A composition, lotus seed protein peptide, compound beverage and preparation method and application thereof for improving immunity

Abstract

This invention belongs to the field of functional food technology, and provides a composition that helps improve immunity, lotus seed protein peptides, a compound beverage, and their preparation methods and applications. The composition for improving immunity contains the following raw materials in parts by weight: 18-23 parts lotus seeds, 15-21 parts astragalus, 15-19 parts poria cocos, 13-17 parts dried tangerine peel, and 10-13 parts malt. This invention uses lotus seeds, astragalus, poria cocos, dried tangerine peel, and malt as raw materials, and combines them with modern enzymatic hydrolysis technology to prepare lotus seed protein peptides. The extracts of traditional Chinese medicine are then combined with lotus seed protein peptides to produce a plant protein peptide beverage. The beverage prepared by this invention has stable processing, controllable quality, and is rich in nutrients. It contains marker components such as astragaloside A, hesperidin, and lotus seed protein peptides, which can increase the thymus index, spleen index, and macrophage phagocytic capacity in immunosuppressed mouse models, and has a good auxiliary effect in improving low immunity.

Description

Technical Field

[0001] This invention relates to the field of functional food technology, and in particular to a composition that helps improve immunity, lotus seed protein peptides, compound beverages, their preparation methods and applications. Background Technology

[0002] Weakened immunity is a common problem among the elderly, post-operative recovery groups, those experiencing chronic fatigue, and those in a sub-healthy state. It typically manifests as decreased resistance, susceptibility to external pathogens, and weakened recovery ability. With the upgrading of health-conscious consumption concepts, food and beverages that combine nutritional supplementation and functional regulation are gradually becoming market hotspots. Among these, substances that are both food and medicine have broad application prospects in functional foods due to their natural origin and readily available dietary base.

[0003] Traditional Chinese medicine theory holds that "the spleen and stomach are the foundation of acquired constitution and the source of qi and blood production." A healthy spleen and stomach ensure sufficient vital energy and strong defensive qi. Fupi San, a traditional formula for strengthening the spleen and replenishing qi, has the effect of tonifying the spleen and assisting vital energy, and is suitable for regulating conditions related to spleen and stomach weakness and impaired digestion. To meet the needs of food product development, it is necessary to optimize and improve the traditional formula, ensuring that the formula conforms to the principle of using medicinal and edible ingredients while also adapting to modern food processing and consumer demands. However, existing functional beverages mostly focus on extracting small-molecule active ingredients from herbs, and the utilization of plant proteins, especially protein peptides, remains insufficient. Therefore, developing a stable, quality-controllable composition with auxiliary immune-enhancing effects and its preparation method is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0004] The purpose of this invention is to provide a composition, lotus seed protein peptide, compound beverage, preparation method and application of which help improve immunity, combining traditional Chinese medicine compound with immunomodulatory peptides, which is expected to synergistically enhance immune efficacy.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: The present invention provides a composition that helps improve immunity, comprising the following ingredients in parts by weight: 18-23 parts lotus seeds, 15-21 parts astragalus, 15-19 parts poria cocos, 13-17 parts dried tangerine peel, and 10-13 parts malt.

[0006] The present invention also provides the use of the described composition in the preparation of products that help improve immunity.

[0007] The present invention also provides a compound extract that helps improve immunity, prepared from the aforementioned composition.

[0008] The present invention also provides a method for preparing the compound extract, comprising the following steps: Mix lotus seeds, astragalus, poria cocos, dried tangerine peel, malt, and water, soak, extract, and filter to obtain the final product.

[0009] Preferably, the amount of water used is 4 to 14 times the total mass of lotus seeds, astragalus, poria cocos, tangerine peel and malt, the soaking time is 10 to 60 minutes, the extraction temperature is 50 to 100°C, the extraction time is 40 to 90 minutes, and the extraction is performed 1 to 3 times.

[0010] The present invention also provides a plant-based beverage that helps improve immunity, containing the following ingredients in parts by weight: 40-60 parts of the compound extract, 5-25 parts of lotus seed protein peptide, 3-7 parts of xylitol, and 0.1-0.5 parts of compound stabilizer.

[0011] Preferably, the composite stabilizer contains the following raw materials in parts by weight: 0.5-1.5 parts gellan gum, 1.5-2.5 parts xanthan gum, and 0.5-1.5 parts sodium alginate; the lotus seed protein peptide has a molecular weight of less than 3 kDa, and the lotus seed protein peptide is obtained by enzymatic hydrolysis of lotus seed protein solution with alkaline protease.

[0012] Preferably, the amount of alkaline protease used is 2000~5500 U, the enzymatic hydrolysis temperature is 35~70℃, the enzymatic hydrolysis time is 2~6 h, and the concentration of lotus seed protein in the lotus seed protein solution is 2.5~7 wt%. The enzymatic hydrolysis further includes inactivation and centrifugation steps, the inactivation temperature is 90~99℃, the inactivation time is 5~15 min, the centrifugation speed is 3000~5000 r / min, and the centrifugation time is 5~15 min.

[0013] The present invention also provides a method for preparing the plant-based beverage, comprising the following steps: Mix the compound stabilizer, xylitol and water, heat to 85~90℃ for 3~7 minutes to hydrate, cool to 35~45℃, add the compound extract and lotus seed protein peptides to obtain the final product.

[0014] Lotus seeds are not only a food and medicine source, but also rich in protein. The small peptides formed from lotus seed protein after enzymatic hydrolysis are more easily absorbed and may possess immunomodulatory and antioxidant activities. Therefore, combining lotus seed protein peptides with extracts from spleen-strengthening and qi-tonifying compound formulas holds promise for creating an immunomodulatory beverage that combines the characteristics of traditional formulations with modern nutritional advantages.

[0015] The beneficial effects of this invention are as follows: This invention, based on traditional Chinese medicine theory, uses lotus seeds, astragalus, poria cocos, tangerine peel, and malt to construct a food-medicine homology composition, which is then compounded with small-molecule lotus seed protein peptides, overcoming the limitations of existing functional beverages with single-ingredient components. The combination of plant active ingredients and protein peptide nutrients enables multi-pathway and multi-target immunomodulatory effects, significantly enhancing the product's overall functionality. However, the compound extract is prone to precipitation and flocculation when combined with proteins / peptides. This invention specifically screened a composite stabilizer composed of gellan gum, xanthan gum, and sodium alginate for this composite system, effectively improving the suspension and dispersion state of the system, solving the problems of stratification and precipitation during product storage, and ensuring the uniformity of taste.

[0016] The composition, lotus seed protein peptide, and compound beverage provided by this invention can significantly increase the thymus index and spleen index of cyclophosphamide-induced immunosuppressed mice; significantly enhance DNFB-induced delayed-type hypersensitivity and improve cellular immune function; significantly increase the phagocytic index of mouse macrophages and enhance monocyte-macrophage function, thus having a good auxiliary effect in improving immunity. Attached Figure Description

[0017] Figure 1 The effect of each experimental group on the organ / body weight ratio in mice; Figure 2 The effects of each experimental group on the cellular immune function of mice (A: mouse spleen lymphocyte transformation experiment; B: delayed-type hypersensitivity experiment). Figure 3 The effect of each experimental group on the phagocytic index of mice; Figure 4 The effects of protein peptides of different molecular weights on the spleen index (A), thymus index (B), ear swelling (C), and phagocytic index (D) in mice; Figure 5 For the establishment and training of BP neural networks; Figure 6 The convergence curve of the genetic algorithm optimization process is shown. Figure 7 The response surface plot and contour plot show the impact of each factor on the overall score; Figure 8 The effect of the proportion of composite stabilizers on the stability coefficient; Figure 9 The effect of the amount of composite stabilizer added on the stability coefficient. Detailed Implementation

[0018] The present invention provides a composition that helps improve immunity, comprising the following ingredients in parts by weight: 18-23 parts lotus seeds, 15-21 parts astragalus, 15-19 parts poria cocos, 13-17 parts dried tangerine peel, and 10-13 parts malt.

[0019] In this invention, the composition is further preferably composed of the following raw materials in parts by weight: 21 parts lotus seeds, 19 parts astragalus, 17 parts poria cocos, 15 parts dried tangerine peel, and 12 parts malt.

[0020] The present invention also provides the use of the described composition in the preparation of products that help improve immunity.

[0021] The present invention also provides a compound extract that helps improve immunity, prepared from the aforementioned composition.

[0022] The present invention also provides a method for preparing the compound extract, comprising the following steps: Mix lotus seeds, astragalus, poria cocos, dried tangerine peel, malt, and water, soak, extract, and filter to obtain the final product.

[0023] In this invention, the amount of water used is preferably 4 to 14 times the total mass of lotus seeds, astragalus, poria cocos, tangerine peel, and malt, more preferably 8 to 10 times the total mass of lotus seeds, astragalus, poria cocos, tangerine peel, and malt; the soaking time is preferably 10 to 60 minutes, more preferably 20 to 50 minutes; the extraction temperature is preferably 50 to 100°C, more preferably 60 to 90°C; the extraction time is preferably 40 to 90 minutes, more preferably 60 to 70 minutes; and the number of extractions is preferably 1 to 3 times, more preferably 2 times.

[0024] The present invention also provides a plant-based beverage that helps improve immunity, containing the following ingredients in parts by weight: 40-60 parts of the compound extract, 5-25 parts of lotus seed protein peptide, 3-7 parts of xylitol, and 0.1-0.5 parts of compound stabilizer.

[0025] In this invention, the plant beverage is further preferably made of the following ingredients in parts by weight: 50 parts of the compound extract, 10-20 parts of lotus seed protein peptide, 5 parts of xylitol, and 0.2-0.4 parts of compound stabilizer.

[0026] In this invention, the composite stabilizer preferably contains the following raw materials in parts by weight: 0.5-1.5 parts gellan gum, 1.5-2.5 parts xanthan gum, and 0.5-1.5 parts sodium alginate; more preferably, it contains the following raw materials in parts by weight: 1 part gellan gum, 2 parts xanthan gum, and 1 part sodium alginate; the molecular weight of the lotus seed protein peptide is preferably less than 3 kDa, more preferably 2 kDa, and the lotus seed protein peptide is preferably obtained by enzymatic hydrolysis of lotus seed protein solution with alkaline protease.

[0027] In this invention, the amount of alkaline protease used is preferably 2000-5500 U, more preferably 3000-4000 U; the enzymatic hydrolysis temperature is preferably 35-70℃, more preferably 45-60℃; the enzymatic hydrolysis time is preferably 2-6 h, more preferably 3-5 h; the concentration of lotus seed protein in the lotus seed protein solution is preferably 2.5-7 wt%, more preferably 5-6 wt%; the enzymatic hydrolysis preferably includes inactivation and centrifugation steps, the inactivation temperature is preferably 90-99℃, more preferably 95℃; the inactivation time is preferably 5-15 min, more preferably 10 min; the centrifugation speed is preferably 3000-5000 r / min, more preferably 4000 r / min; and the centrifugation time is preferably 5-15 min, more preferably 10 min.

[0028] The present invention also provides a method for preparing the plant-based beverage, comprising the following steps: Mix the compound stabilizer, xylitol and water, heat to 85~90℃ for 3~7 minutes to hydrate, cool to 35~45℃, add the compound extract and lotus seed protein peptides to obtain the final product.

[0029] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0030] Example 1

[0031] (1) Preparation of compound extract that helps improve immunity: Weigh 18 g of lotus seeds, 16 g of astragalus, 16 g of poria cocos, 14 g of tangerine peel and 10 g of malt, grind them into the coarsest powder, add water (14 times the total mass of lotus seeds, astragalus, poria cocos, tangerine peel and malt), soak for 30 min, decoct at 95℃ for 90 min, repeat the extraction 3 times to obtain compound extract; (2) Preparation of lotus seed protein peptides that help improve immunity: Lotus seed protein was dissolved in deionized water to prepare a 5.5 wt% lotus seed protein solution. 3524 U of alkaline protease was added, and the solution was enzymatically hydrolyzed at 46℃ for 5 h. After the reaction, the hydrolysate was inactivated in a 95℃ water bath for 10 min, centrifuged at 4000 r / min for 10 min, and the supernatant was collected. The solution was then fractionated using 10 kDa and 3 kDa ultrafiltration tubes, and centrifuged at 4000 r / min for 10 min to obtain lotus seed protein peptides with a molecular weight of less than 3 kDa. (3) Preparation of a compound beverage that helps improve immunity: Weigh 0.3 parts by weight of a compound stabilizer (composed of gellan gum, xanthan gum, and sodium alginate in a weight ratio of 1:2:1) and 6 parts by weight of xylitol, and premix thoroughly in a dry state. Under high-speed shearing, slowly add the mixed powder to purified water for dispersion. Then heat to 87.5±2.5℃ and keep stirring for 5 minutes to completely hydrate all colloids. Cool the above colloidal solution to 40℃, add 46 parts by weight of the compound extract and 10 parts by weight of lotus seed protein peptides while stirring, and finally add purified water to make up to 100 parts by weight to obtain a compound beverage that improves immunity.

[0032] Example 2

[0033] (1) Preparation of compound extract that helps improve immunity: Weigh 18 g of lotus seeds, 16 g of astragalus, 16 g of poria cocos, 14 g of tangerine peel and 10 g of malt, grind them into coarse powder, add water (12 times the total mass of lotus seeds, astragalus, poria cocos, tangerine peel and malt), soak for 60 min, decoct at 60℃ for 40 min, repeat the extraction twice to obtain compound extract; (2) Preparation of lotus seed protein peptides that help improve immunity: Lotus seed protein was dissolved in deionized water to prepare a 2.5 wt% lotus seed protein solution. 2500 U of alkaline protease was added, and the solution was enzymatically hydrolyzed at 40℃ for 2.5 h. After the reaction, the hydrolysate was inactivated in a 95℃ water bath for 10 min, centrifuged at 4000 r / min for 10 min, and the supernatant was collected. The solution was then fractionated using 10 kDa and 3 kDa ultrafiltration tubes, and centrifuged at 4000 r / min for 10 min to obtain lotus seed protein peptides with a molecular weight of less than 3 kDa. (3) Preparation of a compound beverage that helps improve immunity: Weigh 0.3 parts by weight of a compound stabilizer (composed of gellan gum, xanthan gum, and sodium alginate in a weight ratio of 1:2:1) and 7 parts by weight of xylitol, and premix thoroughly in a dry state. Under high-speed shearing, slowly add the mixed powder to purified water for dispersion. Then heat to 87.5±2.5℃ and keep stirring for 5 minutes to completely hydrate all colloids. Cool the above colloidal solution to 40℃, add 55 parts by weight of the compound extract and 10 parts by weight of lotus seed protein peptides while stirring, and finally add purified water to make up to 100 parts by weight to obtain a compound beverage that improves immunity.

[0034] Example 3

[0035] (1) Preparation of compound extract that helps improve immunity: Weigh 18 g of lotus seeds, 16 g of astragalus, 16 g of poria cocos, 14 g of tangerine peel and 10 g of malt, add water (8 times the total mass of lotus seeds, astragalus, poria cocos, tangerine peel and malt), soak for 20 min, decoct at 70℃ for 40 min, repeat the extraction 3 times to obtain compound extract; (2) Preparation of lotus seed protein peptides that help improve immunity: Lotus seed protein was dissolved in deionized water to prepare a 5wt% lotus seed protein solution. 5000 U of alkaline protease was added, and the solution was enzymatically hydrolyzed at 65℃ for 2.5 h. After the reaction, the hydrolysate was inactivated in a 95℃ water bath for 10 min, centrifuged at 4000 r / min for 10 min, and the supernatant was collected. The solution was then fractionated using 10 kDa and 3 kDa ultrafiltration tubes, and centrifuged at 4000 r / min for 10 min to obtain lotus seed protein peptides with a molecular weight of less than 3 kDa. (3) Preparation of a compound beverage that helps improve immunity: Weigh 0.2 parts by weight of a compound stabilizer (composed of gellan gum, xanthan gum, and sodium alginate in a weight ratio of 1:2:1) and 5 parts by weight of xylitol, and premix thoroughly in a dry state. Under high-speed shearing, slowly add the mixed powder to purified water for dispersion. Then heat to 87.5±2.5℃ and keep stirring for 5 minutes to completely hydrate all colloids. Cool the above colloidal solution to 40℃, add 50 parts by weight of the compound extract and 5 parts by weight of lotus seed protein peptides while stirring, and finally add purified water to make up to 100 parts by weight to obtain a compound beverage that improves immunity.

[0036] Experimental Example 1

[0037] Forty SPF-grade ICR mice, half male and half female, were selected. After one week of acclimatization, the mice were divided into four groups: a blank male group, a blank female group, a drug-treated male group, and a drug-treated female group, with 10 mice in each group. The blank group mice were administered an equal volume of purified water by gavage, while the drug-treated groups were given the compound extract prepared in Example 1 at the maximum solubility (1.27 g / mL) and maximum gavage volume (0.35 mL / 10 g). The mice were observed for 14 consecutive days after a single administration, and their general condition, including food intake, activity, hair growth, weight changes, and mortality, was recorded.

[0038] Experimental results are shown in Table 1.

[0039] Table 1 Maximum tolerance test of the extract prepared in Example 1 ( ±S, n=10)

[0040] Table 3 shows that all mice had normal food and water intake during the observation period, their fur was smooth and shiny, their weight increased steadily, and they moved freely. No symptoms of poisoning or death were observed. The maximum dosage of the compound extract in both male and female ICR mice was calculated to be greater than 44.45 g / kg.

[0041] Experimental Example 2

[0042] Animal grouping and administration: Sixty male ICR mice were purchased from the Experimental Animal Center of Ningxia Medical University (License No.: SCXK(Ning)2019-0008), with standard body weight (18-22 g). After one week of acclimatization feeding, the mice were randomly divided into a normal control group, a model group, low (7 g / kg), medium (14 g / kg), and high (28 g / kg) dose groups of the extract, and a compound beverage group prepared in Example 1 (Lianqingyuan protein peptide beverage group), with 10 mice in each group. The mice were fasted for 12 hours before administration. During the experiment, the blank control group and the model group were administered purified water by gavage, while the administration groups were administered the corresponding test drug at a dose of 0.2 mL / 10 g for 30 days. On day 28, after gavage, except for the normal control group, the other groups were injected with 80 mg / kg cyclophosphamide solution for 3 consecutive days to induce an immunodeficient mouse model.

[0043] (1) Determination of organ / body weight ratio: On day 31 of the experiment, after the mice in each group were euthanized, the spleen and thymus of the mice were removed, the blood stains on the surface of the organs were dried with filter paper, and the organs were weighed to determine the organ index.

[0044] Thymus index = thymus weight (mg) / body weight (g); Spleen index = spleen weight (mg) / body weight (g).

[0045] Experimental results: such as Figure 1 As shown in A-B, compared with the blank control group, the organ indices of the model group mice were significantly reduced (P<0.001), indicating that the cyclophosphamide-induced immunosuppression model was successfully established. Compared with the model group, the thymus index and spleen index of each extract dosage group and the compound beverage group were increased to varying degrees, suggesting that the extract can effectively improve immune organ atrophy, promote immune organ development, and enhance the body's immune function.

[0046] (2) Mouse spleen lymphocyte transformation experiment: Mouse spleens were removed under aseptic conditions and a single-cell suspension was prepared. The suspension was filtered through a 200-mesh sieve, washed twice with Hank's solution, and centrifuged at 1000 r / min for 10 min each time. The supernatant was discarded, and the cells were resuspended in RPMI-1640 medium. Trypan blue staining was used to detect cell viability, and the cell concentration was adjusted to 2 × 10⁻⁶ cells / mL. 6Spleen cell suspension of adjusted concentration was added to 96-well plates, 100 µL per well. 50 µL of ConA solution (final concentration 5 μg / mL) was added to the stimulation wells, and an equal volume of RPMI-1640 culture medium was added to the control wells. Each sample was tested in triplicate. The plates were incubated at 37°C in a 5% CO2 incubator for 48 h. Two h before the end of the incubation period, 10% CCK-8 solution was added to each well, and incubation continued for another 2 h. The OD value was then measured at 450 nm using a microplate reader.

[0047] Experimental Results: Primary splenic lymphocytes from mice in each group were isolated, and the lymphocyte transformation rate was detected after 48 h of ConA intervention. The results are as follows: Figure 2 As shown in Figure A, the lymphocyte transformation rate in the model group was significantly lower than that in the normal control group. Compared with the model group, the lymphocyte transformation rates in the medium and high dose groups of Fupi San and the Lianqingyuan beverage group were increased, showing statistically significant differences.

[0048] (3) Delayed-type hypersensitivity test: On day 25 of drug administration, the abdominal hair of mice was removed. 50 µL of freshly prepared DNFB solution (50 mg DNFB added to 5 mL of pre-prepared acetone-sesame oil solution, acetone:sesame oil = 1:1, mixed well and sealed) was applied to induce sensitization. On day 30 of drug administration, a second challenge was performed by evenly applying 10 μL of DNFB solution to both sides of the right ear of the mouse. 24 h later, the mice were sacrificed, and the left and right ear flaps were cut off. Ear pieces with a diameter of 8 mm were taken from both ears using a punch and weighed.

[0049] Experimental results: In delayed-type hypersensitivity reactions, the intensity of the DTH response was reflected by the weight difference between the left and right earpieces, as shown in the following figures. Figure 2 As shown in Figure B, the ear swelling of mice in the model group was significantly reduced compared with that in the blank control group (P<0.01), while the ear swelling of each dose group of Fupi San extract was higher than that in the model group. Among them, the differences in the medium and high dose groups and the Lianqingyuan beverage group were significant (P<0.05). This indicates that Fupi San extract and products can enhance the body's delayed-type hypersensitivity response induced by DNFB and improve the T cell-mediated specific immune response.

[0050] (4) Carbon clearance experiment: Indian ink stock solution was diluted with physiological saline and injected into mice via the tail vein at a dose of 0.1 mL / 10 g body weight. 2 min and 10 min after injection, 20 µL of blood was collected from the venous plexus at the inner canthus of the mice and added to centrifuge tubes containing 2 mL of 0.1% Na2CO3 solution. The mixture was shaken well. Using 0.1% Na2CO3 solution as a blank control, the OD values ​​of the blood samples collected at the two time points were measured at a wavelength of 600 nm. The mice were euthanized, and the liver and spleen were harvested. Blood stains on the surface of the organs were blotted dry with filter paper, and the organs were weighed separately. The phagocytic index was calculated using the following formula: ; .

[0051] Experimental results: such as Figure 3 As shown, the phagocytic index α of the model group mice was significantly lower than that of the blank control group (P<0.01), indicating that the phagocytic function of macrophages was inhibited. Compared with the model group, the phagocytic index α of the medium and high dose groups of Fupi San extract and the Lianqingyuan beverage product group were significantly increased (P<0.05 or P<0.01), indicating that Fupi San extract can enhance the phagocytic capacity of macrophages and improve the body's non-specific immune defense function.

[0052] Experimental Example 3

[0053] Preparation of lotus seed protein peptides with different molecular weights: Lotus seed protein was enzymatically hydrolyzed using alkaline protease, and the resulting hydrolysate was fractionated sequentially using 10 kDa and 3 kDa ultrafiltration tubes. The fractions (Mw>10 kDa, 3-10 kDa, and <3 kDa) were collected by centrifugation at 4500 rpm and 4℃ for 15 min. These fractions were then freeze-dried and stored at -20℃ for later use.

[0054] Animal grouping and administration: Fifty male ICR mice were purchased from the Experimental Animal Center of Ningxia Medical University (License No.: SCXK(Ning)2019-0008) with a standard body weight of 20±2g. After one week of acclimatization, the mice were randomly divided into a blank control group, a model group, and groups with lotus seed protein peptide Mw>10 kD, 3~10 kD, and <3 kD, respectively. The blank control group and the model group were administered purified water by gavage, while the other groups were administered 50 mg / kg lotus seed protein peptide solution by gavage. All mice were given 2 mL / 10 g BW for 30 consecutive days. On day 28, after gavage, except for the normal control group, the other groups were injected with 80 mg / kg cyclophosphamide solution for 3 consecutive days to induce an immunodeficient mouse model.

[0055] (1) Determination of organ / body weight ratio: On day 31 of the experiment, after the mice in each group were euthanized, the spleen and thymus of the mice were removed, the blood stains on the surface of the organs were dried with filter paper, and the organs were weighed to determine the organ index.

[0056] Thymus index = thymus weight (mg) / body weight (g); Spleen index = spleen weight (mg) / body weight (g).

[0057] Experimental results: such as Figure 4As shown in -A and 4-B, compared with the normal control group, the spleen index (3.97±0.27) and thymus index (3.78±0.39) of the model group mice were significantly reduced, indicating that CTX caused damage to immune organs. The spleen index (3.72±0.47) and thymus index (3.47±0.48) of the Mw<3kDa group were significantly different from those of the model group (P<0.05) and closest to those of the normal group. This suggests that lotus seed peptide components can effectively alleviate CTX-induced atrophy of immune organs and have certain immunomodulatory potential.

[0058] (2) Delayed-type hypersensitivity test: On day 25 of drug administration, the abdominal hair of mice was removed. 50 µL of freshly prepared DNFB solution (50 mg DNFB added to 5 mL of pre-prepared acetone-sesame oil solution, acetone:sesame oil = 1:1, mixed well and sealed) was applied to induce sensitization. On day 30 of drug administration, a second challenge was performed by evenly applying 10 μL of DNFB solution to both sides of the right ear of the mouse. 24 h later, the mice were sacrificed, and the left and right ear flaps were cut off. Ear pieces with a diameter of 8 mm were taken from both ears using a punch and weighed.

[0059] Experimental results: such as Figure 4 As shown in Figure C, compared with the normal control group, the ear swelling degree of the model group mice was significantly reduced (P<0.001), indicating that the body's cellular immune response was suppressed after CTX injection, and the immunosuppressive model was successfully established. Among the drug administration groups, the DTH response of mice in the Mw<3 kDa group was significantly higher than that in the model group (P<0.001), indicating that this component can restore the cellular immune response capacity of immunosuppressed mice to a certain extent.

[0060] (3) Carbon clearance experiment: Indian ink stock solution was diluted with physiological saline and injected into mice via the tail vein at a dose of 0.1 mL / 10 g body weight. 2 min and 10 min after injection, 20 µL of blood was collected from the venous plexus at the inner canthus of the mice and added to centrifuge tubes containing 2 mL of 0.1% Na2CO3 solution. The mixture was shaken well. Using 0.1% Na2CO3 solution as a blank control, the OD values ​​of the blood samples collected at the two time points were measured at a wavelength of 600 nm. The mice were euthanized, and the liver and spleen were removed. Blood stains on the surface of the organs were blotted dry with filter paper, and the organs were weighed separately. The phagocytic index was calculated using the following formula: ; .

[0061] Experimental results: such as Figure 4As shown in Figure -D, the phagocytic capacity of mice in the model group was significantly lower than that in the normal control group (P<0.001), indicating that CTX has an inhibitory effect on non-specific immune function. After 30 days of gavage intervention, the phagocytic capacity of mice in each treatment group was improved to varying degrees. Among them, the Mw<3 kDa group was significantly different from the model group (P<0.001), and its phagocytic capacity was close to the normal level, suggesting that this component helps to promote the recovery of macrophage function.

[0062] As can be seen from the above examples and experimental cases, the composition, lotus seed protein peptide and compound beverage provided by the present invention can significantly increase the thymus index and spleen index of cyclophosphamide-induced immunosuppressed mice, improve the function of immune organs; significantly enhance DNFB-induced delayed-type hypersensitivity, enhance cellular immune function; and significantly increase the phagocytic index of mouse macrophages, enhance the function of monocytes and macrophages.

[0063] Experiment Example 4

[0064] I. Optimization of the extraction method for the composition: 1. Uniform experimental design Using U 12 (12) 10 The experimental design investigated the effects of six factors at different levels on the total polysaccharides, total flavonoids, total saponins and the yield of the dry extract in the composition. These factors included soaking time (10 min, 20 min, 30 min, 40 min, 50 min, 60 min), water addition ratio (4 times, 6 times, 8 times, 10 times, 12 times, 14 times), decoction time (40 min, 50 min, 60 min, 70 min, 80 min, 90 min), decoction temperature (40℃, 50℃, 60℃, 70℃, 80℃, 90℃), fineness of powder (uncrushed, coarsest powder, coarse powder), and number of extractions (1 time, 2 times, 3 times).

[0065] The AHP-CRITIC weighted analysis method was used to obtain the comprehensive weight of each indicator. The comprehensive score of the four evaluation indicators (total polysaccharides, total flavonoids, total saponins, and dry extract yield) in each experiment of the uniform experimental design was calculated using the formula.

[0066] .

[0067] Experimental results are shown in Tables 2 and 3.

[0068] Table 2. Determination of total polysaccharide, total flavonoid, and total saponin content and dry extract yield in each experiment under uniform experimental design.

[0069] Table 3. Calculation of the overall score for each experiment in a uniform experimental design.

[0070] 2. BP Neural Network Modeling and Optimization

[0071] (1) Model Establishment: A BP neural network model with a specific layer group of 3 was constructed based on Matlab2025a software, including an input layer, a hidden layer, and an output layer. The input layer contains 6 process parameters: fineness of powder, soaking time, water addition ratio, extraction time, extraction temperature, and number of extractions. The output layer is for optimization purposes, namely, the comprehensive score under different extraction conditions. Twelve sets of data obtained from uniform experiments were used as samples, and 70% of them were randomly assigned as the training set and 30% as the test set.

[0072] (2) BP neural network prediction: By training the neural network, combining the mean squared error (MSE) between the predicted and true values ​​and the correlation coefficient (R), the number of hidden layer nodes was determined. The training iterations were set to 1000, the learning rate to 0.1, the minimum training error to 0.0001, and the momentum factor to 0.01. The leave-one-out method was used to cross-validate the uniformly designed group data and train the network, and then the data was validated.

[0073] Experimental results: such as Figure 5 As shown.

[0074] (3) Verification experiment: The optimal extraction process obtained by the genetic algorithm for the extreme value optimization of the BP neural network was used to conduct a process verification experiment. The contents of total polysaccharides, total flavonoids, total saponins and the yield of dry extract were measured and compared with the optimal extraction process of uniform experimental design.

[0075] Experimental results: such as Figure 6 As shown in Table 4, the optimal extraction process was obtained by using a genetic algorithm to optimize the BP neural network, with a comprehensive score of 96.29. The highest comprehensive score of the uniform experimental design was 93.16. The BP neural network significantly outperformed the uniform experimental design in predicting the optimal extraction process.

[0076] Table 4 GA-BP neural network predicts optimal extraction process

[0077] II. Investigation of the enzymatic hydrolysis process of lotus seed protein peptides

[0078] Using U 8 (12) 10A uniform experimental design was used to investigate the effects of four factors at eight levels on the degree of hydrolysis and the yield of Mw < 3 kD: substrate concentration (2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%), enzyme dosage (2000 U / g, 2500 U / g, 3000 U / g, 3500 U / g, 4000 U / g, 4500 U / g, 5000 U / g, 5500 U / g), hydrolysis temperature (35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃), and hydrolysis time (2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 5.5 h).

[0079] Experimental results: As shown in Tables 5 and 6, the regression equation obtained from the uniform experiment is: Y = 181.4100666 - 0.06984188743X2 + 0.000007294047825X22 + 0.5484418244X42 + 0.005687167019X1X2 - 0.18681976877X1X3 - 0.0007843615644X2X4. The correlation coefficient R=1, the adjusted correlation coefficient Ra=0.9998, the overall significance test value F=3723.3983, the significance level P-value is 0.0039<0.01, the residual standard deviation is S=0.0796, and the Durbin-Watson statistic d=2.09495407, which is close to 2, indicating that the residuals follow a normal distribution, the model is stable, and the reliability is high. The calculated optimal process conditions are: lotus seed protein concentration 5.5wt%, alkaline protease addition 3524U / g, enzymatic hydrolysis temperature 46℃, and enzymatic hydrolysis time 5 h.

[0080] Table 5. Determination of degree of hydrolysis, yield of Mw < 3kD, and overall score in each experiment of the uniform experimental design.

[0081] The process parameters were optimized using a uniform design and then validated. The results showed that the optimal parameters obtained from the uniform design yielded a higher overall score and a lower RSD value compared to Experiment 6. This indicates that the validated process (substrate concentration 5.5%, enzyme dosage 3524 U / g, hydrolysis temperature 46℃, hydrolysis time 5 h) provides better and more stable hydrolysis results than the parameters initially selected in Experiment 6 using the uniform design. Therefore, the optimal combination of conditions obtained using this uniform design is accurate and reliable.

[0082] Table 6 Validation Experiment of Lotus Seed Protein Peptide Enzymatic Hydrolysis Process

[0083] III. Investigation of Compound Beverage Forming Process

[0084] 1. Single-factor experiment on flavoring agents for compound beverages

[0085] Scoring criteria: Referring to the sensory scoring criteria of the National Food Safety Standard for Beverages (GB 7101-2022), 10 judges scored the aroma, color, texture and taste of Lianqingyuan Beverage.

[0086] (1) Effect of the amount of compound extract added on sensory score

[0087] With a fixed addition of 15% lotus seed protein peptides and 5% xylitol, the effects of different extraction liquid additions (40%, 45%, 50%, 55%, and 60%) on the sensory scores of Lianqingyuan beverage were investigated.

[0088] Experimental results are shown in Table 7. With increasing extract dosage, the medicinal aroma of the beverage gradually intensified, and the flavor complexity increased. Based on comprehensive sensory evaluation, the optimal dosage of the herbal compound extract is 50%.

[0089] Table 7 Sensory scores for different amounts of extract added

[0090] (2) Effect of lotus seed protein peptide addition on sensory score

[0091] With a fixed addition of 50% of the compound extract of traditional Chinese medicine and 5% of xylitol, the effects of different additions of lotus seed protein peptides (5%, 10%, 15%, 20%, and 25%) on the sensory scores of Lianqingyuan beverage were investigated.

[0092] Experimental results are shown in Table 8. With increasing amounts of added protein peptides, the peptide flavor became more pronounced, and the richness of the beverage increased. Based on comprehensive sensory evaluation, the optimal amount of lotus seed protein peptides added is 15%.

[0093] Table 8 Sensory scores for different amounts of lotus seed protein peptides added

[0094] (3) Effect of xylitol addition on sensory score

[0095] With a fixed addition of 50% of the compound extract of traditional Chinese medicine and 15% of the lotus seed protein peptide, the effects of different xylitol additions of 3%, 4%, 5%, 6%, and 7% on the sensory scores of Lianqingyuan beverage were investigated.

[0096] Experimental results are shown in Table 9. Xylitol, as a sweetener, mainly functions to improve taste and mask bitterness. Based on comprehensive sensory evaluation, the optimal addition amount of xylitol is 6%.

[0097] Table 9 Sensory scores for different xylitol addition amounts

[0098] 2. Optimization of compound beverage formulation using response surface methodology

[0099] Based on the results of single-factor experiments, the amounts of extract, lotus seed protein peptides, and xylitol added were used as independent variables, and the comprehensive score was used as the response index. A fuzzy mathematics combined with Box-Behnken response surface methodology was employed to conduct the study. A three-factor, three-level central composite experiment was designed, and regression analysis was performed on the comprehensive score. The interaction between experimental factors was also examined to optimize the best formulation process. The factor level coding table is shown in Table 10.

[0100] Table 10. Factor and Level Coding Table for Response Surface Experiments

[0101] Seventeen composite samples were prepared using response surface methodology. Their odor, color, and taste were evaluated, and scores were compiled using fuzzy mathematical sensory evaluation to obtain a final comprehensive score.

[0102] (1) Fuzzy Mathematics Evaluation Method

[0103] Based on the sensory rating results, the following fuzzy evaluation matrix R is constructed, and the results are shown in Table 11. Using the matrix multiplication rule, the comprehensive membership degree Y=A is calculated. R. According to the normalization principle, a 气味 +a 色泽 +a 滋味 =1, determine the weight set A of the sensory rating set. = {a 滋味 a 口感 a 质地 ={0.2, 0.3, 0.5}. The results of the response surface methodology experimental design and the corresponding fuzzy mathematical method comprehensive scoring are shown in Table 12.

[0104] Table 11 Fuzzy Mathematical Sensory Evaluation Results

[0105] The fuzzy evaluation matrix R is established as follows: .

[0106] The overall membership degree Y = A × R is calculated as follows: .

[0107] Table 12 Response Surface Experimental Design and Fuzzy Mathematics Method for Comprehensive Scoring

[0108] (2) Analysis of variance

[0109] The reaction surface methodology (RSM) in Designin-Expert 13 was used to investigate the molding process of Lianqingyuan beverage, yielding the regression equation Y = -419.62 + 484.2A + 937.6B + 1693.2C + 1500AB + 660AC - 140BC - 2042A² - 1722B² - 3122C². The results of the analysis of variance are shown in Table 13. The RSM results indicate that the model is significant, with no significant lack-of-fit terms, and therefore it can be used for optimizing the beverage molding process.

[0110] Table 13 Results of Analysis of Variance

[0111] Response surface methodology software was used to construct response surface curves to analyze the interactions between various factors and their impact on the overall score.

[0112] Experimental results: such as Figure 7 As shown. The final prediction is: A. Extract addition amount: 46.64wt%, B. Lotus seed protein peptide addition amount: 10.05wt%, C. Xylitol addition amount: 5.97%, Y comprehensive score = 88.95.

[0113] 3. Investigation of stabilizers in compound beverages

[0114] (1) Determination of the compound ratio of composite stabilizers

[0115] Gellan gum, xanthan gum, and sodium alginate were selected and formulated at a total weight of 0.3% of the beverage mass in ratios of 1:1:1, 2:1:1, 1:2:1, 1:1:2, and 2:2:1. The effects of different stabilizer ratios on the stability and sensory evaluation of the beverage were determined.

[0116] Experimental results: as shown in Tables 14-15 and Figure 8 As shown: Table 14 Effect of Composite Stabilizer Ratio on Sensory Evaluation

[0117] (2) Determination of the amount of compound stabilizer added

[0118] The compound stabilizers were added to the beverage at a ratio of 1:2:1, with total amounts of 0.1%, 0.2%, 0.3%, 0.4%, and 0.5% of the beverage mass, respectively. The stability coefficient and sensory evaluation of the beverage were then determined.

[0119] Experimental results: as shown in the table and Figure 9As shown, based on the comprehensive stability coefficient and sensory evaluation results, when the total addition of gellan gum, xanthan gum and sodium alginate is 0.3% and the ratio is 1:2:1, the stability coefficient of the beverage is (91.38±1.34)%. The fuzzy mathematical sensory evaluation of the beverage prepared by the optimal process yields a comprehensive score of 89.90, indicating good sensory quality.

[0120] Table 15 Effect of Composite Stabilizer Addition Amount on Sensory Evaluation

[0121] As can be seen from the above examples and experimental cases, the principal ingredient, lotus seed, the assistant ingredients, astragalus root, poria cocos, and the adjuvant ingredients, tangerine peel and malt, in the composition of the present invention work synergistically to enhance immunity. Lotus seed, the principal ingredient, is a high-quality protein resource; through enzymatic hydrolysis, large protein molecules can be converted into small peptides, synergistically enhancing immune function. A compound beverage that enhances immunity is prepared by combining the composition, lotus seed protein peptides, xylitol, and a compound stabilizer. The composition, lotus seed protein peptides, and compound beverage provided by the present invention can significantly increase the thymus index and spleen index in cyclophosphamide-induced immunosuppressed mice; significantly enhance DNFB-induced delayed-type hypersensitivity reactions and improve cellular immune function; and significantly increase the phagocytic index of mouse macrophages and enhance monocyte-macrophage function.

[0122] As can be seen from the above embodiments, the present invention provides a composition that helps improve immunity, lotus seed protein peptides, a compound beverage, and its preparation method and application. The composition that helps improve immunity contains the following raw materials in parts by weight: 18-23 parts lotus seeds, 15-21 parts astragalus, 15-19 parts poria cocos, 13-17 parts dried tangerine peel, and 10-13 parts malt. The present invention uses lotus seeds, astragalus, poria cocos, dried tangerine peel, and malt as raw materials, and combines them with modern enzymatic hydrolysis technology to prepare lotus seed protein peptides. The extracts of traditional Chinese medicine are then combined with lotus seed protein peptides to prepare a plant protein peptide beverage. The beverage prepared by the present invention has stable processing, controllable quality, and is rich in nutrients. It contains marker components such as astragaloside A, hesperidin, and lotus seed protein peptides, which can increase the thymus index, spleen index, and macrophage phagocytic capacity in immunosuppressed mouse models, and has a good auxiliary effect in improving low immunity.

[0123] 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 principle 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 composition for improving immunity, characterized by comprising: The ingredients contain the following parts by weight: lotus seeds 18-23 parts, astragalus 15-21 parts, poria cocos 15-19 parts, dried tangerine peel 13-17 parts, and malt 10-13 parts.

2. The use of the composition of claim 1 in the preparation of products that help improve immunity.

3. A compound extract that helps improve immunity, characterized in that, It is prepared from the composition according to claim 1.

4. The method for preparing the compound extract according to claim 3, characterized in that, Includes the following steps: Mix lotus seeds, astragalus, poria cocos, dried tangerine peel, malt, and water, soak, extract, and filter to obtain the final product.

5. The preparation method according to claim 4, characterized in that, The amount of water used is 4 to 14 times the total mass of lotus seeds, astragalus, poria cocos, tangerine peel and malt. The soaking time is 10 to 60 minutes. The extraction temperature is 50 to 100°C. The extraction time is 40 to 90 minutes. The number of extractions is 1 to 3.

6. A plant-based beverage that helps improve immunity, characterized in that, The raw materials contain the following parts by weight: 40-60 parts of the compound extract as described in claim 3, 5-25 parts of lotus seed protein peptide, 3-7 parts of xylitol, and 0.1-0.5 parts of compound stabilizer.

7. The plant-based beverage according to claim 6, characterized in that, The composite stabilizer contains the following raw materials in parts by weight: 0.5-1.5 parts gellan gum, 1.5-2.5 parts xanthan gum, and 0.5-1.5 parts sodium alginate; the lotus seed protein peptide has a molecular weight of less than 3 kDa and is obtained by enzymatic hydrolysis of lotus seed protein solution with alkaline protease.

8. The plant-based beverage according to claim 7, characterized in that, The amount of alkaline protease used is 2000~5500 U, the enzymatic hydrolysis temperature is 35~70℃, the enzymatic hydrolysis time is 2~6 h, and the concentration of lotus seed protein in the lotus seed protein solution is 2.5~7 wt%. The enzymatic hydrolysis also includes inactivation and centrifugation steps. The inactivation temperature is 90~99℃, the inactivation time is 5~15 min, the centrifugation speed is 3000~5000 r / min, and the centrifugation time is 5~15 min.

9. A method for preparing the plant-based beverage according to any one of claims 6 to 8, characterized in that, Includes the following steps: Mix the compound stabilizer, xylitol and water, heat to 85~90℃ for 3~7 minutes to hydrate, cool to 35~45℃, add the compound extract and lotus seed protein peptides to obtain the final product.

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