Food Control 21 (2010) 12821290

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Food Control
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Preventive effect of tannic acid in combination with modied atmospheric packaging on the quality losses of the refrigerated ground beef
Sajid Maqsood, Soottawat Benjakul *
Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand

a r t i c l e

i n f o

a b s t r a c t
Chemical, microbiological and sensorial changes of ground beef treated without and with tannic acid (200 mg/kg) and stored in air and under modied atmosphere packaging (MAP) (80%O2/20%CO2 or 10%O2/20%CO2/70%N2) were monitored during 15 days of storage at 4 C. During the storage, samples treated with tannic acid and kept under all packaging conditions contained lower peroxide value (PV) and thiobarbituric acid-reactive substances (TBARS) with coincidental lower non-haem iron content, compared with non-treated counterparts (P < 0.05). The sample packed in high oxygen MAP treated without and with tannic acid had the higher oxymyoglobin and a values and received the higher likeness scores for colour, whereas the samples stored in air and under low oxygen MAP showed the lower values, regardless of tannic acid treatment. After 15 days of storage, myosin heavy chain (MHC) and actin of all tannic acid treated samples underwent less degradation than those without tannic acid treatment for all packaging conditions. Degradation of MHC was more pronounced in samples kept under MAP with high oxygen. Psychrophilic bacterial count (PBC) of all tannic acid treated samples was lower, compared with that of non-treated samples (P < 0.05), irrespective of packaging condition. Therefore, tannic acid treated samples stored under high oxygen MAP could maintain the red colour and retard lipid oxidation and microbial growth of ground beef during refrigerated storage. 2010 Elsevier Ltd. All rights reserved.

Article history: Received 20 November 2009 Received in revised form 19 February 2010 Accepted 27 February 2010

Keywords: Lipid oxidation MAP Ground beef Colour Tannic acid Quality

1. Introduction The colour and odour of meat are the most important quality attributes for the consumers by which meat quality is readily assessed (Mancini & Hunt, 2005). Myoglobin (Mb) is the principle haem protein responsible for meat colour. The oxygenated form of Mb or oxymyoglobin (OxyMb) contributes to the cherry red colour associated with fresh meat (Bou et al., 2008). However, OxyMb can change its colour to brown due to the formation of metmyoglobin (MetMb) (Bou et al., 2008). For meat, modied atmosphere packaging with a high level of oxygen (7080%) is required to maintain the acceptable red colour. High oxygen atmosphere preserves the bright red colour of meat (Okayama, Muguruma, Murakami, & Yamada, 1995). Although high oxygen MAP maintains the redness of meat during storage, the rancidity often develops (Jayasingh, Cornforth, Brennand, Carpenter, & Whittier, 2002; Okayama et al., 1995). To alleviate such a drawback, the use of antioxidants, especially phenolic compounds could be an effective means to prevent lipid oxidation in high oxygen packaging (Morrissey, Sheehy, Galvin, Kerry, & Buckley, 1998). Recently, Maqsood and Benjakul (2010a) reported that

tannic acid exhibited the superior radical scavenging activities as well as reducing power and effectively inhibited the lipid oxidation in sh mince and emulsion model systems. Tannic acid is afrmed as Generally Recognized as Safe (GRAS) by the Food and Drug Administration (FDA) for the use as a direct additive in some food products including meat products (21 CFR184. 1097, U.S. Code of Federal Regulations, 2006). Thus, the objective of the present study was to investigate the combined effect of tannic acid and high- and low-oxygen modied atmospheric packaging on the prevention of lipid oxidation and colour stability in the ground beef. 2. Materials and methods 2.1. Chemicals All the chemicals and reagents were of analytical grade and were purchased from Sigma Chemical Co. (St. Louis, MO, USA), Merck (Darmstadt, Germany), Fluka Chemical Co. (Buchs, Switzerland) and Bio-Rad (Richmond, CA, USA). 2.2. Sample preparation Fresh beef tendon loin (5 kg) was purchased from a local slaughter house in Hat Yai, Songkhla, Thailand. The beef was

* Corresponding author. Tel.: +66 7428 6334; fax: +66 7421 2889. E-mail address: soottawat.b@psu.ac.th (S. Benjakul). 0956-7135/$ - see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2010.02.018

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packed in polythene bags and kept in ice during transportation to Department of Food Technology, Prince of Songkla University. Upon arrival, the sample was rinsed with the chilled sterilized distilled water (810 C), cut into small pieces and the connective tissue and depot fat were removed manually. The prepared sample was minced using a mincer with the hole diameter of 3 mm. Ground beef was packed in polythene bags and kept at 4 C until use but was not longer than 2 h. 2.3. Tannic acid treatment and modied atmosphere packaging (MAP) of ground beef Ground beef was divided into six portions of 800 g each. Tannic acid (0.16 g) was dissolved in 25 ml of sterilized distilled water and pH was adjusted to neutral by 2 M NaOH. Three portions of ground beef (800 g) were separately added with 25 ml of neutralized solution of tannic acid to obtain a nal concentration of 200 mg/kg. Other three portions without addition of tannic acid were used as control and the same volume of sterilized distilled water was added instead. The ground beef (60 g) treated without and with tannic acid was placed on the polystyrene trays (20 12 cm2) and inserted in the nylon/LLDPE bag (30 16 cm) (Asian Foams, Hat Yai, Thailand) with the thickness of 0.08 mm and gas permeability (CO2, N2 and O2: 1.7 1010, 0.1 1010 and 0.4 1010 m3 mm/cm2 s cm Hg at 25 C, 1 atm pressure, respectively) and was packed with a sample/gas ratio of 1:3 (w/v) using a Henkovac type 1000 (Tecnovac, Italy). Two gas mixtures containing 10%O2/20%CO2/70%N2 or 80%O2/20% CO2 with a pressure of 5 kg/ cm2 were lled in the bag. Ground beef treated without and with tannic acid and packed in air were designated as A1(T) and A1(+T) respectively. Those packed under MAP with low oxygen concentration (10%) and added without and with tannic acid were referred to as M1(T) and M1(+T). Those packed under MAP with high oxygen concentration (80%) and treated without and with tannic acid were designated as M2(T) and M2(+T), respectively. All samples were stored at 4 C and taken for chemical and sensory analyses every 3 days for 15 days, except sensory analysis was omitted at day 12. For microbiological analysis and SDSPAGE, samples were taken for analyses at day 0 and 15. 2.4. Chemical analysis 2.4.1. Peroxide value Peroxide value was determined as per the method of Maqsood and Benjakul (2010b). A standard curve was prepared using cumene hydroperoxide at the concentration range of 0.52 ppm. Peroxide value was expressed as lmol of peroxide/kg of sample. 2.4.2. Thiobarbituric acid-reactive substances Thiobarbituric acid-reactive substances (TBARS) were determined as described by Maqsood and Benjakul (2010a). Standard curve was prepared using 1,1,3,3-tetramethoxypropane (MAD) at the concentration ranging from 0 to 10 ppm and TBARS were expressed as mg of MAD equivalents/kg of sample. 2.4.3. Determination of haem iron and non-haem iron contents The haem iron and non-haem iron contents were determined according to the method of Gomez-Basauri and Regenstein (1992) and Schricker, Miller, and Stouffer (1982), respectively as described by Maqsood and Benjakul (2010a). The haem and nonhaem iron content was expressed as mg/100 g sample. 2.4.4. Determination of oxymyoglobin and metmyoglobin contents Oxymyoglobin and metmyoglobin contents of ground beef were determined as described by Carlez, Veciana-Nogues, and Cheftel (1995) with a slight modication. Ground beef (2 g) was trans-

ferred into a 50 ml-polypropylene centrifuge tube and 20 ml of cold 40 mM phosphate buffer (pH 6.8) were added. The mixture was homogenised at a speed of 13,500 rpm for 10 s with a Ultra-Turrax T25 homogeniser (Janke & Kunkel, Staufen, Germany). The homogenate was centrifuged using a RC-5B plus centrifuge (Beckman, JE-AVANTI, Fullerton, CA, USA) at 3000 for 30 min at 4 C. The supernatant was ltered with Whatman No.1 lter paper (Whatman International, Ltd, Maidstone, England) and the volume was brought up to 25 ml with the same phosphate buffer. Absorbance of the clear supernatant was measured at 525, 545, 565 and 572 nm using a UV-1601 spectrophotometer (Shimadzu, Kyoto, Japan). The oxymyoglobin (OxyMb) and metmyoglobin (MetMb) contents were calculated according to Krywicki (1982), using the following equations:

OxyMb % 0:882R1 1:267R2 0:809R3 0:361 100 MetMb % 2:541R1 0:777R2 0:800R3 1:098 100
where R1, R2 and R3 are the absorbance ratio of A572/A525, A565/A525 and A545/A525, respectively. 2.4.5. Colour determination Colour of the ground beef samples was measured using a colourimeter (Hunter Lab., Model colour Flex, Reston, VIRG, USA) with the port size of 0.50 in. The determination of colour was done at six different positions of the sample. Standardization of the instrument was done using a black and white Minolta calibration plate. The values were reported in the CIE colour prole system as L-value (lightness), a-value (redness/greenness), and b-value (yellowness/blueness). 2.4.6. SDSpolyacrylamide gel electrophoresis (SDSPAGE) Fresh ground beef sample obtained at day 0 and all samples stored for 15 days were subjected to SDSPAGE according to the method of Laemmli (1970) as described by Maqsood and Benjakul (2010b). Quantitative analysis of protein band intensity was performed using a Model GS-700 Imaging Densitometer (Bio-Rad Laboratories, Hercules, CA, USA) with Molecular Analyst Software version 1.4 (image analysis system). The intensity of interested protein bands was expressed, relative to that found in fresh sample at day 0. 2.5. Microbiological analysis Fresh ground beef (day 0) and all samples stored for 15 days (25 g) were collected aseptically in a stomacher bag and 10 volumes of sterile saline solution (0.85 g/100 ml) were added. After homogenising in a Stomacher blender (Stomacher M400, Seward Ltd., Worthington, England) for 1 min, a series of 10-fold dilutions was made using normal saline solution (0.85%) for microbiological analyses. Psychrophilic bacterial counts (PBC) were determined by plate count agar (PCA) with the incubation at 7 C for 7 days (Cousin, Jay, & Vasavada, 1992). PBC was expressed as log cfu/g. 2.6. Sensory analysis The sensory evaluation was performed by 30 untrained panelists, who were the graduate students in Food Science and Technology programme with the age of 2533 years and were familiar with beef consumption. The assessment of raw samples was conducted for the colour and odour using a nine-point hedonic scale (Mailgaad, Civille, & Carr, 1999): 1, dislike extremely; 2, dislike very much; 3, dislike moderately; 4, dislike slightly; 5, neither like nor dislike; 6, like slightly; 7, like moderately; 8, like very much; 9, like extremely. The samples were presented to the panelists just

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after opening the packs. The samples were placed in the sterile petridish and served to panelist. 2.7. Statistical analysis All experiments were run in triplicate. The experimental data were subjected to Analysis of Variance (ANOVA) and the differences between means were evaluated by Duncans New Multiple Range Test (Steel & Torrie, 1980). For pair comparison, T-test was used. Data analysis was performed using a SPSS package (SPSS 14.0 for Windows, SPSS Inc., Chicago, IL, USA). 3. Results and discussion 3.1. Effect of tannic acid in combination without and with MAP on the chemical changes of ground beef during refrigerated storage 3.1.1. Changes in peroxide value (PV) and thiobarbituric acid-reactive substances (TBARS) Changes in PV and TBARS of ground beef treated without and with tannic acid (200 mg/kg) and stored in air and under MAP with different gas mixtures during refrigerated storage are shown in Fig. 1a and b, respectively. Gradual increase in PV was found in all samples throughout the storage period of 15 days (P < 0.05), except for the samples without tannic acid treatment, in which the PV decreased markedly after 9 days of storage (P < 0.05). A decrease in PV was most likely due to the decomposition of hydroperoxide, a primary oxidation product, to the secondary lipid oxidation products (Boselli et al., 2005). During the rst 9 days of storage, the samples without tannic acid treatment underwent a higher lipid oxidation as evidenced by the higher PV. The oxidation of those samples more likely proceeded continuously, however the rate of decomposition of primary oxidation products might be greater. The samples stored under MAP with high oxygen concen-

tration without tannic acid treatment [M2(T)] contained the highest PV, especially within the rst 9 days, while samples treated with tannic acid and kept under MAP with low oxygen concentration [M1(+T)] contained the lowest PV (P < 0.05). Tannic acid was very effective in retarding the propagation stage of lipid oxidation in ground beef, even when the high oxygen level was used in packaging. Phenolic compounds including tannic acid could retard the initiation or propagation steps of lipid oxidation reactions by scavenging lipid radicals (Maqsood & Benjakul, 2010a) and forming low-energy antioxidant radicals that do not readily promote oxidation of unsaturated fatty acids. TBARS values of the ground beef without tannic acid treatment stored in air and under MAP increased rapidly with increasing storage time (P < 0.05). Among those samples, that stored under MAP with high oxygen level had the high TBARS values, compared with others (P < 0.05). For samples treated with tannic acid, the gradual increase in TBARS was noticeable during the storage. It was noted that the much lower TBARS values were obtained in samples treated with tannic acid, regardless of packaging conditions. Jayasingh et al. (2002) reported that after 6 days of storage, MAP ground beef samples (80%O2/20%CO2) had higher TBARS than those stored in air. The higher TBARS were found in ground beef stored under MAP with increasing oxygen concentrations after 10 days of storage (OGrady et al., 2000). High oxygen partial pressure found in high oxygen MAP can promote oxidation processes by enabling oxygen to react with muscle components (OGrady et al., 2000). From the results, tannic acid treatment effectively retarded the oxidation as shown by no differences in TBARS among all samples stored under different atmospheres. Lund, Hviid and Skibsted (2007) found that treatment of beef patties with natural antioxidant (rosemary) retarded the TBARS formation under the MAP with high oxygen concentration (80%). Grape seed extract and pine bark extract containing tannic acid signicantly improved the oxidative stability of cooked beef (Ahn, Grun, & Fernando, 2002). Thus, the

A1(+T)

A1(-T)

M1(+T)

M1(-T)

M2(+T)

M2(-T)

PV (mol of peroxide/kg sample)

4 3 2 1 0 0 3 6 9 12 15

Storage time (days)

(a)
14

A1(+T)

A1(-T)

M1(+T)

M1(-T)

M2(+T)

M2(-T)

TBARS (mg MAD/kg sample)

12 10 8 6 4 2 0 0 3 6 9 12 15

Storage time (days)

(b)
Fig. 1. Changes in peroxide value (a) and thiobarbituric acid-reactive substances (b), of ground beef treated without and with tannic acid stored in air and under MAP (10%O2/ 20%CO2/70%N2 or 80%O2/20%CO2) during 15 days of refrigerated storage. Bars represent the standard deviation (n = 3).

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A1(+T)
8

A1(-T)

M1(+T)

M1(-T)

M2(+T)

M2(-T)

Haem iron content (mg/100g sample)

0 0 3 6 9 12 15

Storage time (days)

(a)
A1(+T)
4

A1(-T)

M1(+T)

M1(-T)

M2(+T)

M2(-T)

Non-haem iron content (mg/100g sample)

0 0 3 6 9 12 15

Storage time (days)

(b)
Fig. 2. Changes in haem iron content (a) and non-haem iron content (b) of ground beef treated without and with tannic acid stored in air and under MAP (10%O2/20%CO2/ 70%N2 or 80%O2/20%CO2) during 15 days of refrigerated storage. Bars represent the standard deviation (n = 3).

tannic acid was proved to be an effective antioxidant in the refrigerated ground beef packed under high oxygen concentration. 3.1.2. Changes in haem and non-haem iron content Haem and non-haem iron contents of ground beef treated without and with tannic acid and stored in air and under MAP with different gas compositions during the refrigerated storage are presented in Fig 2a and b, respectively. Haem iron content of all ground beef samples decreased gradually as the storage time increased (P < 0.05). Haem pigment concentration in lamb meat decreased with increasing storage time (Luciano et al., 2009). Decrease in haem iron content with increasing storage time was probably due to the haem breakdown, resulting in the increase in non-haem iron content (Benjakul & Bauer, 2001). The ground beef treated with tannic acid and packed under MAP with high oxygen concentration [M2(+T)] showed the highest haem iron content, compared with other samples, particularly after 6 days of storage (P < 0.05). In general, the lower haem iron content was found in the samples without tannic acid treatment throughout the storage. Thus, tannic acid might reduce the destruction of haem as indicated by the lowered decrease in haem iron content. Non-haem iron content of all the samples increased gradually as the storage time increased (P < 0.05) (Fig 2b). The increase in non-haem iron content of ground beef was coincidental with the decrease in haem iron in the ground beef (Fig. 2a). Deterioration of sub-cellular, organelles, e.g. mitochondria, and the release of cytochrome-C, could also be responsible for the increase in nonhaem iron (Decker & Hultin, 1990). Decker and Hultin (1990) also suggested that haem pigment or other iron-containing proteins are possibly denatured with increasing storage time, resulting in the release of iron. The sample treated with tannic acid had the lower non-haem iron content, compared with those without tannic acid treatment, particularly after 6 days of storage (P < 0.05). The lower

non-haem iron content in the samples treated with tannic acid was more likely owing to the chelating property of tannic acid (Maqsood & Benjakul, 2010a). Tannic acid is able to chelate iron particularly in free form (Maqsood & Benjakul, 2010a). As a consequence, non-haem iron content in tannic acid treated samples was lower than those without tannic acid treatment (P < 0.05). During storage, non-haem iron was released due to the deterioration by microbial spoilage (Maqsood & Benjakul, 2010a). Those free irons might accelerate the oxidation process in the ground beef. After 15 days of storage, samples stored under MAP with high oxygen concentration without tannic acid treatment M2(T) tended to contain higher content of non-haem iron than other samples. Under high oxygen atmosphere, the spoilage microorganisms grew rapidly and destructed haem, resulting in more release of nonhaem iron. Among the transition metals, iron particularly ferrous form is known as the most important pro-oxidant due to its high reactivity (Love, 1983). The ferrous state of iron accelerates lipid oxidation by breaking down hydrogen peroxide and lipid peroxides to reactive free radicals via the Fenton reaction (Fe2+ + H2O2 ? Fe3+ + OH + OH) (Maqsood & Benjakul, 2010a, 2010b). Therefore, the application of tannic acid having metal chelating ability in conjunction with MAP could lower the lipid oxidation mediated by transition metal ions in ground beef. 3.1.3. Changes in oxymyoglobin and metmyoglobin percentage Changes in oxymyoglobin and metmyoglobin percentages in ground beef treated without and with tannic acid and stored in air and under MAP with different gas compositions during refrigerated storage are presented in Fig. 3a and b, respectively. Oxymyoglobin in all samples decreased gradually throughout the storage of 15 days. The decreases were more pronounced within the rst 3 days of storage. The higher decrease in oxymyoglobin was noticeable in samples stored in the air and under MAP with low

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A1(+T)
80

A1(-T)

M1(+T)

M1(-T)

M2(+T)

M2(-T)

Oxymyoglobin (%)

70 60 50 40 30 20 10 0 0 3 6 9 12 15

Storage time (days)

(a)
A1(+T)
100

A1(-T)

M1(+T)

M1(-T)

M2(+T)

M2(-T)

Metmyoglobin (%)

80 60 40 20 0 0 3 6 9 12 15

Storage time (days)

(b)
Fig. 3. Changes in oxymyoglobin (a) and metmyoglobin (b) percentages in ground beef treated without and with tannic acid stored in air and under MAP (10%O2/20%CO2/ 70%N2 or 80%O2/20%CO2) during 15 days of refrigerated storage. Bars represent the standard deviation (n = 3).

oxygen concentration, compared with those kept under MAP with high oxygen concentration. The results suggested that the atmosphere rich in oxygen could retard the oxidation of oxymyoglobin during the extended storage. Tang et al. (2006) reported the decrease in oxymyoglobin in minced beef patties held in air and under MAP (80%O2/20%CO2) with increasing storage time. MAP with high oxygen was effective in maintaining the myoglobin in its oxygenated form. The higher oxymyoglobin in the ground beef stored under MAP with high oxygen was related with the bright red colour of ground beef during the refrigerated storage. High oxygen atmosphere (6080%) maintains the bright red colour of meat (Okayama et al., 1995), in which myoglobin was present in its oxygenated form (OGrady et al., 2000). Metmyoglobin formation of ground beef increased with increasing storage time (P < 0.05). The increase in metmyoglobin was coincidental with the decrease in oxymyoglobin (Fig. 3a). Tang et al. (2006) also reported the increase in metmyoglobin in minced beef patties held in air and under MAP (80%O2 and 20%CO2) as the time of storage increased. The ground beef stored under MAP with high oxygen concentration showed the lower metmyoglobin formation, compared with those kept in air and under MAP with low oxygen concentration (P < 0.05). Ordonez and Ledward (1977) found that increasing oxygen concentration caused a significant decrease in the metmyoglobin (MetMb) formation in pork meat and less than 30% of MetMb of the total surface pigment concentration was obtained after 15 days of storage in 80%O2/20%CO2. The higher metmyoglobin formation in the ground beef kept in air and under MAP with low oxygen concentration correlated well with lower oxymyoglobin percentage in those samples. Metmyoglobin formation is related with browning in the red meats (Bou et al., 2008). At day 15, the samples stored under MAP with low oxygen showed the highest metmyoglobin formation. In general,

the treatment of tannic acid had no effect on oxymyoglobin and metmyoglobin formation. The ground beef packed under MAP with high oxygen concentration (80%) had the lower metmyoglobin and higher oxymyoglobin throughout the refrigerated storage of 15 days. With the treatment using tannic acid, the samples kept under MAP with high oxygen exhibited the lower metmyoglobin in comparison with non-treated counterparts after 15 days of refrigerated storage (P < 0.05). This was coincidental with the higher oxymyoglobin in the former (P < 0.05). 3.1.4. Changes in colour Changes in colour (L, a and b) of the ground beef treated without and with tannic acid and stored in air and under MAP during the refrigerated storage are depicted in Fig. 4. The a (redness) value of all the samples decreased gradually during the storage (P < 0.05), however the rate of decrease varied with the treatments. The decrease in a value was in agreement with the oxidation of myoglobin and accumulation of metmyoglobin (Mancini & Hunt, 2005). Decreasing a values with increasing storage time were reported in beef burgers during frozen storage (Georgantelis, Blekas, Katikou, Ambrosiadis, & Fletouris, 2007). When meat losses its ability to reduce metmyoglobin to oxymyoglobin, the brown colour of metmyoglobin begins to appear on the surface of beef steaks, and a values begins to decrease (Friedrich et al., 2008). The samples kept under MAP with high oxygen concentration showed the highest a value, regardless of tannic acid treatment. Those packed in air and under MAP with low oxygen concentration displayed the lower a value (P < 0.05). This reconrmed the essential role of oxygen in oxygenation of myoglobin in beef. The refrigerated beef steaks stored under MAP with high oxygen concentration (5080%) had the higher a values when compared with those stored under MAP with low oxygen concentration

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A1(+T)
50

A1(-T)

M1(+T)

M1(-T)

M2(+T)

M2(-T)

L*-value

45 40 35 30 0 3 6 9 12 15

Storage time (days)

(a)
A1(+T)
25

A1 (-T)

M1(+T)

M1(-T)

M2(+T)

M2(-T)

a*-value

20 15 10 5 0 3 6 9 12 15

Storage time (days)

(b)
A1(+T)
25 20 15 10 5 0 0 3 6 9 12 15

A1(-T)

M1(+T)

M1(-T)

M2(+T)

M2(-T)

b*-value

Storage time (days)

(c)
Fig. 4. Changes in L (a), a (b) and b (c) values of ground beef treated without and with tannic acid stored in air and under MAP (10%O2/20%CO2/70%N2 or 80%O2/20%CO2) during 15 days of refrigerated storage. Bars represent the standard deviation (n = 3).

(020%) (Zakrys, Hogan, OSullivan, Allen, & Kerry, 2008). The higher redness (a values) in M2(T) and M2(+T) samples was associated with the higher percentage of oxymyoglobin in these samples (Fig. 3a). Therefore, high-oxygen atmospheres (80%O2) promote the oxygenation of pigments, thereby prolonging the time before metmyoglobin is visible on the muscle surface (Jayasingh et al., 2002). The lower redness in the sample stored in air and under low oxygen MAP was attributed to the rapid oxidation of oxymyoglobin into metmyoglobin, resulting in the increased browning in those samples. No changes in L value were found in all samples during the refrigerated storage of 15 days (P > 0.05). Friedrich et al. (2008) reported slight changes in the L values of the minced beef in the oxygen rich atmosphere during chilled storage. The samples stored under MAP with low oxygen concentration showed the higher L values than those stored under MAP with high oxygen concentration, especially after 9 days of storage (P < 0.05). The b-value indicating yellowness of all samples except A1(+T) increased slightly within the rst 3 days of storage (P < 0.05). Thereafter the b-values remained more or less constant (P > 0.05). Among all samples, M2(T) showed the higher b-values, suggesting the higher yellowness of the sample. Under the high oxygen atmosphere in the absence of tannic acid, lipid oxidation could take place (Fig. 1b). Lipid oxidation products, especially carbonyl compounds, could undergo Maillard reaction in the presence of amino groups in beef. The loss in redness (a) and the increase in yellowness (b) correlated well with the formation of metmyoglobin along with the de-

crease in oxymyoglobin in the beef (Fig. 3). The treatment of ground beef with tannic acid could lower the b-values of samples kept under MAP with high oxygen. This suggested the ability of tannic acid in inhibiting lipid oxidation, where yellowness could be developed in the beef. Thus, the high oxygen MAP could maintain the redness up to 12 days of refrigerated storage, irrespective of tannic acid treatment. 3.1.5. Changes in protein patterns Protein patterns of fresh ground beef (day 0) and ground beef treated without and with tannic acid stored in air and under MAP for 15 days are shown in Fig. 5. All samples contained myosin heavy chain (MHC) and actin as the major proteins. Additionally, all samples contained tropomyosin, troponin-T and troponin-C. MHC and actin underwent degradation after 15 days of storage at 4 C at different degrees, depending upon the treatments. Under MAP with high oxygen concentration, band intensity of MHC of M2(T) sample decreased by 29.76%, whereas that of M2(+T) sample decreased by 25.93% after 15 days of storage. The results suggested that tannic acid could lower the degradation caused by aerobic microorganisms. On the other hand, samples stored under MAP with low oxygen concentration had the lower degradation, particularly with tannic acid treatment. For M1(+T) samples, MHC was degraded by 8.01%. This was mainly due to the synergistic effect of low oxygen MAP and tannic acid on retardation of the growth of microorganisms with proteolytic activity. Apart from MHC, actin was also degraded after 15 days of storage when com-

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A1 M1 M2

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F kDa 200 116 97 66 55 45 36 29 24 20 M +T

MHC (200 kDa)

Actin (44 kDa), Tropomyosin (38 kDa) Troponin-T (32 kDa) Troponin-C (30 kDa)

-T

+T

-T

+T

-T

Fig. 5. SDSPAGE patterns of muscle protein from fresh ground beef (F) and sample treated without and with tannic acid stored in air (A1) and under MAP with 10%O2/ 20%CO2/70%N2 (M1) or 80%O2/20%CO2 (M2) after 15 days of refrigerated storage. M: marker; +T: with tannic acid treatment; T: without tannic acid treatment.

observed (P < 0.05). Under the same packaging atmosphere, the samples treated with tannic acid had the lower PBC, compared with non-treated counterparts (P < 0.05). PBC of ground beef without tannic acid treatment and stored under MAP with high oxygen concentration was similar to the sample kept in air [A1(T)] (P > 0.05). Viana, Gomide, and Vanetti (2005) found the pronounced growth of psychrophilic bacteria Pseudomonas in beef (loin) samples stored under MAP (100%O2) than found in samples kept in air. The result suggested that the tannic acid was effective in retarding the microbial growth even in MAP with high oxygen. Tannic acid could act as antimicrobial agent (Zaidi-Yahiaoui, Zaidi, & Ait Bessai, 2008). Grape seed extract and pine bark extract containing tannin at a level of 1% rapidly reduced the numbers of Escherichia coli O157:H7 in cooked beef within the rst 3 days of storage at 4 C (Ahn, Grun, & Mustapha, 2007). Taguri, Tanaka, and Kouno (2004) reported the antibacterial activity of hydrolysable tannins against Salmonella aureus and Staphylococcus. Thus, the use of tannic acid in combination with high and low oxygen MAP exhibited the inhibitory effect on the growth of psychrophilic bacteria which were dominant at refrigerated storage temperature. 3.3. Effect of tannic acid in combination without and with MAP on the sensorial changes of ground beef during refrigerated storage Scores of colour and odour likeness of ground beef treated without and with tannic acid and stored in air and under MAP are shown in Table. 1. The likeness scores for both attributes continuously decreased with increasing storage time (P < 0.05). During the storage, the highest colour likeness scores were obtained in ground beef stored under high oxygen MAP, while the sample stored in air and under low oxygen MAP had the lower scores in descending order. This was mainly due to the higher percentage of oxymyoglobin present in the ground beef stored under high oxygen MAP (Fig. 3a). Under the same packaging conditions, tannic acid treatment had no impact on colour likeness of ground beef. However, a non-signicantly lower score of colour likeness was found in the samples treated with tannic acid. This can be attribute to the fact that oxidation of oxymyoglobin might take place. Tannic acid might have the interaction with the globin protein chain, thereby altering the tertiary structure and potentially opening the haem cleft. This change could destabilize the haem, resulting in enhanced oxidation of oxymyoglobin along with the formation of metmyoglobin (Hayes et al., 2009). The samples stored in air and under low oxygen MAP received the lowest colour scores and were rejected by the panelists from the day 9 onwards of refrigerated storage. These samples turned to brownish rapidly, which correlated well with the high metmyoglobin formation (Fig. 3b) and lowered redness (a) values (Fig. 4a) in those samples. Carpenter, Cornforth, and Whittier (2001) stated that panelist description of brown beef is associated with a decrease in redness (a) values. Similar nding
M1 (-T)
a c d

pared with that found in the fresh sample. Actin was degraded by 25.9% in samples stored under MAP with high oxygen concentration without tannic acid treatment M2(T) and by 1.85% for the samples kept under MAP with low oxygen concentration and treated with tannic acid, M1(+T). The higher degradation of MHC and actin in sample kept under MAP with high oxygen without tannic acid treatment, M2(T) might be due to the higher degree of proteolysis associated with the pronounced microbial growth. Moreover, protein oxidation (Park, Xiong, & Alderton, 2007) and endogenous and microbial proteinases such as cathepsin and calpain can also result in protein degradation (Masniyom, Benjakul, & Visessanguan, 2004). Lund et al. (2007) reported that MHC was found to be slightly less intense in the high oxygen stored beef due to cross linking caused by protein oxidation. Troponin and tropomyosin were also degraded to a higher extent when the samples were stored under MAP with high oxygen concentration. Thus, the tannic acid could prevent protein degradation of refrigerated ground beef to some degree and packaging atmosphere played more important role in protein degradation in beef during the refrigerated storage. 3.2. Effect of tannic acid in combination without and with MAP on the microbiological changes of ground beef during refrigerated storage Psychrophilic bacterial count (PBC) of ground beef treated without and with tannic acid and stored in air and under MAP at the day 0 and 15 of refrigerated storage is shown in Fig. 6. PBC of all samples increased from an initial value of 2.69 101 cfu/g to 7.69.96 108 cfu/g at the end of storage (day 15) (P < 0.05). When MAP with low oxygen concentration was used, the lower PBC was
A1 (+T) A1 (-T) M1(+T)

M2 (+T)

M2 (-T)
a b c

Psychrophilic Bacterial Count (CFU/g)

12 10 8 6 4 2 0 0

15

Storage time (days)

Fig. 6. Changes in psychrophilic bacterial count (PBC) in fresh ground beef and sample treated without and with tannic acid stored in air and under MAP (10%O2/20%CO2/ 70%N2 or 80%O2/20%CO2) after 15 days of refrigerated storage. Bars represent the standard deviation (n = 3). Different letters on the bar within the same storage time denote the signicant differences (P < 0.05).

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Table 1 Likeness scores of ground beef without and with tannic acid treatment and stored in air and under MAP (10%O2/20%CO2/70%N2 or 80%O2/20%CO2) during 15 days of refrigerated storage. Attributes Colour Samples A1(+T) A1(T) M1(+T) M1(T) M2(+T) M2(T) A1(+T) A1(T) M1(+T) M1(T) M2(+T) M2(T) Day 0 9.20 0.34Aa 9.40 0.45Aa 9.30 0.49Aa 9.10 0.29Aa 9.40 0.48Aa 9.70 0.42Aa 10.0 0.38Aa 9.90 0.49Aa 9.80 0.57Aa 9.90 0.22Aa 9.80 0.38Aa 9.80 0.43Aa Day 3 6.40 0.44Bc 6.70 0.36Bc 6.20 0.51Bc 6.30 0.48Bc 8.40 0.44Bb 8.50 0.39Bb 9.10 0.41Ba 9.30 0.48Ba 8.80 0.33Bab 8.70 0.30Bab 8.90 0.39Bab 9.00 0.37Ba Day 9 5.10 0.48Ce 5.60 0.37Ce 4.20 0.28Ce 4.60 0.46Ce 6.90 0.42Cad 7.10 0.43Ccd 7.70 0.45Cc 7.20 0.39Cbc 6.90 0.42Cab 6.60 0.38Ca 6.80 0.41Ca 6.50 0.39Ca Day 15 3.40 0.41Dd 3.60 0.64Dd 3.00 0.37Dd 3.20 0.30Dd 5.30 0.38Dab 5.80 0.31Da 5.10 0.33Db 4.50 0.29Dc 5.00 0.28Dac 4.10 0.39Dc 5.30 0.33Dab 4.60 0.39Dc

Odour

Values with the same capital superscript in the same row do not differ signicantly (P > 0.05). Values with same superscript in the same column within same attribute do not differ signicantly (P > 0.05).

was also reported by Jayasingh et al. (2002) who found that ground beef packed in high oxygen MAP maintained a bright red colour for 10 days. John et al. (2005) reported that beef steaks packed under high oxygen atmosphere had a desirable red colour on day 7 of storage, but some browning was evident by day 14 and steaks were completely brown and unappealing by day 21. The likeness scores for odour of all samples also decreased with the increasing storage time (P < 0.05). The samples without tannic acid treatment, regardless of the packaging conditions, received the lower odour scores, comparable with those treated with tannic acid (P < 0.05). This was attributed to higher TBARS formation (Fig. 1b) and higher PBC (Fig. 6) in those samples without tannic acid treatment. The panelists detected off-odour mainly in the sample stored in high oxygen MAP, which was mainly due to the oxidative rancidity. St. Angelo, Crippen, Dupuy, and James (1990) reported that lipid oxidation causes the deterioration of avour in beef stored under atmospheres enriched in O2 and this deterioration can be closely related to TBARS. The odour scores were higher for all ground beef samples treated with tannic acid under all packaging conditions (P < 0.05). Tannic acid could lower the oxidation of lipids in muscle foods (Maqsood & Benjakul, 2010a) and retard the microbial growth (Zaidi-Yahiaoui et al., 2008). Thus, the development of rancidity and off-odour in samples without tannic acid treatment was in accordance with the increased PBC (Fig. 6) and increased TBARS values (Fig. 1b). 4. Conclusion Tannic acid at a level of 200 ppm in combination with high oxygen concentration MAP (80%O2 and 20%CO2) could be effective in maintaining the bright red colour and retarding the lipid oxidation and microbial growth in ground beef up to 15 days of refrigerated storage. Nevertheless, the protein degradation still proceeded to some degree. Therefore, tannic acid can be a natural additive with multiple preservative functions in meat and meat products. Acknowledgements The authors would like to express their sincere thanks to Graduate School of Prince of Songkla University and The Commission of Higher education, Ministry of Education, Thailand for the nancial support. References
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