INTRODUCTIONFish meat is an ideal source of animal protein whichhas a high nutritional value. Nowadays, consumers areinterested in healthy food [1]. Nevertheless, conveniencefood, including burgers, has remained common all overthe world [2]. Ready-to-cook fish products is becomingpopular among consumers due to their high nutritionalvalue and short time of cooking [2]. Still, to preserve itsquality, fish meat should be processed properly [3].In recent years, changing socioeconomic factors,namely, an increase in the number of employed women,have led to an increased demand for convenienceproducts. Therefore, some efforts have been made toextend the shelf life of ready-to-eat food [4, 5].Surimi, stabilised myofibrillar proteins of fishmuscle, can be made of both sea-water and fresh-waterfish. To obtain surimi, fish fillet is minced, washed bywater, and stabilised by blending with cryoprotectants.A cryoprotectant mix, containing sugar, sorbitol, andphosphates, is added to the minced fish [6]. Surimi isan important ingredient for food production in manycountries due to its technological properties [6].Currently, there are a number of ways to controlthe growth of pathogenic microorganisms in foodproducts. One of the ways is the use of essential oils(EOs). EOs are aromatic oily extracts obtained fromdifferent parts of plants, such as flowers, leaves, wood,bark, roots, seeds, or peel, which exhibit bactericidalor bacteriostatic properties [7]. EOs are consideredas natural preservatives for raw or mildly processedfood [8]. EOs have a wide spectrum of antimicrobialproperties. As an antimicrobial agent, EOs destroy boththe lipid bi-layer of cell membranes and enzyme systemsas well as inactivate the genetic material of bacteria [9].EOs display their antimicrobial action againstpathogenic microorganisms, including gram-positive andgram-negative, as well as mold and parasites [10–14].In addition, EOs are reported to have antioxidantproperties [15–17]. Natural antioxidants have anadvantage over artificial ones because of their highcontent in phenolic compounds as well as other active302Rashidimehr A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. 301–310components which can effectively inhibit oxidativereactions [17, 18].Cumin (Cuminum cyminum L.) is a flowering plant inthe family Apiaceae. Its seeds have been commonly usedfor centuries as a spice [19]. Thyme (Zataria MultifloraBoiss.) is an aromatic perennial evergreen herb beloningto the family Labiateae and used in cooking [20]. Inaddition, there is data on the successful use of thyme EOas an antimicrobial agent in chicken meat patties [21].The aim of this work was to find a way to prolongthe characteristics and shelf life of novel burgers madeof chicken meat and surimi, as well as to investigatechemical and microbiological changes in the burgersstored at 2°C for 27 days.STUDY OBJECTS AND METHODSPreparation of minced chicken meat and surimi.Fresh chicken and silver carp (Hypophthalmichthysmolitrix L.) were purchased from a local market inAhvaz, Khuzestan Province, Iran. The chicken wasminced and then kept at –18°C until used. Fresh fish wastransported on ice into a laboratory, washed, beheaded,gutted, and filleted. The fillet obtained was thoroughlywashed, put through a meat mincer with 4 mm diameterholes (EG-1200-EBS, Jahan Ava, Iran) for 2 min.The minced fish was washed with a triple volume ofwater (4°C) and stirred for 10 min. The washed mincedfish was filtered through two layers of cheesecloth andthen subsequently dewatered by using a manual juicerextractor. Washing was performed three times. Thethird washing was carried out with 0.5% NaCl (Merck,Germany) solution. A ratio of the minced fish to NaClwas 1:3 (w/w).After dewatering, the minced fish was mixed withcryoprotectants, i.e. sucrose 3% (Merck, Germany) andsorbitol 3% (Merck, Germany), for 60 s and frozen usinga blast freezer. The surimi obtained was kept at –18°Cuntil used.Preparation of combined burgers and treatments.Before burgers preparation, frozen surimi and mincedchicken meat were put in a refrigerator (at 4°C) at night.Meat for burgers was prepared from surimi (63%) andminced chicken meat (37%).The meat was then blended with toasted flour, 8.2%;wheat flour, 2%; soy flour, 3%; sunflower oil, 1%; freshlygrated onion, 7%; garlic powder, 1%; sodium chloride,1%; white pepper, 0.5%; lemon juice, 1%; and sodiumtri-polyphosphate, 0.3% (Merck, Germany).All the ingredients in combination with 125 mg/Lof nisin (Sigma Aldrich, England) were ground througha blender with a 5 mm plate (Gosonic, Turkey) for4–5 min. Nisin solution, which was added to avoidthe growth of Clostridium botulinum, was preparedby dissolving a required amount of nisin powder insterilised 0.02N HCl solution. Burgers (25 g in weight,50–60 mm in diameter, and 1 cm in thickness) wereformed by a burger-maker according to [22].RSM (response surface methodology) was used tooptimise the formulation. The results were analysedusing Design Expert 6.0.2 software, and each of thedependent variables in the form of a quadratic regressionmodel was presented as follows:y = β0 +Σ βi Xi+ Σ βii Xi2+ ΣΣi<j βij XiXjki=1ki=1% Cooking yield = cooked weightraw weight × 100 (2)% Shrinkage = ( raw thickness− cooked thickness)+ (raw diameter− raw thickness+ raw diameter % Moisture retention = (cooked weight× % moisture in cooked burger)raw weight× % moisture in raw burger × (1)where β0, βi, βii and βij are regression coefficients,and Xi and Xj are coded independent variables. Theformula was selected based on the results of the sensoryevaluation of the burgers that were stored at 2°C beforetesting. The test was performed with the help of RSMsoftware.As control samples were used burgers made withoutessential oils. They were objected to analyses ofproximate composition and cooking characteristics. Thecontrol samples included burgers with 100, 300, andTable 1 Composition of thyme essential oilNumber ofcomponentComponent Retentiontime, minAmount,%1 α-Thujene 15.24 0.492 α-Pinene 15.42 2.283 Camphene 15.73 0.154 β-Pinene 16.37 0.525 3-Octanone 16.65 0.826 β-Myrecene 16.80 0.917 3-Octanol 17.00 0.208 α-Phellandrene 17.56 0.159 α-Terpinene 18.15 1.2010 p-Cymene 20.31 16.1311 Limonene 20.56 0.6512 1,8-Cineole 21.76 0.9213 β-ocimene 21.86 0.0814 γ-Terpinene 22.07 2.4315 Trans-sabinene hydrate 23.44 0.1916 Linalool 24.69 6.9217 Hotrienol 25.75 0.1118 Borneol 26.84 0.4219 4-Trpineol 29.16 0.8120 α-Trpineol 30.63 0.6421 Thymol methyl ether 32.11 1.5122 Carvacrol methyl ether 33.13 2.6423 Thymol 35.88 20.4824 Carvacrol 36.23 29.6125 Thymol acetate 37.52 0.1326 Carvacryl acetate 39.05 0.1527 β-caryophyllene 41.52 2.3728 Aromadendrene 42.77 1.1829 α-humulene 43.23 0.1330 Allo-Aromadendrene 43.89 0.3331 Ledene 45.05 0.5632 Spatulenol 47.24 0.5833 Caryophyllene oxide 47.96 1.34Total: 98.03303Rashidimehr A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. 301–310500 mg/L of both cumin essential oil (Barij Essen, Iran)and thyme essential oil (Barij essence, Iran). Based onthe sensory evaluation results, an optimal concentrationfor each of the EOs was selected.Tables 1 and 2 demonstrate results of thecomposition analysis of thyme and cumin EOs. Theanalysis was carried out by Barij Essence Company(Iran) by means of gas chromatography-massspectrometry (GC-MS).The burgers were subdivided into two groups.One group was packaged in high density polyethyleneMicrobiological analyses. Twenty five gramsof burger sample was added into 225 mL of sterilepeptone water and blended using a Stomacher labblender (Interscience Bag Mixer, China) for 1 min.Homogenates of various concentrations were preparedfor the microbial test. Cultured Plate Count Agar (PCA)(Merck, Darmstadt, Germany) was incubated at 7°C for10 days for psychrotrophic bacteria count and at 30°Cfor 48 h for total viable count (TVC) [26]. Lactic acidbacteria (LAB) were determined on de Man RogosaSharpe Agar (MRS) (Q Lab, Canada) incubated at 30°Cfor 72 h [27]. Sulfite-reducing clostridia were grownon Sulphite Polymyxin Sulfadiazine Agar (Merck,Darmstadt, Germany) [28] incubated at 30°C for 48 hin a plastic anaerobic AnaeroGen sachet (Anaerobicgas pack A, Merck, Darmstadt, Germany). Allmicrobiological analyses were performed in triplicate,Table 2 Composition of cumin essential oilNumber ofcomponentsComponent Retentiontime, minAmount,%1 β-Pinene 9.362 10.522 β-Myrcene 9.796 0.753 δ-3-Carene 10.30 0.364 α-Terpinene 10.523 1.205 o-Cymene 10.855 16.036 Phellandral 10.912 0.337 1,8-Cineole 10.975 4.908 γ-Terpinene 11.856 20.899 α-Thujene 12.640 0.2110 Terpinene-4-ol 15.593 0.7511 Cuminlaldehyde 17.338 38.4812 Carvacrol 18.872 0.2013 Trans-β-Farnesene 22.717 0.2614 Caryophyllene oxide 25.881 0.1015 Carotol 26.201 0.5816 Trans-Caryophyllene 27.870 0.19Total: 95.75Table 3 Experimental design of burgers with thyme and cuminEOs in air and vacuum packagingSample PackagingAir packaging(AP)Vacuumpackaging (VP)Control (without EOs)With thyme EO, mg/LWith cumin EO, mg/L–500500–500500y = β0 +Σ βi Xi+ Σ βii Xi2+ ΣΣi<j βij XiXjki=1ki=1% Cooking yield = cooked weightraw weight × 100 (2)% Shrinkage = ( raw thickness− cooked thickness)+ (raw diameter− cooked diameter)raw thickness+ raw diameter × 100 (3)% Moisture retention = (cooked weight× % moisture in cooked burger)raw weight× % moisture in raw burger × 100 (4)(3)y = β0 +Σ βi Xi+ Σ βii Xi2+ ΣΣi<j βij XiXjki=1ki=1% Cooking yield = cooked weightraw weight × 100 (2)% Shrinkage = ( raw thickness− cooked thickness)+ (raw diameter− cooked diameter)raw thickness+ raw diameter × 100 (3)% Moisture retention = (cooked weight× % moisture in cooked burger)raw weight× % moisture in raw burger × 100 (4)(4)(HDPE) bags under vacuum, and another group – inbags (aerobically), six burgers in each bag. Each groupincluded control, thyme EO and cumin EO samples.The packaged burger samples were stored at 2°C for27 days. Microbiological and chemical evaluation ofthree different batches was carried out on day 0, 3, 6, 9,12, 15, 18, 21, 24, and 27 of storage.Sensory analysis. Sensory evaluation was performedby a panel of seven experienced (laboratory-trained)judges. To optimise the fish burger formulation, thepanellists were asked to evaluate taste, colour, aroma,and overall quality of burgers on a nine-point scale. Thescale points were: excellent, 9; very good, 8; good, 7;acceptable, 5–6; unacceptable, 1–4 [23].Proximate composition. Protein, moisture, ash andfat contents were measured by AOAC method [24].Cooking characteristics. The thickness anddiameter of raw burgers were estimated at roomtemperature. The burgers were fried in sunflower oil at170°C for 5 min until an inner temperature of 72°C wasreached [25]. Cooking yield, shrinkage and moisturereyt e=n tβio0n + wΣereβ di eXteir+m Σinedβ biiy X thi2e +fo ΣlloΣwi<inj βgi je qXuiXatjions:ki=1ki=1% Cooking yield = cooked weightraw weight × 100 (2)% Shrinkage = ( raw thickness− cooked thickness)+ (raw diameter− cooked raw thickness+ raw diameter % Moisture retention = (cooked weight× % moisture in cooked burger)raw weight× % moisture in raw burger × 100 (2)and results were expressed as logarithm colony formingunit (log CFU)/g sample.Mold and yeast were counted on Yeast Extract Agar(Merck, Darmstadt, Germany) incubated at 25°C for5 days [29]. The experiment was performed in duplicate.Chemical analysis. pH value was determinedusing a digital pH meter on the first homogenisedconcentration of samples (Sartorius, USA) [30]. Totalvolatile base nitrogen (TVB-N) content was quantifiedby the method of Malle and Poumeyrol [31], whilethiobarbituric acid (TBA) amount was calculated by themethod of Tsironi et al. [32].Peroxide value (PV) was determined according to themethod described by AOAC [33]. All chemical analyseswere performed in triplicate.Statistical Analysis. Statistical analysis was carriedout with the help of SPSS 19 (SPSS, 2010) software304Rashidimehr A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. 301–310and one-way variance. Results were expressed asmean values and standard deviation (S.D.). Analysis ofvariance (ANOVA) data were subjected to determiningsignificant differences (P < 0.05).RESULTS AND DISCUSSIONSensory analysis. Average scores of sensorycharacteristics were evaluated using RSM method. Theresults of the analysis are shown in Table 4. The optimalburger formulation was selected, which contained 63% ofsurimi and 37% of minced chicken meat. Also, based onaverage scores of sensory evaluation, a concentration of500 mg/L for each EO was selected as optimal (Table 5).Proximate analysis. Proximate composition wasperformed in burgers made without EOs before storage.Samples had moisture of 70.40% and contained 19.98%of protein, 4.27% carbohydrate, 3.35% fat, and 2.0% ash.Our results are in good agreement with those obtainedby Vanitha et al. [34].Cooking characteristics. The cookingcharacteristics of samples with no EOs were determinedbefore storage. Cooking yield, shrinkage, and moistureretention were found to be 94.73, 10.19, and 80.98%,respectively. These data are in accordance with thoseof Heydari et al., who measured cooking properties incamel burgers during freezer storage [25].Microbiological analysis. Analysis of varianceshowed that both packaging and EOs used had asignificant effect on the microbial characteristics ofburgers (P < 0.001).Figure 1a demonstrates changes in TVC of theburgers under study during storage. Results indicatea significant effect (P < 0.001) of storage time, EOsaddition and packaging conditions on TVC. Themaximum TVC value obtained (107 CFU/g) wasacceptable for fresh and frozen fish [35].The initial (day 0) TVC of burgers in air packaging,with and without the EOs, was 4.05–4.38 log CFU/g.For burgers in vacuum packaging, with or withoutcumin/thyme EO, these values were 4.46–4.82 logCFU/g. These results are consistent with those obtainedby Cózar et al. for fish burgers (4 log CFU/g) andindicate a good burger quality [36]. Eventually, by day27, TVC was 8.39–8.78 and 6.13–6.74 log CFU/g in airpackaged and in vacuum packaged burgers, respectively.As one can see in Fig. 1a, burgers with thyme EOin vacuum packaging demonstrated the least microbialgrowth, which indicates inhibitory properties ofthyme EO. Similar results were found in an edible filmcontaining 0.10% of oregano and 0.15% of thyme EO infresh chicken sausages [17, 21].Initial counts of psychrotrophic bacteria in samplesin air and vacuum packaging were 4.34–4.76 log CFU/g,which reached 7.04–8.79 log CFU/g by day 27 of storage(Fig. 1b).Kilinc et al. observed an increase in TVC andpsychrotrophic bacteria count in sardine patties from2.50 and 2.60 log CFU/g to 6.72 and 6.98 log CFU/g onday 7 of storage [37]. According to Pavelková et al., theinitial TVC value in control chicken breast was 4.72 logCFU/g, while after 18 days of storage at 4 ± 0.5°C, it was3.68 and 4.05 log CFU/g for samples with oregano andthyme EOs in vacuum packaging [38].In our research, thyme EO acted as a synergist tovacuum packaging, combinations of air packaging +cumin EO and air packaging + thyme EO were lesseffective in inhibiting microbial growth. Soni et al. alsoreported lower psychrophilic bacteria counts in chickenpatties containing 0.10% of oregano and 0.15% of thymeEos [21]. Similar results were obtained by Sharma et al.in fresh chicken sausages during storage [17].This inhibitory effect was also apparently due tolarge amounts of phenolic substances and flavonoids inthyme and cumin EOs.Initially, a lacto acid bacteria (LAB) amount was3.16 log CFU/g. By the end of the storage, it wasrecorded to be 7.47–7.98 for burgers in air packagingand 4.15–4.40 log CFU/g for those in vacuum packaging(Fig. 1c). In [39], the initial LAB concentration in controlminced goat meat was 2.75 log CFU/g, which increasedto 6 log CFU/g by the end of vacuum storage at 4°C.Also, Fratianni et al. reported that thyme essential oildecreased total viable bacteria count and lactic acidbacterial growth in chicken breast; total microbialcontent reduced down to 50% compared to the controlsamples [40].In the work of Pavelková et al, the LAB count in acontrol chicken breast fillet was within the range from4.31 (day 3) to 2.62 log CFU/g (day 15), while the bestresult was observed in the vacuum packing + thyme EOTable 4 Average scores of sensory characteristics of burgersamples (surimi percentages predicted by RSM)Surimi, % Colour Taste Aroma Texture Overall quality50 7.17 6.00 7.57 5.43 6.14100 7.57 5.14 8.29 7.71 5.4375 7.83 5.57 7.00 6.14 5.710 7.85 5.71 7.14 6.86 5.71100 7.42 5.29 8.14 7.57 5.570 7.71 5.57 7.28 6.71 5.8525 7.30 5.57 8.28 5.43 5.71Table 5 Average scores of sensory characteristics of burgersamples for selecting proper concentrations of cumin andthyme EOsEssential oil Concentration, mg/L Taste AromaCumin EO 100 5.71 7.71300 6.43 7.29500 6.43 8.14Thyme EO 100 6.86 7.00300 7.14 7.71500 7.00 8.14305Rashidimehr A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. 301–310group (the highest count was 4.29 log CFU/g, on day 3,and the lowest count was 1.43 log CFU/g, on day 6) [38].The authors found that addition of 0.20% (v/w) of thymeEO and storage of samples in vacuum allowed shelf lifeof the chicken breast fillet to be extended.Clearly, it can be concluded that vacuum packaginginhibits LAB growth. Of the samples examined in thisstudy, the vacuum packaging + thyme EO sample hadthe maximum impact on the LAB growth. LAB are oneof the main components of meat product microflora thatdecreases pH of meat product through carbohydratefermentation [41].We found that, due to the antibacterial propertiesof cumin and thyme EOs, the shelf life of burgers withthe EOs in vacuum packaging increased. The cause ofthat can be the presence of phenolic compounds such asthymol and carvacrol in thyme and cuminaldehyde incumin.In this study, initial mold and yeast counts wereapproximately 2 log CFU/g and reached 6.49–6.95and 2.03–3.08 log CFU/g in samples stored in air andvacuum packaging, respectively (Fig. 1d). Lower moldand yeast counts in test samples compared to controlindicates the presence of EOs antifungal constituents inmeat products [42].As for sulfite-reducing clostridia, they were notdetected in any of the samples throughout the storageperoid.Chemical analysis. Figure 2a demonstrates asignificant decrease in pH values of control and treatedsamples during storage (P < 0.001). The initial pHvalue in burger samples was 6.41. By day 27, their pHvalues were 4.34–4.53 for all samples in air packagingand 4.71–4.98 for all samples in vacuum packaging.This decrease can be due to a reduced oxygencontent as a result aerobic microflora growth and CO2production. Another cause of the pH decrease can besugar contained in the burgers, which is utilised as acryoprotectant.According to Bingol and Ergun, pH diminishes bythe end of storage [43]. They also reported that the pHof meat is influenced by various factors however the(a) (b)D21 D24 D27AP + thyme EOVP + thyme EO02468D0 D3 D6 D9 D12 D15 D18 D21 D24 D27Mold and yeast count, log 10 CFU/gStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EOD21 D24 D27AP + thyme EOVP + thyme EO0246810D0 D3 D6 D9 D12 D15 D18 D21 D24 D27Total viable count,log 10 CFU/gStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO024681012D0 D3 D6 D9 D12 D15 D18 D21 D24 D27Psychrotrophic bacteria count,log 10 CFU/gStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO0510152025D0 TVN-B, mg/100 g burgerAP VP 0.20.40.60.8TBA, mg MDA/kg of burgerAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO0246810D 0 PV, meq /kg lipidAP VP 0246D0 D3 D6 D9 D12 D15 D18 D21 D24 D27pHStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO02468D0 D3 D6 D9 D12 D15 D18 D21 D24 D27Mold and yeast count, log 10 CFU/gStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO02468D0 D3 D6 D9 D12 D15 D18 D21 D24 D27Lactic acid bacteria, log 10 CFU/gStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO0246810D0 D3 D6 D9 D12 D15 D18 D21 D24 D27Total viable count,log 10 CFU/gStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO0246D0 D3 D6 D9 D12 D15 D18 D21 D24 D27pHStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO02468D0 D3 D6 D9 D12 D15 D18 D21 D24 D27Mold and yeast count, log 10 CFU/gStorage time, daysAP + thyme EOVP VP + cumin EO VP + thyme EO02468D0 D3 D6 D9 D12 D15 D18 D21 D24 D27Lactic acid bacteria, log 10 CFU/gStorage time, days0246810D0 D3 D6 D9 D12 D15 D18 D21 D24 D27Total viable count,log 10 CFU/gStorage time, days(c) (d)Figure 1 Effects of vacuum packaging (VP) and thyme and cumin essential oils on: (a) TVC, (b) psychrophilic count, (c) LAB,and (d) mold and yeast count in burgers stored at 2°C024681012D0 D3 D6 D9 Psychrotrophic bacteria count,log 10 CFU/gAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO0510152025D0 D3 D6 D9 D12 D15 D18 D21 D24 D27TVN-B, mg/100 g burgerStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO00.20.40.60.8D0 D3 D6 D9 D12 D15 D18 D21 D24 D27TBA, mg MDA/kg of burgerStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO0246810D 0 D 3 D 6 D 9 D 12 D 15 D 18 D 21 D 24 D 27PV, meq /kg lipidStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO024681012D0 D3 D6 D9 D12 D15 D18 D21 D24 D27Psychrotrophic bacteria count,log 10 CFU/gStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EOTVN-B, mg/100 g burger00.20.40.60.8D0 D3 D6 D9 D12 D15 D18 D21 D24 D27TBA, mg MDA/kg of burgerStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EOPV, meq /kg lipid024681012D0 D3 D6 D9 D12 D15 D18 D21 D24 D27Psychrotrophic bacteria count,log 10 CFU/gStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO10152025TVN-B, mg/100 g burger00.20.40.60.8D0 D3 D6 D9 D12 D15 D18 D21 D24 D27TBA, mg MDA/kg of burgerStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO10PV, meq /kg lipid024681012D0 D3 D6 D9 D12 D15 D18 D21 D24 D27Psychrotrophic bacteria count,log 10 CFU/gStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO0510152025D0 D3 D6 D9 D12 D15 D18 D21 D24 D27TVN-B, mg/100 g burgerStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO00.20.40.60.8D0 D3 D6 D9 D12 D15 D18 D21 D24 D27TBA, mg MDA/kg of burgerStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO0246810D 0 D 3 D 6 D 9 D 12 D 15 D 18 D 21 D 24 D 27PV, meq /kg lipidStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO(1) (2)(3) (4) (5)(1) (2) (3)(4) (5)024681012D0 D3 D6 D9 D12 D15 D18 D21 D24 D27Psychrotrophic bacteria count,log 10 CFU/gStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO0510152025D0 D3 D6 D9 D12 D15 D18 D21 D24 D27TVN-B, mg/100 g burgerStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO00.20.40.60.8D0 D3 D6 D9 D12 D15 D18 D21 D24 D27TBA, mg MDA/kg of burgerStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO0246810D 0 D 3 D 6 D 9 D 12 D 15 D 18 D 21 D 24 D 27PV, meq /kg lipidStorage time, daysAP AP + cumin EO AP + thyme EO(6) VP VP + cumin EO VP + thyme EO(6)024681012D0 D3 D6 D9 D12 D15 D18 D21 D24 D27Psychrotrophic bacteria count,log 10 CFU/gStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO10152025TVN-B, mg/100 g burgerAP VP 00.20.40.60.8D0 D3 D6 D9 D12 D15 D18 D21 D24 D27TBA, mg MDA/kg of burgerStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO10PV, meq /kg lipid(1) (2)(3)(4) (5) (6)(1) (2) (3)(4) (5) (6)(1)(2)(5) (3)(4)(6)(1) (2) (3)(4) (5) (6)(1) (2)(3)(5)(4)(6)(1) (2) (3)(4) (5) (6)306Rashidimehr A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. 301–310major one is lactic acid bacteria growth resulted fromlactic acid production. Similar results were also obtainedby Soni et al. in regard to chicken patties stored atrefrigerator temperature [21].Total volatile base nitrogen (TVB-N) content isoften used as an index to determine a degree of meatdecomposition. As one can see in Figure 2b, TVB-Nvalues of burgers increased significantly during storage(P < 0.001). TVN concentration was determined to bebetween 5 and 25 mg N/100g [44].TVB-N content was the highest (P < 0.001) insamples in air packaging, which indicates that airpackaging alone, even without EOs, can significantlyincrease TVB-N formation. Erkan investigated TVB-Nin vacuum-packaged filleted hot smoked rainbow trout[45]. By day 27 of storage at 2°C, the TVB-N contentincreased to 33.82 and 24.16 mg/100 g flesh in untreatedand treated with thyme EO samples, respectively. Also,Eskandari et al. reported that a TVB-N value in fishsamples treated with black cumin remained below itsacceptable limit by day 27 [46].According to hygienic standards, the TVB-Nacceptable limit in fish muscle is 20 mg/100 g. Thus, theresults of this study demonstrated that TVB-N values invacuum packaged samples with thyme and cumin EOswere below the limit during storage.Fat oxidation is the main cause of fish putrefaction;an increasing amount of thiobarbituric acid (TBA) andperoxide leads to rancidity. A steady increase in TBAin burgers was observed during 27 days of storage(Fig. 2c). Vacuum packaging effectively protected theburgers from zero days, keeping TBA scores lowerthan 1 mg MDA/kg during the storage period. EOsin the combination with vacuum packaging displayeda positive effect on the inhibition of oxidation. Köseet al. found that a TBA level in surimi was acceptableup to day 15, while a TVB-N concentration reached38.2 mg/100 g by day 13, which exceed the limit ofacceptability [5].Karabagias et al. reported that thyme did not protectlamb meat in air packaging from oxidation, at leastnot within its normal shelf life [47]. This finding is incontrast to the results of Botsoglou et al. who observeda three-fold reduction in a degree of lipid oxidation inturkey in air packaging [48].0246D0 D3 D6 D9 D12 D15 D18 D21 D24 D27pHStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO02468D0 D3 D6 D9 D12 D15 D18 D21 D24 D27Mold and yeast count, log 10 CFU/gStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO02468D0 D3 D6 D9 D12 D15 D18 D21 D24 D27Lactic acid bacteria, log 10 CFU/gStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO0246810D0 D3 D6 D9 D12 D15 D18 D21 D24 D27Total viable count,log 10 CFU/gStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO024681012D0 D3 D6 D9 D12 D15 D18 D21 D24 D27Psychrotrophic bacteria count,log 10 CFU/gStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO0510152025D0 D3 D6 D9 D12 D15 D18 D21 D24 D27TVN-B, mg/100 g burgerStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO00.20.40.60.8D0 D3 D6 D9 D12 D15 D18 D21 D24 D27TBA, mg MDA/kg of burgerStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO0246810D 0 D 3 D 6 D 9 D 12 D 15 D 18 D 21 D 24 D 27PV, meq /kg lipidStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO024681012D0 D3 D6 D9 D12 D15 D18 D21 D24 D27Psychrotrophic bacteria count,log 10 CFU/gStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO0510152025D0 D3 D6 D9 D12 D15 D18 D21 D24 D27TVN-B, mg/100 g burgerStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO00.20.40.60.8D0 D3 D6 D9 D12 D15 D18 D21 D24 D27TBA, mg MDA/kg of burgerStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO0246810D 0 D 3 D 6 D 9 D 12 D 15 D 18 D 21 D 24 D 27PV, meq /kg lipidStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO024681012D0 D3 D6 D9 D12 D15 D18 D21 D24 D27Psychrotrophic bacteria count,log 10 CFU/gStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO0510152025D0 D3 D6 D9 D12 D15 D18 D21 D24 D27TVN-B, mg/100 g burgerStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO00.20.40.60.8D0 D3 D6 D9 D12 D15 D18 D21 D24 D27TBA, mg MDA/kg of burgerStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO0246810D 0 D 3 D 6 D 9 D 12 D 15 D 18 D 21 D 24 D 27PV, meq /kg lipidStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO(a) (b)(c) (d)Figure 2 Effects of vacuum packaging (VP) and thyme and cumin essential oils on: (a) pH, (b) TVN-B, (c) TBA,and (d) PV of burgers at 2°C024681012D0 D3 D6 D9 D27Psychrotrophic bacteria count,log 10 CFU/gAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO0510152025D0 D3 D6 D9 D12 D15 D18 D21 D24 D27TVN-B, mg/100 g burgerStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO00.20.40.60.8D0 D3 D6 D9 D12 D15 D18 D21 D24 D27TBA, mg MDA/kg of burgerStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO02468100 D 3 D 6 D 9 D 12 D 15 18 21 D 24 D 27PV, meq /kg lipidStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO024681012D0 D3 D6 D9 D12 D15 D18 D21 D24 D27Psychrotrophic bacteria count,log 10 CFU/gStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EOTVN-B, mg/100 g burger00.20.40.60.8D0 D3 D6 D9 D12 D15 D18 D21 D24 D27TBA, mg MDA/kg of burgerStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EOPV, meq /kg lipid024681012D0 D3 D6 D9 D12 D15 D18 D21 D24 D27Psychrotrophic bacteria count,log 10 CFU/gStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EOTVN-B, mg/100 g burger00.20.40.60.8D0 D3 D6 D9 D12 D15 D18 D21 D24 D27TBA, mg MDA/kg of burger Storage time, daysAP AP + cumin EO AP + thyme EOVP + thyme EOPV, meq /kg lipid024681012D0 D3 D6 D9 D12 D15 D18 D21 D24 D27Psychrotrophic bacteria count,log 10 CFU/gStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO0510152025D0 D3 D6 D9 D12 D15 D18 D21 D24 D27TVN-B, mg/100 g burgerStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO00.20.40.60.8D0 D3 D6 D9 D12 D15 D18 D21 D24 D27TBA, mg MDA/kg of burgerStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO0246810D 0 D 3 D 6 D 9 D 12 D 15 D 18 D 21 D 24 D 27PV, meq /kg lipidStorage time, daysAP AP + cumin EO AP + thyme EOVP VP + cumin EO VP + thyme EO(1) (2)(3)(5)(4)(6)(1) (2) (3)(4) (5) (6)(1)(2)(3) (5)(4)(6)(1) (2) (3)(4) (5) (6)(1)(2) (3)(5)(4)(6)(1) (2) (3)(4) (5) (6)(1)(2)(3)(4) (5)(6)(1) (2) (3)(4) (5) (6)307Rashidimehr A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. 301–310According to Liu et al., TBA increased from0.16 mg/kg (day 0) to 0.42 mg/kg (day 35) in samplesstored at –1°C. In [45], the initial TBA index value forhot smoked rainbow trout fillets was 0.77 mg MDA/kgand reached 1.5 mg MDA/kg by day 27. The lowerproduction of TBA in vacuum packing + thyme samplescan contribute to the antioxidant properties of thyme oil.Soni et al. noticed lower TBA values in chicken pattiescontaining 0.10% of oregano and 0.015% of thyme EOs.Jayawardana et al. suggested that a cause of the reductionof TBA values could be polyphenols present in EOs [49].In our research, TBA values did not exceed theacceptable limit in all samples. Similar results wereobtained by Eskandari et al. in fish treated with blackcumin [46]. Therefore, TBA cannot be used as a reliablequality index for burgers. TBA of 2–4 mg MDA/kgindicates a good quality of fish. TBA values in thisstudy were lower than 1 mg MDA/kg in all treatmentsthroughout the storage period. It was apparently due to arelatively low fat content in fish (surimi).We revealed that the antioxidant properties of cuminand thyme EOs prolonged significantly the burgershelf life. Sarıçoban and Yilmaz also confirmed theantioxidant effect of cumin and thyme on TBA, whichis due to the antioxidant activity of phenolic compoundscontained in different parts of plants [44]. The maincompounds of cumin are gammaterpinene, 2-methyl-3-phenyl-propanal, myrtenal, and glucopyranosides [44].Figure 2d demonstrates an effect of packaging andthyme and cumin EOs on a PV value in the burgersunder study. The initial PV value was 0.16–0.18 meq/kgof lipid in all the burgers, while, by day 27, it reached5.82–8.75 and 1.11–2.35 meq/kg of lipid in samples in airand vacuum packaging, respectively (P < 0.001).In this study, PV was increasing up to day 21 ofstorage in all samples and then, by day 27, decreased.At the end of the storage time, PV in all vacuumpackagedsamples did not reach the acceptablelimit (5 meq/kg). Similar findings were obtained byÇoban and Keleştemur in catfish burger treated withthyme [50]. Such findings are an evidence of EOsinhibitory effect on microorganisms which causeburger spoilage. The reduction of PV after day 21 canbe due to hydroperoxide degradation. The decay ofhydroperoxides results in the formation of degradationproducts [51]. The reduction in PV in samples withcumin EO can be due to cumin aldehyde, which preventslipid peroxidation [52].CONCLUSIONWe found that the shelf life of the novel burgers fromsurimi and minced chicken meat could be extended byusing essential oils and vacuum packaging. According tothe results of the microbiological analysis, the shelf lifeof the burgers was as follows: 9 days for burgers in airpackaging, 12 days for burgers with cumin and thymeEOs in air packaging, 18 days for burgers in vacuumpackaging, and 21 days for burgers with cumin andthyme EOs in vacuum packaging.The shelf life for vacuum-packed burgers treatedwith thyme and cumin EOs was established as 18 daysat 2°C, in compared to that for untreated burgers, whichwas 6 days. In addition, vacuum packaging alone wasfound to maintain burger freshness during 15 days.Thus, burger shelf life was extended by 9 days forthe combination of thyme/cumin EO + air packaging,15 days for vacuum-packaged samples, and 18 daysfor the combination of thyme/cumin EO + vacuumpackaging. Overall, the combined use of vacuumpackaging and thyme/cumin EO demonstrated theirsynergistic effect on the shelf life of the novel burgers.These results allowed us to suggest that surimi could besuccessfully used as an alternative ingredient to mincedmeat in burgers production.CONFLICT OF INTERESTThe authors declare no conflict of interest.ACKNOWLEDEMENTSThis article was written based on the Ph.D. thesis ofDr. Azadeh Rashidimehr. The authors wish to expresstheir gratitude to the research council of the ShahidChamran University of Ahvaz and to Mrs. P. Esfahani,for her kind technical assistance in the food hygienelaboratory.FUNDINGThis study was financially supported by theShahid Chamran University of Ahvaz (GrantNo.: 97/3/02/26247).