CN114656578A

CN114656578A - Method for preparing high-ester pectin rich in RG-I by enzyme-assisted method

Info

Publication number: CN114656578A
Application number: CN202210427688.6A
Authority: CN
Inventors: 齐军茹; 刘紫薇
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2022-04-22
Filing date: 2022-04-22
Publication date: 2022-06-24

Abstract

The invention belongs to the technical field of pectin, and discloses a method for preparing RG-I-rich high-ester pectin by an enzyme-assisted method. The method comprises the following steps: 1) mixing the orange peel with enzyme activating solution, performing enzymolysis treatment, and inactivating enzyme; 2) adding acid into the solution after enzyme deactivation to adjust the pH value to 1.5-3, stirring and extracting for 1-3 hours at the temperature of 65-80 ℃, and centrifuging; 3) precipitating the centrifuged supernatant with ethanol, and drying the precipitate to obtain high-ester pectin; the enzyme activation solution consists of enzyme and buffer solution; the enzyme is at least one of xylanase and cellulase. The method shortens the acid treatment time, reduces the equipment loss, and greatly improves the product yield and the extraction efficiency; the pectin prepared by the method disclosed by the invention not only retains a high esterification degree, but also retains a relatively more complete RG-I structure domain, the RG-I content is 30-45%, the esterification degree is 70-85%, the viscosity is higher, and the gelling performance is good.

Description

Method for preparing high-ester pectin rich in RG-I by enzyme-assisted method

Technical Field

The invention belongs to the technical field of pectin production, relates to a pectin extraction method, and particularly relates to a method for preparing RG-I-rich high-ester pectin by an enzyme-assisted method.

Background

Citrus fruits are the most consumed fruits in the world, and in addition to direct consumption, citrus is used for producing freshly squeezed juice, citrus beverages, jelly, and the like. According to the adopted technology and different fruit varieties, the yield of the processed citrus peel waste is about 50-70% of the yield of the processed fruits, and the annual yield in the world can be close to 1000 ten thousand tons. Pectin is one of main functional components in citrus peel, the functional components extracted from the citrus peel can greatly improve the added value of citrus processed products, and the method is a commonly used citrus peel waste treatment mode.

Pectin is a natural biological macromolecule with a complex structure, has high structural diversity, is mainly present in the intercellular layer of the cell wall of a dicotyledonous plant, and has close relation with the connection among cells, the porosity of the cell wall and the regulation and control of the ionic environment in the cell wall. Pectin is widely used in the food, pharmaceutical and cosmetic industries due to its good emulsifying, thickening, stability and gelling properties. In recent years, pectin has received much attention for its physiological health activity, because it can reduce the risk of cancer, diabetes, obesity, regulate cholesterol and blood glucose and maintain intestinal health. The high-ester pectin refers to pectin with an esterification degree of more than 50%, and the morphological structure of the high-ester pectin is closest to that of natural pectin molecules in industrially produced pectin. The esterification degree of pectin has important influence on the structure and functional properties of pectin, and high-ester pectin, which is a common type in pectin production, has good application prospect due to the natural structure. Pectin is present in the primary cell wall and the middle lamella of the plant, and protopectin is embedded in the network formed by the mutual cross-linking of cellulose and cellulose to form the load-bearing structure of the cell wall. Extracting pectin is the process of hydrolyzing cell walls, dissolving protopectin, and further extracting pectin. If the degradation of pectin is reduced in the whole extraction process, the complete structure of the pectin is kept to the maximum extent, and the pectin has good performance and can be well applied to various fields related to food.

Pectin contains rhamnogalacturonan (rhamnose and galacturonic acid are alternately connected to form RG-I main chain), and pectin rich in RG-I has effects in preventing cancer by inducing cancer cell apoptosis, and has potential blood sugar control function, immune activity and prebiotic property. Therefore, how to scientifically extract pectin with complete molecular configuration is worthy of research.

At present, common extraction methods of pectin include an acid extraction method, an alkali extraction method, an enzyme extraction method and the like, but the acid extraction method and the alkali extraction method generally use strong acid and strong alkali, have long extraction time, high extraction temperature, strong corrosiveness on equipment, and great pollution of produced wastewater to the environment, and the enzyme extraction method generally has low extraction rate and low quality of extracted pectin because enzymes cannot completely decompose cell walls.

One of the problems to be solved is how to prepare high-ester pectin which is rich in RG-I structure, good in quality and high in yield.

Disclosure of Invention

Aiming at the defects and shortcomings of the prior art, the invention aims to provide a method for preparing RG-I-rich high-ester pectin by an enzyme-assisted method. The pectin obtained by the method is citrus high-ester pectin which is rich in RG-I structure, good in quality and high in yield. The method has low energy consumption and low pollution.

In order to achieve the purpose, the invention provides the following technical scheme:

a method for preparing RG-I-rich high-ester pectin by an enzyme-assisted method comprises the following steps:

(1) mixing the orange peel with an enzyme activating solution, carrying out enzymolysis treatment at 30-55 ℃ for 0.5-3 hours, and inactivating the enzyme;

(2) adding acid into the solution after enzyme deactivation in the step (1) to adjust the pH value to 1.5-3, stirring and extracting for 1-3 hours at the temperature of 65-80 ℃, and centrifuging;

(3) and (3) carrying out alcohol precipitation on the supernatant obtained after centrifugation in the step (2), and drying the precipitate to obtain the high-ester pectin.

The precipitating agent for alcohol precipitation in the step (3) is an ethanol solution; the volume ratio of the supernatant to the ethanol solution is 1: 1-1: 3. the alcohol precipitation time is 0.5-1.5 hours. The volume fraction of the ethanol solution is not less than 95%.

The number of times of alcohol precipitation is 1-3.

And (3) when the ethanol precipitation is carried out for multiple times, mixing the supernatant obtained after the centrifugation in the step (2) with a precipitator for ethanol precipitation, separating out precipitates, then continuously mixing the precipitates with the precipitator for separating out the precipitates, carrying out the ethanol precipitation for multiple times, and finally drying the precipitates.

The separation refers to centrifugal separation or suction filtration. The centrifugal separation is carried out for 15-30 min under the condition of 4000-8000 r/min, and the suction filtration is vacuum filtration.

The citrus peel in the step (1) is one or more of lemon peel, orange peel, shaddock peel and the like.

The enzyme activating solution in the step (1) consists of enzyme and buffer solution, wherein the buffer solution is sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution, and the pH value of the sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution is 4-6; the mass volume ratio of the enzyme to the enzyme activation liquid is (0.0015-0.0075) g to (20-40) mL, the mass volume ratio of the citrus peel to the enzyme activation liquid is 1g to (20-40) mL, and the mass ratio of the enzyme to the citrus peel in the enzyme activation liquid is (0.15-0.75) to 100.

The enzyme is at least one of xylanase and cellulase

In the step (2), the acid is one of nitric acid, hydrochloric acid, sulfuric acid, oxalic acid and citric acid.

The centrifugation in the step (2) is performed for 10-30 min under the condition of 5000-8000 r/min.

And (3) the alcohol precipitation in the step (3) is to add the supernatant into an ethanol solution and to perform standing precipitation.

The drying in the step (3) is forced air drying, rake drying or fluidized bed drying. The drying temperature is 50-60 ℃, and the drying time is 0.5-4 h.

The esterification degree of the pectin prepared by the method is 70-85%. The proportion (mass ratio) of the pectin RG-I area prepared by the method is 30-45%. The pectin yield is 20-30%.

The invention breaks the covalent link between pectin and cellulose and hemicellulose in the cell wall through enzyme, separates the pectin in the primary cell wall and the middle lamella of the plant cell wall from the cell wall, and releases the pectin in the cell wall to a certain extent. The xylanase is adopted to act on the hemicellulose in the cell wall, covalent bonds between the pectin in the RG-I region and the cellulose and the hemicellulose are hydrolyzed, the RG-I region structure of the pectin is more reserved, and the high-ester pectin rich in RG-I is extracted.

According to the invention, the high-ester pectin (the esterification degree is more than or equal to 50%) prepared by processing the citrus peel by adopting an enzyme-assisted acid method is subjected to enzymolysis time, temperature and pH control, covalent cross-linking between an RG-I region of the pectin and cellulose and hemicellulose in cell walls is destroyed, the network structure of the cell walls is bulked, the contact area between a solvent and a matrix is increased, the acid extraction time, the temperature and the pH are controlled, and the pectin is extracted by adopting inorganic acid or organic acid.

Compared with the prior art, the extraction method and the obtained product have the following advantages:

(1) the method of the invention reserves RG-I area structure of pectin more, extracts high ester pectin rich in RG-I;

(2) the pectin is extracted from the raw material by acid after the citrus peel is treated by enzyme, and more protopectin is converted into soluble pectin to be extracted. The pectin yield can be significantly improved by an enzyme-assisted acid extraction method.

(3) The pectin extracted from the orange peel by the enzyme-assisted acid method has the esterification degree of 70-85 percent, belongs to high-ester pectin, has the viscosity and the gel strength equivalent to those of the pectin extracted by the acid method, and is superior to the pectin extracted by the enzyme method.

(4) Compared with the current industrialized pectin extraction method, the method greatly reduces the acid extraction time, and simultaneously reduces the energy consumption and equipment loss.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.

In the following examples:

(1) the viscosity was measured by using a rotary viscometer to measure a 1% (w/v) pectin-based polysaccharide solution, and the type of the viscometer was an IKA ROTAVSC lo-vi Complete rotary viscometer.

(2) The method for measuring the degree of esterification is carried out by referring to a titration method in national food safety standard GB 25533-2010.

(3) Monosaccharide was measured by TFA method, and a polysaccharide sample was accurately weighed at 5mg, and 1ml of 2MTFA acid solution was added and heated at 121 ℃ for 2 hours. Introducing nitrogen and drying. Adding intoWashing with methanol, drying by blowing, and repeating washing with methanol for 2-3 times. Dissolving in sterile water, transferring into chromatographic flask, detecting with Thermo ICS5000 ion chromatographic system (ICS5000, Thermo Fisher Scientific, USA), analyzing and detecting monosaccharide components with electrochemical detector, and detecting with Dionex^TM CarboPac^TMPA20(150 x 3.0mm, 10um) liquid chromatography column; the sample injection amount is 5uL, and the flow rate is 0.5 ml/min; the column temperature was 30 ℃. The RG-I content was calculated according to the following formula

RG-I(％)＝2Rha(％)+Ara(％)+Gal(％)

(4) Gel strength was determined according to the jelly SAG method (SAG method) specified in light industry Standard QB 2484-2000.

Example 1

(1) Activating xylanase in a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution with the pH value of 5.5 for 20min under the condition of constant-temperature water bath at 50 ℃ to obtain an enzyme activation solution (the mass-to-volume ratio of the enzyme to the enzyme activation solution is 0.0045 g: 40mL), wherein the xylanase: the mass ratio of the orange peel is 0.45% (w/w); 20g of citrus peel and enzyme activating solution are mixed according to the mass volume ratio of 1 g: 40mL of the pectin is mixed, the mixture is kept warm in water bath at 55 ℃ and stirred for 0.5h, after the reaction is finished, the pectin suspension is subjected to enzyme deactivation treatment (heating is carried out for 5min at 100 ℃), and the mixture is cooled to room temperature;

(2) adjusting the pH of the pectin suspension obtained in the step (1) to 1.7 by using a saturated oxalic acid solution, stirring and extracting for 1h in a constant-temperature water bath at the temperature of 80 ℃, and centrifuging (6000r/min, 20min) to obtain a pectin extracting solution;

(3) and (3) pouring 3 times volume of ethanol (95% ethanol solution) into the pectin extracting solution obtained in the step (2) for ethanol precipitation, standing for 0.5h, performing vacuum filtration, performing secondary ethanol precipitation, filtering, and performing fluidized bed drying (the drying temperature is 50 ℃, and the drying time is 0.5h) to obtain 5.09g of pectin, wherein the yield is 25.45%, the viscosity is 165.9mPa & s, the esterification degree is 75.05%, and the RG-I content is 40.06%.

Example 2

(1) Activating xylanase in a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution with the pH value of 5.0 for 0.5h under the condition of constant-temperature water bath at 50 ℃ to obtain an enzyme activation solution, wherein the xylanase comprises the following components in percentage by weight: the mass ratio of the orange peel is 0.30% (w/w); mixing 15g of orange peel and an enzyme activating solution according to a mass volume ratio of 1 g: mixing 30mL of the pectin and the pectin, keeping the temperature of the mixture in a water bath at 50 ℃, stirring the mixture for 1.5 hours, carrying out enzyme deactivation treatment on the pectin suspension liquid after the reaction is finished, and cooling the pectin suspension liquid to room temperature;

(2) adjusting the pH of the pectin suspension obtained in the step (1) to 2.3 by using a dilute nitric acid solution, stirring and extracting for 2 hours in a constant-temperature water bath at the temperature of 75 ℃, and centrifuging (8000r/min, 10min) to obtain a pectin extracting solution;

(3) and (3) adding 2 times volume of ethanol (absolute ethanol) into the pectin extracting solution in the step (2) for alcohol precipitation, standing for 20min, centrifuging at 5000r/min for 20min, performing secondary alcohol precipitation, filtering, and performing forced air drying (drying temperature is 60 ℃, drying time is 4h) to obtain 4.92g of pectin, wherein the yield is 28.62%, the viscosity is 148.5mPa & s, the esterification degree is 72.61%, and the RG-I content is 35.72%.

Example 3

(1) Activating cellulase in sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution with pH of 4.0 in 30 deg.C constant temperature water bath for 40min to obtain enzyme activation solution, cellulase: the mass ratio of the orange peel is 0.30% (/ w); 10g of citrus peel and enzyme activating solution are mixed according to the mass volume ratio of 1 g: mixing 20mL of the pectin and the pectin, keeping the mixture in a water bath at 30 ℃, stirring for 3 hours, carrying out enzyme deactivation treatment on the pectin suspension liquid after the reaction is finished, and cooling to room temperature;

(2) adjusting the pH value of the pectin suspension liquid obtained in the step (1) to 3.0 by using a saturated oxalic acid solution, stirring and extracting for 3 hours in a constant-temperature water bath at the temperature of 65 ℃, and centrifuging (5000r/min, 20min) to obtain a pectin extracting solution;

(3) and (3) adding 2 times volume of ethanol into the pectin extracting solution in the step (2) for alcohol precipitation, standing for 0.5h, centrifuging at 4000r/min for 30min, performing secondary alcohol precipitation, filtering, and performing rake drying (the drying temperature is 55 ℃, and the drying time is 50min) to obtain 2.38g of pectin, wherein the yield is 23.80%, the viscosity is 156.3mPa & s, the esterification degree is 83.65%, and the RG-I content is 36.41%.

Comparative example 1

This comparative example was directly subjected to acid treatment without enzyme treatment.

Specifically, 20g of citrus peel is taken, and the mass volume ratio of the citrus peel to distilled water is 1 g: adding distilled water into 40mL of the pectin, adjusting the pH value to 1.7 by using a nitric acid solution, stirring and heating the mixture in a constant-temperature water bath at 75 ℃ for 2 hours, cooling the mixture to room temperature, adding ethanol with the volume being 3 times that of the pectin, carrying out alcohol precipitation, standing the mixture for 0.5 hour, carrying out vacuum filtration, carrying out secondary alcohol precipitation, filtering the mixture, and carrying out forced air drying to obtain 3.94g of the pectin, wherein the yield is 20.70%, the viscosity is 131.9mPa & s, the esterification degree is 71.17%, and the RG-I content is 31.5%.

Comparative example 2

This comparative example did not add an enzyme preparation.

Specifically, the method comprises the following steps: taking 15g of citrus peel, and mixing the citrus peel with distilled water according to the mass volume ratio of 1 g: adding 40mL of distilled water, stirring for 1h in a water bath at 50 ℃, adjusting the pH value to 2.0 by using a saturated oxalic acid solution, stirring and heating for 1h in a constant-temperature water bath at 70 ℃, cooling to room temperature, adding 2 times of ethanol for alcohol precipitation, standing for 0.5h, centrifuging for 25min at 7000r/min, performing secondary alcohol precipitation, filtering, and performing forced air drying to obtain 4.68g of pectin, wherein the yield is 18.9%, the viscosity is 148.4mPa & s, the esterification degree is 70.48%, and the RG-I content is 30.93%.

The physical and chemical parameters of the pectins prepared in examples 1 to 3 and low ratios 1 to 2 are shown in Table 1.

TABLE 1 physical and chemical indexes of pectins prepared in examples 1-3 and low ratios 1-2

Claims

1. A method for preparing RG-I-rich high-ester pectin by an enzyme-assisted method is characterized by comprising the following steps: the method comprises the following steps:

(3) precipitating the supernatant obtained in the step (2) by alcohol, and drying the precipitate to obtain high-ester pectin;

the enzyme activating solution in the step (1) consists of enzyme and buffer solution; the enzyme is at least one of xylanase and cellulase.

2. The enzyme-assisted method for preparing RG-I high-ester pectin according to claim 1, which comprises the following steps: the buffer solution in the step (1) is a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution, and the pH value of the sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution is 4-6;

the mass volume ratio of the enzyme to the enzyme activation liquid is (0.0015-0.0075) g to (20-40) mL, the mass volume ratio of the citrus peel to the enzyme activation liquid is 1g to (20-40) mL, and the mass ratio of the enzyme to the citrus peel in the enzyme activation liquid is (0.15-0.75) to 100.

3. The method for preparing RG-I-rich high-ester pectin by the enzyme-assisted method according to claim 1, which is characterized in that: the citrus peel in the step (1) is one or more of lemon peel, orange peel and shaddock peel;

4. The method for preparing RG-I-rich high-ester pectin by the enzyme-assisted method according to claim 1, which is characterized in that: the precipitating agent for alcohol precipitation in the step (3) is an ethanol solution; the volume ratio of the supernatant to the ethanol solution is 1: 1-1: 3; the alcohol precipitation time is 0.5-1.5 hours.

5. The enzyme-assisted method for preparing RG-I high ester pectin according to claim 4, which comprises the following steps: the volume fraction of the ethanol solution is not less than 95%;

and the number of times of alcohol precipitation is 1-3.

6. The method for preparing RG-I-rich high-ester pectin by the enzyme-assisted method according to claim 5, which is characterized in that: when the alcohol precipitation is carried out for multiple times, the supernatant obtained after the centrifugation in the step (2) is mixed with a precipitator for alcohol precipitation, the precipitate is separated out, then the precipitate is continuously mixed with the precipitator for separating out the precipitate, the alcohol precipitation is carried out for multiple times, and finally the precipitate is dried;

the separation refers to centrifugal separation or suction filtration; the centrifugal separation is performed for 15-30 min under the condition of 4000-800 r/min, and the suction filtration is vacuum filtration.

7. The method for preparing RG-I-rich high-ester pectin by the enzyme-assisted method according to claim 1, which is characterized in that: the centrifugation in the step (2) is performed for 10-30 min under the condition of 5000-8000 r/min;

the drying in the step (3) is forced air drying, rake drying or fluidized bed drying; the drying temperature is 50-60 ℃, and the drying time is 0.5-4 h.

8. An RG-I rich high ester pectin obtainable by the process of claim 1.

9. The RG-I rich high ester pectin of claim 1, characterized in that: the esterification degree of the pectin is 70-85%; the proportion of the pectin RG-I area is 30-45%.

10. Use of RG-I rich high ester pectin according to claim 8 or 9, characterized in that: the RG-I-rich high ester pectin is used in the fields of yogurt products, fruit juices, jams and/or fondants.