Application of probiotic-fructo-oligosaccharide-lycium ruthenicum microecological preparation for targeted hippocampal neuron repair in neuroprotection and composite product of probiotic-fructo-oligosaccharide-lycium ruthenicum microecological preparation

Abstract

The invention provides application of a probiotic-fructo-oligosaccharide-lycium ruthenicum microecological preparation for targeted hippocampal neuron repair in neuroprotection and a composite product, and relates to the technical field of microorganisms. The probiotic oligosaccharide composition provided by the invention comprises lycium ruthenicum powder, fructo-oligosaccharide and compound probiotic powder, the compound probiotic powder comprises plant lactobacillus, lactobacillus rhamnosus and bifidobacterium animalis subsp. Lactis, and the probiotic composition provided by the invention can significantly improve the neuron morphology and arrangement of a hippocampus area of a model group; the neuron damage is reduced.

Description

Technical Field

[0001] The present invention belongs to the field of microorganisms, and specifically relates to an application of a probiotic-oligofructose-black wolfberry microecological preparation targeting hippocampal neuron repair in neuroprotection and a composite product. Background Art

[0002] Insomnia refers to the patient's subjective description of persistent difficulties in sleep initiation, duration, consolidation or quality, which leads to some form of daytime impairment. Sleep quality is closely related to human health. Poor sleep quality may affect the body's health level by affecting lipid metabolism and endocrine status. At the same time, it may increase the risk of cardiovascular and cerebrovascular diseases and mental illnesses, affect the patient's quality of life, and even threaten their life safety.

[0003] Studies have found that prolonged sleep disturbances can cause an imbalance in the antioxidant and oxidative systems, leading to excessive oxidative stress and causing neuronal metabolic disorders and apoptosis. Previous studies using the Morris water maze have shown that chronic sleep deprivation can significantly reduce learning and memory abilities in mice. Chronic sleep deprivation can also increase oxidative stress in the hippocampus, causing structural and functional changes in synapses, reducing neuronal plasticity, and ultimately impairing cognitive function. Insufficient sleep can also inhibit cell proliferation and reduce the number of hippocampal neurons, leading to decreased cognitive ability.

[0004] Currently, the main medications used in the clinical treatment of insomnia include benzodiazepine receptor agonists, melatonin receptor agonists, hypnotic antidepressants, and atypical antipsychotics. Long-term use of these medications can lead to side effects such as dependence and tolerance, cognitive decline, impaired daytime function, and effects on the central nervous system. In contrast, natural food and medicinal resources are increasingly popular for improving sleep, especially for those who are sensitive to medication side effects or who desire long-term sleep improvement.

[0005] Black wolfberry ( Lycium ruthenicumMurry, also known as black wolfberry, is a perennial, deciduous, thorny shrub belonging to the genus Lycium in the Solanaceae family. Rich in proanthocyanidins, anthocyanidins, lycium barbarum polysaccharides, amino acids, B vitamins, and minerals, it boasts the highest anthocyanin content ever discovered in any plant, earning it the nickname "King of Anthocyanidins." Anthocyanidins in black wolfberries regulate the nervous system, alleviating nervous tension and anxiety, thereby improving sleep quality. Lycium barbarum polysaccharides in black wolfberries alleviate depression by reducing brain inflammation and abnormal activity in the lateral habenula. Furthermore, anthocyanidins and lycium barbarum polysaccharides can regulate levels of monoamine neurotransmitters (such as serotonin and dopamine), exerting antidepressant effects. Black wolfberries also possess antioxidant, immune-enhancing, vision-protecting, and anti-aging benefits. These combined health benefits provide additional support for improving sleep. Therefore, they have broad application in the pharmaceutical, functional food, health food, and cosmetics industries.

[0006] Probiotics improve sleep quality through multiple mechanisms. They synthesize and regulate neurotransmitters such as serotonin, GABA, and melatonin, regulating the sleep-wake cycle and alleviating insomnia. Furthermore, probiotics can reduce the low-grade inflammation associated with sleep disorders, modulate immune function, and reduce inflammatory factors, further improving sleep quality. Probiotics are safe and have minimal side effects, making them particularly suitable for patients intolerant to traditional medications, offering a new strategy for treating sleep disorders.

[0007] Chinese invention patent CN113855657A discloses a method for preparing anthocyanin extract and freeze-dried powder from Lycium ruthenicum and its use in sleep-enhancing products. This method involves extraction, high-speed centrifugation, ceramic membrane filtration, organic membrane degumming and concentration, simulated moving bed chromatography, cryogenic concentration, and freeze-drying to produce a freeze-dried powder of Lycium ruthenicum anthocyanins with high anthocyanin content, excellent color, and no residual exogenous acidic substances. Experimental results showed that this freeze-dried powder significantly prolonged sleep time in mice, increased sleep incidence, and shortened sleep latency, demonstrating a significant sleep-improving effect. However, the preparation process utilizes a macroporous adsorption resin separation and purification process. According to current Chinese regulations, this freeze-dried powder is only suitable for use as a health food ingredient, not as a general food ingredient. Furthermore, the process involves the use of large amounts of ethanol, which places high demands on factory equipment and production safety management, requiring the safe storage, use, and emergency handling of ethanol.

[0008] Chinese invention patent CN117814423A discloses a probiotic-fermented black wolfberry composition and its use in the preparation of anti-aging products. By combining lactic acid bacteria with cold-pressed black wolfberry juice for fermentation, this patent increases the content of polyphenols, flavonoids, anthocyanins, and other nutrients in the juice. The fermented black wolfberry composition is rich in small molecule nutrients, making it easier for the human body to absorb and exhibiting significant anti-aging effects. However, the low-temperature sterilization of cold-pressed black wolfberry juice does not meet commercial sterility requirements, posing a high food safety risk. Further optimization of the sterilization process (such as combining ultrahigh pressure and acidic environment control) is needed, as well as verification of the fermentation strain's protective effect on anthocyanins and improved shelf life testing.

[0009] Therefore, there is an urgent need to develop an innovative food formula that scientifically combines black wolfberry with probiotics to achieve a significant improvement in sleep efficacy while ensuring the safety and legality of the product. Summary of the Invention

[0010] In order to solve the above problems, the present invention provides an application of a probiotic oligosaccharide composition in neuroprotection and a composite product.

[0011] In one aspect, the present invention provides an application of a probiotic-oligofructose-black wolfberry microecological preparation targeting hippocampal neuron repair in neuroprotection and a composite product, wherein the product is an oral product; The neuroprotective function includes any one or more of: improving neuronal morphology and arrangement, improving the synthesis and secretion of neurotransmitters, and reducing neuronal damage; The composition comprises, by weight: 40-50 parts of black wolfberry powder, 12-18 parts of oligofructose and 2-4 parts of compound probiotics; The probiotics include: Lactobacillus plantarum, Lactobacillus rhamnosus and Bifidobacterium animalis subsp. lactis.

[0012] Specifically, the improvement of neuron morphology and arrangement includes: restoring neurons from abnormal cell morphology to normal cell morphology; restoring neurons from disorderly arrangement to regular arrangement.

[0013] Specifically, the improvement of the synthesis and secretion of neurotransmitters includes: increasing the synthesis and secretion of GABA and 5-HT in neuronal cells.

[0014] Specifically, the reduction of neuronal damage includes: restoring the neuronal cells from dark nuclear staining and irregular shape to normal nuclear staining and morphology.

[0015] Specifically, in the composite probiotic powder, the mass ratio of Lactobacillus plantarum, Lactobacillus rhamnosus and Bifidobacterium animalis subsp. lactis is 1-10:1-10:1-10.

[0016] Preferably, in the composite probiotic powder, the mass ratio of Lactobacillus plantarum, Lactobacillus rhamnosus and Bifidobacterium animalis subsp. lactis is 4.5-5.5:2.5-3.5:1.5-2.5.

[0017] Further preferably, the preparation method of the probiotic composition comprises the following steps: (1) Preparation of black wolfberry powder: Take dried wolfberries, add deionized water, soak and pulp, let stand, remove the floating matter on the liquid surface, add ascorbic acid, adjust the pH of black wolfberry juice to 3.0-4.0, stir evenly, filter, vacuum freeze-dry, crush, and sieve to obtain black wolfberry powder; (2) Fully mix the black wolfberry powder, oligofructose and probiotic compound powder to obtain a probiotic composition.

[0018] In certain specific embodiments of the present invention, the amount of deionized water added is 10-25 times the weight of the dry wolfberry, the soaking time is 30-60 minutes, and the amount of ascorbic acid added is 0.05-0.08%.

[0019] In certain specific embodiments of the present invention, the total viable count of the composite probiotic powder is ≥ 2.0×10 11 CFU / g.

[0020] In another aspect, the present invention provides a probiotic composition composite product for protecting nerves, comprising the probiotic composition described above.

[0021] Specifically, the probiotic composition composite product further includes auxiliary materials.

[0022] More specifically, the excipients include but are not limited to: lactose, mannitol, methylcellulose, microcrystalline cellulose, benzoic acid, sorbic acid, food coloring and / or talc.

[0023] Compared with the prior art, the present invention has the following advantages: (1) The probiotic composition provided by the present invention can significantly improve the morphology and arrangement of neurons in the hippocampus of the model group and reduce neuronal damage.

[0024] (2) The preparation steps of the probiotic composition of the present invention are simple, safer and more compliant, and conducive to large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Changes in sleep duration before and after modeling in mice.

[0026] Figure 2 are the concentrations of GABA and 5-HT in the hypothalamus tissue of mice in each group.

[0027] Figure 3Figure 2 shows the pathological changes in the hippocampus and cortex of mice in each group. DETAILED DESCRIPTION

[0028] The present invention will be further described in detail below with reference to specific examples. The following examples are not intended to limit the present invention but are merely intended to illustrate the present invention. The experimental methods used in the following examples are generally based on conventional conditions unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified.

[0029] The mouse model used in the present invention was constructed using the optimal experimental conditions for insomnia mouse modeling studied in the laboratory. The specific parameters are as follows: Fifty healthy male KM mice aged 6 weeks were purchased from Hangzhou Qizhen Laboratory Animal Technology Co., Ltd. Mice were housed in the animal room of the Experimental Animal Center of East China University of Science and Technology at an ambient temperature of 25 ± 2°C, a humidity of 50 ± 5%, and a light-dark cycle every 12 h.

[0030] Except for the normal group, mice in each group were intraperitoneally injected with 300 mg / kg of para-chlorophenylalanine (PCPA) starting from the first day for two consecutive days. On the third day, a suprathreshold dose of pentobarbital sodium was performed to induce a reversal experiment. The sleep duration of the mice was compared with that of the same mice before modeling to judge the modeling effect. On the tenth day, PCPA was intraperitoneally injected again at a concentration of 200 mg / kg to consolidate the modeling effect.

[0031] from Figure 1 It can be seen that the sleep duration of mice after modeling was significantly different from that of mice before modeling (p<0.05), thus confirming that the insomnia mouse model was successfully established.

[0032] Example 1 Preparation of probiotic oligosaccharide composition The sources of probiotics used in the present invention are shown in Table 1. Table 1

[0033] S1: Preparation of black wolfberry powder: First, remove bad fruits and impurities, select dry wolfberries with bright color and no physical damage, add 15 times their weight of deionized water, soak for 45 minutes and beat for pulping. After beating, let it stand for 3 hours, remove the floating matter on the liquid surface and add 0.06% ascorbic acid. Use 1 mol / L citric acid solution to adjust the pH of the black wolfberry juice to 3.5, stir evenly, filter and vacuum freeze-dry at -40℃, crush, and pass through a 60-mesh sieve to obtain black wolfberry powder.

[0034] S2: Fully mix 45 parts of black wolfberry powder, 15 parts of oligofructose, and 3 parts of probiotic compound powder (the mass ratio of Lactobacillus plantarum, Lactobacillus rhamnosus, and Bifidobacterium animalis subsp. lactis is 5:3:2) to obtain the black wolfberry probiotic composition of the present invention.

[0035] Comparative Example 1 Preparation of black wolfberry powder Preparation of black wolfberry powder: First, remove bad fruits and impurities, select dry wolfberries with bright color and no physical damage, add 15 times their weight of deionized water, soak for 45 minutes and beat for pulping. After beating, let it stand for 3 hours, remove the floating matter on the liquid surface and add 0.06% ascorbic acid. Use 1 mol / L citric acid solution to adjust the pH of the black wolfberry juice to 3.5, stir evenly, filter and vacuum freeze-dry at -40°C, crush, and pass through a 60-mesh sieve to obtain black wolfberry powder.

[0036] Comparative Example 2 Preparation of probiotics Lactobacillus plantarum, Lactobacillus rhamnosus, and Bifidobacterium animalis subsp. lactis (mass ratio: 5:3:2) were thoroughly mixed. Batch number: W2406080.

[0037] Effect Experiment Example 1: Experiment on the Protection of Neurons by Probiotic Composition in Mice 1. Animal grouping After one week of adaptive feeding, the mice were randomly divided into a normal group, a model group, a diazepam group (drug positive control), a low-dose combination group, a high-dose combination group, a low-dose black wolfberry group, a high-dose black wolfberry group, a low-dose probiotic group, and a high-dose probiotic group according to their body weight.

[0038] 2. Drug treatment of insomnia mice The drug-treated group received oral administration once daily from day 1 to day 14. Ten minutes after oral administration on day 14, a sodium pentobarbital reversal test was performed, and on day 16, changes in the concentrations of 5-hydroxytryptamine (5-HT) and gamma-aminobutyric acid (GABA) neurotransmitters in the hypothalamus were measured as indicators of drug efficacy.

[0039] The groups and drug administration of mice are shown in Table 2.

[0040] Table 2 Mouse grouping and modeling methods

[0041] 3. Data Analysis: All numerical values ​​in the experiment are expressed as (mean ± standard deviation). The results were compared with the differences between different groups using one-way analysis of variance (ANOVA) using the statistical software GraphPad Prism 10, with the significance level set at p < 0.05.

[0042] 4. Experimental results (1) Effects of the composition on GABA and 5-HT neurotransmitters in the hypothalamus of mice The experimental results are shown in Table 3 and Figure 2 .

[0043] Table 3

[0044] Figure 2 This study shows changes in hypothalamic γ-aminobutyric acid (GABA) concentrations across the different groups. Compared with the normal group, the model group showed significantly lower GABA concentrations (*p < 0.05). The high-dose black wolfberry probiotic composition group showed significantly higher GABA concentrations than the model group (*p < 0.05), while the low-dose composition group showed no significant difference in GABA concentrations compared with the model group (p > 0.05). Figure 2 This study demonstrates changes in 5-hydroxytryptamine (5-HT) concentration in the hypothalamus. The 5-HT concentration in the model group was significantly lower than that in the normal group (*p < 0.05). The 5-HT concentration in the high-dose black wolfberry probiotic composition group was significantly higher than that in the model group (*p < 0.05). The 5-HT concentration in the low-dose composition group was also higher than that in the model group, but the difference did not reach significance (p > 0.05).

[0045] The decrease in GABA and 5-HT concentrations in the model group may be related to the establishment of a depression model, which simulates the decrease in these neurotransmitters in patients with depression. Diazepam, as a known antidepressant drug, can increase the concentrations of GABA and 5-HT, thereby exerting an antidepressant effect. The increase in GABA and 5-HT concentrations in the high-dose group of the black wolfberry probiotic composition indicates that the composition may have an antidepressant effect similar to that of diazepam, improving depressive symptoms by increasing the concentrations of these neurotransmitters, showing its potential antidepressant effect. However, the antidepressant effect of the low-dose group of the black wolfberry probiotic composition was not significant, indicating that dosage may be a key factor affecting its efficacy.

[0046] (2) Effects of the composition on mouse hippocampal and cortical pathology The neuronal morphology and arrangement in the hippocampus of mice in different groups were analyzed in detail by histological staining. Figure 3 As shown, the hippocampus of the normal group mice, including the dentate gyrus (DG), CA1, CA3, and cortex, all showed good neuronal structure and orderly cell arrangement. These neurons had regular morphology and uniform nuclear staining, reflecting a normal physiological state. In contrast, the hippocampus of the model group mice showed significant pathological changes. In the dentate gyrus, CA1, CA3, and cortex, the neurons were arranged in a disorganized manner, the cell morphology was abnormal, the nuclear staining was dark, and the shape was irregular. These changes suggest that the neurons in the hippocampus of the model group mice may have suffered damage, which is associated with cognitive dysfunction.

[0047] Mice treated with a high dose of the black wolfberry anthocyanin combination showed significant improvements in the morphology and arrangement of neurons in the hippocampus. The arrangement of neurons in these mice became more regular, and cell morphology and nuclear staining gradually returned to normal, suggesting that the black wolfberry anthocyanin combination may have a protective effect on neurons in the hippocampus. Furthermore, mice in the diazepam group showed similar improvements, further validating the potential neuroprotective effects of the black wolfberry anthocyanin combination.

[0048] The research results show that the black wolfberry anthocyanin composition can significantly improve the neuronal morphology and arrangement in the hippocampus of model group mice at high doses and reduce neuronal damage, which provides a strong experimental basis for its application in neuroprotection and improvement of cognitive function.

[0049] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. Application of a probiotic-oligofructose-black wolfberry microecological preparation targeting hippocampal neuron repair in neuroprotection and application of the composite product in the preparation of a product with neuroprotective function, characterized in that: The product described is an oral product; The neuroprotective function includes: improving neuronal morphology and arrangement, improving the synthesis and secretion of neurotransmitters, and reducing neuronal damage. The microecological preparation comprises, by weight: 40-50 parts of black wolfberry powder, 12-18 parts of oligofructose and 2-4 parts of probiotics; The probiotics include: Lactobacillus plantarum, Lactobacillus rhamnosus and Bifidobacterium animalis subsp. lactis.

2. The use according to claim 1, characterized in that The improvement of neuron morphology and arrangement includes: restoring neurons from abnormal cell morphology to normal cell morphology; restoring neurons from disorderly arrangement to regular arrangement.

3. The use according to claim 1, characterized in that The improvement of the synthesis and secretion of neurotransmitters includes: increasing the synthesis and secretion of GABA and 5-HT in neuronal cells.

4. The use according to claim 1, characterized in that The reduction of neuronal damage includes: restoring the neuronal cells from dark nuclear staining and irregular shape to normal nuclear staining and morphology.

5. The use according to claim 1, characterized in that In the composite probiotic powder, the mass ratio of Lactobacillus plantarum, Lactobacillus rhamnosus and Bifidobacterium animalis subsp. lactis is 1-10:1-10:1-10.

6. The use according to claim 5, characterized in that In the composite probiotic powder, the mass ratio of Lactobacillus plantarum, Lactobacillus rhamnosus and Bifidobacterium animalis subsp. lactis is 4.5-5.5:2.5-3.5:1.5-2.

5.

7. The use according to claim 6, characterized in that The preparation method of the probiotic composition comprises the following steps: (1) Preparation of black wolfberry powder: Take dried wolfberries, add deionized water, soak and pulp, let stand, remove the floating matter on the liquid surface, add ascorbic acid, adjust the pH of black wolfberry juice to 3.0-4.0, stir evenly, filter, vacuum freeze-dry, crush, and sieve to obtain black wolfberry powder; (2) Fully mix the black wolfberry powder, oligofructose and probiotic compound powder to obtain a probiotic composition.

8. The use according to claim 7, characterized in that In step (1), the amount of deionized water added is 10-25 times the weight of the dry wolfberry, the soaking time is 30-60 minutes, and the amount of ascorbic acid added is 0.05-0.08%.

9. The use according to claim 7, characterized in that In step (2), the total viable count of the composite probiotic powder is ≥ 2.0 × 10 11 CFU / g.

10. A probiotic composition product for protecting nerves, characterized in that: The product comprises the probiotic composition according to any one of claims 1 to 9.

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