The addition of non-meat proteins into processed meat products has been done for increase of binding ability, reduction of raw material loss, and prevent loss of moisture and fat [1,2]. Protein acts as a bridge between fat and water to form meat emulsion [3,4]. Various proteins derived from soy, milk, egg, and animal skin are commonly used in the manufacturing of meat products [5–7]. It has also been studied that blood proteins including blood plasma and blood components resulting from animal slaughter can be used as protein sources for food materials [8,9].
According to the statistics of American soybean association, in 2015, 320.2 million metric tons of soybeans were produced worldwide, of which 218.7 million metric tons were available for consumption , and it accounts for 71% of the total plant protein source of humanity. Soy proteins also have outstanding emulsifying capacity and stability in meat products [11–13]. Thus, soy protein has long been widely used in meat products. Soy proteins are divided into soy protein concentrate (more than 70% protein content) and soy protein isolate (SPI, more than 90% protein content) .
Sodium caseinate (SC) is produced by attaching sodium to casein separated from skimmed milk with acid treatment (citric or carbonic acid) . The casein protein in combination with sodium is more soluble in water than pure casein proteins.
Casein accounts for approximately 80% of milk protein, and it has excellent emulsifying capacity and emulsion stability. SC is excellent in solubility, surface activity, heat resistance and water retention was used as an emulsifier . However, SC has the disadvantage is expensive compared to other non-meat proteins . Currently, consumers are gradually reluctant to purchase food containing SC because it is classified as a synthetic additive in the process of attaching sodium.
Meat consumption is constantly increasing globally . Therefore, slaughtering of livestock is also increasing every year. Slaughter blood production has been estimated to be 6.7 liters per pig (100 kg) and 27 liters per cattle (450 kg) . In 2015, world total slaughter heads of swine and cattle were 1,246 million and 231 million respectively . If the total amount of slaughter blood is calculated using slaughtered heads of swine and cattle, the total blood volume is approximately 15 billion litters. Assuming the yield of approximately 50%, total slaughter blood volume is calculated to be 7,294.5 million liters. Currently, some researchers have attempted to recycle unused blood as a functional material and food resource, but it was not practical due to the high cost of blood processing [21,22]. Animal blood is composed of 79.14% moisture, 19.40% of organic matter, and 1.46% of inorganic matter, and protein of organic matter is 18.22% including albumin 2.08%, globulin 1.99%, fibrinogen 0.12% and hemoglobin 14.02% . These blood proteins can be used as an emulsifier, colorant, fertilizer, medicine, and so on.
In previous studies, the addition of 2% blood plasma to meat product improved the yield by 4%–5% , and blood plasma could also be used as an alternative binding agent for egg white protein, and in particular, it showed higher binding ability than egg white protein . In addition, there was also an important study that heat-induced gelation could be changed by pH because of the different protein components (serum and albumin) in blood plasma . However, no studies have logically compared and analyzed the physicochemical characteristics of pork batters according to the horizontal levels of addition of non-meat proteins, which are mainly used most frequently in the meat processing industry.
Thus, the purpose of this study was to determine effect of porcine blood plasma (PBP) on physico-chemical characteristics of pork emulsion as emulsifier compared to that of commercial additives, SPI or SC, and to determine whether slaughter blood protein could be used in meat products as a binder.
MATERIALS AND METHODS
Commercial SPI and SC were bought from Dongbang foodmaster (Eumseong, Korea). Porcine blood was freshly taken right after slaughter and immediately moved to laboratory for plasma preparation. Ethylenediaminetetraacetic acid was added to obtained blood at 2 g per liter. Blood was placed immediately in an ice slash and transferred to the laboratory within 30 min. Then, samples were centrifuged (SUPRA 25K, Hanil Science, Gimpo, Korea) at 8,000×g for 15 mins at 4°C. Separated plasma was completely freeze-dried (PVTFD10R, Ilshinlab, Dongducheon, Korea) at −50°C. Fresh pork (Biceps femoris) and backfat from Landrace × Yorkshire × Duroc pigs were purchased from a local slaughter house. The pork was ground twice into 5 mm pieces with excess connective tissue removed.
Both PBP and commercial proteins used in this study were prepared in 10 kg units of emulsion. The basic recipe of emulsified pork batter consisted of 72.4% meat, 11.2% backfat, 14.9% ice, and 1.5% refined salt. Minced meat was ground for 1 min in a bowl cutter (Talsa K30, DSL Food Machinery, Burton-on-Trent, UK), then, 1.5% white salt and half of ice (1/2) were subsequently added to the batter and mixed for 2 min. As the experimental design (Control, no added; SPI 0.5%, 1%, 2%; SC 0.5%, 1%, 2%; and, PBP 0.5%, 1%, 2%), respective groups were prepared (Table 1). After 1 min, fat was added and mixed for 1 min. The remained ice (1/2) was put into a batter, and further mixed for 3 min at a high speed (bowl speed: 24 rpm; knife shaft speed: 2,840 rpm) to take a final emulsion batter. At this time, the temperature of batter was below at 11.5°C. The batters were stuffed into fibrous casings (Nalo Top, Kalle GmbH, Wiesbaden, Germany; 70-mm diameter) using a stuffer (IS-8, Sirman, Pieve PD, Italy). The stuffed emulsions were heated in a cooking chamber (Smoker 851, Thematec Food Industry, Seongnam, Korea) until the internal temperature going up 75°C. The cooked emulsion samples were then cooled at 4°C and used for subsequent physicochemical analyses.
The ES was analyzed by the modified method of Bloukas and Honikel . The emulsion batter was filled in to a pre-weighed graduated test tubes (Pyrex, Chojalab, Seoul, Korea, volume: 15 mL, graduated units: 0.2 mL). The test tubes were capped and heated for 30 min in a water bath to a core temperature of 75±1°C. After cooling down 4±1°C, water and fat separated in each graduated test tube were read out. The ES values were calculated as separation of water, fat and sum of them. The separated water and fat were calculated as a percentage to the total loss of the emulsified pork batter.
A 10 g of pork emulsion was weighted into small pieces to which 90 mL of distilled water was added, and slurry was mixed by a homogenizer (T25B, IKA Sdn. Bhd., Selangor, Malaysia) and the pH was measured using a pH meter. The pH meter was used to measure the pH of emulsified pork batters with calibration daily by standard buffers of pH 4.0 (9863 pH buffer solution, Mettler Toledo, Greifensee, Swizerland) and 7.0 (9865 pH buffer solution, Mettler Toledo) at 25°C. Shear force of the sample was measured by the Instron 3343 (US/MX50, A&D, San Jose, CA, USA) with a Warner Bratzler shearing device, in a 100 mm/min crosshead speed. Five cores (2 × 2 × 1 cm) of each emulsion were measured, in a crosshead speed of 100 mm/min. The CIE lightness (L*), redness (a*), and yellowness (b*) of emulsified pork batters were measured by a Minolta colorimeter (CR-400, Minolta, Osaka, Japan). Whiteness (W) was determined using the following formula: L*-3b*. The instrument was calibrated using a white plate (L* = 93.5, a* = 0.3132, b* = 0.3198) and D65 illuminant source. The chroma (C*) and hue angle were calculated as (a*2 + b*2) and Tan−1(b*/a*), respectively . The color measurements were replicated at five times on the surface of the slices randomly.
The textural properties analysis of the cooked batters were measured by the EZTest-500N texture analyzer (TA-XTZ-5, Shimadzu, Kyoto, Japan) with a cylindrical plunger (5 mm diameter, depression speed = 60 mm/min) and a 500N load cell. Texture profile variables were obtained from the hardness, cohesiveness, springiness, gumminess and chewiness, adhesiveness.
Randomized complete block design (RCBD) was used confirmation of insight. The data was analyzed by the orthogonal contrast test to find difference between the treatments (Control versus means of PBP + SPI + SC; PBP versus SPI; PBP versus SC), and regressions were used to evaluate the addition levels of non-meat ingredient (NMI). Duncan’s multiple range test was used to determine the statistical significance among the means at a 95% significance level. All data analysis was performed using SAS program for Windows 7.0, version 9.1.4 .
RESULTS AND DISCUSSION
Results of emulsion stability and physico-chemical properties of pork emulsions added with NMI are summarized in Table 2. The addition of NMI significantly increased the emulsion stability of pork emulsion. The total loss, percentages of moisture and fat losses in the total loss values of pork emulsions were lower in the treatment groups than control (p < 0.01). Especially each 2% addition group of NMI greatly were improved in the emulsion stability, and the 0.5% and 2% PBP additions further reduced the total loss, the percentages of moisture and fat losses in the total loss values of pork emulsion than those of SPI and SC (p < 0.05). Of the results of linear and quadratic regressions analysis, the emulsion stability values significantly were decreased as the PBP addition increases (p < 0.05). The pH values of pork emulsions added with NMI were lower than control (p < 0.01), and the pH value of pork emulsion added with PBP was lower than those of SPI and SC significantly. In the regression of pH values of pork emulsions, the NMI addition also showed to decrease all the pH values of treatments. The addition of NMI significantly affected the shear force values of pork emulsions compared to the control (1 vs 2–10), and the shear force values of pork emulsions were increased by the addition of 0.5% NMI (p < 0.01). However, the addition of 1% and 2% NMI did not show any remarkable results compared with the shear force values of 0.5% addition, because the regression results of shear force were not significant by the addition levels (1%–2%) of NMI. The shear force value of pork emulsion with PBP was higher than that of pork emulsion with SPI (p < 0.01), whereas there was no significantly different shear force between PBP and SC treatments.
In the instrumental color measurements of pork emulsions added with NMI, all color traits of pork emulsions added with NMI showed significant differences with those of control (control vs NMI). In each group, the addition of SPI significantly increased the lightness, yellowness, chroma and hue angle values, and the addition of SC significantly increased the lightness, whiteness, yellowness, and hue angle values and decreased redness and chroma values. The addition of PBP significantly increased redness and whiteness values, and decreased yellowness, chroma and hue angle values. The quadratic regression of lightness (L*) were significant in the SPI and SC treatments, and the PBP showed a not significant tendency (p-value = 0.065). In the redness (a*), the linear regression was significant in the SC treatment (p < 0.01), and the quadratic regression was significant in the SC and PBP treatments (p < 0.01). In the yellowness, significant p-values of regressions were observed in the PBP (Quadratic) and SPI (Linear and Q) emulsions. The whiteness was significant at quadratic regression in all treatments (p < 0.05), and only significant at linear regression in SPI treatment. The chroma was significant in the quadratic regression of SPI and PBP treatments, and only SPI treatment was significant in the linear regression. In the hue angle, the three NMI (SPI, SC, and PBP) were significant in the quadratic regression, and the linear regression was significant only in the emulsion with SPI or SC except for PBP. Meanwhile, in the comparison of the PBP and SPI or SC, the pork emulsion with PBP significantly was higher in the lightness, yellowness, and hue angle than those of pork emulsion with SPI or SC, but the redness of pork emulsion with PBP were lower than those of pork emulsion with SPI or SC (p < 0.01).
The textural properties of pork emulsion with NMI were presented in the Table 3. The addition of NMI did not show significant effect on the textural property traits of pork emulsions. But, the hardness (p-value = 0.05) of pork emulsion tended to increase by the addition of NMI compared to the control (control vs NMI), whereas the other textural traits were not affected by the addition of NMI (p > 0.05). On the other hand, there was a significant difference in the gumminess between PBP and SC treatments (p < 0.05), the gumminess of pork emulsion with PBP was higher than that of pork emulsion with SC, as well as the cohesiveness, chewiness, and adhesiveness values of pork emulsion added with PBP showed higher tendency than those of pork emulsion added with SC. Additionally there were no significant differences between the PBP and SPI pork emulsions. In the results of regressions, significances were only observed in the SPI treatment. Especially the hardness value of pork emulsion added with SPI was significant in the linear regression (p < 0.05), and the gumminess and chewiness values were significant in both linear and quadratic regressions (p < 0.05). Additionally, the addition of PBP had tendencies of significance on the regression results of hardness, gumminess, and chewiness.
Many researchers reported that soy proteins can improve emulsifying ability and stability of processed meat products by gel formation between the soy proteins and components of meat [15,28]. Fukushima  described that soy proteins are composed of the major globulins such as β-conglycinin (7S) and glycinin (11S) proteins. However, Zhang et al.  found that the 7s and 11s proteins were denatured at 70°C and 90°C, respectively. Lin and Mei  also reported that salt soluble and water soluble proteins in SPI were denatured by heating temperature above 76.7°C, and protective effect of soy proteins on meat proteins was not observed at high heating temperature (Approximately 68% denaturation rate of proteins at 82.2°C). SC is composed of the major proteins such as αs1-casein and β-casein which can be used to prepare stable emulsion by the covering of fat globule, because both proteins exhibit a net negative charge at neutral pH and, are substantially amphiphilic with a high proportion of accessible non-polar residues, thus have a strong tendency to absorb at hydrophobic surfaces . According to the study of Hurtado et al. , water holding capacity and cooking loss of pork sausage with added porcine plasma protein were not different with the sausages added SC and pentasodium tripolyphosphate. Also, Cofrades et al.  reported a similar result with the study of Hurtado et al. . This is because that PBP protein contains necessary proteins such as albumin and globulin for emulsification. Feiner  described that blood plasma has a pH value of 7.4 to 7.8, and addition of plasma for improvement of physicochemical properties slightly raises the pH in the meat product, resulting in increase of water-holding capacity. In this study, the concentrate of blood proteins was further increased unlike previous studies because of the freeze-dried blood plasma. In the study of Pietrasik et al. , cooking loss was not different among the pork gels added respective non-meat proteins (SC, PBP, and isolated soy protein at 2 g/100 g), and the pH values of pork gels also ranged from 5.87 to 6.26, the highest pH values were observed in the pork gels containing sodium caseinateSC and porcine blood plasmaPBP. Previously, Chin et al.  reported that addition of 2.2% SPI in the low-fat bologna sausage increased pH of sausage significantly. However, the pH result of this study was not similar to those of Chin et al. . It is believed that this is due to the increase of pH by the addition of 14.9% fat in our emulsion formulation. On the other hand, Hsu & Sun  indicated that the color values of pork meatball added with non-meat proteins. The lightness, redness and yellowness in the pork meatballs by addition of 4% SC and 4% SPI were not different compared with control significantly. However, in the result of Cofrades et al. , they reported that an increase in the proportion of plasma protein (0%–5%) significantly increase lightness and yellowness values of pork sausage.
According to the study of Herrero et al. , pork emulsion added with 2% NaCl and 3% PBP powder showed higher hardness, springiness, and breaking force than pork emulsion with no addition, as well as frankfurters with 33% PBP were significantly harder and chewer than those control sausages with 1.5% SC and 0.15% tripolyphosphate, contrary to hardness and chewiness, control was more adhesive than frankfurters with 33% pork blood plasma . In addition, Cofrades et al.  indicated that the hardness (p < 0.01), cohesiveness (p < 0.01) and chewiness (p < 0.001) in the pork sausage by addition of plasma protein (0–5%) were significantly increased. Meanwhile, Suter et al.  concluded that the addition of 1% beef plasma protein increased binding strength in cooked ground beef, but the addition of 2% plasma protein had no significant difference compared with that. These trends were similar with regression results of texture properties in this study, although significances were rare in the linear and quadratic regressions. In addition, in the comparison of pork meatballs added with of 10 non-meat proteins including SC, whey protein, skimmed milk powder, soybean flour, soy proteins, wheat gluten, egg white powder, and gelatin, the hardness, adhesiveness, viscosity, chewiness, and gumminess values were higher in the meatballs added with SC or SPI than those of the others . Until now, there were many studies that described the improvement effect of texture properties of each non-meat protein. But, it was not easy to find the study comparing the texture properties of SPI, SC, and PBP in pork emulsion. It is judged that additional experiments through different addition levels and processing methods are necessary.
To enhance quality characteristics such as binding ability, water holding capacity, and emulsifying ability of processed meat products, NMI are usually added to meat emulsion in the meat product industry. In this study, the emulsifying ability and quality characteristics of pork emulsions added with PBP or commercial NMI (SPI and SC) were compared. Our results revealed that pork emulsion added with PBP had similar emulsion stability and texture properties (based on the results of cooking loss, hardness, cohesiveness, gumminess, and chewiness) compared to pork emulsions added with SPI or SC. Our results revealed that pork emulsion added with PBP had similar emulsifying ability and texture properties compared to control and those added with SPI or SC. Therefore, PBP could be used as an emulsifying agent for pork emulsion products if its mass production system is in place industrially.