Ekachai Chukeatirote， Novi Arfarita，， Piyanuch Niamsup， and Anittaya Kanghae

1School of Science， Mae Fah Luang University， Chiang Rai 57100， Thailand

2Faculty of Agriculture， Brawijaya University， Malang 65145， Indonesia

3Faculty of Science， Maejo University， Chiang Mai 50290， Thailand

Phenotypic and Genetic Characterization of Bacillus Species Exhibiting Strong Proteolytic Activity Ιsolated from Terasi， An Ιndonesian Fermented Seafood Product

Ekachai Chukeatirote1， Novi Arfarita1，2， Piyanuch Niamsup3， and Anittaya Kanghae1

1School of Science， Mae Fah Luang University， Chiang Rai 57100， Thailand

2Faculty of Agriculture， Brawijaya University， Malang 65145， Indonesia

3Faculty of Science， Maejo University， Chiang Mai 50290， Thailand

In this study， two bacilli strains namely S2-3 and S4-5， isolated from Terasi， a traditional fermented seafood product of Indonesia， were studied in terms of their phenotypic and genotypic properties. Both strains are of great interests due to their high proteolytic activity. Initially， they were subjected to morphological determination and a series of biochemical tests. These bacteria were Gram-positive， endospore-forming bacilli. Based on 16S rRNA gene sequence analysis， the identities of the strains S2-3 and S4-5 were confirmed as Bacillus thuringiensis and B. subtilis， respectively. Additionally， the two strains were also evaluated for their antibiogram profiles. It was found that they were susceptible to chloramphenicol， erythromycin， kanamycin， tetracycline and vancomycin and resistant to ampicillin and intermediately susceptible to bacitracin.

Bacillus， fermented seafood， proteolytic activity， Terasi

Ιntroduction

Terasi， an indigenous fermented seafood product of Indonesia， is made from fish and/or shrimp. The product is popular and widely used as condiments in various local dishes （Surono and Hosono， 1994a）. Traditionally， Terasi is practically produced using underutilised fish and/or shrimp. Typical production process consists of Ⅰ） preparation of raw materials； Ⅱ） addition of salt （at high concentration）； andⅢ） natural fermentation under drying condition （often by the means of sunlight exposure）. The fermentation time is between 1 and 4 weeks； the fermented products can then be packed for sales （Surono and Hosono，1994a）. Various forms of Terasi are present because of difference in used raw materials， fermentationcondition， and fermenting microbiota. The common colour of Terasi is either dark brown， grey or red. Terasi has a distinct taste and strong aroma. Similar products are found in several countries including Bagoong （Philippines）， Belachan （Malaysia）， Kapi（Thailand）， and Ngapi （Myanmar）.

Considering that these products are diverse and important in social culture of people in many Asian countries， scientific literature dealing with these foods is scant. For Terasi in particular， some previous studies have been performed generating the data of its nutritional data （Surono and Hosono， 1994a）， and microflora （Surono and Hosono， 1994a， b； Kobayashi et al.， 2003； Setyorini et al.， 2006）. Terasi's research on microflora presents a diverse group of bacteria， including those in the genera Bacillus， Kurthia， Micrococcus，Pseudomonas， Sporolactobacillus， and Tetragenoco-ccus （Surono and Hosono， 1994a， b； Kobayashi et al.，2003； Setyorini et al.， 2006）. Bacillus species， known to be involved in several fermented food products， also appear to be one of the predominant bacterial groups isolated from Terasi. In this study， we further documented the presence of bacterial community isolated from Terasi focusing on those capable of producing extracellular protease enzymes. Two isolates named S2-3 and S4-5 showing high proteolytic activities were subsequently characterised and reported.

Materials and Methods

Terasi samples and bacterial strains

Four Terasi samples were used in this study. Samples A and B were from Cirebon （north coast of West Java）； sample C from Malang （East Java）； and sample D from Tuban （north coast of East Java）. Bacillus strains used in this study were isolated from Terasi.

Screening and isolation of protease-producing bacteria

Ten grams of Terasi were suspended in 90 mL of sterile 0.1% peptone and homogenised in a stomacher. The suspension （1 mL） was then serially diluted in 9 mL of sterile peptone and suitable decimal solutions（0.1 mL） were spreaded on casein agar. After incubating at 37℃ for 24 h， the colonies exhibiting clear zones were selected and subcultured to obtain the pure cultures. The presence of a clear zone was used to indicate the bacterial ability to produce protease enzymes.

Subsequently， these bacterial strains were screened for their proteolytic activities. For this， a single colony of these bacteria was inoculated into a test tube containing 3 mL of nutrient broth containing 1% peptone，0.5% beef extract， and 0.5% NaCl. The culture was then grown at 37℃ for 24 h. Fifty microlitres of the overnight culture was transferred to a new test tube containing 3 mL of the nutrient broth and the culture was further incubated at 37℃ for 24 h. After incubation， the bacterial cells were harvested at 14 000 r · min-1for 15 min at 4℃. The supernatant was collected for further use as the source of the protease enzymes and thus referred to the "crude extract". For an assay， 10 µL of the crude extract were used to spot on a skim milk agar plate （containing 0.5% peptone，0.3% beef extract， 0.5% skim milk， and 1.5% agar）. The inoculated plate was then incubated at 37℃ for 24 h. The presence of a clear zone was recorded. In addition， further investigation was performed using two different protein-based media: skim milk agar（1% skim milk， 2% agar） and nutrient agar supplement with 1% gelatin. For this， the selected bacterial isolates were cultured and their supernatants were used to determine the proteolytic activity as mentioned. The proteolytic activity assay was carried out at least three times. The value of the relative index of the protease activity was also calculated， based on the ratio between the diameter of the clear zone and that of the bacterial colony.

Morphology and biochemical profiles

The selected bacterial strains were characterised by morphological， and biochemical properties. These included Gram-staining， presence of spore， oxygen requirement， catalase test， lecithinase test， ability to growth in 5% and 7% NaCl， growth at 50℃ and 65℃，IMViC test， nitrate reduction， fermentation of glucose，arabinose， xylose and sucrose， and starch hydrolysis（Slepecky and Hemphill， 1992）. In addition， the bacterial species identification was performed using API-50 CHB kit （bioMerieux， Inc.）.

Molecular characterisation

Genomic DNA was isolated from the bacteria based on the standard protocol of Sambrook and Green （2012）. 16S rRNA genes were then amplified using primers 27F （5'-AGA GTT TGA TCC TGG CTC AG-3'）and 520R （5'-ACC GCG GCK GCT GGC-3'） （Lane，1991）. The polymerase chain reaction was performed in a 50 µL reaction consisting of 5 µL 10X buffer，3 µL 25 mmol · L-1MgCl2， 5 µL 2 mmol · L-1dNTPs，1 µL Taq polymerase （Promega）， 1 μL of each primer（1 pmol）， 33 μL of sterile water， and 1 μL of 500ng · μL-1bacterial DNA. Amplification protocol consisted of an initial denaturation at 94℃ for 5 min，followed by 25 cycles of 94℃ for 1 min， 55℃ for 1 min， and 72℃ for 1 min， and a final extension at 72℃ for 5 min. The amplified products were electrophoresed in 1.5% agarose gel， and purified using TaKaRa SUPRECTM-PCR （TaKaRa， Japan）. The purified products were then sent to 1st Base Laboratories Company （Malaysia） for sequencing. The sequencing data were analyzed using BLAST （Altschul et al.，1990） and the closest known species were determined based on the percentages of sequence similarity. The accession numbers of Bacillus isolates used were as follows: FJ217159 and FJ217160 for strains S2-3 and S4-5， respectively. Sequence alignment and phylogenetic analysis were then carried out using the Phylogeny.fr software （Dereeper et al.， 2010）.

Susceptibility tests

Antimicrobial susceptibility was performed using the disc agar diffusion method （NCCLS， 1997）. Initially，a bacterial culture was prepared in 5 mL of Mueller-Hinton broth and incubated at 37℃ for 24 h with shaking 150 r · min-1. A sterile cotton swab was dipped into the inoculum （0.5 MacFarland） and applied on the surface of the agar plate. After drying， the antibiotic discs （Oxoid， England） were placed and the plates were incubated at 37℃ for 24 h. The zones were measured and expressed as sensitive， intermediate，and resistance as described by Hong et al （2008）. Antibiotic discs tested included vancomycin （30 µg），tetracycline （30 µg）， chloramphenicol （30 µg）， erythromycin （15 µg）， kanamycin （30 µg）， ampicillin （30 µg），bacitracin （10 U）， and streptomycin （10 µg）.

Results and Discussion

Proteolytic activity of Terasi bacteria

In this study， 117 bacterial strains were randomly isolated from four Terasi samples and screened for their protease productions using skim milk agar （Table 1）. The initial data revealed that， of 117 bacterial strains tested， 71 isolates （ca. 61%） were able to produce the protease enzymes as indicated by the presence of the clear zones. It was also evident that these bacteria had different proteolytic activities. The relative index values were calculated and used to group these protease-producing bacteria （Table 2）. The majority of the testing bacteria （42 isolates representing ca. 59%） had the relative index values between 1.01 and 1.50. Microflora of Terasi were studied and composed of a diverse group of bacteria（Surono and Hosono， 1994a， b）. Terasi is unique considered thatⅠ） its raw material is derived from shrimp and/or fish， and Ⅱ） its production process has a high salt concentration. These two distinct characteristics are of great interests and thus are major issues for scientific study. Setyorini et al.（2006） described halotolerant proteases obtained from Bacillus subtilis strain FP-133. Two Tetragenococcus species were also isolated from Terasi and described as halophilic lactic acid bacteria （Kobayahsi et al.，2003）. Some enzymatic profiles of Terasi bacteria were determined and most bacteria had a high activity of esterase and lipase enzymes （Surono and Hosono，1994b）. This study was further documented the bacterial diversity of Terasi focusing on those capable of producing the protease enzyme. As shown in Table 3， two isolates namely S2-3 and S4-5 exhibiting strong proteolytic activity on both skim milk agar and nutrient gelatin agar as shown by their relative index values were selected for further experiment.

Table 1 Distribution of protease-producing bacteria isolated from from Terasi samples

Table 2 Group of protease-producing bacteria isolated from Terasi based on the relative index value of protease activity

Morphological and biochemical characteristics of Bacilli isolated

Two bacterial strains namely S2-3 and S4-5 were subject to a series of biochemical tests as shown in Table 4.

They were Gram-positive， endospore-forming， and rod-shaped bacteria. Based on the following taxonomic criteria （i.e.， facultative anaerobic， catalase and nitrate reductase positive reactions， production of acetylmethylcarbinol and acid from glucose）， S2-3 isolated was identified as Bacillus thuringiensis， and S4-5 as B. licheniformis （Slepecky and Hemphill，1992）.

Evaluation of substrate utilisation patterns of the two bacterial strains was also determined by using API-50 CHB kit （Table 5）.

Table 3 Relative index values （calculated by the ratio of the diameter of the clear zone and the bacterial colony） of protease activity of the bacilli strains when cultured in skim milk agar and nutrient gelatin agar （nutrient agar supplement with 1% gelatin）

Table 4 Morphological， physiological and biochemical characteristics of bacterial strains S2-3 and S4-5 isolated from Terasi

Table 5 Fermentation of carbohydrates of S2-3 and S4-5 isolated from Terasi using API 50 CHB

Identification of strains S2-3 and S4-5

To further verify their identities， a DNA fragment containing 16S rRNA genes of the strains S2-3 and S4-5 were amplified and sequenced. This sequence information was initially determined using a BLAST search GenBank database （Zhang et al.， 2000）. 16S rRNA gene sequence of the strain S2-3 showed the highest similarity （99.86%） with B. anthracis， B. cereus， and B. thuringiensis， whereas that of the strain S4-5 was closely related to B. subtilis （99.56%）. Phylogenetic tree was then constructed to evaluate the phylogeny of the strains S2-3 and S4-5 compared with the type strains of the closely related Bacillus species （Table 6 and Fig. 1）. Based on the dendogram， the strain S2-3 was grouped with B. anthracis and B. thuringiensis，and the strain S4-5 was closely related to B. subtilis and B. amyloliquefaciens. It should be noted， however，that the genus Bacillus was complex simply defined as low G+C Gram-positive， endospore-forming bacilli. Recent taxonomy using molecular approach revealed the difficulty particularly the use of the 'universal' 16S rRNA-based method. For example， B. subtilis group is heterogenous and consists of several closely related members （i.e.， B. amyloliquefaciens， B. licheniformis，B. mojavensis， and B. sonorensis） （Rooney， 2009）. Bacillus anthracis， B. cereus， and B. thuringiensis are also close relatives and often considered as members of B. cereus group （Helgason et al.， 2000）. These closely related to the species， albeit showing very high degree of similarity of DNA sequences， demonstrate widely different phenotypes （as well as pathological effects especially the pathogenic Bacillus species）（Maughan and Van der Auwera， 2011）. Therefore，this present study was to provide a description of phenotypic and genotypic characteristics of Bacillus strains S2-3 and S4-5 which would be 'essential' either as a fundamental dataset or as a useful information for comparative study. In terms of their identities， we would like to propose that the strain S2-3 was classified in B. anthracis/B. thuringiensis species complex， whereas the strain S 4-5 was in B. subtilis group.

Table 6 GenBank accession numbers for 16S rRNA gene sequence of Bacillus species used in this study

Fig. 1 Phylogenetic relationships of bacterial strains S2-3 and S4-5 with type strains of other Bacillus speciesThe dendogram is constructed based on the similarity of 16S rRNA gene sequences （accession numbers are given in parentheses）. Bootstrap values greater than 60% are illustrated. The branch length is proportional to the number of nucleotide substitutions per site.

Antimicrobial susceptibility

Both bacterial strains （S2-3 and S4-5） were also tested for their antimicrobial susceptibility using the agar disc-diffusion assay as recommended by NCCLS（1997）. The main objective of this antimicrobial susceptibility test was to detect possible drug resistance in common pathogens （Jorgensen and Ferraro， 2009）. Apart from this， the antimicrobial susceptibility profile was also important as phenotypic data of the bacteria which could be useful when considering them for using in industrial application. Table 7 showed the antimicrobial susceptibility profiles of Terasi bacterial strains S2-3 and S4-5. In general， both strains were sensitive to chloramphenicol， erythromycin， kanamycin， streptomycin， tetracycline and vancomycin. They were resistant to ampicillin and were intermediately susceptible to bacitracin （Table 7）. The information of the antibiotic susceptibility of Bacillus species（especially for the non-medical group） is scarce. This is possibly because the non-medical Bacillus species（i.e.， B. subtilis and B. amyloliquefaciens） are generally regarded as safe （GRAS status）， besides several Bacillus species of the non-medical group are being used as probiotics （Cutting， 2011）， and for producing fermented soybeans and industrial enzymes （Schallmey et al.， 2004）. A few studies including the present studies dealing with the antimicrobial susceptibility ofthis particular Bacillus group have been reported （Hong et al.， 2008； Jorgensen and Ferraro， 2009）. Based on the data obtained， using the strain S4-5 in food industry was promising considering from its origin and strong proteolytic activity； however， using of the strain S2-3 was prohibited due to its pathogenic nature.

Table 7 Antibiotic susceptibility of bacilli strains isolated from Terasi

Conclusions

Most Terasi bacteria （ca. 61%） exhibited proteolytic activity. The bacilli strains S2-3 and S4-5 showing strong proteolytic activity were studied in terms of their phenotypic， biochemical， and genetic properties. Such data are provided to point out some key characteristics of the two Terasi bacterial strains. These can be useful for comparative purpose （or study）， when investigated the closely related bacteria especially those isolated from similar food products.

Acknowledgemet

NA thanks the Ministry of Education and Culture （Indonesia） and MFU for a scholarship.

Altschul S F， Gish W， Miller W， et al. 1990. Basic local alignment search tool. J Mol Biol， 215: 403-410.

Cutting S M. 2011. Bacillus probiotics. Food Microbiol， 28: 214-220.

Dereeper A， Audic S， Claverie J M， et al. 2010. BLAST-EXPLORER helps you building datasets for phylogenetic analysis. BMC Evol Biol， 12: 8.

Helgason E， Okstad O A， Caugant D A， et al. 2000. Bacillus anthracis，Bacillus cereus， and Bacillus thuringiensis — one species on the basis of genetic evidence. Appl Env Microbiol， 66: 2627-2630.

Hong H A， Huang J M， Khaneja R， et al. 2008. The safety of Bacillus subtilis and Bacillus indicus as food probiotics. J Appl Microbiol，105: 510-520.

Jorgensen J H， Ferraro M J. 2009. Antimicrobial susceptibility testing: a review of general principles and contemporary practices. Clin Inf Dis，49: 1749-1755.

Kobayashi T， Kajiwara M， Wahyuni M， et al. 2003. Isolation and characterisation of halophilic lactic acid bacteria isolated from "Terasi" shrimp paste: a traditional fermented seafood product in Indonesia. J Gen Appl Microbiol， 49: 279-286.

Lane D J. 1991. 16S/23S rRNA sequencing. In: Stackebrandt E，Goodfellow M. Nucleic acids techniques in bacterial systematics. Wiley， New York. pp. 115-175.

Maughan H， Van der Auwera G. 2011. Bacillus taxonomy in the genomic era finds phenotypes to be essential though often misleading. Inf Gen Evol， 11: 789-797.

NCCLS. 1997. Performance standards for antimicrobial disk susceptibility tests: approved standard M2-A7. National Committee for Clinical Laboratory Standards， Wayne.

Rooney A P， Price N P C， Ehrhardt J L， et al. 2009. Phylogeny and molecular taxonomy of the Bacillus subtilis species complex and description of Bacillus subtilis subsp. inaquosorum subsp. nov. Int J Sys Evol Microbiol， 59: 2429-2436.

Sambrook J， Green M R. 2012. Molecular cloning: a laboratory manual. 4th ed. Cold Spring Harbor Laboratory Press， New York.

Schallmey M， Singh A， Ward O P. 2004. Developments in the use of Bacillus species. Can J Microbiol， 50: 1-17.

Setyorini E， Takenaka S， Murakami S， et al. 2006. Purification and characterisation of two novel halotolerant extracellular proteases from Bacillus subtilis strain FP-133. Biosci Biotechnol Biochem， 70: 433-440.

Slepecky R A， Hemphill H E. 1992. The genus Bacillus—nonmedical. In: Balows A， Truper H G， Dworkin M， et al. The prokaryotes. 2nd ed. Springer-Verlag Inc， New York. pp. 1663-1696.

Surono I S， Hosono A. 1994a. Chemical and aerobic bacterial composition of "Terasi"， a traditional fermented product from Indonesia. J Food Hyg Soc Jap， 35: 299-304.

Surono I S， Hosono A. 1994b. Microflora and their enzyme profile in "Terasi" starter. Biosci Biotechnol Biochem， 58: 1167-1169.

Zhang Z， Schwartz S， Wagner L， et al. 2000. A greedy algorithm for aligning DNA sequences. J Comp Biol， 7: 203-214.

Q81 Document code： A Article lD： 1006-8104（2015）-04-0015-08

Received 28 August 2015

Supported by Mae Fah Luang University （MFU） （57101010027）Ekachai Chukeatirote， E-mail: ekachai@mfu.ac.th