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Effect of calcium chloride marination on bovine Cutaneus trunci muscle
Meat Science 57 (2001) 251±256
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Eect of calcium chloride marination on bovine Cutaneus trunci muscle Claudia B. Gonzalez *, Valeria A. Salitto, Fernando J. Carduza, Adriana A. Pazos, Jorge A. Lasta Instituto TecnologõÂa de Alimentos, Centro de Agroindustrias, CNIA, Instituto Nacional de TecnologõÂa Agropecuaria (INTA), Casilla de Correo 77, (1708), MoroÂn, Buenos Aires, Argentina Received 27 March 2000; received in revised form 31 July 2000; accepted 31 July 2000
Abstract The aim of this investigation was to determine the possibility of using calcium chloride solution in tough muscles (Cutaneus trunci) to reduce the aging period required to increase tenderness, without introducing undesirable ¯avors. Muscles were marinated in 0.25 M CaCl2 solution for 2 h and after that aged for 0, 1, 2, 3, 4, 5 and 7 days. Tenderness was evaluated by the myo®brillar fragmentation index (MFI), Warner Bratzler shear force (WBS) and sensory panel evaluation. MFI values showed signi®cant differences between treated and control samples aged for 1, 2 and 3 days (P<0.05). MFI values of treated samples aged 3 days were similar to those obtained for the control samples but aged seven days. WBS values were not signi®cantly dierent between samples. Consumer panelists preferred treated samples aged 3 days to the control ones aged 7 days. It was concluded that calcium chloride treatment can be used in Cutaneus trunci muscle to reduce the aging time required to increase tenderness. # 2000 Elsevier Science Ltd. All rights reserved. Keywords: Calcium chloride; Tough muscle; Tenderness; Cutaneus trunci; Ageing
1. Introduction Consumers consider meat tenderness as the most important palatability trait of meat quality (Cross, Savell & Francis, 1986) and its variability is an area of major concern to the meat industry (Koohmaraie, Wheeler & Shackelford, 1998; Morgan et al., 1991; Smith et al., 1992). Over the years, many studies have been carried out in relation to the myo®brillar protein changes during post-mortem storage and their causes, and the relationship between these alterations and meat tenderness (Olson, Parrish, Dayton & Goll, 1977; Penny & Drans®eld, 1979). Dierent factors have been reported to aect tenderness of aged beef, such as animal age, percentage of Bos indicus inheritance, carcass pH and temperature, muscular calpain activity, sarcomere length, amount and type of collagen, muscular ®bre type * Corresponding author at present address: Tinogasta 3101, 7 A, CP1417, Capital Federal, Buenos Aires, Argentina. Fax: +54-114621-2012. E-mail addresses: [email protected] or [email protected]. gov.ar (C.B. Gonzalez)
and size (Gonzalez, Gallinger, Pazos & Lasta, 1997; Hostetler, Link, Landmann & Fitzhugh, 1972; Koohmaraie et al., 1998). With regard to these factors, Drans®eld and Jones (1981), found that the intensity and the rate of these modi®cations are not only speciesdependent but also, within a given species, muscledependent (Ouali, 1981; Penny & Drans®eld, 1979). On the other hand, Savell and Shackelford (1992) found a direct relationship between muscle tenderness and market price. Even more, Koohmaraie et al. (1998) and Drans®eld (1997) reported that consumers preferred to pay a premium for high quality products. Consequently, it is essential to improve and develop dierent postmortem processes to increase meat tenderness, particularly in those muscles that are traditionally tough and that are usually punished with lower prices. One of these postmortem methodologies is the use of calcium chloride solution. The calcium ion activates natural endogenous calpain proteinases: m-calpain, mcalpain and calpastatin Ð a speci®c inhibitor of both calpains Ð which are responsible for the breakdown of myo®brillar proteins (Koohmaraie et al., 1998). The endogenous calcium concentration present immediately
0309-1740/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S0309-1740(00)00099-1
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C. B. Gonzalez et al. / Meat Science 57 (2001) 251±256
postmortem is enough to activate m-calpain but not mcalpain. Then, when the calcium concentration increases, due to a gradually released of calcium ions from the sarcoplasmic reticulum and mitochondria, m-calpain is activated and produces a further tenderization (Drans®eld, 1994). A similar mechanism can be used to explain the calcium chloride postmortem tenderization process applied within 36 h after slaughter. In this procedure the meat is treated with higher concentration of calcium ion than the endogenous one in order to accelerate the tenderization process (Drans®eld, 1994). However, some of the sensory properties, such as color and ¯avor, can be altered by this treatment depending on the calcium concentration (Lansdell, Miller, Wheeler, Koohmaraie & Ramsey, 1995; Wheeler, Koohmaraie, Lansdell, Siragusa & Miller, 1993). There is a very good understanding of the biological basis that determines the tenderness in most of the muscles, particularly in the case of Longissimus dorsi, Semitendinosous, Biceps femoris, etc. (Koohmaraie, 1996). It was also demonstrated that the use of calcium salts improves meat tenderness in those muscles, however, there is no much information about the eciency of this treatment on tough muscles. It is known that Cutaneus trunci muscle is composed mainly of the fast twitch white ®ber type (70%) (Egelandsdal, Martinsen & Autio, 1995). This muscle type has the characteristic of exhibiting a low calpain content, however this muscle responds to aging treatment at faster rate than red muscles (Ouali, 1990). Considering that the lack of tenderness is a common complaint in the meat industry, the Cutaneus trunci muscle was selected in this study as a pattern of tough muscle. The objective was to establish the optimal conditions required to improve meat tenderization in this tough muscle by using calcium chloride solution and consequently to reduce the time required for the postmortem tenderization, which will result in a cost bene®t for the meat industry. 2. Materials and methods 2.1. Experimental design A three steps study was conducted to examine the eect of calcium chloride solution in Cutaneus trunci muscle tenderness. Step I consisted in a preliminary assay designed to determine the optimal conditions for the calcium salt tenderization process. In step II, the Cutaneus trunci muscle tenderness was measured by using two dierent methodologies: chemical measurement of Mio®brillar Fragmentation Index (MFI) and Warner Bratzler (Instron) shear force determination. Finally in step III a consumer panel was carried out to
evaluate if the level of tenderness achieved with the calcium salt treatment was enough to make Cutaneus trunci muscle acceptable to consumers. 2.1.1. Step I: preliminary assay Eight Cutaneus trunci muscles from four steers (36±42 months of age) were used in the preliminary assay. Steers were slaughtered under regular commercial procedures with Good Manufacture Practice. Muscles were removed 24 h postmortem and submitted to the laboratory assays. For the incorporation of calcium chloride solution the marination procedure was selected because the anatomic characteristics of the Cutaneus trunci muscle (thin muscle) made it dicult to apply the injection process. The marination-time was set up as the necessary time to incorporate 10% (w/w) of the calcium chloride solution. The selection of the maximum concentration of calcium chloride solution that does not promote o-¯avors (metallic ¯avor) was made by an eight-member trained sensory panel. For this purpose, Cutaneus trunci muscles were marinated with 0.20, 0.25 and 0.30 M calcium chloride solution. Samples were wrapped in aluminum foil and cooked in an electric oven at 180 C during a ®xed time depending on their weight (40 min/kg of muscle) and then submitted to the sensory panel evaluation. 2.1.2. Step II: tenderization procedure Cutaneus trunci muscles from 42 steers (36±42 months of age) were removed 24 h postmortem; right and left sides were assigned randomly to the marination procedure (10% w/w of a 0.25 M CaCl2 solution for 2 h) or were kept as control. After the marination process, all the muscles were vacuum packaged and aged at 11 C for 0, 1, 2, 3, 4, 5 and 7 days. After each aging period samples from both muscles (treated and control samples) were taken from the refrigerator to perform the tenderness evaluation. 2.1.2.1. Myofibril fragmentation index (MFI). For the evaluation of tenderness MFI was chosen as a biochemical assay. This determination was considered as a good predictor of beef tenderness because calciumdependent enzymes produce the myo®brillar proteins degradation. Myo®brils were extracted from the muscle following the procedure described by Olson, Parrish Jr. and Stromer (1976) and the protein concentration was determined by the Biuret method of Gornall, Bardawill and David (1949). MFI was carried out on three replicate experiments per quadruplicate and was applied to the dierent periods of storage (0, 1, 2, 3, 5 and 7 days) in both, control and treated samples. 2.1.2.2. Warner Bratzler (Instron). The objective evaluation of tenderness was performed applying the modi®ed
C. B. Gonzalez et al. / Meat Science 57 (2001) 251±256
Warner Bratzler shear force procedure (Bouton & Harris, 1978) by using the Instron Universal Testing Machine. Since the anatomical characteristics of Cutaneus trunci muscle made impossible to obtain the typical cores for the Warner Bratzler procedure, it was necessary to cut it in cubes and use the planar shear instead of the triangular one. Muscle samples were wrapped in aluminum foil and cooked in an electric oven at 180 C for a ®xed time depending on their weight (40 min/kg of muscle). Sample pieces (10 replicates) of 0.6 cm thick1.5 cm wide3 cm long were cut from the cooked muscles and kept at 8 C for approximately one hour until assaying. 2.1.3. Step III: sensory evaluation-consumer panel To determine if the improvement of meat tenderness could be detected by consumers, a panel session was carried out. It was composed of 120 consumers with ages between 20 and 60 years (58% female, 42% male). Muscle samples, control (7 days-aged) and treated (calcium-treated and 3 days-aged), from 44 animals (36±45 months of age) were prepared as described above (Step II). After cooking, samples were cut in 2 cm3 cubes and served in closed recipients with a tomato sauce added just before degustation. A paired comparison assay (IRAM Instituto Argentino de NormalizacioÂn, Norm 20002, 1985) was utilized between control and treated samples. In this assay, consumers were asked to choose between both samples giving the reasons for their choice. 2.2. Statistical analysis MFI and Warner Bratzler data were analyzed for signi®cant dierences by analysis of variance using the GLM procedure of the Statistical Analysis Systems Institute (SAS, 1996). When signi®cant (P<0.05) main eects were observed without interactions, mean separation was accomplished by the use of Tukey's means comparison test. The paired comparison data in the preference test were analyzed using expanded tables of the accumulative binomial probability distribution (two-tailed test) (Roessler, Pangborn, Sidel & Stone, 1978) following the methodology included in the IRAM norm cited previously. 3. Results and discussion 3.1. Step I: preliminary assay The time selected for the marination procedure was 2 h since the incorporation of the 10% (w/w) of the salt was already completed. There were not changes on quality characteristics.
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The results of the sensory trained panel for samples marinated with the three calcium concentrations did not show signi®cant dierences (data not shown), however some panelists found a ¯avor described as salty in samples treated with 0.3 M calcium solution. Despite this, the ¯avor was not described as unpleasant, the 0.25 M solution was chosen because it maintained more accurately the ¯avor characteristics of control samples. 3.2. Step II: tenderization procedure 3.2.1. Myo®bril fragmentation index (MFI) A comparison of MFI values at each aging day between control and treated samples (Table 1, control vs. treated), indicates that there is a signi®cant increase in MFI values (P<0.05) in treated samples on days 1, 2 and 3 of aging. There are not signi®cant dierences between control and treated samples for the rest of the aging days. Most of the studies related to the action of calcium chloride solution on beef tenderness have utilized sensory and objective evaluations to score tenderness (BenitoDelgado, Marriott, Claus, Wang & Graham, 1994; Morgan et al., 1991; Wheeler, Koohmaraie & Shackelford, 1997), however, in this study not only Warner± Bratzler shear force values and sensory panel evaluation but also MFI values were used as tenderness predictors. The reason for the selection of this methodology was that many authors (Culler, Parrish Jr., Smith & Cross, 1978; Davey & Gilbert, 1969; Olson et al., 1976; Parrish, Jr., Vandell & Culler, 1979) have demonstrated the correlation between MFI values and tenderness, providing a potential tool for identifying tough and tender muscles. Consequently, the higher levels of myo®bril fragmentation found in samples treated with calcium could be considered as a result of the action of endogenous proteolytic enzymes that are activated at an accelerated rate reducing the time necessary for the post mortem aging (Cottin, Poussard, Desmazes, Georgescauld & Ducastaing, 1991; PeÂrez, Escalona & Guerrero, 1998). Our results demonstrated that calcium chloride treatment produced higher tenderness level on day 1, 2 and 3 of the aging period than the tenderness achieved by the conditioning process alone. It means that the combination of aging and marination is more eective in increasing MFI values in this type of muscle than aging alone, at least for the ®rst stage of the aging period when the tenderness achieved is not the highest. The data in Table 1 (MFI) show that, in control samples the aging process produced a signi®cant increase (P<0.05) of MFI values on days 3 and 4 of the aging period. A second signi®cant increase (P<0.05) occurred on the last two days (days 5 and 7). In the case of marinated samples (treated samples) the increase in MFI values was noticed on the 2nd day of aging (P<0.05), after that a gradual increase occurred during
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Table 1 Myo®brillar fragmentation index (MFI) values for control and treated samples MFI valuesa
Aging days
0 1 2 3 4 5 7 CV2g a b c d e f g
Control vs. treated
Controlb
Treatedb
Signi®cant dierences
CV1f
74.4a 66.2a 80.6a 103.2b 99.9b 127.7c 124.1c 19.5
74.4a 71.3a 101.8b 117.9bc 110.2bc 131.5c 132.5c 19.6
nse 0.05 0.05 0.05 ns ns ns
18.1 8.3 12.8 7.5 30.6 24.2 13.8
3.2.2. Warner Bratzler (Instron) Table 2 shows the shear force values obtained with the Warner Bratzler machine (Instron), these values represent an average of 10 samples from each of the 42 muscles. Shear force values between control and treated Table 2 Warner Bratzler shear force Ð INSTRON Ð (WBS) values for control and treated samples WBS values (kg)
Control vs treated
Aging days
Control
Treated
Signi®cant dierences
CV1b
0 1 2 3 4 5 7 CV2c
5.3 3.62 4.33 3.8 6.69 4.02 4.61 30
3.95 3.36 4.51 3.65 4.59 3.26 3.83 34
nsa ns ns ns ns ns ns
26.8 28.5 41.7 41.1 20 16.2 37
b c
%Td
± 0 8.3 38.7 34.3 71.6 66.8
± 0 36.8 58.5 48.1 76.7 78.1
Dierent letters on each column indicate signi®cant dierences among aging time. Average of three replicates. %C: Indicates percentage of increase of each aging day with respect to day 0 for control samples. %T: Indicates percentage of increase of each aging day with respect to day 0 for treated samples. ns: Not signi®cant dierences. CV1: Coecient of variance for ANOVA between columns. CV2: Coecient of variance for ANOVA between rows.
the rest of the aging period until day 5. However, there were not signi®cant dierences between days 5 and 7 of the aging period. Evidently, the activation of both calpains by the calcium salt produces a higher and earlier increase in muscle tenderness compared to the nontreated samples. The percentage of MFI increment of each aging day with respect to day 0 (Table 1, %C and %T) shows more clearly the action of calcium treatment beyond aging processing, since treated samples on day 3 achieved 58.5% of increased tenderness, almost the same value obtained with 7 days of aging in control samples (66.8%).
a
%Cc
ns: Not signi®cant dierences. CV1: Coecient of variance for ANOVA between columns. CV2: Coecient of variance for ANOVA between rows.
samples showed no signi®cant dierences among each aging day. These results are dierent from those published by Morgan et al. (1991) and Wheeler et al. (1993), who found that the use of CaCl2 solution lowered shear force values compared with control samples. The dierent results obtained in this study could be explained by sample variability (CV 16.2 to 41.7%, Table 2) found in this particular muscle, Cutaneus trunci. 3.3. Step III: sensory evaluation-consumer panel Consumer panel evaluation was conducted considering the results of the tenderness evaluation by MFI. The test was carried out including samples submitted to marination and aging during three days, and samples submitted to aging alone during 7 days, since the MFI values were similar. The results of the consumer panel indicated that there are no dierences between treated samples (aged 3 days) and control ones (aged 7 days). As it was expected the use of calcium chloride marination resulted in tenderness improvement and in the reduction of the postmortem storage time (from 7 to 3 days) required to achieve an acceptable level of tenderness. These results are in accordance with PeÂrez et al. (1998) and Whipple and Koohmaraie (1992) who found that marination with calcium chloride solution improved tenderness in post rigor muscles from dierent species, however, PeÂrez et al. (1998) found that calcium solution produces after taste and bitter ¯avor. Our preliminary results (trained panel of step I) demonstrated no negative eects upon meat odor and ¯avor. The dierent results found by PeÂrez et al. (1998) are probably due to a higher incorporation of the calcium salt. Although they used a 0.075 M of calcium solution the samples were marinated for a longer period, 48 h. In our experience, the marination
C. B. Gonzalez et al. / Meat Science 57 (2001) 251±256
procedure applied for 24 h or longer produced bitter ¯avor, and undesirable texture and color changes in the samples (data obtained with a trained panel and not shown). The comparison between treated samples (3 days aged) and control samples (7 days aged) showed nonsigni®cant dierences between genders (data not shown). Even more, when they were asked to choose one of the two samples, both sexes preferred the treated one because the tenderness was higher. These results are consistent with those published by Hoover et al. (1995), who showed that 62 restaurant consumers found that CaCl2 injection improved both tenderness and ¯avor intensity. Moreover, those consumers preferred treated steaks when they were asked to choose between control and treated samples. 4. Conclusions The results of this study show that even for a tough and thin muscle as Cutaneus trunci, calcium chloride treatment is eective in increasing tenderness and reducing the postmortem storage time necessary to achieve an acceptable level of tenderness. This procedure has an important implication for the beef industry since this tough muscle has the potential of being marketed at better price if a higher level of tenderness is guaranteed. Another contribution to the beef industry is that the incorporation of calcium to the meat will also provide an additional source of this mineral, required in certain nutritional diets (Heaney & Barger-Lux, 1991), without introducing undesirable ¯avors or any chemical additives that could attempt to human health. References Benito-Delgado, J., Marriott, N. G., Claus, J. R., Wang, H., & Graham, P. P. (1994). Chuck longissimus and infraspinatus muscle characteristics as aected by rigor state, blade tenderization and calcium chloride injection. Journal of Food Science, 59(2), 295± 299. Bouton, P. E., & Harris, P. V. (1978). Factors aecting tensile and Warner-Bratzler shear values of raw and cooked meat. Journal of Texture Studies, 9, 395±413. Cottin, P., Poussard, J. P., Desmazes, D., Georgescauld, H., & Ducastaing, A. (1991). Free calcium and calpain I activity. Biochimica et Biophysica Acta, 1079, 139±140. Cross, H. R., Savell, J. W., & Francis, J. J. (1986). National consumer retail beef study. In Proceedings 39th Annual Reciprocal Meat Conference, 39, 112±114. Culler, R. D., Parrish Jr., F. C., Smith, G. C., & Cross, H. R. (1978). Relationship of myo®bril fragmentation index to certain chemical, physical and sensory characteristics of bovine longissimus muscle. Journal of Food Science, 43, 1177±1180. Davey, C. L., & Gilbert, K. V. (1969). Studies in meat tenderness. 7. Changes in the ®ne structure of meat during aging. Journal of Food Science, 34, 69±74.
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Drans®eld, E. (1994). Optimisation of tenderization aging and tenderness. Meat Science, 36, 105±121. Drans®eld, E. (1997). Beef Ð What price for tenderness? Meat International, 7, 24±26. Drans®eld, E., & Jones, R. C. (1981). Relationship between tenderness of three muscles. Journal of Science and Food Agriculture, 32, 300± 304. Egelandsdal, B., Martinsen, B., & Autio, K. (1995). Rheological parameters as predictors of protein functionality: a model study using myo®brils of dierent ®bre-type composition. Meat Science, 39, 97±111. Gonzalez, C. B., Gallinger, M. M., Pazos, A. A., & Lasta, J. A. (1997). Tenderness in beef Longissimus dorsi muscle of Hereford (H), Aberdeen Angus (A) and Brahman cross-breed steers. The in¯uence of postmortem aging. In Proceedings 43rd International Congress of Meat Science and Technology (pp. 564±565). 27 July±1 August, Auckland, New Zealand. Gornall, A. G., Bardawill, C. T., & David, M. M. (1949). Determination of serum proteins by means of the Biuret reagent. Journal of Biological Chemistry, 177, 751±766. Heaney, R. P., & Barger-Lux, M. J. (1991). Food and Nutrition News (Vol. 63, p. 2). Chicago, IL: National Live Stock & Meat Board. Hoover, L. C., Cook, K. D., Miller, M. F., Human, K. L., Wu, C. K., Lansdell, J. L., & Ramsey, C. B. (1995). Restaurant consumer acceptance of beef loin strip steaks tenderized with calcium chloride. Journal of Animal Science, 73(12), 3633±3638. Hostetler, R. L., Link, B. A., Landmann, W. A., & Fitzhugh, H. A. (1972). Eect of carcass suspension on sarcomere length and shear force of some major bovine muscles. Journal of Food Science, 37, 132±137. IRAM Instituto Argentino de NormalizacioÂn, Norm 20002. (1985). AnaÂlisis Sensorial. Directivas Generales para la MetodologõÂa. Koohmaraie, M., Wheeler, T. L., & Shackelford, S. D. (1998). Beef tenderness regulation and prediction. In Proceedings International Live Stock Congress, (pp. 25±27). February 1998, Houston, Texas. Koohmaraie, M. (1996). Biochemical factors regulating the toughening and tenderization processes of meat. Meat Science, 43(S), S193± S201. Lansdell, J. L., Miller, M. F., Wheeler, T. L., Koohmaraie, M., & Ramsey, C. B. (1995). Postmortem injection of calcium chloride eects on beef quality traits. Journal of Animal Science, 73, 1735± 1740. Morgan, J. B., Savell, J. W., Hale, D. S., Miller, R. K., Grin, D. B., Cross, H. R., & Shackelford, S. D. (1991). National beef tenderness survey. Journal of Animal Science, 69, 3274±3283. Olson, D. G., Parrish Jr., F. C., & Stromer, M. M. (1976). Myo®bril fragmentation and shear resistance of three bovine muscles during postmortem storage. Journal of Food Science, 41, 1036±1041. Olson, D. G., Parrish Jr., F. C., Dayton, W. R., & Goll, D. G. (1977). Eect of postmortem storage and calcium activated factor on the myo®brillar proteins of bovine skeletal muscle. Journal of Food Science, 42, 117±124. Ouali, A. (1981). Variation between muscles of the eect of aging on myo®brilar ATPase activity. Science des Aliments, 1, 1±6. Ouali, A. (1990). Meat tenderization: possible causes and mechanisms. A review. Journal of Muscle Foods, 1, 129±165. Parrish Jr., F. C., Vandell, C. J., & Culler, R. D. (1979). Eect of maturity and marbling on the myo®bril fragmentation index of bovine longissimus muscle. Journal of Food Science, 44, 1668± 1671. Penny, I. F., & Drans®eld, E. (1979). Relationship between toughness and troponine T in conditioned beef. Meat Science, 3, 135±141. PeÂrez, M. L., Escalona, H., & Guerrero, I. (1998). Eect of calcium chloride marination on calpain and quality characteristics of meat from chicken, horse, cattle and rabbit. Meat Science, 48, 125±134. Roessler, E. B., Pangborn, R. M., Sidel, J. L., & Stone, H. (1978). Expanded statistical tables for estimating signi®cance in paired-
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preference, paired-dierence, duo-trio and triangle tests. Journal of Food Science, 43(3), 940±943. Statistical Analysis Systems Institute (1996). SAS User's Guide for PC Computers. Cary, NC: Author. Savell, J. W., & Shackelford, S. D. (1992). Signi®cance of tenderness to the meat industry. In Proceedings 45th Annual Reciprocal Meat Conference (pp. 43±46). Smith, G. C., Savell, J. W., Clayton, R. P., Field, T. G., Grin, D. B., Hale, D. S., Miller, M. F., Montgomery, T. H., Morgan, J. B., Tatum, J. D., & Wise, J. W. (1992). The ®nal report of the national beef quality audit. Colorado State University, Ft. Collins and Texas A & M University, College Station.
Wheeler, T. L., Koohmaraie, M., Lansdell, J. L., Siragusa, G. R., & Miller, M. F. (1993). Eects 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. (1997). Eect of postmortem injection time and post injection aging time on the calcium-activated tenderization process in beef. Journal of Animal Science, 75(10), 2652±2660. Whipple, G., & Koohmaraie, M. (1992). Freezing and calcium chloride marination eects on beef tenderness and calpastatin activity. Journal of Animal Science, 70, 3081±3085.
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Eect of calcium chloride marination on bovine Cutaneus trunci muscle Claudia B. Gonzalez *, Valeria A. Salitto, Fernando J. Carduza, Adriana A. Pazos, Jorge A. Lasta Instituto TecnologõÂa de Alimentos, Centro de Agroindustrias, CNIA, Instituto Nacional de TecnologõÂa Agropecuaria (INTA), Casilla de Correo 77, (1708), MoroÂn, Buenos Aires, Argentina Received 27 March 2000; received in revised form 31 July 2000; accepted 31 July 2000
Abstract The aim of this investigation was to determine the possibility of using calcium chloride solution in tough muscles (Cutaneus trunci) to reduce the aging period required to increase tenderness, without introducing undesirable ¯avors. Muscles were marinated in 0.25 M CaCl2 solution for 2 h and after that aged for 0, 1, 2, 3, 4, 5 and 7 days. Tenderness was evaluated by the myo®brillar fragmentation index (MFI), Warner Bratzler shear force (WBS) and sensory panel evaluation. MFI values showed signi®cant differences between treated and control samples aged for 1, 2 and 3 days (P<0.05). MFI values of treated samples aged 3 days were similar to those obtained for the control samples but aged seven days. WBS values were not signi®cantly dierent between samples. Consumer panelists preferred treated samples aged 3 days to the control ones aged 7 days. It was concluded that calcium chloride treatment can be used in Cutaneus trunci muscle to reduce the aging time required to increase tenderness. # 2000 Elsevier Science Ltd. All rights reserved. Keywords: Calcium chloride; Tough muscle; Tenderness; Cutaneus trunci; Ageing
1. Introduction Consumers consider meat tenderness as the most important palatability trait of meat quality (Cross, Savell & Francis, 1986) and its variability is an area of major concern to the meat industry (Koohmaraie, Wheeler & Shackelford, 1998; Morgan et al., 1991; Smith et al., 1992). Over the years, many studies have been carried out in relation to the myo®brillar protein changes during post-mortem storage and their causes, and the relationship between these alterations and meat tenderness (Olson, Parrish, Dayton & Goll, 1977; Penny & Drans®eld, 1979). Dierent factors have been reported to aect tenderness of aged beef, such as animal age, percentage of Bos indicus inheritance, carcass pH and temperature, muscular calpain activity, sarcomere length, amount and type of collagen, muscular ®bre type * Corresponding author at present address: Tinogasta 3101, 7 A, CP1417, Capital Federal, Buenos Aires, Argentina. Fax: +54-114621-2012. E-mail addresses: [email protected] or [email protected]. gov.ar (C.B. Gonzalez)
and size (Gonzalez, Gallinger, Pazos & Lasta, 1997; Hostetler, Link, Landmann & Fitzhugh, 1972; Koohmaraie et al., 1998). With regard to these factors, Drans®eld and Jones (1981), found that the intensity and the rate of these modi®cations are not only speciesdependent but also, within a given species, muscledependent (Ouali, 1981; Penny & Drans®eld, 1979). On the other hand, Savell and Shackelford (1992) found a direct relationship between muscle tenderness and market price. Even more, Koohmaraie et al. (1998) and Drans®eld (1997) reported that consumers preferred to pay a premium for high quality products. Consequently, it is essential to improve and develop dierent postmortem processes to increase meat tenderness, particularly in those muscles that are traditionally tough and that are usually punished with lower prices. One of these postmortem methodologies is the use of calcium chloride solution. The calcium ion activates natural endogenous calpain proteinases: m-calpain, mcalpain and calpastatin Ð a speci®c inhibitor of both calpains Ð which are responsible for the breakdown of myo®brillar proteins (Koohmaraie et al., 1998). The endogenous calcium concentration present immediately
0309-1740/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S0309-1740(00)00099-1
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C. B. Gonzalez et al. / Meat Science 57 (2001) 251±256
postmortem is enough to activate m-calpain but not mcalpain. Then, when the calcium concentration increases, due to a gradually released of calcium ions from the sarcoplasmic reticulum and mitochondria, m-calpain is activated and produces a further tenderization (Drans®eld, 1994). A similar mechanism can be used to explain the calcium chloride postmortem tenderization process applied within 36 h after slaughter. In this procedure the meat is treated with higher concentration of calcium ion than the endogenous one in order to accelerate the tenderization process (Drans®eld, 1994). However, some of the sensory properties, such as color and ¯avor, can be altered by this treatment depending on the calcium concentration (Lansdell, Miller, Wheeler, Koohmaraie & Ramsey, 1995; Wheeler, Koohmaraie, Lansdell, Siragusa & Miller, 1993). There is a very good understanding of the biological basis that determines the tenderness in most of the muscles, particularly in the case of Longissimus dorsi, Semitendinosous, Biceps femoris, etc. (Koohmaraie, 1996). It was also demonstrated that the use of calcium salts improves meat tenderness in those muscles, however, there is no much information about the eciency of this treatment on tough muscles. It is known that Cutaneus trunci muscle is composed mainly of the fast twitch white ®ber type (70%) (Egelandsdal, Martinsen & Autio, 1995). This muscle type has the characteristic of exhibiting a low calpain content, however this muscle responds to aging treatment at faster rate than red muscles (Ouali, 1990). Considering that the lack of tenderness is a common complaint in the meat industry, the Cutaneus trunci muscle was selected in this study as a pattern of tough muscle. The objective was to establish the optimal conditions required to improve meat tenderization in this tough muscle by using calcium chloride solution and consequently to reduce the time required for the postmortem tenderization, which will result in a cost bene®t for the meat industry. 2. Materials and methods 2.1. Experimental design A three steps study was conducted to examine the eect of calcium chloride solution in Cutaneus trunci muscle tenderness. Step I consisted in a preliminary assay designed to determine the optimal conditions for the calcium salt tenderization process. In step II, the Cutaneus trunci muscle tenderness was measured by using two dierent methodologies: chemical measurement of Mio®brillar Fragmentation Index (MFI) and Warner Bratzler (Instron) shear force determination. Finally in step III a consumer panel was carried out to
evaluate if the level of tenderness achieved with the calcium salt treatment was enough to make Cutaneus trunci muscle acceptable to consumers. 2.1.1. Step I: preliminary assay Eight Cutaneus trunci muscles from four steers (36±42 months of age) were used in the preliminary assay. Steers were slaughtered under regular commercial procedures with Good Manufacture Practice. Muscles were removed 24 h postmortem and submitted to the laboratory assays. For the incorporation of calcium chloride solution the marination procedure was selected because the anatomic characteristics of the Cutaneus trunci muscle (thin muscle) made it dicult to apply the injection process. The marination-time was set up as the necessary time to incorporate 10% (w/w) of the calcium chloride solution. The selection of the maximum concentration of calcium chloride solution that does not promote o-¯avors (metallic ¯avor) was made by an eight-member trained sensory panel. For this purpose, Cutaneus trunci muscles were marinated with 0.20, 0.25 and 0.30 M calcium chloride solution. Samples were wrapped in aluminum foil and cooked in an electric oven at 180 C during a ®xed time depending on their weight (40 min/kg of muscle) and then submitted to the sensory panel evaluation. 2.1.2. Step II: tenderization procedure Cutaneus trunci muscles from 42 steers (36±42 months of age) were removed 24 h postmortem; right and left sides were assigned randomly to the marination procedure (10% w/w of a 0.25 M CaCl2 solution for 2 h) or were kept as control. After the marination process, all the muscles were vacuum packaged and aged at 11 C for 0, 1, 2, 3, 4, 5 and 7 days. After each aging period samples from both muscles (treated and control samples) were taken from the refrigerator to perform the tenderness evaluation. 2.1.2.1. Myofibril fragmentation index (MFI). For the evaluation of tenderness MFI was chosen as a biochemical assay. This determination was considered as a good predictor of beef tenderness because calciumdependent enzymes produce the myo®brillar proteins degradation. Myo®brils were extracted from the muscle following the procedure described by Olson, Parrish Jr. and Stromer (1976) and the protein concentration was determined by the Biuret method of Gornall, Bardawill and David (1949). MFI was carried out on three replicate experiments per quadruplicate and was applied to the dierent periods of storage (0, 1, 2, 3, 5 and 7 days) in both, control and treated samples. 2.1.2.2. Warner Bratzler (Instron). The objective evaluation of tenderness was performed applying the modi®ed
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Warner Bratzler shear force procedure (Bouton & Harris, 1978) by using the Instron Universal Testing Machine. Since the anatomical characteristics of Cutaneus trunci muscle made impossible to obtain the typical cores for the Warner Bratzler procedure, it was necessary to cut it in cubes and use the planar shear instead of the triangular one. Muscle samples were wrapped in aluminum foil and cooked in an electric oven at 180 C for a ®xed time depending on their weight (40 min/kg of muscle). Sample pieces (10 replicates) of 0.6 cm thick1.5 cm wide3 cm long were cut from the cooked muscles and kept at 8 C for approximately one hour until assaying. 2.1.3. Step III: sensory evaluation-consumer panel To determine if the improvement of meat tenderness could be detected by consumers, a panel session was carried out. It was composed of 120 consumers with ages between 20 and 60 years (58% female, 42% male). Muscle samples, control (7 days-aged) and treated (calcium-treated and 3 days-aged), from 44 animals (36±45 months of age) were prepared as described above (Step II). After cooking, samples were cut in 2 cm3 cubes and served in closed recipients with a tomato sauce added just before degustation. A paired comparison assay (IRAM Instituto Argentino de NormalizacioÂn, Norm 20002, 1985) was utilized between control and treated samples. In this assay, consumers were asked to choose between both samples giving the reasons for their choice. 2.2. Statistical analysis MFI and Warner Bratzler data were analyzed for signi®cant dierences by analysis of variance using the GLM procedure of the Statistical Analysis Systems Institute (SAS, 1996). When signi®cant (P<0.05) main eects were observed without interactions, mean separation was accomplished by the use of Tukey's means comparison test. The paired comparison data in the preference test were analyzed using expanded tables of the accumulative binomial probability distribution (two-tailed test) (Roessler, Pangborn, Sidel & Stone, 1978) following the methodology included in the IRAM norm cited previously. 3. Results and discussion 3.1. Step I: preliminary assay The time selected for the marination procedure was 2 h since the incorporation of the 10% (w/w) of the salt was already completed. There were not changes on quality characteristics.
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The results of the sensory trained panel for samples marinated with the three calcium concentrations did not show signi®cant dierences (data not shown), however some panelists found a ¯avor described as salty in samples treated with 0.3 M calcium solution. Despite this, the ¯avor was not described as unpleasant, the 0.25 M solution was chosen because it maintained more accurately the ¯avor characteristics of control samples. 3.2. Step II: tenderization procedure 3.2.1. Myo®bril fragmentation index (MFI) A comparison of MFI values at each aging day between control and treated samples (Table 1, control vs. treated), indicates that there is a signi®cant increase in MFI values (P<0.05) in treated samples on days 1, 2 and 3 of aging. There are not signi®cant dierences between control and treated samples for the rest of the aging days. Most of the studies related to the action of calcium chloride solution on beef tenderness have utilized sensory and objective evaluations to score tenderness (BenitoDelgado, Marriott, Claus, Wang & Graham, 1994; Morgan et al., 1991; Wheeler, Koohmaraie & Shackelford, 1997), however, in this study not only Warner± Bratzler shear force values and sensory panel evaluation but also MFI values were used as tenderness predictors. The reason for the selection of this methodology was that many authors (Culler, Parrish Jr., Smith & Cross, 1978; Davey & Gilbert, 1969; Olson et al., 1976; Parrish, Jr., Vandell & Culler, 1979) have demonstrated the correlation between MFI values and tenderness, providing a potential tool for identifying tough and tender muscles. Consequently, the higher levels of myo®bril fragmentation found in samples treated with calcium could be considered as a result of the action of endogenous proteolytic enzymes that are activated at an accelerated rate reducing the time necessary for the post mortem aging (Cottin, Poussard, Desmazes, Georgescauld & Ducastaing, 1991; PeÂrez, Escalona & Guerrero, 1998). Our results demonstrated that calcium chloride treatment produced higher tenderness level on day 1, 2 and 3 of the aging period than the tenderness achieved by the conditioning process alone. It means that the combination of aging and marination is more eective in increasing MFI values in this type of muscle than aging alone, at least for the ®rst stage of the aging period when the tenderness achieved is not the highest. The data in Table 1 (MFI) show that, in control samples the aging process produced a signi®cant increase (P<0.05) of MFI values on days 3 and 4 of the aging period. A second signi®cant increase (P<0.05) occurred on the last two days (days 5 and 7). In the case of marinated samples (treated samples) the increase in MFI values was noticed on the 2nd day of aging (P<0.05), after that a gradual increase occurred during
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Table 1 Myo®brillar fragmentation index (MFI) values for control and treated samples MFI valuesa
Aging days
0 1 2 3 4 5 7 CV2g a b c d e f g
Control vs. treated
Controlb
Treatedb
Signi®cant dierences
CV1f
74.4a 66.2a 80.6a 103.2b 99.9b 127.7c 124.1c 19.5
74.4a 71.3a 101.8b 117.9bc 110.2bc 131.5c 132.5c 19.6
nse 0.05 0.05 0.05 ns ns ns
18.1 8.3 12.8 7.5 30.6 24.2 13.8
3.2.2. Warner Bratzler (Instron) Table 2 shows the shear force values obtained with the Warner Bratzler machine (Instron), these values represent an average of 10 samples from each of the 42 muscles. Shear force values between control and treated Table 2 Warner Bratzler shear force Ð INSTRON Ð (WBS) values for control and treated samples WBS values (kg)
Control vs treated
Aging days
Control
Treated
Signi®cant dierences
CV1b
0 1 2 3 4 5 7 CV2c
5.3 3.62 4.33 3.8 6.69 4.02 4.61 30
3.95 3.36 4.51 3.65 4.59 3.26 3.83 34
nsa ns ns ns ns ns ns
26.8 28.5 41.7 41.1 20 16.2 37
b c
%Td
± 0 8.3 38.7 34.3 71.6 66.8
± 0 36.8 58.5 48.1 76.7 78.1
Dierent letters on each column indicate signi®cant dierences among aging time. Average of three replicates. %C: Indicates percentage of increase of each aging day with respect to day 0 for control samples. %T: Indicates percentage of increase of each aging day with respect to day 0 for treated samples. ns: Not signi®cant dierences. CV1: Coecient of variance for ANOVA between columns. CV2: Coecient of variance for ANOVA between rows.
the rest of the aging period until day 5. However, there were not signi®cant dierences between days 5 and 7 of the aging period. Evidently, the activation of both calpains by the calcium salt produces a higher and earlier increase in muscle tenderness compared to the nontreated samples. The percentage of MFI increment of each aging day with respect to day 0 (Table 1, %C and %T) shows more clearly the action of calcium treatment beyond aging processing, since treated samples on day 3 achieved 58.5% of increased tenderness, almost the same value obtained with 7 days of aging in control samples (66.8%).
a
%Cc
ns: Not signi®cant dierences. CV1: Coecient of variance for ANOVA between columns. CV2: Coecient of variance for ANOVA between rows.
samples showed no signi®cant dierences among each aging day. These results are dierent from those published by Morgan et al. (1991) and Wheeler et al. (1993), who found that the use of CaCl2 solution lowered shear force values compared with control samples. The dierent results obtained in this study could be explained by sample variability (CV 16.2 to 41.7%, Table 2) found in this particular muscle, Cutaneus trunci. 3.3. Step III: sensory evaluation-consumer panel Consumer panel evaluation was conducted considering the results of the tenderness evaluation by MFI. The test was carried out including samples submitted to marination and aging during three days, and samples submitted to aging alone during 7 days, since the MFI values were similar. The results of the consumer panel indicated that there are no dierences between treated samples (aged 3 days) and control ones (aged 7 days). As it was expected the use of calcium chloride marination resulted in tenderness improvement and in the reduction of the postmortem storage time (from 7 to 3 days) required to achieve an acceptable level of tenderness. These results are in accordance with PeÂrez et al. (1998) and Whipple and Koohmaraie (1992) who found that marination with calcium chloride solution improved tenderness in post rigor muscles from dierent species, however, PeÂrez et al. (1998) found that calcium solution produces after taste and bitter ¯avor. Our preliminary results (trained panel of step I) demonstrated no negative eects upon meat odor and ¯avor. The dierent results found by PeÂrez et al. (1998) are probably due to a higher incorporation of the calcium salt. Although they used a 0.075 M of calcium solution the samples were marinated for a longer period, 48 h. In our experience, the marination
C. B. Gonzalez et al. / Meat Science 57 (2001) 251±256
procedure applied for 24 h or longer produced bitter ¯avor, and undesirable texture and color changes in the samples (data obtained with a trained panel and not shown). The comparison between treated samples (3 days aged) and control samples (7 days aged) showed nonsigni®cant dierences between genders (data not shown). Even more, when they were asked to choose one of the two samples, both sexes preferred the treated one because the tenderness was higher. These results are consistent with those published by Hoover et al. (1995), who showed that 62 restaurant consumers found that CaCl2 injection improved both tenderness and ¯avor intensity. Moreover, those consumers preferred treated steaks when they were asked to choose between control and treated samples. 4. Conclusions The results of this study show that even for a tough and thin muscle as Cutaneus trunci, calcium chloride treatment is eective in increasing tenderness and reducing the postmortem storage time necessary to achieve an acceptable level of tenderness. This procedure has an important implication for the beef industry since this tough muscle has the potential of being marketed at better price if a higher level of tenderness is guaranteed. Another contribution to the beef industry is that the incorporation of calcium to the meat will also provide an additional source of this mineral, required in certain nutritional diets (Heaney & Barger-Lux, 1991), without introducing undesirable ¯avors or any chemical additives that could attempt to human health. References Benito-Delgado, J., Marriott, N. G., Claus, J. R., Wang, H., & Graham, P. P. (1994). Chuck longissimus and infraspinatus muscle characteristics as aected by rigor state, blade tenderization and calcium chloride injection. Journal of Food Science, 59(2), 295± 299. Bouton, P. E., & Harris, P. V. (1978). Factors aecting tensile and Warner-Bratzler shear values of raw and cooked meat. Journal of Texture Studies, 9, 395±413. Cottin, P., Poussard, J. P., Desmazes, D., Georgescauld, H., & Ducastaing, A. (1991). Free calcium and calpain I activity. Biochimica et Biophysica Acta, 1079, 139±140. Cross, H. R., Savell, J. W., & Francis, J. J. (1986). National consumer retail beef study. In Proceedings 39th Annual Reciprocal Meat Conference, 39, 112±114. Culler, R. D., Parrish Jr., F. C., Smith, G. C., & Cross, H. R. (1978). Relationship of myo®bril fragmentation index to certain chemical, physical and sensory characteristics of bovine longissimus muscle. Journal of Food Science, 43, 1177±1180. Davey, C. L., & Gilbert, K. V. (1969). Studies in meat tenderness. 7. Changes in the ®ne structure of meat during aging. Journal of Food Science, 34, 69±74.
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