Method for preparing inonotus obliquus fermentation liquor by taking ginseng leaves as raw materials

Abstract

The invention relates to the technical field of biology, in particular to a method for preparing inonotus obliquus fermentation liquor by taking ginseng leaves as raw materials. According to the method for preparing the inonotus obliquus fermentation liquor by taking the ginseng leaves as the raw material, the scheme is different from the scheme that the inonotus obliquus is independently fermented and then liquid is discarded to take mycelia through optimization of a culture medium and fermentation parameters, and all the fermentation liquor obtained after fermentation has good utilization value. Moreover, the obtained fermentation product not only contains a large amount of triterpenoids, but also contains very rich total saponins, and also contains rare saponins which are not contained in the fresh ginseng leaves.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a method for preparing inonotus obliquus fermentation liquid by taking ginseng leaves as raw materials. Background Art

[0002] Ginseng (Latin name: Panax ginseng) is a perennial herbaceous plant of the Araliaceae family and the genus Panax. The traditional medicinal part of ginseng is the root. As a by-product of ginseng cultivation, most of the ginseng leaves and flowers are abandoned, and only a small part is used in the form of substitute tea or cosmetic raw materials. However, studies have found that the ingredients contained in ginseng leaves and flowers are similar to ginseng, and the total amount of some ginsenoside monomers and 10 major ginsenosides is much greater than that of ginseng roots. However, due to the limitations of traditional processing technology, the utilization rate of ginseng leaves and flowers has always been low, and a large amount of precious resources have been wasted.

[0003] Inonotus obliquus, also known as Chaga, is a medicinal fungus that grows on birch trees. In recent years, people have conducted extensive research on Chaga from the perspective of pharmacological effects and clinical applications. Inonotus obliquus is rich in triterpenoids, which are well-known for their powerful antioxidant, anti-tumor, antibacterial, and blood sugar and lipid regulation activities. Against the backdrop of the rapid development of modern biotechnology and the health industry, the research and application of Inonotus obliquus and its fermentation products are gradually deepening, showing broad market prospects.

[0004] With the continuous development of technology, obtaining the mycelium of edible fungi through biological deep fermentation technology has become a hot topic of research, but the substrate used in the fermentation of mycelium of Inonotus birch is still mainly carbon source and nitrogen source. The obtained product also only has the metabolites of Inonotus birch itself, and if the fermentation conditions are not carefully controlled, the content of triterpenoids is not as good as that of Inonotus birch fruiting body. In addition, the appearance and taste of the fermentation product are poor, resulting in poor market recognition of the fermentation broth obtained by deep fermentation. Summary of the invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a method for preparing Inonotus obliquus fermentation liquid using ginseng leaves as raw materials.

[0006] The present invention provides a method for preparing Inonotus obliquus fermentation liquid by using ginseng leaves as raw materials, which comprises the following steps:

[0007] Step 1: Activate the Inonotus birchii strain and make it into liquid strain.

[0008] Step 2: Inoculate the liquid bacteria into the fermentation medium, at 25-30°C, with a shaking speed of 100-200 rpm, and ferment for 8-12 days to obtain the fermentation liquid.

[0009] Based on the addition of ginseng leaves, the present invention optimizes the culture medium of Inonotus obliquus in detail, so that the use of carbon source, nitrogen source and inorganic salt can best match the ginseng leaves to obtain a fermentation liquid with richer secondary metabolites. In addition, by optimizing the culture medium, more saponins in ginseng are converted into rare saponins.

[0010] In the present invention, the activated culture medium is a PDA solid culture medium.

[0011] In the present invention, the liquid culture medium is made of 30.0 g / L glucose, 3.0 g / L peptone, 3.0 g / L yeast powder, 3.0 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate and water;

[0012] In the present invention, the fermentation medium is prepared from 10-30 g / L ginseng leaves, 20-30 g / L glucose, 10-25 g / L bran, 3.0 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate and water.

[0013] In the present invention, the activation is performed twice, and after two activations, the culture is inoculated in a liquid seed culture medium for cultivation, and then transferred to a liquid seed culture medium for secondary cultivation to obtain a liquid strain.

[0014] The present invention optimizes the fermentation culture conditions, and the obtained conditions are more suitable for the growth of Inonotus obliquus and the accumulation of secondary metabolites. Preferably, the fermentation conditions of the fermentation medium are 28°C, the shaking speed is 150 rpm, and the fermentation is for 10 days.

[0015] In the embodiment of the present invention, the fermentation medium is prepared from 20 g / L ginseng leaves, 25 g / L glucose, 20 g / L bran, 3.0 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate and water.

[0016] In a specific embodiment, the preparation of the fermentation medium includes: crushing fresh ginseng leaves into a paste, adding water, glucose, bran, potassium dihydrogen phosphate and magnesium sulfate, sterilizing at 121° C. for 25 min, cooling to room temperature, and filtering through two stages of 0.45 μm and 0.22 μm.

[0017] In an embodiment of the present invention, the method further comprises the step of ultrasonically disrupting the fermentation broth, and the parameters of the ultrasonic disruption include: working for 5 seconds, resting for 3 seconds, power of 400W, frequency of 25kHz, and ultrasonication for a total of 12 minutes.

[0018] In the embodiment of the present invention, after the ultrasonic disruption, a filtering and sterilization step is also included.

[0019] The method provided by the present invention is applicable to the fermentation culture of various Inonotus obliquus. In a specific embodiment, the Inonotus obliquus is Inonotus obliquus BNCC117822.

[0020] The present invention also provides a fermentation broth prepared by the method described above. The fermentation broth contains a large amount of ginsenosides and triterpenoids, especially rare saponins, and has good application prospects. A variety of products can be obtained by further extracting and / or blending the fermentation product described above.

[0021] Furthermore, the present invention also provides a fermentation culture solution of Inonotus obliquus, which is prepared from 10-30 g / L ginseng leaves, 20-30 g / L glucose, 10-25 g / L bran, 3.0 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate and water.

[0022] Furthermore, the present invention also provides a culture medium combination for Inonotus obliquus, which includes a PDA solid culture medium, a liquid seed culture medium and a fermentation culture medium;

[0023] The liquid culture medium is made of 30.0 g / L glucose, 3.0 g / L peptone, 3.0 g / L yeast powder, 3.0 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate and water;

[0024] The fermentation medium is prepared from 10-30 g / L ginseng leaves, 20-30 g / L glucose, 10-25 g / L bran, 3.0 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate and water.

[0025] The present invention provides a method for preparing a fermentation liquid of Inonotus obliquus using ginseng leaves as raw materials. Through the optimization of culture substrate and fermentation parameters, different from the solution of fermenting Inonotus obliquus alone and discarding the liquid to obtain mycelium, all fermentation products after fermentation in the present invention have good utilization value. In addition, the obtained fermentation product not only contains a large amount of triterpenoid substances, but also has a very rich content of total saponins, and also contains rare saponins not contained in fresh ginseng leaves. DETAILED DESCRIPTION

[0026] The present invention provides a method for preparing a fermentation liquid of Inonotus obliquus using ginseng leaves as raw materials. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve the method. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0027] The test materials used in the present invention are all common commercial products, and can be purchased on the market. Among them, the birch inonotus is birch inonotus BNCC117822, which is purchased on the market.

[0028] The present invention achieves a good culture effect by optimizing the composition in the culture medium. In the preliminary experiment, attempts were made to add ginseng leaves. For example, in addition to the following examples, attempts were made to use dried ginseng leaves, add part of ginseng roots or only add ginseng roots. However, dried ginseng leaves are not only difficult to dissolve after crushing, but also the content of ginsenosides after fermentation is not as good as directly adding fresh leaves. The addition of ginseng roots also does not bring a higher content of ginsenosides.

[0029] The present invention also screens the carbon source and nitrogen source in the culture medium. Ginseng leaves contain certain carbon sources and nitrogen sources, but as a fermentation matrix, the carbon source and nitrogen source therein are insufficient. Therefore, additional supplementation is required. In addition to the glucose, sucrose and starch shown in the following examples, the alternative carbon sources also tried corn flour, wheat flour, fructose or maltose, but the biomass of the obtained birch inonotus was less than 20 mg / mL, and in some cases (such as using wheat flour), the mycelium could not form a mass, which may be related to the culture solution being too viscous, and will not be repeated here. In addition to the bran, peptone and yeast powder shown in the following examples, the alternative nitrogen sources also tried soybean powder and bean dregs, but these two substances are very difficult to dissolve, and high biomass is not obtained during fermentation. Instead, a large amount of particles are wrapped in the formed mycelium, so they are eliminated.

[0030] In addition, attempts have been made to isolate strains of Inonotus obliquus on our own, but the strains obtained so far are not as strong as commercially available strains. In the future, we can further screen excellent strains and try fermentation.

[0031] The present invention will be further described below in conjunction with embodiments:

[0032] Example 1 Activation of Inonotus obliquus and preparation of liquid fungus

[0033] 1. Activation: Inoculate the purchased Inonotus obliquus into PDA solid culture medium, culture at 28°C for 8 days, then transfer to fresh PDA solid culture medium, and culture again at 28°C for 8 days.

[0034] 2. Prepare liquid fungus: Take about 1cm of activated birch fungus 3 The size was inoculated into liquid seed culture medium and cultured in a shaking incubator at 28°C for 8 days with a shaking speed of 130 rpm; then, the inoculation ratio was 10% and inoculated into liquid seed culture medium again, and the culture was carried out in a shaking incubator at 28°C for 8 days with a shaking speed of 130 rpm to obtain liquid strains.

[0035] PDA solid medium includes: 200.0 g / L potato, 25.0 g / L glucose, 5.0 g / L peptone, 3.0 g / L yeast powder, 1.0 g / L potassium dihydrogen phosphate, and 25.0 g / L agar. Sterilize at 121°C for 25 min and then cool to solidify.

[0036] Liquid seed culture medium: 30.0 g / L glucose, 3.0 g / L peptone, 3.0 g / L yeast powder, 3.0 g / L potassium dihydrogen phosphate, and 0.5 g / L magnesium sulfate. Sterilize at 121°C for 25 min.

[0037] Example 2 Preparation of fermentation medium

[0038] Fresh ginseng leaves were crushed into a paste and fermentation medium was prepared according to the following formula.

[0039] Ginseng leaf mud 20g / L, glucose 25g / L, bran 20g / L, potassium dihydrogen phosphate 3.0g / L, magnesium sulfate 0.5g / L, prepared fermentation medium with purified water and sterilized at 121℃ for 25 min.

[0040] Preliminary experiments have found that directly inoculating and fermenting the sterilized culture medium will cause some impurities to be entangled in the mycelium. In order to facilitate the crushing and extraction after fermentation, the culture medium sterilized by high temperature and high pressure is cooled and then filtered through two stages of 0.45μm and 0.22μm.

[0041] Example 3 Preparation of fermentation medium

[0042] Fresh ginseng leaves were crushed into a paste and fermentation medium was prepared according to the following formula.

[0043] 10g / L ginseng leaf mud, 30g / L glucose, 25g / L bran, 3.0g / L potassium dihydrogen phosphate, and 0.5g / L magnesium sulfate were used to prepare the fermentation medium with purified water, sterilized at 121℃ for 25 min, cooled, and then filtered through two stages of 0.45μm and 0.22μm.

[0044] Example 4 Preparation of fermentation medium

[0045] Fresh ginseng leaves were crushed into a paste and fermentation medium was prepared according to the following formula.

[0046] 30 g / L ginseng leaf mud, 20 g / L glucose, 10 g / L bran, 3.0 g / L potassium dihydrogen phosphate, and 0.5 g / L magnesium sulfate were used to prepare the fermentation medium with purified water, sterilized at 121℃ for 25 min, cooled, and then filtered through two stages of 0.45μm and 0.22μm.

[0047] Example 5 Preparation of fermentation medium

[0048] Ginseng roots were crushed into a paste and fermentation medium was prepared according to the following formula.

[0049] 20g / L ginseng root mud, 25g / L glucose, 20g / L bran, 3.0g / L potassium dihydrogen phosphate, and 0.5g / L magnesium sulfate were used to prepare the fermentation medium with purified water, sterilized at 121℃ for 25 min, cooled, and then filtered through two stages of 0.45μm and 0.22μm.

[0050] Example 6 Preparation of fermentation medium

[0051] Fresh ginseng leaves were crushed into a paste and fermentation medium was prepared according to the following formula.

[0052] 20g / L ginseng leaf mud, 25g / L sucrose, 20g / L bran, 3.0g / L potassium dihydrogen phosphate, and 0.5g / L magnesium sulfate were used to prepare the fermentation medium with purified water, sterilized at 121℃ for 25 min, cooled, and then filtered through two stages of 0.45μm and 0.22μm.

[0053] Example 7 Preparation of fermentation medium

[0054] Fresh ginseng leaves were crushed into a paste and fermentation medium was prepared according to the following formula.

[0055] 20g / L ginseng leaf mud, 25g / L starch, 20g / L bran, 3.0g / L potassium dihydrogen phosphate, and 0.5g / L magnesium sulfate were used to prepare the fermentation medium with purified water, sterilized at 121℃ for 25 min, cooled, and then filtered through two stages of 0.45μm and 0.22μm.

[0056] Example 8 Preparation of fermentation medium

[0057] Fresh ginseng leaves were crushed into a paste and fermentation medium was prepared according to the following formula.

[0058] 20g / L ginseng leaf mud, 25g / L glucose, 20g / L peptone, 3.0g / L potassium dihydrogen phosphate, and 0.5g / L magnesium sulfate were used to prepare the fermentation medium with purified water, sterilized at 121℃ for 25 min, cooled, and then filtered through two stages of 0.45μm and 0.22μm.

[0059] Example 9 Preparation of fermentation medium

[0060] Fresh ginseng leaves were crushed into a paste and fermentation medium was prepared according to the following formula.

[0061] 20g / L ginseng leaf mud, 25g / L glucose, 20g / L yeast powder, 3.0g / L potassium dihydrogen phosphate, and 0.5g / L magnesium sulfate were used to prepare the fermentation medium with purified water, sterilized at 121℃ for 25 min, cooled, and then filtered through two stages of 0.45μm and 0.22μm.

[0062] Example 10 Small scale fermentation

[0063] The liquid strain activated in Example 1 was inoculated with the fermentation medium prepared in Examples 2 to 9 at a 10% inoculation rate, and a small test was carried out in a 150 mL Erlenmeyer flask. Three bottles of each group of culture medium were inoculated, and samples of each group of culture medium were taken before fermentation to detect the type and content of saponins.

[0064] Each group was fermented in the dark at 28°C and 150 rpm on a shaker. Samples were taken on the 10th day to observe the morphology of the mycelium and measure the dry weight.

[0065] Then the fermentation broth was ultrasonically broken (working 5s, resting 3s, power 400W, frequency 25kHz, total ultrasonic 12min), filtered, and the filtrate was taken to detect the polysaccharide content and total ginsenosides. The detection of triterpenes and total ginsenosides was carried out by HPLC.

[0066] Table 1 Ginsenoside content in culture medium before fermentation (mg / mL)

[0067]

[0068] It can be seen that the culture medium before fermentation contained ginsenosides due to the addition of ginseng leaves, but since the content of rare saponins in fresh leaves is extremely low, the presence of rare saponins is mostly not detected in the culture medium.

[0069] Table 2 Mycelium and active substance contents in the filtrate on the 10th day of shake flask culture

[0070]

[0071] From the above results, we can see that:

[0072] In terms of biomass, Examples 2 to 4 and Examples 8 and 9 can obtain higher biomass than other groups, but considering that yeast powder or peptone is expensive and difficult to achieve food grade, the schemes of Examples 8 and 9 are abandoned. The culture medium of Example 5 uses ginseng root as the matrix for fermentation, but the growth state of Inonotus birchii in it is poor, and the resulting biomass is low. Different carbon sources are used in Examples 6 and 7, and the results are also quite different from those of the culture medium of Example 2. Therefore, the products obtained by culture in the culture medium of Examples 2 to 4 have the best effect. After statistical analysis, compared with other groups, there is a significant difference in the mycelium content in the product obtained by culture in Example 2 (p<0.05). The content of total triterpenes in the product obtained by culture comes from the mycelium of Inonotus birchii, and the content of triterpenes is also positively correlated with the biomass, and the triterpenoid content of Example 2 is the highest.

[0073] As for the total saponin content, it can be seen that the total saponin content in some groups before and after fermentation has undergone very obvious changes. This is mainly because after several days of fermentation, the saponins in some groups have been degraded, but the saponin content in some groups has maintained a certain level. It is speculated that this may be related to the antioxidant substances produced by the fermentation of Inonotus obliquus.

[0074] Table 3 Ginsenoside content in the fermentation broth obtained after 10 days of shake flask fermentation (mg / mL)

[0075]

[0076] The fermentation broth obtained from each group of cultures was tested for the types and contents of ginsenosides. The results showed that rare saponins were generated during the fermentation process, which may be due to the metabolites promoting the change of saponin configuration during the fermentation process. After fermentation of each group, the culture medium with the highest content of rare saponins was Example 2 and Example 4-5. Combined with the results of Table 2, it can be seen that the solution of Example 2 is the best solution.

[0077] Example 11: Expanded system

[0078] The culture medium prepared in Example 2 was used for culture in a 1 L shake flask. The activation and inoculation steps were the same as before. The fermentation temperature, time, and rotation speed were investigated (3 bottles per group). The results are as follows:

[0079] Table 4 Mycelium and active substance contents in the filtrate obtained by culture under different conditions

[0080]

[0081] From the above results, it can be seen that the conditions of Group 4: 28°C, fermentation for 10 days, and rotation speed of 150 rpm are the most suitable fermentation conditions. Under this condition, the highest biomass and active substance yield can be obtained. After analysis, the dry weight, total triterpene content and total ginsenoside content of this group were significantly different from those of all groups except Group 5, p<0.05.

[0082] Although the fermentation conditions of group 5 can also produce good results, it can be seen that with the extension of fermentation time, the total biomass will not increase, and the content of secondary metabolites will also decrease, indicating that further extension of time has no positive effect on the increase of product content. Therefore, group 4 conditions were selected as the preferred conditions.

[0083] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing inonotus obliquus fermentation liquid using ginseng leaves as raw materials, characterized in that: The steps include: Step 1: Activate the Inonotus birchii strain and make it into liquid strain. Step 2: inoculate the liquid bacteria into the fermentation medium, ferment at 25-30°C, shake at 100-200 rpm, for 8-12 days to obtain a fermentation liquid; The activated culture medium is a PDA solid culture medium; The liquid culture medium is made of 30.0 g / L glucose, 3.0 g / L peptone, 3.0 g / L yeast powder, 3.0 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate and water; The fermentation medium is prepared from 10-30 g / L ginseng leaves, 20-30 g / L glucose, 10-25 g / L bran, 3.0 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate and water.

2. The method according to claim 1, characterized in that The fermentation medium was fermented at 28° C., with a shaking speed of 150 rpm, for 10 days.

3. The method according to claim 1, characterized in that The fermentation medium is prepared from 20 g / L of ginseng leaves, 25 g / L of glucose, 20 g / L of bran, 3.0 g / L of potassium dihydrogen phosphate, 0.5 g / L of magnesium sulfate and water.

4. The method according to claim 1 or 3, characterized in that: The preparation of the fermentation medium comprises: crushing fresh ginseng leaves into mud, adding water, glucose, bran, potassium dihydrogen phosphate and magnesium sulfate, sterilizing at 121° C. for 25 min, cooling to room temperature, and filtering at two levels of 0.45 μm and 0.22 μm.

5. The method according to claim 1, characterized in that The method further comprises the step of ultrasonically crushing the fermentation liquid, wherein the parameters of the ultrasonic crushing include: working for 5 seconds, resting for 3 seconds, power of 400W, frequency of 25kHz, and ultrasonication for a total of 12 minutes.

6. The method according to claim 5, characterized in that After the ultrasonic disruption, the method further comprises a filtering and sterilizing step.

7. The method according to any one of claims 1 to 6, characterized in that: The Inonotus obliquus is Inonotus obliquus BNCC117822.

8. The fermentation broth prepared by the method according to any one of claims 1 to 7.

9. The fermentation culture liquid of Inonotus obliquus is characterized in that: It is made of 10~30g / L ginseng leaves, 20~30g / L glucose, 10~25g / L bran, 3.0g / L potassium dihydrogen phosphate, 0.5g / L magnesium sulfate and water.

10. A culture medium combination for Inonotus obliquus, characterized in that: Including PDA solid culture medium, liquid seed culture medium and fermentation culture medium; The liquid culture medium is made of 30.0 g / L glucose, 3.0 g / L peptone, 3.0 g / L yeast powder, 3.0 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate and water; The fermentation medium is prepared from 10-30 g / L ginseng leaves, 20-30 g / L glucose, 10-25 g / L bran, 3.0 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate and water.

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