CN110684070B - A method of extracting cholesterol from tripe - Google Patents

Abstract

The invention belongs to the technical field of cholesterol extraction, and particularly relates to a method for extracting cholesterol from beef tripe, which comprises the following steps: the method has the characteristics of high extraction rate, high purity, few process steps and no need of expensive precision equipment, is simple and convenient, avoids the use of precious membrane products such as nanofiltration, ultrafiltration membranes and the like or precision equipment such as silica gel column separation and the like, and has simple separation process and low requirement.

Description

A method of extracting cholesterol from tripe

technical field

The invention belongs to the technical field of cholesterol extraction, in particular to a method for extracting cholesterol from tripe.

Background technique

Tripe is the stomach of cattle, which is one of the by-products of cattle. There are four stomachs in cattle, namely abomasum, reticulum, rumen and ovum. Among them, the rumen, the reticulum and the ovum are formed by the variation of the esophagus of cattle, while the abomasum is the true stomach. Tripe can be used as a dish after soaking in alkaline water; at the same time, the famous dishes such as "Tripe Hot Pot" and "Husband Lung Slices" use the rumen to tear off the beef serosal membrane and eat them. The application of the reticulum is the same as that of the rumen, and the ovum is also called the louver stomach because it has many leaves inside, and the leaves are arranged alternately. Ovum and abomasum can be shredded and eaten as a dish. The most commonly used tripe is the belly collar and the louver. Its main protein is collagen. Collagen has a unique triple helix structure, which can keep the molecular structure of collagen stable. Tripe is crisp and delicious, rich in nutrients, and the nutrient content is shown in Table 1:

Table 1

Cholesterol, also known as cholesterol, is an indispensable and important component of animal tissue cells. Its main physiological functions are to form cell membranes, form bile acids, and synthesize hormones. Cholesterol has a wide market demand and is mainly used in the industry as raw materials for artificial bezoar, vitamin D3 and synthetic hormones, and is also the main raw material for cosmetics and liquid crystals. Cholesterol is mainly extracted from animal brain and lanolin, and there is currently no literature report on the extraction of cholesterol from tripe; the method of extraction with organic reagents is usually used, which has long immersion time and low extraction cholesterol yield (≤90%). ), and the defects of residual toxic organic solvents and large amounts of organic reagents.

SUMMARY OF THE INVENTION

In order to solve the above-mentioned technical problems, the present invention provides a method for extracting cholesterol from tripe.

Specifically, this is achieved through the following technical solutions:

A method for extracting cholesterol from tripe, comprising the following steps:

1) Ball milling treatment: the tripe and the pretreated compound enzyme solution are placed in a ball mill at a mass ratio of 1:(1-2) and ground at a rate of 300-500 rev/min for 10-20mins;

2) Ultrasonic treatment: the ball milled product is placed under the condition that the power is 100-120W and the frequency is 20-40kHz and the ultrasonic treatment is performed for 10-20min;

3) Hydrolysis: add the modified diatomite of 1-5% of the mass of the ultrasonic treatment and (2-4) times of distilled water to the ultrasonic treatment result, and then treat it in a constant temperature water bath for 30-45min;

4) Filtration and separation: remove the hydrolyzed diatomite and elute 2-3 times with ethanol with a mass concentration of 40-60%. In each elution process, the amount of ethanol is (2-3) times the mass of diatomite. Add magnetic adsorbent material to the deliquoring, stir at 1000-2000 r/min for 5-10 min, filter, take the filtrate and place it in a centrifuge, stir at 5000-10000 r/min for 3-5 min, and dry. .

The compound enzyme solution includes pectinase, xylanase, mannanase, mica powder, potassium permanganate; wherein, the activity of the polygalacturonase in the compound enzyme solution is: 1200u/ml-1500u/ml, xylanase activity is 2000u/ml-3000u/ml, mannanase activity is 800u/ml-1200u/ml, mica powder is 20-30μg/ml, permanganate Potassium 1-4 μg/ml.

The preparation method of the composite enzyme solution is as follows: taking pectinase, xylanase and mannanase, placing them in an aqueous solution, stirring magnetically for 60-120s at a temperature of 30-40° C., Add mica powder and potassium permanganate, and stir at a speed of 300-500 rpm for 3-7min.

For the pretreated composite enzyme solution, the composite enzyme solution is placed under the condition of a constant intensity magnetic field of 1-3T for 10-15s, and then borneol with a mass of 0.1-0.3% of the composite enzyme solution is added and stirred evenly.

The working conditions of the constant temperature water bath are as follows: the temperature is 30-45°C.

The modified diatomite is to mix and react chitosan and copper gluconate at a temperature of 45-65°C for 10-15min, then add diatomite to mix, and process at a steam temperature of 120-140°C 10-15min, then hold for 10-15min.

The mass ratio of chitosan, copper gluconate and diatomite in the modified diatomite is (23-27):1:(73-77).

The magnetic adsorption material is obtained by mixing magnetic biochar and ferric tetroxide in a mass ratio of (13-23):1.

Beneficial effects:

The method of the invention has the characteristics of high extraction rate, high purity, few process steps, and no need for expensive precision equipment. Use, the separation process is simple and low in requirements.

In this application, the ball milling treatment is used to make the compound enzyme liquid penetrate into the tripe, and the rotation speed of the ball mill can be controlled, which can generate mechanical heat, which in turn helps to promote penetration and improve enzyme activity; if the rotation speed is too large, the enzyme system will be damaged; Too small and not enough heat to penetrate. Combined with the action of the pretreated compound enzyme solution, borneol can not only promote penetration, but also enable potassium permanganate and mica powder to penetrate before the compound enzyme system, thereby making the tripe sterilized and ensuring the effect of the compound enzyme. The powder is subjected to the action of a constant-strength magnetic field and is magnetic, which can carry potassium permanganate to penetrate first, and at the same time adsorb the components that are not conducive to the growth of the compound enzyme, so that the components in the tripe that affect the action of the compound enzyme are adsorbed and separated from the compound enzyme.

The present application uses ultrasonic cavitation to destroy the tissue structure of tripe, so that the composite enzyme can enter the tripe through the destroyed channel, and is separated from the mica powder, so that the composite enzyme can better destroy the stable structure of collagen.

The application uses modified diatomite for hydrolysis, which can not only dissolve cholesterol, but also form physical and chemical adsorption with cholesterol. At the same time, the copper gluconate contained in the application can catalyze the enzymatic hydrolysis system, thereby making the enzymatic hydrolysis system stable and improving the extraction of cholesterol. Rate.

The modified diatomite used in this application is a porous solid, which has physical adsorption and chemical adsorption on cholesterol. In the process of physical adsorption, the surface of diatomite and cholesterol first form single molecules, and then change to It has become a multi-molecular layer adsorption, and the chemical adsorption of cholesterol is mainly based on the combination of active groups on the surface and cholesterol molecules.

The present application utilizes the magnetic adsorption material, which can adsorb the organic solvent and compound enzyme liquid of crops attached to cholesterol, improve the safety of cholesterol, and ensure the physiological activity and quality of cholesterol.

The applicant uses modified diatomite to carry out adsorption verification on cholesterol, and the experiment is as follows:

Weigh 0.1 g of modified diatomite samples, soak them in glacial acetic acid with a mass of 40 times, respectively, for 2 hours, and then remove the glacial acetic acid by suction filtration to obtain the samples. The membrane was sealed, shaken and adsorbed at 30 °C for 90 min. After suction filtration, 0.5 mL of filtrate was drawn, and 3.5 mL of glacial acetic acid was added. After complete mixing, 2 mL of jarosite color developing solution was added along the tube wall. Colorimetric at 560nm wavelength, measure its absorbance, and calculate the quality of the cholesterol solution, and then calculate the adsorption amount according to the formula of "Q=V(A-B/V)/m", where: Q is the adsorption amount, mg/g; V is the volume of the cholesterol solution (that is, the volume of the filtrate absorbed during the reaction), mL; m is the dry weight of the modified diatomite sample, g; A is the mass concentration of the cholesterol solution before adsorption, mg/mL; B is the adsorption equilibrium The mass of cholesterol, mg; at the same time, unmodified diatomite and chitosan were separately weighed as the control group. The results are shown in Table 1:

Table 1

Modified Diatomaceous Earth Unmodified diatomaceous earth Chitosan Adsorption capacity mg/g 19.93 12.15 9.67

Detailed ways

The specific embodiments of the present invention are described in further detail below, but the present invention is not limited to these embodiments, and any improvement or substitution in the basic spirit of the present embodiment still belongs to the scope of protection of the claims of the present invention.

Example 1

A method for extracting cholesterol from tripe, comprising the following steps:

1) ball milling treatment: the tripe and the pretreated compound enzyme solution are placed in a ball mill at a mass ratio of 1:2 and ground at a rate of 500 rev/min for 20mins;

2) Ultrasound treatment: the ball milled product was placed under the condition that the power was 120W and the frequency was 40kHz and the ultrasonic treatment was carried out for 20min;

3) Hydrolysis: adding the modified diatomite of 5% by mass of the ultrasonic treatment and 4 times of distilled water to the ultrasonic treatment result, and then treating it in a constant temperature water bath for 45 minutes;

4) Filtration and separation: remove the hydrolyzed diatomite and elute 3 times with ethanol with a mass concentration of 60%. In each elution process, the amount of ethanol used is 3 times the mass of diatomite, and a magnetic adsorption material is added to the eluent, After stirring for 10 minutes at a speed of 2000 rev/min, filter, take the filtrate, place it in a centrifuge, stir at a speed of 10,000 rev/min for 5 min, and then dry it;

The compound enzyme solution includes pectinase, xylanase, mannanase, mica powder, potassium permanganate; wherein, the activity of the polygalacturonase in the compound enzyme solution is: 1500u/ml, the activity of xylanase is 3000u/ml, the activity of mannanase is 1200u/ml, the mica powder is 30μg/ml, and the potassium permanganate is 4μg/ml;

The preparation method of the compound enzyme solution is as follows: take pectinase, xylanase and mannanase, put them in an aqueous solution, stir magnetically for 120s at a temperature of 40°C, and then add mica powder, Potassium permanganate was stirred at a rate of 500 rpm for 7 min;

For the pretreated composite enzyme solution, the composite enzyme solution is placed under the condition of a constant-strength magnetic field of 3T for 15s, and then borneol with a mass of 0.3% of the composite enzyme solution is added and stirred evenly;

The working conditions of the constant temperature water bath are: the temperature is 45°C;

The modified diatomite is to mix and react chitosan and copper gluconate at a temperature of 65°C for 15min, then add diatomite to mix, treat at a steam temperature of 140°C for 15min, and then keep the temperature for 15min;

The mass ratio of chitosan, copper gluconate and diatomite in the modified diatomite is 27:1:77;

The magnetic adsorption material is a mixture of magnetic biochar and ferric tetroxide in a mass ratio of 23:1.

Example 2

A method for extracting cholesterol from tripe, comprising the following steps:

1) Ball milling treatment: the tripe and the pretreated compound enzyme solution are placed in a ball mill at a mass ratio of 1:1 and ground at a rate of 300 rev/min for 10 mins;

2) Ultrasonic treatment: the ball milled product is placed under the condition that the power is 100W and the frequency is 20kHz and the ultrasonic treatment is performed for 10min;

3) Hydrolysis: adding the modified diatomite of 1% by mass of the ultrasonic treatment and 2 times of distilled water to the ultrasonic treatment result, and then treating it in a constant temperature water bath for 30 min;

4) Filtration and separation: remove the hydrolyzed diatomite and elute twice with ethanol with a mass concentration of 40%. In each elution process, the amount of ethanol used is twice the mass of diatomite, and a magnetic adsorption material is added to the eluent, After stirring for 5 minutes at a speed of 1000 rev/min, filter, take the filtrate, place it in a centrifuge, stir for 3 min at a speed of 5000 rev/min, and dry it;

The compound enzyme solution includes pectinase, xylanase, mannanase, mica powder, potassium permanganate; wherein, the activity of the polygalacturonase in the compound enzyme solution is: 1200u/ml, the activity of xylanase is 2000u/ml, the activity of mannanase is 800u/ml, the mica powder is 20μg/ml, and the potassium permanganate is 1μg/ml;

The preparation method of the composite enzyme solution is as follows: taking pectinase, xylanase and mannanase, placing them in an aqueous solution, stirring magnetically for 60s at a temperature of 30° C., then adding mica powder, Potassium permanganate, stirred for 3 min at a rate of 300 rpm;

For the pretreated composite enzyme solution, the composite enzyme solution is placed under the condition of a constant-strength magnetic field of 1T for 10s, then borneol with 0.1% mass of the composite enzyme solution is added, and the mixture is evenly stirred;

The working conditions of the constant temperature water bath are: the temperature is 30°C;

The modified diatomite is to mix and react chitosan and copper gluconate at a temperature of 45°C for 10 minutes, then add diatomite to mix, treat at a steam temperature of 120°C for 10 minutes, and then keep the temperature for 10 minutes;

The mass ratio of chitosan, copper gluconate and diatomite in the modified diatomite is 23:1:73;

The magnetic adsorption material is a mixture of magnetic biochar and ferric tetroxide in a mass ratio of 13:1.

Example 3

A method for extracting cholesterol from tripe, comprising the following steps:

1) Ball milling treatment: the tripe and the pretreated compound enzyme solution are placed in a ball mill at a mass ratio of 1:1.5 and ground at a rate of 400 rev/min for 15 mins;

2) Ultrasound treatment: the ball milled product was placed under the condition of power of 110W and frequency of 30kHz for ultrasonic treatment for 15min;

3) Hydrolysis: adding the modified diatomite of 3% by mass of the ultrasonic treatment and 3 times of distilled water to the ultrasonic treatment result, and then treating it in a constant temperature water bath for 38 minutes;

4) Filtration and separation: remove the hydrolyzed diatomite and elute twice with 50% ethanol. In each elution process, the amount of ethanol used is 2.5 times the mass of diatomite, and a magnetic adsorption material is added to the eluate. After stirring for 8 minutes at a speed of 1500 rev/min, filter, take the filtrate, place it in a centrifuge, stir at a speed of 8000 rev/min for 4 min, and dry it;

The compound enzyme solution includes pectinase, xylanase, mannanase, mica powder, potassium permanganate; wherein, the activity of the polygalacturonase in the compound enzyme solution is: 1300u/ml, xylanase activity is 2500u/ml, mannanase activity is 1000u/ml, mica powder is 25μg/ml, potassium permanganate is 2μg/ml;

The preparation method of the composite enzyme solution is as follows: taking pectinase, xylanase and mannanase, placing them in an aqueous solution, stirring magnetically for 90s at a temperature of 35° C., then adding mica powder, Potassium permanganate, stirred for 5 min at a rate of 400 rpm;

For the pretreated composite enzyme solution, the composite enzyme solution is placed under the condition of a constant-strength magnetic field of 2T for 12 s, and then borneol with a mass of 0.2% of the composite enzyme solution is added, and the mixture is evenly stirred;

The working conditions of the constant temperature water bath are: the temperature is 37°C;

In the modified diatomite, chitosan and copper gluconate are mixed and reacted at a temperature of 55° C. for 12 minutes, then diatomite is added for mixing, treated at a steam temperature of 130° C. for 12 minutes, and then kept for 12 minutes;

The mass ratio of chitosan, copper gluconate and diatomite in the modified diatomite is 25:1:75;

The magnetic adsorption material is a mixture of magnetic biochar and ferric tetroxide in a mass ratio of 18:1.

Example 4

A method for extracting cholesterol from tripe, comprising the following steps:

1) Ball milling treatment: the tripe and the pretreated compound enzyme solution are placed in a ball mill at a mass ratio of 1:1 and ground at a rate of 500 rev/min for 10 mins;

2) Ultrasonic treatment: the ball milled product is placed under the condition that the power is 100W and the frequency is 40kHz and the ultrasonic treatment is carried out for 20min;

3) Hydrolysis: adding the modified diatomite of 5% by mass of the ultrasonic treatment result and 3 times of distilled water to the ultrasonic treatment result, and then treating it in a constant temperature water bath for 30 minutes;

4) Filtration and separation: remove the hydrolyzed diatomite and elute twice with ethanol with a mass concentration of 60%. In each elution process, the amount of ethanol used is 3 times the mass of diatomite, and a magnetic adsorption material is added to the eluent, After stirring for 10 minutes at a speed of 2000 rev/min, filter, take the filtrate, place it in a centrifuge, stir for 3 min at a speed of 5000 rev/min, and dry it;

The compound enzyme solution includes pectinase, xylanase, mannanase, mica powder, potassium permanganate; wherein, the activity of the polygalacturonase in the compound enzyme solution is: 1200u/ml, the activity of xylanase is 3000u/ml, the activity of mannanase is 1100u/ml, the mica powder is 25μg/ml, and the potassium permanganate is 3μg/ml;

The preparation method of the composite enzyme solution is as follows: taking pectinase, xylanase and mannanase, placing them in an aqueous solution, stirring magnetically for 60s at a temperature of 40°C, then adding mica powder, Potassium permanganate was stirred at a rate of 300 rpm for 7 min;

For the pretreated composite enzyme solution, the composite enzyme solution is placed under the condition of a constant-strength magnetic field of 3T for 10s, and then borneol with a mass of 0.3% of the composite enzyme solution is added and stirred evenly;

The working conditions of the constant temperature water bath are: the temperature is 40°C;

In the modified diatomite, chitosan and copper gluconate are mixed and reacted at a temperature of 55° C. for 10 minutes, then diatomite is added for mixing, treated at a steam temperature of 120° C. for 10 minutes, and then kept for 15 minutes;

The mass ratio of chitosan, copper gluconate and diatomite in the modified diatomite is 23:1:76;

The magnetic adsorption material is a mixture of magnetic biochar and ferric oxide in a mass ratio of 19:1.

Comparative Example 1

The difference from Example 3 is that borneol was not added to the pretreated composite enzyme solution.

Comparative Example 2

The difference from Example 3 is that the composite enzyme solution does not include mica powder.

Comparative Example 3

The difference from Example 3 is that copper gluconate is not included in the modified diatomaceous earth.

Comparative Example 4

The difference from Example 3 is that it is not subjected to ultrasonic treatment.

Comparative Example 5

The difference from Example 3 is that no magnetic adsorption material is added during the separation and filtration process.

Test Example 1

Weigh the cholesterol extraction amount in the gains of Example and Comparative Examples 1-4, and the test is as follows:

1.1 Preparation of reference substance: Precisely weigh about 5.0 mg of cholesterol standard substance, place it in a 100-mL volumetric flask, add an appropriate amount of methanol for ultrasonic dissolution, and adjust the volume to the mark with methanol, that is, the concentration of cholesterol reference substance solution is 0.05 mg/mL;

1.2 Preparation of the test solution: Take 10g of the obtained products of Examples and Comparative Examples 1-4, dissolve them with an appropriate amount of methanol, put them in a 10mL volumetric flask, adjust the volume of methanol to the mark, and shake well, that is, the test solution;

1.3 Chromatographic conditions: the chromatographic column is AgilentTC-C18 (250mm×4.6mm, 5μm), the mobile phase is methanol:water (V/V, 98:2), the flow rate is 1mL/min, the detection wavelength is 205nm, and the column temperature is 30 ℃, the injection volume was 10 μL. The number of theoretical plates calculated according to cholesterol is not less than 3000, and under this condition, the separation degree of cholesterol and other substances is greater than 1.5;

1.4 Test method: Precisely draw 10 μL each of the reference substance and the test solution, inject them into a liquid chromatograph, measure the content, record the chromatographic peak area, and calculate the cholesterol content in the test solution according to the external standard method. The results are shown in Table 2:

Table 2

project Example 1 Example 2 Example 3 Example 4 Contentmg/100g 95.3 94.8 97.6 95.9 project Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Contentmg/100g 90.3 88.7 92.6 86.4

Claims (4)

1. A method for extracting cholesterol from beef tripe is characterized by comprising the following steps:

1) ball milling treatment: mixing beef tripe and the pretreated compound enzyme solution according to the proportion of 1: the mass ratio of (1-2) is put into a ball mill and ground for 10-20mins at the speed of 300-500 r/min;

2) ultrasonic treatment: subjecting the ball-milled product to ultrasonic treatment for 10-20min under the conditions of power of 100-120W and frequency of 20-40 kHz;

3) hydrolysis: adding modified diatomite with the mass of 1-5% of that of the product obtained by ultrasonic treatment and distilled water with the mass of 2-4 times of that of the product obtained by ultrasonic treatment into the product obtained by ultrasonic treatment, and then carrying out constant-temperature water bath treatment for 30-45 min;

4) filtering and separating: removing hydrolyzed diatomite, eluting with 40-60% ethanol for 2-3 times, wherein the amount of ethanol is 2-3 times of the diatomite in each elution process, adding magnetic adsorption material into the eluate, stirring at the speed of 1000-;

the compound enzyme solution comprises pectin polygalacturonase, xylanase, mannase, mica powder and potassium permanganate; wherein the activity of polygalacturonase in the compound enzyme solution is 1200-1500 u/ml, the activity of xylanase is 2000-3000 u/ml, the activity of mannase is 800-1200 u/ml, the activity of mica powder is 20-30 mu g/ml, and the activity of potassium permanganate is 1-4 mu g/ml.

The preparation method of the compound enzyme solution comprises the following steps: placing pectin polygalacturonase, xylanase and mannase in water solution, magnetically stirring at 30-40 deg.C for 60-120s, adding mica powder and potassium permanganate, and stirring at 300-500 rpm for 3-7 min;

the pretreated compound enzyme solution is prepared by treating the compound enzyme solution for 10-15s under the condition of a constant-intensity magnetic field of 1-3T, adding borneol accounting for 0.1-0.3% of the mass of the compound enzyme solution, and uniformly stirring;

the modified diatomite is prepared by mixing chitosan and copper gluconate at 45-65 deg.C for reaction for 10-15min, adding diatomite, mixing, treating at 120-140 deg.C for 10-15min, and maintaining the temperature for 10-15 min.

2. The method for extracting cholesterol from beef tripe according to claim 1, wherein the working conditions of the thermostatic water bath are as follows: the temperature is 30-45 ℃.

3. The method for extracting cholesterol from beef tripe according to claim 1, wherein the modified diatomite comprises chitosan, copper gluconate and diatomite in a mass ratio of (23-27) to 1 (73-77).

4. The method for extracting cholesterol from beef tripe according to claim 1, wherein the magnetic adsorption material is formed by mixing magnetic biochar and ferroferric oxide according to the mass ratio of (13-23) to 1.