Isolation of Lactic Acid Bacteria Strain Staphylococcus Piscifermentans from Malaysian Traditional Fermented Shrimp Cincaluk
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International Food Research Journal 20(1): 125-129 (2013) Journal homepage: http://www.ifrj.upm.edu.my Isolation of lactic acid bacteria strain Staphylococcus piscifermentans from Malaysian traditional fermented shrimp cincaluk Hajar, S. and *Hamid, T. H. T. A. Department of Biotechnology, Kulliyyah of Science, International Islamic University Malaysia, Kuantan Campus, Jalan Istana, Bandar Indera Mahkota, 25200, Kuantan, Pahang, Malaysia Article history Abstract Received: 11 November 2011 Lactic acid bacteria is well known for it uses as starter culture in various fermented food, and Received in revised form: it functions as a good natural antimicrobial agent. Cincaluk, a Malaysian fermented shrimp 12 July 2012 product commonly found in traditional dishes is commonly enriched with LAB. Out of 50 Accepted:17 July 2012 colonies from a local cincaluk, 7 strains were successfully isolated and shown to be positive in lactose utilization and catalase tests. The majority of the isolates from cincaluk showed Gram-positive cocci morphology and belonged to the group Staphyloccoccus spp. By using Keywords agar disc diffusion method, the anti-bacterial properties of these isolates (namely isolate 1, 2, 3, 4, 5, 6, and 7) moderately inhibited the growth of several pathogenic strains, i.e., Escherichia cincaluk coli, Staphylococcus aureus, Salmonella typhimurium and Bacillus subtilis which were used fermented shrimp as indicator bacteria. Other than isolates 1, 2, 3 and 5; the 16S rRNA gene for isolate 6 and lactic acid bacteria 7 were successfully amplified. The 16S rRNA gene fragment from isolate 7 was successfully Staphylococcus cloned and sequenced. Based on rRNA sequences, both isolates 6 and 7 belonged to the group piscifermentans Staphylococcus piscifermentans, a rare strain previously reported to be specifically isolated exclusive from fish sources. © All Rights Reserved Introduction preservation, they also produce variety of compounds with antimicrobial activity, including organic acids, Consumer interests in food that are natural, hydrogen peroxide and bacteriocin (Choi et al., 1997; fresh and healthy are increasing in demand. There O’Sullivan et al., 2002). Bacteriocin produced by is a growing concern on the nutritional food losses LAB could inhibit not only closely related species upon processing and possible health risk of chemical but also the growth of pathogenic bacteria (Jack et preservatives. Despite of improved manufacturing al., 1995; Klaenhammer, 1988; Tagg et al., 1976). standard and effective legislative control on processing Many LABs have important roles in the production procedures, the number of food borne illness still of fermented foods, and some of the bacteria were remains a public concern (Tharmaraj and Shah, 2009). capable of inhibiting the growth of a wide variety The contamination and growth of psychrotropic and of food spoilage microorganisms (Lindgren and pathogenic spoilage microorganisms in refrigerated Dobrogosz, 2006). Thus, LABs are an attractive foods is a major risk in food industry. Consequently, source of inhibitory compounds with promising the use of naturally produced anti-microbial agents natural food preservatives for improved food quality without any adverse effect on human health to inhibit and safety. The purpose of the study is to isolate LAB the proliferation of pathogenic microorganisms in from a Malaysian fermented shrimp product known food has been a prime concern. as cincaluk. Cincaluk is a sauce in traditional Malay Lactic acid bacteria or (LAB) is a group of delicacies produced by fermenting small shrimp related bacteria producing lactic acid as the result of called Acetes sp., or locally known as Udang geragau, carbohydrate fermentation (Ali et al., 2009). LAB Udang bubok or Udang gari (Ali et al., 2009). The is widely used as starter cultures for dairy, meat sauces, in various varieties, are also consumed in and vegetable fermentations (Jeppesen and Huss, other Southeast Asian countries. Various strains 1993). In addition to flavour development and food from Lactobacillus sp. and Lactococcus sp. were *Corresponding author. Email: [email protected] 126 Hajar, S. and * Hamid, T. H. T. A./IFRJ 20(1): 125-129 the dominant LAB strains found in Thai fish product (Kirby et al., 1966). LAB isolate was propagated in (Østergaard et al., 1998; Paludan-Muller et al., MRS broth medium and incubated anaerobically at 1999). Similar works on various types of fermented 30°C for 48 h. The propagated cell was pipetted onto shrimp products had previously isolated LAB from filter paper disc (diameter of 5 mm, Whatman No.1) other genera such as Leuconostoc mesenteroids, and dried for 10 min. The paper discs were put on Streptococcus faecalis and Pediococcus halophilus the agar surface pre-spread with 500 µl of indicator (Tanasupawat et al., 1998), Tretragenococcus sp. organism. Four indicator strains; Bacillus subtilis, (Kobayashi et al., 2003) and Leuconostoc durionis E. coli ATCC 35215, Staphylococcus aureus and sp. nov (Leisner et al., 2005). In an attempt to isolate Salmonella Typhimurium obtained from Institute of γ-amino butyric acid (GABA) producing LAB, Ali et Medical Research, Malaysia (IMR) were used. The al. (2009) isolated Leuconostoc NC5 from cincaluk. test was performed in triplicates. Then, the disc on In another work on Malaysian local fermented fish the plates were incubated at 37°C for 24 h and the product or budu, the isolation of Lactobacillus spp. zone of inhibitions formed surrounding the disc were was reported (Liasi et al., 2009). In this work, we observed. report on the isolation of LAB strain from a local cincaluk. However, further identification has led to 16S Ribosomal (rRNA) gene amplification, the isolation of Staphylococcus piscifermentans a sequencing and analysis rare LAB strain which was never been reported from The 16S rRNA gene fragment of ~1.5 kb was this source. amplified by using a pair of universal primers pA forward (5’-AGAGTTTGATCCTGGCTCAG-3’) and Materials and Methods pE reverse (5’-CCGTCAATTCCTTTGAGTTT-3’) (Edwards, 1989). The amplified product was purified LAB samples and characterisation with spin columns (Qiagen, USA). The amplifications A bottle of fermented shrimp product, cincaluk were performed with initial denaturation at 94°C for used as the sample source was purchased from 4 min, and with 29 cycles of denaturation at 94°C for domestic and home-made supplier at Melaka, 2 min; annealing at 55°C for 1 min and extension at Malaysia. Approximately, 25 ml of sauce sample 72°C. All DNA templates used were approximately was mixed with 225 ml of buffered peptone water at 5 ng DNA. The amplified fragments were then to obtain a 1:10 dilution. Serial dilution of the cloned by using blunt end of PCR product into samples was made using 0.1% peptone water. The pJET1.2/blunt cloning vector (Fermentas, Lithuania) diluted samples were spread on the De Man, Rogosa and transformed into Escherichia coli competent and Sharpe (MRS) agar plates and incubated, cells (JM101). The DNA was analysed by using anaerobically, for 48 h at 37oC. Single colonies were 1.0% (w/v) agarose gel electrophoresis in 1x TAE picked out using sterile tooth pick and subjected to buffer at 90 V for 65 min; and was visualized by further studies. The morphological, physiological using gel documentation system (Alpha Imager). For and biochemical characterization of the isolates successful transformant, the recombinant plasmid were determined by the standard procedure of Gram was extracted (Qiagen, USA). Then, the extracted staining, and tested for catalase and gas production. plasmid was further verified by PCR using vector In lactose utilization test, the selected colonies were targeted primer. The insert was then sequenced with streaked onto nutrient agar (NA) with the addition of vector-targeted primers by using sequencing service lactose containing 0.005 g/L of bromo-cresol purple, available at 1st Base Laboratory Sdn. Bhd, Malaysia. as pH indicator dye. The plates were incubated at The purified PCR product was also sequenced directly 30°C for 24 h. Isolates utilizing lactose and producing with 16S rRNA primers and further primer walked to acid were differentiated by the change in media color get the complete rRNA sequence. The sequences of from violet to yellow. Catalase activity was tested by the whole gene fragment were used for the similarity adding a drop of 30% hydrogen peroxide solution search against NCBI GenBank database using the onto the culture smears. Positive reaction was seen BLAST program, available at website http://blast. as bubbles or froths generated from the colonies, ncbi.nlm.nih.gov/Blast.cgi. indicating a rapid production of oxygen gas from hydrogen peroxide. Results and Discussion Antagonist test using disc diffusion method As shown in Table 1, out of 50 colonies, isolates The antagonistic activity of the isolated LAB 1, 2, 3, 4, 5, 6 and 7 appeared to be positive in lactose against Salmonella typhimurium was determined utilisation test. These isolates were able to ferment by using agar disc diffusion method based on Kirby lactose to produce lactic acid that lowers the pH of the Hajar, S. and * Hamid, T. H. T. A./IFRJ 20(1): 125-129 127 Table 1. Morphological and biochemical tests for isolates 1 to 7 Table 2. Top five similarity searches hits using BLAST for Characterization LAB isolates rRNA sequences for Isolate 6 and 7. The highest similarity Strain Strain 2 Strain 3 Strain 4 Strain Strain Strain 1 5 6 7 was found to be Staphyloccus piscifermentans, followed