1. Introduction

In recent years, trends toward the consumption of fast and convenient food have led to an increased production of ground meat and various meat products. Thus, the quality of ground meat products during processing and subsequent storage has attracted much attention. Oxidative reactions are considered to be closely associated with the quality of meat and meat products. Lipid oxidation can lead to the deterioration of color, texture and flavor, together with a reduction of nutritive values and the formation of toxic compounds in meat and meat product ^[1]. Protein oxidation can affect the water-holding capacity and the tenderness of meat products resulting in quality deterioration ^{[2, 3]}. In addition, meat and meat products are susceptible to microbial contamination during storage which may bring about decrease in their shelf life resulting in undesirable sensory attributes and even foodborne diseases ^[4].

Synthetic additives having antioxidative or antibacterial activities, including butylated hydroxy toluene (BHT), butylated hydroxy anisole and benzoic acid, are commonly used to minimize oxidative reactions, inhibit microbial growth and extend the shelf life of meat products. However, the potential toxicity of synthetic additives has recently attracted extensive attention leading to renewed interest toward natural antioxidants and preservatives ^[5]. The use of plant extracts from fruits, vegetables, herbs and spices as antioxidants or antibacterial agents in meat systems are becoming increasingly popular ^{[6, 7]}.

Rosemary essential oils have been reported to have great capacities of scavenging free radical and resisting bacterial growth in vitro ^[8]. The abundant phenolic diterpenes in rosemary including carnosic acid, carnosol, rosmarinic acid and rosmanol may contribute to its antioxidative and antibacterial activities as a functional spice ^{[8, 9]}. Previous investigations have indicated the presence of antioxidative properties of rosemary extract (RE) when used in meat and meat products such as cooked pork meat, beef meatballs, ground ostrich meat and fresh chicken sausages ^{[10, 11, 12, 13]}. However, little is known about the antioxidative and antibacterial activities of RE powder in raw ground pork products. In addition, the composition of these components of rosemary from different regions and environments may vary, leading to diverse functional properties ^[14] and inconsistent effectiveness. Nevertheless, few studies have investigated the functional properties of rosemary grown in China. Consequently, the current study was aimed to investigate the antioxidative and antibacterial activities of RE from China in pallet-packaged raw ground pork patties during chilled storage.

2. Materials and Methods

RE was obtained from Zelang Medical Technology Co., Ltd (Nanjing, Jiangsu, China). It was extracted from dried rosemary leaves grown in Nanjing (Jiangsu, China) using a refluxing process with 70% (v/v) ethanol at 70°C. The extraction efficiency was 10% (w/w). The brown extract powder was vacuum-packaged and stored at 4°C until used. Lean pork taken from the rear leg and backfat purchased from Sushi Food Group (Nanjing, Jiangsu, China) was stored at 4°C for 24 h postmortem. Seasonings were obtained from the National Center of Meat Quality and Safety Control of China. Chemicals including 2-thiobarbituric acid (TBA), guanidine hydrochloride, trichloroacetic acid (TCA), 2, 4-dinitrophenylhydrazine (DNPH) and plate count agar (PCA) were purchased from Sinopharm Chemical Reagent Co., Ltd (Beijing, China). BHT was supplied by Yuzhong Bio-technology Co., Ltd (Zhengzhou, Henan, China). All other chemicals were obtained from Aladdin Industrial Corporation (Shanghai, China).

The content of carnosic acid, carnosol and rosmarinic acid was determined by high-performance liquid chromatography (HPLC) as described by Munné-Bosch et al ^[15] with some modifications. Prior to HPLC analysis, various amounts of RE powder were mixed with 90% (v/v) ethanol to a total volume of 100 mL, and 37.26 mg, 6.34 mg and 20.70 mg were for the determination of carnosic acid, carnosol and rosmarinic acid. The contents of each solution were separated by a reverse-phase column (Shim-Pack C₁₈, 4.6 mm × 150 mm, 5 μm, Shimadzu Corp., Kyoto, Japan) with a corresponding eluent at a flow rate of 1 mL/min. Eluents consisting of methanol and 0.1% phosphoric acid (90:10, v:v), methanol and 0.1% phosphoric acid (75:25, v:v) and methanol and 0.05% phosphoric acid (45:55, v:v) were used for the determination of the contents of carnosic acid, carnosol and rosmarinic acid, respectively. The volume of injection was 10 µL and the wavelengths used for the determination of contents of carnosic acid, carnosol and rosmarinic acid were 230 nm, 230 nm and 283 nm, respectively.

Boneless lean pork was ground twice through a grinder (TC 12E, Sirman, Venezia, Italy) using 4 mm plates after being trimmed of connective tissues, while backfat was separately ground once. The ground lean pork and the backfat were mixed at a ratio of 4:1 (w:w). Then, basic ingredients including 6 g kg^-1 carrageenan, 30 g kg^-1 starch, 15 g kg^-1 salt, 2 g kg^-1 sodium tripolyphosphate, 5 g kg^-1 sugar, 1 g kg^-1 monosodium glutamate, 5 g kg^-1 soy sauce, 19 g kg^-1 rice wine and 130 g kg^-1 ice water were uniformly added into the mixture of lean pork and backfat. Five different treatments, each containing the basic ingredients, were used in this study. The first was the control (without any RE extract or BHT) and to each of the other four was added either 0.1 g kg^-1, 0.2 g kg^-1 or 0.3 g kg^-1 RE, or 0.2 g kg^-1 BHT respectively. The mixture was blended using a chopper (BZBJ-15, Expro Stainless Steel Mechanical & Engineering Co., Ltd., Hangzhou, China) for 1 min at the cutting speed of 1,400 r/min. Following blending, the thoroughly mixed pork batter (100 g for each patty) was shaped into patties using a round-shaped mold with a diameter of 8 cm and a height of 1.5 cm. Six replications were conducted for each treatment. Using the method of Jia et al., ^[16] patties were put into polypropylene trays (Sealed Air Co., Ltd., Shanghai, China) and tightly covered by polyethylene film (Surong Plastic Products Co., Ltd., Suzhou, China). Pallet-packaged patties were put into a 4°C refrigeration house and stored for 1, 4, 7 or 10 d under fluorescent lamps simulating the conditions present in supermarkets. Six batches of patties for each treatment were prepared among which each batch was considered as one replication for each treatment.

The extent of protein oxidation was assessed by measurement of the protein carbonyl content as described by Rodríguez-Carpena et al. ^[17] using a UV-visible spectrophotometer (UV-2450, Shimadzu Corp., Kyoto, Japan). Protein oxidation was expressed as nanomole of carbonyl per milligram of protein using an absorption coefficient of 21 mM^-1 cm^-1.

The measurement of thiobarbituric acid reactive substance (TBARS) values was used to evaluate the extent of lipid oxidation as described by Zhang et al ^[18]. TBARS values were expressed as milligrams of malondialdehyde (MDA) per kilogram of patty.

Microbial analysis was carried out as described by Moroney et al. ^[6] with some modifications. Ten grams of pork were removed from the center of a patty and put into a stomacher bag and stomached in 90 mL of physiological saline using a bagmixer (BagMixer 400, Interscience Corp., Saint-Nom-la-Breteche, France) at the speed of 3 for 2 min, giving a 10^-1 dilution. This 10^-1 dilution was then serially diluted 10 times and 1 mL of each dilution was added to a petri dish containing 20 mL of 2.35% PCA (w/v). The plates were incubated at 37°C for 48 h to determine total bacterial counts. Results were expressed as Log₁₀ CFU (colony forming units) per gram of patty.

The pH of patties was measured according to Moroney et al. ^[6] using a desktop pH meter (FE20, Mettler-Toledo International Inc., Zurich, Switzerland).

The surface color (lightness, redness and yellowness; L*, a* and b*) of patties was measured using a portable colorimeter (CR-400, Konica Minolta, Inc., Tokyo, Japan) consisting of an 8 mm diameter measuring area, a 2° observer, a D65 illuminant and a data processor (DP-400). The colorimeter was calibrated on CIE color space system with a white tile (D65: Y = 94.0, x = 0.3156, y = 0.3321).

Patties were cooked in a preheated oven (VTO-34A, North America Appliance, Zhuhai Co., Ltd., Guangdong, China) at 190°C for 10 min until the internal temperature reached 75°C. The cooked patties were cooled to room temperature and then cut into cylindrical pieces with 1 cm thickness and 2.5 cm diameter by a cylindrical sampler and a two-edged knife as described by Gao et al. ^[19] Each piece was used for a two-cycle compression test at room temperature using a texture analyzer (TA.XTPlus, Stable Micro Systems Ltd., Godalming, Surrey, UK). The parameters were as follows: probe P/50, pre-test speed 2 mm s^-1, test speed 2 mm s^-1, post-test speed 5 mm s^-1, strain (compression ratio) 50%, trigger force 5 g and testing interval 5 s. Results of TPA were analyzed by TPA-macro. The textural attributes were expressed as hardness, springiness, resilience and chewiness.

The cooking yield of patties was determined by the procedure of Gao et al ^[19]. Cooking yield was calculated using the following formula:

All tests were performed in triplicate for each replication. Data was analyzed by one-way analysis of variance (ANOVA) using SAS statistical software (Version 8.1, SAS Institute Inc., North Carolina, USA). Differences were compared by Duncan’s multiple range test. Significant differences were considered at P < 0.05. Values were expressed as means ± standard deviation (SD).

3. Results and Discussion

The contents of carnosic acid, carnosol and rosmarinic acid in RE used in the current study were 53, 33 and 55 g kg^-1 respectively. Richheimer et al. ^[14] reported that dried rosemary leaves from different regions (United States, Spain, Morocco, Albania, Turkey and Tunisia) showed various concentrations of carnosol (2 g kg^-1 to 4 g kg^-1) and carnosic acid (17 g kg^-1 to 39 g kg^-1). Munné-Bosch et al. ^[15] indicated that seasonal variation had a strong impact on the content of phenolic diterpenes. However, phenolic diterpenes, especially carnosic acid, carnosol and rosmarinic acid, are considered to be mainly responsible for the antioxidative and antibacterial activities of rosemary ^[11].

As shown in Table 1, patties treated with 0.1, 0.2 and 0.3 g kg^-1 RE exhibited significantly lower TBARS values than control samples throughout the 10 d of storage (P < 0.05). These results are consistent with previous studies which have shown the remarkable ability of RE to inhibit lipid oxidation in meat systems ^{[7, 13]}. Lipid oxidation is the result of a free radical chain reaction which produces a number of compounds that influence sensory attributes and cause quality loss in meat and meat products ^[20]. In particularly, polyunsaturated fatty acids in lipids are readily attacked by free radicals leading to the generation of odors ^[21]. Phenolic diterpenes in RE, including carnosic acid and carnosol, are believed to have the capacity of inhibiting free radical reactions through chelating transition metal ions which otherwise behave as catalysts ^{[10, 22]}.

However, no effect of RE on these properties was observed by Haak et al. ^[23] in the range of 0.05 g kg^-1 to 0.2 g kg^-1. In addition, Rojas et al. ^[21] indicated that addition of 0.2 g kg^-1 rosemary had no ability to retard lipid oxidation in cooked beef and pork patties. In the current study, the additions of 0.2 g kg^-1 and 0.3 g kg^-1 RE showed significantly lower TBARS values than addition of 0.1 g kg^-1 RE at 4 and 7 d of storage. Patties with 0.3 g kg^-1 RE displayed significantly lower TBARS values than those with 0.1 g kg^-1 and 0.2 g kg^-1 RE at 10 d of storage (P < 0.05, Table 1). The inconsistency in these studies may be explained by the different amounts and compositions of the active antioxidant components in RE leading to their various abilities to retard lipid oxidation ^[24].

Table 1. Effects of RE on TBARS values, carbonyl contents and total bacterial counts of raw ground pork patties during chilled storage

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Compared to the BHT-treated patties, patties prepared with RE had similar TBARS values at each of the storage times. Patties supplemented with 0.2 g kg^-1 extract showed a reduction in lipid oxidation at 7 d of storage compared with the controls and those supplemented with 0.3 g kg^-1 extract displayed significantly greater activities in retarding lipid oxidation (P < 0.05, Table 1) at 7 and 10 d of storage. As a synthetic phenolic antioxidant, BHT plays an important role in the meat industry. Nevertheless, an increasing concern about the negative health effects of BHT results in increased attention toward natural antioxidants with phenolic structures in meat products ^[25]. Sebranek et al. ^[26] reported that 0.25 g kg^-1 RE was as effective as 0.2 g kg^-1 BHT in inhibiting lipid oxidation of chilled and cooked-frozen sausages. In raw-frozen sausages, RE was even more efficient in retarding lipid oxidation than BHT which is in accordance with the current result. However, Ahn et al. ^[27] found that BHT was more effective than rosemary in retarding lipid oxidation in cooked ground beef. Further investigations are required to determine the underlying mechanism for the differences in their effectiveness against lipid oxidation when obtained from different sources.

The addition of 0.3 g kg^-1 RE showed higher activity than control in protecting proteins against oxidation at 1 d of storage. At 10 d of storage, treatments with 0.2 g kg^-1 and 0.3 g kg^-1 extract significantly inhibited the carbonyl formation (P < 0.05, Table 1). Patties with 0.1 g kg^-1, 0.2 g kg^-1 or 0.3 g kg^-1 RE or 0.2 g kg^-1 BHT exhibited significantly lower carbonyl contents compared to control at 4 and 7 d of storage (P < 0.05, Table 1). There was no significant difference between treatments of RE and BHT in inhibiting protein oxidation of pork patties throughout the chilled storage period (P > 0.05, Table 1).

Protein oxidation, especially oxidation of myofibrillar proteins, can result in quality loss of meat through its ability to affect moisture retention, leading to lower water holding capacity, higher toughness and decreased textural properties ^{[2, 28]}. Protein oxidation occurs through free radical chain reactions caused by reactive oxygen species (ROS) in a similar way to lipid oxidation ^[29]. It is thought that phenolic compounds may scavenge ROS and have the ability to chelate metals, thus retarding protein degradation and causing aggregation of proteins to form complexes thereby leading to reduced protein oxidation ^{[30, 31]}. It is likely that the phenolic components in RE may be oxidized to phenoxyl radicals which have poor reactivity, and therefore result in the inhibition of the radical-mediated oxidation of proteins. ^[32] Furthermore, the covalent binding of the phenoxyl radicals and proteins may reduce the formation of protein carbonyls. However, Haak et al. ^[23] found that protein oxidation in pork patties was not be retarded by the addition of RE which was similar to the findings of Lund et al. ^[33] in beef patties. These conflicting results may be due to various chemical structures of phenolics in the different extracts, interactions between phenolics and proteins, and the conformation of the proteins ^{[31, 34]}. Additionally, characteristics and components of meat systems, the concentration of antioxidants and production technologies would influence the effects of inhibiting protein oxidation of different additives ^[35].

Compared to control, markedly lower total bacterial counts were observed in patties containing RE during chilled storage (P < 0.05) apart from the additions of 0.1 and 0.2 g kg^-1 RE at 1 d of storage which showed similar total bacterial counts to the control (P > 0.05, Table 1). Georgantelis et al. ^[4] and Liu et al. ^[12] reported similar positive effects of rosemary on retarding microbial growth in various meat systems. Essential oils of rosemary were also found to have antibacterial activity on both Gram-positive and Gram-negative bacteria ^[9]. Active substances present in rosemary essential oils is thought to inhibit the growth of bacteria by disrupting cell membrane integrity and hence altering membrane permeability ^[8]. These active substances in rosemary were phenolic diterpenoids, especially carnosic acid and rosmarinic acid ^{[36, 37]}. In our study, patties blended with either 0.1 g kg^-1 RE or 0.2 g kg^-1 BHT exhibited greater inhibiting effect on microbial growth than the control at 4, 7 and 10 d of storage due to its phenolic structures (P < 0.05, Table 1). In addition to phenolic compounds, flavonols and anthocyanins in plant extracts have also been reported to contribute to the retarding of bacterial growth. This is believed to occur through the inhibition of certain enzyme activities involved in bacterial metabolism and for the regulation of the synthesis of compounds necessary for their growth, together with the chelation of metal ions essential for enzymatic reactions ^[38].

At variance with our results, no significant effects of RE on inhibiting the microbial growth of ostrich and beef patties were observed by Sánchez-Escalante et al. ^[39] and by Seydim et al. ^[13]. Fernández-López et al. ^[11] stated that RE showed significant effects against bacteria when contacted with bacteria directly while no significant antibacterial properties were observed when added into meat systems. The reduction of antibacterial activity by spices in meat systems might be caused by the presence of fat and/or protein ^[40]. Dispersed fat may cover the bacteria cells thereby preventing active substances in spices from entering the cells, while protein is likely to bind to other active substances which may decrease its antibacterial effect. In addition, Rožman and Jeršek ^[41] pointed out that different components and their concentrations in RE would likewise impact the effectiveness of RE against bacterial growth.

As shown in Table 2, patties containing 0.2 g kg^-1 and 0.3 g kg^-1 RE showed lower pH values than the control during chilled storage (P < 0.05). The pH values of patties containing 0.1 g kg^-1 RE were significantly lower than the controls at 7 d of storage (P < 0.05). No significant differences were observed between BHT-treated patties and controls at 1, 7 and 10 d of storage, while the pH values of BHT-treated patties at 4 d were higher than control samples (P < 0.05). Choi et al. ^[42] found that pH values in pork patties with Laminaria japonica powder declined significantly which is similar to our finding. The decrease in pH values could be ascribed to the presence of strong acid components present in this powder including fucoidan and alginic acid. Lara et al. ^[43] also attributed the decrease in pH values in cooked pork patties treated with RE to the presence of carnosic acid present in the extract. However, carnosic acid is considered to be the major antibacterial component in rosemary while low pH would weaken the activity of RE against bacteria ^{[36, 37]}. The interaction between pH, antibacterial effect and acid components in RE appears complicated and requires further investigation.

Table 2. Effects of RE on pH values and cooking yields of raw ground pork patties during chilled storage

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With increasing amount of RE in pork patties, there were small decrease in L* values. A large decrease was observed with the addition of 0.3 g kg^-1 RE compared to control throughout the storage period, while supplementation of 0.2 g kg^-1 RE showed a lower L* value than the control at 1 d of storage (P < 0.05, Figure 1A). It is thought that the loss of pigmented materials present in extracts including chlorophylls and anthocyanins could lead to the observed reduction of L* values ^{[16, 18]}. Phenolics in rosemary can be oxidized to quinones by polyphenol oxidases and quinones are likely to be condensed to form darkened compounds. This browning reaction may result in the change of lightness of RE-treated patties ^[12]. Moreover, lipid oxidation would affect L* values of meat and rosemary could alter L* values through its lipid oxidation retarding ability ^[10]. Patties with 0.2 and 0.3 g kg^-1 RE showed higher b* values than control throughout the storage period which might attributable to the presence of pigmented materials in the extract (Figure 1C).

In this study, patties with 0.2 g kg^-1 RE exhibited greater a* values than the control at 4 and 7 d of storage (P < 0.05, Figure 1B). This result is consistent with some previous investigations which showed that rosemary or RE contributed to a large improvement in color stability of various meat systems ^{[12, 39]}. Lara et al. ^[43] and Sebranek et al. ^[26] stated that RE was more efficient in stabilizing the redness of pork products compared with BHT. The redness of meat is determined by the chemical status of myoglobin, heme proteins and hemoglobin which are red in the reduced form but become brown with oxidized form ^[44]. The protective effect of RE on color stability may be ascribed to its inhibitory effect on the formation of metmyoglobin resulting from the oxidation of myoglobin ^[10]. In addition, lipid oxidation results in the formation of metmyoglobin which contributes to the discoloration of meat ^[16]. Therefore, phenolic components present in RE might protect meat systems against discoloration through retarding lipid oxidation.

As shown in Table 2, those patties blended with 0.3 g kg^-1 RE showed significantly higher cooking yields than the controls during the first 7 d of storage, while the cooking yield of patties containing 0.2 g kg^-1 RE increased at 4 and 7 d of storage (P < 0.05). With 7 d of storage, the cooking yield of patties containing 0.1 g kg^-1 RE was also improved (P < 0.05). Fat and moisture retention capacity are closely linked to the cooking yield ^[45]. Protein integrity is regarded as being a major factor in managing the amount of moisture retained in the meat matrix. RE may protect the loss of water in pork patties by inhibiting protein denaturation and oxidation ^[43]. In addition, Reihani et al. ^[46] suggested that phenolics present in plant extracts would protect the collagen stability in beef patties resulting in improved cooking yield.

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Supplementation with 0.1 and 0.3 g kg^-1 RE or 0.2 g kg^-1 BHT gave increased hardness compared with the control during chilled storage, whereas those patties containing 0.2 g kg^-1 RE had higher hardness values at 4 d (P < 0.05, Figure 2A). Patties containing RE or BHT showed higher resilience and chewiness values in comparison to control, while no significant differences were observed among the treatments with different amounts of RE (P > 0.05, Figure 2D, Figure 2C). Moroney et al. ^[6] found similar results in that addition of seaweed extracts caused an increase in the hardness of fresh pork patties. The increased hardness might be due to swelling, gelling and thickening properties and greater water-retention ability of dietary fibers present in the extract. Yi et al. ^[45] indicated that the meat texture was affected by fat and moisture contents present in meat matrix. The antioxidative ability of plant extracts may retain membrane integrity of muscle fibers and decrease moisture loss through retarding lipid oxidation resulting in improved texture ^[46]. Aleson-Carbonell et al. ^[47] reported that the protein matrix is also closely associated with meat texture due to its ability to retain moisture and improve binding of fat. The antioxidative ability of plant extracts might protect the integrity of protein matrix by inhibiting protein oxidation. However, no significant differences were found for springiness among all treatments during chilled storage which is in accordance with the finding of Moroney et al. ^[6] (Figure 2B).

4. Conclusions

RE exerted an effective ability of retarding protein and lipid oxidation and inhibiting microbial growth in raw ground pork patties. The addition of 0.3 g kg^-1 RE was more effective than 0.1 and 0.2 g kg^-1 BHT as an antioxidant and antibacterial agent. RE also improved the color stability, cooking yields and textural attributes of pork patties. Therefore, RE can be considered as an effective additive with both antioxidative and antibacterial activities to replace BHT in meat products given its potential to enhance the qualities and shelf life of meat products.

Acknowledgements

This research was conducted with finance support by The National Key Research and Development Program of China (2016YFD0400703) and Open Funding for Young Scientists between Nanjing Agricultural University and Tarim University (KYLH201501, KYLH201501-2).

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