Impact of gamma irradiation and poppy seed extract on quality and storage stability of beef patties

ABSTRACT The present research was conducted to measure the impact of poppy seed extract (PSE) and gamma irradiation on the storage stability and antioxidant profile of beef patties during storage at different packaging. Different doses of gamma irradiation (2.5 kGy and 5 kGy) were applied alone and with combination of 2% PSE. The PSE enriched beef patties were packed (aerobically and vacuum) and kept at refrigeration temperature. The results show that higher value of TBARS and POV was found in 5 kGy aerobically packed samples at 14 day, while the lower value of TVBN was observed in 0 kGy+PSE2% at 0 day. However, the lower value of TBARS, POV and TBVN was found in vacuum packed samples (0kGy +2%PSE) at 0 day. The higher total phenolic contents, ferric reducing antioxidant power and DPPH value were found in 0kGy +2%PSE and lower value was found in samples (5 kGy). The microbial results were observed to decrease with the increase in dose of gamma radiation. The significant changes were observed in the hunter color and sensory attributes of beef patties on different treatment at storage intervals under vacuum and aerobic packaging. Conclusively, PSE, gamma irradiation and both packaging is improved the quality, stability and antioxidants profile of enriched patties.


Introduction
Ground beef patties are the most popular beef products for consumers preparing meals at the home.The limited shelf life of fresh ground beef has less than 7 days in ordinary refrigerators that can protect it from microorganisms spoilage.Pseudomonads and lactic acid are two bacteria that are recognized as the main spoilage microbes.They can initiate the deterioration of chilled or stored minced beef. [1]Beef meat is categorized as highly valuable food, and it comprises minerals such as zinc and selenium and also contains B complex vitamin.The food products that are easily prepared and ready-to-eat food items are increasing with the demands, and on another hand the major, concerns for food security and safety also increasing quality.Lipid peroxidation is one of the important key factors in meat that affects meat acceptability.It decreases the meat freshness and also affects its color.Lipid peroxidation in beef not only reduced meat value but also produced other free radical that causes mutagenesis, inflammation, cardiovascular disease, aging and carcinogenesis.Therefore, it is necessary to reduce lipid peroxidation arising from natural resources. [2]Basically, lipid oxidation is the process in which unsaturated fatty acid makes the section of membrane phospholipids.Furthermore, oxidized are sensitive in the oxidation of meat due to more unsaturated fatty acids as compared to other lipids.However, the level of oxidative degradation can be decreased by manipulation with natural antioxidants which preserve lipids from oxidation and secure oxmyoglobin.There is a significant increase in utilization of antioxidants to reduce the chemical deterioration of meat products.The antioxidants obtained from plants can be combined with meat or meat items during processing.Ascorbic acid has antioxidant properties that can be used in meat products to preserve basic quality. [3]oppy (Papaver somniferum) is an ornamental plant that is cultivated all over the world.The poppy seeds (Papaver somniferum) are a multiuse yield that is utilized in pharmaceutical purpose, bakery and confectionary products and seed oil. [4]Poppy seeds have high antioxidant activity. [5]Although alkaloids from poppy capsules and straw are generally used in the pharmaceutical industry, whereas its seeds are used largely in various baked products. [6]In addition to alkaloids, phenyl propanoids flavonoid capacity in poppy seed extracts was identified by. [7]A recent study suggests that poppy seeds can be used as food and to produce edible oil. [8]Previous studies showed that poppy seeds were incorporated into the different types of meat products as a fat replacer and antioxidants. [9,10]The newly developed technologies make assured the microbiological safety of meat with radiation handling. [11]Food irradiation eliminates the microbial population from food that are injurious for human health.It is the physical procedure involving the handling of foods between ionizing radiation. [12]The effects of the combined treatment of turmeric powder and irradiations in chicken meat and observed that with the increase in gamma radiation dose, the microbial load decreases.Composition of gamma rays and bioactive composite is helpful in increasing the stability and quality of chicken as well as its products.It can also be useful for bringing down the quantity of gamma rays. [13]amma rays (GR) are mostly used in commercial plants to handle pre-packed products due to high penetrating power.However, electron beam (EB) has limited penetration power and depth only up to 8 cm in food.The low dosage of gamma radiation (3 kGy) are helpful in instantly the shelf life of ground beef by reducing the growth of microbes.The 3 kGy gamma radiation dose would reduce 99% of bacterial loads (Salmonella spp., and Listeria spp.,) from meat products as well as from ground beef. [14]he main objective was to minimize the quality loss of beef patties as well as enhancing the antioxidant profile of poppy seed extract enriched beef patties during storage.The significance of this study shows that the combination of poppy seed extract and irradiation increased the shelf stability of beef patties at different storage and packaging.

Procurement of materials and preparation of samples
All the raw materials including beef and poppy seed were procured from local market of Faisalabad, Pakistan.The chemicals and glassware are purchased from Sigma-Aldrich®, USA.Poppy seeds were sun dried for one day and then crushed to form fine powder by means of grinding machine.After that grinding powder was preserved in air tight bags to prevent moisture gain prior to extraction.Patties were formed when beef was thawed and minced.

Extraction of PSE and solvent filtration and evaporation
Poppy seeds powder was put in 500 ml beaker with 70% ethanol at 1:10 solid to solvent ratio.The extract was carried out at room temperature for 10 min. [15]The extractions were clarified by using Whatman No.1 filter paper.The filtrate of all samples were evaporated with a rotary evaporator attached with a vacuum pump and a frozen cooling system.All solvent was dried in water bath at high temperature (79°C) and vacuum pressure (0.07 MPa).

Development of beef patties
Ground beef weighed (100 g of each) and shaped into patties.The extract with different concentration was added to beef patties and then patties were stored in airtight plastic bags at refrigeration temperature.

Gamma irradiation
Gamma irradiation was applied at Nuclear Institute for Agriculture and Biology, Faisalabad, which is under the Pakistan Atomic Energy Commission.There were total six samples with vacuum and aerobic packaging.Two doses of gamma irradiation (2.5kGy and 5kGy) were used alone and with combination of poppy seed extract (2%).

Hunter color (Lab)
The PSE enriched beef patties surface color was evaluated by Hunter colorimeter, with respect to calibration plate (L = 89.2, a = 0.921and b = 0.783).The CIE L (lightness), CIE a(redness) and CIE b (yellowness) color values were evaluated and examined statistically.The Hunter color of beef patties was measured according to the method of American Meat Science Association. [16]

Determination of total phenolic contents (TPC)
Total phenolic content of enriched beef patties was evaluated by spectrophotometric with using Folin-Ciocalteu reagent. [15]Briefly 1 ml of 10% Folin-Ciocalteu reagent was added to 0.5 ml of a recognized concentration of a sample.The mixture was left for 6 min after mixing well and then add 2 ml of 20% sodium carbonate solution in the above mixture.At 760 nm the phenols were measured for 60 min at 30°C using the spectrophotometer after reacting.A standardized bend was set using standard solution of gallic acid and the results of total phenols was expressed as 1 g of gallic acid equivalents (GAE) per gram of sample.

DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging assay
DPPH is a firm extremely colored free radical that can abstract labile hydrogen atoms from phenolic antioxidants with related shape of a colorless hydrazine (DPPH-H|). [17]The scavenging activity of free radical (FRSA) of the PSE enriched patties was determined according to the technique explained by the [18] with some changes.Using spectrophotometer homogenized sample of 0.1 ml was added in freshly prepared at 517 nm.The total FRSA of each sample was expressed as the % age of DPPH decreased and calculated as follows: FRSA = 100-(initial absorbance -final absorbance)/initial absorbance.The absorbance values of the initial and final absorbance are of DPPH at time zero and after 60 min respectively.

Ferric Reducing Antioxidant Power (FRAP) Assay
Homogenized sample of 10 µl was added to 30 µl of distilled water and 300 µl of FRAP reagent at room temperature.After that the following solution was incubated for 5 min.After the power of the blue hued complex formed at that point was expected spectrophotometrically utilizing Jenway UV-Vis Spectrophotometer at 593 nm.The FRAP reagent was prepared by mixing 25 ml of acetate buffer (300 mM Sodium acetate at pH 3.6) and 2.5 ml of TPTZ (10 mM 2,4,6-Tripyridyl-s-triazine in 40 mM HCl) with 2.5 ml of Ferric chloride solution (20 mM FeCl3.6H2O in distilled water).FRAP reagent was warmed at 37°C and prepared fresh before use.The decreasing intensity of each sample at that point was determined as equal to that of 1 mM of Fe (II) (FRAP unit).All the samples were analyzed in triplicates (n = 3). [19]

Total Volatile Basic Nitrogen (TVB-N)
The TVBN was performed according to the Conway micro-diffusion method described by Pearson. [20]Samples were mixed with trichloroacetic acid (TCA) in the quantity of 1:2 (w/v) and homogenized using mixer (Waring Commercial Blender, USA).The samples were centrifuged (3000 rpm for 5 minutes) and filtered through Whatman No.1 channel paper.The 5 ml of 10% sodium hydroxide was added to the mixture.Steam cleansing was performed using distillated.Until the shade turns pink the distillate was titrated against 0.05 M sulfuric corrosive.

Thiobarbituric Reactive Substances (TBARS)
The 2-thiobarbituric reactive substances values were measured according to the method of Schmedes et al. [21] with few modifications.The 5 g sample was mixed with 25 ml of 20% trichloroacetic acid (200 g/L) in 135 ml/L phosphoric acid solution in a homogenizer for 30 s.The homogenized samples were separated through Whatman filter paper number 4 to dispose of solid particles from the filtrate.2 ml of 0.02 M watery TBA arrangement (3 g/L) was added to 2 ml filtrate in a test tube at point.The cylinders were incubated at 100°C for 30 min and cooled in running tap water.The absorbance of supernatant solutions was estimated at 532 nm utilizing UV-VIS spectrophotometer.Standard curve was determined the TBA and communicated as mg malonaldehyde per kilogram (MDA/kg) of sample.mg malondialdehydes perkg meat ¼ Sample absorbance À blankÞ � Total sample vol:0:000156 � 1000

Peroxide value (POV)
The peroxide value of beef patties was measured according to the method of Salam. [22]Total 3 g samples were weighed in Erlenmeyer flask and warmed for 3 minutes at 60°C to liquefy the fat in a water bath.After that the jar agitated for 3 min with 30 ml acetic acid-chloroform solution (3:2 v/v) to break down the fat.To extract the solid particles from the filtrate, whatman channel paper number 1 was utilized.The procedure was proceeded with the addition of starch as indicator after the addition of potassium iodide solution (0.5 ml) to filtrate.The titration was sustained against usual solution of sodium thiosulfate POV was calculated by following equation.
S is the volume of titrant (ml), N is the normality of sodium thiosulfate solution (N = 0.01) and W is the weight of sample (g).

Total bacterial and coliform count
The total bacterial and coliform of beef patties were measured by using different treatmentsMeat samples were placed in enrichment broth, and then meat samples were incubated in most suitable condition. [23]

Sensory evaluation of beef patties
Sensory evaluation of the different treated beef patties was measured on the basis of color, flavor, texture, taste, and overall acceptability.The beef patties were evaluated by a panel of trained judges using a 9-point hedonic scale ranging from 9 = like extremely to 1 = dislike extremely. [24]All panelists were asked to evaluate flavor, color, taste, odor and texture.Water was given to all experienced panelists to rinse their mouths between the samples

Statistical analysis
The collected data was evaluated statistically using the Statistical Package, Statistic 8.1.according to respected method. [25]The levels of significance (P ≤ .05)were checked (ANOVA) using 3 factor factorial under CRD.The means were compared by using LSD.

Results and discussion
The present research was intended to the impact of different doses of gamma irradiation and poppy seed extract (PSE) for exploitation as a preservative and antioxidant in beef patties.Afterwards impact of various combinations of gamma irradiation and poppy seed extract were analyzed on the sensory and quality characteristics of beef patties.It was contrived at various levels (0kGy, 2.5kGyand 5kGy) of gamma radiation in combination with poppy seed extract (2%).The PSE enriched beef patties were assessed physically (Hunter colorimeter), physicochemical (TBARS, POV and TVBN), functionally (DPPH, FRAP, and TPC).Microbial analysis was done by TBC and Coliform.Beef patties were significant effects of TBARS, POV and TBVN value on treatments, packaging and storage interval.

Thiobarbituric acid reactive substances (TBARS)
TBARS are formed as a by-product of lipid peroxidation.Malondialdehyde (MDA) is a reactive aldehyde formed by the lipid peroxidation of different fatty acids.The results were found to change regarding the TBARS value of PSE beef patties with different storage intervals.Beef patties have significant effects of TBARS value on treatments, packaging and storage interval.The results depicted that the higher value of TBARS (0.49 ± 0.03MDA/kg) was found in samples treated with (5 kGy +PSE2%) aerobically packed samples on 14 days storage interval.However the (Table 1) represents that the lower value (0.22 ± 0.04 MDA/kg) was found in the vacuum-packaged sample (0 kGy +PSE2%) on 0 day of storage interval.Irradiation is a method of meat preservation and possess excellent potential to improve and enhance meat safety and extend shelf life of meat.Poppy seed extract has antioxidant properties which slows down the mechanism of oxidation.Ham et al. [26] conducted a study who examined that the beef patties had higher values of TBARS when treated (10 kGy) on 0 day storage time.On the other hand, Brewer, [27] stated that the results of TBARS values was increased in beef meat when treated with 1.25 kGy gamma irradiation on 7 days of storage time.Meanwhile, Rababah et al. [28] examined that the results of TBARS values were increased in chicken breast meat when treated with irradiation for 0 − 12 days of storage at 5°C.In another study examined that a high amount of TBARS in the irradiation dose in vacuum-packaged beef sausage patties on 10 days of storage period. [29]Rima et al. [30] examined that the raw broiler meat had higher values of TBARS when treated (3.5 kGy) at 60 day storage time.The outcomes of this study also depicted that with the increase in storing interval, the amount of TBARS was increased.An et al. [31] who examined that the smoked duck meat had maximum values of TBARS when treated (4.5 kGy) on 40 day storage period, while a minimum value of TBARS was analyzed in untreated samples.Our findings showed the similarity with the Arshad et al. [13] who reported the impact of turmeric powder and gamma irradiations in chicken meat and examined that the chicken meat had higher values of TBARS when treated (2 kGy) in aerobically packed samples on 14 days storage period, while a minimum value was analyzed in untreated samples.Kamal et al. [32] depicted that the TBARS value of crushed poppy seed paste addition in chevon nugget.The TBARS value was minimum as compared to untreated chevon nugget.The recorded TBARS value depicted that the untreated chevon nugget was spoiled and was not suitable for human intake till 21st day of refrigeration storage.Moreover, poppy seed paste enriched chevon nugget was analyzed to be fit for human intake even on 21st day of refrigeration storage.

Peroxide value (POV)
The statistical results regarding the POV value of PSE beef patties were significant effect with respect to treatments, packaging and storage interval as showed in Table 2. Higher value of POV (0.53 ± 0.01 meq peroxide/kg) was analyzed in PSE beef patties treated with (5kGy) aerobically packed on the 14 th day of storage duration, followed by vacuum packed beef patties samples (0.42 ± 0.03 meq peroxide/ kg) in treated (5kGy) on 14 days of storage, whereas Table 2 presented that the lower value (0.20 ± 0.0 meq peroxide/kg) was found in beef patties with 2% PSE vacuum -packaged samples treated with 0 kGy on day 0. Poppy seed extract contains natural anti-oxidants which limits the mechanism of lipid oxidation.The results depicted that the peroxide value was lower in vacuum packed beef patties samples as compare to aerobically packed samples.Arshad et al. [33] conducted a study on frozen duck meat that was treated with 0, 3 and 7 kGy at the storage period.The higher finding of POV was observed at the end of storage and on high dose of E-bean.Arshad et al. [13] depicted that POV values were increased significantly with 14 days storage interval in broiler meat.Although with the higher irradiation doses POV values in aerobic and in vacuum packaging also increases.On the other hand, when samples treated with moringa leaf powder the POV values decreases.Rima et al. [30] depicted that POV values were increased significantly in broiler meat with higher irradiation doses treated with (2kGy) as well as storage time of 30 day.Dzudie et al. [34] stated that POV value significantly increases with 10 day storage interval although the beef patties treated with 0.2% of ginger essential oil had shown reduction in lipid oxidation.The results indicated that the treated beef patties sample had a minimum value in vacuum packaging and maximum peroxide value in treated samples.Our results are in agreement with the results of Kim et al. [35] who declared with the increase in dosage of gamma radiation and increase in storage period POV also increases.

Total volatile basic nitrogen (TVBN)
The statistical results regarding the TBVN value of PSE beef patties with different storage intervals are given in Table 3. Beef patties results were significant effects of TVBN value on treatments, packaging, and storage interval.The results depicted that the highest value of TVBN (12.56 ± 0.39 mg/100 mL) was analyzed in (0 kGy) aerobically-packed beef patties samples on 14 day of storage period, followed by vacuum-packaging fresh beef patties samples at (0 kGy) showed the value (12.96 ± 0.41 mg/100 mL) on 14th day of storage interval.However, the results depicted that the lowest value (8.03 ± 0.24 mg/ 100 mL) was analyzed in the vacuum-packaged beef patties samples treated with (0 kGy+ PSE 2%) on 0 day of storage interval.The results portrayed that the TVBN value in vacuum-packaged beef patties samples significantly decreased as compared to aerobically packed beef patties samples.Moreover, the TVBN value in raw beef patties samples (control) become higher with the increase in storage period, while irradiated beef patties with poppy seed extract and without poppy seed extract suppressed the higher TBVN value with the storage period.By the addition of poppy seed extract, the TBVN value decreased in both the aerobically packed beef patties and vacuum packed beef patties samples.The results designated that the treated (0kGy +2%PSE) samples of beef patties have a minimum value of TVBN in vacuum packaged samples, whereas maximum value of TVBN was analyzed in treated beef patties samples, which are in reliable with the results of [13] who reported that the total volatile basic nitrogen content was found higher with the increase in storing time but irradiation treatment controls the development of TVBN during storage time.Minimizing the level of bacteria and by enhancing the applied dose of gamma radiation can decrease the TBVN content during storage period.Yang et al. [36] who examined that the TVBN value was higher at the start of the storage duration However, different irradiation doses significantly suppressed TVBN value during 12 days of storage interval.These findings showed that higher the dose of irradiation lower the rate of TVBN formation during storage period by reducing the levels of spoilage microorganisms.Li et al. [15] depicted that the volatiles substances become higher in amount with the increase dose of irradiation in pork samples as compared to lower dose irradiated samples during storage interval.Ahn et al. [37] stated that increase in TVBN level by irradiation process may be due to the breakdown of nitrogenous complexes/ compounds present in meat samples.An et al. [31] reported that, TVBN had maximum value at 0-day storage without the radiation treatment but the minimum value of TBVN was in irradiated samples Our findings showed the similarity with [38] who depicted that TVBN value in chicken meat untreated samples become higher with the increase in storage period, while irradiated chicken samples with moringa leaf powder reduced the higher TBVN value with the storage period.By adding moringa leaf powder the TBVN value decreased in both the aerobically and vacuum packed chicken meats samples.Our outcome is related to Yun [39] who stated that volatile compounds were enhanced by the ionization process in ready-to-eat chicken breast, whereas TVBN values were formed slowly as compared to unirradiated meat at the time of storage.However, another study showed that volatile substances were found to increase in pork meat when subjected to a high dose of irradiation. [40]

DPPH (2,2-diphenyl-1-picrylhydrazyl)
The statistical results regarding the DPPH value of PSE beef patties with different storage intervals are given in Table 4. Beef patties had significant effects of DPPH value on treatments, packaging and storage interval.The results depicted that the higher value of DPPH (72.54 ± 2.935%) was found in samples treated with (0 kGy + 2%PSE) vacuum packed samples on 0-day storage However, the outcome represents that the minimum value (48.52 ± 1.00%) was found in the aerobically-packaged samples treated with (5 kGy) on 14 day of storage interval.The results showed the higher value of DPPH in vacuum packed beef patties samples and lower value in aerobically packaged beef patties samples.Furthermore, with the increase in storage time, the DPPH value in untreated (control) beef patties samples also decreased.With the addition of (PSE) poppy seed extract, the DPPH value increased in aerobically packaged beef patties samples as well as in vacuum packaged samples.The result depicted that the treated beef patties samples had a lower value of DPPH in aerobically packaged beef samples, while a higher value of DPPH was found in untreated (control) beef patties samples.Our results are similar to Falowo et al. [41] that the extract of Bidenspilosa leaf exhibited maximum antiradical activity against 2,2-diphenyl-2-picrylhydrazyl (DPPH) and radicals of 2,2íazinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) than Moringaoleifera leaf extract and standard butylated hydroxyl toluene (BHT) fresh ground beef during 6 days of cold storage.The results of our experiment is relating to the study conducted by Arshad et al. [13] who depicted that the DPPH value significantly decreased with the passage of time but increased by adding (TP) turmeric powder in aerobically packaged chicken meat samples as well as vacuum packaged samples.

Total phenolic content (TPC)
The statistical results regarding the TPC value of PSE beef patties with different storage intervals are given in Table 5. Beef patties results had significant effects of TPC value on treatments, packaging, and storage interval.The results depicted that the highest value of TPC (127.14 ± 3.93 mg/g GAE) was observed in (0 kGy + 2%PSE) vacuum-packed beef patties samples on 0 day of storage period, followed by aerobicallypackaging of beef patties samples at (0kGy +2%PSE) showed the value (124.32 ± 3.31 mg/g GAE) on 0 day of storage interval.However the results depicted that the lowest value (92.52 ± 1.89 mg/g GAE) was analyzed in the aerobically-packaged beef patties samples treated with (5 kGy) on 14 day of storage interval.The results portrayed that the TPC value in vacuum-packaged beef patties samples significantly increases as compared to aerobically packed beef patties samples.Furthermore, the TPC value in untreated beef patties samples (control) decreases with the increase in storage period, while irradiated beef patties with poppy seed extract and without poppy seed extract suppressed the higher TBVN value with the storage period.By the addition of poppy seed extract, the TPC value increased in both the aerobically packed beef patties and vacuum packed beef patties samples.The results designated that the treated (0kGy +2%PSE) samples of beef patties have a higher value of TPC in vacuum packaged samples, whereas lower value of TPC was analyzed in treated beef patties samples aerobically packed.Moreover, Ergezer & Serdaroğlu [42] reported that the total phenolic content of all the raw beef patties samples decreased significantly by the expansion in storage interval.Our findings showed the similarity with the results of [31] who stated that the TPC value significantly increased in vacuum packaged samples as compare to aerobically packed samples.Sharma & Bhat [43] depicted that during the storage time TPC total phenolic content significantly decreased in raw chicken sausage products at each interval of storage duration.

Ferric Reducing Antioxidant Power (FRAP)
To evaluate the total antioxidant content of a sample, the ferric decreasing/antioxidant power (FRAP) is one of the most generally utilized strategies, which is moderately basic, fast, delicate, and reasonable to perform.The statistical results regarding the FRAP values of PSE extract beef patties with different storage interval are given in Table 6.The results depicted that the FRAP values were significantly different among all the treatments.The higher values of FRAP (7.89 ± 0.59 μM TE/g) was found in samples treated with (0 kGy + 2%PSE) vacuum packaged samples on 0 days.However, the outcomes represents that the minimum value (2.89 ± 0.48 μM TE/g) was found in the vacuum-packaged samples treated with (5 kGy) on 14th day of storage interval.Wojdyło et al. [44] reported that the medicinal herbs can be categorized according to their antioxidant capability.Vuong et al. [45] created the evidence of anti-oxidant and anti-cancer capacities of saponin enriched papaya leaf extracts.Moreover, Rady et al. [46] reported that the antioxidant activity of faba been seed extract significantly increased by applying different doses of gamma radiation. [41]stated that shrimp patties treated with GLE 2%+PLE 2% were showed lower FRAP value at the start and end of storage interval.

Total aerobic bacteria and coliforms count
The statistical results regarding the total aerobic bacteria and coliform value of beef patties with different storage intervals are given in Table 7.The treated samples with PSE extract and gamma radiation were analyzed regarding to coliform count and total aerobic bacteria.The results depicted that the bacterial load/microbial population decreases with increased dose of gamma radiation.Moreover, bacterial contamination increased in aerobic packaging and decreased in vacuum packaging.The results indicated that the higher bacterial count of (11.28 ± 0.37 log CFU/g) of total aerobic bacteria was analyzed in control samples (0kGy) in aerobically packaged samples on the 14 th day of storage interval.The lower total bacterial count (2.88 ± 0.16 log CFU/g) was analyzed in (5kGy gamma radiation + PSE 2%) vacuum packaged samples on 0 th day of storage.The findings depicted that the higher coliform count of (8.45 ± 0.34 log CFU/g) was analyzed in untreated samples (0kGy) in aerobically packaged samples on the 14 th day of storage interval.The lower coliform count (2.23 ± 0.1 log CFU/g) was analyzed in (5kGy gamma radiation + PSE 2%) vacuum packaged samples on 0 th day of storage.The findings indicated that the treated beef patties samples had maximum values of total bacterial count and coliforms in aerobically packaged samples, while minimum values were analyzed in the treated vacuum packaged samples.Our findings indicated the similarity with the results of 4 reported the effects of combined treatment of turmeric powder and gamma irradiations in chicken meat and examined that the chicken meat had maximum values of Total aerobic bacteria and coliform in untreated samples (0kGy gamma radiation) on 14 th day of storage period while a minimum value of TAB and coliform was found in control samples.The TAB and coliform count increased significantly with the increased storage period but decreases with the increase in radiation dose.However, our finding is similar to Arshad et al. [13] who examined that the smoked duck meat had higher values of total aerobic bacteria and coliform in untreated samples (0kGy) on 40 day storage period, while a lower value of TAB and coliform was found in control samples.Dzudie et al. [34] reported that the broiler meat had higher coliform count in (0kGy) at 60 day of storage period but the coliform count decreased with the increase in radiation dose.In another study in which Nisar et al. [35] reported that effects of combined treatment of moringa leaf powder and gamma irradiations in chicken meatballs and examined that the meatballs had higher value of TAB and coliform (control) untreated samples (0kGy) on 14 th day of storage period while a minimum value of total aerobic bacteria and coliform was analyzed in control samples.Cunha et al. [47] who stated that antioxidants play important role in preserving and enhancing shelf life of different meat products.

Hunter color (Lab)
Hunter L, a, b scales are created on the basis of Opponent-Color Theory.This theory accepts that the receptors in the human eye distinguish color as the following pairs of opposites.The L value indicates the level of light or dark, a value redness or greenness, and the b value yellowness or blueness.All three values are required to completely describe an object's color.The statistical results regarding the color parameter of PSE beef patties are shown up in Table 8.The outcomes depicted that the L* value of beef patties have significant effects with respect to treatments, packaging and storage interval.The outcomes depicted that the higher value of L* (59.76 ± 3.75) was analyzed in those beef patties samples treated with (0 kGy + 2% PSE) aerobically packed samples on 14 days" storage interval followed by vacuum packaged treated beef patties samples showed the value (57.10 ± 2.29) on 14 th day of storage period, however the (Table 8) represents that the minimum L* value (50.40 ± 2.53) was analyzed in the vacuum-packaged samples treated with (2.5 kGy + 2%PSE) on 14th day of storage interval.The results depicted that L* value decreased significantly in vacuum packed beef patties samples as contrast to aerobically packaged samples.Furthermore, with the passage of time the L* value in (control) trials increased.With the addition of poppy seed extract, the L* value increased in aerobically packaged samples and vacuum packaged samples.The results depicted that treated beef patties samples has maximum value in aerobically packaged samples, while minimum value was analyzed in in treated beef patties samples.Our findings showed the similarity with the findings of Arshad et al. [10] who reported that with the increase in dose of gamma radiation 0 to 2 kGy, L* value of gamma irradiated chicken meat also increases.In another study in which An et al [31] stated that L* value was maximum with the higher the dose of gamma radiation (4.5kGy)of smoked duck meat.The results depicted that the a* value of PSE beef patties have significant effects with respect to treatments, packaging and storage interval.The outcomes depicted that the higher value of a* (20.80 ± 1.19) was found in samples treated with (0kGy +2%PSE) aerobically packed samples on 0 days storing interval followed by vacuum packaged treated beef patties samples showed the value (20.39 ± 1.87) on 7th day of storage period, however Table 8 represents that the minimum a* value (13.22 ± 1.31) was analyzed in the aerobically-packaged samples on 14th day of storage interval.The outcomes depicted that a* value decreased significantly in vacuum packaged beef patties samples as contrast to aerobically packaged samples.Moreover, a* value in untreated (control) samples decreased with the passage of time.With the addition of poppy seed extract, a* value become higher in aerobically packed samples and vacuum packaged samples.Our findings depicted that treated beef patties samples has maximum value in vacuum packaged samples, while minimum value was analyzed in aerobically packaged treated beef patties samples.Our findings showed the similarity with the results of An et al. [31] who reported that with the increase in dose of gamma radiation 0 to 2kGy, the a* value of gamma irradiated chicken meat also increases.In another study in which Rima et al. [30] stated that higher the dose of gamma radiation 0 to 4.5kGy, higher will the a* value of gamma irradiated smoked duck meat.Our findings are in line with Nam et al. [48] who depicted that the fresh chicken breast meat and turkey treated with gamma irradiation had significant increase in redness.
The results depicted that the b* value of PSE beef patties were significant effects with respect to treatments, packaging and storing interval.The results found that the higher value of b* (14.59 ± 1.30) was found in samples treated with (0kGy +2%PSE) aerobically packed samples on 0 days storage interval followed by vacuum packaged treated beef patties samples showed the value (14.29 ± 1.30) on 7th day of storage period.However, Table 8 represents that the minimum b*value (8.25 ± 0.75) was analyzed in the vacuum-packaged samples on 14 th day of storage interval.The outcomes depicted that b* value decreased significantly in vacuum packaged beef patties samples as contrast to aerobically packaged samples.Moreover, the b* value in untreated (control) samples decreased with the passage of time.With the addition of poppy seed extract, the b* value increased in aerobically packaged samples and vacuum packaged samples.The results depicted that treated beef patties samples has maximum value in aerobically packaged samples, while minimum value was analyzed in vacuum packaged treated beef patties samples.Our findings showed the similarity with the findings of An et al. [31] who reported that with the increase in dose of gamma radiation (0 to 2kGy), the b* value of gamma irradiated chicken meat also increases.In another study Rima et al. [30] stated that higher the dose of gamma radiation (0 to 4.5kGy) was changed the b* value of gamma irradiated smoked duck meat.Our results are in line with Nam et al. [48] who depicted that the fresh chicken breast meat and turkey treated with gamma irradiation had significant increase in b × .Sommers et al. [49] who reported that the combined treatment of meat with gamma radiation and citric acid significantly effects the values of L*and b × .

Heme pigment
The results regarding the myoglobin content of PSE beef patties samples which are significantly affected by with treatments, packaging and storage interval as showed in Tables 4-9.The results indicated that the maximum amount of (Mb) myoglobin (45.98 ± 2.23%) content was analyzed in samples treated with (0kGy +2%PSE) in vacuum packaged beef patties samples on 0 day of storage period, followed by aerobically packaged beef patties samples treated with (0kGy +2%PSE) showed the value (44.89 ± 2.45%) on 0 day storage whereas, the lower value of Mb content (28.09 ± 2.18%) was analyzed in vacuum packaged beef patties samples treated with (2.5kGy +2%PSE) on the 14 th day of storage period as shown in Table 9.The outcomes depicted that the myoglobin content decreased significantly in vacuum packaged beef patties samples as compared to aerobically packed samples.Furthermore, the myoglobin content of untreated (control) beef patties samples reduced gradually.With the addition of poppy seed extract, the myoglobin content increased in aerobically packed samples as well as in vacuum packaged samples.The outcomes showed the higher amount of (MbO2) oxymyoglobin (20.90 ± 0.32%) content was analyzed in samples treated with 2.5kGy in aerobically packaged beef patties samples on 14th day of storage period, followed by vacuum packaged beef patties samples treated with 2.5kGy showed the value (19.06 ± 0.725%) on 14 th day storage whereas, the lower value of MbO2 content (9.38 ± 0.21%) was analyzed in vacuum packaged beef patties samples treated with (2.5kGy+PSE 2%) on the 14 th day of storage period.The findings depicted that the oxymyoglobin content decreased significantly in vacuum packaged beef patties samples as compared to aerobically packed samples.Moreover, with the passage of time the oxymyoglobin content of untreated (control) beef patties samples increased.With the addition of poppy seed extract, the oxymyoglobin content decreased in aerobically packed samples as well as in vacuum packaged samples The results showed that the higher amount of (MMb) metmyoglobin (66.11 ± 4.05%) content was analyzed in samples treated with (2.5KGy)in aerobically packaged beef patties samples on 14th day of storage period, followed by vacuum packaged beef patties samples treated with(2.5kGy)showed the value (62.26 ± 4.02%) on 14 th day storage whereas, the lower value of MMb content (40.69 ± 2.13%) was analyzed in vacuum packaged beef patties samples treated with (0kGy+PSE 2%) on the 0 th day of storage period as shown in Table 9.The outcomes indicated that the metmyoglobin content decreased significantly in vacuum packaged beef patties samples as compared to aerobically packed samples.Furthermore, with the passage of time the metmyoglobin content of untreated (control) beef patties samples increased.With the addition of poppy seed extract, the metmyoglobin content decreased in aerobically packed samples as well as in vacuum packaged samples.The findings depicted that the treated beef patties samples have minimum myoglobin content in aerobically packaged samples, whereas in oxymyoglobin and met myoglobin minimum content were analyzed in vacuum packaging, however the maximum content of myoglobin was found in the control samples (untreated) but in oxymyoglobin and met myoglobin maximum content was found in treated samples.Our outcomes have similarity with the findings of [34] who stated that under aerobic packaging and 0 day of storage period the myoglobin content was higher but when the samples treated with different dose of radiation lower content was found in samples treated with high dose (4.5kGy).Oxymyoglobin and met myoglobin found to have higher content in aerobically packaged samples when treated with (4.5kGy) on the 40 th day of storage period.On the other hand, control samples (0kGy) nonirradiated found to have lower content of oxymyoglobin and met myoglobin on 0 day of storage period.Our findings are in agreement with [31] who stated that chicken meat samples treated with (2 kGy) found to have lower content of myoglobin in aerobically packaged samples at 14 days of storage interval, whereas, the samples that were treated with 2 kGy gamma radiation vacuum packaged have lower levels of oxymyoglobin and metmyoglobin on the 14th day of storage period.

Sensory evaluation PSE enriched beef patties
The sensory attributes of a food product are very important in consumer demand.The sensory evaluation of PSE enriched beef patties was carried by trained panelists.The sensory scores of PSE enriched beef patties are shown in the Table 10.The statistical result of sensory score for appearance, taste, texture, odor, and overall acceptability of irradiated PSE beef patties is shown in Table 10.Sensory attributes indicated a significant variation with respect to the dose of gamma irradiation but changed significantly with storage period.The maximum score for appearance (7.8 ± 0.29), taste (7.6 ± 0.28), texture (7.9 ± 0.30), odor (7.6 ± 0.27) and overall acceptability (7.96 ± 0.28) was analyzed in the control (0KGy) on the 0 day of storage period whereas, the minimum score for appearance (6.02 ± 0.07), taste (7.6 ± 0.28), texture (5.9 ± 0.8), odor (5.2 ± 0.03) and overall acceptability (5.03 ± 0.6) was analyzed on the 14 day of storage period.With the increase in storage period score for various sensory features significantly decreased.During storage there was decrease in the sensory score because of higher level of lipid oxidation initiated by the irradiation, which results in less acceptability, but the overall acceptability was in the range of acceptance.Our findings is similarity with the results of Lewis et al. [50] who depicted that the sensory score in chicken meat decreased with irradiation during the storage duration of 28 days.Moreover, our results are in agreement with [31] who reported that with the increase in storage duration there was significant decrease in all the sensory score of gamma irradiated chicken meat.Another study is in agreement with our finding in which ostrich and chicken meat were treated with kale leaf powder and gamma irradiation.The slight changes in sensory parameters of both types of meat were observed during storage on different treatments. [51]ble 10.Sensory evaluation of beef patties treated with gamma irradiation and Poppy seed extract (PSE) at different storage periods (0,7th and 14th days).6.90 ± 0.7 6.6 ± 0.6 6.5 ± 0.05 6.3 ± 0.04 6.7 ± 0.22b 7.20 ± 0.12 6.9 ± 0.07 6.7 ± 0,13 6.5 ± 0.24 6.98 ± 0.24b 6.5 ± 0.24 6.4 ± 0.6 6.3 ± 0.4 6.1 ± 0.5 6.48 ± 0.12c 7.2 ± 0.11 6.4 ± 0.05 6.1 ± 0.5 6.0 ± 0.05 6.76 ± 0.09a 7.4 ± 0.22 6.6 ± 0.6 6.3 ± 0.4 6.2 ± 0.03 6.93 ± 0.11a 6.9 ± 0.7 6.0 ± 0.05 5.7 ± 0.4 5.7 ± 0.4 6.36 ± 0.07b 7.00 ± 0.25 6.3 ± 0.08 6.0 ± 0.5 5.9 ± 0.06 6.58 ± 0.08b 6.5 ± 0.06 5.6 ± 0.4 5.2 ± 0.03 5.4 ± 0.05 5.95 ± 0.04c

Conclusion and limitations
It is concluded that different doses of gamma irradiation significantly improved the quality of beef patties with some insignificant changes in physicochemical properties during storage period.The preservative and antioxidant potential of (PSE) poppy seed extract patties during storage were revealed by physical, physicochemical and microbial analysis.The results showed that the samples treated with 5kGy+PSE were in well condition after the storage interval.There was decline in the antioxidant profile during storage, but it was higher in vacuum packaged samples as compared to aerobic packaged samples.Moreover, the TBVN value was lower in vacuum packaged samples as compared to aerobic packaged samples during the storage period.The influence of PSE was positive on the storage of beef patties as well as their antioxidant profile was good as compared to controls.The gamma irradiation and phenolic nature of PSE efficiently enhanced the physical and chemical profile of beef patties.The irradiation technology keeps the ability to eliminate the microorganism from meat and meat products but high dose of irradiation cannot suitable because during irradiation processing the oxidation process start.Poppy seeds contain different type of antioxidants that can help to improve the stability of meat products but it also sources of morphine and codeine.High concentration of poppy seeds extract may be harmful for human health.

Table 2 .
Per oxide value (POV) value of beef patties treated with gamma irradiation and poppy seed extract at different storage periods (0, 7th, 14th days).
PSE: Poppy seed extract: The values are mean ±SD of three independent determinations Means carrying different letters in a columns differed significantly.

Table 3 .
Total volatile basic nitrogen (TVBN) of beef patties treated with gamma irradiation and poppy seed extract at different storage periods (0,7 th and 14thdays).
PSE: Poppy seed extract: The values are mean ±SD of three independent determinations Means carrying different letters in a columns differed significantly.
PSE: Poppy seed extract: The values are mean ±SD of three independent determinations Means carrying different letters in a columns differed significantly.

Table 5 .
Total phenolic content (TPC) of beef patties treated with gamma irradiation and Poppy seed extract (PSE) at different storage periods (0,7 th and 14th days).

Table 6 .
Ferric Reducing Antioxidant Power (FRAP) of beef patties treated with gamma irradiation and Poppy seed extract (PSE) at different storage periods (0,7 th and 14th days).

Table 7 .
Total aerobic bacteria and coliforms count of beef patties treated with gamma irradiation and Poppy seed extract (PSE) at different storage periods (0,7 th and 14th days).

Table 8 .
Hunter color of beef patties treated with gamma irradiation and Poppy seed extract (PSE) at different storage periods (0,7 th and 14th days).

Table 9 .
Heme pigment (%) of beef patties treated with gamma irradiation and Poppy seed extract (PSE) at different storage periods (0,7 th and 14th days).
Poppy seed extractValue are Mean ± SD of 10 independent determinations; different letters represent significant differences (p < .05).