CN111662862A - Application of edible vegetable oil in improving content of poria triterpene in poria mycelium cells - Google Patents

Abstract

The invention discloses an application of edible vegetable oil, which is characterized in that it is applied to increase the content of Poria cocos triterpenes in the mycelium of Poria cocos. In the present invention, edible vegetable oil is added to the culture medium of the fully grown Poria mycelium, which can significantly increase the content of the Poria cocos triterpenes in the cells of the Poria mycelium. The optimal solution can even increase the content of Poria cocos triterpenes by more than 4 times. Edible vegetable oil is safe to eat, and triterpenoid synthesis can be induced in trace amounts. In the present invention, the edible vegetable oil is used as the inducer to realize the high-level expression of the intracellular triterpenoid content of Poria cocos.

Description

Application of edible vegetable oil in increasing the content of Poria cocos triterpenoids in the mycelium of Poria cocos

technical field

The invention belongs to the field of biotechnology, and more particularly, relates to the application of edible vegetable oil in increasing the content of Poria cocos triterpenes in the mycelium of Poria cocos.

Background technique

Poria (Poria cocos (Schw.) Wolf) is a large medicinal fungus in the phylum of fungi, Basidiomycetes, Polyporia, Polyporaceae, Poria cocos (Poria), and has been used in my country for thousands of years. Modern science has confirmed that the chemical constituents of Poria cocos are polysaccharides, triterpenes, sterols, alkaloids, amino acids, etc. One of the most important medicinal components is Poria triterpenes, which have anti-tumor, liver-protecting, regulating immunity, etc. Increasing the triterpenoid content of Poria cocos plays an important role in the wide application of Poria cocos.

Current studies have shown that methyl jasmonate, acetic acid, phenobarbital, miconazole, aspirin, copper ions, manganese ions, etc. can induce the synthesis of tuckahoe triterpenes. These works provide good materials and phenotypes for studying the regulatory mechanism of triterpenoid biosynthesis in Poria cocos. However, considering that Poria cocos is a kind of traditional Chinese medicine with homology of medicine and food, if we want to promote its widespread use in the medical field, we must consider the safety and economy of fermentation. Methyl jasmonate, as a plant hormone, has not been practically used due to potential health and safety risks; phenobarbital and miconazole are central nervous system depressants, and aspirin is an antipyretic and analgesic. And have adverse effects on healthy people; copper ions and manganese ions are heavy metals, which may cause human poisoning. Therefore, it is necessary to develop more ideal methods, conditions and inducers for liquid fermentation of Poria cocos to increase the content of triterpenoids in the mycelium stage of Poria cocos.

SUMMARY OF THE INVENTION

In view of the above defects or improvement needs of the prior art, the present invention provides an application of edible vegetable oil in increasing the content of Poria cocos triterpenoids in the mycelium of Poria cocos, the purpose of which is to use edible vegetable oil with food safety level as an inducing liquid fermentation The inducer for increasing the content of tuckahoe triterpenes in the tuckahoe mycelium package can obtain the obvious effect of inducing the expression of tuckahoe triterpenes by adding a low dose, thereby solving the existing inducers for increasing the content of tuckahoe triterpenes in the tuckahoe mycelium. Residual, there are technical problems of potential safety hazards.

In order to achieve the above object, according to one aspect of the present invention, an application of edible vegetable oil is provided, which is applied to increase the intracellular content of Poria cocos triterpenes in the mycelium of Poria cocos.

Preferably, the application of the edible vegetable oil is to increase the intracellular content of Poria cocos triterpenes in liquid fermentation of Poria mycelium.

Preferably, in the application of the edible vegetable oil, the iodine value of the edible vegetable oil is above 50g/100g, preferably 50 to 110g/100g; the saponification value of the edible vegetable oil is above 180mgKOH/g, preferably 180 to 220mgKOH/ g; the edible vegetable oil is preferably olive oil, peanut oil, rapeseed oil, palm oil, soybean oil, and/or coix seed oil, more preferably olive oil.

Preferably, in the application of the edible vegetable oil, the liquid fermentation of the Poria mycelium includes a growth stage and an induction stage;

In the growth stage, it is fully grown until the mycelium of Poria cocos is flocculent, and the mycelium is vigorous;

In the induction stage, edible vegetable oil is added to the fermentation broth to induce the Poria mycelium to increase and increase the intracellular content of Poria cocos triterpenes.

Preferably, in the application of the edible vegetable oil, in the induction stage, 1% to 4% of the edible vegetable oil is added according to the volume ratio.

Preferably, in the application of the edible vegetable oil, edible vegetable oil is added between the start of the induction phase and 96 hours, the fermentation temperature is 28-30° C., the rotation speed is 120-150 r/min, the fermentation time is 4 to 12 days, the mycelium is Body density increases and fermentation ends before the mycelium browns.

Preferably, in the application of the edible vegetable oil, in the growth stage, the liquid strain of Poria cocos is inoculated in the culture medium, and cultured for 5 to 6 days at a temperature of 28-30° C. and a rotation speed of 120-150 r/min.

Preferably, in the application of the edible vegetable oil, in the growth stage, the liquid strain of Poria cocos is inoculated in the medium according to 4% to 8% of the volume of the medium.

Preferably, the application of described edible vegetable oil, its described Poria liquid strain is prepared according to the following method:

(1) Activating the Poria cocos mycelium with a PDA liquid medium, at a temperature of 28-30° C. and a rotating speed of 120-150 r/min, for 6 to 7 days, to obtain the activated Poria cocos mycelium;

(2) Inoculate the Poria cocos mycelium obtained in step (1) into the Poria cocos seed medium according to 10% of the culture volume of the Poria seeds, at a temperature of 28-30° C., at a rotational speed of 120-150 r/min, and culture for 5- On the 6th day, the Poria cocos liquid strain was obtained.

Preferably, the application of the edible vegetable oil, the Poria cocos seed medium is a PDA medium, prepared according to the following method:

Cut 200 grams of potatoes into small pieces, boil for 20-30 minutes, filter with eight layers of gauze, add 20 grams of glucose to the filtrate, dilute to 1 liter, and sterilize with high-pressure steam at 121°C for 20-25 minutes.

Preferably, the application of the edible vegetable oil, the medium of its described growth stage is CYM medium, and is prepared according to the following method:

Add 35 grams of glucose, 5 grams of peptone, 5 grams of yeast powder, 0.883 grams of potassium dihydrogen phosphate, and 0.5 grams of magnesium sulfate heptahydrate, dilute to 1 liter with distilled water, and sterilize with high-pressure steam at 121°C for 20-25 minutes.

In general, compared with the prior art, the above technical solutions conceived by the present invention can achieve the following beneficial effects:

In the present invention, edible vegetable oil is added to the culture medium of the fully grown Poria mycelium, which can significantly increase the content of the Poria cocos triterpenes in the cells of the Poria mycelium. The optimal solution can even increase the content of Poria cocos triterpenes by more than 4 times. Edible vegetable oil is safe to eat, especially olive oil can induce triterpenoid synthesis in trace amounts. In the present invention, the edible vegetable oil is used as the inducer to realize the high-level expression of the intracellular triterpenoid content of Poria cocos. The use of edible vegetable oil as an inducer is safe and efficient, and triterpenoid synthesis can be induced in a trace amount, and it is safe to eat, and can significantly increase the intracellular triterpenoid content of Poria cocos.

Description of drawings

Fig. 1 is the result diagram that application different kinds of edible vegetable oil provided by embodiment 1 improves the content of tuckahoe triterpenes in the tuckahoe mycelium bag;

Fig. 2 is the application different concentration olive oil that embodiment 2 provides improves Poria cocos mycelium intracellular Poria triterpenoid content result figure;

Fig. 3 is that the application olive oil that embodiment 3 provides improves Poria cocos mycelium intracellular Poria triterpenoid content result graph at different treatment times;

Fig. 4 is the application olive oil that embodiment 4 provides improves the Poria cocos triterpenoid content result figure in different fermentation time lengths;

Fig. 5 is the result diagram of the content of tuckahoe triterpenes fermented by tuckahoe mycelium under different inoculation amounts provided by the comparative example.

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, but not to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

The application of the edible vegetable oil provided by the present invention is applied to increase the intracellular content of Poria cocos triterpenes in the mycelium of Poria cocos, and preferably is applied to increase the content of intracellular triterpenes of Poria cocos mycelium by liquid fermentation; the specific steps are as follows:

In the growth stage, fully grow until the mycelium of Poria cocos is flocculent distribution, and the mycelium is vigorous; inoculate the liquid strain of Poria cocos in the medium, the inoculation amount is preferably 4% to 8% of the volume of the medium, and the temperature is 28-30 ℃. , preferably at 28°C, rotating speed 120r/min-150r/min, cultured for 5-6 days, preferably cultured for 5 days. The medium of described growth stage is preferably CYM medium, and is prepared according to the following method:

Add 35 grams of glucose, 5 grams of peptone, 5 grams of yeast powder, 0.883 grams of potassium dihydrogen phosphate, and 0.5 grams of magnesium sulfate heptahydrate, dilute to 1 liter with distilled water, and sterilize with high-pressure steam at 121°C for 20-25 minutes.

Described Poria liquid strain is prepared according to the following method:

(1) Activating the Poria cocos mycelium with the PDA liquid medium, at a temperature of 28-30 ° C and a rotating speed of 120-150 r/min, for 6-7 days, preferably for 7 days, to obtain the activated Poria mycelium;

(2) Inoculate the Poria cocos mycelium obtained in step (1) into the Poria cocos seed medium according to 10% of the culture volume of the Poria seeds, at a temperature of 28-30° C., at a rotational speed of 120-150 r/min, and culture for 5- 6 days, preferably 5 days, to obtain Poria liquid strain. Described Poria seed culture medium is PDA culture medium, prepared according to the following method: cut 200 grams of potato into small pieces, boil for 20-30 minutes, filter with eight layers of gauze, add 20 grams of glucose to the filtrate, set the volume to 1 liter, pressurize Sterilize with steam at 121°C for 20-25 minutes.

In the induction stage, edible vegetable oil was added to the fermentation broth to induce the mycelium of Poria cocos to increase the intracellular triterpenoid content of Poria cocos. In the induction stage, the edible vegetable oil is added according to the mass ratio of 1% to 4%, preferably the mass ratio is 2%; the edible vegetable oil is added between the beginning and 96 hours, and the edible vegetable oil is preferably added at the beginning of the induction stage, and the fermentation temperature is 28-30 Between °C, preferably 28 °C, rotating speed of 120-150r/min, preferably 150r/min, fermentation time of 4 to 12 days, the density of mycelium increases, and the fermentation ends before the browning of the mycelium.

Edible vegetable oil is safe to eat, and triterpenoid synthesis can be induced in trace amounts. In the present invention, the edible vegetable oil is used as the inducer to realize the high-level expression of the intracellular triterpenoid content of Poria cocos. The iodine value of the edible vegetable oil is above 50g/100g, preferably 50 to 110g/100g; the saponification value of the edible vegetable oil is above 180mgKOH/g, preferably 180 to 220mgKOH/g; the edible vegetable oil is preferably olive oil, Peanut oil, rapeseed oil, palm oil, soybean oil, and/or coix seed oil, more preferably olive oil. The iodine value of oil indicates its degree of unsaturation, and the saponification value is related to the molecular weight of the oil, both of which affect the participation of oil in the intracellular secondary metabolism of Poria cocos mycelium. We found that edible vegetable oils with suitable saponification value and iodine value showed good performance. Induction of triterpenoid synthesis in the mycelial envelope of Poria cocos.

The following are examples:

Example 1

Preparation of Poria liquid strain:

(1) Poria cocos mycelium was activated with PDA liquid medium, at a temperature of 28-30° C. and a rotation speed of 120-150 r/min, for 7 days.

(2) Poria cocos seed medium was inoculated with 10% (v:v) of Poria cocos mycelium in (1), and cultured for 5 days at a temperature of 28-30° C. and a rotation speed of 120-150 r/min.

Growth stage culture:

Inoculate the Poria cocos liquid strain (2) into the CYM medium, the seed liquid inoculum amount is 1%, at a temperature of 28-30° C., a rotation speed of 120-150 r/min, and cultured for 3 days, the first stage of fermentation is carried out to make the Poria mycelium. Rapid growth and reproduction.

Induction culture stage:

After the fermentation of the growth segment, under sterile conditions, 1% (v/v) of peanut oil, rapeseed oil, palm oil, soybean oil, olive oil, and coix seed oil were added to the 6 edible vegetable oils as the treatment group (inoculation). amount of 1%, fermentation for 6 days), control (no oil, inoculum size of 1%, fermentation for 6 days). The second-stage fermentation continued with three replicate replicates for each treatment. The iodine value of 6 kinds of vegetable oils was determined by Webster's method, and the saponification value of 6 kinds of plants was determined according to "Determination of Saponification Value of Animal and Vegetable Oils and Fats (GB/T 5534-2008)".

After the fermentation, the mycelium balls were collected with a Buchner funnel, the fermentation broth was centrifuged at 3000 r/min for 3 min, the bacterial cells and the fermentation broth were separated, the vegetable oil suspended in the upper layer was removed, and then four layers of medical gauze were used on the Buchner funnel to filter, Repeat washing with deionized water for three times, and then perform centrifugation and sedimentation for 3 min again, separate the mycelium with Buchner funnel again, and repeatedly wash with deionized water until it is colorless. Weight, weighing is the biomass, and the content of Poria cocos triterpenes was determined by spectrophotometry. Take 0.05 g of dried mycelium powder, add 5 mL of 50% (v/v) ethanol, and extract by ultrasonic for 2 h (repeat once), each time with 5 mL of 50% (v/v) ethanol. After centrifugation, the supernatant was obtained, and the two supernatants were combined with a total volume of 10 mL, placed in a rotary evaporator, and evaporated to dryness at 50°C. The residue after evaporation to dryness was resuspended with 3 mL of ddH2O, and extracted twice with 5 mL of chloroform; the chloroform layers were combined, the total volume was 10 mL, dried at 45°C under vacuum, and dissolved in 3 mL of methanol. The test results are shown in Figure 1. Compared with the comparative example, the content increased significantly, and the percentage increase in the content of Poria triterpenes is shown in Table 1.

Table 6 The effects of adding different oils and fats in the second stage after optimization on the triterpenes of Poria cocos

Example 2

Preparation of Poria liquid strain:

(1) Poria cocos mycelium was activated with PDA liquid medium, at a temperature of 28-30° C. and a rotation speed of 120-150 r/min, for 7 days.

(2) Poria cocos seed medium was inoculated with 10% (v:v) of Poria cocos mycelium in (1), and cultured for 5 days at a temperature of 28-30° C. and a rotation speed of 120-150 r/min.

Growth stage culture:

Inoculate the liquid strain of Poria cocos into the CYM medium, the seed liquid inoculation amount is 1%, cultivate for 3 days at a temperature of 28-30 ° C and a rotation speed of 120-150 r/min, and carry out fermentation in the growth stage, so that the mycelium of Poria cocos grows rapidly. .

Induction phase culture:

After the fermentation of the growth stage, olive oil with final concentrations of 1%, 2%, 3%, and 4% was added to the fermentation broth under aseptic conditions to continue the second-stage fermentation, with three replicates for each treatment. ; Temperature 28-30 ℃, rotating speed 120-150r/min, cultured for 6 days, the density of mycelium increased, and the fermentation was finished.

After the fermentation, the mycelium balls were collected with a Buchner funnel, the fermentation broth was centrifuged at 3000 r/min for 3 min, the bacterial cells and the fermentation broth were separated, the vegetable oil suspended in the upper layer was removed, and then four layers of medical gauze were used on the Buchner funnel to filter, Repeat washing with deionized water three times, and then perform centrifugation and sedimentation for 3 min again, separate the mycelium with Buchner funnel again, and repeatedly wash it with deionized water until it becomes colorless. Weight, weighing is the biomass, and the content of Poria cocos triterpenes was determined by spectrophotometry. Take 0.05 g of dried mycelium powder, add 5 mL of 50% (v/v) ethanol, and extract by ultrasonic for 2 h (repeat once), each time with 5 mL of 50% (v/v) ethanol. After centrifugation, the supernatant was obtained, and the two supernatants were combined with a total volume of 10 mL, placed in a rotary evaporator, and evaporated to dryness at 50°C. The residue after evaporation to dryness was resuspended with 3 mL of ddH ₂ O, and extracted twice with 5 mL of chloroform; the chloroform layers were combined, the total volume was 10 mL, dried at 45°C under vacuum, and dissolved with 3 mL of methanol. The test results are shown in Figure 2. Compared with the comparative example, the content increased significantly, and the percentage increase in the content of Poria cocos triterpenes is shown in Table 2.

Table 2 Effects of different concentrations of olive oil on the growth and triterpenoid biosynthesis of Poria cocos in liquid fermentation

Induction phase olive oil concentration % Poria triterpenoid content mg/100ml The content of tuckahoe triterpenes increased by % 0 18.03 - 1 34.06 88.85 2 42.01 132.95 3 41.58 130.55 4 31.28 73.48

Example 3

Preparation of Poria liquid strain:

(1) Poria cocos mycelium was activated with PDA liquid medium, at a temperature of 28-30° C. and a rotation speed of 120-150 r/min, for 7 days.

(2) Poria cocos seed medium was inoculated with 10% (v:v) of Poria cocos mycelium in (1), and cultured for 5 days at a temperature of 28-30° C. and a rotation speed of 120-150 r/min.

Growth stage culture:

Inoculate the Poria cocos liquid strain (2) into the CYM medium, the seed liquid inoculation amount is 1%, the temperature is 28-30°C, the rotation speed is 120-150r/min, and the culture is carried out for 3 days, and the first stage fermentation is carried out to make the Poria mycelium. Rapid growth and reproduction.

Induction phase culture:

After the fermentation in the growth stage, under aseptic conditions, the 0h, 24h, 48h, 72h, and 96h of the liquid fermentation started as the time points for adding 2% olive oil. Continue to the second stage of fermentation with three replicates for each treatment; temperature at 28-30°C, rotation speed at 120-150r/min, culture for 6 days, the density of mycelium increases, and the fermentation ends.

After the fermentation, the mycelium balls were collected with a Buchner funnel, the fermentation broth was centrifuged at 3000 r/min for 3 min, the bacterial cells and the fermentation broth were separated, the vegetable oil suspended in the upper layer was removed, and then four layers of medical gauze were used on the Buchner funnel to filter, Repeat washing with deionized water for three times, and then perform centrifugation and sedimentation for 3 min again, separate the mycelium with Buchner funnel again, and repeatedly wash with deionized water until it is colorless. Weight, weighing is the biomass, and the content of Poria cocos triterpenes was determined by spectrophotometry. Take 0.05 g of dried mycelium powder, add 5 mL of 50% (v/v) ethanol, and extract by ultrasonic for 2 h (repeat once), each time with 5 mL of 50% (v/v) ethanol. After centrifugation, the supernatant was obtained, and the two supernatants were combined with a total volume of 10 mL, placed in a rotary evaporator, and evaporated to dryness at 50°C. The residue after evaporation to dryness was resuspended with 3 mL of ddH2O, and extracted twice with 5 mL of chloroform; the chloroform layers were combined, the total volume was 10 mL, dried at 45°C under vacuum, and dissolved in 3 mL of methanol. The test results are shown in Figure 3. Compared with the comparative example, the content increased significantly, and the percentage increase in the content of Poria cocos triterpenes is shown in Table 3.

Table 3 Effects of different olive oil addition times on the biosynthesis of Poria cocos in the second stage

olive oil addition time hr Poria triterpenoid content mg/100mL The content of tuckahoe triterpenes increased by % - 16.38 - 0 40.94 149.99 twenty four 36.71 124.17 48 31.30 91.12 72 29.58 80.63 96 28.61 74.72

Example 4

Preparation of Poria liquid strain:

(1) Poria cocos mycelium was activated with PDA liquid medium, at a temperature of 28-30° C. and a rotation speed of 120-150 r/min, for 7 days.

(2) Poria cocos seed medium was inoculated with 10% (v:v) of Poria cocos mycelium in (1), and cultured for 5 days at a temperature of 28-30° C. and a rotation speed of 120-150 r/min.

Growth stage culture:

The Poria cocos liquid strain (2) is inoculated into the CYM medium, cultured for 3 days at a temperature of 28-30° C. and a rotation speed of 120-150 r/min, and the first stage of fermentation is carried out, so that the Poria mycelium rapidly grows and propagates.

Induction phase culture:

After the fermentation of the growth stage, under aseptic conditions, two groups of treatments are set, and one group is to add olive oil 2%, and the 0th day of fermentation is added, and the seed liquid inoculum is 8%; one group is the treatment of not adding oil as a contrast ( That is, no olive oil is added, and the seed liquid inoculum is 8%). Six fermentation time gradients were set on days 2, 4, 6, 8, 10, and 12. The second-stage fermentation continued with three replicate replicates for each treatment.

After the fermentation, the mycelium balls were collected with a Buchner funnel, the fermentation broth was centrifuged at 3000 r/min for 3 min, the bacterial cells and the fermentation broth were separated, the vegetable oil suspended in the upper layer was removed, and then four layers of medical gauze were used on the Buchner funnel to filter, Repeat washing with deionized water for three times, and then perform centrifugation and sedimentation for 3 min again, separate the mycelium with Buchner funnel again, and repeatedly wash with deionized water until it is colorless. Weight, weighing is the biomass, and the content of Poria cocos triterpenes was determined by spectrophotometry. Take 0.05 g of dried mycelium powder, add 5 mL of 50% (v/v) ethanol, and extract by ultrasonic for 2 h (repeat once), each time with 5 mL of 50% (v/v) ethanol. After centrifugation, the supernatant was obtained, and the two supernatants were combined with a total volume of 10 mL, placed in a rotary evaporator, and evaporated to dryness at 50°C. The residue after evaporation to dryness was resuspended with 3 mL of ddH2O, and extracted twice with 5 mL of chloroform; the chloroform layers were combined, the total volume was 10 mL, dried at 45°C under vacuum, and dissolved in 3 mL of methanol. The test results are shown in Figure 4. Under the same 8% inoculation amount, the content of olive oil was significantly increased compared with that of the comparative example, and the percentage of increase in the content of Poria triterpenes is shown in Table 4.

Table 4 The process of optimizing the process of inducing the increase in the yield of Poria cocos triterpenes by liquid fermentation with olive oil added in the second stage

Example 6

Preparation of Poria liquid strain:

(1) Poria cocos mycelium was activated with PDA liquid medium, at a temperature of 28-30° C. and a rotation speed of 120-150 r/min, for 7 days.

(2) Poria cocos seed medium was inoculated with 10% (v:v) of Poria cocos mycelium in (1), and cultured for 5 days at a temperature of 28-30° C. and a rotation speed of 120-150 r/min.

Growth stage culture:

The Poria cocos liquid strain (2) is inoculated into the CYM medium, cultured for 3 days at a temperature of 28-30° C. and a rotation speed of 120-150 r/min, and the first stage of fermentation is carried out, so that the Poria mycelium rapidly grows and propagates.

Induction culture stage:

After the fermentation of the growth segment, under sterile conditions, the optimized group (the olive oil addition time was the 0th hour of fermentation, and the seed liquid inoculum was 8%; the fermentation time was 8 days), the control (no oil, the seed liquid inoculum was 1%) , fermented for 6 days). The second-stage fermentation continued with three replicate replicates for each treatment.

After the fermentation, the mycelium balls were collected with a Buchner funnel, the fermentation broth was centrifuged at 3000 r/min for 3 min, the bacterial cells and the fermentation broth were separated, the vegetable oil suspended in the upper layer was removed, and then four layers of medical gauze were used on the Buchner funnel to filter, Repeat washing with deionized water for three times, and then perform centrifugation and sedimentation for 3 min again, separate the mycelium with Buchner funnel again, and repeatedly wash with deionized water until it is colorless. Weight, weighing is the biomass, and the content of Poria cocos triterpenes was determined by spectrophotometry. Take 0.05 g of dried mycelium powder, add 5 mL of 50% (v/v) ethanol, and extract by ultrasonic for 2 h (repeat once), each time with 5 mL of 50% (v/v) ethanol. After centrifugation, the supernatant was obtained, and the two supernatants were combined with a total volume of 10 mL, placed in a rotary evaporator, and evaporated to dryness at 50°C. The residue after evaporation to dryness was resuspended with 3 mL of ddH2O, and extracted twice with 5 mL of chloroform; the chloroform layers were combined, the total volume was 10 mL, dried at 45°C under vacuum, and dissolved in 3 mL of methanol. Compared with the comparative example, the content increased significantly, and the percentage increase in the content of Poria cocos triterpenes is shown in Table 5.

Table 5 The effect of adding olive oil on the triterpenes of Poria after optimization in the second stage

Comparative Example 4

Preparation of Poria liquid strain:

(1) Poria cocos mycelium was activated with PDA liquid medium, at a temperature of 28-30° C. and a rotation speed of 120-150 r/min, for 7 days.

(2) Poria cocos seed medium was inoculated with 10% (v:v) of Poria cocos mycelium in (1), and cultured for 5 days at a temperature of 28-30° C. and a rotation speed of 120-150 r/min.

Growth and culture stage:

Inoculate the Poria cocos liquid strain (2) into the CYM medium, determine the optimal inoculum amount in the fermentation medium (without oil), set 1%, 2%, 4%, 6%, 8%, 10% (v/v ) a total of 6 seed solution inoculum gradients. Proceed to second stage fermentation with three replicates for each treatment. The temperature is 28-30°C, the rotation speed is 120-150r/min, and the culture is carried out for 3 days, and the first stage fermentation is carried out, so that the Poria mycelium grows rapidly. Continue to carry out the second stage fermentation, the temperature is 28-30 ℃, the rotation speed is 120-150 r/min, and the culture is cultured for 6 days, the density of mycelium increases, and the fermentation is ended.

After the fermentation, collect the mycelial balls with a Buchner funnel, centrifuge the fermentation liquid at 3000 r/min for 3 min, separate the bacteria and the fermentation liquid, filter with four layers of medical gauze on the Buchner funnel, and repeatedly wash with deionized water three times. , carry out a centrifugal sedimentation for 3 min, separate the mycelium with a Buchner funnel again, wash it with deionized water until it is colorless, and dry the obtained mycelium in an oven at 60 °C to a constant weight, and the weighing is the biomass , Determination of the triterpenoid content of Poria cocos by spectrophotometry. Take 0.05 g of dried mycelium powder, add 5 mL of 50% (v/v) ethanol, and extract by ultrasonic for 2 h (repeat once), each time with 5 mL of 50% (v/v) ethanol. After centrifugation, the supernatant was obtained, and the two supernatants were combined with a total volume of 10 mL, placed in a rotary evaporator, and evaporated to dryness at 50°C. The residue after evaporation to dryness was resuspended with 3 mL of ddH2O, and extracted twice with 5 mL of chloroform; the chloroform layers were combined with a total volume of 10 mL, dried at 45°C under vacuum, and dissolved in 3 mL of methanol. The test results are shown in Figure 5. The inoculum amount increased most obviously at 8%, and the percentage increase of the triterpenoid content of Poria cocos is shown in Table 6.

Table 6 Influence of inoculum size on Poria cocos triterpenes

Second stage bacterial solution inoculum % Poria triterpenoid content mg/100mL 1 18.03 2 17.65 4 20.34 6 22.47 8 24.12 10 22.34

As can be seen from Table 6, with the increase of inoculation amount, the content of Poria cocos triterpenes increased, but even with a large amount of inoculation (the inoculation amount was 8%), the content of Poria triterpenes was still at a low level. In the example of adding the inducer, even if the inoculation amount is only 1%, the content of Poria cocos triterpenes is still significantly increased.

Those skilled in the art can easily understand that the above are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention, etc., All should be included within the protection scope of the present invention.

Claims (10)

1. an application of edible vegetable oil, is characterized in that, is applied to improve Poria cocos triterpenoid content in mycelium of Poria cocos. 2. the application of edible vegetable oil as claimed in claim 1 is characterized in that, is applied to improve the intracellular Poria triterpenoid content of Poria mycelium liquid fermentation. 3. the application of the edible vegetable oil as described in any one of claim 1 or 2, it is characterized in that, described edible vegetable oil iodine value is more than 50g/100g, is preferably 50 to 110g/100g; The saponification value of described edible vegetable oil Above 180 mgKOH/g, preferably 180 to 220 mgKOH/g; the edible vegetable oil is preferably olive oil, peanut oil, rapeseed oil, palm oil, soybean oil, and/or coix seed oil, more preferably olive oil. 4. the application of edible vegetable oil as described in any one of claim 1 to 3, is characterized in that, described Poria mycelium liquid fermentation comprises growth stage and induction stage; In the growth stage, it is fully grown until the Poria mycelium is flocculent distributed; In the induction stage, edible vegetable oil is added to the fermentation broth to induce the Poria mycelium to increase and increase the intracellular content of Poria cocos triterpenes. 5. The application of edible vegetable oil according to claim 4, characterized in that, edible vegetable oil is added according to the volume ratio of 1% to 4% in the induction stage. 6. the application of edible vegetable oil as claimed in claim 4, is characterized in that, adds edible vegetable oil between described induction phase beginning to 96 hours, fermentation temperature is 28-30 ℃, rotating speed 120-150r/min, until bacteria The mycelium density increases and the fermentation ends before the mycelium browns. 7. the application of edible vegetable oil as claimed in claim 4 is characterized in that, described growth stage inoculates Poria liquid strain in culture medium, under temperature 28-30 ℃, rotating speed 120-150r/min, cultivates 5 to 6 days. 8. The application of edible vegetable oil as claimed in claim 4, characterized in that, in the growth stage, the liquid strain of Poria cocos is inoculated in the culture medium according to 4% to 8% of the volume of the culture medium. 9. the application of edible vegetable oil as claimed in claim 8, is characterized in that, described Poria liquid bacterial classification is prepared according to the following method: (1) Activating the Poria cocos mycelium with a PDA liquid medium, at a temperature of 28-30° C. and a rotating speed of 120-150 r/min, for 6 to 7 days, to obtain the activated Poria cocos mycelium; (2) Inoculate the activated Poria cocos mycelia obtained in step (1) into the Poria cocos seed medium according to 10% of the culture volume of the Poria seeds, at a temperature of 28-30° C., at a rotating speed of 120-150 r/min, for 5 to On the 6th day, the Poria cocos liquid strain was obtained. 10. the application of edible vegetable oil as claimed in claim 9, is characterized in that, described Poria seed medium is PDA medium, prepared according to the following method: 200 grams of potatoes are cut into small pieces, boiled 20-30 minutes, take eight Filter through layers of gauze, add 20 grams of glucose to the filtrate, dilute to 1 liter, and sterilize with high-pressure steam at 121°C for 20-25 minutes.

Images