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Sensory and Flavor Chemistry Characteristics of Australian Beef; the Influence of Intramuscular Fat, Feed and Breed. Damian Conrad Frank, Alex J Ball, Joanne M Hughes, Udayasika Piyasiri, Janet Stark, Peter Watkins, and Robyn Dorothy Warner J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00160 • Publication Date (Web): 27 Apr 2016 Downloaded from http://pubs.acs.org on May 6, 2016

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Journal of Agricultural and Food Chemistry

Sensory and Flavor Chemistry Characteristics of Australian Beef; the Influence of Intramuscular Fat, Feed and Breed.

Damian Frank*1, Alex Ball2, Joanne Hughes3, Raju Krishnamurthy1, Udayasika Piyasiri1, Janet Stark3, Peter Watkins4 and Robyn Warner4,5 1

Commonwealth Scientific Industrial Research Organisation (CSIRO), 11 Julius Ave, North

Ryde, NSW, 2113, Australia. 2

Meat & Livestock Australia (MLA), Level 1, 40 Mount Street, North Sydney, NSW, 2060,

Australia. 3

Commonwealth Scientific Industrial Research Organisation (CSIRO), 39 Kessels Rd,

Coopers Plains, Qld. 4108, Australia 4

Commonwealth Scientific Industrial Research Organisation (CSIRO), 671 Sneydes Rd.,

Werribee, Vic. 3030, Australia.

5

Current details: Faculty of Veterinary and Agricultural Science, The University of Melbourne, Royal Parade, Parkville, Vic 3010

*Corresponding author: Damian Frank, CSIRO, 11 Julius Ave, North Ryde, NSW 2113. Tel: +61 2 9490 8584 Fax: +61 2 9490 8499 E-mail: [email protected]

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Intramuscular fat and beef flavor

1

Abstract

2

The sensory attributes and flavor chemistry of grilled beef striploins (M. Longissimus

3

lumborum, n=42) varying widely in marbling from commercial production types typical for

4

Southern Australia, were extensively characterized. Striploins from Angus grass-fed

5

yearlings (5.2% - 9.9% intramuscular fat), Angus grain-finished steers (10.2% - 14.9%) and

6

Wagyu grass-fed heifers (7.8% - 17.5%) were evaluated. Inherent differences between

7

samples from grass and grain fed Angus cattle were minimal when the intramuscular fat

8

content was above ~ 5%. Wagyu samples had more intense flavor, higher tenderness and

9

juiciness compared to Angus grass fed samples. Grilled beef flavor, dairy fat and

10

sweetness increased with the marbling level and sourness and astringency decreased.

11

Tenderness and juiciness increased with marbling level and were correlated with Warner-

12

Bratzler peak force measurements. Trained panel sensory differences in flavor

13

corresponded with increases in aroma volatiles and changes non-volatile flavor

14

compounds. Unsaturated fatty acids with potential health benefits (vaccenic, rumenic acids)

15

increased with the level of marbling.

16 17 18 Keywords: Beef, flavor, Wagyu, Angus, marbling, pasture, Warner-Bratzler, olfactometry,

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Intramuscular fat and beef flavor 19

INTRODUCTION

20

Grilled beef flavor arises through a combination of thermally generated aroma volatiles and

21

non-volatile taste components delivered in a matrix of muscle fiber, connective tissue

22

(collagen), warmed-meat juices and partly dissolved fat. The amount of fat within the

23

muscle — the intramuscular fat (IMF) — plays a critical role in the beef eating experience.

24

While a positive relationship between IMF and palatability (tenderness and juiciness) is

25

well-established1-5, its impact on beef flavor is less certain6, although recent studies indicate

26

a positive association.4, 7

27

The amount of IMF within beef muscle is typically assessed as visual marbling on the

28

surface of the meat. In Australia, the Meat Standards Australia (MSA) marbling score

29

(MSA-MB) system is used to score the level of IMF, using a fine visual scale ranging from

30

100 (no visible fat) to a maximum of 1190, in increments of 10 units. The fatty acid

31

composition of IMF is known to be affected by feed, which may in turn affect meat flavor.

32

Previous research has demonstrated distinct grass-fed (pasture) or grain-fed (feedlot,

33

concentrate) flavors in beef.8, 9 Grass-fed beef is the dominant production system used in

34

Australia, although a substantial proportion of cattle are finished on a high energy grain diet.

35

Extensive research has been devoted to understanding the genetics of IMF deposition and

36

marbling.10,

37

eating quality, other breeds such as Angus can also attain high marbling levels, especially

38

on a high nutritional plane.10 In addition, a better understanding of breed-related sensory

39

differences with different levels of IMF would be useful.

40

Sensory “halo-effects” are known to play a confounding role in assessing meat flavor;

41

untrained or naïve consumers tend to rate flavor high, when other attributes such as

42

tenderness and juiciness are also high.4 A primary aim of this research was to objectively

43

evaluate marbling effects on beef flavor using a trained panel to minimize confounding

11

While the Wagyu breed is synonymous with high marbling and excellent



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sensory interactions as well as to identify objective chemical markers that may underpin

45

sensory differences. The impact of animal diet (pasture vs. grain) and breed (Wagyu vs.

46

Angus) on beef flavor, after taking into account differences in the fat content, was also an

47

important question.

48

MATERIALS AND METHODS

49

Chemicals

50

Solvents were chromatography grade and purchased from Merck-Millipore (Bayswater,

51

Australia). The GLC-20 fatty acid methyl ester standard, C7-C30 saturated alkane linear

52

retention index mix, the glucose oxidase assay kit, sodium L-lactate-3-13C, methyl

53

tricosanoate, 1,1,3,3-tetraethoxypropane (>96%) and methyl chloroformate reagents were

54

obtained from Sigma-Aldrich (Castle Hill, Australia). Volatile standard reference compounds

55

of greater than 95% purity were also supplied by Sigma-Aldrich except, 2-methylpropanal,

56

3-methylbutanal,

57

dimethylpyrazine, 2-ethyl-3,5-dimethylpyrazine, trimethylpyrazine, furfural, 2-phenylethanal,

58

2-nonanone, which were supplied by Givaudan (ex-Quest), Baulkham Hills, NSW, Australia.

59

2-ethylhexanol, decanal, benzaldehyde and 4-methylphenol were purchased from Fluka

60

(Darmstadt, Germany). Norvaline, individual amino acids and organic acid standards were

61

greater than 95% purity (Sigma-Aldrich).

2,3-pentanedione,

2-methylpyrazine,

2,6-dimethylpyrazine,

2,3-

62 63

Collection of Beef Samples

64

Animals from three typical “production types” were identified from commercial farms for use

65

in

66

(“WagyuGrass”), Robbins Island, Northwest Tasmania (Hammond Farms) (2), 100% full-

67

blood Angus steers finished on a mixed ration including wheat and potato waste for 150

the

study;

(1)

100%

full-blood Wagyu

(Japanese

Black)

grass-fed

heifers


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days (“AngusGrain”), Tasmanian Feedlot Pty. Ltd., (Perth, Tasmania) and (3) 100% full-

69

blood Angus grass-fed yearlings (“AngusGrass”), Muirhead Enterprises, (Cape Grim,

70

Tasmania). The cattle were slaughtered in December 2012 at the Greenham Tasmania

71

Pty. Ltd. abattoir (Smithton, Tasmania). After overnight chilling, carcasses were graded by

72

meat inspectors and assigned MSA marbling scores (MSA-MB). Pasture-fed Wagyu is a

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relatively unique product compared to traditional grain-finished Wagyu. The latter was not

74

available in Tasmania at the time of sample collection. Meat was purchased at commercial

75

wholesale prices. Replicate carcasses for each production type were selected according to

76

nominal marbling bands — low (n=5), medium (n=4) and high (n=5), within each breed/feed

77

combination, giving a total of n = 42 carcasses. These carcasses were labelled and tracked

78

into the boning room and striploins (M. Longissimus lumborum) were boned from the right

79

side of each carcass. Subcutaneous fat was removed and striploins were wet-aged in

80

vacuum for 28 days in a chiller (1 ± 1 oC), before freezing at –20 oC. Frozen striploins were

81

fabricated into standardized steaks (25 x 25 x 75 mm) using a band saw; steaks were

82

vacuum packed and stored at -20 oC until use.12

83

Carcass and Meat Physicochemical Measurements

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Carcass data were collected as part of routine processing and MSA grading.12-14 These

85

included MSA-MB, hot carcass weight (HCWT), eye muscle area (EMA), dentition, and

86

ossification score, a measure of physiological maturity13. Ultimate pH (upH) and meat color,

87

lightness (L*), redness (a*) and yellowness (b*) were measured approximately 24-hours

88

post-mortem in the chiller according to published protocols.15 Total collagen (TC) and heat

89

soluble collagen (HSC) content in the muscle was determined by measuring the

90

hydroxyproline content in lyophilized muscle (~2 g) expressed as a percentage of wet

91

weight (total) or of the total (heat soluble) fraction.16 HSC samples were defatted with

92

chloroform/methanol and hydrolysate and standards were neutralized with 0.6M NaOH 5 ACS Paragon Plus Environment


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solution prior to the assay. The IMF (% w/w) content in the raw meat samples was

94

estimated using the method described in Thornton.17

95 96

Thiobarbituric acid reactive substances (TBARS) were measured in duplicate raw meat

97

samples (~2 g) according to published methods.15 The residual glycogen content of the

98

frozen muscle subsamples was measured using a rapid assay modification using H2SO4

99

addition.18 Samples were homogenized (1:10 w/v) in 30 mM HCl for 2 x 15 sec bursts,

100

centrifuged (3,000 rpm, 4°C, 10 minutes). Samples were analyzed for total glucosyl units by

101

incubating 50 µL (37°C, 90 minutes) with the addition of 500 µL of hydrolyzing enzyme

102

amyloglucosidase (Sigma-Aldrich, 1:200 dilution in 40 mM acetate buffer, pH 4.8). Total

103

glucosyl units (mg/g) (considered to be glycogen content) was determined by absorbance

104

at 540 nm in duplicate using a glucose assay kit.

105 106

Warner-Bratzler shear (WBS) force provides an objective measure of meat tenderness.19

107

After overnight defrosting at 4˚C, WBS was determined according to established

108

procedures20. Samples were weighed and cooked in plastic bags in a water bath (70˚C, 60

109

min) and cooled prior to measurement. The amount of water lost during cooking— WBS-

110

cook loss — was calculated by mass balance, expressed as a percent of initial weight (%

111

w/w).

112 113

Beef Grilling Protocol

114

Frozen steaks were thawed overnight on plastic trays at 4 °C and grilled at 220 °C on a

115

commercial clamshell grill (Silex, Marrickville, Australia), according to published protocols 12,

116

13

117

type, the lid was closed and samples were grilled to a final internal temperature of 57 oC

. A thermocouple probe was inserted into the middle of the first of five steaks of the same


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(defined as “medium doneness”).21 “Grilling time” was recorded using a stopwatch for each

119

set of five steaks (in seconds). Moisture lost at various steps in grilling and resting was

120

recorded for replicate batches of steaks. The moisture lost during grilling — “grill cook loss”

121

(% w/w), was determined by weight difference using a calibrated balance before and

122

immediately after grilling. “Grill rest loss” (% w/w) was determined after resting grilled meat

123

for 3 minutes under loosely placed aluminum foil and measuring the mass of liquid left in

124

the foil . Grilling data for low (n=10) and high (n=10) IMF samples for each production type

125

(total n=60) were obtained. After resting, steaks were cut into small pieces (~10 g) and

126

immediately placed into a standard wine glass (labelled with unique 3-digit code) and

127

covered with a watch glass, before serving to panelists in individual sensory booths.

128

Sensory Descriptive Analysis

129

Human ethics approval was obtained (CSIRO LR15-2012-C) for the sensory testing.

130

Experienced assessors (nine females and one male, 51± 6 years) participated in five two-

131

hour training sessions conducted over a two week period to generate and define the

132

sensory vocabulary. Published beef lexicons and attributes were considered in the

133

development of the final sensory vocabulary.8, 22, 23 Reference standards were used to help

134

illustrate some attributes (supporting information, Table S1). Assessors were equally

135

exposed to samples representing the experimental design variables. Impressions of meat

136

tenderness and juiciness were given after 3 and 10 chews. Remaining undissolved

137

connective tissue and total number of chews were rated at the point just before swallow.

138

Attributes (except number of chews to swallow) were rated using a 100 mm line scale on a

139

computer screen using the Compusense® five sensory software (Release 4.6, Compusense

140

Inc., Guelph, ON, Canada). Performance was monitored and regular feedback was given

141

until panelists had a clear understanding of all attributes. Sensory descriptive profiling was

142

performed on all samples (n=42) in triplicate over a two week period, hence a total of 30 7 ACS Paragon Plus Environment


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sensory assessments for each attribute and beef sample. Randomization of presentation

144

order was determined using CycDesigN Software (VSN International, Hemel Hempstead,

145

United Kingdom). A total of 32 attributes were generated by the trained panel to measure

146

grilled beef sensory properties, mostly in agreement with those reported by others.3,

147

Panelists removed the cover of the wineglass and first assessed nine odor attributes

148

orthonasally in the headspace; odor impact, grilled beef, livery, metallic, bloody, caramel,

149

barnyard, hay/grainy and fishy. Panelist then placed one piece of grilled meat in their

150

mouth and after two chews assessed nine flavor attributes retronasally; flavor impact,

151

grilled beef, livery, metallic, bloody, dairy-fat, grassy, hay/grainy and fishy and three taste

152

attributes; sour/acidic, sweet and salty. After swallowing, five aftertaste attributes were

153

rated; acidic, metallic, astringency, oily mouth-coating and lingering. The second piece of

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meat was used for rating texture attributes; tenderness and juiciness after three and ten

155

chews, number of chews to swallow and amount of connective tissue before swallowing.

156

Fatty Acid Methyl Ester Analysis

157

Subsamples (~2 g) of raw ground meat (from ~30 g sample) were homogenized in

158

chloroform:methanol (2:1) and left at room temperature for 2 hr. Saline (0.73% NaCl) was

159

added and samples were centrifuged (1000 rpm, 5 min, 25 °C). The organic layer was

160

removed and reduced in volume under vacuum for ~16 hr. One mL of tetrahydrofuran, 5 %

161

H2SO4 in methanol, internal standard (IS) — 2 mg/mL methyl tricosanoate in heptane —

162

were respectively added, the mixture vortexed and then heated at 70 °C for 2 hr. After

163

cooling, heptane (2 mL) and saturated NaCl solution (1 mL) was added and, after mixing,

164

the fatty acid methyl esters (FAMEs) were extracted with heptane (2 x 2 mL). The combined

165

organic extract was washed with NaHCO3 solution (5%, 1 mL). The FAMEs (1 µL, split

166

1:50) were separated using a Supelco SP-2560 capillary column (100 m, 0.25 mm, 0.2 µm)

167

in an Agilent 6890 gas chromatograph. The GC oven was isothermally heated at 180 °C

4, 7, 8


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with helium as carrier (flow rate = 1.2 mL/min) with the injector heated at 250 °C. An FID

169

(250 °C) was used for detection (flow rates for H2, air and N2 were 45, 450 and 45 mL/min,

170

respectively). Identification was made using a standard FAME mix and standard anhydrous

171

milkfat (prepared in house). Reference samples were also analyzed using mass

172

spectrometry (Agilent 5793 mass selective detector) to facilitate identification.

173

replicates were used for each sample and mean values (n=42) used to calculate total

174

amount of each FAME in the total extracted fat (mg/g). Total amount of each lipid in the IMF

175

was calculated for each sample and expressed as amount of FAME (mg) per 100 g serving

176

(raw).

177

Volatile Extraction and Analysis by Gas Chromatography Mass Spectrometry

178

The collection method for volatiles was designed to mimic dynamic ‘in mouth’ volatile

179

release.24 Separate individual replicate freshly grilled steaks were prepared from low (n=6)

180

and high (n=6) IMF levels for each of the three production types (n=36 samples in total).

181

After resting, middle sections of each steaks of the same type were removed and pooled

182

and (60 g) was suspended in Milli-Q water (1:2 ratio, ~37oC) and homogenized to a fine

183

slurry. The meat suspension, with the addition of an internal standard (4-methyl-1-pentanol,

184

40 ng/g) were concentrated onto Tenax-TA traps (60/80 mesh size, 100 mg) for 30 minutes

185

at 37 oC and analyzed by gas chromatography-mass spectrometry (GC-MS) using an

186

Agilent (ex-Varian GC-MS 4000 ion trap system) according to published protocols.24 To

187

facilitate identification, selected samples were also analyzed by methanol chemical

188

ionization (CI) to obtain the mass of the [M+H]+ parent ion, where applicable. Mass spectral

189

matches were conducted with the NIST-Mass Spectral Search database (Version 2.0,

190

2002). Reference standards (St) were used to confirm the identity of most compounds.

191

Integrated area data were normalized to the IS and semi-quantitative data (mg/kg) were

192

estimated.

Two



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Gas Chromatography-Olfactometry

194

Grilled beef volatile Tenax extracts described in the previous section were also

195

simultaneously evaluated by gas chromatography-olfactometry (GC-O) with time intensity

196

(TI) sensory analysis as described previously.24 Six trained assessors evaluated the effluent

197

of each of the six sample types individually; giving a total of 36 sniffs. Odor intensity data

198

were acquired at 1 second intervals by a computer mouse and a 10-point scale using the TI

199

function in the software package SensoMaker® (Version 1.7).25 TI responses had both

200

maximum intensity (height) and duration (width). Integrated area under the curve (AUC)

201

data for each defined odor peak was calculated. The statistical average of AUC values for

202

each odor peak was estimated to obtain an average representative aromagram.

203

Derivatization of Free Amino Acids and Analysis by GC-MS

204

Quantification of free amino acids (FAAs) (except arginine), carnosine and other non-

205

volatile compounds (organic acids and fatty acids) was achieved by methyl chloroformate

206

derivatization and subsequent GC-MS analysis according to published protocols.26, 27 Raw

207

and corresponding grilled low and high IMF samples from each production type were

208

prepared. The purpose of analyzing raw and grilled samples was to measure potential

209

changes in non-volatiles within the surface layer of the meat which may affect the flavor

210

intensity. Slices (~ 4 cm wide) were excised from the middle of two separate steaks and the

211

top surface (~5 mm depth) were reduced into small pieces. A total of 3 animals x 2

212

replicates x 2 marbling levels (low and high) = 12 samples were prepared for each

213

production type (n=36 raw, n=36 grilled). The small pieces of raw or freshly grilled meat (2

214

g) were immediately suspended in ice-cold methanol solution (70%), homogenized,

215

centrifuged and the supernatant was filtered before derivatization. Relative response factors

216

were determined for quantitative ions (m/z) for each analyte and concentrations of FAAs


Page 11 of 39



(mg/100g) were estimated against the internal standard norvaline (100 µg/mL, m/z 130).

218

Lactic acid was quantified against L-lactate-3-13C internal standard isotopomer (1000

219

µg/mL, m/z 46). Chloroformate derivatives (1 µL) were injected at 250 oC (splitless) into the

220

GC-MS (QP-2010-Plus, Shimadzu) and separated on a Sol-Gel Wax column (SGE,

221

Australia, 30 m, 0.25 id, 0.25 µm film) using temperature programming; initial temperature

222

45 oC (held 2 minutes) and then heated at 9 °C/min to 180 °C (held 5min), 40°C/min to

223

220°C (held 5 min). Reference compounds were used to confirm compounds, which were

224

quantified using characteristic ion fragments (m/z). Data for raw and grilled samples from

225

each production type and corresponding IMF data were used in the statistical analysis.

226 227

Statistical Analyses

228

Statistical analyses were performed using GenStat® 16th Edition (VSN International Ltd,

229

Hemel Hempstead, United Kingdom). For statistical purposes, samples were either

230

classified as three “production types” (AngusGrass, AngusGrain or WagyuGrass) or as nine

231

distinct “sample types”, e.g. AngusGrass low marbling (AGL), AngusGrass medium

232

marbling (AGM), AngusGrass high marbling (AGH), and similarly, AngusGrain (AGRNL,

233

AGRNM, AGRNH) and WagyuGrass (WGL, WGM, WGH). Sensory differences were

234

assessed by multivariate analysis of variance (MANOVA) comparing the nine distinct

235

samples, using ‘sample type’ and ‘panelist’ as a fixed effects; different marbling levels were

236

not taken into consideration. A separate MANOVA analysis was conducted using the three

237

‘production types’ and the IMF (for each individual sample) as a covariate term, to correct

238

for differences in marbling level. To ascertain feed effects, AngusGrass and AngusGrain

239

production types were compared by MANOVA using ‘feed’ and ‘panelist’ as fixed factors

240

and IMF as a covariate. Similarly, for breed effects, AngusGrass was compared to

241

AngusGrain using ‘breed’ and ‘panelist’ as fixed factors and IMF as a covariate. Similar



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MANOVA comparisons were made for various replicate chemical data using fixed effects of

243

production type, sample type, feed and breed and the covariate IMF. Mean sensory and

244

chemical data values were used to determine Pearson’s correlation coefficients and

245

subjected to a two-sided test for significance.

246

RESULTS AND DISCUSSION

247

Carcass Characteristics

248

All carcasses were eligible for MSA grading according to specified criteria12,

249

covered a typical range of values (Table 1). In accordance with the experimental design,

250

differences in MSA-MB and IMF were measured between the nominal marbling bands.

251

AngusGrass represented the lower end, AngusGrain the middle, whereas WagyuGrass

252

samples were at the high end of the marbling range. Hot carcass weight (HCWT) and eye

253

muscle area (EMA) were significantly different between production types. The Wagyu breed

254

was generally lower in carcass weight than Angus. EMA generally increased with marbling

255

level. AngusGrain carcasses were heaviest in carcass weight, consistent with being from

256

animals on a higher energy diet during the finishing phase. Dentition and ossification scores

257

indicated that WagyuGrass heifers were older than the AngusGrain steers and AngusGrass

258

yearlings. Other factors being equal, marbling typically increases with maturity; age

259

differences are inevitable across such a broad marbling range and are typical for beef

260

production in Australia.23, 29, 30 The upH was < 5.7 for all carcasses, eliminating high pH dark

261

cutting meat as a potential negative factor affecting meat sensory quality.31,

262

(a* and b* values) indicated typical values and did not vary significantly according to sample

263

type, except for (L*), which was higher in the AngusGrain especially compared to the

264

AngusGrass.15

265

TBARS values were higher in WagyuGrass (p < 0.001); for the WagyGrass, TBARS was

266

positively correlated with IMF (r = 0.62, P < 0.007) (Table 1). There were no feed-related

32

13, 28

and

Meat color


Page 13 of 39



difference in TBARS between the AngusGrain and AngusGrass and a correlation between

268

TBARS and IMF was not found in these samples. The higher TBARS for the WagyuGrass

269

but not the AngusGrass compared to the AngusGrain, may have been due to differences in

270

overall antioxidant status in these samples, e.g. selenium and vitamin E (not measured).33

271

Although no differences were observed in upH, there were significant differences in the

272

muscle glycogen stores at 24-hrs post-mortem. This is expected as usually residual

273

glycogen remains in the muscle after glycolysis ceases post-mortem.34, 35 Average residual

274

glycogen content for AngusGrass, AngusGrain and WagyuGrass was 16.5 mg/g, 11.9 mg/g

275

and 14.42 mg/g, respectively (p = 0.033).

276

Warner-Bratzler Shear Force, Soluble Collagen and Insoluble Collagen

277

As expected, WBS was negatively correlated with MSA-MB (r = -0.66, p < 0.001) and IMF (r

278

= -0.53, p < 0.001). WagyuGrass had lower WBS values compared to AngusGrass (p

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