Bacteriocin‐capped silver nanoparticles for enhanced antimicrobial efficacy against food pathogens

Abstract

Bacteriocins produced by lactic acid bacteria are safer alternatives to the more popularly used chemical preservatives which exhibit several adverse effects. The bacteriocins have an advantage of being efficient in controlling food pathogens without possessing any side‐effects. However, the bacteriocins have a limitation of exhibiting a narrow antimicrobial spectrum and having a high‐dosage requirement. With an aim to combat these limitations, the present study involved the biosynthesis of bacteriocin‐capped nanoparticles, using two bacteriocins (Bac4463 and Bac22) extracted and purified from Lactobacillus strains. Nanoconjugates synthesised at optimum conditions were characterized using various physico‐chemical techniques. The interaction of bacteriocin‐capped silver nanoparticles with the pathogenic bacteria was observed using scanning electron microscopy, wherein the deformed and elongated cells were clearly visible. In vitro antimicrobial efficacy of both Bac4463‐capped silver nanoparticles and Bac22‐capped silver nanoparticles against different food pathogens was observed to be enhanced in comparison to the antimicrobial activity of bacteriocins alone. Minimum inhibitory concentration was observed to be as low as 8 μg/ml for Bac4463‐capped silver nanoparticles against Staphylococcus aureus, and 2 μg/ml for Bac22‐capped silver nanoparticles against Shigella flexneri. This study, therefore, recommends the use of bacteriocin‐capped nanoparticles as food preservatives to control the growth of food spoiling bacteria.

1 Introduction

Food‐borne illnesses are infections caused by the consumption of food contaminated with bacteria, viruses or the toxins released by them. More than 250 food‐borne diseases are identified to date. According to World Health Organization (WHO), almost 600 million people in the world fall ill after consumption of contaminated food and every year an estimated 420,000 people die leading to a loss of almost 33 million people [1]. Bacteria are known to be one of the major causes of food‐borne diseases and have become a great concern for human health [2, 3]. Major food spoiling bacteria include Listeria monocytogenes, Escherichia coli, Staphylococcus aureus, Clostridium botulinum, Bacillus cereus, Vibrio cholerae, Klebsiella sp., Enterobacter sp., Pseudomonas sp. and Shigella sp. [4, 5, 6, 7]. Although antibiotics are known to be efficient in inhibiting the growth of disease‐causing food spoilage bacteria; however, their overuse has led to an emergence of antibiotic‐resistant bacteria, rendering the treatment of food‐borne diseases ineffective in humans. Due to an increase in cases of this antibiotic‐resistance over a period of time, prevention of food from food‐borne pathogens and its long‐term storage has become undeniably a serious issue worldwide. Therefore, to effectively counteract this problem; researchers have constantly been in the look‐out for biological preservatives or antimicrobials which are both compatible and safe for human health. In this direction, bacteriocins produced by lactic acid bacteria, have since some time emerged as potential antibacterials.

Bacteriocins are ribosomally synthesised peptides which are highly efficient in inhibiting the growth of similar or closely related microorganisms. These peptide molecules inhibit the growth of food pathogens, either by inducing pore‐formation in the bacterial cell‐surface or, by interfering with the DNA replication, transcription and translation processes in pathogens. The bacteriocins, thus, have the potential of finding application in the control of food‐spoiling pathogens during various forms of processing of different food items, hence aiding in enhancing the shelf life of food items [8, 9, 10]. Bacteriocins produced by lactic acid bacteria are also considered to be generally regarded as safe for consumption by the Food and Drug Administration. Most of the bacteriocins isolated and characterised from lactic acid bacteria are found to be heat stable, non‐toxic and susceptible to degradation by proteolytic enzymes present in the human gut. Thus, the use of bacteriocins as a food preservative can meet the demands of consumers for a safe and minimally processed food devoid of any chemical additive. However, despite having several advantages, certain drawbacks such as a high‐dosage requirement, low production yield and a narrow antimicrobial spectrum, limit the usage of bacteriocins as effective bio‐preservatives. In this context, the amalgamation of nanotechnology and biotechnology which has already proven to be beneficial in various fields such as medicine and agriculture; has also been discovered recently to provide new hopes in dealing with various issues related to food preservation and packaging.

Nanoparticles which display unique physical, chemical and biological properties have been proposed to be suitable candidates capable of encountering some of the drawbacks of bacteriocins, and have therefore caught the attention of food biologists. Nanoparticles are known to be synthesised by two methods, viz. chemical method and the green method. However, chemical synthesis of nanoparticles, in most of the cases, proves to be toxic to the environment. Therefore, to circumvent the side‐effects associated with this method, researchers are more interested in the greener and safer method of nanoparticle synthesis. Green methods make use of biocompatible and non‐toxic compounds, hence, have become a preferred choice for materials seeking applications in the food sector. Since the past decade, various metallic nanoparticles like silver, gold, copper oxide and zinc oxide are being explored owing to their potential antibacterial and antifungal properties. However, among these, silver nanoparticles (SNPs) are the ones which have been the most investigated and are known for their high antimicrobial responses. Silver in nano‐form has exceptional magnetic, antibacterial and antifungal properties [11, 12]; and the SNPs are known to impart higher toxicity against both Gram‐positive and Gram‐negative bacteria in comparison to the other metallic nanoparticles. Remarkably, although they have been observed to be toxic to microorganisms; but at the same time, they have also been touted to be the least toxic to mammalian cells [13]. SNPs act by attaching to the bacterial cell surface, and penetrating the cell membrane, owing to their small size.

However, since nanoparticles undergo fast oxidation and are also un‐stabilised at times, and have a tendency of forming aggregates in solutions, their use in various areas of food science becomes slightly unfavourable. In this direction, to counter‐act this problem, more recently, biopolymer‐encapsulated nanoparticles have attracted the interest of researchers. It has been proposed in a few studies that conjugation of nanoparticles with bacteriocins may aid in increasing the antimicrobial activity, enhancing the stability, and also in increasing the shelf life of food [14, 15, 16]. Metal nanoparticles, because of their properties such as a broad antimicrobial spectrum and a large surface area are known to be promising candidates for conjugation with bacteriocins. Silver, followed by gold is the most commonly used metal for the synthesis of bacteriocin‐conjugated nanoparticles [15, 16]. Since both bacteriocins and SNPs exhibit good antibacterial properties; hence conjugation of bacteriocins with SNPs can be used to enhance the antimicrobial responses against both Gram‐ positive and Gram‐negative food spoiling bacteria. Thus, the development of such nano‐conjugates as antimicrobial tools involving green methods hold a higher potential for use in the food sector.

The present study reports the synthesis of bacteriocin‐capped SNPs (Bac‐SNPs) and evaluation of their antibacterial activity and minimum inhibitory concentration (MIC) against various Gram‐negative and Gram‐positive food pathogens.

2 Material and methods

2.1 Bacteriocin‐producing strains

One bacteriocin‐producing strain (L. herbarum) was isolated from raw milk using dilution‐plating method and plating on de Man, Ragosa and Sharpe (MRS) agar medium. Another bacteriocin‐producing strain (L. brevis MTCC 4463) procured from Institute of Microbial Technology (IMTECH) Chandigarh, India was used as a reference strain. Both the bacteriocin‐producing strains were cultured and maintained on MRS agar medium.

2.2 Indicator test strains

To study the antibacterial efficacy of bacteriocins and bacteriocin‐capped nanoparticles, a total of six pathogenic indicator test strains including three Gram‐positive bacteria (B. cereus MTCC 1272, S. aureus MTCC 11949 and L. monocytogenes MTCC 657), and three Gram‐negative bacteria (E. coli MTCC 9721, Shigella flexneri MTCC 1457 and Pseudomonas aeruginosa MTCC 3542) were procured from IMTECH Chandigarh, India.

2.3 Production and partial purification of bacteriocins

The production and partial purification of bacteriocin were carried out according to the method described by Patil et al. [11], and Sharma et al. [17] with slight modifications. An overnight grown culture of L. brevis MTCC 4463 (standard strain) and the isolate L. herbarum was centrifuged at 10,000 rpm for 15 min at 4°C and the cell‐free supernatant was collected. To neutralise the effect of organic acids produced by the producer strains, the pH of the cell‐free supernatants was adjusted to 6.0 using 1 N NaOH, and the supernatants were then filtered through a 0.22 μm membrane filter. The protein precipitation was carried out with 80% ammonium sulphate at 4°C under continuous overnight stirring conditions. The precipitates obtained were centrifuged at 10,000 rpm for 10 min at 4°C, and the pellet obtained was dissolved in 20 mM phosphate buffer. These pellets represented the crude bacteriocins. To remove salt and other impurities from the crude bacteriocins, the pellets were dialysed overnight at 4°C through 1 kDa dialysis membrane (Himedia, India) against the same buffer. The antibacterial activity of both the crude bacteriocins was then assayed against all the six indicator test strains (as per the methodology detailed in Section 2.7).

2.4 Biosynthesis of Bac‐SNPs

Silver nitrate (AgNO₃) was used for the synthesis of Bac‐SNP suspension. A 2 mM solution of silver nitrate was prepared with continuous stirring for 5–6 h on a magnetic stirrer at room temperature. An aliquot of 5 ml of each purified bacteriocin; Bac4463 produced by L. brevis MTCC 4463 (reference strain), and Bac22 produced by the isolate L. herbarum, was added to 45 ml of freshly prepared 2 mM silver nitrate solution. The resultant mixed solution was incubated under UV light for 50–60 min at room temperature. A change in the colour from light yellow to reddish‐brown indicated the formation of SNPs. The obtained suspension was then centrifuged at 15,000 rpm for 20 min to pellet down the Bac4463‐capped SNPs and Bac22‐capped SNPs. The capped‐SNPs were washed thrice with Milli‐Q water to remove the unbound silver nitrate and the free Bac4463 and Bac22.

2.5 Optimisation of synthesis conditions

Synthesis parameters like temperature, pH and concentration of bacteriocins are known to affect the biosynthesis of Bac‐SNPs. These parameters were therefore analysed for the optimum synthesis of bacteriocin‐capped SNPs. Both the purified bacteriocins were mixed separately with silver nitrate in varying ratios (0.5:9.5, 1:9, 1.5:8.5 and 2:8) and then incubated at room temperature for 24 h, and thereafter the results were recorded as spectrophotometric measurements. Effect of temperature on the synthesis was analysed by incubating the reaction at varying temperatures, viz. 25, 35, 45, 55, 65 and 75°C for 24 h. To study the effect of pH, silver nitrate was mixed with each of the two bacteriocins (Bac4463 and Bac22) in the ratio of 1:9 and then adjusting the pH of the mixed solution at different pH values, viz. 3, 5, 7, 9, 11 and 13 using 1 N NaOH or 1 N HCl. UV‐Spectroscopy was performed in the wavelength range of 200–800 nm, and the spectra were plotted using OriginPro 8 software.

2.6 Characterisation of Bac‐SNPs

Both the bacteriocin‐capped SNPs were characterised by using various physico‐chemical techniques such as Fourier‐transform infrared spectroscopy (FTIR), X‐ray diffraction (XRD), Transmission electron microscopy (TEM), Zeta potential and Dynamic light scattering.

2.6.1 Fourier‐transform infrared spectroscopy

FTIR analysis is used to identify the organic and inorganic materials responsible for the reduction of silver ions, capping and stabilisation of nanoparticle suspensions. The purified liquid suspension of Bac4463‐capped SNPs and Bac22‐capped SNPs was scanned in FTIR spectrum in the range of 4000–450 cm⁻¹ using Perkin Elmer FTIR Spectrophotometer.

2.6.2 X‐ray diffraction

XRD is performed to study the crystalline or amorphous nature of nanoparticles. The XRD spectrum aids in confirming the synthesis of SNPs. For XRD analysis, Bac4463‐capped SNPs and Bac22‐capped SNPs were centrifuged at 10,000 rpm for 20 min, and the obtained pellet was air‐dried on a glass plate at 40°C. The dried sample was then collected to make a fine powder and analysed using XRD. The spectra were obtained using Ultima VI (Rigaku, Japan) in the 2θ range of 20°–70° degrees with an X‐ray wavelength of 1.5406 Å.

2.6.3 Differential light scattering (DLS) and zeta potential

DLS was performed to measure the hydrodynamic size of the particle, and zeta potential was performed to determine the stability of the synthesised capped nanoparticles. Both the zeta potential and zeta size of nanoparticles was measured using Zetasizer Nano‐ZS (Malvern Instruments, Worcestershire, UK). Both the analyses were performed at 25°C and measurement was recorded as a function of time.

2.6.4 Transmission electron microscopy

TEM analysis was performed to investigate the size and morphology of both the bacteriocin‐capped SNPs. For TEM analysis, the prepared powdered samples were diluted to obtain a less turbid suspension and then sonicated for 5 min. A single drop of the suspension was placed on the copper grid and allowed to dry for 15 min. The dried sample grid was loaded on the sample holder and TEM analysis was performed using TELOS bright field SA 150kx electron microscope at a high‐tension voltage of 200 kV to deduce the size and morphology of nanoparticles. The images were visualised using an Olympus soft imaging system.

2.7 Antibacterial activity of partially purified bacteriocins and Bac‐SNPs

Antibacterial activity of Bac4463‐capped SNPs, Bac22‐capped SNPs, Bac4463 alone and Bac22 alone, were examined using agar well diffusion assay. All the six food spoiling indicator bacterial strains were grown in nutrient broth overnight and freshly grown cultures were used for antibacterial analysis. An aliquot of 100 μl of each culture was spread evenly on fresh nutrient agar plates, and three wells of 7 mm diameter were bored in each plate using sterile well borer. In each plate, 50 μl of the test solution (Bac4463‐capped SNPs or Bac22‐capped SNPs, Bac4463 alone and Bac22 alone) was added in one well, 20 μl of 50 μg/ml tetracycline was used as a positive control in the second well, and water was added in the third well as a negative control. The plates were then incubated in an upright position at 37°C for 24 h. The zone of inhibition obtained was recorded using a scale.

2.8 Determination of MIC and minimum bactericidal concentration (MBC)

MIC of synthesised Bac‐SNPs was determined using the broth micro‐dilution method [18]. MIC corresponds to the minimum amount of test sample required to inhibit the growth of the target bacteria; lower the MIC, stronger is the antibacterial activity. All the indicator organisms were grown overnight and the bacterial growth was adjusted to the optical density value of 0.1 ± 0.001 at wavelength 600 nm using a spectrophotometer. These diluted suspensions were used as inoculum for the micro‐dilution assay. For calculating MIC, 2 ml of media (nutrient broth) was first poured in sterile tubes and 2 ml of nanoparticle suspensions (256 μg/ml) was further added in the first tube, which was then used for two‐fold dilution of consecutively numbered tubes. An amount of 2 ml of the solution from the last tube in the series was discarded. The tubes were then seeded with 50 μl of the diluted bacterial suspension and incubated for 24 h at 37°C. Presence and absence of bacterial growth were checked visually and MIC values were determined by spectrophotometer readings at 600 nm. MBC is the lowest concentration of the antibacterial agent which is efficient enough to completely kill the bacteria. MBC test was performed by plating the suspension from each tube on to the nutrient agar plates. The plates were then incubated for 24 h at 37°C. The lowest concentration with no visible growth was then considered as MBC value.

2.9 Study of the effect of Bac‐SNPs on the bacterial cell surface using scanning electron microscopy (SEM)

For studying the changes resulting in the bacterial cell morphology of the pathogens, as a result of their interaction with the Bac‐SNPs, two representative food‐spoiling bacteria, one Gram‐positive (B. cereus), and one Gram‐negative (S. flexneri) were selected. Growing cultures (food pathogens) were treated with a sub‐MIC dose of bacteriocin‐capped SNPs for 3 h and then centrifuged at 10,000 rpm for 10 min. The cells were fixed with 2% glutaraldehyde overnight at room temperature, centrifuged at 8000 rpm for 5 min., washed with phosphate buffer twice, and finally resuspended in 0.1 M phosphate buffer (pH 7.2). The cells were then treated with a series of ethanol (30–70%) washings for 5 min each and critical drying was performed at 100% ethanol. SEM (ZEISS EVO18, Carl Zeiss, India) was used to visualise the morphological changes resulting due to interaction of bacteriocin‐capped SNPs with bacterial cells.

3 Results and discussion

3.1 Biosynthesis of Bac‐SNPs

The synthesis of bacteriocin‐capped SNPs using the two bacteriocins (Bac4463 and Bac22) was confirmed by recording a change in colour from light yellow to reddish‐brown. This gave the preliminary indication of the synthesis of Bac‐SNPs. This colour change could be attributed to the surface plasmon resonance of SNP suspension, which is primary and visible evidence for the synthesis of SNPs [19].

3.2 Optimisation of synthesis parameters

Different parameters such as varying concentrations of bacteriocins, varying temperature and varying pH range were studied for obtaining the optimum conditions for the synthesis of bacteriocin‐capped nanoparticles. Different ratios of bacteriocins (Bac4463 and Bac22) and silver nitrate were tested to optimise the biosynthesis process. Bacteriocins and silver nitrate were added in the ratio of 0.5:9.5, 1:9, 1.5:8.5 and 2:8 in a total of 10 ml reaction mixture. It was observed that for both the reaction mixtures; Bac4463‐capped SNPs and Bac22‐capped SNPs, the concentration ratio 1:9 gave a sharp and intense peak at 450 and 430 nm, respectively. A further increase in the bacteriocin concentration in silver nitrate led to the broadening of peak and a decrease in absorbance (Fig. 1 a). Similar results were observed by Jamdagni et al. [20] for Elettaria cardamomum, wherein 1:9 ratio concentrations of the plant extract and SNPs was observed to be optimal for biogenic synthesis and a further increase in concentration led to diminishing effects.

Fig. 1.

UV–Visible spectrum of synthesis parameters for Bac4463‐capped SNPs and Bac22‐capped SNPs

(a) Effect of bacteriocin concentration, (b) Effect of temperature, (c) Effect of pH

The effect of temperature on the synthesis of nanoparticles was studied by incubating the reaction mixture at different temperatures (25–75°C) for 24 h and the results were recorded in the form of UV–Vis absorption spectra. It was observed that increasing the temperature from 25 to 65°C for both Bac4463‐capped SNPs and Bac22‐capped SNPs led to an increase in absorbance and peak sharpening; however, a further increase in temperature exhibited a decrease in absorbance for both the bacteriocin‐capped nanoparticles. At temperatures <65°C, a less intense peak and broadening were observed which may depict inefficient synthesis. At optimum temperature, i.e. 65°C, Bac4463‐capped SNPs and Bac22‐capped SNPs showed a significant peak at 452 and 431 nm, respectively (Fig. 1 b). In consonance with the results of our study, Singh et al. [21] also reported the optimum temperature for the synthesis of monodispersed SNPs from Acinetobacter calcoaceticus to be 70°C. Similar results were also obtained by Song and Kim [22].

The pH of a solution has also been known to be an important factor which can affect the synthesis of nanoparticles. Bac4463‐capped SNP and Bac22‐capped SNP synthesis was carried out by maintaining the reaction mixture at different pH values viz., 3, 5, 7, 9, 11 and 13. A shift in pH towards basic range by the addition of 1 N NaOH showed an increase in the rate of reaction. Normally the colour change occurs in 4–5 h of incubation, the same was observed within a few minutes of adding NaOH to the reaction mixture. Increase in absorbance and peak sharpening was also noted with an increase in pH till 11 in both the Bac4463‐capped SNPs and Bac22‐capped SNPs. Further increase in pH showed a decrease in absorbance. Bac4463‐capped SNPs and Bac22‐capped SNPs showed the absorbance at 453 and 432 nm, respectively at pH 11(Fig. 1 c). Similar results have been reported by Ndikau et al., wherein they observed pH 10 to be the optimum pH for the synthesis of SNPs from the extract of Citrullus lanatus fruit rind [23].

The synthesis of Bac4463‐capped SNPs and Bac22‐capped SNPs at the optimal conditions resulted in an increase in absorbance, accompanied by sharp and intense peaks at 453 and 428 nm, respectively (Fig. 2). The peaks at higher wavelength may attribute to the wavelength shift caused by capping of bacteriocins on the SNPs. These biosynthesis results are in consonance with similar studies conducted by different researchers. The absorption peak at 420 nm was reported by Patil et al. [11] using Bacillus species RPT0001 in conjugation with SNPs. Singh et al. [24] reported absorption peak at 450 nm using partially purified bacteriocins extracted from lactic acid bacteria. Sarvana and Annalakshmi [25] also reported the synthesis of nisin‐capped SNPs at 450 nm. Pandit et al. [26] reported an absorption peak at 457 nm for nisin–silver nanoconjugate. Similar results were also reported by various other researchers [17, 27, 28].

Fig. 2.

UV–Visible spectrum of synthesised Bac4463‐capped SNP and Bac22‐capped SNP at optimised conditions

3.3 Characterisation of Bac‐SNPs

The capping of Bac4463 and Bac22 on SNPs was ascertained by the results of FTIR spectroscopy (Fig. 3). The peaks for Bac4463‐capped SNPs were obtained at 3354.07, 2140.14, 1638.64, 1365.57, 1217.03 cm⁻¹ and the peaks for Bac22‐capped SNPs were obtained at 3361.15, 2139.86, 1639.12, 1365.87, 1217.16. The peaks in the region, 3400–3250 cm⁻¹ indicates the presence of hydrogen‐bonded OH stretch and NH₂ group stretching. Bending at 2140.14 corresponds to C ≡ C stretch of alkynes. The sharp and peculiar peak in the region 1650–1580 and 1370–1350 cm⁻¹ attributes to the peptide linkage in amides and C–H stretching, respectively. The peaks in the region 1250–1020 cm⁻¹ might be due to C–O and C–N stretching [29]. The results depict the presence of amide group, OH group and C = O as major groups involved in reduction and stabilisation of Bac4463‐capped SNPs and Bac22‐capped SNPs. The spectrum of both Bac4463‐capped SNPs and Bac22‐capped SNPs was almost similar to that of Bac4463 and Bac22, respectively, indicating that the protein structure was not affected by the interaction of bacteriocins with SNPs during the synthesis of capped nanoparticles (Fig. 3). Sharma et al. [17] also reported that no major difference was observed in the spectrum of enterocin‐capped SNPs to that of free enterocin. The results are also in agreement with Rasheed [28] and Monowar et al. [30] who also reported no change in protein structure upon their interaction with silver ions.

Fig. 3.

FTIR spectrum of

(a) Bac4463‐capped SNP, (b) Bac22‐capped SNP

Crystalline nature of both the Bac‐SNPs was ascertained by XRD analysis (Fig. 4). XRD results for Bac4463‐capped SNPs showed peaks at 2θ values of 38.32, 44.28, 64.48, and 77.82. Similarly, the XRD pattern for Bac22‐capped SNPs showed peaks at 38.44, 44.74, 64.42 and 77.42, which were in accordance with the JCPDS file for crystalline silver 04–0783. This shows that the above 2θ values correspond to (111), (200), (220) and (311) planes of Braggs reflections for face‐centred cubic facets. XRD peaks observed in the present study clearly stated the crystalline nature of synthesised nanoparticles. These results were also in accordance with the study reported by Pandit et al. [26] and Bhople et al. [31]. In the present study, two intense and unidentified peaks at 2θ values of 54 and 57 were also observed, which may be attributed to the capping agent present on SNPs [26, 28, 32]. Particle size was calculated using the Debye–Scherrer formula. Particle size deciphered from the intense peak (111) for Bac4463‐capped SNPs and Bac22‐capped SNPs was found to be 34.59 and 28.22 nm, respectively (Fig. 4).

Fig. 4.

XRD spectrum of Bac4463‐capped SNP and Bac22‐capped SNP

DLS technique was used to calculate the size of the synthesised nanoparticles. DLS for Bac4463‐capped SNPs and Bac22‐capped SNPs gave an average hydrodynamic size of 93.23 and 77.36 nm, respectively. Similar Z ‐average values were also obtained in a study involving the synthesis of bacteriocin‐mediated SNPs suggesting that the nanoparticles were monodispersed, monodomal and spherical [33]. The hydrodynamic size of 82.1 nm was reported by Hong et al. [34]. The increase in size observed by DLS in comparison to XRD data in the present study can be attributed to the capping of Bac4463 and Bac22 on the surface of the nanoparticles, since hydrodynamic diameter is calculated as total size of core metallic particle and the shell or layer that envelops the core during reduction process (Fig. 5 a). Zeta potential was performed to determine the charge present on Bac4463‐capped SNPs and Bac22‐capped SNPs. Zeta potential of Bac4463‐capped SNPs was recorded to be −20.12 ± 1.33 mV, which shows that nanoparticles are fairly stable over a period of time. Bac22‐capped SNPs showed zeta potential of −32.12 ± 1.23 mV, which shows that the nanoparticles are highly stable and have less tendency to form aggregates (Fig. 5 b), since zeta potential values <−30 mV and >+30 mV are generally considered highly stable and are not prone to agglomeration due to more repulsion between the particles [35]. The negative charge on the nanoparticles also confirms the stability and repulsion between the nanoparticles [36]. The TEM analysis of purified Bac4463‐capped SNPs and Bac22‐capped SNPs showed irregularly shaped spherical nanoparticles with a size range of 16–36 and 15–29 nm, respectively, which are very similar to the particle size reported by XRD (Fig. 5 c). These results are in agreement with previous studies carried out using biological compounds [37]. Hence, the results of physicochemical techniques performed in the present study confirm the efficient synthesis of bacteriocin‐capped nanoparticles.

Fig. 5.

Bac4463‐capped SNPs and Bac22‐capped SNPs

(a ) DLS analysis, (b) Zeta potential, (c) TEM images

3.4 Antibacterial activity of partially purified bacteriocins and Bac‐SNPs

In‐vitro antibacterial activity of partially purified bacteriocins (Bac4463 and Bac22) alone, and bacteriocin‐capped SNPs (Bac4463‐capped SNP and Bac22‐capped SNP) was evaluated against six bacterial food pathogens. Nanoparticles are generally reported to act on bacterial membrane leading to membrane deformation, DNA and protein damage. Membrane damage caused by SNPs is basically induced by their interaction with the cell membrane which causes the formation of pores on the surface and eventually leads to cell death. Similarly, bacteriocins are also known to act on the target cell membrane by pore formation. Therefore it was hypothesised that bacteriocins in conjugation with nanoparticles would be more effective in killing the target organisms.

The results of antibacterial activity showed an increase in the activity of Bac4463‐capped SNPs and Bac22‐capped SNPs in comparison to Bac4463 and Bac22 alone (Table 1). Bac4463 showed inhibition against S. aureus, L. monocytogenes, P. aeruginosa and B. cereus with the zone of inhibition in the range of 14–24 nm. On the other hand, Bac4463‐capped SNPs were found to show enhanced antibacterial activity (1.0–1.5 fold) against all the tested indicator test strains as compared to Bac4463 alone, which might be because of the additional antibacterial and antifungal properties exhibited by silver ions. Bac22 showed inhibition against S. aureus, P. aeruginosa, S. flexneri and B. cereus with the zone of inhibition in the range of 12–23 nm. However, Bac22‐capped SNPs in comparison to Bac22 alone showed ∼1.5–2.3‐fold increase in inhibitory activity against all the indicator test strains. Enhanced activity of Bac22‐capped SNPs in comparison to Bac4463‐capped SNPs may be attributed to their small particle size and improved efficiency. Similar results for enhancement in activity of bacteriocin nano‐conjugates were reported by Sarvanna and Annalakshmi [25] and Gomaa [38] for bacteriocin‐capped SNPs. Thirumurugan et al. [39] also reported the improved activity of bacteriocins when combined with gold nanoparticles in comparison to bacteriocin and nanoparticle alone. On similar lines, Sharma et al. [17] also reported a better antibacterial activity of enterocin‐capped SNPs in comparison to the chemically synthesised citrate‐capped SNPs in his study. Singh et al. [40] also reported increased anti‐listerial activity of Gold nanoparticles‐pediocin‐LAP conjugate. Morales‐Avila et al. [41] also reported an increase in antibacterial activity of ubiquicidin–conjugated SNPs with regard to SNPs alone and ubiquicidin‐conjugated gold nanoparticles. Also, no inhibition by gold nanoparticles alone was reported.

Table 1.

Antibacterial activity of bacteriocin‐capped SNPs and bacteriocins alone against six food‐borne pathogens

Bacterial strains	Mean zone of inhibition (mm) ± SD
	Bac4463	Bac4463‐capped SNPs	Bac 22	Bac22‐capped SNPs
S. aureus	24.6 ± 0.66	30.3 ± 0.88	14.6 ± 0.33	21 ± 0.57
L. monocytogenes	20 ± 0.57	26.3 ± 0.33	—	12.3 ± 0.66
P. aeruginosa	14.6 ± 0.33	19.3 ± 0.88	18.3 ± 0.33	26.6 ± 0.66
B. cereus	20.6 ± 0.33	27.6 ± 0.8	12.6 ± 0.88	21.3 ± 0.33
S. flexneri	—	9.3 ± 0.88	23 ± 0.57	34.6 ± 0.88
E. coli	—	11.3 ± 0.33	—	13.3 ± 0.33

3.5 Determination of MIC and MBC

The MIC and MBC of Bac4463‐capped SNPs and Bac22‐capped SNPs were determined for all the six food spoiling Gram‐negative and Gram‐positive bacteria (Table 2). Table 2 shows that the MIC value of Bac4463‐capped SNPs was least for S. aureus (8 μg/ml) followed by B. cereus and L. monocytogenes which showed inhibition at 16 μg/ml. The highest MIC value of 128 μg/ml was exhibited against E. coli and S. flexneri. The value of MBC for S. aureus, B. cereus, L. monocytogenes, E. coli, P. aeruginosa and S. flexneri was found to be 8, 16, 32, 128, 64 and 128 μg/ml, respectively. On the other hand, Bac22‐capped SNPs showed least MIC value of 2 μg/ml against S. flexneri, followed by B. cereus and P. aeruginosa which were inhibited at a MIC value of 4 and 8 μg/ml, respectively. Highest inhibition at 64 μg/ml was shown for E. coli and L. monocytogenes. The MIC and MBC value of E. coli showed that E. coli was less susceptible to both synthesised Bac‐capped SNPs. Similarly, Loo et al. [42] also reported the low susceptibility of E. coli to green synthesised SNPs. Both the synthesised SNPs displayed good antibacterial activity, but Bac22‐capped SNPs were found to exhibit better inhibitory activity against S. flexneri, which is a potent food pathogen.

Table 2.

MIC (μg/ml) values of synthesised Bac4463‐capped SNPs and Bac22‐capped SNPs against pathogenic indicator test strains

Bacterial strains	Bac4463‐capped SNPs		Bac22‐capped SNPs
	MIC, μg/ml	MBC, μg/ml	MIC, μg/ml	MBC, μg/ml
S. aureus	8	16	8	8
B. cereus	16	16	8	16
L. monocytogenes	16	32	64	64
E. coli	128	128	64	128
P. aeruginosa	32	64	4	8
S. flexneri	128	128	2	4

The results of the present study displayed strong antibacterial efficiency of synthesised Bac‐SNPs in comparison to the bacteriocins alone against both Gram‐negative and Gram‐positive food spoiling bacteria. Similar results were obtained by Sharma et al. [17] who reported ∼2–16‐fold increase in the inhibitory activity of enterocin‐capped SNPs when compared with same concentration of citrate‐capped SNPs against a range of food pathogens used in the study. Pandit et al. [26] also reported that the MIC values of silver bio‐conjugate were lower than that of SNPs alone.

3.6 Effect of Bac‐SNPs on bacterial morphology

SEM analysis was carried out to visualise the changes in the bacterial cell morphology upon interaction with the synthesised capped‐SNPs. For this, two bacterial strains (one Gram‐positive and one Gram‐negative) were selected. The interaction of Bac4463‐capped SNP was observed with the cell wall of B. cereus, and that of Bac 22‐capped SNP was studied on S. flexneri. The results show a significant loss of membrane integrity when treated with sub‐MIC values. Pore formation and cell deformation could be observed in B. cereus cell membrane. Moreover, cell deformation and cell elongation due to stress response were observed in S. flexneri cells. These effects may be attributed to the combined effect of SNPs and bacteriocins (Fig. 6).

Fig. 6.

Interaction of Bac4463‐capped SNPs and Bac22‐capped SNPs with bacterial cells as visualised by SEM

(a) B. cereus untreated cells, (b) B. cereus cells treated with Bac4463‐capped SNPs, (c) S. flexneri untreated cells, (d) S. flexneri cells treated with Bac22‐capped SNPs

4 Conclusion

The present study deals with the biogenic synthesis of SNPs from antimicrobial peptides (bacteriocins) produced by lactic acid bacteria having the potential to act against food‐spoiling organisms. The antimicrobial activity shown by synthesised bacteriocin nanoconjugates was significantly higher than the activity of bacteriocin alone. Both the synthesised bacteriocin‐nanoconjugates were better than their non‐conjugated counterparts, but Bac22‐capped SNPs were found to be smaller in size, highly stable with better antimicrobial activity against both Gram‐negative and Gram‐positive bacteria in comparison to Bac4463‐capped SNPs. This safer and easy method of Bac‐SNP synthesis ensures enhanced antimicrobial efficiency against food pathogens and has the potential of being used for long‐term food preservation and in packaging materials. Hence, we may conclude that the amalgamation of nanotechnology and biotechnology opens the doors to a new prospect for dealing with bacterial food pathogens. However, their direct addition into food items still needs to be verified by detailed toxic studies.