Application of plant lactobacillus and composition thereof for relieving anxiety, soothing nerves and helping sleep in solid beverage

Abstract

The present application relates to a kind of plant lactobacillus rhamnosus for relieving anxiety, tranquilizing sleep aid and its composition in solid beverage, the preservation number of the plant lactobacillus rhamnosus is CGMCC No.30265, it is isolated from traditional yogurt, it is strong in safety, no pathogenicity, without acute toxicity;And after culture screening, it is verified by experiment, combined with plant extract, promote the production of 5-hydroxytryptamine, enhance the effect of relieving anxiety, tranquilizing sleep aid.The present application also provides a kind of probiotic solid beverage for tranquilizing sleep aid, it can promote the rapid and efficient planting of plant lactobacillus rhamnosus by adding prebiotic combination, while adding plant extract combination, synergistic effect with probiotic, greatly enhance the effect of relieving anxiety, tranquilizing sleep aid.The composition component is safe, nontoxic and harmless, tranquilizing sleep aid effect is remarkable, with broad market prospect.

Description

TECHNICAL FIELD

[0001] The application relates to application of Lactobacillus plantarum and a composition thereof for relieving anxiety, soothing nerves and helping sleep in a solid beverage, and belongs to the technical field of microorganisms and application thereof. BACKGROUND

[0002] With the rapid development of society, the living habits of Chinese people have changed, and the life pressure has increased, accompanied by a series of health problems such as anxiety and insomnia. Related research shows that more than 300 million people in China have sleep disorders, and the proportion of people with sleep disorders is 10% higher than the world average. Sleep disorders are a manifestation of abnormal sleep and abnormal behavior during sleep, and are also a manifestation of the normal rhythmic alternation of sleep and wakefulness. Sleep disorders are accompanied by anxiety and depression, and 40% of insomnia patients have one or more mental disorders, of which anxiety disorders account for 24%.

[0003] Existing anti-anxiety and depression drugs mainly include amitriptyline, doxepin and the like, most of which are nerve drugs, and long-term use can destroy the balance of intestinal flora, cause imbalance of intestinal microbial ecology, and further affect human health. Some stress-reducing products can only have a psychological relief effect and cannot bring any effect to the patient's body. Long-term use may form dependence, reduce reaction ability and memory, and even cause mental abnormalities and depression. How to develop an effective sleep-promoting and anxiety-relieving beverage to replace drugs is a technical problem that needs to be solved. SUMMARY

[0004] (I) Technical problem to be solved

[0005] In order to solve the above problems in the prior art, the application provides application of Lactobacillus plantarum for relieving anxiety, soothing nerves and helping sleep in a solid beverage.

[0006] (II) Technical scheme

[0007] In order to achieve the above purpose, the main technical scheme adopted by the application comprises:

[0008] A Lactobacillus plantarum ZQ15 for relieving anxiety, soothing nerves and helping sleep, which was preserved in the China General Microbiological Culture Collection Center on April 7, 2024, and the preservation number is CGMCC No. 30265.

[0009] The application of the Lactobacillus plantarum in the preparation of the product for relieving anxiety, soothing nerves and helping sleep is described above. Further, the product is a solid beverage, an oral liquid, a granular substance or the like.

[0010] A composition for relieving anxiety and helping sleep, comprising, by mass fraction, a prebiotic combination 10-30 parts, plant lactobacillus 5-10 parts, and plant extract mixture 5-10 parts.

[0011] The composition as described above, preferably, the prebiotic combination comprises fructooligosaccharide 5-15 parts, resistant dextrin 1-10 parts, and isomaltooligosaccharide 5-15 parts;

[0012] The plant lactobacillus has a number of CGMCC No. 30265, and the number of live bacteria is 1×10 10 ~ 8×10 11 CFU / g.

[0013] The plant extract mixture is jujube seed extract 1-25 parts, lily extract 1-15 parts, and poria cocos extract 1-10 parts.

[0014] The composition as described above further comprises auxiliary materials 3-10 parts, which are a mixture of erythritol, sorbitol, and skim milk powder in a mass ratio of (5-15):(1-10):(10-15).

[0015] Further, the extraction method of the jujube seed extract, lily extract, and poria cocos extract is as follows: after jujube seeds, lilies, and poria cocos are respectively crushed, 8-10 times the mass of 75% ethanol is added, and after 2-3 hours of ultrasonic water bath, the first filtrate is obtained; then 4-6 times the mass of 75% ethanol is added to the residue, and after 0.5-2 hours of ultrasonic water bath, the second filtrate is obtained; the second filtrate and the first filtrate are mixed, and the ethanol is removed by rotary evaporation, and the plant extract powder is obtained by freeze-drying.

[0016] Preferably, the conditions of the ultrasonic water bath are that the water bath temperature is 50-60°C, and the ultrasonic power is 20-40 KHZ; the conditions of freeze-drying are that the freezing temperature is -25°C for 8-12 hours, the cold trap temperature is -60°C to -80°C, the vacuum degree is 50-150 Pa, and the drying time is 20-24 hours.

[0017] A sleep-aiding probiotic solid beverage, comprising, by mass fraction: jujube seed extract 1-25 parts, lily extract 1-15 parts, poria cocos extract 1-10 parts, plant lactobacillus powder 1-10 parts, fructooligosaccharide 5-15 parts, resistant dextrin 1-10 parts, isomaltooligosaccharide 5-15 parts, erythritol 5-15 parts, sorbitol 1-10 parts, and skim milk powder 10-15 parts.

[0018] Further, preferably, the plant lactobacillus powder has a number of CGMCC No. 30265, and the number of live bacteria is 1×10 10 ~ 8×10 11 CFU / g.

[0019] The probiotic solid beverage as described above, preferably, the extract of Semen Ziziphi Spinosae is 3-5 parts, the extract of Lili is 1-3 parts, the extract of Poria cocos is 1-2 parts, the powder of Bacillus lactis is 2-5 parts, the fructooligosaccharide is 4-6 parts, the resistant dextrin is 3-5 parts, the isomaltooligosaccharide is 4-7 parts, the erythritol is 1-3 parts, the sorbitol is 1-2 parts and the skimmed milk powder is 2-4 parts.

[0020] Most preferably, the extract of Semen Ziziphi Spinosae is 3.3 parts, the extract of Lili is 1.4 parts, the extract of Poria cocos is 1.3 parts, the powder of Bacillus lactis is 3 parts, the fructooligosaccharide is 5 parts, the resistant dextrin is 4 parts, the isomaltooligosaccharide is 6 parts, the erythritol is 2 parts, the sorbitol is 1.4 parts and the skimmed milk powder is 2.6 parts.

[0021] The probiotic solid beverage as described above, preferably, the water activity of the fructooligosaccharide, the resistant dextrin, the isomaltooligosaccharide, the erythritol, the sorbitol and the skimmed milk powder are all less than 0.05.

[0022] The preparation method of the probiotic solid beverage as described above, preferably, comprises the following steps:

[0023] S1, crushing Semen Ziziphi Spinosae, Lili and Poria cocos respectively, adding ethanol, filtering after 2-3 hours of ultrasonic water bath to obtain the first filtrate, then adding ethanol to the residue again, filtering after 1 hour of ultrasonic water bath to obtain the second filtrate, mixing the first filtrate and the second filtrate, removing the ethanol by rotary evaporation, and freeze-drying to obtain the plant extract powder;

[0024] S2, drying the fructooligosaccharide, the resistant dextrin and the isomaltooligosaccharide respectively, and mixing the dried fructooligosaccharide, the resistant dextrin and the isomaltooligosaccharide with the plant extract to obtain a mixture; wherein the water activity of the dried fructooligosaccharide, the resistant dextrin and the isomaltooligosaccharide is ≤0.05 respectively;

[0025] S3, mixing the mixture, the excipient and the Bacillus lactis ZQ15 to obtain the probiotic solid beverage.

[0026] The preparation method as described above, preferably, in step S1, the amount of ethanol added in the first filtrate is 8-10 times the mass of the plant powder, and the amount of ethanol added in the second filtrate is 4-6 times the mass of the plant powder, and the ethanol is preferably 75% ethanol;

[0027] The ultrasonic water bath is at a water bath temperature of 50-60°C and an ultrasonic power of 20-40 KHZ;

[0028] The freeze-drying is at a cold trap temperature of -60 to -80°C, a vacuum degree of 50-150 Pa and a drying time of 20-24 hours;

[0029] The plant extract powder is a mixture of Zizyphus jujuba extract, lily extract and Poria cocos extract mixed in a mass ratio of 1-25:1-15:1-10, and the optimal ratio is 3.3:1.4:1.4.

[0030] The preparation method described above, preferably, in step S2, the fructooligosaccharides, resistant dextrin and isomaltooligosaccharides are mixed in a mass ratio of 5-15:1-10:5-15 to obtain a prebiotic combination; and the mass ratio of the prebiotic combination and the plant extract powder in the mixture is 10-30:5-10.

[0031] The preparation method described above, preferably, in step S3, the auxiliary material is a mixture of erythritol, sorbitol and skimmed milk powder in a mass ratio of 5-15:1-10:10-15.

[0032] The live bacterial concentration of the Lactobacillus plantarum ZQ15 bacterial powder is 1x10 10 ~ 5x10 11 The amount is in a mass ratio of 10-30:5-15:10-25 of the prebiotic combination, the Lactobacillus plantarum ZQ15 bacterial powder and the auxiliary material.

[0033] Further, the mass ratio of the fructooligosaccharides, resistant dextrin and isomaltooligosaccharides is 5:4:6, the mass ratio of the prebiotic combination and the plant extract powder is 25:10, and the auxiliary material is a mixture of erythritol, sorbitol and skimmed milk powder in a mass ratio of 10:7:13.

[0034] (Three) Beneficial effects

[0035] The beneficial effects of the present application are:

[0036] (1) The live bacteria of the Lactobacillus plantarum ZQ15 provided by the present application can produce a large amount of 5-hydroxytryptamine in the intestinal environment, and can enter the nerve cells with the blood, bind to the 5-hydroxytryptamine receptors between neurons, and play a role in regulating emotions, thereby playing a role in preventing sleep disorders, improving sleep quality and relieving anxious emotions.

[0037] (2) The Lactobacillus plantarum ZQ15 provided by the present application can also produce 5-hydroxytryptamine through metabolic production of the culture medium, so the strain can be fermented in vitro to prepare products for relieving anxiety and improving sleep quality using the fermentation product or fermentation extract.

[0038] (3) The Lactobacillus plantarum ZQ15 provided by the present application is isolated from traditional yogurt, has strong safety, no pathogenicity and no acute toxicity; and after culture and screening, it is verified through experiments that the combination with plant extracts promotes the production of 5-hydroxytryptamine and enhances the effects of relieving anxiety, soothing the nerves and improving sleep.

[0039] The prebiotic combination added in the present application can promote the rapid and efficient colonization of Lactobacillus plantarum, and the combination of plant extracts can synergize with the probiotics to greatly enhance the effect of relieving anxiety, calming the nerves and helping sleep. The composition for relieving anxiety, calming the nerves and helping sleep provided in the present application is safe, non-toxic and harmless, has a significant effect of calming the nerves and helping sleep, and has a broad market prospect. DETAILED DESCRIPTION

[0040] The present application improves the MRS stress culture of Lactobacillus plantarum, induces the tolerance of the strain to plant extracts (a mixture of acid jujube extract, lily extract and Poria cocos extract), and the extract can promote the large production of 5-hydroxytryptamine by Lactobacillus plantarum ZQ15. The preferred fructooligosaccharides, resistant dextrin and isomaltooligosaccharides can rapidly promote the colonization and growth of Lactobacillus plantarum ZQ15 in the intestinal tract.

[0041] In the preparation method, the ultrasonic water bath step can achieve a high success rate. The water bath temperature is preferably 50-60℃, and the ultrasonic power is 20-40KHZ. If it is higher than 60℃, ethanol may be excessively volatilized, resulting in the loss of part of the active substances with ethanol; if it is lower than 50℃, the extraction time will be prolonged, and therefore the water bath temperature is preferably 50-60℃.

[0042] In order to better explain the present application and facilitate understanding, the present application is described in detail below through specific embodiments.

[0043] Example 1

[0044] This embodiment is the screening of Lactobacillus plantarum and the preparation of its fermentation agent.

[0045] 1. Strain screening

[0046] The strain screening was carried out by taking Qinghai yak yogurt as a sample.

[0047] (1) 2mL of the sample was taken into 100mL of MRS medium, and cultured at 37℃ for 18-24h for enrichment.

[0048] (2) Stress culture: 2mL of the enriched sample was taken into 100mL of modified MRS medium, and cultured at 37℃ for 18-24h for successive activation of three generations.

[0049] (3) Gradient dilution: 0.5mL of the activated sample was taken into 4.5mL of sterile physiological saline to obtain 10 -1 dilution, then 0.5mL of the 10 -1 dilution was taken into 4.5mL of physiological saline to obtain 10 -2 dilution, and the operation was sequentially performed to obtain 10 -3 , 10 -4,10 -5 Dilution liquid.

[0050] (4) Plate culture: 100 μL of gradient dilution liquid was taken and coated on MRS agar plate, 10 -3 ,10 -4 ,10 -5 One plate per gradient; incubated at 37°C for 48h.

[0051] (5) Streak isolation: according to the shape, size, edge, transparency and other selection of colonies coated plate with typical characteristics of colonies, the colonies were picked up with inoculation ring and streaked on selective medium plate, and incubated at 37°C for 48h.

[0052] (6) Obtained purified single colony, picked single colony inoculated into 5 mL of modified MRS medium, and incubated for 18-24h. Meanwhile, each strain was numbered and strain identification was performed.

[0053] The formula of the modified MRS medium is as follows: 10 g of peptone, 10 g of beef extract, 20 g of glucose, 5 g of yeast extract, 2 g of anhydrous sodium acetate, 0.25 g of manganese sulfate monohydrate, 1 mL of Tween 80, 2.6 g of dipotassium hydrogen phosphate trihydrate, 0.5 g of magnesium sulfate heptahydrate, and 2 g of diammonium citrate were added to 1 L of distilled water, pH 6.2-6.5, and 5% (w / v, g / ml) of plant extract (a mixture of Zizyphus jujuba Mill. extract, Lilium brownii Mill. extract and Poria cocos (Schw.) Wolf extract in a mass ratio of 3.3:1.4:1.3) was added. The Zizyphus jujuba Mill. extract, Lilium brownii Mill. extract and Poria cocos (Schw.) Wolf extract were first crushed, 75% ethanol was added at 10 times the mass of the raw material, and the mixture was extracted under ultrasonic power of 40 KHZ and water bath temperature of 55°C for 2.5h, and then filtered to obtain the first filtrate. Then 5 times the mass of 75% ethanol was added to the residue, and the mixture was extracted under the same conditions for 1h, and then filtered to obtain the second filtrate. The second filtrate and the first filtrate were mixed, and the ethanol was removed by rotary evaporation. The plant extract powder was obtained by freeze-drying, and the freeze-drying method was as follows: freezing at -25°C for 10h, cold trap temperature -70°C, vacuum degree 100 Pa, and drying time 24h.

[0054] 2. Screening results

[0055] DNA was extracted from the isolated single colony strains, and PCR amplification of 16S rRNA gene was performed using 27F (SEQ ID NO. 1: 5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (SEQ ID NO. 2: 5'-GGTTACCTTGTTACGACTT-3') universal primers. The PCR product was sent to Meigene sequencing company for sequencing. The strains numbered ZQ15, HY6-2 and DP1-12, which were identified as Lactobacillus plantarum and grew better, were selected for subsequent experiments. The better growth refers to the absorbance value OD 600nm at 600 nm of the strains was the largest.

[0056] 3. Morphological characteristics

[0057] The obtained Lactobacillus plantarum ZQ15 has the following characteristics:

[0058] (1) Morphological characteristics:

[0059] Gram-positive staining, cell rod-shaped, cell body about 0.4-1.2 μm wide, 1.5-4 μm long, single, paired or short chain, no spores, both ends round;

[0060] (2) Culture traits:

[0061] Formed obvious colonies on MRS medium, diameter between 0.3-3.0 mm, round, edge neat, milky white, opaque, surface smooth, no pigment produced;

[0062] (3) Physiological and biochemical characteristics:

[0063] Can utilize arabinose, cellobiose, fructose, lactose, galactose, raffinose, sorbose, trehalose, sucrose; no reduction of nitrate, no liquefaction of gelatin, both contact enzyme and oxidase negative; has excellent simulated gastrointestinal survival ability and cell adhesion ability.

[0064] 4. Preparation of leavening agent

[0065] In a case we actually operated, the leavening agent was obtained by drying the bacterial liquid containing strains numbered ZQ15, HY6-2 and DP1-12, respectively. It was determined that the leavening agent contained more than 10 10 CFU / g of activity. The powder can be obtained by conventional freeze-drying or other drying methods that do not affect the activity.

[0066] In one of the embodiments used by us, the specific fermentant preparation process is as follows:

[0067] (1) Preparation of culture medium: the formulation of the culture medium is as follows by total weight: water: 85.7%; skimmed milk: 11.5%; glucose: 0.7%; tryptone: 1.8%; yeast extract: 0.3%. After dissolving the above ingredients, the pH is adjusted to 6.7.

[0068] (2) Preparation of protective agent: dissolve the following into 1 L of pure water system: skimmed milk powder: 110 g; glycerol: 25 mL; trehalose: 200 g; sterilize the above raw materials at 65°C for 30 min.

[0069] (3) Respectively inoculate Lactobacillus plantarum HY6-2, Lactobacillus plantarum DP1-12, and Lactobacillus plantarum ZQ15 into the culture medium of step (1) at an inoculation amount of 2-4% by weight of the culture medium, and then culture at a temperature of 37°C for 24 h; then, uniformly mix them with the protective agent of step (2) at a volume ratio of 7:3 (v:v), and after 6 h of fermentation, the viable cell concentration is increased to 10 10 CFU / mL, and after cold drying, Lactobacillus plantarum HY6-2 powder, Lactobacillus plantarum DP1-12 powder, and Lactobacillus plantarum ZQ15 powder are obtained.

[0070] Example 2

[0071] This example is to explore the simulated gastrointestinal survival ability of the Lactobacillus plantarum screened in Example 1.

[0072] 1 Test method

[0073] (1) Preparation of simulated gastric juice: dissolve pepsin in sterilized normal saline (0.9% w / v) to a final concentration of 0.3% (w / v), and adjust the pH to 2.5 with hydrochloric acid. Filter with a 0.22 μm sterile filter membrane, and prepare fresh every time.

[0074] (2) Preparation of simulated intestinal juice: dissolve trypsin in sterilized normal saline (0.9% w / v) to a final concentration of 1 g / L, adjust the pH to 8.0 with NaOH, and add bile salt to a final concentration of 1.8% (w / v). Filter with a 0.22 μm sterile filter membrane, and prepare fresh every time.

[0075] (3) After three generations of continuous activation (20 h each time) of different Lactobacillus plantarum, the experiment is performed. Measure the OD value of the strains after three generations of continuous activation at a wavelength of A600nm, and calculate the amount of bacterial solution required for OD 600nm = 5.

[0076] (4) Mix the bacterial solution required for OD 600nmThe bacterial liquid required for 5 was centrifuged at 8000 x g for 10 min, the supernatant was discarded, and the bacterial liquid was resuspended in 1 mL of simulated gastric juice. After incubation at 37 °C for 3 h, plate viable count was performed.

[0077] (5) The bacterial liquid treated with simulated gastric juice was centrifuged at 8000 x g for 10 min, the supernatant was discarded, and the bacterial liquid was resuspended in an equal volume of simulated intestinal juice. After incubation at 37 °C for 4 h, plate viable count was performed.

[0078] The survival rate after tolerance to gastric juice (%) = (the number of viable bacteria in the bacterial liquid after tolerance to simulated gastric juice / the number of original viable bacteria in the bacterial liquid) x 100%.

[0079] The survival rate after tolerance to intestinal juice (%) = (the number of viable bacteria in the bacterial liquid after tolerance to simulated intestinal juice / the number of viable bacteria in the bacterial liquid after tolerance to simulated gastric juice) x 100%.

[0080] The survival rate after tolerance to gastric juice (%) = (the number of viable bacteria in the bacterial liquid after tolerance to simulated intestinal juice / the number of original viable bacteria in the bacterial liquid) x 100%.

[0081] 2 Experimental results

[0082] Table 1 Tolerance ability of different plant lactobacillus in simulated gastrointestinal tract

[0083]

[0084] Plant lactobacillus DP1-12 was used as a control, and * indicates a significant difference (P < 0.05), ** and ** indicates a very significant difference (P < 0.01).

[0085] After 2 h of incubation in physiological saline containing pepsin (simulated gastric juice) at pH 2.5, the acid tolerance of plant lactobacillus was good, and the survival rates of plant lactobacillus DP1-12 and plant lactobacillus ZQ15 were 96.96 ± 2.08% and 96.23 ± 1.98%, respectively. After 4 h of incubation in physiological saline containing trypsin and bile salt (simulated intestinal juice) at pH 8, the survival rates of plant lactobacillus HY6-2 and plant lactobacillus ZQ15 were 9.17 ± 1.08% and 17.67 ± 2.25%, respectively. After 2 h of incubation in physiological saline containing pepsin at pH 2.5 and then 4 h of incubation in physiological saline containing trypsin and bile salt at pH 8, i.e. 6 h of simulated gastric and intestinal juice, the survival rate of plant lactobacillus ZQ15 was the highest, being 15.99 ± 2.47%. This indicates that plant lactobacillus ZQ15 has excellent tolerance to the gastrointestinal tract and has the potential to exert a probiotic function in the intestinal tract.

[0086] The plant lactobacillus ZQ15 obtained above was preserved in China General Microbiological Culture Collection Center (address: No. 1, Beichen West Road, No. 3, Chaoyang District, Beijing, Institute of Microbiology of Chinese Academy of Sciences, postcode: 100101) on April 7, 2024, and the strain classification name was Lactobacillus plantarum, and the preservation number was CGMCC No. 30265 (it is necessary to point out that "Lactobacillus plantarum" has been renamed "Lactobacillus plantarum" in the latest national standard, and the applicant has submitted a renaming application to the preservation center, and after that, the classification name in the survival certificate will be changed to "Lactobacillus plantarum").

[0087] Wherein, the sequencing result of the plant lactobacillus ZQ15 is as follows: SEQ ID NO. 3: GGCTGGTTCC TAAAAGGTTACC CACCGACTTT GGGTTACAAA CTCTCATGGT GTGACGGGCG GTGTGTACA AGGCCCGGGA ACGTATTCAC CGCGGCATGC TGATCCGCGA TTACTAGCGA TTCCGACTTC ATGTAGGCGA GTTGCAGCC TACAATCCGA ACTGAGAATG GGCTTTAAG AGATTAGCTT ACTCTCGCGA GTTCGCAACT CGTTGTACCA TCATTGTAGC ACGTGTGTAG CCCAGGTCAT AAGGGGCATG ATGATTTGA CGTCATCCCC ACCTTCCTCG GTTTGTCACC GGCAGTCTCA CCAGAGTGCC CAACTTAATG CTGGCAACTG ATAATAAGGG TTGCGCTCGT TGCGGGACTT AACCCAACAT CTCACGACAC GAGCTGACGA CCACCATGCA CCACCTGTAT CCATGTCCCC GAAGGGAACG TCTAATCTCT TAGATTTGCA TAGTATGTCA AGACCTGGTA AGGTTCTTCG CGTAGCTTCG AATTAAACCA TGCTCCACCG CTTGTGCGGG CCCCGTCAAT TCCTTTGAGT TTCAGCCTTG CGGCCGTACT CCCCGGCGGA ATGCTTAATG CGTTAGCTGC AGCACTGAAG GGCGGAAACCT CCACACTTAG CATTCATCGT TTACGGTATG GACTACCAGG GTATCTAATC CTGTTTGCTA CCCATACTTT CGAGCCTCAG CGTCAGTTAC AGACCAGACA GCCGCCTTCG CCACCGGTGT TCTTCCATAT ATCTACGCAT TTCACCGCTA CATATGGAGT TCCACTGTCC TCTTCTGCAC TCAAGTTTCC CAGTTTCCGA TGCACTTCTT CGGTTGAGCC GAAGGCTTTC ACATCAGACT TAAAAAACCG CCGTGGCTCG CTTTACGCCC AATAAATCCC GGACAACGC.

[0088] Example 3

[0089] This embodiment is to explore the in vitro 5-hydroxytryptamine production ability of the plant lactobacillus screened in embodiment 1.

[0090] 1 Test method

[0091] 1) Take plant lactobacillus HY6-2 bacterial powder, plant lactobacillus DP1-12 bacterial powder, and plant lactobacillus ZQ15 bacterial powder, inoculate them into MRS culture medium at an inoculation amount of 1%, and take another portion of plant lactobacillus ZQ15 bacterial powder, inoculate it into modified MRS added with plant extract combination at an inoculation amount of 1%, and culture it at 37°C for 24h. Among them, 5g of plant extract combination is added to every 100ml of modified MRS, and the plant extract combination is a mixture of sour jujube extract, lily extract, and fuzi extract mixed in a mass ratio of 3.3:1.4:1.3.

[0092] 2) Centrifuge the bacterial solution at 8000rpm per minute for 10 minutes, take the supernatant, and use high-performance liquid chromatography to determine the content of 5-hydroxytryptamine, and the results are shown in Table 2.

[0093] 2 Experimental results

[0094] Table 2 In vitro 5-hydroxytryptamine production of different plant lactobacillus

[0095]

[0096] With plant lactobacillus DP1-12 as the control, * indicates significant difference (P<0.05), and ** indicates extremely significant difference (P<0.01).

[0097] As can be seen from the results, after 24h of culture, the 5-hydroxytryptamine production of the plant lactobacillus HY6-2 group is 33.79±4.18, the 5-hydroxytryptamine production of the plant lactobacillus DP1-12 group is 39.37±3.61, the 5-hydroxytryptamine production of the plant lactobacillus ZQ15 group is 63.95±2.13ng / mL, and the 5-hydroxytryptamine production of the plant lactobacillus ZQ15+plant extract (sour jujube, lily, and fuzi) group is 148.18±4.57ng / mL. It shows that plant lactobacillus ZQ15 can have a synergistic effect with plant extract (sour jujube, lily, and fuzi), and greatly improves the production of 5-hydroxytryptamine.

[0098] Example 4

[0099] A preparation method of a probiotic composition solid beverage (except for special requirements, the raw materials used are ordinary market raw materials), the specific operation is as follows:

[0100] 1) Take raw materials: fructooligosaccharide, resistant dextrin, isomalto-oligosaccharide, and auxiliary materials: erythritol, sorbitol and skimmed milk powder, and then test the water activity of the above raw materials and auxiliary materials by a water activity tester;

[0101] 2) Put fructooligosaccharide, resistant dextrin, isomalto-oligosaccharide, erythritol, sorbitol and skimmed milk powder with water activity higher than 0.05 after the water activity test in step 1) into an oven, and then dry them at 35-45℃ with circulating low-temperature air for 1-4h;

[0102] 3) Take out each raw material and auxiliary material in step 2) every 30min, and then test the water activity of fructooligosaccharide, resistant dextrin, isomalto-oligosaccharide, erythritol, sorbitol and skimmed milk powder again by a water activity tester;

[0103] 4) Repeat the drying treatment and water activity test of fructooligosaccharide, resistant dextrin, isomalto-oligosaccharide, erythritol, sorbitol and skimmed milk powder according to the method of step 2) and step 3), and finally control the water activity of each raw material and auxiliary material to be below 0.05; weigh each component according to the raw material formula in Table 3;

[0104] Table 3 A solid beverage formula of probiotic composition

[0105]

[0106] The auxiliary materials are prepared according to the same method as in Example 1.

[0107] 5) Put fructooligosaccharide, resistant dextrin, isomalto-oligosaccharide, erythritol, sorbitol and skimmed milk powder with water activity below 0.05 obtained in step 4) and plant extract combination into a stirrer, and then stir at a speed of 200rpm / min for 3min to make each raw material and auxiliary material fully mixed and uniform, to obtain a mixture, for standby;

[0108] 6) Take Lactobacillus plantarum ZQ15 powder with a viable bacterial concentration of 5×10 11 CFU / g, and add it into the mixture obtained in step 5), and then continue to stir at a speed of 200rpm / min for 2min to make the probiotic powder, raw materials, auxiliary materials and plant extract combination fully mixed and uniform, to obtain a probiotic mixture, for standby;

[0109] 7) Package the probiotic mixture obtained in step 6) into small bags, 3g per bag, seal, and the solid beverage of probiotic composition of the present application is obtained.

[0110] Example 5

[0111] This example is to detect the efficacy of the probiotic composition solid beverage prepared in Example 4, as follows:

[0112] 1. Establishment of mouse model

[0113] Seventy ICR male mice were raised in a mouse room with relative humidity of 40% to 60% and temperature of (20-23) °C. After adaptive feeding for 7 days, the mice were randomly divided into negative control group, positive control group, probiotic group and experimental group. The modeling method was as follows: the mice were intraperitoneally injected with PCPA (0.1 g / mL) 0.3 mL / 100 g at 10:00-11:00 am every day for 3 days to replicate insomnia model. The mice were considered to be successfully modeled when they showed behaviors such as disappearance of circadian rhythm, increased activity during the day, abnormal sensitivity to sound, easy to huddle, increased aggressiveness, etc.

[0114] The experiment was divided into 7 groups in total, i.e. negative control group (given normal saline), positive control group (diazepam 2.6 mg per kg of mouse body weight, which is the adult clinical equivalent dose), Lactobacillus plantarum HY6-2 group (given Lactobacillus plantarum HY6-2 powder 50 mg per kg of mouse body weight dissolved in 0.2 mL of normal saline), Lactobacillus plantarum DP1-12 group (given Lactobacillus plantarum DP1-12 powder 50 mg per kg of mouse body weight dissolved in 0.2 mL of normal saline), Lactobacillus plantarum ZQ15 group (given Lactobacillus plantarum ZQ15 powder 50 mg / kg dissolved in 0.2 mL of normal saline), prebiotic + plant extract group (a mixture of the components other than Lactobacillus plantarum in the formulation of Example 4, given at 50 mg / kg dissolved in 0.2 mL of normal saline), and experimental group (given Lactobacillus plantarum ZQ15 powder 50 mg per kg of mouse, and also added with the other components of the solid beverage prepared according to the formulation of Example 4, dissolved in 0.2 mL of normal saline). Each group contained 10 mice. The gavage time was 08:00-09:00 every day, once a day. The mice were raised for 30 days, during which the mouse body weight was recorded every 6 days. After 30 days, the gavage was stopped and the subsequent experiments were prepared. The mice could freely eat and drink during the whole experiment.

[0115] 2. Sleep rate experiment of mice

[0116] The subthreshold experiment of sodium pentobarbital was carried out 1 hour after the last gavage. Each group of mice was intraperitoneally injected with sodium pentobarbital 35 mg / kg, with injection volume of 0.1 mL / 10 g of body weight. The disappearance of the mouse righting reflex was taken as the judgment standard of sleep, and the time from the completion of intraperitoneal injection to the disappearance of the righting reflex was taken as the sleep latency. The sleep of each group of mice was counted and calculated to obtain the sleep rate, and the results are shown in Table 4.

[0117] Table 4. Results of subthreshold experiment of sodium pentobarbital

[0118]

[0119] Compared with the negative control group, the superscript * indicates that the difference is significant (P<0.05), and the superscript ** indicates that the difference is extremely significant (P<0.01).

[0120] The results show that compared with the negative control group, the sleep latency of the remaining groups is significantly shortened (P<0.05), and compared with the negative control group, the sleep rate of the probiotic group and the experimental group is significantly increased, indicating that the solid beverage containing plant lactobacillus ZQ15 can significantly shorten the sleep latency of the experimental mice, improve the sleep rate, and the effect is obviously enhanced when combined with plant extracts. It is proved that plant lactobacillus ZQ15 colonizes in the intestinal tract of mice and secretes 5-hydroxytryptamine in the growth and metabolism process, and the bacteria and the probiotic composition solid beverage have the effect of soothing nerves and helping sleep.

[0121] The above is only a preferred embodiment of the present application, and is not intended to limit the present application in other forms. Any person skilled in the art can modify or change the above disclosed technical content to equivalent embodiments. However, any simple modification, equivalent change and modification made on the basis of the technical essence of the present application to the above embodiments without departing from the technical scheme of the present application still belongs to the protection scope of the present application.

Claims

1. A plant lactobacillus that produces high levels of 5-hydroxytryptamine ( Lactobacillus plantarum ZQ15, characterized in that, The preservation number of the strain is CGMCC No. 30265.

2. A method of increasing serotonin production, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of claim 1. The plant lactobacillus (as described in claim 1) Lactobacillus plantarum ZQ15 was cultured in MRS containing a combination of added plant extracts at 37°C for 24 hours. The bacterial culture was then centrifuged at 8000 rpm for 10 minutes, and the supernatant was collected to obtain 5-hydroxytryptamine. The plant extract combination was a mixture of jujube seed extract, lily extract, and poria cocos extract in a mass ratio of 3.3:1.4:1.

3. The MRS containing the plant extract combination contains 5g of the plant extract combination per 100mL; the extract of Semen Ziziphi Spinosae, the extract of Fritillaria and the extract of Poria cocos are prepared as follows: Semen Ziziphi Spinosae, Fritillaria and Poria cocos are respectively pulverized, 8-10 times the mass of 75% ethanol is added according to the mass ratio, and after 2-3h of ultrasonic water bath, the first filtrate is obtained; then 4-6 times the mass of 75% ethanol is added to the residue, and after 0.5-2h of ultrasonic water bath, the second filtrate is obtained; the second filtrate and the first filtrate are mixed, and the ethanol is removed by rotary evaporation, and the plant extract powder is obtained by freeze-drying.

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