Composite probiotic composition as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of probiotic and amino acid compositions, and particularly relates to a compound probiotic composition as well as a preparation method and application thereof. The compound probiotic composition is prepared from the liposome, the compound probiotics, the compound amino acid, the vitamin and the complexing agent, the liposome serves as a carrier, the 12 probiotics, the compound amino acid and the vitamin serve as active ingredients, the complexing agent enables the active ingredients and the carrier to be fully fused and bonded, and therefore the compound probiotic composition is prepared by utilizing the carrier property and the fusion property of the liposome. And the wrapped active components can safely pass through the stomach and are absorbed by the intestinal tract, so that the effective components can be quickly absorbed and utilized. The compound probiotic composition prepared by the invention not only has the effects of protecting brain nerves, soothing nerves and promoting sleep, but also can effectively repair tissues such as amygdala kernel, hippocampus, hypothalamus and pineal body, nourish nerves and help to stabilize emotion.

Description

Technical Field

[0001] The present invention belongs to the technical field of probiotics and amino acid compositions, and particularly relates to a compound probiotic composition, a preparation method thereof, and an application thereof. Background Art

[0002] Common symptoms of depression include low mood, slow thinking, reduced volitional activity, cognitive impairment, somatic symptoms, etc. Cognitive impairment is closely related to the degree of depression. By promoting the proliferation of endogenous neural stem cells and promoting nerve regeneration, cognitive dysfunction can be improved. The incidence of depression is increasing year by year worldwide and has become a public health problem that cannot be ignored. Research shows that depression can cause subacute aging in animals. The large accumulation in cells can cause disorders in cell metabolic functions, resulting in an increase in reactive oxygen species in the body, thereby damaging the functions and structures of biological membranes and other macromolecules, and leading to the decline of multiple organs and multiple systems in the body. In addition, obvious peroxidation reactions occur in the brain tissue of depression patients, causing oxidative stress damage and imbalance of the antioxidant mechanism by generating excessive reactive oxygen species and oxygen free radicals, accelerating the degradation process of brain tissue, and forming a vicious cycle.

[0003] It is reported that the human body becomes slow in movement, less intelligent in mind, and has cognitive impairment after lacking relevant amino acids. Active peptides can act on the nervous system and activate endogenous stem cells, thereby alleviating cognitive impairment. In the brains of adult mammals, there are still two regions where neural stem cells exist, and these neural stem cells can continuously produce new neurons and integrate into local neural circuits. One region is the ventricular-subventricular zone near the lateral ventricle. The neural stem cells in this region can generate neurons in the olfactory bulb. The other region is the subgranular layer of the hippocampal region. The neural stem cells in this region can generate granular neurons in the dentate gyrus. Since the hippocampal region itself is a key brain region involved in learning and memory, many current studies mainly focus on neural regeneration in the hippocampal region.

[0004] The etiology of depression is complex and diverse, and its pathogenesis is not very clear. There are currently no effective treatment methods. Research shows that relevant amino acids have good antidepressant effects, which can make them functional food ingredients or excipients with antidepressant effects, and can have a neuroprotective effect on the brain through the WNT / β-catenin signaling pathway. However, there are currently no related products or drugs for effectively treating depression based on the amino acid level.

[0005] Therefore, there is an urgent need to study a related product or drug that can effectively stabilize emotions and improve sleep quality. Summary of the Invention

[0006] The purpose of the present invention is to provide a compound probiotic composition. The compound probiotic composition provided by the present invention can not only effectively regulate human emotions but also improve sleep quality.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides a composite probiotic composition, and the composition comprises the following components by mass fraction: 30% - 40% liposome, 20% - 25% composite probiotics, 10% - 20% composite amino acids, 5% - 10% composite vitamins, and 5% - 10% complexing agent;

[0009] The liposome comprises lecithin, cholesterol, and vitamin E;

[0010] The composite probiotics include Lactobacillus reuteri - 1, Lactobacillus reuteri - 2, Lactobacillus salivarius, Lactobacillus acidophilus, Bifidobacterium lactis, Bifidobacterium adolescentis, Lactobacillus helveticus, Lactobacillus bulgaricus, Bifidobacterium breve, Lactobacillus plantarum, Lactobacillus rhamnosus, and Streptococcus thermophilus;

[0011] The composite amino acids include γ - aminobutyric acid, 5 - hydroxytryptamine precursor, tryptophan, phenylalanine, and tyrosine;

[0012] The composite vitamins include nicotinamide, vitamin B6, and vitamin C;

[0013] The complexing agent includes sodium hyaluronate and yeast fermentation broth.

[0014] Preferably, the mass ratio of lecithin, cholesterol, and vitamin E in the liposome is: 5 - 10:0.5 - 2.0:0.1 - 0.5;

[0015] In the composite probiotics, by mass fraction, it includes Lactobacillus reuteri - 1: 5% - 15%, Lactobacillus reuteri - 2: 5% - 10%, Lactobacillus salivarius: 3% - 5%, Lactobacillus acidophilus: 5% - 10%, Bifidobacterium lactis: 5% - 10%, Bifidobacterium adolescentis: 5% - 10%, Lactobacillus helveticus: 4% - 8%, Lactobacillus bulgaricus: 5% - 10%, Bifidobacterium breve: 5% - 8%, Lactobacillus plantarum: 5% - 10%, Lactobacillus rhamnosus: 5% - 10%, and Streptococcus thermophilus: 4% - 8%;

[0016] In the composite amino acids, by mass fraction, it includes: γ - aminobutyric acid: 25% - 35%, tryptophan: 15% - 25%, tyrosine: 10% - 20%, 5 - hydroxytryptamine precursor 10% - 15%, and phenylalanine 15% - 25%;

[0017] In the composite vitamins, by mass fraction, it includes: nicotinamide: 15% - 30%, vitamin B6: 20% - 30%, and vitamin C: 10% - 40%;

[0018] The complexing agent includes, by mass fraction: sodium hyaluronate: 40% - 55% and yeast fermentation broth: 45% - 60%.

[0019] The present invention also provides a preparation method of the above composition, which specifically includes the following steps:

[0020] S1. Add the above-mentioned compound probiotics and compound amino acids into a solvent, and perform the first mixing to obtain an activated amino acid solution; the solvent is a mixed emulsion of 65% - 75% water, 15% - 20% maltose, and 10% - 15% fish protein;

[0021] S2. Purify the above-mentioned liposomes, break the membranes and dilute them to obtain a liposome solution;

[0022] S3. Pour the activated amino acid solution described in step S1 into the liposome solution described in step S2, and at the same time add the above-mentioned compound vitamins and complexing agent, perform the second mixing, and obtain a compound probiotic composition after filtration and drying.

[0023] Preferably, the first mixing in step S1 is stirring mixing, the stirring speed of the first mixing is 2000 - 2500 r / min, and the time is 15 - 25 min.

[0024] Preferably, the liposomes in step S2 are prepared by dissolving lecithin, cholesterol, and vitamin E in absolute ethanol, with lecithin: 30% - 40%, cholesterol: 25% - 35%, and vitamin E: 35% - 45%; after evaporation and drying, hydration and homogenization are carried out; the solution used for hydration is a 0.2% - 0.8% (mass fraction) aqueous solution of Tween - 80, and the pressure of the homogenization is 100 - 150 kPa;

[0025] The purification in step S2 includes: purifying with a Sephadex G - 75 solution; the membrane breaking includes: breaking the membranes with a 0.5% - 1.5% (mass fraction) Triton X - 100 solution.

[0026] Preferably, the second mixing in step S3 is ultrasonic mixing, the power of the second mixing is 100 - 150 kW, and the time is 1 - 2 h.

[0027] The present invention also provides the application of the above compound probiotic composition or the compound probiotic composition prepared by the above preparation method in the preparation of products or drugs related to preventing depression, calming the nerves, or improving sleep.

[0028] The present invention also provides the application of the above compound probiotic composition or the compound probiotic composition prepared by the above preparation method in repairing pyramidal cells in the hippocampal region.

[0029] Preferably, the composite probiotic composition reduces the expression of GSK-3β protein in the hippocampal tissue and increases the expression of β-catenin protein in the hippocampal tissue.

[0030] The present invention also provides the application of the above composite probiotic composition or the composite probiotic composition prepared by the above preparation method in nourishing nerves and stabilizing emotions.

[0031] Beneficial effects of the present invention:

[0032] (1) By utilizing the synergistic effect between probiotics and the mixture of amino acids and vitamins, the present application effectively improves the emotional stability and thinking ability of mice. Therefore, the composition of the present application has the effect of regulating human emotions and improving human sleep quality to a certain extent.

[0033] (2) By using the liposome encapsulation technology and a complexing agent, the liposome can fully encapsulate probiotics and amino acids, and then through the transportation of the liposome, it can fully act on nerve cells, effectively providing nerve nutrition and improving the nerve excitability of the body.

[0034] (3) The composition provided by the present application can be used as an additive for mood foods or drugs, and has the effects of being non-toxic, harmless and fully absorbable by the human body. Description of the Drawings

[0035] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0036] Figure 1 It is a strip chart of the expression of β-catenin and GSK-3β proteins in the hippocampal tissues of rats in each group determined by the Western blotting method. Detailed Embodiments

[0037] The present invention provides a composite probiotic composition, which is prepared from liposomes, composite probiotics, composite amino acids, vitamins and complexing agents. By mass fraction, the composition includes: 30% - 40% of liposomes, 20% - 25% of probiotics, 10% - 20% of composite amino acids, 5% - 10% of vitamins and 5% - 15% of complexing agents.

[0038] In the present invention, the mass ratio of lecithin, cholesterol and vitamin E in the liposome is: 5-10: 0.5-2: 0.1-0.5; in the compound probiotics, by mass fraction, it includes Lactobacillus reuteri-1: 5%-15%, Lactobacillus reuteri-2: 5%-10%, Lactobacillus salivarius: 3%-5%, Lactobacillus acidophilus: 5%-10%, Bifidobacterium lactis: 5%-10%, Bifidobacterium adolescentis: 5%-10%, Lactobacillus helveticus: 4%-8%, Lactobacillus bulgaricus: 5%-10%, Bifidobacterium breve: 5%-8%, Lactobacillus plantarum: 5%-10%, Lactobacillus rhamnosus: 5%-10% and Streptococcus thermophilus: 4%-8%; in the compound amino acids, by mass fraction, it includes: γ-aminobutyric acid 25%-35%, tryptophan 15%-25%, tyrosine 10%-20%, 5-hydroxytryptamine precursor 10%-15% and phenylalanine 15%-25%; in the compound vitamins, by mass fraction, it includes; nicotinamide: 15%-30%, vitamin B6: 20%-30%, vitamin C: 10%-40%; in the complexing agent, by mass fraction, it includes: sodium hyaluronate 40%-55% and yeast fermentation broth 45%-60%.

[0039] The positive effect of the mass fraction of the liposome in the present invention being 30%-40% is that within this mass fraction range, it can effectively encapsulate the remaining components and form a stable structure; when the value of the mass fraction is greater than the maximum value of the range endpoint, the adverse effect will be that the liposome content is too high, which not only causes waste of raw materials, but also leads to the formation of multi-layer structures of liposomes, affecting the release of the internal compound amino acids. When the value of the mass fraction is less than the minimum value of the range endpoint, the adverse effect will be that too low liposomes cannot effectively encapsulate the compound amino acids.

[0040] The compound probiotic composition of the present invention can help regulate the intestinal flora, improve anti-inflammatory and antioxidant capabilities, and can also promote nutrient absorption and growth and development.

[0041] The mass fraction of the compound amino acids in the present invention is 10%-20%. Its positive effect is that within this mass fraction range, it can release sufficient components to improve the mood of the human body and improve sleep quality; when the value of the mass fraction is greater than the maximum value of the range endpoint, the adverse effect will be that too much compound amino acids are added, affecting the encapsulation effect of the liposome, and it is easy to cause insufficient encapsulation. At the same time, excessive intake of amino acids by the human body will cause waste. When the value of the mass fraction is less than the minimum value of the range endpoint, the adverse effect will be that the amount of compound amino acids entering human cells is insufficient, resulting in the ineffective improvement of the mood and sleep quality of the human body.

[0042] The mass fraction of the compound vitamin in the present invention is 5% - 10%. Among them, nicotinamide, vitamin B6, and vitamin C can promote digestion and absorption, enhance gastrointestinal function, and are beneficial to the digestion and absorption metabolism of food. And it has the effect of improving nerve stability and promoting nerve repair.

[0043] The mass fraction of the complexing agent in the present invention is 5% - 15%. Its positive effect is that within this mass fraction range, the liposome can fully encapsulate the compound amino acid component; when the value of the mass fraction is greater than the maximum value of the range endpoint, too high a content of the complexing agent will lead to a stronger binding force between the compound amino acid component and the liposome, resulting in the aggregation of liposomes around the compound amino acid component and affecting the encapsulation of the liposome. When the value of the mass fraction is less than the minimum value of the range endpoint, it will cause the liposome to be unable to fully encapsulate the compound amino acid component.

[0044] The compound amino acid in the present invention includes, by mass fraction: 25% - 35% of γ-aminobutyric acid, 15% - 25% of tryptophan, 10% - 20% of tyrosine, 10% - 15% of 5-hydroxytryptamine precursor, and 15% - 25% of phenylalanine.

[0045] Among them, the positive effect of the mass fraction of γ-aminobutyric acid being 25% - 35% is that within this mass fraction range, γ-aminobutyric acid can improve the sleep quality of the human body while reducing blood pressure. At the same time, γ-aminobutyric acid can also cooperate with theanine in green tea extract to improve the mood of the body and increase the permeability of 5-hydroxytryptamine in nerve cells; when the value of the mass fraction is greater than the maximum value of the range endpoint, the adverse effect will be that due to the regulatory effect of γ-aminobutyric acid on hormones, excessive γ-aminobutyric acid will lead to disorder of human hormone regulation and excessive decrease in human blood pressure, affecting the balance of human blood pressure. When the value of the mass fraction is less than the minimum value of the range endpoint, the adverse effect will be that due to the too low content of γ-aminobutyric acid, it will affect the improvement of sleep quality.

[0046] The positive effect of the mass fraction of tryptophan being 15% - 25% is that within this mass fraction range, since tryptophan is a precursor substance of melatonin, sufficient tryptophan can promote the body to produce sufficient melatonin. At the same time, tryptophan can also promote the synthesis of niacin and heme, and further improve the nervous system of the body and promote the mood regulation of the body; when the value of the mass fraction is greater than the maximum value of the range endpoint, the adverse effect will be that too much tryptophan will lead to too high contents of niacin and heme in the body, affecting the normal activities of the body. At the same time, excessive tryptophan will increase the production cost of the composition. When the value of the mass fraction is less than the minimum value of the range endpoint, the adverse effect will be that the content of tryptophan is too low, resulting in a decrease in the contents of melatonin, niacin, and heme, and being unable to effectively regulate the nervous system of the body, affecting the improvement of the sleep quality of the composition.

[0047] The positive effect of phenylalanine with a mass fraction of 15% - 25% is that within this mass fraction range, it has the effects of synthesizing important neurotransmitters and hormones, and participating in the body's carbohydrate and lipid metabolism, playing a role in preventing mental sluggishness and treating phenylketonuria. Phenylalanine is one of the essential amino acids that cannot be synthesized by the human body. Phenylalanine is an important component in protein synthesis, which is beneficial to the body's carbohydrate and lipid metabolism, and can help the body maintain healthy growth and tissue repair functions. Abnormal phenylalanine metabolism may lead to phenylketonuria. For people suffering from this disease, regular intake of phenylalanine can effectively help patients recover to normal. Normal phenylalanine metabolism produces tyrosine. Feeding children a low dose of L-phenylalanine to maintain the blood drug concentration at 15 - 40 mg / L can prevent mental sluggishness.

[0048] The positive effect of tyrosine with a mass fraction of 10% - 20% is that within this mass fraction range, it plays a protective role in synthesizing neurotransmitters in the brain, regulating emotions, protecting the nervous system, etc.

[0049] The positive effect of serotonin with a mass fraction of 10% - 15% is that within this mass fraction range, it can play the following positive roles: 1. Serotonin can bind to different serotonin receptors on the surface of human immune cells to regulate the immune system. 2. Appropriately increasing the serotonin level in the brain can effectively relieve the symptoms of depression. 3. It can directly affect the functions of multiple organs in the digestive system. The lack of serotonin in the esophagus is somewhat related to gastroesophageal reflux disease and reflux esophagitis.

[0050] In the present invention, by mass fraction, the complexing agent includes: sodium hyaluronate: 40% - 55% and yeast fermentation broth: 45% - 60%.

[0051] In this application, the positive effect of sodium hyaluronate with a mass fraction of 40% - 55% is that within this mass fraction range, it can effectively complex compound amino acids and liposomes, and at the same time complex other raw materials of the composition to form a stable complex. Since sodium hyaluronate can also regulate the permeability of the blood vessel wall, the active ingredients of the composition can be quickly diffused between cells and absorbed; when the value of the mass fraction is greater than the maximum value of the range endpoint, the adverse effect will be that too high sodium hyaluronate will cause the liposomes or compound amino acids to agglomerate, so that the two cannot be bonded, affecting the formation of the composition. When the value of the mass fraction is less than the minimum value of the range endpoint, the adverse effect will be that too low sodium hyaluronate will not be able to effectively complex the remaining raw materials in the composition, resulting in incomplete encapsulation of the liposomes and causing leakage.

[0052] The positive effect of the yeast fermentation broth with a mass fraction of 45% to 60% is that within this mass fraction range, small molecule proteins, small molecule acids, etc. in the yeast fermentation broth can all increase the complexing ability of sodium hyaluronate. At the same time, the yeast fermentation broth can improve the human digestive environment, especially the digestive environment of the small intestine, increase the absorption ability of the small intestine for nutrients, accelerate the absorption of the composition by the human body, so that the components of the composition can be effectively absorbed. When the value of the mass fraction is greater than the maximum value of the range endpoint, the adverse effect is that too high a yeast fermentation broth will lead to an increase in the acidity of the composition, affecting the release of the composite amino acid components. At the same time, the complexing ability of hyaluronic acid intensifies, resulting in the direct aggregation of small particle raw materials and affecting the release of the effective components of the composition. When the value of the mass fraction is less than the minimum value of the range endpoint, the adverse effect is that too low a yeast fermentation broth will not be able to provide small molecule proteins and small molecule acids, and at the same time cannot effectively improve the human digestive environment.

[0053] The molecular weight of the sodium hyaluronate of the present invention is 10 to 600 kDa. Its positive effect is that by using low molecular weight sodium hyaluronate, the hydrolyzed hyaluronic acid not only has a low molecular weight, but also can carry the active components of the composite amino acids into the cells, so that the composition can be fully absorbed. When the value of the mass fraction is greater than the maximum value of the range endpoint, the adverse effect is that too high molecular weight sodium hyaluronate cannot be effectively absorbed by the cells, affecting the absorption degree of the composition. When the value of the mass fraction is less than the minimum value of the range endpoint, the adverse effect is that although the molecular weight of too low sodium hyaluronate can quickly diffuse into the cells, because low molecular weight sodium hyaluronate is generally oligomeric sodium hyaluronate, it cannot effectively complex liposomes and other raw materials, so that the liposomes cannot complex the active components of the composite amino acids and whey protein degradation polypeptides, resulting in leakage and affecting the absorption of the active components by the human body.

[0054] The liposomes of the present invention include lecithin, cholesterol and vitamin E, and the mass ratio of lecithin, cholesterol and vitamin E is: 5 to 10: 0.5 to 2: 0.1 to 0.5. The positive effect of the liposomes including lecithin, cholesterol and vitamin E is that by using phospholipids as the skeleton of the liposomes, and then using cholesterol as a filler and vitamin E as a fat-soluble vitamin, it can not only increase the diffusibility of the liposomes, but also prevent the liposomes from being oxidized, thus obtaining stable liposomes. At the same time, the mass ratio of each substance is limited to: 5 to 10: 0.5 to 2: 0.1 to 0.5, so as to obtain stable liposomes, which is convenient for the composition to be quickly absorbed by the human body.

[0055] The preparation method of the composite probiotics of the present invention is to add the composite probiotics and composite amino acids into a solvent composed of 65% - 75% water, 15% - 20% maltose, and 10% - 15% fish protein, and perform the first mixing to obtain an activated amino acid solution; purify the liposome, break the membrane and dilute it to obtain a liposome solution; pour the obtained activated amino acid solution into the liposome solution, and at the same time add composite vitamins and complexing agents, and perform the second mixing, and filter and dry to obtain the composite probiotics.

[0056] Further, in the preparation process of the present invention, the first mixing is stirring mixing, the stirring speed of the first mixing is 2000 - 2500 r / min, and the time of the first mixing is 15 - 25 min; the second mixing is ultrasonic mixing, the power of the second mixing is 100 - 150 kW, and the time of the second mixing is 1 - 2 h.

[0057] The positive effect of the stirring speed of 2000 - 2500 r / min in the first mixing of the present invention is that under this stirring condition, the composite amino acids and probiotics can be mixed sufficiently, making each component in the composite amino acids become dispersed and activated, and becoming easy to be absorbed by the human body; when the value of the rotation speed is greater than the maximum value of the range endpoint, the adverse effect will be that too fast rotation speed will not be able to mix the composite amino acids and probiotics sufficiently, and it is also easy to cause material loss. When the value of the rotation speed is less than the minimum value of the range endpoint, the adverse effect will be that too slow rotation speed will not be able to mix the composite amino acids and probiotics completely.

[0058] The positive effect of the time of the first mixing being 15 - 25 min is that within this time range, the composite amino acids and probiotics can be mixed sufficiently, making each component in the composite amino acids become dispersed and activated, and becoming easy to be absorbed by the human body; when the value of the time is greater than the maximum value of the range endpoint, the adverse effect will be that too long activation time will lead to an extension of the overall process time. When the value of the time is less than the minimum value of the range endpoint, the adverse effect will be that too short mixing time will lead to insufficient mixing of the composite amino acids and probiotics.

[0059] The positive effect of the power of the second mixing being 100 - 150 kW is that under this ultrasonic power condition, the complexing agent in the activated components can be mixed sufficiently with the other components, and at the same time, the liposome is fully diffused by ultrasonic waves to form a uniform liposome, which is convenient for the liposome to encapsulate the active components of the composite amino acids and probiotics; when the value of the power is greater than the maximum value of the range endpoint, the adverse effect will be that the ultrasonic power is too high, the liposome diffuses violently, and it is easy to be dispersed into small particle liposomes, and a liposome with a suitable area cannot be obtained. When the value of the oscillation range is less than the minimum value of the range endpoint, the adverse effect will be that the ultrasonic power is insufficient and a liposome with a suitable area cannot be obtained.

[0060] The positive effect of the second mixing time being 1.5 - 2 h is that within this time range, the liposomes can fully encapsulate the active ingredients of the compound amino acids and probiotics; when the value of the time is greater than the maximum value of the end point of this range, the adverse effect is that too long an ultrasonic time will lead to an extension of the overall process time, and when the value of the time is less than the minimum value of the end point of this range, the adverse effect is that too short a mixing time will lead to insufficient encapsulation of the liposomes.

[0061] The liposomes of the present invention are prepared by dissolving the lecithin, cholesterol and vitamin E in absolute ethanol, followed by evaporation drying and vacuum drying, and then hydration and homogenization. The solution used for hydration in the preparation process is a 0.2% - 0.8% (by mass) aqueous solution of Tween - 80, and the pressure of the homogenization is 100 - 150 kPa. Among them, the Tween - 80 solution is a solution of polyoxyethylene sorbitan monooleate.

[0062] In the present invention, the positive effect of the homogenization pressure being 100 - 150 kPa is that under this pressure condition, liposomes with appropriate small particle sizes can be obtained; when the value of the pressure is greater than the maximum value of the end point of this range, the adverse effect is that too high a pressure will result in liposomes with smaller particle sizes, affecting the encapsulation effect of the final composition, and when the value of the pressure is less than the minimum value of the end point of this range, the adverse effect is that too low a pressure will result in liposomes with too large particle sizes, and the particles of the formed composition for encapsulation are too large.

[0063] Furthermore, the present invention performs membrane rupture treatment and dilution on the liposomes to obtain a liposome solution, which specifically includes: purifying the liposomes, and then performing membrane rupture and dilution to obtain a liposome solution;

[0064] Among them, the purification includes: purifying with a Sephadex G - 75 solution, and the membrane rupture includes: rupturing with a 0.5% - 1.5% (by mass) Triton - 100 solution. Among them, the Sephadex G - 75 solution is a solution of G - 75 crosslinked dextran gel, and the Triton - 100 solution is a solution of polyethylene glycol octyl phenyl ether.

[0065] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below in conjunction with the drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0066] In this invention, male Sprague-Dawley rats were supplied by the Experimental Animal Management Center of Xinjiang Medical University, with a qualified quality code [SCXK(Xin) 2019-0002]. Main drugs and reagents: a composition of probiotics and amino acids, D-galactose (D-Gal), hydrochloric acid, and clomipramine were purchased from Shenzhen Zhenqiang Biotechnology Co., Ltd.; β-catenin antibody was purchased from Abcam Company in the UK, and glycogen synthase kinase 3β (GSK-3β) antibody and GAPDH antibody were both purchased from Beijing Boao Ocean Bioscience and Technology Co., Ltd.; the animal tissue total RNA extraction kit, the first-strand synthesis kit, and the fluorescence quantitative kit were all purchased from Beijing TianGen Biochemical Research Co., Ltd., and the BCA protein content determination kit and the SDS-PAGE gel preparation kit were both purchased from Beijing Solarbio Science & Technology Co., Ltd. Main instruments: the QutantStudio 6 real-time fluorescence PCR instrument and the Nanddrop 2000 / 2000C spectrophotometer were both purchased from Thermo Company in the United States.

[0067] In the embodiments of this invention, for the production processes, experimental methods, or detection methods involved, unless otherwise specified, they are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and definite in the relevant application fields. Those skilled in the art can understand the conventional process steps according to the names and apply the corresponding equipment, and implement them under conventional conditions or the conditions recommended by the manufacturer.

[0068] For various instruments, equipment, raw materials, or reagents used in the embodiments of this invention, there are no special restrictions on their sources. They are all conventional products that can be obtained through regular commercial channels, and can also be prepared according to the conventional methods well-known to those skilled in the art.

[0069] Example 1 Preparation of Compound Probiotics

[0070] 1. Preparation of raw materials

[0071] The compound probiotics include: Lactobacillus reuteri-1: 15%, Lactobacillus reuteri-2: 10%, Lactobacillus salivarius: 3%, Lactobacillus acidophilus: 10%, Bifidobacterium lactis: 10%, Bifidobacterium adolescentis: 10%, Lactobacillus helveticus: 4%, Lactobacillus bulgaricus: 5%, Bifidobacterium breve: 5%, Lactobacillus plantarum: 10%, Lactobacillus rhamnosus: 10%, and Streptococcus thermophilus: 8%.

[0072] The compound amino acids include, by mass fraction: γ-aminobutyric acid: 35%, tryptophan: 15%, tyrosine: 15%, 5-hydroxytryptamine precursor: 10%, and phenylalanine: 25%; the compound vitamins include, by mass fraction: nicotinamide: 30%, vitamin B6: 30%, and vitamin C: 40%; the complexing agent includes, by mass fraction: sodium hyaluronate: 55% and yeast fermentation broth: 45%.

[0073] 2. Preparation process

[0074] (1) Preparation of liposomes: Dissolve lecithin (EG): 40%, cholesterol (OE): 25%, and vitamin E: 35% in absolute ethanol, perform evaporation drying and vacuum drying, with a drying time of 3 h and a temperature of 80 °C. Hydrate with a 0.6% (mass fraction) aqueous Tween-80 solution and homogenize under a pressure of 150 kPa to obtain liposomes; Purify the liposomes with a Sephadex G-75 solution, and perform membrane rupture and dilution with a 1.5% (mass fraction) Triton-100 solution to obtain a liposome solution;

[0075] (2) Add compound amino acids and compound probiotics to an emulsion composed of 65% water, 15% maltose, and 10% fish protein, stir and mix at a stirring speed of 2000 r / min for 20 min to obtain an activated amino acid solution;

[0076] (3) Pour the obtained activated amino acid solution into the liposome solution, and simultaneously add compound vitamins and complexing agents, perform ultrasonic mixing with a power of 150 kW for 1.5 h, and filter and dry to obtain compound probiotics.

[0077] Example 2 Toxicity experiment

[0078] Experimental animals: Select 180 mice at 4 weeks old with a body weight of 18 - 20 g, randomly divide them into 18 groups, with 10 mice in each group, and label them as Group 1 - Group 18.

[0079] Acute toxicity experiment: Intragastrically administer the compound probiotics with antidepressant effects to the mice in Group 1 - Group 18 in sequence, with a gavage dose of 25 g / kg·BW, and then continuously observe for 14 days.

[0080] Experimental results: No obvious poisoning manifestations were observed in the mice during the experiment, and no deaths occurred during the observation period. The acute oral toxicity (MTD) of the sample to mice was greater than 25 g / kg·BW. According to the acute toxicity classification in "Food Safety Toxicological Evaluation Procedures and Methods" (2003 edition), it belongs to the non-toxic level.

[0081] Example 3 CRS model experiment on SD rats

[0082] The CRS model of SD rats refers to an animal model that mimics human depressive symptoms using Sprague-Dawley (SD) rats as experimental animals and established by the method of Chronic Restraint Stress (CRS). This model is mainly used to study the pathogenesis of depression, evaluate the efficacy of drugs, and explore new treatment methods.

[0083] 3.1. Preliminary preparation

[0084] Forty Sprague-Dawley rats (200 - 250 g) were from the Key Laboratory of Shanghai Mental Health Center. The animals were individually housed under standard colony conditions with a 12-hour light / dark cycle and had free access to food and water. They were given at least 7 days to acclimatize to the colony before any experiments were conducted.

[0085] 3.2. Construction of the CRS model of SD rats

[0086] The SD rats were placed in restraint tubes made of 50-ml centrifuge tubes (the centrifuge tubes were pre-heated to make air holes, which were dispersed throughout the tube body, and there was a small hole in the middle of the lid through which the rat's tail passed and was exposed to the air. The rats were continuously restrained for 21 days, 6 hours per day, and were fasted and water-deprived during the restraint period).

[0087] After the rat model was established, the experimental rats were immediately transferred to the behavioral testing room for the sucrose preference test, elevated plus maze test, and forced swimming test. The behavioral tests were completed within 3 days. Before the behavioral tests began, the animals to be tested were placed in the test room for 1 hour for environmental adaptation. After the rat behavioral test experiment was completed, the rats were moved back to the breeding room.

[0088] 3.3. Animal grouping

[0089] All the animals were randomly divided into four groups, with 8 rats in each group: the control group was given ordinary rat feed; the ordinary group had 0.5 g of whey protein added to the ordinary rat feed; the low, medium, and high intervention dose groups of the compound probiotic composition had the compound probiotic composition of the present invention added to the ordinary rat feed, and the content of the compound probiotic composition in each group of feed was 0.1 g / kg, 0.3 g / kg, and 0.5 g / kg per rat respectively.

[0090] 3.4. Detection of rat body weight (BW)

[0091] The four groups of rats were continuously fed the feed for four weeks to detect the effect of the compound probiotic composition on rat body weight. As shown in Table 1, the body weight of the rats in the 0.3 g / kg and 0.5 g / kg dose groups increased significantly higher than that of the control group in the 1st - 4th weeks (P < 0.01).

[0092] Table 1 Effects of the compound probiotic composition on the body weight (BW) of SD rats

[0093]

[0094]

[0095] Note: *. Compared with the control group, P < 0.05; ** Compared with the control group, P < 0.01.

[0096] 3.5. Sucrose preference test

[0097] The sensitivity of mice to rewards and the degree of anhedonia can be reflected by the degree of preference for sucrose solution. 48 hours before the experiment, mice were trained with 1% sucrose solution and water for sucrose adaptation, that is, in the first 24 hours, two bottles of 1% sucrose solution were placed at the drinking place in the mouse cage for the mice to drink freely. In the second 24 hours, one of the sucrose solution bottles was replaced with water. Before the formal measurement of sucrose preference, all mice were fasted and water-deprived for 24 hours. During the measurement, 1% sucrose solution and water were weighed in advance and recorded. After the mice had free drinking for 24 hours, the sucrose solution bottle and the water bottle were removed and weighed and recorded respectively.

[0098] As shown in Table 2, before modeling, the sucrose consumption was similar among groups. During the whole process, the sucrose consumption changed significantly with time. The CRS model significantly reduced the sucrose consumption. The compound probiotic composition could significantly alleviate the decrease in sucrose consumption in animals with the CRS model. However, in the control group, no similar effect was found. Compared with the control group, the sucrose consumption in the dose groups increased significantly after intervention. But the increase in sucrose consumption in the normal group was not obvious. (P > 0.05)

[0099] Table 2 Effects of the compound probiotic composition on the sucrose consumption of rats with the CRS model

[0100] Group n Sugar water consumption before modeling / g Sugar water consumption in CRS model / g Sugar water consumption after intervention / g Control group 8 14.67±1.58 6.69±1.67** 7.87±2.04 Normal group 8 14.49±1.47 7.01±1.22** 12.63±2.22 0.1 g / kg 8 14.22±1.70 7.31±1.44* 17.06±2.11** 0.3 g / kg 8 14.76±1.23 7.12±1.31* 19.11±1.83** 0.5 g / kg 8 14.88±1.18 6.94±2.17** 20.08±2.35*

[0101] Note: *. Compared with the control group, P < 0.05; ** Compared with the control group, P < 0.01.

[0102] 3.6. Elevated plus maze (EPM) experiment

[0103] The elevated plus maze (EPM) is one of the indicators for evaluating depressive-like behaviors. During the test, the rats were placed at the central intersection of two open arms and two closed arms (head facing the open arms), and the activity time of the rats in the open arms was recorded for 5 minutes. After each rat completed the test, the feces and urine of the rat were cleaned up in time, and the inner wall and the bottom surface were disinfected with 75% alcohol and wiped clean to avoid the interference of the smell of the previous rat.

[0104] The results are shown in Table 3. There were significant differences in the percentage of time spent by rats in the open arms of the maze, demonstrating that the compound probiotic composition had antidepressant-like activity. It was not difficult to find that the percentage of time spent by CRS model rats in the open arms of the maze was significantly reduced compared with the control group. Compared with the control group, the percentage of time spent by rats in the normal group in the open arms of the maze recovered but not significantly (P > 0.05), while the intervention in the dosage groups showed that the larger the dosage, the more significant the recovery of the percentage of time spent by rats in the open arms of the maze.

[0105] Table 3 Effects of the compound probiotic composition on the percentage of time spent by CRS model rats in exploring the open arms

[0106] Group n Exploration time in open arm before modeling / % Exploration time in open arm in CRS model / % Exploration time in open arm after intervention / % Control group 8 7.00±0.98 3.84±0.59* 4.01±1.04 Normal group 8 6.91±0.56 3.68+0.68* 5.24±1.11 0.1 g / kg 8 7.21±0.65 3.77+1.03* 6.26±0.68* 0.3 g / kg 8 6.89±1.14 3.94±0.84* 6.57±0.87* 0.5 g / kg 8 7.11±1.18 3.76±0.77* 7.66±1.24**

[0107] Note: *. Compared with the control group, P < 0.05; **. Compared with the control group, P < 0.01.

[0108] 3.7 Forced swimming test (FST)

[0109] The forced swimming test is used to evaluate whether animals have despair behavior and is also an evaluation criterion for depressive behavior. Rats were individually placed in a plexiglass cylinder with a height of 50 cm and a diameter of 20 cm. The cylinder was filled with water at 22 ± 1 °C to a depth of 40 ± 1.5 cm, making it impossible to escape. After 15 minutes, the rats were removed and dried, and then returned to the cage. Fresh water was used for each rat, and the cylinder was also cleaned between trials. The immobility time, swimming time, and climbing time for 2 - 6 minutes were recorded by a camera on the side of the cylinder, and the cylinder was illuminated from the other side.

[0110] The results are shown in Tables 4 - 6. Before modeling, the immobility time, swimming time, and climbing time of the animals were uniform. After modeling, significant changes occurred in the immobility time, swimming time, and climbing time of CRS model rats. After 4 weeks of antidepressant intervention, the immobility time in each dosage group (0.1 g / kg, 0.3 g / kg, 0.5 g / kg) was significantly shortened, and it became more obvious with the increase in dosage; the swimming time in all three groups increased significantly; the climbing time also showed a significant difference compared with the control group. There were changes in the immobility time, swimming time, and climbing time in the normal group, but not as significant as those in each dosage group. (P > 0.05)

[0111] Table 4 Effects of the compound probiotic composition on the immobility time in the forced swimming test of CRS model rats

[0112] Group n Immobile time before modeling / s Immobile time in CRS model / s Immobile time after intervention / s Control group 8 46.56±5.15 88.67±9.66* 101.94±7.87 Normal group 8 44.61±5.58 96.27±8.97* 88.46±7.08 0.1 g / kg 8 43.83±6.31 110.54±8.63* 53.58±6.88** 0.3 g / kg 8 48.24±5.66 126.51±10.46* 37.24±6.25** 0.5 g / kg 8 53.47±4.68 125.33±8.58* 28.37±5.82**

[0113] Note: * Compared with the control group, P < 0.05; ** Compared with the control group, P < 0.01.

[0114] Table 5 Effects of the compound probiotic composition on the swimming time of rats in the forced swimming test of the CRS model

[0115]

[0116]

[0117] Note: *. Compared with the control group, P < 0.05; ** Compared with the control group, P < 0.01.

[0118] Table 6 Effects of the compound probiotic composition on the climbing time of rats in the forced swimming test of the CRS model

[0119] Group n Climbing time before modeling / s Climbing time in CRS model / s Climbing time after intervention / s Control group 8 105.22±8.66 10.16±1.87* 55.46±6.10 Normal group 8 99.66±8.44 12.52±1.21* 27.76±2.20 0.1 g / kg 8 93.48±7.86 5.88±1.06* 11.19±1.26** 0.3 g / kg 8 90.11±6.89 6.13±0.87* 12.08±0.47** 0.5 g / kg 8 91.33±7.59 4.16±1.12* 8.09±0.86**

[0120] Note: *. Compared with the control group, P < 0.05: ** Compared with the control group, P < 0.01.

[0121] Example 4 Hippocampal tissue experiment

[0122] 4.1. Materials Experimental animals: 84 male SD rats, 12 - 16 weeks old, body weight (250 ± 50) g. The rats were conventionally raised in an SPF - level laboratory, maintaining sufficient ventilation and lighting conditions. 3 - 4 rats were housed in each cage, with free access to food and water.

[0123] 4.2. Establishment of the CUMS depression model in rats

[0124] 84 SD rats were adaptively raised for 1 week and randomly divided into a blank group, a model group, a clomipramine group, a whey protein group, a low - dose group (0.1 g / kg) of the compound probiotic composition, a medium - dose group (0.3 g / kg), and a high - dose group (0.5 g / kg), with 12 rats in each group.

[0125] Except for the blank group, the CUMS depression model of rats in other groups was made as follows:

[0126] ① The cage was tilted at 45° for 7 h; ② The tail was clamped 1 cm from the tip of the rat's tail for 1 min; ③ Food was withheld for 24 h; ④ Water was withheld for 24 h; ⑤ The rats swam in cold water at 4°C for 5 min; ⑥ The light - dark cycle was reversed for 24 h; ⑦ The rats were shaken horizontally at 200 revolutions per minute for 5 min; ⑧ Different noises were applied for 6 h; ⑨ Foreign objects were placed in the cage for 12 h; ⑩ The bedding was made wet for 12 h; ⑪ No stimulation was given for 24 h; ⑫ The rats swam in hot water at 40°C for 5 min. The above 12 kinds of stimulations were randomly arranged within 21 days, with 1 kind of stimulation per day, and each kind of stimulation could not occur more than 3 times, and the same kind of stimulation could not occur continuously, so that the animals could not predict the occurrence of the stimulation. On the 22nd day, behavioral evaluation was performed on all animals. If the data had statistical differences (P < 0.05) before and after treatment, it indicated that the model was successfully established.

[0127] 4.3. For the rats with established models, after the control group and experimental groups in the blank group, model group, clomipramine group, whey protein group, low-dose group (0.1 g / kg), medium-dose group (0.3 g / kg), and high-dose group (0.5 g / kg) of the compound probiotic composition were raised for a period of time, hippocampal tissue experiments were conducted.

[0128] 4.4. Pathological observation of hippocampal tissue

[0129] HE staining was used. After 60 days of grouping treatment for each group, 4 rats were randomly selected from each group. They were anesthetized by intraperitoneal injection of 0.45 mL / 100 g of 1% sodium pentobarbital. After the rats were unconscious, they were placed on a dissection tray, and their hearts were exposed. The perfusion needle was inserted and fixed in the left ventricle; the right auricle was incised, and 150 mL of sterile normal saline pre-frozen at 4°C was slowly perfused until the rat's nose tip turned pale, the colors of the liver and lungs changed from red to white, and clear liquid flowed out from the right auricle. Then, 200 mL of 4% paraformaldehyde was perfused; when the rats' limbs twitched and spasm, their heads were cut off to remove the brain, and the bilateral hippocampi of the brain were dissected on an ice table, fixed with 4% paraformaldehyde, and prepared for sectioning.

[0130] After gradient ethanol dehydration, tissue clearing, wax infiltration, and embedding in sequence, the hippocampal tissue was coronally cut into paraffin sections with a thickness of 4 μm. After the sections were baked for several hours, they were dewaxed, cleared, dehydrated with a dewaxing solution, washed in sterile water, stained with hematoxylin for 1 min, differentiated with 1% hydrochloric acid ethanol, and rinsed thoroughly again; stained with 1% eosin for 1 min, dehydrated in gradient, cleared with a dewaxing solution, and sealed with neutral balsam. The pathological changes of the hippocampal tissue in each group were observed under an optical microscope.

[0131] Comparison of pathological changes in the hippocampal tissue of rats in each group. Compared with the control group, the arrangement of pyramidal cells in the hippocampal region of rats in the model group was disordered, the volume of many nerve cells decreased, nuclear pyknosis occurred, and the number of neurons decreased significantly, and there were also many damages to the pyramidal cells. Compared with the model group, the number of neurons in the clomipramine group and the low-, medium-, and high-dose groups of the compound probiotic composition increased significantly and their morphology was relatively intact. Among them, in the high-dose group and the clomipramine group, most of the pyramidal cells in the hippocampal region had relatively normal morphology, the cell arrangement was relatively dense, the nucleolus structure was clear, and the staining was uniform.

[0132] 4.5. Observation of the positive expression of β-catenin in hippocampal tissue

[0133] Using immunohistochemistry, coronal sections of hippocampal tissues from each group were taken. After rehydration, the sections were soaked in 3% hydrogen peroxide solution at room temperature for 10 min, washed with distilled water for 3 min, and thoroughly washed with phosphate buffer (PBS) three times, 5 min each time; placed in citrate buffer, microwave repaired (92 - 98 °C) for 12 min; cooled to room temperature at room temperature, rinsed with PBS three times; after antigen repair, placed in 5% BSA for blocking for 30 min; the blocking solution was removed, and diluted polyclonal rabbit anti-β-catenin (1:500) was added dropwise, and incubated overnight in a 4 °C refrigerator. The next day, it was rinsed with PBS three times, diluted horseradish peroxidase-labeled secondary antibody was added dropwise on the surface of the section tissue, and incubated in an incubator at 37 °C for 30 min; rinsed with PBS three times, DAB chromogenic solution was added dropwise evenly on the tissue surface for chromogenesis for 60 s until brown-yellow color appeared in the tissue under the microscope, and terminated with tap water; counterstained with hematoxylin for 1 min, differentiated with hydrochloric acid, dehydrated with gradient ethanol, and sealed with neutral gum. The localization and quantity of β-catenin protein in the hippocampal tissues of each group were observed under the microscope.

[0134] The comparison results of the positive expression of β-catenin in the hippocampal tissues of rats in each group are shown. The β-catenin protein in the hippocampal tissues of rats in each group was mainly located in the cytoplasm, and the positive cells were yellow-brown. Compared with the control group, the positive cells of β-catenin protein in the hippocampal tissues of rats in the model group were lightly stained, and the number of positive cells was small and the cells were more dispersed. Compared with the model group, the number of positive cells of β-catenin protein in the hippocampal tissues of rats in the clomipramine group and the low, medium, and high-dose groups of the compound probiotic composition increased, and the staining was deeper.

[0135] 4.6. Detection of the expression of GSK-3β and β-catenin mRNA in hippocampal tissues

[0136] Using the Real-time PCR method, the remaining rats in each group after 60 days of grouping treatment were taken. After cardiac perfusion, the brain was removed, the hippocampal tissue was isolated, quickly frozen in liquid nitrogen and transferred to an -80 °C refrigerator for storage.

[0137] Weighed 20 mg of hippocampal tissue, added lysis buffer and grinding beads, ground several times in an automatic grinder, and extracted total hippocampal mRNA. The purity of mRNA in each sample was detected. When the absorbance A260 / A280 measured at 260 and 280 nm was 1.9 - 2.0, reverse transcription was carried out.

[0138] Amplification was carried out using a real-time fluorescence quantitative PCR instrument. The primer sequences of GSK-3β, β-catenin and the internal reference GAPDH were designed and synthesized by Shanghai Sangon Biotech Co., Ltd., and the primer sequences are shown in Table 7.

[0139] Table 7 Primer sequences of GSK-3B, β-catenin and internal reference GAPDH

[0140]

[0141] PCR amplification conditions: 95°C, 15 min, 1 cycle; 95°C, 10 s, 60°C, 32 s, 40 cycles; 65 - 95°C for melting curve plotting. The relative expression level of the target gene was calculated by the 2-ΔΔCt method.

[0142] The comparison results of the expressions of GSK-3β and β-catenin mRNA in the hippocampal tissues of rats in each group are shown in Table 8. Compared with the blank group, the expression of GSK-3β mRNA in the hippocampal tissues of rats in the model group increased, while the expression of β-catenin mRNA decreased (both P < 0.05); compared with the model group, the expressions of GSK-3β mRNA in the hippocampal tissues of rats in the normal group, the clomipramine group, and the low, medium, and high-dose groups of the compound probiotic composition decreased, and the expressions of β-catenin mRNA increased (both P < 0.05). There were no statistically significant differences in the expressions of GSK-3β and β-catenin mRNA in the hippocampal tissues of rats between the clomipramine group and the high-dose group of the compound probiotic composition (both P > 0.05).

[0143] Statistical methods used the SPSS 22.0 statistical software. For measurement data, the Shapiro-Wilk normality test method was adopted. If it showed a normal distribution, it was expressed as For multiple-group comparisons, analysis of variance was used, and for two-group comparisons, the t-test was used; if it was not normally distributed, it was expressed as M(P25, P75), and the rank sum test was used for group comparisons. P < 0.05 was considered statistically significant.

[0144] Table 8 Comparison of the expressions of GSK-3β, β-catenin, and mRNA in the hippocampal tissues of rats in each group

[0145]

[0146]

[0147] Note: Compared with the blank group, *P < 0.05; compared with the model group, #P < 0.05; compared with the clomipramine group, △P < 0.05; compared with the low and medium-dose groups of the bioactive peptide, ▲P < 0.05.

[0148] 4.7. Detection of the expressions of GSK-3β and β-catenin proteins in the hippocampal tissues

[0149] Using the Western blotting method, the hippocampal tissues of rats in each group stored in step 1.5 were taken out, cut into tissues weighing 200 - 500 mg, and 250 - 500 μL of RIPA lysis buffer, protease inhibitor, and phosphatase inhibitor were added, followed by thorough homogenization using a grinder. Centrifuge at 14000 r / min for 10 min at 4°C, and take the supernatant.

[0150] The protein concentration was determined using a BCA protein quantification kit. A standard curve was plotted, the concentration of each sample was calculated, and RIPA was added to normalize the concentration of each sample. Polyacrylamide gel (SDS-PAGE) was prepared for gel electrophoresis, and the proteins were transferred to a PVDF membrane using the wet transfer method. The PVDF membrane was blocked with 5% skim milk for 2 h. The PVDF membrane was immersed in the diluted primary antibodies of β-catenin (1:5000) and GSK-3β (1:1000), and incubated overnight on a shaker at 4°C. The next day, the washed PVDF membrane was immersed in the secondary antibody (1:5000) and incubated at room temperature for 1 h. After washing the membrane, the PVDF membrane was immersed in the ECL chemiluminescent reagent, taken out, and exposed and developed using a gel imager. The gray value of the band was analyzed using ImageJ software, and GAPDH was used as an internal reference to calculate the relative protein expression.

[0151] The comparison results of the protein expressions of GSK-3β and β-catenin in the hippocampal tissues of rats in each group are as Figure 1 shown in Table 9. Compared with the blank group, the protein expression of GSK-3β in the hippocampal tissues of rats in the model group increased, while the protein expression of β-catenin decreased (P < 0.05 for both). Compared with the model group, the protein expression of GSK-3β in the hippocampal tissues of rats in the normal group, the clomipramine group, and the low, medium, and high-dose groups of the compound probiotic composition decreased, while the protein expression of β-catenin increased (P < 0.05 for both). There were no statistically significant differences in the comparison of the protein expressions of GSK-3β and β-catenin in the hippocampal tissues of rats between the clomipramine group and the high-dose group of the prepared compound probiotic composition (P > 0.05 for both).

[0152] The statistical method used the SPSS 22.0 statistical software. For measurement data, the Shapiro-Wilk normality test method was used. If it showed a normal distribution, it was expressed as , one-way ANOVA was used for comparison among multiple groups, and the t-test was used for comparison between two groups; if it did not show a normal distribution, it was expressed as M(P25, P75), and the rank sum test was used for comparison between groups. P < 0.05 was considered statistically significant.

[0153] Table 9 Comparison of the protein expressions of GSK-3β, β-catenin in the hippocampal tissues of rats in each group

[0154]

[0155]

[0156] Note: Compared with the blank group, *P < 0.05; compared with the model group, #P < 0.05; compared with the clomipramine group, △P < 0.05.

[0157] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A composite probiotic composition, characterized in that: The composition comprises the following components by mass: 30% to 40% of liposomes, 20% to 25% of compound probiotics, 10% to 20% of compound amino acids, 5% to 15% of compound vitamins and 5% to 10% of complexing agents; The liposome comprises lecithin, cholesterol and vitamin E; The composite probiotics include Lactobacillus reuteri-1, Lactobacillus reuteri-2, Lactobacillus salivarius, Lactobacillus acidophilus, Bifidobacterium lactis, Bifidobacterium adolescentis, Lactobacillus helveticus, Lactobacillus bulgaricus, Bifidobacterium breve, Lactobacillus plantarum, Lactobacillus rhamnosus and Streptococcus thermophilus; The complex amino acids include gamma-aminobutyric acid, 5-hydroxytryptamine precursor, tryptophan, phenylalanine, and tyrosine; The multivitamin comprises niacinamide, vitamin B6 and vitamin C; The complexing agent includes sodium hyaluronate and yeast fermentation broth.

2. The composition according to claim 1, characterized in that The mass ratio of lecithin, cholesterol and vitamin E in the liposome is: 5-10: 0.5-2.0: 0.1-0.5; The composite probiotics include, by mass fraction, 5% to 15% of Lactobacillus reuteri-1, 5% to 10% of Lactobacillus reuteri-2, 3% to 5% of Lactobacillus salivarius, 5% to 10% of Lactobacillus acidophilus, 5% to 10% of Bifidobacterium lactis, 5% to 10% of Bifidobacterium adolescentis, 4% to 8% of Lactobacillus helveticus, 5% to 10% of Lactobacillus bulgaricus, 5% to 10% of Bifidobacterium breve, 5% to 8% of Lactobacillus plantarum, 5% to 10% of Lactobacillus rhamnosus and 4% to 8% of Streptococcus thermophilus. The composite amino acids include, by mass fraction, 25% to 35% of gamma-aminobutyric acid, 15% to 25% of tryptophan, 10% to 20% of tyrosine, 10% to 15% of 5-hydroxytryptamine precursor, and 15% to 25% of phenylalanine; The compound vitamins include, by mass fraction, niacinamide: 15%-30%, vitamin B6: 20%-30%, and vitamin C: 10%-40%; The complexing agent comprises, by mass fraction, 40% to 55% of sodium hyaluronate and 45% to 60% of yeast fermentation liquid.

3. The method for preparing the composition according to claim 1, characterized in that: The specific steps include: S1. Add the composite probiotics and composite amino acids described in claim 1 into a solvent, perform a first mixing, and obtain an activated amino acid solution; the solvent is a mixed emulsion of 65% to 75% water, 15% to 20% maltose, and 10% to 15% fish protein; S2, purifying the liposomes according to claim 1, breaking the membrane and diluting to obtain a liposome solution; S3, pouring the activated amino acid solution described in step S1 into the liposome solution described in step S2, and adding the complex vitamins and complexing agent described in claim 1, performing a second mixing, filtering and drying to obtain a composite probiotic composition.

4. The preparation method according to claim 3, characterized in that: In the step S1, the first mixing is stirring mixing, and the stirring speed of the first mixing is 2000-2500 r / min, and the stirring time is 15-25 min.

5. The preparation method according to claim 3, characterized in that: The liposomes in step S2 are prepared by dissolving lecithin, cholesterol and vitamin E in anhydrous ethanol, wherein the lecithin is 30%-40%, the cholesterol is 25%-35% and the vitamin E is 35%-45%; evaporating and drying, and then hydrating and homogenizing; the hydration solution is a Tween-80 aqueous solution with a mass fraction of 0.2%-0.8%, and the homogenization pressure is 100-150 kPa; The purification in step S2 includes: using glucose gel G-75 solution for purification; the membrane breaking includes: using Triton X-100 solution with a mass fraction of 0.5% to 1.5% for membrane breaking.

6. The preparation method according to claim 3, characterized in that: In the step S3, the second mixing is ultrasonic mixing, the power of the second mixing is 100-150 kW, and the time is 1-2 hours.

7. Use of the composite probiotic composition according to any one of claims 1 to 2 or the composite probiotic composition prepared by the preparation method according to any one of claims 3 to 6 in preparing products or medicines related to preventing depression, calming the nerves or improving sleep.

8. Use of the composite probiotic composition according to any one of claims 1 to 2 or the composite probiotic composition prepared by the preparation method according to any one of claims 3 to 6 in repairing pyramidal cells in the hippocampus.

9. The use according to claim 8, characterized in that: The composite probiotic composition reduces the expression of GSK-3β protein in hippocampal tissue and increases the expression of β-catenin protein in hippocampal tissue.

10. Use of the composite probiotic composition according to any one of claims 1 to 2 or the composite probiotic composition prepared by the preparation method according to any one of claims 3 to 6 in nourishing nerves and stabilizing emotions.

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