Preparation method of probiotic preparation and application of probiotic preparation in sleep improvement

Abstract

The invention discloses a probiotic preparation, the preparation is composed of an effective amount of anti-TGF-beta1 monoclonal antibody, an effective amount of probiotics and other auxiliary materials, and amino acid sequences of a heavy chain variable region and a light chain variable region of the anti-TGF-beta1 monoclonal antibody are shown as SEQ ID NO.1 and SEQ ID NO.2. The invention further discloses a preparation method of the probiotic preparation. The probiotic preparation disclosed by the invention can effectively improve sleep quality and solve the problem of insomnia generally faced by modern people. By combining the probiotics with the anti-TGF-beta1 monoclonal antibody, the preparation not only can regulate the intestinal microecology, but also can promote sleep by influencing the nervous system, and has a remarkable improvement effect. Meanwhile, the preparation method disclosed by the invention is simple, economical and suitable for large-scale production, and has a good market application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a preparation method of a probiotic preparation and its application in improving sleep. Background Art

[0002] With the acceleration of the modern life rhythm and the increase of social pressure, insomnia has become a global health problem. According to the statistics of the World Health Organization, approximately one-third of the global population will encounter sleep disorders of varying degrees at some stage. Insomnia not only affects an individual's physical health but also significantly reduces their quality of life and work efficiency. Common types of insomnia include difficulty falling asleep, difficulty maintaining sleep, and early awakening. Severe insomnia symptoms may even trigger psychological problems such as anxiety and depression, further exacerbating health risks.

[0003] Currently, common methods for treating insomnia mainly include drug treatment and cognitive behavioral therapy (CBT). However, drug treatment is usually accompanied by side effects such as drug dependence, drug resistance, drowsiness, etc., which makes many people develop dependence or encounter weakened drug efficacy when using drugs for a long time. To avoid these side effects, more and more consumers and researchers have begun to focus on natural and side-effect-free treatment methods.

[0004] In recent years, probiotics, as a natural and side-effect-free biological preparation, have gradually attracted the attention of the scientific community. Probiotics refer to live bacteria that are beneficial to the host and can enhance intestinal health and immune function and improve the state of the digestive system by regulating the intestinal microbiota. Studies have found that the intestinal microbiota not only directly affects intestinal health but also interacts with the function of the brain through the gut-brain axis. Probiotics in the intestine can regulate the synthesis and release of neurotransmitters through various mechanisms, thereby affecting mood, anxiety, stress coping ability, and sleep quality.

[0005] Existing studies have shown that some specific probiotic strains, such as Lactobacillus casei, Bifidobacterium bifidum, and Lactobacillus acidophilus, have the potential to improve sleep, reduce anxiety, and enhance mental health by regulating the intestinal microbiota, enhancing the synthesis of neurotransmitters such as 5-HT (5-hydroxytryptamine) and GABA (γ-aminobutyric acid), and regulating the level of stress hormones such as cortisol. However, the effects of only these probiotics are still limited. Therefore, it is necessary to further develop better probiotic preparations. Summary of the Invention

[0006] The purpose of the present invention is to provide a probiotic preparation, its preparation method, and its application in improving sleep.

[0007] Therefore, on the one hand, the present invention discloses a probiotic preparation, which is composed of an effective amount of anti-TGF-β1 monoclonal antibody, an effective amount of probiotics and other excipients, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-TGF-β1 monoclonal antibody are shown as SEQ ID NO.1 and SEQ ID NO.2.

[0008] Preferably, the probiotics of the present invention are composed of Lactococcus lactis, Bifidobacterium and Lactobacillus acidophilus, and Lactococcus lactis, Bifidobacterium and Lactobacillus acidophilus are mixed in an equal mass ratio.

[0009] Preferably, the concentration of the anti-TGF-β1 monoclonal antibody of the present invention is 10-50 μg / ml.

[0010] Preferably, the concentration of the probiotics of the present invention is maintained at 10^8 - 10^9 CFU / ml.

[0011] Preferably, the other excipients of the present invention are vitamin C solution and glycerol solution.

[0012] Preferably, the final concentration of the vitamin C solution of the present invention is 20 mg / ml, and the final concentration of the glycerol solution is 50 mg / ml.

[0013] On the other hand, the present invention also discloses the application of the anti-TGF-β1 monoclonal antibody in the preparation of a probiotic preparation.

[0014] The present invention provides a new probiotic preparation, which can effectively improve sleep quality and solve the insomnia problem commonly faced by modern people. By combining probiotics with anti-TGF-β1 monoclonal antibody, this preparation can not only regulate the intestinal microecology, but also promote sleep by affecting the nervous system, with significant improvement effects. At the same time, the preparation method of the present invention is simple and economical, suitable for large-scale production, and has good market application prospects.

[0015] The probiotic preparation of the present invention not only improves intestinal health by regulating the intestinal microbiota, but also improves sleep quality by affecting neurotransmitter and hormone levels, with a relatively comprehensive effect. The use of the probiotics of the present invention helps to improve sleep quality in the long term and has a positive effect on maintaining long-term health. The preparation of the present invention combines probiotics with anti-TGF-β1 monoclonal antibody, and enhances the sleep improvement effect through interaction, with innovation.

[0016] TGF-β1 (Transforming Growth Factor-β1) is a multifunctional cytokine that is widely involved in immune regulation, cell proliferation, differentiation, and fibrosis processes. TGF-β1 has an inhibitory effect on the immune system and plays an important role in chronic inflammation, immune tolerance, tissue repair, etc. In sleep regulation, the overactivation of TGF-β1 has been found to be associated with sleep disorders (such as insomnia, anxiety, etc.). Anti-TGF-β1 monoclonal antibody can specifically bind to TGF-β1, inhibit its binding to receptors, and thus prevent the activation of the TGF-β1 signaling pathway. In this way, anti-TGF-β1 monoclonal antibody can alleviate the immune and nervous system dysfunction caused by excessive TGF-β1 activity and indirectly improve sleep quality.

[0017] TGF-β1 plays an immunosuppressive role in the immune system, can promote immune tolerance and inhibit the activation of immune cells. In some cases, the overactivation of TGF-β1 may lead to an excessive immune response and induce chronic inflammation, which is closely related to sleep disorders (such as anxiety, stress-induced insomnia). By using anti-TGF-β1 monoclonal antibody, the excessive response of the immune system can be reduced, and long-term immune activation and inflammatory responses can be avoided, thus alleviating sleep disorders caused by immune dysregulation.

[0018] Chronic inflammation has been proven to be associated with various sleep disorders, especially with symptoms of anxiety, depression, and insomnia. TGF-β1 is involved in the regulation of immune cells, especially promoting the activation of T cells, macrophages, etc., which play important roles in the process of chronic inflammation. Excessive TGF-β1 activity may promote the release of inflammatory factors (such as IL-6, TNF-α), which will affect the nervous system and lead to a decline in sleep quality. Anti-TGF-β1 monoclonal antibody can reduce the TGF-β1-mediated immune response and inhibit the generation of inflammatory factors, thus alleviating sleep problems caused by chronic inflammation. Experiments have shown that anti-TGF-β1 antibody can significantly reduce the inflammation level, which is helpful for the recovery of sleep.

[0019] The gut-brain axis refers to the interaction between the gut microbiota, the immune system, and the brain. In the case of imbalance of the gut microbiota or overactivation of the immune response, it may lead to disorders of the neuroendocrine system, which in turn affects sleep quality. TGF-β1 not only plays a role in the immune system but is also closely related to the stability of the gut microbiota and the function of the nervous system. Anti-TGF-β1 monoclonal antibody can regulate the immune response, improve the imbalance of the gut microbiota, and thus contribute to the normal operation of the gut-brain axis. In this way, anti-TGF-β1 monoclonal antibody can regulate the sleep-wake cycle and improve sleep quality.

[0020] TGF-β1 also plays an important role in the fibrosis process and collagen synthesis of the skin. Excessive TGF-β1 activity may lead to excessive hyperplasia and fibrosis of the skin (such as scar formation), which in turn affects the health and appearance of the skin. The excessive action of TGF-β1 is also closely related to factors such as skin aging and wrinkle formation. By inhibiting the excessive activity of TGF-β1, anti-TGF-β1 monoclonal antibodies can help alleviate the fibrosis and aging processes of the skin, improve the skin condition, and further enhance the sleep improvement effect, because the improvement of skin health is often accompanied by the improvement of mental state and sleep quality. Description of the Drawings

[0021] Figure 1 SDS-PAGE detection results of anti-TGF-β1 monoclonal antibody, where 1 is anti-TGF-β1 monoclonal antibody. Detailed Implementation Modes

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this invention belongs. The terms used in the description of this invention herein are only for the purpose of describing specific embodiments and are not intended to limit the invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0023] Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0024] Example 1: Preparation Method of Probiotic Preparation

[0025] 1. Preparation of Anti-TGF-β1 Monoclonal Antibody

[0026] (1) Select SPF-grade BALB / c mice at about 6 weeks old and inject recombinant human TGF-β1 protein (MCE, catalog number: HY-P73616) to induce an immune response. Each mouse is injected with 50 μg of TGF-β1 protein during the primary immunization, which is mixed with complete Freund's adjuvant. Then, booster immunization injections are carried out every two weeks, repeated 3 - 4 times until the mice produce a strong immune response.

[0027] (2) Seven days after the last immunization, the mice were sacrificed, and B lymphocytes were extracted from the spleens of the immunized mice. The spleen cells were fused with anti-mouse myeloma cells using polyethylene glycol (PEG) as the fusogen. The fused hybridoma cells were screened by medium to inhibit unfused cells, and monoclonal hybridoma cells secreting anti-TGF-β1 antibody were selected. Monoclonal antibodies against TGF-β1 were screened from the hybridoma cells by ELISA method. Clones with high affinity and high specificity were selected from the screened positive clones for amplification culture.

[0028] (3) The above-selected hybridoma cell line with high antibody-producing ability was expanded to an appropriate cell number (about 1×10 7 to 1×10 8 cells). Then these hybridoma cells were injected into the peritoneal cavities of 6-week-old SPF-grade BALB / c mice by intraperitoneal injection. The injection dose was usually 1×10 7 to 1×10 8 cells per mouse. After injecting the hybridoma cells, 7 - 14 days later, ascites fluid was extracted by aseptic technique through peritoneal puncture using a syringe and a slender needle.

[0029] (4) The monoclonal antibody was purified from the ascites supernatant by affinity chromatography (such as Protein A / G column) purification method. The purity of the purified antibody was verified by SDS-PAGE. The results showed ( Figure 1 ) that the SDS-PAGE purity of this monoclonal antibody could reach more than 95%.

[0030] (5) By sequencing and analyzing the above-prepared monoclonal antibody, the amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-TGF-β1 monoclonal antibody prepared by the present invention are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0031] (6) An ELISA experiment was performed using the anti-TGF-β1 monoclonal antibody prepared by the present invention and a commercial antibody (ab215715) to measure its affinity for TGF-β1. The results showed (Table 1): The anti-TGF-β1 monoclonal antibody prepared by the present invention had a lower IC50 value (2.0 ng / mL), showing strong affinity and being superior to the commercially available antibody.

[0032] Table 1 Detection results of monoclonal antibody activity

[0033]

[0034] 2. Cultivation of probiotics

[0035] (1) Take the lyophilized powders of Lactobacillus casei, Bifidobacterium bifidum, and Lactobacillus acidophilus, and culture the probiotics in specific MRS medium (De Man, Rogosa, Sharpe agar) respectively. The culture conditions are set as: 37 °C, pH 6.0 - 6.5, anaerobic conditions, and the culture time is 24 - 48 hours. Use the optical density (OD) measurement method (OD600) to monitor the growth of the probiotics. When the OD of the probiotic culture reaches 0.8 - 1.2, it indicates that the concentration of the bacterial solution has reached the optimal harvest period.

[0036] (2) After the probiotics grow to the logarithmic phase, use low-speed centrifugation (3000 rpm, 10 minutes) to remove impurities in the medium and collect the probiotic precipitate. Ensure that the activity of each strain reaches 10^9 CFU / ml, and use the acidity test or antibiotic resistance test to evaluate the stability and tolerance of the probiotics. These strains all have good growth, acid resistance, and bile tolerance.

[0037] 3. Preparation of the probiotic - anti - TGF - β1 monoclonal antibody complex preparation

[0038] (1) Ratio of anti - TGF - β1 monoclonal antibody to probiotics: According to the experimental results and optimized dosage, mix the anti - TGF - β1 monoclonal antibody with probiotics (the three probiotics are first mixed in equal mass ratio). The concentration of anti - TGF - β1 monoclonal antibody in the final preparation is 10 - 50 μg / ml. The concentration of probiotics is maintained at 10^8 - 10^9 CFU / ml to ensure sufficient probiotic activity.

[0039] (2) Mixing of the complex preparation

[0040] First, mix the probiotic culture solution and the anti - TGF - β1 monoclonal antibody solution in a certain proportion (where the final concentration of anti - TGF - β1 monoclonal antibody is 30 μg / ml and the final concentration of probiotics is 10^9 CFU / ml), and then slowly add the vitamin C solution (final concentration of 20 mg / ml) and the glycerol solution (final concentration of 50 mg / ml), and stir evenly to ensure that each component is fully dissolved and evenly distributed.

[0041] Keep the temperature at 2 - 8 °C during the mixing process to prevent the inactivation of active ingredients. Use a magnetic stirrer or a rotary mixer, and keep the stirring speed between 100 - 200 rpm to avoid the generation of bubbles. Ensure the uniformity of the mixture and let each component be fully dissolved.

[0042] (3) Low - temperature freeze - drying treatment

[0043] Dispense the mixed solution into lyophilization bottles. First, pre-freeze it at -20°C for 12 hours to ensure the stability of probiotics and antibodies during the drying process.

[0044] Start the lyophilizer, set the low-temperature freezing (-40°C) and vacuum conditions, and the drying process lasts for 24 - 48 hours to ensure the complete removal of moisture.

[0045] After the lyophilization process is completed, immediately store the dried composite preparation at -20°C or lower temperature to ensure the long-term stability of probiotics and anti-TGF-β1 antibody.

[0046] (4) Quality control and stability testing: Conduct stability testing on the prepared composite preparation. Sample and detect parameters such as probiotic survival rate, activity of anti-TGF-β1 monoclonal antibody, pH value, and colony count every week to ensure its stability at room temperature for at least 12 months (as shown in Table 2). Confirm the effectiveness and activity of the preparation through cell proliferation tests and immunological activity tests.

[0047] (5) Packaging and storage of the preparation: Under aseptic conditions, dispense the above preparation into acid-resistant capsules, and at the same time ensure that it is not contaminated during transportation and storage. Store the packaged capsules in a low-temperature (2 - 8°C) environment for standby.

[0048] Table 2 Stability testing of probiotic composite preparation

[0049]

[0050] 5. Conclusion: Through the above preparation steps, a composite preparation containing anti-TGF-β1 monoclonal antibody and probiotics can be successfully prepared. The experimental results show that the preparation performs excellently in improving the activity of probiotics, maintaining the activity of anti-TGF-β1 antibody and overall stability, and has a significant effect on improving sleep. This provides strong technical support for the application of probiotic preparations in the health field.

[0051] Example 2: Application of probiotic preparation

[0052] 1. Experimental animals: C57BL / 6J mice, 6 weeks old, weighing about 20 g, in good health.

[0053] 2. Grouping: The mice are randomly divided into 4 groups, with 10 mice in each group, as follows:

[0054] Experimental group 1: Add probiotic preparation (containing probiotics and anti-TGF-β1 monoclonal antibody),

[0055] Experimental group 2: Add probiotic preparation (containing only probiotics, without anti-TGF-β1 monoclonal antibody),

[0056] Control group: The basal medium (without probiotics) was added.

[0057] Blank group: No substances were added, and only conventional feed and drinking water were provided.

[0058] 3. Experimental design: Mice were adaptively fed for 3 days before the experiment. After the experiment started, all mice were monitored for sleep every day, and indicators such as sleep onset time, sleep duration, and deep sleep ratio were measured. The sleep patterns of mice were recorded by polysomnography (PSG), and the sleep onset time (from entering the stationary state to the start of sleep), sleep duration (total sleep time), and deep sleep ratio were calculated.

[0059] (1) The results of experimental group 1 showed (Table 3):

[0060] Sleep onset time: The average sleep onset time was 12.4 ± 3.5 minutes, significantly lower than that of the control group (22.3 ± 5.2 minutes), p < 0.01.

[0061] Sleep duration: The sleep duration of mice in experimental group 1 was 8.0 ± 1.2 hours, significantly higher than that of the control group (6.2 ± 1.3 hours), p < 0.05.

[0062] Deep sleep ratio: The deep sleep ratio of mice in experimental group 1 was 38.5% ± 5.2%, and that of the control group was 27.4% ± 6.3%, p < 0.01.

[0063] (2) The results of experimental group 2 showed (Table 3):

[0064] Sleep onset time: It was 15.0 ± 4.0 minutes, showing improvement compared with the control group (22.3 ± 5.2 minutes), but the difference was not significant (p > 0.05).

[0065] Sleep duration: It was 7.2 ± 1.0 hours, showing a significant increase compared with the control group, p < 0.05.

[0066] Deep sleep ratio: It was 32.5% ± 4.3%, showing an increase compared with the control group, but lower than that of experimental group 1.

[0067] Table 3 Summary of experimental results

[0068]

[0069] 4. Conclusion: The combination of probiotics + anti-TGF-β1 monoclonal antibody significantly improved the sleep quality of mice, including shortening the sleep onset time, increasing the sleep duration, and increasing the deep sleep ratio, indicating that this preparation has good sleep improvement effects and is suitable for large-scale applications.

[0070] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A probiotic preparation, characterized in that: The preparation consists of an effective amount of anti-TGF-β1 monoclonal antibody, an effective amount of probiotics and other excipients, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-TGF-β1 monoclonal antibody are shown in SEQ ID NO.1 and SEQ ID NO.

2.

2. The preparation according to claim 1, characterized in that The probiotics consist of Lactococcus lactis, Bifidobacterium and Lactobacillus acidophilus, wherein Lactococcus lactis, Bifidobacterium and Lactobacillus acidophilus are mixed in equal weight ratio.

3. The preparation according to claim 1, characterized in that The concentration of the anti-TGF-β1 monoclonal antibody is 10-50 μg / ml.

4. The preparation according to claim 1, characterized in that The concentration of the probiotics is maintained at 10^8 to 10^9 CFU / ml.

5. The preparation according to claim 1, characterized in that The other auxiliary materials are vitamin C solution and glycerol solution.

6. The preparation according to claim 5, characterized in that The final concentration of the vitamin C solution is 20 mg / ml, and the final concentration of the glycerol solution is 50 mg / ml.

7. Use of the anti-TGF-β1 monoclonal antibody according to claim 1 in the preparation of a probiotic preparation.

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