Curcumin powder (Cod No. C7727), polyethylene glycol, chitosan (Cod No. 448877), gelatin, 2,2-diphenyl-1-picrylhydrazyl (DPPH), Folin–Ciocalteu, 95% ethanol, casein, sodium carbonate, sodium nitrite, sodium hydroxide, acetic acid, gallic acid, methanol, and acetonitrile were bought from Sigma–Aldrich (St. Louis, MO, USA). The raw chicken filet was supplied from a supermarket in Cairo, Egypt and then moved to the laboratory within 15 min, minced, and kept in the refrigerator. Wheat flour, oat flour, and fat were obtained from a local market in Cairo, Egypt.

Three bacterial strains, E. coli (ATCC 25922), S. aureus (ATCC 6538), and B. cereus (ATCC 6633), were supplied from the Animal Health Research Institute, Dokki, Giza, Egypt. The bacteria were activated in tryptic soy broth (TSB; Biolife, Italy) and cultivated in the same media at 37 ± 1°C for 24 h. The bacterial population of ~6 log₁₀ CFU ml⁻¹ (colony-forming unit) confirmed the count by enumeration on tryptic soy agar.

Then, 200 g of curcumin was minimized, according to the study of Khataee et al. (2017). Briefly, the curcumin powder was crushed by a grinder (Model MC300, Moulinex, France) and then milled by a planetary ball mill (PM 2400, Iran) at 310 rpm/2 h. Under ambient conditions, the ball-milling procedure was used with the following ball mass-to-powder ratio of 10:1. After that, the curcumin was transformed into nanoparticles through a solid dispersion technique. Then, 100 mg of curcumin was dissolved in 5 mL of a polyethylene glycol (PEG) solution (80% w/v). Under constant magnetic stirring (~2 h), the curcumin-PEG complex was added drop-wise into chitosan (3%)-gelatin (1%) nanoparticle (CS-G-NPs; 1:1; v/v) solutions (polysaccharide mixture was 50 ml). The chitosan-gelatin nanoparticle (CS-GNPs) was used as a carrier and improved the solubility of curcumin. Subsequently, NPs were centrifuged at 10,000 rpm for 10 min (Farnia et al., 2016). The size of curcumin nanoparticles (Curcumin-NPs) was measured by a Zetasizer (NS300, UK). The sample was placed in a glass cuvette (1 ml) to avoid bubbles and then placed in a Zetasizer at a temperature of 10–90°C. Curcumin-NPs were kept at −80°C and applied in the experiments.

Total phenolic (TP) was measured based on the study of Hajimahmoodi et al. (2010). TP was expressed as mg of gallic acid equivalent (GAE) per g of curcumin. The total flavonoid (TF) content of curcumin was measured colorimetrically at 510 nm (Formagio et al., 2014). TF was computed as mg rutin equivalents (RE) per gram of curcumin. The analysis was performed in triplicate. The antioxidant ability of curcumin was estimated using DPPH radical scavenging at 517 nm (Liu et al., 2007). The results were expressed as IC₅₀ (μg ml⁻¹) (Ebrahimzadeh et al., 2010).

HPLC-Agilent Technologies (Santa Clara, CA, USA) was used to analyze the phenolic and flavonoid fractions (Ruslay et al., 2007). One gram of curcumin-NPs was added to 50 ml of methanol (70%), sonicated for 30 min, and then cooled. The curcumin extract was filtrated (Whatman paper No. 1) and concentrated to dryness by a rotary evaporator (IKA-WERKE; Germany) at the following conditions (speed 250 rpm, temperature 40°C, under vacuum). The sample was reconstituted with 100 ml of the mobile phase and filtered through an acrodisc syringe filter (0.45 μm; Gelman Laboratory, Ann Arbor, MI, USA), and then, 10 μl injected into the HPLC system. A detector (DAD) with a C₁₈ guard column, the HPLC column was an Agilent Eclipse XDB-C18 with an ID of 4.6 mm, a length of 150 mm, and a particle size of 5 μm (4.6 m × 5 μm). Two solvents formed the mobile phase: the first solvent was acetonitrile, and the second was acetic acid in acetonitrile (0.5%: 99.5%; v/v). Gradient elution was used to elute the solution, beginning with 100% solvent (1) and ending with 100% solvent (2). A DAD detector frequency of 425 nm was utilized. The flow rate was set at 0.8 mL/min⁻¹, and the run time was ~65 min. Consistent retention times and UV spectra were used to identify peaks and compare standards.

The antimicrobial ability of curcumin-NPs was determined by the disc diffusion method (Khezerlou et al., 2018). Briefly, tryptic soy agar plates were poured with 10 ml of semi-soft tryptic agar seeded with 100 ml of E. coli O157:H7 or S. aureus and/or B. cereus (~6 log₁₀ CFU ml⁻¹). The CFU was confirmed using the following equation:

Concentration _(start) × Volume _(start) = Concentration _(final) × Volume _(final)

The discs, which were placed over the plates, included curcumin-NPs at 2, 5, and 10 μg ml-1 (equivalent to 2, 5, and 10 ppm). The plates were examined for inhibition zones after 48 h at 37 ± 1°C. The inhibition zones were expressed as mm.

The morphology of curcumin-NPs and their mode of action against E. coli, S. aureus, and B. cereus were estimated. Briefly, 2 ml of sterilized TSB, 1 ml of bacterial, and 1 ml of curcumin-NPs were added in a falcon tube. The tubes were placed at 37 ± 1°C for 24 h. The pellets were recovered by centrifuging at 2,500 rpm min⁻¹ for 10 min. The pellets were placed on a carbon-coated copper grid, spread, and then dried under a lamp before being examined using transmission electron microscopy (TEM; JEOL JEM 1400, USA) at a voltage of 200 Kv with negative staining of phosphor tungstic acid (PTA 1%) (He et al., 2016). A Malvern Zetasizer (ZS90, Malvern Instruments, Worcester, UK) was used to measure the size and distribution of the nanoparticles.

The chicken finger was made using the technique described by Sharma et al. (2015) with slight modifications. Briefly, chicken filet (72%; w/w), wheat flour (7%; w/w), oat flour (8%; w/w), casein (2.5%; w/w), and fat (7.5%; w/w) were mixed and shaped. The chicken fingers were treated with UV-C (254 nm) for 15 min (Mcleod et al., 2018) to reduce background bacteria (Morsy et al., 2018). The bacterial cultures (~6 log CFU cm⁻²) were spread on the surface of the chicken finger.

After inoculation, samples were placed at room temperature for 10 min to allow cell attachment. The best concentration of antimicrobials in the challenge study was chosen according to Section 2.6, Antimicrobial Activity Assay (2, 5, and 10 μg g⁻¹). Curcumin-NPs at a level of 10 μg g⁻¹ were placed on the chicken finger surface and spread consistently. The control sample was chicken fingers without curcumin addition. Every sample was put into a sterile bag separately and tightly closed. For 27 days, the packed samples were placed at 4 ± 1°C. On days 0, 3, 6, 9, 12, 15, 18, 21, 24, and 27, the samples were assessed for any remaining microbes.

After being opened aseptically, we placed 10 g of each chicken sample in 90 ml of purified peptone water (PPW; Biolife; 0.1%) and shaken for 1 min. After preparing 10-fold serial dilutions, 1 ml of the supernatant was spread on Eosin methylene blue (EMB; Biolife) for E. coli and Paired Parker agar (Biolife) for S. aureus, and B. cereus agar base (Biolife). After 24 h of incubation at 37 ± 1°C, the colonies were counted (log₁₀ CFU g⁻¹) (Mangalassary et al., 2007).

A digital pH meter (Consort, Belgium) was used to measure the pH value. Total volatile base nitrogen (TVB-N; N 100 g⁻¹ of the sample) was estimated according to the approach of AOAC (2005). Thiobarbituric acid-reactive substances (TBARS; MDA kg⁻¹) were determined using spectrophotometry (AOAC, 2005). The results were expressed in terms of optical density at a wavelength of 538 nm, measured using a digital spectrophotometer (CE 599 Universal, USA).

Statistical evaluation was conducted using a one-way ANOVA to assess physicochemical properties and bacterial populations in the experiments (P ≤ 0.05) using SPSS 22 (IBM's SPSS Statistics Software). The data were analyzed using a completely random design (Armonk, New York, USA). For each treatment, each challenge experiment was conducted in triplicate. The LSD and Tukey's tests were utilized for multiple mean comparisons (Steel and Torrie, 1980).

The characteristics of curcumin NPs were assessed. TEM was used to confirm the curcumin's morphology and droplet size (Figure 1). The results showed that curcumin-NPs were spherical, smooth, and had a particle size of 80 ± 2 nm. The polydispersity index (PDI) was 0.14, and the zeta potential was 4.5 mV. In addition, it was observed that curcumin-NPs had a higher size distribution compared with nanoparticles produced by Bhawana et al. (2011). The curcumin-NP powder had high physical stability and chemical reliability and was easily dispersible in water. No curcumin residues remained after dissolution and uniform dispersion. The larger surface area of nano-sized curcumin particles, which encourages dissolution, may be the reason for their improved aqueous solubility (Stefan and Monchaud, 2019). Similar outcomes have been noted in previous studies, where active ingredients' efficacy, solubility, and bioavailability were enhanced by reducing their particle size to nanoparticles (Nishimoto-Sauceda et al., 2022).

The antagonistic effects of curcumin-NPs at concentrations of 2, 5, and 10 μg g⁻¹ were evaluated against E. coli, S. aureus, and B. cereus, as reported in Table 1. It has been shown that curcumin-NPs inhibit all foodborne pathogens. The inhibition zone diameters gradually increased when the concentration of curcumin-NPs increased. Curcumin-NPs at 10 μg g⁻¹ were found to be more active against S. aureus than E. coli and B. cereus. Additionally, the antimicrobial ability of Curcumin-NPs was examined in the literature (Zorofchian Moghadamtousi et al., 2014). It has been established that free curcumin has antimicrobial properties against a variety of bacteria, including the Gram-positive bacteria, i.e., B. cereus, B. subtilis, and S. aureus, as well as the Gram-negative bacteria, i.e., E. coli, P. aeruginosa, and Yersinia entero (Mukhtar and Ghori, 2012; Rai et al., 2020; Dai et al., 2022). One study by Sankhwar et al. (2021) demonstrated that nano-curcumin is more effective against Gram-positive bacteria than Gram-negative bacteria. Pandit et al. (2015) found nano-curcumin (in vitro) antibacterial activity against E. coli and S. aureus with diameters of 12 and 15 mm, respectively.

Polyphenols and flavonoids exhibit functional properties such as antimicrobial and/or antioxidant activities. Table 2 presents the phenolic profiles of curcumin and curcumin-NPs. A total of 19 phenolics were discovered, and the main compounds were salicylic acid, e-vanillic, ellagic, rutin, caffeine, benzoic acid, p-coumaric, and pyrogallol. However, eight flavonoid compounds were identified: apigenin, hispertin, luteolin, rosmarinic, kaempferol, rutin, quercetin, and quercetrin. It was observed that the curcumin-NPs contain higher levels of phenolic and flavonoid compounds compared to bulk curcumin. The results demonstrated that curcumin-NPs contain higher levels of salicylic acid (305.58 mg 100 g⁻¹), e-vanillic acid (244.76 mg 100 g⁻¹), and ellagic acid (107.35 mg 100 g⁻¹) compared to bulk curcumin. The phenolic compounds are of considerable interest in dietary antioxidant supplementation. In contrast, the major flavonoid compound is apegnin (239.03 mg/100 g⁻¹). These results are consistent with those mentioned by Shalaby et al. (2016), who reported that curcumin-NPs are rich in phenolic compounds, especially salicylic acid, e-vanillic acid, ellagic, rutin, caffeine, benzoic acid, p-coumaric, and pyrogallol.

In Table 3, the curcumin-NPs had a higher total phenolic content (67.48 mg g⁻¹ as gallic acid equivalent) than curcumin (58.33 mg g⁻¹ as gallic acid equivalent). Curcumin-NPs also had a higher total flavonoid content than curcumin. Faid (2021) found phenolic and flavonoid contents of 55.35 and 40.12 mg g⁻¹gallic acid in the ethanol extract of curcumin. However, Shalaby et al. (2016) reported that phenolic contents ranged from 18.4 to 20 mg g⁻¹gallic acid, and flavonoid content ranged from 36.07 to 40.48 mg g⁻¹ rutin in water and ethanol extracts of turmeric. These results are consistent with those reported by Hettiarachchi et al. (2022). The presence of polyphenols and flavonoids illustrates the antimicrobial and antioxidant activities of curcumin nanoparticles. When in nanoform, curcumin dissolves in water and is just as effective as regular curcumin (with increased phenolic and flavonoid levels). Circumin-NPs's greater efficacy compared to curcumin is attributed to its smaller particle size. It is worth remembering that, as curcumin becomes nanoparticles, its size decreases to 80 nm, which is significantly smaller than the size of curcumin particles, which allows for improved cell penetration and uptake.

Furthermore, Table 3 shows the antioxidant activity of curcumin-NPs (scavenging DPPH free radicals). The curcumin-NPs had a powerful ability to eliminate free radicals (IC₅₀; 22.47 μg ml⁻¹) compared to α-tocopherol and BHT (IC₅₀; 15.58 and 5.16 μg ml⁻¹, respectively), considering that the antioxidant capacity and the IC₅₀ amount have a negative correlation. IC₅₀ denotes the concentration of the sample required to scavenge 50% of DPPH radicals. Curcumin nanoparticles showed better IC₅₀ than curcumin, possibly due to particle size, speed solubility, and distribution. The study by Ak and Gülçin (2008) reported that curcumin extract has a high free radical-scavenging capacity compared to trolox, and the EC₅₀ for curcumin was 34.86 g ml⁻¹. Faid (2021) found that 50 μg ml⁻¹ of nano-curcumin extract had the highest scavenging capacity in ethanol extract compared to its chloroform and aqueous counterparts. Moreover, the IC₅₀ value was 22.35 μg ml⁻¹.

The mechanism of curcumin-NPs against food-borne pathogens appears necessary. Figure 2 depicts the TEM images of cells of E.coli, S. aureus, and B.cereus. It was observed that, during the growth of the pathogens in the presence of curcumin-NPs, these compounds appeared as dark, electron-dense spheres anchored to the bacterial cell wall. They disrupted the peptidoglycan layer and penetrated the cell, leading to cell lysis and resulting in cell death and the disruption of organelles.Curcumin-NPs were highly effective against both Gram-positive (G+) and Gram-positive (G–) bacteria. This is illustrated in Figure 3. The NPs alter the shape of the bacteria's cells and interact with their membrane, such as (a) the disruption of the membrane walls of specific bacteria, (b) the damaging effect on bacterial DNA, and (c) membrane leakage of the bacterium, producing a lot of reactive oxygen species, either as singlet oxygen or hydroxyl radicals. Our findings are in line with earlier research that demonstrated the mobilization of nanoparticles made of various materials within the bacterial cell. Previous research on B. subtilis 168 has shown that curcumin's ability to inhibit microbes involves interfering with the GTPase activity of FtsZ protofilaments, which are essential for bacterial cytokinesis (Jain et al., 2009). This dysfunction kills bacteria and prevents bacterial cell division by inhibiting the cluster dynamics of FtsZ in the Z ring. In another study by Bhawana et al. (2011), it was found that curcumin extract prevents cell adhesion to fibronectin and positively inhibits the bacterial surface protein sortase A, thereby demonstrating its antibacterial activity against S. aureus.

Another study reported that the antimicrobial ability of curcumin-NPs could be due to (i) the formation of transmembrane cell pores, which results in the leakage of essential metabolites, and (ii) the disruption of the structure of the bacterial cell wall, such as ergosterol, which is responsible for the synthesis and maintenance of cell wall rigidity (Rai et al., 2020). In the current study, curcumin-NPs are believed to exhibit an antibacterial effect by adhering to the bacterial cell wall, rupturing it, entering the cell, and interfering with the organization of cell organelles.

As is known, the most important bacteria related to chicken are Salmonella and Campylobacter. However, in recent years, the literature in Egypt and around the world has isolated E. coli, S. aureus, and B. cereus from chickens. Our study also aimed to evaluate the antibacterial effect of nano-curcumin against different foodborne pathogens, a representative type of bacteria from each of Gram +ve (S.aureus), Gram –ve (E. coli), and spore-forming (B. cereus). The antimicrobial effectiveness and modes of action of curcumin-NPs at a concentration of 10 μg g⁻¹ against E. coli, S. aureus, and B. cereus in chicken fingers stored under refrigeration for up to 27 days. Figure 3 demonstrates the antimicrobial effectiveness of curcumin-NPs against pathogens found on chicken fingers. The bacterial community developed persistently during the 27 days of refrigerated storage in the control. Conversely, treated samples with Curcumin-NPs revealed a decrease of nearly 2 log units in populations of E. coli and S. aureus after 3 days, while a 1 log decrease in B. cereus after 6 days stayed fixed for the rest of the challenge study. E. coli and S. aureus were sensitive to the Curcumin-NPs, as evidenced by the decline in population (~3 to 4 log₁₀ CFU g⁻¹). However, compared to control, B. cereus (~2 to 3 log10 CFU g⁻¹) was found on chicken fingers after 27 days of storage (Figure 4). These findings demonstrate that curcumin nanoparticles applied to processed chicken can restrain foodborne microorganisms for up to 27 days of refrigerated stockpiling. The outcomes concur with those detailed by Rai et al. (2020), who found that curcumin-stacked nanoparticles are more successful against foodborne microbes such as E. coli and S. aureus.

The pH, TVB-N, and TBARS contents are important freshness indices of meat quality. Figure 5A shows the progression of the pH value of chicken fingers during refrigerated storage. There were no huge contrasts (P ≥ 0.05) in pH values between the treated chicken samples and the control at time zero. However, a significant difference (P ≤ 0.05) was observed during storage at 4°C. After 9 days, the control sample's pH value quickly increased between 6.6 and 6.7. However, samples treated with curcumin-NPs at 10 μg g⁻¹ demonstrated only a slight increase in pH during storage. The pH score of chicken samples incorporating curcumin-NPs ranged from 6.33 to 6.44 after 21 days. The findings reveal that nitrogenous compounds are broken down by endogenous or microbial enzymes, thereby increasing the pH (Fijałkowska et al., 2015). Untreated chicken fingers began to deteriorate and were rejected on day 12 of cold storage as a result of this pH increase.

Figure 5B shows the changes in the TVB-N content of chicken fingers, including curcumin-NPs, during storage at 4°C for 27 days. Curcumin-NP addition and storage period significantly affected protein quality (P ≤ 0.05). In the control sample, the TVB-N level rapidly increased from 2.86 to 21.59 mg N₂100 g⁻¹ on the 9 day of storage due to the presence of various bacteria. The chicken fingers that included curcumin-NPs had the lowest TVB-N score, indicating that curcumin-NPs inhibit microbial growth, particularly proteolytic bacteria, which break down proteins into volatile nitrogen compounds. The increase in TVB-N during the storage of chicken fingers (control) might be attributed to a breakdown of nitrogenous mixtures by microbial action (El-Nashi et al., 2015).

Figure 5C, at time zero, the TBARS scores in various chicken finger samples ranged from 0.08 to 0.11 mg MDA kg⁻¹. However, during storage time, a rapid increase in the MDA level was recorded in the control samples (with different bacteria), ranging from 0.88 to 1.09 mg MDA kg⁻¹, after 9 days. These outcomes are consistent with previous data on chicken meat (Khajeh Bami et al., 2020; Panahi et al., 2022). In general, the storage of the chicken finger samples prompted a remarkable increase in the TBARS scores in most treatments, but it could be observed that there was a higher increase in TBARS in the control samples than in the treated samples. The samples included Curcumin-NPs with the lowest TBARS value due to their antioxidant activity. The antioxidant effect of the addition of curcumin to meat products was studied, such as rabbit burgers (Mancini et al., 2015), beef meatballs (Milon et al., 2016), lamb sausage (De Carvalho et al., 2020), and chicken mince (Sharma et al., 2012). Mughal (2019) has shown that turmeric's antioxidant properties prevent peroxide formation in food. Curcumin, which is found in turmeric, has a unique structure that enables it to act as an antioxidant and break the chains by trapping oxygen-free radicals (Urošević et al., 2022).