阅读说明：本技术 一种果蔬中不可萃取多酚的制备方法 (Preparation method of non-extractable polyphenol in fruits and vegetables ) 是由 程安玮 罗凯云 崔文玉 丰程凤 夏智慧 郭时印 吴卫国 于 2021-03-05 设计创作，主要内容包括：本发明属于食品精加工领域,具体提供一种果蔬中不可萃取多酚的制备方法,本发明通过果胶酶和单宁酶复合酶解的作用,将NEPP从果胶中释放出来,并通过单宁酶转换成方便提取的多酚,本发明多酚提取率高,方法简单,不需要辅以酸碱。危害小,污染物少,适合操作以及大规模工业化生产。(The invention belongs to the field of food finishing, and particularly provides a preparation method of non-extractable polyphenol in fruits and vegetables. The harm is small, the pollutant is less, and the method is suitable for operation and large-scale industrial production.)

1. A method for preparing non-extractable polyphenol in fruits and vegetables is characterized by comprising the following steps:

(1) pulping or drying fruit and vegetable raw materials, extracting extractable polyphenol with ethanol, collecting the residual residue, and drying at below 50 deg.C to obtain residue dry powder;

(2) adding buffer solution of tannase and pectinase into the dry residue powder, and performing enzymolysis treatment to obtain enzymolysis solution;

(3) centrifuging the enzymolysis solution, collecting supernatant, washing the residue for 1 time, centrifuging and mixing the two supernatants to obtain non-extractable polyphenol crude extract, and heating at 105 deg.C for 5min for inactivating enzyme;

(4) further concentrating the non-extractable polyphenol crude extract to 1/2-1/3 of the original volume to obtain a non-extractable polyphenol crude extract concentrated solution;

(5) extracting the non-extractable polyphenol crude extract concentrate with a mixed solution of ethyl acetate and ethanol, standing for layering, collecting water phase, and mixing to obtain non-extractable polyphenol extract;

(6) further concentrating the non-extractable polyphenol extract to 1/3-1/5 of the original volume, filtering and collecting filtrate, and freeze-drying to obtain the non-extractable polyphenol freeze-dried powder.

2. The preparation method according to claim 1, wherein in the step (1), the fruit and vegetable raw materials are dried, namely, the dry substances of the fruit and vegetable are pulverized, and the pulverized particle size is 40-80 meshes.

3. The preparation method according to claim 1, wherein the step (1) is carried out in the following manner: after being treated, the fruit and vegetable raw materials are mixed with 50-80% ethanol uniformly according to the proportion of 1:20-40 (g: mL), then ultrasonic treatment is carried out for 1-2h at the ultrasonic frequency of 200-.

4. The method according to claim 1, wherein in the step (2), the buffer solution of tannase and pectinase is: dissolving with 0.05-0.2mol/L sodium acetate buffer solution, wherein the pH of the buffer solution is 5.0-6.0, the concentration of tannase is 50-100U/mL, and the concentration of pectinase is 10-20U/mL.

5. The preparation method according to claim 1, wherein in the step (2), the enzymolysis temperature is 35-42 ℃, the time is 15-25h, and the oscillation speed is 10-20 rpm.

6. The preparation method as claimed in claim 1, wherein in the step (3), the enzymatic hydrolysate is cooled and then centrifuged at the rotation speed of 4000-6000r/min for 10-15min, the supernatant is collected, the residue is washed with 2-4 times of pure water by shaking for 10-15min and then centrifuged at the rotation speed of 4000-6000r/min for 10-15min, the two supernatants are combined and then heated at 105 ℃ for 5min for enzyme deactivation treatment and then cooled.

7. The preparation method according to claim 1, wherein in the step (5), the volume ratio of the ethyl acetate to the ethanol in the mixed liquid is 1:1-3, and the number of extraction times is 4-6.

Technical Field

The invention belongs to the field of food fine processing, and particularly relates to a preparation method of non-extractable polyphenol in fruits and vegetables.

Background

Plant polyphenols are a generic term for a class of polyhydroxy compounds present in plants, mainly in the skin, roots, leaves, shells, fruits and seeds of plants, and are considered to be the 7 th major nutrient for humans. The active phenolic hydroxyl in the polyphenol enables the polyphenol to have unique chemical and physiological activities, and mainly has the functions of resisting oxidation, removing free radicals and the like.

The fruit and vegetable raw materials are rich in polyphenol, and are divided into extractable polyphenol and non-extractable polyphenol according to different polyphenol combination modes and extraction methods. The extractable polyphenol is mainly polyphenol in a free state obtained by simple organic solvent extraction or water extraction, and the non-extractable polyphenol is mainly polyphenol which is bound to some hydrated tannins, procyanidins and the like on cell walls, and is separated by breaking chemical bonds between polyphenol substances and cell walls through a chemical or enzyme treatment and other methods.

At present, researches on polyphenol in fruit and vegetable raw materials mainly focus on extractable polyphenol, but pay little attention to non-extractable polyphenol. If non-extractable polyphenols are considered, the content of beneficial polyphenols such as proanthocyanidins, ellagic acid, and catechins contained in the fruit and vegetable raw materials is far beyond the people's estimation. At present, the non-extractable polyphenol is not listed as the nutritional composition of food, and actually the non-extractable polyphenol is a bioactive component in food and can be fermented by bacteria in colon, and the produced metabolite is very beneficial to human bodies.

In the prior art, the extraction method of the non-extractable polyphenol mainly comprises an acid method and an alkaline method, wherein the acid method mainly comprises the steps of mixing sulfuric acid and methanol according to a certain proportion to serve as an extraction solvent, and dissociating the non-extractable polyphenol by a heating hydrolysis method. The alkaline method is to use 1-5mol/L NaOH as an extraction solvent to hydrolyze and free the non-extractable polyphenol, both methods need strong acid or strong alkali, the generated waste liquid is easy to cause environmental pollution, and the safety of the waste liquid needs to be very careful.

Disclosure of Invention

Aiming at the technical defects of the preparation of the non-extractable polyphenol in the prior art, the invention provides a preparation method of the non-extractable polyphenol in fruits and vegetables.

A method for preparing non-extractable polyphenol in fruits and vegetables comprises the following steps:

(1) pulping or drying fruit and vegetable raw materials, extracting extractable polyphenol with ethanol, collecting the residual residue, and drying at below 50 deg.C to obtain residue dry powder;

(2) adding buffer solution of tannase and pectinase into the dry residue powder, and performing enzymolysis treatment to obtain enzymolysis solution;

(3) centrifuging the enzymolysis solution, collecting supernatant, washing the residue for 1 time, centrifuging and mixing the two supernatants to obtain non-extractable polyphenol crude extract, and heating at 105 deg.C for 5min for inactivating enzyme;

(4) further concentrating the non-extractable polyphenol crude extract to 1/2-1/3 of the original volume to obtain a non-extractable polyphenol crude extract concentrated solution;

(5) extracting the non-extractable polyphenol crude extract concentrate with a mixed solution of ethyl acetate and ethanol, standing for layering, collecting water phase, and mixing to obtain non-extractable polyphenol extract;

(6) further concentrating the non-extractable polyphenol extract to 1/3-1/5 of the original volume, filtering and collecting filtrate, and freeze-drying to obtain the non-extractable polyphenol freeze-dried powder.

Preferably, the drying of the fruit and vegetable raw materials in the step (1) is implemented by powdering the dry matter of the fruit and vegetable, and the crushed grain size is 40-80 meshes.

Preferably, step (1) is performed in the following manner: after being treated, the fruit and vegetable raw materials are mixed with 50-80% ethanol according to the proportion of 1:20-40 (g: mL), then ultrasonic treatment is carried out for 1-2h at the ultrasonic frequency of 200-.

Wherein the buffer solution of the tannase and the pectinase in the step (2) is as follows: dissolving with 0.05-0.2mol/L sodium acetate buffer solution, wherein the pH of the buffer solution is 5.0-6.0, the concentration of tannase is 50-100U/mL, and the concentration of pectinase is 10-20U/mL.

Preferably, in the step (2), the enzymolysis temperature range is 35-42 ℃, the enzymolysis time is 15-25h, and the oscillation speed is 10-20 rpm.

Wherein, in the step (3), the enzymatic hydrolysate is centrifuged for 10-15min at the rotating speed of 4000-6000r/min after being cooled, the supernatant is collected, the residue is washed by pure water with the volume of 2-4 times of that of the supernatant for 10-15min in a shaking way, and is centrifuged for 10-15min at the rotating speed of 4000-6000r/min, the two supernatants are combined, and then the enzymatic hydrolysate is heated for 5min at 105 ℃ for enzyme deactivation treatment and then is cooled.

In the step (5), the volume ratio of the ethyl acetate to the ethanol in the mixed liquid is 1:1-3, preferably 1: 1.

Wherein, in the step (5), the extraction times are 4-6.

The important one of the non-extractable polyphenol is tannin, and most of the tannin is attached to pectin. Tannins are polyphenol compounds, tannic acid is not a single compound, and the chemical composition is relatively complex and roughly divided into two types: one is condensed tannic acid, which is a flavanol derivative; the second is hydrolysable tannic acid, which has an ester bond in the molecule. Tannase is strictly enzymatically defined as a tannyl hydrolase, meaning that the hydrolase has a serine active site which has both esterase activity and depside activity, meaning that it hydrolyzes both ester and depside bonds in tannins.

According to the invention, tannase is added in enzymolysis, so that condensed tannic acid and hydrolyzed tannic acid can be hydrolyzed to release free catechol, gallic acid and other components, and thus the extraction rate of the non-extractable polyphenol is increased.

The invention has the beneficial effects

The invention provides a method for extracting non-extractable polyphenol by using tannase and pectinase buffer solution for hydrolysis. Compared with the prior art acid method (sulfuric acid/methanol) heating hydrolysis extraction process:

the method does not need high-temperature heating, does not need dangerous chemical reagents such as sulfuric acid and the like or strong alkaline conditions, and has the characteristics of mild experimental conditions, high safety, no generation of a large amount of dangerous wastes, simple operation steps, high extract purity and the like.

Detailed Description

The following embodiments are merely exemplary embodiments of the present invention, which are not intended to limit the present invention in any way, and any simple modification, equivalent change and modification made to the above embodiments according to the technical spirit of the embodiments of the present invention are still within the scope of the technical solution of the embodiments of the present invention.

Example 1

Selecting the residual pomace after apple juicing, and mixing the pomace and the pomace according to the weight ratio of 1: adding 50% ethanol water solution at a ratio of 25 (g/mL); ultrasonic extraction at 500W for 1h, and centrifugation at 5000r/min for 10 min.

Filtering, oven drying the obtained fruit residue in an oven at 50 deg.C, and pulverizing to 60 mesh. The tannase and the pectinase were dissolved in 0.1mol/L sodium acetate buffer (pH 5.5) to a tannase concentration of 50U/mL and a pectinase concentration of 20U/mL.

Mixing the pomace with mixed enzyme buffer solution at a ratio of 1:25 (g/mL) uniformly, wherein the enzymolysis temperature is 38 ℃, the enzymolysis time is 20h, and the shaking speed is 20 r/min. Centrifuging at 6000r/min for 10min after enzymolysis, collecting supernatant, washing residue with 2 times volume of pure water for 10min, centrifuging at 6000r/min for 10min, mixing the two supernatants, heating at 105 deg.C for 5min for inactivating enzyme, and cooling.

The combined supernatants were concentrated to 1/3 of the original volume to give a crude non-extractable polyphenol concentrate. Preparing an extraction solvent of ethyl acetate and ethanol in a ratio of 1:1, mixing and shaking the non-extractable polyphenol crude extract concentrated solution and the extraction solvent (ethyl acetate and ethanol) in a ratio of 1:1, standing until layering, collecting a lower-layer water phase, repeatedly extracting for 6 times, combining the water phases, concentrating to 1/5 of the original volume, and freeze-drying to obtain the non-extractable polyphenol freeze-dried powder.

The content of non-extractable polyphenol was determined to be 9.2mg/g using Folin-Ciocalteu method at 765 nm.

Example 2

Fresh and mature blueberries are selected as raw materials, rotten and rotten fruits, sick and insect fruits and impurities are removed, and silt and floating materials are removed in clear water. Crushing and pulping the drained blueberries, and then mixing the crushed blueberries according to the weight ratio of 1: adding 50% ethanol solution at a ratio of 30 (g/mL), ultrasonic extracting at 200W for 2h, and centrifuging at 3000r/min for 20 min.

Dissolving tannase and pectinase with 0.1mol/L sodium acetate buffer (pH = 5.0) until the concentration of tannase is 100U/mL and the concentration of pectinase is 10U/mL, filtering, and mixing the obtained pomace with mixed enzyme buffer according to the ratio of 1:25 (g/mL), wherein the enzymolysis temperature is 38 ℃, the enzymolysis time is 20h, and the shaking speed is 20 r/min. Heating at 105 ℃ for 5min after complete reaction for enzyme deactivation, cooling the enzymolysis liquid, centrifuging at 6000r/min for 10min, collecting supernatant, shaking and washing the residue for 10min by using pure water with 2 times of volume, centrifuging at 6000r/min for 10min, combining the two supernatants, heating at 105 ℃ for 5min for enzyme deactivation, and cooling.

The combined supernatants were concentrated to 1/3 of the original volume to give a crude non-extractable polyphenol concentrate. Preparing an extraction solvent ethyl acetate and ethanol in a ratio of 1:1, then shaking and uniformly mixing the non-extractable polyphenol crude extract concentrated solution and the extraction solvent in a ratio of 1:1, standing to separate layers, collecting a lower-layer water phase, repeatedly extracting for 6 times, combining the water phases, then concentrating to 1/5 of the original volume, and then freeze-drying to obtain the non-extractable polyphenol freeze-dried powder.

The content of non-extractable polyphenols was determined to be 11.5mg/g using Folin-Ciocalteu method at 765 nm.

Example 3

Selecting residues left after wine brewing, removing grape stalks and seeds, leaving grape skin residues, crushing the grape skin residues to 60 meshes, and then, mixing the grape skin residues with the crushed grape skin residues according to the weight ratio of 1: adding 50% ethanol water solution at a ratio of 40 (g/mL), performing 400W ultrasonic extraction for 2h, and centrifuging at 4000r/min for 20 min. The extraction was repeated twice.

The selected tannase and pectinase are dissolved in 0.05mol/L sodium acetate buffer (pH 6.0) until the concentration of tannase is 75U/mL and the concentration of pectinase is 15U/mL. Filtering, and mixing the obtained pomace with mixed enzyme buffer solution at a ratio of 1:30 (g/mL) uniformly, wherein the enzymolysis temperature is 35 ℃, the enzymolysis time is 24h, and the shaking speed is 20 r/min. Heating at 105 ℃ for 5min after complete reaction for enzyme deactivation, cooling the enzymatic hydrolysate, centrifuging at 4000r/min for 15min, collecting supernatant, washing residues with 2 times of pure water by shaking for 10min, centrifuging at 4000r/min for 15min, combining the two supernatants, heating at 105 ℃ for 5min for enzyme deactivation, and cooling.

The combined supernatants were concentrated to 1/3 of the original volume to give a crude non-extractable polyphenol concentrate. Preparing an extraction solvent ethyl acetate and ethanol in a ratio of 1:1, then shaking and uniformly mixing the non-extractable polyphenol crude extract concentrated solution and the extraction solvent according to a ratio of 1:1, standing to separate layers, collecting a lower-layer water phase, repeatedly extracting for 6 times, combining the water phases, then concentrating to 1/5 of the original volume, and then freeze-drying to obtain the non-extractable polyphenol freeze-dried powder.

The content of non-extractable polyphenol was determined to be 10.9mg/g using Folin-Ciocalteu method at 765 nm.

Comparative example 1

The pomace was only subjected to enzymatic hydrolysis with pectinase, the pectinase concentration was 50U/ml, the other steps and parameters were as in example 2, and Folin-Ciocalteu was used to determine the non-extractable polyphenol content at 765nm, which was found to be 6.3 mg/g.

Comparative example 2

The pomace was subjected to enzymatic hydrolysis with pectinase and cellulase at a pectinase concentration of 50U/ml and cellulase concentration of 5U/ml, and the content of non-extractable polyphenols was determined to be 7.2mg/g, using Folin-Ciocalteu method at 765nm, using the same procedure and parameters as example 2.