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Food
Chemistry
Food Chemistry 107 (2008) 714721
www.elsevier.com/locate/foodchem
Received 26 March 2007; received in revised form 23 July 2007; accepted 27 August 2007
Abstract
Mechanically deboned meat (MDM) contains about 10 times more polyunsaturated fatty acids (PUFAs) and also more hemoproteins
than hand deboned meat (HDM) and is essentially more susceptible to both chemical and biochemical oxidation. The oxidation, leading
to the formation of potentially mutagenic and carcinogenic derivatives of PUFAs, can be inhibited by berry extracts rich in antioxidant
polyphenols. Using the 2-thiobarbituric acid reactive substances (TBARS) method, we have established that the ethanol slurry of the
juice-free solid residue of sea buckthorn (Hippophae rhamnoides SB) berries inhibits oxidation of unsaturated fatty acids, of both
chicken and turkey MDM. The polyphenols, mainly avonols, responsible for this inhibition, are comparatively stable during short-term
cooking and 6-day storage of cooked SB-MDMs at +6 C. About half of the polyphenols are lost, obviously oxidised, during the storage
of the uncooked samples of turkey 2%SB-MDM at +6 C. The loss of polyphenols is much smaller in the case of chicken MDM, which is
characterised by an in situ lower content of fatty acids, including the polyunsaturated ones. The liquid chromatographydiode array
detectiontandem mass spectrometry (LCDADESI-MS/MS) method was used for identication and ranking of the potent polyphe-
nolic antioxidants in the berry residue.
2007 Elsevier Ltd. All rights reserved.
Keywords: Mechanically deboned meat (MDM); Functional food; Sea buckthorn; Polyphenols; LCMS/MS-ESI; TBARS
0308-8146/$ - see front matter 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2007.08.090
T. Pussa et al. / Food Chemistry 107 (2008) 714721 715
cooked SB-MDM compositions were stored for 6 days The HPLC 2 D ChemStation Software with a ChemStation
at a temperature of +6 C. The initial moisture content, Spectral SW module was used for the process guidance. The
68.8 g/100 g, was reduced to 60.1 g/100 g in the course of conditions of MS/MS detection: m/z interval 501000 in
cooking. Alteration of the moisture content during the fol- negative ionisation mode; target mass 400; number of
lowing sample storage was insignicant. Reduction of the fragmented ions 2; maximal accumulation time 100
water content, during cooking, was taken into account ms; compound stability 100%; drying gas N2 from
when integrating the areas under the curves (Table 2), generator ow rate 10 l/min, gas temperature 350 C; neb-
extracted ion chromatogram (EIC) peak relative heights ulizer pressure 30 psi, collision gas He pressure 6 106
(Table 3) and the construction of Figs. 2 and 4. Every mbar.
experiment was repeated twice and two secondary samples DAD was working at the interval 200600 nm and the
were taken, from every sample, for polyphenol analysis. eluate optical density was continuously monitored at wave-
lengths 250 (phenolic acids), 280 (avanols), 306 (trans-
2.4. Assay of thiobarbituric acid reactive substances stilbenes) and 370 (avonols) nm.
(TBARS)
3. Results and discussion
The modied extraction method of Esterbauer and
Cheeseman (1990) was used. Ten grams of a MDM or 3.1. Fatty acid composition of the original MDM-s
HDCM composition were homogenised, during 1 min at
10,000 rpm in 40 ml of 4% perchloric acid, containing The MDMs studied contained, in comparison with
90 mg/100 g BHT (0.5 ml 7.2% solution of BHT) to retard HDM, up to 10 times higher quantities of PUFAs, mainly
the oxidative processes. The mixture which was obtained linoleic (cis,cis-9,12-octadecadienoic) acid (see Table 1).
was ltrated through a glassbre lter. Five millilitres of PUFAs are excellent substrates for lipid peroxidation
the ltrate and 5 ml of 0.02 M 2-thiobarbituric acid solution because of the presence of active bis-allylic methylene
were pipetted into the test tubes, which were closed with groups in their molecules. The carbonhydrogen bonds of
stoppers and heated using a water bath at 80 0.2 C, dur- these activated methylene units have lower bond dissocia-
ing 1 h. The tubes were cooled in a cold water bath for 1 min tion energies, making these hydrogen atoms more easily
and the absorption of the solution was determined using a abstractable in the radical reactions. The susceptibility of
spectrophotometer Specord 200 (Analytik Jena, Germany), PUFAs toward peroxidation increases with an increase in
at k 532 nm. Respective quantities of malonic dialdehyde the number of unsaturated sites in the hydrocarbon chain
(mg/kg of sample) were calculated using calibration curves, of the fatty acids. Although the main target of oxidation
obtained with standard compound 1,1,3,3-tetraethoxypro- in MDMs is linoleic acid with two double bonds, oxidation
pane (TEP). The analysis of every item was repeated twice. of arachidonic (all-cis-5,8,11,14-eicosatetraenoic) acid con-
taining four double bonds also makes a weighty contribu-
2.5. Determination of polyphenol content by HPLCDAD tion to the summary peroxidation process of PUFAs.
ESI-MS/MS
3.2. Thiobarbituric acid reactive substances (TBARS)
Sample preparation (Weisburger et al., 2002): 2:00
0:01 g of SB-MDM was extracted with 4 ml of methanol The dynamics of TBARS values (mg/kg), indicating to
by shaking for 30 min and then centrifuged, at 20 C, using malonic dialdehyde (MDA) formation via oxidation of
a cooling centrifuge Eppendorf 5810R equipped with a unsaturated fatty acids, during both microwaving and stor-
swinging bucket rotor for 10 min at 978g. The supernatant age of MDCMs is illustrated in Fig. 1.
was treated twice with 2 ml of hexane and the methanol It can be stated that during cooking of MDCM a
layer was passed through a 100 mg reversed phase SPE col- remarkable oxidation of fatty acids with formation of
umn and kept at 40 C until analysed. MDA already takes place. This oxidation is slightly
For chromatographic analysis of the SB polyphenols, a
tandem liquid chromatographydiode arraymass spec-
trometry (LCDADESI-MS/MS) method was developed. Table 1
Fatty acid composition of the meats studied (FAs, fatty acids; MUFAs,
For separation of compounds, a Zorbax 300SB-C18 col-
monounsaturated and PUFAs, polyunsaturated fatty acids)
umn 2:1 150 mm; 5 lm was used, in a stepwise mobile
Type of meat MDCM MDTM HDCM
phase gradient of 0.1% formic acid (solvent A) and acetoni-
trile (solvent B), at velocity 0.3 ml/min at 35 C. The sample Total fat (g/100 g of meat) 14.7 20.3 1.4
% of various fatty acids
injection volume was 15 ll. For detection and quantitation
Saturated FAs 29.7 31.2 28.4
of substances, the Agilent 1100 Series UVVis diode array MUFAs 48.6 47.2 42.5
detector (DAD) and LC/MSD Trap-XCT with an electro- PUFAs, 21.5 21.3 29.2
spray (ESI) interface were connected to an Agilent 1100 Ser- Linoleic acid 17.8 18.4 24.3
ies HPLC instrument, consisting of an autosampler, solvent a-Linolenic acid 2.7 2.3 1.3
Arachidonic acid 0.4 0.3 1.8
membrane degasser, binary pump and column thermostat.
T. Pussa et al. / Food Chemistry 107 (2008) 714721 717
Raw MDCM with different supplements Cooked MDCM with different supplements
10
10
9
9
8
8
TBARS mg MDA/kg
TBARS mg MDA/kg
7
7
6
6
5 1st day 1st day
5
4 6th day 6th day
4
3
3
2
2
1
1
0
0
SB
SB
SB
SB
SB
SB
H
H
0
0
O
O
%
%
Et
Et
+1
+2
+4
+1
+2
+4
H
H
O
O
Et
Et
Et
Et
Et
Et
Fig. 1. Dynamics of TBARS values in the course of aging of raw and cooked MDCMs at +6 C. EtOH ethanol.
retarded by the added ethanol but completely stopped by obvious correlation between the AUC numbers and the ini-
the SB extracts, starting from the lowest studied level tial concentration of the SB in the respective SM-MDMs
(1%) of the supplement (see Fig. 1b). Ethanol has no eect should be noted. Pearsons r value is 0.83 and 0.96
on the oxidation of the raw MDCM, this process is again for the linear correlation between percentage (1, 2 and 4)
remarkably, but in this case not entirely, inhibited by the of SB supplement in MDM and the DS 370 nm values in the
SB supplements (Fig. 1a). case of raw and cooked HDCM, respectively. It shows a
MDTM, used for the experiments, was although still sucient quality of the sample preparation as well as the
ocially utilisable and without any obvious signs of putre- analytical process in total.
faction, extensively and heterogenously oxidised, with The numbers in Table 2 indicate that the total avonol
TBARS numbers in the interval of 35 mg MDA/kg. Nev- glycoside content in SB-MDMs is reduced in average by
ertheless, it was decided to use just this MDTM as a proper 9.5% during cooking and by 50.8% and 21.4% during stor-
matrix to reveal whether organoleptically allowable, rela- age of uncooked SB-MDTM and SB-MDM samples,
tively low amounts of plant antioxidant that change neither respectively. The content of polyphenols is only slightly
the texture nor taste of the meat composition, can manage reduced in the course of storage of the cooked samples.
with a high initial oxidative potential of the lipid material. Various identied avonol glucosides behave dierently
during the storage of uncooked SB-MDTM (see Fig. 3).
3.3. Stability of SB polyphenols in the compositions Derivatives of isorhamnetin (except rhamnoside) and
kaempferol are decidedly more persistent than quercetin
A functional food is capable of causing desired health derivatives (see Table 3 and Fig. 4).
eects only in the case of containing the health-promoting It has been published that plant polyphenols, like querce-
compounds in the nal (ready-for-eating) product, after a tin, are rather stable during short-term cooking or microwa-
denite storage. Sea buckthorn berries contain large ving (Liazid, Palma, Brigui, & Barroso, 2007; Lombard,
amounts of avonol glycosides, characterised by a specic Peey, Georiau, Thompson, & Herring, 2005). Our results
UV absorption maximum around k 370 nm as the main conrm these ndings (see Tables 2 and 3).
group of polyphenols (Guliyev et al., 2004). Using tandem A hypothesis can be set up that the dierent stability of
UVVis and MS/MS detection, 11 major polyphenols, all various avonol glycosides during storage is mostly caused
avonol glucosides, were identied in the SB-MDM sam- by dierences in their antioxidative capacities (free radical
ples (see Fig. 2 and Table 3). Since the method used does scavenging abilities). More potent catchers of free radicals
not allow determination of the exact location of a glyco- must also have been more completely consumed during
sidic group in the aglycone molecule, the position numbers both microwaving and storage. Rosch with co-authors
are indicated only for the glycosides identied earlier in sea has shown that the antioxidant capacities of avonol glyco-
buckthorn berries (Rosch et al., 2003). Example UV-chro- sides depend rst of all on the availability of free OH-
matograms at 370 nm of the raw 2% SB-MDTM and SB- groups (o-diphenolic arrangement) in the positions 30 and
MDCM, before and after 6-day storage, are presented in 40 of the aglycone molecule, enabling formation during oxi-
Fig. 2. As an approximate measure of the total avonol dation of o-quinonic moiety, stabilised by conjugation
content, S 370 , the area under the HPLC chromatogram (Rosch et al., 2003). This situation is realised in the mole-
(AUC) between the 7th and 19th minutes at wavelength cule of quercetin and its identied glycosides but not in
370 nm (see Fig. 2), which is the sum of the areas of peaks the case of derivatives of isorhamnetin and kaempferol.
of individual avonols, can be taken (see Table 2). An The second site where the o-diphenolic moiety can be
718 T. Pussa et al. / Food Chemistry 107 (2008) 714721
a Intens.
mAU
5
6
8
2 6
4
2
1 3
11
9 10
0
8 10 12 14 16 18 Time (min)
b Intens. 5
mAU
4
8
4 6
11
2
2
3
1
9 10
0
8 10 12 14 16 18 Time (min)
Fig. 2. HPLCUV chromatogram of the extracts of raw 2% SB-MDCM (a) and SB-MDTM (b) at k 370 nm at 1st (light line) and 6th day (dark line)
days of storage at +6 C. For the names of polyphenols behind the peak numbers, see Table 3.
Table 2 R1
Areas under the curves of HPLCDAD chromatograms k 370 nm of 3
2 OH
raw and cooked SB-MDMs on the 1st day and after storage during 6 days 4
R1 R2 Compound 8 1
O 1
Type of % of Raw Cooked H H Kaempferol HO 7 5 R2
meat SB OH H Quercetin 2 6
1st day 6th day 1st day 6th day
OMe H Isorhamnetin 6 4 3 OH
MDTM 1 242 30 109 18 227 15 183 10 5
OH O
2 551 42 231 32 463 33 396 32
4 1073 88 651 43 973 40 884 45
Fig. 3. Structure of avonols.
MDCM 1 291 30 190 29 239 15 247 17
2 589 44 500 37 584 32 474 30
4 1227 75 1051 55 1147 50 983 40
cosides this situation is realised in the case of isorhamne-
a a
tin-7-rhamnoside, which has the highest antioxidant
HDCM 1 316 25 372 30
a a capacity among isorhamnetin derivatives (Rosch et al.,
2 692 48 669 50
a
2003) and which is also clearly the most depleted non-quer-
Not estimated.
cetin avonol in the case of MDTM in our investigation.
The generally smaller disappearance of avonol derivatives
during aging of cooked rather than uncooked MDMs dem-
formed during oxidation is around positions 3 and 4, pro- onstrates that the oxidation process of meat unsaturated
vided that the OH-group in position 3 is free (non-substi- fatty acids is a combination of both enzymatic (including
tuted). This is the situation with quercetin-7-rhamnoside, pseudoenzymes like hemoproteins) and non-enzymatic
that is indeed the most consumed during oxidation of both reactions. These results are in harmony with the data
the MDTM and MDCM quercetin derivative. As it can be obtained by Rosch and co-authors and prove our initial
seen in Table 4, among isorhamnetin and kaempferol gly- hypothesis.
Table 3
Mass data and relative heights of the EIC peaks of the main identied polyphenols determined in 2% SB-MDTM and SB-MDCM (amu atom mass units)
Comp. no. in Fig. 2 Flavonol O-glucoside [MH] amu Main daughter ions (in the order EIC-peak relative height (%)a
of decrease of intensity)
Raw 6th day Cooked, 1st day Cooked, 6th day Cooked 6th day/cooked 1st dayb
MDTM, quercetin-
1 -Diglucoside-rhamnoside 771 625; 301; 300; 446; 445; 463 0 97 68 70
3 -Glucoside-rhamnoside 609 447; 301; 463; 519 0 97 50 52
5 -3-Rutinoside 609 301; 271; 344; 255 0 108 40 37
6 -3-Glucoside 463 301; 306; 300; 151; 179 45 84 43 52
9 -7-Rhamnoside 447 301; 151; 427; 177; 285 0 119 0 0
MDTM, kaempferol-
10 -Glucoside-rhamnoside 593 285; 447; 307 80 95 87 92
MDCM, quercetin-
1 -Diglucoside-rhamnoside 771 625; 301; 300; 446; 445; 463 85 89 104 100
3 -Glucoside-rhamnoside 609 447; 301; 463; 519 90 83 80 96
5 -3-Rutinoside 609 301; 271; 344; 255 55 71 55 77
6 -3-Glucoside 463 301; 306; 300; 151; 179 57 77 74 96
9 -7-Rhamnoside 447 301; 151; 427; 177; 285 47 106 92 87
MDCM, isorhamnetin-
2 -Diglucoside-rhamnoside 785 639; 315; 610; 623; 733 86 96 70 73
4 -3-Glucoside-7-rhamnoside 623 477; 461; 315; 300 96 78 79 100
7 -3-Rutinoside 623 315; 300; 271; 255 76 67 77 100
8 -3-Glucoside 477 314; 315; 271; 285; 357 84 92 68 74
11 -7-Rhamnoside 461 315; 446; 300; 151; 287 46 89 94 100
MDCM, kaempferol-
10 -Glucoside-rhamnoside 593 285; 447; 307 97 122 121 100
a
The height of respective polyphenol peak in the raw meat at the 1st day is taken as 100%.
b
Per cent of the particular polyphenol preserved during storage of cooked MDM.
719
720 T. Pussa et al. / Food Chemistry 107 (2008) 714721
intens. x 10-3
intens. x 10-3
5000
20000
4000
1.day 15000 1.day
3000
6.day 10000 6.day
2000
5000
1000
0
0
1%SB(c)
2%SB(c)
4%SB(c)
MDM+
MDM+
MDM+
MDM+
MDM+
MDM+
1%SB(r)
2%SB(r)
4%SB(r)
1%SB(c)
2%SB(c)
4%SB(c)
MDM+
MDM+
MDM+
MDM+
MDM+
MDM+
1%SB(r)
2%SB(r)
4%SB(r)
[M-H]-609(11.4 min) [M-H]-477(14.5 min)
Fig. 4. Comparison of concentration dynamics of two selected avonol glucosides in the course of 6-day storage of SB-MDTM (r raw, c cooked).
Numbers on the y-axis are the intensities of the respective EIC peaks.
storage at +6 C. The loss of polyphenols is much smaller natural extracts: application in beef meatballs. Meat Science, 69,
in the case of SB-MDCM, which contains less fatty acids 371380.
Folch, J., Lees, M., & Sloane-Stanley, G. H. (1957). A simple method for
and is initially less oxidated than MDTM. The cooking the isolation and purication of total lipids from animal tissue. Journal
process itself has little eect on polyphenol integrity in of Biological Chemistry, 226, 497509.
the SB-MDMs. Guliyev, V. B., Gul, M., & Yildrim, A. (2004). Hippophae rhamnoides L:
Consequently, it is safe to say that the processing residue chromatographic methods to determine chemical composition, use in
of sea buckthorn juice is a good functional supplement to traditional medicine and pharmacological eects. Journal of Chroma-
tography B, 812, 291307.
MDM or HDM products, guaranteeing inhibition of the Hassan, O., & Fan, L. S. (2005). The anti-oxidant potential of polyphenol
oxidation of fatty acids as well as enriching the meat extract from cocoa leaves on mechanically deboned chicken meat
products with plant-derived health-benecial polyphenols. (MDCM). Lebensmittel-Wissenschaft und -Technologie (LWT), 38,
In addition, the optimal 2% supplement of berry powder 315321.
does not deteriorate the organoleptic properties like taste, Liazid, A., Palma, M., Brigui, J., & Barroso, G. G. (2007). Investigation of
phenolic compounds stability during microwave-assisted extraction.
avour or texture of the patties prepared from the poultry Journal of Chromatography A, 1140, 2934.
MDM. Lombard, K., Peey, E., Georiau, E., Thompson, L., & Herring, A.
(2005). Quercetin in onion (Allium cepa L) after heat treating
Acknowledgements simulating home preparation. Journal of Food Composition and
Analysis, 18, 571581.
Mielnik, M. B., Aaby, K., & Skrede, K. (2003). Commercial antioxidants
Financial support of the Ministry of Education and Re- control lipid oxidation in mechanically deboned turkey meat. Meat
search of Estonia (Pilot project RP0091VL04) is greatly Science, 65, 11471155.
acknowledged. Sirje Kuusik and Leno Matas are acknowl- Mitsumoto, M., OGrady, M. N., Kerry, J. P., & Buckley, D. J. (2005).
edged for their excellent technical assistance. Addition of tea catechins and vitamin C on sensory evaluation, colour
and lipid stability during chilled storage in cooked or raw beef and
chicken patties. Meat Science, 69, 773779.
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