Meat Science 133 (2017) 95–102

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Meat Science
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Supplementing entire male pig diet with hydrolysable tannins: Effect on MARK
carcass traits, meat quality and oxidative stability
Vida Rezara, Janez Salobira, Alenka Levarta, Urška Tomažinb, Martin Škrlepb,
Nina Batorek Lukačb, Marjeta Čandek-Potokarb,c,⁎
a
Department of Animal Science, Biotechnical Faculty, University of Ljubljana, Groblje 3, 1230 Domžale, Slovenia
b
Agricultural Institute of Slovenia, Hacquetova Ulica 17, 1000 Ljubljana, Slovenia
c
University of Maribor, Faculty of Agriculture and Life Sciences, Pivola 10, 2311 Hoče, Slovenia

A R T I C L E I N F O A B S T R A C T

Keywords: The purpose of the present study was to investigate the potential impact on carcass and meat quality of a sweet
Pig chestnut wood extract (SCWE)diet supplement for pigs, in particular on oxidative stability and fatty acid
Tannin composition. Entire (non-castrated) male pigs (n = 24) were assigned to treatment groups within litter and
Carcass offered one of 4 finisher diets on an ad libitum basis: T0 (control), T1, T2 or T3, supplemented with 0, 1, 2 or 3%
Meat
of commercially available SCWE, respectively. The highest SCWE supplementation reduced carcass fat deposi-
Oxidative stability
tion and water holding capacity of meat (higher thawing loss). In fresh meat, SCWE supplementation increased
lipid (malondialdehyde) and protein oxidation (carbonyl groups in myofibril isolates). With regard to fat tissue,
SCWE supplementation increased the proportion of polyunsaturated fatty acids.

1. Introduction demonstrated that hydrolysable tannins, besides reducing total protein

Tannins are secondary plant metabolites with great structural di-
versity (classified as hydrolysable, condensed or complex tannins) ex-
erting different physiological effects according to their form, the
amount ingested and the animal species involved (for detailed review
see Mueller-Harvey, 2006). In certain amounts tannins are considered
anti-nutritive substances because they precipitate proteins, inhibit di-
gestive enzymes and affect the utilisation of vitamins and minerals
(Chung, Wei, & Johnson, 1998). In addition they reduce feed palat-
ability and consequently feed intake and animal performance
(Jansman, 1993; Mueller-Harvey, 2006). On the other hand, certain
tannins are, due to their antimicrobial properties, commonly fed to
monogastric animals as antihelmintic, antimicrobial and antiviral
agents, and as supportive treatment for diarrhoea (Mueller-Harvey,
2006; Redondo, Chacana, Dominguez, & Fernandez Miyakawa, 2014).
Tannins may work as antioxidants to scavenge free radicals (Riedl,
Carando, Alessio, McCarthy, & Hagerman, 2002). Sweet chestnut (Cas-
tanea sativa Mill.) wood extract (SCWE), consisting mainly of hydro-
lysable tannins, was shown to possess reducing and antioxidant capa-
city in in vitro trials (Lampire et al., 1998), was able to reduce in vivo
oxidative stress in pigs and poultry (Frankič & Salobir, 2011;Voljč,
Levart, Žgur, & Salobir, 2013) and increased oxidative stability of meat
through preserving vitamin E (Voljč et al., 2013). In pigs, it was
digestibility (Antongiovanni, Minieri, & Petacchi, 2007; Salobir,
Kostanjevec, Štruklec, & Salobir, 2005), inhibit protein fermentation in
the colon (Biagi, Cipollini, Paulicks, & Roth, 2010) and reduce intestinal
production of skatole, a boar taint compound (Čandek-Potokar et al.,
2015). Because of the possible effects on the digestion of protein and
other nutrients, and due to its antioxidant properties, tannin supple-
mentation is likely to affect carcass and meat quality. However, scien-
tific literature on this topic is still lacking, especially in relation to pigs.
Apart from the research dealing with pigs fed with acorns and chestnuts
(García-Valverde, Nieto, Lachica, & Aguilera, 2007; Pugliese et al.,
2009; Tejerina, García-Torres, Cabeza de Vaca, Vázquez, & Cava, 2011),
other literature data refers either to ruminants (Luciano et al., 2009,
2011; Vasta, Nudda, Cannas, Lanza, & Priolo, 2008), rabbits (Gai et al.,
2009; Liu, Dong, Tong, & Zhang, 2011; Liu, Zhou, Tong, & Vaddella,
2012; Liu et al., 2009) or poultry (Schiavone et al., 2008). Moreover, so
far the reported dosages have been low, and despite the fact that pigs
(e.g. Iberian) can ingest relatively high levels of tannins, information
about the effect of tannins (type and concentration) is lacking. There-
fore, the present study was conducted to investigate the potential im-
pact on carcass and meat quality of supplementing the diet of pigs with
SCWE rich in hydrolysable tannins, considering also fatty acid com-
position and oxidative stability.

⁎
Corresponding author at: Agricultural Institute of Slovenia, Hacquetova Ulica 17, 1000 Ljubljana, Slovenia.
E-mail address: meta.candek-potokar@kis.si (M. Čandek-Potokar).

http://dx.doi.org/10.1016/j.meatsci.2017.06.012
Received 10 April 2017; Received in revised form 8 June 2017; Accepted 22 June 2017
Available online 23 June 2017
0309-1740/ © 2017 Elsevier Ltd. All rights reserved.
V. Rezar et al. Meat Science 133 (2017) 95–102

Table 1 (AOAC method 920.39), crude fibre (fritted glass crucible method,
Chemical and fatty acid composition (% of total fatty acids) of feed mixtures. AOAC method 978.10) and crude ash (AOAC method 942.05). Animals
were fed experimental diets for 70 days, including a 5-day transitional
Treatment groupa
period. At the age of 193 days and weight of 122.5 kg, the experimental
T0 T1 T2 T3 pigs were slaughtered in one batch using routine abattoir procedure
(CO2 stunning, dehairing). Feed was withdrawn one day prior to
Dry matter (g/kg) 892 885 881 885 slaughter.
Crude ash (g/kg) 48 45 46 45
Crude protein (g/kg) 175 169 166 164
Crude fat (g/kg) 26 29 28 27 2.2. Carcass and meat quality measurements
Crude fibre (g/kg) 52 47 49 50
Nitrogen free extract (g/kg) 593 595 591 599 After the slaughter, pigs were eviscerated, leaf (i.e. subperitoneal)
α-tocopherol (mg/kg) 67.3 70.4 71.4 69.6
fat was removed, carcasses split apart, weighed and classified by the
γ-tocopherol (mg/kg) 20.9 21.3 21.7 21.9
Main fatty acid composition (% of the official classification body, using a method approved for Slovenia (OJ
total fatty acids)b EU L56/28, 2008). The method consists of measuring minimal fat
C 12:0 < 0.01 < 0.01 < 0.01 < 0.01 thickness over gluteus medius muscle (backfat thickness) and the
C 14:0 0.09 0.08 0.08 0.08 shortest distance between cranial end of gluteus medius and dorsal edge
C 16:0 14.0 13.7 13.6 13.7
C 18:0 2.2 2.4 2.3 2.3
of vertebral canal at the carcass split line with a digital caliper and
Σ C18:1 27.9 27.7 28.0 27.9 enables calculation of lean meat content. Measurement of pH (pH 3)
C 18:2 n − 6 51.2 50.9 51.1 51.0 was taken in longissimus lumborum muscle (LL) at the level of last rib 3 h
C 18:3 n − 3 2.7 3.3 3.1 3.1 post mortem using a MP120 Mettler-Toledo pH meter (Mettler-Toledo
Σ SFA 17.4 17.2 17.1 17.2
GmbH, Schwarzenbach, Switzerland). The carcasses were cooled
Σ MUFA 28.7 28.5 28.7 28.6
Σ PUFA 53.9 54.2 54.2 54.1 overnight at 0–2 °C until the internal temperature dropped below 7 °C.
Σ n − 3 PUFA 2.7 3.3 3.1 3.1 A day after the slaughter the carcasses were cut at the level of last rib
Σ n − 6 PUFA 51.3 50.9 51.1 51.0 perpendicularly to the spine. The measurements of CIE L*, a*, b* colour
Σ n − 6/Σ n − 3 PUFA 19.2 15.5 16.4 16.6 parameters (using Minolta Chroma Meter CR-300, Minolta Co. Ltd.,
a Osaka, Japan) and ultimate pH were performed on a freshly cut surface
The control group (T0) received feed without supplementation, while the experi-
mental groups T1, T2 and T3 were offered the same diet supplemented with 1%, 2% and of LL. A digital photo of the cross-section was taken and the measure-
3% of commercially available sweet chestnut wood extract, respectively. Diet T0 was ments of loin eye area and area of the corresponding fat performed
composed of maize (62%), soya meat (13%), wheat meal (8%), rapeseed meal (7%), using LUCIA.NET 1.16.5 software (Laboratory Images s.r.o., Prague,
sunflower meal (5%), molasses (2%), CaCO3 (1.1%), NaCl (0.6%), lysine (1%), methio- Czech Republic) performed as described in Batorek et al. (2012).
nine (0.3%), Ca(H2PO4)2 (0.17%).
b
Only predominant fatty acids are listed, but the sum of saturated fatty acids (SFAs),
Caudally from last rib, two 2.5 cm thick chops of LL were taken,
monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) are trimmed of epimysium and external fat and used for the determination
computed using all analysed fatty acids. of drip loss, chemical composition, thawing loss, cooking loss and shear
force. Drip loss was determined according to the EZ method
2. Materials and methods (Christensen, 2003). In short, two cylindrical samples of LL were ex-
cised, weighed and stored in plastic containers at 4 °C. Drip loss was
2.1. Animals and diets expressed as the difference (%) from the initial sample weight after 24
and 48 h. For determination of chemical composition, LL samples were
Experimental design, animals and feed composition are described in minced and the moisture, intramuscular fat (IMF) and protein content
detail elsewhere (Čandek-Potokar et al., 2015). As explained therein, were determined using near-infrared spectral analysis (NIR Systems
the work was undertaken with full owner compliance and within the 6500, Foss NIR System, Silver Spring, MD, USA) using internal cali-
normal running of the farm. The study was performed following the bration (Prevolnik et al., 2005). The second LL chop was weighed,
Slovenian law on animal protection (Zakon o zaščiti živali, 2007) and vacuum packed and frozen at − 20 °C until analysis. Samples were
was not subject to ethical protocols according to Directive 2010/63/EU thawed (overnight at 4 °C), gently drained with a paper towel, weighed
(2010), i.e. approved feed additives were used (European Union and the difference in weights used for thawing loss calculation. The
Register of Feed Additives, 2013) and tissue samples were taken after same samples were then cooked in a thermostatic bath (ONE 7-45,
the slaughter. Briefly, 24 crossbred (Large White × Landrace) entire Memmert GmbH, Schwabach, Germany) until the internal temperature
male pigs were allocated within litters to four treatment groups, housed reached 72 °C, drained and reweighed for cooking loss evaluation, and
individually with ad libitum access to feed and water. All the animals cooled overnight at 4 °C. The next day, shear force was measured on
were fed a commercial feed mixture (Table 1) that was calculated to two 2.5-cm-wide cylindrical cores excised from the sample using TA
contain 13.2 MJ ME/kg and to meet requirements according to NRC Plus texture analyzer (Ametek Lloyd Instruments Ltd., Bognor Regis,
(2012). The control group (T0) received feed without supplementation, UK) equipped with a 60° V-shaped blade at a crossheaf speed of
while the experimental groups T1, T2 and T3 were offered the same 3.3 mm/s.
feed supplemented with 1%, 2% and 3% of commercially available
SCWE (Farmatan®, Tanin Sevnica d.d., Sevnica, Slovenia), respectively. 2.3. Preparation of meat and subcutaneous fat samples for chemical
Sweet chestnut wood extract is rich in hydrolysable tannins, mainly analysis
gallotannins (Bee et al., 2016; Biagi et al., 2010). The analysis of SCWE
for total phenols – determined by the Folin-Ciocalteau colourimetric Muscle and fat samples were trimmed of other tissues. Each muscle
method (Rigo et al., 2000) and expressed as gallic acid equivalents – sample was divided into two parts: one was homogenised in liquid ni-
showed the content of 43.6%. Feed samples were taken at the beginning trogen, the other was transferred to 50-mL polypropylene containers
and at the end of the experiment, pooled and used for proximate ana- with covers, cooked in a water bath (LCS 0081 Lauda, Bartelt GmbH,
lysis and determination of fatty acid composition. Proximate analysis Graz, Austria) for 60 min at 90 °C, and then cooled to room temperature
(moisture, crude ash, crude protein, crude fat, crude fibre) of feed was in a cold water bath. All fat and muscle samples were frozen in liquid
performed according to standard procedures (AOAC, 2000): dry matter nitrogen and homogenised using a laboratory knife mill (Grindomix
(in oven drying at 95–100 °C AOAC method 934.01), crude protein (the GM200, Retsch GmbH and Co., Haan, Germany). Concentration of
copper catalyst Kjeldahl method AOAC method 984.13), crude fat carbonyls was determined in myofibril isolates from fresh muscle

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V. Rezar et al. Meat Science 133 (2017) 95–102

samples, while tocopherols, malondialdehyde (MDA), and antioxidant
capacity of the lipid-soluble compounds (ACL) were determined in both
fresh and thermally treated meat. Fatty acid composition of meat
(fresh) and subcutaneous fat were also determined.
2.3.1. Determination of vitamin E (α- and γ-tocopherol)
Concentrations of tocopherols in feed and meat samples were
measured according to the methodology of Abidi and Mounts (1997)
and Rupérez, Martín, Herrera, and Barbas (2001). Samples were treated
with ethanol and the tocopherols were extracted using hexane. Hexane
phases were transferred to clean tubes and evaporated under a gentle
stream of nitrogen. Residues were subsequently dissolved in ethanol.
Samples were transferred to autosampler vials and analysed by reverse-
phase HPLC using a Prodigy 5 μ ODS(2) column (250 × 4.6 mm, Phe-
nomenex Inc., USA). The mobile phase consisted of methanol and had a
flow rate of 1.5 ml/min. Results of the analysis were evaluated using
the Agilent HPLC (Agilent Technologies, Wilmington, DE, USA). The
instrument was calibrated using external standards (Tocopherol set,
Sigma-Aldrich Co.).
2.3.2. Determination of antioxidant capacity of the lipid-soluble compounds
(ACL)
The ACL in meat samples was measured using a photo-
chemiluminescence method by PhotoChem® (Analytik Jena, Jena,
Germany) and is presented as Trolox equivalent. Lipid- soluble anti-
oxidants from samples were extracted with hexane (0.6 g of homo-
genised meat sample in 500 μl of water and 500 μl of hexane), mixed on
a vortex and centrifuged (10.000 ×g, 10 min, 4 °C). The supernatants
were analysed in accordance with ACL Kit protocol (Analytik Jena,
Jena, Germany). Briefly, 10 μl of supernatant was mixed with 2300 μl
of R1 (methanol), 200 μl of R2 (buffer) and 25 μl or R3 (photosensitizer
and detection reagent containing luminol), and analysed. Inhibition of
luminescence signal (integration over 250 s) in comparison to blank
was calculated (PCLsoft). For calibration of instrument, trolox was used
in 0.5 to 2.0 nmol range.
2.3.3. Determination of malondialdehyde (MDA)
Oxidation of meat lipids was monitored by measurement of MDA
concentration in individual fresh and heat-processed meat samples. A
modified version of the methodology of Vila, Jaradat, Marquardt, and
Frohlich (2002) was implemented to determine MDA concentration in
samples using HPLC. Homogenised samples (0.3 g) were mixed with
1.5 ml of 2.5% trichloroacetic acid in 2 ml microcentrifuge tubes, left
for 10 min and then centrifuged (15,000 × g for 15 min at 4 °C). One ml
of supernatant was mixed with 1.5 ml of 0.6% thiobarbituric acid and
heated at 90 °C for 1 h. After cooling, the samples were filtered through
Millipore filters (0.22 μm) into auto-sampler vials. The Agilent HPLC
system was used with a 1260 Infinity fluorescence detector. For se-
paration, a reverse-phase HPLC chromatography column (HyperClone 5
u ODS (CI8) 120A, 150 × 4.6 mm; Phenomenex Inc., USA) and CI8
ODS guard column (4 × 3 mm; Phenomenex Inc., USA) were used. The
mobile phase consisted of a 65% 50 mM KH2P04 buffer (pH 6.9) and
35% methanol. The mobile phase flow rate was 1.0 ml/min.
50% trichloroacetic acid. After centrifugation (4000 × g for 15 min at
4 °C), the pellets were washed three times with a 1 ml of mixture of
ethanol:ethyl acetate (1:1) to eliminate traces of residual DNPH. The
pellets were afterwards dissolved in 2 ml of 6 M guanidine HCl with
20 mM sodium phosphate buffer (pH 6.5) and centrifuged for 15 min at
4000 ×g. The protein and carbonyl concentrations were spectro-
photometrically determined in the supernatant (BioSpectrometer
Fluorescence, Eppendorf, Hamburg, Germany). The concentration of
protein was calculated from absorbance at 280 nm according to the
standard concentrations of BSA in 6 M guanidine HCl while the con-
centration of carbonyls was measured in the samples treated with
DNPH at 370 nm (considering that the extinction coefficient for DNPH
at 370 nm is 21/mM− 1 cm− 1).
2.3.5. Fatty acids composition
The fatty acid composition of the diets, intramuscular fat and sub-
cutaneous fat was determined using gas chromatography following
transesterification of lipids (Fidler, Salobir, & Stibilj, 2000). Approxi-
mately 0.5 g of each sample was transmethylated in situ (Park & Goins,
1994) using 0.5 M NaOH in methanol followed by 14% BF3 in me-
thanol. Fatty acid methyl ester (FAME) was extracted using hexane. For
FAME separation, an Agilent GC (Agilent, Santa Clara, CA; USA)
equipped with an Omegawax 320 column (30 m × 0.32 mm
i.d. × 0.25 μm; Supelco, Bellefonte, PA, USA) and FID detector was
used. An Agilent GC ChemStation (Agilent, Santa Clara, CA; USA) was
used for data acquisition and processing. Individual FAMEs were
identified using standard mixtures (Nu Chek Prep Inc., Elysian, MN,
USA).
2.4. Statistical analysis
An analysis of variance (one-way ANOVA) for the effect of treat-
ment group was performed by MIXED procedure of SAS statistical
software 9.2 (2011) (SAS Inst., Inc., Cary, NC, USA). In the case of
significant treatment group effect, the differences (between least-
squares means) were tested with the Dunnett test (i.e. comparing each
treatment group with the control) and in the case of significance, results
are presented in Figs. 1–4. Additionally, carcass weight and backfat
thickness -were tested as a covariate in the case of carcass traits and
fatty acid composition, respectively; results are not shown, but are used
for discussion.
3. Results and discussion
3.1. Carcass traits
There was no major effect of the treatment groups noted for carcass
traits (Table 2) except for the reduced fat deposition in the group with
the highest (3%) addition of tannin extract (Fig. 1). In T3 group, backfat
Table 2
Carcass traits - least squares means and effect of treatment group.
Treatment groupa

2.3.4. Concentration of carbonyl groups in myofibril isolates T0 T1 T2 T3 SEM P-value

Protein oxidation was measured according to the method of Oliver,
Ahn, Moerman, Goldstein, and Stadtman (1987) as modified by Warm carcass weight, kg 93.6 95.4 96.7 86.3 3.40 0.18
Mercier, Gatellier, Viau, Remignon, and Renerre (1998) in myofibril Dressing percentage, % 75.2 76.5 76.8 74.8 0.62 0.11
Loin thickness, mm 74.7 75.7 73.8 72.5 2.34 0.81
isolates which were prepared according to Pietrzak, Greaser, and Backfat thickness, mm 11.2 11.0 10.6 7.5 0.77 0.001
Sosnicki (1997). The concentration of carbonyl groups (nmol/mg of Lean meat content, % 61.9 62.5 62.1 64.2 0.74 0.15
protein) was detected by 2,4-dinitrophenylhydrazine (DNPH) to form Loin eye area, cm2 47.8 50.0 51.1 47.8 1.99 0.64
protein hydrazones. Two aliquots (300 μl) of myofibrillar suspension LL overlying fat area, cm2 13.4 13.5 13.2 10.8 0.54 0.005
were treated with 1 ml of 2 N HCl (to determine the concentration of
LL – longissimus lumborum muscle, SEM – standard error of mean, n = 24.
proteins), while two aliquots were treated with an equal volume of a
The control group (T0) received feed without supplementation, while the experi-
0.2% (w/v) DNPH in 2 N HCl. Samples were incubated for 1 h at room mental groups T1, T2 and T3 were offered the same diet supplemented with 1%, 2% and
temperature under agitation and afterwards precipitated by 300 μl of 3% of commercially available sweet chestnut wood extract, respectively.

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V. Rezar et al. Meat Science 133 (2017) 95–102

Fig. 1. Effect of tannin supplementation on carcass and meat quality traits. Only traits significantly affected (P < 0.05) by dietary treatment are depicted. The values shown represent
differences between the control and supplemented groups. The levels of significance are denoted as: NS – P > 0.10; †P > 0.05; *P < 0.05; **P < 0.01. T0 – control group, no sweet
chestnut wood extract supplement; T1–1% sweet chestnut wood extract; T2–2% sweet chestnut wood extract; T3–3% sweet chestnut wood extract.

thickness and fat area over LL were reduced by 32% and 20%, re-
Table 3
spectively (P < 0.05) compared to T0 group. The results reflect the
Meat quality traits of longissimus lumborum muscle - least squares means and effect of
differences in fattening performance as previously reported (Čandek- treatment group.
Potokar et al., 2015). However, it is worth noting that although smaller,
the difference in fat deposition remained significant also when groups Treatment groupa
were compared at the same carcass weight, whereas no effect was noted
T0 T1 T2 T3 SEM P-value
for muscular deposition. Previous findings suggest that tannins not only
affect food consumption and digestion but also decrease the efficiency Water, % 74.1 74.9 74.1 74.3 0.3 0.47
of converting the absorbed nutrients to new body substances (Chung, Intramuscular fat, % 1.23 1.07 1.07 1.06 0.09 0.49
Protein, % 24.1 23.8 23.9 23.7 0.13 0.14
Wong, Wei, Huang, & Lin, 1998). Since the concentration of tannins in
pH 3 h 6.04 5.87 5.71 5.89 0.11 0.31
our experiment was two to six times higher than in the previously pH 24 h 5.29 5.34 5.33 5.30 0.03 0.48
mentioned studies it was interesting to observe rather limited impact on Drip loss 24 h, % 5.2 5.7 6.2 6.5 0.69 0.64
growth performance and body composition. It is possible that negative Drip loss 48 h, % 7.2 8.2 8.8 8.9 0.81 0.47
effects on nutrient utilisation were counterbalanced by the positive Thawing loss, % 11.0 14.7 15.4 15.7 1.17 0.044
Cooking loss, % 35.4 35.4 34.0 33.5 0.89 0.36
influence on gut health (Biagi et al., 2010; Chung, Wei, & Johnson,
Shear force, N 185.3 196.1 161.6 170.5 13.0 0.30
1998) or by adaptation ability of salivary glands (Čandek-Potokar et al., L* (lightness) 57.9 55.6 56.1 57.5 0.8 0.20
2015) producing increased amounts of tannin-binding proline-rich a* (redness) 7.6 7.1 7.9 8.0 0.5 0.56
proteins (Cappai, Wolf, Pinna, & Kamphues, 2013). Recent publications, b* (yellowness) 4.8 3.9 4.3 4.9 0.4 0.33
testing similar supplementations with hydrolysable tannin extract, also a
The control group (T0) received feed without supplementation, while the experi-
show no major effect on pig performance (Bee et al., 2016; Bilić-Šobot
mental groups T1, T2 and T3 were offered the same diet supplemented with 1%, 2% and
et al., 2016), proving that pigs are relatively resistant to high doses of 3% of commercially available sweet chestnut wood extract, respectively. SEM – standard
ingested tannins. As for the effect of dietary tannin addition on body error of mean, n = 24.
fatness (i.e. reducing fat deposition), the two mentioned studies showed
no effects. However, numerous studies on various dietary polyphenols
show their anti-obesity potential (Lei et al., 2007), interfering with Table 4
Concentration of α- and γ-tocopherol, antioxidant capacity of lipid-soluble compounds
biosynthesis of cholesterol and utilisation of lipids (Axling et al., 2012)
(ACL), malondialdehyde (MDA) and carbonyls in meat (longissimus lumborum muscle) -
and affecting carbohydrate metabolism (Hanhineva et al., 2010). As least squares means and effect of treatment group.
reviewed by Wang et al. (2014), in vitro cellular studies demonstrated
that polyphenols reduce adipocyte viability and preadipocyte pro- Treatment groupa
liferation, suppress adipocyte differentiation and triglyceride accumu-
T0 T1 T2 T3 SEM P-value
lation, and stimulate lipolysis and β-oxidation. In compliance, studies
on laboratory animals also showed lower body weight, fat mass, and α-Tocopherol, μg/100 g muscle
triglyceride concentration through enhancing energy expenditure and Fresh 268.2 267.9 241.1 202.5 21.3 0.036
Cooked 242.2 256.2 258.2 252.9 16.3 0.91
fat utilisation in relation to consuming polyphenols.
γ-Tocopherol, μg/100 g muscle
Fresh 8.19 9.15 7.60 6.31 0.69 0.058
Cooked 7.58 9.45 8.38 7.63 0.81 0.36
3.2. Meat quality ACL, nmol/g muscle
Fresh 3.69 3.73 3.77 3.87 1.10 0.61
Cooked 3.27 3.11 3.06 3.12 0.24 0.94
Dietary treatments with tannins did not result in any major differ-
MDA, nmol/g muscle
ences in observed physical-chemical properties of meat quality or Fresh 0.23 0.28 0.26 0.39 0.03 0.014
chemical composition (Table 3). Although carcass adiposity of T3 group Cooked 7.38 5.92 3.33 5.14 0.60 0.002
was significantly lower than in other groups, no effect on IMF content Carbonyls, nmol/mg proteins 1.20 1.83 1.95 2.58 0.13 < 0.001
was observed. This result could be related to the fact that IMF was
SEM – standard error of mean, n = 24.
generally very low, as experimental pigs were entire (non-castrated) a
The control group (T0) received feed without supplementation, while the experi-
males which exhibit higher leanness than their castrated counterparts mental groups T1, T2 and T3 were offered the same diet supplemented with 1%, 2% and
(Batorek et al., 2012). The only statistically important difference ob- 3% of commercially available sweet chestnut wood extract, respectively.
served among the groups was in thawing loss (Fig. 1); in T3 group it
was 43% higher than in T0 group (P < 0.05). The thawing loss in T1 3.3. Tocopherols, ACL, MDA and carbonyl levels in meat
and T2 was also notably higher than in T0 (34 and 40% respectively),
and close to significance (P = 0.16 and P = 0.09, respectively). Al- The effect of dietary tannin supplementation was observed on α-
though the differences were less notable (P > 0.10), other water and γ-tocopherol, MDA and carbonyl concentrations of LL (Table 4).
holding capacity (WHC) indicators (drip loss after 24 and 48 h) showed However, it should be emphasised that the effect of treatment on α- and
the same trend, (i.e. values increasing with higher tannin doses). γ-tocopherol concentrations was noted only in T3 and is presumably an
artefact, as it was not confirmed in cooked samples (higher values than

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V. Rezar et al. Meat Science 133 (2017) 95–102

Fig. 2. Effect of tannin supplementation on indicators of muscle oxidation. Only traits significantly affected (P < 0.05) by dietary treatment are depicted. The values shown represent
differences between the control and supplemented groups. The levels of significance are denoted as: NS – P > 0.10; *P < 0.05; **P < 0.01. T0 – control group, no sweet chestnut wood
extract supplement; T1–1% sweet chestnut wood extract; T2–2% sweet chestnut wood extract; T3–3% sweet chestnut wood extract; MDA – malondialdehyde.

threshold level (0.50 mg/kg) for rancidity detection by trained sensory

Table 5 panellists (Dunshea, D'Souza, Pethick, Harper, & Warner, 2005). Low
Fatty acid composition (% of total fatty acids) of subcutaneous and intramuscular fat -
levels of MDA in fresh meat samples can be related to low amounts of
least squares means and effect of treatment group.
polyunsaturated fatty acids (PUFAs) and sufficient vitamin E con-
Treatment groupa centration for adequate PUFA protection (the minimal physiological
requirement of vitamin E needed for protection of PUFA is 112–131 μg/
T0 T1 T2 T3 SEM P-value 100 g; Muggli, 1994). Increased MDA concentration along with in-
Subcutaneous fat creased concentration of carbonyl groups indicates prooxidative effect
Σ SFA 36.69 38.14 37.90 33.90 0.75 0.003 of tannin supplementation denoting that meat oxidation could be fur-
Σ MUFA 44.01 41.05 41.28 41.30 0.58 0.005 ther increased during storage (Filgueras et al., 2010; Mercier et al.,
Σ PUFA 19.30 20.82 20.82 24.81 0.86 0.002 1998) or processing (i.e. in dry-cured meat products) (Koutina,
Σ n − 6 PUFA 18.27 19.47 19.53 23.33 0.80 0.002
Σ n − 3 PUFA 0.97 1.29 1.24 1.41 0.06 < 0.001
Jongberg, & Skibsted, 2012). As suggested by Traore et al. (2012) and
n − 6/n − 3 PUFA 18.86 15.10 15.81 16.60 0.21 < 0.001 Lonergan, Huff-Lonergan, Rowe, Kuhlers, and Jungst (2001), inferior
Σ LC PUFA 1.48 1.49 1.49 1.81 0.07 0.006 WHC may be directly associated with higher oxidation level of either
Σ LC n − 6 PUFA 1.25 1.22 1.23 1.50 0.06 0.006 proteins (i.e. carbonyls) or lipids (i.e. MDA), leading to protein dena-
Σ LC n − 3 PUFA 0.23 0.27 0.26 0.31 0.01 0.003
turation, thus lowering their ability to bind water (Joo, Kauffman,
Σ LC n − 6/Σ n − 3 PUFA 5.45 4.53 4.73 4.90 0.08 < 0.001
Kim, & Park, 1999; Offer & Knight, 1988), as can be observed also in the
Intramuscular fat present study. With regard to the possible effects of tannins on vitamin
Σ SFA 36.81 38.09 38.54 37.16 0.40 0.029
Σ MUFA 41.24 37.99 38.53 36.81 1.36 0.17
E content and oxidative stability of meat in monogastrics, most research
Σ PUFA 21.94 23.91 22.92 26.03 1.55 0.32 was done in chickens and rabbits and shows mostly no effect, or some
Σ n − 6 PUFA 20.90 22.68 21.79 24.71 1.50 0.35 positive effects, on vitamin E concentration and oxidative stability of
Σ n − 3 PUFA 1.03 1.23 1.11 1.31 0.07 0.039 meat. However, the concentrations of tannins used were much lower in
Σ n − 6/Σ n − 3 PUFA 20.26 18.36 19.49 18.84 0.47 0.056
comparison to our experiment. Liu et al. (2009) found significantly
Σ LC PUFA 6.58 7.09 7.03 7.87 0.66 0.59
Σ LC n − 6 PUFA 5.89 6.29 6.28 7.00 0.60 0.63 lower iron-induced lipid oxidation in rabbit meat with 0.5% dietary
Σ LC n − 3 PUFA 0.69 0.80 0.75 0.88 0.06 0.24 tannin supplementation. In contrast, Schiavone et al. (2008) observed
Σ LC n − 6/Σ n − 3 PUFA 8.51 7.85 8.35 7.96 0.25 0.23 no effect of dietary tannin supplementation (0.15 to 0.25%) on lipid
a
oxidation in raw or heat-treated breast muscle of broilers. Similarly,
The control group (T0) received feed without supplementation, while the experi-
Voljč et al. (2013) showed that tannins at 0.3% in broiler diet had no
mental groups T1, T2 and T3 were offered the same diet supplemented with 1%, 2% and
3% of commercially available sweet chestnut wood extract, respectively. SEM - standard effect on concentration of vitamin E in raw, and MDA in raw and
error of mean, SFA – saturated fatty acids, MUFA – monounsaturated fatty acids, PUFA – cooked, breast muscle. According to Surai (2014), polyphenols do not
polyunsaturated fatty acids, LC – long chain (C ≥ 20), n = 24. seem to offer in vivo antioxidant protection similar to that of vitamin E,
since they are not efficiently absorbed from the gastrointestinal tract,
they are quickly metabolised and excreted from the organism. How-
in fresh meat). The muscle antioxidant capacity indicator (ACL) was not ever, Luciano et al. (2009, 2011) showed significant accumulation of
affected by treatment, whereas increased carbonyl groups' concentra- phenolic compounds after dietary supplementation of condensed
tion and MDA (T3 group only) in fresh meat are indicative of the pro- tannin-rich quebracho in lambs, resulting in improved overall muscle
oxidant effect of tannin supplementation (Fig. 2). On the other hand, in antioxidant status but with no effect on lipid oxidation.
cooked meat samples there was a significant decrease of MDA in T2 and
T3 (55% and 30%, respectively)compared to the control. It should be
noted that the observed MDA concentrations detected in fresh meat 3.4. Fatty acid composition of meat and subcutaneous fat tissue
(from 16.6 μg/kg in T0 to 28.1 μg/kg in T3) and cooked meat (from
0.24 mg/kg in T2 to 0.53 mg/kg in T0) were mainly well below the Important differences in the fatty acid profile occurred to a much
greater extent in subcutaneous fat than in IMF (Table 5). In the case of

Fig. 3. Effect of tannin supplementation on fatty acid composi-

tion of intramuscular fat (% of the total fatty acids). Only traits,
significantly affected (P < 0.05) by dietary treatment are de-
picted. The values shown represent differences between the
control and supplemented groups. The levels of significance are
denoted as: NS – P > 0.10; *P < 0.05. T0 – control group, no
sweet chestnut wood extract supplement; T1–1% sweet chestnut
wood extract; T2–2% sweet chestnut wood extract; T3–3% sweet
chestnut wood extract; SFA – saturated fatty acids; PUFA –
polyunsaturated fatty acids.

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V. Rezar et al. Meat Science 133 (2017) 95–102

Fig. 4. Effect of tannin supplementation on fatty acid composition of subcutaneous fat (% of the total fatty acids). Only traits significantly affected (P < 0.05) by dietary treatment are
depicted. The values shown represent differences between the control and supplemented groups. The levels of significance are denoted as: NS – P > 0.10; *P < 0.05; **P < 0.01;
***P < 0.0001. T0 – control group, no sweet chestnut wood extract supplement; T1–1% sweet chestnut wood extract; T2–2% sweet chestnut wood extract T3–3% sweet chestnut wood
extract; SFA – saturated fatty acids; MUFA – monounsaturated fatty acids; PUFA – polyunsaturated fatty acids; LC – long chain (C ≥ 20).

IMF, dietary treatment influenced total n − 3 PUFAs (mostly marked
by α-linolenic acid) that were present in a higher proportion in T3
compared to T0 (Fig. 3). At the same time, an increased proportion of
saturated fatty acids (SFAs) was observed in T2. In subcutaneous fat,
the effect of hydrolysable tannins supplementation was most pro-
nounced in T3 (Fig. 4). In comparison to T0, the proportion of SFA and
monounsaturated fatty acids (MUFAs), especially palmitic (C:16:0) and
oleic acid (C18:1 n − 9) was lower in T3, whereas the proportion of
almost all fatty acids from n − 6 and n − 3 families was higher (for
more details see Supplementary Table 2). i.e. linoleic (C18:2 n − 6), α-
linolenic acid (C18:3 n − 3), arachidonic (C20:4 n − 6), doc-
osapentaenoic (C22:5 n − 3) and docosahexaenoic (C22:6 n − 3)
acids, and total n − 6 and n − 3 PUFA and long chain (LC) PUFA. In
addition, n − 6/n − 3 PUFA and LC-PUFA ratios were also reduced.
The effects in T1 and T2 groups followed the same, but less pronounced,
trend. In these two groups, a significantly reduced proportion of oleic
acid and total MUFA, and increased proportion of α-linolenic acid and
total n − 3 PUFA were observed in addition to reduced n − 6/n − 3
PUFA ratio (P < 0.05) (Table 5). It is difficult to know to what extent
the tannins could have a direct or indirect effect on the observed fatty
acid composition. As the body is able to synthesise only SFAs and
MUFAs, the reduced proportion of both in T3 can be simply a con-
sequence of lower feed intake and thus reduced energy availability for
fat synthesis. Alternatively, it could be due to lower synthesis of SFA
and MUFA when fat deposition is reduced (Wood et al., 2008), which
can also explain increased proportions of linoleic acid in tannin sup-
plemented groups. Similarly in IMF, the gradual increase in linoleic
acid proportion can be attributed to low IMF content and consequently
more pronounced contribution of PUFA (linoleic acid) rich phospho-
lipid membrane lipids to overall fatty acid composition of IMF. How-
ever, when backfat thickness was included in the statistical model, the
proportions of SFA and PUFA in subcutaneous fat and n − 3 PUFA
proportion in IMF were still affected by the diet, indicating an effect of
tannin supplementation regardless of the effect of fat deposition.
4. Conclusions
According to the results of the present study, there is an indication
that supplementation with hydrolysable tannins reduces carcass fat
deposition, leading also to a higher percentage of PUFA in fat tissue.
With regard to meat quality, the results on fresh meat (increased
markers of lipid and protein oxidation accompanied by reduced water
holding capacity) are indicative of increased prooxidative potential.
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.meatsci.2017.06.012.

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