Production of monosaccharides and bio-active compounds derived from marine polysaccharides using subcritical water hydrolysis

Article abstract of DOI:10.1016/j.foodchem.2014.08.097

Polysaccharides are the major components of brown seaweed, accounting for approximately 40-65% of the total mass. The majority of the brown seaweed polysaccharides consists of alginate (40% of dry matter), a linear hetero-polysaccharides commonly developed in fields. However, depolymerisation of alginate is required to recover high-value compounds. In this report, depolymerisation was performed using subcritical water hydrolysis (SWH) at 180-260 °C, with a ratio of material to water of 1:25 (w/v) and 1% formic acid as a catalyst. Sugar recovery was higher at low temperatures in the presence of catalyst. The antioxidant properties of Saccharina japonica showed the best activity at 180 °C in the presence of a catalyst. The mass spectra produced using MALDI-TOF showed that polysaccharides and oligosaccharides were produced during hydrothermal treatment. Hydrolysis treatment at 180 °C in the presence of a catalyst may be useful for modifying the structure of S. japonica and purified alginate.

Full text of DOI:10.1016/j.foodchem.2014.08.097

Food Chemistry 171 (2015) 70–77
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Production of monosaccharides and bio-active compounds derived
from marine polysaccharides using subcritical water hydrolysis
Aviannie Meillisa a, Hee-Chul Woo b, Byung-Soo Chun a,
⇑
a Department of Food Science and Technology, Pukyong National University, 608-737, 45 Yongso-ro, Nam-gu, Busan, Republic of Korea
b Department of Chemical Engineering, Pukyong National University, 608-739, 365 Sinseon-ro, Nam-gu, Busan, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 March 2014
Received in revised form 21 July 2014
Accepted 22 August 2014
Available online 2 September 2014
Polysaccharides are the major components of brown seaweed, accounting for approximately 40–65% of
the total mass. The majority of the brown seaweed polysaccharides consists of alginate (40% of dry mat-
ter), a linear hetero-polysaccharides commonly developed in fields. However, depolymerisation of algi-
nate is required to recover high-value compounds. In this report, depolymerisation was performed
using subcritical water hydrolysis (SWH) at 180–260 °C, with a ratio of material to water of 1:25 (w/v)
and 1% formic acid as a catalyst. Sugar recovery was higher at low temperatures in the presence of cat-
alyst. The antioxidant properties of Saccharina japonica showed the best activity at 180 °C in the presence
of a catalyst. The mass spectra produced using MALDI-TOF showed that polysaccharides and oligosaccha-
rides were produced during hydrothermal treatment. Hydrolysis treatment at 180 °C in the presence of a
catalyst may be useful for modifying the structure of S. japonica and purified alginate.
Keywords:
Saccharina japonica
Alginate
Subcritical water hydrolysis
Monosaccharides
Bioactive compounds
Ó 2014 Published by Elsevier Ltd.
1. Introduction
acids, b-D-mannuronic acid (ManA or M) and a-L-guluronic acid
(GulA or G), which are linked by 1–4 bonds. The physical, chemical,
and structural properties of alginic acid (e.g., viscosity, solubility,
interaction with metal cations) are directly attributable to the
molecular weight and guluronic/mannuronic (G/M) ratio, both of
which show important variations among species (Clementi,
Crudele, Parente, Mancini, & Moresi, 1999). The biochemical and
biophysical properties of alginate are also dependent on the molec-
ular weight and G/M ratio. Alginate has been used in the food
industry as gelatinisers and thickeners, as wound coatings and
antifouling agents, for gastric parietal protection in various indus-
tries (e.g., food and pharmaceuticals), and for the biosorption of
heavy metals (Lodeiro et al., 2005; Renn, 1997). Alginate has a
molecular weight between 32 and 200 kDa, and the G/M ratio var-
ies by species, age, portion of the plant, and distance from the
shore. For applications using alginate, depolymerisation to adjust
the molecular weight is required.
The decomposition of carbohydrate into a reducing sugar,
monosaccharides, and polysaccharides offers economic value.
Many studies have been performed to convert carbohydrates from
marine macroalgae into monosaccharides, polysaccharides, and
other compounds. Alnaief, Alzaitoun, García-González, and
Smirnova (2011) proposed using alginate as a nanoporous biode-
gradable material, and Fitton, Irhimeh, and Falk (2007) produced
marine cosmetics using fucoidan fractions and phloroglucinol. Glu-
cose, mannose, and galactose in brown seaweed belong to the
Seaweed (also known as marine macroalgae) is a heterogeneous
assemblage of organisms with a long fossil history, contributing to
85% of the total global production of aquatic plants. Seaweed can
be classified into three groups based on pigmentation: green sea-
weed (Chlorophyta), red seaweed (Rhodophyta), and brown sea-
weed (Phaeophyta). Seaweeds, multicellular algae rich in
minerals and vitamins, are found in marine waters. Seaweed is
an important component of food, feed, and medicine, and is consid-
ered a high-value marine plant because of its biotechnological
properties. The seaweed species most commonly used for its bioac-
tive compounds is brown seaweed. The major constituents of
brown seaweed include carbohydrates (45–55%), proteins (10–
20%) and lipids (1–10%) (Kumar, Ganesan, Suresh, & Bhaskar,
2008).
Brown seaweed (Phaeophyta) is one of the most abundant sea-
weed groups of economic importance. The major constituent of
brown seaweed is polysaccharide. Alginate or alginic acid is an
unbranched hetero-polysaccharide consisting of two hexuronic
⇑
Corresponding author. Address: Department of Food Science and Biotechnology,
Pukyong National University, 599-1 Daeyeon-3dong, Nam-Gu, Busan 608-737,
Republic of Korea. Tel.: +82 51 629 5830; fax: +82 51 629 5824.
E-mail address: bschun@pknu.ac.kr (B.-S. Chun).
http://dx.doi.org/10.1016/j.foodchem.2014.08.097
0308-8146/Ó 2014 Published by Elsevier Ltd.

A. Meillisa et al. / Food Chemistry 171 (2015) 70–77
71
reducing sugar group, which contain essential compounds that can
be converted into valuable intermediate products.
140 rpm. The time to reach the desired temperature was 30–
75 min. The hydrolysate samples from the reactor were collected
Alginate depolymerisation methods have been widely investi-
gated. Several methods have been used to adjust the molecular
weight and G/M ratio, including acid hydrolysis (Haug, Myklestad,
Larsen, & Smidsrod, 1967), base hydrolysis (Rigouin, Delbarre,
Sinquin, Colliec-Jouault, & Dion, 2009), enzymatic depolymerisa-
tion (Kim et al., 2012), photolytic depolymerisation (Burana-Osot
et al., 2009), and hydrothermal conditions (Aida, Yamagata,
Watanabe, & Smith, 2010). Traditional methods use organic sol-
vents such as ethanol, methanol, or hexane during the extraction
process (Demirel, Yilmaz-Koz, Karabay-Yavagoslu, Ozdemir, &
Sukatar, 2009; Osman, Abushady, & Elshobary, 2010). Although
these methods are useful, they have several disadvantages, includ-
ing the use of harsh chemicals, the need for stable environmental
conditions during enzymatic depolymerisation, long reaction
times, and high experimental costs. To address these disadvantages,
hydrothermal conditions with subcritical water are being used to
break down the complex polysaccharide alginate in brown sea-
weed. Subcritical water is liquid water under pressure at tempera-
tures between the usual boiling point (100 °C) and the critical
temperature (374 °C), also known as superheated water and
pressurised hot water.
and filtered using Whatman nylon membrane filter (0.45 lm)
and stored at 4 °C.
2.4. Total glucose
Total glucose measurements were performed using the phenol
sulphuric acid method (Dubois, Gilles, Hamilton, Rebers, & Smith,
1956) with minor modifications. Briefly, hydrolysate samples
(0.75 ml) were mixed with 2.25 ml of concentrated sulphuric acid.
Then, 0.45 ml of 40% phenol was added and the mixture heated in a
water bath. The mixture was cooled at room temperature and its
absorbance at 490 nm measured using a spectrophotometer (Shi-
madzu 1240 UV–Vis spectrophotometer). D-Glucose was used as
a standard to generate a calibration curve. Each hydrolysate was
analysed in triplicate, and the results are expressed in milligrams
per litre (mg/l).
2.5. Reducing sugar
Reducing sugar analysis was conducted using the 3,5-dinitro-
salicylic (DNS) acid method (Miller, 1959). The reagent solution
was prepared by mixing 10 g of sodium hydroxide and 700 ml of
water until the mixture completely dissolved. Then, 300 g of potas-
sium sodium tartrate was added to the mixture followed by 10 g of
3,5-dinitrosalicylic (DNS) acid. After all of the components were
fully dissolved, 0.5 g sodium sulfite was added followed by 2 g of
phenol. The volume of the mixture was adjusted to 1 L and pro-
tected from light. Reducing sugar analysis was performed by mix-
ing 0.5 ml of hydrolysate water and 0.5 ml of the reagent solution,
heating the mixture for 10 min, and immediately adding 5 ml of
cold water. Finally, the absorbance at 540 nm was measured.
2. Materials and methods
2.1. Materials
The brown seaweed Saccharina japonica was collected from
Guemil-eup, Wando-gun, Jeollanam-do, South Korea. Purified algi-
nate from brown algae was provided by Sigma Aldrich (United
Kingdom). High-purity nitrogen gas (99.99%) was supplied by
KOSEM (Yangsan, Republic of Korea). Standards of 2,2-diphenyl-
1-picrylhydrazyl (DPPH), gallic acid, catechin, D-gulose, and L-man-
2.6. HPLC analysis
nose were purchased from Sigma Aldrich Chemical Co. (St. Louis,
Mo. USA). Distilled water was used in these experiments. All
reagents used in this study were ofanalytical or high performance
liquid chromatography (HPLC) grade and obtained from Sigma
Aldrich Chemical Co.
Gulose and mannose were quantified using high performance
liquid chromatography (HPLC) with an evaporative light scattering
detector (ELSD). HPLC analysis was performed using a Jasco HPLC
(Easton, USA) model 400 equipped with ChromNav analysis soft-
ware. High-purity nitrogen (99.99%) from KOSEM Co. was used as
a carrier gas. A Shodex (Japan) SUGAR column (SP0810) of 300 mm
with 8 mm i.d., thermostated to 80 °C, was used to analyse gulose
and mannose compounds. Hydrolysate samples were diluted four-
fold using filtered and sonicated water (HPLC grade). The water
used for elution was filtered using a Whatman nylon membrane
2.2. Sample preparation
After washing fresh S. japonica samples with fresh water,
unused materials, attached salt, and minerals were removed, and
the samples were cut into small pieces. The pieces were dried at
À80 °C for 3 days in a freeze dryer (Eyela FDU-2100, Tokyo Rikaki-
kai Co., LTD, Japan) equipped with a square-type drying chamber
(Eyela DRC-1000, Tokyo Rikakikai Co., LTD, Japan). The dried sam-
ples were collected into sealed plastic bags. The samples were then
finely ground using a mechanical blender (PN SMKA-4000 mixer)
filter (0.45 lm) and sonicated. The flow rate of the eluent was
maintained at 0.6 ml/min. Gulose and mannose standards (pur-
ity > 98%) were purchased from Sigma Aldrich (United Kingdom).
2.7. Matrix assisted laser desorption/ionisation-time of flight (MALDI-
TOF) analysis
and sieved through a 710-lm stainless steel sieving mesh.
2.3. Subcritical water hydrolysis
The mass spectra of the hydrolysate solutions were obtained
using the MALDI-TOF Bruker Autoflex-III, as described by Aida
et al. (2010) with minor modifications. Briefly, the device was
equipped with a smart beam laser and the spectra were collected
with an accelerating voltage of 19.8 kV, according to the polarity
of the recording mode. The measurement was performed using 2,5
dihydroxybenzoic acid (DHBA) as a matrix and acetonitrile:water
Subcritical water hydrolysis was performed in a 200-cm3 batch
reactor made of 276 Hastelloy with temperature control (Meillisa,
Chun, & Woo, 2012). A total of 6 g of material samples were loaded
into the reactor. Formic acid (1%), which is used as a catalyst, was
suspended separately in 150 ml of distilled water. The reactor was
then closed and heated using an electric heater to the required
temperature (180–260 °C). Pressures were estimated based on sat-
urated steam to be between 15 and 65 bar for the temperature
range studied. The temperature and pressure in the reactor were
controlled using a temperature controller and pressure gauge,
respectively. The sample was stirred using a four-blade stirrer at
(75:25) as a solvent. Samples were prepared by mixing 0.5
ll of
the product and 0.5 l of the matrix solution. The matrix solution
l
was prepared by mixing a norharmane–acetonitrile solution and
trifluoroacetic acid at a ratio of 7:3. The norharmane–acetonitrile
solution was prepared by dissolving norharmane (10 mg) in
acetonitrile (1 ml).

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A. Meillisa et al. / Food Chemistry 171 (2015) 70–77
2.8. DPPH radical scavenging activity
water, followed by 112
ll of 5% sodium nitrite. The mixture was
allowed to stand for 6 min and 225
ll of 10% aluminium chloride
DPPH assays were performed as described by Sharma and Bhat
was then added. A total of 0.75 ml of 1 M sodium hydroxide and
0.41 ml of distilled water were added, and the absorbance at
510 nm was measured. Catechin was used as a standard. Each
hydrolysate was analysed in triplicate and the results were
expressed in milligrams per litre (mg/l).
(2009) with minor modifications. Briefly, 100
ll of sample was
mixed with 3.9 ml of methanolic DPPH (50 M). The mixture was
l
vortexed for 1 min, wrapped in aluminium foil, and incubated at
30 °C for 30 min in darkness. The absorbance of the negative con-
trol was measured by replacing the sample with methanol
(100 ll). Spectrophotometric measurements of hydrolysate water
2.12. pH measurement
and the negative control were performed at 517 nm (UVmini
1240, Shimadzu Corp., Japan). The DPPH radical scavenging activity
(%) was calculated using the following equation: scavenging activ-
ity = [1À(As/Ac)] Â 100%, where As is the absorbance of the crude
extract at 517 nm and Ac is the absorbance of the blank at
517 nm. Analyses were performed in triplicate, and the DPPH
activity was expressed as% activity.
The pH of the hydrolysate water was measured using a Mettler
Toledo Five Easy Plus pH metre at 20 °C. Prior to the measure-
ments, the pH metre was calibrated using technical buffer
solutions of pH 4.01 0.02, 7.00 0.02, and 9.2 0.02.
3. Results and discussion
2.9. Ferric reducing assay power (FRAP)
3.1. Total glucose and reducing sugar
The FRAP assay was performed as described by Chu, Chang, and
Hsu (2000) with minor modifications. Briefly, 2.5 ml of 0.1 M
potassium phosphate buffer (pH 6.6) was mixed with 2.5 ml of
1% potassium ferricyanide. Then, 1 ml of sample was added and
the mixture incubated at 50 °C for 20 min. Next, 2.5 ml of 10% tri-
chloroacetic acid, 2.5 ml of water, and 0.5 ml of 0.1% of iron (III)
chloride were added to the mixture. The mixture was then incu-
bated at room temperature for 30 min and its absorbance at
700 nm measured using a UV spectrophotometer (UVmini 1240,
Shimadzu Corp.). Gallic acid was used as a standard. Analyses were
performed in triplicate, and the FRAP of each condition was
described in mg/l.
The carbohydrate content in seaweed can be converted into a
wide range of water-soluble sugars (such as poly-, oligo-, and
monosaccharides) by hydrolysis under subcritical water condi-
tions. The subcritical water technique uses hot water and high
pressure to maintain water in the liquid state. Under these condi-
tions, the physical characteristics of water change, with reduced
solvent viscosity and surface tension and a lower dielectric con-
stant. Table 1 shows the amounts of total glucose that can be
obtained as a function of temperature. The highest recovery was
achieved at 180 °C and 13 bar in the absence of a catalyst with
purified alginate or S. japonica. The amounts of glucose obtained
from purified alginate were higher (0.58 g/l) than from S. japonica
(0.43 g/l). Differentiation of total soluble sugars in hydrolysate
water is caused by changes in the physical properties of water,
which change in the subcritical state (Daneshvar, Salak, Ishii, &
Otsuka, 2012). Glucose recovery was monitored at high concentra-
tions with lower temperatures and pressures, and decreased at
higher temperatures and pressures. In the subcritical state, poly-,
oligo-, and monosaccharides depolymerised into other byproduct
compounds. In fact, the addition of a catalyst increased the decom-
position rate. However, the addition of acid during hydrolysis pro-
duced byproduct compounds, such as hydroxymethylfurfural
(HMF), levulinic acid, and furan aldehyde. Meinita et al. (2012)
and Omari, Besaw, and Kerton (2012) described that under certain
conditions in which monosaccharides were depolymerised into
byproduct compounds, the monosaccharide concentrations in
hydrolysate decreased and byproduct compounds increased.
The amounts of reducing sugars (Table 1) obtained from puri-
fied alginate in the absence of a catalyst were higher than from S.
japonica, approximately 0.08–0.12 g/l at 180 °C/13 bar to 260 °C/
49 bar. At higher temperatures and pressures, the recovery of
reducing sugars in hydrolysate water decreased. Purified alginate
2.10. Total phenolic content (TPC)
The TPC of hydrolysate water was determined with Folin–Cio-
calteu reagent as described by Chew et al. (2011) with minor mod-
ifications. Briefly, hydrolysate water was diluted using distilled
water. A total of 1 ml of diluted sample was mixed with 1 ml of
Folin–Ciocalteu reagent (1:10). The mixture was incubated at room
temperature for 4 min. Next, 0.8 ml of 7.5% sodium carbonate
anhydrous solution was added and the mixture was incubated in
darkness for 2 h, after which the absorbance at 765 nm was mea-
sured. Gallic acid dissolved in ethanol was used as a standard. Each
hydrolysate was analysed in triplicate, and the results were
expressed in milligrams per litre (mg/l).
2.11. Total flavonoid content (TFC)
The TFC of hydrolysate water was determined as described by
Ozsoy, Can, Yanardag, and Akev (2008) with minor modifications.
Briefly, 0.375 ml of hydrolysate water was mixed with 1.88 ml of
Table 1
Total glucose and reducing sugar recovery (g/l) in different hydrolysis conditions.
Temperature (°C)/
Purified alginate (g/l)
S. japonica (g/l)
pressure (bar)
Total glucose
Reducing sugar
Total glucose
Reducing sugar
With catalyst
180/13
200/17
220/25
240/34
260/49
0.45 0.01
0.35 0.01
0.26 0.03
0.17 0.00
0.11 0.00
0.10 0.00
0.07 0.00
0.05 0.00
0.05 0.00
0.05 0.00
0.34 0.02
0.22 0.02
0.14 0.01
0.09 0.00
0.07 0.00
0.05 0.00
0.04 0.00
0.04 0.00
0.04 0.00
0.04 0.00
Without catalyst
180/13
200/17
220/25
240/34
260/49
0.58 0.00
0.51 0.03
0.46 0.02
0.46 0.00
0.39 0.00
0.12 0.00
0.12 0.00
0.11 0.00
0.10 0.00
0.08 0.00
0.43 0.02
0.38 0.00
0.26 0.01
0.22 0.00
0.21 0.00
0.06 0.00
0.06 0.00
0.05 0.00
0.05 0.00
0.04 0.00

A. Meillisa et al. / Food Chemistry 171 (2015) 70–77
73
consists of high-purity alginate; at increased temperatures, algi-
nate as a polysaccharide was converted into simpler chains such
as mono-sugars. Because S. japonica is composed of complex
compounds (oligosaccharides, fat, protein, minerals, and other
compounds), its decomposition rate was lower than that of puri-
fied alginate. Moreover, the presence of other compounds during
subcritical water hydrolysis affected the carbohydrate decomposi-
tion rate. Water-soluble sugars are valuable products of fermenta-
tion reactions. Therefore, subcritical water treatment could play an
costs for R&D efforts and basic research of $900/g and $240/g,
respectively (Woodyer, Christ, & Deweese, 2010). Mannose is a
type of sugar most commonly used as a source for bio-ethanol
production (Saeman, 1945).
3.3. Antioxidant properties
The antioxidant properties of hydrolysate water were examined
using antioxidant assays, including total phenolic content (TPC),
total flavonoid content (TFC), DPPH radical scavenging activity,
and ferric reducing antioxidant power (FRAP). TPC and TFC
describe the total amount of phenolics and flavonoids, respectively,
in hydrolysate water. The DPPH radical scavenging activity mea-
sures the ability of a substance to prevent free radical molecule for-
mation. FRAP can be used to determine total antioxidant power.
As shown in Figs. 1 and 2, the antioxidant properties were
higher in the presence of a catalyst. The optimal temperature for
antioxidant properties was 180 °C. When purified alginate was
used as a starting material, the highest total phenolic, flavonoid,
FRAP, and DPPH levels were observed in the presence of catalyst,
at 4.06 0.023 mg/l, 1.39 0.002 mg/l, 6.30 0.002 mg/l, and
96.92%, respectively. Similarly, the antioxidant properties (TPC,
TFC, FRAP, and DPPH) when S. japonica was used as a starting mate-
rial were 9.63 0.013 mg/l, 1.92 0.05 mg/l, 3.11 0.01 mg/l, and
97.17%, respectively.
The antioxidant properties of hydrolysate water are affected by
bio-compounds such as phenolics, flavonoids, and minerals. The
growth environment, harvest time, and storage conditions also
affected the amounts of these compounds (Roh, Salim-Uddin, &
Chun, 2008). The addition of formic acid as a catalyst is known to
increase the antioxidant properties of hydrolysate water. Subcriti-
cal water has a high ion product H+ and OHÀ value which presents
both acidic and basic, and can hydrolyse biomass catalysed by both
H+ and OHÀ without adding any acid or base catalysts (Matsumura
et al., 2006). H+ might be increased due to add of acid on subcritical
water and produced more bioactive compound. Koivikko, Eränen,
Loponen, and Jormalainen (2008) reported that the presence of
acid yields more protection from oxidation and damage by antiox-
idant compounds, and also changes the solubility and density of
water.
important role as
production.
a
pretreatment for marine bioethanol
3.2. Quantification of gulose and mannose by HPLC
The dominant molecule in brown seaweed is carbohydrate,
with alginate identified as a major constituent. Alginate is found
in all brown seaweeds as a structural component of the cell wall
in the form of insoluble mixed salts of mainly calcium, with lesser
amounts of magnesium, sodium, and potassium, and is concen-
trated in the intracellular space (Venugopal, 2009). Alginate con-
tains three kinds of polymer units: mannuronic (M), guluronic
(G), and alternates of the M and G units. In addition to mannuronic
and guluronic units of seaweed, it also contains monosaccharides,
which have known bioactive activities. The recovery of monosac-
charides is required to increase the economic value of seaweed.
The depolymerisation method using liquefied hydrothermal pro-
cesses can be used to break down the polysaccharide polymer in
purified alginate or S. japonica. The results in Table 2 describe the
M and G recoveries in hydrolysate water. The recovery of G and
M under all hydrolysate conditions was enhanced in the presence
of formic acid as a catalyst. Increased amounts of M and G were
obtained from purified alginate in the presence of a catalyst; 3.16
and 6.50 g/l, respectively at 180 °C and 13 bar. The highest recov-
ery from S. japonica was 3.24 g/l for mannose and 9.07 g/l for
gulose. The highest recovery of G and M also occurred at 180 °C
and 13 bar. After increasing the temperature and pressure, G and
M concentration of purified alginate and S. japonica hydrolysate
decreased. Therefore, we can say that G and M is not stable at
higher temperature and pressure.
Gulose is an aldohexose sugar belonging to the rare sugars. The
International Rare Sugar Society (ISRS) defines a rare sugar as a
monosaccharide and derivative that occurs rarely in nature.
Although it has been found at low frequencies, this rare sugar
has been reported to be a bio-valuable compound. Gulose has been
reported to be a building block in the synthesis of nucleoside ana-
logues, which are useful as potential anticancer compounds
(Gumina, Song, & Chu, 2001; Sugiura et al., 2007), and a starting
material for antiviral medications (Jeong et al., 1993; Tang,
2012). The limited availability of rare sugars have led to estimated
The majority of antioxidant compounds found in brown sea-
weed belong to a group of polar compounds. The general principle
of solvent extraction is ‘‘like dissolves like’’, or in other words, sol-
vents only dissolve substances with similar polarities. Hence, those
antioxidant compounds dissolved in water as the solvent. The tem-
peratures above 180 °C used in this process decreased the antiox-
idant properties of the compounds. It is possible that the
antioxidant ability of hydrolysate water was not stable at temper-
atures above 180 °C.
Table 2
The amounts of gulose and mannose (g/l) in different hydrolysis conditions.
Temperature (°C)/
Purified alginate (g/l)
S. japonica (g/l)
pressure (bar)
Mannose
Gulose
Mannose
Gulose
With catalyst
180/13
200/17
220/25
240/34
260/49
3.20 0.2
2.50 0.1
2.60 0.1
2.80 0.1
2.70 0.1
6.50 0.1
5.90 0.2
5.90 0.0
5.40 0.2
5.50 0.1
3.20 0.1
3.20 0.1
2.90 0.1
2.60 0.2
2.00 0.0
9.10 0.2
7.60 0.1
7.50 0.0
6.90 0.1
6.30 0.1
Without catalyst
180/13
200/17
220/25
240/34
260/49
0.20 0.0
0.20 0.0
0.10 0.0
0.05 0.0
0.03 0.0
2.70 0.1
2.10 0.0
2.00 0.0
2.00 0.0
2.20 0.1
1.50 0.1
1.20 0.1
1.10 0.0
1.20 0.0
1.00 0.0
9.80 0.1
8.30 0.1
9.00 0.2
8.50 0.0
9.10 0.1

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A. Meillisa et al. / Food Chemistry 171 (2015) 70–77
(a)
(b)
(d)
(c)
Fig. 1. Antioxidant properties of purified alginate hydrolysate samples under different conditions. Tags: (a) total phenolic content (TPC); (b) total flavonoid content (TFC); (c)
DPPH radical scavenging activity; (d) Ferric reducing antioxidant power (FRAP). Data represent the mean values of three replicates S.D.
(a)
(b)
(c)
(d)
Fig. 2. Antioxidant properties of S. japonica hydrolysate samples under different conditions. Tags: (a) total phenolic content (TPC); (b) total flavonoid content (TFC); (c) DPPH
radical scavenging activity; (d) Ferric reducing antioxidant power (FRAP). Data represent the mean values of three replicates S.D.
3.4. MW determination of carbohydrate by MALDI TOF mass spectra
experimental conditions that resulted in high antioxidant activities
and sugar recovery. Depolymerisation was accompanied by a
reduction in the molecular weight. Fig. 3 shows representative
hydrolysate mass spectra from S. japonica and purified alginate.
Various sizes of MW containing carbohydrate compounds were
produced in hydrolysate which were 574, 642, 658, 805, 1221
and 1237 dalton for purified alginate and 574, 658, 674 and
MW determination of carbohydrate compounds in S. japonica
and purified alginate hydrolysate were determined based on
MALDI TOF mass spectra. Based on a previous analysis of hydroly-
sate water, mass spectral analysis was performed during hydroly-
sis at 180 °C and 13 bar in the presence of catalyst, the

A. Meillisa et al. / Food Chemistry 171 (2015) 70–77
75
Fig. 3. MALDI-TOF mass spectra of hydrolysate water in the presence of catalyst at 180 °C/13 bar of (a) purified alginate and (b) S. japonica.
(4-1)
(a)
(b)
(4-2)
(a)
(b)
Fig. 4. (4-1) Antioxidant properties and (4-2) sugar recovery of (a) purified alginate and (b) S. japonica hydrolysate water affected by pH and temperature.

76
A. Meillisa et al. / Food Chemistry 171 (2015) 70–77
821 dalton for S. japonica. These results show that diversified lower
molecular weight carbohydrate compounds were produced during
the hydrolysis process.
4. Conclusions
Subcritical water hydrolysis is known to produce monosaccha-
rides and bioactive compounds from marine macroalgae. The anti-
oxidant properties were higher under experimental conditions in
the presence of a catalyst, whereas a higher sugar recovery was
achieved under experimental conditions lacking a catalyst. In the
subcritical state, a chemical reaction occurs between the material
and water, changing the pH. The monosaccharides produced dur-
ing subcritical water hydrolysis process were confirmed as a
renewable source of biofuel and bioactive compounds with antiox-
idant, anticancer, and antiviral properties.
3.5. Antioxidant properties affect by pH and temperature
Antioxidant properties (TPC, TFC, and FRAP) were examined by
measuring the pH in hydrolysate with presence of 1% formic acid at
various temperature and pressure shown in Fig. 4-1. The pH values
of purified alginate (3.61–4.29) and S. japonica (2.98–3.31) hydrol-
ysates were increased at elevated temperatures and pressures. The
ions produced in the subcritical state are three orders of magnitude
higher than ions in water at ambient conditions and therefore, its
potential to act as an acid or base like catalyst. So, the pH values
were changed in hydrolysate using various temperature and pres-
sure applied. The antioxidant properties of both purified alginate
and S. japonica hydrolysates showed an inverse relationship with
pH and temperature: when the pH value was greater, produced
in hydrolysate at elevated temperature, the antioxidant properties
were lower, and vice versa. Therefore, lower pH values or higher
acidity levels of hydrolysate water provided the best protection
against antioxidant compounds. This was also observed by Ragan
and Glombitza (1986) and Santos-Buelga and Williamson (2003),
and the addition of acid has been used to prevent the oxidation
of polyphenolic groups. Pokorny (1986) reported that heating
accelerates the initiation reactions and decreases antioxidant
activities. The relationship between antioxidant activities and tem-
perature can be understood based on the oxidisability of the anti-
oxidant. Oxidisability is the capability to undergo a chemical
reaction with oxygen, namely, the ability to lose hydrogen atoms.
The reactivity of antioxidants against free radicals is characterised
by the bond dissociation energy (BDE) of the O–H bonds in antiox-
idant compounds. The easily oxidisable antioxidant compounds
showed decreasing antioxidant activities with increasing tempera-
tures. This phenomenon is caused by a decrease in the ability of
antioxidants to react with free radicals at higher temperatures.
Hence, less oxidisable antioxidants lose their antioxidant activity
sooner than easily oxidisable antioxidants (Reblova, 2012).
Acknowledgements
This work was supported by the Ministry of Oceans and Fisher-
ies, South Korea (contract no. 20131039449).
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