TWI785245B - Liquid culturing method for cordyceps cicadae - Google Patents

Abstract

A liquid culturing method for Cordyceps cicadae is provided, including culturing a Cordyceps cicadae strain in a liquid medium to obtain a mycelium of the Cordyceps cicadae strain, wherein the sequence of the Cordyceps cicadae strain is shown as SEQ ID NO:1, wherein the liquid medium includes a basic medium.

Description

The liquid culture method of cicadae

The invention relates to a liquid culture method of cicadae, in particular to a liquid culture method of cicada cultured by using a liquid culture medium.

With the development of medical technology, in addition to western medicine, traditional Chinese medicine has also become a hot topic. The literature states that Cordyceps sinensis is a popular traditional Chinese medicine in Taiwan that has been used as a remedy for more than hundreds of years.

However, in recent years, the output of Cordyceps sinensis has not only shown a downward trend, but also the source of acquisition has begun to be restricted. Therefore, the development of alternative materials for Cordyceps sinensis or other species of Cordyceps sinensis has attracted much attention. Therefore, since Cicadae has good pharmacological activity like Cordyceps sinensis, it has received attention accordingly.

Therefore, in order to avoid long-term overharvesting of wild cicadae in the future, wild cicadae will be in short supply similar to Cordyceps sinensis in the market. Still need a kind of liquid culture method of the cicadae that can reduce culture time, reduce field bacterial contamination ratio.

In view of the above problems, the present invention provides a liquid culture method of cicadae, which comprises: cultivating the cicadae strain in a liquid medium, and the sequence of the cicadae strain as shown in SEQ ID NO: 1, to obtain the cicadae strain Mycelia; wherein the liquid medium comprises a basal medium.

Optionally, the basal medium comprises a carbon source, and the carbon source comprises glucose, fructose, xylose, sucrose, maltose, lactose, dextrin, carboxymethylcellulose, soluble starch, mannitol, or a combination thereof.

Optionally, the carbon source accounts for 0.5%-5% of the total volume ratio of the basal medium.

Optionally, the basal medium comprises a nitrogen source, and the nitrogen source comprises protein, yeast extract, malt extract, beef extract, casein, sodium nitrate, potassium nitrate, ammonium sulfate, or combinations thereof.

Optionally, the nitrogen source accounts for 0.1%-3% of the total volume ratio of the basal medium.

Optionally, the basal medium comprises mineral elements, and the mineral elements comprise magnesium sulfate, calcium sulfate, ferrous sulfate, ferric sulfate, manganese sulfate, zinc sulfate, copper sulfate, nickel sulfate, or a combination thereof.

Optionally, the biomass content of the mycelia of cicadae is 2-9g/L.

Optionally, the uracil, uridine, adenine and adenosine contents of the mycelium of the cicada flower are respectively 0.5~3mg/mg, 0.05~0.6mg/mg, 0.02~0.9mg/mg and 0.1~2.91 mg/mg.

Optionally, the contents of total flavonoids and total phenols in cicada flower are 0.1-37 mg/mg and 1.5-25 mg/mg respectively.

The liquid culture method of cicadae of the present invention has the following advantages:

(1) Since the cicadae of the present invention is Cordyceps cicadae Wu-BFP14 (SEQ ID NO: 1), it is possible to obtain extracts with different ratios than those of the conventional cicadae.

(2) Compared with cultivating cicadae in the field, the liquid culture method of cicadae of the present invention can achieve the purpose of reducing cultivation time, reducing the rate of bacterial contamination in the field, and improving the morphological stability of cicadae mycelium.

(3) In addition, because the liquid culture method of cicadae of the present invention can adjust the height of the composition that extract is obtained by adjusting parameters such as culture time, culture humidity, carbon source type, nitrogen source type, mineral element, therefore is It is a cultivation method that can be adjusted according to the needs.

S10~S20: steps

Figure 1 is a schematic flow chart of the cultivation method of the present invention.

Figure 2 is an image of the cicadae of the present invention.

Fig. 3 is the solid-state culture diagram of cicadae of the present invention.

Fig. 4 is the liquid culture diagram of cicadae of the present invention.

Fig. 5 is a solid-state culture diagram of cicadae of the present invention.

Fig. 6 to Fig. 8 are analysis diagrams of examples 1 to 11 of the present invention.

Fig. 9 to Fig. 11 are analysis diagrams of examples 12 to 21 of the present invention.

Fig. 12 to Fig. 14 are analysis diagrams of examples 22 to 30 of the present invention.

Fig. 15 to Fig. 18 are analysis diagrams of examples 31 to 28 of the present invention.

Fig. 19 to Fig. 22 are analysis diagrams of examples 39 to 45 of the present invention.

Fig. 23 to Fig. 26 are analysis diagrams of examples 46 to 53 of the present invention.

Fig. 27 to Fig. 30 are analysis diagrams of examples 54 to 73 of the present invention.

In order to make the above-mentioned purpose, technical features and benefits after actual implementation easier for those skilled in the art to understand, it will be described in more detail below with examples and drawings.

Referring to Fig. 1, it is a schematic flow chart of the liquid culture method of the present invention.

In step S10, culturing Cicadae species in liquid culture medium. The cicadae species is Cordyceps cicadae Wu-BFP14, and its sequence is shown in SEQ ID NO:1.

Liquid media may contain a carbon source. The carbon source may comprise glucose, fructose, xylose, sucrose, maltose, lactose, dextrin, carboxymethylcellulose, soluble starch and mannitol, or combinations thereof. Preferably, the carbon source accounts for 0.5%-5% of the total volume ratio of the basal medium, more preferably 1%-3%.

Liquid media can contain a nitrogen source. The nitrogen source may comprise protein, yeast extract, malt extract, beef extract, casein, sodium nitrate, potassium nitrate, ammonium sulfate, or combinations thereof. Preferably, the nitrogen source accounts for 0.1%-3% of the total volume ratio of the basal medium, more preferably 0.3-0.8%.

Liquid media may contain mineral elements. The mineral elements may include magnesium sulfate, calcium sulfate, ferrous sulfate, ferric sulfate, manganese sulfate, zinc sulfate, copper sulfate, nickel sulfate, or combinations thereof.

In one embodiment, the biomass content of the mycelia of Cicada japonica is 2-9 g/L. In one embodiment, the contents of uracil, uridine, adenine and adenosine in the mycelia of cicadae are 0.5-3 mg/mg, 0.05-0.6 mg/mg, 0.02-0.9 mg/mg and 0.1-2.91 mg/mg, respectively. mg/mg. In one embodiment, the contents of total flavonoids and total phenols in cicada flower are 0.1-37 mg/mg and 1.5-25 mg/mg, respectively.

In step S20, the mycelium of Cicadae sp. is obtained. Preferably, the incubation time is 7-14, more preferably 7-9. Preferably, the culture temperature is 20-32, more preferably 23-28. Preferably, the cultivation humidity is 100.

Each example is described in detail below.

research method

1.1 strain screening

The cicadae bacterial classification that the present invention selects is screened by Taiwan's Sanxia Mountain Area voluntarily, the bacterial classification of screening gained is cultivated, and its mycelium is collected, then extracts its genome DNA, utilizes PCR technology to make DNA Amplify. After selection and desequencing, its sequence was compared with other cicadae species in the NCBI gene bank by BLAST.與本發明進行比較之蟬花菌種包含習知之KF740422、KF373077、EU807996、KJ173474、FJ765285、KJ173475、FJ765283、KJ173461、FJ765282、AB916360、KP771879、AY245627、KJ857272、EU573331、JQ283963、EU573333、AB086631、FJ765284、KJ173448 , FJ765280, JX488475 and KJ173447 cicadae strains.

1.2 Liquid culture of cicada mycelium

Take the cicada mycelium in the petri dish, take a piece of bacteria slice with a diameter of 5mm, insert it into the sterilized liquid medium, and explore the effects of parameters such as different environmental factors, carbon source types and concentrations, nitrogen source types and concentrations, and trace elements. Effects on the growth of cicadae strains. The process is: use a metal tube with a diameter of 5mm to inoculate bacteria from the plate (the amount of bacteria inoculated is a piece of bacteria with a diameter of 5mm) to a 125mL flat-bottomed conical flask containing a medium, and cultivate it at a constant temperature of 28°C and 130rpm. The mycelium was filtered with a filter paper with a pore size of 0.45 μm and dried. Then, steps such as subpackaging and freezing bacteria can be carried out.

1.3 Strain activation

Re-inoculate the frozen strains into a culture dish containing potato dextrose medium (Potato dextrose agar, PDA), then use a milling cutter to cut out two PDAs with a diameter of 1 cm on the culture dish that has grown colonies, and inoculate Into 250mL strain liquid activation medium, cultivated at 25°C in a constant temperature shaking incubator at 150rpm for 7 days. Wherein, the liquid activation medium is potato dextrose medium (Potato Dextrose Broth, PDB).

1.4 Different carbon sources

Based on the above activated medium formula, the analysis was carried out without adding any carbon source and adding 2% of different carbon sources. Among them, the carbon source includes glucose (Glucose, Glu), fructose (Fructose, Fru), xylose (Xylose, Xyl), sucrose (Sucrose, Sur), maltose (Maltose, Mal), lactose (Lactos, Lac), dextrin (Dextrin, Dex), carboxymethylcellulose (carboxymethylcellulose, CMC), soluble starch (Soluble Starch, SS) and mannitol (Mannitol, Man). Wherein, the culture time is 7 days to 14 days; the culture temperature is 23° C. to 28° C.; the culture humidity is 100%.

1.5 Different nitrogen sources

Taking glucose as the carbon source for comparison, 0.5% of different nitrogen sources were added for analysis. Among them, the nitrogen source includes protein (Peptone, PT), yeast extract (YT), malt extract (Malt Extract, ME), beef extract (Beef extract, BE) and casein (Casein, Cas) and inorganic nitrogen sources such as sodium nitrate (SN), potassium nitrate (PN), ammonium sulfate (AS), sodium nitrite and urea Nitrogen source. Wherein, the culture time is 7 days to 14 days; the culture temperature is 23° C. to 28° C.; the culture humidity is 100%.

In addition, since mineral elements are also a major element for the growth of mycelia, glucose (Glucose, Glu) and yeast extract (Yeast extract, YT) were selected as carbon and nitrogen sources, and 2mM magnesium sulfate (MgSO ₄ ) was added respectively. , calcium sulfate (CaSO ₄ ), ferrous sulfate (FeSO ₄ ), iron sulfate (Fe ₂ (SO ₄ ) ₃ ), manganese sulfate (MnSO ₄ ), zinc sulfate (ZnSO ₄ ), copper sulfate (CuSO ₄ ), sulfuric acid Mineral elements of nickel (NiSO ₄ ) and 5mM magnesium sulfate were analyzed. Wherein, the culture time is 7 days to 14 days; the culture temperature is 23° C. to 28° C.; the culture humidity is 100%.

1.6 Cicada solid-state culture

With the aforementioned liquid-cultured cicadae strain liquid strains, take 10% of the total volume of the solid culture substrate and insert it into different sterilized grains, wherein the grains include red beans (RB), mung beans (GB), soybeans, etc. (YB), black beans (BB), rice beans (MB), legumes and grains, such as red barley (RY), white barley (WY), brown rice (RR), white rice (WR), purple rice (PR), millet ( MR), rice grains of ten grains (Ten), such as red Quinoa (TR), Peruvian quinoa (MUR), oats (YM), oatmeal (OL), wheat (MM), quinoa-like grains such as buckwheat (CM), and others such as sorghum (H), corn (PC) cereals.

1.7 Different light intensity

Take 30g of wheat as a solid-state culture substrate, add 1:1.5 solid-to-liquid ratio of the culture medium to sterilize, then insert 5% liquid strains, first cultivate in a dark room until white mycelium grows on the surface, and transpose different light intensities (0, 50, 100, 250, 500, 1000 and 1500Lux), and cultured at 25°C for 42 days.

1.8 Different light sources

Take 45g of wheat as the solid-state culture substrate, add 1:2 solid-to-liquid ratio medium for sterilization, then add 5% liquid strains, first cultivate in a dark room until white mycelium grows on the surface, and then transpose different light sources at a constant temperature of 25°C for 42 sky. Among them, the light source is 5 kinds of cold light fluorescent lamps (Cw) with different light quality in the micro tissue culture system (MEL, Mini E Light, Guangyin Biotechnology), 100% red light, 70% red light, 30% blue light, 30% Red light, 70% blue light, and 100% blue light are light-emitting diode light sources, and the light source parameters of the light source are shown in Table 1.

The above parameters are shown in Table 2 and Table 3. Among them, "--" means not added.

1.9 Extraction method

Use 1g of cicadae mycelium or solid-state culture to dry the powder, add solvent for extraction, then centrifuge at 9000rpm for 10 minutes, and take the supernatant as the extract. Wherein, the solvent can be deionized water, alcohol, acid or lye.

1.10 Sample handling

Take 1 g of freeze-dried mycelium and add it to 10 mL of distilled water, put it in an autoclave at 121°C for hot water extraction for 20 min, and filter to obtain the extract. The supernatant of the culture medium (the supernatant described in the extraction method) and the intracellular extract (the extract of the mycelia after extraction) were filtered through a 0.45 μm microporous membrane and analyzed by HPLC.

2.1 Analysis of polysaccharide content

Take 0.5mL of sample, add 0.5mL of phenol (Phenol, 10%, v/w), then add 2.5mL of sulfuric acid (Sulfuric acid, 36N), mix well and react at room temperature for 10 minutes, then place it in 25°C water for cooling reaction After 15 minutes, the sample after the reaction was recorded at a wavelength of 490 nm in the absorption spectrum.

2.2 Content analysis of cordycepin and adenosine

Take 10 mg of standard substances of cordycepin and adenosine, dissolve them in 10 mL of methanol (Methanol, 15%), analyze by high performance liquid chromatography (High Performance Liquid Chromatography, HPLC) to make calibration curves respectively, and then add cordycepin and The peak area of adenosine was substituted into the linear regression equation to obtain the concentrations of both.

2.3 Content analysis of uracil, uridine, adenine and adenosine

Standards of Uracil, Uridine, Adenine and Adenosine were prepared and dissolved in 10 mL of deionized water, and analyzed by high performance liquid chromatography to make calibration curves respectively. The operating conditions of HPLC are as follows: the column is Phenomenex Luna 5μ C18 100A; the mobile phase (Mobile phase) is 0.02M potassium dihydrogen phosphate (KH ₂ PO ₄ ): Methanol (85:15); the flow rate is 0.8mL/min; UV detector with a wavelength of 254nm; and the injection volume is 20μ.

2.4 Total phenols content (TPC) content

Take 1mL of sample solution, add 1mL of Folin-Ciocalteu reagent, shake and mix for 3 minutes, then add 1mL of sodium carbonate (Sodium carbonate, 10%, v/w), and react in the dark for 30 minutes at room temperature. Make a record. Using gallic acid (Gallic acid) as a standard, and making a calibration line, use the interpolation method to obtain the relative gallic acid equivalent (Gallic acid equivalent, GAE mg/g) in each gram of sample, which means the The total amount of phenolic compounds.

2.5 Total flavonoids content (TFC) content

Take 250 μL sample solution, add 1.25 mL deionized water and 75 μL sodium nitrite (NaNO ₂ , 5%, v/w), mix and react for 6 minutes, add 150 μL aluminum chloride (AlCl ₃ , 10%, v/w), Mix and react for 5 minutes, then add 0.5 mL of 1M sodium hydroxide (NaOH) and 275 μL of deionized water, and centrifuge at 9,000 rpm for 3 minutes in a high-speed centrifuge, take the supernatant and record the absorption spectrum at a wavelength of 510 nm. Use quercetin (Quercetin) standard substance and make a calibration line, use the interpolation method to obtain the relative quercetin equivalent (Quercetin equivalent, QE mg/g) in each gram of sample, indicating the total amount of flavonoids in the sample .

Discussion of results

3.1 Morphological characteristics of cicadae

With reference to Fig. 2, it is the cicadae image figure of the present invention. Among them, part (a) represents the whole picture of wild cicadae; part (b) represents the fruiting body; part (c) represents the insect body; part (d) represents the microscopic photo of the fruiting body; part (e) represents the microscopic photo of the fruiting body; (f ) part represents the microscopic photo of fruiting body; part (g) represents the microscopic photo of fruiting body; part (h) represents the microscopic photo of mycelium; and part (i) represents the microscopic photo of mycelium.

The cicadae disclosed by the present invention is Taiwan's native cicadae which is a pure species of cicadae isolated from bamboo forests in mountainous areas of Taiwan. The surface of fresh cicadae is covered by white or off-white hyphae, which will turn light yellow to brown after drying. Except for the epidermis, compound eyes and three pairs of thoracic legs, all the organs of the insect body remain unchanged. All will be covered by hyphae, forming a long oval sclerotia with a size of about 3 cm and a diameter of about 1-1.1 cm. The sclerotium grows 3-4 branches with a length of about 4-10 cm, antler-shaped or rod-shaped. Observing the fruiting bodies of wild cicadas with an optical microscope, it can be found that there are many branches and oval conidia in the fruiting bodies of cicadas, while only many filamentous structures are found in the cicadas.

With reference to Fig. 3, it is the cicadae solid-state culture figure of the present invention. Wherein, part (a) represents plate culture; part (b) represents the close-up of plate culture; part (c) represents the microscope photo of mycelia; and part (d) represents the microscope photo of mycelia.

After 10 days of culturing the fruiting bodies of cicadae in a PDA petri dish, a white to off-white felt-like to fluffy mycelium of cicadae can grow, and the back of the colony shows a pattern of color-releasing grooves. Half-yellow to orange-yellow hyphae, the inner wall of the hyphae of cicadae is smooth and transparent tube, showing many dense and irregular branches, and the thickness of these branches is about 2.0μm-3.5μm.

With reference to Fig. 4, it is the liquid culture figure of cicadae of the present invention. Among them, part (a) represents liquid shake flask culture; part (b) represents plate culture; part (c) represents close-up of liquid shake flask culture; part (d) represents microscopic photos of mycelia; part (e) represents mycelium Part (f) represents a microphotograph of mycelium; part (g) represents a microphotograph of mycelium; part (h) represents a microphotograph of mycelium; and part (i) represents a microphotograph of mycelium photomicrograph.

After the cicada is cultured in a liquid state, it will gradually turn from a light yellow medium to a light pink to lavender culture medium. Observing with an optical microscope, it can be found that the mycelium of the cicada is fermented in a liquid state. Under low magnification, it appears densely packed Such as filaments in the shape of the hair structure, and then enlarged to 500 times with the magnification, it can be found that the liquid cultured hyphae of cicadae are similar to the mycelia cultured on the petri dish, with a transparent tube with smooth inner wall, showing There are many dense and irregular branches, and the thickness of these branches is about 1.5 μm-3.0 μm, which is smaller than the cicadae petri dish, but the density is higher than that of the cicadae petri dish mycelium. After the liquid culture medium was left to stand for 1 to 2 weeks, the surface of the medium changed to form a thin layer of cicada mycelium film. Observing the surface of the mycelium film with an optical microscope can find that the surface of the mycelium film has a rather multi-layered filamentous structure. With the magnification of the microscope, you can gradually find dense transparent filaments in the shape of hair structure. After magnifying to 500 times, you can also find that the mycelium is similar to the mycelium and the mycelium of the petri dish, with an inner wall. Light Smooth transparent tube with many dense and irregular branches, and the thickness of these branches is about 5.0μm-10μm.

With reference to Fig. 5, it is the solid-state culture figure of cicadae of the present invention. Among them, part (a) represents the solid-state culture of cicadae; part (b) represents the close-up of the fruit body; part (c) represents the close-up of the base surface; part (d) represents the micrograph of the fruit body; and part (e) represents the mycelium body micrographs.

Inoculate cicadae strains into whole grains and cultivate them for about 1 to 2 months to produce artificial solid-state cultured fruiting bodies and mycelia. The artificially cultivated fruiting bodies are as fresh as wild fruiting bodies The appearance of the entity is white or off-white, the length is about 4-10 cm, and the thickness is about 0.1-0.5 cm. After drying, it will turn light yellow to brown, but the length and thickness of the fruiting body will shrink obviously. Further use of optical microscope observation, it can be found that the artificial fruiting body presents a multi-level structure, and the distribution of many hyphae can be observed around the fruiting body. The artificial mycelium had similar results to the wild cicada, with multi-layered filamentous structures.

3.2 Identification of Cicadae species

The native cicadae was first judged by its appearance, and then cultured in liquid and solid state respectively. Generally, the classification of fungi is mainly based on ribosomal DNA. For the 18SRrna gene, 5.8Rrna gene, or 28SRrna gene ribosomal DNA has a very high retention in fungi, it is mainly used for research on the classification of families and genus. The 5.8S Rrna gene and the 18S and 28S Rrna genes respectively have an internal transcribed spacer (internal transcribed spacer, ITS), divided into ITS1 and ITS2, and the evolution rate of ITS1 and ITS2 is relatively fast. The sequence will be significantly different. Therefore, the ITS sequence of the cicadae strain is analyzed, and its ITS sequence is compared with the ITS sequences of 22 different cicadae ( Cordyceps cicadae ) on the gene database (NCBI GenBank), and it can be found that its sequence is similar to that of Cordyceps cicadae The accuracy reached more than 99%, confirming that the screened bacterial strain is the cicadae strain, and this bacterial strain was named Cordyceps cicadae Wu-BFP14, and its gene sequence was uploaded to the genetic data database, the accession number (Accession number) is KX289580, in the following Abbreviated herein as BFP14 (SEQ ID NO: 1).

3.3 Different carbon sources

Referring to Fig. 6, it is an analysis diagram of Example 1 to Example 11 of the present invention. Among them, parts (a) to (d) respectively represent the pH value, the content of exopolysaccharides (Exo polysaccharides, EPS, unit: g/L), the content of intracellular polysaccharides (Intracellular polysaccharides, IPS, unit: g/L), and Biomass (Biomass, unit: g/L) content.

There is not much difference in the pH of BFP14 fermented with various carbon sources, and they are all maintained at around pH5-pH6. Only when CMC is used as the carbon source, the pH is slightly higher, at around pH7. It can be found that BFP14 can be used in monosaccharides (Gly, Fru and Xyl), disaccharides (Sur, Mal and Lac), polysaccharides (Dex, CMC and SS) and even polyols (Man).

In terms of biomass of BFP14 mycelium, the biomass of mycelium was 2.11g/L under the condition of no carbon source added. After adding various carbon sources, the biomass of mycelium increased significantly. Among them With Gly as the better carbon source for cultivation, the biomass of mycelium can reach 6.55g/L, which is more than three times higher than that without adding carbon source, followed by Man, with about 6.21g/L of mycelium Biomass, again 6.15g/L of SS. In terms of intracellular polysaccharide content, Gly is also the best and can reach 6.19g/L, followed by lactose (Lac) which can reach 5.25g/L. In terms of extracellular polysaccharides, mannitol (Man) is the best up to 17.8g/L, followed by Glu up to 14.51g/L, and there is a significant difference in the production of extracellular polysaccharides from other carbon sources.

With reference to Fig. 7, it is the analytical diagram of example 1 to example 11 of the present invention. Wherein, parts (a) to (d) respectively represent the content (mg/mg) of uracil, uridine, adenine and adenosine.

It can be found that when Xyl is used as the carbon source, it has the highest content of uracil, uridine and adenine, which are 2.91mg/g, 0.53mg/g and 0.10mg/g respectively; Glycosides are The content of carbon source is second only to xylose, which are 2.49mg/g and 0.44mg/g respectively; followed by Sur, which are 2.39mg/g and 0.48mg/g respectively, and then glucose and fructose. Since Mal and Sur belong to disaccharides formed by the polymerization of two molecules of glucose, one molecule of glucose and one molecule of fructose respectively, it is speculated that Glu and Fru can promote the formation of uracil and uridine. In terms of adenosine, it can be clearly found that the preference is CMC, which has a content of about 2.91 mg/g, followed by Lac, which has a content of 2.65 mg/g, and then Glu, which has a content of 2.22 mg/g. .

Referring to Fig. 8, it is an analysis diagram of Example 1 to Example 11 of the present invention. Wherein, parts (a) and (b) respectively represent the content (mg/mg) of total flavonoids and total phenols.

In terms of total phenol content, it can be found that when Xyl is used as the carbon source, it is the highest, with a gallic acid content of 9.87mg/g, followed by Dex with a gallic acid content of 6.93mg/g, and then followed by Glucose had a gallic acid content of 5.35 mg/g, while none of the remaining carbon sources yielded a gallic acid higher than 4 mg/g. In terms of flavonoid content, the same Xyl as the carbon source is the highest, with a quercetin content of 19.76mg/g, followed by Lac with a quercetin content of 15.5mg/g.

Based on the above results, Glu is the carbon source used by most bacteria and fungi. It is relatively easy to obtain and relatively cheap in cost. It is also superior to other active ingredients such as uracil, uridine, adenine and adenosine. For most of the carbon source, glucose is selected as the carbon source used in the following examples.

Referring to Fig. 9, it is an analysis diagram of Example 12 to Example 21 of the present invention. Wherein, parts (a) to (d) respectively represent pH value, exopolysaccharide content, intracellular polysaccharide content, and biomass content.

All five organic nitrogen sources could be utilized by BFP14, but only SN, PN and AS could be utilized among inorganic nitrogen sources. Overall, the culture conditions with organic nitrogen sources were better than inorganic nitrogen sources. Among the organic nitrogen sources, the biomass of BFP14 mycelium is better than YT, and the biomass of 8.37g/L can be obtained, which is nearly three times higher than that of the condition without nitrogen source (3.11g/L). times, followed by Cas (7.32g/L), while MT The lowest mycelium biomass is only 4.50g/L. Among the inorganic nitrogen sources, SN was the best, and 5.05g/L mycelium biomass could be obtained, followed by PN (5.01g/L), and ammonium sulfate was the most unsuitable for the growth of BFP14 mycelium biomass.

In terms of polysaccharides, MT is the most preferred, with the contents of intracellular polysaccharides and exopolysaccharides being 17.54g/L and 54.96g/L respectively, and polysaccharides in inorganic nitrogen sources such as mycelium biomass, and SN (11.53g/L, 50.45g/L) is the best, followed by PN (10.17g/L, 46.37g/L). In the present invention, whether BFP14 is mycelia biomass, intracellular polysaccharides or exopolysaccharides, organic nitrogen sources are superior to inorganic nitrogen sources, and it can be found in inorganic nitrogen sources that nitrate nitrogen sources are superior to ammonium salts Nitrogen source.

Referring to Fig. 10, it is an analysis diagram of Example 12 to Example 21 of the present invention. Wherein, parts (a) to (d) respectively represent the content (mg/mg) of uracil, uridine, adenine and adenosine.

In terms of biologically active ingredients, among the organic nitrogen sources, YT has the highest four biologically active ingredients of uracil, uridine, adenine and adenosine, with 1.69mg/g, 0.17mg/g, and 0.91mg respectively /g and 0.98mg/g content, followed by Cas, PT, BT and MT, in which the presence of uridine was not detected in MT, and in the inorganic nitrogen source, the same nitric acid The salt nitrogen source is better than the ammonium salt nitrogen source, and the four bioactive components have their own advantages in SN and PN, but SN is better than PN.

Referring to Fig. 11, it is an analysis diagram of examples 12 to 21 of the present invention. Wherein, parts (a) and (b) respectively represent the content (mg/mg) of total flavonoids and total phenols.

In terms of total phenol content, when YT is used as the nitrogen source, there will be a better total phenol content, with a gallic acid content of 24.21mg/g, followed by PT with a gallic acid content of 23.6mg/g, and then The reason is that Cas has a gallic acid content of 22.36 mg/g, and the total phenolic content of the other two organic nitrogen sources is less than 20 mg/g of gallic acid content. Among the inorganic nitrogen sources, SN is the best, which is equivalent to 21.62 mg/g of gallic acid content, followed by PN (20.29 mg/g), which is much higher than AS (1.7 mg/g). In terms of flavonoid content, the organic nitrogen source is YE For the best, there is 24.36mg/g quercetin content, followed by PT with 23.96mg/g quercetin content, and Cas with 23.93mg/g quercetin content, and the other two organic nitrogen sources All of them have a total flavonoid content greater than 20mg/g. Among the inorganic nitrogen sources, AS has the highest total flavonoid content, up to 12.83mg/g.

Referring to Fig. 12, it is an analysis diagram of examples 22 to 30 of the present invention. Wherein, parts (a) to (d) respectively represent pH value, exopolysaccharide content, intracellular polysaccharide content, and biomass content.

With the addition of various mineral elements, BFP14 mycelium can grow. Under the conditions of no mineral elements added and magnesium sulfate and calcium sulfate added, the fermentation broth will appear red, while under the conditions of other mineral elements, different color changes will appear. BFP14 mycelium biomass is better without adding mineral elements, up to 6.35g/L mycelium biomass, followed by adding 2mM magnesium sulfate (6.18g/L), and other trace elements are higher than 6g/L The mycelium biomass above L, nickel sulfate is the most unfavorable to the growth of BFP14 mycelium, only 2.73g/L mycelium biomass, followed by iron sulfate (3.43g/L). In terms of intracellular polysaccharides, it can be clearly observed that the addition of magnesium sulfate is the best, up to 7.12g/L intracellular polysaccharides, followed by calcium sulfate with 6.40g/L intracellular polysaccharides, and the rest of the conditions are all The intracellular polysaccharide yield is higher than 6g/L, and the iron sulfate is the lowest, and the intracellular polysaccharide yield is only 3.79g/L. Adding different mineral elements has no significant difference in the exopolysaccharide. Nickel sulfate is the best, and the exopolysaccharide can be obtained at 55.19g/L, and the exopolysaccharide output of the other conditions is maintained at 48 to 53g/L. between.

Referring to Fig. 13, it is an analysis diagram of examples 22 to 30 of the present invention. Wherein, parts (a) to (d) respectively represent the content (mg/mg) of uracil, uridine, adenine and adenosine.

For the content of uracil, uridine, adenine and adenosine in BFP14 mycelium extract, ferric sulfate is the best in terms of uracil content, which can reach 2.75mg/g, but its uridine content is relatively low good. Under the condition of copper sulfate, the contents of uridine, adenine and adenosine are the worst, obviously copper sulfate may be less conducive to the production of biologically active ingredients. In adenine, manganese sulfate is preferred, which can reach 0.52mg/g, followed by iron sulfate (0.49mg/mg), but in adenosine, zinc sulfate is better, up to 2.46mg/g, followed by nickel sulfate (2.44mg/g).

Referring to Fig. 14, it is an analysis diagram of examples 22 to 30 of the present invention. Wherein, parts (a) and (b) respectively represent the content (mg/mg) of total flavonoids and total phenols.

In terms of total phenol content, magnesium sulfate is the best, which is equivalent to 9.73mg/g of gallic acid content, and the content of flavonoids can be clearly observed that manganese sulfate is the highest, which is equivalent to 36.98mg/g of quercetin. Followed by manganese sulfate.

The addition of different mineral elements in the present invention will not only affect the biomass of mycelia and intracellular polysaccharides, but also affect the composition of different bioactive components, and the proportions of bioactive components contained in different element extracts are quite different. In terms of mycelium biomass, no mineral elements are added, followed by magnesium sulfate, and intracellular polysaccharides are better than magnesium sulfate. In terms of biological active ingredients, they are different. Among them, the flavonoid ingredients added with manganese sulfate are the best. However, its mycelium biomass does not perform well. Considering the antioxidant activity and flavonoid content in the future, manganese sulfate is a key point worthy of attention.

Referring to Fig. 15, it is an analysis diagram of Examples 31 to 38 of the present invention, wherein, parts (a) and (b) represent moisture content (%) and yield (Product yield) respectively. The calculation method of the yield is = product (g) / raw material (raw material) (g).

In the solid-state culture of BFP14 with oatmeal as the grain with different culture days, it can be found that after 7 days of culture, the mycelium can cover the entire grain substrate, and after 14 days of culture, it can grow 0.3-0.5cm The primordia, when cultured for 21 days, can grow fruiting bodies of 1-3cm. When cultivating for 28 days, the fruiting body height can reach 3-5 centimeters, and when cultivating for 35 days, the fruiting body grows to about 5-7 cm, and then when cultivating for 42 days, spores form, and the fruiting body shrinks gradually. Among them, the cultivation humidity is 60-80% around, and as the number of days of cultivation increases, the humidity will also increase. In terms of productivity, there is also a phenomenon that productivity decreases as the number of days of culture increases.

Referring to Fig. 16, it is an analysis diagram of Examples 31 to 38 of the present invention, which represents the analysis of polysaccharide (Total sugars content, TSC, unit: glucose equivalent (glucose equivalent) g/L) content.

It can be found that as the number of days of culture increases, the total polysaccharide content shows a downward trend. On the seventh day of solid-state culture of BFP14 grain matrix, the total polysaccharide content is 27.55g/L. After 56 days of culture, the polysaccharide content is only 8.88g/L.

With reference to Fig. 17, it is the analytical diagram of example 31 to example 38 of the present invention. Wherein, parts (a) to (d) respectively represent the content (mg/mg) of uracil, uridine, adenine and adenosine.

The four biologically active components of uracil, uridine, adenine and adenosine all increased with the number of days of culture. On the 35th day of culture, which is also the most mature stage of the fruiting body, the highest content was 0.42 and 0.09 respectively. , 0.07 and 0.63mg/g content.

Referring to Fig. 18, it is an analysis diagram of examples 31 to 38 of the present invention. Wherein, parts (a) and (b) respectively represent the content (mg/mg) of total flavonoids and total phenols.

Total flavonoids and total phenols also increased with the number of days of culture. On the 42nd day of culture, there was the highest total phenol content, which was equivalent to the gallic acid content of 9mg/g, compared to 7 days of culture (2.62mg/g The total phenolic content of g) can be increased by more than 3 times, while the flavonoid content has been showing an increase along with the number of days of culture. There is a quercetin content equivalent to 10.74mg/g in 7 days of culture, and after 56 days of culture, Then it can reach the content of quercetin equivalent to 49.83mg/g, which is nearly five times higher.

With reference to Fig. 19, it is the analytical chart of example 39 to example 45 of the present invention, wherein, part (a) and part (b) represent moisture content and yield respectively.

It can be observed that under the condition of light intensity of 0Lux, a layer of white mycelium film grows along the edge of the culture container, and no fruiting bodies are produced. Under the light intensity of 50Lux, fruiting bodies of 5-7 cm can grow. With the increase of light intensity, there is not much change. When the light intensity is 1500Lux, it can be found that the fruiting bodies are gradually shrinking. In the solid-state culture of BFP14 grains, a little light is necessary to stimulate the fruiting bodies. growth, and the light intensity should not be too high. If the light intensity is too high, spores will form and the fruiting bodies will gradually shrink.

In the cultivation of different light intensities, the cultivation humidity without light is about 70%, and the cultivation humidity of other light intensities is maintained at about 80%. In terms of yield, under the condition of no light, the yield of 70% can be obtained , After 30g of grains are fermented, 21g of fermentation products can be obtained. Under the light conditions of 50 to 1500Lux, the yield is about 50%, and about 15g of fermentation products can be obtained.

Referring to Fig. 20, it is an analysis diagram of Example 39 to Example 45 of the present invention, which represents the analysis of polysaccharide content.

It can be found that under the culture condition of 0Lux, there is the highest polysaccharide content (14.86g/L), but with the increase of light intensity, the polysaccharide content gradually shows a downward trend.

With reference to Fig. 21, it is the analytical diagram of example 39 to example 45 of the present invention. Wherein, parts (a) to (d) respectively represent the content (mg/mg) of uracil, uridine, adenine and adenosine.

Light intensity does not have much effect on the biologically active components of uracil, uridine, adenine and adenosine, but it can be observed that after light culture, the biologically active components are higher than those without light, especially in the content of uridine , it can be clearly observed that the content of uridine cannot be detected under the light conditions of 0 to 100Lux. As the light intensity increases, the content increases. At 500Lux, there is the highest uridine content (0.09mg /g), and then decreased.

With reference to Fig. 22, it is the analytical diagram of example 39 to example 45 of the present invention. Wherein, parts (a) and (b) respectively represent the content (mg/mg) of total flavonoids and total phenols.

In terms of total phenol content, it can be observed that there is no significant difference under the light conditions of 50 to 1000Lux white light; under no light and 1500Lux light, there are 9.18mg/g and 10.67mg/g respectively. Gallic acid content, other conditions are greater than 12mg/g gallic acid content, and 1000Lux is the highest, equivalent to 13.24mg/g gallic acid content. In terms of flavonoid content, there is also no significant difference under the light of 50-1000Lux; while under the light of no light and 1500Lux, there are quercetin contents equivalent to 13.17mg/g and 13.77mg/g respectively, and the rest The conditions are all greater than 15mg/g quercetin content, and 500Lux is the highest, which is equivalent to 16.77mg/g quercetin content.

In terms of fruiting body shape, the light intensity is between 50 and 1000Lux, which does not have much effect on the growth of fruiting bodies. Except for the high content of uracil at 500Lux, other biologically active ingredients are also between 50-1000Lux There is not much difference between them. The content of total phenols and flavonoids also has this phenomenon. However, there is no significant difference in the antioxidant activity. It can be found that no light and high light intensity (1500Lux) are more unfavorable, and 1000Lux is better. Based on the above results, it is considered that the light intensity of 500Lux is a better culture condition, and the follow-up research will be carried out with a light intensity of 500Lux. Wherein, the follow-up research refers to the use of white light as the illumination light source condition is 500 Lux, while the light source of other light sources is not 500 Lux due to different light source types.

With reference to Fig. 23, it is the analytical chart of example 46 to example 53 of the present invention, wherein, (a) and (b) part represent moisture content and yield respectively.

Under various light conditions, BFP14 grain solid-state culture can produce fruiting bodies. It can be observed that under 6R3B and 9IR, the growth height of fruiting bodies is relatively high, about 5 cm. However, the height of fruiting bodies under other light source conditions, It is about 3-4 cm.

The cultivation humidity of different light sources is about 80%, and in terms of yield, it is about 40-50%, and it is higher than 50% under 5000K and 6R3B, which are 53% and 54% respectively.

Referring to Fig. 24, it is an analysis diagram of Example 46 to Example 53 of the present invention, which represents the analysis of polysaccharide content.

In terms of total sugar content, it can be found that there is not much difference, and the polysaccharide content is maintained at 9-11g/L. 6R3B is the best, with a polysaccharide content of 11.41g/L.

Referring to Fig. 25, it is an analysis diagram of examples 46 to 53 of the present invention. Wherein, parts (a) to (d) respectively represent the content (mg/mg) of uracil, uridine, adenine and adenosine.

In uracil, the control group (white light of 500 Lux) is the best, with a content of 1.4 mg/g, followed by 3R3B3IR with a content of 1.31 mg/g, and 9IR with a content of 1.27 mg/g. For uridine, 9B is preferred, with a content of 0.19 mg/g, followed by 9IR with a content of 0.15 mg/g, and 3R3B3IR with a content of 0.14 mg/g. For adenine, 3R3B3IR is the best, with a content of 0.49 mg/g, followed by 6R3B with a content of 0.41 mg/g, and 9R with a content of 0.4 mg/g. Adenosine is preferably 9B, with a content of 1.05 mg/g, followed by 9IR with a content of 0.92 mg/g, and 3R3B3IR with a content of 0.87 mg/g. It can be found that far infrared rays can stimulate the content of uracil, blue light can stimulate the content of uridine and adenosine, and red light can stimulate the content of adenine.

With reference to Fig. 26, it is the analytical diagram of example 46 to example 53 of the present invention. Wherein, parts (a) and (b) respectively represent the content (mg/mg) of total flavonoids and total phenols.

Under different light source conditions, there is a gallic acid content equivalent to nearly 10mg/g, and 2700K is the best, with a gallic acid content of 12.32mg/g. In terms of flavonoid content, it can be found that 3R3B3IR is the best, which is equivalent to 41.36mg/g quercetin content, followed by 9IR which is equivalent to 36.61mg/g quercetin content. Representing that far-infrared rays can stimulate the flavonoid content of BFP14 grain solid-state culture.

With reference to Fig. 27, it is the analytical chart of example 54 to example 73 of the present invention, wherein, part (a) and part (b) represent moisture content and yield respectively.

It can be found that, among the 20 different grains used, the BFP14 grain solid-state culture can grow. With the exception of a few types of grains, most solid-state cultures of grains are capable of growing fruiting bodies. In bean grains, except for YB and BB that do not grow fruiting bodies, other bean grains can grow fruiting bodies of about 1-2 cm. Among rice grains, except for RR, which grow a thick layer of mycelium, the rest The six kinds of rice grains can grow fruiting bodies, and WR is the best among them. Among Limai grains, TR and MUR also only grow thick mycelium, and the other four Limai grains are all It can grow fruiting bodies, and the H and PC of other grains can also grow fruiting bodies. Among these grains, MM is the best fruiting body type, followed by WR. The fruiting body types of other grains have at least 3-4 cm fruiting bodies, but the fruiting body types of legumes are poor.

Except for the poor growth patterns in YB and BB, the moisture content is about 60%, while the moisture content of the other 18 grains is between 70% and 80%. In terms of yield, it can be found that the yield of bean grains is higher than 50%, which is higher than other types of grains, especially the two types of bean grains YB and BB, which are 74% and 79% respectively, and can be obtained 22.4 g and 23.7 g of fermentation product. However, the yield of rice grains is relatively low, with an average yield of about 40%.

Referring to Fig. 28, it is an analysis diagram of Examples 54 to 73 of the present invention, which represents the analysis of polysaccharide content.

In terms of total sugar content, different grains have different polysaccharide content. Among rice grains, MR is the highest, with a polysaccharide content equivalent to 18.75g/L, and WR has a polysaccharide content equivalent to 13.14g/L. For the sugar content, MB is the highest in beans and grains, with a polysaccharide content equivalent to 13.59g/L, and MM is the highest in Limai cereals, with a polysaccharide content equivalent to 11.71g/L.

With reference to Fig. 29, it is the analytical diagram of example 54 to example 73 of the present invention. Wherein, parts (a) to (d) respectively represent the content (mg/mg) of uracil, uridine, adenine and adenosine.

In legumes and grains, uracil is the highest in MB, with a content of 0.99 mg/g, followed by GB with a content of 0.94 mg/g. In addition, uridine and adenine were not detected in RB and BB. Uridine has the highest content of YB with a content of 0.02mg/g, but for adenine, it is better to use GB, with a content of 0.02mg/g, while for adenosine, it is better to use MB, with a content of 0.47mg/g g content. In rice grains, Ten is the highest content of uracil, and the content of 1.23mg/g can be obtained. In addition, in the content of uridine, WR, PR and MR have not been detected, and the highest content of RR is 0.21mg/g However, for adenine, PR is the best, with a content of 0.12 mg/g, while for adenosine, RR is the best, with a content of 0.97 mg/g. In Limai grains, uracil and uridine are better in TR, with contents of 1.13mg/g and 0.21mg/g respectively. YM is preferred, with a content of 0.08 mg/g, and YM is preferred for adenosine, with a content of 0.76 mg/g. In other types of grains, the content of these four bioactive components is relatively small, followed by Limai type of grains.

Referring to Fig. 30, it is an analysis diagram of examples 54 to 73 of the present invention. Wherein, parts (a) and (b) respectively represent the content (mg/mg) of total flavonoids and total phenols.

In terms of the total phenolic content of beans and grains, GB is the best, with a gallic acid content of 7.8mg/g, while PR is the best for rice cereals, with a gallic acid content of 12.77mg/g , followed by RR has a gallic acid content of 11.02mg/g, and in Limai cereals, OL has a gallic acid content of 10.92mg/g, while the H and PC of other cereals are respectively The gallic acid content was 8.45 and 6.63mg/g. In terms of flavonoid content, RB is the highest in bean grains, with a quercetin content of 31.88 mg/g, while rice grains are There is a big difference, the content of quercetin in WR is only 9.9mg/g, but the content of quercetin in PR is 41.22mg/g, which is more than four times worse, and the content of Limai grains is better in TR , with a quercetin content of 40.9 mg/g, while H and PC of other cereals have a quercetin content of 22.77 and 9.94 mg/g, respectively.

BFP14 can grow in 20 kinds of grains. In terms of fruiting body type, MM and WR are the best, but the content of bioactive components, total phenols and flavonoids is not the best. Each different grain has different advantages. For example, RR has the highest uridine and adenosine content, but its total phenolic and flavonoid content is not better, and its antioxidant activity is not better than other grains. PR has the highest The content of total phenols and flavonoids is high, and it also has quite good DPPH free radical scavenging activity, but the content of uridine has not been detected, and other antioxidant activities are not as expected. Here, it is only proved that BFP14 can grow in various grains, and the selected grains and the ratio can be adjusted according to the desired characteristics.

4 Conclusion

Cicadae belongs to the medicinal fungus of the genus Cordyceps. It is rich in biologically active components related to Cordyceps sinensis and Cordyceps militaris such as nucleotides, cordycepin and ergosterol, etc., and has antioxidant and anti-inflammatory effects. The present invention screened out the Taiwan native cicadae strain and named it Cordyceps cicadae Wu-BFP14 (NCBI: KX289580) after 18S rDNA identification, and explored the effects of carbon and nitrogen sources on the growth and biological activity of cicadae liquid fermentation mycelium .

The results of carbon source research showed that using glucose had better mycelium production (6.55g/L), and had the highest intracellular polysaccharide production (6.19g/L). In addition, the results also showed that when xylose was used as the carbon source, it had the highest content of uracil, uridine and adenine, which were 2.912mg/g, 0.527mg/g and 0.100mg/g, respectively. At the same time, xylose is also preferred in terms of total phenol and flavonoid content. In the study of nitrogen sources, the yield of BFP14 mycelium is better with yeast extract, which has a yield of 8.37g/L, and also has better uracil, uridine, adenine and adenosine, which are respectively 1.690mg /g, 0.172mg/g, 0.905mg/g and 0.976mg/g. The contents of total phenols and flavonoids were 24.21mgGAE/g and 23.96mg QE/g respectively.

The composition of the liquid medium for optimal mycelium growth, polysaccharide and bioactive component content is: glucose 50g/L, yeast extract 5g/L and 5mM manganese sulfate added.

In the solid-state culture, wheat was used as the culture substrate to explore the influence of various culture conditions on the biological activity. The best biological activity yield was obtained after 42 days of culture with a solid-liquid ratio of 2:1 and a light intensity of 500 Lux. Tested with different LED light sources, the contents of uracil (0.19mg/g), adenosine (1.05mg/g), total phenols (12.32mg GAE/g) and flavonoids (41.36mg QE/g) were respectively in blue light, Blue light, white light 2700K and red light, blue light, far infrared mixed light source are the best. Using 20 kinds of grains as the solid-state culture substrate of BFP14, observing the growth morphology and biological activity of different grains, the best grain substrate for the production of uridine and adenosine is brown rice, while the best content of phenols and flavonoids is purple rice. Meter.

The above descriptions are illustrative only, not restrictive. Any equivalent modification or change made without departing from the spirit and scope of the present invention shall be included in the scope of the patent application.

<110> Daye University

<120> Solid state culture method of cicadae

<130> DY-19007-TWI

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 549

<212>DNA

<213> Cicada (Cordyceps bifusispora)

<400> 1

S10~S20: steps

Claims (1)

A liquid culture method for cicadae, which comprises: cultivating a cicadae strain in a liquid medium, the cultivation time of the cicadae strain is 7-14 days, the cultivation temperature is 23-28°C, and the cultivation humidity is 100%, and The sequence of the cicadae species is shown in SEQ ID NO: 1, to obtain a mycelium of the cicadae species; wherein, the biomass content of the mycelium is 2 ~ 9g/L, and the biomass contains One uracil, one uridine, one adenine, one adenosine, a kind of flavonoid compound and one phenolic compound, the contents of the uracil, the uridine, the adenine and the adenosine are respectively 0.5~3mg/mg, 0.05 ~0.6mg/mg, 0.02~0.9mg/mg and 0.1~2.91mg/mg, the flavonoid compound (total flavonoid) content and the phenolic compound (total phenol) content are 0.1~37mg/mg and 1.5~ 25mg/mg; and the liquid medium comprises a basal medium, the basal medium comprises carbon source, nitrogen source and mineral elements, wherein the carbon source comprises glucose, fructose, xylose, sucrose, maltose, lactose, dextrin, carboxymethyl Cellulose, soluble starch, mannitol or a combination thereof, and the carbon source accounts for 0.5% to 5% of the total volume ratio of the basal medium; the nitrogen source includes protein, yeast extract, malt extract, beef extract, Casein, sodium nitrate, potassium nitrate, ammonium sulfate or a combination thereof, and the nitrogen source accounts for 0.1% to 3% of the total volume ratio of the basal medium; and the mineral elements include magnesium sulfate, calcium sulfate, ferrous sulfate, sulfuric acid Iron, manganese sulfate, zinc sulfate, copper sulfate, nickel sulfate or combinations thereof.

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