CN100412186C - Method for preparing Phyllopyreum thallus by Phyloporus xylophilus and liquid submerged fermentation - Google Patents

Abstract

The invention discloses a fungus fungus and a method for preparing fungus body by submerged fermentation of liquid. The aim is to prepare Phylophora xylophilus cells by large-scale liquid submerged fermentation. Said Phyloporus chrysogenum is preserved in the General Microorganism Center of China Microorganism Culture Collection Management Committee, and the preservation number is: CGMCC No.1658, which belongs to Basidiomycetes (Basidiomycetes), Polyporales (Polyporales), Polyporaceae (Polyporaceae) ), Fomes. In the present invention, artificially isolated and cultivated Phylophorus xylophyllum is used as the starting strain, and it is cultivated on a solid inclined plane, shaker seed culture, and fermenter tank successively, and the mycelium in the fermentation liquid can reach 10-15g/kg by dry weight. L. The liquid submerged aerated fermentation medium of the present invention has a reasonable ratio of nutrient components, can ensure the growth needs of P. xylophilus, and is rich in medium materials. In the whole batch fermentation process, only one batching and one sterilization are required, and it has the advantages of It saves time and labor, and reduces the chance of bacterial contamination due to feeding in the middle.

Description

Method for preparing Phyllopyreum thallus by Phyloporus xylophilus and liquid submerged fermentation

technical field

The invention belongs to the technical field of microbial engineering, and relates to a fungus and a method for preparing fungus cells by liquid submerged fermentation.

Background technique

Due to the scarcity of wild resources and the deterioration of the growth environment, many valuable medicinal fungi are on the verge of extinction. How to save these precious medicinal resources and meet the needs of drug development is particularly important. Active strains are isolated and screened using microbial technology, and modern fermentation technology is used to carry out Large-scale preparation of medicinal materials is necessary.

Fomes fomentarius is a rare Chinese herbal medicine, which is commonly used by folks in Northeast my country and has a long history. Its fruiting bodies are rich in terpenoids, polyphenols, alkaloids and polysaccharides. It has high medicinal value in the treatment of human malignant tumors. It is mainly used to treat esophageal cancer, gastric cancer and other digestive system tumors. It also has a significant effect in treating children's food accumulation, eliminating accumulation and removing blood stasis.

The modern fermentation technology developed in the middle and late last century has been successfully applied to the large-scale preparation of various medicinal fungi, among which the liquid fermentation technology is the most mature. This application of liquid fermentation technology to the preparation of the mycelium of Phyllostachys chinensis is the first case in China, and the research results show that the scheme is feasible.

Contents of the invention

The object of the present invention is to provide a method for preparing the fungus body of the fungus fungus and the submerged fermentation of the liquid.

Fomes fomentarius (Fomes fomentarius) is preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee, and the preservation number is: CGMCC No.1658, which belongs to Basidiomycetes (Basidiomycetes), Polyporales (Polyporales), Polyporaceae ( Polyporaceae), Fomes.

The method for preparing Phyloporus thalli cells by liquid submerged fermentation: the Phyloporus thorax strain obtained by separating and purifying from wild woody fruit bodies, using the strain as the starting strain, successively cultured on a solid slant, cultivated on a shaker seed, Culture in a fermenter to prepare the mycelia of Phyllostachys spp.

The internal transcribed spacer ribosomal DNA sequence of described bacterial strain is as follows:

CCTCCGCTTATTGATATGCTTAAGTTCAGCGGGTAGTCCTACCTGATTTGAGGTCAGAGTTCATAAAA

GCTGTCTCTGACGAGACCATTAGAAGCTCTCCAAACGCTTCACGGTCGCGGCGTAGACATTATCACAC

CGACAGCCGATCCGCAAGGAACCAAGCTAATGCATTTGAGAGGAGCTGACTCAAACGAGGGCCAGCAA

AAGCCTCCAATAAGCCAACCCTACAAACCCGCAAAGGTTTATAGGTTGAGAATTTCATGACACTCAAA

CAGGCATGCTCCTCGGAATACCAAGGAGCGCAAGGTGCGTTCAAAGATTCGATGATTCACTGAATTCT

GCAATTCACATTACTTATCGCATTTCGCTGCGTTTCTTCATCGATGCGAGAGCCAAGAGATCCGTTGCT

GAAAGTTGTATTATAGATGCGTTACATCAATAAACATTCTGTTACTTCAATAGTTTGTAAAGAAAACG

TGGGGCCAAGTGACGCCGCCGCAAAGCGACGCACCTGAAATCCCACAGTAAGTGCACAGGTGTAGAGT

GGATGAGCAGGGCGTGCACATGCCTCGGAAGGCCAGCTACAACCCATGTCAAAAACTCGTTAATGATCC

TTCCGCAGGTTCACCTACGGAAACCTTGTTACGACTTTACTTCCA.

The conditions for solid slant culture are as follows: inoculate the prepared slant culture medium from the preserved strains, at a temperature of 20-30° C., and cultivate for 160-240 hours.

The main components of the slant medium per 1000 ml are: 100-300 grams of potatoes, 10-30 grams of glucose and 15-25 grams of agar, with a natural pH.

The condition of shaker seed culture is: after culturing the bacteria on a solid slope for 160-240°C, the mycelium is inoculated in a triangular flask filled with liquid seed medium, cultured at 20-30°C, with a rotation speed of 150-200rpm, and cultured at 62-96°C. Hour.

The main ingredients of the seed medium per 1000 ml are: 10-60 grams of glucose, 5-30 grams of peptone, 3-20 grams of yeast powder or yeast extract, 3-20 grams of malt extract powder, and the pH is natural.

The method of fermentor cultivation is that liquid seeds are inoculated into fermentors equipped with fermentation medium after 62-96 hours of shaker culture, inoculum size is 5-15% (V/V), and the tank temperature is maintained at 20-30°C. The pressure is 0.4-0.8kg/cm, the stirring rate is 100-200rpm, the air flow is 1:0.3-1:1.2, and the fermentation time is 120-200 hours.

The main components of the fermentation medium per 1000 ml are: 20-80 grams of glucose, 10-40 grams of peptone, 5-20 grams of yeast powder or yeast extract, 0.2-1 gram of MgSO ₄ and 0.2-1 gram of KH ₂ PO ₄ , The pH value of the culture medium is 4.0-7.0.

The fermenter culture adopts liquid submerged aerated fermentation culture.

The tank standard of the present invention is that the fermented liquid becomes viscous, and the microscopic examination finds that the mycelium begins to age.

Through comparison with other cultivation techniques and repeated test verification, the present invention has remarkable effect:

(1) The seed medium in the present invention provides the nutrients needed for the rapid growth of the mycelia in a short period of time, and submerged fermentation can be carried out after about 62 hours of cultivation.

(2) The liquid submerged aerated fermentation medium in the present invention has a reasonable nutrient distribution ratio, which can ensure the growth needs of P. xylophilus, and the medium material is rich. In the whole batch fermentation process, only one batching and one sterilization are needed. Yes, it saves time and labor, and reduces the chance of bacterial contamination due to feeding in the middle.

(3) The liquid submerged aerated fermentation process of the present invention is adopted to carry out batch fermentation, and 10-15 grams of dry mycelium can be obtained per liter of fermentation broth.

(4) The present invention is the first domestic study on the fermentation process of P. xylophilus, which has great guiding significance for the subsequent experimental research and industrial preparation of the bacterium.

Detailed ways

The above-mentioned Fomes fomentarius was isolated from wild Fomes fomentarius collected from the Changbai Mountains in Jilin, my country. It is now preserved in the General Microbiology Center of the China Microbiological Culture Collection Management Committee. Fomes).

The present invention will be further described in detail below in conjunction with specific embodiments.

Embodiment 1: Isolation and cultivation of bacterial strains

According to the isolation and culture in the laboratory according to the following conditions, the Phylopora xylophilus described in the present invention can be obtained. Wooden hooves collected from the Changbai Mountains in Jilin, my country, sterilized the surface with 0.1% mercury chloride for 3 minutes, rinsed with sterile water, placed on sterile filter paper to absorb the water, cut off part of the outer cortex with a sterile blade, and picked a small piece Tissues were transplanted onto 2% water agar plates containing 100 μg/ml ampicillin and 100 μg/ml streptomycin. Cultivate at a temperature of 20° C. to 30° C. After the hyphae grow out, the top of the mycelium is transplanted onto the PDA medium, and the purification is repeated 3 to 5 times continuously to obtain a pure culture.

Embodiment 2: the preparation of slant medium

(1) Take 100 grams of fresh peeled potatoes, cut them into cubes of about 0.5 cubic centimeters, rinse them with water immediately, boil them with 1000 milliliters of distilled water for 20 minutes, filter them with gauze, take the filtrate, add 15 grams of agar and 10 grams of glucose to dissolve, add water to set The capacity is up to 1000 ml, and it is a slant medium after sterilization.

(2) Take 200 grams of fresh peeled potatoes, cut them into cubes of about 0.5 cubic centimeters, rinse them with water immediately, boil them with 1000 milliliters of distilled water for 20 minutes, filter them with gauze, take the filtrate, add 20 grams of agar and 20 grams of glucose to dissolve, add water to set The capacity is up to 1000 ml, and it is a slant medium after sterilization.

(3) Take 300 grams of fresh peeled potatoes, cut them into cubes of about 0.5 cubic centimeters, rinse them with water immediately, boil them with 1000 milliliters of distilled water for 20 minutes, filter them with gauze, take the filtrate, add 25 grams of agar and 30 grams of glucose to dissolve, add water to set The capacity is up to 1000 ml, and it is a slant medium after sterilization.

Embodiment 3: the preparation of seed culture medium

(1) Take 10 grams of glucose, 5 grams of peptone, 3 grams of yeast powder (paste), and 3 grams of malt extract powder, mix and dissolve each component, add water to make the volume 1000 ml, and sterilize it as the seed medium.

(2) Take 30 grams of glucose, 20 grams of peptone, 10 grams of yeast powder (paste), and 10 grams of malt extract powder, mix and dissolve each component, add water to make the volume 1000 ml, and sterilize it to form the seed medium.

(3) Take 60 grams of glucose, 30 grams of peptone, 20 grams of yeast powder (paste), and 20 grams of malt extract powder, mix and dissolve each component, add water to make the volume 1000 ml, and sterilize it as a seed medium.

Embodiment 4: the preparation of liquid submerged fermentation medium

(1) Take 20 grams of glucose, 10 grams of peptone, 5 grams of yeast powder (cream), 0.2 grams of MgSO ₄ , and 0.2 grams of KH ₂ PO ₄ . Mix the above ingredients and add water to 1000 ml, and add 0.6 ml of foam diol as an antifoaming agent to adjust the pH to 4.0. After sterilization, it becomes the fermentation medium.

(2 Take 40 grams of glucose, 20 grams of peptone, 10 grams of yeast powder (paste), 0.5 grams of MgSO ₄ , and 0.5 grams of KH ₂ PO ₄ . Mix the above ingredients and add water to 1000 ml, and add 0.6 ml of foam enemy as a defoamer , adjust the pH to 5.0, and after sterilization, it becomes the fermentation medium.

(3) Take 80 grams of glucose, 40 grams of peptone, 20 grams of yeast powder (cream), 1 gram of MgSO ₄ , and 1 gram of KH ₂ PO ₄ . Mix the above ingredients and add water to 1000 ml, and add 0.6 ml of foam diol as an antifoaming agent, adjust the pH to 7.0, and sterilize it to become the fermentation medium.

Embodiment 5: liquid submerged culture method one

(1) Take the fresh slant medium prepared according to Example 2, pick the mycelia of the preserved strains and inoculate them on the slant medium by coating or streaking, and cultivate at 20° C. for 240 hours.

(2) Select the luxuriant slant culture bacterial classification of mycelium, pick mycelium from above or dig out bacterial lawn and inoculate in the shake flask that the liquid seed culture medium prepared by embodiment 3 is housed, at temperature 20 ℃, Under the condition of rotating speed 150rpm, cultivate for 96 hours.

(3) Get the seed culture that is in the middle and late stages of logarithmic growth, and transfer it to the fermenter equipped with the fermentation medium prepared in Example 4 with an inoculum size of 15% (V/V), keep the tank temperature at 20°C , tank pressure 0.5kg/cm, stirring rate 100rpm, ventilation rate 1:0.3, fermentation and cultivation time 200 hours. When the fermented liquid becomes viscous, when the mycelium begins to age under microscope observation, the fermentation is stopped, the tank is placed, and the mycelium and fermented liquid are collected respectively. More than 10 g of dried mycelium can be obtained per liter of fermented liquid.

Embodiment 6: liquid submerged culture method two

(1) Take the fresh slant medium prepared according to Example 2, pick the mycelia of the preserved strains and inoculate them on the slant medium by coating or streaking, and cultivate them at 25° C. for 200 hours.

(2) select the luxuriant slant culture bacterial classification of mycelium, pick mycelium from above or dig out bacterial lawn and inoculate in the shake flask that the liquid seed culture medium prepared by embodiment 3 is housed, at 25 ℃ of temperature, Cultivate for 62 hours under the condition of rotating speed 180rpm.

(3) Get the seed culture in the middle and late stages of logarithmic growth, transfer it to the fermenter equipped with the fermentation medium prepared in Example 4 with an inoculum size of 5% (V/V), keep the tank temperature at 25°C , Tank pressure 0.6kg/cm, stirring rate 150rpm, air flow 1:0.6, fermentation time 120 hours. When the fermented liquid becomes viscous, when the mycelium begins to age under microscope observation, the fermentation is stopped, the tank is placed, and the mycelium and fermented liquid are collected respectively. More than 10 g of dried mycelium can be obtained per liter of fermented liquid.

Embodiment 7: liquid submerged culture method three

(1) Take the fresh slant medium prepared according to Example 2, pick the mycelia of the preserved strains and inoculate them on the slant medium by coating or streaking, and cultivate at 30° C. for 160 hours.

(2) Select the luxuriant slant culture bacterial classification of mycelium, pick mycelium from above or dig out bacterial lawn and inoculate in the shake flask that the liquid seed culture medium prepared by embodiment 3 is housed, at 30 ℃ of temperature, Cultivate for 80 hours under the condition of rotating speed 200rpm.

(3) Get the seed culture in the middle and late stages of logarithmic growth, transfer it to the fermenter equipped with the fermentation medium prepared in Example 4 with an inoculum size of 10% (V/V), and keep the tank temperature at 30°C , the tank pressure is 0.8kg/cm, the stirring rate is 200rpm, the ventilation volume is 1:1.2, and the fermentation is about 180 hours. When the fermented liquid becomes viscous, when the mycelium begins to age under microscope observation, the fermentation is stopped, the tank is placed, and the mycelium and fermented liquid are collected respectively. More than 10 g of dried mycelium can be obtained per liter of fermented liquid.

Finally, it should also be noted that what is listed above are only specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and many variations are possible.

The present invention may be embodied in other specific forms without departing from the spirit and main characteristics of the invention. Therefore, no matter from which point of view, the above-mentioned embodiments of the present invention can only be regarded as descriptions of the present invention and cannot limit the present invention, and the claims have pointed out the scope of the present invention, and the above description does not point out the scope of the present invention. Therefore, any change within the meaning and scope equivalent to the claims of the present invention should be considered to be included in the scope of the claims.

Claims (6)

1. A method for liquid submerged fermentation to prepare Phyloporus thalline, characterized in that, the Phylofora thallus strain obtained by separating and purifying from wild woody sap fruiting bodies, using the bacterial strain as the starting strain, is subjected to solid Slope culture, shaker seed culture, and fermenter culture to prepare the mycelium of Phyllostachys xylophilus, the method of fermenter culture is to inoculate the liquid seed shaker culture for 62 to 96 hours and then inoculate to the fermenter containing fermentation medium. In the tank, the inoculation amount is 5-15% (V/V), the tank temperature is maintained at 20-30°C, the tank pressure is 0.4-0.8kg/cm, the stirring rate is 100-200rpm, the ventilation rate is 1:0.3-1:1.2, and the fermentation time is 120-200 hours; the main ingredients per 1000 ml of fermentation medium are: 20-80 grams of glucose, 10-40 grams of peptone, 5-20 grams of yeast powder or yeast extract, 0.2-1 gram of MgSO ₄ and KH ₂ PO ₄ 0.2-1 g, the pH of the culture medium is 4.0-7.0. 2. a kind of liquid submerged fermentation according to claim 1 prepares the method for Phylophora xylophilus thalline, it is characterized in that: the internal transcriptional spacer ribosomal DNA sequence of described bacterial strain is as follows: CCTCCGCTTATTGATATGCTTAAGTTCAGCGGGTAGTCCTACCTGATTTGAGGTCAGAGTTCATAAAAGCTGTCTCTGACGAGACCATTAGAAGCTCTCCAAACGCTTCACGGTCGCGGCGTAGACATTATCACACCGACAGCCGATCCGCAAGGAACCAAGCTAATGCATTTGAGAGGAGCTGACTCAAACGAGGGCCAGCAAAAGCCTCCAATAAGCCAACCCTACAAACCCGCAAAGGTTTATAGGTTGAGAATTTCATGACACTCAAACAGGCATGCTCCTCGGAATACCAAGGAGCGCAAGGTGCGTTCAAAGATTCGATGATTCACTGAATTCTGCAATTCACATTACTTATCGCATTTCGCTGCGTTCTTCATCGATGCGAGAGCCAAGAGATCCGTTGCTGAAAGTTGTATTATAGATGCGTTACATCAATAAACATTCTGTTACTTCAATAGTTTGTAAAGAAAACGTGGGGCCAAGTGACGCCGCCGCAAAGCGACGCACCTGAAATCCCACAGTAAGTGCACAGGTGTAGAGTGGATGAGCAGGGCGTGCACATGCCTCGGAAGGCCAGCTACAACCCATGTCAAAACTCGTTAATGATCCTTCCGCAGGTTCACCTACGGAAACCTTGTTACGACTTTTACTTCCA。 3. A kind of liquid submerged fermentation according to claim 1 prepares the method for the thallus of Phylophora xylophilus, it is characterized in that, the condition of described solid slant culture is: inoculate from preserved bacterial classification to the prepared slant culture medium above, at a temperature of 20-30°C, and cultivate for 160-240 hours. 4. According to claim 3, a method for preparing Phyllostachys spp. by submerged fermentation, wherein the main components of the slant culture medium per 1000 ml are: 100-300 grams of potatoes, 10 grams of glucose ～30 grams and 15～25 grams of agar, the pH is natural. 5. The method for preparing Phylophora thalum by submerged fermentation according to claim 3, characterized in that, the conditions for culturing seeds in a shaker are: after 160 to 240 hours of culturing on a solid slant surface of the strain, The mycelium is inoculated in a conical flask equipped with a liquid seed medium, cultivated at 20-30° C., with a rotating speed of 150-200 rpm, and cultivated for 62-96 hours. 6. The method for preparing Phylophora xylophilus thallus by submerged liquid fermentation according to claim 5, wherein the main components of the seed culture medium per 1000 milliliters are: 10-60 grams of glucose, peptone 5-30 grams, 3-20 grams of yeast powder or yeast extract, 3-20 grams of malt extract powder, natural pH.