Method for increasing lentinan content by adding codonopsis pilosula extract

Abstract

The invention discloses a method for increasing the content of lentinan by adding a codonopsis pilosula extract, which comprises the following steps: the codonopsis pilosula extract is added into a liquid culture medium during liquid fermentation of lentinus edodes mycelia, and the concentration of the codonopsis pilosula extract in the liquid culture medium is 10-50g / L. The inoculation amount of the mushroom mycelia during liquid fermentation of the mushroom mycelia is 4-6%, and the mushroom mycelia are cultured in a solid culture medium for 6-10 days before inoculation, or the mushroom mycelia are cultured in a solid culture medium with the concentration of the codonopsis pilosula extract being 10-50g / L for 5-6 days before inoculation. According to the application, the codonopsis pilosula extract is added in the liquid fermentation of the mushroom mycelium, so that the growth of the mushroom mycelium can be promoted, and the polysaccharide content of the mushroom mycelium is remarkably increased; the combined configuration of mushroom mycelium solid culture medium culture and liquid fermentation can improve the productivity of liquid fermentation and the production efficiency of lentinan; the fermented product can be used for preparing functional lentinan, and an effective way is provided for large-scale preparation of functional lentinan.

Description

Technical Field

[0001] The present application relates to the technical field of polysaccharide preparation, and in particular to a method for increasing the content of Lentinan polysaccharide by adding Codonopsis pilosula extract. Background Art

[0002] Codonopsis pilosula is one of the traditional tonic medicines commonly used in China. Its chemical composition types include sugars, alkynes, phenylpropanoids, alkaloids, triterpenes, steroids, flavonoids, volatile oils, organic acids, etc. It has the effects of enhancing immunity, protecting gastrointestinal mucosa, promoting hematopoiesis, regulating blood sugar, anti-fatigue, chronic obstructive pulmonary disease, anti-tumor, and anti-aging. The main active ingredient of shiitake mushrooms is shiitake polysaccharide, which is the main functional ingredient of shiitake mushrooms to exert various pharmacological activities. Shiitake polysaccharide has the effects of antibacterial, enhancing immunity, promoting appetite, invigorating qi, strengthening spleen, anti-tumor, anti-inflammatory, anti-viral, scavenging free radicals and reducing liver damage. It has been widely studied and applied in the fields of biochemistry and medicine. Shiitake mushrooms are mostly cultivated using fruiting bodies, but the application of liquid deep fermentation technology in fungal seed production, expanded culture, and comprehensive utilization is becoming more and more extensive, and the liquid deep fermentation technology of shiitake mushrooms is gradually maturing. Liquid fermentation of shiitake mushrooms does not require wooden culture media, has a short culture cycle, and produces a large amount of intracellular and extracellular polysaccharides during the fermentation process, which not only reduces the cost of preparing shiitake mushroom polysaccharides, but also reduces damage to the environment.

[0003] There is no report on using Codonopsis pilosula extract to prepare functional lentinan from deep fermentation of shiitake mushrooms. Therefore, providing a method for increasing the content of lentinan by adding Codonopsis pilosula extract, fermenting shiitake mycelium with Codonopsis pilosula extract, thereby increasing the content of lentinan and preparing functional lentinan has become a technical problem that needs to be solved urgently by those skilled in the art. Summary of the invention

[0004] The purpose of the present application is to provide a method for increasing the content of lentinan by adding codonopsis extract, by adding codonopsis extract during the liquid fermentation of lentinan mycelium to increase the content of lentinan and production efficiency.

[0005] A method for increasing the content of lentinan by adding codonopsis extract comprises: adding codonopsis extract to a liquid culture medium during liquid fermentation of lentinan mycelium, wherein the concentration of codonopsis extract in the liquid culture medium is 10-50 g / L.

[0006] Furthermore, the concentration of Codonopsis pilosula extract in the liquid culture medium is 20 g / L.

[0007] Furthermore, during the liquid fermentation of the shiitake mycelium, the inoculation amount of the shiitake mycelium is 4-6%, and the shiitake mycelium is cultured in a solid culture medium for 6-10 days before inoculation.

[0008] Furthermore, during the liquid fermentation of the shiitake mycelium, the inoculation amount of the shiitake mycelium is 4-6%, and before inoculation, the shiitake mycelium is cultured in a solid culture medium with a concentration of 10-50 g / L of Codonopsis pilosula extract for 5-6 days.

[0009] Furthermore, the liquid fermentation time of the shiitake mushroom mycelium is 4-12 days.

[0010] Furthermore, the liquid fermentation time of the shiitake mushroom mycelium is 8 days.

[0011] Furthermore, the preparation steps of the Codonopsis pilosula extract are as follows:

[0012] After soaking the Codonopsis pilosula powder, the powder is boiled and filtered at 80-100°C, and the filtrate is collected. The above boiling and filtering steps are repeated for the filtered medicinal residue. The two filtrates are combined, concentrated and frozen, and vacuum freeze-dried into powder.

[0013] Furthermore, the particle size of the Codonopsis pilosula extract is no more than 60 meshes.

[0014] Furthermore, the shiitake mycelium and fermentation liquid obtained after the shiitake mycelium liquid fermentation can be used to prepare functional shiitake polysaccharide.

[0015] Beneficial effects: The present application promotes the growth of shiitake mycelium and significantly increases the polysaccharide content of shiitake mycelium by adding codonopsis extract to the liquid fermentation of shiitake mycelium. The productivity of liquid fermentation and the production efficiency of shiitake polysaccharide can be improved by combining solid culture medium culture of shiitake mycelium and liquid fermentation of shiitake mycelium. The liquid fermentation product with codonopsis extract added can be used to prepare functional shiitake polysaccharide, providing an effective way for large-scale preparation of functional polysaccharides. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The growth of Lentinus edodes mycelium in solid culture without adding Codonopsis pilosula extract;

[0017] Figure 2 This is the growth of Lentinus edodes mycelium in solid culture when 20g / L of Codonopsis pilosula extract was added;

[0018] Figure 3 It is the shiitake mycelium fermented in liquid without adding Codonopsis pilosula extract;

[0019] Figure 4 It is shiitake mycelium fermented with 20g / L Codonopsis pilosula extract in liquid. DETAILED DESCRIPTION

[0020] The technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the accompanying drawings of the present application. Obviously, the embodiments described in the present application are only some embodiments of the present application, rather than all embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present application. The following is a specific implementation method of the method for adding Codonopsis pilosula extract to increase the content of Lentinan in the present application. It should be noted that the Lentinus edodes mycelium, Codonopsis pilosula, Astragalus membranaceus and Poria cocos in the present application are all purchased on the market.

[0021] Preparation of Codonopsis pilosula extract

[0022] Crush the Codonopsis pilosula until it can pass through a 60-mesh sieve. Soak the Codonopsis pilosula powder in eight times the volume of distilled water for 30 minutes, then boil it at 80-100℃ for 30 minutes, filter the residue with four layers of gauze, and collect the filtrate. Repeat the above steps to boil and filter the filtered residue, and combine the two filtrates. After concentrating and freezing the two combined filtrates, freeze-dry them in a vacuum freeze dryer for 24 hours into a powdered form, which is the freeze-dried powder of Codonopsis pilosula extract.

[0023] According to the preparation method of Codonopsis pilosula extract, freeze-dried powder of Astragalus membranaceus extract and freeze-dried powder of Poria cocos extract were prepared respectively.

[0024] Solid Culture Medium for Lentinus edodes Mycelium

[0025] Select potatoes, wash and peel them, weigh about 50 g, cut them into small uniform pieces, add 200 mL of distilled water, boil them for 30 minutes, and filter them with four layers of gauze. Add 5 g of glucose and 5 g of agar powder to the filtrate. Prepare 19 copies of the above PDA culture medium. Add dangshen extract freeze-dried powder, dangshen and astragalus extract freeze-dried powder combination (mass ratio of 1:1), dangshen and poria extract freeze-dried powder combination (mass ratio of 1:1), and adjust the volume to the scale to make 0g / L, 10g / L, 20g / L, 30g / L, 40g / L, 50g / L, 60g / L dangshen extract solution, 10g / L, 20g / L, 30g / L, 40g / L, 50g / L, 60g / L dangshen and astragalus combined extract solution, 10g / L, 20g / L, 30g / L, 40g / L, 50g / L, 60g / L dangshen and poria combined extract solution. After sealing, put it into a high-pressure steam sterilizer together with the culture dish for sterilization. Inoculate the mushroom strains on the above solid PDA culture medium in the clean bench, and keep aseptic operation throughout the process. After inoculation, the cells were placed in a biochemical incubator at 28°C and the growth status of the mycelium was recorded.

[0026] The growth of mycelium colonies of Lentinus edodes in solid culture medium is shown in Table 1. It can be seen that the combination of Codonopsis pilosula and Astragalus membranaceus extracts and the combination of Codonopsis pilosula and Poria cocos extracts have no obvious effect on the growth and reproduction of Lentinus edodes mycelium and even have an inhibitory effect. Figure 1 and 2 The growth of mycelium of shiitake mushrooms can be seen from the results that the concentration of Codonopsis pilosula extract solution in the range of 10-50g / L has a promoting effect on the growth of mycelium of shiitake mushrooms, and the concentration of Codonopsis pilosula extract solution at 20g / L has the strongest promoting effect on the growth of mycelium of shiitake mushrooms. The concentration of Codonopsis pilosula extract solution at 60g / L has an inhibitory effect on the growth of mycelium of shiitake mushrooms.

[0027] Table 1 Colony morphology of Lentinus edodes when cultured on solid medium

[0028]

[0029]

[0030]

[0031] Example

[0032] Preparation of liquid culture medium for Lentinus edodes mycelium: Take 200g peeled potatoes, cut them into pieces, add 1000mL distilled water, boil them for 30min, filter them with four layers of gauze, add glucose, yeast extract, KH 2 PO 4 MgSO 4 , NaOH solution, Codonopsis pilosula extract, fixed volume to 1000mL, pH 6, 20g / L glucose, 5g / L yeast extract, 2g / L KH 2 PO 4 , 1g / L MgSO 4 The above solution was prepared into 6 portions, and the concentrations of Codonopsis pilosula extract were 0g / L, 10g / L, 20g / L, 30g / L, 40g / L and 50g / L respectively.

[0033] In the liquid culture medium with the concentration of Codonopsis pilosula extract of 0g / L, 10g / L, 20g / L, 30g / L, 40g / L and 50g / L, the shiitake mycelium cultured on the ordinary potato solid culture medium for 7 days was inoculated at an inoculation amount of 5%, that is, the shiitake mycelium cultured on the solid culture medium of shiitake mycelium with the concentration of Codonopsis pilosula extract of 0g / L for 7 days. At this time, the shiitake mycelium is in the logarithmic growth phase, and the setting of the inoculation amount can make the shiitake mycelium reproduce at a high speed, thereby improving the productivity of liquid fermentation. It should be noted that in order to improve the production efficiency of shiitake polysaccharide, the shiitake mycelium inoculated by liquid fermentation can be the shiitake mycelium cultured on the solid culture medium with the concentration of Codonopsis pilosula extract of 10-50g / L for 5-6 days, which does not affect the activity of the inoculated shiitake mycelium and the solid culture time is shorter.

[0034] The mycelia inoculated in the liquid fermentation in the embodiment are all Lentinus edodes mycelia cultured in ordinary solid culture medium, so that there is a better comparison between the fermentation with the Codonopsis pilosula extract and the fermentation without the Codonopsis pilosula extract.

[0035] After inoculation, the culture was carried out in a shaking incubator at 28°C and 150 r / min. After 12 days of culture, the mycelia of Lentinus edodes cultured without adding Codonopsis pilosula extract and the mycelia of Lentinus edodes cultured with 20 g / L Codonopsis pilosula extract were as follows: Figure 3 and 4 As shown. It can be seen that the shiitake mycelium obtained by fermentation with the addition of 20g / L Codonopsis pilosula extract is significantly more than the shiitake mycelium obtained by fermentation without the addition of Codonopsis pilosula extract. The biomass of shiitake mycelium was determined by drying the filtered mycelium at 60°C until it was dried to a constant weight, which is the biomass of shiitake mycelium. The results are shown in Table 2. The content of intracellular and extracellular polysaccharides of shiitake was determined by the phenol-sulfuric acid method, and the results are shown in Tables 3 and 4. The protein content and polyphenol content of shiitake mushrooms fermented in liquid were determined by the Coomassie Brilliant Blue method and the Folin phenol method, respectively, and the results are shown in Tables 5 and 6. The activity of PGI enzyme (triosephosphate isomerase) was determined by the enzymatic reaction method, and the activity of PGM enzyme (phosphoglucomutase) was determined by the fluorescence method. The results are shown in Tables 7 and 8. The activities of UGD enzyme (UGD dehydrogenase), GK enzyme (glucokinase) and UGP enzyme (UGD pyrophosphorylase) were measured using the corresponding activity detection kits, and the results are shown in Tables 9-11. The OH free radical scavenging ability of Lentinus edodes was measured using a UV-visible spectrophotometer, and the DPPH scavenging ability of Lentinus edodes was measured using spectrophotometry, and the results are shown in Tables 12-13.

[0036] Table 2 Biomass of Lentinus edodes mycelium during liquid fermentation (g / L)

[0037]

[0038] Table 3 Polysaccharide content in Lentinus edodes during fermentation (g / L)

[0039]

[0040] Table 4 Extracellular polysaccharide content during fermentation (g / L)

[0041]

[0042] Table 5 Protein content during fermentation (mg / g)

[0043]

[0044] Table 6 Total phenol content during fermentation (mg / g)

[0045]

[0046] Table 7 PGI activity during fermentation (U / g)

[0047]

[0048] Table 8 PGM activity during fermentation (U / g)

[0049]

[0050] Table 9 UGD activity during fermentation (U / g)

[0051]

[0052] Table 10 GK activity during fermentation (U / g)

[0053]

[0054] Table 11 UGP activity during fermentation (U / g)

[0055]

[0056] Table 12 DPPH scavenging capacity during fermentation (%)

[0057]

[0058] Table 13 OH radical scavenging ability during fermentation (%)

[0059]

[0060] It can be seen from Tables 2-4 that adding 10-50 g / L of Codonopsis extract during liquid fermentation can increase the biomass of Lentinus edodes mycelium and the content of Lentinan, especially when the Codonopsis extract is 20 g / L, the biomass of Lentinus edodes mycelium and the content of Lentinan are significantly increased compared with not adding Codonopsis extract, and the fermentation time is short, and liquid fermentation can be completed in 8 days, and the liquid fermentation productivity is significantly improved. The results of the determination of the content of Lentinus edodes protein and total phenol in the fermentation process in Tables 5 and 6 also show that when the Codonopsis extract is 20 g / L, it has a significant promoting effect on the liquid fermentation of Lentinus edodes.

[0061] The activities of PGI enzyme, PGM enzyme, UGD enzyme, GK enzyme and UGP enzyme affect polysaccharide metabolism. Tables 7-11 show that adding 20g / L Codonopsis extract can increase the enzyme activity of key enzymes of polysaccharide metabolism in liquid fermented shiitake mycelium, especially the activities of PGI enzyme and PGM enzyme increase significantly, promote the decomposition of polysaccharides, and promote the production of polysaccharides along the direction of synthesis. DPPH scavenging ability and OH free radical scavenging ability reflect the antioxidant effect of cells. Tables 12 and 13 show that the antioxidant capacity of shiitake cells is significantly improved when the Codonopsis extract is 20g / L compared with that without adding Codonopsis extract.

Claims

1. A method for increasing the content of Lentinan by adding Codonopsis pilosula extract, characterized in that: include: During the liquid fermentation of Lentinus edodes mycelium, the Codonopsis pilosula extract is added to the liquid culture medium, and the concentration of the Codonopsis pilosula extract in the liquid culture medium is 10-50 g / L.

2. The method for increasing the content of Lentinan by adding Codonopsis pilosula extract according to claim 1, characterized in that: The concentration of the Codonopsis pilosula extract in the liquid culture medium is 20 g / L.

3. The method for increasing the content of Lentinan by adding Codonopsis pilosula extract according to claim 1, characterized in that: During the liquid fermentation of the shiitake mycelia, the inoculation amount of the shiitake mycelia is 4-6%, and the shiitake mycelia is cultured in a solid culture medium for 6-10 days before inoculation.

4. The method for increasing the content of Lentinan by adding Codonopsis pilosula extract according to claim 1, characterized in that: During the liquid fermentation of the shiitake mycelium, the inoculation amount of the shiitake mycelium is 4-6%, and before the inoculation, the shiitake mycelium is cultured in a solid culture medium with a concentration of 10-50 g / L of codonopsis pilosula extract for 5-6 days.

5. The method for increasing the content of Lentinan by adding Codonopsis pilosula extract according to any one of claims 1 to 4, characterized in that: The liquid fermentation time of the shiitake mushroom mycelium is 4-12 days.

6. The method for increasing the content of Lentinan by adding Codonopsis pilosula extract according to claim 5, characterized in that: The liquid fermentation time of the shiitake mushroom mycelium is 8 days.

7. The method for increasing the content of Lentinan by adding Codonopsis pilosula extract according to claim 1, characterized in that: The preparation steps of the Codonopsis pilosula extract are as follows: After soaking the Codonopsis pilosula powder, the powder is boiled and filtered at 80-100°C, and the filtrate is collected. The above boiling and filtering steps are repeated for the filtered medicinal residue. The two filtrates are combined, concentrated and frozen, and vacuum freeze-dried into powder.

8. The method for increasing the content of Lentinan by adding Codonopsis pilosula extract according to claim 1 or 7, characterized in that: The particle size of the Codonopsis pilosula extract particles is no more than 60 meshes.

9. The method for increasing the content of Lentinan by adding Codonopsis pilosula extract according to claim 1, characterized in that: The shiitake mycelium and fermentation liquid after the shiitake mycelium liquid fermentation can be used to prepare functional shiitake polysaccharide.

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