TWI395813B - Manufacturing mmethod for obtaining polysaccharide from the solid fermentation product of antrodia camphorate - Google Patents

Description

Preparation method of crude polysaccharide of solid fermentation product of Antrodia camphorata mycelium

The present invention relates to a method for preparing a crude polysaccharide, and more particularly to a method for preparing a crude polysaccharide extracted from a solid fermentation product of Antrodia camphorata mycelium.

Antrodia camphorata , also known as Antrodia camphorata or Burdock mushroom, belongs to the fungi community (Fungi), Basidiomycota, Basidiomycotina, and Homobasidiomycetes. Aphyllophorales, Polyporaceae, Antrodia, Antrodia camphorata grown on the inner wall of the heartwood of Cinnamoum kanehirai Hay in Taiwan, the periphery is curved inward and can be decomposed The cellulose of wood causes the wood to decay and is a decaying wood. The growth environment of wild Antrodia camphorata belongs to the dark, humid and low temperature middle altitude areas, especially the wild Antrodia camphora grows slowly, so it takes a long time to produce fruiting bodies. Because the burdock tree is a kind of conservation tree species and the distribution is extremely rare, coupled with the artificial piracy, the number of wild burdock is quite rare, making the burdock an expensive price, so it has the reputation of "the ruby in the forest".

The body of Ox sylvestris is perennial, sessile and cork-to-wood. It has a strong aroma of eucalyptus and has a variety of shapes. It has a plate shape, a bell shape, a horseshoe shape or a tower shape. It is close to the surface of the wood and has a pore shape on the surface of the mushroom. It is bright red at the beginning of life, and becomes milky white, reddish brown, light brown or yellowish brown as the growth progresses. The periphery is often radial and spreads to the periphery, showing a semicircular or irregular shape.

Some species of Antrodia camphorata have been found to have pharmacological activity. In Taiwan folk medicine therapy, the body of A. sylvestris is considered to have certain medical effects. The fruiting body is finely ground into a dry powder or taken orally in a boiled way, which is effective for folk circulation. The edible mushroom has the activities of detoxification, anti-inflammatory, anti-tumor, inhibition of vascular proliferation and treatment of liver diseases.

As a result of the above-mentioned knowledge of the pharmacological activity of Antrodia camphorata, there is no shortage of health products of the Antrodia camphorata series. At present, the related health products of Niuzhizhi sold in the market are processed by a mycelium from the body of the burdock. The main purpose is to eat the mycelium for health care. However, Antrodia camphorata is a complex rot fungus. Some studies have indicated that when cultured and screened for the mycelium of Antrodia camphorata, the carcinogenic strains will be screened, indicating that not all ingredients are beneficial to human health after eating, and may also contain certain Toxins, if consumed rashly, can affect health, produce unpredictable side effects, and lose health care. Furthermore, in order to destroy the health hazard of non-pharmacological active substances in Antrodia camphorata, a high-temperature heating step is selected to ensure the safety of eating. However, the high-temperature heating step can also lose the activity of the active ingredients in Antrodia camphorata, and lose the Antrodia camphorata. The value of pharmacological activity, so finding and isolating an effective and safe strain of mycelium of A. angustifolia (isolated strain) is an important issue.

At present, from many studies on Antrodia camphorata, it is known that the composition of Antrodia camphorata is very complicated. It is found that the body complex of A. angustifolia contains unique and different skeletons of triterpenoids. It has also been found that the composition of A. annuum contains polysaccharides. The mechanism of the polysaccharide and the regulation of the immune response indicates that the structure of the polysaccharide rapidly activates the antigen-presenting cells, and has the functions of enhancing immunity, anti-tumor, anti-inflammatory and antibacterial activity. If a pharmacologically active polysaccharide can be found from the extract of Antrodia camphorata to promote immunity, and the polysaccharide can be cultured in large quantities by artificial culture, it can be applied to enhance the human immune system, which will bring great benefits to human health. help.

The main object of the present invention is to provide a method for preparing a crude polysaccharide of a solid fermentation product of the mycelium of Antrodia camphorata, and to isolate the mycelium of Antrodia camphorata which has both safety and pharmacological activity, thereby producing a crude solid fermentation product of Antrodia camphorata mycelium. Polysaccharide.

A second object of the present invention is to provide a method for preparing a crude polysaccharide of a solid fermentation product of Antrodia camphorata mycelium, which can be cultured by artificially cultivating a solid mash of Mycelium of Antrodia camphorata, and obtained by extraction, separation and purification. Crude polysaccharide.

A further object of the present invention is to provide a method for preparing a crude polysaccharide of a solid fermentation product of the mycelium of Antrodia camphorata, which can effectively activate immune cells.

The invention relates to a method for preparing a crude polysaccharide of a solid fermentation product of Antrodia camphorata mycelium according to the invention, relating to preparing a large-scale extraction of a pharmacologically active crude polysaccharide from a solid fermentation culture of A. angustifolia mycelium. The method, in particular, the preparation method is established by temperature regulation during culture, time control, selection of a medium, and separation of a crude polysaccharide to obtain a crude polysaccharide of a solid fermentation product of the mycelium of Antrodia camphorata.

The above and other objects, features and advantages of the present invention will become more <RTIgt; For example, the crude polysaccharide system of the mycelium of Astragalus membranaceus mycelium is obtained from the solid mash of a NPU-50 strain. First, how to screen the NPU-50 strain by artificial culture, and identify the NPU-50 strain as A. The mycelial strain, further, obtained a crude polysaccharide (ACP) by extracting the solid mash of the A. niger mycelium NPU-50 strain, and found that the crude polysaccharide has immunological activity and can proliferate immune cells by immunological tests.

I. Preparation of N. gingivalis mycelium NPU-50 strain

First, the wild A. sylvestris fruit body is ground into a fruit body powder. In a preferred embodiment of the present invention, the fruit body powder is suspended in 0.1% peptone and diluted into a suspension, and the suspension is applied to an MEA plate medium containing antibiotics. The MEA plate medium comprises a substrate, a carbon source and a nitrogen source, and the substrate is selected from a seaweed, the carbon source being selected from the group consisting of glucose, fructose, and galactose. a group consisting of lactose, starch, cellulose, and sucrose selected from the group consisting of peptone, malt extract, and yeast extract ( The MEA plate medium of the present invention preferably uses acai as a substrate, glucose as a carbon source, peptone and malt extract as a nitrogen source, and the MEA plate medium is cultured. The conditions are preferably selected to be 27 ° C for 2-3 days. During the cultivation process, a single colony was inoculated into the new MEA plate medium, and the strains of A. angustifolia mycelium having different traits were isolated by this method. The invention separates a strain with a bright red appearance color, a special aroma and a growth rate according to the above method, and the strain is named as NPU-50.

2. Identification of NPU-50 strain of Mycelium of Antrodia camphorata

After obtaining the NPU-50 strain, it is necessary to identify whether the strain is indeed a mycelium of Antrodia camphorata. In a preferred embodiment of the present invention, the NPU-50 strain is selected to be inoculated into a MEB liquid medium comprising a carbon source and a nitrogen source selected from the group consisting of glucose and fructose. a group consisting of galactose, lactose, starch, cellulose, and sucrose, selected from peptone, malt extract ), a mixture of yeast extract and tripeptone. Preferably, the MEB liquid medium of the present invention selects glucose as a carbon source, peptone and malt extract as a nitrogen source, and the culture condition of the MEB liquid medium is shaking culture at 25-30 ° C for 5-12 days, preferably in the present invention. The system was selected to be shake culture at 27 ° C for 7 days. The shock system was set at about 300 rpm, preferably at a rate of 120 rpm, and the cultured bacteria system was selected for centrifugation using an ultra-high speed refrigerated centrifuge. The NPU-50 mycelium was obtained by centrifugation at rpm for 20 minutes, and then filtered by a Buchner funnel.

There are three main methods for judging the authenticity of Antrodia camphorata: (1) judging the appearance of color, (2) observing the existence of arthrospores in type, and (3) molecular bioassay system. The most commonly used 18S ribosomal DNA gene sequence is identified by species classification. The 18S ribosomal DNA (18 rDNA) gene sequence is used as a classification basis between fungal genus, with high copy number and high retention in evolution. Features. The invention selects a molecular biological identification system, and the mycelium of the NPU-50 is freeze-ground by liquid nitrogen, and the DNA extraction of the NPU-50 is performed, and the NPU-50 DNA is used as a template, and then added to identify 18S. The primers NS3 (5'-GCAAGTCTGGTGCCAGCAGCC-3') and NS6 (5'-GCATCACAGACCTGTTATTGCCTC-3') of the rDNA gene sequence are subjected to a polymerase chain reaction (PCR) to obtain a polymerase chain reaction product. After denaturation at 93 ° C to 99 ° C for 3 to 5 minutes, reaction at a denaturation temperature of 93 ° C to 99 ° C for 30 to 60 seconds, reaction at a reaction temperature of 50 ° C to 57 ° C for 30 to 60 seconds and The reaction temperature of 70 ° C to 75 ° C is 1 to 3 minutes, and the number of cycles of DNA denaturation, refining and stretching is 10 to 50 cycles, and then reacted at an elongation temperature of 70 ° C to 75 ° C for 7 to 12 minutes, and finally stored in At 4 ° C; preferred polymerase chain reaction conditions of the present invention are selected as the initial denaturation temperature of 98 ° C, 2 minutes, denaturation temperature 95 ° C, 45 seconds, synthesis temperature 52 ° C, 45 seconds, extension temperature 72 ° C, 2 minutes A total of 35 cycles were performed, and the final extension temperature was 72 ° C for 10 minutes. Then, the electrophoresis analysis is used to determine whether there is a polymerase chain reaction product. If the polymerase chain reaction product is seen in the electrophoresis analysis, the polymerase chain reaction product is reordered, and finally the sequencing result is compared with the NCBI database. The alignment was compared with the Antrodia camphorata strain and the similarity was 100%. It was confirmed that the NPU-50 strain of the present invention was indeed the mycelium of Antrodia camphorata , which is hereinafter referred to as N. chinensis mycelium NPU-50. In the present invention, the strain of N. gingivalis NPU-50 was deposited in the Republic of China Food Industry Research Institute's Culture Collection and Research Center (BCRC) on March 19, 1999. The strain registration number is BCRC 930127. Named as Ophiopogon japonicus mycelium NPU-50.

From the above, it is known that the NPU-50 strain is a mycelium of Antrodia camphorata, which is hereinafter referred to as a strain of N. gingivalis NPU-50, and then the strain of N. gingivalis NPU-50 is taken for liquid culture and solid state. The yeast is fermented into a solid mash, and the solid mash is extracted to obtain the crude polysaccharide (ACP) of the solid fermentation product of the mycelium of Antrodia camphorata, and the effect of the crude polysaccharide of the solid fermentation product of the mycelium of Astragalus membranaceus is studied.

3. Method for preparing crude polysaccharide (ACP) of the solid fermentation product of Antrodia camphorata mycelium of the present invention

Next, it is studied whether the solid lysate of the A. niger mycelium NPU-50 strain has pharmacologically active ingredients. First, the liquid culture and solid fermentation of the A. sinensis mycelium NPU-50 strain into a solid mash is carried out. The solid mash is extracted to further study the solid mash.

The liquid culture and the solid fermentation step can be carried out by liquid culture and solid state fermentation on the liquid medium and the solid medium. The preferred embodiment of the present invention selects the mycelium of Antrodia camphorata. The NPU-50 strain is initially fermented in a liquid medium to form a mash, which is then inoculated into a solid medium to complete a solid mash. The solid medium contains a preferred growth component, and the nutrient is supplied to form the solid mash. The solid mash has a strong aroma of burdock, and the solid mash has a sponge-like structure inside, and the sponge-like structure is wild. The structure of the growth of Antrodia camphorata, and the solid yeast contains a crude polysaccharide.

First, the above-mentioned A. sinensis mycelium NPU-50 strain is cultured in the MEB liquid medium at 27 ° C, cultured for 7 days, and the homogenizer is used to homogenize the broth, and then the homogenized mash is used. Inoculate onto a solid medium. The solid medium comprises a substrate, a carbon source and a nitrogen source, and the substrate is rice, which is selected from the group consisting of brown rice, germ rice, glutinous rice, Penglai rice, rice or glutinous rice, and the carbon source is selected. a group of free glucose, fructose, galactose, lactose, starch, cellulose, and sucrose, which is selected from peptone ( A group consisting of peptone), malt extract, yeast extract, and tripeptone. Preferably, the solid medium of the present invention selects germ rice as a substrate, glucose as a carbon source, peptone and malt extract as a nitrogen source, and the solid culture fermentation conditions are cultured at 25 ° C to 30 ° C for 2 to 6 months. Preferably, the present invention is selected to be fermented at 27 ° C for 3 months to form a solid mash.

Next, the solid mash is extracted with water or an organic solvent to obtain an aqueous extract or an organic solvent extract of the solid lyophilized product of the genus Antrodia camphorata, and the crude polysaccharide is obtained from the extract, wherein the organic solvent It is selected from the group consisting of alcohols, esters, alkanes or molars, but is not limited thereto. In a preferred embodiment of the present invention, the water extraction method is selected. First, the solid mash system is selectively added to distilled water, and after homogenization, the ultrasonic wave is oscillated in an environment of 75 ° C to 90 ° C. After shaking at 80 ° C for 2 hours, after cooling, a supernatant was selected by centrifugation at 10,000 rpm for 20 minutes in an ultra-high speed refrigerated centrifuge, and the supernatant was selected to be filtered by a Buchner funnel, and the supernatant was filtered to 95%. The alcohol was precipitated at 4 ° C for one night to obtain a precipitate, and the precipitate was collected by suction filtration, and the precipitate was washed twice with 70% alcohol, and the precipitate was the NPU-50 solid mash of the Astragalus membranaceus mycelium. The crude polysaccharide is called Antrodia camphorata polysaccharides (ACP), hereinafter referred to as ACP. Please refer to Figure 1 for the crude fermentation product of the mycelium of Astragalus membranaceus. Polysaccharide ACP produced a wave front retention time map by HPLC-RI chromatography.

At present, studies on polysaccharides indicate that it is related to the mechanism of modulating the immune response, and may be a receptor for TLR-4, Dentin 1, MR or MRL of the antigen-presenting cells (APC) on the polysaccharide. APC quickly transmits the message to the unactivated T lymphocytes, and the activated T lymphocytes will carry out immunity, anti-tumor, anti-inflammatory and antibacterial activities through special routes, and will be tested below to prove the use of the present invention. The crude polysaccharide (ACP) of the crude extract of Astragalus membranaceus mycelium extracted by the method has the ability to stimulate the proliferation of immune cells.

4. The effect of the proliferation of the crude polysaccharides of the solid fermentation product of the mycelium of Antrodia camphorata in the present invention

In the study of edible fungi, it is known that Ganoderma lucidum polysaccharide can inhibit tumor growth and stimulate T lymphocyte and B lymphocyte activation, and Brazilian mushroom polysaccharide can resist tumor and enhance immunity activity, so the preferred embodiment of the present invention is taken The invented Astragalus membranaceus mycelium solid fermentation product crude polysaccharide (ACP) was subjected to an immunocyte proliferation test.

First, the peripheral blood mononuclear cells of the SD rats and the immune cells in the spleen are isolated. In the preferred embodiment of the present invention, the immune cells in the peripheral blood mononuclear cells and the spleen of the rat are separately added to the 96-well plate. In each well, 50 μL of 2×10 ⁶ cells/mL cells were added to each well, and a dose of 1 μg/mL lipopolysaccharides (LPS), 10 μg/mL concanalin A (ConA), ACP containing 1 μg/mL LPS, and ACP containing 10 μg/mL ConA was selected in a 5% CO ₂ incubator at 37 ° C. After incubating the cells for 72 hours, add cell proliferation assay reagent Alamar Blue ^TM 10 μL per well at 5% CO ₂ 37 °C. After reacting for four hours in the incubator, the absorbance values were separately measured at 570 nm and 600 nm using an ELISA reader, and the percentage increase of the immune cells was obtained by the calculation formula. As shown in Figure 2, the proliferation of mononuclear cells in the peripheral blood of mice is as shown in Fig. 2. The value-added effect of ACP and LPS is about twice that of the control group. The effect of co-stimulation of ACP and ConA is about 5.8 times that of the control group. As a result of the proliferation of immune cells in the spleen of mice, as shown in Fig. 3, the proliferation effect of the ACP and LPS or ConA was about twice as high as that of the control group.

According to the results of the above immunoassay, the crude polysaccharide ACP of the Astragalus membranaceus mycelium is mixed with LPS, or the crude polysaccharide ACP of the A. sinensis mycelium is mixed with ConA to stimulate the peripheral blood. The proliferation of mononuclear cells and spleen immune cells is good. The cells can be activated by the activation of the antigen on the structure of the polysaccharide, and the message can be quickly transmitted to the unactivated T lymphocytes, thereby activating T lymphocytes to enhance immunity; It will strengthen the efficacy of LPS or ConA, and achieve the common promotion of immune cell proliferation, so it can effectively trigger the immune response, and can stimulate the proliferation and activation of immune cells. It can be verified that the strain of N. gingivalis NPU-50 is screened by the preparation method of the preferred embodiment of the present invention, which is a pharmacologically effective and safe strain of Mycelium of Astragalus membranaceus, and the Mycelium of Asteris sinensis NPU-50 The strain is fermented into a solid mash by solid culture, and then a crude polysaccharide is obtained by separation and purification. The crude polysaccharide has pharmacological activity and can effectively proliferate immune cells.

The invention separates a safe and pharmacologically active strain of N. gingivalis NPU-50, and uses the preparation method of the crude polysaccharide of the solid fermentation product of the mycelium of Astragalus membranaceus, from the solid lysate of the mycelium NPU-50 strain Extraction can obtain a pharmacologically active crude polysaccharide.

The method for preparing the crude polysaccharide of the solid fermentation product of the mycelium of Antrodia camphorata according to the invention, the artificially mass-cultivating the mycelium NPU-50 into a solid mash to replace the wild Antrodia camphorata, and the solid mash is directly extracted and separated After purification, the crude polysaccharide of the solid fermentation product of the mycelium of A. angustifolia can be obtained in a large amount.

The preparation method of the crude polysaccharide of the solid fermentation product of the mycelium of Antrodia camphorata according to the invention, the crude polysaccharide of the mycelium of the mycelium of the mycelium of the Antrodia camphorata induces the proliferation of peripheral blood mononuclear cells and the immune cells of the spleen, and stimulates immunity Cell-activated LPS or ConA co-stimulation can effectively enhance the activation of immune cells, effectively elicit an immune response, and achieve the effect of stimulating the proliferation of immune system cells.

While the invention has been described in connection with the preferred embodiments described above, it is not intended to limit the scope of the invention. The technical scope of the invention is protected, and therefore the scope of the invention is defined by the scope of the appended claims.

Fig. 1 is a HPLC-RI spectrum of the crude polysaccharide of Astragalus membranaceus mycelium solid-state fermentation product ACP after extraction.

Fig. 2 is a diagram showing the proliferation reaction of the peripheral blood mononuclear cells of the rat with the crude polysaccharide ACP of the Astragalus membranaceus mycelium of the present invention and LPS or with ConA.

Fig. 3 is a diagram showing the spleen immune cell proliferation reaction of the crude polysaccharide ACP of the Astragalus membranaceus mycelium of the present invention with LPS or with ConA.

Claims (23)

The invention relates to a method for preparing a crude polysaccharide of a solid fermentation product of Antrodia camphorata, which comprises: carrying out liquid culture and solid state fermentation of A. sinensis mycelium NPU-50 strain to obtain a solid mash, and the A. sinensis mycelium NPU- The 50 strain was deposited in the Center for Culture and Preservation and Research (BCRC) of the Republic of China Food Industry Development Institute under the registration number BCRC 930127; and the solid mash of the strain was isolated and purified to obtain a crude solid fermentation product of Antrodia camphorata mycelium. Polysaccharide. The method for preparing a crude polysaccharide of a solid fermentation product of the mycelium of Antrodia camphorata according to the first aspect of the patent application, wherein the strain of N. gingivalis NPU-50 is cultured in a liquid medium for liquid culture into one The bacterial solution is then subjected to solid state fermentation on a solid medium to obtain a solid mash. The method for preparing a crude polysaccharide of the solid solution of the mycelium of Antrodia camphorata according to the second aspect of the patent application, wherein the solid medium comprises a substrate, a carbon source and a nitrogen source. A method for preparing a crude polysaccharide of a solid fermentation product of Antrodia camphorata according to claim 3, wherein the substrate is rice. A method for preparing a crude polysaccharide of a solid fermentation product of Antrodia camphorata according to claim 4, wherein the rice variety of the substrate is selected from the group consisting of brown rice, germ rice, glutinous rice, and Penglai rice. One of rice and white. A method for preparing a crude polysaccharide of a crude solid fermentation product of Antrodia camphorata according to claim 3, wherein the carbon source is selected from the group consisting of glucose, fructose, galactose, a group consisting of lactose, starch, cellulose, and sucrose. A method for preparing a crude polysaccharide of a solid fermentation product of Antrodia camphorata according to claim 3, wherein the nitrogen source is selected from peptone, malt extract, and yeast. A group of yeast extracts and tripeptone. A method for preparing a crude mycelium solid fermentation product polysaccharide of Antrodia camphorata according to claim 3, wherein the ratio of the carbon source to the nitrogen source is 1:1 to 5:1. The method for preparing a crude polysaccharide of the crude extract of Antrodia camphorata mycelium according to the third aspect of the patent application, wherein the weight percentage of the carbon source is 0.7% to 5%, which is the total weight of the solid medium. For 100%. The method for preparing a crude polysaccharide of the solid solution of the mycelium of Antrodia camphorata according to the third aspect of the patent application, wherein the weight percentage of the nitrogen source is 1% to 10%, which is the total weight of the solid medium. For 100%. The method for preparing a crude polysaccharide of the solid solution of the mycelium of Antrodia camphorata according to the second aspect of the patent application, wherein the temperature of the bacterial solution in solid state fermentation is 25 ° C to 30 ° C. A method for preparing a crude polysaccharide of a solid fermentation product of Antrodia camphorata according to claim 2, wherein the bacterial solution is subjected to solid fermentation on a solid medium for 2 to 6 months. A method for preparing a crude polysaccharide of a solid fermentation product of Antrodia camphorata according to claim 2, wherein the liquid medium comprises a carbon source and a nitrogen source. The method for preparing a crude polysaccharide of the crude fermentation product of Antrodia camphorata according to claim 13 , wherein the carbon source is selected from the group consisting of glucose, fructose, galactose, a group consisting of lactose, starch, cellulose, and sucrose. A method for preparing a crude polysaccharide of a solid fermentation product of Antrodia camphorata according to claim 13 wherein the nitrogen source is selected from peptone, malt extract, and yeast. A group of yeast extracts and tripeptone. The method for preparing a crude polysaccharide of the crude extract of Astragalus membranaceus mycelium according to the second aspect of the patent application, wherein the A. sinensis NPU-50 strain is cultured in a liquid medium at a temperature of 25 ° C to 30 ° C. The method for preparing a crude polysaccharide of the crude extract of Antrodia camphorata mycelium according to the second aspect of the patent application, wherein the A. sinensis NPU-50 strain is cultured in a liquid medium for 5 to 12 days. The method for preparing a crude polysaccharide of the solid fermentation product of Antrodia camphorata according to the second aspect of the patent application, wherein the strain of A. sylvestris NPU-50 is cultured in a liquid medium for shaking culture. The method for preparing a crude polysaccharide of the solid fermentation product of the mycelium of Antrodia camphorata according to the first aspect of the patent application, wherein the crude polysaccharide system of the solid fermentation product of the mycelium of A. angustifolia is extracted from the solid fermentation product of the mycelium of Antrodia camphorata The substance is isolated and produced. a method for preparing a crude polysaccharide of a solid fermentation product of Antrodia camphorata according to claim 19, wherein the crude polysaccharide system of the solid fermentation product of the mycelium of A. angustifolia is solidified from the mycelium of Antrodia camphorata The water extract is obtained by separation. A method for preparing a crude polysaccharide of a solid fermentation product of Antrodia camphorata according to claim 20, wherein the water extraction temperature is from 75 ° C to 90 ° C. a method for preparing a crude polysaccharide of a solid fermentation product of Antrodia camphorata according to claim 19, wherein the crude polysaccharide system of the solid fermentation product of the mycelium of A. angustifolia is solidified from the mycelium of Antrodia camphorata The organic solvent extract is isolated and obtained. A method for preparing a crude polysaccharide of a crude solid fermentation product of Antrodia camphorata according to claim 22, wherein the organic solvent is selected from the group consisting of esters, alcohols, alkanes and halogenated alkanes.