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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).
R
C 2014 Institute of Food Technologists
doi: 10.1111/1750-3841.12387
Further reproduction without permission is prohibited
M: Food Microbiology
& Safety
Kook-Il Han, Bharat Bhusan Patnaik, Yong Hyun Kim, Hyun-Jung Kwon, Yeon Soo Han, and Man-Deuk Han
M: Food Microbiology
& Safety
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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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 (%)
+
+
+
+
Stormy fermentation
+
+
Alkaline reaction
6.5
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Figure 1Scanning electron micrographs of the isolated chitinase-producing strains SCH-1 (A and B) and SCH-2 (C and D).
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
Carbohydrates
+
+
+
+
+
+
+
+
+
+
+
+
+
+
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.
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.
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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.
Discussion
M: Food Microbiology
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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.
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
M: Food Microbiology
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Saturated acids
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.
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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References
M: Food Microbiology
& Safety