阅读说明：本技术 猴耳环多肽的制备方法和猴耳环多肽脂质体的制备方法 (Preparation method of pithecellobium clypearia polypeptide and preparation method of pithecellobium clypearia polypeptide liposome ) 是由 陈东明 孙葵英 龙孝军 于 2020-08-24 设计创作，主要内容包括：本发明公开了一种猴耳环多肽的制备方法,包括以下步骤：S1、取猴耳环,灭菌后移至发酵罐；S2、往发酵罐中加入酵母菌粉、乳酸菌粉和适量水后,密封发酵；S3、将纤维素酶、果胶酶分别加入4～5倍猴耳环重量的水中,真空抽取酶液至发酵罐中,使发酵罐中的混合液在40～50℃保温18～36h；S4、往发酵罐中加入酸液调节混合液的pH为3～5,过滤得到滤液；S5、将滤液进行浓缩、干燥,得到猴耳环多肽干粉。通过酶解法破坏猴耳环的细胞壁,避免细胞壁吸附猴耳环多肽和多酚从而影响得率,利用表面活性剂吐温80,解决了脂质体难成膜且容易溶散的问题,且得到的猴耳环多肽脂质体包封率高。(The invention discloses a preparation method of pithecellobium clypearia polypeptide, which comprises the following steps: s1, taking the pithecellobium clypearia, sterilizing and transferring to a fermentation tank; s2, adding yeast powder, lactic acid bacteria powder and a proper amount of water into the fermentation tank, and sealing and fermenting; s3, adding cellulase and pectinase into water with the weight 4-5 times that of the pithecellobium clypearia, and vacuumizing enzyme liquid into a fermentation tank to keep the temperature of mixed liquid in the fermentation tank at 40-50 ℃ for 18-36 hours; s4, adding acid liquor into the fermentation tank to adjust the pH of the mixed liquor to 3-5, and filtering to obtain filtrate; and S5, concentrating and drying the filtrate to obtain the pithecellobium clypearia polypeptide dry powder. The cell wall of the pithecellobium clypearia is damaged by an enzymolysis method, the problem that the pithecellobium clypearia polypeptide and polyphenol are adsorbed by the cell wall to influence the yield is avoided, the problem that the liposome is difficult to form a film and easy to dissolve is solved by using the surfactant Tween 80, and the encapsulation rate of the obtained pithecellobium clypearia polypeptide liposome is high.)

1. A method for preparing a pithecellobium clypearia polypeptide is characterized by comprising the following steps:

s1, taking the pithecellobium clypearia, sterilizing and transferring to a fermentation tank;

s2, adding yeast powder, lactic acid bacteria powder and a proper amount of water into the fermentation tank, and sealing and fermenting for 72-108 hours; the weight of the yeast powder is 0.1-1.0% of that of the pithecellobium clypearia, and the weight of the lactic acid bacteria powder is 0.1-0.5% of that of the pithecellobium clypearia;

s3, adding cellulase and pectinase into water with the weight being 4-5 times that of the pithecellobium clypearia, and vacuumizing enzyme liquid into the fermentation tank to keep the temperature of the mixed liquid in the fermentation tank at 40-50 ℃ for 18-36 hours;

s4, adding acid liquor into the fermentation tank to adjust the pH of the mixed liquor to 3-5, and filtering to obtain filtrate;

and S5, concentrating and drying the filtrate to obtain the pithecellobium clypearia polypeptide dry powder.

2. The method for preparing pithecellobium clypearia polypeptide of claim 1, wherein in step S5, the filtrate is first concentrated to a density of 1.15-1.35 g/cm³And then spray-drying to obtain dry powder.

3. The method for producing the pithecellobium clypearia polypeptide of claim 1, wherein the cellulase and pectinase are added in an amount of 0.01-0.02% based on the pithecellobium clypearia weight in step S3.

4. The method for producing a simian ear loop polypeptide according to claim 1, wherein the acidic solution in step S4 is tartaric acid or citric acid.

5. The method of claim 1, wherein the mixture in the fermentor is cooled to room temperature before the pH is adjusted in step S4.

6. The method for producing a pithecellobium clypearia polypeptide of claim 1, wherein the yeast powder and the lactic acid bacteria powder are dissolved in water in advance in step S2.

7. A method for preparing a liposome of a monkey ear loop polypeptide, comprising the step of preparing the monkey ear loop polypeptide of any one of claims 1 to 6, further comprising the steps of:

s6, crushing the pithecellobium clypearia polypeptide dry powder to 500-800 meshes to obtain pithecellobium clypearia polypeptide fine powder, and adding the pithecellobium clypearia polypeptide fine powder into propylene glycol containing Tween 80 for dissolving, wherein the weight of the Tween 80 is 0.01-0.08% of that of the pithecellobium clypearia polypeptide fine powder;

s7, dissolving the soybean phospholipid and the liquid cholesterol by using ether; the weight of the soybean lecithin is 1-5% of that of the pithecellobium clypearia polypeptide fine powder; the weight of the liquid cholesterol is 1-5% of the weight of the pithecellobium clypearia polypeptide fine powder;

s8, uniformly mixing the propylene glycol solution obtained in the step S6 with an ether solution, adding hydroxypropyl methylcellulose, uniformly mixing, and performing spray drying to obtain the pithecellobium clypearia polypeptide liposome; the weight of the hydroxypropyl methylcellulose is 0.1-1% of the weight of the pithecellobium clypearia polypeptide fine powder.

8. The method for preparing pithecellobium clypearia polypeptide liposome of claim 7, wherein in step S8, the propylene glycol solution and the ether solution are stirred and mixed until no ether smell is generated, and then hydroxypropyl methylcellulose is added.

Technical Field

The invention relates to the field of traditional Chinese medicine pharmacy, in particular to a preparation method of pithecellobium clypearia polypeptide and a preparation method of pithecellobium clypearia polypeptide liposome.

Background

Earrings, named Bischoob, folk name California, Cor-Membranaceae, Sanzheng, Ningpo-Mao, shampoo, Fang jin, etc., are perennial arbors, and the former plant was identified as Mimosaceae by the south China plant institute classification room in 1984 in 7 months, and then classified into Leguminosae and Macaca by the International health organization, named Pithecellobium typoria (belonging to perennial arbor kindline tree). The pithecellobium clypearia is bitter and cold in nature, has the effects of clearing heat and removing toxicity, and astringing dampness and healing sores, and is a southern medicinal material unique for treating various heat-toxin symptoms.

Currently, most of the extraction methods for pithecellobium clypearia are organic extraction methods, for example, chinese patent application No. 2013103140324 discloses a preparation method of pithecellobium clypearia extract, which is to extract with water or ethanol water solution to obtain extract, however, the yield of pithecellobium clypearia polypeptide is low. In addition, the targeted synthesis or the specific proteolysis requires specific pharmaceutical intermediates and specific proteins, which have extremely high requirements for raw materials. Therefore, a method for producing a monkey-ear loop polypeptide with high yield and low requirement for raw materials is needed.

Disclosure of Invention

In view of the above, the present invention provides a method for preparing a pithecellobium clypearia polypeptide.

A method for preparing a monkey ear loop polypeptide comprises the following steps:

s1, taking the pithecellobium clypearia, sterilizing and transferring to a fermentation tank;

s2, adding yeast powder, lactic acid bacteria powder and a proper amount of water into the fermentation tank, and sealing and fermenting for 72-108 hours; the weight of the yeast powder is 0.1 to 1 percent of the weight of the pithecellobium clypearia, and the weight of the lactic acid bacteria powder is 0.1 to 0.5 percent of the weight of the pithecellobium clypearia;

s3, adding cellulase and pectinase into water with the weight 4-5 times that of the pithecellobium clypearia, and vacuumizing enzyme liquid into a fermentation tank to keep the temperature of mixed liquid in the fermentation tank at 40-50 ℃ for 18-36 hours;

s4, adding acid liquor into the fermentation tank to adjust the pH of the mixed liquor to 3-5, and filtering to obtain filtrate;

and S5, concentrating and drying the filtrate to obtain the pithecellobium clypearia polypeptide dry powder.

In one embodiment of the present invention, in step S5, the filtrate is first concentrated to a density of 1.15-1.35 g/cm³And then spray-drying to obtain dry powder. And concentrating the filtrate to the corresponding density, so that the spray drying of the step S5 can be ensured to be smoothly carried out, and the blockage of a spray head is avoided.

In one embodiment of the present invention, the cellulase and pectinase are added in the amount of 0.01-0.02 wt% of the pithecellobium clypearia in step S3.

In one embodiment of the present invention, the acid solution in step S4 is tartaric acid or citric acid. The pH adjustment by tartaric acid or citric acid can be used for preparing the medicine without removing residual tartaric acid or citric acid except for adjusting the pH.

In one embodiment of the present invention, the mixture in the fermentor is cooled to room temperature before the pH is adjusted in step S4. The separation of the pithecellobium clypearia polypeptide caused by the reduction of the temperature in the filtering process is avoided.

In one embodiment of the present invention, the yeast powder and the lactic acid bacteria powder are dissolved in water in advance in step S2. The yeast powder and the lactic acid bacteria powder are dissolved in water in advance, so that the dispersion of the yeast powder and the lactic acid bacteria powder is facilitated, and the consistency of the fermentation degree in a fermentation tank is ensured.

Based on the preparation method of the pithecellobium clypearia polypeptide, the invention further provides a preparation method of pithecellobium clypearia polypeptide liposome, which comprises the following steps on the basis of the steps S1-S5:

s6, crushing the pithecellobium clypearia polypeptide dry powder to 500-800 meshes to obtain pithecellobium clypearia polypeptide fine powder, and adding the pithecellobium clypearia polypeptide fine powder into propylene glycol containing Tween 80 for dissolving, wherein the weight of the Tween 80 is 0.01-0.08% of that of the pithecellobium clypearia polypeptide fine powder;

s7, dissolving the soybean phospholipid and the liquid cholesterol by using ether; the weight of the soybean lecithin is 1 to 5 percent of the weight of the pithecellobium clypearia polypeptide fine powder; the weight of the liquid cholesterol is 1 to 5 percent of the weight of the pithecellobium clypearia polypeptide fine powder;

s8, uniformly mixing the propylene glycol solution obtained in the step S6 with an ether solution, adding hydroxypropyl methylcellulose, uniformly mixing, and performing spray drying to obtain the pithecellobium clypearia polypeptide liposome; the weight of the hydroxypropyl methylcellulose is 0.1-1% of the weight of the pithecellobium clypearia polypeptide fine powder.

In one embodiment of the present invention, in step S8, the propylene glycol solution and the ether solution are stirred and mixed until no ether smell is generated, and then hydroxypropylmethylcellulose is added. Ensures that the ether has no large residue, and prevents the molding of the pithecellobium clypearia polypeptide liposome from being influenced because the ether and the hydroxypropyl methylcellulose are not mutually soluble.

Compared with the prior art, the invention has the beneficial effects that: destroy the cell wall of monkey ear ring through enzymolysis method, thereby avoid the cell wall to adsorb monkey ear ring polypeptide and polyphenol influence the yield, and adjust pH before filtering, improve the solubility of monkey ear ring polypeptide in the zymotic fluid, avoid too much monkey ear ring polypeptide to be filtered out along with monkey ear ring residue. The surfactant Tween 80 is used for solving the problems that the liposome is difficult to form a film and is easy to dissolve, and the obtained pithecellobium clypearia polypeptide liposome has high encapsulation rate.

Detailed Description

The technical solution of the present invention will be further described with reference to the following examples.