Evaluation of Quality Characteristics of Low-Nitrite Pork Sausages with Paprika Oleoresin Solution during Refrigerated Storage

Raw pork ham lean and back fat were purchased from a local meat market (Hyundai Retail Meat Market, Gwangju, Korea). After the external fat and connective tissue were removed from pork ham, and it was ground with a meat chopper (M-12S, Korea Fuji Kogyo, Busan, Korea) and stored frozen at –50°C until used for sausage manufacturing. The frozen ground raw meat and back fat were thawed and refrigerated at 4°C for 24 h before sausages manufacturing. Paprika oleoresin was provided by Kalsec (Kalamanzoo, MI, USA). Sunflower seed oil was purchased from Sajo (Seoul, Korea) and used for preparing diluted paprika oleoresin solution (POS; 20 times and 100 times).

Formulation of pork sausages is listed in Table 1. In this experiment, POS was prepared by diluting paprika oleoresin and sunflower seed oil to avoid excessive increase of redness and yellowness from undiluted paprika oleoresin. Sunflower seed oil was extracted using organic solvents similar to paprika oleoresin (Salgin et al., 2006). It had no trans-fatty acids. However, it had linolenic acid, linoleic acid, unsaturated fatty acids, vitamin A, and vitamin D (Lee and Park, 2010). Pork ESs were prepared for the following four treatments: 1) REF, 150 ppm NaNO₂; 2) CTL, 75 ppm NaNO₂; 3) TRT1, 75 ppm NaNO₂ and 0.1% POS (1% paprika oleoresin+99% sunflower seed oil); and 4) TRT2, with 75 ppm NaNO₂ and POS 0.1% (5% paprika oleoresin+95% sunflower seed oil). Physicochemical chracteristics, textural properties, antioxidant activities, and antimicrobial activities of pork emulsified-sausages (ESs) at 0, 3, 7, 14, 21, 28, and 35 days after manufacture were then determined.

Ground raw meat was mixed with an ice water for 30 sec using a hood mixer (Mixer, HMC-401, Hanil Electric, Seoul, Korea). The mixture and curing agents were then mixed with an ice water for 1 min. After the addition of curing agents, back fat and POS were added. The mixture was then emulsified for 1 min. Finally, the meat batter was ground for 1 min. Stuffed meat batter in 50 mL conical tube (SPL Life Science, Pocheon, Korea) was heated in a water bath (WB-22, Daihan Scientific, Seoul, Korea) at 75°C for 30 min, vacuum packaged, and refrigerated stored at 4°C until analyzed.

pH values were measured five times for the inside of each treatment using a pH meter (Model 340, Mettler-Toledo, Columbus, OH, USA). pH calibration was performed based on optimum slope measuring by pH 4.01 and 7.00 buffer.

Meat color was measured six times. Lightness (CIE L*), redness (CIE a*), and yellowness (CIE b*) of cross-sections of sausages were measured using a Minolta Color Reader (CR-10, Minolta, Tokyo, Japan). White color plate (CIE L*=94.8, CIE a*=1.0, CIE b*=0.1) was used as the standard.

Homogenized sausage sample (10 g) and double-distilled (dd) water (90 mL) were mixed with a stomacher (BagMixer^® 400 CC^®, Interscience, Saint-Nom-la-Bretèche, France) for microbial counts. Total plate count (TPC) and violet red bile (VRB) agar plates were used for measuring numbers of total bacteria and Enterobacteriaceae, respectively. After spreading samples onto agar plates in petri dishes, plates were incubated at 37°C in an incubator for 48 h.

Lipid oxiatation products were measured using the method of Sinnhuber and Yu (1977). Briefly, each sample was prepared by adding 1% of thiobarbituric acid solution (3 mL) to ground sausage (2 g). Then 2.5% of trichloroacetic acid solution (17 mL) was blended with the mixture in a glass tube. The tube was heated at 100°C in a water bath (WB-22, Daihan Scientific, Seoul, Korea) for 30 min. After heating, the supernatant of the heated mixture (5 mL) and 5 mL of chloroform were mixed together for 1 min and centrifuged at 1,660×g (Model VS-5500, Vision Science, Korea) for about 5 min. The supernatant of the sample (3 mL) was blended with petroleum ether (3 mL) for 1 min. The mixture was then centrifuged at 1,660×g for 10 min. The absorbance of the reaction product was determined using a spectrophotometer (Model UV-1601, Shimadzu, Kyoto, Japan) at a wavelength of 532 nm. TBARS value was derived by calculating the measured absorbance with the following equation:

The constant 9.48 was obatained from the reaction product of red thiobarbituric acids’s sample dilution factor and its absorption coefficient (152,000 M⁻¹ cm⁻¹).

For EM, each sample (1.5 g) was prepared by shaping into a rectangular shape. The sample was covered with three pieces of filter paper (Whatman #3, GE Healthcare, Little Chalfont, UK), placed in the bottom of a conical tube, and centrifuged at 1,660×g (VS-5500, Vision Science, Gyeongsan, Korea) for 15 min. After the centrifugation was completed, EM was determined based on changed weight of the sample and the filter paper using the following formula:

Proximate composition was performed according to methods of the Association of Offical Analytical Chemists (AOAC, 2000). Moisture content (%) was measured with the dry oven method. Briefly, 2 g of each sample in a paper thimble was dried for 18 h in a dry oven at 102°C and the weight of the samples was measured after drying. Crude fat content (%) was measured with the Soxhlet extraction. Briefly, the paper thimble after moisture drying was placed in Soxhlet’s extractor, and fat in the samples was extracted with petroleum ether to measure the weight loss. Crude protein content (%) was determined based on the Kjeldahl method.

Cooking loss (CL) was expressed in percentage (%) by calculating the weight of sausage before and after heating with the following formula.

For texture profile analysis, sausage samples with 1.3 cm in height and 1.25 cm in diameter were prepared. Texture properties such as hardness (gf), springiness (mm), gumminess, chewiness, and cohesiveness of each sample were determined with a bellow cross head compression probe (cross speed of 300 mm/min and load cell of 50 kg) using an Instron Universal Machine (Model 3344, Instron, Canton, MA, USA).

Residual nitrite content was measured by a modified method of AOAC (2000). Briefly, homogenized sausage sample (5 g) was blended with double-distilled water (300 mL), and heated in a water bath (WB-22, Daihan Scientific, Seoul, Korea) for 1 h at 100°C. After heating, filteration was performed using a funnel and a filter paper (Whatman #2, GE Healthcare, Little Chalfont, UK) to remove sausage grounds. Filtrated sample was diluted with distilled water to make a mixture of 500 mL. Sulfanilamide solution (2.5 mL) was added to 25 mL of this mixture, blended, and incubated at room temperature for 5 min for reactions to occur. Then, N-(1-naphthyl)ethylene dihydrochloride solution (2.5 mL) was added to the reactant, blended, and incubated at room temperature for 15 min. The absorbance of the final reaction product was measured with a spectrophotometer (Model UV-1601, Shimadzu, Kyoto, Japan) at wavelength of 540 nm. A standard curve was obtained by measuring the absorbance of nitrite solution to derive residual amount of nitrite. It was used to determine residual amount of nitrite based on the measured absorbance of the sample.

All experiments for this study were carried out in triplicate for each sample of treatments. Data were represented as mean and standard deviation and were analyzed using two-way analysis of variance (ANOVA) and IBM SPSS Statistics 23 (SPSS, Chicago, IL, USA) with Duncan's multiple range test at a significance level of 5% (p<0.05). If a significant interaction between treatments and storage time was observed, means and standard deviations were separated by treatments within each storage time and by storage time within each treatment. When the interaction was not significant (p>0.05), data were pooled to test the primary effect by each main factor.

Table 2 shows results of pH of pork ESs added with POS during refrigerated storage. Since no interaction between treatment and storage time was found (p>0.05), data were pooled by treatment within storage time and storage time within treatment. For pork ESs, their pH values ranged from 6.06 to 6.09, showing no difference among ESs (p>0.05). In a study of Eskandari et al. (2013), pH values of frankfurters added with 0.1% or 1.5% of paprika extract were not different from the pH values of frunfurters without adding paprika extract (p>0.05). Similar to results of their study, our results also showed that the addition of paprika extract did not affect pH values of meat products (p>0.05).

As shown in Table 2, results of color determination were pooled by treatment within storage time and storage time within treatment since no interaction between treatment and storage time was found (p>0.05). Lightness (L*) values of sausages were higher for the CTL than those of the treatments added with 75 ppm of nitrite alone (p<0.05). However, lightness (L*) values of sausages in REF and TRT2 were not different from each other (p>0.05), although they were lower than those of other treatments (p<0.05). The reason for the difference in L* values among CTL, TRT1, and TRT2 with the same amount of nitrite added might be partially due to a decrease in lightness with the addition of diluted POS. A previous study has reported that 15 or 30 g/kg of paprika powder decreased L* values of dry-cured sausages at 20 mm of meat mincing level (Fernández-López et al., 2002). Redness (a*) values of sausages in the TRT2 added with 75 ppm of nitrite and 0.1% of POS (paprika oleoresin 5% and sunflower seed oil 95%) were higher than those of the REF added with 150 ppm of nitrite alone (p<0.05). The addition of paprika oleoresin resulted in higher redness values (p<0.05). Thus, the addition of paprika oleoresin could increase the redness of pork ES, however, redness values showed no change during the storage time (p>0.05). The addition of paprika powder (0.1%, 0.5%, or 2%) into fresh pork sausages also increased a* values in the study of Martínez et al. (2006). Paprika has a high amount of ketocarotenoids such as capsorubin and capsanthin (Levy et al., 1995). These carotenoids, called red xanthophylls (Minguez-Mosquera et al., 1992), are specific red compounds that can increase the redenss of foods including meat products. Yellowness (b*) values of TRT2 were the highest in the TRT2 among all treatments (p<0.05). The addition of diluted paprika oleoresin also increased yellowness (p<0.05), which showed no change during the storage time (p>0.05). According to a study of Jokanović et al. (2011), the addition of 1% of paprika oleoresin increased the yellowness of marinated chicken breast. Thus, meat products with paprika oleoresin increased their yellowness values, as well.

As shown in Table 3, there was no interaction between treatments and storage time in results of microbiological analysis (p>0.05). TPC in the CTL were higher than those in other treatment groups (p<0.05). However, TRT1 and TRT2 were lower than those with TPC, indicating that paprika oleoresin might have an antibacterial activity. Salih (2006) reported that oil extract from paprika possessed antimicrobial activity against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae due to the presence of capsanthin from paprika. In addition, oleoresins including paprika extract at concentration above 5,000 ppm had antimicrobial activity by fracturing bacterial membrane. This mechanism can inhibit the growth of foodborne pathogens such as Listeria monocytogens, Staphylococcus aureus, Bacillus cerus,Salmonella, and Pseudomosnas aeruginosas (Dussault et al., 2014). However, microbial counts of Enterobacteriaceae (VRB) were not changed during storage for any treatment (p>0.05). Both TPC and VRB anlaysis results increased with increasing storage time (p<0.05).

Table 3 shows results of TBARS of pork ESs added with POS during storage time. Data were pooled by treatment within storage time and storage time within treatment since no interaction between treatment and storage time was found (p>0.05). Although no difference in the TBARS values were observed among REF, TRT1, and TRT2 (p>0.05), TBARS values of the CTL were the highest among treatments (p<0.05). These results indicated that the addition of paprika oleoresin could inhibit lipid oxidation during storage. Antioxidant activity and lipid oxidaition inhibited the ability of paprika as reported by previous studies. Capsicum annuum group including paprika contained a great antioxidant activity from rich polyphenol pattern (Marín et al., 2004). When paprika extract is added to meat products, it can be expected to inhibit lipid oxidation. In fact, Shim and Chin (2013) reported that pork patties containing oven-dried paprika powder (0.5% and 1.0%) had lower TBARS values than control without paprika powder. Kim et al. (2013) have also reported that the lipid oxidation of pork patties added with 0.323% of paprika oleoresin for nitrite replacement was lower than those of the control without adding paprika oleoresin during refrigerated storage. Carotenoids, pigments present in paprika, have a radical scavenging ability. They can function as antioxidants by physically trapping singlet oxygen (Kiritsakis and Dugan, 1985). Carotenoids in liposome systems reported to increase antioxidant acitivity with a decrease of oxygen content (Kiokias and Gordon, 2004). TBARS value started to increase rapdily from day 14 of storage (p<0.05). They continued to increase and tended to be the highest at day 35 of storage (p<0.05).

Results of EM are shown in Table 3. There was no interaction between treatments and storage time in results of EM (p>0.05). EMs of sausages in REF and CTL were not significantly different(p>0.05). However, EM values in the TRT2 were higher than those of REF and CTL (p<0.05). During storage, EM (%) showed an increase from day 21 (p<0.05). It continued to increase up to day 35 during storage. Bázan-Lugo et al. (2012) have reported that sausage batter added with more than 1% of paprika powder have higher EM values than those without adding paprika powder. These results were different from our results due to the differences in the structure of paprika powder and oleoresin type.

Table 4 shows proximate composition. Moisture, fat and protein contents (%) were not different among treatments (p>0.05). The moisture content of ESs ranged from 62.2% to 62.9%, fat content ranged from 18.1% to 18.8%, and the protein content ranged from 13.9% to 14.0%. These results indicated that lower level (0.1%) of POS might not affect the proximate composition of ES. Yusop et al. (2012) have reported that the addition of 1% to 3% of paprika oleoresin did not affect moisture or fat contents of marinated chicken breasts. Therefore, the addition levels of paprika oleoresin at 0.1% might not be enough to change proximate composition.

CL values of pork ESs added with POS are shown in Table 4. There was no differences in CL among the treatments (p>0.05). Kim and Chin (2018) have also reported that CLs of pork low-fat sausages added with paprika powder (0.05% or 0.1%) were not different from those of the control without the addition of paprika powder. These results indicate that paprika pigment does not affect CL of pork ESs.

Results of texture profile analysis of pork ESs added with POS are shown in Table 4. There were no differences in all textural properties (hardness, springiness, gumminess, chewiness and cohesiveness) among treatments (p>0.05). Bázan‐Lugo et al. (2012) have also detected that there were no differences in hardness value between sausage added with 1% of paprika powder and control (0% of paprika powder). However, treatment added with 2% of paprika powder had a higher hardness than control and 1% treatment. It indicates that the ingredients of paprika changed texture of sausage due to retain the water levels during the heating. Thus, the addition of 0.1% of POS into ESs might not be enough to change texture properties of those in our study.

Table 5 shows residual nitrite levels in pork ESs added with POS. As expected, residual nitrite levels (ppm) in the REF were the highest among all treatments (p<0.05), and those in the CTL, TRT1, and TRT2 showed no differences until 21 days (p>0.05). Residual nitrite levels in the TRT2 at 28 and 35 days were lower than those of the CTL (p<0.05), indicating that the paprika oleoresin could reduce residual nitrite levels in the TRT2. Jeong et al. (2006) have reported that paprika with higher antioxidant capacity possess higher nitrite scavenge activity. Paprika contains phenolic compounds, ascorbic acid, flavonoid, and carotenoid pigments including capsanthin, capsorubin, and cryptocapsin (Baliga et al., 2003; Zhang and Hamauzu, 2003). Since they are effective in scavengering free radicals, they could inhibit oxidation during storage due to their antioxidant activity. These results indicate that antioxidants in paprika oleoresin can scavenge residual nitrite, thus decreasing residual nitrite levels.