Platycladus orientalis extract as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of microbial fermentation, and discloses a platycladus orientalis extract as well as a preparation method and application thereof. The platycladus orientalis extract is prepared by fermenting an Alternaria sp. C-1 strain on the basis of conventionally extracting quercitrin from platycladus orientalis leaves, and the yield of quercitrin in the finally obtained platycladus orientalis extract is increased by 25%. The preservation number of the strain of the Alternaria sp. (Alternaria sp.) C-1 is GDMCC No: 65881, and the preservation number of the strain is GDMCC No: 65881. According to the method for fermenting the Alternaria sp., a liquid culture medium is inoculated with a seed solution of the Alternaria sp. C-1 for fermentation culture, the strain has a relatively short growth cycle and a high propagation speed, quercitrin can be continuously produced, the highest quercitrin content in a strain fermentation product can reach 75 mg / L, and the quercitrin content in the strain fermentation product can reach 75 mg / L; the obtained platycladus orientalis extract has the beneficial technical effects of high quercitrin content, stable quercitrin yield, reduction of consumption of the platycladus orientalis leaves, environmental friendliness and the like.

Description

Technical Field

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

[0002] Platycladus orientalis leaves, also known as cypress leaves, cluster cypress leaves, and flat cypress leaves, are the dried young branches and leaves of the cypress plant Platycladus orientalis L. The main active ingredient in Platycladus orientalis leaves is quercitrin. According to the Chinese Pharmacopoeia 2025 edition, calculated on a dry product basis, the content of quercitrin in Platycladus orientalis leaves shall not be less than 0.1%. Quercitrin is a flavonoid monomer compound widely present in plants and has various pharmacological effects such as anti - hair loss, antioxidant, anti - tumor, hypoglycemic, and hypolipidemic effects. In addition, quercitrin also has certain anti - inflammatory activity, mainly manifested as an inhibitory effect on pro - inflammatory factors.

[0003] Most of the existing technologies directly extract quercitrin from Platycladus orientalis leaves using water or alcohol - containing solutions, and there are mainly the following problems: (1) The low content of quercitrin in Platycladus orientalis leaves: The conventional extraction method is to crush Platycladus orientalis leaves and extract quercitrin using an alcohol - containing solution, resulting in a low content of quercitrin in the obtained Platycladus orientalis extract; (2) The unstable content of quercitrin in the extract: Factors such as environmental pollution, seasonal cyclic changes, and soil quality will affect the growth of Platycladus orientalis, hinder the stable production of secondary metabolites in Platycladus orientalis, greatly increase the difficulty of obtaining quercitrin, and cause the unstable content of quercitrin in the obtained Platycladus orientalis extract; (3) The huge consumption of Platycladus orientalis leaves: Calculated based on a quercitrin content of 0.1%, about 1000 g of Platycladus orientalis leaves are required to extract 1 g of quercitrin. The large demand for quercitrin will lead to the over - exploitation of Platycladus orientalis resources. Summary of the Invention

[0004] The present invention provides a strain of Alternaria sp. C - 1, and a fermentation product containing quercitrin can be obtained after fermentation of the Alternaria sp. C - 1 strain.

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

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

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

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

[0009] To solve the above technical problems, the technical solution provided by the present invention is as follows: An Alternaria sp. strain C-1, with the preservation number of GDMCC No: 65881.

[0010] The Alternaria sp. strain C-1 has the following properties: 1. Morphological characteristics On the PDA medium at 28°C, after the Alternaria sp. strain C-1 grows for about one week, it covers the whole plate. Its colony is flat, with regular edges, yellowish-brown in the middle of the front, white around, and there is no obvious difference in color between the front and the back. The hyphae are sparse. After growing for about one month, the bottom layer of the colony is yellowish-brown, and the upper layer is grayish-white in hyphal form, and the conidia are dark brown.

[0011] 2. Characteristics on various media (1) Potato Dextrose Agar medium (PDA): The hyphae cover the whole plate, with white and brown hyphae alternating, yellowish-brown in the middle of the front, and little difference in color between the front and the back, and a large number of conidia are produced; (2) Malt Extract Agar medium (MEA): The plate is covered with colonies of different sizes, with brown in the middle of the colony and yellow or white around, the colony is flat, and there are cracks on the back; (3) Rose Bengal Agar medium (REA): The strain grows for seven days and almost covers the whole plate, with white and grayish-brown hyphae alternating, a large number of conidia are produced, and the aerial hyphae are less.

[0012] 3. Metabolic characteristics Regarding the carbon source utilization of Alternaria sp. strain C-1 Well utilized: Starch, Glucose, Fructose Generally utilized: Sucrose, Lactose The differentiating characteristics of the Alternaria sp. strain C-1 from known Alternaria are: The characteristics of members of the genus Alternaria are as follows: The conidiophores are unbranched or sometimes branched, erect or curved, solitary or clustered, arising from the hyphae or borne on simple stromata, sometimes swollen at the base, brown of varying shades; the scars of the spores are clearly visible, with a darker color at the edge and a lighter color in the center; the conidia are ovoid, obclavate, or obpyriform, with an obvious hilum, the conidial bodies being brown of varying shades or bluish yellowish brown, smooth or verrucose on the surface, with several transverse and longitudinal or oblique true septa, not constricted or significantly constricted at the septa. In most cases, the body of the spore tapers gradually towards the apex, without a beak or with a short beak, columnar or slender and long beak, the beak septate or non-septate, and the color is significantly lighter than that of the spore body. The beak sometimes becomes a secondary conidiophore (pseudo-beak), producing secondary conidia and forming short or long chains of spores.

[0013] The C-1 strain described in the present invention belongs to the genus Alternaria, but there are some differences in morphological characteristics. The edge of this fungus is regular, yellowish brown in the middle of the front and white around, the hyphae grow prostrate, the bottom layer of the colony is yellowish brown and the upper layer is grayish white after growing on PDA for 30 days, and there is less aerial hyphae; a large number of conidia are produced about one week after growing on PDA. The spores have transverse and longitudinal septa, are oblong in shape, the conidial wall is brick-shaped, with 3 - 5 transverse septa, significantly brown, of different sizes, mostly 30 - 36 microns long and 10 - 25 microns wide, and often dozens form a chain.

[0014] The present invention ferments Alternaria sp. C-1 to produce a fermentation product, which has the following advantages: 1. High content of quercitrin: The C-1 strain of Alternaria sp. 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. Through fermentation, it can continuously produce quercitrin using Platycladus orientalis medicinal residues. The highest content of quercitrin in the fermentation product can reach 75 mg / L.

[0015] 2. Stable quercitrin yield: The C-1 strain of Alternaria sp. has relatively loose requirements for culture conditions and can grow and metabolize using a variety of conventional carbon and nitrogen sources. The content of quercitrin in the fermentation product of the strain is stable, which is expected to solve the problem of unstable quercitrin yield in the obtained extracts caused by the influence of environmental pollution, seasonal periodic changes, land quality, etc. on the growth of Platycladus orientalis in nature.

[0016] 3. Reduction of the consumption of Platycladus orientalis leaves: The suitable fungus - C-1 strain of Alternaria sp. screened from Platycladus orientalis leaves by the present invention is rich in quercitrin in its fermentation product, which can, to a certain extent, get rid of the dependence on Platycladus orientalis plant resources and reduce the phenomenon of overexploitation of plants.

[0017] The present invention also protects a Platycladus orientalis extract, which contains a fermentation product prepared by self-fermentation of the Alternaria sp. C-1, and the fermentation product contains quercitrin.

[0018] The Platycladus orientalis extract described in the present invention also contains a Platycladus orientalis extract solution.

[0019] The present invention also protects a method for preparing the Platycladus orientalis extract, which includes the following steps: S1. Inoculate the seed liquid of the Alternaria sp. C-1 into a culture medium for fermentation culture to obtain a strain fermentation product.

[0020] Preferably, the method for preparing the Platycladus orientalis extract described in the present invention further includes the following step: S0. Extract quercitrin from Platycladus orientalis leaves to obtain a Platycladus orientalis extract solution.

[0021] The method for preparing the Platycladus orientalis extract described in the present invention further includes the following step: S2. Mix, filter, concentrate, and refine the Platycladus orientalis extract solution and the strain fermentation product to obtain the Platycladus orientalis extract.

[0022] Preferably, the culture medium in S1 is a mixture composed of rice and Platycladus orientalis leaf residue.

[0023] More preferably, in the mixture composed of rice and Platycladus orientalis leaf residue in S1, the mass ratio of rice is 40% - 60%.

[0024] The Platycladus orientalis leaf residue in S1 of the present invention is the residue left after extracting quercitrin from Platycladus orientalis leaves.

[0025] The present invention can realize the secondary utilization of Platycladus orientalis leaf residue.

[0026] The existing method for extracting quercitrin from Platycladus orientalis leaves is to extract quercitrin with ethanol of a certain concentration. After ethanol extraction, a Platycladus orientalis extract solution containing quercitrin and Platycladus orientalis leaf residue are obtained. The extraction rate of extracting quercitrin from Platycladus orientalis leaves by heating with ethanol can reach up to 90%, that is, there is still about 10% content of quercitrin remaining in the Platycladus orientalis leaf residue.

[0027] The present invention uses a mixture composed of rice and Platycladus orientalis leaf residue as the culture medium. On the one hand, it realizes the secondary utilization of Platycladus orientalis leaf residue and reduces resource waste; on the other hand, since there are still a small amount of quercitrin and various intermediate substances for generating quercitrin in the Platycladus orientalis leaf residue, it is beneficial for the C-1 strain to ferment and produce quercitrin.

[0028] The culture medium described in the present invention adds rice on the basis of Platycladus orientalis leaf residue, provides sufficient nutrients for the growth of the C-1 strain, and further increases the yield of quercitrin in the strain fermentation product.

[0029] Preferably, the inoculum size of the Alternaria sp. C-1 seed solution in the medium in S1 is 1-5%.

[0030] Preferably, the fermentation temperature in S1 is 26-30 °C In the present invention, the fermentation time in S1 is 25-65 days.

[0031] S2 specifically includes the following steps: S21. Combine the strain fermentation product obtained in S1 with the Platycladus orientalis extract obtained in S0, and then filter and concentrate to obtain a crude Platycladus orientalis extract rich in quercitrin. S22. Purify and concentrate the crude Platycladus orientalis extract successively through a D101 macroporous resin column and an ultrafiltration membrane to obtain a Platycladus orientalis extract.

[0032] Specifically, the process in S21 is to combine the strain fermentation product in S1 and the alcohol-extracted Platycladus orientalis extract in S0, filter through a 10 μm filter membrane, concentrate to 1 / 10 of the solution, and then carry out purification.

[0033] Specifically, the purification process in S22 can use an ultrafiltration membrane with a molecular weight lower than that of quercitrin for concentration and impurity removal, and collect the retentate.

[0034] More specifically, the purification process in S22 can use a macroporous resin material to adsorb and remove impurities on the extract.

[0035] In addition, methods such as silica gel column chromatography, polyamide column chromatography, and Sephadex gel column chromatography can also be used for purification and impurity removal of the fermentation product.

[0036] The present invention also protects the application of the above fermentation product as an anti-hair loss, soothing, and antioxidant component in cosmetics.

[0037] Preservation of biological materials: The Alternaria sp. C-1 strain of the present invention was deposited at the Guangdong Provincial Microbial Culture Collection Center on February 14, 2025. Its abbreviation is GDMCC, and the deposit number of the collection center is GDMCC No: 65881. Address of the collection center: 5th Floor, Building 59, 100th Yard, Xianlie Middle Road, Guangzhou. Description of the drawings

[0038] Figure 1 It is a two-month growth state diagram of the Alternaria sp. C-1 strain on a PDA medium, where (a) is the front view and (b) is the back view.

[0039] Figure 2Growth status diagrams of Alternaria sp. strain C-1 on PDA medium at various time periods. Among them, figure (a) is the front side after 7 days of growth, figure (b) is the back side after 7 days of growth, figure (c) is the front side after 14 days of growth, figure (d) is the back side after 14 days of growth, figure (e) is the front side after 30 days of growth, and figure (f) is the back side after 30 days of growth.

[0040] Figure 3 Growth status diagrams of Alternaria sp. strain C-1 on MEA medium at various time periods. Among them, figure (a) is the front side after 7 days of growth, figure (b) is the back side after 7 days of growth, figure (c) is the front side after 14 days of growth, figure (d) is the back side after 14 days of growth, figure (e) is the front side after 30 days of growth, and figure (f) is the back side after 30 days of growth.

[0041] Figure 4 Growth status diagrams of Alternaria sp. strain C-1 on REA medium at various time periods. Among them, figure (a) is the front side after 7 days of growth, figure (b) is the back side after 7 days of growth, figure (c) is the front side after 14 days of growth, figure (d) is the back side after 14 days of growth, figure (e) is the front side after 30 days of growth, and figure (f) is the back side after 30 days of growth.

[0042] Figure 5 Detection chromatogram of quercitrin content in the fermentation product obtained in Example 2. Detailed implementation manners

[0043] The following further elaborates on the present invention in detail in conjunction with the specific implementation manners.

[0044] The components of the PDA solid medium in the examples are: 300 g of potatoes, 20 g of glucose, 15 g of agar, and 0.1 g of chloramphenicol per 1 L of PDA medium.

[0045] In the examples, the Platycladus orientalis residue is obtained by extracting quercitrin twice from dried Platycladus orientalis with 50% ethanol at 70 °C.

[0046] The components of the Alternaria sp. strain C-1 seed liquid medium used in the examples and comparative examples are: 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 per 1 L of seed liquid; the pH of the seed liquid medium is 5 - 6.

[0047] In the preparation methods of the Platycladus orientalis extracts in each example and comparative example, the specific extraction method of the S0 Platycladus orientalis extract is as follows: S01. Crush 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. After extraction, separate the extract from the residue to obtain a Platycladus orientalis extract containing quercitrin. The quercitrin content in the Platycladus orientalis leaves used in each example and comparative example of this application is 0.3%. S02. Combine the two Platycladus orientalis alcohol extracts, and the quercitrin content in the extract is about 135 mg / L.

[0048] Example 1 This example is a screening method for Alternaria sp. strain C-1, which specifically includes the following steps: S1. Collect and isolate strains: Fresh Platycladus orientalis leaves are 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) respectively.

[0049] First, cut the Platycladus orientalis leaves into small pieces of 0.5 cm × 0.5 cm, inoculate them on the prepared PDA solid medium, and evenly inoculate 3 explants in each petri dish. Streptomycin (10 mg / L) and tetracycline (3 mg / L) are added to the medium. Then, place them in a constant temperature incubator at 28 °C for dark incubation. When hyphae grow from the Platycladus orientalis leaf explants inoculated on the PDA medium, use an inoculation needle to pick the tip hyphae at the edge of the colony with different appearances in time and transfer them to a new PDA plate. Finally, invert the plate and culture it in a constant temperature incubator at 28 °C in the dark for 3 - 7 d, observe and record the growth of hyphae every day. When colonies with different colors or morphologies grow out, continue the purification operation by the hyphal tip purification method until the colony is a single pure culture. A total of 21 strains are isolated from the Platycladus orientalis leaves from the three production areas, and the strain codes are marked as C-1, C-2... C-3 respectively.

[0050] S2. Strain culture and identification: The 21 strains obtained in S1 were inoculated into a 500 mL conical flask containing rice culture medium, and placed in a constant temperature incubator. After culturing at 28 °C for a period of time, the fermentation culture of the strain was obtained, and then the culture was ultrasonically extracted with 75% ethanol for 30 min. The aluminum chloride colorimetric method was used to detect whether flavonoid components were generated in the solution, and then HPLC was used to detect whether quercetin was present in the solution containing flavonoid components. The test found that the fermentation broth of the C-1 strain isolated from Guizhou could form a yellow complex with aluminum chloride solution (1 mL of the solution was taken, and 2% aluminum chloride ethanol solution was added, showing that a yellow complex was generated), and the flavonoid components produced contained quercetin. The strain was identified by molecular biology and was identified as Alternaria sp. C-1 was deposited in the Guangdong Microbiological Culture Collection Center, and the registration number of the collection center is GDMCC 65881, with the following characteristics: Morphological characteristics / colony characteristics: After Alternaria sp. C-1 strain was grown on PDA medium at 28°C for about 2 months, the colony morphology was Figure 1 .

[0051] 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 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 dark brown.

[0052] Characteristics on various media (1) PDA medium: After Alternaria sp. C-1 strain was grown on PDA medium at 28°C for about 7 days, the colony morphology was 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, and a large number of conidia are produced; after 14 days of growth, the colony morphology is Figure 2 From (c) and (d) in the figure, 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 of the 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 and the part was still white. The back of the strain was not much different from that on the 14th day of growth. (2) MEA medium: After Alternaria sp. C-1 strain was grown on MEA medium at 28°C for about 7 days, the colony morphology was Figure 3 From (a) and (b) in Figure 1, we can see that the plate is covered with colonies of different sizes, and the colonies are brown in the middle and yellow or white around them. The colonies are flat and have cracks on the back. After 14 days of growth, the colony morphology is Figure 3 From (c) and (d) in Figure 1, we can see that the strain has spread all over the 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, and the back of the plate is black with 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 became larger, and no more droplets were produced. The surface of the strain covering the entire plate had pore-like structures similar to stomata visible to the naked eye, the color of the hyphae was bluish-brown, and there was no obvious change on the back of the strain. (3) REA medium: After Alternaria sp. C-1 strain was grown on MEA medium at 28°C for about 7 days, the colony morphology was Figure 4 From (a) and (b) in Figure 1, we can see that the strain almost covered 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 was Figure 4 In (c) and (d), it can be seen that the mycelium has completely 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 obvious boundaries, and the back has begun to change from the unique red color of the culture medium to black and red. After 30 days of growth, the colony morphology is Figure 4 From (e) and (f), we can see that: there are a large number of holes distributed on the surface of the strain, and the color has not changed significantly compared with 15 days, but 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.

[0053] Example 2 This embodiment is the second embodiment of the present invention, and its purpose is to prepare a Platycladus orientalis extract, comprising the following steps: S0. Extracting quercetin from dried Platycladus orientalis leaves to obtain Platycladus orientalis extract; S1. Inoculate the seed solution of Alternaria sp. C-1 into the culture solution for fermentation to obtain the fermentation solution of the strain. The specific steps are as follows: Strain fermentation: Cut the Alternaria sp. C-1 strain grown on a PDA plate into small pieces about 0.5 cm × 0.5 cm and inoculate them into the seed liquid medium. Place the seed liquid medium on a shaker, set it at 120 rpm, and culture it at 28°C with shaking for one week to obtain the seed liquid; then inoculate the seed liquid into the fermentation medium, with a culture temperature of 28°C and a culture time of 43 days; the inoculation amount of the seed liquid is 3%; the medium consists of water, rice, and Platycladus orientalis leaf residue, where the mass ratio of rice is 50%, and the ratio of Platycladus orientalis residue to the fermentation broth is 1:10. After culturing, the strain fermentation product is obtained, and the quercitrin content in the strain fermentation product can reach 75 mg / L; S2. Preparation of Platycladus orientalis extract S21. Filter the strain fermentation product with a 10-μm filter membrane and combine it with the Platycladus orientalis extract solution. Concentrate it under reduced pressure to remove ethanol and continue to concentrate it to 1 / 10 of the original solution to obtain the crude Platycladus orientalis extract; S22 First, purify the crude Platycladus orientalis extract obtained in S21 using D101 macroporous resin. Determine the resin dosage according to 1 g of D101 adsorbing 30 mg of quercitrin. After the macroporous resin material is completely adsorbed, first wash it with about 5 BV of pure water to remove water-soluble impurities, and then elute it with 30% ethanol for 3 - 4 BV, and combine the ethanol eluate; then, utilize the physicochemical property that the molecular weight of the quercitrin compound is 448.38. Pass the ethanol eluate passing through the macroporous resin through an ultrafiltration membrane, which can remove some impurities and also has a concentration effect; finally, use a rotary evaporator to further concentrate the solution passing through the ultrafiltration membrane to 10% of the resin loading amount to obtain the Platycladus orientalis extract.

[0054] Example 3 This example is the 3rd example of the present invention. Different from Example 2, the strain culture temperature in S1 is 26°C.

[0055] Example 4 This example is the 4th example of the present invention. Different from Example 2, the strain culture temperature in S1 is 30°C.

[0056] Example 5 This example is the 5th example of the present invention. Different from Example 2, the medium in S1 consists of rice and Platycladus orientalis leaf residue, where the mass ratio of rice is 40%.

[0057] Example 6 This example is the 6th example of the present invention. Different from Example 2, the medium in S1 consists of rice and Platycladus orientalis leaf residue, where the mass ratio of rice is 60%.

[0058] Example 7 This example is the 7th example of the present invention. Different from Example 2, the inoculation amount of the Alternaria sp. C-1 seed liquid in S1 is 1%.

[0059] Example 8 This example is the 8th example of the present invention. Different from Example 2, the inoculation amount of the Alternaria sp. C-1 seed liquid in S1 is 5%.

[0060] Comparative Example 1 This comparative example is the 1st comparative example of the present invention. Different from Example 2, in this comparative example, the preparation method of the Platycladus orientalis extract only has the S0 Platycladus orientalis leaf extraction step, and there is no S1 step of fermenting and preparing the fermentation product with the Alternaria sp. C-1 strain.

[0061] Comparative Example 2 This comparative example is the 2nd comparative example of the present invention. Different from Example 2, in the S1 step of this comparative example, a general strain, Lactobacillus plantarum, is used for fermentation and extraction. The specific preparation steps include: S11. After mixing the extracted Platycladus orientalis residue, milk, glycerol, and deionized water, a plant composite liquid is obtained; the mass ratio of Platycladus orientalis : milk : glycerol : deionized water is 15:25:15:300; S12. Mix 200 g of the plant composite liquid, 2 mL of the Lactobacillus plantarum liquid (concentration 10^8 CFU / mL), and 4 g of MRS medium, 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 a speed of 6000 rpm / min for 20 min, and take the supernatant after centrifugation to obtain the strain fermentation product containing quercetin.

[0062] Comparative Example 3 This comparative example is the 3rd 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.

[0063] Comparative Example 4 This comparative example is the 4th 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.

[0064] Comparative Example 5 This comparative example is the 5th comparative example of the present invention. Different from Example 2, the addition amount of the Alternaria sp. C-1 strain seed liquid in S1 is 0.5%.

[0065] Comparative Example 6 This comparative example is the 6th comparative example of the present invention. The difference from Example 2 is that the addition amount of the Alternaria sp. C-1 strain seed liquid in S1 is 6%.

[0066] Comparative Example 7 This comparative example is the 7th comparative example of the present invention. The difference from Example 2 is that the culture medium in S1 is rice and the culture days are 25 days.

[0067] Comparative Example 8 This comparative example is the 8th 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.

[0068] Detection of active ingredient content: The quercetin content of the Platycladus orientalis extract in each example was detected by HPLC, and two products were prepared from this extract. One is a liquid with a quercetin content of 500 - 1500 mg / L and the system is a 40 - 60% butanediol or 1,3 - propanediol system; the other is a powder obtained by spray drying with a centrifugal spray dryer.

[0069] (1) Detection of quercetin content in the fermentation product of Alternaria sp. C-1 strain: HPLC conditions for detecting quercetin: Among them, the chromatographic column: Inertsil ODS-HL 5μm 4.6mm * 250mm; Mobile phase: 0.1% phosphoric acid water is phase A, acetonitrile is phase B, gradient elution, and the 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.

[0070] Table 1 HPLC mobile phase elution conditions for detecting quercetin

[0071] The detection chromatogram of quercetin in the fermentation product of the strain obtained in Example 2 is shown in Figure 5 , where t R = 30.631 min, the quercetin content in the fermentation product obtained in Example 2 is 75 mg / L, and the quercetin contents in the fermentation products of the strains obtained in the other examples and comparative examples are shown in Table 2.

[0072] (2) The calculation method of quercetin content in the Platycladus orientalis extract is as follows: Quercetin content (mg / L) =

[0073] Among them, Y - peak area; a, b - coefficients of the linear equation; P - purity of the standard product; N - dilution factor The quercetin content in the Platycladus orientalis extracts obtained in each example and comparative example is shown in Table 2.

[0074] The calculation method for the increase in the yield of quercetin in the Platycladus orientalis extract is as follows: Increase in the yield of quercetin in the Platycladus orientalis extract = (mass of obtained quercetin ÷ mass of quercetin in raw material) × 100%, where the quercetin content in the raw material is about 0.3% i.e., 300 mg / L.

[0075] The increase in the yield of quercetin in the Platycladus orientalis extracts obtained in each example and comparative example is shown in Table 2.

[0076] Table 2 Quercetin content in examples and comparative examples

[0077] As described above, this is only the implementation mode of the invention, and the patent protection scope is not limited thereby. Those skilled in the art make non-substantive changes or substitutions based on the invention, and still fall within the patent protection scope.

Claims

1. An Alternaria sp. strain C-1 with the preservation number of GDMCC No: 65881.

2. A Platycladus orientalis extract, characterized in that, It contains a fermentation product prepared by self-fermentation of the Alternaria sp. strain C-1 described in claim 1, and the fermentation product of this strain contains quercitrin.

3. The Platycladus orientalis extract according to claim 2, wherein, It also contains Platycladus orientalis extract.

4. The preparation method of the Platycladus orientalis extract according to claim 2, characterized in that, It includes the following steps: S1. Inoculate the seed liquid of the Alternaria sp. strain C-1 described in claim 1 into a culture medium for fermentation culture to obtain a strain fermentation product.

5. The preparation method of Platycladus orientalis extract according to claim 4, characterized in that, It also includes the following steps: S0. Extract quercitrin from Platycladus orientalis leaves to obtain Platycladus orientalis extract.

6. The preparation method of Platycladus orientalis extract according to claim 4, characterized in that, In the S1, the culture medium is a mixture composed of rice and Platycladus orientalis leaf residue.

7. The preparation method of the Platycladus orientalis extract according to claim 6, wherein, In the mixture composed of rice and Platycladus orientalis leaf residue, the mass ratio of rice is 40% - 60%.

8. The preparation method of the Platycladus orientalis extract according to claim 4, characterized in that, In the S1, the inoculation amount of Alternaria sp. strain C-1 in the culture medium is 1% - 5%.

9. The preparation method of the Platycladus orientalis extract according to claim 1, wherein, The fermentation culture temperature in the S1 is 26 - 30 °C.

10. Application of the fermentation product described in claim 2 as an anti-hair loss, soothing, and antioxidant ingredient in cosmetics.

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