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Metabolomics Profiling to Determine the Effect of Postmortem Aging on Color and Lipid Oxidative Stabilities of Different Bovine Muscles Danyi Ma, Yuan H. Brad Kim, Bruce R. Cooper, Ji-Hwan Oh, Hyonho Chun, Juhui Choe, J. P. Schoonmaker, Kolapo Matthew Ajuwon, and Byungrok Min J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02175 • Publication Date (Web): 12 Jul 2017 Downloaded from http://pubs.acs.org on July 12, 2017

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Journal of Agricultural and Food Chemistry

Metabolomics Profiling to Determine the Effect of Postmortem Aging on Color and Lipid Oxidative Stabilities of Different Bovine Muscles Danyi. Ma, † Yuan H. Brad Kim*, † Bruce Cooper, § Ji–Hwan. Oh, ‡ Hyonho Chun, ‡ Ju–Hui Choe, † Jon P. Schoonmaker, † Kolapo Ajuwon, † Byungrok Min, # †

Department of Animal Sciences, Purdue University, West Lafayette, IN 47907, USA. ‡ Department of Statistics, Purdue University, West Lafayette, IN 47907, USA. § Bindley Bioscience Center, Purdue University, West Lafayette, IN 47907, USA. # Food Science and Technology Program, University of Maryland Eastern Shore, Princess Anne, MD 21853, USA

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* To whom correspondence should be addressed. Phone: +1–765–496–1631; E–mail address: [email protected]

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ABSTRACT: The objective of this study was to identify the metabolites that could be associated

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with oxidative stability of aged bovine muscles. Three muscles (longissimus lumbrum (LL),

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semimembranosus (SM), and psoas major (PM)) from 7 beef carcasses at 1 day postmortem

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were divided into three sections and assigned to three aging periods (9, 16 and 23 days). While

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an increase in discoloration was found in all muscles with aging, LL was the most color/lipid

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oxidative stable, followed by SM and PM (P < 0.05). Lower myoglobin and non–heme iron

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contents were observed in LL compared to SM and PM (P < 0.05). The HPLC–ESI–MS based

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metabolomics analysis identified metabolites that were significantly responsive to aging and/or

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muscle type, such as acyl carnitines, free amino acids, nucleotides, nucleosides, and

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glucuronides. The results from the current study suggested that color and oxidative stability is

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inversely associated with aging, but is also muscle–specific. Further studies determining the

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exact role of the identified metabolites in color and oxidative stability of beef muscles should be

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warranted.

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KEYWORDS: Beef, aging, metabolomics, meat color, lipid oxidation 1 ACS Paragon Plus Environment


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INTRODUCTION

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Postmortem aging has been extensively practiced in the meat industry mainly due to its

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beneficial impacts on improving eating quality attributes. In particular, significant improvements

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in meat tenderness, flavor, or juiciness occur through muscle protein degradation by endogenous

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proteases during extended aging. However, prolonged postmortem aging may have an adverse

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impact on color and/or lipid oxidation. A few studies reported that long–term chilled meat

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products had inferior oxidative stability, resulting in a decrease in display shelf–life and an

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increase in rancid off–flavor development, when repackaged from vacuum packs into a retail

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display format

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on surface meat color to determine the degree of freshness for their meat purchasing decisions.

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Consumers would likely discriminate fresh meat products appearing brown–red color (due to

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formation of metmyoglobin; oxidized form of myoglobin) even if meat products have been

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chilled for long–term to enhance eating quality characteristics 3.

1-2

. This can be a significant economic problem because consumers heavily rely

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A few studies suggested that the accumulation of pro–oxidants, such as heme and non–

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heme iron 4, and/or the depletion of endogenous antioxidants 5-6 could be attributed to the aging–

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induced oxidation. Further, the extent of compounds or enzyme activities that impart antioxidant

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properties would be varied among specific muscle types 7-8, which could result in different levels

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of oxidative stability of muscles during postmortem aging. This postulation warrants further

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examination because understanding the underlying mechanisms of oxidative stability of beef

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muscles is a crucial step to develop a practical aging strategy to prevent oxidation–related quality

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defects, while maintaining beneficial aging impacts on eating quality attributes.

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Metabolomics is an emerging technique to analyze small molecule compounds (M.W. less

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than 1 kDa) in complex biological systems such as cell, tissue or bio–fluids 9. Mass spectrometry 2 ACS Paragon Plus Environment

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(MS)–based metabolomics, in particular, is gaining wide acceptance in the food science

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discipline due to its potential in analyzing molecular composition, safety and quality properties,

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and health and nutritional properties of food matrix 10. In muscle foods, several studies have been

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recently published in obtaining metabolome profiles of meat samples including beef, pork,

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chicken and lamb. Those studies determined effects of various pre–and post–harvest factors on

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meat metabolites, such as packaging

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type

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have been conducted to identify muscle–specific metabolites associated with oxidative stability

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of different muscles during postmortem aging.

15

11

, chilling

and postmortem processing conditions

12

, feed authentication

11, 16

13

, genetics

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, muscle

. However, there is little or no studies that

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Therefore, the objectives of the current study were to determine the effect of postmortem

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aging on color and lipid oxidative stability of different bovine muscles, and to identify the

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metabolites that were related to oxidation during postmortem aging. In the current study, three

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beef muscles including longissimus lumborum (LL), semimembranosus (SM), and psoas major

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(PM) were selected, because those muscles were known to have distinct differences in fiber

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composition, color and lipid oxidative stability 7.

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MATERIALS AND METHODS

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Materials. Seven beef steers (Angus x Simmental crossbred steers, 14 months of age,

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average slaughter weight 624 kg), which were fed a high concentrate feedlot diet (about 10%

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forage), were slaughtered at Purdue University Meat Laboratory. At one day postmortem, three

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muscles (LL, SM, and PM) were separated from one side of each carcass. Each muscle was

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divided into three equal sections, vacuum packaged, and randomly assigned to three aging

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periods (9, 16, and 23 days postmortem) at 1 °C. After the assigned aging period, one thin slice

3 ACS Paragon Plus Environment


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cut (about 1 mm) of each section was chopped, snap-frozen, powdered, vacuum packaged, and

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stored at –80 °C until sample extraction for metabolomics profiling;

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thickness) was cut from each section, placed on a plastic tray, packaged with oxygen permeable

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polyvinylchloride film (23,000 cm3 O2/m2/24h at 23 °C) and displayed under fluorescent white

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light (approximately 1,450 lx, Color temperature = 3,500 K) for 7 days at 2.5 °C. For SM, the

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deep portion (the medial inner 1/4 closest to the femur) and the superficial portion (the lateral

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outer 1/4 closest to the surface of the carcass) of the muscle were cut and discarded. The

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remaining portion of SM steak was used in order to limit any confounding locational effects on

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quality and/or chemical attributes within the muscle 17. The packaged steaks were relocated on a

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display table on a daily basis to minimize any confounding effect associated with different

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display location. After display, the steak samples were vacuum packaged and stored at –80 °C

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until further chemical analyses.

one steak (2.54 cm

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Color Measurement. During the simulated retail display, instrumental color characteristics

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of the steak surface were measured using a Minolta CR–400 colorimeter (D65, 1 cm diameter

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aperture 2° standard observer; Konica Minolta Photo Imaging Inc., Tokyo, Japan) at display

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days 1, 4, and 7. Calibration was performed using a standard white tile (CIE L*=97.06, CIE

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a*=0.41, CIE b*=1.72) prior to the color measurement. CIE L*, a* and b* values were obtained

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by the average of 3 spot measurements per steak. Chroma [(a*2+b*2)1/2] and hue angle [(b*/a*)

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tan–1] values were estimated from CIE L*, a* and b* values 18.

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Visual color evaluation of steak samples was conducted according to the AMSA Color 18

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Measurement Guideline

. The sensory color panelists (n=10), who passed a Farnsworth—

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Munsell 100 Hue screening test, were trained multiple–times. The extent of lean color change


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was evaluated using eight scale points (1=extremely dark red, 2=dark red, 3=moderately dark

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red, 4=slightly dark red, 5=slightly bright red, 6=moderately bright red, 7=bright red, and

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8=extremely bright red). The extent of discoloration was assessed based on seven scale points

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(1=no discoloration (0%), 2=slight discoloration (1–19%), 3=small discoloration (20–39%),

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4=modest

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discoloration (80–99%), 7=total discoloration (100%)).

discoloration

(40–59%),

5=moderate

discoloration

(60–79%),

6=extensive

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Lipid Oxidation: Conjugated diene (CD) and TBARS. The extent of diene conjugation

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(primary lipid oxidation product) was determined after 7 days of retail display of beef samples

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by following the procedure as described by Srinivasan, Xiong, and Decker

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modifications. In duplicate, 0.5 g of meat sample was homogenized with 5 ml of distilled water.

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Then, 0.5 ml of homogenate was mixed with 5 ml of extraction solution (isopropanol and

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hexane, 3:1 ratio) and centrifuged at 2,000 × g for 5 min. The absorbance of the supernatant at

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233 nm was measured against a blank (extraction solution) using a spectrophotometer (Epoch,

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BioTek Instrument Inc.) in a 1cm quartz cuvette. The concentration of CD was calculated using a

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molar extinction coefficient of 25,200 M–1cm–1 and the results were expressed as µmol/mg meat

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lipid sample.

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with minor

The extent of secondary lipid oxidation products was measured in duplicate, following 2– 20

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thiobarbituric reactive substances acid (TBARS) assay

. Beef samples displayed for 7 days

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were frozen powdered and homogenized in distilled water (1:3 ratio) with butylated hydroxyl

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anisole (BHA) solution (10% v/v with 90% ethanol solution) using an Ultra Turrax (Ultra–

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Turrax T25, Janke & Kunkel IKA–Labortechnik, Staufen, Germany) at 6,000 rpm for 30s and

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centrifuged at 2,000 × g for 10 min at 4 °C. The supernatant (2 ml) was mixed with 4 ml of



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TBA/TCA reagent (20 mmol TBA with 15% trichloroacetic acid solution), heated for 15 min in

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a water bath (80°C) to develop the chromogen, then cooled for 10 min in ice water. The

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absorbance of each sample at 538 nm was measured using a microplate spectrophotometer

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(Epoch, BioTek Instrumrtnts Inc.) against a blank (2 ml distilled water + 4 ml TCA/TBA

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solution). TBARS value was expressed as mg malondialdehyde (MDA)/kg muscle.

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Myoglobin Content and Non–Heme Iron. Myoglobin contents of beef samples were

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measured in duplicate by following the procedure of Warris, Trout, and Rickansrud and

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Henrickson

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mmol potassium phosphate buffer (pH 6.8) and held on ice for 1 h. After centrifuging the

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homogenates at 35,000 × g for 30 min at 4 °C, supernatant was collected and filtered. The

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filtered supernatant was scanned in a cuvette for the absorbance spectra at 400 to 700 nm

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measured by VWR UV–1600 PC spectrophotometer (VWR International, San Francisco, CA).

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The myoglobin concentration (mg/g meat) was calculated using the absorbance difference

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between 525 nm and 700 nm multiplied by 2.303 and a dilution factor.

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21-23

with a few modifications. In brief, samples (2.5 g) were homogenized in 40

Non–heme iron content was determined following the described procedure

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with minor

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modifications. Powdered meat samples from steaks displayed for 7 day were homogenized in 0.1

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M citrate–phosphate buffer (pH 5.5), mixed with ascorbic acid (1% in 0.2 N HCl, w/v) and

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11.3% TCA solution (w/v, 1 ml) and held for 5 min at room temperature. The mixture was

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centrifuged at 3,000 × g for 15 min at 20 °C, and supernatant was mixed with 10% ammonium

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acetate (w/v) and ferrozine at 22 °C for 10 min for color development. The absorbance was

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determined at 562 nm against a blank. The non–heme iron content was expressed as mg non–

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heme iron per 100 gram of meat.


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Metabolomics Sample Preparation and Extraction. Protein removal and sample 25

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extraction were performed using a Bligh–Dyer extraction protocol

. Chloroform (200 uL)

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mixed with an equal volume of methanol was added to 100 mg of meat powder. Samples were

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extracted in a Precellys 24 tissue homogenizer. Two hundreds microliter of water was mixed

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with the extract and was centrifuged at 16,000 × g for 8 minutes. The upper methanol and water

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phase contained the polar metabolites, which was transferred to separate vials and were

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evaporated to dryness in a SpeedVac Concentrator. The dried polar fraction was reconstituted in

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50 uL of a diluent composed of 95% water and 5% acetonitrile containing 0.1% formic acid.

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HPLC–MS Analysis. Separations were performed on an Agilent 1100 system (Palo Alto,

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CA), with a mobile phase flow rate of 0.3 mL/min. The metabolites were assayed using a Waters

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Atlantis T3 column (3 µm, 2.1 × 50 mm), where the mobile phase A and B were 0.1% formic

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acid in ddH2O and acetonitrile, respectively. Initial conditions were 100:0 A:B, held for 1

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minute, followed by a linear gradient to 5:95 at 21 min and was held until 26 min. Column re–

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equilibration was performed by returning to 100:0 A:B at 30 minutes, and holding until 35

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minutes. The mass spectrometry chromatograms were obtained using an Agilent MSD–TOF

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spectrometer with ESI capillary voltage 3.5 kV, nitrogen gas temperature 350 °C, drying gas

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flow rate 9.0 L/min, nebulizer gas pressure 35 psig, fragmentor voltage 135 V, skimmer 60 V,

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and OCT RF 250 V. Mass data (from m/z 70–1100) were collected using Agilent MassHunter

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Acquisition software (v. B.03). Mass accuracy was improved by infusing Agilent Reference

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Mass Correction Solution.



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Peak deconvolution was performed using Agilent MassHunter Qualitative Analysis (v.

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B.06). Chromatographic peaks were aligned across all samples. Peak areas were normalized by

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sample weights, converting to log2 and applying a 75% percentile shift. Peak annotations were

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performed based on mass assignment and retention behavior using the METLIN

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(www.metlin.scripps.edu) and HMDB (www.hmdb.ca) metabolite databases with a mass error of

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less than 10 ppm.

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Statistical Analysis. The experimental design of this study was split–split plot design, with

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muscle type effect (LL, SM and PM) as the whole plot and aging time effect (9, 16 and 23 days

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of aging) as a subplot. For color characteristics, the display effect (1, 4, and 7) was set as sub–

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sub plot. Animals were considered as a random effect. Color characteristics, pH, lipid oxidation

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(CD and TBARS), and non–heme iron were analyzed by the PROC MIXED procedure of SAS

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9.4 software (SAS Institute Inc.).

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For the metabolomics profiling, statistical analysis was performed by using split–plot

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ANOVA or Kruskal–Wallis test (KWT) via R software (www.r–project.org) with Benjamini–

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Hochberg FDR correction. Metabolites with P < 0.05 were considered as significantly affected

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by aging or muscle type effect. Then the data were further analyzed collectively via principal

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component analysis (PCA) using R software. The corresponding false discovery rate (FDR)

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values were reported as further reference. Associations of metabolites and color characteristics,

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lipid oxidation, non–heme iron and myoglobin content were analyzed by using Spearman’s rank

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correlation. Correlation with statistical significance (P < 0.05) was considered for further

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biological interpretation.

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RESULTS

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Color, Oxidative Stability and Pro–Oxidant Species. Instrumental color (CIE L*, a*, b*,

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chroma and hue angle) and visual color (lean color and discoloration) attributes were assessed to

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determine the effects of postmortem aging on color and color stability of different beef muscles.

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In general, LL and SM maintained higher redness, color intensity, and lower hue angle values

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compared to PM throughout display (P < 0.05) irrespective of aging periods (Table 1), which

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was consistent with the visual color evaluation. Furthermore, postmortem aging significantly

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affected color and color stability of beef muscles. As aging time increased, a decrease in redness

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of surface beef color was found as indicated by a*, chroma, and visual lean color (P < 0.05;

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Table 2).

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In particular, a significant interaction of muscle by aging was found in hue angle and

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discoloration, where PM showed significantly higher discoloration and hue angle values after 16

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days of aging, while LL and SM maintained the similar values throughout the whole postmortem

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aging (P < 0.05). There was a significant aging × display interaction in a*, chroma, hue angle,

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lean color and discoloration (Table 2). No significant interactions between muscle, aging and

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display duration were found in L* values (lightness). The lightness values were only affected by

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muscle (P < 0.05), where SM had the highest L* values (lightest) followed by LL and PM

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(darkest) (Table 1).

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Changes in primary oxidation (CD formation) and secondary lipid oxidation (TBARS)

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were assessed after 7 days of the simulated retail display. There were no significant interactions

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between muscle and aging on CD and TBARS. CD concentration was significantly affected by

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muscle type (P < 0.001), but not aging (P = 0.1; Table 3). LL maintained lower CD value than

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SM and PM after display, regardless of aging treatment. Conversely, TBARS was significantly



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affected by both aging time (P < 0.05) and muscle type (P < 0.01). An increase in TBARS was

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observed in the 23 day aged group compared with 9 and 16 day aged beef samples, and the

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highest TBARS was found in the SM muscle, followed by PM and LL (Table 3). A trend of

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aging × muscle interaction (P=0.06) showed that LL was lower in TBARS value than the other

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two muscles in 9 and 16 day aged groups (data not shown).

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There were no significant interactions between muscle and aging in myoglobin content and

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non-heme iron. Myoglobin content was affected by muscle type (P < 0.05), but not postmortem

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aging (P > 0.05; Table 3). Myoglobin content was significantly higher in PM compared with LL

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and SM (P < 0.001), which was likely due to high content of type I red muscle fiber in PM

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muscle. Non–heme iron was significantly affected by muscle type (P < 0.001) and postmortem

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aging (P < 0.001). Non–heme iron was lowest at 9 day of aging, and accumulated as aging time

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prolonged. LL maintained the lowest non–heme iron level followed by SM and PM, regardless

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of the aging treatment (Table 3).

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The chemical attributes related to oxidative stability were correlated to color

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characteristics of beef samples (Table 4). CD was moderately correlated to all the color

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characteristics except CIE L* value (Table 4), suggesting a potential involvement of primary

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oxidation in meat color deterioration. Myoglobin and non–heme iron content were strongly

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correlated with CD (r= 0.78 and 0.66 respectively, P < 0.001) and also exhibited significant

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correlations with color characteristics including CIE a* (r = -0.49 and -0.76, P < 0.001), hue

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angle (r = 0.59 and 0.63, P < 0.01), lean color score (r = 0.57 and -0.7, P < 0.001), and

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discoloration score (r = 0.57 and 0.73, P < 0.001).

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Metabolome Profiles. The untargeted metabolome profiling initially detected 1695

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different compounds in meat samples. Metabolites were checked with the assumption of equal


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variance and normality of error term to determine the applicability of conducting ANOVA,

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where 702 metabolites that satisfied the two assumptions were tested for ANOVA, and the

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remaining metabolites were tested using the Kruskal–Wallis method (KWT).

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For the effect of aging, 222 out of 702 metabolites were significantly affected by the aging

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(P < 0.05); 150 of which showed strong signals (FDR < 0.05, P < 0.05) after Benjamin–

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Hochberg multiple testing correction, an adjustment of P–values to control the level of Type I

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error. From the 222 compounds, the major groups included free amino acids, fatty acetyl

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carnitines, vitamins and coenzymes, and nucleotide–related metabolites as listed in Table 5.

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For the effect of muscle type, 35 of the metabolites were identified as significant. Primary

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metabolites being identified from this group were presented in Table 5. Overall, the LL and SM

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muscles were characterized with more abundant in β–alanine–histidyl dipeptides, fatty acetyl

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carnitines, phenylalanine and niacinamide. L–carnitine, xanthine and hypoxanthine were tested

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using non–parametric method (KWT). The fold changes of these compounds indicate a decrease

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of these metabolites with aging (data not shown).

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Principal Component Analysis (PCA). The principal component analysis (PCA) was

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used to visualize the extent of distinct differences of the identified metabolomes of the muscles

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with aging. Two PCA models have been built based on the different subsets of the original

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dataset, namely the 243 metabolites that showed statistical significance indicated by ANOVA or

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KWT (Figure 1A and 1B) and the 150 metabolites that satisfied both P < 0.05 and FDR < 0.05

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criteria of ANOVA (Figure 1C and 1D). The PCA plots based on the further selected data

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(Figure 1C and 1D) clearly showed improved efficacy compared to those on the initially selected

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data (Figure 1A and 1B), as the total variance explained by first principal component (PC1)



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increased from 33% in the first model to 41% in the second model. As showed in the both

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figures, the first two principal components have already specified more than 50% of the observed

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variance. In particular, PC1 mostly accounted for the difference in aging treatments and clearly

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separated muscle samples that aged for 9 days, 16 days, and 23 days respectively. Because PC1

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is the axis where the data set has the largest variation

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differences of the metabolomes among muscle samples were caused by postmortem aging. In

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addition, PC2 could explain 21% of the total variance of the data set, which separated the PM

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samples from the cluster formed by overlapped LL and SM samples. Moreover, the loading

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analysis indicated some possible associations between metabolites and each PC (Table S1,

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Figure 1E). The top 10 metabolites with positive or negative scores in each PC were listed in

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Table S1, which included NAD/NADH, carnitines, and peptides. As reflected in their scores,

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these metabolites may be more closely affected by the aging process. More biological

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interpretations will be discussed in later sections.

26

, in the current study, the major

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Correlation. The heatmaps demonstrated the correlations between key metabolites that

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were significantly affected by either muscle type or aging time and major color characteristics

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and pro–oxidation related indexes (Figure. S1). Metabolites that were significantly correlated to

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the color or oxidation related chemical attributes were mainly NAD, acyl–carnitines, free amino

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acids, assorted nucleotides, and glucuronides (Table S2). In particular, NAD showed significant

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positive correlations to CIE a* (r = 0.672) and negative correlation to discoloration (r = -0.535),

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TBARS (r = -0.554), and non–heme iron (r = -0.667). Acyl carnitines, nucleotides xanthine and

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hypoxanthine, free amino acids phenylalanine and tryptophan, and glucuronides were, in general,

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positively associated with discoloration characteristics and non–heme iron accumulation.


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DISCUSSION

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Effect of Aging on Oxidation Stabilities of Different Muscles. Meat color stability is a

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muscle–specific trait. The observed muscle differences in color stability were in agreement with

287

several other studies

288

impacts on beef color stability is also a muscle–specific trait, where PM, color labile muscle, is

289

more susceptible to aging–induced discoloration compared to other color stable muscles (e.g.

290

LL).

7, 27

. Particularly, the current observation indicates that the adverse aging

291

The current study found moderate correlations between CD and myoglobin content, non-

292

heme iron, and all the color characteristics except CIE L* value (Table 4), suggesting that

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primary oxidation could affect meat color deterioration mainly due to its impact on pigment

294

redox stability. Previous studies showed that lipid oxidation–induced myoglobin oxidation was

295

more likely due to primary rather than secondary products

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lipid hydroperoxide was reported to trigger myoglobin into a transient ferryl status. This leads to

297

formation of a covalent bond between the heme group and protein moiety

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myoglobin contains heme iron, which is highly reactive to oxidation processes. During aging,

299

exposure of heme group caused by myoglobin deformation would result in non–heme iron

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accumulation 30, which may further facilitate meat discoloration.

28

, with the supporting evidence that

29

. Furthermore,

301 302

Effect of Aging on Muscle Metabolome and Oxidative Stabilities. During postmortem

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aging, muscle cells and cell apparatuses lose structural integrity and enzyme activities, resulting

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in a decreased ability to maintain reducing conditions. Such changes may lead postmortem

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muscles to be more oxidation susceptible 31. The multivariate analysis results in the current study



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clearly indicated the substantial aging impact on the metabolome profiling. In particular, the

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current study identified that oxidative stability of bovine muscles could be related to key

308

metabolites including NAD, assorted free amino acids, nucleotides and its degradation products,

309

and phenolic compounds as illustrated in Figure S1 and Table S2. However, it should be noted

310

that the current study used reversed-phase HPLC-MS, which provides better retention on semi-

311

polar metabolites rather than polar metabolites including sugars and organic acids 32. This could

312

likely explain why little to no detection of metabolites related to glycolytic or TCA was present

313

in the current study.

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Nicotinamide adenine dinucleotide (NADH) and its oxidized form NAD+ was identified by

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the m/z values 665.1249 and 663.1092 respectively. The ANOVA results indicated that both

316

NADH and NAD+ were significantly decreased with prolonged aging period (P < 0.05, FDR

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