Application of pachymaran derivative in functional food for improving intestinal flora and calming heart and tranquilizing mind

Abstract

The invention relates to the technical field of functional food, in particular to application of a pachymaran derivative in functional food for improving intestinal flora and calming heart and tranquilizing mind. The invention relates to application of a pachymaran derivative in functional food for improving intestinal flora and calming heart and tranquilizing mind. The pachymaran derivative is prepared by adopting the following method: S1, sterilizing a carboxymethyl pachymaran solution prepared from pure water at high temperature and high pressure; s2, adjusting the pH value of the obtained solution to 4.5, and adding a biological enzyme into the solution to carry out an enzymatic reaction; s3, carrying out enzyme deactivation, concentration and freeze drying on the sample solution obtained in S2 to obtain the pachymaran derivative. The pachymaran derivative provided by the invention has a remarkable sleep improving effect, and the mechanism of the pachymaran derivative is possibly related to the regulation of the neurotransmitter level in the brain and the improvement of intestinal flora.

Description

Technical Field

[0001] This invention relates to the field of functional food technology, and in particular to the application of a Poria cocos polysaccharide derivative in functional foods that improve intestinal flora and calm the mind. Background Technology

[0002] With the continuous development of society, people are experiencing increasing pressure in their work, study, and life, leading to many people being unable to have high-quality sleep. The interaction between stress and sleep disorders has resulted in more and more people experiencing symptoms such as insomnia, depression, melancholy, and neurasthenia. Sub-health issues have long plagued people, and these symptoms are gradually becoming more prevalent among young people, significantly impacting their physical and mental health, quality of life, and social productivity.

[0003] Currently, the treatment of depression mainly relies on Western medicine. However, existing Western medicine treatments generally suffer from incomplete treatment effects and a high relapse rate. Moreover, most antidepressants also cause side effects such as gastrointestinal discomfort, nervous system reactions, and burden on liver and kidney function, resulting in poor tolerability. As for sleep problems, clinically and daily-used sleep aids also have significant drawbacks. Not only are they subject to significant side effects, but they also easily lead to dependence. Long-term use can further disrupt normal sleep structure, making it impossible to fundamentally guarantee sleep quality, and may even aggravate negative emotions such as anxiety and depression, creating a vicious cycle.

[0004] Poria cocos, a traditional Chinese medicine and food, possesses effective components with calming, spleen-strengthening, and mood-regulating effects, giving it a natural advantage in treating nervous system-related discomfort. Chinese patent CN115068509A discloses a pharmaceutical composition containing highly substituted carboxymethyl poria cocos polysaccharide and its application; however, it requires combination with other antimicrobial agents to regulate intestinal flora, and its effects on regulating the nervous system are not explicitly disclosed. Currently, there is a lack of functional foods with carboxymethyl poria cocos polysaccharide as the main component that improve intestinal flora and calm the mind. Summary of the Invention

[0005] The purpose of this invention is to address the aforementioned shortcomings of the prior art by proposing an application of a Poria cocos polysaccharide derivative in functional foods that improve intestinal flora and calm the mind.

[0006] The present invention relates to the application of a Poria cocos polysaccharide derivative in functional foods that improve intestinal flora and calm the mind. The Poria cocos polysaccharide derivative is prepared by the following method: S1. Sterilize the carboxymethyl poria polysaccharide solution prepared with pure water under high temperature and high pressure; S2. Adjust the pH of the obtained solution to 4.5, and add biological enzymes to the solution to carry out an enzymatic reaction; S3. The sample solution obtained in S2 is inactivated by enzyme, concentrated, and freeze-dried to obtain the Poria cocos polysaccharide derivative.

[0007] Furthermore, the concentration of the carboxymethyl poria cocos polysaccharide solution is 20-30 mg / mL.

[0008] Furthermore, sterilize at 110-130℃ and 0.18-0.3 MPa for 15-25 minutes.

[0009] Furthermore, the bioenzyme is one or more of cellulase, hemicellulase, amylase, and pectinase.

[0010] Furthermore, the dosage of the bioenzyme is 0.4-0.6% w / w.

[0011] Furthermore, the enzymatic reaction temperature is 40-45 ℃, and the time is 22-26 hours.

[0012] Furthermore, enzyme inactivation is performed in a boiling water bath for 8-12 minutes.

[0013] Furthermore, the degree of substitution (DS) of carboxymethyl Poria cocos polysaccharide is 0.6-0.7, the weight-average molecular weight is 706.4 ± 38.4 kDa, and the number-average molecular weight is 285.0 ± 28.4 kDa.

[0014] Furthermore, the degree of substitution (DS) of the Poria cocos polysaccharide derivative is 0.6-0.7, the weight-average molecular weight is 129.9 ± 5.1 kDa, and the number-average molecular weight is 75.86 ± 3.61 kDa.

[0015] The poria cocos polysaccharide derivative provided by this invention has significant sleep-improving effects, and its mechanism may be related to regulating neurotransmitter levels in the brain and improving gut microbiota. Animal experiments show that this poria cocos polysaccharide derivative can effectively prolong sleep time, and has high safety with no obvious toxic side effects, showing broad clinical application prospects. Attached Figure Description

[0016] Figure 1 The time for the positive reflection to disappear and recover; Figure 2-4 The detection data are for 5-HT, γ-aminobutyric acid, and dopamine, respectively. Figure 5 NMDS plot; Figure 6 For PCoA plot; Figure 7 The abundance of Lactobacillus genus. Detailed Implementation

[0017] The following are specific embodiments of the present invention, which are described in conjunction with the accompanying drawings. However, the present invention is not limited to these embodiments.

[0018] Example 1 ① Sample preparation for high-temperature treatment: Prepare a carboxymethyl poria polysaccharide solution (20 mg / mL) with pure water and sterilize it under high temperature and high pressure at 125℃ and 0.2 MPa for 20 min.

[0019] ② Sample preparation by enzymatic hydrolysis: Adjust the pH of the sample solution obtained in step ① to 4.5, and add cellulase (dosage: 0.5% w / w) to the solution. Initiate the enzymatic reaction at 40 °C for 24 hours.

[0020] ③ Enzyme inactivation and freeze drying: The sample solution obtained in step ② was concentrated in a boiling water bath for 10 min and then freeze-dried to obtain the Poria cocos polysaccharide derivative HTEC-24.

[0021] Table 1 shows the molecular weights of the carboxymethyl poria cocos polysaccharide used and the poria cocos polysaccharide derivative HTEC-24 obtained.

[0022] Table 1

[0023] Example 2: Evaluation of the efficacy in improving sleep Animal model establishment (1) Kunming mice (female, 20.0±2g) were randomly divided into blank control group, model group, melatonin group (2mg / ml) and HTEC-24 group, with 10 mice in each group; HTEC-24 group was prepared by dissolving the freeze-dried Poria cocos polysaccharide derivative HTEC-24 prepared in Example 1 in physiological saline to 20mg / mL.

[0024] (2) Except for the blank control group, the other groups were injected intraperitoneally with 350 mg / kg PCPA solution for 2 days to establish an insomnia model.

[0025] Dosing regimen After modeling, the blank control group and the model group were administered physiological saline by gavage, the melatonin group was administered melatonin by gavage, and the HTEC-24 group was administered HTEC-24 solution by gavage for 7 consecutive days.

[0026] Indicator Testing (1) Righting reflex test: 30 minutes after the last administration, sodium pentobarbital (60 kg / kg) was injected intravenously, and the recovery time of the righting reflex was recorded. The results are as follows: Figure 1 As shown, from Figure 1 It can be seen that after HTEC-24 administration, the righting reflex recovery time increased from 249 s to 342 s, which is a significant improvement compared to the model group. p<0.05), indicating that HTEC-24 has the effect of prolonging sleep time.

[0027] (2) Neurotransmitter detection: Brain tissue was collected, and 5-HT (serotonin), DA (dopamine), and GABA (gamma-aminobutyric acid) were measured by ELISA. The results are as follows: Figure 2-4 As shown, from Figure 2-4 It can be seen that the brain 5-HT level was significantly increased in the HTEC-24 group. p <0.05), GABA levels increased to some extent, while DA levels decreased to some extent.

[0028] Example 3: Evaluation of improved gut microbiota 1. Animal model establishment: Establish the model and administer the drug according to the method in Example 1.

[0029] 2. Intestinal contents sampling: Seven days after administration, the animals were euthanized by cervical dislocation. Five animals were randomly selected from each group, and the contents of the cecum were collected by opening the abdomen and mixed in a preservation tube. The samples were then frozen and stored at -80°C.

[0030] 3. Total DNA extraction from the microbiome: The most suitable total DNA extraction method was selected for microbiome samples from various sources. At the same time, Nanodrop was used to quantify the DNA, and the quality of DNA extraction was detected by 1.2% agarose gel electrophoresis.

[0031] 4. Target fragment PCR amplification: Using target sequences that can reflect the composition and diversity of the microbial community, such as microbial ribosomal RNA or specific gene fragments, as targets, corresponding primers are designed according to the conserved regions in the sequence, and sample-specific barcode sequences are added to amplify the variable region (single or multiple consecutive) of the rRNA gene or specific gene fragments by PCR.

[0032] 5. Purification and recovery of amplification products using magnetic beads: (1) Add 0.8 times the volume of magnetic beads (Vazyme VAHTS™ DNA CleanBeads) to 25 μl of PCR product, shake to suspend fully, and then adsorb on a magnetic rack for 5 min. Carefully aspirate the supernatant with a pipette. (2) Add 20 μl of 0.8 times magnetic bead washing solution, shake to suspend fully, place on a magnetic rack for adsorption for 5 min, and carefully aspirate the supernatant; (3) Add 200 μl of 80% ethanol, place it upside down on the magnetic rack, and use magnetic beads to adsorb onto the other side of the PCR tube. After full adsorption, aspirate the supernatant. (4) Let it stand at room temperature for 5 minutes until the alcohol has completely evaporated and cracks appear on the magnetic beads; (5) Add 25 μl of Elution Buffer to elute; (6) Place the PCR tube on the adsorption rack for 5 min to allow it to fully adsorb, then remove the supernatant into a clean 1.5 ml centrifuge tube for storage.

[0033] 6. Quantitative PCR of Amplified Products: The recovered PCR amplified products were quantitatively analyzed using the Quant-iTPicoGreen dsDNA Assay Kit as the fluorescent reagent and a Microplate reader (BioTek, FLx800) as the quantification instrument. Based on the quantitative PCR results, the samples were mixed in appropriate proportions according to the sequencing volume requirements of each sample.

[0034] 7. Sequencing library preparation: Sequencing libraries were prepared using the TruSeq Nano DNA LT Library Prep Kit from Illumina.

[0035] 8. High-throughput sequencing: Ribosomal RNA contains multiple conserved and highly variable regions. We typically use conserved regions to design primers to amplify single or multiple variable regions of the rRNA gene, followed by sequencing analysis to assess microbial diversity. This is used to assess changes in the gut microbiota, and the results are as follows: Figure 5-7 As shown, from Figure 5-6 It can be seen that after modeling, the composition of the gut microbiota shifted compared to the normal group of mice. However, after HTEC-24 administration, the gut microbiota composition became close to that of the normal group, indicating that HTEC-24 has a regulatory effect on the gut microbiota. Figure 7 It can be seen that the level of Lactobacillus spp. in the gut microbiota of the HTEC-24 group is higher.

[0036] For any points not covered above, existing technologies shall apply.

[0037] Although specific embodiments of the present invention have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them, without departing from the direction of the invention or exceeding the scope defined by the appended claims. Those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc., made to the above embodiments based on the technical essence of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a Poria cocos polysaccharide derivative in functional foods that improve intestinal flora and calm the mind, characterized in that, The Poria cocos polysaccharide derivative was prepared using the following method: S1. Sterilize the carboxymethyl poria polysaccharide solution prepared with pure water under high temperature and high pressure; S2. Adjust the pH of the obtained solution to 4.5, and add biological enzymes to the solution to carry out an enzymatic reaction; S3. The sample solution obtained in S2 is inactivated by enzyme, concentrated, and freeze-dried to obtain the Poria cocos polysaccharide derivative.

2. The application as described in claim 1, characterized in that, The concentration of the carboxymethyl poria polysaccharide solution is 20-30 mg / mL.

3. The application as described in claim 1, characterized in that, Sterilize at 110-130℃ and 0.18-0.3 MPa for 15-25 minutes.

4. The application as described in claim 1, characterized in that, The bioenzyme is one or more of cellulase, hemicellulase, amylase, and pectinase.

5. The application as described in claim 1, characterized in that, The dosage of the bioenzyme is 0.4-0.6% w / w.

6. The application as described in claim 1, characterized in that, The enzymatic reaction temperature is 40-45 ℃, and the time is 22-26 hours.

7. The application as described in claim 1, characterized in that, Enzyme inactivation is performed in a boiling water bath for 8-12 minutes.

8. The application as described in claim 1, characterized in that, The degree of substitution (DS) of carboxymethyl Poria cocos polysaccharide was 0.6-0.7, the weight-average molecular weight was 706.4 ± 38.4 kDa, and the number-average molecular weight was 285.0 ± 28.4 kDa.

9. The application as described in claim 1, characterized in that, The degree of substitution (DS) of the Poria cocos polysaccharide derivative is 0.6-0.7, the weight-average molecular weight is 129.9 ± 5.1 kDa, and the number-average molecular weight is 75.86 ± 3.61 kDa.

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