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Effect of Gaeddongssuk (Artemisia annua L.) Powder on Quality and Shelf Stability of Emulsion Sausages during Refrigerated Storage

The objective of this study was to evaluate effects of Gaeddongssuk powder (GP) on quality characteristics and shelf stability of emulsion sausages during storage. Proximate composition properties showed no significant differences in all treatment (p>0.05). Control showed the highest cooking loss while the treatment with GP showed decreased cooking loss depending on increasing GP content (p<0.05). Apparent viscosity of batter was increased as the amount of GP increased, whereas hardness of emulsion sausages was decreased with increasing GP level. In sensory evaluation, emulsion sausage with 0.1% GP resulted in the highest score in overall acceptability. The pH values of all treatments decreased at the early storage stage, followed by gradual increase. The lightness and redness of treatments were decreased when the level of GP was increased. However, the yellowness of sausages with GP were higher than that of control (p<0.05). The addition of GP inhibited lipid oxidation of emulsion sausages during storage depending on its level. The aerobic bacteria population and VBN was unaffected by addition of GP during the storage (p>0.05). Therefore, Gaeddongssuk powder up to 0.1% has a potential as a natural antioxidant for meat products because it can inhibit lipid oxidation of sausages without decreasing their sensory properties.


Introduction
Recently, improvement in the economic status and educational level of consumers has led to healthy food choices. Consumers will consider the impact of food on their health as well as the nutritional value of food. Consumers have been avoided synthetic additives that could potentially be harmful to health. So the physiological activity of natural products and their derivatives as functional materials have been intensively studied (Lee and An, 2010; Lee et al., 2002;Ryu and Kwon, 2012).
Most meat products contain lipid and lipid oxidation causes loss of its nutritional value. Various oxidation products can be produced by peroxide oxidation, including aldehydes, hydrogen peroxide. They are potentially toxic to humans and animals (Horton et al., 1987). They can damage DNA and cause cancer. In addition, oxidation products are associated with aging (Zanardi et al., 2004). Synthetic antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) have been used to prevent lipid oxidation. However, these are known that cause of disease such as liver hepatomegaly with increased microsomal enzyme activity has been reported to be toxic or carcinogenic (Branen, 1975). Therefore, it is very important to develop safe and healthy natural antioxidants.
Gaeddongssuk (Artemisia annua L.) is an annual plant belonging to family of Chrysanthemum (Compositae). This plant is mostly distributed in Asia at roadside and riverside (Räth et al., 2004). This plant has been used as traditional herbal medicine. Gaeddongssuk contains various bioactive compounds. It is especially rich in artemisinin (Räth et al., 2004;Towler and Weathers, 2015). In addition, Gaeddongssuk contains flavonoids, phenolics, and purines (Iqbal et al., 2012;Weathers et al., 2011). The antioxidant activity of phenolic compounds in Gaeddongssuk has been reported (Brisibe et al., 2009). It can protect lipid against peroxidation by inhibiting oxidizing chain reactions (Laguerre et al., 2007). Some studies have reported the effect of Gaeddongssuk added to food and feed (Kang et al., 2012;Lee et al., 2014). However, Gaeddongssuk powder (GP) was not used as natural antioxidants to inhibit lipid oxidation in meat products.
Therefore, the objective of this study was to determine the effect of GP on quality characteristics during refrigerated storage of emulsion sausages as well as to provide effective uses of GP as a natural and functional ingredient in meat products.

Materials and Methods
Preparation of raw materials Commercial samples of hot air-dried Gaeddongssuk powder (GP) (L* = 47.23±1.33, a* = -6.82±0.11, b* = 24.52 ±0.34) were purchased from a local market (Jirisan honghwain, Korea). Fresh pork ham, chicken breast and pork back-fat were obtained from a local commercial processor (Korea) 24 h after slaughter.

Manufacturing of emulsion sausage
All subcutaneous, intramuscular fat and visible connective tissues were removed from the fresh ham muscles. Lean meat and pork back fat were ground through a 3 mm plate (Model 7548, Biro MFG. Co, USA). Pork ham, chicken meat, and pork back fat were mixed with curing ingredients (salt, sugar, phosphate) and stored at 4°C overnight (Fig. 1). Five formulations of emulsion sausages; control, one with 0.02% butylated hydroxytoluene (BHT) and three with different GP levels (0.1, 0.3 and 0.5%) and were prepared. Pork ham, chicken meat, water and other ingredients were emulsified by using a silent cutter (CR-40, Spain). After emulsification, the meat batter was stuffed into PVDC casings (Ø 65 mm) using a stuffer, and then were heated until the core temperature reached 71°C for 30 min in smoke house (IT-90404, Intertek, Korea). The cooked sausages were cooled with cold water. The produced samples were aerobically packed in polyethylene bags, and stored at 4°C until testing.

Proximate composition
The moisture, protein and fat contents of emulsion sausage samples were analyzed using a Food Scan™Lab 78810 (Foss Tecator Co., Ltd., DK), according to the method of Anderson et al. (2007).

Cooking loss
As mentioned above, all emulsion sausages were cooked using water bath at 75±2 o C for 30 min. The cooking loss of emulsion sausages were determined by calculating the difference in weight after and before cooking as follows: Cooking loss (%) = [the weight before cooking (g) − the weight after cooking (g)] / the weight before cooking (g) × 100

Apparent viscosity
The meat batter viscosity was measured in triplicate with a rotational viscometer (HAKKE Viscotester® 500, Thermo Elctron Corporation, Germany) set at 10 rpm. The standard cylinder sensor (SV-2) was positioned in a 25 mL metal cup filled with batter and allowed to rotate under a constant shear rate at (s -1 ) for 30 s before each reading was taken. Apparent viscosity values in centipoises were obtained. The temperature of each sample at the time (18±1 o C) of viscosity testing was also recorded.

Texture profile analysis (TPA)
The texture properties of the emulsion sausages were analyzed using a puncture probe (7 mm diameter) attached to a texture analyzer (Model 4465, Instron Corp, UK) (Seo et al., 2015). For texture analysis, the samples were taken from the central portion of each treatment and cut into 2.54 cm long pieces in cylinder shape. The samples were axially compressed twice until reaching each time 80% of its initial height. The speed of load cell was set at 120 mm/min and the following parameters were calculated: hardness (kg), springiness (mm), and cohesiveness (kg* mm), gumminess (kg) and chewiness (kg*mm).

Sensory evaluation
The sensory evaluations were performed on each evaluation of emulsion sausages by panelist. The panels consisted of 7 trained members (3 females, 4 males; ages 27-32) from Animal Products Processing Division of the National Institute of Animal Science, Suwon, Korea. The samples were cut into 1.5 cm and served to the panels when samples were at room temperature (about 25°C). The emulsion sausages were evaluated for color (1=extremely undesirable, 9=extremely desirable), Gaeddongssuk flavor (1=extremely undesirable, 9=extremely desirable), flavor (1=extremely undesirable, 9=extremely desirable), texture (1=extremely undesirable, 9=extremely desirable) and overall acceptability (1=extremely undesirable, 9=extremely desirable) using a 9-point scales. After evaluation of each sample, the panelists were asked to rinse their mouth with the distilled drinking water and salt-free crackers.
Shelf life of emulsion sausages during storage for 4 weeks at 4°C

pH measurement
The pH values of emulsion sausages samples were determined in triplicates using a pH meter (Model 340, Mettler-Toledo GmbH, Switzerland). The pH was measured after homogenizing 3 g of each sample with 27 mL of distilled water for 30 s using a homogenizer.

Color measurement
Color was determined at 4 defined areas on the cut surface of each emulsion sausages sample using a Minolta Chroma Meter CR-400 (Minolta Camera Co., Ltd., Japan) that was standardized with a white plate (Y = 93.5, X = 0.3132 and y = 0.3198). Color was expressed according to the Commission International d'Eclairage (CIE) system and reported as CIE L* (lightness), CIE a* (redness), CIE b* (yellowness).

Thiobarbituric acid reactive substances (TBARS)
Lipid oxidation was investigated in the emulsion sausages samples by measuring TBARS value using the distillation method, with slight modifications (Pikul et al., 1989). A 10 g sample was blended with 35 mL of trichloroacetic acid (5%) and 1 mL of butylated hydroxytoluene (0.5%) and then transferred to a round-bottom flask. After addition of 100 mL of distilled water, the mixture was distilled using a distillation column and 50 mL of distillate was collected. Five milliliters of distillate was added to a tube containing 5 mL of 2-thiobarbituric acid (0.02 M) and vortexed. The tubes were capped and heated in a boiling water bath for 60 min to develop the pink color and then cooled to room temperature. The absorbance was measured at 532 nm against a blank containing 5 mL of water and 5 mL of TBA (0.02 M) solution. The TBARS values were expressed as milligram malondialdehyde (MDA) per kilo-gram of sample. TEP (1,1,3,3-tetraethoxypropane, Merck) was used for the preparation of standard curve and calculation of TBARS value.

Volatile basic nitrogen (VBN)
Volatile basic nitrogen was determined by the microdiffusion method according to Conway and Byrne (1933), and was express as mg% of the sample using the following steps: 10 g of samples were mixed with 90 mL of distilled water for 1 min by filtering through Whatman paper No. 1 (Ø150mm, Cat. No. 1001 150, Whatman Intermational Ltd., England). One mL of filtrate was placed in outer space of Conway unit, and 1 mL of 0.01 N H 3 BO 3 and 200 µL of Conway regent (0.066% methyl red in ethanol : 0.066% bromocresol green in ethanol = 1:1) were added in inner space. The Conway unit was sealed immediately after adding 1 mL of 50% K 2 CO 3 to the outer space. The sealed Conway unit was shaken slowly and incubated at 37 o C for 2 h. The 0.02 N H 2 SO 4 was added to inner space for titration. The VBN was calculated using the following equation.
a = titer for sample, b = titer for blank, f = factor of reagent, N = normality, S = sample weight (g).

Microbial analysis
Emulsion sausages (25 g) were taken aseptically from each batch in each treatment, transferred to sterile plastic pouches and homogenized for 1 min at room temperature with 225 mL of 1% peptone water using a stomacher Lab-Blender 400 (Seward Medical, London). Serial dilutions were made in sterilized diluted solution and aerobic mesophilic bacteria were determined by spreading 1 mL of appropriate dilutions on to 3M Petrifilm Aerobic Count Plate (3M Health Care., USA) incubated for 48 h at 35 o C. Each sample was done in duplicates and total aerobic mesophilic bacteria were expressed as log 10 numbers of colony forming units/gram (CFU/g).

Statistical analysis
All tests were done at three times for each experimental condition and mean values were reported. The statistical analysis of all data was performed by SPSS Ver. 18.0 (SPSS Inc., USA). One-way analysis of variance (ANOVA) tests were carried out on quality characeristics. Two-way analysis of variance (ANOVA) tests were carried out on each variable of treatments and weeks. Duncan's multiple range comparison were used to find the level of significant differences (p<0.05).

Quality characteristics of emulsion sausage
Proximate composition Proximate composition of emulsion sausages with GP is shown in Table 2. Moisture content of emulsion sausages of control and treatments were, 58.95-59.53%, showing no significant differences among control and treatments (p>0.05). Fat and protein contents of emulsion sausages did not shown significantly different at all treatments (p>0.05). Moon et al., (2003) showed no differences on proximate composition of pork patty among treatment groups within/without addition mugwort powder, as a similar result. This study indicated that addition of GP had no effect on proximate composition.

Cooking loss
Cooking loss of emulsion sausages with GP additive is summarized in Table 3. Cooking loss of the control was higher than T4 (p<0.05). It has been reported that a large amount of dietary fiber (dry 40.09%, wet 6.87%) is contained in mugwort (Hwang et al., 1996). Generally, cooking loss is affected by the types of dietary fiber added to the meat product. Cooking loss of meat products with dietary fiber can be decreased when its contents are increased (Choi et al., 2010a). Jeon et al. (2012) reported that cooking loss of pork patties was decreased when the content of seaweed powder (sea mustard, sea lettuce, and hijikia) was increased, resulting from increased water holding capacity due to dietary fiber in seaweeds. And addition of louts root or louts leaf powder to pork patties result in the low cooking loss compared with control. (Choi et al., 2012). Therefore, dietary fiber in GP added to the sausages might have increased the water absorption ability of BHT: Butylated hydroxytoluene. All values are mean±standard deviation.

Apparent viscosity
Apparent viscosity of meat batter with GP is shown in Fig. 2. The control and treatments with GP showed reduction in apparent viscosity as turning time was increased, which is a characteristic of thixotropic material. Initial viscosity of the control had the lowest value at 111 Pa*s while that of the treatment with 0.5% GP (T4) had the highest value at 213 Pa*s. This is thought to be due to the effect of GP on increased viscosity of the emulsion. Nuria et al. (1999) reported that dietary fiber, fat, and water contents can affect the viscosity of meat emulsions. This study showed that decreased fat and high dietary fiber content in batter increased viscosity. Dogan et al. (2005) reported that viscosity of emulsions can exert the largest effect on water absorption of additives. Akras and Genccelep (2006) reported that increased emulsion viscosity is related to increased emulsion stability. Such relationship can serve as a useful standard for manufacturing high-quality emulsion sausages.

Texture profile analysis (TPA)
Changes in texture properties of emulsion sausages with GP are shown in Table 4. Hardness was decreased as the amount of GP was increased (p<0.05). The springiness value was the highest (18.41 mm) in the control (p<0.05). Han (2010) reported that the hardness of meat products is decreased as the added amount of salted bamboo shoot powder is increased, similar to the results of this study. However, cohesiveness, gumminess, and chewiness showed no significant (p>0.05) differences among treatments. The quality of raw meat and types of additives as well as differences in the extent of protein denaturation based on differences in heating temperature during processing process can lead to changes in histological characteristics . In addition, dietary fiber in the GP may have effect on hardness and springiness of emulsion sausages by retaining more moisture due to its high water holding capacity as reported in previous studies (

Sensory evaluation
Sensory characteristics (color, Gaeddongssuk flavor, flavor, texture, and overall acceptability) of emulsion sau- All values are mean±standard deviation.

A , B
Means within a row with different letters are significantly different (p<0.05). sages with various GP levels are summarized in Table 5. There was significant (p<0.05) difference in color, Gaeddongssuk flavor, and overall acceptability among treatments. However, there was no significant (p<0.05) difference in flavor or texture among treatments. The color and Gaeddongssuk flavor of emulsion sausages were increased as the GP concentration increased. Overall acceptability of treatment with 0.1% GP showed the highest score while the treatment with 0.5% GP (T4) had the lowest score (p<0.05). Kim et al. (2009) reported that excessive dietary fiber may cause a decrease in the quality attributes of meat products such as physicochemical, rheological, and sensory properties. Therefore, during manufacture of the sausage, it is preferable to adding GP to less than 0.3% without negative effects.

Shelf life of emulsion sausages during storage for 4 weeks at 4 o C pH measurement
The pH of emulsion sausages with GP added under different storage durations are shown in Table 6. As the storage duration was increased, significant (p<0.05) decrease in pH was found in all treatments. After 4 weeks of storage, the treatments without GP (Control) had significantly the lowest pH value at 6.62 while the treatment All values are mean±standard deviation.

A -C
Means within a row with different letters are significantly different (p<0.05).

)
Treatments: Control, no added ingredient; T1, sausage with 0.02% BHT(butylated hydroxytoluene); T2, sausage with 0.1% Gaeddongssuk powder; T3, sausage with 0.3% Gaeddongssuk powder; T4, sausage with 0.5% Gaeddongssuk powder. with 0.5% GP (T4) had significantly the highest pH value at 6.82 (p<0.05). These higher pH of treatment with GP resulted from higher pH value of GP than raw meat. Okonkwo et al. (1992) reported a decreasing trend of pH with an increase in storage duration for meat products. It has been reported that the decrease in pH with increase in storage duration is due to the effect of salt concentration on ionic strength (Bower et al., 2003). Choi et al. (2007) reported that pH of meat products is generally decreased as the storage duration is increased, similar to the results of this study. Affected by manufacture and storage conditions, the pH values of meat products during storage are decreased due to lactic acid produced during the growth of microorganisms (Langlosis and Kemp, 1974).

Color evaluations
The additives added to sausage products have been shown to play important roles in meat color during storage (Choi et al., 2010b). Colors of emulsion sausages with GP during storage are shown in Table 7. The CIE L* value indicating the lightness of emulsion sausages in the control was significantly (p<0.05) higher than that of treatments with GP added. As the amount of GP in sausages was increased, the lightness was significantly decreased (p<0.05). The CIE a* value indicating the redness was also higher in the control compared to that in the treatments with GP added. As GP content increased, redness of sausages was significantly decreased (p<0.05). The CIE b* value indicating the yellowness was higher in the treatments with GP compared to that of the control. Lee et al. (2004) reported that lightness and redness values of sausages showed significantly decreasing tendency, when increasing amount of Wormwood (Artemisia absinthium L.) powder added during manufacture of sausages, similar to the results of this study. This was in contrast to the report of Moon et al. (2003) that the addition of 0.3% mugwort powder to patties failed to affect meat product color. The changes in lightness and redness observed in sausages with different amounts of GP in this study could be due to color of GP itself.

Thiobarbituric acid reactive substances (TBARS)
To measure the lipid oxidation of emulsion sausages  All values are mean±standard deviation.

)
Treatments: Control, no added ingredient; T1, sausage with 0.02% BHT(butylated hydroxytoluene); T2, sausage with 0.1% Gaeddongssuk powder; T3, sausage with 0.3% Gaeddongssuk powder; T4, sausage with 0.5% Gaeddongssuk powder. with GP added during storage, TBARS values were measured (Fig. 4). During storage, TBARS value of the control was the highest (p<0.05) at 0.99 mg MDA/kg, while the treatment with 0.5% GP (T4) had the lowest TBARS value at 0.32 mg MDA/kg. It has been reported that mugwort contains large amounts of caffeic acid, catechol, and protocatechinic acid, all of which have strong antioxidant properties, allowing for the delay in lipid oxidation (Lee et al., 1992). As storage duration was increased, lipid oxidation of all treatments was significantly increased (p< 0.05). Turner (1954) have stated that meat with TBARS value of 1.2 mg/kg or higher is generally considered as rotten. In this study, no treatments showed TBARS value over 1.2 mg/kg during storage. GP might have suppressed lipid oxidation since the TBARS value of the control was higher than that of the treatments GP added. Jung et al. (2003) reported that large amount of antioxidants such as polyphenol in mugwort can lower the TBARS values of sausages when mugwort powder added. Heating of mug-wort extracts can contribute to the generation of antioxidants and the conversion of bound polyphenol into free polyphenol, leading to its increased antioxidant activity (Kang and Lee, 2013).

Volatile basic nitrogen (VBN)
VBN is commonly used to evaluate the freshness of meat. Biochemical changes in meat can occur due to enzymes present in muscles and those produced by microorganisms that induce catabolism of proteins into peptides or free amino acids and further breakdown into ammonia, amines, mercaptan, and hydrogen sulfide, causing unpleasant odor (Huang et al., 2015). Such changes in inorganic nitrogen level can reflect the freshness of meat products. In terms of the relationship between VBN formation and food edibility limit, 5-10 mg% indicates fresh state whereas 30-40 mg% indicates early decomposition state (Witte et al., 1970). VBN values of emulsion sausages with GP during storage are shown in Table 8. As storage duration  All values are mean±standard deviation.

Microbial analysis
The count of aerobic bacteria (AB) of emulsion sausages refrigerated for 4 wk is summarized in Fig. 5. As expected, the number of AB of all treatment groups were constantly increased when storage days were increased. The initial AB in emulsion sausages ranged from 1.8-2.6 Log CFU/g. It was increased to 4.5-6.9 Log CFU/g after 28 d. The maximal recommended limit of microbial content is 7 Log CFU/g in meat (Nottingham, 1982). None of the treatments exceeded the permitted limit of AB at 7 Log CFU/g at the end of storage. Result that AB of emulsion sausage containing GP was slightly lower compared with the control. Jung et al., (1989) observed terpinen-4ol in essential oils of mugwort is a leaded food microbiology S. cerevisiae, L. mesenteroides, L. plantarum, A. oryzae and B. subtilis and the count of E. coli, the indicator of food contamination also inhibited by far 1000 ppm, so that demonstrated great antimicrobial effect of mugwort. Also, Kim (2011) reported similar result that AB of emulsified sausage containing mugwort was slightly lower compared with the control.

Conclusion
In this study, the addition of GP increased pH and yellowness but decreased lightness, redness and cooking loss on emulsion sausages. In sensory evaluation, the addition of 0.1% GP to emulsion sausage obtained the highest score. Also, emulsion sausage with GP added inhibited lipid oxidation during refrigerated storage. These effects were dependent on the amount of GP added. The AB population and VBN was not significantly different among treatments (p>0.05). In addition, GP in emulsion sausages could retard lipid oxidation. GP has the potential to increase shelf stability of meat products. Thus, GP could be used as a natural antioxidant to prevent oxidative damage and improve the sensory properties of meat products.