Immune enhancement capsule based on directional synergy of hirsutella hepialid strain and rhodiola rosea

Abstract

The invention relates to the technical field of health food, in particular to an immune enhancement capsule based on directional synergy of a hirsutella hepialid strain and rhodiola rosea. The mycelium adenosine content is increased through a hirsutella hepialid strain micro-aerobic directional fermentation process; efficient extraction and stability control of the salidroside are realized by combining a gradient water extraction technology with a composite antioxidant; after being wrapped by a nano-carrier technology, the astragalus extract is compounded with adenosine, salidroside, a codonopsis pilosula fermentation product and ganoderma spore wall-broken powder, so that the immune synergistic effect is enhanced; and microbial residues are controlled through an ozone and low-dose irradiation sterilization process, so that the loss of active ingredients is reduced. The adenosine and rhodiola rosea compound product can solve the problems of low active ingredient content, poor stability and insufficient synergistic effect in the adenosine and rhodiola rosea compound product, and is suitable for development of high-activity immunoregulation functional food.

Description

Technical Field

[0001] The invention relates to the technical field of health foods, in particular to an immune-enhancing capsule based on the directional synergy of Hirsutella batriaceae strains and Rhodiola rosea. Background Art

[0002] Cordyceps sinensis, a rare traditional medicinal herb, has been included in the pharmacopoeias of many countries for its immune-enhancing effects. To alleviate resource pressures, artificially cultivated mycelia of Cordyceps militaris and Hirsutella hepiali have been extensively studied. However, significant drawbacks remain in their industrial application: the adenosine content of commercially available mycelia is generally less than 100 mg / 100 g; the mycelial biomass of conventional liquid fermentation processes typically does not exceed 10 g / L, with production cycles lasting 10-15 days; and long-term propagation of strains is prone to genetic degeneration, with expression levels of adenosine biosynthesis gene clusters (such as the adenosine kinase encoding gene) decreasing by more than 50%, resulting in efficacy fluctuations exceeding 30% between product batches.

[0003] At the same time, Rhodiola rosea, as a plateau specialty medicinal plant, has a confirmed immune-enhancing mechanism for its core active ingredient, salidroside, but the chemical instability of this ingredient severely restricts product development. The traditional water extraction process uses continuous decoction at 100°C, which causes the glycosidic bond of salidroside to hydrolyze and break at high temperatures, resulting in a loss rate of over 30%. The extract is exposed to light and oxygen-induced oxidative degradation during storage, and the retention rate after 6 months in the accelerated test (40°C, RH75%) is less than 70%. Existing purification technologies, such as AB-8 macroporous resin, have an adsorption rate of only 60-70% for salidroside, and 70% ethanol elution can easily cause structural damage to heat-sensitive flavonoids.

[0004] In the field of compound preparation development, although some studies have attempted to combine Cordyceps mycelium with Rhodiola rosea to enhance the immunomodulatory effect, the existing technology still has systemic defects: most products simply physically mix Cordyceps powder and Rhodiola rosea extract, and clinical trials have shown that the IgG antibody level is increased by less than 30%; the differences in the physical and chemical properties of the raw materials make the preparations prone to stratification and agglomeration, and the disintegration time generally exceeds 45 minutes; Chinese patent CN101301413A discloses Cordyceps Rhodiola rosea capsules and their preparation methods, but the patent is vague about the ratio range of Cordyceps mycelium and Rhodiola rosea, causing the ratio of adenosine to salidroside to fluctuate by more than ±40%. In addition, the contradiction between the sterilization process and the retention of active ingredients is another technical pain point: conventional moist heat sterilization results in a loss of more than 25% of salidroside and a loss of more than 15% of adenosine; ozone or ethylene oxide treatment may generate toxic byproducts such as bromate; and high doses 60 Co irradiation caused the polysaccharide chain to break and the liposome encapsulation structure to collapse, resulting in a decrease of more than 50% in the in vitro release of functional ingredients. Summary of the Invention

[0005] The purpose of the present invention is to provide an immune-enhancing capsule based on the directional synergy of the Hirsutella batriaceae strain and Rhodiola rosea, so as to solve the problems raised in the above background technology.

[0006] In order to solve the above technical problems, the present invention provides the following technical solution: an immune-enhancing capsule based on the directional synergistic effect of Hirsutella batriaceae strain and Rhodiola rosea, comprising the following components by weight:

[0007] 50-55 parts of Hirsutella batriacea mycelium powder, 6-10 parts of Rhodiola rosea extract, 10-12 parts of astragalus extract nanoliposomes, 15-18 parts of Codonopsis pilosula fermentation product, 8-12 parts of Ganoderma lucidum spore wall-broken powder, and 0.8-1 part of magnesium stearate.

[0008] Furthermore, the preparation method of the immune enhancement capsule based on the directional synergistic effect of the Hirsutella batriacea strain and Rhodiola rosea is characterized by comprising the following steps:

[0009] S1: Inoculating a culture medium with a strain of Hirsutella batrix; culturing the culture medium under microaerobic conditions of 26±0.5°C, pH 6.5±0.1, and a dissolved oxygen saturation of 5-10% for 144±2 hours; collecting the mycelium, freezing and pulverizing the mycelium at -40--30°C to a particle size of ≤50 μm and a mycelial cell wall rupture rate of ≥99%, to obtain Hirsutella batrix mycelium powder having an adenosine content of ≥250 mg / 100 g;

[0010] S2: extracting Rhodiola rosea by gradient water extraction technology, first adding 10 times the weight of the Rhodiola rosea medicinal material to the medicinal material, adjusting the pH to 5.5±0.1, adding a tea polyphenol-chitosan complex aqueous solution, extracting at 80-85°C, filtering out the medicinal residue and crushing it into 100 mesh; again adding 8 times the weight of the medicinal material to the medicinal residue, adjusting the pH to 6.0±0.1, adding a tea polyphenol-chitosan complex aqueous solution again, extracting at 70-75°C, filtering out the medicinal residue; combining the two extracts, first purifying with resin, then eluting with ethanol solution, and drying to obtain a Rhodiola rosea extract, wherein the salidroside content is ≥7wt.%;

[0011] S3: Astragalus extract, soybean lecithin, and cholesterol are mixed in a mass ratio of 4:1:0.5, and astragalus extract nanoliposomes are prepared by thin-film ultrasound, with a particle size distribution D90 ≤ 100 nm and an encapsulation efficiency ≥ 95%; the astragalus extract contains astragaloside IV and flavonoids;

[0012] S4: fermenting Codonopsis pilosula with Lactobacillus plantarum, drying, and crushing to obtain a Codonopsis pilosula fermentation product having a β-glucan content of ≥5 wt.%;

[0013] S5: breaking the spores of Ganoderma lucidum to obtain a spore breaking rate of ≥98%, thereby obtaining broken spore powder of Ganoderma lucidum;

[0014] S6: Evenly mix the mycelial powder of Trichosporon batriaceae, Rhodiola rosea extract, Astragalus extract nanoliposomes, Codonopsis pilosula fermentation product, and Ganoderma lucidum spore wall-broken powder, dry them, grind them into fine powder, and pass them through a 180-200 mesh sieve to obtain a mixed powder;

[0015] S7: Sterilization: After the mixed powder is treated with ozone for 30 to 40 minutes, 60 Coγ-ray irradiation sterilization;

[0016] S8: Add magnesium stearate to the sterilized mixed powder, mix evenly, and fill to obtain immune-enhancing capsules.

[0017] Furthermore, in S1, the culture medium contains 30-40 g / L of glucose, 2-3 g / L of silkworm pupa peptide, 10-15 g / L of soybean meal powder, and 5-8 g / L of yeast extract powder.

[0018] Furthermore, in S2, citric acid or acetic acid is used to adjust the pH during the first extraction, and acetic acid-sodium acetate buffer is used to adjust the pH during the second extraction.

[0019] Furthermore, in S2, the concentration of the tea polyphenol-chitosan complex aqueous solution is 1-2% w / v, which is prepared by mixing tea polyphenol and chitosan in a mass ratio of 2:1 and then adding a volume concentration of 1-2% glacial acetic acid aqueous solution; each time the extraction is performed, the amount of the tea polyphenol-chitosan complex aqueous solution added is 0.1-0.2% of the volume of the extraction solvent.

[0020] Furthermore, in S3, the astragalus extract has an astragaloside IV content of ≥0.5 wt.%, and a flavonoid content of ≥8 wt.%.

[0021] Furthermore, in S4, the fermentation process is to control the fermentation temperature at 24-26° C., the initial pH of the culture medium at 5.5-6.0, the moisture content at 40-50%, add 0.2-0.5% of the substrate mass of trehalose, and ferment for 72-96 hours.

[0022] Furthermore, in S7, the ozone concentration is 5 to 6 ppm, 60 The Coγ irradiation dose is 3 to 3.5 kGy.

[0023] Furthermore, in S8, 100 g of the immune enhancement capsule contains salidroside ≥ 350 mg, adenosine ≥ 100 mg, and β-glucan ≥ 700 mg.

[0024] The immune-enhancing capsule prepared by the present invention has the effect of regulating the function of the intestinal immune-endocrine axis. Compared with the existing technology, the beneficial effects achieved by the present invention are:

[0025] 1. The present invention significantly improves the active ingredient content, stability and immunomodulatory efficacy of the compound capsules through stabilized extraction, nanoencapsulation and low-damage sterilization processes. In the mycelium fermentation process, a specific culture medium containing silkworm pupa peptides and soybean meal powder is used, combined with a low-temperature dissolved oxygen control process, so that the biomass of the mycelium of the bat moth hair spore is significantly improved compared with the traditional process, and the adenosine content is stabilized at more than 250mg / 100g. Rhodiola rosea extraction uses double crushing and pH gradient water extraction technology, and adds tea polyphenol-chitosan complex to inhibit oxidation. The extract contains salidroside ≥7wt.%, and the accelerated test (40℃, RH75%, 6 months) has a retention rate of ≥95%. Astragalus extract is encapsulated using nanoliposome technology, with a particle size D90≤100nm and an encapsulation rate ≥95%, which can achieve intestinal targeted release. The release rate in the intestinal fluid exceeds 90% in 6 hours, greatly improving bioavailability.

[0026] 2. In terms of immune efficacy, animal experiments confirmed that the compound capsules of this invention can synergistically enhance multiple immune functions. The high-dose 900 mg / kg group showed significantly higher splenic lymphocyte proliferation compared to the control group, stronger delayed-type hypersensitivity reactions, and increased numbers of antibody-producing cells and carbon clearance phagocytosis index, without causing abnormalities in immune organ weights.

[0027] 3. In terms of production process, the ozone pretreatment combined with irradiation sterilization technology provided by the present invention has a higher retention rate of salidroside and adenosine. Compared with high-temperature sterilization, the loss rate of active ingredients is significantly reduced, and the total colony count is far below the national standard limit; the disintegration time is shortened to 23 minutes, which meets the requirements of the Chinese Pharmacopoeia. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0029] Figure 1 It is a process flow chart of the present invention.

[0030] Figure 2 This is a photo of the finished product of the immune enhancement capsule prepared by the present invention. DETAILED DESCRIPTION

[0031] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0032] Materials and sources used in the present invention:

[0033] Hirsutella batriacea strain: China Industrial Culture Collection Administration Center (CICC), strain number: CICC14106; Lactobacillus plantarum: China Industrial Culture Collection Administration Center (CICC), strain number: CICC 25125; AB-8 resin: Tianjin Nankai Hecheng Technology Co., Ltd.; No. 0 gelatin capsule: Suzhou Capsule Co., Ltd., model: 0#; Astragalus extract: Xi'an Tianyi Biotechnology Co., Ltd.

[0034] Example 1: A method for preparing an immune-enhancing capsule based on the synergistic effect of Hirsutella batrifolia strain and Rhodiola rosea, comprising the following steps:

[0035] Step 1: Preparation of mycelial powder of Hirsutella batriata: inoculate Hirsutella batriata into liquid culture medium (containing 30 g / L glucose, 2 g / L silkworm pupa peptide, 10 g / L soybean meal powder, and 5 g / L yeast extract powder), and ferment at 26°C, pH 6.5, and 5% dissolved oxygen for 144 hours.

[0036] The mycelium biomass reached 16.2 kg / m 3 , an increase of 62% compared with the control group without silkworm pupa peptide; it was frozen and crushed at -40℃ to D50=50μm, and the adenosine content was determined by HPLC according to the 2020 edition of the "Chinese Pharmacopoeia" to be 258mg / 100g.

[0037] Preparation of Rhodiola rosea extract: Take the rhizome of Tibetan Rhodiola rosea, crush it to 40 mesh, add 10 times its weight of deionized water, adjust the pH to 5.5 with citric acid, extract at 80°C for 2h, filter the residue and crush it to 100 mesh, add 8 times its weight of deionized water, adjust the pH to 6.0 with acetic acid-sodium acetate buffer, extract at 70°C for 2h; add tea polyphenol-chitosan complex aqueous solution (the tea polyphenol-chitosan complex is equivalent to 0.1% of the extraction solvent volume) in both extractions. The invention relates to a polysaccharide complex solution prepared by dissolving tea polyphenols and chitosan in a 2:1 mass ratio in an aqueous solution containing 2% glacial acetic acid by volume (the total concentration of the tea polyphenol-chitosan complex is 1% w / v); the combined extracts are purified by AB-8 resin and then eluted with 30% ethanol by mass to obtain a rhodiola rosea extract; ultraviolet spectrophotometry is used to determine the purity of salidroside to be 96.5%, and the extraction rate to be 92.3%, equivalent to obtaining 7.2g of salidroside per 100g of raw material.

[0038] Astragalus Extract Nanoliposomes: Astragalus extract (containing saponin IV ≥ 0.5 wt.%, flavonoids ≥ 8 wt.%), soybean lecithin, and cholesterol were dissolved in a chloroform-methanol solution (v / v 2:1) at a mass ratio of 4:1:0.5. The resulting film was rotary evaporated and then hydrated. Ultrasonication at 300W for 30 minutes at 40°C was performed. Dynamic light scattering determined the resulting liposome particle size (D90) to be 100 nm. Ultrafiltration-HPLC analysis demonstrated an encapsulation efficiency of 95.2%. In vitro release revealed a 21.5% release in artificial gastric fluid (2 hours) and a cumulative release of 91.5% in artificial intestinal fluid (6 hours).

[0039] Preparation of Codonopsis pilosula fermentation product: Codonopsis pilosula was fermented with Lactobacillus plantarum, the fermentation temperature was controlled at 25°C, the initial pH of the culture medium was 6.0, the moisture content was 44%, 0.4% of the substrate weight of trehalose was added, fermentation was carried out for 96 hours, and the Codonopsis pilosula fermentation product was obtained by drying and crushing.

[0040] The dry powder sample of Codonopsis pilosula fermentation product was extracted with boiling water, and the crude polysaccharide was precipitated with 80% ethanol and washed repeatedly. The precipitate was dissolved in alkaline copper reagent to selectively precipitate the glucan structure. After centrifugation, the residue was dissolved in sulfuric acid, and the phenol-sulfuric acid color reaction was used to quantitatively calculate the β-glucan content in the Codonopsis pilosula fermentation product, which was 5.4wt.%, using a glucan standard curve.

[0041] Preparation of Ganoderma lucidum spore wall-broken powder: Ganoderma lucidum spores are subjected to wall-breaking treatment, with a wall-breaking rate of ≥98%, to obtain Ganoderma lucidum spore wall-broken powder.

[0042] Compound molding and sterilization: 50 kg of bat moth mycelium powder, 8 kg of Rhodiola rosea extract, 10 kg of Astragalus extract nanoliposomes, 15 kg of Codonopsis pilosula fermentation product and 8 kg of Ganoderma lucidum spore wall-broken powder were weighed and mixed evenly according to the mass ratio, dried and crushed into fine powder to obtain a mixed powder. After being treated with 5 ppm ozone for 30 min, the mixture was sterilized with 3 kGy 60 The mixture was sterilized by Co irradiation, 0.8 kg of magnesium stearate was added to the sterilized mixed powder, mixed in a V shape for 30 min, and filled into size 0 gelatin capsules to obtain the finished product.

[0043] The various components in the finished compound capsules prepared in this example were tested, and the experimental results are shown in Table 1. The disintegration time was tested with reference to the 2020 edition of the Chinese Pharmacopoeia, and the total bacterial count was tested with reference to GB 4789.2; the adenosine and salidroside contents were detected by HPLC, and the β-glucan content was detected by the phenol-sulfuric acid method.

[0044] Table 1 Key quality parameters of compound capsule finished products

[0045] index Numerical Test methods / standards Adenosine content (mg / 100g) 127 HPLC in the 2020 edition of the Chinese Pharmacopoeia Salidroside content (mg / 100g) 453 HPLC in the 2020 edition of the Chinese Pharmacopoeia β-glucan (mg / 100g) 792 Phenol-sulfuric acid method Disintegration time (min) 23 Chinese Pharmacopoeia 2020 Edition Total colony count (CFU / g) 80 GB4789.2

[0046] Example 2: The immunomodulatory efficacy of the immune-enhancing capsule prepared in Example 1 was verified.

[0047] SPF-grade Kunming male mice weighing 18-22 g were selected and randomly divided into a negative control group (oral administration of pure water), a low-dose group (225 mg / kg BW), a medium-dose group (450 mg / kg BW), a high-dose group (900 mg / kg BW) and a positive control group (levamisole 25 mg / kg BW) used only in ConA experiments, with 10 mice in each group. With reference to the "Technical Specifications for Inspection and Evaluation of Health Foods", all mice were orally administered for 30 consecutive days. The test indicators included cellular immune functions such as ConA-induced spleen lymphocyte transformation ability and delayed-type hypersensitivity reaction (DTH), humoral immune functions such as serum hemolysin HC5 level and the number of antibody-producing cells, and macrophage functions such as carbon clearance ability and the phagocytic ability of macrophages to chicken red blood cells.

[0048] In terms of immune organs, the data in Table 2 show that there was no significant difference in weight gain, thymus / body weight ratio, and spleen / body weight ratio of mice in each dose group compared with the negative control group (P>0.05), that is, the samples had no significant effect on the body weight and immune organ weight of mice.

[0049] Table 2 Ratio of immune organs to body weight of mice ( n=10)

[0050] Dosage group (mg / kg BW) Thymus / body weight (%) P-value Spleen / body weight (%) P-value 900 0.194±0.023 0.403 0.379±0.037 0.780 450 0.206±0.024 0.996 0.399±0.054 0.981 225 0.194±0.021 0.433 0.387±0.024 0.977 Negative control 0.207±0.021 - 0.393±0.048 -

[0051] In terms of cellular immune function, the data in Table 3 show that the proliferation capacity (OD value) of spleen lymphocytes in each dose group of the sample was significantly higher than that in the negative control group, among which the high-dose group (900 mg / kg) reached 0.325±0.077 (P<0.01); the data in Table 4 show that the weight difference of delayed-type hypersensitivity ear pieces in the high-dose group was 20.50±6.51 mg (P<0.05), showing a promoting effect on cellular immunity.

[0052] Table 3 Splenic lymphocyte proliferation ability (OD value, )

[0053] Dosage group (mg / kg BW) Lymphocyte proliferation ability (OD value) P-value 900 0.325±0.077 <0.01 450 0.288±0.051 <0.01 225 0.250±0.066 <0.05 Negative control 0.177±0.047 -

[0054] Table 4 Delayed allergic reaction (ear piece weight difference, mg)

[0055] Dosage group (mg / kg BW) Ear piece weight difference (mg) P-value 900 20.50±6.51 <0.05 450 19.17±4.80 0.086 225 17.75±3.97 0.261 Negative control 13.80±6.10 -

[0056] In the humoral immune function test, the data in Table 5 showed that the number of antibody-producing cells (hemolytic plaque number) in the high-dose and medium-dose groups were 226.9±40.8×10 6 / 10 6 Spleen cells, 205.1±23.6×106 / 10 6 spleen cells (P<0.01, P<0.05); the data in Table 6 showed that the serum hemolysin antibody volume counts in the high-dose and medium-dose groups were 189.0±21.6 and 179.9±12.2, respectively (P<0.01, P<0.05), which were significantly higher than those in the control group.

[0057] Table 5 Antibody-producing cell numbers (×10 6 / 10 6 splenocytes)

[0058] Dosage group (mg / kg BW) <![CDATA[溶血空斑数(×10 6 / 10 6 splenocytes)]]> P-value 900 226.9±40.8 <0.01 450 205.1±23.6 <0.05 225 195.6±27.8 0.062 Negative control 164.6±21.8 -

[0059] Table 6 serum hemolysin antibody volume (HC 50 )

[0060] Dosage group (mg / kg BW) Anti-volume number P-value 900 189.0±21.6 <0.01 450 179.9±12.2 <0.05 225 168.7±12.3 0.324 Negative control 157.6±18.5 -

[0061] Regarding monocyte-macrophage function, data in Table 7 indicate that the carbon clearance phagocytosis index in the high-dose group was 9.05±0.99 (P<0.05); while data in Table 8 indicate that the phagocytic rate and phagocytic index of peritoneal macrophages were not significantly different from those in the control group (P>0.05). Furthermore, there were no significant differences in NK cell activity between the various dose groups and the control group (P>0.05).

[0062] The positive results of three key indicators, namely, cellular immunity, humoral immunity and monocyte-macrophage function, indicate that the capsule of the present invention has a clear function of enhancing immunity.

[0063] Table 7 Carbon clearance phagocytosis index of monocytes and macrophages (α)

[0064] Dosage group (mg / kg BW) Phagocytic index P-value 900 9.05±0.99 <0.05 450 8.71±1.00 0.137 225 8.60±1.42 0.199 Negative control 7.65±1.33 -

[0065] Table 8 Phagocytic rate of peritoneal macrophages and NK cell activity

[0066]

[0067] Comparative Example 1: Rhodiola rosea was extracted using a traditional single water extraction process, with the remaining parameters being the same as in Example 1.

[0068] After the rhizome of Rhodiola rosea was crushed, 10 times the amount of water (pH = 7.0) was directly added, and it was decocted at 100°C for 3 hours. The extract was spray-dried (inlet air temperature 180°C) to obtain a powder. The results showed that the content of salidroside in the extract was 3.2wt.%, and the loss rate was 56% compared with Example 1. The color of the extract was dark brown, indicating that the Maillard reaction occurred. The disintegration time of the compound capsule reached 45 minutes, exceeding the upper limit of 30 minutes stipulated in the pharmacopoeia, and the subjects reported a significant bitter taste. This is because its Maillard reaction product - melanoidin is highly hydrophobic, and after being added to the capsule, it hinders water penetration, resulting in excessive disintegration; at the same time, the bitter by-products generated by the Maillard reaction (such as burnt bitter melanoidin, furfural, etc.) and the high temperature destroys the taste-masking components, which together aggravate the bitterness problem of the capsule.

[0069] Comparative Example 2: No tea polyphenol-chitosan complex was added during the extraction of Rhodiola rosea, and the other parameters were the same as those in Example 1.

[0070] The results showed that compared with Example 1, the purity of salidroside in Comparative Example 2 decreased from 96.5% to 82.4%. After 6 months of accelerated testing (40°C, RH75%), the purity of salidroside in Example 1 was 95.2%, while the retention rate in Comparative Example 2 was only 68.5%. In addition, the improvement in NK cell activity of the compound preparation decreased from 66.2% to 42.5%, demonstrating that the tea polyphenol-chitosan complex plays a key role as a stabilizer in protecting ingredients and maintaining efficacy.

[0071] Comparative Example 3: The sterilization process adopts moist heat sterilization at 121° C. for 20 minutes, and the other parameters are the same as those in Example 1.

[0072] The test found that the salidroside content dropped from 453mg / 100g to 340mg / 100g, a decrease of 24.9% compared with Example 1; the adenosine content dropped from 127mg / 100g to 96mg / 100g, with a retention rate of only 75.6%; the β-glucan content dropped from 792mg / 100g to 676mg / 100g, a decrease of 14.7% compared with Example 1; the total colony count was 1200CFU / g, and the capsule contents were clumped, with poor fluidity, an angle of repose ≥50°, and could not be filled normally. This is because the high temperature and high humidity environment can cause the heat-sensitive components to decompose, and the steam causes the material to clump and lose fluidity, and the sterilization is not thorough.

[0073] Example 3: Large-scale production verification.

[0074] The production was expanded in a 500L fermenter, and the culture medium formulation and fermentation conditions were the same as in Example 1. Three consecutive batches of tests showed that the mycelial biomass was stable at 15.8-16.5 kg / m 3The adenosine content is 255-260mg / 100g, the salidroside extraction rate is ≥92%, and the qualified rate of finished capsules is ≥98.5%. Microbiological testing showed that the total colony counts of the three batches of products were 75 CFU / g, 82 CFU / g, and 79 CFU / g, respectively, all meeting national standards and confirming the process stability and industrial feasibility.

[0075] The present invention achieves a salidroside content of ≥350 mg / 100 g and adenosine ≥100 mg / 100 g in the capsule through strain-specific fermentation, gradient water extraction and nano-encapsulation technology; compared with traditional processes, the immune index is improved by more than 50%, the total retention rate of active ingredients (adenosine, salidroside, β-glucan) after sterilization is ≥98%, the disintegration time is shortened to 23 minutes, and the large-scale production cost is reduced, which has clear clinical application value and market competitiveness.

[0076] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus.

[0077] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for preparing an immune-enhancing capsule based on the directional synergistic effect of Hirsutella batrifolia strain and Rhodiola rosea, characterized in that: The following steps are involved: S1: inoculating a culture medium with a strain of Hirsutella batrix, and microaerophilically fermenting the culture at 26±0.5°C, pH 6.5±0.1, and a dissolved oxygen saturation of 5-10% for 144±2 hours. Mycelia were collected and frozen at -40--30°C to a particle size of ≤50 μm and a mycelial cell wall rupture rate of ≥99%, thereby obtaining Hirsutella batrix mycelial powder having an adenosine content of ≥250 mg / 100 g. S2: extracting Rhodiola rosea by gradient water extraction technology, first adding 10 times the weight of the Rhodiola rosea medicinal material to the medicinal material, adjusting the pH to 5.5±0.1, adding a tea polyphenol-chitosan complex aqueous solution, extracting at 80-85°C, filtering out the medicinal residue and crushing it into 100 mesh; again adding 8 times the weight of the medicinal material to the medicinal residue, adjusting the pH to 6.0±0.1, adding a tea polyphenol-chitosan complex aqueous solution again, extracting at 70-75°C, filtering out the medicinal residue; combining the two extracts, first purifying with resin, then eluting with ethanol solution, and drying to obtain a Rhodiola rosea extract with a salidroside content of ≥7wt.%; S3: Mixing astragalus extract, soybean lecithin, and cholesterol, and preparing astragalus extract nanoliposomes by thin-film ultrasound, controlling the particle size distribution D90 to be ≤100 nm and the encapsulation efficiency to be ≥95%; the astragalus extract contains astragaloside IV and flavonoids; S4: fermenting Codonopsis pilosula with Lactobacillus plantarum, drying, and crushing to obtain a Codonopsis pilosula fermentation product having a β-glucan content of ≥5 wt.%; S5: breaking the spores of Ganoderma lucidum to obtain a spore breaking rate of ≥98%, thereby obtaining broken spore powder of Ganoderma lucidum; S6: Evenly mix the mycelial powder of Trichosporon batriaceae, Rhodiola rosea extract, Astragalus extract nanoliposomes, Codonopsis pilosula fermentation product, and Ganoderma lucidum spore wall-broken powder, dry them, grind them into fine powder, and pass them through a 180-200 mesh sieve to obtain a mixed powder; S7: Sterilization: After the mixed powder is treated with ozone for 30 to 40 minutes, 60 Coγ-ray irradiation sterilization; S8: Add magnesium stearate to the sterilized mixed powder, mix evenly, and fill to obtain immune-enhancing capsules.

2. The method for preparing an immune-enhancing capsule based on the directional synergistic effect of Hirsutella batrifolia strain and Rhodiola rosea according to claim 1, characterized in that: In S1, the culture medium contains 30-40 g / L of glucose, 2-3 g / L of silkworm pupa peptide, 10-15 g / L of soybean meal powder, and 5-8 g / L of yeast extract powder.

3. The method for preparing an immune-enhancing capsule based on the directional synergistic effect of Hirsutella batrifolia strain and Rhodiola rosea according to claim 1, characterized in that: In S2, citric acid or acetic acid was used to adjust the pH during the first extraction, and acetic acid-sodium acetate buffer was used to adjust the pH during the second extraction.

4. The method for preparing an immune-enhancing capsule based on the directional synergistic effect of Hirsutella batrifolia strain and Rhodiola rosea according to claim 1, characterized in that: In S2, the concentration of the tea polyphenol-chitosan complex aqueous solution is 1-2% w / v, which is prepared by mixing tea polyphenol and chitosan in a mass ratio of 2:1 and then adding a volume concentration of 1-2% glacial acetic acid aqueous solution; each time the extraction is performed, the amount of the tea polyphenol-chitosan complex aqueous solution added is 0.1-0.2% of the volume of the extraction solvent.

5. The method for preparing an immune-enhancing capsule based on the directional synergy of Hirsutella batrifolia strain and Rhodiola rosea according to claim 1, characterized in that: In S3, astragalus extract, soybean lecithin and cholesterol are mixed in a mass ratio of 4:1:0.5; in the astragalus extract, the content of astragaloside IV is ≥0.5wt.%, and the content of flavonoids is ≥8wt.%.

6. The method for preparing an immune-enhancing capsule based on the directional synergistic effect of Hirsutella batrifolia strain and Rhodiola rosea according to claim 1, characterized in that: In S4, the fermentation temperature is 24-26° C., the initial pH of the culture medium is 5.5-6.0, the moisture content is 40-50%, 0.2-0.5% of the substrate weight of trehalose is added, and the fermentation is carried out for 72-96 hours.

7. The method for preparing an immune-enhancing capsule based on the directional synergy of Hirsutella batrifolia strain and Rhodiola rosea according to claim 1, characterized in that: In S7, the ozone concentration is 5-6 ppm. 60 The Coγ irradiation dose is 3 to 3.5 kGy.

8. The method for preparing an immune-enhancing capsule based on the directional synergistic effect of Hirsutella batrifolia strain and Rhodiola rosea according to claim 1, characterized in that: In S8, the contents of each component in the immune enhancement capsule are calculated by weight: 50-55 parts of Hirsutella batriata mycelium powder, 6-10 parts of Rhodiola rosea extract, 10-12 parts of Astragalus extract nanoliposomes, 15-18 parts of Codonopsis pilosula fermentation product, 8-12 parts of Ganoderma lucidum spore wall-broken powder, and 0.8-1 part of magnesium stearate.

9. The method for preparing an immune-enhancing capsule based on the directional synergy of Hirsutella batrifolia strain and Rhodiola rosea according to claim 1, characterized in that: In S8, 100g of immune enhancement capsules contains salidroside ≥350mg, adenosine ≥100mg, and β-glucan ≥700mg.

10. The immune-enhancing capsule based on the directional synergy of Hirsutella batriaceae strain and Rhodiola rosea prepared by the preparation method according to any one of claims 1 to 9.

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