A method for preparing a high-content shikimic acid bacterial metabolite

Abstract

The application discloses a method for preparing high-content shikimic acid bacterial substance, which comprises the following steps: adding one or more of rice bean powder, yam powder, peptone and beef extract into star anise powder and ginkgo leaf powder, and adding pure water, sterilizing to prepare a solid culture medium; inoculating activated cordyceps liquid bacterial seed into the solid culture medium, and culturing at 15-35 DEG C in dark to obtain the solid bacterial substance with increased shikimic acid content; and the method uses star anise, ginkgo leaf and other raw medicinal materials as fermentation substrates, generates bacterial substance through one-time fermentation, and the shikimic acid content in the bacterial substance is obviously increased compared with blank control medium, so that a new way is provided for deep development and utilization of star anise, ginkgo leaf and other traditional Chinese medicine resources.

Description

Technical Field

[0001] This invention relates to a method for preparing mycelium with high shikimic acid content using star anise and ginkgo leaves. Background Technology

[0002] Shikimic acid, also known as toxic octanoic acid, has the chemical name 3,4,5-trihydroxy-1-cyclohexene-1-carboxylic acid and the molecular formula C7H. 10 O5, widely found in higher plants such as star anise (Magnoliaceae) and ginkgo (Ginkgoaceae) and microorganisms, is an important natural organic acid. Shikimic acid possesses various pharmacological activities, including antitumor, antibacterial, analgesic, antithrombotic, and anti-cerebral ischemia activities. It is a crucial raw material for the synthesis of oseltamivir phosphate, the only drug used to treat avian influenza and influenza A. Star anise ( True allium *Hook. f.* is a plant of the Magnoliaceae family, widely distributed in southern my country. Its fruit, star anise, is a medicinal and edible herb with warming and dispersing properties, as well as regulating qi and relieving pain. Studies have shown that different parts of star anise contain various chemical components with insecticidal, antibacterial, anti-inflammatory, analgesic, anti-fatigue, anti-anxiety, and anti-atherosclerotic biological activities. The shikimic acid content in star anise is approximately 7%. Ginkgo (… Ginkgo biloba Ginkgo biloba (L.) is one of the world's most precious relict plants, with its leaves being its main medicinal part. In recent years, there has been considerable research on the chemical composition of ginkgo leaves. Flavonoids and lactones have received widespread attention due to their effects on improving the cardiovascular system, regulating blood lipids, anti-oxidation, and anti-aging. However, research on organic acids is still relatively limited. The shikimic acid content in ginkgo is approximately 5%. Currently, shikimic acid is mainly extracted from star anise, but this method is affected by factors such as the harvesting time and yield of star anise. Further research into increasing the shikimic acid content has significant research and application value.

[0003] Cordyceps militaris ( Cordyceps tenuipes ), also known as giant cordyceps, Japanese cordyceps, snowflake cordyceps, was once named Penicillium sclerotium ( Paecilomyces tenuipes ) and fine-footed fascicles ( Isaria tenuipes The active ingredients of Cordyceps militaris include polysaccharides, cordycepic acid, cytidine, uridine, guanosine, inosine, thymidine, adenosine, proteins, and amino acids. Due to its significant effects in scavenging free radicals, regulating immunity, improving sleep quality, anti-tumor activity, and anti-inflammatory and antibacterial properties, Cordyceps militaris has been developed into various products in Japan and South Korea as a substitute for Cordyceps sinensis.

[0004] Currently, there are some reports on using bacteria such as Salmonella and engineered Escherichia coli to ferment star anise to increase the shikimic acid content in the fermentation product, but there are no research reports on using Cordyceps fungi to ferment and cultivate star anise. Summary of the Invention

[0005] This invention provides a method for preparing high-shikimic acid content bacterial culture. The method uses star anise and ginkgo leaves as fermentation substrates and Cordyceps militaris to prepare bacterial culture with increased shikimic acid content.

[0006] The operation steps of the method of the present invention are as follows:

[0007] (1) Add one or more of the following to star anise powder and ginkgo leaf powder: coix seed powder, yam powder, peptone, and beef extract, add pure water, and sterilize at 121℃ for 30-40 minutes to obtain star anise and ginkgo leaf solid culture medium;

[0008] The Job's tears flour, yam flour, peptone, and beef extract mentioned are conventional commercially available products;

[0009] Star anise powder is made by crushing star anise (a traditional Chinese medicine) into powder and passing it through a 20-mesh sieve to obtain a particle size of less than or equal to 20 mesh; ginkgo leaf powder is made by crushing ginkgo leaves (a traditional Chinese medicine) into powder and passing it through a 30-mesh sieve to obtain a particle size of less than or equal to 30 mesh.

[0010] The mass ratio of star anise powder to ginkgo leaf powder is 1:0.5-1; the mass ratio of star anise powder to coix seed powder is 20:2-5; the mass ratio of star anise powder to yam powder is 20:2-5; the mass ratio of star anise powder to peptone is 100:1-3; the mass ratio of star anise powder to beef extract is 100:1-3; and the mass ratio of star anise powder to pure water is 1:2-6.

[0011] (2) Inoculate Cordyceps militaris into PDA slant culture medium and culture it at 20-35℃ until the colony diameter reaches 2.0-3.0cm; pick the colonies into PDA liquid culture medium and culture them in a shaker at 120r / min for 4-8 days to obtain Cordyceps militaris liquid culture. Inoculate the Cordyceps militaris liquid culture into the solid culture medium of star anise and ginkgo leaves in step (1) and culture it in the dark at 15-30℃ to obtain solid culture medium with increased shikimic acid content.

[0012] The Cordyceps militaris strain mentioned is a commercially available strain, purchased from the China Microbial Culture Collection Network, with the number bio-81548.

[0013] (3) The solid mycelium obtained in step (2) was dried at 60°C, ground into powder, passed through an 80-mesh sieve, and set aside for use. The content of shikimic acid was detected by high performance liquid chromatography.

[0014] The advantages of this invention are:

[0015] Using Cordyceps militaris to biotransform raw medicinal materials such as star anise and ginkgo leaves, through a single solid-state fermentation, can increase the content of shikimic acid. The operation is simple, energy-efficient, and pollution-free, and this fermentation technology has good application prospects. Detailed Implementation

[0016] The technical solution of the present invention will be further described in detail below through embodiments, but the content of the present invention is not limited thereto. Unless otherwise specified, the methods in this embodiment are conventional methods, and the materials and reagents used are obtained from commercial sources unless otherwise specified.

[0017] Example 1: The method for increasing the shikimic acid content in star anise and ginkgo leaves is as follows:

[0018] (1) After crushing the star anise, pass it through a 20-mesh sieve to obtain star anise powder with a particle size of less than or equal to 20 mesh; crush the ginkgo leaves and pass them through a 30-mesh sieve to obtain ginkgo leaf powder with a particle size of less than or equal to 30 mesh.

[0019] Weigh out 20g of star anise powder, 12g of ginkgo leaf powder, 3.5g of coix seed powder, 3.5g of yam powder, 0.5g of peptone, 0.5g of beef extract, and 60g of pure water. Place them in a culture bottle and stir well. Seal the bottle with a breathable plastic cap. Prepare a total of 6 bottles. After sterilizing at 121℃ for 40 minutes, you will get solid culture medium for star anise and ginkgo leaves.

[0020] (2) Preparation, inoculation and fermentation culture of Cordyceps militaris liquid culture

[0021] Cordyceps militaris was inoculated into PDA slant culture medium and incubated at 20℃ until the colony diameter reached approximately 2 cm; 0.5 cm samples were then picked. 2 The colonies were transferred to PDA liquid culture medium and cultured on a shaker at 120 r / min for 8 days to obtain Cordyceps militaris liquid culture. 4 mL of Cordyceps militaris liquid culture was inoculated into 3 bottles of star anise and ginkgo leaf solid culture medium prepared in step (1), and the other 3 bottles were left uninoculated as blank controls (4 mL of PDA liquid culture medium was added to each bottle). The cultures were cultured in the dark at 20℃ for 45 days to obtain solid mycelium through fermentation. The fermented mycelium was dried at 60℃, powdered, and passed through an 80-mesh sieve for later use.

[0022] (3) The content of shikimic acid in the solid mycelium obtained in step (2) was detected by high performance liquid chromatography;

[0023] Determination of shikimic acid content in the sample: Accurately weigh 0.5000 g of the above sample and place it in a 15 mL centrifuge tube. Accurately add 8.0 mL of 50% methanol solution to the centrifuge tube, mix well, and extract by sonication for 30 min. Centrifuge at 4000 r / min for 10 min. Transfer the supernatant to a 25 mL volumetric flask. Repeat the extraction of the lower solid sediment twice as described above. Combine the supernatants and make up to 25 mL with 50% methanol solution. Then filter through a 0.45 µm filter membrane. Detect the shikimic acid content in the filtrate using HPLC. Each sample is measured three times, and the average value of the three samples is taken as the shikimic acid content in the sample.

[0024] Tests showed that the shikimic acid content in the culture medium of star anise and ginkgo leaves that were not inoculated with Cordyceps militaris was 5.52%, while the shikimic acid content in the solid mycelium produced by Cordyceps militaris fermentation was 8.63%, with an increase rate of 56.34%.

[0025] Increase rate = (Shikimic acid content in fermented sample - Shikimic acid content in blank culture sample) / Shikimic acid content in blank culture sample × 100%;

[0026] Meanwhile, star anise powder was used as a separate inoculation fermentation to prepare mycelium. 20g of star anise powder, 3.5g of coix seed powder, 3.5g of yam powder, 0.5g of peptone, 0.5g of beef extract, and 60g of pure water were placed in a culture bottle and stirred until well mixed. The bottle was then sealed with a breathable plastic cap and sterilized at 121℃ for 40 minutes to obtain star anise solid culture medium. The culture medium was then inoculated and fermented according to step (2). The results showed that Cordyceps militaris could not grow on this culture medium.

[0027] Example 2: The method for increasing the shikimic acid content in star anise and ginkgo leaves is as follows:

[0028] (1) After crushing the star anise, pass it through a 20-mesh sieve to obtain star anise powder with a particle size of less than or equal to 20 mesh; crush the ginkgo leaves and pass them through a 30-mesh sieve to obtain ginkgo leaf powder with a particle size of less than or equal to 30 mesh.

[0029] Weigh out 20g of star anise powder, 15g of ginkgo leaf powder, 4.5g of coix seed powder, 0.25g of peptone, 0.25g of beef extract, and 65g of pure water. Place them in a culture bottle and stir well. Seal the bottle with a breathable plastic cap. Prepare a total of 6 bottles. After sterilizing at 121℃ for 40 minutes, you will get solid culture medium for star anise and ginkgo leaves.

[0030] (2) Preparation, inoculation and fermentation culture of Cordyceps militaris liquid culture

[0031] Cordyceps militaris was inoculated into PDA slant culture medium and incubated at 25°C until the colony diameter reached approximately 2.5 cm; 0.5 cm samples were then picked. 2 The colonies were transferred to PDA liquid culture medium and cultured on a shaker at 120 r / min for 7 days to obtain Cordyceps militaris liquid culture. The Cordyceps militaris liquid culture and PDA liquid culture medium were inoculated into star anise and ginkgo leaf solid culture medium prepared in step (1) respectively (same as in Example 1), and cultured at 25°C in the dark for 30 days to obtain solid mycelium through fermentation. The fermented mycelium was dried at 60°C, powdered, and passed through an 80-mesh sieve for later use.

[0032] (3) The shikimic acid content in the solid mycelium obtained in step (2) was detected by high performance liquid chromatography, the method being the same as in Example 1;

[0033] Tests showed that the shikimic acid content in the culture medium of star anise and ginkgo leaves that were not inoculated with Cordyceps militaris was 5.80%, while the shikimic acid content in the solid mycelium produced by Cordyceps militaris fermentation was 9.85%, with an increase rate of 69.83%.

[0034] Example 3: The method for increasing the shikimic acid content in star anise and ginkgo leaves is as follows:

[0035] (1) The preparation of star anise powder and ginkgo leaf powder is the same as in Example 1. Weigh 20g of star anise powder, 17g of ginkgo leaf powder, 2.5g of yam powder, 0.5g of peptone, and 55g of pure water, place them in a culture bottle and stir to mix. Seal with a breathable plastic cap. A total of 6 bottles were prepared. After sterilization at 121℃ for 40min, star anise and ginkgo leaf solid culture medium was obtained.

[0036] (2) Preparation, inoculation and fermentation culture of Cordyceps militaris liquid culture

[0037] Cordyceps militaris was inoculated into PDA slant culture medium and incubated at 30℃ until the colony diameter reached approximately 2.5 cm; 0.5 cm of the colony was then picked. 2 The colonies were transferred to PDA liquid culture medium and cultured on a shaker at 120 r / min for 5 days to obtain Cordyceps militaris liquid culture. The Cordyceps militaris liquid culture and PDA liquid culture medium were respectively inoculated into the star anise and ginkgo leaf solid culture medium prepared in step (1) (same as in Example 1), and cultured at 30°C in the dark for 20 days to obtain solid mycelium through fermentation. The fermented mycelium was dried at 60°C, powdered, and passed through an 80-mesh sieve for later use.

[0038] (3) The content of shikimic acid in the solid mycelium obtained in step (2) was detected by high performance liquid chromatography;

[0039] Tests showed that the shikimic acid content in the star anise and ginkgo leaf culture medium without Cordyceps militaris was 6.30%, while the shikimic acid content in the solid mycelium produced by Cordyceps militaris fermentation was 9.42%, with an increase rate of 49.52%.

[0040] The above experimental results show that the shikimic acid content in the solid mycelium produced by fermenting star anise and ginkgo leaves with Cordyceps militaris is significantly higher than that in the blank control sample without Cordyceps militaris inoculation. This invention provides a new approach for the development and utilization of star anise and ginkgo leaves and is an effective method for increasing natural shikimic acid.

Claims

1. A method for preparing high-content shikimic acid bacteria, characterized in that, Includes the following steps: (1) Add one or more of the following to star anise powder and ginkgo leaf powder: coix seed powder, yam powder, peptone, beef extract, and pure water. Sterilize at 121℃ for 30-40 minutes to obtain a solid culture medium. (2) The activated Cordyceps militaris liquid culture was inoculated into the solid culture medium of step (1) and cultured in the dark at 15-35°C to obtain solid culture medium with increased shikimic acid content.

2. The method for preparing high-content shikimic acid bacteria according to claim 1, characterized in that: The mass ratio of star anise powder to ginkgo leaf powder is 1:0.5-1; the mass ratio of star anise powder to coix seed powder is 20:2-5; the mass ratio of star anise powder to yam powder is 20:2-5; the mass ratio of star anise powder to peptone is 100:1-3; the mass ratio of star anise powder to beef extract is 100:1-3; and the mass ratio of star anise powder to pure water is 1:2-6.

3. The method for preparing high-content shikimic acid bacteria according to claim 1, characterized in that: The activated Cordyceps militaris liquid culture is prepared by inoculating Cordyceps militaris into PDA slant medium and incubating at a constant temperature of 20-35℃ until the colony diameter reaches 1.5-3.0 cm; then picking the colonies and incubating them in PDA liquid medium for 4-8 days on a shaker.

4. The method for preparing high-content shikimic acid bacteria according to claim 1, characterized in that: The incubation period is 20 to 45 days in the dark.