Appl Microbiol Biotechnol (2006) 71: 234237
DOI 10.1007/s00253-005-0126-3
APPLIED MICRO BIAL AND CELL PHYSIOLOGY
W. J. Jung . G. H. Jo . J. H. Kuk . K. Y. Kim .
R. D. Park
Extraction of chitin from red crab shell waste by cofermentation
with Lactobacillus paracasei subsp. tolerans KCTC-3074
and Serratia marcescens FS-3
Received: 23 February 2005 / Revised: 20 June 2005 / Accepted: 1 August 2005 / Published online: 2 September 2005
# Springer-Verlag 2005
Abstract For one-step extraction of chitin from red crab
shell waste, cofermentation with Lactobacillus paracasei
subsp. tolerans KCTC-3074, a lactic-acid-producing bac-
terium, and Serratia marcescens FS-3, a protease-produc-
ing bacterium, was conducted. Fermentation with single
strain (L. 3074 or FS-3) was also conducted. At day 7, the
pH in L. 3074, FS-3, and L. 3074+FS-3 (1:1) treatment
decreased from 6.90 to 3.30, 5.88, and 3.48, respectively.
Ash content in the residue after fermentation treatment of
crab shells in L. 3074 and L. 3074+FS-3 (1:1) treatment
drastically decreased from 41.2% to 3.19 and 1.15%,
respectively. In L. 3074+FS-3 (1:1) cofermentation, the
level of demineralization was the highest value of 97.2%,
but the level of deproteinization in the cofermentation was
52.6% at day 7. Protein content in the treatment of FS-3
alone reduced from 22.4 to 3.62%. These results indicate
that cofermentation of the shells using the two strains is
efficient and applicable for the one-step extraction of crude
chitin from red crab shell waste.
Introduction
The sources of raw material for the production of chitin are
shells of various crustaceans, principally crabs, scampi,
crayfish, prawn, and shrimps.
The traditional processes of chitin production consisted
of the use of strong acids and bases for demineralization
and deproteinization, respectively. These processes may
cause hydrolysis of the polymer (Simpson et al. 1994;
W. J. Jung . G. H. Jo . J. H. Kuk . K. Y. Kim . R. D. Park (*)
Glucosamine Saccharide Materials-National
Research Laboratory (GSM-NRL),
Division of Applied Bioscience and Biotechnology,
Institute of Agricultural Science and Technology,
Chonnam National University,
Gwangju, 500-757, South Korea
e-mail: rdpark@chonnam.ac.kr
Tel.: +82-62-5302133
Fax: +82-62-5300876
Healy et al. 1994), inconsistent physical properties (Gagne
and Simpson 1993), and a source of pollution (Allan et al.
1978).
As an alternative to the chemical process, biological
process using microorganisms has been evaluated for
demineralization (Hall and Silva 1992) and deproteiniza-
tion (Shirai et al. 1998). Lactic acid bacterial fermentation
of shrimp waste for demineralization was studied with
added carbohydrate source such as cassava or molasses
(Hall and Silva 1992), organic acids (Rao et al. 2000), and
salt supply (Rao et al. 2002). Deproteinization of crus-
tacean shell wastes was reported using protease-produc-
ing bacteria such as Pseudomonas aeruginosa K-187
(Wang and Chio 1998), Pseudomonas maltophilia LC-102
(Shimahara et al. 1984), and Bacillus subtilis (Yang et al.
2000). Deproteinization process with the demineralization
also took place from the crustacean shells (Shirai et al.
2001).
However, a combined study using lactic-acid-producing
bacteria and protease-producing bacteria for extraction of
chitin from crustacean shells has not been tried. This is
because the optimum conditions for the growth of different
strains are usually different, and, as a result, the fermen-
tation may not be efficient. In the present work, we tried the
one-step extraction of crude chitin from red crab shell
waste by cofermentation with Lactobacillus paracasei
subsp. tolerans KCTC-3074, a lactic acid bacterium, and
Serratia marcescens FS-3, an isolate as high proteolytic
bacterium.
Materials and methods
Red crab shell
Red crab (Chionoecetes japonicus) was purchased from
Yeongdeok crab store, Korea. The leg shell part was
separated after steaming the crab and washed with tap
water. The shells were cut (23 cm length) and used for
fermentation.
 235

Microorganisms 100 ml with distilled water. In a test tube, 0.25 ml of

The lactic acid bacterium, L. paracasei subsp. tolerans
KCTC-3074 (L. 3074), was obtained from Korean Collec-
tion Type Cultures (KCTC). Protease-producing bacteri-
um, S. marcescens FS-3 (FS-3), was isolated and identified
from a disposal site for crab shells on the west coast of
Korea. The isolate grew fast and produced clear zone on
LuriaBertani (LB) agar medium containing 1% skim
milk, showing strong proteolytic activity. The nucleotide
sequence of 16S rRNA gene was determined by an ABI
Prism 377 DNA Sequencer (PE Applied Biosystems, USA)
and compared with published 16S rRNA sequences using a
Blast search at NCBI. The strains in 50% glycerol as
cryoprotectant were stored in a deep freezer at 70C.
Preparation of inoculum
sample solution, 2.5 ml of 0.5 M acetate buffer (pH 5.1),
and 2.5 ml of ninhydrin-hydrindantin solution are added
and mixed. After incubation in boiling water for 10 min,
absorbance was measured at 564 nm. The protein content
P was calculated from Eq. (1), where A564 stands for
absorbance at 564 nm and W for sample mass.
P% 2:37A564 =W (1)
Statistical analysis
The Tukeys studentized range test was used to compare
the means of separate replicates. Unless otherwise stated,
conclusions are based on differences between means
significant at p0.05.

In order to prepare a starter culture, the cells from the

freezer were transferred into 100 ml of sterile Man Rogosa
Sharpe (MRS) broth for L. 3074 or LB broth for FS-3 and
incubated at 30C for 2 days. To prepare an inoculum for
fermentation, 2.0 ml of the starter culture was transferred to
100 ml sterile MRS broth (2% inoculation) or LB broth and
incubated with shaking incubator (180 rpm) at 30C for 2
days. The inoculum prepared yielded a cell concentration
of approximately 108 cfu/ml.
Fermentation
For single-strain fermentation, crab leg shells (FW 2.5 g)
were added to 50 ml of 10% glucose concentration and
inoculated with 10% L. 3074 or FS-3. For cofermentation
Results
The isolate L. 3074 and FS-3 were cultured for organic acid
and protease production in MRS and LB medium, respec-
tively, at 30C. The highest level of proteolytic activity of
FS-3 was 60 U/ml in the cultural supernatant after 3 days
culture and decreased thereafter (Fig. 1a). Cell growth of
L. 3074 increased rapidly to a maximum level within 2
days, and then the same density was maintained (Fig. 1b).
TTA of L. 3074 was 22.5% in the cultural supernatant after
3 days culture and slightly increased thereafter.
The changes in pH, TTA, dry weight, protein, and ash
contents were followed during 7 days cofermentation with
5 70

Protease activity (U/ml) ( )

with L. 3074 and FS-3, crab leg shells (FW 2.5 g) were A
)

60
4
added to 50 ml of 10% glucose concentration and
Cell growth (A660) (

50
inoculated with 5% L. 3074 and 5% FS-3 (1:1) together. 3 40
The fermentation was carried out at optimum temperature
30
of 30C for both microorganisms in a shaking incubator 2
(180 rpm) for 7 days. 20
1
10
0 0
Analysis 0 1 2 3 4 5
3 25
Dry weight was measured after drying at 60C for 48 h in B
)

20
an oven. Ash content was determined after combustion at
Cell growth (A660)(

500C for 3 h in electric furnace (A.O.A.C. 1990). The pH 2

15
TTA (%) (

was measured with a pH meter (Beckman, PHI 34, USA).

Total titratable acidity (TTA) was determined in the diluted 10
samples by titration with 0.1N NaOH to pH 8.4 and 1
expressed as a percentage of lactic acid (Pearson 1976). 5
Protein content was determined by modified method of
0 0
Shimahara and Takiguchi (1988). That is, 150 mg of dried
0 1 2 3 4 5
material was added to 25 ml of 10 N NaOH in a 100-ml
Fermentation time (day)
flask. The flask was covered with aluminum foil and heated
at 121C for 60 min in an autoclave. The reaction mixture Fig. 1 Cell growth and proteolytic activity of protease-producing S.
was then cooled rapidly, neutralized with HCl in an ice marcescens FS-3 (a) and cell growth and TTA content of organic-
bath, and filtered. Final volume of filtrate was adjusted to acid-producing L. paracasei KCTC-3074 (b)
236
Table 1 Changes of pH, total titratable acidity (TTA), dry weight (DW), and ash and protein contents during fermentation
Treatment pH TTA (%) DW (g) Ash (%) Protein (%)
Fermentation Days

L. paracasei KCTC-3074 alone 0 6.900.10a 1.500.03a 41.162.50a 22.361.82a

1 5.530.18c 1.080.09e 1.250.10b 35.230.73cd 19.400.50b
3 3.520.15de 10.900.46c 0.630.05f 12.010.42g 19.071.02b
5 3.310.08e 10.990.19bc 0.610.02f 3.660.11h 18.501.30b
7 3.300.09e 11.350.16b 0.600.02f 3.190.07h 18.010.70b
S. marcescens FS-3 alone 1 6.100.10b 1.300.06e 1.170.08bc 40.300.41ab 17.500.50b
3 6.100.08b 1.400.09e 1.020.06cd 34.400.90d 8.900.80fg
5 5.890.04b 1.500.07e 0.890.05de 23.700.98e 7.420.40g
7 5.880.05b 1.700.08e 0.830.06e 22.000.41ef 3.620.30h
L. 3074+FS-3 (1:1) cofermentation 1 5.980.16b 1.530.09e 1.010.09bcd 38.070.80bc 14.510.68c
3 5.810.11bc 3.330.09d 0.710.02ef 19.932.02f 12.990.29cd
5 3.670.09d 11.891.05b 0.600.06f 2.170.07h 11.711.02de
7 3.480.08de 15.131.01a 0.560.04f 1.150.03h 10.621.05ef
Values in a vertical column followed by different superscripted letters are significantly different at p0.05 by Tukeys studentized range
(HSD) test

the two strains L. 3074 and FS-3 (Table 1). In L. 3074
alone, the pH decreased most rapidly from pH 6.90 to
3.52 in 3 days. At day 7, the pH in L. 3074, FS-3, and L.
3074+FS-3 (1:1) treatments were 3.30, 5.88, and 3.48,
respectively.
TTA at the end of fermentation with L. 3074 and L.
3074+FS-3 (1:1) increased to 11.35 and 15.13%, respec-
tively. TTA in FS-3 alone showed little change during
fermentation.
Ash contents in L. 3074, FS-3, and L. 3074+FS-3 (1:1)
treatments were 3.19, 22.00, and 1.15%, respectively, at
day 7. Cofermentation with L. 3074+FS-3 (1:1) was most
effective for removal of mineral ash from the shells (97.2%
100
A
80
Demineralization (%)
demineralization) (Fig. 2a). Demineralization in protease-
producing bacterium FS-3 was much lower than that in
L. 3074.
Protein contents in residual crab shell decreased rapidly
from 22.36 to 8.90% in 3 days in FS-3 (60% deprotein-
ization) (Fig. 2b). At day 7, the protein contents in L. 3074,
FS-3, and L. 3074+FS-3 (1:1) treatment were 18.01, 3.62,
and 10.62%, respectively. The level of deproteinization in
FS-3 treatment was the highest value of 83.8%, while that
in L. 3074+FS-3 (1:1) cofermentation was 52.6% (Fig. 2b).
Dry weight contents decreased from 1.50 to 0.63 g in 3
days in L. 3074. At day 7, the dry weights in L. 3074, FS-3,
and L. 3074+FS-3 (1:1) treatments were 0.60, 0.83, and
0.56 g, respectively.

Discussion
60
The L. 3074 effectively removed mineral CaCO3 by pro-
40 ducing organic acid such as lactic acid (Jung et al. 2005).
The newly isolated S. marcescens FS-3 was also effective
20 for removal of proteins from the shells by producing
0 extracellular proteases (unpublished data). Thus, we sup-
0 1 2 3 4 5 6 7
posed that cofermentation with the two distinct strains
100 together in a fermenter makes the process simple, easy, and
B
effective in extracting chitin from crustacean shells.
Deproteinization (%)

80
The pH decreased from pH 6.90 to below 4.0 in the L.
60 3074 alone and L. 3074+FS-3 (1:1), respectively, after 3
and 5 days of fermentation (Table 1). In an experiment with
40
scampi waste, pH of the liquor achieved a minimum value
20 of 5.0 over the first 48 h of fermentation with 10% glucose
and 10% inoculum L. paracasei strain A3 (Zakaria et al.
0 1998). The acidification of crab shell waste below pH 5.0 is
0 1 2 3 4 5 6 7
important for biological processes because the acidic pH
Days after fermentation
suppressed the growth of spoilage organisms (Shirai et al.
Fig. 2 Changes in demineralization (a) and deproteinization (b) 2001). An increase in TTA coincided with the decreases in
during fermentation. L. paracasei KCTC-3074 (), S. marcescens pH and ash content with fermentation time for L. 3074
FS-3 (), S. marcescens. FS-3+L. paracasei KCTC-3074 (1:1)() alone and L. 3074+FS-3 (1:1) cofermentation. Deminerali-

zation level of L. 3074+FS-3 (1:1) cofermentation increased
up to 97.2% after 7 days of fermentation, suggesting that
cofermentation with L. 3074+FS-3 (1:1) was most effec-
tive for removal of ash from the shells (Fig. 2).
Demineralization was 81.4% during scampi waste fer-
mentation in 2% glucose and 5.5% inoculum Lactobacillus
plantarum 541 (Rao et al. 2002). Ash content was 7.2
and 8.1% in shrimp waste with 10% inoculum and 1%
enzyme (commercial Bacillus protease)+10% L. plantarum
541 strain, respectively (Rao et al. 2001). Protein contents
of FS-3 alone and L. 3074+FS-3 (1:1) cofermentation
were 3.62% and 10.62%, respectively, after days of
fermentation.
Deproteinization level of 52.6% in L. 3074+FS-3 (1:1)
cofermentation was lower than that of FS-3 alone (83.8%)
after 7 days of fermentation but higher than that of L. 3074
alone (19.5%) (Fig. 2). In cofermentation, FS-3 is less
contributing to deproteinization. This was probably due to
the lower pH than the optimum neutral pH for the pro-
teolytic activity in the culture supernatant because of
organic acids produced by the cocultured L. 3074.
Deproteinization was 80.3 and 90.5% in shrimp waste
with 10% inoculum and 10% enzyme+10% L. plantarum
541, respectively (Rao et al. 2001). Deproteinization was
77.06% during prawn waste fermentation in 10% glucose
and 5% inoculum Lactobacillus sp. B2 (Shirai et al. 1998).
All together, it turned out that cofermentation process
with L. 3074 and FS-3 is efficient and applicable for one-
step extraction of crude chitin from red crab shell waste by
simultaneously removing ash and protein in a fermenter but
needs improvement in deproteinization. Studies are cur-
rently being undertaken to improve the efficiency of
deproteinization and demineralization from the shells using
the cofermentation process.
Acknowledgements This study was supported by National
Research Laboratory (NRL) program from the Ministry of Science
and Technology (MOST), Korea.
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