Potential of bacteriocins produced by probiotic bacteria isolated from tiger shrimp and prawns as antibacterial to Vibrio, Pseudomonas, and Aeromonas species on fish

Sample collection

A total of ten isolates of probiotic bacteria were collected, consisting of five isolates from tiger shrimp and five isolates from prawn⁹, which were from the collection of the Marine Microbiology Laboratory of the Faculty of Fisheries, University of Riau, Indonesia. ID numbers for each isolates are (GenBank): H1: KY995544, H2: KY995545, H3: KY995546, H4: KY995547, H5: KY995548, W1: KY995549, W2: KY995550, W3: KY995551, W4: KY995552 and W5: KY995553. This study was conducted from April-September 2016. Pathogenic gram-negative bacteria, Vibrio alginolyticus was ontained from Brackish water Research Institute in Jepara, Indonesia, while Aeromonas hydrophila and Pseudomonas stutzeri were purchased from Quarantine Centre in Pekanbaru, Indonesia.

The Nutrient Agar (NA; Merck, Kenilworth, NJ, USA, Cat.No. 1.05450.0500), Nutrient Broth (NB; Thermo Scientific Oxoid, Waltham, MA, USA, Cat.No.CM0001), Thiosulfate Citrate Bile Sucrose (TCBS; Merck, Cat.No.1.10263.0500), Trypticase Soy Agar, (TSA;Merck, Cat.No. 1.05458.0500), Trypticase Soy Broth (TSB, Merck, Cat.No. 1.05459.0500) were used in this study.

Glycerol, glasswool, paper disc 6mm (Macharey-Nagel, Düren, Germany, MN827ATD), gel filtration chromatography using Sephadex LH-20 (Sigma-Aldrich, St-Louis, MO, USA - Offer sigma-GE17-0090-01), methanol and other chemicals were used in accordance with the laboratory procedures.

Refreshment of probiotic bacteria

Probiotic bacteria can grow in acidic conditions so each process was performed in physiological acidic pH solutions. In this study, probiotic bacteria were grown at pH 4. The probiotic bacteria was cultured in the following manner: a tube filled with the 500μL physiological solution of pH 4 was inoculated with probiotic bacteria aseptically at concentration of 10⁸ cfu/mL. The mixture was homogenized and left to stand for 5 minutes. One loop full of the mixture was streaked on NA media. Media containing the bacteria were incubated at 37 °C for ± 24 hours.

Production of bacteriocins

Liquid medium, nutrient broth of 300 mL volume was used for the production of bacteriocins¹⁰. The Medium was sterilized in an autoclave at a pressure of 15 psi, at 121°C for 15 minutes. After one day at room temperature, the medium was inoculated with 5% of probiotic bacteria that had previously been incubated overnight at the optical density (OD) at 600nm ~ 0.1 (v/v) which was equivalent to 10⁷ CFU/mL (OD measured with Thermo Scientific GENESYS 10S Uv-Visible)¹¹. The inoculated media was then fermented in a shaking incubator at a speed of 150 rpm for ± 24 hours at 37 °C. After incubation, the fermented medium was cooled in a refrigerator at 5–10°C for ± one hour. The crude bacteriocin extract from the medium by centrifugation (Hitachi, Tokyo, Japan – CS150FNX) at 10,000 rpm for 10 min at 4°C. The supernatant was then separated by filtration through glasswool. The cell-free supernatant (extracted bacteriocins) produced, was tested for activity against pathogenic bacteria by using a disc diffusion method. Some of the supernatants was precipitated by the addition of 80% salt ammonium sulfate [(NH4) 2SO4], and then was tested for activity in the same manner, and finally was analyzed by using High-performance liquid chromatography (HPLC).

Purification of bacteriocins

Bacteriocin was precipitated from the crude extract by the addition of 80% salt ammonium sulfate [(NH4) 2SO4] in a cold condition (temperatures of 5 °C to 10 °C) while stirring gently to achieve 80% saturation and was then left overnight. The precipitate was then separated from the filtrate by centrifugation (Hitachi – CS150FNX) in a cold state at 13,000 rpm for 10 minutes. After centrifugation, the precipitate was added with 0.05 M phosphate buffer solution at pH 7.0, and it was ready to be used for the bacteriocins activity test. The rest was put in microtubes and was stored in a freezer (-20 °C).

Bacteriocin activity test

Bacteriocin activity was tested by the diffusion agar method by using a paper disc of 6 mm pore size against pathogenic bacteria V. alginolyticus, A. halophyla, and P. stutzeri. One mL of each of pathogen inoculum (OD 600nm ~ 0.1, which is equivalent to 10⁷ CFU/mL¹¹ was inoculated into a test tube containing 15 mL of NA medium and was then vortexed. The inoculated medium was poured into a Petri dish and was allowed to solidify. A total of 50 mL of bacteriocins (bacteriocin extract both before and after precipitation) was dropped on paper discs and was allowed to dry. Amoxsan® 30μg from local pharmacy in Pekanbaru Indonesia was used as positive control and sterile liquid medium as the negative control. The dried paper discs were placed on pathogenic-inoculated NA media. After incubation for ± 24 hours, the bacteriocin activities were indicated by clear zone formed around the discs. The diameter of the clear zones was measured by using calipers. Inhibitory activity against pathogenic bacteria of extracellular fluid was calculated as AU (Activity Unit). One AU/mL was the area of inhibition zone per unit volume of bacteriocinsamples tested (mm²/ml). Bacteriocin activity can be calculated using the following equation⁵:

Bacteriocin Activity (mm²/mL) = $\frac{Lz–Lc}{V}$

Gel filtration chromatography

Gel filtration or gel permeation is a protein separation technique based on molecular size¹². This technique used a column measuring 2.5 × 50 cm (Sephadex LH-20) as steady phase and methanol as mobile phase. A total of 9 g Sephadex LH-20 was weighed and then was added to 50 mL of methanol. The mixture was stirred gently until dissolved. The dissolved Sephadex was poured into the column until the marked limit. The eluent was collected in a beaker containing a mixture of Sephadex, it was then poured again into the column until Sephadex expanded and solidified for 20 minutes. Afterward, the bacteriocin was inserted into the column through the column wall carefully and waited until the sample penetrated through pores of the gel Sephadex LH-20 pores. The eluate was then collected in a marked vial as the first fraction, and the next eluent was collected in vials containing 20 drops until final-clear eluent reached. Small molecules will enter the pores of the gel Sephadex and move slowly, while the large molecules will move faster because it cannot enter and retaining the gel. Thus, the large molecules will emerge as the initial component. Products of each of separated-bacteriocin fractions were finally analyzed by using FTIR spectroscopy.

HPLC (high-performance liquid chromatography) analysis

Bacteriocins produced after precipitation in ammonium sulphate was then analyzed using HPLC. A spectrophotometer ultraviolet (UV) detector (Thermo Scientific Genesys 10S) was used for the analysis of bacteriocins at a wavelength of 210 nm and 250 nm. Wavelength selection was based on preliminary measurement using UV spectrophotometer in The Research Laboratory of Enzymes, Fermentation, and Biomolecular, following the research performed by Masuda et al.,¹³. The analysis used Shimadzu HPLC system of UFLC Shim Pack C18 series with column size of 4.6 mm × 250 mm (Shimadzu, Kyoto, Japan).

Statistical analysis

The data were subjected to one-way analysis of Variance (ANOVA)¹⁴ followed by Duncan multiple range testat significance levels of 95%. The analysis was performed using a SPSS ver.20 software.

Bacteriocins activities

Inhibitory activity of bacteriocins was expressed as the inhibition zone per unit volume of samples tested (mm²/mL). Table 1 shows the activity of bacteriocins extract of tiger shrimp against pathogenic bacteria V. alginolyticus, A. hydrophila and P. stutzeri. Statistical analysis indicated that the activity was not significantly different (P> 0.05), the highest activity was indicated by bacteria isolate H1 against V. alginolyticus. However, the activity was not significantly different (P> 0.05) from isolates H2 and H5, which was 674.65 mm²/mL, but it was significantly different (P> 0.05) from isolates H3 and H4 (Table 1). The highest activity of bacteriocin crude extract against A. hydrophila was found at 319.91 mm²/mL from isolate H4, which was not significantly different (P> 0.05) from isolates H2, H3, and H5. However, it was significantly different (P> 0.05) from isolate H1 (P <0.05). The highest inhibitory activity against P. stutzeri was indicated by isolate H5 which was significantly different (P <0.05) from four other bacteria.

After precipitation with ammonium sulphate [(NH4)2SO4], the activity of each of bacteriocins tends to change. The highest activity was performed by isolate H3 against V alginolyticus, that was 896.50 mm²/mL (Table 2). Meanwhile, the lowest activity was indicated by isolate H1 that was 321.47 mm²/mL. This condition was different from that which was before been precipitated with ammonium sulphate. The highest activity was indicated by isolate H1 against V alginolyticus (Table 1). This concluded that precipitation process resulted in the different effect on bacteriocin produced by each of tested bacteria.

The activity of the bacteriocins-crude extract of bacterial isolates from prawns against pathogens V. alginolyticus, A. hydrophila and P. stutzeri was not significantly different (P>0.05), and the highest activity was indicated by isolate W4 against A. hydrophila (746.95 mm²/mL). In comparison to bacteriocin produced by bacteria isolated from black tiger shrimp, of which the highest was indicated by isolate H1 against A. hydrophila, that was 674.65 mm²/mL. Therefore, the activity of bacteriocin produced by bacteria from prawns was higher than that of black tiger shrimp.

Values for W4 were not significantly different from isolates W2 and W5 (P>0.05), but it was significantly different from W1 and W3 (P <0.05). The highest activity of the bacteriocins-crude extract against V. alginolyticus was shown by isolate W4 (645.76 mm²/mL), which was significantly different (P<0.05) from other four isolates. Meanwhile, the highest activity of bacteriocins-crude extract against P. stutzeri was indicated by isolate W2 (412.73 mm²/mL), which was significantly different (P <0.05) from the other four isolates.

Bacteriocinactivities of bacterial isolates from prawns against all pathogens (V. alginolyticus, A. hydrophila, and P. stutzeri) were not significantly different (P>0.05). The highest activity was produced by isolate W2 against P. stutzeri, and the highest inhibitory activity was 1466.96 mm²/mL which was significantly (P> 0.05) different from other four bacteria isolates (Table 4). However, the activity of isolate W1 was not significantly different from isolate W5 (P> 0.05) against P. stutzeri (Table. 4). Overall, the activity of bacteriocin precipitated in ammonium sulphate {(NH4)2SO4} was higher than bacteriocin before precipitation in ammonium sulphate.

A quantity of 270 mL of bacteriocin-crude extract was obtained after centrifugation, and then it was precipitated with ammonium sulphate salt [(NH4)₂SO₄]. In the deposition process at salt saturation level of 80%, a total of 139.32 g of salt ammonium sulphate [(NH4) 2SO4] was added to the crude extract of bacteriocins. The precipitated bacteriocin was added with buffer in order to reach a volume of 3,375 mL bacteriocins.

HPLC analysis. High-Performance Liquid Chromatography (HPLC) is a chromatographic method that uses a reversed-phase system as its working system. This method was used to determine the purity level of a compound to be analyzed. Bacteriocins of tiger shrimp and prawns with high bacteriocin-activity values (H4 and W2), were then analyzed by HPLC as shown in Figure 1 and Figure 2. The figures showed that bacteriocin produced by each of probiotic bacteria was not a pure product indicated by the number of chromatogram peaks.

Figure 1. Chromatogram bacteriocins H4 of probiotic bacteria isolated from black tiger shrimp.

Figure 2. Chromatogram of bacteriocins W2 from probiotic bacteria isolated from prawns.

Gel filtration chromatography

Bacteriocins as a group of proteins, were separated by column chromatography on Sephadex LH-20 using methanol. The resulted eluate was initially appeared light brown in color in the column, and was collected in a vial which was then marked as fraction 1. After that, as the sample was in the middle of the column, the next eluate was collected as fraction 2, and followed by collecting every 20 drop samples until the sample sappeared faded, and finally clear in vial 25. This indicates that the protein samples in the column Sephadex LH-20 have been completely eluted by methanol. The eluate of 25 fractions was allowed to evaporate at room temperature to remove the solvent. The solids obtained were observed to look like thin films in some of the fractions. Five fractions contained thin films of 25 fractions produced, those were fractions 6, 9, 14, 18 and 25, which were then analyzed using FTIR spectroscopy to observe the functional groups containing the fraction.

FTIR analysis

Fourier Transform Infra-Red (FTIR) was used to find out bonding vibration and functional groups contained by bacteriocins. FTIR analysis results from one of the bacteriocins produced by bacteria H4 are shown in Figure 3. The spectrum figure shows the comparison of fractions that produced films (fractions 6, 9, 14, 18 and 25) as the separation products of bacteriocins H4 using gel filtration column, and after being precipitated in ammonium sulphate [(NH4) 2SO4]. Each fraction showed absorption bands at similar wave numbers.

Figure 3. FTIR spectrum bacteriocin H4 precipitated in ammonium sulfate [(NH4) 2SO4] in comparation to the purification product of bacteriocins of some fractions (fraction 6,9,14,18 and 25).

The infrared spectrum of bacteriocins for fraction 6 shows absorption at wave numbers (cm-1), 978, 1340, 1355, 1648, 2831, 2921, 3420 and 3595. Fraction 9 shows absorption at wave numbers (cm-1) of 977, 1089, 1339, 1363, 1404, 1457, 1647, 3239, and 3587. Fraction 14 showed absorption at wave numbers (cm-1) of 978, 1097, 1312, 1363, 1405, 1457, 1647, 2836, and 3502. Fraction 18 shows absorption at wave numbers (cm-1) of 988, 1334, 1358, 1400, 1460, 1649, 2835, 2954, 3415 and 3588. Finally, fraction 25 shows absorption at wave numbers (cm-1) of 971, 1082, 1312, 1339, 1355, 1416, 1457, 1647, 2834, 2988, 3336, 3443 and 3593. Meanwhile, bacteriocins precipitated by salts of ammonium sulphate [(NH4) 2SO4] shows an absorption band at wave number (cm-1) 979, 1339, 1355, 1418, 1456, 3336, 3445, and 3592.