Biogenic fabrication of CuNPs, Cu bioconjugates and in vitro assessment of antimicrobial and antioxidant activity

Abstract

In the present study, the authors synthesised copper nanoparticles (CuNPs) by using extract of Zingiber officinale (ginger) and later the NPs were bioconjugated with nisin, which shows antimicrobial activity against food spoilage microorganisms. CuNPs and its bioconjugate were characterised by ultraviolet–vis spectroscopy, NP tracking analysis, Zetasizer, transmission electron microscopy analysis, X‐ray diffraction and Fourier transform infra‐red (FTIR) spectroscopy. Zeta potential of CuNPs and its bioconjugate were found to be very stable. They evaluated in vitro efficacy of CuNPs and its bioconjugate against selected food spoilage bacteria: namely, Staphylococcus aureus, Pseudomonas fluorescens, Listeria monocytogenes and fungi including Fusarium moniliforme and Aspergillus niger. Antimicrobial activity of CuNPs was found to be maximum against F. moniliforme (18 mm) and the least activity was noted against L. monocytogenes (13 mm). Antioxidant activity of CuNPs and ginger extract was performed by various methods such as total antioxidant capacity reducing power assay, 1‐1‐diphenyl‐2‐picryl‐hydrazyl free radical scavenging assay and hydrogen peroxide assay. Antioxidant activity of CuNPs was higher as compared with ginger extract. Hence, CuNPs and its bioconjugate can be used against food spoilage microorganisms.

1 Introduction

Owing to the excessive use of antibiotics, microorganisms are developing resistance, and hence there is a need to search therapeutic agents, which can solve the problem of antibiotics resistance. Nanoparticles (NPs) have been known for its antimicrobial activity and can serve as an alternative to the antibiotics. Metal NPs have wide applications owing to their unique physical, chemical, catalytic, electrical and optical properties [1]. From the previous research, it has been revealed that CuNPs have several applications such as antimicrobial agents, sensors, heat transfer system and for the development of super strong materials [2]. Biosynthesis of NPs is a bottom up approach, which uses the biological system such as plants, fungi, bacteria, myxobacteria, actinomycetes etc. It is safe, cost effective and easily reproducible [3, 4, 5, 6, 7, 8, 9].

Phytochemicals present in the plants exhibit various antioxidants and reducing agents such as terpenoids, flavones, ketones, aldehydes and amides which accounts for the reduction process of metal ions leading to the synthesis of NPs [10]. Synthesis of NPs from plants is more advantageous than other biological systems, because it is easy and eco‐friendly. Moreover, the tedious work of isolation, identification and maintenance of culture is not involved [10, 11]. Lee et al. [12] synthesised CuNPs from Magnolia kobus leaves extract and copper (Cu) sulphate was used as a precursor salt solution. It was stated that synthesised CuNPs have potential antibacterial activity [12]. Phytofabrication of CuNPs has been reported from the plants such as Capparis zeylanica [13], citron juice extract [14], Gloriosa superba [15], Vitis vinifera [16], Punica granatum [17], Ocimum sanctum [18] Nerium oleander [19] and flower extract of Aloe vera [10].

Ingle et al. [20] reviewed that CuNPs exhibited antibacterial, antiviral, antiparasital and anticancerous activities. Nanocomposite film containing CuNPs was prepared and polylactic acid was used as polymer. Fabricated nanocomposite film was evaluated against one of the most common food contaminating bacterium, i.e. Pseudomonas spp. [21]. Many bacteria and other food‐borne pathogens form biofilm resulting into the food spoilage [22]. Listeria monocytogenes, Escherichia coli and Staphylococcus aureus are known worldwide for the food spoilage. These organisms adhere and form biofilm on the food surface and are responsible for the food‐borne infections. Biofilms are a unique, complex and dense assembly of microorganisms, which are interdependent on each other and adhere by an exopolysaccharides, proteins and nucleic acids. Biofilms are resistant to various antimicrobials, disinfectants and sanitisers [23]. Many researchers reported that metal and metal oxide NPs are known for its antimicrobial activity and wide range of other applications. Silver (Ag), zinc and Cu, act as antimicrobial agents, which help in the prevention of biofilm formation [24, 25, 26, 27, 28].

Nisin is composed of 34 amino acid residues known for its activity against food spoilage organisms. Food and Drug Administration (FDA) approved it as a safe food [29]. A recent research demonstrated that antimicrobial activity can be enhanced when nisin is loaded with pectin NPs [30]. Gomashe and Dharmik [31] reported nisin loaded gold NPs and demonstrated antimicrobial activity against food spoilage microorganisms such as E. coli, Micrococcus luteus, S. aureus, Proteus mirabilis and Klebsiella pneumoniae can be used in the prevention of food spoilage microorganisms. In the present paper, we synthesised CuNPs from ginger rhizome. Zingiber officinale (ginger) is one of the most commonly used condiments for flavouring food from ancient times. It is cultivated in southeastern Asia [32]. CuNPs were bioconjugated with nisin and their antimicrobial activity was evaluated against common food spoilage microorganisms such as S. aureus, Pseudomonas fluorescens, L. monocytogenes, Fusarium moniliforme and Aspergillus niger. Anti‐biofilm activity of CuNPs was tested after 24 h at different concentrations (0.50, 0.75, 1 mg/ml) against S. aureus, P. fluorescens and L. monocytogenes.

2 Materials and methods

2.1 Materials

Copper sulphate pentahydrate, sodium potassium tartarate and sodium hydroxide, polyethylene glycol (PEG 6000) were purchased from Hi‐Media, Mumbai, India.

2.2 Preparation of extract from Z. officinale rhizome

Rhizomes of Z. officinale were collected from the local market of Amravati, Maharashtra, India. The extract was prepared by boiling eight grams of rhizome in 100 ml of distilled water, filtered through Whatman's filter paper No. 41 and centrifuged at 4000 rpm for 30 min. The filtrate was then used for the synthesis of CuNPs.

2.3 Synthesis of CuNPs

Two solutions were prepared for the synthesis of CuNPs. A solution of Cu sulphate was prepared by dissolving seven grams of Cu sulphate pentahydrate in 100 ml distilled water. Another solution was prepared in distilled water by dissolving five grams of sodium potassium tartarate and eight grams of sodium hydroxide. Both the solutions were taken in 1:1 proportion and stirred vigorously. After that rhizome filtrate was added to the solution and heated at 50 °C for 20 min. Then, 500 µl of L‐ascorbic acid (1 mg/ml) was added to the mixture. After 20 min, red colour precipitate was obtained. It was centrifuged, washed thrice with distilled water, then dried in an oven to obtain powder. It was dissolved in liquid ammonia for further studies.

2.4 Bioconjugation of CuNPs

About 5 ml (1 mg/ml stock solution) of CuNPs solution was prepared in liquid ammonia, and therafter it was diluted with 5 ml of sterile distilled water. 5 ml (1 mg/ml) of PEG (PEG 6000) solution was added to it and stirred for 2 h. After that the stock solution of antimicrobial peptide nisin was prepared (1 mg/ml). 5 ml of the nisin solution was added to the above solution and stirred for 3 h. It was then centrifuged at 10,000 rpm for 20 min, washed twice with distilled water, to remove the unbounded peptide. Cu bioconjugate was characterised by ultraviolet–visible (UV–vis) spectroscopy, Nanoparticle tracking and analysis (NTA), Zetasizer and transmission electron microscopy (TEM).

2.5 Characterisation of CuNPs

2.5.1 UV–vis spectroscopy

Detection of the synthesised CuNPs and Cu biocojugate was made by subjecting the reaction mixture to UV–vis spectrophotometer (Shimadzu UV‐1700, Japan) in the range of 200–800 nm.

2.5.2 Nanoparticle tracking analysis

To determine the average size and size distribution of CuNPs and Cu bioconjugate, NTA analysis was performed by linear monolithic 20 (Nanosight Pvt. Ltd., UK). For determining the size of NPs, 5 µl of NPs solution and Cu bioconjugate were diluted in 2 ml of sterile distilled water. LM 20 is based on light scattering system, in which particles suspended in distilled water were injected into the LM viewing unit and viewed in the close proximity to the optical element. Brownian motion of particles was recorded (at 30 fps) by charge‐coupled device camera and the video and images of size distribution of particles were captured.

2.5.3 Zetasizer analysis

Zetasizer analysis was carried out by Zetasizer Nano zetasizer 90 (Malvern Instrument Ltd., UK) for measuring zeta potential and polydispersity index of synthesised NPs. Zeta potential was measured to determine the charges present on the surface of NPs. About 30 µl of NPs solution and Cu bioconjugate was diluted in 2 ml of sterile distilled water in order to determine the zeta potential of CuNPs and Cu bioconjugate. Distilled water was used as a diluting solvent for measuring the zeta potential charge present on the surface of CuNPs and Cu bioconjugate. The refractive index of solvent used for the analysis was 1.33 (distilled water).

2.5.4 Fourier transform infra‐red (FTIR) spectroscopy

FTIR data gives an idea about the biomolecules, which were responsible for the reduction of ions and stabilisation of CuNPs. FTIR study was performed on a Perkin‐Elmer FTIR‐1600, USA in the range of 500–4000 cm⁻¹ to determine the presence of capping agent and role of biomolecules involved in the synthesis of CuNPs. For sample preparation, colloidal NPs solution was mixed with potassium bromide in clean crucible, till it becomes a fine powder. The samples were prepared and dried in oven to remove the traces of moisture present at the time of NPs preparation.

2.5.5 X ray diffraction (XRD) analysis

XRD spectra were recorded on a Rigaku Miniflex II desktop X‐ray diffractometer instrument using CuKa radiation (0.15416 nm). It depicted a number of Bragg reflections indexed on the basis of the face‐centred cubic (FCC) structure of CuNPs. XRD data obtained after analysis was compared with Joint Committee on Powder Diffraction Standard file no. 04‐0836.

2.5.6 Transmission electron Microscopy

TEM analysis was performed to determine the size and shape of CuNPs. CuNPs were characterised by TEM (Philips, CM 12), on conventional carbon‐coated Cu grids. About 5 µl of the NPs sample and Cu bioconjugate were placed on the carbon‐coated grid and it was dried at room temperature for 1 h. The sample was inspected at 120 kV. Images of the sample were recorded.

2.6 Assessment of antimicrobial assay of CuNPs

2.6.1 Test organisms

The pure cultures of the food‐borne microorganisms P. fluorescens (Microbial Type Culture Collection 2269), L. monocytogenes (MTCC 1143) and F. moniliforme (MTCC 6636) were procured from Microbial Type Culture Collection and Gene Bank MTCC, Chandigarh, India. S. aureus (American Type Culture Collection 333591) was obtained from American Type Culture Collection, Manassas, VA, USA. A. niger was isolated from peanut (Department of Biotechnology 606) and procured from Department of Biotechnology, Sant Gadge Baba Amravati University Amravati, India.

2.6.2 In vitro evaluation of antimicrobial activity

The antimicrobial activity of CuNPs was evaluated against P. fluorescens, L. monocytogenes, S. aureus, A. niger and F. moniliforme by Kirby–Bauer disc diffusion [33]. The standard antibiotics discs of gentamicin and ketoconazole were purchased from Hi‐Media Laboratories Pvt. Ltd., Mumbai, India. Kirby–Bauer disc diffusion assay was performed against P. fluorescens, L. monocytogenes and S. aureus on nutrient agar, brain heart infusion agar and Muller–Hinton agar plates, respectively. A single colony forming unit of individual test organism was grown overnight in respective broth. For P. fluorescens, 24 h old culture was taken and maintained at 37 °C, whereas for L. monocytogenes and S. aureus 24 h old cultures were used at 37 °C. About 100 μl of inoculum (approximate concentration 10⁵ colony forming unit/ml) of each bacterium was streaked onto the respective agar plates to assess the activity of CuNPs, bioconjugate and sterile dics were impregnated with 20 μl of CuNPs (1 mg/ml), CuNPs, Cu bioconjugate, nisin were placed onto the surface of agar in respective plates. Antibiotic gentamicin was used as a standard antibiotic. The plates containing P. fluorescens were incubated at 30 °C for 24 h other containing L. monocytogenes and S. aureus were kept at 37 °C for 24 h. The zones of inhibition were recorded in millimetres against all the organisms. The above assay was performed in triplicates.

For evaluation of antifungal activity of CuNPs against F. moniliforme and A. niger, potato dextrose agar was used. About 20 µl spore suspension was streaked on the surface of agar in plates. The combination of discs as mentioned above were placed onto the agar surface and incubated at 28 °C for 48 h, followed by the measurement of the zones of inhibition. The assays were performed in triplicates.

2.6.3 Determination of minimum inhibitory concentration

Microbroth dilution method was used for determining minimum inhibitory concentration (MIC) of CuNPs against P. fluorescens, L. monocytogenes and S. aureus in 96 wells microtitre plate. About 24 h old bacterial inocula having 1 × 10⁸ CFU/ml was used for determining MIC of bacteria. About 10–100 µg/ml concentration of CuNPs was used for determining the MIC. For the visual observation, the microtitre plates of S. aureus and L. monocytogenes and P. fluorescens were incubated at 37 °C for 24 h. After complete incubation, 40 µl of tetrazolium salt was added to each microwell and the colour change was observed. MIC was performed in triplicates.

For determining MIC of CuNPs against A. niger and F. moniliforme. Microbroth dilution method was used in 96 wells microtitre plate. Fungi were inoculated in Rosewell Park Memorial Institute medium and incubated for 7–8 days at 25 ± 2 °C. The optical density (OD) of the fungal suspension was adjusted between 0.15 and 0.17 by using Rosewell Park Memorial Institute medium. The absorbance was measured by using colorimeter at 530 nm and the fungal load was maintained at 0.4 × 10⁴ –5 × 10⁴ CFU/ml. This suspension was used for evaluation of MIC. MIC of CuNPs and antifungal agent (ketoconazole) was determined by the guidelines of National Committee for Clinical Laboratory Standard/Clinical and Laboratory Standard Institute M27‐A [34]. About 10–80 µg/ml concentration of CuNPs was used for determining the MIC of CuNPs and the assay was performed in triplicates.

2.7 Antioxidant properties of ginger extract and CuNPs

Antioxidant activity of ginger extract and CuNPs was evaluated by total antioxidant capacity, reducing power assay, 1‐1‐diphenyl‐2‐picryl‐hydrazyl (DPPH) method radical scavenging assay and hydrogen peroxide assay. All the antioxidant assays were performed in multiple sets.

2.7.1 Determination of total antioxidant capacity

For performing total antioxidant capacity of ginger extract and CuNPs, 0.3 ml of samples was mixed with 3 ml of (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The mixture was incubated at 95 °C for 3 h in water bath. The absorbance was measured at 695 nm. Ascorbic acid was used as a standard [35].

2.7.2 Determination of reducing power

Antioxidant assay was performed by reducing power method [36]. In this method, 4 ml of CuNPs solution and ginger extract were taken in phosphate buffer solution (0.2 M, pH 6.6), potassium ferricyanide (1%w/v) and incubated for 20 min at 50 °C in water bath. The above solution was then mixed with distilled water and ferric chloride solution. The absorbance was noted at 700 nm. Ascorbic acid was used as standard.

2.7.3 DPPH assay

The free radical scavenging activity of ginger extract and CuNPs was measured by DPPH method. 0.1 mM DPPH solution was prepared in methanol. About 1 ml of this solution was added to 3 ml of the ginger extract and CuNPs solution. It was incubated for 10 min and after that absorbance was measured at 517 nm. Ascorbic acid was used as standard [37, 38].

2.7.4 Hydrogen peroxide assay

The free radical scavenging activity of ginger extract and CuNPs was performed by hydrogen peroxide assay (10 mM). Hydrogen peroxide was prepared in phosphate buffer solution (0,1 M, pH 7.4). About 1 ml of colloidal CuNPs and 0.6 ml of hydrogen peroxide solution were mixed and later incubated at 37 °C for 10 min. The absorbance was recorded at 230 nm [35, 39]. Ascorbic acid was used as standard. Antioxidant activity was calculated as below

$$\mathrm{antioxidant}\mathrm{effect}=\frac{\text{absorbanc}{\mathrm{e}}_{\mathrm{control}}-\text{absorbanc}{\mathrm{e}}_{\mathrm{sample}}}{\text{absorbanc}{\mathrm{e}}_{\mathrm{control}}}\times 100$$

2.8 Biofilm inhibition assay

For determining biofilm assay of CuNPs, 24 well (polyvinyl chloride) microtitre plates were used. The assay was performed against biofilm producing bacteria such as P. fluorescens, L. monocytogenes and S. aureus. Different concentrations of CuNPs (0.50, 0.75, 1 mg/ml) were tested after 24 h. About 24 h old cultures of P. fluorescens and L. monocytogenes were inoculated in nutrient broth and brain heart infusion broth, respectively. S. aureus was inoculated in Muller–Hinton broth. About 1 ml of inoculum of each organism (10⁵ CFU/ml) was used in the assay. This biofilm assay was performed after 24 h, growth medium was discarded from well without disturbing biofilm and 1 ml of 0.1% crystal violet was added and kept at room temperature for 30 min. Then, the solution was washed with distilled water and the remaining stain was removed by the addition of 2 ml of 95% alcohol. Biofilm inhibition was quantified by measuring OD at 580 nm. Wells without NPs act as control, while wells with CuNPs act as experimental.

Biofilm inhibition was calculated by the formula

$$\mathrm{biofilm}\mathrm{inhibition}(\text{\%})=\frac{\mathrm{O}{\mathrm{D}}_{\mathrm{control}}-\mathrm{O}{\mathrm{D}}_{\mathrm{experiment}}}{\mathrm{O}{\mathrm{D}}_{\mathrm{control}}}\times 100$$

3 Results and discussion

Biological synthesis of CuNPs was performed by using ginger extract. The extract was treated with copper sulphate and mixture of sodium hydroxide and sodium potassium tartarate. The reaction mixture was heated at 50 °C and it resulted in the formation of red colour precipitate which indicates the formation of CuNPs. It was centrifuged at 4000 rpm and the supernatant was discarded and pellet was washed thrice with distilled water which was dried in oven at 60 °C for 3 h. NPs were dissolved in liquid ammonia and blue colour colloidal suspension of NPs was obtained (Fig. 1). CuNPs were bioconjugated with nisin and PEG was used as linker molecule for the bioconjugation of CuNPs and antimicrobial peptide nisin. CuNPs and Cu bioconjugate were subjected to UV–vis spectrophotometer analysis. The synthesised CuNPs showed absorbance band at 618 nm (Fig. 2) which was due to the characteristic surface plasmon resonance of CuNPs. The present results showed resemblance with the studies of Shende et al. [14], who demonstrated the biological synthesis of CuNPs from Citrus medica juice extract and its absorbance peak was noted at 630 nm. UV–vis extinction of bioconjugate was recorded at 648 nm. UV‐vis absorbance value of nisin. It showed absorbance at 214 nm. (Fig. 2). After bioconjugation of CuNPs NPs with nisin, UV–vis spectra of CuNPs show red shift in the spectra that is UV–vis absorbance band of CuNPs shifted toward higher wavelength and the maximum absorbance was observed at 648 nm. The study was in accordance with the finding of Golubeva et al. [40], who bioconjugated AgNPs with peptide G Bac3.4 and it was found that the absorbance peak of Cu bioconjugate shifted toward higher wavelength. UV–vis absorbance spectra shifted toward higher wavelength. This present study corroborates with the study of Gaikwad et al. [4], who reported that larger is the size of NPs, higher is the wavelength (red shift), smaller is the size of NPs, less is the absorbance spectra (blue shift) [4].

Fig. 1.

Synthesis of CuNPs

Fig. 2.

UV–vis spectra of CuNPs synthesised from Z. officinale

(a) Spectra of nisin, (b) CuNPs, (c) Cu bioconjugate

NTA analysis was made to determine the size and concentration of CuNPs and bioconjugate. The size of CuNPs was found to be 29 nm and total concentration of CuNPs was found to be 0.52 × 10⁸ CFU/ml (Fig. 3 a). From NTA, size of Cu bioconjugate was determined and it was 63 nm and the concentration of was 0.67 × 10⁸ particles/ml (Fig. 3 b). After bioconjugation of NPs, it was found that the size of NPs increased. LM 20 is a laser‐based light scattering instrument, when a laser light is passed through the liquid suspension of NPs, particles scattered because of the Brownian motion of NPs. NTA analysis depends on the particles by particles size distribution [41].

Fig. 3.

Size distribution by NTA

(a) NTA (Nanosight LM 20) histogram showing size of CuNPs, (b) NTA (Nanosight LM 20) histogram showing size of Cu bioconjugate

After synthesis of NPs and Cu bioconjugate, zeta potential was measured by zetasizer which can determine the acquired charge present on the surface of CuNPs and Cu bioconjugate. In the present study, it was found that both NPs and Cu bioconjugates showed negative zeta potential, this might be due to the capping of NPs with proteins and metabolites of ginger extract. The zeta potential value of CuNPs and bioconjugate was found to be −44.3 and –39.3 mV, respectively, which demonstrated that CuNPs and bioconjugate were stable (Figs. 4 a and b). Higher is the zeta potential value, more is the repulsive force which prevents the aggregation of NPs. More is the zeta potential charge of the NPs, more is the stability of NPs [4]. If the zeta potential value lies between −30 and +30 mV that NPs are stable [42]. It was observed that after bioconjugation zeta potential value of bioconjugate increased by 5 mV. This study was in accordance with the result of Sadiq et al. [43] who investigated that after bioconjugation of nisin along with monolaurin, zeta potential value of bioconjugate increases by 5 mV.

Fig. 4.

Zeta potential value of CuNPs and bioconjugate

(a) Zeta potential measurement of CuNPs showing zeta potential value −44.3 mV, (b) Zeta potential of Cu bioconjugate showing zeta potential value −39.3 mV

The morphology and crystalline nature of CuNPs was determined by TEM and selected area electron diffraction (SAED). TEM analysis confirms the size and morphology of biosynthesised CuNPs and bioconjugate. Size of CuNPs was around 50 nm (Fig. 5 a). The crystalline nature of CuNPs was confirmed from SAED image. Debye–Sherrer rings of bright dots demonstrated crystalline and face cubic centred structure of biofabricated CuNPs ( Fig. 5 b). After TEM analysis, it was found that the size of Cu bioconjugate increases significantly and the size was near about 80 nm (Fig. 5 c). SAED pattern confirms the crystalline nature of the bioconjugate (Fig. 5 d). From TEM analysis, it was found that CuNPs and bioconjugate were not uniform in size but were spherical in shape.

Fig. 5.

Size of CuNPs

(a) TEM micrograph showing the size of CuNPs, (b) Diffraction pattern of CuNPs, (c) TEM micrograph showing the size of Cu bioconjugate, (d) Diffraction pattern of Cu bioconjugate

FTIR study was performed to understand which biomolecules act as capping agent on the surface of NPs. They are supposed to play vital role in the stabilisation of CuNPs. FTIR spectra of CuNPs were analysed and compared with the spectra of rhizome extract (control) (Fig. 6). FTIR spectra of control showed absorbance peaks at 1635, 1592, 1387, 1346, 832 and 758 cm⁻¹. FTIR spectra of CuNPs showed absorbance bands at 1632, 1180, 1113, 777 and 623 cm⁻¹. When the absorbance band of CuNPs was compared with control, it was found that the shifting of the band occurred as compared with control. The shifting of bands mainly occurs because of the stretching vibration of different functional group. The absorbance at 1592, 1387 and 1346 cm⁻¹ was due to stretching vibrations of the nitrogen dioxide (NO₂) group [44]. The sharp peak at 832 cm⁻¹ and weak band at 758 cm⁻¹ may be attributed to the C–H bending vibrations of –HCCH‐links of molecules present in the rhizome of ginger [45]. This was due to stretching vibrations of the NO₂ group, 1180 cm⁻¹, 1113 cm⁻¹ were attributed due to C–N stretching of amines [46, 47]. The absorbance peak at 1632 cm⁻¹ can be assigned to N–H bending of primary amines or it may appear due to –C=C– or aromatic groups [47]. The results reported in this study showed similarity with the previous studies they demonstrated that the functional group or bonds C=C, N–H are derived from biomolecules such as amino acids. Amino acids are nothing but the building block of proteins, which are composed of peptide linkage between various amino acids which are present in the plant extract [46, 47].

Fig. 6.

FTIR spectra of Z. officinale extract and synthesised CuNPs

(a) Z. officinale extract (control), (b) CuNPs (experimental)

Furthermore, the confirmation of CuNPs was made by XRD analysis. Bragg's reflection peaks generated due to XRD analysis, which depicted the crystalline nature of NPs. For CuNPs, sharp Bragg's reflections peak was observed at 2θ value of 44.5°, 52.2° and 74.4° corresponds to (111), (200) and (220) lattice planes which indicate face cubic centred crystalline nature of CuNPs (Fig. 7). The intense and sharp peaks of XRD showed that synthesised CuNPs was found to be crystalline in nature (Joint Committee on Powder Diffraction file no. 04‐784). The obtained peaks were compared with the JCPDS file no. 04‐784. The present study showed resemblance with the results of Shende et al. [14].

Fig. 7.

XRD pattern of CuNPs synthesised from Z. officinale extract showing FCC structure

In vitro antimicrobial activity of CuNPs and Cu bioconjugate was evaluated against S. aureus, L. monocytogenes and P. fluorescens, A. niger and F. moniliforme. It was found that synthesised CuNPs and Cu bioconjugate showed significant activity against all the tested microorganisms. Among all the microbes tested, F. moniliforme was found to be the most sensitive followed by A. niger, P. fluorescens, S. aureus and the least activity of NPs was noted against L. monocytogenes. It was demonstrated that antimicrobial activity of CuNPs and nisin both enhances after bioconjugation. Bioconjugate was found to be most effective against F. moniliforme and least against L. monocytogenes (Fig. 8). Many researchers demonstrated the antimicrobial activity of CuNPs [14]. It was reported that metal NPs possess antimicrobial activity against food spoilage organisms [48]. Moreover, it was also reported that after bioconjugation of gold NPs with antimicrobial peptides, the antimicrobial activity of both NPs and antimicrobial peptide was increased [49].

Fig. 8.

Antimicrobial activity of the synthesised CuNPs against microorganisms

(a) CuNPs, (b) Cu bioconjugate, (c) Nisin, (d) Antibiotics (for bacteria – gentamicin, for fungi – ketoconazole)

The biofilm inhibition activity of CuNPs was studied at different concentration against S. aureus, L. monocytogenes and P. fluorescens. It was found that biofilm inhibition activity of CuNPs increases as the concentration of NPs increased. The percentage of biofilm inhibition was found to be maximum against P. fluorescens followed by S. aureus and L. monocytogenes. The percentage of biofilm inhibition at different concentration is shown in Fig. 9. The present study also demonstrated the antimicrobial activity of CuNPs against certain food spoilage microorganisms and also showed biofilm inhibition activity against L. monocytogenes, S. aureus and P. fluorescens. Biofilm inhibition effect of CuNPs was reported by Ghasemian et al. [50] against biofilm forming P. aeruginosa and L. monocytogenes. It was reported that CuNPs not only act as antimicrobial agent but also as anti‐biofilm agents [51]. Recently, Ahmed et al. [52] investigated that bimetallic NPs Ag and CuNPs (Ag–Cu) act as excellent antimicrobial agent against food spoilage microorganisms which can prevent the deterioration of food. FDA recommended that bimetallic Ag–Cu NPs can be used as the suitable packaging material which can inhibit the food spoilage [52]. Tamayo et al. [53] reviewed that CuNPs based nanocomposite film can act as active food packaging material. It is cost‐effective material and it can be used for the preservation of food.

Fig. 9.

Biofilm inhibition assay of CuNPs synthesised from Z. officinale against bacteria at different concentrations

(a) Concentration of CuNPs was 0.50 mg/ml, (b) Concentration of CuNPs was 0.75 mg/ml, (c) Concentration of CuNPs was 1 mg/ml

MIC of CuNPs was evaluated against selected test microorganisms. MIC of CuNPs was least for F. moniliforme and maximum for L. monocytogenes (Table 1). When MIC of bacteria was performed, TTC was added in 96 wells plate. The appearance of pink colour in the microtitre plate was due to the reduction of Triphenyl tetrazolium chloride and formation of red colour complex formazan which represent the viable active growth of bacteria. The well which did not retain colour and became colourless was due to the inhibition of bacterial growth. MIC value of NPs is the lowest value at which bacterial growth is inhibited. MIC of CuNPs was found to be 20 µg/ml against F. moniliforme, whereas it was 55 µg/ml against L. monocytogenes. Antioxidant activity of ginger extract and CuNPs was calculated by four methods such as total antioxidant capacity, reducing power assay, DPPH free radical scavenging assay and hydrogen peroxide assay. Both ginger extract and CuNPs exhibited antioxidant property but as compared with ginger extract, antioxidant activity of CuNPs was more. Maximum antioxidant activity of CuNPs was noted for DPPH free radical scavenging assay (Table 2).

Table 1.

MIC of CuNPs synthesised from Z. officinale against tested microorganisms

Sl. no.	Name of the strain	MIC, µg/ml
1	F. moniliforme	20 ± 0.93
2	A. niger	25 ± 0.29
3	L. monocytogenes	55 ± 1.25
4	S. aureus	40 ± 0.87
5	P. fluroscens	35 ± 1.21

Table 2.

Antioxidant activity of Z. officinale extract and synthesised CuNPs

Sl. no.	Name of assay	Percentage of scavenging activity of ginger extract	Percentage of scavenging activity of CuNPs
1	total antioxidant capacity	40 ± 0.63	48 ± 061
2	reducing power assay	57 ± 1.03	68 ± 1.21
3	DDPH free radical scavenging assay	75 ± 0.87	81 ± 1.50
4	hydrogen peroxide assay	68 ± 0.74	74 ± 1.09

3.1 Hypothesis for antioxidant activity of Cu nanoparticles conjugated with nisin

Cu is converted into CuNPs by the reduction of organic molecule present in ginger extract. CuNPs possess higher antioxidant activity as compared with extract of ginger. This might be possible due to the coating of antioxidant compounds on the surface of NPs from plant extract. NPs have small surface to volume ratio so, they might have higher antioxidant activity as compared with the extract. When CuNPs are bioconjugated with nisin, the antioxidant activity maybe higher than CuNPs alone. From the previous report, it was found that when nisin is combined with the seed extract, the antioxidant capacity of the extract can be enhanced and it can be used against food spoilage microorganisms. In case of CuNPs bioconjugated with nisin, antioxidant activity of Cu bioconjugate maybe more. A few components of nisin may have antioxidant property which may enhance the antioxidant activity of the CuNPs. Free radical formation capacity maybe increased and so antioxidant capacity maybe enhanced.

4 Conclusions

To sum up, bioconjugation of CuNPs with nisin is possible and the properties of CuNPs and bioconjugate differ from each other when characterised by UV–vis spectroscopy, zeta potential, NTA analysis and TEM analysis. Furthermore, CuNPs can be utilised as anti‐biofilm agent against food‐borne pathogens such as S. aureus, P. fluorescens and L. monocytogenes. Antimicrobial and biofilm inhibition activity of CuNPs was remarkable against all the test microorganisms such as S. aureus, P. fluorescens, L. monocytogenes and fungi including F. moniliforme and A. niger. CuNPs can be used as a novel candidate for the prevention of food contamination. In addition, CuNPs and ginger extract exhibited potential antioxidant property. Bioconjugate exhibited superior antimicrobial activity against food spoilage microbes as compared with CuNPs alone. CuNPs and its bioconjugate exhibit excellent antimicrobial activity. Bioconjugated NPs can act as novel antimicrobial agent.