TWI765156B - Solid culturing method for cordyceps cicadae - Google Patents

Abstract

A solid culturing method for Cordyceps cicadae is provided, including culturing a Cordyceps cicadae strain in a liquid medium as a liquid strain, wherein the sequence of the Cordyceps cicadae strain is shown as SEQ ID NO: 1; inoculating the liquid strain with a specific inoculating ratio in a solid medium; and culturing with a specific light source and a specific light intensity for a predetermined time to obtain a mycelium and fruiting bodies of the Cordyceps cicadae strain.

Description

Solid-state culture method of cicada flower

The present invention relates to a method for culturing Cicada flower, in particular to a solid-state culture method for Cicada flower that is cultivated by using a solid medium.

Medicinal fungi are popular in the global market due to their medicinal properties, making research on medicinal fungi a very important topic. From ancient times to the present, most of the secondary metabolites produced by medicinal fungi have many different mechanisms. In humans, these secondary metabolites may be harmful, but may also have biological activities that can improve health or prevent disease.

Among them, cicada ( Cordyceps cicadae , C.cicadae) belongs to the ergot family of Cordyceps genus, and it is parasitic in the cicada pupa or cicada larvae, and the cicada pupae or cicada larvae as the host are used as a source of nutrition to transform A cicada flower mycelium grows, and then a bud-shaped fruiting body grows. Because of this, cicada flowers contain many biologically active secondary metabolites.

At present, cicada flowers can be divided into natural collection or artificial cultivation according to the source. However, the way of natural collection will be affected by timeliness, regionality, etc., and a large number of collections will also damage the environment. However, the conventional artificial culture methods mainly focus on the liquid culture of mycelium, so there is almost no relevant research on artificial solid state culture of cicada fruiting bodies. Therefore, there is still a need for a method capable of culturing Cicada fruiting bodies in a solid medium.

In view of the above-mentioned problems, the present invention provides a solid-state culture method of Cicada flower, which comprises: culturing Cicada flower strains in a liquid medium as liquid strains, and the sequence of the Cicada flower strains is shown in SEQ ID NO: 1; The specific inoculation ratio is used to inoculate the liquid bacterial strain in the solid medium; and the specific light source type is used for culturing under a specific light intensity for a predetermined time, so as to obtain the mycelium and fruiting body of the cicada species.

Optionally, the solid medium comprises red bean, mung bean, soybean, black bean, rice bean, red barley, white barley, brown rice, white rice, purple rice, millet, ten-grain rice, red quinoa, Peruvian quinoa, oat, oatmeal, wheat, buckwheat , sorghum, corn, or a combination thereof.

Optionally, the predetermined time is 0 to 60 days.

Optionally, the inoculation ratio is 3% to 15% of the total volume of the solid medium.

Optionally, the light source type includes red light, blue light, white light, infrared light or a combination thereof.

Optionally, the light intensity is 0~2000Lux.

Optionally, the total polysaccharide content of the mycelium and fruiting body of Cicada flower is 4-28 g/L.

Optionally, the content of uracil, uridine, adenine and adenosine in mycelium and fruiting body of Cicada flower are respectively 0～0.42mg/mg, 0～0.09mg/mg, 0～0.07mg/mg and 0～0.07mg/mg. ~1.05mg/mg.

Optionally, the total flavonoids and total phenolic contents of the mycelium and fruiting body of Cicada flower are 8-50 mg/mg and 4-14 mg/mg, respectively.

The solid-state culture method of the cicada flower of the present invention has the following advantages:

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

(2) being different from the liquid culturing method mainly can only cultivate the mycelium of Cicada, the solid-state culture method of Cicada of the present invention can cultivate the fruiting body of Cicada, thereby obtaining the mycelium of Cicada in different proportions The content of polysaccharides, active ingredients, total phenols and flavonoids.

(3) In addition, since the solid-state cultivation method of the cicada flower of the present invention can adjust the level of the components obtained by the extract by adjusting the parameters such as cultivation time, cultivation humidity, light source type, light intensity, and solid-state cultivation grain type, it is For a cultivation method that can be adjusted according to needs.

S10~S30: Steps

FIG. 1 is a schematic flow chart of the solid-state culture method of the present invention.

Figure 2 is an image of the cicada flower of the present invention.

Fig. 3 is a solid-state culture diagram of the cicada flower of the present invention.

Fig. 4 is a diagram showing the liquid culture of cicada flower of the present invention.

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

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

Figures 9 to 11 are analysis diagrams of Examples 12 to 21 of the present invention.

Figures 12 to 14 are analysis diagrams of Examples 22 to 30 of the present invention.

Fig. 15 to Fig. 18 are analysis diagrams of Example 31 to Example 38 of the present invention.

Figures 19 to 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 purposes, technical features and benefits after practical implementation easier for those skilled in the art to understand, the following will be described in more detail with examples and drawings.

Referring to FIG. 1, it is a schematic flow chart of the solid-state culture method of the present invention.

In step S10, the Cicada species is cultivated in a liquid medium as a liquid strain. The species of Cicadae is Cordyceps cicadae Wu-BFP14, and its sequence is shown in SEQ ID NO: 1.

In step S20, liquid bacteria are inoculated into the solid medium with a specific inoculation ratio. Preferably, the inoculation ratio can be 3%-15% of the total volume of the solid medium, more preferably 5%-10%. Preferably, the solid medium can comprise red bean, mung bean, soybean, black bean, rice bean, red barley, white barley, brown rice, white rice, purple rice, millet, ten-grain rice, red quinoa, Peruvian quinoa, oat, oatmeal, wheat, Buckwheat, sorghum, corn, or a combination thereof.

In step S30, culture is performed for a predetermined time under a specific light intensity with a specific light source type, so as to obtain the mycelium and fruiting body of the Cicada species. Preferably, the predetermined time may be 0-60 days, more preferably 7-56 days. Preferably, the types of light sources include red light, blue light, white light, infrared light or a combination thereof. Preferably, the light intensity is 0~2000Lux, more preferably 0~1500Lux.

In one embodiment, the total polysaccharide content of the mycelium and fruiting body of Cicada flower is 4-28 g/L. In one embodiment, the content of uracil, uridine, adenine and adenosine in the mycelium and fruiting body of Cicada flower are 0~0.42mg/mg, 0~0.09mg/mg, 0~0.07mg/mg respectively and 0~1.05mg/mg. In one embodiment, the total flavonoids and total phenolic contents of the mycelium and fruiting body of Cicada flower are 8-50 mg/mg and 4-14 mg/mg, respectively.

Each example is described in detail below.

Research methods

1.1 strain screening

The selected cicada species in the present invention are screened by themselves in the Three Gorges Mountains of Taiwan, the strains obtained by screening are cultured, their mycelia are collected, and then their genomic DNA is extracted, and the DNA is amplified by PCR technology. After being cloned and unsequenced, its sequences were BLAST compared with other species of Cicada species in the NCBI gene bank.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 Cicada species.

1.2 Liquid culture of cicada flower mycelium

Take a piece of cicada flower mycelium from the petri dish, take a piece of 5mm diameter, and insert it into a sterilized liquid medium to discuss parameters such as different environmental factors, types and concentrations of carbon sources, types and concentrations of nitrogen sources, and trace elements. Effects of Cicada species on the growth of strains. The process is as follows: use a metal tube with a diameter of 5mm to inoculate bacteria from a bacterial plate (the amount of bacteria inoculated is a piece of bacteria with a diameter of 5mm) to a 125mL flat-bottom conical flask containing a medium, and culture at 28°C with constant temperature shaking at 130rpm. The mycelia were filtered and dried with filter paper with a pore size of 0.45 μm. Then, steps such as subpackaging and freezing bacteria can be performed.

1.3 Strain activation

The bacterial classification after the frozen bacteria is re-inoculated into the petri dish containing potato dextrose solid medium (Potato dextrose agar, PDA) and cultivated, then the milling cutter is cut out the PDA of 2 diameters 1cm on the petri dish of the long bacterium colony, It was inoculated into 250 mL of bacterial liquid activation medium, and cultured at 25°C in a constant temperature shaking incubator at 150 rpm for 7 days. Wherein, the liquid medium is Potato dextrose broth (PDB).

1.4 Different carbon sources

Based on the above activated medium formulation, the analysis was performed with different carbon sources without any carbon source and with 2% added. 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). Among them, the culture time is 7 days to 14 days; the culture temperature is 23°C to 28°C; and the culture humidity is 100%.

1.5 Different nitrogen sources

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

In addition, since mineral elements are also a main element of mycelium growth, glucose (Glu) and yeast extract (Yeast extract, YT) were selected as carbon and nitrogen sources, and magnesium sulfate (MgSO ₄ ) at a concentration of 2 mM 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 5 mM magnesium sulfate were analyzed. Among them, the culture time is 7 days to 14 days; the culture temperature is 23°C to 28°C; and the culture humidity is 100%.

1.6 Solid-state culture of cicadas

The cicada flower strain liquid strain of the aforementioned liquid culture is taken, and 10% of the bacterial liquid of the total volume of the solid culture substrate is inserted into the sterilized different grains, wherein the grains include red bean (RB), mung bean (GB), Soybeans (YB), Black Beans (BB), Rice Beans (MB) and grains such as Red Barley (RY), White Barley (WY), Brown Rice (RR), White Rice (WR), Purple Rice (PR), Millet (MR), ten-grain rice (Ten) rice grains, such as red quinoa (TR), Peruvian quinoa (MUR), oat (YM), oatmeal (OL), wheat (MM), buckwheat (CM) quinoa Wheat grains, and other grains like sorghum (H), corn (PC).

1.7 Different light intensity

Take 30g of wheat as solid-state culture medium, add medium with a solid-liquid ratio of 1:1.5 to sterilize and 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 1500 Lux), incubated at 25°C for 42 days.

1.8 Different light sources

Take 45 g of wheat as the solid-state culture medium, add a medium with a solid-liquid ratio of 1:2 for sterilization, and then insert 5% liquid strains. First, cultivate in a dark room until white mycelium grows on the surface. sky. Among them, the light source is 5 different light quality cold fluorescent lamps (Cw), 100% red light, 70% red light and 30% blue light, 30% blue light, 30% blue light The light-emitting diode light sources of red light, 70% blue light, and 100% blue light, 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

The dried powder was cultured with 1 g of cicadae mycelium or solid-state, added with a solvent for extraction, and then centrifuged at 9000 rpm for 10 minutes, and the supernatant was taken as the extract. Wherein, the solvent can be deionized water, alcohol, acid solution or alkali solution.

1.10 Sample processing

1 g of freeze-dried mycelium was added to 10 mL of distilled water, placed in an autoclave at 121° C. for hot water extraction for 20 min, and the extract was obtained by filtration. The supernatant of the culture medium (supernatant described in the extraction method) and the intracellular extract (extract after the mycelium was extracted) were filtered through a 0.45 μm microporous membrane and analyzed by HPLC.

2.1 Analysis of polysaccharide content

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

2.2 Analysis of Cordycepin Content

Take 10 mg of the standard cordycepin each, dissolve it in 10 mL of methanol (Methanol, 15%), and analyze it by High Performance Liquid Chromatography (HPLC) to make calibration lines respectively, and then substitute the peak area of cordycepin into Linear regression equation to obtain the concentration of both.

2.3 Analysis of uracil, uridine, adenine and adenosine content

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

2.4 Total phenolics content (TPC) content

Take 1 mL of the sample solution, add 1 mL of Folin-Ciocalteu reagent, shake and mix for 3 minutes, then add 1 mL of sodium carbonate (10%, v/w), and react at room temperature for 30 minutes in the dark. The wavelength of the absorption spectrum is 735 nm. record. Taking gallic acid as the standard, and making the calibration line, the relative gallic acid equivalent (GAE mg/g) in each gram of the sample is obtained by interpolation, which means the amount of gallic acid in the sample. The total amount of phenolic compounds.

2.5 Total flavonoids (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 using a high-speed centrifuge. Take the supernatant and record it with an absorption spectrum wavelength of 510 nm. The relative quercetin equivalent (QEmg/g) in each gram of the sample was obtained by using the standard substance of Quercetin and the calibration curve was made, which represented the total amount of flavonoids in the sample.

Results discussion

3.1 Morphological characteristics of cicada flowers

Referring to Figure 2, it is an image of the cicada flower of the present invention. Among them, (a) part represents the whole picture of wild cicada flower; (b) part represents fruiting body; (c) part represents parasite; (d) part represents fruiting body microscope image; (e) represents fruiting body microscope image; (f) represents fruiting body microscope image Part ) represents a micrograph of fruiting bodies; part (g) represents a micrograph of fruiting bodies; part (h) represents a micrograph of mycelium; and part (i) represents a micrograph of mycelium.

The cicada flower system disclosed in the present invention is a Taiwan native giant cicada flower, which is a pure strain of cicada flower isolated after being collected from bamboo forests in the mountainous area of Taiwan. The surface of fresh cicada flowers is covered by white or grayish-white mycelium, which turns light yellow to brown after drying. Except for the shape of the epidermis, compound eyes and three pairs of thoracic feet, all the organs of the insect body remain unchanged. All are covered by mycelium, forming a long oval sclerotia with a size of about 3 cm and a diameter of about 1-1.1 cm. The sclerotia grows into 3-4 antler-shaped or rod-shaped branches with a length of about 4-10 cm. Observation of wild cicada fruiting body by optical microscope shows that many branches and oval conidia exist in the cicada fruiting body part, but only many filamentous structures are found in the cicada cicada.

Referring to Figure 3, it is a solid-state culture diagram of the cicada flower of the present invention. Wherein, part (a) represents plate culture; part (b) represents plate culture close-up; part (c) represents micrograph of mycelium; and part (d) represents micrograph of mycelium.

After culturing the cicada flower fruiting body in a PDA petri dish for 10 days, the cicada flower mycelium with a diameter of about 6-8 cm, white to off-white felt-like to fluffy, and the back of the colony shows a color-like groove. Half-yellow to orange-yellow hyphae, the hyphae of cicada flower are smooth and transparent tubular, showing many dense and irregular branches, and the thickness of these branches is about 2.0μm-3.5μm.

Referring to Figure 4, it is a diagram of the liquid culture of Cicada flower of the present invention. Among them, (a) part represents liquid shake flask culture; (b) part represents plate culture; (c) part represents liquid shake flask culture close-up; (d) part represents mycelium photomicrograph; (e) part represents mycelium Part (f) represents mycelium; part (g) represents mycelium; (h) represents mycelium; and (i) represents mycelium photomicrograph.

After the cicada flower is cultured in liquid state, it will gradually change from light yellow medium to light pink to light purple medium. The mycelium of cicada flower liquid fermentation can be found by optical microscope observation. At low magnification, it is densely packed. For example, the filaments in the shape of the hair structure can be found after magnification to 500 times. There are many dense and irregular branches, and the thickness of these branches is about 1.5μm-3.0μm, which is smaller than that of the Petri dish, but the density is higher than that of the hyphae of the Petri dish. After the liquid culture medium was left standing for 1 to 2 weeks, the surface of the medium changed to form a thin layer of cicada flower mycelial film. Observing the surface of the mycelial film with an optical microscope can find that the surface of the mycelial film has a considerable multi-layered filamentous structure. With the magnification of the microscope, you can gradually find transparent filaments in the shape of a hair-like structure. After magnifying to 500 times, you can also find that the shape of the mycelium is similar to that of the mycelium and the petri dish, with an inner wall. Smooth and transparent tubular, showing many dense and irregular branches, and the thickness of these branches is about 5.0μm-10μm.

Referring to Figure 5, it is a solid-state culture diagram of the cicada flower of the present invention. Among them, (a) part represents the solid-state culture of cicada flower; (b) part represents the close-up of the fruiting body; (c) part represents the close-up of the basal surface; (d) part represents the photomicrograph of the fruiting body; and (e) part represents the hyphae Body micrographs.

Inoculate the cicada flower strain into the whole grains, and cultivate it for about 1 to 2 months, then the artificial solid-state cultured fruiting body and mycelium can be reared, and the artificially cultured fruiting body is as fresh as the wild fruiting body. The appearance of the body is white or gray-white, about 4-10 cm in length, and about 0.1-0.5 cm in thickness. After drying, it will turn light yellow to tan, but the length and thickness of the fruiting body will shrink significantly. Further observation with optical microscope showed that the artificial fruiting body presented a multi-layered structure, and the distribution of many hyphae could be observed around the fruiting body. The artificial mycelium has similar results with the wild cicada, with the existence of multi-layered filamentous structure.

3.2 Identification of Cicada species

The native cicada flower was first judged by its appearance, and cultured in liquid and solid state respectively. Generally, the classification of fungi is mainly based on ribosomal DNA. For 18SRrna gene, 5.8Rrna gene, or 28SRrna gene, ribosomal DNA has a very high retention in fungi, and is mainly used for research on classifications above families and genus. There is an internal transcribed spacer (ITS) between the 5.8S Rrna gene and the 18S and 28S Rrna genes, respectively, which are divided into ITS1 and ITS2, and the evolution rate of ITS1 and ITS2 is faster. The sequence will be significantly different. Therefore, the ITS sequence of the cicadae strain was analyzed, and its ITS sequence was compared with the ITS sequence of 22 different cicadae ( Cordyceps cicadae ) on the gene database (NCBI GenBank). The strain was confirmed to be a cicada flower strain, and the strain was named Cordyceps cicadae Wu-BFP14, and its gene sequence was uploaded to the genetic database, and the accession number (Accession number) was KX289580. The following It is abbreviated as BFP14 (SEQ ID NO: 1) herein.

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, (a) to (d) respectively represent pH value, exopolysaccharides (Exo polysaccharides, EPS, unit: g/L) content, intracellular polysaccharides (Intracellular polysaccharides, IPS, unit: g/L) content, and Biomass (Biomass, unit: g/L) content.

The pH of BFP14 was not significantly different when using various carbon sources to cultivate the fermented liquid, and they were all maintained at pH5-pH6. Only when CMC was used as the carbon source, it was slightly higher, at pH7. It can be found that BFP14 can be used in both monosaccharides (Glu, Fru and Xyl), disaccharides (Sur, Mal and Lac), polysaccharides (Dex, CMC and SS) and even polyols (Man).

The biomass of BFP14 mycelium was 2.11g/L without adding carbon source. After adding various carbon sources, the biomass of mycelium increased significantly. With Glu as the best carbon source for cultivation, the biomass of mycelium can reach 6.55g/L, which is more than three times higher than that without carbon source, followed by Man, with a mycelium of about 6.21g/L Biomass, and then 6.15g/L of SS. In terms of intracellular polysaccharide content, Glu is the best and can reach 6.19g/L, followed by lactose (Lac), which can reach 5.25g/L. In the extracellular polysaccharide, mannitol (Man) is the best and can reach 17.8g/L, followed by Glu, which can reach 14.51g/L, and there is a significant difference with other carbon sources in the production of extracellular polysaccharides.

Referring to FIG. 7, it is an analysis diagram of Example 1 to Example 11 of the present invention. Wherein, parts (a) to (d) represent the contents (mg/mg) of uracil, uridine, adenine and adenosine, respectively.

It can be found that when Xyl is used as 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; when Mal is used as carbon source, uracil and urine Glycosides are second only to xylose as the carbon source, which are 2.49mg/g and 0.44mg/g respectively; followed by Sur, which are 2.39mg/g and 0.48mg/g, respectively, followed by glucose and fructose . Since Mal and Sur belong to two It is speculated that Glu and Fru can promote the production of uracil and uridine from a molecule of glucose and a disaccharide formed by the polymerization of a molecule of glucose and a molecule of fructose. In terms of adenosine, CMC was clearly found to be preferred, with a content of about 2.91 mg/g, followed by Lac with a content of 2.65 mg/g, followed by Glu with 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) represent the content (mg/mg) of total flavonoids and total phenols, respectively.

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

Based on the above results, Glu is the carbon source utilized by most bacteria and fungi, and it is relatively easy to obtain, and the cost is relatively cheap. For most carbon sources, glucose was 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) represent pH value, exopolysaccharide content, intracellular polysaccharide content, and biomass content, respectively.

All five organic nitrogen sources can be utilized by BFP14, but among the inorganic nitrogen sources, only SN, PN and AS can be utilized. In general, the cultivation conditions of 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 mycelium of 8.37g/L can be obtained, which is nearly three times higher than the condition without nitrogen source (3.11g/L). times, followed by Cas (7.32g/L), and MT was the lowest mycelial biomass of only 4.50g/L. Among the inorganic nitrogen sources, SN is preferred, which can be obtained 5.05g/L mycelial biomass, followed by PN (5.01g/L), and ammonium sulfate was the least suitable for BFP14 mycelial biomass growth.

In terms of polysaccharides, MT is mainly preferred, the content of intracellular polysaccharide or exopolysaccharide is 17.54g/L and 54.96g/L respectively, and polysaccharide of inorganic nitrogen source is 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, the organic nitrogen source of BFP14 is superior to the inorganic nitrogen source in terms of mycelial biomass, intracellular polysaccharide and exopolysaccharide, and it can be found that the nitrate nitrogen source is superior to the ammonium salt in the inorganic nitrogen source. 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) represent the contents (mg/mg) of uracil, uridine, adenine and adenosine, respectively.

In terms of biologically active components, among the organic nitrogen sources, YT has the highest four biologically active components of uracil, uridine, adenine and adenosine, with 1.69mg/g, 0.17mg/g and 0.91mg respectively. /g and 0.98mg/g, followed by Cas, PT, BT and MT, in which uridine was not detected in MT, while in inorganic nitrogen sources, the same nitric acid was found. 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 Example 12 to Example 21 of the present invention. Wherein, parts (a) and (b) represent the content (mg/mg) of total flavonoids and total phenols, respectively.

In terms of total phenolic content, when YT is the nitrogen source, there will be a better total phenolic content, with a gallic acid content of 24.21 mg/g, followed by PT with a gallic acid content of 23.6 mg/g, and then The one 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 gallic acid content. Among the inorganic nitrogen sources, SN is the best, which is equivalent to 21.62 mg/g of gallic acid, followed by PN (20.29 mg/g), which is much higher than AS (1.7 mg/g). In terms of flavonoid content, YE is the better organic nitrogen source, with a quercetin content of 24.36 mg/g, followed by PT with a quercetin content of 23.96 mg/g, and then It means that Cas has a quercetin content of 23.93mg/g, and the other two organic nitrogen sources have a total flavonoid content of more 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 Example 22 to Example 30 of the present invention. Wherein, parts (a) to (d) represent pH value, exopolysaccharide content, intracellular polysaccharide content, and biomass content, respectively.

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

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

The content of uracil, uridine, adenine and adenosine in BFP14 mycelium extract is better than ferric sulfate in 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 bioactive components. Manganese sulfate is preferred in adenine, which can reach 0.52mg/g, followed by ferric sulfate (0.49mg/mg). However, in adenosine, zinc sulfate is better, which can reach 2.46mg/g, followed by nickel sulfate (2.44mg/g).

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

The total phenolic content is preferably magnesium sulfate, which is equivalent to the gallic acid content of 9.73 mg/g, and the flavonoid content can be obviously observed to be the highest with manganese sulfate, which is equivalent to 36.98 mg/g quercetin content, Next is manganese sulfate.

The addition of different mineral elements in the present invention not only affects the biomass of mycelium and intracellular polysaccharide, but also affects the composition of different biologically active components, and the proportions of biologically active components contained in different element extracts are very different. In terms of the biomass quality of mycelium, no mineral elements are added, magnesium sulfate is the next, and intracellular polysaccharide is preferably magnesium sulfate. In terms of bioactive components, they are different. Among them, the flavonoids added with manganese sulfate are the best. However, its mycelial biomass did not perform well. If the antioxidant activity and flavonoid content are considered in the future, manganese sulfate is a very important focus.

Referring to Figure 15, which is an analysis diagram of Example 31 to Example 38 of the present invention, where (a) and (b) parts represent moisture content (%) and yield (Product yield), respectively. Yield is calculated as = product (g)/raw material (g).

For BFP14 in the solid-state culture of cereals with different culture days, it can be found that after 7 days of culture, the mycelium can cover the entire grain matrix, and after 14 days of culture, it can grow 0.3-0.5cm. Primordium, in 21 days of culture, can grow 1-3cm fruit body. After 28 days of culture, the height of the fruit body can reach 3-5 cm, and after 35 days of culture, the fruit body grows to about 5-7 cm, and then at 42 days of culture, the sporophyte is formed, and the fruit body gradually shrinks. Among them, the culture humidity is 60-80% around, and as the number of days of culture increases, the humidity will also increase. In terms of productivity, there is also a phenomenon that the productivity decreases as the number of days of culture increases.

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

It can be found that with the increase of culture days, the total polysaccharide content showed a downward trend. On the 7th day of solid-state culture of BFP14 grain matrix, the total polysaccharide content was 27.55g/L. After 56 days of culture, the polysaccharide content was only left. 8.88g/L.

Referring to FIG. 17, it is an analysis diagram of Example 31 to Example 38 of the present invention. Wherein, parts (a) to (d) represent the contents (mg/mg) of uracil, uridine, adenine and adenosine, respectively.

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

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

Total flavonoids and total phenols also increased with the number of culture days, and on the 42nd day of culture, there was the highest total phenolic content, equivalent to 9 mg/g gallic acid content, compared to 7 days of culture (2.62 mg/g). The total phenolic content of g) can be increased by more than 3 times, while the flavonoid content has been increasing with the number of days of culture. Then the quercetin content equivalent to 49.83mg/g can be achieved, which is nearly five times higher.

Referring to FIG. 19 , which is an analysis diagram of Examples 39 to 45 of the present invention, parts (a) and (b) represent moisture content and yield, respectively.

It can be observed that under the condition of light intensity of 0Lux, a layer of white mycelial film grows along the edge of the culture vessel, and no fruit bodies are produced. Under the light intensity of 50Lux, fruit bodies of 5-7 cm can grow. With the increase of the light intensity, there is not much change. When the light intensity is 1500Lux, it can be found that the fruiting body gradually shrinks. In the solid-state culture of BFP14 grains, a little light is required to stimulate the fruiting body. However, 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 humidity of the culture without light is about 70%, and the humidity of the culture with other light intensities is maintained at about 80%. In terms of yield, under the condition of no light, a yield of 70% can be obtained , After 30g of grain is fermented, 21g of fermented product can be obtained. Under the light condition of 50 to 1500Lux, about 50% of the yield can be obtained, and about 15g of fermented product can be obtained.

Referring to Figure 20, which is an analysis chart of Examples 39 to 45 of the present invention, it represents the analysis of polysaccharide content.

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

Referring to FIG. 21, it is an analysis diagram of Example 39 to Example 45 of the present invention. Wherein, parts (a) to (d) represent the contents (mg/mg) of uracil, uridine, adenine and adenosine, respectively.

Light intensity does not have much effect on the bioactive components of uracil, uridine, adenine and adenosine, but it can be observed that the bioactive components are higher than those without light after culturing with 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. With the increase of light intensity, the content increases. At 500Lux, the highest uridine content (0.09mg /g), and then decreased.

Referring to FIG. 22, it is an analysis diagram of Examples 39 to 45 of the present invention. Among them, parts (a) and (b) respectively represent the content of total flavonoids and total phenols (mg/mg).

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

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

Referring to FIG. 23 , which is an analysis diagram of Examples 46 to 53 of the present invention, parts (a) and (b) represent moisture content and yield, respectively.

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

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

Referring to Figure 24, which is an analysis chart of Examples 46 to 53 of the present invention, it represents the analysis of polysaccharide content.

In the total sugar content, it can be found that the difference is not big, and the polysaccharide content is maintained at 9-11g/L, with 6R3B being the better, 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) represent the contents (mg/mg) of uracil, uridine, adenine and adenosine, respectively.

Among the uracils, the control group (white light of 500Lux) is the best, with a content of 1.4mg/g, followed by 3R3B3IR with a content of 1.31mg/g, and 9IR with a content of 1.27mg/g. Uridine is preferably 9B with a content of 0.19mg/g, followed by 9IR with a content of 0.15mg/g, and 3R3B3IR with a content of 0.14mg/g. Adenine is preferably 3R3B3IR with a content of 0.49mg/g, followed by 6R3B with a content of 0.41mg/g, and 9R with a content of 0.4mg/g. Adenosine is preferably 9B with a content of 1.05mg/g, followed by 9IR with a content of 0.92mg/g, and 3R3B3IR with a content of 0.87mg/g. It can be found that far infrared 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.

Referring to FIG. 26, it is an analysis diagram of Examples 46 to 53 of the present invention. Wherein, parts (a) and (b) represent the content (mg/mg) of total flavonoids and total phenols, respectively.

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, equivalent to 41.36mg/g quercetin content, followed by 9IR equivalent to Quercetin content of 36.61 mg/g. represents that far-infrared rays can stimulate the flavonoid content of BFP14 grains in solid-state culture.

Referring to Fig. 27, which is an analysis diagram of Examples 54 to 73 of the present invention, in which parts (a) and (b) represent moisture content and yield, respectively.

It was found that of the 20 different grains used, BFP14 grains were grown in solid-state culture. With the exception of a few grains, most solid-state cultures of grains produce fruiting bodies. In bean grains, except for YB and BB, which do not grow fruit bodies, the rest of the bean grains can grow fruit bodies of about 1-2 cm. In rice grains, except for RR, which grows a thick layer of mycelium, the rest Six kinds of rice grains can grow fruiting bodies, among which WR is the better one. Among the Limai grains, TR and MUR only grow thick mycelium, and the other four kinds of Limai grains are all Fruiting bodies can be grown, and fruiting bodies can also be grown in H and PC of other grains. Among these grains, MM was the best, followed by WR. The rest of the grains had at least 3-4 cm of fruiting bodies, but the grains of legumes had poor fruiting bodies.

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

Referring to Figure 28, which is an analysis chart of Example 54 to Example 73 of the present invention, it 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. In terms of sugar content, MB is the highest in legume cereals, with a polysaccharide content equivalent to 13.59g/L, while Limai cereals are highest in MM, with a polysaccharide content equivalent to 11.71g/L.

Referring to FIG. 29, it is an analysis diagram of Example 54 to Example 73 of the present invention. Wherein, parts (a) to (d) represent the contents (mg/mg) of uracil, uridine, adenine and adenosine, respectively.

Among legumes, 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, the content of uridine and adenine was not detected in RB and BB. In uridine, the content of YB has the highest content of 0.02mg/g, but in adenine, GB is better, with 0.02mg/g, but in adenosine, MB is better, with 0.47mg/g g content. Among rice grains, Ten is the highest uracil, which can be obtained at 1.23 mg/g. In addition, WR, PR and MR are not detected in the content of uridine, and the highest content of RR is 0.21 mg/g. However, in adenine, PR is better, with a content of 0.12mg/g, while in adenosine, RR is better, with a content of 0.97mg/g. In Limai grains, TR is the best for uracil and uridine, with 1.13mg/g and 0.21mg/g respectively, but no uridine was detected in OL, and uridine was not detected in adenine. YM is preferred, with a content of 0.08 mg/g, and in the same way as adenosine, YM is preferred, with a content of 0.76 mg/g. In other types of grains, the content of these four bioactive components is relatively low, followed by grains of Limai.

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

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

BFP14 could grow in 20 kinds of grains, and MM and WR were the better ones in terms of fruit body types, but the contents of bioactive components, total phenols and flavonoids were not better. 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, and PR has the highest The content of total phenols and flavonoids was also very good, but it also had quite good DPPH free radical scavenging activity, but it did not detect the content of uridine, and other antioxidant activities were not as expected. Here, it is only demonstrated that BFP14 can be grown in a variety of grains, and the choice of grains and proportions can be adjusted according to the desired characteristics.

4 Conclusion

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

The results of the carbon source study showed that the use of glucose had the best mycelium yield (6.55g/L) and the highest intracellular polysaccharide yield (6.19g/L). In addition, the results also showed that xylose had the highest content of uracil, uridine and adenine, which were 2.912 mg/g, 0.527 mg/g and 0.100 mg/g, respectively. At the same time, the content of total phenols and flavonoids is also better than xylose. In the nitrogen source study, the yield of BFP14 mycelium was better than yeast extract, with a yield of 8.37g/L, and also had better uracil, uridine, adenine and adenosine, which were 1.690mg respectively /g, 0.172 mg/g, 0.905 mg/g and 0.976 mg/g. The contents of total phenols and flavonoids were 24.21mgGAE/g and 23.96mg QE/g, respectively.

The liquid medium with the best mycelium growth, polysaccharide and bioactive components content was composed of: glucose 50g/L, yeast extract 5g/L and 5mM manganese sulfate added.

In the solid-state culture, wheat was used as the culture substrate, and the effects of various culture conditions on the biological activity were discussed. 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 in blue light, Blue light, white light 2700K and mixed light source of red light, blue light and far infrared light are the best. Using 20 kinds of grains as the solid-state culture medium of BFP14 to observe the growth morphology and biological activity of different grains, brown rice is the best grain medium for the production of uridine and adenosine, while purple rice is the best grain medium for the production of uridine and adenosine. Meter.

The above description is exemplary only, not limiting. Any equivalent modifications or changes without departing from the spirit and scope of the present invention should be included in the scope of the patent application.

<110> Daye University

<120> Solid-state culture method of cicada flower

<130> DY-19006-TWI

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 549

<212> DNA

<213> Cicada (Cordyceps bifusispora)

<400> 1

S10~S30: Steps

Claims (8)

A solid-state culture method for Cicada flower, comprising: cultivating a Cicada flower strain (SEQ ID NO: 1) in a liquid medium as a liquid strain; inoculating the liquid strain in a solid medium with an inoculation ratio ; And cultivate a predetermined time under a light intensity with a light source type to obtain a mycelium and a fruit body of the cicadae species; wherein the solid medium comprises 2% glucose, fructose, xylose, sucrose, At least one of maltose, lactose, dextrin, carboxymethyl cellulose, soluble starch and mannitol as a carbon source, and magnesium sulfate, calcium sulfate, ferrous sulfate, iron sulfate, manganese sulfate, zinc sulfate at a concentration of 2~5mM , at least one mineral element in copper sulfate and nickel sulfate; wherein the total flavonoid and total phenolic content of the mycelium and the fruiting body of the cicada flower are 8-50 mg/mg and 4-14 mg/mg, respectively. The solid-state culture method according to claim 1, wherein the solid-state medium comprises red bean, mung bean, soybean, black bean, rice bean, red coix seed, white coix seed, brown rice, white rice, purple rice, millet, ten-grain rice, red quinoa, Peruvian Quinoa, oats, oatmeal, wheat, buckwheat, sorghum, corn, or a combination thereof. The solid-state culture method according to claim 1, wherein the predetermined time is 0 to 60 days. The solid-state culture method according to claim 1, wherein the inoculation ratio is 3% to 15% of the total volume of the solid-state medium. The solid-state culture method as claimed in claim 1, wherein the light source type comprises red light, Blue light, white light, infrared light, or a combination thereof. The solid-state culture method as claimed in claim 1, wherein the light intensity is 0-2000 Lux. The solid-state culture method according to claim 1, wherein the total polysaccharide content of the mycelium and the fruiting body of the cicada flower is 4-28 g/L. The solid-state culture method according to claim 1, wherein the content of uracil, uridine, adenine and adenosine in the mycelium and the fruiting body of the cicada flower are 0-0.42 mg/mg, 0-0.09 mg, respectively /mg, 0~0.07mg/mg and 0~1.05mg/mg.

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