Talaromyces purpureogenus AUMC2603 was acquired from Asuit University's Mycological Center for Culture Collection, http://www.aun.edu.eg/aumc.htm. This strain was chosen as a potential source of red pigment in this study. The stock culture was kept at 4 °C on Potato Dextrose Agar (PDA) slopes and subcultured at regular intervals for the subsequent studies.
Culture condition for pigment production
Maximum pigment yield was produced under optimum condition obtained from statistical optimization methodology in our initial study  as follow: initial medium pH 6, temperature 25 °C for 18 day incubation period on Yeast Malt Broth (YMB) of the following composition (g/l): malt extract 3; yeast extract 3; peptone 5; glucose 10. The fermentation process was maintained under static dark condition. The un-inoculated media was used as control. These conditions were achieved using response surface methodology.
Extraction and estimation of extracellular pigment
One-factor-at-A-time (OFAT) strategy was used to determine the best solvent for extraction of pigment. Following the end of the incubation, the biomass and fermented broth were separated by filtration using filter paper, and the supernatant containing the pigment was taken out. Different organic solvents, including diethyl ether, chloroform, hexane, n-butanol, ethanol, and ethyl acetate, were used separately, to extract the crude red pigment under shaking at 100 rpm for 24 h at 25 ± 3 °C. To get rid of the solvents, the pigment extracts were concentrated at 45C in a rotary evaporator under decreased pressure.
The extracted pigment under each extraction solvent was tested for antibacterial activity to determine the most effective extraction. Eight pathogenic bacterial species were used as test organisms for the antibacterial activity. Bacillus cereus GST4, Bacillus subtilis BW2 (obtained from Faculty of Medicine's Microbiology Department at Zagazig University in Egypt), Escherichia coli ATCC 11,229, Salmonella typhi ATCC 14,028, Klebsiella pneumonia ATCC 13,883, Staphylococcus aureus ATCC 25,923, Pseudomonas aeruginosa ATCC 15,442, and Listeria inanovii ATCC 19,119 strains (obtained from The National Research Center, Giza, Egypt). The disc diffusion method was used to assess the antibacterial activity, according to Bennett . As a check, organic solvents were utilized to eliminate the solvent-related inhibition. 20 μl of dissolved extract was loaded on 5 mm sterilized disc of Whatman filter paper. Plates were then incubated at 37 °C for 24 h. Control for each organic solvent was also taken simultaneously to eliminate the solvent-related inhibition. The inhibition zone (mm) surrounding the filter was measured after the incubation. The yield of the pigment was expressed as optical density at 510 nm (OD510) of the extract which is the appropriate wavelength for maximum light absorption by produced pigment. In this study only extracellular pigments were considered.
LC mass spectrometry
In this investigation, 10 mL of HPLC grade 50:50 water:ethanol (Fisher Scientific, Ontario) were used to dissolve 2.5 mg of dried crude pigments. A LC and Q-Exactive HF Quadrupole-Orbitrap mass spectrometer were used for the analyses (Thermo-Fisher, Mississauga, ON). A Kinetex 1.7 m C18 LC column (100 2.1 mm) from Phenomenex, Torrance, California, was used for the separation. Gradient elution with 95:5% water:ethanol (A) and 100% ethanol (B), unbuffered for the negative mode and 0.1 percent formic acid for the positive mode, was used to produce the separation. The column was heated to 40 °C and the solvent flow rate was 0.1 mL/min. The gradient elution started at 5 percent B, climbed linearly to 100 percent B over 20 min, held at 100 percent B for 7 min, and then re-equilibrated to 5 percent B for 8 min over the course of the 35-min run. The following source settings for positive/negative ions were used to ionise the samples: sheath gas flow was 35/30, aux gas flow was 10/8, sweep gas flow was 1, aux gas heater was 400/300 °C, spray voltage was 3.8/2.7 kV, S-lens RF was 60, and capillary temperature was 350 °C. The following scan parameters were used: 120,000 resolution, 1 × 106 AGC target, 100 ms maximum injection duration, and 100–1000 m/z entire MS scan range. Thermo Fisher's Compound Discoverer 2.1 SP1 was used to locate unidentified peaks and produce elemental compositions based on precise mass data with a 1 ppm mass tolerance. Untargeted research workflow without statistics: Find and identify unknown compounds was the built-in workflow template that was employed. It performs retention time alignment, unknown compound detection, and compound grouping across all samples. All compounds' elemental compositions are anticipated, chemical background is obscured by utilizing blank samples, and ChemSpider can be used to determine the structures of compounds (exact mass or formula). 2560 and 973 distinct features were found in the positive and negative modes, respectively. To decrease the amount of features, the post-analysis filtering listed below was carried out: 1) Peaks observed only in sample extracts and not in blanks were taken into account; Only discovered peaks with area ratios (10 ppm/100 ppm) ranging between 0.08 and 0.12 were taken into consideration. Minimum peak areas are 1 × 105.
Fourier transform infrared spectroscopy FTIR
Fourier transform infrared spectroscopy FTIR was used to identify and characterize pigments structure in the crude extract by using (VERTEX 80v, BRUKER, Germany) at 4 cm−1 resolution and measurement scale range of 4000–400 cm−1.
Food is exposed to ionizing radiation during the food irradiation process in order to increase food safety and shelf life. In this experiment, it was also looked into how gamma radiation affected the extracted pigment. At the Nuclear Research Center in Inshas, Egypt, gamma radiation was administered. Cobalt-60 (60Co) source irradiations were carried out in a gamma irradiation cell at a dosage rate of 476.26 Gy/hr. Different irradiation dosages were used to radioactively treat pigment samples (1, 3, and 5 kGy).
Impact of irradiation on the biological functions of pigment
The disc diffusion assay was used as reported above to test the antimicrobial activity of irradiation pigment against bacteria and fungi. On agar plates, a 24-h-old bacterial culture and a 5-day-old fungus culture were alternately distributed on the suitable cultivation medium (Nutrient agar for bacteria and PDA for fungi). Each disc was inoculated by 20 µl of irradiation pigment, and left to diffuse for 2 h at a refrigerator. Following that, the plates were incubated for 24 h for bacteria and 5 days at 37 °C for fungal strains at 25 °C. As a control, discs inoculated with the same volume of unirradiated pigment were also tested concurrently. Following incubation, the growth inhibition zones' widths were measured. For every pigment, three replicates were run against each test organism. A mean and standard deviation were used to express the data.
DPPH and ABTS radical scavenging activity
The antioxidants activity of crude pigments, crude pigments exposed to gamma rays1, gamma rays2, gamma rays3 were evaluated using DPPH and ABTS assays at different concentration (0.05, 0.01, 0.15, 0.2, 0.5 and 1 mg/ml) as previously described by [49, 50].
DNA damage protection
To determine relevant impact of gamma ray treatment on DNA damage protection potency, the crude pigments treated with and without Gamma ray were subjected damaged DNA plasmid induces by Fenton's reagent as previously reported by Leba . In brief, 2 µg/ml of each sample were added to a mixture containing 3 μl of Ribonuclease Inhibitor (RNH1) plasmid DNA (60 μg/μl), Fenton's reagent composed of 5 mM of H2O2 and 0.35 mM of FeSO4 and 0.60 mM of EDTA and the final volume was completed to 20 μl with phosphate buffer 8.3 mM, pH 7.4. After 20 min of incubation at room temperature. The samples were separated at 1.5% agarose gel electrophoresis % and separated bands were analyzed. Plasmide DNA treated with fenton's reagent and 3 μl of RNH1 plasmid DNA (20 μg/μl) were used as positive and negative controls.
The samples were tested for their cytotoxic activity against two non-cancerous cell lines, BJ-1 skin fibroblast (ATCC® CRL-2522™) and MCF-12 epithelial breast (ATCC® CRL-10782™). The seeding, culturing and sub-culturing were carried out as previously described by [52, 53], with slightly modifications. In brief, 1 × 105/well cells were plated into 100 µl of medium/well in 96-well plates (Hi media) containing different concentration of crude pigments exposed or not γ-ray at three different doses. After 48 h incubation the cell viability was detected using MTT assay as previously reported by % . The concentration causes 50% inhibition of viability (IC50) was determined by plotting the cytotoxicity % against concentration. All experiments were performed in triplicate.
Preparation of beef burger samples
Fresh beef meat was obtained from the local market, Zagazig city, Sharqia Governorate in the day before experiment. The meat was stored in a refrigerator at 4 ± 1 °C overnight. Burger was prepared by the common method according to Modi  the mixture divided into four groups as follow:
The first group was the control (no additives), the second group had 200 ppm of synthetic BHT added to it as an antioxidant, the third group was split into three portions with AQP concentrations of 0.5, 1 and 2 percent, and the fourth group was exposed to gamma radiation at dose levels of 3 and 5 kGy. All groups were formed and put into polyethylene bags before being stored at (4 ± 1 °C) and periodically analyses.
The irradiation treatment was carried out using a 60Co Russian gamma chamber (dose rate 1.3 kGy/h) belonging to Cyclotron Project, Nuclear Research Center, Atomic Energy Authority, Cairo, Egypt. The applied irradiation doses were 3 and 5 kGy for beef burger.
Total viable bacterial count
Two identical Petri dish sets were used, together with pipette-diluted 1 ml portions of standard plate count agar (PCA, Biolife cod. No. 402145) from 10–1 to 10–6, which were then melted in the steam that followed. Agar is placed into Petri dishes after being cooled to 44–46 °C. As soon as possible, aliquots were rotated and tilted in the Petri dishes to combine with the agar medium. The Petri dishes were inverted and incubated at 37 °C for 48 h after solidification. The dilution factor was used to count and multiply the expanding aerobic colonies .
Psychrophilic bacterial count
According to the American Public Health Association , total psychrophilic bacteria were counted using plate count media estimated using the total bacteria count method, with the exception that incubation was carried out at 7 °C for 5 days in refrigeration.
Coliform bacterial count
In accordance with the instructions outlined in the Difco Manual, the coliform bacteria were identified using violet red bile agar media. The plates were incubated for 24 h at 37 °C .
Yeasts and molds
On potato-dextrose agar medium, yeast and mold counts were determined according to the Difco Manual protocols . The plates were incubated for 5 days at 20 to 25 °C.
Total volatile basic nitrogen (TVBN)
Total volatile basic nitrogen (TVBN) was measured as follows: 50 g of the minced sample were soaked in 600 ml of distillate water for 24 h in the refrigerator. The mixture was then filtered through cotton, and the volume was increased to 1 L in a flask using distillate water. A total of 400 ml was used, followed by the addition of 30 ml of ethanol, 2 g of magnesium oxide, and 25 ml of 0.1 N sulfuric acid to obtain a final volume of 150 ml in a distillation flask. To eliminate carbon dioxide, the distillate (150 ml) was heated for 10 to 15 min. The mixture was allowed to cool at room temperature before the addition of 0.2 ml of the 0.2% rosolic acid indicator and the quick titration back of the surplus sulfuric acid with sodium hydroxide. mg TVNB per 100 g of material was used to represent the results .
Thiobarbituric acid-reactive substances (TBARS)
The following values for thiobarbituric acid-reactive compounds (TBARS) were calculated using the Kirk and Sawyer method . Ten grams of the sample were combined with 50 ml of water for two minutes, then were washed into a distillation flask with 47.5 ml of water. 2.5 ml of 4 M hydrochloric acid was then added to the mixture to lower the pH to 1.5, and then an antifoaming agent and some glass beads were added. The flask was heated using an electric mantle, and after 10 min of boiling, 50 °C of distillate had been obtained. A glass-stopper tube was filled with five millilitres of distillate, or five ml T.B. Glacial acetic acid at a concentration of 0.2883 g/100 ml was added, stoppered, shacked, and heated for 35 min in boiling water. In parallel, a blank was made by mixing 5 ml of distilled water with 5 ml of reagent. The tubes were then cooled in water for 10 min, and the absorbance (O.D.) against the blank at 538 nm was measured using 1 cm cells using a spectrophotometer. TBARS = O.D. × 7.8 (as mg malonaldehyde per kg sample).
According to the procedure outlined by Watts et al., , sensory evaluation of beef burger samples (untreated and treated) was done in real time and regularly every three days for their appearance, odour, texture, and general acceptability during cold storage at (41 °C) for 24 days. Ten members of the Nuclear Research Center's food irradiation team are on the panel for sensory evaluation.
All independent analysis were carried out in triplicates (n = 3) for which the results were expressed as mean ± standard error. Data were analyzed using SPSS analytical software version 18.0 (SPSS Inc., Illinois, USA). Data were subjected to one-way analysis of variance (ANOVA) followed by Duncan test for comparison of means as a post-hoc test. Significant levels were based on the confidence level of 95% (p < 0.05). All methods were carried out in accordance with relevant guidelines.