A composition and method for a flaxseed omega-3 drink

Abstract

The present invention belongs to the field of biotechnology, and relates to a composition and method for a flaxseed omega-3 drink. The composition is preferably prepared by using a herbaceous plant of the genus Rorippa in the cruciferous family as an extraction raw material. The active ingredient is a composition obtained by separating and extracting from a herbaceous plant of the genus Rorippa in the cruciferous family. The fermented enzymolysis polypeptide products, SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 significantly increase the immunoglobulin IgG and IgD in the blood, significantly increase the total antioxidant capacity and SOD enzyme activity in the antioxidant indexes, significantly decrease TNF-α, decrease the total blood glucose density, and improve the antioxidant capacity and immune capacity of the body. It is confirmed that the extract has the effects of anti-diabetes and enhancing physical fitness.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a composition and method for a flaxseed omega-3 drink. Background Art

[0002] Diabetes is a metabolic disease. Due to the destruction or deterioration of pancreatic cells that secrete insulin in the body, it leads to the destruction or deterioration of insulin secretion, as well as the rejection of insulin receptors in tissues. Blood sugar is excreted through urine without being transported into tissues, and it is characterized by hyperglycemia and high diabetes. When diabetes is induced, the secretion of insulin and glucagon is disturbed, resulting in abnormal metabolic regulation functions in the body, such as protein and lipid metabolism and carbohydrates, leading to various metabolic diseases. If the symptoms of diabetes persist for a long time, the capillary epithelial membrane thickens, causing many complications in the circulatory system.

[0003] The most important goal of diabetes treatment is to control blood sugar levels as close to normal as possible. Treatment regimens include medications, diet, and exercise. Currently, oral hypoglycemic drugs for type 1 and type 2 diabetes patients include glucosidase inhibitors, sulfonylurea preparations, and biguanide preparations. Glucosidase inhibitors are effective in treating diabetes by delaying the digestion and absorption of carbohydrates in the diet and reducing the increase in blood sugar and insulin in the blood after meals. Glucosidase inhibitors do not cause hyperinsulinemia or hypoglycemia, but have the advantages of stimulating insulin secretion and stimulating the secretion of glucagon-like peptide-1 in the small intestine, and inhibiting glucagon secretion (Mooradian, A.D., Thurman, J.E., Drugs, 57, pp19 - 29, 1999; Baron, A.D. Diabetes Research and Clinical Practice, 40, ppS54 - S55, 1998). Currently, the clinical use of acarbose, voglibose, and miglitol is limited, but long-term use of glucosidase inhibitors may cause side effects such as abdominal distension, vomiting, and diarrhea in some patients (Hanefeld M., Journal of Diabetes and Its Complications, 12, pp228 - 237, 1998). Omega-3 fatty acids have the effect of enhancing the ability of cells to respond to insulin and reducing insulin resistance, which is of great value to type II diabetes patients. The improvement of insulin sensitivity can help patients more precisely control blood sugar levels, thereby reducing the potential risk of hyperglycemia. Actively exploring a safer and more effective way to use natural products for the combined prevention and treatment of diabetes has positive significance. Summary of the Invention

[0004] The object of the present invention is to prepare a composition for a flaxseed omega-3 drink, the composition comprising the following: (i) the polypeptides of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or sequences having at least 70% homology with their polypeptide sequences; (ii) or the polynucleotide sequences encoding the polypeptides of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or sequences having at least 70% homology with their nucleotide sequences; (iii) or derivative chemical additives processed from the sequences described in (i) and (ii).

[0005] Furthermore, the small molecule polypeptide is preferably prepared using a cruciferous Cardamine herb as the extraction raw material.

[0006] Furthermore, the cruciferous Rorippa genus is Cardamine flexuosa With.

[0007] Furthermore, the preparation method of the polypeptide comprises:

[0008] (1) Pretreatment; washing, freeze-drying and pulverizing Cardamine flexuosa.

[0009] (2) Concentration treatment: at room temperature, mix 1,500 mL of 70% Ethanol solution with the powder at a dilution ratio of 1:10 (W / V), extract within 24 hours, filter using Whatman No 2 filter paper, and then perform concentration treatment using a rotary vacuum evaporator in a 45°C water bath.

[0010] (3) Enzymatic hydrolysis: Next, mix the concentrated solution with a hydrolytic enzyme and then carry out an enzymatic reaction at 50°C for 2 hours.

[0011] (4) Fermentation treatment: After the reaction, sterilize at 95°C for 10 minutes, mix with fermentation bacteria for fermentation, filter the culture medium using filter paper and gauze, and concentrate using a reflux evaporator in a 45°C water bath, and then place it in a -45°C cryogenic chamber for 12 hours.

[0012] (6) Nanofiltration: Ultra-nanofiltrate the fermentation liquid, and after concentration, freeze-dry to obtain small molecule active peptides;

[0013] (7) Use high performance liquid chromatography-mass spectrometry to determine and select small molecule sequences.

[0014] Furthermore, the hydrolytic enzyme is subtilisin and papain.

[0015] Furthermore, the fermentation bacteria are Saccharomyces cerevisiae, Aspergillus oryzae, and Aspergillus niger.

[0016] Furthermore, the polypeptide preferably has the following amino acid sequences:

[0017] SEQ ID NO.1: LPGASPKPSGAKISGALAIL,

[0018] SEQ ID NO.2: AKLASLKEAPLPALAPSK,

[0019] SEQ ID NO.3: LAGALGSASPAILGEKLPAK.

[0020] Furthermore, the application of the composition in powders, tablets, and liquids.

[0021] Advantages of the Invention

[0022] The present invention provides a simple method for the screening, enzymatic hydrolysis, and evaluation of small molecule peptides of herbal extracts, as well as drug evaluation. This method is applicable to herbal extracts. After fermentation and enzymatic hydrolysis, the recovery rate of raw materials is quite high. The herbal extracts do not cause harm to cells. The fermented and enzymatically hydrolyzed polypeptide products, SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, significantly increase the immunoglobulin IgG and IgD in the blood, significantly increase the total antioxidant capacity and SOD enzyme activity in the antioxidant index, significantly decrease TNF-α, and decrease the total blood glucose density. The antioxidant capacity and immune capacity of the body are both improved. This will confirm that our extracts have the effects of anti-diabetes and enhancing physical fitness. Brief Description of the Drawings

[0023] Figure 1 Measurement of blood glucose density in streptozotocin-induced diabetic (rats). Detailed Description of the Invention

[0024] To make the above objects, features, and advantages of the present invention more obvious and understandable, the following detailed description of the specific embodiments of the present invention will be given with reference to the accompanying drawings of the specification.

[0025] Example 1

[0026] (1) Pretreatment; washing, freeze-drying, and pulverizing Cardamine flexuosa;

[0027] (2) Concentration treatment: At room temperature, mix 1,500 mL of 70% Ethanol solution with the powder at a dilution ratio of 1:10 (W / V), extract for 24 hours, filter using Whatman No 2 filter paper, and then concentrate using a rotary vacuum evaporator in a water bath at 45°C.

[0028] (3) Enzymatic hydrolysis: Next, the concentrated solution is mixed with the hydrolytic enzyme, and then an enzymatic reaction is carried out at 50 °C for 2 hours.

[0029] (4) Fermentation treatment: After the reaction, it is sterilized at 95 °C for 10 minutes, mixed with fermenting bacteria for fermentation, the culture medium is filtered with filter paper and gauze, and concentrated using a reflux evaporator in a water bath at 45 °C, and then placed in a deep freezer at -45 °C for 12 hours.

[0030] (6) Nanofiltration: The fermented liquid is ultra-nanofiltrated, and after concentration, it is freeze-dried to obtain small molecule active peptides;

[0031] (7) Use high performance liquid chromatography-mass spectrometry to determine and select small molecule sequences.

[0032] Furthermore, the hydrolytic enzyme is subtilisin and papain.

[0033] Furthermore, the fermenting bacteria are Saccharomyces cerevisiae, Aspergillus oryzae, and Aspergillus niger in a weight ratio of 8:10:3.

[0034] Furthermore, the preferred amino acid sequence of the polypeptide is:

[0035] SEQ ID NO.1: LPGASPKPSGAKISGALAIL,

[0036] SEQ ID NO.2: AKLASLKEAPLPALAPSK,

[0037] SEQ ID NO.3: LAGALGSASPAILGEKLPAK.

[0038] (4) The enzyme-treated solution is concentrated to 100 mL using a 1000 kD hollow fiber membrane column, purified through an anion exchange chromatography column, and then filtered through a 0.1 μm membrane to remove bacteria to obtain a sterile mesenchymal stem cell exosome solution, which is concentrated and then freeze-dried to obtain small molecule active peptides;

[0039] (5) Use high performance liquid chromatography-mass spectrometry to determine and select small molecule sequences. For the purity and amino acid sequence determination, the collected small molecule peptides are detected as a single peak by liquid chromatography, and further analyzed using a mass spectrometer, and the results are analyzed and identified by combining with mass spectrometry software. The mass spectrometry results are used to perform blast alignment and analysis and identification on the substance, and multiple peptide segments are identified, and short peptides with safety and easy absorption are selected from them.

[0040] The hydrolytic enzyme is subtilisin and papain in a weight ratio of 10:3.

[0041] The fermentation bacteria mentioned above are Aspergillus giganteus, Candida utilis, and Bacillus cereus, and their weight ratio is 10:5:8.

[0042] The water solubility, antigenicity, and other physicochemical properties of these peptides were predicted through the online databases AllerTOP v.2.0 and Innovagen. Then, their toxicological and safety properties were predicted using the online database ADMETLab 2.0. Finally, unreported peptides with inhibitory activity against glycoside hydrolases were screened out through the online database BIOPEP-UMW database. The selected amino acid sequences are as follows:

[0043] SEQ ID NO.1: LPGASPKPSGAKISGALAIL,

[0044] SEQ ID NO.2: AKLASLKEAPLPALAPSK,

[0045] SEQ ID NO.3: LAGALGSASPAILGEKLPAK.

[0046] Example 2

[0047] Four chemical substances are essential components for making effervescent beverages: The first is the micropeptide extract used in Example 1, and 200 mg is required; the second is lactose, and 200 mg is needed; the third is starch, and 200 mg is required; the last is magnesium stearate, and the corresponding dose is used; in the liquid formula, the amount of the micropeptide extract used in Example 1 is doubled to 1500 mg, and the usage amount of another key chemical substance CMC-Na (referred to as CMC-Na in English) is also adjusted to 50 g. When manufacturing various reagents, it is necessary to follow the relevant safety operating procedures to ensure the safety of oneself and others. For the preparation of liquids and capsules, it is necessary to ensure that all containers are clean and hygienic to prevent contamination.

[0048] Example 3

[0049] Seven-week-old Sprague-Dawley (SD) rats weighing 300 - 350 g were selected and housed in an environment with a specially formulated standard diet and citrate buffer. After one week of acclimation to the environment, their weights were evenly grouped. On the seventh day of environmental acclimation, after a 10-hour fast, they were injected with 1 mL of STZ (streptozotocin) [65 mg / kg body weight], which was pre-prepared with 0.01 M citrate buffer (pH 4h). After injecting STZ, they were not given water and food for the first four hours after administration, and then food was provided again at the fourth hour. Approximately 72 hours later, their fasting blood glucose was measured, and rats with blood glucose below 250 mg / dL were considered to have diabetes. On the first day after administration, these rats were allowed free access to food for the first week of the experiment. Then, 30 minutes after administration, they were orally administered glucose (3 g / kg). After that, they were placed in a constant temperature environment at 40°C, and different groups of rats (including untreated control groups and control groups of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and omega-3*) were tested. Approximately 30 minutes later, they were orally administered glucose again. Then, we observed the blood glucose levels at 0, 30, 60, 120, and 180 minutes after administration and recorded them in heparin-treated culture tubes. Figure 1 , it can be seen that by comparing the blood glucose levels of different groups of rats, the blood glucose levels of STZ-induced diabetic rats decreased after ingesting glucose, especially after 10 days of treatment with SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and omega-3. The blood glucose density of streptozotocin (STZ)-induced diabetes in SD (rats) was measured, in which fermented enzymatically hydrolyzed polypeptide products, SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and omega-3 (500 mg / kg) were administered. Compared with the control group of distilled water, these fermented enzymatically hydrolyzed polypeptide products, SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 could reduce the blood glucose level, and there was a significant synergistic effect when the fermented enzymatically hydrolyzed polypeptide product and omega-3 were administered in combination;

[0050] By the 30th day, the immunoglobulin IgG and IgD in the blood of the test samples fed with the fermented enzymatically hydrolyzed polypeptide products, SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 of the embodiments of the present invention were significantly increased, the total antioxidant capacity and SOD enzyme activity in the antioxidant indexes were significantly increased, and TNF-α was significantly decreased. The antioxidant capacity and immune capacity of the body were both improved. This will confirm that our extract has the effects of anti-diabetes and enhancing physical fitness.

[0051] Example 4

[0052] Meanwhile, the concentrated processed extract in step (2), the enzymatically hydrolyzed product in step (3); the fermented processed product in step (4), the nanofiltration small molecule active peptide in step (6); the small molecule sequence in step (7) in Example 1 were also compared. For the reaction products of each step, according to the test method of Example 3, the sugar control activity of the product optimized according to the reaction steps is better than that of the crude extract in the previous steps.

[0053] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention. The structures, devices and operation methods not specifically described and explained in the present invention are implemented according to the conventional means in the art without special explanation and limitation.

Claims

1. A composition for a flaxseed omega 3 drink, characterized in that: The composition contains the following polypeptide as shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3, and the amino acid sequence of the polypeptide is: SEQ ID NO.1:LPGASPKPSGAKISGALAIL; SEQ ID NO.2: AKLASLKEAPLPALAPSK; SEQ ID NO. 3: LAGALGSASPAILGEKLPAK.

2. The composition according to claim 1, characterized in that: The polypeptide is prepared by using the herbaceous plant of the genus Cardamine of the cruciferous family as an extraction raw material.

3. The composition according to claim 2, characterized in that: The Cruciferae family Cardamine is Cardamine bent ( Cardamine flexuosa With).

4. The composition according to claim 1, characterized in that: The preparation method of the polypeptide comprises: (1) Pretreatment: washing, freeze-drying and crushing of Cardamine flexuosa; (2) Concentration: Extract and filter the ethanol at room temperature, and then concentrate it in a water bath using a rotary vacuum evaporator; (3) Enzymatic hydrolysis: Next, the concentrate is mixed with a hydrolase for reaction; (4) Fermentation treatment: fermentation with fermentation bacteria, filtration and concentration; (5) Nanofiltration: The fermentation liquid is ultra-nanofiltered, concentrated, and freeze-dried to obtain small molecular active peptides; (6) Use high performance liquid chromatography-mass spectrometry to determine and select small molecule sequences.

5. The composition according to claim 4, characterized in that: The hydrolase is subtilisin and papain.

6. The composition according to claim 4, characterized in that: The fermentation bacteria are saccharomyces cerevisiae, Aspergillus oryzae and Aspergillus niger.

7. Use of the composition according to claims 1-6, characterized in that: The composition is applied in powder, effervescent agent and liquid.

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