Quinoa pancreatic lipase inhibitory peptide and its application in preparing health products for weight loss

Abstract

The present application provides a quinoa pancreatic lipase inhibitory peptide NVEGSALLPA and its application in the preparation of a weight loss health product. The quinoa pancreatic lipase inhibitory peptide NVEGSALLPA of the present application is derived from the food enzyme hydrolysis product of quinoa protein, has a significant pancreatic lipase inhibitory effect and good safety.

Description

[0001] The present application belongs to the field of food and metabolic disease treatment. Specifically, the present application provides quinoa pancreatic lipase inhibitory peptide and its application in the preparation of health products for weight loss. Background Art

[0002] With the improvement of nutrition level and the change of lifestyle, obesity has become an important issue affecting the health of a large number of people around the world. In addition to a few genetic and endocrine causes, the main cause of obesity is excessive intake of energy, such as triglycerides, in the daily diet. Therefore, inhibiting enzymes related to lipid absorption and metabolism is one of the common strategies to inhibit obesity, among which pancreatic lipase is one of the most popular targets.

[0003] The only pancreatic lipase inhibitor currently available on the market is orlistat, which has a definite weight loss effect, but also has adverse reactions such as oily stool, diarrhea, headache, rectal discomfort, fatigue, etc. Therefore, finding new pancreatic lipase inhibitors, especially natural and safe pancreatic lipase inhibitors, is one of the research directions for obesity treatment.

[0004] Quinoa ( Chenopodium quinoa Willd. Although it is called "wheat", it is actually a plant of the genus Chenopodiaceae, which is native to South America. Quinoa is rich in nutrients, with higher protein and fat content than general grain crops and lower glucose and fructose content than general grain crops. It is an ideal food for regulating sugar and lipid metabolism. At present, the development of quinoa is basically focused on its processing as a grain crop, and there is no precedent for in-depth research on the high-content component quinoa protein to provide quinoa pancreatic lipase inhibitory peptide.

[0005] Small peptides of 2 to 10 amino acids. They have good solubility, are stable, and can easily avoid gastrointestinal digestion, overcoming the disadvantage that proteins are destroyed by digestive enzymes and cannot be taken orally. They have ideal oral or injection effects, and have small relative molecular weight, are easy to modify and regulate, have low synthesis costs, and can be prepared in large quantities. They are a type of pancreatic lipase inhibitor with good application potential. Summary of the invention

[0006] In response to the above situation, the applicant used a variety of food enzymes to hydrolyze quinoa protein and isolated a pancreatic lipase inhibitory peptide NVEGSALLPA from the hydrolysate with good pancreatic lipase inhibitory effect. The peptide is stable, safe and has excellent pancreatic lipase inhibitory effect, and can be used to prepare a variety of weight loss health products, especially beverages.

[0007] On the one hand, the present application provides a pancreatic lipase inhibitory peptide derived from quinoa, whose amino acid sequence is NVEGSALLPA.

[0008] Furthermore, the quinoa pancreatic lipase inhibitory peptide is prepared by hydrolyzing quinoa protein with pepsin and ficin.

[0009] Furthermore, the enzymatic hydrolysis system comprises 5000U / mL of pepsin and 5000U / mL of ficin, and 4% w / v of calcium lactate or 2% w / v of sodium pyrophosphate.

[0010] Furthermore, the enzymolysis is carried out at 37 degrees Celsius for 4 hours.

[0011] Furthermore, the quinoa pancreatic lipase inhibitory peptide is prepared using the Fmoc solid phase synthesis method.

[0012] On the other hand, the present application provides the use of the above-mentioned quinoa pancreatic lipase inhibitory peptide in the preparation of health products for weight loss.

[0013] Furthermore, the health product for weight loss is a liquid beverage or a solid beverage.

[0014] On the other hand, the present application provides a health product for weight loss, which comprises the above-mentioned quinoa pancreatic lipase inhibitory peptide.

[0015] Furthermore, the health product for weight loss is a liquid beverage or a solid beverage.

[0016] In the case of known sequences, those skilled in the art can prepare peptides of desired purity using existing knowledge. These methods include but are not limited to extraction after enzymatic hydrolysis, or solid phase synthesis methods. Those skilled in the art can ensure that the peptide product does not contain harmful substances and ensure its safety by controlling parameters and using appropriate reagents and purification methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 These are the results of pancreatic lipase inhibition activity test of quinoa protein products hydrolyzed by different enzymes.

[0018] Figure 2 This is the mass spectrum of the solid phase synthesized peptide product. DETAILED DESCRIPTION

[0019] The present invention is described in detail below in conjunction with specific embodiments, but the protection scope of the present invention is not limited to the following embodiments.

[0020] Materials and basic methods

[0021] Quinoa Varieties:

[0022] The experiment used quinoa No. 1 (red), a common variety in northern my country.

[0023] The above-mentioned quinoa is produced in Shanxi and Inner Mongolia.

[0024] Enzyme preparations:

[0025] Food-grade pepsin (200,000 U / g), trypsin (100,000 U / g), papain (100,000 U / g), chymotrypsin (100,000 U / g), ficin (100,000 U / g), and bromelain (50,000 U / g) were purchased from Dongheng Huadao Biotechnology Co., Ltd.

[0026] Detection of pancreatic lipase inhibitory activity:

[0027] A 2.0 mg / mL pancreatic lipase solution (SIGMA L3126) was prepared using PBS at pH 7.0, and the supernatant was collected by centrifugation.

[0028] A 10 mM p-nitrophenylbutyrate solution was prepared using PBS, pH 7.0.

[0029] After mixing 40 μl of the test solution (5 mg / mL hydrolyzate sample solution or distilled water control), 40 μl of the above pancreatic lipase solution and 20 μl of the above p-nitrophenylbutyrate solution, incubate in a 37°C water bath for 30 minutes, and use a microplate reader to record the blank 405 nm absorbance of the incubated system and the sample and control.

[0030] The pancreatic lipase activity inhibition rate (%) was calculated using the following formula:

[0031] Pancreatic lipase activity inhibition rate (expressed in %) = 1-(sample absorbance-sample blank absorbance) / (control absorbance-control blank absorbance)

[0032] Unless otherwise specified, the inhibition rate listed is the average inhibition rate of three parallel experiments.

[0033] LC-MS / MS analysis:

[0034] The fractions smaller than 3 kDa in the quinoa protein hydrolysate were separated by centrifugal ultrafiltration tubes. The obtained fractions were desalted by column and analyzed by LC-MS / MS (Agilent StreamSelect, online nanospray ion source). Parameters: injection volume: 5 μL, column flow rate: 500 nL / min, column temperature: 40°C, electrospray voltage: 2 kV. Mobile phase A: 0.1% formic acid in water, phase B: 0.1% formic acid in 80% ACN solution. The gradient started from 5% phase B, equilibrated for 2 minutes, and increased to 95% with a nonlinear gradient; MS: scanning range (m / z): 350-1550, resolution: 120000, maximum injection time: 100 milliseconds; HCD-MS / MS: resolution: 30000, dynamic exclusion time: 30 seconds, maximum injection time: 54 milliseconds. PEAKS DB searched the uniprot-Setaria_italica database.

[0035] Peptide performance prediction:

[0036] Biological activity prediction: PeptideRanker

[0037] Pepsin and trypsin digestion prediction: PeptideCutter

[0038] Toxicity prediction: ToxinPrep (SVM)

[0039] Allergy prediction: AllerTOP

[0040] Hydrophilicity prediction: ExPasy

[0041] Instability prediction: ExPasy

[0042] Peptide safety testing

[0043] In general feeding experiments, Wistar rats with a body weight of 180-240g were used. The test peptide (8mg / kg) or an equal amount of distilled water (10 rats per group, half male and half female) was given by gavage every day for 10 consecutive days. The rats were fed with normal drinking water and feeding, and the observation period was 3 weeks. The observation and inspection items included daily activities, diet, behavior; alanine aminotransferase, aspartate aminotransferase, creatinine (weekly testing); and the liver and kidney weight and organ condition observation after dissection at the end of the observation period.

[0044] The high-dose experiment used Wistar rats, weighing 200-220g, and gavaged the rats with 100mg / kg and 200mg / kg of the test peptide (10 rats for each dose, half male and half female), and the observation period was 7 days. The observation items were the animal's death, daily activities, diet, and behavior.

[0045] Example 1 Extraction of Quinoa Protein

[0046] Referring to the existing practices and previous explorations in this field, the extraction method of quinoa protein is as follows:

[0047] The quinoa seeds were washed, dried, ground and passed through a 100-mesh sieve; stirred with 95% edible ethanol at a solid-liquid ratio of 1:30 (g / mL) at room temperature for 1 hour and allowed to stand for 8 hours; the supernatant was discarded and the defatted quinoa powder was dried; purified water was added to the defatted quinoa powder at a solid-liquid ratio of 1:30 (g / mL); the pH was adjusted to 9.5 with 1M NaOH, and extracted in a water bath at 40 degrees Celsius for 3 hours; the supernatant was obtained by centrifugation, the pH was adjusted to 4.5 with 1M HCl, allowed to stand for 30 minutes, and the precipitate was obtained by centrifugation; the precipitate was washed with purified water and freeze-dried to obtain quinoa protein powder.

[0048] The extraction rate of each batch is about 70%, and the protein purity is above 69%.

[0049] Example 2 Enzymatic hydrolysis of quinoa protein

[0050] Basic enzymatic hydrolysis experimental methods

[0051] The quinoa protein powder prepared by the method of Example 1 was dissolved in pure water at a ratio of 5% w / v; the enzyme was added after adjusting the pH; the enzyme was hydrolyzed at 37 degrees Celsius for 4 hours on a shaker at 150 rpm; the enzyme was inactivated in a boiling water bath for 10 minutes; the supernatant was taken as the hydrolyzate after cooling, and it could be stored in a liquid or solid state as needed.

[0052] After preliminary experiments, among several proteases, only the enzymatic hydrolysis product of pepsin can provide relatively obvious pancreatic lipase inhibitory activity. Therefore, in subsequent experiments, pepsin is used as the basis for configuring the composite enzymatic hydrolysis system, and the pH at which pepsin and other proteases can act together is appropriately selected (the specific screening experiment is not shown. Since the enzymatic hydrolysis experiment involves many variables, such as enzymes, dosage, pH, temperature, auxiliary reagents, etc., only some representative schemes and results are shown below):

[0053] Pepsin (5000 U / mL), pH 5.0;

[0054] Pepsin (5000 U / mL), trypsin (5000 U / mL), pH 5.5;

[0055] pepsin (5000 U / mL), papain (2000 U / mL), pH 5.0;

[0056] Pepsin (5000U / mL), chymotrypsin (3000U / mL), pH 5.5;

[0057] pepsin (5000U / mL), ficin (5000U / mL) pH 5.0;

[0058] pepsin (5000U / mL), bromelain (3000U / mL) pH 5.5;

[0059] The results of pancreatic lipase inhibition rate of enzymatic hydrolysate are as follows Figure 1 As shown in the results, various combined enzymatic hydrolysis schemes can provide better pancreatic lipase inhibitory activity than pepsin alone, among which the combination of pepsin and ficin, and pepsin and papain can provide significantly better inhibitory effects than other combinations (inhibition rate of more than 40%).

[0060] Example 3 Effect of emulsifier on enzymatic hydrolysis

[0061] In order to further improve the enzymatic hydrolysis effect, the applicant verified more complex enzyme schemes, and the results showed that more combinations of proteases did not provide better inhibition effects. Subsequently, the applicant considered that quinoa has a higher fat content, which is significantly higher than other food crops, and some of the fat exists in the form of lipoproteins, which cannot be removed by degreasing with general organic solvents. The barrier of these lipids in the subsequent alkaline extraction and enzymatic hydrolysis may affect the enzymatic hydrolysis process. Therefore, it is considered to add emulsifiers to the enzymatic hydrolysis system to improve the enzymatic hydrolysis effect. The results of some emulsifiers and chelating agents are shown in Table 1 (due to the large number of types and concentration factors, only some representative results are shown):

[0062] Table 1 Pancreatic lipase inhibition rate after adding emulsifier to the enzymatic hydrolysis system (%)

[0063] Pepsin (5000U / mL), Ficin (5000U / mL) pH 5.0 Pepsin (5000U / mL), papain (2000U / mL) pH 5.0 Calcium lactate (2% w / v) 50.2% 44.6% Calcium lactate (4% w / v) 54.1% 46.8% Sucrose fatty acid esters (4% w / v) 46.8% 48.8% Sucrose fatty acid esters (6% w / v) 48.2% 50.3% Sodium pyrophosphate (2% w / v) 56.9% 46.6%% Sodium pyrophosphate (4% w / v) 51.5% 49.5%

[0064] The results showed that various emulsifiers such as calcium lactate, sucrose fatty acid esters, and sodium pyrophosphate could enhance the pancreatic lipase inhibitory effect of the hydrolysate, among which 4% w / v calcium lactate and 2% w / v sodium pyrophosphate were particularly effective against pepsin plus ficin.

[0065] Example 4 Peptide Analysis of Enzymatic Hydrolysates

[0066] The best hydrolyzate of pepsin (5000 U / mL), ficin (5000 U / mL) pH 5.0, sodium pyrophosphate (2% w / v) in Example 3 was used to identify peptides according to the method in the "Materials and Basic Methods" section, and the properties were predicted using the software listed in the "Peptide Performance Prediction" section. The identified peptides with predicted biological activities greater than 0.6 are shown in Table 2 below:

[0067] Table 2 Some identified peptides and their properties

[0068] The hydrophilicity of several peptides with expected high biological activity was average, but considering that the hydrophilicity issue has no major impact in the preparation of most health products, NVEGSALLPA, which is predicted to have the highest biological activity and better stability, was selected for further study.

[0069] To further clarify the properties and safety of the peptide, we commissioned Nanjing GenScript Biotech Co., Ltd. to prepare the pure peptide NVEGSALLPA (purity above 96%, mass spectrum see Figure 2 ; The pure product was precipitated with ether and then washed with ether several times, dissolved in pure water, desalted by HPLC and freeze-dried).

[0070] The pancreatic lipase inhibition rate was detected according to the method described in "Materials and Basic Methods". The inhibition rate of 5 mg / mL peptide NVEGSALLPA can reach 69.2%.

[0071] Example 5 Safety test of peptide

[0072] General feeding experiments and high-dose experiments were performed as described in the peptide safety experiments section in the "Materials and Basic Methods" section.

[0073] In the general feeding experiment, there were no obvious abnormalities in the daily activities, diet, and behavior of the two groups; there were no obvious abnormalities in the organs after dissection; other parameters are as shown in Table 3 below

[0074] Table 3 Blood biochemical results in general feeding experiments (average values)

[0075]

[0076] Whether from the trend of changes after feeding the peptide or from the comparison with the control, the peptide NVEGSALLPA has no obvious liver and kidney toxicity, and has no significant effect on the daily behavior and organ condition of the animals.

[0077] In the large-dose experiment, none of the rats died, and there was no obvious abnormality in their daily activities. It can be expected that the median lethal dose of this peptide is above 500 mg / kg, reaching the level of general health care raw materials.

[0078] The peptide NVEGSALLPA is derived from the enzymatic hydrolysis of the common grain quinoa, and the above experiments have verified the safety of the peptide NVEGSALLPA in detail. It can be considered that the peptide NVEGSALLPA can be used to prepare health products, especially suitable for the preparation of liquid or solid beverages, lozenges, candies and other health products.

[0079] Example 6 Preparation of health care products

[0080] Slimming tea (tea bag): 5 parts of green tea powder, 2 parts of oolong tea powder, 5 parts of hawthorn powder, 3 parts of lotus leaf powder, 1 part of perilla leaf powder, and 0.05 parts of peptide NVEGSALLPA.

[0081] Weight loss drink: 5 parts of green tea powder, 5 parts of hawthorn powder, 3 parts of orange peel powder, 2 parts of coix seed powder, appropriate amount of sugar substitute, 0.05 parts of peptide NVEGSALLPA, add 50-70 parts of water to prepare.

[0082] Slimming tea candy: 2 parts of carrageenan, 24 parts of sorbitol, 40 parts of maltitol, 18 parts of isomaltooligosaccharide, 30 parts of oolong tea powder, 0.5 parts of peptide NVEGSALLPA, 80 parts of water; mix the syrup into tea water after boiling, mix in the peptide when the temperature is below 50 degrees Celsius, and mold it into shape.

Claims

1. A quinoa pancreatic lipase inhibitory peptide, characterized in that: The amino acid sequence of the quinoa pancreatic lipase inhibitory peptide is NVEGSALLPA.

2. The quinoa pancreatic lipase inhibitory peptide according to claim 1, wherein the quinoa pancreatic lipase inhibitory peptide is prepared by enzymolysis of quinoa protein with pepsin and ficin.

3. The quinoa pancreatic lipase inhibitory peptide according to claim 2, wherein the enzymatic hydrolysis system comprises 5000U / mL of pepsin and 5000U / mL of ficin, and 4% w / v of calcium lactate or 2% w / v of sodium pyrophosphate.

4. The quinoa pancreatic lipase inhibitory peptide according to claim 3, wherein the enzymatic hydrolysis is carried out at 37 degrees Celsius for 4 hours. 5 . The quinoa pancreatic lipase inhibitory peptide according to claim 1 , wherein the quinoa pancreatic lipase inhibitory peptide is prepared by using the Fmoc solid phase synthesis method.

6. Use of the quinoa pancreatic lipase inhibitory peptide according to any one of claims 1 to 5 in the preparation of a health product for weight loss.

7. The use according to claim 6, wherein the health product for weight loss is a liquid beverage or a solid beverage.

8. Health products for weight loss, characterized by: The health product for weight loss comprises the quinoa pancreatic lipase inhibitory peptide according to any one of claims 1-5.

9. The health-care product for weight loss according to claim 8, wherein the health-care product for weight loss is a liquid beverage or a solid beverage.

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