TWI596210B - Medium for culture of Antrodia camphorata and culture method - Google Patents

Description

Medium and culture method for cultivating Antrodia camphorata

The present invention relates to a solid medium for culturing Antrodia camphorata, and a culture method using the above medium.

Antrodia cinnamomea belongs to the genus Aphyllophorales, Polyporaceae, and Antrodia . It is a medicinal fungus endemic to Taiwan, also known as Antrodia camphorata, Oyster mushroom, Poria, and cave. Nervous and god mushrooms.

The strain of Antrodia camphorata can be divided into two parts: mycelium and fruiting body. In research, the mycelium of Antrodia camphorata grows faster and has better artificial culture effect, so it is often used as research material, but it is generally used to study The active ingredient content of the silk body is far less than that of the fruit body, and the drug is poor.

The body shape of Antrodia camphorata is rich and varied, and it has a plate shape, a bell shape, a horseshoe shape or a tower shape. It is bright red at the time of birth and gradually turns into a reddish brown, light brown or yellowish brown. In Taiwanese folk medicine, the fruiting body of Niu Zhizhi is often used in hepatic disease, nameless swollen fever, fever, diarrhea, cold, diabetes, high blood pressure and hangover. So far, the fruiting body of Antrodia camphorata has been confirmed to have hepatoprotective effect, detoxication, hangover, anti-tumor, anti-inflammation, and prevention of renal disease. Prevention) and immune modulation (immunomodulation) and other effects.

The physiologically active components known to the body of A. angustifolia are: polysaccharides, triterpenoids, suproxide dismutase (SOD), adenosines, proteins (including immune proteins), vitamins. , ergosterols, metal trace elements, nucleic acid, amino acids, lignin (Lignin) and blood pressure stabilizing substances (such as Antrodia acid). Among them, triterpenoids have been shown to inhibit cancer cell growth, inhibit histamine release, preserve liver function, promote platelet aggregation and lower blood lipids, and its immune system regulation and anti-tumor activity have been studied by domestic and foreign scholars. Extensive attention is one of the important indicators for judging the effective composition of Antrodia camphorata.

In the natural environment, Antrodia camphorata grows only on the hollow heartwood of the trunk of Cinnamomum kanehirae , which is also endemic to Taiwan. The calf itself is an endangered conservation plant, and the number is scarce, making wild Antrodia camphorata very difficult to obtain. The development of artificial breeding methods for Antrodia camphorata is an important direction in both academia and industry. At present, the domestic artificial breeding methods for burdock are mainly: segment wood cultivation method, liquid fermentation method and solid culture method.

The wood cultivation method is to use the native host burdock of Oryza sativa or other wood of similar species as the medium to cultivate the fruit body of Antrodia camphorata. Since the growth conditions are closest to the original environment, the Antrodia camphorata fruit body can be cultivated by this method. The same or similar active ingredients and efficacy as the wild A. angustifolia fruit body, but the disadvantage is that the medium is not easy to obtain, and the cultivation cost is high. And the cultivation time is longer.

The liquid fermentation method utilizes a liquid medium to carry out fermentation culture of the mycelium of Antrodia camphorata in a fermentation tank, which has a short cultivation time and a relatively low cost, and can enlarge the culture scale and has high industrial utilization. However, the physiologically active ingredient content of the obtained mycelium of Antrodia camphorata is much lower than that obtained by the segmental wood or solid culture method, and the triterpenoids unique to the wild A. angustifolia cannot be obtained.

The solid culture method is to culture mycelium in a solid medium such as a space bag containing a fiber, a saccharide or a whole grain, or a medium containing agar, and can induce fruit body formation in the process. The method has the advantages that the culture medium is easy to obtain and there is no shortage of raw materials, and the obtained active ingredients of the mycelium, fruit body and the fruiting body-like structure of the Antrodia camphorata, the culture time and the culture cost are all introduced. Between the above two methods, it is one of the currently feasible artificial culture methods of Antrodia camphorata. The above description is summarized as shown in Table 1:

Therefore, in the first aspect, the present invention provides a medium for cultivating Antrodia camphorata which is easy to obtain, easy to prepare, and low in cost, and capable of cultivating a triterpenoid containing a high content of a physiologically active substance.

In a second aspect, the present invention provides a culture method for culturing Antrodia camphorata which is carried out using the above-mentioned medium, which is simple and effective, can save time and labor costs, and is easy to be popularized.

For the purpose of unambiguously understanding the present specification, it should be noted that the term "comprising" means "including but not limited to" and the term "comprises" has the same meaning.

Unless otherwise defined, all technical and scientific terms used in the specification have the same meaning Those skilled in the art to which the invention pertains can equivalently substitute the methods and materials described in the specification, and can also be used to practice the invention. Of course, the invention is in no way limited to the particular methods and materials described in the specification.

Thus, in a first aspect, the present invention provides a medium for culturing Antrodia camphorata, the medium comprising a cereal as a matrix and a nutrient solution added to the cereal, the nutrient solution comprising 100 parts by weight of water and 30 parts by weight or less A carbon source, 30 parts by weight or less of a nitrogen source, 0.1 to 2.0 parts by weight of a peptide, and 0.1 to 2.0 parts by weight of a flavonoid. The weight of the cereal is 30 to 70 parts by weight based on 100 parts by weight of the nutrient solution added to the cereal.

According to the invention, the cereals include, but are not limited to, rice ( Oryza sativa ), millet ( Setaria italica ), sorghum bicolor , panicum miliaceum , wheat ( Triticum aestivum ), barley ( Horaeum vulgare ), barley ( Hordeum) vulgare), oats (Avena sativa), rye (Secale cereale), Job's tears (Coix lacryma-jobi), maize (Zea mays), buckwheat (Fagopyrum esculentum), India buckwheat (Fagopyrum tataricum), wild buckwheat (Polygonum nepalense), Chenopodium quinoa , Glycine max , Glycine max , Vicia faba , Pisum sativum , Vigna radiata , Vigna angularis , Arachis hypogea or One of the combinations.

In one embodiment of the invention, the cereals used are pearl barley and wheat mixed in a weight ratio of 2:1. Pearl barley refers to barley kernels that remove bran and alfalfa.

According to the invention, the pH of the nutrient solution is between 5 and 7; in a particular embodiment of the invention, the pH of the nutrient solution is 6.

According to the present invention, the carbon source used includes, but is not limited to, glucose, fructose, sucrose, maltose, or a combination thereof.

According to the present invention, the nitrogen source used includes, but is not limited to, yeast extract, casein hydrolysate, tryptone, peptone, various amino acids (amino Acids), urea (urea), amine acetate (CH ₃ COONH ₄ ), ammonium chloride (NH ₄ Cl), ammonium nitrate (NH ₄ NO ₃ ), potassium nitrate (KNO ₃ ), sodium nitrate (NaNO ₃ ) or this One of the combinations.

The selection of these carbon sources and nitrogen sources falls within the professional literacy and routine technology of those skilled in the art. In one embodiment of the invention, the carbon source and nitrogen source used are glucose and yeast extracts, respectively.

In order to enable Antrodia camphorata to produce more physiologically active substances, especially triterpenoids of the active ingredient of the indicator, in addition to the carbon source and nitrogen source necessary for growth, in the present invention, a peptide which is a physiological regulator is added to the medium. Flavonoid compounds.

According to the present invention, the peptide used includes, but is not limited to, a natural extract peptide, a synthetic peptide, a peptide fragment obtained by proteolysis of the protein, and a protein obtained by microbial decomposition. Peptide fragments, peptides obtained by microbial fermentation processes, or a combination of these.

It is to be noted that the term "peptide" as used in the present invention refers to a short-chain molecule polymerized from an amino acid monomer via a peptide bond, generally comprising 2 to 50 Amino acids are not equivalent to a single molecule of "amino acid" nor to a "protein" with a relatively large molecular weight.

According to the present invention, the peptide used is a vegetable peptide, and in one embodiment of the invention, the peptide used is a soybean peptide.

According to the invention, the flavonoid compounds used include, but are not limited to, flavones, flavonols, dihydroflavones, dihydroflavonols, anthocyanidins ), flavans, benzophenone Xanthones, chalcones, biflavonoids, isoflavones, aurones, or a combination thereof.

In one embodiment of the invention, the flavonoid compound used is Citrus Flavonoids.

In a second aspect of the present invention, there is provided a culture method for cultivating Antrodia camphorata, the culture method comprising the steps of: (1) mixing a composition of a medium in proportion, the composition of the medium comprising a cereal as a matrix, and A nutrient solution added to the cereal. The nutrient solution includes 100 parts by weight of water, 30 parts by weight or less of a carbon source, 30 parts by weight or less of a nitrogen source, 0.1 to 2.0 parts by weight of a peptide, and 0.1 to 2.0 parts by weight of a flavonoid compound. 100 parts by weight of the cereal is mixed with 30 to 70 parts by weight of the nutrient solution to obtain a medium for cultivating Antrodia camphorata; (2) the medium is stirred and allowed to stand, and the cereal is completely absorbed by the nutrient solution. (3) the medium is subjected to a sterilization procedure; and (4) Antrodia camphorata is inoculated to the medium and cultured at 20 to 30 °C.

According to the present invention, the composition and characteristics of the medium in the step (1) are completely the same as those mentioned in the first aspect of the present invention, and will not be described herein.

According to the present invention, in the step (2), the cereal which is saturated with the nutrient solution is further divided; more preferably, it is dispensed into a space bag or a glass. In a bottle or a plastic bottle; in one embodiment of the invention, it is dispensed into a space bag.

According to the present invention, the step (4) can be cultured under ordinary room temperature conditions.

In the process of cultivation, the effective active ingredients of Antrodia camphorata are affected by two factors, such as different parts and different growth stages. For example, the content of triterpenoids is much more in the fruit body than in the semi-mycelium and mycelium; If the culture time is longer, the content of triterpenoids in Antrodia camphorata will increase, and the content of polysaccharides will decrease. Therefore, according to the present invention, the length of the culture time in the step (4) depends on the growth state of the Antrodia camphorata and the desired part and the growth period to determine when to stop the culture and harvest. Generally, the culture period is 2~ 4 months.

Other features and effects of the present invention will be apparent from the detailed description of the preferred embodiments of the accompanying drawings, wherein: Figure 1 is an HPLC chromatogram of a wild A. niger fruit body; and Figure 2 is a present invention for culturing The HPLC chromatogram of the culture medium of the Antrodia camphorata and the culture method of the Astragalus membranaceus mycelium, fruit body and half body.

Detailed description of specific examples

The invention will be further illustrated by the following specific examples, but it should be understood that the specific examples are for illustrative purposes only and should not be construed as limiting The state of the implementation of the Ming Dynasty.

experimental method

Specific Example 1 Preparation of solid medium for culturing Antrodia camphorata

The dried grains of pearl barley and wheat are mixed at a weight ratio of 2:1 to obtain a cereal composition. 2 parts by weight of glucose (Sigma, Louisiana, USA), 5 parts by weight of yeast extract (Sigma, Louisiana, USA), 0.5 parts by weight of soybean peptide (10 liters) were added to 100 parts by weight of sterile water. Enterprise Co., Ltd., Taiwan) and 0.5 parts by weight of citrus flavonoids (Lifeng Industrial Co., Ltd., Taiwan), adjusted the pH to 6 to obtain a nutrient solution.

The total weight of the cereal composition is set to 100 parts by weight, and 60 parts by weight of the nutrient solution is added to the cereal composition, and then the two are uniformly mixed and allowed to stand until the cereal composition completely absorbs the nutrient solution. Thus, a solid medium for cultivating Antrodia camphorata was obtained.

Specific Example 2: Culture of Antrodia camphorata in solid medium

The solid medium obtained in the specific example 1 was dispensed into a plurality of space packs, and the space packs were subjected to high temperature and high pressure in a sterilizer (angular steam sterilizer A4-1, Yongdaming Instrument Co., Ltd., Taiwan). A sterilization procedure under the conditions of high temperature autoclaving at a temperature of 121 ° C and a pressure of 1.2 kg/cm ² for 60 minutes.

After the sterilized space bag is cooled, the vaccination of the burdock strain can be carried out. After the vaccination, the space bag is placed at room temperature for 2 to 4 months, and then the mycelium and fruit body of the burdock can be harvested. With half of the sub-entities.

Combining the above-mentioned mycelium, fruit body and half fruit body together with culture After the base was harvested, the components were analyzed by high performance liquid chromatography (HPLC, HP/Agilent 1100, USA) (refer to Zhang Dongzhu et al. in Taiwan Journal For Science 26(2): 125-133, 2011); The aldehyde-perchloric acid test method is used to test the content of the indicator substance in the physiologically active component: triterpenoids (refer to Jiang Shaolin et al., published in Journal of Jiangxi Agricultural University, Vol. 28, No. 4, pp. 634-636); The content of polysaccharides was examined by phenol-sulfuric acid detection method (refer to Xue Mei et al., 2003, pp. 267-268, No. 24, No. 3, Shaanxi Traditional Chinese Medicine).

result

1 is an HPLC chromatogram of a wild A. angustifolia fruiting body (refer to Zhang Dongzhu et al., Taiwan Journal For Science 26(2): 125-133, 2011); FIG. 2 is a culture medium and a culture method for cultivating Antrodia camphorata according to the present invention. HPLC chromatograms obtained by mixing the mycelium, fruiting bodies and half fruit bodies of Antrodia camphorata.

Comparing Fig. 1 with Fig. 2, it can be seen that the mycelium, fruit body and half fruit body obtained by the invention are not completely identical to the components contained in the wild A. angustifolia fruit body, and it is seen from Fig. 2 A considerable number of peaks were precipitated between 130 and 180 minutes, indicating that the composition of the Antrodia camphorata obtained by the present invention may be more diverse.

Comparing the results of the content of the polysaccharides of the Antrodia camphorata and the triterpenoids obtained by the present invention and the traditional segmental wood cultivation method and the solid culture method are as follows:

From the results, it can be seen that the content of the triterpenoids of the anthracnose cultivated by the present invention is much higher than that obtained by the general solid culture method, and the value is even the same as that of the fruit body obtained by the wood cultivation method. It is a scale; because the cultivation time of the present invention is shorter than that of the wood cultivation method, the content of the polysaccharide is much higher than the polysaccharide content of the fruit body obtained by the wood cultivation method.

It is worth mentioning that the culture method for cultivating Antrodia camphorata according to the present invention is characterized in that it is possible to determine when to stop the culture and harvest according to the type, amount and ratio of the physiologically active ingredients desired, for example, if the polysaccharide content is required to be compared The cultivation time of the high person needs to be shortened, and the culture time of the higher the triterpenoid content can be prolonged, whereby the present invention can regulate the physiologically active ingredient obtained.

In summary, the culture medium for cultivating Antrodia camphorata uses readily available dry cereals as a culture substrate, and the peptides and flavonoid compounds additionally added as physiological regulators are also abundant in natural products, and are produced or purified in various ways. The characteristics of the means, so the present invention is used to cultivate calves The medium of Zhizhi has the advantages of no shortage of raw materials and low price, and its preparation method is also quite simple.

In addition, the culture method for cultivating Antrodia camphorata is cultured by using the above medium, the operation steps are simple, and the required materials and instruments are common and easy to obtain; further, the culture method for cultivating Antrodia camphorata is not required Strict environmental parameter control, can be carried out smoothly at room temperature, compared with the expensive and difficult to obtain wood culture method, and the equipment is expensive and requires constant monitoring during cultivation to maintain its strict environmental conditions. The liquid fermentation method of the parameter can effectively save time, manpower and cost, and thus is easy to popularize.

The analysis results show that the invention can change the shortcomings of the low content of the physiological active ingredients of the general mycelium and the half fruit body, and the medicinal properties of the cultured A. angustifolia mycelium and the half fruit body are greatly enhanced, and the present invention not only contains the unique characteristics of the wild A. angustifolia. The triterpenoids, the physiologically active ingredients of the index can also compete with wild Antrodia camphorata, and even better than wild Antrodia camphorata.

It is worth mentioning that the present invention can regulate the type, amount and proportion of the physiologically active ingredients obtained by adjusting the length of the culture time, and harvest the same period of growth of the Antrodia camphorata (mycelium, fruit body). And the semi-sub-entity), can also obtain metabolites unique to different growth stages of O. chinensis, so the species of physiologically active ingredients can be even richer and more diverse than the O. chinensis fruiting body cultivated by the segmental wood cultivation method. Thus, the present invention It can stimulate the growth of A. sinensis to produce more and more effective physiologically active ingredients, thereby improving the health and disease treatment effect of Antrodia camphorata, and thus it is indeed possible to achieve the object of the present invention.

The above is only the preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, that is, the simple equivalent changes and modifications made by the patent application scope and patent specification content of the present invention, All remain within the scope of the invention patent.

Claims (6)

A culture medium for cultivating Antrodia cinnamomea , comprising a cereal as a matrix and a nutrient solution added to the cereal, the nutrient solution comprising 100 parts by weight of water, 2 to 30 parts by weight of a carbon source, and 5 to 30 parts by weight a nitrogen source, 0.1 to 2.0 parts by weight of the soybean peptide, and 0.1 to 2.0 parts by weight of the citrus flavonoid, the weight of the cereal being 30 to 70 parts by weight based on 100 parts by weight of the nutrient solution added to the cereal . The medium for cultivating Antrodia camphorata according to claim 1, wherein the cereal is at least one selected from the group consisting of rice, millet, sorghum, alfalfa, wheat, barley, barley, oat, rye, alfalfa, maize, buckwheat , Indian buckwheat, wild buckwheat, buckwheat, soybean, black beans, broad beans, peas, mung beans, red beans and peanuts. The culture medium for cultivating Antrodia camphorata according to claim 1, wherein the nitrogen source is at least one selected from the group consisting of yeast extract, whey protein hydrolyzate, tryptone, peptone, various amino acids, urea, Ammonium acetate, ammonium chloride, ammonium nitrate, potassium nitrate and sodium nitrate. A culture method for cultivating Antrodia cinnamomea comprises the steps of: (1) mixing a composition of a medium in proportion, the composition of the medium comprising a cereal as a matrix, and a nutrient solution added to the cereal, The nutrient solution comprises 100 parts by weight of water, 2 to 30 parts by weight of a carbon source, 5 to 30 parts by weight of a nitrogen source, 0.1 to 2.0 parts by weight of a soybean peptide, and 0.1 to 2.0 parts by weight of citrus flavonoids, and 100 weights. a portion of the cereal, mixing 30-70 parts by weight of the nutrient solution, that is, a medium for cultivating Antrodia camphorata; (2) mixing the medium and allowing the whole liquid to completely absorb the nutrient solution; (3) The medium is subjected to a sterilization procedure; and (4) Antrodia camphorata is inoculated to the medium and cultured at 20 to 30 °C. The culture method for cultivating Antrodia camphorata according to claim 4, wherein the cereal of the step (1) is at least one selected from the group consisting of rice, millet, sorghum, alfalfa, wheat, barley, barley, oats, black Wheat, alfalfa, maize, buckwheat, Indian buckwheat, wild buckwheat, buckwheat, soybean, black bean, broad bean, pea, mung bean, red bean and peanut. The culture method for cultivating Antrodia camphorata according to claim 4, wherein the nitrogen source used in the step (1) is at least one selected from the group consisting of yeast extract, whey protein hydrolyzate, tryptone, Peptone, various amino acids, urea, ammonium acetate, ammonium chloride, ammonium nitrate, potassium nitrate and sodium nitrate.