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Appl Microbiol Biotechnol (2006) 71: 234237

DOI 10.1007/s00253-005-0126-3


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 Healy et al. 1994), inconsistent physical properties (Gagne
shell waste, cofermentation with Lactobacillus paracasei and Simpson 1993), and a source of pollution (Allan et al.
subsp. tolerans KCTC-3074, a lactic-acid-producing bac- 1978).
terium, and Serratia marcescens FS-3, a protease-produc- As an alternative to the chemical process, biological
ing bacterium, was conducted. Fermentation with single process using microorganisms has been evaluated for
strain (L. 3074 or FS-3) was also conducted. At day 7, the demineralization (Hall and Silva 1992) and deproteiniza-
pH in L. 3074, FS-3, and L. 3074+FS-3 (1:1) treatment tion (Shirai et al. 1998). Lactic acid bacterial fermentation
decreased from 6.90 to 3.30, 5.88, and 3.48, respectively. of shrimp waste for demineralization was studied with
Ash content in the residue after fermentation treatment of added carbohydrate source such as cassava or molasses
crab shells in L. 3074 and L. 3074+FS-3 (1:1) treatment (Hall and Silva 1992), organic acids (Rao et al. 2000), and
drastically decreased from 41.2% to 3.19 and 1.15%, salt supply (Rao et al. 2002). Deproteinization of crus-
respectively. In L. 3074+FS-3 (1:1) cofermentation, the tacean shell wastes was reported using protease-produc-
level of demineralization was the highest value of 97.2%, ing bacteria such as Pseudomonas aeruginosa K-187
but the level of deproteinization in the cofermentation was (Wang and Chio 1998), Pseudomonas maltophilia LC-102
52.6% at day 7. Protein content in the treatment of FS-3 (Shimahara et al. 1984), and Bacillus subtilis (Yang et al.
alone reduced from 22.4 to 3.62%. These results indicate 2000). Deproteinization process with the demineralization
that cofermentation of the shells using the two strains is also took place from the crustacean shells (Shirai et al.
efficient and applicable for the one-step extraction of crude 2001).
chitin from red crab shell waste. 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
Introduction because the optimum conditions for the growth of different
strains are usually different, and, as a result, the fermen-
The sources of raw material for the production of chitin are tation may not be efficient. In the present work, we tried the
shells of various crustaceans, principally crabs, scampi, one-step extraction of crude chitin from red crab shell
crayfish, prawn, and shrimps. waste by cofermentation with Lactobacillus paracasei
The traditional processes of chitin production consisted subsp. tolerans KCTC-3074, a lactic acid bacterium, and
of the use of strong acids and bases for demineralization Serratia marcescens FS-3, an isolate as high proteolytic
and deproteinization, respectively. These processes may bacterium.
cause hydrolysis of the polymer (Simpson et al. 1994;

Materials and methods

W. J. Jung . G. H. Jo . J. H. Kuk . K. Y. Kim . R. D. Park (*)
Glucosamine Saccharide Materials-National
Research Laboratory (GSM-NRL), Red crab shell
Division of Applied Bioscience and Biotechnology,
Institute of Agricultural Science and Technology, Red crab (Chionoecetes japonicus) was purchased from
Chonnam National University, Yeongdeok crab store, Korea. The leg shell part was
Gwangju, 500-757, South Korea
e-mail: separated after steaming the crab and washed with tap
Tel.: +82-62-5302133 water. The shells were cut (23 cm length) and used for
Fax: +82-62-5300876 fermentation.

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

sample solution, 2.5 ml of 0.5 M acetate buffer (pH 5.1),
The lactic acid bacterium, L. paracasei subsp. tolerans and 2.5 ml of ninhydrin-hydrindantin solution are added
KCTC-3074 (L. 3074), was obtained from Korean Collec- and mixed. After incubation in boiling water for 10 min,
tion Type Cultures (KCTC). Protease-producing bacteri- absorbance was measured at 564 nm. The protein content
um, S. marcescens FS-3 (FS-3), was isolated and identified P was calculated from Eq. (1), where A564 stands for
from a disposal site for crab shells on the west coast of absorbance at 564 nm and W for sample mass.
Korea. The isolate grew fast and produced clear zone on
LuriaBertani (LB) agar medium containing 1% skim P% 2:37A564 =W (1)
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 Statistical analysis
Blast search at NCBI. The strains in 50% glycerol as
cryoprotectant were stored in a deep freezer at 70C. The Tukeys studentized range test was used to compare
the means of separate replicates. Unless otherwise stated,
conclusions are based on differences between means
Preparation of inoculum 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 Results
Sharpe (MRS) broth for L. 3074 or LB broth for FS-3 and
incubated at 30C for 2 days. To prepare an inoculum for The isolate L. 3074 and FS-3 were cultured for organic acid
fermentation, 2.0 ml of the starter culture was transferred to and protease production in MRS and LB medium, respec-
100 ml sterile MRS broth (2% inoculation) or LB broth and tively, at 30C. The highest level of proteolytic activity of
incubated with shaking incubator (180 rpm) at 30C for 2 FS-3 was 60 U/ml in the cultural supernatant after 3 days
days. The inoculum prepared yielded a cell concentration culture and decreased thereafter (Fig. 1a). Cell growth of
of approximately 108 cfu/ml. 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
Fermentation 3 days culture and slightly increased thereafter.
The changes in pH, TTA, dry weight, protein, and ash
For single-strain fermentation, crab leg shells (FW 2.5 g) contents were followed during 7 days cofermentation with
were added to 50 ml of 10% glucose concentration and
inoculated with 10% L. 3074 or FS-3. For cofermentation 5 70

Protease activity (U/ml) ( )

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

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

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

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

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

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
effective in extracting chitin from crustacean shells.
Deproteinization (%)

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
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-

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up to 97.2% after 7 days of fermentation, suggesting that facilitate recovery of chitin from shrimp wastes. Food Bio-
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