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Proximate composition, antihypertensive and antioxidative properties of the semimembranosus muscle from pork and beef after cooking and in vitro digestion
Meat Science 96 (2014) 916–921
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Proximate composition, antihypertensive and antioxidative properties of the semimembranosus muscle from pork and beef after cooking and in vitro digestion Ida-Johanne Jensen a,⁎, Junio Dort b, Karl-Erik Eilertsen a a b
Norwegian College of Fishery Science, University of Tromsø, N-9037 Tromsø, Norway Department of Food Science and Nutrition, Laval University, Quebec Qc G1V 0A6, Canada
a r t i c l e
i n f o
Article history: Received 12 February 2013 Received in revised form 4 October 2013 Accepted 8 October 2013 Keywords: Amino acid Angiotensin converting enzyme ORAC Bioactive
a b s t r a c t The aims of this study were to evaluate and compare proximate composition, antihypertensive activity and antioxidative capacity of the semimembranosus muscle from pork and beef and to study how these characteristics were affected by household preparation and subsequent digestion. The proximate composition was similar between pork and beef. Both pork and beef contained protein with the essential amino acids. Cooking in a heated pan did not affect the retention of lipid or sum of amino acids, but reduced the amount of the free amino acid taurine. The antihypertensive effect did not differ significantly between pork and beef, whereas the antioxidative capacity did. Cooking affected the antioxidative capacity negatively. The results from this study show that pork and beef are equally good sources of protein and bioactive properties, and whereas the nutritional composition is not affected, bioactive properties may be reduced after household preparations. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Hypertension is a major independent risk factor for CVD affecting people world-wide (Harris, Cook, Kannel, Schatzkin, & Goldman, 1985; WHO, 2011). Angiotensin converting enzyme (ACE) which plays an important role in increasing blood pressure and hypertension is normally pharmacologically treated by inhibition of ACE. Pharmaceuticals currently in use are, however, associated with adverse effects such as dry cough, taste alterations, skin rashes and renal dysfunction (Atkinson & Robertson, 1979). Moderate hypertension may be controlled by a nutritional approach and numerous studies have documented antihypertensive and ACE inhibitory effects of different food sources (Fujita, Yokoyama, & Yoshikawa, 2000; Wijesekara & Kim, 2010). Oxidative stress, a condition of oxidant overload in relation to antioxidants, has also been connected to different diseases including CVD (Halliwell & Gutteridge, 2007). A higher intake of antioxidants has indeed been linked to a lower occurrence of oxidative stress and antioxidative capacity (AOC) has been documented in different peptide sources (Samaranayaka & Li-Chan, 2011).
⁎ Corresponding author. Tel.: +47 776 46 721; fax: +47 776 46 020. E-mail address: [email protected] (I.-J. Jensen). 0309-1740/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.meatsci.2013.10.014
In recent years studies have focused on bioactive food derived components, exerting a positive physiological effect in the body, beyond that of nutrition. As major parts of proteins, peptides are examples of such bioactive components, which have been demonstrated to be hypotensive, antioxidative, opioid-like and anti-inflammatory (Ryan, Ross, Bolton, Fitzgerald, & Stanton, 2011). In order to exert any effect, the peptides need to be released during processing, fermentation or digestion. Lean red meat is a high quality protein source with low fat content. It is also an important source of the B group of vitamins, and minerals such as iron, copper, zink and manganese (Friedman, 1996). Red meat has in the past been considered a major dietary risk factor for cardio-metabolic diseases and type 2 diabetes mellitus. However, such an association has only been confirmed with processed meat (Micha, Michas, & Mozaffarian, 2012), demonstrating that processing may alter the nutritional value of meat. In fact, culinary preparation and processing of meat may result in both advantages, such as enhanced taste, killed pathogens, inactivated anti-nutrient enzymes and increased digestibility and bioavailability and disadvantages such as browning reactions, racemisation, destructed valuable components (Meade, Reid, & Gerrard, 2005) and lost water soluble-potentially bioactive-components (Larsen, Stormo, Dragnes, & Elvevoll, 2007). Although several studies have evaluated the nutritional and bioactive properties of meat, the effect of household preparation has been disregarded. The aims of this study were to evaluate the proximate composition, antihypertensive activity and antioxidative capacity of pork and beef and to study the effect of household preparation and subsequent digestion on these parameters.
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2. Materials and methods 2.1. Materials Pork (Sus scrofa domesticus) (n = 20) of the breeds Yorkshire, Duroc and Norwegian countrypork, male and female, 5–6 months of age, and approximately 100 kg live weights and beef cattle (Bos taurus) (n = 20), of the breed Norwegian red cattle, both male and female, 6 months of age and approximately 300 kg live weights, were slaughtered in Troms County, Norway in September 2010. The carcasses were kept at 4°C for 3days and at day 4 post mortem, the semimembranosus muscles (approximately 2.5 kg) from twenty animals of each species were dissected and brought to the laboratory. 2.2. Experimental design From each of the semimembranosus muscle samples, two pieces of approximately 100 g (10 cm × 15 cm× 1.5 cm) were cut from the central portion, labelled and randomly distributed in two groups; raw and cooked. Cooking was performed by placing the pieces on a pre-heated pan without oil and heated until thoroughly cooked, 10 min on each side reaching an internal temperature of 75 ± 3 °C. The pieces, raw and cooked, were weighed and minced before storage in sealed plastic bags at −50 °C. All samples were analysed for moisture, ash and fat content and 10 samples were analysed for total amino acids. After a simulated digestion (GI), antihypertensive activity and AOC were evaluated in ten samples for ACE inhibitory activity and oxygen radical absorbance capacity (ORAC), respectively. Cooking loss was determined as the percentage weight loss after cooking. True retention (TR) of selected components was calculated to evaluate compositional changes during processing according to Murphy, Criner, and Gray (1975): TR; % ¼
nutrient content per g cooked meat g meat after cooking nutrient content per g raw meat g meat before cooking 100:
2.3. Analytical methods 2.3.1. Proximate composition The AOAC 925.04 and AOAC 938.08 (Cunniff, 1995) were used to determine the water and ash contents. Approximately 10 g sample was dried at 105 °C until constant weight and water content was determined gravimetrically. The water free sample was thereafter combusted at 500 °C for 12 h and ash content determined gravimetrically. Total lipids were determined gravimetrically after extraction (Folch, Lees, & Stanley, 1957), replacing chloroform/ methanol with dichloromethane/methanol due to safety reasons. 2.3.2. Amino acid analysis For analysis of total amino acids except tryptophan, approximately 200 μg sample was mixed with 900 μL 20 mM norleucine (Sigma Chemicals Co, St. Louis, MO, USA) and 1200 μL 12 M HCl and hydrolysed at 110 °C for 24 h (Maehre, Hamre, & Elvevoll, 2012). The hydrolysates (100 μL) were dried under nitrogen and dissolved in lithium citrate buffer (pH2.2) at a suitable concentration and analysed using a Biochrom 30 Amino Acid Analyzer (Biochrom Limited, Cambridge, UK) with a lithium citrate equilibrated column and post column derivatization with ninhydrine. Norleucine was used as internal standard and the signal was analysed with Chromeleon software (Dionex, Sunnyvale, CA, USA). The A9906 (Sigma) physiological amino acid standard was used for identification and quantification of the amino acids. 2.3.3. In vitro gastrointestinal digestions The in vitro GI model used to digest samples for analysis of ACE inhibitory activity is described by Dragnes, Stormo, Larsen, Ernstsen,
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and Elvevoll (2009). Sample (12.5 g) was mixed with 50 mL water and the pH was adjusted to 2 with 3 M HCl. The stomach phase was simulated by adding 50 mg pepsin (Sigma P6887) dissolved in 1 mL water, and the mixture was incubated 2 h at 37 °C on a magnetic stirrer. The pH was thereafter adjusted to 6.5 with 3 M NaOH and the intestinal phase was simulated by adding 50mg trypsin (Sigma T1426) and 50mg chymotrypsin (Sigma C4129) dissolved in 2 mL water. After a total of 4.5 h of incubation, the digests were frozen and freeze-dried. The freeze-dried samples were finely ground and kept frozen at −55 °C until determination of ACE inhibitory effect. The in vitro digestion described by Jensen, Abrahamsen, Maehre and Elvevoll (2009) was used to digest samples for analysis of AOC. The digestion was simulated by adding a pepsin solution containing 0.462% pepsin (Sigma P6887) (w/v), 49 mM NaCl, 12 mM KCl, 10 mM CaCl2, 2.4 mM MgCl2, and 2.5 mM K2HPO4, representing the gastric phase and bile/pancreatic solution (containing 0.2 g pancreatine (Sigma P1750), 1.25 g bile extract (Sigma B8631) and 0.1 M NaHCO3 in 50 mL distilled water) representing the intestinal phase, along with gradient pH adjustment. An amount of 1 g muscle (or 50 mM Na2HPO4 with 0.9% NaCl and pH 6.75 as control) was mixed with 15 mL pepsin solution. The pH was adjusted to 5.5 and a 3 mL sample was collected (0 min). The remaining reaction mixtures were incubated at 220 rpm and 37 °C for 30 min, before the pH was adjusted to 3.8. Further incubation was continued for 30 min before another pH adjustment to 2.0. After 15 min of incubation, another 3 mL sample was collected (75 min). Thereafter 1.5 mL bile and pancreatic solution was added the reaction mixtures. The pH was adjusted to 5.0 and the reaction mixtures were continuously incubated for 30 min before the pH was adjusted to 6.5. The reaction mixtures were incubated for another 60 min and the last sample was collected (165 min). The samples were centrifuged at 4500 g at 4 °C for 15 min to remove large particles. The digested samples were frozen and kept at −55 °C until analysis of AOC.
2.3.4. Angiotensin-converting enzyme inhibitory assay Angiotensin converting enzyme inhibitory activity assay was performed based on a previous method (Cushman & Cheung, 1971) with modifications. The freeze-dried sample was dissolved in 100 mM sodium borate buffer (pH 8.3) and 25 μL was pre incubated with 100 μL substrate (2 mM hippuryl-L-histidyl-L-leucine (HHL, Sigma H1635) in 100 mM sodium borate buffer), at 37 °C for 10 min. The enzymatic reaction was initiated by addition of 50 μL ACE (Sigma A6778) (5 mU, final concentration) and carried out on a shaker at 37 °C. After 30 min, the reaction was stopped by addition of 215 μL 1 M HCl and the end product hippuric acid was measured by quantitative HPLC analyses (Dragnes et al., 2009). A dilution series from 100 to 2000 μg/mL was used for the samples and the amount of sample/ACE inhibitor needed to inhibit 50% ACE activity was defined as the IC50% value and presented as μg/mL hence a lower IC50% value signified a higher ACE inhibitory activity.
2.3.5. Oxygen radical absorbance capacity The ORAC assay was carried out according to Dávalos, GómezCordovés, and Bartolomé (2004) in a 75 mM phosphate buffer at pH 7.4, which was also used as a blank. An amount of 20 μL sample was mixed with 120 μL 117 nM fluorescein sodium salt (Sigma F6377) and incubated at 37 °C for 15 min before 60 μL 40 mM 2,2′-azobis-2methyl-propanimidamide dihydrochloride (AAPH, Sigma) was added. The AOC is defined as the net difference between the area under the fluorescence decay curve of the sample and the blank, as a consequence of the AAPH radical attack. The water-soluble vitamin E equivalent Trolox (Sigma) was used as standard. The fluorescence was measured at 485 and 520 nm (Spectramax Gemini EM fluorimeter, Molecular Devices, Sunnyvale, USA) and the results were expressed as μmol Trolox equivalents (TE)/g dry weight.
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Table 1 Proximate composition (% of wet weight) and cooking loss (% of wet weight) of semimembranosus muscle samples of raw and cooked pork (n = 19) and beef (n = 20) (values are presented as mean ± standard error). Proximates
Water Sum of AA Fat (n = 20) Ash (n = 20) Cooking loss
Pork
Beef
Effect of species (p-value)
Raw
Cooked
Effect of cooking (p-value)
Raw
Cooked
Effect of cooking (p-value)
Raw
Cooked
73.6 ± 0.2 18.3 ± 0.4 4.5 ± 0.3 1.1 ± 0.01
60.6 ± 0.8 29.0 ± 0.7 6.9 ± 0.4 1.4 ± 0.03 31.4 ± 1.7
b0.001 b0.001 b0.001 b0.001
73.9 ± 0.4 18.9 ± 0.5 3.2 ± 0.4 1.0 ± 0.01
59.0 ± 1.3 31.0 ± 1.2 6.1 ± 0.8 1.3 ± 0.04 38.3 ± 1.4
b0.001 b0.001 b0.001 b0.001
0.003 0.531 b0.001 b0.001
0.334 0.791 0.422 0.149 0.005
AA, amino acids.
2.4. Statistical analysis
than in beef (1.0 and 3.2%) (p = 0.001) and water content slightly lower in pork (74%) than in beef (p b 0.003).
Data was analysed with the MIXED procedure of the Statistical Analysis System (SAS Institute, version 9.2, Cary, NC, USA). Normality was evaluated using the Shapiro–Wilk test and an analysis of variance (ANOVA) was then performed on normal distributed parameters (lipid, water, sum of AA, TR, cooking loss, ACE, ORAC and the amino acids taurine, proline, lysine, asparagine, serine, glutamic acid, cysteine, β-alanine and histidine). Tukey's post hoc test was used to determine the effects of species (pork vs beef) nested within both raw and cooked samples or to compare the conditions (raw vs cooked) nested within each species. For the ORAC values, the effect of time, conditions (raw, cooked) and the interaction terms was also tested for each species. Non normal distributed variables (ash and the amino acids threonine, glycine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine and arginine) were analysed with the Kruskal–Wallis non parametric test. Results are presented as mean ± standard error of the means and the significance level was set at 5%. 3. Results and discussion 3.1. Proximate composition The proximate compositions of pork and beef were evaluated (Table 1). Sum of AA (19%) content was similar for pork and beef, whereas the ash and fat content was higher in pork (1.1 and 4.5%)
3.2. Total amino acids The quality of a dietary protein source may be defined as to what extent its amino acid content cover our daily requirement for essential amino acids. Analysis of total amino acids (Tables 3 and 4) showed that pork and beef were similar in amino acid composition and an amount of 100 g meat of both pork and beef contained all essential amino acids, excluding tryptophan, in equal amounts as 100 g whole eggs which is considered an ideal protein source (FAO/WHO, 2007). Glycine, alanine, cysteine, β-alanine and histidine were significantly different; glycine, alanine and cysteine being lower and β-alanine and histidine being higher in pork than in beef. Glutamic acid was the most abundant amino acid in both pork and beef, accounting for approximately 30 mg/g muscle. Aspartic acid, alanine, leucine, lysine and arginine were also abundant in equal amounts in both pork and beef. When evaluating the distribution of amino acids in per cent of all amino acids, glutamic acid and lysine were also significantly different between pork and beef. Taurine is referred to as an exclusively free amino acid which is involved in many physiological processes and human clinical studies, epidemiological data and animal studies have all indicated that taurine is beneficial for CVD, hypertension, diabetes, infant development and other medical conditions (Larsen, Eilertsen,
Table 2 Amino acids (mg/g meat and % of sum of amino acid) of semimembranosus muscle samples from raw and cooked pork and beef (n = 10) (values are expressed as mean ± standard error). Amino acid
Pork
Beef
Raw
Taurine Asparagine Threonine Serine Glutamic acid Proline Glycine Alanine Valine Cysteine Methionine Isoleucine Leucine Tyrosine b-Alanine Phenylalanine Lysine Histidine Arginine AA, amino acid.
Cooked
Raw
Cooked
mg/g meat
% of sum of AA
mg/g meat
% of sum of AA
mg/g meat
% of sum of AA
mg/g meat
% of sum of AA
0.4 ± 0.0 13.5 ± 0.4 9.2 ± 0.2 8.6 ± 0.2 29.3 ± 0.8 11.8 ± 2.2 8.8 ± 0.2 12.0 ± 0.3 8.3 ± 0.2 0.4 ± 0.0 5.3 ± 0.1 7.8 ± 0.2 16.1 ± 0.4 6.3 ± 0.7 2.2 ± 0.1 7.8 ± 0.1 17.8 ± 0.5 7.5 ± 0.2 12.2 ± 0.3
0.2 ± 0.0 7.4 ± 0.1 5.0 ± 0.1 4.7 ± 0.1 16.0 ± 0.2 6.2 ± 1.0 4.8 ± 0.1 6.6 ± 0.1 4.6 ± 0.01 0.2 ± 0.0 2.9 ± 0.1 4.3 ± 0.1 8.9 ± 0.2 3.4 ± 0.4 1.2 ± 0.1 4.3 ± 0.1 9.8 ± 0.2 4.1 ± 0.1 6.7 ± 0.2
0.5 ± 0.0 21.2 ± 0.8 14.7 ± 0.6 13.8 ± 0.5 46.7 ± 1.8 15.8 ± 2.4 14.4 ± 0.7 19.4 ± 0.7 13.5 ± 0.5 1.2 ± 0.1 8.7 ± 0.4 12.6 ± 0.5 25.7 ± 1.0 11.0 ± 0.5 2.6 ± 0.1 12.4 ± 0.5 28.1 ± 1.2 10.7 ± 0.2 20.5 ± 0.8
0.2 ± 0.0 7.3 ± 0.1 5.1 ± 0.1 4.7 ± 0.1 16.1 ± 0.2 5.5 ± 0.8 5.0 ± 0.1 6.7 ± 0.1 4.7 ± 0.1 0.4 ± 0.0 3.0 ± 0.1 4.3 ± 0.0 8.8 ± 0.1 3.8 ± 0.1 0.9 ± 0.1 4.3 ± 0.1 9.7 ± 0.2 3.7 ± 0.1 7.0 ± 0.1
0.3 ± 0.1 13.6 ± 0.5 9.2 ± 0.4 8.7 ± 0.3 31.8 ± 1.3 8.5 ± 0.4 10.8 ± 0.6 13.0 ± 0.5 8.2 ± 0.4 0.6 ± 0.0 5.5 ± 0.2 7.6 ± 0.5 16.4 ± 0.7 6.8 ± 0.3 1.6 ± 0.1 7.8 ± 0.3 17.5 ± 0.7 6.5 ± 0.3 12.9 ± 0.4
0.1 ± 0.1 7.2 ± 0.1 4.9 ± 0.1 4.6 ± 0.1 16.8 ± 0.2 4.5 ± 0.2 5.7 ± 0.3 6.9 ± 0.2 4.3 ± 0.1 0.3 ± 0.0 2.8 ± 0.0 4.0 ± 0.1 8.7 ± 0.1 3.6 ± 0.1 0.8 ± 0.1 4.1 ± 0.0 9.3 ± 0.1 3.4 ± 0.1 6.8 ± 0.1
0.4 ± 0.1 22.6 ± 7.1 15.2 ± 4.8 14.6 ± 4.6 51.6 ± 16.3 14.6 ± 4.6 18.4 ± 5.8 21.5 ± 6.8 13.6 ± 4.3 1.2 ± 0.4 8.9 ± 2.8 12.5 ± 4.0 27.0 ± 8.5 11.3 ± 3.6 1.8 ± 0.6 12.9 ± 4.1 28.1 ± 8.9 9.5 ± 3.0 21.9 ± 6.9
0.1 ± 0.0 7.2 ± 0.1 4.9 ± 0.1 4.7 ± 0.1 16.6 ± 0.2 4.8 ± 0.2 6.0 ± 0.3 6.9 ± 0.2 4.4 ± 0.1 0.4 ± 0.0 2.9 ± 0.1 4.0 ± 0.2 8.7 ± 0.1 3.6 ± 0.1 0.6 ± 0.0 4.1 ± 0.0 9.0 ± 0.1 2.7 ± 0.3 7.0 ± 0.1
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Table 3 Significance of difference in amino acids (mg/g meat and % of sum of amino acid) of semimembranosus muscle samples from raw and cooked pork and beef. Amino acid
Taurine Asparagine Threonine Serine Glutamic acid Proline Glycine Alanine Valine Cysteine Methionine Isoleucine Leucine Tyrosine b-Alanine Phenylalanine Lysine Histidine Arginine
Effect of cooking, pork
Effect of cooking, beef
Effect of species raw
Effect of species, cooked
mg/g meat
% of sum of AA
mg/g meat
% of sum of AA
mg/g meat
% sum of AA
mg/g meat
% sum of AA
0.008 b0.001 b0.001 b0.001 b0.001 0.149 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 0.008 b0.001 b0.001 b0.001 b0.001
0.013 0.606 0.920 0.507 0.924 0.714 0.654 0.546 0.269 b0.001 0.265 0.691 0.986 0.725 b0.001 0.987 0.728 0.006 0.370
0.511 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 0.002 b0.001 0.202 b0.001 b0.001 b0.001 b0.001
0.044 0.761 0.790 0.503 0.308 0.344 0.616 0.535 0.817 0.199 0.766 0.974 0.855 0.770 0.003 0.562 0.079 b0.001 0.022
0.867 0.828 0.969 0.741 0.119 0.337 0.006 0.01 0.582 b0.001 0.833 0.435 0.836 0.356 b0.001 0.754 0.699 0.005 0.434
0.745 0.313 0.062 0.405 0.010 0.493 0.011 0.181 0.091 0.002 0.290 0.137 0.299 0.119 b0.001 0.190 0.035 b0.001 0.747
0.428 b0.001 0.791 0.500 0.209 0.423 b0.001 0.157 0.630 0.792 0.571 0.407 0.763 0.354 b0.001 0.520 0.987 0.046 0.833
0.205 0.720 0.038 0.593 0.058 0.657 0.002 0.229 0.038 0.608 0.080 0.096 0.365 0.056 b0.001 0.351 0.016 b0.001 0.832
Maehre, Jensen, & Elvevoll, in press). The taurine content of pork and beef were 0.4 and 0.3 mg/g muscle, respectively. This is in accordance with previous results on semimembranosus muscles of pork where taurine content was 40 mg/100 g meat (Purchas, Morel, Janz, & Wilkinson, 2009). Taurine content has also been documented to 0.23 mg/g muscle (Aristoy & Toldra, 1998) in pork and 0.73 mg/g in beef muscle (Franco et al., 2010). It should, however, be acknowledged that the amino acid content, and taurine content in particular (Larsen et al., 2007), vary between muscle parts, and the results obtained in this study represents only the semimembranosus muscle of pork and beef.
3.3. Changes in biochemical composition after cooking Cooking of meat resulted in loss of water content and thus a relative increase in fat, sum of amino acids (Table 1) and individual amino acids (Tables 2 and 3). However, when amino acids were presented as percentage of the sum of amino acids, only taurine, cysteine, β-alanine and histidine were significantly higher after cooking of pork and taurine, β-alanine, histidine and arginine significantly higher after cooking of beef. To evaluate compositional changes and the losses of components after cooking, TR of selected components was calculated (Fig. 1). The components negatively affected by cooking were ash and
Fig. 1. True retention (%) of protein, lipid, ash and taurine after cooking of semimembranosus muscle samples from pork and beef (values are expressed as mean ± standard error).
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Table 4 Concentrations (μg/mL) of digested raw and cooked pork (n = 10 (9–10)) and beef (n = 10 (7–10)) semimembranosus muscle samples needed for 50% inhibition (IC50) of 1 mU angiotensin converting enzyme (ACE). Higher values indicate lower ACE inhibitory activity (values are expressed as mean ± standard error). Pork
IC50
Beef
Effect of species (p-value)
Raw
Cooked
Effect of cooking (p-value)
Raw
Cooked
Effect of cooking (p-value)
Raw
Cooked
60 ± 3.2
80 ± 3.2
0.009
70 ± 6.3
90 ± 3.2
0.082
0.269
0.133
the free amino acid taurine. No reduction in sum of amino acids was observed. True lipid retention was higher than 100%. This may be explained by a higher extractability of lipids from cooked samples compared to raw samples, as muscle lipoproteins can be denatured under heat and bound lipids released (Woolsey & Pauline, 1969).
acid sequences of 2–12 amino acids (Pihlanto, 2006) incorporated in proteins. During cooking, proteins are subjected to denaturation which may alter the digestion into smaller peptide and this may explain the lower ACE inhibitory capacity after preparation. 3.5. Oxygen radical absorbance capacity
3.4. Angiotensin-converting enzyme inhibition assay The GI digests from hydrolysed muscle samples were subjected to in vitro ACE inhibitory screening and the IC50 values were determined (Table 4). There was no significant difference between the ACE inhibitory activity of pork and beef (p = 0.269), exhibiting IC50 values of 60 and 70 μg/mL, respectively. The hydrolysate control, diluted to the same concentration as the samples, did not exhibit any activity (results not shown). The ACE inhibitory activity of cod and salmon has previously been determined (Dragnes et al., 2009) with IC50 values corresponding to 64 and 88 μg/mL. This indicates that both pork and beef muscle are just as adequate source of antihypertensive peptides as cod and salmon. Katsuobushi oligopeptide (K.O.) is a dry powder, produced by Nippon Supplement Inc., which is on the market as a functional food to inhibit ACE and hypertension. This product has previously been shown to exhibit an IC50 value equivalent to 52 μg/mL (Dragnes et al., 2009), which is comparable to that of pork and beef. After cooking, the IC50 values were 80 and 90 μg/mL for pork and beef, corresponding to respective losses in ACE inhibitory capacity of 33% and 29%. Naturally occurring ACE inhibitory peptides are small amino
Peptides formed during digestion of proteins have previously been shown to exhibit AOC (Samaranayaka & Li-Chan, 2011) in different assays. In this study, the AOC was assessed by analysing the ORAC of samples collected during a simulated GI digestion of raw and cooked pork and beef semimembranosus muscle samples (Fig. 2 and Table 5). The pattern of changes was similar for all samples. In general, the positive effect of conditions (raw vs cooked) was more pronounced with increasing digestion, as revealed by a significant interaction between time and conditions for both beef (p = 0.012) and for pork (p b 0.0001) (Table 5). At the start of digestion pork exhibited approximately 100 μmol TE/g dry weight, while beef exhibited 50 μmol TE/g dry weight (p = 0.004). After gastric digestion (75 min), the AOC had increased six times compared to the start of digestion for all samples. At this point, the ORAC values were approximately 600 and 400 μmol TE/g dry weights of raw pork and beef, respectively and less than 400 μmol TE/g dry weights for the cooked samples. A further increase was seen throughout the digestion, however only significant for raw samples (p = 0.002). This development during GI digestion was similar to that previously reported for saithe and shrimp (Jensen,
Fig. 2. Antioxidative capacity (ORAC) at the start of digestion (0 min), after in vitro gastric digestion (75 min), and after in vitro gastric plus intestinal digestion (165 min) of 1 g raw and cooked pork and beef semimembranosus muscle samples (n = 10) (values are expressed as mean ± standard error). Columns without a common letter between time intervals differ significantly (p b 0.001). p-Values for differences between 75 and 165 min were 0.002, 0.096, 0.002, and 0.894 for raw beef, cooked beef, raw pork and cooked pork, respectively.
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Table 5 Significance of difference in oxygen radical absorbance capacity (ORAC) of raw and cooked pork and beef semimembranosus muscle samples, at different time points during a simulated gastrointestinal digestion. Effect of species (p-value)
Start of digestion (0 min) Gastric digestion (75 min) Gastrointestinal digestion (165 min) Time × condition (raw, cooked)
Effect of cooking (p-value)
Interaction
Raw
Cooked
Pork
Beef
0.0043 0.0881 0.012
0.2224 0.0350 0.5066
0.0102 0.0709 0.0001
0.501 0.0578 0.0011
Abrahamsen, Maehre, & Elvevoll, 2009). The AOC after gastric digestion of saithe and shrimp were reported to be 150 and 160 μmol TE/g fresh weight (approximately 700 and 800μmolTE/g dry weight), respectively, which is slightly higher than for meat. The AOC of blueberry after gastric digestion were reported to be approximately 100 μmol TE/g fresh weight (approximately 500 μmol TE/g dry weight). At the end of the GI digestion, the AOC had increased to approximately 8 times the start values of all investigated samples. At this point, raw sample of pork was significantly higher in AOC than raw sample of beef (p b 0.001), while the cooked samples of both pork and beef exhibited equal AOC. 4. Conclusion Proximate composition of meat was not affected by plate cooking; taurine content was however reduced. The ACE inhibitory activity and AOC were similar in pork and beef and cooking significantly reduced the ACE inhibitory activity of pork and the AOC of both pork and beef after GI digestion. Hence, both pork and beef are good sources of protein and some bioactive properties, but after cooking potentially bioactive properties may be reduced. Acknowledgement The authors acknowledge Klaus Renmaelmo at Nortura Målselv for kindly preparing the semimembranosus muscles and providing detailed information about the material. References Aristoy, M. C., & Toldra, F. (1998). Concentration of free amino acids and dipeptides in porcine skeletal muscles with different oxidative patterns. Meat Science, 50, 327–332. Atkinson, A.B., & Robertson, J. I. S. (1979). Captopril in the treatment of clinical hypertension and cardiac-failure. Lancet, 2, 836–839. Cunniff, P. (Ed.). (1995). Official methods of analysis of AOAC International, (edition). Gaithesburg: AOAC International. Cushman, D. W., & Cheung, S. H. (1971). Spectrophotometric assay and properties of the angiotensin-converting enzyme of rabbit lung. Biochemical Pharmacology, 20, 1637–1648. Dávalos, A., Gómez-Cordovés, C., & Bartolomé, B. (2004). Extending applicability of the oxygen radical absorbance capacity (ORAC-fluorescein) assay. Journal of Agricultural and Food Chemistry, 52, 48–54. Dragnes, B. T., Stormo, S. K., Larsen, R., Ernstsen, H. H., & Elvevoll, E. O. (2009). Utilisation of fish industry residuals: Screening the taurine concentration and angiotensin converting enzyme inhibition potential in cod and salmon. Journal of Food Composition and Analysis, 22, 714–717.
Pork
Beef
b0.0001
0.012
FAO/WHO (2007). Protein and amino acid requirements in human nutrition. WHO technical report series, 935, Rome, Italy: Food and Agricultural Organization of the United Nations. Folch, J., Lees, M., & Stanley, G. H. S. (1957). A simple method for the isolation and purification of total lipides from animal tissues. Journal of Biological Chemistry, 226, 497–509. Franco, D., Gonzalez, L., Bispo, E., Rodriguez, P., Garabal, J. I., & Moreno, T. (2010). Study of hydrolyzed protein composition, free amino acid, and taurine content in different muscles of Galician blonde beef. Journal of Muscle Foods, 21, 769–784. Friedman, M. (1996). Nutritional value of proteins from different food sources. A review. Journal of Agricultural and Food Chemistry, 44, 6–29. Fujita, H., Yokoyama, K., & Yoshikawa, M. (2000). Classification and antihypertensive activity of angiotensin I-converting enzyme inhibitory peptides derived from food proteins. Journal of Food Science, 65, 564–569. Halliwell, B., & Gutteridge, J. M. C. (2007). Free radicals in biology and medicine (4th ed.) Oxford, New York: Oxford University Press Inc. Harris, T., Cook, E. F., Kannel, W., Schatzkin, A., & Goldman, L. (1985). Blood-pressure experience and risk of cardiovascular-disease in the elderly. Hypertension, 7, 118–124. Jensen, I. J., Abrahamsen, H., Maehre, H. K., & Elvevoll, E. O. (2009). Changes in antioxidative capacity of saithe (Pollachius virens) and shrimp (Pandalus borealis) during in vitro digestion. Journal of Agricultural and Food Chemistry, 57, 10928–10932. Larsen, R., Eilertsen, K. E., Maehre, H., Jensen, I. J., & Elvevoll, E. O. (in press). Taurine content in marine foods — Beneficial health effects. In M. Herrero, & B. Hernandez (Eds.), Bioactive compounds from marine foods: Plant and animal sources. Oxford, UK: Wiley-Blackwell. http://dx.doi.org/10.1002/9781118412893.ch12 (in press). Larsen, R., Stormo, S. K., Dragnes, B. T., & Elvevoll, E. O. (2007). Losses of taurine, creatine, glycine and alanine from cod (Gadus morhua L.) fillet during processing. Journal of Food Composition and Analysis, 20, 396–402. Maehre, H. K., Hamre, K., & Elvevoll, E. O. (2012). Nutrient evaluation of rotifers and zooplankton: Feed for marine fish larvae. Aquaculture Nutrition, 19, 301–311. Meade, S. J., Reid, E. A., & Gerrard, J. A. (2005). The impact of processing on the nutritional quality of food proteins. Journal of AOAC International, 88, 904–922. Micha, R., Michas, G., & Mozaffarian, D. (2012). Unprocessed red and processed meats and risk of coronary artery disease and type 2 diabetes — An updated review of the evidence. Current Atherosclerosis Reports, 14, 515–524. Murphy, E. W., Criner, P. E., & Gray, B. C. (1975). Comparisons of methods for calculating retentions of nutrients in cooked foods. Journal of Agricultural and Food Chemistry, 23, 1153–1157. Pihlanto, A. (2006). Antioxidative peptides derived from milk proteins. International Dairy Journal, 16, 1306–1314. Purchas, R. W., Morel, P. C. H., Janz, J. A.M., & Wilkinson, B. H. P. (2009). Chemical composition characteristics of the longissimus and semimembranosus muscles for pigs from New Zealand and Singapore. Meat Science, 81, 540–548. Ryan, J. T., Ross, R. P., Bolton, D., Fitzgerald, G. F., & Stanton, C. (2011). Bioactive peptides from muscle sources: Meat and fish, 3, 765–791. Samaranayaka, A. G. P., & Li-Chan, E. C. Y. (2011). Food-derived peptidic antioxidants: A review of their production, assessment, and potential applications. Journal of Functional Foods, 3, 229–254. WHO (2011). Hypertension fact sheet. Department of Sustainable Development and Healthy Environments. Wijesekara, I., & Kim, S. K. (2010). Angiotensin-I-converting enzyme (ACE) inhibitors from marine resources: Prospects in the pharmaceutical industry. Marine Drugs, 8, 1080–1093. Woolsey, A. P., & Pauline, C. P. (1969). External fat cover influence on raw and cooked beef. 1. Fat and moisture content. Journal of Food Science, 34, 554–557.
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Meat Science journal homepage: www.elsevier.com/locate/meatsci
Proximate composition, antihypertensive and antioxidative properties of the semimembranosus muscle from pork and beef after cooking and in vitro digestion Ida-Johanne Jensen a,⁎, Junio Dort b, Karl-Erik Eilertsen a a b
Norwegian College of Fishery Science, University of Tromsø, N-9037 Tromsø, Norway Department of Food Science and Nutrition, Laval University, Quebec Qc G1V 0A6, Canada
a r t i c l e
i n f o
Article history: Received 12 February 2013 Received in revised form 4 October 2013 Accepted 8 October 2013 Keywords: Amino acid Angiotensin converting enzyme ORAC Bioactive
a b s t r a c t The aims of this study were to evaluate and compare proximate composition, antihypertensive activity and antioxidative capacity of the semimembranosus muscle from pork and beef and to study how these characteristics were affected by household preparation and subsequent digestion. The proximate composition was similar between pork and beef. Both pork and beef contained protein with the essential amino acids. Cooking in a heated pan did not affect the retention of lipid or sum of amino acids, but reduced the amount of the free amino acid taurine. The antihypertensive effect did not differ significantly between pork and beef, whereas the antioxidative capacity did. Cooking affected the antioxidative capacity negatively. The results from this study show that pork and beef are equally good sources of protein and bioactive properties, and whereas the nutritional composition is not affected, bioactive properties may be reduced after household preparations. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Hypertension is a major independent risk factor for CVD affecting people world-wide (Harris, Cook, Kannel, Schatzkin, & Goldman, 1985; WHO, 2011). Angiotensin converting enzyme (ACE) which plays an important role in increasing blood pressure and hypertension is normally pharmacologically treated by inhibition of ACE. Pharmaceuticals currently in use are, however, associated with adverse effects such as dry cough, taste alterations, skin rashes and renal dysfunction (Atkinson & Robertson, 1979). Moderate hypertension may be controlled by a nutritional approach and numerous studies have documented antihypertensive and ACE inhibitory effects of different food sources (Fujita, Yokoyama, & Yoshikawa, 2000; Wijesekara & Kim, 2010). Oxidative stress, a condition of oxidant overload in relation to antioxidants, has also been connected to different diseases including CVD (Halliwell & Gutteridge, 2007). A higher intake of antioxidants has indeed been linked to a lower occurrence of oxidative stress and antioxidative capacity (AOC) has been documented in different peptide sources (Samaranayaka & Li-Chan, 2011).
⁎ Corresponding author. Tel.: +47 776 46 721; fax: +47 776 46 020. E-mail address: [email protected] (I.-J. Jensen). 0309-1740/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.meatsci.2013.10.014
In recent years studies have focused on bioactive food derived components, exerting a positive physiological effect in the body, beyond that of nutrition. As major parts of proteins, peptides are examples of such bioactive components, which have been demonstrated to be hypotensive, antioxidative, opioid-like and anti-inflammatory (Ryan, Ross, Bolton, Fitzgerald, & Stanton, 2011). In order to exert any effect, the peptides need to be released during processing, fermentation or digestion. Lean red meat is a high quality protein source with low fat content. It is also an important source of the B group of vitamins, and minerals such as iron, copper, zink and manganese (Friedman, 1996). Red meat has in the past been considered a major dietary risk factor for cardio-metabolic diseases and type 2 diabetes mellitus. However, such an association has only been confirmed with processed meat (Micha, Michas, & Mozaffarian, 2012), demonstrating that processing may alter the nutritional value of meat. In fact, culinary preparation and processing of meat may result in both advantages, such as enhanced taste, killed pathogens, inactivated anti-nutrient enzymes and increased digestibility and bioavailability and disadvantages such as browning reactions, racemisation, destructed valuable components (Meade, Reid, & Gerrard, 2005) and lost water soluble-potentially bioactive-components (Larsen, Stormo, Dragnes, & Elvevoll, 2007). Although several studies have evaluated the nutritional and bioactive properties of meat, the effect of household preparation has been disregarded. The aims of this study were to evaluate the proximate composition, antihypertensive activity and antioxidative capacity of pork and beef and to study the effect of household preparation and subsequent digestion on these parameters.
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2. Materials and methods 2.1. Materials Pork (Sus scrofa domesticus) (n = 20) of the breeds Yorkshire, Duroc and Norwegian countrypork, male and female, 5–6 months of age, and approximately 100 kg live weights and beef cattle (Bos taurus) (n = 20), of the breed Norwegian red cattle, both male and female, 6 months of age and approximately 300 kg live weights, were slaughtered in Troms County, Norway in September 2010. The carcasses were kept at 4°C for 3days and at day 4 post mortem, the semimembranosus muscles (approximately 2.5 kg) from twenty animals of each species were dissected and brought to the laboratory. 2.2. Experimental design From each of the semimembranosus muscle samples, two pieces of approximately 100 g (10 cm × 15 cm× 1.5 cm) were cut from the central portion, labelled and randomly distributed in two groups; raw and cooked. Cooking was performed by placing the pieces on a pre-heated pan without oil and heated until thoroughly cooked, 10 min on each side reaching an internal temperature of 75 ± 3 °C. The pieces, raw and cooked, were weighed and minced before storage in sealed plastic bags at −50 °C. All samples were analysed for moisture, ash and fat content and 10 samples were analysed for total amino acids. After a simulated digestion (GI), antihypertensive activity and AOC were evaluated in ten samples for ACE inhibitory activity and oxygen radical absorbance capacity (ORAC), respectively. Cooking loss was determined as the percentage weight loss after cooking. True retention (TR) of selected components was calculated to evaluate compositional changes during processing according to Murphy, Criner, and Gray (1975): TR; % ¼
nutrient content per g cooked meat g meat after cooking nutrient content per g raw meat g meat before cooking 100:
2.3. Analytical methods 2.3.1. Proximate composition The AOAC 925.04 and AOAC 938.08 (Cunniff, 1995) were used to determine the water and ash contents. Approximately 10 g sample was dried at 105 °C until constant weight and water content was determined gravimetrically. The water free sample was thereafter combusted at 500 °C for 12 h and ash content determined gravimetrically. Total lipids were determined gravimetrically after extraction (Folch, Lees, & Stanley, 1957), replacing chloroform/ methanol with dichloromethane/methanol due to safety reasons. 2.3.2. Amino acid analysis For analysis of total amino acids except tryptophan, approximately 200 μg sample was mixed with 900 μL 20 mM norleucine (Sigma Chemicals Co, St. Louis, MO, USA) and 1200 μL 12 M HCl and hydrolysed at 110 °C for 24 h (Maehre, Hamre, & Elvevoll, 2012). The hydrolysates (100 μL) were dried under nitrogen and dissolved in lithium citrate buffer (pH2.2) at a suitable concentration and analysed using a Biochrom 30 Amino Acid Analyzer (Biochrom Limited, Cambridge, UK) with a lithium citrate equilibrated column and post column derivatization with ninhydrine. Norleucine was used as internal standard and the signal was analysed with Chromeleon software (Dionex, Sunnyvale, CA, USA). The A9906 (Sigma) physiological amino acid standard was used for identification and quantification of the amino acids. 2.3.3. In vitro gastrointestinal digestions The in vitro GI model used to digest samples for analysis of ACE inhibitory activity is described by Dragnes, Stormo, Larsen, Ernstsen,
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and Elvevoll (2009). Sample (12.5 g) was mixed with 50 mL water and the pH was adjusted to 2 with 3 M HCl. The stomach phase was simulated by adding 50 mg pepsin (Sigma P6887) dissolved in 1 mL water, and the mixture was incubated 2 h at 37 °C on a magnetic stirrer. The pH was thereafter adjusted to 6.5 with 3 M NaOH and the intestinal phase was simulated by adding 50mg trypsin (Sigma T1426) and 50mg chymotrypsin (Sigma C4129) dissolved in 2 mL water. After a total of 4.5 h of incubation, the digests were frozen and freeze-dried. The freeze-dried samples were finely ground and kept frozen at −55 °C until determination of ACE inhibitory effect. The in vitro digestion described by Jensen, Abrahamsen, Maehre and Elvevoll (2009) was used to digest samples for analysis of AOC. The digestion was simulated by adding a pepsin solution containing 0.462% pepsin (Sigma P6887) (w/v), 49 mM NaCl, 12 mM KCl, 10 mM CaCl2, 2.4 mM MgCl2, and 2.5 mM K2HPO4, representing the gastric phase and bile/pancreatic solution (containing 0.2 g pancreatine (Sigma P1750), 1.25 g bile extract (Sigma B8631) and 0.1 M NaHCO3 in 50 mL distilled water) representing the intestinal phase, along with gradient pH adjustment. An amount of 1 g muscle (or 50 mM Na2HPO4 with 0.9% NaCl and pH 6.75 as control) was mixed with 15 mL pepsin solution. The pH was adjusted to 5.5 and a 3 mL sample was collected (0 min). The remaining reaction mixtures were incubated at 220 rpm and 37 °C for 30 min, before the pH was adjusted to 3.8. Further incubation was continued for 30 min before another pH adjustment to 2.0. After 15 min of incubation, another 3 mL sample was collected (75 min). Thereafter 1.5 mL bile and pancreatic solution was added the reaction mixtures. The pH was adjusted to 5.0 and the reaction mixtures were continuously incubated for 30 min before the pH was adjusted to 6.5. The reaction mixtures were incubated for another 60 min and the last sample was collected (165 min). The samples were centrifuged at 4500 g at 4 °C for 15 min to remove large particles. The digested samples were frozen and kept at −55 °C until analysis of AOC.
2.3.4. Angiotensin-converting enzyme inhibitory assay Angiotensin converting enzyme inhibitory activity assay was performed based on a previous method (Cushman & Cheung, 1971) with modifications. The freeze-dried sample was dissolved in 100 mM sodium borate buffer (pH 8.3) and 25 μL was pre incubated with 100 μL substrate (2 mM hippuryl-L-histidyl-L-leucine (HHL, Sigma H1635) in 100 mM sodium borate buffer), at 37 °C for 10 min. The enzymatic reaction was initiated by addition of 50 μL ACE (Sigma A6778) (5 mU, final concentration) and carried out on a shaker at 37 °C. After 30 min, the reaction was stopped by addition of 215 μL 1 M HCl and the end product hippuric acid was measured by quantitative HPLC analyses (Dragnes et al., 2009). A dilution series from 100 to 2000 μg/mL was used for the samples and the amount of sample/ACE inhibitor needed to inhibit 50% ACE activity was defined as the IC50% value and presented as μg/mL hence a lower IC50% value signified a higher ACE inhibitory activity.
2.3.5. Oxygen radical absorbance capacity The ORAC assay was carried out according to Dávalos, GómezCordovés, and Bartolomé (2004) in a 75 mM phosphate buffer at pH 7.4, which was also used as a blank. An amount of 20 μL sample was mixed with 120 μL 117 nM fluorescein sodium salt (Sigma F6377) and incubated at 37 °C for 15 min before 60 μL 40 mM 2,2′-azobis-2methyl-propanimidamide dihydrochloride (AAPH, Sigma) was added. The AOC is defined as the net difference between the area under the fluorescence decay curve of the sample and the blank, as a consequence of the AAPH radical attack. The water-soluble vitamin E equivalent Trolox (Sigma) was used as standard. The fluorescence was measured at 485 and 520 nm (Spectramax Gemini EM fluorimeter, Molecular Devices, Sunnyvale, USA) and the results were expressed as μmol Trolox equivalents (TE)/g dry weight.
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Table 1 Proximate composition (% of wet weight) and cooking loss (% of wet weight) of semimembranosus muscle samples of raw and cooked pork (n = 19) and beef (n = 20) (values are presented as mean ± standard error). Proximates
Water Sum of AA Fat (n = 20) Ash (n = 20) Cooking loss
Pork
Beef
Effect of species (p-value)
Raw
Cooked
Effect of cooking (p-value)
Raw
Cooked
Effect of cooking (p-value)
Raw
Cooked
73.6 ± 0.2 18.3 ± 0.4 4.5 ± 0.3 1.1 ± 0.01
60.6 ± 0.8 29.0 ± 0.7 6.9 ± 0.4 1.4 ± 0.03 31.4 ± 1.7
b0.001 b0.001 b0.001 b0.001
73.9 ± 0.4 18.9 ± 0.5 3.2 ± 0.4 1.0 ± 0.01
59.0 ± 1.3 31.0 ± 1.2 6.1 ± 0.8 1.3 ± 0.04 38.3 ± 1.4
b0.001 b0.001 b0.001 b0.001
0.003 0.531 b0.001 b0.001
0.334 0.791 0.422 0.149 0.005
AA, amino acids.
2.4. Statistical analysis
than in beef (1.0 and 3.2%) (p = 0.001) and water content slightly lower in pork (74%) than in beef (p b 0.003).
Data was analysed with the MIXED procedure of the Statistical Analysis System (SAS Institute, version 9.2, Cary, NC, USA). Normality was evaluated using the Shapiro–Wilk test and an analysis of variance (ANOVA) was then performed on normal distributed parameters (lipid, water, sum of AA, TR, cooking loss, ACE, ORAC and the amino acids taurine, proline, lysine, asparagine, serine, glutamic acid, cysteine, β-alanine and histidine). Tukey's post hoc test was used to determine the effects of species (pork vs beef) nested within both raw and cooked samples or to compare the conditions (raw vs cooked) nested within each species. For the ORAC values, the effect of time, conditions (raw, cooked) and the interaction terms was also tested for each species. Non normal distributed variables (ash and the amino acids threonine, glycine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine and arginine) were analysed with the Kruskal–Wallis non parametric test. Results are presented as mean ± standard error of the means and the significance level was set at 5%. 3. Results and discussion 3.1. Proximate composition The proximate compositions of pork and beef were evaluated (Table 1). Sum of AA (19%) content was similar for pork and beef, whereas the ash and fat content was higher in pork (1.1 and 4.5%)
3.2. Total amino acids The quality of a dietary protein source may be defined as to what extent its amino acid content cover our daily requirement for essential amino acids. Analysis of total amino acids (Tables 3 and 4) showed that pork and beef were similar in amino acid composition and an amount of 100 g meat of both pork and beef contained all essential amino acids, excluding tryptophan, in equal amounts as 100 g whole eggs which is considered an ideal protein source (FAO/WHO, 2007). Glycine, alanine, cysteine, β-alanine and histidine were significantly different; glycine, alanine and cysteine being lower and β-alanine and histidine being higher in pork than in beef. Glutamic acid was the most abundant amino acid in both pork and beef, accounting for approximately 30 mg/g muscle. Aspartic acid, alanine, leucine, lysine and arginine were also abundant in equal amounts in both pork and beef. When evaluating the distribution of amino acids in per cent of all amino acids, glutamic acid and lysine were also significantly different between pork and beef. Taurine is referred to as an exclusively free amino acid which is involved in many physiological processes and human clinical studies, epidemiological data and animal studies have all indicated that taurine is beneficial for CVD, hypertension, diabetes, infant development and other medical conditions (Larsen, Eilertsen,
Table 2 Amino acids (mg/g meat and % of sum of amino acid) of semimembranosus muscle samples from raw and cooked pork and beef (n = 10) (values are expressed as mean ± standard error). Amino acid
Pork
Beef
Raw
Taurine Asparagine Threonine Serine Glutamic acid Proline Glycine Alanine Valine Cysteine Methionine Isoleucine Leucine Tyrosine b-Alanine Phenylalanine Lysine Histidine Arginine AA, amino acid.
Cooked
Raw
Cooked
mg/g meat
% of sum of AA
mg/g meat
% of sum of AA
mg/g meat
% of sum of AA
mg/g meat
% of sum of AA
0.4 ± 0.0 13.5 ± 0.4 9.2 ± 0.2 8.6 ± 0.2 29.3 ± 0.8 11.8 ± 2.2 8.8 ± 0.2 12.0 ± 0.3 8.3 ± 0.2 0.4 ± 0.0 5.3 ± 0.1 7.8 ± 0.2 16.1 ± 0.4 6.3 ± 0.7 2.2 ± 0.1 7.8 ± 0.1 17.8 ± 0.5 7.5 ± 0.2 12.2 ± 0.3
0.2 ± 0.0 7.4 ± 0.1 5.0 ± 0.1 4.7 ± 0.1 16.0 ± 0.2 6.2 ± 1.0 4.8 ± 0.1 6.6 ± 0.1 4.6 ± 0.01 0.2 ± 0.0 2.9 ± 0.1 4.3 ± 0.1 8.9 ± 0.2 3.4 ± 0.4 1.2 ± 0.1 4.3 ± 0.1 9.8 ± 0.2 4.1 ± 0.1 6.7 ± 0.2
0.5 ± 0.0 21.2 ± 0.8 14.7 ± 0.6 13.8 ± 0.5 46.7 ± 1.8 15.8 ± 2.4 14.4 ± 0.7 19.4 ± 0.7 13.5 ± 0.5 1.2 ± 0.1 8.7 ± 0.4 12.6 ± 0.5 25.7 ± 1.0 11.0 ± 0.5 2.6 ± 0.1 12.4 ± 0.5 28.1 ± 1.2 10.7 ± 0.2 20.5 ± 0.8
0.2 ± 0.0 7.3 ± 0.1 5.1 ± 0.1 4.7 ± 0.1 16.1 ± 0.2 5.5 ± 0.8 5.0 ± 0.1 6.7 ± 0.1 4.7 ± 0.1 0.4 ± 0.0 3.0 ± 0.1 4.3 ± 0.0 8.8 ± 0.1 3.8 ± 0.1 0.9 ± 0.1 4.3 ± 0.1 9.7 ± 0.2 3.7 ± 0.1 7.0 ± 0.1
0.3 ± 0.1 13.6 ± 0.5 9.2 ± 0.4 8.7 ± 0.3 31.8 ± 1.3 8.5 ± 0.4 10.8 ± 0.6 13.0 ± 0.5 8.2 ± 0.4 0.6 ± 0.0 5.5 ± 0.2 7.6 ± 0.5 16.4 ± 0.7 6.8 ± 0.3 1.6 ± 0.1 7.8 ± 0.3 17.5 ± 0.7 6.5 ± 0.3 12.9 ± 0.4
0.1 ± 0.1 7.2 ± 0.1 4.9 ± 0.1 4.6 ± 0.1 16.8 ± 0.2 4.5 ± 0.2 5.7 ± 0.3 6.9 ± 0.2 4.3 ± 0.1 0.3 ± 0.0 2.8 ± 0.0 4.0 ± 0.1 8.7 ± 0.1 3.6 ± 0.1 0.8 ± 0.1 4.1 ± 0.0 9.3 ± 0.1 3.4 ± 0.1 6.8 ± 0.1
0.4 ± 0.1 22.6 ± 7.1 15.2 ± 4.8 14.6 ± 4.6 51.6 ± 16.3 14.6 ± 4.6 18.4 ± 5.8 21.5 ± 6.8 13.6 ± 4.3 1.2 ± 0.4 8.9 ± 2.8 12.5 ± 4.0 27.0 ± 8.5 11.3 ± 3.6 1.8 ± 0.6 12.9 ± 4.1 28.1 ± 8.9 9.5 ± 3.0 21.9 ± 6.9
0.1 ± 0.0 7.2 ± 0.1 4.9 ± 0.1 4.7 ± 0.1 16.6 ± 0.2 4.8 ± 0.2 6.0 ± 0.3 6.9 ± 0.2 4.4 ± 0.1 0.4 ± 0.0 2.9 ± 0.1 4.0 ± 0.2 8.7 ± 0.1 3.6 ± 0.1 0.6 ± 0.0 4.1 ± 0.0 9.0 ± 0.1 2.7 ± 0.3 7.0 ± 0.1
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Table 3 Significance of difference in amino acids (mg/g meat and % of sum of amino acid) of semimembranosus muscle samples from raw and cooked pork and beef. Amino acid
Taurine Asparagine Threonine Serine Glutamic acid Proline Glycine Alanine Valine Cysteine Methionine Isoleucine Leucine Tyrosine b-Alanine Phenylalanine Lysine Histidine Arginine
Effect of cooking, pork
Effect of cooking, beef
Effect of species raw
Effect of species, cooked
mg/g meat
% of sum of AA
mg/g meat
% of sum of AA
mg/g meat
% sum of AA
mg/g meat
% sum of AA
0.008 b0.001 b0.001 b0.001 b0.001 0.149 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 0.008 b0.001 b0.001 b0.001 b0.001
0.013 0.606 0.920 0.507 0.924 0.714 0.654 0.546 0.269 b0.001 0.265 0.691 0.986 0.725 b0.001 0.987 0.728 0.006 0.370
0.511 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 0.002 b0.001 0.202 b0.001 b0.001 b0.001 b0.001
0.044 0.761 0.790 0.503 0.308 0.344 0.616 0.535 0.817 0.199 0.766 0.974 0.855 0.770 0.003 0.562 0.079 b0.001 0.022
0.867 0.828 0.969 0.741 0.119 0.337 0.006 0.01 0.582 b0.001 0.833 0.435 0.836 0.356 b0.001 0.754 0.699 0.005 0.434
0.745 0.313 0.062 0.405 0.010 0.493 0.011 0.181 0.091 0.002 0.290 0.137 0.299 0.119 b0.001 0.190 0.035 b0.001 0.747
0.428 b0.001 0.791 0.500 0.209 0.423 b0.001 0.157 0.630 0.792 0.571 0.407 0.763 0.354 b0.001 0.520 0.987 0.046 0.833
0.205 0.720 0.038 0.593 0.058 0.657 0.002 0.229 0.038 0.608 0.080 0.096 0.365 0.056 b0.001 0.351 0.016 b0.001 0.832
Maehre, Jensen, & Elvevoll, in press). The taurine content of pork and beef were 0.4 and 0.3 mg/g muscle, respectively. This is in accordance with previous results on semimembranosus muscles of pork where taurine content was 40 mg/100 g meat (Purchas, Morel, Janz, & Wilkinson, 2009). Taurine content has also been documented to 0.23 mg/g muscle (Aristoy & Toldra, 1998) in pork and 0.73 mg/g in beef muscle (Franco et al., 2010). It should, however, be acknowledged that the amino acid content, and taurine content in particular (Larsen et al., 2007), vary between muscle parts, and the results obtained in this study represents only the semimembranosus muscle of pork and beef.
3.3. Changes in biochemical composition after cooking Cooking of meat resulted in loss of water content and thus a relative increase in fat, sum of amino acids (Table 1) and individual amino acids (Tables 2 and 3). However, when amino acids were presented as percentage of the sum of amino acids, only taurine, cysteine, β-alanine and histidine were significantly higher after cooking of pork and taurine, β-alanine, histidine and arginine significantly higher after cooking of beef. To evaluate compositional changes and the losses of components after cooking, TR of selected components was calculated (Fig. 1). The components negatively affected by cooking were ash and
Fig. 1. True retention (%) of protein, lipid, ash and taurine after cooking of semimembranosus muscle samples from pork and beef (values are expressed as mean ± standard error).
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Table 4 Concentrations (μg/mL) of digested raw and cooked pork (n = 10 (9–10)) and beef (n = 10 (7–10)) semimembranosus muscle samples needed for 50% inhibition (IC50) of 1 mU angiotensin converting enzyme (ACE). Higher values indicate lower ACE inhibitory activity (values are expressed as mean ± standard error). Pork
IC50
Beef
Effect of species (p-value)
Raw
Cooked
Effect of cooking (p-value)
Raw
Cooked
Effect of cooking (p-value)
Raw
Cooked
60 ± 3.2
80 ± 3.2
0.009
70 ± 6.3
90 ± 3.2
0.082
0.269
0.133
the free amino acid taurine. No reduction in sum of amino acids was observed. True lipid retention was higher than 100%. This may be explained by a higher extractability of lipids from cooked samples compared to raw samples, as muscle lipoproteins can be denatured under heat and bound lipids released (Woolsey & Pauline, 1969).
acid sequences of 2–12 amino acids (Pihlanto, 2006) incorporated in proteins. During cooking, proteins are subjected to denaturation which may alter the digestion into smaller peptide and this may explain the lower ACE inhibitory capacity after preparation. 3.5. Oxygen radical absorbance capacity
3.4. Angiotensin-converting enzyme inhibition assay The GI digests from hydrolysed muscle samples were subjected to in vitro ACE inhibitory screening and the IC50 values were determined (Table 4). There was no significant difference between the ACE inhibitory activity of pork and beef (p = 0.269), exhibiting IC50 values of 60 and 70 μg/mL, respectively. The hydrolysate control, diluted to the same concentration as the samples, did not exhibit any activity (results not shown). The ACE inhibitory activity of cod and salmon has previously been determined (Dragnes et al., 2009) with IC50 values corresponding to 64 and 88 μg/mL. This indicates that both pork and beef muscle are just as adequate source of antihypertensive peptides as cod and salmon. Katsuobushi oligopeptide (K.O.) is a dry powder, produced by Nippon Supplement Inc., which is on the market as a functional food to inhibit ACE and hypertension. This product has previously been shown to exhibit an IC50 value equivalent to 52 μg/mL (Dragnes et al., 2009), which is comparable to that of pork and beef. After cooking, the IC50 values were 80 and 90 μg/mL for pork and beef, corresponding to respective losses in ACE inhibitory capacity of 33% and 29%. Naturally occurring ACE inhibitory peptides are small amino
Peptides formed during digestion of proteins have previously been shown to exhibit AOC (Samaranayaka & Li-Chan, 2011) in different assays. In this study, the AOC was assessed by analysing the ORAC of samples collected during a simulated GI digestion of raw and cooked pork and beef semimembranosus muscle samples (Fig. 2 and Table 5). The pattern of changes was similar for all samples. In general, the positive effect of conditions (raw vs cooked) was more pronounced with increasing digestion, as revealed by a significant interaction between time and conditions for both beef (p = 0.012) and for pork (p b 0.0001) (Table 5). At the start of digestion pork exhibited approximately 100 μmol TE/g dry weight, while beef exhibited 50 μmol TE/g dry weight (p = 0.004). After gastric digestion (75 min), the AOC had increased six times compared to the start of digestion for all samples. At this point, the ORAC values were approximately 600 and 400 μmol TE/g dry weights of raw pork and beef, respectively and less than 400 μmol TE/g dry weights for the cooked samples. A further increase was seen throughout the digestion, however only significant for raw samples (p = 0.002). This development during GI digestion was similar to that previously reported for saithe and shrimp (Jensen,
Fig. 2. Antioxidative capacity (ORAC) at the start of digestion (0 min), after in vitro gastric digestion (75 min), and after in vitro gastric plus intestinal digestion (165 min) of 1 g raw and cooked pork and beef semimembranosus muscle samples (n = 10) (values are expressed as mean ± standard error). Columns without a common letter between time intervals differ significantly (p b 0.001). p-Values for differences between 75 and 165 min were 0.002, 0.096, 0.002, and 0.894 for raw beef, cooked beef, raw pork and cooked pork, respectively.
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Table 5 Significance of difference in oxygen radical absorbance capacity (ORAC) of raw and cooked pork and beef semimembranosus muscle samples, at different time points during a simulated gastrointestinal digestion. Effect of species (p-value)
Start of digestion (0 min) Gastric digestion (75 min) Gastrointestinal digestion (165 min) Time × condition (raw, cooked)
Effect of cooking (p-value)
Interaction
Raw
Cooked
Pork
Beef
0.0043 0.0881 0.012
0.2224 0.0350 0.5066
0.0102 0.0709 0.0001
0.501 0.0578 0.0011
Abrahamsen, Maehre, & Elvevoll, 2009). The AOC after gastric digestion of saithe and shrimp were reported to be 150 and 160 μmol TE/g fresh weight (approximately 700 and 800μmolTE/g dry weight), respectively, which is slightly higher than for meat. The AOC of blueberry after gastric digestion were reported to be approximately 100 μmol TE/g fresh weight (approximately 500 μmol TE/g dry weight). At the end of the GI digestion, the AOC had increased to approximately 8 times the start values of all investigated samples. At this point, raw sample of pork was significantly higher in AOC than raw sample of beef (p b 0.001), while the cooked samples of both pork and beef exhibited equal AOC. 4. Conclusion Proximate composition of meat was not affected by plate cooking; taurine content was however reduced. The ACE inhibitory activity and AOC were similar in pork and beef and cooking significantly reduced the ACE inhibitory activity of pork and the AOC of both pork and beef after GI digestion. Hence, both pork and beef are good sources of protein and some bioactive properties, but after cooking potentially bioactive properties may be reduced. Acknowledgement The authors acknowledge Klaus Renmaelmo at Nortura Målselv for kindly preparing the semimembranosus muscles and providing detailed information about the material. References Aristoy, M. C., & Toldra, F. (1998). Concentration of free amino acids and dipeptides in porcine skeletal muscles with different oxidative patterns. Meat Science, 50, 327–332. Atkinson, A.B., & Robertson, J. I. S. (1979). Captopril in the treatment of clinical hypertension and cardiac-failure. Lancet, 2, 836–839. Cunniff, P. (Ed.). (1995). Official methods of analysis of AOAC International, (edition). Gaithesburg: AOAC International. Cushman, D. W., & Cheung, S. H. (1971). Spectrophotometric assay and properties of the angiotensin-converting enzyme of rabbit lung. Biochemical Pharmacology, 20, 1637–1648. Dávalos, A., Gómez-Cordovés, C., & Bartolomé, B. (2004). Extending applicability of the oxygen radical absorbance capacity (ORAC-fluorescein) assay. Journal of Agricultural and Food Chemistry, 52, 48–54. Dragnes, B. T., Stormo, S. K., Larsen, R., Ernstsen, H. H., & Elvevoll, E. O. (2009). Utilisation of fish industry residuals: Screening the taurine concentration and angiotensin converting enzyme inhibition potential in cod and salmon. Journal of Food Composition and Analysis, 22, 714–717.
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