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Nghiên cứu điều chế alginate khối lượng phân tử thấp dùng làm thực phẩm chức năng hỗ trợ phòng chống đông máu tt tiếng anh 2

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

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The project was completed at Nha Trang University


The scientific advisors:
1. Assoc. Prof. Dr. Vu Ngoc Boi
2. 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

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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:

1. Assoc. Prof. Dr. Vu Ngoc Boi
2. 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 3 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 3 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
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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 4 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

3

Nguyen Van Thanh


LIST OF PUBLISHED ARTICLES
1. 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.
2. 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.
3. 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.
4. 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

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PREFACE
1. 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.
2. 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.
3. 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
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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.
4. 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.
5. The structure of the dissertation
The dissertation has 148 pages, including 3 pages of introduction, 35 pages of
overview, 21 pages of research methods, 87 pages of research results, 2 pages of summary,
22 tables, 63 images, 246 references ( 27 Vietnamese documents, 219 English documents)
and 52 pages of appendix.

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Chapter 1. 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
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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
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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 do 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
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alginate is relatively large, not only in high molecular weight but also in low molecular
weight alginate. With low molecular weight alginates, it has shown many valuable
biological activities and is completely applicable to functional foods and medicine.
Up to now, in Vietnam, there has not been any scientific research published on low
molecular weight alginate research with anticoagulant activity as raw material for
functional food production. Based on the results of the literature review, it is highly feasible
to study the production of low molecular weight alginate from our brown seaweed
resources. Therefore, the orientation of research on low molecular weight alginate with
human activity to support blood coagulation is a new study and promising field.
1.5. THE BLOOD CLOT PROCESS AND ANTICOAGULANT ACTIVITY OF
ALGINATE
Normally, in the blood and in the tissues, there are blood coagulants and
anticoagulants, but the blood coagulants exist in the form of precursors, which are not
active. When the blood coagulant factors are activated, chain reactions that cause the blood
to coagulate will be promoted. Blood coagulation can be triggered by endogenous pathway
by contact of the blood with the negative surface, or by the exogenous pathway, due to the
interference of organizational elements. Both pathways lead to the activation of factor X
into Xa, which change prothrombin into thrombin. This assists to change fibrinogen into
fibrin. Fibrin is like a net containing platelets that cause blood clots.
Alginate has no anti-coagulant activity but the alginate sulfate derivative (sulfated
alginate) is similar in structure and characteristics to the anticoagulant produced in
hepatocytes. When being sulfated, low molecular weight alginate will have anti-coagulant
activity. However, the anticoagulant activity of low molecular weight alginate depends on
the average molecular weight and its concentration. Studies in the world have shown that
alginate sulfate has a major effect on prolonging Activated Partial Thromhoplastin Time
(APTT) and Thrombin Time (TT) and has little effect on prolonging Prothrombin Time
(PT).

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Chapter 2. MATERIALS AND METHODS
2.1. MATERIALS
Brown seaweed material composes two species belong to branch of Sargassum C.
Agardh 1821 (S. mcclurei Setchell and S. polycystum C. Agardh) and one species belong
to branch of Turbinaria Lamouroux 1828 (T. ornata (Turner) J. Agardh). Sampling
locations: in Hon Tre, Hon Rua and Bai Nam in Nha Trang, Khanh Hoa.
We first collected fresh seaweed samples of each species. When harvesting, we used
the sickle cut to the roots of seaweed, washed the seaweed residues with seawater. Later,
the seaweed was transported to the laboratory of the Nha Trang Institute for Research and
Applied Technology. In the laboratory, seaweeds are dried by the freeze drying method at
a temperature of 450C, wind speed of 2 m/s until the seaweed reaches a moisture content
of 14-15%, then ending the drying process. Then dried seaweeds were wrapped in vacuum
packaging PA and stored at normal temperature, high temperature, cool to use as raw
materials during the study.
Mice: guinea pig (Cavia porcellus), average weight from 300 to 350g, there are 80
mice for study.
2.2. METHODS
2.2.1. Diagram of access to research contents: presented in Figure 2.1
2.2.2. The analysis methods
+ Determining the raw fucoidan content: According to the method of Usov et al.
(2001); Determining alginate and alginic acid content: According to the method of Haug
et al. (1968); Determining the humidity according to TCVN 4326 - 2001; Determining the
raw protein content according to TCVN 3705 - 1990; Determining the raw fat content
according to TCVN 3703 - 1990; Determining the ash content according to TCVN 5611 1991; Determining the content of heavy metals Hg, As, Cd and Pb by atomic absorption
spectrophotometric - graphite furnace - chemical modifier (CM-GF-AAS) according to
AOAC999.11:2011
+ Determining the microbial content by colony counting method: determining the
total aerobic bacteria according to TCVN 4884 - 2005; determining the total number of
yeast spores, molds according to TCVN 4993 - 1989; determining Coliforms according to
TCVN 4882: 2007; determining Salmonella according to TCVN 4829:2005.

7


Brown seaweed

Research on the sources
of brown seaweed
material for current
fucoidan and alginate
production

Fucoidan extraction

Sodium alginate
extraction
Hydrolysis of
sodium alginate
with acid
Receive low
molecular weight
alginate (LMWA)

Research on the
production process
of sodium
guluronate sulfate
(SGS)

determine harvesting time
suitable for brown
seaweed
Selection of seaweed
species for research
Research on alginate
extraction processed
Deteminating the ethanol
contents for precipitable
alginate
Suggest technological
process of sodium alginate
extraction

* pH (8÷12), temperature
(50oC ÷ 80oC), time (1÷4 giờ)
* The highest level of sodium
alginate content and viscosity

* Ethanol content (50 ÷ 80%)
* The highest alginate
content

Quality equalization of
alginate produced
Determination of content of
low molecular weight
alginate
Quality equalization of low
molecular weight alginate

Investigating the reaction
conditions to prepare
sulfating agent
Investigating the synthesis
reaction conditions of SGS
Propose SGS production
process from sodium alginate
and evaluate the
characteristics of SGS

Evaluation of
anticoagulant activity
and toxicity
assessment

* Harvesting time: from
month 1/2013 ÷ 7/2013
* The highest alginate content
and fucoidan content

Evaluation of anticoagulant
activity of SGS
Evaluation of toxicity
assessment of SGS

*Temperature (30oC÷90oC);
time (15÷120 phút), ratio of
NaHSO3/NaNO2 (3/1÷4,75/1
mol/g)
* DS of SGS is highest
* Time (1÷6 hours),
temperature (30oC÷50oC);
ratio of NaNO2/SG (1/198
÷3,5/198 mol/g), pH (3÷11)
* DS of SGS is highest

* An average molecular weight
(15÷35kDa);
* Concentration (25÷75μg/mL)
* High dose (2 mg/day),
medium dose (1 mg/day), low
dose (0,5 mg/day).
* Clinical observation; mass;
urine test; Hematology test and
blood chemistry; surgery and
microscopic observation.

Figure 2.1. Diagram of access to research contents

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+ Viscosity determination method: 1% alginate solution at 22°C is measured viscosity
on Brookfield viscometer, using spindle LV-02, speed 50 rpm.
+ Determining the alginate averages molecular weight: by Gel Permeation
Chromatography (GPC) method at the University of Natural Sciences, Vietnam National
University Ho Chi Minh City.
+ Alginate quality assessment method: According to QCVN 4-16: 2010 / Ministry of
Health: "National Technical Regulation on Food Additives - Fillers"
+ Measurement of degree of substitution (DS): the DS values were determined by
barium sulfate nephelometry (using reflective light to measure particle density in liquid)
according to the method of Dodgson et al. (1962).
+ Structural analysis method: InfraRed (IR) spectroscopy is measured on Bruker FTIR; The nuclear magnetic resonance (NMR) spectroscopy is measured at a the temperature
of 70°C with a D2O solvent on a 500 MHz Bruker AVANCE at the Institute of Chemistry
- Academy of Science and Technology of Vietnam; ESIMS spectroscopy is measured on
Waters Xevo TQMS.
+ Methods of examining anticoagulant activity of sodium guluronate sulfate (SGS):
according to Fan et al. (2011). The experiment was conducted at Army Medical Academy
- Military Central Hospital 108, Hanoi.
+ Method for determining toxicant of sodium guluronate sulfate: by oral route, with
three levels: high (2mg/day/mice), medium (1mg/day/mice) and low (0,5mg/day/mice),
control group (physiological saline). Each level of study on one group of mice was assessed
20 mice and the two waves, each of the 10 mice. 1st determine acute toxicity, performed
immediately after the end of treatment and 2nd determine the extent of the recovery or the
expression of toxic potential may occur after the end of treatment for 3 weeks.
2.3. STATISTICAL ANALYSIS
Experimental data were collected by observation and statistical method. Each
experiment was repeated three times independently and the data were the mean of the
repetitions. The differences in statistics were checked by software Statgraphics Centurion
XVII trial. Optimizing experiments by experimental planning method. Differences in the
regression equation coefficients were assessed according to the t-Student standard with a
significance level of α = 0.05 and a degree of freedom of 2. Validation of the regression
equation by Fisher standard.
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Chapter 3. RESULTS AND DISCUSSIONS
3.1. SCREENING THE BROWN SEAWEED SPECIES FOR FUCOIDAN AND
SODIUM ALGINATE PRODUCTION
3.1.1. Evaluation of fucoidan and sodium alginate content in some seaweed species
3.1.1.1. Evaluation of fucoidan content in 3 seaweed species

Content of fucoidan (%)

3.0
S. mcclurei
S. polycystum
T. ornata

2.8
2.6

2.72c4

2.72c4
2.63c5

2.61c3

2.53b4

2.53b4
2.44b5

2.4

2.25c6

2.2

2.03c2
1.92b3

2.0

1.92a4

1.87a4

1.72b2

1.8

1.72a5

1.67b6

1.61a3

1.6

1.46b1
1.33a1 1.33c1

1.4

1.51a2

1.45a6

1.2
1.0
1

2

3

4

5

6

7

Time (month)

Figure 3.1. The variation on fucoidan contents in three researched seaweed species
following the time of sampling
The results of Figure 3.1 show that the time of harvesting algae is suitable for use as
fucoidan production material for all 3 seaweed species (S. mcclurei, S. polycystum and T.
ornata) in Nha Trang bay, Khanh province is in April and May. Among the 3 brown algae
studied, T. ornata species have higher fucoidan content than S. mcclurei and S. polycystum
species.
3.1.1.2. Evaluation of sodium alginate content in 3 seaweed species

Content of alginate (%)

45

S. mcclurei
S. polycystum
T. ornata

40
35
30

28.73a1

31.42a2
30.09c1

34.21a3
32.64c2

24.54 b2

25

38.93c4
36.47c3
35.44a4

25.02 b3

33.89 b5 34.18
32.12a5

25.79 b4

c5

32.11c6

23.64a6
23.64 b6

22.67c7

21.41 b1

20
13.87a7 b7
14.27

15
10
1

2

3

4

5

6

7

Time (month)

Figure 3.2. The variation on alginat contents in three researched seaweed species
following the time of sampling
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Sodium alginate content of both S. mcclurei and T. ornata species was highest in
April, while S. polycystum species was highest in May. The content of sodium alginate in
T. ornata (38.93 ± 0.026%) was higher than in S. mcclurei (35.44 ± 0.046%) and S.
polycystum (33.89 ± 0.026%). T. ornata species is superior to use for production materials
of sodium alginate, therefore, it should be selected as raw materials for the production of
sodium alginate.
From the analysis of the content of fucoidan and sodium alginate in 3 brown algae S.
mcclurei, S. polycystum and T. ornata showed that T. ornata species had higher content of
fucoidan and alginate compared to S. mcclurei and S. polycystum species. Therefore, the
dissertation decided to select T. ornata species to be used as fucoidan material and at the
same time to receive sodium alginate.
3.1.2. Some basic chemical compositions of Turbinaria ornata
Table 3.1. Some basic chemical compositions of Turbinaria ornata
No

Criteria

unit

Content

1

Humidity

% dry weight

10,26 ± 0,06

2

Protein

% dry weight

8,65 ± 0,08

3

Lipid

% dry weight

0,91 ± 0,03

4

Ash

% dry weight

18,85 ± 0,23

5

Alginate

% dry weight

38,93 ± 0,03

6

Fucoidan

% dry weight

2,73 ± 0,01

7

As

mg/kg

2,8

8

Cd

mg/kg

1,4

9

Hg

mg/kg

1,2

10

Pb

mg/kg

0,9

The results show that brown seaweed T. ornata collected in Nha Trang Bay, Khanh
Hoa Province have relatively higher fucoidan and sodium alginate content than other
seaweeds in the world. Thus, it is a suitable source of material for fucoidan and sodium
alginate production. Therefore, the dissertation will conduct simultaneous extraction of
fucoidan before studying alginate extraction to avoid wasting natural resources from brown
algae. In other hand, the content of heavy metals is much lower than that of other seaweeds
in the world and fully meets the standards of Ministry of Health. This result shows that the
11


dissertation can use the T. ornata species collected in Nha Trang bay, Khanh Hoa province
as a source of obtaning both fucoidan and sodium alginate.
3.2. RESEARCH ON OPTIMIZING THE ALGINATE EXTRACTION
STAGE FROM TURBINARIA ORNATA
* Optimizing to maximize the sodium alginate content: The optimum conditions for
alginate extraction to obtain the highest alginate content are: the alkaline solution to pH =
10.7, the extraction temperature 68oC and time 2.7 hours. At this point, the extracted
alginate content is 37.379 ± 0.129% of dried seaweed weight.
* Optimizing to maximize the viscosity of sodium alginate: The optimum
conditions for the alginate extraction process to obtain the highest viscosity alginate are:
the alkaline solution pH = 11, the temperature 57.6°C and time 1.5 hours. At that condition,
the viscosity of alginate reaches 720 ± 5,508 cP.
* Optimizing to achieve simultaneously the highest level of sodium alginate
content and viscosity
The optimum conditions for alginate extraction from T. ornata in order to achieve
simultaneously the two highest levels of alginate content and viscosity are: Na 2CO3
solution with pH = 11; extraction temperature 59oC; extraction time 1.5 hours. With these
conditions, the alginate content extracted from the residues of brown seaweed T. ornata is
34.231 ± 0.21% of dried seaweed weight, alginate production process performance reached
87.93%. The viscosity of alginate extracted from the residues of brown seaweed T. ornata
is 734 ± 6,245 cP, it should be classified into relatively high viscosity alginate and should
be used for food and pharmaceutical industry.
3.3. RESEARCH ON EXTRACTING SODIUM ALGINATE FROM THE
RESIDUES OF MARINE BROWN SEAWEED AND QUALITY EQUALIZATION
OF SODIUM ALGINATE
3.3.1. Deteminating the ethanol contents for precipitable alginate
Ethanol content 70% is an appropriate content used for precipitating so as to obtain
sodium alginate from T. ornata.

12


Content of sodium alginate
(%)

40
35
30

29.137b

34.150e

34.177e

34.190e

70

75

80

32.153d

30.247c

27.143a

25
20
15
10
5
0
50

55

60

65

Ethanol content (%)

Figure 3.3. The effect of ethanol content on sodium alginate content
3.3.2. Suggest technological process of sodium alginate extraction from T. Ornata
* Explaining the process:
+ Treatment: brown seaweed raw material impurities are chopped (0.5 ÷ 1 cm) and
then treat with solvent mixture (Ethanol 96%/Chloroform/H2O = 89/1/10 (v/v)) with ratio
w/v = 1/10 from 10 to 15 days. Brown seaweed is washed three times with water RO with
pH- neutral (about 6.5) and then dried.
+ Extract fucoidan: brown seaweed is extracted 3 times with HCl 0.1 N solution
(pH 2 ÷ 2.5), the ratio of seaweed/solution is 1/20 (w/v), the temperature extraction is 60°C
in 2 hours for each extraction. The filtrate obtained fucoidan is filtered through a filtering
cloth. The residue of brown seaweed is recovered to extract alginate.
+ Washing: seaweed residue is washed three times with water with pH- neutral and
then dried.
+ Extract alginate: preparing Na2CO3 solution to pH = 11, the volume of the
alkaline solution is 25 times higher than dried seaweed weight. The extraction process is
carried out at temperature is 59°C within 1.5 hours.
+ Filtering: after finishing the extraction, a crude filtration is carried out through
many filter sieves from small size to large size to recover the extract. The seaweed residues
are stirred in a clean water solution at 55 ÷ 60°C to recover the extract that remains on the
seaweed residues and equipments.
13


Brown seaweed

Treatment

Crude fucoidan

Extract fucoidan

The ratio of ethanol
96%/Cloroform/H2O = 89/1/10
(v/v); The ratio of dry seaweed/Sol.
= 1/10 (w/v); Time: 10-15 days
HCl 0,1N (pH=2-2,5); The ratio of dry
seaweed/Sol. = 1/20 (w/v); Temp.
60oC; Time: 2 hours/times; No: 3 times

Washing

Extract alginate

The ratio of dried seaweed/sol. =
1/25 (w/v); pH = 11; temp. 59oC;
Time: 1.5 hours

Crude filtration
The residues of
brown seaweed

Refining filtration

Washing residues

Alginic acid

HCl 10 %

Neutralizing

Na2CO3 10 %

Crude filtration
Seaweed residues

Dialyzing

Fertilizer
Concentrating

Precipitating

Vacuum-drying

Ethanol content 70%

Temp. 50 oC
Time: 4 hours

Packaging, storing
Figure 3.4. Flow chart of processing sodium alginate extraction from the marine
brown seaweed

14


After a crude filtration, the filtrate continues to be refined through the vacuum
filtration whatman filter-paper type 6 (3 microns) with diatomite filter aid.
+ Alginic acid precipitation:
Using HCl 10% solution adjusts pH of the filtrate to pH = 1.8 and stir the filtrate at
the same time. Alginic acid is formed and floats on the surface, then the press filter stage
is conducted to obtain alginic acid.
+ Neutralizing:
Adding Na2CO3 10% solution slowly to alginic acid, stirring within 8 hours. The
process is terminated when the pH of the filtrate reaches 7.0 to 7.5, and obtains a colloidal
solution.
+ Dialyzing: conducting dialysis with distilled water after 72 hours.
+ Concentrating: after dialysis, concentrating by the rotavap at 50°C until the
volume is 1/10.
+ Precipitating:
Using ethanol content 70% to make precipitant reaction the amount of alginate
extraction. Slowly adding the alginate solution to the ethanol content 70%, stirring and
keeping within 4 hours, then filtering to recover alginate.
+ Drying:
Alginate is spread into layers of 0.5÷1cm thickness, vacuum-drying at 50oC within
4 hours to obtain dried sodium alginate, humidity <10%.
+ Packaging, storing:
Depending on the purposes of use, it is possible to grind alginate into powder. Dried
sodium alginate is packaged polyamid, vacuumed and stored under normal conditions.
3.3.3. The production and quality equalization of sodium alginate
Table 3.8. Sensory quality of sodium alginate
No

Criteria

1

Shape

QCVN 4 - 16: 2010/BYT

Result
Homogeneous powder

Granular, powdered or filiform
materials

2

Colour

White to slightly ivory-white

3

Flavour Nonspecific

White to brown - yellow
-

15


Table 3.9. Physical and chemical properties of sodium alginate
No

Criteria

Unit

Result

1

Humidity

%

9,01 ± 0,11

2

Ash content

%

12,67 ± 0,23

3

Alginic acid content

%

78,62 ± 0,99

QCVN 4 - 16: 2010/BYT

(humidity 8.75%)
4

Solubility

%

> 98

5

Viscosity

cP

734 ± 6,25

6

Heavy metals
- Lead (Pb)

mg/kg

0,02

< 5,0 (3,0*)

- Arsen (As)

mg/kg

0,03

< 3,0

- Cadimi (Cd)

mg/kg

0,002

< 3,0*

- Mercury (Hg)

mg/kg

0,015

< 0,1*

(*) QCVN 8-2:2011/BYT: National technical regulation on the safety limits of heavy
metals contaminants in functional foods.
Table 3.10. The microbiological criteria of the sodium alginate
No

Criteria

Unit

Result

QCVN 4 - 16: 2010/BYT

1

Total aerobic microorganisms

CFU/g

15

< 5000

2

Salmonella

CFU/g

Neg

Neg

3

Coliforms

CFU/g

Neg

Neg

4

Total number of yeast and

CFU/g

Neg

< 500

mold spores
(Neg: Negative)
Sodium alginate products meet the sensory criteria, the physical and chemical
criteria, and the microbiological criteria according to QCVN 4-16: 2010 / BYT: National
Technical Regulation on Food Additives - Filler. So, it is very suitable for food processing
and others field.

16


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

2

H H
OH

O
OH

1

H

H

H

HOOC
H

1

H2
H

H

H

HO O
O

O HO

O

3

HOOC
4H

1

H

5

H5

HOOC
H

HOOC
OH

HO O

3

OH
O

OH

OH

4

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

0
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


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