Culture method for increasing intracellular polysaccharide content of phellinus igniarius mycelium

Abstract

The invention provides a culture method for rapidly increasing the content of polysaccharide in phellinus igniarius mycelium cells, and belongs to the technical field of phellinus igniarius culture.The culture method comprises the steps that mulberry leaves are cleaned, subjected to fixation and microwave drying and then crushed into 200-mesh mulberry leaf powder, then the mulberry leaf powder, cane sugar, corn starch and other components are prepared into a specific liquid culture medium, and the liquid culture medium is prepared into the phellinus igniarius mycelium cells. The phellinus igniarius mycelium grows rapidly through liquid culture, and the content of polysaccharide active ingredients in phellinus igniarius mycelium cells is remarkably increased. The phellinus igniarius thallus liquid culture method is rapid and efficient, the content of intracellular polysaccharide in the cultured phellinus igniarius mycelia is high, extraction is convenient and rapid, and the phellinus igniarius thallus liquid culture method can be suitable for large-scale industrial production.

Description

Technical Field

[0001] The present invention belongs to the technical field of Sanghuang cultivation, and particularly relates to a cultivation method for increasing the intracellular polysaccharide content of Sanghuang mycelium. Background Art

[0002] Sanghuang (Sanghuangporus sanghuang), belonging to the genus Sanghuangporus of the family Hymenochaetaceae, also known as Sang'er, grows on the trunks of Morus plants and is a medicinal fungus. Chen Jiamo's "Ben Cao Meng Quan" in the Ming Dynasty recorded that Sanghuang with different appearances has different effects. "The soft and wet one" can "disperse blood like a god and stop bleeding very quickly". "The black one is mainly used for treating women's abdominal masses, metrorrhagia and leukorrhea, and sudden breast swelling". "If it is yellow, ripe, old and white, it can stop diarrhea and tonify the primordial yang". Existing research shows that the main active ingredients in Sanghuang are polysaccharides, flavonoids and triterpenoid compounds, which have medicinal functions such as anti-tumor, antioxidant, anti-inflammatory, hypoglycemic, liver protection and immune enhancement. The research by Lin et al. in 2017 has confirmed that the antioxidant, anti-inflammatory and anti-tumor activities of Sanghuang from Morus are superior to those of Sanghuang from Populus and Phellodendron amurense. Although Sanghuang from Morus has extremely high medicinal value, its fruiting body is perennial and grows slowly, resulting in a scarcity of wild Sanghuang from Morus. Moreover, it is currently very difficult to cultivate artificially. Most of the so-called cultivated Sanghuang fruiting bodies on the market are actually Sanghuang from Populus, not Sanghuang from Morus. Therefore, how to achieve rapid artificial cultivation of Sanghuang fruiting bodies with high biological activity from Morus has become a severe challenge. Artificial cultivation requires simulating the wild living conditions of Sanghuang, and it takes at least three years to optimize the active ingredients in artificially cultivated Sanghuang fruiting bodies, which is time-consuming, laborious and has a high cultivation cost. The polysaccharide of Sanghuang mycelium has similar biological activities to the polysaccharide of Sanghuang fruiting body, and has functions such as antibacterial, anti-tumor, anti-inflammatory, hypoglycemic, liver protection and immune enhancement. Compared with the time-consuming and laborious cultivation of Sanghuang fruiting body, a large amount of mycelium can be obtained in a short time through liquid culture. However, how to rapidly and effectively improve the intracellular active substances such as polysaccharide components in Sanghuang mycelium by optimizing the culture medium components and cultivation methods, and achieve the cultivation of mycelium with short cycle, low cost and high yield still needs to be continuously explored.

[0003] Two-way liquid fermentation using fungi as fermentation strains and natural medicinal materials as medicinal matrices is an effective method to increase the content of bioactive components in medicinal fungi. Cultivating fungi with the active substances released from natural medicinal matrices and producing new bioactive substances through the secondary metabolism of fungi can effectively improve the extraction efficiency. During the fermentation process, the fungi may produce new active ingredients, further enhancing the medicinal effect. Mulberry leaves are the leaves of deciduous trees or shrubs (Morus alba L.) of the genus Morus in the Moraceae family. Its powder contains various nutritional components such as rich lignin fiber, crude protein, polysaccharides, and flavonoids. However, mulberry leaves are only used for sericulture and animal feed in traditional production. In recent years, with the continuous development of mulberry resources, mulberry leaves have been processed into various forms. Among them, mulberry leaf powder is being increasingly used in medicine and food due to its higher soluble dietary fiber content, total polyphenol content, and antioxidant properties. However, there is no report on how to use mulberry leaves as nutrients to promote the rapid growth of Phellinus igniarius mycelium and increase the content of intracellular active substances in Phellinus igniarius mycelium.

[0004] In summary, by optimizing the culture conditions and using mulberry leaf powder as a raw material to prepare a liquid fermentation medium, the present invention can not only promote the growth of mycelium and increase the content of intracellular Phellinus igniarius polysaccharides with biological activity, but also provide high-vitality mycelium raw materials for subsequent artificial cultivation of fruiting bodies, realizing the rapid cultivation and collection of Phellinus igniarius mycelium, meeting the requirements of the current Phellinus igniarius industry. Summary of the Invention

[0005] The object of the present invention is to provide a liquid medium, a culture method, and their applications for cultivating Phellinus igniarius. The liquid medium uses mulberry leaf powder, sucrose, corn flour, etc. as raw materials, which can efficiently promote the growth of Phellinus igniarius mycelium of Morus alba, and then further increase the polysaccharide content of Phellinus igniarius mycelium, realizing the large-scale cultivation of Phellinus igniarius mycelium while achieving the efficient utilization of mulberry leaves and meeting the market demand for Phellinus igniarius polysaccharides.

[0006] The present invention provides a Phellinus igniarius liquid medium for promoting the rapid growth of mycelium, including the following steps: (1) Medium preparation: The mass percentages of the raw materials for preparing the medium are as follows: Based on preparing 1 L of Phellinus igniarius liquid medium, 30 - 50 g / L of sucrose, 3 - 5 g / L of corn flour, 1 - 2 g / L of mulberry leaf powder, 2 - 4 g / L of peptone, 1 - 3 g / L of yeast powder, 1 - 3 g / L of KH2PO4, and 0.1 - 0.6 g / L of MgSO4 are required; the preparation steps of the medium are: A. Heating and dissolving: Add 30 - 50 g of sucrose, 3 - 5 g of corn flour, 2 - 4 g of peptone, 1 - 3 g of yeast powder, 1 - 3 g of KH2PO4, and 0.1 - 0.6 g of MgSO4 that have been weighed respectively, then add water to mix to a total volume of 1000 ml, and heat to boiling with a low gear on an induction cooker while constantly stirring to prevent sticking to the bottom or overflowing; B. Addition of mulberry leaf powder: Weigh 1 - 2 g / L of ultrafine mulberry leaf powder and slowly add it to the liquid in the boiling pot while constantly stirring to prevent caking and gelatinization, and keep it boiling until before subpackaging. C. Adjustment of pH value: Use a pH meter to measure the pH value of the liquid medium between 80 - 90 °C, and adjust the pH value to 5.5 - 7.5 with 2M NaOH or HCl. D. High-pressure steam sterilization: Divide the prepared liquid medium into 10 250 - ml conical flasks. After plugging the mouths of the flasks with cotton plugs, wrap kraft paper around the mouths and tie them tightly. Then put them into the autoclave and sterilize at a high pressure of 0.1 MPa for 20 min. After cooling to room temperature, take them out and place them in the laminar flow hood for standby.

[0007] (2) Inoculation of strains: In the laminar flow hood, inoculate the mulberry yellow hyphae into the medium in step (1), and culture at 28 - 37 °C and 100 - 180 r / min for 5 - 8 d to obtain mycelia.

[0008] Preferably, in the medium, the mass ratio of mulberry leaf powder to water is (0.1 - 0.2):100.

[0009] The beneficial effects of the present invention are as follows: (1) The present invention adopts a natural medium made of components such as mulberry leaf powder, sucrose, and corn flour, which can effectively promote the rapid germination of mulberry yellow mycelia and the growth of hyphae.

[0010] (2) The mulberry yellow hyphae cultured by the present invention have a fast cell proliferation rate. They can form hyphal pellets within 24 h at 30 ± 1 °C under lightless conditions, and continuously and stably increase, and reach a stable form in 6 - 7 d. Description of the drawings Figure 1 It is a control chart of hyphal growth of Examples 1, 2, 3 and the control group when cultured for 6 days with different mixing ratios of mulberry leaf powder and other raw materials provided in Comparative Example 1 of the present invention.

[0012] Figure 2 It is a statistical chart of the dry weight of mycelia of Examples 1, 2, 3 and the control group when cultured for 6 days with different mixing ratios of mulberry leaf powder and other raw materials provided in Comparative Example 1 of the present invention.

[0013] Figure 3 It is a standard curve of polysaccharides measured by the phenol-sulfuric acid method for the polysaccharide content per gram of dry mycelia after culturing with different mixing ratios of mulberry leaf powder and other raw materials provided in Comparative Example 1 of the present invention.

[0014] Figure 4Statistical chart of polysaccharide content per gram of dry mycelium after culturing mulberry leaf powder and other raw materials in different mixing ratios provided in Comparative Example 1 of the present invention, and the polysaccharide content per gram of dry mycelium in Examples 1, 2, 3 and the control group. Detailed implementation mode

[0015] The following describes the implementation mode of the present invention in combination with the accompanying drawings and specific specific examples. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation modes. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. The protection scope of the present invention is not limited to the following.

[0016] Source of raw materials: (1) The Sanghuang strain is Sanghuangporus sanghuang, purchased from Zhejiang Qianjifang Medical Technology Co., Ltd. (2) Potato dextrose agar, sucrose, corn flour, peptone, MgSO4, KH2PO4 are all purchased from Chongqing Taixin Chemical Co., Ltd. (3) The ultrafine mulberry leaf powder is provided by Chongqing Kuncheng Agricultural Technology Co., Ltd. Example 1 Inoculate 4 - 5 pieces (each piece has an area of about 0.05 ± 0.01 cm 2 ) of the Sanghuang mycelium cultured on the potato dextrose agar plate into the Sanghuang liquid medium (sucrose 40 g / L, corn flour 4 g / L, mulberry leaf powder 0.10 g / L, peptone 3 g / L, yeast powder 2 g / L, KH2PO4 1 g / L, MgSO4 0.5 g / L, pH 7.0). After inoculation, culture at 30 °C with a rotation speed of 120 rpm for 7 d, and collect the cultured mycelium.

[0017] The collected mycelium is filtered on filter paper and washed 3 - 4 times with water, and then placed in an oven at 60 °C and dried to a constant weight.

[0018] Determination of the total polysaccharide content of Phellinus igniarius mycelium: Grind the dried Phellinus igniarius mycelium into powder and sieve it through a 100-mesh sieve. Take 5 g of mycelium and add 100 ml of water, then ultrasonicate for 1 h. After that, centrifuge at 10,000 r / min for 8 min. Take the supernatant and add absolute ethanol to make the ethanol concentration 65%. Refrigerate at 4°C for 24 h, then centrifuge at 10,000 r / min for 8 min. Dissolve the precipitate in 20 ml of deionized water to obtain the sample solution to be measured. Precisely pipette 50 μL of the sample solution to be measured into a stoppered test tube (set three replicates), then successively add 950 μL of deionized water, 1 ml of 6% phenol solution and 5 ml of concentrated sulfuric acid. Shake well and let stand for 20 min. Place the test tube in a boiling water bath and let stand for 15 min, then take it out and cool for 30 min. Measure the OD value at the maximum wavelength of 490 nm, and calculate the total polysaccharide content of the sample by substituting it into the glucose standard curve. As Figure 2 In B (Effect of different concentrations of mulberry leaf powder on the intracellular polysaccharide content), the total polysaccharide content of the mycelium measured by this method was 1.83%, compared with 1.51% of the control, an increase of 21.2%. Example 2

[0019] Inoculate 4 - 5 pieces (each piece with an area of about 0.05 ± 0.01 cm 2 ) of the Phellinus igniarius mycelium cultured on a potato dextrose agar plate into the Phellinus igniarius liquid medium (sucrose 40 g / L, corn flour 4 g / L, mulberry leaf powder 0.15 g / L, peptone 3 g / L, yeast powder 2 g / L, KH2PO4 1 g / L, MgSO4 0.5 g / L, pH 7.0). After inoculation, culture at 30°C and a rotation speed of 150 rpm for 6 d, and collect the cultured mycelium.

[0020] The collected mycelium is filtered on filter paper and washed 3 - 4 times with clear water, then placed in an oven and dried at 60°C to constant weight.

[0021] Determination of the total polysaccharide content of Phellinus igniarius mycelium: The method for determining the total polysaccharide of Phellinus igniarius is the same as that in Example 1. As Figure 2 In B (Effect of different concentrations of mulberry leaf powder on the intracellular polysaccharide content), the total polysaccharide content measured by this method was 1.90%, compared with 1.51% of the control, an increase of 25.8%. Example 3

[0022] Inoculate 4 - 5 pieces (each piece with an area of about 0.05 ± 0.01 cm 2 ) of the Phellinus igniarius mycelium cultured on a potato dextrose agar plate into the Phellinus igniarius liquid medium (sucrose 40 g / L, corn flour 4 g / L, mulberry leaf powder 0.20 g / L, peptone 3 g / L, yeast powder 2 g / L, KH2PO4 1 g / L, MgSO4 0.5 g / L, pH 7.0). After inoculation, culture at 30°C and a rotation speed of 150 rpm for 6 d, and collect the cultured mycelium.

[0023] The collected mycelia were filtered on filter paper and then washed 3 - 4 times with clear water, and then placed in an oven and dried at 60 °C to constant weight.

[0024] Determination of the total polysaccharide content of the Phellinus igniarius mycelia: The method for determining the total polysaccharide of Phellinus igniarius was the same as that in Example 1. As Figure 2 in B (the effect of different concentrations of mulberry leaf powder on the intracellular polysaccharide content), the content of the total polysaccharide measured by this method was 1.71%, compared with 1.51% in the control, an increase of 13.2%.

[0025] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the purpose and scope of the present technical solution, and they should all be covered by the scope of the claims of the present invention.

Claims

1. A liquid medium for increasing the intracellular polysaccharide content of Phellinus igniarius mycelium, characterized in that: Comprising the following raw materials in parts by weight, based on 100% of the total amount of raw materials: Sucrose 3% - 6%, corn flour 0.3% - 0.5%, mulberry leaf powder 0.1% - 0.2%, peptone 0.2% - 0.4%, yeast powder 0.1% - 0.3%, KH2PO4 0.1% - 0.3%, MgSO4 0.01% - 0.06%.

2. The mulberry leaf powder is obtained by washing, blanching, microwave drying and wall-breaking and pulverizing fresh mulberry leaves, then filtered through a 200-mesh sieve, and the water content is less than 6%; the mass ratio of mulberry leaf powder to water is (0.1 - 0.15):100; the pH value is adjusted to 5 - 7 with a pH regulator; After inoculating the mycelium, culture for 6 - 7 days. The raw materials for preparing the medium include the following components in parts by weight, based on 100% of the total amount of raw materials: mulberry leaf powder 0.1% - 0.2%, corn flour 0.3% - 0.5%, peptone 0.2% - 0.4%, yeast powder 0.1% - 0.3%, KH2PO4 0.1% - 0.3%, MgSO4 0.01% - 0.06%; using corn flour, sucrose, peptone, yeast powder, KH2PO4, MgSO4 as raw materials, prepare a high-sugar corn flour medium; mix the high-sugar corn flour medium and mulberry leaf powder to obtain a medium for cultivating Phellinus igniarius.

3. A liquid culture method for Phellinus linteus mycelium, characterized in that, Inoculate Phellinus igniarius into the medium described in claim 6 and culture to obtain Phellinus igniarius mycelium; the temperature of the culture is 28 - 37 °C.

4. Application of mulberry leaf powder in the liquid culture of Phellinus igniarius.

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