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Effects of calcium salts on beef longissimus quality
Meat Science 64 (2003) 299–308 www.elsevier.com/locate/meatsci
Effects of calcium salts on beef longissimus quality§ T.E. Lawrencea, M.E. Dikemana,*, M.C. Hunta, C.L. Kastnera, D.E. Johnsonb a
Department of Animal Sciences & Industry, Kansas State University, Manhattan, KS 66506-0201, USA b Department of Statistics, Kansas State University, Manhattan, KS 66506-0802, USA Received 6 May 2002; received in revised form 22 July 2002; accepted 22 July 2002
Abstract The objective of this research was to evaluate the effects of injection marination with calcium salts on beef longissimus quality traits. Strip loins were injected (11% by weight) with distilled water or a 0.1, 0.2, or 0.3 M solution of calcium ascorbate, calcium chloride, or calcium lactate. Non-injected loins served as controls. Visual and instrumental color evaluations indicated that calcium ascorbate accelerated myoglobin oxidation, and increasing molar concentration of any calcium salt caused faster (P< 0.05) discoloration. Aerobic microbial plate counts were lower (P< 0.05) for treatments containing calcium lactate than those with calcium chloride or calcium ascorbate. Calcium ascorbate inhibited lipid oxidation whereas calcium lactate and calcium chloride appeared to be pro-oxidants of lipid oxidation. No differences for Warner-Bratzler shear force or sensory panel tenderness were found among the calcium salts; however, 0.3 M treatments had lower shear values and were judged more tender than 0.1 M treatments. Calcium ascorbate and calcium chloride treatments resulted in less (P< 0.05) beef flavor and more (P< 0.05) off-flavors than calcium lactate treatments. In addition, 0.1 M treatments had higher (P< 0.05) beef flavor scores while 0.3 M treatments had higher (P <0.05) offflavor scores. Considering the effects on color life, microbial inhibition, shear force, and sensory traits, we recommend injecting beef longissimus with a 0.1 M solution of calcium lactate to enhance both uncooked and cooked quality. # 2003 Elsevier Science Ltd. All rights reserved. Keywords: Beef; Calcium; Ascorbate; Chloride; Lactate; Tenderness; Color
1. Introduction Previous research has shown that injecting beef and lamb with calcium chloride improves tenderness (Koohmaraie, Crouse, & Mersmann, 1989; Wheeler, Koohmaraie, & Crouse, 1991; Kerth, Miller, & Ramsey, 1995). However, some research has shown that calcium chloride induces bitter, metallic, and livery off-flavors (Morgan, Miller, Mendez, Hale, & Savell, 1991; Wheeler, Koohmaraie, Lansdell, Siragusa, & Miller, 1993; Wheeler, Koohmaraie, & Shackelford, 1997). In addition, calcium chloride injection has induced muscle darkening and increased aerobic plate counts (Wheeler et al., 1993), and caused faster discoloration (Kerth et al., 1995) than untreated controls. Ideally, calcium marination technology would improve other economically §
Contribution No. 02-422-J from the Kansas Agricultural Experiment Station. * Corresponding author. Tel.: +1-785-532-1225; fax: +1-785-5327059. E-mail address: [email protected] (M.E. Dikeman).
important traits in addition to tenderness, such as lengthening color life, or inhibiting microbial growth. Previous calcium marination research was conducted nearly exclusively using calcium ions from the dissociation of calcium chloride. No research has been published on the use of calcium ascorbate in marination to improve tenderness, and only one report (Got, Rousset-Akrim, Bayle, & Culioli, 1996) was found on the use of calcium lactate. Therefore, the objective of this research was to compare the effects of calcium ascorbate, calcium chloride, and calcium lactate on beef color and palatability traits.
2. Materials and methods 2.1. Muscles Beef strip loins (IMPS 180, n=26, NAMP (1997)) from USDA Select carcasses were selected from the fabrication line of a commerical processor. Loins were transported at 4 C to the Kansas State University meat laboratory, trimmed of all external fat and accessory
0309-1740/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved. PII: S0309-1740(02)00201-2
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muscles, and cut transversely into three equal longissimus sections. 2.2. Injection marination Using an incomplete block design, the 78 loin sections were allocated randomly to the 11 marination treatments: (1) non-marinated control; (2) distilled water; (3) 0.1 M calcium ascorbate; (4) 0.2 M calcium ascorbate; (5) 0.3 M calcium ascorbate; (6) 0.1 M calcium chloride; (7) 0.2 M calcium chloride; (8) 0.3 M calcium chloride; (9) 0.1 M calcium lactate; (10) 0.2 M calcium lactate; or (11) 0.3 M calcium lactate. The treatments will be referred to, respectively, as Control, Dwater, 1CaAsc, 2CaAsc, 3CaAsc, 1CaCl, 2CaCl, 3CaCl, 1CaLac, 2CaLac, and 3CaLac. Also, contrast statements comparing the three molar concentrations will be referred to as 0.1 M, 0.2 M, or 0.3 M. Calcium ascorbate (Ashland Nutritional Products, La Mirada, CA), calcium lactate (PURAC America, Inc., Lincolnshire, IL), and calcium chloride (TETRA Chemicals, The Woodlands, TX) solutions were made using pre-chilled distilled water. Marinade temperature was 4 C during injection. Loin sections were injected at 11% by weight (Schro¨der N50, Werther, Germany) with their respective treatment, vacuum packaged (Multivac C500, Wolfertschwenden, Germany), and tumbled (Food Processing Equipment Company, model VT85, Sante Fe Springs, CA) for 15 min. Surface temperature (measured using an infrared thermometer, Kane-May KM800S, Hertfordshire, England) of the loin sections was 3 C before tumbling and 6 C after tumbling. After injection marination, loins were stored at 1 C until 14 days postmortem. The pH of loin sections was measured before injection and after aging 14 days with a Sentron Argus w pH meter connected to a Sentron Red-Line LanceFET probe (Sentron, Gig Harbor, WA). 2.3. Sample processing At 14 days postmortem, each loin section was weighed, and then cut into two 2.54-cm-thick steaks, one for shear force and one for sensory panel evaluation. An additional 1.91-cm thick steak was cut for simulated retail display. The remaining portion was divided into thirds and utilized for initial microbiological evaluation, initial TBARS analysis, and uncooked calcium quantification. Shear force and sensory panel steaks were vacuum packaged and frozen ( 38 C). Retail display steaks were placed on 17S white foam trays (Tenneco Packaging Specialty and Consumer Products, Lake Forest, IL) and overwrapped with heat shrinkable PVC film (MAPAC M—23,250 cc O2/m2/24 h—72 gauge, Borden Packaging and Industrial Products, Borden, Inc., North Andover, MA). Initial TBARS and uncooked calcium samples were vacuum packaged and frozen ( 80 C).
2.4. Simulated retail display Steaks were displayed for 5 days at 3 C in a retail display case (Tyler Refrigeration Corporation, model DMF8, Niles, MI) under 1614 lux of fluorescent light (Philips UltralumeTM 30U, Somerset, NJ). Trained color panelists (all panelists passed the FarnsworthMunsell 100 Hue color test) evaluated color deterioration (1=very bright cherry red, 2=bright cherry red, 3=slightly dark red to tannish red, 3.5=borderline acceptable, 4=moderately grayish tan to brown, 5=tan to brown) and percentage of surface discoloration (1=0%, 2=1–10%, 3=11–25%, 4=26–50%, 5=51– 75%, 6=76–99%, 7=100%). Instrumental data (L* a* b*) were measured at three locations on each steak using a MiniScanTM XE (Hunter Associates Laboratory, Inc., Reston, VA) with a 35-mm aperture set for Illuminant A, and a 10 observer. Instrumental color was measured through the packaging film and the instrument was calibrated with the same packaging film over the tiles. Chroma and hue angle (Little, 1975) were calculated. Steaks were evaluated instrumentally and by the visual panel at 0, 24, 48, 72, and 96 h. 2.5. Microbiological analysis Two surface cores (2.54 cm diameter, 3 mm thick) were aseptically cut from steaks, placed in filtered stomacher bags (Spiral Biotech, Norwood, MA) to which 99 ml of 0.1% peptone buffer (NutraMax Products, Inc., Gloucester, MA) was added, and stomached (Seward stomacher 400, Seward Medical, London, UK) for 2 min. Duplicate initial (at 0 h of retail display) and final (after 96 h of retail display) samples were plated at 1, 10 1, 10 2, and 10 3 on Aerobic Plate Count Petrifilm (Microbiology Products—3 M Health Care, St. Paul, MN) and incubated for 48 h at 36 C. Microbiological growth was counted and converted to log colony-forming units per cm2. 2.6. Warner-Bratzler shear force determinations Steaks were thawed at 3 C for 40 h. They were cooked on an electric belt grill (model TBG-80 Magigrill, MagiKitch’n Inc., Quakertown, PA) set at 117 C using the following settings: top platen=110 C, bottom platen=109.4 C, preheat disconnected, platen gap=1.905 cm, cook time=9.0 min. This temperature was chosen because previous research (Lawrence, King, Obuz, Yancey, & Dikeman, 2001) indicated that it provided the highest repeatability for Warner-Bratzler shear force of longissimus steaks. Steaks were cooked to an internal temperature of 70 C, cooled in a refrigerator for 24 h at 0 C (AMSA, 1995), then eight cores (1.27 cm diameter) were removed from each steak parallel to muscle fiber orientation using a mechanical
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coring device. Cores were sheared once through the center by a V-notch Warner-Bratzler shear attachment connected to an Instron Universal Testing Machine (model 4201, Instron Corp., Canton, MA) using a 50 kg compression load cell and a 250 mm/min crosshead speed. The peak force and total energy required to shear each core were recorded. Cooking time and percentage of cooking loss [((raw wt-cooked wt)/raw wt) 100] were recorded for each steak. 2.7. 2-Thiobarbituric acid reactive substances (TBARS) analysis TBARS analysis was conducted using the extraction method of Witte, Krause, and Bailey (1970). Initial (at 0 h of retail display) and final (after 96 h of retail display) samples were immersed in liquid nitrogen and pulverized. Duplicate 10-g samples were weighed into stainless steel mixers, to which 15 ml of 9% perchloric acid and 20 ml of distilled-deionized water were added. Samples were blended for 15 s, and then filtered through Whatman no.1 filter paper into test tubes. Five millilitres of TBA reagent were added to the filtrate and stored overnight before spectral absorbance was measured at 530 nm (Bausch & Lomb, Spectronic 21, Rochester, NY). A standard curve was developed using 0, 0.2, 0.4, 0.8, 1.0, 2.0, 4.0, 6.0, and 8.010 8 mol/ml of 1,1,3,3tetraethoxypropane. 2.8. Descriptive attribute sensory panel evaluation Steaks were thawed in a refrigerator at 3 C for 40 h, and cooked as previously described for Warner-Bratzler shear force determinations. An untreated longissimus steak aged 14 days postmortem was served to the panelists as a warm-up sample. Using an eight-point scale, trained panelists (AMSA, 1995) scored samples for myofibrillar and overall tenderness, juiciness, connective tissue amount, beef flavor intensity, and off-flavor intensity (1=extremely tough, dry, abundant, bland, and intense; 8=extremely tender, juicy, none, intense, and none). In addition, panelists were instructed to describe off-flavors as astringent, bitter, bloody/serumy, fatty, livery, metallic, salty, soapy, or sour, or to provide their own descriptor. 2.9. Calcium quantification Uncooked and cooked (Warner-Bratzler shear steaks) samples were immersed in liquid nitrogen and pulverized. Duplicate 2-g samples were placed in ceramic crucibles and ashed in a muffle furnace (Neytech 85A, Bloomfield, CT) at 600 C for 4 h, digested with 25 ml of 2.5 N hydrochloric acid for 1 h, then diluted to 100 ml with distilled, deionized water. One ml of diluted sample was added to 9 ml of 1% strontium chloride
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before absorbance was measured at 422.7 nm using an atomic absorption spectrophotometer (AAnalyst 100, Perkin Elmer, Norwalk, CT). 2.10. Experimental design and statistical analysis The treatment structure was a 33 factorial (three calcium salts at three molar concentrations) with a negative (non-marinated) and a positive (distilled water) control. The design structure was an incomplete block design. Loin was the blocking factor and one-third of a loin was the experimental unit. Data were analyzed using the MIXED procedure of SAS (1999). The model included the fixed effect of treatment, and the random effect of loin. Repeated measures data (pH, color deterioration, discoloration, L*, a*, b*, chroma, hue angle, TBARS, total plate counts) were analyzed using an unstructured covariance model. Treatment means were generated using the LSMEANS option. In addition, pre-determined single degree of freedom contrasts were generated using the ESTIMATE option to test main effects of calcium salt and molar concentration.
3. Results and discussion 3.1. pH and yield The distilled water from which all solutions were made had a pH of 6.9. The CaAsc solutions were slightly acidic (formation of ascorbic acid) and slightly increased in acidity as molar concentration increased (5.9, 5.8, 5.8). The CaCl solutions were basic and increased in alkalinity (9.7, 10.0, 10.1) as molar concentration increased. The CaLac solutions were slightly alkaline (7.5, 7.4, 7.4). Contrast differences for initial muscle pH (Table 1) were not significant. After 14 days of postmortem aging, final muscle pH values generally increased slightly above pre-marination values, which was expected because the pH of the marination solutions generally was above that of the loin sections. However, the final muscle pH value for the 3CaCl treatment was lower (P < 0.05) than the pre-marination value. Because the pH of the 3CaCl solution was 10.1, the final muscle pH was expected to increase rather than decrease. Final pH values of CaAsc and CaLac treatments were higher (P < 0.05) than CaCl treatments. In addition, 0.1 M treatments had higher (P < 0.05) final pH values than 0.3 M treatments. Contrasts for pumped yield (Table 1) indicated that marination with CaAsc resulted in higher (P < 0.05) yields than CaCl, which had higher (P < 0.05) yields than CaLac. Among the individual marination treatments, 3CaAsc had the highest pumped yield and Dwater the lowest. Pumped yield increased in a linear
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Table 1 Least squares means and significant contrasts of pH and pumped yield of marinateda beef longissimus Marination treatment
Initial pHb
Final pH after agingc
Pumped yield (%)d
0.1 M CaAsc 0.2 M CaAsc 0.3 M CaAsc 0.1 M CaCl 0.2 M CaCl 0.3 M CaCl 0.1 M CaLac 0.2 M CaLac 0.3 M CaLac Control Dwater SEM
5.5 5.5 5.4 5.4 5.4 5.5 5.4 5.4 5.5 5.5 5.4 0.03
5.5 5.5 5.5 5.5 5.4 5.4 5.5 5.5 5.4 5.6 5.5 0.02
104.6 105.8 108.3 102.8 105.0 106.9 103.8 103.3 103.8 – 102.5 0.70
* Asc
* Asc * Asc * Cl
Contrasts CaAsc vs. CaCl CaAsc vs. CaLac CaCl vs. CaLac CaAsc vs. Control CaCl vs. Control CaLac vs. Control CaAsc vs. Dwater CaCl vs. Dwater CaLac vs. Dwater Control vs. Dwater 0.1 M vs. 0.2 M 0.1 M vs. 0.3 M 0.2 M vs. 0.3 M
* Lac * Con * Con * Con * Dw
*1
* Asc * Cl * Lac
*3 *3
* Contrast difference P <0.05; Asc, Cl, Lac, Con, Dw, 1, 2, or 3 indicates which treatment in the contrast has the higher value. a 0.1 M calcium ascorbate; 0.2 M calcium ascorbate; 0.3 M calcium ascorbate; 0.1 M calcium chloride; 0.2 M calcium chloride; 0.3 M calcium chloride; 0.1 M calcium lactate; 0.2 M calcium lactate; 0.3 M calcium lactate; Non-marinated control; Distilled water. b pH of loin sections immediately before injection marination. c pH of loin sections after 14-day aging period. d Pumped yield=(Pumped, aged loin wt./green loin wt.) 100.
manner as molar concentration of CaAsc or CaCl increased; however, this effect was not evident for CaLac. Clearly, contrasts indicated that pumped yield increased (P < 0.05) as molar concentration increased, apparently due to increased ionic strength of increasing molar concentrations. 3.2. Visual color evaluations Contrasts for average color scores (Table 2) indicated that steaks marinated with CaCl, CaLac, Dwater, or Control treatments were less (P < 0.05) deteriorated than those marinated with the CaAsc treatment throughout the 96 h display period. Throughout the 96 h display period, 1CaLac, 1CaCl, and Dwater treatments retained color the best. In addition, steaks marinated with 0.1 M solutions had less (P < 0.05) surface browning than those marinated with 0.2 or 0.3 M solutions, with no differences in color between 0.2 and 0.3 M solutions. Steaks marinated with CaCl, CaLac, Dwater, or Control treatments were less (P < 0.05) discolored than those marinated with the CaAsc treatment throughout
the 96 h of display. Steaks marinated with CaLac or those from the Control treatment were also less (P < 0.05) discolored than those marinated with CaCl. These data agree with those of Kerth et al. (1995) and Wheeler, Koohmaraie, and Shackelford (1996) who reported that marination with calcium chloride increased surface browning over that of non-marinated controls. In our study, 1CaLac and Control treatments had the greatest color stability during 96 h of simulated display. In addition, 0.1 M treatments had less (P < 0.05) discoloration than 0.2 or 0.3 M treatments. Kerth et al. (1995) reported that marination with 0.25 M calcium chloride caused faster discoloration than a 0.20 M solution. The increase in discoloration caused by the calcium solutions and by increased molar concentrations likely resulted from the oxidative catalyzing characteristics of the salts. However, this effect was not as pronounced for the 1CaLac or 1CaCl treatments as it was for all other calcium treatments. In summary, visual color evaluations indicated that CaAsc marination resulted in less color life than CaCl or CaLac treatments because of greater myoglobin
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Table 2 Least squares means and significant contrasts of visual and instrumental color evaluations of marinateda beef longissimus steaks during a 96 h simulated retail display Marination treatment
Visual Color b
Discoloration c
L*
a*
b*
Chroma
Hue angle
0.1 M CaAsc 0.2 M CaAsc 0.3 M CaAsc 0.1 M CaCl 0.2 M CaCl 0.3 M CaCl 0.1 M CaLac 0.2 M CaLac 0.3 M CaLac Control Dwater SEM
3.3 4.1 4.3 2.3 2.8 3.0 2.3 2.7 2.7 2.8 2.5 0.2
2.5 3.6 3.9 1.8 2.5 2.5 1.6 2.1 2.0 1.7 1.9 0.2
43.3 40.0 39.2 45.5 44.6 42.6 45.4 43.3 44.4 42.7 45.3 1.1
25.2 22.5 21.0 30.0 27.7 26.4 30.2 29.0 28.6 30.6 30.2 0.7
19.6 17.8 17.0 23.2 22.0 20.7 23.4 22.3 22.1 23.3 23.7 0.5
31.9 28.7 27.0 38.0 35.4 33.6 38.2 36.7 36.1 38.5 38.4 0.8
37.8 38.2 39.0 37.7 38.6 38.2 37.2 37.6 37.8 37.2 38.2 0.4
* Asc * Asc
* Asc * Asc * Cl * Asc * Cl
* Cl * Lac
* Cl * Lac
* * * * *
* Asc
* Asc
* Dw
* * * * * * * *
*2 *3
*2 *3
*1 *1
Contrasts CaAsc vs. CaCl CaAsc vs. CaLac CaCl vs. CaLac CaAsc vs. Control CaCl vs. Control CaLac vs. Control CaAsc vs. Dwater CaCl vs. Dwater CaLac vs. Dwater Control vs. Dwater 0.1 M vs. 0.2 M 0.1 M vs. 0.3 M 0.2 M vs. 0.3 M
* Asc
Cl Lac Lac Con Con Con Dw Dw
* Con * Con
Cl Lac Lac Con Con
* Dw * Dw * Dw
* Dw * Dw
*1 *1 *2
*1 *1 *2
* Asc * Asc * Cl
* Dw *1 *1 *2
*3
* Contrast difference P <0.05; Asc, Cl, Lac, Con, Dw, 1, 2, or 3 indicates which treatment in the contrast has the higher value. a 0.1 M calcium ascorbate; 0.2 M calcium ascorbate; 0.3 M calcium ascorbate; 0.1 M calcium chloride; 0.2 M calcium chloride; 0.3 M calcium chloride; 0.1 M calcium lactate; 0.2 M calcium lactate; 0.3 M calcium lactate; Non-marinated control; Distilled water. b 1=very bright cherry red, 2=bright cherry red, 3=slightly dark red to tannish red, 3.5=borderline acceptable, 4=moderately grayish tan to brown, 5=tan to brown. c 1=0%, 2=1–10%, 3=11–25%, 4=26–50%, 5=51–75%, 6=76–99%, 7=100%.
oxidation. These results were unexpected because we hypothesized that the ascorbate would act as a sequestrant of free electrons, thereby lessening metmyoglobin formation. Ascorbic acid has been used as an antioxidant to inhibit pigment oxidation in fresh beef systems (Harbers, Harrison, & Kropf, 1981; Mitsumoto, Faustman, Cassens, Arnold, Schaefer, & Scheller, 1991; Okayama, Imae, & Yamanoue, 1987; Shivas, Kropf, Hunt, Kastner, Kendall, & Dayton, 1984). However, it has also been a pro-oxidant in the presence of iron (Kanner & Mendel, 1977; Liu & Watts, 1970) or copper (Mahoney & Graf, 1986). The addition of increased levels of calcium ions may also induce effects similar to iron and copper. Furthermore, Sato and Hegarty (1971) investigated ascorbic acid levels from 0 to 10,000 ppm and reported the greatest amount of oxidation occurred in the 50–100 ppm range. That may provide an explanation for the drastic oxidation that occurred with all of our CaAsc treatments because the levels of 1CaAsc, 2CaAsc, and 3CaAsc injected into the longissimus were
43, 86, and 129 ppm, respectively. Marination with CaLac treatments retained red color better than other calcium treatments. The mechanism for CaLac increasing the color stability of muscle appears to be through the influx of lactate into the system. Lactate can be converted to NADH, H+, and pyruvate by lactate dehydrogenase. Increasing the supply of NADH allows for the ability to donate electrons to ferric iron (Fe+++), thereby reducing it to ferrous iron (Fe++). However, 0.2 and 0.3 M marination treatments of CaAsc and CaCl resulted in significantly more color deterioration than 0.1 M treatments. As molar concentration increases, the number of free radical electrons likely increases, thereby providing more catalysts for myoglobin oxidation. 3.3. Instrumental color evaluations Contrasts for luminescence (L*) values (Table 2) indicated that steaks marinated with CaCl, CaLac, or
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Dwater were lighter (P < 0.05) (higher L* values) than those marinated with CaAsc throughout the 96 h display period, further indicating the degree of muscle darkening caused by marination with CaAsc. However, CaCl and CaLac treatments were similar to Control and Dwater treatments. Marination with 1CaCl, 1CaLac, or Dwater resulted in the highest L* values throughout the display period. In addition, 0.1 M treatments had higher (P < 0.05) L* values than 0.2 or 0.3 M treatments. Steaks marinated with the CaCl, CaLac, Dwater or Control treatments were more red (P< 0.05) (higher a* values) than CaAsc treatments throughout display. The CaLac, Dwater, and Control treatments were more red (P < 0.05) than the CaCl treatment throughout display. Furthermore, 0.1 M treatments were more red (P < 0.05) than 0.2 M and 0.3 M treatments, and 0.2 M treatments were more red (P < 0.05) than 0.3 M treatments. Steaks marinated with CaCl, CaLac, Dwater, or Control treatments were more yellow (P < 0.05) (higher b* values) than CaAsc treatments. Furthermore, Dwater and Control treatments were more yellow (P < 0.05) than the CaCl treatment throughout display. In addition, 0.1 M treatments were more yellow (P < 0.05) than both 0.2 M treatments and 0.3 M treatments throughout display. Moreover, 0.2 M treatments were more yellow (P < 0.05) than 0.3 M treatments. Steaks marinated with CaCl, CaLac, Dwater, or Control treatments had more intense red color (higher chroma values, P < 0.05) than CaAsc treatments. The CaLac, Dwater, and Control treatments had a more intense red color (P< 0.05) than CaCl treatments. In addition, 0.1 M treatments had a more intense red color (P < 0.05) than 0.2 M or 0.3 M treatments throughout display, and the 0.2 M treatments had a more intense red color (P < 0.05) than 0.3 M treatments. Steaks marinated with CaLac or Control treatments were less brown (P < 0.05) (lower hue-angle) than those marinated with CaAsc treatments. The Control treatment was less brown (P < 0.05) than the CaCl or Dwater treatments. In addition, 0.1 M treatments were less brown (P < 0.05) than 0.3 M treatments. In summary, instrumental color determinations confirmed visual scores. CaAsc resulted in darker steaks that were less red, less yellow, and more brown than CaCl- or CaLac-marinated steaks. Marination with CaCl resulted in steaks that were less red than those marinated with CaLac. In addition, steaks darkened and became less red and less yellow as molar concentration increased from 0.1 to 0.2 to 0.3 M. 3.4. Microbiological evaluations Initial aerobic plate counts (log cfu/cm2) were not different among the 11 marination treatments (Table 3); however, counts increased (P < 0.05) for all treatments after 5 days of simulated retail display, except 2CaLac
and the non-marinated Control. The CaLac treatments inhibited microbial growth (P < 0.05) more than CaAsc or CaCl treatments. Although not evident in our data, Benito-Delgado et al. (1994) and Wheeler et al. (1993) reported that marination with CaCl increased total aerobic plate counts more than non-marinated controls. In our study, increasing molar concentrations of CaAsc or CaCl treatments did not consistently influence final counts. However, increasing concentration of CaLac resulted in numerically lower, but statistically similar values. In support of Chen and Shelef (1992), these data indicate that marination with CaLac may be useful for inhibiting growth of aerobic microflora during retail display. 3.5. TBARS analysis Initial 2-Thiobarbituric acid reactive substances values (an index of lipid oxidation, Table 3) did not differ among the 11 treatments. Final TBARS values were highest for the 3CaCl, 2CaCl, 3CaLac, and 2CaLac treatments. Contrasts of TBARS values indicated that CaAsc treatments had lower (P < 0.05) final values than CaCl, CaLac, or Dwater treatments. It appears that all CaAsc treatments inhibited lipid oxidation, whereas all CaCl and CaLac treatments enhanced lipid oxidation. CaCl and CaLac treatments caused more lipid oxidation (higher TBARS values, P < 0.05) than Dwater or Control treatments. These data are in agreement with those of St. Angelo, Koohmaraie, Crippen, and Crouse (1991) and Harris, Huff-Lonergan, Lonergan, Jones, and Rankins (2001) who reported that CaCl marination resulted in increased TBARS values. Furthermore, 0.1 M marination treatments resulted in less (P < 0.05) lipid oxidation than 0.2 or 0.3 M treatments. In contrast to the visual and instrumental color data, which indicated a myoglobin prooxidant effect for CaAsc, it appears that injection of a CaAsc solution into muscle inhibits the extent of lipid oxidation. 3.6. Calcium quantification Calcium concentration (mg/g) of loins marinated with calcium treatments was 400–1350% higher than the non-marinated Control treatment (Table 3). As expected, contrasts indicated no difference in uncooked or cooked calcium concentration among calcium salts. There also was no difference between Dwater and the non-marinated Control. Also, as expected, uncooked or cooked calcium concentration increased as molar concentration increased. Previous uncooked calcium quantification of CaCl marinated steaks (Koohmaraie et al. 1989; Morgan et al. 1991; Polidori, Marinucci, Fantuz, Renieri, & Polidori, 2000; Wheeler et al., 1993) yielded much lower values than in our experiment ( 500 vs. 1252.0 mg/g for a 10% pump of a 0.3 M solution).
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Table 3 Least squares means and significant contrasts of aerobic plate counts (APC)a, 2-thiobarbituric acid (TBARS)b evaluations, of marinatedc beef longissimus steaks initially and after 5 days of simulated retail display and of uncooked and cooked calcium concentrationd Marination treatment
Initial APC
Final APC
Initial TBARS
Final TBARS
Uncooked calcium
Cooked calcium
0.1 M CaAsc 0.2 M CaAsc 0.3 M CaAsc 0.1 M CaCl 0.2 M CaCl 0.3 M CaCl 0.1 M CaLac 0.2 M CaLac 0.3 M CaLac Control Dwater SEM
0.9 1.4 1.3 1.5 0.9 1.3 1.5 1.4 1.1 1.6 1.1 0.3
3.0 3.5 2.9 3.3 2.2 3.7 2.4 2.2 2.1 2.2 2.6 0.4
0.12 0.15 0.18 0.06 0.06 0.06 0.05 0.05 0.07 0.05 0.06 0.09
0.14 0.17 0.23 0.47 0.99 1.05 0.43 0.82 0.85 0.23 0.43 0.09
517.0 934.0 246.0 486.5 850.9 1333.4 460.2 831.8 1176.1 91.8 72.3 56.8
498.6 1025.5 1354.0 481.3 1008.6 1330.9 352.4 727.3 1475.2 77.3 68.7 70.5
* Asc * Cl * Lac * Asc * Cl * Lac
* * * * * *
*2 *3 *3
*2 *3 *3
Contrasts CaAsc vs. CaCl CaAsc vs. CaLac CaCl vs. CaLac CaAsc vs. Control CaCl vs. Control CaLac vs. Control CaAsc vs. Dwater CaCl vs. Dwater CaLac vs. Dwater Control vs. Dwater 0.1 M vs. 0.2 M 0.1 M vs. 0.3 M 0.2 M vs. 0.3 M
* Asc * Cl
* Cl * Lac
* Cl * Lac * Dw * Cl * Lac *2 *3
Asc Cl Lac Asc Cl Lac
* Contrast difference P <0.05; Asc, Cl, Lac, Con, Dw, 1, 2, or 3 indicates which treatment in the contrast has the higher value. a log CFU/cm2. b mg of malonaldehyde/kg of muscle. c 0.1 M calcium ascorbate; 0.2 M calcium ascorbate; 0.3 M calcium ascorbate; 0.1 M calcium chloride; 0.2 M calcium chloride; 0.3 M calcium chloride; 0.1 M calcium lactate; 0.2 M calcium lactate; 0.3 M calcium lactate; Non-marinated control; Distilled water. d mg calcium/g muscle tissue.
This discrepancy may be explained by analytical procedural differences. Previous experiments of this type used a water-extractable-calcium procedure whereby muscle tissue was minced, blended with water, and centrifuged, with calcium concentration being measured on the supernatant only rather than the whole muscle tissue. Therefore, previous data did not account for calcium that was remaining in the pellet. In addition, previous data did not report the calcium concentration in cooked steaks. Except for the 3CaLac treatment, which increased in calcium concentration during cooking, no differences were found between uncooked and cooked calcium measurements. This finding suggests that some calcium is lost during cooking so that the concentration after cooking remained similar to the concentration before cooking. Dietary reference intakes for calcium suggest that adult males and females should consume 1000 to 1300 mg/day. Using our data, an 85g (3 oz.) uncooked portion of non-marinated beef longissimus would contain 7.8 mg of calcium or 0.8% of the recommended daily intake for calcium. In contrast, beef longissimus mari-
nated with 0.1, 0.2, or 0.3 M calcium solutions would contain 41.5, 74.2, and 106.6 mg of calcium and account for 4.2, 7.4, and 10.7% respectively of the recommended daily intake for calcium. Because one serving of the 0.3 M calcium-injected steaks contains between 10 and 19% of the recommended daily value for calcium, they could be labeled as a ‘‘Good source’’ of calcium. In summary, these data suggest that calcium-marinated steaks could be marketed as a calcium-enhanced product to further improve salability and consumer acceptance. 3.7. Warner-Bratzler shear and cooking trait evaluations Contrasts of Warner-Bratzler shear values (Table 4) indicated that CaAsc, CaCl, and CaLac treatments were equal in their tenderizing effect and had lower (P < 0.05) peak force and total energy values than Dwater or Control treatments. Only 4.2% of calcium-marinated steaks were tough (shear values 54.5 kg) whereas 37.5% of Control steaks were classified as tough. Increasing molar concentration of CaAsc, CaCl, or CaLac resulted in numerically lower peak force values
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Table 4 Least squares means and significant contrasts of Warner-Bratzler shear values and cooking traits of marinateda beef longissimus steaks Marination treatment
Purge loss (%)
Peak force (kg)
Total energyb
Cooking loss (%)
Cooking time (min)
0.1 M CaAsc 0.2 M CaAsc 0.3 M CaAsc 0.1 M CaCl 0.2 M CaCl 0.3 M CaCl 0.1 M CaLac 0.2 M CaLac 0.3 M CaLac Control Dwater SEM
5.8 5.8 5.2 5.7 5.2 5.2 5.2 6.1 5.8 3.8 5.5 0.4
2.97 2.84 2.46 3.07 2.87 2.64 3.17 3.11 2.91 4.29 3.87 0.26
34.66 32.49 30.39 33.68 32.65 30.83 35.48 36.59 33.34 45.31 40.87 2.6
19.5 18.8 18.7 19.9 21.5 19.4 20.7 20.5 19.7 18.4 24.3 1.5
6.9 6.8 7.0 6.4 7.1 6.8 6.7 6.7 6.8 7.1 6.9 0.2
* Asc * Cl * Lac
* Con * Con * Con * Dw * Dw * Dw
* Con * Con * Con * Dw * Dw * Dw
Contrasts CaAsc vs. CaCl CaAsc vs. CaLac CaCl vs. CaLac CaAsc vs. Control CaCl vs. Control CaLac vs. Control CaAsc vs. Dwater CaCl vs. Dwater CaLac vs. Dwater Control vs. Dwater 0.1 M vs. 0.2 M 0.1 M vs. 0.3 M 0.2 M vs. 0.3 M
* Dw
* * * *
Dw Dw Dw Dw
*1
* Contrast difference P <0.05; Asc, Cl, Lac, Con, Dw, 1, 2, or 3 indicates which treatment in the contrast has the higher value. a 0.1 M calcium ascorbate; 0.2 M calcium ascorbate; 0.3 M calcium ascorbate; 0.1 M calcium chloride; 0.2 M calcium chloride; 0.3 M calcium chloride; 0.1 M calcium lactate; 0.2 M calcium lactate; 0.3 M calcium lactate; Non-marinated control; Distilled water. b Total energy=force (kg) distance traveled (mm).
and, overall, 0.3 M treatments had lower (P < 0.05) peak force values than 0.1 M treatments. Our data agree with numerous previous studies that reported a reduction in shear force values following marination in CaCl (Kerth et al., 1995; Morgan et al., 1991; Pringle, Harrelson, West, Williams, & Johnson, 1999; Wheeler et al., 1993, 1997). However, our data are unique in that they demonstrate the ability of calcium salts other than CaCl to induce a calcium tenderizing effect. Purge losses (Table 4) were higher (P< 0.05) for CaAsc, CaCl, CaLac, or Dwater treatments than for the non-marinated Control treatment. Cooking times were not different among the 11 treatments, which is in agreement with data presented by Morgan et al. (1991). In contrast, Wheeler et al. (1997) reported that nonmarinated control steaks cooked significantly faster than those marinated with CaCl. Contrasts for percentages of cooking loss indicated that Dwater marination resulted in higher (P< 0.05) cooking losses than Control, CaAsc, CaLac, or CaCl treatments. The depressed water retention of the Dwater treatment was likely due to a lower ionic strength than all of the calcium marination treatments. The addition of a salt (e.g. a calcium salt) to
a solution increases ionic strength, thereby increasing the number of hydrophilic protein interactions, which causes an increase in the binding of free water. During cooking of Warner-Bratzler shear force steaks, a dull green surface appeared on the cooked steaks marinated with CaAsc. This cooked surface discoloration is likely due to formation of cholemyoglobin (H2O2 bound to heme). L-Ascorbate has functional groups that could likely be donated to the formation of H2O2 during the heating process. This coloration would likely be objectionable to most consumers. 3.8. Descriptive attribute sensory panel evaluations Myofibrillar and overall tenderness values of all calcium marination treatments were rated higher (P < 0.05) than Dwater or Control treatments (Table 5). All three calcium salts equally increased sensory-tenderness scores; however, the 0.3 M treatments were judged more tender (P < 0.05) than the 0.1 or 0.2 M treatments. Other reports of increased sensory-panel tenderness due to CaCl marination include Kerth et al. (1995), Morgan et al. (1991), and Wheeler et al. (1993, 1997).
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Marination treatment Myofibrillar tendernessb Juicinessc Beef flavor intensityd Connective tissue amounte Overall tendernessb Off-flavor intensityf 0.1 M CaAsc 0.2 M CaAsc 0.3 M CaAsc 0.1 M CaCl 0.2 M CaCl 0.3 M CaCl 0.1 M CaLac 0.2 M CaLac 0.3 M CaLac Control Dwater SEM Contrasts CaAsc vs. CaCl CaAsc vs. CaLac CaCl vs. CaLac CaAsc vs. Control CaCl vs. Control CaLac vs. Control CaAsc vs. Dwater CaCl vs. Dwater CaLac vs. Dwater Control vs. Dwater 0.1 M vs. 0.2 M 0.1 M vs. 0.3 M 0.2 M vs. 0.3 M
5.8 6.0 6.5 5.7 5.9 6.4 5.9 6.0 6.4 4.7 5.0 0.2
4.8 5.3 5.3 5.1 5.0 5.1 5.2 5.3 5.2 5.1 4.9 0.2
5.0 4.9 4.8 5.1 4.7 4.5 5.2 5.2 5.0 5.0 4.9 0.1
7.0 7.0 7.4 7.0 7.1 7.4 7.1 7.2 7.2 6.3 6.8 0.2
6.1 6.2 6.7 6.1 6.2 6.7 6.2 6.3 6.6 5.0 5.3 0.2
* Lac * Lac * * * * * *
Asc Cl Lac Asc Cl Lac
* Asc * Cl * Lac
* * * * * *
Asc Cl Lac Asc Cl Lac
7.2 6.9 5.8 7.0 6.1 4.9 6.9 7.4 7.4 7.4 7.4 0.3
* Asc * Lac * Lac * Con * Con * Dw * Dw
* Dw *3 *3
*1
*3 *3
*3 *3
*1 *2
* Contrast difference P <0.05; Asc, Cl, Lac, Con, Dw, 1, 2, or 3 indicates which treatment in the contrast has the higher value. a 0.1 M calcium ascorbate; 0.2 M calcium ascorbate; 0.3 M calcium ascorbate; 0.1 M calcium chloride; 0.2 M calcium chloride; 0.3 M calcium chloride; 0.1 M calcium lactate; 0.2 M calcium lactate; 0.3 M calcium lactate; Non-marinated control; Distilled water. b 4=slightly tough, 5=slightly tender, 6=moderately tender, 7=very tender. c 4=slightly dry, 5=slightly juicy. d 4=slightly bland, 5=slightly intense. e 6=traces, 7=practically none. f 4=slightly intense, 5=slight, 6=traces, 7=practically none.
No differences in juiciness scores were detected among the 11 treatments. Intensity of beef flavor was judged lowest for the 3CaCl, 2CaCl, and 3CaAsc treatments and highest for the CaLac, Control, and 1CaCl treatments. Contrasts indicated that beef-flavor intensity was higher (P < 0.05) for CaLac than for CaAsc or CaCl treatments and was higher (P < 0.05) for 0.1 M treatments than for 0.3 M treatments. Off-flavor scores indicated that 0.3 M CaCl induced the most intense offflavors followed by 0.3 M CaAsc and 0.2 M CaCl. Overall, CaCl treatments (characterized by panelists as bitter, metallic, sour, soapy, and astringent) had more (P < 0.05) off-flavor development than all other treatments. These data support previous reports of CaCl marination causing undesirable off-flavor characteristics. Morgan et al. (1991) reported increased metallic, bitter, and livery flavors; Wheeler et al. (1993) reported increased sour and bitter flavors; Morris, Theis, Miller, Acuff, and Savell (1997) reported increased livery, soured, and oniony flavors. Moreover, 0.3 M treatments
resulted in higher (P < 0.05) off-flavor intensity scores than 0.1 or 0.2 M treatments. Differences in connective tissue amount were detected among the treatments, with the Control treatment having lower scores (more connective tissue) than the calcium marination and Dwater treatments. Because longissimus muscle was used and all steaks were cooked similarly, differences in connective tissue are likely an artifact of variation in myofibrillar tenderness rather than real differences in connective tissue quality or quantity.
4. Conclusions Our study investigated the effects of calcium ascorbate, calcium chloride, and calcium lactate at three molar concentrations on color, microbiological, lipid oxidation, tenderness, and palatability traits of beef longissimus muscle. Our findings suggest that an increase in tenderness and palatability traits concurrent
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with acceptable display color life and microbial inhibition will result from injection marination of a 0.1 M solution of calcium lactate. Conversely, due to unacceptable color deterioration during retail display and the incidence of off-flavors, we do not recommend the use of calcium ascorbate or calcium chloride solutions or the injection (11% by weight) of a solution with a concentration greater than 0.1 M to improve the quality traits of beef longissimus. References AMSA. (1995). Research guidelines for cookery, sensory evaluation and instrumental tenderness measurements of fresh meat. Chicago, IL: American Meat Science Association and National Live Stock and Meat Board. (now the National Cattlemen’s Beef Assoc.). Benito-Delgado, J., Marriott, N. G., Claus, J. R., Wang, H., & Graham, P. P. (1994). Chuck longissimus and infraspinatus muscle characteristics as affected by rigor state, blade tenderization and calcium chloride injection. Journal of Food Science, 59, 295–299. Chen, N., & Shelef, L. A. (1992). Relationship between water activity, salts of lactic acid, and growth of Listeria monocytogenes in a meat model system. Journal of Food Protection, 55, 574–578. Got, F., Rousset-Akrim, S., Bayle, M. C., & Culioli, J. (1996). Effects of injection of calcium as a lactate salt on tenderness and flavour of beef. International Congress of Meat Science and Technology, 42, 394–395. Harbers, C. A. Z., Harrison, D. L., & Kropf, D. H. (1981). Ascorbic acid effects on bovine muscle pigments in the presence of radiant energy. Journal of Food Science, 46, 7–12. Harris, S. E., Huff-Lonergan, E., Lonergan, S. M., Jones, W. R., & Rankins, D. (2001). Antioxidant status affects color stability and tenderness of calcium chloride-injected beef. Journal of Animal Science, 79, 666–677. Kanner, J., & Mendel, H. (1977). Prooxidant and antioxidant effects of ascorbic acid and metal salts in a b-carotene-linoleate model system. Journal of Food Science, 42, 60–64. Kerth, C. R., Miller, M. F., & Ramsey, C. B. (1995). Improvement of beef tenderness and quality traits with calcium chloride injection in beef loins 48 hours postmortem. Journal of Animal Science, 73, 750–756. Koohmaraie, M., Crouse, J. D., & Mersmann, H. J. (1989). Acceleration of postmortem tenderization in ovine carcasses through infusion of calcium chloride: Effect of concentration and ionic strength. Journal of Animal Science, 67, 934–942. Lawrence, T. E., King, D. A., Obuz, E., Yancey, E. J., & Dikeman, M. E. (2001). Evaluation of electric belt grill, forced-air convection oven, and electric broiler cookery methods for beef tenderness research. Meat Science, 58, 239–246. Little, A. C. (1975). Off on a tangent. Journal of Food Science, 40, 410– 411. Liu, H. P., & Watts, B. M. (1970). Catalysis of lipid peroxidation in meats. 3. Catalysts of oxidative rancidity in meats. Journal of Food Science, 35, 596–598.
Mahoney, J. R. Jr, & Graf, E. (1986). Role of alpha-tocopherol, ascorbic acid, citric acid and EDTA as oxidants in model systems. Journal of Food Science, 51, 1293–1296. Mitsumoto, M., Faustman, C., Cassens, R. G., Arnold, R. N., Schaefer, D. M., & Scheller, K. K. (1991). Vitamins E and C improve pigment and lipid stability in ground beef. Journal of Food Science, 56, 194–197. Morgan, J. B., Miller, R. K., Mendez, F. M., Hale, D. S., & Savell, J. W. (1991). Using calcium chloride injection to improve tenderness of beef from mature cows. Journal of Animal Science, 69, 4469– 4476. Morris, C. A., Theis, R. L., Miller, R. K., Acuff, G. R., & Savell, J. W. (1997). Improving the flavor of calcium chloride and lactic acid injected mature beef top round steaks. Meat Science, 45, 531–537. NAMP. (1997). The meat buyers guide. Reston, VA: North American Meat Processors Association. Okayama, T., Imai, T., & Yamanoue, M. (1987). Effect of ascorbic acid and alpha-tocopherol on storage stability of beef steaks. Meat Science, 21, 267–273. Polidori, P., Marinucci, M. T., Fantuz, F., Renieri, C., & Polidori, F. (2000). Tenderization of wether lambs meat through pre-rigor infusion of calcium ions. Meat Science, 55, 197–200. Pringle, T. D., Harrelson, J. M., West, R. L., Williams, S. E., & Johnson, D. D. (1999). Calcium-activated tenderization of strip loin, top sirloin, and top round steaks in diverse genotypes of cattle. Journal of Animal Science, 77, 3230–3237. SAS. (1999). The SAS system for Windows. Release 8.01. Cary, NC. Sato, K., & Hegarty, G. R. (1971). Warmed-over flavor in cooked meats. Journal of Food Science, 36, 1098–1102. Shivas, S. D., Kropf, D. H., Hunt, M. C., Kastner, C. L., Kendall, J. L. A., & Dayton, A. D. (1984). Effects of ascorbic acid on display life of ground beef. Journal of Food Protection, 47, 11–15. St. Angelo, A. J., Koohmaraie, M., Crippen, K. L., & Crouse, J. (1991). Acceleration of tenderization/inhibition of warmed-over flavor by calcium chloride-antioxidant infusion into lamb carcasses. Journal of Food Science, 56, 359–362. Wheeler, T. L., Koohmaraie, M., & Crouse, J. D. (1991). Effects of calcium chloride injection and hot boning on the tenderness of round muscles. Journal of Animal Science, 69, 4871–4875. Wheeler, T. L., Koohmaraie, M., Lansdell, J. L., Siragusa, G. R., & Miller, M. F. (1993). Effects of postmortem injection time, injection level, and concentration of calcium chloride on beef quality traits. Journal of Animal Science, 71, 2965–2974. Wheeler, T. L., Koohmaraie, M., & Shackelford, S. D. (1996). Effect of vitamin C concentration and co-injection with calcium chloride on beef retail display color. Journal of Animal Science, 74, 1846– 1853. Wheeler, T. L., Koohmaraie, M., & Shackelford, S. D. (1997). Effect of postmortem injection time and postinjection aging time on the calcium-activated tenderization process in beef. Journal of Animal Science, 75, 2652–2660. 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.
Effects of calcium salts on beef longissimus quality§ T.E. Lawrencea, M.E. Dikemana,*, M.C. Hunta, C.L. Kastnera, D.E. Johnsonb a
Department of Animal Sciences & Industry, Kansas State University, Manhattan, KS 66506-0201, USA b Department of Statistics, Kansas State University, Manhattan, KS 66506-0802, USA Received 6 May 2002; received in revised form 22 July 2002; accepted 22 July 2002
Abstract The objective of this research was to evaluate the effects of injection marination with calcium salts on beef longissimus quality traits. Strip loins were injected (11% by weight) with distilled water or a 0.1, 0.2, or 0.3 M solution of calcium ascorbate, calcium chloride, or calcium lactate. Non-injected loins served as controls. Visual and instrumental color evaluations indicated that calcium ascorbate accelerated myoglobin oxidation, and increasing molar concentration of any calcium salt caused faster (P< 0.05) discoloration. Aerobic microbial plate counts were lower (P< 0.05) for treatments containing calcium lactate than those with calcium chloride or calcium ascorbate. Calcium ascorbate inhibited lipid oxidation whereas calcium lactate and calcium chloride appeared to be pro-oxidants of lipid oxidation. No differences for Warner-Bratzler shear force or sensory panel tenderness were found among the calcium salts; however, 0.3 M treatments had lower shear values and were judged more tender than 0.1 M treatments. Calcium ascorbate and calcium chloride treatments resulted in less (P< 0.05) beef flavor and more (P< 0.05) off-flavors than calcium lactate treatments. In addition, 0.1 M treatments had higher (P< 0.05) beef flavor scores while 0.3 M treatments had higher (P <0.05) offflavor scores. Considering the effects on color life, microbial inhibition, shear force, and sensory traits, we recommend injecting beef longissimus with a 0.1 M solution of calcium lactate to enhance both uncooked and cooked quality. # 2003 Elsevier Science Ltd. All rights reserved. Keywords: Beef; Calcium; Ascorbate; Chloride; Lactate; Tenderness; Color
1. Introduction Previous research has shown that injecting beef and lamb with calcium chloride improves tenderness (Koohmaraie, Crouse, & Mersmann, 1989; Wheeler, Koohmaraie, & Crouse, 1991; Kerth, Miller, & Ramsey, 1995). However, some research has shown that calcium chloride induces bitter, metallic, and livery off-flavors (Morgan, Miller, Mendez, Hale, & Savell, 1991; Wheeler, Koohmaraie, Lansdell, Siragusa, & Miller, 1993; Wheeler, Koohmaraie, & Shackelford, 1997). In addition, calcium chloride injection has induced muscle darkening and increased aerobic plate counts (Wheeler et al., 1993), and caused faster discoloration (Kerth et al., 1995) than untreated controls. Ideally, calcium marination technology would improve other economically §
Contribution No. 02-422-J from the Kansas Agricultural Experiment Station. * Corresponding author. Tel.: +1-785-532-1225; fax: +1-785-5327059. E-mail address: [email protected] (M.E. Dikeman).
important traits in addition to tenderness, such as lengthening color life, or inhibiting microbial growth. Previous calcium marination research was conducted nearly exclusively using calcium ions from the dissociation of calcium chloride. No research has been published on the use of calcium ascorbate in marination to improve tenderness, and only one report (Got, Rousset-Akrim, Bayle, & Culioli, 1996) was found on the use of calcium lactate. Therefore, the objective of this research was to compare the effects of calcium ascorbate, calcium chloride, and calcium lactate on beef color and palatability traits.
2. Materials and methods 2.1. Muscles Beef strip loins (IMPS 180, n=26, NAMP (1997)) from USDA Select carcasses were selected from the fabrication line of a commerical processor. Loins were transported at 4 C to the Kansas State University meat laboratory, trimmed of all external fat and accessory
0309-1740/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved. PII: S0309-1740(02)00201-2
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muscles, and cut transversely into three equal longissimus sections. 2.2. Injection marination Using an incomplete block design, the 78 loin sections were allocated randomly to the 11 marination treatments: (1) non-marinated control; (2) distilled water; (3) 0.1 M calcium ascorbate; (4) 0.2 M calcium ascorbate; (5) 0.3 M calcium ascorbate; (6) 0.1 M calcium chloride; (7) 0.2 M calcium chloride; (8) 0.3 M calcium chloride; (9) 0.1 M calcium lactate; (10) 0.2 M calcium lactate; or (11) 0.3 M calcium lactate. The treatments will be referred to, respectively, as Control, Dwater, 1CaAsc, 2CaAsc, 3CaAsc, 1CaCl, 2CaCl, 3CaCl, 1CaLac, 2CaLac, and 3CaLac. Also, contrast statements comparing the three molar concentrations will be referred to as 0.1 M, 0.2 M, or 0.3 M. Calcium ascorbate (Ashland Nutritional Products, La Mirada, CA), calcium lactate (PURAC America, Inc., Lincolnshire, IL), and calcium chloride (TETRA Chemicals, The Woodlands, TX) solutions were made using pre-chilled distilled water. Marinade temperature was 4 C during injection. Loin sections were injected at 11% by weight (Schro¨der N50, Werther, Germany) with their respective treatment, vacuum packaged (Multivac C500, Wolfertschwenden, Germany), and tumbled (Food Processing Equipment Company, model VT85, Sante Fe Springs, CA) for 15 min. Surface temperature (measured using an infrared thermometer, Kane-May KM800S, Hertfordshire, England) of the loin sections was 3 C before tumbling and 6 C after tumbling. After injection marination, loins were stored at 1 C until 14 days postmortem. The pH of loin sections was measured before injection and after aging 14 days with a Sentron Argus w pH meter connected to a Sentron Red-Line LanceFET probe (Sentron, Gig Harbor, WA). 2.3. Sample processing At 14 days postmortem, each loin section was weighed, and then cut into two 2.54-cm-thick steaks, one for shear force and one for sensory panel evaluation. An additional 1.91-cm thick steak was cut for simulated retail display. The remaining portion was divided into thirds and utilized for initial microbiological evaluation, initial TBARS analysis, and uncooked calcium quantification. Shear force and sensory panel steaks were vacuum packaged and frozen ( 38 C). Retail display steaks were placed on 17S white foam trays (Tenneco Packaging Specialty and Consumer Products, Lake Forest, IL) and overwrapped with heat shrinkable PVC film (MAPAC M—23,250 cc O2/m2/24 h—72 gauge, Borden Packaging and Industrial Products, Borden, Inc., North Andover, MA). Initial TBARS and uncooked calcium samples were vacuum packaged and frozen ( 80 C).
2.4. Simulated retail display Steaks were displayed for 5 days at 3 C in a retail display case (Tyler Refrigeration Corporation, model DMF8, Niles, MI) under 1614 lux of fluorescent light (Philips UltralumeTM 30U, Somerset, NJ). Trained color panelists (all panelists passed the FarnsworthMunsell 100 Hue color test) evaluated color deterioration (1=very bright cherry red, 2=bright cherry red, 3=slightly dark red to tannish red, 3.5=borderline acceptable, 4=moderately grayish tan to brown, 5=tan to brown) and percentage of surface discoloration (1=0%, 2=1–10%, 3=11–25%, 4=26–50%, 5=51– 75%, 6=76–99%, 7=100%). Instrumental data (L* a* b*) were measured at three locations on each steak using a MiniScanTM XE (Hunter Associates Laboratory, Inc., Reston, VA) with a 35-mm aperture set for Illuminant A, and a 10 observer. Instrumental color was measured through the packaging film and the instrument was calibrated with the same packaging film over the tiles. Chroma and hue angle (Little, 1975) were calculated. Steaks were evaluated instrumentally and by the visual panel at 0, 24, 48, 72, and 96 h. 2.5. Microbiological analysis Two surface cores (2.54 cm diameter, 3 mm thick) were aseptically cut from steaks, placed in filtered stomacher bags (Spiral Biotech, Norwood, MA) to which 99 ml of 0.1% peptone buffer (NutraMax Products, Inc., Gloucester, MA) was added, and stomached (Seward stomacher 400, Seward Medical, London, UK) for 2 min. Duplicate initial (at 0 h of retail display) and final (after 96 h of retail display) samples were plated at 1, 10 1, 10 2, and 10 3 on Aerobic Plate Count Petrifilm (Microbiology Products—3 M Health Care, St. Paul, MN) and incubated for 48 h at 36 C. Microbiological growth was counted and converted to log colony-forming units per cm2. 2.6. Warner-Bratzler shear force determinations Steaks were thawed at 3 C for 40 h. They were cooked on an electric belt grill (model TBG-80 Magigrill, MagiKitch’n Inc., Quakertown, PA) set at 117 C using the following settings: top platen=110 C, bottom platen=109.4 C, preheat disconnected, platen gap=1.905 cm, cook time=9.0 min. This temperature was chosen because previous research (Lawrence, King, Obuz, Yancey, & Dikeman, 2001) indicated that it provided the highest repeatability for Warner-Bratzler shear force of longissimus steaks. Steaks were cooked to an internal temperature of 70 C, cooled in a refrigerator for 24 h at 0 C (AMSA, 1995), then eight cores (1.27 cm diameter) were removed from each steak parallel to muscle fiber orientation using a mechanical
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coring device. Cores were sheared once through the center by a V-notch Warner-Bratzler shear attachment connected to an Instron Universal Testing Machine (model 4201, Instron Corp., Canton, MA) using a 50 kg compression load cell and a 250 mm/min crosshead speed. The peak force and total energy required to shear each core were recorded. Cooking time and percentage of cooking loss [((raw wt-cooked wt)/raw wt) 100] were recorded for each steak. 2.7. 2-Thiobarbituric acid reactive substances (TBARS) analysis TBARS analysis was conducted using the extraction method of Witte, Krause, and Bailey (1970). Initial (at 0 h of retail display) and final (after 96 h of retail display) samples were immersed in liquid nitrogen and pulverized. Duplicate 10-g samples were weighed into stainless steel mixers, to which 15 ml of 9% perchloric acid and 20 ml of distilled-deionized water were added. Samples were blended for 15 s, and then filtered through Whatman no.1 filter paper into test tubes. Five millilitres of TBA reagent were added to the filtrate and stored overnight before spectral absorbance was measured at 530 nm (Bausch & Lomb, Spectronic 21, Rochester, NY). A standard curve was developed using 0, 0.2, 0.4, 0.8, 1.0, 2.0, 4.0, 6.0, and 8.010 8 mol/ml of 1,1,3,3tetraethoxypropane. 2.8. Descriptive attribute sensory panel evaluation Steaks were thawed in a refrigerator at 3 C for 40 h, and cooked as previously described for Warner-Bratzler shear force determinations. An untreated longissimus steak aged 14 days postmortem was served to the panelists as a warm-up sample. Using an eight-point scale, trained panelists (AMSA, 1995) scored samples for myofibrillar and overall tenderness, juiciness, connective tissue amount, beef flavor intensity, and off-flavor intensity (1=extremely tough, dry, abundant, bland, and intense; 8=extremely tender, juicy, none, intense, and none). In addition, panelists were instructed to describe off-flavors as astringent, bitter, bloody/serumy, fatty, livery, metallic, salty, soapy, or sour, or to provide their own descriptor. 2.9. Calcium quantification Uncooked and cooked (Warner-Bratzler shear steaks) samples were immersed in liquid nitrogen and pulverized. Duplicate 2-g samples were placed in ceramic crucibles and ashed in a muffle furnace (Neytech 85A, Bloomfield, CT) at 600 C for 4 h, digested with 25 ml of 2.5 N hydrochloric acid for 1 h, then diluted to 100 ml with distilled, deionized water. One ml of diluted sample was added to 9 ml of 1% strontium chloride
301
before absorbance was measured at 422.7 nm using an atomic absorption spectrophotometer (AAnalyst 100, Perkin Elmer, Norwalk, CT). 2.10. Experimental design and statistical analysis The treatment structure was a 33 factorial (three calcium salts at three molar concentrations) with a negative (non-marinated) and a positive (distilled water) control. The design structure was an incomplete block design. Loin was the blocking factor and one-third of a loin was the experimental unit. Data were analyzed using the MIXED procedure of SAS (1999). The model included the fixed effect of treatment, and the random effect of loin. Repeated measures data (pH, color deterioration, discoloration, L*, a*, b*, chroma, hue angle, TBARS, total plate counts) were analyzed using an unstructured covariance model. Treatment means were generated using the LSMEANS option. In addition, pre-determined single degree of freedom contrasts were generated using the ESTIMATE option to test main effects of calcium salt and molar concentration.
3. Results and discussion 3.1. pH and yield The distilled water from which all solutions were made had a pH of 6.9. The CaAsc solutions were slightly acidic (formation of ascorbic acid) and slightly increased in acidity as molar concentration increased (5.9, 5.8, 5.8). The CaCl solutions were basic and increased in alkalinity (9.7, 10.0, 10.1) as molar concentration increased. The CaLac solutions were slightly alkaline (7.5, 7.4, 7.4). Contrast differences for initial muscle pH (Table 1) were not significant. After 14 days of postmortem aging, final muscle pH values generally increased slightly above pre-marination values, which was expected because the pH of the marination solutions generally was above that of the loin sections. However, the final muscle pH value for the 3CaCl treatment was lower (P < 0.05) than the pre-marination value. Because the pH of the 3CaCl solution was 10.1, the final muscle pH was expected to increase rather than decrease. Final pH values of CaAsc and CaLac treatments were higher (P < 0.05) than CaCl treatments. In addition, 0.1 M treatments had higher (P < 0.05) final pH values than 0.3 M treatments. Contrasts for pumped yield (Table 1) indicated that marination with CaAsc resulted in higher (P < 0.05) yields than CaCl, which had higher (P < 0.05) yields than CaLac. Among the individual marination treatments, 3CaAsc had the highest pumped yield and Dwater the lowest. Pumped yield increased in a linear
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Table 1 Least squares means and significant contrasts of pH and pumped yield of marinateda beef longissimus Marination treatment
Initial pHb
Final pH after agingc
Pumped yield (%)d
0.1 M CaAsc 0.2 M CaAsc 0.3 M CaAsc 0.1 M CaCl 0.2 M CaCl 0.3 M CaCl 0.1 M CaLac 0.2 M CaLac 0.3 M CaLac Control Dwater SEM
5.5 5.5 5.4 5.4 5.4 5.5 5.4 5.4 5.5 5.5 5.4 0.03
5.5 5.5 5.5 5.5 5.4 5.4 5.5 5.5 5.4 5.6 5.5 0.02
104.6 105.8 108.3 102.8 105.0 106.9 103.8 103.3 103.8 – 102.5 0.70
* Asc
* Asc * Asc * Cl
Contrasts CaAsc vs. CaCl CaAsc vs. CaLac CaCl vs. CaLac CaAsc vs. Control CaCl vs. Control CaLac vs. Control CaAsc vs. Dwater CaCl vs. Dwater CaLac vs. Dwater Control vs. Dwater 0.1 M vs. 0.2 M 0.1 M vs. 0.3 M 0.2 M vs. 0.3 M
* Lac * Con * Con * Con * Dw
*1
* Asc * Cl * Lac
*3 *3
* Contrast difference P <0.05; Asc, Cl, Lac, Con, Dw, 1, 2, or 3 indicates which treatment in the contrast has the higher value. a 0.1 M calcium ascorbate; 0.2 M calcium ascorbate; 0.3 M calcium ascorbate; 0.1 M calcium chloride; 0.2 M calcium chloride; 0.3 M calcium chloride; 0.1 M calcium lactate; 0.2 M calcium lactate; 0.3 M calcium lactate; Non-marinated control; Distilled water. b pH of loin sections immediately before injection marination. c pH of loin sections after 14-day aging period. d Pumped yield=(Pumped, aged loin wt./green loin wt.) 100.
manner as molar concentration of CaAsc or CaCl increased; however, this effect was not evident for CaLac. Clearly, contrasts indicated that pumped yield increased (P < 0.05) as molar concentration increased, apparently due to increased ionic strength of increasing molar concentrations. 3.2. Visual color evaluations Contrasts for average color scores (Table 2) indicated that steaks marinated with CaCl, CaLac, Dwater, or Control treatments were less (P < 0.05) deteriorated than those marinated with the CaAsc treatment throughout the 96 h display period. Throughout the 96 h display period, 1CaLac, 1CaCl, and Dwater treatments retained color the best. In addition, steaks marinated with 0.1 M solutions had less (P < 0.05) surface browning than those marinated with 0.2 or 0.3 M solutions, with no differences in color between 0.2 and 0.3 M solutions. Steaks marinated with CaCl, CaLac, Dwater, or Control treatments were less (P < 0.05) discolored than those marinated with the CaAsc treatment throughout
the 96 h of display. Steaks marinated with CaLac or those from the Control treatment were also less (P < 0.05) discolored than those marinated with CaCl. These data agree with those of Kerth et al. (1995) and Wheeler, Koohmaraie, and Shackelford (1996) who reported that marination with calcium chloride increased surface browning over that of non-marinated controls. In our study, 1CaLac and Control treatments had the greatest color stability during 96 h of simulated display. In addition, 0.1 M treatments had less (P < 0.05) discoloration than 0.2 or 0.3 M treatments. Kerth et al. (1995) reported that marination with 0.25 M calcium chloride caused faster discoloration than a 0.20 M solution. The increase in discoloration caused by the calcium solutions and by increased molar concentrations likely resulted from the oxidative catalyzing characteristics of the salts. However, this effect was not as pronounced for the 1CaLac or 1CaCl treatments as it was for all other calcium treatments. In summary, visual color evaluations indicated that CaAsc marination resulted in less color life than CaCl or CaLac treatments because of greater myoglobin
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Table 2 Least squares means and significant contrasts of visual and instrumental color evaluations of marinateda beef longissimus steaks during a 96 h simulated retail display Marination treatment
Visual Color b
Discoloration c
L*
a*
b*
Chroma
Hue angle
0.1 M CaAsc 0.2 M CaAsc 0.3 M CaAsc 0.1 M CaCl 0.2 M CaCl 0.3 M CaCl 0.1 M CaLac 0.2 M CaLac 0.3 M CaLac Control Dwater SEM
3.3 4.1 4.3 2.3 2.8 3.0 2.3 2.7 2.7 2.8 2.5 0.2
2.5 3.6 3.9 1.8 2.5 2.5 1.6 2.1 2.0 1.7 1.9 0.2
43.3 40.0 39.2 45.5 44.6 42.6 45.4 43.3 44.4 42.7 45.3 1.1
25.2 22.5 21.0 30.0 27.7 26.4 30.2 29.0 28.6 30.6 30.2 0.7
19.6 17.8 17.0 23.2 22.0 20.7 23.4 22.3 22.1 23.3 23.7 0.5
31.9 28.7 27.0 38.0 35.4 33.6 38.2 36.7 36.1 38.5 38.4 0.8
37.8 38.2 39.0 37.7 38.6 38.2 37.2 37.6 37.8 37.2 38.2 0.4
* Asc * Asc
* Asc * Asc * Cl * Asc * Cl
* Cl * Lac
* Cl * Lac
* * * * *
* Asc
* Asc
* Dw
* * * * * * * *
*2 *3
*2 *3
*1 *1
Contrasts CaAsc vs. CaCl CaAsc vs. CaLac CaCl vs. CaLac CaAsc vs. Control CaCl vs. Control CaLac vs. Control CaAsc vs. Dwater CaCl vs. Dwater CaLac vs. Dwater Control vs. Dwater 0.1 M vs. 0.2 M 0.1 M vs. 0.3 M 0.2 M vs. 0.3 M
* Asc
Cl Lac Lac Con Con Con Dw Dw
* Con * Con
Cl Lac Lac Con Con
* Dw * Dw * Dw
* Dw * Dw
*1 *1 *2
*1 *1 *2
* Asc * Asc * Cl
* Dw *1 *1 *2
*3
* Contrast difference P <0.05; Asc, Cl, Lac, Con, Dw, 1, 2, or 3 indicates which treatment in the contrast has the higher value. a 0.1 M calcium ascorbate; 0.2 M calcium ascorbate; 0.3 M calcium ascorbate; 0.1 M calcium chloride; 0.2 M calcium chloride; 0.3 M calcium chloride; 0.1 M calcium lactate; 0.2 M calcium lactate; 0.3 M calcium lactate; Non-marinated control; Distilled water. b 1=very bright cherry red, 2=bright cherry red, 3=slightly dark red to tannish red, 3.5=borderline acceptable, 4=moderately grayish tan to brown, 5=tan to brown. c 1=0%, 2=1–10%, 3=11–25%, 4=26–50%, 5=51–75%, 6=76–99%, 7=100%.
oxidation. These results were unexpected because we hypothesized that the ascorbate would act as a sequestrant of free electrons, thereby lessening metmyoglobin formation. Ascorbic acid has been used as an antioxidant to inhibit pigment oxidation in fresh beef systems (Harbers, Harrison, & Kropf, 1981; Mitsumoto, Faustman, Cassens, Arnold, Schaefer, & Scheller, 1991; Okayama, Imae, & Yamanoue, 1987; Shivas, Kropf, Hunt, Kastner, Kendall, & Dayton, 1984). However, it has also been a pro-oxidant in the presence of iron (Kanner & Mendel, 1977; Liu & Watts, 1970) or copper (Mahoney & Graf, 1986). The addition of increased levels of calcium ions may also induce effects similar to iron and copper. Furthermore, Sato and Hegarty (1971) investigated ascorbic acid levels from 0 to 10,000 ppm and reported the greatest amount of oxidation occurred in the 50–100 ppm range. That may provide an explanation for the drastic oxidation that occurred with all of our CaAsc treatments because the levels of 1CaAsc, 2CaAsc, and 3CaAsc injected into the longissimus were
43, 86, and 129 ppm, respectively. Marination with CaLac treatments retained red color better than other calcium treatments. The mechanism for CaLac increasing the color stability of muscle appears to be through the influx of lactate into the system. Lactate can be converted to NADH, H+, and pyruvate by lactate dehydrogenase. Increasing the supply of NADH allows for the ability to donate electrons to ferric iron (Fe+++), thereby reducing it to ferrous iron (Fe++). However, 0.2 and 0.3 M marination treatments of CaAsc and CaCl resulted in significantly more color deterioration than 0.1 M treatments. As molar concentration increases, the number of free radical electrons likely increases, thereby providing more catalysts for myoglobin oxidation. 3.3. Instrumental color evaluations Contrasts for luminescence (L*) values (Table 2) indicated that steaks marinated with CaCl, CaLac, or
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Dwater were lighter (P < 0.05) (higher L* values) than those marinated with CaAsc throughout the 96 h display period, further indicating the degree of muscle darkening caused by marination with CaAsc. However, CaCl and CaLac treatments were similar to Control and Dwater treatments. Marination with 1CaCl, 1CaLac, or Dwater resulted in the highest L* values throughout the display period. In addition, 0.1 M treatments had higher (P < 0.05) L* values than 0.2 or 0.3 M treatments. Steaks marinated with the CaCl, CaLac, Dwater or Control treatments were more red (P< 0.05) (higher a* values) than CaAsc treatments throughout display. The CaLac, Dwater, and Control treatments were more red (P < 0.05) than the CaCl treatment throughout display. Furthermore, 0.1 M treatments were more red (P < 0.05) than 0.2 M and 0.3 M treatments, and 0.2 M treatments were more red (P < 0.05) than 0.3 M treatments. Steaks marinated with CaCl, CaLac, Dwater, or Control treatments were more yellow (P < 0.05) (higher b* values) than CaAsc treatments. Furthermore, Dwater and Control treatments were more yellow (P < 0.05) than the CaCl treatment throughout display. In addition, 0.1 M treatments were more yellow (P < 0.05) than both 0.2 M treatments and 0.3 M treatments throughout display. Moreover, 0.2 M treatments were more yellow (P < 0.05) than 0.3 M treatments. Steaks marinated with CaCl, CaLac, Dwater, or Control treatments had more intense red color (higher chroma values, P < 0.05) than CaAsc treatments. The CaLac, Dwater, and Control treatments had a more intense red color (P< 0.05) than CaCl treatments. In addition, 0.1 M treatments had a more intense red color (P < 0.05) than 0.2 M or 0.3 M treatments throughout display, and the 0.2 M treatments had a more intense red color (P < 0.05) than 0.3 M treatments. Steaks marinated with CaLac or Control treatments were less brown (P < 0.05) (lower hue-angle) than those marinated with CaAsc treatments. The Control treatment was less brown (P < 0.05) than the CaCl or Dwater treatments. In addition, 0.1 M treatments were less brown (P < 0.05) than 0.3 M treatments. In summary, instrumental color determinations confirmed visual scores. CaAsc resulted in darker steaks that were less red, less yellow, and more brown than CaCl- or CaLac-marinated steaks. Marination with CaCl resulted in steaks that were less red than those marinated with CaLac. In addition, steaks darkened and became less red and less yellow as molar concentration increased from 0.1 to 0.2 to 0.3 M. 3.4. Microbiological evaluations Initial aerobic plate counts (log cfu/cm2) were not different among the 11 marination treatments (Table 3); however, counts increased (P < 0.05) for all treatments after 5 days of simulated retail display, except 2CaLac
and the non-marinated Control. The CaLac treatments inhibited microbial growth (P < 0.05) more than CaAsc or CaCl treatments. Although not evident in our data, Benito-Delgado et al. (1994) and Wheeler et al. (1993) reported that marination with CaCl increased total aerobic plate counts more than non-marinated controls. In our study, increasing molar concentrations of CaAsc or CaCl treatments did not consistently influence final counts. However, increasing concentration of CaLac resulted in numerically lower, but statistically similar values. In support of Chen and Shelef (1992), these data indicate that marination with CaLac may be useful for inhibiting growth of aerobic microflora during retail display. 3.5. TBARS analysis Initial 2-Thiobarbituric acid reactive substances values (an index of lipid oxidation, Table 3) did not differ among the 11 treatments. Final TBARS values were highest for the 3CaCl, 2CaCl, 3CaLac, and 2CaLac treatments. Contrasts of TBARS values indicated that CaAsc treatments had lower (P < 0.05) final values than CaCl, CaLac, or Dwater treatments. It appears that all CaAsc treatments inhibited lipid oxidation, whereas all CaCl and CaLac treatments enhanced lipid oxidation. CaCl and CaLac treatments caused more lipid oxidation (higher TBARS values, P < 0.05) than Dwater or Control treatments. These data are in agreement with those of St. Angelo, Koohmaraie, Crippen, and Crouse (1991) and Harris, Huff-Lonergan, Lonergan, Jones, and Rankins (2001) who reported that CaCl marination resulted in increased TBARS values. Furthermore, 0.1 M marination treatments resulted in less (P < 0.05) lipid oxidation than 0.2 or 0.3 M treatments. In contrast to the visual and instrumental color data, which indicated a myoglobin prooxidant effect for CaAsc, it appears that injection of a CaAsc solution into muscle inhibits the extent of lipid oxidation. 3.6. Calcium quantification Calcium concentration (mg/g) of loins marinated with calcium treatments was 400–1350% higher than the non-marinated Control treatment (Table 3). As expected, contrasts indicated no difference in uncooked or cooked calcium concentration among calcium salts. There also was no difference between Dwater and the non-marinated Control. Also, as expected, uncooked or cooked calcium concentration increased as molar concentration increased. Previous uncooked calcium quantification of CaCl marinated steaks (Koohmaraie et al. 1989; Morgan et al. 1991; Polidori, Marinucci, Fantuz, Renieri, & Polidori, 2000; Wheeler et al., 1993) yielded much lower values than in our experiment ( 500 vs. 1252.0 mg/g for a 10% pump of a 0.3 M solution).
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Table 3 Least squares means and significant contrasts of aerobic plate counts (APC)a, 2-thiobarbituric acid (TBARS)b evaluations, of marinatedc beef longissimus steaks initially and after 5 days of simulated retail display and of uncooked and cooked calcium concentrationd Marination treatment
Initial APC
Final APC
Initial TBARS
Final TBARS
Uncooked calcium
Cooked calcium
0.1 M CaAsc 0.2 M CaAsc 0.3 M CaAsc 0.1 M CaCl 0.2 M CaCl 0.3 M CaCl 0.1 M CaLac 0.2 M CaLac 0.3 M CaLac Control Dwater SEM
0.9 1.4 1.3 1.5 0.9 1.3 1.5 1.4 1.1 1.6 1.1 0.3
3.0 3.5 2.9 3.3 2.2 3.7 2.4 2.2 2.1 2.2 2.6 0.4
0.12 0.15 0.18 0.06 0.06 0.06 0.05 0.05 0.07 0.05 0.06 0.09
0.14 0.17 0.23 0.47 0.99 1.05 0.43 0.82 0.85 0.23 0.43 0.09
517.0 934.0 246.0 486.5 850.9 1333.4 460.2 831.8 1176.1 91.8 72.3 56.8
498.6 1025.5 1354.0 481.3 1008.6 1330.9 352.4 727.3 1475.2 77.3 68.7 70.5
* Asc * Cl * Lac * Asc * Cl * Lac
* * * * * *
*2 *3 *3
*2 *3 *3
Contrasts CaAsc vs. CaCl CaAsc vs. CaLac CaCl vs. CaLac CaAsc vs. Control CaCl vs. Control CaLac vs. Control CaAsc vs. Dwater CaCl vs. Dwater CaLac vs. Dwater Control vs. Dwater 0.1 M vs. 0.2 M 0.1 M vs. 0.3 M 0.2 M vs. 0.3 M
* Asc * Cl
* Cl * Lac
* Cl * Lac * Dw * Cl * Lac *2 *3
Asc Cl Lac Asc Cl Lac
* Contrast difference P <0.05; Asc, Cl, Lac, Con, Dw, 1, 2, or 3 indicates which treatment in the contrast has the higher value. a log CFU/cm2. b mg of malonaldehyde/kg of muscle. c 0.1 M calcium ascorbate; 0.2 M calcium ascorbate; 0.3 M calcium ascorbate; 0.1 M calcium chloride; 0.2 M calcium chloride; 0.3 M calcium chloride; 0.1 M calcium lactate; 0.2 M calcium lactate; 0.3 M calcium lactate; Non-marinated control; Distilled water. d mg calcium/g muscle tissue.
This discrepancy may be explained by analytical procedural differences. Previous experiments of this type used a water-extractable-calcium procedure whereby muscle tissue was minced, blended with water, and centrifuged, with calcium concentration being measured on the supernatant only rather than the whole muscle tissue. Therefore, previous data did not account for calcium that was remaining in the pellet. In addition, previous data did not report the calcium concentration in cooked steaks. Except for the 3CaLac treatment, which increased in calcium concentration during cooking, no differences were found between uncooked and cooked calcium measurements. This finding suggests that some calcium is lost during cooking so that the concentration after cooking remained similar to the concentration before cooking. Dietary reference intakes for calcium suggest that adult males and females should consume 1000 to 1300 mg/day. Using our data, an 85g (3 oz.) uncooked portion of non-marinated beef longissimus would contain 7.8 mg of calcium or 0.8% of the recommended daily intake for calcium. In contrast, beef longissimus mari-
nated with 0.1, 0.2, or 0.3 M calcium solutions would contain 41.5, 74.2, and 106.6 mg of calcium and account for 4.2, 7.4, and 10.7% respectively of the recommended daily intake for calcium. Because one serving of the 0.3 M calcium-injected steaks contains between 10 and 19% of the recommended daily value for calcium, they could be labeled as a ‘‘Good source’’ of calcium. In summary, these data suggest that calcium-marinated steaks could be marketed as a calcium-enhanced product to further improve salability and consumer acceptance. 3.7. Warner-Bratzler shear and cooking trait evaluations Contrasts of Warner-Bratzler shear values (Table 4) indicated that CaAsc, CaCl, and CaLac treatments were equal in their tenderizing effect and had lower (P < 0.05) peak force and total energy values than Dwater or Control treatments. Only 4.2% of calcium-marinated steaks were tough (shear values 54.5 kg) whereas 37.5% of Control steaks were classified as tough. Increasing molar concentration of CaAsc, CaCl, or CaLac resulted in numerically lower peak force values
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Table 4 Least squares means and significant contrasts of Warner-Bratzler shear values and cooking traits of marinateda beef longissimus steaks Marination treatment
Purge loss (%)
Peak force (kg)
Total energyb
Cooking loss (%)
Cooking time (min)
0.1 M CaAsc 0.2 M CaAsc 0.3 M CaAsc 0.1 M CaCl 0.2 M CaCl 0.3 M CaCl 0.1 M CaLac 0.2 M CaLac 0.3 M CaLac Control Dwater SEM
5.8 5.8 5.2 5.7 5.2 5.2 5.2 6.1 5.8 3.8 5.5 0.4
2.97 2.84 2.46 3.07 2.87 2.64 3.17 3.11 2.91 4.29 3.87 0.26
34.66 32.49 30.39 33.68 32.65 30.83 35.48 36.59 33.34 45.31 40.87 2.6
19.5 18.8 18.7 19.9 21.5 19.4 20.7 20.5 19.7 18.4 24.3 1.5
6.9 6.8 7.0 6.4 7.1 6.8 6.7 6.7 6.8 7.1 6.9 0.2
* Asc * Cl * Lac
* Con * Con * Con * Dw * Dw * Dw
* Con * Con * Con * Dw * Dw * Dw
Contrasts CaAsc vs. CaCl CaAsc vs. CaLac CaCl vs. CaLac CaAsc vs. Control CaCl vs. Control CaLac vs. Control CaAsc vs. Dwater CaCl vs. Dwater CaLac vs. Dwater Control vs. Dwater 0.1 M vs. 0.2 M 0.1 M vs. 0.3 M 0.2 M vs. 0.3 M
* Dw
* * * *
Dw Dw Dw Dw
*1
* Contrast difference P <0.05; Asc, Cl, Lac, Con, Dw, 1, 2, or 3 indicates which treatment in the contrast has the higher value. a 0.1 M calcium ascorbate; 0.2 M calcium ascorbate; 0.3 M calcium ascorbate; 0.1 M calcium chloride; 0.2 M calcium chloride; 0.3 M calcium chloride; 0.1 M calcium lactate; 0.2 M calcium lactate; 0.3 M calcium lactate; Non-marinated control; Distilled water. b Total energy=force (kg) distance traveled (mm).
and, overall, 0.3 M treatments had lower (P < 0.05) peak force values than 0.1 M treatments. Our data agree with numerous previous studies that reported a reduction in shear force values following marination in CaCl (Kerth et al., 1995; Morgan et al., 1991; Pringle, Harrelson, West, Williams, & Johnson, 1999; Wheeler et al., 1993, 1997). However, our data are unique in that they demonstrate the ability of calcium salts other than CaCl to induce a calcium tenderizing effect. Purge losses (Table 4) were higher (P< 0.05) for CaAsc, CaCl, CaLac, or Dwater treatments than for the non-marinated Control treatment. Cooking times were not different among the 11 treatments, which is in agreement with data presented by Morgan et al. (1991). In contrast, Wheeler et al. (1997) reported that nonmarinated control steaks cooked significantly faster than those marinated with CaCl. Contrasts for percentages of cooking loss indicated that Dwater marination resulted in higher (P< 0.05) cooking losses than Control, CaAsc, CaLac, or CaCl treatments. The depressed water retention of the Dwater treatment was likely due to a lower ionic strength than all of the calcium marination treatments. The addition of a salt (e.g. a calcium salt) to
a solution increases ionic strength, thereby increasing the number of hydrophilic protein interactions, which causes an increase in the binding of free water. During cooking of Warner-Bratzler shear force steaks, a dull green surface appeared on the cooked steaks marinated with CaAsc. This cooked surface discoloration is likely due to formation of cholemyoglobin (H2O2 bound to heme). L-Ascorbate has functional groups that could likely be donated to the formation of H2O2 during the heating process. This coloration would likely be objectionable to most consumers. 3.8. Descriptive attribute sensory panel evaluations Myofibrillar and overall tenderness values of all calcium marination treatments were rated higher (P < 0.05) than Dwater or Control treatments (Table 5). All three calcium salts equally increased sensory-tenderness scores; however, the 0.3 M treatments were judged more tender (P < 0.05) than the 0.1 or 0.2 M treatments. Other reports of increased sensory-panel tenderness due to CaCl marination include Kerth et al. (1995), Morgan et al. (1991), and Wheeler et al. (1993, 1997).
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Marination treatment Myofibrillar tendernessb Juicinessc Beef flavor intensityd Connective tissue amounte Overall tendernessb Off-flavor intensityf 0.1 M CaAsc 0.2 M CaAsc 0.3 M CaAsc 0.1 M CaCl 0.2 M CaCl 0.3 M CaCl 0.1 M CaLac 0.2 M CaLac 0.3 M CaLac Control Dwater SEM Contrasts CaAsc vs. CaCl CaAsc vs. CaLac CaCl vs. CaLac CaAsc vs. Control CaCl vs. Control CaLac vs. Control CaAsc vs. Dwater CaCl vs. Dwater CaLac vs. Dwater Control vs. Dwater 0.1 M vs. 0.2 M 0.1 M vs. 0.3 M 0.2 M vs. 0.3 M
5.8 6.0 6.5 5.7 5.9 6.4 5.9 6.0 6.4 4.7 5.0 0.2
4.8 5.3 5.3 5.1 5.0 5.1 5.2 5.3 5.2 5.1 4.9 0.2
5.0 4.9 4.8 5.1 4.7 4.5 5.2 5.2 5.0 5.0 4.9 0.1
7.0 7.0 7.4 7.0 7.1 7.4 7.1 7.2 7.2 6.3 6.8 0.2
6.1 6.2 6.7 6.1 6.2 6.7 6.2 6.3 6.6 5.0 5.3 0.2
* Lac * Lac * * * * * *
Asc Cl Lac Asc Cl Lac
* Asc * Cl * Lac
* * * * * *
Asc Cl Lac Asc Cl Lac
7.2 6.9 5.8 7.0 6.1 4.9 6.9 7.4 7.4 7.4 7.4 0.3
* Asc * Lac * Lac * Con * Con * Dw * Dw
* Dw *3 *3
*1
*3 *3
*3 *3
*1 *2
* Contrast difference P <0.05; Asc, Cl, Lac, Con, Dw, 1, 2, or 3 indicates which treatment in the contrast has the higher value. a 0.1 M calcium ascorbate; 0.2 M calcium ascorbate; 0.3 M calcium ascorbate; 0.1 M calcium chloride; 0.2 M calcium chloride; 0.3 M calcium chloride; 0.1 M calcium lactate; 0.2 M calcium lactate; 0.3 M calcium lactate; Non-marinated control; Distilled water. b 4=slightly tough, 5=slightly tender, 6=moderately tender, 7=very tender. c 4=slightly dry, 5=slightly juicy. d 4=slightly bland, 5=slightly intense. e 6=traces, 7=practically none. f 4=slightly intense, 5=slight, 6=traces, 7=practically none.
No differences in juiciness scores were detected among the 11 treatments. Intensity of beef flavor was judged lowest for the 3CaCl, 2CaCl, and 3CaAsc treatments and highest for the CaLac, Control, and 1CaCl treatments. Contrasts indicated that beef-flavor intensity was higher (P < 0.05) for CaLac than for CaAsc or CaCl treatments and was higher (P < 0.05) for 0.1 M treatments than for 0.3 M treatments. Off-flavor scores indicated that 0.3 M CaCl induced the most intense offflavors followed by 0.3 M CaAsc and 0.2 M CaCl. Overall, CaCl treatments (characterized by panelists as bitter, metallic, sour, soapy, and astringent) had more (P < 0.05) off-flavor development than all other treatments. These data support previous reports of CaCl marination causing undesirable off-flavor characteristics. Morgan et al. (1991) reported increased metallic, bitter, and livery flavors; Wheeler et al. (1993) reported increased sour and bitter flavors; Morris, Theis, Miller, Acuff, and Savell (1997) reported increased livery, soured, and oniony flavors. Moreover, 0.3 M treatments
resulted in higher (P < 0.05) off-flavor intensity scores than 0.1 or 0.2 M treatments. Differences in connective tissue amount were detected among the treatments, with the Control treatment having lower scores (more connective tissue) than the calcium marination and Dwater treatments. Because longissimus muscle was used and all steaks were cooked similarly, differences in connective tissue are likely an artifact of variation in myofibrillar tenderness rather than real differences in connective tissue quality or quantity.
4. Conclusions Our study investigated the effects of calcium ascorbate, calcium chloride, and calcium lactate at three molar concentrations on color, microbiological, lipid oxidation, tenderness, and palatability traits of beef longissimus muscle. Our findings suggest that an increase in tenderness and palatability traits concurrent
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with acceptable display color life and microbial inhibition will result from injection marination of a 0.1 M solution of calcium lactate. Conversely, due to unacceptable color deterioration during retail display and the incidence of off-flavors, we do not recommend the use of calcium ascorbate or calcium chloride solutions or the injection (11% by weight) of a solution with a concentration greater than 0.1 M to improve the quality traits of beef longissimus. References AMSA. (1995). Research guidelines for cookery, sensory evaluation and instrumental tenderness measurements of fresh meat. Chicago, IL: American Meat Science Association and National Live Stock and Meat Board. (now the National Cattlemen’s Beef Assoc.). Benito-Delgado, J., Marriott, N. G., Claus, J. R., Wang, H., & Graham, P. P. (1994). Chuck longissimus and infraspinatus muscle characteristics as affected by rigor state, blade tenderization and calcium chloride injection. Journal of Food Science, 59, 295–299. Chen, N., & Shelef, L. A. (1992). Relationship between water activity, salts of lactic acid, and growth of Listeria monocytogenes in a meat model system. Journal of Food Protection, 55, 574–578. Got, F., Rousset-Akrim, S., Bayle, M. C., & Culioli, J. (1996). Effects of injection of calcium as a lactate salt on tenderness and flavour of beef. International Congress of Meat Science and Technology, 42, 394–395. Harbers, C. A. Z., Harrison, D. L., & Kropf, D. H. (1981). Ascorbic acid effects on bovine muscle pigments in the presence of radiant energy. Journal of Food Science, 46, 7–12. Harris, S. E., Huff-Lonergan, E., Lonergan, S. M., Jones, W. R., & Rankins, D. (2001). Antioxidant status affects color stability and tenderness of calcium chloride-injected beef. Journal of Animal Science, 79, 666–677. Kanner, J., & Mendel, H. (1977). Prooxidant and antioxidant effects of ascorbic acid and metal salts in a b-carotene-linoleate model system. Journal of Food Science, 42, 60–64. Kerth, C. R., Miller, M. F., & Ramsey, C. B. (1995). Improvement of beef tenderness and quality traits with calcium chloride injection in beef loins 48 hours postmortem. Journal of Animal Science, 73, 750–756. Koohmaraie, M., Crouse, J. D., & Mersmann, H. J. (1989). Acceleration of postmortem tenderization in ovine carcasses through infusion of calcium chloride: Effect of concentration and ionic strength. Journal of Animal Science, 67, 934–942. Lawrence, T. E., King, D. A., Obuz, E., Yancey, E. J., & Dikeman, M. E. (2001). Evaluation of electric belt grill, forced-air convection oven, and electric broiler cookery methods for beef tenderness research. Meat Science, 58, 239–246. Little, A. C. (1975). Off on a tangent. Journal of Food Science, 40, 410– 411. Liu, H. P., & Watts, B. M. (1970). Catalysis of lipid peroxidation in meats. 3. Catalysts of oxidative rancidity in meats. Journal of Food Science, 35, 596–598.
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