CN115960276A - Novel pectin oligosaccharide and preparation method and application thereof - Google Patents
Novel pectin oligosaccharide and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a novel fruitA colloidooligosaccharide and a preparation method and application thereof. The structural formula of the novel pectin oligosaccharide is shown as the formula (1):
formula (1). The novel pectin oligosaccharide is rich in RG-I galactose side chain, has low molecular weight and low methyl ester degree, has good intestinal flora probiotic function, and can be used in products for improving the intestinal probiotic function; the method for preparing the pectin oligosaccharide is simple to operate, low in production cost, beneficial to fully utilizing fruit and vegetable wastes, capable of improving the added value of the fruit and vegetable wastes, capable of realizing green production and efficient preparation of the pectin oligosaccharide, easy to realize industrial production and capable of improving economic benefits.
Description
Technical Field
The invention relates to the technical field of food processing, in particular to novel pectin oligosaccharide and a preparation method and application thereof.
Background
The pectin is a byproduct derived from fruit and vegetable peel residues, has good gelling property and emulsifying property, and can be used as a gelling agent, an emulsifying agent and a thickening agent to be applied to food; and has antiinflammatory, immunity enhancing and anticancer effects. Pectin oligosaccharide is a low molecular weight component based on pectin, and researches in recent years show that the pectin oligosaccharide has better biological activity and multiple health promotion functions such as pathogen inhibition, cancer cell apoptosis induction, immunoregulation, cardiovascular protection, anti-inflammation and the like due to small molecular weight and is easier to be absorbed by organisms, and can be used in health food. The pectin oligosaccharide is safe and free of mutagenesis, and can be used in children food such as milk powder. In addition, pectin oligosaccharides, as a novel prebiotic, have also received much attention for their regulatory function on intestinal microorganisms.
The prior method for preparing pectin oligosaccharide mainly comprises a physical method, a chemical method, a biological enzymolysis method and the like. Wherein the physical method adopts ultrasonic and microwave modes, and utilizes ultrasonic cavitation and high frequency to break the glycosidic bond in pectin molecule, thereby reducing the molecular weight; chemical methods using acid hydrolysis (trifluoroacetic acid, hydrochloric acid), redox reactions, e.g. Smith degradation, periodate oxidation, fenton's reaction (hydrogen peroxide and Fe) 2+ Mixing to generate redox reaction to generate free radical, and attacking glycosidic bond to break) to hydrolyze or oxidize pectin molecules to generate low molecular weight pectin oligosaccharide. However, these two methods have the disadvantages of high cost, high equipment requirement, high loss, easy generation of processing pollutants and difficult realization of productionIndustrialization and the like. Compared with a physical method and a chemical method, the method has the advantages of safety in enzyme method, mild reaction condition, lower cost, greenness and environmental protection, and can realize industrial production. However, how to prepare pectin oligosaccharide with specific structure and function by using an enzymatic method promotes the further development and application of pectin oligosaccharide, so that the realization of high-value utilization of fruit and vegetable wastes becomes a problem to be solved urgently.
Disclosure of Invention
The invention aims to provide a novel pectin oligosaccharide, a preparation method and an application thereof, wherein the pectin oligosaccharide is rich in galactose side chains, low in molecular weight and low in methyl ester degree, can improve the content of short-chain fatty acids in a fermentation process, and has a probiotic function of regulating intestinal microorganisms.
In order to achieve the above objects, the first aspect of the present invention provides a novel pectin oligosaccharide having a structural formula represented by formula (1):
formula (1).
Optionally, the molecular weight of the novel pectin oligosaccharide is 500 to 3500 g/mol.
In a second aspect, the present invention provides a method for preparing the novel pectin oligosaccharide, which comprises the following steps:
s1, dissolving a pectin raw material in a buffer solution to obtain a first raw material solution;
s2, adding an enzyme preparation into the first raw material liquid for carrying out enzymolysis reaction to obtain an enzymolysis liquid; inactivating the enzymolysis liquid, and then carrying out first centrifugal treatment to obtain a first supernatant;
s3, carrying out first concentration treatment on the first supernatant to obtain a first concentrated solution, and carrying out first semi-permeable membrane dialysis on the first concentrated solution to obtain a first dialysate and a first retentate; carrying out second concentration treatment on the first retentate to obtain a second concentrated solution, and dialyzing the second concentrated solution through a second semipermeable membrane to obtain a second dialyzate and a second retentate;
s4, performing third concentration treatment and freeze drying on the second dialysate respectively;
wherein the pH value of the buffer solution is 4.0-5.0, and the buffer solution is selected from at least one of citrate-phosphate buffer solution, acetic acid-sodium acetate buffer solution, phosphate buffer solution and potassium hydrogen phthalate-sodium hydroxide; the molecular weight cut-off of the first semi-permeable membrane is 500-1000Da, and the molecular weight cut-off of the second semi-permeable membrane is 3500-4000 Da.
Optionally, the pectin material is selected from at least one of citrus pectin material, apple pectin material, beet pectin material and fig pectin material, preferably citrus pectin material; further preferably, the preparation method of the citrus pectin raw material comprises the following steps: crushing orange peel, and sieving the crushed orange peel to obtain a first orange peel raw material; adding deionized water into a first orange peel raw material, wherein the ratio of the weight of the first orange peel raw material to the volume of the deionized water is 1:20 to 35, and adjusting the pH value to be 1.5 to 3 to obtain a first mixed solution; stirring the first mixed solution, and then carrying out second centrifugal treatment to obtain a second supernatant; adding ethanol into the second supernatant, wherein the volume ratio of the second supernatant to the ethanol is 1:2~3 to obtain a second mixed solution; carrying out precipitation treatment on the second mixed solution, and then carrying out suction filtration treatment to obtain a first precipitate; and drying and crushing the first precipitate.
Optionally, in step S2, the enzyme preparation is a composite pectinase preparation, and the enzyme preparation contains polygalacturonase, beta-galactosidase, arabinanase and arabinofuranosidase; the mass ratio of the polygalacturonase to the beta-galactosidase to the arabinanase to the arabinofuranosidase is 23 to 28:10 to 15:10 to 15:20 to 25.
Optionally, the enzyme activity of the enzyme preparation is 7700U/mL; the amount of the enzyme preparation added is 0.2 to 0.6mL per liter of the first raw material liquid.
Optionally, in step S2, the conditions of the enzymatic hydrolysis reaction include: the temperature is 40 to 50 ℃, preferably 43 to 47 ℃; the time is 2 to 3h, preferably 2.5 to 3h; the step of inactivating comprises: heating the enzymolysis liquid in a boiling water bath for 10 to 15 min, and cooling to 20 to 30 ℃; the conditions of the first centrifugation treatment include: the revolution number is 8000-10000 r/min; the time is 10 to 20min.
Optionally, the buffer is a citrate-phosphate buffer; the pH value of the buffer solution is 4.3 to 4.7;
optionally, the citrate in the citrate-phosphate buffer is selected from one or more of citric acid, sodium citrate and citric acid monohydrate; the phosphate is one or more selected from disodium hydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate dihydrate and dipotassium hydrogen phosphate dihydrate.
Optionally, in step S3, the first concentration treatment is vacuum cryoconcentration, the temperature of the first concentration treatment is 45 to 55 ℃, and the volume ratio of the first concentrated solution to the supernatant is 1:3~5, preferably 1:4; the second concentration treatment is vacuum low-temperature concentration, the temperature of the second concentration treatment is 45-55 ℃, and the volume ratio of the second concentrated solution to the supernatant is 1:5 to 15, preferably 1:10; the third concentration treatment is vacuum low-temperature concentration, the temperature of the third concentration treatment is 45-55 ℃, and the volume ratio of a third concentrated solution obtained by the third concentration treatment to the supernatant is 1:5 to 15, preferably 1:10; the conditions for freeze-drying include: the temperature is-60 to-50 ℃, and the time is 36 to 72 hours.
The third aspect of the invention provides an application of the novel pectin oligosaccharide in a product for increasing intestinal short-chain fatty acids and improving intestinal probiotic functions; optionally, the product is selected from at least one of a food, a beverage, a nutraceutical, and a pharmaceutical.
Through the technical scheme, the invention provides novel pectin oligosaccharide and a preparation method and application thereof. The pectin oligosaccharide prepared by the method has the molecular weight of 500-3500 Da and is rich in galactose side chains, the novel pectin can obviously improve the content of short-chain fatty acids in the fermentation process, can adjust the intestinal microecology, and increases the relative abundance of beneficial bacteria such as bacteroides, coriolus, parahaemobacter dychii, corynebacterium and the like. Has the probiotic function of regulating intestinal microorganisms; meanwhile, the method is simple to operate, low in production cost, beneficial to fully utilizing fruit and vegetable wastes, capable of improving the added value of the fruit and vegetable wastes, capable of realizing green production and efficient preparation of pectin oligosaccharide, easy to realize industrial production and capable of improving economic benefits.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 is a flow chart of the steps for preparing different pectin enzymatic products in accordance with the present invention.
FIG. 2 is a SDS-PAGE pattern of a complex pectinase used in the present invention; wherein: the molecular weights of polygalacturonase, beta-galactosidase, arabinosidase, arabinofuranosidase and rhamnogalacturonase are 25-50 kDa, 125 kDa, 37 kDa, 62 kDa and 76.14 k Da, respectively.
FIG. 3 shows the enzymatic hydrolysis process using the enzyme preparation in example 1 of the present invention.
FIG. 4 is a graph showing the elution profile of S-5 in example 1 of the present invention.
FIG. 5 is an infrared spectrum of the pectin enzymatic hydrolysate.
FIG. 6 is a graph of the acetic acid content of the pectin enzymatic hydrolysate in vitro fermentation.
FIG. 7 is a propionic acid content diagram of in vitro fermentation of the pectin enzymatic hydrolysate.
FIG. 8 is a graph of butyric acid content of the pectin enzymatic hydrolysate in vitro fermentation.
FIG. 9 is a diagram of the total short-chain fatty acid content of the pectin enzymatic hydrolysate in vitro fermentation.
FIG. 10 shows the taxonomic level of gut microbial changes during in vitro fermentation of pectin enzymatic hydrolysate.
FIG. 11 is the nuclear magnetic hydrogen spectrum of the component S-5 with the highest short-chain fatty acid production performance in the pectin enzymolysis product.
FIG. 12 is the nuclear magnetic carbon spectrum of the component S-5 with the highest short-chain fatty acid production performance in the pectin enzymolysis product.
FIG. 13 is the HMQC spectrum of the fraction S-5 with the highest short-chain fatty acid production performance in the pectin enzymatic hydrolysate.
FIG. 14 is a COSY spectrogram of a component S-5 with the highest short-chain fatty acid production performance in the pectin enzymolysis product.
FIG. 15 is the HMBC spectrum of the fraction S-5 with the highest short-chain fatty acid production performance in the pectin enzymatic hydrolysate.
FIG. 16 shows the structure of the component S-5 with the highest short-chain fatty acid production in the pectin enzymolysis product.
Detailed Description
The following detailed description of embodiments of the invention refers to the accompanying drawings. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.
The first aspect of the present invention provides a novel pectin oligosaccharide having a structural formula represented by formula (1):
formula (1).
Wherein "alpha-GalpA' represents a galactopyranosyl acid in the alpha configuration; "alpha-Rhap"represents pyranorhamnose of the alpha configuration; "beta-Galp"represents galactopyranose in the beta configuration; the numbers at the arrows represent the attachment positions between the saccharides, e.g., "1 → 4" represents the attachment between galacturonic acid via α -1,4; "1 → 2" represents the glycosidic linkage between galacturonic acid and rhamnose via α -1,2; "3 → 1" represents galactose connected by β -1,3 glycosidic linkages.
In one embodiment of the invention, the molecular weight of the novel pectin oligosaccharide is 500 to 3500 g/mol.
In the present invention, the novel pectin oligosaccharide consists of galactose, galacturonic acid and rhamnose.
The novel pectin oligosaccharide disclosed by the invention is low in molecular weight and low in methyl ester degree, the content of short-chain fatty acids in a fermentation process can be obviously improved, and after 72h is fermented in vitro, the total concentration of the short-chain fatty acids is 123.49 +/-1.10 mmol/L, which is far higher than that of recognized prebiotic inulin (93.32 +/-0.74 mmol/L) and original pectin (40.55 +/-1.83 mmol/L). Meanwhile, the structure of intestinal micro-ecology can be adjusted, the relative abundance of beneficial bacteria such as bacteroides, coriolus, parabacteroides dieselii and cladosporium is increased, and the probiotic function of intestinal micro-organisms is adjusted. The enzyme used in the present invention is a complex pectinase containing a polygalacturonase enzyme that can cleave the alpha-glycosidic bonds between galacturonic acids in the HG region of pectin to produce an RG-I rich fraction; the enzyme also contains arabinosidase and galactosidase (figure 2), and can break arabinose side chain and galactose side chain in pectin RG-I to generate novel pectin oligosaccharide rich in RG-I side chain.
In a second aspect, the present invention provides a method for preparing the novel pectin oligosaccharide, which comprises the following steps:
s1, dissolving a pectin raw material in a buffer solution to obtain a first raw material solution;
s2, adding an enzyme preparation into the first raw material liquid for carrying out enzymolysis reaction to obtain an enzymolysis liquid; inactivating the enzymolysis liquid, and then carrying out first centrifugal treatment to obtain a first supernatant;
s3, carrying out first concentration treatment on the first supernatant to obtain a first concentrated solution, and carrying out first semi-permeable membrane dialysis on the first concentrated solution to obtain a first dialysate and a first retentate; carrying out second concentration treatment on the first retentate to obtain a second concentrated solution, and dialyzing the second concentrated solution through a second semipermeable membrane to obtain a second dialyzate and a second retentate;
s4, performing third concentration treatment and freeze drying on the second dialysate respectively;
wherein the pH value of the buffer solution is 4.0-5.0, and the buffer solution is selected from at least one of citrate-phosphate buffer solution, acetic acid-sodium acetate buffer solution, phosphate buffer solution and potassium hydrogen phthalate-sodium hydroxide; the molecular weight cutoff of the first semi-permeable membrane is 500 to 1000Da, and the molecular weight cutoff of the second semi-permeable membrane is 3500 to 4000Da.
The preparation method of the novel pectin oligosaccharide provided by the invention adopts an enzymolysis method, the specific method is shown in figure 1, the method is simple to operate and low in production cost, and the method can realize the preparation of citrus pectin oligosaccharides with different molecular weights, is suitable for realizing industrial production and improves economic benefits; the prepared novel pectin oligosaccharide has the function of improving intestinal probiotic, and after the pectin oligosaccharide is fermented in vitro for 72 hours, the concentration of total short-chain fatty acid is 123.49 +/-1.10 mmol/L, meanwhile, the structure of intestinal microecology can be adjusted, and the relative abundance of beneficial bacteria such as bacteroides, corynebacteria, paradisea dychii and Scutellaria is increased. In the present invention, the enzymolysis process is shown in FIG. 3; in one embodiment of the present invention, as shown in fig. 4, the enzymatic hydrolysate is purified, and polygalacturonase contained in the enzyme preparation can break the α -glycosidic bond between galacturonic acid in HG region of pectin; the arabinase, beta-galactosidase and arabinofuranosidase contained in the enzyme preparation can break arabinose side chains and galactose side chains in pectin RG-I, and pectin oligosaccharide rich in rhamnogalacturonan I (RG-I for short) side chains is generated after grading according to molecular weight.
In one embodiment of the invention, in the pectin raw material, the concentration of pectin can be 0.5 to 1.5wt%; for example, it may be 0.5%, 1.0%, 1.5%, or any value therebetween. Optionally, the pectin material is selected from at least one of citrus pectin material, apple pectin material, beet pectin material and fig pectin material, preferably citrus pectin material; in a preferred embodiment of the present invention, the method for preparing the citrus pectin raw material comprises: crushing orange peel, and sieving the crushed orange peel to obtain a first orange peel raw material; adding deionized water into a first orange peel raw material, wherein the ratio of the weight of the first orange peel raw material to the volume of the deionized water is 1:20 to 35, and adjusting the pH to 1.5 to 3 to obtain a first mixed solution; stirring the first mixed solution, and then carrying out second centrifugal treatment to obtain a second supernatant; adding ethanol into the second supernatant, wherein the volume ratio of the second supernatant to the ethanol is 1:2~3 to obtain a second mixed solution; carrying out precipitation treatment on the second mixed solution, and then carrying out suction filtration treatment to obtain a first precipitate; and drying and crushing the first precipitate.
In the invention, the pectin raw material is preferably prepared by taking citrus peel as a raw material, and the high-ester pectin prepared by an acid extraction method has good gelling property, is the same as the preparation method of commercial pectin widely applied in the food industry, and is beneficial to industrial production and application. In the above embodiment, hydrochloric acid may be used to adjust the pH, and the concentration of hydrochloric acid may be 0.1mol/L.
In a specific embodiment of the present invention, in step S2, the enzyme preparation is a composite pectinase preparation, and the enzyme preparation contains polygalacturonase, β -galactosidase, arabinanase and arabinofuranosidase; the mass ratio of the polygalacturonase to the beta-galactosidase to the arabinanase to the arabinofuranosidase is 23 to 28:10 to 15:10 to 15:20 to 25.
The compound pectinase preparation used in the invention is a novel enzyme preparation, mainly comprises pectinases such as polygalacturonase, beta-galactosidase, arabinosidase and arabinofuranosidase (shown in figure 2), and the enzyme activity is 7700U/mL. The inventor of the invention finds that the enzymolysis liquid obtained by enzymolysis of the complex enzyme preparation provided by the application contains RG-I galactan structural domain.
In one embodiment of the present invention, the enzyme preparation is added in an amount of 0.2 to 0.6mL per liter of the first raw material solution.
In one embodiment of the present invention, in step S2, the conditions of the enzymatic hydrolysis reaction include: the temperature is 40 to 50 ℃, preferably 43 to 47 ℃; the time is 2 to 3h, preferably 2.5 to 3h;
the step of inactivating comprises: heating the enzymolysis liquid in a boiling water bath for 10 to 15 min, and cooling to 20 to 30 ℃;
the conditions of the first centrifugation treatment include: the revolution number is 8000-10000 r/min; the time is 10 to 20min.
In one embodiment of the invention, the buffer is a citrate-phosphate buffer; the pH value of the buffer solution is 4.3 to 4.7; in a preferred embodiment of the present invention, the citrate in the citrate-phosphate buffer is selected from one or more of citric acid, sodium citrate, and citric acid monohydrate; the phosphate is one or more selected from disodium hydrogen phosphate, dipotassium hydrogen phosphate dihydrate and disodium hydrogen phosphate dihydrate.
In a specific embodiment of the present invention, in step S2, the conditions of the enzymatic hydrolysis reaction include: the temperature is 40-50 ℃, preferably 43-47 ℃; the time is 2-3 h, and the time is preferably 2.5 to 3h; the step of inactivating comprises: heating the enzymolysis liquid in a boiling water bath for 10 to 15 min, and cooling to 20 to 30 ℃; the conditions of the centrifugation treatment include: the revolution number is 8000-10000 r/min; the time is 10 to 20min.
In the invention, after the enzymolysis reaction is finished, the used pectinase preparation can be inactivated by placing the reacted feed liquid in a boiling water bath for heat preservation. The "centrifugation" may be performed using a centrifuge conventional in the art.
In one embodiment of the invention, in step S3, the first concentration process is vacuum cryoconcentration, the temperature of the first concentration process is 45 to 55 ℃, and the volume ratio of the first concentrated solution to the supernatant is 1:3~5, preferably 1:4; the second concentration treatment is vacuum low-temperature concentration, the temperature of the second concentration treatment is 45-55 ℃, and the volume ratio of the second concentrated solution to the supernatant is 1:5 to 15, preferably 1:10.
in a specific embodiment of the invention, the third concentration treatment is vacuum low-temperature concentration, the temperature of the third concentration treatment is 45 to 55 ℃, and the volume ratio of a third concentrated solution obtained by the third concentration treatment to the supernatant is 1:5 to 15, preferably 1:10; the conditions for freeze-drying include: the temperature is-60 to-50 ℃, and the time is 36 to 72 hours.
The third aspect of the invention provides an application of the novel pectin oligosaccharide in a product for increasing intestinal short-chain fatty acids and improving intestinal probiotic functions; in one embodiment of the present invention, the product is at least one selected from the group consisting of food, beverage, health product and pharmaceutical product.
The invention is further illustrated by the following examples, but is not to be construed as being limited thereto.
The starting materials used in the examples and comparative examples were obtained by a commercial route without specific indication.
The centrifugation in the invention adopts a conventional centrifuge.
The high performance exclusion chromatograph of the present invention is model L2130 (Hitachi, japan).
The ion chromatograph of the present invention is model ICS-3000 (Sunyvale, USA).
The nitrogen blower of the present invention was model DCY-24B (Shen Bei scientific instruments (Suzhou) Inc.).
The Fourier Infrared spectrometer of the present invention is a TENSOR 27 FTIR (Bruker, germany).
Example 1
This example illustrates the preparation of pectin oligosaccharides having a molecular weight of 500 to 3500 Da.
(1) And (3) pectin enzymolysis: pectin as a raw material was dissolved in 1L of 0.1mol/L citric acid-disodium hydrogen phosphate buffer solution at pH 4.5 to give a pectin concentration of 1.0%. Adding a compound pectinase preparation (enzyme activity is 7700U/mL) into the system for enzymolysis, wherein the dosage of the enzyme is 0.2 mL compound pectinase is added into a pectin solution with the concentration of 1% per 1000 mL, the enzymolysis temperature is 45 ℃, and the enzymolysis time is 2.5 h. After the enzymolysis is finished, the system is put into a boiling water bath to be heated for 10 min so as to inactivate the enzyme, after the system is cooled to the room temperature, the reaction system is centrifuged for 15 min at the rotating speed of 8000 r/min so as to take supernatant.
(2) And (3) grading enzymolysis products: and (3) performing vacuum rotary concentration on the centrifuged supernatant at 50 ℃ to 1/4 of the original volume to obtain a concentrated solution, dialyzing the concentrated solution by using a semipermeable membrane with the molecular weight cutoff of 500 Da to remove inorganic salt in a buffer solution system and small molecular oligosaccharides generated in the enzymolysis process, concentrating the solution in a dialysis bag to 1/10 of the original volume after dialysis is finished, then dialyzing by using a semipermeable membrane with the molecular weight cutoff of 3500 Da, collecting the solution outside the dialysis bag after dialysis is finished, concentrating the solution to 1/10 of the original volume, and then performing freeze drying to obtain pectin oligosaccharides with the molecular weight of 500-3500 Da, wherein the name of the pectin oligosaccharides is S-5.
Example 2
This example illustrates the isolation and purification of citrus pectin oligosaccharides having a molecular weight of 500 to 3500 Da.
And (3) separating and purifying pectin oligosaccharide: the citrus pectin oligosaccharide S-5 having a molecular weight of 500 to 3500 Da prepared in example 1 was further isolated and purified by Sephadex G-15 gel column (10 mm. Times.40 cm). A50 mgS-5 sample was dissolved in 2mL of ultrapure water to a concentration of 25 mg/mL. Loading the pectin oligosaccharide on an activated gel column, eluting with ultrapure water at the flow rate of 1.5 mL/min, collecting 1 tube of eluent every 2min, measuring the absorbance at 490 nm by adopting a phenol-sulfuric acid method, collecting the components with the absorbance being more than 0.3, mixing, and freeze-drying to obtain the separated and purified pectin oligosaccharide, which is named as PS-5 for subsequent fine structure identification.
Comparative example 1
This comparative example illustrates the preparation of a low molecular weight pectin having a molecular weight greater than 500 Da.
(1) And (3) pectin enzymolysis: pectin is used as raw material, and is dissolved in 1L, 0.1mol/L, and pH 4.5 citric acid-disodium hydrogen phosphate solution to make pectin concentration be 1%. Adding screened pectinase preparation (compound pectinase preparation, with the enzyme activity of 7700U/mL) into the system for enzymolysis, wherein the dosage of the enzyme is 0.2 mL compound pectinase preparation added into 1% pectin solution per 1000 mL, and the enzymolysis time is 2.5 h at 45 ℃. After the enzymolysis is finished, the system is put into a boiling water bath to be heated for 10 min to inactivate the enzyme, and after the system is cooled to the room temperature, the reaction system is centrifuged for 15 min at the rotating speed of 8000 r/min to obtain supernatant.
(2) Grading enzymolysis products: and rotationally concentrating the centrifuged supernatant to 1/4 of the original volume under vacuum at 50 ℃ to obtain a concentrated solution, dialyzing the concentrated solution by using a semipermeable membrane with the molecular weight cutoff of 500 Da to remove inorganic salt in a buffer solution system and micromolecule oligosaccharide generated in the enzymolysis process, concentrating the solution in a dialysis bag to 1/10 of the original volume after dialysis is finished, and freeze-drying the concentrated solution to obtain a low-molecular pectin enzymolysis product with the molecular weight of more than 500 Da, which is named as SP.
Comparative example 2
This comparative example illustrates the preparation of a low molecular weight pectin having a molecular weight greater than 3500 Da.
(1) And (3) pectin enzymolysis: pectin as a raw material was dissolved in 1L of 0.1mol/L citric acid-disodium hydrogen phosphate buffer solution at pH 4.5 to give a pectin concentration of 1%. Adding pectinase (compound pectinase preparation, with the enzyme activity of 7700U/mL) into the system for enzymolysis, wherein the dosage of the pectinase is 0.2 mL compound pectinase preparation r per 1% pectin solution with the concentration of 1000 mL, the enzymolysis temperature is 45 ℃, and the enzymolysis time is 2.5 h. After the enzymolysis is finished, the system is put into a boiling water bath to be heated for 10 min so as to inactivate the enzyme, after the system is cooled to the room temperature, the reaction system is centrifuged for 15 min at the rotating speed of 8000 r/min so as to take supernatant.
(2) Grading enzymolysis products: and (3) performing vacuum rotary concentration on the centrifuged supernatant at 50 ℃ to 1/4 of the original volume to obtain a concentrated solution, dialyzing the concentrated solution by using a semipermeable membrane with the molecular weight cutoff of 500 Da to remove inorganic salts in a buffer solution system and micromolecule oligosaccharides generated in the enzymolysis process, concentrating the solution in a dialysis bag to 1/10 of the original volume after dialysis, then dialyzing by using the semipermeable membrane with the molecular weight cutoff of 3500 Da, collecting the solution in the dialysis bag after dialysis, concentrating the solution to 1/10 of the original volume, and freeze-drying to obtain a low-molecular pectin enzymolysis product with the molecular weight of more than 3500 Da, which is named as S-35.
Comparative example 3
This comparative example serves to illustrate the preparation of pectin products enzymatically digested using polygalacturonase.
The preparation method was the same as in example 1, except that the enzyme preparation in step S2 was a polygalactosidase.
Grading enzymolysis products: concentrating the centrifuged supernatant at 50 deg.C under vacuum rotary concentration to 1/4 of the original volume to obtain concentrated solution, and dialyzing with semi-permeable membrane with molecular weight cutoff of 3500 Da to obtain pectin product 3 with molecular weight of 3500 Da.
Comparative example 4
This comparative example serves to illustrate the preparation of an enzymatic pectin product using beta-glucanase.
The preparation method was the same as in example 1, except that the enzyme preparation in step S2 was β -glucanase.
Grading enzymolysis products: and (3) rotationally concentrating the centrifuged supernatant under vacuum at 50 ℃ to 1/4 of the original volume to obtain a concentrated solution, and dialyzing the concentrated solution by using a semi-permeable membrane with the molecular weight cutoff of 3500 Da to obtain a pectin product 4 with the molecular weight of 3500 Da.
Test example 1
This test example serves to illustrate the structural characterization of the pectin oligosaccharides.
Physical and chemical property characterization: the yield of the pectin enzymolysis product is = M1/M0 × 100%, wherein M1 is the mass of the pectin enzymolysis product, and M0 is the mass of the original pectin.
A sample of 5 mg was dissolved in 1mg/mL 0.1M NaCl and filtered through a 0.22 μ M microporous membrane before loading and molecular weight determination by high performance exclusion chromatography.
The monosaccharide composition of pectin enzymolysis product is determined by ion chromatography, and 10 mg sample is hydrolyzed by adding 2M trifluoroacetic acid 5mL, charging nitrogen gas at 120 deg.C, and hydrolyzing 1.5 h. Blowing and drying by using a nitrogen blowing instrument after the hydrolysis is finished, re-dissolving the hydrolysate to 1mg/mL by using ultrapure water, diluting by 10 times, passing through a 0.45 mu m microporous filter membrane, and detecting on a machine, wherein the result is shown in Table 1. Mixing the sample and potassium bromide powder by 1 (m/m), fully grinding and tabletting, and measuring by a Fourier infrared spectrometer at 4000-400 cm -1 In-range scanning to analyze the structure. The results are shown in FIG. 5.
TABLE 1
The monosaccharide composition and the molecular weight of the pectin enzymatic hydrolysis product are shown in table 1, and the content of the pectin enzymatic hydrolysis product RG-I in the embodiment 1 of the invention can be as high as 83.95%, and the pectin enzymatic hydrolysis product is a pectin enzymatic hydrolysis product which is low in molecular weight and rich in RG-I structural domain. Infrared spectroscopy (FIG. 5) shows that it contains-OH, -CH 2 、-CH 3 -COOH and-COOCH 3 Absorption of vibration by polysaccharide representative functional group, and esterification of carboxyl group (-COOCH) 3 ) All absorption peaks are lower than the carboxylic acid groupThe absorption peak of the group (-COOH) shows that the degree of methyl ester is low, and the degree of esterification is 24.57-37.54%.
Test example 2
The method for researching the in vitro fermentation characteristics of the pectin enzymolysis product comprises the following steps:
and (3) fermentation performance characterization: fresh stool samples were collected from 4 healthy volunteers (age 22-30 years, no history of antibiotic use over four months, no gastrointestinal disease) and mixed in equal amounts. The feces were diluted and homogenized with sterile PBS (0.1m, ph 7.2) at a rate of 1:9, the mixture was centrifuged (300 xg, 4C, 5 min), and the supernatant was collected as an inoculum. Samples of 300 mg (commercial pectin, SP, S-5, S-35, and positive control inulin) were dissolved in 27 mL sterile medium (121C, 15 min), 3 mL fecal inoculum was added to 100 mL sterile anaerobic bottles containing 27 mL fermentation medium in a sterile anaerobic incubator with basal medium without additional carbon source used for blank Control (CK), three experiments were performed on each sample, 5mL fermentation samples were collected at 0, 6, 12, 24, 48, 72h and transferred quickly to ice bath to stop fermentation, and then centrifuged at 13000 xg, 4 ℃ for 15 min. The supernatant and pellet were frozen in liquid nitrogen and kept at-80 ℃ in a refrigerator for further analysis. Wherein the supernatant is subjected to gas chromatography to determine the content of short-chain fatty acids; the pellet was used to sequence the microorganisms using 16S rRNA. The results of short-chain fatty acids (fig. 6-9) show that, compared with the original commercial pectin, the content of acetic acid, propionic acid and total short-chain fatty acids in the prepared citrus pectin enzymatic hydrolysate is remarkably increased in the in vitro fermentation process, especially the content of acetic acid and total short-chain fatty acids in the pectin oligosaccharide S-5 with the lowest molecular weight is remarkably higher than that of the accepted prebiotics, namely inulin, so that the low-molecular-weight pectin enzymatic hydrolysate has better fermentation performance and has potential as a prebiotic. The results of the intestinal microbial composition (fig. 10) show that the pectin enzymatic hydrolysate can change the structure of intestinal microbes at different taxonomic levels, thereby exerting influence on the body.
The fine structure analysis method of the pectin oligosaccharide with high short-chain fatty acid yield is as follows:
fine structure characterization: use S-5 as SAnd (3) after the ephadex G-15 column chromatography is analyzed and purified, adopting one-dimensional nuclear magnetism (hydrogen spectrum and carbon spectrum) and two-dimensional nuclear magnetism (COSY, HMQC and HMBC) to represent the fine structure of the pectin oligosaccharide S-5. A30 mg sample was completely dissolved in 1.5mL of heavy water (D) 2 O), freeze-drying 28 h, repeating this procedure three times to maximize deuterium exchange from interfering with hydrogen atoms in the solution. The resulting mixture was reconstituted with repeated water to a concentration of 20 mg/mL and placed in a 5 mm NMR tube for testing using a nuclear magnetic spectrometer. Nuclear magnetic results (FIGS. 11-15) indicate that pectin oligosaccharides of the present invention are purified to contain predominantly → 1) - α -GalAp-(4→、→2)-α-Rhap-(1→、→4)-α-GalpA、→3)-β -Galp- (1 →. Based on the two-dimensional nuclear magnetic results, the main chain structure thereof is presumed to be → 4) - α -GalAp-(1→4)-α-GalAp-(1→4)-α-GalAp-(1→4)-α-GalAp-(1→2)-α-Rhap-(1→4)-α-GalApMeanwhile, the O-3 position of Rha is replaced by a galactan side chain connected with a beta-1,3 glycosidic bond, and the structure is shown in figure 16.
The preferred embodiments of the present invention have been described in detail with reference to the accompanying drawings, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications all fall within the protection scope of the present invention.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.
In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.
Claims (10)
1. A novel pectin oligosaccharide is characterized in that the structural formula of the novel pectin oligosaccharide is shown as a formula (1):
formula (1).
2. The novel pectin oligosaccharide according to claim 1, wherein the molecular weight of the novel pectin oligosaccharide is 500 to 3500 g/mol.
3. A method for preparing a novel pectin oligosaccharide according to claim 1 or 2, comprising the steps of:
s1, dissolving a pectin raw material in a buffer solution to obtain a first raw material solution;
s2, adding an enzyme preparation into the first raw material liquid for carrying out enzymolysis reaction to obtain an enzymolysis liquid; inactivating the enzymolysis liquid, and then carrying out first centrifugal treatment to obtain a first clear liquid;
s3, carrying out first concentration treatment on the first supernatant to obtain a first concentrated solution, and carrying out first semi-permeable membrane dialysis on the first concentrated solution to obtain a first dialysate and a first retentate; carrying out second concentration treatment on the first retentate to obtain a second concentrated solution, and dialyzing the second concentrated solution through a second semipermeable membrane to obtain a second dialyzate and a second retentate;
s4, performing third concentration treatment and freeze drying on the second dialysate respectively;
the pH value of the buffer solution is 4.0 to 5.0, and the buffer solution is selected from at least one of a citrate-phosphate buffer solution, an acetic acid-sodium acetate buffer solution, a phosphate buffer solution and a potassium hydrogen phthalate-sodium hydroxide buffer solution; the molecular weight cut-off of the first semi-permeable membrane is 500-1000Da, and the molecular weight cut-off of the second semi-permeable membrane is 3500-4000 Da.
4. The method according to claim 3, wherein the concentration of pectin in the first raw material liquid is 0.5 to 1.5wt%;
optionally, the pectin material is selected from at least one of citrus pectin material, apple pectin material, beet pectin material and fig pectin material, preferably citrus pectin material;
further preferably, the preparation method of the citrus pectin raw material comprises the following steps: crushing orange peel, and sieving the crushed orange peel to obtain a first orange peel raw material; adding deionized water into a first orange peel raw material, wherein the ratio of the weight of the first orange peel raw material to the volume of the deionized water is 1:20 to 35, and adjusting the pH value to be 1.5 to 3 to obtain a first mixed solution; stirring the first mixed solution, and then carrying out second centrifugal treatment to obtain a second supernatant; adding ethanol into the second supernatant, wherein the volume ratio of the second supernatant to the ethanol is 1:2~3 to obtain a second mixed solution; carrying out precipitation treatment on the second mixed solution, and then carrying out suction filtration treatment to obtain a first precipitate; and drying and crushing the first precipitate.
5. The method according to claim 3, wherein in step S2, the enzyme preparation is a composite pectinase preparation containing polygalacturonase, beta-galactosidase, arabinanase and arabinofuranosidase;
the mass ratio of the polygalacturonase to the beta-galactosidase to the arabinanase to the arabinofuranosidase is 23 to 28:10 to 15:10 to 15:20 to 25.
6. The method of claim 3, wherein the enzyme activity of the enzyme preparation is 7700U/mL; the addition amount of the enzyme preparation is 0.2-0.6mL per liter of the first raw material liquid.
7. The method of claim 6, wherein in step S2, the conditions of the enzymatic reaction include: the temperature is 40 to 50 ℃, preferably 43 to 47 ℃; the time is 2 to 3h, preferably 2.5 to 3h;
the step of inactivating comprises: heating the enzymolysis liquid in a boiling water bath for 10 to 15 min, and cooling to 20 to 30 ℃;
the conditions of the first centrifugation treatment include: the revolution number is 8000-10000 r/min; the time is 10 to 20min.
8. The method of claim 3, wherein the buffer is a citrate-phosphate buffer; the pH value of the buffer solution is 4.3 to 4.7;
optionally, the citrate in the citrate-phosphate buffer is selected from one or more of citric acid, sodium citrate and citric acid monohydrate; the phosphate is one or more selected from disodium hydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate dihydrate and dipotassium hydrogen phosphate dihydrate.
9. The method according to claim 3, wherein in the step S3, the first concentration treatment is vacuum low-temperature concentration, the temperature of the first concentration treatment is 45-55 ℃, and the volume ratio of the first concentrated solution to the supernatant is 1:3~5, preferably 1:4;
the second concentration treatment is vacuum low-temperature concentration, the temperature of the second concentration treatment is 45-55 ℃, and the volume ratio of the second concentrated solution to the supernatant is 1:5 to 15, preferably 1:10;
the third concentration treatment is vacuum low-temperature concentration, the temperature of the third concentration treatment is 45-55 ℃, and the volume ratio of a third concentrated solution obtained by the third concentration treatment to the supernatant is 1:5 to 15, preferably 1:10;
the conditions for freeze-drying include: the temperature is-60 to-50 ℃, and the time is 36 to 72h.
10. Use of a novel pectin oligosaccharide according to claim 1 or 2 or prepared by the method according to any one of claims 3-9 in a product for increasing intestinal short-chain fatty acids and improving intestinal probiotic function; optionally, the product is selected from at least one of a food, a beverage, a nutraceutical, and a pharmaceutical.
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Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996006163A1 (en) * | 1994-08-18 | 1996-02-29 | Novo Nordisk A/S | Novel rhamnogalacturonan rhamnosidases |
| KR20060004008A (en) * | 2004-07-08 | 2006-01-12 | 주식회사 이코바이오 | Development of plant growth stimulant using uronic acid oligosaccharides |
| WO2015192247A1 (en) * | 2014-06-21 | 2015-12-23 | The Royal Institution For The Advancement Of Learning/Mcgill University | Enzymatic production of prebiotic oligosaccharides from potato pulp |
| CN106460020A (en) * | 2014-04-30 | 2017-02-22 | 帝斯曼知识产权资产管理有限公司 | Process for enzymatic hydrolysis of lignocellulosic material and fermentation of sugars |
| WO2019061018A1 (en) * | 2017-09-26 | 2019-04-04 | 浙江大学 | Method for extracting pectin rich in rg-i |
| WO2019081525A1 (en) * | 2017-10-23 | 2019-05-02 | Nutrileads B.V. | Method of producing a pectic polysaccharide isolate enriched in rhamnogalacturonan-i |
| CN110511298A (en) * | 2019-09-09 | 2019-11-29 | 浙江大学 | A method of extracting the RG-I pectin polysaccharide for being rich in galactose side |
| CN110652013A (en) * | 2019-09-25 | 2020-01-07 | 中国农业科学院农产品加工研究所 | Method for efficiently preparing citrus pectin capable of relieving type II diabetes |
| CN110801028A (en) * | 2019-12-06 | 2020-02-18 | 中国农业科学院农产品加工研究所 | Modified apple pectin with excellent colonic fermentation characteristic and preparation method thereof |
| CN111171179A (en) * | 2020-01-15 | 2020-05-19 | 中国农业科学院农产品加工研究所 | Citrus pectin with intestinal probiotic function and preparation method and application thereof |
-
2023
- 2023-03-10 CN CN202310252342.1A patent/CN115960276B/en active Active
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996006163A1 (en) * | 1994-08-18 | 1996-02-29 | Novo Nordisk A/S | Novel rhamnogalacturonan rhamnosidases |
| KR20060004008A (en) * | 2004-07-08 | 2006-01-12 | 주식회사 이코바이오 | Development of plant growth stimulant using uronic acid oligosaccharides |
| CN106460020A (en) * | 2014-04-30 | 2017-02-22 | 帝斯曼知识产权资产管理有限公司 | Process for enzymatic hydrolysis of lignocellulosic material and fermentation of sugars |
| WO2015192247A1 (en) * | 2014-06-21 | 2015-12-23 | The Royal Institution For The Advancement Of Learning/Mcgill University | Enzymatic production of prebiotic oligosaccharides from potato pulp |
| WO2019061018A1 (en) * | 2017-09-26 | 2019-04-04 | 浙江大学 | Method for extracting pectin rich in rg-i |
| WO2019081525A1 (en) * | 2017-10-23 | 2019-05-02 | Nutrileads B.V. | Method of producing a pectic polysaccharide isolate enriched in rhamnogalacturonan-i |
| CN111683540A (en) * | 2017-10-23 | 2020-09-18 | 纽崔莱公司 | Method for producing pectin polysaccharide isolate rich in rhamnogalacturonan-I |
| CN110511298A (en) * | 2019-09-09 | 2019-11-29 | 浙江大学 | A method of extracting the RG-I pectin polysaccharide for being rich in galactose side |
| CN110652013A (en) * | 2019-09-25 | 2020-01-07 | 中国农业科学院农产品加工研究所 | Method for efficiently preparing citrus pectin capable of relieving type II diabetes |
| CN110801028A (en) * | 2019-12-06 | 2020-02-18 | 中国农业科学院农产品加工研究所 | Modified apple pectin with excellent colonic fermentation characteristic and preparation method thereof |
| CN111171179A (en) * | 2020-01-15 | 2020-05-19 | 中国农业科学院农产品加工研究所 | Citrus pectin with intestinal probiotic function and preparation method and application thereof |
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