Strain zjxqj001 of spirulina platensis with high protein amino acid content and application thereof

Abstract

The application discloses a high-protein amino acid content strain of Exidia glandulosa ZJXQJ001 and application thereof, the preservation number of which is CGMCC NO.40778, and the gene sequence is shown as SEQ NO.1. The strain of Exidia glandulosa ZJXQJ001 is higher than other varieties of existing Exidia glandulosa in protein, various amino acid polysaccharides, adenosine and triterpenes, and has higher industrial application value, and can meet the production needs of various downstream products. The strain has good antioxidant, anti-inflammatory, antiviral, immune and metabolic regulation effects, and also has good treatment and protection effects on a cardiovascular system, and is a very excellent low-fat high-protein health food.

Description

Technical Field

[0001] This application relates to the field of microbial technology, and in particular to a *Hydrangea hydrangea* strain ZJXQJ001 with high protein amino acid content and its applications. Background Technology

[0002] Hydrangea ( Sparassis crispa (Wulfen) Fr. It belongs to the kingdom Fungi, phylum Basidiomycota, class Agaricomycetes, order Polyporales, family Sprassidaceae, and genus Sprassis.

[0003] The fruiting body of *Hydrangea macrophylla* is fleshy, branching out from a stout stem with numerous intricate petals at the tips, hence its name, resembling a giant hydrangea. *Hydrangea macrophylla* requires more than 10 hours of sunlight daily to grow, making it the world's only "sunshine mushroom." Wild *Hydrangea macrophylla* grows in spruce, fir, or pine forests and mixed forests. The base of the stem is root-like and connected to the tree roots, 20-50 cm in diameter, white to dirty yellow; the petals are fan-shaped or ginkgo leaf-shaped, thin with uneven, curved edges, darkening in color when dry, and becoming hard and brittle. *Hydrangea macrophylla* is characterized by high β-glucan content, high protein, high potassium and low sodium, and is rich in vitamins, minerals, ergosterol, etc., possessing anti-inflammatory, antibacterial, anti-tumor, immunomodulatory, hematopoietic-promoting, blood pressure-lowering, and skin-whitening effects, making it an ideal healthy edible and medicinal fungus.

[0004] Currently, only a few institutions or companies in countries such as Japan, South Korea, and China have achieved artificial cultivation of *Hydrangea macrophylla*. Research on *Hydrangea macrophylla* in my country began in the 1980s, with artificial cultivation achieved in 2005 and industrialized cultivation in 2010. In recent years, cultivation techniques have gradually caught up with those abroad, but there are still shortcomings in its pharmacology, polysaccharide extraction, omics, preservation, and deep processing, indicating a very broad research prospect. Due to the unique biological morphology and cultivation methods of *Hydrangea macrophylla*, only a few companies in China currently cultivate it. The types and quantities of *Hydrangea macrophylla* on the market are limited, the classification is unclear, and the yield and quality vary greatly. Therefore, developing a precise and effective identification system for *Hydrangea macrophylla* strains using molecular biology techniques is extremely important.

[0005] Meanwhile, the existing *Hydrangea macrophylla* strains contain low levels of bioactive proteins and amino acids, which cannot meet the requirements for downstream extraction, resulting in high consumption of raw materials and hindering the development and production of related downstream products. Current technologies primarily address this issue by improving the extraction methods for *Hydrangea macrophylla*.

[0006] The information disclosed in the background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art. Summary of the Invention

[0007] This application addresses the aforementioned technical problems by providing a *Hydrangea hydrangea* strain ZJXQJ001 with high protein amino acid content and its application. The protein content of this strain is 22.1%, which is significantly higher than that of existing *Hydrangea hydrangea* varieties, and can meet the production needs of various downstream products.

[0008] This application provides a *Hydrangea hydrangea* strain ZJXQJ001 with high protein amino acid content, accession number CGMCC NO.40778, and its gene sequence is shown in SEQ NO.1.

[0009] Preferably, the fruiting body of strain ZJXQJ001 has the following protein content: 25.1 g / 100g, aspartic acid 1.32 g / 100g, threonine 0.64 g / 100g, serine 0.67 g / 100g, glutamic acid 1.60 g / 100g, alanine 0.76 g / 100g, valine 0.66 g / 100g, methionine 0.137 g / 100g, isoleucine 0.46 g / 100g, leucine 0.80 g / 100g, tyrosine 0.48 g / 100g, phenylalanine 0.64 g / 100g, and histidine 0.46 g / 100g. The content of lysine is 0.90g / 100g, arginine is 0.72g / 100g, and proline is 0.89g / 100g. Preferably, the fruiting body of strain ZJXQJ001 contains 718 mg / kg adenosine, 0.75 g / 100g crude fiber, 0.25 g / 100g crude fat, 6.38 g / 100g crude polysaccharide, and 2.41% total triterpenoids.

[0010] Preferably, the fruiting rate of strain ZJXQJ001 is 96-97%, and the biological efficiency is 66-71.45%.

[0011] Another aspect of this application provides an SSR molecular marker for identifying the *Hydrangea hydrangea* strain ZJXQJ001 with high protein amino acid content as described above, characterized in that it is an XQJSSR005 molecular marker pair, an XQJSSR031 molecular marker pair, an XQJSSR085 molecular marker pair, an XQJSSR115 molecular marker pair, an XQJSSR145 molecular marker pair, and an XQJSSR160 molecular marker pair; The number of alleles in the XQJSSR005 molecular marker pair is 3; The number of alleles in the XQJSSR031 molecular marker pair is 4; The number of alleles in the XQJSSR085 molecular marker pair is 3; The number of alleles in the XQJSSR115 molecular marker pair is 5; The number of alleles in the XQJSSR145 molecular marker pair is 4; The number of alleles in the XQJSSR160 molecular marker pair is 4.

[0012] Preferably, the XQJSSR005 molecular marker pair includes: a forward primer as shown in SEQ NO.2 and a reverse primer as shown in SEQ NO.3; The XQJSSR031 molecular marker pair includes: a forward primer as shown in SEQ NO.4 and a reverse primer as shown in SEQ NO.5; The XQJSSR085 molecular marker pair includes: a forward primer as shown in SEQ NO.6 and a reverse primer as shown in SEQ NO.7; The XQJSSR115 molecular marker pair includes: a forward primer as shown in SEQ NO.8 and a reverse primer as shown in SEQ NO.9; The XQJSSR145 molecular marker pair includes: a forward primer as shown in SEQ NO.10 and a reverse primer as shown in SEQ NO.11; The XQJSSR160 molecular marker pair includes: a forward primer as shown in SEQ NO.12 and a reverse primer as shown in SEQ NO.13.

[0013] Another aspect of this application provides a detection reagent for the *Hydrangea hydrangea* strain ZJXQJ001 with high protein amino acid content as described above, characterized in that it includes: an SSR molecular marker as described above for the identification of *Hydrangea hydrangea* strain ZJXQJ001 with high protein amino acid content.

[0014] Another aspect of this application provides a method for identifying the *Hydrangea hydrangea* strain ZJXQJ001 with high protein amino acid content as described above, characterized by the following steps: extracting DNA from the analyte and then performing SSR-labeled PCR amplification on the obtained DNA using the SSR molecular marker described above for identifying *Hydrangea hydrangea* strain ZJXQJ001 with high protein amino acid content; taking 2 μL of the PCR product for agarose gel electrophoresis and fluorescence capillary electrophoresis detection, comparing the obtained fluorescence peak patterns, and obtaining the identification result; Preferably, the strain that conforms to the SSR allelic fragment numbering combination of 3 / (3+4) / (1+2) / (2+3) / 3 / (3+4) is the *Hydrangea hydrangea* strain ZJXQJ001.

[0015] Another aspect of this application provides a method for cultivating the *Hydrangea hydrangea* strain ZJXQJ001 with high protein amino acid content as described above, comprising the following steps: 1) After preparing the cultivation substrate, add water and mix well. The resulting cultivation substrate should have a moisture content of 60-65%. 2) Mechanized bagging of the prepared cultivation medium into 17cm×35cm×0.05cm plastic bags, punching holes in the center, with each bag containing 350g of material (dry weight). Autoclave and cool to room temperature. 3) Use liquid inoculum for inoculation, with an inoculation volume of 25-30 mL / bag, and incubate indoors under light for 40-50 days at 22-25℃ and 60%-70% humidity. 4) Once the mycelium has grown to more than 70% and primordia have begun to appear, it can be transferred to the fruiting room for management. It should be kept at 20-22℃, with an air humidity of 80-90%, 10 hours of light per day, and a light intensity of 200 lx for 8-12 days. 5) Once the primordia have covered the surface of the cultivation medium at the bag opening, adjust the temperature to 18-20℃, provide 12 hours of light per day, with a light intensity of 500-1000 lx, and intermittently expose the primordia to blue and white light. After 10-15 days of cultivation, the primordia will have basically differentiated into leaves. 6) Open the bag, adjust the temperature to 16-18℃ and the air humidity to 90-95%, increase ventilation, and cultivate for 15-20 days until the fruiting bodies emerge from the bag opening. Once the leaves unfold and turn from white to pale yellow, they are ready for harvesting.

[0016] Preferably, the cultivation substrate is prepared from 76 parts sawdust, 18 parts wheat bran, 2 parts corn flour, 1.5 parts glucose, 1.5 parts gypsum, and 1 part superphosphate.

[0017] Preferably, the cultivation substrate is prepared from 50 parts sawdust, 25 parts cottonseed hulls, 16 parts wheat bran, 6 parts corn flour, 1 part calcium carbonate, 1.5 parts lime, and 0.5 parts potassium dihydrogen phosphate.

[0018] The beneficial effects that this application can produce include: 1) This application provides the *Hydrangea hydrangea* strain ZJXQJ001 with high protein amino acid content and its applications. This strain ZJXQJ001 contains higher levels of various amino acids, polysaccharides, adenosine, and triterpenes than other existing *Hydrangea hydrangea* varieties, exhibiting high industrial application value and meeting the production needs of various downstream products. It possesses excellent antioxidant, anti-inflammatory, antiviral, immune, and metabolic regulatory effects, and also has good therapeutic and protective effects on the cardiovascular system, making it an excellent low-fat, high-protein health food.

[0019] 2) This application provides the *Hydrangea macrophylla* strain ZJXQJ001 with high protein amino acid content and its application. The *Hydrangea macrophylla* SSR molecular marker exhibits specificity for *Hydrangea macrophylla* ZJXQJ001 among the eight tested *Hydrangea macrophylla* cultivated strains (including ZJXQJ001, G, GZ, RK, S, SL, SX, and YN). Using the primer combination provided by this invention, *Hydrangea macrophylla* strains can be conveniently and quickly distinguished from the other seven strains, achieving advantages such as cost savings, improved efficiency, convenient operation, and accurate results. It can be used for the rapid identification of *Hydrangea macrophylla* strain ZJXQJ001 and has good application prospects.

[0020] 3) The accurate identification method of the *Hydrangea fuciformis* strain ZJXQJ001 can prevent the selected superior strains of *Hydrangea fuciformis* from being mixed with other varieties in the production, operation and market circulation process, thereby effectively protecting its intellectual property rights. It is of great significance for the authenticity identification of varieties in the production process of *Hydrangea fuciformis*.

[0021] A new strain of *Hydrangea macrophylla*, ZJXQJ001, was deposited on August 14, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and identified as *Hydrangea macrophylla*. Sparassis sp The accession number is CGMCC NO.40778. Attached Figure Description

[0022] Figure 1 The relative molecular weight peaks of allelic sites obtained by sequentially detecting the primer XQJSSR005 provided for this application in the selected *Hydrangea spp.* ZJXQJ001 and seven *Hydrangea spp.* strains G, GZ, RK, S, SL, SX, and YN are shown in the diagrams. a) *Hydrangea spp.* ZJXQJ001; b) *Hydrangea spp.* strain G; c) *Hydrangea spp.* strain GZ; d) *Hydrangea spp.* strain RK; e) *Hydrangea spp.* strain S; f) *Hydrangea spp.* strain SL; g) *Hydrangea spp.* strain SX; h) *Hydrangea spp.* strain YN; Figure 2 The relative molecular weight peaks of allelic sites obtained by sequentially detecting the primer XQJSSR031 provided for this application in the selected Hydrangea spp. ZJXQJ001 and seven Hydrangea spp. strains G, GZ, RK, S, SL, SX, and YN were obtained. Figure 3 The primer XQJSSR085 provided for this application was used to detect the relative molecular mass peaks of allelic sites in the selected *Hydrangea spp.* ZJXQJ001 and seven *Hydrangea spp.* strains G, GZ, RK, S, SL, SX, and YN, respectively; a) *Hydrangea spp.* ZJXQJ001; b) *Hydrangea spp.* strain G; c) *Hydrangea spp.* strain GZ; d) *Hydrangea spp.* strain RK; e) *Hydrangea spp.* strain S; f) *Hydrangea spp.* strain SL; g) *Hydrangea spp.* strain SX; h) *Hydrangea spp.* strain YN; Figure 4 The primer XQJSSR115 provided for this application was used to detect the relative molecular mass peaks of allelic sites in the selected *Hydrangea spp.* ZJXQJ001 and seven *Hydrangea spp.* strains G, GZ, RK, S, SL, SX, and YN, respectively; a) *Hydrangea spp.* ZJXQJ001; b) *Hydrangea spp.* strain G; c) *Hydrangea spp.* strain GZ; d) *Hydrangea spp.* strain RK; e) *Hydrangea spp.* strain S; f) *Hydrangea spp.* strain SL; g) *Hydrangea spp.* strain SX; h) *Hydrangea spp.* strain YN; Figure 5 The relative molecular weight peaks of allelic sites obtained by sequentially detecting the primer XQJSSR145 provided for this application in the selected Hydrangea spp. ZJXQJ001 and seven Hydrangea spp. strains G, GZ, RK, S, SL, SX, and YN were obtained. Figure 6 The relative molecular weight peaks of allelic sites obtained by sequentially detecting the primer XQJSSR160 provided for this application in the selected Hydrangea spp. ZJXQJ001 and seven Hydrangea spp. strains G, GZ, RK, S, SL, SX, and YN were obtained. Figure 7 The UPGMA clustering tree of genetic distances of eight Hydrangea spp. strains constructed based on SSR molecular markers is provided for this application. Detailed Implementation

[0023] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but this does not limit the present invention in any way. Any modifications or improvements made based on the teachings of the present invention shall fall within the protection scope of the present invention. Example

[0024] Unless otherwise specified, all materials and instruments used in the following embodiments were obtained through commercial channels; and all detection methods used are existing methods unless otherwise specified.

[0025] Example 1: Obtaining the *Hydrangea hydrangea* strain ZJXQJ001 (1) After cleaning the wild hydrangea fruiting bodies, place them in a clean bench; the wild hydrangea fruiting bodies used were collected from Diqing Tibetan Autonomous Prefecture, Yunnan Province on July 20, 2023.

[0026] (2) Cut the fruiting body into small pieces with a clean scalpel and place the fruiting body upside down in a sterilized PDA plate; (3) The Petri dish was placed at a constant temperature of 25℃ and protected from light for 20 days to obtain the *Hydrangea hydrangea* strain ZJXQJ001. It was deposited at the China General Microbiological Culture Collection Center (CGMCC) on August 14, 2023; the accession number is CGMCCNO.40778.

[0027] Example 2 Molecular identification of *Hydrangea hydrangea* strain ZJXQJ001 (1) DNA extraction The tested strains were transferred to potato dextrose agar solid medium and cultured at 25°C for 20 days before mycelia were collected. Nucleic acid was extracted from the samples using a magnetic bead genomic extraction kit, and the purity, concentration, and integrity of the extracted nucleic acids were determined using NanoDrop and agarose gel electrophoresis.

[0028] (2) PCR amplification and detection PCR amplification was performed using universal ITS primers ITS1 and ITS4. The PCR amplification system consisted of a total volume of 30 μL, including: 15 μL of 2×Taq PCR Master Mix (GeneTech), 2 μL each of 5 pmol / μL forward and reverse primers, 1 μL of 20 ng genomic template DNA, and 10 μL of ddH2O. The PCR amplification program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; and 72℃ extension for 5 min. After PCR, 2 μL of the PCR product was analyzed by agarose gel electrophoresis (1% concentration). After passing the analysis, Sanger sequencing was performed using an ABI 3730xL DNA Analyzer sequencer.

[0029] (3) Sequence comparison and phylogenetic analysis The well-sequentially assembled sequence was compared with the nt / nr database in NCBI, showing the best match with the *Hydrangea spp.* sequence in the database, with a similarity of 99.82%. The ITS sequence is shown in SEQ NO.1: gcgaggttgt agctggcctt ctcggaggca tcgtgcacgc cctgcccgtc ccatatcata 60 cctgtgaact ttttggtagg cgggtttgtg tcggcctcga aaggggtcgg ccggccctcc 120 ggccgtcttt atatacacac catacgagtc tttagaatgt ttgtgcgtct cgacgcatct 180 tatatataac tttcagcgac ggatctcttg gctctcgcat cgatgaagaa cgcagcgaaa 240 cgcgataagt aatgtgaatt gcagaattca gtgaatcatc gaatctttga acgcaccttg 300 cgctcctcgg tattccgagg agcatgcctg tttgagtgtc atgaaattat caacccctcc 360 tccttcatcg gcggtggggc ttggacttgg aggctttgcg ggcttttaac gagtcggctc 420 ctctcaaatg cattagctcg aaccccctgc ggatcggccg tcggtgtgat ataatgtcta 480 cgtcgtggtc gtgagcgtcg gatcggcttc taatggtccc ctttcggagg cggaatttga 540 acttgt 546 Based on the collected fruiting body morphology, it can be concluded that strain ZJXQJ001 belongs to the family Sprassidaceae and the genus Sprassidaceae. Sparassis ) of the physalis ( Sparassis crispa The ITS sequence is shown in SEQ NO.1.

[0030] Example 3: Identification of *Hydatida fuciformis* strain ZJXQJ001 using the SSR molecular markers provided by this invention. I. Experimental Methods (1) DNA extraction The tested strains were transferred to potato dextrose agar solid medium and cultured at 25°C for 20 days before mycelia were collected. Nucleic acid was extracted from the samples using a magnetic bead genomic extraction kit, and the purity, concentration, and integrity of the extracted nucleic acids were determined using NanoDrop and agarose gel electrophoresis.

[0031] (2) PCR amplification Six pairs of SSR molecular markers were used to perform SSR-labeled PCR amplification on the extracted DNA; The SSR molecular marker pairs used were XQJSSR005, XQJSSR031, XQJSSR085, XQJSSR115, XQJSSR145, and XQJSSR160. The primer sequence information of the SSR molecular markers is shown in Table 1.

[0032] Table 1 SSR marker primer information The number and molecular weight of alleles in the above 6 pairs of SSR primers are shown in Table 2: Table 2. Allelic fragment information amplified by six pairs of SSR marker primers for identifying *Hydrangea rubra* cultivar ZJXQJ001. The PCR amplification system consisted of a total volume of 10 μL, including: 5 μL of 2×Taq PCR Master Mix (GeneTech), 0.5 μL each of 10 pmol / μL SSR-labeled forward and reverse primers, 1 μL of 20 ng genomic DNA, and 3 μL of ddH2O. The reaction was performed on a Veriti 384 PCR instrument. The PCR amplification program was set as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, gradient annealing from 62 to 52℃ for 30 s, extension at 72℃ for 30 s, 10 cycles; 95℃ denaturation for 30 s, 52℃ annealing for 30 s, extension at 72℃ for 30 s, 25 cycles; and a final extension at 72℃ for 20 min.

[0033] (3) Electrophoretic identification of PCR products and dilution After PCR amplification, 2 μL of PCR product was taken for agarose gel electrophoresis detection (1% concentration). The fluorescent PCR products were diluted according to the sample detection concentration requirements of the ABI3730XL sequencer to obtain fluorescent PCR products with uniform concentration.

[0034] (4) Fluorescent capillary electrophoresis detection Add the diluted fluorescent PCR product to the PCR plate, and add the following reagents according to the following ratio: 1 μL fluorescent PCR product, 0.5 μL GeneScan™ 500 LIZ, and 8.5 μL Hi-Di™ Formamide. Centrifuge the plate containing the sample and reagents, then place it on the PCR instrument and run the denaturation program (95℃, 3 min). Cool immediately after denaturation. Following the ABI 3730xl PCR procedure, select the detection file corresponding to the plate name and run the SSR sample analysis detection program.

[0035] II. Results Analysis: (1) Comparative analysis of fluorescence detection peak diagrams in capillary electrophoresis The electrophoretic peak values ​​of the other seven *Hydrangea hydrangea* strains were compared with those of ZJXQJ001. Figures 1-6 In this study, the peaks of each fluorescent marker appearing in *Hydrangea hydrangea* ZJXQJ001 and the other 7 strains were clearly distinguishable.

[0036] Figure 1The results of amplification using primer XQJSSR005 show that the fragment sizes corresponding to ZJXQJ001 and seven *Hydrangea hydrangea* strains G, GZ, RK, S, SL, SX, and YN are 155, (149+152), (149+152), 152, (149+152), (149+152), (149+152), and (149+152), respectively. Figure 2 The results of amplification with primer XQJSSR031 show that the fragment sizes corresponding to ZJXQJ001 and the seven *Hydrangea spp.* strains G, GZ, RK, S, SL, SX, and YN are (156+159), (144+147), (144+147), 147, (144+147), 147, (144+147), and (144+147) respectively. Figure 3 The results of amplification using primer XQJSSR085 show that the fragment sizes corresponding to ZJXQJ001 and the seven *Hydrangea hydrangea* strains G, GZ, RK, S, SL, SX, and YN are (200+203), 203, 203, 200, 203, (200+206), 203, and 203, respectively. Figure 4 The results of amplification using primer XQJSSR115 show that the fragment sizes corresponding to ZJXQJ001 and seven *Hydrangea hydrangea* strains G, GZ, RK, S, SL, SX, and YN are (216+220), (226+257), (226+257), (213+226), (226+257), 213, (226+257), and (226+257), respectively. Figure 5 The results of amplification using primer XQJSSR145 show that the fragment sizes corresponding to ZJXQJ001 and seven *Hydrangea hydrangea* strains G, GZ, RK, S, SL, SX, and YN are 192, (186+195), (186+195), (186+195), (186+195), (183+186), (186+195), and (186+195), respectively. Figure 6 The results of amplification using primer XQJSSR160 show that the fragment sizes corresponding to ZJXQJ001 and the seven *Hydrangea hydrangea* strains G, GZ, RK, S, SL, SX, and YN are (187+193), 185, 185, 185, 185, (175+193), 185, and 185, respectively.

[0037] Figures 1-6 The results show that the six pairs of SSR molecular marker primers provided by this invention can accurately distinguish the *Hydrangea hydrangea* strain ZJXQJ001 from the other seven tested *Hydrangea hydrangea* strains.

[0038] (2) Analysis of band combinations amplified by 6 pairs of SSR primers Allelic fragments amplified by 6 pairs of SSR primers in 8 tested *Hydrangea spp.* strains were encoded according to molecular weight and then numbered. The numbering combinations corresponding to G, GZ, RK, S, SL, SX, and YN are (1+2) / (1+2) / 2 / (4+5) / (2+4) / 2, (1+2) / (1+2) / 2 / (4+5) / (2+4) / 2, 2 / 2 / 1 / (1+4) / (2+4) / 2, (1+2) / (1+2) / 2 / (4+5) / (2+4) / 2, (1+2) / 2 / (1+3) / 1 / (1+2) / (1+4), (1+2) / (1+2) / 2 / (4+5) / (2+4) / 2, and (1+2) / (1+2) / 2 / (4+5) / (2+4) / 2. The strain that matches the SSR allelic fragment numbering combination of 3 / (3+4) / (1+2) / (2+3) / 3 / (3+4) is *Hydrangea hydrangea* strain ZJXQJ001.

[0039] (3) Cluster analysis Genetic diversity analysis was performed on eight *Hydrangea hydrangea* strains based on polymorphic fragments amplified from six pairs of SSRs. UPGMA clustering trees for the eight *Hydrangea hydrangea* strains were constructed based on Nei genetic distance. Figure 7 ).from Figure 7 It can be seen that the six primer pairs provided by this invention cluster *Hydrangea hydrangea* strains G, GZ, S, SX, and YN into one group, while *Hydrangea hydrangea* strains RK, SL, and ZJXQJ001 each form a separate branch and are completely distinguishable from other strains. Therefore, the six SSR primer pairs provided by this invention can be used to differentiate the aforementioned eight different *Hydrangea hydrangea* strains.

[0040] Example 4: Cultivation method of *Hydrangea rubra* strain ZJXQJ001 1) Prepare the cultivation substrate: Weigh the raw materials according to the following proportions: 70 parts sawdust, 15 parts wheat bran, 5 parts corn flour, 5 parts wheat flour, 3 parts glucose, 1 part gypsum, and 1 part potassium dihydrogen phosphate. Add water and mix well to make the moisture content 60-65%. 2) Sterilization of substrate: The prepared cultivation substrate is mechanically filled into 17cm×35cm×0.05cm plastic bags, with holes punched in the center. Each bag contains 350 grams of substrate (dry weight). The substrate is then autoclaved and cooled to room temperature. 3) Inoculation: Use liquid inoculum for inoculation, with an inoculation volume of 25-30 mL / bag. Incubate indoors under light for 40-50 days at 22-25℃ and 60%-70% humidity. 4) Fruiting Management: Once the mycelium has grown to more than 70% and primordia have begun to appear, it can be transferred to the fruiting room for management. The room should be kept at 20-22℃, with an air humidity of 80-90%, 10 hours of light per day, and a light intensity of 200 lx for 8-12 days.

[0041] 5) Once the primordia have covered the surface of the substrate at the bag opening, adjust the temperature to 18-20℃, provide 12 hours of light per day with a light intensity of 500-1000 lx, and intermittently irradiate with blue and white light. After 10-15 days of cultivation, the primordia will have basically differentiated into leaves.

[0042] 6) Open the bag, adjust the temperature to 16-18℃ and the air humidity to 90-95%, increase ventilation, and cultivate for 15-20 days until the fruiting bodies emerge from the bag opening. Once the leaves unfold and turn from white to pale yellow, they are ready for harvesting.

[0043] Statistics show that the entire process from inoculation to harvest takes about 95 days, with a fruiting rate of 97%, an average fresh mushroom weight of 250g / bag, and a biological efficiency of 71.45%.

[0044] Example 5: Cultivation method of *Hydrangea rubra* strain ZJXQJ001 The difference from Example 4 is as follows: 1) Preparation of cultivation material: Weigh the raw materials according to the cultivation material ratio of 76 parts sawdust, 18 parts wheat bran, 2 parts corn flour, 1.5 parts glucose, 1.5 parts gypsum and 1 part superphosphate, add water and mix well to make the moisture content 60-65%; other steps are the same as in Example 4.

[0045] According to statistics, the fruiting rate was 96% and the biological efficiency was 69%.

[0046] Example 6: Cultivation method of *Hydrangea fusiforme* strain ZJXQJ001 The difference from Example 4 is as follows: 1) Preparation of cultivation material: Weigh the raw materials according to the cultivation material ratio of 50 parts sawdust, 25 parts cottonseed hulls, 16 parts wheat bran, 6 parts corn flour, 1 part calcium carbonate, 1.5 parts lime, and 0.5 parts potassium dihydrogen phosphate, add water and mix well to make the moisture content 65%; other steps are the same as in Example 4.

[0047] According to statistics, the fruiting rate was 97% and the biological efficiency was 66%.

[0048] Example 7: Determination of nutritional components in the fruiting body of *Hydrangea hydrangea* strain ZJXQJ001 1) Adenosine was determined using high performance liquid chromatography (see NY / T 2116-2012 for details). Weigh 0.5 g of the uniformly pulverized fruiting body sample obtained from Example 4 into a 100 mL volumetric flask, add approximately 80 mL of water, and sonicate for 3 h in an ultrasonic extractor. After extraction, dilute to volume with water and mix well. Centrifuge 1 mL of the sample solution, filter the supernatant through a 0.45 μm microporous membrane, and use the filtrate for high performance liquid chromatography analysis.

[0049] 2) Protein content was determined using the Kjeldahl method (see GB 5009.5-2016 for details). Weigh 0.2 g of the thoroughly mixed solid sample into a digestion tube, then add 0.4 g of copper sulfate, 6 g of potassium sulfate, and 20 mL of sulfuric acid to the digestion furnace for digestion. After the digestion furnace temperature reaches 420 °C, continue digestion for 1 h. After cooling, add 50 mL of water and perform automatic liquid addition, distillation, titration, and recording of titration data on an automatic Kjeldahl nitrogen analyzer.

[0050] 3) Crude polysaccharides were determined using spectrophotometry (see NY / T 1676-2023 for details). Weigh 0.2–0.5 g of the sample into the bottom of a microwave digester, add 20 mL of water, mix thoroughly with a vortex mixer, refrigerate overnight at 4°C, mix again thoroughly, and microwave extract at 140°C for 2 h. Then remove monosaccharides to obtain the test solution. Measure the absorbance of 0.2–1 mL of the test solution and plot a standard curve to calculate the crude polysaccharide content.

[0051] 4) Total triterpenes were determined by spectrophotometry (see NY / T 3676-2020 for details). Weigh 0.5 g of the sample into a 250 mL stoppered conical flask, add 50 mL of anhydrous alcohol, tighten the stopper, shake well, and place in an ultrasonic extractor for ultrasonic extraction for 1 h, shaking frequently during extraction. After extraction, mix well, take an appropriate volume and centrifuge at 8000 r / min for 10 min, take the supernatant to measure the absorbance and plot a standard curve, and calculate the total triterpenoid content.

[0052] 5) Amino acids were determined using a ninhydrin post-column derivatization ion exchange chromatography system (see GB 5009.124-2016 for details). Weigh a certain amount of pulverized sample and place it in a hydrolysis tube. Based on the protein content of the sample, add appropriate amounts of hydrochloric acid and phenol to the hydrolysis tube, then place it in a refrigerant and freeze for 3-5 minutes. Connect it to the vacuum pump's suction pipe, evacuate, and then fill with nitrogen. Repeat this evacuation and nitrogen filling process three times. Seal the tube or tighten the screw cap while it is under nitrogen purging. Place the sealed hydrolysis tube in an electric heating oven or hydrolysis furnace at 110℃±1℃ for 22 hours. After hydrolysis, remove it and cool to room temperature. Open the hydrolysis tube and filter the hydrolysate into a 50 mL volumetric flask. Rinse the hydrolysis tube several times with a small amount of water, transferring the washings to the same 50 mL volumetric flask. Finally, dilute to the mark with water and shake to mix. Take the filtrate, dry it under reduced pressure, and evaporate it to dryness. Add an appropriate amount of sodium citrate buffer solution, shake to mix, and then filter through a 0.22 μm filter membrane to obtain the sample assay solution. The amino acid standard working solution and the sample determination solution were injected into the amino acid analyzer in the same volume for determination.

[0053] Table 3. Partial nutritional components of *Hydrangea rubra* strain ZJXQJ001 The test results show that the content of polysaccharides, adenosine, triterpenes, and various amino acids in the fruiting body of *Hydrangea caudatus* ZJXQJ001 is higher than that in other existing *Hydrangea caudatus* varieties. It exhibits excellent antioxidant, anti-inflammatory, antiviral, immune, and metabolic regulatory effects, and also has good therapeutic and protective effects on the cardiovascular system. It is an excellent low-fat, high-protein health food and can be used as a raw material for various downstream industrial products.

[0054] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A type of *Hydrangea macrophylla* with high protein amino acid content (… Sparassis sp .) strain ZJXQJ001, characterized in that, The accession number is CGMCC NO.40778, and the gene sequence is shown in SEQ NO.

1.

2. The *Hydrangea hydrangea* strain ZJXQJ001 according to claim 1, characterized in that, The fruiting body of strain ZJXQJ001 contains the following protein contents: 25.1 g / 100g, aspartic acid 1.32 g / 100g, threonine 0.64 g / 100g, serine 0.67 g / 100g, glutamic acid 1.60 g / 100g, alanine 0.76 g / 100g, valine 0.66 g / 100g, methionine 0.137 g / 100g, isoleucine 0.46 g / 100g, leucine 0.80 g / 100g, tyrosine 0.48 g / 100g, and phenylalanine 0.64 g / 100g. The content of histidine is 0.46g / 100g, the content of lysine is 0.90g / 100g, the content of arginine is 0.72g / 100g, and the content of proline is 0.89g / 100g.

3. The *Hydrangea hydrangea* strain ZJXQJ001 according to claim 1, characterized in that, The fruiting body of strain ZJXQJ001 contained 718 mg / kg adenosine, 0.75 g / 100g crude fiber, 0.25 g / 100g crude fat, 6.38 g / 100g crude polysaccharide, and 2.41% total triterpenes.

4. The *Hydrangea hydrangea* strain ZJXQJ001 according to claim 1, characterized in that, The fruiting rate of strain ZJXQJ001 was 96-97%, and the biological efficiency was 66-71.45%.

5. An SSR molecular marker for identifying the *Hydrangea hydrangea* strain ZJXQJ001 with high protein amino acid content as described in any one of claims 1-4, characterized in that, For the molecular marker pairs XQJSSR005, XQJSSR031, XQJSSR085, XQJSSR115, XQJSSR145 and XQJSSR160; The number of alleles in the XQJSSR005 molecular marker pair is 3; The number of alleles in the XQJSSR031 molecular marker pair is 4; The number of alleles in the XQJSSR085 molecular marker pair is 3; The number of alleles in the XQJSSR115 molecular marker pair is 5; The number of alleles in the XQJSSR145 molecular marker pair is 4; The number of alleles in the XQJSSR160 molecular marker pair is 4; The XQJSSR005 molecular marker pair includes: a forward primer as shown in SEQ NO.2 and a reverse primer as shown in SEQ NO.3; The XQJSSR031 molecular marker pair includes: a forward primer as shown in SEQ NO.4 and a reverse primer as shown in SEQ NO.5; The XQJSSR085 molecular marker pair includes: a forward primer as shown in SEQ NO.6 and a reverse primer as shown in SEQ NO.7; The XQJSSR115 molecular marker pair includes: a forward primer as shown in SEQ NO.8 and a reverse primer as shown in SEQ NO.9; The XQJSSR145 molecular marker pair includes: a forward primer as shown in SEQ NO.10 and a reverse primer as shown in SEQ NO.11; The XQJSSR160 molecular marker pair includes: a forward primer as shown in SEQ NO.12 and a reverse primer as shown in SEQ NO.

13.

6. A detection reagent for the *Hydrangea hygroscopica* strain ZJXQJ001 with high protein amino acid content as described in any one of claims 1 to 4, characterized in that, include: The SSR molecular marker for the identification of the *Hydrangea hydrangea* strain ZJXQJ001 with high protein amino acid content as described in claim 5.

7. A method for identifying the *Hydrangea hygroscopica* strain ZJXQJ001 with high protein amino acid content as described in any one of claims 1 to 4, characterized in that, Includes the following steps: After extracting the DNA of the sample, the SSR molecular marker described in claim 5 for the identification of the *Hydrangea hydrangea* strain ZJXQJ001 with high protein amino acid content was used to perform SSR-labeled PCR amplification on the obtained DNA. Two μL of the PCR product was subjected to agarose gel electrophoresis and fluorescence capillary electrophoresis. The obtained fluorescence peak patterns were compared to obtain the identification results.

8. The identification method according to claim 7, characterized in that, The strain that matches the SSR allelic fragment numbering combination of 3 / (3+4) / (1+2) / (2+3) / 3 / (3+4) is *Hydrangea hydrangea* strain ZJXQJ001.

9. A method for cultivating the *Hydrangea sylvestris* strain ZJXQJ001 with high protein amino acid content as described in any one of claims 1 to 4, characterized in that, Includes the following steps: 1) After preparing the cultivation substrate, add water and mix well. The resulting cultivation substrate should have a moisture content of 60-65%. 2) Pack the prepared cultivation material into 17 cm × 35 cm × 0.05 cm plastic bags using mechanized bagging, punch holes in the middle, and fill each bag with 350 grams of material (dry weight); autoclave and cool to room temperature; 3) Use liquid inoculum for inoculation, with an inoculation volume of 25-30 mL / bag, and incubate indoors under light for 40-50 days at 22-25℃ and 60%-70% humidity. 4) Once the mycelium has grown to more than 70% and primordia have begun to appear, it can be transferred to the fruiting room for management. It should be kept at 20-22℃, with an air humidity of 80-90%, 10 hours of light per day, and a light intensity of 200 lx for 8-12 days. 5) Once the primordia have covered the surface of the cultivation medium at the bag opening, adjust the temperature to 18-20℃, provide 12 hours of light per day, with a light intensity of 500-1000 lx, and intermittently expose the primordia to blue and white light. After 10-15 days of cultivation, the primordia will have basically differentiated into leaves. 6) Open the bag, adjust the temperature to 16~18℃ and the air humidity to 90~95%, strengthen ventilation, and cultivate for 15~20 days until the fruiting bodies grow out of the bag; when the leaves unfold and the color changes from white to light yellow, they can be harvested.

10. The cultivation method according to claim 9, characterized in that, The cultivation substrate is prepared from 76 parts sawdust, 18 parts wheat bran, 2 parts corn flour, 1.5 parts glucose, 1 part gypsum, and 1 part potassium dihydrogen phosphate.

11. The cultivation method according to claim 9, characterized in that, The cultivation substrate is prepared from 50 parts sawdust, 25 parts cottonseed hulls, 16 parts wheat bran, 6 parts corn flour, 1 part calcium carbonate, 1.5 parts lime, and 0.5 parts potassium dihydrogen phosphate.

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