WO2004099427A1

WO2004099427A1 - Method of extracting crude beta glucan from phellinus linteus

Info

Publication number: WO2004099427A1
Application number: PCT/KR2003/001723
Authority: WO
Inventors: Kwan Ho Kim; Il Keun Roh
Original assignee: Kwan Ho Kim; Il Keun Roh
Priority date: 2003-05-07
Filing date: 2003-08-26
Publication date: 2004-11-18
Also published as: KR20040096000A; AU2003256111A1; WO2004099428A1

Abstract

Disclosed is a method of extracting beneficial ingredients inclusive of crude beta glucan from Phellinus Linteus. The fruit body of Phellinus Linteus is decomposed by ultrasonic wave, the cell walls of the fruit body are disrupted by hydrothermal process, and then enzyme fermentation is performed, whereby crude beta glucan can be extracted from Phellinus Linteus with increased yield rate.

Description

METHOD OF EXTRACTING CRUDE BETA GLUCAN FROM PHELLINUS

LINTEUS

TECHNICAL FIELD The present invention is directed to a method of extracting crude beta glucan from

Phellinus Linteus with high yield rate, and crude beta glucan extracted by the method.

BACKGROUND ART

Phellinus Linteus is a kind of perennial mushroom growing on mulberry trees and broad- leaved trees in an alpine region, which is classified to scores of species.

The typical species of the mushroom Phellinus are Phellinus Linteus and Phellinus Baumi, which grow on the stalks of the mulberry trees and the broad-leaved trees. Phellinus Linteus has been generally used as a medical material, but is also used as a food material recently. Phellinus Linteus contains magnificent nutrition beneficial to a human body such as potassium, calcium, magnesium, vitamin B2, B3 and C, fibroid material, amino acids, etc., and also contains beta glucan that is a kind of polysaccharide. Among such ingredients, beta glucan is especially beneficial to a human body in a medical point of view, and the immunity enhancement and cancer resistance effects of the beta glucan have been mentioned on a variety of classical medical books in the Orient.

Furthermore, the recent research results show that Phellinus Linteus has the immunity improvement effect by suppressing virus, bacteria, mildew, and vermin, cancer resistance effect resisting the cancer with the level of 96.7%, and anti-oxidation effect influencing on noxious oxygen and radical. Beta glucan in Phellinus Linteus exists as proteins cohesive on the cell walls, and surrounded by plenty of non-water-soluble fibroid materials. Furthermore, the fruit body of Phellinus Linteus is hardened thickly with a great amount of cell layers.

Korean Patent Laid-Open Publication No. 2003-30636 dated April 18, 2003., discloses the method for obtaining the protein polysaccharide by adding inorganic compound and organic nutrition to the fruit body of a mushroom to manufacture ferment compound, fermenting after inoculating Basidiomycota hypha or yeast to the ferment compound, and performing hydrothermal process, dialysis, centrifugal separation, and freeze drying.-^""

According to this method, the yield rate of the protein polysaccharide increases as the tissues of the fruit body is softened by the fermenting process to make the extracting process of the protein polysaccharide easy, however, such a method has a shortcoming in that the original ingredients of the mushroom are generated together with other ferment metabolic products, so the functional substance in a natural mushroom cannot be extracted as it is.

Furthermore, the robust fruit body of Phellinus Linteus is, even when it is crushed into powder, too voluminous to be a substrate of enzyme generated during the fermentation by the Basidiomycota hypha and yeast, and it also shows limited solubility, so it is inappropriate to be used in extracting the beta glucan in large quantities. Moreover, many kinds of foreign substances other than beta glucan are included.

The Korean Patent No. 308392 dated August 28, 2001 discloses a method for obtaining an extract from natural Phellinus Linteus, in which method Phellinus Linteus undergoes the ^"extraction process wherein the crushed Phellinus is mixed with extraction solvent and then is warmed up in a double boiler with being decompressed, and in such a situation, ultrasonic wave is applied for about 24 hours to increase the extraction efficiency. However, such a method is time-consuming, and the organic solvent such as 1,3- butylenechloride, propyleneglicol, or isopropanol has to be used besides water as the extraction solvent, so such a method is not preferable for use in the extraction of the beta glucan of high polarity included in the cell wall in a large quantities, and in the extraction of the original ingredient as it is.

Furthermore, the overall extraction rate just reaches to 5% to 12%, and the remaining after the extraction is not used again. Considering the fact that the price of Phellinus Linteus is as high as hundreds to thousands of dollars per kilogram, the above hydrothermal extraction cannot be said economical.

Moreover, the beta glucan that is beneficial in the medical point of view exists on the hardened cell layers, and it is a bulky molecule of which molecular weight is more than a million Dalton. As the quantity measurement of such a bulky beta glucan has not been set yet, mere hydrothermal extraction has to be reconsidered.

Therefore, the research to extract the beneficial ingredients including the beta glucan from Phellinus Linteus will make it possible that such a superior material is supplied to the patients with low cost, and the clinical results can be brought about scientifically.

DISCLOSURE OF THE INVENTION

With the above-noted problems in mind, it is an object of the present invention to provide a method of extracting a beneficial ingredient, including crude beta glucan, from Phellinus Linteus with high yield rate. Another object of the invention is to provide crude beta glucan extracted from

Phellinus Linteus by the method set forth just above.

In accordance with one aspect of the invention, there is provided a method of extracting beneficial ingredients inclusive of crude beta glucan from Phellinus Linteus, comprising the steps of: (a) mixing crushed powder of Phellinus Linteus with distilled water of which weight is 20 through 50 times greater than the weight of Phellinus Linteus; (b) applying ultrasonic wave to the mixture obtained at said step (a); (c) subjecting the ultrasonically treated material to a hydrothermal process; (d) filtering the hydrothermally treated material; (e) causing the filtered material to be fermented with cellulase and alcalase; and (f) subjecting the fermented material to ultra-filtration to thereby obtain crude beta glucan.

In accordance with another aspect of the invention, there is provided crude beta glucan extracted from Phellinus Linteus by means of the above-referenced method BRIEF DESCRIPTION OF THE DRAWINGS

FIGS, la through le show the change of structure of the fruit body of Phellinus Linteus observed with an optical microscope of 1250 magnifications, each of the fruit body obtained in Example 1 and Comparative Examples 1 and 2, in which, FIG. la sEows Phellinus Linteus as it is;

FIG. lb depicts powdery Phellinus Linteus; FIG. 1 c shows Phellinus Linteus crushed by ultrasonic wave;

FIG. 1 d illustrates Phellinus Linteus after going through a high pressure hydrothermal process; and FIG. le shows Phellinus Linteus after subjected to fermentation.

BEST MODE FOR CARRYING OUT THE INVENTION

According to the present invention, the hardened cells of Phellinus Linteus are disrupted through an ultrasonic wave process and a hydrothermal process, thus enabling protein decomposing enzyme and cellulose decomposing enzyme to act in such a manner that beta glucan and other beneficial ingredients are extracted with high yield rate. Furthermore, the content of soluble ingredients is confirmed by a drying method to measure the total extraction efficiency, while the extraction efficiency of crude beta glucan is analyzed quantitatively by virtue of an ultra-filtration method which takes advantage of the molecular size of beta glucan.

The present invention will no be described in greater detail. It should be apreciated that the following explanation is not to limit the scope of the invention but to show a preferred example of the invention.

Comparative Example 1: Hydrothermal Extraction and Ultra-filtration of a Sample.

Initially, 300 ml of distilled water is mixed with lOg of powder of Phellinus Linteus, and then the mixture is heated at 100°C for one hour, two hours, three hours, and for hours, respectively. Then, the centrifugal -separation is performed against the resultant material, and then the supernatant obtained through the centrifugal separation is concentrated with heat into the quantity of 15 ml. Subsequently, 5 ml of the concentrated material is allowed to dry and then the weight of the dried material is multiplied by 3 and recorded in Table 1 as the data for the total dried extract. The remaining 10 ml of the concentrated material is ultra-filtrated by use of a membrane filter with one hundred thousand pore-size, and 10 ml of phosphoric acid buffering solution with pH 5.8 is added to the ultra-filtrated material. Then, 1.2 ml of cellulase(Fluka 22180 0.3U/mg) is added to the resultant material, which material is then cultured for one day. The acidity of the resultant material is changed into 9.0 with potassium phosphate, and an enzyme reaction is performed with cellulase. The resultant material is added with 1 ml of alcalase and cultured at 60 °C for one day. Then, the resultant material is ultra-filtrated with the membrane filter of one hundred thousand pore-size to obtain 1 ml of concentrated supernatant liquid, and then the concentrated liquid is ultra-filtrated once again with the use of 20 ml of distilled water, the resultant material of which is allowed to dry under a vacuum condition to obtain dark brown-colored crude beta glucan. Then, the weight of the crude beta glucan is multiplied by one and a half, and the calculated result is shown in Table 1 in terms of extraction efficiency of crude beta glucan.

TABLE 1

Comparative Example 2: High Pressure Hydrothermal Extraction and Ultra- filtration of a Sample.

300 ml of distilled water is mixed with lOg of Phellinus Linteus powder, and then the mixture is heated at 120°C for one hour, two hours, three hours, and four hours, respectively, under 2 atm.. Then, the same steps as in Comparative Example^"l is carried out, the result of which is recorded in Table 2.

TABLE 2

Example 1: Ultrasonic Hydrothermal Extraction under High Pressure and Ultra- filtration of a Sample

300 ml of distilled water is mixed with lOg of Phellinus Linteus powder, and then the ultrasonic wave (Power 6, Sonifier Cell Disrupter 350, Branson Sonic Power Co., Ltd.) is applied to the mixture for 10 minutes, 20 minutes, 30 minutes, and 60 minutes, respectively. The ultrasonically treated mixture is then subjected to a hydrothermal extraction process at 120^°C under 2 atm. for three hours. Subsequently, the same steps as in Comparative Example 1 is carried out, the result of which is indicated in Table 3. TABLE 3

As is apparent from the above table, the maximum yield of the total dried extract reaches as high as 34.6% and the maximum yield of crude beta glucan reaches up to 5.2% according to Example 1 wherein the fruit body of Phellinus Linteus is decomposed through the ultrasonic wave process and extracted with the high pressure hydrothermal process, which result is better than the result in Comparative Examples 1 and 2.

Such a result means that the total yield of the dried extract in the present invention is five times greater than, and the yield of the cancer-resistant crude beta glucan is twelve times greater than the yield according to the hydrothermal extract method.

Experiment for Confirming the Content of Crude Beta Glucan in the Dried Extract Obtained in Example 1.

The content of crude beta glucan in the dried extract obtained in Example 1 is confirmed in accordance with the methods proposed by Mannors. D. J. et. al. (1973, The structure of a β -(l,6)-D-glucan from yeast cell walls, Biochem. J. 135, 19-30), and Catley, B. J. et. al. (1988. Isolation and analysis of cell walls yeast (A practical approach), IRL press, pp. 163-183). The content of crude beta glucan in the total dried extract is measured by calculating the quantity of glucose in glucan fractions obtained through the chemical process with NaOH solution.

Further, the structural change of Phellinus Linteus at the respective process steps is shown in FIGS.- la through le, respectively. Referring to the figures, it can be appreciated that the fruit body of Phellinus Linteus becomes loose, thus assuring easier extraction.

INDUSTRIAL APPLICABILITY

As set forth hereinabove, the inventive extraction method makes it possible to extract beneficial components, including crude beta glucan, in a cost-effective and highly quantitative manner and therefore can be broadly used in manufacturing medicines, cosmetics and foodstuffs.

Claims

1. A method of extracting beneficial ingredients inclusive of crude beta glucan from Phellinus Linteus, comprising the steps of:

(a) mixing crushed powder of Phellinus Linteus with distilled water of which weight is 20 through 50 times greater than the weight of Phellinus Linteus;

(b) applying ultrasonic wave to the mixture obtained at said step (a);

(c) subjecting the ultrasonically treated material to a hydrothermal process;

(d) filtering the hydrothermally treated material;

(e) causing the filtered material to be fermented with cellulase and alcalase; and (f) subjecting the fermented material to ultra-filtration to thereby obtain crude beta glucan.

2. The method as recited in claim 1, wherein said step (c) is performed under the pressure of 2 through 3 atm.

3. The method as recited in claim 1, wherein said steps (d) and (f) are carried out by use of a membrane filter.

4. Crude beta glucan extracted by the method as recited in any one of claims 1, 2 and 3.