A probiotic composition, preparation method and application thereof in promoting sleep

Abstract

The present invention provides a probiotic composition, a preparation method and an application thereof in promoting sleep, comprising probiotics and a sleep-promoting active peptide, wherein the amino acid sequence of the active peptide is shown in SEQ ID NO: 1, and the probiotics are selected from lactobacillus and bifidobacterium. The composition can effectively improve sleep conditions, regulate neurotransmitter secretion, improve body immunity, and maintain internal environment stability.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology research and development, and specifically provides a probiotic composition, a preparation method and application thereof in promoting sleep. Background Art

[0002] Sleep is an important biological function that plays an important role in promoting physical recovery and repair, supporting neural development, enhancing immune and metabolic function, and improving cognition and mood. Therefore, adequate sleep time and sufficient quality sleep are essential for maintaining physical and mental health and overall quality of life (see Chattu VK, Manzar MD, Kumary S, et al. The global problem of insufficient sleep and its serious public health implications. Healthcare. 2018;7:1–16). Insufficient sleep increases the risk of chronic diseases, such as obesity, diabetes, heart disease, cancer, and mental illness (see Elder BL, Ammar EM, Pile D. Sleep duration, activity levels, and measures of obesity in adults. Public Health Nurs. 2016;33:200–5; Ferrie JE, Kivimaki M, Akbaraly TN, et al. Change in sleep duration and type 2 diabetes: the Whitehall II study. Diabetes Care. 2015;38:1467–72; Tobaldini E, Costantino G, Solbiati M, et al. Sleep, sleep deprivation, autonomic nervous system and cardiovascular diseases. Neurosci Biobehav Rev. 2017;74:321–9). Therefore, sleep disorders are increasingly recognized as a global public health problem, with typical symptoms including decreased ability to sleep and maintain sleep, increased stress, and even anxiety and depression, which seriously affect people's normal life and health.

[0003] Currently, a variety of therapies have been developed to treat sleep disorders, including non-drug strategies and drug treatments to improve sleep. Commonly used drugs are benzodiazepine compounds, such as diazepam, triazolam, flurazepam, oxazepam, clonazepam and lorazepam. Although these drugs can relieve insomnia symptoms, they have serious adverse reactions including addiction (see Krystal AD. A compendium of placebo-controlled trials of the risks / benefits of pharmacological treatments for insomnia: The empirical basis for US clinical practice. Sleep Med Rev.2009;13:265–74.). At the same time, non-drug strategies have slow effects and are difficult to meet the needs of patients.

[0004] Recent studies have shown that probiotics can relieve insomnia symptoms, inhibit sleep disorders, and improve sleep quality. Probiotics are defined as "live microorganisms that, when administered in adequate amounts, confer a health benefit on the host" (see Hill C, Guarner F, Reid G, et al. Expert consensus document. The international scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic. Nat Rev Gastroenterol Hepatol. 2014;11:506–514). The health benefits of probiotics include regulating immune responses, maintaining the intestinal barrier, antagonizing pathogen adhesion to host tissues, and producing different metabolites, such as vitamins, short-chain fatty acids (SCFAs), and molecules that act as neurotransmitters involved in gut-brain axis communication (see Duranti S, Ferrario C, van Sinderen D, et al. Obesity and microbiota: an example of an intricate relationship. Genes Nutr. 2017;12:18). It has been reported that supplementation with 4 × 10 9CFU / viable cells of fermentative lactic acid bacteria LF16, rhamnosus lactic acid bacteria LR06, plant lactic acid bacteria LP01 and Bifidobacterium longum BL04 can improve mood and sleep quality, reduce depressive mood, anger and fatigue (see Marotta A, Sarno E, Del Casale A, et al. Effects of probiotics on cognitive reactivity, mood, and sleep quality. Front Psychiatry. 2019;10:164). Other studies have shown that serum TNF-α concentrations in vivo after probiotic supplementation were significantly lower than those in the placebo group, and elevated TNF-α was associated with protein synthesis inhibition, sleep disorders and impaired muscle performance (see Main LC, Dawson B, Heel K, et al. Relationship between inflammatory cytokines and self-report measures of training overload. Res Sports Med (Print) 2010;18:127–139). However, probiotics also have the disadvantages of slow onset and unstable sleep-promoting effects.

[0005] Polypeptides have multiple physiological functions and are widely used in the field of sleep. For example, CN117482214B discloses a hydrolyzed casein peptide for improving insomnia in the middle-aged and elderly, and its preparation method and application. Casein is used as a raw material, and the hydrolyzed casein peptide is obtained by directional and controllable enzymatic hydrolysis, which has a significant sleep-promoting function; CN117964692A discloses a pentapeptide for people with sleep deprivation or sleep disorders. The pentapeptide is TPTLN, which can activate GABAA receptors, thereby playing a role in helping sleep. 5 mg / kg of TPTLN can significantly shorten the sleep latency of mice and prolong the sleep time of mice; CN117338905A discloses a hydrolyzed casein peptide with a sleep-promoting effect, and its preparation method and application. The obtained hydrolyzed casein peptide has the effects of prolonging the total sleep time, reducing the number of awakenings, increasing the serum melatonin content, and increasing the serum tryptophan content.

[0006] In order to overcome the defects of using probiotics alone to help sleep, the present invention is to develop a probiotic composition, a preparation method and its application in promoting sleep, which can effectively improve sleep conditions, regulate neurotransmitter secretion, improve body immunity and maintain internal environment stability. Summary of the invention

[0007] A first aspect of the present invention provides a sleep-promoting composition, characterized in that the composition comprises probiotics and a sleep-promoting active peptide, and the amino acid sequence of the active peptide is shown in SEQ ID NO: 1.

[0008] The composition includes probiotics that can regulate intestinal flora and the sleep system through the brain-gut circulation pathway, and also includes active peptides derived from casein that can combat insomnia symptoms, improve sleep quality, and ensure physical and mental health through different signal transduction mechanisms.

[0009] Furthermore, the probiotics are selected from at least one of Lactobacillus, Lactococcus and Bifidobacterium.

[0010] Furthermore, the probiotics are selected from lactobacilli and bifidobacteria.

[0011] Furthermore, the lactobacillus is Lactobacillus paracasei, and its preservation number is CGMCC 1.2884.

[0012] Furthermore, the bifidobacterium is Bifidobacterium longum, and its preservation number is CGMCC No.1.2186.

[0013] Furthermore, the ratio of Lactobacillus to Bifidobacterium is 1:10-10:1.

[0014] Furthermore, the ratio of Lactobacillus to Bifidobacterium is 1:1.

[0015] Furthermore, the viable counts of Lactobacillus and Bifidobacterium are 1.0×10 11 CFU / g and above.

[0016] The viable count may be 1.0×10 9 CFU / g-1.0×10 12 CFU / g, preferably 1.0×10 11 CFU / g.

[0017] Furthermore, the preparation method of the probiotics comprises: inoculating lactobacillus and bifidobacterium into MRS culture medium respectively, culturing to the logarithmic growth phase, and mixing them in equal proportions according to the number of live bacteria.

[0018] A second aspect of the present invention provides a use of the sleep-promoting composition according to any one of claims 1 to 9 in the preparation of a sleep-promoting health product.

[0019] Beneficial Effects

[0020] The present invention provides a probiotic composition, a preparation method and application thereof in promoting sleep, with the following beneficial effects:

[0021] 1. Screened and obtained new active peptides that can improve sleep conditions;

[0022] 2. Choose the best probiotic combination to overcome sleep disorders;

[0023] 3. The probiotics are used in combination with active peptides to effectively regulate sleep conditions and improve the expression levels of 5-HT, GABA, and TPH. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 : 5-HT expression level in brain tissue;

[0025] Figure 2 : GABA expression level in brain tissue;

[0026] Figure 3 : Expression levels of TPH in serum. DETAILED DESCRIPTION

[0027] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents, biological materials, and detection kits are all commercially available unless otherwise specified.

[0028] Example 1

[0029] 1.1 Obtaining sleep-promoting active peptides

[0030] Animal milk, such as human milk and cow's milk, has a strong sleep-promoting effect, but the composition is complex, the therapeutic effect of direct drinking is not good, and the source is greatly limited, which is not conducive to large-scale extraction and use. Hydrolyzed casein peptides are produced by controlled enzymatic hydrolysis of bovine casein, which has been shown to reduce anxiety and stress and improve insomnia (see Thiagarajah K, Chee HP, Sit WW. Effect of alpha-S1-casein tryptic hydrolysateand L-theanine on poor sleep quality: a double blind, randomized placebo-controlled crossover trial. Nurtrients, 2022;14(3):652). In the present invention, based on casein hydrolysate, a new active peptide with strong sleep-promoting function was screened, and its amino acid sequence is shown in SEQ ID NO: 1. The polypeptide is prepared by solid phase synthesis.

[0031] 1.2 Preparation of sleep-promoting composition

[0032] It is reported that a variety of probiotics have a promoting effect on sleep. In the preliminary experiments of the present invention, a variety of lactobacilli, lactococci and bifidobacteria were selected for sleep experimental tests, and it was found that the use of Lactobacillus paracasei (CGMCC 1.2884, purchased from the General Microbiology Center of China National Microbiological Culture Collection Committee) and Bifidobacterium longum (CGMCC No.1.2186, purchased from the General Microbiology Center of China National Microbiological Culture Collection Committee) had the best effect.

[0033] The above microorganisms were inoculated and cultured in MRS medium. The components of the medium were: peptone 10 g / L, beef extract 10 g / L, glucose 15 g / L, lactose 15 g / L, yeast powder 5 g / L, diammonium hydrogen citrate 2 g / L, K2PO4·3H2O 2.6 g / L, MgSO4·7H2O 0.1 g / L, MnSO4 0.05 g / L, Tween 80 1 mL / L, cysteine ​​0.5 g / L. After culturing to the logarithmic growth phase, the concentration of the microorganisms was adjusted and mixed in a 1:1 ratio so that the viable counts of Lactobacillus paracasei and Bifidobacterium longum reached 1.0×10 11 CFU / g and above.

[0034] Example 2 Probiotic composition promotes animal sleep

[0035] 2.1 Animal modeling and drug administration

[0036] After the SPF mice were adaptively raised for 1 week, they were intraperitoneally injected with chlorophenylalanine (PCPA) suspension for 2 consecutive days at a dose of 450 mg / kg. The normal group was intraperitoneally injected with the same volume of saline. When the modeling group mice showed significant behavioral changes such as circadian rhythm disorders, significant weight loss, dry and frizzy fur, increased excitability, and increased aggression, it indicated that the mouse insomnia model was successfully prepared.

[0037] The successfully modeled animals were randomly divided into 4 groups, 10 in each group, including the probiotic group: 0.2 mL of mixed bacterial solution was gavaged daily for 2 weeks; the active peptide group: 5 mg / kg of active peptide was gavaged daily for 2 weeks; the mixed group: 0.2 mL of mixed bacterial solution and 5 mg / kg of active peptide were gavaged daily for 2 weeks; the control group: the same amount of saline was gavaged daily for 4 weeks. Another 10 normal mice were taken as the normal control group, and the same amount of saline was gavaged daily for 2 weeks.

[0038] 2.2 Animal sleep experiments

[0039] 30 min after the last administration, sodium pentobarbital aqueous solution (45 mg / kg) was intraperitoneally injected, and the sleep latency (the time from the injection of sodium pentobarbital to the disappearance of the righting reflex of the mouse) and the sleep duration (the time from the disappearance of the righting reflex to its recovery) were measured and recorded, taking the disappearance of the righting reflex as the standard. The results are shown in Table 1. After modeling, the sleep time of the animals was significantly shortened, and the sleep latency was significantly prolonged. After treatment, the sleep time and sleep treatment of the animals were improved. The effect of probiotics was roughly equivalent to that of active peptides, and there was no significant difference between the two. However, after simultaneous use, there was a significant improvement in sleep time, indicating that the occurrence of insomnia can be significantly inhibited:

[0040]

[0041] 2.3 Expression of signaling factors in brain tissue

[0042] Sleep is regulated by a variety of signal factors in the brain. In the present invention, after the sleep experiment, the subjects were anesthetized with sodium pentobarbital, the brain was removed by decapitation, the hypothalamic tissue was separated, and 9 times PBS buffer was added to make a homogenate. After standing for 30 minutes, the mixture was centrifuged at 4 ℃ and 3500r / min for 15 minutes, and the supernatant was collected. The ELISA method (ELISA kit purchased from Ruixin Bio Co., Ltd.) was used to determine the content of 5-HT and γ-aminobutyric acid (GABA) in the hypothalamic tissue homogenate.

[0043] 5-HT is a monoamine neurotransmitter in the brain that can regulate the sleep-wake cycle, promote slow-wave sleep, and achieve a sedative and hypnotic effect. Figure 1 As shown in the figure, the 5-HT content in brain tissue was significantly reduced after modeling, but after treatment, its expression level was restored, and the expression level was the highest in the mixed group, which was close to the normal level.

[0044] GABA is an inhibitory neurotransmitter with anti-anxiety, anticonvulsant, and sedative effects. Its content varies with the body's sleep-wake cycle. GABA content increases during sleep and decreases during wakefulness. Figure 2 As shown, GABA in brain tissue decreased after modeling, which was physiologically manifested as shorter sleep time and longer sleep latency; after treatment, GABA content increased, but the effect of active peptides was significant, and probiotics seemed to have little effect on GABA, indicating that it may regulate sleep through other mechanisms.

[0045] 2.4 Serological testing

[0046] After the sleep experiment, the mice were decapitated and blood was collected. The blood was allowed to stand for 2 h and centrifuged at 4 °C and 3 000 r / min for 15 min to separate the serum. An appropriate amount of mouse serum was taken and the tryptophan hydroxylase (TPH) content in the serum was detected according to the instructions of the ELISA kit (purchased from Jiangsu Enzyme-Free Industry Co., Ltd.).

[0047] TPH can catalyze tryptophan to produce 5-hydroxytryptophan, which is an important neurotransmitter. TPH expression is reduced in patients with depression or insomnia. Figure 3 As shown, the TPH content in the model group was significantly reduced. After treatment, the content in the probiotics and active peptide groups began to recover, and the increase in the mixed group seemed to be greater, indicating that insomnia symptoms can be effectively suppressed.

[0048] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may still be modified, or some or all of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A sleep-promoting active peptide, characterized in that: The amino acid sequence of the active peptide is shown in SEQ ID NO:

1.

2. A sleep-promoting composition, characterized in that: The composition comprises probiotics and the sleep-promoting active peptide according to claim 1, wherein the probiotics are selected from at least one of lactobacillus, lactococcus and bifidobacterium.

3. The sleep-promoting composition according to claim 2, characterized in that: The probiotics are selected from lactobacilli and bifidobacteria.

4. The sleep-promoting composition according to claim 3, characterized in that: The lactobacillus is Lactobacillus paracasei, and its preservation number is CGMCC No. 1.2884.

5. The sleep-promoting composition according to claim 3, characterized in that: The bifidobacterium is Bifidobacterium longum, and its preservation number is CGMCC No.1.2186.

6. The sleep-promoting composition according to claim 3, characterized in that: The ratio of the lactobacillus to the bifidobacterium is 1:10-10:

1.

7. The sleep-promoting composition according to claim 6, characterized in that: The ratio of the lactobacillus to the bifidobacterium is 1:

1.

8. The sleep-promoting composition according to claim 3, characterized in that: The viable counts of Lactobacillus and Bifidobacterium were 1.0×10 11 CFU / g and above.

9. The sleep-promoting composition according to claim 7, characterized in that: The preparation method of the probiotics comprises: inoculating lactobacillus and bifidobacterium into MRS culture medium respectively, culturing to the logarithmic growth phase, and then mixing them according to the number of live bacteria in equal proportions.

10. Use of the sleep-promoting composition according to any one of claims 2 to 9 in the preparation of a sleep-promoting health product.

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