Compound probiotics with effects of soothing nerves and helping sleep and application of compound probiotics

Abstract

The invention provides a compound probiotic with the effects of soothing the nerves and helping sleep and application of the compound probiotic. The compound probiotic comprises lactobacillus plantarum ZQ15 (the preservation number is CGMCC (China General Microbiological Culture Collection Center) NO.30265) and lactobacillus rhamnosus AlcoKer0067 (the preservation number is CGMCC NO.31221). The invention further provides a preparation method of the compound probiotic. Research finds that the lactobacillus plantarum ZQ15 and the lactobacillus rhamnosus AlcoKer0067 form a compound probiotic (or a compound probiotic microbial agent), the two bacteria are compounded and can be mutually matched and promoted, and when the lactobacillus plantarum ZQ15 and the lactobacillus rhamnosus AlcoKer0067 are applied to preparation of the nerve soothing and sleep aiding product, the generation of 5-HT and GABA can be remarkably promoted, and the effects of relieving anxiety, soothing the nerves and aiding sleep are enhanced. The compound probiotics composed of lactobacillus plantarum ZQ15 and lactobacillus rhamnosus AlcoKer0067 further form a composition with traditional Chinese medicine extracts, prebiotics, small molecule peptides and the like, the content of 5-HT and GABA in blood of mice can be obviously increased, and the effect is most remarkable after the compound probiotics, the traditional Chinese medicine extracts, the prebiotics, the small molecule peptides and the like are combined for administration.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to a composite probiotic with the effects of calming nerves and promoting sleep and an application thereof. Background Art

[0002] Insomnia is a common clinical disease, mainly manifested by prolonged sleep latency, sleep maintenance disorders and daytime fatigue. Clinical evidence-based medicine shows that long-term insomnia will significantly increase the risk of heart disease, hypertension, type 2 diabetes, obesity, Alzheimer's disease, menopausal syndrome, etc. At the same time, long-term insomnia is also a high-risk factor for the onset of mental system diseases, seriously affecting human health and quality of life.

[0003] At present, the safer treatment options for insomnia mainly include traditional Chinese medicine and the use of probiotics, small molecule peptides and other substances to regulate the body's metabolism and physiological functions. Traditional Chinese medicine focuses on the overall concept and syndrome differentiation in the treatment of sleep disorders. Drug therapy is more common in clinical practice. Practice has shown that Chinese date seeds, lilies, and Poria cocos have the ability to regulate the nervous system and improve sleep quality. They can regulate the level of melatonin in the body and promote the body to return to a healthier sleep cycle; Poria cocos and Bupleurum have antioxidant and anti-inflammatory effects, which help to alleviate sleep problems caused by chronic inflammation. Many studies have shown that probiotics can improve insomnia symptoms. For example, Lactobacillus plantarum and Bifidobacterium lactis have good interactions and affinity in the intestinal ecosystem, and their metabolites may have a positive effect on the nervous system, immune system and inflammatory response, thereby improving sleep. Small molecule peptides also have a positive effect on sleep quality. For example, wheat oligopeptides can promote the repair of damaged skeletal muscle cells, maintain the integrity of skeletal muscle cells, reduce the generation of oxygen free radicals in myocardial mitochondria, maintain the normal structure and function of myocardial mitochondria, protect the heart, and improve oxygen tolerance, thereby promoting sleep by reducing physical and mental fatigue; the glutamate in pecan oligopeptides can promote the synthesis of inhibitory neurotransmitters such as 5-hydroxytryptamine and γ-aminobutyric acid in the brain, thereby playing a positive role in improving sleep; the rich tryptophan in sturgeon skin oligopeptides and cow's milk oligopeptides is the main raw material for the brain to synthesize 5-hydroxytryptamine, which can improve pre-sleep mood, inhibit neural activity, and shorten sleep latency.

[0004] Most of the existing patents use a single method to treat insomnia symptoms, which is difficult to achieve the ideal therapeutic effect. For example, patent CN118903328A discloses a Chinese medicine composition for improving sleep and its preparation method and application; patent CN112056482A discloses a sleep-aiding small molecule peptide solid beverage and its preparation method and application; patent CN115226773A discloses a composite probiotic sleep-aiding solid beverage and its preparation method. The above patents use the single action of Chinese medicine, probiotics or small molecule peptides, and lack the technical means of the three to synergistically treat each other. This is a problem that needs to be solved. Summary of the invention

[0005] In order to overcome the problems of the prior art, the present invention proposes a composite probiotic with a calming and sleep-inducing effect and its application. Studies have found that the composite probiotic (or composite probiotic agent) composed of Lactobacillus plantarum ZQ15 and Lactobacillus rhamnosus AlcoKer0067 can cooperate with and promote each other, and can be used in the preparation of calming and sleep-inducing products, which can significantly promote the production of 5-HT and GABA, and enhance the effects of relieving anxiety, calming the nerves and helping sleep.

[0006] The object of the present invention is achieved in that: The first aspect of the present invention provides a composite probiotic with a calming effect and a sleep-inducing effect, wherein the composite probiotic comprises Lactobacillus plantarum ( Lactobacillus plantarum ) ZQ15 and Lactobacillus rhamnosus ( Lactobacillus buckthorn )AlcoKer0067; Lactobacillus plantarum ZQ15, deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit number CGMCC NO. 30265 and the deposit date April 7, 2024; Lactobacillus rhamnosus AlcoKer0067, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number of CGMCC NO. 31221 and the deposit date of July 8, 2024; The mass ratio of Lactobacillus plantarum ZQ15 and Lactobacillus rhamnosus AlcoKer0067 in the composite probiotics is (5-10): (1-5).

[0007] The second aspect of the present invention provides a composite probiotic agent having a calming effect and a sleep-inducing effect, wherein the composite probiotic agent comprises the composite probiotic described in the first aspect.

[0008] Furthermore, the effective live bacterial concentration of Lactobacillus plantarum ZQ15 in the composite probiotic agent is not less than 5×10 11 CFU / g, the effective live bacterial concentration of Lactobacillus rhamnosus AlcoKer0067 is not less than 5×10 10 CFU / g.

[0009] The third aspect of the present invention provides a method for preparing the composite probiotic agent having the effect of calming the nerves and promoting sleep as described in the second aspect, comprising the following steps: Lactobacillus plantarum ZQ15 and Lactobacillus rhamnosus AlcoKer0067 are inoculated, proliferated, fermented and dried to prepare a composite probiotic agent; The fermentation conditions are as follows: pH before fermentation is 6.20-6.80, fermentation temperature is 35-40°C, fermentation speed is 60rpm, and fermentation time is 14-16 h.

[0010] The fourth aspect of the present invention provides a traditional Chinese medicine, compound probiotics, and small molecule peptide composition for calming nerves and promoting sleep, wherein the active ingredients of the composition include traditional Chinese medicine extracts, compound probiotics, prebiotics, small molecule peptides, γ-aminobutyric acid, and N-acetylneuraminic acid; The Chinese medicine extracts include Ziziphus jujuba seed extract, lily extract and Poria cocos extract; The composite probiotics are the composite probiotics with the effect of calming the nerves and promoting sleep as described in the first aspect, or the composite probiotic agent with the effect of calming the nerves and promoting sleep as described in the second aspect; The prebiotics include oligofructose, resistant dextrin and isomaltooligosaccharide; The small molecule peptides include sturgeon skin oligopeptides, wheat oligopeptides, pecan oligopeptides and cow milk oligopeptides.

[0011] Further, the weight parts of each component are: 15-30 parts of Chinese herbal medicine extract, 15-30 parts of compound probiotics, 30-90 parts of prebiotics, 30-60 parts of small molecule peptides, 3-15 parts of γ-aminobutyric acid, and 3-15 parts of N-acetylneuraminic acid.

[0012] Furthermore, the preparation method of the Chinese medicine extract comprises the following steps: (1) After crushing the spiny jujube seeds, lily bulbs, and poria cocos respectively, add 10 times the mass of 75% ethanol according to the mass ratio, and filter after ultrasonic water bath for 2-3 hours to obtain the first filtrate; (2) Then, 5 times the mass of 75% ethanol was added to the filter residue again, and the mixture was filtered after being ultrasonically bathed for 1 hour to obtain the second filtrate; (3) The second filtrate is mixed with the first filtrate, and the ethanol is removed by rotary evaporation. The mixture is freeze-dried to obtain a Chinese medicine extract powder.

[0013] Furthermore, in steps (1) and (2), the conditions of the ultrasonic water bath are: the ultrasonic power is 20 to 40 kHz, and the water bath temperature is 50° C. to 60° C.; In step (3), the drying method is freeze drying, and the conditions are: freezing at -25°C for 8 to 12 h, cold trap temperature of -60°C to -80°C, vacuum degree of 50 to 150 Pa, and drying time of 20 to 24 h.

[0014] The fifth aspect of the present invention provides the use of the composite probiotics described in the first aspect, the composite probiotic agent described in the second aspect, or the traditional Chinese medicine, composite probiotics, and small molecule peptide composition described in the fourth aspect in the preparation of a tranquilizing and sleep-aiding product.

[0015] Furthermore, the products include food, health products, and medicines. Strain deposit information:

[0016] Lactobacillus plantarum ZQ15 is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration (CGMCC), address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, with a deposit number of CGMCC NO.30265 and a deposit date of April 7, 2024; the classification name is: Lactobacillus plantarum Lactobacillus plantarum .

[0017] Lactobacillus rhamnosus AlcoKer0067 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC), address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, with a deposit number of CGMCC NO. 31221 and a deposit date of July 8, 2024; the classification name is: Lactobacillus rhamnosus Lactobacillus buckthorn . The advantages and beneficial effects of the present invention are:

[0018] 1. The plant lactobacillus ZQ15 described in the present invention is separated from traditional yogurt, has strong safety, is non-pathogenic, and has no acute toxicity; the rhamnosus lactobacillus AlcoKer0067 is separated from fermented kimchi, and the live bacteria of both can colonize in the intestines and promote the production of a large amount of 5-hydroxytryptamine and GABA, and can enter nerve cells with the blood, bind to the 5-hydroxytryptamine receptors between neurons, and play a role in regulating emotions, thereby preventing sleep disorders, improving sleep quality, and relieving anxiety; further research has found that the composite probiotics (or composite probiotic agents) composed of plant lactobacillus ZQ15 and rhamnosus lactobacillus AlcoKer0067 can cooperate with and promote each other, and can be used in the preparation of tranquilizing and sleeping products, which can significantly promote the production of 5-HT and GABA, and enhance the effects of relieving anxiety and tranquilizing and sleeping (significantly better than a single strain); 2. The composite probiotics composed of Lactobacillus plantarum ZQ15 and Lactobacillus rhamnosus AlcoKer0067 described in the present invention are further combined with Chinese medicine extracts, prebiotics and small molecule peptides to form a composition, which can significantly increase the 5-HT and GABA content in the blood of mice. The combined administration of the three has the most significant effect. DETAILED DESCRIPTION

[0019] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0020] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0021] The present invention will be described in detail below by way of examples. It should be understood that the following examples are only used to further explain and illustrate the content of the present invention in detail, and are not used to limit the present invention. Example 1: Screening and domestication of Lactobacillus plantarum ZQ15

[0022] This example provides the screening and domestication process of Lactobacillus plantarum ZQ15.

[0023] 1.1 Strain Isolation The bacterial flora was isolated from commercially available yogurt. The specific isolation process was as follows: (1) Pipette 2 mL of yogurt sample into 100 mL of MRS medium and culture at 37℃ for 16-22 h for enrichment.

[0024] (2) Gradient dilution: Pipette 0.5 mL of the acclimated sample into 4.5 mL of sterile saline to obtain 10 -1 diluent, then pipette 0.5 mL 10 -1 Dilute in 4.5 mL saline to obtain 10 -2 Dilution, follow this procedure to obtain 10 -3 , 10 -4 , 10 -5 diluent.

[0025] (3) Plate culture: Pipette 100 μL of gradient dilution solution and plate on MRS agar plate for 10 -3 , 10 -4 , 10 -5 One plate per gradient was cultured at 37°C for 48 h.

[0026] (4) Streak separation: Select colonies with typical characteristics on the coated plate based on their shape, size, edge, transparency, etc. Use an inoculation loop to pick up colonies and streak them on the selective culture medium plate. Incubate at 37°C for 48 h.

[0027] (5) Activation culture: After obtaining purified single colonies, pick a single colony and inoculate it into 5 mL of MRS medium. After culturing for 16 to 22 h, transfer it to a new medium at a ratio of 1:50 and continue culturing. At the same time, number each strain and obtain the strain by centrifugation.

[0028] (6) The strain was resuspended in physiological saline, screened through mouse experiments, and administered orally at a dose of 10 billion CFU / mouse. The enzyme-linked immunosorbent assay was used to screen out strains with high production of 5-hydroxytryptamine (5-HT) and γ-aminobutyric acid (GABA).

[0029] 1.2 Strain screening Four strains with better growth were selected and numbered as ZQ15, ZQ17, AL13 and SL23, respectively, and continuously activated and cultured with AlcoKer0067. Among them, ZQ15 and ZQ17 are Lactobacillus plantarum, AL13 and AlcoKer0067 are Lactobacillus rhamnosus, and SL23 is Bifidobacterium.

[0030] The levels of 5-HT and GABA in the blood of mice after administration were determined by enzyme-linked immunosorbent assay.

[0031]

[0032] The results of one-way ANOVA showed that among the different strains, the serum 5-HT and GABA production of mice was significantly higher after combined administration of Lactobacillus plantarum ZQ15 and Lactobacillus rhamnosus AlcoKer0067 (mixing ratio 1:1). Embodiment 2:

[0033] The present embodiment provides a traditional Chinese medicine, composite probiotics and small molecule peptide composition solid beverage for calming nerves and promoting sleep, comprising the following components in parts by weight: 10 parts of jujube seed extract, 10 parts of lily extract, 10 parts of Poria extract, 15 parts of Lactobacillus plantarum powder, 15 parts of Lactobacillus rhamnosus powder, 15 parts of sturgeon skin oligopeptides, 10 parts of wheat oligopeptides, 10 parts of pecan oligopeptides, 10 parts of milk oligopeptides, 15 parts of γ-aminobutyric acid, 15 parts of N-acetylneuraminic acid, 10 parts of oligofructose, 10 parts of resistant dextrin, 10 parts of isomaltose, 2 parts of erythritol, 1 part of sorbitol, and 3 parts of skim milk powder.

[0034] The preparation method of the solid beverage of the traditional Chinese medicine, composite probiotics and small molecule peptide composition for calming nerves and promoting sleep comprises the following steps: (1) Take prebiotics: oligofructose, resistant dextrin, oligomaltose, and auxiliary materials: erythritol, sorbitol, and skim milk powder, and then test the water activity of the above raw materials and auxiliary materials respectively using a water activity meter; After the water activity test, the oligofructose, resistant dextrin, isomalt oligosaccharide, erythritol, sorbitol and skimmed milk powder are placed in an oven respectively, and then dried at a low temperature of 35 to 45°C with circulating air for 1 to 4 hours; During the above drying process, each raw material and auxiliary material being dried was taken out every 30 minutes, and then the water activity of oligofructose, resistant dextrin, oligomaltose, erythritol, sorbitol and skimmed milk powder was tested again by a water activity tester; Furthermore, the oligofructose, resistant dextrin, isomalt oligosaccharide, erythritol, sorbitol and skim milk powder were repeatedly dried and tested for water activity according to the above method, and finally the water activity of each raw material and auxiliary material was controlled below 0.05; The obtained fructooligosaccharides, resistant dextrin, isomalt oligosaccharides, erythritol, sorbitol and skimmed milk powder with a water activity of less than 0.05 are put into a blender, and then stirred at a speed of 200 rpm / min for 3 minutes to fully mix the raw materials and auxiliary materials to obtain a mixed material for use.

[0035] (2) Lactobacillus plantarum ZQ15 and Lactobacillus rhamnosus AlcoKer0067 were inoculated, proliferated, fermented and dried to obtain composite probiotic powder; The fermentation conditions are as follows: pH before fermentation is 6.20-6.80, fermentation temperature is 35-40°C, fermentation speed is 60rpm, and fermentation time is 14-16 h; The composite probiotic powder was added to the mixture obtained in step (1), and then stirred at 200 rpm / min for 2 min to uniformly mix the composite probiotic powder, raw materials and auxiliary materials to obtain a probiotic mixture for later use.

[0036] (3) The probiotic mixture prepared in step (2) is mixed with a small molecule peptide component (sturgeon skin oligopeptide, wheat oligopeptide, pecan oligopeptide, and milk oligopeptide) to obtain a probiotic and small molecule peptide composition.

[0037] The sturgeon skin oligopeptide, wheat oligopeptide, pecan oligopeptide and milk oligopeptide can be prepared by the following method: S31, soak the washed wheat in 2 to 3 times of clean water for 20 to 30 minutes, and then homogenize to obtain wheat homogenate. Then add 0.3% to 0.5% of the raw material mass of alkaline protease (papain: trypsin = 1:1, enzyme activity is 400,000 to 500,000 u / g) to the corresponding homogenate, and perform enzymolysis at a temperature of 45 to 55°C for 1 to 2 hours. After enzymolysis, adjust the temperature to 85 to 90°C to inactivate the enzyme for 15 to 20 minutes. Filter and centrifuge the above enzymolysis solution, and take the supernatant for ultrafiltration centrifugation (retention molecular weight 30ku) to remove small molecule peptides. Specifically, the mass of the added alkaline protease is 0.3% of the raw material mass, the enzymolysis temperature is 45°C, and the enzymolysis is 2 hours.

[0038] S32, soak the washed pecans in 2 to 3 times of clean water for 20 to 30 minutes, and then homogenize to obtain a walnut homogenate. Then add 0.3% to 0.5% of the raw material mass of alkaline protease (papain: trypsin = 1:1, enzyme activity of 400,000 to 500,000 u / g) to the corresponding homogenate and milk, and perform enzymolysis at a temperature of 45 to 55°C for 1 to 2 hours. After enzymolysis, adjust the temperature to 85 to 90°C to inactivate the enzyme for 15 to 20 minutes. Filter and centrifuge the above enzymolysis solution, and take the supernatant for ultrafiltration centrifugation (retention molecular weight 30ku) to obtain small molecule peptides. Specifically, the mass of the added alkaline protease is 0.4% of the raw material mass, the enzymolysis temperature is 50°C, and the enzymolysis is 1.5 hours.

[0039] S33, soak the washed sturgeon skin in 2 to 3 times of clean water for 20 to 30 minutes, and then homogenize to obtain a sturgeon skin homogenate. Then add 0.3% to 0.5% of the raw material mass of alkaline protease (papain: trypsin = 1:1, enzyme activity of 400,000 to 500,000 u / g) to the corresponding homogenate, and perform enzymolysis at a temperature of 45 to 55°C for 1 to 2 hours. After enzymolysis, adjust the temperature to 85 to 90°C to inactivate the enzyme for 15 to 20 minutes. Filter and centrifuge the above enzymolysis solution, and take the supernatant for ultrafiltration centrifugation (retention molecular weight 30ku) to remove small molecule peptides. Specifically, the mass of the added alkaline protease is 0.4% of the raw material mass, the enzymolysis temperature is 50°C, and the enzymolysis is 1.5 hours.

[0040] S34, add 0.3% to 0.5% of the raw material mass of alkaline protease (papain: trypsin = 1:1, enzyme activity of 400,000 to 500,000 u / g) to the raw milk, and perform enzymolysis at a temperature of 45 to 55°C for 1 to 2 hours. After enzymolysis, adjust the temperature to 85 to 90°C to inactivate the enzyme for 15 to 20 minutes. Filter and centrifuge the above enzymolysis solution, and take the supernatant for ultrafiltration centrifugation (molecular weight cutoff 30ku) to remove small molecule peptides. Specifically, the mass of the added alkaline protease is 0.5% of the raw material mass, the enzymolysis temperature is 55°C, and the enzymolysis is performed for 1 hour.

[0041] (4) The probiotics and small molecule peptide composition prepared in step (3) is further mixed with Ziziphus jujuba seed extract, Lilium sibiricum extract, and Poria cocos extract to obtain a probiotics, small molecule peptide, and Chinese medicine extract composition.

[0042] The Chinese date seed extract, lily extract and Poria cocos extract can be prepared by the following method: After crushing the spiny jujube seeds, lily and poria respectively, 10 times the mass of 75% ethanol are added according to the mass ratio, and the mixture is filtered after being in an ultrasonic water bath for 2 to 3 hours to obtain a first filtrate; then 5 times the mass of 75% ethanol is added to the filter residue again, and the mixture is in an ultrasonic water bath for 1 hour to obtain a second filtrate; the second filtrate is mixed with the first filtrate, and the ethanol is evaporated to remove the ethanol, and the Chinese medicine extract powder is obtained by drying; the drying is freeze-drying, freezing at -25°C for 8 to 12 hours, the cold trap temperature is -60°C to -80°C, the vacuum degree is 50 to 150 Pa, and the drying time is 20 to 24 hours.

[0043] (5) The probiotics, small molecule peptides and Chinese medicine extract composition prepared in step (4) are mixed with γ-aminobutyric acid and N-acetylneuraminic acid to obtain a solid beverage of a Chinese medicine, composite probiotics and small molecule peptide composition that can calm nerves and help sleep. Test Example 1: Effects of different compositions on 5-HT and GABA levels in mouse blood

[0044] In order to explore the effects of different compositions on the content of 5-HT and GABA in the blood of mice, 80 healthy SPF Kunming mice (20±2g) were randomly selected in this test case. After one week of adaptive feeding under the same conditions, they were randomly divided into 8 groups, namely blank group, Chinese medicine group, small molecule peptide group, compound probiotic group, Chinese medicine + small molecule peptide, Chinese medicine + compound probiotic, small molecule peptide + compound probiotic, and Chinese medicine + compound probiotic + small molecule peptide. Except for the blank group, which was gavaged with normal saline, the other groups were gavaged with the same dose (5g / kg / d) for 14 consecutive days. After the experiment, the mice in each group were killed, serum was taken, and the 5-HT and GABA contents were determined according to the instructions of the ELISA kit.

[0045]

[0046] The results of one-way analysis of variance in Table 2 showed that Chinese medicine, small molecule peptides, and compound probiotics could significantly increase the levels of 5-HT and GABA in the blood of mice after administration separately, and the effect was most significant after the combination of the three. Test Example 2: Effect of a solid beverage containing a calming and sleep-inducing traditional Chinese medicine, a compound probiotic, and a small molecule peptide composition on the sleep-inducing effect on experimental mice

[0047] In order to explore the calming and sleep-inducing effect of the solid beverage composed of traditional Chinese medicine, composite probiotics and small molecule peptides prepared in Example 2, this test example conducted a subthreshold hypnotic dose of sodium pentobarbital mouse sleep test and a sodium pentobarbital-induced sleep time extension test for mice.

[0048] 1. Subthreshold hypnotic dose of sodium pentobarbital in mice sleep test 1.1 Experimental animals and methods 100 healthy SPF Kunming mice (20±2g) were randomly selected and randomly divided into 5 groups after one week of adaptive feeding under the same conditions, namely blank control group (distilled water), positive control group (diazepam 2.6 mg / kg, which is the clinical equivalent dose for adults), low-dose solid beverage group (0.5g / kg / d), medium-dose solid beverage group (5g / kg / d), and high-dose solid beverage group (10g / kg / d), with 20 mice in each group, and the drugs were continuously administered by gavage for 28 days. After the last administration of each dose group, the blank control group was given an equal volume of distilled water. 30 minutes later, each group was intraperitoneally injected with 150mg / kg of sodium pentobarbital. The disappearance of the righting reflex for more than 1 minute was used as the sleep judgment standard. The effects of each experimental group on the sleep rate of mice injected with subthreshold sodium pentobarbital were observed.

[0049] 1.2 Experimental Results

[0050] 2. Experiment on prolonging the sleep time of mice induced by sodium pentobarbital 2.1 Experimental methods After administration of each dose group, the blank control group was given an equal volume of distilled water. 30 minutes later, each group was intraperitoneally injected with 300 mg / kg sodium pentobarbital. The disappearance of the righting reflex for more than 1 minute was used as the sleep judgment standard. The recovery of the righting reflex indicated that the mouse was awake. The time from the disappearance of the righting reflex to the recovery was the sleep time of the mouse. The effect of each experimental group on the extension of sleep time by sodium pentobarbital was observed.

[0051] 2.2 Experimental Results

[0052] The above experimental results show that compared with the blank group, the medium and high dose groups of the solid beverage of the Chinese medicine, compound probiotics and small molecule peptide composition can significantly shorten the sleep latency of mice with sodium pentobarbital and prolong the sleep time of mice induced by sodium pentobarbital, among which the solid beverage of the high dose group can significantly improve the sleep rate of mice injected with subthreshold sodium pentobarbital. The above results show that the solid beverage of the Chinese medicine, compound probiotics and small molecule peptide composition provided by the present invention has a significant effect of calming the nerves and helping sleep.

[0053] Finally, it should be noted that the above is only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred arrangement scheme, a person skilled in the art should understand that the technical solution of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A composite probiotic with the effect of calming the nerves and promoting sleep, characterized in that: The composite probiotics include Lactobacillus plantarum ( Lactobacillus plantarum ) ZQ15 and Lactobacillus rhamnosus ( Lactobacillus rhamnosus )AlcoKer0067; Lactobacillus plantarum ZQ15, deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit number CGMCC NO. 30265 and the deposit date April 7, 2024; Lactobacillus rhamnosus AlcoKer0067, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number of CGMCC NO. 31221 and the deposit date of July 8, 2024; The mass ratio of Lactobacillus plantarum ZQ15 and Lactobacillus rhamnosus AlcoKer0067 in the composite probiotics is (5-10): (1-5).

2. A composite probiotic agent with the effect of calming the nerves and promoting sleep, characterized in that: The composite probiotic agent comprises the composite probiotic according to claim 1.

3. The composite probiotic agent with the effect of calming nerves and promoting sleep according to claim 2, characterized in that: The effective live bacterial concentration of Lactobacillus plantarum ZQ15 in the composite probiotic agent is not less than 5×10 11 CFU / g, the effective live bacterial concentration of Lactobacillus rhamnosus AlcoKer0067 is not less than 5×10 10 CFU / g.

4. A method for preparing the composite probiotic agent having the effect of calming nerves and promoting sleep as claimed in claim 2 or 3, characterized in that: The preparation method comprises the following steps: Lactobacillus plantarum ZQ15 and Lactobacillus rhamnosus AlcoKer0067 are inoculated, proliferated, fermented and dried to prepare a composite probiotic agent; The fermentation conditions are as follows: pH before fermentation is 6.20-6.80, fermentation temperature is 35-40°C, fermentation speed is 60 rpm, and fermentation time is 14-16 h.

5. A Chinese medicine, compound probiotics, and small molecule peptide composition for calming nerves and promoting sleep, characterized in that: The active ingredients of the composition include Chinese herbal medicine extracts, compound probiotics, prebiotics, small molecule peptides, γ-aminobutyric acid and N-acetylneuraminic acid; The Chinese medicine extracts include Ziziphus jujuba seed extract, lily extract and Poria cocos extract; The composite probiotics are the composite probiotics with a calming effect and a sleep-inducing effect as claimed in claim 1, or the composite probiotic agent with a calming effect and a sleep-inducing effect as claimed in claim 2 or 3; The prebiotics include oligofructose, resistant dextrin and isomaltooligosaccharide; The small molecule peptides include sturgeon skin oligopeptides, wheat oligopeptides, pecan oligopeptides and cow milk oligopeptides.

6. The composition of traditional Chinese medicine, compound probiotics and small molecule peptides for calming nerves and promoting sleep according to claim 5, characterized in that: The weight parts of each component are: 15-30 parts of Chinese herbal medicine extract, 15-30 parts of compound probiotics, 30-90 parts of prebiotics, 30-60 parts of small molecule peptides, 3-15 parts of γ-aminobutyric acid, and 3-15 parts of N-acetylneuraminic acid.

7. The Chinese medicine, compound probiotics, and small molecule peptide composition for calming nerves and promoting sleep according to claim 5 or 6, characterized in that: The preparation method of the Chinese medicine extract comprises the following steps: (1) After crushing the spiny jujube seeds, lily bulbs, and poria cocos respectively, add 10 times the mass of 75% ethanol according to the mass ratio, and filter after ultrasonic water bath for 2-3 hours to obtain the first filtrate; (2) Then, 5 times the mass of 75% ethanol was added to the filter residue again, and the mixture was filtered after being ultrasonically bathed for 1 hour to obtain the second filtrate; (3) The second filtrate is mixed with the first filtrate, and the ethanol is removed by rotary evaporation. The mixture is freeze-dried to obtain a Chinese medicine extract powder.

8. The composition of traditional Chinese medicine, compound probiotics and small molecule peptides for calming nerves and promoting sleep according to claim 7, characterized in that: In steps (1) and (2), the conditions of the ultrasonic water bath are: the ultrasonic power is 20 to 40 kHz, and the water bath temperature is 50° C. to 60° C.; In step (3), the drying method is freeze drying, and the conditions are: freezing at -25°C for 8 to 12 h, cold trap temperature of -60°C to -80°C, vacuum degree of 50 to 150 Pa, and drying time of 20 to 24 h.

9. Use of the composite probiotics according to claim 1, the composite probiotic agent according to claim 2 or 3, or the traditional Chinese medicine, composite probiotics, and small molecule peptide composition according to claims 5 to 8 in the preparation of a product for calming the nerves and aiding sleep.

10. The use according to claim 9, characterized in that: The products include food, health products and medicines.

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