CN113249226A - Fermentation method for improving biomass and total triterpene content of phellinus igniarius - Google Patents
Fermentation method for improving biomass and total triterpene content of phellinus igniarius Download PDFInfo
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Abstract
The invention provides a fermentation method for improving the biomass of Phellinus igniarius and the content of total triterpenes, which adopts Phellinus sp YC-1 strain for fermentation, the strain is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No.20222 and the preservation time of 2020, 7 and 13 days, and comprises the following steps: step 1, inoculating the subcultured phellinus igniarius into a seed culture medium for culture, and then inoculating into a secondary seed culture medium for culture to obtain a secondary seed solution; and 2, mixing the liquid fermentation culture medium with the volume V and the secondary seed liquid with the volume of 10-20% in a container, fermenting for the first time at a set rotating speed, oxygen introduction amount and fermentation temperature to obtain primary fermentation liquid, adding an extracting agent with the volume of 10-30% in the container, and continuously fermenting for the second time at the same rotating speed, oxygen introduction amount and fermentation temperature to obtain the fermentation liquid.
Description
Technical Field
The invention belongs to the field of fermentation industry, and particularly relates to a fermentation method for improving phellinus igniarius biomass and total triterpene content.
Background
Phellinus linteus is a rare medicinal fungus for many years, is named as forest gold in the name of beauty, is a famous and precious Chinese medicinal material, and is mainly used for treating diseases such as dysentery, night sweat, rectocele and bloody diarrhea in the traditional Chinese medicine of China. In recent years, research shows that phellinus igniarius contains various active ingredients such as polysaccharide, flavone, terpenoid, steroids and the like, shows various pharmacological actions such as anti-tumor, anti-bacterial and anti-virus, anti-inflammatory, immunoregulation, anti-oxidation and the like, and has wide application prospect.
The phellinus igniarius is used as an important medicinal fungus resource and has good medicinal value and market application prospect. However, since there are few natural fruit bodies and a large amount of collection of the natural fruit bodies will cause damage to resources, researches on culture and artificial cultivation techniques of Phellinus linteus mycelia are gradually developed, and medicinal active substances are obtained by methods such as solid culture, liquid fermentation, artificial cultivation of fruit bodies and the like. Liquid state fermentation is a common filamentous fungus culture method, and has the advantages of easy realization of fermentation regulation, high automation degree and the like. The liquid fermentation of phellinus igniarius is researched more, the production of mycelium, intracellular polysaccharide, extracellular polysaccharide, flavone and other active substances is mainly focused on, and few research reports on the total triterpenoids of phellinus igniarius are reported. Moreover, the current research is limited to the culture medium optimization stage, the yield of the produced active product is low, the yield of mycelium is also low, and the method is not suitable for large-scale industrialization. This also limits the popularization and application of Phellinus linteus in health promotion and medicinal application.
Disclosure of Invention
The present invention has been made to solve the above problems, and an object of the present invention is to provide a fermentation method for increasing the biomass of phellinus linteus and the total triterpene content.
The invention provides a fermentation method for improving the biomass of Phellinus igniarius and the content of total triterpenes, which adopts a Phellinus sp YC-1 strain for fermentation, the strain is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No.20222 and the preservation time of 2020, 7 and 13 days, and has the characteristics that the method comprises the following steps: step 1, inoculating the subcultured phellinus igniarius into a seed culture medium for culture, and then inoculating into a secondary seed culture medium for culture to obtain a secondary seed solution; and 2, mixing the liquid fermentation culture medium with the volume V and the secondary seed liquid with the volume of 10-20% V in a container, fermenting for the first time at a set rotating speed, oxygen introduction amount and fermentation temperature, adding an extracting agent with the volume of 10-30% V in the container, and continuously fermenting for the second time at the same rotating speed, oxygen introduction amount and fermentation temperature to obtain a fermentation liquid.
The fermentation method for improving the biomass and the total triterpene content of the phellinus igniarius provided by the invention can also have the following characteristics: wherein in the step 2, the rotating speed is 130-280 r/min, the oxygen introducing amount is 0.5-1.5 v/(v.m), the fermentation temperature is 25-32 ℃, and the first time is 48-168 hours.
The fermentation method for improving the biomass and the total triterpene content of the phellinus igniarius provided by the invention can also have the following characteristics: wherein, in the step 2, the second time is 70-240 h.
The fermentation method for improving the biomass and the total triterpene content of the phellinus igniarius provided by the invention can also have the following characteristics: wherein the total fermentation time of the first time and the second time is 12 d.
The fermentation method for improving the biomass and the total triterpene content of the phellinus igniarius provided by the invention can also have the following characteristics: wherein, in the step 2, the extracting agent is one of oleyl alcohol, oleic acid, butyl oleate, coconut oil, vegetable oil and petroleum ether.
Action and Effect of the invention
According to the fermentation method for improving the biomass and the total triterpene content of the phellinus igniarius, a certain amount of extractant is added into fermentation liquor for simultaneous fermentation, so that most of the total triterpene in the phellinus igniarius is transferred into the extractant, the product inhibition is reduced, the total triterpene yield is obviously improved, the content of phellinus igniarius mycelium is obviously improved, and the content of other active ingredients such as active polysaccharide is also obviously improved. The extraction agent adopted by the invention has simple fermentation process and easy control, and the extraction solvent can be recovered and reused by simple adsorbent treatment, thereby reducing the production cost and realizing large-scale industrial application.
Furthermore, the fermentation method for improving the biomass and the total triterpene content of the phellinus igniarius is started from a fermentation system, a phellinus igniarius bacterial solvent extraction fermentation system is established, the quantity and the total triterpene content of the phellinus igniarius bacterial liquid state fermentation bacterial can be obviously improved, and due to the adoption of solvent extraction fermentation, active products are highly enriched in an extraction solvent, so that the separation and purification of later-stage products are facilitated.
Drawings
FIG. 1 is a schematic diagram of the construction of a phylogenetic tree of YC-1 based on ITS sequences in an embodiment of the present invention;
FIG. 2 is a schematic diagram of the morphology of Phellinus sp.YC-1 liquid fermentation mycelium pellets in an embodiment of the present invention.
Detailed Description
In order to make the technical means and functions of the present invention easy to understand, the present invention is specifically described below with reference to the embodiments and the accompanying drawings.
The experimental methods used in the examples of the present invention are conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available unless otherwise specified.
PDA slant culture medium (g/L): 10 parts of tryptone, 5 parts of yeast powder, 10 parts of NaCl and 15 parts of agar powder.
Liquid seed medium (g/L): glucose 20, tryptone 20, corn steep liquor 4, MgSO4 & 7H2O 0.5.5 and NaH2 PO40.5.
Liquid fermentation medium (g/L): 50 parts of starch, 8 parts of yeast powder, 4 parts of corn steep liquor, MgSO4 & 7H2O 0.5.5 parts of corn steep liquor and NaH2PO40.5 parts of corn steep liquor.
The Phellinus sp YC-1 strain in the following examples was deposited in China general microbiological culture Collection center (CGMCC) with a collection number of CGMCC No.20222 for 7/13 days at 2020, and was deposited at Beijing Corp-Korea Beichen Xilu No. 1 Hospital No. 3.
Example 1: separation of Phellinus sp.YC-1 strain from wild Phellinus linteus fruiting body
(1) Culture medium: PDA slant culture medium (g/L): tryptone 10, yeast powder 5, NaCl 10 and agar powder 15.
(2) And (3) strain screening and separation: under aseptic environment, puncturing with an inoculating needle to take out internal tissues of wild Phellinus linteus fruiting body, dibbling on PDA plate, culturing at 28 deg.C for 6d, picking off part of mycelia after bacterial colony grows out, inoculating on new PDA plate, repeating for 3 times, and retaining the plate with uniform colony morphology, wherein the plate is separated Phellinus linteus strain. And extracting DNA of the phellinus igniarius strain, and identifying an ITS sequence.
(3) Colony morphology and ITS sequence identification
The strain is cultured on a PDA culture medium at 28 ℃ for 8 hours, the colony is brownish yellow, hyphae grow radially, and the hyphae are thick and dense.
Sequencing an ITS universal primer PCR product of the strain YC-1 to obtain an ITS rDNA sequence, comparing and analyzing through NCBI, wherein the result shows that the similarity with Phellinus sp.SA03 reaches 100%, further, through phylogenetic tree analysis shown in figure 1, the strain YC-1 is identified as Phellinus sp.YC-1, and the ITS sequence of YC-1 is SEQ ID No: 1 is shown.
Example 2: phyllinus sp.YC-1 fermentation blank control experiment
Step 1, inoculating Phellinus sp.YC-1 on a PDA slant culture medium, culturing at 28 ℃ for 15d, and then inoculating in a liquid seed culture medium. The liquid seed culture conditions are as follows: the culture temperature is 28 ℃, the rotating speed of a shaking table is 150r/min, and the culture time is 5d, so as to obtain the secondary seed liquid.
And 2, inoculating the secondary seed liquid into a fermentation culture medium according to the inoculation amount of 15%, wherein the culture temperature is 28 ℃, the rotating speed of a shaking table is 180r/min, the culture time is 12d, and the ventilation volume is 1.2 v/(v.m).
And 3, after fermentation is finished, centrifugally collecting thalli, drying at low temperature, crushing to prepare ultramicro phellinus igniarius hypha powder, and measuring the content of total triterpenoids and polysaccharides.
Under the condition, the amount of the Phellinus sp.YC-1 zymophyte is 16.21g/L, the content of the total triterpene is 1.24g/L, and the content of the active polysaccharide is 0.68 g/L.
Example 3: phyllinus sp.YC-1 vegetable oil extraction fermentation experiment
Step 1, inoculating Phellinus sp.YC-1 on a PDA slant culture medium, culturing at 28 ℃ for 15d, and then inoculating in a liquid seed culture medium. The liquid seed culture conditions are as follows: the culture temperature is 28 ℃, the rotating speed of a shaking table is 150r/min, and the culture time is 5d, so as to obtain the secondary seed liquid.
And 2, inoculating the secondary seed liquid into a fermentation culture medium according to the inoculation amount of 10%, wherein the culture temperature is 25 ℃, the rotating speed of a shaking table is 130r/min, the ventilation volume is 0.5v/(v.m), the vegetable oil is added after the culture time is 48h, the addition amount is 10%, and then the fermentation is continued to 12 d.
Step 3, after fermentation is finished, collecting a plant oil layer, and measuring the content of the triterpene; centrifuging to collect thallus, drying at low temperature, pulverizing to obtain ultramicro Phellinus Linteus mycelium powder, and measuring the content of triterpene and active polysaccharide; adding the triterpene contents in the plant oil layer and the ultramicro hypha powder to obtain the total triterpene content.
Under the condition, the amount of the Phellinus sp.YC-1 zymophyte is 17.96g/L, the total triterpene content is 11.34g/L, and the active polysaccharide content is 0.89 g/L.
Example 4: phyllinus sp.YC-1 vegetable oil extraction fermentation experiment
Step 1, inoculating Phellinus sp.YC-1 on a PDA slant culture medium, culturing at 28 ℃ for 15d, and then inoculating in a liquid seed culture medium. The liquid seed culture conditions are as follows: the culture temperature is 28 ℃, the rotating speed of a shaking table is 150r/min, and the culture time is 5d, so as to obtain the secondary seed liquid.
And 2, inoculating the secondary seed liquid into a fermentation culture medium according to the inoculation amount of 15%, wherein the culture temperature is 28 ℃, the rotating speed of a shaking table is 180r/min, the ventilation volume is 1.0v/(v.m), the vegetable oil is added after the culture time is 120h, the addition amount is 20%, and then the fermentation is continued to 12 d.
Step 3, after fermentation is finished, collecting a plant oil layer, and measuring the content of the triterpene; centrifuging to collect thallus, drying at low temperature, pulverizing to obtain ultramicro Phellinus Linteus mycelium powder, and measuring the content of triterpene and active polysaccharide; adding the triterpene contents in the plant oil layer and the ultramicro hypha powder to obtain the total triterpene content.
Under the condition, the amount of the Phellinus sp.YC-1 zymophyte is 33.53g/L, the content of the total triterpene is 27.56g/L, and the content of the active polysaccharide is 1.53 g/L.
Example 5: phyllinus sp.YC-1 vegetable oil extraction fermentation experiment
Step 1, inoculating Phellinus sp.YC-1 on a PDA slant culture medium, culturing at 28 ℃ for 15d, and then inoculating in a liquid seed culture medium. The liquid seed culture conditions are as follows: the culture temperature is 28 ℃, the rotating speed of a shaking table is 150r/min, and the culture time is 5d, so as to obtain the secondary seed liquid.
And 2, inoculating the secondary seed liquid into a fermentation culture medium according to the inoculation amount of 20%, wherein the culture temperature is 32 ℃, the rotating speed of a shaking table is 280r/min, the ventilation volume is 1.5v/(v.m), the vegetable oil is added after the culture time is 168h, the addition amount is 30%, and then the fermentation is continued to 12 d.
Step 3, after fermentation is finished, collecting a plant oil layer, and measuring the content of the triterpene; centrifuging to collect thallus, drying at low temperature, pulverizing to obtain ultramicro Phellinus Linteus mycelium powder, and measuring the content of triterpene and active polysaccharide; adding the triterpene contents in the plant oil layer and the ultramicro hypha powder to obtain the total triterpene content.
Under the condition, the amount of Phellinus sp.YC-1 zymophyte is 24.38g/L, the content of total triterpene is 20.17g/L, and the content of active polysaccharide is 1.31 g/L.
Example 6: phyllinus sp.YC-1 oleic acid extraction fermentation experiment
Step 1, inoculating Phellinus sp.YC-1 on a PDA slant culture medium, culturing at 28 ℃ for 15d, and then inoculating in a liquid seed culture medium. The liquid seed culture conditions are as follows: the culture temperature is 28 ℃, the rotating speed of a shaking table is 150r/min, and the culture time is 5d, so as to obtain the secondary seed liquid.
And 2, inoculating the secondary seed liquid into a fermentation culture medium according to the inoculation amount of 15%, wherein the culture temperature is 28 ℃, the rotating speed of a shaking table is 180r/min, the ventilation amount is 1.0v/(v.m), the oleic acid is added after the culture time is 120h, the addition amount is 20%, and then the fermentation is continued to 12 d.
Step 3, after fermentation is finished, collecting an oleic acid layer, and measuring the content of triterpene; centrifugally collecting the mycelia, drying at low temperature, pulverizing to obtain ultramicro Phellinus Linteus mycelium powder, and measuring the content of triterpene and active polysaccharide; adding the triterpene contents of the oleic acid layer and the ultramicro mycelium powder to obtain the total triterpene content.
Under the condition, the amount of the Phellinus sp.YC-1 zymophyte is 30.52g/L, the total triterpene content is 16.84g/L, and the active polysaccharide content is 1.45 g/L.
Effects and effects of the embodiments
According to the embodiments 2 to 6, when the triterpene is extracted into the extracting agent, the product inhibition is reduced, the yield of the total triterpene reaches 11.34 to 27.56g/L, the yield of the triterpene is greatly improved, and the yield of the active polysaccharide is also obviously improved to 0.89 to 1.53g/L, wherein when the inoculation amount of the secondary seed liquid is 15 percent, the culture temperature is 28 ℃, the rotating speed of a shaking table is 180r/min, the ventilation volume is 1.0v/(v.m), and the amount of the Phellinus sp.YC-1 fermentation bacteria, the content of the total triterpene and the active polysaccharide are respectively the highest when the addition amount of the vegetable oil is 20 percent, and the total triterpene content and the active polysaccharide are respectively 33.53g/L, 27.56g/L and 1.53 g/L.
According to the fermentation method for improving the biomass and the total triterpene content of the phellinus igniarius, a certain amount of extractant is added into fermentation liquor for simultaneous fermentation, so that most of the total triterpene in the phellinus igniarius is transferred into the extractant, the product inhibition is reduced, the total triterpene yield is obviously improved, the content of phellinus igniarius mycelium is obviously improved, and the content of other active ingredients such as active polysaccharide is also obviously improved. The extraction agent adopted by the invention has simple fermentation process and easy control, and the extraction solvent can be recovered and reused by simple adsorbent treatment, thereby reducing the production cost and realizing large-scale industrial application.
Furthermore, the fermentation method for improving the biomass and the total triterpene content of the phellinus igniarius is started from a fermentation system, a phellinus igniarius bacterial solvent extraction fermentation system is established, the quantity and the total triterpene content of the phellinus igniarius bacterial liquid state fermentation bacterial can be obviously improved, and due to the adoption of solvent extraction fermentation, active products are highly enriched in an extraction solvent, so that the separation and purification of later-stage products are facilitated.
The above embodiments are preferred examples of the present invention, and are not intended to limit the scope of the present invention.
Sequence listing
<110> Shanghai university of science and engineering
<120> fermentation method for improving phellinus igniarius biomass and total triterpene content
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 639
<212> DNA
<213> Phellinus baumii (Phellinus baumii)
<400> 1
cgggggcaat cggacctgat ttgaggtcaa ggtgtcaaga aaaagaaagg aggtaacaac 60
tccttgtccg accacgcgga ccgttagaag caagctcgtt aggcaagcgc tcgttggtga 120
acggaatcaa ctattacacc gtaagcgcga gccaaagccc agctaatgta tttaagagga 180
gccgaccccc ttcgaaaggc ggccagcagt aaacctccaa gtccaaacct caagcccttc 240
agttaaagaa agacgagcgg tttgagataa acatgacact caaacaggca tgcccctcgg 300
aataccaagg ggcgcaaggt gcgttcaaag attcgatgat tcactgaatt ctgcaattca 360
cattacttat cgcatttcgc tgcgttcttc atcgatgcga gagccaagag atccgttgtt 420
gaaagttgta ttgattttcg cccacgagga gcattacatt cacaaagaca atataagggg 480
gggtttgtaa caacaagccc aagtcttcac ccgacgcact cccgctcgct ttcattttcg 540
aagggttacc taacgagcaa cactcgctcg ctttcgccct tctactcatt acttacaaga 600
cagacctcag actactaact tcgactcgcg atataataa 639
Claims (5)
1. A fermentation method for improving the biomass and the total triterpene content of Phellinus linteus is characterized in that Phellinus linteus (Phellinus sp.) YC-1 strain is adopted for fermentation, the strain is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No.20222, and the preservation time is 13 days at 7 months after 2020, and the fermentation method comprises the following steps:
step 1, inoculating the subcultured phellinus igniarius into a seed culture medium for culture, and then inoculating into a secondary seed culture medium for culture to obtain a secondary seed solution;
and 2, mixing the liquid fermentation culture medium with the volume of V and the secondary seed liquid with the volume of 10-20% V in a container, fermenting for the first time at a set rotating speed, oxygen introduction amount and fermentation temperature, adding an extracting agent with the volume of 10-30% V into the container, and continuously fermenting for the second time at the same rotating speed, oxygen introduction amount and fermentation temperature to obtain fermentation liquid.
2. The fermentation method for increasing phellinus igniarius biomass and total triterpene content according to claim 1, which comprises the following steps:
in the step 2, the rotating speed is 130-280 r/min, the oxygen introduction amount is 0.5-1.5 v/(v.m), the fermentation temperature is 25-32 ℃, and the first time is 48-168 hours.
3. The fermentation method for increasing phellinus igniarius biomass and total triterpene content according to claim 1, which comprises the following steps:
wherein, in the step 2, the second time is 70-240 h.
4. The fermentation method for increasing phellinus igniarius biomass and total triterpene content according to claim 1, which comprises the following steps:
wherein, in the step 2, the total fermentation time of the first time and the second time is 12 d.
5. The fermentation method for increasing phellinus igniarius biomass and total triterpene content according to claim 1, which comprises the following steps:
in the step 2, the extracting agent is one of oleyl alcohol, oleic acid, butyl oleate, coconut oil, vegetable oil and petroleum ether.
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