阅读说明：本技术 水果中三种多酚加合物的连续提取方法及其应用 (Continuous extraction method of three polyphenol adducts in fruits and application thereof ) 是由 苏东晓 罗楠 辛婷 唐淑英 舒彬 于 2021-08-13 设计创作，主要内容包括：本发明公开了水果中三种多酚加合物的连续提取方法及其应用,包括以下步骤：在果浆中加入乙醇,进行醇沉,固液分离,收集固相A；将固相A溶解后,进行水提,收集液相A和固相B；液相A脱除蛋白后,加入乙醇,醇沉后,所得的沉淀即为水溶性多酚加合物；固相B中加入碳酸钠溶液,固液分离,收集固相C和液相B,所述液相B为碱溶性多酚加合物提取液；所述固相C为不溶性多酚加合物。通过本发明的方法,可以从水果中连续提取高纯度的水溶性多酚加合物、不溶性多酚加合物和碱溶性多酚加合物,方法简单高效,工艺可操作性强,成本低,易实现工业化生产。(The invention discloses a continuous extraction method of three polyphenol adducts in fruits and application thereof, which comprises the following steps: adding ethanol into the fruit pulp, carrying out alcohol precipitation, carrying out solid-liquid separation, and collecting a solid phase A; dissolving the solid phase A, performing water extraction, and collecting a liquid phase A and a solid phase B; removing protein from the liquid phase A, adding ethanol, and precipitating with ethanol to obtain precipitate as water-soluble polyphenol adduct; adding a sodium carbonate solution into the solid phase B, carrying out solid-liquid separation, and collecting a solid phase C and a liquid phase B, wherein the liquid phase B is an alkali-soluble polyphenol adduct extracting solution; the solid phase C is an insoluble polyphenol adduct. The method can continuously extract the water-soluble polyphenol adduct, the insoluble polyphenol adduct and the alkali-soluble polyphenol adduct with high purity from the fruits, has the advantages of simple and high-efficiency method, strong process operability, low cost and easy realization of industrial production.)

1. A continuous extraction method of three polyphenol adducts in fruits is characterized by comprising the following steps:

s1, adding ethanol into the fruit pulp, carrying out alcohol precipitation, carrying out solid-liquid separation, and collecting a solid phase A;

s2, dissolving the solid phase A obtained in the step S1, then carrying out water extraction, and collecting a liquid phase A and a solid phase B;

s3, removing protein from the liquid phase A obtained in the step S2, adding ethanol, and precipitating with ethanol to obtain a precipitate, namely the water-soluble polyphenol adduct;

s4, adding a sodium carbonate solution into the solid phase B obtained in the step S2, carrying out solid-liquid separation, and collecting a solid phase C and a liquid phase B, wherein the liquid phase B is an alkali-soluble polyphenol adduct extracting solution; the solid phase C is an insoluble polyphenol adduct.

2. The method according to claim 1, wherein in step S1, the mass-to-volume ratio of pulp to ethanol is 1: 1 to 10.

3. The method according to claim 1, wherein in step S1, the mass fraction of ethanol is 95% -100%.

4. The method according to claim 1, wherein in step S1, the temperature of the alcohol precipitation is 1-4 ℃.

5. The method of claim 1, wherein the water extraction is performed by heating in a water bath in step S2.

6. The method of claim 1, wherein in step S3, the removing protein is performed by using a protein precipitation reagent; preferably, the protein precipitation reagent is Sevag reagent.

7. The method according to claim 1, wherein in step S4, the mass-to-volume ratio of the solid phase B to the added sodium carbonate solution is 1: 4 to 8.

8. The method according to claim 1, wherein in step S4, the concentration of the sodium carbonate solution is 0.05-0.10 mol/L; preferably, the sodium carbonate solution further comprises 20-40 mmol/L sodium borohydride.

9. Use of the method according to any one of claims 1 to 8 for extracting polyphenols.

10. Use of a method according to any one of claims 1 to 8 for the preparation of a food or pharmaceutical product for promoting digestion.

Technical Field

The invention belongs to the technical field of natural product extraction, and particularly relates to a continuous extraction method and application of three polyphenol adducts in fruits.

Background

Recent studies have shown that plant polysaccharides have various biological activities, such as reducing cardiovascular diseases and metabolic syndrome. The phenolic substances are secondary metabolites of plants, and epidemiology shows that the phenolic substances also have biological activities beneficial to human bodies, such as antioxidation, immunity regulation, bacteriostasis and the like. Phenolics can be further divided into free phenols and bound phenols. The bound phenol is covalently bound with the polysaccharide and can be protected from gastrointestinal digestion and absorption, thereby achieving the effect of the colon on playing the probiotic effect of regulating intestinal flora.

At present, the extraction of plant polysaccharide is diversified, such as chemical extraction, microwave extraction, ultrasonic extraction and the like. Researchers shorten the extraction time, reduce the extraction energy consumption and improve the extraction efficiency through different technologies, thereby obtaining the polysaccharide with higher quality and purer purity. However, plant polysaccharides have various fractions, and polysaccharides in different fractions have different contents and compositions of bound phenol, so that the plant polysaccharides have different antioxidant functions and biological activities, and the current extraction research only focuses on the extraction and preparation of a certain polysaccharide. The prior art does not aim at a method for separating and extracting polyphenol adducts of different fractions.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides a continuous extraction method of three polyphenol adducts in fruits, which can simply and efficiently separate the polyphenol adducts of different fractions.

The invention also provides an application of the method.

According to one aspect of the present invention, there is provided a continuous process for the extraction of three polyphenol adducts from fruit, the process comprising the steps of:

s1, adding ethanol into the fruit pulp, carrying out alcohol precipitation, carrying out solid-liquid separation, and collecting a solid phase A;

s2, dissolving the solid phase A obtained in the step S1, then carrying out water extraction, and collecting a liquid phase A and a solid phase B;

s3, removing protein from the liquid phase A obtained in the step S2, adding ethanol, and precipitating with ethanol to obtain a precipitate, namely the water-soluble polyphenol adduct;

s4, adding a sodium carbonate solution into the solid phase B obtained in the step S2, carrying out solid-liquid separation, and collecting a solid phase C and a liquid phase B, wherein the liquid phase B is an alkali-soluble polyphenol adduct extracting solution; the solid phase C is an insoluble polyphenol adduct.

In some embodiments of the invention, in step S1, the preparing of the fruit pulp comprises: peeling and removing kernels of fruits, crushing and pulping.

In some embodiments of the invention, the polyphenol adduct is a complex of a polysaccharide and a polyphenol.

In some embodiments of the invention, the fruit is one or more of lychee, apple, kiwi, blueberry, mulberry, grape, pineapple, strawberry, banana, peach, pear, apricot, cantaloupe and watermelon.

In some embodiments of the invention, in step S1, the mass-to-volume ratio of pulp to ethanol is 1: 1 to 10.

In some embodiments of the invention, in step S1, the mass fraction of ethanol is 95% to 100%.

In some embodiments of the present invention, in step S1, the temperature of the alcohol precipitation is 1-4 ℃ and the time is 8-12 hours.

In some embodiments of the present invention, in step S1, the solid-liquid separation method is filtration.

In some embodiments of the invention, in step S2, the water extraction is performed by heating in a water bath at 85-95 ℃ for 4-6 hours.

In some embodiments of the present invention, in step S2, the solid-liquid separation method is centrifugation, the centrifugation rate is 4000-6000 rpm, and the time is 10-15 min.

In some embodiments of the present invention, in step S3, the protein removing means employs a protein precipitating agent; preferably, the protein precipitation reagent is Sevag reagent.

In some embodiments of the invention, the composition of the Sevag reagent comprises chloroform and n-butanol; the volume ratio of the trichloromethane to the n-butanol is 4: 1;

in some embodiments of the invention, the ratio of liquid phase to Sevag reagent is 1: 2-6 times of 10-20 min and 9-15 times of extraction. The extraction temperature is 55-65 ℃.

In some embodiments of the present invention, the step of adding Sevag reagent in step S3 further comprises a stirring step, wherein the stirring speed is 100 to 500rpm, and the stirring time is 1 to 5 min.

In some embodiments of the present invention, the step of removing the organic solvent by using a rotary evaporator at 60-70 ℃ is further included after the Sevag reagent is added in step S3.

In some embodiments of the present invention, the protein precipitate is removed by centrifugation at 4000-6000 rpm for 10-15 min.

In some embodiments of the invention, in step S3, the mass fraction of ethanol is 95% to 100%.

In some embodiments of the present invention, in step S3, the water-soluble polyphenol adduct further comprises a drying step of drying at 50-75 ℃ under vacuum to a constant weight.

In some embodiments of the present invention, in step S3, the temperature of the alcohol precipitation is 1-4 ℃ and the time is 8-12 hours.

In some embodiments of the invention, in step S4, the mass-to-volume ratio of the solid phase B to the added sodium carbonate solution is 1: 4 to 8.

In some embodiments of the invention, in step S4, the concentration of the sodium carbonate solution is 0.05-0.10 mol/L.

In some embodiments of the invention, in step S4, the sodium carbonate solution further includes 20 to 40mmol/L sodium borohydride.

In some embodiments of the present invention, in step S4, the solid-liquid separation method is centrifugation, the centrifugation rate is 4000-6000 rpm, and the time is 10-15 min.

In some embodiments of the present invention, the step S4 further includes a stirring step after the sodium carbonate solution is added, wherein the stirring speed is 100-500 rpm and the stirring time is 1-5 min.

According to a second aspect of the present invention, there is provided the use of the above method in the extraction of polyphenols.

A method for extracting litchi polyphenol comprises the following steps: adding the extracted litchi polyphenol adduct into an alkaline solution, and stirring for 12-24 hours under the protection of an inert atmosphere; adjusting the pH of the mixture to be neutral, adding ethyl acetate for extraction, and separating to obtain an ethyl acetate extract phase; and (3) concentrating the obtained extract phase at 30-40 ℃ in vacuum until ethyl acetate is completely volatilized to obtain the litchi polyphenol.

In some embodiments of the invention, the alkaline solution is one of sodium hydroxide and potassium hydroxide.

In some embodiments of the invention, the use is in the preparation of a food or pharmaceutical product for promoting digestion.

According to a preferred embodiment of the present invention, at least the following advantages are provided: the method can continuously extract the water-soluble polyphenol adduct, the insoluble polyphenol adduct and the alkali-soluble polyphenol adduct with high purity from the fruits, basically has no loss of polysaccharide and polyphenol, is simple and efficient, has strong process operability and low cost, and is easy to realize industrial production.

Drawings

The invention is further described with reference to the following figures and examples, in which:

FIG. 1 is a high performance liquid chromatogram of three polyphenol adducts in test examples of the present invention;

FIG. 2 is a graph of bound phenol content of three polyphenol adducts of the present invention in test examples.

Detailed Description

The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention. The test methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available reagents and materials unless otherwise specified.

Example 1

The embodiment provides a continuous extraction method of three polyphenol adducts in fruits, which comprises the following specific processes:

(1) soaking litchi pulp 200g in anhydrous ethanol, and removing small molecular substances such as fat and pigment at 4 deg.C overnight (12 hr);

(2) centrifuging at 4500r/min for 10min, and collecting precipitate;

(3) adding the precipitate obtained in the step (2) into 1000mL of distilled water, and extracting for 4h in a water bath at 90 ℃. Extracting the filter residue again and combining the filtrates;

(4) vacuum concentrating the filtrate obtained in the step (3) at 60 ℃, deproteinizing for 8 times by a Sevag method, adding 4 times of volume of absolute ethyl alcohol, and standing overnight at 4 ℃; filtering, collecting the precipitate and drying to obtain the water-soluble polyphenol adduct.

(5) Adding 500mL of 0.05mol/L Na into the filter residue obtained in the step (3)₂CO₃Shaking and extracting the solution (containing 20mmol/L sodium borohydride) for 4h, and centrifuging at 4000rpmFor 10min, obtaining supernatant and precipitate; freeze-drying the supernatant to obtain alkali-soluble polyphenol adduct; the precipitate was freeze-dried to obtain an insoluble polyphenol adduct.

Example 2

This example separately extracts bound phenols from the water-soluble polyphenol adduct, insoluble polyphenol adduct and alkali-soluble polyphenol adduct extracted in example 1 using the following procedure.

The polyphenol adduct (water-soluble polyphenol adduct, insoluble polyphenol adduct or alkali-soluble polyphenol adduct) was added to a 2M sodium hydroxide solution and stirred at room temperature for 18 hours with a magnetic stirrer under a nitrogen atmosphere. The mixture was then neutralized with 6M hydrochloric acid and extracted 6 times with ethyl acetate. The combined organic phases were evaporated to dryness in vacuo at 35 ℃ and redissolved in 85% methanol and then made to volume of 10mL to give bound phenol which was stored in a refrigerator at-20 ℃.

Test examples

1. Detection of three polyphenol adducts

The content of bound phenol in the 3 polyphenol adducts prepared in example 2 was individually identified using High Performance Liquid Chromatography (HPLC) -Diode Array Detector (DAD); identifying the phenolic compositions of the three polyphenol adducts by adopting a TOF5600+ liquid chromatography high-resolution tandem mass spectrometer; and identifying the phenolic compounds in the three polyphenol adducts by adopting a reversed phase high performance liquid chromatography. The method comprises the following steps:

the 3 polyphenol adducts prepared in example 1 were chromatographed using YMC-Pack ODS-A column (250X 4.6mml. D, 5 μm particle size). The mobile phase consisted of 0.4% aqueous acetic acid (solution a) and acetonitrile (solvent B). The gradient curves are as follows: 0-40min, 5-25% B; 40-45min, 25-35% B; the total running time is 50min, the balance is 5min, the flow rate is 1.0mL/min, the sample injection amount is 20 mu L, and the column incubator is 30 ℃. All samples were filtered through a 0.45 μm filter prior to analysis. (as shown in fig. 1).

The phenolic compounds of the three polyphenol adducts prepared in example 1 were identified using a mass spectrometer using a TOF5600+ liquid chromatography high resolution tandem mass spectrometer equipped with a turbine ion spray ionization source operating in negative ion ESI mode using an eclipse plus C18 column (2.1 × 100mm, 1.8 μm, agilent, germany). The capillary voltage was set at 4500v (negative) and the mobile phase was 0.4% formic acid in water (a) and acetonitrile (B). Analysis was performed with an elution gradient: 0-16min, 5-25% B; 16-18min, 25-35% B; 18-20min, 35-50% B. The flow rate was 0.4mL/min, the column temperature was 35 deg.C, the ion source temperature was 500 deg.C, and the sample volume was 4. mu.L. Before analysis, all samples were filtered through a 0.22 μm filter. Scanning within the range of 100-1000 m/z by 10 times of multi-channel acquisition. (as shown in tables 1-3).

TABLE 1 Water-soluble polyphenol adducts

TABLE 2 alkali soluble polyphenol adducts

TABLE 3 insoluble polyphenol adducts

And (3) identifying the bound phenol content in the three polyphenol adducts by adopting a reversed phase high performance liquid chromatography. All samples were filtered through a 0.45 μm filter prior to analysis. The high performance liquid chromatography adopts YMC-Pack ODS-A column (250X 4.6mml.D., 5 μm particle diameter); a column oven 30 ℃; the sample volume is 20 mu L; a detector: DAD; performing high performance liquid chromatography analysis on Agilent 1260 liquid chromatograph at a mobile phase flow rate of 1.0 mL/min. The mobile phase was solvent a (water/acetic acid, 996:4) and solvent B (acetonitrile). The elution gradient was as follows: 0-40min of solvent B: 5% -25%; 40-45min of solvent B: 25 to 35 percent; 45-50 min of solvent B: 35% -55%; the total operation is carried out for 50min, and the balance is carried out for 5 min. Chromatographic data were collected under a 280nm uv detector and the content of phenolic compounds in the samples was analyzed according to the retention time of the reference standards (gallic acid, (+) -catechin, caffeic acid, procyanidin B2, plateau catechin, quercetin, syringic acid) as the average of three replicates obtained from the three samples.

TABLE 4

The results of the hplc data of the three polyphenol adducts are shown in fig. 1, and it can be seen from the figure that 3 polyphenol adducts were successfully prepared according to the present invention, the mass spectrum data is shown in tables 1-3, the content of bound phenol in the 3 polyphenol adducts is shown in table 4, and it can be seen from the table that 3 polyphenol adducts were successfully prepared according to the present invention, and the polyphenol adducts prepared according to the present invention were identified to contain various polyphenols, such as gallic acid, catechin, protocatechin, protocatechuic aldehyde, rutin, (+) -gallocatechin, caffeic acid, procyanidin B2, and the like, at high contents.

2. Determination of the Total phenol content of bound phenols in three Polyphenol adducts

To 125. mu.L of each of the 3 bound phenol samples prepared in example 2 diluted 20 times was added 125. mu.L of the forskolin's reagent and distilled water (0.50 mL). After vortex shaking uniformly and light-shielding reaction for 6min, sequentially adding 1.25mL of Na₂CO₃Solution (7%, m/v) and 1mL of distilled water. Vortex and shake evenly, and then react for 90min in dark. Absorbance was measured at 760nm with Shimadzu UV-1800 Spectrophotometer. The total phenol content is expressed as Gallic Acid Equivalent (GAE) content per 100 g Dry Weight (DW) of litchi pulp, using gallic acid as a standard.

The results of the experiment are shown in fig. 2, from which it can be seen that the insoluble polyphenol adduct of the present invention has the highest bound phenol content and the lowest alkali-soluble polyphenol adduct content, and the results show that the scheme of the present application successfully extracts the water-soluble polyphenol adduct, the insoluble polyphenol adduct and the alkali-soluble polyphenol adduct.

In conclusion, the scheme of the invention can continuously extract the water-soluble polyphenol adduct, the insoluble polyphenol adduct and the alkali-soluble polyphenol adduct from the fruits, and the method is simple and efficient and has strong process operability.

The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present invention. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.