Effect of sage (Salvia officinalis) on the oxidative stability of Chinese-style sausage during refrigerated storage

Meat Science 95 (2013) 145–150

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Effect of sage (Salvia officinalis) on the oxidative stability of Chinese-style sausage during refrigerated storage L. Zhang a, Y.H. Lin b, X.J. Leng a, M. Huang a,⁎, G.H. Zhou a a b

Key Laboratory of Meat Processing and Quality Control, Ministry of Education, College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China Hormel China Idea & Innovation Center, 30 Nanda Road, Shanghai 200436, China

a r t i c l e

i n f o

Article history: Received 8 December 2012 Received in revised form 8 May 2013 Accepted 8 May 2013 Keywords: Sage Chinese-style sausage Protein oxidation Lipid oxidation Color Texture

a b s t r a c t The objective of this study was to assess the effect of sage, at levels of 0.05%, 0.1% and 0.15% (w/w), on the oxidative stability of Chinese-style sausage stored at 4 °C for 21 days. The results showed that inclusion of sage in sausages resulted in lower L* values (P b 0.05) and higher a* values (P b 0.05) compared to the control. During refrigerated storage, sausages containing sage showed significantly retarded increases in TBARS values, and in the formation of protein carbonyls (P b 0.05), but showed accelerated losses of thiol groups (P b 0.05). Addition of sage to the sausages at levels of 0.1% and 0.15% reduced textural deterioration during refrigerated storage (P b 0.05). Sage used in this study had no negative effects on the sensory properties of sausages. © 2013 Elsevier Ltd. All rights reserved.

1. Introduction Chinese-style sausage is famous for its unique cured meat flavor that comes from natural fermentation. Since natural maturation is time consuming, the method of choice for commercial manufacturing has been to use a high temperature drying procedure in order to minimize the process time. However, high temperature can accelerate lipid oxidation, which is a major cause of off-flavor in meat and meat products, and can result in a product that is unacceptable for human consumption. It is known that protein oxidation occurs simultaneously with lipid oxidation in meat systems, and protein oxidation also has detrimental effects on meat quality in terms of texture traits, color, aroma, flavor, water-holding capacity and biological functionality (Estévez, 2011). Hence, it is necessary to control and minimize the extent of lipid and protein oxidation in meat products. Addition of antioxidants is one of the accepted methods to retard lipid oxidation in processed meat products. Synthetic antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tert-butylhydroquinone (TBHQ) are widely used in industrial processing to improve the quality and to prolong the shelf life of meat product. However, the use of such compounds has been related to potential safety problems, which has restricted its application in the food industry (Ito et al., 1986).

⁎ Corresponding author. Tel.: +86 25 84396808; fax: +86 25 84395939. E-mail address: [email protected] (M. Huang). 0309-1740/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.meatsci.2013.05.005

Consequently, the use of naturally-occurring substances is becoming increasingly popular as alternatives to the synthetic additives. Of particular interest, extracts derived from fruits (Du, Li, Ma, & Liang, 2009), vegetables (Ismail, Marjan, & Foong, 2004), cereals (Yu, Perret, Davy, Wilson, & Melby, 2002), herbs (Dorman, Peltoketo, Hiltunen, & Tikkanen, 2003) and spices (Su et al., 2007) have been reported for their high antioxidant activities, presumably due to their abundant contents of phenolic compounds. Sage (Salvia officinalis) is a variety of aromatic herb which has been planted widely throughout much of the world. It is not only used as raw material in the pharmaceutical and cosmetic industries but also used to improve flavors of foods (Tepe, Sokmen, Akpulat, & Sokmen, 2006). Sage has been reported to have excellent activities in scavenging radicals, reducing metal ions and inhibiting lipid peroxidation (Dorman et al., 2003; Grzegorczyk, Matkowski, & Wysokińska, 2007). The phenolic compounds, such as carnosol, carnosic acid and rosmarinic acid, in the plant may account for the antioxidant activity of sage. Some researchers have reported that sage, or sage extracts, can effectively retard lipid oxidation of muscle foods (Fasseas, Mountzouris, Tarantilis, Polissiou, & Zervas, 2007; McCarthy, Kerry, Kerry, Lynch, & Buckley, 2001a; Tanabe, Yoshida, & Tomita, 2002). However, to our knowledge, there is no information available regarding the effectiveness of the inclusion of sage to inhibit lipid and protein oxidation in Chinese-style sausage. Consequently, the objective of the present study was to investigate the effects of addition of different levels of sage to Chinese-style sausage on retarding lipid and protein oxidation, as well as determining its impact on quality deterioration during refrigerated storage.

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2. Materials and methods

standard for malondialdehyde (MDA) and the amounts of TBARS were expressed as milligrams of MDA per 100 g of sample.

2.1. Sage preparation Ground sage (S. officinalis) leaves (approximate 0.5 mm at length), of a brown and green appearance, was purchased from Hela Schwarz Food., Ltd. (Jinan, China). It was dried in an oven at 60 °C for 8 h prior to use. 2.2. Sausage preparation Fresh boneless chilled pork hams and fresh chilled pork backfat were obtained from a commercial meat processing company (Sushi Group, Jiangsu, China) in which pigs were slaughtered according to the requirements of National Standards of China “Pig Slaughter and Quarantine Regulations”. Collagen casings were obtained from Shenguan Protein Casing Co. Ltd, Guangxi, China. The hams were trimmed free of connective tissue and subcutaneous fat and then ground (TC-12E, Sirman, Italy) through a 10 mm plate. The backfat was manually sliced into cubes (approximate 0.5 cm3). The ground meat was blended thoroughly with non-meat ingredients, including 8% sugar (on the base of raw meat weight), 1.6% salt, 0.5% monosodium glutamate, 0.15% five-spice powder, and 2% white wine. Depending on the experimental batch, different levels of ground sage were added to the formula: 0% (C), 0.05% (S5), 0.10% (S10), and 0.15% (S15). Then, diced pork backfat (20%) was added to the mixture and mixed thoroughly prior to stuffing (VF608, Stuffer, Germany) into collagen casings. Raw sausages were linked into approximate 10 cm lengths manually and dried in a pre-heated oven (KBF 115-pgm, Binder, Germany) at 55 °C for 48 h. The sausages were then taken and cooled to room temperature, vacuum-packaged (DC-800, Promarks Inc., USA) and then stored at 4 °C. Each treatment of packaged sausage was randomly taken for analyses at days 0, 7, 14 and 21. The entire sausage processing procedure was replicated three times on different occasions. 2.3. Instrumental color measurement Sausage samples were first ground with a grinder (GM200, Retsch, Germany) and then placed in a circular template enabling a thickness of 1 cm. Color of the samples was then measured using a colorimeter (CR-400, Chroma Meter, Japan). The instrument was calibrated by a standard plate (Y = 94.0, x = 0.3156, y = 0.3321) prior to measuring. The values were reported in the CIE color profile system as L value (L* = 0 darkness, L* = 100 lightness), a value (+60 = red, −60 = green), and b value (+60 = yellow, −60 = blue). 2.4. Lipid oxidation: determination of thiobarbituric acid-reactive substances (TBARS) value The TBARS value was measured by the method of Yoshikawa et al. (1979) with slight modifications. Briefly, 1 g of minced sausage (GM200, Retsch, Germany) was homogenized with 10 mL distilled water for two periods of 30 s at speed of 12,000 rpm. Then, 0.2 mL of the sausage homogenate was placed in a test tube and 0.2 mL of 8.1% sodium dodecylsulphate, 1.5 mL of 20% acetate buffer pH 3.5), 1.5 mL of 0.8% (w/v) thiobarbituric acid (TBA) solution and 0.6 mL distilled water were added. The mixtures were incubated at 95 °C in a water bath (ZKSY-600, Keer, China) for 1 h to develop a pink color. After cooling the tubes in running tap water, 1 mL distilled water and 4 mL of n-butanol-pyridine solution (butanol/pyridine:15/1) were added and then the test tubes were vortexed (MX-F, SCILOGEX, USA) for 30 s. After centrifuging for 10 min at 4000 rpm, the color of the butanol-pyridine layer was determined at 532 nm in a spectrophotometer (SpectraMax M2 e, Molecular Devices Corporation, USA). A standard curve was prepared using 1,1,3,3-tetraethoxypropane (TEP) as a

2.5. Protein oxidation 2.5.1. Determination of protein carbonyls Protein carbonyl content was evaluated according to the method of Rodríguez-Carpena, Morcuende, and Estévez (2011). Carbonyl groups were reacted with 2,4-dinitrophenylhydrazine (DNPH) to develop protein hydrazones, which were detected by measuring the absorbance at 370 nm in a spectrophotometer (UV-2450, SHIMADZU, Japan). Protein concentrations were calculated by measuring the absorbance at 280 nm, using BSA as standard. The content of carbonyl groups was expressed as nmol carbonyl/mg protein using an extinction coefficient of 21.0 mM −1 × cm −1.

2.5.2. Determination of protein thiol groups Protein thiol groups were measured using 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) according to the method described by Lund, Hviid, and Skibsted (2007). The protein thiol concentration was calculated using a standard curve prepared from a 200 μM L-cysteine stock solution. Protein concentrations were determined using a BSA standard curve. The results were expressed as nmol thiol/mg protein.

2.6. Texture profile analysis The texture profile analysis of the sausages was determined according to the procedures of Mercadante, Capitani, Decker, and Castro (2010) with a slight modification. Firstly, the sausage samples (about 2 cm in diameter) were removed from casings and cut into 2.5 cm length section, and then subjected twice to compression tests with a Texture Analyzer (TA-XT Plus, Stable Microsystems, UK). Each section was compressed to 50% of its original height with a cylindrical probe of 5 cm diameter at the speed of 1 mm/s. The textural properties of sausage were expressed as hardness, springiness, cohesiveness, gumminess and chewiness.

2.7. Sensory evaluation Sensory analysis was performed on day 0 according to the method of Maqsood, Benjakul, and Balange (2012) with modifications. The sausage samples were placed in polythene bags and boiled in water for 15 min. The samples were drained and allowed to cool to room temperature. They were then sliced into pieces 0.25–0.30 cm thick and served to 15 experienced panelists who were familiar with the sensory profiles of Chinese-style sausage. Panelists were asked to evaluate the sensory attributes of sausage samples, including color, flavor, texture and overall-acceptability. A 9-point hedonic scale was used to determine these four attributes: 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.

2.8. Statistical analyses The entire experiment was carried out in triplicate for each treatment. The effects of different treatments, storage times and interaction between treatment and storage time on the variables were evaluated by the analyses of variance (ANOVA) procedure using the linear model package program of SAS 8.0 software, and differences among the individual means were compared using the Fisher's LSD test. Differences were considered significant at P b 0.05.

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3. Results and discussion

3.2. Lipid oxidation determined by TBARS value

3.1. Instrumental color measurement

The TBARS values of sausages significantly (P b 0.05) increased during 21 days of storage in all sausage samples (Table 1), indicating that lipid oxidation was occurring during refrigerated storage. Sage-treated sausages showed significantly lower TBARS values (P b 0.05) compared to the control on each of the sampling days (Table 2). Significant differences (P b 0.05) were also found among the three sage treatments; the greater the level of addition, the lower was the TBARS value of the sample. The results showed that addition of sage retarded lipid oxidation in Chinese-style sausage. A similar positive effect of sage or sage extracts in meat systems has been reported by others. Sage reduced the TBARS values of raw and cooked pork patties (McCarthy et al., 2001b) as well as in fresh and frozen pork patties (McCarthy et al., 2001a) during storage at 4 °C. Addition of 3% (w/w) essential oil from sage efficiently inhibited lipid oxidation in raw pork and in cooked bovine meat (Fasseas et al., 2007). Estévez, Ventanas, and Cava (2006) found that 0.1% essential oil from sage was more effective in inhibiting the generation of MDA in liver pâté during 90 days of storage at 4 °C than 0.02% BHT. Our findings are in agreement with these previous results, demonstrating that sage can act as a natural antioxidant in meat products. It appears to be the phenolic compounds in sage that contribute to its antioxidant activity through reductive, free radical-scavenging and lipid oxidation inhibiting activities (Dorman et al., 2003; Grzegorczyk et al., 2007). The compounds in sage showing the greatest antioxidant activity have been identified as carnosol, rosmarinic acid, and carnosic acid, followed by caffeic acid, rosmanol, rosmadial, genkwanin, and cirsimaritin (Cuvelier, Richard, & Berset, 1996). Purified carnosol and carnosic acid have been reported to be powerful inhibitors of lipid peroxidation in microsomal and liposomal systems and good scavengers of peroxyl radicals (CCl3O2) and hydroxyl radicals (•OH) (Aruoma, Halliwell, Aeschbach, & Löligers, 1992). Rosmarinic acid and its oligomeric derivatives are excellent DPPH (1,1-Diphenyl-2-picrylhydrazyl) radical scavengers and super oxide anion radical (O 2−) scavengers (Lu & Foo, 2001). Phenolic compounds serve as antioxidants by donating hydrogen atoms to radical species, and in effect being oxidized to a phenoxyl radical themselves. Phenoxyl radicals are rather stable radicals that prevent further initiation of lipid or protein oxidation, but may be oxidized again to yield quinone structures, or in high concentrations, enter into termination reactions with other phenoxyl radicals to form dimers and polymerized phenols of higher degrees (Jongberg, Tørngren, Gunvig, Skibsted, & Lund, 2013). It should be noted that the TBARS values of samples for all treatments in this study were comparatively higher than values reported

The L* values of the sausages significantly decreased (P b 0.05) during the refrigerated storage (Table 1). The sausages containing sage showed significantly lower L* values (P b 0.05) compared to the control treatment. The greater the amount of sage added to the sausage, the lower was the L* value of the samples. The lower L* values of the sausages with sage may have resulted from the presence of pigmented materials which comes from the plant itself, such as chlorophylls (Li-wen et al., 2012). The polyphenols contained in sage are likely to be oxidized to corresponding quinines by polyphenol oxidases, which are widespread in plant materials; those quinines may condense to form darkened compounds, which will result in an intense color of sausage (Liu, Tsau, Lin, Jan, & Tan, 2009). At day 0, the control sausage visually appeared more red, but this was not significantly different (P > 0.05) from the sausages containing sage when measured by a* values (Table 2). Where sage had been added to the formulations, the a* values were significantly higher (P b 0.05) in comparison with control at days 7, 14 and 21. The samples treated with sage maintained stable a* values, while the a* value of control treatment significantly decreased (P b 0.05) during the refrigerated storage. It was observed that sage showed a protective effect on the deterioration of color during refrigerated storage. McCarthy, Kerry, Kerry, Lynch, and Buckley (2001b) reported that patties manufactured from fresh pork containing 0.05% sage showed significantly higher a* values during 6 days of chilled storage. However, in their study, the a* values of sage-treated patties suffered a significant decline during storage. They found that the type of package (patties overwrapped in an oxygen permeable cling film) caused the patties to be more sensitive to oxidation, which may have accounted for the decrease in a* values. The redness of meat products results from the presence of the heme proteins, hemoglobin and myoglobin. These proteins are red when they exist in the reduced forms and brown in the oxidized forms (Sabeena Farvin, Grejsen, & Jacobsen, 2012). Lipid oxidation also contributes to the deterioration of redness (Faustman, Sun, Mancini, & Suman, 2010). Lara, Gutierrez, Timón, and Andrés (2011) linked the protective effect of natural antioxidants on color deterioration in meat products to the antioxidant activities of phenolic compounds in plants. The phenolic compounds have been shown to inhibit lipid (Liu et al., 2009) and protein oxidation (Ganhão, Morcuende, & Estévez, 2010; Jongberg, Skov, Tørngren, Skibsted, & Lund, 2011) in muscle foods. Consequently, it is likely that sage exerts its protective effect on color stability of meat products through provision of phenolics.

Table 1 Effects of sage and storage time on measured variables (P values of A NOVA). SEMA

Treatment

L* a* TBA. Carb. Thio. Hard. Spri. Cohe. Gum. Chew.

C

S5

S10

S15

40.0a 7.6b 8.6a 2.5a 56.5a 107.3a 0.68 0.37 38.7a 26.6a

37.0b 8.0a 3.4b 2.3b 56.0ab 98.8b 0.69 0.37 36.5a 25.2a

36.3b 8.0a 2.0c 2.2c 55.4b 94.4c 0.68 0.37 32.3b 22.0b

35.1c 8.1a 1.5d 2.2c 54.1c 87.4d 0.68 0.38 31.8b 21.7b

0.36 0.03 0.37 0.02 0.82 1.22 0.002 0.004 0.73 0.53

Storage time 0d

7d

14d

21d

38.3a 8.0a 3.0d 2.2d 64.4a 96.3b 0.67c 0.35b 30.7c 20.8c

38.8a 7.8b 3.6c 2.3c 57.5b 95.3b 0.68b 0.38a 35.5ab 24.1b

37.4b 7.9ab 4.3b 2.4b 52.1c 95.7b 0.69ab 0.38a 34.9b 23.8b

33.9c 7.9ab 4.6a 2.5a 47.9d 100.6a 0.69a 0.39a 38.2a 26.8a

SEMB

P value T1

T2

T1 × T2

0.36 0.03 0.37 0.02 0.82 1.22 0.002 0.004 0.73 0.53

b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 0.82 0.69 b0.001 b0.001

b0.001 0.09 b0.001 b0.001 b0.001 0.048 b0.001 0.002 b0.001 b0.001

0.30 b0.001 b0.001 0.77 b0.001 0.02 0.82 0.99 0.03 0.26

T1: Treatment; T2: Storage Time; T1 × T2: Treatment × Time; TBA.: TBARS Value; Carb.: Carbonyl Content; Thio.: Thiol Groups; Hard.: Hardness; Spri.: Springiness; Cohe.: Cohesiveness; Gum.: Gumminess; Chew.: Chewiness. C: control sausage without sage; S5: sausage treated with 0.05% sage; S10: sausage treated with 0.1% sage; S15: sausage treated with 0.15% sage. Within each treatment or within each storage time, means with different superscripts (a–c) in each row differ significantly (P b 0.05). A Standard error of the mean within the treatments (n = 48). B Standard error of the mean within the storage days (n = 48).

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Table 2 Effect of sage on the color, lipid oxidation and protein oxidation of Chinese-style sausage during refrigerated storage. Storage time

C L*

a*

TBARS valueA

Carbonyls contentB

Thiol groupsC

Day0 Day7 Day14 Day21 SEME Day0 Day7 Day14 Day21 SEM Day0 Day7 Day14 Day21 SEM Day0 Day7 Day14 Day21 SEM Day0 Day7 Day14 Day21 SEM

SEMD

Treatment S5 axy

41.2 42.7ax 40.4ay 35.8az 0.71 8.1x 7.6by 7.4by 7.4by 0.08 7.2az 8.7ay 8.9axy 9.7ax 0.27 2.3az 2.4az 2.6ay 2.7ax 0.04 61.6cx 58.9y 55.1az 50.2aw 1.16

S10 bx

38.7 38.5bx 37.6bx 33.9by 0.52 8.0 7.8ab 8.0a 8.0a 0.05 2.1bw 3.0bz 3.9by 4.4bx 0.23 2.2aby 2.3abxy 2.4bx 2.5bx 0.04 65.6abx 56.5y 52.9abz 48.9abw 1.61

S15 bcx

37.5 38.0bx 36.4bcx 33.2bcy 0.54 7.9 7.9a 8.0a 8.0a 0.04 1.5cy 1.6cy 2.5cx 2.6cx 0.14 2.1bz 2.2byz 2.3bxy 2.4bx 0.03 66.7ax 57.8y 50.4bz 46.8bw 1.98

36.3cx 36.4bx 35.2cx 32.7cy 0.44 7.9 8.0a 8.1a 8.2a 0.06 1.2cy 1.2cy 1.7dx 1.8dx 0.10 2.1bz 2.2by 2.3bx 2.4bx 0.03 63.7bcx 57.1y 49.8bz 45.8bw 1.80

0.51 0.67 0.57 0.36 – 0.04 0.05 0.07 0.09 – 0.64 0.78 0.72 0.79 – 0.03 0.03 0.03 0.04 – 0.54 0.34 0.67 0.54 –

C: control sausage without sage; S5: sausage treated with 0.05% sage; S10: sausage treated with 0.1% sage; S15: sausage treated with 0.15% sage. Means with different superscripts (a–c) within a row of the same storage day are significantly different (P b 0.05). Means with different superscripts (x–w) within a column of the same batch are significantly different (P b 0.05). A Expressed as mg MDA/100 g samples nmol hydrazones/mg protein. B Expressed as nmol Carbonyls/mg protein. C Expressed as nmol Thiol Groups/mg protein. D Standard error of the mean within the same storage day (n = 12). E Standard error of the mean within the same treatment (n = 12).

by others. According to Ganhão, Estévez, and Morcuende (2011), there are two possible reasons that may account for this phenomenon. Firstly, in the method we used to measure TBARS value, the TBA reagent was added directly to the meat sample. It is known that other substances in the meat matrix such as ketones, steroids ketones, acids, fat, carbohydrates, imides, amide, amino acids, pyridine, pyrimidine and vitamins may be rapidly produced during heating procedure and all of these substances can react with TBA, thus contributing to an increase of TBARS values. Secondly, the large amount of sugars (8%, on the basis of raw meat weight) added to the sausage, may decompose to a variety of aldehydes during processing and storage, which may also react with TBA and intensify the TBARS values. 3.3. Protein oxidation Protein oxidation is known to have a number of negative effects on attributes of meat quality including texture, color, aroma, flavor, water-holding capacity and biological functionality (Estévez, 2011). The oxidative deterioration of proteins has been mainly evaluated by determining the formation of protein carbonyls as well as the loss of thiol groups. 3.3.1. Determination of protein carbonyls The amount of protein carbonyls increased significantly (P b 0.05) during refrigerated storage (Table 1) and the control batch showed the highest values on each of the sampling days (Table 2). Compared to the control treatment, addition of 0.1% and 0.15% sage significantly inhibited (P b 0.05) the formation of protein carbonyls in sausages throughout the storage, while addition of 0.05% sage was only effective at days 14 and 21.

These results demonstrated the ability of sage to inhibit protein oxidation in the sausage. Similarly, Estévez et al. (2006) reported that addition of 0.1% sage extract significantly inhibited the increase of protein carbonyls in porcine liver pâté during refrigerated storage. This protecting effect of sage, against protein oxidation, can be attributed to the phenolic compounds in the plant. Carnosol, which was one of the most active phenolic compounds in sage, has been shown to inhibit Cu2+ induced LDL (low-density lipoprotein) oxidation and lipid free radicals in mouse liver microsomes (Zeng et al., 2001). Phenolic compounds in sage might scavenge ROS (reactive oxygen species) and inhibit protein degradation in muscle foods during cooking or storage (Estévez et al., 2006). In addition, phenolic compounds prevent protein oxidation by acting as metal chelators and radical scavengers (Estévez & Heinonen, 2010), by retarding lipid oxidative reactions, and by binding to the proteins to form complexes (Estévez, Ventanas, & Cava, 2005). Recently, Jongberg et al. (2013) suggested that the covalent binding of phenolic compounds to the meat proteins may act as a protective mechanism against protein carbonyl formation, by stabilizing and accumulating protein-bound phenoxyl radicals and thereby decelerating the radical-mediated production of protein carbonyls. It is known that the varieties of phenolic compounds in rosemary are similar to those in sage. However, the reported effects of rosemary on protein oxidation are not all consistent with our findings. Rosemary extract was found to be ineffective in inhibiting protein oxidation in beef patties (Lund, Hviid, et al., 2007). Estévez and Cava (2006) also found that addition of rosemary extract showed antioxidant effects on protein oxidation in frankfurters prepared from Iberian pigs, while displaying a pro-oxidant effect on protein oxidation in frankfurters prepared from white pigs. These conflicting results may be ascribed to the differences in the food matrix including proteins types, the content of α-tocopherol, the presence of metal ions and how the oxidation is initiated (Estévez & Cava, 2006; Jia, Kong, Liu, Diao, & Xia, 2012; Lund, Hviid, et al., 2007). Moreover, plant phenolics are known as redox-active compounds that display antioxidant and pro-oxidant actions depending on their concentrations and the presence of other redox compounds (Sabeena Farvin et al., 2012). The exact mechanisms between phenolic compounds and the formation of proteins carbonyls are not well known. Accordingly, further research is required to shed light on the precise interactions between phenolic compounds and protein oxidation. 3.3.2. Determination of protein thiol groups The levels of protein thiol groups rapidly decreased during 21 days of refrigerated storage (Table 1). At day 0, sausages containing 0.05% and 0.1% sage displayed significantly higher values (P b 0.05) compared to the control (Table 2). No differences were found among sausages in any treatments at day 7. However, at days 14 and 21, the control sample showed the highest values, which were significantly higher (P b 0.05) than the sausages containing additions of 0.1% and 0.15% sage. Protein oxidation is also associated with the loss of thiol groups. The oxidation of cysteine thiol groups can contribute to the formation of intermolecular disulfide bridges, which is responsible for the decrease of thiol groups (Lara et al., 2011). Thus, determination of thiol groups is one of the important methods widely used to measure the extent of protein oxidation. The content of thiol groups of the control on day 0 was lower than that of any of the batches treated with sage, indicating that sage reduced the loss of thiol groups, suggesting that protein oxidation was inhibited during the heating process. This is similar to the finding of Lara et al. (2011) who suggested that cooking promoted protein oxidation in pork meat, and the presence of rosemary extract reduced the loss of thiol groups. There is little information on the effect of sage to reduce the loss of thiol groups in meat products. The protective effect against the loss of protein thiol groups by other natural antioxidants has been observed in several meat systems, such as with the use of black currant extract in raw pork patties (Jia et al., 2012), and with pomegranate juice in chicken meat (Vaithiyanathan, Naveena, Muthukumar, Girish, & Kondaiah, 2011). The antioxidant activities of these natural

L. Zhang et al. / Meat Science 95 (2013) 145–150

antioxidants, including sage in the present study, may relate to the phenolic compounds in these plants, which can scavenge free radicals and compete with thiol groups for trapping free radicals (Jia et al., 2012). It is evident that the values of thiol groups in sausages containing sage decreased faster than those of the control (Table 2). The results demonstrate that during refrigerated storage, the presence of sage in sausages caused an increase in the loss of thiol groups, which is inconsistent with our results for the protein carbonyl groups. A similar phenomenon has been reported by Jongberg et al. (2011) who found that addition of white grape extract accelerated the loss of thiol groups but reduced the formation of myosin cross-links in beef patties. The authors explained that there could be interactions between thiol groups and the polyphenols in the extract. Ortho-phenolic structures are highly susceptible to adduct formation with nucleophilic thiols to form thiol-quinone adducts (Jongberg et al., 2011). A variety of ortho-phenolic compounds in sage, such as carnosol, carnosic acid and rosmarinic acid, may react with thiol groups resulting in lower amounts of thiol groups in the sausage where sage has been added. The present findings regarding the changes of thiol groups in sausages with addition of sage are likely to result from a balance between the antioxidant and the pro-oxidant effects of phenolic compounds in sage. Nevertheless, these findings on the contents of the protein carbonyls suggest that sage does possess the ability to inhibit protein oxidation in Chinese-style sausage. 3.4. Texture profile analysis The hardness of the sausage increased significantly (P b 0.05) during refrigerated storage (Table 1). Initially (day 0), there were no differences in any textural properties among any of the treatments (Table 3). However, the hardness of the control sausages significantly increased (P b 0.05) during 21 days of refrigerated storage, while there were no significant changes in any of the sage-treated groups.

Table 3 Effect of sage on the texture properties of Chinese-style sausage during refrigerated storage. Storage time

Hardness (N)

Springiness (cm)

Cohesiveness

Gumminess (N)

Chewiness (N × cm)

Day0 Day7 Day14 Day21 SEMB Day0 Day7 Day14 Day21 SEM Day0 Day7 Day14 Day21 SEM Day0 Day7 Day14 Day21 SEM Day0 Day7 Day14 Day21 SEM

SEMA

Treatment C

S5

S10

S15

99.5y 105.7ay 103.9ay 119.9ax 2.56 0.67 0.68 0.69 0.69 b0.001 0.34 0.37 0.38 0.37 0.007 30.4y 41.2ax 39.5ax 43.9ax 1.81 21.8y 26.1y 27.0axy 31.5ax 1.26

98.8 96.3ab 99.0ab 101.1b 1.30 0.67 0.69 0.69 0.70 b0.001 0.35 0.38 0.39 0.40 0.009 30.1z 36.5aby 38.1axy 42.5ax 1.41 20.2z 25.7y 26.2axy 28.9ax 1.00

96.8 92.6b 93.7bc 94.5b 1.18 0.68 0.69 0.68 0.69 b0.001 0.36 0.39 0.38 0.37 0.008 30.5 32.0b 30.6b 33.8b 0.95 19.9 22.4 21.6b 24.0b 0.83

90.1 86.3b 86.3c 86.9c 1.27 0.67 0.69 0.68 0.69 b0.001 0.35 0.39 0.39 0.38 0.006 31.9 31.4b 31.5b 32.4b 0.66 21.3 22.4 20.4b 22.6b 0.48

1.56 2.35 2.04 3.43 – 0.004 0.002 0.003 0.003 – 0.009 0.006 0.005 0.007 – 0.89 1.35 1.29 1.63 – 0.69 0.85 0.94 1.17 –

C: control sausage without sage; S5: sausage treated with 0.05% sage; S10: sausage treated with 0.1% sage; S15: sausage treated with 0.15% sage. Means with different superscripts (a–c) within a row of the same storage day are significantly different (P b 0.05). Means with different superscripts (x–z) within a column of the same batch are significantly different (P b 0.05). A Standard error of the mean within the same storage day (n = 12). B Standard error of the mean within the same treatment (n = 12).

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The highest values of hardness at each sampling day were observed for the control sausages. Among the sage-treated samples, the higher the level of sage, the lower was the hardness. The textural parameters, such as hardness, chewiness and gumminess, have a great impact on consumer acceptability. The results indicated that the addition of sage inhibits the increase of hardness of sausages during refrigerated storage that was observed in the control. Previously, Estévez et al. (2006) also reported that the addition of 0.1% sage extract significantly reduced the increase of hardness in porcine liver pâté at day 30 of refrigerated storage. The increase of hardness in meat products during refrigerated storage is thought to result from emulsion destabilization caused by water and fat separation from the protein matrix (Estévez et al., 2005). Protein oxidation may also lead to an increase of hardness through the formation of carbonyls, the loss of protein functionality and the formation of protein cross-linking via disulphide bonding (Ganhão et al., 2010). For these reasons, natural antioxidants may reduce the hardness of the meat products by increasing emulsion stability and reducing the cross-linking through their protective role on proteins against oxidation. Moreover, natural antioxidants may protect muscle membranes from lipid oxidation, thus maintaining the membrane integrity of muscle fibers and therefore reduce moisture loss, which in turn would have a beneficial effect on the textural properties of sausages (Maqsood et al., 2012). In combination with the previous findings of the inhibitory effect of sage on lipid and protein oxidation, it is plausible that phenolic-rich sage reduced the hardness increase in sausages through protecting lipid and protein from oxidation during refrigerated storage. The changes of gumminess and chewiness of sausages during refrigerated storage were similar to the hardness. The gumminess and chewiness values of the sausages containing of 0.1% and 0.15% sage showed significantly lower (P b 0.05) values than those of the control during refrigerated storage. None of the treatments had any effect on the springiness and cohesiveness values of the sausages during storage compared to the control. These results suggest that sage can minimize texture deterioration of sausage caused by oxidation.

3.5. Sensory evaluation The sensory properties of Chinese-style sausages, including color, flavor, texture and overall-acceptability, with and without the addition of sage at day 0, are presented in Table 4. Lower scores were found for the color of sausages containing sage, although the differences were not significant, and this is consistent with the changes in L* values; possibly due to the color of the phytochemicals itself. No off-flavors were detected in the sausages by the panelists in the sensory evaluation, although the TBARS values of samples were high. Even though the texture profile analysis indicated a decrease in hardness with increasing content of sage, the panelists were unable to detect any differences in texture with the addition of greater amounts of sage, or compared with the controls. Also, the overall-acceptability was not affected by addition of sage. These results indicate that sage can be incorporated into Chinese-style sausage without having any detrimental effects on the sensory attributes.

Table 4 Effect of sage on the sensory attributes of Chinese-style sausage. Treatment

Color Flavor Texture Overall-acceptability

C

S5

S10

S15

7.0 7.0 6.8 6.6

6.9 7.2 6.8 6.9

6.8 6.4 6.9 6.7

6.5 6.9 7.1 6.8

SEMA

P value

0.16 0.16 0.15 0.14

0.62 0.31 0.89 0.87

C: control sausage without sage; S5: sausage treated with 0.05% sage; S10: sausage treated with 0.1% sage; S15: sausage treated with 0.15% sage. A Standard error of the mean within the same row (n = 60).

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