A preparation method of probiotics and application in treating insomnia

Abstract

The present invention provides a preparation method of probiotics and application thereof in treating insomnia. More specifically, a polypeptide composition containing probiotics is provided, and the composition can effectively treat insomnia symptoms and has good application value.

Description

Technical Field

[0001] The present application relates to the biological field, and more specifically to a preparation method of probiotics and application of the probiotics in treating insomnia. Background Art

[0002] With the acceleration of modernization, people are facing increasing pressure and competitiveness, and more and more people are suffering from insomnia. According to statistics, the incidence of insomnia among adults in my country is 9% to 12%, and the incidence among the elderly is 30% to 40%. Insomnia seriously affects human physical and mental health and quality of life. How to effectively treat insomnia has become an important research direction for major pharmaceutical companies and research institutions.

[0003] The human microbiome is a general term for the trillions of microorganisms that reside in and on the body, such as bacteria, archaea, eukaryotes, fungi, viruses and their genes. There is about 1kg of bacteria in an adult, which is approximately equal to the mass of the human brain. The human intestine contains a large number of microorganisms, the total number of which is about 10 times the total number of human cells. The genes contained in these microorganisms are 100 times the number of human coding genes, and are called the "second genome" of the human body. Among them, Bacteroidetes and Firmicutes are the most common intestinal bacterial genera, accounting for about 3 / 4 of the entire flora. There are individual differences in the types and distribution of intestinal microorganisms, and the types and distribution of flora in the same individual at different stages of growth are also different. In addition, factors such as diet, exercise, drugs, stress, and environment can all affect the intestinal flora. The intestinal flora has been shown to regulate the health of the body and brain function in a variety of ways. Studies have shown that intestinal microorganisms can affect host health by participating in food digestion and decomposition, regulating bile acid metabolism, resisting pathogen invasion, and participating in immune response. In recent years, a large number of studies have confirmed that the intestinal flora is related to sleep. The study found that disrupting the sleep pattern of mice can change the structure and diversity of their intestinal flora, and similar findings were obtained in shift workers who were sleep-deprived. The abundance of some bacterial genera in the feces of children with autism who suffer from sleep disorders decreased, the levels of 3-hydroxybutyrate and melatonin decreased, and the levels of serotonin increased. These changes, in turn, may aggravate the sleep problems and autism of children with autism. Regulating the intestinal flora has been shown to prolong the sleep time and improve the quality of sleep in patients with insomnia. γ-Aminobutyric acid (GABA) is a common inhibitory neurotransmitter in the brain. Its main function is to maintain the balance of excitation and inhibition in the brain. If it is not synthesized enough, it will cause insufficient inhibition in the brain and insomnia. GABA is strongly associated with complex nervous system functions and memory and sleep-related factors. There are also studies that treat insomnia by giving probiotics to increase the production of GABA.

[0004] At present, the drugs used in clinical treatment of insomnia mainly include benzodiazepine receptor (γ-aminobutyric acid A receptor) agonists (such as diazepam, etc.), non-benzodiazepine sedatives (such as zolpidem, etc.), melatonin receptor agonists (such as agomelatine, etc.) and antidepressants with hypnotic effects (such as mirtazapine, etc.). In 2014, the US FDA approved suvorexant (trade name Belsomra) developed by Merck for the treatment of insomnia, making it the first orexin receptor antagonist on the market. The significance of suvorexant in clinical use is that it is a drug specifically used to treat insomnia. This type of drug has a completely new target and mechanism of action. Orexin-1 receptor (OX1R) and orexin-2 receptor (OX2R), as well as two neurotransmitter peptide agonists: orexin-A (OX-A) and orexin-B (OX-B). OX-A and OX-B, as neurotransmitter peptide agonists, are produced in the hypothalamus. OX-A is a polypeptide with a sequence of 33 amino acids, which can activate OX1R and OX2R with the same intensity; while OX-B is a polypeptide with a sequence of 28 amino acids, which can only selectively activate OX2R to a moderate degree. Both orexin receptors are widely expressed throughout the central nervous system. The loss of OX2R function is inevitably linked to narcolepsy, which has led people to transition their understanding of insomnia from sleep disorders to the level of symptoms, that is, insomnia is also a disease. Many subsequent studies have shown that the influence of the orexin system extends to sleep, addiction, appetite, stress, anxiety, pain, and reward. The orexin system is widely present in mammals, and its structural and functional commonalities make it possible to develop drugs using this system as a target. Orexin can keep people awake. If the drug can competitively block the binding of orexin and orexin receptors, it can make people fall asleep as soon as possible, and can be used to treat insomnia and sleep disorders.

[0005] At present, effective drugs for treating insomnia are either expensive or have complex ingredients and mediocre therapeutic effects, and their value in promotion and application needs to be improved. Summary of the invention

[0006] In one aspect, the present invention provides a preparation method of probiotics and application of the probiotics in treating insomnia.

[0007] More specifically, the present invention also provides a probiotic composition, which is prepared by mixing freeze-dried powder of Lactobacillus casei CICC6104 and Lactobacillus plantarum CICC 22708 in a ratio of 3:1.

[0008] Specifically, a method for preparing a probiotic composition is also provided, comprising mixing freeze-dried powders of Lactobacillus casei CICC6104 and Lactobacillus plantarum CICC 22708 in a ratio of 3:1 to prepare the probiotic composition.

[0009] Furthermore, the present invention also provides a polypeptide SM-18S targeting orexin-1 receptor (OX1R) and orexin-2 receptor (OX2R), the amino acid sequence of the polypeptide being shown in SEQ ID NO:1.

[0010] Specifically, according to the report of Nanjing Ruiyuan Biological, the polypeptide SM-18S is water-soluble, and the toxicity experiment confirmed that the polypeptide has no biological toxicity. The overdose experiment of mice with 100 mg / mouse / day for 30 consecutive days proved that there was no damage to the mouse organs.

[0011] Specifically, the present invention also provides a polypeptide probiotic composition for treating insomnia, which is prepared by mixing freeze-dried powder of Lactobacillus casei CICC6104 and Lactobacillus plantarum CICC 22708 at a live bacteria count of 3:1 to obtain a probiotic composition; further mixing the mixed probiotic composition with SM-18S polypeptide at a mass ratio of 50:1 to obtain a polypeptide probiotic composition.

[0012] The present invention also provides use of a polypeptide probiotic composition in preparing a health product for improving sleep quality. The polypeptide probiotic composition is prepared by mixing freeze-dried powder of Lactobacillus casei CICC6104 and Lactobacillus plantarum CICC 22708 at a live bacterial count of 3:1 to obtain a probiotic composition; and further mixing the mixed probiotic composition with an SM-18S polypeptide at a mass ratio of 50:1 to obtain a polypeptide probiotic composition.

[0013] Furthermore, the preparation method of the freeze-dried powder of the present invention is to mix and resuspend the bacteria with a freeze-dried protective agent (containing 100 g / L trehalose, 100 g / L skimmed milk powder, and the solvent is purified water) (the mass ratio of the freeze-dried protective agent to the bacteria is 2:1) to obtain a resuspension; and freeze-dry the resuspension by a vacuum freezing method to obtain a freeze-dried powder.

[0014] Furthermore, the composition of the present invention may be an oral preparation.

[0015] Further, for oral, buccal and sublingual administration, acceptable solid dosage forms include: powder, suspension, granule, tablet, pill, capsule, soft capsule and capsule. Its preparation method is, for example, mixing one or more compounds of the present invention, or pharmaceutically acceptable salt or its tautomer, adding at least one additive, such as starch or other additives. Suitable additives include: sucrose, lactose, cellulose sugar, mannitol, maltitol, dextran, starch, agar, alginate, chitin, chitosan, pectin, tragacanth gum, gum arabic, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymer or glyceride. Alternatively, oral dosage forms may contain other components to assist administration, such as inert diluents, or lubricants such as magnesium stearate, or preservatives such as parabens or sorbic acid, or antioxidants such as ascorbic acid, vitamin E or cysteine, disintegrants, binders, thickeners, buffers, sweeteners, flavoring agents or aromatics. Tablets and pills may be further treated with suitable coating materials known in the art.

[0016] Furthermore, the composition of the present invention also includes a suitable carrier. Specifically, examples of suitable pharmaceutically acceptable excipients include one or more polymers, wetting agents or surfactants, pH regulators, isotonicity regulators, preservatives, buffers and chelating agents, or any combination thereof.

[0017] Examples of suitable pharmaceutically acceptable pH adjusting agents include, but are not limited to, sodium hydroxide, citric acid, hydrochloric acid, boric acid, acetic acid, phosphoric acid, succinic acid, sodium hydroxide, potassium hydroxide, ammonium hydroxide, magnesium oxide, calcium carbonate, magnesium carbonate, magnesium aluminum silicate, malic acid, potassium citrate, sodium citrate, sodium phosphate, lactic acid, gluconic acid, tartaric acid, 1,2,3,4-butanetetracarboxylic acid, fumaric acid, diethanolamine, monoethanolamine, sodium carbonate, sodium bicarbonate, triethanolamine, or any combination thereof. In one embodiment, the pharmaceutically acceptable pH adjusting agent is present in an amount of about 0.01% to about 2.0% (weight / volume), preferably about 0.05% to about 1% (weight / volume).

[0018] Furthermore, the present invention also provides a use of a polypeptide and a probiotic in preparing a health product for improving insomnia, wherein the health product is prepared by mixing freeze-dried powder of Lactobacillus casei CICC6104 and Lactobacillus plantarum CICC 22708 at a live bacterial count of 3:1 to obtain a probiotic composition; the mixed probiotic composition is further mixed with the SM-18S polypeptide according to claim 1 at a mass ratio of 50:1 to obtain a health product; the freeze-dried powder is prepared by resuspending the bacterial body with a freeze-dried protective agent: 100 g / L trehalose, 100 g / L skimmed milk powder, and a solvent of purified water, the mass ratio of the freeze-dried protective agent to the bacterial body being 2:1, to obtain a resuspension; and the resuspension is freeze-dried by a vacuum freezing method to obtain a freeze-dried powder.

[0019] Beneficial Effects

[0020] The present invention provides a preparation method of probiotics and application thereof in treating insomnia. More specifically, a polypeptide composition containing probiotics is provided, and the composition can effectively treat insomnia symptoms and has good application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Effects of each group on the total distance of open field exercise in rats DETAILED DESCRIPTION

[0022] Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described by preferred embodiments, and relevant personnel can obviously change or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and apply the technology of the present invention. The methods, equipment, and materials in the following implementation cases, if not specifically stated, are all conventional methods, equipment, and materials in the art, and can be purchased from the market.

[0023] As used herein, the terms "purified" or "isolated" in connection with a polypeptide or nucleic acid means that the polypeptide or nucleic acid is not in its native medium or native form. Thus, the term "isolated" includes a polypeptide or nucleic acid removed from its original environment, such as if it is naturally occurring, from its natural environment. For example, an isolated polypeptide is generally free of at least some of the proteins or other cellular components with which it is normally associated or with which it is normally mixed or in solution. Isolated polypeptides include naturally produced polypeptides contained in cell lysates, polypeptides in purified or partially purified form, recombinant polypeptides, polypeptides expressed or secreted by cells, and polypeptides in heterologous host cells or cultures.

[0024] Example 1 Preparation and efficacy verification of peptides specifically targeting orexin-1 receptor (OX1R) and orexin-2 receptor (OX2R)

[0025] Nanjing Ruiyuan Biotechnology was commissioned to screen and identify peptide libraries based on the receptor structures of OX1R and OX2R, and obtained SEQ ID NO: 1 and SEQ ID NO: 2 peptides with good receptor inhibition activity, named SM-18S and SM-43F, respectively. After the peptides were artificially synthesized, the orexin receptor antagonist activity of the peptides was determined by the following experimental methods known in the art.

[0026] Chinese hamster ovary (CHO) cells expressing human orexin-1 receptor and human orexin-2 receptor were cultured in medium containing 300 μg / ml G418, 100 U / ml penicillin, 100 μg / ml streptomycin and 10% heat-inactivated fetal calf serum (FCS). Cells were seeded at 20,000 cells / well on sterile plates. The seeded plates were incubated overnight at 37°C in 5% CO2. Human orexin-A as an agonist was prepared as a 1 mM stock solution in MeOH: water (1:1) and diluted to a final concentration of 3 nM in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g / l and 2 mM HEPES for assay. The peptide was prepared as a 10 mM stock solution. During the assay, 50 μl of staining buffer (HBSS containing 1% FCS, 2 mM HEPES, NaHCO3: 0.375 g / l, 5 mM probenecid (Sigma) and 3 μM fluorescent calcium indicator fluo-4AM (1 mM stock solution in DMSO with 10% pluronic acid)) was added to each microwell. The well plate was incubated at 37°C in 5% CO2 for 50 minutes and then equilibrated at room temperature for 30-120 minutes before measurement. In a fluorescent imaging plate reader (FLIPR2), a volume of 10 μl / microwell of polypeptide was added to the plate, incubated for 10 minutes and finally 10 μl / microwell of agonist was added. The fluorescence of each microwell was detected at intervals of 1 second, and the height of each fluorescence peak was compared with the height of the fluorescence peak induced by 3 nM orexin-A when the antagonist was replaced by the solvent. The IC50 value (the concentration of the compound required to inhibit 50% of the agonist response) of each polypeptide was detected. The results are shown in Table 1:

[0027] Table 1 shows the antagonistic activity of selected peptides

[0028] ;

[0029] As can be seen from Table 1, the screened SM-18S has good inhibitory activity against both OX1 and OX2, especially against OX1. SM-18S was selected for subsequent experiments.

[0030] Example 2 Preparation of probiotic composition

[0031] Lactobacillus casei CICC6104 was purchased from Beijing Yuwei Technology Co., Ltd. The CICC6104 strain was inoculated into skim milk and cultured at 37°C for 20 hours for activation to obtain an activated solution; the activated solution was inoculated into MRS liquid culture medium at an inoculum rate of 3% (v / v), and cultured at 37°C for 24 hours to obtain a bacterial solution; the bacterial solution was centrifuged to obtain bacterial cells; the bacterial cells were mixed and resuspended with a freeze-drying protective agent (containing 100 g / L trehalose, 100 g / L skim milk powder, and the solvent was purified water) (the mass ratio of freeze-drying protective agent to bacterial cells was 2:1) to obtain a resuspension; the resuspension was freeze-dried by vacuum freezing to obtain freeze-dried powder of Lactobacillus casei CICC6104.

[0032] Lactobacillus plantarum CICC 22708 was purchased from Beijing Yuwei Technology Co., Ltd. The strain preservation tube was taken out, and the CICC 22708 strain was inoculated into skim milk and cultured at 37°C for 20 hours for activation to obtain an activated solution; the activated solution was inoculated into MRS liquid culture medium at an inoculum amount of 2.5% (v / v), and cultured at 37°C for 24 hours to obtain a bacterial solution; the bacterial solution was centrifuged to obtain bacterial cells; the bacterial cells were mixed and resuspended with a freeze-drying protective agent (containing 100 g / L trehalose and 100 g / L skim milk powder, and the solvent was purified water) (the mass ratio of freeze-drying protective agent to bacterial cells was 2:1) to obtain a resuspension; the resuspension was freeze-dried by vacuum freezing to obtain freeze-dried powder of Lactobacillus plantarum CICC 22708.

[0033] The freeze-dried powders of Lactobacillus casei CICC6104 and Lactobacillus plantarum CICC 22708 were mixed at a ratio of 3:1 of viable bacteria to prepare a probiotic composition.

[0034] Furthermore, the mixed freeze-dried powder composition was mixed with the SM-18S polypeptide at a mass ratio of 50:1 to obtain a polypeptide probiotic composition.

[0035] Example 3 Efficacy verification of probiotic composition

[0036] The present invention uses the PCPA method commonly used in the art to construct an insomnia model. DL-4-chlorophenylalanine (PCPA) is an inhibitor of tryptophan hydroxylase (TDH), and TDH is the rate-limiting enzyme for the synthesis of 5-HT (serotonin). After the PCPA-induced insomnia rat model is successfully replicated, the neurotransmitter 5-HT in the brain of the insomnia rat decreases significantly, resulting in the disappearance of the rat's circadian rhythm and insomnia in the rat.

[0037] After one week of acclimatization, rats in all groups except the normal control group were intraperitoneally injected with 300 mg / kg PCPA daily for 2 consecutive days to obtain the rat insomnia model. The normal control group was intraperitoneally injected with the same volume of weakly alkaline 0.9% sodium chloride injection as PCPA.

[0038] After the rat insomnia model was successfully replicated, the drug-administered groups were given samples by gavage at regular intervals every day; the specific drug-administered groups were SM-18S low-dose peptide group, SM-18S high-dose peptide group, probiotic composition group, peptide probiotic composition group, and positive control group. Among them, the low-dose peptide group was administered at a dose of 1 mg / kg·d; the high-dose peptide group was administered at a dose of 3 mg / kg·d; the probiotic composition group was administered at a dose of 3 mg / kg·d; the peptide probiotic composition group was administered at a dose of 3 mg / kg·d; the diazepam positive control group was administered at a dose of 3 mg / kg·d, and the model group was only given the same amount of saline as the positive control group. The drug was administered for 7 consecutive days, and fasting for 12 hours before the last dose (dose 7).

[0039] After the last administration of the above groups, the open field test was used to evaluate the anxiety of the rats in the new environment. The open field reaction box is 50 cm high and 50 cm long at the bottom. The inner area of ​​the open field is set as a 4×4 grid. The 4 middle grids are combined into the central area, and the outer grids are combined into the peripheral area. The open field is placed directly under the incandescent lamp, and the camera is adjusted to be directly above the open field. The open field experiment is carried out in a quiet environment. The experimenter puts the animal into the center of the bottom of the box at a uniform angle and then films and times it. The rat's activities are spontaneously recorded by the camera. Each rat is tested for 10 minutes, and the total distance of the rat's open field movement is recorded. The results are as follows: Figure 1 shown.

[0040] from Figure 1 It can be seen that the total distance traveled by rats in the insomnia model group was significantly different from that in the blank group (p<0.05), and the total distance traveled by rats in the positive control group and each treatment group was reduced compared with the model group, especially in the high-concentration peptide group and the peptide combined with probiotics group. This result suggests that each treatment group can improve the total distance traveled by rats with insomnia, thereby improving the anxiety of rats in the open field caused by insomnia.

[0041] Five mice were taken from each group of mice after the open field test, and a sleep model was established by intraperitoneal injection of sodium pentobarbital (45 mg / kg). Sodium pentobarbital was used to induce rats to sleep, and the animals were considered to be in a sleeping state when the righting reflex disappeared for more than 1 minute. The total time from intraperitoneal injection to the disappearance of the righting reflex was the sleep latency. The total time for the rats to recover from the righting reflex disappearance was the sleep time. The time for the righting reflex to disappear and recover was recorded for each group. The results are shown in Table 2:

[0042] Table 2 Results of sleep latency and sleep time in each group

[0043] ;

[0044] From the results in Table 2, it can be seen that the sleep latency of each experimental group of the present invention is shortened compared with the model control group, and the sleep time is also significantly prolonged, especially after the polypeptide combined with probiotics treatment, the effect is more significant. This shows that in addition to the polypeptide itself, the polypeptide combined with probiotics has a better effect in treating insomnia.

[0045] Take 5 mice from each group that have completed the open field test, take the hypothalamus of the rats on a low-temperature operating table, put them in a 5mL centrifuge tube, add tissue lysis solution at a ratio of 200μL tissue lysis solution per 10mg brain, homogenize them in a frozen bead mill, incubate them in a -20℃ refrigerator for 20min, freeze centrifuge for 20min (14000r / min, 4℃), filter them with a 0.22μm filter membrane, and use the conventional ultra-high performance liquid chromatography-tandem mass spectrometry in the field to determine the content of neurotransmitter components. The specific results are shown in Table 3:

[0046] Table 3 Effects of each group on neurotransmitters in the hypothalamus of insomnia model rats

[0047] ;

[0048] As can be seen from Table 3, compared with the model group, the inhibitory neurotransmitter GABA content in the rat hypothalamus in each treatment group and the positive control group was significantly increased (p<0.05), and the excitatory neurotransmitter NE content was significantly decreased (p<0.05); in addition, the 5-HT content was also significantly increased (p<0.05). The above results indicate that peptides and probiotics can achieve the sedative and hypnotic effect by regulating neurotransmitters in the rat hypothalamus.

[0049] At the same time, the organs of mice in each group were tested and no obvious toxic side effects were found, indicating that the polypeptide and probiotics of the present invention have no obvious toxic side effects.

[0050] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. Although embodiments of the present invention have been shown and described, those skilled in the art will appreciate that various changes, modifications, substitutions and variations may be made to these embodiments without departing from the principles and purpose of the present invention, and that the scope of the present invention is defined by the claims and their equivalents.

Claims

1. A polypeptide SM-18S targeting orexin-1 receptor and orexin-2 receptor, characterized in that The amino acid sequence of the polypeptide is shown in SEQ ID NO:

1.

2. Use of the polypeptide according to claim 1 in the preparation of a drug for treating insomnia.

3. A polypeptide probiotic composition for treating insomnia, characterized in that The composition is prepared by mixing freeze-dried powders of Lactobacillus casei CICC6104 and Lactobacillus plantarum CICC 22708 at a live bacterial count of 3:1 to obtain a probiotic composition; further mixing the mixed probiotic composition with the SM-18S polypeptide described in claim 1 at a mass ratio of 50:1 to obtain a polypeptide probiotic composition; the freeze-dried powder is prepared by resuspending bacterial bodies with a freeze-dried protective agent: 100 g / L trehalose, 100 g / L skimmed milk powder, and a solvent: purified water, wherein the mass ratio of the freeze-dried protective agent to the bacterial bodies is 2:1 to obtain a resuspended liquid; and freeze-drying the resuspended liquid by a vacuum freezing method to obtain a freeze-dried powder.

4. Use of polypeptides and probiotics in the preparation of health products for improving insomnia, wherein: The health care product is prepared by mixing freeze-dried powders of Lactobacillus casei CICC6104 and Lactobacillus plantarum CICC 22708 at a live bacterial count of 3:1 to obtain a probiotic composition; further mixing the mixed probiotic composition with the SM-18S polypeptide according to claim 1 at a mass ratio of 50:1 to obtain a health care product; the freeze-dried powder is prepared by resuspending the bacterial body with a freeze-drying protective agent: 100 g / L trehalose, 100 g / L skimmed milk powder, and a solvent of purified water, wherein the mass ratio of the freeze-drying protective agent to the bacterial body is 2:1 to obtain a resuspension; and freeze-drying the resuspension by using a vacuum freezing method to obtain a freeze-dried powder.

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