MINISTRY OF EDUCATION AND TRAINING NHA TRANG UNIVERSITY NGUYEN VAN THANH RESEARCH ON PREPARATION OF LOW MOLECULAR WEIGHT ALGINATE FOR USING FUNCTIONAL FOODS IN PREVENTING BLOOD COAGULATION SUMMARY OF THE DOCTORAL DISSERTATION Speciality : Aquatic Products Processing Code : 9540105 KHANH HOA - 2019 The project was completed at Nha Trang University The scientific advisors: Assoc Prof Dr Vu Ngoc Boi Assoc Prof Dr Tran Thi Thanh Van Reviewer 1: Assoc Prof Dr Ngo Đang Nghia Reviewer 2: Assoc Prof Dr Ngo Đai Nghiep Reviewer 3: Assoc Prof Dr Nguyen Huu Đai The dissertation was evaluated by the Scientific Committee at Nha Trang University on The dissertation can be found at: The National Library The Library of Nha Trang University SUMMARY OF THE DOCTORAL DISSERTATION’S NEW CONTRIBUTIONS Dissertation topic: Research on preparation of low molecular weight alginate for using functional food in preventing blood coagulation Major: Aquatic Products Technology Code: 9540105 Ph.D Candidate: Nguyen Van Thanh Course: 2012 The scientific advisors: Assoc Prof Dr Vu Ngoc Boi Assoc Prof Dr Tran Thi Thanh Van School: Nha Trang University Content: The dissertation has obtained some new results added to the field of research, production of alginate and low molecular weight alginate from marine brown seaweed: 1) The dissertation has identified species of seaweed S mcclurei, S polycystum and T ornata with the highest fucoidan content when they are harvested in April and May In addition, two species of S mcclurei and T ornata obtained the highest alginate content when they are harvested in April, S polycystum had the highest alginate content when being harvested in May Of the studied species of seaweed, T ornata is a suitable seaweed for the production of both fucoidan and alginate 2) The dissertation has identified the optimal parameters for the extraction procedures for alginate has high viscosity from marine brown seaweed T ornata: solution to Na2CO3 was pH 11, the temperature was 59oC, time was 1.5 hours Sodium alginate precipitated in concentrations of ethanol appropriate was 70% Sodium alginate from T ornata has high purity, the M/G ratio of 1.06, an average molecular weight of 648.32 kDal, an average degree of polymerization of 1037, the polydispersity index indicator of 3.56, the process performance reached 87,93% Alginate products are produced according to the process meeting the criteria of sensory, chemistry and microorganism according to the current regulations of Ministry of Health 3) The dissertation has prepared low molecular weight alginate by hydrolysis with acid After hydrolysis products obtained: sodium guluronate fraction, sodium mannuronate fraction and fraction of sodium guluronate - mannuronate content was 49.17 ± 1.21%, 38.13 ± 1.16% and 3.96 ± 1.08% of alginate weight, respectively The results indicated that the low molecular weight alginate has high purity An average molecular weight of sodium guluronate and sodium mannuronate of 21.661 kDa and 33.759 kDa, an average degree of polymerization of 89 and 128, the polydispersity index indicator of 1.38 and 1.49, respectively 4) The dissertation has determined the appropriate conditions for preparing for low molecular weight sodium guluronate sulfate (SGS): the conditions of the sulfonation agent reaction have been optimized: the NaHSO3/NaNO2 concentration rate is 4.25/1 mol/g, the reaction temperature is 90°C and the reaction time is 90 minutes The conditions of sodium guluronate sulfate synthesis reaction were surveyed under appropriate conditions of pH = 9, the sulfonation agent/sodium guluronate concentration rate is 2/198 mol/g, the reaction temperature is 40°C and synthetic reaction time is hours The dissertation has built the process of producing SGS from alginate of T ornata Since then, the process of producing SGS from sodium alginate of brown seaweed T ornata has been established Sodium guluronate sulfate has high purity, an average molecular weight of 25.408 kDa, an average degree of polymerization of 107, the polydispersity index indicator of 1.35 5) The dissertation has evaluated the anticoagulant activity of low molecular weight sodium guluronate sulfate The results have showed that sodium guluronate sulfate depend on its an average molecular weight and contents The study results the anticoagulant activity of sodium guluronate sulfate have showed that it can extend the activated partial thromboplastin time (APTT) and the thrombin time, but it cann’t almost be able to expend the prothrombin time (PT) Sodium guluronate sulfate is not toxic to mice So it is usable for producing functional foods support anticoagulant activity in humans The scientific advisors Ph.D Candidate Assoc Prof Dr Vu Ngoc Boi Assoc Prof Dr Tran Thi Thanh Van Nguyen Van Thanh LIST OF PUBLISHED ARTICLES Nguyen Van Thanh, Vu Ngoc Boi, Tran Thi Thanh Van and Nguyen Dinh Thuat (2017), “Determination of the optimum conditions for the synthesis of polyguluronate sunfate”, Journal of science - Aquatic products processing technology, NO 1/2017, Nha Trang University, pp 82-90 Nguyen Van Thanh, Bui Van Nguyen, Nguyen Dinh Thuat, Tran Thi Thanh Van and Vu Ngoc Boi (2017), “Optimizing the alginate extraction procedures from the brown seawees residues Turbinaria ornata (TURNER) J AGARDH”, Can Tho University Journal of Science, 49B, Can Tho University, pp 116-121 Nguyen Van Thanh, Do Thi Thanh Xuan, Ngo Van Quang, Vu Ngoc Boi, Bui Minh Ly, Tran Thi Thanh Van and Thanh Thi Thu Thuy (2014), “Structure and antimicrobial activity of alginate from brown seaweed Turbinaria ornata”, Journal of chemistry, 52 (6A), Vietnam Academy of Science and Technology, pp 149-152 Do Thi Thanh Xuan, Nguyen Van Thanh, Dang Vu Luong, Bui Minh Ly, Tran Thi Thanh Van and Thanh Thi Thu Thuy (2014), “Study on the separation and chemical structure of alginate and its fractions from brown seaweed Turbinaria ornata (TURNER) J AGARDH”, Journal of science and technology, 52 (5A), Vietnam Academy of Science and Technology, pp 35 PREFACE The necessary of the dissertation Alginate is one of the hydrocolloid compounds widely used in a wide variety of industries, especially in food, cosmetic and pharmaceutical industries In recent years, many studies in the world have shown that low molecular weight alginates show many valuable biological activities more than high molecular weight alginates such as antioxidant activity, anti-inflammatory activity, allergy resistance, anti-bacterian, antiobesity, anti-cancer, hypertension prevention, cholesterol and blood sugar reduction, etc Alginate does not have the anticoagulant activity, but alginate sulfate (alginate were sulfated), especially low molecular weight alginate sulfate is highly compatible with blood because its structure is similar to the structure of heparin and its anticoagulant activity has been researched The anticoagulant activity of alginate sulfate depend on the molecular weight, the M/G ratio, the uronic sequencing, the seaweed species, etc Therefore, low molecular weight alginate sulfate has opened up potential applications for the pharmaceutical and functional food industries The dissertation “Research on preparation low molecular weight alginate for using functional food in preventing blood coagulantion” from the source of the raw algae after the fucoidan extract is extracted, it is a very necessary requirement, in order to enhance the exploitation and efficient use of algae resources, complete the research on alginate of Vietnamese algae according to regulations, search direction, detect its anticoagulant activity Since then, open studies on low molecular weight alginate application for functional food production to actively support care for community health and socioeconomic development in the future The purposes of the dissertation Preparation of low molecular weight sodium alginate from brown algae in Nha Trang Bay which has anticoagulant activity as a raw material for functional food production Materials and methods 3.1 Materials: Alginate from brown seaweed, collected in Nha Trang Bay, Khanh Hoa province 3.2 The scope of research (1) Research on the sources of brown seaweed material for current fucoidan and alginate production; (2) Research on optimization of alginate extraction processed from marine brown seaweed and quality equalization of alginate produced; (3) Research on preparation of low molecular weight alginate and evaluate its characteristics; (4) Research on the production process of sodium guluronate sulfate (SGS) from alginate of T ornata and evaluate the characteristics of SGS; (5) In vitro evaluation of anticoagulant activity and toxicity assessment of SGS as a raw material for functional food production Experimental method Using modern analytical methods: Analysis of the content of heavy metals such as Hg, As, Cd and Pb by atomic absorption spectrometric (AAS) - graphite furnaces (GF) – chemical modifier (CM); Determinate average molecular weight of alginate by gel permeation chromatography (GPC) at the University of Natural Sciences, National University Ho Chi Minh City; Determinate sulfate contents by Sulfate Barium Nephtlometry (using reflective light to measure the particle density in liquid); Methods for determining the structure of alginate: determinate Infrared Spectroscopy (IR) by FT-IR Bruker and Nuclear Magnetic Resonance Spectrum (NMR) measured at 70°C with D2O solvent on Bruker AVANCE 500 MHz at the Institute of Chemistry - Vietnam Academy of Science and Technology; At the same time, the dissertation also uses mathematical methods in order to optimize the experimental process and process the collected statistical data aiming to assure highly reliable experimental results The structure of the dissertation The dissertation has 148 pages, including pages of introduction, 35 pages of overview, 21 pages of research methods, 87 pages of research results, pages of summary, 22 tables, 63 images, 246 references ( 27 Vietnamese documents, 219 English documents) and 52 pages of appendix Chapter OVERVIEW 1.1 SOURCES OF THE BROWN SEAWEED IN THE WORLD AND IN VIETNAM Around 2,060 species of brown seaweed and more than 95% of brown seaweed have originated in the sea The Fucales are the most common and economical species, represented by the Sargassaceae, with two branch Sargassum and Turbinaria China is the largest brown seaweed producer in the world with over 667,000 tonnes of dried seaweed per year South Korea, Japan, Norway and Chile has the brown seaweed yield about 96,000; 51,000; 40,000 and 27,000 tonnes of dried seaweed per year, respectively Vietnam has discovered over 120 brown seaweed species Brown seaweed harvesting season is mainly from April to June every year Annual yield is between 15,000 and 35,000 tonnes of dried seaweed Among them, Sargassum seaweed species has the largest reserves with about 68 species, harvesting yield about 10,000 tonnes of dried seaweed per year In Khanh Hoa province, more than 39 seaweed species in Sargassum genus are classified, however, they are gathering and have the largest reserves in Nha Trang Bay with over 21 common species Yield is estimated more than 4,800 tonnes of dried seaweed per year In the cell of brown seaweed, the major components are anionic polysaccharides such as alginate, fucoidin, fucin In particular, alginate content is the highest, up to 40% of dried seaweed weight; Fucoidan accounts for up to 8% of dried seaweed weight; Mannitol content can reach 30% of dried seaweed weight; Laminaran content can reach 30% of dried seaweed weight; The content of minerals in brown seaweed is 10 to 20 times higher than terrestrial plants, especially it is rich in calcium, potassium, phosphorus, magnesium and iodine; In addition, brown seaweed contains many other biological compounds such as pigment, vitamins, phenolic compounds, etc In general, the chemical compositions of brown seaweed vary with the seaweed species, season, weather, habitat, geographic locations, etc In our country, brown seaweeds are mainly used for raw fucoidan production with an annual output of about 400 ÷ 800 tonnes of raw fucoidan In addition, this material is sold as raw materials to China with low economic value or used for fertilizers, food for animals After being used for fucoidan production, the rest of seaweed is used for fertilizers or being discarded It can be seen that we have wasted the alginate resources almost intact in seaweed after fucoidan extraction Thus, the study on collecting alginate from brown seaweed after fucoidan extraction is essential, which enhances the value of brown seaweed resources Therefore, the dissertation will be studied on alginate collection from brown seaweed after extract fucoidan aim to increase the efficiency of using seaweed resources in Vietnam 1.2 OVERVIEW OF ALGINATE Alginate is a common term for salts of alginic acid, it is also an anionic polysaccharide, a straight chain copolymer formed from (1 4) glycosit bond of β-Dmannuronic acid (M) and α-L-guluronic acid (G) Alginate is extracted from brown seaweed In addition, alginate is also produced from a number of bacterial species The physical, chemical and biological properties of alginate vary with the molecular weight, viscosity and M/G ratio as well as the uronic arrangement in the polymer The solubility of alginate depends on the type of metal salt, with metal salt has valence I then they dissolve in water In contrast, with metal salt has valence II then they are not soluble in water There are many studies on alginate extraction technology worldwide However, it can be seen that the study results of various seaweeds are different to the seaweed processing system, extraction system, extraction efficiency of alginate, and the cooking mode will affect the viscosity of alginate The viscosity of alginate is one of the important parameters reflecting the quality of sodium alginate obtained and affecting the alginate collection efficiency If the viscosity of low alginate means alginate short circuit When the alginate circuit structure is cuting short for the extraction process, the alginate recovery efficiency is low The reason that the short molecular circuit alginate is difficult to precipitate by alcohol The extraction efficiency and viscosity of alginate are closely related in the process of extracting alginate from brown algae In Vietnam, the number of studies on alginate extraction technology is rather rare, they have not been paid much attention At now, there haven’t been any alginate production companies All alginate products of the market are mainly imported from other countries The research on alginate extraction from brown seaweed residues of the fucoidan production process was studied in the country from 2008 to 2010 by Nha Trang Research and Applied Technology Institute However, there are some following limitations: (1) The seaweed residues extracted from fucoidan are dried before the alginate extraction This process is not necessary because it will lead to costly energy, extraction solvent, while seaweed residue can be used immediately for alginate extraction (2) The use of formol discolors seaweed residues within 24 hours prior to alginate extraction, which will have significant effects on health and environmental sanitation, as well as on viscosity, alginate molecular weight (3) The obtained product is the calcium alginate which is insoluble in water It is also quite complicated to shift into sodium alginate which is soluble in water, the production process needs more stage (4) The process of producing alginate is not concerned with the viscosity of alginate, alginate quality has not been assessed and the structural properties of alginate have not been determined yet 1.3 PREPARATION OF LOW MOLECULAR WEIGHT ALGINATE Low molecular weight alginate can be prepared by different methods such as physical method, chemical method or method of enzyme use The physical method (commonly used by irradiation) requires the equipment that normal laboratories not have The method of enzyme used to hydrolyze alginate into low molecular weight alginate This method does not cause environmental pollution, the products are produced by the clean technology, so it is easy to purify and obtain However, the method of using enzymes is still relatively new to Vietnam On the other hand, there is currently no enzyme hydrolyzed alginate in commercial form, so it is difficult to research and use enzymes in alginate hydrolysis The chemical method used for alginate hydrolysis have the advantage that the cost is not high but there are disadvantages after hydrolysis which requires product refining However, the use of chemical methods of alginate hydrolysis are easy to implement and the probability of success is quite high, in accordance with domestic research conditions Although low molecular weight alginate is prepared by hydrolysis method (acid) which has not been extensively studied in the country, it has been extensively studied in the world The ability to apply low molecular weight alginate relates to the modulation method The results of the literature review show that low molecular weight alginate prepared by the irradiation method is mainly used in agricultural production (plants, livestock), there aren’t researches applied in food production field Meanwhile, low molecular weight alginates prepared by chemical methods and using hydrolysis enzymes have been studied and applied for food, cosmetics and medicine Therefore, to prepare low molecular weight alginate as a raw material for functional food production, the dissertation aims to select the alginate hydrolysis method by acid agent 1.4 THE APPLICATIONS OF ALGINATE AND LOW MOLECULAR WEIGHT ALGINATE Alginate is a biopolymer considered as a new material, it is widely used in various industries The potential for alginate application for the country as well as in the world is very great, especially in food, cosmetic and pharmaceutical field The applicability of 3.3.4 The average molecular weight of alginate in T ornata seaweed Figure 3.5 Gel Permeation Figure 3.6 The molecular weight distribution Chromatography of alginate of alginate The obtained sodium alginate of T ornata has high purity; the average molecular weight ̅̅̅̅̅ Mw is 648.32 kDa; an average degree of polymerization ̅̅̅̅̅̅ DPn is 1,037; Poly index PI indicator 3.56 The average molecular weight meet criteria according to QCVN 4-16: 2010/ BYT: National Technical Regulation on Food Additives - Filler (average from 10 kDa to 600 kDa) 3.3.5 Determining the structural characteristics of sodium alginate from T ornata Table 3.11 Result of IR spectrum of alginate extracted from T ornata Guluronic Mannuronic acid (cm-1) acid (cm-1) –OH 3383 3383 –CH 2923 2923 COO– 1622 1735 C–O–C 1037 1090 Vibration Figure 3.7 IR spectrum of alginate extracted from T ornata 17 Figure 3.8 ESI-MS spectrum of alginate extracted from T ornata Figure 3.9 ESI-MS spectrum of disaccharide at m/z 369 Figure 3.10 13C-NMR spectrum of alginate extracted from T ornata Figure 3.11 1H -NMR spectrum of alginate extracted from T ornata 18 Figure 3.12 COSY spectrum of alginate extracted from T ornata Figure 3.13 HSQC spectrum of alginate extracted from T ornata Figure 3.14 HMBC spectrum of alginate extracted from T ornata Figure 3.15 ROESY spectrum of alginate extracted from T ornata 1H HOOC HO OH H H OH O OH H H H HOOC H H2 H H H HO O O O HO O HOOC 4H H H5 HOOC H HOOC OH HO O OH O OH OH 4 G G M M G Figure 3.16 The interaction of protons on ROESY spectrum From the analysises of the sodium alginate structural characteristics of the seaweed Turbinaria ornata, we can see that: alginate is a straight chain co-polymer formed from (14) glycosidic bond of β-D-mannuronic acid (M) and α-L-guluronic acid (G); The glycosidic bond at carbon anomer of guluronate is the  bond, carbon anomer of 19 mannuronate is  bond; M/G ratio of alginate 1.06 shows that in the alginate component, the mannuronic acid content is higher than guluronic acid content; Extracted sodium alginate has high purity 3.4 RESEARCH ON PREPARATION OF LOW MOLECULAR WEIGHT ALGINATE AND DETERMINATION OF STRUCTURAL PROPERTIES OF LOW MOLECULAR WEIGHT ALGINATE 3.4.1 Determination of content of low molecular weight alginate The Content of alginate (%) 60 50 40 guluronate 49.17 obtained (SG) sodium content is relatively high, accounting for 38.13 49.17 ± 1.21% of alginate weight The sodium mannuronate (SM) 30 content separated from T ornata 20 is 38.13 ± 1.16% of alginate 10 weight The sodium mannuronate- 3.96 guluronate SM SG SMG Fractions of alginate (SMG) content separated from T ornata is relatively low, only 3.96 ± 1.08% Figure 3.17 The content of low molecular weight alginate fractions of alginate weight 3.4.2 Determination of structural properties of low molecular weight alginate 3.4.2.1 The structural properties of sodium guluronate Figure 3.18 13C-NMR spectrum of sodium guluronate 20 Figure 3.19 1H-NMR spectrum of sodium guluronate On the Nuclear Magnetic Resonance Spectrum 13C-NMR carbon of sodium guluronate, there are only six peaks representing for carbons of acid guluronic Concurrently, Nuclear Magnetic Resonance Spectrum 1H-NMR proton in anomer area has only high intensity peak This proves that the sodium guluronate was separated with high purity The bond on the sodium guluronate is a α- anomer 3.4.2.2 Structural properties of sodium mannuronate On the Nuclear Magnetic Resonance Spectrum 13 C-NMR carbon of sodium mannuronate, there are only six peaks representing for carbons of mannuronic acid Concurrently, the Nuclear Magnetic Resonance Spectrum 1H-NMR proton nucleus in the anomer area has only high intensity peak This proves that the sodium mannuronate was separated with high purity The bond on the sodium mannuronate is a - anomer Figure 3.20 13C-NMR spectrum of sodium mannuronate 21 Figure 3.21 1H-NMR spectrum of sodium mannuronate 3.4.3 Average molecular weight of low molecular weight alginate 3.4.3.1 The average molecular weight of sodium guluronate Figure 3.22 Gel Permeation Chromatography of Sodium guluronate Figure 3.23 The molecular weight distribution of sodium guluronate The obtained sodium guluronate has high purity; It has an average molecular weight ̅̅̅̅̅ ̅̅̅̅̅̅ is 89; the polydispersity Mw is 21.661 kDa; an average degree of polymerization DPn index PI indicator 1.38 3.4.3.2 The average molecular weight of sodium mannuronate The obtained sodium mannuronate has high purity It has an average molecular weight ̅̅̅̅̅ Mw is 33.759 kDa; an average degree of polymerization ̅̅̅̅̅̅ DPn is 128; the polydispersity index indicator 1.49 22 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 1.445f The degree of subtitution (DS) The degree of subtitution (DS) Figure 3.24 Gel Permeation Figure 3.25 The molecular weight Chromatography of sodium distribution of sodium mannuronate mannuronate 3.5 PREPARATION OF SODIUM GULURONATE SULFATE (SGS) AND DETERMINING ITS STRUCTURAL PROPERTIES 3.5.1 Preparation of SGS from sodium alginate extracted T ornata 3.5.1.1 Investigating the reaction conditions to prepare sulfating agent 1.514g 1.319e 0.984d 0.478c 0.250a 30 0.323b 40 50 60 70 80 90 Figure 3.26 The effect of temperature to reaction for preparation of sulfating agent to DS of SGS The degree of subtitution (DS) 1.755f 1.8 1.564e 1.6 f 1.719e 1.783 1.707e 1.676g 1.409c 1.182b 0.742a 15 30 45 60 75 90 105 120 Figure 3.27 The effect of time to reaction for preparation of sulfating agent to DS of SGS 1.609e 1.348d 1.4 1.079c 1.2 0.917b 1.0 0.8 1.673g 1.623d Time (minute) Temperature (oC) 2.0 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 0.754a 0.6 0.4 0.2 Figure 3.28 The effect of ratio of NaHSO3/NaNO2 to reaction for preparation of sulfating agent to DS of SGS 0.0 3.00/13.25/13.50/13.75/14.00/14.25/14.50/14.75/1 Ratio of NaHSO3/NaNO2 (mol/g) 23 The optimal conditions for the preparing reaction of sulphation agent: NaHSO3/NaNO2 concentration rate is 4.25/1, the reaction temperature is 90oC and the reaction time is 90 minutes 1.6 1.468d 1.338e 1.4 The degree of subtitution (DS) The degree of subtitution (DS) 3.5.1.2 Investigating the synthesis reaction conditions of sodium guluronate sulfate 1.275f 1.2 1.0 0.856c 0.8 0.696b 0.6 0.4 0.314a 0.2 0.0 1.8 1.512c 1.6 1.2 0.963b 1.0 0.8 0.708a 0.6 0.4 0.2 30 35 40 1.764c 1.608d 50 Figure 3.30 The effect of temperature to synthesis reaction to DS of SGS The degree of subtitution (DS) Figure 3.29 The effect of time to synthesis reaction to DS of SGS 1.628d 45 Temperature (oC) Time (hour) The degree of subtitution (DS) 1.356e 0.0 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 1.424d 1.4 1.606d 0.964b 0.721a 1,0/198 1,5/198 2,0/198 2,5/198 3,0/198 3,5/198 2.0 1.845d 1.8 1.6 1.4 1.243c 1.2 0.922e 1.0 0.8 0.745b 0.576a 0.6 0.4 0.2 0.0 Ratio of NaNO2/SG (mol/g) 11 pH Figure 3.31 The effect of ratio NaNO2/SG Figure 3.32 The effect of pH to to synthesis reaction to DS of SGS synthesis reaction to DS of SGS The optimal conditions for the synthesis reaction of SGS are pH=9, the sulfation agent/sodium guluronate concentration ratio is 2/198, the reaction temperature is 40°C and the synthesis reaction time of sodium guluronate sulfate is hours 3.5.2 Propose SGS production process from sodium alginate of T ornata and evaluate the characteristics of SGS 3.5.2.1 Propose SGS production process from sodium alginate of T ornata * Explaining the process: + Sodium alginate: Sodium alginate extracted from brown seaweed Turbinaria ornata It has high purity, the humidity is 9.01%, the viscosity is 734 cP, the average molecular weight is 648.32 kDa, an average degree of polymerization is 1037, the poly index indicator 3.56 24 Sodium alginate Acid Hydrolysis SMG SG SM Sulfating Dialyzing Concentrating Precipitating Vacuum-drying Packaging, storing Figure 3.33 Flow chart of processing sodium alginate sulfate from sodium alginate + Acid hydrolysis: A 1% aqueous solution of sodium alginate was made up to 0.3M in HCl by addition of 3.0M HCl and heated for 0.5 h at 100oC under nitrogen The mixture was cooled and centrifuged (10.000 x g, 20 min) The solid was suspended in 0.3M HCl and heated for h at 100oC under nitrogen The mixture was cooled and centrifuged (10.000 x g, 20 min) For the solution of two times centrifuged was neutralization by addition of 1M NaOH, conducting dialysis with distilled water after 72 hours, concentrating by the rotavap at 50°C until the volume is 1/10, using five volumes of ethanol to make precipitant reaction The precipitate were collected by centrifugation, vacuumdrying at 50oC within h (SMG) For the precipitate collected after 2nd centrifugation was neutralization by addition of 1M NaOH The solution was adjusted to pH 2.85 by addition of M HCl The soluble fraction and the insoluble fraction was separated by centrifugation (10.000 x g, 20 min), neutralization by addition of 1M NaOH, conducting dialysis with distilled water after 72 hours, concentrating by the rotavap at 50°C until the volume are 25 1/10, using five volumes of ethanol to make precipitant reaction The precipitates were collected by centrifugation, vacuum-drying at 50oC within h They are SM and SG + Sulfating: The sulfating agent was prepared from NaHSO and NaNO2 in water The optimizing conditions as follows: ratio of concentration NaHSO3/NaNO2 of 4,25/1 mol/g, temperature of 90oC and time of 90 minutes The synthesis reaction conditions of sodium guluronate sulfate were determined as follows: pH = 9, ratio of concentration sulfating agent/ sodium guluronate of 2/198 mol/g, temperature of 40oC and time of hours + Dialyzing: Conducting dialysis with distilled water after 72 hours to remove all impurities including excess sulfate + Concentrating: After dialysis, concentrating by the rotavap at 50°C until the volume is 1/10 + Precipitating: Using ethanol 96% with volume more times than solution obtained SGS to make precipitant reaction the amount of SGS Slowly adding the solution to the ethanol 96%, stirring and keeping within hours, then centrifuged (10.000 x g, 20 min) to recover SGS + Drying: Sodium guluronate sulfate is spread into layers of 0.5÷1cm thickness, vacuum-drying at 50oC within hours to obtain dried SGS, humidity