- Email: [email protected]
Combined effects of gamma irradiation and aging on tenderness and quality of beef from Nellore cattle
Journal Pre-proofs Combined effects of gamma irradiation and aging on tenderness and quality of beef from Nellore cattle Lorena Mendes Rodrigues, Luana Aparecida Sales, Paulo Rogério Fontes, Robledo de Almeida Torres Filho, Monalisa Pereira Dutra Andrade, Alcinéia de Lemos Souza Ramos, Eduardo Mendes Ramos PII: DOI: Reference:
S0308-8146(19)32289-7 https://doi.org/10.1016/j.foodchem.2019.126137 FOCH 126137
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
4 August 2019 16 November 2019 27 December 2019
Please cite this article as: Mendes Rodrigues, L., Aparecida Sales, L., Rogério Fontes, P., de Almeida Torres Filho, R., Pereira Dutra Andrade, M., de Lemos Souza Ramos, A., Mendes Ramos, E., Combined effects of gamma irradiation and aging on tenderness and quality of beef from Nellore cattle, Food Chemistry (2019), doi: https:// doi.org/10.1016/j.foodchem.2019.126137
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Elsevier Ltd. All rights reserved.
1
Combined effects of gamma irradiation and aging on tenderness and quality of beef from Nellore
2
cattle
3 4
Lorena Mendes RODRIGUESa, Luana Aparecida SALESa, Paulo Rogério FONTESb, Robledo de Almeida
5
TORRES FILHOc, Monalisa Pereira Dutra ANDRADEd, Alcinéia de Lemos Souza RAMOSa, Eduardo
6
Mendes RAMOSa*
7 8
a Departamento
9
Gerais, 37200-000, Brasil.
de Ciência dos Alimentos, Universidade Federal de Lavras, P.O. Box 3037, Lavras, Minas
10
b
11
900, Brasil.
12
c Instituto
13
Minas Gerais, 35690-000, Brasil.
14
d Instituto
15
Diamantina, Minas Gerais, 30161-970, Brasil.
Departamento de Tecnologia de Alimentos, Universidade Federal de Viçosa, Viçosa, Minas Gerais, 36570-
de Ciências Exatas e Tecnológicas, Universidade Federal de Viçosa, Campus Florestal, Florestal,
de Ciência e Tecnologia, Universidade Federal dos Vales do Jequitinhonha e Mucuri, Campus JK,
16 17 18
* Corresponding author tel.: +55 35 3829 1403; fax: +55 35 3829 1401; email: [email protected] (E. M. Ramos).
19
1
21
Abstract
22
Combined effects of gamma irradiation (0, 3, 6 and 9 kGy) and aging (1 and 14 days) on quality
23
attributes of vacuum-packaged beef from Nellore cattle were evaluated. The meat water holding capacity was
24
affected by irradiation, increasing (p < 0.05) purge and cooking loss regardless of the dose used. Irradiation
25
negatively affected myofibrillar fragmentation, but samples irradiated at 9 kGy had (p < 0.05) higher soluble
26
collagen and lower shear force values. The meat metmyoglobin reducing activity was reduced (p < 0.05) by
27
the irradiation process, inducing the metmyoglobin accumulation with increasing dose applied. Samples
28
irradiated at 9 kGy presented (p < 0.05) higher lipid oxidation and lower oxymyoglobin proportion and color
29
redness and chroma values. It was concluded that irradiation at 9 kGy combined with aging can be used as an
30
effective tool for improving the tenderness of Nellore beef, but resulted in a discoloration of the beef.
31
Keywords: Shear force; fragmentation index; collagen; lipid oxidation; color.
32 33
1. Introduction
34 35
According to the Brazilian Association of Meat Exporting Industries (ABIEC), Brazil exported 1.64
36
million tons of beef in 2018, consolidating its position as the world's largest exporter, but over 80% of
37
Brazilian bovine herd consists of zebu (Bos indicus) cattle, whit Nellore maintaining 90% of this share.
38
Although the wet aging system (vacuum packed beef) is used by the meat industry to improve tenderness,
39
zebu beef is still considered less tender than that of European (Bos taurus) beef (Aroeira et al., 2016). Since,
40
tenderness stands out as a quality attribute of the beef, being considered the most influential sensory
41
characteristic on acceptance of meat by consumers (Delgado et al., 2006), improving this quality attribute
42
remains a challenge for the Brazilian industry.
43
Among studies aiming at finding ways to improve meat tenderness, the gamma irradiation process
44
seems to have great potential, as besides being considered an excellent method for microbial control in foods,
45
it has been reported that relative low- to medium doses of 5 to 10 kGy are efficient in reducing shear force in
46
non-aged bovine (Yook, Lee, Lee, Kim, Song, & Byun, 2001), lamb and buffalo (Kanatt, Chawla, & Sharma,
47
2015) meat. However, only Kanatt et al. (2015) evaluated the effects of gamma irradiation after rigor mortis
48
process and yet they did not evaluate the effects after aging. Moreover, Rowe, Maddock, Lonergan, and Huff2
49
Lonergan (2004) reported that beef steaks irradiated at relatively low dose (6.4 kGy) of electron beam and
50
aged by 14-days showed significantly higher shear force values than non-irradiated steaks, while, more
51
recently, Kim, Yong, Nam, Jung, Yim, and Jo (2018) observed no adverse effect on 14-days aged beef
52
tenderness due to the pre-rigor irradiation (5 kGy) by electron beam or X-ray. Therefore, the combined effects
53
of aging and irradiation on meat proteolysis and tenderness are still conflicting and scarce.
54
Despite possible effects on tenderness and its usefulness to ensure the microbial safety and extend shelf
55
life of meat without loss of nutritional quality (WHO, 1999), the irradiation technology can influence lipid
56
oxidation, color changes, and off-odor of meat, which may generate negative consumer responses (Brewer,
57
2004; Millar, Moss, & Stevenson, 2000). Of these changes, the formation of undesirable or unexpected colors
58
from the consumer's viewpoint are critical, as meat color is the most important attribute that consumers use as
59
purchase criterion (Aroeira et al., 2017), and are related to the content and chemical form of heme pigments,
60
muscle condition (pH, reducing equivalents, etc.) and to applied irradiation dose. In this context, the
61
irradiation of vacuum-packed beef is interesting to overcome or reduce the lipid oxidation and color
62
discoloration, since is the presence of oxygen during irradiation that often results in unacceptable radiolytic
63
changes (Brewer, 2004). The effects of low- to medium dose irradiation on aged beef quality are not well
64
defined, since most research has been conducted on ground beef, which reacts differently to irradiation in
65
terms of lipid oxidation and color changes when compared to intact beef.
66
Faced with the expectation of improving the tenderness of zebu animal’s meat and, consequently,
67
improving the quality of Brazilian beef, new techniques are being increasingly evaluated. In this context, the
68
interaction of irradiation and aging on beef tenderness seems to require further investigation to maximize
69
commercial application, especially in meat from Nellore cattle, where very limited data are currently
70
available. Moreover, any process that negatively affects the color of fresh beef can lead to lower consumer
71
appeal and marketability. Thus, the aim of this study was to verify the gamma irradiation as a novel technique
72
to improve beef tenderness of Nellore animal’s, evaluating the application of different doses and its effects on
73
the parameters associated to the meat tenderness and color before and after the aging process.
74 75
2. Material and methods
76 3
77
2.1 Collection and preparation of samples
78 79
Samples of Longissimus lumborum muscle (LL), from eight Nellore bovines, with an average age of 30
80
months old and similar livestock and slaughter system, were obtained 48 hours postmortem directly from a
81
slaughterhouse (Plena Alimentos Ltda.) with Federal Inspection (SIF) in the State of Minas Gerais, Brazil.
82
Of each LL muscles, eight pieces approximately 5.0 cm thick (beef section) were obtained, individually
83
weighed, identified, vacuum-packed (90 µm-thick nylon-polyethylene, with an oxygen transmission rate of
84
30–60 cm3/m2/day/atm) and randomly distributed in duplicate in the four radiation doses treatments: control
85
(non-irradiated), 3, 6 and 9 kGy. The beef sections were placed in coolers and were subjected to the ionizing
86
radiation in the Gamma Radiator IR-214 (MDS, Nordion; cobalt-60 source and 1925.8 Gy/h rate) at the
87
Nuclear Technology Development Center of the National Commission of Nuclear Energy (CDTN/CNEN),
88
Belo Horizonte, Minas Gerais, Brazil. Non-irradiated samples (control) were kept in coolers for time periods
89
like the irradiated samples. The whole irradiation process (up to 9 kGy dose) lasted 4.5 hours. After
90
irradiation, the coolers were taken to Laboratory of Meat and Meat Products Technology (LabCarnes), at the
91
Federal University of Lavras (UFLA), and the beef sections of each irradiation dose were randomly
92
distributed into two aging time (1 and 14 days) conducted in a climatic chamber (model EL202; EletroLab
93
Inc., São Paulo, Brazil) at 1.0 °C.
94 95
2.2 pH and soluble proteins
96 97 98
The pH was measured by an insertion electrode in three different locations (triplicate), using a portable pH meter (model HI 99163; Hanna Instruments, Woonsocket, RI, USA).
99
Extractable sarcoplasmic proteins (in potassium phosphate buffer 25 mM; pH 7.2) and total proteins (in
100
potassium iodate 1.1M solution in potassium phosphate buffer 100 mM; pH 7.2) were determined as described
101
by Joo, Kauffman, Kim, and Park (1999). The protein concentration of the extracts was determined by the
102
Biuret method, being expressed in mg/g of beef. The concentration of myofibrillar proteins was estimated by
103
the difference between the concentration of total and sarcoplasmic proteins.
104 4
105
2.3 Water losses
106 107
At each aging time (1 and 14 days), the beef sections have been removed from the packaging, dried in
108
paper towels and weighed again to determine the purge (measured by mass difference between the samples
109
before and after packing), being expressed as a percentage.
110
The water holding capacity (WHC) was measured by the method of pressure in filter paper (MPPF)
111
described by Aroeira et al. (2016). Samples of approximately 300 mg of meat were placed on previously dry
112
filter paper, and the set was pressed for 5 min with a 5 kg weight. After pressing, the areas in the filter paper
113
delimited by the pressed meat (Ap) and by the exudate liquid (Ae) were obtained using ImageJ software ®
114
1.42 q (National Institute of Health, USA), and the WHC expressed as the ratio Ap/Ae.
115
One steak of 2.54 cm thickness was obtained from each beef section and an inner standard-sized
116
rectangular sample (8.0 x 4.0 x 2.5 cm) removed, weighed, vacuum-packed and cooked in water bath at 80 °C
117
until internal temperature of 71 °C (monitored by a digital thermometer inserted in the steak center). After
118
cooking, the samples were cooled (4 °C) for 3 h, removed from the vacuum-packed bags, dried with a paper
119
towel and weighed again. The cooking loss was determined by the difference between the weighing before
120
and after cooking, being expressed as a percentage.
121 122
2.4 Collagen content
123 124
The soluble and insoluble collagen fractions were separated after heating at 77 °C for 70 min and the
125
collagen content was quantified by determining the hydroxyproline amino acid (Bergman & Loxley, 1963), as
126
described by Ramos and Gomide (2017). The hydroxyproline content was obtained using an analytical curve,
127
and the collagen content (mg/g) was calculated using a factor of 7.52 for soluble and 7.25 for insoluble
128
fractions (Cross et al., 1973). The total collagen content was obtained from the sum of the soluble and
129
insoluble fractions.
130 131
2.5 Fragmentation index
132 5
133
The degree of myofibrillar fragmentation in samples was assessed by the fragmentation index (FI)
134
method described by Aroeira et al. (2016), with minor modifications. All samples in the stipulated aging time
135
were previously frozen and stored (-18 °C) until analyzed and the sample was homogenized in extraction
136
solution at a ratio of 1:5 (w / v) instead of 1:10 (w / v) originally used. After vacuum filtration (Vacuum pump
137
NOF-650, New Pump, Brazil) of the homogenate using a 250 µm-pores nylon screen, the residue weight
138
(RW) was determined and the FI was expressed as 100 × RW.
139 140
2.6 Shear force
141 142
Shear force was determined according to method Warner-Bratzler square Shear Force (WBsSF)
143
described by Silva, Torres Filho, Cazedey, Fontes, Ramos, and Ramos (2015), using cooked samples from the
144
cooking loss measurement (section 2.3). Of each standard-sized rectangular sample cooked, six cuboids (1.0
145
cm x 1.0 cm square cross-section) cores were obtained in the muscle fiber direction and sheared transversely
146
(across the predominant muscle fiber orientation) at 200 mm/min by a Warner-Bratzler blade coupled to a TA.
147
XTplus texturometer (Stable Micro Systems Ltd., Godalming, Surrey, UK). Maximum force (N) was
148
measured and the average value of each beef was used in statistical analysis.
149 150
2.7 Lipid oxidation
151 152
The oxidative stability of meat was evaluated by the number of 2-thiobarbituric acid reactive substances
153
(TBARS index), measured according to the methodology proposed by Raharjo, Sofos, and Schmidt (1992),
154
with modifications described by Cardoso, Dutra, Fontes, Ramos, Gomide, and Ramos (2016). The TBARS
155
values were reported in milligrams of malonaldehyde (MDA) per kilogram of the sample (mg MDA/kg) by
156
means of a standard calibration curve using 1,1,3,3-tetraethoxypropane (TEP).
157 158
2.8 Oxygen consumption rate and metmyoglobin reducing activity
159
6
160
The oxygen consumption rate (OCR) and the metmyoglobin reducing activity (MRA) were measured
161
by determination of the myoglobin redox forms, using a CM-700 spectrophotometer (Konica Minolta;
162
Sensing Inc., Osaka, Japan) with an 8-mm aperture size, 10º observer angle and in the specular component
163
included (SCI) mode. Each index was measured in two (replicates) pieces (5 x 5 x 1.25 cm) of each sample, as
164
described by Cardoso et al. (2019).
165
The OCR was measured as the conversion of the surface oxymyoglobin (OMb) to deoxymyoglobin
166
(DMb) after 10 min under vacuum (Madhavi & Carpenter, 1993), being expressed as the percentage of time-
167
zero surface OMb consumed. The metmyoglobin reducing activity (MRA) was determined by the nitric oxide
168
metmyoglobin reducing activity (NORA) method (Watts, Kendrick, Zipser, Hutchins, & Saleh, 1966). The
169
proportion of surface metmyoglobin (MMb) immediately formed after oxidation with 0.3% sodium nitrite
170
solution was recorded as initial MMb formation (IMF) and the reducing ability was reported as the percentage
171
decrease in surface MMb concentration after an incubation period of 2 hours at ambient temperature
172
(approximately 20 °C).
173
The relative concentration of the pigments heme OMb and MMb were estimated from the surface
174
reflectance data (recorded from 400 to 710, in a 10-nm interval), using the mathematical method developed by
175
Krzywicki (1979).
176 177
2.9 Myoglobin redox forms and color evaluation
178 179
The myoglobin redox forms and the CIE color indexes of beef samples were determined using a CM-
180
700 spectrophotometric colorimeter (Kônica Minolta Sensing Inc., Osaka, Japan), with 8 mm aperture,
181
illuminant A, 10º observer angle and with both specular component included (SCI) and excluded (SCE)
182
modes. The color measurements were taken on the surface of a one 2.54-cm thickness steak, obtained from the
183
beef section, after being exposed 30 min in atmospheric air for blooming. Meat surface reflectance data (from
184
400 to 710, in a 10-nm interval) were recorded using an average of five consecutive measurements
185
representing the entire surface of each sample.
186
The relative content of the pigments heme (OMb, DMb, and MMb) were estimated by the Krzywicki
187
(1979) mathematical method. Based on the readings taken on the SCE mode, the lightness (L*), redness (a*) 7
188
and yellowness (b*) values were obtained. Chroma (C*) and hue angle (h) were also determined as: C* = (a*2
189
+ b*2)1/2; and h = tan-1 (b*/a*).
190 191
2.10 Statistical analysis
192 193
The statistical analyses were performed in SAS software, version 9.2 (Statistical Analysis System-SAS
194
Institute Inc., Cary, NC, USA), at a significance level of 5%, using SAS GLM procedure. The experiment was
195
conducted in a randomized block design, with the blocks consisted of different animals (8 replications), in a
196
split-plot scheme, with the four treatments (0, 3, 6, and 9 kGy) in the whole plot and the aging time (1 and 14
197
days) in the split-plot. The main effects and their interaction on the meat quality indicators were determined
198
by analysis of variance (ANOVA) and, when necessary, means were separated by Tukey's test.
199 200
3. Results and discussion
201 202
3.1 pH and extractable proteins
203 204 205
The effects of gamma irradiation doses and aging time on pH and extractable proteins are described in the Table 1. Interaction effects (treatment x aging time) were not observed (p > 0.05) for any characteristic.
206
For pH values, no significant effect (p > 0.05) was observed for gamma irradiation doses or aging time
207
factors (mean value of 5.60 ± 0.12) and was consistent with the mean values observed for beef. The absence
208
of irradiation effect on the pH values of the beef steaks agrees with the observation of other authors (Kanatt et
209
al., 2015; Luchsinger et al., 1997). Moreover, the absence of effect by aging time could be due to the short
210
period of aging, since Aroeira et al. (2016) also did not observe a significant difference of pH in bovine LL
211
muscles after 14-days aging.
212
As for the extractable proteins, a significant effect (p < 0.05) was observed only of aging time on
213
sarcoplasmic and myofibrillar proteins. The amount of soluble protein extracted from meat can give an
214
indication of the relative level of distortion that can occur during a process or treatment (Kanatt et al., 2015).
215
The solubility of the sarcoplasmic proteins decreased with aging time, while the myofibrillar proteins 8
216
solubility increased (Table 1). Sarcoplasmic proteins are known to be more susceptible to early denaturation,
217
due to the rapid postmortem glycolysis, although the effects of postmortem aging on the protein’s solubility
218
are still not clear. However, aging-related changes in the extraction of proteins are probably due to changes in
219
protein conformation, molecular size and intra and inter-molecular bonds that occur with postmortem aging
220
(Bowker, Fahrenholz, Paroczay, & Solomon, 2008). These authors also observed that postmortem aging (up to
221
8 days) decreased solubility of sarcoplasmic proteins and increased solubility of myofibrillar proteins in
222
bovine striploins.
223
The irradiation process did not affect (p > 0.05) the solubility of meat proteins, which does not agree
224
with the observations reported by Kanatt et al. (2015). These authors observed an increase for total and
225
myofibrillar extractable protein from buffalo meat irradiated with 2.5 to 10 kGy. Lee, Yook, Lee, Kim, Kim,
226
and Byun (2000) reported that myosin subunits were structurally modified, increasing its solubility when
227
higher doses of irradiation were applied. According to these authors, irradiation can affect the solubility of
228
proteins through the formation of free radicals, generated due to the radiolysis of the water molecule,
229
catalyzing reactions like: deamination; decarboxylation; reduction of disulfide bonds; oxidation of sulfhydryl
230
groups; hydrolysis of peptide bonds; and changes in the valence of the metal ions of enzymes. However,
231
changes in the proteins do not depend only on their structure and state, but also on the conditions of the
232
radiation process, like dose and rate applied, temperature, and absence or presence of oxygen.
233 234
3.2 Water losses
235 236
The effects of gamma irradiation dose and aging time on water losses values of bovine LL muscles are
237
described in the Table 1. As observed for pH and extractable proteins, interaction effects were not observed (p
238
> 0.05) for any of the characteristics.
239
The irradiation process increased (p < 0.05) the purge values, regardless of the applied dose. This is
240
possibly due to a denaturation of beef proteins caused by the free radicals generated by the radiolysis of the
241
water molecule (Zabielski, Kijowski, Fiszer, & Niewiarowicz, 1984), although an irradiation effect was not
242
observed on the proteins solubility. Higher purge (p < 0.05) values were also observed in the samples with
243
longer aging time (14 days) and can be due to the negative pressure of the vacuum packaging, that induces 9
244
progressive drainage of the water present in the extracellular spaces through the drip channels formed during
245
aging (Aroeira et al., 2016; Huff-Lonergan & Lonergan, 2005).
246
The meat water holding capacity (WHC), measured by the filter paper pressure method (FPPM), was
247
affected (p < 0.05) only by the gamma irradiation doses. Overall, the radiation process reduced the FPPM
248
from values of 0.30 in the non-irradiated samples to 0.25 in irradiated ones. This may indicate a negative
249
effect on proteolysis, which would reduce the degradation of cytoskeletal proteins and, consequently, the
250
WHC. The degradation of the cytoskeletal proteins results in increased muscle fiber diameter, thereby
251
increasing its ability to retain the water expelled by the myofibrils (Huff-Lonergan & Lonergan, 2005). Kanatt
252
et al. (2015), measuring the WHC (by centrifugation of minced meat in a salt solution) of buffalo meats also
253
observed a reduction on WHC values with higher irradiation doses. However, these authors observed that the
254
WHC loss was dose-dependent, being, probably, due the rupture of muscle fibers membranes and, or
255
denaturation of meat protein induced by the radiation process.
256
The absence of effect by aging time on the FPPM could also be due to the short period of aging,
257
especially in Nellore meats. Aroeira et al. (2016) did not observe a statistical difference of FPPM in bovine
258
LL muscles after 14-days aging, reporting significative higher values only after 21 days of storage.
259
Unlike observed for the WHC, the cooking loss was affected (p < 0.05) by gamma irradiation and by
260
aging time individually, as described in the Table 1. The irradiated samples, regardless of the dose applied,
261
presented greater values (27.83%) of cooking loss than non-irradiated ones (25.17%). This is possibly due to
262
the effects of the free radicals generated by the radiolysis of the water molecule, as discussed for purge.
263
Kanatt et al. (2015), evaluating the effect of different irradiation doses (up to 10 kGy) in buffalo, chicken and
264
lamb meat also observed that the irradiation increased sample's cooking losses. Relative to aging effects on
265
cooking losses, the results reported in the literature are conflicting; while some studies reported an increase in
266
the cooking loss values (Aroeira et al., 2016; Boakye & Mittal, 1993), other studies reported no aging effect
267
(Vieira, Diaz, Martínez, & García-Cachán, 2009; Wheeler, Miller, Savell, & Cross, 1990).
268 269
3.3 Collagen content
270
10
271
The effects of gamma irradiation and aging on collagen content of bovine LL muscles are described in
272
the Table 2. The total collagen content and its insoluble fraction, including the percentage of soluble collagen,
273
were affected (p < 0.05) only by the irradiation process. Irradiation doses of 3 and 6 kGy resulted in greater
274
proportions of the insoluble collagen fraction, while samples irradiated at a 9 kGy doses did not differ from
275
the non-irradiated samples. Moreover, irradiation doses above 3 KGy resulted in greater amounts of the
276
soluble collagen fraction. Kanatt et al. (2015) did not observe an effect of radiation process on collagen
277
content in the three types of meat (chicken, lamb and buffalo) evaluated. The lack of effect of aging times (up
278
to 14 days) on the collagen fractions was also reported by Koohmaraie, Seidemann, Schollmeyer, Dutson, and
279
Crouse (1987) for bovine Longissimus muscle.
280
However, for the soluble collagen percentage, samples irradiated at 9 kGy had higher (p < 0.05) values
281
than non-irradiated samples. Bailey and Rhodes (1964) reported that the radiation process increase the
282
collagen molecule solubility due to rupture of the molecule's peptide bonds by direct absorption of radiation
283
energy, forming low molecular weight fragments which are, consequently, more soluble. Kanatt et al. (2015)
284
observed an increase in collagen solubility from 3.58% in non-irradiated to 6.02% in irradiated (at 10 kGy)
285
lamb meat, while 48% increasing in solubility was reported when buffalo meat was irradiated at same dose.
286 287
3.4 Fragmentation and shear force values
288 289
A was observed a significant effect (p < 0.05) of irradiation and aging time on the degree of
290
myofibrillar fragmentation (Table 2), measured by the fragmentation index (FI) method. Greater
291
fragmentation (lower FI values) of meat structure was observed in samples after 14 days of aging, which are
292
expected since the proteolysis of key myofibrillar proteins is the principal reason for improvement in meat
293
tenderness during post mortem storage (Koohmaraie et al., 1987).
294
Overall, irradiation resulted in higher FI values than the non-irradiated samples, indicating that the
295
gamma radiation negatively affected the meat fragmentation. This could be due to a reduction in the
296
proteolytic activity of the enzymes during ageing . According to Brewer (2004), the formation of free radicals
297
by radiation process creates highly oxidizing conditions, which can affect the meat's endogenous enzymes.
298
Rowe et al. (2004) reported that the calpains have an oxidizable cysteine residue in their active site, requiring 11
299
reducing conditions to be activated. These authors observed that the irradiation of meat with 6.4 kGy doses
300
decreased the proteolytic action of calpain I and reduced the rate of inactivation of its inhibitor, calpastatin,
301
within a period of 14 days of aging. These changes are consistent with less fragmentation observed in the
302
irradiated samples.
303
However, samples irradiated with higher doses (6 and 9 kGy) did not differ from the non-irradiated
304
samples. Some authors (Lee et al., 2000; Yook et al., 2001; Yoon, 2003) suggested that the irradiation process
305
can induce physical rupture of myofibrils directly by gamma rays (by breaking the actomyosin) or by
306
denaturation of structural proteins such as desmin. Thus, the inhibitory effect on the proteases could be
307
counterbalanced by the fragmentation arising from higher doses of irradiation.
308
For the shear force (WBsSF) values, both isolated effects (irradiation and aging time) were also
309
significant (p < 0.05). As expected, aging improved the samples tenderness. Moreover, lower shear force was
310
observed in samples irradiated at 9 kGy, which agrees with the observation of some authors that the radiation
311
process increased meat tenderness. Kanatt et al. (2015) reported lower shear force values in chicken
312
(Pectoralis major), lamb (Biceps femoris) and buffalo (Biceps femoris) meat with increasing irradiation dose
313
(2.5, 5 and 10 kGy) and this tenderization was attributed to increased collagen solubility. This is consistent
314
with the highest values of soluble collagen content observed in samples irradiated with 9 kGy in this
315
experiment.
316
In the scientific literature, however, the effects of relative low dose irradiation on meat tenderness are
317
still contradictory. As Kanatt et al. (2015), Yook et al. (2001) reported a significant reduction in shear force of
318
bovine Sternomandibularis muscle irradiated pre-rigor at low doses (3 and 5 kGy), being attributed by these
319
authors to a physical disruption of the myofibrils and, or, protein denaturation caused by gamma radiation.
320
This was not supported by the data of this experiment, since lower shear force seems not to be related to the
321
differences in protein solubility or myofibrillar fragmentation. Otherwise, Yoon (2003), evaluating the effect
322
of gamma irradiation (0.5 to 5 kGy) on tenderness of refrigerated chicken breasts, reported that the irradiated
323
samples had higher shear force (11.8 kgf) than the non-irradiated ones (6.81 kgf). These differences could be
324
due to the differences in muscle type and species, or even the moment of irradiation (pre- or post-rigor), and
325
it’s also observed in experiment with different irradiation source. Rowe et al. (2004) reported that beef steaks
326
irradiated at relatively low dose (6.4 kGy) of electron beam and aged by 14 days showed significantly higher 12
327
shear force values than non-irradiated steaks, while Kim et al. (2018) observed no adverse effect on 14-days
328
aged beef tenderness due to the pre-rigor irradiation (5 kGy) by electron beam or X-ray. Luchsinger et al.
329
(1997) also reported that shear forces 14-days aged beef were not influenced by X-ray irradiation up to 3.5
330
kGy.
331 332
3.5 Lipid oxidation
333 334
The effect of irradiation on lipid oxidation of beef during aging was monitored by the TBARS index
335
and was affected (p < 0.05) only by the gamma radiation treatments (Table 3). Beef irradiation at doses up to
336
6 kGy did not affect (p > 0.05) the TBARS values, but when 9 kGy were applied, a significant increase was
337
observed. This is consistent with the observation that ionizing radiation induces lipid oxidation by the
338
hydroxyl radicals generated in water radiolysis, which is dose-dependent (Brewer, 2004). Nevertheless, a high
339
dose of irradiation was required to induce lipid oxidation. This could be due to the fact that packaging (aerobic
340
vs vacuum) appears to have a greater effect on meat quality than does the process of irradiation alone.
341
Differences between irradiated and nonirradiated meat products occurred primarily when these products were
342
irradiated in air. The oxygen present during or after irradiation combine with free radicals generated to form
343
hydroperoxides, accelerating lipid oxidation in irradiated meat (Ahn, Olson, Jo, Chen, Wu, & Lee, 1998;
344
Luchsinger et al., 1997).
345
The TBARS results observed in this experiment agree with the observation of other authors for intact
346
muscles (Kim, Nam, & Ahn, 2002b; Luchsinger et al., 1997), who reported that TBARS numbers of 7- and
347
14-aged vacuum-packaged beefs steaks were not influenced by electron beam (3.0 kGy) or X-ray (2.0 and 3.5
348
kGy) when irradiated at relative low-dose.
349 350
3.5 Oxygen consumption rate and metmyoglobin reducing activity
351 352
The effects of gamma irradiation and aging on inherent metmyoglobin reducing capacity (MRA) and
353
oxygen consumption rates (OCR) of bovine LL muscles are described in the Table 3. These biochemical
13
354
characteristics have been implicated as important determinants of muscle color stability and both were
355
affected (p < 0.05) only by gamma irradiation doses.
356
The MRA was measured by the muscle-resistance to induce metmyoglobin (MMb) formation in the
357
presence of an oxidizing agent, in this case sodium nitrite. Therefore, the initial amount of MMb (IMF)
358
formed by oxidation in sodium nitrite solution and the percentage decrease in surface MMb concentration
359
during the incubation period (NORA) were related to the sample MRA. By the NORA, the MRA decreased (p
360
< 0.05) with irradiation, regardless of the dose applied, while the reducing capacity of the meat by IMF was
361
not affected (p > 0.05) by the application of up to 3 kGy.
362
The loss of MRA by irradiation could be due to the depletion and/or degradation of substrates and
363
cofactors, such as NADH, essential to the muscle reducing system (O'Keeffe & Hood, 1982). Kim, Keeton,
364
Smith, Maxim, Yang, and Savell (2009) reported a decreased in the NADH concentration of beef steaks when
365
irradiated by 2.5 kGy dose, being hypothesized as probably due to oxidation. An effect of an oxidation
366
environment is compatible with our results for lipid oxidation, although the TBARS values were only
367
significant at high irradiation doses.
368
Furthermore, the loss of MRA could be due to the loss of mitochondrial structural integrity and
369
functionality. It was suggested that mitochondria are involved in MMb reduction by regenerating the cofactor
370
NADH, by endogenous enzymes or by reversing electron transport, and to promote an anaerobic environment,
371
by oxygen consumption, that favors MMb reduction (Sammel, Hunt, Kropf, Hachmeister, & Johnson, 2002).
372
This is also consistent with the reduction (p < 0.05) in the OCR values observed in this experiment when
373
irradiation doses greater than 3 kGy were applied and could be due to the oxidative conditions generated by
374
higher irradiation doses. According to O'Keeffe and Hood (1982), besides degradation of enzymes involved in
375
mitochondrial respiration, the OCR also decreases due to depletion of substrates and coenzymes.
376 377
3.6 Myoglobin redox forms and CIE color
378 379 380
The effects of gamma irradiation and aging on the myoglobin chemical forms and CIE color of bovine LL muscles are described in the Tables 4 and 5.
14
381
For the myoglobin redox forms, both deoxymyoglobin (DMb) and oxymyoglobin (OMb) were affected
382
(p < 0.05) by the interaction of irradiation doses and aging, while metmyoglobin (MMb) was affected (p <
383
0.05) by these factors alone. Overall, the irradiation doses affected the myoglobin proportions in the samples.
384
At 1-day of aging, only a reduction in the OMb content was observed in samples irradiated by 9 kGy, but after
385
14-days of aging a simultaneous reduction in OMb and increase in DMb was observed as higher irradiation
386
doses were applied (Fig. 1). The MMb content also increased with higher doses of irradiation (Table 4). These
387
changes indicate the oxidation of the heme pigments during the irradiation process and are related to the
388
effects observed on the lipid oxidation and mainly on the MRA and OCR.
389
Ionizing radiation is a well-known pro-oxidant towards lipids and pigments. The highly reactive
390
substances, such as hydroxyl radicals, produced by water radiolysis during ionizing radiation can oxidize
391
myoglobin directly or they can oxidize lipids producing lipid radicals which subsequently oxidize myoglobin
392
(Brewer, 2004; Millar et al., 2000). Regarding MRA and OCR, as described above, meat color stability is
393
dependent on the residual enzymic activity in meat which, directly or indirectly, controls myoglobin
394
oxygenation, oxidation and reduction. The oxygen consumption plays a significant role in metmyoglobin
395
formation initially, but as oxygen consumption decreases, reducing activity becomes the predominant factor in
396
maintaining stability (King, Shackelford, Rodriguez, & Wheeler, 2011). Therefore, the reduction in the MRA
397
values explains the observed increase in MMb values with higher irradiation doses.
398
For the aging effects, higher OMb content was observed in non-irradiated samples aged by 14-days than
399
in 1-day. This OMb increase during aging could be explained by a reduction in OCR, which increase the beef
400
oxygen penetration during blooming, inducing a thicker layer of oxymyoglobin form (O'Keeffe & Hood,
401
1982). However, the OMb content reduced with the application of higher doses of irradiation, so that the
402
difference between aging also decreased. Moreover, an accumulation of MMb was also observed during
403
aging, which can be attributed to the loss of meat reduction capacity (MacDougall, 1982), although no
404
significant effects of aging was observed for MRA values.
405
Despite the observed changes in the myoglobin redox forms, irradiation doses affected (p < 0.05) only
406
the redness (a*) and chroma (C*, saturation index) values of the CIE color indices (Table 5). Moreover, the a*
407
and C* values had a similar behavior, decreasing (p < 0.05) when samples were irradiated at 9 kGy. Color is a
408
three-dimensional attribute being better described by its hue (h), lightness (L*) and saturation properties 15
409
(Ramos & Gomide, 2017), but although redness is a chromacity coordinate that, together with yellowness
410
(b*), are used to calculate hue and chroma, Holman, van de Ven, Mao, Coombs, and Hopkins (2017) reported
411
that a* value provided the most simple and robust prediction of beef color acceptability. Chroma represents
412
the color intensity, describing how vivid or dull the color is, and is a good indicator of the oxygenation of
413
meat recently exposed to air (Ramos & Gomide, 2017). Therefore, the a* and C* values reduction agrees with
414
the observed reduction in OMb content (and MMb increase) with higher irradiation doses (Table 4) and could
415
be attributed to the increase on the lipid oxidation and reduction on the MRA and OCR due to irradiation, as
416
previously discussed.
417
The results reported in the literature for the effects of irradiation on the color indices of beef steaks (not
418
ground) are variable. Luchsinger et al. (1997) reported that X-ray dose level (up to 3.5 kGy) did not affect CIE
419
colors indices in 14-days vacuum-packaged aged beef (L. lumborum), which corresponds to that observed in
420
this experiment for this applied dose range. Similarly, although working with pre-rigor meat, Kim et al. (2018)
421
showed that 5 kGy of electron beam (EB) and X-ray irradiation had no effect on the overall color (CIE
422
L*a*b*) of beef (Semimembranosus) after 14 days of storage at 4 °C, although a decrease in L* and a* values
423
were detected at day 0 in irradiated samples when compared to non-irradiated samples. Other works, however,
424
report different results. Nanke, Sebranek, and Olson (1999) reported that EB irradiation dose had no
425
significant effects on beef (L. dorsi) L*, b* and h values, but the a* and C* values decreased as irradiation
426
dose levels increased from 0 kGy to 4.5 kGy and increased as irradiation dose increased from 4.5 kGy to 10.5
427
kGy. These authors concluded that significant decreases in redness and increases in brown pigments would
428
develop at dose levels as low as 1.5 kGy in vacuum-packaged beef. Kim, Nam, and Ahn (2002a) also did not
429
observe a 3 kGy dose (EB) effect on beef (L. dorsi) L* and b* values after 7-days aging, but irradiated
430
samples had lower a* values than non-irradiated ones.
431
Despite the differences regarding the type of muscle (Longissimus or Semimembranosus), irradiation
432
type/source (electron beam, X-rays or gamma) and moment of irradiation (pre- and post-rigor), data from the
433
literature and from this experiment seem to point to an effect of irradiation on color intensity of vacuum-
434
packaged beef only when doses as high as 6 kGy are applied.
435
Regarding the aging effects, all CIE color indexes were affected by aging time (Table 5), with 14-aged
436
samples were lighter (higher L* values) and with a red hue more yellowish (higher h values) and intense 16
437
(higher C* values). These changes are consistent with those observed in Nellore beef after 14-days aging by
438
Aroeira et al. (2016).
439 440
4. Conclusion
441 442
The results of this current study found that the gamma radiation process adversely affected the Nellore
443
beef's shear force when doses of 3 or 6 kGy were applied, most often by reduction in proteolysis during aging.
444
However, higher doses of irradiation (9 kGy) increase the collagen solubility, reducing the samples shear
445
force. Although these results suggest that the application of gamma irradiation doses as high as 9 kGy
446
combined to aging can be used as an effective tool to improve tenderness of vacuum-packaged Nellore beef,
447
this radiation level also implied in some change in the meat color. Overall, samples irradiated at 9 kGy
448
exhibited significant surface discoloration, with higher proportion of metmyoglobin. However, it remains to
449
be known if these differences are perceptible by the consumers.
450 451
5. Acknowledgements
452 453
The authors would like to thank the Nuclear Technology Development Center (CDTN/CNEN), in the
454
person of researcher Marcio Tadeu Teixeira, for the irradiation samples, to the National Council for Scientific
455
and Technological Development (CNPq; 430206 / 2016-0) and the Minas Gerais State Research Support
456
Foundation (FAPEMIG; CVZ APQ-02015-15) for their financial support. Also, they thanks to the CNPq and
457
the Higher Education Personnel Improvement Coordination (CAPES) for the scholarship granting to the first
458
(master's degree/CNPq), second (master's degree/CNPq) and third (post-doctoral PNPD/CAPES) authors.
459 460
References
461 462
Ahn, D. U., Olson, D. G., Jo, C., Chen, X., Wu, C., & Lee, J. I. (1998). Effect of muscle type, packaging, and
463
irradiation on lipid oxidation, volatile production, and color in raw pork patties. Meat Science, 49(1),
464
27-39. https://doi.org/10.1016/S0309-1740(97)00101-0 17
465
Aroeira, C. N., Torres Filho, R. A., Fontes, P. R., Gomide, L. A. M., Ramos, A. L. S., Ladeira, M. M., &
466
Ramos, E. M. (2016). Freezing, thawing and aging effects on beef tenderness from Bos indicus and
467
Bos taurus cattle. Meat Science, 116, 118-125. https://doi.org/10.1016/j.meatsci.2016.02.006
468
Aroeira, C. N., Torres Filho, R. A., Fontes, P. R., Ramos, A. L. S., Gomide, L. A. M., Ladeira, M. M., &
469
Ramos, E. M. (2017). Effect of freezing prior to aging on myoglobin redox forms and CIE color of
470
beef
471
https://doi.org/10.1016/j.meatsci.2016.11.010
472
from
Nellore
and
Aberdeen
of
474
https://doi.org/10.1002/jsfa.2740150712
meat.
Journal
of
the
determination
477
https://doi.org/10.1021/ac60205a053
479
Meat
Science,
125,
16-21.
Science
of
Food
and
Agriculture,
15,
504-508.
Bergman, I., & Loxley, R. (1963). Two improved and simplified methods for the spectrophotometric
476
478
cattle.
Bailey, A. J., & Rhodes, D. N. (1964). Treatment of meats with ionising radiations. XI - changes in the texture
473
475
Angus
of
hydroxyproline.
Analytical
Chemistry,
35,
1961-1965.
Boakye, K., & Mittal, G. S. (1993). Changes in pH and water holding properties of Longissimus dorsi muscle during beef ageing. Meat Science, 34(3), 335-349. https://doi.org/10.1016/0309-1740(93)90082-S
480
Bowker, B. C., Fahrenholz, T. M., Paroczay, E. W., & Solomon, M. B. (2008). Effect of hydrodynamic
481
pressure processing and aging on sarcoplasmic proteins of beef strip loins. Journal of Muscle Foods,
482
19(2), 175-193. https://doi.org/10.1111/j.1745-4573.2007.00104.x
483 484
Brewer, S. (2004). Irradiation effects on meat color - a review. Meat Science, 68(1), 1-17. https://doi.org/10.1016/j.meatsci.2004.02.007
485
Cardoso, G. P., Andrade, M. P. D., Rodrigues, L. M., Massingue, A. A., Fontes, P. R., Ramos, A. d. L. S., &
486
Ramos, E. M. (2019). Retail display of beef steaks coated with monolayer and bilayer chitosan-gelatin
487
composites. Meat Science, 152, 20-30. https://doi.org/10.1016/j.meatsci.2019.02.009
488
Cardoso, G. P., Dutra, M. P., Fontes, P. R., Ramos, A. L. S., Gomide, L. A. M., & Ramos, E. M. (2016).
489
Selection of a chitosan gelatin-based edible coating for color preservation of beef in retail display.
490
Meat Science, 114, 85-94. https://doi.org/10.1016/j.meatsci.2015.12.012
18
491
Cross, H. R., Carpenter, Z. L., & Smith, G. C. (1973). Effects of intramuscular collagen and elastin on bovine
492
muscle tenderness. Journal of Food Science, 38(6), 998-1003. https://doi.org/10.1111/j.1365-
493
2621.1973.tb02133.x
494
Delgado, E. F., Aguiar, A. P., Ortega, E. M. M., Spoto, M. H. F., & Castillo, C. J. C. (2006). Brazilian
495
consumers' perception of tenderness of beef steaks classified by shear force and taste. Scientia
496
Agricola, 63, 232-239. https://doi.org/10.1590/S0103-90162006000300004.
497
Holman, B. W. B., van de Ven, R. J., Mao, Y., Coombs, C. E. O., & Hopkins, D. L. (2017). Using
498
instrumental (CIE and reflectance) measures to predict consumers' acceptance of beef colour. Meat
499
Science, 127, 57-62. https://doi.org/10.1016/j.meatsci.2017.01.005
500
Huff-Lonergan, E., & Lonergan, S. M. (2005). Mechanisms of water-holding capacity of meat: The role of
501
postmortem
502
https://doi.org/10.1016/j.meatsci.2005.04.022
biochemical
and
structural
changes.
Meat
Science,
71(1),
194-204.
503
Joo, S. T., Kauffman, R. G., Kim, B. C., & Park, G. B. (1999). The relationship of sarcoplasmic and
504
myofibrillar protein solubility to colour and water-holding capacity in porcine longissimus muscle.
505
Meat Science, 52(3), 291-297. https://doi.org/10.1016/S0309-1740(99)00005-4
506 507
Kanatt, S. R., Chawla, S. P., & Sharma, A. (2015). Effect of radiation processing on meat tenderisation. Radiation Physics and Chemistry, 111(0), 1-8. https://doi.org/10.1016/j.radphyschem.2015.02.004
508
Kim, S. Y., Yong, H. I., Nam, K. C., Jung, S., Yim, D.-G., & Jo, C. (2018). Application of high temperature
509
(14°C) aging of beef M. semimembranosus with low-dose electron beam and X-ray irradiation. Meat
510
Science, 136, 85-92. https://doi.org/10.1016/j.meatsci.2017.10.016
511
Kim, Y. H., Keeton, J. T., Smith, S. B., Maxim, J. E., Yang, H. S., & Savell, J. W. (2009). Evaluation of
512
antioxidant capacity and colour stability of calcium lactate enhancement on fresh beef under highly
513
oxidising
514
https://doi.org/10.1016/j.foodchem.2008.12.008
conditions.
Food
Chemistry,
115(1),
272-278.
515
Kim, Y. H., Nam, K. C., & Ahn, D. U. (2002a). Color, Oxidation-Reduction Potential, and Gas Production of
516
Irradiated Meats from Different Animal Species. Journal of Food Science, 67(5), 1692-1695.
517
https://doi.org/10.1111/j.1365-2621.2002.tb08707.x
19
518
Kim, Y. H., Nam, K. C., & Ahn, D. U. (2002b). Volatile profiles, lipid oxidation and sensory characteristics
519
of
520
https://doi.org/10.1016/S0309-1740(01)00191-7s
irradiated
meat
from
different
animal
species.
Meat
Science,
61(3),
257-265.
521
King, D. A., Shackelford, S. D., Rodriguez, A. B., & Wheeler, T. L. (2011). Effect of time of measurement on
522
the relationship between metmyoglobin reducing activity and oxygen consumption to instrumental
523
measures
524
https://doi.org/10.1016/j.meatsci.2010.08.013
of
beef
longissimus
color
stability.
Meat
Science,
87,
26–32.
525
Koohmaraie, M., Seidemann, S. C., Schollmeyer, J. E., Dutson, T. R., & Crouse, J. D. (1987). Effect of post-
526
mortem storage on Ca++-dependent proteases, their inhibitor and myofibril fragmentation. Meat
527
Science, 19(3), 187-196. https://doi.org/10.1016/0309-1740(87)90056-8
528 529 530
Krzywicki, K. (1979). Assessment of relative content of myoglobin, oxymyoglobin and metmyoglobin at the surface of beef. Meat Science, 3(1), 1-10. https://doi.org/10.1016/0309-1740(79)90019-6 Lee, J. W., Yook, H. S., Lee, K. H., Kim, J. H., Kim, W. J., & Byun, M. W. (2000). Conformational changes
531
of
532
https://doi.org/10.1016/S0969-806X(99)00466-1
myosin
by
gamma
irradiation.
Radiation
Physics
and
Chemistry,
58(3),
271-277.
533
Luchsinger, S. E., Kropf, D. H., García Zepeda, C. M., Hunt, M. C., Stroda, S. L., Marsden, J. L., & Kastner,
534
C. L. (1997). Color and oxidative properties of irradiated whole muscle beef. Journal of Muscle
535
Foods, 8(4), 427-443. https://doi.org/10.1111/j.1745-4573.1997.tb00730.x
536 537 538
MacDougall, D. B. (1982). Changes in the colour and opacity of meat. Food Chemistry, 9(1), 75-88. https://doi.org/10.1016/0308-8146(82)90070-X Madhavi, D. L., & Carpenter, C. E. (1993). Aging and Processing Affect Color, Metmyoglobin Reductase and
539
Oxygen
540
https://doi.org/10.1111/j.1365-2621.1993.tb06083.x
Consumption
of
Beef
Muscles.
Journal
of
Food
Science,
58(5),
939-942.
541
Millar, S. J., Moss, B. W., & Stevenson, M. H. (2000). The effect of ionising radiation on the colour of beef,
542
pork and lamb. Meat Science, 55(3), 349-360. https://doi.org/10.1016/S0309-1740(99)00164-3
543
Nanke, K. E., Sebranek, J. G., & Olson, D. G. (1999). Color characteristics of irradiated aerobically packaged
544
pork, beef, and turkey. Journal of Food Science, 64(2), 272-278. https://doi.org/10.1111/j.1365-
545
2621.1998.tb15842.x 20
546
O'Keeffe, M., & Hood, D. E. (1982). Biochemical factors influencing metmyoglobin formation on beef from
547
muscles of differing colour stability. Meat Science, 7(3), 209-228. https://doi.org/10.1016/0309-
548
1740(82)90087-0
549
Raharjo, S., Sofos, J. N., & Schmidt, G. R. (1992). Improved speed, specificity, and limit of determination of
550
an aqueous acid extraction thiobarbituric acid-C18 method for measuring lipid peroxidation in beef.
551
Journal of Agricultural and Food Chemistry, 40 (11), 2182-2185. https://doi.org/10.1021/jf00023a02
552 553
Ramos, E. M., & Gomide, L. A. M. (2017). Avaliação da Qualidade de Carnes: Fundamentos e Metodologias (2a ed.). Viçosa: Editora UFV.
554
Rowe, L. J., Maddock, K. R., Lonergan, S. M., & Huff-Lonergan, E. (2004). Oxidative environments decrease
555
tenderization of beef steaks through inactivation of μ-calpain. Journal of Animal Science, 82(11),
556
3254-3266. https://doi.org/10.2527/2004.82113254x
557
Sammel, L. M., Hunt, M. C., Kropf, D. H., Hachmeister, K. A., & Johnson, D. E. (2002). Comparison of
558
Assays for Metmyoglobin Reducing Ability in Beef Inside and Outside Semimembranosus Muscle.
559
Journal of Food Science, 67(3), 978-984. https://doi.org/10.1111/j.1365-2621.2002.tb09439.x
560
Silva, D. R. G., Torres Filho, R. A., Cazedey, H. P., Fontes, P. R., Ramos, A. L. S., & Ramos, E. M. (2015).
561
Comparison of Warner–Bratzler shear force values between round and square cross-section cores
562
from
563
https://doi.org/10.1016/j.meatsci.2014.12.009
cooked
beef
and
pork
Longissimus
muscle.
Meat
Science,
103,
1-6.
564
Vieira, C., Diaz, M. T., Martínez, B., & García-Cachán, M. D. (2009). Effect of frozen storage conditions
565
(temperature and length of storage) on microbiological and sensory quality of rustic crossbred beef at
566
different
567
https://doi.org/10.1016/j.meatsci.2009.06.013
568 569
states
of
ageing.
Meat
Science,
83(3),
398-404.
Watts, B. M., Kendrick, J., Zipser, M. W., Hutchins, B., & Saleh, B. (1966). Enzymatic Reducing Pathways in Meat. Journal of Food Science, 31(6), 855-862. https://doi.org/10.1111/j.1365-2621.1966.tb03261.x
570
Wheeler, T. L., Miller, R. K., Savell, J. W., & Cross, H. R. (1990). Palatability of Chilled and Frozen Beef
571
Steaks. Journal of Food Science, 55(2), 301-304. https://doi.org/10.1111/j.1365-2621.1990.tb06748.x
572
WHO. (1999). High-dose irradiation: wholesomeness of food irradiated with doses above 10 kGy. Report of a
573
Joint FAO/IAEA/WHO Study Group. World Health Organ Tech Rep Ser, 890. 21
574
Yook, H. S., Lee, J. W., Lee, K. H., Kim, M. K., Song, C. W., & Byun, M. W. (2001). Effect of gamma
575
irradiation on the microstructure and post-mortem anaerobic metabolism of bovine muscle. Radiation
576
Physics and Chemistry, 61(2), 163-169. https://doi.org/10.1016/S0969-806X(00)00392-3
577 578
Yoon, K. S. (2003). Effect of gamma irradiation on the texture and microstructure of chicken breast meat. Meat Science, 63(2), 273-277. https://doi.org/whip10.1016/S0309-1740(02)00078-5
579
Zabielski, J., Kijowski, J., Fiszer, W., & Niewiarowicz, A. (1984). The effect of irradiation on technological
580
properties and protein solubility of broiler chicken meat. Journal of the Science of Food and
581
Agriculture, 35(6), 662-670. https://doi.org/10.1002/jsfa.2740350612
582 583
Conflict of interest
584 585
The authors declare that there is no conflict of interest.
586
22
588
Figure 1. Gamma irradiation × aging time interaction on myoglobin redox forms of bovine Longissimus
589
lumborum muscle: (A) oxymyoglobin (OMb); and (B) deoxymyoglobin (DMb). Means with different letters
590
(a-d), for irradiation dose within each aging time, differs (P < 0.05). ns = difference between means of day 1
591
and day 14 are not significant (P > 0.05). Bars represent the standard error of means.
592 593
Research Highlights
594 595
Gamma irradiation reduces WHC and increases purge and cooking loss
596
Gamma irradiation negatively affected myofibrillar fragmentation
597
Irradiation at 9 kGy induces higher soluble collagen and lower shear force
598
Higher irradiation dose oxidizes myoglobin to metmyoglobin
599
Irradiation at 9 kGy induce lipid oxidation and surface discoloration
600 601 602 603
SAMPLE CREDIT AUTHOR STATEMENT
604 605 606
Lorena Mendes Rodrigues: Conceptualization, Methodology, Investigation, Writing- Original draft preparation.
607
Luana Aparecida Sales: Conceptualization, Investigation.
608
Paulo Rogério Fontes: Validation.
609
Robledo de Almeida Torres Filho: Formal analysis, Validation.
610
Monalisa Pereira Dutra Andrade: Validation.
611
Alcinéia de Lemos Souza Ramos: Formal analysis, Writing- Reviewing and Editing.
612
Eduardo Mendes Ramos: Supervision, Funding acquisition, Writing- Reviewing and Editing.
613 614 615 23
616
Table 1. Gamma irradiation dose (G) and aging time (A) effects on pH, extractable proteins and water losses
617
values (means ± standard deviations) of bovine Longissimus lumborum muscle. Soluble proteins (mg/g) Source of variation
Effects
Purge
pH
Sarcoplasmic
Myofibrillar
Gamma irradiation Non-irradiated
5.58±0.14
45.68±8.53
70.40±22.74
2.24±1.37a
dose (G)
3 kGy
5.62±0.11
47.57±17.13
66.58±15.13
3.40±2.02b
6 kGy
5.61±0.16
49.12±8.85
73.34±16.50
3.35±2.06b
9 kGy
5.61±0.09
49.32±11.18
64.78±15.75
3.38±2.15b
1 day
5.61±0.12
51.68±12.80x
63.92±17.47x
1.91±1.03x
14 days
5.59±0.14
44.16±5.64y
73.05±16.88y
4.28±1.93y
G
0.864
0.748
0.525
0.038
A
0.636
0.006
0.037
<0.001
G×A
0.961
0.387
0.340
0.069
Aging time (A)
Pr > F1
(%)
618
FPPM = filter paper press method.
619
1 Significant
620
a-b Means
in the same column, into gamma irradiation effect, followed by different letters differ (P < 0.05).
621
x-y Means
in the same column, into aging effect, followed by different letters differ (P < 0.05).
probabilities (P < 0.05) were placed in bold.
622
24
624
Table 2. Gamma irradiation dose (G) and aging time (A) effects on collagen content, myofibrillar
625
fragmentation and shear force values (means ± standard deviations) of bovine Longissimus lumborum
626
muscle. Collagen (mg/g) Source of variation
Effects
Collagen soluble
Total
Insoluble
Soluble
Gamma irradiation Non-irradiated
2.23±0.35a
1.84±0.32a
0.39±0.09a
17.77±3.53a
dose (G)
3 kGy
2.72±0.88b
2.29±0.86b
0.43±0.11ab
17.07±6.46a
6 kGy
2.84±0.96b
2.36±1.01b
0.48±0.12b
18.52±6.34a
9 kGy
2.10±0.26a
1.60±0.22a
0.49±0.09b
23.59±3.48b
1 day
2.54±0.91
2.08±0.88
0.46±0.12
19.44±6.29
14 days
2.40±0.61
1.96±0.59
0.44±0.10
19.04±5.05
G
0.002
0.001
0.008
<0.001
A
0.338
0.383
0.359
0.699
G×A
0.463
0.539
0.082
0.324
Aging time (A)
Pr > F1
(%)
627
FI = fragmentation index; WBsSF = Warner-bratzler square shear force.
628
1 Significant
629
a-b Means
in the same column, into gamma irradiation effect, followed by different letters differ (P < 0.05).
630
x-y Means
in the same column, into aging effect, followed by different letters differ (P < 0.05).
probabilities (P < 0.05) were placed in bold.
631
25
633
Table 3. Gamma irradiation dose (G) and aging time (A) effects on lipid oxidation, metmyoglobin reducing
634
activity (MRA), oxygen consumption rate (OCR) and myoglobin chemical forms (means ± standard
635
deviations) of bovine Longissimus lumborum muscle. MRA (%) Effects
Source of variation
Gamma irradiation
Non-irradiated
dose (G)
Aging time (A)
Pr > F1
TBARS
Myog OCR
IMF
NORA
0.21±0.31a
24.19±17.98a
40.05±15.82a
98.07±4.02a
15.60±6.62
3 kGy
0.39±0.36a
28.07±18.46a
31.61±6.27b
86.35±11.51a
15.70±4.32
6 kGy
0.36±0.31a
39.79±3.05b
32.17±11.68b
45.89±24.18b
17.06±3.67
9 kGy
0.87±0.66b
40.98±1.68b
25.89±4.73b
38.61±21.49b
19.34±3.62
1 day
0.45±0.50
32.78±14.74
33.06±14.08
68.66±32.58
19.53±4.29
14 days
0.47±0.58
33.74±14.78
31.79±8.38
65.80±29.15
14.32±3.92
G
<0.001
<0.001
0.001
<0.001
<0.001
A
0.833
0.713
0.573
0.476
<0.001
G×A
0.487
0.884
0.227
0.683
0.002
(mg MAD/kg)
DMb
(%)
636
MAD = malonaldehyde; IMF = initial metmyoglobin formed; NORA = nitric oxide metmyoglobin reducing
637
activity; DMb = deoxymyoglobin; OMb = oxymyoglobin; and MMb = metmyoglobin.
638
1 Significant
639
a-b Means
in the same column. into gamma irradiation effect. followed by different letters differ (P < 0.05).
640
x-y Means
in the same column. into aging effect. followed by different letters differ (P < 0.05).
probabilities (P < 0.05) were placed in bold.
26
642
Table 4. Gamma irradiation dose (G) and aging time (A) effects on CIE color (means ± standard deviations) of
643
bovine Longissimus lumborum muscle.
Effects
Source of variation
Gamma irradiation dose (G)
Aging time (A)
Pr > F1
L*
a*
b*
Non-irradiated
43.62±3.16
22.40±2.20a
15.08±2.28
27.01
3 kGy
43.80±2.78
22.37±1.63a
15.14±1.71
27.03
6 kGy
44.32±2.45
22.15±1.45ab
15.06±1.59
26.79
9 kGy
44.24±2.69
21.32±1.93b
14.46±1.86
25.77
1 day
42.80±2.43x
21.24±1.80x
14.08±1.79x
25.5
14 days
45.19±2.52y
22.87±1.50y
15.79±1.51y
27.8
G
0.232
0.020
0.157
0.
A
<0.001
<0.001
<0.001
<0
G×A
0.547
0.651
0.581
0.
644
L* = lightness; a* = redness; b* = yellowness; C* = chroma; and h = hue angle.
645
1 Significant
646
a-b Means
in the same column. into gamma irradiation effect. followed by different letters differ (P < 0.05).
647
x-y Means
in the same column. into aging effect. followed by different letters differ (P < 0.05).
C
probabilities (P < 0.05) were placed in bold.
648 649
27
S0308-8146(19)32289-7 https://doi.org/10.1016/j.foodchem.2019.126137 FOCH 126137
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
4 August 2019 16 November 2019 27 December 2019
Please cite this article as: Mendes Rodrigues, L., Aparecida Sales, L., Rogério Fontes, P., de Almeida Torres Filho, R., Pereira Dutra Andrade, M., de Lemos Souza Ramos, A., Mendes Ramos, E., Combined effects of gamma irradiation and aging on tenderness and quality of beef from Nellore cattle, Food Chemistry (2019), doi: https:// doi.org/10.1016/j.foodchem.2019.126137
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Elsevier Ltd. All rights reserved.
1
Combined effects of gamma irradiation and aging on tenderness and quality of beef from Nellore
2
cattle
3 4
Lorena Mendes RODRIGUESa, Luana Aparecida SALESa, Paulo Rogério FONTESb, Robledo de Almeida
5
TORRES FILHOc, Monalisa Pereira Dutra ANDRADEd, Alcinéia de Lemos Souza RAMOSa, Eduardo
6
Mendes RAMOSa*
7 8
a Departamento
9
Gerais, 37200-000, Brasil.
de Ciência dos Alimentos, Universidade Federal de Lavras, P.O. Box 3037, Lavras, Minas
10
b
11
900, Brasil.
12
c Instituto
13
Minas Gerais, 35690-000, Brasil.
14
d Instituto
15
Diamantina, Minas Gerais, 30161-970, Brasil.
Departamento de Tecnologia de Alimentos, Universidade Federal de Viçosa, Viçosa, Minas Gerais, 36570-
de Ciências Exatas e Tecnológicas, Universidade Federal de Viçosa, Campus Florestal, Florestal,
de Ciência e Tecnologia, Universidade Federal dos Vales do Jequitinhonha e Mucuri, Campus JK,
16 17 18
* Corresponding author tel.: +55 35 3829 1403; fax: +55 35 3829 1401; email: [email protected] (E. M. Ramos).
19
1
21
Abstract
22
Combined effects of gamma irradiation (0, 3, 6 and 9 kGy) and aging (1 and 14 days) on quality
23
attributes of vacuum-packaged beef from Nellore cattle were evaluated. The meat water holding capacity was
24
affected by irradiation, increasing (p < 0.05) purge and cooking loss regardless of the dose used. Irradiation
25
negatively affected myofibrillar fragmentation, but samples irradiated at 9 kGy had (p < 0.05) higher soluble
26
collagen and lower shear force values. The meat metmyoglobin reducing activity was reduced (p < 0.05) by
27
the irradiation process, inducing the metmyoglobin accumulation with increasing dose applied. Samples
28
irradiated at 9 kGy presented (p < 0.05) higher lipid oxidation and lower oxymyoglobin proportion and color
29
redness and chroma values. It was concluded that irradiation at 9 kGy combined with aging can be used as an
30
effective tool for improving the tenderness of Nellore beef, but resulted in a discoloration of the beef.
31
Keywords: Shear force; fragmentation index; collagen; lipid oxidation; color.
32 33
1. Introduction
34 35
According to the Brazilian Association of Meat Exporting Industries (ABIEC), Brazil exported 1.64
36
million tons of beef in 2018, consolidating its position as the world's largest exporter, but over 80% of
37
Brazilian bovine herd consists of zebu (Bos indicus) cattle, whit Nellore maintaining 90% of this share.
38
Although the wet aging system (vacuum packed beef) is used by the meat industry to improve tenderness,
39
zebu beef is still considered less tender than that of European (Bos taurus) beef (Aroeira et al., 2016). Since,
40
tenderness stands out as a quality attribute of the beef, being considered the most influential sensory
41
characteristic on acceptance of meat by consumers (Delgado et al., 2006), improving this quality attribute
42
remains a challenge for the Brazilian industry.
43
Among studies aiming at finding ways to improve meat tenderness, the gamma irradiation process
44
seems to have great potential, as besides being considered an excellent method for microbial control in foods,
45
it has been reported that relative low- to medium doses of 5 to 10 kGy are efficient in reducing shear force in
46
non-aged bovine (Yook, Lee, Lee, Kim, Song, & Byun, 2001), lamb and buffalo (Kanatt, Chawla, & Sharma,
47
2015) meat. However, only Kanatt et al. (2015) evaluated the effects of gamma irradiation after rigor mortis
48
process and yet they did not evaluate the effects after aging. Moreover, Rowe, Maddock, Lonergan, and Huff2
49
Lonergan (2004) reported that beef steaks irradiated at relatively low dose (6.4 kGy) of electron beam and
50
aged by 14-days showed significantly higher shear force values than non-irradiated steaks, while, more
51
recently, Kim, Yong, Nam, Jung, Yim, and Jo (2018) observed no adverse effect on 14-days aged beef
52
tenderness due to the pre-rigor irradiation (5 kGy) by electron beam or X-ray. Therefore, the combined effects
53
of aging and irradiation on meat proteolysis and tenderness are still conflicting and scarce.
54
Despite possible effects on tenderness and its usefulness to ensure the microbial safety and extend shelf
55
life of meat without loss of nutritional quality (WHO, 1999), the irradiation technology can influence lipid
56
oxidation, color changes, and off-odor of meat, which may generate negative consumer responses (Brewer,
57
2004; Millar, Moss, & Stevenson, 2000). Of these changes, the formation of undesirable or unexpected colors
58
from the consumer's viewpoint are critical, as meat color is the most important attribute that consumers use as
59
purchase criterion (Aroeira et al., 2017), and are related to the content and chemical form of heme pigments,
60
muscle condition (pH, reducing equivalents, etc.) and to applied irradiation dose. In this context, the
61
irradiation of vacuum-packed beef is interesting to overcome or reduce the lipid oxidation and color
62
discoloration, since is the presence of oxygen during irradiation that often results in unacceptable radiolytic
63
changes (Brewer, 2004). The effects of low- to medium dose irradiation on aged beef quality are not well
64
defined, since most research has been conducted on ground beef, which reacts differently to irradiation in
65
terms of lipid oxidation and color changes when compared to intact beef.
66
Faced with the expectation of improving the tenderness of zebu animal’s meat and, consequently,
67
improving the quality of Brazilian beef, new techniques are being increasingly evaluated. In this context, the
68
interaction of irradiation and aging on beef tenderness seems to require further investigation to maximize
69
commercial application, especially in meat from Nellore cattle, where very limited data are currently
70
available. Moreover, any process that negatively affects the color of fresh beef can lead to lower consumer
71
appeal and marketability. Thus, the aim of this study was to verify the gamma irradiation as a novel technique
72
to improve beef tenderness of Nellore animal’s, evaluating the application of different doses and its effects on
73
the parameters associated to the meat tenderness and color before and after the aging process.
74 75
2. Material and methods
76 3
77
2.1 Collection and preparation of samples
78 79
Samples of Longissimus lumborum muscle (LL), from eight Nellore bovines, with an average age of 30
80
months old and similar livestock and slaughter system, were obtained 48 hours postmortem directly from a
81
slaughterhouse (Plena Alimentos Ltda.) with Federal Inspection (SIF) in the State of Minas Gerais, Brazil.
82
Of each LL muscles, eight pieces approximately 5.0 cm thick (beef section) were obtained, individually
83
weighed, identified, vacuum-packed (90 µm-thick nylon-polyethylene, with an oxygen transmission rate of
84
30–60 cm3/m2/day/atm) and randomly distributed in duplicate in the four radiation doses treatments: control
85
(non-irradiated), 3, 6 and 9 kGy. The beef sections were placed in coolers and were subjected to the ionizing
86
radiation in the Gamma Radiator IR-214 (MDS, Nordion; cobalt-60 source and 1925.8 Gy/h rate) at the
87
Nuclear Technology Development Center of the National Commission of Nuclear Energy (CDTN/CNEN),
88
Belo Horizonte, Minas Gerais, Brazil. Non-irradiated samples (control) were kept in coolers for time periods
89
like the irradiated samples. The whole irradiation process (up to 9 kGy dose) lasted 4.5 hours. After
90
irradiation, the coolers were taken to Laboratory of Meat and Meat Products Technology (LabCarnes), at the
91
Federal University of Lavras (UFLA), and the beef sections of each irradiation dose were randomly
92
distributed into two aging time (1 and 14 days) conducted in a climatic chamber (model EL202; EletroLab
93
Inc., São Paulo, Brazil) at 1.0 °C.
94 95
2.2 pH and soluble proteins
96 97 98
The pH was measured by an insertion electrode in three different locations (triplicate), using a portable pH meter (model HI 99163; Hanna Instruments, Woonsocket, RI, USA).
99
Extractable sarcoplasmic proteins (in potassium phosphate buffer 25 mM; pH 7.2) and total proteins (in
100
potassium iodate 1.1M solution in potassium phosphate buffer 100 mM; pH 7.2) were determined as described
101
by Joo, Kauffman, Kim, and Park (1999). The protein concentration of the extracts was determined by the
102
Biuret method, being expressed in mg/g of beef. The concentration of myofibrillar proteins was estimated by
103
the difference between the concentration of total and sarcoplasmic proteins.
104 4
105
2.3 Water losses
106 107
At each aging time (1 and 14 days), the beef sections have been removed from the packaging, dried in
108
paper towels and weighed again to determine the purge (measured by mass difference between the samples
109
before and after packing), being expressed as a percentage.
110
The water holding capacity (WHC) was measured by the method of pressure in filter paper (MPPF)
111
described by Aroeira et al. (2016). Samples of approximately 300 mg of meat were placed on previously dry
112
filter paper, and the set was pressed for 5 min with a 5 kg weight. After pressing, the areas in the filter paper
113
delimited by the pressed meat (Ap) and by the exudate liquid (Ae) were obtained using ImageJ software ®
114
1.42 q (National Institute of Health, USA), and the WHC expressed as the ratio Ap/Ae.
115
One steak of 2.54 cm thickness was obtained from each beef section and an inner standard-sized
116
rectangular sample (8.0 x 4.0 x 2.5 cm) removed, weighed, vacuum-packed and cooked in water bath at 80 °C
117
until internal temperature of 71 °C (monitored by a digital thermometer inserted in the steak center). After
118
cooking, the samples were cooled (4 °C) for 3 h, removed from the vacuum-packed bags, dried with a paper
119
towel and weighed again. The cooking loss was determined by the difference between the weighing before
120
and after cooking, being expressed as a percentage.
121 122
2.4 Collagen content
123 124
The soluble and insoluble collagen fractions were separated after heating at 77 °C for 70 min and the
125
collagen content was quantified by determining the hydroxyproline amino acid (Bergman & Loxley, 1963), as
126
described by Ramos and Gomide (2017). The hydroxyproline content was obtained using an analytical curve,
127
and the collagen content (mg/g) was calculated using a factor of 7.52 for soluble and 7.25 for insoluble
128
fractions (Cross et al., 1973). The total collagen content was obtained from the sum of the soluble and
129
insoluble fractions.
130 131
2.5 Fragmentation index
132 5
133
The degree of myofibrillar fragmentation in samples was assessed by the fragmentation index (FI)
134
method described by Aroeira et al. (2016), with minor modifications. All samples in the stipulated aging time
135
were previously frozen and stored (-18 °C) until analyzed and the sample was homogenized in extraction
136
solution at a ratio of 1:5 (w / v) instead of 1:10 (w / v) originally used. After vacuum filtration (Vacuum pump
137
NOF-650, New Pump, Brazil) of the homogenate using a 250 µm-pores nylon screen, the residue weight
138
(RW) was determined and the FI was expressed as 100 × RW.
139 140
2.6 Shear force
141 142
Shear force was determined according to method Warner-Bratzler square Shear Force (WBsSF)
143
described by Silva, Torres Filho, Cazedey, Fontes, Ramos, and Ramos (2015), using cooked samples from the
144
cooking loss measurement (section 2.3). Of each standard-sized rectangular sample cooked, six cuboids (1.0
145
cm x 1.0 cm square cross-section) cores were obtained in the muscle fiber direction and sheared transversely
146
(across the predominant muscle fiber orientation) at 200 mm/min by a Warner-Bratzler blade coupled to a TA.
147
XTplus texturometer (Stable Micro Systems Ltd., Godalming, Surrey, UK). Maximum force (N) was
148
measured and the average value of each beef was used in statistical analysis.
149 150
2.7 Lipid oxidation
151 152
The oxidative stability of meat was evaluated by the number of 2-thiobarbituric acid reactive substances
153
(TBARS index), measured according to the methodology proposed by Raharjo, Sofos, and Schmidt (1992),
154
with modifications described by Cardoso, Dutra, Fontes, Ramos, Gomide, and Ramos (2016). The TBARS
155
values were reported in milligrams of malonaldehyde (MDA) per kilogram of the sample (mg MDA/kg) by
156
means of a standard calibration curve using 1,1,3,3-tetraethoxypropane (TEP).
157 158
2.8 Oxygen consumption rate and metmyoglobin reducing activity
159
6
160
The oxygen consumption rate (OCR) and the metmyoglobin reducing activity (MRA) were measured
161
by determination of the myoglobin redox forms, using a CM-700 spectrophotometer (Konica Minolta;
162
Sensing Inc., Osaka, Japan) with an 8-mm aperture size, 10º observer angle and in the specular component
163
included (SCI) mode. Each index was measured in two (replicates) pieces (5 x 5 x 1.25 cm) of each sample, as
164
described by Cardoso et al. (2019).
165
The OCR was measured as the conversion of the surface oxymyoglobin (OMb) to deoxymyoglobin
166
(DMb) after 10 min under vacuum (Madhavi & Carpenter, 1993), being expressed as the percentage of time-
167
zero surface OMb consumed. The metmyoglobin reducing activity (MRA) was determined by the nitric oxide
168
metmyoglobin reducing activity (NORA) method (Watts, Kendrick, Zipser, Hutchins, & Saleh, 1966). The
169
proportion of surface metmyoglobin (MMb) immediately formed after oxidation with 0.3% sodium nitrite
170
solution was recorded as initial MMb formation (IMF) and the reducing ability was reported as the percentage
171
decrease in surface MMb concentration after an incubation period of 2 hours at ambient temperature
172
(approximately 20 °C).
173
The relative concentration of the pigments heme OMb and MMb were estimated from the surface
174
reflectance data (recorded from 400 to 710, in a 10-nm interval), using the mathematical method developed by
175
Krzywicki (1979).
176 177
2.9 Myoglobin redox forms and color evaluation
178 179
The myoglobin redox forms and the CIE color indexes of beef samples were determined using a CM-
180
700 spectrophotometric colorimeter (Kônica Minolta Sensing Inc., Osaka, Japan), with 8 mm aperture,
181
illuminant A, 10º observer angle and with both specular component included (SCI) and excluded (SCE)
182
modes. The color measurements were taken on the surface of a one 2.54-cm thickness steak, obtained from the
183
beef section, after being exposed 30 min in atmospheric air for blooming. Meat surface reflectance data (from
184
400 to 710, in a 10-nm interval) were recorded using an average of five consecutive measurements
185
representing the entire surface of each sample.
186
The relative content of the pigments heme (OMb, DMb, and MMb) were estimated by the Krzywicki
187
(1979) mathematical method. Based on the readings taken on the SCE mode, the lightness (L*), redness (a*) 7
188
and yellowness (b*) values were obtained. Chroma (C*) and hue angle (h) were also determined as: C* = (a*2
189
+ b*2)1/2; and h = tan-1 (b*/a*).
190 191
2.10 Statistical analysis
192 193
The statistical analyses were performed in SAS software, version 9.2 (Statistical Analysis System-SAS
194
Institute Inc., Cary, NC, USA), at a significance level of 5%, using SAS GLM procedure. The experiment was
195
conducted in a randomized block design, with the blocks consisted of different animals (8 replications), in a
196
split-plot scheme, with the four treatments (0, 3, 6, and 9 kGy) in the whole plot and the aging time (1 and 14
197
days) in the split-plot. The main effects and their interaction on the meat quality indicators were determined
198
by analysis of variance (ANOVA) and, when necessary, means were separated by Tukey's test.
199 200
3. Results and discussion
201 202
3.1 pH and extractable proteins
203 204 205
The effects of gamma irradiation doses and aging time on pH and extractable proteins are described in the Table 1. Interaction effects (treatment x aging time) were not observed (p > 0.05) for any characteristic.
206
For pH values, no significant effect (p > 0.05) was observed for gamma irradiation doses or aging time
207
factors (mean value of 5.60 ± 0.12) and was consistent with the mean values observed for beef. The absence
208
of irradiation effect on the pH values of the beef steaks agrees with the observation of other authors (Kanatt et
209
al., 2015; Luchsinger et al., 1997). Moreover, the absence of effect by aging time could be due to the short
210
period of aging, since Aroeira et al. (2016) also did not observe a significant difference of pH in bovine LL
211
muscles after 14-days aging.
212
As for the extractable proteins, a significant effect (p < 0.05) was observed only of aging time on
213
sarcoplasmic and myofibrillar proteins. The amount of soluble protein extracted from meat can give an
214
indication of the relative level of distortion that can occur during a process or treatment (Kanatt et al., 2015).
215
The solubility of the sarcoplasmic proteins decreased with aging time, while the myofibrillar proteins 8
216
solubility increased (Table 1). Sarcoplasmic proteins are known to be more susceptible to early denaturation,
217
due to the rapid postmortem glycolysis, although the effects of postmortem aging on the protein’s solubility
218
are still not clear. However, aging-related changes in the extraction of proteins are probably due to changes in
219
protein conformation, molecular size and intra and inter-molecular bonds that occur with postmortem aging
220
(Bowker, Fahrenholz, Paroczay, & Solomon, 2008). These authors also observed that postmortem aging (up to
221
8 days) decreased solubility of sarcoplasmic proteins and increased solubility of myofibrillar proteins in
222
bovine striploins.
223
The irradiation process did not affect (p > 0.05) the solubility of meat proteins, which does not agree
224
with the observations reported by Kanatt et al. (2015). These authors observed an increase for total and
225
myofibrillar extractable protein from buffalo meat irradiated with 2.5 to 10 kGy. Lee, Yook, Lee, Kim, Kim,
226
and Byun (2000) reported that myosin subunits were structurally modified, increasing its solubility when
227
higher doses of irradiation were applied. According to these authors, irradiation can affect the solubility of
228
proteins through the formation of free radicals, generated due to the radiolysis of the water molecule,
229
catalyzing reactions like: deamination; decarboxylation; reduction of disulfide bonds; oxidation of sulfhydryl
230
groups; hydrolysis of peptide bonds; and changes in the valence of the metal ions of enzymes. However,
231
changes in the proteins do not depend only on their structure and state, but also on the conditions of the
232
radiation process, like dose and rate applied, temperature, and absence or presence of oxygen.
233 234
3.2 Water losses
235 236
The effects of gamma irradiation dose and aging time on water losses values of bovine LL muscles are
237
described in the Table 1. As observed for pH and extractable proteins, interaction effects were not observed (p
238
> 0.05) for any of the characteristics.
239
The irradiation process increased (p < 0.05) the purge values, regardless of the applied dose. This is
240
possibly due to a denaturation of beef proteins caused by the free radicals generated by the radiolysis of the
241
water molecule (Zabielski, Kijowski, Fiszer, & Niewiarowicz, 1984), although an irradiation effect was not
242
observed on the proteins solubility. Higher purge (p < 0.05) values were also observed in the samples with
243
longer aging time (14 days) and can be due to the negative pressure of the vacuum packaging, that induces 9
244
progressive drainage of the water present in the extracellular spaces through the drip channels formed during
245
aging (Aroeira et al., 2016; Huff-Lonergan & Lonergan, 2005).
246
The meat water holding capacity (WHC), measured by the filter paper pressure method (FPPM), was
247
affected (p < 0.05) only by the gamma irradiation doses. Overall, the radiation process reduced the FPPM
248
from values of 0.30 in the non-irradiated samples to 0.25 in irradiated ones. This may indicate a negative
249
effect on proteolysis, which would reduce the degradation of cytoskeletal proteins and, consequently, the
250
WHC. The degradation of the cytoskeletal proteins results in increased muscle fiber diameter, thereby
251
increasing its ability to retain the water expelled by the myofibrils (Huff-Lonergan & Lonergan, 2005). Kanatt
252
et al. (2015), measuring the WHC (by centrifugation of minced meat in a salt solution) of buffalo meats also
253
observed a reduction on WHC values with higher irradiation doses. However, these authors observed that the
254
WHC loss was dose-dependent, being, probably, due the rupture of muscle fibers membranes and, or
255
denaturation of meat protein induced by the radiation process.
256
The absence of effect by aging time on the FPPM could also be due to the short period of aging,
257
especially in Nellore meats. Aroeira et al. (2016) did not observe a statistical difference of FPPM in bovine
258
LL muscles after 14-days aging, reporting significative higher values only after 21 days of storage.
259
Unlike observed for the WHC, the cooking loss was affected (p < 0.05) by gamma irradiation and by
260
aging time individually, as described in the Table 1. The irradiated samples, regardless of the dose applied,
261
presented greater values (27.83%) of cooking loss than non-irradiated ones (25.17%). This is possibly due to
262
the effects of the free radicals generated by the radiolysis of the water molecule, as discussed for purge.
263
Kanatt et al. (2015), evaluating the effect of different irradiation doses (up to 10 kGy) in buffalo, chicken and
264
lamb meat also observed that the irradiation increased sample's cooking losses. Relative to aging effects on
265
cooking losses, the results reported in the literature are conflicting; while some studies reported an increase in
266
the cooking loss values (Aroeira et al., 2016; Boakye & Mittal, 1993), other studies reported no aging effect
267
(Vieira, Diaz, Martínez, & García-Cachán, 2009; Wheeler, Miller, Savell, & Cross, 1990).
268 269
3.3 Collagen content
270
10
271
The effects of gamma irradiation and aging on collagen content of bovine LL muscles are described in
272
the Table 2. The total collagen content and its insoluble fraction, including the percentage of soluble collagen,
273
were affected (p < 0.05) only by the irradiation process. Irradiation doses of 3 and 6 kGy resulted in greater
274
proportions of the insoluble collagen fraction, while samples irradiated at a 9 kGy doses did not differ from
275
the non-irradiated samples. Moreover, irradiation doses above 3 KGy resulted in greater amounts of the
276
soluble collagen fraction. Kanatt et al. (2015) did not observe an effect of radiation process on collagen
277
content in the three types of meat (chicken, lamb and buffalo) evaluated. The lack of effect of aging times (up
278
to 14 days) on the collagen fractions was also reported by Koohmaraie, Seidemann, Schollmeyer, Dutson, and
279
Crouse (1987) for bovine Longissimus muscle.
280
However, for the soluble collagen percentage, samples irradiated at 9 kGy had higher (p < 0.05) values
281
than non-irradiated samples. Bailey and Rhodes (1964) reported that the radiation process increase the
282
collagen molecule solubility due to rupture of the molecule's peptide bonds by direct absorption of radiation
283
energy, forming low molecular weight fragments which are, consequently, more soluble. Kanatt et al. (2015)
284
observed an increase in collagen solubility from 3.58% in non-irradiated to 6.02% in irradiated (at 10 kGy)
285
lamb meat, while 48% increasing in solubility was reported when buffalo meat was irradiated at same dose.
286 287
3.4 Fragmentation and shear force values
288 289
A was observed a significant effect (p < 0.05) of irradiation and aging time on the degree of
290
myofibrillar fragmentation (Table 2), measured by the fragmentation index (FI) method. Greater
291
fragmentation (lower FI values) of meat structure was observed in samples after 14 days of aging, which are
292
expected since the proteolysis of key myofibrillar proteins is the principal reason for improvement in meat
293
tenderness during post mortem storage (Koohmaraie et al., 1987).
294
Overall, irradiation resulted in higher FI values than the non-irradiated samples, indicating that the
295
gamma radiation negatively affected the meat fragmentation. This could be due to a reduction in the
296
proteolytic activity of the enzymes during ageing . According to Brewer (2004), the formation of free radicals
297
by radiation process creates highly oxidizing conditions, which can affect the meat's endogenous enzymes.
298
Rowe et al. (2004) reported that the calpains have an oxidizable cysteine residue in their active site, requiring 11
299
reducing conditions to be activated. These authors observed that the irradiation of meat with 6.4 kGy doses
300
decreased the proteolytic action of calpain I and reduced the rate of inactivation of its inhibitor, calpastatin,
301
within a period of 14 days of aging. These changes are consistent with less fragmentation observed in the
302
irradiated samples.
303
However, samples irradiated with higher doses (6 and 9 kGy) did not differ from the non-irradiated
304
samples. Some authors (Lee et al., 2000; Yook et al., 2001; Yoon, 2003) suggested that the irradiation process
305
can induce physical rupture of myofibrils directly by gamma rays (by breaking the actomyosin) or by
306
denaturation of structural proteins such as desmin. Thus, the inhibitory effect on the proteases could be
307
counterbalanced by the fragmentation arising from higher doses of irradiation.
308
For the shear force (WBsSF) values, both isolated effects (irradiation and aging time) were also
309
significant (p < 0.05). As expected, aging improved the samples tenderness. Moreover, lower shear force was
310
observed in samples irradiated at 9 kGy, which agrees with the observation of some authors that the radiation
311
process increased meat tenderness. Kanatt et al. (2015) reported lower shear force values in chicken
312
(Pectoralis major), lamb (Biceps femoris) and buffalo (Biceps femoris) meat with increasing irradiation dose
313
(2.5, 5 and 10 kGy) and this tenderization was attributed to increased collagen solubility. This is consistent
314
with the highest values of soluble collagen content observed in samples irradiated with 9 kGy in this
315
experiment.
316
In the scientific literature, however, the effects of relative low dose irradiation on meat tenderness are
317
still contradictory. As Kanatt et al. (2015), Yook et al. (2001) reported a significant reduction in shear force of
318
bovine Sternomandibularis muscle irradiated pre-rigor at low doses (3 and 5 kGy), being attributed by these
319
authors to a physical disruption of the myofibrils and, or, protein denaturation caused by gamma radiation.
320
This was not supported by the data of this experiment, since lower shear force seems not to be related to the
321
differences in protein solubility or myofibrillar fragmentation. Otherwise, Yoon (2003), evaluating the effect
322
of gamma irradiation (0.5 to 5 kGy) on tenderness of refrigerated chicken breasts, reported that the irradiated
323
samples had higher shear force (11.8 kgf) than the non-irradiated ones (6.81 kgf). These differences could be
324
due to the differences in muscle type and species, or even the moment of irradiation (pre- or post-rigor), and
325
it’s also observed in experiment with different irradiation source. Rowe et al. (2004) reported that beef steaks
326
irradiated at relatively low dose (6.4 kGy) of electron beam and aged by 14 days showed significantly higher 12
327
shear force values than non-irradiated steaks, while Kim et al. (2018) observed no adverse effect on 14-days
328
aged beef tenderness due to the pre-rigor irradiation (5 kGy) by electron beam or X-ray. Luchsinger et al.
329
(1997) also reported that shear forces 14-days aged beef were not influenced by X-ray irradiation up to 3.5
330
kGy.
331 332
3.5 Lipid oxidation
333 334
The effect of irradiation on lipid oxidation of beef during aging was monitored by the TBARS index
335
and was affected (p < 0.05) only by the gamma radiation treatments (Table 3). Beef irradiation at doses up to
336
6 kGy did not affect (p > 0.05) the TBARS values, but when 9 kGy were applied, a significant increase was
337
observed. This is consistent with the observation that ionizing radiation induces lipid oxidation by the
338
hydroxyl radicals generated in water radiolysis, which is dose-dependent (Brewer, 2004). Nevertheless, a high
339
dose of irradiation was required to induce lipid oxidation. This could be due to the fact that packaging (aerobic
340
vs vacuum) appears to have a greater effect on meat quality than does the process of irradiation alone.
341
Differences between irradiated and nonirradiated meat products occurred primarily when these products were
342
irradiated in air. The oxygen present during or after irradiation combine with free radicals generated to form
343
hydroperoxides, accelerating lipid oxidation in irradiated meat (Ahn, Olson, Jo, Chen, Wu, & Lee, 1998;
344
Luchsinger et al., 1997).
345
The TBARS results observed in this experiment agree with the observation of other authors for intact
346
muscles (Kim, Nam, & Ahn, 2002b; Luchsinger et al., 1997), who reported that TBARS numbers of 7- and
347
14-aged vacuum-packaged beefs steaks were not influenced by electron beam (3.0 kGy) or X-ray (2.0 and 3.5
348
kGy) when irradiated at relative low-dose.
349 350
3.5 Oxygen consumption rate and metmyoglobin reducing activity
351 352
The effects of gamma irradiation and aging on inherent metmyoglobin reducing capacity (MRA) and
353
oxygen consumption rates (OCR) of bovine LL muscles are described in the Table 3. These biochemical
13
354
characteristics have been implicated as important determinants of muscle color stability and both were
355
affected (p < 0.05) only by gamma irradiation doses.
356
The MRA was measured by the muscle-resistance to induce metmyoglobin (MMb) formation in the
357
presence of an oxidizing agent, in this case sodium nitrite. Therefore, the initial amount of MMb (IMF)
358
formed by oxidation in sodium nitrite solution and the percentage decrease in surface MMb concentration
359
during the incubation period (NORA) were related to the sample MRA. By the NORA, the MRA decreased (p
360
< 0.05) with irradiation, regardless of the dose applied, while the reducing capacity of the meat by IMF was
361
not affected (p > 0.05) by the application of up to 3 kGy.
362
The loss of MRA by irradiation could be due to the depletion and/or degradation of substrates and
363
cofactors, such as NADH, essential to the muscle reducing system (O'Keeffe & Hood, 1982). Kim, Keeton,
364
Smith, Maxim, Yang, and Savell (2009) reported a decreased in the NADH concentration of beef steaks when
365
irradiated by 2.5 kGy dose, being hypothesized as probably due to oxidation. An effect of an oxidation
366
environment is compatible with our results for lipid oxidation, although the TBARS values were only
367
significant at high irradiation doses.
368
Furthermore, the loss of MRA could be due to the loss of mitochondrial structural integrity and
369
functionality. It was suggested that mitochondria are involved in MMb reduction by regenerating the cofactor
370
NADH, by endogenous enzymes or by reversing electron transport, and to promote an anaerobic environment,
371
by oxygen consumption, that favors MMb reduction (Sammel, Hunt, Kropf, Hachmeister, & Johnson, 2002).
372
This is also consistent with the reduction (p < 0.05) in the OCR values observed in this experiment when
373
irradiation doses greater than 3 kGy were applied and could be due to the oxidative conditions generated by
374
higher irradiation doses. According to O'Keeffe and Hood (1982), besides degradation of enzymes involved in
375
mitochondrial respiration, the OCR also decreases due to depletion of substrates and coenzymes.
376 377
3.6 Myoglobin redox forms and CIE color
378 379 380
The effects of gamma irradiation and aging on the myoglobin chemical forms and CIE color of bovine LL muscles are described in the Tables 4 and 5.
14
381
For the myoglobin redox forms, both deoxymyoglobin (DMb) and oxymyoglobin (OMb) were affected
382
(p < 0.05) by the interaction of irradiation doses and aging, while metmyoglobin (MMb) was affected (p <
383
0.05) by these factors alone. Overall, the irradiation doses affected the myoglobin proportions in the samples.
384
At 1-day of aging, only a reduction in the OMb content was observed in samples irradiated by 9 kGy, but after
385
14-days of aging a simultaneous reduction in OMb and increase in DMb was observed as higher irradiation
386
doses were applied (Fig. 1). The MMb content also increased with higher doses of irradiation (Table 4). These
387
changes indicate the oxidation of the heme pigments during the irradiation process and are related to the
388
effects observed on the lipid oxidation and mainly on the MRA and OCR.
389
Ionizing radiation is a well-known pro-oxidant towards lipids and pigments. The highly reactive
390
substances, such as hydroxyl radicals, produced by water radiolysis during ionizing radiation can oxidize
391
myoglobin directly or they can oxidize lipids producing lipid radicals which subsequently oxidize myoglobin
392
(Brewer, 2004; Millar et al., 2000). Regarding MRA and OCR, as described above, meat color stability is
393
dependent on the residual enzymic activity in meat which, directly or indirectly, controls myoglobin
394
oxygenation, oxidation and reduction. The oxygen consumption plays a significant role in metmyoglobin
395
formation initially, but as oxygen consumption decreases, reducing activity becomes the predominant factor in
396
maintaining stability (King, Shackelford, Rodriguez, & Wheeler, 2011). Therefore, the reduction in the MRA
397
values explains the observed increase in MMb values with higher irradiation doses.
398
For the aging effects, higher OMb content was observed in non-irradiated samples aged by 14-days than
399
in 1-day. This OMb increase during aging could be explained by a reduction in OCR, which increase the beef
400
oxygen penetration during blooming, inducing a thicker layer of oxymyoglobin form (O'Keeffe & Hood,
401
1982). However, the OMb content reduced with the application of higher doses of irradiation, so that the
402
difference between aging also decreased. Moreover, an accumulation of MMb was also observed during
403
aging, which can be attributed to the loss of meat reduction capacity (MacDougall, 1982), although no
404
significant effects of aging was observed for MRA values.
405
Despite the observed changes in the myoglobin redox forms, irradiation doses affected (p < 0.05) only
406
the redness (a*) and chroma (C*, saturation index) values of the CIE color indices (Table 5). Moreover, the a*
407
and C* values had a similar behavior, decreasing (p < 0.05) when samples were irradiated at 9 kGy. Color is a
408
three-dimensional attribute being better described by its hue (h), lightness (L*) and saturation properties 15
409
(Ramos & Gomide, 2017), but although redness is a chromacity coordinate that, together with yellowness
410
(b*), are used to calculate hue and chroma, Holman, van de Ven, Mao, Coombs, and Hopkins (2017) reported
411
that a* value provided the most simple and robust prediction of beef color acceptability. Chroma represents
412
the color intensity, describing how vivid or dull the color is, and is a good indicator of the oxygenation of
413
meat recently exposed to air (Ramos & Gomide, 2017). Therefore, the a* and C* values reduction agrees with
414
the observed reduction in OMb content (and MMb increase) with higher irradiation doses (Table 4) and could
415
be attributed to the increase on the lipid oxidation and reduction on the MRA and OCR due to irradiation, as
416
previously discussed.
417
The results reported in the literature for the effects of irradiation on the color indices of beef steaks (not
418
ground) are variable. Luchsinger et al. (1997) reported that X-ray dose level (up to 3.5 kGy) did not affect CIE
419
colors indices in 14-days vacuum-packaged aged beef (L. lumborum), which corresponds to that observed in
420
this experiment for this applied dose range. Similarly, although working with pre-rigor meat, Kim et al. (2018)
421
showed that 5 kGy of electron beam (EB) and X-ray irradiation had no effect on the overall color (CIE
422
L*a*b*) of beef (Semimembranosus) after 14 days of storage at 4 °C, although a decrease in L* and a* values
423
were detected at day 0 in irradiated samples when compared to non-irradiated samples. Other works, however,
424
report different results. Nanke, Sebranek, and Olson (1999) reported that EB irradiation dose had no
425
significant effects on beef (L. dorsi) L*, b* and h values, but the a* and C* values decreased as irradiation
426
dose levels increased from 0 kGy to 4.5 kGy and increased as irradiation dose increased from 4.5 kGy to 10.5
427
kGy. These authors concluded that significant decreases in redness and increases in brown pigments would
428
develop at dose levels as low as 1.5 kGy in vacuum-packaged beef. Kim, Nam, and Ahn (2002a) also did not
429
observe a 3 kGy dose (EB) effect on beef (L. dorsi) L* and b* values after 7-days aging, but irradiated
430
samples had lower a* values than non-irradiated ones.
431
Despite the differences regarding the type of muscle (Longissimus or Semimembranosus), irradiation
432
type/source (electron beam, X-rays or gamma) and moment of irradiation (pre- and post-rigor), data from the
433
literature and from this experiment seem to point to an effect of irradiation on color intensity of vacuum-
434
packaged beef only when doses as high as 6 kGy are applied.
435
Regarding the aging effects, all CIE color indexes were affected by aging time (Table 5), with 14-aged
436
samples were lighter (higher L* values) and with a red hue more yellowish (higher h values) and intense 16
437
(higher C* values). These changes are consistent with those observed in Nellore beef after 14-days aging by
438
Aroeira et al. (2016).
439 440
4. Conclusion
441 442
The results of this current study found that the gamma radiation process adversely affected the Nellore
443
beef's shear force when doses of 3 or 6 kGy were applied, most often by reduction in proteolysis during aging.
444
However, higher doses of irradiation (9 kGy) increase the collagen solubility, reducing the samples shear
445
force. Although these results suggest that the application of gamma irradiation doses as high as 9 kGy
446
combined to aging can be used as an effective tool to improve tenderness of vacuum-packaged Nellore beef,
447
this radiation level also implied in some change in the meat color. Overall, samples irradiated at 9 kGy
448
exhibited significant surface discoloration, with higher proportion of metmyoglobin. However, it remains to
449
be known if these differences are perceptible by the consumers.
450 451
5. Acknowledgements
452 453
The authors would like to thank the Nuclear Technology Development Center (CDTN/CNEN), in the
454
person of researcher Marcio Tadeu Teixeira, for the irradiation samples, to the National Council for Scientific
455
and Technological Development (CNPq; 430206 / 2016-0) and the Minas Gerais State Research Support
456
Foundation (FAPEMIG; CVZ APQ-02015-15) for their financial support. Also, they thanks to the CNPq and
457
the Higher Education Personnel Improvement Coordination (CAPES) for the scholarship granting to the first
458
(master's degree/CNPq), second (master's degree/CNPq) and third (post-doctoral PNPD/CAPES) authors.
459 460
References
461 462
Ahn, D. U., Olson, D. G., Jo, C., Chen, X., Wu, C., & Lee, J. I. (1998). Effect of muscle type, packaging, and
463
irradiation on lipid oxidation, volatile production, and color in raw pork patties. Meat Science, 49(1),
464
27-39. https://doi.org/10.1016/S0309-1740(97)00101-0 17
465
Aroeira, C. N., Torres Filho, R. A., Fontes, P. R., Gomide, L. A. M., Ramos, A. L. S., Ladeira, M. M., &
466
Ramos, E. M. (2016). Freezing, thawing and aging effects on beef tenderness from Bos indicus and
467
Bos taurus cattle. Meat Science, 116, 118-125. https://doi.org/10.1016/j.meatsci.2016.02.006
468
Aroeira, C. N., Torres Filho, R. A., Fontes, P. R., Ramos, A. L. S., Gomide, L. A. M., Ladeira, M. M., &
469
Ramos, E. M. (2017). Effect of freezing prior to aging on myoglobin redox forms and CIE color of
470
beef
471
https://doi.org/10.1016/j.meatsci.2016.11.010
472
from
Nellore
and
Aberdeen
of
474
https://doi.org/10.1002/jsfa.2740150712
meat.
Journal
of
the
determination
477
https://doi.org/10.1021/ac60205a053
479
Meat
Science,
125,
16-21.
Science
of
Food
and
Agriculture,
15,
504-508.
Bergman, I., & Loxley, R. (1963). Two improved and simplified methods for the spectrophotometric
476
478
cattle.
Bailey, A. J., & Rhodes, D. N. (1964). Treatment of meats with ionising radiations. XI - changes in the texture
473
475
Angus
of
hydroxyproline.
Analytical
Chemistry,
35,
1961-1965.
Boakye, K., & Mittal, G. S. (1993). Changes in pH and water holding properties of Longissimus dorsi muscle during beef ageing. Meat Science, 34(3), 335-349. https://doi.org/10.1016/0309-1740(93)90082-S
480
Bowker, B. C., Fahrenholz, T. M., Paroczay, E. W., & Solomon, M. B. (2008). Effect of hydrodynamic
481
pressure processing and aging on sarcoplasmic proteins of beef strip loins. Journal of Muscle Foods,
482
19(2), 175-193. https://doi.org/10.1111/j.1745-4573.2007.00104.x
483 484
Brewer, S. (2004). Irradiation effects on meat color - a review. Meat Science, 68(1), 1-17. https://doi.org/10.1016/j.meatsci.2004.02.007
485
Cardoso, G. P., Andrade, M. P. D., Rodrigues, L. M., Massingue, A. A., Fontes, P. R., Ramos, A. d. L. S., &
486
Ramos, E. M. (2019). Retail display of beef steaks coated with monolayer and bilayer chitosan-gelatin
487
composites. Meat Science, 152, 20-30. https://doi.org/10.1016/j.meatsci.2019.02.009
488
Cardoso, G. P., Dutra, M. P., Fontes, P. R., Ramos, A. L. S., Gomide, L. A. M., & Ramos, E. M. (2016).
489
Selection of a chitosan gelatin-based edible coating for color preservation of beef in retail display.
490
Meat Science, 114, 85-94. https://doi.org/10.1016/j.meatsci.2015.12.012
18
491
Cross, H. R., Carpenter, Z. L., & Smith, G. C. (1973). Effects of intramuscular collagen and elastin on bovine
492
muscle tenderness. Journal of Food Science, 38(6), 998-1003. https://doi.org/10.1111/j.1365-
493
2621.1973.tb02133.x
494
Delgado, E. F., Aguiar, A. P., Ortega, E. M. M., Spoto, M. H. F., & Castillo, C. J. C. (2006). Brazilian
495
consumers' perception of tenderness of beef steaks classified by shear force and taste. Scientia
496
Agricola, 63, 232-239. https://doi.org/10.1590/S0103-90162006000300004.
497
Holman, B. W. B., van de Ven, R. J., Mao, Y., Coombs, C. E. O., & Hopkins, D. L. (2017). Using
498
instrumental (CIE and reflectance) measures to predict consumers' acceptance of beef colour. Meat
499
Science, 127, 57-62. https://doi.org/10.1016/j.meatsci.2017.01.005
500
Huff-Lonergan, E., & Lonergan, S. M. (2005). Mechanisms of water-holding capacity of meat: The role of
501
postmortem
502
https://doi.org/10.1016/j.meatsci.2005.04.022
biochemical
and
structural
changes.
Meat
Science,
71(1),
194-204.
503
Joo, S. T., Kauffman, R. G., Kim, B. C., & Park, G. B. (1999). The relationship of sarcoplasmic and
504
myofibrillar protein solubility to colour and water-holding capacity in porcine longissimus muscle.
505
Meat Science, 52(3), 291-297. https://doi.org/10.1016/S0309-1740(99)00005-4
506 507
Kanatt, S. R., Chawla, S. P., & Sharma, A. (2015). Effect of radiation processing on meat tenderisation. Radiation Physics and Chemistry, 111(0), 1-8. https://doi.org/10.1016/j.radphyschem.2015.02.004
508
Kim, S. Y., Yong, H. I., Nam, K. C., Jung, S., Yim, D.-G., & Jo, C. (2018). Application of high temperature
509
(14°C) aging of beef M. semimembranosus with low-dose electron beam and X-ray irradiation. Meat
510
Science, 136, 85-92. https://doi.org/10.1016/j.meatsci.2017.10.016
511
Kim, Y. H., Keeton, J. T., Smith, S. B., Maxim, J. E., Yang, H. S., & Savell, J. W. (2009). Evaluation of
512
antioxidant capacity and colour stability of calcium lactate enhancement on fresh beef under highly
513
oxidising
514
https://doi.org/10.1016/j.foodchem.2008.12.008
conditions.
Food
Chemistry,
115(1),
272-278.
515
Kim, Y. H., Nam, K. C., & Ahn, D. U. (2002a). Color, Oxidation-Reduction Potential, and Gas Production of
516
Irradiated Meats from Different Animal Species. Journal of Food Science, 67(5), 1692-1695.
517
https://doi.org/10.1111/j.1365-2621.2002.tb08707.x
19
518
Kim, Y. H., Nam, K. C., & Ahn, D. U. (2002b). Volatile profiles, lipid oxidation and sensory characteristics
519
of
520
https://doi.org/10.1016/S0309-1740(01)00191-7s
irradiated
meat
from
different
animal
species.
Meat
Science,
61(3),
257-265.
521
King, D. A., Shackelford, S. D., Rodriguez, A. B., & Wheeler, T. L. (2011). Effect of time of measurement on
522
the relationship between metmyoglobin reducing activity and oxygen consumption to instrumental
523
measures
524
https://doi.org/10.1016/j.meatsci.2010.08.013
of
beef
longissimus
color
stability.
Meat
Science,
87,
26–32.
525
Koohmaraie, M., Seidemann, S. C., Schollmeyer, J. E., Dutson, T. R., & Crouse, J. D. (1987). Effect of post-
526
mortem storage on Ca++-dependent proteases, their inhibitor and myofibril fragmentation. Meat
527
Science, 19(3), 187-196. https://doi.org/10.1016/0309-1740(87)90056-8
528 529 530
Krzywicki, K. (1979). Assessment of relative content of myoglobin, oxymyoglobin and metmyoglobin at the surface of beef. Meat Science, 3(1), 1-10. https://doi.org/10.1016/0309-1740(79)90019-6 Lee, J. W., Yook, H. S., Lee, K. H., Kim, J. H., Kim, W. J., & Byun, M. W. (2000). Conformational changes
531
of
532
https://doi.org/10.1016/S0969-806X(99)00466-1
myosin
by
gamma
irradiation.
Radiation
Physics
and
Chemistry,
58(3),
271-277.
533
Luchsinger, S. E., Kropf, D. H., García Zepeda, C. M., Hunt, M. C., Stroda, S. L., Marsden, J. L., & Kastner,
534
C. L. (1997). Color and oxidative properties of irradiated whole muscle beef. Journal of Muscle
535
Foods, 8(4), 427-443. https://doi.org/10.1111/j.1745-4573.1997.tb00730.x
536 537 538
MacDougall, D. B. (1982). Changes in the colour and opacity of meat. Food Chemistry, 9(1), 75-88. https://doi.org/10.1016/0308-8146(82)90070-X Madhavi, D. L., & Carpenter, C. E. (1993). Aging and Processing Affect Color, Metmyoglobin Reductase and
539
Oxygen
540
https://doi.org/10.1111/j.1365-2621.1993.tb06083.x
Consumption
of
Beef
Muscles.
Journal
of
Food
Science,
58(5),
939-942.
541
Millar, S. J., Moss, B. W., & Stevenson, M. H. (2000). The effect of ionising radiation on the colour of beef,
542
pork and lamb. Meat Science, 55(3), 349-360. https://doi.org/10.1016/S0309-1740(99)00164-3
543
Nanke, K. E., Sebranek, J. G., & Olson, D. G. (1999). Color characteristics of irradiated aerobically packaged
544
pork, beef, and turkey. Journal of Food Science, 64(2), 272-278. https://doi.org/10.1111/j.1365-
545
2621.1998.tb15842.x 20
546
O'Keeffe, M., & Hood, D. E. (1982). Biochemical factors influencing metmyoglobin formation on beef from
547
muscles of differing colour stability. Meat Science, 7(3), 209-228. https://doi.org/10.1016/0309-
548
1740(82)90087-0
549
Raharjo, S., Sofos, J. N., & Schmidt, G. R. (1992). Improved speed, specificity, and limit of determination of
550
an aqueous acid extraction thiobarbituric acid-C18 method for measuring lipid peroxidation in beef.
551
Journal of Agricultural and Food Chemistry, 40 (11), 2182-2185. https://doi.org/10.1021/jf00023a02
552 553
Ramos, E. M., & Gomide, L. A. M. (2017). Avaliação da Qualidade de Carnes: Fundamentos e Metodologias (2a ed.). Viçosa: Editora UFV.
554
Rowe, L. J., Maddock, K. R., Lonergan, S. M., & Huff-Lonergan, E. (2004). Oxidative environments decrease
555
tenderization of beef steaks through inactivation of μ-calpain. Journal of Animal Science, 82(11),
556
3254-3266. https://doi.org/10.2527/2004.82113254x
557
Sammel, L. M., Hunt, M. C., Kropf, D. H., Hachmeister, K. A., & Johnson, D. E. (2002). Comparison of
558
Assays for Metmyoglobin Reducing Ability in Beef Inside and Outside Semimembranosus Muscle.
559
Journal of Food Science, 67(3), 978-984. https://doi.org/10.1111/j.1365-2621.2002.tb09439.x
560
Silva, D. R. G., Torres Filho, R. A., Cazedey, H. P., Fontes, P. R., Ramos, A. L. S., & Ramos, E. M. (2015).
561
Comparison of Warner–Bratzler shear force values between round and square cross-section cores
562
from
563
https://doi.org/10.1016/j.meatsci.2014.12.009
cooked
beef
and
pork
Longissimus
muscle.
Meat
Science,
103,
1-6.
564
Vieira, C., Diaz, M. T., Martínez, B., & García-Cachán, M. D. (2009). Effect of frozen storage conditions
565
(temperature and length of storage) on microbiological and sensory quality of rustic crossbred beef at
566
different
567
https://doi.org/10.1016/j.meatsci.2009.06.013
568 569
states
of
ageing.
Meat
Science,
83(3),
398-404.
Watts, B. M., Kendrick, J., Zipser, M. W., Hutchins, B., & Saleh, B. (1966). Enzymatic Reducing Pathways in Meat. Journal of Food Science, 31(6), 855-862. https://doi.org/10.1111/j.1365-2621.1966.tb03261.x
570
Wheeler, T. L., Miller, R. K., Savell, J. W., & Cross, H. R. (1990). Palatability of Chilled and Frozen Beef
571
Steaks. Journal of Food Science, 55(2), 301-304. https://doi.org/10.1111/j.1365-2621.1990.tb06748.x
572
WHO. (1999). High-dose irradiation: wholesomeness of food irradiated with doses above 10 kGy. Report of a
573
Joint FAO/IAEA/WHO Study Group. World Health Organ Tech Rep Ser, 890. 21
574
Yook, H. S., Lee, J. W., Lee, K. H., Kim, M. K., Song, C. W., & Byun, M. W. (2001). Effect of gamma
575
irradiation on the microstructure and post-mortem anaerobic metabolism of bovine muscle. Radiation
576
Physics and Chemistry, 61(2), 163-169. https://doi.org/10.1016/S0969-806X(00)00392-3
577 578
Yoon, K. S. (2003). Effect of gamma irradiation on the texture and microstructure of chicken breast meat. Meat Science, 63(2), 273-277. https://doi.org/whip10.1016/S0309-1740(02)00078-5
579
Zabielski, J., Kijowski, J., Fiszer, W., & Niewiarowicz, A. (1984). The effect of irradiation on technological
580
properties and protein solubility of broiler chicken meat. Journal of the Science of Food and
581
Agriculture, 35(6), 662-670. https://doi.org/10.1002/jsfa.2740350612
582 583
Conflict of interest
584 585
The authors declare that there is no conflict of interest.
586
22
588
Figure 1. Gamma irradiation × aging time interaction on myoglobin redox forms of bovine Longissimus
589
lumborum muscle: (A) oxymyoglobin (OMb); and (B) deoxymyoglobin (DMb). Means with different letters
590
(a-d), for irradiation dose within each aging time, differs (P < 0.05). ns = difference between means of day 1
591
and day 14 are not significant (P > 0.05). Bars represent the standard error of means.
592 593
Research Highlights
594 595
Gamma irradiation reduces WHC and increases purge and cooking loss
596
Gamma irradiation negatively affected myofibrillar fragmentation
597
Irradiation at 9 kGy induces higher soluble collagen and lower shear force
598
Higher irradiation dose oxidizes myoglobin to metmyoglobin
599
Irradiation at 9 kGy induce lipid oxidation and surface discoloration
600 601 602 603
SAMPLE CREDIT AUTHOR STATEMENT
604 605 606
Lorena Mendes Rodrigues: Conceptualization, Methodology, Investigation, Writing- Original draft preparation.
607
Luana Aparecida Sales: Conceptualization, Investigation.
608
Paulo Rogério Fontes: Validation.
609
Robledo de Almeida Torres Filho: Formal analysis, Validation.
610
Monalisa Pereira Dutra Andrade: Validation.
611
Alcinéia de Lemos Souza Ramos: Formal analysis, Writing- Reviewing and Editing.
612
Eduardo Mendes Ramos: Supervision, Funding acquisition, Writing- Reviewing and Editing.
613 614 615 23
616
Table 1. Gamma irradiation dose (G) and aging time (A) effects on pH, extractable proteins and water losses
617
values (means ± standard deviations) of bovine Longissimus lumborum muscle. Soluble proteins (mg/g) Source of variation
Effects
Purge
pH
Sarcoplasmic
Myofibrillar
Gamma irradiation Non-irradiated
5.58±0.14
45.68±8.53
70.40±22.74
2.24±1.37a
dose (G)
3 kGy
5.62±0.11
47.57±17.13
66.58±15.13
3.40±2.02b
6 kGy
5.61±0.16
49.12±8.85
73.34±16.50
3.35±2.06b
9 kGy
5.61±0.09
49.32±11.18
64.78±15.75
3.38±2.15b
1 day
5.61±0.12
51.68±12.80x
63.92±17.47x
1.91±1.03x
14 days
5.59±0.14
44.16±5.64y
73.05±16.88y
4.28±1.93y
G
0.864
0.748
0.525
0.038
A
0.636
0.006
0.037
<0.001
G×A
0.961
0.387
0.340
0.069
Aging time (A)
Pr > F1
(%)
618
FPPM = filter paper press method.
619
1 Significant
620
a-b Means
in the same column, into gamma irradiation effect, followed by different letters differ (P < 0.05).
621
x-y Means
in the same column, into aging effect, followed by different letters differ (P < 0.05).
probabilities (P < 0.05) were placed in bold.
622
24
624
Table 2. Gamma irradiation dose (G) and aging time (A) effects on collagen content, myofibrillar
625
fragmentation and shear force values (means ± standard deviations) of bovine Longissimus lumborum
626
muscle. Collagen (mg/g) Source of variation
Effects
Collagen soluble
Total
Insoluble
Soluble
Gamma irradiation Non-irradiated
2.23±0.35a
1.84±0.32a
0.39±0.09a
17.77±3.53a
dose (G)
3 kGy
2.72±0.88b
2.29±0.86b
0.43±0.11ab
17.07±6.46a
6 kGy
2.84±0.96b
2.36±1.01b
0.48±0.12b
18.52±6.34a
9 kGy
2.10±0.26a
1.60±0.22a
0.49±0.09b
23.59±3.48b
1 day
2.54±0.91
2.08±0.88
0.46±0.12
19.44±6.29
14 days
2.40±0.61
1.96±0.59
0.44±0.10
19.04±5.05
G
0.002
0.001
0.008
<0.001
A
0.338
0.383
0.359
0.699
G×A
0.463
0.539
0.082
0.324
Aging time (A)
Pr > F1
(%)
627
FI = fragmentation index; WBsSF = Warner-bratzler square shear force.
628
1 Significant
629
a-b Means
in the same column, into gamma irradiation effect, followed by different letters differ (P < 0.05).
630
x-y Means
in the same column, into aging effect, followed by different letters differ (P < 0.05).
probabilities (P < 0.05) were placed in bold.
631
25
633
Table 3. Gamma irradiation dose (G) and aging time (A) effects on lipid oxidation, metmyoglobin reducing
634
activity (MRA), oxygen consumption rate (OCR) and myoglobin chemical forms (means ± standard
635
deviations) of bovine Longissimus lumborum muscle. MRA (%) Effects
Source of variation
Gamma irradiation
Non-irradiated
dose (G)
Aging time (A)
Pr > F1
TBARS
Myog OCR
IMF
NORA
0.21±0.31a
24.19±17.98a
40.05±15.82a
98.07±4.02a
15.60±6.62
3 kGy
0.39±0.36a
28.07±18.46a
31.61±6.27b
86.35±11.51a
15.70±4.32
6 kGy
0.36±0.31a
39.79±3.05b
32.17±11.68b
45.89±24.18b
17.06±3.67
9 kGy
0.87±0.66b
40.98±1.68b
25.89±4.73b
38.61±21.49b
19.34±3.62
1 day
0.45±0.50
32.78±14.74
33.06±14.08
68.66±32.58
19.53±4.29
14 days
0.47±0.58
33.74±14.78
31.79±8.38
65.80±29.15
14.32±3.92
G
<0.001
<0.001
0.001
<0.001
<0.001
A
0.833
0.713
0.573
0.476
<0.001
G×A
0.487
0.884
0.227
0.683
0.002
(mg MAD/kg)
DMb
(%)
636
MAD = malonaldehyde; IMF = initial metmyoglobin formed; NORA = nitric oxide metmyoglobin reducing
637
activity; DMb = deoxymyoglobin; OMb = oxymyoglobin; and MMb = metmyoglobin.
638
1 Significant
639
a-b Means
in the same column. into gamma irradiation effect. followed by different letters differ (P < 0.05).
640
x-y Means
in the same column. into aging effect. followed by different letters differ (P < 0.05).
probabilities (P < 0.05) were placed in bold.
26
642
Table 4. Gamma irradiation dose (G) and aging time (A) effects on CIE color (means ± standard deviations) of
643
bovine Longissimus lumborum muscle.
Effects
Source of variation
Gamma irradiation dose (G)
Aging time (A)
Pr > F1
L*
a*
b*
Non-irradiated
43.62±3.16
22.40±2.20a
15.08±2.28
27.01
3 kGy
43.80±2.78
22.37±1.63a
15.14±1.71
27.03
6 kGy
44.32±2.45
22.15±1.45ab
15.06±1.59
26.79
9 kGy
44.24±2.69
21.32±1.93b
14.46±1.86
25.77
1 day
42.80±2.43x
21.24±1.80x
14.08±1.79x
25.5
14 days
45.19±2.52y
22.87±1.50y
15.79±1.51y
27.8
G
0.232
0.020
0.157
0.
A
<0.001
<0.001
<0.001
<0
G×A
0.547
0.651
0.581
0.
644
L* = lightness; a* = redness; b* = yellowness; C* = chroma; and h = hue angle.
645
1 Significant
646
a-b Means
in the same column. into gamma irradiation effect. followed by different letters differ (P < 0.05).
647
x-y Means
in the same column. into aging effect. followed by different letters differ (P < 0.05).
C
probabilities (P < 0.05) were placed in bold.
648 649
27