CN113564094A

CN113564094A - Method for increasing intracellular polyphenol accumulation of phellinus igniarius

Info

Publication number: CN113564094A
Application number: CN202110738974.XA
Authority: CN
Inventors: 赵艳霞; 郑维发; 袁雯雯; 孙梦妮; 周蓉
Original assignee: Jiangsu Normal University
Current assignee: Jiangsu Normal University
Priority date: 2021-06-30
Filing date: 2021-06-30
Publication date: 2021-10-29
Anticipated expiration: 2041-06-30
Also published as: CN113564094B

Abstract

The method for increasing accumulation of intracellular polyphenol of phellinus linteus by using unsaturated fatty acid comprises the following steps: cutting solid cultured Phellinus linteus into small pieces, transferring to fermentation medium, culturing in constant temperature shaking table, taking out shake flask, crushing under aseptic condition, and culturing in constant temperature shaking table to obtain mother liquor for subsequent liquid fermentation. Adding unsaturated fatty acid into culture solution of fermented Phellinus linteus. The invention takes unsaturated fatty acid as an exciton during fermentation culture of phellinus igniarius, and can obviously improve the accumulation of phellinus igniarius biomass and the yield of the bristletin polyphenol.

Description

Method for increasing intracellular polyphenol accumulation of phellinus igniarius

Technical Field

The invention relates to the technical field of biological fermentation engineering, in particular to a method for increasing accumulation of intracellular polyphenol of phellinus linteus by utilizing unsaturated fatty acid.

Background

Phellinus igniarius belongs to the medicinal fungi of Basidiomycota and is a precious medicinal material in China. It is recorded in Bencao gang mu that sang Huang has the functions of benefiting five internal organs, ventilating intestines and stomach qi, expelling toxin and qi, etc. Research shows that phellinus igniarius contains various active ingredients such as polysaccharide, triterpene, polyphenol, organic acid and the like, and the ingredients have remarkable activities in the aspects of resisting tumors, protecting livers, resisting oxidation, resisting bacteria, diminishing inflammation, enhancing the immunity of organisms and the like [1-4 ]. The polyphenol compounds are composed of one or more hydroxyl groups, are mainly divided into flavonoid compounds, pyrone compounds, furanone compounds and the like, and have high antioxidant activity [5 ]. The flavonoids have antioxidant, antitumor, blood vessel dilating and antibacterial effects; the pyrone compound has stronger capability of eliminating free radicals and inhibits the bioactivity of NF-kB pathway; furanone compounds have antibacterial, antiviral and anticomplementary activities [6-7 ].

Although wild-type phellinus linteus can produce various active substances, the growth of phellinus linteus is environmentally critical and the growth of fruit body is slow in nature. With the continuous increase of market demand, wild resources are less and less, and artificial cultivation of phellinus igniarius becomes a hot spot concerned by people. However, under the condition of artificial fermentation culture, the accumulation of secondary metabolites of phellinus igniarius is very low, and the production requirement cannot be met. This is because all the related genes of Phellinus linteus for synthesizing secondary metabolites are under low expression or silent state under artificial culture conditions. Researches show that after fungi are stimulated by external environment, thalli can generate a series of response reactions, so that the expression level of gene organisms is influenced, the enzyme activity is changed, and the accumulation amount of secondary metabolites is finally increased.

The liquid fermentation culture has the characteristics of controllable conditions, short growth period and the like, however, the problem of low yield of active ingredients exists in the fermentation process of the phellinus igniarius, so that how to improve the content of the active ingredients of the phellinus igniarius through an exciton becomes an important research content under the condition of liquid fermentation.

Reference documents:

1.Ge Qing,Mao Jianwei,Zhang Anqiang,Wang Yongjiang,Sun Peilong.Purification,chemical characterization,and antioxidant activity of a polysaccharide from the fruiting bodies of sanghuang mushroom(Phellinus baumii Pilát)[J].Food Sci Biotech,2013,22(2):384-389.

2.Yayeh T,Lee WM,Ko D,Park SC,Cho JY,Park HJ,Lee IK,Kim SH,Hong SB,Kim S,Yun BS,Rhee MH.Phellinus baumii ethyl acetate extract alleviated collagen type II induced arthritis in DBA/1mice[J].J Nat Med,2013,67(4):807-813.

3.Hwang BS,Lee IK,Choi HJ,Yun BS.Anti-influenza activities of polyphenols from the medicinal mushroom Phellinus baumii[J].Bioorg Med Chem Lett,2015,25(16):3256-3260.

4.Noh JR,Lee IK,Kim YH,Gang GT,Hwang JH,Ly SY,Yun BS,Lee CH.Antiatherogenic effect of antioxidant polyphenols from Phellinus baumii in apolipoprotein e-deficient mice[J].Ann Nutr Metab,2011,59(2-4):145-153.

5. vertical peak, Bahaiying, Lidanhua, research on polyphenol substances in "phellinus igniarius" fungi and their biological activities progress [ J ] research and development of natural products, 2016, (1): 147-.

6. Huangxianxiao, Shu Zhong, Dongyu, etc. the application of phellinus igniarius in ancient Chinese medical book is recorded and the progress of modern medicinal research [ J ] Chinese materia medica journal of TCM 2014,032(009) 2249-.

7. Wu chang, study of chemical composition of medicinal fungus phellinus igniarius [ D ]. university of shandong, 2011.

Disclosure of Invention

The invention aims to provide a method for improving the accumulation of intracellular polyphenol of phellinus igniarius cultured by fermentation.

In order to achieve the above purpose, the technical scheme of the invention is as follows:

the method for increasing accumulation of intracellular polyphenol of phellinus linteus by using unsaturated fatty acid comprises the following steps: adding unsaturated fatty acid into Phellinus linteus culture medium.

Further, the medium is a liquid medium.

Further, the adding proportion of the unsaturated fatty acid is 0.5 g/L-1 g/L.

Further, the unsaturated fatty acid is linoleic acid and/or linolenic acid.

Further, the culturing of phellinus linteus specifically comprises:

inoculating a solid cultured phellinus igniarius strain into a liquid culture medium, performing activated culture, then inoculating the phellinus igniarius strain into a seed fermentation culture medium according to the inoculation amount of 10% of the volume percentage, performing seed culture, and performing shake culture at constant temperature for 5 days to obtain a seed solution; inoculating the seed liquid into a liquid fermentation culture medium according to the inoculation amount of 5 percent by volume, adding unsaturated fatty acid to ensure that the concentration of the unsaturated fatty acid in the fermentation liquid is 0.5-1 g/L, carrying out shake culture at constant temperature for 13 days, collecting fermentation culture, and filtering to obtain mycelium.

Further, the rotation speed of the constant temperature shaking table is 150 rpm, and the culture temperature is 26 ℃.

Further, the seed culture medium is a YPD culture medium, and the formula is as follows: 10 parts of yeast extract, 20 parts of peptone and 20 parts of glucose, and fully stirring until the yeast extract, the peptone and the glucose are dissolved.

Further, the formula of the liquid fermentation medium is as follows: weighing MgSO₄0.5 part of (NH)₄)₂SO₄0.2 part of glucose, 20 parts of CaCl₂0.1 part of yeast extract, 10 parts of KH₂PO₄1 part of the mixture is put into a container, deionized water is added, the mixture is fully stirred until the mixture is dissolved, and the pH value is adjusted to 5.5.

Compared with the prior art, the invention has the beneficial technical effects that:

the invention takes unsaturated fatty acid as an exciton during fermentation culture of phellinus igniarius, thereby remarkably improving the accumulation of phellinus igniarius biomass and the yield of the bristletin polyphenol.

Drawings

FIG. 1 shows the experimental results of the effect of linoleic acid on the biomass of fermentation mycelium of Phellinus vannamei;

FIG. 2 shows the results of experiments on the effect of linoleic acid on intracellular polyphenols of Phellinus vannamei;

FIG. 3 shows the results of experiments on the effect of linoleic acid on the content of three polyphenols;

FIG. 4 shows the experimental results of the effect of linolenic acid on the biomass of fermentation mycelium of Phellinus vannamei;

FIG. 5 shows the experimental results of the effect of linolenic acid on intracellular polyphenols of Phellinus vannamei;

FIG. 6 shows the experimental results of the effect of linolenic acid on the content of three polyphenols.

Detailed Description

The present invention will be further illustrated with reference to the following examples.

Example 1

1. Test strain and culture medium

The strain of Phellinus vannamei (Sanghuangporos vanninii) was preserved in paraffin medium. Liquid medium of phellinus igniarius: MgSO (MgSO)₄ 0.5g，(NH₄)₂SO₄0.2g, glucose 20g, CaCl₂0.1g, 10g of yeast extract and KH₂PO₄Adding 800mL of deionized water into a 1L beaker by 1g, fully stirring until the deionized water is dissolved, adjusting the pH value to 5.5, and fixing the volume to 1L. The solid culture medium is prepared by adding 15g agar powder per liter liquid culture medium.

2. Liquid fermentation of Phellinus linteus

The solid cultured Floweringtonia phellinus is cut into small pieces and transferred to a fermentation medium. Culturing in a constant temperature shaking table at a temperature of 26 ℃ for 5 days at 150 rpm. The shake flask was removed and broken up thoroughly in a hand-held homogenizer under sterile conditions. Continuously placing in a constant temperature shaking table, culturing at 26 deg.C and 150 rpm for 5 days. Namely the mother solution. Can be used for subsequent liquid fermentation. Liquid fermentation of Fleckedflesh polypore (S.vaninii) was divided into three groups, i.e., a control group containing no unsaturated fatty acid in the medium, and experimental groups containing 0.5g/L and 1g/L unsaturated fatty acid. Samples were taken while culturing 5d, 7d, 9d, 11d and 13 d.

3. Determination of biomass

Collecting the fermentation culture, filtering to obtain mycelium, drying the mycelium to constant weight, and weighing.

4. Extraction of polyphenols

The fermentation broth was filtered, and the mycelia were collected and placed in centrifuge tubes. Adding methanol with the volume of 1 time of the thallus into a centrifuge tube: acetone ═ 1: 1 extracting the liquid, smashing the thalli by using a flash extractor, fully contacting the thalli with the extracting liquid, and soaking the thalli at room temperature overnight. The sample tube was removed at 6000rmp, centrifuged for 5 minutes and the supernatant collected. This was repeated three times. The supernatant was collected. The sample was concentrated using a rotary evaporator and dissolved with methanol.

5. Determination of polyphenol content

And (3) determining the polyphenol content by using gallic acid as a standard substance by utilizing a Folin-Ciocalteu method and drawing a standard curve. Determination of polyphenol content in samples: and (3) determining the content of polyphenol in different samples by utilizing a Folin-Ciocalteu method, substituting into a standard curve equation, and calculating the polyphenol concentration of the samples.

6. Detection of Polyphenol composition by HPLC

The HPLC detection conditions are as follows:

(1) a chromatographic column: atlantis dC 183 μm 2.1X 150nm Column

(2) Chromatographic conditions are as follows: mobile phase: a: 0.01% formic acid plus 5% acetonitrile in water, B: 0.01% formic acid acetonitrile; flow rate: 0.2 mL/min; detection wavelength: 300 nm; sample introduction amount: 2 mu L of the solution; column oven: 35 ℃; the gradient elution procedure is as follows in table 1:

TABLE 1 gradient elution procedure

Example 2: influence of linoleic acid on Phellinus Linteus biomass accumulation and polyphenol synthesis

1. Variation of mycelium Biomass

Adding different amounts of linoleic acid into a phellinus igniarius liquid culture medium to ensure that the concentrations of the linoleic acid in the culture medium are respectively as follows: 0. 0.5, 1g/L, using the culture medium without linoleic acid as a control. Both 0.5g/L and 1g/L linoleic acid can promote the increase of biomass of the mycelium of the phellinus igniarius, and the mycelium with the concentration of 0.5g/L linoleic acid grows at the fastest speed in the fifth day of growth. With the increase of the culture time, after the seventh day, the 1g/L linoleic acid concentration had a more significant promoting effect, and the biological amount thereof was higher than that of the control group and the concentration of 0.5 g/L. By day 13, when the linoleic acid concentration was 1g/L, the biomass of Phellinus linteus reached 6.9g/L, which was 53.33% higher than that of the control group (FIG. 1).

2. Changes in accumulation of polyphenols in mycelia

In the early stage of growth of phellinus igniarius, linoleic acid showed no promoting effect on the accumulation of intracellular polyphenols. However, the content of intracellular polyphenol in the experiment group added with linoleic acid is gradually higher than that in the control group (no linoleic acid is added) from the 7 th day of growth, the linoleic acid promoting effect at the concentration of 0.5g/L is more obvious, and at the 11 th day, the intracellular polyphenol in the linoleic acid group at the concentration of 0.5g/L reaches 5.37mg/g and is increased by 48.75 percent compared with the intracellular polyphenol at the concentration of 3.61mg/g in the control group (figure 2).

3. Accumulation of the Compound Hispidin

Hispidin is used as a standard substance, and the difference of polyphenol compositions under different culture conditions is detected by an HPLC method. As can be seen from FIG. 3, the increase in Hispidin content was more evident on days 5-7 after treatment with 1g/L linoleic acid, with concentrations of 0.5g/L and 1g/L reaching maximum values on

days

9 and 13, which were 43.08. mu.g/mg and 39.45. mu.g/mg, respectively.

Example 2 Effect of linolenic acid on Phellinus Linteus Biomass accumulation and Polyphenol Synthesis

1. Influence on biomass accumulation

Adding different amounts of linolenic acid into a phellinus igniarius liquid culture medium, so that the concentrations of the linolenic acid in the culture medium are respectively as follows: 0. 0.5g/L and 1g/L, and a culture medium without linolenic acid is taken as a control. Selecting the growth time of 5d, 7d, 11d and 13d according to the growth condition of the mycelia for sampling, taking out 3 groups of samples, filtering and separating fermentation liquor and the mycelia, fully drying, weighing the dry weight of the mycelia, recording, and finishing to obtain the graph 4. As can be seen from FIG. 4, the mycelium biomass was in a steady increase, and increased with the addition of 0.5g/L and 1g/L linolenic acid, compared to the control without linolenic acid. And on day 13, the dry weight of the mycelium reaches the highest under each concentration, the control group is 3.99g/L, the 0.5g/L linolenic acid experimental group is 4.76g/L, and the 1g/L linolenic acid experimental group is 5.16g/L, wherein the experimental group added with 1g/L linolenic acid has the best growth promoting effect, and the dry weight is increased by 1.17g/L compared with the dry weight of the control group.

2. Changes in accumulation of polyphenols

By day 5, the total polyphenol content was highest in the three groups of samples at 0.5 g/L. The data from day 7 to 14 show that the effect of the stimulation is most pronounced in the test group with 1g/L linolenic acid added, whereas the content increases after stimulation with 0.5g/L linolenic acid compared to the control group, although the content increases less than 1 g/L. Finally, the maximum was reached on day 13 for each group and the total polyphenol content was about the same for both groups, 0.5g/L and 1g/L linolenic acid addition, which was increased by 0.46mg/g compared to the control group at that time (FIG. 5).

3. Accumulation of the Compound Hispidin

FIG. 6 shows that the content of Hispidin in the linolenic acid-added experimental group is higher than that in the control group from day 9, and the content of Hispidin in the linolenic acid-added experimental group at 0.5g/L is highest at day 13 and reaches 8.17. mu.g/mg.

Claims

1. The method for increasing accumulation of intracellular polyphenol of phellinus linteus by using unsaturated fatty acid is characterized by comprising the following steps: adding unsaturated fatty acid into Phellinus linteus culture medium.

2. The method of claim 1, wherein the culture medium is a liquid culture medium.

3. The method according to claim 2, wherein the unsaturated fatty acid is added at a ratio of 0.5g/L to 1 g/L.

4. The method according to claim 1, characterized in that the unsaturated fatty acid is linoleic acid and/or linolenic acid.

5. The method according to claim 1, wherein the culturing of Phellinus linteus specifically comprises:

inoculating a solid cultured phellinus igniarius strain into a liquid culture medium, performing activated culture, then inoculating the phellinus igniarius strain into a seed fermentation culture medium according to the inoculation amount of 10 percent of the volume percentage, performing seed culture, and performing shake cultivation for a period of time at a constant temperature to obtain a seed solution; inoculating the seed liquid into a liquid fermentation culture medium according to the inoculation amount of 5 percent by volume, adding unsaturated fatty acid to ensure that the concentration of the unsaturated fatty acid in the fermentation liquid is 0.5-1 g/L, carrying out shake culture at constant temperature for a period of time, collecting the fermentation culture, and filtering to obtain the mycelium.

6. The method of claim 5, wherein the rotation speed of the constant temperature shaking culture is 150 rpm, and the culture temperature is 26 ℃.

7. The method according to claim 5, wherein the seed medium is YPD medium with a formula of: 10 parts of yeast extract, 20 parts of peptone and 20 parts of glucose, and fully stirring until the yeast extract, the peptone and the glucose are dissolved.

8. The method of claim 5, wherein the liquid fermentation medium is formulated as: weighing MgSO₄0.5 part of (NH)₄)₂SO₄0.2 part of glucose, 20 parts of CaCl₂0.1 part of yeast extract, 10 parts of KH₂PO₄1 part of the mixture is put into a container, deionized water is added, the mixture is fully stirred until the mixture is dissolved, and the pH value is adjusted to 5.5.