Meat Science 98 (2014) 569–573Contents lists available at ScienceDirect Meat Science journal homepage: www.elsevier.com/locate/meatsci TBARs distillation method: Revision to minimize the interference from yellow pigments in meat products P. Díaz, M.B. Linares, M. Egea, S.M. Auqui, M.D. Garrido ⁎ Tecnología de los Alimentos, Facultad de Veterinaria, Universidad de Murcia, Campus de Espinardo, 30100 Murcia, Spain a r t i c l e i n f o Article history: Received 22 April 2013 Received in revised form 9 April 2014 Accepted 14 June 2014 Available online 21 June 2014 Keywords: TBA distillation method Yellow pigment Interference Meat products Sucrose a b s t r a c t The aim was to study the effect of the incubation method and TBA reagent (concentration/solvent) on yellow pigment interference in meat products. Distillates from red sausage, sucrose, malondialdehyde and a mixture of sucrose–malondialdehyde were reacted with four different TBA solutions at five different temperature/time relations. Two TBA solutions were prepared at 20 mM using 90% glacial acetic acid or 3.86% perchloric acid. In addition, an 80 mM TBA solution was prepared using distilled water adjusted to pH 4 and another using 0.8% TBA in distilled water. The temperature/time relations were: (1) 35 min in a boiling water bath; (2) 70 °C/30 min; (3) 40 °C/90 min; (4) room temperature (r.t.) (24 °C) in dark conditions for 20 h; and (5) 60 min in a boiling water bath. The results showed that aqueous or diluted acid solutions of TBA reagent and the application of 100 °C for less than 1 h provided the best conditions to minimize the presence of yellow pigments and maximize pink pigment formation in meat products. © 2014 Elsevier Ltd. All rights reserved. 1. Introduction The 2-thiobarbituric acid (TBA) reaction as an index of lipid oxidation was first described by Kohn and Liversedge (1944) in their studies of the oxidation products in animal tissues. Until then, the nature of the substance reacting with TBA and the cause of pink colour formation were unknown. Bernheim et al. (1948) suggested a structure composed of three carbon atoms and an aldehyde or ketone group, and it was not until the early 1950s (Patton & Kutz, 1951) that malondialdehyde (MDA) was established as being responsible for the formation of a pink complex with TBA that showed maximum absorbance at 532 nm. MDA, the principal product of lipid peroxidation, has since become the centre of attention when monitoring the oxidation of meat and meat products (Raharjo & Sofos, 1993). However, the reaction between TBA and MDA is not specific and other carbonyl compounds resulting from lipid peroxidation may react with TBA to form a pink complex, leading to an overestimation of the results (Rey et al., 2005). Schmidt (1959) established a generic formula for these compounds: HO–(CR_CH)x–CHO, where x = 0, 1, and 2 and R may be any substituent; hence the term thiobarbituric acid reactive substances (TBARs). To the problem related with the lack of specificity, may be added the existence of other molecules that may react with TBA to generate yellow or orange complexes with an absorbance maximum at 440– 460 nm. The overlapping of peaks also leads to overestimation of absorbance at 532 nm, which corresponds to the pink complex. Indeed, several compounds present in meat products can react with TBA to ⁎ Corresponding author. Tel.: +34 868884708; fax: +34 868887167. E-mail address:
[email protected] (M.D. Garrido). http://dx.doi.org/10.1016/j.meatsci.2014.06.012 0309-1740/© 2014 Elsevier Ltd. All rights reserved. form yellow or orange complexes, such as water soluble proteins and peptides (Schmedes & Holmes, 1989; Shamberger et al., 1977), aldehydes (Kosugi et al., 1988), sugars (Wang et al., 2002), nitrites and nitrates (Ulu, 2004), metal chelators (Gutteridge & Quinlam, 1983), polyphenolicantioxidants (Raharjo & Sofos, 1993), pigments (Bird & Draper, 1984; Shamberger et al., 1977), amino acids, additives and fatty acids (Hoyland & Taylor, 1991). Various alternatives exist to eliminate the interference produced by these pigments and to increase specificity and detection limits; for example, the quantification of MDA by high-performance liquid chromatographic (HPLC) (Candan & Tuzmen, 2008), the addition of sulphanilamide to eliminate the interference produced by nitrites in cured meat products (Ulu, 2004) or filtration of the extract in solidphase cartridges (C18) before the TBA-MDA reaction takes place (Raharjo et al., 1993). Also, aldehydes can react with free amino groups of protein to give fluorescent Schiff bases which can be measured by spectroscopy (Gatellier et al., 2007). There are also mathematical methods based on the differences in absorbance between pink and yellow pigments (Yu & Russell, 1962) and on the third-derivative signal at 532 nm (Botsogloum, Fletouris, Papageorgiu, Vassilopoulos, Mantis & Trakatellis, 1994). However, the most widely used methods to quantify the TBARs index are distillation (Tarladgis et al., 1960) and aqueous acid extraction (Botsoglou et al., 1994). In the case of interference caused by the nonvolatile compounds present in meat products e.g. colourants such as paprika, distillation is recommended even though it is less accurate and slower (Ganhao et al., 2011; Raharjo & Sofos, 1993; Raharjo & Sofos, 1993; Yu & Russell, 1962). Although these methods are widely cited in the bibliography, the solvent and temperature/time relations applied The spice mixture was composed of paprika. were those involving acid solutions (glacial acetic acid and perchloric acid) of TBA (20 mM) and incubation temperatures lower than 100 °C. (4) room temperature (r.. 1959. is one such compound. Sucrose. 2011). (2002). (2011) used room temperature for periods of up to 20 h. 1960).. Díaz et al. the matrix of meat products is complex and it is difficult to identify any particular compound as being solely responsible for the interference. At the same time. garlic. (1960) and Botsoglou et al.2. The reactivity of TBA with substances that produce yellow pigments is similarly affected by these factors. Then. especially when perchloric acid (3. (2) 70 °C/30 min (Botsoglou et al. Results and discussion Table 1 shows the mean values and standard deviations of the absorbance recorded at 444 nm and 532 nm for the reaction between TBA and the distillate from red sausage stored for 15 days. They used the aqueous acid extraction method and standard solutions of sucrose and MDA to reproduce the development of yellow and pink pigments. 2011). an 80 mM TBA solution was prepared using distilled water adjusted to pH 4 (Wang et al.. Table 2 shows the mean values and standard deviations of the absorbance measurements at 444 and 532 nm for the reaction of TBA and distillates from the standard dissolutions. 1979).. 2. However. After incubation. 1990). 2 ml of distillate and 2 ml of TBA solution were added to a test tube and incubated at specific temperature/time relations (Ganhao et al. black pepper. and therefore the least advisable. the yellow colour developed . San Sebastian.570 P. while Ganhao et al. 1958). Spain). 1965. three different samples were prepared by adding 2. mainly paprika. Although some of these treatments also show maximum absorbance at 532 nm.. 2. 3.4. leading Wang et al. 2002) and another using 0. At room temperature.1.86% perchloric acid (Panreac. Selecta) and added to a spherical flask. Deventer. such as sucrose.2 mg) of TEP (Acros Organics. All these variables must be taken into account when choosing the most suitable incubation treatment to be used in cases where substances may exist that will interfere with the determination. (3) 40 °C/90 min (Wang et al. Sociedad de Envases Alimentarios. which were dissolved in water before use. 1994). 1948.5 ml of sucrose (BDH AnalaR) solution (10%) (S). Spain) without film and stored at room temperature for 15 days to ensure high levels of lipid oxidation. The temperature/time relations were: (1) 35 min in a boiling water bath (Tarladgis et al. the reactivity with the peroxidized lipids present in meat is produced at a pH of between 3 and 4 and is affected by the type of acid present during incubation (Asakawa & Matsushita. and the results were expressed as absorbance units (Wang et al. USA) was added. they were discarded because of the high values recorded at 444 nm..t. A relation 60/40% of pork lean/fat was minced (5 mm) using a P3298 cutter (Braher International..1.5 ml HCl (4 N) (Panreac. Ottolenghi. The MDA working solution (2. The samples were packaged in a transparent polystyrene tray BA85 (Sena. (1994) proposed higher incubation temperatures and shorter times than Wang et al.1 N HCl (Panreac. (2002) recommended treatment at 40 °C/90 min since above 50 °C sucrose may react with TBA. All ingredients plus minced meat were homogenized for 5 min using an RM-60 mixer (MaincaGranollers.. The samples used in this study were manufactured simulating commercial processing conditions. a reagent blank was prepared for each TBA solution and incubation treatment through the addition of 3 ml of distilled water and 3 ml of TBA solution. Spain). The hydrolyzed TEP solution was transferred into a 100 ml volumetric flask and diluted to volume with water to obtain the MDA stock solution (239 μl/ml). Although the specific reactivity of TBA with MDA is not affected by pH or the type of acid used (Janero. the solvent used in preparing the TBA reagent may also affect the reaction. Baker. 1994).. (1960). 2.P. In addition. 5 ml of MDA working solution (M) or a mixture of both (S + M). the test tubes were cooled in a water bath for 10 min.39 μl/ml) was obtained by pipetting a 1 ml aliquot into another 100 ml volumetric flask and diluting to volume with distilled water.86%) was used.. but for maximal colour development from peroxidized lipids. The samples were distilled in triplicate following the procedure described by Tarladgis et al. 2002). USA) was weighed into a screw-capped test tube and 10 ml of 0. Aduna. (1994). 2002). and the effect may further differ depending on whether distillation or the aqueous acid extraction method is used. / Meat Science 98 (2014) 569–573 differ between authors. Samples and distillation method A 10 g sample of meat product and 50 ml of distilled water were homogenized (Heidolph Silent Crusher. exposure to high temperatures (80–120 °C) for time periods ranging from 10 min to over 1 h is necessary (Bernheim et al. respectively. 1962)..) (24 °C) in the dark for 20 h (Ganhao et al. The samples were then distilled and the first 50 ml of distillate was collected.. which is elaborated with pork lean/fat and a mixture of spices.3. MDA working solution The MDA working solution was prepared through acid hydrolysis of 1. which may exceed 4% of the content of meat products. one drop of silicone antifoaming agent was added plus 2. New Jersey. (2002) to study the effect of TBA concentration and the temperature/time relation on the interference caused by sucrose in cooked ground beef meat. Tarladgis et al. The test tube was immersed in a boiling water bath for 5 min and quickly cooled with water bath. salt. The objective of the present study therefore was to establish the optimal conditions (incubation temperature/time and TBA reagent) that minimize the interference produced by sucrose and maximize the formation of pink pigment during TBARs determination using the distillation method in a meat product containing paprika. and (5) 60 min in a boiling water bath (Tarladgis et al. This wide range of absorbances measured at 444 and 532 nm reflects the need to unify the criteria used for TBA reagent and the incubation treatment when there are potential interferants. Material and methods 2..The fresh sausages meat was stuffed into lamb casings weighing 40 g. These results show that the reaction not only depends on temperature but also on the pH of the TBA solution. Netherlands) (Tarladgis et al. TBA solution and temperature/time of incubation Four different TBA solutions were tested at five different temperature/ time relations. The temperature is another factor which affects TBA reactivity. A quantity (73. 2011). 1960).3. The treatments that favoured the TBA–sucrose reaction. Then. USA) (Ganhao et al. Two TBA solutions were prepared at 20 mM using 90% glacial acetic acid (J. MDA may react with TBA to produce a pink pigment (Tarladgis et al. Pimpinellaanisum.8% TBA in distilled water (Botsoglou et al. Fresh red sausage Fresh red sausage is a typical Spanish meat product. USA) before diluting to 100 ml with distilled water.3-tetraethoxypropane (TEP) following the procedure described by Botsoglou et al. 2. TBA reagent (concentration/solvent) and incubation method are parameters which must be analysed together if we are to know the conditions that favour the formation of pink pigments and minimize the formation of yellow pigments in meat products. However. It is also known that the reaction is faster as the TBA concentration increases (Wang et al. J. lactose and dextrose. Also. 1960) or 3. and the absorption spectrum was scanned from 400 to 600 nm with an UV2 UNICAM UV/Vis Spectrometer. McKnight & Hunter.T. Sinnhuber et al. In all the treatments where the pH of TBA was below 4. The yellow colour of the sucrose–TBA and pink colour of the MDA–TBA were measured at 444 and 532 nm.. 2002). Wang et al.. 013 yz 0.159 0. xyz 0.003 a.030 0. z 0.022 z 0.211 0.226 0. y. the absorbance at 444 nm for the sucrose distillate did not significantly (P N 0.34 in the meat product and 0.017 c.003 d.16 for the standards).132 −1.007 c. b.516 0.009 a.051 a.539 0.013 b 0.050 y.181 0. y 0. y 0.028 a. y 0. y 0.082 −0.510 0.250 0.265 0. y 0.006 z 0.012 c.028 c.121 bc.171 0.183 0. yz 0.617 2.011 0. y 0.006 c. z 0.111 0.05) of TBA solution for the same treatment. especially in the meat product.008 a.05) of absorbance at 532 nm being observed for the distillate of MDA and the sucrose/MDA mixture.P.304 20 mM acetic acid glacial 90% 80 mM distilled water pH = 4 20 mM perchloric acid 3.013 0.368 0.173 0.014 b 0.545 2.016 a. yz 0. TBA 0.019 b. y 0.05) differences between treatments. x 0.011 0.014 1.975 0.018 z 0.411 a.695 0.011 0.052 b.161 0. the temperature of 100 °C for 35–60 min being the most effective at minimizing the interference at 444 nm.225 0.044 b. xy 0. yz 0. x 0.137 0.007 c.020 b. x 0. x 0.054 0. xy 0.029 0.064 0.074 b 0.083 −0.087 b.025 c.035 0.150 0.017 bc.027 a.015 a.335 0.010 0. x 0. x 0. x 0. y 0.004 a.300 0.003 a.756 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 70 °C/30 min 0. x 0.021 0.117 a.008 x 0.104 c. when the aqueous TBA solution (80 mM) was adjusted to pH = 4.013 bc.129 4. b.209 3. x 0.028 0. x 0. w: Effect (P b 0.063 −0. which showed statistically significant (P b 0.260 0.073 1. xy 0. y 0. y 0.159 0.155 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.058 a. x 0. a 0.012 c. z 0.224 0.521 0.039 b. these treatments fulfilled the criteria of A444/A532 close to zero (0.003 b.037 a.032 b.007 b.043 1. z 1.491 0.030 0.030 b.185 0.007 a.018 b. the first treatments to be selected were those using a solution of 0.096 0.198 −0. Choosing a priori the TBA–TREATMENT combinations with a A444/A532 b 1 and the maximum positive value for A532 − A444.029 b.039 0.136 a.006 a. y 0.001 c.059 0. y 0.000 a. z 0.016 a 0. w 0.032 ab 0.187 0.030 a.001 c. w 0. z. z 0.180 0.008 c.039 b.178 0.004 b. w 0.055 ab.008 b. ab 0.195 0.223 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 100 °C/60 min 0.088 0. x 0.156 0.007 b.015 b.006 c 0. w 0.005 0.874 0. xy 0.081 0.006 b. To select the most suitable TBA–TREATMENT combination. xy 0.023 b 0. z 0.212 0.005 1.006 y 0. z 0.280 −0.239 0. xy 0.142 −0.004 b. x 0.020 b.86% perchloric acid) and the treatments at 100 °C lasting 35 min and 60 min.029 a 0.227 0.206 2.017 b.011 d. x 0. z.057 0.015 c. we established that the difference between the absorbances at 532 nm and 444 nm (A532 − A444) should be the maximum with regard to the other treatments and greater than zero to minimize interference from the yellow pigment.079 0. The effect of the TBA–TREATMENT interaction on these variables was determined by ANOVA (see Table 3).013 b.525 1.281 0. x 0. y 0. y 0./20 h 0.069 −0.215 0.128 −0. z 0.007 b.177 0.004 0. x 0.062 0. y 0.023 c.201 0. x 0.311 2.381 0. x 0. Figs.001 b.002 z 0.008 w 0. the lowest values (P b 0.009 d.05) differ between the different incubation treatments and was always below 0.064 0.005 1.171 0. MDA and mixture of both. xy 0.756 0.899 0.041 ab. However.424 0.054 1. y 0. x 0.060 0.175 0.044 c. xy 0. 1 and 2 show the absorbance spectra for the distillates of red sausage and the sucrose/MDA mixture.209 0.007 b. although the pink pigments were still not maximal if we compare it with other TBA– TREATMENT combinations.001 b.336 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± r.024 a. z 0.8% distilled water A444 A532 A532 − A444 A444/A532 A444 A532 A532 − A444 A444/A532 A444 A532 A532 − A444 A444/A532 A444 A532 A532 − A444 A444/A532 0.158 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± r. ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 40 °C/90 min b. x 0.596 0.003 b.023 b.816 0.011 b.012 a.003 a.029 0.023 a 0.095 0.137 0.021 b. x 0. w 0.001 c 0. x 0.062 0.004 a.043 0.031(Table 2). z 0.013 b.077 0. x 0.111 0.311 0.013 bc. Furthermore.047 y . c: Effect (P b 0.012 a. w 0. x 0.429 0. z 0.007 c. / Meat Science 98 (2014) 569–573 571 Table 1 Mean values and standard deviations of the absorbance at 444 nm and 532 nm for the reaction between TBA and the distillate of red sausage stored for 15 days.001 a 0.003 b. y 0.008 c.168 0.031 0.016 0.079 b. less as the temperature increased.399 0.008 a.205 0. z 0.164 0.180 0.145 0.016 c.006 b.008 b. xy 0.14–0. x 0.008 c. xy 0.011 c.008 c. TBA Absorbance 100 °C/35 min 0. The use of aqueous solutions of TBA (80 mM) adjusted to pH 4 produced low interference on the part of the yellow pigments. x 0. w 0. c: Effect (P b 0.004 ab 0.609 0.007 a. y 0. y 0.918 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 100 °C/60 min c.011 a.194 0. x 0.118 1. x 0.005 b.116 0. Table 2 Mean values and standard deviations of the absorbance at 444 nm and 532 nm for the reaction between TBA and the distillates of sucrose. w 0.593 0.474 −0.192 1. z 2.923 6. the behaviour was similar in both cases. although the values were also low (below 0.014 a.013 b.007 b.05) of treatment for the same TBA solution.214 0.008 a.022 0.182 0.8% distilled water 20 mM acetic acid glacial 90% 80 mM distilled water pH = 4 20 mM perchloric acid 3.351 0. x 0.351 −0. As can be seen.t.021 0. x 0.014 8. x 0. y 0.007 a.089 0.30–0.008 a.067 0.8% perchloric acid for the different treatments assayed.006 a.023 b.102 0.038).026 a 0.007 b.292 0.097 x 0.016 0.042 −0.034 a.86% Absorbance S444 M532 (S + M)444 (S + M)532 (S + M)532 − (S + M)444/(S S444 M532 S + M444 S + M532 (S + M)532 − (S + M)444/(S S444 M532 S + M444 S + M532 (S + M)532 − (S + M)444/(S S444 M532 S + M444 S + M532 (S + M)532 − (S + M)444/(S 100 °C/35 min (S + M)444 + M)532 (S + M)444 + M)532 (S + M)444 + M)532 (S + M)444 + M)532 0. y 0.085 0.022 b.776 0. x 0.136 0.003 ab.114 2.z 0.720 0. z 0. z 0. y 0. xy 0.160 0./20 h xy 0.030 a 0.030 ab. y a. zw 0.044 0.020 a.009 b.007 d.148 0.t. z 0.006 a.126 0. z 0. z 0.620 0. z 0.008 d. y 0. w 0. yz 0. x 0.007 0. x 0. z 0. we established the following condition: that the ratio between the absorbances at 444 nm and 532 nm (A444/A532) should be close to zero to ensure that the combination favoured the formation of pink rather than yellow pigments.033 0. x. y 0. y 0.334 0.688 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.968 0.396 0.017 w. z 0.058 0.457 0.174 0. x 0.012 b.265 −0.377 0.013 y 0.020 a.211 0.166 0. y 0.224 0.019 0.061 −0. y 0. y 0.006 a. x 0.05) of TBA solution for the same treatment. x 0.568 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 70 °C/30 min c. z 0.013 0.05) of treatment for the same TBA solution.009 a.011 b. x 0.003 1.162 0.003 a.015 c. using the 20 mM TBA solution in 3.006 d. y 0. xy 0.008 d. y 0.038 0.003 b.146 0.035 b.007 c. This low reactivity between TBA and sucrose was also observed between TBA and MDA. z 0.006 a. Jointly.001 a.413 0.798 a. w: Effect (P b 0.011 0.039 a 0.031 a 0.000 b.031 b. z 0.099 0. w 0.009 c.530 −0.006 a.609 0.483 0.154 −0. y 0.472 0.013 ab. as used by Wang et al. x 0.006 a. x 0.006 a. z 0.012 a.025 ab 0.053 0.007 a. y 0.990 0. yz 0. w 0.192 0.031 0. as a complement to this condition.044 0.117 0. x 0. y 0.059 ab 0.228 a 0.052 b 0.204 0.261 0.155 −0.006 c 0. x 0.012 b.023 a 0.003 a.006 b.007 0. However.006 b. x 0.228 0.199 0.86% ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 40 °C/90 min 0. y 0. x 0.648 0.007 b. h 0.004 a. y 0. x 0.078 0.056 ab. y. xy 0.062 0.015 b.064 0.05) differences in the value of A532 − A444 compared with the other treatments.004 c. x 0.064 0.173 0.011 b. x 0. z 2.046 0.359 0.097 b. z 0.014 c.z 0. y 0. x 0. w 0. in the sucrose/MDA mixture there were statistically significant (P b 0. x. respectively.008 b. (2002). x 0.014 0. Díaz et al.057 0.003 a. y 0.021 a 0. x 0. w 0.209 0.204 0.8% TBA (distilled water) and 20 mM TBA (3.230 0.070 0.012 w 0.011 b. yz 0. x 0.074 0.837 14.016 c.090 ab. xy 0.009 b. 76 2. Botsoglou et al. the choice of incubation treatment differed from that made by Ganhao et al. Y = Treatment (1 = 100 °C/35 min.13 0.40 0. The use of a blank in the same conditions ruled out the possibility of an increase in absorbance as a result of changes in the TBA molecule (Tarladgis et al. involving the appearance of a yellow-orange colour with maximum absorbance at 450 and 490 nm (Tarladgis et al.57 1. 1962). Absorbance spectra for the distillate of sucrose and MDA using a solution of TBA (20 mM) in perchloric acid (3. in our case. which have a similar pink colour to the TBA– MDA complex. who concluded that the r.04 −0. X = TBA (1 = TBA 0.78 8.31 1.31 A AB AB AB AB AB AB AB ABC ABC ABC ABCD ABCD ABCD BCD CDE DEF DEF EF F 22 23 42 45 25 43 33 31 15 12 21 13 32 44 41 34 11 35 14 24 4.t. while absorbance at 532 nm was maximal.69 1.21 −0.20 0.62 0.97 2. The aqueous solutions of 0.86% perchloric acid for distillates of sucrose. The use of acids and a temperature of 100 °C may produce changes in the TBA molecule.01 A A B BC BC C D DE DEF DEF EF F G H H I J K L M 22 25 23 15 42 12 45 33 43 32 35 13 21 31 24 11 34 14 44 41 14.42 2. These findings do not agree with those of Ganhao et al.07 1.11 0./20 h treatment with the same type of TBA was best.22 0. ANOVA: Different indices reflect statistically significant differences (P b 0.84 −0. 2 = 40 °C/90 min.00 −0. the distillates obtained from red sausage and the standard solutions were transparent and there were no coloured pigments.21 0. However.12 −0. However.30 A B B C C C C C C C C C C C C C C C C C 41 44 24 11 13 14 12 21 35 15 34 31 32 33 43 25 23 22 45 42 0.13 −0.. .99 3.8% TBA gave acceptable results based on the established criteria. To avoid this problem.21 −0. MDA and a mixture of both.62 −0.18 0. (2011).05 0. which was not further analysed by the authors. the only problem with this being its instability and the possibility of precipitation with room temperature incubations.52 0.30 0. making them unadvisable.03 0.17 0.34 0. 4 = 100 °C/60 min. Absorbance spectra for the distillate of red sausage using a solution of TBA (20 mM) in perchloric acid (3.12 0. / Meat Science 98 (2014) 569–573 Table 3 ANOVA of variables A532 − A444 and A444/A532 as a function of the combination TBA × Treatment.48 0. (2002) recommended adjusting the solution to pH = 4. treatments that involved glacial acetic acid (90%) or perchloric acid (3. Such changes may increase the absorbance of the TBA–sucrose complex in treatments involving these conditions. using in their case a concentration similar to that used by Botsoglou et al.300 between 420 and 460 nm.37 0.17 0. These results were similar to those obtained with 20 mM TBA in 3.86%) and a temperature of 100 °C led to lower absorbance values at 444 nm than the other temperature/time combinations studied.43 0.41 0.92 −1.572 P. (1994).82 0.01 −0.72 6.16 0. Díaz et al.8%). The treatments with acid solutions of TBA and mild temperatures for long times favoured the formation of TBA-sucrose.16 0.92 0.06 0.49 0./20 h). (2011). especially at 100 °C.19 −0.06 0. For this reason.52 0.16 0.08 0. 1962)..86%) and different incubation treatments.54 −0. The absorbance of these pigments not only showed a maximum at 532 nm but also showed a mean absorbance of 0.19 0.08 A AB AB AB AB AB B B B B B B B B B B B B B B T × T: TBA × Treatment: XY.18 1. 2 = TBA 20 mM glacial acetic acid 90%. 2. (1994) used an aqueous solution of TBA (0.61 2.02 −0.11 0.52 2. 1.00 0.14 0.8% distilled water.11 −0. However the results of this paper were based on the interference caused by coloured pigments (flavonoids) from red fruit.t.22 1.08 0.20 0.05 −0.05). Wang et al. and 4 = TBA 20 mM perchloric acid 3. 5 = r.09 0. 3 = 70 °C/30 min.14 0.87 0.03 0.22 0.21 0.86%) and different incubation treatments.36 0.16 0. Fig.19 0.00 0.15 1. 3 = TBA 80 mM distilled water pH = 4.86%. Fresh red sausage Sucrose + MDA A532 − A444 A532 − A444 A444/A532 A444/A532 T×T Mean ANOVA T×T Mean ANOVA T×T Mean ANOVA T×T Mean ANOVA 41 44 14 24 11 34 21 31 13 35 32 33 43 12 15 45 42 22 23 25 0. In Fig. Taking into consideration the interference produced by the yellow pigments. Bernheim. Malonaldehyde content of food.M. W.. E. (2007). (1988). 29. 683–687. J. N. Tarladgis. sensitive. & Dugan. Rey. Meat Science. 39. 293–299. and feedstuff samples. Food Chemistry and Toxicology.. 44–48. (1962). Rapid. L. (1979). S. B. 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