Bio-control of Vibrio species in cultured milk by in situ bacteriocin production from lactic acid bacteria

The growth of resistance to antibiotic by Vibrio signifies a possible risk to human health; hence, there is a need to deploy another technique for controlling species of Vibrio. This study was undertaken to demonstrate the antagonistic activity of bacteriocin-producing Lactic acid bacteria against Vibrio alginolyticus, Vibrio vulnificus, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio fluvialis, and Vibrio cholera in vitro and in situ. Lactic acid bacteria (LAB) were isolated from milk products and identified phenotypically. They were initially screened for antagonistic activity against the Vibrio species by the agar well diffusion assay, bacteriocins produced by the LAB were characterized with respect to pH, enzymes and temperatures. The effect of in situ bacteriocin production by LAB on the survival of Vibrio species was determined in Nono, after fermentation of milk during the storage period of 72 h (12 h interval). Of the 112 strains of LAB tested for antagonistic activity against Vibrio species, only twelve were selected based on the bacteriocin production and large zone of inhibition against Vibrio species. They were characterised phenotypically and identified to be Pediococcus damnosus, Pediococcus acidilactici, Lactobacillus brevis and Lactobacillus plantarum. The bacteriocins produced by the LAB were heat stable at 90°C for 20 min, active over a wide pH range (2 to 6), stable in the present of catalase but lost their activity in the present of proteolytic enzymes. Bacteriocins produced by the LAB showed antagonistic activity against Vibrio species with zones of inhibition ranges from 12 to 20mm. Vibrio species counts were reduced significantly to different extents in all samples of Nono and undetectable within 48 to 60 hours of Nono storage. On the contrary, Vibrio species survived for 72 h of storage in the control experiment that lack bacteriocin producing LAB. This work demonstrates that the use of selected bacteriocin-producing starter in milk fermentation might contribute to safety of dairy products.


Introduction
Lactic Acid Bacteria are gram-positive, catalase negative, usually non-motile, non-spore-forming rods and cocci which produce lactic acid as the major end product of carbohydrate fermentation [1]. They cannot generate ATP because they lack the ability to synthesize cytochromes and porphyrins which are components of respiratory chains. They can only obtain ATP by fermentation, usually from sugars. They are facultative anaerobes and are protected from oxygen byproducts (e.g. H2O2) because they possess peroxidases [2]. Lactic Acid Bacteria are usually found in the environment related with rich nutrients, such as different food products (milk, meat, vegetables), although some of their members are of the flora of the intestine, mouth and vagina of mammals [3; 4]. One of the most important and significant values of lactic acid bacteria to humans is their beneficial role in health and inhibition of pathogenic bacteria. Some LAB are reputed to act directly as probiotics when ingested or incorporated into food products [5], or indirectly by action of the antimicrobial substances they produce in foods. Several antimicrobial substances are produced by various starter cultures of LAB, which include organic acids, hydrogen peroxide, CO2, diacetyl and bacteriocins [6] which have received increasing attention during the past two decades.
Bacteriocins are definitely regarded as antimicrobial peptides that are produced from their ribosomes (ribosomally) which are active against other bacteria, either of the same species or across genera [7 ; 8] . They may be produced by both Gram negative and Gram positive bacteria [9]. In recent years, bacteriocin producing LAB have attracted significant attention due to their Generally Recognized as Safe (GRAS) status and potential use as safe additives for preservation of food [10] . Bacteriocins have potent antagonistic effect against important clinical pathogens as observed by [11]. Different types of bacteriocins from food-associated lactic acid bacteria have been characterised and identified, of which the important ones are nisin, diplococcin, acidophilin, bulgarican, helveticins, lactacins, and plantaricins [12]. Quite a lot of studies showed that LAB starter cultures or co-cultures are able to produce bacteriocins in food media, and as a result exhibited inhibitory potentials towards labile foods capable of spoilage or pathogenic bacteria. The genus Vibrio is oxidase-positive, Gram-negative, rod or curved shaped and facultative anaerobes that are motile, which produces cholera enterotoxin. They are responsible for food-borne disease, morbidity, life threatening and mortality [13; 14]. Several foods include vegetables, fruits, dairy products, sea foods, poultry, milk, meat products and others can become contaminated with Vibrio species as a result of improper handling, undercooking, washing with unhygienic water and by the use of untreated soil [15;16]. These organisms are capable of producing a thermostable toxin known as hemolysin.
The principal cause of mortality in the developed, developing and the rest of the world is the Vibro vulnificus because of its association with sea food consumption [17]. However, the use of antibiotics in regulating Vibrio species is not encouraging as a result of the growth of antibiotic-resistance and the negative impact of antibiotic consumption on the host. An efficient microbiological protection and safety of milk is vital to ensure its purity and to avoid adverse human heath implication. However, research in this area has not been fully explored, therefore, the focus of this work centered on the testing of Lactic Acid Bacteria against multiple-drug resistance Vibrio species in milk and its products.

Sample collection
Samples of milk products ("Nono", "Wara" and Yoghurt) used in this study were obtained from local markets in Ibadan, Oyo State. Two replicate samples were collected and transported to the laboratory in sterile bottles. The samples were refrigerated at 4°C and analyzed within 24 hours of collection [18]. The multidrug resistance Vibrio species used as indicator organisms in this work had been previously phenotypic and molecular characterised as reported by [19].

Sterilization
All media were sterilized in an autoclave at 121°C for 15 minutes, except otherwise stated. Used glass wares were soaked in solutions containing antiseptic (Jik) overnight. They were then washed with liquid soap and rinsed in several changes of tap water. The glass wares were arranged on the drain board to drip off.

Isolation procedure
One mL/1g of the samples was homogenized with 9ml of sterile distilled water to make an initial dilution 10 -1 . The suspensions were used for making suitable serial dilutions up to10 -6 by incorporating 1ml into 9ml of sterile distilled water in sterile tubes. Using different sterile 1.0ml pipette, 0.1ml of 10 -4 and 10 -6 dilutions of the various samples were plated out.

Culture preservation
The isolates of Lactic acid bacteria were sub-cultured onto maintenance medium consisting of MRS broth with 12% (v/v) glycerol and incubated at 30°C until growth becomes visible. The stock cultures were stored at 4°C for subsequent use for a period of 2 to 4 weeks before sub-culturing into fresh medium. Vibrio species was stored in Tryptone Soy broth (TSB) and maintained at room temperature for further studies.

Characterization of isolates
This was carried out by employing macroscopic, microscopic and biochemical tests (all Gram positive, catalase negative and non-spores forming isolates were selected for sugar fermentation test).

Identification of Isolates
The isolates were identified based on the results of the various biochemical tests using Bergey's Manual of Systematic Bacteriology [20].

Antimicrobial activity of bacteriocin producing LAB
The agar diffusion assay described by [21]. Freshly prepared Vibrio species were used for this assay. One (1) ml of the indicator organism (Vibrio species) was inoculated into 15 ml of semisolid Mueller Hinton agar (MHA plus 0.75% bacteriological agar) and then poured into a petri dish. After solidification, three wells (7 mm diameter) were cut and 50 μl of cell-free supernatant (CFS) from each LAB isolate were added to each well. Cell-free supernatant was prepared as follows; one ml of frozen LAB isolate was cultured overnight in 20 ml MRS broth, then 1 ml culture was sub-cultured for 72 hours in 20 ml MRS broth. Cells were removed by centrifuging at 14,000g for 5mins and 50 μl of the unadjusted aliquot of cell-free supernatant was added to the wells. . The plates were incubated at 37°C aerobically for 24 h. Inhibition zones were measured and recorded appropriately.

Treatment of antimicrobial compounds with NaOH and catalase enzyme
The isolates that exhibited antagonistic activity against the pathogenic organism were investigated for their antimicrobial compounds using a modified method of [22]. The cell-free supernatant was adjusted to pH 6.0 with 1 mol l -1 NaOH in order to rule out possible inhibition effects due to organic acids. 50 μl of the pH adjusted cell-free supernatant were filtered (0.2 μm pore-size cellulose acetate filter) and added to the second well. The neutralized cell-free supernatant was then treated with 1 mg ml-1 of catalase at 25 °C for 30 min to eliminate the possible inhibitory action of H2O2 and then was placed in the third well. The Mueller Hinton plates were incubated at 37°C aerobically for 24 h. Inhibition zones were measured and recorded appropriately. If inhibition zones are found in the third well, the isolates were considered to be able to produce bacteriocin-like substance.

Sensitivity of bacteriocin produced by LAB to proteinase
To confirm production of a proteinaceous compound, cell-free supernatant displaying antimicrobial potential after acid neutralization and H2O2 elimination were treated with 1 mg ml-1 of proteolytic enzymes, including Pepsin and Trypsin at 37 °C for 2 h [23 ; 24]. Each enzyme is dissolved in plug phosphate buffer and sterilized by filtration (0.2 μm). Antimicrobial activity of treated culture broth was determined by the agar diffusion bioassay as described above.

Thermal stability of bacteriocins produced by LAB isolates:
Sensitivity of the bacteriocins to heat was investigated using a modified method of [25]. The pH-adjusted and H2O2 eliminated cell-free supernatant described above were treated at 60 °C, 90 °C for 20 mins and at 121 °C for 15 mins. pH -adjusted and H2O2-eliminated cell-free supernatant without any heat treatments served as control. Residual antimicrobial activity of heat-treated culture broth was determined by the agar diffusion bioassay.

Effects of pH on the bacteriocins:
In order to determine the sensitivity of the bacteriocins to pH, a modified method of [25] was employed. The cell-free supernatant of each strain was adjusted to pH levels ranging from 2 to 10 (intervals of 2) with HCl and NaOH, incubated at 37 °C for 5h and then tested for bacteriocin activity using the agar well diffusion assay. The supernatant of unadjusted pH served as controls.

Production of a model cultured milk (Nono)
The effect of in situ bacteriocin production on the behavior of Vibrio spp in Nono was determined during storage using a modified method of [26]. Fresh milk was aseptically collected from the teat of a cow and transported to the laboratory. The milk was pasteurised at 72°C for 20 minutes. After pasteurisation the milk was cooled to 40 -45°C. The milk was then inoculated with the bacteriocin producing LAB (1.5 X 10 8 cfu/ml) and stirred well for 3-5 minutes to ensure uniform distribution of starter culture. The milk was fermented at 30°C for a period of 24 hours. None inoculated milk served as control. The fermented milk was then artificially contaminated with Vibrio species (1.5 X 10 6 cfu/ml). The food product was tested at selected intervals (12 h) for 72 hours for the presence or absence of Vibrio species. Counts of Vibrio species were performed on TCBS (Thiosulphate citrate bile salt sucrose) agar after incubation at 37°C for 48 hours.

Results
A total of 112 LAB strains were isolated from fermented milk products and were initially screened for antagonistic activity against six Vibrio species (V. alginolyticus, V. parahaemolyticus, V. cholera, V. fluvialis, V. harveyi, V. vulnificus) by the agar well diffusion assay using cell free supernatant of broth culture (Table 1). Of the 112 strains tested, 13 produced bacteriocin that inhibited Vibrio spp. Subsequently, only twelve were selected based on their zone of inhibition for identification to species level. Table 1 Antagonistic activity of LAB metabolites isolated from milk products against Vibrio species All the LAB isolates were Gram positive, catalase negative, non-spores forming, non-motile, cocci and rods. Carbohydrate utilization pattern of the isolates was used to differentiate and identified the isolates to species level. The isolates were identified as Pediococcus damnosus, Pediococcus acidilactici, Lactobacillus brevis and Lactobacillus plantarum. The cell free supernatant of the 12 strains of LAB were treated with catalase, NaOH, proteolytic enzymes (trypsin and pepsin) and tested by the agar well diffusion assay against the Vibrio species as shown in Table 2. Catalase and NaOH had no effect on the inhibition, that is, the antimicrobial activity was still maintained, indicating that hydrogen peroxide and organic acids respectively did not account for the observed inhibition. However, the antimicrobial activity from all the strains was completely inactivated by treatment with trypsin and pepsin ( Table 2). The bacteriocins produced by these isolates were heat-treated at 60°C, 90°C for 20 mins and at 121°C for 15 mins and were observed to be stable at 60°C -90°C conditions as indicated by their inhibitory effects against Vibrio species (Table 3).
Stability of the bacteriocin was evaluated at different pH values ranging from 2 to 10 at 37 °C for 5 h, it was observed that the antimicrobial activity against Vibrio species was retained in pH ranges form 2-6, however the inhibitory activity was lost at alkaline pH (Table 4).

Discussion
This study was undertaken to demonstrate the antagonistic property of bacteriocin-producing Lactic acid bacteria on Vibrio cholera, Vibrio alginolyticus, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio harveyi, and Vibrio fluvialis in vitro and in situ The fermented milk products (yoghurt, nono, wara) analyzed was found contained lactic acid bacteria (LAB) in different numbers. Nono had higher lactic acid bacteria counts than wara and yoghurt. This is in conformity with the report of [27] and [28] that in the locally fermented foods analyzed, nono had higher LAB count than wara, fufu and akamu. Phenotypic and biochemical identification of the twelve selected isolates were carried out.
A total of 112 LAB strains were isolated from fermented milk products and were initially screened for antagonistic activity against the Vibrio species by the agar well diffusion assay. Of the 112 strains tested, 13 produced an inhibition zone against the Vibrio spp. In this step, the possible inhibitory effect of the organic acids and hydrogen peroxide was not excluded. Subsequently, only twelve were selected by the large zone of inhibition for their identification at species level. The cell free supernatant of the 12 strains were treated with catalase, NaOH, proteolytic enzymes (trypsin and pepsin) and tested by the agar well diffusion assay against the Vibrio spp. Catalase and Sodium hydroxide (NaOH) treatment of cell free supernatant had no effect on the inhibitory activity, that is, the antimicrobial activity was still maintained, indicating that hydrogen peroxide and organic acids did not account for the observed inhibition. However, the antimicrobial activity from all the strains was completely inactivated by treatment of the cell free supernatant with trypsin and pepsin. This confirms that the inhibition is as a result of a proteinacious compound and provides evidence that growth inhibition of the Vibrio species was caused by a bacteriocin. Bacteriocins have been reported to be inhibitory against several other bacteria [11; 29; 30; 31; 25; 22].
Phenotypic, morphological and biochemical characterization of the LAB isolates identified the organisms as Pediococcus damnosus, Pediococcus acidilactici, Lactobacillus brevis and Lactobacillus plantarum. The characterization of these isolates agreed with Bergey's Manual of Systematic Bacteriology [20].
The bacteriocins produced by LAB isolates were observed to be stable at 60°C, 90°C for 20 mins but not at 121°C for 15 mins. This is in agreement with the work of Andersson [32] who reported the loss of bacteriocin activity after heating at 121°C for 10 mins. Also, Ogunbanwo et al. [11] recorded the loss of inhibitory activity of bacteriocin produced by L. plantarum at 121°C for 10 mins. The thermal stability of the bacteriocins produced by these LAB isolates may constitute an advantage for potential use as biopreservatives in combination with thermal processing in order to preserve food products [22]. This resistance is also known for other bacteriocin produced by lactic acid bacteria: lactacin B [33], lactacin F [34], nisin [35] and bacteriocin ST15 [36].
Bacteriocins were stable at different pH values ranging from 2 to 6 (intervals of 2) at 37°C for 5h, it was observed that the antimicrobial activity against Vibrio species was retained in pH range 2-6. However the inhibitory activity was lost at pH 8 and 10. This is in agreement with the work of Ogunbanwo et al. [11] who reported that the activity of bacteriocin elaborated by the test isolates was pH dependent and recorded that the highest antibacterial activity was exhibited in an acidic pH range of 2 to 6, while inactivation occurred at pH 8 to 12. The work carried out by Lade et al.
[37] also stated that bacteriocin were stable in acidic to neutral range i.e. from pH 4.0 to 7.0, but, inactive in the alkaline range. These data suggest that the bacteriocin described in this study could be applied in both low and medium-acidic food products with final pH values in such range; this includes a number of fermented and ripened dairy and meat products [25].
The effect of in situ bacteriocin production on the survival of Vibrio species in Nono was determined after fermentation during the storage period of 72 hours. It was observed that Vibrio species counts were reduced to different extents in all samples of Nono and undetetable within 48 to 60 hours in Nono produced with bacteriocin producing LAB during storage periods. On the contrary, Vibrio species survived for 72 hours of storage in the control experiment (unfermented milk) that contained no bacteriocin-producing LAB. This is in consonance with the work of Benkerroum et al.
[38] that reported a significant decrease in the amount of L. monocytogenes in a 1-mL sample within 24 h of storage at 7°C in lben fermented with the bacteriocin-producing starter culture. The work demonstrates that the use of selected bacteriocin-producing starter in milk fermentation might contribute to ensuring the safety of dairy products, especially when they are obtained from raw or minimally processed milk.

Conclusion
From the present study, bacteriocin-producing LAB were obtained from fermented milk products and were able to inhibit the growth of Vibrio species with multiple antibiotic drug resistance. The selected bacteriocin-producing LAB isolated from milk fermentation could be used to inhibit Vibrio species and act as biopreservation of milk and its products. Vibrio species have shown to be sensitive to the bacteriocin produced by LAB in vitro and in situ.