CN103304678B - A kind of method utilizing multiplex-enzyme extraction Alfalfa Polysaccharide - Google Patents

Abstract

The present invention provides one to utilize the method that compound enzyme (cellulase, papain and pectase) extracts Alfalfa Polysaccharide, it is possible to reduce the Extracting temperature of Alfalfa Polysaccharide, improves the extraction ratio of Alfalfa Polysaccharide, is suitable for promoting the use of.The present invention determines the trial stretch of the factor affecting Alfalfa Polysaccharide extraction ratio by single factor experiment, orthogonal test is used to determine the optimum proportioning of compound enzyme, then Central Composite test is used to combine the relational expression that the response surface (RSM) meets with a response between value and influence factor, and the level of influence factor when being calculated maximum response, so that it is determined that the optimum extraction condition of multiplex-enzyme extraction Alfalfa Polysaccharide, i.e. cellulase 2.5% (weight), papain 2.0% (weight), pectase 3.0% (weight), Extracting temperature 52.7 DEG C, extract pH value 3.87, extract solid-to-liquid ratio 78.91, 2.73 hours extraction times.Under the conditions of empirical tests obtains this, the extraction ratio of Alfalfa Polysaccharide is more than 5.0%.

Description

A method for extracting polysaccharides from alfalfa using compound enzymes

technical field

The invention relates to a method for extracting plant active ingredient polysaccharides, in particular to a method for extracting polysaccharides from alfalfa by using cellulase, papain and pectinase.

Background technique

Alfalfa polysaccharides (Alfalfa Polysaccharides) are water-soluble polysaccharides extracted from alfalfa stems and leaves, which have various biological activities. A large number of experiments have proved that within a certain dosage range, alfalfa polysaccharide can promote the growth of livestock and poultry, and improve the immunity, antioxidant capacity and disease resistance of livestock and poultry. In addition, alfalfa polysaccharides also have a certain curative effect on the treatment of high cholesterol, high blood sugar, arteriosclerosis and myocardial infarction, and more importantly, alfalfa polysaccharides have no toxic and side effects on the body. Based on the above advantages, health products and additives based on alfalfa polysaccharides will become an important part of new health food and feed additives, thereby improving people's lives and promoting healthy breeding of animals.

As a perennial high-quality herbaceous leguminous forage, alfalfa is known as the "king of pastures". It is widely planted in various countries in the world, and its resources are very rich. It provides sufficient and cheap raw materials for the production of alfalfa polysaccharides. However, the extraction process of alfalfa polysaccharides is not yet perfect. The current extraction method is mainly hot water alcohol precipitation method. Through continuous heating, the plant cell walls are softened and ruptured, and the polysaccharides overflow. This method not only takes a long time to operate and the extraction rate is low, but also the continuous high-temperature heating will destroy the polysaccharide structure and reduce the biological function of the polysaccharide, so the extraction process needs to be improved urgently.

Contents of the invention

The object of this invention is to provide a kind of method utilizing compound enzyme (cellulase, papain, pectinase) to extract alfalfa polysaccharide, according to the composition characteristic of plant cell wall, utilize cellulase, papain, pectinase to make alfalfa cell wall Cracking breaks down and helps polysaccharide leaching. The method can not only reduce the temperature used in polysaccharide extraction, shorten the extraction time, save energy, but also significantly improve the polysaccharide extraction efficiency, and is suitable for application and popularization.

The technical scheme provided by the present invention is characterized in that it comprises the following process steps:

1. Crush the air-dried or dried alfalfa powder, pass through a 40-mesh sieve, and degrease it with petroleum ether or ether on a Soxhlet extractor until the supernatant is transparent and clear, then dry it at 40°C to obtain defatted grass powder;

2. The cellulase, papain and pectinase used in this method were purchased from Sinopharm Chemical Reagent Co., Ltd., and the enzyme activities were 15000U/g, 6000U/mg and 50U/g respectively.

3. Take defatted grass powder, add 1.5-2.5% cellulase, 1.0-3.0% papain, 1.0-3.0% pectinase according to the weight of grass powder, and then add dihydrogen phosphate 60-80 times the weight of grass powder Sodium-citrate buffer (pH=3.0-5.0);

4. Put it in a constant temperature water bath, raise the temperature to 40-60°C, then keep the constant temperature, extract for 2.0-3.0h, stir once every 20min during this period, filter with a vacuum pump after the action is over, and collect the filtrate to obtain polysaccharide extract;

5. Boil the polysaccharide extract for 5-10 minutes to inactivate the enzyme activity;

6. Concentrate the polysaccharide extract with a rotary evaporator at 60°C to 1/5 of the original volume, then add 4 times the volume of absolute ethanol, and precipitate at 4°C for 12 hours;

7. Centrifuge the alcohol precipitation solution at 4000rpm/min for 10min, and collect the lower sediment, that is, alfalfa polysaccharide;

8. Dissolve the polysaccharide precipitate, and use the phenol-sulfuric acid method to measure the polysaccharide content at 490nm.

The ethanol used in the above scheme is absolute ethanol. In order to calculate the polysaccharide extraction rate as accurately as possible, the container used during extraction and suction filtration was washed 3 times with distilled water and incorporated into the polysaccharide solution. The absolute ethanol used in the above steps can be distilled and recovered for next use.

The present invention takes the extraction rate of alfalfa polysaccharide as an index through multiple tests, and utilizes a single factor test to sequentially determine the extraction temperature, extraction pH value, solid-liquid ratio, extraction time, cellulase ratio, and papain ratio when the polysaccharide extraction rate is maximum. and pectinase ratio. Then, the optimal ratio of cellulase, papain and pectinase was determined by orthogonal experiment. The optimal extraction conditions of alfalfa polysaccharides were determined by optimizing the four factors of extraction temperature, extraction pH value, solid-liquid ratio, and extraction time through response surface experiments (central composite design), which provided a basis for the development and production of alfalfa polysaccharides.

The beneficial effect of the invention is that the compound enzyme (cellulase, papain, pectinase) is used to crack and decompose the plant cell wall, which facilitates the leaching of polysaccharides, thereby greatly improving the extraction efficiency of polysaccharides and reducing the extraction time of polysaccharides. In addition, the temperature used during extraction is reduced, which saves available input. The operation is simple and easy, and it is suitable for large-scale production. It is conducive to promoting the development and application of alfalfa polysaccharides as health products and feed additives.

Description of drawings

Below, describe embodiment of the present invention in detail in conjunction with accompanying drawing, wherein:

Figure 1 is a three-dimensional distribution diagram of the response surface of Y=f(X ₁ , X ₂ ) (X ₃ =70, X ₄ =2.5h);

Fig. 2 is a contour map of Y=f(X ₁ , X ₂ ) (X ₃ =70, X ₄ =2.5h);

Fig. 3 is a three-dimensional distribution diagram of the response surface of Y=f(X ₁ , X ₃ ) (X ₂ =4.0, X ₄ =2.5h);

Fig. 4 is a contour map of Y=f(X ₁ , X ₃ ) (X ₂ =4.0, X ₄ =2.5h);

Fig. 5 is a three-dimensional distribution diagram of the response surface of Y=f(X ₁ , X ₄ ) (X ₂ =4.0, X ₃ =70);

Fig. 6 is a contour map of Y=f(X ₁ , X ₄ ) (X ₂ =4.0, X ₃ =70);

Fig. 7 is a response surface stereo distribution diagram of Y=f(X ₂ , X ₃ ) (X ₁ =50°C, X ₄ =2.5h);

Fig. 8 is a contour map of Y=f(X ₂ , X ₃ ) (X ₁ =50°C, X ₄ =2.5h);

Fig. 9 is a response surface stereo distribution diagram of Y=f(X ₂ , X ₄ ) (X ₁ =50°C, X ₃ =70);

Fig. 10 is a contour map of Y=f(X ₂ , X ₄ ) (X ₁ =50°C, X ₃ =70);

Figure 11 is a response surface stereo distribution diagram of Y=f(X ₃ , X ₄ ) (X ₁ =50°C, X ₂ =4.0);

Figure 12 is a contour map of Y=f(X ₃ , X ₄ ) (X ₁ =50°C, X ₂ =4.0);

In Figure 1 to Figure 12, X ₁ represents the extraction temperature (°C), X ₂ represents the extraction pH value, X ₃ represents the solid-liquid ratio, X ₄ represents the extraction time (h), and Y represents the polysaccharide extraction rate.

detailed description

1. Crush the air-dried or dried alfalfa powder, pass through a 40-mesh sieve, and degrease it with petroleum ether or ether on a Soxhlet extractor until the supernatant is transparent and clear, then dry it at 40°C to obtain defatted grass powder;

2. The cellulase, papain and pectinase used in this method were purchased from Sinopharm Chemical Reagent Beijing Co., Ltd., and the enzyme activities were 15000U/g, 6000U/mg and 50U/g respectively.

3, get defatted grass powder, by grass powder weight, add 2.5% cellulase, 2.0% papain, 3.0% pectinase, then add disodium hydrogen phosphate-citric acid buffer solution (pH =3.87);

4. Put it in a constant temperature water bath, raise the temperature to 52.7°C, then keep the constant temperature, extract for 2.73 hours, stir once every 20 minutes during this period, filter with a vacuum pump after the action, collect the filtrate, and obtain the polysaccharide extract;

5. Boil the polysaccharide extract for 6 minutes to inactivate the enzyme activity;

6. Concentrate the polysaccharide extract with a rotary evaporator at 60°C to 1/5 of the original volume, then add 4 times the volume of absolute ethanol, and precipitate at 4°C for 12 hours;

7. Centrifuge the alcohol precipitation solution at 4000rpm/min for 10min, and collect the lower precipitate;

8. Dissolve the polysaccharide precipitate in distilled water, and use the phenol-sulfuric acid method to measure the polysaccharide content to be greater than 5.0% at 490 nm.

Claims (6)

1. A method utilizing compound enzymes to extract polysaccharides from alfalfa, the method utilizes cellulase, papain, and pectinase to process alfalfa powder simultaneously to break and decompose alfalfa cell walls and simultaneously extract with disodium hydrogen phosphate-citric acid buffer Obtain the polysaccharide extract, and then obtain the alcohol precipitation solution through ethanol precipitation, and obtain the alfalfa polysaccharide after centrifugation; Among them, based on the weight of grass powder, the addition ratio of cellulase with an enzyme activity of 15000U/g is 1.5%-2.5%, the addition ratio of papain with an enzyme activity of 6000U/mg is 1.0%-3.0%, and the enzyme activity is 50U/g. The addition ratio of pectinase in g is 1.0%-3.0%; Wherein, the extraction temperature is 40-60° C., and the temperature is kept constant. 2. The method according to claim 1, characterized in that, the disodium hydrogen phosphate-citric acid buffer is added in an amount of 60-80 times the grass powder weight in the leaching step. 3. The method according to claim 2, characterized in that the pH of disodium hydrogen phosphate-citric acid buffer is 3.0-5.0. 4. The method according to claim 1, characterized in that the leaching time is 2.0-3.0h. 5. The method according to claim 4, characterized in that the polysaccharide extract is concentrated to 1/5 of the original volume at 60°C with a rotary evaporator, and then 4 times the volume of absolute ethanol is added, at 4°C Precipitation for 12h. 6. The method according to claim 5, characterized in that, the alcohol precipitation solution is centrifuged at 4000rpm/min for 10min, and the lower sediment is collected.