The Brazil nut is an important product from the Amazonian region and its productive chain is an income source for local communities. The effect of combinations of packaging atmospheres (loose or vacuum-packed) and storage temperatures (4±1 °C or 24±2 °C) on the tendency of lipid radical formation and on volatiles was investigated for the first time in shelled Brazil nut kernels. It was observed that refrigeration, whether combined with lose packing or vacuum packing, was effective to reduce the tendency for lipid radical formation, as detected by spin-trapping electron spin resonance (ESR) spectroscopy, as well as peroxides, conjugated dienes and 3-octen-2-one. However, the combination of refrigeration with vacuum packing, even using low-density polyethylene (LDPE) pouches with a high oxygen transmission rate (OTR), also reduced the formation of hexanal, which is a major off-flavor volatile, and thus should be recommended for the storage of Brazil nut kernels for the studied period.
Brazil nuts are the seeds of the
Regarding biochemical composition, the Brazil nut kernel is the greatest food source of selenium, which plays a key role as cofactor for antioxidant glutathione peroxidase (Rotruck
As Brazil nut kernels have high lipid contents (60-70%), of which around 40% is linoleic acid (USDA,
Therefore, the objective of the present study was to investigate for the first time the effect of retail storage conditions on the tendency for lipid radical formation and on volatiles in shelled Brazil nut kernels.
One metalized vacuum-packed bag of 20 kg of fresh and shelled Brazil nut kernels of small size (at least 68 kernels in 453 g) was purchased from a local market. After thorough mixing, portions of the kernels (300 g) were placed in low-density polyethylene (LDPE) pouches, which are commonly used as packaging material, vacuum or loose, for nuts in Brazil, with an estimated oxygen transmission rate (OTR) of 9843 cm3/m2/24h (at 23 °C and 0% relative humidity). The Kernels were vacuum or loose-packed on a sealing machine (model 300 B, Selovac, São Paulo, Brazil) and stored in the dark at ambient temperature (24±2 °C) and under refrigeration (4±1 ºC). Therefore, four treatments were evaluated:
Ambient temperature, loose packing (treatment AL)
Refrigerated temperature, loose packing (treatment RL)
Ambient temperature, vacuum packing (treatment AV)
Refrigerated temperature, vacuum packing (treatment RV)
Temperature was monitored using a thermo hygrometer (Incoterm, Porto Alegre, Brazil), and the effectiveness of the sealing was checked by visually evaluating the formation of air bubbles when submerging an extra pouch (not used in the study) in water after sealing each three pouches. Every 30 days and during four months, samples were collected and 200 g were chopped in a domestic mixer and stored under vacuum in LDPE pouches, while 100 g were cold pressed with a hydraulic press (Carver, Wabash, USA) under up to 172 MPa and the obtained oil was filtered and stored in Eppendorf flasks. The chopped kernels and the cold pressed oils were kept at −18 °C in the dark until analysis. All chemicals were analytical grade or higher as required.
The composition of fatty acid methyl esters (FAME) in the cold-pressed oil was assessed at time zero and after four months of storage. FAME were prepared based on the method described by Hartman and Lago (
GC analysis: FAME solution (1 mL) was added to 0.1 mL of a solution of methyl tridecanoate (T0627, Sigma-Aldrich, St. Louis, USA) diluted in
The spin-trapping ESR method was based on procedures described by Thomsen
Overlap of the ESR spectra of the treatments after 4 months and the peak used to determine the tendency of lipid radical formation (indicated by the vertical arrow).
AL: Ambient conditions and loose packing; RL: Refrigerated temperature and loose packing; AV: Ambient conditions and vacuum packing; RV: Refrigerated temperature and vacuum packing.
Peroxide value (PV) was measured according to the method described by Shantha and Decker (
Specific absorption at 232 nm (K232) was determined according to standard method ISO 3656:2011, and the solvent was
The procedures to concentrate and measure VAC formation were based on a method described by de Camargo
A panel composed of seven assessors evaluated the samples of chopped kernels. Criteria to choose assessors were to be familiar with sensory analysis, Brazil nuts and rancid odor in foods. A type I incomplete Latin square (t = 21; k = 5; r = 5; b = 21; ʎ = 1, E = 0.84) design was used (Cochran and Cox,
In order to choose the most appropriate statistical tests, all data were first checked for normality, which is the likelihood that the data be distributed normally, by Ryan-Joyner’s test, and for homoscedasticity, which is homogeneity of variances, by Bartlett’s test. As all data were parametric, mean values were evaluated by analysis of variance (one-way ANOVA), and, in case of mean differences, Tukey’s test was used. Pearson correlation was tested between PV, K232 and ESR analysis data. The level of confidence of 0.05 was considered and all statistical analyses were determined using Minitab® 17 software (Minitab Inc., State College, USA).
The composition of major fatty acids was determined at time zero and after four months of storage (
Composition of major fatty acids in shelled Brazil nuts stored under different temperatures and atmospheres
Fatty acid | Time zero | 4 months |
|||
---|---|---|---|---|---|
AL | RL | AV | RV | ||
Palmitic (C16:0) | 15.91 ± 0.03 | 17.09 ± 0.02 | 16.23 ± 0.00 | 16.44 ± 0.02 | 15.94 ± 0.02 |
Palmitoleic (C16:1) | 0.37 ± 0.00 | 0.38 ± 0.00 | 0.37 ± 0.00 | 0.43 ± 0.00 | 0.35 ± 0.00 |
Stearic (C18:0) | 11.30 ± 0.01 | 12.17 ± 0.02 | 10.78 ± 0.02 | 11.33 ± 0.03 | 11.67 ± 0.01 |
Oleic (C18:1) | 32.83 ± 0.02 | 30.94 ± 0.00 | 32.56 ± 0.00 | 32.60 ± 0.01 | 32.44 ± 0.05 |
Linoleic (C18:2) | 39.35 ± 0.00 | 39.15 ± 0.04 | 39.82 ± 0.02 | 38.96 ± 0.06 | 39.35 ± 0.04 |
Arachidic (C20:0) | 0.25 ± 0.00 | 0.26 ± 0.00 | 0.23 ± 0.00 | 0.24 ± 0.00 | 0.25 ± 0.00 |
∑SFA | 27.45 ± 0.02 | 29.53 ± 0.04 | 27.24 ± 0.02 | 28.01 ± 0.04 | 27.85 ± 0.01 |
∑MUFA | 33.20 ± 0.02 | 31.32 ± 0.00 | 32.94 ± 0.00 | 33.03 ± 0.01 | 32.80 ± 0.05 |
∑PUFA | 39.35 ± 0.00 | 39.15 ± 0.04 | 39.82 ± 0.02 | 38.96 ± 0.06 | 39.35 ± 0.04 |
Results expressed as mean ± standard deviation (n = 2) of percent mass of total fatty acid mass. SFA: Total saturated fatty acids; MUFA: Total monounsaturated fatty acids; PUFA: Total polyunsaturated fatty acids. AL: Ambient conditions and loose packing; RL: Refrigerated temperature and loose packing; AV: Ambient conditions and vacuum packing; RV: Refrigerated temperature and vacuum packing.
After four months of storage, the fatty acid profile showed small alterations when compared with time zero (
To the best of our knowledge, this was the first time ESR spectroscopy was used to measure radical species in Brazil nut kernels and in nuts in general (Andersen and Skibsted,
Results for the tendency for radical formation are shown in
Tendency of lipid radical formation, peroxide value and specific extinction at 232 nm in Brazil nuts stored under different temperatures and atmospheres
Tendency of lipid radical formation (a.u.) |
||||
---|---|---|---|---|
Time (months) | AL | RL | AV | RV |
0 | 5.37 ± 0.44D | 5.37 ± 0.44B | 5.37 ± 0.44D | 5.37 ± 0.44B |
1 | 9.37 ± 0.28Ca | 5.16 ± 0.07Bb | 8.94 ± 0.00Ca | 5.03 ± 0.00Bb |
2 | 10.04 ± 0.10Cb | 4.07 ± 0.11Cc | 11.40 ± 0.07Ba | 4.79 ± 0.02Bc |
3 | 13.70 ± 0.11Ba | 4.42 ± 0.10Cb | 13.31 ± 0.33Aa | 5.71 ± 0.49Bb |
4 | 18.06 ± 0.21Aa | 6.09 ± 0.11Ac | 13.61 ± 0.04Ab | 6.93 ± 0.40Ac |
|
|
|
|
|
0 | 2.68 ± 0.18C | 2.68 ± 0.18B | 2.68 ± 0.18C | 2.68 ± 0.18A |
1 | 3.88 ± 0.27Ba | 2.16 ± 0.11CDbc | 2.64 ± 0.21Cb | 2.17 ± 0.10Bc |
2 | 4.68 ± 0.31Bb | 2.32 ± 0.03Cc | 5.47 ± 0.34Ba | 2.03 ± 0.19Bc |
3 | 4.44 ± 0.24Bb | 3.16 ± 0.14Ac | 5.91 ± 0.50Ba | 1.46 ± 0.06Cd |
4 | 9.86 ± 0.70Aa | 1.98 ± 0.10Dc | 8.12 ± 0.07Ab | 2.77 ± 0.00Ac |
|
|
|
|
|
0 | 3.16 ± 0.03E | 3.16 ± 0.03B | 3.16 ± 0.03D | 3.16 ± 0.03C |
1 | 3.95 ± 0.03Da | 3.13 ± 0.00Bc | 3.81 ± 0.09Cb | 3.13 ± 0.02Cc |
2 | 4.38 ± 0.09Cb | 2.81 ± 0.10Cc | 5.00 ± 0.09Ba | 2.91 ± 0.09Dc |
3 | 5.15 ± 0.02Ba | 3.05 ± 0.13Bc | 5.38 ± 0.00Aa | 3.37 ± 0.12Bb |
4 | 6.00 ± 0.14Aa | 3.61 ± 0.04Ac | 5.62 ± 0.16Ab | 3.56 ± 0.04Ac |
Results expressed as mean ± standard deviation (n = 3). Means followed by different superscript upper-case letters within the same column are significantly different. Means followed by different superscript lower-case letters within the same row are significantly different. a.u.: adimensional unit. Statistical tests used: one-way ANOVA and Tukey’s test, with a level of confidence of 0.05. AL: Ambient conditions and loose packing; RL: Refrigerated temperature and loose packing; AV: Ambient conditions and vacuum packing; RV: Refrigerated temperature and vacuum packing.
Although the identification of the trapped free radicals are hindered due to their addition to the PBN molecule (Andersen and Skibsted,
During the first month, PV slightly increased for AL (3.88±0.27 meq O2/kg) and remained low for the other treatments, with values varying from 2.16±0.11 meq O2/kg for RL to 2.64±0.21 meq O2/kg for AV. Thereafter, the PV for kernels stored under refrigeration (RL and RV) tended to remain low during the entire storage, not exceeding 3.16 meq O2/kg. For AL, the PV remained low from the first to the third month (from 3.88 to 4.68 meq O2/kg) and then sharply increased during the fourth month, reaching 9.86±0.70 meq O2/kg. Meanwhile, it continued to increase, reaching 8.12±0.07 meq O2/kg after four months for AV. Thus, as expected, refrigeration prevented peroxide formation in stored kernels, which is in agreement with Ribeiro
In contrast, vacuum did not show the expected protective effect for AV, most likely due to the relatively high OTR of the LDPE pouches used, which might not have properly maintained a vacuum atmosphere within the package over time, although OTR was not estimated during storage to confirm this hypothesis. Chun
Another method for measuring primary lipid oxidation products is K232, which is based on the property of conjugated diene hydroperoxides formed in oils containing linoleic acid to give rise to an absorption peak at 232 nm in the ultraviolet region. The results for K232 are comprised in
However, the K232 results were similar to those found for the tendency of lipid radical formation, and this similarity was confirmed by high correlation coefficients between these methods for all treatments at a level of confidence of 0.01 (AL: 0.980; AV: 0.975; RL: 0.876, RV: 0.851). Stronger correlations between K232 and the ESR analysis than between PV and the ESR analysis were also observed for cold-pressed Brazil nut oils stored in brown glass bottles at room temperature (Sartori
The VACs detected by HS-SPME-GC-MS in the kernels were aldehydes, ketones, alcohols and pyrroles, as shown at
Volatile aroma compounds in shelled Brazil nuts stored under different temperatures and atmospheres
Volatile aroma compounds | LRI | Treatment | Time zero | 1 month | 2 months | 3 months | 4 months |
---|---|---|---|---|---|---|---|
Hexanal |
800.8 | AL | 2878 ± 129d | 4029 ± 170Ac | 4568 ± 142Ab | 5391 ± 231Aa | 5268 ± 320Ba |
RL | 2878 ± 129d | 3760 ± 289Ac | 4227 ± 324Abc | 4513 ± 233Bb | 5760 ± 293Aa | ||
AV | 2878 ± 129c | 4279 ± 141Ab | 4443 ± 304Aab | 4489 ± 232Bab | 4959 ± 233Ba | ||
RV | 2878 ± 129c | 2829 ± 165Bc | 3198 ± 126Bb | 3525 ± 6Ca | 2836 ± 320Cc | ||
960.3 | AL | 390 ± 15b | 373 ± 15BCb | 401 ±13Bb | 451 ±13Aa | 472 ± 18Ba | |
RL | 390 ± 15b | 377 ± 20Bb | 385 ± 10BCb | 465 ± 33Aa | 422 ± 23Cab | ||
AV | 390 ± 15b | 476 ± 19Aa | 467 ± 17Aa | 462 ± 23Aa | 507 ± 3Aa | ||
RV | 390 ± 15a | 325 ± 21Cb | 349 ± 23Cab | 310 ± 6Bb | 368 ± 13Da | ||
1060.9 | AL | 51 ± 4e | 90 ± 6Bd | 110 ± 5Ac | 158 ± 12Aa | 129 ± 5Bb | |
RL | 51 ± 4d | 91 ± 7Bc | 118 ± 15Abc | 146 ± 14Ab | 201 ± 16Aa | ||
AV | 51 ± 4c | 123 ± 8Aab | 106 ± 2Ab | 135 ± 5Aa | 124 ± 13Bab | ||
RV | 51 ± 4c | 104 ± 15Aba | 101 ± 1Aa | 81 ± 0Bb | 92 ± 2Cab | ||
Nonanal | 1105.0 | AL | 88 ± 5bc | 100 ± 8Bbc | 102 ± 6Bb | 133 ± 7Aa | 86 ± 4Cc |
RL | 88 ± 5b | 99 ± 11Bb | 107 ± 1ABb | 135 ± 13Aa | 136 ± 2Aa | ||
AV | 88 ± 5c | 127 ± 3Aab | 115 ± 4Ab | 128 ± 7Aa | 131 ± 4Aa | ||
RV | 88 ± 5c | 82 ± 2Bcd | 77 ± 2Cd | 127 ± 7Aa | 99 ± 8Bb | ||
3-Octen-2-one | 1041.7 | AL | 255 ± 25d | 314 ± 17ABc | 390 ± 23Ab | 461 ± 27Aa | 472 ± 23Ba |
RL | 255 ± 25ab | 255 ± 22BCab | 229 ± 23Cb | 313 ± 31Ba | 291 ± 14Cab | ||
AV | 255 ± 25d | 339 ± 21Ac | 427 ± 9Ab | 463 ± 8Ab | 508 ± 10Aa | ||
RV | 255 ± 25abc | 238 ± 33Cbc | 282 ± 4Bab | 226 ± 14Cc | 291 ± 20Ca | ||
2-Nonanone | 1093.4 | AL | 49 ± 9d | 170 ± 7Bc | 167 ± 12Bc | 335 ± 21Ca | 207 ± 1Ab |
RL | 49 ± 9d | 121 ± 4Cc | 148 ± 19Bc | 310 ± 29Ca | 219 ± 19Ab | ||
AV | 49 ± 9e | 275 ± 6Ac | 413 ± 35Ab | 583 ± 70Ba | 163 ± 4Bd | ||
RV | 49 ± 9c | 124 ± 3Cb | 109 ± 6Cb | 2170 ± 194Aa | 121 ± 13Cb | ||
2-Decanone | 1192.0 | AL | Nd | 33 ± 2Ac | 42 ± 3Ab | 52 ± 3Ba | 39 ± 0Ab |
RL | Nd | 29 ± 4Ab | 33 ± 5Bb | 51 ± 5Ba | 34 ± 4Bb | ||
AV | Nd | 28 ± 1Abc | 29 ± 1Bb | 52 ± 4Ba | 24 ± 0Cc | ||
RV | Nd | 11 ± 4Bc | 20 ± 1Cb | 126 ± 23Aa | 22 ± 0Cb | ||
1-Octen-3-ol | 982.4 | AL | 266 ± 43b | 429 ± 29Bb | 500 ± 25Aba | 565 ± 39Aa | 541 ± 27Ba |
RL | 266 ± 43d | 406 ± 19Bc | 427 ± 54Bbc | 514 ± 46ABab | 559 ± 26Ba | ||
AV | 266 ± 43c | 545 ± 29Ab | 521 ± 11Ab | 545 ± 32Ab | 698 ± 23Aa | ||
RV | 266 ± 43b | 409 ± 63Ba | 443 ± 27Aba | 425 ± 15Ba | 442 ± 24Ca | ||
1-Pentanol | 770.4 | AL | 174 ± 7d | 265 ± 13Bc | 333 ± 10Ab | 420 ± 35Aa | 368 ± 14Ab |
RL | 174 ± 7d | 225 ± 19Cc | 252 ± 16Bbc | 279 ± 26Bb | 403 ± 19Aa | ||
AV | 174 ± 7c | 322 ± 11Ab | 307 ± 34ABb | 381 ± 23Aab | 441 ± 78Aa | ||
RV | 174 ± 7b | 257 ± 14BCa | 256 ± 28Ba | 197 ± 12Cb | 240 ± 10Ba | ||
2-Nonanol | 1100.3 | AL | 4 ± 1c | 18 ± 1Bb | 20 ± 0Ab | 24 ± 3Ca | 20 ± 0Bab |
RL | 4 ± 1e | 12 ± 2Cd | 19 ± 1Ac | 31 ± 2Ca | 26 ± 1Ab | ||
AV | 4 ± 1c | 22 ± 1Ab | 20 ± 1Ab | 85 ± 17Ba | 19 ± 0Cb | ||
RV | 4 ± 1d | 7 ± 1Dc | 10 ± 0Bb | 813 ± 63Aa | 10 ± 2Db | ||
1-Methyl-1 |
746.2 | AL | 179 ± 9c | 265 ± 18Ab | 240 ± 3Bb | 273 ± 28Bb | 495 ± 12Aa |
RL | 179 ± 9d | 261 ± 19Ac | 290 ± 21Abc | 435 ± 33Aa | 327 ± 25Bb | ||
AV | 179 ± 9b | 187 ± 16Bb | 183 ± 12Bc | 268 ± 2Ba | 327 ± 49Ba | ||
RV | 179 ± 9c | 225 ± 13ABab | 199 ± 17Cbc | 188 ± 4Cc | 245 ± 10Ca |
Results expressed as mean ± standard deviation (n = 3) of the peak area (adimensional unit). Means followed by different superscript upper-case letters within the same column are significantly different. Means followed by different superscript lower-case letters within the same row are significantly different. Statistical tests used: one-way ANOVA and Tukey’s test, with a level of confidence of 0.05. LRI: Linear retention index. AL: Ambient conditions and loose packing; RL: Refrigerated temperature and loose packing; AV: Ambient conditions and vacuum packing: RV: Refrigerated temperature and vacuum packing.
Identification confirmed by comparing mass spectrum and retention time with reference standard.
Aldehydes are important VACs related to (off)-flavor in foods, and some of them, such as hexanal, which is a major product from linoleic acid oxidation, are used as markers for secondary lipid oxidation product formation (Barriuso
Except for hexanal, there is a lack of studies which monitor the formation of other volatiles during storage in foods (Barriuso
On the other hand, the formation of
Ketones generally have low thresholds and may contribute to the flavor profile of foods. The formation of 3-octen-2-one was continuously increased in the kernels stored under ambient conditions (AL and AV); while it remained low in the kernels stored under refrigeration (RL and RV), which indicates a clear effect of temperature. 3-Octen-2-one occurs as unsaturated ketone formed during linoleic acid autoxidation (Ullrich and Grosch,
The formation of 1-octen-3-ol and 1-pentanol increased for all treatments, but less for RV (
Pyrrole-derivatives are heterocyclic aromatic compounds that may be products of the interaction between amino acids and aliphatic aldehydes (Adams
Overall, with the exception for 3-octen-2-one, the effects of the considered storage time, temperature and atmosphere on VAC formation were shown to be quite different from the tendency of lipid free radicals and primary lipid oxidation product formation, which were affected only by temperature. For off-flavor VACs, packing and temperature conditions may act synergistically, since vacuum packing with refrigeration lowered the contents of some of them (hexanal,
Sensory analysis was conducted to verify the effect of undergoing chemical changes on rancidity and the results for rancid odor are shown in
Rancid odor attributed by the trained assessors to Brazil nut samples with respect to storage conditions and period
Treatment | Time zero | 1 month | 2 months | 3 months | 4 months |
---|---|---|---|---|---|
AL | 1.7 ± 0.3 | 1.4 ± 0.9 | 1.3 ± 0.4 | 2.0 ± 0.7 | 1.6 ± 0.5 |
RL | 1.4 ± 0.5 | 1.2 ± 0.4 | 2.0 ± 0.7 | 1.3 ± 0.4 | |
AV | 1.8 ± 0.8 | 1.5 ± 0.5 | 1.8 ± 0.8 | 1.8 ± 0.8 | |
RV | 1.4 ± 0.5 | 2.0 ± 1.0 | 1.3 ± 0.4 | 1.6 ± 0.9 |
No significant changes were detected. Statistical tests used: one-way ANOVA and Tukey’s test, with a level of confidence of 0.05. Data from time zero is an average of the five samples used. AL: Ambient conditions and loose packing; RL: Refrigerated temperature and loose packing; AV: Ambient conditions and vacuum packing; RV: Refrigerated temperature and vacuum packing.
The use of refrigeration, whether combined with lose packing or vacuum packing, was effective in reducing the tendency of radical formation, as well as peroxides and conjugated dienes in Brazil nut kernels during storage. The formation of off-flavor VACs was also affected by storage conditions and the use of refrigeration reduced the formation of one VAC, the 3-octen-2-one. However, the combination of refrigeration and vacuum packing, even when LDPE pouches with high OTR were used, reduced the formation of hexanal, which is a major contributor to flavor deterioration, as well as the formation of other off-flavor VACs, and thus should be recommended for Brazil nut kernel storage.
The authors acknowledge financial support from the Coordination for the Improvement of Higher Education Personnel (CAPES); and the National Council of Technological and Scientific Development (CNPq) [grant number 201635/2015-1].