Preparation method of spleen-tonifying and stomach-nourishing nutritional preparation

Abstract

The invention provides a spleen and stomach conditioning factor lactobacillus reuteri obtained by fermenting poria cocos powder. The strain has good acid resistance, cholate resistance and antibacterial ability. The invention further provides a probiotic preparation which has an obvious effect on improving the spleen and the stomach. In addition, the invention also provides a composite preparation composition containing Lactobacillus reuteri, and the composition effectively promotes normal peristalsis and digestion and absorption functions of gastrointestinal tracts by regulating the secretion level of gastrointestinal hormones, enhances the immunity of the organism, protects intestinal mucosa barriers, comprehensively improves the symptoms of weakness of the spleen and the stomach, and improves the overall health level.

Description

Technical Field

[0001] The present invention belongs to the field of probiotics, and particularly relates to Lactobacillus mucosae roigii, its fermentation product, a probiotic preparation and their applications. Background Art

[0002] With the rapid development of modern society, people's eating habits and lifestyles have changed significantly. These changes have led to the problem of weak spleen and stomach becoming increasingly common in modern society, with symptoms such as stomachache and spleen deficiency becoming more prevalent and younger. In the visceral theory of traditional Chinese medicine, the "spleen" is regarded as a conceptual structure covering a wide range of functions. It is not only closely connected with the digestive system but also involves multiple aspects such as the nervous system, immune function, hematopoiesis mechanism, muscle metabolism, and liver metabolism function. Zhang Zhongjing, a famous medical scientist in the Eastern Han Dynasty, pointed out profoundly in his classic work "Treatise on Febrile Diseases": "The spleen assists the stomach qi in digesting food. If the spleen qi does not function properly, the food in the stomach cannot be digested." This statement emphasizes that spleen deficiency can lead to disorders of digestion and transportation functions. Further, "On the Spleen and Stomach" also states: "Once the spleen and stomach are damaged, all kinds of diseases will occur", highlighting the close connection between the spleen and the body's immune function. In the clinical practice of traditional Chinese medicine, spleen deficiency syndrome is a common syndrome. Therefore, exploring and discovering substances that can effectively tonify the spleen is of great significance.

[0003] The "spleen" in traditional Chinese medicine theory is a multi-functional concept that is related to multiple physiological systems of the human body, especially digestion and immunity. When spleen deficiency occurs, it will affect the normal operation of these systems, and finding effective substances to tonify the spleen is crucial for maintaining human health. Discomfort in the spleen and stomach is often manifested as dull pain in the stomach, poor appetite, thick, enlarged tongue, or tooth marks on the tongue; dull, sallow skin, dark circles under the eyes, and large eye bags; being overly obese or overly thin; flatulence, acid reflux, frequent belching, bad breath and dry mouth; often drooling while sleeping; pale, dry lips; poor digestion, frequent diarrhea, or frequent constipation and difficult defecation; feeling uncomfortable in the stomach after eating something cold or getting cold. It not only affects work and study but also hinders health. In recent years, many researchers have emerged, but most of them have started with specific prescriptions for specific diseases.

[0004] The concept of "medicine and food sharing the same origin" is an extremely valuable contribution of traditional Chinese original medicine to humanity. In traditional ancient prescriptions, there are many prescriptions for regulating the spleen and stomach, such as Si Jun Zi Tang, Liu Jun Zi Tang, Shen Ling Bai Zhu San, Xiao Jian Zhong Tang, etc.

[0005] With the increasing awareness of people's health, the combination of traditional Chinese medicinal materials and modern biotechnology has become a research hotspot. Poria cocos, which promotes diuresis and percolates dampness, invigorates the spleen, and calms the heart, is mainly used for oliguria with edema, dizziness and palpitation due to phlegm retention, poor appetite due to spleen deficiency, loose stools and diarrhea, restlessness, palpitation, and insomnia. However, the existing processing technologies lack unified standards. For example, there are no clear regulations on the steaming time, temperature, etc., which may lead to inconsistent contents of active ingredients in the products and uneven quality. If the medicinal materials are not properly processed during the processing, it may cause the loss of nutrients such as vitamins and minerals.

[0006] With the pursuit of natural and healthy foods by people and the scientific verification of the role of probiotics in regulating intestinal health, enhancing immunity, etc., the development of Poria cocos products combined with probiotic fermentation technology can not only enhance the biological activity of Poria cocos but also improve its taste and flavor, meeting the market's demand for efficient, safe, and healthy foods.

[0007] As a biotransformation technology, probiotic fermentation can improve the nutritional value of raw materials, change their flavor characteristics, and even produce new functional components through the metabolic activities of microorganisms. Existing studies have shown that specific probiotics can break down plant cell walls, release more active ingredients, and simultaneously produce metabolites beneficial to the human body, such as short-chain fatty acids.

[0008] The process of microbial fermentation has been proven to improve the biological properties of plants and herbs. The application of microorganisms in traditional Chinese medicine processing mainly refers to the means of making the drug coated through various chemical reactions such as catalytic decomposition promoted by various intracellular and extracellular enzymes produced during the growth and metabolic activities of microorganisms under specific fermentation conditions such as temperature and humidity, using the processed traditional Chinese medicinal materials and various additives. Through probiotic fermentation technology, the efficient transformation and quality improvement of Poria cocos have been achieved, providing a new way for the development of medicine-food homologous products with unique health effects and good market acceptance.

[0009] For example, the Chinese patent application for invention with the publication number CN109430837A discloses a composition of ginger, cinnamon, jujube, stachyose, milk basic protein, adzuki bean, corn, oats, taro, yam, and pumpkin, which contains a large amount of carbohydrates, fats, and proteins, making the nutritional ratio in the large intestine moderate; and this miscellaneous grain powder contains a large amount of dietary fiber, providing sufficient nutrients for the spleen and stomach; it contains a large amount of pectin that can protect the gastrointestinal wall and activate the probiotics in the intestine, helping to regulate the gastrointestinal function and prevent constipation.

[0010] The Chinese invention patent application with the publication number CN103431261A combines "coarse grains" perfectly with traditional Chinese medicines of "medicinal and edible homology" and "new resource foods", organically combines diet and health preservation, regulates, prevents and resists spleen and stomach cold deficiency. On the basis of inheriting the treatment methods of the motherland's traditional Chinese medicine treasures, it innovatively uses a variety of probiotics as the vanguard to enhance the motor function of the spleen and stomach. At the same time, according to the physical characteristics of patients with spleen and stomach cold deficiency, it adds a variety of natural and original ecological medicinal and edible homologous ingredients, integrating the triple effects of diet therapy, prevention and conditioning into one unique advantage.

[0011] In the invention patent application with the publication number CN106177885A, one or several of Saccharomyces cerevisiae, Bacillus subtilis and Lactobacillus are used to ferment traditional Chinese medicine raw materials: Gentiana scabra, Dry Ginger, Eucommia ulmoides, Malt, Tangerine Peel, Atractylodes lancea, Magnolia officinalis, Licorice Root, Atractylodes macrocephala, Areca catechu. The compound fermented traditional Chinese medicine preparation for strengthening the stomach and invigorating the spleen is safe, effective, residue-free and drug-resistant.

[0012] In the invention patent application with the publication number CN118077899A, Lactobacillus animalis subsp. lactis Bb-12 and / or a mixture of Lactobacillus acidophilus are used to ferment Hawthorn Fruit, Malt, Chinese Yam, Tangerine Peel, White Hyacinth Bean, Poria cocos and Endothelium corneum gigeriae galli. Probiotics can regulate and maintain the balance of intestinal flora, promote gastrointestinal peristalsis, so as to achieve the effect of promoting digestion. Through the internal conditioning of the two, the digestive organs of children are regulated fundamentally to ensure the normal operation of the organs and stabilize the growth and development of children.

[0013] In the invention patent application with the publication number CN118077893A, Lactiplantibacillus plantarum and Streptococcus salivarius subsp. thermophilus are used to ferment Cannabis seed, Cassia seed, Hovenia dulcis, Black Sesame Seed, Sterculia lychnophora, Mulberry Fruit, Polygonatum odoratum, Hawthorn Fruit, Chinese Yam, Coix seed, Gorgon fruit, Lotus seed, Poria cocos, White Hyacinth Bean, Fructus citri sarcodactylis, Red Date, Dark Plum, Citrus aurantium L., Fructus foeniculi, Alpinia officinarum Hance, Amomum villosum Lour., Endothelium corneum gigeriae galli, Fructooligosaccharide, etc., and then by adding spleen aminopeptide concentrate Tremella polysaccharide, Taurine, food flavoring agent, etc. The composition can be used for people with disharmony between the spleen and stomach and loss of appetite, and has the advantage of good spleen-strengthening effect. Summary of the Invention

[0014] The first object of the present invention is to provide a Limosilactobacillus reuteri HX-LRE501.

[0015] The second object of the present invention is to provide a Limosilactobacillus reuteri that can improve the biological characteristics of Poria cocos.

[0016] The third object of the present invention is to provide a fermentation product of Limosilactobacillus reuteri.

[0017] The fourth object of the present invention is to provide a probiotic preparation comprising Limosilactobacillus reuteri.

[0018] The fifth object of the present invention is to provide an application of Limosilactobacillus reuteri.

[0019] The present invention is achieved by the following technical solutions:

[0020] A Limosilactobacillus reuteri HX-LRE501 was deposited with the China General Microbiological Culture Collection Center (CGMCC) on December 6, 2024, and its deposit number is CGMCC No. 32956.

[0021] The Limosilactobacillus reuteri HX-LRE501 as described above is used to ferment Poria cocos to obtain a fermentation product.

[0022] The fermentation product as described above includes a spleen and stomach conditioning factor;

[0023] The structural formula of the spleen and stomach conditioning factor is as follows:

[0024]

[0025] A probiotic preparation includes the Limosilactobacillus reuteri HX-LRE501 as claimed in claim 1, the fermentation product as described above, and a lyoprotectant.

[0026] The lyoprotectant includes 10-15% skim milk powder, 0.6-2.5% calcium alginate, 2-4% pectin, 1-2% inulin, and the balance is water.

[0027] The application of the Limosilactobacillus reuteri is used to prepare a food or drug for restoring the D-xylose content in serum.

[0028] The application of the Limosilactobacillus reuteri is used to prepare a food or drug for restoring the gastrin content in serum; or used to prepare a food or drug for restoring the motilin content in serum.

[0029] The application of the Limosilactobacillus reuteri is used to prepare a food or drug for restoring the vasoactive intestinal peptide content in serum. The application of the Limosilactobacillus reuteri is used to prepare a food or drug for restoring the serum amylase content in serum.

[0030] The application of the Limosilactobacillus reuteri is used to prepare a food or drug for restoring the immunoglobulin content in serum. Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] The present invention provides a Limosilactobacillus reuteri with a deposit number of CGMCC No. 32956. The Limosilactobacillus reuteri HX-LRE501 provided by the present invention can ferment Poria cocos, and its fermentation product contains a spleen and stomach protectant.

[0032] In the animal model with symptoms of weak spleen and stomach, the probiotic preparation and probiotic ferment of the present invention improve the weakness of the spleen and stomach by regulating the secretion of gastrointestinal hormones, promoting the normal peristalsis, digestion and absorption functions of the gastrointestinal tract, enhancing immunity, and protecting the intestinal mucosa. The present invention also provides a strain of Limosilactobacillus reuteri with unique fermentation ability, and also develops a probiotic preparation and its fermentation product based on this strain. These products have broad application prospects in improving the weakness of the spleen and stomach and promoting digestive system health.

[0033] (1) A strain of Limosilactobacillus reuteri HX-LRE501 was isolated from centenarians. It was found through research that this strain has good acid and bile salt tolerance and antibacterial ability. (2) This strain can obtain spleen and stomach conditioning factors by fermenting Poria cocos powder; (2) The probiotic preparation, ferment, and gastrointestinal conditioning factors of the present invention have obvious improvement effects on mice with weak spleen and stomach; (3) After fermenting Poria cocos powder with Limosilactobacillus reuteri HX-LRE501, the active ingredients in the Chinese herbal medicine can stimulate the peristalsis of the gastrointestinal tract and promote the secretion of digestive juices after fermentation, thereby improving the function of the spleen and stomach. At the same time, the probiotics and metabolites produced by fermentation can also regulate the balance of the intestinal flora and maintain intestinal health. The preparation after probiotic fermentation, as an immunomodulator, can activate and enhance the immune function of the body and improve resistance. Description of the Drawings

[0034] Figure 1 HE staining microscopic examination image of the spleen of mice in the normal control group

[0035] Figure 2 HE staining microscopic examination image of the spleen of mice in the model group

[0036] Figure 3 HE staining microscopic examination image of the spleen of mice in the probiotic preparation group

[0037] Figure 4 HE staining microscopic examination image of the spleen of mice in the ferment group

[0038] Figure 5 HE staining microscopic examination image of the spleen of mice in the spleen and stomach conditioning factor group

[0039] Figure 6 HE staining microscopic examination image of the spleen of mice in the probiotic group

[0040] Figure 7 HE staining microscopic examination image of the spleen of mice in the Sijunzi Decoction group Detailed Description of the Invention

[0041] Example 1

[0042] Isolation and Identification of Limosilactobacillus reuteri HX-LRE501

[0043] 1. Strain isolation, purification, and determination of gastric acid and bile salt tolerance

[0044] Take 2 g of feces from the elderly over 80 years old, put it into a centrifuge tube with 8 mL of sterile 0.85% normal saline, shake well, and enrich and culture at 37 °C for 30 min to obtain an enriched culture solution. The enriched culture solution is diluted 10-fold in gradients and diluted to 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 with 0.85% normal saline. After shaking and mixing evenly, take 100 μL of the dilution and spread it on the surface of MRS solid medium, and anaerobically culture at 37 °C for 48 h. Pick out single colonies with regular milky white edges and inoculate them on the surface of MRS solid medium for repeated streak purification. After culturing at 37 °C for 24 h, 10 pure strains are obtained. The in vitro gastric acid and bile salt tolerance and the utilization of Poria cocos powder of the 10 strains are determined. The activated bacterial solution is inoculated into the prepared artificial gastric juice and artificial intestinal juice containing 0.3% bile salt at a volume fraction of 10%, mixed evenly and cultured at 37 °C for 3 h. The viable bacteria counts at 0 h and 3 h are measured respectively. The results of the culture medium are shown in Table 1. Part of the glucose in the culture medium is replaced with Poria cocos powder. After culturing at 37 °C for 24 h, the growth of probiotics at different Poria cocos addition amounts is detected. The absorbance values of OD600 after fermentation are shown in Table 1.

[0045] The preparation method of the reagent is as follows:

[0046] Composition of MRS solid medium: 10.0 g of casein peptone, 2.0 g of ammonium citrate, 10.0 g of beef extract powder, 0.2 g of magnesium sulfate, 4.0 g of yeast extract powder, 0.05 g of manganese sulfate, 5.0 g of sodium acetate, 2.0 g of dipotassium hydrogen phosphate, 20.0 g of glucose, 1.08 g of Tween-80, 18 g of agar, made up to 1 L, final pH: 5.7 ± 0.2, sterilized at 121 °C for 15 min.

[0047] Composition of MRS liquid medium: 10.0 g of casein peptone, 2.0 g of ammonium citrate, 10.0 g of beef extract powder, 0.2 g of magnesium sulfate, 4.0 g of yeast extract powder, 0.05 g of manganese sulfate, 5.0 g of sodium acetate, 2.0 g of dipotassium hydrogen phosphate, 20.0 g of glucose, 1.08 g of Tween-80, made up to 1 L, final pH: 5.7, sterilized at 121 °C for 15 min.

[0048] Fermentation of Poria cocos powder: 10.0 g of caseinase digest, 2.0 g of ammonium citrate, 10.0 g of beef extract powder, 0.2 g of magnesium sulfate, 4.0 g of yeast extract powder, 0.05 g of manganese sulfate, 5.0 g of sodium acetate, 2.0 g of dipotassium hydrogen phosphate, 10.0 g of glucose, 10.0 g of Poria cocos powder, 1.08 g of Tween-80, made up to 1 L, final pH: 5.5, sterilized at 121 °C for 15 min.

[0049] Artificial gastric juice: 20 ml of sterilized normal saline plus 0.06 g of pepsin (3 g / L), adjusted to pH 3.0 with 0.1 mol / L HCl, and filtered through a 0.22-μm filter membrane into a sterilized empty conical flask for use.

[0050] Artificial intestinal juice (bile salt): 20 ml of sterilized normal saline plus 0.02 g of trypsin (1 g / L) and 0.06 g of bile salt (0.3%), adjusted to pH 8.0 with 0.1 mol / L NaOH, and filtered through a 0.22-μm filter membrane into a sterilized empty conical flask for use.

[0051] Table 1 Results of determination of acid and bile salt tolerance of strains

[0052]

[0053] According to the above test results, BS501-1 (HX-LRE501), BS501-3, and BS501-5 were selected for the next test to determine their hydrophobicity and self-aggregation.

[0054] 2. Determination of hydrophobicity and self-aggregation

[0055] Take the cultured bacterial solution, centrifuge at 6000 r / min for 10 min to collect the bottom bacteria. Wash the bacteria twice with PBS buffer. The blank control is PBS buffer, and the concentration of the bacteria is adjusted with PBS buffer to make the OD600 nm value about 1.00 (A0). Pipette 3 mL of the adjusted bacterial solution into a 10-mL sterile centrifuge tube, add 1 mL of xylene, shake for 2 min, then stand at 37 °C for 1 h. Pipette the lower aqueous phase and measure the OD600 nm value (A) with PBS buffer as the blank control. The calculation formula is:

[0056]

[0057] Take the cultured bacterial solution, centrifuge at 4000 r / min for 10 min to collect the bottom bacteria. Wash the bacteria twice with PBS buffer. The blank control is PBS buffer. Adjust the bacterial concentration with PBS buffer to make its OD600 nm value about 1.00 (A0). Let it stand at 37 °C, and take samples every 1 h to measure the optical density value (At) of the sample supernatant at 600 nm at different incubation times until 6 h. The calculation formula is:

[0058]

[0059] The specific screening results are shown in Table 2 below.

[0060] Table 2 Results of the hydrophobicity and self-aggregation of bacteria

[0061]

[0062] 3. Evaluation of the performance of inhibiting pathogenic bacteria

[0063] Select BS501-1 (HX-LRE501) and BS501-3 to measure the antibacterial ability. Study the inhibitory effects on Salmonella, Escherichia coli, Staphylococcus aureus, and Shigella flexneri. Activate the conditional pathogenic bacteria and test strains according to the corresponding culture conditions until the OD600nm is about 2. Take 100 μl of the bacterial solution of the conditional pathogenic bacteria and spread it on the MRS medium with glass beads. After the bacterial solution is absorbed (30 min), place the Oxford cup on the plate with sterile forceps, and then add 100 μl of the test strain to the Oxford cup. Use normal saline and antibiotics as controls. Place the plate upright and seal it, soak it at 4 °C for 2 h, and culture it at 37 °C for 24 h. Measure and record the diameter (mm) of the antibacterial circle of the test strains with a vernier caliper. The results are shown in Table 3.

[0064] Table 3 Results of the antibacterial ability determination

[0065] Strain Escherichia coli Salmonella Shigella flexneri Staphylococcus aureus 3BS501-1(HX-LRE501) 21.37 16.53 17.80 16.32 BS501-3 17.21 14.21 16.33 13.78

[0066] As can be seen from the above examples, the acid and bile salt tolerance ability, the nutritional utilization rate of poria powder, the self-aggregation ability, and the antibacterial ability of the strain 3BS501-1 (HX-LRE501) are relatively ideal. Therefore, Lactobacillus mucosae 3BS501-1 (HX-LRE501) is selected as the experimental strain for fermenting poria.

[0067] The strain was sequenced using 16S rDNA universal primers. The gene sequence of Lactobacillus mucosae HX-LRE501 is as follows: GCGGCGAGGCGATCAGCTATAATGCAGTCGTACGCACTGGCCCAACTGATTGATGGTGCTTGCACCTGATTGACGATGGATCACCAGTGAGTGGCGGACGGGTGAGTAACACGTAGGTAACCTGCCCCGGAGCGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAACAACAAAAGCCGCATGGCTTTTGTTTGAAAGATGGCTTTGGCTATCACTCTGGGATGGACCTGCGGTGCATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCGATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACAATGGAACTGAGACACGGTCCATACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGGCGCAAGCCTGATGGAGCAACACCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTGGAGAAGAACGTGCGTGAGAGTAACTGTTCACGCAGTGACGGTATCCAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTGCTTAGGTCTGATGTGAAAGCCTTCGGCTTAACCGAAGAAGTGCATCGGAAACCGGGCGACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGGAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTGCGCTAACCTTAGAGATAAGGCGTTCCCTTCGGGGACGCAATGACAGGTGGTGCATGGTCATCAACAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTACTAGTTGCCAGCATTAAGTTGGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAGATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTCGCAAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCGTTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCTCTCCATGGGAGTTTGTAACGCCCAAAGTCGGTGGCCTAACCTTATGGAGGCAGCCGCCTAAGACAGATAGTGGTCA

[0068] Example 2

[0069] Determination of the concentration of fermented Poria cocos by Lactobacillus mucosae HX-LRE501 and preparation method of its probiotic preparation. Influence of Poria cocos addition amount on the fermentation ability of bacteria: According to the culture medium formula, casein peptone 10.0 g, trisodium citrate 2.0 g, beef extract powder 10.0 g, magnesium sulfate 0.2 g, yeast extract powder 4.0 g, manganese sulfate 0.05 g, sodium acetate 5.0 g, dipotassium hydrogen phosphate 2.0 g, glucose 10.0 g, Tween-80 1.08 g, Poria cocos powder 5 - 20 g, make up the volume to 1 L with water, and adjust the pH to 5.8.

[0070] The addition amounts of Poria cocos were 5 g, 10 g, 15 g, and 20 g respectively. After the bacterial solution was cultured at 37 °C for 24 h, the absorbance value of the bacterial solution at OD600 was measured, and the OD values were compared. The results are shown in the table. When the addition amount was 15 g, the OD value was the highest.

[0071] Table 4 OD values with different addition amounts of Poria cocos

[0072] Strain Poria powder addition amount OD value HX-LRE501 5g 0.965 HX-LRE501 10g 1.012 HX-LRE501 15g 1.235 HX-LRE501 20g 1.110

[0073] The activated HX-LRE501 bacterial solution (3% by volume) was inoculated into a liquid fermentation medium (10.0 g of casein digest, 2.0 g of ammonium citrate, 10.0 g of beef extract powder, 0.2 g of magnesium sulfate, 4.0 g of yeast extract powder, 0.05 g of manganese sulfate, 5.0 g of sodium acetate, 2.0 g of dipotassium hydrogen phosphate, 10 g of glucose, 15 g of Poria cocos powder, 1.08 g of Tween-80, pH 5.8, made up to 1 L with water), and fermented at 37 °C for 48 hours to obtain a fermentation broth. After centrifugation, the supernatant and bacterial sludge precipitate were obtained.

[0074] Preparation of the probiotic preparation: The bacterial sludge precipitate and the probiotic freeze-drying protectant were thoroughly mixed and then freeze-dried (for subsequent animal experiments). The freeze-drying protectant includes: 10-15% skim milk powder, 0.6-2.5% calcium alginate, 2-4% pectin, 1-2% inulin, and the balance being water.

[0075] Example 3

[0076] Determination of the spleen and stomach conditioning factor of Lactobacillus mucosae HX-LRE501 fermented Poria cocos powder. After testing the fermentation product with a human gastrin (GAS) ELISA kit, it was found that the fermentation product of strain HX-LRE501 had an agonist effect on the gastrin receptor. To extract, isolate, and identify the spleen and stomach protectant from the fermentation broth, the present invention purified the supernatant of the fermentation broth. The specific steps are as follows:

[0077] First, this part of the bacterial solution was concentrated. Then, after concentration, it was freeze-dried to obtain a fermentation preparation (for subsequent animal experiments). It was dissolved in 75% ethanol to obtain a clear liquid. Subsequently, this clear liquid was slowly added to a column filled with HP20 macroporous adsorption resin at a rate of 0.5 times the volume per hour (BV / h) for adsorption treatment. Then, 10 times the volume of pure water and a 50% ethanol aqueous solution were used to elute the resin column at a flow rate of 1.5 times the volume per hour (BV / h) respectively. The purpose of these two elution steps was to remove some impurities and pigments adsorbed on the resin to ensure the purity of the subsequent extraction. Finally, 4 times the volume of 95% ethanol was used for elution, and this part of the eluate was collected and concentrated to obtain a paste-like crude extract.

[0078] 3. Detect the active components of the collected eluate by HPLC, combine the same components, concentrate under reduced pressure in a vacuum, dissolve the collected concentrate in DMSO, load it onto a DAC dynamic axial compression system, combine the target products after HPLC detection, and obtain the pure compound after vacuum drying. HPLC analysis conditions: mobile phase acetonitrile (A) - 0.1% formic acid in water (B) (1:9), isocratic elution at a flow rate of 1 mL / min. Chromatographic column: ZORBAX SB-C18 (250×4.6 mm, 25 μm); column temperature: 25 °C; wavelength: 242 nm; injection volume: 10 μL;

[0079] For the DAC dynamic axial compression system mentioned above, the packing material is C18, the mobile phase is 85% acetonitrile - aqueous solution, the flow rate is controlled at 10 ml / min, and the detection wavelength is 242 nm.

[0080] Among the active components extracted by the above method, the following compound - the spleen and stomach conditioning factor exhibits the effect of spleen conditioning.

[0081]

[0082] Determine the hydrogen spectrum 1H-NMR (600 MHz, CDCl3) and carbon spectrum 13C-NMR (600 MHz, CDCl3) of the compound by a nuclear magnetic resonance spectrometer to identify its chemical structure.

[0083] 1H-NMR (600 MHz, CDCl3)

[0084] 7.45 (1H in sec amine), 6.52 (1H in benzene), 6.07 (1H in benzene), 4.57 (1H in 1-ethylene), 4.50 (1H in 1-ethylene), 3.78 (2H in methylene), 3.61 (2H in methylene), 3.35 (2H in methylene), 3.01 (2H in methylene), 2.90 (2H in methylene), 2.68 (1H, in methine), 2.53 (2H in methylene), 2.51 (1H in methine), 2.38 (1H in methine), 2.18 (2H in methylene), 1.98 (3H in methyl), 1.80 (2H in methylene), 1.66 (2H in methylene), 1.38 (2H in methylene), 1.29 (2H in methylene), 1.28 (2H in methylene), 0.88 (3H in methyl), 0.86 (6H in methyl).

[0085] 13C-NMR (600 MHz, CDCl3)

[0086] 209.5, 154.2, 143.7, 127.3, 115.4, 112.1, 109.1, 105.1, 103.1, 70.7, 56.2, 56.2, 56.0, 55.9, 53.8, 46.8, 33.9, 32.2, 30.3, 29.2, 29.0, 26.3, 25.7, 23.3, 22.4, 21.5, 21.5, 14.1.

[0087] Furthermore, the agonist activity test of the above compound on the gastrin receptor is as follows:

[0088] Inoculate antral G cells corresponding to the human gastrin receptor gene into a 24-well culture plate at an appropriate cell density (such as 4×10 4 holes), culture them in serum-free medium for 12 h, set up groups, blank control (only medium), positive control (GRP10 - 7M), experimental group (spleen and stomach conditioning factor), and then place them in a constant temperature incubator at 37 °C for 1 hour.

[0089] Detection was carried out according to the method of the human gastrin (Gas) ELISA kit. The OD value was read at 450 nm with an enzyme-labeled instrument, and the gastrin concentration was calculated. Using the blank group as the baseline, the percentage increase or decrease in the secretion amount of the experimental group was calculated.

[0090] When the test concentration was 0.92 mM, the compound had a significant agonistic effect on the gastrin receptor, and the agonistic rate reached 93.2%. The agonistic rate of the positive control group was 95.3%. This result indicates that the compound can be used as a novel gastrin receptor agonist and has potential application value, such as improving the health function of the spleen and stomach.

[0091] Comparative Example 1

[0092] The pachymaran in Example 2 was replaced with glucose in the same proportion, and all other processes and parameters were carried out according to Example 2, resulting in the probiotic group.

[0093] Comparative Example 2

[0094] Sijunzi Decoction: The traditional Chinese medicine composition: 12 g of Codonopsis pilosula, 12 g of Poria cocos, 12 g of stir-fried Atractylodes macrocephala, and 6 g of roasted licorice. It was soaked in 8 times the amount of water for 1 h, decocted over low heat for 1 h, then decocted with 6 times the amount of water for another 1 h, and the medicinal liquids were combined and freeze-dried.

[0095] Example 4

[0096] Animal experiment performance test

[0097] 1. Animal experiment design: After 70 ICR mice were adaptively fed for 1 week, 60 of them were intragastrically administered senna at a dose of 0.4 g / d for modeling. When the mice showed symptoms of spleen and stomach weakness such as loose stools, huddling, arched backs, laziness, anal soiling, sparse and dull dorsal hair, while the normal control group (10 mice) had normal activities, shiny and thick coats, sensitive responses, and granular feces. The 50 successfully modeled mice were randomly divided into 6 groups, with 10 mice in each group, namely the model group, the probiotic preparation group, the fermented product group, the spleen and stomach conditioning factor group, Comparative Example 1 (probiotic group), and Comparative Example 2. The model group and the normal control group were intragastrically administered normal saline.

[0098] Probiotic preparation group: From the first day, intragastrically administered a probiotic preparation at a dose of 100 million CFU / kg / d;

[0099] Fermented product group: From the first day, intragastrically administered the concentrated raw material after fermentation at a dose of 1 g / kg / d;

[0100] Spleen and stomach conditioning factor group: From the first day, intragastrically administered the spleen and stomach conditioning factor after fermentation extraction at a dose of 1 g / kg / d;

[0101] Comparative Example 1 (probiotic group): From the first day, intragastrically administered probiotics at a dose of 100 million CFU / kg / d.

[0102] Control group 2: The mice were intragastrically administered with 1 g / kg / d of freeze-dried Sijunzi Decoction from the first day.

[0103] After 6 weeks of continuous intragastric administration, the dosage and efficacy of each preparation were evaluated.

[0104] Sampling of mice: After the successful establishment of the model, the mice were fasted but allowed to drink water for 24 hours, then intragastrically administered with 0.2 g / kg of D-xylose. One hour later, the animals were sacrificed by eye bleeding after enucleation. After the spleen of the mice was removed and quickly frozen, it was stored in a -80 °C refrigerator for later use. After the blood was allowed to stand for 1 hour, it was centrifuged at 4 °C and 3000 r / min for 10 min to obtain the serum.

[0105] Evaluation indicators: ① Body weight

[0106] ② Macroscopic manifestations

[0107] The mice in the blank group were sensitive in activity, had shiny hair, and their feces were granular. After the model was established in the model group, the mice showed symptoms of spleen deficiency such as loose stools, huddling together, arched backs, laziness in movement, perianal soiling, sparse and dull back hair, etc., indicating the successful preparation of the model.

[0108] ③ In the serum, D-xylose and its fermentation products can significantly improve the symptoms of spleen-deficiency mice and restore the levels of D-xylose, gastrin (GAS), motilin (MTL), vasoactive intestinal peptide (VIP), serum amylase (AMS), immunoglobulin G (IgG) and immunoglobulin M (IgM) in the serum to normal levels. At the same time, the structural changes of the spleen were observed by HE staining.

[0109] Explanation of evaluation indicators: Although the spleen-deficiency animal model shows a certain degree of agreement with the clinical diagnosis of spleen-deficiency syndrome in traditional Chinese medicine in terms of macroscopic manifestations, this consistency is still limited by the lack of an objective evaluation system. Spleen deficiency, as a complex traditional Chinese medicine syndrome covering multiple systems and organs, requires us to go beyond the surface and deeply explore objective quantitative indicators that can accurately capture the essence of spleen deficiency and are based on the core theory of the spleen. Only by organically integrating the identification of traditional Chinese medicine syndromes with these objective indicators can a widely recognized and scientific evaluation system be constructed.

[0110] As one of the key indicators for measuring the absorption efficiency of the small intestine, the content of serum D-xylose plays an important role in the construction and verification of the spleen-deficiency syndrome animal model, and the detection of its level is often used as a standard to evaluate the success of the model. This indicator is also included in the "Guiding Principles for Clinical Research of New Chinese Medicines" of the country as a reference for assisting the clinical diagnosis of spleen-deficiency syndrome.

[0111] On the other hand, there is a close connection between gastrin and the dysfunction of the spleen and stomach. It can not only promote gastric acid secretion, optimize the nutrition and blood supply of the gastric mucosa, but also accelerate the repair of the gastrointestinal mucosa and stimulate gastrointestinal motility. When the function of the spleen is abnormal and the transportation and transformation ability of the spleen and stomach decreases, it is often accompanied by abnormal changes in serum gastrin secretion.

[0112] In summary, although the spleen deficiency animal model corresponds to the clinical manifestations of spleen deficiency syndrome in traditional Chinese medicine at the macroscopic level, the lack of sufficient objectivity remains an urgent problem to be solved. Therefore, based on the basic theory of the spleen, screening and establishing a series of quantitative indicators that can comprehensively and objectively reflect the essence of spleen deficiency syndrome is crucial for deepening the scientific understanding and diagnostic accuracy of traditional Chinese medicine syndromes.

[0113] Model verification

[0114] ① Body weight

[0115] Table 5 Body weight of normal control group and model group

[0116] Group Body weight before modeling (g) Body weight 5 weeks after modeling (g) Normal control group 21.33 35.31 Model group 21.55 30.93

[0117] ② Macroscopic manifestations

[0118] In the normal control group, mice showed agile locomotor ability, shiny hair, and their feces were granular, indicating healthy signs. In contrast, in the model group, mice after specific modeling showed a series of spleen deficiency-related symptoms: the feces became thin, mice tended to huddle together, their backs were curved, activity decreased, the area around the anus was unclean, and the hair on the back became sparse and lost its luster.

[0119] ③ Detect the changes in the content of D-xylose and gastrin (GAS) in serum to verify whether the model is successfully established

[0120] Detect using xylose kit and gastrin ELISA kit.

[0121] Table 6 Comparison of D-xylose content and GAS content after modeling

[0122]

[0123] Detection of various indicators of spleen deficiency mice after continuous administration for 6 weeks

[0124] ① Effects of the preparation on the body weight and behavior of spleen deficiency mice

[0125] The mice in the normal control group showed agile locomotor ability, shiny hair, and their feces were granular in shape, indicating healthy signs. In the model group, the feces became thin, the mice tended to huddle together, their backs were bent, their activities decreased, the area around the anus was unclean, and the hair on their backs became sparse and lost its luster. After 6 weeks of administration, the activities of the mice in the probiotic preparation group, fermented product group, spleen and stomach conditioning factor group, probiotic group, and Sijunzi Decoction group returned to normal. Compared with the normal control group, the body weight of the mice in the model group decreased significantly, and the body weight of the spleen and stomach conditioning factor group was significantly higher than that of the model group. The body weight results are shown in Table 7.

[0126] Table 7 Effects of various preparations on the body weight of spleen-deficient mice

[0127] Group Body weight Normal control 38.76 Model group 34.57 Probiotic preparation group 39.48 Fermentation product group 38.65 Example 3 (Spleen and stomach conditioning factor group) 39.48 Comparative example 1 (Probiotic group) 37.71 Comparative example 2 (Sijunzi decoction group) 39.00

[0128] ② Determination of each index in serum

[0129] Compared with the normal control group, the content of D-xylose (D-xylose kit) decreased significantly, while the serum serotonin content of D-xylose in the probiotic preparation, fermented product, spleen and stomach conditioning factor, and Sijunzi Decoction groups of mice was significantly higher than that in the model group.

[0130] In the comparison of gastrin (GAS, Elisa kit), motilin (MTL, Elisa kit), and vasoactive intestinal peptide (VIP, Elisa kit) among gastrointestinal hormones in mice, compared with the normal control group, the levels of GAS and MTL in the serum of the model group were significantly increased, while VIP was significantly decreased. Compared with the model group, the levels of GAS and MTL in the serum of mice in the probiotic preparation, fermented product, spleen and stomach conditioning factor, and Sijunzi Decoction groups were significantly decreased, while VIP was significantly increased, and the trend returned to the level of the normal control group.

[0131] Compared with the normal control group, the level of serum amylase (AMS) in the model group was significantly decreased. Compared with the model group, the probiotic preparation, fermented product, spleen and stomach conditioning factor, and Sijunzi Decoction could all increase the content of AMS and gradually restore it to the normal level.

[0132] Compared with the normal control group, the levels of serum IgG and IgM in the model group were significantly decreased. Compared with the model group, the probiotic preparation, fermented product, spleen and stomach conditioning factor, and Sijunzi Decoction could all increase the content of IgG and IgM in mice.

[0133]

[0134]

[0135] ③ Observation of the structural changes of the spleen by HE staining

[0136] Compared with the normal control group, the model group had fewer splenic corpuscles, increased splenic blood sinuses, a larger white pulp area, and a relatively reduced red pulp area. The overall size of the spleen was half smaller than that of the control. Compared with the model group, the probiotic preparation, fermentate, spleen and stomach conditioning factor, Sijunzi Decoction, and probiotic group all had obvious restorative effects on the spleen.

Claims

1. A Lactobacillus reuteri HX-LRE501, characterized in that: Its deposit number is CGMCC No.32956.

2. A fermentation product obtained by fermenting Poria cocos with Lactobacillus reuteri HX-LRE501 as claimed in claim 1.

3. The fermentation product according to claim 2, characterized in that: Including spleen and stomach regulating factors; The structural formula of the spleen and stomach regulating factor is as follows:

4. A probiotic preparation, characterized in that: The invention comprises the Lactobacillus reuteri HX-LRE501 as claimed in claim 1, the fermentation product as claimed in claim 2 and a freeze-drying protective agent.

5. The probiotic preparation according to claim 4, characterized in that: The freeze-drying protective agent comprises 10-15% skim milk powder, 0.6-2.5% calcium alginate, 2-4% pectin, 1-2% inulin and the balance water.

6. The use of Lactobacillus reuteri as claimed in claim 1, characterized in that: The invention is used for preparing food or medicine for restoring the D-xylose content in serum.

7. The use of Lactobacillus reuteri as claimed in claim 1, characterized in that: For use in the preparation of food or medicine for restoring gastrin content in serum; or The invention is used for preparing food or medicine for restoring the content of motilin in serum.

8. The use of Lactobacillus reuteri as claimed in claim 1, characterized in that: The method is used for preparing food or medicine for restoring the content of vasoactive intestinal peptide in serum.

9. The use of Lactobacillus reuteri as claimed in claim 1, characterized in that: The invention is used for preparing food or medicine for restoring the serum amylase content in serum.

10. The use of Lactobacillus reuteri as claimed in claim 1, characterized in that: The invention is used for preparing food or medicine for restoring the immunoglobulin content in serum.

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