阅读说明：本技术 一种低温酶解提取马齿苋的方法 (Method for extracting purslane by low-temperature enzymolysis ) 是由 韩德平 于 2021-08-11 设计创作，主要内容包括：本发明公开了一种低温酶解提取马齿苋的方法。所述马齿苋的低温酶解提取方法,包括如下步骤：S1、马齿苋经粉碎得到马齿苋粉末,将所述马齿苋粉末加入至胃蛋白酶水溶液中进行浸提Ⅰ；S2、所述浸提Ⅰ结束后,加入胰蛋白酶进行浸提Ⅱ；S2、所述浸提Ⅱ结束后,依次经过滤、离心,收集上清液,即得到马齿苋提取液。本发明提供的低温酶解提取马齿苋有效成分的方法,可以保护马齿苋的有效活性成分,并且操作步骤简便,每一步操作时间容易控制,且无需高精尖设备,可以保障不同批次操作的稳定性,便于其在畜牧兽医临床中的应用。(The invention discloses a method for extracting purslane by low-temperature enzymolysis. The low-temperature enzymolysis extraction method of the purslane comprises the following steps: s1, crushing purslane to obtain purslane powder, and adding the purslane powder into a pepsin water solution for extraction I; s2, adding trypsin to carry out leaching II after the leaching I is finished; and S2, after the extraction II is finished, sequentially filtering and centrifuging, and collecting supernate to obtain the purslane extracting solution. The method for extracting the effective components of the purslane by low-temperature enzymolysis can protect the effective active components of the purslane, is simple and convenient in operation steps, is easy to control the operation time of each step, does not need high-precision equipment, can ensure the stability of different batches of operation, and is convenient for application in animal husbandry and veterinary clinic.)

1. A low-temperature enzymolysis extraction method of purslane comprises the following steps:

s1, crushing purslane to obtain purslane powder, and adding the purslane powder into a pepsin water solution for extraction I;

s2, adding trypsin to carry out leaching II after the leaching I is finished;

and S2, after the extraction II is finished, sequentially filtering and centrifuging, and collecting supernate to obtain the purslane extracting solution.

2. The extraction method according to claim 1, characterized in that: crushing stems, leaves and seeds of the overground part of the purslane;

the particle size of the purslane powder is 80-150 meshes.

3. The extraction method according to claim 1 or 2, characterized in that: in step S1, the pepsin aqueous solution is prepared with physiological saline;

in the pepsin water solution, the concentration of the pepsin is 1-1.2 g/L;

and adjusting the pH value of the pepsin water solution to 1.5-2.0 by adopting hydrochloric acid.

4. The extraction method according to any one of claims 1 to 3, characterized in that: in step S1, the ratio of the pepsin aqueous solution to the purslane is: 10-15 ml: 1g of the total weight of the composition.

5. The extraction method according to any one of claims 1 to 4, characterized in that: in step S1, the leaching conditions of the first leaching solution are as follows:

the temperature is 37-38 ℃, the time is 2-2.2 h, and the process is carried out in water bath.

6. The extraction method according to any one of claims 1 to 5, characterized in that: in the reaction system of the step S2, the concentration of the trypsin is 1-1.2 g/L;

in the step S2, after the trypsin is added, adjusting the pH of the reaction system to 8.0-9.0 by using sodium hydroxide;

in step S2, the leaching conditions of ii are:

the temperature is 37-38 ℃, the time is 1-1.2 h, and the process is carried out in water bath.

7. The extraction method according to any one of claims 1 to 6, characterized in that: in step S3, filtering the dregs of a decoction by a stainless steel sieve, wherein the aperture of the stainless steel sieve is 20 microns;

the conditions of the centrifugation were as follows:

the rotating speed is 4500-5500 r/min, and the time is 10-20 min;

the extraction method further comprises the following steps:

and (3) positively-pressure filtering the purslane extracting solution by adopting a filter membrane with the aperture of 0.2 mu m.

8. The purslane extractive solution obtained by the extraction method of any one of claims 1-7.

9. The use of the purslane extractive solution of claim 8 as or in the preparation of a feed additive for preventing and treating livestock and poultry diarrhea.

10. A feed for preventing and treating diarrhea of livestock and poultry, which comprises the purslane extractive solution of claim 8.

Technical Field

The invention relates to a method for extracting purslane by low-temperature enzymolysis, and belongs to the technical field of biological medicines.

Background

Purslane (Portulaca oleracea L.) is an annual herb plant of Portulaca genus of Portulacaceae family, and belongs to an important medicinal and edible plant. In addition to wild purslane, which is widely cultivated worldwide, purslane is eaten as a wild vegetable since ancient times. As for the medicinal value, the medical books such as Ben Cao gang mu are recorded early, and the purslane has the efficacies of dispersing blood and reducing swelling, benefiting intestines and smoothing fetus, detoxifying and treating stranguria, treating postpartum sweating due to debility and the like. Modern medicine finds that purslane has a plurality of biological characteristics such as bacteria resistance, coccidium resistance, oxidative stress resistance, inflammation resistance, cancer resistance and the like, is widely developed and used in livestock and poultry breeding, natural medicines and a plurality of cosmetics at present, and is a Chinese herbal medicine which is very much concerned.

All purslane has important pharmaceutical effects, and the plants of the purslane are rich in compound components such as polysaccharide, alkaloid, organic acid, terpenes, flavonoids and the like, and contain hormone components such as melatonin, norepinephrine, dopamine and the like. Except for eating the whole plant, the active ingredients of the purslane are extracted at present, and the most common extraction is water decoction or ethanol heating extraction. Although the two methods are simple and convenient to operate, the temperature requirement in the extraction process is too high, and the effective components of the purslane can be seriously damaged; ethanol is also used as a flammable chemical, and its use is limited. In the prior art, purslane polysaccharide and flavonoid compounds are extracted under the assistance of microwave or ultrasonic, although the extraction efficiency is improved, the temperature is high in the extraction process, the effective components can be damaged to a certain extent, and the equipment is inconvenient to use and operate, so that the cost in industrialization is high. Therefore, it is an urgent problem to establish an extraction method which is simple and easy to operate, does not contain other chemical components in the extraction process, and can retain the active ingredients to the maximum extent.

Disclosure of Invention

The invention aims to provide a method for extracting purslane by low-temperature enzymolysis, which can protect active ingredients of purslane from being damaged and realize high-efficiency extraction by utilizing methods of digestive enzyme enzymolysis and low-temperature extraction, so that the purslane can be fully absorbed and utilized when in use, and the drug effect can be exerted to the maximum extent.

The low-temperature enzymolysis extraction method of the purslane provided by the invention comprises the following steps:

s1, crushing purslane to obtain purslane powder, and adding the purslane powder into a pepsin water solution for extraction I;

s2, adding trypsin to carry out leaching II after the leaching I is finished;

and S2, after the extraction II is finished, sequentially filtering and centrifuging, and collecting supernate to obtain the purslane extracting solution.

In the extraction method, the stems, leaves and seeds of the overground part of the purslane are crushed;

the particle size of the purslane powder is 80-150 meshes.

In the above extraction method, in step S1, the pepsin aqueous solution is prepared with normal saline, such as 0.9% normal saline;

in the pepsin water solution, the concentration of the pepsin is 1-1.2 g/L;

adjusting the pH value of the pepsin aqueous solution to 1.5-2.0 by using hydrochloric acid (such as 0.1 mol/L).

In the above extraction method, in step S1, the ratio of the pepsin aqueous solution to the purslane is: 10-15 ml: 1g of the total weight of the composition.

In the above extraction method, in step S1, the leaching conditions of the first leaching solution are as follows:

the temperature is 37-38 ℃, the time is 2-2.2 h, the operation is carried out in a water bath, and the preferable time is 2 h;

in the extraction method, in the reaction system of the step S2, the concentration of the trypsin is 1-1.2 g/L;

in step S2, after adding the trypsin, adjusting the pH of the reaction system to 8.0-9.0 by using sodium hydroxide (such as 0.1 mol/L);

in step S2, the leaching conditions of ii are:

the temperature is 37-38 ℃, the time is 1-1.2 h, the reaction is carried out in a water bath, and the preferable time is 1 h;

in the above extraction method, in step S3, the residue is filtered by a stainless steel sieve, the aperture of the stainless steel sieve is 20 μm;

the conditions of the centrifugation were as follows:

the rotating speed is 4500-5500 r/min, and the time is 10-20 min;

the extraction method further comprises the following steps:

and (3) positively-pressure filtering the purslane extracting solution by adopting a filter membrane with the aperture of 0.2 mu m.

The invention also provides a feed for preventing and treating livestock and poultry diarrhea, which comprises the purslane extract, wherein the mass addition concentration of the purslane extract is 1-10%, preferably 1-3%, 1% or 3%.

The extracting solution prepared by the method disclosed by the invention keeps various effective components of the purslane, can be used as a feed additive for preventing and treating livestock and poultry diarrhea, and can be used for well improving the excrement viscosity of laying hens in high-temperature summer and relieving the diarrhea symptoms of the laying hens; after the lamb milk is added with different concentrations, the diarrhea symptom of lambs can be well improved, and the 1% addition concentration can have a good treatment effect. Therefore, the purslane extracting solution can be added into daily ration of livestock and poultry to replace silage, and can play a role more quickly and efficiently.

The method for extracting the effective components of the purslane by low-temperature enzymolysis can protect the effective active components of the purslane, is simple and convenient in operation steps, is easy to control the operation time of each step, does not need high-precision equipment, can ensure the stability of different batches of operation, and is convenient for application in animal husbandry and veterinary clinic.

Drawings

FIG. 1 is a flow chart of the low-temperature enzymolysis extraction method of purslane.

FIG. 2 shows the sun-dried purslane plant and the leaching liquor after low-temperature enzymolysis extraction.

FIG. 3 is a statistical result of the cure rate of the purslane extractive solution for diarrhea lambs.

Fig. 4 is a morphological observation result of jejunum mucosa of a cured diarrhea lamb with a purslane extracting solution with an adding concentration of 3%.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 method for extracting effective components of purslane by low-temperature enzymolysis

Purslane (Portulaca oleracea L.) is collected in autumn, and the overground part of the purslane (Portulaca oleracea L.) is extracted according to the flow shown in figure 1, wherein the overground part comprises stems, leaves and seeds:

1. collecting the whole overground part of the purslane plant in 9-10 months, wherein the whole overground part of the purslane plant comprises stems, leaves and seeds, and the seeds are not completely mature and are not easy to fall off.

2. Cleaning dust on the surface of the purslane with clear water, and then airing the purslane in a shady and ventilated place to avoid direct sunlight.

3. A crushing step: accurately weighing 100g of purslane by using an electronic balance, and grinding purslane plants into powder with the granularity of 100 meshes by adopting a stainless steel grinder at the rotating speed of 200 rpm.

4. Preparing an extraction solvent 1: 9g of sodium chloride is weighed and added into 1000mL of distilled water, and the sodium chloride is used as normal saline for standby after being fully dissolved. 1g of pepsin (Beijing Virginian Oriental Co., Ltd.) was weighed, added to 1000mL of physiological saline, dissolved at room temperature, and then the pH of the solution was adjusted to about 1.5 with 0.1mol/L hydrochloric acid.

5. And (3) homogenizing: adding 100g of ground purslane powder into the extraction solution 1, and slowly stirring the purslane powder in the same direction by using a glass rod to fully and uniformly mix the purslane powder to form a suspension state.

6. Low-temperature extraction: transferring the mixed purslane extractive solution 1 into a sealed glass bottle with a bottle cap. The glass vials were then placed in a 37 ℃ water bath for 2 h.

7. Preparing an extraction solution 2: the glass bottle after 2 hours of reaction was taken out, 1g of trypsin (Beijing Hui Tian Oriental Co., Ltd.) was immediately weighed and added to the glass bottle, and slowly stirred in the same direction with a glass rod, and the pH was adjusted to 8.5 with 0.1mol/L sodium hydroxide.

8. After sealing, the glass vials were placed in a 37 ℃ water bath for a further 1 h.

9. And (3) filtering: filtering the herba Portulacae solution after low temperature extraction with common 20 μm stainless steel sieve, collecting filtrate, and removing residue.

10. A centrifugation step: and subpackaging the collected filtrate into 50mL centrifuge tubes, centrifuging for 15min at 5000r/min by using a low-speed centrifuge, and collecting supernatant.

11. And (3) sterilization step: the collected supernatant was aspirated with a syringe and sterilized by filtration through a 0.2 μm filter (Beijing, Hui Tian, Oriental Co., Ltd.).

The photographs of the dried purslane plant and the leaching liquor extracted by the low-temperature enzymolysis in the embodiment are shown in fig. 2, and it can be seen that the dried purslane can well retain the whole plant. After low-temperature enzymolysis extraction, the extracting solution is dark green, which shows that water in purslane plants volatilizes in the drying process, but chlorophyll and other components are not damaged, and the extraction method can well release all the components of the purslane.

In the embodiment, enzymolysis is carried out for 2 hours under pepsin and 1 hour under trypsin, if the enzymolysis time is shortened, the effective components of the purslane are not completely extracted, and if the enzymolysis time is prolonged, the effective components cannot be damaged, but the content cannot be increased continuously.

Example 2 application of Low-temperature enzymolysis to extract effective components of Portulaca oleracea L

1. Application in laying hens.

200 laying hens of the same day age in the same batch are selected and randomly divided into a control group and a purslane administration group, and each group contains 100 laying hens.

A500 mL watering can was selected to prepare a purslane working solution at a concentration of 0.2% (1mL of purslane extract prepared in example 1 mixed with 500mL of drinking water). After uniformly mixing, spraying the purslane extract into a trough in a spraying mode, and stirring with conventional daily ration. After feeding in the morning every day, spraying herba Portulacae working solution to ensure that hen is fed freely, and administering only 1 time every day for 30 days continuously.

The excrement form of the laying hens is observed every day after administration, the diarrhea rate of the laying hen group is reflected, and the statistics of the results are shown in table 1.

TABLE 1 statistics of treatment results of purslane extract on diarrhea-causing layer chicken

As can be seen from the results in Table 1, the purslane extract prepared by the invention can well improve the excrement viscosity of laying hens in high-temperature summer seasons and relieve the diarrhea symptoms of the laying hens.

2.2 application of lamb in lactation period.

50 lactation lambs with diarrhea clinically are selected as treatment objects and are randomly divided into 5 groups, and 10 lambs in each group.

Working solutions were prepared from the purslane extract prepared in example 1 and drinking water at volume concentrations of 0.1%, 1%, 3%, 5%, and 10%, respectively. After the lambs are manually fixed, the lambs are orally administrated by using a syringe without a needle, 1mL of the medicine is administrated to each lamb, 2 times a day, and the treatment is continuously carried out for 1 week.

After 1 week of treatment, the diarrhea cure rate of the lambs is observed and counted, and the result is shown in fig. 3, so that the purslane extracting solution extracted by the method can well improve the diarrhea symptoms of the lambs after being added at different concentrations, the diarrhea occurrence rate is obviously reduced, and the good treatment effect can be achieved by adding 1% of the purslane extracting solution.

The observation result of the purslane extracting solution with the addition concentration of 3% on the morphology of the jejunal mucosa of the cured diarrhea lambs is shown in fig. 4, and it can be seen that the purslane extracting solution plays a good role in protecting the intestinal mucosa and can relieve the inflammatory reaction of the intestinal tract and the shedding of epithelial cells of the mucosa.