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WO2005118830A1 - Procede rentable de preparation d'agarose a partir de gracilaria spp. - Google Patents

Procede rentable de preparation d'agarose a partir de gracilaria spp. Download PDF

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Publication number
WO2005118830A1
WO2005118830A1 PCT/IB2004/001792 IB2004001792W WO2005118830A1 WO 2005118830 A1 WO2005118830 A1 WO 2005118830A1 IB 2004001792 W IB2004001792 W IB 2004001792W WO 2005118830 A1 WO2005118830 A1 WO 2005118830A1
Authority
WO
WIPO (PCT)
Prior art keywords
agarose
seaweed
extractive
gracilaria
alkali
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.)
Ceased
Application number
PCT/IB2004/001792
Other languages
English (en)
Inventor
Arup Kumar Siddhanta
Ramavatar Meena
Kamalesh Prasad
Bharatkumar Kalidas Ramavat
Pushpito Kumar Ghosh
Karuppanan Eswaran
Sangaiya Thiruppathi
Vaibhav Ajit Mantri
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Council of Scientific and Industrial Research CSIR
Original Assignee
Council of Scientific and Industrial Research CSIR
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Council of Scientific and Industrial Research CSIR filed Critical Council of Scientific and Industrial Research CSIR
Priority to GB0624561A priority Critical patent/GB2429209A/en
Priority to CNA200480043673XA priority patent/CN1993474A/zh
Priority to RU2006146982/13A priority patent/RU2381276C2/ru
Priority to CA002569495A priority patent/CA2569495A1/fr
Priority to AU2004320343A priority patent/AU2004320343A1/en
Priority to PCT/IB2004/001792 priority patent/WO2005118830A1/fr
Priority to JP2007514150A priority patent/JP2008501816A/ja
Publication of WO2005118830A1 publication Critical patent/WO2005118830A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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

Definitions

  • 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 34°-35° Celsius to minimize possible 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 gem "2 under standard conditions of measurement. Reference may be made to the Fluka Catalog of 2003-2004 wherein the specifications are provided for a range of agar products.
  • 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 ( 1 % solution) with gel point in the range of 36-42°C are described.
  • One drawback of such products is their high cost, which is presumably due to the elaborate purification processes involved.
  • 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, 40:63-69) where agars from Gelidiella acerosa having gelling temperatures of 38°C and 47°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.
  • Agardh (Rhodophyta, Gracilariaceae) in the west coast of India 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 1 N sulfuric acid followed by neutralization with 1.5% alkali.
  • Agar having gel strength of 260 g/cm 2 was obtained through this process.
  • 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°C), low gelling temperature ( ⁇ 35°C), low ( 0.25) sulfate residue and low ash 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 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. 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. Yet another object of this invention is to demonstrate that the seaweed is amenable to cultivation, even at locations distant from its natural origin.
  • 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 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 thawed liquid and thereafter squeezing to expel residual liquid to the extent possible to obtain agarose, and a agarose thereof.
  • 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 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 thawed liquid and thereafter squeezing to expel residual liquid to the extent possible to obtain agarose.
  • the invention relates to an agarose of following characteristics: i. about 1% (> 1900 g/cm 2 ) gel strength at about 20°C, ii. 35 to 35.5°C gelling temperature, iii. ⁇ 0.25 % sulphate content, and iv. ⁇ 0.9 % ash content,
  • the agarose is obtained from Gracilaria spp. more particularly Gracilaria dura.
  • the agarose gel has melting temperature ranging between 98-100°C.
  • 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 comprising steps of: • obtaining dry seaweed Gracilaria spp, • 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, • 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 1 15- 125°C for time duration ranging between 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 95°C to obtain hot extractive, • vacuum-filtering the hot extractive over a Celite bed
  • alkali is sodium hydroxide. In still another embodiment of the present invention, wherein the concentration of alkali is about 10%.
  • time duration of autoclaving at step (d) is preferentially about 1.5 hours.
  • the agarose yield is ranging between 20-23% of dry seaweed weight.
  • pre-treating the seaweed at temperature preferably about 85 C.
  • concentration of charcoal is about 0.06%. In still another embodiment of the present invention, wherein concentration of Celite is about 12.5%.
  • the present invention describes the preparation of agarose from Gracilaria dura occurring sparsely in the
  • Gracilaria dura was harvested from Veraval coast in western India situated at 20°54'N, 70°22'E.
  • alkali pretreatment was carried out for
  • the concentration of alkali for pretreatment was in the range of 0-15% (w/v) and preferably 10%.
  • the volume of alkali taken was 300 mL for every 10 g of seaweed.
  • 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.
  • the wet seaweed after pretreatment was taken in water in an autoclave wherein the water quantity was 300 mL for every 10 g of dry seaweed taken initially.
  • the pretreated seaweed was cooked in water for 1.5-2.0 h at 120 °C.
  • the hot extractive was discharged at 70-80 °C, following which charcoal and celite were added into the extractive and then taken to boiling at atmospheric pressure.
  • the boiling extractive was vacuum filtered over a celite bed.
  • the clear hot filtrate was poured into flat steel trays and allowed to cool to room temperature so as to form a gel.
  • 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.
  • the process of freeze-thaw was repeated if required.
  • the agarose obtained under optimum extraction conditions had gel strength of > 1900 gem "2 at 1 % concentration and 20°C temperature.
  • gel prepared from the agarose obtained under optimum extraction conditions had a melting, temperature of 98- 100 °C whereas the sol had a gelling temperature of 35.0-35.5°C.
  • the agarose obtained under optimum processing conditions had a sulphate content of 0.25% and ash content of
  • the hot agarose sol was spray dried for easy dissolution on heating.
  • Gracilaria dura was amenable to cultivation in polythene bag or raft in the Gulf of Mannar at the southeast coast of India with up to 5% daily growth rate for cultivation on raft in Krusadai island (9°16'N, 79°19'E).
  • 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.
  • the quality of agarose extracted from dry seaweed stored in plastic bags for up to 1 year was similar to that of agarose prepared from freshly dried seaweed.
  • the present invention describes the preparation of agarose of high gel strength and low gelling temperature from naturally occurring or cultivated Gracilaria 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°C for 2 h, (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°C for 1.5 h, (v) treating the extractive with 0.06% charcoal and 12.5% Celite (percentages w.r.t.
  • 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 temperatures were measured following the method described by Craigie et al. (Hand Book Of Phycological Methods, 1978 (Eds. Hellebust.
  • ash content was measured by incinerating the solid at 800 °C for 6h, sulphate content was estimated by treating the ash with concentrated nitric 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 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 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.
  • 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 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 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 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.
  • 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 cultivation may not be yearlong activity.
  • Gracilaria dura from Veraval, India (20°54' N and 70°22' E) was harvested in April 2003 and sun-dried.
  • 15 g of the seaweed was soaked in tap water for 1 h at ambient temperature (30-35°C) and the water then discarded.
  • the 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°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.
  • Gracilaria dura of Example 1 was initially soaked in water, the water then discarded and the wet seaweed treated with 5% NaOH at 80-85°C for 2 h followed by washing 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 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. Table 1.
  • Table 1 Agar obtained with alkali treatment and neutralization of residual alkali with acid
  • Gracilaria dura collected at different times from Veraval, India 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.
  • Table 5 Agars obtained from Gracilaria dura harvested in different times of the year
  • 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.
  • 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.

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Abstract

Un procédé simple direct et rentable de préparation d'agarose à force en gelée élevée et à faible température de gélatinisation à partir de Gracilaria spp. naturelle ou cultivée, plus particulièrement Gracilaria dura. Le procédé comprend les étapes de prétraitement de l'algue séchée avec un alcalin, le rinçage de l'algue prétraitée jusqu'à ce que le lavage montre un pH compris entre 7 et 8, l'ajout d'eau, la stérilisation afin d'obtenir un extrait, le traitement de l'extrait avec du charbon et Célite afin d'obtenir un extrait chaud, le filtrage sous vide de l'extrait chaud sur un lit Célite, la congélation du filtrat en une masse et le ramollissement de celle-ci, la redissolution de la masse dans l'eau par chauffage dans un autoclave, la répétition du cycle congélation-dégel, la percolation du produit afin de retirer le liquide et la compression pour extraire le liquide résiduel dans la mesure du possible afin d'obtenir une agarose et l'agarose ainsi obtenue.
PCT/IB2004/001792 2004-06-03 2004-06-03 Procede rentable de preparation d'agarose a partir de gracilaria spp. Ceased WO2005118830A1 (fr)

Priority Applications (7)

Application Number Priority Date Filing Date Title
GB0624561A GB2429209A (en) 2004-06-03 2004-06-03 A cost-effective process for preparing agarose from gracilaria spp
CNA200480043673XA CN1993474A (zh) 2004-06-03 2004-06-03 一种划算的用于从江蓠属藻类制备琼脂糖的方法
RU2006146982/13A RU2381276C2 (ru) 2004-06-03 2004-06-03 Агароза и способ ее получения
CA002569495A CA2569495A1 (fr) 2004-06-03 2004-06-03 Procede rentable de preparation d'agarose a partir de gracilaria spp.
AU2004320343A AU2004320343A1 (en) 2004-06-03 2004-06-03 A cost-effective process for preparing agarose from Gracilaria SPP
PCT/IB2004/001792 WO2005118830A1 (fr) 2004-06-03 2004-06-03 Procede rentable de preparation d'agarose a partir de gracilaria spp.
JP2007514150A JP2008501816A (ja) 2004-06-03 2004-06-03 オゴノリ属の種（ｇｒａｃｉｌａｒｉａｓｐｐ．）からアガロースを調製するための費用対効果の高い方法

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/IB2004/001792 WO2005118830A1 (fr) 2004-06-03 2004-06-03 Procede rentable de preparation d'agarose a partir de gracilaria spp.

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WO2005118830A1 true WO2005118830A1 (fr) 2005-12-15

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JP (1) JP2008501816A (fr)
CN (1) CN1993474A (fr)
AU (1) AU2004320343A1 (fr)
CA (1) CA2569495A1 (fr)
GB (1) GB2429209A (fr)
RU (1) RU2381276C2 (fr)
WO (1) WO2005118830A1 (fr)

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CN101891835A (zh) * 2010-07-26 2010-11-24 华侨大学 一种用聚乙二醇沉淀法从琼脂中分离制备琼脂糖的方法
CN101138414B (zh) * 2007-10-22 2011-11-30 天津科技大学 一种从龙须菜等含胶海藻中同时提取藻红蛋白和琼胶的方法
WO2020100124A1 (fr) * 2018-11-16 2020-05-22 Sea6 Energy Private Limited Fibre gélifiante à base de plantes, procédé et application associés
CN112175110A (zh) * 2020-09-18 2021-01-05 福建省绿麒食品胶体有限公司 一种琼脂胶液的制备方法

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CN102439047B (zh) * 2009-03-24 2014-05-07 科学与工业研究会 用于从海藻提取物制备琼脂糖聚合物的方法
CN102775524A (zh) * 2011-05-12 2012-11-14 高金华 一种从海藻直接生产琼脂糖的方法
CN102775815B (zh) * 2012-08-06 2014-03-26 青岛德慧海洋生物科技有限公司 一种缓释可控的凝胶助剂的制备方法
CN102964469B (zh) * 2012-10-08 2014-12-10 福建省绿麒食品胶体有限公司 一种超声波辅助制备高强度江蓠琼脂的方法
KR101875115B1 (ko) * 2017-06-30 2018-07-10 농업회사법인 주식회사 오션푸드코리아 해조류 유래 아가로스를 이용한 하이드로겔 폼의 제조방법 및 이의 용도
CN109054053B (zh) * 2018-06-28 2020-12-25 福建天源兴达生物科技有限公司 一种高凝胶强度琼脂的加工工艺
CN109503729A (zh) * 2018-12-12 2019-03-22 湖南医药学院 一种提取多糖的方法
JP7333980B2 (ja) * 2021-03-31 2023-08-28 伊那食品工業株式会社 低強度高粘性寒天
JP7350271B1 (ja) 2022-08-10 2023-09-26 伊那食品工業株式会社 寒天含有食品、および品質改良剤

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CN101138414B (zh) * 2007-10-22 2011-11-30 天津科技大学 一种从龙须菜等含胶海藻中同时提取藻红蛋白和琼胶的方法
CN101891835A (zh) * 2010-07-26 2010-11-24 华侨大学 一种用聚乙二醇沉淀法从琼脂中分离制备琼脂糖的方法
WO2020100124A1 (fr) * 2018-11-16 2020-05-22 Sea6 Energy Private Limited Fibre gélifiante à base de plantes, procédé et application associés
CN112175110A (zh) * 2020-09-18 2021-01-05 福建省绿麒食品胶体有限公司 一种琼脂胶液的制备方法

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GB0624561D0 (en) 2007-01-17
JP2008501816A (ja) 2008-01-24
CA2569495A1 (fr) 2005-12-15
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CN1993474A (zh) 2007-07-04

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