A kind of Platycladus orientalis extract and its preparation method and application

Abstract

The present invention belongs to the field of microbial fermentation technology, and discloses a Platycladus orientalis extract, and its preparation method and application. The Platycladus orientalis extract is obtained by fermenting an Alternaria sp. C-1 strain on the basis of conventionally extracting quercetin from Platycladus orientalis leaves, and the quercetin yield in the obtained Platycladus orientalis extract is increased by 25%. The Alternaria sp. C-1 strain has a strain preservation number of GDMCC No: 65881. The fermentation method of Alternaria of the present invention is to inoculate the seed liquid of the Alternaria sp. C-1 into a liquid culture medium for fermentation and culture. The strain has a relatively short growth cycle and a fast reproduction rate, and can continuously produce quercetin. The quercetin content in the strain fermentation product can reach up to 75 mg / L. The obtained Platycladus orientalis extract has the beneficial technical effects of high quercetin content, stable quercetin yield, reduced consumption of Platycladus orientalis leaves, and environmental friendliness.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial fermentation, and more particularly to a Platycladus orientalis extract, a preparation method thereof, and an application thereof. Background Art

[0002] Platycladus orientalis leaves, also known as cypress leaves, bush cypress leaves, and cypress leaves, are the dried young branches and leaves of Platycladus orientalis L., a plant of the Cupressaceae family. The main active ingredient in Platycladus orientalis leaves is quercetin. The 2025 edition of the Chinese Pharmacopoeia stipulates that the quercetin content in Platycladus orientalis leaves, calculated on a dry basis, must not be less than 0.1%. Quercetin is a flavonoid monomer compound widely found in plants and has multiple pharmacological effects, including anti-hair loss, antioxidant, anti-tumor, and hypoglycemic and lipid-lowering properties. Quercetin also exhibits certain anti-inflammatory activity, primarily through its inhibitory effect on pro-inflammatory factors.

[0003] Most existing technologies use water or alcoholic solutions to directly extract quercetin from Platycladus orientalis leaves, which mainly have the following problems: (1) The quercetin content in Platycladus orientalis leaves is low: the conventional extraction method is to crush the Platycladus orientalis leaves and use alcoholic solutions to extract quercetin, and the quercetin content in the obtained Platycladus orientalis extract is low; (2) The quercetin content in the extract is unstable: environmental pollution, seasonal changes, and land quality will affect the growth of Platycladus orientalis, hinder the stable generation of secondary metabolites in Platycladus orientalis, greatly increase the difficulty of obtaining quercetin, and cause the quercetin content in the obtained Platycladus orientalis extract to be unstable; (3) The consumption of Platycladus orientalis leaves is huge: calculated based on a quercetin content of 0.1%, it takes about 1000g of Platycladus orientalis leaves to extract 1g of quercetin. The large demand for quercetin will lead to the over-exploitation of Platycladus orientalis resources. Summary of the Invention

[0004] The invention provides an Alternaria sp. C-1 strain. After fermentation of the Alternaria sp. C-1 strain, a fermentation product containing quercetin can be obtained.

[0005] Another object of the present invention is to provide a fermentation product of Alternaria sp. C-1 strain.

[0006] Another object of the present invention is to provide the use of Alternaria sp. C-1 in the preparation of Platycladus orientalis extract.

[0007] Another object of the present invention is to provide a method for preparing the Platycladus orientalis extract.

[0008] Another object of the present invention is to provide the use of a fermentation product of Alternaria sp. C-1 strain in cosmetics.

[0009] In order to solve the above technical problems, the technical solution provided by the present invention is:

[0010] An Alternaria sp. C-1 strain, with a strain deposit number of GDMCC No: 65881.

[0011] Alternaria sp. C-1 strain has the following properties:

[0012] 1. Morphological characteristics

[0013] Alternaria sp. C-1 strain grows on PDA medium at 28°C for about a week and then covers the entire plate. Its colonies are flat with regular edges, yellow-brown in the middle of the front and white around it, with no obvious difference in color between the front and back. The hyphae are sparse. After about a month of growth, the bottom layer of the colony is yellow-brown, the upper layer is grayish-white, and the conidia are black-brown.

[0014] 2. Characteristics on various culture media

[0015] (1) Potato dextrose agar (PDA): The mycelium covers the entire plate. The mycelium is white and brown, with the front and back being yellow-brown in the middle. There is little difference in color between the front and back. A large number of conidia are produced.

[0016] (2) Malt extract agar (MEA): The plate is covered with colonies of varying sizes, with the center of the colony being brown and the surrounding area being yellow or white. The colony is flat and has cracks on the back.

[0017] (3) Red Bengal agar (REA): The strain almost covers the entire plate after seven days of growth. The mycelium is white and gray-brown, and a large number of conidia are produced, with fewer aerial mycelium.

[0018] 3. Metabolic characteristics

[0019] About Alternaria sp. C-1 Carbon Source Utilization

[0020] Well utilized: starch, glucose, fructose

[0021] Commonly used: sucrose, lactose

[0022] The distinguishing characteristics of Alternaria sp. C-1 strain from known Alternaria species are:

[0023] Members of the genus Alternaria are characterized by unbranched or sometimes branched conidiophores, erect or curved, solitary or clustered, arising from hyphae or on simple stromata, sometimes swollen at the base, and varying shades of brown. Spore scars are clearly visible, with dark margins and a lighter center. Conidia are ovate, obclaudiculate, or obpyriform, with a distinct umbilicus. The conidial body is varying shades of brown or greenish-yellow-brown, smooth or tuberculous, with several transverse, longitudinal, or oblique true septa, with no or marked constrictions at the septa. In most cases, the body tapers to the apex, lacking a beak or possessing a short, columnar, or slender beak, which may or may not be septate and is noticeably lighter in color than the body. The beak sometimes transforms into a secondary conidiophore (false beak), which produces secondary conidia in short or long chains.

[0024] The C-1 strain of the present invention belongs to the genus Alternaria, but has some differences in morphological characteristics. The strain has regular edges, the middle of the front is yellow-brown, the periphery is white, the hyphae grow in a creeping manner, and after growing on PDA for 30 days, the bottom layer of the colony is yellow-brown, the upper layer is grayish-white, and there are fewer aerial hyphae. After growing on PDA for about a week, a large number of conidia are produced. The spores have transverse and longitudinal septa, are oblong, and are brick-shaped with 3-5 transverse septa. They are obviously brown and vary in size. Most of them are 30-36 microns long and 10-25 microns wide, and dozens of them often form chains.

[0025] The present invention utilizes Alternaria sp. C-1 fermentation to prepare a fermentation product, which has the following advantages:

[0026] 1. High quercetin content: The Alternaria sp. C-1 strain provided by the present invention is an endophytic fungus isolated and screened from Platycladus orientalis leaves. It has a relatively short growth cycle and a fast reproduction rate. It can continuously produce quercetin using Platycladus orientalis residues through fermentation. The quercetin content in the fermentation product can reach up to 75 mg / L.

[0027] 2. Stable quercetin production: The Alternaria sp. C-1 strain has relatively loose requirements for culture conditions and can utilize a variety of conventional carbon and nitrogen sources for growth and metabolism. The quercetin content in the strain's fermentation products is stable, which is expected to solve the problem of unstable quercetin production in the resulting extract caused by environmental pollution, seasonal changes, land quality, etc. that affect the growth of natural arborvitae.

[0028] 3. Reduce the consumption of Platycladus orientalis leaves: The present invention screened out a suitable fungus - Alternaria sp. C-1 strain from Platycladus orientalis leaves. Its fermentation product is rich in quercetin, which can to a certain extent get rid of the dependence on Platycladus orientalis plant resources and reduce the phenomenon of over-exploitation of plants.

[0029] The present invention also protects a Platycladus orientalis extract, which contains a fermentation product prepared by self-fermentation of Alternaria sp. C-1, and the fermentation product contains quercetin.

[0030] The Platycladus orientalis extract of the present invention further contains Platycladus orientalis extract solution.

[0031] The present invention also provides a method for preparing the Platycladus orientalis extract, comprising the following steps:

[0032] S1. The seed liquid of the Alternaria sp. C-1 is inoculated into a culture medium for fermentation to obtain a fermentation product of the strain.

[0033] Preferably, the method for preparing the Platycladus orientalis extract of the present invention further comprises the following steps: S0. extracting quercetin from Platycladus orientalis leaves to obtain a Platycladus orientalis extract.

[0034] The preparation method of the Platycladus orientalis extract of the present invention further comprises the following steps: S2. mixing the Platycladus orientalis extract and the fermentation product of the strain, filtering, concentrating and refining to obtain the Platycladus orientalis extract.

[0035] Preferably, the culture medium in S1 is a mixture of rice and Platycladus orientalis leaf residue.

[0036] More preferably, S1 is a mixture of rice and Platycladus orientalis leaf residue, in which rice accounts for 40% to 60% by mass.

[0037] The Platycladus orientalis leaf residue described in S1 of the present invention is the residue left after quercetin is extracted from Platycladus orientalis leaves.

[0038] The invention can realize the secondary utilization of Platycladus orientalis leaf residue.

[0039] The existing method for extracting quercetin from Platycladus orientalis leaves is to use a certain concentration of ethanol to extract quercetin. After ethanol extraction, a Platycladus orientalis extract containing quercetin and Platycladus orientalis leaf residue are obtained. The extraction rate of quercetin from Platycladus orientalis leaves by heating with ethanol can reach up to 90%, that is, about 10% of quercetin remains in the Platycladus orientalis leaf residue.

[0040] The present invention utilizes a mixture of rice and Platycladus orientalis leaf residue as a culture medium, which, on the one hand, realizes the secondary utilization of Platycladus orientalis leaf residue and reduces resource waste; on the other hand, since the Platycladus orientalis leaf residue also contains a small amount of quercetin and a variety of intermediate substances for producing quercetin, it is beneficial for the C-1 strain to ferment and produce quercetin.

[0041] The culture medium of the present invention adds rice to the Platycladus orientalis leaf residue, provides sufficient nutrients for the growth of the C-1 strain, and further improves the yield of quercetin in the fermentation product of the strain.

[0042] Preferably, the inoculation amount of Alternaria sp. C-1 seed solution in the culture medium of S1 is 1-5%.

[0043] Preferably, the fermentation temperature in S1 is 26-30°C

[0044] The fermentation culture time described in S1 of the present invention is 25 to 65 days.

[0045] The S2 specifically includes the following steps:

[0046] S21. The fermentation product of the strain obtained in S1 was combined with the Platycladus orientalis extract obtained in S0, and then filtered and concentrated to obtain a crude extract of Platycladus orientalis rich in quercetin;

[0047] S22. The crude extract of the Platycladus orientalis extract is purified and concentrated sequentially through a D101 macroporous resin column and an ultrafiltration membrane to obtain the Platycladus orientalis extract.

[0048] Specifically, the process in S21 is to combine the fermentation product of the strain S1. and the alcohol-extracted Platycladus orientalis extract of S0., filter through a 10 μm filter membrane, and concentrate to 1 / 10 of the solution, and then purify.

[0049] Specifically, the purification process in S22 can use an ultrafiltration membrane with a molecular weight lower than that of quercetin to perform concentration and impurity removal, and collect the retentate.

[0050] More specifically, the purification process in S22 may use a macroporous resin material to adsorb and remove impurities on the extract.

[0051] In addition, silica gel column chromatography, polyamide column chromatography, and dextran gel column chromatography can also be used to purify and remove impurities from the fermentation product.

[0052] The present invention also protects the use of the fermentation product as an anti-hair loss, soothing and antioxidant ingredient in cosmetics.

[0053] Deposit of biological materials:

[0054] The Alternaria sp. C-1 strain described in the present invention was deposited with the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on February 14, 2025, with the registration number GDMCC No. 65881. The address of the collection center is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 This is a diagram of the growth status of Alternaria sp. C-1 strain on PDA culture medium for two months, where (a) is the front side and (b) is the back side.

[0056] Figure 2 These are growth status diagrams of Alternaria sp. C-1 strain on PDA culture medium at different time periods, where (a) is the front side after 7 days of growth, (b) is the back side after 7 days of growth, (c) is the front side after 14 days of growth, (d) is the back side after 14 days of growth, (e) is the front side after 30 days of growth, and (f) is the back side after 30 days of growth.

[0057] Figure 3 These are growth status diagrams of Alternaria sp. C-1 strain on MEA culture medium at different time periods, where (a) shows the front side after 7 days of growth, (b) shows the back side after 7 days of growth, (c) shows the front side after 14 days of growth, (d) shows the back side after 14 days of growth, (e) shows the front side after 30 days of growth, and (f) shows the back side after 30 days of growth.

[0058] Figure 4 These are growth status diagrams of Alternaria sp. C-1 strain on REA culture medium at different time periods, where (a) shows the front side after 7 days of growth, (b) shows the back side after 7 days of growth, (c) shows the front side after 14 days of growth, (d) shows the back side after 14 days of growth, (e) shows the front side after 30 days of growth, and (f) shows the back side after 30 days of growth.

[0059] Figure 5 This is the detection graph of quercetin content in the fermentation product obtained in Example 2. DETAILED DESCRIPTION

[0060] The present invention is further described in detail below in conjunction with specific embodiments.

[0061] The components of the PDA solid culture medium in the embodiment are: 300 g of potato, 20 g of glucose, 15 g of agar, and 0.1 g of chloramphenicol per 1 L of PDA culture medium.

[0062] The Platycladus orientalis leaf residue in the embodiment is obtained by extracting quercetin twice from dried Platycladus orientalis leaves by heating them at 70° C. with 50% ethanol.

[0063] The composition of the Alternaria sp. C-1 seed culture medium used in the Examples and Comparative Examples is as follows: per liter of seed culture medium, 20 g of maltose, 10 g of sodium glutamate, 0.5 g of KH2PO4, 0.3 g of MgSO4·7H2O, 10 g of glucose, 3 g of yeast extract, and 20 g of mannitol; the pH of the seed culture medium is 5-6.

[0064] In the preparation methods of the Platycladus orientalis extracts described in the various embodiments and comparative examples, the specific extraction method of the SO Platycladus orientalis extract is as follows:

[0065] S01. Crush the Platycladus orientalis leaves, mix the Platycladus orientalis leaf powder with 50% ethanol solution at a solid-liquid ratio of 1:10, stir and extract at 70 ° C for 1.5 h, and separate the extract from the residue after the extraction to obtain a Platycladus orientalis extract containing quercetin; the quercetin content in the Platycladus orientalis leaves used in each embodiment and comparative example of the present application was 0.3%;

[0066] S02. Combine the two thujinol extracts, and the quercetin content in the extract is about 135 mg / L.

[0067] Example 1

[0068] This embodiment is a method for screening Alternaria sp. C-1 strain, which specifically includes the following steps:

[0069] S1. Collection and isolation of strains: Fresh Platycladus orientalis leaves were collected from Sinan County, Tongren City, Guizhou Province (108°9′ E, 27°37′ N), Zengcheng District, Guangzhou City (113°32′ E, 23°5′ N), and Tanghe County, Nanyang City, Henan Province (112°28′ E, 32°21′ N).

[0070] First, the Platycladus orientalis leaves were cut into 0.5 cm × 0.5 cm segments and inoculated onto the prepared PDA solid medium, with three explants evenly planted per dish. Streptomycin (10 mg / L) and tetracycline (3 mg / L) were added to the medium.

[0071] Then, the cells were placed in a 28°C constant temperature incubator in the dark for incubation. When hyphae grew from the Platycladus orientalis leaf explants inoculated in the PDA medium, the tips of the hyphae at the edge of the colony with different appearances were promptly picked with an inoculation needle and transferred to a new PDA plate.

[0072] Finally, the plate was inverted and cultured in a constant temperature incubator at 28°C in the dark for 3-7 days. The growth of the mycelium was observed and recorded every day. When colonies of different colors or shapes grew out, the mycelium tip purification method was continued until the colonies were a single pure culture. A total of 21 strains were isolated from the Platycladus orientalis leaves from the three production areas, and the strain codes were marked as C-1, C-2...C-3.

[0073] S2. Strain Cultivation and Identification: The 21 strains obtained in S1 were inoculated into 500 mL Erlenmeyer flasks containing rice culture medium and incubated in a constant-temperature incubator at 28°C. The fermentation culture was then ultrasonically extracted with 75% ethanol for 30 minutes. The solution was assayed for flavonoid formation using an aluminum chloride colorimetric assay. The flavonoid-containing solution was then assayed for the presence of quercetin using HPLC. The fermentation broth of strain C-1, isolated from Guizhou, was found to form a yellow complex with aluminum chloride solution (1 mL of the solution, plus 2% aluminum chloride in ethanol, showed the presence of a yellow complex). The flavonoids produced contained quercetin. Molecular biological characterization of the strain revealed that it belongs to the genus Alternaria sp. C-1 is deposited with the Guangdong Provincial Microbiological Culture Collection under the registration number GDMCC 65881 and exhibits the following characteristics:

[0074] Morphological characteristics / colony characteristics: After Alternaria sp. C-1 strain was grown on PDA medium at 28℃ for about 2 months, the colony morphology was Figure 1 .

[0075] according to Figure 1 It can be seen that the Alternaria sp. C-1 strain covers the entire plate, with flat colonies and regular edges. The middle of the front side is yellow-brown, the surrounding area is white, and there is no difference in color between the front and back sides. The hyphae are sparse, the bottom layer of the colony is yellow-brown, and the upper layer is grayish-white hyphae. The conidia are black-brown.

[0076] Characteristics on various culture media

[0077] (1) PDA medium: After Alternaria sp. C-1 strain grows on PDA medium at 28°C for about 7 days, the colony morphology is Figure 2 In (a) and (b), it can be seen that the mycelium covers the entire plate, the mycelium is white and brown, the front is yellow-brown in the middle, and the front and back are not much different in color. A large number of conidia are produced; after 14 days of growth, the colony morphology is Figure 2 In (c) and (d), it can be seen that the aerial hyphae have reached the top of the plate, most of the hyphae are gray with white hyphae, some hyphae are intertwined into balls, and the back is dark black; after 30 days of growth, the colony morphology is Figure 2 In (e) and (f), it can be seen that: a large number of silvery-white hyphae appeared, with tiny water droplets attached to the hyphae, which were obviously produced by respiratory metabolism. The part of the strain close to the culture medium was gray-brown, while the other part was still white. The back of the strain did not change much from that at 14 days of growth.

[0078] (2) MEA medium: After Alternaria sp. C-1 strain grows on MEA medium at 28°C for about 7 days, the colony morphology is Figure 3 In (a) and (b), it can be seen that the plate is covered with colonies of different sizes, and the center of the colony is brown, and the surrounding is yellow or white. The colony is flat and there are cracks on the back. After 14 days of growth, the colony morphology is Figure 3 In (c) and (d), it can be seen that the strain has spread over the entire plate, the hyphae are yellow-brown, a large amount of black liquid appears on the surface of the hyphae and condenses into droplets of different sizes, the surface of the strain is wrinkled, the back of the plate is black, and there are obvious cracks; after 30 days of growth, the colony morphology is Figure 3 In (e) and (f), it can be seen that the droplets condensed on the surface of the strain have become larger, but no more droplets have been produced. Porous structures similar to stomata are visible to the naked eye on the surface of the strain covering the entire plate, the mycelium is bluish-brown in color, and there is no obvious change on the back of the strain.

[0079] (3) REA medium: After Alternaria sp. C-1 strain is grown on MEA medium at 28°C for about 7 days, the colony morphology is Figure 4 In (a) and (b), it can be seen that the strain almost covers the entire plate in seven days, with white and gray-brown hyphae, producing a large number of conidia and fewer aerial hyphae; after 14 days of growth, the colony morphology is Figure 4 In (c) and (d), it can be seen that the mycelium has changed from white and gray to gray, and almost covers the entire plate. The mycelium is in a mass, similar to mushrooms, close together with clear boundaries. The back has changed from the unique red color of the culture medium to black and red. After 30 days of growth, the colony morphology is as follows: Figure 4 From (e) and (f), we can see that there are a large number of pores distributed on the surface of the strain, and the color has not changed significantly compared with 15 days. However, the mycelium is closely attached to the culture medium, showing that the strain is malnourished. The strain should be in the decline stage at this time, resulting in the accumulation of harmful metabolites, slowing growth, and enlarged cracks on the back of the strain. The color and state are similar to the back of the PDA plate.

[0080] Example 2

[0081] This embodiment is the second embodiment of the present invention, and its purpose is to prepare a Platycladus orientalis extract, comprising the following steps:

[0082] S0. Extracting quercetin from dried Platycladus orientalis leaves to obtain Platycladus orientalis extract;

[0083] S1. Inoculate Alternaria sp. C-1 seed solution into culture medium for fermentation to obtain a fermentation broth. The specific steps are as follows:

[0084] Strain fermentation: Alternaria sp. C-1 strain growing on a PDA plate was divided into small pieces of approximately 0.5 cm × 0.5 cm and inoculated into a seed liquid culture medium. The seed liquid culture medium was placed on a shaker, set at 120 rpm, and shaken at 28°C for one week to obtain seed liquid. The seed liquid was then inoculated into a fermentation medium, cultured at 28°C for 43 days; the seed liquid inoculation volume was 3%. The culture medium consisted of water, rice, and Platycladus orientalis leaf residue, of which rice accounted for 50% by weight, and the Platycladus orientalis residue to fermentation liquid ratio was 1:10. After the culture was completed, a fermentation product of the strain was obtained, and the quercetin content in the fermentation product of the strain could reach 75 mg / L.

[0085] S2. Preparation of Platycladus orientalis extract

[0086] S21. The fermentation product of the strain was filtered through a 10 μm filter membrane and combined with the Platycladus orientalis extract. The ethanol was removed by concentrating under reduced pressure and the solution was further concentrated to 1 / 10 of the original solution to obtain a crude Platycladus orientalis extract.

[0087] S22 First, the crude extract of the Platycladus orientalis extract obtained in S21 was purified using D101 macroporous resin, and the resin dosage was determined based on the adsorption of 30 mg of quercetin by 1 gram of D101. After the macroporous resin material was completely adsorbed, it was first eluted with about 5BV of pure water to remove water-soluble impurities, and then eluted with 30% ethanol for 3-4BV, and the alcohol eluates were combined; then, using the physical and chemical properties of the quercetin compound with a molecular weight of 448.38, the alcohol eluate that passed the macroporous resin was passed through an ultrafiltration membrane, which not only removed some impurities but also had the effect of concentrating; finally, a rotary evaporator was used to further concentrate the solution that passed the ultrafiltration membrane to 10% of the resin loading amount to obtain the Platycladus orientalis extract.

[0088] Example 3

[0089] This embodiment is the third embodiment of the present invention. Different from embodiment 2, the culture temperature of the strain in S1 is 26°C.

[0090] Example 4

[0091] This embodiment is the fourth embodiment of the present invention. Different from embodiment 2, the culture temperature of the strain in S1 is 30°C.

[0092] Example 5

[0093] This embodiment is the fifth embodiment of the present invention. Different from embodiment 2, the culture medium in S1 is composed of rice and Platycladus orientalis leaf residue, wherein the rice accounts for 40% by weight.

[0094] Example 6

[0095] This embodiment is the sixth embodiment of the present invention. Different from embodiment 2, the culture medium in S1 is composed of rice and Platycladus orientalis leaf residue, of which the rice accounts for 60% by weight.

[0096] Example 7

[0097] This embodiment is the seventh embodiment of the present invention. Different from the embodiment 2, the inoculation amount of Alternaria sp. C-1 seed solution in S1 is 1%.

[0098] Example 8

[0099] This embodiment is the eighth embodiment of the present invention. Different from the embodiment 2, the inoculation amount of Alternaria sp. C-1 seed solution in S1 is 5%.

[0100] Comparative Example 1

[0101] This comparative example is the first comparative example of the present invention. Different from Example 2, the preparation method of the Platycladus orientalis extract in this comparative example only has the step of S0 extracting Platycladus orientalis leaves, and does not have the step of S1 fermenting the fermentation product with Alternaria sp. C-1 strain.

[0102] Comparative Example 2

[0103] This comparative example is the second comparative example of the present invention. Unlike Example 2, step S1 of this comparative example adopts the general strain Lactobacillus plantarum for fermentation extraction. The specific preparation steps include:

[0104] S11. The extracted Platycladus orientalis leaf residue, milk, glycerol, and deionized water were mixed to obtain a plant composite solution; the Platycladus orientalis leaf: milk: glycerol: deionized water mass ratio of 15:25:15:300;

[0105] S12. Mix 200 g of the plant complex liquid, 2 mL of Lactobacillus plantarum liquid (concentration of 10 8 CFU / mL), and 4 g of MRS medium, and ferment at 30°C for 72 h. After fermentation, sterilize the fermentation liquid at 121°C for 20 min. Then, centrifuge the sterilized fermentation liquid at 6000 rpm / min for 20 min. Collect the supernatant after centrifugation to obtain the fermentation product of the strain containing quercetin.

[0106] Comparative Example 3

[0107] This comparative example is the third comparative example of the present invention. Different from Example 2, the culture temperature of the Alternaria sp. C-1 strain in S1 is 33°C.

[0108] Comparative Example 4

[0109] This comparative example is the fourth comparative example of the present invention. Different from Example 2, the culture temperature of the Alternaria sp. C-1 strain in S1 is 23°C.

[0110] Comparative Example 5

[0111] This comparative example is the fifth comparative example of the present invention. The difference from Example 2 is that the amount of the seed liquid of Alternaria sp. C-1 strain added to S1 is 0.5%.

[0112] Comparative Example 6

[0113] This comparative example is the sixth comparative example of the present invention. The difference from Example 2 is that the amount of the seed liquid of Alternaria sp. C-1 strain added to S1 is 6%.

[0114] Comparative Example 7

[0115] This comparative example is the seventh comparative example of the present invention. Different from Example 2, the culture medium in S1 is rice, and the culture days are 25 days.

[0116] Comparative Example 8

[0117] This comparative example is the eighth comparative example of the present invention. The difference from Example 2 is that the culture medium in S1 is composed of Platycladus orientalis leaf residues, and the culture days are 65 days.

[0118] Active ingredient content detection:

[0119] The quercetin content of the Platycladus orientalis extract in each example was detected by HPLC, and two products were prepared from the extract: one was a liquid with a quercetin content of 500-1500 mg / L, and the system was a 40-60% butanediol or 1,3-propylene glycol system; the other was a powder obtained by spray drying in a centrifugal spray dryer.

[0120] (1) Detection of quercetin content in fermentation products of Alternaria sp. C-1 strain:

[0121] HPLC conditions for the detection of quercetin: Column: Inertsil ODS-HL 5μm 4.6mm*250mm;

[0122] Mobile phase: 0.1% phosphoric acid water as phase A, acetonitrile as phase B, gradient elution. Specific elution conditions are shown in Table 1, where: flow rate: 1 mL / min; column temperature: 35°C; detection wavelength: 256 nm; injection volume: 10 μL.

[0123] Table 1 HPLC mobile phase elution conditions for detecting quercetin

[0124]

[0125] The detection spectrum of quercetin in the fermentation product of the strain obtained in Example 2 is shown in Figure 5 , where t R=30.631min, the quercetin content in the fermentation product obtained in Example 2 was 75 mg / L, and the quercetin content in the fermentation products obtained from the strains in other examples and comparative examples is shown in Table 2.

[0126] (2) The calculation method of quercetin content in Platycladus orientalis extract is as follows:

[0127] Quercetin content (mg / L)

[0128] Where, Y is the peak area; a and b are the linear equation coefficients; P is the purity of the standard; N is the dilution factor.

[0129] The quercetin content in the Platycladus orientalis extracts obtained in various examples and comparative examples is shown in Table 2.

[0130] The calculation method for the increase in the yield of quercetin in Platycladus orientalis extract is as follows:

[0131] The increase in the yield of quercetin in the Platycladus orientalis extract = (mass of quercetin obtained ÷ mass of quercetin in the raw material) × 100%, wherein the content of quercetin in the raw material is about 0.3%, i.e., 300 mg / L.

[0132] The increase in quercetin yield in the Platycladus orientalis extracts obtained in the various examples and comparative examples is shown in Table 2.

[0133] Table 2 Quercetin content in Examples and Comparative Examples

[0134]

[0135] The above is only an embodiment of the present invention and does not limit the scope of patent protection. Those skilled in the art can make non-substantial changes or substitutions based on the present invention and still fall within the scope of patent protection.

Claims

1. An Alternaria sp. C-1 strain, deposited with GDMCC No: 65881.

2. A method for preparing a Platycladus orientalis extract, characterized in that: The following steps are involved: S0 extracted from the leaves of Platycladus orientalis quercetin, to obtain a Platycladus orientalis extract containing quercetin; S1. The seed solution of Alternaria sp. C-1 according to claim 1 is inoculated into a culture medium for fermentation to obtain a fermentation product of the strain; S2. The Platycladus orientalis extract and the fermentation product of the strain were mixed, filtered, concentrated, and refined to obtain a Platycladus orientalis extract; The culture medium in S1 is a mixture of rice and Platycladus orientalis leaf residue, with rice accounting for 40% to 60% by weight of the mixture; In the S1, the inoculation amount of Alternaria sp. C-1 in the culture medium is 1-5%.

3. The method for preparing the Platycladus orientalis extract according to claim 2, wherein The S1 fermentation culture temperature is 26-30°C.

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