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Meat Science 96 (2014) 719728

Contents lists available at ScienceDirect

Meat Sc

l sLopez-Rituerto et al., 2012), an NMR metabolomic investigation onbovine milk revealed milk metabolites of importance for thetechnological properties of milk (Sundekilde, Frederiksen, Clausen,

Footed Iberian and Mangalitza (Straadt, Aaslyng, & Bertram, 2013).The study was conducted by raising a large number of pigs (n= 100)under identical and controlled conditions and consumer evaluations ofscience. Thus, metabolomics has been applied to study fruits,beverages and wine (Clausen, Pedersen, Bertram, & Kidmose,2011; Cuthbertson, Andrews, Reganold, Davies, & Lange, 2012;

We recently investigated if new pork prosensory attributes could be obtained by crossing pused in the Danish pig production with the alte2012). New terminologies such as Nutrimetabolomics (Llorach,Garcia-Aloy, Tulipani, Vazquez-Fresno, & Andres-Lacueva, 2012)and Foodomics (Cifuentes, 2012) have emerged to dene studiesin the food and nutrition domains through the application ofadvanced omics technologies, and many recent studies havedemonstrated the usefulness of metabolomic approaches in food

meat, numerous biochemical and biophysical processes take place thatinuence the nal sensory attributes of pork. (Mottram, 1998).Variations in these important meat quality characteristics are alsoclosely related to breed and genetic variations (Estevez, Morcuende,Ventanas, & Cava, 2003; Ramirez & Cava, 2007; Ventanas, Ventanas,Jurado, & Estevez, 2006).Larsen, & Bertram, 2011) and it has also been s

Corresponding author. Tel.: +45 87158353; fax: +45E-mail address: HanneC.Bertram@agrsci.dk (H.C. Bertr

0309-1740/$ see front matter 2013 Elsevier Ltd. All rihttp://dx.doi.org/10.1016/j.meatsci.2013.10.006dical areas, but are alsotechnology, agricultureak, 2012; Weckwerth,

about the changes that occur during ageing of meat (Graham et al.,2012, 2010). However, in addition to ageing, many other parametersare decisive for meat quality. During handling, storage, and cooking ofemerging as powerful approaches in bioand food science (Patti, Yanes, & Siuzd1. Introduction

The beginning of the 21st centuryopening of the so-called postgenTremendous improvements in the deniques and platforms mean that omgaining wide use in pharmaceutical ameat avor/taste, while IMP and inosine were in general not correlated with sensory attributes related to meatavor/taste.

2013 Elsevier Ltd. All rights reserved.

en announced to be thera (Cifuentes, 2012).ent of analytical tech-chniques are not only

can be applied to study processing-induced changes in foods (Beleggiaet al., 2011).

Metabolomic studies on meat have shown that metabolomicsproles could differentiate different cow breeds (Ritota, Casciani,Failla, & Valentini, 2012), as well as the geographical origin of beef(Jung et al., 2010), and that metabolomics could provide informationMeat avor precursorsProton NMR relaxometryWater-holding capacity

thh

e membrane properties and glycolytic potential of muscle bres and differences in lipolysis and proteolysis. Aigh content of carnosine in the meat was associated with a low value of many sensory attributes related toCarnosine specic differences in the mAn NMR-based metabolomics study of porand relation to sensory perception

Ida K. Straadt a, Margit D. Aaslyng b, Hanne Christine Ba Aarhus University, Dept. Food Science, Research Centre Aarslev, Kirstinebjergvej 10, DK-5792b Danish Meat Research Institute, Technological Institute, DK-4000 Roskilde, Denmark

a b s t r a c ta r t i c l e i n f o

Article history:Received 6 August 2013Received in revised form 24 September 2013Accepted 4 October 2013

Keywords:Meat metabolitesPig breeds

Meat extracts from ve diffeIberian/Duroc/Landrace (ILY)by nuclear magnetic resonanand sensory analyses inorderof importance for technoloisoleucine, methionine, pheglycerol and choline-contain

j ourna l homepage: www.ehown thatmetabolomics

87154812.am).

ghts reserved.from different crossbreeds

tram a,slev, Denmark

pig crossbreeds including Duroc/Landrace/Yorkshire (DLY), Iberian/Duroc (ID),angalitza/Duroc (MD), and Mangalitza/Landrace/Yorkshire (MLY) were analysedNMR)-basedmetabolomics. The results were compared with technological traitslucidate the potential of NMR-basedmetabolomics to highlightmeatmetabolitesl and sensory attributes of meat. Amino acids including alanine, carnosine,lanine, and valine, as well as lactate, inosine monophosphate (IMP), inosine,compounds were found to be signicantly affected by crossbreed. The breed-olome were ascribed to differences in ante mortem metabolism, differences in

ience

ev ie r .com/ locate /meatsc ithe obtained pork products revealed that the alternative crossbreedsobtained positive associations compared with conventional DLYcrossbreed (Straadt et al., 2013). Especially texture attributes of thealternative cross-breeds were found to be improved in the alternativecrossbreeds compared with conventional DLY crossbreed (Straadtet al., 2013). The breed-specic differences in the consumer and

4096 echoes were acquired as 16 scan repetitions. The repetitiontime between the scans was 3 s. The measurements were

720 I.K. Straadt et al. / Meat Science 96 (2014) 719728performed on a total of 297 meat samples, and the samples werestored at 20 C for subsequent high-resolution proton NMRmeasurements (see below).

The obtained T2 data were analysed using distributedexponential tting analysis (Menon & Allen, 1991), which wascarried out in MatLab (The Mathworks Inc., Natick, MA,USA)version 7.01 using in-house scripts. Distributed exponential ttingresults in a plot of relaxation amplitude versus relaxation time overa predened range of characteristic relaxation times. In this studywe t 256 logarithmically distributed relaxation times from0.5 ms to 3000 ms, and the area of the T22 relaxation population2.2. NMR transverse relaxation (T2) measurements

Freshmeat samples of approximately 11cm in cross-sectional areaand approximately 5cm long (~5g)were cut along thebre direction ofthe loin using a scalpel either on day 2 or day 3 after slaughter. Threemeat samples were cut for each of the 20 pigs for each of the vecrossbreeds, except for the ID crossbreed where 19 pigs were used.The samples were placed with a vertical bre direction in cylindricalglass tubes (14 mm in diameter and 5 cm high). The meat sampleswere t into the NMR probe with a diameter of 18 mm. Beforemeasurements, all the samples were equilibrated to 25 C in a waterbath for 30min.

The NMR T2 relaxation measurements were performed on a MaranBenchtop Pulsed NMR analyser (Resonance Instruments, Witney, UK)operating at a resonance frequency of 23.2MHz. Transverse relaxation,T2, was measured using the CarrPurcellMeiboomGill (CPMG)sequence. The T2 measurements were performed with a -value(time between 90 pulse and 180 pulse) of 150 s. Data fromsensory evaluations could not be ascribed to differences in aromaproles obtained from gas-chromatography mass spectrometry(GS-MS) (Straadt et al., 2013). Therefore, the aim of the presentstudy was i) to investigate if nuclear-magnetic resonance (NMR)-based metabolomics could identify breed-specic differences inthe metabolite prole of meat extracts and freeze-thaw drip frommeat, and ii) to investigate if these breed-specic differences inthe metabolite proles could be associated with differences intechnological properties (water-holding capacity and amount ofextramyobrillar water as determined by NMR T2 relaxation) andthe sensory perception of the meat.

2. Materials and methods

2.1. Animals and sampling

Crossbreeds were produced by the traditional crossingbetween Duroc boars and Landrace/Yorkshire sows (DLY), and byproducing four alternative crossbreeds derived from crossingsbetween Black Footed Iberian and Mangalitza boars and Durocand Landrace/Yorkshire sows, respectively (ID, ILY, MD, MLY)(Straadt, Aaslyng, & Bertram, 2011). The slaughter pigs wereproduced on a conventional Danish farm and fed ad libitum. Theywere slaughtered at a live weight of approximately 110 kg. Theday after slaughter the loin (longissimus dorsi) was excised,vacuum-packed and aged for 23 days at 5 C, and frozen andstored at 20 C until further analysed. One hundred animalswere included in the present study with 20 animals (10 femaleand 10 castrates) from each of the ve crossbreeds.

The pigs were treated according to EU legislation (Reg. CE n. 1099/2009).was calculated.2.3. pH and drip loss measurements

The pH was determined 22 hours after slaughter (Knick PortamesspH-meter no 751, Berlin, Germany) between 4th and 5th lumbarvertebrae. Water holding capacity (WHC) was measured the day afterslaughter by using the EZ drip loss method (Christensen, 2003). Inshort, for each 20 pigs for each of the ve crossings a 2 cm thick sliceof loin was excised the day after slaughter. Two cubes, each 2.2 cm indiameter were drilled from the core of the chop and placed in a plasticcontainer collecting the drip, and the drip loss was determined after24h by weighing.

2.4. 1H NMR spectroscopy

High-resolution proton NMR spectroscopy was performed onextractions of the loins and on freeze-thaw drip from the loins. Forextraction of the meat samples, a methanol/water extraction methodwas applied. The loin samples from the proton NMR relaxation analysiswere thawed and 100mg samples were cut for each of the 99 pigs andthe samples were transferred to eppendorf tubes. A volume of 300L ofmethanol and 300 L of water were added to each sample, which wassubsequently homogenized on ice for 10 s (Ultra-Turrax Yellow line DIbasic, Bie & Berntsen, Aarhus, Denmark), followed by vortexing for10 seconds. The samples were centrifuged at 10,000 g for 10 min at4 C. The supernatant was transferred to an eppendorf tube and driedwith nitrogen. The samples were frozen at 80 C until proton NMRanalysis. On the day of proton NMR measurements the pellet wasdissolved in 500l water and 100l D2O containing 0.1 % (w/v) sodiumtrimethylsilyl[2,2,3,3-D4]-1-propionate (TSP) and transferred to a 5mmNMR tube.

For the freezethaw drip samples chops of loins of approximately7 7 3 cm (~130 g) were thawed and all the drip from each samplewas collected for each of the 99 pigs. Oneml of the drip was centrifugedat 14,000 g for 2 min at 25 C, and 500 L of the supernatant wastransferred and mixed with 100 L D2O in a 5mm NMR tube.

Proton NMR spectra were recorded at 25C on a Bruker Avance 600spectrometer, operating at a 1H frequency of 600.13 MHz, equippedwith a 5 mm 1H TXI probe (Bruker BioSpin, Rheinstetten, Germany).Standard one-dimensional (1D) proton NMR spectra were acquiredusing a single 90 pulse experiment, and each spectrum was the sumof 64 FIDs. Water suppression was achieved by irradiating the waterpeak during the relaxation delay of 2 s, and 32 K data points spanninga spectral width of 17.36ppm were collected.

All the spectra from the meat extraction were referenced to TSP at0ppm, whereas the spectra from the freezethaw drip were referencedto -glucose at 5.24 ppm. Assignment of the 1H NMR spectra wasobtained by literature data (Brescia et al., 2002; Graham et al., 2010;Jung et al., 2010) or by adding standard compounds. Furthermore, 1DJRES, 2D 1H-1H TOCSY and 2D 1H13C HSQC experiments wereperformed on selected samples to aid in conrming the assignment ofmetabolites. Quantication of selected 1H resonances was carried outby integration of peak areas using the Topspin 3.1 software (BrukerBioSpin, Rheinstetten, Germany). The integrals were normalized to atotal intensity of 1000 to reduce the effect of concentration differencesbetween samples.

2.5. Sensory analysis

Sensory analysis was performed using a trained sensory panelconsisting of 9 menbers with experience in assessing meat asdescribed in Straadt et al. (2013). Briey, for each of the 100 animalsve chops of 2 cm thickness were fried on a pan smeared with a thinlayer of grape seed oil at 170C. The chops were turned every secondminute until a core temperature of 6568 C was reached and thetemperature wasmeasured by using a thermometer with a handheld

probe (Testo 926, Testoterm, Buhl and Bundsoe, Virum, Denmark).

The following attributes were assessed on a 15 cm unstructured linescale going from no intensity to very high intensity, with an anchorone cm from each side. The references used during training of theassessors are given in brackets. Fried pork odour (the odour of thesurface of a pork chop fried at high versus low pan temperature),piggy odour (the odour of melted pig fat), brown surface, meatcolour on the cutting surface, degree of cooking, fried meat avour(reference as for odour), piggy avour (reference as for odour),sweet taste (sucrose), nutty avour (hazelnuts), oily/fatty avour,acidic taste (apple-acetic cider, 10% diluted in water), metallic taste(copper coins), bitter taste (85% bitter dark chocolate), chewing

thaw drip

et al., 2011). Sixteen different metabolites were identied in the meatextraction and freeze-thaw drip samples and assigned (Table 2).

The metabolites identied in the meat extraction and the freezethaw drip samples were quantied by integrating peaks for selected1H NMR resonances (Figs. 1 and 2). The selection of 1H NMR resonancesfor quantication was generally based on the largest peaks and peakswith the least degree of overlap with other peaks. Seven amino acids(alanine, carnosine, isoleucine, methionine, phenylalanine, tyrosineand valine) were quantied, and comparing the levels of the aminoacids between the different crossbreeds reveals that ID has a higherlevel of all these amino acids when compared with the traditionalDLY crossing in both the meat extraction and in the drip samples(Fig. 1ag). In addition, carnosine is higher or signicantly higherfor all the alternative crossbreeds compared with DLY in both themeat extraction and in the drip samples. Signicantly higher levels

Table 2Assignment of metabolites identied in the 1H NMR spectra of the meat extractions andthe freezethaw drip.

Metabolite Chemical shift (ppm) Assignment Multiplicity (Hz)

Amino acidsAla 1.48 CH3 dCarnosine 2.7 CH2 m

3.11 C6H dd7.25 CH4 s8.53 CH2 s

Carnosine (IMP) 4.51 CH mIle 0.96 -CH3 t

1.01 -CH3 d1.91 -CH m

Met 2.14 SCH3 sPhe-Ala 3.14 -CH2 dd

3.97 -CH dd7.38 C2H, C6H, Ring m

C4H, RingC5H, Ring

Tyr 6.9 H3,H5 d7.2 H2,H6 d

Val 0.99 CH3 d1.04 CH3 d

Compounds related to nucleotidesIMP 4.33 C2H m

4.39 C4H m6.15 H1 d8.23 CH-8 s

IMP (Carnosine) 4.51 CH2 ddInosine 4.28 C2H q

4.44 C4H dd6.10 -CH d8.24 CH-8 s8.35 CH-3 s

Organic acidsAcetate 1.93 CH3 s

721I.K. Straadt et al. / Meat Science 96 (2014) 719728When visually inspecting the 1HNMR spectra of themeat extractionsand freeze-thaw drip from the loins, they appear very similar (Straadt

Table 1Meat quality parameters for the loins from the ve crossbreeds (means).

DLY ID ILY MD MLY SEM p-value

pH 5.7 5.7 5.6 5.7 5.6 0.05 0.0491Drip loss (%) 2.9a 0.8b 2.0ab 1.6b 1.7b 0.37 b0.0001T22 area 2.4a 0.8b 1.5b 1.1b 1.3b 0.30 b0.0001

Means within a row with different superscript letters (ab) differ signicantly (P b 0.05).Signicant p-values (b0.05) are shown in bold.The area of the T22 population is calculated from the distributed exponential tting of thePCA analyses were performed. In addition, partial least squaresregression (PLS) analyses were carried out with the polar metabolitesfrom the meat extraction and from the freeze-thaw drip, respectively,as x-variables, and the evaluation by the sensory panel as y-variables.Cross validation was performed with ve cross validation groups, onefor each of the different crossbreeds. The analyses were carried out byusing the SIMCA-P 13.0.0.0 software (Umetrics, Ume, Sweden).

3. Results

3.1. Technological meat quality traits

Results from NMR T2 relaxation, pH and drip loss measurements areshown in Table 1. An overall signicant difference in pH was foundbetween the ve crossbreeds, but no signicant difference was foundwhen comparing the different crossbreeds. The traditional DLY cross-breed had a higher drip loss than all of the alternative crossbreedsexcept ILY. The T22 area was also higher in the DLY compared to all ofthe alternative crossbreeds.

3.1.1. 1H NMR spectroscopic analysis of extraction of meat and freezeThe data were analysed by using the linear mixed-effects model tby maximum likelihood by application of the R statistical software,version 2.14.2 (Department of Statistics, University of Auckland,Auckland, NZ, http://www.r-project.org). Data were analysed with thecrossbreed as xed effect and the replicates within the crossbreed asrandom effect. Data are presented as means +/ standard errors ofthe means (SEM). For testing correlation between two variables,Pearson correlations were calculated by the R statistical software.

Multivariate data analyses were performed by using mean-centreddata for the attributes evaluated by the sensory panel, and mean-centred and unit-variance (UV) scaled data for the polar metabolitesdetected by NMR spectroscopy.resistance, crunchy bres, juiciness after 5 chews, brousness,crumbliness, tenderness and acidic aftertaste after 1015 s.

2.6. StatisticsNMR T2 relaxation measurements.Lac 1.33 CH3 d

Other compoundsBetaine 3.27 N-(CH3)3 s

3.90 CH2 sChols 3.23 N-(CH3)3 sCreatine 3.04 CH3 s

3.93 N-CH2 sGlc 3.49 C2H m

3.74 C3H m3.85 C5H m3.89 C5H d3.91 C6H d4.65 C1H d5.24 C1H d

Glycerol 3.66 CH2 ddUnassigned compound 5.13 NI* d

Ala, alanine, IMP, inosinic acid, Ile, isoleucine, Met, methionine, Phe-Ala, phenyl-alanine,Tyr, tyrosine, Val, valine, Lac, lactate, Chols, choline-containing compounds, Glc, Glucose.

NI: Not identied.

of IMPwere found in all the alternative crossbreeds compared to DLYin both the meat extraction and in the drip samples (Fig. 1h).Conversely for the energy and avour metabolite inosine, lowerlevels of inosine were found in all the alternative crossbreeds

compared to DLY in both the meat extraction and in the dripsamples, however, the difference was only signicant for ILY in themeat extraction samples (Fig. 2a). For lactate the most apparentdifference is that ID has a lower level compared with DLY (Fig. 2c).

a b

c d

e f

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Tyr (6.90 ppm)

DLY ID ILY MD MLY

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Phe-Ala (3.97 ppm)

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abab ab

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P (cine

722 I.K. Straadt et al. / Meat Science 96 (2014) 719728DLY ID ILY MD MLY0

Fig. 1. Relative integral intensities (means) of selected 1H resonances for amino acids and IMFreezethaw drip samples. a) Ala, alanine (1.48ppm), b) carnosine (7.26ppm), c) Ile, isoleu

tyrosine (6.90 ppm), g) Val, valine (0.99 ppm), and h)IMP, inosine monophosphate (4.33 ppmDLY ID ILY MD MLY0

from Table 2) for the ve cross-breeds. Black bars, Extraction of meat samples, White bars,(0.96ppm), d)Met, methionine (2.14ppm), e) Phe-Ala, phenylalanine (3.97ppm), f) Tyr,

).

723I.K. Straadt et al. / Meat Science 96 (2014) 719728aInosine (4.28 ppm)

inte

nsitie

s

4

5

a

b

abab ab3.1.2. Correlations between technological quality (drip loss and T22 area)and the polar metabolites in meat/freezethaw drip

The highest correlationwas found between drip loss and the T22 area(r2=0.70, data not shown). Correlations between drip loss and T22 areaand the metabolites identied in the meat extraction and in the dripsamples are shown in Tables 3 and 4. In general drip loss/T22 area

c

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Laca (1.33 ppm)

DLY ID ILY MD MLY

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ab

Fig. 2. Relative integral intensities (means) of selected 1H resonances for inosine, organic acids asamples, White bars, Freezethaw drip samples. a) inosine (4.28ppm), b) acetate (1.93ppm), c(3.23 ppm), f) creatine (3.04 ppm), g) Glc, Glucose (3.74 ppm) and h) glycerol (3.66 ppm).bAcetate (1.93 ppm)

inte

nsitie

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2.0

2.5

ashowed high negative correlations with the amino acids in themeat extractions and in the freezethaw drip samples, however,for phenylalanine and tyrosine the correlations to drip/T22 areawere not high. For methionine poor negative correlations werefound with drip loss/T22 area in the meat extraction samples,whereas very high negative correlations were found between

d

f

h

DLY ID ILY MD MLY

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Betaine (3.27 ppm)

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babab

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Glycerol (3.66 ppm)

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14a

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nd other compounds (fromTable 2) for theve cross-breeds. Black bars, extraction ofmeat) Lac, lactate (1.33ppm), d) betaine (3.27ppm), e), Chols, choline-containing compounds

Table 3Pearsons correlations between drip loss, T22 area and sensory attributes of the meat and metabolites identied in the meat extracts.

Compounds related to nucleotides

Amino acids Organic acids Other compounds

Metabolite Chem. shift (ppm) Ala Carnosine Ile Met Phe Tyr Val IMP Inosine Acetate Lac Betaine Chols Creatine Glc Glycerol

1.48 7.26 0.96 2.14 3.97 6.9 0.99 4.33 4.28 1.93 1.33 3.27 3.23 3.04 5.24 3.66

Drip loss 0.36 0.55 0.25 0.06 0.07 0.14 0.27 0.43 0.11 0.15 0.23 0.09 0.59 0.12 0.63 0.47T22 area 0.44 0.59 0.43 0.25 0.16 0.26 0.41 0.45 0.03 0.23 0.15 0.22 0.62 0.01 0.56 0.39Acidic avour 0.29 0.43 0.22 0.05 0.04 0.23 0.26 0.22 0.23 0.15 0.11 0.07 0.38 0.18 0.45 0.34Acidic aftertaste 0.42 0.43 0.27 0.12 0.02 0.24 0.30 0.37 0.17 0.15 0.21 0.22 0.58 0.02 0.51 0.38Chewing resistance 0.24 0.57 0.34 0.09 0.14 0.23 0.30 0.38 0.18 0.13 0.01 0.24 0.56 0.05 0.41 0.36Crunchy bers 0.15 0.21 0.19 0.08 0.14 0.13 0.19 0.22 0.05 0.01 0.11 0.14 0.31 0.03 0.27 0.22Fried pork odour 0.01 0.26 0.15 0.02 0.10 0.16 0.17 0.11 0.10 0.01 0.04 0.07 0.18 0.17 0.17 0.25Piggy odour 0.09 0.08 0.03 0.02 0.03 0.07 0.02 0.04 0.04 0.09 0.14 0.10 0.03 0.12 0.10 0.07Brown surface 0.08 0.21 0.29 0.20 0.14 0.24 0.30 0.21 0.13 0.19 0.04 0.06 0.22 0.21 0.35 0.16Meat colour 0.01 0.06 0.04 0.05 0.14 0.04 0.06 0.05 0.10 0.05 0.01 0.27 0.20 0.42 0.16 0.14Degree of cooking 0.27 0.40 0.30 0.16 0.26 0.17 0.29 0.18 0.06 0.25 0.06 0.18 0.24 0.07 0.27 0.15Fried meat avour 0.15 0.25 0.24 0.08 0.14 0.21 0.25 0.15 0.05 0.08 0.06 0.09 0.21 0.19 0.17 0.20Piggy avour 0.05 0.21 0.05 0.05 0.10 0.01 0.01 0.02 0.08 0.03 0.16 0.11 0.08 0.08 0.16 0.13Sweet taste 0.07 0.27 0.12 0.05 0.14 0.11 0.11 0.15 0.00 0.09 0.02 0.09 0.17 0.00 0.16 0.09Nutty avour 0.11 0.12 0.15 0.18 0.11 0.12 0.10 0.12 0.02 0.11 0.01 0.01 0.11 0.19 0.00 0.00Oily-fatty avour 0.10 0.11 0.15 0.07 0.26 0.13 0.19 0.08 0.01 0.05 0.10 0.06 0.05 0.12 0.07 0.01Metallic taste 0.13 0.32 0.12 0.02 0.29 0.10 0.14 0.19 0.14 0.02 0.08 0.04 0.26 0.04 0.29 0.21Bitter taste 0.03 0.04 0.04 0.08 0.06 0.02 0.00 0.09 0.00 0.05 0.12 0.01 0.11 0.07 0.11 0.02Juiciness 0.20 0.09 0.15 0.09 0.13 0.08 0.11 0.03 0.07 0.12 0.06 0.04 0.08 0.13 0.02 0.02Fibrousness 0.11 0.52 0.22 0.04 0.04 0.11 0.19 0.39 0.27 0.03 0.04 0.21 0.64 0.00 0.38 0.35Crumbliness 0.04 0.43 0.11 0.08 0.07 0.09 0.08 0.28 0.34 0.05 0.11 0.13 0.55 0.03 0.35 0.39Tenderness 0.25 0.57 0.29 0.04 0.08 0.16 0.25 0.38 0.26 0.06 0.08 0.25 0.58 0.05 0.42 0.43

Signicant correlations (p b 0.05) are shown in bold.Abbreviations:Ala, alanine, IMP, inosinic acid, Ile, isoleucine, Met, methionine, Phe-Ala, phenyl-alanine, Tyr, tyrosine, Val, valine, Lac, lactate, Chols, choline-containing compounds, Glc, Glucose.

724 I.K. Straadt et al. / Meat Science 96 (2014) 719728methionine and drip loss/T22 area in the freezethaw drip samples.High negative correlations were found between drip loss/T22 areaand IMP, whereas no correlations were found between drip loss/T22 area and inosine in the meat extraction and the freeze-thawTable 4Pearson's correlations between drip loss, T22 area and sensory attributes of the meat and meta

Compounds related to nucleotides

Metabolite Chem. shift (ppm) Amino acids

Ala Carnosine Ile Met Phe Tyr Val

1.48 7.26 0.96 2.14 3.97 6.9 0.99

Drip loss 0.35 0.52 0.43 0.66 0.15 0.08 0.T22 area 0.52 0.56 0.59 0.64 0.39 0.15 0.Acidic avour 0.36 0.40 0.41 0.59 0.16 0.04 0.Acidic aftertaste 0.43 0.40 0.49 0.60 0.25 0.05 0.Chewing resistance 0.40 0.56 0.50 0.57 0.23 0.21 0.Crunchy bers 0.27 0.23 0.28 0.26 0.05 0.21 0.Fried pork odour 0.13 0.13 0.11 0.19 0.02 0.05 0.Piggy odour 0.00 0.14 0.05 0.07 0.02 0.21 0.Brown surface 0.28 0.16 0.27 0.31 0.10 0.11 0.Meat colour 0.06 0.03 0.01 0.02 0.00 0.21 0.Degree of cooking 0.28 0.27 0.36 0.42 0.15 0.18 0.Fried meat avour 0.32 0.12 0.26 0.33 0.04 0.23 0.Piggy avour 0.04 0.17 0.01 0.07 0.11 0.27 0.Sweet taste 0.19 0.18 0.25 0.24 0.12 0.03 0.Nutty avour 0.12 0.05 0.04 0.09 0.09 0.08 0.Oily-fatty avour 0.18 0.03 0.25 0.35 0.01 0.18 0.Metallic taste 0.25 0.21 0.25 0.38 0.14 0.00 0.Bitter taste 0.10 0.13 0.01 0.11 0.04 0.07 0.Juiciness 0.12 0.14 0.18 0.13 0.05 0.14 0.Fibrousness 0.31 0.48 0.40 0.51 0.14 0.14 0.Crumbliness 0.15 0.41 0.25 0.31 0.14 0.02 0.Tenderness 0.35 0.55 0.48 0.54 0.25 0.12 0.

Signicant correlations (p b 0.05) are shown in bold.Abbreviations:Ala, alanine, IMP, inosinic acid, Ile, isoleucine, Met, methionine, Phe-Ala, phenyl-alanine, Tyr, tydrip samples. Only modest and negative correlations were foundbetween drip loss/T22 area and lactate in the meat extractionsamples, whereas good positive correlations were found betweendrip loss/T22 area and lactate in the freezethaw drip samples.bolites identied in freeze-thaw drip.

Organic acids Other compounds

IMP Inosine Acetate Lac Betaine Chols Creatine Glc Glycerol

4.33 4.28 1.93 1.33 3.27 3.23 3.04 5.24 3.66

50 0.34 0.18 0.33 0.43 0.18 0.57 0.29 0.56 0.2359 0.50 0.11 0.43 0.56 0.26 0.59 0.12 0.42 0.1244 0.28 0.01 0.42 0.43 0.24 0.46 0.24 0.36 0.1754 0.43 0.03 0.37 0.42 0.29 0.59 0.21 0.40 0.0750 0.50 0.06 0.36 0.59 0.40 0.65 0.14 0.28 0.0532 0.13 0.22 0.18 0.26 0.23 0.35 0.18 0.19 0.0212 0.18 0.02 0.05 0.18 0.14 0.24 0.12 0.22 0.1906 0.02 0.04 0.08 0.02 0.06 0.03 0.00 0.02 0.0229 0.19 0.03 0.22 0.23 0.00 0.23 0.23 0.32 0.0604 0.26 0.23 0.03 0.09 0.10 0.12 0.40 0.25 0.2137 0.21 0.01 0.37 0.38 0.32 0.35 0.14 0.20 0.0128 0.20 0.01 0.20 0.34 0.26 0.33 0.21 0.21 0.0703 0.13 0.09 0.07 0.09 0.06 0.10 0.01 0.09 0.1326 0.13 0.02 0.20 0.21 0.17 0.18 0.08 0.13 0.0001 0.08 0.01 0.02 0.04 0.01 0.10 0.11 0.00 0.0428 0.04 0.02 0.18 0.19 0.15 0.16 0.20 0.13 0.0226 0.13 0.07 0.28 0.41 0.20 0.31 0.17 0.18 0.0902 0.14 0.07 0.10 0.14 0.07 0.07 0.03 0.03 0.0915 0.01 0.06 0.19 0.22 0.21 0.04 0.15 0.01 0.0439 0.51 0.16 0.25 0.48 0.33 0.67 0.11 0.25 0.1025 0.45 0.14 0.05 0.30 0.15 0.53 0.01 0.22 0.1947 0.51 0.07 0.29 0.54 0.40 0.64 0.13 0.27 0.14

rosine, Val, valine, Lac, lactate, Chols, choline-containing compounds, Glc, Glucose.

3.2. Correlations between sensory attributes and the polar metabolites inmeat/freezethaw drip

In Fig. 3 an overview of the relation between the sensory attributesand the polarmetabolites is displayed fromPLS analyses.Most apparentis that the polar metabolites from the meat extraction and from thefreezethaw drip are related to the sensory attributes in a very similarway, with DLY somewhat separated from the alternative crossbreeds.

Correlations between the sensory attributes and the metabolitesidentied in the meat extraction and freezethaw drip samples areshown in Tables 3 and 4. Carnosine was the amino acid that correlatedto most sensory attributes. Generally negative correlations were foundbetween acidic avour/acidic aftertaste and the seven amino acidsidentied in the meat extraction and freezethaw drip samples.Likewise, negative correlations were found between acidic avour/acidic aftertaste and IMP, whereas no correlations were found betweenacidic avour/acidic aftertaste and inosine in the meat extraction andthe freezethaw drip samples. Poor negative correlations were foundbetween acidic avour/acidic aftertaste and lactate in the meatextraction samples, whereas high positive correlations were foundbetween acidic avour/acidic aftertaste and lactate in the freezethawdrip samples.

The textural attribute juiciness only showedweak positive correlationto alanine in the meat extractions and weak negative correlation tolactate and betaine in the freezethaw drip samples. The attributeschewing resistance, crunchy bres and brousness displayed signicantnegative correlationswith some of the amino acids in themeat extractionand freezethaw drip samples. Negative correlations were found be-tween chewing resistance, crunchy bres and brousness and IMP, andconversely, positive correlations were found between crumbliness and

tenderness and IMP in the meat extraction and the freezethaw dripsamples. No correlations were found between the textural attributesand inosine, except for brousness, which were positively correlatedwith inosine, and for crumbliness and tenderness, which is negativelycorrelated with inosine in the meat extraction samples. No correlationswere found between the textural attributes and lactate in the meatextraction samples, whereas positive correlations were found betweenchewing resistance, crunchy bres and brousness and lactate, andconversely negative correlations were found between crumbliness andtenderness and lactate in the freezethaw drip samples.

4. Discussion

4.1. Energy metabolism and stress susceptibility in the crossbreeds

In the present study the metabolite prole of pork originating fromve different crossbreedswere elucidated byNMR-basedmetabolomics.NMR analyses were conducted on methanol extract of meat and onfreeze-thaw drip to elucidate polar metabolites, and these two extractswere chosen to minimize time and labour for sample preparation.Signicant effects of crossbreed were found on several of the polarmetabolites that were identied in the meat extracts and in freezethaw drip (Figs. 12). The alternative crossbreeds, except ILY, hadsignicantly lower drip loss, compared with the traditional DLYcrossbreed (Table 1). The alternative crossbreeds also had smaller T22areas (Table 1), which represents extramyobrillar water susceptibleto be lost as drip (Bertram, Dnstrup, Karlsson, & Andersen, 2002;Bertram et al., 2001), demonstrating an improved water-holdingcapacity for the alternative crossbreeds. Post mortem formation oflactate caused by glycolysis under anaerobic condition is known to be

rost tw9% o

725I.K. Straadt et al. / Meat Science 96 (2014) 719728Fig. 3. PLS score (a and b) and loading (c and d) plots showing the means of the sensory p(b and d) from the ve crossbreeds. For the metabolites from the meat extraction, the r(c). Whereas for the metabolites from the freezethaw drip PC1 and PC2 explain 83 and

show metabolites, and magenta circles show attributes assessed by the sensory panel. Abbrevile and the polar metabolites from the meat extraction (a and c) and the freezethaw dripo principal components (PC1 and PC2) explain 81 and 11% of the variance, respectivelyf the variance, respectively (d). O, odour, F, avour, and AT, aftertaste. Grey boxes

ations as in Table 2.

726 I.K. Straadt et al. / Meat Science 96 (2014) 719728important for subsequent development in water-holding capacity ofpork (Bertram, Whittaker, Andersen, & Karlsson, 2003). A metabolomicstudy conducted on blood collected at the time of slaughter revealedthat lactate was the best candidate marker for pre-slaughter stressexposure in slaughter pigs (Bertram, Oksbjerg, & Young, 2010). In thepresent study, the NMR-based metabolomic approach revealed thatlactate levels in extractions of the meat from the crossbreeds were notsignicantly different. This nding is in agreement with the fact thatno signicant differences were found in the ultimate pH between thedifferent crossbreeds. However, the lactate levels in the freeze-thawdrip were signicantly lower for ID when compared to DLY. This couldindicate a higher production of lactate in the DLY pigs which has beenliberated from the muscles and lost in the drip exudate. The resultimplicates that lactate levels measured in the drip exudate or bloodplasma (Bertram et al., 2010) may be a better indicator of post mortemlactate formation than lactate levelsmeasured in themeat. Furthermore,the present ndings indicate that despite nding no differences in pHbetween the different crossbreeds, a lower lactate production aroundthe time of slaughter may still have occurred in the alternativecrossbreeds compared with DLY, and also less drip loss was found inthe alternative crossbreeds. This may indicate that the alternativecrossbreeds are less susceptible to stress.

In the present study generally lower levels of inosine and converselysignicantly higher levels of IMPwere found in both themeat and in thefreezethaw drip of the alternative crossbreeds compared with the DLYcrossbreed. These ndings suggest a less accelerated post mortemmetabolism in the alternative crossbreeds compared with DLY, andalso indicate that the alternative crossbreeds may be less susceptibleto stress, and hence give rise to an improvedmeat quality. Furthermore,in skeletal muscle it has been found that the extent of nucleotidedephosphorylation during ischemia was strongly correlated with theextent of muscle necrosis after 48 h of reperfusion in vivo. Hence, itwas found that accumulation of inosine and hypoxanthine and notIMP after 5 h of ischemia were indicators of less protection duringischemia (Rubin, Liauw, Tittley, Romaschin, & Walker, 1992; Rubin,Romaschin, Walker, Gute, & Korthuis, 1996). These ndings alsosupport that the alternative crossbreeds are less stress-susceptiblecompared with DLY.

Intriguingly, the present metabolomic study revealed a strongdifference in the level of choline-containing compounds, whichwas increased in the alternative crossbreeds compared with theDLY crossbreed. As choline is a membrane constituent, a possibleexplanation for the increased level of choline-containing compoundsin the alternative crossbreedsmay be linked to differences inmembraneproperties, which is in agreement with proposed theories suggestingthat membrane disintegration play a major role in the post mortemformation of drip loss (Bertram, Stagsted, Young, & Andersen, 2004).Possibly, the differences in choline-containing compound can also beascribed to a difference in the size of the muscle bres, which affectthe amount of membrane material present in the meat. However, theunderlying biochemical mechanisms giving rise to this breed-specicdifference in the level of choline-containing compounds remainunknown and further research is needed to understand the exact roleof choline-containing compounds in relation to post mortem musclephysiology.

A signicantly lower amount of glycerol was found in the meatextract from the alternative breeds, except for ID, compared with theDLY crossbreed. Intramuscular fat (IMF) content was higher in thealternative crossbreeds (Straadt et al., 2013). Thus, even though thealternative crossbreeds deposited more IMF than the DLY, the lowerlevel of glycerol could indicate a lower lipolysis in the alternative breeds.This nding could seem surprising as the Iberian pig breed is commonlyused for production of dry-cured ham (Pugliese & Sirtori, 2012).Possibly, the higher content of glycerol found in meat of DLY can beascribed to the fact that glycerol is not cleared so quickly in DLY, or

possibly, around slaughter the DLY crossbreeds use more lipids as fuelsubstrates and rely on fat oxidation and lipolysis to sustain the stress-induced increases in metabolic requirements.

The histidine dipeptide carnosine was found to be signicantlyaffected by crossbreed. This nding is of interest from a health per-spective as evidence has been presented that carnosine has anti-oxidative and anti-inammatory effects (Alpsoy, Akcayoglu, & Sahin,2011; Bellia, Vecchio, Cuzzocrea, Calabrese, & Rizzarelli, 2011; Hipkiss,2011; Tsai, Kuo, Liu, & Y, 2010). Previous studies on different muscletypes have revealed that the content of natural dipeptides includingcarnosine is signicantly lower in oxidative muscles (Aristoy & Toldra,1998; Mora, Sentandreu, & Toldra, 2008). It has also been shown thecontent of several amino acids in muscle are closely related to themetabolic type of bers (Cornet & Bousset, 1998), and carnosinecontent is also affected by animal age (Carnegie, Hee, & Bell, 1982).Breeding for leaner pig carcasses results in higher content of glycolyticbres (Brocks, Hulsegge, & Merkus, 1998), and it has been shown thatthe Iberian pig breed has a lower content of glycolytic bers and acorresponding higher content of oxidative bers than the Landrace pigbreed (Serra et al., 1998). Consequently, DLY is expected to representthe crossbreed with the highest content of glycolytic bers and fromthe previous studies showing that carnosine is signicantly lower inoxidativemuscles (Aristoy & Toldra, 1998;Mora et al., 2008), carnosinecould be expected to be higher in the DLY breed compared with thealternative breeds. However, since the present study showedcontrasting results, lower carnosine content in DLY, it must beconcluded that carnosine does not constitute an indicator of muscleglycolytic activity across different pig breeds. Glycolytic bers aregenerally also associated with decreased water-holding capacity(Karlsson, Klont, & Fernandez, 1999). Consequently, the fact thatcarnosine was negatively correlated with drip loss and amount ofextra-myobrillar water (T22 area) is also contradictory to carnosineas an indicator of muscle glycolytic activity across different pig breeds.

4.2. Meat metabolites and avor

The avor of meat develops mainly during the cooking process andthe Maillard reaction plays a central role in the fried avours (Mottram,1998). However, fresh meat contains non-volatile constituents such asamino acids and nucleotides that are essential avor precursors andcontribute to the development of cooked meat taste (Mottram, 1998).Hence, differences in the amount of these non-volatile avour precursorsincluding sugars, amino acids, creatine, carnosine and nucleotides couldbe expected to inuence the avor development in meat (Meinertet al., 2009; Tikk et al., 2006). In the present study correlation analysesbetween the polar metabolites in the fresh meat and sensory attributesclearly showed that the amino acid carnosine displayed the strongestcorrelations to the sensory attributes of the meat in general, and mostof the correlations between carnosine and sensory attributes such asfriedmeat avorwere negative. This nding is in agreementwith studieson model mixture systems that showed that the addition of carnosineinto the reaction mixtures in general caused a reduction in importantmeaty avor compounds such as 2-methyl-3-furanthiol, 2-furfurylthioland their associated dimers (Chen & Ho, 2002). On the other hand,addition of carnosine also facilitated the generation of several importantnitrogen-containing volatiles such as pyrazine, methylpyrazine, 2,6-dimethylpyrazine, and other alkyl pyrazines and thiazoles, which areknown to elicit roasty and nutty avor notes (Chen & Ho, 2002), whichmay explain that positive correlations were found between carnosineand the sensory attributes sweet and nutty in the present study.

In the present study differences in the levels of several avorprecursor metabolites were found between the alternative crossbreeds,and the DLY. Higher levels of carnosine and IMP and for ID also higherlevels of the amino acids were found in both the meat and the freeze-thaw drip when compared with DLY. Conversely, generally lowerlevels of inosine, creatine and glucose were found in the alternative

crossbreeds in both meat and freeze-thaw drip, compared with

727I.K. Straadt et al. / Meat Science 96 (2014) 719728DLY. In spite of these differences in these avour precursors, nosignicant differences between the different crossbreeds werefound in the avour/taste of the meat except for acidic aftertaste(Straadt et al., 2013). Hence, by application of NMR-basedmetabolomicsit is possible to detect/identify differences in avour precursors,however, apparently these differences were not sufciently strong tobe detectable by sensory analysis.

4.3. Meat metabolites and texture

In the present metabolomic study on the 5 different pig crossbreedsseven amino acids (alanine, carnosine, isoleucine, methionine, phenyl-alanine, tyrosine and valine) were quantied, and comparing the levelsof the amino acids between the different crossbreeds revealed that IDhas a higher level of all these amino acids when compared with thetraditional DLY crossing in both the meat extraction and in the dripsamples. Even though post mortem tenderization probably can beascribed to proteolysis of larger protein structures within the meat(Kemp, Sensky, Bardsley, Buttery, & Parr, 2010), the amount of aminoacids in the meat and in the freezethaw drip can probably beconsidered as markers of proteolysis (Graham et al., 2010; Grahamet al., 2012; Koohmaraie & Geesink, 2006), which is in agreement withthe fact that the individual amino acid contents were positivelycorrelated with meat tenderness (Table 3). However, it should benoted that NMR analyses were carried out two-three days afterslaughter while sensory evaluation was performed 4 days afterslaughter, which may have inuenced the correlations.

5. Conclusions

Overall, the present study demonstrated the potential of NMR-basedmetabolomics for exploringmetabolites of importance for sensorymeatquality. The study revealed differences in several amino acids andavor and aroma precursors in pork from the unique crossbreedsof Duroc/Landrace/Yorkshire (DLY), Iberian/Duroc (ID), Iberian/Landrace/Yorkshire (ILY), Mangalitza/Duroc (MD), and Mangalitza/Duroc/Yorkshire (MLY) and demonstrated how the combination ofNMR spectroscopy and sensory analysis can be coupled through ametabolomics approach to unravel metabolites of importance for meatquality.

Acknowledgement

The authors whish to thank the Danish Ministry of Food, Agricultureand Fisheries for nancial support through the project New Gourmetpork products obtained throughmolecular understanding of alternativepig breeds and high pressure technology (project no. 3304-FVFP-08-K-21-04) The Danish research council FTP for nancial supportthrough the project 'Advances in food and nutrition research throughimplementation of metabolomics technologies (#274-09-107) and TheDanish Pig Levy-Fund for nancial support. Nina Eggers is thanked forher assistance in theNMRanalyses, andMaiken Baltzer, JonnaAndersenand Camilla Bejerholm are thanked for assisting in the sensory analyses.

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An NMR-based metabolomics study of pork from different crossbreeds and relation to sensory perception1. Introduction2. Materials and methods2.1. Animals and sampling2.2. NMR transverse relaxation (T2) measurements2.3. pH and drip loss measurements2.4. 1H NMR spectroscopy2.5. Sensory analysis2.6. Statistics

3. Results3.1. Technological meat quality traits3.1.1. 1H NMR spectroscopic analysis of extraction of meat and freezethaw drip3.1.2. Correlations between technological quality (drip loss and T22 area) and the polar metabolites in meat/freezethaw drip

3.2. Correlations between sensory attributes and the polar metabolites in meat/freezethaw drip

4. Discussion4.1. Energy metabolism and stress susceptibility in the crossbreeds4.2. Meat metabolites and flavor4.3. Meat metabolites and texture

5. ConclusionsAcknowledgementReferences