International Food Research Journal 22(4): 1410-1416 (2015) Journal homepage: http://www.ifrj.upm.edu.my

Improving the quality of citric acid and calcium chloride marinated culled cow

1*Klinhom, P., 2Klinhom, J., 2Senapa, J. and 1Methawiwat, S.

1Agricultural Science Program, Faculty of Science and Technology, Kamphaeng Phet Rajabhat University, Kamphaeng Phet, 62000, Thailand 2Department of Product Development Technology, Faculty of Agro-Industry, Chiang Mai University, Chiang Mai, 50100, Thailand

Article history Abstract

Received: 2 June 2014 The objective of this research was to evaluate the effects of immersion with citric Received in revised form: acid, calcium chloride and combined mixture of citric acid and calcium chloride solutions on 1 December 2014 Semimembranosus meat quality traits. Meat cuts obtained from culled cows were marinated Accepted: 18 December 2014 with 0.05 M citric acid, 0.2 M calcium chloride or the combined mixture of these two compounds for 24 hr. Non- marinated meat served as control. Application of calcium chloride Keywords to the marination (either solely or in combination) improved water holding capacity (WHC) thus reduced cooking loss and shear force values. Meat marinated in citric acid solution showed Meat quality high exudative during cooking and was not significantly different in shear force value compared Marination to the unmarinated samples. Lightness was higher (paler) in all marinated meat samples. The Culled cow a* values showed that marinated meat with citric acid solution had less red intensity. Lower of yellow intensity (b* value) appeared in the meat samples marinated with calcium chloride and the combined mixture. The sensory attribute evaluation showed that the highest scores for juiciness and tenderness were for calcium chloride and the combined mixture. The lowest flavor score was found in the meat marinated with citric acid solution and reported as sour. Sample treated with calcium chloride revealed no detrimental effect on meat flavor and odour. The evidence in the present study indicates that calcium chloride marinate solution increases juiciness and tenderness of meat from culled cow. © All Rights Reserved

Introduction of acidic marinades is believed to involve in the weakening of structures due to swelling of the meat Tenderness is considered to be the most important and increased conversion of collagen to gelatin at low trait of meat acceptability by the consumers (Miller pH during cooking (Offer and Knight, 1988; Berge et et al., 1995). Meat tenderness is related principally al., 2001). to the two structural elements: the connective tissue Calcium chloride has been identified as a means and the myofibrillar protein components of muscle for increasing tenderness (Koohmaraie et al., (Lawrie, 1998). Basically, the mechanism responsible 1990; Morgan et al., 1991). Benefit of marinating for increased meat tenderness is connected with the beef cuts with calcium chloride to increase tenderness weakening of connective tissue and the weakening is due to the enhancing calcium activated proteolysis of myofibrillar protein component (Takahashi, 1996). (endogenons calpain proteinase system) which is Marination involves the immersion of meat in responsible for the breakdown of myofibrillar proteins organic acid or salt solutions are a practical method of (Whipple and Koohmaraie, 1992). However, some of altering meat tenderness for many years (Gault, 1991). the sensory properties such as colour and flavour can Marination process not only improves tenderness but be altered by calcium chloride treatment (Wheeler et also increases juiciness of meat, due to the retention al., 1993; Eilers et al., 1994). of added water (Offer and Trinick, 1983). Thus, the Numerous experiments have demonstrated marinades with a tenderising capacity are important the use of organic acid and salts marinade solution means for applications involving in low-value of improve meat tenderness. However, most of the meat cut or muscle with rich in connective tissue. investigations have been documented with growing Citric acid, a food acidulant is often used in meat finishing animals where rather tender beef could be marination to improve the water holding capacity expected anyway. In Thailand, beef originating from (WHC) and tenderness of beef muscle (Ke et al., culling cattle is prevalent in local markets with most 2009). The mechanism of the tenderising action

*Corresponding author. Email: [email protected] 1411 Klinhom et al./IFRJ 22(4): 1410-1416 of them being Bos indicus origin (Jaturasitha et al., after marinating. 2004). Meat from Bos indicus cattle is known to be more degree of toughness than beef from Bos tauras Color measurement origin (Lansdell et al., 1995). Limited work has been Meat color was measured using a colorimeter done on marinated beef from culling Bos indicus cow. (Hunter Lab, Hunter Associates Laboratory, Inc., Therefore, the objective of this study was to USA) in the CIE L*, a*, b* mode. Before each compare the effects of citric acid, calcium chloride measurement, the apparatus was standardized against and the combined mixture of citric acid and calcium black and white plate. chloride marinate on the sensory attributes such as marination mass gain, cooking loss, tenderness, Cooking loss colour and flavor of meat from culling cows. The meat samples were cooked by placing in plastic bags and immersing in a water bath at 80oC Materials and Methods until an internal temperature of 70oC was reached (about 1 hr). Cooked meat strips were cooled to room Meat samples temperature, blotted and reweighed. Cooking loss The meat samples used in this research were was calculated by the different weight before and obtained from six culling Thai native cows (about after cooking. 7 years of age) reared in Tak province, Thailand. After slaughter, the samples of muscles from topside Shear force determination of hind legs (M. semimembranosus muscles) were Three cores (1.3 cm diameters) were removed collected and the visual connective tissues were from each cooked meat samples. Each core was manually removed as much as possible. The muscles sheared once perpendicular with the muscle fiber (the were then stored at 4oC over night. longitudinal axis of the sample) using a computer controlled Texture Analyser (TA-XT plus, Stable Sample marination Micro Systems, UK) equipped with a Warner-Bratzler Meat samples were cut parallel to the muscle fiber type shear blade. The crosshead speed of 200 mm/ direction (a cutting thickness of 5 mm) using a meat min with a 5 kN load cell was used. slicer (Sirman snc. Padova, Italy). The meat strips from each cow were allocated randomly submitted to Sensory evaluation the four treatments as follow: 1) control (Co, without For sensory characteristics assessment, the marination) 2) 0.05 M citric acid marination (Cc) cooked meat samples were randomly served warm 3) 0.2 M calcium chloride marination (Ca) and 4) to a 50-member sensory panel in individual booth. combined mixture in equal volume of 0.05 M citric Panelists assessed meat sample for juiciness acid and 0.2 M calcium chloride (Cc + Ca). tenderness, odor, flavor and general acceptability in Meat strips were dipped in marinade solutions an eight point scale, where 8 = extremely desirable at a ratio of 1:1 (meat: liquid) in plastic boxes and juicy, tender, intense in meat flavor and very good keep in refrigerator at 4oC for 24 hr. The untreated appraisal and a score of 1 = extremely dry, tough, samples (control) were only kept in the boxed without devoid of meat flavor and extremely undesirable. addition. Statistical analysis pH measurement The experiment was conducted according to After marinating, meat samples (3 g) were cut completely randomized blocks design with six into small pieces, made up to 30 ml with 5 mM Na- replicates. Analysis of variance was used in analyzing iodoacetate in 150 mM KCl and then homogenized. all data (Steel and Torrie, 1980). Significant means were Slurry was then filtrated through Whatman paper separated using Tukey’s w procedure test at P< 0.05. no. 4. The pHs of filtrates were determined using a glass electrode pH meter (Sartorius AG, Gottingen, Results and Discussion Germany). Effect of marination on pH Uptake of marinade Low pH (4.5) was observed in the meat samples Following marination, excess marinade was marinated with citric acid and the combined mixture removed by applying paper towel lightly to samples of citric acid and calcium chloride solutions (Table surface. %.uptake of marinade was taken by the 1). Citric acid marinades decreased meat pH to below difference between the sample weights before and the isoelectric point (about 5.2-5.3 in ). In Klinhom et al./IFRJ 22(4): 1410-1416 1412

Table 1. Effect of marinated solution treatments on pH, marinade uptake and cooking loss of the Semimembranosus muscle from culled cows

a,b,c Values in a row with different letters are significantly different (P<0.05). 1Co, control (unmarinated); Cc, 0.05 M citric acid; Ca, 0.2 M Calcium Chloride; Cc + Ca, combined mixture of 0.05 M citric acid and 0.2 M calcium chloride . 2Standard error of means calcium chloride marinated meat, the pH revealed no marination, the anions (Cl- ) of the calcium chloride different from the unmarinated control meat (pH 5.5) could form ionised salts with the available cationic (P>0.05) (Table 1). Koohmariae et al. (1989) reported + side groups (R- NH3 ) on the protein molecules. This the application of meat with calcium chloride do not results in an unequal distribution of free ions between alter the meat pH. the myofibrillar protein and the external solution. This could induce the difference in osmotic pressure. Marinade uptake The difference in osmotic pressure lead the flow of Uptake of marinade after immersion in the water into the protein structure results in a reduction treatment marinade solutions are shown in Table 1. of this difference. It was found that the meat samples marinated with When the mixture of citric acid and calcium citric acid solution and calcium chloride solution chloride was applied to meat, the lower of marinade revealed higher uptake than the samples treated uptake was found in comparison with the treatments with combined mixture of citric acid and calcium where two compounds were used separately. Goli chloride solution(P<0.05). In the citric acid treatment, et al. (2014) also found the limitation of marinade the high marinade uptake could be attributed to the uptake when salt was applied to the acid marinate strong effect of lower pH on protein as indicated by solution in turkey breast meat. Their experiment the results reported by Rao et al. (1989). The addition demonstrated the presence of salt in water acid of acid below the isoelectric point of meat proteins marination considerably reduced the swelling led to the protonation of negatively charged (–COO- observed upon the fall in pH compared with the ) groups on protein molecules. The increase in net swelling that would have been observed in a water positive charges on myofibrillar protein resulted in acid solution. This phenomenon could be explained repulsive forces among protein groups of similar by the reduction in osmotic difference as described charge in the myofibrils. This repulsive force push the by Medynski et al. (2000). Addition of salt with thick and thin filaments apart laterally and creating acid at the same time increases the amount of free space for the immobilization of extra water (Ke et al., anions in the external solution. This could reduce the 2009). Previous study also reported the immersion of difference in anion concentration between the protein meat in acid marinade solution increased marinade structure and external solution, hence reduction in the absorption (Burke and Monahan, 2003). osmotic difference. Therefore, the swelling potential In the calcium chloride medium, the high of protein fibers is reduced. marinade uptake may be explained by the combination of 2 phenomena: (1) degradation of the Cooking loss cytoskeleton in muscle cell. During the marination, Differences in the cooking loss in the meat calcium ion activates endogenous calpain proteinases samples have been seen in Table 1. Cooking loss (µ- and m- calpains) which their activities specific was lower in the calcium chloride and in the mixture degraded the proteins include those involved in inter- of citric and calcium chloride treatments than in the (eg. desmin and vinculin) and intra- myofibril (eg. citric acid marinate solution (P<0.05). The difference titin and nebulin). The function of these proteins is to may be due to conformational changes at the protein maintain the structural integrity of myofibrils (Pearce water interface. Lawrence et al. (2003) reported the et al., 2001). Thus, the proteolytic degradation of addition of a salt (e.g. a calcium salt) to a solution these proteins causes a weakening of myofibrillar increased ionic strength, thereby increasing the and then creates the space available for water within number of hydrophilic protein interactions which the muscle cell. (2) osmotic pressure created by the caused an increase in the binding of free water. Thus, difference in the free ions concentration. During the calcium chloride when applied solely or added to 1413 Klinhom et al./IFRJ 22(4): 1410-1416

Table 2. Effect of marinated solution treatments on hunter colour and shear force of the Semimembranosus muscle from culled cows

a,b Values in a row with different letters are significantly different (P<0.05). 1Co, control (unmarinated); Cc, 0.05 M citric acid; Ca, 0.2 M Calcium Chloride; Cc + Ca, combined mixture of 0.05 M citric acid and 0.2 M calcium chloride . 2Standard error of means acid water marinade solution may alter the affinity Marriot (1989) concluded that the acid treatment of the proteins to bind more extent of the free water appeared to enhance the conversion of myoglobin to molecules leading lower in cooking loss. metmyoglobin via oxidative reaction which turned On the contrary, meat sample marinated with red colour to brown and lower colour intensity. citric acid exhibit a high exudation when cooked. Shikama (1998) also reported that the formation of Previous researches have reported the acid treatment the oxidized forms of hemeproteins (metmyoglobin) decreased cooking loss of beef cores (Oreskovich might contribute from the rapid oxidation of muscle et al., 1992; Önenc et al., 2004). The contrast result at acidic pH values. Thus, the lower pH in citric acid may be due to the difference in types of connective marinate solution could provide the oxidant effect tissue. Meat samples in this present study derived condition for metmyoglobin formation. from the old cows (about 7 yrs of age) which the In general, the lower the pH values, the stronger maturation of collagen fibers could be expected. At the prooxidant effect. However, it could be noticed maturity, collagen developed the cross links result in that the meat samples in the combination of citric the formation of rigid three dimensional architecture acid and calcium chloride solution treatment revealed collagen fibers of considerable tensile strength. Such no difference in a* value from the control (P>0.05), structure probably prevented the unfold at the protein eventhough the lower pH has been seen. This could be water interface within the collagen fibers, thus reduce assumed that the myoglobin in this medium was less the number of hydrophilic protein interaction. Meat susceptible to oxidation. It seems to us that calcium present with this type of collagen, which has retained chloride addition might be responsible for decreasing its integrity, contributes to the exudation through the oxidatant effect of citric acid under low pH. Citric its shrinkage when cooked, which compress the acid has been reported as strong metal chelators muscle fibers. Miller et al. (1983) also reported that (Decker, 1998). Thus, the combination of citric acid collagenous tissue with more cross linkages would and calcium chloride solutions could inhibit oxidation be more resistant to swelling and have a lower water by binding the metal via bonds formed between the holding capacity. metal and the carbonyl or hydroxyl groups of the citric acid molecule as has been described by Francis Hunter colour values et al. (1992). The effects of marinating meat on hunter colour Meat marinated with calcium chloride solely values are showed in Table 2. It was found that the or combine with the acid revealed lower b* values L* value (lightness) was higher or paler in marinate (P<0.05) (less yellow) than the control. Calcium treated meat samples than in control (P<0.05) which chloride treated meat demonstrated less in yellow agreed with results published previously (Pérez et has also been reported by Jaturasith et al. (2004) al., 1998; Önenc et al., 2004). The increase in L* and Bunmee et al. (2014). They proposed that the values could be due to the water uptake of meat discolouration of meat in Ca salt application could during marination. Aktas and Kaya (2001) reported be due to the continuous action of oxygen which is that the amount of water dispersed among the muscle facilitated by the catalyzing properties of the salts. fibers could affect the reflectance ability of meat. However, the mechanism in detail did not elucidate. Our resulted supported to this with more extend of marinate uptake, the paler of meat subject. Shear value Marinating meat with citric acid solution had a Tenderness, an important attribute of meat, was significant effect on *a value which was less red colour evaluated by a shear force. The data obtained here compared to the control (Table 2). Similar result was demonstrated that there were significant reductions in obtained by Aktas and Kaya (2001). Arganosa and shear force for meat marinated with calcium chloride Klinhom et al./IFRJ 22(4): 1410-1416 1414

Table 3. Effect of marinated solution treatments on sensory panel scores of the Semimembranosus muscle from culled cows

a,b,c Values in a row with different letters are significantly different (P<0.05). 1Co, control (unmarinated); Cc, 0.05 M citric acid; Ca, 0.2 M Calcium Chloride; Cc + Ca, combined mixture of 0.05 M citric acid and 0.2 M calcium chloride. 2Standard error of means either solely or in the combination with acid compared scores were significantly higher (P<0.05) for the to the control (Table 2). Previous studies (Koohmaraie calcium chloride and the combined mixture compared and Shackelford, 1991; Morgan et al., 1991; Whipple to the citric acid treatments. Since juiciness reflects and Koohmaraie, 1992) supported the effectiveness WHC (Pearce et al., 2011), therefore the increase of calcium chloride in decreasing shear force values. in water binding ability of the meat marinated with The reduction in shear force with calcium chloride calcium chloride (either solely or in combination salt applied could be attributed to the weakening of the with acid) could be corresponding to this situation. myofibrillar structure and the increased water content Meat samples were more tender when calcium of the samples. Calcium chloride application into chloride was applied and showed a highly significant meat increases intracellular calcium concentration difference between untreated and meat samples sufficiently to activate the calcium dependent calpain marinated with citric acid (P<0.05). This result was system. The enzyme degrades the protein around fully corresponding to the shear values (Table 2) and Z-disk of myofibril results in myofibrils fragmenting consistent with the study by Whipple and Koohmarai into smaller segment (Koohmaraie et al., 1986). The (1992) who found that marination with calcium fragmentation of myofibrils leads to the structural chloride solution improved tenderness. weakening of myofibrils thus, increased tenderness. When the samples were rated for flavor, the citric Furthermore, the application of calcium chloride acid marinated meat was described as sour by the promoted myofibril swelling that could retain as panelists and was judged as off flavor with the lowest much added water upon cooking as evidenced by low score (Table 3 ). Similar result was also reported for cooking loss of the treatments seen in this study. The beef treated with citric acid (Aktas and Kaya, 2001). ability of meat to retain water is related to tenderness No difference in odour scores was detected among by dilute the load bearing material due to the swelling treatments. For this point of view, previous reports of the myofibrils across their main axis (Gault, 1985; of calcium chloride marination caused undesiable Rao and Gault, 1990). meat odour and flavor (metallic, livery, astringent On the contrary, the shear force value of meat and bitter tastes) (Morgan et al., 1991; Wheeler et marinated with citric acid showed no statistical al., 1993; Lawrence et al., 2003). However, results difference to the control (P>0.05) (Table 2). This in the present study found no difference in flavor and indicated that citric acid marinade did not improve odour with respect to the unmarinated meat. This meat tenderness. Previous studies reported the contrast results are probably due to the difference in achievement of meat tenderization in acid marination concentration of calcium salt solution and marination was due to the swelling of muscle fiber under low duration. It could be stated that the potentially pH medium (Burke and Monahan, 2003; Aktas et adverse effect on flavor may have been increased al., 2007). In this study, the citric acid marinated with increasing calcium chloride concentration and meat revealed high exudation during cooking. This long marination duration. For example, Morris et indicated the low ability in water retention of the meat al. (1997) reported the 0.3 M of calcium chloride sample. It is suggested that the swelling of muscle treatments resulted in higher off flavor intensity was restricted to the citric acid at this concentration. scores than 0.1 or 0.2 M treatments. Furthermore, Gonzalez et al. (2001) reported the calcium marinade Sensory panel evaluations procedure applied longer than 24 hr. produced bitter After cooking, all the treatments samples were flavor. rated for sensory attributes and the results are shown Concerning the overall acceptability attribute, in Table 3. Among the marinated samples, juiciness the scores that rated with the calcium chloride and 1415 Klinhom et al./IFRJ 22(4): 1410-1416 the combination revealed no difference from the connective tissue and sensory properties of beef. unmarinated meat, even though tenderness score European Food Research and Technology 213: 88-94. suggested the better acceptation. This could be due Arganosa, G. C. and Marriot, N. G. 1989. Organic acids as to the fact that the meat samples from culled cow tenderizers of collagen in restructured beef. Journal of Semimembranosus muscle was not tender which Food Science 54: 1173-1176. 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