Referenced pathogenic bacterial strains: E. coli (lot no: 020090, Des: NCTC: 12241 and ATCC: 25922), S. aureus (lot no: 460074, Des: NCTC: 10788 and ATCC: 6538), and B. cereus (lot no: 02900402, Des: NCTC: 10400 and ATCC: 6633) were obtained from Media Unit, Food Hygiene Department, Animal Health Research Institute, Dokki, Giza, Egypt.
The pathogenic strains were prepared and adjusted to obtain a population of 6 log10 CFU/mL.
Preparation of nano-curcumin (NC)
Curcumin powder from Curcuma longa (turmeric) (diferuloylmethane, (E,E)-1,7-bis(4-Hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione, natural yellow 3) was purchased from Sigma-Aldrich (CAS 458-37-7).
The nanoparticles were produced at the National Research Center, Physics Division, according to A Khataee, S Fathinia and M Fathinia  with some modifications. Briefly, powder was ground using a Moulinex grinder (Model MC300, France) and then crushed using a high-energy planetary ball-mill (Model PM 2400, Iran) at 320 rpm/2 h rotation speed to prepare NC. The ball-milling process was applied under cold atmospheric conditions (25°C and 1 atm.). Finally, the homogenous NC was measured using transmission electron microscopy (TEM).
All processes used for producing NC were controlled to keep its antioxidant and antibacterial properties.
Assessment of in vitro antimicrobial activity of NC against different food poisoning bacteria
The disk diffusion method was used according to N Padmavathy and R Vijayaraghavan  to assess the inhibitory range of NC at different concentrations (i.e., 2, 5, and 10 ppm) against each inoculated pathogenic bacterium: E. coli, S. aureus, and B. cereus at a concentration of 106 CFU/mL.
Evaluation of the antimicrobial property of NC using TEM
The morphology and size of NC and its biocide effect on E. coli, S. aureus, and B. cereus were analyzed using TEM with negative staining of phosphor tungstic acid (PTA 1%). Nano-emulsions were diluted according to K Kaur, R Kumar and S Mehta . The nanoemulsion samples were left for 3 h to dry. The grid was analyzed using TEM (JEOL JEM 1400, USA) with a working voltage of 200 kV at the TEM Unit of Cairo University Research Park, Egypt.
Assessment of the antimicrobial effect of NC on food poisoning bacteria inoculated into chicken kofta
- Preparation of chicken kofta
Chicken kofta was prepared according to the method described by . Chicken kofta prepared from minced chicken meat (72%) and refined wheat flour (7%), and the quantities of oat flour (8%), casein (2.5%), and hydrogenated fat (7.5%) were optimized.
Before the experiment, the meat was surface treated with ultraviolet light (UV) for 15 min for each side to minimize background micro-flora according to MK Morsy, R Elsabagh and V Trinetta .
The prepared mixture was divided into six groups and then inoculated with the prepared cultured bacteria adjusted at 106 bacteria as follows: 1st group: E. coli-inoculated samples (EC); 2nd group: E. coli-inoculated kofta treated with NC (10 ppm) (NCEC); 3rd group: S. aureus-inoculated samples (SA); 4th group: S. aureus-inoculated samples treated with NC (10 ppm) (NCSA); 5th group: B. cereus-inoculated samples (BS); and 6th group: B. cereus-inoculated samples treated with NC (10 ppm) (NCBS).
After inoculation, the samples were maintained at room temperature for 15 min to allow for cell attachment and stuffed into a sterile polyethylene casing. The samples were kept at 4°C ± 1°C for 27 days, and on days 0, 3, 6, 9, 12, 15, 18, 21, 24, and 27, the remaining microbial populations were analyzed. This experiment was repeated in triplicates to obtain the mean values for statistical analysis (n = 3).
The samples were aseptically opened, and approximately 10 g from each sample was transferred into 90-mL buffered peptone water 0.1% (BPW; Biolife) and then stomached (model G-560E, Bohemia) for approximately 1 min. Ten-fold serial dilutions were prepared, and then, 1 mL was spread plated over EMB (Biolife) according to ISO21150  for E. coli, paired parker (LO, Biolife) according to ISO6888-1  for S. aureus, and B. cereus Agar Base-MYP (BC-MYP, Biolife) with polymyxin B sulfate supplement (Code 4240001) and egg yolk emulsion (Code 42111601) following  for B. cereus. The obtained colonies were counted after 24 h from incubation at 37°C and expressed as log10 CFU/gm.
Physicochemical evaluations were applied according to AOAC . pH values were monitored using a digital pH-meter (model P107, Consort, Belgium), total volatile base nitrogen TVB-N (N/100 g of sample). Meanwhile, TBARS (MDA kg−1) was evaluated using spectrophotometry (CE 599 Universal, USA).
Sensory assessment for chicken kofta was evaluated under controlled conditions of temperature and humidity by seven well-trained panelists working at the Food Hygiene and Control Department of the Animal Health Research Institute. The criteria used as the basis of the descriptive organoleptic assessment (i.e., color, odor, and texture) using the triangle test and hedonic rating system. The scale points were used in the evaluation as follows: (9: excellent, 8: very very good, 7: very good, 6: good, 5: medium, 4: fair, 3: poor, 2: very poor, and 1: very very poor) .
Data on bacteriological and physicochemical properties and sensory attributes were tested for normality and homogeneity. The values were expressed as means ± standard errors of the mean.
Concerning the results of the effect of NC on food poisoning bacteria, the results were statistically analyzed using Student’s t-test according to R Steele and J Torrey .
To know whether the “P” value is significant or not, the calculated “P” value was compared with the tabulated “P” value at the level of degree of freedom at P ≤ 0.05 from the tables.