阅读说明：本技术 一种方格星虫非变性胶原蛋白的制备方法 (Preparation method of sipunculus nudus non-denatured collagen ) 是由 汤须崇 赵丽茹 代均涵 邓爱华 黄志宏 吴雅清 宋嘉怡 黄韵霏 于 2019-10-21 设计创作，主要内容包括：本发明公开了一种方格星虫非变性胶原蛋白的制备方法,以方格星虫为原料,建立了以低温匀浆技术处理后采用超声辅助酶法提取非变性胶原蛋白提取工艺,采用亲水超滤技术快速分离纯化方格星虫胶原蛋白,得到高纯度的胶原蛋白产品。本发明具有制备工艺简单,胶原蛋白提取率高,纯度高、资源利用率高,提取经济效益高等特点,有效提高方格星虫产品附加值,实现方格星虫高值化利用为胶原蛋白的制备提供新来源。(The invention discloses a preparation method of Sipunculus nudus non-denatured collagen, which takes Sipunculus nudus as a raw material, establishes an extraction process for extracting the non-denatured collagen by adopting an ultrasonic-assisted enzyme method after low-temperature homogenization treatment, and quickly separates and purifies the Sipunculus nudus collagen by adopting a hydrophilic ultrafiltration technology to obtain a high-purity collagen product. The method has the characteristics of simple preparation process, high collagen extraction rate, high purity, high resource utilization rate, high extraction economic benefit and the like, effectively improves the additional value of Sipunculus nudus products, realizes high-value utilization of Sipunculus nudus, and provides a new source for the preparation of collagen.)

1. A preparation method of Sipunculus nudus non-denatured collagen is characterized by comprising the following steps: the method comprises the following steps:

(1) removing viscera of Sipunculus nudus, cleaning, cutting into pieces of 0.8-1.2mm × 0.8-1.2mm, and removing soluble non-collagen protein in Sipunculus nudus by treatment with isopropanol solution at 0-4 deg.C and sodium chloride solution to obtain pretreated Sipunculus nudus;

(2) mixing the pretreated Sipunculus nudus with phosphate buffer solution with pH of 5-10, and dispersing at 0-5 deg.C at 12000-18000rpm for 10-30 min;

(3) adding 8000U/g of protease 2000-8000U/g into the material obtained in the step (2), wherein the protease comprises at least one of flavourzyme, alkaline protease, papain, trypsin and pepsin, performing enzymolysis for 3-5h at 30-50 ℃ after ultrasonic treatment, and centrifuging for 15-25min at 10000-12000rpm at 0-4 ℃ to obtain supernatant and precipitate; repeatedly treating the precipitate for several times by using the process parameters of the step, and combining the obtained supernatant to obtain collagen mucus; then filtering the collagen mucus through a ceramic membrane with the aperture of 0.7-0.9 μm to obtain a permeate;

(4) separating and purifying the permeate at 0-4 deg.C with ultrafiltration membrane with cut-off molecular weight of 10-50kDa until the conductivity and pH value of the permeate become stable to remove excessive acid and salt ions;

(5) concentrating the material obtained in the step (4) by adopting a nanofiltration membrane with the molecular weight cutoff of 300-;

(6) and (5) freeze-drying the material obtained in the step (5) to obtain the sipunculus nudus non-denatured collagen.

2. The method of claim 1, wherein: the step (1) is as follows: removing viscera of Sipunculus nudus, cleaning, and cutting into pieces of 0.8-1.2mm × 0.8-1.2 mm; soaking the fragments in 5-10% isopropanol solution at a ratio of 1 g: 10-30mL, defatting at 0-4 deg.C for 12-24 hr, and cleaning with distilled water to obtain defatted fragments; soaking the degreased fragments in 5-10% sodium chloride solution at a ratio of 1 g: 10-20mL at 0-4 deg.C for 12-24h, washing with distilled water, and draining to obtain pretreated Sipunculus nudus.

3. The method of claim 1, wherein: in the step (2), the ratio of the pretreated Sipunculus nudus to the phosphate buffer solution with the pH value of 5-10 is 1 g: 5-30 mL.

4. The method of claim 1, wherein: the protease is at least one of flavourzyme, alkaline protease, papain, trypsin and pepsin.

5. The method of claim 1, wherein: the inlet pressure of the ultrafiltration membrane in the step (4) is 0.5-3.0 MPa.

6. The method of claim 1, wherein: the initial inlet pressure of the nanofiltration membrane in the step (5) is 0.5-2.0 MPa.

7. The method of claim 1, wherein: the freeze drying in the step (6) is as follows: pre-freezing at-20 deg.C to-40 deg.C for 24-48h, and freeze-drying at-80 deg.C for 36-72 h.

Technical Field

The invention belongs to the technical field of collagen extraction, and particularly relates to a preparation method of sipunculus nudus non-denatured collagen.

Background

Sipunculus nudus (Sipunculus nudus) belongs to Sipunculus, Sipunculus nudus, and Sipunculus arenarius, also known as Sipunculus nudus, and Shachang, and is commonly distributed in Atlantic ocean, Indian ocean, Pacific coast, Guangdong, Guangxi, Hainan and Taiwan coastal areas in China, and generally lives in sandy areas of coastal beaches. Sipunculus nudus not only has delicious taste and is widely popular with people on both sides of the strait, but also is one of the precious marine medicines in China. Sipunculus nudus is cold in nature, sweet in taste and salty in taste, has effects of nourishing yin to reduce pathogenic fire, clearing lung-heat, tonifying deficiency, invigorating kidney and caring skin, can be used for treating hectic fever due to yin deficiency, night sweat, cough due to lung deficiency, chest distress, excessive phlegm, and milk deficiency after parturition, and can be used for treating hepatitis, pulmonary tuberculosis cough, neurasthenia and infantile spleen deficiency. Therefore, the Sipunculus nudus has the beautiful name of 'animal ginseng' in Minnan. Sipunculus nudus contains abundant protein (83.6% -86.5%), polysaccharide, various amino acids and microelements, and has effects of resisting oxidation, regulating immunity, resisting bacteria, radiation, virus, fatigue and delaying aging.

Collagen is a white, opaque, unbranched fibrous protein macromolecular substance, is the main component of extracellular matrix, and is a protein with the largest content and the widest distribution in animals. It is widely distributed in connective tissue, skin, bone, etc. and has the functions of supporting organs and protecting organism, and is the main factor determining the toughness of connective tissue. The structure is characterized in that a triple-helix structure is formed by 3 alpha chains with left-handed helix configuration, and then the triple-helix structure is mutually wound into a right-handed supercoiled structure. Collagen has the functions of lubricating joints, strengthening bones, protecting blood vessels, promoting metabolism, enhancing immunity, stopping bleeding, supporting a stent and the like, and is widely applied to the fields of food industry, biological pharmaceutical industry, cosmetic industry and the like.

At present, researches on the preparation of collagen by using aquatic products as raw materials are mostly focused on aquatic products, and most aquatic collagen has low content of imide acid (proline and hydroxyproline) and thus has poor thermal stability, such as: the thermal denaturation temperature of the cod skin collagen is only 15 ℃ and the thermal denaturation temperature of the grass carp skin collagen (34 ℃). The traditional extraction method of collagen is a process with high energy consumption, high solvent consumption and high time consumption, the damage to the environment is large, the extraction process is difficult to control, and most of the obtained collagen products are denatured collagen products. CN101381411A discloses that the body wall of a sipunculus nudus from which non-collagen is removed is taken as a raw material, and is extracted by acidic protease or acid with the molar concentration of 0.2-2.0mol/L, and the sipunculus nudus collagen is obtained through the steps of centrifugation, salting out, re-centrifugation, dialysis and freeze-drying. CN106636275A discloses that after passing through ultrasonic waves and an electromagnetic field, the cell of the insect pulp is broken to be beneficial to the enzymolysis of protein in Sipunculus nudus, then the high-pressure enzymolysis extraction is carried out, pepsin and papain are used in a compounding way, the extraction rate of the protein in the Sipunculus nudus is obviously improved, and in the method, enzyme deactivation is carried out at 100-120 ℃, the triple helix structure of collagen is destroyed, and the denaturation of the collagen leads to the inactivation of the collagen.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a preparation method of sipunculus nudus non-denatured collagen.

The technical scheme of the invention is as follows:

a preparation method of Sipunculus nudus non-denatured collagen comprises the following steps:

(1) removing viscera of Sipunculus nudus, cleaning, cutting into pieces of 0.8-1.2mm × 0.8-1.2mm, and removing soluble non-collagen protein in Sipunculus nudus by treatment with isopropanol solution at 0-4 deg.C and sodium chloride solution to obtain pretreated Sipunculus nudus;

(2) mixing the pretreated Sipunculus nudus with phosphate buffer solution with pH of 5-10, and dispersing at 0-5 deg.C at 12000-18000rpm for 10-30 min;

(3) adding 8000U/g of protease 2000-8000U/g into the material obtained in the step (2), wherein the protease comprises at least one of flavourzyme, alkaline protease, papain, trypsin and pepsin, performing enzymolysis for 3-5h at 30-50 ℃ after ultrasonic treatment, and centrifuging for 15-25min at 10000-12000rpm at 0-4 ℃ to obtain supernatant and precipitate; repeatedly treating the precipitate for several times by using the process parameters of the step, and combining the obtained supernatant to obtain collagen mucus; then filtering the collagen mucus through a ceramic membrane with the aperture of 0.7-0.9 μm to obtain a permeate;

(4) separating and purifying the permeate at 0-4 deg.C with ultrafiltration membrane with cut-off molecular weight of 10-50kDa until the conductivity and pH value of the permeate become stable to remove excessive acid and salt ions;

(5) concentrating the material obtained in the step (4) by adopting a nanofiltration membrane with the molecular weight cutoff of 300-;

(6) and (5) freeze-drying the material obtained in the step (5) to obtain the sipunculus nudus non-denatured collagen.

In a preferred embodiment of the present invention, the step (1) is: removing viscera of Sipunculus nudus, cleaning, and cutting into pieces of 0.8-1.2mm × 0.8-1.2 mm; soaking the fragments in 5-10% isopropanol solution at a ratio of 1 g: 10-30mL, defatting at 0-4 deg.C for 12-24 hr, and cleaning with distilled water to obtain defatted fragments; soaking the degreased fragments in 5-10% sodium chloride solution at a ratio of 1 g: 10-20mL at 0-4 deg.C for 12-24h, washing with distilled water, and draining to obtain pretreated Sipunculus nudus.

In a preferred embodiment of the present invention, in the step (2), the ratio of the pretreated sipunculus nudus to the phosphate buffer solution with pH of 5-10 is 1 g: 5-30 mL.

In a preferred embodiment of the present invention, the protease is at least one of flavourzyme, alcalase, papain, trypsin and pepsin.

In a preferred embodiment of the present invention, the inlet pressure of the ultrafiltration membrane in the step (4) is 0.5 to 3.0 MPa.

In a preferred embodiment of the invention, the initial inlet pressure of the nanofiltration membrane in step (5) is 0.5 to 2.0 MPa.

In a preferred embodiment of the present invention, the freeze-drying in the step (6) is: pre-freezing at-20 deg.C to-40 deg.C for 24-48h, and freeze-drying at-80 deg.C for 36-72 h.

The invention has the beneficial effects that:

1. the invention adopts a low-temperature homogenization technology, greatly improves the Sipunculus nudus collagen yield, and shortens the collagen enzymolysis time.

2. The invention adopts an ultrasonic-assisted enzymolysis technology, and the ultrasonic waves generate physical actions after cavitation generated by a medium in a solution, such as shock waves, micro-jet, shearing force, high temperature and high pressure actions, and chemical actions after ultrasonic cavitation, so that free radicals appear in the solution, and the acting force for stabilizing collagen molecules can be destroyed (such as hydrophobic interaction force), thereby causing the expansion of collagen, further causing the dispersion of collagen aggregates, further causing the collagen aggregates to be more evacuated on the spatial structure, and causing the collagen to present relatively higher solubility in the solution, thereby increasing the yield and shortening the extraction time.

3. The invention adopts the hollow fiber ultrafiltration membrane treatment technology, and the collagen is separated, purified and concentrated, and the separation time is compressed to be within 3 hours from 1 to 3 days of the traditional process; the treatment capacity of the collagen is improved from less than 1L in the traditional dialysis process to 45L of collagen solution which can be treated at one time. The traditional dialysis method is time-consuming and water-consuming, the concentration efficiency of the collagen is not high, and the ultrafiltration membrane treatment technology can realize the concentration and purification of the collagen with high efficiency and low energy consumption under the drive of certain pressure, so that the production efficiency is improved, the energy consumption is reduced, the production cost is reduced, the recovery rate of the collagen can be effectively improved while the disposable treatment capacity is large, and the method is suitable for industrial production.

Drawings

FIG. 1 is an electron micrograph (500X and 1000X) of a Sipunculus nudus non-denatured collagen prepared according to an example of the present invention.

Detailed Description

The technical solution of the present invention is further illustrated and described by the following detailed description.