Investigating the effect of incubation time and starter culture addition level on quality attributes of indirectly cured, emulsified cooked sausages

Meat Science 88 (2011) 454–461

Contents lists available at ScienceDirect

Meat Science j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m e a t s c i

Investigating the effect of incubation time and starter culture addition level on quality attributes of indirectly cured, emulsified cooked sausages Matthew J. Terns, Andrew L. Milkowski, James R. Claus, Jeffrey J. Sindelar ⁎ University of Wisconsin, Department of Animal Sciences, Meat Science & Muscle Biology Laboratory, 1805 Linden Drive, Madison, WI 53706, USA

a r t i c l e

i n f o

Article history: Received 7 December 2010 Received in revised form 20 January 2011 Accepted 21 January 2011 Keywords: Uncured Residual nitrate Residual nitrite Emulsified Starter culture

a b s t r a c t A process of “natural curing” utilizes vegetable juice/powder and a nitrate reducing starter culture to generate cured meat characteristics. The objective was to determine the effect varying levels of a mixed-strain bacterial starter culture (SC) and incubation time (INC) had on the quality characteristics of indirectly cured sausages. Four treatments (TRT) (TRT 1: 0.01% SC, 0 min INC; TRT 2: 0.01% SC, 90 min INC; TRT 3: 0.02% SC, 0 min INC; TRT 4: 0.02% SC, 90 min INC) and a control (C) were investigated. TRTs 2 and 4, and C revealed higher (P b 0.05) CIE a* redness values and greater (P b 0.05) cured pigment concentrations than TRTs 1 and 3 at days 0 and 14 while TRTs 2, 3, 4, and C were also redder (P b 0.05) than TRT 1 at days 28, 56, and 84. The results indicated the use of an incubation step was more critical than increasing the level of SC. Published by Elsevier Ltd.

1. Introduction Increasing consumer demands for foods perceived to be “healthy” and “wholesome” have resulted in an increased presence of natural and organic meats in the meat cases of retail stores in recent years (Major 2006; National Meat Case Study 2007). Mainstream grocers and retailers now account for more than half (54%) of organic food sales, surpassing farmers' markets, co-ops, and local organic food stores (Organic Trade Association 2010). Consumer purchasing trends of organic foods have increased dramatically in the last two decades. Organic food sales have been estimated to have increased almost 20% annually since 1990 (Winter & Davis 2006). In recent years, organic food sales have slowed with 5.1% growth in 2009 (Organic Trade Association 2010), yet organic meat sales were the fastest growing organic food sales sector in 2005 with 51% growth (Mitchell 2006). United States Department of Agriculture (USDA) regulations require products labeled “natural” are minimally processed and contain no artificial flavoring, artificial color, or chemical preservatives (USDA, 2005). As a result, synthetic chemical ingredients such as sodium nitrate or nitrite are prohibited from being included in foods labeled “natural”. Further, products labeled “organic”, governed by the USDA Organic Foods Production Act, also prohibit the addition of synthetic curing ingredients (Winter & Davis 2006). The addition of nitrite is responsible for many of the important and distinctive characteristics unique to cured meats. Cured color is the most visually noticeable cured meat quality that nitrite provides

⁎ Corresponding author. Tel.: + 1 608 262 0555; fax: + 1 608 265 3110. E-mail address: [email protected] (J.J. Sindelar). 0309-1740/$ – see front matter. Published by Elsevier Ltd. doi:10.1016/j.meatsci.2011.01.026

(MacDougall, Mottram, & Rhodes 1975; Fox 1966). Nitrite is also responsible for the formation of the characteristic cured meat flavor (Gray, Macdonald, Pearson, & Morton 1981; Mottram & Rhodes 1974) and the antioxidant protection that effectively controls the oxidation of unsaturated fatty acids responsible for the development of rancid and warmed over flavors (Vasavada & Cornforth 2005; Zubillaga, Maerker, & Foglia 1984). Arguably the most important role that nitrite addition plays in cured meats is antimicrobial activity, specifically against the growth of Clostridium botulinum (Bowen, Cerveny, & Deibel 1974; Hustad et al. 1973). Without the inclusion of nitrate or nitrite, natural and organic products are devoid of the typical appearance and flavor and perhaps safety aspects that consumers have come to expect from the traditionally cured equivalents. Sebranek and Bacus (2007) discuss a process of indirect curing of meats without the addition of synthetic nitrate or nitrite. This process, referred to as “natural curing” utilizes ingredients with relatively high amounts of naturally occurring nitrate in conjunction with a bacterial starter culture with specific nitrate reducing ability. Several vegetables, such as celery, have been reported to possess high amounts of naturally occurring nitrate (Walker 1990) and are commonly used as a natural curing ingredient. When in the presence of a starter culture with nitrate reductase activity, a portion of the nitrate from vegetables or other natural ingredients is reduced to nitrite when temperatures for optimal starter culture function exist. The longer these optimal temperature conditions are maintained, the greater the opportunity that exists for maximizing conversion and associated nitrite generation. The typical recommended holding temperature for commercial nitrate reducing cultures is 38–42 °C to minimize the time necessary for adequate nitrite formation. Nitrite generated from the reduction of nitrate by the starter culture is then available to participate in normal curing reactions which

M.J. Terns et al. / Meat Science 88 (2011) 454–461

can subsequently result in cured meat properties similar to nitriteadded cured meats. Sindelar, Cordray, Sebranek, Love, and Ahn (2007a,b) have shown the successfulness of this natural curing process as a method to create natural and organic cured meats similar to conventional nitrite cured versions. No nitrite-added, emulsified-frankfurter-style-cooked sausages and hams manufactured with varying levels of vegetable juice powder and incubation times utilizing a single-strain starter culture were found to result in cured meat properties similar to those of a sodium nitriteadded control. The authors reported that the length of incubation at 38 °C was more critical than the amount of naturally-added nitrate for development of cured meat properties in frankfurters and hams. Since less nitrite is typically generated during natural curing compared to tradition curing, maximizing the amount of nitrate conversion is critical for the success of creating products with cured meat properties. Recent advances in natural curing technologies have resulted in the development of mixed-strain bacterial starter cultures with a broader temperature range of nitrate reductase activity. This offers the ability for conversion to take place at lower temperatures, such as during manufacture, with a potential benefit of increasing total conversion time. However, little is known about the impact these mixed-strain starter cultures may have on the generation of nitrite from nitrate or the subsequent impact on cured meat quality. Further, it is unclear whether the amount of starter culture added has an impact on the magnitude of nitrate-to-nitrite conversion. Therefore, the first objective of this research was to determine the effects increasing levels of starter culture and incubation time had on quality characteristics including color and cured pigment concentrations of emulsified cooked sausages over an extended storage period. The second objective was to assess the impact a mixed-strain bacterial starter culture, with a broader temperature range of nitrate reductase activity, had on quality characteristics such as color and cured pigment concentration. A third objective was to determine the effects a mixedstrain bacterial starter culture had on nitrate and nitrite concentrations during product manufacture and throughout an ensuing storage period.

2. Materials and methods 2.1. Experimental design and data analysis Varying levels of Staphylococcus carnosus/Staphylococcus vitulinus starter culture (SC) and incubation times (INC) for the manufacture of naturally cured, emulsified, cooked sausages (NCEC sausage) were investigated. Four treatments (TRTs) and a control (C) were used in this study. TRTs were as follows: TRT 1: 0.01% SC, 0 min INC; TRT 2: 0.01% SC, 90 min INC; TRT 3: 0.02% SC, 0 min INC; TRT 4: 0.02% SC, 90 min INC. Statistical analysis of all measurements was performed by the SAS (Version 9.2, SAS Institute Inc., Cary, N.C., U.S.A.) mixed model procedure (SAS Institute Inc 2009). The experimental design was a 2 (SC level) × 2 (INC time) factorial design. The main plot consisted of 3 blocks (replication) and four emulsified, cooked sausage TRTs and a control, resulting in 15 observations for proximate composition, pH, nitrate, and nitrite. The model included the fixed main effects of treatment and replication. The random effect was the interaction of treatment × replication. Within the main factorial design was a split plot for measurements over time. The split plot contained five sampling days during storage (0, 14, 28, 56, and 84) in combination with the main plot to result in 75 overall observations for pH, color, nitrate, nitrite, total pigment, and cured pigment. The fixed main effects of the model were replication, treatment, day, and the interaction of treatment × day. The random effect was the interaction of replication × treatment. The significant main effects for all experiments were separated and least significant differences were found using Tukey–Kramer multiple pairwise comparison method. Significance levels were determined at

455

P b 0.05. For all other experiments, main effects were tested for significance using a mixed effects model.

2.2. Product procurement and manufacture Ready-to-eat NCEC sausages were manufactured using 90% lean fresh beef trimmings (Abbyland Foods Inc., Abbotsford, Wis., U.S.A.) and 42% lean fresh pork trimming (Seaboard Farms, Shawnee Mission, Kansas, U.S.A.) obtained from a local supplier. Both the 90% beef trimmings and 42% pork trimming were coarse-ground (Hobart Model 4732, Hobart Corporation, Troy, Ohio, U.S.A.) using a 12.7 mm plate. Finished emulsified cooked sausages were formulated to 80% lean content using a 60% beef and 40% pork raw material mixture. Beef trimmings and pork trimmings were randomly separated into 5 batches of 11.34 kg each. Batches were randomly assigned to treatments (TRTs 1–4) and control (C). The mixed-strain SC (Texel NatuRed LT, Danisco, New Century, Kansas, U.S.A.) used contained S. vitulinus and S. carnosus bacterial strains with a total cell count of at least 1011 cfu/g. The optimal nitrate reducing temperature of S. vitulinus was 11 °C and the optimal nitrate reducing temperature of S. carnosus was approximately 32 °C (according to manufacturer specifications). The freeze-dried SC was stored at −20 °C until used and was mixed with approximately 250 ml of distilled, de-ionized water immediately prior to addition. The emulsified cooked sausage formulation for TRTs 1 and 2 consisted of 48.1% beef trimmings, 32.1% pork trimmings, 16.0% ice/water, 1.81% salt, 1.61% dextrose, 0.40% spices (Product 60187990 V1 [dextrose and spice extractives], Saratoga Food Specialties, Bolingbrook, Ill., U.S.A.), 0.20% VJP (VegStable 502, Florida Food Products Inc., Eustis, Fla., U.S.A.), 0.16% garlic powder (Product 4011620, Saratoga Food Specialties, Bolingbrook, Ill., U.S.A.), and 0.01% starter culture (Texel NatuRed LT, Danisco, New Century, Kansas, U.S.A.). TRTs 3 and 4 consisted of 48.1% beef trimmings, 32.1% pork trimmings, 16.0% ice/water, 1.81% salt, 1.61% dextrose, 0.40% spices (Product 60187990 V1, Saratoga Food Specialties, Bolingbrook, Ill., U.S.A.), 0.20% VJP (VegStable 502, Florida Food Products Inc., Eustis, Fla., U.S.A.), 0.16% garlic powder (Product 4011620, Saratoga Food Specialties, Bolingbrook, Ill., U.S.A.), and 0.02% starter culture (Texel NatuRed LT, Danisco, New Century, Kansas, U.S.A.). The control consisted of 48.2% beef trimmings, 32.1% pork trimmings, 16.1% ice/water, 1.81% salt, 1.61% dextrose, 0.40% spices (Product 60187990 V1, Saratoga Food Specialties, Bolingbrook, Ill., U.S.A.), 0.201% cure (6.25% sodium nitrite and 93.75% salt; 156 ppm meat block basis), 0.16% garlic powder (Product 4011620, Saratoga Food Specialties, Bolingbrook, Ill., U.S.A.), and 0.0439% sodium erythorbate (547 ppm meat block basis). The total batch weights for TRTs 1–4 and C were 14.12 kg. Phosphates were not added to any TRTs or C because the NCEC sausages were intended to replicate natural and organic products that restrict phosphate use. Emulsions were manufactured using the methods described by Rust (1987). Manufacture of emulsified cooked sausages was performed in a bowl chopper (Krämer & Grebe 67–225, Krämer & Grebe GmbH & Co. KG., Biendenkopf-Wallau, Germany). The beef trimmings were added to the bowl chopper along with salt, VJP or nitrite (depending on treatment), and half of ice/water and chopped until a temperature of 2.2 °C was achieved. Pork trimmings were then added to the bowl chopper along with dextrose, spices, garlic powder, starter culture or sodium erythorbate (depending on treatment), and the remaining ice/water. The contents of the bowl chopper were chopped until a temperature of 14 °C was achieved. Meat batter was removed from the bowl chopper and transferred into a rotary vane vacuum-filler machine (Handtman VF608 plus Vacuum Filler, Handtmann CNC Technologies Inc, Buffalo Grove, Ill., U.S.A.). Emulsified batter was stuffed into 45 mm impermeable plastic casings (HC5 Red 45 Micron, World Pac U.S.A. International Inc., Sturtevant, Wis., U.S.A.). The impermeable casings were used to control cross-contamination effects that nitric oxide gas generated in the cooking oven could have on the TRTs during thermal processing. The casings had

456

M.J. Terns et al. / Meat Science 88 (2011) 454–461

an O2 permeability rate of 6–7cm3/m2/24 h at 1 atm and a water vapor permeability of 130 g/m2/24 h. In order to minimize any quality difference effects that could result from lower temperature nitrate conversion during product manufacture, time conscious manufacturing and thermal processing procedures were followed. TRTs requiring incubation were manufactured first and were immediately transferred into a single truck thermal processing oven (Alkar Model 450 MiniSmoker, Alkar Engineering Corp., Lodi, Wisc., U.S.A.). Incubation for TRTs 2 and 4 took place for 90 min at 40.6 °C dry bulb and 39.4 °C wet bulb temperatures and timing started when the internal temperature of the NCEC sausages reached 37.8 °C. The time needed for the internal temperature of the TRTs 2 and 4 to reach optimum incubation temperatures (37.8 °C) ranged from 17.5 to 23.3 min (data not shown). TRTs not requiring incubation and the C were manufactured after incubation for TRTs 2 and 4 began and were added to the thermal processing oven when incubation was nearly completed. The manufacture of TRTs 1 and 3 had a total elapsed time of 35 min. The C was manufactured last and the total time elapsed of TRTs 1, 3, and the C was 55 min. TRTs 1, 3 and the C were then collectively placed on the same smokehouse rack as TRTs 2 and 4 approximately 10 min prior to the end of the 90 min incubation to allow a come-up time for approaching equal internal temperatures of all TRTs and C prior to the start of cooking. Cooking was accomplished using a common frankfurter thermal processing schedule reaching an internal temperature of 71.1 °C. After thermal processing, the NCEC sausages were chilled to below 4.4 °C, placed in 3 mil nylon/polyethylene vacuum pouches (Prime Source Vacuum Pouches Items 75001817 and 75001830, Koch Supplies Inc., Kansas City, Missouri, U.S.A.), and vacuum packaged (UltraVac Vacuum Packager 2100-C, UltraVac Solutions, Koch Supplies Inc., Kansas City, Missouri, U.S.A.). Samples were stored at 2 °C prior to sampling at pre-determined dates.

Residual nitrite was determined by the AOAC method (1990c). All residual nitrite assays were done in duplicate and all treatments within a block were analyzed at the same time to minimize variation in the analysis due to time.

2.3. Proximate composition

2.8. Residual nitrate analysis

Proximate composition was determined for the NCEC sausages including crude fat (AOAC, 1990a), moisture (AOAC, 1990b), and crude protein (AOAC, 1993).

Sample preparation and determination methods were modifications of Ahn and Maurer (1987). Five grams of sample were weighed in a 50 mL test tube and homogenized with 20 mL of DDW using a Polytron Homogenizer (Type PT/35, Brinkmann Instruments Inc., Westbury, N.Y., U.S.A.) for 10 s at high speed. The homogenate was heated for 1 h in 80 °C water bath. After cooling in cold water for 10 min, 2.5 mL of the homogenate was transferred to a disposable test tube (16 × 100 mm). Carrez II (10.6 g potassium ferrocyanide in 100 mL DDW) and Carrez I (23.8 g zinc acetate in 50 mL DDW, 3 mL glacial acetic acid, diluted to 100 mL with DDW) reagents were added (0.1 mL each) to precipitate proteins. The solution was diluted with 2.3 mL of DDW and mixed well. After precipitation, the supernatant was centrifuged at 10,000 ×g for 20 min and the clear upper layer was used for nitrate measurement by high performance liquid chromatography (Agilent 1100 Series HPLC System, Agilent Technologies, Wilmington, Del., U.S.A.). The column used was Agilent Zorbax SAX (analytical 4.6 × 150 mm, 5 μm) (Agilent, Wilmington, Del., U.S.A.) and the elution buffer was 15 mM phosphate buffer, pH 2.35, with isocratic elution. Flow rate was 1.0 mL/min and sample volume was 25 μL. The wavelength used was 210 nm. The area of the nitrate peak was used to calculate nitrate concentration (ppm) using a nitrate standard curve. Duplicates were averaged together for use in data analysis.

2.4. Color measurements Color measurements were taken using a colorimeter (Model CR-300, Minolta Camera Co., Ltd., Osaka, Japan; 1 cm aperture, illuminant C, 2o observer angle). The colorimeter was standardized with the same packaging material as used on the samples, placed over a white calibration plate. Values for the white calibration plate were CIE L*=97.06, a*= −0.14, and b*=1.93). Color was measured using the Commission Internationale de l'Eclairage (CIE) L* (lightness), a* (redness), and b* (yellowness) system. Interior color measurements on the NCEC sausages were taken immediately after three samples (n= 3) were sliced lengthwise and placed inside packaging materials. Measurements were taken at two randomly selected locations on each of the three samples and the resulting average of the 6 measurements was used in data analysis. 2.5. pH measurement Samples for pH measurement were blended in a 1:9 ratio of sample to distilled, de-ionized water (DDW) and homogenized with a Polytron mixer (P10/35, Kinematica, GmbH, AG, Switzerland) at speed setting 7 for 45 s. Whatman #1 filter paper was folded and pushed into the beaker slurry to allow for separation of the fat free solution. The tip of the electrode was inserted into the fat free solution of the slurry and was measured with a pH meter (Accumet AB 15 plus, Fisher Scientific, Fair Lawn, N.J., U.S.A.) equipped with an electrode (Accumet combination pH electrode with Ag/AgCl reference Model 13-620-285, Fisher Scientific, Fair Lawn, N.J., U.S.A.) calibrated with 4.0 and 7.0 buffer solutions. For each treatment, the pH was measured in duplicate.

2.6. Total and cured pigment analysis Cured pigment analysis was conducted using a modified method of Hornsey (1956). Duplicate 10 g samples were added to 40 mL of acetone and 3 mL of DDW and blended with a Polytron mixer (P10/35, Kinematica, GmbH, AG, Switzerland) for 1 min at speed setting 7. The sample was immediately filtered through Whatman 42 filter paper and the absorbance (540 nm) measured on the filtrate. Nitrosylhemochrome concentration calculated as A540 ×290 was reported in parts per million (ppm). The analysis, including sample preparation, was performed in reduced light to prevent light induced pigment fading. Samples were finely ground/chopped with a food processor (Fresh Chop Model 72600, Hamilton Beach Brands Inc., Washington, N.C., U.S.A. Hamilton) prior to extraction. Total pigment analysis was also conducted using a modified method of Hornsey (1956). Duplicate 10 g finely ground/chopped samples were mixed with 40 mL of acetone, 2 mL of DDW, and 1 mL of concentrated hydrochloric acid (HCl) and blended with a Polytron mixer (P10/35, Kinematica, GmbH, AG, Switzerland) at speed setting 7 for 1 min. The samples were allowed to stand for 1 h, then filtered through Whatman 42 filter paper and immediately analyzed. Absorbance was measured at 640 nm. Total pigment concentration calculated as A640 × 680 was reported in parts per million (ppm). 2.7. Residual nitrite analysis

3. Results and discussion 3.1. Product processing attributes Fresh beef trimmings revealed means for pH of 5.91 and temperature of 1.9 °C while pork trimmings had means for pH of 6.11 and temperature of 1.9 °C. The pH of TRTs and C batches were measured after stuffing and before incubation (pre-incubate) as well as after incubation but prior to cooking steps (post-incubate). The main effect of treatment was observed to be significant (Pb 0.05) for the pre-incubate pH of NCEC sausages

M.J. Terns et al. / Meat Science 88 (2011) 454–461

(Table 1). Differences in pH values at pre-incubate were likely attributed to slight variations in raw materials. Although not of practical importance, a significant (Pb 0.05) difference was observed between post-incubate pH values for TRTs 2 and 4. The time needed, at incubation temperatures, for the internal temperature of TRTs 2 and 4 to reach optimum incubation temperatures (37.8 °C) ranged from 17.5 to 23.3 min (data not shown).

Table 2 Least squares means for the interaction of treatment combination (TRTs 1–4, C) with storage time (days 0, 14, 28, 56, and 84) for objective color (a*)a of indirectly cured (TRT 1–4) and conventionally cured control (C) emulsified cooked sausages. Dayc TRT

3.3. Color measurements The least squares means of NCEC sausages, for which a significant (P b 0.05) interaction of treatment× storage time was observed for internal CIE a* (redness) values are listed in Table 2. TRT 1 a* redness values were observed to be lower (P b 0.05) than TRTs 2, 4, and C for all days and lower (P b 0.05) than TRT 3 at days 28, 56, and 84. In addition, TRTs 2 and 4 displayed higher a* redness values than TRTs 1 and 3 for all days, although they were only found to be significantly higher (P b 0.05) at days 0 and 14. This indicates that increased a* redness values were dependent on the inclusion of an incubation step regardless of starter culture level. Sindelar et al. (2007b) also reported significantly higher (P b 0.05) a* redness values in TRTs of no nitrite-added emulsifiedfrankfurter-style-cooked (EFSC) when longer incubation took place. The authors stated that incubation time was the most important factor in developing higher a* redness values in no nitrite-added EFSC sausages. Lower a* redness values for TRTs 1 (low SC+ no INC) and 3 (high SC +no INC) compared to a* redness values for TRTs 2 (low SC+ INC) and 4 (high SC +INC) may be further explained by the significantly greater amount (P b 0.05) of nitrate-to-nitrite conversion that occurred for TRTs 2 and 4 compared to TRTs 1 and 3, which likely existed from incubation related differences (Table 3). This suggests nitrite associated curing reactions occurred to a greater extent for TRTs 2 and 4. When an incubation step was not utilized, the effect of doubling SC level for TRT 3 (high SC+ no INC) resulted in higher a* redness values over all days compared to TRT 1 (low SC+ no INC), although TRT 3 was only significantly (P b 0.05) higher on days 28, 56, and 84 (Table 2). Further, TRT 3 a* redness values were not statistically different (P N 0.05) than the C on days 28, 56, and 84. These results indicate that doubling the amount of SC in the absence of an incubation step could achieve a* redness values that closely resemble those of a nitrite-added C. It was Table 1 Least squares means for main effect of treatment combination (TRTs 1–4, C) for pre-incubate pHa and post-incubate pHb of indirectly cured (TRTs 1–4) and conventionally cured control (C) emulsified cooked sausages. TRTc 1 Pre-incubate pHa Post-incubate pHb

f 6.32 –e

2 g f

6.16 6.29

3 6.21 –e

4 6.21 6.31

g

C 6.22 –e

b

1 2 3 4 C SEMd = 0.57

3.2. Proximate composition Proximate analysis of moisture, fat, and protein were determined for NCEC sausage samples (data not shown). Average moisture content was 62.5% and values ranged from 60.4% to 63.7%. Average fat content was 21.5% and values ranged from 20.4% to 23.5%. Average protein content was 12.2% and values ranged from 12.1% to 12.3%. These results indicate that TRTs and C were uniform in proximate composition.

0

14

28

56

84

9.1h 15.4i e 11.7h 15.6i 16.1i

9.3h 15.0i ef 13.3h 15.2i 15.8i

9.2h 14.9i fg 12.0i 15.0i 15.7i

9.7h 14.7i fg 13.4i 14.6i 15.5i

10.4h 14.9i g 14.2i 15.1i 15.5i

a

Commission Internationale de l'Eclairage (CIE) L*, a*, b*, where a* = redness on a 1 to 100 white scale for internal surface color of emulsified cooked sausages. Treatment combinations: TRT 1 = low SC + no INC; TRT 2 = low SC + INC; TRT 3 = high SC + no INC; TRT 4 = high SC + INC; C = 156 ppm (mg/kg) sodium nitrite. c Day = 0, 14, 28, 56, and 84 day vacuum-packaged samples held at 2 °C. d SEM = standard error of the means for no nitrite-added and nitrite-added emulsified cooked sausages. e–g Means within same row with different superscripts are different (P b 0.05). h–i Means within same column with different superscripts are different (P b 0.05). b

also observed that a* redness values for TRTs 1 and 3 increased over storage time (day). Increased a* redness values were also reported by Sindelar et al. (2007b) in no nitrite-added EFSC sausages. The authors explained residual nitrate in these products may serve as a reservoir for nitrite related reactions to occur during storage. Although not significant (P N 0.05), a* redness values for TRTs 2, 4, and C were observed to generally decrease over storage time (day). This data agrees with Fernandez-Gines, Fernandez-Lopez, Sayas-Barbera, Sendra, and PerezAlvarez (2003), who suggested decreased a* redness values over storage are the result of nitrosylhemochrome pigment degradation. No significance (P N 0.05) was found for the interaction of treatment× storage time for CIE L* and b* values, but the main effect of day was observed to be significant (P b 0.05). The corresponding least squares combined means for TRTs and C are reported in Table 4. Both L* lightness values and b* yellowness values generally increased over storage time (day). CIE L* lightness values significantly (P b 0.05) increased from day 0 to days 18 and 28, and again from days 56 and 84 when compared to all other days. CIE b* yellowness values were significantly (P b 0.05) higher at days 56 and 84 than at days 0 and 14. CIE b* values was also significant (P b 0.05) for the main effect of treatment. Table 5 lists the least squares combined means for all storage days (0, 14, 28, 56, and 84). Significantly (P b 0.05) higher b* yellowness values were observed for TRT 1 compared to all other TRTs and C. 3.4. pH measurements No significant differences were observed for the interaction of treatment and storage time, but the main effect of storage time was Table 3 Least squares means for the interaction of treatment combination (TRTs 1–4, C) with storage time (days 0 and 84) for residual nitrate (PPM)a of indirectly cured (TRT 1–4) and conventionally cured control (C) emulsified cooked sausages. TRTc

SEMd Day

0.03 0.0028

b

0 84 SEMd = 2.37

a

Pre-incubate pH of emulsified cooked sausage samples collected randomly during stuffing and before incubation (INC). b Post-incubate pH of emulsified cooked sausage samples collected randomly after incubation (INC) prior to cooking. c Treatment combinations: TRT 1 = low SC + no INC; TRT 2 = low SC + INC; TRT 3 = high SC + no INC; TRT 4 = high SC + INC; C = 156 ppm (mg/kg) sodium nitrite. d SEM = standard error of the means for no nitrite-added and nitrite-added emulsified cooked sausages. e No measurements was taken as this TRT did not undergo INC. f–g Means within same row with different superscripts are different (P b 0.05).

457

a

1

2

3

4

g

e

g

e

116.5 fg 116.7

27.0 e 45.7

106.3 g 117.2

24.6h e 50.4i

C f

f 64.8h 103.5i

Residual nitrate determination reported in ppm (mg/kg) of sample. Day = 0 and 84 day vacuum-packaged samples held at 2 °C. c Treatment combinations: TRT 1 = low SC + no INC; TRT 2 = low SC + INC; TRT 3 = high SC + no INC; TRT 4 = high SC + INC; C = 156 ppm (mg/kg) sodium nitrite. d SEM = standard error of the means for no nitrite-added and nitrite-added emulsified cooked sausages. e–g Means within same row with different superscripts are different (P b 0.05). h–i Means within same column with different superscripts are different (P b 0.05). b

458

M.J. Terns et al. / Meat Science 88 (2011) 454–461

Table 4 Least squares means for main effect of storage time (days 0, 14, 28, 56, and 84) for pHa, total pigment concentrationb, and objective color (L*)c and (b*)d of indirectly cured (TRT 1–4) and conventionally cured control (C) emulsified cooked sausages.

Table 6 Least squares means for the interaction of treatment combination (TRTs 1–4, C) with storage time (days 0, 14, 28, 56, and 84) for cured pigment concentrationa of indirectly cured (TRT 1–4) and conventionally cured control (C) emulsified cooked sausages.

Daye

pHa Total Pigmentb L* c b* d

Dayc

0

14

28

6.32 h 137.5 g 64.53 g 8.97

6.34 g 134.9 h 65.33 g 9.13

6.32 136.1 h 65.32 9.16

56

84

g

h

6.34 h 137.5 i 65.82 h 9.38

6.31 136.4 i 66.16 h 9.40

SEM

f

TRT

a

b

significant (P b 0.05) for pH of NCEC sausages (Table 4). As expected, pH values post-thermal processing changed very little over the duration of storage at 2 °C. 3.5. Total and cured pigment analysis The main effect of storage time was observed to be significant (P b 0.05) for the total pigment concentration of NCEC sausages. Total pigment concentrations (ppm) listed in Table 4 revealed slight fluctuations throughout storage. It would not be expected to see total pigment concentration change, but the observed differences could be due to slight raw material differences or experimental error because measurements were carried out on different days (0, 14, 28, 56, and 84). Least squares means for the significant (P b 0.05) interaction of treatment and storage time observed for cured pigment concentration are listed in Table 6. TRTs 2, 4, and C revealed significantly higher (P b 0.05) cured pigment concentrations than TRTs 1 and 3 at days 0 and 14 and, although not significant (P N 0.05), were also higher at days 28, 56, and 84. No significant differences (P N 0.05) were observed between TRTs 2, 4, and C at anytime throughout storage (day). This indicated that the addition of an incubation step produced cured pigments concentrations similar to the C, regardless of the level of SC. In addition, cured pigment concentrations for TRT 3 were significantly (P b 0.05) greater than TRT 1 for all days. Thus, in the absence of an incubation step, a higher level of SC was shown to have a noteworthy impact in resulting in cured pigment concentrations approaching those of a nitrite-added C. It was observed that cured pigment concentrations for TRTs 2 and 4 (Table 6) decreased over time, while TRTs 1 and C showed unexpected fluctuation. Further, TRT 3 was found to increase over time which is in

0

1 20.9h e 2 109.5j e 3 55.8i e 4 113.0j C 116.7j SEMd = 6.32

0.024 1.62 0.15 0.068

pH of emulsified cooked sausage samples. Total pigment analysis reported in ppm. c Commission Internationale de l'Eclairage (CIE) L*, a*, b*, where L* = lightness on a 1 to 100 white scale for internal surface color of emulsified cooked sausages. d Commission Internationale de l'Eclairage (CIE) L*, a*, b*, where b* = yellowness on a 1 to 100 white scale for internal surface color of emulsified cooked sausages. e Day = 0, 14, 28, 56, and 84 day vacuum-packaged samples held at 2 °C. f SEM = standard error of the means for no nitrite-added and nitrite-added emulsified cooked sausages. g–i Means within same row with different superscripts are different (P b 0.05).

b

14

28

56

84

15.6h e 108.8j ef 71.6i 108.0j 114.9j

16.8h 103.6i fg 85.9i 100.9i 112.5i

21.2h 95.9ij fg 83.5i 94.5ij 114.6j

19.6h 87.4i g 91.7i f 88.4i 111.0i f

a

Cured pigment (nitrosylhemochrome) analysis reported in ppm. Treatment combinations: TRT 1 = low SC + no INC; TRT 2 = low SC + INC; TRT 3 = high SC + no INC; TRT 4 = high SC + INC; C = 156 ppm (mg/kg) sodium nitrite. c Day = 0, 14, 28, 56, and 84 day vacuum-packaged samples held at 2 °C. d SEM = standard error of the means for no nitrite-added and nitrite-added emulsified cooked sausages. e–g Means within same row with different superscripts are different (P b 0.05). h–j Means within same column with different superscripts are different (P b 0.05). b

agreement with a previous study by Sindelar et al. (2007b), where cured pigment concentrations of no nitrite-added EFSC sausages were also observed to increase over storage time. The observed decrease in cured pigment concentration for TRTs 2 and 4 may be explained by the fact that residual nitrate levels of TRTs 2 and 4 were lower than all other TRTs following thermal processing (Table 3; day 0). Since this data would suggest TRTs 2 and 4 had greater nitrate-to-nitrite conversion, supported by higher levels of cured pigment, less residual nitrite would be available for regeneration of cured pigment losses during storage. Therefore, a loss of cured pigment over time could be expected if cured pigment concentrations were possibly not able to be maintained. This is also supported by the trend of decreasing residual nitrite levels over storage time (Table 7). 3.6. Residual nitrite analysis Residual nitrite levels were determined before incubation (preincubate) and after incubation (post-incubate) (Table 8), following thermal processing (day 0), and throughout an 84 day storage period (days 14, 28, 56, and 84) (Table 7). Post-incubation residual nitrite levels for TRTs 1, 3, and C were not measured because no incubation occurred for those TRTs. No residual nitrite was detected for any TRTs (TRTs 1–4) prior to incubation (pre-incubate) and thermal processing, but the C showed significantly (P b 0.05) greater residual nitrite compared to all TRTs as nitrite was added directly to the C during manufacture. Following incubation (post-incubate), residual nitrite Table 7 Least squares means for the interaction of treatment combination (TRTs 1–4, C) with storage time (days 0, 14, 28, 56, and 84) for residual nitrite (PPM)a of indirectly cured (TRT 1–4) and conventionally cured control (C) emulsified cooked sausages. DAYc

Table 5 Least squares means for main effect of treatment combination (TRTs 1–4, C) for objective color (b*)a of indirectly cured (TRT 1–4) and conventionally cured control (C) emulsified cooked sausages.

TRT

1 2 3 4 C SEMd = 4.92

TRTb

b* a

a

1

2

3

4

C

SEMc

d

e

e

e

e

0.085

9.75

9.09

9.10

8.90

9.20

Commission Internationale de l'Eclairage (CIE) L*, a*, b*, where b* = yellowness on a 1 to 100 white scale for internal surface color of emulsified cooked sausages. b Treatment combinations: TRT 1 = low SC + no INC; TRT 2 = low SC + INC; TRT 3 = high SC + no INC; TRT 4 = high SC + INC; C = 156 ppm (mg/kg) sodium nitrite. c SEM = standard error of the means for no nitrite-added and nitrite-added emulsified cooked sausages. d–e Means within same row with different superscripts are different (P b 0.05).

b

a

0

14

28

56

3.0i g 43.8j 17.8i g 43.4j h 73.2k

2.2i fg 38.6j 11.8i fg 37.3j gh 59.7k

2.7i fg 35.3j 17.7k fg 34.5j fg 54.9m

0.0i ef 27.3j 10.6i ef 26.6j f 43.0k

84 1.1ik 21.3jmo 10.8kmp e 19.7op e 26.8o e

Residual nitrite determination reported in ppm (mg/kg) of sample. Treatment combinations: TRT 1 = low SC + no INC; TRT 2 = low SC + INC; TRT 3 = high SC + no INC; TRT 4 = high SC + INC; C = 156 ppm (mg/kg) sodium nitrite. c Day = 0, 14, 28, 56, and 84 day vacuum-packaged samples held at 2 °C. d SEM = standard error of the means for no nitrite-added and nitrite-added emulsified cooked sausages. e–h Means within same row with different superscripts are different (P b 0.05). i–p Means within same column with different superscripts are different (P b 0.05). b

M.J. Terns et al. / Meat Science 88 (2011) 454–461 Table 8 Least squares means for main effect of treatment combination (TRTs 1–4, C) for residual nitrate (ppm)a and residual nitrite (ppm)b of indirectly cured (TRT 1–4) and conventionally cured control (C) during the manufacture of emulsified cooked sausages. ppm Residual nitratea TRT

c

1 2 3 4 C SEMf

Pre-incubate i

121.0 122.3i 120.9i 122.9i 57.7h 2.09

d

Post-incubate g

– 42.3 –g 43.5 –g 1.38

ppm Residual nitriteb e

Pre-incubated h

0.0 0.0h 0.0h 0.0h 13.8i 1.50

Post-incubatee –g 14.70 –g 14.45 –g 0.21

a

Residual nitrate determination reported in ppm (mg/kg) of sample. Residual nitrite determination reported in ppm (mg/kg) of sample. c Treatment combinations: TRT 1 = low SC + no INC; TRT 2 = low SC + INC; TRT 3 = high SC + no INC; TRT 4 = high SC + INC; C = 156 ppm (mg/kg) sodium nitrite. d Pre-incubate = samples collected randomly during stuffing and before incubation (INC). e Post-incubate = samples collected randomly after incubation (INC) prior to cooking. f SEM = standard error of the means for no nitrite-added and nitrite-added emulsified cooked sausages. g — indicates that no measurements was taken as this TRT did not undergo INC. h–i Means within same row with different superscripts are different (P b 0.05). b

459

This indicates that the use of an incubation step was more effective in increasing the rate of nitrate-to-nitrite conversion than by increasing the level of SC. The impact of using a mixed-strain SC was also investigated for its ability in facilitating more rapid nitrate-to-nitrite conversion. The two bacterial strains of the mixed-strain SC were S. vitulinus and S. carnosus, which optimally reduce nitrate at temperatures near 11 °C and 32 °C, respectively. The use of a mixed-strain nitrate reducing SC would then be expected to begin reducing nitrate immediately upon addition to the meat system. As such, the mixed-strain would be expected to provide nitrate reductase activity during both product manufacture (colder temperatures) and during warmer incubation temperatures allowing a longer window of conversion time. However, since the residual nitrite level for TRT 1 after thermal processing was 3.0 ppm (Table 7), it does not appear that the mixed-strain SC generated a large amount of nitrite in the absence of an incubation step in this experiment. This may be explained by shorter processing times related to this type of product or due to the quick come-up time at the during incubation and thermal processing for a small diameter product. It should, however, be noted that a TRT with a higher level of SC and no incubation did reveal noticeably higher (P N 0.05) levels of nitrite generation compared to the TRT comprising a low level of SC and no incubation. 3.7. Residual nitrate levels

levels for TRTs 2 and 4 were not observed to be significantly (P N 0.05) different suggesting the presence of an incubation step had a greater impact on nitrate to nitrite reduction than the level of SC used. Levels of post-incubation residual nitrite for TRTs 2 and 4 were slightly lower to those observed in similar TRTs by Sindelar et al. (2007b) who reported 24.5 ppm post-incubation residual nitrite in TRTs containing 0.20% vegetable juice powder (VJP). A significant (P b 0.05) difference for the interaction of treatment × storage time was found for residual nitrite of NCEC sausages. The least squares means for the residual nitrite values are listed in Table 7. As expected, residual nitrite levels declined over time for all TRTs and C. The trend of declining residual nitrite levels over storage time was also observed by Sindelar et al. (2007a) in TRTs of no nitrite-added RTE ham and has also been found to be affected by storage temperatures reported by Hustad et al. (1973) in wieners. Further, Ahn, Kim, Jo, Lee, and Byun (2002) reported that an anaerobic vacuum packaged environment reduced residual nitrite levels over time more rapidly than aerobic packaging conditions. The authors explained that vacuum conditions maintain a low redox potential environment in which nitrite is more quickly converted to nitric oxide, thus depleting residual nitrite levels. Interestingly, residual nitrite levels for the C were observed to be higher than expected following the cooking process (day 0). Hustad et al. (1973) also high reported high residual nitrite levels of 51 ppm in wieners following thermal processing yet our research revealed even higher levels. The higher reported residual nitrite levels may be explained by the absence of vacuum during nitrite addition while manufacturing, which is important for creating favorable nitrite reducing conditions. Since mixing, chopping, etc. under vacuum conditions can create increased reducing environment conditions, a more efficient conversion of nitrite to nitric oxide could take place, thereby decreasing residual nitrite levels measured post-processing (Tantikarnjathep, Sebranek, Topel, & Rust 1983). Levels of residual nitrite (Table 7) for TRTs 1 and 3 were significantly (P b 0.05) lower than TRTs 2, 4, and C for days 0, 14, and 56 and although not significant (P N 0.05), were also lower at days 28 and 56. In addition, TRT 3 revealed higher (P b 0.05) levels of residual nitrite than TRT 1 at days 28 and 56 while these trends also continued (P N 0.05) at days 56 and 84. This suggests that nitrate-to-nitrite reduction for TRT 1 may have occurred at a very slow rate. These results also imply that higher SC levels did have an impact on nitrite generation, although a significant impact was not realized. Residual nitrite levels for TRTs 2 and 4 were significantly (P b 0.05) higher than TRTs 1 and 3 for days 0, 14, 28, and 56.

Residual nitrate levels were determined before incubation (preincubate) and after incubation (post-incubate) (Table 8), following thermal processing (day 0), and at day 84 of storage (Table 3). Postincubation residual nitrate levels for TRTs 1, 3, and C were not measured because no incubation occurred for these TRTs. No significant differences (P N 0.05) were observed for pre-incubation levels of residual nitrate for all TRTs (Table 8), which was expected since all TRTs were formulated with identical amounts of VJP. Post-incubation levels of residual nitrate were also not observed to be significantly (P N 0.05) different, which indicates the incubation of TRTs decreased nitrate levels to a relatively similar extent, regardless of SC level. Levels of post-incubation residual nitrate for TRTs 2 and 4 decreased from pre-incubation residual nitrate levels. Trends of decreasing nitrate levels as a result of incubation in combination with the presence of post-incubation residual nitrite confirm that nitrate-to-nitrite reduction did in fact occur. Nitrate reduction has been observed by the natural flora of meat (Kramlich, Pearson, & Tauber 1973; Sebranek 1979), but can also occur from the intentional addition of nitrate reducing bacteria such as S. carnosus (Sindelar et al. 2007a,b). These authors corroborated that adding S. carnosus reduced nitrate which was inherently present in VJP to nitrite, which was then available for curing reactions. Residual nitrate levels were significant (P b 0.05) for the interaction of treatment× storage time, with least squares means listed in Table 3. At days 0 and 84, TRTs 2 and 4 were observed to have significantly (P b 0.05) lower levels of residual nitrate than TRTs 1 and 3, indicating that a greater amount of nitrate may have been converted to nitrite in TRTs 2 and 4. This supports that the inclusion of an incubation step resulted in a greater extent of nitrate-to-nitrite reduction than by increasing the level of SC. The mixed-strain SC was expected to reduce a greater portion of nitrate in treatments not containing an incubation step than what was actually observed for TRTs 1 and 3. One of the strains has optimal nitrate reductase activity at 11 °C, which means it would be able to reduce nitrate in meat at colder temperatures (i.e. immediately after addition) and prior to thermal processing. The second strain's optimal nitrate reductase activity was 32 °C, allowing reduction of nitrate to occur during the incubation step of thermal processing. However, large amounts of residual nitrate were measured in TRTs 1 and 3 after thermal processing (day 0), which suggests that the mixed-strain SC had a limited amount of reduction. Residual nitrate levels were also detected in the C, to which only nitrite had been added (Table 3, Table 8). Perez-Rodriguez, Bosch-Bosch,

460

M.J. Terns et al. / Meat Science 88 (2011) 454–461

and Garcia-Mata (1996) also reported finding detectable levels of nitrate in frankfurters to which only nitrite had been added. The authors found that approximately 10–15% of ingoing nitrite was recovered in the finished product as nitrate. Dethmers, Rock, Fazio, and Johnston (1975) also reported the formation of nitrate in manufactured thuringer from a portion of added nitrite. Herring (cited in Dethmers et al. 1975) proposed that nitrate regeneration was the result of the simultaneous oxidation and reduction of nitrous acid to yield nitric oxide and nitrate and from the oxidation of nitric acid to give nitrite, which was subsequently reacted with water to yield nitrate and nitrite. 4. Conclusions Varying levels of a mixed-strain starter culture with and without the inclusion of an incubation step were investigated in NCEC sausages to determine the ability to result in products with cured meat properties similar to those of a sodium nitrite-added control. The sodium nitrite-added control in this study generally achieved and maintained the greatest level of cured meat properties. Color and cured pigment measurements indicated that TRTs 2 and 4 had cured meat properties most similar to a sodium nitrite-added control. Measurements of objective color, cured pigment, residual nitrate, and residual nitrite substantiated that this “natural curing” system, where naturally occurring nitrates in VJP are reduced to nitrite by a S. carnosus/S. vitulinus bacterial starter culture, could successfully result in adequate curing reactions and acceptable cured meat characteristics. Objective color, cured pigment, and residual nitrate levels also indicated that the use of an incubation step, when a mixed-strain starter culture was utilized in small diameter sausages with short processing times, was more critical than increasing starter culture levels in producing cured meat properties similar to a sodium nitrite-added control. Treatments including an incubation step (TRTs 2 and 4) reduced a greater amount of nitrate from vegetable juice powder to nitrite, which subsequently created more available nitrite for ensuing curing reactions resulting in a greater extent of cured meat properties. As it has been demonstrated, the presence of an incubation step (in this research) and an increased length of incubation time (120 min) (research by Sindelar et al. 2007a,b) can produce more acceptable cured meat properties. Therefore, future work could indentify whether increasing the length of incubation time further allows for even greater cured meat properties in naturally cured meats. In addition, longer process/ manufacturing times should be investigated to determine the impact mixed-strain starter cultures, with the ability to reduce nitrate-to-nitrite a lower temperatures, may have on nitrite generation and the ensuring cured meat properties. In the absence of an incubation step, objective color, cured pigment, and residual nitrite measurements indicated that increasing the level of starter culture can improve cured meat properties, but not to the level of a sodium nitrite-added control. The treatment comprising a low level of starter culture and no incubation (TRT 1) was ineffective in producing many cured meat properties comparable to those of the control and thus indicates that either the inclusion of an incubation step or increased levels of starter culture are necessary to more effectively approach cured meat properties found in a sodium nitrite-added control. It would be of interest to investigate whether increasing the level of starter culture to higher yet levels would continue to increase cured meat properties or if a maximum level of starter culture addition has been reached thus resulting in a diminishing returns situation. Future research should also investigate the microbial safety of naturally cured products. Although high standardized levels of nitratecontaining vegetable juices and powders are now common (exceeding 30,000 ppm), it is still unclear what impact these high nitrate levels might have on the presence and growth of microorganisms. As a result of the markedly high ingredient levels, ingoing levels of nitrate can feasibly approach 100 ppm; however, even if 100% conversion of nitrate-to-nitrite

occurs, adequate levels of nitrite needed for providing antimicrobial properties, especially for C. botulinum appear to be questionable. However, since residual nitrite levels from natural curing have been shown to approach those of a nitrite added control, it is unclear whether sufficient levels of nitrite is, in fact, present for C. botulinum control. Additional studies are thus needed to better determine the antimicrobial and shelf life characteristics of naturally cured products compared to nitrite-added versions. In conclusion, this research has demonstrated that mixed-strain bacterial starter cultures can have an impact on improving nitrate-tonitrite conversion and subsequent curing related reactions; however, more work is needed to better understand how this improvement can be further increased. Acknowledgement This work was financially supported by the College of Agricultural and Life Sciences, University of Wisconsin-Madison, HATCH Project WIS01309. References Ahn, D. U., & Maurer, A. J. (1987). Concentration of nitrate and nitrite in raw turkey breast meat and the microbial conversion of added nitrate to nitrite in tumbled turkey breast meat. Poultry Science, 66(12), 1957−1960. Ahn, H. J., Kim, J. H., Jo, C., Lee, C. H., & Byun, M. W. (2002). Reduction of carcinogenic nnitrosamines and residual nitrite in model system sausage by irradiation. Journal of Food Science, 67(4), 1370−1373. Association of Official Analytical Chemists [AOAC] (1990a). Fat (crude) or ether extract in meat. Official method 960.39. Official Methods of Analysis (15th ed). Arlington: AOAC. Association of Official Analytical Chemists [AOAC] (1990b). Moisture in meat. Official method 950.46. Official Methods of Analysis (15th ed). Arlington: AOAC. Association of Official Analytical Chemists [AOAC] (1990c). Nitrites in cured meat. Official method 973.31. Official Methods of Analysis (15th ed). Arlington: AOAC. Association of Official Analytical Chemists [AOAC] (1993). Crude protein in meat and meat products. Official method 992.15. Official methods of analysis (15th ed. 4th suppl). Arlington: AOAC. Bowen, V. G., Cerveny, J. G., & Deibel, R. H. (1974). Effect of sodium ascorbate and sodium nitrite on toxin formation of Clostridium botulinum in wieners. Applied Microbiology, 27(3), 605−606. Dethmers, A. E., Rock, H., Fazio, I., & Johnston, R. W. (1975). Effect of added sodium nitrite and sodium nitrate on sensory qualities and nitrosamine formation in thuringer sausage. Journal of Food Science, 40(3), 491−495. Fernandez-Gines, J. M., Fernandez-Lopez, J., Sayas-Barbera, E., Sendra, E., & Perez-Alvarez, J. A. (2003). Effect of storage conditions on quality characteristics of bologna sausages made with citrus fiber. Journal of Food Science, 68(2), 710−715. Fox, J. B. (1966). Chemistry of meat pigments. Journal of Agricultural and Food Chemistry, 14(3), 207−210. Gray, J. I., Macdonald, B., Pearson, A. M., & Morton, I. D. (1981). Role of nitrite in cured meat flavor — a review. Journal of Food Protection, 44(4), 302−312. Hornsey, H. C. (1956). The colour of cooked cured pork. 1. — Estimation of the nitric oxide-haem pigments. Journal of the Science of Food and Agriculture, 7(8), 534−540. Hustad, G. O., Cerveny, J. G., Trenk, H., Deibel, R. H., Kautter, D. A., Fazio, T., et al. (1973). Effect of sodium nitrite and sodium nitrate on botulinal toxin production and nitrosamine formation in wieners. Applied Microbiology, 26(1), 22−26. Kramlich, W. E., Pearson, A. M., & Tauber, F. W. (1973). Processed meats. Westport, CT: AVI Publishing Co 348pp. MacDougall, D. B., Mottram, D. S., & Rhodes, D. N. (1975). Contribution of nitrite and nitrate to color and flavor of cured meats. Journal of the Science of Food and Agriculture, 26(11), 1743−1754. Major, M. (2006). Natural meat: stampede! Progressive Grocer. http://www.lexisnexis. com/hottopics/lnacademic/?rand=0.7543170581003872&cookieState=0&ipcounter=1&bhcp=1 Accessed 25.10.10. Mitchell, R. (2006). Outrunning supplies. Meat & Deli Retailer. http://www.meatanddeliretailer.com/ Accessed 22.9.10. Mottram, D. S., & Rhodes, D. N. (1974). Nitrite and the flavour of cured meat. Proceedings of the Symposium on Nitrite in Meat Products (pp. 161−171). National Meat Case Study (2007). Today's retail meat case. The 2007 National Meat Case Study http://www.sealedair.com/products/food/nmcs.html Accessed 22.9.10. Organic Trade Association (2010). OTA manufacturer survey — summary. http://www. ota.com Accessed 28.9.10. Perez-Rodriguez, M. L., Bosch-Bosch, N., & Garcia-Mata, M. (1996). Monitoring nitrite and nitrate residues in frankfurters during processing and storage. Meat Science, 44 (1–2), 65−73. Rust, R. E. (1987). Sausage products. In J. F. Price, & B. S. Schweigert (Eds.), The science of meat and meat products (pp. 473−485). (3rd ed). Westport, CT: Food and Nutrition Press, Inc. SAS Institute Inc (2009). SAS/STAT User's Guide Version 9.2 (Second Edition). N.C: Cary. Sebranek, J. G. (1979). Advances in the technology of nitrite use and consideration of alternatives.Food Technology, 33(7), 58−62 93.

M.J. Terns et al. / Meat Science 88 (2011) 454–461 Sebranek, J., & Bacus, J. (2007). Natural and organic cured meat products: regulatory, manufacturing, marketing, quality and safety issues. American Meat Science Association White Paper Series, Number 1, 1−15. Sindelar, J. J., Cordray, J. C., Sebranek, J. G., Love, J. A., & Ahn, D. U. (2007a). Effects of varying levels of vegetable juice powder and incubation time on color, residual nitrate and nitrite, pigment, pH, and trained sensory attributes of ready-to-eat uncured ham. Journal of Food Science, 72(6), S388−S395. Sindelar, J. J., Cordray, J. C., Sebranek, J. G., Love, J. A., & Ahn, D. U. (2007b). Effects of vegetable juice powder concentration and storage time on some chemical and sensory quality attributes of uncured, emulsified cooked Sausages. Journal of Food Science, 72(5), S324−S332. Tantikarnjathep, K., Sebranek, J. G., Topel, D. G., & Rust, R. E. (1983). Use of vacuum during formation of meat emulsions. Journal of Food Science, 48(4), 1039−1041.

461

USDA (2005). Food standards and labeling policy book www.fsis.usda.gov/OPPDE/larc/ Policies/Labeling_Policy_Book_082005 pdf Accessed 23.9.2010. Vasavada, M. N., & Cornforth, D. P. (2005). Evaluation of milk mineral antioxidant activity in beef meatballs and nitrite-cured sausage. Journal of Food Science, 70(4), C250−C253. Walker, R. (1990). Nitrates, nitrite, and n-nitroso compounds — a review of the occurrence in food and diet and the toxicological implications. Food Additives and Contaminants, 7(6), 717−768. Winter, C. K., & Davis, S. F. (2006). Organic foods. Journal of Food Science, 71(9), R117−R124. Zubillaga, M. P., Maerker, G., & Foglia, T. A. (1984). Antioxidant activity of sodium nitrite in meat. Journal of the American Oil Chemists Society, 61(4), 772−776.