Isolation and Characterization of

Chitinase-Producing Bacillus and Paenibacillus

Strains from Salted and Fermented
Shrimp, Acetes japonicus
Abstract: Chitinases catalyze the conversion of chitin and are produced by a wide range of bacteria. The biological
applications of these enzymes have been exploited in food and pharmaceutical industries. We isolated 2 halophilic
chitinase-producing novel strains of bacteriaSCH-1 and SCH-2 from Saeu-jeot, a traditional Korean salted and fermented
food made with shrimp (Acetes japonicus). The isolated strains- SCH-1 and SCH-2 were Gram-positive, rod-shaped,
endospore-forming facultative anaerobes, with strain SCH-2 showing peritrichous flagella. Molecular characterization of
the 16S rRNA gene identified the strains SCH-1 and SCH-2 as Bacillus sp. and Paenibacillus sp. respectively. Basic Local
Alignment Search Tool and subsequent phylogenetic analysis of strain SCH-1 showed an identity of 97.83% with Bacillus
cereus ATCC 14579 (NR_074540), whereas strain SCH-2 showed an identity of 99.16% with Paenibacillus lautus JCM
9073 (NR_040882). Furthermore, the SCH-1 strain could use glucose, N-acetyl glucosamine, esculin, and maltose as
carbon source substrates. Cellular fatty acid analysis showed that iso-C15:0 and anteiso-C15:0 are the major acids in strain
SCH-1 and SCH-2, respectively. The SCH-1 strain showed a higher chitinase activity at 15.71 unit/mg protein compared
with SCH-2 strain. Chitinase isozymes of Bacillus sp. SCH-1was expressed as 2 bands having sizes of 41 and 50 kDa, and
as 4 bands with sizes of 30, 37, 45.7, and 50 kDa in Paenibacillus sp. SCH-2. The rich chitinase activity with the isozyme
profiles of the isolated Bacillus and Paenibacillus strains provide advancement in the study of fermentation and may play
putative functions in the chitin bioconversion of sea crustacean foods.
Keywords: Bacillus sp., cellular fatty acids, chitinase, isozyme profile, Paenibacillus sp., salted and fermented shrimp,
16S rRNA gene

Practical Application: This is the 1st report for the isolation of chitinolytic Bacillus and Paenibacillus sp. from the Korean
traditional food, the jeotgal, made of salted and fermented shrimp (SFS), Acetes japonicus. The novel isolates available now
under Korean Collection for Type Cultures (KCTC) strains 33049 and 33051 could be beneficial for starter culture design
and preservation of SFS.

Introduction
Chitinase, the chitin degrading enzyme have been found distributed in organisms as diverse as fungi, plants, insects, crustaceans, and bacteria and is involved in the process of producing
mono- and oligosaccharides from chitin (Ajit and others 2006;
Song and others 2012). Chitinase-producing marine bacteria play
an important role in the degradation of chitin in the oceans
(Orikoshi and others 2005). Fungi and bacteria are thought to be
important degraders of chitin in soil and thereby contribute toward
the recycling of carbon and nitrogen resources in soil ecosystems.
In bacteria, the primary role of the chitinase is thought to be the
digestion and utilization of chitin as a carbon and energy source
(Cohen-Kupiec and Chet 1998).

MS 20131559 Submitted 10/28/2013, Accepted 1/7/2014. Authors K.-I. Han,

Kim, Kwon, and M.-D. Han are with Dept. of Biology, Soonchunhyang Univ., Asan,
Chungnam, 336-745, Republic of Korea. Authors Patnaik and Y.S. Han are with
Div. of Plant Biotechnology, College of Agriculture and Life Science, Chonnam Natl.
Univ., Gwangju, 500-757, Republic of Korea. Direct inquiries to author M.-D. Han
(E-mail: mdhan@sch.ac.kr).

R

C 2014 Institute of Food Technologists

doi: 10.1111/1750-3841.12387
Further reproduction without permission is prohibited

Chitinase genes have been cloned from diverse bacterial groups

(Shekhar and others 2006; Song and others 2012). Bacterial chitinase members have been subdivided into 3 groups (group A, B,
and C), based on the amino acid sequence similarity in the Cterminal catalytic domain (Suzuki and others 1999). They have a
size range of approximately 20 to 60 kDa and are typically smaller
than the plant (approximately 25 to 40 kDa) and insect chitinases
(approximately 40 to 85 kDa). Their stability over a wide range
of temperature (approximately 28 to 80 C) and pH (4.5 to 10),
make them excellent candidates for applications under different
conditions. The foremost application have been in inhibiting the
phytopathogenic fungal growth by disorganization of their cell
walls, serving as biocontrol agents in agriculture (Jung and others 2003). Transgenic technologies that include the expression of
bacterial chitinase genes into cereal crops have provided success in
resistance to common phytopathogenic fungal species (BarbozaCorona and others 2003). The biotechnological applications of
Bacillus thuringiensis for the control of pests and fungi have been
richly explored with the engineering of heterologous chitinase
genes from wide bacterial resources (Ramirez-Reyes and others
2004). Other major applications of bacterial and viral chitinases

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Kook-Il Han, Bharat Bhusan Patnaik, Yong Hyun Kim, Hyun-Jung Kwon, Yeon Soo Han, and Man-Deuk Han

Chitinase-rich isolates from shrimp food . . .

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have been their inherent property to dissolve the chitin-containing

cell wall (Barboza-Corona and others 2003; Oh and others 2013),
and the acceleration of protoplast generation leading to the development of economically viable strains for industrial use (Shimosaka
and others 2001). Furthermore, generation of bio-pharmaceutical
products as chitobiose and N-acetyl d-glucosamine by bacterial
chitinases has been seriously explored (Felse and Panda 2000).
Chitinase also play an important role in the bioconversion of shellfish waste to obtain value-added products (Wang and others 2006).
For the conversion of shrimp shell into commercially valued food
and chitosan products, it is imperative to isolate study chitinases
from high-salt inhabiting bacterial populations and their activity
in the degradation of marine crustaceans.
Salted and fermented food, either as an additive or as food in
itself, forms a delight in Korean cuisine. It is made by adding 20%
to 30% (w/w) salt to various types of seafood such as shrimp,
oyster, shellfish, fish, fish eggs, and fish intestines and becomes
palatable through subsequent preservation and fermentation. The
jeotgal is a traditional recipe from Korea that is made from tiny
shrimps (Acetes japonicus) and rock salt (Guan and others 2011).
Salted and fermented shrimp (SFS) constitutes chitin-rich food
that serves good protein and chitosan sources, due to the decomposition of shrimp shells and its body by chitinase-producing bacteria from oceans (Ghorbel-Bellaaj and others 2012; Halder and
others 2012). To gain rich insights into commercially valuable food
preservation industry, isolation of chitinase and efficient bacteria
from SFS is necessary. To our knowledge, there are no available
reports on identification of such bacterial isolates toward elucidating the microbial community dynamics and chitinase-producing
bacteria from SFS. In addition, such microbial chitinases could
provide for broad-spectrum applications in industrial and scientific
environments. An earlier report has isolated Paenibacillus tyraminigens sp. from Myeolchi-jeotgal, a traditional salted and fermented
anchovy (Engraulis japonicus), having high tyramine activity (Mah
and others 2008). In this study, phenotypic, molecular, and biochemical characterization of novel chitinase-rich bacterial strains
have been reported from salted and fermented food made with
small prawns (A. japonicus). These chitinases would be able to efficiently degrade shrimp shell to obtain useful chitosan for humans.
This is the 1st description for chitinase-producing Bacillus and
Paenibacillus sp. from Korean traditional food, jeotgal. The strains
SCH-1 and SCH-2 have been deposited to Korean Collection for
Type Cultures (KCTC) with No. 33049 and 33051, respectively.

Materials and Methods

Samples, culture, and isolation of chitinase-producing
bacterial strains
Fresh shrimp (A. japonicus), fermented with 20% to 25% salt for
12 wk at 15 C were collected, serially diluted, and spread on 0.5%
colloidal chitin marine agar (CCMA) (Difco, Mich., U.S.A.). The
composition of modified CCMA agar (per liter) was as follows:
peptone 5.0 g, yeast extract 1.0 g, ferric citrate 0.1 g, NaCl
19.45 g, MgCl2 8.8 g, Na2 SO4 3.24 g, CaCl2 1.8 g, KCl
0.55 g, NaHCO3 0.16 g, KBr 80 mg, SrCl2 34 mg, H3 BO3
22 mg, Na2 SiO3 4 mg, NaF 2.4 mg, NH4 NO3 1.6 mg,
Na2 HPO4 8 mg, 0.5% (w/v) colloidal chitin, 20 g agar at pH 7.0
(Roberts and Selitrennikoff 1988). After 5 d of incubation at 37 C,
the isolates capable of degrading chitin with distinct zone of clearance on CCMA were selected. All experiments for the enzymatic
tests were replicated 3 times. Typically, 5 different types of colonies
were collected from each plate based on differences in their morM666 Journal of Food Science r Vol. 79, Nr. 4, 2014

phological, biochemical, and genetic characteristics. The collected

colonies were selected by successive transfer on CCMA medium.

Morphological and phenotypic characterizations

The chitinase-rich bacterial isolates were treated according to
the procedure described by Weise and Rheinheimer (1978) and
Novitsky and MacSween (1989). Bacterial motility tests were
observed using a phase-contrast microscope. For morphological
characterization, the strains were cultivated for 24 h at 37 C
on marine agar medium and were subsequently prefixed in 2.5%
glutaraldehyde for 1 h. The prefixed samples were washed and
postfixed in 1% osmium tetroxide (pH 7.2, 0.1 M phosphate
buffer) solution for 90 min. Following postfixation, the samples
were dehydrated in a graded ethanol series (60%, 70%, 80%,
90%, and 100% with each change for 10 min), and subsequently
in hydroxymexamethyldisilazane. After drying, the grains were
attached to Scanning Electron Microscope stubs using doublesided conductive tape and sputter coated with gold. The samples
were examined using Hitachi S-4700 Field Emission Scanning
Electron Microscope (Hitachi High-Technologies Corp., Japan)
with an acceleration tension of 40 kV.
The bacterial isolates were phenotypically characterized by using the API 50CH system tests (bioMerieux, Inc, Hazelwood,
Mo., U.S.A.) as described by Logan and Berkeley (1984). The
API 50CH strips were inoculated with 2 McFarland standard suspensions of bacterial cells in CHB/E medium (bioMerieux, Inc,
Durham, N.C., U.S.A.) as recommended by the manufacturer
and incubated at 37 C for 2 d. Carbohydrate fermentation test
was performed as described previously (Wauters and others 1998).
Other phenotypic tests included catalase activity and the effect of
salinity on growth (Snibert and Krieg 1994).
Phylogenetic analysis of 16S rRNA sequences
Molecular procedures were carried out as described by
Sambrook and Rusell (2001). Genomic DNA of the isolates was
extracted using genomic DNA preparation Kit (SolGent, Daejeon,
Korea), according to manufacturers instructions. The 16S rRNA
gene was polymerase chain reaction (PCR)-amplified using the
universal primers, 27f (5-AGAGTTTGATCCTGGCTCAG-3)
and 1492r (5-GGTTACCTTGTTACGACTT-3) in an UNO
II Thermo cycler (Biometra, Gottingen, Germany). The PCR
reaction mixture consisted of the template DNA, 0.5 mM of
each primer, 1 U of Taq polymerase (SolGent, Daejeon, Korea),
100 mM dNTPs, and 2.5 mM MgCl2 . Samples were preheated
for 15 min at 95 C and then amplified for 30 cycles at 95 C
for 20 s, 50 C for 40 s, and 72 C for 90 s. Subsequent to PCR
amplification, 5 mL of each reaction was run on a 1% agarose
gel, and the DNA was visualized by UV illumination followed
with ethidium bromide staining. The amplified PCR products
were purified using the DNA clean up system (SolGent, Daejeon,
Korea) according to manufacturers instructions. DNA sequences
were determined directly from the purified PCR products with
automated fluorescent Taq cycle sequencing using an ABI 3730XL
DNA Analyzer (Applied Biosystems, Foster City, Calif., U.S.A.).
The primers for sequencing used in this study were 27f and 1492r
(Johnson 1994).
Small-subunit rRNA sequences of Bacillus and Paenibacillus
reference strains were obtained from GenBank and the Ribosomal Database Project (RDP; Maidak and others 1999). The
16S rRNA sequence similarities of the chitinase-rich new isolates
were inferred by comparison with other gene sequences of Bacillus
and Paenibacillus sp., using the Basic Local Alignment Search

Chitinase-rich isolates from shrimp food . . .

Whole-cell fatty acid (FAME) analyses

For fatty acid compositional analysis (Van der Velde and others 2006), the chitinase-rich isolates were grown on marine agar
(Difco, Becton, Dickinson Co., N.J., U.S.A.) and incubated for
24 h in an oxygen incubator. Approximately, 50 mg of the cell
mass were harvested and transferred to a Teflon-lined screw cap
tube (13 100 mm, Coning, Inc, Corning, N.Y., U.S.A.) using
inoculation loops. To release the fatty acids, 1 mL of saponification
reagent (sodium hydroxide 45 g, methanol 150 mL, distilled
water 150 mL) was added, and the tubes were heated at 100 C
for 5 min. The methyl ester formation was cooled and 2 mL of
methylation reagent (6N hydrochloric acid 325 mL, methanol
27 mL) was added. The mixture was then heated for 10 min
at 80 C and after cooling, the fatty acid methyl esters (FAMEs)
were extracted by adding 1.25 mL of extraction solvent (hexane
20 mL, methyl-tert butyl ether 200 mL). After mixing for
10 min, the aqueous layer was removed and the organic layer was
washed with 3 mL of base (sodium hydroxide 10.8 g, distilled water 900 mL). After mixing the samples for 5 min, the upper layer
was transferred to a gas chromatograph vial. A total of 2 L of the
FAMEs were then analyzed using a HP6890 gas chromatograph
(Agilent Technologies, Inc. Wilmington, Del., U.S.A.), with 5%
phenyl methyl silicone capillary column, 25 m 0.2 mm (Agilent Technologies, Diegem, Belgium) and Sherlock Microbial
Identification System version 6.1 (MIS, Microbial ID, Newark,
Del., U.S.A.). Sherlock MIS uses fatty acids of 9 to 20 carbons
in length. The peaks are automatically named and quantitated by
the system. The parameter settings of the gas chromatograph were
as follows: injection volume of 2 L, column split ratio 1:100,
injection port temperature 250 C, detector temperature 300 C,
column temperature 170 to 270 C at 5 C/min, and run time
of 22 min. Identification and comparison were made using the
Aerobe (TSBA version 3.9) database of the Sherlock MIS.
Growth kinetics
The cell growth was monitored at 620 nm using an UVspectrophotometer (Libra S12 UV spectrophotometer, Biochrom
Ltd, Cambridge, UK) to determine the temperature and pH range
of growth. Test strains were routinely grown using marine agar at
37 C, and maintained as a glycerol suspension (10%, w/v) at
80 C. The isolated strains were cultured at 37 C for 6 d in
colloidal chitin (CMB) medium (having 0.5% colloidal chitin).
Samples were collected daily for a period of 7 d for studying the
cell growth, pH, total protein, and chitinase enzyme activity.

Preparation of colloidal chitin

Colloidal chitin was prepared by partial hydrolysis of chitin
(Sigma-Aldrich, St. Louis, Mo., U.S.A.) using 10 N HCl and
left at 4 C overnight (Roberts and Selitrennikoff 1988). Subsequently, the mixture was added to 95% ethanol and kept at
20 C overnight. The precipitate was collected by centrifugation
at 4000 g for 20 min at 4 C. The colloidal chitin was washed
several times with sterile distilled water till pH 7.0. It was freezedried to powder and stored at 4 C. A total of 2% colloidal chitin
was used for the chitinase activity experiments.
Determination of chitinase activity
Chitinase assay mixture consisted of 0.6 mL of sample and
0.4 mL of 2% colloidal chitin in 50 mM sodium acetate buffer
(pH 5.0) (Monreal and Reese 1969). The reaction was maintained
at 37 C for 30 min. Subsequently, the mixture was heated in boiling water bath for 10 min and centrifuged at 10000 g for 1 min,
to remove the insoluble chitin. The resultant adduct of reducing
sugar was calculated using the dinitrosalicylic acid method (Miller
1959). The standard curve was generated from known concentrations of GlcNAc (0 to 100 g). One unit of chitinase activity was
defined as the amount of enzyme that released 1 mol of GlcNAc
per hour. Protein concentrations were measured using the Pierce
BCA assay kit (Pierce Biotechnology, Rockford, Ill., U.S.A.).
Activity staining of chitinase using gel electrophoresis
Bacillus sp. strain SCH-1 and Paenibacillus sp. strain SCH-2 were
incubated in marine agar medium containing 0.5% colloidal chitin
at 37 C for 4 d. To investigate the expression patterns of chitinase
isozymes, the culture media were loaded on 12% sodium dodecyl
sulfatepolyacrylamide gel electrophoresis (SDSPAGE) gels and
electrophoresis was conducted using a Bio-Rad Mini-PROTEAN
gel system (Bio-Rad Laboratories, Hercules, Calif., U.S.A.), according to the method described by Laemmli (1970). For assessing
the active staining of chitinase, a 12% SDSPAGE gel containing
0.01% glycol chitin was used (Trudel and Asselin 1989). After the
electrophoresis step, the gel was incubated in 100 mM sodium acetate buffer (pH 5.0), containing 1% (v/v) Triton X-100 and 1%
skim milk at 37 C for 2 h. This was followed with incubation under the same conditions, but without skim milk. Subsequently, the
gel was stained in a solution of 500 mM Tris-HCl (pH 8.9) containing 0.01% Calcofluor white M2R (Daihan Sci. Co., WGD-30,
Korea).

Results
Isolation of chitinase-producing bacterial strains
A total of 63 morphologically different chitinolytic bacterial
colonies were isolated from 10 samples of SFS. On the basis of
colloidal chitin degradation and zone of clearance (>0.2 cm) on
Colloidal Chitin Agar plates, 2 colonies were selected for secondary screening in broth media. A parallel assessment was conducted for the testing of enzyme activity. These potential isolates,
named as SCH-1 and SCH-2 had the maximum chitinolytic activity on agar medium containing 0.5% (w/v) colloidal chitin, showing clear zones around colonies. The morphological, biochemical,
genetic, and fatty acid analysis of the isolates were investigated.
Initial observations suggested a mixed culture; thus, cells were
separated by streaking them into marine agar plates and finally bacteria displaying 2 different colony morphologies were obtained.
Scanning electron microscopic investigations revealed the morphology of the isolated strains (Figure 1). No major differences
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Tool program at Natl. Center for Biotechnology Information.

Multiple-sequence alignment of 16S rRNA was conducted using
the Clustal W2 editor at www.ebi.ac.uk/tools/msa/clustalw2/.
The 5- and 3-gaps were edited using the BioEdit program
at (http://www.mbio.ncsu.edu/BioEdit/bioedit.html/). On the
basis of the molecular identification of the isolated strains SCH-1
and SCH-2, the nucleotide sequence information was submitted
to GenBank with accession nr. KC878876 and KC878877,
respectively.
Neighbor-joining (NJ) analysis (Saitou and Nei 1987) was performed using PHYLIP suite to find the phylogenetic positions
of the isolates. Evolutionary distances were calculated by using
the MEGA 5.0 Software (Tamura and others 2011) and bootstrap
analysis was used to evaluate the NJ tree topology, performing
1000 replicates and marked into branching points. The evolutionary distance matrix was estimated using the Kimuras 2-parameter
method (Kimura 1980).

Chitinase-rich isolates from shrimp food . . .

Table 1Taxonomic characteristics of the isolated chitinolytic Table 2Phenotypic characteristics of the isolated chitinolytic
strains from salted and fermented shrimp.
strains from salted and fermented shrimp.
Bacillus sp. SCH-1
Colony shape
Colony color
Shape of cell
Cell size (m)
Motility
Gram stain

Round
Cream
Rod
(2.77 to 3.69) (1.23
to 1.36)

Paenibacillus sp.
SCH-2
Round
Cream
Rod
(3.38 to 3.76) (0.63
to 0.66)
+
+

Catalase
Oxidase
Methyl red
Voges proskauer
Gelatin hydrolysis
Litmus
Nitrate reduction
Salt tolerance (%)

Bacillus sp. SCH-1

Paenibacillus sp. SCH-2

+
+

+
+
Stormy fermentation

+
+

Alkaline reaction

6.5

were observed between SCH-1 and SCH-2 isolates, with respect

to colony shape and color, although the rod-shaped cells of SCH2 isolate were longer in size as compared with SCH-1 isolate
(Table 1; Figure 1A and 1B). SCH-2 isolate showed peritrichous
flagellation and was motile (Figure 1C and 1D). CCMA agar plates
were the sole carbon source and after incubation for 7 d at 37 C
on the medium, the strain colonies turned into circular form.

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lase, methyl red, Vogesproskauer tests with alkaline reaction in the

litmus milk reaction test, although it was negative with the oxidase,
gelatin hydrolysis, and nitrate reduction tests. Furthermore, biochemical characteristics of the isolated strains were complemented
with studies on the utilization of a variety of substrates as carbon
source. The strain SCH-1 (Bacillus sp.) was able to utilize glucose, N-acetylglucosamine, esculin, and maltose as a substrate for
their growth. The SCH-2 strain (Paenibacillus sp.) showed propensity to utilize a variety of carbon sources for its growth including
Phenotypic characterization of the isolates
In further investigating the identity of the isolates, we con- arabinose, glucose, N-acetylglucosamine, starch, and other sugars
ducted a complete series of biochemical tests that includes catalase (Table 3).
test, oxidase test, methyl red test, VogesProskauer test, gelatin
liquefaction test, nitrate reduction test, and litmus milk reaction Molecular identity and phylogenetics of the isolates
(Table 2). The isolate Bacillus sp. SCH-1 showed a positive reMolecular identification of the isolated strains showing chitisponse with catalase, oxidase, Vogesproskauer, and gelatin hy- nolytic activity was carried out based on 16S rDNA sequence
drolysis test with stormy fermentation in the litmus milk reaction analysis. The nearly complete 16S rDNA sequence for SCH-1
and showed negative response to methyl red and nitrate reduction (1450 bases; GenBank accession nr. KC878876) and SCH-2 strains
tests. The isolate Paenibacillus sp. SCH-2 was positive in the cata- (1453 bases; GenBank accession nr. KC878877) were determined.

Figure 1Scanning electron micrographs of the isolated chitinase-producing strains SCH-1 (A and B) and SCH-2 (C and D).

M668 Journal of Food Science r Vol. 79, Nr. 4, 2014

Chitinase-rich isolates from shrimp food . . .

Carbohydrates
Erythritol
d-Arabinose
l-Arabinose
Ribose
d-Xylose
l-Xylose
Adonitol
Methyl--d-xylopyranoside
Galactose
Glucose
Fructose
Mannose
Sorbose
Rhamnose
Dulcitol
Inositol
Mannitol
Sorbitol
Methyl-, d-mannopyranoside
Methyl-, d-glucoside
N-Acetyl-glucosamine
Amygdalin
Arbutin
Esculin

Bacillus sp. SCH-1

Paenibacillus sp. SCH-2

Carbohydrates

Bacillus sp. SCH-1

Paenibacillus sp. SCH-2

+
+
+
+

+
+
+
+
+

+
+
+
+
+

Salicin
Cellobiose
Maltose
Lactose
Melibiose
Sucrose
Trehalose
Inulin
Melezitose
Raffinose
Starch
Glycogen
Xylitol
Gentiobiose
d-Turanose
d-Lyxose
d-Tagatose
d-Fucose
l-Fucose
d-Arabitol
l-Arabitol
Gluconate
2-keto-Gluconate
5-keto-Gluconate

+
+
+
+
+
+
+

+
+
+
+

+
+

+, positive; , negative.

The sequence match option from the RDP and Basic Local Alignment Search Tool analysis of the GenBank was used to identify
the most similar sequences available in the database. Phylogenetic
trees based on the 16S rDNA sequence data of SCH-1 and SCH-2
strains were constructed (Figure 2). The tree topology inferred
that the SCH-1 strain belonged to the genus Bacillus, whereas
the SCH-2 strain was classified within the Paenibacillus genus.
Figure 2A shows the relationship between strain SCH-1 and representatives of the genus Bacillus. Strain SCH-1 closely resembled
its nearest neighbors, Bacillus cereus ATCC 14579 (NR_074540),
Bacillus thuringiensis IAM 12077 (NR_043403), and Bacillus
weihenstephanensis DSM 11821 (NR_024697) with sequence similarities of 97.83%, 97.69%, and 97.48% respectively. Strain SCH-2
formed a highly significant clade with the members of the genus
Paenibacillus, and closely resembled Paenibacillus lautus JCM 9073
(NR_040882) with a sequence similarity of 99.16% (Figure 2B).
The SCH-2 strain also has about 97.57% and 96.81% similarity
with Paenibacillus glucanolyticus DSM 5162 and Paenibacillus lactis
MB1871, respectively. The Paenibacillus clade is also supported by
a high bootstrap value of 95%. On the basis of pairwise 16S rDNA
gene similarities, it was evident that chitinolytic strains SCH-1 and
SCH-2 represent novel genomic species in the genus Bacillus and
Paenibacillus, respectively. This is the 1st report of identification of
novel Bacillus and Paenibacillus strains showing chitinase activity;
isolated from the Korean traditional foodthe jeotgal.
Hence, the morphological, physio-biochemical and molecular
evidence presented in the study, classifies the strains SCH-1 and
SCH-2 to be belonging to the genus Bacillus and Paenibacillus,
respectively.

Cellular fatty acid analysis of the strains

The cellular fatty acids composition in both the aerobic,
endospore-forming, rod-shaped strains isolated from SFS were
determined using the gas chromatograph. Table 4 lists the fatty
acid compositions of the isolates, and the reference type strains (B.

cereus JCM 2152 and P. lautus NRRL NRS-666). Most importantly, the fatty acids profile for both SCH-1 and SCH-2 strain
shows saturated iso- and anteiso-methyl-branched fatty acids. The
most often encountered fatty acids and with structures of this type
have 14 to 18 carbons in the chain are common constituents in
bacteria. Four fatty acids were present in abundant amounts in the
gas chromatograms profile of SCH-1 strain: iso-C15:0 , iso-C16:0 ,
C16:0 , and iso-C17:0 fatty acid. The predominant cellular fatty acid
of the Bacillus strain SCH-1 was the 15:0 iso fatty acid (31.92%),
followed with the 16:0 iso fatty acid (16.31%). The most abundant
fatty acid in the Paenibacillus strain SCH-2 was the C15:0 anteiso
fatty acid (39.39%), followed by C16:0 iso fatty acid (16.79%). This
is indicative of the genus Paenibacillus (Shida and others 1997). The
fatty acids iso-C12:0 , iso-C13:0 , anteiso-C13:0 , anteiso A-C17:0 , and
iso-C17:1 w5c were not found in the gas chromatograph profile
of the Paenibacillus SCH-2 strain. These were detected in a lower
significant percentage in the Bacillus SCH-1 strain. The unsaturated fatty acids were found in trace amounts (>0.2%) in both
the strains. The differences in the levels of the C14:0 , iso-C15:0 ,
anteiso-C15:0 , iso-C16:0 , and C16:0 were sufficient to differentiate
the 2 novel strains.

Growth kinetics and chitinase activity

The isolated strainsBacillus sp. SCH-1 and Paenibacillus sp.
SCH-2- were grown aerobically in CMB medium (0.5% colloidal
chitin) at 37 C for 6 d to assess the cell growth, optimum pH
conditions, total protein content, and the chitinase activity. The
time course profile for the cell growth, pH, and total protein
content for Bacillus sp. SCH-1 and Paenibacillus sp. SCH-2 has
been depicted in Figure 3A and 3B, respectively. The Bacillus
strain SCH-1 grew rapidly for 2 d and subsequently showed a
marginal decline toward the end of the cultivation time. The pH
of the medium for the optimal growth of the strain was about 6.0
to 7.0. In the same medium, the protein content was found to
get declined as the growth rates of the strain increased. At 2 d of
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Table 3Physiological and biochemical characteristics of the isolates Bacillus sp. SCH-1 and Paenibacillus sp. SCH-2 based on the
API 50 CH system and additional tests described in Materials and Methods section.

Chitinase-rich isolates from shrimp food . . .

cultivation time, the total protein declined appreciably from the
start of the culture. In case of the Paenibacillus strain SCH-2, the
cell growth was high till 1 d of cultivation but thereafter showed a
steady decline till 2 d of cultivation, followed by a marked decline
at the later stages of cultivation. The optimal pH for growth of
the strain was 6.0 to 7.0. The total protein content of the medium
decreased with the cultivation time of Paenibacillus sp.
The chitinase activity of the Bacillus sp. strain SCH-1 increased
along with the cell growth and reached maximum (12.52 unit/mg
protein) when the cell growth reached stationary phase at about 4 d
of incubation. The chitinase activity for the Paenibacillus sp. SCH2 strain also increased with the cell growth and reached maximum
(5.12 unit/mg protein), when the cell growth reached death phase
at 4 d of incubation (Figure 4). The chitinase isozymes in chitin
medium showed 2 bands at 41 and 50 kDa for the Bacillus sp. strain
SCH-1. In contrast we observed 4 chitinase isozymic bands with
sizes of 30, 37, 45.7, and 50 kDa with the Paenibacillus sp. SCH-2
(Figure 5).

Discussion

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Sixty-three chitinase-producing bacterial colonies were isolated

from traditional Korean SFS on the selective medium containing colloidal chitin. Strains KC878876 (SCH-1) and KC878877
(SCH-2) were selected for further study of chitinase activity as
they formed the largest clear zone on the chitin agar plate. We
confirmed the strains by studying their morphological, biochemical, and 16S rRNA-based molecular characteristics. Strain SCH-1

was identified as Bacillus sp. and strain SCH-2 was identified as

Paenibacillus sp. It is known that the Gram-positive bacterial genus
Bacillus secretes a number of degradative enzymes (Schallmey and
others 2004). Therefore, it was not surprising to isolate chitinaserich proteolytic strains of Bacillus in our study. Also in agreement
to our study, a Gram-positive, rod-shaped, endospore-forming
strain of P. tyraminigens sp. has been characterized from traditional
Korean salted and fermented anchovy (E. japonicus) (Mah and
others 2008). It is expected that SFS includes other halotolerant
and/or halophilic bacteria along with the spore-forming bacteria. But the chitin degrading activity of other bacterial isolates in
this study was not considered to be significant. Some earlier reports have isolated Salimicrobiums and Lactobacillus from the salted
and fermented anchovy (Lee and others 2012; Belfiore and others
2013). Paenibacillus chitinolyticus strain MP-306 has been isolated
from the cast-off shells of cicads with strong chitinase activity
(Song and others 2012). A proteolytic but chitinase-deficient microbial culture has been isolated from shrimp shell waste that was
characterized as Bacillus licheniformis (Waldeck and others 2006).
Apart from the above, chitinase-producing marine bacteria have
been isolated that play important roles in degradation of chitin in
oceans (Annamalai and others 2010, 2011).
Physiological tests (API) (bioMerieux, Inc., Hazelwood, Mo.,
U.S.A.), 16S rRNA gene sequence comparison (Goto and others 2000), as well as microscopic and macroscopic investigations,
revealed differences between the bacterial isolates SCH-1 and
SCH-2. Our observations can be considered reliable, as we have

Figure 2Phylogenetic tree based on 16S rRNA sequence showing the position of strains SCH-1 (A) and SCH-2 (B) and their relationships within the
Bacillus and Paenibacillus group. The tree was constructed using the neighbor-joining method with Kimura 2-parameter distance matrix and pairwise
deletion. Numbers at nodes represent the bootstrap percentage (based on 1000 replicates). The scale bar indicates 0.005 substitutions per nucleotide
position.

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Chitinase-rich isolates from shrimp food . . .

Table 4Fatty acid composition of the isolates Bacillus sp. SCH-1 and Paenibacillus sp. SCH-2 and the reference test species (Bacillus
cereus JCM 2152T and Paenibacillus lautus NRRL NRS-666T ).
Fatty acid composition (%)

Bacillus sp. SCH-1

Bacillus cereus JCM 2152T

Paenibacillus sp. SCH-2

Paenibacillus lautus NRRL NRS-666T

C13:0
C14:0
C15:0
C16:0
C17:0
C18:0
C16:1 w7c alcohol
C16:1 w9c
C16:1 w11c
iso-C12:0
iso-C13:0
anteiso-C13:0
iso-C14:0
iso-C15:0
anteiso-C15:0
iso-C16:0
anteiso A-C17:1
iso-C17:0
anteiso-C17:0
iso-C17:1 w5c
iso-C17:1 w10c
iso-C18:0

Tr
3.62
ND
11.22
0.46
Tr

0.8
3.1
4.9
2.4
Tr
Tr
Unsaturated acids

Tr
1.08
ND
15.67
0.27
0.37

Tr
1.1
0.3
15.6
Tr
Tr

Tr
Tr
Tr

Tr
1.1
4.4
Branched acids

Tr
Tr
Tr

Tr
Tr
2.0

Tr
Tr
Tr
2.03
7.20
39.39
16.79
Tr
6.03
10.88
Tr
Tr
0.29

Tr
Tr
Tr
0.8
1.5
57.3
7.4
Tr
1.2
9.7
Tr
0.2
ND

0.75
2.25
0.67
3.90
31.92
4.06
16.31
0.85
10.03
3.33
1.04
Tr
Tr

Tr
7.8
0.6
2.4
48.7
3.8
2.7
Tr
6.2
0.7
Tr
2.8
ND

Values are percentages of total fatty acids.

Tr, trace (<0.2%); ND, not detected.

Figure 3Time course of cell growth, pH, and total protein

in a defined culture (CMB) medium. (A) Bacillus sp. SCH-1
and (B) Paenibacillus sp. SCH-2. 0.1% seed culture was
transferred into the CMB medium and further grown at
37 C in a shaker at 100 rpm. () Cell growth (OD620 ); ()
pH; (); total protein (g/mL).

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Saturated acids

Chitinase-rich isolates from shrimp food . . .

conducted 16S rRNA gene sequence comparisons in conjunction with a number of phenotypic and phylogenetic properties.
It is important to note that 2 distinct species may have identical
16S rRNA gene sequences, and therefore are unreliable without
other evidences (Stackerbrandt and others 2002).
The SCH-1 strain has shown highest homology with B.
cereus ATCC 14579 (NR_074540) and also substantially high
relatedness with other Bacillus sp. B. cereus is a Gram-positive,

facultative anaerobic rod-shaped bacterium seen ubiquitously in

soil and also found in many raw and processed foods such as
rice, milk, dairy products, spices, and vegetables (de Vries and
others 2004). The mixed culture consisting of 2 B. licheniformis
strains, isolated from shrimp shell waste were found to encode a
frameshift mutated chitinase, thus showing the absence of chitinolytic activities (Waldeck and others 2006). Phylogenetic analysis
for SCH-2 strain revealed its existence within the Paenibacillus
Figure 4Determination of chitinase activity by Bacillus sp.
SCH-1 and Paenibacillus sp. SCH-2 cultivated in CMB medium
at 37 C for 6 d. () Bacillus sp. SCH-1; () Paenibacillus sp.
SCH-2.

M: Food Microbiology
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Figure 5Chitinase activity staining of Bacillus
sp. strain SCH-1 and Paenibacillus strain
SCH-2 on SDSPAGE after M2R staining. The
bacterial isolates were grown in marine agar
medium containing 0.5% colloidal chitin.

M672 Journal of Food Science r Vol. 79, Nr. 4, 2014

clade and showed highest 16S rRNA gene similarities with P.

lautus strain JCM 9073 (NR_040882). P. lautus JCM 9073 have
been isolated from sources that include soil, water, plant rhizosphere, plant materials, food, and fodder (Daane and others 2002;
Lee and others 2013). The P. tyraminigens strain earlier reported
from E. japonicus also formed a highly significant monophyletic
clade with the members of the genus Paenibacillus (Mah and others 2008). The bootstrap value (95%) shown was in agreement
with our study. The genus Paenibacillus is phenotypically related
to the Bacillus, and is generally characterized as rod-shaped and
endospore-forming bacteria with DNA G+C content of 39 to 54
mol% (Saha and others 2005).
Cellular fatty acid analysis falls under the category chemotaxonomy and has been applied for the bacterial identification process.
We utilize the gas chromatograph to understand fatty acid profile
of both the chitinolytic strains. The major cellular fatty acid was
iso-C15:0 in Bacillus sp. and the anteiso-C15:0 in Paenibacillus sp. We
also found other iso- and anteiso- forms of branched cellular fatty
acids. As per the literature, the composition of Bacillus sp. seems
to be complex with typically 7 or more iso- or anteiso- forms
(Ehrhardt and others 2010). In case of B. cereus, large amounts
of C16:0 , iso-C15:0 , iso-C15:0 , iso-C17:0 , iso-C14:0 , C15:0 , anteisoC15:0 , and anteiso-C13:0 fatty acids are found (Lawrence and others 1991). Bacillus strain SCH-1 characterized in our study also
showed a higher proportion of C16:0 iso and iso-C17:0 that agrees
to the available report. Chromatographic analysis of FAMEs has
also been developed that gives an added advantage, as over 1500
species of bacteria, including B. anthrax, used as potential agents in
biological terrorism have been identified (Teska and others 2001).
This was attributed to the unique cellular fatty acid profile of B.
anthracis that distinguished it from other related Bacillus sp. The
anteiso-C15:0 fatty acid was also the predominant fatty acid in the
Paenibacillus sp. isolated from salted and fermented anchovy that is
very relevant to our findings (Mah and others 2008). The anteisoC15:0 value was lower at 39.39% in our study, although it can be
useful to differentiate the bacterial strains.
We also made a study on the cell growth characteristics and pH
development of the 2 chitinolytic strainsSCH-1 and SCH-2isolated from our SFS samples. The total protein in the media
at the start of the cultivation period and its utilization for cell
growth has also been observed. Both the Bacillus and Paenibacillus
sp. cell growth increased rapidly for 2 d and then maintained
in the medium although at a lower level. Similar observations
have been recorded for P. chitinolyticus grown in Luria Bertani
(LB) medium with 0.5% colloidal chitin (Song and others 2012).
The pH of the culture dropped during the exponential phase
of growth, but subsequently increased, although the cell growth
declined over the cultivation period. This decrease in pH can be
attributed to the fermentation of glucose as subsequently when the
glucose level depleted, the pH of the culture started rising again.
Upon the substrate and protein exhaustion from the medium, the
cells entered the stationary phase and may form aggregates. Cell
aggregation is a special event within the B. cereus group and may
play a role in the transfer of genetic material (Helgason and others
2000). The growth characteristics and the sporulation process in
B. cereus ATCC 14579 have been extensively studied (de Vries and
others 2004).
Chitinase activity of Bacillus strain SCH-1 and Paenibacillus strain
SCH-2 increased rapidly and was maximum at about 4 d of cultivation in colloidal chitin medium. The trend of chitinase activity for
both the isolated strains were found similar, although Bacillus strain
SCH-1 would have a stronger chitinolytic activity than the Paeni-

bacillus strain SCH-2. It is known that under lower environmental pH and depleted protein, the cell growth stopped increasing.
With a subsequent upsurge in pH, a sharp increment of chitinase secretion is observed, indicating an alteration to secondary
metabolism (Yan and others 2011). The reasons for decreased production may be the lack of nutrients in the medium resulting in
the inactivation of the enzyme synthesis machinery (Nochur and
others 1993). Furthermore, according to the adaptations (alkaline
and acidic conditions) of the isolated strains, the difference in the
chitinase enzyme activity is noticed. Bacillus sp. K2914 showed
optimal chitinase production in 0.5% concentration of colloidal
chitin and crustacean waste powder in combination after 8 d of
cultivation (Uria and others 2005). Paenibacillus sp. D1 showed the
highest chitinase activity in pH 5.0 at 50 C (Singh and Chhatpar 2011). Previous reports have suggested that B. laterosporous
(Shanmugaiah and others 2008) and Aeromonas punctata (Kuddus
and Ahmed 2013) are capable for producing highest chitinase at
alkaline conditions. It is known that chitinases from B. cereus provide the best activity at an acidic or near neutral pH (Wang and
others, 2001; Chang and others 2003). Chitinase activity in B.
cereus SV1 was found maximum at 55 C and pH 7.0 in media containing shrimp shell powder (Ghorbel-Bellaaj and others
2012). In an earlier report, chitinases produced from B. amyloliquefaciens have been reported to show a pH optimum close to 7
(Wang and others 2002). Other bacterial chitinases isolated from
marine sources, show broader pH optima, with maximal activity
in neutral or slightly alkaline conditions (Wang and Chang 1997;
Park and others 2000). In our study on the chitinase isozymes, we
observed 4 bands of chitinase from Paenibacillus strain, in contrast
to 2 bands from Bacillus strain isolated in this study. Recent report has suggested differential expression patterns of P. chitinolyticus
MP-306 chitinase isozymes in both colloidal chitin and LB media.
Three chitinase isozyme bands were observed irrespective of the
medium in Native-gel (Song and others 2012). Three chitinase
isozymes (63, 54, and 38 kDa) were observed in a SDSPAGE
gel for P. illinoisensis KJA-424 having antifungal activity (Jung and
others 2005).

Conclusions
We have noticed that in recent years scientific thrust on chitinase
purification and production has led to exploring the Korean traditional fermented food, including the vegetable kimchi, soybean
paste and most recently the salted and beneficial microorganisms.
The results of this study, advances our knowledge on the isolated bacterial strains having high chitinase activity from SFS. This
would be extremely useful in the development of starter culture
design and preservation of SFS. These novel identified bacterial
isolates would be useful in improving the traditional fermented
food.

Acknowledgments
This work was supported in part by the Soonchunhyang Univ.
Research Fund.

Author Contributions
Kook-Il Han performed the experiments, analyzed the data, and
wrote the paper. Bharat Bhusan Patnaik wrote the paper and analyzed the data. Yong Hyun Kim and Hyun-Jung Kwon performed
the experiments. Yeon Soo Han provided technical details. ManDeuk Han designed the experiments, analyzed the data, provided
technical details, and wrote the paper.
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Chitinase-rich isolates from shrimp food . . .

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