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Effects of pyruvate, succinate, and lactate enhancement on beef longissimus raw color
Meat Science 88 (2011) 424–428
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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
Effects of pyruvate, succinate, and lactate enhancement on beef longissimus raw color R. Ramanathan, R.A. Mancini ⁎, G.A. Dady Department of Animal Science, University of Connecticut, Storrs, CT 06269-4040, USA
a r t i c l e
i n f o
Article history: Received 8 November 2010 Received in revised form 20 January 2011 Accepted 21 January 2011 Keywords: Pyruvate Succinate Lactate Beef color Myoglobin Lipid oxidation
a b s t r a c t Beef strip loins (n = 30) were divided into halves, and each half was assigned randomly to one of four injection enhancements: (1) non-enhanced control, (2) 3% pyruvate, (3) 3% succinate, and (4) 3% lactate. Steaks were cut and packaged in either vacuum, high oxygen (80% O2/20% CO2), or PVC. Color and lipid oxidation were measured on days 0, 5, and 13 of storage at 1 °C. Enhancement had a significant effect on steak pH. On day 13 of storage, steaks enhanced with lactate, pyruvate, and succinate were less discolored (P b 0.05) than control steaks in PVC and high oxygen. Enhancement darkened steaks (P b 0.05) compared with control steaks. Succinate had the greatest and pyruvate had the least metmyoglobin-reducing activity (P b 0.05). Lactate and pyruvate decreased the TBARS values of steaks packaged in PVC (P b 0.05) whereas pyruvate was most effective for lowering lipid oxidation in high-oxygen packaging. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction Maintenance of fresh meat color is critical for extending the shelf life of beef. Although numerous extrinsic and intrinsic factors influence myoglobin redox stability during both storage and display, meat has inherent metmyoglobin-reducing systems that utilize NADH to delay the accumulation of surface metmyoglobin (Giddings, 1974; Ledward, 1985). Nevertheless, depletion of substrates in postmortem muscle influences the regeneration of reducing equivalents (Saleh & Watts, 1968; Watts, Kendrick, Zipser, Hutchins, & Saleh, 1966). Caseready technology provides an opportunity to enhance meat with various ingredients that can serve as substrates for reducing systems and therefore, influence color life. Lactate is commonly added to case-ready meat and is often described as a color stabilizer in fresh beef. Researchers have reported that NADH formed via lactate dehydrogenase activity can increase color stability by increasing metmyoglobin reduction (Kim et al., 2006). Moreover, various in vitro studies suggest that other glycolytic and tricarboxylic intermediates also can influence myoglobin redox stability. For example, Mohan, Hunt, Barstow, Houser, and Muthukrishnan (2010) reported that addition of pyruvate and malate improved color stability of muscle homogenates. In addition, Ramanathan and Mancini (2010) reported that pyruvate addition to isolated mitochondria resulted in electron transport mediated metmyoglobin reduction. Tang, Faustman, Mancini, Seyfert, and
⁎ Corresponding author. Tel.: +1 860 486 1775; fax: +1 860 486 4375. E-mail address: [email protected] (R.A. Mancini). 0309-1740/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2011.01.021
Hunt (2005) reported that the addition of succinate to isolated cardiac mitochondria increased oxygen consumption and electron transport mediated metmyoglobin reduction. More specifically, Tang et al. (2005) hypothesized that this succinate-mediated increase in oxygen consumption will lower oxygen partial pressure, thereby increasing the amount of available electrons that can be transferred to metmyoglobin. Furthermore, the ability of pyruvate and succinate to minimize lipid oxidation has been reported in various non-meat model systems (Puntel et al., 2007; Valentovic & Minigh, 2003). Although fundamental research suggests that pyruvate and succinate can influence myoglobin redox chemistry, no research has assessed the effects of pyruvate and succinate enhancement on beef color stability. Therefore, the objectives of the present study were to assess the comparative effects of pyruvate, succinate, and lactate enhancement on the color of beef longissimus lumborum packaged in either vacuum, PVC, or high oxygen and stored for 0, 5, and 13 days at 1 °C.
2. Materials and methods 2.1. Raw materials and processing Beef strip loins (NAMP 180; NAMP, 2002; n = 30) were obtained locally from a beef purveyor at 7 days postmortem. Each loin was divided into two halves, and each experimental unit within a loin was assigned randomly to one of four injection enhancements: (1) nonenhanced control, (2) 3% pyruvate, (3) 3% succinate, and (4) 3% lactate, resulting in n = 15 replications per treatment. Enhancement solutions contained deionized distilled water and either 33% sodium pyruvate (Nutriscience, Trumbull, CT), 33% lactate (PURASAL HiPure P, 78% potassium lactate and 22% water; PURAC
R. Ramanathan et al. / Meat Science 88 (2011) 424–428
America, Inc., Lincolnshire, IL), or 33% sodium succinate (Fisher Scientific, Pittsburg, PA). Loins were pumped to 110% of green weight using a multi-needle injector (Koch, Kansas City, MO) to achieve an ingredient concentration of 3% w/w in the final product. For control samples, no enhancement solution was injected into the individual loin units. Samples were weighed before and 30 min after injection to confirm target enhancement levels: calculated as [(weight after injection − green weight)/green weight] × 100. From each experimental unit, seven 1.91-cm thick steaks were cut and one steak was assigned to day 0 analyses (no packaging or storage time) for surface color, metmyoglobin-reducing activity, lipid oxidation, and pH measurements. The remaining six steaks were individually packaged in either vacuum, high-oxygen (80% O2/20% CO2), or aerobic packaging (PVC; over-wrapped with oxygen-permeable polyvinyl chloride fresh meat film; 15,500–16,275 cm3 O2/m2/24 h at 23 °C, E-Z Wrap Crystal Clear Polyvinyl Chloride Wrapping Film, Koch Supplies, Kansas City, MO). Both vacuum and high-oxygen packaging were performed using a Koch MultiVac 500 (Kansas City, MO), Prime Source vacuum pouches (4 mil, standard barrier nylon/ polyethylene, 0.6 cm3 O2/645.16 cm2/24 h at 0 °C; Bunzl Koch Supplies Inc., Kansas City, MO), and certified gas blends (Airgas, Cheshire, CT). Using a headspace analyzer (Model 6600 Headspace Oxygen/Carbon Dioxide Analyzer, Illinois Instruments, Ingleside, IL), the percentage O2 in extra modified atmosphere packages was determined 24 h after packaging. Packages were stored in the dark at 1 °C. 2.2. Instrumental color analysis The fresh-cut surface color of each day 0 steak was measured 1 h after fabrication (before packaging, bloom at 1 °C). Of the two steaks assigned to each packaging type, one steak was used for day 5 analyses and the other steak was used for day 13 analyses. On days 5 and 13 of storage, surface color was measured immediately after each steak was removed from its respective packaging. All instrumental color measurements were performed using a HunterLab MiniScan XE Plus spectrophotometer (Model 45/0 LAV, 2.54-cm diameter aperture, illuminant A, 10° observer). Both reflectance spectra from 400 to 700 nm (10 nm increments) and CIE L* and a* were measured in triplicate for each steak and subsamples were averaged for statistical analyses. Reflectance data from 400 to 700 nm also was used to estimate % metmyoglobin during storage (AMSA, 1991).
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calculate metmyoglobin-reducing activity. The following equation was used: %Metmyoglobin =
K=S 572 K=S 525 K=S 572 K=S 525
for 100% deoxy or oxymyoglobin
for 100% deoxy or oxymyoglobin
K=S 572 K=S 525
K=S 572 K=S 525
for sample
for 100% metmyoglobin
2.4. Metmyoglobin-reducing activity Following surface color measurement, each steak was cut in half perpendicular to the surface. One half was used for metmyoglobinreducing activity and the other half was used for lipid oxidation and pH measurements. Metmyoglobin-reducing activity (MRA) was determined using a sample from the interior portion of each steak on days 0, 5, and 13, according to a procedure described by Sammel, Hunt, Kropf, Hachmeister, and Johnson (2002). Samples were submerged for 20 min in a 0.3% solution of sodium nitrite at 25 °C to facilitate metmyoglobin formation, and then removed, blotted dry, vacuum packaged (Prime Source Vacuum Pouches, 4 mil, KOCH Supplies Inc., Kansas City, MO), and scanned twice with a HunterLab MiniScan XE Plus Spectrophotometer to determine preincubation metmyoglobin values (AMSA, 1991). Each sample was incubated at 30 °C for 2 h (Fisher Scientific, Model 630F, NY) to induce metmyoglobin reduction. Upon removal from the incubator, samples were rescanned twice to determine the percentage of remaining surface metmyoglobin. The following equation was used to calculate metmyoglobin-reducing activity: [(% surface metmyoglobin pre-incubation − % surface metmyoglobin post-incubation) /% surface metmyoglobin preincubation]× 100. 2.5. Determination of pH Samples from steaks assigned to days 0, 5, and 13 of storage, visually devoid of fat and connective tissue, were used to determine pH after instrumental color analysis. Using a Waring table-top blender (Dynamics Corp. of America, New Hartford, CT), 10 g of sample was combined with 90 ml of deionized water and mixed for 30 s, and the pH values were determined using an Accumet 50 pH meter (Fisher Scientific, Fairlawn, NJ). 2.6. Lipid oxidation
2.3. Measuring raw metmyoglobin percentage Reflectance at isobestic wavelengths was used to estimate raw surface metmyoglobin content. Reference standards for 100% of oxyand metmyoglobin were created for both control and enhanced steaks. To create 100% metmyoglobin, additional steaks not used for color analysis were placed in 1.0% potassium ferricyanide for 1 min, drained, blotted dry, and packaged in oxygen-permeable film (PVC; over-wrapped with oxygen-permeable polyvinyl chloride fresh meat film; 15,500–16,275 cm3 O2/m2/24 h at 23 °C, E-Z Wrap Crystal Clear Polyvinyl Chloride Wrapping Film, Koch Supplies, Kansas City, MO). Samples were stored at 4 °C for 12 h, removed from the package, and then reflectance was measured from 700 to 400 nm. To create 100% oxymyoglobin, steaks were placed in a pouch (Prime Source Vacuum Pouches, 4 mil, Koch Supplies Inc., Kansas City, MO) and flushed with 100% oxygen (Airgas, Cheshire, CT). Each pouch was sealed, kept for 2 h at 4 °C, and samples were removed from the package and reflectance was measured from 700 to 400 nm. Reflectance at 525 and 572 nm for each myoglobin form were converted to K/S values using the following equation: K/S = (1 − R)2 / 2R. These values were then substituted into the appropriate equation outlined in AMSA (1991) to calculate the percentage of metmyoglobin on the surface of raw steaks. Percentage metmyoglobin also was used to
Thiobarbituric acid reactive substances (TBARS) values were measured according to the procedure of Witte, Krause, and Bailey (1970) as an indicator of lipid oxidation. Five grams from each steak were blended with 25 ml tricholoracetic acid (TCA) solution (20%) and 20 ml distilled water. The mixture was homogenized using a Waring table-top blender (Dynamics Corp. of America, New Hartford, CT) for 30 s and filtered through Whatman (#1) filter paper. One milliliter of filtrate was mixed with 1 ml thiobarbituric acid (TBA) solution (20 mM) and incubated at 25 °C for 20 h. After incubation, absorbance was measured using a Shimadzu UV-2101 PC spectrophotometer (Shimadzu Inc., Columbia, MD, USA) at 532 nm against a blank consisting of 2 ml acid/water mix (TCA/water 1:1 v/v) and 2 ml TBA solution. 2.7. Experimental design and analysis The combined effects of enhancement, modified atmosphere packaging, and storage time were evaluated using a split-plot design. Within the whole plot, loin halves were considered experimental units (two experimental units per loin). Each loin half was assigned to one of four enhancement treatments (non-injected negative control, 3% pyruvate, 3% lactate, and 3% succinate in the final product).
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Injection treatments were replicated n = 15 times using a balanced incomplete block [(30 loins × 2 units per loin) / 4 treatments]. Within the sub-plot, each loin half was fabricated into seven steaks after injection. Non-injected units also were fabricated into seven steaks. Each steak within a loin half was assigned to a packaging × storage time combination in a completely randomized design. The seven combinations included: (1) Initial day 0 (prior to packaging and storage), (2) 5 days of storage in vacuum, (3) 5 days of storage in high oxygen, (4) 5 days of storage in PVC, (5) 13 days of storage in vacuum, (6) 13 days of storage in high oxygen, and (7) 13 days of storage in PVC. Type-3 tests of fixed effects for injection, packaging, storage time, and their interactions were performed using the MIXED procedure of SAS. Least square means for protected F-tests (P b 0.05) were separated using the diff option and were considered significant at P b 0.05. 3. Results and discussion 3.1. Raw material injection, packaging, and pH The average percentage of injection levels for pyruvate, lactate, and succinate loins were 11.1, 10.2, and 10.8, respectively. Headspace analysis of high-oxygen packages showed an oxygen concentration of 79.2% oxygen. The pH of enhancement solutions were 6.1 (pyruvate), 7.4 (lactate), and 9.1 (succinate). As a result, the enhancement treatments had a significant effect on steak pH [succinate (5.91) N lactate (5.78) = pyruvate (5.74) N control (5.60)]. 3.2. Surface redness (a*) and percentage metmyoglobin There was a significant enhancement × packaging × storage time interaction for a* values and percentage metmyoglobin on the surface of beef longissimus steaks stored at 1 °C (Tables 1 and 2). At the end of storage (day 13), steaks enhanced with lactate, pyruvate, and succinate were more red (P b 0.05; greater a* and less metmyoglobin) than control steaks in PVC and high-oxygen packaging. Of the three injection enhancement treatments, lactate-treated steaks in high oxygen were the least discolored on day 13 (greatest a* and least% MMb). In vacuum packaging, succinate and lactate steaks were more red than control steaks (P b 0.05) whereas pyruvate enhanced steaks were more discolored (P b 0.05; lower a* values and greater% MMb) than control steaks. Improved color stability via lactate enhancement has been reported by various researchers (Kim et al., 2006; Lawrence, Dikeman, Hunt, Kastner, & Johnson, 2003; Maca, Miller, & Acuff, 1997; Maca, Miller, Bigner, Luvia, & Acuff, 1999; Mancini, Suman, Konda, & Ramanathan, 2009). Research assessing the mechanisms of lactatemediated color stability suggests that NADH formed via lactate dehydrogenase activity results in metmyoglobin reduction. Moreover, in vitro studies have reported that reducing equivalents necessary for color stability can be generated by mitochondria after the addition of pyruvate, lactate, and succinate (Ramanathan, Mancini, & Naveena, 2010; Tang et al., 2005). The current research is the first to report the comparative effects of pyruvate, lactate, and succinate on postmortem beef color stability. In vacuum packaged steaks, pyruvate addition may have resulted in discoloration due to the consumption of NADH via enzyme-mediated conversion of pyruvate to lactate. Kim, Keeton, Smith, Berghman, and Savell (2009) reported the presence of LDH 5 (an LDH isoform that converts pyruvate to lactate) in bovine longissimus. 3.3. Surface darkening (L* value) There was a significant enhancement × packaging interaction for L* values (Table 3). Regardless of packaging type, injection enhancement darkened steaks (P b 0.05) compared with control steaks. Of the three
Table 1 Effects of enhancement, packaging, and storage time on the a* values (redness) of beef longissimus steaks stored at 1 °C. Days of storage
Enhancement
0 5
Control Control Pyruvate Succinate Lactate Control Pyruvate Succinate Lactate
13
Packaging VP 29.5 20.5 10.3 22.4 21.4 18.4 15.5 23.5 22.4
PVC b,x c,x a,x ab,x b.x c,x a,x a,x
29.5 28.7 23.8 26.4 25.0 16.7 19.9 18.1 20.5
HiOx a,y d,y b,y c,y c,y a,y b,y a,y
29.5 33.2 28.7 31.3 31.2 21.3 22.9 24.1 26.3
a,z c,z b,z b,z c,z b,z b,z a,z
Least square means in a column within a day with a different letter (a–d) are significantly different (P b 0.05). Least square means within a row with a different letter (x–z) are significantly different (P b 0.05). Standard error for a* = 0.6. Control = non-enhanced steaks. Pyruvate, lactate, and succinate were enhanced to 3% by weight of the final product. PVC = over-wrapped in oxygen-permeable film (atmospheric oxygen); VP = vacuum packaging; high oxygen (HiOx) = 80% O2 + 20% CO2.
ingredients, pyruvate resulted in the most surface darkening for steaks packaged in PVC and high oxygen (P b 0.05). The mechanism by which pyruvate and succinate darken steaks is not clear. Salts, including potassium lactate, can influence the reflectance properties of muscle (Ramanathan, Mancini, Naveena, & Konda, 2010; Swatland & Barbut, 1999). Moreover, increased pH following enhancement also can influence water holding capacity and mitochondrial activity, both of which darken surface color. Studies have shown that increased meat hydration will promote a relatively closed muscle structure and decrease oxygen diffusion to intracellular proteins (Bate-Smith, 1948; GasÏperlin, ZÏlender, & Abram, 2000; Lawrie, 1958). This will result in tightly packed muscle fibers and less light scatter (Hamm, 1960). In support, Knock et al. (2006) reported less surface glossiness and shine for lactate-enhanced beef longissimus steaks. Nevertheless, pyruvate and succinate used for mitochondrial oxygen consumption also can decrease myoglobin oxygenation, therefore darkening muscle color (Ramanathan, Mancini, & Konda, 2009; Ramanathan, Mancini, & Konda, 2010; Tang et al., 2005).
Table 2 Effects of enhancement, packaging, and storage time on the surface metmyoglobin (%) of beef longissimus steaks stored at 1 °C. Days of storage
Enhancement
0 5
Control Control Pyruvate Succinate Lactate Control Pyruvate Succinate Lactate
13
Packaging VP
PVC
5.2 14.0 b,x 94.9 a,x 1.2 d,x 10.4 c,x 16.4 b,x 58.3 a,x 6.4 c,x 13.2 b,x
5.2 21.8 32.7 30.4 37.1 66.5 56.0 58.6 55.4
HiOx c,y b,y b,y a,y a,y b,y b,y b,y
5.2 6.0 c,z 21.4 a,z 8.3 bc,z 11.6 b,x 60.9 a,z 46.3 b,z 41.9 c,z 32.8 d,z
Least square means in a column within a day with a different letter (a–d) are significantly different (P b 0.05). Least square means within a row with a different letter (x–z) are significantly different (P b 0.05). Standard error for % metmyoglobin = 1.8. Control = non-enhanced steaks. Pyruvate, lactate, and succinate were enhanced to 3% by weight of the final product. PVC = over-wrapped in oxygen-permeable film (atmospheric oxygen); VP = vacuum packaging; high oxygen (HiOx) = 80% O2 + 20% CO2.
R. Ramanathan et al. / Meat Science 88 (2011) 424–428 Table 3 Effects of enhancement and packaging on the L* values of beef longissimus steaks stored at 1 °C. Enhancement
dehydrogenase and subsequently regenerated NADH. The increase in MRA during storage of pyruvate-enhanced steaks packaged in vacuum might be due to this NADH formation.
Packaging VP
Control Pyruvate Succinate Lactate
39.4 34.6 36.1 34.4
PVC a,x bc,y b,y c,x
43.8 36.6 41.7 40.2
HiOx a,y c,x b,x b,y
45.0 35.8 43.1 40.6
a,z d,xy b,x c,y
Least square means in a column with a different letter (a–d) are significantly different (P b 0.05). Least square means within a row with a different letter (x-z) are significantly different (P b 0.05). Standard error for L* = 0.8. Control = non-enhanced steaks. Pyruvate, lactate, and succinate were enhanced to 3% by weight of the final product. PVC = over-wrapped in oxygen-permeable film (atmospheric oxygen); VP = vacuum packaging; high oxygen (HiOx) = 80% O2 + 20% CO2.
3.4. Metmyoglobin-reducing activity There was a significant enhancement × packaging × storage time interaction for metmyoglobin-reducing activity (Table 4). Regardless of packaging type, succinate had the greatest and pyruvate had the least metmyoglobin-reducing activity during storage (succinate N lactate N control N pyruvate; P b 0.05). In general, steaks in vacuum packaging had the greatest metmyoglobin-reducing activity compared with steaks in PVC and high oxygen (P b 0.05). As expected, reducing activity decreased during storage (P b 0.05; day 0 N day 5 N day 13). Increased metmyoglobin-reducing activity of succinate-enhanced steaks is possibly due to increased pH and mitochondrial activity, which promotes enzymatic metmyoglobin-reducing systems and myoglobin stability (Reddy & Carpenter, 1991; Tang et al., 2005). However, pyruvate-enhanced steaks had the least metmyoglobinreducing activity, even though the pH of pyruvate steaks was greater than control steaks. Hence, pyruvate-enhanced steaks were expected to have more reducing activity than non-enhanced control samples. Conversely, metmyoglobin-reducing activity was the least in pyruvate-injected steaks (P b 0.05). Increased metmyoglobin-reducing activity in lactate enhanced steaks can be due to regeneration of NADH via lactate dehydrogenase activity and has been reported by previous researchers (Kim et al., 2006; Seyfert, Hunt, Ahnstrom, & Johnson, 2007). During prolonged storage (day 5 to day 13), lactate formed from pyruvate might have served as a substrate for lactate Table 4 Effects of enhancement, packaging, and storage time on the metmyoglobin-reducing activity of beef longissimus steaks stored at 1 °C. Days of storage
Enhancement
0 5
Control Control Pyruvate Succinate Lactate Control Pyruvate Succinate Lactate
13
427
Packaging VP
PVC
HiOx
70.5 46.4 a,x 2.0 b,x 63.4 c,x 52.4 d,x 38.2 a,x 16.9 b,x 53.8 c,x 46.3 d,x
70.5 40.5 a,y 4.2 b,x 56.3 c,y 46.7 d,y 16.8 a,y 6.2 b,y 48.6 c,y 30.3 d,y
70.5 35.4 a,z 2.1 b,x 50.4 c,z 42.7 d,z 18.5 a,z 5.8 b,y 40.4 c,z 34.5 d,y
Least square means in a column within a day with a different letter (a–d) are significantly different (P b 0.05). Least square means within a row with a different letter (x-z) are significantly different (P b 0.05). Standard error for metmyoglobin-reducing activity = 1.9. Control = non-enhanced steaks. Pyruvate, lactate, and succinate were enhanced to 3% by weight of the final product. PVC = over-wrapped in oxygen-permeable film (atmospheric oxygen); VP = vacuum packaging; high oxygen (HiOx) = 80% O2 + 20% CO2.
3.5. Lipid oxidation There was a significant enhancement × packaging × storage time interaction for TBARS values as indicated by absorbance at 532 nm (Table 5). As expected, control steaks in vacuum packaging had less lipid oxidation (P b 0.05) than control steaks in PVC and high oxygen. Moreover, there was no effect (P N 0.05) of injection enhancement on lipid oxidation of steaks packaged in vacuum. Conversely, lactate and pyruvate decreased the TBARS values of steaks packaged in PVC (P b 0.05; compared with control steaks). Although all injection enhancement treatments decreased lipid oxidation (P b 0.05) compared with control steaks in high oxygen; pyruvate was the most effective for lowering lipid oxidation on day 13 (TBARS: control N succinate N lactate N pyruvate; P b 0.05). As a result, storage time had no effect on lipid oxidation in high oxygen when steaks were enhanced with pyruvate. A similar trend was noted for steaks in PVC enhanced with either pyruvate or lactate (no significant increase in TBARS during storage). The antioxidant activity of pyruvate has been reported in nonmeat systems (Herz, Blake, & Grootveld, 1997). More specifically, the keto-enol group present in pyruvate can directly scavenge hydrogen peroxide and other reactive oxygen species (Bassenge, Sommer, Schwemmer, & Bünger, 2000). Pyruvate also has the ability to regenerate glutathione (a potent antioxidant; Valentovic & Minigh, 2003). Lactate's role in minimizing lipid oxidation may be due to its ability to scavenge superoxide and hydroxyl radicals (Groussard et al., 2000). The ability of lactate enhancement to decrease lipid oxidation in ground beef, pork, and chicken has been reported previously (Mancini et al., 2010; Naveena, Sen, Muthukumar, Vaithiyanathan, & Babji, 2006; Nnanna, Ukuku, McVann, & Shelef, 1994). In model systems, studies have shown that succinate can prevent lipid oxidation induced by iron (Takeshige & Minakami, 1975). The antioxidant effect of succinate has been attributed to the formation of ubiquinol (a potent biological antioxidant; Vianello, Macri, Cavallini, & Bindoli, 1986). 4. Conclusion Injection enhancement of beef loins with succinate, pyruvate, and lactate increased muscle pH. Enhancing steaks with lactate, pyruvate, Table 5 Effects of enhancement, packaging, and storage time on the TBARS values of beef longissimus steaks stored at 1 °C. Days of storage
Enhancement
0 5
Control Control Lactate Pyruvate Succinate Control Lactate Pyruvate Succinate
13
Packaging VP
PVC
0.04 0.04 0.02 0.05 0.03 0.05 0.03 0.07 0.06
0.04 0.18 0.05 0.05 0.11 0.28 0.06 0.09 0.24
a,x a,x a,x a,x a,x a,x a,x a,x
HiOx a,y c,x c,x b,y a,y b,x b,x a,y
0.04 0.21 0.07 0.04 0.10 0.41 0.22 0.08 0.29
a,z b,x c,x b,y a,z c,y d,x b,y
Least square means in a column within a day with a different letter (a–d) are significantly different (P b 0.05). Least square means within a row with a different letter (x-z) are significantly different (P b 0.05). Standard error for TBARS value = 0.03. Control = non-enhanced steaks. Pyruvate, lactate, and succinate were enhanced to 3% by weight of the final product. PVC = over-wrapped in oxygen-permeable film (atmospheric oxygen); VP = vacuum packaging; high oxygen (HiOx) = 80% O2 + 20% CO2.
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and succinate can improve beef steak color stability during storage in PVC and high-oxygen packaging. However, this injection enhancement also will darken beef steaks. Pyruvate will minimize lipid oxidation of steaks packaged in both PVC and high oxygen. Addition of substrates such as pyruvate and succinate has the potential to improve the color of beef packaged in PVC and high oxygen. Future studies should further evaluate the mechanism(s) by which substrates such as pyruvate, succinate, and lactate influence meat color. Acknowledgements This project was funded, in part, by beef producers and importers through The Beef Checkoff and The National Cattlemen's Beef Association. References AMSA (1991). Guidelines for meat color evaluation. Reciprocal Meat Conference Proceedings, 44, 1−17. Bate-Smith, E. C. (1948). The physiology and chemistry of rigor mortis with special reference to the aging of beef. Advances in Food Research, 1, 1−38. Bassenge, E., Sommer, O., Schwemmer, M., & Bünger, R. (2000). Antioxidant pyruvate inhibits cardiac formation of reactive oxygen species through changes in redox state. American Journal of Physiology. Heart and Circulatory Physiology, 279, 2431−2438. GasÏperlin, L., ZÏlender, B., & Abram, V. (2000). Colour of normal and high pH beef heated to different temperatures as related to oxygenation. Meat Science, 54, 391−398. Giddings, G. G. (1974). Reduction of ferrimyoglobin in meat. Critical Reviews in Food Technology, 5, 143−173. Groussard, C., Morel, I., Chevanne, M., Monnier, M., Cillard, J., & Delamarche, A. (2000). Free radical scavenging and antioxidant effects of lactate ion: An in vitro study. Journal of Applied Physiology, 89, 169−175. Hamm, R. (1960). Biochemistry of meat hydration. Advances in Food Research, 10, 355−463. Herz, H., Blake, D. R., & Grootveld, M. (1997). Multicomponent investigations of the hydrogen peroxide- and hydroxyl radical scavenging antioxidant capacities of biofluids: The roles of endogenous pyruvate and lactate. Free Radical Research, 26, 19−35. Kim, Y. H., Hunt, M. C., Mancini, R. A., Seyfert, M., Loughin, T. M., Kropf, D. H., et al. (2006). Mechanism for lactate-color stabilization in injection-enhanced beef. Journal of Agricultural and Food Chemistry, 54, 7856−7862. Kim, Y. H., Keeton, J. T., Smith, S. B., Berghman, L. R., & Savell, J. W. (2009). Role of lactate dehydrogenase in metmyoglobin reduction and color stability of different bovine muscles. Meat Science, 83, 376−382. Knock, R. C., Seyfert, M., Hunt, M. C., Dikeman, M. E., Mancini, R. A., Unruh, J. A., et al. (2006). Effects of potassium lactate, sodium chloride, and sodium acetate on surface shininess/gloss and sensory properties of injection-enhanced beef striploin steaks. Meat Science, 74, 319−326. Lawrie, R. A. (1958). Physiological stress in relation to dark-cutting beef. Journal of the Science of Food and Agriculture, 11, 721−725. Lawrence, T. E., Dikeman, M. E., Hunt, M. C., Kastner, C. L., & Johnson, D. E. (2003). Effects of calcium salts on beef longissimus quality. Meat Science, 64, 299−308. Ledward, D. A. (1985). Post-slaughter influences on the formation of metmyoglobin in beef muscles. Meat Science, 15, 149−171. Maca, J. V., Miller, R. K., & Acuff, G. R. (1997). Microbiological, sensory and chemical characteristics of vacuum packaged ground beef patties treated with salts of organic acids. Journal of Food Science, 62, 591−596. Maca, J. V., Miller, R. K., Bigner, M. E., Luvia, L. M., & Acuff, G. R. (1999). Sodium lactate and storage temperature effects on shelf life of vacuum packaged beef top rounds. Meat Science, 53, 23−29.
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, 71−76. Mancini, R. A., Ramanathan, R., Suman, S. P., Konda, M. K. R., Joseph, P., Dady, G. A., et al. (2010). Effects of lactate and modified atmospheric packaging on premature browning in cooked ground beef patties. Meat Science, 85, 339−346. Mohan, A., Hunt, M. C., Barstow, T. J., Houser, T. A., & Muthukrishnan, S. (2010). Effects of malate, lactate, and pyruvate on myoglobin redox stability in homogenates of three bovine muscles. Meat Science, 86, 304−310. NAMP (2002). The meat buyers guide. Reston, VA: North American Meat Processors Association. Naveena, B. M., Sen, A. R., Muthukumar, M., Vaithiyanathan, S., & Babji, Y. (2006). The effect of lactates on the quality of microwave-cooked chicken patties during storage. Journal of Food Science, 71, 603−608. Nnanna, I. A., Ukuku, D. O., McVann, K. B., & Shelef, L. A. (1994). Antioxidant activity of sodium lactate in meat and model systems. Lebensmittel-Wissenschaft und Technologie, 27, 78−85. Puntel, R. L., Roos, D. H., Grotto, D., Garcia, S. C., Nogueira, C. W., & Rocha, J. B. T. (2007). Antioxidant properties of Krebs cycle intermediates against malonate pro-oxidant activity in vitro: A comparative study using the colorimetric method and HPLC analysis to determine malondialdehyde in rat brain homogenates. Life Sciences, 81, 51−62. Ramanathan, R., & Mancini, R. A. (2010). Effects of pyruvate on bovine heart mitochondria-mediated metmyoglobin reduction. Meat Science, 86, 738−741. Ramanathan, R., Mancini, R. A., & Konda, M. K. (2009). Effects of lactate on beef heart mitochondrial oxygen consumption and muscle darkening. Journal of Agricultural and Food Chemistry, 57, 1550−1555. Ramanathan, R., Mancini, R. A., & Konda, M. K. (2010a). Effect of lactate enhancement on myoglobin oxygenation of beef longissimus steaks overwrapped in PVC and stored at 4C. Journal of Muscle Foods, 21, 669−684. Ramanathan, R., Mancini, R. A., Naveena, B. M., & Konda, M. K. (2010b). Effects of lactate-enhancement on surface reflectance and absorbance properties of beef longissimus steaks. Meat Science, 84, 219−226. Ramanathan, R., Mancini, R. A., & Naveena, B. M. (2010c). Effects of lactate on bovine heart mitochondria-mediated metmyoglobin reduction. Journal of Agricultural and Food Chemistry, 58, 5724−5729. Reddy, I. M., & Carpenter, C. E. (1991). Determination of metmyoglobin reductase activity in bovine skeletal muscles. Journal of Food Science, 56, 1161−1164. Saleh, B., & Watts, B. M. (1968). Substrates and intermediates in the enzymatic reduction of metmyoglobin in ground beef. Journal of Food Science, 33, 353−358. Sammel, L. M., Hunt, M. C., Kropf, D. H., Hachmeister, K. A., & Johnson, D. E. (2002). Comparison of assays for reducing ability in beef inside and outside semimembranosus muscle. Journal of Food Science, 67, 978−984. Seyfert, M., Hunt, M. C., Ahnstrom, M. L., & Johnson, D. E. (2007). Efficacy of lactic acid salts and sodium acetate on ground beef colour stability and metmyoglobinreducing activity. Meat Science, 75, 134−142. Swatland, H. J., & Barbut, S. (1999). Sodium chloride levels in comminuted chicken muscle in relation to processing characteristics and Fresnel reflectance detected with a polarimetric probe. Meat Science, 51, 377−381. Takeshige, K., & Minakami, S. (1975). Reduced nicotinamide adenine-dinucleotide phosphate-dependent lipid peroxidation by beef heart submitochondrial particles. Journal of Biochemistry, 77, 1067−1073. Tang, J., Faustman, C., Mancini, R. A., Seyfert, M., & Hunt, M. C. (2005). Mitochondrial reduction of metmyoglobin: dependence on the electron transport chain. Journal of Agricultural and Food Chemistry, 53, 5449−5455. Valentovic, M. A., & Minigh, J. (2003). Pyruvate attenuates myoglobin in vitro toxicity. Toxicological Sciences, 74, 345−351. Vianello, A., Macri, F., Cavallini, L., & Bindoli, A. (1986). Induction of lipid peroxidation in soybean mitochondria and protection by respiratory substrates. Journal of Plant Physiology, 125, 217−224. Watts, B. M., Kendrick, J., Zipser, M. W., Hutchins, B., & Saleh, B. (1966). Enzymatic reducing pathways in meat. Journal of Food Science, 31, 855−862. Witte, V. C., Krause, G. F., & Bailey, M. E. (1970). A new extraction method for determining 2-thiobarbituric acid values of pork and beef during storage. Journal of Food Science, 35, 582−585.
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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
Effects of pyruvate, succinate, and lactate enhancement on beef longissimus raw color R. Ramanathan, R.A. Mancini ⁎, G.A. Dady Department of Animal Science, University of Connecticut, Storrs, CT 06269-4040, USA
a r t i c l e
i n f o
Article history: Received 8 November 2010 Received in revised form 20 January 2011 Accepted 21 January 2011 Keywords: Pyruvate Succinate Lactate Beef color Myoglobin Lipid oxidation
a b s t r a c t Beef strip loins (n = 30) were divided into halves, and each half was assigned randomly to one of four injection enhancements: (1) non-enhanced control, (2) 3% pyruvate, (3) 3% succinate, and (4) 3% lactate. Steaks were cut and packaged in either vacuum, high oxygen (80% O2/20% CO2), or PVC. Color and lipid oxidation were measured on days 0, 5, and 13 of storage at 1 °C. Enhancement had a significant effect on steak pH. On day 13 of storage, steaks enhanced with lactate, pyruvate, and succinate were less discolored (P b 0.05) than control steaks in PVC and high oxygen. Enhancement darkened steaks (P b 0.05) compared with control steaks. Succinate had the greatest and pyruvate had the least metmyoglobin-reducing activity (P b 0.05). Lactate and pyruvate decreased the TBARS values of steaks packaged in PVC (P b 0.05) whereas pyruvate was most effective for lowering lipid oxidation in high-oxygen packaging. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction Maintenance of fresh meat color is critical for extending the shelf life of beef. Although numerous extrinsic and intrinsic factors influence myoglobin redox stability during both storage and display, meat has inherent metmyoglobin-reducing systems that utilize NADH to delay the accumulation of surface metmyoglobin (Giddings, 1974; Ledward, 1985). Nevertheless, depletion of substrates in postmortem muscle influences the regeneration of reducing equivalents (Saleh & Watts, 1968; Watts, Kendrick, Zipser, Hutchins, & Saleh, 1966). Caseready technology provides an opportunity to enhance meat with various ingredients that can serve as substrates for reducing systems and therefore, influence color life. Lactate is commonly added to case-ready meat and is often described as a color stabilizer in fresh beef. Researchers have reported that NADH formed via lactate dehydrogenase activity can increase color stability by increasing metmyoglobin reduction (Kim et al., 2006). Moreover, various in vitro studies suggest that other glycolytic and tricarboxylic intermediates also can influence myoglobin redox stability. For example, Mohan, Hunt, Barstow, Houser, and Muthukrishnan (2010) reported that addition of pyruvate and malate improved color stability of muscle homogenates. In addition, Ramanathan and Mancini (2010) reported that pyruvate addition to isolated mitochondria resulted in electron transport mediated metmyoglobin reduction. Tang, Faustman, Mancini, Seyfert, and
⁎ Corresponding author. Tel.: +1 860 486 1775; fax: +1 860 486 4375. E-mail address: [email protected] (R.A. Mancini). 0309-1740/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2011.01.021
Hunt (2005) reported that the addition of succinate to isolated cardiac mitochondria increased oxygen consumption and electron transport mediated metmyoglobin reduction. More specifically, Tang et al. (2005) hypothesized that this succinate-mediated increase in oxygen consumption will lower oxygen partial pressure, thereby increasing the amount of available electrons that can be transferred to metmyoglobin. Furthermore, the ability of pyruvate and succinate to minimize lipid oxidation has been reported in various non-meat model systems (Puntel et al., 2007; Valentovic & Minigh, 2003). Although fundamental research suggests that pyruvate and succinate can influence myoglobin redox chemistry, no research has assessed the effects of pyruvate and succinate enhancement on beef color stability. Therefore, the objectives of the present study were to assess the comparative effects of pyruvate, succinate, and lactate enhancement on the color of beef longissimus lumborum packaged in either vacuum, PVC, or high oxygen and stored for 0, 5, and 13 days at 1 °C.
2. Materials and methods 2.1. Raw materials and processing Beef strip loins (NAMP 180; NAMP, 2002; n = 30) were obtained locally from a beef purveyor at 7 days postmortem. Each loin was divided into two halves, and each experimental unit within a loin was assigned randomly to one of four injection enhancements: (1) nonenhanced control, (2) 3% pyruvate, (3) 3% succinate, and (4) 3% lactate, resulting in n = 15 replications per treatment. Enhancement solutions contained deionized distilled water and either 33% sodium pyruvate (Nutriscience, Trumbull, CT), 33% lactate (PURASAL HiPure P, 78% potassium lactate and 22% water; PURAC
R. Ramanathan et al. / Meat Science 88 (2011) 424–428
America, Inc., Lincolnshire, IL), or 33% sodium succinate (Fisher Scientific, Pittsburg, PA). Loins were pumped to 110% of green weight using a multi-needle injector (Koch, Kansas City, MO) to achieve an ingredient concentration of 3% w/w in the final product. For control samples, no enhancement solution was injected into the individual loin units. Samples were weighed before and 30 min after injection to confirm target enhancement levels: calculated as [(weight after injection − green weight)/green weight] × 100. From each experimental unit, seven 1.91-cm thick steaks were cut and one steak was assigned to day 0 analyses (no packaging or storage time) for surface color, metmyoglobin-reducing activity, lipid oxidation, and pH measurements. The remaining six steaks were individually packaged in either vacuum, high-oxygen (80% O2/20% CO2), or aerobic packaging (PVC; over-wrapped with oxygen-permeable polyvinyl chloride fresh meat film; 15,500–16,275 cm3 O2/m2/24 h at 23 °C, E-Z Wrap Crystal Clear Polyvinyl Chloride Wrapping Film, Koch Supplies, Kansas City, MO). Both vacuum and high-oxygen packaging were performed using a Koch MultiVac 500 (Kansas City, MO), Prime Source vacuum pouches (4 mil, standard barrier nylon/ polyethylene, 0.6 cm3 O2/645.16 cm2/24 h at 0 °C; Bunzl Koch Supplies Inc., Kansas City, MO), and certified gas blends (Airgas, Cheshire, CT). Using a headspace analyzer (Model 6600 Headspace Oxygen/Carbon Dioxide Analyzer, Illinois Instruments, Ingleside, IL), the percentage O2 in extra modified atmosphere packages was determined 24 h after packaging. Packages were stored in the dark at 1 °C. 2.2. Instrumental color analysis The fresh-cut surface color of each day 0 steak was measured 1 h after fabrication (before packaging, bloom at 1 °C). Of the two steaks assigned to each packaging type, one steak was used for day 5 analyses and the other steak was used for day 13 analyses. On days 5 and 13 of storage, surface color was measured immediately after each steak was removed from its respective packaging. All instrumental color measurements were performed using a HunterLab MiniScan XE Plus spectrophotometer (Model 45/0 LAV, 2.54-cm diameter aperture, illuminant A, 10° observer). Both reflectance spectra from 400 to 700 nm (10 nm increments) and CIE L* and a* were measured in triplicate for each steak and subsamples were averaged for statistical analyses. Reflectance data from 400 to 700 nm also was used to estimate % metmyoglobin during storage (AMSA, 1991).
425
calculate metmyoglobin-reducing activity. The following equation was used: %Metmyoglobin =
K=S 572 K=S 525 K=S 572 K=S 525
for 100% deoxy or oxymyoglobin
for 100% deoxy or oxymyoglobin
K=S 572 K=S 525
K=S 572 K=S 525
for sample
for 100% metmyoglobin
2.4. Metmyoglobin-reducing activity Following surface color measurement, each steak was cut in half perpendicular to the surface. One half was used for metmyoglobinreducing activity and the other half was used for lipid oxidation and pH measurements. Metmyoglobin-reducing activity (MRA) was determined using a sample from the interior portion of each steak on days 0, 5, and 13, according to a procedure described by Sammel, Hunt, Kropf, Hachmeister, and Johnson (2002). Samples were submerged for 20 min in a 0.3% solution of sodium nitrite at 25 °C to facilitate metmyoglobin formation, and then removed, blotted dry, vacuum packaged (Prime Source Vacuum Pouches, 4 mil, KOCH Supplies Inc., Kansas City, MO), and scanned twice with a HunterLab MiniScan XE Plus Spectrophotometer to determine preincubation metmyoglobin values (AMSA, 1991). Each sample was incubated at 30 °C for 2 h (Fisher Scientific, Model 630F, NY) to induce metmyoglobin reduction. Upon removal from the incubator, samples were rescanned twice to determine the percentage of remaining surface metmyoglobin. The following equation was used to calculate metmyoglobin-reducing activity: [(% surface metmyoglobin pre-incubation − % surface metmyoglobin post-incubation) /% surface metmyoglobin preincubation]× 100. 2.5. Determination of pH Samples from steaks assigned to days 0, 5, and 13 of storage, visually devoid of fat and connective tissue, were used to determine pH after instrumental color analysis. Using a Waring table-top blender (Dynamics Corp. of America, New Hartford, CT), 10 g of sample was combined with 90 ml of deionized water and mixed for 30 s, and the pH values were determined using an Accumet 50 pH meter (Fisher Scientific, Fairlawn, NJ). 2.6. Lipid oxidation
2.3. Measuring raw metmyoglobin percentage Reflectance at isobestic wavelengths was used to estimate raw surface metmyoglobin content. Reference standards for 100% of oxyand metmyoglobin were created for both control and enhanced steaks. To create 100% metmyoglobin, additional steaks not used for color analysis were placed in 1.0% potassium ferricyanide for 1 min, drained, blotted dry, and packaged in oxygen-permeable film (PVC; over-wrapped with oxygen-permeable polyvinyl chloride fresh meat film; 15,500–16,275 cm3 O2/m2/24 h at 23 °C, E-Z Wrap Crystal Clear Polyvinyl Chloride Wrapping Film, Koch Supplies, Kansas City, MO). Samples were stored at 4 °C for 12 h, removed from the package, and then reflectance was measured from 700 to 400 nm. To create 100% oxymyoglobin, steaks were placed in a pouch (Prime Source Vacuum Pouches, 4 mil, Koch Supplies Inc., Kansas City, MO) and flushed with 100% oxygen (Airgas, Cheshire, CT). Each pouch was sealed, kept for 2 h at 4 °C, and samples were removed from the package and reflectance was measured from 700 to 400 nm. Reflectance at 525 and 572 nm for each myoglobin form were converted to K/S values using the following equation: K/S = (1 − R)2 / 2R. These values were then substituted into the appropriate equation outlined in AMSA (1991) to calculate the percentage of metmyoglobin on the surface of raw steaks. Percentage metmyoglobin also was used to
Thiobarbituric acid reactive substances (TBARS) values were measured according to the procedure of Witte, Krause, and Bailey (1970) as an indicator of lipid oxidation. Five grams from each steak were blended with 25 ml tricholoracetic acid (TCA) solution (20%) and 20 ml distilled water. The mixture was homogenized using a Waring table-top blender (Dynamics Corp. of America, New Hartford, CT) for 30 s and filtered through Whatman (#1) filter paper. One milliliter of filtrate was mixed with 1 ml thiobarbituric acid (TBA) solution (20 mM) and incubated at 25 °C for 20 h. After incubation, absorbance was measured using a Shimadzu UV-2101 PC spectrophotometer (Shimadzu Inc., Columbia, MD, USA) at 532 nm against a blank consisting of 2 ml acid/water mix (TCA/water 1:1 v/v) and 2 ml TBA solution. 2.7. Experimental design and analysis The combined effects of enhancement, modified atmosphere packaging, and storage time were evaluated using a split-plot design. Within the whole plot, loin halves were considered experimental units (two experimental units per loin). Each loin half was assigned to one of four enhancement treatments (non-injected negative control, 3% pyruvate, 3% lactate, and 3% succinate in the final product).
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R. Ramanathan et al. / Meat Science 88 (2011) 424–428
Injection treatments were replicated n = 15 times using a balanced incomplete block [(30 loins × 2 units per loin) / 4 treatments]. Within the sub-plot, each loin half was fabricated into seven steaks after injection. Non-injected units also were fabricated into seven steaks. Each steak within a loin half was assigned to a packaging × storage time combination in a completely randomized design. The seven combinations included: (1) Initial day 0 (prior to packaging and storage), (2) 5 days of storage in vacuum, (3) 5 days of storage in high oxygen, (4) 5 days of storage in PVC, (5) 13 days of storage in vacuum, (6) 13 days of storage in high oxygen, and (7) 13 days of storage in PVC. Type-3 tests of fixed effects for injection, packaging, storage time, and their interactions were performed using the MIXED procedure of SAS. Least square means for protected F-tests (P b 0.05) were separated using the diff option and were considered significant at P b 0.05. 3. Results and discussion 3.1. Raw material injection, packaging, and pH The average percentage of injection levels for pyruvate, lactate, and succinate loins were 11.1, 10.2, and 10.8, respectively. Headspace analysis of high-oxygen packages showed an oxygen concentration of 79.2% oxygen. The pH of enhancement solutions were 6.1 (pyruvate), 7.4 (lactate), and 9.1 (succinate). As a result, the enhancement treatments had a significant effect on steak pH [succinate (5.91) N lactate (5.78) = pyruvate (5.74) N control (5.60)]. 3.2. Surface redness (a*) and percentage metmyoglobin There was a significant enhancement × packaging × storage time interaction for a* values and percentage metmyoglobin on the surface of beef longissimus steaks stored at 1 °C (Tables 1 and 2). At the end of storage (day 13), steaks enhanced with lactate, pyruvate, and succinate were more red (P b 0.05; greater a* and less metmyoglobin) than control steaks in PVC and high-oxygen packaging. Of the three injection enhancement treatments, lactate-treated steaks in high oxygen were the least discolored on day 13 (greatest a* and least% MMb). In vacuum packaging, succinate and lactate steaks were more red than control steaks (P b 0.05) whereas pyruvate enhanced steaks were more discolored (P b 0.05; lower a* values and greater% MMb) than control steaks. Improved color stability via lactate enhancement has been reported by various researchers (Kim et al., 2006; Lawrence, Dikeman, Hunt, Kastner, & Johnson, 2003; Maca, Miller, & Acuff, 1997; Maca, Miller, Bigner, Luvia, & Acuff, 1999; Mancini, Suman, Konda, & Ramanathan, 2009). Research assessing the mechanisms of lactatemediated color stability suggests that NADH formed via lactate dehydrogenase activity results in metmyoglobin reduction. Moreover, in vitro studies have reported that reducing equivalents necessary for color stability can be generated by mitochondria after the addition of pyruvate, lactate, and succinate (Ramanathan, Mancini, & Naveena, 2010; Tang et al., 2005). The current research is the first to report the comparative effects of pyruvate, lactate, and succinate on postmortem beef color stability. In vacuum packaged steaks, pyruvate addition may have resulted in discoloration due to the consumption of NADH via enzyme-mediated conversion of pyruvate to lactate. Kim, Keeton, Smith, Berghman, and Savell (2009) reported the presence of LDH 5 (an LDH isoform that converts pyruvate to lactate) in bovine longissimus. 3.3. Surface darkening (L* value) There was a significant enhancement × packaging interaction for L* values (Table 3). Regardless of packaging type, injection enhancement darkened steaks (P b 0.05) compared with control steaks. Of the three
Table 1 Effects of enhancement, packaging, and storage time on the a* values (redness) of beef longissimus steaks stored at 1 °C. Days of storage
Enhancement
0 5
Control Control Pyruvate Succinate Lactate Control Pyruvate Succinate Lactate
13
Packaging VP 29.5 20.5 10.3 22.4 21.4 18.4 15.5 23.5 22.4
PVC b,x c,x a,x ab,x b.x c,x a,x a,x
29.5 28.7 23.8 26.4 25.0 16.7 19.9 18.1 20.5
HiOx a,y d,y b,y c,y c,y a,y b,y a,y
29.5 33.2 28.7 31.3 31.2 21.3 22.9 24.1 26.3
a,z c,z b,z b,z c,z b,z b,z a,z
Least square means in a column within a day with a different letter (a–d) are significantly different (P b 0.05). Least square means within a row with a different letter (x–z) are significantly different (P b 0.05). Standard error for a* = 0.6. Control = non-enhanced steaks. Pyruvate, lactate, and succinate were enhanced to 3% by weight of the final product. PVC = over-wrapped in oxygen-permeable film (atmospheric oxygen); VP = vacuum packaging; high oxygen (HiOx) = 80% O2 + 20% CO2.
ingredients, pyruvate resulted in the most surface darkening for steaks packaged in PVC and high oxygen (P b 0.05). The mechanism by which pyruvate and succinate darken steaks is not clear. Salts, including potassium lactate, can influence the reflectance properties of muscle (Ramanathan, Mancini, Naveena, & Konda, 2010; Swatland & Barbut, 1999). Moreover, increased pH following enhancement also can influence water holding capacity and mitochondrial activity, both of which darken surface color. Studies have shown that increased meat hydration will promote a relatively closed muscle structure and decrease oxygen diffusion to intracellular proteins (Bate-Smith, 1948; GasÏperlin, ZÏlender, & Abram, 2000; Lawrie, 1958). This will result in tightly packed muscle fibers and less light scatter (Hamm, 1960). In support, Knock et al. (2006) reported less surface glossiness and shine for lactate-enhanced beef longissimus steaks. Nevertheless, pyruvate and succinate used for mitochondrial oxygen consumption also can decrease myoglobin oxygenation, therefore darkening muscle color (Ramanathan, Mancini, & Konda, 2009; Ramanathan, Mancini, & Konda, 2010; Tang et al., 2005).
Table 2 Effects of enhancement, packaging, and storage time on the surface metmyoglobin (%) of beef longissimus steaks stored at 1 °C. Days of storage
Enhancement
0 5
Control Control Pyruvate Succinate Lactate Control Pyruvate Succinate Lactate
13
Packaging VP
PVC
5.2 14.0 b,x 94.9 a,x 1.2 d,x 10.4 c,x 16.4 b,x 58.3 a,x 6.4 c,x 13.2 b,x
5.2 21.8 32.7 30.4 37.1 66.5 56.0 58.6 55.4
HiOx c,y b,y b,y a,y a,y b,y b,y b,y
5.2 6.0 c,z 21.4 a,z 8.3 bc,z 11.6 b,x 60.9 a,z 46.3 b,z 41.9 c,z 32.8 d,z
Least square means in a column within a day with a different letter (a–d) are significantly different (P b 0.05). Least square means within a row with a different letter (x–z) are significantly different (P b 0.05). Standard error for % metmyoglobin = 1.8. Control = non-enhanced steaks. Pyruvate, lactate, and succinate were enhanced to 3% by weight of the final product. PVC = over-wrapped in oxygen-permeable film (atmospheric oxygen); VP = vacuum packaging; high oxygen (HiOx) = 80% O2 + 20% CO2.
R. Ramanathan et al. / Meat Science 88 (2011) 424–428 Table 3 Effects of enhancement and packaging on the L* values of beef longissimus steaks stored at 1 °C. Enhancement
dehydrogenase and subsequently regenerated NADH. The increase in MRA during storage of pyruvate-enhanced steaks packaged in vacuum might be due to this NADH formation.
Packaging VP
Control Pyruvate Succinate Lactate
39.4 34.6 36.1 34.4
PVC a,x bc,y b,y c,x
43.8 36.6 41.7 40.2
HiOx a,y c,x b,x b,y
45.0 35.8 43.1 40.6
a,z d,xy b,x c,y
Least square means in a column with a different letter (a–d) are significantly different (P b 0.05). Least square means within a row with a different letter (x-z) are significantly different (P b 0.05). Standard error for L* = 0.8. Control = non-enhanced steaks. Pyruvate, lactate, and succinate were enhanced to 3% by weight of the final product. PVC = over-wrapped in oxygen-permeable film (atmospheric oxygen); VP = vacuum packaging; high oxygen (HiOx) = 80% O2 + 20% CO2.
3.4. Metmyoglobin-reducing activity There was a significant enhancement × packaging × storage time interaction for metmyoglobin-reducing activity (Table 4). Regardless of packaging type, succinate had the greatest and pyruvate had the least metmyoglobin-reducing activity during storage (succinate N lactate N control N pyruvate; P b 0.05). In general, steaks in vacuum packaging had the greatest metmyoglobin-reducing activity compared with steaks in PVC and high oxygen (P b 0.05). As expected, reducing activity decreased during storage (P b 0.05; day 0 N day 5 N day 13). Increased metmyoglobin-reducing activity of succinate-enhanced steaks is possibly due to increased pH and mitochondrial activity, which promotes enzymatic metmyoglobin-reducing systems and myoglobin stability (Reddy & Carpenter, 1991; Tang et al., 2005). However, pyruvate-enhanced steaks had the least metmyoglobinreducing activity, even though the pH of pyruvate steaks was greater than control steaks. Hence, pyruvate-enhanced steaks were expected to have more reducing activity than non-enhanced control samples. Conversely, metmyoglobin-reducing activity was the least in pyruvate-injected steaks (P b 0.05). Increased metmyoglobin-reducing activity in lactate enhanced steaks can be due to regeneration of NADH via lactate dehydrogenase activity and has been reported by previous researchers (Kim et al., 2006; Seyfert, Hunt, Ahnstrom, & Johnson, 2007). During prolonged storage (day 5 to day 13), lactate formed from pyruvate might have served as a substrate for lactate Table 4 Effects of enhancement, packaging, and storage time on the metmyoglobin-reducing activity of beef longissimus steaks stored at 1 °C. Days of storage
Enhancement
0 5
Control Control Pyruvate Succinate Lactate Control Pyruvate Succinate Lactate
13
427
Packaging VP
PVC
HiOx
70.5 46.4 a,x 2.0 b,x 63.4 c,x 52.4 d,x 38.2 a,x 16.9 b,x 53.8 c,x 46.3 d,x
70.5 40.5 a,y 4.2 b,x 56.3 c,y 46.7 d,y 16.8 a,y 6.2 b,y 48.6 c,y 30.3 d,y
70.5 35.4 a,z 2.1 b,x 50.4 c,z 42.7 d,z 18.5 a,z 5.8 b,y 40.4 c,z 34.5 d,y
Least square means in a column within a day with a different letter (a–d) are significantly different (P b 0.05). Least square means within a row with a different letter (x-z) are significantly different (P b 0.05). Standard error for metmyoglobin-reducing activity = 1.9. Control = non-enhanced steaks. Pyruvate, lactate, and succinate were enhanced to 3% by weight of the final product. PVC = over-wrapped in oxygen-permeable film (atmospheric oxygen); VP = vacuum packaging; high oxygen (HiOx) = 80% O2 + 20% CO2.
3.5. Lipid oxidation There was a significant enhancement × packaging × storage time interaction for TBARS values as indicated by absorbance at 532 nm (Table 5). As expected, control steaks in vacuum packaging had less lipid oxidation (P b 0.05) than control steaks in PVC and high oxygen. Moreover, there was no effect (P N 0.05) of injection enhancement on lipid oxidation of steaks packaged in vacuum. Conversely, lactate and pyruvate decreased the TBARS values of steaks packaged in PVC (P b 0.05; compared with control steaks). Although all injection enhancement treatments decreased lipid oxidation (P b 0.05) compared with control steaks in high oxygen; pyruvate was the most effective for lowering lipid oxidation on day 13 (TBARS: control N succinate N lactate N pyruvate; P b 0.05). As a result, storage time had no effect on lipid oxidation in high oxygen when steaks were enhanced with pyruvate. A similar trend was noted for steaks in PVC enhanced with either pyruvate or lactate (no significant increase in TBARS during storage). The antioxidant activity of pyruvate has been reported in nonmeat systems (Herz, Blake, & Grootveld, 1997). More specifically, the keto-enol group present in pyruvate can directly scavenge hydrogen peroxide and other reactive oxygen species (Bassenge, Sommer, Schwemmer, & Bünger, 2000). Pyruvate also has the ability to regenerate glutathione (a potent antioxidant; Valentovic & Minigh, 2003). Lactate's role in minimizing lipid oxidation may be due to its ability to scavenge superoxide and hydroxyl radicals (Groussard et al., 2000). The ability of lactate enhancement to decrease lipid oxidation in ground beef, pork, and chicken has been reported previously (Mancini et al., 2010; Naveena, Sen, Muthukumar, Vaithiyanathan, & Babji, 2006; Nnanna, Ukuku, McVann, & Shelef, 1994). In model systems, studies have shown that succinate can prevent lipid oxidation induced by iron (Takeshige & Minakami, 1975). The antioxidant effect of succinate has been attributed to the formation of ubiquinol (a potent biological antioxidant; Vianello, Macri, Cavallini, & Bindoli, 1986). 4. Conclusion Injection enhancement of beef loins with succinate, pyruvate, and lactate increased muscle pH. Enhancing steaks with lactate, pyruvate, Table 5 Effects of enhancement, packaging, and storage time on the TBARS values of beef longissimus steaks stored at 1 °C. Days of storage
Enhancement
0 5
Control Control Lactate Pyruvate Succinate Control Lactate Pyruvate Succinate
13
Packaging VP
PVC
0.04 0.04 0.02 0.05 0.03 0.05 0.03 0.07 0.06
0.04 0.18 0.05 0.05 0.11 0.28 0.06 0.09 0.24
a,x a,x a,x a,x a,x a,x a,x a,x
HiOx a,y c,x c,x b,y a,y b,x b,x a,y
0.04 0.21 0.07 0.04 0.10 0.41 0.22 0.08 0.29
a,z b,x c,x b,y a,z c,y d,x b,y
Least square means in a column within a day with a different letter (a–d) are significantly different (P b 0.05). Least square means within a row with a different letter (x-z) are significantly different (P b 0.05). Standard error for TBARS value = 0.03. Control = non-enhanced steaks. Pyruvate, lactate, and succinate were enhanced to 3% by weight of the final product. PVC = over-wrapped in oxygen-permeable film (atmospheric oxygen); VP = vacuum packaging; high oxygen (HiOx) = 80% O2 + 20% CO2.
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and succinate can improve beef steak color stability during storage in PVC and high-oxygen packaging. However, this injection enhancement also will darken beef steaks. Pyruvate will minimize lipid oxidation of steaks packaged in both PVC and high oxygen. Addition of substrates such as pyruvate and succinate has the potential to improve the color of beef packaged in PVC and high oxygen. Future studies should further evaluate the mechanism(s) by which substrates such as pyruvate, succinate, and lactate influence meat color. Acknowledgements This project was funded, in part, by beef producers and importers through The Beef Checkoff and The National Cattlemen's Beef Association. References AMSA (1991). Guidelines for meat color evaluation. Reciprocal Meat Conference Proceedings, 44, 1−17. Bate-Smith, E. C. (1948). The physiology and chemistry of rigor mortis with special reference to the aging of beef. Advances in Food Research, 1, 1−38. Bassenge, E., Sommer, O., Schwemmer, M., & Bünger, R. (2000). Antioxidant pyruvate inhibits cardiac formation of reactive oxygen species through changes in redox state. American Journal of Physiology. Heart and Circulatory Physiology, 279, 2431−2438. GasÏperlin, L., ZÏlender, B., & Abram, V. (2000). Colour of normal and high pH beef heated to different temperatures as related to oxygenation. Meat Science, 54, 391−398. Giddings, G. G. (1974). Reduction of ferrimyoglobin in meat. Critical Reviews in Food Technology, 5, 143−173. Groussard, C., Morel, I., Chevanne, M., Monnier, M., Cillard, J., & Delamarche, A. (2000). Free radical scavenging and antioxidant effects of lactate ion: An in vitro study. Journal of Applied Physiology, 89, 169−175. Hamm, R. (1960). Biochemistry of meat hydration. Advances in Food Research, 10, 355−463. Herz, H., Blake, D. R., & Grootveld, M. (1997). Multicomponent investigations of the hydrogen peroxide- and hydroxyl radical scavenging antioxidant capacities of biofluids: The roles of endogenous pyruvate and lactate. Free Radical Research, 26, 19−35. Kim, Y. H., Hunt, M. C., Mancini, R. A., Seyfert, M., Loughin, T. M., Kropf, D. H., et al. (2006). Mechanism for lactate-color stabilization in injection-enhanced beef. Journal of Agricultural and Food Chemistry, 54, 7856−7862. Kim, Y. H., Keeton, J. T., Smith, S. B., Berghman, L. R., & Savell, J. W. (2009). Role of lactate dehydrogenase in metmyoglobin reduction and color stability of different bovine muscles. Meat Science, 83, 376−382. Knock, R. C., Seyfert, M., Hunt, M. C., Dikeman, M. E., Mancini, R. A., Unruh, J. A., et al. (2006). Effects of potassium lactate, sodium chloride, and sodium acetate on surface shininess/gloss and sensory properties of injection-enhanced beef striploin steaks. Meat Science, 74, 319−326. Lawrie, R. A. (1958). Physiological stress in relation to dark-cutting beef. Journal of the Science of Food and Agriculture, 11, 721−725. Lawrence, T. E., Dikeman, M. E., Hunt, M. C., Kastner, C. L., & Johnson, D. E. (2003). Effects of calcium salts on beef longissimus quality. Meat Science, 64, 299−308. Ledward, D. A. (1985). Post-slaughter influences on the formation of metmyoglobin in beef muscles. Meat Science, 15, 149−171. Maca, J. V., Miller, R. K., & Acuff, G. R. (1997). Microbiological, sensory and chemical characteristics of vacuum packaged ground beef patties treated with salts of organic acids. Journal of Food Science, 62, 591−596. Maca, J. V., Miller, R. K., Bigner, M. E., Luvia, L. M., & Acuff, G. R. (1999). Sodium lactate and storage temperature effects on shelf life of vacuum packaged beef top rounds. Meat Science, 53, 23−29.
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, 71−76. Mancini, R. A., Ramanathan, R., Suman, S. P., Konda, M. K. R., Joseph, P., Dady, G. A., et al. (2010). Effects of lactate and modified atmospheric packaging on premature browning in cooked ground beef patties. Meat Science, 85, 339−346. Mohan, A., Hunt, M. C., Barstow, T. J., Houser, T. A., & Muthukrishnan, S. (2010). Effects of malate, lactate, and pyruvate on myoglobin redox stability in homogenates of three bovine muscles. Meat Science, 86, 304−310. NAMP (2002). The meat buyers guide. Reston, VA: North American Meat Processors Association. Naveena, B. M., Sen, A. R., Muthukumar, M., Vaithiyanathan, S., & Babji, Y. (2006). The effect of lactates on the quality of microwave-cooked chicken patties during storage. Journal of Food Science, 71, 603−608. Nnanna, I. A., Ukuku, D. O., McVann, K. B., & Shelef, L. A. (1994). Antioxidant activity of sodium lactate in meat and model systems. Lebensmittel-Wissenschaft und Technologie, 27, 78−85. Puntel, R. L., Roos, D. H., Grotto, D., Garcia, S. C., Nogueira, C. W., & Rocha, J. B. T. (2007). Antioxidant properties of Krebs cycle intermediates against malonate pro-oxidant activity in vitro: A comparative study using the colorimetric method and HPLC analysis to determine malondialdehyde in rat brain homogenates. Life Sciences, 81, 51−62. Ramanathan, R., & Mancini, R. A. (2010). Effects of pyruvate on bovine heart mitochondria-mediated metmyoglobin reduction. Meat Science, 86, 738−741. Ramanathan, R., Mancini, R. A., & Konda, M. K. (2009). Effects of lactate on beef heart mitochondrial oxygen consumption and muscle darkening. Journal of Agricultural and Food Chemistry, 57, 1550−1555. Ramanathan, R., Mancini, R. A., & Konda, M. K. (2010a). Effect of lactate enhancement on myoglobin oxygenation of beef longissimus steaks overwrapped in PVC and stored at 4C. Journal of Muscle Foods, 21, 669−684. Ramanathan, R., Mancini, R. A., Naveena, B. M., & Konda, M. K. (2010b). Effects of lactate-enhancement on surface reflectance and absorbance properties of beef longissimus steaks. Meat Science, 84, 219−226. Ramanathan, R., Mancini, R. A., & Naveena, B. M. (2010c). Effects of lactate on bovine heart mitochondria-mediated metmyoglobin reduction. Journal of Agricultural and Food Chemistry, 58, 5724−5729. Reddy, I. M., & Carpenter, C. E. (1991). Determination of metmyoglobin reductase activity in bovine skeletal muscles. Journal of Food Science, 56, 1161−1164. Saleh, B., & Watts, B. M. (1968). Substrates and intermediates in the enzymatic reduction of metmyoglobin in ground beef. Journal of Food Science, 33, 353−358. Sammel, L. M., Hunt, M. C., Kropf, D. H., Hachmeister, K. A., & Johnson, D. E. (2002). Comparison of assays for reducing ability in beef inside and outside semimembranosus muscle. Journal of Food Science, 67, 978−984. Seyfert, M., Hunt, M. C., Ahnstrom, M. L., & Johnson, D. E. (2007). Efficacy of lactic acid salts and sodium acetate on ground beef colour stability and metmyoglobinreducing activity. Meat Science, 75, 134−142. Swatland, H. J., & Barbut, S. (1999). Sodium chloride levels in comminuted chicken muscle in relation to processing characteristics and Fresnel reflectance detected with a polarimetric probe. Meat Science, 51, 377−381. Takeshige, K., & Minakami, S. (1975). Reduced nicotinamide adenine-dinucleotide phosphate-dependent lipid peroxidation by beef heart submitochondrial particles. Journal of Biochemistry, 77, 1067−1073. Tang, J., Faustman, C., Mancini, R. A., Seyfert, M., & Hunt, M. C. (2005). Mitochondrial reduction of metmyoglobin: dependence on the electron transport chain. Journal of Agricultural and Food Chemistry, 53, 5449−5455. Valentovic, M. A., & Minigh, J. (2003). Pyruvate attenuates myoglobin in vitro toxicity. Toxicological Sciences, 74, 345−351. Vianello, A., Macri, F., Cavallini, L., & Bindoli, A. (1986). Induction of lipid peroxidation in soybean mitochondria and protection by respiratory substrates. Journal of Plant Physiology, 125, 217−224. Watts, B. M., Kendrick, J., Zipser, M. W., Hutchins, B., & Saleh, B. (1966). Enzymatic reducing pathways in meat. Journal of Food Science, 31, 855−862. Witte, V. C., Krause, G. F., & Bailey, M. E. (1970). A new extraction method for determining 2-thiobarbituric acid values of pork and beef during storage. Journal of Food Science, 35, 582−585.