Identification, Characterization and Genetic Improvement of Bacteriocin Producing Lactic Acid Bacteria

MATERIALS AND METHODS

LAB isolates: Ten LAB isolates were purified from Egyption traditional dairy products according to the method adopted by Lavanya et al.¹⁴.

Indicator strains: Eight bacterial indicator strains were used for study the bacteriocin antimicrobial activity included Staphylococcus aureus ATCC 25923, E. coli ATCC 19404, Salmonella typhimurium ATCC 14028, Bacillus cereus ATCC 33018, Bacillus subtilis ATCC 6633, Pseudomonas aeruginosa ATCC 9027, Listeria monocytogenes ATCC 7644 and Lactococcus lactis ATCC 11454.

Morphological and physiological identification of LAB isolates: Identification of LAB isolates were performed by examination for cell morphology, Gram staining, catalase activity, oxidase, optimum pH, temperatures and salt tolerance. Profile matching method based on Bergey’s manual of systematic bacteriology were used for characterization and identification of LAB isolates¹⁵.

Antimicrobial activity: Detection of antimicrobial activity was carried out using disc diffusion method according to Tejero-Sarinena et al.¹⁶. Each assay was performed in triplicate.

Quantification of bacteriocin activity: Arbitrary Units (AU mL^–1) of bacteriocin activities were calculated according to Yamamoto et al.¹⁷.

Carbohydrate fermentation profile of LAB isolates: The API 50 CHL and API 20 strips kit (API system, Biomerieux, France) were used to identify the LAB isolates based on carbohydrate fermentation profile according to Adebayo-Tayo and Onilude¹⁸ and Maqsood et al.¹⁹. The results were analyzed by API soft ware (API systems, Biomerieux, France).

Extraction of genomic DNA: Extraction of genomic DNA was carried out using Qiagen kit (Qiagen Sciences, Maryland, USA) according to the manufacturer’s instruction manual.

PCR amplification of 16S rDNA gene: The PCR reactions were done to amplify the 16S rDNA gene from LAB isolates. The 16S rDNA region was amplified by using the universal primer set; 8F (5ʹ-AGAGTTTGATCCTGGCTCAG-3ʹ) and 1492R (5ʹ-GGTTACCTTGTTACGACTT-3ʹ), according to Weisburg et al.²⁰. Reaction was analyzed on 1% (w/v) agarose gel.

Conjugation procedure: The two different conjugating techniques, filter and broth mating were carried out according to Gevers et al.²¹.

Random Amplified Polymorphic DNA (RAPD): The RAPD-PCR technique was done using 13 oligonucleotide primers (Table 1). The PCR was performed according to Plengvidhya et al.²² in a total of 25 μL reaction volume and amplification was programmed to 40 cycles after an initial denaturation cycle for 2 min at 94°C. Each cycle consisted of a denaturation step at 94°C for 1 min, an annealing step at 36°C for 1 min and an extension step at 72°C for 2 min, followed by extension for 10 min at 72°C in the final cycle.

Computer software: The phylogenetic tree for different LAB were deigned according to the blast result in GenBank database (www.ncbi.nlm.nih.gov).

RAPD analysis: The analysis RAPD and phylogenetic tree for transconjugants, donor and recipient was generated using SPSS 16 (SPPS Inc., Chicago, Il, USA) and Genomes.urv.cat/UPGMA/index.php?entrada.

Table 1:	RAPD primers name and sequence

Statistical analysis: Numerical data were obtained from three independent experiments and these data carried out with MS-excel and the data presented as means. The significant differences between means were analyzed using one way ANOVA with a significance level of p<0.05.

RESULTS AND DISCUSSION

Isolation and characterization of LAB: Ten local LAB isolates were purified from milk and traditional dairy product samples from Egypt and named as I₁-I₁₀. Ten bacterial isolates were pre-characterized based on their phenotypic and morphological characterizations. The colony morphology of the isolates was tested at different growth media. Three specific media were used for isolation for specific bacterial genus; MRS for used for isolation Lactobacillus species²³, (KAA) for streptococci (Enterococci) in foodstuffs²⁴ and M17 for lactic streptococci²⁵.

Table 2 summarizes the morphological characterization of ten LAB isolates. These isolates were grouped on the basis of Gram stain reaction. The bacterial isolates were Gram positive, catalase negative and cocci except I₇ and I₈ rods. A similar observation was reported by Khalid²⁶ who also noted that LAB were Gram positive without spore, with negative catalase, microaerophilic, resistant to acid and could be fermentation. Also, Holzapfel et al.²⁷ found that LAB were bacteria in rod or coccus shapes, with negative catalase, non motile, homo fermentative or hetero fermentative and growing in low acid condition.

Antimicrobial spectrum of crude bacteriocin from LAB isolates: The production of antimicrobial compound such as bacteriocin were more effective criteria used to characterize probiotics. The antimicrobial activity of the ten bacterial isolates were tested (Table 3) all the isolates give variable degree of inhibition zone and activity on different tested strains specially I₁, I₂ and I₃ against Gram positive bacteria, Listeria monocytogenes and Staphylococcus aureus as target propose. The bacterial isolates (I₁, I₂ and I₃) were the highest bacteriocin activity (320, 640 and 2560 AU mL^–1) and large clear inhibition zone (20, 22 and 25 mm), while I₆ and I₉ were the lowest bacteriocin activity (20 AU mL^–1) with hazy clear zone.

Table 2:	Morphological characterization of the ten bacterial isolates
1: MRS, 2: KAA and 3: MI7

Table 3:	Bacteriocin activity of LAB isolates and inhibition zone

Table 4:	Morphological and physiological characterization of three isolates that highly bacteriocin activity

Figure 1 show the inhibition zone of (I₁, I₂, I₃ and I₄) against Staphylococcus aureus.

These results agreement with Buntin et al.²⁸ who reported that three strains of lactic acid bacteria, P. pentosaceus APa4, P. pentosaceus AIa1 and Enterococcus faecium ARa1 were able to inhibit growth of S. aureus, Salmonella sp., E. coli and L. monocytogenes by agar well diffusion method.

Fig. 1:	Staphyloccus ATCC 25923 clear zone for different bacterial isolates (I1, I2, I3 and I4) on agar plate

Bacteriocins produced by lactic acid bacteria can inhibit growth of Gram positive bacteria more than Gram negative bacteria because the outer membranes of Gram positive bacteria that play as barrier for bacteriocins to penetrate into their cell membranes²⁸. Also, Bassyouni et al.²⁹ and Du Toit et al.³⁰ conferred that bacteriocins produced by the E. faecium strains showing a broader spectrum of activity, against indicator strains of Enterococcus spp., Listeria spp., Clostridium spp. and Propionibacterium spp., compared with those from E. faecalis strains showed a narrow spectrum of activity, mainly against other Enterococcus spp. Another study were isolated LAB from raw fruits and vegetables inhibited E. coli isolated from human sources³¹. Because of the high production of bacteriocin by the I₁, I₂ and I₃ isolates the three bacterial isolates (I₁, I₂ and I₃) were identified at the morphological, biochemical and molecular level.

Morphological and physiological characterization: Table 4 summarizes the morphological and physiological characterization of three important isolates (I₁, I₂ and I₃). The three isolates were cell shape cocci, non-motility, Gram positive, which not forming spores and negative catalase and oxidase. The bacterial isolates I₁ and I₂ grow between 30-45°C but isolate I₃ grow at 30-40°C. These isolates grow at various concentrations of NaCl ranging from 2-10% (w/v). On the other hand, isolate I₃ was grow at 6% (w/v) NaCl. The selected LAB isolates were tolerate to acidic pH and resist to high concentration of NaCl which important and crucial criteria for potential probiotic and important starter culture in dairy product. These results agreement with Axelsson³² who determined that LAB were belong to Gram positive group, with negative catalase, coccus or round shaped without spore and non-motile without respiration, but can grow on aerobic or microaerotolerant conditions and can produce lactate acid as main metabolic product from carbohydrate fermentation. The API 20 strep and API CHL 50 kits were used for biochemical identification of I₁, I₂ and I₃ bacterial isolates (Table 5).

The results indicated that isolates classified as follows: I₁-I₂ were Enterococcus faecium with 94.4% identical rate and I₃ Pediococcus pentosaceus with 99.5% identical rate. These results were preliminary identification of isolates by standard biochemical and morphological testes.

Molecular characterization: Universal primers designed for the amplification of 16S rDNA used to amplify ~1.5 kb size fragment in all of the selected isolates I₁, I₂ and 1₃ in Fig. 2. Amplicons of 16S rDNA were column purified and sequenced for ~1.5 kb fragment using a set of primers. The 16S rDNA sequences were aligned using blast algorithm (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) and compared with the published sequences of 16S rDNA gene of different LAB strains deposited in NCBI databases.

The molecular identify were confirmed to be I₁ (99%) similarity with Enterococcus faecium strain L3-23, I₂ (98%) similarity with Enterococcus faecium strain gp 34, I₃ (97%) similarity with Pediococcus pentosaceus strain LAB2. Figure 3-5 show the phylogenetic tree of isolates and neighbor joining according to blast results. All sequences were deposited in the GenBank nucleotide databases under accession number LC063691.1, LC063692.1 and LC063861.1. These results indicated that the importance of using molecular methods for typing newly isolates microorganisms, phenotypic and genotypic identifications are part of the first step in the selection of potential probiotic bacteria³³.

The identification of LAB based on the morphological, physiological and biochemical characteristics are often considered as unreliable, since different species may have similar morphological and nutritional requirements⁶.

Fig. 2:	PCR-amplification of 16S rDNA gene using 8F and 1492R primers, Lane M: Gene ruler DNA ladder 1 kb, Lane 1: 16s rDNA gene fragment of isolate I1, Lane 2: 16s rDNA gene fragment of isolate I2, Lane 3: 16s rDNA gene fragment of isolate I3

Table 5:	Biochemical identification of LAB isolates

Table 6:	Conjugation between Lactococcus lactis sub sp. lactis and Enterococcus faecium AH2

The phenotypic characterization based on sugar fermentation profile may be used as a presumptive identification, it may not provide reliable identification, but genotype-based methods such as 16S rDNA are robust to identify bacteria as a complement or alternative to phenotypic methods²⁰. Genotype methods are independent from variation of growth conditions, if species-specific primers or probes are available; these offer a very fast way to detect the target organism.

Traditional genetic improvement of selected isolates via conjugation: Table 6 summarized the mating experiments were carried out in broth and filter mating techniques. The experiments were assessed using Lactococcus lactis sub sp. lactis (nisin producer) as a donor strain with tetracycline resistance markers, located on transferable genetic elements and Enterococcus faecium AH2 (LC063692.1) erythromycin resistance as a recipient strain. The results show low frequency of gene transfer approximately (2.4×10^–5) progeny per recipient occurred during broth mating, but (4.6×10^–5) progeny per recipient by filter mating, these results indicated that the filter technique facilitates a greater degree of donor-recipient contact than the broth method.

Fig. 3:	Phylogenetic tree based on partial 16S rDNA sequences, showing the relationship between isolate (I1) and other species. The tree was constructed using the neighbor-joining method. Enterococcus faecium strain: AH2 (LC063692.1)

Fig. 4:	Phylogenetic tree based on partial 16S rDNA sequences, showing the relationship between isolate 1and other species. The tree was constructed using neighbor-joining method. Enterococcus faecium: AH3 (LC063861.1)

These might be due to the concentration of cells in the broth system which considerably less and therefore contact between donor and recipient would be more sporadic. These results agreement with Lampkowska et al.³⁴. The resulted transconjugants were confirmed using antibiotic selection and activity of bacteriocin (Table 7). The transconjugants T1, T2 and T3 showed increased of bacteriocin activity (1280, 1280 and 2560 AU mL^–1), respectively more than the recipient (460 AU mL^–1).

These results may be resulted from transfer of some genetic element from donor to recipient strain or genetic recombination lead to increase in bacteriocin production and activity. Moreover, when transposable integrated into cells, the transconjugation has a new genetic makeup or unique DNA sequences not completely like either parent, making it possible for the new cells with new characters³⁵.

Fig. 5:	Phylogenetic tree based on partial 16S rDNA sequences, showing the relationship between isolate (I3) and other species. The tree was constructed using neighbor-joining method. Pediococcus pentosaceus, strain: AH1 (LC063691.1)

Table 7:	Activity of bacteriocin (AU mL–1) produced by donor (Lactococus lactis sub sp. lactis), recipient (Enterococcus AH2) and transconjugants strains (T1, T2 and T3)

Table 8:	Total number of scorable bands and the polymorphic among strains

Table 9:	Similarity matrix computed with Jaccard coefficient
R: Recipient, D: Donor and T: Transconjugants strains

In order to study the genetic difference among the transconjugants lines, donor and recipient, DNA samples were subjected to RAPD analysis using 13 selected primers. Eight of the 13 primers produced reproducible PCR products with a clear pattern for each strain and showing informative and easily scrabble RAPD profiles (Fig. 6). A total of 109 bands were detected among the transconjugants lines, donor and recipient (Table 8 and 9). Only 9 of them were polymorphic markers (8.25%). These primers produced multiple band profiles with a number of amplified DNA fragments varying from 11-18. The highest number of bands (18 bands) was generated by using the primer OPD-03, while the lowest two were 11 bands and generated with primer OPD-06 and OPD-07. Donor and recipient strains gave distinct DNA fingerprint patterns, while transconjugants showed patterns matching the corresponding recipient strain. The distance matrix based on RAPD data sets was used to construct a dendrogram (Fig. 7). The results also indicated that the closest relationship between T3, T2 and T1 were closely related to recipient Enterococcus faecium AH2 0.500, 0.506 and 0.494 similarity. These results agreement with Toomey et al.³⁶ who used another technique (PFGE) but found the same results.

Resistance to temperature and sodium chloride: The bacterial capability to grow at high temperature and high osmotic is important characteristics of LAB that should be evaluated when selecting strains used as probiotics.

Fig. 6:	RAPD-PCR image of the donor, recipient and resulted transconjugants. Lane M: 1 kb ladder, Lane 1, 2, 3, 6, 7, 8: Transconjugants, Lane 4-9: Lactococcus lactis donor, Lane 5-10: Enterococcus AH2 recipient

Fig. 7:	Dendrogram demonstrating the relationships among the donor, recipient and transconjugants strains. Based on RAPD-PCR analysis using Jaccard coefficient

Results showed that all transconjugants strains and recipient were grow at temperatures of 45°C due to an increased growth rate, while donor strain decreased in growth rate. The growth rate was determined as OD at 600 nm. The data collected were examined with the ANOVA technique; differences between strains were statistically analysis. Transconjugants strains gave significant variance with donor and high similarity with recipient at the significance level of LSD p<0.05 = 0.0859 (Table 10). On the other hand, all transconjugants strains and recipient tolerate to high osmotic concentrations of NaCl (2-10%), while donor could be tolerate to (2-6%).

Table 10:	Growth rate of donor, recipient and transconjugated strains OD at 600 nm at 45°C
p<000***, LSD 0.05 = 0.0859, ***ANOVA analysis, Means have the same letter are not significant according to LSD, p<0.05

These examinations gave an indication of the osmotolerance level of the LAB strains resulted from this experiment. These results accepted with Ibourahema et al.³⁷ bacterial cells cultured with a high osmotolerance would be a requirement of LAB strains to be used as commercial strains, because when lactic acid is produced by the strain, alkali would be pumped into the broth to prevent an excessive reduction in pH and the free acid would be converted to its salt form, increasing the osmotic pressure on the bacterial cells.

CONCLUSION

The results of the present study clearly suggest that three bacteriocin producing LAB isolates Enterococcus faecium (I₁-I₂) and Pediococcus pentosaceus (I₃) with probiotic properties. These LAB isolates could provide significant health benefits and enhance safety of the product and shelf life. However, they need further isolation, identification of new isolates of LAB producing bacteriocin and genetic improvement to use these isolates in different industrial application. Consumers are very concerned of chemical preservatives and processed foods, but they accept easily LAB as a natural way to preserve food and promote their health bacteriocinogenic LAB inhibit the growth of spoilage and pathogen bacteria in foods.

ACKNOWLEDGMENT

The authors acknowledge the National Research Center, Egypt and for funding this study. Funding source PhD thesis code 11/6/2 and Microbial Genetic lab Department of Genetics, Faculty of Agriculture, Cairo University, Giza, Egypt.