

[0249] This example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which differs from Example 35 in that the amount of L-glutamic acid sodium added is 4 g;

[0250] The other ingredients and the method of operation are the same as in Example 35.

[0251] Example 54

[0252] This example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which differs from Example 35 in that the pH is adjusted to 5;

[0253] The other ingredients and the method of operation are the same as in Example 35.

[0254] Example 55

[0255] This example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which differs from Example 35 in that the pH is adjusted to 5.5;

[0256] The other ingredients and the method of operation are the same as in Example 35.

[0257] Example 56

[0258] This example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which differs from Example 35 in that the pH is adjusted to 6;

[0259] The other ingredients and the method of operation are the same as in Example 35.

[0260] Example 57

[0261] The embodiment provides a fermentation product of Lactobacillus plantarum CX1-3-2 strain, which is different from that in Embodiment 35 in that the pH value is adjusted to 7.

[0262] Other components and operation methods and Embodiment 35 are the same.

[0263] The viable cell count of Lactobacillus plantarum CX1-3-2 in the fermentation products of Embodiments 35-57 is determined, and the results are shown in Table 3. When tomato juice is used as the fermentation medium, the optimal formula of Lactobacillus plantarum CX1-3-2 is that the carbon source is glucose, and the addition amount is 1 g / 100 mL to 3 g / 100 mL; the nitrogen source is corn syrup powder, and the addition amount is 1.5 g / 100 mL to 2.5 g / 100 mL; the addition amount of L-glutamic acid sodium is 0 to 2 g / 100 mL; and the pH value is 6 to 7. Under the fermentation condition, the viable cell count of Lactobacillus plantarum CX1-3-2 in the fermentation product reaches 10 8 CFU / mL to 10 9 CFU / mL.

[0264] Table 3 Viable cell count of Lactobacillus plantarum CX1-3-2 in fermentation products

[0265]

[0266]

[0267] Embodiment 58

[0268] The embodiment provides a fermentation product of Lactobacillus plantarum CX1-3-2 strain, which is prepared by fermentation of Lactobacillus plantarum CX1-3-2 strain, and the fermentation specifically comprises the following steps.

[0269] (1) Tomato is washed and cut into small pieces, and then juice is extracted by using a juicer, and then supernatant is obtained by centrifugation at 4 ℃ and 4000 r for 10 min, and the supernatant is used for standby;

[0270] (2) 2 g of glucose, 1 g of corn syrup powder and 1 g of L-glutamic acid sodium are added into 100 mL of tomato juice, and then the mixture is uniformly mixed, and then the pH value is adjusted to 6.5, and then the mixture is sterilized at 115 ℃ for 15 min, so as to obtain a fermentation medium;

[0271] 10 9 CFU / mL of Lactobacillus plantarum CX1-3-2 seed liquid is inoculated into the fermentation medium in a 2% inoculation amount to perform fermentation culture, the fermentation culture temperature is 37 ℃, and the fermentation culture time is 48 h.

[0272] Embodiment 59

[0273] The embodiment provides a fermentation product of Lactobacillus plantarum CX1-3-2 strain, which is different from that in Embodiment 58 in that the inoculation amount is 1%.

[0274] The other ingredients and the method of operation are the same as in Example 58.

[0275] Example 60

[0276] This example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which differs from Example 58 in that the inoculum is 3%;

[0277] The other ingredients and the method of operation are the same as in Example 58.

[0278] Example 61

[0279] This example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which differs from Example 58 in that the inoculum is 4%;

[0280] The other ingredients and the method of operation are the same as in Example 58.

[0281] Example 62

[0282] This example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which differs from Example 58 in that the inoculum is 5%;

[0283] The other ingredients and the method of operation are the same as in Example 58.

[0284] Example 63

[0285] This example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which differs from Example 58 in that the temperature of the fermentation culture is 27°C;

[0286] The other ingredients and the method of operation are the same as in Example 58.

[0287] Example 64

[0288] This example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which differs from Example 58 in that the temperature of the fermentation culture is 32°C;

[0289] The other ingredients and the method of operation are the same as in Example 58.

[0290] Example 65

[0291] This example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which differs from Example 58 in that the temperature of the fermentation culture is 42°C;

[0292] The other ingredients and the method of operation are the same as in Example 58.

[0293] Example 66

[0294] The present example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which is different from example 58 in that the fermentation culture temperature is 47°C.

[0295] The other components and the operation method are the same as example 58.

[0296] Example 67

[0297] The present example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which is different from example 58 in that the fermentation culture time is 24h.

[0298] The other components and the operation method are the same as example 58.

[0299] Example 68

[0300] The present example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which is different from example 58 in that the fermentation culture time is 36h.

[0301] The other components and the operation method are the same as example 58.

[0302] Example 69

[0303] The present example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which is different from example 58 in that the fermentation culture time is 60h.

[0304] The other components and the operation method are the same as example 58.

[0305] Example 70

[0306] The present example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which is different from example 58 in that the fermentation culture time is 72h.

[0307] The other components and the operation method are the same as example 58.

[0308] The GABA content of the fermentation products of examples 58-70 was determined, and the results are shown in table 4. When tomato juice was used as the fermentation medium, the optimal fermentation conditions for Lactobacillus plantarum CX1-3-2 were: inoculum amount of 2%-4%; fermentation temperature of 32°C-42°C; and fermentation time of 48h-72h. Under these conditions, the GABA yield in the fermentation product reached 641.25mg / L.

[0309] Table 4 GABA content in the fermentation product of Lactobacillus plantarum CX1-3-2

[0310] Group GABA content in fermentation product (mg / L) pH value of fermentation product Example 58 532.40 3.81 Example 59 496.35 3.78 Example 60 589.63 3.85 Example 61 511.26 3.90 Example 62 445.21 3.88 Example 63 403.60 3.84 Example 64 489.55 3.80 Example 65 510.75 3.90 Example 66 205.44 4.83 Example 67 399.64 3.91 Example 68 458.92 3.84 Example 69 587.51 3.79 Example 70 641.25 3.75

[0311] Example 71

[0312] The present embodiment provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which is prepared by fermentation of Lactobacillus plantarum strain CX1-3-2, and the fermentation specifically comprises the following steps:

[0313] (1) 12 g of full-fat milk powder was dissolved in 88 g of deionized water, and the solution was stirred and dissolved for standby.

[0314] (2) 2 g of glucose, 1 g of yeast extract powder and 1 g of L-glutamic acid sodium were added to the full-fat milk powder solution prepared in the above (1), and the mixture was uniformly mixed, and the pH value was adjusted to 6.5, and sterilized at 115°C for 15 min as a fermentation medium;

[0315] 10 9 CFU / mL of Lactobacillus plantarum CX1-3-2 seed liquid was inoculated into the fermentation medium at an inoculation amount of 2% for fermentation culture, and the fermentation culture temperature was 37°C, and the fermentation culture time was 48 h.

[0316] Example 72

[0317] The present embodiment provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which is prepared by fermentation of Lactobacillus plantarum strain CX1-3-2, and the fermentation specifically comprises the following steps:

[0318] Other ingredients and operation methods are the same as those of Example 71.

[0319] Example 73

[0320] The present embodiment provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which is prepared by fermentation of Lactobacillus plantarum strain CX1-3-2, and the fermentation specifically comprises the following steps:

[0321] Other ingredients and operation methods are the same as those of Example 71.

[0322] Example 74

[0323] The present embodiment provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which is prepared by fermentation of Lactobacillus plantarum strain CX1-3-2, and the fermentation specifically comprises the following steps:

[0324] Other ingredients and operation methods are the same as those of Example 71.

[0325] Example 75

[0326] The present embodiment provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which is prepared by fermentation of Lactobacillus plantarum strain CX1-3-2, and the fermentation specifically comprises the following steps:

[0327] Other ingredients and operation methods are the same as those of Example 71.

[0328] Example 76

[0329] The present example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which is different from example 71 in that the pH value of the fermentation medium is 6;

[0330] The other ingredients and the method of operation are the same as example 71.

[0331] Example 77

[0332] The present example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which is different from example 71 in that the temperature of the fermentation culture is 42℃;

[0333] The other ingredients and the method of operation are the same as example 71.

[0334] Example 78

[0335] The present example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which is different from example 71 in that the fermentation time is 60h;

[0336] The other ingredients and the method of operation are the same as example 71.

[0337] Example 79

[0338] The present example provides a fermentation product of Lactobacillus plantarum strain CX1-3-2, which is different from example 71 in that the time of the fermentation culture is 72h;

[0339] The other ingredients and the method of operation are the same as example 71.

[0340] The fermentation products provided in examples 71-79 were subjected to viable cell count and GABA content determination, and the results are shown in Table 5. Under the fermentation conditions, the GABA yield in the fermentation product reached 846.41 mg / L.

[0341] Table 5 Viable cell count and GABA content of Lactobacillus plantarum CX1-3-2 fermentation product

[0342]

[0343] Example 80

[0344] The present example provides a bacterial powder, which is obtained by mixing the fermentation product prepared in example 79 and trehalose, the mass volume ratio of trehalose and fermentation product being 6g: 100mL, and then vacuum freeze-drying;

[0345] The specific operation of vacuum freeze-drying is as follows: pre-freezing at -40℃ for 8h, setting the vacuum degree to 0.20Mbar, the cold trap temperature to -80℃, and freeze-drying for 24h.

[0346] Example 81

[0347] This example provides a bacteria powder, which differs from example 80 in that trehalose is replaced by an equal amount of skimmed milk powder;

[0348] The other ingredients and methods of operation are the same as in example 80.

[0349] Example 82

[0350] This example provides a bacteria powder, which differs from example 80 in that trehalose is replaced by an equal amount of whole milk powder;

[0351] The other ingredients and methods of operation are the same as in example 80.

[0352] Example 83

[0353] This example provides a bacteria powder, which differs from example 80 in that trehalose is replaced by an equal amount of lactose;

[0354] The other ingredients and methods of operation are the same as in example 80.

[0355] Example 84

[0356] This example provides a bacteria powder, which differs from example 80 in that vacuum freeze-drying is replaced by spray-drying;

[0357] The spray-drying is carried out with an inlet temperature of 150°C and an outlet temperature of 75°C.

[0358] Example 85

[0359] This example provides a bacteria powder, which differs from example 84 in that trehalose is replaced by an equal amount of skimmed milk powder;

[0360] The other ingredients and methods of operation are the same as in example 84.

[0361] Example 86

[0362] This example provides a bacteria powder, which differs from example 84 in that trehalose is replaced by an equal amount of whole milk powder;

[0363] The other ingredients and methods of operation are the same as in example 84.

[0364] Example 87

[0365] This example provides a bacteria powder, which differs from example 84 in that trehalose is replaced by an equal amount of lactose;

[0366] The other ingredients and methods of operation are the same as in example 84.

[0367] The viable cell count and GABA content of the bacterial powder prepared from Examples 80-87 were determined, and the strain survival rate and GABA retention rate were calculated, and the results are shown in Table 6.

[0368]

[0369] Table 6 Viable cell count and GABA content of Lactobacillus plantarum CX1-3-2 in bacterial powder

[0370]

[0371]

[0372] As shown in Table 6, the bacterial powder was prepared by adding additives to the fermentation product of Lactobacillus plantarum CX1-3-2 fermented fruit and vegetable juice, wherein the viable cell count of Lactobacillus plantarum CX1-3-2 was 10 7 CFU / g ~ 10 8 CFU / g, the strain survival rate was 37% ~ 84%, the GABA content was 7 mg / g ~ 9 mg / g, and the GABA retention rate was 81% ~ 99%.

[0373] Example 88

[0374] Evaluation of the sleep-improving effect of the bacterial powder provided in Example 82 in mice:

[0375] 1. Animal feeding and grouping

[0376] SPF level 6-week-old male Kunming (KM) mice were purchased, weighing 18 g ~ 22 g, and after one week of adaptive feeding, the study was carried out. All mice were divided into 3 batches according to weight, 40 in each batch, and there was no statistical difference in quality. Among them, the first batch carried out the prolonged sodium pentobarbital sleep time experiment; the second batch carried out the subthreshold dose of sodium pentobarbital hypnotic experiment; the third batch carried out the barbital sodium sleep latency experiment; the above experiments were carried out directly sleep experiment at the same time.

[0377] The initial body weight was measured before the experiment, and each batch of animals was randomly divided into four groups according to weight (10 in each group, 5 / cage): blank group (NC group, normal saline), positive control group (PC group, diazepam 2 mg / kg·BW), bacterial powder low dose group (LD group, 3 g / kg·BW), bacterial powder high dose group (HD group, 9 g / kg·BW). Intragastric administration was performed once a day, with a gavage volume of 10 mL / kg·BW, and continuous intervention for 30 days, with body weight changes recorded every 6 days. The experimental environment temperature was appropriate, quiet and ventilated, 12 h light and dark alternation, free feeding and drinking water.

[0378] 2. Sleep improvement experiment

[0379] Direct sleep experiment, pentobarbital sodium sleep time extension experiment, pentobarbital sodium threshold dose sleep experiment and barbital sodium sleep latency experiment were carried out according to Health Food Function Test and Evaluation Methods (2023 Edition) on mice.

[0380] (1) Direct sleep experiment

[0381] After daily intragastric administration, whether the mice in each group appeared direct sleep phenomenon was observed. The judgment of sleep state was based on the disappearance and recovery of righting reflex: when the mice were placed in dorsal recumbency, if they could not recover to normal body position within 1 min (i.e. the righting reflex disappeared), it was considered to enter sleep state; otherwise, when the mice regained the righting ability, it was determined that the sleep ended. The number of mice falling asleep in each group and the sleep time (the time interval from the disappearance to the recovery of righting reflex) were recorded.

[0382] Within 30 min after daily intragastric administration, the mice in NC group, PC group, LD group and HD group did not appear sleep phenomenon, indicating that the test substances in each group had no direct hypnotic effect on mice.

[0383] (2) Pentobarbital sodium sleep time extension experiment

[0384] After 30 min of the last intragastric administration, pentobarbital sodium (50 mg / kg·BW) was injected intraperitoneally to the mice, with an injection volume of 0.1 mL / 10 g. The time interval between the disappearance (sleep initiation) and recovery (awakening) of righting reflex was observed, and the sleep duration was calculated.

[0385] The results of pentobarbital sodium sleep time extension experiment of mice in each group are shown in Table 6. Figure 4 Compared with the NC group, the PC group, LD group and HD group significantly prolonged the pentobarbital sodium-induced sleep time of mice, among which the sleep time of mice in the PC group was prolonged by 55.65 min, and the sleep time of mice in the LD group and HD group was prolonged by 40.99 min and 44.11 min, respectively. There was no significant difference between the LD group and the HD group (p>0.05).

[0386] (3) Pentobarbital sodium threshold dose sleep experiment

[0387] After 30 min of the last intragastric administration, pentobarbital sodium (30 mg / kg·BW) was injected intraperitoneally to the mice, with an injection volume of 0.1 mL / 10 g. The mice were considered to fall asleep successfully if the disappearance of righting reflex lasted more than 1 min, and the number of animals falling asleep within 30 min was recorded.

[0388] The results of pentobarbital sodium threshold dose experiment of mice in each group are shown in Table 7. The sleep rate of mice in the PC group was 70%, the sleep rate of mice in the HD group was 70%, and the sleep rate of mice in the LD group was 20%. Compared with the NC group, the PC group and the HD group significantly improved the sleep rate of mice (p<0.05).

[0389] Table 7 Effect of Lactobacillus plantarum CX1-3-2 powder on the sleep rate of mice induced by sodium pentobarbital

[0390] Group Total number of mice (number) Number of sleeping mice (number) Sleeping rate (%) Negative control group (NC) 10 0 0 Positive control group (PC) 10 7 70* Low-dose group (LD) 10 2 20 High-dose group (HD) 10 7 70*

[0391] Note: * indicates significant difference (p < 0.05) from the blank group.

[0392] (4) Sodium barbital sleep latency experiment

[0393] The mice were intraperitoneally injected with sodium pentobarbital (280 mg / kg·BW) 30 min after the last gavage, with an injection volume of 0.1 mL / 10 g. The time from sodium pentobarbital injection to the disappearance of the righting reflex was recorded to determine the sleep latency.

[0394] The results of the sodium barbital sleep latency experiment for the mice in each group are shown in Table 7. Figure 5 Compared with the NC group, the sleep latency of the mice in the PC, LD, and HD groups was significantly shortened, with the sleep latency of the PC group shortened by 13.26 min, the sleep latency of the LD group shortened by 7.64 min, and the sleep latency of the HD group shortened by 11.89 min. Compared with the LD group, the sleep latency of the mice in the HD group was more significantly shortened (p < 0.05).

[0395] 3. Changes in mouse body weight

[0396] The body weight of the mice was measured every 6 days after gavage. The changes in the body weight of the mice within 30 days are shown in Table 8. Figure 6 The body weight of the mice in the four groups showed basically the same trend, and the body weight of the mice in each group reached about 42 g on the 30th day. There was no significant difference between the groups (p > 0.05), indicating that the Lactobacillus plantarum CX1-3-2 powder did not affect the normal growth of the mice.

[0397] Example 89

[0398] Organic acid content of Lactobacillus plantarum CX1-3-2 fermentation product

[0399] Lactobacillus plantarum CX1-3-2 was inoculated into MRS liquid medium at an inoculation amount of 2%, and incubated at 37°C for 24 h and 48 h. After incubation, the fermentation supernatant was obtained by centrifugation at 8000 r / min for 10 min. The organic acid content of the fermentation supernatant was determined by high performance liquid chromatography. The results are shown in Table 8. The fermentation metabolites of Lactobacillus plantarum CX1-3-2 included γ-aminobutyric acid, hydroxyphenyllactic acid, citric acid, malic acid, succinic acid, lactic acid, formic acid, acetic acid, propionic acid, isobutyric acid, and phenyllactic acid. The yield of lactic acid was the highest, reaching 7.62 mg / mL, and the yield of acetic acid was 3.57 mg / mL.

[0400] Table 8 Organic acid detection of Lactobacillus plantarum CX1-3-2 fermentation product supernatant

[0401]

[0402] Example 90

[0403] Functional properties of Lactobacillus plantarum CX1-3-2

[0404] 1. Bacteriostatic property

[0405] Lactobacillus plantarum CX1-3-2 was inoculated into MRS liquid medium at an inoculation amount of 2%, and incubated at 37°C for 24 h. The supernatant was obtained by centrifugation at 8000 r / min for 10 min. The bacterial pellet was washed with physiological saline for 3 times and resuspended in physiological saline. The OD value of the bacterial suspension was adjusted to 1.0, and was used as needed. 600 (1) Bacteriostatic effect of fermentation supernatant: The bacteriostatic activity of fermentation supernatant was determined by Oxford cup agar diffusion method. The NA medium was prepared, 100 μL of Lactobacillus plantarum CX1-3-2 fermentation supernatant was added to the Oxford cup, and was diffused at 4°C for 10-12 h. The size of the bacteriostatic circle was measured after incubation at 37°C for 6-12 h. 6 (2) Bacteriostatic effect of bacterial cells: The bacteriostatic activity of bacterial cells was determined by double-layer plate method. The bacterial cell solution was prepared, 1 μL was spotted on the MRS solid medium, and was naturally dried. The NA medium with a final concentration of 1 x 10

[0406] Lactobacillus plantarum CX1-3-2 and its fermentation product supernatant had bacteriostatic property on Escherichia coli, Staphylococcus aureus, Bacillus cereus, and Pseudomonas fluorescens. The fermentation supernatant had obvious inhibitory effect on Escherichia coli, Staphylococcus aureus, and Pseudomonas fluorescens, and the diameter of the bacteriostatic circle was greater than 17 mm.

[0407] Table 9 Bacteriostatic property results

[0408]

[0409] 2. Antioxidant property

[0410] Lactobacillus plantarum CX1-3-2 seed liquid was inoculated into MRS liquid medium at an inoculation amount of 2%, and incubated at 37°C for 24 h. The fermentation supernatant was obtained by centrifugation at 8000 r / min for 10 min. The DPPH free radical scavenging rate and ABTS free radical scavenging rate of the fermentation supernatant were detected. The DPPH free radical scavenging rate of the fermentation supernatant was 96.4%, and the ABTS free radical scavenging rate was 78.3%, indicating that the fermentation product of Lactobacillus plantarum CX1-3-2 had good antioxidant capacity.

[0411] The above descriptions are only the preferred embodiment of the application, not intended to limit the application and any modification, equivalent replacement and improvement made within the spirit and principle of the application shall be included in the protection scope of the application.

Claims

1. A Lactobacillus plantarum CX1-3-2 having a gamma-aminobutyric acid production and sleep improvement effect, characterized by, and has a preservation number of CGMCC No. 34340.

2. Use of the Lactobacillus plantarum CX1-3-2 strain of claim 1 in production of gamma-aminobutyric acid.

3. Use of the Lactobacillus plantarum CX1-3-2 strain of claim 1 in preparation of sleep-improving, antioxidant and bacteriostatic products.

4. A fermentation product, characterized in that, fermented by the Lactobacillus plantarum CX1-3-2 strain of claim 1.

5. The fermentation product of claim 4, wherein, The preparation method comprises the following steps: seed liquid of the Lactobacillus plantarum CX1-3-2 strain is inoculated into a culture medium for fermentation culture; the culture medium is a fruit and vegetable juice culture medium or a milk powder culture medium.

6. The fermentation product of claim 5, wherein, the fruit and vegetable juice culture medium comprises water, fruit and vegetable juice, a carbon source, a nitrogen source and L-sodium glutamate, and has a pH value of 5-7; wherein, based on the sum of the volumes of water and fruit and vegetable juice being 100%, the addition amount of fruit and vegetable juice is 20%-100%, and the balance is water; the concentration of the carbon source in the fruit and vegetable juice culture medium is 0 g / 100 mL-5 g / 100 mL; the concentration of the nitrogen source in the fruit and vegetable juice culture medium is 0.5 g / 100 mL-2.5 g / 100 mL; the concentration of the L-sodium glutamate in the fruit and vegetable juice culture medium is 0 g / 100 mL-4 g / 100 mL.

7. The fermentation product of claim 5, wherein, the milk powder culture medium comprises water, whole milk powder or skimmed milk powder, a carbon source, a nitrogen source and L-sodium glutamate, and has a pH value of 5-7; wherein, based on the sum of the masses of water and milk powder being 100%, the addition amount of milk powder is 4%-20%, and the balance is water; the concentration of the carbon source in the milk powder culture medium is 0 g / 100 g-5 g / 100 g; the concentration of the nitrogen source in the milk powder culture medium is 0.5 g / 100 g-2.5 g / 100 g; the concentration of the L-sodium glutamate in the milk powder culture medium is 0 g / 100 g-4 g / 100 g.

8. The fermentation product of claim 5, wherein, The inoculation amount of the seed liquid of the Lactobacillus plantarum CX1-3-2 strain is 1% to 5%, and the viable cell count in the seed liquid of the Lactobacillus plantarum CX1-3-2 strain is 10 8 CFU / mL to 10 9 CFU / mL; and / or the fermentation culture conditions are as follows: temperature is 27°C-47°C, and time is 24 h-72 h.

9. A bacterial powder, characterized by, at least one of the Lactobacillus plantarum CX1-3-2 strain of claim 1 or the fermentation product of any one of claims 4-8.

10. Use of the Lactobacillus plantarum CX1-3-2 strain of claim 1, the fermentation product of any one of claims 4-8 or the bacterial powder of claim 9 in preparation of food, medicine, health care product, feed, food additive or feed additive.

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