Bioresource Technology 84 (2002) 287290

Flocculation properties of pectin in various suspensions

H. Yokoi *, T. Obita, J. Hirose, S. Hayashi, Y. Takasaki
Department of Applied Chemistry, Faculty of Engineering, Miyazaki University, Miyazaki 889-2192, Japan
Received 25 August 2001; received in revised form 28 December 2001; accepted 3 January 2002

Abstract
Pectin had a occulating activity and its occulating activities in various suspensions were investigated. Flocculating activity of
pectin in a kaolin suspension was markedly stimulated by the addition of Al3 and Fe3 to the suspension. Optimum temperature for
occulating activity of pectin in the kaolin suspension was around 30 C and high occulating activity was obtained when 30 mg/l
of pectin and 0.2 mM Fe3 were added to the suspension. Other inorganic suspensions of activated carbon and acid clay were
occulated by pectin in the presence of Al3 or Fe3 . Flocculation of organic suspensions such as cellulose and yeast by pectin
occurred when 0.10.2 mM Fe3 was present in the suspensions. 2002 Elsevier Science Ltd. All rights reserved.

Keywords: Pectin; Flocculation; Flocculating activity; Flocculant; Properties; Suspension

1. Introduction as inorganic occulants, manufactured organic high-

Pectin is a heterogeneous polysaccharide occurring in
the cell walls and intracellular layers of land plants. The
main sources of pectin in commercial production are
agricultural and food-industrial wastes of fruits, i.e., the
peel of citrus fruits such as lemon, lime, orange and
grapefruit, and apple pomace. The major component of
pectin is a polymer of a-D -galacturonic acid and the
carboxyl groups of the a-D -galacturonic acid are either
free or esteried with methyl groups. Pectin is a water-
soluble biopolymer and an aqueous solution of pectin is
viscous. Furthermore, pectin has a gel-forming ability.
Based on these chemical properties, pectin is widely used
in the preparation of jams, jellies, canned fruits, fruit
juices and confectionery products in the food industry
(Thakur et al., 1997). Other applications of pectin have
been investigated to enlarge its utilization as a useful
material. For instance, pectin is used to reduce blood
cholesterol levels in humans (Judd and Truswell, 1982).
Pectin is useful as a sizing agent for paper and textiles
because of its lm-forming properties (Miers et al.,
1953), and applicable to removal of toxic heavy metals
from aqueous solution (Paskins-Hurlburt et al., 1977).
In the elds of wastewater treatment, fermentation
processes and food industries, various occulants such
*
Corresponding author. Fax: +81-985-58-7323.
E-mail address: yokoi@cc.miyazaki-u.ac.jp (H. Yokoi).
polymer occulants and naturally occurring occulants
have been used. Although the manufactured organic
high-polymer occulants such as polyacryl amide are
widely used, they give rise to environmental problems in
that some of them are not readily biodegradable and
intermediate products of their degradation are harmful
to humans. To solve these environmental problems,
utilization of the naturally occurring occulants ob-
tained from microorganisms and biomass has been
suggested due to their biodegradability and the harm-
lessness to the environment of their degradation inter-
mediates (Kurane et al., 1986; Yoon et al., 1998). Most
occulants produced by microorganisms are usually
high molecular weight polymers, and are polysaccha-
rides (Fujita et al., 2000; Kwon et al., 1996; Suh et al.,
1997; Yokoi et al., 1996b; Yokoi et al., 1997), proteins
(Takeda et al., 1991; Takeda et al., 1992), glycoproteins
(Lee et al., 1995) and others (Yokoi et al., 1996a). The
occulants obtained from plants and other biological
resources are also promising since biomass and agri-
cultural wastes can be reutilized as raw materials. For
instance, chitosan obtained from the waste of the sea-
food processing industry is used as a occulant (Knorr,
1991).
The authors found that pectin had a occulating ac-
tivity, however, there has so far been no report about
occulating activity of pectin and its applicability as a
occulating agent. If occulation properties of pectin
were claried, it could be utilized as a new and harmless

0960-8524/02/$ - see front matter 2002 Elsevier Science Ltd. All rights reserved.
PII: S 0 9 6 0 - 8 5 2 4 ( 0 2 ) 0 0 0 2 3 - 8
288 H. Yokoi et al. / Bioresource Technology 84 (2002) 287290

biopolymer occulant, since it is biodegradable, edible
and nontoxic toward humans and the environment. In
this study, the occulation properties of pectin in vari-
ous suspensions and its applicability as a new occu-
lating agent were investigated.
2. Methods
2.1. Pectin
Pectin prepared from apples was obtained from
Wako Pure Chemical Industries, Ltd. (Osaka, Japan).
Pectin dissolved in distilled water was used as a pectin
solution.
2.2. Assay of occulating activity
was inuenced by addition of cations to the kaolin
suspension. Fig. 1 shows the eects of cations added to
the reaction mixture on occulating activity of pectin.
Concentrations of cations and pectin in the reaction
mixture were 5 mM and 20 mg/l, respectively. Solutions
of AlCl3  6H2 O, FeCl3  6H2 O, FeSO4  7H2 O, CaCl2 ,
MgCl2 , NaCl and KCl were used as the source of ca-
tions. Although occulating activity of pectin was not
observed in the absence of cations, occulation of the
kaolin suspension occurred by addition of Al3 and Fe3
at some concentrations.
The eects of Al3 and Fe3 concentrations on the
occulating activity of pectin in the kaolin suspension
are shown in Fig. 2. Pectin concentration in reaction
mixtures was 15 mg/l. Optimum concentrations of Al3
and Fe3 for the occulating activity of pectin in the

The assay of occulating activity of pectin in a kaolin

suspension was carried out as follows. One-tenth ml of
the pectin solution, 0.25 ml of cation solution and 4.65
ml of 5 g/l kaolin suspension were mixed in a test tube
(i.d. 8 mm  height 90 mm). The mixture was stirred
with a Vortex mixer and left standing for 5 min. Two
milliliters of supernatant was carefully removed from
the upper layer in the test tube, and its absorbance at
550 nm (A) was measured. A control experiment with-
out the pectin solution was carried out in the same
manner and absorbance at 550 nm (B) was measured.
Flocculating activity was calculated using the following
equation (Toeda and Kurane, 1991):
Flocculating activity 1=OD 1=A  1=B
Flocculating activities in activated carbon (Wako Pure
Chemical Industries Ltd.), acid clay (Wako Pure
Chemical Industries Ltd.), cellulose (AVCEL SF, Fu- Fig. 1. Eects of cations on occulating activity of pectin in kaolin
suspension. Cation concentration in the kaolin suspension was 5 mM.
nakoshi Co. Ltd., Tokyo, Japan) and yeast suspensions
were also measured. Activated carbon, acid clay and
cellulose were suspended in distilled water and the ab-
sorbance at 550 nm of the suspensions was adjusted to
1.4. The cells of Saccharomyces cerevisiae IFO2044 were
used to prepare the yeast suspension. After cultivation
aerobically in a medium (pH 6.0) consisting of 0.5%
polypepton, 0.3% yeast extract, 0.3% malt extract and
1.0% glucose at 30 C for 12 h, the yeast cells were
harvested from the culture broth by centrifugation at
8500 g for 15 min. The harvested cells were suspended in
distilled water and the absorbance at 550 nm of the
suspension was adjusted to 1.4.

3. Results and discussion

3.1. Flocculation properties in inorganic suspensions

Flocculation properties of pectin in a kaolin suspen- Fig. 2. Eects of Al3 () and Fe3 (d) concentrations on occulating
sion were rst examined. Flocculation activity of pectin activity of pectin in kaolin suspension.
 H. Yokoi et al. / Bioresource Technology 84 (2002) 287290 289

kaolin suspension were 0.2 mM, respectively. It is

known that addition of cations to kaolin suspension is
necessary to induce the eective occulating activity of
negative-charged biopolymer occulants produced by
Rhodococcus erythropolis (Takeda et al., 1991), Alcalig-
enes cupidas (Toeda and Kurane, 1991), Nocardia ama-
rae (Takeda et al., 1992), Phormidium J-1 (Fattom and
Shilo, 1984), Aspergillus sp. JS-42 (Nam et al., 1996) and
Bacillus subtilis TB11 (Yoon et al., 1998). Compared
with these microbial occulants, the eects of cations
on the occulating activity of pectin in a kaolin sus-
pension were dierent. It is assumed that Al3 and Fe3
ions stimulate occulation by neutralization and stabi-
lization of residual negative charges of carboxyl groups
of galacturonic acid in pectin forming bridges which
bind kaolin particles to each other.
Flocculating activity of pectin was inuenced by re- Fig. 4. Eects of Al3 () and Fe3 (d) concentrations on occulating
activity of pectin in activated carbon (solid line) and acid clay (dotted
action temperature. Optimum region of the reaction
line) suspensions.
temperature for the occulating activity of pectin in the
kaolin suspension containing 0.2 mM Fe3 was 2040
C, and the highest occulating activity was obtained
lationship between pectin concentration and occulating
at 30 C (data not shown). The eect of temperature on
activity is similar to that of a protein occulant pro-
occulating activity of pectin was dierent from that of
duced by Rhodococcus erythropolis (Takeda et al., 1991),
occulants from Phormidium J-1 (Fattom and Shilo,
a polysaccharide occulant by Pestalotiopsis sp. (Kwon
1984) and Citrobacter sp. (Fujita et al., 2000).
et al., 1996) and poly(c-glutamic acid) by Bacillus sp.
Flocculating activity of pectin was also inuenced by
PY-90 (Yokoi et al., 1995).
reaction pH of a kaolin suspension. The maximum
Pectin could occulate other inorganic suspensions.
occulating activity of pectin was obtained at pH 3.0
As shown in Fig. 4, the highest activities were obtained
and the activity decreased with increasing pH (data not
at 0.2 mM of Fe3 and 2 mM of Al3 in activated car-
shown).
bon and acid clay suspensions containing 15 mg/l of
Fig. 3 shows the eect of pectin concentration on
pectin, respectively. Optimum pH for occulation of the
occulating activity in the kaolin suspension containing
suspensions was 3.0 (data not shown).
0.2 mM Fe3 . High occulating activity of pectin was
observed at 1040 mg/l and an optimum concentration
of pectin in the kaolin suspension was 30 mg/l. The re- 3.2. Flocculation properties in organic suspensions

Organic suspensions such as cellulose and yeast could

Fig. 3. Eect of pectin concentration on occulating activity of pectin
in kaolin suspension.
be occulated by pectin. Cellulose and yeast suspensions
were occulated in the presence of Fe3 , but not in the
presence of Al3 . The eects of Fe3 concentration on
occulating activity of pectin in the cellulose and yeast
suspensions containing 15 mg/l of pectin are shown in
Fig. 5. Optimum concentration of Fe3 for the occu-
lating activity of pectin existed also in the organic sus-
pensions and the highest occulating activities were
obtained at 0.1 and 0.2 mM of Fe3 in the cellulose and
yeast suspensions, respectively.
Optimum reaction pH and reaction temperature for
occulation of the cellulose suspension by pectin were
3.0 and 30 C, respectively (data not shown).
From these ndings, it can be concluded that pectin
has occulating activity in inorganic and organic sus-
pensions, and is applicable as a biodegradable, edible
and harmless biopolymer occulant like chitin (Knorr,
1991), xanthan (Yokoi et al., 1996b) and poly(c-glu-
tamic acid) (Yokoi et al., 1996a).
290 H. Yokoi et al. / Bioresource Technology 84 (2002) 287290
Fig. 5. Eects of Fe3 concentration on occulating activity of pectin
in cellulose () and yeast (d) suspensions.
References
Fattom, A., Shilo, M., 1984. Phormidium J-1 bioocculant: production
and activity. Arch. Microbiol. 139, 421426.
Fujita, M., Ike, M., Tachibana, S., Kitada, G., Kim, S.M., Inoue, Z.,
2000. Characteristics of a bioocculant produced by Citrobacter sp.
YKF04 from acetic and propionic acids. J. Biosci. Bioeng. 89, 4046.
Judd, P.A., Truswell, A.S., 1982. Comparison of the eects of high and
low methoxyl pectins on blood and faecal lipids in man. Br. J.
Nutr. 48, 451458.
Knorr, D., 1991. Recovery and utilization of chin and chitosan in food
processing waste management. Food Technol. 45, 114122.
Kurane, R., Takeda, K., Suzuki, T., 1986. Screening for and charac-
teristics of microbial occulants. Agr. Biol. Chem. 50, 23012307.
Kwon, G.S., Moon, S.H., Lee, H.M., Kim, H.S., Oh, H.M., Yoon,
B.D., 1996. A novel occulant biopolymer produced by Pestaloti-
opsis sp. KCTC 8637P. Biotechnol. Lett. 18, 14591464.
Lee, H.L., Lee, S.O., Jang, K.L., Lee, T.H., 1995. Microbial
occulant from Arcuadendron sp. TS-49. Biotechnol. Lett. 17, 95
100.
Miers, J.C., Swenson, H.A., Schultz, T.H., Owens, H.S., 1953.
Pectinates and pectate coatings. I. General requirements and
procedures. Food Technol. 7, 229231.
Nam, J.S., Kwon, G.S., Lee, S.O., Hwang, J.S., Lee, J.D., Yoon, B.D.,
Lee, T.H., 1996. Bioocculant produced by Aspergillus sp. JS-42.
Biosci. Biotech. Biochem. 60, 325327.
Paskins-Hurlburt, A.J., Tanaka, Y., Skoryna, S.C., Moore, W., Stara,
J.F., Stara, J.R., 1977. The binding of lead by a pectic polyelec-
trolyte. Environ. Res. 14, 128140.
Suh, H.H., Kwon, G.S., Lee, C.H., Kim, H.S., Oh, H.M., Yoon, B.D.,
1997. Characteristics of bioocculant produced by Bacillus sp. DP-
152. J. Ferment. Bioeng. 84, 108112.
Takeda, M., Koizumi, J., Matsuoka, H., Hikuma, M., 1992. Factors
aecting the activity of a protein bioocculant produced by
Nocardia amarae. J. Ferment. Bioeng. 74, 408409.
Takeda, M., Kurane, R., Koizumi, J., Nakamura, I., 1991. A protein
bioocculant produced by Rhodococcus erythropolis. Agr. Biol.
Chem. 55, 26632664.
Thakur, B.R., Singh, R.K., Handa, A.K., 1997. Chemistry and uses of
pectin: a review. Crit. Rev. Food Sci. Nutr. 37, 4773.
Toeda, K., Kurane, R., 1991. Microbial occulant from Alcaligenes
cupidas KT201. Agr. Biol. Chem. 55, 27932799.
Yokoi, H., Natsuda, O., Hirose, J., Hayashi, S., Takasaki, Y., 1995.
Characteristics of a biopolymer occulant produced by Bacillus sp.
PY-90. J. Ferment. Bioeng. 79, 378380.
Yokoi, H., Arima, T., Hirose, J., Hayashi, S., Takasaki, Y., 1996a.
Flocculation properties of poly(c-glutamic acid) produced by
Bacillus subtilis. J. Ferment. Bioeng. 82, 8487.
Yokoi, H., Shiraki, M., Hirose, J., Hayashi, S., Takasaki, Y., 1996b.
Flocculation properties of xanthan produced by Xanthomonas
campestris. Biotechnol. Techniq. 10, 789792.
Yokoi, H., Yoshida, T., Mori, S., Hirose, J., Hayashi, S., Takasaki, Y.,
1997. Biopolymer occulant produced by Enterobacter sp. Bio-
technol. Lett. 19, 569573.
Yoon, S.H., Song, J.K., Go, S.J., Ryu, J.C., 1998. Production of
biopolymer occulant by Bacillus subtilis TB11. J. Microbiol.
Biotechnol. 8, 606612.