<i>Hericium erinaceus </i>strain HE015, molecular markers and uses thereof

Abstract

The present invention discloses a new Hericium erinaceus strain HE015, and a molecular marker and use thereof. Wild fungi are widely collected throughout China and subjected to systematic strain breeding to obtain a new fine-quality Hericium erinaceus strain HE015. The strain HE015 was preserved in Guangdong Microbial Culture Collection Center on May 12, 2022 with an accession number of GDMCC No: 62464. The genetic background of the strain is obviously different from that of the existing Hericium erinaceus species; therefore, the new strain is of important value and significance in supplementing fine-quality cultivated Hericium erinaceus varieties, meeting the market demand and promoting high-quality development of the industry. The present invention further provides a specific molecular marker and a rapid detection method for detecting and identifying the strain HE015.

Claims

1. A method of protecting gastric mucosa or improving alcohol-induced gastric mucosal damage, comprising: administering an effective amount of Hericium erinaceus strain HE015 extract, wherein the Hericium erinaceus strain HE015's representative sample is deposited at Guangdong Microbial Culture Collection Center under accession number of GDMCC No: 62464, and the Hericium erinaceus HE015 extract comprises Hericium erinaceus HE015 fruiting body polysaccharide extract, small-molecule Hericium erinaceus HE015 fruiting body extract, solid-cultured Hericium erinaceus HE015 mycelia polysaccharide extract, or solid-cultured small-molecule Hericium erinaceus HE015 mycelia extract.

2. A Hericium erinaceus inoculant, comprising Hericium erinaceus strain HE015 and a PDA medium, wherein the Hericium erinaceus strain HE015's representative sample is deposited at Guangdong Microbial Culture Collection Center under accession number of GDMCC No: 62464.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows a specimen picture (upper) and growing environment picture (lower) of a new Hericium erinaceus strain HE015.

(2) FIG. 2 shows a phenotype of a fruiting body of the strain HE015 after being domesticated.

(3) FIG. 3 shows an amplification result of a primer pair HE015-F8/R8 in the strain HE015 and market species.

(4) FIG. 4A and FIG. 4B show specific amplification results of primers; FIG. 4A denotes polymorphic analysis of the primer HE015-F4/R4; FIG. 4B denotes polymorphic analysis of the primer HE015-F2/R2.

(5) FIG. 5A and FIG. 5B show effects of the HE015 extract on body weight of mice with chronic alcohol-induced gastric mucosal damage (FIG. 5A) and survival curve (FIG. 5B); Control denotes normal group; Model denotes model group; Positive denotes positive drug group; HEF denotes liquid-fermented HE015 culture group; HEFP denotes HE015 fruiting body polysaccharide extract group; HEFX denotes small-molecule HE015 fruiting body extract group; HESP denotes solid-cultured HE015 mycelia polysaccharide extract group; and HESX denotes solid-cultured small-molecule HE015 mycelia extract group.

(6) FIG. 6 shows effects of the HE015 extracts on the pathology of chronic alcohol-induced gastric mucosal damage.

DESCRIPTION OF EMBODIMENTS

(7) The present invention will be further described with reference to the accompanying drawings and detailed embodiments of the description, but the embodiments are not construed as limiting the present invention in any form. Reagents, methods and devices used in the present invention, unless otherwise specified, are conventional reagents, methods and devices in the art. Reagents and materials used in the following examples, unless otherwise specified, are commercially available.

(8) Synthetic PDA-enriched medium used in the examples contains, in mass fraction, 20% potato+2% glucose+1% peptone+2% agar+0.3% potassium dihydrogen phosphate +0.15% magnesium sulfate+0.001% vitamin B.sub.1, water as the remaining. The preparation method is as follows: potato was first cleaned and peeled, and then 200 g of the potato was weighed and cut into small pieces, added with water and well-cooked (boiled for 20-30 min, capable of being poked with a glass rod), filtered with 8-layered gauze; 20 g glucose, 10 g peptone, 20 g agar, 3 g potassium dihydrogen phosphate, 1.5 g magnesium sulfate, and 0.01 g vitamin B.sub.1 were added to the filtrate, stirred well, slightly cooled and complemented with water to 1000 mL, and subjected to moist heat sterilization for 30 min at high temperature of 121 C. and high pressure of 0.11 MPa.

(9) Mother strain medium (PDA medium) used in the examples: 200 g/L potato, 20 g/L glucose, 20 g/L agar, and water as the remaining. The preparation method is as follows: potato was peeled; 200 g were weighed, cut into small pieces and put to a boiler, added with 1000 mL water, heated and boiled in an induction cooker and kept for 20-30 min, filtered with 2-layered gauze; dregs were discarded; filtrate was supplemented to 1000 mL and added with 20 g glucose and 20 g agar, and then heated by soft fire, continuously stirred with a glass rod to prevent agar from sticking onto the bottom of the boiler or overflow; after the agar was completely dissolved, water was supplemented to the needed. Subpackage and sterilization: the solid medium was about of the height of the test tube, a funnel may be used during subpackage to prevent the medium from staining the opening of the tube or bottle neck to avoid contamination. After subpackaging, the test tubes were plugged and 7 were bound up and sterilized for 20 min at 121 C., after temperature dropped to 80 C. around, the test tubes were put in an inclined plane.

(10) Stock culture medium: 98% sorghum and 2% calcium carbonate in weight ratio. Preparation method is as follows: sorghum was added with water and boiled until there was no white core and not well-cooked, moisture was dried in the air; the boiled sorghum was added with calcium carbonate and stirred well, subjected to high-pressure sterilization for 90 min at 126 C.

(11) Cultivation material: 58% cottonseed hull, 30% hardwood saw dust, 10% wheat bran and 2% gypsum in weight ratio. Water content was about 65%.

(12) Information and sources of other Hericium erinaceus market species and wild strains used in the examples are shown in Table 1:

(13) TABLE-US-00001 TABLE 1 Strain information Strain number Strain source Way to obtain Market I044 Heilongjiang Provincial Purchase species Jiagedaqi Academy of Agriculture and Forestry Sciences MC-HE-1 Luhou No.1, Shandong MC-JN-1 Guangzhou Jiangnan Fruits and Vegetables Wholesale Market Wild HE015 Mount Arxan, Inner Mongolia The strains were species M178 Mount Mangshan, Hunan isolated and purified, W461 Mount Fanjing, Guizhou and identified E108 Mount Arxan, Inner Mongolia by ITS; the 793 Baotianman, Henan ITS sequence was subjected to ncbi sequence alignment to obtain 100% ITS similarity with Hericium erinaceus

Example 1 Isolation and Identification of the New Hericium erinaceus HE015 Strain

(14) 1. Sample: in August 2017, our research team conducted large-scale collections and surveys of fungus resources in Mount Arxan, Inner Mongolia to obtain a Hericium erinaceus sample (FIG. 1), clustered on apricus dead woods.

(15) 2. Isolation process: a pure strain was isolated from the fruiting body of the above obtained Hericium erinaceus sample by tissue isolation and recorded as HE015.

(16) Specific method was as follows: surface of the fruiting body of the Hericium erinaceus collected outdoor was wiped with 75% ethanol, torn down under aseptic conditions, and 0.2-0.5 mm0.2-0.5 mm of internal bacterial context tissue was clamped and inoculated onto a PDA medium. The medium was placed into a 25 C. incubator and cultured in dark at a constant temperature, after mycelia overgrew the slope, pointed mycelia were taken, transferred, and purified to obtain the pure strain.

(17) 3. Identification of the strain HE015

(18) (1) Morphological identification: macroscopic morphology and microscopic features of the strain HE015 strain were observed:

(19) The fruiting body is stemless or has very short lateral stems; it is fleshy when fresh, soft leathery in later period, odorless and tasteless, and becomes cheese-like texture or soft suberin after dried and smells slightly sour.

(20) Cap of the fungus is near spherical and its surface is snowy white to milky white, light milky yellow later on; it is rough and has a color of wood and microvillus without a texture of concentric ring after dried.

(21) Tooth of the fungus is snowy white or cream color when fresh and becomes tawny after dried, contracted strongly; it is cylinder-shaped and tapers from bottom to top, fleshy when fresh and hard fibrous after dried, lengthens 10 mm and 1-2 pieces per millimeter.

(22) The bacterial context has a color of wood, cheese-like texture or soft suberin after dried, and has pores without a ring zone.

(23) Stem is white to milk white and soft suberin after dried.

(24) Basidiospore is 5.8-74.8-5.9 m, oval, colorless, thick-wall, and its surface has fine bosses and starch-like texture, cyanophilous.

(25) (2) Molecular identification: the purified HE015 mycelia were transferred onto a plating medium covered with a cellophane film on the surface (synthetic PDA-enriched medium), and placed and cultured in the dark at a constant temperature of 25 C. After mycelia overgrew the plate and fresh mycelia were collected and ground at room temperature; genomic DNA was extracted by a full-automatic nucleic acid isolation machine matched with a genomic DNA extraction kit (magnetic beads) (Mabio, Art. No.: DNF628-05B), to obtain a DNA solution (DNA template), then the DNA solution was cold stored at 20 C. for further use.

(26) The above obtained DNA template was subjected to ITS-PCR detection by universal primers ITS1/ITS4 (ITS1 as SEQ ID NO.11: TCCGTAGGTGAACCTGCGG, ITS4 as SEQ ID NO.12: TCCTCCGCTTATTGAT ATGC, synthesized by Sangon Biotech (Shanghai) Co., Ltd.) of ribosomal DNA internal gene region in fungi.

(27) Composition of the PCR reaction system (30 l in total) is shown in Table 2. PCR procedure is shown in Table 3.

(28) PCR related reagents were purchased from Nanjing Vazyme Biotech Co., Ltd.

(29) TABLE-US-00002 TABLE 2 ITS PCR system of the fungus Composition Amount (L) 2 Taq Master Mix 15 DNA template 2 PCR primer 1 (10 mol/L) 1.5 PCR primer 2 (10 mol/L) 1.5 ddH.sub.2O 10

(30) TABLE-US-00003 TABLE 3 ITS PCR procedure of the fungus Temperature Time 94 C. 5 min 94 C. 30 s 55 C. 30 s 72 C. 30 s 34 72 C. 5 min 25 C. 1 min

(31) The PCR product was directly sent back to Beijing Genomics Institute for bi-directional sequencing. The sequencing results are shown in SEQ ID NO.10.

(32) TABLE-US-00004 ITSsequencesofthestrainHE015(SEQIDNO.10): GAAGGATCATTAATGAATTTGAAAGGAGTTGTTGCTGGCCTGAAACCCA GGCATGTGCACGCTCCAATCTCATCCATCTTACACCTGTGCACCCTTGC GTGGGTCCGTCGGCTTTGCGGTCGATGGGCTTGCGTTTTTCATAAACTC TTATGTATGTAACAGAATGTCATAATGCTATAAACGCATCTTATACAAC TTTCAACAACGGATCTCTTGGCTCTCGCATCGATGAAGAACGCAGCGAA ATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTT GAACGCACCTTGCGCCCCTTGGTATTCCGAGGGGCACGCCTGTTCGAGT GTCGTGAAATTCTCAACTCAATCCTCTTGTTATGAGAGGGCTGGGCTTG GACTTGGAGGTCTTGCCGGTGCTCCCTCGGGAAGTCGGCTCCTCTTGAA TGCATGAGTGGATCTCTTTTGTAGGGTTTGCCCTTGGTGTGATAATTAT CTACGCCGCGGGTAGCCTTGCGTTGGTCTGCTTCTAACCGTCTTCGGAC AACTTTCATCTCAACTTGACCTCGAATCAGGCGGGACTACCCGCTGAAC TTAAGC

(33) The sequencing results were subjected to sequence alignment on NCBI GenBank to find that it has 100% similarity with Hericium erinaceus.

(34) The above macroscopic and microscopic features are integrated and combined with the identification results of the ITS sequence to determine that HE015 is Hericium erinaceus. The strain was preserved in Guangdong Microbial Culture Collection Center on May 12, 2022 with an accession number of GDMCC No: 62464 and address of 5th Floor, Building 59, 100 Mid. Xianlie Road, Guangzhou.

Example 2 HE015 Cultivation and Quality Comparison

(35) In this study, two common market species (I044 and MC-HE-1) and four wild species of Hericium erinaceus strains (HE015, M178, W461, and E108) were cultivated and compared.

(36) I. Experimental Method:

(37) 1. Medium Preparation

(38) (1) Mother strain medium (PDA medium): potato was peeled; 200 g were weighed, cut into small pieces and put to a boiler, added with 1000 mL water, heated and boiled in an induction cooker and kept for 20-30 min, filtered with 2-layered gauze; dregs were discarded; filtrate was supplemented to 1000 mL and added with 20 g glucose and 20 g agar, and then heated by soft fire, continuously stirred with a glass rod to prevent agar from sticking onto the bottom of the boiler or overflow; after the agar was completely dissolved, water was supplemented to the needed. Subpackage and sterilization: the solid medium was about of the height of the test tube, a funnel may be used during subpackage to prevent the medium from staining the opening of the tube or bottle neck, to avoid contamination. After subpackaging, the test tubes were plugged and 7 were bound up and sterilized for 20 min at 121 C., after temperature dropped to 80 C. around, the test tube were put in an inclined plane.

(39) (2) Stock culture medium: 98% sorghum and 2% calcium carbonate in weight ratio. In the afternoon of the previous day of seed production, components in the formula were weighed; sorghum was washed with fresh water for 2-3 times, and soaked with fresh water over the night. After soaking, sorghum was washed with fresh water and fished out, then boiled with water and continuously stirred to prevent from sticking at the bottom of the boiler until there was no white core and not well-cooked, heating was stopped, the sorghum was poured into a sieve mesh and washed with fresh water; after thick substances were removed and the sorghum was spread out to dry surface moisture in the air; calcium carbonate was added and stirred manually well, and bagged. The medium was subjected to autoclaved sterilization for 90 min at 126 C., naturally cooled and put into an inoculation chamber for inoculation.

(40) (3) Cultivation material: 58% cottonseed hull, 30% hardwood saw dust, 10% wheat bran and 2% gypsum in weight ratio. Raw materials were weighed according to the formula, and water was added in a material/water ratio of 1:(1.4-1.5), retention for 2 h to absorb adequate water, and finally, water content was regulated to about 65%.

(41) 2. Plating Methods (to Determine the Growth Rate of Mycelia):

(42) A PDA medium sterilized at high temperature was taken, and about 15 mL of the medium was poured into a sterile plate; when the medium was solidified and cooled to room temperature, 2 mm2 mm of a mother strain piece was inoculated in the center of the plate and cultured at a constant temperature of 25 C. 6 repeats were set per strain and cultured for 7-8 d; growth vigor, color, and growth rate of mycelia were observed and recorded every day.

(43) 3. Bag Culture Method:

(44) 15560.05 cm of polypropylene plastic bags was prepared; 2 kg of cultivation materials were packaged per bag and compressed moderately, and then the openings of the culture bags were tightened. The culture bags were subjected to autoclaved sterilization for 90 min at 126 C., and cooled naturally; cover of the autoclave was opened, and the bags were taken out and put to an inoculation chamber. The stock culture piece was inoculated and then placed into a 25 C. culture room and cultured in the dark; the growth situation, growth vigor, color, growth rate, and whether there was living contaminants of mycelia were recorded every week. 20 bags were prepared per variety.

(45) 4. Comparison of fruiting body yields: mycelia were implanted into a mushroom house after overgrowing the culture bag and primordium emerged, placed onto frames and standing on the ground; temperature was controlled within 16 C.-20 C., not lower than 12 C. or higher than 23 C.; air humidity was controlled to about 90%; ground watering and space spraying may be available, but water was not allowed to be directly sprayed onto the fruiting body. Scattered light was ensured in the growth stage of the fruiting body, 200 lx-400 lx was appropriate, and fine ventilation environment was provided. After fruiting, shapes, average yield and other indicators of each variety of mushrooms were recorded in detail, respectively.

(46) 5. Analysis on the Active Ingredients of the Fruiting Body

(47) The fruiting body of each variety of the harvested Hericium erinaceus was dried and crushed, and then the polysaccharide content in the Hericium erinaceus was determined with reference to the standard of the Ministry of Agriculture NY T1676-2008 Determination of Crude Mushroom Polysaccharides.

(48) II. Experimental Result:

(49) 1. Growth Situation of Mycelia

(50) As can be seen from the experimental results (Table 4), the strain HE015 mother strain has the fastest average growth rate of mycelia, and its growth vigor is equivalent to that of the market species MC-HE-1. The growth vigor of M178 is the sparsest and its growth rate is slower.

(51) As can be seen from Table 5, the stock cultures of the 6 Hericium erinaceus varieties were inoculated and cultivated, and mycelia could be overgrown after 21-30 d of growth; there was no major difference between the growth rate and color of mycelia; the strain HE015 had the fastest growth rate and mycelia were overgrown only for 21 d; each variety had good growth vigor of mycelia, of which the strain HE015 and MC-HE-1 achieved better growth vigor; there was no living contaminant except W461 and MC-HE-1.

(52) TABLE-US-00005 TABLE 4 Growth vigor and growth rate of mother strain mycelia of each Hericium erinaceus variety Growth Average vigor of Color of growth rate Variety mycelia mycelia (mm/d) Contamination I044 +++ Pure white 6.5 No HE015 ++++ Pure white 7.3 No M178 ++ Pure white 6.2 No W461 +++ Milky white 6.7 No E108 +++ Pure white 6.8 No MC-HE-1 ++++ Pure white 7.1 No

(53) Note: growth vigor of mycelia depends upon the white and dense degrees of mycelia; +, ++, +++, and ++++ denote that the growth vigor of mycelia enhances gradually.

(54) TABLE-US-00006 TABLE 5 Growth vigor and growth rate of mycelia of each Hericium erinaceus variety at stages of cultivation Days of Growth over- vigor of Color of Variety growing/d mycelia mycelia Contamination I044 30 +++ Milky white No HE015 21 ++++ Pure white No M178 23 +++ Pure white No W461 25 +++ Milky white 2 bags E108 23 +++ Pure white No MC-HE-1 22 ++++ Pure white 1 bag

(55) Note: growth vigor of mycelia depends upon the white and dense degrees of mycelia; +, ++, +++, and ++++ denote that the growth vigor of mycelia enhances gradually.

(56) 2. Growth Situation of Fruiting Body

(57) As can be seen from Table 6, the fruiting bodies of different varieties of strains greatly differ in appearance, biological character and yield. The first harvesting days of the 6 Hericium erinaceus varieties are within 14-18 d; mushroom transverse diameter is within 7.8-10.6 cm; mushroom longitudinal diameter is within 6.5-8.2 cm; average yield per bag is within 0.525-0.706 kg.

(58) The strain HE015 has the shortest harvesting time of first mushrooms, the largest individual mushroom (transverse diameter and longitudinal diameter), and the highest average yield, near 10% higher than that of the MC-HE-1.

(59) The variety M178 has the longest harvesting time of first mushrooms, smaller individual mushroom (transverse diameter and longitudinal diameter), and the lowest average yield.

(60) TABLE-US-00007 TABLE 6 Biological character and yield of different varieties of Hericium erinaceus Echinu- Trans- Longi- First lation verse tudinal Average harvesting length/ diameter/ diameter/ yield Variety days/d cm cm cm (kg/bag) I044 15 Long 8.4 6.9 0.554 0.068 HE015 14 Moderate 10.6 8.2 0.706 0.095 M178 18 Long 7.9 6.5 0.525 0.070 W461 14 Short 8.5 7.1 0.612 0.055 E108 16 Moderate 7.8 7.3 0.594 0.072 MC-HE-1 15 Short 9.0 7.8 0.650 0.081
3. Analysis on the Active Ingredients of the Fruiting Body

(61) As can be seen Table 7, in the fruiting bodies of the 6 Hericium erinaceus varieties, the polysaccharide content is M178>MC-HE-1>W461>HE015>E108>I044 from high to low in turn.

(62) TABLE-US-00008 TABLE 7 Measured results of the polysaccharide content in the fruiting body of each Hericium erinaceus variety Variety Polysaccharide content/% I044 4.85 0.21 HE015 5.21 0.36 M178 5.68 0.29 W461 5.27 0.35 E108 5.13 0.22 MC-HE-1 5.38 0.21

(63) To sum up, the plating method was used; growth rate and growth vigor of mycelia of the strains to be selected were determined to assess the advantages and disadvantages of the different production strains; afterwards, culture bags were prepared and strains were inoculated by bag cultivation; growth rate and growth vigor of mycelia were observed as well as the fruiting body yield and active ingredients in the fruiting body were determined to further assess the quality and production performance of the strains to be selected; the optimal production strain of Hericium erinaceus (dense and sturdy mycelia, fast growth, high yield, and high content of target active ingredients of fruiting body) is preferred. Research results show that the average growth rate of mycelia of the 6 collected strains is 6.3-7.2 mm/d; mycelia were overgrown the culture bag for 21-30 d; the harvesting time of first mushrooms is 14-18 d; the average yield is 0.525-0.706 kg; the polysaccharide content of fruiting body is 4.85%-5.68%; the production performance greatly varies from the different quality of strains. During the selection of strains, our team is focused on the strain with dense and sturdy mycelia, fast growth, strong living contaminants resistance, high yield of fruiting body, and high content of active ingredients of polysaccharide on the basis of the data collected at present. By synthetic analysis, the strain HE015 has the advantages such as dense and sturdy mycelia of its mother strain, fast growth of its stock culture, strong living contaminants resistance, short harvesting time of first mushrooms, and high yield of fruiting body; even though its polysaccharide content is slightly lower than other strains, the strain HE015 strain is the optimal production strain via comprehensive comparison.

(64) Phenotype of the HE015 fruiting body is shown in FIG. 2.

Example 3 HE015 Genomic Sequencing and Development of its Specific Molecular Marker

(65) The third-generation (Nanopore)+the next-generation of sequencing strategies were applied in the present invention to obtain the strain HE015 genomic sequence information. The genome size is 39.45 Mb; N50 is 2.79 Mb; 17.23% of the sequences are repeated sequences; 11756 functional genes, 367 tRNAs and 25 rRNAs were obtained by annotation. Moreover, the whole genome re-sequencing (WGS) was adopted to obtain genomic sequence information of the widely collected market cultivated strains and wild species collected in the wild. The obtained data was aligned to genome of the strain HE015 to obtain a set of InDel variation sites; specific primers were designed purposefully and PCR amplified to obtain specific DNA fragments of the wild Hericium erinaceus HE015 strain, i.e., the molecular marker of the Hericium erinaceus HE015 strain. The specific experiment is as follows:

(66) 1. Genomic Sequencing and Assembly

(67) Echinulation on the mature fruiting body of the strain HE015 strain was collected, and spores on the echinulation were washed out with sterile water; monokaryotic mycelia germinated by basidiospores were screened out and subjected to high quality DNA extraction. The sequencing was performed on the basis of the Illumina next-generation sequencing platform and Nanopore third-generation sequencing platform. For the next-generation sequencing library, the insertion size was 350 bp and the double-ended sequencing length was 150 bp. 300 ng of genomic DNA was fragmented by restriction enzyme digestion. The fragmented DNA was screened by magnetic beads to obtain a mean size of 200-400 bp. The selected fragments were repaired at ends, 3adenylated and linked to an adapter; the PCR products were purified with magnetic beads. For the third-generation sequencing library, the genomic DNA was 26 Gneedle fragmented; more than 20 kb of fragments were selected by BluePippin, repaired at ends and added with a tail A, and linked with adapters at both ends of the fragments, thus preparing the DNA library. The constructed library was subjected to Qubit concentration quantitation and qualified after check; based on the effective concentration of the library and data output demand, the Nanopore platform was used for sequencing.

(68) 2 softwares of Nextdenovo and Necat were used to assemble the third-generation sequenced data, approximately divided into three parts: 0 Nanopore data was assembled using Nextdenovo and Necat, respectively; Nextdenovo used the following parameters: read cuffoff=3k, seed-cutoff=9k; Necat used the following parameters: GENOME_SIZE=45000000, MIN_READ_LENGTH=2000, PREP_OUTPUT_COVERAGE=40, NUM_ITER=3, others were default parameters. OO Output results were polished with Medaka, default parameters; 0 the assembled results were corrected according to the next generation data: pilon default parameters. Assembly results from different softwares were subjected to statistics; the assembled sequences were aligned and homologous sequences were assessed; the assembled sequence with the best assembly effect was selected as the strain HE015 genomic sequence.

(69) 2. WGS, Access and Screening of a Set of Indel Variation Sites

(70) The widely collected Hericium erinaceus market species and wild Hericium erinaceus strains (HE015, W461, 1044, 793, E108, MC-JN-1, MC-HE-1, and M178) were inoculated onto plating media (synthetic PDA-enriched media); mycelia were collected and total DNA was extracted; after the genomic DNA was qualified and then fragmented by restriction enzyme digestion, afterwards, the fragmented DNA was purified, repaired at ends, added with A at a 3 end, and linked to a sequencing adapter, and subjected to selection of fragment size by magnetic beads, and then PCR amplified to form a sequencing library. The constructed library was subjected to quality testing, and the qualified library was sequenced at both ends by Illumina; reads were 150 bp to generate more than 5G of data.

(71) Raw reads obtained by the sequencing were filtered to obtain Clean Reads for use in the following information analysis. Data filtering mainly includes the following steps: (1) reads with an adapter were removed; (2) reads having N content of greater than 10% were filtered; and (3) reads whose bases having a mass of lower than 10 exceed 50% were removed. The obtained clean reads were positioned onto the Hericium erinaceus HE015 genome by software BWA; InDel variations were detected by HaplotypeCaller (local haplotype assembly) of GATK, to obtain the final set of variation sites. Coverage of the variation sites and sites with high genotype quality were selected and primers were designed.

(72) 3. Specific Primer Design and PCR Amplification

(73) Coverage of the variation sites and sites with high genotype quality were selected from the set of Indel variation sites, and primers were then designed. Our team screened three sets of primer pairs (HE015-F2/R2, HE015-F4/R4, and HE015-F8/R8). The primer information is as follows:

(74) TABLE-US-00009 HE015-F2(SEQIDNO.4): GCAGTGGTCTCAAAGGCCAT, HE015-R2(SEQIDNO.5): GAAACTTGGTGCTGCAGAGC; HE015-F4(SEQIDNO.6): CGGGGTCTGGGATGAGACC, HE015-R4(SEQIDNO.7): GAATGGGCAAATGAGGTCGGG; HE015-F8(SEQIDNO.8): CCTGCAAGCCATCGGACGTA, HE015-R8(SEQIDNO.9): CTTGGTCTACGCTACGTCC.

(75) The primer pairs were synthesized by Sangon Biotech (Shanghai) Co., Ltd. PCR amplification was conducted by a BioRad PCR amplifier. Composition (30 L in total) of the PCR reaction liquid is shown in Table 8.

(76) TABLE-US-00010 TABLE 8 PCR system of the strain HE015 molecular marker Composition Amount (L) 2 Taq Master Mix 15 DNA template (50 ng/uL) 2 Forward primer (10 mol/L) 1 Reverse primer (10 mol/L) 1 ddH.sub.2O 11

(77) PCR procedure is as follows: the primers were pre-degenerated at 95 C., degenerated for 15 sec at 95 C., annealed for 15 sec at 58 C., extended for 15 sec at 72 C., and then GOTO step 2; 32 cycles were performed, and extension was performed for 10 min at 72 C.

(78) The PCR products were put to 1-1.5% of an agarose gel for electrophoresis (5 v/cm) for 25 min, and then placed into a gel imaging system and photographed.

(79) 4. PCR Amplification Result and Analysis

(80) (1) Amplification result of the primer pair HE015-F8/R8 is shown in FIG. 3.

(81) As shown in FIG. 3, the primer pair HE015-F8/R8 amplifies a single amplified band with a size of 400-500 bp in the strain HE015; there is no band in other market strains or wild strains (FIG. 3).

(82) The PCR product amplified by HE015-F8/R8 was sent to BGI Genomics for bi-directional sequencing. The obtained specific sequence information is shown in SEQ ID NO.1, with a size of 457 bp. The specific DNA fragment is the InDel molecular marker of the new Hericium erinaceus HE015 strain in the present invention.

(83) TABLE-US-00011 SequenceofSEQIDNO.1(sequenceoftheHE015- F8/R8amplifiedproduct): CCTGCAAGCCATCGGACGTATTGAGAAGTCCAAAATGAAGACGAAGAAA GAAATCAGTAGATTGACAAGCAAGTGTCATAAGAGTTGGTCTCGTTGAA GGAGAGCCGGACTTGTGCGCGATATCGCAGAGCAATGGAAGCGAAAAGA CGCGCATCGAGGGAGACGACGAAGGGACGAAAAAAAGAAGGAGGGAAGG GGGGGATGAAGATGAAGAAATTGAGAAAACAGCATCGGTGGCCGCAATA ATCCCGTCGGCGTAGACACGGAAGCGAACAGGAAGCGAGCGCAGCGGGT GTCTGAAGATACAGACAGTAATAGCAAAATGAACGCAGCAATGTTCCGG TCGGAAGAAATGGCAATATGGGGGCAACGAATGGAAGGACAACTCACAA TGAAACGCACTGATGCCAATTAATTTGCAGATTGAGATCATTCGTAGGA CGTAGCGTAGACCAAG

(84) (2) Amplification results of the primer pairs HE015-F2/R2 and HE015-F4/R4 are shown in FIG. 4A and FIG. 4B.

(85) As shown in FIG. 4A, the amplified band of the HE015-F4/R4 in the Hericium erinaceus MC-JN-1 is close to that of the strain HE015 (the amplified product of HE015-F4/R4 in the strain HE015 is subjected to sequencing, the sequence information is shown in SEQ ID NO.2). The remaining 6 control strains have no band or have different bands. Therefore, the primer pair may distinguish the strain HE015 and MC-JN-1 from the rest 6 Hericium erinaceus strains.

(86) As shown in FIG. 4B, the amplified band of the HE015-F2/R2 in the Hericium erinaceus M178 is close to that of the strain HE015 (the amplified product of HE015-F2/R2 in the strain HE015 is subjected to sequencing, the sequence information is shown in SEQ ID NO.3). The remaining control strains have no band or have different bands. Therefore, the primer pair may distinguish the strain HE015 and M178 from the rest 6 Hericium erinaceus strains.

(87) HE015-F2/R2 and HE015-F4/R4 were integrated to effectively distinguish the strain HE015 from other strains.

(88) TABLE-US-00012 SequenceofSEQIDNO.2(sequenceoftheHE015- F4/R4amplifiedproduct): CATAGGCCTGTGTCCACCCGGGCAGGCCGCCCGGGTTTTCGGGGTGTAA GATCATAGCTCGCTCCCGGCGGGGCCGGTTTTCGGGGTTCAGGGTGTCG AACCTTTACCGGGATTATCAGGGGTCCTCGTTACACTCGCGTTACAGTT ATTTATTTATTTAACGTTAGGCGCTCAGGACCCGACAGTGTTCGGGAGT TTACCGGTTTTGAACATATATATGTACCTCCCTCAATACGCTAAACAAC TGTCTTCTGTGTGAGGTCAGCAACGGAGATCAGATACCTCCCAGCTAAA TATTCGAACTGTTCAATTTCTCGGGCAGATTTATCTAACCGGTTATATG CTATGGACGAGCAGATAAGCGTCTACTTAATCCTTCTGGCCCTTGACGT CCAACAGCCATTTCCCGACCTCATTTGCCCATTC SequenceofSEQIDNO.3(sequenceoftheHE015- F2/R2amplifiedproduct): GCAGTGGTCTCAAAGGCCATCCCAGGCAGCTATGACGAGGTTCAGGTGA AATATCGCAGCCAAGGCACCTCTTGCCCATTGTATCTACAAAGCATACT GAAGGACGAGGCACACATTGCCTAGTGCCACAAGTAGAATCAATTTTAA GACAGCTGCCCCAGCAGGTGGTATCAAACGTTTCAAGAATAGATCACAA ATGAGGTGTTATCTTACAATCCCAACATGTCTGCATGCCTACATCTCAT CAACAAGACCACTTTCCCGACCAGTGCCCTACAATCTATCCAATATCTC CTTCCATTCCTTCACAAATTCTTGTTCACTTGGCAGCATTTTGACCTTC CATTCGACAATACCTAGCTTTCCATGTGAATTGCACACTGCAAGCTCTC TCAACTTCTTCTGATGGGCACCTTTGTTGCTCTGCAGCACC

(89) Results are summarized below:

(90) (1) The primer pair HE015-F8/R8 (corresponding to the molecular marker SEQ ID NO.1) is used alone to completely distinguish the strain HE015 from other Hericium erinaceus strains.

(91) (2) HE015-F2/R2 and HE015-F4/R4 (corresponding to the molecular markers SEQ NO.3 and SEQ ID NO.2) are used together to completely distinguish the strain HE015 from other Hericium erinaceus strains; the strain that the corresponding identical bands may be expanded by these two sets of primer pairs is the strain HE015.

(92) Based on the above results, the present invention provides a product for detecting or identifying the strain HE015, which provides a technical basis for the quality control of the strain HE015.

Example 4 Preparation of the HE015 Extract

(93) 1. Preparation of the liquid-fermented HE015 culture:

(94) The HE015 mother strain was enlarged step by step and then inoculated onto a liquid PDA medium, then placed into a table concentrator at 25 C. and 150 rmp, and cultured until the medium became yellowish-brown and was overgrown with mycelia, isolated to obtain mycelia; culture solution was concentrated under reduced pressure by a rotary evaporator; the mycelia and the fermentation concentrated solution were mixed and homogenated, frozen dried and crushed to obtain the liquid-fermented HE015 culture (HEF).

(95) 2. Preparation of the small-molecule HE015 fruit body extract (HEFX), and HE015 fruiting body polysaccharide extract (HEFP):

(96) The HE015 fruiting body was crushed, added with 20 times of purified water, stirred and extracted for 2 h at 95 C. and filtered; filtrate was concentrated under reduced pressure to a relative density of 1.10; and then slowly added with 95% ethanol such that the alcohol concentration was up to 80%, standing for 24 h at 4 C. to obtain a supernatant and a lower precipitate; the supernatant was concentrated under reduced pressure, frozen dried and crushed to obtain the small-molecule HE015 fruit body extract (HEFX); the lower precipitate was redissolved with 5 times of water and added with 95% ethanol to regulate the alcohol concentration to 80%, standing for 24 h at 4 C.; and then cleaned with 80% ethanol and acetone, frozen dried and crushed to obtain the HE015 fruiting body polysaccharide extract (HEFP).

(97) 3. Preparation of the solid-cultured small-molecule HE015 mycelia extract (HESX) and the solid-cultured HE015 mycelia polysaccharide extract (HESP):

(98) The HE015 mother strain was enlarged step by step and then inoculated onto a sterilized solid PDA medium, then placed into a 25 C. culture room until mycelia were overgrown; the culture was taken out and the portion with thick mycelia growth was selected and torn into small pieces with the addition of 20 times of pure water, and stirred and extracted for 2 h at 95 C. and filtered to obtain a filtrate; the filtrate was concentrated under reduced pressure to a relative density of 1.10; and then slowly added with 95% ethanol such that the alcohol concentration was up to 80%, standing for 24 h at 4 C. to obtain a supernatant and a lower precipitate; the supernatant was concentrated under reduced pressure, frozen dried and crushed to obtain the solid-cultured small-molecule HE015 mycelia extract (HESX); the lower precipitate was redissolved with 5 times of water and added with 95% ethanol to regulate the alcohol concentration to 80%, standing for 24 h at 4 C.; and then cleaned with 80% ethanol and acetone, frozen dried and crushed to obtain the solid-cultured HE015 mycelia polysaccharide extract (HESP).

Example 5 Effect of the HE015 Extract on the Improvement of Alcohol-Induced Gastric Mucosal Damage

(99) 1. Laboratory Animal

(100) Male KM mice, SPF grade, body weight of 18-22 g, 5-6 weeks of age, feeding temperature and humidity: 231 C. and 5510%; 12 h day-and-night discontinuous illumination was used; conditions of the raising room were always kept stable, thereby ensuring the reliability of the test result. The mice took food and drank water freely.

(101) 2. Experimental Grouping

(102) Male KM mice were adaptively raised for 7 d, and then randomly divided into the following 8 groups: Blank control group 1: Control Model control group 1: Model Administration group 1: Positive Administration group 2: HEF Administration group 3: HEFP Administration group 4: HEFX Administration group 5: HESP Administration group 6: HESX.

(103) There were 14 mice in the Model group, and 13 mice in the other groups, and the mice were fed with an ordinary fodder.

(104) 3. Experimental Process

(105) Experimental duration is 2 weeks:

(106) First week: the mice in each administration group were prophylactically administered intragastrically once every day; and its administration concentration is shown in Table 9; meanwhile, the mice in the Control and Model groups were administered 0.3 mL of pure water intragastrically.

(107) Second week: the mice in the Model and each administration groups were administered 0.3 mL of 60% ethanol intragastrically once per day for modeling, to induce a chronic alcohol-induced gastric mucosal damage mice model, and modeling was performed for a week; meanwhile, the mice in the administration groups were administered (administration concentration and dosage were the same as those in the first week), the protective effects of different administration groups on gastric mucosa were observed.

(108) TABLE-US-00013 TABLE 9 Administration concentration and dosage of each group Admini- stration Group name concentration Volume Control Pure water 0.3 mL Model 60% ethanol 0.3 mL Lansoprazole (Positive) 0.35 mg/mL 0.3 mL Liquid-fermented HE015 culture (HEF) 10 mg/mL 0.3 mL HE015 fruiting body polysaccharide 10 mg/mL 0.3 mL extract (HEFP) Small-molecule HE015 fruit body 10 mg/mL 0.3 mL extract (HEFX) Solid-cultured HE015 mycelia 10 mg/mL 0.3 mL polysaccharide extract (HESP) Solid-cultured small-molecule HE015 10 mg/mL 0.3 mL mycelia extract (HESX)

(109) Administration was stopped the night before the end of the experiment; the mice were not allowed to take food, but allowed to drink water. Eyeballs were picked out and blood was collected, and then the mice were killed by cervical dislocation; gastric tissues were taken and placed into 4% paraformaldehyde stationary solution for immobilization, for use in pathological examination.

(110) 4. Experimental Results

(111) (1) Effects of the Hericium erinaceus HE015 Extract on Body Weight of the Mice With Chronic Alcohol-Induced Gastric Mucosal Damage

(112) As shown in FIG. 5A, during the prophylactic administration: by comparison, there is no significant difference (P>0.05) in the body weight among each group of mice, showing a trend of increasing. During modeling, the body weight of the mice in the Control shows a trend of increasing, while the body weight of the mice in other groups shows a downtrend; the body weight of the mice in the Model, administration groups and Positive is lower than that in the Control (P<0.01), indicating that the modeling greatly affects body weight.

(113) At the end of the experiment, the body weight of the mice in each administration group is higher than that in the Model, of which the body weight of the mice in the HESX, HEFX, HEFP, and Positive is significantly greater than that in the Model (P<0.05), and the body weight of the mice in the HESX and HEFP is higher than that in the Positive, but there is no significant difference (P>0.05). The results indicate that the HE015 extract may reduce the effects on the body weight of the mice with chronic alcohol-induced gastric mucosal damage, especially groups HESX, HEFP, and HEFX achieve a significant effect, and even superior to the Positive.

(114) (2) Effects of the Hericium erinaceus HE015 Extract on the Survival Curve of the Mice with Chronic Alcohol-Induced Gastric Mucosal Damage

(115) As shown in FIG. 5B, compared with the Control, survival rate of the mice in other groups shows a downtrend, indicating that the modeling lays a significant impact on the survival rate.

(116) At the end of the experiment, death count of the mice in the Model, Positive and HESP groups is obviously higher than that in other groups; and survival rates of the Positive and HESP groups are slightly higher than that of the Model group. The survival rate of the mice in the groups HEF, HEFP, HEFX, and HESX is significantly higher than that in the Model group, indicating that the HEF, HEFP, HEFX, and HESX have better efficacy and may prolong the survival time of individuals.

(117) (3) Hematoxylin-Eosin (HE) Staining Results of Mice Gastric Tissues

(118) The HE staining results of mice gastric tissues are shown in FIG. 6; mice in the Control have an intact and clear gastric mucosa structure of gastric tissues, intact cellular morphology, and regular gland arrangement, free of abnormal lesions such as tissue defect, bleeding, and inflammation. Mice in the Model suffer serious disseminated necrosis exfoliation of gastric mucosal cells, swelling cells, loose arrangement of cells and infiltration of inflammatory cells. Mice in the Positive suffer necrosis exfoliation of partial mucosal epithelial cells of the gastric tissue, loose arrangement of partial cells and infiltration of a small amount of inflammatory cells. Compared with the Model, mice in the HE015 extract administration groups have different degrees of improved gastric mucosa morphology, and the improvement is superior to the Positive.

(119) In conclusion: the HE015 extracts HESX, HEFX, and HEFP as well as the positive drug Lansoprazole may increase the body weight of the mice with chronic alcohol-induced gastric mucosal damage, and decrease the effects of the chronic alcohol-induced gastric mucosal damage on the body weight of the mice; the extracts HEF, HEFP, HEFX, and HESX may extend the survival time of individuals; the extracts HEF, HEFP, HEFX, HESP, and HESX may improve the gastric mucosa of the mice with chronic alcohol-induced gastric mucosal damage. The results indicate that the HE015 extracts of the present invention may be used for improving chronic alcohol-induced gastric mucosal damage, and particularly, its fruiting body achieves a better effect.

(120) The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples. Any other changes, modifications, replacements, combinations, and simplifications made within the spiritual essence and principle of the present invention shall be equivalent substitution modes, and shall fall within the protection scope of the present invention.