Peptides of pea proteins, methods of preparation and use in the inhibition of tumour cell growth

Abstract

This invention discloses pea protein peptides, their preparation method, and their application in anti-tumor cell growth, relating to the technical field of complete nutritional formula foods for tumors for special medical purposes. The pea protein peptides disclosed in this invention include one or more of polypeptides LY-1, LY-2, LY-3, LY-4, LY-5, LY-6, LY-7, and LY-8.

Description

Technical Field

[0001] This invention relates to the field of complete nutritional formula foods for tumors for special medical purposes, specifically to pea protein peptides, their preparation methods, and their application in anti-tumor cell growth. Background Technology

[0002] Malignant tumors are one of the major diseases seriously threatening human health, and their occurrence and spread are closely related to the imbalance between cell proliferation and apoptosis. Under normal physiological conditions, cell division, differentiation, and death maintain a dynamic balance; when the body is affected by external environmental factors (such as radiation, chemical pollutants, etc.) or internal genetic factors, gene mutations may occur, giving cells uncontrolled proliferative capacity, leading to the formation of malignant tumors, which may metastasize to other parts of the body. According to the "Global Cancer Statistics Report 2025" released by the International Agency for Research on Cancer (IARC) of the World Health Organization, there are nearly 22 million new cancer cases and approximately 10.3 million cancer deaths worldwide, with lung cancer, breast cancer, and colorectal cancer being the most common types, and the cancer burden continues to increase. Currently, more and more cancer patients are in a state of severe metabolic disorders and weakened immune function after receiving radiotherapy or chemotherapy, and their physical condition is extremely weak. Ordinary foods are difficult to meet their needs for key nutrients and energy, and are not easily digested and absorbed. Therefore, developing a food-derived protein peptide that can provide the body with essential nutrients and energy while inhibiting tumor cell growth and inducing apoptosis is of great clinical and nutritional significance.

[0003] Currently, several studies have reported that protein hydrolysates from different sources possess activity in inhibiting tumor cell proliferation. Mao-Cheng Sun et al. published a review in the journal *Probiotics and Antimicrobial Proteins*, systematically summarizing the sources, mechanisms of action, and therapeutic potential of plant-derived anticancer peptides. The study indicates that anticancer peptides derived from legumes, grains, and medicinal plants possess high specificity, multi-target mechanisms, and good safety. For example, a patent titled "Application of Pea Active Peptides in Inhibiting Cancer Cell Growth and its Preparation Method" (patent number CN 106075384 A) discloses the application of pea active peptides in inhibiting cancer cell growth and its preparation method. This peptide powder is composed of peptides with a molecular weight less than 1 kDa and has inhibitory effects on liver cancer cells, breast cancer cells, and gastric cancer cells, promoting research progress on plant peptides in inhibiting cancer cell growth. However, the patent only conducts sufficient research at the cellular level and has not yet conducted animal experiments, so its inhibitory effect in live animals cannot be directly demonstrated; furthermore, there are no reports of this preparation method being used for large-scale production.

[0004] Regarding plant-derived protein peptides, peas ( Pisum sativumPea, as an important legume crop, is rich in protein, has a balanced amino acid composition, and exhibits low allergenicity and good processing characteristics, making it a high-quality raw material for preparing plant protein peptides. Existing studies have shown that pea protein hydrolysates possess various biological activities, including antioxidant, antihypertensive, and immunomodulatory effects. However, research on the inhibitory effect of pea protein peptides on tumor cell growth is relatively limited, and existing reports mostly focus on the activity evaluation of crude pea protein hydrolysates, lacking systematic separation and purification processes targeting specific molecular weight ranges or specific active components. This leads to problems such as unclear active ingredients and difficulty in achieving protein content exceeding 95%. Furthermore, existing methods for preparing antitumor pea protein peptides are mostly conducted in the laboratory, resulting in low yields and hindering large-scale industrial production of pea protein peptides that inhibit tumor cell growth. Therefore, developing a stable and scalable method for preparing pea protein peptides, and obtaining pea protein peptide components with significant tumor cell growth inhibitory activity through targeted enzymatic hydrolysis and multi-stage separation and purification techniques, is of great significance for expanding the high-value applications of pea protein peptides and developing novel, specialized medical-purpose, tumor-specific nutritional formula foods. The purpose of this invention is to provide a pea protein peptide with the effect of inhibiting tumor cell growth and its preparation method, so as to solve the problems in the prior art where the anti-tumor active ingredients of pea protein peptides are unclear, the protein content is difficult to exceed 95%, and large-scale production is not possible. Summary of the Invention

[0005] The purpose of this invention is to provide a pea protein peptide with inhibitory effects on tumor cell growth and its preparation method, thereby solving the problems in the prior art where the antitumor active ingredients of pea protein peptides are unclear, the protein content is difficult to exceed 95%, and large-scale production is not possible. The objective of this invention can be achieved through the following technical solutions: Pea protein peptides, comprising one or more of polypeptides LY-1, LY-2, LY-3, LY-4, LY-5, LY-6, LY-7, and LY-8, wherein the amino acid sequence of polypeptide LY-1 is shown in SEQ ID NO.1, the amino acid sequence of polypeptide LY-2 is shown in SEQ ID NO.2, the amino acid sequence of polypeptide LY-3 is shown in SEQ ID NO.3, the amino acid sequence of polypeptide LY-4 is shown in SEQ ID NO.4, the amino acid sequence of polypeptide LY-5 is shown in SEQ ID NO.5, the amino acid sequence of polypeptide LY-6 is shown in SEQ ID NO.6, the amino acid sequence of polypeptide LY-7 is shown in SEQ ID NO.7, and the amino acid sequence of polypeptide LY-8 is shown in SEQ ID NO.8; the pea protein peptides are obtained by enzymatic hydrolysis of pea protein powder; the molecular weight of the pea protein peptides is 1000-3000 Da.

[0006] The preparation method of pea protein peptides includes the following steps: The slurry containing protein powder was sequentially treated with medium-temperature amylase, alkali and ultrasonic treatment, dynamic high-pressure microfluidic treatment, alkaline protease hydrolysis, and compound protease hydrolysis to obtain an enzymatic hydrolysate containing pea active peptides. The enzymatic hydrolysate containing pea protein peptides was separated and purified to obtain the pea protein peptides.

[0007] As a further aspect of the present invention: the enzymatic hydrolysate containing pea protein peptides is subjected to enzyme inactivation treatment before separation and purification.

[0008] As a further aspect of the present invention, the preparation method of the liquid includes the following steps: mixing pea protein powder with pure water at a ratio of 1g:6-12mL to obtain a liquid containing pea protein powder.

[0009] As a further aspect of the present invention: the activity of the mesophilic amylase is 2000-10000 U / g, the amount of mesophilic amylase is 0.5-1% of the total mass of the liquid, the temperature is controlled at 65-80℃ and the pH is 5.5-6.5, and the reaction time is 30-120 min; after the mesophilic amylase hydrolysis, the pH of the liquid is adjusted to 4.5, centrifuged, and the protein-containing precipitate is collected.

[0010] As a further embodiment of the present invention: the alkali and ultrasonic treatment steps are as follows: the protein-containing precipitated solid phase is redissolved in water at a material-to-liquid ratio of 1:3-6, 2-6 mol / L sodium hydroxide aqueous solution is added to adjust the pH to 7.5-9.5, and ultrasonic treatment is performed for 10-30 min.

[0011] As a further aspect of the present invention, the specific steps of dynamic high-pressure microjet treatment are as follows: homogenization pressure 80-120MPa, cyclic 1-3 times.

[0012] As a further embodiment of the present invention: the alkaline protease hydrolysis step is as follows: the amount of alkaline protease is 1-2% of the total mass of the liquid, the enzyme activity is 50000U / g-100000U / g, the pH value is adjusted to 7.5-9.5, the hydrolysis temperature is 50-60℃, and the hydrolysis time is 30-60 min.

[0013] As a further aspect of the present invention: the pH of the pea protein hydrolysate after alkaline protease hydrolysis is adjusted to 7.0-8.0; a complex protease is added, the hydrolysis temperature is 55-60℃, and the hydrolysis time is 30-90 min; the amount of the complex protease accounts for 0.25-2.0% of the total mass of the liquid; the complex protease is composed of trypsin, papain, and chymotrypsin in a mass ratio of 2.0-1.0:1-0.5:2-1.

[0014] As a further aspect of the present invention: separation and purification includes microfiltration membrane separation, nanofiltration membrane concentration and desalting, sterilization, and spray drying to obtain pea active peptides.

[0015] The application of pea protein peptides prepared by the above-mentioned method or any of the above-mentioned methods in the preparation of foods that inhibit the growth of tumor cells, including human pancreatic cancer cells, human breast cancer cells, human non-small cell lung cancer cells, human liver cancer cells, human gastric cancer cells, and human colon cancer cells.

[0016] The beneficial effects of this invention are: This invention provides bioactive peptides with anti-tumor cell growth properties. The bioactive peptides include one or more of polypeptides LY-1, LY-2, LY-3, LY-4, LY-5, LY-6, LY-7, and LY-8. The amino acid sequence of polypeptide LY-1 is shown in SEQ ID NO.1, the amino acid sequence of polypeptide LY-2 is shown in SEQ ID NO.2, the amino acid sequence of polypeptide LY-3 is shown in SEQ ID NO.3, the amino acid sequence of polypeptide LY-4 is shown in SEQ ID NO.4, the amino acid sequence of polypeptide LY-5 is shown in SEQ ID NO.5, the amino acid sequence of polypeptide LY-6 is shown in SEQ ID NO.6, the amino acid sequence of polypeptide LY-7 is shown in SEQ ID NO.7, and the amino acid sequence of polypeptide LY-8 is shown in SEQ ID NO.8. The pea protein peptides of this invention (hereinafter referred to as pea peptide powder) are isolated from pea protein powder and have anti-tumor cell growth effects, especially a strong inhibitory effect on liver cancer cells. They can be used to prepare products with anti-tumor cell growth properties. Attached Figure Description

[0017] The invention will now be further described with reference to the accompanying drawings.

[0018] Figure 1 This is a typical fluorescence intensity diagram of zebrafish pancreatic cancer cells after treatment with pea peptide powder; Figure 2 It is the effect of pea peptide powder on zebrafish in fighting pancreatic cancer; Figure 3 This is a typical image of fluorescence intensity in zebrafish breast cancer cells after treatment with pea peptide powder; Figure 4 The effect of pea peptide powder on the anti-breast cancer effect of zebrafish; Figure 5 This is a typical image of fluorescence intensity in zebrafish lung cancer cells after treatment with pea peptide powder; Figure 6 The effect of pea peptide powder on the anti-lung cancer activity of zebrafish; Figure 7 This is a typical fluorescence intensity diagram of zebrafish human liver cancer cells after treatment with pea peptide powder; Figure 8 The effect of pea peptide powder on the anti-liver cancer activity of zebrafish; Figure 9This is a typical fluorescence intensity diagram of zebrafish human gastric cancer cells after treatment with pea peptide powder; Figure 10 The effect of pea peptide powder on zebrafish's anti-gastric cancer activity; Figure 11 This is a typical fluorescence intensity diagram of zebrafish human colon cancer cells after treatment with pea peptide powder; Figure 12 It is the effect of pea peptide powder on the anti-colon cancer effect of zebrafish. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] The preparation method of pea peptide powder in this example includes the following steps: Dissolve pea protein powder in water at a ratio of 1g:10mL, adjust the pH to 6.0, add medium-temperature amylase at 0.5% of the total amount, control the temperature at 70℃, and hydrolyze for 60 minutes. After hydrolysis, adjust the pH of the solution to 4.5, then centrifuge at 4000g to separate the contents, removing the supernatant containing hydrolysate and collecting the protein-containing precipitate. The protein-containing precipitate was redissolved in water at a material-to-liquid ratio of 1g:4mL. The pH of the solution was adjusted to 8.5 by adding 6mol / L sodium hydroxide solution. The solution was then ultrasonically treated at 30kHz frequency and 120W / L power and extracted with alkali for 30min. The protein slurry, after being treated with alkali and ultrasound, is then subjected to dynamic high-pressure micro-jet homogenization at a pressure of 120 MPa and circulated 3 times. Alkaline protease was added to the pea protein solution after dynamic high-pressure microfluidic treatment for enzymatic hydrolysis. The amount of alkaline protease added accounted for 1% of the total mass of the solution, the enzyme activity was 50000U / g, the pH value was adjusted to 8.5, the hydrolysis time was 60min, and the hydrolysis temperature was 60℃. After the pea protein hydrolysate was treated with alkaline protease, the pH was adjusted to 8.0; a complex protease was added and reacted for 2 hours; the complex protease consisted of trypsin, papain and chymotrypsin in a mass ratio of 1:0.5:1, and the amount used accounted for 0.5% of the total mass of the liquid. The temperature was 60℃ and the enzymatic hydrolysis time was 90 minutes to obtain the hydrolysate. The hydrolyzed liquid obtained above is then subjected to microfiltration membrane separation, nanofiltration desalination, sterilization, and spray drying to obtain pea peptide powder, which has a protein content of 98.3%.

[0021] Performance testing Different amounts of pea peptide powder were added to zebrafish for anti-tumor experiments.

[0022] (1) Anti-tumor effects 1. Testing materials 1.1 Sample Preparation Information Prepare a 5.00 mg / mL stock solution of pea peptide powder with standard dilution water and use immediately after preparation.

[0023] 1.2 Experimental Animals and Cells Zebrafish were raised in aquarium water at 28℃ (water quality: 200mg of instant sea salt added per 1L of reverse osmosis water, conductivity 450-550μS / cm; pH 6.5-8.5; hardness 50-100mg / L CaCO3), bred and provided by our aquarium. The laboratory animal use license number is: SYXK (Zhejiang) 2012-0171, and the husbandry and management meet the requirements of international AAALAC certification (certification number: 001458).

[0024] Wild-type AB strain zebrafish were bred through natural pair mating. Zebrafish aged 2 days post-fertilization (2 dpf) were used to determine the maximum detectable concentration (MTC) of pea peptide powder for antitumor efficacy and to evaluate its efficacy.

[0025] Human pancreatic cancer cells, human breast cancer cells, human non-small cell lung cancer cells, human liver cancer cells, human gastric cancer cells, and human colon cancer cells were cultured in 1640 medium at 37°C with 5% CO2.

[0026] 2. Testing Content Please see Figure 1-12Human pancreatic cancer cells, human breast cancer cells, human lung cancer cells, human liver cancer cells, human gastric cancer cells, and human colon cancer cells were labeled with red fluorescent dye and microinjected into the yolk sacs of 2-day-flight (dpf) wild-type AB strain zebrafish, with approximately 200 cells per fish, to establish a zebrafish tumor transplantation model. The zebrafish injected with these cells were cultured at 35°C until 3 dpf. At 3 dpf, zebrafish with good tumor cell uniformity were selected under a microscope and randomly assigned to 6-well plates, with 30 fish per well (experimental group). Pea peptide powder (concentration shown in Table 1) was administered in water, and gemcitabine was injected at a dose of 20.0 ng / fish as a positive control. A model control group was also included, with a volume of 3 mL per well. The samples were treated at 35℃ for 48 hours. Ten zebrafish were randomly selected from each experimental group and photographed under a fluorescence microscope. Data were analyzed and collected using NIS-Elements D 3.20 advanced image processing software. The fluorescence intensity (S) of tumor cells was analyzed, and the antitumor efficacy of pea peptide powder was evaluated based on the statistical analysis results of this index. Statistical results are expressed as mean ± SE. The formula for calculating antitumor efficacy is as follows: Anti-tumor efficacy (%) = [S 模型对照组 -S 样品组 / S 模型对照组 ]×100% Statistical analysis was performed using SPSS 26.0 software. A p-value < 0.05 indicated statistical significance. The results are shown in Tables 1-6. Table 1: Experimental results evaluating the anti-pancreatic cancer efficacy of pea peptide powder (n=10)

[0027] Compared with the model control group,

[0028] Table 2: Experimental results evaluating the anti-breast cancer efficacy of pea peptide powder (n=10)

[0029] Compared with the model control group,

[0030] Table 3: Experimental results evaluating the anti-lung cancer efficacy of pea peptide powder (n=10)

[0031] Compared with the model control group,

[0032] Table 4: Experimental results evaluating the anti-liver cancer efficacy of pea peptide powder (n=10)

[0033] Compared with the model control group,

[0034] Table 5: Experimental results evaluating the anti-gastric cancer efficacy of pea peptide powder (n=10)

[0035] Compared with the model control group,

[0036] Table 6: Experimental results evaluating the anti-colon cancer efficacy of pea peptide powder (n=10)

[0037] Compared with the model control group,

[0038] 3. Perform peptide fragment detection on pea peptide powder. Desalination using ZipTip C18 1) Weigh 10 mg of sample and add 30 μL of 0.1% TFA to dissolve by sonication. 2) Rinse tip 10 times with 50uL of 60%ACN / 0.1% TFA.

[0039] 3) Clean the tip 10 times with 10uL of 0.1% TFA.

[0040] 4) Inhale and exhale the sample 20 times to expel the liquid.

[0041] 5) Clean the tip five times with 10uL of 0.1% TFA.

[0042] 6) Elute the peptides into a new EP tube with 10 μL of 60% ACN and 0.1% TFA, and then vacuum dry.

[0043] 7) On-machine testing The peptide was dissolved in 20 μL of dissolving buffer (0.1% formic acid, 5% acetonitrile), vortexed thoroughly, and centrifuged at 13500 rpm at 4°C for 20 min. The supernatant was transferred to a sample tube, and 8 μL was taken for mass spectrometry identification. Liquid chromatography parameter settings: Mobile phase A: 0.1% formic acid; Mobile phase B: 0.1% formic acid, 80% ACN; Table 7: Mobile Phase Parameters

[0044] Table 8: Mass Spectrometry Settings

[0045] The experimental results are shown in Table 9: Table 9: Peptide sequences of SEQ ID NO.1-SEQ ID NO.8

[0046] The foregoing has provided a detailed description of one embodiment of the present invention, but this description is merely a preferred embodiment and should not be construed as limiting the scope of the invention. All equivalent variations and modifications made within the scope of the claims of this invention should still fall within the patent coverage of this invention.

Claims

1. Pea protein peptides, characterized in that, The protein peptides include one or more of polypeptides LY-1, LY-2, LY-3, LY-4, LY-5, LY-6, LY-7, and LY-8. The amino acid sequence of polypeptide LY-1 is shown in SEQ ID NO.1, the amino acid sequence of polypeptide LY-2 is shown in SEQ ID NO.2, the amino acid sequence of polypeptide LY-3 is shown in SEQ ID NO.3, the amino acid sequence of polypeptide LY-4 is shown in SEQ ID NO.4, the amino acid sequence of polypeptide LY-5 is shown in SEQ ID NO.5, the amino acid sequence of polypeptide LY-6 is shown in SEQ ID NO.6, the amino acid sequence of polypeptide LY-7 is shown in SEQ ID NO.7, and the amino acid sequence of polypeptide LY-8 is shown in SEQ ID NO.

8. The pea protein peptides are obtained by enzymatic hydrolysis of pea protein powder. The molecular weight of the pea protein peptides is 1000-3000 Da.

2. The method for preparing pea protein peptides according to claim 1, characterized in that, Includes the following steps: The slurry containing protein powder was sequentially subjected to medium-temperature amylase pretreatment, alkali extraction and ultrasonic treatment, dynamic high-pressure microfluidic treatment, alkaline protease hydrolysis, and compound protease hydrolysis to obtain an enzymatic hydrolysate containing pea protein peptides. The enzymatic hydrolysate containing pea protein peptides was separated and purified to obtain the pea protein peptides.

3. The method for preparing pea protein peptides according to claim 2, characterized in that, The preparation method of the liquid includes the following steps: mixing pea protein powder with pure water at a ratio of 1g:6-12mL to obtain a liquid containing pea protein powder.

4. The method for preparing pea protein peptides according to claim 2, characterized in that, The mesophilic amylase hydrolysis step is as follows: the mesophilic amylase activity is 2000-10000 U / g, the amount of mesophilic amylase used accounts for 0.5-1% of the total mass of the liquid, the temperature is controlled at 65-80℃ and the pH is 5.5-6.5, and the reaction time is 30-120 min; after the mesophilic amylase hydrolysis, the pH of the liquid is adjusted to 4.5, centrifuged, and the protein-containing precipitate is collected.

5. The method for preparing pea protein peptides according to claim 4, characterized in that, The steps of alkali treatment and ultrasonic treatment are as follows: the protein-containing precipitated solid phase is redissolved in water at a material-to-liquid ratio of 1g:3-6mL, 2-6mol / L sodium hydroxide aqueous solution is added to adjust the pH to 7.5-9.5, and ultrasonic treatment is performed for 10-30min.

6. The method for preparing pea protein peptides according to claim 2, characterized in that, The specific steps of the dynamic high-pressure microjet treatment are as follows: homogenization pressure 80-120MPa, cycle 1-3 times.

7. The method for preparing pea protein peptides according to claim 2, characterized in that, The alkaline protease hydrolysis step is as follows: the amount of alkaline protease used accounts for 1-2% of the total mass of the liquid, the enzyme activity is 50000U / g-100000U / g, the pH value is adjusted to 7.5-9.5, the hydrolysis temperature is 50-60℃, and the hydrolysis time is 30-60 min.

8. The method for preparing pea protein peptides according to claim 2, characterized in that, After alkaline protease hydrolysis, the pH of the pea protein hydrolysate is adjusted to 7.0-8.0; a complex protease is added, and the hydrolysis temperature is 55-60℃ for 30-90 minutes; the amount of the complex protease accounts for 0.25-2.0% of the total mass of the solution; the complex protease is composed of trypsin, papain, and chymotrypsin in a mass ratio of 1.0-3.0:0.5-1.0:1.0-2.

0.

9. The application of the pea protein peptide as described in claim 1 or the pea protein peptide prepared by the preparation method according to any one of claims 2-8 in the preparation of food products that inhibit tumor cell growth, characterized in that, The tumor cells include human pancreatic cancer cells, human breast cancer cells, human non-small cell lung cancer cells, human liver cancer cells, human gastric cancer cells, and human colon cancer cells.

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