CN104988129A - Extraction method of efficient active enzymes of fig latex - Google Patents

Abstract

The invention discloses an extraction method of efficient active enzymes of fig latex. The method comprises the steps that acetone precipitation, gel chromatography separation, anion chromatography and vacuum freeze-drying are sequentially conducted on the fig latex which is pretreated at the temperature of 4 DEG C to obtain efficient active enzyme powder. Due to the fact that pretreatment and heat exchange are conducted and the extraction process is conducted under a low-temperature condition, the influence of conditions such as long-term high temperature on reducing enzyme activity can be effectively reduced; by means of milling action of a colloid mill, fig enzymes are dissolved in material liquid as many as possible, and the subsequent extraction is facilitated. By means of continuous separation through a disc-type centrifuge and a tubular bowl centrifuge, clear fig protease material liquid is obtained. The vacuum freezing and drying technology is adopted, due to the fact that when water in materials becomes ice, the water directly sublimates to be discharged, it is guaranteed that drying is conducted on the enzymes in the low-temperature condition, and the loss of enzyme mass and reducing of the enzyme activity in the drying process are reduced.

Description

A kind of high-efficiency active enzyme extraction method of fig milk

technical field

The invention relates to the field of food. Specifically, it relates to a method for extracting high-efficiency active enzymes from fig milk.

Background technique

Fig (Ficus carica Linn.) is a flowering plant belonging to the genus Moraceae, which mainly grows in some tropical and temperate regions and belongs to subtropical deciduous small trees. There are currently 800 known varieties of figs, most of which are evergreen varieties, and only those that grow in temperate regions are deciduous varieties. The fruit is bulbous with a small hole in the tail, and the pollen is spread by wasps. Because of its good medicinal value, it is more and more popular.

Ficin (Papain) widely exists in the stems, leaves and fruits of figs, and the content of immature fruit milk is the highest. It can be prepared by chemical methods (precipitation method) and biotechnology methods. Ficin is a plant protease, referred to as ficin, because of its good thermal stability, it has a very strong ability to decompose animal and plant proteins, fats, amides, etc., and can be widely used in medicine and health products, food, cosmetics, and chemical leather. and other industries and fields.

In recent years, there have been many reports on the extraction and application of ficin, but most of the crude enzymes used in the market are crude ficins. Because the extraction process of crude enzymes is simple and the production cost is low, hot air drying is generally used to obtain ficins. Vitality is only about 10000u/g; at the same time, due to the complex production process and high production cost of other refined ficinase, it is not conducive to the industrial production of refined ficinase, which also limits the application of ficinase in high-end medicine and health care products and beauty cosmetics. industry applications.

Contents of the invention

To solve the above problems, the present invention proposes a method for extracting high-efficiency active enzymes from fig milk.

In order to realize the foregoing invention object, the present invention adopts following technical scheme:

The present invention adopts following technical scheme:

A method for extracting high-efficiency active enzymes from fig milk. The pretreated fig milk is subjected to acetone precipitation, gel chromatography separation, anion chromatography, and vacuum freeze-drying to obtain high-efficiency active enzymes at a temperature capable of maintaining enzyme activity. Enzyme powder.

In actual research, it is found that ethanol has a poor precipitation effect on the active enzymes of fig milk, which cannot meet the requirements of high-efficiency extraction. Precipitating proteins with acetone must be carried out at low temperature, and protein precipitation is faster at lower temperatures. The action of acetone and water can destroy the hydration film of the protein, so that the protein can be separated in a certain concentration of organic solvent due to the difference in solubility. Compared with ethanol, the protein precipitation is faster and the effect is obvious at the same low temperature, and the protein precipitation is more thorough.

The temperature capable of maintaining enzyme activity is 4°C.

The present invention adopts anion exchange resin SefinoseFastFlow and gel filtration resin SephadexG-75 to separate hemolytic toxin protein, has the characteristics of fast flow rate, high resolution and high yield, and has few separation steps, and can be obtained only through two-step column chromatography separation The purer protease effectively shortens the separation time and reduces the loss of protease activity.

Further, the pretreatment conditions are as follows: collect fresh latex from the incision of fig branches, centrifuge the latex at 10,000-12,000 rpm for 30-40 minutes, take the supernatant and store it at 4-6°C to obtain the product.

Further, the specific steps of the acetone precipitation are as follows: the latex is precipitated in a 20-100% (v/v) acetone solution, centrifuged, the precipitate is removed and dried, and dissolved in 10-12mM, pH8.0-8.5 Tris- HCl buffer. The pKa value of Tris-HCl buffer is affected by the concentration of the buffer and the temperature of use, which can protect and participate in various enzyme reactions. The pKa value of the existing PBS solution will change with the dilution of the buffer, and it can inhibit the activity of most enzymes, including kinases, phosphorylases, dehydrogenases, etc.

Further, the separation conditions of the gel chromatography are as follows: the enzyme liquid after the acetone precipitation is passed through the gel chromatography column SephadexG-75 using 25-30mM, pH3-9, containing 5/1000 polyethylene glycol octyl Phenyl ether (w/v) was separated and purified in Tris-HCl buffer at a flow rate of 20-25mL/h to detect protein peaks. There were 2 protein peaks in total. The first protein solution was collected after enzyme activity detection. Polyethylene glycol octyl phenyl ether is a non-ionic surfactant, good lubricant, and can also play a role in separating enzyme components. But the concentration should be as low as possible to avoid decomposing enzyme protein. Therefore, the preferred content of polyethylene glycol octylphenyl ether in the present invention is 0.5% (w/v) of Tris-hydrochloric acid buffer.

Further, the gel chromatographic column Sephadex G-75 is replaced by Sephadex G-50, Sephadex G-100, Sephadex G-150.

Further, the specific steps of the anion chromatography are that the eluent collected by gel chromatography passes through an anion exchange column containing Sefinose FastFlow, and the eluent is purified and separated at a flow rate of 50-60mL/h, and the eluent is collected in the Concentrate with a 3-5K ultrafiltration tube at low temperature.

Further, the specific steps of vacuum freeze-drying are as follows: put the protease concentrated solution from anion chromatography into a vacuum freeze dryer or an ultra-low temperature refrigerator (freezer) to quickly freeze until the temperature is -30～-25°C; start vacuuming, Continue to maintain refrigeration for 2 to 2.5 hours; change refrigeration to heating, and slowly heat to increase the temperature of the feed liquid to 30 to 35°C to obtain the dried solid of ficin.

Beneficial effects of the present invention:

(1) Pretreatment, heat exchange and keeping the extraction process carried out under low temperature conditions can effectively reduce the impact of long-term high temperature and other conditions on the reduction of enzyme activity;

(2) Dissolving the fig enzyme into the feed liquid as much as possible through the grinding effect of the colloid mill is beneficial to the subsequent extraction.

(3) Obtain clarified ficinase feed solution through continuous separation of a disc centrifuge and a tube centrifuge.

(4) Vacuum freeze-drying technology is adopted to directly sublimate and drain the water in the material when it turns into ice, so as to ensure that the enzyme is carried out under low temperature conditions during the drying process, and reduce the loss of enzyme quality and enzyme activity during the drying process reduce.

(5) The ficin protease extracted by this method has high purity, high activity (enzyme activity 25700U/mg, final yield 9.25%), good thermal stability, and indicators such as appearance, physicochemical, and microbial limits all reach or exceed relevant food and drug at home and abroad Standards, products of different specifications can be produced through modulation, and can be widely used in industries and fields such as medicine and health products, food, beauty cosmetics, and chemical leather.

(6) The method has simple process, low production cost, high yield and can be industrialized.

(7) in the present invention, synergistic cooperation and mutual promotion between each step have realized the synchronous improvement of ficin purity, vigor and extraction rate. From the characteristics of ficin, select the corresponding extraction process, and in conjunction with the characteristics of the previous step of the extraction process, The subsequent extraction method is selected and adjusted to maximize the extraction efficiency.

Detailed ways

The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. For the experimental methods not indicating specific conditions in the following examples, select according to conventional methods and conditions.

Example 1

Fresh latex was collected from the incision of fig branches, and the latex was centrifuged at 10,000 rpm for 30 minutes to remove gum and other insoluble components. The remaining clear liquid was natural latex, which was stored at 4°C for enzyme purification. All subsequent steps were carried out at 4°C to prevent enzyme degradation and depolymerization.

The protein content was determined by the Coomassie brilliant blue method, and bovine serum albumin was used as the standard curve.

For the detection of enzyme activity, tyrosine was used as the standard curve, casein was used as the substrate, and Folin’s phenol reagent was used for color reaction.

1. Acetone precipitation method

Latex is first precipitated with acetone. The enzymes in the latex were precipitated in 20–40% (v/v) acetone solution, and then centrifuged at 10,000 rpm. The enzyme was vacuum-dried to remove acetone and dissolved in 10 mM Tris-HCl buffer (pH 8.0). Determine protein concentration. The enzyme yield was 85.4% (based on the clarified latex liquid protein after centrifugation above). At this time 15200U/mg.

2. Chromatographic separation

The enzyme solution is filtered through a Sephadex G-75 (2cm*90) column to remove impurities and inorganic salts. Equilibrium buffer solution A (25mM Tris-hydrochloric acid buffer solution, pH7.6.0, containing 5/1000 polyethylene glycol octylphenyl ether). The sample was passed through the column using the same buffer A at a speed of 25 mL/h, and the protein content (absorbance at 595 nm) was measured at the same time, and the protein yield was 25%. Detect protein peaks, a total of 2 protein peaks, after enzyme activity detection, collect the first protein solution. At this time, the enzyme activity was 17650U/mg.

3. Anion chromatography

Collect the protein eluate with combined active ingredients, put it on Sefinose Fast Flow anion exchange chromatography column to continue separation, use buffer B (25mM Tris-hydrochloric acid buffer, pH7.6), flush the column, flow rate 50mL/h. The enzyme activity is 25700U/mg, and the final yield is 9.25%.

Quaternary ammonium salt anion column purification removes metal cations and further purifies the enzyme.

4. Vacuum freeze-drying to obtain enzyme powder.

Example 2

The specific extraction steps are the same as in Example 1, except that the Sephadex G-50 column is used for filtration in step 2. The enzyme activity is 25600U/mg, and the final yield is 9.15%.

Example 3

The specific extraction steps are the same as in Example 1, except that the Sephadex G-100 column is used for filtration in step 2. The enzymatic activity of the finally extracted ficin was 25650U/mg, and the final yield was 9.21%.

Example 4

The specific extraction steps are the same as in Example 1, except that the Sephadex G-150 column is used for filtration in step 2. The enzymatic activity of the finally extracted ficin was 25760U/mg, and the final yield was 9.22%.

Example 5

The specific extraction steps are the same as in Example 1, except that the Sephadex G-150 column is used for filtration in step 2. The enzymatic activity of the finally extracted ficin was 25770U/mg, and the final yield was 9.26%.

Example 6

The specific extraction steps are the same as in Example 1. The difference is that fresh latex is collected from the cut of the fig branch, and the latex is centrifuged at 10,000 rpm for 30 minutes to remove gum and other insoluble components. The remaining clear liquid is natural latex, and the pH value of the latex is adjusted. 6.6, store at 5°C for enzyme purification. All subsequent steps were carried out at 5°C to prevent enzyme degradation and depolymerization. The enzyme activity of the finally extracted ficin was 25870U/mg, and the final yield was 9.29%.

Although the specific implementation of the present invention has been described above, it is not a limitation to the protection scope of the present invention. Those skilled in the art should understand that on the basis of the technical solution of the present invention, those skilled in the art can do it without creative work. Various modifications or deformations are still within the protection scope of the present invention.

Claims (7)

1. A method for extracting high-efficiency active enzymes from fig milk, characterized in that, the fig milk after pretreatment is successively subjected to acetone precipitation, gel chromatography separation, anion chromatography, and vacuum freezing at a temperature that can keep enzyme activity. After drying, high-efficiency active enzyme powder is obtained. 2. method as claimed in claim 1, is characterized in that, described pretreatment condition is: collect fresh latex from fig branch otch, latex is centrifugal 30～35min under 10000-12000rpm, gets supernatant and preserves in 4～30min. 6 ℃, that is. 3. The method according to claim 1, characterized in that, the specific steps of the acetone precipitation are: latex is precipitated in 20-100% (v/v) acetone solution, centrifuged, desettled and dried, dissolved in 10-15mM, pH8.0-8.5 Tris-HCl buffer. 4. the method for claim 1, is characterized in that, the condition of described gel chromatography separation is: the enzyme liquid after acetone precipitation adopts 25-30mM, pH3-9 through gel chromatographic column Sephadex G-75 , Tris-hydrochloric acid buffer solution containing five thousandths of polyethylene glycol octylphenyl ether (w/v) was separated and purified at a flow rate of 20-25 mL/h, and the eluate of the first protein peak was collected. 5. method as claimed in claim 4, is characterized in that, described gel chromatographic column Sephadex G-75 replaces with SephadexG-50, Sephadex G-100, Sephadex G-150 type. 6. method as claimed in claim 1, is characterized in that, the concrete step of described anion chromatography is that the eluent collected by gel chromatography contains Sefinose Fast Flow anion exchange column, adopts eluent with 50- Purify and separate at a flow rate of 60mL/h, collect the eluate and concentrate it with a 3-5K ultrafiltration tube at low temperature. 7. The method according to claim 1, characterized in that, the specific steps of vacuum freeze-drying are: putting the protease concentrated solution of anion chromatography into a vacuum freeze dryer or an ultra-low temperature freezer until the temperature is -30 ~-25°C; start vacuuming, and continue to maintain refrigeration for 2 to 2.5 hours; change refrigeration to heating, and slowly heat to increase the temperature of the feed liquid to 30 to 35°C to obtain the dried solid of ficin.