In recent years much attention has focused on bacteriocin-producing LAB isolated from various sources, because bacteriocins are considered safe as food biopreservatives and can be degraded by gastrointestinal proteases
. However, LAB species present in traditional foods of Southeast Asian countries have not been widely studied
. In this study, 11 LAB strains isolated from traditional fermented milk products and cocoa beans from rural areas of Malaysia and Iran were found to produce antimicrobial substances. These LAB isolates were characterized, and two of the strains (Kp8 and Kp10) produced substances active against Listeria monocytogenes (888.56 AU/mL).
Phenotypic characterization based on sugar fermentation reveals biochemical properties of the microorganisms
 but may not always provide a strong basis for LAB identification
. Although 16S rDNA sequence analysis is a powerful technique for identifying microorganisms and determining phylogenetic relationships
, further analysis is needed for positive identification
. Therefore, we used both of these methods to identify the isolates. All 11 isolates were able to ferment ribose, galactose, glucose, fructose, mannose, n-acetyl-glucosamine, esculin, salicin, cellobiose and gentiobiose. Three different LAB species (Lactococcus lactis, Lactobacillus plantarum, and Pediococcus acidilactici) were identified using the API 50 CHL system and 16S rDNA analysis. Identification of Kp10 as P. acidilactici was confirmed by phylogenetic analysis (Figure
In addition, β-galactosidase activity, tolerance to bile salts and acid conditions, and antimicrobial activity were to evaluate the probiotic properties of Kp10 (P. acidilactici). The isolate was able to grow in the presence of 2% NaCl, but growth was inhibited by 3% NaCl. Homofermentative LAB are more resistant than heterofermentative LAB to NaCl
. Pediococci strains are homofermentative, and tolerance to pH, temperature, and NaCl is species- and strain-dependent
. Bacterial cells cultured in high salt concentrations experience a loss of turgor pressure, which affects cell physiology, enzyme and water activities, and metabolism
; however, some bacteria overcome this effect by regulating osmotic pressure on both sides of the cell membrane
. Optimum temperature can also be used to differentiate among LAB strains
. Our results indicated that Kp10 (P. acidilactici) is a mesophile, which is in agreement with the findings of Ronald
LAB are found in many natural environments; however, antibiotic resistance in these bacteria is a growing concern
. Thus, sensitivity to antibiotics must be determined before LAB strains can be used in food production
. Antibiotic-resistant strains can be detrimental to the health of humans and animals
, because they are capable of transferring antibiotic resistance genes to pathogenic bacteria
, which can contaminate raw food products such as meat or milk.
Data on the antibiotic susceptibility of Pediococcus spp. isolated from food are limited. Penicillin G, imipenem, gentamicin, netilmicin, erythromycin, clindamycin, rifampin, chloramphenicol, daptomycin, and ramoplanin are generally active against Pediococcus species
[24–27]. However, susceptibility is thought to be species-dependent. We found that isolate Kp10 (P. acidilactici) was susceptible to ß-lactam antibiotics (penicillin G and ampicillin), as well as erythromycin, chloramphenicol, nitrofurantoin, and tetracycline. In contrast, previous studies have reported that LAB are often resistant to commonly used antibiotics such as β-lactams, cephalosporins, aminoglycosides, quinolone, imidazole, nitrofurantoin, and fluoroquinolones
[23, 28]. ß-lactams, which are bactericidal, are the most widely used class of antimicrobial agent because of their broad spectrum of action and excellent safety profile. ß-lactams inhibit bacteria cell wall synthesis and have a lethal effect on gram-positive bacteria. Erythromycin is a macrolide antibiotic with a range of action and efficacy similar to that of penicillin. Macrolides, which are bacteriostatic, bind to ribosomes to block protein synthesis and are effective against gram-positive microorganisms
. The rationale for this contradictory finding with those of Halami, et al. and Herreros et al. is not known. Lactobacillus and Lactococcus were previously reported to be susceptible to β-lactam antibiotics
, which is in agreement with the findings of this study. It is possible that the reports of Halami et al. and Herreros et al. referred to LAB in general, whereas the present study specifically analyzed the species P. acidilactici.
The isolate Kp10 (P. acidilactici) was susceptible to a gram-negative antibiotic (nalidixic acid) and aminoglycosides (amikacin, kanamycin, neomycin, and streptomycin). In contrast, Zhou et al. and Temmerman et al. reported that most Lactobacillus, Enterococcus, and Pediococcus strains used as probiotics are resistant to gram-negative and aminoglycoside antibiotics. Thus, susceptibility to gram-negative antibiotics may be specific for this LAB species.
Vancomycin, an inhibitor of cell wall synthesis, is an important antibiotic because it is the last agent broadly effective against multi-drug resistant pathogens
. Kp10 (P. acidilactici) was not resistant to vancomycin, making it potentially useful for applications in the food industry
. Kp10 (P. acidilactici) was also susceptible to sulfonamide. Resistance to this antibiotic is caused by mutations in the gene encoding dihydropteroate synthase or by acquisition of plasmid-borne genes carrying sulfonamide-resistant forms of the enzyme
Our results also showed that Kp10 (P. acidilactici) produced blue/green colonies when grown on M17 agar supplemented with X-gal and IPTG, demonstrating β-galactosidase activity. β-galactosidase is involved in lactose digestion and is used in the production of lactose-free milk. β-galactosidase–producing bacteria may also be potential probiotics to reduce lactose intolerance
Mean bile concentration in the human gastrointestinal tract is 0.3% (w/v), with a residence time of about 4 h
. Therefore, we tested tolerance to bile salts at a concentration of 0.3%, which revealed 11% survival after 4 h. Bile salts interact with bacterial cell membranes, which are composed of lipids and fatty acids, inhibiting growth and killing many bacteria. The protonated (non-dissociated) form of bile salt exhibits toxicity by a mechanism similar to that of organic acids. This is involves intracellular acidification and collapse of the proton motive force, which in turn, inhibits the nutrient transport. However, some LAB strains are able to hydrolyze bile salts with bile salt hydrolase
Resistance to low pH is one of the major criteria for selecting strains for probiotic applications
. Survival of Kp10 (P. acidilactici) at pH 3 exceeded 97%, suggesting its potential for use as a probiotic.
Several strains of P. acidilactici isolated from the intestine of healthy dairy cows and characterized using methods similar to those used in the present study were found to inhibit Escherichia coli.
. The authors reported that P. acidilactici was resistant to acid and bile salts, indicting the ability to survive and colonize in the intestine. In the present study, we found that Kp10 (P. acidilactici) was active against the pathogen L. monocytogenes. It is interesting to note that P. acidilactici from two different agricultural sources (intestine of dairy cows and a traditional milk product) showed promising prophylactic properties.
We found that the BLIS from Kp10 (P. acidilactici) was stable in a wide range of pH (2–9), suggesting that its antimicrobial activity was not due to the pH of the cell-free supernatant. The reduced activity at high pH was probably due to denaturation of the protein. A similar result was also observed for an antimicrobial compound produced by Lactococcus lactis, which was active at the pH range 2 to 10 and completely inactivated at pH 12
Since bacteriocins are proteinaceous substances, they must be sensitive to at least one proteolytic enzyme
. Therefore, bacteriocins can be identified in part by exposure to proteolytic enzymes
. We found proteolytic enzyme treatment reduced the activity of the antimicrobial compound secreted by Kp10 (P. acidilactici). However, activity was not reduced by catalase, indicating that H2O2 was not responsible for microbial inhibition, or α-amylase activity, indicating that the compound was not glycosylated, which is characteristic of most bacteriocins
. Complete inactivation activity was observed after treatment with proteinase K and trypsin, in accordance with a report by Albano et al. of pediocin PA-1 activity
. Treatment with pepsin did not alter the antimicrobial activity of the BLIS in this study; however, proteolytic enzymes do not always reduce the antimicrobial activity of a bacteriocin
. Stability in the presence of a proteolytic enzyme could be due to unusual amino acids in the bacteriocin structure or cyclic N-terminal or C-terminal protected peptides
We conclude that isolate Kp10 (P. acidilactici) is a potential probiotic that may exert beneficial positive effects on intestinal flora, because the strain is tolerant of bile salts (0.3%) and acidic conditions (pH 3). To better understand its potential as a probiotic, future studies are needed to characterize the interactions of this P. acidilactici strain to the intestinal mucosal epithelium.