CN110106122B - Lactobacillus plantarum capable of improving sleep and use thereof - Google Patents

Abstract

The invention discloses lactobacillus plantarum capable of improving sleep and application thereof, and belongs to the technical field of microorganisms. The lactobacillus plantarum GDMCC No.60604 has excellent capability of simulating gastrointestinal tract survival and cell adhesion; can prolong the sleep time induced by the sodium pentobarbital and shorten the sleep latency induced by the sodium barbital. The lactobacillus plantarum GDMCC No.60604 is used for preparing a pharmaceutical composition and a fermented food for improving sleep and has very wide application prospect.

Description

Lactobacillus plantarum capable of improving sleep and use thereof

technical field

The invention relates to a Lactobacillus plantarum capable of improving sleep and use thereof, belonging to the technical field of microorganisms.

Background technique

Sleep is one of the important physiological processes of the body, and it has an important recovery function to eliminate physical fatigue and restore body damage. But with the increase of life stress or under the influence of various diseases, sleep disturbance has now become a long-term problem. Studies have shown that about 50% of people in my country have experienced sleep disorders of varying degrees. Mild sleep disturbances may lead to memory impairment, attention changes and learning difficulties, resulting in a decline in one’s own health and quality of life. In severe cases, it can lead to pathological changes such as diabetes, cardiovascular disease, and cancer, or aggravate existing diseases.

类、非苯二氮

类、其它类型安眠药物等阶段。药物治疗虽然见效快，效果明显，但患者长期服用会产生依赖性和耐受性，一旦停止使用，可能反而会出现失眠加重的不良反应。鉴于传统治疗方法存在的多种问题，所以找到一种副作用小、效果好的干预或治疗方法是现在亟待解决的问题。At present, the treatment of sleep disorders is mainly divided into two types: drug treatment and non-drug treatment. Non-drug treatment mainly includes sleep hygiene education, traditional Chinese medicine therapy, psychotherapy and other methods, which have the advantages of simplicity, economy and safety. However, when the insomnia is severe, non-drug therapy has little effect, and drug therapy becomes the first choice. Pharmacological treatment experienced early sleeping pills, benzodiazepines

class, non-benzodiazepines

Class, other types of sleeping drugs and other stages. Although drug treatment is quick and effective, long-term use by patients will cause dependence and tolerance. Once the drug is stopped, adverse reactions such as aggravating insomnia may occur. In view of the many problems existing in traditional treatment methods, it is an urgent problem to find an intervention or treatment method with less side effects and good effect.

Growing evidence suggests that the interplay between gut microbes, gut and central nervous system (CNS) operates through the microbiome-gut-brain axis. Microbes regulate brain function mainly through immune regulation, neuroendocrine and vagal pathways. Gut microbes can use ingested food as a substrate to form neurochemicals, or respond to neuroactive food components themselves, which are then absorbed from the gut into the portal circulation, or can interact directly with receptors in the enteric nervous system, ultimately affecting the brain . Animal studies now show that gut microbes can activate the vagus nerve, and this activation plays a crucial role in modulating effects on the brain and behavior. Even in the absence of overt inflammation, the vagus nerve appears to discriminate between non-pathogenic and potentially pathogenic bacteria, and these signals can induce anxiety and anxiolytic effects depending on the stimulus. Certain vagal signals from the gut can trigger anti-inflammatory reflexes in the brain, releasing mediators (eg, acetylcholine) that attenuate inflammation through interactions with immune cells. This immunomodulatory role of the vagus nerve may also have implications for the regulation of brain function and mood. So harnessing the interaction between gut microbes and the brain to improve sleep brings us a new therapeutic strategy for intervention.

But due to the presence of digestive juices in the gastrointestinal tract, many lactic acid bacteria are already dead by the time they reach the intestine, unable to play their own role. Therefore, the premise of lactic acid bacteria to exert its probiotic function is that it has excellent acid resistance and bile salt resistance, can survive after passing through the gastrointestinal tract, and has strong cell adhesion ability, which can play a role in intestinal colonization.

At present, some patent documents relate to strains or compositions that improve sleep. For example, patent CN102960447A discloses a nutritional composition containing probiotics and improving sleep patterns, which can be used to reduce sleep disturbances and improve sleep in humans or animals; patent US6444203 uses The combination of Lactobacillus acidophilus, Lactobacillus helveticus, Streptococcus thermophilus increases the duration of non-rapid eye movement (NREM) sleep and reduces the duration of rapid eye movement (REM) sleep; patent EP2438821 discloses a sleep-improving Probiotic strains or nutritional compositions that quality and induce more mature sleep patterns, which have the function of reducing REM sleep time, increasing non-REM sleep time and reducing the number of awakening episodes. However, the above-mentioned patent does not deal with the acid resistance and bile salt tolerance and colonization ability of lactic acid bacteria, while probiotic bacteria can tolerate the acid and bile salt environment of the gastrointestinal tract and colonize the intestinal tract, which are the necessary prerequisites for probiotic bacteria to function in the body. Therefore, it is necessary to develop a lactic acid bacteria with excellent simulating gastrointestinal tract survival ability and cell adhesion ability and explore the effect of lactic acid bacteria in improving sleep, so as to further explore the function of probiotic bacteria and develop lactic acid bacteria with higher health care value. Using dietary strategies to improve sleep opens up new avenues and solutions.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a kind of Lactobacillus plantarum GDMCCNo.60604.

The present invention as described above relates to a kind of lactic acid bacteria, and utilizes the microbiological characteristics such as morphological characteristics, culture characters and physiological and biochemical characteristics to identify this lactic acid bacteria as Lactobacillus plantarum GDMCCNo.60604, and this bacterial strain has been preserved in Guangdong microbial strains Central deposit, its deposit number is GDMCC No.60604.

Lactobacillus plantarum of the present invention has the following characteristics:

1. Morphological characteristics: Gram-positive, rod-shaped cells, the cells are about 0.5-1.0 μm wide, 2-4 μm long, single, paired or chained, no spores are formed, and both ends are rounded;

2. Cultivation traits:

Form obvious colonies on MRS medium, the diameter is between 0.3~2.0mm, round, with neat edges, milky white, opaque, moist and smooth surface, no pigment;

3. Physiological and biochemical characteristics:

Can utilize arabinose, cellobiose, fructose, lactose, galactose, raffinose, sorbose, trehalose, sucrose; contact enzyme, oxidase, nitrate reduction, gelatin liquefaction, hydrogen sulfide, motility are all negative; It has excellent simulated gastrointestinal tract survival ability and cell adhesion ability;

4. The effect of gavage in mice:

The sleep time induced by sodium pentobarbital can be significantly prolonged by 140.60%, and the sleep latency induced by sodium barbital can be significantly shortened by 21.31%.

The second object of the present invention is to provide a starter containing the Lactobacillus plantarum GDMCC No.60604.

In one embodiment, the starter is a powder prepared by using the bacterial liquid containing the Lactobacillus plantarum GDMCC No.60604, which contains more than 10 ⁶ CFU/g of active Lactobacillus plantarum GDMCC No.60604.

In one embodiment, the powder is obtained by preparing the bacterial liquid containing the Lactobacillus plantarum GDMCC No. 60604 through a conventional freeze-drying process or other processes.

In one embodiment, the starter is obtained by the following preparation steps:

A. Preparation of medium: 10% enzymatically hydrolyzed skim milk, 0.5% glucose, 1.5% tryptone and 0.3% yeast extract were dissolved in 87.7% water based on the total weight of the medium, and then adjusted to pH 6.8 , so obtain described culture medium;

B, the preparation of protective agent: use water and protective agent raw material to mix and prepare to contain 100g/L skimmed milk powder, 30mL/L glycerol, 100g/L maltodextrin, 150g/L trehalose, 10g/L L-sodium glutamate protective agent;

C. Inoculate the Lactobacillus plantarum GDMCC No.60604 strain according to the inoculum amount of 2 to 4% by weight of the medium into the medium sterilized for 8 to 12 minutes at a temperature of 110 to 120° C. Incubate at 37°C for 18h, wash with pH 7.2 phosphate buffer for 2 to 4 times, and resuspend with the protective agent to a concentration of 10 ¹⁰ CFU/mL; then, let the suspension at a temperature of 37°C Pre-cultured for 60 min under conditions, and then freeze-dried to obtain the starter.

In one embodiment, the starter also contains food-usable microorganisms. Optionally, it also contains one or more of Lactobacillus acidfast, Lactobacillus brevis, and Lactobacillus bulgaricus.

The third object of the present invention is to provide the application of the Lactobacillus plantarum GDMCC No.60604.

Optionally, the application is for the preparation of fermented foods. The fermented food is produced by using the Lactobacillus plantarum GDMCC No. 60604 of the present invention or the starter containing the strain of Lactobacillus plantarum GDMCC No. 60604.

In one embodiment, the fermented food is a dairy product, a soy product or a fruit and vegetable product. Optionally, the dairy product is milk, sour cream or cheese. Optionally, the soy product is soy milk, tempeh or soybean paste. Optionally, the fruit and vegetable products are cucumber, carrot, beet, celery or cabbage products.

Optionally, the application is for preparing a pharmaceutical composition with a sleep-improving function.

In one embodiment, the pharmaceutical composition is composed of Lactobacillus plantarum GDMCC No. 60604 bacterial agent and a pharmaceutically acceptable carrier.

In one embodiment, the pharmaceutically acceptable carrier is one or more selected from fillers, binders, wetting agents, disintegrants, lubricants or flavoring agents commonly used in pharmacy vector. Optionally, the pharmaceutical composition is in the form of granules, capsules, tablets, pills or oral liquids.

The beneficial effects and advantages of the present invention are as follows:

The Lactobacillus plantarum GDMCC No. 60604 of the present invention has excellent ability to simulate gastrointestinal tract survival and cell adhesion; can significantly prolong the sleep time induced by sodium pentobarbital (the prolongation ratio is 140.60%), shorten the barbiturate Sodium-induced sleep latency (21.31% shortened). The Lactobacillus plantarum GDMCC No. 60604 is used for preparing sleep-improving pharmaceutical compositions and fermented foods, and has very broad application prospects.

biological material preservation

Lactobacillus plantarum, classified as Lactobacillus plantarum, has been preserved in the Guangdong Provincial Microbial Culture Collection Center on March 12, 2019. The preservation address is 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou City, and the preservation number is For GDMCC No: 60604.

Description of drawings

Fig. 1 is the effect of test substance on pentobarbital sodium-induced sleep time experiment.

Figure 2 is the effect of the test substance on the barbital sodium-induced sleep latency experiment.

(Note: *p<0.05, **p<0.01)

Detailed ways

The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and biological materials can be obtained from commercial sources unless otherwise specified.

The screening of embodiment 1 Lactobacillus plantarum

1 Experimental method

The strains were screened with fresh human feces and kimchi water as samples.

(1) Draw 0.5 mL of the sample into 5 mL of MRS medium, and culture at 37°C for 18 to 24 hours for enrichment.

(2) Gradient dilution: draw 0.5 mL of the enriched sample into 4.5 mL of sterile normal saline to obtain 10 ^-1 dilution, then draw 0.5 mL of 10 ^-1 dilution into 4.5 mL of normal saline to obtain 10 ^-2 dilution According to this operation, 10 ^-3 , 10 ^-4 , 10 ^-5 , and 10 ^-6 dilutions were obtained in turn.

(3) Plate culture: draw 100 μL of gradient dilution solution and spread it on MRS plates, 1 plate for each gradient of 10 ^-4 , 10 ^-5 , and 10 ^-6 ; culture at 37° C. for 48 hours.

(4) Streak separation: Select colonies with typical characteristics on the coating plate according to the shape, size, edge, transparency, etc. of the colonies, pick the colonies with an inoculation loop and streak them on the selective medium plate, and cultivate at 37°C for 48h.

(5) To obtain a purified single colony, pick a single colony and inoculate it into a corresponding 5 mL liquid medium, and cultivate for 18-24 hours. Each strain was numbered, and experiments such as strain identification, Gram staining, physiology and biochemistry were carried out.

Among them, the formula of MRS medium is: add 10g peptone, 10g beef extract, 20g glucose, 5g yeast extract, 2g anhydrous sodium acetate, 0.25g manganese sulfate monohydrate, 1mL Tween 80, 2.6g trihydrate to 1L distilled water Hydrated dipotassium hydrogen phosphate, 0.5g magnesium sulfate heptahydrate, 2g diammonium citrate, pH 6.2～6.5.

2 Experimental results

The isolated strains were subjected to PCR amplification of 16S rDNA, and the PCR products were sent to BGI Sequencing Co., Ltd. for sequencing. DP1-12, HY6-2, HY8-2, HY9-1, DL3-1, GDMCC from different sample sources The identification result of No.60604 (the number after being preserved in the Guangdong Provincial Microbial Culture Collection Center) is Lactobacillus plantarum, wherein the characteristics of Lactobacillus plantarum GDMCC No.60604 are:

1. Morphological characteristics:

Gram-positive, rod-shaped cells, about 0.5-1.0 μm wide, 2-4 μm long, single, paired or chained, no spores formed, rounded at both ends.

2. Cultivation traits:

On the MRS medium, obvious colonies were formed, the diameter was between 0.3 and 2.0 mm, round, with neat edges, milky white, opaque, moist and smooth surface, and no pigment was produced.

3. Physiological and biochemical characteristics:

Can utilize arabinose, cellobiose, fructose, lactose, galactose, raffinose, sorbose, trehalose, sucrose; contact enzyme, oxidase, nitrate reduction, gelatin liquefaction, hydrogen sulfide, and motility are all negative.

Example 2 Exploration on the survival ability of different Lactobacillus plantarum to simulate the gastrointestinal tract

1 Test method

(1) Configuration of simulated gastric juice: Dissolve pepsin in sterilized physiological saline (0.9% w/v, adjusted to pH 3.0 with hydrochloric acid) to make the final concentration 3 g/L. Filtered with 0.22μm sterile filter membrane, ready-to-use.

(2) Configuration of simulated intestinal juice: Dissolve trypsin in sterilized normal saline (0.9% w/v, adjusted to pH 8.0 with NaOH) to make the final concentration 1 g/L, and add bile salt to make the final concentration 0.3 %. Filtered with 0.22μm sterile filter membrane, ready-to-use.

(3) Different Lactobacillus plantarum were continuously activated for three generations (18h each time) to carry out the experiment. Measure the OD value of the strains continuously activated for three generations under A ₆₀₀ , and calculate the amount of bacterial liquid required for OD 5.

(4) Centrifuge the OD 5 bacterial solution at 8000 × g for 10 min, discard the supernatant, resuspend in 1 mL of simulated gastric juice, and culture the plate at 37 °C for 3 h to count the viable bacteria on the plate.

(5) Centrifuge the bacterial liquid treated with simulated gastric juice at 8000 × g for 10 min, discard the supernatant, resuspend in an equal volume of simulated intestinal juice, and culture for 4 hours at 37°C to count the viable bacteria on the plate.

Wherein, survival rate (%) after tolerance to gastric juice=(number of viable bacteria in bacterial liquid after tolerance to simulated gastric juice/original viable number of bacteria in bacterial liquid)×100%. Survival rate (%) after tolerance to intestinal fluid = (number of viable bacteria in bacterial fluid after tolerance to simulated intestinal fluid/number of viable bacteria in bacterial fluid after tolerance to simulated gastric fluid)×100%. Survival rate (%) after tolerance to gastrointestinal fluid = (number of viable bacteria in bacterial solution after tolerance to simulated intestinal fluid/original viable number of bacteria in bacterial solution)×100%.

2 Experimental results

Table 1 Tolerability of different Lactobacillus plantarum in simulated gastrointestinal tract

The acid resistance of Lactobacillus plantarum was better after culturing in physiological saline containing pepsin at pH 3 for 3 hours, among which Lactobacillus plantarum DP1-12, Lactobacillus plantarum HY9-1 and Lactobacillus plantarum GDMCC No.60604 survived The rate was higher, 96.01±2.45%, 95.17±3.24% and 95.92±3.35%, respectively. Lactobacillus plantarum HY6-2 and Lactobacillus plantarum GDMCC No.60604 had higher survival rates of 10.50±1.04% and 16.04±1.61%, respectively, after 4 h incubation in normal saline containing trypsin and bile salts at pH 8 . After culturing in physiological saline containing pepsin at pH 3 for 3 hours, and continuing to culture in physiological saline containing trypsin and bile salts at pH 8 for 4 hours, Lactobacillus plantarum GDMCC No.60604 had the highest survival rate of 15.36 ±1.12%. It shows that Lactobacillus plantarum GDMCC No.60604 has excellent gastrointestinal tolerance and has the potential to exert probiotic functions in the intestinal tract.

Example 3 In vitro adhesion ability of different Lactobacillus plantarum to HT-29 cells

1 Test method

Normal HT-29 cells were cultured and passaged, and different Lactobacillus plantarum were continuously activated for 3 passages. The cultured cells were collected, counted with a hemocytometer, and resuspended to 2×10 ⁵ cells/mL with 1640 medium (supplemented with double antibody and fetal bovine serum). Add acid-washed and sterilized coverslips to 6-well plates. Inoculate 2 mL of cell suspension, and wash 3 times with sterile PBS after the cells adhere to the coverslip (about 12 h). Take a certain amount of bacterial liquid to collect the bacterial cells by centrifugation, wash the bacterial cells with sterile PBS once and then centrifuge to collect the bacterial cells again, and resuspend to a bacterial concentration of 10 ⁸ CFU/ mL. Add 2 mL of 1640 medium bacterial suspension to each well of the 6-well plate and incubate for 2 h. After the incubation, the culture medium was discarded and washed 3-6 times with sterile PBS. After cleaning, 2 mL of methanol was added to each well for 1 h at room temperature. After fixation, methanol was discarded, Gram (crystal violet) staining, and observation under a microscope.

2 Experimental results

Table 2 In vitro adhesion ability of different Lactobacillus plantarum to HT-29 cells

After the in vitro adhesion test of HT-29 cells, the microscopic observation results are shown in Table 2. It was found that the cell adhesion number of Lactobacillus plantarum HY6-2 was 22.67±2.08 cells/cell, and the cells of Lactobacillus plantarum GDMCC No.60604 The number of adherents was 27.67±3.06/cell, which was at a high level. The better cell adhesion ability of these two strains is conducive to their colonization in the intestinal tract and exert their own beneficial functions.

The preparation of embodiment 4 a kind of Lactobacillus plantarum freeze-dried bacteria powder

(1) Strain activation

Lactobacillus plantarum was inoculated into MRS medium with 2% inoculum, and cultured at 37°C for 18 hours to obtain activated Lactobacillus plantarum strains;

(2) Preparation of freeze-dried bacteria powder

The above-mentioned activated strains were inoculated into MRS medium at 2% of the inoculum. After culturing for 18 hours at 37°C, the supernatant was discarded after centrifugation at 8000 × g for 10 minutes, and the bacteria were collected after washing with sterile saline for 3 times. The collected bacteria are resuspended with a protective agent, freeze-dried to obtain freeze-dried bacteria powder, and the protective agent can be skimmed milk powder, trehalose or sucrose, etc.

Example 5 Evaluation of sleep improvement effect of Lactobacillus plantarum GDMCC No.60604

1 Design of animal experiments

80 healthy 6-week-old male ICR mice of 20-25 g were randomly divided into 8 groups: blank control group, positive control group (diazepam group), Lactobacillus plantarum DP1-12 group, Lactobacillus plantarum HY6-2 group, Lactobacillus plantarum HY8-2 group, Lactobacillus plantarum HY9-1 group, Lactobacillus plantarum DL3-1 group, Lactobacillus plantarum GDMCC No.60604 treatment group. The blank control group was gavaged with sterile saline daily; the Lactobacillus plantarum group was gavaged with the Lactobacillus plantarum skim milk suspension prepared in Example 4 of this specification at a concentration of 3×10 ⁹ CFU/mL every day, and the positive control group was gavaged with 3 mg/mL Lactobacillus plantarum skim milk suspension. kg.bw of diazepam solution. The corresponding doses of the test substances were administered orally to the mice once a day, and the mice were given 20 mL/kg.bw by gavage for 30 days. The sleep improvement experiment was carried out with reference to the standard part of improving sleep function evaluation in "Technical Specifications for Inspection and Evaluation of Health Foods (2003 Edition)".

1.1 Prolonged pentobarbital sodium sleep time experiment

Pre-experiment was carried out before the formal experiment, and the sodium pentobarbital dose (30-60 mg/kg.bw), which made the animals fall asleep 100%, but did not sleep too long, was used for the formal experiment. 30 minutes after the last sample administration, the animals in each group were intraperitoneally injected with sodium pentobarbital, the injection volume was 0.2 mL/20 g, and the disappearance of righting reflex was used as an indicator to observe whether the test samples could prolong the sleep time of sodium pentobarbital. The experimental results are listed in Figure 1.

1.2 Pentobarbital sodium subthreshold hypnosis experiment

Before the formal experiment, conduct a preliminary experiment to determine the subthreshold hypnotic dose of pentobarbital sodium (16-30 mg/kg.bw), that is, the maximum subthreshold dose of pentobarbital sodium that does not disappear in 80-90% of mice . 30 minutes after the last sample administration, animals in each group were intraperitoneally injected with the maximum subthreshold hypnotic dose of sodium pentobarbital, and the number of animals that fell asleep within 30 minutes (those whose righting reflex disappeared for more than 1 minute) was recorded. The experiments were carried out in a quiet environment at 24-25°C.

1.3 Barbital sodium sleep latency experiment

The pre-experiment was carried out before the formal experiment to determine the dose of sodium barbital (200-300 mg/kg.bw) that made the animals fall asleep 100%, but did not make the sleep time too long, and used this dose for the formal experiment. 30 minutes after the last administration of the sample, the animals in each group were intraperitoneally injected with sodium barbiturate, the injection volume was 0.2 mL/20g, and the disappearance of righting reflex was used as an indicator to observe the effect of the test substance on the sleep latency of sodium barbital. The experimental results are listed in Figure 2.

2 Experimental results

2.1 The experiment of prolonging the sleep time of sodium pentobarbital Through the pre-experiment, the intraperitoneal injection dose of sodium pentobarbital was determined to be 49 mg/kg.bw. As shown in Figure 1, compared with the blank group, diazepam (3 mg/kg.bw) as a positive control group significantly prolonged the duration of pentobarbital sodium-induced sleep. Compared with the blank group, the sleep time of mice fed with Lactobacillus plantarum HY6-2 and Lactobacillus plantarum GDMCCNo.60604 was significantly prolonged, and the prolongation ratio was 65.33% and 140.60%, indicating that Lactobacillus plantarum HY6-2 and Lactobacillus plantarum GDMCC No.60604 can effectively increase the sleep time of mice and has the effect of improving sleep.

2.2 Pentobarbital sodium subthreshold hypnosis experiment

Table 3 Effects of test substances on pentobarbital sodium-induced sleep rate in mice

The experimental results are shown in Table 3. After intraperitoneal injection of pentobarbital sodium (33 mg/kg.bw) into mice, the sleep rate of mice in the positive control group was 50%, which was significantly different from that in the blank group. The sleep rate of mice fed with Lactobacillus plantarum HY9-1 and Lactobacillus plantarum GDMCCNo.60604 was 30%, which was significantly improved compared with the blank group, but there was no statistical difference.

2.3 Barbital sodium sleep latency experiment

The dose of barbital sodium intraperitoneal injection was determined to be 320 mg/kg.bw by pre-experiment. As shown in Figure 2, compared with the blank group, diazepam (3 mg/kg.bw) as a positive control group significantly shortened the latency of sodium barbital-induced sleep. The sleep latency of mice in each group fed with Lactobacillus plantarum decreased, and the Lactobacillus plantarum GDMCC No.60604 group was significantly different from the blank group, indicating that Lactobacillus plantarum GDMCC No.60604 could shorten the sleep latency of mice. The shortening ratio is 21.31%, making it fall asleep faster, with the effect of improving sleep.

To sum up, according to the requirements of the "Technical Specifications for Inspection and Evaluation of Health Foods (2003 Edition)" to improve sleep function evaluation specifications, the pentobarbital sodium sleep time extension experiment, pentobarbital sodium subthreshold hypnosis experiment, barbital sodium Two of the three experiments of the sodium sleep latency test were positive, and there was no obvious direct sleep effect. It can be determined that Lactobacillus plantarum GDMCC No.60604 has the effect of improving sleep function.

Example 6: Application of Lactobacillus plantarum GDMCC No.60604

(1) Production of Lactobacillus milk beverage using Lactobacillus plantarum GDMCC No.60604

The raw milk skimmed milk was heat sterilized at 95°C for 20min, then cooled to 4°C, and then added Lactobacillus plantarum GDMCCNo.60604 working starter to make its concentration reach 10 ⁶ CFU/mL or more, and stored at 4°C to obtain plant-containing Lactobacillus GDMCC No.60604 live bacteria whole milk drink.

(2) Manufacture of soybean milk using Lactobacillus plantarum GDMCC No.60604

Soak the soybeans in soft water, the water volume is three times the original soybean volume, soak at a temperature of 80 ℃ for 1-2 hours, and then remove the soybean hulls. Next, drain the soaking water, add boiling water to refine the slurry, and keep the temperature higher than 80° C. for 10 to 15 minutes. The slurry is filtered with a 150-mesh filter membrane and then centrifuged, and the obtained centrifuge is crude soy milk, which is then heated to a temperature of 140-150 ° C, and then the hot crude soy milk is quickly introduced into a vacuum cooling chamber for vacuuming. The odorous substances in soy milk are quickly discharged with the water vapor. After vacuum degassing, the temperature was lowered to about 37°C, and then the working starter of Lactobacillus plantarum GDMCCNo.60604 was added to make its concentration reach 10 ⁶ CFU/mL or more, and the Lactobacillus plantarum containing Lactobacillus plantarum was obtained by refrigerated storage at 4°C. GDMCC No.60604 Soymilk with live bacteria.

(3) Using Lactobacillus plantarum GDMCC No.60604 to manufacture fruit and vegetable beverages

The fresh vegetables are washed and squeezed, followed by high-temperature instant sterilization. After high-temperature heat sterilization at a temperature of 140°C for 2s, the temperature is immediately lowered to about 37°C, and then the Lactobacillus plantarum GDMCC No.60604 starter is added to make the concentration The fruit and vegetable beverage containing Lactobacillus plantarum GDMCC No. 60604 viable bacteria can be obtained when it reaches 10 ⁶ CFU/mL or more, and is refrigerated and stored at 4°C.

(4) Using Lactobacillus plantarum GDMCC No.60604 to make capsule products

Lactobacillus plantarum GDMCC No.60604 was cultured on MRS medium for 24h, centrifuged for 20min at a temperature of 4°C and 4000r/min, rinsed twice with PBS, and then added to finally obtain a solution containing Lactobacillus plantarum GDMCC No.60604. Mix 4% skimmed milk powder and 6% lactose by weight for 10 minutes, then add sterile 2% calcium chloride and 3% sodium alginate, stir at 150 r/min for 10 minutes, and then statically solidify for 30 minutes, and finally wash and filter to obtain the filtrate. Carry out freeze-drying for 20h to obtain a powder containing Lactobacillus plantarum GDMCC No.60604, which is loaded into currently marketed medicinal microcapsules to obtain the capsule product.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone who is familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, The protection scope of the present invention should be defined by the claims.

Claims (12)

1. a Lactobacillus plantarum GDMCC No.60604, is characterized in that, described bacterial strain has been preserved in Guangdong Province Microorganism Culture Collection Center on March 12, 2019, and preservation address is No. 100, Xianlie Middle Road, Guangzhou The 5th floor of Building 59 of the compound, the preservation number is GDMCC No: 60604. 2 . A starter, characterized in that it contains the Lactobacillus plantarum GDMCC No.60604 of claim 1 . 3 . 3. starter according to claim 2, is characterized in that, described starter is the powder prepared by utilizing the bacterial liquid containing described Lactobacillus plantarum GDMCC No.60604, and it contains more than 10 ⁶ CFU/g Active Lactobacillus plantarum GDMCC No. 60604. 4. The starter according to any one of claims 2-3, wherein the starter contains one or more of Lactobacillus acidfast, Lactobacillus brevis, and Lactobacillus bulgaricus. 5. Lactobacillus plantarum GDMCC No.60604 according to claim 1, or the application of the starter according to claim 2 in the preparation of fermented food. 6. The application according to claim 5, wherein the fermented food is a dairy product, a soy product or a fruit and vegetable product. 7. The application according to claim 6, wherein the dairy product is milk, sour cream or cheese. 8. The application according to claim 6, wherein the soybean product is soybean milk, tempeh or soybean paste. 9. The application according to claim 6, wherein the fruit and vegetable products are cucumber, carrot, beet, celery or cabbage products. 10. The application of Lactobacillus plantarum GDMCC No. 60604 according to claim 1 in the preparation of a pharmaceutical composition with improved sleep function. The application according to claim 10, wherein the pharmaceutical composition is composed of Lactobacillus plantarum GDMCC No. 60604 inoculum and a pharmaceutically acceptable carrier. 12. The application according to claim 10 or 11, wherein the pharmaceutically acceptable carrier is one or more selected from fillers, binders, wetting agents, disintegrating agents commonly used in pharmacy The carrier of dissolving agent, lubricant or flavoring agent; the pharmaceutical composition is in the form of granule, capsule, tablet, pill or oral liquid.

Images