Plant polypeptide composition for promoting increase of hemoglobin and preparation process of plant polypeptide composition

Abstract

The invention provides a plant polypeptide composition for promoting hemoglobin increase and a preparation process thereof, and belongs to the technical field of health care products. The preparation process comprises the following steps: preparing the rubus parvifolius extract and the tartary buckwheat leaf extract; preparing hemoglobin peptide powder; preparing black bean peptide powder; and preparing the plant polypeptide composition. The preparation method comprises the following steps: preparing rubus parvifolius fruits into rubus parvifolius powder, adding the rubus parvifolius powder into an ethanol solution, carrying out ultrasonic-assisted extraction so as to obtain a rubus parvifolius extract, crushing tartary buckwheat leaves, adding the crushed tartary buckwheat leaves into the ethanol solution, carrying out heating reflux extraction so as to obtain a tartary buckwheat leaf extract, adding the rubus parvifolius extract and the tartary buckwheat leaf extract into a purified hemoglobin solution, and carrying out uniform mixing so as to obtain the rubus parvifolius extract. When the compound enzyme agent is added for enzymolysis, the enzymolysis of the compound enzyme agent on the hemoglobin can be promoted together, so that the efficiency of preparing the hemoglobin peptide through enzymolysis of the hemoglobin by the compound enzyme agent is synergistically improved.

Description

Technical Field

[0001] This invention relates to the field of health product technology, specifically to a plant polypeptide composition that promotes the increase of hemoglobin and its preparation process. Background Technology

[0002] Hemoglobin, a key protein in red blood cells that carries oxygen, plays an irreplaceable role in human physiological activities. It is responsible for taking in oxygen from the lungs and transporting it to all tissues and cells throughout the body, ensuring that cells can carry out normal aerobic respiration and metabolism. Normal hemoglobin levels are crucial for maintaining human health. When hemoglobin levels fall below the normal range, it can trigger a series of health problems. Hemoglobin deficiency leads to anemia, and anemic patients often experience symptoms such as dizziness, weakness, fatigue, and paleness, severely impacting their quality of life and work efficiency. Long-term anemia can also damage vital organs such as the heart and liver, increasing the risk of cardiovascular disease.

[0003] Currently, improving hemoglobin levels in clinical practice mainly relies on iron supplementation. Iron is a key raw material for hemoglobin synthesis, and iron deficiency leads to iron-deficiency anemia. Iron supplementation is a common method for treating iron-deficiency anemia. However, traditional iron supplements have many drawbacks. For example, while inorganic iron supplements such as ferrous sulfate have significant iron-supplementing effects, they are highly irritating to the gastrointestinal tract, easily causing adverse reactions such as nausea, vomiting, and constipation, resulting in poor patient compliance. Organic iron supplements, such as ferrous fumarate, have relatively fewer gastrointestinal reactions, but their bioavailability is limited, and long-term, high-dose use may cause iron overload, damaging organs such as the liver and heart.

[0004] Hemoglobin peptides are small molecule peptide products obtained by enzymatic hydrolysis of hemoglobin. They not only have the potential to promote hemoglobin synthesis, but also are less likely to cause adverse reactions or iron overload when taken orally. Furthermore, they can effectively promote the increase of hemoglobin in the body. However, the efficiency of preparing hemoglobin peptides by enzymatic hydrolysis of hemoglobin is not high, and the output is difficult to meet market demand, due to the influence of various factors on the hydrolysis process, such as enzyme specificity, hydrolysis conditions, and enzyme activity limitations.

[0005] Therefore, there is a need to propose a plant polypeptide composition that promotes hemoglobin production by improving the efficiency of hemoglobin enzymatic hydrolysis and its preparation process. Summary of the Invention

[0006] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a plant polypeptide composition that promotes the increase of hemoglobin and its preparation process.

[0007] A process for preparing a plant polypeptide composition that promotes hemoglobin increase includes the following steps: S1: Preparation of raspberry extract and buckwheat leaf extract Raspberry and buckwheat leaves were extracted separately with ethanol solution to prepare raspberry extract and buckwheat leaf extract; S2: Preparation of hemoglobin peptide powder Fresh animal blood was collected to prepare purified red blood cells. The purified red blood cells were then suspended in pure water, stirred and ruptured, and hemoglobin was collected. The hemoglobin was then purified by dialysis to obtain a purified hemoglobin solution. Finally, the above-mentioned strawberry extract and buckwheat leaf extract were added for enzymatic hydrolysis to prepare hemoglobin peptide powder. S3: Preparation of black bean peptide powder Black soybeans are made into black soybean milk, sodium hydroxide solution is added to prepare black soybean protein extract, hydrochloric acid is added and the crude black soybean protein is collected by centrifugation, then a complex protease is added and incubated for enzymatic hydrolysis to prepare black soybean peptide powder. S4: Preparation of plant polypeptide compositions Pomegranate juice, longan powder, and jujube extract were prepared separately, and then thoroughly mixed with the above-mentioned hemoglobin peptide powder, black bean peptide powder, purified water, tremella polysaccharide, heme iron, sodium iron EDTA, sucralose, and citric acid to obtain a plant polypeptide composition.

[0008] Furthermore, S1 specifically includes the following steps: S1.1: Wash the ripe raspberries, dry them at low temperature, and then crush and grind them to obtain raspberry powder; S1.2: Add the above raspberry powder to a 70% ethanol solution at a solid-liquid ratio of 1g:(20-30)mL, and extract with ultrasonic-assisted heating and stirring at 200-300W and 40-50℃ for 40-50min. After filtration, vacuum concentration and drying, the raspberry extract is obtained. S1.3: Wash, dry and crush fresh buckwheat leaves, then add them to 60% ethanol solution at a solid-liquid ratio of 1g:(20-30)mL, heat and stir under reflux at 70-80℃ for 1-2h, cool, centrifuge, concentrate under reduced pressure and dry to obtain buckwheat leaf extract.

[0009] Furthermore, S2 specifically includes the following steps: S2.1: Collect fresh animal blood, add anticoagulant, and centrifuge at 3000-4000 r / min for 10-15 min at 4℃. Collect red blood cells, wash with physiological saline 2-3 times to obtain purified red blood cells. S2.2: The purified red blood cells were suspended in 0.1% sodium chloride solution and stirred until fully ruptured. The mixture was then centrifuged at 3000-4000 r / min for 8-10 min and the supernatant was collected. Ammonium sulfate was added until the ammonium sulfate saturation reached 40-60%. After standing for 6-8 h, the mixture was centrifuged at 4000-5000 r / min for 10-15 min and the precipitate was collected to obtain hemoglobin. S2.3: Dissolve the above hemoglobin in phosphate buffer solution with pH 7-7.5 to prepare a hemoglobin solution with a concentration of 10 mg / mL. Then dialyze the hemoglobin solution with a dialysis bag for 20-24 hours, changing the dialysate every 4 hours to obtain a purified hemoglobin solution. S2.4: Add the raspberry extract obtained in step S1.2 and the buckwheat leaf extract obtained in step S1.3 to the above purified hemoglobin solution, heat to 40-45℃ and keep warm, then add the compound enzyme and keep warm for 4-6 hours for enzymatic hydrolysis. After enzyme inactivation, centrifuge at 4000-5000 r / min for 10-15 min, collect the supernatant for ultrafiltration, collect the permeate, and obtain hemoglobin peptide powder by dialysis, concentration and drying.

[0010] Furthermore, S3 specifically includes the following steps: S3.1: Wash the plump black beans that are free from pests, diseases, and mold, then soak them in clean water for 8-12 hours. After draining the water, grind them into a paste to obtain black bean milk. S3.2: Add 0.1mol / L sodium hydroxide solution to the above black soybean milk to adjust the pH to 8-9, and extract with ultrasonic assistance at 200-300W and 40-60℃ for 1-2 hours. After centrifugation to remove the residue, black soybean protein extract is obtained. S3.3: Add 0.1 mol / L hydrochloric acid to the above black bean protein extract, adjust the pH to 4-5, then centrifuge to separate the precipitate and obtain crude black bean protein; S3.4: Add the above black bean crude protein to distilled water at a solid-liquid ratio of 1g:(40-50)mL, stir and mix thoroughly, heat to 40-50℃ and keep warm, then add compound protease, heat and enzymatically hydrolyze for 2-3 hours, then heat to 80-90℃ and keep warm to inactivate the enzyme for 10-15 minutes to obtain black bean enzymatic hydrolysate. S3.5: Filter the above black bean enzymatic hydrolysate, then use an ultrafiltration membrane to ultrafilter the filtrate, and after vacuum concentration and freeze drying, obtain black bean peptide powder.

[0011] Furthermore, S4 specifically includes the following steps: S4.1: Wash fresh, smooth-skinned, and disease-free pomegranates, remove the pomegranate seeds, juice the pomegranate seeds, filter the juice, and obtain pomegranate juice. S4.2: Remove the shells and pits from the dried longan, dry the longan pulp at 60-80℃ for 2-4 hours, then crush, grind and sieve it to obtain longan powder; S4.3: After cleaning the dried red dates, remove the pits and grind them into powder. Then add them to purified water at a solid-liquid ratio of 1g:(10-20)mL. Heat at 60-70℃ for 1-2 hours and extract. Repeat the extraction 2-3 times. Combine the extracts, filter, concentrate and dry to obtain red date extract. S4.4: Add the hemoglobin peptide powder obtained in step S2.4 and the black bean peptide powder obtained in step S3.5 to purified water, stir and mix thoroughly, then add the above-mentioned jujube extract, pomegranate juice, longan powder, tremella polysaccharide, heme iron, sodium ferric EDTA, sucralose and citric acid, continue to stir and mix evenly and homogenize to obtain the plant polypeptide composition.

[0012] Furthermore, the animal blood includes pig blood, cow blood, sheep blood, or chicken blood, and the anticoagulant is a 3-4% sodium citrate solution, with the amount of anticoagulant added being 10% of the animal blood volume.

[0013] Furthermore, the amount of strawberry extract and buckwheat leaf extract added is 3-5% and 2-3% of the mass of the purified hemoglobin solution, respectively, and the amount of compound enzyme added is 2-4% of the mass of the purified hemoglobin solution. The compound enzyme is composed of trypsin, papain and neutral protease mixed in a mass ratio of 1:(1-3):(2-3).

[0014] Furthermore, the amount of the complex protease added is 1-3% of the crude protein content of black beans, and the complex protease is a mixture of papain and trypsin in a mass ratio of (2-3):1.

[0015] Further, by weight, the plant polypeptide composition comprises: 15-20 parts hemoglobin peptide powder, 13-15 parts black bean peptide powder, 10-12 parts jujube extract, 6-8 parts pomegranate juice, 3-5 parts longan powder, 3-5 parts tremella polysaccharide, 1-2 parts heme iron, 0.3-0.5 parts sodium ferric EDTA, 0.6-1 part sucralose, 1-2 parts citric acid, and 80-90 parts purified water.

[0016] Furthermore, a plant polypeptide composition that promotes the increase of hemoglobin is prepared by the preparation process of a plant polypeptide composition that promotes the increase of hemoglobin described in any one of the above claims.

[0017] Compared with the prior art, the present invention has at least the following beneficial effects: 1. This invention first prepares raspberry powder from raspberry fruit and then extracts it with ultrasound in an ethanol solution to obtain raspberry extract. Next, pulverize buckwheat leaves and add them to an ethanol solution for reflux extraction to obtain buckwheat leaf extract. Then, add the raspberry extract and buckwheat leaf extract to a purified hemoglobin solution and add a compound enzyme for enzymatic hydrolysis. The polyphenols in the raspberry extract bind to the hydrophobic regions of hemoglobin, inducing the unfolding of its α-helix structure and exposing more cleavage sites. The saponins in the buckwheat leaves have surfactant properties, disrupting the aggregated state of hemoglobin and increasing enzyme accessibility. The synergistic effect of both promotes the activity of different enzymes, achieving full coverage of cleavage sites, reducing large fragment residues, and jointly promoting the enzymatic hydrolysis of hemoglobin by the compound enzyme, thereby synergistically improving the efficiency of the compound enzyme in preparing hemoglobin peptides from hemoglobin.

[0018] 2. This invention first processes black beans into black bean milk, then adds sodium hydroxide solution to prepare black bean protein extract, and adds hydrochloric acid to the black bean protein extract to precipitate crude black bean protein. Then, a complex protease is used to enzymatically hydrolyze the crude black bean protein to prepare black bean peptide powder. This black bean peptide powder is then mixed with hemoglobin peptides to form a plant polypeptide composition. Since the heme iron in the hemoglobin peptides can be directly taken up by intestinal epithelial cells, after entering the cells, the heme iron releases iron ions under the action of heme oxygenase. Black bean peptides can regulate the intestinal pH, making it more conducive to the dissolution and absorption of iron. They can also form soluble complexes with iron ions, preventing the formation of poorly absorbed precipitates such as ferric hydroxide in the intestines, thus increasing the bioavailability of iron. Simultaneously, they can promote the synthesis and activity of transferrin, which is responsible for transporting iron ions to hematopoietic tissues such as bone marrow. Therefore, the combination of these two can synergistically promote the increase of hemoglobin levels in the body.

[0019] 3. This invention, by first preparing pomegranate juice and longan powder separately, and then mixing them together with tremella polysaccharide and hemoglobin peptides to form a plant polypeptide composition, utilizes the antioxidants such as pomegranate polyphenols and anthocyanins, which can scavenge free radicals in the body, reduce oxidative stress damage to hematopoietic cells and hemoglobin, and protect the stability of the hematopoietic microenvironment. This allows the hemoglobin peptides to better exert their hematopoietic-promoting effects. Longan powder can promote the proliferation and differentiation of bone marrow hematopoietic stem cells, increase the number of erythroid progenitor cells, improve hematopoietic efficiency, and interact with hemoglobin... The combination of protein peptides can promote hemoglobin production from different angles, enhancing the blood-tonifying effect. Tremella polysaccharide can regulate the intestinal microbiota, promote the growth of beneficial bacteria, improve the intestinal microecological environment, and help improve the intestinal absorption capacity of iron, providing sufficient iron for hemoglobin synthesis. This assists hemoglobin peptides in playing a blood-tonifying role. Therefore, when pomegranate juice, longan powder and tremella polysaccharide are used together, they can synergistically assist the blood-tonifying effect of hemoglobin peptides and further enhance the efficacy of the prepared plant polypeptide composition in promoting hemoglobin increase. Attached Figure Description

[0020] The accompanying drawings, which are incorporated herein and form part of the specification, illustrate embodiments of the present disclosure and, together with the specification, further serve to explain the principles of the present disclosure and enable those skilled in the art to implement and use the present disclosure.

[0021] Figure 1 This is a flowchart illustrating the preparation process of the plant polypeptide composition that promotes hemoglobin increase used in the embodiments of the present invention. Detailed Implementation

[0022] The following describes in detail, with reference to the accompanying drawings and specific embodiments, a plant polypeptide composition that promotes the increase of hemoglobin and its preparation process provided by the present invention.

[0023] Example 1 A preparation process for a plant polypeptide composition that promotes hemoglobin increase, such as... Figure 1 As shown, it includes the following steps: S1: Preparation of raspberry extract and buckwheat leaf extract S1.1: Wash the ripe raspberries, dry them at low temperature, and then crush and grind them to obtain raspberry powder; S1.2: Add the above raspberry powder to a 70% ethanol solution at a solid-liquid ratio of 1g:20mL, and extract with ultrasonic-assisted heating and stirring at 200W and 40℃ for 40min. After filtration, vacuum concentration and drying, the raspberry extract is obtained. S1.3: Fresh buckwheat leaves were washed, dried and crushed, and then added to 60% ethanol solution at a solid-liquid ratio of 1g:20mL. The mixture was heated and stirred under reflux at 70℃ for 1h. After cooling, the extract was obtained by centrifugation, vacuum concentration and drying. S2: Preparation of hemoglobin peptide powder S2.1: Collect fresh pig blood, add anticoagulant, and centrifuge at 3000 r / min for 10 min at 4℃. Collect red blood cells, wash twice with physiological saline to obtain purified red blood cells. The anticoagulant is a 3% sodium citrate solution, and the amount of anticoagulant added is 10% of the volume of pig blood. S2.2: The purified red blood cells were suspended in 0.1% sodium chloride solution and stirred until fully ruptured. The mixture was then centrifuged at 3000 r / min for 8 min and the supernatant was collected. Ammonium sulfate was added until the ammonium sulfate saturation reached 40%. After standing for 6 h, the mixture was centrifuged at 4000 r / min for 10 min and the precipitate was collected to obtain hemoglobin. S2.3: Dissolve the above hemoglobin in phosphate buffer solution with pH 7 to prepare a hemoglobin solution with a concentration of 10 mg / mL. Then dialyze the hemoglobin solution with a dialysis bag for 20 h, changing the dialysate every 4 h to obtain a purified hemoglobin solution. S2.4: Add the raspberry extract obtained in step S1.2 and the buckwheat leaf extract obtained in step S1.3 to the above purified hemoglobin solution, heat to 40℃ and keep warm, then add the compound enzyme and keep warm for 4 hours for enzymatic hydrolysis. After enzyme inactivation, centrifuge at 4000 r / min for 10 min, collect the supernatant for ultrafiltration, collect the permeate, and obtain hemoglobin peptide powder by dialysis, concentration and drying. The amount of raspberry extract and buckwheat leaf extract added is 3% and 2% of the mass of purified hemoglobin solution, respectively, and the amount of compound enzyme added is 2% of the mass of purified hemoglobin solution. The compound enzyme is composed of trypsin, papain and neutral protease mixed in a mass ratio of 1:1:2. S3: Preparation of black bean peptide powder S3.1: Wash the plump black beans that are free from pests, diseases, and mold, then soak them in clean water for 8 hours. After draining the water, grind them into a paste to obtain black bean milk. S3.2: Add 0.1 mol / L sodium hydroxide solution to the above black soybean milk to adjust the pH to 8, and extract with ultrasonic assistance at 200W and 40℃ for 1 hour. After centrifugation to remove the residue, black soybean protein extract is obtained. S3.3: Add 0.1 mol / L hydrochloric acid to the above black bean protein extract, adjust the pH to 4, then centrifuge to separate the precipitate and obtain crude black bean protein; S3.4: Add the above black bean crude protein to distilled water at a solid-liquid ratio of 1g:40mL, stir and mix thoroughly, heat to 40℃ and keep warm, then add the complex protease, heat and enzymatically hydrolyze for 2h, then heat to 80℃ and keep warm to inactivate the enzyme for 10min to obtain black bean enzymatic hydrolysate. The amount of complex protease added is 1% of the black bean crude protein, and the complex protease is a mixture of papain and trypsin at a mass ratio of 2:1. S3.5: Filter the above black bean enzymatic hydrolysate, then use an ultrafiltration membrane to ultrafilter the filtrate, and after vacuum concentration and freeze drying, obtain black bean peptide powder; S4: Preparation of plant polypeptide compositions S4.1: Wash fresh, smooth-skinned, and disease-free pomegranates, remove the pomegranate seeds, juice the pomegranate seeds, filter the juice, and obtain pomegranate juice. S4.2: Remove the shells and pits from the dried longan, dry the longan pulp at 60℃ for 2 hours, then crush, grind and sieve it to obtain longan powder; S4.3: After cleaning the dried red dates, remove the pits and grind them into powder. Then add the powder to purified water at a solid-liquid ratio of 1g:10mL, heat at 60℃ for 1 hour, and repeat the extraction twice. Combine the extracts, filter, concentrate and dry to obtain red date extract. S4.4: Add 15 parts by weight of the hemoglobin peptide powder obtained in step S2.4 and 13 parts by weight of the black bean peptide powder obtained in step S3.5 to 80 parts by weight of purified water, stir and mix thoroughly. Then add 10 parts by weight of the above-mentioned jujube extract, 6 parts by weight of the above-mentioned pomegranate juice, 3 parts by weight of the above-mentioned longan powder, 3 parts by weight of tremella polysaccharide, 1 part by weight of heme iron, 0.3 parts by weight of EDTA iron sodium, 0.6 parts by weight of sucralose and 1 part by weight of citric acid, continue to stir and mix evenly and homogenize to obtain a plant polypeptide composition.

[0024] Example 2 A preparation process for a plant polypeptide composition that promotes hemoglobin increase, such as... Figure 1 As shown, it includes the following steps: S1: Preparation of raspberry extract and buckwheat leaf extract S1.1: Wash the ripe raspberries, dry them at low temperature, and then crush and grind them to obtain raspberry powder; S1.2: Add the above raspberry powder to a 70% ethanol solution at a solid-liquid ratio of 1g:25mL, and extract with ultrasonic-assisted heating and stirring at 250W and 45℃ for 45min. After filtration, vacuum concentration and drying, the raspberry extract is obtained. S1.3: Fresh buckwheat leaves were washed, dried and crushed, and then added to 60% ethanol solution at a solid-liquid ratio of 1g:25mL. The mixture was heated and stirred under reflux at 75℃ for 1.5h. After cooling, the extract was obtained by centrifugation, vacuum concentration and drying. S2: Preparation of hemoglobin peptide powder S2.1: Collect fresh bovine blood, add anticoagulant, and centrifuge at 3500 r / min for 12 min at 4℃. Collect red blood cells, wash twice with physiological saline to obtain purified red blood cells. The anticoagulant is a 3.5% sodium citrate solution, and the amount of anticoagulant added is 10% of the volume of bovine blood. S2.2: The purified red blood cells were suspended in 0.1% sodium chloride solution and stirred until fully ruptured. The mixture was then centrifuged at 3500 r / min for 9 min and the supernatant was collected. Ammonium sulfate was added until the ammonium sulfate saturation reached 50%. After standing for 7 h, the mixture was centrifuged at 4500 r / min for 12 min and the precipitate was collected to obtain hemoglobin. S2.3: Dissolve the above hemoglobin in phosphate buffer solution with pH 7.2 to prepare a hemoglobin solution with a concentration of 10 mg / mL. Then dialyze the hemoglobin solution with a dialysis bag for 22 hours, changing the dialysate every 4 hours to obtain a purified hemoglobin solution. S2.4: Add the raspberry extract obtained in step S1.2 and the buckwheat leaf extract obtained in step S1.3 to the above purified hemoglobin solution, heat to 42℃ and keep warm, then add the compound enzyme and keep warm for 5 hours of enzymatic hydrolysis. After enzyme inactivation, centrifuge at 4500 r / min for 12 min, collect the supernatant for ultrafiltration, collect the permeate, and obtain hemoglobin peptide powder by dialysis, concentration and drying. The amount of raspberry extract and buckwheat leaf extract added is 4% and 2.5% of the mass of purified hemoglobin solution, respectively, and the amount of compound enzyme added is 3% of the mass of purified hemoglobin solution. The compound enzyme is composed of trypsin, papain and neutral protease mixed in a mass ratio of 1:2:2.5. S3: Preparation of black bean peptide powder S3.1: Wash the plump black beans that are free from pests, diseases, and mold, then soak them in clean water for 10 hours. After draining the water, grind them into a paste to obtain black bean milk. S3.2: Add 0.1 mol / L sodium hydroxide solution to the above black soybean milk to adjust the pH to 8.5, and extract with ultrasonic assistance at 250W and 50℃ for 1.5h. After centrifugation to remove the residue, black soybean protein extract is obtained. S3.3: Add 0.1 mol / L hydrochloric acid to the above black bean protein extract, adjust the pH to 4.5, then centrifuge to separate the precipitate and obtain crude black bean protein; S3.4: Add the above-mentioned crude black bean protein to distilled water at a solid-liquid ratio of 1g:45mL, stir and mix thoroughly, heat to 45℃ and keep warm, then add the complex protease, heat and enzymatically hydrolyze for 2.5h, then heat to 85℃ and keep warm to inactivate the enzyme for 12min to obtain black bean enzymatic hydrolysate. The amount of complex protease added is 2% of the crude black bean protein, and the complex protease is a mixture of papain and trypsin at a mass ratio of 2.5:1. S3.5: Filter the above black bean enzymatic hydrolysate, then use an ultrafiltration membrane to ultrafilter the filtrate, and after vacuum concentration and freeze drying, obtain black bean peptide powder; S4: Preparation of plant polypeptide compositions S4.1: Wash fresh, smooth-skinned, and disease-free pomegranates, remove the pomegranate seeds, juice the pomegranate seeds, filter the juice, and obtain pomegranate juice. S4.2: Remove the shells and pits from the dried longan, dry the longan pulp at 70℃ for 3 hours, and then crush, grind and sieve it to obtain longan powder; S4.3: After cleaning the dried red dates, remove the pits and grind them into powder. Then add the powder to purified water at a solid-liquid ratio of 1g:15mL, heat at 65℃ for 1.5h, and repeat the extraction twice. Combine the extracts, filter, concentrate and dry to obtain red date extract. S4.4: Add 17.5 parts by weight of the hemoglobin peptide powder obtained in step S2.4 and 14 parts by weight of the black bean peptide powder obtained in step S3.5 to 85 parts by weight of purified water, stir and mix thoroughly. Then add 11 parts by weight of the above-mentioned jujube extract, 7 parts by weight of the above-mentioned pomegranate juice, 4 parts by weight of the above-mentioned longan powder, 4 parts by weight of tremella polysaccharide, 1.5 parts by weight of heme iron, 0.4 parts by weight of sodium iron EDTA, 0.8 parts by weight of sucralose and 1.5 parts by weight of citric acid, continue to stir and mix evenly and homogenize to obtain the plant polypeptide composition.

[0025] Example 3 A preparation process for a plant polypeptide composition that promotes hemoglobin increase, such as... Figure 1 As shown, it includes the following steps: S1: Preparation of raspberry extract and buckwheat leaf extract S1.1: Wash the ripe raspberries, dry them at low temperature, and then crush and grind them to obtain raspberry powder; S1.2: Add the above raspberry powder to a 70% ethanol solution at a solid-liquid ratio of 1g:30mL, and extract with ultrasonic-assisted heating and stirring at 300W and 50℃ for 50min. After filtration, vacuum concentration and drying, the raspberry extract is obtained. S1.3: Fresh buckwheat leaves were washed, dried and crushed, and then added to 60% ethanol solution at a solid-liquid ratio of 1g:30mL. The mixture was heated and stirred under reflux at 80℃ for 2h. After cooling, the extract was obtained by centrifugation, vacuum concentration and drying. S2: Preparation of hemoglobin peptide powder S2.1: Collect fresh sheep blood, add anticoagulant, and centrifuge at 4000 r / min for 15 min at 4℃. Collect red blood cells, wash three times with physiological saline to obtain purified red blood cells. The anticoagulant is a 4% sodium citrate solution, and the amount of anticoagulant added is 10% of the sheep blood volume. S2.2: The purified red blood cells were suspended in 0.1% sodium chloride solution and stirred until fully ruptured. The mixture was then centrifuged at 4000 r / min for 10 min and the supernatant was collected. Ammonium sulfate was added until the ammonium sulfate saturation reached 60%. After standing for 8 h, the mixture was centrifuged at 5000 r / min for 15 min and the precipitate was collected to obtain hemoglobin. S2.3: Dissolve the above hemoglobin in phosphate buffer solution with pH 7.5 to prepare a hemoglobin solution with a concentration of 10 mg / mL. Then dialyze the hemoglobin solution with a dialysis bag for 24 hours, changing the dialysate every 4 hours to obtain a purified hemoglobin solution. S2.4: Add the raspberry extract obtained in step S1.2 and the buckwheat leaf extract obtained in step S1.3 to the above purified hemoglobin solution, heat to 45℃ and keep warm, then add the compound enzyme and keep warm for 6 hours of enzymatic hydrolysis. After enzyme inactivation, centrifuge at 5000 r / min for 15 min, collect the supernatant for ultrafiltration, collect the permeate, and obtain hemoglobin peptide powder by dialysis, concentration and drying. The amount of raspberry extract and buckwheat leaf extract added is 5% and 3% of the mass of purified hemoglobin solution, respectively, and the amount of compound enzyme added is 4% of the mass of purified hemoglobin solution. The compound enzyme is composed of trypsin, papain and neutral protease mixed in a mass ratio of 1:3:3. S3: Preparation of black bean peptide powder S3.1: Wash the plump black beans that are free from pests, diseases, and mold, then soak them in clean water for 12 hours. After draining the water, grind them into a paste to obtain black bean milk. S3.2: Add 0.1 mol / L sodium hydroxide solution to the above black soybean milk to adjust the pH to 9, and extract with ultrasonic assistance at 300W and 60℃ for 2 hours. After centrifugation to remove the residue, black soybean protein extract is obtained. S3.3: Add 0.1 mol / L hydrochloric acid to the above black bean protein extract, adjust the pH to 5, then centrifuge to separate the precipitate and obtain crude black bean protein; S3.4: Add the above black soybean crude protein to distilled water at a solid-liquid ratio of 1g:50mL, stir and mix thoroughly, heat to 50℃ and keep warm, then add the complex protease, heat and enzymatically hydrolyze for 3h, then heat to 90℃ and keep warm to inactivate the enzyme for 15min to obtain black soybean enzymatic hydrolysate. The amount of complex protease added is 3% of the black soybean crude protein, and the complex protease is a mixture of papain and trypsin in a mass ratio of 3:1. S3.5: Filter the above black bean enzymatic hydrolysate, then use an ultrafiltration membrane to ultrafilter the filtrate, and after vacuum concentration and freeze drying, obtain black bean peptide powder; S4: Preparation of plant polypeptide compositions S4.1: Wash fresh, smooth-skinned, and disease-free pomegranates, remove the pomegranate seeds, juice the pomegranate seeds, filter the juice, and obtain pomegranate juice. S4.2: Remove the shells and pits from the dried longan, dry the longan pulp at 80℃ for 4 hours, and then crush, grind and sieve it to obtain longan powder; S4.3: After cleaning the dried red dates, remove the pits and grind them into powder. Then add the powder to purified water at a solid-liquid ratio of 1g:20mL, heat at 70℃ for 2 hours, and repeat the extraction 3 times. Combine the extracts, filter, concentrate and dry to obtain red date extract. S4.4: Add 20 parts by weight of the hemoglobin peptide powder obtained in step S2.4 and 15 parts by weight of the black bean peptide powder obtained in step S3.5 to 90 parts by weight of purified water, stir and mix thoroughly. Then add 12 parts by weight of the above-mentioned jujube extract, 8 parts by weight of the above-mentioned pomegranate juice, 5 parts by weight of the above-mentioned longan powder, 5 parts by weight of tremella polysaccharide, 2 parts by weight of heme iron, 0.5 parts by weight of sodium iron EDTA, 1 part by weight of sucralose and 2 parts by weight of citric acid, continue to stir and mix evenly and homogenize to obtain a plant polypeptide composition.

[0026] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that the raspberry extract and buckwheat leaf extract in step S2.4 are removed.

[0027] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that the raspberry extract in step S2.4 is replaced with an equal amount of buckwheat leaf extract.

[0028] Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that the buckwheat leaf extract in step S2.4 is replaced with an equal amount of raspberry extract.

[0029] Comparative Example 4 The difference between Comparative Example 4 and Example 1 is that the black bean peptide powder in step S4.4 is replaced with an equal amount of hemoglobin peptide powder.

[0030] Comparative Example 5 The difference between Comparative Example 5 and Example 1 is that the hemoglobin peptide powder in step S4.4 is replaced with an equal amount of black bean peptide powder.

[0031] Comparative Example 6 The difference between Comparative Example 6 and Example 1 is that the longan powder and tremella polysaccharide in step S4.4 are replaced with an equal amount of pomegranate juice.

[0032] Comparative Example 7 The difference between Comparative Example 7 and Example 1 is that the pomegranate juice and tremella polysaccharide in step S4.4 are replaced with an equal amount of longan powder.

[0033] Comparative Example 8 The difference between Comparative Example 8 and Example 1 is that the pomegranate juice and longan powder in step S4.4 are replaced with an equal amount of Tremella polysaccharide.

[0034] Test case Test 1: The content of small molecule peptides (molecular weight ≤3000Da) in the hemoglobin peptides prepared in Examples 1-3 and Comparative Examples 1-3 was determined by high performance liquid chromatography. The results are shown in Table 1.

[0035] Table 1: Percentage of small molecule peptides (molecular weight ≤3000 Da) in hemoglobin peptides

[0036] As shown in Table 1, in Comparative Example 1, when no raspberry extract or buckwheat leaf extract was added to the purified hemoglobin solution for enzymatic hydrolysis, the content of small molecular weight hemoglobin peptides in the resulting hemoglobin peptide powder was approximately 27.5%, which was much lower than in Example 1. This indicates that adding raspberry extract and buckwheat leaf extract during hemoglobin enzymatic hydrolysis is beneficial for promoting the enzymatic hydrolysis of hemoglobin. Furthermore, in Comparative Examples 2 and 3, when only raspberry extract or buckwheat leaf extract was added to participate in hemoglobin enzymatic hydrolysis, the content of small molecular weight peptides in the resulting hemoglobin peptides was lower than in Example 1. Therefore, it can be seen that raspberry extract and buckwheat leaf extract can synergistically improve the efficiency of hemoglobin peptide preparation by enzymatic hydrolysis of hemoglobin with compound enzymes.

[0037] Test 2: Zebrafish embryonic erythrocyte promotion test.

[0038] (1) Test organisms: Zebrafish embryos obtained by peeling off the shells of wild-type AB strain zebrafish 1 day old were selected as test organisms.

[0039] (2) Experimental grouping: Zebrafish embryos were randomly divided into 8 groups, namely blank control group (culture medium: 0.2 mL phenylthiourea working solution), model control group (culture medium: 0.2 mL phenylthiourea + 100 mg / L doxorubicin) and experimental group. Among them, the experimental group was Example 1 group (culture medium: 0.1 mL phenylthiourea + 100 mg / L doxorubicin + 0.1 mL plant polypeptide composition prepared in Example 1), Comparative Example 4 group (culture medium: 0.1 mL phenylthiourea + 100 mg / L doxorubicin + 0.1 mL plant polypeptide composition prepared in Comparative Example 4), and Comparative Example 5 group (culture medium: 0.1 mL phenylthiourea + 100 mg / L doxorubicin + 0.1 mL plant polypeptide composition prepared in Comparative Example 4). The following groups were included: 1 mL phenylthiourea + 100 mg / L doxorubicin + 0.1 mL of the plant polypeptide composition prepared in Comparative Example 5; Comparative Example 6 (culture medium: 0.1 mL phenylthiourea + 100 mg / L doxorubicin + 0.1 mL of the plant polypeptide composition prepared in Comparative Example 6); Comparative Example 7 (culture medium: 0.1 mL phenylthiourea + 100 mg / L doxorubicin + 0.1 mL of the plant polypeptide composition prepared in Comparative Example 7); and Comparative Example 8 (culture medium: 0.1 mL phenylthiourea + 100 mg / L doxorubicin + 0.1 mL of the plant polypeptide composition prepared in Comparative Example 8); each group contained 24 zebrafish embryos.

[0040] (3) Experimental method: Zebrafish embryos were transferred to 96-well plates, with one zebrafish embryo and culture medium in each well. After being cultured in a constant temperature incubator at 28℃ for 24 hours, the zebrafish embryos were stained with o-dianisidine staining solution, fixed with paraformaldehyde, and washed with PBST. The washed zebrafish embryos were then placed abdomen-up and photographed under a microscope. The images were then opened with ImageJ software to mark the distribution range of blood cells in the cardiac region of each embryo. The average gray value was then selected as the red blood cell content index, and the promotion rate was calculated using the following formula: Promotion rate (%) = (average mean gray value of zebrafish embryo red blood cells in the experimental group - average mean gray value of zebrafish embryo red blood cells in the model control group) / (average mean gray value of zebrafish embryo red blood cells in the blank control group - average mean gray value of zebrafish embryo red blood cells in the model control group) × 100%, and the results are shown in Table 2.

[0041] Table 2: Zebrafish Embryo Erythrocyte Promotion Rate Test

[0042] As shown in Table 2, the plant polypeptide compositions prepared by using single hemoglobin peptide powder and single black bean peptide powder in Comparative Examples 4 and 5 had a lower promotion rate of zebrafish erythrocytes than the plant polypeptide compositions prepared by using both hemoglobin peptide powder and black bean peptide powder in Example 1. This indicates that the combination of hemoglobin peptide and black bean peptide can synergistically promote the increase of hemoglobin levels in the body. Furthermore, in Comparative Examples 6-8, the plant polypeptide compositions prepared using only pomegranate juice, longan powder, and tremella polysaccharide showed lower rates of promoting zebrafish erythrocytes compared to the plant polypeptide compositions prepared using pomegranate juice, longan powder, and tremella polysaccharide simultaneously in Example 1. This indicates that the combined use of pomegranate juice, longan powder, and tremella polysaccharide can synergistically enhance the blood-replenishing effect of hemoglobin peptides, further improving the hemoglobin-increasing efficacy of the prepared plant polypeptide compositions.

[0043] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A preparation process for a plant polypeptide composition that promotes hemoglobin increase, characterized in that, Includes the following steps: S1: Preparation of raspberry extract and buckwheat leaf extract Raspberry and buckwheat leaves were extracted separately with ethanol solution to prepare raspberry extract and buckwheat leaf extract; S2: Preparation of hemoglobin peptide powder Fresh animal blood was collected to prepare purified red blood cells. The purified red blood cells were then suspended in pure water, stirred and ruptured, and hemoglobin was collected. The hemoglobin was then purified by dialysis to obtain a purified hemoglobin solution. Finally, the above-mentioned strawberry extract and buckwheat leaf extract were added for enzymatic hydrolysis to prepare hemoglobin peptide powder. S3: Preparation of black bean peptide powder Black soybeans are made into black soybean milk, sodium hydroxide solution is added to prepare black soybean protein extract, hydrochloric acid is added and the crude black soybean protein is collected by centrifugation, then a complex protease is added and incubated for enzymatic hydrolysis to prepare black soybean peptide powder. S4: Preparation of plant polypeptide compositions Pomegranate juice, longan powder, and jujube extract were prepared separately, and then thoroughly mixed with the above-mentioned hemoglobin peptide powder, black bean peptide powder, purified water, tremella polysaccharide, heme iron, sodium iron EDTA, sucralose, and citric acid to obtain a plant polypeptide composition.

2. The preparation process of the plant polypeptide composition for promoting hemoglobin increase according to claim 1, characterized in that, S1 specifically includes the following steps: S1.1: Wash the ripe raspberries, dry them at low temperature, and then crush and grind them to obtain raspberry powder; S1.2: Add the above raspberry powder to a 70% ethanol solution at a solid-liquid ratio of 1g:(20-30)mL, and extract with ultrasonic-assisted heating and stirring at 200-300W and 40-50℃ for 40-50min. After filtration, vacuum concentration and drying, the raspberry extract is obtained. S1.3: Wash, dry and crush fresh buckwheat leaves, then add them to 60% ethanol solution at a solid-liquid ratio of 1g:(20-30)mL, heat and stir under reflux at 70-80℃ for 1-2h, cool, centrifuge, concentrate under reduced pressure and dry to obtain buckwheat leaf extract.

3. The preparation process of a plant polypeptide composition that promotes hemoglobin increase according to claim 2, characterized in that, S2 specifically includes the following steps: S2.1: Collect fresh animal blood, add anticoagulant, and centrifuge at 3000-4000 r / min for 10-15 min at 4℃. Collect red blood cells, wash with physiological saline 2-3 times to obtain purified red blood cells. S2.2: The purified red blood cells were suspended in 0.1% sodium chloride solution and stirred until fully ruptured. The mixture was then centrifuged at 3000-4000 r / min for 8-10 min and the supernatant was collected. Ammonium sulfate was added until the ammonium sulfate saturation reached 40-60%. After standing for 6-8 h, the mixture was centrifuged at 4000-5000 r / min for 10-15 min and the precipitate was collected to obtain hemoglobin. S2.3: Dissolve the above hemoglobin in phosphate buffer solution with pH 7-7.5 to prepare a hemoglobin solution with a concentration of 10 mg / mL. Then dialyze the hemoglobin solution with a dialysis bag for 20-24 hours, changing the dialysate every 4 hours to obtain a purified hemoglobin solution. S2.4: Add the raspberry extract obtained in step S1.2 and the buckwheat leaf extract obtained in step S1.3 to the above purified hemoglobin solution, heat to 40-45℃ and keep warm, then add the compound enzyme and keep warm for 4-6 hours for enzymatic hydrolysis. After enzyme inactivation, centrifuge at 4000-5000 r / min for 10-15 min, collect the supernatant for ultrafiltration, collect the permeate, and obtain hemoglobin peptide powder by dialysis, concentration and drying.

4. The preparation process of a plant polypeptide composition for promoting hemoglobin increase according to claim 3, characterized in that, S3 specifically includes the following steps: S3.1: Wash the plump black beans that are free from pests, diseases, and mold, then soak them in clean water for 8-12 hours. After draining the water, grind them into a paste to obtain black bean milk. S3.2: Add 0.1mol / L sodium hydroxide solution to the above black soybean milk to adjust the pH to 8-9, and extract with ultrasonic assistance at 200-300W and 40-60℃ for 1-2 hours. After centrifugation to remove the residue, black soybean protein extract is obtained. S3.3: Add 0.1 mol / L hydrochloric acid to the above black bean protein extract, adjust the pH to 4-5, then centrifuge to separate the precipitate and obtain crude black bean protein; S3.4: Add the above black bean crude protein to distilled water at a solid-liquid ratio of 1g:(40-50)mL, stir and mix thoroughly, heat to 40-50℃ and keep warm, then add compound protease, heat and enzymatically hydrolyze for 2-3 hours, then heat to 80-90℃ and keep warm to inactivate the enzyme for 10-15 minutes to obtain black bean enzymatic hydrolysate. S3.5: Filter the above black bean enzymatic hydrolysate, then use an ultrafiltration membrane to ultrafilter the filtrate, and after vacuum concentration and freeze drying, obtain black bean peptide powder.

5. The preparation process of a plant polypeptide composition for promoting hemoglobin increase according to claim 4, characterized in that, S4 specifically includes the following steps: S4.1: Wash fresh, smooth-skinned, and disease-free pomegranates, remove the pomegranate seeds, juice the pomegranate seeds, filter the juice, and obtain pomegranate juice. S4.2: Remove the shells and pits from the dried longan, dry the longan pulp at 60-80℃ for 2-4 hours, then crush, grind and sieve it to obtain longan powder; S4.3: After cleaning the dried red dates, remove the pits and grind them into powder. Then add them to purified water at a solid-liquid ratio of 1g:(10-20)mL. Heat at 60-70℃ for 1-2 hours and extract. Repeat the extraction 2-3 times. Combine the extracts, filter, concentrate and dry to obtain red date extract. S4.4: Add the hemoglobin peptide powder obtained in step S2.4 and the black bean peptide powder obtained in step S3.5 to purified water, stir and mix thoroughly, then add the above-mentioned jujube extract, pomegranate juice, longan powder, tremella polysaccharide, heme iron, sodium ferric EDTA, sucralose and citric acid, continue to stir and mix evenly and homogenize to obtain the plant polypeptide composition.

6. The preparation process of a plant polypeptide composition for promoting hemoglobin increase according to claim 3, characterized in that, Animal blood includes pig blood, cow blood, sheep blood, or chicken blood. The anticoagulant is a 3-4% sodium citrate solution, and the amount of anticoagulant added is 10% of the animal blood volume.

7. The preparation process of a plant polypeptide composition for promoting hemoglobin increase according to claim 3, characterized in that, The amount of strawberry extract and buckwheat leaf extract added is 3-5% and 2-3% of the mass of the purified hemoglobin solution, respectively. The amount of compound enzyme added is 2-4% of the mass of the purified hemoglobin solution. The compound enzyme is composed of trypsin, papain and neutral protease mixed in a mass ratio of 1:(1-3):(2-3).

8. The preparation process of a plant polypeptide composition for promoting hemoglobin increase according to claim 4, characterized in that, The amount of compound protease added is 1-3% of the crude protein content of black beans, and the compound protease is a mixture of papain and trypsin in a mass ratio of (2-3):

1.

9. The preparation process of a plant polypeptide composition for promoting hemoglobin increase according to claim 5, characterized in that, By weight, the plant polypeptide composition comprises: 15-20 parts hemoglobin peptide powder, 13-15 parts black bean peptide powder, 10-12 parts jujube extract, 6-8 parts pomegranate juice, 3-5 parts longan powder, 3-5 parts tremella polysaccharide, 1-2 parts heme iron, 0.3-0.5 parts sodium EDTA, 0.6-1 part sucralose, 1-2 parts citric acid, and 80-90 parts purified water.

10. A plant polypeptide composition that promotes hemoglobin increase, characterized in that, It is prepared by the preparation process of a plant polypeptide composition that promotes the increase of hemoglobin as described in any one of claims 1-9.

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