Experimental design and product preparation

Three product formulations of all-beef, large-diameter sandwich bologna were manufactured in the Iowa State University Meat Laboratory, Ames, Iowa, to provide 3 different concentrations of ingoing nitrite. The 3 formulations were packaged in conventional and nitrite-embedded film films as follows. (1) The first formulation (“CON-CF”) was a conventionally cured control (nitrite from Modern Cure, with sodium erythorbate, both provided by A.C. Legg Inc., Calera, AL), which was vacuum packaged with conventional, high-barrier film that is commercially available (Sealed Air Corporation, Duncan, SC). The second (“CJP-CF”) and third (“CJP-NEF”) formulations were an alternatively cured bologna formulation utilizing nitrite from cultured celery juice powder (VegStable 506) and cherry powder (VegStable 515) supplied by Florida Food Products, Inc. (Eustis, FL), with (2) one-half of the batch (CJP-CF) vacuum packaged in conventional film and (3) the second half of the batch (CJP-NEF) vacuum packaged in nitrite-embedded film (Bemis Company Inc., a division of Amcor Flexibles North America, Oshkosh, WI) pouches. An additional alternatively cured formulation was produced using Natpre T-10 EML Plus S (Productos Sur, S.A. [Prosur], San Ginés, Murcia, Spain), with (4) one-half of the batch (“NT10-CF”) vacuum packaged in conventional film and (5) the second half of the batch (“NT10-NEF”) vacuum packaged in nitrite-embedded film pouches. The Natpre T-10 EML Plus S was chosen for this study because it was a commercially available alternative cure that provided a very low ingoing nitrite concentration for the bologna. The nitrite concentration for the Modern Cure (CON-CF) ingredient was 62,500 parts per million (ppm) sodium nitrite, which resulted in 156 ppm ingoing sodium nitrite for the bologna as formulated; the cultured celery juice powder (VegStable 506) ingredient (CJP-CF and CJP-NEF groups) contained 22,500 ppm sodium nitrite, which resulted in 99 ppm ingoing sodium nitrite; and the Natpre T-10 EML Plus S ingredient (NT10-CF and NT10-NEF groups) contained 1,700 ppm sodium nitrite, which resulted in 21 ppm ingoing sodium nitrite. Nitrite concentrations of Modern Cure and celery juice powder were provided by respective commercial ingredient suppliers, and the nitrite concentration of Natpre T-10 EML Plus S was determined by analysis using AOAC Method 993.30 (AOAC International, 2005a). The ingoing nitrite concentrations were determined by calculation based on quantities of the respective ingredients used (USDA, 1995).

All nitrite concentrations were determined and expressed as sodium nitrite. Treatment formulations are listed in Table 1. The experiment was replicated twice.

The beef used for this study was harvested from market weight animals obtained from university farms, fabricated and frozen by the Iowa State University Meat Laboratory. The meat was stored frozen at −20°C until use, then thawed at 4.4°C for 2 d and moved into refrigerated storage at 1°C for 24 to 72 h. All treatments were processed separately using the same procedure. Replications were manufactured on separate, consecutive days, and the treatment processing sequence was randomized prior to production. Manufacturing and thermal processing of all treatments occurred on the same day for each replication. The slicing and packaging order of each treatment was conducted in the same order as production.

Beef raw materials were first ground through a 12.7-mm plate (Biro Manufacturing Company, Marblehead, OH). Each formulation consisted of 45.36 kg of meat including 14.97 kg of beef 50s and 30.39 kg of beef 80s. Lean (beef 80s) and fat (beef 50s) portions were mixed separately using a double action mixer (Leland Southwest, Fort Worth, TX) to ensure uniform distribution of fat and lean after grinding and to create a uniform blend of each. The lean ground beef (beef 80s) was then added to a vacuum bowl chopper (Kramer & Grebe GmbH and Co., KG, Biendenkopf-Wallau, Germany) along with salt, curing ingredient, half of the water/ice mixture, spices (A.C. Legg Inc., Calera, AL), and cure accelerator (if needed). For the experimental treatments of CON-CF, CJP-CF, and CJP-NEF, the cure accelerator (sodium erythorbate or cherry powder) was added to the spice blend and mixed prior to use. A cure accelerator was not included in the Natpre T-10 EML Plus S formulation. The mixtures were chopped until the batter temperature reached 4.4°C. The fat trimmings (beef 50s) were then added, along with remaining water/ice mixture, and chopping continued until the batter temperature reached 13°C. The meat batters were then moved to a vacuum stuffer (Handtmann VF 608 Plus, Lake Forest, IL) and stuffed into 14.25 cm × 114.3 cm pre-stuck fibrous casings (Kalle: 6.5 × 45 Fibrous N Clear, Kalle, Wiesbaden, Germany). Each bologna log was individually weighed, placed horizontally on a smoke rack, and moved into a smokehouse (Alkar, The Middleby Corporation, Elgin, IL) for thermal processing, which included application of smoke. All treatments were thermally processed together in the smokehouse for each replication. Thermal processing utilized a standard large-diameter bologna thermal processing schedule with stepwise temperature increases to reach internal product temperature of 71°C (approximately 6 h).

After cooking, the bologna logs were chilled overnight (approximately 19 h) at 1 ± 2°C. Subsequently, bologna logs were weighed for cooked and chilled yield measurements, casings were removed, and the product was sliced (Bizerba, Piscataway, NJ) into 6.35-mm-thick slices and packaged. Four slices were stacked and placed into either conventional vacuum packages (CON-CF, CJP-CF, NT10-CF) (2-mil thick with an oxygen transmission rate of 1.5–3.5 cm³/0.06 m²/24 h/1 atmosphere (atm) at 5°C and 0% relative humidity [RH]; water vapor transmission rate of 0.3–0.6 g/0.06 m²/24 h/1 atm at 38°C and 100% RH; Cryovac, Sealed Air Corporation, Duncan, SC) or nitrite-embedded film vacuum packages (CJP-NEF, NT10-NEF) (7-mil thick with an oxygen transmission rate of < 0.3 cm³/0.06 m²/24 h/1 atm at 23°C and 0% RH; water vapor transmission rate of < 0.5 g/0.06 m²/24 h/1 atm at 38°C and 100% RH; FreshCase, Bemis Company Inc., a division of Amcor Flexibles North America, Oshkosh, WI). The nitrite-embedded film was the same as that approved for use with fresh meat and included 113 mg of sodium nitrite per square meter of film. Both package types (conventional film, nitrite-embedded film) were vacuum sealed (Ultravac UV 2100 vacuum chamber packaging machine, UltraSource LLC, Kansas City, MO) and immediately stored at 1 ± 2°C under continuously lighted (24 h/d) simulated retail display conditions using white fluorescent lights (32W, 120V; Sylvania, Danvers, MA) for the duration of the study. Packages were placed in single layers on shelves, with each shelf having fluorescent lights suspended immediately above the packages. The light source was placed approximately 254 mm from the package surface, and illuminance was 2,200 ± 500 lx. Illuminance was measured using an URCERI Light Meter MT-912 (URCERI, Shenzhen Huanhui E-commerce company, Ltd, Shenzhen, China). Multiple locations throughout the storage area were selected for illuminance measurements. Sample locations were routinely rotated (every 3 wk) to provide uniform light exposure. Product packaging day was considered day 0 of the experiment.

Analytical procedures: Color

Surface color and internal color of the stacks of slices were measured in 2 ways using Commission Internationale de l’Eclairage (CIE) L*a*b* color space (L* = lightness; a* = redness; b* = yellowness). For in-package color, measurements were done with the packaging film in place on the products. Unpackaged color was measured on the products after opening the packages and removing the packaging film. In both cases, color measurements were done on days 1, 6, 13, 27, 41, 55, 69, 83, 97, 111, and 125 following packaging. All color measurements were taken at a 10° observer angle, using illuminant D65 (daylight at 6,500 K), and a 2.4-cm aperture size, with a HunterLab MiniScan EZ 4500L colorimeter (Hunter Associates Laboratory Inc., Reston, VA). For unpackaged color measurements, a standard instrument calibration (with no film covering the black or white tiles) was used, and measurements were conducted by placing the HunterLab MiniScan EZ instrument nose cone directly onto the product surface. For the in-package surface measurements, a modified colorimeter standardization procedure was utilized, in which the white calibration tile was covered with the respective packaging material (conventional film or nitrite-embedded film). Product measurements were then taken by placing the nose cone of the HunterLab MiniScan EZ instrument directly onto the package. This process was used to more accurately measure the visual color of the packaged product as observed by consumers.

For unpackaged products, both surface and internal color were measured. One package per treatment (5), per sample day (11), per replication (2) was randomly selected for both surface and internal color measurement for a total of 110 packages measured during the display period. Measurements were done by opening the package, removing the slices, and subsequently measuring the surface of the top slice (slice directly exposed to light source) for unpackaged surface color as well as measuring the color at the interface of slices 2 and 3 of the 4-slice stack for the internal color measurement.

For in-package surface color (with the packaging film intact), 22 packages of each treatment and each replication were selected for repeated measurement of the same designated packages on each sample date for the duration of the display period. This generated a total of 220 measurements (22 packages × 5 treatments × 2 replications) of in-package color on each sampling day of the 125-d display period. The mean of the 3 measurements on each of the 220 packages for both in-package and unpackaged products was calculated and used to represent the color values of each package at each time point of the display period.

Analytical procedures: Residual nitrite

Residual nitrite analysis was done in accordance with AOAC Method 973.31 (AOAC International, 2005b). Sampling was conducted in duplicate on days 1, 6, 13, 27, 41, 55, 69, 83, 97, 111, and 125 post packaging. Samples were prepared by separating the 2 exterior slices (slices 1 and 4 in the stack) from the 2 interior slices (slices 2 and 3 in the stack) of each package. The exterior and interior slices were finely chopped separately using a food processor (KitchenAid, St. Joseph, MI), and 5.0 g (± 0.01 g) was weighed into a beaker. Each 5.0 g sample was then added to a 500 mL volumetric flask with approximately 300 mL hot (approximately 50°C–80°C) distilled water and placed into a 100°C water bath. Flasks remained in the water bath for 2 h and were swirled every 30 min. After heating, the flasks were cooled to room temperature (approximately 23°C), then filled to volume (500 mL) with distilled water. Approximately 30 mL was filtered through Whatman #1 filter paper (Whatman Grade 1, GE Health Care Life Sciences, Pittsburgh, PA) into a 50 mL volumetric flask. Next, reagents were added as described in the AOAC International procedure, flasks were filled to volume, and absorbance at 540 nm was recorded using a Beckman DU 640 spectrophotometer (Model 4320940; Beckman, Fullerton, CA).

Analytical procedures: Residual nitrate

Residual nitrate analysis was conducted by Hormel Laboratories (Division of Hormel Foods, LLC, Austin, MN) using AOAC Method 993.30 (AOAC International, 2005a). Sampling was conducted on days 1, 6, 13, 27, 41, 55, 69, 83, 97, 111, and 125 post packaging. On each sampling day, collected samples were immediately frozen at −20°C ± 5°C and shipped to Hormel Laboratories in insulated shipping containers with ice packs. The samples were held frozen until analysis and thawed overnight prior to sample preparation. Thawed samples were prepared by first separating the 2 exterior slices from the two interior slices. The exterior and interior slices were homogenized separately using a commercial grade food processor. One gram of each sample was weighed into a 100 mL Kohlrausch volumetric flask, and 50 mL of distilled hot water (approximately 50°C–80°C) was added. Flasks were placed on a 100°C steam bath for 1 h and chilled in ice water (approximately 0°C) for 15 min. After chilling to room temperature (approximately 23°C), the flasks were filled to volume and shaken. Approximately 30 mL of sample was filtered through Whatman GF/C filter paper (Whatman Grade 1, GE Health Care Life Sciences, Pittsburgh, PA), and the filtrate was again filtered under vacuum through a Dionex OnGuard II RP 2.5 cc cartridge (Thermo Fisher Scientific, Inc., Waltham, MA) equipped with a 10-mL syringe pretreated with 10 mL of methanol and 15 mL of distilled water. The first 6 mL of sample filtrate was discarded to ensure proper flushing of methanol and distilled water. A multisample Restek Resprep 12-Port Vacuum Manifold (Restek Corporation, Bellefonte, PA) was used under vacuum to ensure proper sample flow rate. Sample flow rate through the cartridge was approximately 1 mL per minute to provide a 1:100 dilution factor. The samples were then added to a Dionex High Pressure Ion Chromatography system (Thermo Fisher Scientific, Inc., Waltham, MA). Residual nitrate peaks from the resulting chromatograms were plotted, and the area under each peak used to calculate concentration in ppm.

Analytical procedures: Proximate composition

Duplicate samples of raw batters and cooked products from each production batch were finely chopped using a food processor (KitchenAid, St. Joseph, MI) for measurement of fat, moisture, and protein content. Fat content was analyzed by the CEM ORACLE System (AOAC International, 2013; CEM Corporation Matthews, NC), moisture was evaluated with the CEM SMART 6 System (AOAC International, 2013; CEM Corporation), and protein content was measured with the CEM Sprint Rapid Protein Analyzer (AOAC International, 2011; CEM Corporation).

Analytical procedures: pH

Samples of raw batters and cooked products from each treatment were finely chopped using a food processor, as done for the proximate composition measurements. Ten grams of each sample were weighed into a beaker, and 90 mL of ambient-temperature distilled water were added. The meat and water were mixed thoroughly by hand with a glass stirring rod for 1 min, after which a filter paper disc (Whatman Grade 1, GE Health Care Life Sciences, Pittsburgh, PA) was folded and submerged into the sample. The pH of the filtered solution was measured with a Mettler Toledo SevenMulti pH meter (Mettler Toledo, Columbus, OH). Duplicate measurements of raw batters were done on the day of production, whereas the cooked products were measured in duplicate on day 14 following thermal processing and chilling of the products. The pH of some of the nonmeat ingredients included in the formulations was measured by first dispersing 5 g of each ingredient in 90 mL of distilled water, then measuring the pH as described previously.

Analytical procedures: Microbiology

Microbial populations of aerobic bacteria and of lactic acid bacteria on the products were monitored utilizing a shelf life procedure in which all treatments and replications were stored at 1°C ± 2°C. The analyses were conducted on days 0, 7, 14, 30, 60, 90, and 120. Eleven grams of sample was aseptically removed from each package, and 99 mL of 0.1% peptone water (Hardy Diagnostics, Category Number D299, Santa Maria, CA) was added, creating a 1:10 dilution. The samples were then placed into a WhirlPak filter stomaching bag (Nasco, Ft. Atkinson, WI), homogenized in a stomacher with the peptone water for 1 min, and serially diluted, with the first 10-fold dilution coming from the stomaching bag and the remaining dilutions (to extinction) consisting of 1 mL into 9 mL of 0.1% peptone water. Subsequently, total aerobic bacterial populations were enumerated in duplicate on Aerobic Count Petrifilm (3M Health Care, St. Paul, MN) following the manufacturer’s instructions. Petrifilms were incubated aerobically at 21°C for 72 h to assess total aerobes, and populations were determined following the manufacturer’s instructions. For lactic acid bacteria populations, enumeration was conducted anaerobically on the same days as the total aerobic populations and followed the procedure previously described, but with 0.1 mL of homogenized sample surface plated onto DeMan, Rogosa and Sharpe (MRS) agar (Becton, Dickinson and Company, Sparks, MD) in duplicate. Plates were incubated anaerobically at 31°C ± 2°C for 72 h before counting.

Due to the low bacterial growth observed during conventional storage at 1°C, an additional accelerated shelf life analysis was conducted to test the impact of the packaging environment on potential growth of spoilage bacteria. To achieve this, a spoilage inoculum of lactic acid bacteria was isolated from samples of commercial bologna. To create the inoculum, packages of 5 different commercial bologna products were purchased from retail stores. Two slices (approximately 60 g) were removed from each package, added to a bag with 1 L of tryptic soy broth (Becton, Dickinson and Company), and allowed to incubate at 20°C for 72 h. After incubation, 1 mL of broth was removed, added to 9 mL of MRS broth (Becton, Dickinson and Company), and incubated at 30°C for 48 h; 1 mL of the inoculated broth was then transferred to an additional 9 mL of MRS broth and again incubated at 30°C for 48 h. This bacterial transfer process was repeated 4 times. Finally, 1 mL of culture was added to 40 mL of MRS broth and incubated at 30°C for 48 h. Serial dilutions (to extinction) were made from the final culture and plated onto MRS agar to enumerate the population of the lactic acid bacteria inoculum. The culture was held at 1°C during this time. The culture was then diluted to achieve an approximate inoculation population of log₁₀3 colony-forming units (CFU)/g in the packages. The packages were inoculated with 1 mL of the spoilage inoculum by aseptic injection through a self-closing foam adhesive septum placed on each package. The inoculated packages were held at 10°C for the duration of the inoculation study. Inoculation occurred on day 158 post processing of the previously described products. For the inoculation study (denoted as I), day 158 post processing was designated as day 0(I). Analyses were conducted on days 0(I), 3(I), 6(I), 9(I), 12(I), 18(I), and 21(I) of storage at 10°C ± 2°C.

All microbiological data (conventional and inoculated shelf life) were collected and reported logarithmically as CFU/g.

Raw composition, processing attributes, and final composition

Mean treatment effects on proximate composition of raw and cooked bologna in addition to cooked yields (Table 2) showed no significant difference (P = 0.133; Table 3) for protein content of the raw products between any treatments. Fat content of raw CJP-CF and CJP-NEF treatments were not different, but both treatments had higher fat content than CON-CF, NT10-CF, and NT10-NEF treatments. Moisture content was higher for raw CON-CF than for all other treatments (CJP-CF, CJP-NEF, NT10-CF, and NT10-NEF), but there were no significant differences (P > 0.20) between the other treatments.

For cooked products, fat, moisture, and protein content in samples of the treatment groups followed trends similar to those of the raw product composition. Because there was relatively little numerical difference between treatments for raw or cooked composition, the composition was not expected to affect the outcome of this study. Cooked and chilled yield results were all within approximately 1% among the treatments, though the Natpre T-10 EML Plus S treatments resulted in a small but significant reduction of the cooked yield for those products.

Table 4 shows that there was no difference (P = 0.115) between treatments for pH of the raw products. However, cooked pH was significantly lower (P < 0.001) in NT10-CF and NT10-NEF treatments than in the other treatments. The pH measurements of the nonmeat ingredients showed that the Natpre T-10 EML Plus S ingredient was considerably more acidic (pH 4.76) than Modern Cure (pH 7.84), sodium erythorbate (pH 7.50), celery juice powder (pH 8.67), or cherry powder (pH 6.03) and probably resulted in the lower pH of the cooked products. The reduced product pH may have contributed to the lower cooking yields observed (Table 2) for those treatments.

In-package surface color

Table 5 shows the main effects for treatments during 125-d display, which were significant (P < 0.001); however, main effects are only briefly described later since the interaction was also significant (P < 0.001). Nitrite-embedded film resulted in significantly greater in-package surface a* values (redness) in both the celery juice powder and Natpre T-10 EML Plus S formulations. Further, the CJP-NEF treatment was also significantly redder than the control despite a lower ingoing nitrite concentration (99 ppm vs. 156 ppm). However, the nitrite-embedded film treatment had the greatest impact on redness in the case of the two Natpre T-10 EML Plus S products in which ingoing nitrite was extremely low (21 ppm). The decreased redness resulting from the low nitrite concentration in the conventional film package of the NT10-CF (a* = 7.34) in combination with the observed increased redness in the NT10-NEF treatment (a* = 12.02) highlights the benefit of nitrite-embedded film. There was no difference for in-package surface a* values for the conventional film treatments (CON-CF and CJP-CF) with ingoing nitrite concentrations of 156 and 99 ppm, respectively. The relative treatments effects for in-package surface a* values were in the following order: CJP-NEF > CJP-CF and CON-CF >NT10-NEF > NT10-CF (Table 5).

Figure 1 shows the in-package surface a* values for treatment × day effects. The impact of the nitrite-embedded film package on the very low ingoing nitrite formulation (NT10-NEF) compared to the conventional film package (NT10-CF) is clear, with a* values for the NT10-NEF treatment approaching the other treatments that included much greater ingoing nitrite in the formulations. While not specifically denoted in Figure 1, the increase in NT10-NEF a* value between 1 d and 27 d was significant (P = 0.010), suggesting an increase in cured pigment content during the first 27 d while in contact with the film for NT10-NEF. CJP-CF most notably showed significantly (P < 0.001) reduced a* values between 1 d and 83–125 d of storage, while its nitrite-embedded film counterpart did not show any significant reduction from 1-d a* value until after day 111. There was a significant decline (from day 1) toward the end of lighted display period (as would be expected) for CJP-NEF (after day 111), CON-CF (after day 83), and CJP-CF (after day 55). Treatment means for in-package surface L* were significantly different as a result of the curing ingredients (NT10-CF and NT10-NEF > CON-CF > CJP-CF and CJP-NEF; Table 5) but not as a result of the packaging film. In-package surface L* values for treatment × day effects did not differ over time for any treatment (data not shown).

Unpackaged surface and internal color

The results for unpackaged surface and internal CIE L*, a*, and b* measurements for treatment effects are shown in Table 6 and are, in general, similar to the results observed for in-package surface color measurements. There were no differences between CJP-NEF, CJP-CF, and CON-CF or between CJP-CF and NT10-NEF for unpackaged surface a* value. The Natpre T-10 EML Plus S treatments with very low ingoing nitrite again demonstrated the impact of nitrite-embedded film packaging for increasing product redness, but the nitrite-embedded film in this case did not have a significant effect on the formulation with celery juice powder, as it did for the in-package a* values.

Unpackaged surface a* values for treatment × day effects are displayed in Table 7. There were no significant differences for CJP-NEF, CON-CF, or NT10-CF over the lighted display period (125 d). Most notable, again, is the effect of the nitrite-embedded film on the low nitrite product (Natpre T-10 EML Plus S). The nitrite-embedded film package resulted in greater a* value than the conventional film for the Natpre T-10 EML Plus S formulation at all time points after day 1. Figure 2 shows the visual difference between NT10-CF and NT10-NEF unpackaged surface redness between day 1 and 41. The visual appearance of both external and internal slice surfaces from nitrite-embedded film packages appear to be slightly redder at day 1 but are clearly redder at day 41, suggesting that further formation of nitrosylhemochrome pigment from nitrite and cooked meat pigments occurred during storage in the nitrite-embedded film package.

The internal a* values for treatment effects are also presented in Table 6 and show similar results as for unpackaged surface a* values. The nitrite-embedded film again resulted in significantly greater internal a* value than the conventional film for the Natpre T-10 EML Plus S (very low nitrite formulation) but lower internal a* values than the CJP-NEF, CON-CF, and CJP-CF treatments, all of which included greater ingoing nitrite concentrations.

Internal a* values for treatment × day effects also did not differ among CJP-NEF, CJP-CF, CON-CF, or NT10-CF treatments during the display period (125 d) (Table 8), but internal a* values for NT10-NEF increased from day 1 to day 27, with no further increase following day 27. It is notable that the nitrite-embedded film package resulted in increased a* values over time in the cooked, finished product with very low nitrite ingoing concentration, suggesting significant formation of cured pigment from the nitrite embedded in the film, after packaging was completed.

There were no differences between NT10-CF, NT10-NEF, CON-CF, or CJP-CF for unpackaged external surface L* value treatment effects (Table 6). However, CJP-NEF was darker than either NT10-CF or NT10-NEF. Internal L* values followed a similar trend with NT10-CF significantly lighter than CON-CF, CJP-CF, and CJP-NEF but not different than NT10-NEF.

Unpackaged surface b* value (Table 6) for NT10-CF was higher than for all other treatments; however, there was no difference between the other treatments. Additionally, NT10-CF was also greater than the other treatments for internal b* values. However, for the internal b* values, although NT10-CF was again highest, there were differences between the other treatments. In this case, NT10-NEF was second highest, while CJP-CF and CJP-NEF followed, with CON-CF having the lowest b* value.

Unpackaged surface and internal L* values for treatment × day effects during lighted display showed no difference (P > 0.90) between any other treatments during the display period (125 d) (data not shown). Internal b* values for treatment × day effects also did not differ in CJP-NEF, CJP-CF, CON-CF, or NT10-CF during the display period (125 d) but were lower for NT10-NEF for days 1 through 125 (data not shown).

Surface and internal residual nitrite

Slices that were in direct contact with the packaging material (slices 1 and 4 of each package) and internal slices that were in the center of each package (slices 2 and 3) were analyzed separately for residual nitrite. Slices 1 and 4 were combined to represent the residual nitrite in the surface slices, while slices 2 and 3 were combined to represent the internal residual nitrite concentration.

Surface residual nitrite values for treatment × day effects are displayed in Figure 3A. The impact of formulated ingoing nitrite is clear with the control (CON-CF) and celery juice powder formulations resulting in the greatest concentration of residual nitrite on day 1. The CJP-NEF was slightly higher than the CJP-CF initially, but this was significant only on day 6. This does, however, again suggest that the nitrite-embedded film package transfers a small amount of nitrite to the product to potentially improve and extend cured color stability during product storage. NT10-CF and NT10-NEF, on the other hand, were consistently very low in residual nitrite and did not differ at any day of the display period. It is notable that the nitrite-embedded film package did not increase the measurable residual nitrite in the product with very low ingoing nitrite concentration. CJP-CF was not different from CON-CF or CJP-NEF except at day 6. None of the treatments were different at day 27 and after.

Internal residual nitrite concentrations as a result of treatments are shown in Figure 3B, and the results are very similar to those for surface residual nitrite. Table 9 shows the means for treatment effects and confirms the similarity of the surface and internal nitrite concentrations. Consequently, while the nitrite-embedded film packages impacted the redness (a* value) of the product produced with very low ingoing nitrite concentration, the film did not contribute to the residual nitrite concentration in the product.

Surface and internal residual nitrate

Surface and internal residual nitrate for each treatment are shown in Table 10. The internal nitrate concentration for celery juice powder formulations was greater than all the other products, probably due to increased nitrate concentrations commonly found in cultured celery juice powder. Surface nitrate concentrations followed a similar pattern though differences are not as clearly defined. There were no treatment or day effects for either surface or internal residual nitrate during the 125-d display period (data not shown).

Microbiological results

The total aerobic bacterial populations (log CFU/g) in conventional shelf life conditions (1°C) showed essentially no growth in any of the treatments during the 120 d of storage (data not shown). However, there was a treatment effect at day 30, with NT10-NEF being significantly higher for total aerobic counts than CON-CF but not significantly different from NT10-CF, CJP-CF, or CJP-NEF. Given that this does not reoccur after day 30, this was most likely due to sampling error or some other procedural effect rather than a treatment effect. Lactic acid bacteria populations (log CFU/g) for conventional shelf life conditions also showed no measurable counts for any of the treatments (data not shown).

Table 11 shows the overall lactic acid bacteria populations (log CFU/g) for the inoculated study (10°C for 21 d). The CON-CF, CJP-CF, and CJP-NEF treatments had significantly greater mean lactic acid bacterial populations than NT10-CF and NT10-NEF, though the differences were less than 0.37 log CFU/g. For treatment × day effects, while growth occurred in all treatments for total aerobic and lactic acid bacteria populations (log CFU/g), there were no significant differences between any treatment at any specific day (data not shown). Lactic acid bacterial populations increased from log 3.70 CFU/g–3.95 CFU/g initially among the treatments to log 8.70 CFU/g–8.90 CFU/g, and total aerobic bacterial populations increased from log 2.80 CFU/g–3.25 CFU/g initially among treatments to log 7.90 CFU/g–8.20 CFU/g after 21 d at 10°C, respectively.