Cordyceps militaris compound walnut protein health conditioning product and preparation method thereof

Abstract

The present invention provides a method for preparing a Cordyceps militaris composite walnut protein health conditioning product. Fresh Cordyceps militaris is used as the raw material, which is treated with an electron beam, pulped, and then a polysaccharide monooxygenase is added to the product. The product is freeze-dried to obtain modified Cordyceps militaris powder, which is then fused with walnut protein using a stage-controlled technique. As a nutritionally functional ingredient, the modified Cordyceps militaris powder overcomes the defects of walnut milk, which are prone to stratification and quality deterioration. The use of fresh Cordyceps militaris increases the nutritional value of the product, improving its content of dietary fiber, Cordyceps polysaccharides, cordycepin, and ergosterol. The active ingredients, particularly ergosterol, are effectively maintained during digestion, thereby improving absorption and bioavailability. The preparation technology and product not only help control blood sugar but also neutralize gastric acid and improve indigestion.

Description

Technical Field

[0001] The present invention relates to the technical field of biotechnology and healthy food, and in particular to a cordyceps militaris compound walnut protein health conditioning product and a preparation method thereof. Background Art

[0002] Cordyceps is rich in nutritional value, and its medicinal uses date back thousands of years in traditional medicine. In Traditional Chinese Medicine, Cordyceps sinensis is the most widely known species. However, climate change and overharvesting have severely damaged its microbial environment, leading to a gradual decline in production. Furthermore, the environment in which Cordyceps sinensis is cultivated is polluted, seriously impacting consumer safety. Studies have shown that Cordyceps sinensis powder and pure powder tablets contain arsenic levels of 4.4-9.9 mg / kg. Long-term consumption of Cordyceps sinensis, its powder, and pure powder tablets can lead to excessive arsenic intake, which can accumulate in the human body, posing a high risk.

[0003] As a cultivated species, Cordyceps militaris shares or has similar chemical compositions and medicinal properties to Cordyceps sinensis, offering a solution to the shortage of wild Cordyceps sinensis resources. In recent years, it has garnered significant attention and recognition in both scientific research and industry. Recognized by both Eastern and Western scientists, Cordyceps militaris has become a new hotspot in global life science research and development, ushering in new application prospects in the food, pharmaceutical, and health product industries. Studies have demonstrated its safety and broad spectrum. A large number of deeply processed Cordyceps militaris products have been launched and gained market acceptance. Most of these products utilize processes such as drying, crushing, impregnation, extraction, and spraying. These products, such as Cordyceps tea, Cordyceps biscuits, Cordyceps capsules, oral liquids, and Cordyceps wine, are less intensively processed and are often used in pharmaceutical products. Health benefits are often focused on components such as cordycepin and Cordyceps polysaccharides, while the fat-soluble component ergosterol has been less explored and utilized. A major drawback of these products is that they use dried Cordyceps as raw material, and the drying process often results in the loss of active ingredients such as ergosterol.

[0004] Walnut milk is a pure, natural plant-based protein beverage. Crafted using modern technology and scientifically formulated, it boasts a delicate taste and a distinctive, rich walnut aroma. It can be enjoyed hot or cold, boasting excellent brain-boosting benefits and rich nutritional value. However, walnut milk is prone to stratification, sedimentation, and odor deterioration during storage. Numerous chemical additives are added to maintain liquid stability, which can adversely affect human health. This issue has garnered industry attention, but few relevant solutions are available.

[0005] Therefore, it is urgent to provide a new conditioning product that combines the advantages of Cordyceps militaris and walnut protein and avoids the disadvantages of both. Summary of the Invention

[0006] In order to solve the above problems, the present invention deeply analyzes the instability of walnut milk and the system deterioration mechanism caused by it, utilizes the comprehensive nutritional and functional components of fresh Cordyceps, and explores a method of precision nutrition that is more in line with human health needs. On the one hand, it improves the antioxidant effect of walnut milk, and on the other hand, it develops fresh Cordyceps-derived foods and health products, which are of great benefit to practicing the health concept of "safety, universal benefits, and nutrition" and promoting human health. To achieve the above purpose, the present invention provides a method for preparing a Cordyceps militaris composite walnut protein health conditioning product, which specifically comprises the following steps:

[0007] Step 1: Preparation of modified Cordyceps militaris powder

[0008] Fresh Cordyceps militaris is used as raw material, is treated with an electron beam and then pulped, and then polysaccharide monooxygenase is added for enzymatic hydrolysis, followed by freeze-drying to obtain modified Cordyceps militaris powder;

[0009] Preferably, the electron beam treatment is performed under helium protection, at a temperature of 4°C, with an electron beam dose of 500-700 Gy; the beating is performed by adding water, with the ratio of Cordyceps militaris to water being 1 g:5 mL.

[0010] Preferably, the amount of the polysaccharide monooxygenase added is: 0.2% of the dry mass of Cordyceps militaris; the enzymatic hydrolysis time is 2-4h, and the enzymatic hydrolysis temperature is 45-50°C;

[0011] Step 2: Prepare composite material

[0012] The modified Cordyceps militaris powder, walnut protein powder and water are fully mixed, an inert gas is introduced and stirred, and then a controlled treatment is performed in stages, and after the treatment is completed, the temperature is raised to room temperature to obtain a composite material;

[0013] Preferably, the inert gas is helium, the stirring speed is 300 r / min, and the staged control process is:

[0014] Stage 1: heating from 25°C to 40°C, gas flow rate 6m / s, stirring for 10-30min;

[0015] The second stage: cooling from 40°C to 35°C, gas flow rate 15m / s, stirring for 1-3h;

[0016] The third stage: cooling from 35°C to 25°C, gas flow rate 5m / s, stirring for 1-2h;

[0017] Stage 4: Cooling from 25°C to 15°C, gas flow rate 2m / s, stirring for 0.5-1h;

[0018] The fifth stage: cooling from 15°C to 10°C, gas flow rate 2m / s, stirring for 10-30min;

[0019] Stage 6: Cooling from 10°C to 5°C, gas flow rate 1m / s, stirring for 5-10min;

[0020] Then warm to room temperature within 2 h;

[0021] Step 3: Prepare the finished product

[0022] Resistant starch is added to the composite material, followed by high-speed shearing, pH adjustment, and then guar gum is added and high-speed shearing is performed to obtain the finished product;

[0023] Preferably, the time of the two high-speed shearing operations is 1-3 min, and the rotation speed is 8000-10000 r / min;

[0024] Preferably, the pH is adjusted to 9-10 with KOH;

[0025] Preferably, based on parts by mass, 10 parts of water, 1-2 parts of modified Cordyceps militaris powder, 1-3 parts of walnut protein powder, and 1-2 parts of resistant starch, the amount of guar gum added is: 0.25-0.5% of the total mass of the modified Cordyceps militaris powder, walnut protein powder, and resistant starch;

[0026] The present invention has the following advantages:

[0027] (1) The modified Cordyceps militaris powder can be effectively reduced in an inert gas atmosphere by electron beam treatment, such as the activity of enzymes such as glucanase, chitinase, polyphenol oxidase and lipoxygenase, inhibiting the degradation of Cordyceps militaris cells and their active ingredients by these enzymes. The loss of Cordyceps militaris active ingredients is small, especially the sensitive ingredient ergosterol is preserved, which is more conducive to the preservation of Cordyceps militaris nutrients. The addition of polysaccharide monooxygenase is conducive to breaking down the cell wall, releasing the active ingredients, exposing more hydrophilic groups in the fiber to water, converting more water-soluble dietary fiber, promoting the loosening of the Cordyceps militaris dietary fiber structure, and improving the hydration of dietary fiber to a certain extent, which can effectively improve the adhesion and adhesiveness of the conditioning products.

[0028] (2) When Cordyceps militaris is added to walnut protein milk and inert gas is introduced, it helps isolate oxygen, reduces protein stratification in later products, and improves food flavor. Stage-by-stage regulation is beneficial to the fusion of cordyceps pulp fiber and protein, especially the formation of protein molecular complexes. The emulsification stability is significantly improved. The added bioactive ingredients enhance the nutritional properties of walnut milk and reduce its calories, which meets the health needs of modern consumers.

[0029] (3) The hydroxyl groups of cordyceps fiber will interact with some groups of protein polypeptide chains to form hydrogen bonds. The complex will wrap cordyceps ergosterol and other substances, which is beneficial to the preservation of active substances. The protein molecular complex will slow down the oxidative degradation of cordyceps ergosterol through physical hindrance, so that it will not be degraded when passing through the stomach and upper digestive tract, thereby better exerting its efficacy and improving the stability and bioavailability of the product; cordyceps pulp fiber can combine with the internal hydrophobic helix of starch molecules through hydrophobic interaction. This complex has positive significance for the application of resistant starch in the development of nutritional and health products;

[0030] (4) The Cordyceps militaris compound walnut protein health care product prepared by the present invention has good stability, high quality, delicate taste, good texture and flavor in terms of sensory quality. Alkaline guar gum is conducive to the fusion of cordyceps pulp fiber, walnut protein powder and resistant starch, which is conducive to the formation of the texture of the product. When the product enters the human stomach through the digestive tract, it balances gastric acid secretion and relieves adverse reactions such as nausea and heartburn.

[0031] (4) The present invention uses modified Cordyceps militaris as a dietary substitute and adds it to walnut milk. The modification effect of fresh Cordyceps militaris is analyzed, and the binding effect between Cordyceps militaris components and walnut protein is investigated. This provides a theoretical basis for the development and utilization of Cordyceps militaris as a nutritional and health product. It has certain physiological effects such as regulating gastric function and lowering blood sugar.

[0032] (5) The Cordyceps militaris compound walnut protein health care product prepared by the present invention is rich in Cordyceps militaris polysaccharides, cordycepin, ergosterol, protein, etc., promotes digestion and absorption, and meets the consumption needs of the elderly, pregnant women and other groups. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0034] Figure 1 The figure shows the effect of different irradiation doses on the activity changes of glucanase, chitinase, polyphenol oxidase, lipoxygenase and the content changes of nutritional functional substances;

[0035] Figure 2 This is a graph showing the effects of different temperatures and enzymatic hydrolysis times on the water holding capacity, water swelling capacity and oil holding capacity of modified Cordyceps militaris powder;

[0036] Figure 3 The microstructure diagram of the composites prepared in Examples 1-4 and Comparative Example 1;

[0037] Figure 4 UV spectra of the composites prepared in Examples 1-4 and Comparative Examples 1-2;

[0038] Figure 5 Graph showing the effects of different treatments on the subunits of the complex molecule in Examples 1-4 and Comparative Example 2;

[0039] Figure 6 This is a graph showing the effects of different treatments on the free thiol and tyrosine contents of the complexes in Examples 1-4 and Comparative Example 2;

[0040] Figure 7 The Zeta point change diagram of Example 1 and Comparative Example 3 during storage;

[0041] Figure 8 This is a graph showing changes in pH during gastric digestion;

[0042] Figure 9 This is the degradation release amount and first-order kinetic curve of ergosterol. DETAILED DESCRIPTION

[0043] The following is a clear and complete description of the technical solutions in the embodiments of the invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0044] Test Example 1

[0045] Fresh Cordyceps militaris was harvested at the Hubei Gugufang Ecological Agriculture Co., Ltd. base. Damaged and defective Cordyceps militaris were removed, and 300 g of the sample was placed in a PE insulated box at 4°C and filled with helium. The Cordyceps militaris was then treated with an electron beam from a 10 MeV-20 kW electron linear accelerator production line at doses of 400, 500, 600, 700, and 800 Gy, respectively.

[0046] The changes in the activities of glucanase, chitinase, polyphenol oxidase, and lipoxygenase and the content changes of nutritional and nutritional functional substances such as protein, polysaccharide, cordycepin, and ergosterol in Cordyceps militaris after electron beam treatment were detected. Figure 1 shown.

[0047] according to Figure 1Electron beam irradiation doses of 400 to 800 Gy inhibited the activities of glucanase, chitinase, polyphenol oxidase, and lipoxygenase, with 500 to 800 Gy exhibiting a significantly greater inhibitory effect than 400 Gy (P < 0.05). At a dose of 800 Gy, nutritional and functional components of Cordyceps militaris, such as protein, polysaccharides, cordycepin, and ergosterol, decreased significantly. This is due to the increased intensity of electron beam irradiation and the enhanced ionization effect, which degraded and damaged the intracellular components of fresh Cordyceps militaris.

[0048] Test Example 2

[0049] 300g of electron beam-treated Cordyceps militaris was slurried in 1500mL of distilled water. The mixture was then slurried with polysaccharide monooxygenase (0.2% of the dry weight of Cordyceps militaris). The modified Cordyceps militaris powder was obtained after enzymatic hydrolysis and freeze-drying. Four holding times (40°C, 45°C, 50°C, and 55°C) and four enzymatic hydrolysis times (1h, 2h, 4h, and 6h) were set for a total of 16 experiments.

[0050] The 16 experimental groups were labeled as CMP-40℃+1h, CMP-40℃+2h, CMP-40℃+4h, CMP-40℃+6h, CMP-45℃+1h, CMP-45℃+2h, CMP-45℃+4h, CMP-45℃+6h, CMP-50℃+1h, CMP-50℃+2h, CMP-50℃+4h, CMP-50℃+6h, CMP-55℃+1h, CMP-55℃+2h, CMP-55℃+4h, CMP-55℃+6h;

[0051] The calculation methods of water holding capacity, water swelling capacity and oil holding capacity are as follows:

[0052] Accurately weigh 1g (accurate to 0.001g) of modified Cordyceps militaris powder into a 50mL centrifuge tube and add 20mL of distilled water. After standing at room temperature for 24 hours, centrifuge (3000g, 15min), remove the supernatant liquid, drain the water, and weigh again. The water holding capacity is expressed by the following formula:

[0053]

[0054] Where: M2 is the total weight of the sample after absorbing water in the centrifuge tube (g); M1 is the weight of the centrifuge tube (g); M0 is the weight of the sample (g).

[0055] Accurately weigh 0.2g (accurate to 0.001g) of modified Cordyceps militaris powder into a 10mL graduated cylinder and record the sample volume as V1. Then add 10mL of distilled water, mix thoroughly, and let stand at room temperature for 24 hours. Observe and record the final volume of the sample as V2. The water absorption and expansion capacity is expressed by the following calculation formula:

[0056]

[0057] Where: V2 is the volume of the sample after absorbing water (mL); V1 is the volume of the sample before adding water (mL); M0 is the mass of the sample (g).

[0058] Accurately weigh 0.5g (accurate to 0.001g) of modified Cordyceps militaris powder into a 10mL centrifuge tube and add 5mL of soybean oil. Mix thoroughly, let stand for 24 hours, and then centrifuge (3000g, 5 minutes) to remove the upper layer of oil. The oil on the centrifuge tube wall is then absorbed with oil-absorbing filter paper. Let stand for 5 minutes and then weigh. The oil holding capacity is calculated using the following formula:

[0059]

[0060] Where: M2 is the total weight of the sample after oil absorption in the centrifuge tube (g); M1 is the weight of the centrifuge tube (g); M0 is the weight of the sample (g).

[0061] The water holding capacity, water absorption and swelling capacity of the modified Cordyceps militaris powder prepared in 16 groups of experiments were analyzed. The results are as follows: Figure 2 As shown;

[0062] according to Figure 2 The results show that the modified Cordyceps militaris powder exhibits optimal water-holding, water-swelling, and oil-holding properties at a hydrolysis temperature of 45-50°C. This is the optimal temperature range for the cleavage of polysaccharide monooxygenase. However, as the temperature rises to 55°C, the water-holding capacity of dietary fiber decreases. The modified Cordyceps militaris powder exhibits optimal water-holding, water-swelling, and oil-holding properties at a hydrolysis time of 2-4 hours, but these values decrease significantly after 6 hours. This is because a longer hydrolysis time further degrades the cell wall, fragmenting the Cordyceps militaris fiber and disrupting its structural integrity. This causes some water-soluble dietary fiber to dissolve in water, breaking away from the cellulose skeleton. This increases the cellulose pores, making it less able to bind more water, leading to a decrease in water-holding and water-swelling capacity. Furthermore, as the temperature rises, the cleavage of polysaccharide monooxygenase loses its activity, hindering the cell decomposition process. The lipophilic groups do not fully expand, limiting oil absorption.

[0063] The preparation conditions of the modified Cordyceps militaris powder used in Examples 1-4 and Comparative Examples 2-3 are: helium protection, temperature 4°C, electron beam dose 500 Gy, 0.2% of the dry mass of Cordyceps militaris, enzymolysis time 3 h, and enzymolysis temperature 45°C.

[0064] Example 1

[0065] 100 mL of distilled water, 10 g of modified Cordyceps militaris powder, and 10 g of walnut protein powder were added, and helium was introduced and stirred at a stirring speed of 300 r / min;

[0066] In the first stage, the temperature was raised from 25°C to 40°C, the gas flow rate was 6m / s, and stirring was carried out for 10min;

[0067] In the second stage, the temperature was lowered from 40°C to 35°C, the gas flow rate was 15m / s, and stirring was carried out for 3h;

[0068] The third stage: cooling from 35°C to 25°C, gas flow rate 5m / s, stirring for 2h;

[0069] Stage 4: Cooling from 25°C to 15°C, gas flow rate 2m / s, stirring for 40min;

[0070] Stage 5: Cooling from 15°C to 10°C, gas flow rate 2m / s, stirring for 20min;

[0071] Stage 6: Cooling from 10°C to 5°C, gas flow rate 1m / s, stirring for 5min;

[0072] After the treatment is completed, the temperature is slowly raised to room temperature within 2 hours to obtain a composite material of modified Cordyceps militaris powder and walnut protein powder; then 10g of resistant starch is added, high-speed shearing (time 1min, speed of 10000r / min), pH=9 is adjusted with KOH, 0.075g of guar gum is added, and high-speed shearing (time 1min, speed of 10000r / min) is obtained.

[0073] Example 2

[0074] 100 mL of distilled water, 10 g of modified Cordyceps militaris powder, 30 g of walnut protein powder, introduce helium and stir at a speed of 300 r / min;

[0075] In the first stage, the temperature was raised from 25°C to 40°C, the gas flow rate was 6m / s, and stirring was carried out for 10min;

[0076] In the second stage, the temperature was lowered from 40°C to 35°C, the gas flow rate was 15m / s, and stirring was carried out for 3h;

[0077] The third stage: cooling from 35°C to 25°C, gas flow rate 5m / s, stirring for 2h;

[0078] Stage 4: Cooling from 25°C to 15°C, gas flow rate 2m / s, stirring for 40min;

[0079] Stage 5: Cooling from 15°C to 10°C, gas flow rate 2m / s, stirring for 20min;

[0080] Stage 6: Cooling from 10°C to 5°C, gas flow rate 1m / s, stirring for 5min;

[0081] After the treatment is completed, the temperature is slowly raised to room temperature within 2 hours to obtain a composite material of modified Cordyceps militaris powder and walnut protein powder; then 20g of resistant starch is added, high-speed shearing (time 2min, speed is 9000r / min), pH=10 is adjusted with KOH, 0.18g of guar gum is added, and high-speed shearing (time 2min, speed is 9000r / min) is obtained.

[0082] Example 3

[0083] Add 30 g of walnut protein powder to 100 mL of distilled water and 15 g of modified Cordyceps militaris powder, introduce helium and stir at a speed of 300 r / min.

[0084] In the first stage, the temperature was raised from 25°C to 40°C, the gas flow rate was 6m / s, and stirring was carried out for 10min;

[0085] In the second stage, the temperature was lowered from 40°C to 35°C, the gas flow rate was 15m / s, and stirring was carried out for 3h;

[0086] The third stage: cooling from 35°C to 25°C, gas flow rate 5m / s, stirring for 2h;

[0087] Stage 4: Cooling from 25°C to 15°C, gas flow rate 2m / s, stirring for 40min;

[0088] Stage 5: Cooling from 15°C to 10°C, gas flow rate 2m / s, stirring for 20min;

[0089] Stage 6: Cooling from 10°C to 5°C, gas flow rate 1m / s, stirring for 5min;

[0090] After the treatment is completed, the temperature is slowly raised to room temperature within 2 hours to obtain a composite material of modified Cordyceps militaris powder and walnut protein powder; then 10g of resistant starch is added, high-speed shearing (time 3min, speed of 8000r / min), pH=9 is adjusted with KOH, 0.1925g of guar gum is added, and high-speed shearing (time 3min, speed of 8000r / min) is obtained.

[0091] Example 4

[0092] 100 mL of distilled water, 20 g of modified Cordyceps militaris powder, 20 g of walnut protein powder, introduce helium and stir at a speed of 300 r / min;

[0093] In the first stage, the temperature was raised from 25°C to 40°C, the gas flow rate was 6m / s, and stirring was carried out for 10min;

[0094] In the second stage, the temperature was lowered from 40°C to 35°C, the gas flow rate was 15m / s, and stirring was carried out for 3h;

[0095] The third stage: cooling from 35°C to 25°C, gas flow rate 5m / s, stirring for 2h;

[0096] Stage 4: Cooling from 25°C to 15°C, gas flow rate 2m / s, stirring for 40min;

[0097] Stage 5: Cooling from 15°C to 10°C, gas flow rate 2m / s, stirring for 20min;

[0098] Stage 6: Cooling from 10°C to 5°C, gas flow rate 1m / s, stirring for 5min;

[0099] After the treatment is completed, the temperature is slowly raised to room temperature within 2 hours to obtain a composite material of modified Cordyceps militaris powder and walnut protein powder; then 20g of resistant starch is added, high-speed shearing (time 1min, speed of 8500r / min), pH=10 is adjusted with KOH, 0.3g of guar gum is added, and high-speed shearing (time 1min, speed of 8500r / min) is obtained.

[0100] Comparative Example 1

[0101] Different from Example 1, this comparative example did not add modified Cordyceps militaris powder, and the specific steps were as follows:

[0102] 100 mL of distilled water, 10 g of walnut protein powder, introduce helium and stir at a speed of 300 r / min;

[0103] In the first stage, the temperature was raised from 25°C to 40°C, the gas flow rate was 6m / s, and stirring was carried out for 10min;

[0104] In the second stage, the temperature was lowered from 40°C to 35°C, the gas flow rate was 15m / s, and stirring was carried out for 3h;

[0105] The third stage: cooling from 35°C to 25°C, gas flow rate 5m / s, stirring for 2h;

[0106] Stage 4: Cooling from 25°C to 15°C, gas flow rate 2m / s, stirring for 40min;

[0107] Stage 5: Cooling from 15°C to 10°C, gas flow rate 2m / s, stirring for 20min;

[0108] Stage 6: Cooling from 10°C to 5°C, gas flow rate 1m / s, stirring for 5min;

[0109] After the treatment is completed, slowly warm to room temperature within 2 hours, then add 10g of resistant starch, high-speed shear (time 1min, speed 10000r / min), adjust pH=9 with KOH, add 0.05g of guar gum, and high-speed shear (time 1min, speed 10000r / min) to obtain the finished product.

[0110] Comparative Example 2

[0111] Different from Example 1, this comparative example does not adopt the staged control technology, and the specific steps are as follows:

[0112] 100 mL of distilled water, 10 g of modified Cordyceps militaris powder, 10 g of walnut protein powder were added, helium was introduced and stirred at a stirring speed of 300 r / min, and the mixture was treated at room temperature for 2 h to obtain a composite material of modified Cordyceps militaris powder and walnut protein powder; then 10 g of resistant starch was added, and high-speed shearing (time 1 min, speed 10000 r / min) was performed. The pH was adjusted to 9 with KOH, and 0.075 g of guar gum was added. The mixture was then high-speed shearing (time 1 min, speed 10000 r / min) was performed to obtain the finished product.

[0113] Comparative Example 3

[0114] Unlike Example 1, the pH value was not adjusted in this comparative example. The specific steps were as follows:

[0115] 100 mL of distilled water, 10 g of modified Cordyceps militaris powder, and 10 g of walnut protein powder were added, and helium was introduced and stirred at a stirring speed of 300 r / min.

[0116] In the first stage, the temperature was raised from 25°C to 40°C, the gas flow rate was 6m / s, and stirring was carried out for 10min;

[0117] In the second stage, the temperature was lowered from 40°C to 35°C, the gas flow rate was 15m / s, and stirring was carried out for 3h;

[0118] The third stage: cooling from 35°C to 25°C, gas flow rate 5m / s, stirring for 2h;

[0119] Stage 4: Cooling from 25°C to 15°C, gas flow rate 2m / s, stirring for 40min;

[0120] Stage 5: Cooling from 15°C to 10°C, gas flow rate 2m / s, stirring for 20min;

[0121] Stage 6: Cooling from 10°C to 5°C, gas flow rate 1m / s, stirring for 5min;

[0122] After the treatment is completed, the temperature is slowly raised to room temperature within 2 hours to obtain a composite material of modified Cordyceps militaris powder and walnut protein powder; then 10g of resistant starch is added, and high-speed shearing (time 1min, speed of 10000r / min) is performed, and 0.075g of guar gum is added, and high-speed shearing (time 1min, speed of 10000r / min) is performed to obtain the finished product.

[0123] Test Example 3

[0124] The composites prepared in Examples 1-4 and Comparative Example 1 were placed on a glass slide and gently covered with a cover glass. The microstructure of the emulsions was observed using an XDS-600C inverted phase contrast microscope. The microstructure of the composites was as follows: Figure 3 .

[0125] Depend on Figure 3 As can be seen, in Comparative Example 1 (no modified Cordyceps militaris powder was added), the protein particles within the field of view were clear and had a single composition. In Examples 1-4, after adding modified Cordyceps militaris powder, as the concentration of modified Cordyceps militaris powder increased, the degree of fusion between the Cordyceps militaris powder slurry and the walnut protein milk gradually increased. Compared with the composites of Examples 1 and 2, the composites of Examples 3 and 4 were more evenly distributed and finely divided.

[0126] The addition of modified Cordyceps militaris powder, in synergistically with the stage-modulation technology, increases the soluble dietary fiber content of Cordyceps militaris, strengthens the protein's water-binding capacity, modifies intermolecular forces, effectively reduces protein interfacial tension, and promotes a more uniform distribution of the complex in the liquid environment. Furthermore, the modulation technology promotes the solubilization of macromolecules such as cell wall polysaccharides in the Cordyceps militaris slurry, exposing more accessible groups and reducing the steric hindrance of walnut protein, thereby enhancing the interaction between the Cordyceps militaris fiber, polysaccharides, and walnut protein.

[0127] Test Example 4

[0128] Since the aromatic amino acids in the protein cause the protein to have a unique ultraviolet absorption at a wavelength of 270-280nm, the degree of covalent interaction can be determined by analyzing the absorption peak intensity, and the analysis of the maximum absorption / emission wavelength can be used to determine the changes in the tertiary structure of the protein. Therefore, ultraviolet spectrophotometry was used to detect Examples 1-4 and Comparative Examples 1-2, and the results are as follows: Figure 4 .

[0129] Depend on Figure 4 Walnut protein's absorption peak is concentrated at wavelengths between 260 and 290 nm, corresponding to the π-π* transitions of tyrosine, tryptophan, and phenylalanine residues. The results from the complexes of Examples 1-4 show that upon binding to Cordyceps militaris dietary fiber, walnut protein's UV absorption is significantly enhanced, accompanied by a blue shift. This alters the protein's spatial structure, with some specific amino acids shifting in position and becoming exposed to the surrounding solvent.

[0130] Test Example 5

[0131] Sodium dodecyl sulfate-PAGE (SDS-PAGE) was used to analyze the effects of different treatments in Examples 1-4 and Comparative Example 2 on the molecular subunits of the Cordyceps militaris walnut protein milk complex. The mass fractions of the concentrated gel and the separation gel were 12.5% and 5%, respectively, and the gel thickness was 1.5 cm. The sample was dissolved in ultrapure water, the sample volume was 15 μL per well, the voltage was adjusted to 80 V, and after the bromophenol blue indicator moved to the separation gel, the voltage was adjusted to 120 V until the electrophoresis was completed. After the electrophoresis was completed, Coomassie Brilliant Blue R-250 was used for staining, and after decolorization, the GS-900 gel imaging system was used for scanning and analysis. The results are shown in the figure. Figure 5 .

[0132] According to the difference in migration rate, the main walnut protein subunits studied include α- / β-subunit, γ-subunit and ω-subunit. Figure 5 It can be seen that there is no obvious change in the subunit distribution of the complexes of Examples 1-4, indicating that no small molecule subunits are produced after the regulation technology treatment. The color of the bands greater than 175 kDa in the complexes of Examples 1-4 deepens. This is because the regulation technology promotes the cross-linking of disulfide bonds between proteins, increases the stability of the protein molecular structure, and does not cause macromolecular aggregation; the subunits of the complexes all show obvious offsets, which is due to the increase in the relative molecular mass after the modified Cordyceps militaris powder is combined with walnut protein; compared with the protein migration bands of the complexes of Examples 1-4, the complex of Comparative Example 2 is missing in the 43 kDa to 58 kDa range, indicating that the failure to adopt the staged control technology leads to insufficient binding between the modified Cordyceps militaris powder and walnut protein, and some protein molecules in the system do not participate in the formation of the complex structure.

[0133] Test Example 6

[0134] The interaction of the Cordyceps militaris-walnut protein complex can be analyzed by detecting the changes in the free thiol and tyrosine contents. Figure 6 shown.

[0135] Depend on Figure 6 It can be seen that compared with the complex prepared in Comparative Example 2, the free thiol and tyrosine contents of the complexes prepared in Examples 1-4 are significantly reduced. This is because the side chain amino acids of the protein are oxidized by free radicals to form reaction sites and undergo nucleophilic addition reactions with soluble dietary fiber hydroxyl groups to form linkages.

[0136] Test Example 7

[0137] The Zata potential of the composites of Example 1 and Comparative Example 3 was measured using a Malvern Zetasizer NanoZS90 at 5, 15, 40, 60, 90, 110, 150, and 175 days of storage, respectively. Figure 7 .

[0138] Depend on Figure 7It can be seen that compared with the complex of Example 1, the absolute value of the Zeta potential of the complex of Comparative Example 3 gradually decreased by 43.21% within the storage period of 180 days, indicating that the binding of the Cordyceps militaris walnut protein complex without pH adjustment is not stable, and its stability is reduced during subsequent storage.

[0139] Test Example 8

[0140] The loading and sustained-release delivery of active nutrients are inextricably linked to human digestion and absorption. Digestion experiments can fully predict and evaluate the comprehensive utilization of complex solutions rich in active nutrients. In vitro simulated digestion experiments are widely used to study the digestive behavior and mechanisms of active nutrients in the gastrointestinal tract.

[0141] In order to investigate the digestion and absorption characteristics of Cordyceps militaris-walnut protein complex, an in vitro simulated gastrointestinal digestion experiment was carried out, and the degradation changes of ergosterol, the most sensitive nutritional function of the complex, were taken as the main investigation indicators.

[0142] (1) In vitro simulation of oral gastrointestinal digestion

[0143] Prepare the simulated stomach and intestine electrolyte solutions according to Table 1. Carry out the simulated digestion test according to the following process:

[0144] Simulated oral digestion stage: Prepare simulated oral digestive fluid, take 15 mL of sample and add 15 mL of oral digestive fluid, adjust the pH value to 6.8-7.0, keep the temperature at 37°C and stir at 120 r / min for continuous incubation, and take samples at 0, 30, 60, 120, and 180 s respectively.

[0145] Simulated gastric digestion stage: Prepare simulated gastric digestion solution, preheat the solution to 37°C before the experiment and maintain this temperature during the experiment. Take 15mL of the oral digestion sample and add 15mL of gastric digestion solution, keep the temperature at 37°C and stir at 180r / min for continuous incubation. Take samples at 0, 30, 60, 90, and 120 minutes respectively, and measure their pH values. The results are shown in Figure 8 .

[0146] Simulated intestinal digestion stage: Prepare simulated intestinal digestive fluid, take 15 mL of the sample after gastric digestion and add 15 mL of intestinal digestive fluid, keep the temperature at 37°C and stir at 150 r / min for continuous incubation, use 0.1 mol / L NaOH solution to maintain the intestinal fluid pH = 7.0, take samples at 0, 30, 60, 120, and 180 min, respectively, and react at 95°C for 5 min to inactivate the enzyme.

[0147] Table 1

[0148]

[0149] *Oral digestive fluid: Add salivary α-amylase to 400 mL of distilled water to an enzyme activity of 75 U / mL in the solution. At the same time, add CaCl2 to a final concentration of 0.75 mmol / L in the solution.

[0150] *Gastric digestion fluid: Add pepsin to 400 mL of gastric stock solution. The pepsin concentration in the digestion mixture is 2000 U / mL and the pH is 3.

[0151] *Intestinal digestion solution: Add trypsin and pancreatic enzymes to 400 mL of intestinal stock solution to achieve a final trypsin concentration of 100 U / mL, pancreatic enzyme concentration of 4 U / mL, bile salt concentration of 10 mmol / L, and pH of 7.

[0152] (2) Ergosterol degradation rate

[0153] 100 μL of simulated oral, gastric, and intestinal digestion samples were taken at different sampling times, and the degradation reaction was terminated with 100 μL of 0.1 mol / L hydrochloric acid. The oil phase in the sample was extracted with 300 μL of n-hexane, the mixed sample was vortexed for 10 seconds, and centrifuged at 10,000 × g for 4 minutes. The upper clear organic phase was collected, dried, and 0.05 g of the sample was weighed and added to 5 mL of a methanol / chloromethane (volume ratio 3:1) mixed solution. The sealed container was sealed and extracted in the dark for 2 hours (the sample extract was ultrasonically vibrated for 15 minutes intermittently). The supernatant was separated, and the remaining sample residue was added to 5 mL of a methanol / dichloromethane (volume ratio 3:1) mixed solution. The above steps were repeated. After 2 hours, the two extracts were mixed and fixed to volume in a 10 mL volumetric flask. The sample extract was filtered through a 0.45 gm filter membrane and then tested by HPLC. During the intestinal digestion stage, ergosterol gradually decreased with digestion time and reached the maximum degradation release rate ( ). Calculate according to the following formula, through ln[ ] plotted against time, such as Figure 9 .

[0154]

[0155] Where: k is the first-order kinetic rate constant of ergosterol degradation / s-1; t is the degradation amount over digestion time (t); b is the reference constant value.

[0156] (3) Ergosterol bioavailability

[0157] In Example 1, the mass of ergosterol in the complex is m0 (g). After digestion, the sample is collected, and the mass of ergosterol in the digestion fluid is recorded as m1 (g). Centrifuge at 10,000 × g for 40 min at 4°C to obtain a thin top layer of oil or emulsion, a middle layer of micellar phase, and a dense, insoluble material at the bottom. 5 mL of the micellar phase is filtered through a 0.22 μm microporous membrane. The filtrate or emulsion is mixed with dimethyl sulfoxide in a 1:1 volume ratio and vortexed to break the emulsion. The ergosterol content in the micellar phase is recorded as m2 (g). Dichloromethane / n-hexane (1:4 volume ratio) is added, vortexed for 30 seconds, and centrifuged at 3,000 × g for 5 min. The supernatant is removed and extracted three or more times. The sample is dried under nitrogen and redissolved in 200 μL of n-hexane containing 0.1% 2,6-di-tert-butyl-p-cresol. The ergosterol concentration is quantified using high-performance liquid chromatography. The bioavailability was calculated according to the following formula. The results are shown in Table 2.

[0158]

[0159] Table 2

[0160]

[0161] Depend on Figure 8 It can be seen that the addition of guar gum helps maintain the stability of ergosterol in acidic media and delays ergosterol degradation during in vitro gastric digestion. In addition, the high viscosity of guar gum can reduce contact and collision between interfacial molecules, which helps to improve the stability of the emulsion.

[0162] Depend on Figure 9 As shown in Table 2, ergosterol release approaches maximum value during initial intestinal digestion. Ergosterol degradation slows between 30 and 40 minutes, reaching a plateau after 60 minutes, reaching a degradation rate of (62.39 ± 5.27)%. This is because, on the one hand, as ergosterol concentration decreases, its contact with the relevant enzymes decreases; on the other hand, steric hindrance of the complex increases in the later stages of intestinal digestion, affecting the interfacial stability of the degrading enzymes. In summary, during the gastric digestion phase, the complex dissolves the Cordyceps militaris walnut protein complex (cordyceps fiber, protein, starch, etc.) under the action of pepsin and gastric peristalsis, gradually releasing ergosterol and transporting it to the intestine. Ergosterol is then degraded by pancreatic juice and bile salts. Bile salts participate in and promote the dissociation and release of ergosterol during intestinal digestion, resulting in a degradation and release rate of ergosterol in the complex reaching (73.25 ± 6.37)% and a bioavailability of (61.28 ± 8.31)%.

[0163] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a Cordyceps militaris compound walnut protein health-care product, characterized in that: The following steps are involved: Step 1: Preparation of modified Cordyceps militaris powder Fresh Cordyceps militaris is used as raw material, is treated with an electron beam and then pulped, and then polysaccharide monooxygenase is added for enzymatic hydrolysis, followed by freeze-drying to obtain modified Cordyceps militaris powder; Step 2: Prepare composite material The modified Cordyceps militaris powder, walnut protein powder and water are fully mixed, an inert gas is introduced and stirred, and a controlled treatment is performed in stages. After the treatment is completed, the mixture is heated to room temperature to obtain a composite material; Step 3: Prepare the finished product Resistant starch is added to the composite material, followed by high-speed shearing, pH adjustment, and then guar gum is added and high-speed shearing is performed to obtain the finished product; The electron beam treatment in step 1 is performed under helium protection, at a temperature of 4° C., with an electron beam dose of 500-700 Gy; the beating is performed by adding water, with the ratio of Cordyceps militaris to water being 1 g:5 mL; The amount of the polysaccharide monooxygenase added in step 1 is 0.2% of the dry mass of Cordyceps militaris; the enzymatic hydrolysis time is 2-4 hours, and the enzymatic hydrolysis temperature is 45-50° C.; In step 2, the inert gas is helium, the stirring speed is 300 r / min, and the staged control process is as follows: Stage 1: heating from 25°C to 40°C, gas flow rate 6m / s, stirring for 10-30min; The second stage: cooling from 40°C to 35°C, gas flow rate 15m / s, stirring for 1-3h; The third stage: cooling from 35°C to 25°C, gas flow rate 5m / s, stirring for 1-2h; Stage 4: Cooling from 25°C to 15°C, gas flow rate 2m / s, stirring for 0.5-1h; The fifth stage: cooling from 15°C to 10°C, gas flow rate 2m / s, stirring for 10-30min; Stage 6: Cooling from 10°C to 5°C, gas flow rate 1m / s, stirring for 5-10min; Then warm to room temperature within 2 h; In step 3, the pH is adjusted to 9-10 with KOH; Calculated by mass, the invention comprises 10 parts of water, 1-2 parts of modified Cordyceps militaris powder, 1-3 parts of walnut protein powder, and 1-2 parts of resistant starch, and the added amount of guar gum is 0.25-0.5% of the total mass of the modified Cordyceps militaris powder, walnut protein powder and resistant starch.

2. The method for preparing a Cordyceps militaris compound walnut protein health conditioning product according to claim 1, characterized in that: In step 3, the time of the two high-speed shearing operations is 1-3 minutes, and the rotation speed is 8000-10000 r / min.

3. A Cordyceps militaris compound walnut protein health care product prepared by the method according to any one of claims 1-2.

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