Implication of modified molecular structure of lipid through heat-related process to fatty acids supply in Brassica carinata seed

Industrial Crops and Products 62 (2014) 204–211

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Implication of modified molecular structure of lipid through heat-related process to fatty acids supply in Brassica carinata seed Hangshu Xin a,1 , Katerina Theodoridou a , Peiqiang Yu a,b,∗,2 a b

Department of Animal and Poultry Science, College of Agriculture and Bioresources, University of Saskatchewan, Saskatoon, SK, S7N 5A8, Canada Department of Animal Science, College of Animal Science and Animal Veterinary, Tianjin Agricultural University, Tianjin 300384, China

a r t i c l e

i n f o

Article history: Received 25 May 2014 Received in revised form 26 July 2014 Accepted 14 August 2014 Keywords: Carinata seed Heat Lipid Fatty acid Structures Correlation

a b s t r a c t This study was conducted to explore the effect of different autoclave heating times (30, 60 and 90 min) on fatty acids supply and molecular stability in Brassica carinata seed. Multivariate spectral analyses and correlation analyses were also carried out in our study. The results showed that autoclaving treatments significantly decreased the total fatty acids content in a linear fashion in B. carinata seed as heating time increased. Reduced concentrations were also observed in C18:3n3, C20:1, C22:1n9, monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), omega 3 (␻-3) and 9 (␻-9) fatty acids. Correspondingly, the heated seeds showed dramatic reductions in all the peak intensities within lipid-related spectral regions. Results from agglomerative hierarchical cluster analysis (AHCA) and principal component analysis (PCA) indicated that the raw oilseed had completely different structural make-up from the autoclaved seeds in both CH3 and CH2 asymmetric and symmetric stretching region (ca. 2999–2800 cm−1 ) and lipid ester C O carbonyl region (ca. 1787–1706 cm−1 ). However, the oilseeds heated for 30, 60 and 90 min were not grouped into separate classes or ellipses in all the lipid-related regions, indicating that there still exhibited similarities in lipid biopolymer conformations among autoclaved B. carinata seeds. Moreover, strong correlations between spectral information and fatty acid compositions observed in our study could imply that lipid-related spectral parameters might have a potential to predict some fatty acids content in oilseed samples, i.e. B. carinata. However, more data from large sample size and diverse range would be necessary and helpful to draw up a final conclusion. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Brassica carinata (Ethiopian mustard) seed contains high oil content (25–47%; Pan et al., 2012; Warwick et al., 2006). The fatty acids concentration in this kind of seed varied in different oilseed breeding programs which depend on the breeding purposes. For instance, B. carinata seed free of erucic acid is bred for human consumption whereas the seed with high content of erucic acid or linoleic acid is developed for applications in bio-fuel processing industries (Bouaid et al., 2009; Cardone et al., 2003). Therefore, B. carinata is regarded as a potential and promising feedstock for the emerging bio-economy as reviewed by Taylor et al. (2010). Heating process is capable of altering chemical values. Results from previous studies indicated that heat treatments could

∗ Corresponding author. E-mail address: [email protected] (P. Yu). 1 Postdoctoral Fellow; Present address: College of Animal Science and Technology, Northeast Agricultural University, Harbin, 150030, China. 2 Principal Investigator (PI). http://dx.doi.org/10.1016/j.indcrop.2014.08.025 0926-6690/© 2014 Elsevier B.V. All rights reserved.

influence biological characteristics in feed/food samples (Deacon et al., 1998; Doiron et al., 2009; Yu et al., 2002). Heat processing of oilseeds has various ways to influence the functional groups such as changes in their chemical profiles and their inherent molecular structures (Abeysekara et al., 2012; Doiron et al., 2009; Yu, 2012). In bio-oil industry, oilseed has to be exposed to a high environment (80–105 ◦ C) for 15–20 min during the oil extraction processing; then the defatted oilseed is usually heated to 95–115 ◦ C for about 30 min, which is the last processing step (desolventization-toasting) to produce the byproduct named as oilseed meal. Different heating parameters such as temperature and heating duration adopted in the industrial process may affect nutritive values and quality of oilseeds as well as oilseed meals. Protein ␣-helix and ␤-sheet ratio and total intestinally absorbed protein supply in Vimy flaxseed were linearly increased as heating time increased from 20 to 60 min (Doiron et al., 2009). Similarly, Mustafa et al. (2003a) found that heating treatment could improve amino acids concentrations as well as polyunsaturated fatty acids availability in the small intestine. However, few studies have been reported on how different heating times affect oilseed fatty acids profile and chemical structure characterization. Since the moist

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heating is better than dry heating (Petit et al., 2002), an autoclave heating treatment was therefore applied (121 ◦ C for 30, 60 and 90 min) in B. carinata seed. The changes in both fatty acids profile and internal biopolymer conformation were detected by using Fourier transform infrared (FTIR) spectroscopic technique with attenuated total reflectance (ATR). Furthermore, in order to compare the spectra of the different heat processes used in this study and to determine if they were underlying structural differences, multivariate spectral analyses were employed. Consequently, the objective of this study was to investigate the effect of different autoclave heating times (30, 60 and 90 min) on lipid nutrients supply and molecular stability in B. carinata seed. 2. Materials and methods 2.1. Seed samples and heat treatments Yellow-seeded B. carinata (n = 2 sources, harvested in 2011) used in our study were obtained from field grown plots at the Saskatoon Research Centre-Agriculture and Agri-Food Canada (AAFC) Research Farm in Saskatoon (Saskatchewan, Canada). Seed samples were heated in an autoclave with a steam pressure at 121 ◦ C in gravity mode for 30, 60 and 90 min (Amsco Eagle SG-3031, Steris Corp., Mentor, OH), named as T1, T2 and T3, respectively. All the heated seed samples were subsequently cooled at room temperature for 1 h and then ground in a coffee grinder (PC770, Loblaws Inc., Toronto, Canada) for 20 s. The reason of using a coffee grinder instead of a common feed grinder was clearly stated in our previous reports (Xin et al., 2013a; Yu and Damiran, 2011). The raw B. carinata seeds were used as a control. All the ground seed samples were sealed in labeled plastic sample bottles. 2.2. Oil content and fatty acid profile determination The samples were analyzed according to AOAC procedures for oil content (AOAC official method 920.39) and fatty acid profile (AOAC official method 969.33 for preparation and method 996.06 for quantification). Briefly, accurately weighed ground seed samples (0.4000 g) were put into a labeled Mojonnier flask and then 110 mg pyrogallic acid, 2.0 mL C21:0 (internal standard), 2.0 mL ethanol and 10.0 mL HCl (8.3 M) were added with a few boiling granules and incubated in a shaking water bath (75 ◦ C) for 40 min. For complete extraction, the solution was mixed well very 10 min on vortex mixer and then cooled to room temperature (25 ◦ C). Subsequently, diethyl ether (25 mL) was added to the flask and shacked for 5 min on a wrist-action shaker (8286A25, Model BT 115V 60CYC, Thomas Scientific, NJ, UAS), followed by adding petroleum ether (25 mL) and shaking for another 15 min. The whole mixture solution was then centrifuged at 600 × g for 5 min. Nitrogen stream was used to evaporate ether in the flask to get the remaining residue containing extracted fat. As for the methylation procedure, chloroform (3 mL) and diethyl ether (3 mL) were used to dissolve the extracted fat and then evaporated again in the water bath (40 ◦ C) under N stream in a glass vial. After dryness completed, the vial was sealed and heated at 100 ◦ C for 45 min after adding 2.0 mL BF3 and 1.0 mL toluene. Cooled the vial to room temperature (25 ◦ C) and added H2 O (5.0 mL), hexane (1.0 mL) and Na2 SO4 (1 g) to allow layer separation. The top layer was transferred to another vial containing 1 g Na2 SO4 for GC analysis (Agilent 6890, Agilent Technologies Inc., CA, USA). The column used in this study was a DB 225 capillary column (0.10 mm ID, 10 m in length and 0.10 um film thickness). The column temperature program was set up as follows: Initial temperature: 35 ◦ C, 25 ◦ C/min to 195 ◦ C, 3 ◦ C/min to 205 ◦ C, 8 ◦ C/min to 230 ◦ C. The detector temperature was at 250 ◦ C.

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2.3. Lipid molecular structure by ATR-FTIR spectroscopy In order to avoid effects of oxidation and storage on lipid molecular structure (Mohamed et al., 2013), the ground seed samples were scanned as soon as heating treatments were done. The mid-IR spectral data (ca. 4000–800 cm−1 ) of both unheated and heated oilseed samples (Fig. 1) were obtained using JASCO FT/IR 4200 with ATR (JASCO Corporation, Tokyo, Japan) in University of Saskatchewan. Each spectrum was produced by 128 co-added scans at a resolution of 4 cm−1 . The OMNIC 7.2 software (Madison, WI, USA) was then used to do the spectral analysis. The identification of lipid-related molecular structural characteristics was performed according to the information reported previously in the literatures (Abeysekara et al., 2012; Himmelsbach et al., 1998; Yu and Damiran, 2011). The present study focused on six spectral parameters: asymmetric CH3 (CH3 -as, ca. 2957 cm−1 ), asymmetric CH2 (CH2 -as, ca. 2921 cm−1 ), symmetric CH3 (CH3 -s, ca. 2872 cm−1 ), symmetric CH2 (CH2 -s, ca. 2852 cm−1 ), unsaturated lipid band (ULB, ca. 3007 cm−1 ) and lipid ester C O carbonyl (LECC, ca. 1743 cm−1 ). Two functions (the second derivative and Fourier self-deconvolution (FSD)) in OMNIC 7.2 software were adopted to identify individual CH3 and CH2 stretching bands (CH3 -as, CH2 -as, CH3 -s and CH2 -s) in the region ca. 2999–2800 cm−1 . Also, spectral peak height and area ratios were calculated based on the original spectral data. 2.4. Multivariate spectral analyses According to wide and successful applications of agglomerative hierarchical cluster analysis (AHCA) and principal component analysis (PCA) in IR spectrum analysis (Jackson and Mantsch, 2000; Liu and Yu, 2011; Yu, 2005), it was decided to employ these two methods to clarify whether there was any difference between the raw and autoclaved oilseed on lipid structural traits within regions of CH3 and CH2 stretching bands (ca. 2999–2800 cm−1 ), unsaturated lipid bands (ca. 3039–2991 cm−1 ) and lipid ester C O carbonyl (ca. 1787–1706 cm−1 ). 2.5. Statistical analysis Data of oil content and fatty acid compositions were statistically analyzed using Mixed Model procedure of SAS 9.2 and the models was: Yij =  + Ti + eij where Yij was the observation of the dependent variable ij;  was the fixed effect of population mean of the variable; Ti was a fixed effect of heating treatment (i = 4; the control, T1, T2 and T3), each seed sources were used as replications; eij was the random error associated with the observation ij. The model for lipid spectral data analysis in B. carinata seeds was: Yijk =  + Ti + S(T )j + eijk where Yijk was the observation of the dependent variable ijk;  was the fixed effect of population mean of the variable; Ti was a fixed effect of heating treatment (i = 4; the control, T1, T2 and T3); S(T)j was a random effect of seed source nested within seed; eijk was the random error associated with the observation ijk. Relationships between the oil content, fatty acid compositions and lipid-related functional groups in all the kinds of B. carinata seed were analyzed using the PROC CORR procedure of SAS using the Pearson correlation method, since the original data were normally distributed as tested by the PROC UNIVERIATE procedure.

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Fig. 1. Typical ATR-FT/IR molecular spectrum of the raw and the heated Brassica carinata seeds in the mid-IR region ca. 4000–800 cm−1 .

Moreover, orthogonal polynomial contrasts were carried out to investigate the linear, quadratic and cubic effects of autoclave heating time (0, 30, 60 and 90 min). The contrast coefficients were calculated by SAS program. Statistical significance was declared and detected at P < 0.05 while trends were declared at P ≤ 0.10.

3. Results and discussion 3.1. Changes in oil content and fatty acids profile in the raw and heated B. carinata seeds The content of ether extract (EE), as presented in Table 1, was not changed by autoclaving treatment (P = 0.11); however, it tended to linearly increased as heating time increased (P = 0.04). The EE concentration in B. carinata seed was slightly higher than that reported by Taylor et al. (2010) who found that B. carinata seed grown in Saskatoon contained 37.8% oil; but it was still within the variation range of 25–47% (Pan et al., 2012; Warwick et al., 2006). As for EE changes response to heating treatment in oilseeds, although numerous results could be referred to from previous literatures (Doiron et al., 2009; Mustafa et al., 2003a, 2003b; Petit et al., 2002), they failed to give a consistent conclusion. Some researchers found an increase in EE concentration upon thermal treatment, which is beneficial to the bio-oil industry, of the flaxseed (Doiron et al., 2009; Oomah and Kenaschuk, 1995), soybean and cottonseed (Mohamed et al., 1988). One possible reason for this phenomenon might be the denaturation of protein mixture or oleosins surrounding the oil droplets which resulted in oil cells rupture followed by combination of larger oil bodies (Booth, 2004; Doiron et al., 2009). In contrast, Petit et al. (2002) demonstrated that EE content declined dramatically from 40% DM (unheated seed) to 11% DM (treated at 160 ◦ C for 0.5 h) in flaxseeds during micronization treatments and this result was explained by the authors from the fatty acid oxidation. In agreement with our results, another studies showed that it was not possible to detect the differences in EE content between unheated and heated (autoclaving or extrusion) oilseed samples (Mustafa et al., 2003a, 2003b). As suggested by Leszkiewicz and Kasperek (1988), the extent of changes in oil content largely depend

on the selected oil type and the applied heating procedure (i.e. thermal method, heating temperature and duration). Autoclaving treatment significantly decreased total fatty acids content in a linear fashion (P = 0.004) in B. carinata seed (Table 1), which resulted from the changes in individual fatty acids. The contents of C18:1n9, C18:1, C20:2n6 and C22:2n6 were not changed by autoclaving; but they were linearly decreased (P < 0.05) as heating time increased. Significantly decreased concentrations (P < 0.05) were observed in C18:3n3 (linearly and quadratically), C20:1 (linearly) and C22:1n9 (linearly); and all these measured values fell into the variation range of fatty acid compositions in B. carinata seed as mentioned in previous publications (Taylor et al., 2010; Velasco et al., 1998). In the light of theoretical description (Somerville et al., 2000), C18:3n3 (linolenic acid) is biosynthesized by microsomal delta-15 desaturases in catalyzation of a third double bond into C18:2 precursors in the oilseed. Therefore, higher temperature would denature the desaturases and then inhibit the catalyzation which might be one of the possible reasons for the slight but significant decrease in the level of C18:3 in our seed samples. Similarly, the baking flaxseed (178 ◦ C for 90 min), extruded and roasted soybeans presented lower fatty acids concentrations compared with the raw seed (Chen et al., 1994; Reddy et al., 1994). Erucic acid (C22:1) is an important feedstock and has more than 1000 applications for various industrial purposes (Scarth and Tang, 2006). In our study, the 1.2–2.8% lower content found in C22:1 upon autoclaving, indicating that heating treatment might alter the yield of this fatty acid in industrial manufactures. In an early study conducted by Leszkiewicz and Kasperek (1988) using raw oil from high erucic rapeseed, an increase in CH CH proton was observed as heating time increased from 0 to 4 h at 110 ◦ C. This might partially explain the changes in the fatty acids content observed in our study. As a result, the concentrations of total monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), omega 3 (␻-3) and 9 (␻-9) fatty acids were also declined in both linear and quadratic fashions (P < 0.05). Although some individual fatty acid contents varied under thermal treatments, the proportions of each individual fatty acid in total fatty acids were not changed, suggesting that autoclaving could keep a stable pattern in fatty acids in B. carinata seed.

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Table 1 Oil content and fatty acids profiles: comparison of raw (control) versus autoclave-treated B. carinata seed. Treatmenta

Control

Ether extract (EE), %DM Total fatty acids, mg/g C16, Palmitic acid C16:1n7, Palmitoleic acid C17, Margaric acid C18, Stearic acid C18:1n9, Oleic acid C18:1, Octadecenoic acid C18:2, Linoleic acid C18:3n3, alpha-Linolenic acid C20, Arachidic acid C20:1, Eicosenoic acid C20:2n6, Eicosadienoic acid C20:3n3, Eicosatrienoic acid C22, Behenic acid C22:1n9, Erucic acid C22:2n9, Docosadienoic acid C22:2n6, Docosadienoic acid C24, Lignoceric acid C24:1n9, Nervonic acid Others fatty acids Total SFAb Total MUFA Total PUFA Total ␻-3 fatty acids Total ␻-6 fatty acids Total ␻-9 fatty acids a b

41.4 914.7a 27.3 1.7 1.1 9.4 66.2 5.5 133.9 115.7a 9.0 69.8a 8.7 1.5 8.6 404.4a 6.5 15.3 5.0 16.8 8.0 60.8 564.4a 281.5a 117.2a 157.9 563.6a

T1

T2

T3

42.8 903.5ab 28.0 1.8 1.1 9.5 65.8 5.4 132.1 111.9b 8.9 68.8ab 8.6 1.5 8.6 399.5ab 6.6 15.1 5.0 16.8 8.4 61.6 557.9b 275.7ab 113.4b 155.7 557.4a

44.7 891.9b 28.0 1.7 1.1 9.5 64.8 5.3 130.8 110.9b 8.8 67.9b 8.5 1.5 8.5 393.2b 6.7 14.8 4.9 16.4 8.4 61.3 549.3c 273.0b 112.4b 154.0 549.0b

44.1 894.1b 27.8 1.7 1.0 9.5 65.2 5.3 131.1 112.5b 8.9 68.0ab 8.5 1.4 8.5 393.6b 6.5 14.9 4.9 16.5 8.4 60.9 550.1c 274.7ab 113.9ab 154.4 549.7b

SEM

P value

0.75 2.76 0.24 0.04 0.04 0.15 0.27 0.06 0.99 0.54 0.09 0.32 0.05 0.06 0.09 1.36 0.20 0.10 0.05 0.20 0.66 0.57 1.06 1.27 0.59 1.05 1.21

0.11 0.01 0.27 0.38 0.80 0.97 0.07 0.11 0.25 0.01 0.52 0.04 0.12 0.38 0.84 0.01 0.82 0.054 0.38 0.46 0.96 0.78 0.002 0.03 0.02 0.17 0.003

Polynomial contrast Linear

Quadratic

Cubic

0.04 0.004 0.28 0.27 0.48 0.89 0.03 0.03 0.09 0.01 0.24 0.01 0.04 0.14 0.43 0.003 0.80 0.02 0.15 0.19 0.71 1.00 0.0004 0.02 0.01 0.06 0.001

0.24 0.07 0.12 0.23 0.59 0.75 0.17 0.68 0.34 0.01 0.45 0.17 0.37 0.46 0.80 0.13 0.43 0.15 1.00 0.81 0.78 0.39 0.03 0.04 0.01 0.29 0.045

0.44 0.31 0.70 0.56 0.81 0.77 0.17 0.85 0.82 0.94 0.73 0.65 0.68 1.00 0.91 0.25 0.80 0.36 0.42 0.47 0.90 0.71 0.07 0.85 0.94 0.75 0.11

T1: autoclaved at 120 ◦ C for 30 min; T2: autoclaved at 120 ◦ C for 60 min; T3: autoclaved at 120 ◦ C for 90 min. SFA, saturated fatty acids, MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids.

Table 2 Spectral profile of lipid-related functional groups: comparison of raw (control) versus autoclave-treated B. carinata seed. Control

Treatmenta

SEMe

T1

T2

T3

P

Polynomial contrast Linear

Quadratic

Cubic

b

CH3 and CH2 stretching profiles CH3 asymmetric (CH3 -as) height CH2 asymmetric (CH2 -as) height CH3 symmetric (CH3 -s) height CH2 symmetric (CH2 -s) height CH2 and CH3 stretchings (CHS) area

0.025a 0.085a 0.022a 0.053a 5.19a

0.008b 0.022b 0.008b 0.012b 1.58b

0.010b 0.030b 0.010b 0.018b 2.03b

0.008b 0.022b 0.009b 0.013b 1.60b

0.001 0.004 0.000 0.002 0.185

<.0001 0.001 <.0001 0.001 0.0004

<.0001 0.0004 <.0001 0.001 0.0002

0.0002 0.002 0.0002 0.002 0.001

0.001 0.01 0.001 0.01 0.004

Unsaturated lipid bands (ULB) profilesc ULB height ULB area

0.005a 0.086a

0.0003b 0.004b

0.001b 0.012b

0.0002b 0.003b

0.0002 0.002

0.0001 <.0001

<.0001 <.0001

0.0004 0.0001

0.001 0.001

Lipid ester C O carbonyl (LECC) profilesd LECC height LECC area

0.068a 1.436a

0.014b 0.317b

0.021b 0.472b

0.014b 0.286b

0.003 0.067

0.001 0.001

0.0004 0.0004

0.002 0.002

0.01 0.01

Spectral ratio profile Height ratio of CH3 -as: CH2 -as Height ratio of CH3 -as: CH3 -s Height ratio of CH3 -s: CH2 -s Height ratio of CH2 -as: CH2 -s Height ratio of asymmetric: symmetric Area ratio of CHS: LECC Area ratio of ULB: CHS Area ratio of ULB: LECC

0.296 1.14a 0.419 1.61 1.47a 3.63 0.017a 0.060a

0.382 0.91b 0.840 1.91 1.44ab 5.70 0.002b 0.007b

0.340 0.98b 0.583 1.67 1.42ab 4.54 0.006b 0.024b

0.382 0.96b 0.700 1.73 1.41b 6.44 0.001b 0.006b

0.037 0.025 0.133 0.093 0.015 0.651 0.001 0.004

0.42 0.01 0.29 0.25 0.03 0.12 0.001 0.002

0.27 0.01 0.38 0.77 0.004 0.07 0.001 0.001

0.59 0.02 0.32 0.25 0.60 0.90 0.01 0.01

0.28 0.03 0.15 0.11 0.77 0.10 0.004 0.005

T1: autoclaved at 120 ◦ C for 30 min; T2: autoclaved at 120 ◦ C for 60 min; T3: autoclaved at 120 ◦ C for 90 min. Lipid data unit, IR absorbance unit; the CH3 and CH2 stretching (CHS) peak baseline, 2999–2800 cm−1 with CH3 -as, CH2 -as CH3 -s and CH2 -s peaks at 2957, 2921, 2872 and 2852 cm−1 , respectively. And their peak regions were 2960–2954, 2923–2918, 2874–2870 and 2853–2848 cm−1 , respectively. c The unsaturated lipid bands (ULB) peak baseline, 3039–2991 cm−1 , the peak region was 3009–3005 cm−1 . d The lipid ester C O carbonyl (LECC) peak baseline, 1787–1706 cm−1 , the peak region was 1745–1733 cm−1 . e SEM, standard error of the mean. Means with the different letters in the same row are significantly different (P < 0.05). a

b

3.2. Changes in spectral profile of lipid-related functional groups in the raw and heated B. carinata seeds The spectra collected by ATR-FTIR spectroscopy in the raw and heated B. carinata seeds in the region ca. 4000–800 cm−1 are shown

in Fig. 1; and the three highlighted and labeled regions were the main functional groups that this study focused on. As expected, the temperature and heating time designed for the treatments in our study were satisfied enough to damage the lipid part of oilseed samples and make some detectable structural changes under

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H. Xin et al. / Industrial Crops and Products 62 (2014) 204–211 Tree Diagram for 40 Cases Ward`s method Euclidean distances

Projection of the cases on the factor-plane ( 1 x 2) Cases with sum of cosine square >= 0.00 25

3.5 20 15 C D DC D D DC C C B C B DD B C B B D D BDB B BC C B

10 Factor 2: 22.05%

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CCCB BCCB BCDCDDCDDCDDDB B BCDB BDB A A A A AA A A A A

I: CLA spectral analysis of the CH3 and CH2 asymmetric and symmetric stretching regions (2999-2800 cm-1) obtained from four different carinata seed samples [Note: CLA: (1) Region of CH3 and CH2 asymmetric and symmetric stretching ca. 2999-2800 cm-1; (2) Distance method: Euclidean; (3) Cluster method: Ward’s algorithm]

A A

A A

5

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Factor 1: 76.33%

II: Scatter plot of the 1st principal component vs. the 2nd principal component of PCA analysis of spectrum obtained from four different carinata seed samples: the 1st and 2nd principal component explains 76.33% and 22.05% of the total variance, respectively.

Fig. 2. Multivariate molecular spectral analyses of the CH3 and CH2 asymmetric and symmetric stretching (2999–2800 cm−1 ) on a molecular basis among different oilseeds: A = raw seed; B = heated seed for 30 min; C = heated seed for 60 min; D = heated seed for 90 min.

Tree Diagram for 40 Cases Ward`s method Euclidean distances

Projection of the cases on the factor-plane ( 1 x 2) Cases with sum of cosine square >= 0.00 10

2.5

8 B B

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BBB

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A

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0.5 -8 -10 -20

0.0 CCCCDCCCCCCDBDBBDDDDDDDBBBBBBBAAAAAAAAAA

I: CLA spectral analysis of the lipid ester C=O carbonyl regions (1787-1706 cm-1) obtained from four different carinata seed samples [Note: CLA: (1) Region of lipid ester C=O carbonyl ca. 1787-1706 cm-1; (2) Distance method: Euclidean; (3) Cluster method: Ward’s algorithm]

-15

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Factor 1: 85.38%

II: Scatter plot of the 1st principal component vs. the 2nd principal component of PCA analysis of spectrum obtained from four different carinata seed samples: the 1st and 2nd principal component explains 85.38% and 13.43% of the total variance, respectively.

Fig. 3. Multivariate molecular spectral analyses of the lipid ester C O carbonyl (1787–1706 cm−1 ) on a molecular basis among different oilseeds: A = raw seed; B = heated seed for 30 min; C = heated seed for 60 min; D = heated seed for 90 min.

spectroscopic scanning (Table 2). By the identification of each peak height and area as well as a univariate spectral analysis, all the heated oilseeds showed dramatically reduced peak intensities in the regions of CH3 and CH2 symmetric and asymmetric stretching vibrations (CHS; including CH3 -as, CH2 -as, CH3 -s and CH2 -s; ca. 2999–2800 cm−1 ), ULB (ca. 3039–2991 cm−1 ) and LECC (ca. 1787–1706 cm−1 ) in linear and curvilinear responses (P < 0.05). However, no obvious changes were found among heated treatments (P > 0.05). These results indicated that intrinsic structures associated with lipid functional groups in oilseeds were sensitive to wet heating, which might consequently cause modifications in lipid and fatty acid compositions (Table 1). The variation in ULB was also fully supported by the results of CH CH proton changes found in the heated rapeseed oil in a previous report (Leszkiewicz and Kasperek, 1988). Another study targeting on flaxseed samples exhibited that a transformation of unsaturated fatty acids into

saturated fatty acids would occur under micronization treatments (Petit et al., 2002). However, it is of interest to note that the peak intensities of CHS, ULB as well as LECC were remarkably greater in bio-ethanol co-product (corn DDGS) than those in original corn grains (Yu, 2011), which is in contrast to our findings. Similarly, neither dry nor wet heating treatment could significantly affect spectral parameters of any of the lipid-related functional groups in canola seed or Vimy flaxseed (Abeysekara et al., 2012; Yu and Damiran, 2011). These insistent results might be partly explained by the different seed types selected in these studies. The spectral ratios of a functional group could reflect a relative biological component and their nutritive values which are highly associated with food/feed quality (Yu et al., 2005). Table 2 shows the height and area ratios of mid-IR absorbed intensities within lipid-related functional group regions. Compared to the individual spectral characteristics which mentioned above, the spectral

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Tree Diagram for 40 Cases Ward`s method Euclidean distances

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B CC DD B B C BDB B DD B B C C B D CD D CD D C C

A AA A C

-2

0.2

-3

0.1 -4 -25

0.0

-20

-15

-10

-5

DDDCCBDBBBDBBBDBDCCDCBBCDCCDCAAAAAAACAAA

0

5

10

15

20

25

30

Factor 1: 98.52%

II: Scatter plot of the 1st principal component vs. the 2nd principal component of PCA analysis of spectrum obtained from four different carinata seed samples: the 1st and 2nd principal component explains 98.52% and 1.39% of the total variance, respectively.

I: CLA spectral analysis of the unsaturated lipid regions (3039-2991 cm-1) obtained from four different carinata seed samples [Note: CLA: (1) Region of unsaturated lipid ca. 3039-2991 cm-1; (2) Distance method: Euclidean; (3) Cluster method: Ward’s algorithm]

Fig. 4. Multivariate molecular spectral analyses of the unsaturated lipid (3039–2991 cm−1 ) on a molecular basis among different oilseeds: A = raw seed; B = heated seed for 30 min; C = heated seed for 60 min; D = heated seed for 90 min. Table 3 Correlation between lipid-related structural characteristics and oil content as well as fatty acids compositions of raw and heated B. carinata seeds. CHS area

Ether extract (EE), %DM Total fatty acids, mg/g C16, Palmitic acid C16:1n7, Palmitoleic acid C17, Margaric acid C18, Stearic acid C18:1n9, Oleic acid C18:1, Octadecenoic acid C18:2, Linoleic acid C18:3n3, alpha-Linolenic acid C20, Arachidic acid C20:1, Eicosenoic acid C20:2n6, Eicosadienoic acid C20:3n3, Eicosatrienoic acid C22, Behenic acid C22:1n9, Erucic acid C22:2n9, Docosadienoic acid C22:2n6, Docosadienoic acid C24, Lignoceric acid C24:1n9, Nervonic acid Others fatty acids Total SFA Total MUFA Total PUFA Total ␻-3 fatty acids Total ␻-6 fatty acids Total ␻-9 fatty acids

ULB area

LECC area

Area ratio of CHS: LECC

Area ratio of ULB: CHS

Area ratio of ULB: LECC

r

p

r

p

r

p

r

p

r

p

r

p

−0.64 0.82 −0.65 −0.05 0.14 −0.14 0.59 0.72 0.74 0.88 0.59 0.73 0.66 0.32 0.40 0.79 −0.19 0.75 0.42 0.46 −0.37 −0.24 0.79 0.89 0.88 0.77 0.79

0.09 0.01 0.08 0.90 0.73 0.73 0.13 0.045 0.04 0.004 0.13 0.04 0.08 0.43 0.32 0.02 0.66 0.03 0.30 0.26 0.36 0.57 0.02 0.003 0.004 0.03 0.02

−0.67 0.82 −0.68 −0.02 0.17 −0.18 0.63 0.72 0.72 0.89 0.56 0.76 0.69 0.35 0.37 0.79 −0.24 0.75 0.40 0.43 −0.37 −0.27 0.80 0.88 0.89 0.75 0.80

0.07 0.01 0.07 0.96 0.68 0.67 0.10 0.04 0.04 0.004 0.15 0.03 0.06 0.40 0.36 0.02 0.57 0.03 0.32 0.29 0.37 0.52 0.02 0.004 0.004 0.03 0.02

−0.63 0.82 −0.63 −0.04 0.15 −0.13 0.57 0.73 0.75 0.86 0.59 0.72 0.66 0.34 0.41 0.79 −0.16 0.75 0.44 0.47 −0.37 −0.21 0.80 0.88 0.86 0.77 0.80

0.09 0.01 0.10 0.93 0.72 0.75 0.14 0.04 0.03 0.01 0.12 0.04 0.08 0.41 0.31 0.02 0.70 0.03 0.28 0.24 0.37 0.61 0.02 0.004 0.01 0.03 0.02

0.21 −0.61 0.17 −0.04 −0.06 −0.22 −0.17 −0.60 −0.67 −0.48 −0.54 −0.35 −0.28 −0.33 −0.49 −0.62 −0.35 −0.50 −0.49 −0.51 0.33 −0.15 −0.57 −0.65 −0.49 −0.65 −0.59

0.61 0.11 0.69 0.93 0.89 0.60 0.69 0.12 0.07 0.23 0.17 0.39 0.50 0.43 0.22 0.10 0.40 0.21 0.21 0.19 0.42 0.73 0.14 0.08 0.22 0.08 0.13

−0.57 0.76 −0.63 −0.08 0.16 −0.18 0.50 0.68 0.69 0.82 0.53 0.70 0.61 0.39 0.35 0.75 −0.16 0.68 0.37 0.41 −0.42 −0.25 0.75 0.83 0.83 0.71 0.75

0.14 0.03 0.10 0.85 0.70 0.66 0.21 0.06 0.06 0.01 0.18 0.053 0.10 0.34 0.40 0.03 0.70 0.06 0.36 0.32 0.30 0.55 0.03 0.01 0.01 0.047 0.03

−0.52 0.73 −0.58 −0.08 0.17 −0.15 0.44 0.67 0.69 0.78 0.53 0.65 0.57 0.35 0.37 0.71 −0.11 0.65 0.37 0.40 −0.38 −0.21 0.71 0.81 0.78 0.70 0.71

0.18 0.04 0.14 0.85 0.69 0.73 0.27 0.07 0.06 0.02 0.18 0.08 0.14 0.39 0.37 0.048 0.80 0.08 0.37 0.32 0.35 0.62 0.050 0.02 0.02 0.052 0.049

ratios were relatively stable upon heating treatments in B. carinata seed. The heated B. carinata seeds had 2–20% lower (P < 0.05) height ratios of CH3 -as: CH3 -s and asymmetric: symmetric than the raw seed which indicated that the lipid chain length and branching were sensitive to the high temperature. Area ratios of ULB: CHS and ULB: LECC were dramatically reduced by autoclaving in both linear and curvilinear responses (P < 0.05). The other spectral ratios were not changed among treatments (P > 0.05). Similar observations were found in earlier studies for canola seed (Abeysekara et al., 2012) and corn seed (Yu, 2011) but not for wheat grains (Yu, 2011) and Vimy flaxseed (Yu and Damiran, 2011). This implied that

different kinds of feed/food sample had different sensitivities to heating treatments (including dry heating, autoclaving and bioethanol processing). It is expected that all the structural changes should be responsible for the variations in chemical compositions. 3.3. Multivariate spectral analysis By visible observations from AHCA and PCA analyses (Figs. 2–4), the untreated B. carinata seed was fully distinguished from all the heated seeds within CHS region (ca. 2999–2800 cm−1 ) and LECC region (ca. 1787–1706 cm−1 ). The distinctions in spectra within

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these two regions meant the lipid biopolymer conformations were different between the raw B. carinata seed and heated seeds. However, the oilseeds heated for 30, 60 and 90 min failed to be grouped into separate classes or ellipses in any of the lipid-related regions, indicating that there still exhibited similarities in lipid structural make-up among autoclave heated B. carinata seeds. To a great extent, the multivariate results could also support the univariate results (Table 2). Compared to the previous published data, discriminated structural make-up in lipid spectral regions could only be found between corn and corn DDGS (Yu, 2011) while other samples like canola seed and Vimy flaxseed had heavily overlapped groups in PCA results (Abeysekara et al., 2012; Yu and Damiran, 2011). It was demonstrated that different heating methods had different impacts on lipid-related functional groups in different types of bio-material.

Funding

3.4. Correlations between spectral data and lipid profiles in B. carinata seeds

Abeysekara, S., Samadi, Yu, P., 2012. Response and sensitivity of lipid related molecular structure to wet and dry heating in canola tissue. Spectrochim. Acta A 90, 63–71. AOAC, 1990. Official Methods of Analysis. Association of Official Analytical Chemists, Washington, DC. Booth, E.J., 2004. Extraction and refining. In: Gunstone, F.D. (Ed.), Rapeseed and Canola Oil: Production, Processing, Properties and Uses. CRC Press, Boca Raton, FL, pp. 17–35. Bouaid, A., Martinez, M., Aracil, J., 2009. Production of biodiesel from bioethanol and Brassica carinata oil: oxidation stability study. Bioresour. Technol. 100 (7), 2234–2239. Cardone, M., Mazzoncini, M., Menini, S., Rocco, V., Senatore, A., Seggiani, M., Vitolo, S., 2003. Brassica carinata as an alternative oil crop for the production of biodiesel in Italy: agronomic evaluation, fuel production by transesterification and characterization. Biomass Bioenergy 25, 623–636. Chen, Z.Y., Ratnayake, W.M.N., Cunnane, S.C., 1994. Stability of flaxseed during baking. In: Proc. 55th Flax Institute of the United States, Fargo, ND, pp. 24–28. Deacon, M.A., DeBoer, G., Kennelly, J.J., 1998. Influence of jet-sploding and extrusion on ruminal and intestinal disappearance of canola and soybeans. J. Dairy Sci. 71, 745–753. Doiron, K., Yu, P., McKinnon, J.J., Christensen, D.A., 2009. Heat-induced protein structure and subfractions in relation to protein degradation kinetics and intestinal availability in dairy cattle. J. Dairy Sci. 92, 3319–3330. Himmelsbach, D.S., Khalili, S., Akin, D.E., 1998. FT-IR microspectroscopic imaging of flax (Linum usitatissimum L.) stems. Cell. Mol. Biol. 44, 99–108. Jackson, M., Mantsch, H.H., 2000. Infrared spectroscopy ex vivo tissue analysis. In: Meyers, R.A. (Ed.), Encyclopedia of Analytical Chemistry. Wiley, Chichester, UK, pp. 131–156. Leszkiewicz, B., Kasperek, M., 1988. The effect of heat treatment on fatty acids of rapeseed oils. J. Am. Oil Chem. Soc. 65 (9), 1511–1515. Liu, N., Yu, P., 2011. Molecular clustering, interrelationships and carbohydrate conformation in hull and seeds among barley cultivars. J. Cereal Sci. 53, 379–383. Mohamed, O.E., Satter, L.D., Grummer, R.R., Ehle, F.R., 1988. Influence of dietary cottonseed and soybean on milk production and composition. J. Dairy Sci. 71, 2677–2688. Mohamed, N.A., Mariod, A.A., Yagoub, S.O., Dagash, Y.M.I., 2013. Effect of irrigation intervals and fertilizers on chemical composition, minerals and fatty acids of safflower (Carthamus tinctorius L.) seed. Acta Agron. Hung. 61 (3), 227–236. Mustafa, A.F., Gonthier, C., Ouellet, D.R., 2003a. Effects of extrusion of flaxseed on ruminal and postruminal nutrient digestibilities. Arch. Anim. Nutr. 57, 455–463. Mustafa, A.F., Chouinard, Y.P., Ouellet, D.R., Soita, H., 2003b. Effects of moist heat treatment on ruminal nutrient degradability of sunflower seed. J. Sci. Food Agric. 83, 1059–1064. Oomah, B.D., Kenaschuk, E.O., 1995. Cultivars and agronomic aspects. In: Cunane, S., Thompson, L.U. (Eds.), Flaxseed in Human Nutrition. AOCS Press, Champaign, IL, pp. 43–55. Pan, X., Caldwell, C.D., Falk, K.C., Lada, R., 2012. The effect of cultivar, seeding rate and applied nitrogen on Brassica carinata seed yield and quality in contrasting environments. Can. J. Plant Sci. 92, 961–971. Petit, H.V., Tremblay, G.F., Tremblay, E., Nadeau, P., 2002. Ruminal biohydrogenation of fatty acids, protein degradability, and dry matter digestibility of flaxseed treated with different sugar and heat combinations. Can. J. Anim. Sci. 82, 241–250. Reddy, R.V., Morrill, J.L., Nagaraja, T.G., 1994. Release of free fatty acids from raw or processed soybeans and subsequent effects of fiber digestibilities. J. Dairy Sci. 77, 3410–3416. Scarth, R., Tang, J., 2006. Modification of Brassica oil using conventional and transgenic approaches. Crop Sci. 46, 1225–1236. Somerville, C., Browse, J., Jaworski, J.G., Ohlrogge, J.B., 2000. Lipids. In: Buchanan, B.B., Gruissem, W., Jones, R.L. (Eds.), Biochemistry and Molecular Biology of Plants. American Society of Plant Physiologists, Rockville, MD, USA, pp. 456–527. Taylor, D.C., Falk, K.C., Palmer, C.D., Hammerlindl, J., Babic, V., Mietkiewska, E., Jadhav, A., Marillia, E., Francis, T., Hoffman, T., Giblin, E.M., Katavic, V., Keller, W.A., 2010. Brassica carinata – a new molecular farming platform for delivering bio-industrial oil feedstocks: case studies of genetic modifications to improve

As we expected, some significant correlations exist between lipid structural features and fatty acids profile in the raw and treated B. carinata seeds (Table 3). Areas of CHS, ULB and LECC were positively and strongly correlated (P < 0.05) with total fatty acids (r = 0.82), C18:1 (r = 0.72–0.73), C18:2 (r = 0.72–0.75), C18:3n3 (r = 0.86–0.89), C20:1 (r = 0.72–0.76), C22:1n9 (r = 0.79), C22:2n6 (r = 0.75), MUFA (r = 0.79–0.80), PUFA (r = 0.88–0.89), ␻3 (r = 0.86–0.89), ␻-6 (r = 0.75–0.77) and ␻-9 (r = 0.79–0.80) fatty acids. These correlations indicated that B. carinata seed with higher mid-IR spectral intensities would have higher fatty acids content. In regard to spectral ratios, area ratio of CHS: LECC was not related to any of the fatty acids (P > 0.05). However, the rest two area ratios – ULB: CHS and ULB: LECC were in close correlations with total fatty acids (r = 0.73–0.76), C18:3n3 (r = 0.78–0.82), C22:1n9 (r = 0.71–0.75), PUFA (r = 0.81–0.83), ␻-3 (r = 0.78–0.83) and ␻-9 (r = 0.71–0.75) fatty acids. As far as it is known, numerous researches revealed that spectroscopic information such as protein and carbohydrate structures are closely linked to the nutrient profiles in various bio-material samples (Doiron et al., 2009; Liu and Yu, 2011; Xin et al., 2013a, 2013b). However, no published report has been conducted on lipid groups. Our findings illustrated that these lipid spectral traits revealed from FT/IR spectroscopy might be used to predict fatty acids content in the oilseed like B. carinata. Since only four treatments were test in the current study, more data from large sample size and diverse range would be necessary and helpful to draw up a more accurate conclusion. 4. Conclusions The autoclaving heating treatments changed significantly the contents of total fatty acids and some individual fatty acids such as C18:3n3, C20:1, C22:1n9 in B. carinata seed. Also MUFA, PUFA, ␻-3 and ␻-9 fatty acids were decreased in linear and curvilinear fashions after moist heating in the samples from 0 to 90 min. The comparison of the raw with the heated seeds showed dramatic reductions in peak intensities within lipid-related spectral regions. Results from AHCA and PCA indicated that the raw oilseed had completely different structural make-up from the autoclaved seeds in both CHS (ca. 2999–2800 cm−1 ) and LECC region (ca. 1787–1706 cm−1 ). Strong correlations observed in our study implied that lipid-related spectral parameters might have the potential to predict the content of specific fatty acids content in oilseed samples like B. carinata. However, more data from large sample size and diverse range would be necessary and helpful to draw up a final conclusion.

The Chair (Professor Dr. Peiqiang Yu) research programs have been finically supported by Ministry of Agriculture Strategic Research Chair Program, NSERC (Discovery Grant and CRD grant), BCRC-AAFC Science Cluster, SaskCanola, Thousand-Talent-People Research Program, and ADF-Saskatchewan. Acknowledgements The authors are grateful to Zhiyuan Niu (University of Saskatchewan) for helpful assistance in the chemical analysis and the spectral data collection. References

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