CN111778163A - New russula griseofulva endophytic fungus and application of extract thereof in bacteriostasis - Google Patents
New russula griseofulva endophytic fungus and application of extract thereof in bacteriostasis Download PDFInfo
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Abstract
The invention relates to russula griseofulvin endophytic fungi, and particularly relates to a new russula griseofulvin endophytic fungus and application of an extract thereof in bacteriostasis. The novel strain of the Cordyceps javanicus (Cordyceps javanica) is collected from Banana Linn county in Meizhou city of Guangdong province, is identified as endophytic fungus of russula griseofulensis, is organized and separated to obtain an original strain, is named as Cordyceps javanica E190189-1, is preserved in Guangdong province microorganism strain preservation center (GDMCC for short, the address is: Guangdong of China) 12 and 9 days in 2019, and has the preservation number of GDMCC NO: 60924. the novel strain shows stronger and obvious inhibition rate on staphylococcus aureus and pseudomonas aeruginosa, and is a novel strain with development prospect.
Description
Technical Field
The invention relates to russula griseofulvin endophytic fungi, and particularly relates to a new russula griseofulvin endophytic fungus and application of an extract thereof in bacteriostasis.
Background
Russula (Russula) is a kind of edible and medicinal fungi with high economic and research values, has the aspects of resisting oxidation, inhibiting tumor and pathogenic bacteria, reducing blood sugar and blood fat, relieving fatigue and the like, and has been reported in China to be 82 edible, 22 medicinal and 13 toxic Russula fungi.
Russula griseocarpa (Russula griseocarnosa) is an important rare wild edible and medicinal fungus which forms ectomycorrhiza with forest trees in Fagaceae, is widely distributed in southwest, southeast and south China, and is called as "Rhodococcus", "Rhodococcus megaterium", "Russula vinosa" by mushroom farmers, and is often used for cooking soup for eating, and has the effects of moistening skin, enhancing energy and prolonging life, and the existing literature shows that the Russula griseocarpa has rich crude protein, crude fat, crude fiber, reducing sugar, carbohydrate, acid, ester, alcohol, olefin and other compounds. Since the grifola frondosa grows in the wild and coexists with plants, a large number of symbiotic bacteria exist in sporocarp, the endophytes directly or indirectly influence the growth of the grifola frondosa and the generation of active ingredients, and the separation of the pure culture of the endophytes has important significance for obtaining the strain with potential utilization value.
Disclosure of Invention
In order to overcome the defects, the invention provides a novel grifola frondosa endophytic fungus separated by a tissue separation method and application of an extract thereof in bacteriostasis.
In the first aspect, the novel strain of Cordyceps javanicus (Cordyceps java) is collected from Banana Linn county of Meizhou city of Guangdong province, is identified as endophytic fungus of russula vinosa, is subjected to tissue separation to obtain an original strain, is named as Cordyceps javanicus Cordyceps java E190189-1, is preserved in Guangdong province microorganism culture collection (GDMCC for short, address: Guangdong of China) 12 and 9 days in 2019, and has a preservation number of GDMCC NO: 60924.
the invention provides a Java Cordyceps java GDMCC NO: 60924 it is obtained by aseptic separation of fruiting body of Russula subrufescens collected from broad-leaved forest of Ridge county of Meizhou.
Aseptically separating the obtained Java Cordyceps java GDMCC NO: 60924 strains are subjected to fermentation culture and can be used for enrichment or subsequent extraction of extract.
The fermentation medium adopted by the fermentation culture is PD medium (potato 200g, glucose 20g, KH)2PO41.5g,MgSO40.5g，VbTrace, water is added to the solution until the volume is 1000ml, and the pH is natural).
In a third aspect, the present invention provides a Cordyceps javanicus cordyces japonica GDMCC NO: 60924 or its extract, and can be used for treating diseases caused by bacteria.
Preferably, the extract comprises exopolysaccharides or intracellular polysaccharides of Cordyceps javanicus cordyces japonica.
Preferably, the bacteria comprise staphylococcus aureus or pseudomonas aeruginosa.
In a fourth aspect, the present invention provides a Cordyceps javanicus cordyces japonica GDMCC NO: 60924 or its extract, and can be used for preparing medicines for treating diseases caused by bacteria or health products.
Preferably, the extract comprises exopolysaccharides or intracellular polysaccharides of Cordyceps javanicus cordyces japonica.
Preferably, the bacteria comprise staphylococcus aureus or pseudomonas aeruginosa.
In a fifth aspect, the invention provides a medicament for treating related diseases caused by bacteria, which comprises Cordyceps javanicus cordyces javanica GDMCC NO: 60924 or an extract thereof and a carrier.
Preferably, the extract comprises exopolysaccharides or intracellular polysaccharides of Cordyceps javanicus cordyces japonica.
Preferably, the bacteria comprise staphylococcus aureus or pseudomonas aeruginosa.
In a sixth aspect, the invention provides a health product comprising Cordyceps javanicus Cordyceps japonica GDMCCNO: 60924 or an extract thereof.
Preferably, the extract comprises exopolysaccharides or intracellular polysaccharides of Cordyceps javanicus cordyces japonica.
In the invention, the method for extracting the exopolysaccharide of the Cordyceps javanicus Cordyceps japonica comprises the following steps: extracting mycelium filtered from liquid fermentation liquor of Cordyceps javanicus (Cordyceps japonica) with water under reflux, precipitating the filtrate with ethanol, dissolving in water, and drying.
The water reflux extraction adopts twice water reflux extraction, and the filtrate is combined and precipitated by ethanol.
In the invention, the method for extracting intracellular polysaccharide from Cordyceps javanicus cordyces japonica comprises the following steps: concentrating the filtered fermentation liquid of Cordyceps javanicus (Cordyceps japonica) liquid fermentation liquid, precipitating with ethanol, dissolving in water, and drying.
The novel strain of the invention has strong and obvious inhibition rate on staphylococcus aureus and pseudomonas aeruginosa, and is a novel strain with development prospect.
Drawings
FIG. 1 is a graph of the inhibitory effect of water-soluble intracellular polysaccharides on Staphylococcus aureus.
FIG. 2 is a graph showing the inhibitory effect of intracellular polysaccharides solubilized by DMSO (dimethyl sulfoxide) on Staphylococcus aureus.
FIG. 3 is a graph of the inhibitory effect of water-soluble exopolysaccharides on Staphylococcus aureus.
FIG. 4 is a graph showing the inhibitory effect of DMSO-solubilized exopolysaccharide on Staphylococcus aureus.
FIG. 5 is a graph of the inhibitory effect of water-soluble intracellular polysaccharides on Pseudomonas aeruginosa.
FIG. 6 is a graph of the inhibitory effect of DMSO-solubilized intracellular polysaccharides on P.aeruginosa.
FIG. 7 is a graph of the inhibitory effect of water-soluble intracellular polysaccharides on Pseudomonas aeruginosa.
FIG. 8 is a graph of the inhibitory effect of DMSO-solubilized intracellular polysaccharides on P.aeruginosa.
Detailed Description
The present invention is further illustrated by the following specific examples.
Example 1: strain acquisition
In the emergence season of the grifola frondosa, go to the collection of broad-leaved forest in Banana Ling county of Meizhou to obtain grifola frondosa fruiting bodies, select the fruiting bodies of which the pileus is not opened and the appearance is complete and free from worm damage, dig up together with the soil of 5-10cm around the fruiting bodies and the depth during collection, and immediately return to the aseptic separation in a laboratory:
firstly picking off the fruiting body of the grifola frondosa to separate the fruiting body from soil, cutting the basal part of a stipe by using an aseptic scalpel, then spraying 75% alcohol on the surface of the grifola frondosa, lightly wiping the surface by using a 75% alcohol cotton ball, then cutting the surface by using an aseptic blade within the range of 3-5cm around the flame of an alcohol lamp, clamping the joint of the stipe and a pileus by using aseptic tweezers, transferring the part of the pileus (about soybean size) which is not contacted with air before cutting to a test tube (18 x 180mm) inclined culture medium (PDA), and culturing for 8-10 days to fully grow the test tube to obtain the strain.
Example 2: strain identification
The endophyte mycelium DNA obtained in the example 1 is extracted by adopting a genome DNA extraction kit of an Ezup type epiphyte, ITS1 and ITS4 are used as primers to carry out ITS region PCR test to obtain a sequence with the base number of 568, the sequence is submitted to GenBank to obtain a registration number MT138575, and the result is Cordyceps japonica after the sequence comparison and analysis of the GenBank.
Wherein:
ITS1 is:
GGCTACGGGCTTTTTCACTCCCTAACCCTTTGTGACATACCTATCGTTGCTTCGGCGGACTCGCCCCGGCGTCCGGACGG
CCCTGCGCCGCCCGCGACCCGGACCCAGGCGGCCGCCGGAGACCCACAAATTCTGTTTCTATCAGTCTTTCTGAATCCGC
CGCAAGGCAAAACAAATGAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGC
GATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGCATTCTGGCGGGC
ATGCCTGTTCGAGCGTCATTTCAACCCTCGACACCCCTTCGGGGGAGTCGGCGTTGGGGACCGGCAGCATACCGCCGGCC
CCGAAATACAGTGGCGGCCCGTCCGCGGCGACCTCTGCGTAGTACTCCAACGCGCACCGGGAACCCGACGCGGCCACGCC
GTAAAACACCCAACTTCTGAACGTTGACCTCGGATCAGGTAGGACTACCCGCTGAACTTAAGCATATCAAAAGGCGGAGG
AA
ITS4 is:
ACGGGGTGTCTACCTGATCCGAGGTCACGTTCAGAAGTTGGGTGTTTTACGGCGTGGCCGCGTCGGGTTCCCGGTGCGCG
TTGGAGTACTACGCAGAGGTCGCCGCGGACGGGCCGCCACTGTATTTCGGGGCCGGCGGTATGCTGCCGGTCCCCAACGC
CGACTCCCCCGAAGGGGTGTCGAGGGTTGAAATGACGCTCGAACAGGCATGCCCGCCAGAATGCTGGCGGGCGCAATGTG
CGTTCAAAGATTCGATGATTCACTGAATTCTGCAATTCACATTACTTATCGCATTTCGCTGCGTTCTTCATCGATGCCAG
AACCAAGAGATCCGTTGTTGAAAGTTTTGATTCATTTGTTTTGCCTTGCGGCGGATTCAGAAAGACTGATAGAAACAGAA
TTTGTGGGTCTCCGGCGGCCGCCTGGGTCCGGGTCGCGGGCGGCGCAGGGCCGTCCGGACGCCGGGGCGAGTCCGCCGAA
GCAACGATAGGTATGTTCACAAAGGGTTAGGGAGTTGAAAAAACTCGTTAATGATCCCTCCGCAT
example 3: characteristics of the Strain
The strain Cordyceps javanicus (cordyces japonica) E190189-1 of example 1 is characterized in that: the growth speed is high, hyphae can grow over the test tube slant (18 x 180mm) in about 10 days, the optimal temperature for hyphae growth is 25-28 ℃, the optimal pH for hyphae growth is 6.5, and the preservation culture medium is a common PDA culture medium. The strain can be preserved in a test tube inclined plane at 4 ℃ for a short time or preserved for a long time by freezing with liquid nitrogen. The strain is sent to a Guangdong province microorganism strain preservation center for preservation, and the preservation number is GDMCC No: 60924.
the common PDA culture medium comprises agar 20g, potato 200g, glucose 20g, and KH2PO41.5g,MgSO40.5g，VbAdding water to a trace amount to reach 1000 ml.
Example 5: extraction of endophyte strain active polysaccharide
The endophyte strain is subjected to fermentation culture (25 deg.C, 180rpm in dark place, and fermentation culture medium is PD culture medium (potato 200g, glucose 20g, KH)2PO41.5g,MgSO40.5g，VbTrace, adding water to reach 1000ml, and keeping pH natural) for 10 days, and extracting intracellular polysaccharide and extracellular polysaccharide by the following method:
extracting intracellular and extracellular polysaccharides:
1. performing suction filtration on liquid fermentation liquor of Cordyceps javanicus (Cordyceps japonica) with filter paper to obtain mycelium and fermentation liquor;
2. adding mycelium into ultrapure water (sample mass: pure water mass: 1:10), boiling and refluxing for 2 hr, vacuum filtering while hot, and collecting filtrate;
3. adding water (1:5) into the filter residue again, boiling and refluxing for 4 hours, carrying out suction filtration while the filter residue is hot, mixing the filter liquor with the filter liquor obtained in the step 2, and carrying out rotary evaporation and concentration;
4. after cooling, adding absolute ethyl alcohol (volume ratio is 1:4) into the concentrated solution, stirring slowly and quickly, then subpackaging into 50mL centrifuge tubes, and standing overnight at 4 ℃;
6. after overnight, centrifuging (80000rmp, 4 ℃, 10min), discarding the supernatant, adding hot water to dissolve the precipitate, performing rotary evaporation and concentration, placing the concentrated solution on a plate for precooling at-80 ℃, and then performing freeze-drying by using a freeze-dryer to obtain intracellular polysaccharide.
And (3) extraction of extracellular polysaccharide:
and (3) performing rotary evaporation and concentration on the fermentation liquor obtained after hypha filtration in the step (1), cooling, adding absolute ethyl alcohol (the volume ratio is 1:4) into the concentrated solution, stirring slowly and quickly, subpackaging the mixture into 50mL centrifuge tubes, and standing overnight at 4 ℃. After overnight, the mixture was centrifuged (80000rmp, 4 ℃ C., 10min), the supernatant was discarded, and the precipitate was dissolved in hot water and concentrated by rotary evaporation. And (3) placing the concentrated solution on a plate for precooling at-80 ℃, and then freeze-drying by using a freeze dryer to obtain the extracellular polysaccharide.
Example 6 application of bacterial strains to bacteriostasis
1. Selecting single bacterial colony of bacteria (staphylococcus aureus and pseudomonas aeruginosa) to be detected, putting the single bacterial colony into a nutrient broth culture medium, and culturing at 37 ℃ and 180rpm in a dark place for 17 hours;
2. measuring the concentration of bacteria liquid (Staphylococcus aureus, Pseudomonas aeruginosa liquid) with ultraviolet spectrophotometer, and diluting with culture medium according to the concentration to obtain bacteria liquid concentration of about 106cfu/ml for standby;
3. and (3) determining the antibacterial activity by adopting an Oxford cup method, decomposing and dissolving the intracellular polysaccharide and the extracellular polysaccharide obtained in the step (5) by DMSO and sterile water, preparing the mixture into a concentration of 300mg/ml, respectively adding the concentration into a plate culture medium coated with bacteria to be detected, selecting antibiotics as a positive control according to the characteristics of the bacteria to be detected, wherein the positive control is ampicillin when the bacteria to be detected is staphylococcus aureus, and the positive control is gentamycin when the bacteria to be detected is pseudomonas aeruginosa. The solubilized polysaccharide sample was used as a negative control against solvent, and the addition of medium was used as a blank control. The results are shown in FIGS. 1 to 8.
From the results in FIGS. 1 to 8, it can be seen that the hydrolyzed and DMSO-solubilized intracellular and extracellular polysaccharide extracts have inhibitory effects on Staphylococcus aureus and Pseudomonas aeruginosa, whereas the extracellular polysaccharide has a stronger inhibitory effect on both bacteria than the intracellular polysaccharide, the extracellular polysaccharide has an inhibitory effect on Pseudomonas aeruginosa comparable to gentamicin, and a slightly weaker inhibitory effect on Staphylococcus aureus than ampicillin. The solvent (water or DMSO) has little effect on the bacteriostatic effect of the polysaccharide.
Example 7 minimum lethal concentration (MIC) concentration determination
1. Selecting single bacterial colony of bacteria (staphylococcus aureus and pseudomonas aeruginosa) to be detected, putting the single bacterial colony into a nutrient broth culture medium, and culturing at 37 ℃ and 180rpm in a dark place for 17 hours;
2. measuring the concentration of bacteria liquid (Staphylococcus aureus, Pseudomonas aeruginosa liquid) with ultraviolet spectrophotometer, and diluting with culture medium according to the concentration to obtain bacteria liquid concentration of about 106cfu/ml for standby;
3. 96-well cell plate setup:
experimental group, 3 replicates. mu.L of nutrient broth medium was added to each well, 100. mu.L of 300mg/ml polysaccharide (DMSO, water-solubilized intracellular polysaccharide, extracellular polysaccharide, respectively) was added to the first well of each row, and the next well (same row, same below) was added after two-fold dilution. Add 100. mu.L of 10 per well6cfu/ml bacterial liquid of bacteria to be detected;
polysaccharide control: 100 μ L of nutrient broth medium was added to each well, 100 μ L of 300mg/ml polysaccharide (optionally DMSO, water-solubilized intracellular polysaccharide, extracellular polysaccharide) was added to the first well of each row, and the next well was added after two-fold dilution. Adding 100 μ L of culture medium into each well;
negative control: 100 mu L of culture medium and 100 mu L of bacterial liquid to be detected
Positive control: 100 mu L of bacterial liquid of bacteria to be detected and 50 mu L of antibiotic solution (the antibiotic is selected as a positive control according to the characteristics of the bacteria to be detected, the ampicillin is selected when the bacteria to be detected is staphylococcus aureus, the gentamicin is selected when the bacteria to be detected is pseudomonas aeruginosa), and 50 mu L of culture medium
Solvent comparison: 100 mu L of bacterial liquid of bacteria to be detected, 50 mu L of solvent and 50 mu L of culture medium
Blank control: 200. mu.L of the culture Medium
Culturing at 37 deg.C for 16h, measuring OD600 of each well with microplate reader, comparing OD600 values of the measured wells and blank control, and determining minimum inhibitory concentration.
And (3) respectively testing:
endophyte Cordyceps javanicus (Cordyceps japonica) E190189-1 intracellular polysaccharide takes DMSO as solvent to test the MIC of staphylococcus aureus;
endophyte Cordyceps javanicus (Cordyceps japonica) E190189-1 exopolysaccharide with DMSO as solvent for testing MIC of Staphylococcus aureus;
endophyte Cordyceps javanicus (Cordyceps japonica) E190189-1 intracellular polysaccharide using water as solvent to test MIC of Staphylococcus aureus;
the endophyte Cordyceps javanicus (Cordyceps japonica) E190189-1 exopolysaccharide uses water as solvent to test the MIC of staphylococcus aureus;
endophyte Cordyceps javanicus (Cordyceps japonica) E190189-1 intracellular polysaccharide takes DMSO as a solvent to test the MIC of pseudomonas aeruginosa;
endophyte Cordyceps javanicus (Cordyceps japonica) E190189-1 exopolysaccharide with DMSO as solvent for testing MIC of pseudomonas aeruginosa;
endophyte Cordyceps javanicus (Cordyceps japonica) E190189-1 intracellular polysaccharide using water as solvent to test MIC of pseudomonas aeruginosa;
endophyte Cordyceps javanicus (Cordyceps japonica) E190189-1 exopolysaccharide using water as solvent to test MIC of pseudomonas aeruginosa;
the results were:
when DMSO is used as solvent, the minimum concentration of intracellular polysaccharide for inhibiting Staphylococcus aureus is about 75mg/ml, and the concentration of extracellular polysaccharide is about 37.5 mg/ml.
When water is used as solvent, the minimum concentration of intracellular polysaccharide for inhibiting staphylococcus aureus is about 9.375mg/ml, and the concentration of extracellular polysaccharide is about 75 mg/ml.
When DMSO is used as a solvent, the minimum concentration of intracellular polysaccharide for inhibiting the pseudomonas aeruginosa is about 37.5mg/ml, and the minimum concentration of extracellular polysaccharide is about 18.75 mg/ml.
When water is used as a solvent, the minimum concentration of intracellular polysaccharide for inhibiting the pseudomonas aeruginosa is about 75mg/ml, and the minimum concentration of extracellular polysaccharide for inhibiting the pseudomonas aeruginosa is about 37.5 mg/ml.
In conclusion, the crude polysaccharide (intracellular and extracellular) extracts of the endophyte strains have certain inhibiting effect on staphylococcus aureus and pseudomonas aeruginosa, and when the crude polysaccharide (intracellular and extracellular) extracts are applied to antibacterial application, the minimum inhibitory concentration on staphylococcus aureus is 9.375mg/ml (intracellular polysaccharide), and the minimum inhibitory concentration on pseudomonas aeruginosa is 18.75mg/ml (extracellular polysaccharide).
The above results also show that the inhibitory effect on Staphylococcus aureus is particularly remarkable when intracellular polysaccharides in water are used as a solvent, and that the inhibitory effect on Pseudomonas aeruginosa is particularly remarkable when exopolysaccharides in DMSO are used as a solvent.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and their concepts should be considered to be equivalent or modified within the technical scope of the present invention.
Claims (9)
1. A new Hypsizygus marmoreus endophytic fungus is characterized in that the new bacterial strain is Cordyceps javanicus Cordyceps javanica E190189-1, and the preservation number is GDMCC NO: 60924.
2. cordyceps javanicus cordyces javanica GDMCC NO: 60924 or an extract thereof, for the preparation of a medicament against a disease associated with bacterial infection.
3. The use according to claim 2, wherein the extract comprises exopolysaccharides or intracellular polysaccharides of Cordyceps javanicus Cordycepsjavanica.
4. The use according to claim 2, wherein the bacteria comprise staphylococcus aureus or pseudomonas aeruginosa.
5. A medicament for treating diseases related to bacterial infection, which comprises Cordyceps javanicus Cordyceps javanica GDMCC NO: 60924 or an extract thereof and a carrier.
6. The medicament according to claim 5, wherein the extract comprises exopolysaccharides or intracellular polysaccharides of Cordyceps javanicus Cordycepsjavanica.
7. The medicine according to claim 6, wherein the method for extracting exopolysaccharide from Cordyceps javanicus cordyces japonica comprises the following steps: extracting mycelium filtered from liquid fermentation liquor of Cordyceps javanicus (Cordyceps japonica) with water under reflux, precipitating the filtrate with ethanol, dissolving in water, and drying.
8. The medicament as claimed in claim 6, wherein the method for extracting intracellular polysaccharide from Cordyceps javanicus Cordyceps japonica comprises: concentrating the filtered fermentation liquid of Cordyceps javanicus (Cordyceps japonica) liquid fermentation liquid, precipitating with ethanol, dissolving in water, and drying.
9. The medicament of claim 5, wherein the bacteria comprise Staphylococcus aureus or Pseudomonas aeruginosa.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112159766A (en) * | 2020-10-23 | 2021-01-01 | 广东省微生物研究所（广东省微生物分析检测中心） | Aphyllophora fuscopsis new strain, fermentation method thereof and application thereof in bacteriostasis |
| CN112569336A (en) * | 2020-12-16 | 2021-03-30 | 广东省微生物研究所（广东省微生物分析检测中心） | Application of grifola frondosa glycoprotein in preparation of drugs or health foods for enhancing body immunity and preventing and/or treating tumor diseases |
| CN115948251A (en) * | 2022-12-06 | 2023-04-11 | 湖南农业大学 | Wild-type Cordyceps javanicus biocontrol strain CJ01 and application thereof |
| CN116693707A (en) * | 2023-01-17 | 2023-09-05 | 吉林大学 | Russula cinerea polysaccharide with hematopoiesis promoting effect and preparation method and application thereof |
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| CN112569336A (en) * | 2020-12-16 | 2021-03-30 | 广东省微生物研究所（广东省微生物分析检测中心） | Application of grifola frondosa glycoprotein in preparation of drugs or health foods for enhancing body immunity and preventing and/or treating tumor diseases |
| CN112569336B (en) * | 2020-12-16 | 2023-08-11 | 广东省微生物研究所（广东省微生物分析检测中心） | Application of russula cinerea glycoprotein in preparation of medicines or health foods for enhancing organism immunity and preventing and/or treating tumor diseases |
| CN115948251A (en) * | 2022-12-06 | 2023-04-11 | 湖南农业大学 | Wild-type Cordyceps javanicus biocontrol strain CJ01 and application thereof |
| CN115948251B (en) * | 2022-12-06 | 2023-10-20 | 湖南农业大学 | Wild cordyceps javanica biocontrol strain CJ01 and application thereof |
| CN116693707A (en) * | 2023-01-17 | 2023-09-05 | 吉林大学 | Russula cinerea polysaccharide with hematopoiesis promoting effect and preparation method and application thereof |
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