Evaluation of mortadella formulated with carbon monoxide-treated porcine blood

Meat Science 97 (2014) 164–173

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Evaluation of mortadella formulated with carbon monoxide-treated porcine blood A.D. Pereira b, L.A.M. Gomide a,⁎, P.R. Cecon c, E.A.F. Fontes a, P.R. Fontes a, E.M. Ramos d, J.G. Vidigal e a

Departamento de Tecnologia de Alimentos, Universidade Federal de Viçosa, CEP, 36570-000 Viçosa, MG, Brazil Departamento de Nutrição, Universidade Federal do Tocantins, CEP, 77001-090 Palmas, TO, Brazil Departamento de Informática, Universidade Federal de Viçosa, CEP, 36570-000 Viçosa, MG, Brazil d Departamento de Ciência dos Alimentos, Universidade Federal de Lavras, CEP, 37200-000 Lavras, MG, Brazil. e Diretoria do Departamento de Pesquisa e Extensão, Instituto Federal Fluminense, CEP, 24220-900 Niterói, RJ, Brazil. b c

a r t i c l e

i n f o

Article history: Received 12 April 2013 Received in revised form 19 January 2014 Accepted 21 January 2014 Available online 5 February 2014 Keywords: Pork blood Carbon monoxide Sausage color Chemical characteristics

a b s t r a c t The proximate composition and color of mortadellas containing carbon monoxide-treated (COTB), untreated (UNTB), or CO-treated dried blood (CODB) were compared to that of control mortadella. Blood addition did not affect (P N 0.05) the proximate composition and TBARS. The mortadella containing 10% UNTB were brown and those containing COTB or CODB were red. Residual nitrite level, L*, a*, b* and c* values of the mortadella decreased (P b 0.05) with an increase in the amount of blood; TBARs did not vary (P N 0.05). Increasing the amount of blood increased (P b 0.05) the hue angle (h*) and browning index (BI) of the mortadella containing UNTB. Increasing blood addition decreased (P b 0.05) h* and did not affect (P N 0.05) BI. Increasing storage length decreased (P b 0.05) residual nitrite, affected BI and color coordinates and did not affect TBARS (P N 0.05). Addition of CO-treated blood allows the production of better-colored sausages having lower residual nitrite levels. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction The food industry has been making considerable efforts to identify alternatives for food by-products in response to the increase in the world population and the costs of food (particularly protein) production by conventional systems. In this regard, blood, which is one of the main by-products of the meat industry, could prove to be a raw material of excellent quality because it is a rich source of dietary heme iron and proteins and thus presents good nutritional and functional quality (Dill & Landmann, 1988; Gorbatov, 1988; Miller & Menichillo, 1991). Despite its potential for use in human nutrition, the dark brownish color that blood imparts on food formulations is a great restriction for its use as a raw material or ingredient in the food industry (Mielnik & Slinde, 1983; Wismer-Pedersen, 1988). As a result, studies have been conducted in an attempt to solve this color problem (Wismer-Pedersen, 1988) because food color decisively influences consumer preferences and the acquisition decision (Carpenter, Cornforth, & Whittier, 2001; von Elbe & Schwartz, 1996). According to von Elbe and Schwartz (1996), foods achieve their best classifications and greater prices through their desired colors, which consumers relate to the quality of the raw materials. To minimize darkening problems due to the use of blood in food formulations, various solutions have been proposed. However, most of the proposed solutions present some type of drawback and are thus ⁎ Corresponding author. Tel.: +55 31 3899 1754; fax: +55 31 3899 2208. E-mail address: [email protected] (L.A.M. Gomide). 0309-1740/$ – see front matter © 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.meatsci.2014.01.017

not completely satisfactory. Of these, the most adopted solutions have used the plasma fraction alone, the discoloration of globin through heme elimination from the hemoglobin moiety, and their separate or combined utilization in a food product (Caldironi & Ockerman, 1982). However, these techniques impart undesirable flavor to the products and cause decreases in the biological and functional values of blood proteins and in the bioavailability of iron (Ockerman & Hansen, 1994). In a previous attempt to solve this problem, Fontes, Gomide, Ramos, Stringheta, and Parreiras (2004) and Fontes, Gomide, Ramos, Fontes, and Ramos (2010) showed that blood saturated with CO produced a pigment with a stable and desirable color that could allow a greater amount of blood addition to meat products that would not lead to their browning. This approach is based on the high affinity of the CO moiety to heme pigments, which is a concept that has been successfully used for the modified atmosphere packaging of meat cuts (Carpenter et al., 2001; Jayasingh, Cornforth, Carpenter, & Whittier, 2001; Luño, Beltrán, & Roncalés, 1998; Luño, Roncalés, Djenane, & Beltrán, 2000; Mancini & Hunt, 2005; Mancini, Suman, Konda, & Ramanathan, 2009; Sørheim, Aune, & Nesbakken, 1997; Sørheim, Nissen, Aune, & Nesbakken, 2001; Sørheim, Nissen, & Nesbakken, 1999; Sørheim et al., 2006) and was approved in the US, New Zealand, and Australia (Cornforth & Hunt, 2008; Wilkinson, Janz, Morel, Purchas, & Hendriks, 2006). Even so, Mancini and Hunt (2005) suggested that the likelihood of conversion of oxymyoglobin to carboxymyoglobin should receive further attention. In addition to color problems, blood addition to meat products may pose keeping and safety concerns as it may interfere with fat oxidation

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and residual nitrite level (Cammacka et al., 1999; Cassens, 1997; Miller & Menichillo, 1991; Tompkin, Christiansen, & Shaparis, 1978; Verma, Paranjape, & Ledward, 1985), which could diminish its acceptance (TBARS over 1 mg kg−1) and antibotulinical effect (residual nitrite below mg kg−1). However, hemoglobin complexation with carbon monoxide generates a compound of greater chemical stability making it possible that blood use in meat sausages may not be a problem (Antonini & Brunori, 1971; Fontes et al., 2004, 2010; Lanier, Carpenter, Toledo, & Reagan, 1978; NAS, 1977). Based on these findings, the present paper was designed to evaluate the chemical and color characteristics of mortadella formulated by the substitution of the beef forequarter for different levels of liquid or reconstituted dried carbon monoxide-treated blood and CO-untreated blood. 2. Materials and methods 2.1. Blood collection and treatment with CO Swine blood was collected through an aseptic closed system (Fontes et al., 2004, 2010) using a “vampire” knife connected to a non-toxic hose. During each replication, blood from 20 animals was collected into a large stainless-steel can to which a mechanical stirring system was connected. During exsanguination, sodium citrate solution (0.5% relative to the total blood volume) was added to the system to avoid blood clotting. After cooling (3–4 °C), two-thirds of the collected blood was transferred to a closed stainless-steel tank to which a mechanical stirring system was connected. Through a perforated disk located at the bottom of the tank, carbon monoxide gas (99% pure; White Martins) was bubbled until CO saturation was achieved (to generate 100% HbCO) to produce the CO-treated blood (COTB) (Fontes et al., 2004). The other third of the collected blood was left untreated (UNTB). Half of the COTB was dried in a Niro Atomizer spray-dryer to obtain dry blood (CODB) with approximately 8% moisture (Fontes et al., 2010). The dry blood was vacuum-packaged and stored in the dark until sausage processing. The other portion of the COTB and the UNTB was maintained at 3–4 °C until sausage processing. 2.2. Sausage manufacture The frozen deboned beef forequarter (shank and chuck) was thawed for 24 h at 4 °C, and the visible fat and superficial connective tissue were trimmed off. In each of three replications, control mortadella was produced with 7 kg of lean beef forequarter (shank and chuck) and 3 kg of pork backfat (2 kg minced and 1 kg of cubed). To this meat mixture, 0.25% phosphate (Adifós—ADICON IND. e COM. de ADITIVOS LTDA, São Bernardo do Campo, SP, Brazil), 20.0% ice cubes, 2.3% salt, 0.25% ascorbate (Newcor—ADICON IND. e COM. de ADITIVOS LTDA), 0.35% curing salt (Curag—ADICON IND. e COM. de ADITIVOS LTDA), 0.3% mortadella seasoning (Temperex M DAL4-200—ADICON IND. e COM. de ADITIVOS LTDA), 4.0% textured soy protein (Beef Pink—Pink Alimentos do Brasil, Belo Horizonte, MG, BRAZIL), and 5.0% commercial cassava starch (Hikari Alimentos, São Paulo, SP, BRAZIL) were added during cutterization. Replicates of the other twelve mortadella treatments were similarly produced by substituting the lean beef forequarter for fresh CO-untreated blood (UNTB), CO-treated blood (COTB), or reconstituted CO-treated dried blood (CODB) to obtain mortadella formulations containing 5%, 10%, 15%, or 20% added blood (Table 1). The CODB was reconstituted to the same moisture content (approximately 79%) of both the UNTB and the COTB by slowly adding water to the CODB followed by vigorous mixing for approximately 5 min in a household blender. For each of the three replications of the mortadella formulations, refrigerated (4 °C) beef forequarter, ice, and blood (except

165

for the control) were transferred to the cutter (Mainca MD-40 BL), and the mincing started. Phosphate was rapidly added. After 30 s, salt was added, and the mincing process was continued for an additional 30 s, at which point the mortadella seasoning, curing salt, ascorbate, cassava flour, and powdered textured soy protein were added. The mincing was continued until the batter reached 7 °C. Two thirds (2 kg) of pork backfat were then added, and the mincing was continued until the emulsified batter reached 15 to 16 °C. The emulsified batter was transferred to a mixing batch, where the remaining third (1 kg) of the pork backfat (cut into 5-mm cubes) was added to the emulsion batter and mixed. The mixture was then stuffed into 63-mm opaque bi-directed nylon casings (Nalobar APM 63, Kalle Nalo; watervapor permeability of 5 g/m2 d at 23 °C and 85% relative humidity; permeability to oxygen of 12 cm3/m2 d bar at 23 °C and 53% relative humidity) using a hydraulic stuffer (Mainca EM-25) to produce mortadella of approximately 700 g. The mortadellas were cooked in a water bath to an internal temperature of 74 °C (checked in the center of the mortadellas with a flexible thermocouple probe connected to a TESTO 175-T3 data logger) following a previously established cooking set-up: 55 °C for 30 min, 65 °C for 30 min, 75 °C for 30 min, and 85 °C for 30 min. The mortadellas were then immersed in an ice water bath (0 °C) for 10 min and chilled (4 °C) before analysis.

2.3. Proximate composition, TBARS analysis and residual nitrite analysis Proximate composition of the experimental mortadella was conducted in duplicate, for protein (A.O.A.C., 1993), moisture, fat and ash content (A.O.A.C., 1990). Carbohydrate content was obtained by difference to 100% of the moisture, protein, ash and fat percentages (A. A. C. C, 1962). Lipid oxidation (TBARS) was determined in triplicates every two weeks in 10 g of cooked mortadellas following a modification (0.2 mL of 0.03% BHT and 1 mL of Sigma Antifoam A) of the method of Tarladgis, Watts, and Younathan (1960). TBARS (mg malonaldehyde equivalents/kg of sample) was calculated by multiplying Abs530 nm (Hitachi U-2001spectrophotometer) readings by 7.8. Residual nitrite content of each mortadella repetition was evaluated, during 8 weeks, in duplicate in 5 g of the cooked mortadella (A.O.A.C., 1990).

2.4. Color measurements The color of the mortadella was objectively evaluated weekly for up to 56 days of storage. To avoid light and oxygen fading at each storage time, one mortadella of each treatment was opened prior to the color measurements. The mortadella fat cubes were removed, and the emulsified batter was minced and placed inside a rectangular quartz cuvette (20-mm path length) to determine the CIELAB color coordinates using a Color Quest II Sphere spectrophotometer (HunterLab, Reston, VA, USA) connected to a computer with the Universal System software. The following settings were used: D65 illuminant, 10º observer angle, and reflectance specular included (RSIN). The L*, a*, and b* coordinates were obtained using the average values of five readings from different cuvette locations; the completely filled cuvette was placed in the spectrophotometer's reflectance port (1-inch opening), and the color measurements were taken from five different positions. The chroma (C*) and hue angle (h*) were calculated using the following formulas: C* = (a⁎2 + b⁎2)1/2 and h* = tan− 1 (b*/a*) (Hunt et al., 1991). To evaluate the color differences and fading, the browning index (ratio of reflectance at 570 nm and 650 nm) defined by Fontes et al. (2010) was also used.

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Table 1 Experimental mortadellas. Treatment

T1

T2

T3

T4

T5

T6

T7

T8

T9

T10

T11

T12

T13

Lean (beef forequarter)a Minced pork fat Cubed pork fat COTBa UNTBa CODBa

70 20 10 0 0 0

65 20 10 5 0 0

60 20 10 10 0 0

55 20 10 15 0 0

50 20 10 20 0 0

65 20 10 0 5 0

60 20 10 0 10 0

55 20 10 0 15 0

50 20 10 0 20 0

65 20 10 0 0 5

60 20 10 0 0 10

55 20 10 0 0 15

50 20 10 0 0 20

a

UNTB = CO-untreated blood, COTB = CO-treated liquid blood, CODB = dry CO-treated, T1 = control treatment.

2.5. Statistical analysis

3.2. TBARS

Proximate composition was subjected to analysis of variance in a split-plot scheme with the treatment (UNTB, COTB, and CODB) on the plot and the levels of added blood (5, 10, 15, and 20%) on the sub-plot. through a completely randomized design. The results were subjected to variance and regression analyses using the SAS System for Windows™ program (version 9.2, SAS Institute, Inc.) with a 5% significance level. The means of the qualitative factors (blood types) were compared using Tukey's test, whereas the models of the quantitative factors were chosen based on the determination coefficient and the regression coefficient significance using a t-test. The nitrite content, TBARS values, color coordinates and browning index were subjected to analysis of variance in a split-plot scheme with the treatment (UNTB, COTB, and CODB) on the plot, the levels of added blood (5, 10, 15, and 20%) on the sub-plot, and the storage time (0, 7, 14, 21, 28, 35, 42, 49, and 56 days) on the sub-sub-plot through a completely randomized design. The results were subjected to variance and regression analyses using the SAS System for Windows™ program (version 9.2, SAS Institute, Inc.) with a 5% significance level. The means of the qualitative factors (blood types) were compared using Tukey's test, whereas the models of the quantitative factors were chosen based on the determination coefficient and the regression coefficient significance using a t-test. The comparison of the treatments (blood-added samples) with the control (samples without blood addition) of recently processed mortadellas was conducted through analysis of variance. This analysis was performed considering a completely randomized design for a split-plot consisting of thirteen treatments in a factorial scheme (3 × 4 + 1) with the blood type in the plot and the blood level in the sub-plot. The mean of the control was compared with the means of each treatment using Dunnett's test considering a 5% significance level. The comparison of recently processed control mortadella (no blood) against recently produced mortadella with different amounts of each type of blood (UNTB, COTB or CODB) substituted for meat was conducted through analysis of variance considering a completely randomized design with thirteen treatments (3 × 4 + 1) in a splitplot scheme with the blood type in the plot and the blood level in the sub-plot. The mean of the control was compared with the means of each treatment using Tukey's test considering a 5% significance level.

TBARS values were not influenced (P N 0.05) by any of the independent variables or their interactions (Table 3). Carboxyhemoglobin is less oxidative than other hemoglobin pigments (HbO2 and Hb) since, similar to NO binding, CO binding to the sixth coordination state of the heme iron stabilizes the pigment making it less reactive; this results in less pigment oxidation and warmed-over-flavor development in processed meats (Fontes et al., 2004, 2010; Livingston & Brown, 1981; NAS, 1977; Verma et al., 1985). Besides the presence of reducing agents (nitrite and ascorbates), high levels of blood may also explain the abscence of TBARS variation during storage (Ŷ = 0.54 mg malonaldehyde kg− 1) which never reached the threshold value of 1.0 mg malonaldehyde kg−1 (Tarladgis et al., 1960). Regardless of the type and concentration of blood, TBARS values of blood added mortadelas did not differ from that of the control mortadella (Table 3). This may also be explained by the generation of monolipidic complexes that avoids initiation and propagation of oxidation processes (Fox & Benedict, 1987) and to the fact that, rather than releasing the pro-oxidant free iron during the cooking process (Chen, Pearson, Gray, Fooladi, & Ku, 1984; Igene, King, Pearson, & Gray, 1979; Igene & Pearson, 1979), at least part of hemoglobin of the UNTB reacted with added nitrite to yield the stable and less reactive NOHb (Livingston & Brown, 1981; NAS, 1977; Verma et al., 1985).

3. Results and discussion 3.1. Proximate composition Because blood has a proximate composition similar to that of meat (Gorbatov, 1988), independent of the type or amount of blood addition, the proximate composition of the blood added mortadella (11.41% protein, 21.61% fat, 55.90% moisture, 3.30% ash and 8.05% carbohydrate) did not differ (P N 0.05) from that of the control (Table 2).

3.3. Residual nitrite Residual nitrite decreased (P b 0.05) during storage (Fig. 1). Even the initial (week 0) levels were much lower than the amount added (385 mg kg−1) to the formulations (Figs. 1 and 2). This is because the pasteurization process and the addition of reducing agents produces a 50 to 65% decrease in the ingoing amount of nitrite (Honikel, 2008; Mowad, Abozeid, & Nadir, 2012) of sausages and only about 10 mg kg− 1 remains after storage (Cassens, 1997; Pérez-Rodríguez, Bosch-Bosh, & Garcia-Mata, 1996; Pérez-Rodríguez, Garcia-Mata, & Bosch-Bosh, 1998). Tompkin et al. (1978) reported residual nitrite levels of 25 mg kg−1 after the sterilization of cured canned meats containing 1% of added hemoglobin. While the nitrite level of control mortadella dropped from 42.36 mg kg−1 (week 0) to 14.70 mg kg−1 (week 8), those of mortadella containing CODB, COTB and UNTB dropped from 17.97 mg kg−1, 17.23 mg kg− 1 and 11.21 mg kg− 1 to 9.39 mg kg−1, 8.11 mg kg−1 and 7.13 mg kg−1 (Fig. 1). Considering that each hemoglobin molecule is roughly equivalent to 4 myoglobin molecules, the decrease in residual nitrite with blood addition may be explained by the fact that each 5% in blood addition increases heme pigment concentration by an equivalent of 108 g of myoglobin. Blood type did not influence (P N 0.05) residual nitrite levels. However, considering the greater stability and lower dissociation

A.D. Pereira et al. / Meat Science 97 (2014) 164–173

167

Table 2 Proximate composition of newly (week 0) formulated control (no blood added) and of mortadella containing different levels of UNTB, COTB and CODB. Averagesa and standard deviations

Blood type and level

Protein Control (no blood added) CODB—5% CODB—10% COD—15% CODB—20% COTB—5% COTB—10% COTB—15% COTB—20% UNTB—5% UNTB—10% UNTB—15% UNTB—20% a

11.65 11.53 11.39 11.52 11.61 11.48 11.44 11.14 11.09 10.90 11.17 11.77 11.58

± ± ± ± ± ± ± ± ± ± ± ± ±

Fat 1.25 0.22ns 0.10ns 0.93ns 0.87ns 0.43 ns 0.73ns 0.74ns 0.52 ns 0.65 ns 0.03 ns 0.26 ns 0.12 ns

21.26 21.95 22.28 20.19 19.24 22.58 21.55 24.75 20.76 21.94 22.44 21.45 20.54

Moisture ± ± ± ± ± ± ± ± ± ± ± ± ±

1.06 1.57ns 2.26ns 0.89ns 0.73ns 2.29ns 1.88ns 3.32ns 3.77ns 2.75 ns 1.24ns 3.08ns 1.48ns

53.94 55.90 54.90 56.27 55.76 56.00 55.84 54.95 56.53 56.52 57.04 56.29 56.72

± ± ± ± ± ± ± ± ± ± ± ± ±

Ash 2.28 2.52ns 2.78ns 1.96ns 1.98ns 2.31ns 0.90ns 0.97ns 0.85ns 2.52ns 2.78ns 1.96ns 1.98ns

3.03 3.04 3.05 3.04 2.98 3.05 3.01 3.00 3.08 3.03 2.94 3.06 3.12

Carbohydrates ± ± ± ± ± ± ± ± ± ± ± ± ±

0.29 0.05ns 0.14ns 0.17ns 0.09ns 0.15ns 0.24ns 0.28ns 0.19ns 0.04ns 0.32ns 0.27ns 0.18ns

10.12 7.58 ns 8.37 ns 8.98 ns 10.4 ns 6.89 ns 8.17 ns 6.16 ns 8.53 ns 7.59 ns 6.42 ns 7.43 ns 8.05 ns

Averages of triplicate analysis of three repetitions of each treatment.

rate of the carboxyhemoglobin (HbCO) in comparison to deoxy (Hb) and oxyhemoglobins (HbO2) (Fontes et al., 2004, 2010) and the fact that the diamagnetic character of ligand dissociation from the heme iron in COHb makes it more stable than the paramagnetics HbNO and MetHb (Livingston & Brown, 1981), it was expected that mortadella containing COTB and CODB would have higher residual nitrite levels than those containing UNTB. Considering a minimum amount of 10 mg kg−1 of residual nitrite to control Clostridium botulinum (Cassens, 1997), blood-added mortadella may pose a risk of botulism. Mortadella containing COTB and CODB were above this critical nitrite level only until 2 weeks of cold storage (15.60 mg kg− 1 and 13.16 mg kg− 1, respectivelly), whereas those containing UNTB were above this critical residual nitrite level only up to 1 week of cold storage (10.77 mg kg− 1). Unless C. botulinum is controlled by residual nitrite levels below 10 mg kg− 1, blood-added sausages should be manufactured with increased amounts of ingoing nitrite. Pérez-Rodríguez et al. (1998) reported residual nitrite levels of 8–10 mg kg−1 in frankfurters after 12 days of storage. At all storage times mortadella containing 5% blood had higher (P b 0.05) initial (25.48 mg kg− 1) and final (11.92 mg kg− 1) levels of nitrite than that of mortadella containing 10% (13.79 mg kg− 1 and 7.53 mg kg − 1), 15% (13.89 mg kg− 1 and 6.98 mg kg − 1) or 20% (11.21 mg kg− 1 and 6.42 mg kg− 1) of blood addition (Fig. 2).

contains 5 mg Mb g−1 and between 0.2 and 2 mg Hb g−1, while blood contains 150 mg Hb g−1; as a result, the addition of 1% blood increases the hemoglobin content of a recipe by approximately 1.5 mg g− 1 (Mielnik & Slinde, 1983). Although darker, the mortadellas containing COTB or CODB were still red in color even at 20% of blood addition. However, for mortadella containing UNTB, the color darkening resulted in a change in the final color to brown at or above 10% of blood addition (Fig. 3). This darkening promoted the decrease in the reflectance spectra of the blood-added mortadellas at all wavelengths (Fig. 4). For every type of blood added the difference between the reflectance spectra (Fig. 4) of the controls and of blood-added mortadellas increased with blood addition. Similar results have been reported (Ferreira, Fernandes, & Yotsuyanagi, 1994; Mielnik & Slinde, 1983; Oellingrath & Slinde, 1985). The lightness (L* values) of the mortadellas was affected by the amount (Fig. 5A) of blood and by the storage time (Fig. 6A) but not (P N 0.05) by the type of blood used (UNTB, COTB, CODB) or interactions. As reported for other meat products (Ferreira et al., 1994; Guzmán, Mclillin, Bidner, Dugas-Sims, & Godber, 1995; Mielnik & Slinde, 1983; Oellingrath & Slinde, 1985; Zhou, Wang, Wang, Zhang, & Cai, 2012), the increase in heme content due to the addition of UNTB, COTB or CODB yielded (P b 0.05) mortadellas with lower lightness than controls (Fig. 5A).

3.4. Color The substitution of meat by blood yielded darker mortadellas due to the increase in their pigment content (Fig. 3). This is because beef

Blood type and level

TBARS

Control (no blood added) CODB—5% CODB—10% CODB—15% CODB—20% COTB—5% COTB—10% COTB—15% COTB—20% UNTB—5% UNTB—10% UNTB—15% UNTB—20%

0.62 0.67 0.69 0.39 0.50 0.66 0.37 0.32 0.64 0.38 0.63 0.67 0.56

± ± ± ± ± ± ± ± ± ± ± ± ±

Residual nitrite (ppm) 0.48 0.45ns 0.42ns 0.13ns 0.23ns 0.43ns 0.11ns 0.13ns 0.46ns 0.16ns 0.50ns 0.16ns 0.21ns

24.63 17.55 11.85 10.17 7.93 16.27 8.69 10.13 8.27 12.09 6.77 6.87 7.36

± ± ± ± ± ± ± ± ± ± ± ± ±

ns—Non-significant at probability level of 5% by Dunnett test (P N 0,05). Averages and standard deviations of 15 observations in duplicate. Averages and standard deviations of 27 observations in duplicate.

6.25 6.11ns 4.23ns 4.24ns 2.42ns 5.61ns 3.01ns 4.81ns 2.56ns 6.25ns 1.85ns 1.64ns 1.53 ns

Residual nitrite (mg kg-1)

Table 3 TBARS and residual nitrite levels of control mortadella (no blood added) and of mortadella containing different levels of UNTB, COTB and CODB.

50

40

30

20

10

0 0

2

4

6

8

Time (weeks) Fig. 1. Effect of storage on residual nitrite of control mortadellas (Δ) and of mortadellas formulated with CO-treated liquid blood (COTB—○), dry CO-treated blood (CODB—▲), and CO-untreated blood (UNTB—●).

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Residual nitrite ( mg kg-1)

30

25

20

15

10

5 0

2

4

6

8

Time (weeks) Fig. 2. Effect of storage on residual nitrite of mortadellas formulated with 5% (Δ), 10% (○), 15% (▲) and 20% (●) of blood addition.

Despite the greater lightness of COTB and CODB than that of UNTB (Fontes et al., 2004, 2010), the L* values of mortadella containing COTB and CODB did not differ (P N 0.05) from those of mortadella containing UNTB (Fig. 5A). This is in accordance with the slight lightness differences observed in sausages produced with the addition of cured or uncured blood (Mielnik & Slinde, 1983). During storage, there was a slight increase (P b 0.05) in the lightness, which reflects some degree of color fading (Fig. 6A). Because the mortadellas were stuffed in opaque nylon casings and one mortadella of each treatment was used for the color analysis at each storage time, this fading is not a result of exposure to light and/or oxygen (Erdman & Watts, 1957; Larsen, Westad, Shoreim, & Nilsen, 2006; Watts, Erdman, & Wentworth, 1955). Since cured color fading may be prevented, delayed or even reversed as long as there is sufficient nitrite and reducing agents (Cassens, 1997;

Erdman & Watts, 1957; Watts et al., 1955), this fading may be due to the observed reduction in the amount of residual nitrite (Fig. 2) and amount of reducing agents (not analyzed) over time (Erdman & Watts, 1957; Izumi, Cassens, & Greaser, 1989; Vossen et al., 2012; Watts et al., 1955). Furthermore, Vossen et al. (2012) stated that, though capable of avoiding lipid oxidation, nitrite and ascorbates may not prevent protein oxidation and the formation of protein radicals, which are more reactive than lipid radicals. At all storage times, the lightness of the control remained greater than that of the blood-added mortadellas (Fig. 6A). The redness (a*) of the mortadella was affected (P b 0.05) by interactions between the amount and type of blood (Fig. 5B) and between the blood type and the storage time (Fig. 6B). The redness of the mortadellas decreased (P b 0.05) with blood addition (Fig. 5B) and this decrease was more pronounced when UNTB was used than when COTB or CODB were used. Although it may be expected for the mortadella containing UNTB, the decrease in the redness with increasing amounts of COTB or CODB was unexpected and may indicate pigment oxidation (Vossen et al., 2012), which may be a result of some CO displacement by oxygen incorporated into the emulsion batter during the mincing process in the cutter. This mincing may introduce a sufficient amount of oxygen into the batter to overwhelm the greater affinity of CO for the sixth heme iron coordination position (Fontes et al., 2010). This would lead to a conversion of some of the carboxyhemoglobin and carboxyhemochrome into hemichrome, slightly reducing (P b 0.05) the redness of the mortadella after the cooking process. Compared to the control, when UNTB was used the redness of the mortadellas was greater (P b 0.05) at 5% and lower (P b 0.05) at 20% of blood addition; at 10% or 15% of UNTB addition the redness of mortadellas containing UNTB did not differ from that of the control (Fig. 5B). For mortadellas containing COTB or CODB the redness was greater (P b 0.05) than that of the control at any level of blood addition (Fig. 5B).

Fig. 3. Visual color* of mortadellas formulated with different levels of CO-untreated blood (UNTB), CO-treated blood (COTB), or dry CO-treated blood (CODB). * Colored online.

A.D. Pereira et al. / Meat Science 97 (2014) 164–173

A

60

50

Reflectance (%)

Reflectance (%)

B

60

50 40 30 20 10 0 400

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Fig. 4. Initial (week zero) and final (week 8) reflectance spectra of mortadellas formulated with 5% (A), 10% (B), 15% (C) and 20% (D) of meat substitution for UNTB, COTB, CODB and their comparison with the initial spectrum of control mortadellas.

Greater redness of mortadellas containing 5% of UNTB addition is in accordance with observations that the addition of 0.5 to 4% of blood leads to an increase in the redness of meat products (Ferreira et al., 1994; Mielnik & Slinde, 1983; Oellingrath & Slinde, 1985). This may be due to the yield of the pinkish-red nitrosohemochrome from the additional pigments introduced in the formulation when there are enough ascorbate and meat reductants in the system to reduce heme iron and nitrite to nitric oxide (Fox & Nicholas, 1974; Honikel, 2008; Izumi et al., 1989). The yield of nitrosohemochrome is supported by the decrease in residual nitrite in blood-added mortadellas (Fig. 1) and the fact that, compared to recently produced (week 0) mortadellas, at all levels of blood addition, after 8 weeks of storage the reflectance spectra of mortadellas containing UNTB, COTB and CODB is closer to that of controls (Fig. 4). Mielnik and Slinde (1983) showed that the reflectance spectrum of sausage containing cured blood is closer to that of control sausages than that of sausage containing uncured blood. Lower redness in mortadella containing 20% of UNTB blood is probably due to the prevalence of the brownish hemichrome. This is because at this level of UNTB addition there may be insufficient reducing capacity and nitrite in the system to keep all of the additional heme iron in the reduced state and to generate enough NO to bind to them; thus,

a greater amount of the hemoglobin would be oxidized to hemichrome, leading to a decrease in redness. Absence of difference in redness between the control and mortadellas containing 10% or 15% of UNTB may be due to the fact that, at intermediate levels of UNTB addition, there may be a mixture of nitrosohemochrome and hemichrome. Greater redness in mortadellas containing COTB or CODB than in the control was also reported by Zhou et al. (2012). Besides the increase in the heme content this is due to the fact that COTB has an a* value of 16.1 (Fontes et al., 2004), and CODB has an a* value of approximately 19 (Fontes et al., 2010). This increase in redness results from the high amount of a red stable pigment generated by binding CO to the sixth coordination position of the heme iron in the CO-treated bloods, which minimizes its oxidation during the cooking of the emulsion batter (Fontes et al., 2004, 2010; John et al., 2004; Lanier et al., 1978; Livingston & Brown, 1981; Verma et al., 1985). Only at 5% of blood addition did the a* values of the mortadella containing UNTB not differ (P N 0.05) from those of the mortadella containing COTB or CODB (Fig. 5B). At all other levels the mortadellas containing COTB or CODB had (P b 0.05) greater a* values than those containing UNTB.

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Fig. 5. Effect of the amount of added CO-untreated blood (UNTB—▲), CO-treated liquid blood (COTB—○), or dry CO-treated blood (CODB—●) on the color parameters of the resulting mortadellas. * Significantly different (P b 0.05) from the control, as determined by Dunnett's test. ns Not significantly different (P N 0.05) from the control, as determined by Dunnett's test.

Whereas the redness of the control and of the mortadellas containing COTB or CODB did not vary, the redness of the mortadellas containing UNTB increased during storage (Fig. 6B). This increase in the redness of the mortadella containing UNTB may be due to the presence of agents that may reduce both nitrite and heme pigments over time. In this case, at least part of the generated nitric oxide would bind to the reduced hemoglobins to yield nitrosohemocrome, and at least part of the brownish hemichromes would turn into the pinkish-red nitrosohemocrome, increasing the redness of the mortadella containing UNTB; this may be evidenced in the reflectance spectra (Fig. 4). In contrast, the maintenance of the redness of the control and of the mortadella containing COTB or CODB may be due to the greater stability of

nitrosohemochrome and carboxyhemochrome (Fontes et al., 2004, 2010; John et al., 2004). The yellowness of the mortadella was only affected (P b 0.05) by the amount of blood and by the interaction between the type of blood and the storage time. As reported by Guzmán et al. (1995), the yellowness of the mortadella decreased (P b 0.05) with blood addition; at all level of addition, it was lower (P b 0.05) than that of the control (Fig. 5C). However, Zhou et al. (2012) reported yellowness increase in cooked pork sausages, while Mielnik and Slinde (1983) and Oellingrath and Slinde (1985) showed only slight variation in yellowness of meat loaves with up to 7% of blood addition.

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Fig. 6. Effect of storage on the color parameters of control mortadellas (Δ) and of mortadellas formulated with CO-treated liquid blood (COTB—○), dry CO-treated blood (CODB—●), and CO-untreated blood (UNTB—▲).

Only at 5% of blood was the yellowness of the mortadella containing UNTB lower (P b 0.05) than that of mortadellas containing COTB or CODB. At all other levels, there were no differences (P N 0.05) in yellowness (Fig. 5C). Whereas the yellowness (b* values) of the control and of the mortadella containing COTB or CODB blood did not change (P N 0.05), the yellowness of the mortadella containing UNTB increased (P b 0.05) during storage (Fig. 5C). These observations are most likely due to the generation of nitrosohemochrome over time and to the greater stability of carboxy pigments.

Zhou et al. (2012) also observed maintenance of yellowness during storage of sausages containing CO-treated blood. During storage, the control always had greater b* values (P b 0.05) than blood-added mortadellas (Fig. 6C). The chroma of the mortadellas was affected by interactions between the type and the amount of blood and between blood type and storage time. Despite the visual appraisal of increased color intensity of bloodadded mortadellas (Fig. 3), their chroma decreased with blood addition and this was (P b 0.05) more pronounced in mortadella containing

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UNTB (Fig. 5D); the chroma of mortadella containing COTB or CODB did not differ (P N 0.05). The decrease in chromaticity with blood addition may indicate a shift in the predominant pigment chemical form. It is hypothesized that the less-intense decrease in chromaticity of mortadellas containing COTB or CODB reflects the smaller decrease in their redness and may indicate a dilution of the pinkish color of nitrosohemocrome of cured cooked sausages due to the incorporation of great amounts of the reddish carboxy-pigments of CO-treated bloods. On the other hand, the greater intensity of chromaticity decrease in the mortadella containing UNTB may indicate a change in the predominance of the pinkish nitrosohemocrome into the brown color of hemichromogen as reflected by the greater decrease in its redness (Fig. 5B). The decrease in chromaticity could also be due to some pigment oxidation and/or color fading (Vossen et al., 2012), especially when UNTB is used in the formulation. The chromaticity of mortadellas containing 10% or more UNTB or 15% and 20% of COTB or CODB were lower (P b 0.05) from that of the control (Fig. 5D). While the chromaticity of the control and of the mortadella containing COTB or CODB did not change (P N 0.05), those of the mortadella containing UNTB increased (P b 0.05) during storage (Fig. 6D), which is probably due to the conversion of part of the hemichromogen into nitrosohemochrome when nitrite and reducing conditions are still available. This is supported by the increase in the reflectance spectra during storage (Fig. 4). The h* value of the mortadella was affected by the interaction between the type and amount of blood (Fig. 5E) and by the interaction between the type of blood and the storage time (Fig. 6E). While the h* values of the mortadella increased (P b 0.05) upon addition of UNTB, it decreased (P b 0.05) with the addition of COTB or CODB (Fig. 5E). The increase in the hue angle of mortadella containing UNTB is a further support of the hypothesis of a change in the predominance of nitrosohemochrome in the control to hemichrome in mortadella containing UNTB. Additionally, the decrease in the hue angle of the mortadella containing COTB or CODB also supports the hypothesis of a change in the predominance of the pinkish nitrosohemochrome of the control to the reddish carboxyhemochrome. Regardless of the type of blood, at all level of blood addition, the hue angles of the mortadellas were different (P b 0.05) from those of the control (Fig. 5E). Except for mortadella containing 5% blood, the h* values of the mortadella containing COTB or CODB were lower (P b 0.05) than those of mortadella containing UNTB (Fig. 5E). While the hue angles of the control and of the mortadella containing UNTB did not change (P N 0.05), those of mortadellas containing COTB or CODB increased slightly (P b 0.05) during storage (Fig. 6E). This indicates that there may be a partial conversion of carboxyhemochrome to nitrosohemochrome due to the greater affinity of heme for NO than for CO (Antonini & Brunori, 1971). This hypothesis is supported by the observation that, at 8 weeks of storage, the reflectance spectra of blood-added mortadellas gets closer to that of the control (Fig. 4). The browning of the mortadellas was affected (P b 0.05) by the interaction between the type and amount of blood. Increasing the amount of blood produced (P b 0.05) an increase in the browning index of the mortadella containing UNTB, whereas it did not change (P N 0.05) when either COTB or CODB was used. The browning index of the mortadella containing COTB did not differ (P N 0.05) from that of the mortadella containing CODB or 5% of UNTB (Fig. 5F). At or above 10% of blood the browning index of the mortadellas containing COTB or CODB were lower than that of mortadellas containing UNTB (Fig. 5F).

The browning of the mortadella containing any amount of COTB, CODB or 5% UNTB was lower (P b 0.05) than that of the control; the browning of the mortadella containing 10% or more UNTB did not differ (P N 0.05) from that of the control (Fig. 5F). Regardless of the treatment, the browning index did not vary (P N 0.05) during storage (Fig. 5F). 4. Conclusions Addition of COTB and CODB, but not of UNTB, is viable for mortadella production. However, even not promoting major changes in proximate composition and TBARS, blood addition induces strong variations in the residual nitrite levels and in the visual appraisal of the color of mortadella. The decrease in residual nitrite may decrease the shelf-life of mortadellas containing COTB and CODB, may restrict UNTB blood addition into sausages or may require an increase in the ingoing amount of nitrite. Though needing the support of sensorial analysis, the color change may not allow its sale without a change in denomination to blood-enriched mortadella. However, sensorial analysis may only be conducted after the safety of mortadella containing CO-treated blood is verified through toxicological analyses (underway in our laboratories) in recently manufactured mortadella due to residual nitrite concerns.

Acknowledgments The authors express their gratitude for the financial support provided by FAPEMIG and Clariant do Brasil and the scholarship granted by CAPES.

References [AOAC] Association of Official Analytical Chemists (1990). Moisture in meat. Official method 950.46; Fat (crude) or ether extract in meat. Official method 960.39; Ash of meat. Official method 920.153; Nitrites in cured in meat. Official method 973.31. Official methods of analysis (15th ed.). Arlington, VA: AOAC. [AOAC] Association of Official Analytical Chemists (1993). Crude protein in meat and meat products. Official method 992.15. Official methods of analysis (15th ed.). Arlington, VA: AOAC. AMERICAN ASSOCIATION OF CEREAL CHEMISTRY—A. A. C. C. (1962). Cereal laboratory methods (7th ed.), Vol. 1, Saint Paul (Meat Science, 22 (4): 283–292). Antonini, E., & Brunori, M. (1988). Hemoglobin and myoglobin in their reactions with ligands. In E. Antonini, & M. Brunori (Eds.), North-Holland research monographs: Frontiers of biology, 21, 1971, North-Holland Pub. Co.Amsterdam, 436 pp. Caldironi, H. A., & Ockerman, H. W. (1982). Incorporation of blood proteins into sausage. Journal of Food Science, 47(2), 405–407. Cammacka, R., Joannou, C. L., Cui, X. Y., Martinez, C. T., Marajb, S. R., & Hughesb, M. N. (1999). Nitrite and nitrosyl compounds in food preservation. Biochimica et Biophysica Acta, 1411(2–3), 475–488. Carpenter, C. E., Cornforth, D. P., & Whittier, D. (2001). Consumer preferences for beef color and packaging did not affect eating satisfaction. Meat Science, 57(4), 359–363. Cassens, R. G. (1997). Residual nitrite in cured meat. Food Technology, 51(2), 53–55. Chen, C. C., Pearson, A.M., Gray, J. I., Fooladi, M. H., & Ku, P. K. (1984). Some factors influencing the nonheme iron content of meat and its implications in oxidation. Journal of Food Science, 49(2), 581–584. Cornforth, D., & Hunt, M. (2008). Low-oxygen p Incorporation of blood proteins into sausage packaging of fresh meat with carbon monoxide meat quality, microbiology, and safety. AMSA White Paper Series Number 2 (11 pp.). Dill, C. W., & Landmann, W. A. (1988). Food grade proteins from edible blood. In A.M. Pearson, & T. R. Dutson (Eds.), Advances in meat research, 5. (pp. 127–145). New York: Elsevier Applied Science. Erdman, A.M., & Watts, B.M. (1957). Spectrophotometric determination of color change in cured meat. Journal of Agricultural and Food Chemistry, 5(6), 453–455. Ferreira, V. L. P., Fernandes, S. V., & Yotsuyanagi, K. (1994). The colour of chiken and pork meat loaf with added cured bovine blood as evaluated by the Rab, Hunter Lab, L*a*b* and XYZ CIE systems. Revista Española de Ciencia y Tecnología de Alimentos, 34(3), 311–322. Fontes, P. R., Gomide, L. A.M., Ramos, E. M., Fontes, E. A. F., & Ramos, A. L. S. (2010). Composition and color stability of carbon monoxide treated dried porcine blood. Meat Science, 85(3), 472–480. Fontes, P. R., Gomide, L. A.M., Ramos, E. M., Stringheta, P. C., & Parreiras, J. F. M. (2004). Color evaluation of carbon monoxide treated porcine blood. Meat Science, 68(4), 507–513.

A.D. Pereira et al. / Meat Science 97 (2014) 164–173 Fox, J. B., & Benedict, R. C. (1987). The role of heme pigments and nitrite in oxidative processes in meat. In A. J. S. ANGELO, & M. E. BAILEY (Eds.), Warmed-over flavor of meat (pp. 119–140). New York: Academic Press. Fox, J. B., Jr., & Nicholas, R. A. (1974). Nitrite in meat. Effect of various compounds on loss of nitrite. Journal of Agricultural and Food Chemistry, 22(2), 302–306. Gorbatov, V. M. (1988). Collection and utilization of blood proteins for edible purposes in the USSR. In A.M. Pearson, & T. R. Dutson (Eds.), Advances in meat research, 5. (pp. 167–195). New York: Elsevier Applied Science. Guzmán, J. C., Mclillin, K. W., Bidner, T. D., Dugas-Sims, S., & Godber, J. S. (1995). Texture, color and sensory characteristics of ground beef patties containing bovine blood proteins. Journal of Food Science, 60(4), 657–660. Honikel, K. -O. (2008). The use and control of nitrate and nitrite for the processing of meat products. Meat Science, 78(1), 68–76. Hunt, M. C., Acton, J. C., Benedict, R. C., Calkins, C. R., Cornforth, D. P., Jeremiah, L. E., Olson, D. G., Salm, C. P., Savell, J. W., & Shivas, S. D. (1991). Guidelines for meat color evaluation, In: 44th Annual Reciprocal Meat Conference, American Meat Science Association. Manhattan, 232–249. Igene, J. O., King, J. A., Pearson, A.M., & Gray, J. I. (1979). Influence of heme pigments, nitrite, and non-heme iron on development of warmed-over flavor (WOR) Development in Cooked Meat. Journal of Agricultural and Food Chemistry, 27(4), 838–842. Igene, J. O., & Pearson, A.M. (1979). Role of phospholipids and triglycerides in warmed-over flavor development in meat model systems. Journal of Food Science, 44(5), 1285–1290. Izumi, K., Cassens, R. G., & Greaser, M. L. (1989). Reaction of nitrite with ascorbic acid and its significant role in nitrite-cured food. Meat Science, 26(1), 141–153. Jayasingh, P., Cornforth, D. P., Carpenter, C. E., & Whittier, D. (2001). Evaluation of carbon monoxide treatment in modified atmosphere packaging or vacuum packaging to increase color stability of fresh beef. Meat Science, 59(3), 317–324. John, L., Cornforth, D., Carpenter, C. E., Soheim, O., Pettee, B. C., & Whittier, D. R. (2004). Comparison of color and thiobarbituric acid values of cooked hamburguer patties after storage of fresh beef chubs in modified atmospheres. Journal of Food Science, 69(8), C608–C614. Lanier, T. C., Carpenter, J. A., Toledo, R. T., & Reagan, J. O. (1978). Metmyoglobin reduction in beef systems as affected by aerobic, anaerobic and carbon monoxide-containing environments. Journal of Food Science, 43(6), 1788–1796. Larsen, H., Westad, F., Shoreim, O., & Nilsen, L. H. (2006). Determination of critical oxygen level in packages for cooked sliced ham to prevent color fading during illuminated retail display. Journal of Food Science, 71(5), S407–S413. Livingston, D. J., & Brown, W. D. (1981). The chemistry of myoglobin and its reactions. Food Technology, 35(5), 244–252. Luño, M., Beltrán, J. A., & Roncalés, P. (1998). Shelf-life extension and colour stabilisation of beef packaged in a low O2 atmosphere containing CO: loin steaks and ground meat. Meat Science, 48(1/2), 75–84. Luño, M., Roncalés, P., Djenane, D., & Beltrán, J. A. (2000). Beef shelf life in low O2 and high CO2 atmospheres containing different low CO concentrations. Meat Science, 55(4), 413–419. Mancini, R. A., & Hunt, M. C. (2005). Current research in meat color. Meat Science, 71(1), 100–121. Mancini, R. A., Suman, S. P., Konda, M. K. R., & Ramanathan, R. (2009). Effect of carbon monoxide packaging and lactate enhancement on the color stability of beef steaks stored at 1 °C for 9 days. Meat Science, 81(1), 71–76. Mielnik, J., & Slinde, E. (1983). Sausage color measured by integrating sphere reflectance spectrophotometry when whole blood or blood cured by nitrite is added to sausages. Journal of Food Science, 48(5), 1723–1725. Miller, A. J., & Menichillo, D. A. (1991). Blood fraction effects on the antibotulinal efficacy of nitrite in model beef sausages. Journal of Food Science, 56(5), 1158–1160.

173

Mowad, R. K., Abozeid, W. M., & Nadir, A. S. (2012). Effect of nitrite level and tea catechins on residual nitrite and quality indices of raw-cured sausages. Journal of Applied Sciences Research, 8(2), 815–822. NAS (1977). Carbon monoxide. Washington: Division of medical sciences assembly of life sciences National Research Council (239 pp.). Ockerman, H. W., & Hansen, C. L. (1994). Industrialización de subproductos de origem animal. Zaragoza: Acribia (387 pp.). Oellingrath, I. M., & Slinde, E. (1985). Color, pigment and iron content of meat loaves with blood, blood emulsion, or mechanically deboned meat added. Journal of Food Science, 50(6), 1551–1555. Pérez-Rodríguez, M. L., Bosch-Bosh, N., & Garcia-Mata, M. (1996). Monitoring nitrite and nitrate residues in frankfurters during processing and storage. Meat Science, 44(1), 65–73. Pérez-Rodríguez, M. L., Garcia-Mata, M., & Bosch-Bosh, N. (1998). Effect of adding smoke-flavouring to frankfurters on nitrite and nitrate levels. Food Chemistry, 62(2), 201–205. Sørheim, O., Aune, T., & Nesbakken, T. (1997). Technological, hygienic and toxicological aspects of carbon monoxide used in modified-atmosphere packaging of meat. Trends in Food Science & Technology, 8(9), 307–312. Sørheim, O., Langsrud, O., Cornforth, D. P., Johannssen, T. C., Slinde, E., Berg, P., & Nesbakken, T. (2006). Carbon monoxide as a colorant in cooked or fermented sausages. Journal of Food Science, 71(9), C549–C555. Sørheim, O., Nissen, H., Aune, T., & Nesbakken, T. (2001). Use of carbon monoxide in retail meat packaging. Proceedings of 54th Reciprocal Meat Conference, july 24–28, 2001, Indianapolis, US. , Vol. 54. (pp. 47–51). Sørheim, O., Nissen, H., & Nesbakken, T. (1999). The storage life of beef and pork packaged in an atmosphere with low carbon monoxide and high carbon dioxide. Meat Science, 52(2), 157–164. Tarladgis, B. G., Watts, B.M., & Younathan, M. T. (1960). A distillation method for the quantitative determination of malonhaldehyde in rancid foods. Journal of the American Oil Chemists' Society, 37(1), 44–48. Tompkin, R. B., Christiansen, L. N., & Shaparis, A.B. (1978). Causes of variation in botulinal inhibition in perishable canned cured meat. Applied and Environmental Microbiology, 35(5), 886–889. Verma, M. M., Paranjape, V., & Ledward, D. A. (1985). Lipid and hemeprotein oxidation in meat emulsions. Meat Science, 14(2), 91–104. von Elbe, J. H., & Schwartz, S. J. (1996). Colorants. In O. W. Fennema (Ed.), Food chemistry (pp. 651–722). New York: Marcel Dekker, Inc. Vossen, E., Doolaege, E. H. A., Moges, H. D., De Meulenaer, B., Szczepaniak, S., Raes, K., & De Smet, S. (2012). Effect of sodium ascorbate dose on the shelf life stability of reduced nitrite liver pâtés. Meat Science, 91(1), 29–35. Watts, B.M., Erdman, A.M., & Wentworth, J. (1955). Relation of free sulfhydryl groups to cured meat color. Journal of Agricultural and Food Chemistry, 3(2), 147–151. Wilkinson, B. H. P., Janz, J. A.M., Morel, P. C. H., Purchas, R. W., & Hendriks, W. H. (2006). The effect of modified atmosphere packaging with carbon monoxide on the storage quality of master-packaged fresh pork. Meat Science, 73(4), 605–610. Wismer-Pedersen, J. (1988). Use of haemoglobin in foods—A review. Meat Science, 24(1), 31–45. Zhou, C., Wang, J., Wang, H., Zhang, L., & Cai, K. (2012). Effect of carboxy-hemoglobin on color stability of cooked pork. Food Science and Biotechnology, 21(1), 267–272.