Food Chemistry 123 (2010) 456–464

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Food Chemistry

journal homepage: www.elsevier .com/locate / foodchem

Near-infrared oximetry of three post-rigor skeletal muscles for followingmyoglobin redox forms

Anand Mohan a, Melvin C. Hunt a,*, Thomas J. Barstow b, Terry A. Houser a, Dennis M. Hueber c

a Department of Animal Sciences and Industry, Weber Hall, Kansas State University, Manhattan, KS 66506, USAb Department of Kinesiology and Anatomy and Physiology, Kansas State University, Manhattan, KS 66506, USAc ISS Inc., 1602 Newton Drive, Champaign, IL 61822-1061, USA

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

Article history:Received 28 November 2009Received in revised form 28 March 2010Accepted 27 April 2010

Keywords:Near-infrared tissue oximeterReflectance spectroscopyModified atmosphere packagingMeat colourMyoglobin redox forms

0308-8146/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.foodchem.2010.04.068

Abbreviations: LL, M. Longissimus lumborum; PSemitendinosus; DMb, deoxymyoglobin; MMb, metmyFDMD, frequency-domain multidistance; NIR, near inmyoglobin; OC, oxygen consumption; NADH, nicotinHiOx-MAP, high oxygen modified atmosphere; PVvacuum packaging.

* Corresponding author. Tel.: +1 785 532 1232; faxE-mail address: hhunt@ksu.edu (M.C. Hunt).

We investigated the response of frequency-domain multidistance (FDMD) near-infrared (NIR) tissueoximetry for detecting absolute amounts of myoglobin (Mb) redox forms and their relationship to meatcolour stability. Four packaging formats were used to create different blends of Mb redox forms and meatcolours during display. Changes in surface colour and subsurface pigment forms during simulated displaytime (0, 2, 4, and 10 d at 2 �C) were evaluated using surface reflecto-spectrophotometry (both L*a*b* andspecific wavelengths) and FDMD NIR tissue oximetry. Data for both methods of direct measurement ofoxymyoglobin and deoxymyoglobin were strongly related and accounted for 86–94% of the display var-iation in meat colour. Indirect estimates of metmyoglobin ranged from r2 = 59–85%. It appears that NIRtissue oximetry has potential as a noninvasive, rapid method for the assessment of meat colour traitsand may help improve our understanding of meat colour chemistry in post-rigor skeletal muscle.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction forms on the exposed meat surface and a few millimetres below

Meat colour is the most important factor that influences con-sumer meat purchasing decisions (Kropf, 1993). The inability tocontrol factors affecting meat colour and to assess the potentialof meat to have good colour life contributes to millions of dollarslost per year at retail because of discolouration (McKenna et al.,2005). Muscle oxygen uptake, its subsequent utilisation by en-zymes and other cellular compartments, and the ability to reducemetmyoglobin (MMb) determines the colour life of meat (Mancini& Hunt, 2005).

In living skeletal muscle, myoglobin (Mb) undergoes rapid oxy-genation and deoxygenation in response to fluctuations in oxygensupply and tissue demand (Millikan, 1939), whereas post-rigormuscles respond more slowly and differently, resulting in variabledegrees of ‘‘worst point” discolouration on meat surfaces (Mancini& Hunt, 2005). Maintenance of a bright red colour of fresh meat is adelicate interaction between retail cold chain management and theinherent chemistry of the meat, which results in various Mb redox

ll rights reserved.

M, M. Psoas major; ST, M.oglobin; OMb, oxymyoglobin;frared; Hb, haemoglobin; Mb,amide adenine dinucleotide;C, polyvinyl chloride; VAC,

: +1 785 532 7059.

the surface. DeVore and Solberg (1975) found that muscle tissuerespiration accounts for 80% of the oxygen uptake in the first10 h of display. Reducing oxygen tensions within muscle tissuepromote the autoxidation of deoxymyoglobin (DMb) and oxymyo-globin (OMb), causing surface discolouration (Atkinson & Follett,1973). Bendall and Taylor (1972) reported the nicotinamide ade-nine dinucleotide (NADH) linked oxidation of mitochondrial tricar-boxylic cycle metabolites pyruvate–malate and muscle oxygenconsumption rates in pre-rigor beef muscle. Tang et al. (2005) re-ported that Mb serves as the oxygen reservoir and transporterfor mitochondria in meat animal tissue.

Meat scientists have used several methods to determine muscleoxygen uptake and consumption (OC) including the Warburg flask(Urbin & Wilson, 1961), differential respirometry (DeVore &Solberg, 1975), Clark oxygen electrodes (Lanari & Cassens, 1991),reflecto-spectrophotometry (Madhavi & Carpenter, 1993), andheadspace oxygen analyzers (Sammel, Hunt, Kropf, Hachmeister,& Johnson, 2002). None of these methods provide real-time, nonin-vasive monitoring of meat colour and thus have limited practicalapplication in the meat industry.

Interactions between light and muscle pigment in meat offer anopportunity to develop methodology for detecting the redoxdynamics of Mb using near-infrared (NIR; 700–1000 nm) technol-ogy. Frequency-domain multidistance (FDMD) NIR tissue oximetryhas been used in exercise physiology and biomedical applica-tions to provide a direct measure of absolute concentrations of

A. Mohan et al. / Food Chemistry 123 (2010) 456–464 457

haemoglobin (Hb) and Mb oxygen saturation in skeletal muscleand brain tissue (Chance, Dait, Zhang, Hamaoka, & Hagerman,1992). Because Hb and Mb have similar NIR absorptions and be-cause there are minimal amounts of Hb in well-bled animal tissue(Hunt & Hedrick, 1977a), it is possible to distinguish DMb, OMb,and MMb in post-rigor skeletal muscle. This approach has beenused successfully to assess the dynamics of Mb oxygenation anddeoxygenation during step changes in cardiac workload (de Groot,Zuurbier, & van Beek, 1999). Mohan et al. (2009) reported that fibreorientation and Mb redox form affected NIR tissue oximetry(FDMD) data in post-rigor muscle.

We are not aware of research using FDMD-based NIR tissueoximetry to evaluate colour and colour stability of beef skeletalmuscle. Thus, the objectives of this project were to investigate(1) NIR tissue oximeter response from three bovine post-rigormuscles varying in pigment concentration, Mb redox state, andpackaging, (2) NIR tissue oximeter response for redox dynamicsof Mb in post-rigor muscle over time, and (3) relationships ofNIR tissue oximetric and spectrophotometric data for colour andredox forms during retail display.

2. Materials and methods

2.1. Raw materials

Six M. Longissimus lumborum (LL; colour stable), M. Semitendino-sus (ST; intermediate colour stability), and M. Psoas major (PM; col-our unstable) muscles were obtained from USDA Select, A-maturitycarcasses at a commercial plant and were fabricated at 10-d post-mortem into 5.0-cm thick portions with the fibre orientation per-pendicular to the muscles’ cut surface. The thick portionsensured that NIR light did not escape from the muscle tissue.

2.2. Packaging and display

Cut portions of each muscle were assigned randomly to the fol-lowing packaging treatments: (1) vacuum packaging (VAC;62.2 cm Hg, Multivac C500, Multivac Inc., Kansas City, MO, USA),(2) modified atmosphere packaging (MAP) with high oxygen (HiOx;80% O2, 20% CO2, AirGas certified gas, MidSouth, Inc., Tulsa, OK,USA), (3) overwrapped with polyvinyl chloride film (PVC; MAPACL, 21,700 cc O2/m2/24 h, Borden Packaging and Industrial Products,North Andover, MA, USA) on foam trays (17S; McCune Paper Com-pany, Salina, KS, USA) with a Dri-Loc soaker pad (AC-50; Sealed AirCorp, Duncan, SC, USA), and (4) HiOx-MAP converted to PVC after2 d and stored as PVC (HiOx-PVC). Steaks packaged in MAP (RossJr. S-3180, Ross, Midland, VA, USA) were placed in 4.32-cm deep ri-gid plastic trays (CS977, Cryovac Sealed Air Corp., Duncan, SC,USA), covered with oxygen barrier film (Lid 550; 1.0 mils; less than20.0 oxygen transmission cc/24 h/m2 at 4.4 �C with 100% relativehumidity, and moisture vapour transmission less than 0.1 g/24 h/645.2 cm2 at 4.4 �C and 100% relative humidity; Cryovac SealedAir Corp., Duncan, SC, USA) with a soaker pad, flushed with (80%O2 and 20% N2) gas blends, and sealed with shrinkable barrier film.Steaks were displayed at 2 �C for 10 d under 2150 ± 50 lux of con-tinuous fluorescent lighting (bulb F32T8/ADV830, 3000 K, CRI = 86;Phillips, Bloomfield, NJ, USA) in open-top display cases (DMF8; Ty-ler Refrigeration Corp., Niles, MI, USA). Packages were rotated dailyto minimise case location effects.

2.3. Proximate and pH determinations

The pH of steaks was measured on d 14 postmortem by insert-ing the tip of a pH probe (MPI pH probe, glass electrode, MeatProbes Inc., Topeka, KS, USA) into the steaks. A composite sample

of tissue remaining after packaging from each of the 18 muscleswas used for proximate analysis. Samples were analysed in tripli-cate for protein [LECO Combustion Analysis (AOAC Official Method990.03; Thiex, 2009)] and moisture and fat [CEM SMART andSMART Trac systems (AOAC PVM 1:2003; Keeton et al., 2003)].

2.4. Colour analysis

Steaks were scanned in triplicate on display time (0, 2, 4, 10,and 14 d) for instrumental colour (HunterLab MiniScan™ XE PlusSpectrophotometer 45/0 LAV, 2.54-cm-diameter aperture, 10�standard observer; Hunter Associates Laboratory, Inc., Reston, VA,USA). Values for CIE L*, a*, and b* (Illuminant A) were collected,and hue angle (tan�1 b*/a*) and chroma [(a*2 + b*2)1/2] were calcu-lated (Hunt et al., 1991) from instrumental measures. Illuminant A(vs. D-65) is recommended for samples where differentiation ofredness is important (Hunt et al., 1991).

Estimation of DMb and OMb was computed using reflectancedata from 400–700 nm with a HunterLab MiniScan™ XE Plus Spec-trophotometer (D/8-S, 14.3-mm-diameter aperture; Hunter Asso-ciates Laboratory, Inc., Reston, VA, USA). Wavelengths isobesticfor DMb and OMb were measured and converted to K/S values,and ratios of wavelengths were inserted into equations with valuesfor 0% and 100% of the pigment form being calculated (Hunt et al.,1991).

2.5. Application of NIR tissue oximeter on beef skeletal muscles

Myoglobin redox forms present in the muscle portions on dis-play time (0, 2, 4, and 10 d) were evaluated using a NIR tissueoximeter (OxiplexTS model 96208, ISS, Champaign, IL, USA), whichis based on the relative tissue transparency for light in the NIRregion and on the O2-dependent absorption changes of Mb.The OxiplexTS is a FDMD spectrophotometer that permits calcula-tion of absolute (lM) concentrations of OMb and DMb using dy-namic calculation of the reduced scattering coefficient. It was notpossible to distinguish between Hb and Mb because of their nearlyidentical spectral characteristics; however, there is usually lessthan 5–8% Hb in post-rigor muscle. The absorption changes atthe discrete wavelengths were converted into concentrations ofOMb and DMb. The concentration of MMb was estimated as100 � ([OMb] + [DMb]).

The NIR tissue oximeter was calibrated each test day accordingto the manufacturer’s recommendations. Simultaneous NIR oxime-try measurements were made using a single probe consisting ofeight laser diodes operating at two different wavelengths (692and 834 nm, four at each wavelength) and a photomultiplier tube(Ferreira, Townsend, Lutjemeier, & Barstow, 2005). The laser diodesand photomultiplier tube were connected by optical fibres consist-ing of two parallel rows of emitter fibres and one detector fibrebundle arranged in four source-detector separations of 2.0, 2.5,3.0, and 3.5 cm for both wavelengths. Measurements were ob-tained by placing the probe longitudinally on the top of the steakwith packaging material intact.

2.6. Statistical analysis

The experimental design for the experiment was a randomizedcomplete block with a split-plot design, and within the whole plot,carcass served as a random effect (a block) and muscles served asthe treatments. Within the subplot, packaging format served as thetreatment assigned to steaks (subplot experimental units). Datawere analysed separately for each variable (OMb, DMb, andMMb). Multiple oximetry scans on each steak (three per steak)were averaged for statistical analysis. Thus, for each variable,the analysis consisted of 288 total observations [six animals

458 A. Mohan et al. / Food Chemistry 123 (2010) 456–464

(replications) � three muscles per animal � four packaging for-mats per muscle � four display time per packaging format].Type-3 tests of fixed effects for muscle, muscle � packaging, mu-scle � day, and muscle � packaging � day were evaluated usingthe MIXED procedure of SAS (SAS Institute, Inc., Cary, NC, USA).F-test denominator degrees of freedom were estimated using theSatterthwaite adjustment. Least squares means for significant F-tests were separated using least significant differences.

To assess the strength of relationships between reflecto-spec-trophotometric and NIR tissue oximeter measurements, simplecorrelation coefficients were computed using the CORR procedureof SAS. Correlation graphics of fitted data with 95% prediction limitand 95% confidence limits were generated using the ODS functionof SAS.

3. Results

3.1. pH, moisture, protein, fat, and myoglobin

All muscles had a pH between 5.5 and 5.7 (Table 1). The ST hadthe most moisture (74.4%; p < 0.05), least fat (1.5%), and intermedi-ate protein (22.4%; p < 0.05) of the three muscles. The LL had thehighest protein (23.6%) and intermediate fat (4.2%; p < 0.05). The

Table 1Least squares meansa for pH, proximate analysis, and reduced scattering coefficientsb

of three beef muscles.c

Trait Muscles

LL PM ST SEd

pH 5.6x 5.5x 5.7x 0.04Moisture (%) 71.3x 71.2x 74.4y 0.36Protein (%) 23.6z 21.9x 22.4y 0.27Fat (%) 4.2y 5.4z 1.5x 0.12Ash (%) 0.8x 1.4y 1.6y 0.19Myoglobin (mg/g) 6.9z 5.1y 4.2x 0.07ls0 (k = 692 nm) 6.4 2.1 4.6

ls0 (k = 834 nm) 5.2 5.1 5.2

a xyz = Within a row, means with a different letter differ (p < 0.05).b ls

0 = Overall mean (summed over all treatments) for the reduced scatteringcoefficients for tissue oximeter data.

c LL = M. Longissimus lumborum, PM = M. Psoas major, ST = M. Semitendinosus.d Standard error.

Table 2Least squares meansa for percentages of oxymyoglobin and deoxymyoglobin in three beefdisplay time.

Muscle Packaging format Myoglobin redox form

Oxymyoglobin/display time (d)

0 2 4

LL HiOx 28.2fw 78.3jz 71.4ky

HiOx-PVC 27.8fw 77.2jz 70.6ky

PVC 5.7ew 43.7gy 41.6gy

VAC 2.6ew 2.1ew 2.2ew

PM HiOx 31.7gw 62.2iy 56.9ix

HiOx-PVC 29.7gx 62.3iz 51.8hy

PVC 11.7fw 42.3gz 38.5fy

VAC 5.2ew 4.8ew 4.2ew

ST HiOx 24.5fw 52.8hy 60.3jz

HiOx-PVC 24.9fw 51.2hz 50.6hy

PVC 5.7ew 35.0fy 35.5fy

VAC 4.1ew 2.9ew 3.3ew

a efghijk = Within a column across muscles, means with a different letter differ (p < 0differ (p < 0.05). Standard error for packaging � display time (d) or muscle = 3.06 – 3.74

b LL = M. Longissimus lumborum = colour stable, ST = M. Semitendinosus = intermediatec HiOx = 80% oxygen and 20% carbon dioxide; HiOx-PVC = Sample remained in 80% oxy

chloride overwrap; VAC, Vacuum packaging.

PM had the most fat (5.4%), least protein (21.9%), and intermediatemoisture (71.2%; p < 0.05). Myoglobin concentrations were great-est in the LL, intermediate in the PM, and least in the ST(p < 0.05). The overall means for oximeter reduced scattering coef-ficients varied by muscle at 692 nm but not at 834.

3.2. NIR tissue oximetry of DMb and OMb

A muscle � packaging � day interaction (p < 0.05) occurred forNIR tissue oximetry response for OMb and DMb (Table 2). On d0, the LL, PM, and ST portions packaged in HiOx and HiOx-PVCMAP had 25–32% OMb, but samples in PVC and VAC had levelsfrom 2.6% to <6%. By d 2, the HiOx and HiOx-PVC packages con-tained 50–78% OMb, whereas OMb levels in PVC were 35–42%and in VAC were <5%. On d 4, OMb decreased significantly in theHiOx and HiOx-PVC samples for the LL and PM muscles, but OMbin the ST was equal to or higher than d 2 quantities. By d 10,OMb levels in the HIOx and HiOx-PVC further decreased (p < 0.05)but were still higher than on d 0. Oxygenated myoglobin in por-tions from the three muscles packaged in PVC changed (p < 0.05)from 5–12% on d 0 to 35–44% on d 2 and 4. By d 10, PVC-packagedportions had significantly less OMb. Portions packaged in VAC hadthe lowest (generally 3–5%) OMb throughout display.

The LL and PM portions packaged in HiOx-PVC MAP had signif-icantly higher levels of OMb (30%) than the ST on display time d 0,likely because of their greater Mb content. On d 2 of display, Mboxygenation of LL portions increased (p < 0.05) to a greater extentthan in PM and ST (77% vs. 62% in PM and 52% in ST) and continuedto retain the same level through d 4 of display. The most rapid de-crease (p < 0.05) in OMb level occurred in PM and ST muscles pack-aged in HiOx-PVC MAP after d 4. Oxygenation of Mb wasconsiderably less (p < 0.05) for all three muscles (p < 0.05) pack-aged in PVC than for either of the HiOx-MAP systems (Table 2).At the end of display, the LL muscle had the most stable red colour,the PM retained the least OMb, and the ST was intermediate. Por-tions of all three muscles in VAC contained 65% OMb.

The NIR tissue oximetry response of all three muscles for DMbexhibited a similar but opposite pattern (Table 2). All samples ini-tially had relatively high amounts of DMb because they were mea-sured shortly after placement in the packages. Samples in the HiOx

and HiOx-PVC MAP packaging systems contained significantly lessDMb (66–73%) on d 0 than the PVC and VAC packages (85–94%).

musclesb in four packaging formatsc evaluated by NIR tissue oximetry during 10 d of

Deoxymyoglobin/display time (d)

10 0 2 4 10

49.8jx 70.3fz 19.7efw 18.6ew 17.4ew

47.2jx 69.9fy 19.8ew 22.6ew 28.7fx

35.8ix 92.5hy 49.4hx 47.2gx 41.2gw

2.4ew 94.9hw 96.1iw 96.4iw 96.5jw

31.5gw 66.2ez 28.9fy 21.1ex 11.9fw

26.7gw 67.2ex 29.1gw 29.5fw 31.8jw

18.1fx 84.5fx 45.6gw 45.1hw 44.9iw

4.2ew 93.7hw 93.9iw 94.8iw 94.6jw

34.5ix 72.4fy 39.1gx 21.7ew 17.4fw

28.9hx 73.1fy 38.9gx 31.3gw 39.1hx

27.9hx 93.1gy 51.9gx 48.4hwx 46.1hw

4.7ew 94.7hw 96.6iw 95.9iw 92.9jw

.05). wxyz = Within a row and myoglobin redox form, means with a different letter.colour stability, PM = M. Psoas major = colour labile.gen and 20% carbon dioxide for 48 h and was then converted to PVC; PVC, Polyvinyl

A. Mohan et al. / Food Chemistry 123 (2010) 456–464 459

Furthermore, the DMb decreased (p < 0.05) by d 2 to 20% in LL, 29%in PM, and 39% in the ST. The LL, PM, and ST portions in HiOx pack-ages contained relatively the same level of DMb from d 4 to 10.Samples in HiOx-PVC decreased in DMb on d 2 and 4, which accom-panied their removal from the HiOx environment. However, LL por-tions packaged in PVC had 92.5% DMb on d 0, which decreased(p < 0.05) to 49.4% on d 2 of display, and maintained predominatelyDMb throughout display. All the three muscles in VAC contained93–97% DMb and, as expected, maintained predominately DMbthroughout display.

3.3. Reflecto-spectrophotometry of DMb and OMb

A muscle � packaging � day interaction (p < 0.05) also occurredfor reflectance measurements of OMb and DMb (Table 3) on themeat’s surface. Percentages of OMb were higher (p < 0.05) for LLand ST muscles than for the PM in all three aerobic packaging sys-tems throughout display, and, as expected, OMb decreased as timepostmortem increased. The LL samples packaged in HiOx, HiOx-PVC, and PVC contained the most (p < 0.05) OMb from display time(2–10 d), whereas OMb in the ST and PM samples with comparablepackaging was intermediate and lowest, respectively. The OMb ofall three muscles packaged in VAC was essentially the same anddid not change (p > 0.05) during display.

The DMb levels followed essentially the same pattern for thethree aerobic packaging systems during display until d 10, whenless reduced redox forms occurred as colour deteriorated (Table 3).During display, the amount of OMb declined but DMb levels weremaintained in the HiOx-PVC and PVC MAP packages. The VAC pack-ages, compared with HiOx, HiOx-PVC, and PVC for all three muscleshad, as expected, the greatest (p < 0.05) DMb at the beginning ofdisplay; DMb in the VAC packages increased (p < 0.05) on d 2and thereafter remained constant throughout the display period.Reflecto-spectrophotometric measurements demonstrated that ingeneral, LL portions in oxygen-containing packaging had the high-est percentages of OMb, PM portions had the least, and the ST por-tions were intermediate. Percentages of DMb were greater for PMand ST muscles than for the LL.

3.4. NIR tissue oximetry and reflecto-spectrophotometry of MMb

Because FDMD NIR tissue oximeter directly measured only DMband OMb, estimates of MMb were calculated and are presented in

Table 3Least squares meansa for percentages of oxymyoglobin and deoxymyoglobin in three beef m10 d of display time.

Muscle Packaging format Myoglobin redox formOxymyoglobin/display time (d)

0 2 4

LL HiOx 68.0gx 68.3jx 68.3mx

HiOx-PVC 68.9gy 66.7jy 63.0lx

PVC 61.6fy 56.5hx 55.7jx

VAC 0.9ew 0.8ew 0.3ew

PM HiOx 60.4fz 52.9hy 39.8hx

HiOx-PVC 62.0fz 51.2hy 24.0gx

PVC 61.6fy 34.0fx 18.6fw

VAC 3.7ew 0.7ew 0.5ew

ST HiOx 65.7gy 60.3ix 58.0kx

HiOx-PVC 65.7gz 59.9jy 54.1jx

PVC 61.0fy 48.1gx 46.9ix

VAC 2.8ew 0.8ew 1.0ew

a efghijklm = Within a column across muscles, means with a different letter differ (p < 0differ (p < 0.05). Standard error for packaging � display time (d) or muscle = 3.06–3.74.

b LL = M. Longissimus lumborum = colour stable, ST = M. Semitendinosus = intermediatec HiOx = 80% oxygen and 20% carbon dioxide; HiOx-PVC = Sample remained in 80% oxy

chloride overwrap; VAC, Vacuum packaging.

Table 4. At d 0, MMb estimates were <3.8%. On d 2, LL portions inHiOx and all muscles in VAC had low estimates of MMb, whereas LLmuscle in PVC and the PM and ST muscles in all non-VAC packag-ing had increased MMb. At d 4, MMb estimates further increasedfor all non-VAC samples, and the largest estimated values ofMMb occurred by d 10.

Levels of MMb measured on the meat’s surface by reflectance(Table 4) followed a pattern of change similar to MMb estimatedfrom NIR data, but the absolute values were different. Estimatesof MMb by reflectance increased as display time increased, espe-cially between d 4 and 10. Packages containing oxygen had moreMMb than samples in VAC. At the end of display, the PM had themost MMb, the ST was intermediate, and the LL had the least.

3.5. Instrumental meat colour measurements

Values for L* (Fig. 1A, D, and G) during display for all muscles inall packaging formats were more stable than values for a* (Fig. 1B,E, and H) and b* (Fig. 1C, F, and I). Products in VAC were consis-tently darker (lower L* values). Each muscle experienced a loss(p < 0.05) of redness (smaller a* values) during display in aerobicpackaging systems (HiOx, HiOx-PVC, and PVC). The a* values forPM declined (p < 0.05) more rapidly during display than those forthe LL and ST (Fig. 1B, E, and H). Overall, the LL was the most red(greater a*), the ST was intermediate, and the PM was least redthroughout display. Values for a* for all muscles were most stablein VAC. Values for b* were most stable for products in VAC. In gen-eral, b* values declined (p < 0.05) similar to a* values throughoutdisplay in all three aerobic packaging systems (Fig. 1C, F, and I)but to a lesser magnitude. All three muscles packaged in VAChad little or no change in b* throughout display.

Chroma (vivid redness calculated from a* and b* values) de-creased (p < 0.05) for all muscles during display in all three aerobicpackaging systems (Fig. 2A–C). Chroma values of LL and ST werestable and declined gradually (p > 0.05) during display; PM chromavalues declined rapidly by d 4 with only a slight further decrease atd 10. At d 10, chroma values of all three muscles were at their low-est values; the LL, ST, and PM had the highest, intermediate, andlowest values, respectively. Chroma values in the VAC packageschanged little during display. Hue angle, a measure of the loss ofsurface redness of meat, increased (p < 0.05) most for the PM; STwas intermediate, and LL had the least change in hue (Fig. 2D–F).

usclesb in four packaging formatsc evaluated by reflectance spectrophotometry during

Deoxymyoglobin/display time (d)

10 0 2 4 10

39.0iw 13.4ew 10.3ew 10.7ew 7.8ew

28.7hw 13.4ew 10.6ew 11.7ew 24.1fx

31.7hw 23.1gw 21.6gw 21.1fw 21.4fw

1.6ew 85.7hw 90.9ix 95.2hx 94.0gx

19.2fw 19.4fx 27.2hy 25.3gy 8.5ew

17.8fw 19.0fx 21.2gx 19.5fx 7.4ew

17.7fw 18.8fx 22.6gx 18.1fx 8.9ew

1.7ew 84.3hw 96.2ix 97.9hx 96.7gx

29.0kw 18.9fx 24.5gy 19.4fx 8.4ew

23.7gw 18.9fx 17.8fx 18.6fx 11.7ew

29.6hw 14.8ex 18.9fy 18.8fy 10.1ew

2.2ew 84.7hw 98.2ix 96.9hx 95.6gx

.05). wxyz = Within a row and myoglobin redox form, means with a different letter

colour stability, PM = M. Psoas major1colour labile.gen and 20% carbon dioxide for 48 h and was then converted to PVC; PVC, Polyvinyl

05

1015202530

0 2 4 6 8 10

25303540455055

Longissimus lumborum

25303540455055

Psoas major

25303540455055

Semitendinosus

510

15202530

35

5

10

15

20

25

30

35

5101520253035

05

1015202530

0 2 4 6 8 10

Display time (d)HiOx HiOx -PVC PVC VAC

05

1015202530

0 2 4 6 8 10

A

B

C

D

IF

HE

G

Fig. 1. Muscle type � display time (d) effects on colour properties (L*, a*, and b*) of bovine M. Longissimus lumborum (A–C), M. Psoas major (D–F), and M. Semitendinosus (G–I)muscles stored in different packaging environments. HiOx = 80% oxygen and 20% carbon dioxide; HiOx-PVC = Sample remained in 80% oxygen and 20% carbon dioxide for 48 hand was then converted to PVC; PVC = Polyvinyl chloride; VAC = Vacuum packaging. Larger values for L*, a*, and b* indicate a lighter, redder, or more yellow colour,respectively.

Table 4Estimated percentage of metmyoglobina in three beef musclesb in four packaging formatsc evaluated by NIR tissue oximetry and reflectance spectrophotometry during 10 d ofdisplay time.

Muscle Packaging format Detection method

NIR tissue oximetry/display time (d) Reflectance spectrophotometry/display time (d)

0 2 4 10 0 2 4 10

LL HiOx 1.5 2.0 10.0 32.8 18.6 21.4 21.0 53.2HiOx-PVC 2.3 3.0 6.8 24.1 17.7 22.7 25.3 47.2PVC 1.8 6.9 11.2 23.0 15.3 21.9 23.2 46.9VAC 2.5 1.8 1.4 1.1 13.4 8.3 4.5 4.4

PM HiOx 2.1 8.9 22.0 56.6 20.2 19.9 34.9 72.3HiOx-PVC 3.1 8.6 18.7 41.5 19.0 27.6 46.5 74.8PVC 3.8 12.1 16.4 37.0 19.6 43.4 53.3 73.4VAC 1.1 1.3 1.0 1.2 12.0 3.1 1.6 1.6

ST HiOx 1.1 8.1 18.0 48.1 15.4 18.2 22.6 62.6HiOx-PVC 2.0 9.9 18.1 32.0 15.4 22.3 27.3 64.6PVC 1.2 13.1 17.1 26.0 24.2 33.0 34.3 57.3VAC 1.2 0.5 0.8 2.4 2.5 1.0 2.1 2.2

a % metmyoglobin estimated as: 100% � (% oxymyoglobin + % deoxymyoglobin). Standard error for packaging � display time (d) or muscle = 3.57–4.13.b LL = M. Longissimus lumborum = colour stable, ST = M. Semitendinosus = intermediate colour stability, PM = M. Psoas major = colour labile.c HiOx = 80% oxygen and 20% carbon dioxide; HiOx-PVC = Sample remained in 80% oxygen and 20% carbon dioxide for 48 h and was then converted to PVC; PVC, Polyvinyl

chloride overwrap; VAC, Vacuum packaging.

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3.6. Correlation of reflecto-spectrophotometric and NIR tissue oximetrymeasurements

To graphically show the strength of the relationship betweenreflectance and NIR tissue oximeter measurements, data were plot-ted with a fitted line and confidence lines in Fig. 3. Correlations be-tween reflectance and NIR oximetry data were the lowest (r = 0.19,p < 0.27–0.69, p < 0.0001) on d 0 regardless of the Mb redox form

(Fig. 3A, E, and I). For OMb and DMb, significant correlations ofP0.93 were obtained between the two measurements on d 2, 4,and 10 for OMb (Fig. 3B–D) and DMb (Fig. 3F–H), which was lo-cated principally below the meat’s surface. Correlations for MMbat d 2, 4, and 10 were significant but lower than those for OMband DMb.

Overall, the reflecto-spectrophotometric data for Mb redoxforms and colourimetric values (L*a*b*) were highly correlated

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Fig. 2. Muscle type � display time (d) for chroma (A–C) and hue angle (D–F) of bovine M. Longissimus lumborum, M. Semitendinosus, and M. Psoas major muscles stored indifferent packaging environments. HiOx = 80% oxygen and 20% carbon dioxide; HiOx-PVC = Sample remained in 80% oxygen and 20% carbon dioxide for 48 h and was thenconverted to PVC; PVC = Polyvinyl chloride; VAC = Vacuum packaging.

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after d 0 and accounted for 86–94% of the variation between thetwo variables for OMb (located mainly on the surface) and DMb(located mainly subsurface). Correlations of the two measures forMMb (located both below and on the meat’s surface) accountedfor 59–84.6% of the variation.

4. Discussion

The muscles used in this study had a pH, proximate composi-tion, myoglobin concentration, and initial visual colour typicallyexpected for those cuts; therefore, the data collected should be areasonable test for the methodologies applied. The main focus ofthis study was to (A) investigate the potential use of FDMD-basedNIR tissue oximetry as a noninvasive, continuous, direct methodfor determining the oxygenation and hemodynamics of post-rigorbeef skeletal muscle and (B) compare NIR tissue oximetry datawith reflectance measures of meat colour and Mb redox forms cre-ated by various packaging systems and display using musclesinherently different in Mb content and colour stability. The princi-pal new finding of our study is that the quantitative determination

of absolute levels of OMb and DMb and the indirect estimate ofMMb is possible using NIR tissue oximetry.

4.1. NIR tissue oximetry and reflectance of Mb redox forms

Previous research (Mancini & Hunt, 2005; McKenna et al., 2005;O’Keefe and Hood, 1982; Renerre & Labas, 1987; Seyfert et al.,2006) has shown that the colour stability of the three muscles inthe present study would be ranked as LL > ST > PM. Our NIR tissueoximetry and reflectance data confirmed that muscle ranking formeat displayed in aerobic packaging systems. O’Keefe and Hood(1982) reported that the LL had >4 d more colour stability thanthe PM. This seems related to the greater population of white-typemuscle fibres in the LL vs. the PM, which is a muscle with predom-inately red-type fibres. Most of the ST is whiter than the LL, but theST has fewer intermediate type fibres that seem to contribute pos-itively to colour life (Hunt & Hedrick, 1977a). Consequently, the LLand ST, with predominantly glycolytic fibres, are more colour sta-ble, and the PM, a muscle with more mitochondria and aerobicmetabolism is least stable in maintaining oxygenated Mb duringdisplay.

Fig. 3. Scatter plot matrix of correlation between reflecto-spectrophotometric and near-infrared (NIR) tissue oximetry data. Ref-OMb = OMb measured by reflecto-spectrophotometric method. NIR-OMb = OMb measured by NIR tissue oximetry. Ref-DMb = DMb measured by reflecto-spectrophotometric method. NIR-DMb = DMbmeasured by NIR tissue oximetry. Ref-MMb = MMb measured by reflecto-spectrophotometric method. NIR-MMb = MMb measured by NIR tissue oximetry.

462 A. Mohan et al. / Food Chemistry 123 (2010) 456–464

In living animals, different muscles have unique physiologicalroles and differ in their micro-anatomy, biochemical activity, andMb concentration (Hunt & Hedrick, 1977a, 1977b; Lanari & Cas-sens, 1991). Muscles with greater oxidative activity show greateroxygen consumption (Hunt & Hedrick, 1977a; Madhavi & Carpen-ter, 1993; Mancini & Hunt, 2005; Mohan et al., 2009; Seyfert et al.,2006) and are more colour labile than those with predominantlyglycolytic activity (Madhavi & Carpenter, 1993; Mancini & Hunt,2005; McKenna et al., 2005; O’Keefe and Hood, 1982). Post-rigorskeletal muscles differ in fibre types and metabolic function onthe basis of their specific physiological role (DeVore & Solberg,1975; Hunt & Hedrick, 1977b). Thus, each muscle experiences dif-ferent levels of oxygen demand and consumption when exposed toatmospheric oxygen (DeVore & Solberg, 1975; Lanari & Cassens,1991; Renerre & Labas, 1987; Seyfert et al., 2006).

Colour stability is primarily an interaction between a muscle’sOC and its ability to keep metmyoglobin reduced (Mancini & Hunt,2005; Mancini, Hunt, & Kropf, 2003). Generally, muscles withgreater OC are less colour stable during display than those withless OC (Kropf, 1993; Lanari & Cassens, 1991; Tang et al., 2005).Other researchers have found that MMb reduction is also criticallyrelated to colour stability (Kropf, 1993; Mancini & Hunt, 2005;McKenna et al., 2005; Mohan et al., 2009; Seyfert et al., 2006). Dis-parities in the importance of OC vs. MMb reduction are likely re-lated to the unknown nature of the chemical and structuralinteractions for the two processes in post-rigor meat (Mancini &Hunt, 2005).

Tang et al. (2005) reported that there was a direct relationshipbetween the postmortem muscle OC and meat colour stability in LLfrom Holsteins. Colour-labile muscles, such as the PM, have a high-er OC rate than colour-stable muscles (Mancini & Hunt, 2005;McKenna et al., 2005). Tang et al. (2005) reported that, in general,the PM tended to have the greatest OC and the LL had the least.Greater OC also decreases surface oxygen penetration, resultingin a shallow OMb layer and a thinner MMb layer below the surfacethat moves to the surface faster (Bendall & Taylor, 1972; McKennaet al., 2005). Because DMb is more prone to oxidation and less sta-ble than OMb (O’Keefe and Hood, 1982), a higher proportion ofDMb in muscles with greater OC would discolour faster becausethe subsurface layer of MMb would appear more rapidly comparedwith muscles that have less OC. Currently available meat colourmeasurement techniques such as reflectance colourimetry andspectrophotometric methods are limited to surface measurementof meat colour because of the inability of the visible light to pene-trate the meat surface. In contrast, the FDMD NIR tissue oximeterpenetrates several centimeters below the meat surface, and itsability to determine the oxygen-dependent absorption of Mb andHb has been demonstrated (Hueber et al., 2001; Marcinek, Amara,Matz, Conley, & Schenkman, 2007). Bowen (1949) demonstratedthat wavelengths of the NIR lights are differentially absorbed bythe oxygenated and deoxygenated forms of Hb.

Hueber et al. (2001) used a FDMD NIR tissue oximeter for mon-itoring tissue haemoglobin concentration and oxygenation in thebrain of newborn piglets during periods of hypo- and hyperoxia.

A. Mohan et al. / Food Chemistry 123 (2010) 456–464 463

In their study, a strong linear correlation (R2 = 0.98) was observedbetween tissue oxygen saturation and the average of arterial andvenous saturation. Because Mb and Hb absorb at the same wave-length in the NIR region, a similar approach was undertaken in thisstudy to investigate the potential use of FDMD NIR tissue oximeterfor determining Mb oxygen saturation and its redox stability underconditions simulating retail packaging and display to predict thecolour stability of beef skeletal muscles.

4.2. NIR tissue oximetry vs. reflectance spectroscopy

A continuous challenge in the meat industry is obtaining a reli-able technique that can provide important information on meatquality traits throughout production and retail display. Numerousmethods have been developed, but most are either invasive or dif-ficult to implement as a reliable, online, continuous measurementof meat quality. The techniques used for the assessment of meatquality serve as diagnostic tools to optimise and control quality.Reflecto-spectrophotometry is an established method for theassessment of meat discolouration and MMb accumulation onmeat surfaces. Quantification of Mb redox forms using reflectancedata often produces values that are either less or more than 100%,which causes problems with accuracy. Moreover, reflecto-spectro-photometry provides only an indirect estimate of Mb redox formson meat surfaces, and there was no clear trend observed for thebeef muscles of varying colour stability and Mb oxygen status.Reflectance measures may be excellent for describing appearanceof meat colour, but they offer very little information about Mb re-dox chemistry at subsurface levels.

Although qualitative and quantitative reflectance data havebeen very useful in meat colour chemistry and for providing in-sight into meat colour problems, these data are not particularlyuseful for predicting or relating changes in Mb redox status, muscleoxygen status, and muscle-colour stability. For the characterisationof the surface discolouration, decreases in a* values (decrease inred colour) and chroma and/or hue angles are generally acceptedmonitoring meat colour changes. However, a* values obtainedfrom reflecto-spectrophotometric measurements seem to be usefulonly for the qualitative characterisation of the surface colour ofmeats. The use of an NIR tissue oximeter on meat provided compli-mentary information such as Mb redox status, muscle oxygen sta-tus, and potential information about muscle-colour stability duringa simulated retail display. Moreover, the instrumental colour dataobtained for a* values corresponded well to the NIR tissue oxime-ter measurements of Mb redox forms, and the data seem to beadaptable to colour stabilization of postmortem beef skeletal mus-cles in several packaging formats during display. Also, NIR tissueoximetry results seemed to be able to differentiate among high,intermediate, and low colour stable beef skeletal muscles on thebasis of Mb oxygen status and redox dynamics during retail dis-play. Beef skeletal muscles with greater colour stability (LL andST) exhibited greater total muscle oxygen saturation combinedwith a lower OC resulting in a higher OMb concentration thanthe colour-labile muscle (PM). The results presented in this paperclearly demonstrate the advantages of using a FDMD-based NIRtissue oximeter to understand the chemistry associated with thecolour change in postmortem beef skeletal muscles during retaildisplay. The correlation data presented in Fig. 3 are interestingwhere the NIR tissue oximeter measurements show a high, positivecorrelation with reflecto-spectrophotometric data, indicating thatinformation provided by the NIR tissue oximeter is reliable. Therate at which meat discolours during retail display is consideredone of the most important traits of meat retailing. The ability ofthe NIR tissue oximeter to relate with the meat discolouration rateand the relative change in Mb redox forms with a high degree ofaccuracy highlights the potential of using an NIR tissue oximeter

to predict several quality traits important to meat colour chemistryin a rapid, noninvasive manner. Overall, NIR tissue oximetry dataappear to be reliable for determining the colour differences andcolour stability of beef skeletal muscles, and agreement of theNIR tissue oximeter measurements with instrumental colourparameters suggests that NIR tissue oximetry has potential for pre-dicting several traits important to meat colour chemistry.

It is easier to conduct repeated colour measurements of meatpackaged in PVC overwrap than in MAP trays with a headspace be-tween the film and the meat. Colour measurements of MAP pack-aged meat can be taken by inverting the package so that the meatcontacts the film, but repeated measurements taken in this mannerresult in film stains, fat smearing, and unwanted light scatter.Other researchers open packages before instrumental colour mea-surement, allowing direct contact between the meat surface andthe instrument’s aperture, but other packages must be availablefor later storage periods. The NIR tissue oximetry measurementsof meat colour do not circumvent these packaging constraints,but they do expand the data from surface characterisation to inter-nal data that relate to factors believed to be related to colour sta-bility such as Mb oxygen status, NADH pools, and MMbreduction mechanisms (Mancini & Hunt, 2005; Millikan, 1939;Seyfert et al., 2006; Urbin & Wilson, 1961).

5. Conclusions

In this study, we demonstrated for the first time that FDMD NIRtissue oximetry was able to detect and quantitatively measure thedynamic changes in surface and subsurface levels of Mb redoxforms of post-rigor muscle packaged and displayed under simu-lated industry conditions. Additionally, the effects of packaging,postmortem storage, and display conditions on Mb redox stabilitywere highly correlated (r values > 0.9) between the NIR tissueoximetry and reflectance methods. Our data clearly show the po-tential for using a FDMD NIR tissue oximeter for measurement ofOMb and DMb of post-rigor muscle and the indirect estimationof MMb. Furthermore, the NIR tissue oximeter detected a heteroge-neous blend of Mb redox forms on (or in) meat during packagedstorage and display.

Acknowledgement

No. 10-007-J from the Kansas Agricultural Experiment Station,Kansas State University, Manhattan, KS, USA.

References

Atkinson, J., & Follett, M. (1973). Biochemical studies on the discolouration of freshmeat. International Journal of Food Science and Technology, 8(1), 51–58.

Bendall, J. R., & Taylor, A. A. (1972). Consumption of oxygen by the muscles of beefanimals and related species. II. Consumption of oxygen by post-rigor muscle.Journal of the Science of Food and Agriculture, 23(6), 707–719.

Bowen, W. J. (1949). The absorption spectra and extinction coefficients ofmyoglobin. The Journal of Biological Chemistry, 179(1), 235–245.

Chance, B., Dait, M., Zhang, C., Hamaoka, T., & Hagerman, F. (1992). Recovery fromexercise-induced desaturation in the quadriceps muscles of elite competitiverowers. American Journal of Physiology – Cell Physiology, 262(3), 766–775.

de Groot, B., Zuurbier, C. J., & van Beek, J. H. G. M. (1999). Dynamics of tissueoxygenation in isolated rabbit heart as measured with near-infraredspectroscopy. American Journal of Physiology – Heart and CirculatoryPhysiology, 276(5), 1616–1624.

DeVore, D. P., & Solberg, M. (1975). A Study of the rate-limiting factors in therespiratory oxygen consumption of intact post-rigor bovine muscle. Journal ofFood Science, 40(3), 651–652.

Ferreira, L. F., Townsend, D. K., Lutjemeier, B. J., & Barstow, T. J. (2005). Musclecapillary blood flow kinetics estimated from pulmonary O2 uptake and near-infrared spectroscopy. Journal of Applied Physiology, 98(5), 1820–1828.

Hueber, D., Franceschini, M., Ma, H., Zhang, Q., Ballesteros, J., Fantini, S., et al. (2001).Non-invasive and quantitative near-infrared haemoglobin spectrometry in thepiglet brain during hypoxic stress, using a frequency-domain multidistanceinstrument. Physics in Medicine and Biology, 46(1), 41–62.

464 A. Mohan et al. / Food Chemistry 123 (2010) 456–464

Hunt, M., Acton, J., Benedict, R., Calkins, C., Cornforth, D., Jeremiah, L., et al. (1991).American Meat Science Association’s guidelines for meat colour evaluation.Chicago: National Livestock and Meat Board.

Hunt, M., & Hedrick, H. (1977a). Profile of fiber types and related properties of fivebovine muscles. Journal of Food Science, 42(2), 513–517.

Hunt, M., & Hedrick, H. (1977b). Histochemical and histological characteristics ofbovine muscles from four quality groups. Journal of Food Science, 42(3),578–582.

Keeton, J. T., Hafley, B. S., Eddy, S. M., Moser, C. R., McManus, B. J., & Leffler, T. P.(2003). Rapid determination of moisture and fat in meats by microwave andnuclear magnetic resonance analysis – PVM 1:2003. Journal of AOACInternational, 86(6), 1193–1202.

Kropf, D. (1993). Colour stability: factors affecting the colour of fresh meat. MeatFocus International, 2(6), 269–275.

Lanari, M., & Cassens, R. (1991). Mitochondrial activity and beef muscle colourstability. Journal of Food Science, 56(6), 1476–1479.

Madhavi, D., & Carpenter, C. E. (1993). Aging and processing affect colour,metmyoglobin reductase and oxygen consumption of beef muscles. Journal ofFood Science, 58(5), 939–942.

Mancini, R., & Hunt, M. (2005). Current research in meat colour. Meat Science, 71(1),100–121.

Mancini, R., Hunt, M., & Kropf, D. (2003). Reflectance at 610 nanometers estimatesoxymyoglobin content on the surface of ground beef. Meat Science, 64(2),157–162.

Marcinek, D. J., Amara, C. E., Matz, K., Conley, K. E., & Schenkman, K. A. (2007).Wavelength shift analysis: a simple method to determine the contribution ofhemoglobin and myoglobin to in vivo optical spectra. Applied Spectroscopy,61(6), 665–669.

McKenna, D., Mies, P., Baird, B., Pfeiffer, K., Ellebracht, J., & Savell, J. (2005).Biochemical and physical factors affecting discolouration characteristics of 19bovine muscles. Meat Science, 70(4), 665–682.

Millikan, G. (1939). Muscle hemoglobin. Physiological Reviews, 19(4), 503–523.Mohan, A., Hunt, M. C., Barstow, T. J., Houser, T. A., Bopp, C., & Hueber, D. M. (2009).

Effects of fiber orientation, myoglobin redox form, and postmortem storage onNIR tissue oximeter measurements of beef Longissimus muscle. Meat Science,84(1), 79–85.

O’Keefe, M., & Hood, D. (1982). Biochemical factors influencing metmyoglobinformation on beef from muscles of differing colour stability. Meat Science, 7(3),209–228.

Renerre, M., & Labas, R. (1987). Biochemical factors influencing metmyoglobinformation in beef muscles. Meat Science, 19(2), 151–165.

Sammel, L., Hunt, M., Kropf, D., Hachmeister, K., & Johnson, D. (2002). Comparison ofassays for metmyoglobin reducing ability in beef inside and outsidesemimembranosus muscle. Journal of Food Science, 67(3), 978–984.

Seyfert, M., Mancini, R. A., Hunt, M. C., Tang, J., Faustman, C., & Garcia, M. (2006).Colour stability, reducing activity, and cytochrome c oxidase activity of fivebovine muscles. Journal of Agricultural and Food Chemistry, 54(23), 8919–8925.

Tang, J., Faustman, C., Hoagland, T. A., Mancini, R. A., Seyfert, M., & Hunt, M. C.(2005). Postmortem oxygen consumption by mitochondria and its effects onmyoglobin form and stability. Journal of Agricultural and Food Chemistry, 53(4),1223–1230.

Thiex, N. (2009). Evaluation of analytical methods for the determination ofmoisture, crude protein, crude fat, and crude fiber in distillers dried grainswith solubles. Journal of AOAC International, 92(1), 61–73.

Urbin, M., & Wilson, G. (1961). The post-mortem oxygen requirements of bovinetissue a. Journal of Food Science, 26(3), 314–317.