CN111363057A

CN111363057A - Hierarchical preparation method of ganoderan based on multistage membrane separation technology

Info

Publication number: CN111363057A
Application number: CN202010207095.XA
Authority: CN
Inventors: 蔡铭; 孙培龙; 邢浩永; 杨开
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2020-03-23
Filing date: 2020-03-23
Publication date: 2020-07-03

Abstract

The invention provides a hierarchical preparation method of ganoderma lucidum polysaccharide based on a multistage membrane separation technology, the molecular weight of the hierarchical polysaccharide obtained by the method can be regulated, and the hierarchical target products mainly comprise different fractions of ganoderma lucidum polysaccharide with the molecular weights of more than 100-50kDa, 10-5kDa, 1kDa and less than 1kDa, which can be selected according to requirements; the invention has simple process, short production period, mild treatment condition and low energy consumption; the used raw materials and reagents have wide sources and low cost, and the industrial large-scale production is easy to realize.

Description

Hierarchical preparation method of ganoderan based on multistage membrane separation technology

Technical Field

The invention relates to a grading preparation method of ganoderma lucidum polysaccharide, in particular to a method for grading ganoderma lucidum polysaccharide by utilizing a multistage membrane separation technology.

Background

Ganoderma lucidum is rich in chemical components and is considered as a biological production plant for active substances. Polysaccharides and triterpenoids are among the most important functional components. In addition, nucleic acids, furans, sterols, alkaloids, amino acid polypeptides, etc. are also included. Ever more reports have been made on ganoderan since the isolation of highly branched arabinoxyloglucan from the fruiting body of ganoderma lucidum and its confirmation of antitumor activity. Most of reported ganoderma lucidum polysaccharides are heteropolysaccharides with complex structures, mainly formed by connecting glucose, galactose, mannose, fucose, xylose and arabinose by different proportions and different glycosidic bond types, and part of ganoderma lucidum polysaccharides are also compounded with protein or polypeptide residues, phenolic substances and the like.

The existing research shows that the ganoderma lucidum polysaccharide has good physiological functions in the aspects of immunoregulation, tumor resistance, oxidation resistance, blood sugar reduction, intestinal health regulation and the like. Meanwhile, researches show that the ganoderan with different molecular weights has different physiological activities. Therefore, it is necessary to prepare polysaccharides by fractionation. At present, most of the grading preparation of the ganoderma lucidum polysaccharide mainly utilizes ethanol with different concentrations to grade the ganoderma lucidum polysaccharide. The method needs a large amount of ethanol, is time-consuming and material-consuming, and has low efficiency.

The membrane separation technology is a separation process which utilizes a separation membrane with selective sieving function, takes pressure difference, concentration difference, potential difference and the like at two sides of the membrane as driving forces, and achieves the separation purpose by means of the selective difference of mass transfer of all components in the membrane. The membrane separation technology has no phase change and secondary pollution in the process, is convenient to operate, compact in structure, low in maintenance cost and easy to automate, and is a separation means with higher efficiency in the modern separation technology. The membrane separation technology comprises various processes, including microfiltration, ultrafiltration, nanofiltration, reverse osmosis and the like, and the membrane separation technology taking pressure as driving force is widely researched in the aspects of extraction and separation of functional components.

At present, some patents and studies have been directed to polysaccharide fractionation. For example:

application publication No. CN206298538U relates to a plant polysaccharide hierarchical purification device. The device comprises a liquid storage device and an ultrafiltration membrane device, wherein a high-temperature high-pressure reaction kettle is connected in front of the liquid storage device; the ultrafiltration membrane device is connected with a backwashing pump; the back flush pump is connected with the high-temperature high-pressure reaction kettle through the recovery tank. The utility model discloses a combine high temperature high pressure reation kettle and ultrafiltration separation to realize hierarchical purification, establish high temperature high pressure physical degradation and membrane separation integrated device, carry out effective control to the molecular weight of degradation polysaccharide. However, this invention does not disclose a specific method for separating a polysaccharide membrane.

The invention with application publication number CN106146680A relates to a method for preparing lycium barbarum polysaccharides with different molecular weights by ultrasonic coupling membrane separation and classification, which comprises the steps of extracting lycium barbarum residues by ultrasonic waves to obtain a polysaccharide solution, standing overnight, performing solid-liquid separation by a nylon net, centrifuging, performing ultrafiltration by a 6.7 ten thousand molecular weight membrane, performing ultrafiltration by a 10 ten thousand molecular weight membrane, respectively collecting ultrafiltrates with molecular weights of less than 6.7 ten thousand, 6.7-10 ten thousand and molecular weights of more than 10 ten thousand to obtain three lycium barbarum polysaccharide solutions with different molecular weights, concentrating, and freeze-drying to obtain the lycium barbarum polysaccharide powders with different purposes. The method firstly utilizes a membrane with smaller pore diameter to carry out ultrafiltration, thus easily causing the problems of serious membrane pollution, lower separation efficiency and the like, and being incapable of better separating polysaccharide components with different molecular weight grades.

Disclosure of Invention

The invention aims to provide a method for preparing ganoderma lucidum hierarchical polysaccharide based on a membrane separation technology, the molecular weight of the hierarchical polysaccharide obtained by the method can be regulated and controlled, and the hierarchical polysaccharide mainly comprises different fractions of ganoderma lucidum polysaccharide with the molecular weights of more than 100kDa, 100k-10kDa, 10k-1kDa and less than 1kDa, and the preparation process has the advantages of short time consumption, high efficiency, no chemical pollution and low energy consumption, and can obtain different fractions of ganoderma lucidum polysaccharide.

The technical scheme of the invention is as follows:

a method for preparing Ganoderma lucidum polysaccharide by stages based on multi-stage membrane separation technology comprises:

(1) mixing the lucid ganoderma sporocarp with distilled water according to the mass ratio of 1: 10-20 (preferably 1: 15), leaching for 1-3h at 90-95 ℃, performing suction filtration, repeating the leaching operation on filter residues, and combining the two filtrates (filtering insoluble solid matters) to obtain an extracting solution for later use;

(2) placing the extracting solution obtained in the step (1) as a raw material solution into a material liquid tank of a high-pressure flat membrane machine, filling a 100k-50kDa ultrafiltration membrane into the high-pressure flat membrane machine, sealing and isolating oxygen in a container, ensuring that the interior of the container is in an aseptic state, carrying out ultrafiltration at the pressure of 0.5-1.4MPa and the rotating speed of 300-900r/min, collecting a permeate for later use, and carrying out vacuum freeze drying on a trapped fluid to obtain primary graded polysaccharide (polysaccharide with the molecular weight of more than 100 kDa);

preferably, permeate and retentate are collected when the feed solution is ultrafiltered to 1/5 of its original volume;

in addition, ultrafiltration membranes with different combinations can be adopted in the step (2), and ultrafiltration membrane combinations with obvious grading effects can be selected according to requirements; meanwhile, in the step (2), after the volume of the raw material liquid is ultrafiltered to 1/5 of the original volume, water is added for dilution, so that the residual polysaccharide with the pore diameter smaller than that of the ultrafiltration membrane can permeate out of the raw material liquid;

(3) taking the permeate (less than 100k-50kDa) obtained in the step (2) as a raw material, carrying out membrane treatment by using a 10k-5kDa filter membrane, collecting the permeate and the retentate under the same pressure and rotation speed as those in the step (2), and carrying out vacuum freeze drying on the retentate to obtain secondary graded polysaccharide (polysaccharide with the molecular weight of more than 10k-5kDa and less than 100 kDa);

(4) and (3) taking the permeate (less than 10k-5kDa) obtained in the step (3) as a raw material, performing membrane treatment by using a 1kDa filter membrane, collecting the retentate and the permeate under the same pressure and rotation speed as those in the step (2), and respectively performing vacuum freeze drying to obtain three times and four times of fractionated polysaccharides (10k-1kDa polysaccharide and oligosaccharide less than 1 kDa).

Compared with the prior art, the invention has the following beneficial effects:

1. the molecular weight of the obtained ganoderan can be regulated, and the grading target products mainly comprise different fractions of ganoderan with a molecular weight of more than 100kDa, 100k-10kDa, 10k-1kDa and less than 1kDa, and can be selected according to requirements.

2. The invention has simple process, short production period, mild treatment condition and low energy consumption. The used raw materials and reagents have wide sources and low cost, and the industrial large-scale production is easy to realize.

Drawings

FIG. 1 is a flow chart of membrane separation process for preparing Ganoderma lucidum hierarchical polysaccharide.

Detailed Description

The invention will be further illustrated with reference to the following specific examples, without limiting the scope of the invention thereto. The experimental methods in the following examples, which are not specified under specific conditions, are generally performed under conventional conditions.

The Ganoderma encarpium is purchased from Zhejiang shouxian Gu pharmaceutical Co., Ltd.

Example 1

A process for preparing ganoderma lucidum hierarchical polysaccharide based on a membrane separation technology comprises the following specific steps:

(1) weighing about 100.00g of Ganoderma, adding 1500g of distilled water, leaching at 90-95 deg.C for 2 hr, vacuum filtering, leaching the residue once, mixing the two filtrates, and performing membrane separation test.

(2) Placing the suction filtration liquid obtained in the step (1) into a liquid feed tank of a high-pressure flat membrane machine, placing a 100kDa PES ultrafiltration membrane into the high-pressure flat membrane machine, sealing and isolating oxygen in a container, ensuring that the interior of the container is in an aseptic state, operating at the pressure of 0.5MPa and the rotating speed of 600r/min, and collecting trapped liquid and penetrating liquid after the raw material liquid is concentrated to the original 1/5 after ultrafiltration.

(3) And (3) filtering the penetrating fluid obtained in the step (2) serving as a raw material fluid through a PES ultrafiltration membrane of 10kDa, sealing the container to isolate oxygen, ensuring that the interior of the container is in an aseptic state, performing ultrafiltration at the pressure of 0.6MPa and the rotating speed of 600r/min, concentrating the raw material fluid to 1/5 of the original fluid after ultrafiltration, and collecting a trapped fluid and the penetrating fluid.

(4) And (3) filtering the penetrating fluid in the step (3) serving as a raw material fluid through a 1kDa PES ultrafiltration membrane, sealing the container to isolate oxygen, ensuring that the interior of the container is in an aseptic state, performing ultrafiltration at the pressure of 0.8MPa and the rotating speed of 600r/min, concentrating the raw material fluid to 1/5 after ultrafiltration, and collecting a trapped fluid and the penetrating fluid.

(5) The total sugars, reducing sugars, uronic acids and proteins in the different fractions are shown in the following table:

TABLE 1 polysaccharide, protein and uronic acid content of ganoderan with different molecular weights

Example 2

(2) And (2) placing the suction filtration liquid obtained in the step (1) into a liquid feed tank of a high-pressure flat membrane machine, placing a 100kDa PES ultrafiltration membrane into the high-pressure flat membrane machine, sealing the container to isolate oxygen, ensuring that the interior of the container is in an aseptic state, performing ultrafiltration at the pressure of 0.5MPa and the rotating speed of 600r/min, adding 300mL of distilled water after the volume of the raw material liquid is ultrafiltered to 1/5 of the original volume, repeating for three times, and collecting trapped liquid and penetrating liquid.

(3) And (3) filtering the penetrating fluid obtained in the step (2) serving as a raw material fluid through a PES ultrafiltration membrane of 10kDa, sealing the container to isolate oxygen, ensuring that the interior of the container is in an aseptic state, performing ultrafiltration at the pressure of 0.6MPa and the rotating speed of 600r/min, adding 200mL of distilled water after the volume of the raw material fluid is ultrafiltered to 1/5 of the original volume, repeating the steps for three times, and collecting the trapped fluid and the penetrating fluid.

(4) And (3) filtering the penetrating fluid serving as the raw material fluid through a 1kDa PES ultrafiltration membrane, sealing the container to isolate oxygen, ensuring that the interior of the container is in an aseptic state, performing ultrafiltration at the pressure of 0.8MPa and the rotating speed of 600r/min, adding 100mL of distilled water after the volume of the raw material fluid is subjected to ultrafiltration by 1/5 of the original volume, repeating the steps for three times, and collecting trapped fluid and the penetrating fluid.

TABLE 2 polysaccharide, protein and uronic acid content of ganoderan with different molecular weights

Claims

1. A ganoderma lucidum polysaccharide grading preparation method based on a multistage membrane separation technology is characterized by comprising the following steps:

(1) mixing the lucid ganoderma sporocarp with distilled water according to the mass ratio of 1: 10-20, leaching for 1-3 hours at 90-95 ℃, performing suction filtration, repeating the leaching operation on filter residues, and combining filtrates of the two times to obtain an extracting solution for later use;

(2) placing the extracting solution obtained in the step (1) as a raw material solution into a material liquid tank of a high-pressure flat membrane machine, filling a 100k-50kDa ultrafiltration membrane into the high-pressure flat membrane machine, sealing and isolating oxygen in a container, ensuring that the interior of the container is in an aseptic state, carrying out ultrafiltration at the pressure of 0.5-1.4MPa and the rotating speed of 300-900r/min, collecting a permeation solution for later use, and carrying out vacuum freeze drying on a trapped solution to obtain primary graded polysaccharide;

(3) taking the permeate obtained in the step (2) as a raw material, carrying out membrane treatment by using a 10k-5kDa filter membrane, collecting the permeate and the trapped fluid under the same pressure and rotation speed as those in the step (2), and carrying out vacuum freeze drying on the trapped fluid to obtain secondary graded polysaccharide;

(4) and (3) taking the permeate obtained in the step (3) as a raw material, performing membrane treatment by using a 1kDa filter membrane, collecting the trapped fluid and the permeate at the same pressure and rotation speed as those in the step (2), and respectively performing vacuum freeze drying to obtain three-time and four-time fractionated polysaccharides.

2. The method for preparing ganoderan based on multi-stage membrane separation technology according to claim 1, wherein the permeate and retentate are collected when the raw material solution is ultrafiltered to 1/5 of the original volume.