Effect of Different Sources of Dietary Starch on Meat Quality, Oxidative Status and Glycogen and Lactate Kinetic in Chicken <i>pectoralis</i> Muscle

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2.3. Animal performance Parameters

Individual body weight at 45 and at 56 days of age was determined, and weight gain for the experimental period was estimated as the difference between the two measures. Individual daily food consumption and conversion efficiency (kg feed / kg gain) were also calculated. At 56 days, all the birds were slaughtered after fasting for 4 hours according to standards established by CHEA, making the sacrifice by cutting the jugular vein until total bleeding (3 min) to cause the least possible stress to the animal. Immediately after exsanguination, the liver was extracted (3 min) and the gallbladder was removed. The total fresh weight of each organ was measured followed by immediate storage at -80 °C until analysis.

2.4. Meat quality Parameters

The pH was determined at 10, 45, 90 minutes and 24 hours post mortem (maintained at 4°C) in the Pectoralis muscle from both sides, using a penetration pHmeter LT Lutron pH-201. Meat colour was determined (after 30 min bloom time) using the CIELAB method L*, a* b* at 10, 45 minutes and 24 hours post mortem (at 4°C) in the Pectoralis muscle from both sides (two readings on each) using a Minolta Lab CR-10 colourimeter. Measurement conditions were with illuminant D_65,anobservationangle of 10° and an aperture size of 0.31 inches. The chroma (C* = √ a*²+b*²) and hue (H° = arctan b*/a*) were also calculated ^[18]. Water drip loss was determined by the weight difference in 2.5 g of Pectoralis muscle samples at 15, 45, 90 minutes and 24 hours post mortem from both sides ^[19]. Glycogen and lactate content were measured in Pectoralis muscles from both sides at 10, 45, 90 minutes and 24 hours post sacrifice (maintained in freezer at -80°C until analysis) and at 3 min post mortem for the liver. For both determinations, an 8 g meat sample was homogenized and extracted with 8 ml of HCl acid (4 N) for 2 hours at 100°C, filtered and neutralized by adding NaOH (4 N) until pH reached 6.5 – 7 ^[20]. Glycogen was determined as glucose total equivalents following Bergmeyer et al, ^[21] using colourimetric diagnostic kits (1001201, Spinreact, Spain) and expressed as mg glucose/kg fresh meat. Lactate was assayed in the same hydrolysed slurry using commercially available enzymatic colourimetric diagnostic kits (LO-POD; 1001330; Spinreact, Spain) and expressed as mg lactate/kg fresh meat.

For the oxidation of lipids and proteins and Fe haem determination, samples of Pectoralis muscle (both sides at 24 hours post mortem) were stored in polyethylene bags at -30°C until analysis (fresh meat) or were vacuum packed, kept at 4°C for 5 days and then frozen at -30°C until determination (aged). Lipid oxidation was determined by the TBARS method with some modifications ^{[22, 23]}. A 1.5 g sample of Pectoralis muscle was homogenized (1 minute, 8000 g) with 30 ml KCl (Mallinckrodt 6858), 750 µl EDTA and 300 µl BHT. An aliquot of 4 ml was taken and frozen at -20°C to determine protein oxidation. The rest was centrifuged at 2000 g, 10 min, 4°C. One millilitre of the supernatant was taken, added to 1 ml TBA-TCA and boiled for 30 min. It was then placed on ice for 5 min to stop the reaction and left at room temperature for 45 min. Then, 2 ml N-butanol ppa was added, vortexed and centrifuged at 3000 g for 3 min. The supernatant was measured colourimetrically at 535 nm. The results were given as mg of MDA/kg of fresh meat. Protein oxidation was estimated by the reactions between carbonyls and DNPH (2,4-dinitrophenylhydrazine) with the resulting formation of a Schiff base, which produces the corresponding hydrazone, quantified spectrophotometrically at 360 to 385 nm ^[23]. The determination was performed using the method of Mercier et al, ^[24] with some modifications. From the homogenized mixture, 2 ml was taken and centrifuged at 2000 g for 10 min at 4 ° C. Then, 2 ml of 20 mM DNPH in 2 M HCl was added, and the mixture was incubated for 1 hour at room temperature, vortexed every 10 min. Next, 2 ml of 200 g.l^-1 TCA was added and the mixture allowed to stand 15 min, vortexed every 5 minutes, then centrifuged at 2000 g for 10 minutes and the supernatant removed. The pellet was washed 3 times with 4 ml ethanol-ethyl acetate (1:1) and centrifuged after each washing. After the third wash, the pellet was dissolved in 6 ml of guanidine 6 M HCl with 0.02 M KH₂PO₄ and incubated at room temperature for 15 min with regular stirring. The resulting mixture was centrifuged at 2400 g for 10 min, and the absorbance of the supernatant was measured at 370 nm. The concentration of DNPH was calculated using its molar extinction coefficient of 22.000 M^-1 cm^-1, and the results were expressed as nmol DNPH/mg protein. Protein content was determined at 280 nm in the homogenized mixture using bovine serum albumin (BSA) from Sigma Chemical Co. (St. Louis, USA) as a protein standard, as described by Stoscheck, ^[25]. For the Fe haem determination, the Hornsey, ^[26] method was followed with some modifications ^{[5, 27]}. Samples of 1 g Pectoralis muscle were macerated in a mixture of acidified acetone in a glass tube, capped to prevent evaporation, and vortexed for 1 minute. After an hour of incubation at room temperature in darkness, the mixture was filtered through an ashless paper and the absorbance measured at 640 nm against acidified acetone. The Fe haem concentration was calculated from the content of haematin with a factor of 0.082.

2.5. Statistical Analysis

Data were analysed as a complete randomized design using the General Linear Model (GLM) procedure of NCSS (NCSS software release 2006, 329 North 1000 East, Kaysville, UT 84037) with the individual bird as the experimental unit. The model used for the ANOVA of oxidation measures and haem iron included diet, process (fresh or aged) and diet x process interactions as fixed effects. The data on pH, drip loss, glycogen and lactate in muscle at different times post mortem were analysed by repeated measures ANOVA with diet as a between-animal factor and time post mortem as a within-animal factor. Interactions are nor shown if p>0.05.To determine differences for each parameter due to the diet at each time post mortem, we used one-way ANOVA. To analyse the performance parameters, we used one-way ANOVA. When appropriate, the Tukey-Kramer post hoc multiple comparison test (p<0,05) was used.

3. Results and Discussion

3.1. Animal Performance Parameters

To evaluate the effect on the animal response of different starch sources in diet prior to slaughter for a period between 45-56 days of age, 300 g.kg^-1 of the ground corn in a standard corn-soya diet was replaced with starch from broken corn, ground sorghum or pure corn starch, and the growth parameters were measured. The results are shown in Table 2. No significant effects were observed due to the source of starch in the parameters considered (p>0.05). Fernández et al, ^[28], García et al, ^[13] and Nyannor et al, ^[29] reported no changes in weight gain, feed conversion and mortality in birds that received diets with replacement of corn by sorghum in proportions from 0 to 100 %, likely due to the similarity in nutritional value, crude protein and metabolisable energy of the two cereals, which assisted in formulating rations with similar nutritional bases. A possible explanation for both the previous and current results is that a lesser sensitivity to tannins is found in the later periods of the life production of the bird.

Table 2. Effect of dietary starch source on animal performance parameters

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3.2. Meat Quality Parameters

3.2.1. pH. The pH was measured at 10, 45, 90 minutes and 24 hours post mortem in the Pectoralis muscle, in response to the starch sources received during the last 11 days before slaughter, and the data are shown in Figure 1. According to the results obtained and considering all post mortem times, the birds receiving broken corn exhibited a significantly higher average pH (6.34, p<0.05) than the birds receiving ground corn (6.23, p<0.05). Ground sorghum and pure starch showed a similar intermediate average pH (6.28 and 6.30, respectively). We can also observe a clear effect of time on the pH drop, as the values reached were all significantly different at 10, 45, 90 minutes and 24 hours (6.67, 6.42, 6.10 and 5.97, respectively). At 10 minutes post mortem, the muscles of the birds receiving broken corn and pure starch have the highest pH (6.74 and 6.77, p<0.05), whereas sorghum sources show the lowest (6.5, p<0.05). At 45 minutes, the source of sorghum and broken corn resulted in significantly higher pH in the muscles (6.58, 6.52, respectively; p <0. 05) than the other two sources and, as the same results were found at 90 min post mortem, these sources could produce a slower pH descent curve. In birds, the decline in pH for the pectoralis muscle, rich in white fibres, can continue until 6-10 hours post mortem, and the pH_u rises to 5.4-5.6 ^[30].

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Figure 1. Effect of dietary starch source on pH evolution at 10, 45, 90 min and 24 hours post mortem in the Pectoralis muscle. Data are the mean ± SEM for n=6 birds (ground corn, pure starch) or 7 birds (ground sorghum, broken corn). a,b,c,d, indicate significant differences by Tukey-Kramer test (p<0,05). Bars within a time group with a different letter above them differ significantly by one-way ANOVA and Tukey-Kramer test (p>0.05)

In our work, pure starch, which is highly digestible, induced a more rapid fall in pH, whereas starch from sorghum induced a slow fall. Garcia et al, ^[13] also observed lower initial pH values in the pectoralis muscle when birds were fed with diets containing more than 75 % sorghum. No differences were found at 24 hours post mortem time between the sources. At this time, the individual values of the final pH ranged between 5.6 and 6.0, which was consistent with the values reported by Duclos et al, ^[7], Jones & Grey, ^[31] and Sams & Mills, ^[32]. These values could indicate normal glycolysis ^[33]. The pH value at 45 minutes would be related to other quality parameters such as colour and water retention ^[33]. As the pH at 10 and at 45 min would be key to the relationship with water retention and colour, we expected that the two sources that led to slower pH fall also improved water retention and reduced colour loss. The rapid decline in pH in the pectoralis muscle from groups fed with pure starch or ground corn (at 10 and 45 minutes post mortem) was similar to the decline found in birds supplemented with glucose, which could indicate a greater amount of liver glycogen due to feeding with more rapidly degradable starch sources ^[34].

However, Bee et al, ^[14] found no difference in the decline of pH when comparing diets with high and low carbohydrate digestibility. Animals fed with ground sorghum or broken corn presented a pH decrease curve similar to the one found in animals not supplemented with glucose, which leads us to think that both cereals behave as slower availability starch sources, as observed through in vitro digestibility tests (starch availability of 85.30 % for pure starch and 34.16 % for ground sorghum) ^{[34, 35]}. It would be expected that, if the pH decrease curves are sufficiently fast, water retention would be adversely affected as the pH falls below 6 in meat that is still warm, which causes protein deterioration. The early pH drop may be due to a higher rate of degradation of the glycogen deposited in the animals fed diets of ground corn or pure starch or to an alteration in muscle buffer capacity. Henckel et al, ^[36] proposed that significant effects on the meat quality in pigs due to glycogen concentrations at slaughter could be expected when the level is below a critical threshold of 53 μmol.g^-1 of wet tissue.

The ability of sorghum to modulate the pH drop could be due to the action of tannins slowing down the starch digestion in broilers or to the protein in sorghum grain, whose amino acid cysteine in β-and γ-kafirins form disulphide bridges in the mature grain, reducing the digestibility of protein ^{[37, 29, 38, 39]} . The effect on pH of broken corn could be due to the lesser digestibility of coarse particles of corn ^[40].

3.2.2. Glycogen and lactate kinetic. Glycogen and lactate produced in the pectoralis muscle were measured at 10, 45, 90 min and 24 hours post mortem, and the data are shown in Table 3. There is a clear effect of the sources on glycogen content, where the pure starch sources show a higher amount of glycogen (3.52 g glucose.kg^-1 of meat), whereas the ground sorghum gives the lowest value (2.09 g glucose.kg^-1 meat). There is also a time effect, showing a permanent decrease in glucose content from 10 minutes to 24 hours post mortem. No interaction effect was found between sources and time for these parameters. For each time, differences due to starch sources were found at 24 hours post mortem, resulting in higher glycogen with a pure starch source and lower glycogen with ground sorghum (1.29 and 0.67 g glucose.kg^-1 meat, respectively, p<0.05). This higher amount of glycogen in the muscles of birds fed with a highly digestible source could be the cause of a lower pH at 45 min post mortem.

Table 3. Effect of dietary starch source on glycogen (g glucose.kg^-1 fresh meat) and lactate (g lactate.kg^-1 fresh meat) content in Pectoralis muscle at 10, 45 and 90 min and 24 hours post mortem

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The data obtained for lactate (Table 3) show no differences among the sources and no interaction effects, but there was a clear effect of time on the lactate content in the pectoralis muscle. In liver (data not shown), the glycogen content at 3 min post exsanguination was higher in the ground corn diet (7.66 g.kg^-1 meat) and lower in the ground sorghum diet (5.85 g.kg^-1 meat; p<0.05), while the lactate content did not differ between diets (p>0.05). The pectoralis muscle has a high buffer capacity, demonstrated by the fact that the same quantity of lactate is accumulated in muscle with different pH_u values ^[41].

However, in this work, pHu did not differ between diets. Rosenvold et al, ^[11] reported that a small decrease in muscle glycogen stores induced by a diet rich in low digestible carbohydrates resulted in a significant increase in pH_i and concluded that the diet, rather than the glycogen store in the muscle, is the main factor in the glycogen metabolism and/or composition of the muscle. In pigs, it was reported that a diet poor in available starch could decrease the protein content and increase the lipid content in muscle ^[42].

3.2.3. Drip loss. Table 4 shows the effect of including different sources of starch in the diet of chickens on the water loss parameter in the pectoralis muscle, in samples obtained at 10, 45, 90 minutes and 24 hours after slaughter.

Table 4. Effect of dietary starch sources on drip loss (%) at 10, 45, 90 min and 24 hours post mortem in the Pectoralis muscle

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There was no source effect on the drip loss (p>0.05). There was a time effect (p=0.001), as water loss is clearly higher in the first 10 minutes post slaughter (5.38 %) versus 45 to 90 minutes and 24 hours (3.40, 3.21 and 4.06 %, respectively). The water holding capacity and the aptitude for transformation are largely influenced by the final pH and its decay rate after the slaughter, as the decline of pH to its lowest value (isoelectric point of myofibrillar proteins; 5.4-5.6) is accompanied by a decrease in the net charge of proteins, causing a loosening of the myofibrillar protein network related to the decrease in electrostatic forces of repulsion between the protein filaments ^{[43, 44]}. This development results in a transfer of water from the intracellular space to the extracellular space ^[45].

Consequently, a detrimental effect is noted on the taste of fresh or processed products in terms of juiciness and tenderness due to the loss of soluble proteins and haem groups ^{[43, 46, 47]}. The expected impact of the different sources of starch on the water loss as a consequence of the effect on the pH was not observed in this work. Although the pH decrease was moderated by the replacement of corn with ground sorghum or broken corn, the effect was not sufficient to reduce the water drip loss (up to 24 hours post slaughter). Garcia et al, ^[13] reported an effect of sorghum on the drip loss when introduced at levels of 500g.kg^-1 and higher. A possible explanation of the lack of effect found in our work could be that the substitution was only of 300g.kg^-1.

3.2.4. Colour. The variation of colour in the pectoralis muscle of animals that received different sources of starch was measured immediately after slaughter and at 10, 45 minutes and 24 hours post mortem (Table 5).

Table 5. Effect of starch source included in diet on the colour, measured as L , a , b * in muscle Pectoralis at three post mortem times (10, 45 min and 24 hours)

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In poultry as well as in other species, colour is the single most important sensory attribute that affects consumer purchasing decisions. The colour of fresh or cooked meat is a very important factor in the buying decision made by the consumer, and this colour is often seen by consumers as an indicator of the freshness and overall quality of the meat ^{[48, 49]}. The colour of meat is generally characterized by its chromaticity (pigment haem) and its surface brightness. Chromaticity depends on the physico-chemical state of the pigment, and the pigment concentration is dependent on biological factors, while the brightness depends on extrinsic factors (pre-slaughter conditions and manipulations after slaughter) ^{[50, 51]}. Colour is usually expressed by three components: brightness, L*; redness, a*; and yellowness, b*. According to the data obtained in our work for the pectoral muscle, the different starch sources in finishing diets for broilers did not change the brightness or redness of chicken meat at any of the times considered (p>0.05). The values found for redness, a*, increase with time (p<0.05), which could be explained by the chemical changes undergone by the tissues to establish rigor mortis. The a* values for the pectoralis muscle for all dietary starch sources evaluated correspond to the values reported by Fletcher et al,⁴⁸, a*= 1.7-2.2 at 24 hours post slaughter.

As for the yellowish-green colour, determined by the variable b*, we observed that broken corn contributes favourably to the emergence of more yellow at 10 minutes and 24 hours post mortem (p> 0.05), perhaps because the larger particle size remains longer in the animal's digestive tract and promotes the action of enzymes to improve absorption of the corn pigments. In the study of the proportion of red and yellow through the Hue parameter (Table 6), we note that in the pectoralis muscle, there was no effect of the source at any of the times considered. It is noteworthy that the values found are in the same order (76.45 ± 1.63 to 76.03 ± 2.74, in chickens fed diets containing 680g.kg^-1 corn and 244g.kg^-1 sorghum) as the results found by Ponsano et al, ^[52].

Table 6. Effect of dietary starch source (ground sorghum, broken corn, ground corn, pure starch) on colour saturation measured as chroma (C) and color tone measured as angle hue (H°) in Pectoralis* muscle at three times post mortem (10 min, 45 min and 24 hours)

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With respect to saturation or purity of colour, the coordinate Chroma, both at 10 min and 24 hours post mortem, broken corn induced significantly higher values (23.13 and 25.29, respectively) than the other sources (p <0.05) in the pectoralis muscle. One possible explanation is that the carotenoid pigments in broken corn would be more available in the digestive tract, resulting in a more vivid colour in the meat^[52]. Ground sorghum and pure starch produced the lowest colour saturation in the pectoralis muscle. Garcia et al, ^[13] studied the inclusion of sorghum replacing corn in broiler feeds and found a significant negative correlation between meat pH decrease and corn replacement. Sorghum inclusion also affected the meat colour, promoting paler meat.

According to Bressan & Beraquet, ^[53] meat colour is the factor most affected when replacing corn with sorghum. Garcia et al, ^[13] reported no differences in the brightness parameter (L*), but found decreases in redness (a*) and yellowness (b*) with increasing replacement of corn by sorghum. Although the colour in carcasses of chickens fed with sorghum could be affected by the lack of pigments, recent studies have shown that the tannins in the diet may slow the deterioration of colour in lamb under prolonged storage, although in this case the reason would be a retarding effect on the appearance of methaemoglobin ^[54]. However, the study of Bee et al,^[14] showed that using diets with low-digestibility carbohydrates produced darker meat and less yellow colouration in pigs subjected to either transport or increased stress before slaughter but differentially affected different muscles according to their respective glycolytic potential.

3.2.5. Lipid and protein oxidation. Another aspect of our study was the oxidation of lipids and proteins in fresh and aged meat with respect to the source of starch in diet. There was no effect of starch source on the lipid oxidation (Table 7) regardless of the process of preserving poultry meat, expressed as TBARS (mg MDA.kg^-1 meat). These results differ from the findings of Rajasekar at al, ^[55] in rats fed diets with high added fructose, as that study recorded changes in lipid metabolism (significant increase in cholesterol, triglycerides and free fatty acids, lower level of phospholipids) and oxidative stress in their tissues (higher content of TBARS, lipid hydroperoxide and conjugated diene). To test this hypothesis, a profile fatty acids study could be desirable. In cereals, it is likely that a slower release starch allows assimilation and utilization, preventing the accumulation of glucose in plasma and thus reversing the previous effect.

Table 7. Effect of dietary starch source and process (fresh and aged) on the lipid oxidation, measured as TBARS (mgMDA .kg^-1 meat) on the Pectoralis muscle

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Tannins in diets for rats reduced lipid oxidation almost as effectively as vitamin E supplements ^{[56, 57]}. Du et al, ^[58] found that birds fed high-tannin sorghum (Sorghum Red Valpo) had lower TBARS values, so that the presence of sorghum improved the oxidative stability of raw meat, probably due to the polyphenols that act as antioxidants. However, in our work, all of the treatments had the same effect on lipid oxidation. In relation to the conservation process, lipid oxidation is not affected by the conservation process regardless of the dietary source (p>0.05). Poultry meat in cold storage (4°C) or in vacuum packaging has been reported to develop increased TBARS values even within a few hours post slaughter^{[59, 60, 61]}.

However, in our work, the vacuum produced a protective effect on lipid oxidation, showing equal TBARS values to fresh meat. In the study of the effect of starch sources on the oxidative deterioration of proteins (Table 8), it was observed that the diets with pure starch resulted in greater protein oxidation (1.06 nmol DNPH.mg^-1 protein), while broken corn and ground sorghum yielded significantly lower values (0.83 and 0.75 nmol DNPH.mg^-1 protein) regardless of the conservation process. There was no effect of ageing on muscle protein degradation (p> 0.05). In fresh meat, pure starch sources showed higher values of protein oxidation, while broken corn had the lowest value (1.08 and 0.60, p<0.008). It could be concluded that the diets containing more available starch promote oxidative damage to the proteins in muscle. The processes of protein oxidation are linked to pro-oxidant factors similar to the ones observed for the oxidation of lipids, resulting in the loss of muscle function associated with a higher water loss and less stable gels ^{[62, 63]}. As these oxidation processes are affected by the same factors as lipid oxidation, it is expected that the increased availability of starch in the diet favours increased glucose in blood plasma and therefore increased protein oxidative damage ^[64]. In rat skeletal muscle with a low state of glycogen, Philp et al, ^[65] found an increased lipid utilization and the transcription of genes regulating mitochondrial β-oxidation. This could be save the muscle protein oxidation as an adaptation mechanism to do face to stress at the time of slaughter. Then, we can hypothesize that a diet high in carbohydrates and a high level of glycogen in muscle immediately post slaughter lead to increased muscle protein oxidation. These findings are original results that show an effect of ground sorghum and broken corn incorporated in pre-slaughter diets on meat quality, perhaps related to the reduced availability of starch and not only the presence of tannins in sorghum.

3.2.6. Haem iron. Haem iron is an indicator of nutritional content, as it is the most bioavailable form of iron in meat and is also an indicator of loss of pigment during poultry meat processing ^[66]. There was no effect of diet on the haem iron content found in the pectoralis muscle (Table 9) (p> 0.05). We had expected a similar effect of the starch source on haem iron to the case of proteins, i.e., a lower presence of haem iron in meat chickens fed with pure starch, as in meat haem is mainly found in iron-dependent proteins. However, the alteration in muscle protein is not reflected in the haem iron, probably because the deterioration was not sufficient to alter these values. Luciano et al, ^[54] found clear effects of dietary tannins on the conservation of haem iron, but the same result was not found in this work.

Table 8. Effect of dietary starch source and process (fresh and aged) on the protein oxidation (nmolDNPH.mg^-1 protein) of the Pectoralis muscle

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Table 9. Effect of dietary starch source on Fe haem content (mg.kg^-1 fresh meat) in the fresh and aged Pectoralis muscle

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