AU2004320343A1 - A cost-effective process for preparing agarose from Gracilaria SPP - Google Patents
A cost-effective process for preparing agarose from Gracilaria SPP Download PDFInfo
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- AU2004320343A1 AU2004320343A1 AU2004320343A AU2004320343A AU2004320343A1 AU 2004320343 A1 AU2004320343 A1 AU 2004320343A1 AU 2004320343 A AU2004320343 A AU 2004320343A AU 2004320343 A AU2004320343 A AU 2004320343A AU 2004320343 A1 AU2004320343 A1 AU 2004320343A1
- Authority
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- Australia
- Prior art keywords
- agarose
- seaweed
- alkali
- extractive
- gracilaria
- 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.)
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- 229920000936 Agarose Polymers 0.000 title claims description 87
- 241000206581 Gracilaria Species 0.000 title claims description 29
- 238000004519 manufacturing process Methods 0.000 title description 6
- 241001474374 Blennius Species 0.000 claims description 101
- 239000003513 alkali Substances 0.000 claims description 78
- 239000000499 gel Substances 0.000 claims description 76
- 238000000034 method Methods 0.000 claims description 65
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 63
- 241000873911 Gracilaria dura Species 0.000 claims description 40
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 36
- 239000000047 product Substances 0.000 claims description 27
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 25
- 239000002253 acid Substances 0.000 claims description 20
- 239000007788 liquid Substances 0.000 claims description 16
- 238000002360 preparation method Methods 0.000 claims description 15
- 239000003610 charcoal Substances 0.000 claims description 14
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 claims description 11
- 239000000706 filtrate Substances 0.000 claims description 11
- 238000001914 filtration Methods 0.000 claims description 11
- 238000005406 washing Methods 0.000 claims description 11
- 238000010438 heat treatment Methods 0.000 claims description 10
- 238000007710 freezing Methods 0.000 claims description 9
- 230000008014 freezing Effects 0.000 claims description 9
- 229910021653 sulphate ion Inorganic materials 0.000 claims description 9
- 238000010257 thawing Methods 0.000 claims description 9
- 238000002844 melting Methods 0.000 claims description 8
- 230000008018 melting Effects 0.000 claims description 8
- 239000000843 powder Substances 0.000 claims description 8
- 239000007787 solid Substances 0.000 claims description 8
- 238000006386 neutralization reaction Methods 0.000 claims description 7
- 150000007513 acids Chemical class 0.000 claims description 6
- 229920001282 polysaccharide Polymers 0.000 claims description 6
- 238000001694 spray drying Methods 0.000 claims description 6
- 230000015556 catabolic process Effects 0.000 claims description 4
- 238000006731 degradation reaction Methods 0.000 claims description 4
- 239000005017 polysaccharide Substances 0.000 claims description 4
- 150000004676 glycans Chemical class 0.000 claims description 3
- 230000003472 neutralizing effect Effects 0.000 claims description 3
- 239000011543 agarose gel Substances 0.000 claims description 2
- 230000001627 detrimental effect Effects 0.000 claims description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims 3
- 230000015572 biosynthetic process Effects 0.000 claims 1
- 230000009699 differential effect Effects 0.000 claims 1
- 230000008030 elimination Effects 0.000 claims 1
- 238000003379 elimination reaction Methods 0.000 claims 1
- 230000008450 motivation Effects 0.000 claims 1
- 238000002203 pretreatment Methods 0.000 claims 1
- 229920001817 Agar Polymers 0.000 description 66
- 235000010419 agar Nutrition 0.000 description 64
- 239000008272 agar Substances 0.000 description 57
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 17
- 238000000605 extraction Methods 0.000 description 13
- 241000206672 Gelidium Species 0.000 description 12
- 241000195493 Cryptophyta Species 0.000 description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- 239000002028 Biomass Substances 0.000 description 5
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 5
- 210000000988 bone and bone Anatomy 0.000 description 5
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 5
- 229920000573 polyethylene Polymers 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 241001137890 Gelidiella acerosa Species 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000002244 precipitate Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 239000007921 spray Substances 0.000 description 4
- 241001137889 Gelidiella Species 0.000 description 3
- 241000206572 Rhodophyta Species 0.000 description 3
- -1 alkylene glycol Chemical compound 0.000 description 3
- 230000000721 bacterilogical effect Effects 0.000 description 3
- 238000009835 boiling Methods 0.000 description 3
- 230000001143 conditioned effect Effects 0.000 description 3
- 238000004090 dissolution Methods 0.000 description 3
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 3
- 229920003023 plastic Polymers 0.000 description 3
- 239000004033 plastic Substances 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 239000003643 water by type Substances 0.000 description 3
- 241001428219 Gracilariaceae Species 0.000 description 2
- 239000004677 Nylon Substances 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 238000005904 alkaline hydrolysis reaction Methods 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000004744 fabric Substances 0.000 description 2
- 239000006260 foam Substances 0.000 description 2
- 239000000416 hydrocolloid Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 229920001778 nylon Polymers 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- 239000004382 Amylase Substances 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 241001269524 Dura Species 0.000 description 1
- 241001428244 Gelidium pusillum Species 0.000 description 1
- 241001428186 Gracilaria cornea Species 0.000 description 1
- 241000703912 Gracilaria edulis Species 0.000 description 1
- 241001180358 Gracilaria eucheumoides Species 0.000 description 1
- 241000206587 Gracilaria gracilis Species 0.000 description 1
- 241001467331 Gracilaria sp. Species 0.000 description 1
- 241001428221 Gracilariales Species 0.000 description 1
- 241000703939 Gracilariopsis longissima Species 0.000 description 1
- CBENFWSGALASAD-UHFFFAOYSA-N Ozone Chemical compound [O-][O+]=O CBENFWSGALASAD-UHFFFAOYSA-N 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 241001137893 Pterocladia Species 0.000 description 1
- 229910000831 Steel Inorganic materials 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000003957 anion exchange resin Substances 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000012736 aqueous medium Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 239000000679 carrageenan Substances 0.000 description 1
- 235000010418 carrageenan Nutrition 0.000 description 1
- 229920001525 carrageenan Polymers 0.000 description 1
- 229940113118 carrageenan Drugs 0.000 description 1
- 239000012159 carrier gas Substances 0.000 description 1
- 239000003729 cation exchange resin Substances 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 229960001927 cetylpyridinium chloride Drugs 0.000 description 1
- YMKDRGPMQRFJGP-UHFFFAOYSA-M cetylpyridinium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCC[N+]1=CC=CC=C1 YMKDRGPMQRFJGP-UHFFFAOYSA-M 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000008395 clarifying agent Substances 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000012364 cultivation method Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000002354 inductively-coupled plasma atomic emission spectroscopy Methods 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 125000000896 monocarboxylic acid group Chemical group 0.000 description 1
- 238000013386 optimize process Methods 0.000 description 1
- 238000009344 polyculture Methods 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000001932 seasonal effect Effects 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000010959 steel Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 150000003467 sulfuric acid derivatives Chemical class 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 238000003809 water extraction Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- UHVMMEOXYDMDKI-JKYCWFKZSA-L zinc;1-(5-cyanopyridin-2-yl)-3-[(1s,2s)-2-(6-fluoro-2-hydroxy-3-propanoylphenyl)cyclopropyl]urea;diacetate Chemical compound [Zn+2].CC([O-])=O.CC([O-])=O.CCC(=O)C1=CC=C(F)C([C@H]2[C@H](C2)NC(=O)NC=2N=CC(=CC=2)C#N)=C1O UHVMMEOXYDMDKI-JKYCWFKZSA-L 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0036—Galactans; Derivatives thereof
- C08B37/0039—Agar; Agarose, i.e. D-galactose, 3,6-anhydro-D-galactose, methylated, sulfated, e.g. from the red algae Gelidium and Gracilaria; Agaropectin; Derivatives thereof, e.g. Sepharose, i.e. crosslinked agarose
-
- 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
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/10—Process efficiency
- Y02P20/133—Renewable energy sources, e.g. sunlight
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Polymers & Plastics (AREA)
- Medicinal Chemistry (AREA)
- Materials Engineering (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Edible Seaweed (AREA)
Description
WO 2005/118830 PCT/IB2004/001792 1 A COST-EFFECTIVE PROCESS FOR PREPARING AGAROSE FROM GRACILARIA SPP. Field of the Present Invention 5 A simple, direct and cost-effective process for the preparation of agarose of high gel strength and low gelling temperature from naturally occurring or cultivated Gracilaria spp. more particularly Gracilaria dura. Background and Prior art references of the Present Invention Reference may be made to the internet web site www.sporeworks.com which states that 10 Agar primarily finds use in the food, medical/pharmaceutical and cosmetic industries. Agar is also used in other industries such as its recent application in the packaging foam industry for the production of biodegradable packaging foam. The site further describes the salient features of high quality bacteriological agar. It states that such agar should have a gelling temperature of 340-35' Celsius to minimize possible 15 degradation of heat sensitive antibiotics that are added into the culture medium after sterilization. It is further stated that the cooler agar is easier to handle and condensation in petri dish is less of a problem. Another important attribute of a good quality bacteriological agar is that it should possess a minimum gel strength of 800 gcm 2 under standard conditions of measurement. 20 Reference may be made to the Fluka Catalog of 2003-2004 wherein the specifications are provided for a range of agar products. Reference may be made to Sigma Catalog of 2000-2001 wherein it is stated that agarose is a purified linear galactan hydrocolloid isolated from agar or agar-bearing marine algae. A range of agarose in the gel strength range of 650-1200 g/cm 2 (1% 25 solution) with gel point in the range of 36-42 0 C are described. One drawback of such products is their high cost, which is presumably due to the elaborate purification processes involved. Reference may also again be made to www.sporeworks.com which states that Gelidium seaweed produces the highest grade agar and agarose. 30 Reference may be made to the paper on production of bacteriological agar from Gelidium wherein it is reported that the products from this seaweed give high gel 'strength (R. Armisen, J. Apple. Phycology, 1995, 7:231-243, and J. Cosson et al. in Progress in Phycological Research, F.E. Round and D.J. Chapman, Eds., Biopress Ltd. 1995. Vol. 11; pp. 269-324).
WO 2005/118830 PCT/IB2004/001792 2 Mention may be made of the paper by Andres Lemus et al. (Food Hydrocolloids, 1991, 5:469-479) wherein it has been reported that agar from different Gelidium species has gelling temperature from 34.0' to 37.5oC and gel strength 687 to 1470 g/cn 2 obtained by a process which involves alkali pretreatment of the seaweed followed by adjustment 5 of pH (with acid presumably) to 6 to 6.5 before extraction and purification of the extracted agar by two freeze-thaw cycles. Mention may be made to "Process for producing agar-agar from an algae extraction juice" by Lebbar et al. (US 4,780,534; 1988) wherein a process for making agar-agar from algae (Gelidium, Gracilaria and Pterocladia spp.) extraction juices and 10 comprising: (a) placing the extraction juice in the presence of a cationic ion exchange resin conditioned into the Na + form, then placing it (b) in the presence of an anion exchange resin conditioned into the Cl and/or SO42- form, then (c) optionally placing the juice in the presence of a cation ion exchange resin conditioned into the OH- form, (d) thereupon gelling the juice, (e) extracting the agar-agar from the obtained gel, and 15 where called for (f) treating the obtained powder by placing it in contact with an ozone loaded carrier gas. Such a process allows producing at reduced costs high-grade agar agar, useful in particular in the medical, pharmaceutical and bioengineering fields. The gel strength was reported in the range 820-910 g/cm 2 in 1.5% gel. Mention may be made of the paper by M.Y. Roleda et al. (Botanica Marina 1997, 20 40:63-69) where agars from Gelidiella acerosa having gelling temperatures of 38 0 C and 47 0 C, and gel strengths of 493 and 200 g cm 2 , respectively, have been reported to be prepared by a process involving acetic acid pretreatment before extraction. Mention may be made to the article of O. P. Mairh et al. (Botanica Marina 1978, 21:169-174) wherein it is reported that agar from cultured Gelidium pusillum obtained 25 from the Arabian Sea at west coast of India has gel strength of 210 g cm 2 which is considered as poor in the context of the present invention. Moreover, no information is provided on gelling temperature. Reference may be made to Krishnamurthy et al. (Proceedings, symposium on marine algae of Indian Ocean Region, Central Salt & Marine Chemicals Research Institute, 30 Bhavnagar, 1979, p41) wherein it is stated that the maximum gel strength of 325 g cm-2 was obtained from Gelidiella acerosa whereas the corresponding gelling temperature was 38-52 0
C.
WO 2005/118830 PCT/IB2004/001792 3 Reference may also be made to K. S. Pillai (J. Phycol., 13 (Suppl.), 1977, p54) who has attempted optimization of process conditions to maximize quality of agar and reported a yield from Gelidiella acerosa as high as 48% hut with maximum gel strength of 300 g/cm 2 whereas the corresponding numbers were 45-50% and 125g/cm 2 for Gracilaria 5 (species not mentioned). Reference may be made to Patel et al. (J. Phycol. 13 (Suppl.), 1977, p5 2 ) who obtained a yield of 24.3% and gel strength of 790 g/cm 2 from Gelidiella acerosa growing in the Indian coast. It can be seen that the best reported result for Gelidium is a gel point of 37.5 0 C and gel 10 strength of 1470 g/cm 2 for 1.5% gel whereas the best reported gel strength is 790 g/cm 2 in case of Gelidiella species. If products such as agarose with still superior specifications are required, then the agar needs to be further purified for this purpose. It would be desirable if such products can be made from seaweed sources without elaborate purification. 15 Mention may be made to "Purification of agar" by Kiyoshi Arai et al. (JP 7017,130, January 13, 1970; Chemical Abstr. 74, 32889r, 1971) wherein it is reported that crude agar was extracted with DMF to separated high purity agarose. 10 g Agar mixed with 500 ml DMF with stirring, dipped 10 h in hot water, centrifuged, the supernatant poured into 2 liter acetone, and the precipitates are passed through a glass filter, washed 20 with 500 ml acetone, dissolved in hot water and filtered to give agarose powder. Mention may be made to "Isolation of partially purified agarose with a quaternary base" by Craigie and Leigh (in Handbook of Phycological Methods, edited by J A Hellebust and J S Craigie, Cambridge University Press, Cambridge, 1978; p. 126) where 250 mg of crude agar was dissolved in 100 ml of boiling distilled water. 25 mg of L 25 carrageenan was added and 1.0 ml 2% Cetavlon (cetylpyridinium chloride) was added at 80-100oC solution. The hot extractive was filtered with Celite and pressure filtered over membrane (0.8 micron) followed by freezing and thawing of the product to have partially purified agarose. Mention may be made to "Agarose purification method using. glycol" by R. B. 30 Provonchee (US 4,990,611, February 1991) where purified agarose was recovered from agar or impure agarose by dissolving the agar or agarose in a lower alkylene glycol at elevated temperature, cooling the agar or agarose-containing glycol solution to WO 2005/118830 PCT/IB2004/001792 4 induce precipitation of a purified agarose product, and recovering the precipitated agarose product. Mention may also be made to US Patent No. 4,983,268 by Kirkpatrick et al. that describes the preparation purified agarose suitable for rapid electrophoresis, 5 characterized by a sulfate content less than 0.2 wt % and gel strength of at least 1200 g/cm 2 (1%). Agaroses are purified by dissolving agarose or alkali-modified agar in an -aqueous medium buffered at a pH of 6.0 to 8.0 and containing no more than 2.0 nM salt as chloride, and precipitating the agarose by contact with lower alkanol. Reference may also be made to the work of Alfred Polson (Chemical Abstract 65: 10 p5865a; 1965) wherein fractionation of mixture of agarose and agaropectin has been described for preparation of agarose. The mixture is treated with an aqueous solution of poly(ethylene glycol) of molecular weight 300, giving a precipitate enriched in agarose. In this process 80g of lonagar No. 2 was dissolved in 2 liter of water. To the hot solution (80C) was added 2 liters of 40% (wt/vol) polyethylene glycol of molecular 15 weight 6000 and the resultant precipitate separated by filtration through 1.10 mesh nylon cloth. The precipitate then washed at 40 0 C for 2-3 minutes, suspended in water at 15 0 C, stirred overnight in 5 liters of water, collected in nylon mesh, washed with acetone and dried in warm air. Reference may be made to the publication ofR. Armisen (J. Apple. Phycol. 1995, 7:231 20 243) wherein it has been stated that world's first source of agar, from the middle of the seventeenth century, was Gelidium from Japan, but by the beginning of the twentieth century demand for the phycocolloid exceeded supply of this algae which made it necessary to look at alternative seaweed sources. It has also been stated that the development of production process through alkaline hydrolysis of sulphates has 25 allowed good quality food grade agar to be obtained from Gracilaria. Reference may be made to the paper of A.Q. Hurtado-Ponce et al. (Botanica Marina 1988, 31:171-174) which has reported agars having poor gel characteristics, specifically different gelling temperature and gel strengths (low and high and vice versa) from various Gracilaria species, as follows: (i) agar from a Gracilaria sp. (no 30 species details provided) having gelling temperature 41.3C and gel strength of 470 g/cm 2 ; (ii) agar from Gracilaria edulis having gelling temperature of 55 0 C and gel strength of 140 g/cnm 2 ; (iii) agar from Gracilaria verrucosa having gelling temperature WO 2005/118830 PCT/IB2004/001792 5 of 53oC and gel strength of 270 g/cm 2 ; (iv) agar from Gracilaria eucheumtnoides having gelling temperature of 34oC and gel strength of 130 g/cm 2 Reference may be made to the paper of J. Rebello et al. (J. Appl. Phycol. 1997, 8:517 521) where agar of Gracilaria gracilis having high gelling temperature (59oC) with low 5 gel strength (350 g/cm 2 ) has been reported. Mention may be made to the internet website (www.rheofuture.coni) and the paper by Y. Freile-Pelegrin et al. (J. Appl. Phycol. 1997, 9:533-539) wherein it is reported that, in the pretreatment of Gracilaria for agar extraction, the optimum concentration of alkali is species specific. The authors of the latter paper further mention that the alkali 10 was neutralized by soaking the pre-treated seaweed in 0.025% H 3
PO
4 for light alkali pretreatment and in 0.025% H 2
SO
4 for 3% and 5% NaOH pretreatment. Such pretreatment was found to give gel strength of agar in the range of 974-1758 g/cm 2 from Gracilaria cornea of Yucatan, Mexico. However, with increase of gel strength the gelling temperature also increases and the agar having gel strength of 1758 g/cm 2 15 has gelling temperature of 42-43 0 C whereas for many applications there is a requirement of low gelling temperature and high gel strength. Reference may be made to the paper by R. D. Villanueva et al. (Botanica Marina Vol. 40, 1997, pp 369-372), which reports the optimized agar extraction from Gracilaria eucheumoides Harvey. In this method the seaweed is subjected to pretreatment with 20 NaOH and then washed with 0.5% acetic acid. With optimized process conditions, the maximum gel strength obtained was 423+43 g/cm 2 . No mention is made of the corresponding gelling temperature. Ma. R. J. Luhan has.also carried out a study similar to that described above (Botanica Marina, 35, 1992, pp.169-172) for Gracilaria heteroclada collected from Iloilo, 25 Central Philippines and the gel strength was found to be in the range of 510-794 g/cm 2 for seaweed collected during the early dry season and 43-101 g/cm for material collected during the wet season. No mention is made of gelling temperature. E. Marinho-Soriano reported extraction of agar polysaceharides from different Gracilaria species (Rhodophyta, Gracilariaceae) including Gracilaria dura (Journal of 30 Biotechnology 89:81-84, 2001), using hot water extraction at 1 10oC for 1 hr, without any pretreatment of the seaweed. The gel strength of agar was reported to be 318+49 g/cm 2 g/cm .
WO 2005/118830 PCT/IB2004/001792 6 Reference may be made to E. Murano et al. (Hydrobiologia 204/205:567-571, 1990) who have referred to the occurrence of Gracilaria dura in the Adriatic Sea. Reference may be made to E. Murano et al. (Carbohydrate Polymers 1992, 18:171-178) where it was reported that agar was extracted from Gracilaria dura growing in northern 5 Adriatic Sea, both with and without alkali pretreatment. Alkali pretreatment was followed by neutralization with HCI before extraction. Gel strength of native and alkali treated agar were reported to be 160 and 390 g/cm 2 . Reference may be made to E. Murano, C. Brandolin, F. Zanetti, S. Paoletti and R. Rizzo (Hydrobiologia 204/205:567-571, 1990) who reported characterization of an agar 10 fraction extracted from Gracilaria dura (Gracilariales, Rhodophyta)- occurring in northern Adriatic Sea as tentatively cultivated in integrated polyculture systems, using hot water (90'C) and 0.5M NaOH (90'C, 3 hr) followed by enzymatic treatment by means of amylase. Besides the fact that the reported results offer nothing unusual in terms of the quality of agar, this is a more complex method of processing than the 15 conventional method of agar extraction. Reference may also be made to E. Marinho-Soriano (Journal of Biotechnology 89:81 84, 2001) who has reported the occurrence of Gracilaria dura in Thau lagoon (43o24'N; 03'32'E) in the Mediterranean Sea. Reference may be made to R.M. Oza and S.H. Zaidi (A revised checklist of Indian 20 marine algae, National Marine Data Centre on Algae and Marine Chemicals, Department of Ocean Development, Government of India; Central Salt and Marine Chemicals Research Institute, Bhavnagar, Gujarat, India, 2001; p 25) who have reported the natural occurrence of Gracilaria dura (C. Agardh) J. Agardh (Rhodophyta, Gracilariaceae) in the west coast of India 25 Reference may be made to A.K. Siddhanta, et al. (Seaweed Research and Utilisation 19 (1&2): 95-99, 1997) who reported the preparation of agar from the natural stock of Gracilaria dura collected from the west coast of India. The dry seaweed was pretreated with I N sulfuric acid followed by neutralization with 1.5% alkali. Agar having gel strength of 260 g/cm 2 was obtained through this process. 30 It will be evident from the above examples that there are no reports of preparation of agar from any Gracilaria species having simultaneously a high gel strength (>1500 g/cm 2 at 1% level) and low gelling temperature (35-36 0
C).
WO 2005/118830 PCT/IB2004/001792 7 It will further be seen from the above examples that in all instances Gracilaria seaweeds have been either extracted directly with water or subjected to pretreatment to effect alkaline hydrolysis and the excess alkali subsequently neutralized with acids (HCI, H 2
SO
4 , CH 3 COOH and o-phosphoric acid) prior to extraction of agar. 5 Reference may be made to the article by H. H. Selby and R.L. Whistler (Industrial gums - Polysaccharides and their Derivatives, R. L. Whistler and J. N. BeMiller, Eds., 3rd Edition, Academic Press Inc., New York, 1993, pp 87-103). It is mentioned therein that several types of seaweeds may be blended to provide an agar composition of desired characteristics. No mention has been made,'however, of any formulations 10 based on Gracilaria dura. It will be evident from the prior art that the quality of agar claimed to be produced from Gracilaria dura growing in different regions of the world is rather ordinary and there are no reports of agarose type products having high gel strength and low gelling temperature that have been produced from this seaweed. 15 Objects of the Present Invention The main object of the invention is to prepare agarose from Gracilaria dura occurring in Indian waters. Another object is to prepare agarose having very high gel strength '(>1900 g/cm 2 ; 1% gel at 20 0 C), low gelling temperature (-35oC), low ( 0.25) sulfate residue and low ash 20 content (<1%) in a cost-effective manner. Yet another object is to prepare agarose by pretreating the seaweed with an optimum concentration of alkali. Yet another object of the invention is to remove residual alkali (after pretreatment) through water rinses only instead of by the conventional method of neutralization with 25 acid. Yet another object is to demonstrate that the above method of excess alkali removal yields a product with greatly enhanced gel strength. Yet another object is to prepare spray dried agarose for easier dissolution of the product. 30 Yet another object is to demonstrate that there is no significant deterioration in agarose quality when the dry Gracilaria dura seaweed is stored in plastic bags under ambient condition for well over a year.
WO 2005/118830 PCT/IB2004/001792 8 Yet another object of this invention is to demonstrate that the seaweed is amenable to cultivation, even at locations distant from its natural origin. Yet another object of this invention is to demonstrate that the quality of agarose obtained from naturally occurring- and cultivated Gracilaria dura are similar. 5 Another object of the invention is to demonstrate that production of agarose from Gracilaria dura in sufficient quantity is feasible even though its natural occurrence is limited. Summary of the present Invention The present invention relates to a simple, direct and cost-effective process for the 10 preparation of agarose of high gel strength and low gelling temperature from naturally occurring or cultivated Gracilaria spp. more particularly Gracilaria dura, said process comprising steps of pre-treating the dry seaweed with alkali, rinsing the pre-treated seaweed until the washing shows a pH ranging between 7 and 8, adding water, autoclaving to obtain extractive, treating the extractive with charcoal and Celite to 15 obtain hot extractive, vacuum-filtering the hot extractive over a Celite bed, freezing the filtrate into a mass and thawing the mass, redissolving the mass in water by heating in an autoclave, repeating the freeze- thaw cycle, straining the product to remove thawed liquid and thereafter squeezing to expel residual liquid to the extent possible to obtain agarose, and a agarose thereof. 20 Detailed description of the Present Invention Accordingly, the present invention relates to a simple, direct and cost-effective process for the preparation of agarose of high gel strength and low gelling temperature from naturally occurring or cultivated Gracilaria spp. more particularly Gracilaria dura, said process comprising steps of pre-treating the dry seaweed with alkali, rinsing the 25 pre-treated seaweed until the washing shows a pH ranging between 7 and 8, adding water, autoclaving to obtain extractive, treating the extractive with charcoal and Celite to obtain hot extractive, vacuum-filtering the hot extractive over a Celite bed, freezing the filtrate into a mass and thawing the mass, redissolving the mass in water by heating in an autoclave, repeating the freeze- thaw cycle, straining the product to remove 30 thawed liquid and thereafter squeezing to expel residual liquid to the extent possible to obtain agarose. In an embodiment of the present invention, wherein the invention relates to an agarose of following characteristics: WO 2005/118830 PCT/IB2004/001792 9 i. about 1% (> 1900 g/cm 2 ) gel strength at about 20 0 C, ii. 35 to 35.5C gelling temperature, iii. 5 0.25 % sulphate content, and iv. _ 0.9 % ash content, 5 In yet another embodiment of the present invention, wherein the agarose is obtained from Gracilaria spp. more particularly Gracilaria dura. In still another embodiment of the present invention, wherein the agarose gel has melting temperature ranging between 98-100 0 C. In still another embodiment of the present invention, A simple, direct and cost-effective 10 process for the preparation of agarose of high gel strength and low gelling temperature from naturally occurring or cultivated Gracilaria spp. more particularly Gracilaria dura. said process comprising steps of: * obtaining dry seaweed Gracilaria spp, * pre-treating the dry seaweed with about 35 parts (v/w) of about I to 15% alkali 15 at 25 to 95 0 C for 0.5 to 5.0 hours, * rinsing the pre-treated seaweed thoroughly with water to remove excess alkali until the washing shows a pH ranging between 7 and 8, * adding about 35 parts (v/w) of water for about every one part of original seaweed and autoclaving at about I 15-125oC for time duration ranging between 20 1.5 to 2.0 hours to obtain extractive, * treating the extractive with about 0.05 to 0.07% charcoal and about 10-15% Celite at a temperature ranging between 85 and 95oC to obtain hot extractive, * vacuum-filtering the hot extractive over a Celite bed, * freezing the filtrate into a mass at about -20oC for 12 to 15 hours and thawing 25 the mass, * redissolving the mass in about 25 parts of water by heating in an autoclave, * repeating the freeze- thaw cycle if required, * straining the product of step (i) to remove thawed liquid and thereafter squeezing to expel residual liquid to the extent possible to obtain agarose, and 30 * optionally redissolving the solid and spray drying to obtain fine powder. In still another embodiment of the present invention, wherein the alkali is sodium hydroxide.
WO 2005/118830 PCT/IB2004/001792 10 In still another embodiment of the present invention, wherein the concentration of alkali is about 10%. In still another embodiment of the present invention, wherein the time duration of autoclaving at step (d) is preferentially about 1.5 hours. 5 In still another embodiment of the present invention, wherein the agarose yield is ranging between 20-23% of dry seaweed weight. In still another embodiment of the present invention, wherein pre-treating the seaweed at temperature preferably about 85 0 C. In still another embodiment of the present invention, wherein pre-treating the seaweed 10 for preferentially about 2.0 hours. In still another embodiment of the present invention, wherein the autoclaving is at temperature preferentially about 120 0 C. In still another embodiment of the present invention, wherein concentration of charcoal is about 0.06%. 15 In still another embodiment of the present invention, wherein concentration of Celite is about 12.5%. While the prior art reveals that high quality agar and agarose are mainly obtained from Gelidium and Gelidiella seaweeds, and that agar extracted from Gracilaria dura exhibits gel strength only in the range of 160-390 g/cm 2 (1.5%), the present invention 20 describes the preparation of agarose from Gracilaria dura occurring sparsely in the Arabian Sea in the west coast of India and the same seaweed cultivated successfully to increase biomass for practical utility, wherein the fresh seaweed was harvested, dried in the field, re-soaked in the laboratory at ambient temperature, treated with aqueous NaOH, washed with water to remove excess alkali, soaked in excess water, pressure 25 cooked, then the hot extractive was homogenized, then boiled in presence of clarifying aids, filtered over a Celite bed, the filtrate was subjected to freeze-thaw cycle, the solid obtained was re-dissolved and once again subjected to freeze-thaw to further reduce impurities, air dried and ground or preferably redissolved in water and spray dried. In an embodiment of the present invention, Gracilaria dura was harvested from 30 Veraval coast in western India situated at 20 0 54'N, 70o22'E. In another embodiment of the present invention, alkali pretreatment was carried out for 1-2 h at 80-85 oC using sodium hydroxide as the alkali of choice.
WO 2005/118830 PCT/IB2004/001792 11 In another embodiment of the present invention, the concentration of alkali for pretreatment was in the range of 0-15% (w/v) and preferably 10%. In another embodiment of the present invention, the volume of alkali taken was 300 mL for every 10 g of seaweed. 5 In yet another embodiment of the present invention excess alkali after pretreatment was removed by rinsing the seaweed with water thereby avoiding the use of any acid and the pH was ensured to be in the range of 7-8 in the final washing. In yet another embodiment of the present invention, the wet seaweed after pretreatment was taken in water in an autoclave wherein the water quantity was 300 mL for every 10 10 g of dry seaweed taken initially. In yet another embodiment of the present invention, the pretreated seaweed was cooked in water for 1.5-2.0 h at 120 C. In yet another embodiment of the present invention, the hot extractive was discharged at 70-80 oC, following which charcoal and celite were added into the extractive and 15 then taken to boiling at atmospheric pressure. In yet another embodiment of the present invention, the boiling extractive was vacuum filtered over a celite bed. In yet another embodiment of the present invention the clear hot filtrate was poured into flat steel trays and allowed to cool to room temperature so as to form a gel. 20 In yet another embodiment of the present invention the gel was sliced at regular intervals along the x and y axes with a knife and then cooled to -20'C over 2-5 h to freeze the mass and then maintained at the low temperature for 12-15 h. In yet another embodiment of the present invention, the process of freeze-thaw was repeated if required. 25 In yet another embodiment of the present invention, the agarose obtained under optimum extraction conditions had gel strength of > 1900 gcm- 2 at 1% concentration and 20 0 C temperature. In yet another embodiment of the present invention, gel prepared from the agarose obtained under optimum extraction conditions had a melting. temperature of 98-100 oC 30 whereas the sol had a gelling temperature of 35.0-35.5 0 C. In yet another embodiment of the present invention, the agarose obtained under optimum processing conditions had a sulphate content of 0.25% and ash content of 0.9% WO 2005/118830 PCT/IB2004/001792 12 In yet another embodiment of the present invention, the hot agarose sol was spray dried for easy dissolution on heating. In yet another embodiment of the present invention, Gracilaria dura was amenable to cultivation in polythene bag or raft in the Gulf of Mannar at the southeast coast of India 5 with up to 5% daily growth rate for cultivation on raft in Krusadai island (9 0 16'N, 79o19'E). In yet another embodiment of the present invention, naturally occurring and cultivated seaweeds yielded agarose of similar quality making the invention practically feasible in view of the large biomass that can be generated through cultivation. 10 In yet another embodiment of the present invention, the quality of agarose extracted from dry seaweed stored in plastic bags for up to I year was similar to that of agarose prepared from freshly dried seaweed. Accordingly, the present invention describes the preparation of agarose of high gel strength and low gelling temperature from naturally occurring or cultivated Gracilaria 15 dura by a process involving: (i) weighing of the dry seaweed, (ii) pretreatment of the dry seaweed with 35 parts (v/w) of 10% sodium hydroxide at 85 0 C for 2 hi, (iii) rinsing the seaweed thoroughly with water to remove excess alkali until the washing shows a pH of 7-8, (iv) adding 35 parts (v/w) of water for every one part of original seaweed and autoclaving at 120 0 C for 1.5 h, (v) treating the extractive with 0.06% charcoal and 20 12.5% Celite (percentages w.r.t. weight of dry seaweed taken) at a temperature of 85 90oC, (vi) vacuum filtering the hot extractive over a Celite bed, (vii) freezing the filtrate at -20 0 C for 15 h and then thawing the mass, (viii) redissolving the mass in 25 parts of water by heating in an autoclave, (ix) repeating the freeze- thaw cycle if required, (x) straining to remove thawed liquid and thereafter squeezing to expel residual liquid to 25 the extent possible, (xi) redissolving the solid and spray drying to obtain fine powder. Gel strength was measured on a Nikkansui type gel tester in 1.5% agar gel at 20 0 C. TGA was measured on a STAR-Toledo TGA machine, Switzerland. Molecular weight determination was done measuring intrinsic viscosity on an Ostwald Viscometer (cf. C. Rochas and M. Lahaye. Carbohydrate Polymers 1989, 10:289). Gelling and melting 30 temperatures were measured following the method described by Craigie et al. (Hand Book Of Phycological Methods, 1978 (Eds. Hellebust. JA and Craigie JS, Cambridge University Press); pp.127), ash content was measured by incinerating the solid at 800 oC for 6h, sulphate content was estimated by. treating the ash with concentrated nitric WO 2005/118830 PCT/IB2004/001792 13 acid, evaporating to dryness, dissolving the residue in water, filtering, and subjecting to ICP-OES analysis for S. The main inventive step is moving away from the traditional belief that Gelidium and Gelidiella are the only seaweeds occurring in the Indian coast that yield high quality 5 agar. Another inventive step is revisiting of the seaweed, Gracilaria dura, given up originally as of little utility both because of the ordinary quality of agar obtained and limited biomass in nature. Another inventive step is identification of optimum NaOH strength for pretreatment of 10 the seaweed. Another inventive step is avoiding the conventional process of neutralizing excess alkali (after pretreatment) with acid and instead giving water rinses only for removal of such excess alkali and thereby preventing acid-catalysed degradation of the polysaccharide which can occur due to high local concentrations of acid. 15 Another inventive step is being undeterred by the low biomass of the seaweed in nature and taking recourse to cultivation in suitable locations. Another inventive step is identifying raft cultivation as a viable cultivation method that can yield daily growth rate in excess of 5% in suitable coastal locations. Another inventive step is revisiting a seaweed that had was given up originally as of 20 little utility. Another inventive step is being undeterred by the low biomass of the seaweed in nature and taking recourse to cultivation in a conducive environment at a very different location from the location of natural origin. Another inventive step is cultivating the seaweed by vegetative cutting on a raft placed 25 in the open, placid sea and thereby obtaining high daily growth rate. Another inventive step is the recognition from the prior art that different seaweed substrates may require different amounts of alkali in the pretreatment step and subsequent optimization of alkali concentration. Another inventive step is the recognition that the process of removal of alkali followed 30 in the prior art, namely neutralization of excess alkali with acid, may pose difficulties in view of the instantaneous build up of high local concentration of acid in the system that would be detrimental to the stability of the linear galactan molecule.
WO 2005/118830 PCT/IB2004/001792 14 Another inventive step is to make a spray dried product for easy dissolution upon heating. Another inventive step is the systematic study leading to the finding that the seaweed after sun drying has adequate shelf life for prolonged storage that is necessary as 5 cultivation may not be yearlong activity. Another inventive step is the preparation through a solvent-free process. The following examples are given by way of illustration and, therefore, should not be construed to limit the scope of the present invention. Examples I and 2 pertain to the prior art whereas Examples 3-8 illustrate the invention described herein. 10 EXAMPLE 1 Gracilaria dura from Veraval, India (20054 ' N and 70022' E) was harvested in April 2003 and sun-dried. 15 g of the seaweed was soaked in tap water for I h at ambient temperature (30-35oC) and the water then discarded. The wet seaweed was then taken in distilled water (seaweed:water = 1:35, w/v) and autoclaved at 120 0 C for 1.5 h. The 15 extract was homogenized and boiled with clarifying agents (charcoal and Celite) followed by filtration over a Celite bed under reduced pressure. The filtrate was then frozen at -20 0 C for 15 h and then thawed. The contents were then taken in a cloth and the water squeezed out to the maximum extent possible. The residue was then air dried at ambient temperature (30-35oC) and subsequently oven dried at 50 0 C for 2 h. 4.41 g 20 of agar (32.5% yield based on bone-dry seaweed) was obtained having 265 g/cm 2 gel strength (1.5% gel; 20C), 32 0 C gelling temperature, 8.04% ash and 3.26% sulphate. EXAMPLE 2 Gracilaria dura of Example 1 was initially soaked in water, the water then discarded and the wet seaweed treated with 5% NaOH at 80-85C for 2 h followed by washing 25 the seaweed with water to remove excess alkali. Residual alkali was neutralized with 0.5% acetic acid in one case and with 0.025% H 2
SO
4 in another case. The seaweed was then subjected to autoclaving and further worked up as described in Example 1. The results obtained are summarized in Table 1.
WO 2005/118830 PCT/IB2004/001792 15 Table 1. Agar obtained with alkali treatment and neutralization of residual alkali with acid Seaweed Pretreatment Yield (g); Gel strength Gelling T quantity conditions (% yield w.r.to (g/cm 2 ) at (oC) bone dry 20'C; (%gel) seaweed) 20g 5.0% alkali, acid wash 3.6 (21.0) 470 33.5 with 0.025% H 2
SO
4 (1.0%) 10g 5.0% alkali, acid wash 1.31 (15.0) 465 . 33.0 with 0.5%AcOH (1.0%) EXAMPLE 3 5 20 g of dry Gracilaria dura was processed as in Example 2, except that the seaweed after alkali treatment was only subjected to water rinses to remove all the alkali and no acid was used in the process. As can be seen from Table 2 below, a significant enhancement of gel strength was observed as a result of the modification. Table 2. Agar obtained after removal of residual alkali from the seaweed by 10 water wash Seaweed Pretrea Yield (g); Gel strength Gelling T Ash Sulphate quantity tment (% yield (g/cm 2 ) (OC) (%) (%) conditi w.r.to bone at 20oC; ons dry (%gel) seaweed) 20g 5% 3.8 1620 (1.0%) 35.0 2.02 0.50 alkali, (21.0%) water wash EXAMPLE 4 Gracilaria dura was treated with different concentrations of alkali, otherwise, following the process of Example 3 in all the cases. The results of the study are shown in Table 3. It can be seen from the table that 10% alkali is the optimum concentration that yields 15 maximum gel strength while at the same time avoiding alkali concentrations beyond that which is required. Table 3. Properties of agar/agarose for different alkali pretreatment Seaweed Pretreatmen Yield (g); Gel strength Gelling Ash Sulphate quantity t conditions (% yield (g/cm 2 ) at T (C) (%) (%) w.r.to bone 20 0 C; dry seaweed) (%gel) WO 2005/118830 PCT/IB2004/001792 16 15g 1.5% alkali, 3.10 (20.7) 275 (1.0%) 33.0 4.5 >0.5 water wash 10g 3% alkali, 1.98 (22.0) 800(1.0%) 35.0 3.4 >0.5 water wash 20g 5% alkali, 3.80 (21.0) 1620 1.0%) 35.0 2.0 0.50 water wash 2 10Og 7% alkali, 1.80 (20.0) 1875 1.0%) 35.0 <0.50 water wash 1.8 0 10g 10% alkali, 1.85 (20.5) >1920 35.0 < <0.25 water wash 1.0%); 0.9 1450 (0.75%); 900 (0.5%) 15g 15% alkali, 3.20 (23.0) >1920 35.0 < < 0.28 water wash 1.0%); 1465 0.9 (0.75%); 900 (0.5%) EXAMPLE 5 The product of EXAMPLE 4 was compared with the product obtained wherein Gracilaria dura was treated with 10% alkali as in Example 4 but wherein residual alkali was neutralized with 0.5% AcOH as was done in one of the cases of Example 2. 5 It can be seen from Table 4 'that the reduction in gel strength accompanying neutralization with AcOH leads to a concomitant decrease in the molecular weight of the linear galactan. This suggests strongly that acidic catalyzed hydrolytic degradation of the polysaccharide is responsible for lowering of gel strength. Table 4. Agar extracted under various concentrations of alkali pretreatment 10 conditions followed by acid wash Seaweed Pretreatment Yield (g); Gel strength Weight quantity conditions (% yield (g/cm 2 ) averaged w.r.to bone at 20 0 C; (%gel) molecular dry seaweed) weight (Daltons) 10g 10% alkali, acid wash 1.63 (18.1) 1025 (1.0%) 0.199xl0 with 0.5% AcOH 10g 10% alkali, water 1.85 (20.5) > 1920 (1%) 1.23xl0 wash EXAMPLE 6 Gracilaria dura collected at different times from Veraval, Gujarat was processed as in Example 3 using 5% alkali. The data are tabulated in Table 5. It can be seen that seasonal variations of seaweed quality are negligible.
WO 2005/118830 PCT/IB2004/001792 17 Table 5. Agars obtained from Gracilaria dura harvested in different times of the year Seaweed Seaweed Pretreatment Yield (g); Gel strength Gelling Ash (%) Sulphate Collection quantity conditions (% yield (g/cm 2 ) at T (C) (%) data' w.r.to 20oC; (%gel) bone dry seaweed) February 20g 5% alkali 4.1 (22.8) 1650(1.0%) 35.0 2.0 0.50 2003 water wash April 2003 20g 5% alkali 3.8 (21.0) 1620(1.0%) 35.0 2.02 0.50 water wash June 2003 20g 5% alkali 4.2(23.0) 1600(1.0%) 35.0 2.0 0.50 water wash December 10g 5% alkali 1.85 1600(1.0%) 35.0 2.0 0.50 2004 water wash (20.5) January 15g 5% alkali 2.90 1630(1.0%) 35.0 2.0 0.50 2004 water wash (20.0) EXAMPLE 7 5 Gracilaria dura of Example I was harvested in January 2004 and transported live to the Gulf of Mannar, Tamil Nadu (9017' N and 7808' E). Results of cultivation in three different locations (Thonithurai, Ervadi and Krusadai Island) are given in Table 6. The daily growth rate was calculated using the following formula: r = (Wt/ Wo)" t x 100, 10 where r stands for daily growth rate in percent, Wt is the wet weight at day t, Wo is the initial wet weight. It can be seen from Table 6 that cultivation of the seaweed is feasible with daily growth rate as high as 4.34%. Table 6. Cultivation of Gracilaria dura in Perforated Polythene bags and on Rafts at three different locations in Gulf of Mannar, Tamil Nadu, India Method of Cultivation Thonithurai Polythene bags Raft _Ropes Initial weight 250 gm (10 bags x25 gm each) 1800 gm (18 1.5 m (09.01.'04) ropes x 100 gm each) Final weight 883 gm 5076 gm (23.02.'04) DGR% day
-
' 2.8 2.3 WO 2005/118830 PCT/IB2004/001792 18 Erwadi Polythene bags Raft _Ropes Initial weight 100 gm (10 bags x 10 gm in 900 gm (18 ropes x 50 (13.01.'04) each bag) gm in each rope) Final weight 306 gm 2318 (27.02.'04) DGR% day
-
' 2.5 2.1 Krusadai Polythene bags Raft _Ropes island Initial weight 100 gm (10 bags x 10 gm in 800gm( 16 ropes x 50 (12.01.'04) each bag) gm in each rope) Final weight 692 gm I11312 gm (26.02.'04) DGR% day' 4.29 4.34 EXAMPLE 8 5 kg of fresh Gracilaria dura cultivated in Krusadai island as described in Example 7 was sun-dried to yield a weight of 795 g. 15 g of dry seaweed was processed as described in Example 4, using 10% NaOH for pretreatment, yielding 2.84 g (20% 5 yield) of product having >1920 g/cm 2 gel strength (1.0% gel; 20 0 C), 35 0 C gelling temperature, and 0.9% ash content. It can be seen by comparing the present results with the corresponding data in Example 4 that the yield and quality of agarose from cultivated material are the same as those obtained from the naturally occurring seaweed (see Example 4). o10 ADVANTAGES OF PRESENT INVENTION The main advantage of this invention is that agarose of desirable specifications can be produced from Gracilaria dura of Indian waters. Another advantage is that the yield of agarose is as high as 20-23% based on dry seaweed weight. 15 Another advantage is that the dry seaweed has adequate shelf life when stored in plastic bags under ambient conditions. Another advantage is that preparation of the agarose is simple to undertake. Another advantage is that the seaweed is amenable to cultivation in Indian waters.
Claims (8)
1. An agarose of following characteristics: i. about 1% ( 1900 g/cm 2 ) gel strength at about 20 0 C, ii. 35 to 35.5 0 C gelling temperature, 5 iii. < 0.25 % sulphate content, and iv. 5 0.9 % ash content, wherein, the agarose is prepared by a simple, direct and cost-effective process from naturally occurring or cultivated Gracilaria spp. more particularly Gracilaria dura, said process comprising steps of: 10 a. obtaining dry seaweed Gracilaria spp, b. pre-treating the dry seaweed with about 35 parts (v/w) of about I to 15% alkali at 25 to 95oC for 0.5 to 5.0 hours, c. rinsing the pre-treated seaweed thoroughly with water to remove excess alkali until the washing shows a pH ranging between 7 and 8, 15 d. adding about 35 parts (v/w) of water for about every one part of original seaweed and autoclaving at about I115-125 0 C for time duration ranging between 1.5 to 2.0 hours to obtain extractive, e. treating the extractive with about 0.05 to 0.07% charcoal and about 10 15% Celite at a temperature ranging between 85 and 95 0 C to obtain hot 20 extractive, f. vacuum-filtering the hot extractive over a Celite bed, g. freezing the filtrate into a mass at about -20oC for 12 to 15 hours and thawing the mass, h. redissolving the mass in about 25 parts of water by heating in an 25 autoclave, i. repeating the freeze- thaw cycle if required, j. straining the product of step (i) to remove thawed liquid and thereafter squeezing to expel residual liquid to the extent possible to obtain agarose, and 30 k. optionally redissolving the solid and spray drying to obtain fine powder.
2. An agarose as claimed in claim 1, wherein the agarose is obtained from Gracilaria spp. more particularly Gracilaria dura. WO 2005/118830 PCT/IB2004/001792 20
3. An agarose as claimed in claim 1, wherein the agarose gel has melting temperature ranging between 98-100oC.
4. A simple, direct and cost-effective process for the preparation of agarose of high gel strength and low gelling temperature from naturally occurring or 5 cultivated Gracilaria spp. more particularly Gracilaria dura, said process comprising steps of: a. obtaining dry seaweed Gracilaria spp, b. pre-treating the dry seaweed with about 35 parts (v/w) of about I to 15% alkali at 25 to 95oC for 0.5 to 5.0 hours, 10 c. rinsing the pre-treated seaweed thoroughly with water to remove excess alkali until the washing shows a pH ranging between 7 and 8, d. adding about 35 parts (v/w) of water for about every one part of original seaweed and autoclaving at about 115-125 0 C for time duration ranging between 1.5 to 2.0 hours to obtain extractive, 15 e. treating the extractive with about 0.05 to 0.07% charcoal and about 10 15% Celite at a temperature ranging between 85 and 95 0 C to obtain hot extractive, f. vacuum-filtering the hot extractive over a Celite bed, g. freezing the filtrate into a mass at about -20 0 C for 12 to 15 hours and 20 thawing the mass, h. redissolving the mass in about 25 parts of water by heating in an autoclave, i. repeating the freeze- thaw cycle if required, j. straining the product of step (i) to remove thawed liquid and thereafter 25 squeezing to expel residual liquid to the extent possible to obtain agarose, and k. optionally redissolving the solid and spray drying to obtain fine powder. 4. A process as claimed in claim 4, wherein the alkali is sodium hydroxide.
5. A process as claimed in claim 4, wherein the concentration of alkali is about 30 10%.
6. A process as claimed in claim 4, wherein the time duration of autoclaving at step (d) is preferentially about 1.5 hours. WO 2005/118830 PCT/IB2004/001792 21
7. A process as claimed in claim 4, wherein the agarose yield is ranging between
20-23% of dry seaweed weight. 8. A process as claimed in claim 4, wherein pre-treating the seaweed at temperature preferably about 85 0 C. 5 9. A process as claimed in claim 4, wherein pre-treating the seaweed for preferentially about 2.0 hours. 10. A process as claimed in claim 4, wherein the autoclaving is at temperature preferentially about 120 0 C. 11. A process as claimed in claim 4, wherein concentration of charcoal is about 10 0.06%. 12. A process as claimed in claim 4, wherein concentration of Celite is about 12.5%. WO 2005/118830 PCT/IB2004/001792 22 AMENDED CLAIMS [received by the International Bureau on 17 March 2005 (17.03.2005); original claims 1-13 replaced by new claims 1-12 (3 pages)] Claims 1. An agarose of following characteristics: i. - about 1% gel strength of> 1900 g/cm 2 at about 20 0 C, ii. 35 to 35.5 0 C gelling temperature, iii. _ 0.25 % sulphate content, iv. 5 0.9 % ash content, and v. melting temperature ranging between 98-100 0 C. wherein, the agarose is prepared by a simple, direct and cost-effective process from naturally occurring or cultivated Gracilaria spp. more particularly Gracilaria dura, said process comprising steps of: a. obtaining dry seaweed Gracilaria spp, b. pre-treating the dry seaweed with about 35 parts (v/w) of about 1 to 15% alkali at 25 to 95'C for 0.5 to 5.0 hours, c. rinsing the pre-treated seaweed thoroughly with water to remove excess alkali until the washing shows a pH ranging between 7 and 8, d. adding about 35 parts (v/w) of water for about every one part of original seaweed and autoclaving at about 115-125 0 C for time duration ranging between 1.5 to 2.0 hours to obtain extractive, e. treating the extractive with about 0.05 to 0.07% charcoal and about 10 15% Celite at a temperature ranging between 85 and 95 0 C to obtain hot extractive, f. vacuum-filtering the hot extractive over a Celite bed, g. freezing the filtrate into a mass at about -20 0 C for 12 to 15 hours and thawing the mass, h. redissolving the mass in about 25 parts of water by heating in an autoclave, i. repeating the freeze- thaw cycle if required, j. straining the product of step (i) to remove thawed liquid and thereafter squeezing to expel residual liquid to the extent possible to obtain agarose, and k. optionally redissolving the solid and spray drying to obtain fine powder. WO 2005/118830 PCT/IB2004/001792 23 2. An agarose as claimed in claim 1, wherein the agarose is obtained from Gracilaria spp. more particularly Gracilaria dura. 3. A simple, direct and cost-effective process for the preparation of agarose of high gel strength and low gelling temperature from naturally occurring or cultivated Gracilaria spp. more particularly Gracilaria dura, said process comprising steps of: a. obtaining dry seaweed Gracilaria spp, b. pre-treating the dry seaweed with about 35 parts (v/w) of about I to 15% alkali at 25 to 95 0 C for 0.5 to 5.0 hours, c. rinsing the pre-treated seaweed thoroughly with water to remove excess alkali until the washing shows a pH ranging between 7 and 8, d. adding about 35 parts (v/w) of water for about every one part of original seaweed and autoclaving at about 115-125C for time duration ranging between 1.5 to 2.0 hours to obtain extractive, e. treating the extractive with about 0.05 to 0.07% charcoal and about 10 15% Celite at a temperature ranging between 85 and 95cC to obtain hot extractive, f. vacuum-filtering the hot extractive over a Celite bed, g. freezing the filtrate into a mass at about -20 0 C for 12 to 15 hours and thawing the mass, h. redissolving the mass in about 25 parts of water by heating in an autoclave, i. repeating the freeze- .thaw cycle if required, j. straining the product of step (i) to remove thawed liquid and thereafter squeezing to expel residual liquid to the extent possible to obtain agarose, and k. optionally redissolving the solid and spray drying to obtain fine powder. 4. A process as claimed in claim 3, wherein the alkali is sodium hydroxide. 5. A process as claimed in claim 3, wherein the concentration of alkali is about 10%. 6. A process as claimed in claim 3, wherein the time duration of autoclaving at step (d) is preferentially about 1.5 hours. WO 2005/118830 PCT/IB2004/001792 24 7. A process as claimed in claim 3, wherein the agarose yield is ranging between 20 23% of dry seaweed weight. 8. A process as claimed in claim 3, wherein pre-treating the seaweed at temperature preferably about 85 0 C. 9. A process as claimed in claim 3, wherein pre-treating the seaweed for preferentially about 2.0 hours. 10. A process as claimed in claim 3, wherein the autoclaving is at temperature preferentially about 120 0 C. 11. A process as claimed in claim 3, wherein concentration of charcoal is about 0.06%. 12. A process as claimed in claim 3, wherein concentration of Celite is about 12.5%. WO 2005/118830 PCT/IB2004/001792 25 STATEMENT UNDER ARTICLE 19(1) The Examiner has rejected the claims on the basis of both lack of novelty and inventiveness. The Applicant respectfully submits that the claims are sufficiently amended to overcome the rejections. Further, the Applicant provides his comments to establish both novelty and inventiveness of the claims. The Applicant would like to draw the attention of Ld. Examiner to the fact that the melting temperature of the agarose of the instant application is ranging between 98-100 0 C. This property of the agarose of the instant application helps us distinguish our claims from that of the cited arts especially XP-002317325, wherein the agarose of product No. A 0576 is having melting temperature 86 + / - 2.0 0 C. The Examiner must appreciate that each of the products provided in cited art XP-002317325 belong to the same category and have partially overlapping features. However, the very fact that these are classified as different products is due to their differential properties and thereby differential behavior and is therefore sufficient to establish that these products are distinct. When we have a novel agarose, it would have some essential features of conventional agarose and at the same time would have at-least one distinct feature, which would make it a novel agarose product. On the same lines, the inventors of the instant application have come out with a novel agarose having certain characteristics like melting temperature, which makes it novel. Therefore, the Examiner is respectfully requested to withdraw the anticipation rejections. WO 2005/118830 PCT/IB2004/001792 26 Further, we would like to draw Ld. Examiner attention to the fact that the process of the instant application does not involve use of acids for neutralizing excess alkali. Conventionally, the alkali is used in the process at pre-treatment stage and thereafter, acids are used for neutralizing the excess alkali. However, the instant application involves inventive step of using water to remove excess alkali until the washing shows a pH ranging between 7 and 8. Therefore, the inventors have been able to do away with the use of acids in the process for the preparation of agarose. Here the Examiner must appreciate that the conventional use of acids for neutralization leads to degradation of polysaccharides, in particular is detrimental to the stability of linear galactan molecule; which in turn leads to formation of agarose with low gel strength. However, such low strength agarose is not desirable in the instant application. Therefore, the inventiveness of the process involves elimination of acids from the entire process. Now, none of the cited arts both individually and in combination provides any clue or motivation to the persons skilled in the art to use the particular methodology of the instant application and to reach desired results. It is only after much effort and intellectual inputs from the inventors that they arrived at the instant invention. Taking aforementioned aspects of the invention into consideration, we respectfully request the Examiner to withdraw obviousness rejection. Further, the invention is distinct from the cited arts because the invention does not involve using formaldehyde for cleansing as used in D4 at page 534. Furthermore, the invention involves use of charcoal in step (e) of claim 1. This adds to the novelty of the invention. In addition, we would like to draw the Ld. Examiners attention to page No.16 of the description, wherein Table No.4 clearly reflects the difference between the agarose of the instant application from the various kinds of agaroses, which would be obtained by using processes of cited arts and are disclosed therein. The differential gel strength and yield of Table 4 clearly establishes that the process of instant application is much superior to the processes disclosed in the cited arts. The Ld. Examiner is requested to take the aforementioned aspects into consideration and thereafter, rightfully establish both novelty and inventiveness of the invention.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/IB2004/001792 WO2005118830A1 (en) | 2004-06-03 | 2004-06-03 | A cost-effective process for preparing agarose from gracilaria spp. |
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| AU2004320343A1 true AU2004320343A1 (en) | 2005-12-15 |
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| AU2004320343A Abandoned AU2004320343A1 (en) | 2004-06-03 | 2004-06-03 | A cost-effective process for preparing agarose from Gracilaria SPP |
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| JP (1) | JP2008501816A (en) |
| CN (1) | CN1993474A (en) |
| AU (1) | AU2004320343A1 (en) |
| CA (1) | CA2569495A1 (en) |
| GB (1) | GB2429209A (en) |
| RU (1) | RU2381276C2 (en) |
| WO (1) | WO2005118830A1 (en) |
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| CN101138414B (en) * | 2007-10-22 | 2011-11-30 | 天津科技大学 | Method for extracting phycoerythrin and gelose synchronously from gum-contained varek such as gardon asparagus |
| CA2756520C (en) * | 2009-03-24 | 2017-07-11 | Council Of Scientific & Industrial Research | Process for the preparation of agarose polymer from seaweed extractive |
| CN101891835B (en) * | 2010-07-26 | 2012-08-22 | 华侨大学 | Method for separating and preparing agarose from agar by using polyethylene glycol precipitation method |
| CN102775524A (en) * | 2011-05-12 | 2012-11-14 | 高金华 | Method for directly producing agarose from alga |
| CN102775815B (en) * | 2012-08-06 | 2014-03-26 | 青岛德慧海洋生物科技有限公司 | Preparation method of gel additives with controllable slow release |
| CN102964469B (en) * | 2012-10-08 | 2014-12-10 | 福建省绿麒食品胶体有限公司 | Ultrasound-assisted preparation method of high strength Gracilaria agar |
| KR101875115B1 (en) * | 2017-06-30 | 2018-07-10 | 농업회사법인 주식회사 오션푸드코리아 | Manufacturing method for hydrogel foam using agarose form seaweed |
| CN109054053B (en) * | 2018-06-28 | 2020-12-25 | 福建天源兴达生物科技有限公司 | Processing technology of agar with high gel strength |
| US20220007701A1 (en) * | 2018-11-16 | 2022-01-13 | Sea6 Energy Private Limited | Plant based gelling fibre, method and application thereof |
| CN109503729A (en) * | 2018-12-12 | 2019-03-22 | 湖南医药学院 | A method of extracting polysaccharide |
| CN112175110B (en) * | 2020-09-18 | 2022-10-04 | 福建省绿麒食品胶体有限公司 | Preparation method of agar glue solution |
| JP7333980B2 (en) * | 2021-03-31 | 2023-08-28 | 伊那食品工業株式会社 | Low strength highly viscous agar |
| JP7350271B1 (en) | 2022-08-10 | 2023-09-26 | 伊那食品工業株式会社 | Agar-containing foods and quality improvers |
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- 2004-06-03 RU RU2006146982/13A patent/RU2381276C2/en not_active IP Right Cessation
- 2004-06-03 GB GB0624561A patent/GB2429209A/en not_active Withdrawn
- 2004-06-03 CA CA002569495A patent/CA2569495A1/en not_active Abandoned
- 2004-06-03 WO PCT/IB2004/001792 patent/WO2005118830A1/en not_active Application Discontinuation
- 2004-06-03 AU AU2004320343A patent/AU2004320343A1/en not_active Abandoned
- 2004-06-03 CN CNA200480043673XA patent/CN1993474A/en active Pending
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| CA2569495A1 (en) | 2005-12-15 |
| GB0624561D0 (en) | 2007-01-17 |
| RU2006146982A (en) | 2008-07-20 |
| WO2005118830A1 (en) | 2005-12-15 |
| RU2381276C2 (en) | 2010-02-10 |
| JP2008501816A (en) | 2008-01-24 |
| GB2429209A (en) | 2007-02-21 |
| CN1993474A (en) | 2007-07-04 |
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