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. 2022 Oct 29;36:e00772. doi: 10.1016/j.btre.2022.e00772

Characterization of novel bacteriocin PB2 and comprehensive detection of the pediocin gene ped-A1 from Pediococcus pentosaceus PB2 strain isolated from a sorghum-based fermented beverage in Nigeria

Ahmed Adebisi Otunba a, Akinniyi Adediran Osuntoki b, Wahab Okunowo b, Daniel Kolawole Olukoya a, Benjamin Ayodipupo Babalola c,⁎
PMCID: PMC9640346  PMID: 36388845

Highlights

  • •

    Novel bacteriocin PB2 was obtained from Pediococcus pentosaceus PB2 strain isolated from fermented sorghum beverage in Nigeria, Pito.

  • •

    The PB2 isolate exhibited antagonistic activity against E. coli ATCC 25922 and L. monocytogenes ATCC 15313.

  • •

    The specific activity, purification fold, and recovery yield of bacteriocin PB2 from Carboxymethyl-Sephadex C-50 are 2.65 U/mg, 12.62 and 13.3% respectively.

  • •

    The bacteriocin PB2 is a proteinous bacteriocidal agent with a molecular weight of 4.87 kDa, with optimum activity at 40 °C and pH 5.0. Bacteriocin PB2 lost its activity on treatment with proteinase K and exposure to UV radiation (after 6 h) but was observed to have stable activity in the presence of organic solvents.

  • •

    The amplicon size of Pediococcus pentosaceus PB2 was 727 bp. P. pentosaceus was found to harbor two plasmids targeted from primers designed from operon encoding pediocin PA-1 gene. The molecular sizes of the plasmids are 0.9 and 1.2 kb. The curing analysis showed that the bacteriocidal activity of P. pentosaceus is derived from the presence of the plasmid present.

Keywords: Bacteriocin PB2, Pediococcus pentosaceus PB2, Pito production, Sorghum, Characterization

Abstract

Lactic acid bacteria (LAB) have been known to possess bacteriocidal activity resulting from ribosomally synthesized antimicrobial peptides called bacteriocin. This study focused on the characterization of the bactericidal activity of bacteriocin PB2 and comprehensive detection of the pediocin ped-A1 from Pediococcus pentosaceus obtained from fermented sorghum beverage, Pito, in Nigeria against Escherichia coli ATCC 25922 and Listeria monocytogenes ATCC 15313. Bacteriocin PB2 was purified in a 2-step purification using 80% NH4 (SO4)2, and Carboxymethyl-Sephadex G-50 column chromatography to achieve a 12.62% purification fold. The physicochemical properties of purified bacteriocin were characterized being treated at different temperatures (20 – 120 °C), pH (2.0 – 10.0), with different detergents and enzymes (sodium dodecyl sulphate (SDS) urea, ox-gall, and proteinase K and RNase A), organic solvents (ethanol, phenol, acetone, chloroform and isoamyl alcohol), and exposure to ultraviolet (UV) radiation (2–12 h) respectively. The molecular weight of the bacteriocin PB2 was determined to be 4.87 kDa. The antibacterial activity of bacteriocin PB2 was optimum at 40 °C and pH 5.0. The bacteriocin PB2 lost its activity on treatment with proteinase K and exposure to UV radiation (after 6 h) but was observed to have stable activity in the presence of organic solvents. Also, P. pentosaceus PB2 harbored two plasmids, 0.9 and 1.2 kb which when cured resulted in the loss of the antimicrobial activity. The mRNA transcript for pedA was detected in P. pentosaceus PB2, but not in the cured derivative, confirming the expression of the plasmid ped-A1 gene in PB2. This study validates our previous study that the PB2 strain of Pediococcus pentosaceus isolated from fermented sorghum, Pito, may be used as a probiotic toward clinically important enteropathogenic bacteria. This peptide is a potential agent for use as an alternative antibacterial agent for the treatment of drug-resistant strains of bacterial infection.

1. Introduction

Sorghum (Sorghum bicolor) is a cereal for the production of a good number of common staple foods in West Africa. In the international market, especially in the United States and Japan, sorghum has gained headway as it is gluten-free and possesses resistant starch, high fiber content, and bioactive components with antioxidant properties [1]. According to the Systematic Survey of Agricultural Production, in Brazil, the number of tons of cereal produced increased by more than 15% between 2018 and 2019 [2]; in the same country, it is being used as a nutrition source for animals [1]. Chadalavada et al. [3] reported sorghum as one of the staple foods to be drought tolerant, one that can be grown in arid and semiarid regions of the world. In Nigeria, sorghum is grown in the far Northern states under semi-arid conditions—Adamawa, Bauchi, Benue, Borno, Gombe, Jigawa, Kaduna, Kano, Katsina, Kebbi, Kogi, Kwara, Nasarawa, Niger, Plateau, Sokoto, Taraba, and Zamfara States; it is used for the production of fermented foods and beverages such as Koko, Furo-furo, Maasa, Fura, Ogi-baba, and Pito.

In our previous study, we determined microorganisms associated with Pito processing [4]. They were isolated and identified utilizing microbial, biochemical, and genomic techniques to be Lactobacillus plantarum and Pediococcus pentosaceus [4]. The aforementioned organisms are examples of lactic acid bacteria (LAB) [4]. LAB has a long history of application in fermented foods because of its beneficial influence based on its nutritional, organoleptic, safety, and preservative characteristics [4], [5], [6], [7], [8]. The involvements of LAB in Pito and similar traditional sorghum-fermented foods in Nigeria like Ogi-baba is based on the spontaneous fermentation process; a deliberate addition of the isolated microorganisms as starters to the food matrix would result in a high degree of control over the fermentation process to yield a more standardized end product. During fermentation, LAB displays a range of biochemical and metabolic activities such as the synthesis of organic acids and bioactive molecules such as ethanol, formic acid, fatty acids, hydrogen peroxide (H2O2), and diacetyl [4]. The screening of LAB has revealed that besides metabolic activities, they may also exhibit probiotic potentials [4,9,10]. This class of bacteria produces inhibitory substances that impede the growth and survival of other bacteria. Strains of LAB that display antimicrobial activities usually produce bacteriocin that inhibits the growth of pathogenic and food spoilage microorganisms, thereby ensuring the safety of the product [11].

These bacteriocins are known to be ribosomal-synthesized, small, heat-stable antibacterial peptides with diverse modes of bactericidal activities against genetically related strains and species [12,13]. As established, most LAB are bacteriocinogenic, and may be isolated from milk, other dairy products, sorghum, and its products [4,14]. The nomenclature of bacteriocin is sometimes derived from its source and mechanism. Colicin is the name given to bacteriocins from Escherichia coli, wamericin is derived from Staphylococcus wameri, and bacteriocins from LAB are designated as lantibiotics [9]. Also, bacteriocin isolated from Pediococcus spp. is called pediocin. Pediocin AcH/PA-1 has been reported to be the most studied bacteriocin (non-modified, non-lantibiotic peptides) [9]. This further gives an insight that the bacteriocidal expression of P. pentosaceus strains would make them suitable as preservatives for foods, crops, and livestock [15]. Biological treatment towards preservation may sustain the quality and general safety of these products instead of chemical treatment. P. pentosaceus also inhibits enteric pathogenic bacteria, improves gastrointestinal tract microflora balance, and adheres to the mucosal surfaces of hosts [4].

In addition, when our research group previously isolated and identified P. pentosaceus PB2 from the well-known Nigerian Sorghum fermented products, Pito and Ogi-baba [4], the same strain was deposited in GenBank (NCBI KP883297) and submitted to the Laragen Incorporation, 10601 Virginia Ave, Culver City, CA90232, California, USA. We found P. pentosaceus PB2 to be acid tolerant, bile salt tolerant, lactic acid homofermenter, β-galactosidase producer, resistant to Oxacillin 5 μg, Streptomycin 10 μg and Tetracycline 25 μg and Chloramphenicol 25 μg [4]. Also, P. pentasoceus PB2 produced antibacterial chemical agents like lactic acid, diacetyl, and hydrogen peroxide. It also exerted its bactericidal effect on other bacteria, namely, Listeria sp., Bacillus sp., Staphylococcus sp., E. coli, H. pylori, Pseudomonas sp., Klebsiella sp., and Salmonella sp [4]. As a result of the bacteriocidal activity of some bacteria, bacteriocin production is now becoming a viable therapy option for both multidrug-resistant and chronic illnesses [16].

Previous research works have primarily shown the bacteriocidal effect of other strains of Pediococcus isolated from other sources but not from Pito and the PB2 strain. Mathys et al. [9] first reported bacteriocin produced by a human intestine Pediococcus acidilactici isolate, then a new real-time Polymerase Chain Reaction (PCR) assay was successfully developed to indicate the large distribution of pedA-containing strains in newborn faeces samples. Halami et al. [17] purified and characterized bacteriocin from Pediococcus pentosaceus ACCEL from vegetable sources. Recently from a characterization study, it was highlighted that the antimicrobial peptide from Pediococcus pentosaceus N33 strain isolated from pickles contained carotenoid pigments displaying good inhibitory effects against Brevibacillus brevis and Lysinibacillus fusiformis strains [16]. Also, Pediococcus pentosaceus NCDO 990 was found to exert similar effects in reducing the growth of Aeromonas hydrophila CAIM 347 at 12 h of interaction [18]. This manuscript presents the antimicrobial activity of P. pentasoceus PB2, isolated from the sorghum-fermented food, Pito, while detecting the pediocin gene ped-A1. There is currently no report in the open literature on the characterization of bacteriocin PB2 and the detection of the pediocin gene ped-A1 from Pediococcus pentosaceus PB2 strain obtained from Pito.

This work intends to provide a foundational basis for further investigation to improve the quality and medical applicability of the sorghum-fermented product. Results from this study can be instrumental in addressing the increasing rate of antimicrobial resistance (AMR) in Sub-Saharan Africa and globally. For a long time now, practitioners of traditional medicine administer Pito in the treatment of diseases (like digestive tract diseases including infectious diarrhea and inflammatory digestive tract diseases) which are now difficult to treat as a result of AMR. Also, computational studies elucidating the inhibitory potentials of these peptides against protein targets of disease-causing microbes could be done to further gain insights into the mechanism of inhibition from a molecular scale [19,20]. The findings from this paper would be of great relevance to indigenous and international researchers, pharmaceutical, food and nutraceutical industries, medicinal chemists, pharmacologists, pharmacists, medical practitioners, and policymakers in the area of food, protein, and probiotics.

2. Materials and methods

2.1. Bacterial strains

The laboratory probiotic strain Pediococcus pentosaceus PB2 used in this study was isolated from Pito as described in our recent finding [4]. The PB2 strain was identified using genomic, biochemical, and microbial tools. It was then deposited in GenBank (NCBI KP883297) and submitted to the Laragen Incorporation, 10601 Virginia Ave, Culver City, CA90232, California, USA [4]. Two reference strains, Escherichia coli ATCC 25922 and Listeria monocytogenes ATCC 15313, obtained from Microbiologics, Medimark Europe, France, were also used.

2.2. Bacteriocin PB2 purification

2.2.1. Screening for bacteriocin production

Selected Pediococcus pentosaceus PB2 isolates were cultured in MRS broths and screened for bacteriocin production according to the method described by Van Reenen et al. [21]. The isolates were grown in MRS broths with lactose as carbon source at 37 °C for 18 h. The cell-free supernatant (CFS) was obtained by centrifuging at 10,000rpm for 5 min at 4 °C, and was adjusted to pH 7.0 with 1 M NaOH. Antimicrobial activity was monitored by using the agar-well diffusion test method [22]. All strains were then stored at – 4 °C.

2.2.2. Ammonium sulphate precipitation of bacteriocin

Ammonium sulphate NH4 (SO4)2 was added gently to the CFS to obtain 80% saturation with continuous stirring using a high-speed magnetic stirrer at 4 °C. The ammonium sulphate precipitated protein which was obtained by centrifuging at 14,000 rpm for 30 min. The obtained precipitate was collected and dissolved in sterile MiliQ water. Dialysis tubing (Spectrum Labs, USA) was utilized to dialyze samples for 8 h at 4 °C against phosphate buffer. The dialysate was then stored at − 20 °C overnight.

2.2.3. Carboxymethyl-sephadex G-50 column chromatography purification

The dialyzed sample was passed through Carboxymethyl-Sephadex G-50 Column Chromatography. Elution was done by the gradient of 10 mM phosphate buffer and 0.1 – 1 M NaCl buffer (pH 7.0 ± 0.2). Fractions were collected at a flow rate of 0.4 ml/min and were monitored at 280 nm using the UV spectrophotometer for the activity [23].

2.3. Characterization of bacteriocin PB2

2.3.1. Protein estimation and peptide assay

The biuret method was used to determine the protein level of the bacteriocin samples obtained after the purification process (HiPerR Protein Estimation Teaching kit). Agar well diffusion method was used to determine the antibacterial activity of the bacteriocin samples against E. coli ATCC25922 [24]. Antibacterial activity is measured in arbitrary units per milliliter (AU/ml). One arbitrary unit is defined as the reciprocal of the highest dilution that exhibits a minimum detectable zone of inhibition [25]. The activity, yield, and fold purification of bacteriocin obtained after various purification steps were also calculated.

2.3.2. Determination of molecular weight of bacteriocin PB2 by tris-tricine SDS-PAGE

The sample depicting a single peak protein from the CM-ion chromatography was subjected to Tris-Tricine SDS-PAGE analysis to determine its molecular weight [26,27]. The purified protein along with molecular weight markers (Dual Xtra standards, BIORAD, USA) was initially electrophoresed at 40 V for 30 min, thereafter at 100 V for 120 min. After electrophoresis, the gel was stained with Coomassie® Brilliant Blue and destained by washing overnight with a mixture of acetic acid-methyl alcohol-water (5:5:1 v/v).

2.3.3. Determination of the effect of different temperatures on bacteriocin PB2

The effect of temperature on the bacteriocin activity was determined by incubating the purified bacteriocin at pH 4.5 at 20, 30, 37, 40, 60, 80, and 100 °C, respectively for 2 h [28]. After treatment, the bacteriocin PB2 samples were kept at room temperature for 30 min and were then tested for antimicrobial activity against reference strains by using the agar diffusion test [22].

2.3.4. Determination of the effect of different pH on bacteriocin PB2

In determining the effect of pH on the activity of bacteriocin PB2 the pH of the purified bacteriocin PB2 was adjusted from 2.0 to 10.0. After 2 h of incubation at room temperature, the samples were tested for antimicrobial activity against reference strains by using the agar diffusion test [22].

2.3.5. Determination of the effect of enzymes and detergents on bacteriocin PB2

To the bacteriocin PB2, 1% (w/v) detergent including sodium dodecyl sulphate (SDS), urea, ox-gall, and hydrolytic enzymes including proteinase K and RNase A was separately added. The untreated bacteriocin was considered as a control, and its activity was taken as 100%. All reactions containing detergents were incubated at 37 °C for 6 h, then tested for antimicrobial activity against reference strains by using the agar diffusion test [22]. Also, the bacteriocin PB2 was adjusted to pH 5.0 with 1 M NaOH. A 1 ml aliquot of the CFS was incubated for 2 h on adding 1 mg/ml and 0.1 mg/ml of each of proteinase K and RNase (Sigma). The bacteriocin activity was assayed against reference strains by the agar-well diffusion method [22].

2.3.6. Determination of the effect of various organic solvents on bacteriocin PB2

The bacteriocin PB2 was treated with 50% v/v organic solvents including ethanol, phenol, acetone, chloroform, and isoamyl alcohol. The mixture was incubated at 37 °C for 2 h. After the incubation, the solvent was evaporated by aeration for 30min, then the antimicrobial activity against the reference strains was determined using the agar-well diffusion assay [22].

2.3.7. Determination of the effect of ultraviolet (UV) radiation on bacteriocin PB2

The bacteriocin PB2 from the isolate was placed in a sterile glass and exposed to short-waive UV light at a distance of 30 cm [22]. The duration of exposure to UV radiation ranged from 2 to 12 h. At 2 hr intervals, bacteriocin activities were analyzed by the agar-well-diffusion method against the different reference strains.

2.4. Determination of genes encoding bacteriocin production

2.4.1. Screening for the presence of bacteriocin gene

P. pentosaceus PB2 extrachromosomal DNA elements were isolated using a modified approach based on Anderson and McKay [29] for small-scale plasmid separation. A 9.5 μl aliquot of mutanolysin (1500 U ml-1 Sigma-Aldrich Chemie GmbH) was added to the lysis solution (solution B) for cell lysis, and the plasmid DNA was resuspended in 1 x TE buffer. Finally, 10 g RNase A was used to degrade the RNA (Sigma-Aldrich Chemie GmbH). Bacteriocin gene-specific primers, Pedpro (5′-CAA GAT CGT TAA CCA GTT T-3′) and Ped 1041 (5′-CCG TTG TTC CCA TAG TCT AA-3′) were designed from the operon encoding pediocin PA-1 (accession number M83924) and synthesized by Genosys Biotechnologies (Europe) Ltd (Cambridgeshire, United Kingdom) [30]. Bacteriocin PB2 genes were amplified from the extracted genomic DNA of P. pentosaceus PB2 with the identified gene-specific primers. PCR reactions were performed using a BioRad® PCR (BioRad Thermal Cycler, USA). Amplification conditions were as follows: initial denaturation at 94 °C for 5 min, 35 cycles of 5 min at 94 °C, and 10 s at 61 °C, followed by an increase to 72°C for 2 min. The final extension of the amplified product was at 72 °C for 75 min [43]. The obtained amplicons were purified with a QIAquick PCR Purification Kit (Qiagen), following the manufacturer's instructions, and submitted to sequencing at the Laragen Inc. Culver City California. The amplified product was visualized in a 1.2% (w/v) agarose gel stained with ethidium bromide.

2.4.2. Curing conditions

Ethidium bromide at a concentration of 125 µg/ml was added to bacteriocin-producing pediococcal cells to exert environmental pressures on cells to favor plasmid loss at a growth temperature of 37 °C for 48 h. After incubation time, the samples were tested for loss of bacteriocin phenotype (PB2-). Strains found to be PB2- were subjected to plasmid analysis along with PB2+ strains as controls. PB2- variants of P. pentosaceus termed plasmid-free cells were inoculated into the overnight culture of reference isolates.

2.5. Statistical analyses

Results from various sets of experiments were statistically examined by determining the mean, standard deviation (SD), and standard error (SE) from a minimum of three observations whenever necessary. All the statistical analysis was carried out using GraphPad Prism version 5.0 at a significance level of p ≤ 0.05.

3. Results

3.1. Screening of bacteriocin production

Treatment with the CFS of P. pentasoceus PB2 showed inhibitory activity against the reference strains. Fig. 1 illustrates that the CFS of the isolate PB2 isolated from Pito exhibited antagonistic activity against the E. coli ATCC 25922 and L. monocytogenes ATCC 15313 compared to the LAB, L. plantarum which did not inhibit the activity of E. coli ATCC 25922.

Fig. 1.

Fig 1

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In vitro antagonistic activity of P. pentosaceus PB2 against reference bacterial strains OB6 = L. plantarum; PB2 = P. pentosaceus PB2; CTRL = MRS broth without bacterial cells. Plate NA contains reference bacterial strain, L. monocytogenes ATCC 15313; plate B contains reference bacterial strain, E. coli ATCC 25922.

3.2. Characterization of bacteriocin PB2

3.2.1. Carboxymethyl-Sephadex G-50 column chromatography purification

For purification of bacteriocin, the precipitated protein was subjected to ion-exchange/gel-filtration chromatography using Carboxymethyl-Sephadex G-50 and eluted with 10 mM phosphate buffer, then with gradient salt (0.1 – 1 M NaCl; pH 7.0 ± 0.2). The elution profile depicts four distinct peaks at 280 nm; of the four peaks which are proteins, the second peak (fractions 15–43) showed maximum activity at 600 nm (Fig 2). Subsequently, the pooled fraction from the second peak was used in further characterization. The activity of bacteriocin PB2, yield and fold purification obtained after various purification steps are provided in Table 1.

Fig. 2.

Fig 2

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Elution pattern of the bacteriocin PB2 through carboxymethyl-sephadex G-50 chromatography.

Table 1.

Summary of purification table of the bacteriocin PB2.

Volume(Ml) MIC(µg/ml) Total protein(mg/ml) Specific activity(U/mg) Total activity(U) Purification fold Recovery yield(%)
Crude Extract 100 10 48.3 0.21 1,000.00 – 100
Ammonium Sulphate /Dialysate 50 22.22 17.35 1.28 399.96 6.10 40.0
Carboxymethyl-Sephadex C-50 24 5.56 2.10 2.65 133.44 12.62 13.3
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MIC = minimum inhibitory concentration

3.2.2. Determination of molecular weight of bacteriocin PB2 by tris-tricine SDS-PAGE

For determining the molecular weight of bacteriocin, the sample collected from Carboxymethyl-Sephadex G-50 was run on Tris-Tricine SDS PAGE. A distinct band of 4.87 kDa was seen after Coomassie brilliant blue staining. (Fig. 3) Further, to confirm the purity of the protein band corresponding to 4.87 kDa, the crude extract was overlaid on a different lane 3 of the gel in situ and the results are shown in Fig. 3.

Fig. 3.

Fig 3

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Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) (12%) showing the molecular weight of purified bacteriocin from P. pentosaceus PB2 (stained with Coomasie blue) based on the relative mobility of the protein. Lane 1: the molecular weight markers (5 – 250 KDa; BIORAD, USA); Lane 2: active eluent of carboxymethyl-Sephadex G-50 chromatography; Lane 3: peptide bands of crude extract; Lane 4: Phospate buffer without peptide.

3.2.3. Effect of different temperatures on bacteriocin PB2

The effect of temperature ranging from 20 °C to 100 °C on the antibacterial activity of the bacteriocin against reference strains, E. coli and L. monocytogenes, was studied and the results are given in Fig. 4. The effect of temperature was observed to have similar patterns in the reference strains; however the bacteriocin PB2 had higher activity against L. monocytogenes. It was observed that the bacteriocin PB2 was found to be heat sensitive as it could retain the antibacterial potential being treated between 30 and 80 °C (Fig. 4). However, at temperature regimes of 20 °C and 100 °C, the bacteriocin PB2 was observed to be of least activity. At 40 °C, the optimum activity of the bacteriocin was observed.

Fig. 4.

Fig 4

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Effect of different temperatures on the activity of bacteriocin PB2 against reference strains.

3.2.4. Effect of different pH on bacteriocin PB2

The influence of pH ranging from 2 to 10 on the activity of bacteriocin PB2 against reference strains was studied and the results are given in Fig. 5. The effect of pH was observed to have similar patterns in the reference strains. The bacteriocin PB2 had higher activity against E. coli through varying pH. Bacteriocin PB2 retains its antibacterial property at pH 3.0 through pH 9.0. Bacteriocin PB2 demonstrated the maximum bacterial activity at pH 5.0 against E. coli a and L. monocytogenes. However, at extreme pH 10 bacteriocin PB2 lost its activity.

Fig. 5.

Fig 5

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Effect of different pH on the activity of bacteriocin PB2 against reference strains.

3.2.5. Effect of detergents and enzymes on the bacteriocin PB2

The effect of detergents and enzymes on the activity of the bacteriocin PB2 against the reference strains was determined and the results are shown in Fig. 6. It was observed that the bacteriocin lost its antibacterial activity on treatment with Proteinase K. Bacteriocin PB2 demonstrated stability on treatment with SDS, urea, Ox-gall, and Rnase (Fig. 6).

Fig. 6.

Fig 6

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Effect of various detergents/hydrolytic enzymes on the bacteriocin PB2 activity. Blue column represents activity against E. coli; red columns represent activity against L. monocytogenes.

3.2.6. Effect of various organic solvents on the bacteriocin PB2

The effect of various organic solvents on the activity of the bacteriocin against reference strains was determined and the results are shown in Fig. 7. It was observed that the activity of bacteriocin was stable on treatment with the organic solvents (ethanol, phenol, acetone, chloroform, and Isoamyl alcohol) (Fig. 7).

Fig. 7.

Fig 7

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Effect of various organic solvents on the bacteriocin activity. Blue column represents activity against E. coli; red columns represent activity against L. monocytogenes.

3.2.7. Effect of UV radiation on bacteriocin PB2

The influence of UV radiation on bacteriocin PB2 was studied. A similar pattern was observed against the reference strains with the time of exposure to the radiation. An indirect relationship was observed between the duration of exposure to UV radiation and the bacteriocidal activity of bacteriocin PB2 toward the reference strains. Bacteriocin PB2 demonstrated maximum bacteriocidal activity towards L. monocytogenes compared to E. coli (Fig. 8).

Fig. 8.

Fig 8

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Effect of duration of exposure to UV radiation on the activity of bacteriocin PB2.

3.3. Purity of amplicon

Fig. 9 shows the purity and molecular weight of the amplicon from the probiotic strain PB2 which was later sequenced and submitted to Laragen Corporation [4]. A distinct band of 727 bp was observed (Fig. 9) from the PB2 strains (PB2a and PB2b).

Fig. 9.

Fig 9

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Result showing the purity and molecular weight of the amplicon from P. pentosaceus PB2 strain.

3.4. Determination of genes encoding bacteriocin production

3.4.1. Screening for the bacteriocin gene

After the plasmid isolation procedures of cured and uncured P. pentosaceus, amplification with primers of operon encoding pediocin PA-1, then visualization in a 1.2% (w/v) agarose gel stained with ethidium bromide was conducted. Afterward, the sample was run on the agarose gel along with the 1 Kb ladder. In the uncured P. pentosaceus strain (PB2+), plasmid screening revealed the presence of two plasmids with 0.93 and 1.2 kb sizes (Fig. 10). In the cured P. pentosaceus strain (PB2-), plasmid screening revealed that there were no plasmids (Fig. 10). Fig. 11 validates this result. Cured cells of Pediococcus pentosaceus (PB2-) didn't show any bacteriocin activity on the test strains, Listeria monocytogenes and Escherichia coli; while the uncured PB2+, displayed bacteriocin activity on the test strains.

Fig. 10.

Fig 10

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Plasmid profile and curing of plasmid from P. pentosaceus PB2. PB2+ represents uncured strain; PB2- represents cured strain.

Fig. 11.

Fig 11

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Plasmid curing analysis of bacteriocin-producing P. pentosaceus PB2. PB2+: uncured; PB2-: cured.

4. Discussion

There is an increasing emergence of drug-resistant pathogens in the world. According to WHO [31], antimicrobial resistance (AMR) is one of the top ten global public health threats that humanity is facing. In Nigeria, AMR is compromising the treatment of infectious diseases like malaria, urinary tract infections, tuberculosis, digestive tract diseases including infectious diarrhea and inflammatory digestive tract diseases, and others. This is engendering an increased diversion from conventional medicine to traditional medicine. There are so many herbal and indigenous products that are being used and are effective against some of these diseases. A typical example is sorghum-fermented products, Pito and Ogi-baba which have been earlier reported to possess probiotic potentials against enteric-pathogenic diseases resulting from the presence of LAB [4]. This treatment option may be safer and cheaper compared to the high cost and potential toxicity associated with the use of chemical treatment.

LAB are found in fermented food like sorghum, meat, fish, potatoes, dairy products, pickles, fruits, humus, and the intestines of people and animals [32]. They are reported to possess antimicrobial properties [33]. Todorov et al. [13] reported that LAB like Lb. plantarum ST8SH has strong antimicrobial activity against L. monocytogenes ScottA, Lb. sakei ATCC 15521 and E. faecalis ATCC 19433 resulting from its bacteriocin.

Correspondingly, the results of this study revealed that P. pentosaceus PB2 (KP883297) isolated from Pito exhibits antagonistic activity against E. coli ATCC 25922 and L. monocytogenes ATCC 15313. This antagonistic property of P. pentasoceus PB2 defines its ability to inhibit the growth of Gram-positive and Gram-negative bacteria with a clear demonstration of bactericidal activity resulting from the acid production and bacteriocin effects. This is in agreement with reports of both Vizoso et al. [34] and Milette et al. [35] who independently deduced that the bactericidal effect of LAB could be attributed to its production of organic acids and bacteriocin-like proteins. Also, Şimsek et al [36] and Ghosh et al. [10] had similar findings with Pediococcus sp. E5 and P. pentosaceus GS4 respectively. Hence, it can be inferred that the synergistic effect of lactic acid, diacetyl, hydrogen peroxide, and the bacteriocin produced by P. pentosaceus PB2 are responsible for antimicrobial activity.

This will be the first study to report the production of bacteriocin PB2, produced by P. pentosaceous PB2 isolated from Pito, a sorghum-based food in Nigeria. This study validates the claim that LAB from fermented sorghum products may show bacteriocidal activity resulting from antibacterial peptides [4]. Bacteriocin from the PB2 was purified and characterized using enzymology and biotechnology techniques [37]. A 2-step purification using 80% NH4 (SO4)2, and Carboxymethyl-Sephadex G-50 column chromatography was used to achieve a 12.62% purification fold and 13.3% yield of bacteriocin PB2. There are several reported protocols documented for bacteriocin purification some require multiple purification steps, while others do not [13,38,39]. Ladha and Jeevaratnam [39], then Vidhyasage and Jeevaratnam [40] likewise reported a high-fold purification.

Subsequently, the bacteriocin from the isolate PB2 was isolated, purified, and characterized and shown to have specific bacteriocidal activity of 2.64 U/mg with molecular weight (Mw) of 4.87 kDa. The molecular weight (Mw) of bacteriocins isolated from different sources varies, and they range approximately from 2700 to 16,599 Da [10,16]. The Mw shown by the bacteriocin ST8SH was estimated to be 5000 Da as reported by Todorov et al [13]. Similarly, a bacteriocin from Pediococcus pentosaceus CFR B19 had an Mw of ∼4.8 kDa [41], and pediocin PD-1 from Pediococcus damnosus NCFB1832 had an Mw of ∼2.6 kDa [42]. Bacteriocin PB2 is observed to have a larger molecular size when compared to bacteriocin PA-1 from Pediococcus pentosaceus strains [43], and pediocin PA-1 from P. acidilactici UVA1 [9]. Therefore, bacteriocin PB2 may be said to belong to class II of bacteriocins which are of small molecular weight < 10 kDa [12].

Physicochemical characterizations of bacteriocin PB2 showed stability at pH 3, 4, 5, 6, 7, 8, and pH 10, with the optimum pH of 5 while retaining activity after 2 h of incubation. Compared to the report by Todorov and Dicks [30] where the bacteriocin ST44AM was active at pH ranges 2 and 12, and Ladha and Jeevaratnam [39] where bacteriocin LJR1 was active at pH 4, 7, and 10, this study suggests that bacteriocin PB2 is active at a broad range of pH. The results show that PB2 can be stable at neutral, acidic, and basic pH.

Bacteriocin PB2 lost its antimicrobial activity when treated with proteinase K. This indicates the proteinaceous nature of the bacteriocidal agent [39,44,45]. The detergents were found to have no effects on the antimicrobial activity of bacteriocin PB2. Similar results to the detergent effect has been reported previously with bacteriocins ST44AM and LJR1 [39]. Also, the bacteriocin was found to be heat stable at temperatures 30, 40, 50, 60, and 80 °C for 2 h. Barathiraja et al. [46] highlighted that purified bacteriocin from the rumen liquor of a goat was unstable at 100 °C for 30 min, and stable at 30 °C. Bacteriocin PB2 was also observed to be stable upon the addition of organic solvents (ethanol, phenol, acetone, chloroform, and Isoamyl alcohol), surfactants, and detergents. This is in agreement with the findings of Kumar et al. [50] where they found bacteriocin LD4 from L. plantarum LD4 strain retaining activity after treatment with different organic solvents. Likewise, enterocin HDX-2 was treated with different organic solvents, surfactants, and detergents and was observed to be stable [26].

Exposure to UV light can alter the nature, structure, and function of a protein. Contrarily, bacteriocin PB2 retained its activity after six hours of exposure. Mahreen et al. [47] in their study observed that bacteriocin from L. helveticus retained activity following 30 min of UV exposure. Also, Shokri et al. [48] reported that BLIS from E. faecium DSH20 retained its activity after 30 min of exposure to UV light, the activity was retained. The result from this study shows that bacteriocin PB2 has better activity retention upon exposure to UV radiation.

In our study, isolation and amplification of genes encoding for the bactericidal activity of P. pentasoceus PB2 were performed targeting pediocin PA-1 genes in the total DNA. An amplicon of 727 bp was observed on agarose gel revealing its purity (Fig. 9), thereby indicating the production of a type of pediocin PA-1, which is a wide-spectrum bacteriocin from LAB. Murua et al. [49] used a similar method targeting plantaricin W, plantaricin S, plantaricin NC8 and pediocin PA-1 genes in L. plantarum LP08AD, and generated positive results only with the primers for plantaricin W, inferring that the organism is a plantaricin W producer.

Plasmid DNA preparations revealed two bands, from strain PB2, on agarose gel electrophoresis which are the two extrachromosomal DNA elements at 0.93 kb and 1.2 kb (Fig. 10) from the uncured plasmid strain PB2+. Similarly, Mathys et al. [9] associated the antimicrobial activity of Pediococcus acidilactici UVA1 with the presence of plasmids; they reported the presence of two plasmids at 9.5 kb and at > 10 kb, and found that the strain bac- lost its bacteriocidal activity after plasmid curing. In this present study, no extrachromosomal DNA was observed in strain PB2-, a non-inhibitory derivative of P. pentosaceus PB2 produced after plasmid curing. This was verified by conducting a plasmid curing screening on the agar (Fig. 11). The results infer that the plasmid encodes for the bacteriocin PB2 of P. pentosaceus PB2, hence, the bacteriocidal activity if the LAB.

5. Conclusion

Bacteriocin PB2, isolated from fermented sorghum product- Pito, was identified and characterized using a polyphasic approach. To our knowledge, Pediococcus pentosaceus PB2 is the first bacteriocin-producing Pediococcus strain isolated from fermented sorghum-based food in Nigeria, Pito. The results of the study show that P. pentosaceous exhibits antagonistic properties because of its identified bacteriocin. Some of the properties of the purified bacteriocin PB2 are a molecular weight of 4.87 kDa, an optimum temperature and pH of 40 °C, and pH of 5.0. Also, bacteriocin PB2 lost its activity on treatment with proteinase K and was observed to have stable activity in the presence of organic solvents. Remarkably, it retained its activity after six hours of exposure to UV light. P. pentosaceus was found to harbor two plasmids targeted from primers designed from operon encoding pediocin PA-1 gene whose molecular sizes of the plasmids are 0.9 and 1.2 kb. Biocomputational elucidation of the inhibitory potentials of bacteriocin PB2 against clinical protein targets of disease-causing microbes could be investigated for further insight into the mechanism of inhibition. This identified bacteriocin has great potential as an alternative treatment for drug-resistance strains of bacterial infections. This equally has the potential for bio-preservation against food spoilage. More studies on the bacteriocin PB2 will have to be done for the aforementioned to be realized.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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