Assessment of the Stability of Fresh Beef Patties with the Addition of Clove Extract during Frozen Storage

Md. Ashrafuzzaman Zahid1,2, Jin-kyu Seo1,3, Rashida Parvin1,2, Jonghyun Ko1, Jun-Young Park1, Han-Sul Yang1,3,4,*
Author Information & Copyright
1Division of Applied Life Science (BK21 Plus), Gyeongsang National University, Jinju 52828, Korea
2Department of Nutrition and Food Technology, Jashore University of Science and Technology, Jashore 7408, Bangladesh
3Institute of Agriculture and Life Science, Gyeongsang National University, Jinju 52828, Korea
4Department of Agriculture, University of Arkansas at Pine Bluff, AR 71601, USA
*Corresponding author : Han-Sul Yang, Division of Applied Life Science (BK21 Plus), Gyeongsang National University, Jinju 52828, Korea Tel: +82-55-772-1948 Fax: +82-55-772-1949 E-mail:

© Korean Society for Food Science of Animal Resources. This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Received: Feb 07, 2020 ; Revised: Apr 23, 2020 ; Accepted: May 07, 2020

Published Online: Jul 01, 2020


The study assessed the stability for fresh beef patties with the inclusion of clove extract (CE) as a natural antioxidant in comparison to butylated hydroxytoluene (BHT) and ascorbic acid (AA) at frozen storage. Four different patties were made dependent on the added antioxidants: control (added no antioxidants), added with 0.02% BHT, 0.05% AA, and 0.1% CE. Inclusion of BHT, AA, and CE resulted in a significant reduction of thiobarbituric acid reactive substances (TBARS) and hue angle (h°) value and increase of redness (CIE a*) and chroma (C*) values (p<0.05). BHT, AA, and CE were observed effectively to retard lipid oxidation and increase color stability. BHT and AA revealed significantly (p<0.05) higher thiol content than the control and CE. However, the reduction percentage for thiol content in CE treated patties was lower than the control and AA-treated patties from first to last time of storage. Moreover, inclusion of AA and CE led to significantly (p<0.05) increased heme iron content when compared to BHT and the control. In conclusion, CE can replace the application of AA and BHT while improving lipid stability, heme iron content, and color stableness of fresh beef patties throughout frozen storage.

Keywords: fresh beef patties; clove extract; oxidative stability; heme iron content; color value


Meat is an important source of major nutrients and constituent of a daily diet. Change for consumers’ demands and increased market competitions have induced a requirement for improving the quality of meat products as beef patty by developing their nutrient values and advantageous-health qualities (Lopez-Lopez et al., 2011). Nevertheless, fresh meat and meat products are highly responsive to quality deterioration because of the higher nutrient constitution (Shah et al., 2014). Frozen storage has been allowed as the most efficient procedure for preserving the attribute for meat products over an extended period of time. The meat quality can be deteriorated because of the physico-chemical process and, consequently, various researches have been presented that the oxidation of lipid is a prime cause for losing the qualities for several types of meat plus processed meats during frozen storage (Ozer and Saricoban, 2010). In addition, auto-oxidation is occurred broadly leading to a reduced functional quality of meats and meat-based products throughout frozen storage (Mielnik et al., 2003).

One of the important factors for meat quality deterioration is the oxidative process which affects lipid, protein, carbohydrate, vitamins, and pigments. Oxidative deterioration induces mainly lipid oxidation and results in loss of nutritional quality and sensorial attributes and reduction of shelf-life for meat-based products (Soriano et al., 2018). Lipid originated reactive oxygen species and oxidizing myoglobin by-products lead to meat oxidation and dramatically decrease quality properties such as color, flavor, and nutrient value of meat products (Seo et al., 2019). Moreover, protein oxidation results in a change for amino acid structure, tenderness, and water-retention capability for meats and processed meats causing a decrease in meat products quality (Turgut et al., 2016).

The uses of antioxidants supplements have been proven to be an efficient plan for delaying or preventing the oxidizing procedures. Therefore, ascorbic acid (AA) is usually employed as an antioxidant supplement during processing of meats. AA is soluble in water, employed as the additives based on the rule of ‘proper amount’ to hinder deterioration of meat product quality, and is regarded for having no toxic impacts for the consumer (Carballo et al., 2018; Ozer and Saricoban, 2010). Moreover, Butylated hydroxytoluene (BHT) is a widely utilized artificial antioxidant which is efficient to purify peroxyl radical and restrain the origination of free radical. This is allowed to incorporate into meat and meat product to slow or inhibit the oxidation and expand the storage stability (Kumar et al., 2015). Nonetheless, consumer interests and requirements on naturally originated antioxidant substances have been increasing owing to the antinutritional and toxicological impacts for synthetically antioxidant substances like BHT, butylated hydroxyanisole (BHA) or propyl gallate (Carballo et al., 2018; Shah et al., 2014).

Clove (Syzigium aromaticum L.), is belonged to the Myrtaceae family, is a dried-up flower bud and is largely utilized in the food products as it possesses a specific aroma and effective health attributes. Clove contains phenolic constituents like tannins, sesquiterpenes, and triterpenoids which can show antioxidant activities (Ramadan et al., 2013; Zhang et al., 2017). Clove extract (CE) acquired from the entire clove bud has been widely examined to show high antioxidant actions in meat products (Shi et al., 2014). In that context, CEs as natural antioxidant have been employed for meats and meat-based products to increase lipid and protein stability against oxidization, enhance color stabilization and sensorial properties, and lengthen the shelf-life at the storage time (Shi et al., 2014; Zhang et al., 2017).

The uses of natural antioxidant have been expanded for improving the oxidizing stability for meats and meat-based products over the past years (Armenteros et al., 2016). Zhang et al. (2016) has recorded that CE as a natural antioxidant has been used potentially in meat products for improving the oxidizing stability. However, it has been utilized BHT and AA as antioxidants in the meat industries (Carballo et al., 2018; Cunha et al., 2018). Considering this, fresh beef patties were formulated with BHT, AA, and CE, and it was examined the comparison for the impacts of abovementioned antioxidants on oxidizing stability, color stableness, and heme iron content for fresh beef patties. Nonetheless, no comparative analysis has been conducted for the antioxidative effect of CE with AA and BHT on the stability of fresh beef patties, especially at frozen storage.

The purpose for the existing study was to determine the antioxidative effect of CE compared to AA and BHT on the stability of fresh beef patties. Cooking loss, pH, lipid oxidization, protein oxidization, heme iron level, and color values of fresh beef patties were evaluated during 6 months of frozen storage. Fresh beef patties with added no antioxidants were employed as the control.

Materials and Methods

Formulation of clove extract

Clove was purchased from the domestic market. The clove powder extract was got utilizing the method of reflux extraction. After grinding of clove, the powder was mixed into the distilled water (w/v, 1:5 ratios) and carried for extraction at 85°C for 7 h. Likewise, the extraction for residue was conducted using distilled water (1:5 ratios) for 14 h at 85°C. The extracted two solutions were filtrated with filter paper of Whatman No. 1. The eventual CE was collected after condensing the solution through a void rotating evaporator at 85°C. The CE was preserved at −60°C to continue analysis.


BHT, AA, 2-thiobarbituric acid (TBA), perchloric acid (PCA), sodium dodecyl sulfate (SDS), tris (hydroxymethyl) amino methane (TRIS) buffer, and 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) were obtained from Sigma Aldrich (St. Louis, MO, USA). The whole chemicals utilized for the current research was of the highest purities of analyzed properties.

Formulation of fresh beef patties

Fresh beef loin and beef back fat were acquired from the domestic market and ground independently utilizing a meat grinder (GG-22, German Knife, CA, USA) consisting of a plate set in 8-mm diameter holes. The study included 4 treatments, and three batches for each treatment were conducted 3 times for each storage month (mon). The preparation of all fresh beef patties was performed employing the same formulations. The beef loin, back fat, and all fresh constituents were combined absolutely in the correct proportion employing a patty mixer (5K5SS, KitchenAid, Michigan, MI, USA). The primary formulations contained 90.8% beef loin, 8.0% back fat, and 1.2% sodium chloride. The fresh beef patties (4) were prepared as control (without BHT, AA, and CE), added with 0.02% BHT (BHT), 0.05% AA (AA), and 0.1% CE (CE). The patties (45 g) were structured through a hand held patties maker. After packaging in the polyethylene packets, the fresh patties were kept at frozen (−21°C) storage for 0, 2, 4, and 6 mon to conduct the whole experiments in the laboratory of Meat Processing.

pH and cooking loss

The pH value of beef patties was assessed employing an electronic pH meter (MP230, Mettler Toledo, Greifensee, Switzerland). The distilled water (30 mL) mixed patties (3 g) were homogenized utilizing an electronic homogenizer (T25 Ultra-Turrax, IKA, Germany) for 25 s. For evaluating the pH of the patties, the pH meter was calibrated through standard buffers of pH 4.01, 7.00, and 9.21 at 21°C.

The cooking loss (%) for fresh beef patties was measured by the calculation of weight difference between uncooked and cooked patties as followed:

Cooking loss  ( % ) = [ ( Weight of uncooked beef patties Weight of cooked beef patties ) / Weight of uncooked beef patties ] × 100.
Lipid oxidation

Thiobarbituric acid reactive substances (TBARS) of beef patties were assessed to measure the lipid oxidation employing the minor modified method adopting from Cherian et al. (2007). Fresh beef patties (3 g) were homogenized through 3.86% perchloric acid (27 mL) and stored for 1 h at 5°C. The homogenate was centrifuged (1736R, Labogene, Seoul, Korea) at 2,100 g for 10 min. After filtrating the supernatant, filtrate (2 mL) was admixed into 20 mM TBA (2 mL) and allowed to stay at room-temperature for 14 h. The absorbance was evaluated at 531 nm spectrophotometrically (Cary 60 UV-Vis, Agilent Technologies, Santa Clara, CA, USA). The TBARS values were stated as mg malondialdehyde (MDA)/kg patties.

Protein oxidation

Thiol content for different patties was determined for measuring the protein oxidization using the method described by Vossen and De Smet (2015) with some modifications. The patties (2 g) were homogenized through 27 mL of 5% SDS in 0.10 M Tris buffer and transferred to the water-bath at 80°C for 30 min. The coolness formed homogenate was centrifuged at 6,000 g for 20 min. The filtrated supernatant (0.5 mL) was added to 2 mL of 0.1 M TRIS buffer (pH 8.0) and 0.5 mL of 10 mM DTNB (5,5′-dithiobis (2-nitrobenzoic acid) in 0.1 M TRIS buffer to evaluate the thiol content. Filtrated supernatant (0.5 mL) was incorporated into 0.1 M TRIS buffer (2.5 mL) to evaluate the protein content. Moreover, the reagent blank was formed by mixing 5% SDS in TRIS buffer (0.5 mL), 10 mM DTNB (0.5 mL), and 0.1 M TRIS buffer (2.0 mL). All solutions were allowed for reacting in the dark place at 5°C for 30 min. Absorbance for thiol content was then read at 412 nm. The calculation for thiol content was done employing the Lambert-Beer equation of ε412 = 14,000 M−1 cm−1, and the result was indicated in nmol of thiol/mg of protein. Protein content was determined at 280 nm utilizing a BSA standard curve.

Heme iron measurement

Heme iron content of beef patties was measured employing the method explained by Ozer and Saricoban (2010) with minor modification. Beef patties (1 g) were added to 5 mL of acidified acetone solution (acetone:distilled water:HCl=90:8:2) in polypropylene tube. The tube was closed with a cap and permitted for standing in darkness condition at room-temperature for 1 h. The tube content was filtrated using Whatman GFA as glass filter paper, and the absorbance was evaluated at 640 nm.

The calculation for heme-iron content was performed by calculating the whole pigment as hematin employing the following formulas:

Whole pigment  ( mg/kg ) = Absorbance × 680 . Heme-iron  ( mg/kg ) = Whole pigment  ( mg/kg ) × 8.82 / 100.
Color value

Color values like lightness (CIE L*), redness (CIE a*), and yellowness (CIE b*) for several patties were determined utilizing a colorimeter (Konica Minolta CR-400, Tokyo, Japan). The standard white plate (Y=81.2; x=0.3191; y=0.3263) was employed for calibrating the colorimeter, and each patty was measured twice. The measurement for chroma (C*) value and hue angle (h°) value was carried out utilizing two equations of {(a* + b*)1/2} and {tan−1(b*/a*)}, respectively.

Statistical analysis

The experiments contained a sum of 48 observations (four treatments×three batches×four storage periods) to conduct statistical analysis. All data were exhibited as mean values of 3 replications with the standard error of means. The data were examined utilizing a statistical software of Statistical Analysis System (SAS) containing 9.3 version. One-way analysis of variance (ANOVA) accompanied by Duncan’s multiple range tests (p<0.05) was utilized to assess significant differences among different categories for fresh beef patties and to assess the effect of storage period.

Results and Discussion

pH and cooking loss

The measurement of pH and cooking loss was conducted for 6 mon of frozen storage and is displayed in Table 1. Non-significant change for pH value was noticed amongst all fresh beef patties throughout frozen storage times (p>0.05). Nonetheless, the pH value in all beef patty samples was significantly increased (p<0.05) from the first to last time of frozen storage. Ozer and Saricoban (2010) recorded that pH value for meat products increased significantly during frozen storage. This increasing for pH value is due to the production of ammonia arising from amino acids deterioration as protein denaturation in meat products. The results are in accordance with Mokhtar and Youssef (2014), who noticed that the beef burgers treated with BHT/BHA and CE showed no significant changes for pH values compared with the control during storage.

Table 1. Effects of different antioxidants on pH and cooking loss of fresh beef patties at frozen storage
Storage month Con BHT AA CE SEM
pH 0 5.56B 5.53B 5.50B 5.54B 0.03
2 5.64AB 5.64AB 5.60AB 5.63AB 0.04
4 5.63AB 5.62AB 5.59AB 5.60AB 0.03
6 5.76A 5.75A 5.71A 5.74A 0.09
SEM 0.04 0.05 0.05 0.05
Cooking loss (%) 0 19.05 19.82 20.63 21.05 1.35
2 20.01 22.05 23.38 18.50 1.66
4 23.65 24.05 24.51 22.72 2.16
6 19.75 19.81 20.68 18.09 1.43
SEM 1.64 1.75 1.71 1.51

a,b Mean values in the same row with different letters presented significant differences (p<0.05).

A,B Mean values in the same column with different letters presented significant differences (p<0.05).

Con, control; BHT, added 0.02% butylated hydroxytoluene; AA, added 0.05% ascorbic acid; CE, added 0.1% clove extract.

Download Excel Table

The cooking loss considers the loss of moisture and fat after cooking of meat products. The insignificant difference (p>0.05) for cooking loss was seen among all patty samples at all storage periods. Nevertheless, the cooking loss in all patties showed no significant changes among all storage times (p>0.05). It is reported that all antioxidants and storages times had no negative effects in cooking loss. No cooking loss in beef patties formulated with BHT, AA, and CE can be related to no fat and moisture loss. Non-significant change for cooking loss in chicken nugget was seen after formulation with antioxidants of sage, rosemary, and tea catechin (O’Sullivan et al., 2004). Basanta et al. (2018) also reported that patties sample with added plum pulp showed non-significant change for cooking loss in comparison to the control sample.

Lipid oxidation

Lipid oxidization for fresh beef patties was assessed by measuring the TBARS value at frozen storage, and the findings are exhibited in Fig. 1. Incorporated antioxidants and storage months presented substantial (p<0.05) effect on TBARS values. Maximal TBARS was shown in control beef patties throughout the storage months; nonetheless, antioxidants formulated patties exhibited obvious reduction for TBARS values. TBARS value for all kinds of beef patties was significantly (p<0.05) increased from mon 0 to mon 6 of storage. The antioxidants like BHT, AA, and CE formulated fresh beef patties revealed significantly lower TBARS values during whole frozen storage periods when compared with the control (p<0.05). The findings noted that added antioxidants presented a positive effect on the oxidative stability for beef patties. The increase for TBARS value for the control could be due to the origination of increased MDA that has been considered as secondary products for lipid oxidization (Zhang et al., 2016). However, non-significant change for TBARS value was observed amongst BHT, AA, and CE contained beef patties for all storage periods (p>0.05). Ozer and Saricoban (2010) reported that chicken patties with added AA had significantly reduced TBARS value compared to the control patties. The substantial decrease for TBARS values was found in CE treated pork patties (Kong et al., 2010), chicken meat sample (Zhang et al., 2016), buffalo patties (Tajik et al., 2014), beef burgers (Mokhtar and Youssef, 2014), and pork sausages (Zhang et al., 2017). The current study is in agreement with such results and indicates that the natural antioxidant like CE can have been employed for enhancing the shelf-life for any meat product. This antioxidant effectiveness of CE has been performed owing to the phenolic constituents and the capacity for the hydrogen molecule donation to deactivate free radical (Baghshahi et al., 2014).

Fig. 1. Effect of different antioxidants on TBARS (mg MDA/kg of sample) value of fresh beef patties during frozen storage. Error bars present standard deviations. Bar charts with different letters present significant differences among the treatments (a,b) at each storage month (p<0.05) or storage months (A–C) in each treatment (p<0.05). Con, control; BHT, added 0.02% butylated hydroxytoluene; AA, added 0.05% ascorbic acid; CE, added 0.1% clove extract; TBARS, thiobarbituric acid reactive substances.
Download Original Figure
Protein oxidation

Protein oxidation of fresh beef patties was measured by the evaluation of thiol contents, and the findings are presented in Table 2. The significant decline for thiol content in whole beef patty samples were observed from mon 0 and 2 to mon 4 and 6 of frozen storage (p<0.05), indicated the proteins oxidization. A similar result has been found by Feng et al. (2016), who revealed that proteins oxidization caused diminished thiol content for pork sausage at storage time. From first to last time of storage, the reducing rate for thiol content in CE treated beef patties were lower compared to AA supplemented beef patties and the control. The decline percentages for thiol content were shown by ascending: control>AA>CE>BHT (12.31%> 11.20%>9.51%>8.72%, respectively). At the end of the storage, thiol content reduction of 12.31% was seen for the control patties; however, thiol content reduction for BHT, AA, and CE treated beef patties (8.72%, 11.20%, and 9.51%, respectively) was lower than the control patties. After mon 4 and 6 of storage, BHT and AA contained beef patties presented significantly higher thiol content by comparing with CE contained patties and the control patties (p<0.05), nevertheless, no significant change (p>0.05) for thiol content was found between BHT and AA contained beef patties. The CE formulated fresh beef patties presented significantly lower thiol content than all other patties for all storage months (p<0.05). The outcomes are similar to the research analyzed by Zhang et al. (2017), who observed that the significant reduction for thiol content was seen for CE formulated pork sausages compared with the control, and this occurrence might be done because of the balancing of the antioxidants and pro-oxidants effect for phenolic components in CE. Jongberg et al. (2011) stated that white grapes extracts led to a reduction in thiol contents for beef patties, and it might be occurred owing to the presence of ortho-phenolic substances in extracts. This thiol content reduction in beef patties by the addition of CE was presented because of the ortho-phenolic component (eugenol), which could react with thiol contents and produce thiol-quinone admixture; as a result, thiol content was decreased (Zhang et al., 2017). Nonetheless, silver carp fillet formulated with CE was seen to hinder the decline of thiol contents (Shi et al., 2014).

Table 2. Effects of different antioxidants on thiol content of fresh beef patties at frozen storage
Storage month Con BHT AA CE SEM
Thiol content 0 93.60Ab 95.98Aab 98.67Aa 76.02Ac 1.41
2 91.06Aa 93.30ABa 95.43Aa 74.33Ab 1.18
4 86.93Bb 90.51BCa 90.15Ba 71.11Bc 0.69
6 82.08Cb 87.61Ca 87.62Ba 68.79Bc 0.46
SEM 0.79 0.76 1.33 0.86

Values presented as nmol thiol/mg of protein in fresh beef patties

a–c Mean values in the same row with different letters presented significant differences (p<0.05).

A–C Mean values in the same column with different letters presented significant differences (p<0.05).

Con, control; BHT, added 0.02% butylated hydroxytoluene; AA, added 0.05% ascorbic acid; CE, added 0.1% clove extract.

Download Excel Table
Heme iron content

Meats and meat-based products are important sources for heme iron connected to protein. Heme iron content for fresh beef patties at frozen storage is presented in Fig. 2. The significant decrease (p<0.05) of heme-iron content was seen for all types of beef patties at frozen storage time from mon 0 to mon 6. The heme iron content in CE treated patties was significantly increased (p<0.05) when compared to the control and BHT treated beef patties at all storage times, whereas non-significant changes for heme iron contents were found between AA and CE treated beef patties (p>0.05). On mon 6, the beef patties formulated with AA showed significantly increased heme iron content in comparison to BHT formulated patties and the control (p<0.05). The results specified that AA and CE prevented the free of iron from heme-iron. The increase in heme-iron content for antioxidant contained patties could have been due to the increased level of soluble heme pigments and the contribution for increased extractability of heme pigment (Ozer and Saricoban, 2010). The reduction of heme-iron content occurred because of the releasing of iron caused by disruption of heme and the increase of frozen storage times (Benjakul and Bauer, 2001; Ozer and Saricoban, 2010). Purchas et al. (2003) stated that the drips freed from meat throughout storage comprised a substantial quantity of iron, especially soluble heme-iron. Moreover, Buzala et al. (2016) reported that heme-iron is mostly located in meat protein, is involved in the contribution of the bright red color in meat and meat products.

Fig. 2. Effect of different antioxidants on heme iron content (mg heme iron/kg of sample) of fresh beef patties during frozen storage. Error bars present standard deviations. Bar charts with different letters present significant differences among the treatments (a–c) at each storage month (p<0.05) or storage months (A–C) in each treatment (p<0.05). Con, control; BHT, added 0.02% butylated hydroxytoluene; AA, added 0.05% ascorbic acid; CE, added 0.1% clove extract.
Download Original Figure
Color evaluation

Color is a precious quality mostly of meat products and makes influencing the consumer for instant purchasing or refusing the meat products through observation (Soriano et al., 2018). The color values of fresh beef patties were evaluated for all storage months and are shown in Table 3. From mon 0 to mon 6, CIE a* and C* values were significantly reduced for all kinds of beef patties (p<0.05), nevertheless, non-significant change (p>0.05) in CIE L*, CIE b*, and h° values was seen for all beef patties. The CIE L* value for all patties presented non-significant variation (p>0.05) throughout the storage. The patties formulated with CE showed significantly increased (p<0.05) CIE b* value compared to the control at the final storage, while the non-significant change (p>0.05) for CIE b* value was observed in BHT and AA processed patties compared with the control. This result is in accordance with Radha Krishnan et al. (2014), who reported that the CIE b* values of the spice extract incorporated chicken meat samples were substantially higher in comparison with the control. After 4th and 6th mon for frozen storage, BHT, AA, and CE supplemented patties showed significantly increased (p<0.05) CIE a* and C* value by comparing with the control patties. Moreover, a significant decline (p<0.05) for h° value was seen for BHT, AA, and CE incorporated beef patties as compared to the control on mon 4 and 6. The lowered h° value has been linked to a lowered decline for red color (Yousuf and Srivastava, 2017), indicated that BHT, AA, and CE processed beef patties revealed lowered color decline compared to the control. The result indicated that added antioxidants (BHT, AA, and CE) showed preventative effects on discoloring for beef patties during frozen storage moment. This might have been related to a reduction for lipid oxidization by the addition of BHT, AA, and CE, since reduced lipid oxidization can cause reduced discoloration. The previous studies showed that lipid oxidization for meat products caused redness degradation (Hayes et al., 2011; Jung et al., 2012). The significant increase for CIE a* value was also observed in CE incorporated pork patties (Kong et al., 2010), chicken meat sample (Zhang et al., 2016), and pork sausages (Zhang et al., 2017). Moreover, Falowo et al. (2014) stated that a preventative impact for natural plant extract on discolorization for meats and meat-based products was found because of the antioxidative action of phenolic substances.

Table 3. Effects of different antioxidants on color values of fresh beef patties at frozen storage
Storage month Con BHT AA CE SEM
Lightness (CIE L*) 0 41.07Aa 41.25Aa 41.51Aa 40.63Aa 1.98
2 39.70Aa 40.38Aa 39.78Aa 40.28Aa 2.15
4 39.27Aa 41.07Aa 40.54Aa 40.50Aa 1.66
6 39.07Aa 40.65Aa 40.13Aa 38.98Aa 1.14
SEM 1.64 1.44 1.80 2.06
Redness (CIE a*) 0 22.13Aa 23.84Aa 23.13Aa 20.98Aa 0.73
2 21.98Aa 23.13ABa 20.40ABa 22.26Aa 1.17
4 12.56Bb 19.78BCa 18.47Ba 18.44Ba 1.01
6 11.42Bb 17.90Ca 18.56Ba 17.65Ba 0.78
SEM 1.07 1.04 0.84 0.74
Yellowness (CIE b*) 0 15.37Aa 16.22Aa 16.19Aa 15.82Aa 0.75
2 19.22Aa 19.49Aa 19.61Aa 20.76Aa 5.43
4 11.66Aa 13.39Aa 12.69Aa 13.34Aa 0.77
6 11.50Ab 12.43Aab 12.65Aab 12.98Aa 0.38
SEM 1.90 1.67 1.88 1.88
Chroma (C*) 0 26.94Aa 28.85Aa 28.24Aa 26.29Aa 0.97
2 23.58Aa 24.95Ba 22.19Ba 24.54ABa 1.73
4 17.19Bb 23.90Ba 22.45Ba 22.79ABa 1.03
6 16.21Bb 21.82Ba 22.22Ba 21.39Ba 0.75
SEM 1.27 1.06 0.98 1.18
Hue angel (h°) 0 34.83ABa 34.18Aa 34.91Aa 36.92Aa 0.86
2 25.92Ba 26.80Aa 27.15Aa 28.27Aa 7.46
4 43.09Aa 34.14Ab 34.67Ab 35.82Ab 1.86
6 45.36Aa 34.76Ab 34.56Ab 36.35Ab 1.51
SEM 2.90 2.29 3.27 3.23

a,b Mean values in the same row with different letters presented significant differences (p<0.05).

A–C Mean values in the same column with different letters presented significant differences (p<0.05).

Con, control; BHT, added 0.02% butylated hydroxytoluene; AA, added 0.05% ascrobic acid; CE, added 0.1% clove extract.

Download Excel Table


The incorporated BHT, AA, and CE in fresh beef patties prompted a significant decline of TBARS and h° values and increase of CIE a* and C* values at frozen storage for 6 mon as compared with the control (p<0.05). Inclusion of AA and CE led to significantly increased (p<0.05) heme iron content in beef patties when compared to BHT treated patties and the control. Moreover, BHT and AA added patties and the control showed significantly increased (p<0.05) thiol content compared to CE treated patties. Nevertheless, the percentage in a decrease for thiol content of CE treated patties was lower than the control and AA-treated patties from first to last time of storage. It is definitely seen that BHT, AA, and CE showed the antioxidant effect on fresh beef patties. The antioxidant impacts for three antioxidants were more pronounced for lipid oxidizing than protein oxidizing. In Sum, the inclusion for CE could have been employed as a safe and substitution of artificial antioxidants in beef patties preparation to efficiently prevent lipid oxidation and increase heme iron content and color stability. Therefore, the results can be concluded that CE can replace the application of AA and BHT when the formulation of fresh beef patties at frozen storage.

Conflicts of Interest

The authors declare no potential conflicts of interest.


This research was supported by the Korea Institute of Planning and Evaluation for Technology in Food Agriculture, Forestry and Fisheries, Ministry of Agriculture, Food and Rural Affairs (Project No.316064-02-2-HD030).

Author Contributions

Conceptualization: Md. Zahid A, Yang HS. Data curation: Seo JK, Yang HS. Formal analysis: Md. Zahid A, Parvin R, Ko J, Park JY. Methodology: Md. Zahid A, Seo JK. Validation: Md. Zahid A, Yang HS. Writing -original draft: Md. Zahid A, Writing -review & editing: Md. Zahid A, Seo JK, Parvin R, Ko J, Park JY, Yang HS.

Ethics Approval

This article does not require IRB/IACUC approval because there are no human and animal participants.



Armenteros M, Morcuende D, Ventanas J, Estévez M. 2016; The application of natural antioxidants via brine injection protects Iberian cooked hams against lipid and protein oxidation. Meat Sci. 116:253-259


Baghshahi H, Riasi A, Mahdavi AH, Shirazi A. 2014; Antioxidant effects of clove bud (Syzygium aromaticum) extract used with different extenders on ram spermatozoa during cryopreservation. Cryobiology. 69:482-487


Basanta MF, Rizzo SA, Szerman N, Vaudagna SR, Descalzo AM, Gerschenson LN, Pérez CD, Rojas AM. 2018; Plum (Prunus salicina) peel and pulp microparticles as natural antioxidant additives in breast chicken patties. Food Res Int. 106:1086-1094


Benjakul S, Bauer F. 2001; Biochemical and physicochemical changes in catfish (Silirus glanis Linne) muscle as influenced by different freeze-thaw cycles. Food Chem. 72:207-217


Buzala M, Slomka A, Janicki B. 2016; Heme iron in meat as the main source of iron in the human diet. J Elem. 21:303-314.


Carballo DE, Caro I, Andres S, Giraldez FJ, Mateo J. 2018; Assessment of the antioxidant effect of astaxanthin in fresh, frozen and cooked lamb patties. Food Res Int. 111:342-350


Cherian G, Traber MG, Goeger PM, Leonard SW. 2007; Conjugated linoleic acid and fish oil in laying hen diets: Effects on egg fatty acids, thiobarbituric acid reactive substances, and tocopherols during storage. Poult Sci. 86:953-958


Cunha LCM, Monteiro MLG, Lorenzo JM, Munekata PES, Muchenje V, de Carvalho FAL, Conte-Junior CA. 2018; Natural antioxidants in processing and storage stability of sheep and goat meat products. Food Res Int. 111:379-390


Falowo AB, Fayemi PO, Muchenje V. 2014; Natural antioxidants against lipid-protein oxidative deterioration in meat and meat products: A review. Food Res Int. 64:171-181


Feng X, Li C, Jia X, Guo Y, Lei N, Hackman RM, Chen L, Zhou G. 2016; Influence of sodium nitrite on protein oxidation and nitrosation of sausages subjected to processing and storage. Meat Sci. 116:260-267


Hayes JE, Stepanyan V, Allen P, O’grady MN, Kerry JP. 2011; Evaluation of the effects of selected plant derived nutraceuticals on the quality and shelf-life stability of raw and cooked pork sausages. LWT-Food Sci Technol. 44:164-172


Jongberg S, Skov SH, Tørngren MA, Skibsted LH, Lund MN. 2011; Effect of white grape extract and modified atmosphere packaging on lipid and protein oxidation in chill stored beef patties. Food Chem. 128:276-283


Jung EY, Yun IR, Go GW, Kim GD, Seo HW, Joo ST, Yang HS. 2012; Effects of radix puerariae extracts on physicochemical and sensory quality of precooked pork sausage during cold storage. LWT-Food Sci Technol. 46:556-562


Kong B, Zhang H, Xiong YL. 2010; Antioxidant activity of spice extracts in a liposome system and in cooked pork patties and the possible mode of action. Meat Sci. 85:772-778


Kumar Y, Yadav DN, Ahmad T, Narsaiah K. 2015; Recent trends in the use of natural antioxidants for meat and meat products. Compre Rev Food Sci Food Saf. 14:796-812


Lopez-Lopez I, Cofrades S, Caneque V, Diaz MT, Lopez O, Jimenez-Colmenero F. 2011; Effect of cooking on the chemical composition of low-salt, low-fat Wakame/olive oil added beef patties with special reference to fatty acid content. Meat Sci. 89:27-34


Mielnik MB, Aaby K, Skrede G. 2003; Commercial antioxidants control lipid oxidation in mechanically deboned turkey meat. Meat Sci. 65:1147-1155


Mokhtar SM, Youssef KM. 2014; Antioxidant effect of some plant extracts as compared with BHA/BHT on lipid oxidation and some quality properties of fresh beef burgers stored at 4?. J Food Sci. 2:9-18


O’Sullivan CM, Lynch AM, Lynch PB, Buckley DJ, Kerry JP. 2004; Use of antioxidants in chicken nuggets manufactured with and without the use of salt and/ or sodium tripolyphosphate: Effects on product quality and shelf-life stability. Int J Poult Sci. 3:345-353


Ozer O, Saricoban C. 2010; The effects of butylated hydroxyanisole, ascorbic acid, and α-tocopherol on some quality characteristics of mechanically deboned chicken patty during freeze storage. Czech J Food Sci. 28:150-160


Purchas RW, Simcock DC, Knight TW, Wilkinson BHP. 2003; Variation in the form of iron in beef and lamb meat and losses of iron during cooking and storage. Int J Food Sci Technol. 38:827-837


Radha Krishnan K, Babuskin S, Azhagu Saravana Babu P, Sasikala M, Sabina K, Archana G, Sivarajan M, Sukumar M. 2014; Antimicrobial and antioxidant effects of spice extracts on the shelf life extension of raw chicken meat. Int J Food Microbiol. 171:32-40


Ramadan MF, Asker MMS, Tadros M. 2013; Lipids profile, antiradical power and antimicrobial properties of Syzygium aromaticum oil. Grasas y Aceites. 64:509-520


Seo JK, Parvin R, Yim DG, Zahid MA, Yang HS. 2019; Effects on quality properties of cooked pork sausages with Caesalpinia sappan L. extract during cold storage. J Food Sci Technol. 56:4946-4955


Shah MA, Bosco SJD, Mir SA. 2014; Plant extracts as natural antioxidants in meat and meat products. Meat Sci. 98:21-33


Shi C, Cui J, Yin X, Luo Y, Zhou Z. 2014; Grape seed and clove bud extracts as natural antioxidants in silver carp (Hypophthalmichthys molitrix) fillets during chilled storage: Effect on lipid and protein oxidation. Food Control. 40:134-139


Soriano A, Alanon ME, Alarcon M, Garcla-Rulz A, Dlaz-Maroto MC, Perez-Coello MS. 2018; Oak wood extracts as natural antioxidants to increase shelf life of raw pork patties in modified atmosphere packaging. Food Res Int. 111:524-533


Tajik H, Farhangfar A, Moradi M, Rohani SMR. 2014; Effectiveness of clove essential oil and grape seed extract combination on microbial and lipid oxidation characteristics of raw buffalo patty during storage at abuse refrigeration temperature. J Food Process Preserv. 38:31-38


Turgut SS, Soyer A, Isıkcı F. 2016; Effect of pomegranate peel extract on lipid and protein oxidation in beef meatballs during refrigerated storage. Meat Sci. 116:126-132


Vossen E, De Smet S. 2015; Protein oxidation and protein nitration influenced by sodium nitrite in two different meat model systems. J Agric Food Chem. 63:2550-2556


Yousuf B, Srivastava AK. 2017; Flaxseed gum in combination with lemon grass essential oil as an effective edible coating for ready-to-eat pomegranate arils. Int J Biol Macromol. 104:1030-1038


Zhang H, Peng X, Li X, Wu J, Guo X. 2017; The application of clove extract protects Chinese style sausages against oxidation and quality deterioration. Korean J Food Sci Anim Resour. 37:114-122


Zhang H, Wu J, Guo X. 2016; Effects of antimicrobial and antioxidant activities of spice extracts on raw chicken meat quality. Food Sci Hum Wellness. 5:39-48