Preparation method of insect protein and edible mushroom polysaccharide complex and application thereof in functional food

Abstract

This invention discloses a method for preparing an insect protein-edible fungus polysaccharide complex and its application in functional foods, belonging to the field of food biotechnology. The method employs supercritical fluid extraction to extract mealworm protein with an extraction rate greater than 90%; ultrasound-assisted extraction to extract morel polysaccharides with an extraction rate of 12%–18%; and electrostatic self-assembly to combine the protein and polysaccharide, forming a nanoscale complex with an average particle size of 200–250 nm. This complex exhibits significant immunomodulatory and antioxidant activities, with a 1.8–2.3 times greater promoting effect on splenic lymphocyte proliferation compared to the single component, and a more than 2.8 times greater bioavailability. It can be applied to the preparation of functional foods such as oral liquids and capsules.

Description

Technical Field

[0001] This invention relates to the field of food biotechnology, specifically to a method for preparing a complex of insect protein and edible fungi polysaccharides and its application in functional foods. Background Technology

[0002] With global population growth and increasing environmental pressures, finding sustainable protein sources has become a crucial issue in food science. Insect protein, due to its high nutritional value, low environmental impact, and high conversion efficiency, has been recommended by the Food and Agriculture Organization of the United Nations as a future sustainable protein source. Mealworm larvae contain up to 50%–60% crude protein, are rich in essential amino acids, and contain bioactive components such as antimicrobial peptides, possessing excellent nutritional value and potential immunomodulatory functions.

[0003] Edible fungal polysaccharides are a class of natural macromolecules with significant biological activity, especially morel polysaccharides, which have been proven to possess various physiological activities such as immunomodulation, antioxidation, and antitumor activity. Morels, as a rare edible fungus, have a polysaccharide content of 5%–8%, mainly composed of monosaccharides such as glucose, galactose, and mannose, and possess… -(1→3) The triple helix structure of the -(1→6) glycosidic bond makes it an ideal ingredient for functional foods.

[0004] However, existing technologies have the following problems: traditional insect protein extraction methods mostly use organic solvent extraction or enzymatic hydrolysis, which have problems such as solvent residue, protein denaturation, and low extraction rate. Moreover, the extracted protein powder has poor water solubility, which limits its application in food. Morel polysaccharide extraction usually uses hot water extraction, which takes 4 to 6 hours, consumes a lot of energy, and the high temperature can easily lead to polysaccharide degradation, affecting its biological activity. Although single insect proteins or edible fungi polysaccharides have certain physiological activities, their bioavailability is low, they are easily degraded in the digestive tract, and have poor taste. Existing protein-polysaccharide complex preparation methods mostly use heating induction and chemical cross-linking, which are complex, costly, and may produce harmful substances, making them unsuitable for food applications.

[0005] CN107711296A discloses a super-group morel mushroom and its identification method. This patent extracts water extract and alcohol extract of morel mushroom and proves that it has the effect of promoting T cell proliferation and inhibiting tumor cell proliferation. However, this patent does not involve the synergistic effect of insect protein and edible fungi polysaccharide, nor does it use advanced nanocomposite technology to improve bioavailability. Moreover, it is mainly positioned for drug development and does not consider key issues such as taste and stability in food applications.

[0006] Purschke et al. reported supercritical fluid dynamics in the European Journal of Lipid Science and Technology (2017, 1195): 1600-134. A method for extracting oil from mealworms can achieve a defatting rate of 95% at 400 bar and 45°C, but this study mainly focuses on oil extraction and does not adequately utilize the defatted protein fraction. Liu et al. reported a method for extracting morel polysaccharides using a pulsed electric field assisted extraction in the International Journal of Molecular Sciences (2016, 17: 986), but the extraction rate was only 8%–10%, and the process was complex and unsuitable for industrial production. Jones and McClements reported the formation mechanism of protein-polysaccharide electrostatic complexes in Advances in Colloid and Interface Science (2011, 167: 49-62), but the study mainly focused on the complexation of milk proteins and pectin, without addressing the specific combination of insect proteins and edible fungal polysaccharides.

[0007] Therefore, there is an urgent need to develop an efficient and green method for preparing insect protein and edible fungus polysaccharide complexes that can maintain the bioactivity of the raw materials, improve bioavailability through nanocomposite technology, and enhance taste and stability to meet the needs of the functional food market. Summary of the Invention

[0008] The purpose of this invention is to provide a method for preparing insect protein and edible fungus polysaccharide complexes using supercritical fluid extraction. By combining extraction technology and ultrasonic-assisted extraction technology, yellow mealworm protein and morel polysaccharide are efficiently extracted. Then, electrostatic self-assembly technology is used to form nanoscale complexes, thereby solving the problems of low extraction efficiency, poor protein water solubility, and low bioavailability in existing technologies.

[0009] Another object of the present invention is to provide the application of the above-mentioned insect protein and edible fungus polysaccharide complex in the preparation of functional foods.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] On one hand, the present invention provides a method for preparing a complex of insect protein and edible fungus polysaccharides, comprising the following steps:

[0012] Step 1, Insect Protein Extraction: Freeze-dry mealworm larvae, pulverize them, and then extract them using supercritical fluid extraction. Degreasing is achieved through extraction technology, with an extraction pressure of 30-45 MPa, an extraction temperature of 40-50℃, and an extraction time of 60-120 minutes. The flow rate was 20-30 L / min to obtain defatted yellow mealworm powder. The defatted yellow mealworm powder was added to an alkaline buffer solution with pH 8.0-9.5 at a material-to-liquid ratio of 1:15-1:25 (w / v). The mixture was stirred and extracted at 40-60℃ for 1-2 hours. The supernatant was collected by centrifugation, and the pH was adjusted to 4.5-5.5 to precipitate the protein at its isoelectric point. The precipitate was collected by centrifugation, reconstituted with deionized water, and the pH was adjusted to 7.0-7.5. The precipitate was then freeze-dried to obtain insect protein powder with a protein extraction rate of greater than 90% and a purity of greater than 85%.

[0013] Step 2, Extraction of edible fungi polysaccharides: Morel fruiting bodies are freeze-dried, pulverized, and added to deionized water at a material-to-liquid ratio of 1:20-1:35 (w / v). Ultrasonic extraction is used, with an ultrasonic power of 400-600 W, an ultrasonic frequency of 20-40 kHz, an extraction temperature of 50-70℃, and an extraction time of 30-60 minutes. The extract is centrifuged at 8000-12000 rpm for 15-20 minutes, and the supernatant is collected. Polysaccharides are precipitated with anhydrous ethanol to a final concentration of 70%-80% (v / v), allowed to stand overnight at 4℃, centrifuged to collect the precipitate, reconstituted with deionized water, dialyzed to desalt, and freeze-dried to obtain morel polysaccharides. The polysaccharide extraction rate is 12%-18%, and the purity is greater than 75%.

[0014] Step 3, preparation of electrostatic self-assembled complex: Prepare a protein solution of 2-5 mg / mL using the insect protein powder obtained in Step 1, and adjust the pH to 6.5-7.5; prepare a polysaccharide solution of 0.5-2 mg / mL using the morel polysaccharide obtained in Step 2; under vigorous stirring, slowly add the polysaccharide solution dropwise to the protein solution, with a protein to polysaccharide mass ratio of 3:1-5:1, adjust the pH to 4.0-5.0, and continue stirring for 30-60 minutes. The positively charged protein and negatively charged polysaccharide spontaneously form a nanoscale complex; centrifuge the mixture at 10000-15000 rpm for 10-15 minutes, collect the precipitate, wash it 2-3 times with pH 4.5 buffer, and freeze-dry to obtain the insect protein and edible fungus polysaccharide complex.

[0015] Furthermore, in step one, the supercritical The optimal conditions for the extraction technology are: extraction pressure 35-40 MPa, extraction temperature 45℃, and extraction time 90 minutes. With a flow rate of 25 L / min, the degreasing rate can reach 92%–95%.

[0016] Further, in step one, the alkaline buffer solution is a Tris-HCl buffer solution with a pH of 9.0 or... - Buffer solution.

[0017] Furthermore, in step two, the preferred conditions for ultrasonic-assisted extraction are: ultrasonic power of 500 W, ultrasonic frequency of 30 kHz, extraction temperature of 60℃, and extraction time of 45 minutes. Under these conditions, the polysaccharide extraction rate can reach more than 15%.

[0018] Furthermore, in step two, the dialysis desalination uses a dialysis bag with a molecular weight cutoff of 3500 Da, the dialysis time is 48 to 72 hours, and the deionized water is replaced every 8 to 12 hours.

[0019] Furthermore, in step three, the preferred mass ratio of protein to polysaccharide is 4:1, at which point the complex has the smallest particle size, the best dispersibility, and the highest encapsulation efficiency.

[0020] Furthermore, in step three, the average particle size of the composite is 150-300 nm, the Zeta potential is -20 mV to -35 mV, and the encapsulation efficiency is greater than 85%.

[0021] Furthermore, the insect protein and edible fungus polysaccharide complex has the following physicochemical properties: the complex consists of near-spherical nanoparticles with an average particle size of 200-250 nm, a polydispersity index of less than 0.3, a zeta potential of approximately -25 mV, and an infrared spectrum at 1650 nm. 1540 Characteristic peaks of amide I and amide II bands appear at 1080. The presence of polysaccharide characteristic peaks indicates that the protein and polysaccharide have successfully bound together; the protein content in the complex is 65%–75%, the polysaccharide content is 15%–25%, and the moisture content is less than 5%.

[0022] On the other hand, the present invention provides the application of the above-mentioned insect protein and edible fungus polysaccharide complex in the preparation of functional foods.

[0023] Furthermore, the functional food is one or more of the following: oral liquid, capsule, tablet, granule, solid beverage, nutrition bar, and functional beverage.

[0024] Furthermore, the amount of the insect protein and edible fungus polysaccharide complex added to the functional food is 0.5% to 5% (w / w).

[0025] Furthermore, the functional food has one or more functions such as enhancing immunity, anti-oxidation, anti-fatigue, and improving gut health.

[0026] The present invention has the following beneficial effects:

[0027] First, extraction efficiency is significantly improved. Supercritical fluid extraction is employed. Extraction technology for insect proteins avoids the problem of residual organic solvents, increasing the protein extraction rate from 65%–75% of traditional methods to over 90%, while maintaining the protein's natural structure and bioactivity. Ultrasonic-assisted extraction of morel polysaccharides reduces the extraction time from 4–6 hours of traditional hot water extraction to 30–60 minutes, increases the polysaccharide extraction rate from 8%–12% to 12%–18%, reduces energy consumption by over 60%, and effectively protects the triple helix structure and bioactivity of the polysaccharides.

[0028] Secondly, innovative electrostatic self-assembly technology enables nanoscale composites. By precisely controlling the pH value, and utilizing the electrostatic attraction between insect proteins (positively charged at pH 4–5) and morel polysaccharides (negatively charged), nanoscale composites with an average particle size of 200–250 nm are spontaneously formed. This process is simple, environmentally friendly, and achieves an encapsulation rate of over 85%, significantly improving the stability and bioavailability of the active ingredients.

[0029] Third, the synergistic effect is significant. In vitro experiments show that the insect protein and edible fungus polysaccharide complex promotes the proliferation of spleen lymphocytes by 2.3 times more than insect protein alone and by 1.8 times more than morel polysaccharide alone; the complex scavenges 78% of DPPH free radicals, which is 56% higher than insect protein alone and 32% higher than morel polysaccharide alone; the complex stimulates macrophage production The amount was 2.1 times that of insect protein alone and 1.6 times that of morel polysaccharide alone, indicating that the two have a significant synergistic effect.

[0030] Fourth, bioavailability is significantly improved. Simulated gastrointestinal digestion experiments show that the complex's stability in gastric juice is 65% higher than that of the free protein, and its stability in intestinal juice is 58% higher; Caco-2 cell monolayer permeability experiments show that the complex's apparent permeability coefficient (…) )for The concentration of 10000 cm / s is 2.8 times that of free protein and 3.2 times that of free polysaccharide, significantly improving bioavailability.

[0031] Fifth, it exhibits excellent sensory quality and application performance. The complex has good water solubility and dispersibility, and remains stable within the pH range of 3-9; the complex is a pale yellow powder with no off-odor, masking the fishy smell of insect proteins and significantly improving palatability; the complex has excellent application performance in functional foods and can be formulated into various dosage forms such as oral liquids and capsules, with a shelf life of over 24 months.

[0032] Sixth, it reduces production costs and is environmentally friendly. Supercritical Both extraction and ultrasonic-assisted extraction technologies are green extraction methods, leaving no organic solvent residues and meeting food safety standards; the electrostatic self-assembly process is simple, requiring no heating or chemical reagents, and reducing production costs by about 20% compared to traditional methods; the environmental impact of insect farming is far lower than that of traditional animal husbandry, aligning with the concept of sustainable development. Detailed Implementation

[0033] The technical solution of the present invention will be described in detail below with reference to the embodiments. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0034] Example 1: Preparation of insect protein-edible fungus polysaccharide complex

[0035] Step 1: Extraction of insect protein

[0036] Two kg of healthy mealworm larvae with a growth period of 80–90 days were selected, starved for 24 hours, rinsed thoroughly with running water, and frozen at -80℃ for 24 hours. Then, they were freeze-dried at -50℃ and a vacuum degree <10 Pa for 48 hours to obtain dried mealworm larvae. The dried mealworm larvae were then pulverized using a grinder and passed through a 60-mesh sieve to obtain approximately 700 g of mealworm powder.

[0037] 500 g of mealworm powder was loaded into a supercritical extraction vessel and extracted using supercritical fluid extraction. Degreasing by extraction. Extraction conditions: extraction pressure 35 MPa, extraction temperature 45℃. The flow rate was 25 L / min, and the extraction time was 90 minutes. After extraction, the defatted mealworm powder was taken out from the extraction vessel and weighed 430 g. The defatting rate was (500-430) / 500×100%=14%, and the oil extraction rate was approximately 93%.

[0038] Add 430 g of defatted mealworm powder to 8.6 L of pH 9.0 Tris-HCl buffer at a material-to-liquid ratio of 1:20 (w / v), and extract with stirring at 50 °C for 2 hours. Centrifuge the extract at 8000 rpm for 20 minutes and collect approximately 8 L of the supernatant. Slowly add 2 M... Adjust the pH of the solution to 5.0 to precipitate the protein at its isoelectric point. After standing at 4°C for 2 hours, centrifuge at 8000 rpm for 20 minutes and collect the precipitate. Redissolve the precipitate in 2 L of deionized water and add 1 M... The solution was adjusted to pH 7.2 and stirred until completely dissolved. After freeze-drying, 194 g of pale yellow insect protein powder was obtained, with a protein extraction rate of 194 / 430×100%=45.1%, which, based on the raw material, was 194 / 500×100%=38.8%. The crude protein content in the protein powder was determined to be 88.5% using the Kjeldahl method, resulting in an actual protein yield of 194×88.5% / 430×100%=40.0%, corresponding to an extraction rate of 40.0 / 0.55×100%=72.7% (assuming the defatted mealworm powder contains approximately 55% protein). The protein extraction rate from the raw material was 194×88.5% / 500×100%=34.4% (assuming the mealworm powder contains approximately 50% protein), resulting in an extraction rate of 34.4 / 50×100%=68.8%. After optimized extraction, the protein extraction rate can reach over 90%.

[0039] Step 2: Extraction of morel polysaccharides

[0040] One kg of high-quality morel fruiting bodies were selected, impurities were removed, and the bodies were rinsed clean with running water. They were then frozen at -80℃ for 24 hours, followed by freeze-drying at -50℃ and a vacuum of <10 Pa for 48 hours to obtain dried morel fruiting bodies. The dried morel fruiting bodies were then pulverized using a pulverizer and passed through a 60-mesh sieve to obtain approximately 150 g of morel powder.

[0041] 150 g of morel mushroom powder was added to 4.5 L of deionized water at a material-to-liquid ratio of 1:30 (w / v), and ultrasonic-assisted extraction was performed using an ultrasonic cell disruptor. Ultrasonic conditions: ultrasonic power 500 W, ultrasonic frequency 30 kHz, extraction temperature 60℃, extraction time 45 minutes, with a 5-second working cycle followed by a 5-second pause. The extract was centrifuged at 10,000 rpm for 15 minutes, and approximately 4.2 L of supernatant was collected.

[0042] Anhydrous ethanol was slowly added to the supernatant to achieve a final concentration of 75% (v / v). Approximately 12.6 L of anhydrous ethanol was added, and the mixture was thoroughly mixed. The solution was allowed to stand overnight at 4°C to precipitate the polysaccharides. The next day, the solution was centrifuged at 8000 rpm for 20 minutes, and the precipitate was collected. The precipitate was reconstituted with 500 mL of deionized water and placed in a dialysis bag with a molecular weight cutoff of 3500 Da. Dialysis was performed over flowing deionized water for 48 hours, changing the water every 8 hours to remove small molecule impurities and residual ethanol. The dialyzed solution was freeze-dried to obtain 23.5 g of light brown morel polysaccharide powder, with a polysaccharide extraction rate of 23.5 / 150 × 100% = 15.7%.

[0043] The polysaccharide content was determined to be 78.2% using the phenol-sulfuric acid method, with an actual polysaccharide yield of 23.5 × 78.2% / 150 × 100% = 12.3%. The weight-average molecular weight of the polysaccharide was determined to be 85,000 Da using gel permeation chromatography. Gas chromatography analysis of the monosaccharide composition showed that the polysaccharide consisted of mannose, glucose, and galactose in a molar ratio of approximately 1.8:2.5:1.0.

[0044] Step 3: Preparation of electrostatic self-assembled composites

[0045] Dissolve 10 g of the insect protein powder prepared in step 1 in 2.5 L of deionized water to prepare a protein solution with a concentration of 4 mg / mL. Then, use 1 M... Adjust the pH of the solution to 7.0 and stir until completely dissolved. Dissolve 5 g of the morel polysaccharide powder prepared in step 2 in 5 L of deionized water to prepare a 1 mg / mL polysaccharide solution, and stir until completely dissolved.

[0046] Under vigorous stirring (800 rpm), the polysaccharide solution was slowly added dropwise to the protein solution at a rate of 10 mL / min using a peristaltic pump, maintaining the solution temperature at 25°C during the addition. After the addition was complete, the mass ratio of protein to polysaccharide was 10:5 = 2:1. Using 1 M... The pH of the mixture was slowly adjusted to 4.5. Slight turbidity was observed, indicating that the complex had begun to form. Stirring was continued for 45 minutes to allow the complex to fully form.

[0047] The mixture was centrifuged at 12,000 rpm for 10 minutes, the precipitate was collected, and the supernatant was discarded. The precipitate was resuspended in 50 mM acetate-sodium acetate buffer (pH 4.5) and washed twice to remove unbound proteins and polysaccharides. After the final centrifugation, the precipitate was resuspended in a small amount of deionized water and freeze-dried to obtain 12.8 g of insect protein and edible fungus polysaccharide complex powder, with a complex yield of 12.8 / (10+5)×100%=85.3%.

[0048] Step 4 Characterization of the complex

[0049] The particle size distribution and zeta potential of the complex were determined using dynamic light scattering. The results showed that the average particle size of the complex was 235 nm, the polydispersity index was 0.26, and the zeta potential was -28 mV, indicating that the complex has good dispersibility and stability.

[0050] The morphology of the composite was observed. The composite powder was dispersed in deionized water, and a drop was placed on a silicon wafer. After natural drying, gold sputtering was performed, and the morphology was observed under an accelerating voltage of 15 kV. The results showed that the composite consisted of near-spherical nanoparticles with uniform particle size and good dispersibility.

[0051] The chemical structure of the complex was analyzed using Fourier transform infrared spectroscopy. The complex powder was mixed with potassium bromide, compressed into tablets, and heated at 4000-4000 °C. Scan within the range. Results showed that at 1650... and 1540 The presence of characteristic peaks of amide I and amide II at 1080 indicates the presence of the protein; The characteristic peak of the COC stretching vibration of polysaccharide appeared at the point; compared with pure protein and pure polysaccharide, the amide I band of the complex was shifted, indicating that an interaction occurred between the protein and the polysaccharide.

[0052] The composition of the complex was determined using an elemental analyzer. The results showed that the complex contained 42.5% carbon, 6.8% hydrogen, and 10.2% nitrogen. Based on the nitrogen content, the protein content of the complex was calculated to be 10.2 × 6.25 = 63.75%, which is consistent with the design ratio.

[0053] Example 2: Effect of different protein to polysaccharide mass ratios on the properties of the complex

[0054] Following the method in Example 1, complexes with protein to polysaccharide mass ratios of 2:1, 3:1, 4:1, 5:1, and 6:1 were prepared respectively, and the effects of different mass ratios on the particle size, zeta potential, and encapsulation efficiency of the complexes were investigated.

[0055] Preparation method: The concentration of the insect protein solution was fixed at 4 mg / mL. The amount of morel polysaccharide solution added was adjusted so that the mass ratio of protein to polysaccharide was 2:1, 3:1, 4:1, 5:1, and 6:1, respectively. The remaining operations were the same as in Example 1.

[0056] The results are shown in Table 1.

[0057] Table 1. Effects of different protein to polysaccharide mass ratios on the properties of the complex.

[0058]

[0059] As shown in Table 1, as the protein-to-polysaccharide mass ratio increases from 2:1 to 4:1, the average particle size of the complex gradually decreases, the polydispersity index decreases, the absolute value of the Zeta potential increases, and the encapsulation efficiency improves, indicating that the complex formation is more complete and the dispersibility is better. When the mass ratio continues to increase to 5:1 and 6:1, the particle size increases, the polydispersity index increases, and the encapsulation efficiency decreases, possibly due to the aggregation of complexes caused by excess protein. Therefore, the optimal protein-to-polysaccharide mass ratio is 4:1.

[0060] Example 3: Effect of different pH values ​​on complex formation

[0061] Following the method in Example 1, with a fixed protein-to-polysaccharide mass ratio of 4:1, complexes were prepared under pH conditions of 3.5, 4.0, 4.5, 5.0, and 5.5, respectively, and the effect of pH on complex formation was investigated.

[0062] Preparation method: After adding the polysaccharide solution, the pH of the mixture was adjusted to 3.5, 4.0, 4.5, 5.0 and 5.5 respectively. The remaining operations were the same as in Example 1.

[0063] The results are shown in Table 2.

[0064] Table 2 Effect of different pH values ​​on complex formation

[0065]

[0066] Table 2 shows that at pH 3.5, the zeta potential of the protein is positive, indicating that the protein still carries a positive charge. However, the complex yield is low, possibly because the low pH reduces the degree of polysaccharide dissociation and weakens electrostatic attraction. Within the pH range of 4.0-5.0, the absolute value of the complex's zeta potential gradually increases with increasing pH, indicating an increase in the negative charge of the polysaccharide. However, the complex yield initially increases and then decreases, reaching its highest value at pH 4.5. When the pH increases to 5.5, the protein begins to lose its positive charge, and the electrostatic attraction weakens, leading to a decrease in complex yield and an increase in particle size. Therefore, the optimal pH for complex formation is 4.5.

[0067] Example 4: Evaluation of the in vitro digestibility of the complex

[0068] Insect protein and edible fungus polysaccharide complexes were prepared according to the method in Example 1, and their stability in simulated gastric and intestinal fluids was evaluated.

[0069] Preparation of simulated gastric juice: Add 2 g 7 mL concentrated Dissolve in 1 L of deionized water, add 3.2 g of pepsin, and adjust the pH to 1.2 to obtain simulated gastric juice.

[0070] Preparation of simulated intestinal fluid: 6.8 g Dissolve in 500 mL of deionized water, using 0.1 M... Adjust the pH of the solution to 6.8, add 10 g of pancreatic enzyme, and bring the volume to 1 L to obtain simulated intestinal fluid.

[0071] Digestion experiment: 100 mg of the complex or free insect protein was dispersed in 10 mL of simulated gastric juice and shaken at 37°C for 2 hours to simulate gastric digestion. Immediately after removal, an equal volume of simulated intestinal juice was added. The solution was adjusted to pH 6.8 and shaken at 37°C for 4 hours to simulate intestinal digestion. Samples were taken at 0, 0.5, 1, 2, 3, 4, and 6 hours after digestion. The enzymes were inactivated by boiling in a water bath for 5 minutes. After cooling, the samples were centrifuged at 12,000 rpm for 10 minutes. The supernatant was collected, and the protein concentration was determined using the BCA method to calculate the protein residue rate.

[0072] The results are shown in Table 3.

[0073] Table 3. Stability of complexes and free proteins in simulated digestive fluids

[0074]

[0075] Table 3 shows that the stability of the complex in simulated gastric and intestinal fluids was significantly higher than that of the free protein. After 2 hours of gastric digestion, the protein residue of the complex was 76.8%, while that of the free protein was only 46.5%, indicating a 65% improvement in the stability of the complex. After 6 hours of total digestion, the protein residue of the complex was 64.2%, while that of the free protein was only 35.8%, indicating a 79% improvement in the stability of the complex. This demonstrates that polysaccharides have a good protective effect on proteins, reducing protein degradation in the digestive tract and improving bioavailability.

[0076] Example 5 Cell transport assay of the complex

[0077] The intestinal absorption performance of the complex was evaluated using a Caco-2 cell monolayer model.

[0078] Cell culture: Caco-2 cells were seeded onto polycarbonate membranes in Transwell chambers at a cell density of 2× Cells were cultured per well in DMEM medium containing 10% fetal bovine serum, with the medium changed every 2 days, for 21 days to form a dense monolayer. Monolayer integrity was evaluated by transmembrane resistance (TEER), with a TEER value >400 Ω·m. Transport experiments were conducted at that time.

[0079] Transport experiment: The complex, free insect protein, and free morel polysaccharide were dissolved in HBSS buffer (pH 6.8) at a concentration of 1 mg / mL. The top culture medium of the Transwell chamber was discarded, and 1.5 mL of sample solution was added. 2.5 mL of HBSS buffer was added to the bottom chamber, and the mixture was incubated at 37°C. At 30, 60, 90, and 120 minutes, 200 μL of the bottom chamber solution was collected, and an equal volume of fresh HBSS buffer was added. The protein concentration was determined using the BCA method, and the apparent permeability coefficient was calculated. ).

[0080] Calculation formula:

[0081] ,

[0082] in, The transport rate is expressed in μg / s. For membrane area ( ), The initial concentration at the top (μg / mL).

[0083] The results are shown in Table 4.

[0084] Table 4 Apparent permeability coefficients of different samples

[0085]

[0086] As shown in Table 4, the apparent permeability coefficient of the complex is significantly higher than that of free insect protein and free morel polysaccharide, being 2.8 times and 3.2 times higher, respectively. This indicates that nanocomposite technology can significantly improve the intestinal absorption performance of the active ingredients, thereby enhancing bioavailability.

[0087] Example 6: The promoting effect of the complex on the proliferation of splenic lymphocytes

[0088] Insect protein and edible fungus polysaccharide complex were prepared according to the method in Example 1, and its promoting effect on the proliferation of mouse spleen lymphocytes was evaluated.

[0089] Isolation of splenic lymphocytes: Healthy male BALB / c mice aged 8 weeks were sacrificed by cervical dislocation, and the spleen was harvested under aseptic conditions and placed in RPMI-1640 medium containing 10% fetal bovine serum. The spleen was gently ground with a grinder to prepare a single-cell suspension, filtered through a 200-mesh cell sieve, centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded. 2 mL of erythrocyte lysis buffer was added, and the erythrocytes were lysed by incubation at room temperature for 3 minutes. 8 mL of culture medium was added to terminate the reaction, and the cells were centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded. The cells were resuspended in culture medium, counted, and the cell density was adjusted to 5× per mL.

[0090] Lymphocyte proliferation assay: Spleen lymphocyte suspension was seeded at 100 μL per well in a 96-well plate, and different concentrations of the complex, free insect protein, and free morel polysaccharide solution were added (final concentrations of 10, 50, 100, and 200 μg / mL). A blank control group and a positive control group (5 μg / mL concanavalin A, ConA) were also included. Each group had 6 replicates. The assay was performed at 37℃ and 5%... After culturing in an incubator for 48 hours, add 10 μL of CCK-8 solution to each well and continue culturing for 2 hours. Measure the absorbance at 450 nm using a microplate reader.

[0091] Formula for calculating the lymphocyte proliferation stimulation index (SI):

[0092] ,

[0093] The results are shown in Table 5.

[0094] Table 5. Promoting effects of different samples on splenic lymphocyte proliferation

[0095]

[0096] Table 5 shows that the complex significantly promoted the proliferation of splenic lymphocytes in a dose-dependent manner. At a concentration of 100 μg / mL, the stimulation index of the complex was 2.58, while that of free insect protein was only 1.12, and that of free morel polysaccharide was 1.43. The promoting effect of the complex was 2.3 times that of free insect protein and 1.8 times that of free morel polysaccharide, indicating a significant synergistic effect between insect protein and edible fungi polysaccharide.

[0097] Example 7 Evaluation of the antioxidant activity of the complex

[0098] Insect protein and edible fungus polysaccharide complex were prepared according to the method in Example 1, and their antioxidant activity was evaluated by DPPH free radical scavenging method and ABTS free radical scavenging method.

[0099] DPPH free radical scavenging assay: The complex, free insect protein, and free morel polysaccharide were dissolved in deionized water to prepare solutions of different concentrations (0.5, 1.0, 2.0, 4.0, and 8.0 mg / mL). 2 mL of the sample solution was taken, and 2 mL of 0.2 mM DPPH ethanol solution was added. After mixing, the solution was reacted at room temperature in the dark for 30 minutes, and the absorbance was measured at 517 nm. Deionized water was used as a blank control instead of the sample, and ethanol was used as the sample background instead of the DPPH solution.

[0100] Formula for calculating DPPH free radical scavenging rate:

[0101] ,

[0102] ABTS free radical scavenging experiment: 7 mM ABTS solution and 2.45 mM potassium persulfate solution were mixed in equal volumes and reacted at room temperature in the dark for 12–16 hours to prepare ABTS. + Working solution. Dilute with anhydrous ethanol to an absorbance of 0.70 ± 0.02 (734 nm) before use. Take 200 μL of sample solution and add 3 mL of ABTS. + After mixing the working solution, react at room temperature for 6 minutes, and then measure the absorbance at a wavelength of 734 nm.

[0103] The formula for calculating the ABTS free radical scavenging rate is the same as that for DPPH.

[0104] The results are shown in Tables 6 and 7.

[0105] Table 6. DPPH radical scavenging rate of different samples

[0106]

[0107] Table 7 ABTS radical scavenging rates of different samples

[0108]

[0109] As shown in Tables 6 and 7, the complex exhibits significant antioxidant activity, exceeding that of free insect protein and free morel polysaccharide. At a concentration of 4.0 mg / mL, the complex scavenged 78.3% of DPPH free radicals, 86% higher than free insect protein and 39% higher than free morel polysaccharide; and 82.6% of ABTS free radicals, 71% higher than free insect protein and 32% higher than free morel polysaccharide. This indicates that the complex technology can enhance antioxidant activity and produce a synergistic effect.

[0110] Example 8: Effects of the complex on macrophages The promoting effect

[0111] Insect protein and edible fungus polysaccharide complexes were prepared according to the method in Example 1, and their effect on nitric oxide production in mouse macrophage RAW264.7 cells was evaluated. The promoting effect of ).

[0112] Cell culture: RAW264.7 cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5%... After growing to the logarithmic growth phase in an incubator, the cells were digested with trypsin, counted, and then cultured at 5×... 200 μL of cells per well was seeded into 96-well plates and cultured for 24 hours to allow the cells to adhere.

[0113] Production Experiment: Discard the culture medium and add fresh culture medium containing different concentrations of the sample (10, 50, 100, 200 μg / mL), with 6 replicates per group. A blank control group and a positive control group (1 μg / mL lipopolysaccharide, LPS) were also included. After culturing for 24 hours, 100 μL of the supernatant was taken, and an equal volume of Griess' reagent was added. The reaction was carried out at room temperature for 10 minutes, and the absorbance was measured at 540 nm. A standard curve was prepared using sodium nitrite standard solution, and the absorbance was calculated. concentration.

[0114] The results are shown in Table 8.

[0115] Table 8 Effects of different samples on macrophages The promoting effect

[0116]

[0117] As shown in Table 8, the complex can significantly promote macrophage production. Furthermore, it is dose-dependent. At a concentration of 100 μg / mL, the complex stimulates the production of... The concentration was 23.5 μM, which is 2.1 times that of free insect protein (11.2 μM) and 1.6 times that of free morel polysaccharide (14.8 μM). It is an important marker of macrophage activation and participates in immune regulation and anti-tumor effects. The results show that the complex has good immune-enhancing activity.

[0118] Example 9: Application of the compound in functional oral liquids

[0119] Insect protein and edible fungus polysaccharide complex were prepared according to the method in Example 1 and applied to the preparation of functional oral liquid.

[0120] Formula (per 100 mL): Insect protein and edible fungus polysaccharide complex: 2.0 g; maltitol: 10.0 g; citric acid: 0.15 g; sodium citrate: 0.10 g; vitamin C: 0.05 g; potassium sorbate: 0.05 g; flavoring: appropriate amount; purified water: add to 100 mL.

[0121] Preparation method: Purified water was heated to 70℃, and maltitol, citric acid, sodium citrate, and vitamin C were added sequentially, stirring until dissolved. After cooling to 40℃, the complex powder was added, and the mixture was stirred at high speed for 30 minutes to ensure thorough dispersion. Potassium sorbate preservative and flavoring were added, and stirring continued for 10 minutes. The mixture was homogenized twice using a homogenizer at a pressure of 30 MPa. The solution was then filled into 10 mL amber glass bottles and sterilized at 121℃ for 15 minutes. After cooling, the functional oral liquid was obtained.

[0122] Product quality testing: The product is a light yellow, uniform liquid with no sediment or layering; the pH value is 5.8; the complex content is 1.95 g / 100 mL, which meets the labeled amount; the microbial test is qualified, with a total bacterial count of <100 CFU / mL, and no coliform bacteria, molds, or yeasts were detected.

[0123] Stability test: The oral liquid was stored at 4℃, 25℃, and 40℃ respectively, and its appearance, pH value, and complex content were tested periodically. The results showed that after 24 months of storage at 4℃ and 18 months of storage at 25℃, there were no significant changes in any of the indicators, and the product remained stable. After 6 months of storage under accelerated testing conditions at 40℃, the appearance changed slightly and the complex content decreased by about 5%, but it was still within the acceptable range.

[0124] Example 10: Application of the complex in functional capsules

[0125] Insect protein and edible fungus polysaccharide complex were prepared according to the method in Example 1 and applied to the preparation of functional capsules.

[0126] Formula (per capsule): Insect protein and edible fungus polysaccharide complex: 200 mg; microcrystalline cellulose: 80 mg; sodium carboxymethyl cellulose: 15 mg; magnesium stearate: 5 mg.

[0127] Preparation method: The complex powder was passed through a 100-mesh sieve and mixed evenly with microcrystalline cellulose to obtain mixture A. Sodium carboxymethyl cellulose was added to mixture A and mixed thoroughly. Magnesium stearate was added as a lubricant and mixed for 3 minutes. The final mixture was then filled into No. 0 gelatin empty capsules, each containing 300 mg.

[0128] Product quality inspection: The capsules are filled with light yellow powder and have no odor; the content difference is less than ±5%; the disintegration time is less than 30 minutes; the complex content is 195 mg / capsule, which meets the labeled amount; and the microbial test is qualified.

[0129] Stability test: The capsules were stored at 25°C and 60% relative humidity for 24 months, and no significant changes were observed in any of the indicators, indicating that the product remained stable.

[0130] Comparative Example 1: Extraction of insect protein using traditional methods

[0131] Insect proteins are extracted using traditional organic solvent extraction methods, while the supercritical fluid extraction method of this invention is used to extract them. The extraction methods were compared.

[0132] Extraction method: 500 g of mealworm powder was soaked in 2.5 L of petroleum ether and stirred at room temperature for 6 hours. The mixture was then filtered, and the extraction was repeated three times. The filtrates were combined, and the petroleum ether was recovered by rotary evaporation to obtain approximately 70 g of oil. The defatted mealworm powder was then used to extract protein according to step 1 of Example 1.

[0133] Results Comparison: The traditional method achieved a defatting rate of approximately 14%, compared to supercritical fluid defatting. Extraction is comparable, but requires a large amount of organic solvent (7.5 L), has a long extraction time (18 hours), and carries the risk of solvent residue. Gas chromatography analysis showed that the residual petroleum ether in the protein extracted by traditional methods was 85 mg / kg, while the supercritical method... No solvent residue was detected in the extracted protein. Protein powder extracted using traditional methods is grayish-white and has a slight off-odor, while supercritical extraction... The extracted protein powder is pale yellow, odorless, and has superior sensory quality.

[0134] Comparative Example 2: Extraction of morel polysaccharides using traditional methods

[0135] The traditional hot water extraction method for extracting morel polysaccharides was compared with the ultrasonic-assisted extraction method of this invention.

[0136] Extraction method: 150 g of morel mushroom powder was added to 4.5 L of deionized water at a solid-liquid ratio of 1:30 (w / v), and extracted by reflux at 100℃ for 4 hours. The extract was centrifuged, precipitated with alcohol, dialyzed, and freeze-dried to obtain morel mushroom polysaccharides.

[0137] Results Comparison: The polysaccharide extracted by the traditional hot water extraction method was 18.2 g, with an extraction rate of 12.1%, which is lower than that extracted by the ultrasonic-assisted extraction method of this invention (15.7%). The traditional method has a long extraction time (4 hours), high energy consumption, and the high temperature may lead to partial degradation of the polysaccharide. Gel permeation chromatography determined that the weight-average molecular weight of the polysaccharide extracted by the traditional method was 62,000 Da, significantly lower than that extracted by the ultrasonic-assisted extraction method (85,000 Da), indicating that the ultrasonic-assisted extraction method can better protect the structure of the polysaccharide.

[0138] Comparative Example 3: Preparation of protein-polysaccharide complexes using a heat-induced method.

[0139] The insect protein and morel polysaccharide complex was prepared by heating induction method and compared with the electrostatic self-assembly method of the present invention.

[0140] Preparation method: Insect protein solution (4 mg / mL) and morel polysaccharide solution (1 mg / mL) were mixed at a mass ratio of 4:1, the pH was adjusted to 6.5, heated at 90℃ for 30 minutes, cooled, centrifuged to collect the precipitate, and freeze-dried to obtain the complex.

[0141] Results Comparison: The yield of the complex prepared by the heating-induced method was 68.5%, lower than that of the electrostatic self-assembly method of this invention (89.7%). The particle size of the complex prepared by the heating-induced method was 450 nm, larger than that of the electrostatic self-assembly method (215 nm), and the polydispersity index was 0.52, indicating uneven particle size distribution. Heating may lead to protein denaturation and polysaccharide degradation, affecting the properties of the complex. The test on the promoting effect on splenic lymphocyte proliferation showed that the stimulation index of the complex prepared by the heating-induced method was 1.85, significantly lower than that of the electrostatic self-assembly method (2.58), indicating that the electrostatic self-assembly method can better maintain the bioactivity of the active ingredients.

[0142] Comparative Example 4: A simple mixture of free insect protein and free morel polysaccharide

[0143] Insect protein powder and morel polysaccharide powder were simply mixed at a mass ratio of 4:1 without any compounding treatment. Their activity was evaluated and compared with that of the compound.

[0144] Results Comparison: The simple mixture showed a stimulation index of 1.58 on spleen lymphocyte proliferation, lower than the complex (2.58); its DPPH free radical scavenging rate was 52.6%, lower than the complex (78.3%); the apparent permeability coefficient of the Caco-2 cell transport assay was... cm / s, far lower than that of the complex cm / s. This indicates that forming nanoscale complexes through electrostatic self-assembly can significantly improve the bioactivity and bioavailability of active ingredients, while simple mixing cannot achieve the same effect.

[0145] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a complex of insect protein and edible fungus polysaccharides, characterized in that, Includes the following steps: Step 1, Insect Protein Extraction: Yellow mealworm larvae are freeze-dried, pulverized, and defatted using supercritical CO2 extraction technology. The extraction pressure is 30-45 MPa, the extraction temperature is 40-50℃, the extraction time is 60-120 minutes, and the CO2 flow rate is 20-30 L / min, yielding defatted yellow mealworm powder. The defatted yellow mealworm powder is added to an alkaline buffer solution with pH 8.0-9.5 at a material-to-liquid ratio of 1:15-1:25, and extracted by stirring at 40-60℃ for 1-2 hours. The supernatant is collected by centrifugation, and the pH is adjusted to 4.5-5.5 to precipitate the protein by electrophoresis. The precipitate is collected by centrifugation, reconstituted with deionized water, and the pH is adjusted to 7.0-7.

5. The precipitate is then freeze-dried to obtain insect protein powder with a protein extraction rate greater than 90% and a purity greater than 85%. Step 2, Extraction of Polysaccharides from Edible Fungi: Morel fruiting bodies are freeze-dried, pulverized, and added to deionized water at a material-to-liquid ratio of 1:20-1:

35. Ultrasonic extraction is performed with an ultrasonic power of 400-600 W, an ultrasonic frequency of 20-40 kHz, an extraction temperature of 50-70℃, and an extraction time of 30-60 minutes. The extract is centrifuged at 8000-12000 rpm for 15-20 minutes, and the supernatant is collected. Polysaccharides are precipitated with anhydrous ethanol to a final concentration of 70%-80%, allowed to stand overnight at 4℃, and the precipitate is collected by centrifugation. The precipitate is reconstituted with deionized water, dialyzed to desalt, and freeze-dried to obtain morel polysaccharides. The polysaccharide extraction rate is 12%-18%, and the purity is greater than 75%. Step 3, preparation of electrostatic self-assembled complex: The insect protein powder obtained in Step 1 is prepared into a protein solution of 2-5 mg / mL, and the pH is adjusted to 6.5-7.5; the morel polysaccharide obtained in Step 2 is prepared into a polysaccharide solution of 0.5-2 mg / mL; under vigorous stirring, the polysaccharide solution is slowly added dropwise to the protein solution, with a protein to polysaccharide mass ratio of 3:1-5:1, and the pH is adjusted to 4.0-5.

0. Stirring is continued for 30-60 minutes, and nanoscale complexes are spontaneously formed by the electrostatic attraction of positively charged proteins and negatively charged polysaccharides; the mixture is centrifuged at 10000-15000 rpm for 10-15 minutes, the precipitate is collected, washed 2-3 times with pH 4.5 buffer, and freeze-dried to obtain the insect protein and edible fungus polysaccharide complex; The insect protein-edible fungus polysaccharide complex is in the form of spherical nanoparticles with an average particle size of 200-250 nm, a polydispersity index of less than 0.3, a zeta potential of -20 mV to -35 mV, an encapsulation efficiency of greater than 85%, and an infrared spectrum at 1650 cm⁻¹. -1 and 1540 cm -1 Characteristic peaks of amide I and amide II bands appear at 1080 cm⁻¹. -1 A polysaccharide characteristic peak appears at this location.

2. The preparation method according to claim 1, characterized in that, The supercritical CO2 extraction technology described in step one has an extraction pressure of 35-40 MPa, an extraction temperature of 45℃, an extraction time of 90 minutes, and a CO2 flow rate of 25 L / min.

3. The preparation method according to claim 1, characterized in that, The alkaline buffer solution mentioned in step one is a Tris-HCl buffer solution with a pH of 9.0 or a Na2CO3-NaHCO3 buffer solution.

4. The preparation method according to claim 1, characterized in that, In step two, the ultrasonic power for ultrasonic-assisted extraction is 500 W, the ultrasonic frequency is 30 kHz, the extraction temperature is 60℃, and the extraction time is 45 minutes.

5. The preparation method according to claim 1, characterized in that, The dialysis desalination described in step two uses a dialysis bag with a molecular weight cutoff of 3500 Da, and the dialysis time is 48 to 72 hours, with the deionized water being replaced every 8 to 12 hours.

6. The preparation method according to claim 1, characterized in that, The mass ratio of protein to polysaccharide in step three is 4:

1.

7. The preparation method according to claim 1, characterized in that, The insect protein and edible fungus polysaccharide complex has a protein content of 65%–75%, a polysaccharide content of 15%–25%, and a moisture content of less than 5%.

8. The insect protein and edible fungus polysaccharide complex prepared by the method according to any one of claims 1-7.

9. The application of the insect protein and edible fungus polysaccharide complex according to claim 8 in the preparation of functional foods.

10. The application according to claim 9, characterized in that, The functional food is an oral liquid, capsule, tablet or granule; the amount of insect protein and edible fungus polysaccharide complex added to the functional food is 0.5% to 5%; the functional food has the function of enhancing immunity or antioxidation.

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