Modified preparation method of dendrobium polypeptide modified immunoglobulin

Abstract

This invention discloses a method for modifying immunoglobulins with Dendrobium officinale polypeptides, belonging to the field of biomodification technology. This invention uses enzymatic hydrolysis of Dendrobium officinale polypeptides to covalently modify immunoglobulins. By controlling the enzymatic hydrolysis and grafting processes through specific process parameters, Dendrobium officinale polypeptides with molecular weights concentrated in the 3-5 kDa range are obtained. Using EDC / NHS as a cross-linking agent, stable covalent binding between the polypeptides and immunoglobulins is achieved under mild conditions. This invention solves the problems of short in vivo half-life and low bioavailability of existing immunoglobulins. The modified product combines the antioxidant and yin-nourishing effects of Dendrobium officinale with the immune activity of immunoglobulins in neutralizing exogenous pathogens. The immunoglobulin activity retention rate reaches over 90%, the in vivo half-life is increased to 72-96 h, and the bioavailability reaches 45%-60%. This invention also provides a special medical food compound preparation formula containing this modified product. The product has high safety and good stability, meeting the needs of special medical food development and providing a new technical path for the synergistic application of natural active substances.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of biomodification technology and preparation of food raw materials for special medical purposes, and specifically relates to a method for modifying and preparing immunoglobulins with Dendrobium polypeptides. Background Technology

[0002] Immunoglobulins (Ig) are important bioactive immune proteins widely found in mammalian body fluids and colostrum. They play a crucial role in neutralizing exogenous pathogens and regulating the body's immune response, making them one of the core active ingredients in special dietary foods, functional foods, and medical foods used to enhance the body's immune defense capabilities. Especially in today's high-pressure lifestyles, factors such as staying up late, irregular sleep patterns, and mental stress lead to weakened immunity and a growing sub-healthy population, creating an increasingly urgent demand for oral immunomodulatory foods.

[0003] However, existing immunoglobulins face insurmountable technical bottlenecks in practical applications: on the one hand, the molecular structure of natural immunoglobulins is unstable, and they are easily degraded by hydrolytic enzymes such as pepsin and trypsin after entering the gastrointestinal tract, leading to rapid inactivation of active fragments. Their half-life in vivo is usually only 12-24 hours, far from meeting the metabolic cycle requirements for long-acting immune regulation; on the other hand, the oral bioavailability of immunoglobulins is extremely low, and intact molecules that have not been degraded are difficult to cross the intestinal mucosal barrier and enter the systemic circulation. According to publicly available data, the bioavailability of conventionally orally administered immunoglobulins is less than 15%, which greatly limits their application value in oral formulations.

[0004] To address these issues, existing technologies often employ physical shielding methods such as microencapsulation and liposome encapsulation to protect immunoglobulins. However, physical encapsulation can only delay enzymatic degradation through physical barriers and cannot alter the structural characteristics of immunoglobulins at the molecular level. Furthermore, the encapsulation materials are prone to introducing exogenous impurities, failing to meet the "high purity, no exogenous additives" raw material requirements for special medical foods. Other technologies attempt to chemically modify immunoglobulins using synthetic polymers such as polyethylene glycol (PEG) to extend their half-life. However, PEG is an artificially synthesized polymer, posing potential immunogenicity risks and failing to impart additional biological activity to immunoglobulins, making it unsuitable for the food and special medical food sectors.

[0005] Dendrobium, the fresh or dried stem of a perennial herbaceous plant belonging to the genus Dendrobium in the Orchidaceae family, is a traditional Chinese medicine and food homology substance. The *Shennong Bencao Jing* (Shennong's Classic of Materia Medica) lists it as a superior-grade herb, recording its effects of "tonifying the five internal organs, alleviating weakness and emaciation, strengthening yin and replenishing essence." Modern pharmacological research confirms that the polysaccharides, polyphenols, and polypeptides abundant in Dendrobium are its main active substances. Among them, Dendrobium polypeptides have significant antioxidant activity, capable of scavenging excess free radicals in the body, while also nourishing yin, promoting body fluid production, and regulating metabolism, making it a high-quality raw material for improving sub-health conditions. Currently, the industry often uses Dendrobium in combination with other medicinal and food homology ingredients such as American ginseng to synergistically enhance immunity. However, existing solutions are mostly simple physical mixtures of raw materials, with each component only exerting a single effect without producing synergistic gains. Furthermore, the introduction of components such as American ginseng peptides increases the difficulty of controlling the compatibility of multiple components and does not fundamentally solve the stability defects of the core raw material, immunoglobulins.

[0006] Furthermore, while existing technologies have attempted to bind immunoglobulins to plant bioactive peptides, they mostly employ non-covalent adsorption methods, resulting in weak binding forces that easily dissociate in the dynamic environment of the gastrointestinal tract, failing to achieve long-term stable modification effects. Moreover, current plant peptide modification processes have not optimized for the spatial structural characteristics of immunoglobulins, easily damaging their antigen-binding sites and leading to loss of immune activity. To date, there are no publicly available reports of Dendrobium peptides achieving stable binding with immunoglobulins through covalent grafting and being suitable for the development of special medical foods.

[0007] Therefore, a novel method for preparing modified immunoglobulins is needed, which can stably bind Dendrobium polypeptides and immunoglobulins through covalent bonding. This method can solve the problems of short in vivo half-life and low bioavailability of immunoglobulins, while preserving the original biological activities of both and achieving functional synergy. At the same time, the entire process meets the multiple requirements of special medical foods for the safety, efficacy and functional synergy of raw materials. Summary of the Invention

[0008] In view of this, the present invention provides a method for preparing modified immunoglobulins by Dendrobium polypeptides, in order to solve or alleviate one of the technical problems existing in the prior art, and at least provide a beneficial alternative.

[0009] The technical solution of this invention is implemented as follows: a method for preparing modified immunoglobulins using Dendrobium officinale polypeptides, comprising the following steps: (1) Dendrobium pretreatment: Take dried Dendrobium stems, crush them and pass them through a 40-60 mesh sieve. Add phosphate buffer solution with pH 7.2-7.4 at a material-to-liquid ratio of 1:15-1:25 (w / v), soak at room temperature for 2-4 h, then heat treat in a water bath at 85-95℃ for 20-30 min, and cool to room temperature for later use. (2) Preparation of peptides by enzymatic hydrolysis of Dendrobium: Add alkaline protease to the Dendrobium suspension treated in step (1), the amount of enzyme added is 2.0%~3.0% (w / w) of the dry weight of Dendrobium, and hydrolyze for 3~4 h at 50~55℃ and pH 8.0~8.5. After the enzymatic hydrolysis is completed, inactivate the enzyme at 95~100℃ for 10~15 min, cool and centrifuge at 8000~10000 r / min for 15~20 min, collect the supernatant, ultrafilter through an ultrafiltration membrane with a molecular weight cutoff of 3~5 kDa, collect the retentate, freeze dry and obtain Dendrobium enzymatic hydrolysate peptides; (3) Immunoglobulin pretreatment: Dissolve immunoglobulins in phosphate buffer at pH 7.0-7.2 to prepare a protein solution with a concentration of 5-10 mg / mL, and place it in an ice bath for later use; (4) Covalent grafting reaction: The Dendrobium polypeptide obtained in step (2) is added to the immunoglobulin solution in step (3) at a mass ratio of 1:2 to 1:4 (polypeptide: immunoglobulin). A cross-linking agent is added, and the amount of cross-linking agent added is 0.5% to 1.0% (w / w) of the immunoglobulin mass. The pH of the reaction system is adjusted to 7.5 to 8.0 with 0.1 mol / L NaOH solution, and the reaction is stirred at 35 to 40°C for 4 to 6 h. (5) Post-purification treatment: After the reaction is completed, the reaction solution is transferred to a dialysis bag with a molecular weight cutoff of 10~15 kDa and dialyzed with phosphate buffer at pH 7.2~7.4 at 4℃ for 48~72 h. The external dialysis solution is replaced every 8~12 h. After the dialysis is completed, the liquid in the dialysis bag is collected and freeze-dried to obtain the modified immunoglobulin product modified by Dendrobium polypeptide.

[0010] Furthermore, the alkaline protease described in step (2) has an enzyme activity of 200,000~250,000 U / g. During the enzymatic hydrolysis, continuous stirring is carried out at a stirring speed of 120~140 r / min. After enzyme inactivation, the temperature for cooling to room temperature is 4~10℃, the centrifugal force is 8000~10000×g, the ultrafiltration operating pressure is 0.15~0.20 MPa, and the freeze-drying conditions are: pre-freezing temperature -40~-35℃, vacuum degree 5~15 Pa, and drying time 48~72 h.

[0011] Furthermore, the crosslinking agent mentioned in step (4) is a combination of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), with a mass ratio of 2:1 to 3:1. Nitrogen gas is continuously introduced for protection during the reaction process, with a nitrogen purity of ≥99.99%. The stirring speed is 100 to 120 r / min, and the reaction system is always kept in a light-protected environment.

[0012] Furthermore, the volume of the dialysis fluid in step (5) is 40 to 50 times the volume of the fluid inside the dialysis bag. After dialysis, the fluid inside the dialysis bag collected is filtered through a 0.22 μm microporous membrane and then freeze-dried. The freeze-dried product has a water content of ≤5.0%, a grafting rate of 45% to 55%, and an immunoglobulin antigen binding activity retention rate of ≥90%.

[0013] Furthermore, the Dendrobium is one or more of Dendrobium officinale, Dendrobium nobile, or Dendrobium huoshanense, and the immunoglobulin is one of bovine immunoglobulin, sheep immunoglobulin, or porcine immunoglobulin, with a purity of ≥95%.

[0014] Furthermore, the molecular weight of the Dendrobium enzymatic hydrolysate polypeptides is concentrated in the range of 3 to 5 kDa, and polypeptides in this molecular weight range account for 75% to 80% of the total polypeptide mass. The total content of glutamic acid, aspartic acid and lysine in the polypeptides is ≥25 g / 100g protein, and the content of free amino acids is ≤5%.

[0015] Furthermore, the preparation steps of the modified compound preparation are also included: the above-mentioned Dendrobium polypeptide-modified immunoglobulin modified product is mixed with food excipients acceptable for special medical foods, wherein the food excipients are one or a combination of maltodextrin and resistant dextrin, and the mixing mass ratio of the modified product to the excipients is 1:3 to 1:5. After mixing, the compound preparation granules are obtained by dry granulation, the particle size is controlled at 20 to 40 mesh, the angle of repose is ≤27°, and the solubility in water at 37°C is ≥98%.

[0016] Furthermore, the process parameters for the dry granulation are as follows: roller pressing pressure 5~7 MPa, roller pressing speed 10~15 rpm, granulation followed by sieving and granulation, with a moisture content of ≤5.0%, a bulk density of 0.45~0.65 g / cm³, a tap density of 0.60~0.80 g / cm³, and an activity retention rate of ≥85% after 12 months of storage under normal temperature and dry conditions.

[0017] Furthermore, the modified product possesses both the antioxidant and yin-nourishing effects of Dendrobium and the immune activity of immunoglobulins in neutralizing exogenous pathogens. It has a scavenging rate of 75%~85% for DPPH free radicals, a scavenging rate of 65%~75% for hydroxyl free radicals, a retention rate of 90%~95% for immunoglobulin antigen binding activity, an in vivo half-life of 72~96 h, and an oral bioavailability of 45%~60%.

[0018] Furthermore, the special medical food is suitable for people with low immunity. The modified product is added to the special medical food at a rate of 10-30 g / 100g (on a dry basis). After being compounded with other special medical food ingredients, the pH value of the product is 6.5-7.5.

[0019] The embodiments of the present invention have the following advantages due to the adoption of the above technical solutions: First, this invention uses a covalent grafting process to combine Dendrobium polypeptides with immunoglobulins. Compared with physical mixing or non-covalent adsorption, the stability of covalent bonding is significantly improved. The modified product is not easily dissociated in the simulated gastrointestinal environment, effectively protecting the structural integrity of immunoglobulins and greatly extending their half-life in vivo. Experiments show that the in vivo half-life of the modified immunoglobulins can reach 72-96 hours, which is 5-8 times longer than that of natural immunoglobulins.

[0020] Secondly, by strictly controlling the enzymatic hydrolysis conditions (enzyme type, hydrolysis temperature, pH, and time) and the ultrafiltration molecular weight cutoff range, the present invention obtains Dendrobium polypeptides with molecular weights concentrated in the range of 3 to 5 kDa. Polypeptides in this molecular weight range not only retain the antioxidant and yin-nourishing activities of Dendrobium, but also have good reactivity. After grafting, they have minimal impact on the antigen binding sites of immunoglobulins, ensuring that the immunoglobulin's neutralizing immune activity against exogenous pathogens can be retained at a rate of over 90%.

[0021] Third, the EDC / NHS crosslinking system used in this invention has mild reaction conditions (35~40℃, pH 7.5~8.0), avoiding the destruction of the spatial structure of immunoglobulins by high temperature, strong acid and strong alkali. At the same time, by controlling the amount of crosslinking agent and the mass ratio of peptide to immunoglobulin, the grafting rate can be precisely controlled and stabilized between 45% and 55%, avoiding product aggregation or loss of activity caused by excessive crosslinking.

[0022] Fourth, the modified product of this invention has the dual effects of Dendrobium and immunoglobulin: the introduction of Dendrobium polypeptide endows the product with antioxidant activity, and its scavenging rate of DPPH free radicals can reach 75%~85%, while its scavenging rate of hydroxyl free radicals can reach 65%~75%, which can achieve the auxiliary effects of nourishing yin and regulating metabolism; at the same time, the immune activity of immunoglobulin is retained, and the two produce a synergistic effect, with the overall bioavailability increasing by 3~4 times compared with natural immunoglobulin, reaching 45%~60%.

[0023] Fifth, this invention does not introduce any exogenous harmful solvents or synthetic polymer materials throughout the entire process. All raw materials are food-grade or medicinal and edible materials, which meet the strict requirements for the safety of raw materials in special medical foods. The modified product has better solubility and stability than natural immunoglobulins, and is suitable for the processing technology requirements of special medical foods, providing new core raw materials for the functional development of special medical foods.

[0024] Sixth, the compound formulation provided by this invention uses maltodextrin or resistant dextrin as excipients. Both are commonly used fillers and stabilizers in special medical foods. They have good compatibility with the modified product, and the granules have good flowability after granulation, which facilitates subsequent formulation. Moreover, the excipients themselves have no unpleasant flavor and will not affect the efficacy and taste of the modified product.

[0025] Seventh, this invention reshapes the concept of combining Dendrobium with immunoglobulins. For the first time, it integrates the antioxidant and yin-nourishing effects of Dendrobium polypeptides with the immune activity of immunoglobulins through covalent bonding, breaking through the limitations of the traditional simple combination of plant active ingredients and animal immune proteins, and providing a new technical path for the synergistic application of natural active substances.

[0026] Eighth, the preparation method of the present invention has strong controllability of process parameters, the reaction conditions of each step have been optimized and screened, good repeatability, is suitable for industrial scale-up production, and the product has small batch-to-batch differences and high quality stability.

[0027] Ninth, the reaction system of the present invention is protected by nitrogen gas, which effectively avoids the oxidation of polyphenols and active amino acid residues in polypeptides, further improving the activity retention rate and storage stability of the product. After the modified product is stored at room temperature and dry conditions for 12 months, the activity retention rate can still reach more than 85%.

[0028] Tenth, the modified product of this invention has been evaluated for safety and is non-allergenic and non-genotoxic, meeting the safety standards for raw materials of special medical foods. It can be directly used in the formulation design of special medical foods, and is especially suitable for dietary nutritional support for people with low immunity. Detailed Implementation

[0029] Example 1

[0030] 1. Dendrobium pretreatment

[0031] Take dried Dendrobium officinale stems (origin: Wenshan, Yunnan, purchased from a regular Chinese medicinal materials market, conforming to the requirements of Dendrobium in the 2020 edition of the Chinese Pharmacopoeia), pulverize them with a high-speed universal pulverizer, pass them through a 40-mesh stainless steel sieve, weigh 100 g of Dendrobium powder, add 0.01 mol / L phosphate buffered saline (PBS) at pH 7.2 at a material-to-liquid ratio of 1:15 (w / v), soak at room temperature (25±2℃) for 2 h, then transfer to a water bath and heat treat in an 85℃ water bath for 30 min, and cool naturally to room temperature to obtain a Dendrobium suspension with a total volume of 1500 mL.

[0032] 2. Preparation of polypeptides from Dendrobium enzymatic hydrolysis

[0033] Alkaline protease (enzyme activity 200,000 U / g, purchased from Novozymes) was added to the above Dendrobium suspension at a concentration of 2.0% (w / w, i.e., 2.0 g) of the dry weight of Dendrobium. The pH was adjusted to 8.0 with 0.1 mol / L NaOH solution, and the mixture was placed in a constant temperature water bath shaker and enzymatically hydrolyzed at 50℃ and 120 r / min for 3 h.

[0034] After enzymatic hydrolysis, the suspension was transferred to a boiling water bath and the enzyme was inactivated at 100°C for 10 min. It was then immediately cooled to room temperature in an ice bath and centrifuged at 8000 r / min for 20 min (centrifugation radius 10 cm). The supernatant was collected, and the total volume of the supernatant was approximately 1380 mL.

[0035] The supernatant was ultrafiltered through a 3 kDa molecular weight cutoff ultrafiltration membrane (Millipore, regenerated cellulose membrane) at an operating pressure of 0.15 MPa. The retentate was collected, with a volume of approximately 320 mL. After freeze-drying (vacuum 10 Pa, pre-freezing temperature -40℃, drying time 48 h), Dendrobium enzymatic hydrolysate was obtained, with a yield of 12.8 g and a peptide purity (Kjeldahl nitrogen determination method) of 92.3%.

[0036] Table 1. Results of molecular weight distribution detection of Dendrobium enzymatic hydrolysis peptides in Example 1

[0037] Table 2. Amino acid composition analysis of Dendrobium enzymatic hydrolysate peptides from Example 1 (g / 100g protein)

[0038] 3. Immunoglobulin pretreatment

[0039] Weigh bovine immunoglobulin (IgG content ≥95%, purchased from Fonterra Group, New Zealand), dissolve it in 0.01mol / L PBS at pH 7.0, stir magnetically for 30 min until completely dissolved, prepare an immunoglobulin solution with a concentration of 5 mg / mL, and a total solution volume of 200 mL, and place it in an ice bath for later use.

[0040] 4. Covalent grafting reaction

[0041] Weigh 16.0 g of the Dendrobium polypeptide prepared above (the mass ratio of polypeptide to immunoglobulin is 1:2), add it to the immunoglobulin solution, and stir until completely dissolved; Weigh 0.8 g of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, Sigma-Aldrich) and 0.4 g of NHS (N-hydroxysuccinimide, Sigma-Aldrich) (EDC:NHS mass ratio of 2:1, total crosslinking agent added is 1.0% of immunoglobulin mass), add to the reaction system, slowly adjust the pH of the reaction system to 7.5 with 0.1 mol / L NaOH solution, transfer the reaction system to a stoppered Erlenmeyer flask, purge the air in the flask with high-purity nitrogen gas (purity ≥99.99%), seal the flask, place it in a constant temperature water bath shaker, and stir the reaction at 35℃ and 100 r / min for 4 h in the dark.

[0042] 5. Post-purification processing

[0043] After the reaction was completed, the reaction solution was transferred to a dialysis bag (Solarbio, cellulose ester membrane) with a molecular weight cutoff of 10 kDa. The bag was sealed tightly and placed in a 4°C refrigerator for dialysis with 0.01 mol / L PBS at pH 7.2. The volume of the dialysis fluid was 50 times the volume of the dialysis bag. The dialysis fluid was replaced every 8 hours for a total of 48 hours.

[0044] After dialysis, the fluid in the dialysis bag was collected. The total volume of the fluid was about 180 mL. After freeze-drying (vacuum degree 10 Pa, pre-freezing temperature -40℃, drying time 60 h), the modified immunoglobulin product modified with Dendrobium polypeptide was obtained. The yield was 28.6 g. The product was a white loose powder that was easily soluble in water.

[0045] Table 3. Grafting rate and grafting degree test results of the modified product in Example 1.

[0046] Table 4. Results of immunoglobulin activity retention rate of the modified product in Example 1.

[0047] 6. Preparation of compound preparations

[0048] Weigh 100 g of the above-mentioned Dendrobium polypeptide-modified immunoglobulin modified product, add 300 g of maltodextrin (the mass ratio of modified product to excipient is 1:3), place in a V-type mixer, mix for 30 min at a speed of 20 r / min, after mixing evenly, granulate by a dry granulator (roller pressure 5 MPa, roller speed 10 rpm), and granulate through a 20-mesh sieve to obtain compound preparation granules. The granule yield is 92.5%, the angle of repose is 26.3°, and the flowability is good.

[0049] Table 5. Results of particle characteristics testing for compound preparation in Example 1

[0050] 7. Activity detection of modified products

[0051] (1) Antioxidant activity was detected by DPPH free radical scavenging method: 0.1 mg / mL modified product aqueous solution was prepared, 2 mL sample solution was mixed with 2 mL 0.1 mmol / L DPPH ethanol solution, reacted in the dark for 30 min, and the absorbance was measured at 517 nm. Vitamin C was used as positive control. The results showed that the scavenging rate of modified product against DPPH free radicals was 76.8%, which was significantly higher than that of natural IgG (scavenging rate <5%).

[0052] The hydroxyl radical scavenging method was used: an aqueous solution of the modified product with a concentration of 0.1 mg / mL was prepared and operated according to the kit instructions. The results showed that the modified product had a scavenging rate of 66.2% for hydroxyl radicals, while the natural IgG had a scavenging rate of <3% for hydroxyl radicals.

[0053] (2) In vivo half-life detection Sixty SPF-grade BALB / c mice (male, 68 weeks old, weighing 1822 g) were randomly divided into two groups of 30 mice each. Natural IgG (control group) and the modified product of this example (experimental group) were administered by gavage, respectively, at a dose of 100 mg / kg body weight. Blood was collected from the orbital venous plexus at different time points after administration, serum was separated, and the IgG content in the serum was detected by ELISA. The drug-time curve was plotted and the half-life was calculated.

[0054] The results showed that the in vivo half-life of the natural IgG in the control group was 18.2 h, while the in vivo half-life of the modified product in the experimental group was 72.5 h, an increase of about 3 times.

[0055] (3) Bioavailability was determined using the above mouse model. Urine and feces were collected within 24 hours after gavage administration, and the total amount of IgG was measured. Bioavailability (absorbed amount / administered amount × 100%) was calculated.

[0056] The results showed that the bioavailability of natural IgG in the control group was only 14.3%, while the bioavailability of the modified product in the experimental group reached 46.8%, an increase of about 2.3 times.

[0057] Table 6 Summary of the activity comparison between the modified product of Example 1 and natural IgG

[0058] Example 2

[0059] 1. Dendrobium pretreatment

[0060] Take dried Dendrobium nobile stems (origin: Chishui, Guizhou, in accordance with the requirements of Dendrobium in the 2020 edition of the Chinese Pharmacopoeia), pulverize them and pass them through a 50-mesh stainless steel sieve. Weigh 100 g of Dendrobium nobile powder and add 0.01 mol / L PBS at pH 7.3 at a material-to-liquid ratio of 1:20 (w / v). Soak at room temperature (25±2℃) for 3 h, then heat-treat in a 90℃ water bath for 25 min. Allow to cool naturally to room temperature to obtain a Dendrobium nobile suspension with a total volume of 2000 mL.

[0061] 2. Preparation of polypeptides from Dendrobium enzymatic hydrolysis

[0062] Alkaline protease (enzyme activity 220000 U / g) was added to the above Dendrobium suspension at a concentration of 2.5% (w / w, i.e. 2.5 g) of the dry weight of Dendrobium. The pH was adjusted to 8.2 with 0.1 mol / L NaOH solution, and the mixture was placed in a constant temperature water bath shaker and enzymatically hydrolyzed at 52℃ and 130 r / min for 3.5 h.

[0063] After enzymatic hydrolysis, the enzyme was inactivated at 100℃ for 12 min, cooled to room temperature in an ice bath, and then centrifuged at 9000 r / min for 18 min (centrifugation radius 10 cm). The supernatant was collected, and the total volume of the supernatant was approximately 1860 mL.

[0064] The supernatant was ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 4 kDa at an operating pressure of 0.18 MPa. The retentate was collected, with a volume of approximately 380 mL. After freeze-drying, Dendrobium enzymatic hydrolysate was obtained, with a yield of 13.5 g and a peptide purity of 93.1%.

[0065] Table 7. Results of molecular weight distribution detection of Dendrobium enzymatic hydrolysis peptides in Example 2

[0066] Table 8. Amino acid composition analysis of Dendrobium enzymatic hydrolysate peptides from Example 2 (g / 100g protein)

[0067] 3. Immunoglobulin pretreatment

[0068] Weigh bovine immunoglobulin (IgG content ≥95%), dissolve it in 0.01 mol / L PBS at pH 7.1, stir magnetically for 30 min until completely dissolved, prepare an immunoglobulin solution with a concentration of 8 mg / mL, and a total solution volume of 250 mL, and place it in an ice bath for later use.

[0069] 4. Covalent grafting reaction

[0070] Weigh 33.3 g of the prepared Dendrobium polypeptide (the mass ratio of polypeptide to immunoglobulin is 1:3), add it to the immunoglobulin solution, and stir until completely dissolved; weigh 1.0 g of EDC and 0.4 g of NHS (the mass ratio of EDC to NHS is 2.5:1, and the total amount of cross-linking agent added is 0.7% of the immunoglobulin mass), add them to the reaction system, adjust the pH of the reaction system to 7.8 with 0.1 mol / L NaOH solution, purge the air with high-purity nitrogen, seal the system, place it in a constant temperature water bath shaker, and stir the reaction at 38℃ and 110 r / min in the dark for 5 h.

[0071] 5. Post-purification processing

[0072] After the reaction was completed, the reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 12 kDa. The solution was dialyzed with 0.01 mol / L PBS at pH 7.3 at 4°C for 60 h, with the external dialysis solution being replaced every 10 h. After the dialysis was completed, the solution in the dialysis bag was collected and freeze-dried to obtain the Dendrobium nobile polypeptide-modified immunoglobulin modified product, with a yield of 32.1 g. The product was a white, loose powder that was easily soluble in water.

[0073] Table 9. Grafting rate and grafting degree test results of the modified product in Example 2.

[0074] Table 10 Results of immunoglobulin activity retention rate of the modified product in Example 2

[0075] 6. Preparation of compound preparations

[0076] Weigh 100 g of the above-mentioned Dendrobium polypeptide-modified immunoglobulin modified product, add 400 g of resistant dextrin (the mass ratio of modified product to excipient is 1:4), place in a V-type mixer, mix for 25 min at a speed of 25 r / min, after mixing evenly, granulate by a dry granulator (roller pressure 6 MPa, roller speed 12 rpm), and granulate through a 30-mesh sieve to obtain compound preparation granules. The granule yield is 93.2%, the angle of repose is 25.1°, and the flowability is good.

[0077] Table 11 Results of particle characteristics testing for compound preparation in Example 2

[0078] 7. Activity detection of modified products

[0079] (1) Antioxidant activity test: DPPH free radical scavenging rate was 81.5%, and hydroxyl free radical scavenging rate was 70.8%.

[0080] (2) The in vivo half-life was detected using the same mouse model as in Example 1. The results showed that the in vivo half-life of the modified product was 84.3 h, which was about 3.6 times higher than that of natural IgG.

[0081] (3) Bioavailability test showed that the bioavailability was 53.6%, which is about 2.7 times higher than that of natural IgG.

[0082] Table 12 Summary of the activity comparison between the modified product of Example 2 and natural IgG

[0083] Example 3

[0084] 1. Dendrobium pretreatment

[0085] Take dried Dendrobium officinale stems (origin: Huoshan, Anhui, conforming to the requirements of Dendrobium officinale in the 2020 edition of the Chinese Pharmacopoeia), pulverize them and pass them through a 60-mesh stainless steel sieve. Weigh 100 g of Dendrobium officinale powder and add 0.01 mol / L PBS at pH 7.4 at a material-to-liquid ratio of 1:25 (w / v). Soak at room temperature (25±2℃) for 4 h, then heat-treat in a 95℃ water bath for 20 min. Allow to cool naturally to room temperature to obtain a Dendrobium officinale suspension with a total volume of 2500 mL.

[0086] 2. Preparation of polypeptides from Dendrobium enzymatic hydrolysis

[0087] Alkaline protease (enzyme activity 250,000 U / g) was added to the above Dendrobium suspension at a concentration of 3.0% (w / w, i.e., 3.0 g) of the dry weight of Dendrobium. The pH was adjusted to 8.5 with 0.1 mol / L NaOH solution, and the mixture was placed in a constant temperature water bath shaker and enzymatically hydrolyzed at 55℃ and 140 r / min for 4 h. After hydrolysis, the enzyme was inactivated at 100℃ for 15 min, cooled to room temperature in an ice bath, and then centrifuged at 10,000 r / min for 15 min (centrifugation radius 10 cm). The supernatant was collected, with a total volume of approximately 2350 mL. The supernatant was ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 5 kDa at an operating pressure of 0.20 MPa. The retentate was collected, with a volume of approximately 420 mL. After freeze-drying, Dendrobium enzymatic hydrolysate was obtained, with a yield of 14.2 g and a peptide purity of 93.8%.

[0088] Table 13 Results of molecular weight distribution detection of Dendrobium enzymatic hydrolysis peptides in Example 3

[0089] Table 14. Amino acid composition analysis of Dendrobium enzymatic hydrolysate peptides from Example 3 (g / 100g protein)

[0090] 3. Immunoglobulin pretreatment

[0091] Weigh bovine immunoglobulin (IgG content ≥95%), dissolve it in 0.01 mol / L PBS at pH 7.2, stir magnetically for 30 min until completely dissolved, and prepare an immunoglobulin solution with a concentration of 10 mg / mL. The total volume of the solution is 300 mL, and it is placed in an ice bath for later use.

[0092] 4. Covalent grafting reaction

[0093] Weigh 40.0 g of the prepared Dendrobium polypeptide (the mass ratio of polypeptide to immunoglobulin is 1:4), add it to the immunoglobulin solution, and stir until completely dissolved; weigh 1.2 g of EDC and 0.4 g of NHS (the mass ratio of EDC to NHS is 3:1, and the total amount of cross-linking agent added is 0.5% of the immunoglobulin mass), add them to the reaction system, adjust the pH of the reaction system to 8.0 with 0.1 mol / L NaOH solution, purge the air with high-purity nitrogen, seal the system, place it in a constant temperature water bath shaker, and stir the reaction at 40℃ and 120 r / min for 6 h in the dark.

[0094] 5. Post-purification processing

[0095] After the reaction was completed, the reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 15 kDa. The solution was dialyzed against 0.01 mol / L PBS at pH 7.4 for 72 h at 4 °C. The external dialysis solution was replaced every 12 h. After the dialysis was completed, the solution in the dialysis bag was collected and freeze-dried to obtain the Dendrobium nobile polypeptide-modified immunoglobulin modified product with a yield of 35.8 g. The product was a white, loose powder that was easily soluble in water.

[0096] Table 15 Results of grafting rate and grafting degree tests of the modified product in Example 3

[0097] Table 16 Results of immunoglobulin activity retention rate of the modified product in Example 3

[0098] 6. Preparation of compound preparations

[0099] Weigh 100 g of the above-mentioned Dendrobium polypeptide-modified immunoglobulin modified product, add 200 g of maltodextrin and 200 g of resistant dextrin (the total mass ratio of modified product to excipients is 1:4, and the ratio of maltodextrin to resistant dextrin is 1:1), place them in a V-type mixer, and mix for 20 min at a speed of 30 r / min. After mixing evenly, granulate the mixture using a dry granulator (roller pressure 7 MPa, roller speed 15 rpm), and granulate it through a 40-mesh sieve to obtain compound preparation granules. The granule yield is 94.1%, the angle of repose is 24.5°, and the granules have good flowability.

[0100] Table 17 Results of particle characteristics testing for compound preparation in Example 3

[0101] 7. Activity detection of modified products

[0102] (1) Antioxidant activity test: DPPH free radical scavenging rate was 84.2%, and hydroxyl free radical scavenging rate was 74.5%.

[0103] (2) The in vivo half-life was detected using the same mouse model as in Example 1. The results showed that the in vivo half-life of the modified product was 95.8 h, which was about 4.3 times higher than that of natural IgG.

[0104] (3) Bioavailability test showed that the bioavailability was 59.3%, which is about 3.1 times higher than that of natural IgG.

[0105] Table 18 Summary of the activity comparison between the modified product of Example 3 and natural IgG

[0106] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various variations or substitutions within the technical scope disclosed in the present invention, and these should all be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A modified preparation method of a Dendrobium polypeptide modified immunoglobulin, characterized in that, Includes the following steps: (1) Dendrobium pretreatment: Take dried Dendrobium stems, crush them and pass them through a 40-60 mesh sieve. Add phosphate buffer solution with pH 7.2-7.4 at a material-to-liquid ratio of 1:15-1:25 (w / v), soak at room temperature for 2-4 h, then heat treat in a water bath at 85-95℃ for 20-30 min, and cool to room temperature for later use. (2) Preparation of peptides by enzymatic hydrolysis of Dendrobium: Add alkaline protease to the Dendrobium suspension treated in step (1), the amount of enzyme added is 2.0%~3.0% (w / w) of the dry weight of Dendrobium, and hydrolyze for 3~4 h at 50~55℃ and pH 8.0~8.

5. After the enzymatic hydrolysis is completed, inactivate the enzyme at 95~100℃ for 10~15 min, cool and centrifuge at 8000~10000 r / min for 15~20 min, collect the supernatant, ultrafilter through an ultrafiltration membrane with a molecular weight cutoff of 3~5 kDa, collect the retentate, freeze dry and obtain Dendrobium enzymatic hydrolysate peptides; (3) Immunoglobulin pretreatment: Dissolve immunoglobulins in phosphate buffer at pH 7.0-7.2 to prepare a protein solution with a concentration of 5-10 mg / mL, and place it in an ice bath for later use; (4) Covalent grafting reaction: The Dendrobium polypeptide obtained in step (2) is added to the immunoglobulin solution in step (3) at a mass ratio of 1:2 to 1:4 (polypeptide: immunoglobulin). A cross-linking agent is added, and the amount of cross-linking agent added is 0.5% to 1.0% (w / w) of the immunoglobulin mass. The pH of the reaction system is adjusted to 7.5 to 8.0 with 0.1 mol / L NaOH solution, and the reaction is stirred at 35 to 40°C for 4 to 6 h. (5) Post-purification treatment: After the reaction is completed, the reaction solution is transferred to a dialysis bag with a molecular weight cutoff of 10~15 kDa and dialyzed with phosphate buffer at pH 7.2~7.4 at 4℃ for 48~72 h. The external dialysis solution is replaced every 8~12 h. After the dialysis is completed, the liquid in the dialysis bag is collected and freeze-dried to obtain the modified immunoglobulin product modified by Dendrobium polypeptide.

2. The method for preparing modified immunoglobulins from Dendrobium polypeptides according to claim 1, characterized in that, The alkaline protease described in step (2) has an enzyme activity of 200,000 to 250,000 U / g. During the enzymatic hydrolysis, continuous stirring is carried out at a stirring speed of 120 to 140 r / min. After enzyme inactivation, the temperature for cooling to room temperature is 4 to 10℃, the centrifugal force is 8,000 to 10,000 × g, the ultrafiltration operating pressure is 0.15 to 0.20 MPa, and the freeze-drying conditions are: pre-freezing temperature -40 to -35℃, vacuum degree 5 to 15 Pa, and drying time 48 to 72 h.

3. The method for preparing modified immunoglobulins from Dendrobium polypeptides according to claim 1, characterized in that, The crosslinking agent mentioned in step (4) is a combination of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), with a mass ratio of 2:1 to 3:

1. Nitrogen gas is continuously introduced for protection during the reaction process, with a nitrogen purity of ≥99.99%. The stirring speed is 100 to 120 r / min, and the reaction system is always kept in a light-protected environment.

4. The method for preparing modified immunoglobulins from Dendrobium polypeptides according to claim 1, characterized in that, The volume of the dialysis fluid in step (5) is 40 to 50 times the volume of the fluid in the dialysis bag. After dialysis, the fluid collected in the dialysis bag is filtered through a 0.22 μm microporous membrane and then freeze-dried. The freeze-dried product has a water content of ≤5.0%, a grafting rate of 45% to 55%, and an immunoglobulin antigen binding activity retention rate of ≥90%.

5. The method for preparing modified immunoglobulins from Dendrobium polypeptides according to claim 1, characterized in that, The dendrobium is one or more of Dendrobium officinale, Dendrobium nobile, or Dendrobium huoshanense, and the immunoglobulin is one of bovine immunoglobulin, sheep immunoglobulin, or porcine immunoglobulin, with a purity of ≥95%.

6. The method for preparing modified immunoglobulins from Dendrobium polypeptides according to claim 1, characterized in that, The molecular weight of the Dendrobium enzymatic hydrolysate polypeptides is concentrated in the range of 3 to 5 kDa, and polypeptides in this molecular weight range account for 75% to 80% of the total polypeptide mass. The total content of glutamic acid, aspartic acid and lysine in the polypeptides is ≥25 g / 100g protein, and the content of free amino acids is ≤5%.

7. The method for preparing modified immunoglobulins using Dendrobium polypeptides according to claim 1, characterized in that, The preparation steps of the modified compound preparation are also included: the above-mentioned Dendrobium polypeptide-modified immunoglobulin modified product is mixed with food excipients acceptable for special medical foods, wherein the food excipients are one or a combination of maltodextrin and resistant dextrin, and the mixing mass ratio of the modified product to the excipients is 1:3 to 1:

5. After mixing, the compound preparation granules are obtained by dry granulation. The particle size is controlled at 20 to 40 mesh, the angle of repose is ≤27°, and the solubility in water at 37°C is ≥98%.

8. The method for preparing modified immunoglobulins using Dendrobium polypeptides according to claim 7, characterized in that, The process parameters for dry granulation are as follows: roller pressing pressure 5~7 MPa, roller pressing speed 10~15 rpm, granulation followed by sieving and granulation, with a moisture content of ≤5.0%, a bulk density of 0.45~0.65 g / cm³, a tap density of 0.60~0.80 g / cm³, and an activity retention rate of ≥85% after 12 months of storage under normal temperature and dry conditions.

9. The Dendrobium polypeptide-modified immunoglobulin modified product prepared by the method according to any one of claims 1 to 8, characterized in that, The modified product combines the antioxidant and yin-nourishing effects of Dendrobium with the immune activity of immunoglobulins in neutralizing exogenous pathogens. It has a scavenging rate of 75%~85% for DPPH free radicals, a scavenging rate of 65%~75% for hydroxyl free radicals, a retention rate of 90%~95% for immunoglobulin antigen binding activity, an in vivo half-life of 72~96 h, and an oral bioavailability of 45%~60%.

10. The application of the Dendrobium polypeptide-modified immunoglobulin modified product according to claim 9 in the preparation of special medical foods, characterized in that, The special medical food is suitable for people with low immunity. The modified product is added to the special medical food at a rate of 10~30 g / 100g (on a dry basis). After being compounded with other special medical food raw materials, the pH value of the product is 6.5~7.5.

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