US20040259217A1 - Plant mangrove-associated fungus Culvularia lunata and a simple and efficient method of obtaining high yield of pure mannitol from the same - Google Patents
Plant mangrove-associated fungus Culvularia lunata and a simple and efficient method of obtaining high yield of pure mannitol from the same Download PDFInfo
- Publication number
- US20040259217A1 US20040259217A1 US10/464,845 US46484503A US2004259217A1 US 20040259217 A1 US20040259217 A1 US 20040259217A1 US 46484503 A US46484503 A US 46484503A US 2004259217 A1 US2004259217 A1 US 2004259217A1
- Authority
- US
- United States
- Prior art keywords
- mannitol
- fungus
- crude
- leaves
- plant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 title claims abstract description 85
- 235000010355 mannitol Nutrition 0.000 title claims abstract description 83
- 229930195725 Mannitol Natural products 0.000 title claims abstract description 82
- 239000000594 mannitol Substances 0.000 title claims abstract description 82
- 241000233866 Fungi Species 0.000 title claims abstract description 43
- 238000000034 method Methods 0.000 title claims abstract description 39
- 240000002044 Rhizophora apiculata Species 0.000 title claims abstract description 26
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 36
- 239000001965 potato dextrose agar Substances 0.000 claims abstract description 36
- 239000000287 crude extract Substances 0.000 claims abstract description 22
- 239000000843 powder Substances 0.000 claims abstract description 11
- 241000223211 Curvularia lunata Species 0.000 claims abstract description 10
- 239000002904 solvent Substances 0.000 claims abstract description 10
- 239000012223 aqueous fraction Substances 0.000 claims abstract description 9
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 9
- 230000008021 deposition Effects 0.000 claims abstract description 9
- 238000005520 cutting process Methods 0.000 claims abstract description 6
- 239000013535 sea water Substances 0.000 claims description 17
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 16
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 15
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- 239000012153 distilled water Substances 0.000 claims description 11
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 9
- 239000003208 petroleum Substances 0.000 claims description 8
- 229910052799 carbon Inorganic materials 0.000 claims description 7
- 238000004362 fungal culture Methods 0.000 claims description 7
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 6
- 241000196324 Embryophyta Species 0.000 claims description 6
- 239000008121 dextrose Substances 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 6
- 235000000346 sugar Nutrition 0.000 claims description 5
- 229920002472 Starch Polymers 0.000 claims description 4
- 239000002054 inoculum Substances 0.000 claims description 4
- 235000019698 starch Nutrition 0.000 claims description 4
- 239000008107 starch Substances 0.000 claims description 4
- 239000011148 porous material Substances 0.000 claims description 3
- 241000207965 Acanthaceae Species 0.000 claims 2
- 239000002609 medium Substances 0.000 description 21
- 238000000855 fermentation Methods 0.000 description 18
- 230000004151 fermentation Effects 0.000 description 18
- 230000035882 stress Effects 0.000 description 14
- 238000004519 manufacturing process Methods 0.000 description 13
- 229920005862 polyol Polymers 0.000 description 11
- 150000003077 polyols Chemical class 0.000 description 11
- 238000000746 purification Methods 0.000 description 10
- 241000722943 Acanthus Species 0.000 description 9
- 238000000605 extraction Methods 0.000 description 9
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 8
- 230000008569 process Effects 0.000 description 8
- 230000002538 fungal effect Effects 0.000 description 7
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 6
- 239000000203 mixture Substances 0.000 description 5
- 150000003839 salts Chemical class 0.000 description 5
- 239000003054 catalyst Substances 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 229920001592 potato starch Polymers 0.000 description 4
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 4
- 239000003642 reactive oxygen metabolite Substances 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 3
- 229930091371 Fructose Natural products 0.000 description 3
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 3
- 239000005715 Fructose Substances 0.000 description 3
- 238000005481 NMR spectroscopy Methods 0.000 description 3
- 239000004698 Polyethylene Substances 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- 239000002036 chloroform fraction Substances 0.000 description 3
- 239000002038 ethyl acetate fraction Substances 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 239000003960 organic solvent Substances 0.000 description 3
- -1 polyethylene Polymers 0.000 description 3
- 229920000573 polyethylene Polymers 0.000 description 3
- 230000003595 spectral effect Effects 0.000 description 3
- 238000004611 spectroscopical analysis Methods 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 2
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- 208000004880 Polyuria Diseases 0.000 description 2
- 239000007868 Raney catalyst Substances 0.000 description 2
- NPXOKRUENSOPAO-UHFFFAOYSA-N Raney nickel Chemical compound [Al].[Ni] NPXOKRUENSOPAO-UHFFFAOYSA-N 0.000 description 2
- 229910000564 Raney nickel Inorganic materials 0.000 description 2
- 230000021736 acetylation Effects 0.000 description 2
- 238000006640 acetylation reaction Methods 0.000 description 2
- 238000009903 catalytic hydrogenation reaction Methods 0.000 description 2
- 230000006037 cell lysis Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 235000009508 confectionery Nutrition 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 230000007123 defense Effects 0.000 description 2
- 230000035619 diuresis Effects 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000005984 hydrogenation reaction Methods 0.000 description 2
- 229960004903 invert sugar Drugs 0.000 description 2
- 229930000044 secondary metabolite Natural products 0.000 description 2
- 238000000527 sonication Methods 0.000 description 2
- 239000000600 sorbitol Substances 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 150000005846 sugar alcohols Chemical class 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- QIVUCLWGARAQIO-OLIXTKCUSA-N (3s)-n-[(3s,5s,6r)-6-methyl-2-oxo-1-(2,2,2-trifluoroethyl)-5-(2,3,6-trifluorophenyl)piperidin-3-yl]-2-oxospiro[1h-pyrrolo[2,3-b]pyridine-3,6'-5,7-dihydrocyclopenta[b]pyridine]-3'-carboxamide Chemical compound C1([C@H]2[C@H](N(C(=O)[C@@H](NC(=O)C=3C=C4C[C@]5(CC4=NC=3)C3=CC=CN=C3NC5=O)C2)CC(F)(F)F)C)=C(F)C=CC(F)=C1F QIVUCLWGARAQIO-OLIXTKCUSA-N 0.000 description 1
- XILIYVSXLSWUAI-UHFFFAOYSA-N 2-(diethylamino)ethyl n'-phenylcarbamimidothioate;dihydrobromide Chemical compound Br.Br.CCN(CC)CCSC(N)=NC1=CC=CC=C1 XILIYVSXLSWUAI-UHFFFAOYSA-N 0.000 description 1
- 241000223602 Alternaria alternata Species 0.000 description 1
- 241000228245 Aspergillus niger Species 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 206010048962 Brain oedema Diseases 0.000 description 1
- 241000135254 Cephalosporium sp. Species 0.000 description 1
- 208000008853 Ciguatera Poisoning Diseases 0.000 description 1
- 241001149956 Cladosporium herbarum Species 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 241000195493 Cryptophyta Species 0.000 description 1
- 241001506775 Epicoccum nigrum Species 0.000 description 1
- 206010016952 Food poisoning Diseases 0.000 description 1
- 208000019331 Foodborne disease Diseases 0.000 description 1
- 206010017533 Fungal infection Diseases 0.000 description 1
- 241001208371 Fusarium incarnatum Species 0.000 description 1
- 206010019196 Head injury Diseases 0.000 description 1
- FBPFZTCFMRRESA-BXKVDMCESA-N L-mannitol Chemical compound OC[C@H](O)[C@H](O)[C@@H](O)[C@@H](O)CO FBPFZTCFMRRESA-BXKVDMCESA-N 0.000 description 1
- 229930182842 L-mannitol Natural products 0.000 description 1
- 241000186660 Lactobacillus Species 0.000 description 1
- 241000192132 Leuconostoc Species 0.000 description 1
- 208000031888 Mycoses Diseases 0.000 description 1
- 241000202223 Oenococcus Species 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- NJVBTKVPPOFGAT-BRSBDYLESA-N [(2r,3r,4r,5r)-2,3,4,5,6-pentaacetyloxyhexyl] acetate Chemical compound CC(=O)OC[C@@H](OC(C)=O)[C@@H](OC(C)=O)[C@H](OC(C)=O)[C@H](OC(C)=O)COC(C)=O NJVBTKVPPOFGAT-BRSBDYLESA-N 0.000 description 1
- 241000222292 [Candida] magnoliae Species 0.000 description 1
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 description 1
- 239000006286 aqueous extract Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 208000006752 brain edema Diseases 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 229940112822 chewing gum Drugs 0.000 description 1
- 235000015218 chewing gum Nutrition 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000005100 correlation spectroscopy Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000000428 dust Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000006353 environmental stress Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 235000021433 fructose syrup Nutrition 0.000 description 1
- 235000012055 fruits and vegetables Nutrition 0.000 description 1
- 238000000669 high-field nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 201000001421 hyperglycemia Diseases 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 238000007917 intracranial administration Methods 0.000 description 1
- 229940039696 lactobacillus Drugs 0.000 description 1
- 230000002475 laxative effect Effects 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 235000019533 nutritive sweetener Nutrition 0.000 description 1
- XULSCZPZVQIMFM-IPZQJPLYSA-N odevixibat Chemical compound C12=CC(SC)=C(OCC(=O)N[C@@H](C(=O)N[C@@H](CC)C(O)=O)C=3C=CC(O)=CC=3)C=C2S(=O)(=O)NC(CCCC)(CCCC)CN1C1=CC=CC=C1 XULSCZPZVQIMFM-IPZQJPLYSA-N 0.000 description 1
- 230000000426 osmoregulatory effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 230000003032 phytopathogenic effect Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000012306 spectroscopic technique Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000036325 urinary excretion Effects 0.000 description 1
- 238000007738 vacuum evaporation Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
Definitions
- the present invention relates to a plant mangrove-associated fungus Culvularia lunata of International deposition no. ______, and a simple and efficient method of obtaining high yield of pure mannitol from a plant mangrove-associated fungus Curvularia lunata , said method comprising steps of cutting the leaves into small pieces and placing it on potato dextrose agar (PDA) plates for 48 hours, maintaining the culture at temperature ranging between 26-47° C.
- PDA potato dextrose agar
- D-mannitol is a six-carbon sugar alcohol or polyol, which is about half as sweet as sucrose and occurs widely in nature in a variety of organisms including plants, algae, fungi, and certain bacteria. L-mannitol does not occur naturally.
- mannitol is industrially produced by catalytic hydrogenation of fructose/glucose (1:1) mixture such as invert sugar. Raney-nickel is used as a catalyst and hydrogen gas is used at high temperature and pressure (Makkee et al., 1985).
- the disadvantage of this method is that the composition of the hydrogenated mixture consists of only about 25% mannitol and the remaining 75% was sorbitol. This production procedure makes the manufacture cost for mannitol relatively high.
- Mannitol is known to have several applications both in plants and humans. Jennings (1984) stated that polyols including mannitol play several roles in fungi; as carbohydrate reserve, as translocatory compounds, as an osmoregulatory compound as in coenzyme regulation, storage or reducing power and has been also shown to quench reactive oxygen species (ROS). Reactive oxygen species are both signal molecules and direct participants in plant defense against pathogens. There is growing evidence that at least some phytopathogenic fungi use mannitol to suppress ROS mediated plant defenses (Jennings et al 1998).
- Mannitol is a valuable nutritive sweetener because it is non-toxic, non-hygroscopic in its crystalline form and has no teeth decaying effects (Debord et al 1987; Dwivedi 1978). Mannitol does not induce hyperglycemia, which makes it useful for diabetics (Griffin and Lynch, 1972). It is used as a sweet builder in sugar free chewing gum and in pharmaceutical preparations (Soetaert, 1991).
- mannitol is used for the treatment of ciguatera in Australia (Lewis, 1992).
- mannitol is used in the treatment of head injury to decrease cerebral edema and intracranial pressure.
- Administration of mannitol in man induces diuresis (promotion of urinary excretion) in oligourea or forced diuresis in food poisoning cases.
- High doses of mannitol exert a laxative effect in man.
- mannitol Several microorganisms are known to produce mannitol. Among bacteria, heterofermentative species belonging to the genera Leuconostoc, Oenococcus , and Lactobacillus seem to be most effective in producing mannitol (Niklas et al 2002). Several filamentous fungi (moulds) produce mannitol form glucose as well. Mannitol was earlier reported from fungal strains like Aspergillus niger (Muraleedharan, 1988), cotton dust associated fungi viz.
- NIO-FM 1 E#001 shows a considerable good yield ( ⁇ 70% of the total crude extract) and a relatively simple and economical process for the production of mannitol. Since mannitol has been reported to accumulate in response to environmental stress (Kets et al 1996; Stoop and Pharr, 1994), the fungus, NIO-FM 1 E #001 was cultured under thermal and salt stress conditions to obtain maximum yield of polyol. Thus, our patent describes an economical process for the production of mannitol and a novel fungal source (NIO-FM 1 E#001) for the production of the same.
- the main object of the present invention is to isolate a plant mangrove-associated fungus Culvularia lunata of International deposition no. ______.
- Another main object of the present invention is to isolate a fast growing fungus.
- Yet another main object of the present invention is tom develop a simple and efficient method of obtaining high yield of pure mannitol from a plant mangrove-associated fungus Curvularia lunata.
- Still another object of the present invention is to isolate a purified crude mannitol by chromatography to obtain pure mannitol with about 75% of the total crude extract.
- Still another object of the present invention is to isolate high percentage yield of mannitol using stress conditions.
- Still another object of the present invention is to identify novel source for mannitol.
- Still another object of the present invention is to identify novel strains of fungus which are easy to isolate and culture.
- Still another object of the present invention is to use fermentation medium used for optimum yield is of low cost.
- Still another object of the present invention is to develop a method of isolating mannitol, wherein secondary metabolites can be easily separated from the main compound.
- Still another object of the present invention is to develop a method of isolating pure mannitol from fungi using room temperature and pressure and not catalyst is used for the production of mannitol.
- the present invention relates to a plant mangrove-associated fungus Culvularia lunata of International deposition no. ______, and a simple and efficient method of obtaining high yield of pure mannitol from a plant mangrove-associated fungus Curvularia lunata , said method comprising steps of cutting the leaves into small pieces and placing it on potato dextrose agar (PDA) plates for 48 hours, maintaining the culture at temperature ranging between 26-47° C.
- PDA potato dextrose agar
- the present invention relates to a plant mangrove-associated fungus Culvularia lunata of International deposition no. ______, and a simple and efficient method of obtaining high yield of pure mannitol from a plant mangrove-associated fungus Curvularia lunata , said method comprising steps of cutting the leaves into small pieces and placing it on potato dextrose agar (PDA) plates for 48 hours, maintaining the culture at temperature ranging between 26-47° C.
- PDA potato dextrose agar
- starch is of concentration ranging between 3-5 g/l.
- the dextrose is of the concentration ranging between 18-22 g/l.
- the ratio of seawater and distilled water is ranging between 1:5 to 5:1, preferably 1:1.
- the stress conditions is temperature ranging between 40-45° C.
- the leaves are young, and living leaves.
- solvents in increasing polarity are petroleum ether, chloroform, and ethyl acetate.
- the present invention relates to a mangrove-associated fungus NIO-FM 1 E#001, as a novel source for the production of mannitol and a simple, economical, extraction and purification process for the production of the same.
- the fungal culture was isolated from living, young leaves of Acanthus illicifolius .
- the isolated culture was grown in potato dextrose broth containing potato starch and dextrose as carbon source, in sea water:distilled water (1:1).
- the fermentation medium was subjected to thermal and salt stress conditions.
- the polyol was obtained from mycelial mat involving minimum extraction and purification steps.
- the sugar was acetylated and its structure confirmed using spectroscopic techniques using, Mass Spectroscopy, high field NMR Spectroscopy, DEPT and COSY experiments.
- the fungus, NIO-FM 1 E#001 was associated with a mangrove Acanthus illicifolius , and was isolated using the following steps.
- the fungal innoculum was prepared by the following steps.
- the medium uses potato starch (4 g/l) and dextrose (20 g/l) as carbon source.
- the fermentation broth was prepared in seawater:distilled water (1:1).
- Flasks were incubated between 28-30° C. on a rotor shaker for 4 to 5 days.
- the innoculum used to seed the fermentation medium comprising of the following steps.
- the temperature of the fermentation medium during innoculation was between 28-45° C.
- the incubation temperature of the fermentation medium was between 28-45° C.
- the salinity of the fermentation medium was between 5-30 ppt.
- the incubation temperature of the fermentation medium was between 28-30° C.
- salt stress was given by maintaining the salinity of the fermentation medium between 15-17 ppt.
- the crude extract was treated with petroleum ether, chloroform and ethyl acetate to separate the petroleum ether fraction, chloroform fraction and ethyl acetate fraction.
- the aqueous fraction which remained as white powder contained crude polyol ( ⁇ 70% of the total crude extract).
- NMR spectra of the crude aqueous extract showed the polyol to be mannitol. Acetylation of mannitol gave hexacetate of the polyol, which was further used for its confirmation.
- the solvent system used was methanol water (95:5).
- FIG. 1 shows the flow diagram for the extraction and purification of mannitol. Accordingly, the present invention describes a mangrove-associated fungus, Curvularia lunata , NIO-FM 1 E#001 as a novel source for the production of mannitol and a simple and economical process for the extraction and purification of the same.
- the fungus used for the production of mannitol was associated with young leaves of mangrove, Acanthus illicifolius and isolated by the following steps.
- the innoculum for the fungal culture was prepared by the following steps.
- Plugs of agar supporting the mycelial growth was cut aseptically and transferred into 100 ml Erlenmeyer flasks containing 25 ml PDB (potato dextrose broth).
- the medium was prepared in seawater:distilled water (1:1).
- Potato starch (4 g/l) and dextrose (20 g/l) were used as carbon source.
- Flasks were incubated on a rotor shaker between 28-30° C. for 4 to 5 days.
- the above innoculum was used to seed 5 litre Erlenmeyer flasks containing 1 litre of potato dextrose broth prepared in 1:1 seawater: distilled water.
- the fermentation medium during innoculation process was between 28-45° C.
- the salinity of the medium used was between 5-30 ppt.
- temperature stress was given by maintaining the fermentation medium during inoculation between 40-45° C.
- the incubation temperature of the fermentation medium was maintained between 28-30° C.
- salt stress was provided to the culture by maintaining the salinity of the fermentation medium between 15-17 ppt.
- fungal mycelia were recovered by filtration, dried in a lyophilizer and extracted repeatedly with methanol after sonication for cell lysis.
- the crude from the extraction with organic solvent was concentrated on a rotor vacuum evaporator at 30° C.
- the crude extract was treated with petroleum ether to remove the petroleum ether fraction, treated with chloroform to remove the chloroform fraction and finally treated with ethyl acetate to remove the ethyl acetate fraction.
- the aqueous fraction left behind in the flask contained a crude white powder, which constituted about 70% of the total crude extract.
- NMR of this white powder showed it to contain a polyol and comparison of the NMR spectral data with standards showed the polyol to be mannitol.
- Mannitol was acetylated using pyridine and acetic anhydride to yield hexacetate, whose spectral values further confirm the presence of mannitol.
- the mobile phase used comprised of methanol:water (95:5) to obtain pure mannitol.
- the isolated culture was designated as Curvularia lunata , NIO-FM 1 E#001.
- Colonies of NIO-FM 1 E #001 are fast growing, brown to blackish brown with a black reverse.
- Conidia are pale brown with three or more traverse septa and are formed apically through a pore (poroconidia) in a sympodially elongated geniculate conidiophore.
- Conidia are cylindrical or slightly curved, with one of the central cells being larger and darker.
- the purified culture was grown on PDA slants for 7 days. Plugs of agar supporting mycelial growth were cut and transferred aseptically to a 100 ml Erlenmeyer flask containing 25 ml potato dextrose broth (HiMedia Laboratories Ltd.) prepared in seawater:distilled water (1:1). The carbon source in this broth was potato starch 4 g/l and dextrose 20 g/l. Flasks were kept under shaker conditions between 28-30° C. for 4-5 days. Subculture from the above flask was aseptically transferred to 5 litre flasks containing 1 litre of the above-mentioned medium. The innoculation was carried out by keeping the temperature of the fermentation medium between 40-45° C. After innoculation, the flasks were kept under shaker conditions for 18 days between 28-30° C.
- mannitol (30 mg) was dissolved in pyridine (2 ml), to which a solution of 3 ml acetic anhydride in pyridine was added and left overnight. The mixture was taken in chloroform and pyridine was removed by washing with dilute HCl. This was followed by washing with distilled water. Chloroform was evaporated and the residue was chromatographed over silica gel to yield 35 mg of mannitol hexaacetate whose structure was confirmed by spectroscopic methods and comparison of spectra with the reported spectral data (Fairbanks and Sinay, 1995).
- mannitol is still produced by the catalytic hydrogenation of a 50:50 fructose:glucose mixture which uses hydrogen at high temperature and pressure with Raney Nickel as a catalyst.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Mycology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Botany (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to a plant mangrove-associated fungus Culvularia lunata of International deposition no. ______, and a simple and efficient method of obtaining high yield of pure mannitol from a plant mangrove-associated fungus Curvularia lunata, said method comprising steps of cutting the leaves into small pieces and placing it on potato dextrose agar (PDA) plates for 48 hours, maintaining the culture at temperature ranging between 26-47° C. and salinity ranging between 4-32 ppt with occasional stress conditions for about 17-19 days to obtain mycelial mat, sonicating the mat to lyse the cells, extracting crude from the sonicated mat using methanol repeatedly, concentrating the crude, treating the concentrated crude extract with solvents of increasing polarity, obtaining aqueous fraction as white powder residue after the treatment containing crude mannitol, purifying the crude mannitol by chromatography to obtain pure mannitol with about 75% of the total crude extract.
Description
- The present invention relates to a plant mangrove-associated fungusCulvularia lunata of International deposition no. ______, and a simple and efficient method of obtaining high yield of pure mannitol from a plant mangrove-associated fungus Curvularia lunata, said method comprising steps of cutting the leaves into small pieces and placing it on potato dextrose agar (PDA) plates for 48 hours, maintaining the culture at temperature ranging between 26-47° C. and salinity ranging between 4-32 ppt with occasional stress conditions for about 17-19 days to obtain mycelial mat, sonicating the mat to lyse the cells, extracting crude from the sonicated mat using methanol repeatedly, concentrating the crude, treating the concentrated crude extract with solvents of increasing polarity, obtaining aqueous fraction as white powder residue after the treatment containing crude mannitol, purifying the crude mannitol by chromatography to obtain pure mannitol with about 75% of the total crude extract.
- D-mannitol is a six-carbon sugar alcohol or polyol, which is about half as sweet as sucrose and occurs widely in nature in a variety of organisms including plants, algae, fungi, and certain bacteria. L-mannitol does not occur naturally.
- Low levels of mannitol are found in several fruits and vegetables. Presently, mannitol is industrially produced by catalytic hydrogenation of fructose/glucose (1:1) mixture such as invert sugar. Raney-nickel is used as a catalyst and hydrogen gas is used at high temperature and pressure (Makkee et al., 1985). The disadvantage of this method is that the composition of the hydrogenated mixture consists of only about 25% mannitol and the remaining 75% was sorbitol. This production procedure makes the manufacture cost for mannitol relatively high.
- Mannitol is known to have several applications both in plants and humans. Jennings (1984) stated that polyols including mannitol play several roles in fungi; as carbohydrate reserve, as translocatory compounds, as an osmoregulatory compound as in coenzyme regulation, storage or reducing power and has been also shown to quench reactive oxygen species (ROS). Reactive oxygen species are both signal molecules and direct participants in plant defense against pathogens. There is growing evidence that at least some phytopathogenic fungi use mannitol to suppress ROS mediated plant defenses (Jennings et al 1998).
- Mannitol is a valuable nutritive sweetener because it is non-toxic, non-hygroscopic in its crystalline form and has no teeth decaying effects (Debord et al 1987; Dwivedi 1978). Mannitol does not induce hyperglycemia, which makes it useful for diabetics (Griffin and Lynch, 1972). It is used as a sweet builder in sugar free chewing gum and in pharmaceutical preparations (Soetaert, 1991).
- The other uses of mannitol are that it is used for the treatment of ciguatera in Australia (Lewis, 1992). In emergency cases, mannitol is used in the treatment of head injury to decrease cerebral edema and intracranial pressure. Administration of mannitol in man induces diuresis (promotion of urinary excretion) in oligourea or forced diuresis in food poisoning cases. High doses of mannitol exert a laxative effect in man.
- Several microorganisms are known to produce mannitol. Among bacteria, heterofermentative species belonging to the generaLeuconostoc, Oenococcus, and Lactobacillus seem to be most effective in producing mannitol (Niklas et al 2002). Several filamentous fungi (moulds) produce mannitol form glucose as well. Mannitol was earlier reported from fungal strains like Aspergillus niger (Muraleedharan, 1988), cotton dust associated fungi viz. Alternaria alternata, Cladosporium herbarum, Epicoccum purpurascens and Fusarium pallidoroseum (Domelsmith et al 1988), Candida magnoliae (Song et al 2002) and Cephalosporium sp. (Bi et al 2001).
- The prior art biotechnical process for the conversion of sugar to mannitol has not proven entirely satisfactory, as they do not provide an adequate conversion yield and therefore the industrial production is still based on hydrogenation. Thus, there remains a need to improve the bioconversion of fructose and other substrate into mannitol in order to provide an industrially economical and acceptable process.
- Our fungal culture, NIO-FM1E#001 shows a considerable good yield (˜70% of the total crude extract) and a relatively simple and economical process for the production of mannitol. Since mannitol has been reported to accumulate in response to environmental stress (Kets et al 1996; Stoop and Pharr, 1994), the fungus, NIO-FM1E #001 was cultured under thermal and salt stress conditions to obtain maximum yield of polyol. Thus, our patent describes an economical process for the production of mannitol and a novel fungal source (NIO-FM1E#001) for the production of the same.
- The main object of the present invention is to isolate a plant mangrove-associated fungusCulvularia lunata of International deposition no. ______.
- Another main object of the present invention is to isolate a fast growing fungus.
- Yet another main object of the present invention is tom develop a simple and efficient method of obtaining high yield of pure mannitol from a plant mangrove-associated fungusCurvularia lunata.
- Still another object of the present invention is to isolate a purified crude mannitol by chromatography to obtain pure mannitol with about 75% of the total crude extract.
- Still another object of the present invention is to isolate high percentage yield of mannitol using stress conditions.
- Still another object of the present invention is to identify novel source for mannitol.
- Still another object of the present invention is to identify novel strains of fungus which are easy to isolate and culture.
- Still another object of the present invention is to use fermentation medium used for optimum yield is of low cost.
- Still another object of the present invention is to develop a method of isolating mannitol, wherein secondary metabolites can be easily separated from the main compound.
- Still another object of the present invention is to develop a method of isolating pure mannitol from fungi using room temperature and pressure and not catalyst is used for the production of mannitol.
- The present invention relates to a plant mangrove-associated fungusCulvularia lunata of International deposition no. ______, and a simple and efficient method of obtaining high yield of pure mannitol from a plant mangrove-associated fungus Curvularia lunata, said method comprising steps of cutting the leaves into small pieces and placing it on potato dextrose agar (PDA) plates for 48 hours, maintaining the culture at temperature ranging between 26-47° C. and salinity ranging between 4-32 ppt with occasional stress conditions for about 17-19 days to obtain mycelial mat, sonicating the mat to lyse the cells, extracting crude from the sonicated mat using methanol repeatedly, concentrating the crude, treating the concentrated crude extract with solvents of increasing polarity, obtaining aqueous fraction as white powder residue after the treatment containing crude mannitol, purifying the crude mannitol by chromatography to obtain pure mannitol with about 75% of the total crude extract.
- The present invention relates to a plant mangrove-associated fungusCulvularia lunata of International deposition no. ______, and a simple and efficient method of obtaining high yield of pure mannitol from a plant mangrove-associated fungus Curvularia lunata, said method comprising steps of cutting the leaves into small pieces and placing it on potato dextrose agar (PDA) plates for 48 hours, maintaining the culture at temperature ranging between 26-47° C. and salinity ranging between 4-32 ppt with occasional stress conditions for about 17-19 days to obtain mycelial mat, sonicating the mat to lyse the cells, extracting crude from the sonicated mat using methanol repeatedly, concentrating the crude, treating the concentrated crude extract with solvents of increasing polarity, obtaining aqueous fraction as white powder residue after the treatment containing crude mannitol, purifying the crude mannitol by chromatography to obtain pure mannitol with about 75% of the total crude extract.
- In an embodiment of the present invention, wherein a mangrove-associated fungusCulvularia lunata of International deposition no. ______.
- In another embodiment of the present invention, wherein the fungus as claimed in claim1, wherein the mangrove plant is Acanthus illicifolius.
- In yet another embodiment of the present invention, wherein the fungus is fast growing fungus.
- In still another embodiment of the present invention, wherein The fungus as claimed in claim1, wherein the fungus is of color brown to blackish brown with black reverse.
- In still another embodiment of the present invention, wherein The fungus as claimed in claim1, wherein the fungus has conidia of pale brown color with three or more traverse septa and are formed apically through a pore (poroconidia) in a sympodially elongated geniculate conidiophore.
- In still another embodiment of the present invention, wherein The fungus as claimed in claim1, wherein the fungus shows conidia of cylindrical or slightly curve shape, with one of the central cells being larger and darker.
- In still another embodiment of the present invention, wherein A simple and efficient method of obtaining high yield of pure mannitol from a plant mangrove-associated fungusCurvularia lunata of International deposition No. ______, said method comprising steps of:
- obtaining leaves from the plant mangrove,
- rinsing the leaves in sterile seawater and keeping them in sterile, moist culture chamber for about two weeks,
- cutting the leaves into small pieces and placing it on potato dextrose agar (PDA) plates for 48 hours,
- transferring individual colonies aseptically into a sterile fresh PDA plate to obtain pure culture,
- maintaining the isolated fungal cultures on PDA slants for about 7 days to obtain mycelia,
- transferring the mycelia to a fresh PDB with seawater and distilled water using starch and sugar as source of carbon.
- incubating the culture at temperature ranging between 26-32° C. for about 3-6 days to obtain inoculum,
- inoculating the PBD having seawater and distilled with the inoculum,
- maintaining the culture at temperature ranging between 26-47° C. and salinity ranging between 4-32 ppt with occasional stress conditions for about 17-19 days to obtain mycelial mat,
- sonicating the mat to lyse the cells,
- extracting crude from the sonicated mat using methanol repeatedly,
- concentrating the crude,
- treating the concentrated crude extract with solvents of increasing polarity,
- obtaining aqueous fraction as white powder residue after the treatment containing crude mannitol,
- purifying the crude mannitol by chromatography to obtain pure mannitol with about 75% of the total crude extract.
- In still another embodiment of the present invention, wherein method as claimed in claim7, wherein the plant is Acanthus illicifolius.
- In still another embodiment of the present invention, wherein the starch is of concentration ranging between 3-5 g/l.
- In still another embodiment of the present invention, wherein the dextrose is of the concentration ranging between 18-22 g/l.
- In still another embodiment of the present invention, wherein the ratio of seawater and distilled water is ranging between 1:5 to 5:1, preferably 1:1.
- In still another embodiment of the present invention, wherein the stress conditions is temperature ranging between 40-45° C.
- In still another embodiment of the present invention, wherein the stress conditions is salinity ranging between 15-17 ppt.
- In still another embodiment of the present invention, wherein the stress conditions lead to high percentage yield of mannitol.
- In still another embodiment of the present invention, wherein the leaves are young, and living leaves.
- In still another embodiment of the present invention, wherein concentrating the crude using vacuum evaporator at temperature ranging between 28-32° C.
- In still another embodiment of the present invention, wherein the solvents in increasing polarity are petroleum ether, chloroform, and ethyl acetate.
- In still another embodiment of the present invention, wherein the chromatography is on G-10 solid support.
- The present invention relates to a mangrove-associated fungus NIO-FM1E#001, as a novel source for the production of mannitol and a simple, economical, extraction and purification process for the production of the same. The fungal culture was isolated from living, young leaves of Acanthus illicifolius. The isolated culture was grown in potato dextrose broth containing potato starch and dextrose as carbon source, in sea water:distilled water (1:1). For optimum growth the fermentation medium was subjected to thermal and salt stress conditions. The polyol was obtained from mycelial mat involving minimum extraction and purification steps. The sugar was acetylated and its structure confirmed using spectroscopic techniques using, Mass Spectroscopy, high field NMR Spectroscopy, DEPT and COSY experiments.
- A mangrove-associated fungus,Curvularia lunata, NIO-FM1E#001, as a novel source for the production of mannitol and a simple, economical process for the extraction and purification of mannitol.
- The fungus, NIO-FM1E#001 was associated with a mangrove Acanthus illicifolius, and was isolated using the following steps.
- a. Fresh young leaves were collected in sterile polyethylene bags.
- b. The leaves were rinsed in sterile seawater and placed in sterile, moist, culture chamber for two weeks.
- c. The leaves were then cut with the help of sterile scalpel and the pieces were placed on sterile potato dextrose agar (PDA) plates.
- d. Individual colony was picked aseptically after 48 hours and transferred on to sterile PDA plates to obtain a pure culture.
- The fungal innoculum was prepared by the following steps.
- a. The pure isolated fungal culture was maintained on PDA slants for 7 days.
- b. Plugs of agar supporting mycelial growth were cut aseptically and transferred into 100 ml Erlenmeyer flasks containing 25 ml potato dextrose broth (PDB).
- c. The medium uses potato starch (4 g/l) and dextrose (20 g/l) as carbon source.
- d. The fermentation broth was prepared in seawater:distilled water (1:1).
- e. Flasks were incubated between 28-30° C. on a rotor shaker for 4 to 5 days.
- The innoculum used to seed the fermentation medium comprising of the following steps.
- a. Seed innoculum was poured aseptically into 5 liter Erlenmeyer flasks containing 1 litre PDB prepared in 1:1 seawater:distilled water.
- b. The temperature of the fermentation medium during innoculation was between 28-45° C.
- c. The incubation temperature of the fermentation medium was between 28-45° C.
- d. The salinity of the fermentation medium was between 5-30 ppt.
- e. The medium was kept under shaker conditions for 18 days.
- f. The temperature stress was given by maintaining the inoculation temperature of the fermentation medium between 40-45° C.
- g. In another preferred claim as claimed in claim1, the incubation temperature of the fermentation medium was between 28-30° C.
- h. In yet another preferred claim as claimed in claim1, salt stress was given by maintaining the salinity of the fermentation medium between 15-17 ppt.
- The process as claimed in claim1, the extraction involved the following steps.
- The dry mycelial mat was sonicated and repeatedly extracted using methanol.
- The crude from the extraction with organic solvent, was concentrated on a rotary vacuum evaporator at 30° C.
- The crude extract was treated with petroleum ether, chloroform and ethyl acetate to separate the petroleum ether fraction, chloroform fraction and ethyl acetate fraction.
- The aqueous fraction which remained as white powder contained crude polyol (˜70% of the total crude extract).
- NMR spectra of the crude aqueous extract showed the polyol to be mannitol. Acetylation of mannitol gave hexacetate of the polyol, which was further used for its confirmation.
- The crude mannitol was chromatographed on G-10 solid support for purification.
- In a preferred claim as claimed in claim1, the solvent system used was methanol water (95:5).
- FIG. 1 shows the flow diagram for the extraction and purification of mannitol. Accordingly, the present invention describes a mangrove-associated fungus,Curvularia lunata, NIO-FM1E#001 as a novel source for the production of mannitol and a simple and economical process for the extraction and purification of the same.
- In an embodiment of the present invention, the fungus used for the production of mannitol was associated with young leaves of mangrove,Acanthus illicifolius and isolated by the following steps.
- Fresh, young leaves were collected in sterile polyethylene bags.
- They were rinsed in sterile seawater and placed in a sterile, moist, culture chamber for two weeks.
- The leaves were cut using sterile scalpel and transferred aseptically into PDA (potato dextrose agar) plates (HiMedia Laboratories Ltd.).
- After 48 hours the fungal colony was removed and again placed on PDA plates till pure isolates of the culture was obtained.
- In another embodiment of the present invention, the innoculum for the fungal culture was prepared by the following steps.
- Initially the culture was grown on PDA slants for 7 days.
- Plugs of agar supporting the mycelial growth was cut aseptically and transferred into 100 ml Erlenmeyer flasks containing 25 ml PDB (potato dextrose broth).
- The medium was prepared in seawater:distilled water (1:1).
- Potato starch (4 g/l) and dextrose (20 g/l) were used as carbon source.
- Flasks were incubated on a rotor shaker between 28-30° C. for 4 to 5 days.
- In yet another embodiment of the present invention, the above innoculum was used to seed 5 litre Erlenmeyer flasks containing 1 litre of potato dextrose broth prepared in 1:1 seawater: distilled water.
- The fermentation medium during innoculation process was between 28-45° C.
- After inoculation the fermentation medium was placed on a shaker for 18 days between 28-45° C.
- The salinity of the medium used was between 5-30 ppt.
- In still another preferred embodiment of the present invention, temperature stress was given by maintaining the fermentation medium during inoculation between 40-45° C.
- In still another preferred embodiment of the present invention, the incubation temperature of the fermentation medium was maintained between 28-30° C.
- In yet another preferred embodiment of the present invention, salt stress was provided to the culture by maintaining the salinity of the fermentation medium between 15-17 ppt.
- In still another embodiment, fungal mycelia were recovered by filtration, dried in a lyophilizer and extracted repeatedly with methanol after sonication for cell lysis. The crude from the extraction with organic solvent was concentrated on a rotor vacuum evaporator at 30° C. The crude extract was treated with petroleum ether to remove the petroleum ether fraction, treated with chloroform to remove the chloroform fraction and finally treated with ethyl acetate to remove the ethyl acetate fraction. The aqueous fraction left behind in the flask contained a crude white powder, which constituted about 70% of the total crude extract.
- In a further embodiment of the present invention, NMR of this white powder showed it to contain a polyol and comparison of the NMR spectral data with standards showed the polyol to be mannitol. Mannitol was acetylated using pyridine and acetic anhydride to yield hexacetate, whose spectral values further confirm the presence of mannitol.
- In still another embodiment of this invention, crude mannitol was chromatographed on G-10 solid support for purification.
- In a preferred embodiment of the present invention, the mobile phase used comprised of methanol:water (95:5) to obtain pure mannitol.
- The invention is further described in detail with the help of the following examples and should not be construed to limit the scope of this invention.
- The leaves from the mangrove plant,Acanthus illicifolius was collected from Sinquerim, Goa coast, India. This fungal culture inhabit Acanthus illicifolius without causing apparent harm to the host. This fungus was isolated from healthy, young leaves where symptoms of fungal infection could not be detected.
- After collection of fresh young leaves ofAcanthus, they were transferred to the laboratory in sterile polyethylene bags. The leaves were rinsed with sterile filtered seawater to remove adhered particles and detritus material. The leaves were next kept in a sterile culture chamber for two weeks. At the end of two weeks, the leaves were cut with the help of a sterile scalpel and the pieces were placed on PDA plates. Individual colony was picked aseptically and transferred repeatedly on PDA plates to obtain a pure culture.
- The isolated culture was designated asCurvularia lunata, NIO-FM1E#001. Colonies of NIO-FM1E #001 are fast growing, brown to blackish brown with a black reverse. Conidia are pale brown with three or more traverse septa and are formed apically through a pore (poroconidia) in a sympodially elongated geniculate conidiophore. Conidia are cylindrical or slightly curved, with one of the central cells being larger and darker.
- The purified culture was grown on PDA slants for 7 days. Plugs of agar supporting mycelial growth were cut and transferred aseptically to a 100 ml Erlenmeyer flask containing 25 ml potato dextrose broth (HiMedia Laboratories Ltd.) prepared in seawater:distilled water (1:1). The carbon source in this broth was potato starch 4 g/l and dextrose 20 g/l. Flasks were kept under shaker conditions between 28-30° C. for 4-5 days. Subculture from the above flask was aseptically transferred to 5 litre flasks containing 1 litre of the above-mentioned medium. The innoculation was carried out by keeping the temperature of the fermentation medium between 40-45° C. After innoculation, the flasks were kept under shaker conditions for 18 days between 28-30° C.
- At the end of the incubation period, mycelia were recovered by filtration and dried in a lyophilizer to determine biomass weight. Dry fungal mat (cake) was extracted with methanol several times with repeated sonication of the cake for cell lysis. The filtrate thus obtained was concentrated to free it from the organic solvent under vacuum evaporation. This resulted in the crude extract of the sample. The crude extract was treated successively with solvents of increasing polarity like petroleum ether, chloroform and ethyl acetate to separate the petroleum ether fraction, chloroform fraction, and ethyl acetate fraction respectively. The last aqueous fraction contained the sugar alcohol as white powder, which constituted about 70% of the total crude extract.
- The white crude powder was chromatographed on G-10 solid support using methanol: water (95:5%) as eluent, to free it from any possible salt contamination. The pure mannitol thus obtained was identified by comparison of its NMR data with spectra reported in Aldrich Catalogue, Vol. 1, pp 289c.
- A subfraction of the above mannitol was acetylated to obtain the acetyl derivative of the polyol and structure was further confirmed by various spectroscopic methods.
- For acetylation, mannitol (30 mg) was dissolved in pyridine (2 ml), to which a solution of 3 ml acetic anhydride in pyridine was added and left overnight. The mixture was taken in chloroform and pyridine was removed by washing with dilute HCl. This was followed by washing with distilled water. Chloroform was evaporated and the residue was chromatographed over silica gel to yield 35 mg of mannitol hexaacetate whose structure was confirmed by spectroscopic methods and comparison of spectra with the reported spectral data (Fairbanks and Sinay, 1995).
- 1. This is a novel source for mannitol.
- 2. It is a common fungus (found associated with mangrove leaves ofAcanthus illicifolius, collected from Goa coast).
- 3. Easy to isolate and culture.
- 4. The fermentation medium used for optimum yield is of low cost.
- 5. The fermentation conditions are feasible and can be easily carried out in the laboratory.
- 6. The secondary metabolites can be easily separated from the main compound.
- 7. Can be preserved using standard techniques and revived as and when required.
- 8. The extraction process of mannitol from fungal mat and purification of the same involves simple techniques as compared to the commercial preparation of mannitol.
- 9. We used room temperature and pressure and no catalyst is used for the production of mannitol. Commercially, mannitol is still produced by the catalytic hydrogenation of a 50:50 fructose:glucose mixture which uses hydrogen at high temperature and pressure with Raney Nickel as a catalyst.
- 10. We produced good yield of mannitol (70% of crude extract) without any other polyol contamination. Commercially, hydrogenation of fructose syrups or invert sugar resulted in the coproduction of another sugar alcohol, sorbitol and the yield of mannitol is only 25%.
- 11. In the present invention, the crude mannitol was chromatographed using G-10 as solid support, using methanol:water (95:5) as eluent for final purification. On the other hand, the purification steps in the commercial production of mannitol are tedious.
- Bi Y., Wang H., Chen Y., Xie J., (2001). Studies on chemical constituents of mycelium of fungusCephalosporium sp. AL031 (1). Journal of Chinese Medicinal Materials. Vol. 24(8), 568-569.
- Debord B., Lefebvre C., Guyot-Hermann A. M., Hubert J., Bouche R., and Guyot J. C. (1987). Study of different forms of mannitol: Comparative behaviour under compression. Drug Development and Industrial Pharmacy, 13, 1533-1546.
- Domelsmith L. N., Klich M. A., Goynes W. R., (1988). Production of mannitol by fungi form cotton dust. Applied and Environmental Microbiology. Vol. 54(7), 1784-1790.
- Dwivedi B. K., (1978). Low calorie and special dietary foods. West Palm Beach: CRC Press, Inc.
- Fairbanks A. J. and Sinay P., (1995). Synthesis of a peracetylated stereoisomer of DeRosa's Calditol: Some Questions about the Corrections of the Original Structure Assigned to this Natural Product. Tetrahedron Letters, Vol. 36(6), 893-896.
- Griffin W. C. and Lynch M. J., (1972). Polyhydric alcohol. In T. E., Furia (ed) CRC Handbook of food additives. Vol. 1 (2nd ed) (pp 431-455). Cleveland, Ohio: CRC Press Inc.
- Jennings D. H., (1984). Polyol metabolism in fungi. Advances in Microbial Physiology, 25, 149-193.
- Jennings D. B., Ehrenshaft M; Pharr D. M., Williamson J. D., (1998). Proceedings of the National Academy of Science of the United States of America. Vol. 95(25), 15129-15133.
- Kets E. P. W., De Bont J. A. M., and Heipieper H. J. (1996). Physiological response ofPseudomonas putida S., 12 subjected to reduced water activity. FEMS Microbiology Letters, 139, 133-137.
- Lewis R. J., Mannitol: The treatment of choice in the acute phase of ciguatera; Ciguatera Inf. Bull., (1992), 2, 9-10.
- Makkee M., Kieboom A. P. G., and Van Bekkum H., (1985). Production methods of D-mannitol. Starch, 37, 136-141.
- Muraleedharan G. Nair and Basil A. Burke. (1988). A new fatty and methyl ester and other biologically active compound fromAspergillus niger. Phytochemistry. Vol. 27(10), 3169-3173.
- Niklas Von Weymam, Mervi Hujanen and Matti Leisola (2002) Production of D-mannitol by heterofermentative lactic acid bacteria. Process Biochemistry. 37, 1207-1213.
- Soetaert W., (1991). Synthesis of D-mannitol and L-sorbose by microbial hydrogenation and dehydrogenation of monosaccharides. Ph.D. Thesis, University of Gent, Gent, Belgium.
- Song K. H., Lee J. K., Song J. Y., Hong S. G., Back H., Kim S. Y., Hyun H. H., (2002). Production of mannitol by a novel strain ofCandida magnoliae. Biotechnology letters Vol. 24(1), 9-12.
- Stoop J. M. H., and Pharr B. M., (1994). Mannitol metabolism in celery stressed by excess macronutrients. Plant Physiology, 106, 503-511.
- Yun J. W., and Kang S. C and Song S. K. (1996). Microbial transformation of fructose to mannitol byLactobacillus sp. KY-107. Biotechnology Letters. 18, 35-40.
Claims (18)
1. A plant mangrove-associated fungus Culvularia lunata of International deposition no. ______.
2. The fungus as claimed in claim 1 , wherein the mangrove plant is Acanthus illicifolius.
3. The fungus as claimed in claim 1 , wherein the fungus is fast growing fungus.
4. The fungus as claimed in claim 1 , wherein the fungus is of color brown to blackish brown with black reverse.
5. The fungus as claimed in claim 1 , wherein the fungus has conidia of pale brown color with three or more traverse septa and are formed apically through a pore (poroconidia) in a sympodially elongated geniculate conidiophore.
6. The fungus as claimed in claim 1 , wherein the fungus shows conidia of cylindrical or slightly curve shape, with one of the central cells being larger and darker.
7. A simple and efficient method of obtaining high yield of pure mannitol from a plant mangrove-associated fungus Curvularia lunata of International deposition No. ______, said method comprising steps of:
a. obtaining leaves from the plant mangrove,
b. rinsing the leaves in sterile seawater and keeping them in sterile, moist culture chamber for about two weeks,
c. cutting the leaves into small pieces and placing it on potato dextrose agar (PDA) plates for about 48 hours,
d. transferring individual colonies aseptically into a sterile fresh PDA plate to obtain pure culture,
e. maintaining the isolated fungal cultures on PDA slants for about 7 days to obtain mycelia,
f. transferring the mycelia to a fresh PDB with seawater and distilled water using starch and sugar as source of carbon.
g. incubating the culture at temperature ranging between 26-32° C. for about 3-6 days to obtain inoculum,
h. inoculating the PBD having seawater and distilled with the inoculum,
i. maintaining the culture at temperature ranging between 26-47° C. and salinity ranging between 4-32 ppt with occasional stress conditions for about 17-19 days to obtain mycelial mat,
j. sonicating the mat to lyse the cells,
k. extracting crude from the sonicated mat using methanol repeatedly,
l. concentrating the crude,
m. treating the concentrated crude extract with solvents of increasing polarity,
n. obtaining aqueous fraction as white powder residue after the treatment containing crude mannitol,
o. purifying the crude mannitol by chromatography to obtain pure mannitol with about 75% of the total crude extract.
8. A method as claimed in claim 7 , wherein the plant is Acanthus illicifolius.
9. A method as claimed in claim 7 , wherein the starch is of concentration ranging between 3-5 g/l.
10. A method as claimed in claim 7 , wherein the dextrose is of the concentration ranging between 18-22 g/l.
11. A method as claimed in claim 7 , wherein the ratio of seawater and distilled water is ranging between 1:5 to 5:1, preferably 1:1.
12. A method as claimed in claim 7 , wherein the stress conditions is temperature ranging between 40-45° C.
13. A method as claimed in claim 7 , wherein the stress conditions is salinity ranging between 15-17 ppt.
14. A method as claimed in claim 7 , wherein the stress conditions lead to high percentage yield of mannitol.
15. A method as claimed in claim 7 , wherein the leaves are young, and living leaves.
16. A method as claimed in claim 7 , wherein concentrating the crude using vacuum evaporator at temperature ranging between 28-32° C.
17. A method as claimed in claim 7 , wherein the solvents in increasing polarity are petroleum ether, chloroform, and ethyl acetate.
18. A method as claimed in claim 7 , wherein the chromatography is on G-10 solid support.
Priority Applications (9)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US10/464,845 US20040259217A1 (en) | 2003-06-19 | 2003-06-19 | Plant mangrove-associated fungus Culvularia lunata and a simple and efficient method of obtaining high yield of pure mannitol from the same |
JP2005500715A JP2007527192A (en) | 2003-06-19 | 2003-11-04 | Fungal strain and method for obtaining mannitol derived from the same substance |
EP03769746A EP1639094A1 (en) | 2003-06-19 | 2003-11-04 | Fungus strain and a method of obtaining mannitol from the same |
PCT/IB2003/004985 WO2004111204A1 (en) | 2003-06-19 | 2003-11-04 | Fungus strain and a method of obtaining mannitol from the same |
CA002529891A CA2529891A1 (en) | 2003-06-19 | 2003-11-04 | Fungus strain and a method of obtaining mannitol from the same |
AU2003278443A AU2003278443A1 (en) | 2003-06-19 | 2003-11-04 | Fungus strain and a method of obtaining mannitol from the same |
BRPI0318346-7A BR0318346A (en) | 2003-06-19 | 2003-11-04 | family of fungi and a method of obtaining mannitol from them |
CNA2003801103518A CN1788076A (en) | 2003-06-19 | 2003-11-04 | Fungus strain and a method of obtaining mannitol from the same |
US11/364,029 US7341855B2 (en) | 2003-06-19 | 2006-03-01 | Plant mangrove-associated fungus Curvularia lunata and a simple and efficient method of obtaining high yield of pure mannitol from the same |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US10/464,845 US20040259217A1 (en) | 2003-06-19 | 2003-06-19 | Plant mangrove-associated fungus Culvularia lunata and a simple and efficient method of obtaining high yield of pure mannitol from the same |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US11/364,029 Division US7341855B2 (en) | 2003-06-19 | 2006-03-01 | Plant mangrove-associated fungus Curvularia lunata and a simple and efficient method of obtaining high yield of pure mannitol from the same |
Publications (1)
Publication Number | Publication Date |
---|---|
US20040259217A1 true US20040259217A1 (en) | 2004-12-23 |
Family
ID=33517351
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/464,845 Abandoned US20040259217A1 (en) | 2003-06-19 | 2003-06-19 | Plant mangrove-associated fungus Culvularia lunata and a simple and efficient method of obtaining high yield of pure mannitol from the same |
US11/364,029 Expired - Fee Related US7341855B2 (en) | 2003-06-19 | 2006-03-01 | Plant mangrove-associated fungus Curvularia lunata and a simple and efficient method of obtaining high yield of pure mannitol from the same |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US11/364,029 Expired - Fee Related US7341855B2 (en) | 2003-06-19 | 2006-03-01 | Plant mangrove-associated fungus Curvularia lunata and a simple and efficient method of obtaining high yield of pure mannitol from the same |
Country Status (8)
Country | Link |
---|---|
US (2) | US20040259217A1 (en) |
EP (1) | EP1639094A1 (en) |
JP (1) | JP2007527192A (en) |
CN (1) | CN1788076A (en) |
AU (1) | AU2003278443A1 (en) |
BR (1) | BR0318346A (en) |
CA (1) | CA2529891A1 (en) |
WO (1) | WO2004111204A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114106584A (en) * | 2021-12-07 | 2022-03-01 | 甘肃农业大学 | Purification process and application of Erwinia carotovora powder pigment |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102154126B (en) * | 2010-11-19 | 2012-12-05 | 广西大学 | Strain and method for producing mannitol by strain |
CN103131643B (en) * | 2013-03-14 | 2014-03-26 | 江南大学 | Strain for producing mannitol and method for producing mannitol through fermentation of strain |
JP5731082B2 (en) * | 2013-03-19 | 2015-06-10 | オリジンバイオテクノロジー株式会社 | An ergothioneine production method and production apparatus using mushroom basidiomycetes using a submerged culture method. |
CN109797107B (en) * | 2019-01-29 | 2021-06-04 | 西北大学 | Preparation method of fungus mycelium fine powder |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4996149A (en) * | 1985-07-23 | 1991-02-26 | Nativelle S.A. | Microbiological hydroxylation process of quinine, quinidine and derivatives thereof |
US5250424A (en) * | 1991-06-14 | 1993-10-05 | Merck & Co., Inc. | Processes for preparing novel squalene synthetase inhibitors |
-
2003
- 2003-06-19 US US10/464,845 patent/US20040259217A1/en not_active Abandoned
- 2003-11-04 CA CA002529891A patent/CA2529891A1/en not_active Abandoned
- 2003-11-04 WO PCT/IB2003/004985 patent/WO2004111204A1/en not_active Application Discontinuation
- 2003-11-04 CN CNA2003801103518A patent/CN1788076A/en active Pending
- 2003-11-04 EP EP03769746A patent/EP1639094A1/en not_active Withdrawn
- 2003-11-04 BR BRPI0318346-7A patent/BR0318346A/en not_active IP Right Cessation
- 2003-11-04 AU AU2003278443A patent/AU2003278443A1/en not_active Abandoned
- 2003-11-04 JP JP2005500715A patent/JP2007527192A/en active Pending
-
2006
- 2006-03-01 US US11/364,029 patent/US7341855B2/en not_active Expired - Fee Related
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4996149A (en) * | 1985-07-23 | 1991-02-26 | Nativelle S.A. | Microbiological hydroxylation process of quinine, quinidine and derivatives thereof |
US5250424A (en) * | 1991-06-14 | 1993-10-05 | Merck & Co., Inc. | Processes for preparing novel squalene synthetase inhibitors |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114106584A (en) * | 2021-12-07 | 2022-03-01 | 甘肃农业大学 | Purification process and application of Erwinia carotovora powder pigment |
Also Published As
Publication number | Publication date |
---|---|
WO2004111204A1 (en) | 2004-12-23 |
CN1788076A (en) | 2006-06-14 |
US7341855B2 (en) | 2008-03-11 |
EP1639094A1 (en) | 2006-03-29 |
BR0318346A (en) | 2006-07-11 |
CA2529891A1 (en) | 2004-12-23 |
US20060205050A1 (en) | 2006-09-14 |
JP2007527192A (en) | 2007-09-27 |
AU2003278443A1 (en) | 2005-01-04 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN100390271C (en) | Method of producing xylosic alcohol and its special baterial strain | |
US7341855B2 (en) | Plant mangrove-associated fungus Curvularia lunata and a simple and efficient method of obtaining high yield of pure mannitol from the same | |
CN101358173B (en) | Aspergillus niger ZJUT712 and application thereof in arctium fruit preparation by solid-state fermentation | |
EP3845658B1 (en) | Method for preparing vanillin by fermentation with eugenol as substrate | |
CN100999750B (en) | Process of synthesizing gastrodiacin by microorganism cell biological transferring parhydroxy benzyl methylol | |
CN112322687A (en) | Microbial inoculum for preparing diosgenin and application thereof | |
CN106701604B (en) | Dongxiang wild rice endophytic fungus for efficiently converting glycyrrhizic acid to produce GAMG and application thereof | |
CN109456899B (en) | Penicillium and method for producing penicillic acid by fermenting penicillium | |
US20060110811A1 (en) | Novel candida tropicalis cj-fid(kctc 10457bp) and manufacturing method of xylitol thereby | |
CN114032187B (en) | Kluyveromyces marxianus strain from Tibetan yoghurt and application thereof | |
El-Refai et al. | Physiological and chemical studies on the bioconversion of glycyrrhizin by Aspergillus niger NRRL595 | |
Vahidi et al. | Effects of carbon sources on growth and production of antifungal agents by Gymnopilus spectabilis | |
TWI229697B (en) | Microbial production of actinol | |
RU2405829C2 (en) | Method of preparing organic solvents | |
CN113337433B (en) | Pseudomonas capable of producing pyrroloquinoline quinone and application thereof | |
JPH06116583A (en) | Fragrant substance and its production | |
CN109810906B (en) | Endophytic fungi and application thereof in fermentation preparation of phenolic acid compounds | |
CN115521877B (en) | Penicillium strain and application thereof in preparation of brefeldin A | |
RU2186846C2 (en) | Yeast strain saccharomyces cerevisiae used for fermentation of molasses must in production of ethyl alcohol and baking yeast | |
RU2036230C1 (en) | Strain of yeast saccharomyces vini for fruit and berry wine production | |
JP3318416B2 (en) | Method for side chain oxidation of aromatic compounds and microorganisms used therefor | |
US5304485A (en) | Antibiotic producing microorganism | |
CN105602856A (en) | Aspergillus niger An-19 strain and use thereof in production of lovastatin and fermentation process | |
CN109628543A (en) | Thallus system, which is mixed, using recombinant Saccharomyces cerevisiae and Inonotus obliquus prepares betulic acid method | |
WO1995033063A1 (en) | Microorganism-containing composition and method for the production of xylitol |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH, IND Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:DEVI, PRABHA;NAIK, CHANDRAKANT GOVIND;WAHIDULLA, SOLIMABI;AND OTHERS;REEL/FRAME:014521/0871 Effective date: 20030826 |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |