APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1976, p. 479-482 Vol. 32, No.

4
Copyright X) 1976 American Society for Microbiology Printed in U.S.A.

Preparation of Agar-Agar from the Red Seaweed Pterocladia

capillacea off the Coast of Alexandria, Egypt
A. V. RAO* AND INAAM A. BEKHEET'
Central Salt and Marine Chemicals Research Institute, Bhavnagar-364 002, India
Received for publication 21 May 1976

The effect of different treatments on the quality of agar produced from

Pterocladia has been studied, and the conditions for the production of material of
good quality have been standardized. In the modified process, sun-bleached
seaweed was washed well in water, soaked for 24 h, and then ground to a pulp
and rinsed again in water. The pulp was then extracted with water (weed-to-
water ratio. 1:30) under pressure for 2 h after adjusting the pH to 6 by the
addition of acetic acid. The agar gel, after freeze thawing, was bleached with
NaClO before drying in a current of hot air. Pretreatment of the seaweed with
alkali at 80C for 2 h prior to extraction was found to improve the quality of agar
to a very great extent.
The prevailing climatic and hydrographic seaweed is not sun bleached, nor is it soaked
factors allow several genera of agarophytes to and wet ground before extraction; moreover,
grow luxuriantly along the Mediterranean the pH during extraction is not adjusted. Pre-
coast. Huge masses of agarophytes, especially treatment of the seaweed with alkali at 80 to
Pterocladia plants, drift throughout the year. 90C was not included, although such a step is
This alga grows profusely along the sublittoral introduced for production of agarose-rich agar
belt of the rocky coast of Egypt (3) and is the from Gracilaria in industry (1, 5). Percival (4)
raw material for agar manufacture in Alexan- has described the differences in the fine struc-
dria. ture of the algal polysaccharides, which are all
Pterocladia is an excellent seaweed raw ma- polydisperse linear galactans, and discussed
terial for agar-agar production, and it has been the effect of these differences on the conforma-
observed that the gel strength of the agar is tion of the macromolecules and, consequently,
higher than that of all the known agar samples on the physical properties, in particular, the
collected from different countries, including Ja- gelling properties, of the individual polymers.
pan and the United States. The quality of agar The effect of some of these factors on agar qual-
produced in Alexandria is far from satisfactory, ity, namely, (i) sun bleaching, (ii) washing and
since the method adopted for extraction of agar soaking for 16 to 24 h followed by wet grinding,
is rather crude. The Alexandria process con- (iii) pH adjustment of the cooking liquor, (iv)
sists of a single washing of the dried seaweed water-to-weed ratio, and (v) pretreatment with
with hot water at 40C, followed by pressure alkali, is the subject of this study.
cooking for 2 h at 30 lb/in2 with 15 times its
weight of water. The liquor is separated from MATERIALS AND METHODS
the residue through a perforated screen in the Method and treatments. (i) Alexandria process.
bottom of the autoclave, filtered in a filter A total of 300 g of air-dried seaweed was given two
press, and set to gel in trays. After freeze thaw- washings with hot water 40C and then cooked for 2
ing, the agar sheets are rinsed thoroughly in h at 30 lb/in2 using 4.5 liters of water (weed-to-water
fresh water, bleached with NaClO, and then ratio, 1:15). The residue after 2 h of extraction was
treated with a dilute solution of hydrogen per- separated from the solution by filtering through a
oxide to remove the excess bleach liquor. They "double cloth" and squeezing the cloth as much as
are then rinsed again in water, the excess wa- possible to remove liquid. The filtrate was collected
in trays, allowed to set to a gel, and then kept in a
ter is squeezed out, and they are dried in a cold chamber for 24 h. After thawing, the gel was
steam-heated tray drier in which hot air at 60C washed several times, bleached with 1.0% NaClO
is circulated. The dried sheets are then pow- solution, and then treated with dilute H202 solution.
dered in a hammer mill. In this process, the The sheets were then washed several times with
' Present address: Department of Botany and Microbiol- water before being dried.
ogy, University College of Swansea, Swansea, SA, 8PP, (ii) Washing, soaking for 24 h in water, and wet
U.K. grinding. The air-dried seaweed was washed well in
479
480 RAO AND BEKHEET
water to remove sand, dirt, and any other extra-
neous matter and then was kept soaking in water for
24 h. The wet seaweed was ground to a pulp in a
blender and then washed thoroughly in water before
using for extraction as described in (i).
(iii) pH adjustment. The pH of the weed-water
mixture described in (i) was adjusted to 6 by adding
the required quantity of 1 N H2SO4 before extraction
under pressure.
(iv) Washing, soaking, wet grinding, and pH ad-
justment (ii and iii combined). After washing, soak-
ing for 24 h, and then pulping the seaweed, the pH
was adjusted to 6 by the addition of 1 N H2S04 and
then the contents were extracted as usual.
(v) Open boiling. Extraction was carried out by
open boiling, both with and without pH adjustment.
Effect of water-to-weed ratio on the extraction
effi'ciency. The Alexandria process uses a water-to-
weed ratio of 15 for the extraction of agar. However,
it was felt that this ratio was not sufficient for the
complete extraction of agar and hence experiments
were performed in which water-to-weed ratios,
ranging from 15 to 50 were used, and the extraction
was carried out incorporating the modifications
that were found to give better results. These experi-
ments were also repeated with sun-bleached sea-
weeds.
Effect of pretreatment of seaweed with alkali.
Experiments were conducted in which air-dried
seaweed was treated with dilute hot alkaline solu-
tion prior to extraction under pressure. A 300-g
amount of air-dried material was placed in a rotary
washer in a rectangular tank in which 0.5% NaOH
solution heated to 80C was added to one-half the
volume (30 liters) so that the washer was half-sub-
merged in the solution. It was then rotated for 2 h,
during which the temperature was maintained at
80C. After this, the alkali was drained out, and the
seaweed was washed with hot water (60C) by the
rotary washing method until the pH of the seaweed
and the wash water was reduced to 7. The seaweed
was then cooked under pressure as usual, after ad-
justing the pH to 6 by the addition of H2S04 or acetic
acid.
Gel strength measurement. The gel strength of
the agar gels was measured by the "penetrometer"
method, in which weights of 50 g were placed on the
top of the penetrometer at intervals of 30 s until the
penetrometer bottom, which is 1 cm2 in area, slowly
and gradually penetrated into the gel. The total
weight that caused the penetration of the gel was
noted, and the gel strength was expressed as grams
per square centimeter.
RESULTS AND DISCUSSION
Table 1 presents data on the effect of various
treatments introduced in the processing of
agar. The yield of agar in the Alexandria proc-
ess is quite low. The gelling power is quite
good, but color and transparency are poor.
When dissolved in boiling water, the agar
leaves much insoluble matter. In contrast, the
various steps introduced in the process gave a
APPL. ENVIRON. MICROBIOL.
higher yield and resulted in better quality agar
which gave a clear transparent gel, leaving
very little sediments. The yield could be im-
proved from 14 to 20%, an increase of nearly
43%. In the case of sun-bleached seaweed, the
yield was 22%, an increase of nearly 56%. Open-
boiling extraction did not result in good gel,
unlike Gelidium or Gracilaria. Hence, in the
case of Pterocladia, autoclaving or pressure ex-
traction is required to obtain a good gel. The
experiments on the effect of water-to-weed ra-
tios clearly demonstrated that by increasing
the amount of water for extraction, a higher
yield of agar could be obtained. At the same
time, the gel strength was not affected in any
way by increasing the volume of the extracting
liquor. These results are more pronounced
when sun-bleached seaweed was used as the
starting raw material (Table 2). Thus, it is
evident that a water-to-weed ratio of 30 appears
to be the optimum for both unbleached and
sunbleached seaweed.
Pretreatment with alkali yields a superior
quality of agar, which is readily soluble in wa-
ter even by open boiling and leaves very little
insoluble matter (Table 3). The gel was very
clear and transparent, without any haze. The
ash and sulfate contents of this agar were quite
low and gel strength was appreciably high, sug-
gesting thereby that agar prepared by this tech-
nique is richer in agarose. This is probably due
to interconvertibility of 1,4-linked galactose-6-
sulfate into 3,6-anhydrogalactose, which is
readily brought about in the laboratory and in
the factory by alkaline treatment of the poly-
saccharide (4). This view is also supported by
the fact that the yield of agar from the alkali-
treated seaweed was rather low, around 8 to
12%.
Matsuhashi and Hayashi (2) have reported a
20 to 23% yield of agar from unwashed, dry
Gracilaria folifera when it is treated with an
aqueous sodium hydroxide-calcium oxide mix-
ture (pH 13.6) for 3 to 4 h at 95C.
Hence, for bacteriological-grade agar, sun-
bleached seaweed of the Pterocladia sp. was
predigested with about 100 times its weight of
0.5% NaOH solution at 80C for 2 h and then
washed with 60C water until the pH of the
wash water was 7. Food-grade agar was pre-
pared from sun-bleached seaweed that had
been washed, soaked in water for 24 h, ground
to a pulp in a blender, and washed again. Agar
was extracted from the alkaline or regular
pulps by treatment for 2 h at 30 lb/in2 (135C) in
30 times their weight of water acidified to pH 6.
The agar sheets after freeze-thawing were
rinsed well in dilute acetic acid solution (pH 6)
before being bleached.
VIOL. 32, 1976 PREPARATION OF AGAR-AGAR FROM SEAWEED 481

TABLE 1. Effect of different treatments on the extraction of agar from Pterocladia capillacea
Method/treatment strength
Yield of agar (%) Gel(g/cm2) Color and transparency of the agar solu-
tion/insolubles
(i) Alexandria process 14.0 725 Light brown; turbid with insoluble par-
ticles
(ii) Washing, soaking, and wet grind- 16.5 800 Golden yellow; clear and transparent,
ing no insolubles
(iii) Addition of acid for pH adjust- 17.5 800 Golden yellow; clear and transparent,
ment no insolubles
(iv) ii and iii combined 20.0 750 Golden yellow; clear and transparent,
no insolubles
(v) ii and iii combined, using sun- 22.0 550 Golden yellow; clear and transparent,
bleached seaweed no insolubles
(vi) (a) Open-boiling extraction No gel could
(b) pH adjustment to 6 and then be ob-
extraction by open boiling J tained

TABLE 2. Effect of water-to-weed ratio on the extraction of agar from Pterocladia by the modified processa
Treatment no. Wt of (g)
weed sea- Water-to-
weed ratio
Wt of agar
obtained (g) Yield M Gel(g/cm2)b
strength Remarks
Unbleached
seaweed
(i) 300 15 42.5 14 700 Alexandria process used
for comparison; does not
dissolve completely by
open boiling
(ii) 300 15 60.0 20 600 Dissolves almost com-
pletely by open boiling
(iii) 225 20 47.3 21 550 Dissolves almost com-
pletely by open boiling
(iv) 150 30 33.0 22 600 Dissolves almost com-
pletely by open boiling
(v) 112.5 40 26.0 23 600 Dissolves almost com-
pletely by open boiling
Sun-bleached
seaweed
(i) 300 15 66.0 22 Dissolves completely by
550
(ii) 225 20 52.0 23 600
open boiling; no insolu-
(ii) 150 30 39.0 26 600
ble matter left after dis-
(iv) 112.5 40 31.0 27 500
solution; clear and
(v) 90 50 24.0 26.6 550
transparent gel ob-
tained
a
Seaweeds was washed well in water, soaked for 24 h, and then ground to a pulp and rinsed again in
water. Extraction was done using different water-to-weed ratios after adjusting the pH to 6 by the addition
of 1 N sulfuric acid under pressure for 2 h.
b The gel strength measurements were made by dissolving agar in tap water by open boiling. Higher
values (almost double) were obtained when agar was dissolved in distilled water and also when the
autoclaving method was used.
482 RAO AND BEKHEET APPL. ENVIRON. MICROBIOL.
TABLE 3. Experimental data for alkali-treated seaweed8
Solubility in Gel
Method of pH ad- Color and distilled water strength Yield Melting Ash SO4
justment transparency by open-boil- solution temp ( (%)
justment of gel ~~~~ingmethod (g/cm2)
(i) No adjustment Milky-white and Leaves small 1,350 11.5 92 1.7 1.12
hazy amount of in-
soluble matter
(ii) pH of the cooking Milky-white, Little insoluble 750 10.5 88
liquor adjusted but not hazy matter
to 6 with acetic 2.85 1.15
acid
Sameb Very transpar- Almost totally 1,050 9.8 93
ent, clear gel; soluble
no haze
(iii) pH adjusted to 6 Clear gel; no Very little insol- 650 10.2 89
during washing haze uble matter
with H2SO4 3.8 1.56

Samer Clear gel; no Very little insol- 750 10.4 89

haze uble matter
(iv) pH adjusted to 6 Slight haze Little insoluble 900 9.5 90
during washing matter
with acetic acid 2.1 0.93

Sameb Slight haze Little insoluble 1,200 9.3 93

matter
a The seaweed was digested with 0.5% NaOH solution at 80C for 2 h in a rotary digester. It was washed well with hot
water at 60C in the same unit until the pH was reduced to that of water (7.5). The seaweed was then extracted under
pressure as usual. Different experiments were performed by adjusting the pH prior to extraction as indicated.
b After thawing, the gel was rinsed in dilute acetic acid solution (pH 6) and then bleached as usual.

ACKNOWLEDGMENTS from Gracilaria folifera of Florida. Agric. Biol. Chem.

We express our sincere thanks to the authorities of the
Food and Agriculture Organization of the United Nations
for fimancial support for this project.
LITERATURE CITED
1. Funaki, K., and Y. Kojima. 1951. Studies on the prepa-
ration of Agar-Agar from Gracilaria Confervoids. I.
Bull. Jpn. Soc. Sci. Fish. 16:401-404.
2. Matsuhashi, T., and K. Hayashi. 1972. Agar processed
36: 1543-1552.
3. Nasr, A. H., A. F. Mohsen, and I. A. Bekheet. 1966.
Effect of salinity and temperature variations on Pter-
ocladia Capillacea. Hydrobiologia 27:395-400.
4. Percival, E. 1972. Chemistry of agarids, carrageenans
and furcellarans. J. Sci. Food Agric. 23:933-940.
5. Tagawa, S., and Y. Kojima. 1972. The alkali-treatment
of the mucilage of Gracilaria Verrucosa, p. 447-450.
In K. Nisizawa (ed.), Proceedings of the seventh
international seaweed symposium, Sapporo, Japan,
August 8-12, 1971. University of Tokyo Press, Tokyo.