阅读说明：本技术 一种南瓜花天然多肽的制备方法和应用 (Preparation method and application of pumpkin flower natural polypeptide ) 是由 菅晓勇 于 2019-01-21 设计创作，主要内容包括：本发明提供了一种南瓜花天然多肽的制备方法,包括如下步骤：将南瓜花洗净后切成丁,然后在低温下匀浆并于低温下静置12小时,离心后的上清提取液经凝胶层析分离纯化,收集分子量在500-12000道尔顿的部分即为南瓜花天然多肽,该部分天然多肽收集液经真空冷冻干燥,得南瓜花天然多肽粉。本发明提供的制备方法所得南瓜花天然多肽分子量较低,保留了较高的天然活性,可以用于制备保健食品及食品。(The invention provides a preparation method of a pumpkin flower natural polypeptide, which comprises the following steps: cleaning the cushaw flower, cutting the cushaw flower into pieces, homogenizing at low temperature, standing for 12 hours at low temperature, separating and purifying the centrifuged supernatant extracting solution through gel chromatography, collecting the part with the molecular weight of 500 plus 12000 daltons, namely the cushaw flower natural polypeptide, and carrying out vacuum freeze drying on the collected liquid of the part of the natural polypeptide to obtain the cushaw flower natural polypeptide powder. The pumpkin flower natural polypeptide prepared by the preparation method provided by the invention has lower molecular weight, retains higher natural activity, and can be used for preparing health-care food and food.)

1. A preparation method of pumpkin flower natural polypeptide is characterized by comprising the following steps:

(1) cleaning fresh cushaw flowers, dicing, homogenizing at 2-8 ℃ in ice bath, adding 0.02-0.2 mol/L of phosphate buffer solution with pH of 6.0-7.2 which is 1-3 times of the weight of the precooled cushaw flowers for soaking, placing in a refrigerator with the temperature of 2-8 ℃ for 12 hours, and then freezing and centrifuging at the temperature of 2-8 ℃, wherein the supernatant is crude extract containing natural polypeptides of the cushaw flowers;

(2) adding an eluent into the crude extract containing the natural polypeptides of the cushaw flowers obtained in the step (1) to dilute the crude extract into a solution containing 30-100 mg/ml of polypeptides, separating the solution by using sephadex chromatography, and collecting components with the molecular weight of 500-12000 daltons;

(3) and (3) carrying out vacuum freeze drying on the cushaw flower natural polypeptide collecting solution in the step (2) to obtain the cushaw flower natural polypeptide extract freeze-dried powder.

2. The method according to claim 1, wherein the concentration of the phosphate buffer solution in the step (1) is 0.05 mol/liter, the pH of the phosphate buffer solution is 7.0, and the amount of the phosphate buffer solution is 2 times that of the pumpkin flower.

3. The method of claim 1, wherein: in the step (2), the crude pumpkin flower natural polypeptide extract collected in the step (1) is diluted by eluent to be a solution containing 50 mg/ml of polypeptide; the separation conditions of the sephadex chromatography are as follows: the gel chromatography column was Sephadex G-50, and the eluent was 0.05 mol/l of pH7.0 phosphate buffer containing 0.15 mol/l of sodium chloride solution.

4. The pumpkin flower natural polypeptide extract obtained by the preparation method of any one of claims 1-3 retains high natural activity, and can be used for preparing health food and food.

Technical Field

The invention belongs to the field of natural product extraction, and particularly relates to a pumpkin flower natural polypeptide, and a preparation method and application thereof.

Background

The pumpkin is also called wheat melon, pumpkin, white gourd, etc. and belongs to the cucurbitaceae family of pumpkin and annual herb. With the improvement of the living standard of people and the increasing attention on nutritional and healthy foods, the pumpkin is found to be rich in nutrition, has the functions of food therapy, health care and medicine after being eaten for a long time, and a large amount of research and reports are carried out on the nutritional ingredients and the health care function of the pumpkin at home and abroad. Pumpkin flower is the flower of pumpkin, generally apricot yellow, hermaphrodite. The pumpkin flower has a long use history in China, can be eaten as soon as possible and can also be used as a medicine, and is one of medicine and food materials. The traditional Chinese medicine considers that the traditional Chinese medicine has the effects of clearing damp-heat, reducing swelling, dissipating blood stasis, resisting and preventing cancers, treating jaundice, eliminating fatigue, promoting blood vessel and heart functions and the like. The pumpkin flower has high value, but the current development degree is still low, the chemical substances extracted from the pumpkin flower for treating diabetes are reported in documents, and the natural polypeptide extracted from the pumpkin flower is not reported yet.

Peptides are classified into oligopeptides and polypeptides, which are bioactive substances in which two or more amino acids are condensed and linked by peptide bonds. The peptides are characterized by high biological activity and diversity compared with other substances in organisms. In the organism, many bioactive substances exist in the form of the molecular structure and the natural conformation of the polypeptide, and play an important role in the organism in the mode of the natural bioactivity. Polypeptides play a crucial role in the growth and development and metabolism of organisms. The active biological polypeptide can regulate and control the vital activities of cells to ensure normal cell division and normal protein synthesis, thereby playing an important role in cell division, differentiation and repair. Therefore, the active biological polypeptide is closely related to human health, anti-aging, life prolonging and the like and has great significance. Recent scientific studies have demonstrated that the major forms of protein absorbed by the human body are not all amino acids, but active biological peptides, and thus can condition health, fight disease and aging by supplementing active biological polypeptides.

At present, a plurality of methods for extracting and preparing the polypeptide comprise an acid hydrolysis method, an alkali hydrolysis method, an enzyme hydrolysis method, a combination of a plurality of methods and the like. However, extraction of polypeptides that retain their native activity, i.e., trying to separate impurities from the extract without altering the structure and properties of these compounds, requires milder extraction conditions. If the selected solution system is too acidic or alkaline, the extracted substance may be irreversibly denatured or excessively hydrolyzed and the molecular conformation may be significantly changed, so that the biological activity and function of the polypeptide are gradually lost. Furthermore, in order to prevent the hydrolysis of the protease added in the enzymatic method after the extraction of the polypeptide is completed, it is generally necessary to boil for inactivating the enzyme, which may cause a decrease or destruction of the biological activity of the polypeptide to some extent.

Importantly, the polypeptide is obtained by decomposing protein in the methods, the operation steps are complicated on the whole, more media are introduced, and the biological activity of the obtained polypeptide has more problems. The method provides a simple and convenient polypeptide preparation method capable of directly extracting natural polypeptide from biological tissues, the method has mild extraction conditions, physiological conditions are adopted as separation conditions, and the obtained polypeptide can keep the natural activity of the polypeptide.

Disclosure of Invention

The invention provides a simple polypeptide preparation method for directly extracting natural polypeptide from biological tissues and retaining the natural activity of the polypeptide, which at least solves the problems of complex technology of a polypeptide extraction method and natural activity retention in the prior art.

The invention provides a preparation method of a pumpkin flower natural polypeptide, which is characterized by comprising the following steps:

(1) cleaning fresh cushaw flowers, dicing, homogenizing at 2-8 ℃ in ice bath, adding 0.02-0.2 mol/l (mol/L) of phosphate buffer solution with pH of 6.0-7.2 which is 1-3 times of the weight of the precooled cushaw flowers for soaking, placing in a refrigerator at 2-8 ℃ for 12 hours, and then freezing and centrifuging at 2-8 ℃, wherein the supernatant is crude extract containing natural polypeptides of the cushaw flowers;

(2) adding an eluent into the crude extract containing the natural polypeptides of the cushaw flowers obtained in the step (1) to dilute the crude extract into a solution containing 30-100 mg/ml (mg/m L) of the polypeptides, separating the solution by using sephadex chromatography, and collecting components with the molecular weight of 500-12000 daltons (D);

(3) and (3) freeze-drying the cushaw flower natural polypeptide collecting solution in the step (2) to obtain the cushaw flower natural polypeptide extract freeze-dried powder.

Preferably, the concentration of the phosphate buffer is 0.05 mol/L, the pH of the phosphate buffer is 7.0, and the dosage of the phosphate buffer is 2 times of that of the pumpkin flowers.

Preferably, in the step (2), the crude extract of natural polypeptides of sweet potato leaves collected in the step (1) is diluted by an eluent to a solution containing 50mg/m L of polypeptides.

Preferably, the separation conditions of the Sephadex G-50 gel chromatography column, the inner diameter of 0.5-2 cm (cm), the column length of 30-120cm, the eluent of 0.05 mol/L pH7.0 phosphate buffer solution, 0.15 mol/L sodium chloride solution, the flow rate of 0.4-7 ml/min (m L/min), the sample amount of 0.5-1m L and the detection wavelength of 280nm are adopted in the Sephadex column.

The pumpkin flower natural polypeptide extract obtained by the preparation method retains high natural biological activity, and can be used for preparing health food and food.

The preparation method of the pumpkin flower natural polypeptide provided by the invention is simple to operate, the natural active polypeptide can be directly extracted from biological tissues, the extraction condition of the method is mild, the separation condition adopts physiological conditions, and the obtained polypeptide can retain the natural biological activity of the polypeptide. Compared with free amino acid, the pumpkin flower natural active polypeptide prepared by the method has lower osmotic pressure, is easier to absorb, and does not cause adverse reactions such as diarrhea and the like after eating; meanwhile, the pumpkin flower natural bioactive peptide has lower antigenicity and cannot cause anaphylactic reaction; the active polypeptide can participate in the synthesis of in vivo protein in human body, promote mitosis and proliferation differentiation of related cells, and enhance metabolism of organism tissues and cells, so that the active polypeptide has the effects of regulating and controlling cell metabolism at molecular level and repairing abnormal damaged cells. In addition, the natural active polypeptide also has the potential functions of reducing blood pressure, blood fat, oxidation resistance, virus resistance, bacteria resistance, cholesterol reduction, mineral absorption promotion, substance metabolism improvement, body immunity enhancement and the like, so the pumpkin flower natural active polypeptide can effectively regulate the health of a human body at a cellular level and resist aging and diseases.

Drawings

FIG. 1 gel chromatogram of pumpkin flower natural polypeptide, i.e. the picture of example 1.

The following detailed description is intended to further illustrate but not limit the invention, the following example being only one preferred embodiment of the invention.

Detailed Description