Edible polypeptide preparation with diuretic and anti-inflammatory functions as well as preparation method and application thereof

Abstract

The invention discloses an edible polypeptide preparation with diuretic and anti-inflammatory functions as well as a preparation method and application thereof, and the preparation method comprises the following steps: (1) taking fresh pleurotus eryngii, carrying out wet grinding and ball material separation to obtain a pleurotus eryngii aqueous solution, and preparing a primary wet material; (2) performing ultrahigh pressure treatment and centrifugal separation on the first-stage wet material to obtain a pleurotus eryngii extract, and preparing a second-stage wet material; and (3) fully mixing the secondary wet material with water according to a mass ratio of 1: 3-1: 30, adding a compound enzyme preparation, putting the mixture into a radio frequency electromagnetic field for treatment, and purifying and drying after treatment to obtain the polypeptide powder preparation. The polypeptide preparation has no electrolyte disorder / liver and kidney toxicity, has dual effects of diuresis and inflammation diminishing, solves the problems of single effect and poor safety of the existing medicine and complex process of the traditional functional food, provides a safe, efficient and sustainable solution for sub-health and chronic disease patients, and has wide application prospects. And the pharmaceutical composition has remarkable market competitiveness and clinical transformation value.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a kind of edible polypeptide preparation with diuretic anti-inflammatory function and its preparation method and application. BACKGROUND

[0002] With the acceleration of urbanization, the proportion of sub-health population continues to rise globally. According to the data of the World Health Organization (WHO), about 75% of adults have chronic low-grade inflammation, which is closely related to metabolic diseases such as obesity, diabetes, and atherosclerosis. At the same time, the problem of water and sodium retention caused by sedentary behavior, high-salt diet, and environmental pollution is becoming increasingly prominent. Epidemiological data shows that about 30% of adults have hidden edema or urinary dysfunction. For the above health problems, the traditional solutions have obvious limitations: (1) Chemical drug dependence: long-term use of non-steroidal anti-inflammatory drugs (such as ibuprofen) can induce gastrointestinal ulcers, while loop diuretics (such as furosemide) can easily cause hypokalemia, glucose and lipid metabolism disorders, and kidney damage; (2) Traditional Chinese medicine application: represented by Chinese patents CN119157959A (Chinese medicine compound diuretic) and CN117223809B (anti-inflammatory oral liquid), although these methods have therapeutic effects, they have problems such as high cost and bitter taste, which leads to insufficient patient dependence; (3) Simple compounding strategy: the mechanical combination of diuretic herbs and anti-inflammatory ingredients lacks molecular synergistic mechanism, and the long-term safety is questionable, and the bioavailability is generally less than 15%. Functional foods with both diuretic and anti-inflammatory effects are still a market blank and need to be developed and innovated.

[0003] In recent years, food-derived protein polypeptides have become a research hotspot in functional foods due to their multi-target regulation characteristics. For example, casein phosphopeptide (SIER1P) significantly alleviates intestinal inflammatory damage by inhibiting the CaSR / PI3K / NF-κB and CaSR / PLC / NF-κB double signal pathways (Journal of Dairy Science, 2025). The soybean peptide seasoning salt developed by Chinese patent CN113208091A can antagonize the increase in blood pressure induced by high-salt diet; the three-step enzymatic combined microbial fermentation process proposed by CN115011660B successfully prepared metabolically promoting and antioxidant oat protein peptides. Although the above studies have confirmed the potential of protein polypeptides in antioxidant, anti-inflammatory, and metabolic regulation, their industrialization still faces severe challenges: traditional processes rely on complex enzymatic hydrolysis and multi-strain step-by-step fermentation (such as CN115011660B), which increases production costs by more than 40%; in addition, existing functional protein peptides cannot simultaneously meet the synergistic needs of diuresis and anti-inflammatory.

[0004] In summary, the development of a process to simplify, with diuretic anti-inflammatory function of polypeptide preparation (powder / oral liquid), not only can fill the sub-health intervention technology gap, more for preventive medicine and precision nutrition to provide innovative path, has significant social and economic value. SUMMARY

[0005] In view of the problems of significant side effects of chemical drugs, low dependence of traditional Chinese medicine preparations, and complex production process of multifunctional protein polypeptides in the prior art, the application provides an edible polypeptide preparation with diuretic and anti-inflammatory functions and a preparation method and application thereof. The edible polypeptide preparation of the application has no toxic side effects, and the production process is simple, and has good industrial application prospect.

[0006] The technical scheme of the application is as follows:

[0007] The first object of the application is to provide a preparation method of an edible polypeptide preparation with diuretic and anti-inflammatory functions, comprising the following steps:

[0008] (1) Take fresh Pleurotus eryngii, separate the ball material after wet grinding to obtain a Pleurotus eryngii aqueous solution, and prepare a first wet material;

[0009] The dispersing agent for wet grinding is water, and the particle size of the grinding ball is 0.4-0.6mm;

[0010] (2) The first wet material prepared in step (1) is treated by ultrahigh pressure, and centrifugal separation is performed to obtain a Pleurotus eryngii extract, and a second wet material is prepared;

[0011] (3) The second wet material prepared in step (2) is mixed with water at a mass ratio of 1:3-1:30, a composite enzyme preparation is added, and the mixture is treated in a radio frequency electromagnetic field. After the treatment, the mixture is purified, dried, and a polypeptide powder preparation is prepared;

[0012] The composite enzyme preparation comprises alkaline protease and papain;

[0013] The radio frequency electromagnetic field is an alternating electromagnetic wave with an electromagnetic frequency of 2-20 kHz.

[0014] In an embodiment of the application, in step (1), the mass ratio of water to material is 1-10:1; the mass ratio of ball to material is 0.5-3:1; the grinding time is 1-2h, the rotation speed is 400-1200rpm, and the working temperature is 40-60℃.

[0015] In an embodiment of the application, in step (1), the method for ball separation is centrifugal separation, and the centrifugal force is 4000-8000xg.

[0016] In an embodiment of the application, in step (2), the initial pH of the first wet material is 8.5-10.

[0017] In one embodiment of the present application, in step (2), the ultra-high pressure treatment condition is: pressure of 200-800 MPa, time of 10-80 min, and temperature of 40-70℃.

[0018] In one embodiment of the present application, in step (2), after the ultra-high pressure treatment, the pH of the wet material is adjusted to 4.5.

[0019] In one embodiment of the present application, in step (2), the centrifugal force of the centrifugal separation is 5000-11000 x g.

[0020] In one embodiment of the present application, in step (3), the enzyme loading of the alkaline protease is 0.1-5 wt% of the secondary wet material, and the enzyme loading of the papain is 0.2-3.6 wt% of the secondary wet material.

[0021] In one embodiment of the present application, in step (3), the alkaline protease and the papain are all commercially available products.

[0022] In one embodiment of the present application, in step (3), the treatment temperature is 40-60℃, and the treatment time is 12-48 h.

[0023] In one embodiment of the present application, in step (3), the purification method is ultrafiltration or nanofiltration.

[0024] In one embodiment of the present application, in step (3), the drying method is freeze-drying or spray-drying.

[0025] A second object of the present application is to provide a polypeptide powder preparation prepared by the above preparation method.

[0026] A third object of the present application is to provide an oral solution containing the above polypeptide powder preparation.

[0027] In one embodiment of the present application, the oral solution comprises: 0.5-30 parts of the above polypeptide powder preparation, 0.1-50 parts of white granulated sugar, 2-40 parts of honey, 0.1-10 parts of citric acid, 0.1-5 parts of a composite stabilizer, and 100-500 parts of water, each in terms of weight fraction.

[0028] In one embodiment of the present application, the composite stabilizer is sodium carboxymethyl cellulose, carrageenan, and guar gum; and the mass ratio of sodium carboxymethyl cellulose, carrageenan, and guar gum is 2:5:1.

[0029] A fourth object of the present application is to provide an application of the above polypeptide powder preparation or the above oral solution, for preparing food, health products, pharmaceuticals, or medical devices with the functions of relieving inflammation and increasing urine.

[0030] The beneficial technical effect of the present application is that:

[0031] The technical principle of the present application is that 1. fresh Pleurotus eryngii is mixed with grinding balls, under high shear, the tissue structure of Pleurotus eryngii is destroyed, so that the tissue particle size is less than 200 mesh; 2. by applying ultra-high pressure, the protein and polysaccharide are separated, the solubility of Pleurotus eryngii protein in alkaline solution is improved, so that the extraction efficiency of protein is increased; 3. constant alternating electromagnetic waves are used to induce the directional migration and friction of dipoles in the solution, so as to promote the unfolding of protein structure, thereby increasing the enzymolysis efficiency and improving the functional activity of protein polypeptide. Finally, a multifunctional Pleurotus eryngii polypeptide preparation is obtained. The protein polypeptide prepared by the principle has the functions of diuresis and anti-inflammation, and has no toxic side effects, and the production process is simple, which has good industrial application prospect.

[0032] The polypeptide preparation has no electrolyte disorder / liver and kidney toxicity, has the dual functions of diuresis and anti-inflammation, solves the problems of single function, poor safety and complex process of traditional functional food of existing drugs, provides a safe, efficient and sustainable solution for sub-health and chronic disease patients, has significant market competitiveness and clinical transformation value. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 The molecular weight distribution of the edible polypeptide powder of Example 1. DETAILED DESCRIPTION

[0034] The present application will be specifically described below in combination with the drawings and examples.

[0035] In the following examples of the present application, the particle size of the grinding ball is 0.6 mm, and the raw materials used are all commercially available products unless otherwise specified.

[0036] Example 1

[0037] A preparation method of an edible polypeptide preparation with diuretic and anti-inflammatory functions, comprising the following steps:

[0038] Take 100 g of fresh Pleurotus eryngii in 200 mL of ultrapure water, add 300 g of grinding balls, the rotation speed is 800 rpm, grind at 45℃ for 90 min, centrifuge at 6000xg, and obtain A solution;

[0039] Adjust the pH of the A solution to 9.0, place it in an ultra-high pressure of 400 MPa, maintain at 50℃ for 60 min, then adjust the pH of the solution to 4.5, centrifuge and separate at 6000xg, and obtain B precipitate;

[0040] The B precipitate is mixed with water at a mass ratio of 1:25, 1wt% of alkaline protease and 1.1wt% of papain are added relative to the B precipitate, and then the mixture is treated under a radio frequency electric field of 14 kHz for 20 h. After ultrafiltration and spray drying, an edible polypeptide powder is obtained.

[0041] 2g of the polypeptide powder, 0.1g of white granulated sugar, 4g of honey, 0.5g of citric acid, and 0.1g of a composite stabilizer (sodium carboxymethyl cellulose, carrageenan, and guar gum at a mass ratio of 2:5:1) are sequentially weighed into 100mL of water, and then stirred uniformly to obtain edible polypeptide oral solution 1.

[0042] Example 2

[0043] A preparation method of an edible polypeptide preparation with diuretic and anti-inflammatory functions, comprising the following steps:

[0044] 100g of fresh Pleurotus eryngii is taken in 200mL of ultrapure water, 300g of grinding balls are added, the rotation speed is 800rpm, and the mixture is ground at 45℃ for 90min. After centrifugation at 6000xg, an A solution is obtained.

[0045] The pH of the A solution is adjusted to 9.0, and the solution is placed in an ultrahigh pressure of 800MPa and maintained at 40℃ for 50min. Then the pH of the solution is adjusted to 4.5, and the solution is centrifuged at 6000xg to obtain a B precipitate.

[0046] The B precipitate is mixed with water at a mass ratio of 1:25, 1wt% of alkaline protease and 1.1wt% of papain are added relative to the B precipitate, and then the mixture is treated under a radio frequency electric field of 20 kHz for 20 h. After ultrafiltration and spray drying, an edible polypeptide powder is obtained.

[0047] 2g of the polypeptide powder, 0.1g of white granulated sugar, 4g of honey, 0.5g of citric acid, and 0.1g of a composite stabilizer (sodium carboxymethyl cellulose, carrageenan, and guar gum at a mass ratio of 2:5:1) are sequentially weighed into 100mL of water, and then stirred uniformly to obtain edible polypeptide oral solution 2.

[0048] Example 3

[0049] A preparation method of an edible polypeptide preparation with diuretic and anti-inflammatory functions, comprising the following steps:

[0050] 100g of fresh Pleurotus eryngii is taken in 200mL of ultrapure water, 300g of grinding balls are added, the rotation speed is 800rpm, and the mixture is ground at 45℃ for 90min. After centrifugation at 6000xg, an A solution is obtained.

[0051] The pH of the A solution was adjusted to 9.0, and the solution was placed in an ultrahigh pressure of 200 MPa and maintained at 70°C for 80 min. Then, the pH of the solution was adjusted to 4.5, and the solution was centrifuged at 6000 x g to obtain a B precipitate;

[0052] The B precipitate was mixed with water at a mass ratio of 1:25. After 1 wt% of alkaline protease and 1.1 wt% of papain were added relative to the B precipitate, the mixture was treated in a radio frequency electric field of 12 kHz for 10 h. After ultrafiltration and spray drying, an edible polypeptide powder was obtained.

[0053] The polypeptide powder, white sugar, honey, citric acid, and a composite stabilizer (sodium carboxymethyl cellulose, carrageenan, and guar gum at a mass ratio of 2:5:1) were sequentially weighed into 100 mL of water. After stirring, an edible polypeptide oral solution 3 was obtained.

[0054] Example 4

[0055] The same as in Example 1, except that the mass of fresh Pleurotus eryngii was 100 g, the amount of ultrapure water was 100 mL, and the mass of the grinding ball was 100 g.

[0056] Example 5

[0057] The same as in Example 1, except that the amount of added alkaline protease was 5 wt% relative to the B precipitate.

[0058] Example 6

[0059] The same as in Example 1, except that the amount of added alkaline protease was 2 wt% relative to the B precipitate.

[0060] Example 7

[0061] The same as in Example 1, except that the amount of added papain was 3.6 wt% relative to the B precipitate.

[0062] Example 8

[0063] The same as in Example 1, except that the amount of added papain was 0.8 wt% relative to the B precipitate.

[0064] Comparative Example 1

[0065] A method for preparing a Pleurotus eryngii polypeptide oral solution without cross-linking electromagnetic field treatment, comprising the following steps:

[0066] The raw materials were prepared. The weight of Pleurotus eryngii, the solid-liquid ratio, the concentration of the composite enzyme preparation, the oral solution formulation, and the amount used were the same as in Example 1.

[0067] The preparation process is different from example 1 in that the B precipitate is diluted with water, and then only the complex enzyme preparation is used for enzymolysis, and the other treatments are the same as in example 1.

[0068] Comparative example 2

[0069] A preparation method of a low-pressure-treated Pleurotus eryngii polypeptide oral liquid, comprising the following steps:

[0070] Prepare the raw materials, and the Pleurotus eryngii weight, solid-liquid ratio, complex enzyme preparation concentration, oral liquid formula, and dosage are the same as in example 1.

[0071] The preparation process is different from example 1 in that the ultrahigh pressure treatment pressure is 50 MPa, and the other treatments are the same as in example 1.

[0072] Comparative example 3

[0073] A preparation method of a low-frequency radio frequency treated Pleurotus eryngii polypeptide oral liquid, comprising the following steps:

[0074] Prepare the raw materials, and the Pleurotus eryngii weight, solid-liquid ratio, complex enzyme preparation concentration, oral liquid formula, and dosage are the same as in example 1.

[0075] The preparation process is different from example 1 in that the radio frequency treatment frequency is 1 kHz, and the other treatments are the same as in example 1.

[0076] Comparative example 4

[0077] A preparation method of a conventional Pleurotus eryngii polypeptide oral liquid, comprising the following steps:

[0078] Take 100 g of fresh Pleurotus eryngii, dry at 50°C and crush through a sieve, then add 2000 mL of ultrapure water, adjust the pH to 11, ultrasonically treat for 1 h, repeat the operation once, and collect the supernatant by centrifugation at 4000 r / min; adjust the pH to 6.5, add neutral protease (14 mg / mL, 5000 U / g), and enzymolysis at 50°C for 4 h, then obtain a Pleurotus eryngii polypeptide powder by ultrafiltration and drying, and configure the polypeptide oral liquid according to example 1.

[0079] Test example:

[0080] 1. Molecular weight determination

[0081] Take the edible polypeptide powder of Example 1, wash buffer to dissolve the sample after adding to the desalting column, after washing, concentrate, after re-dissolving, use nanoLC-MS / MS to detect. Chromatographic separation uses 150μm ID x 15cm reversed-phase chromatographic column, mobile phase uses acetonitrile-water-formic acid system. Mass spectrometer uses DDA PASEF mode to collect DDA data, scanning range from 100-1700m / z, Ramp time is 100ms, Accu time is 100ms.

[0082] 2, in vitro antioxidant function evaluation

[0083] Take 1mL of the sample to be tested, respectively, using superoxide anion scavenging ability detection kit (Solabio, BC1415), DPPH free radical scavenging ability detection kit (Solabio, BC4755) and hydroxyl radical scavenging ability detection kit (Solabio, BC1325) to evaluate the anti-inflammatory activity of each oral solution.

[0084] 3, sensory evaluation

[0085] Invite 12 professional sensory evaluation personnel (male, female half, youth, middle-aged, old, 4 people each), with the help of 100mm VAS test questionnaire to evaluate the aroma, taste and the willingness to urinate produced by each oral solution. Participants are scored independently, without interference, to ensure the accuracy of the evaluation results.

[0086] 4, diuretic efficacy evaluation

[0087] Take SPF SD rats, male, body weight 220-250g. The rats were fed in metabolic cages for 7 days before the test for environmental adaptation. Then, the rats were fasted for 12h without water before the test, and given 2.5mL / 100g of normal saline, and the urine excreted in two hours was collected. Rats with urine excretion greater than 40% of the gavage weight were selected for subsequent experiments.

[0088] Select qualified rats, randomly divide into blank control group (blank group), positive control group (Jisheng Shengqi pill), edible polypeptide oral solution group (Example 1), conventional Pleurotus eryngii polypeptide oral solution group (Comparative Example 4) according to body weight interval, 10 rats in each group. After fasting for 18h without water, each group was given normal saline by gavage at a dose of 2.5mL / 100g. After 20min, the drugs were given by gavage at the dose, the positive control group (0.21g / mL, 1mL / 100g), the edible polypeptide oral solution group (1.5mL / 100g), the conventional Pleurotus eryngii polypeptide oral solution group (1.5mL / 100g), and the blank group (1.5mL / 100g) pure water. After gavage, the rats were placed in metabolic cages, and the urine excreted in five hours was collected.

[0089] Test results

[0090] As shown in Figure 1 The polypeptide molecular weight of the edible polypeptide powder of Example 1 was in the range of 688.7 Da to 3252.2 Da after nanoLC-MS / MS detection. After statistical analysis, it was found that the polypeptides with molecular weight below 1400 Da accounted for 44.2%, the polypeptides with molecular weight in the range of 1400 Da to 2200 Da accounted for 53.0%, and the polypeptides with molecular weight in the range of 2200 Da to 3400 Da accounted for only 2.8%.

[0091] The antioxidant properties of different polypeptide oral solutions were compared as shown in Table 1. The Pleurotus eryngii polypeptide oral solution (Examples 1-3) constructed by radio frequency electromagnetic field-assisted complex enzymolysis had significantly higher superoxide anion scavenging capacity, DPPH free radical scavenging capacity and hydroxyl radical scavenging capacity than Comparative Examples 1-4, confirming the effectiveness of the method.

[0092] Table 1

[0093]

[0094] The sensory evaluation results of different polypeptide oral solutions are shown in Table 2. The edible polypeptide oral solution obtained by physical field combination and assisted enzymolysis (Example 1) had good appearance and taste, and could significantly improve the volunteers' desire to urinate.

[0095] Table 2

[0096]

[0097] The diuretic efficacy of different polypeptide oral solutions is shown in Table 3. As shown in the animal experiment (Table 3), compared with the control and comparative examples, the edible polypeptide oral solution obtained by physical field combination and assisted enzymolysis (Example 1) could significantly increase the urine volume of rats, indicating its potential for preventing or treating complications caused by sub-health.

[0098] Table 3

[0099] Group 1 h (mL) 3 h (mL) 5 h (mL) Total volume of urination (mL) Blank group 1.43±0.19 1.58±0.26 2.18±0.11 5.21±0.49 Positive control group 2.01±0.42 2.41±0.15 2.50±0.26 6.95±0.36 Example 1 2.11±0.52 2.88±0.79 2.52±0.21 7.49±0.62 Comparative Example 4 Example 2 Comparative Example 5 Example 3 Comparative Example 6 1.59±0.19 1.82±0.37 2.21±0.31 5.62±0.46

[0100] The examples provided above are not intended to limit the scope covered by the present application, and the described steps are not intended to limit the execution order. Those skilled in the art can make obvious improvements to the present application in combination with existing common knowledge, which also falls within the protection scope defined by the claims of the present application.

Claims

1. A method for preparing an edible polypeptide preparation having diuretic and anti-inflammatory functions, characterized in that, The method comprises the following steps: (1) taking fresh Pleurotus eryngii, obtaining Pleurotus eryngii water solution by wet grinding and ball separation to prepare first-stage wet material; The dispersant for wet grinding is water, and the grinding ball has a particle size of 0.4-0.6 mm; (2) treating the first-stage wet material prepared in step (1) by ultrahigh pressure, and centrifuging to obtain Pleurotus eryngii extract to prepare second-stage wet material; (3) mixing the second-stage wet material prepared in step (2) with water at a mass ratio of 1:3-1:30, adding a composite enzyme preparation, and placing in a radio frequency electromagnetic field for treatment, and after the treatment, purifying and drying to prepare a polypeptide powder preparation; The composite enzyme comprises alkaline protease and papain; and the radio frequency electromagnetic field is an alternating electromagnetic wave with an electromagnetic frequency of 2-20 kHz.

2. The production method according to claim 1, characterized by, In step (1), the water-material mass ratio is 1-10:1, the ball-material mass ratio is 0.5-3:1, the grinding time is 1-2 h, the rotating speed is 400-1200 rpm, and the working temperature is 40-60℃.

3. The preparation method according to claim 1, characterized in that, In step (2), the ultrahigh pressure treatment conditions are as follows: the pressure is 200-800 MPa, the time is 10-80 min, and the temperature is 40-70℃; after the ultrahigh pressure treatment, the pH of the wet material is adjusted to 4.

5.

4. The method of claim 1, wherein, In step (3), the enzyme addition amount of alkaline protease is 0.1-5 wt%, the enzyme addition amount of papain is 0.2-3.6 wt%, the temperature is 40-60℃, and the time is 12-48 h.

5. The preparation method according to claim 1, characterized in that, In step (3), the drying method is freeze-drying or spray-drying.

6. A polypeptide powder preparation prepared by the preparation method of any one of claims 1-5.

7. An oral liquid containing the polypeptide powder preparation of claim 6.

8. The oral solution of claim 7, wherein, The oral liquid comprises 0.5-30 parts of the polypeptide powder preparation of claim 6, 0.1-50 parts of white granulated sugar, 2-40 parts of honey, 0.1-10 parts of citric acid, 0.1-5 parts of a composite stabilizer, and 100-500 parts of water, each in terms of weight fraction.

9. The oral solution of claim 8, wherein, The composite stabilizer is sodium carboxymethyl cellulose, carrageenan, and guar gum.

10. The use of a polypeptide powder preparation according to claim 6 or an oral solution according to claim 7, characterized in that, The polypeptide powder preparation is used for preparing food, health products, medicines, or medical devices with the functions of relieving inflammation and increasing urine.

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