ISOLATION AND CHARACTERIZATION OF LACTIC ACID BACTERIA PRODUCING ANTIMICROBIAL COMPOUNDS FROM SMALL INTESTINE OF CHICKEN Arya Widinatha 1, Laksmi Hartayanie 2 and Lindayani 2 1 Undergraduate Program of Food Technology Department-Soegijapranata Catholic University, Semarang 2 Post Graduate Program of Food Technology Department-Soegijapranata Catholic University, Semarang Email: aryawidinatha@yahoo.com ABSTRACT In food industry, Lactic Acid Bacteria (LAB) have important role for food preservation because of the ability of some strains to produce antimicrobial compounds, including organic acids, hydrogen peroxide and bacteriocins. A number of studies have reported that the LAB have antagonist properties by inhibit the growth of enteric pathogens i.e. Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, and Salmonella sp. Lactic acid bacteria are commonly found in the gastrointestinal tract of various endothermic animals and humans. The aim of this research was to isolate and to characterize lactic acid bacteria from small intestine of chickens that have antimicrobial activity against enteric pathogenic i.e. Staphylococcus aureus and Escherichia coli bacteria. Three steps in this research were LAB isolation, characterization and antimicrobial activity testing using diffusion method. Nineteen strains were isolated and identified as lactic acid bacteria based on their morphology and biochemical characteristics. All isolates were Gram-positive, non-sporulating, non-motile, catalase negative, did not produce gas and classified as Lactobacillus and Enterococcus. In antimicrobial testing, all isolates produced antimicrobial compounds which were active against pathogenic bacteria, indicated by the formation of clear zones around the well (9-15 mm diameter zones). The result show that isolate number 411 had the highest inhibitory zone, which produced more than 15 mm clear zone for Eschericia coli and more than 24 mm clear zone for Staphylococcus aureus. Keyword : Lactic Acid Bacteria, antimicrobial compound, chicken small intestine, well diffusion method Yogyakarta, 25 th 26 th January 2013 Page 1
INTRODUCTION Human beings are often infected by microorganisms such as bacteria, molds, yeasts or viruses present in their living environments. In the past year, antibiotics are crucial in treating infections that may be occurred as a consequence of microbial contamination in foods. But today, many researchers report increasing the number of multiple antibiotic-resistant microorganisms, which have been shown to have negative health effects to human being and increase health care costs in many countries. In food industry, Lactic Acid Bacteria (LAB) are known as GRAS, and therefore are widely used for food fermentation products. In addition, they are able to act as natural preservatives because can produce antimicrobial agents that exert strong antagonist activity against many microorganisms, including pathogenic and food spoilage microorganisms. Organic acids, hydrogen peroxide, and bacteriocins are examples of antimicrobial agents produced by LAB (de Vuyst & Vandamme, 1994). A number of studies have reported that the LAB has antagonistic properties by inhibit the growth of enteric pathogens i.e. Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, Clostridium perfringens and Salmonella sp. Gram positive Staphyloccocus aureus and Gram negative Eschericia coli were widely live on the body surface mammals and sometimes occur infection for humans. Lactic Acid Bacteria are commonly found in the gastrointestinal tract of various endothermic animals and humans. A number of Lactobacillus sp, Enterococcus sp, and Bifidobacterium sp have been reported as Lactic Acid Bacteria founded indigenously in gastrointestinal tract and to be used as probiotics in human and animal. Several Lactic Acid Bacteria have been isolated from swine gastrointestinal tracts (Khunajakr et al., 2008), poultry farm (Ibourahema et al., 2008), and chicken intestine (Nitisinprasert et al., 2000). The aims of this research were to isolate and to characterize lactic acid bacteria from small intestine of chickens that have antimicrobial activity against enteric pathogenic i.e. Staphylococcus aureus and Escherichia coli bacteria. MATERIALS AND METHODS Isolation of LAB from Small Intestine of Chicken LAB were isolated according to methods of SNI 19-2897-1992 and Rahayu & Margino (1997). Briefly, small intestine organs of chicken samples (25 g) were homogenized with 225 ml sterile aquadest. Isolation of bacteria was carried out on de Man-Rogosa-Sharp (MRS) agar + 1% CaCO3 from appropriate 10-fold dilutions (10-1 until 10-10 ). The MRS plates were incubated 48h at 37 C. Colonies were selected based on appearance and clear zone and then purified with streak plate methods. Purified strains of LAB were inoculated into MRS broth and incubated 48 h at 37 C. All purified strains were kept in MRS broth containing 20% glycerol at -20 C. Characterization of LAB from Small Intestine of Chicken Yogyakarta, 25 th 26 th January 2013 Page 2
Characterization of LAB were done by biochemical characteristics (gram staining, sporeforming, motility, catalase and gas production), morphology test (cell-shape), and growth capabilities test at various conditions such as temperatures (10, 45 and 50 o C), ph (4.4 and 9.6) and NaCl (6.5% and 18%) (Lay, 1994., Jutono, 1973., Brown, 2005., Rahayu & Margino, 1997). Antimicrobial Activity Agar well diffusion method was used to assess the production of antimicrobial compounds by the selected Lactic Acid Bacteria against 2 pathogens including Gram-positive S. aureus and Gramnegative E. coli. An overnight culture of pathogens including E. coli and S. aureus adjusted with 3.0 McFarland standard with a sterilized 0.85% NaCl solution. The pathogenic bacteria then added into NA with ratio 1:1000. For LAB isolates were adjusted with McFarland 0.5 standard by dissolving it in sterilized 0.85% NaCl solution. 1% solution which contained LAB added into MRS broth. Antimicrobial activity assay was done using well diffusion method. NA which contained pathogenic bacteria was poured into petridish plates. The plates were perforated of 7.0 mm diameter and filled with 70 μl LAB culture. Incubation was done at 37 C for 24-48 hrs. Sterile MRS was used as control.. RESULTS AND DISCUSSION Isolation of lactic acid bacteria from small intestine of chickens Based on isolation and identification Lactic Acid Bacteria, 19 isolates from 26 isolates form clear area in medium were obtained (Figure 1). Tabel 1 show their morphological and biochemical characteristics for 19 isolates. All isolates were Gram-positive, non-sporulating, non-motile, catalase negative, did not produce gas and have cocci and rod-shape. Figure 1. Single colony and clear zone formed by LAB isolate Yogyakarta, 25 th 26 th January 2013 Page 3
(a) Figure 2. Morfological of LAB (a) cocci shape of LAB; (b) rod shape of LAB Tabel 1. Biochemical Characteristics of Lactic Acid Bacteria Isolated From Small Intestine of Chicken Isolate Gram Sporulating Motility Catalase Gas production Morphology Test 211 + - - - - cocci 212 + - - - - cocci 221 + - - - - cocci 222 + - - - - cocci 311 + - - - - cocci 312 + - - - - cocci 313 + - - - - cocci 314 + - - - - cocci 315 + - - - - cocci 324 + - - - - cocci 411 + - - - - cocci 412 + - - - - rod 413 + - - - - cocci 414 + - - - - cocci 421 + - - - - rod 423 + - - - - cocci 424 + - - - - cocci 522 + - - - - cocci 621 + - - - - rod Growth capabilities at various temperatures, ph and NaCl were shown in Table 2. From Table 2 and Figure 2, among 19 isolates, 3 isolates were classified as Lactobacillus and 16 isolates were classified as Enterococcus. Isolate 412, 421 and 621 had rod shape, could grow at temperature 10 and 45 o C (could not grow at temperature 50 o C), ph 4.4 and NaCl 6.5%. In ph 9.6 and NaCl 18% the isolates could not grow. The same research by Hutkins et al., 1987 and Glaasker et al., 1998 reported that Lactobacilli decreased in growth rate with increasing osmotic environment of Yogyakarta, 25 th 26 th January 2013 Page 4 (b)
the medium. 16 isolates had cocci shape and grew well at temperatures 10 and 45 o C, ph 4.4 and 9.6 also at NaCl 6.5%. This characteristics referred to Enteroccocus genus (Rahayu & Margino, 1997). Table 2. Effect of Temperatures, ph and NaCl in Growth Capabilities of Lactic Acid Bacteria Isolates Temperatures ( o C) ph NaCl (%) 10 45 50 4,4 9,6 6,5 18 211 + + - + + + - 212 + + - + + + - 221 + + - + + + - 222 + + - + + + - 311 + + - + + + - 312 + + - + + + - 313 + + - + + + - 314 + + - + + + - 315 + + - + + + - 324 + + - + + + - 411 + + - + + + - 412 + + - + - + - 413 + + - + + + - 414 + + - + + + - 421 + + - + - + - 423 + + - + + + - 424 + + - + + + - 522 + + - + + + - 621 + + - + - + - Antimicrobial Activity Antagonistic activity of the isolated LAB strains against a number of pathogenic bacteria was determined by the agar well diffusion method. In antimicrobial testing, all isolates produced antimicrobial compounds which were active against Staphyloccocus aureus and Eschericia coli, indicated by the formation of clear zones around the well (9-24 mm diameter zones). Isolate number 411 had the highest inhibitory zone, which produced 15.50 mm clear zone for Eschericia coli and 24.17 mm clear zone diameters for Staphylococcus aureus (Figure 3). Yogyakarta, 25 th 26 th January 2013 Page 5
Figure 3. Antimicrobial activity of Lactic Acid Bacteria isolated from small intestine of chicken to Eschericia coli (yellow) and Staphylococcus aureus (purple) Isolate 411 Control (MRS broth) Isolate 411 Control (MRS broth) (a) (b) : well of agar : clear zone Figure 4. Result of the highest antimicrobial activity by isolate 411 towards Escherichia coli (clear zone = 15.50 mm) and Staphylococcus aureus (clear zone = 24.17 mm) CONCLUSION AND FUTURE STUDY Nineteen strains of LAB were isolated from gastrointestinal chicken intestines, sixteen isolates were included in genera of Enterococcus, three isolates were included in genera of Lactobacillus. Among all isolates, isolate number 411 had the highest inhibitory ability towards S. aureus. Future study needed to identify LAB isolate number 411 using API 50 CHL test kit and API 20 STREP test kit. ACKNOWLEDGEMENT The author would like to thank Binardo Adi Seno and Amelia Juwana for their helping in finishing this paper. LITERATURE REVIEW Brown, A. E. 2005. Microbiological Applications, Ninth Edition. Mc Graw Hill. Auburn University. New York. Yogyakarta, 25 th 26 th January 2013 Page 6
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