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 bacteria—SCH-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: [email protected]).

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 (bioM´erieux, 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 (bioM´erieux, 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 manufacturer’s 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 manufacturer’s 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 (ø13 × 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 μL 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 μL, 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 μg). One unit of chitinase activity was defined as the amount of enzyme that released 1 μmol 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 sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) 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% SDS–PAGE 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 Vol. 79, Nr. 4, 2014 r Journal of Food Science M667

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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 Kimura’s 2-parameter method (Kimura 1980).

Chitinase-rich isolates from shrimp food . . . Table 1–Taxonomic characteristics of the isolated chitinolytic Table 2–Phenotypic 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 (μm) 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 − 7

+ − + + − 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, Voges–proskauer 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, Voges–Proskauer 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, Voges–proskauer, 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 1–Scanning electron micrographs of the isolated chitinase-producing strains SCH-1 (A and B) and SCH-2 (C and D).

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Chitinase-rich isolates from shrimp food . . .

Carbohydrates Erythritol d-Arabinose l-Arabinose Ribose d-Xylose l-Xylose Adonitol Methyl-β-d-xylopyranoside Galactose Glucose Fructose Mannose Sorbose Rhamnose Dulcitol Inositol Mannitol Sorbitol Methyl-α, d-mannopyranoside Methyl-α, 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 food—the 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 strains—Bacillus 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 Vol. 79, Nr. 4, 2014 r Journal of Food Science M669

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Table 3–Physiological 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 2–Phylogenetic 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 4–Fatty 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 (

Isolation and characterization of chitinase-producing Bacillus and Paenibacillus strains from salted and fermented shrimp, Acetes japonicus.

Chitinases catalyze the conversion of chitin and are produced by a wide range of bacteria. The biological applications of these enzymes have been expl...
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