Using Near Infrared Reflectance Spectroscopy (NIRS) for ANFs Analysis in oilseed Brassicas

R. Font, M. del Ro & A. De Haro-Bailn Instituto de Agricultura Sostenible, Departa ento de Agrono a ! Me"ora #en$tica %egetal, Finca Ala eda del &bispo s'n, ()*+* ,rdoba, Spain. -- ail. de/aro0cica.es

Summary
Oilseed Brassicas are grown primarily for the oil and protein contained in seed making them of great value for human and animal nutrition, and also for industry. However, the antinutritive factors (ANFs) contained in the oil (erucic acid) and meal (glucosinolates and fi re), makes Brassica seed unaccepta le for human and animal nutrition. !tandard methods for ANFs analysis are e"pensive and time#consuming. $n contrast, Near $nfrared %eflectance !pectroscopy (N$%!) is a rapid, non#destructive and economical method of analysis that works without the use of ha&ardous chemicals. $n this paper, we test the possi ilities of N$%! for determining ANFs on the intact Brassica sp. seed. 'he r( and %)* shown y the e+uations for erucic acid (,A), glucosinolates (-!.) and acid detergent fi re (A*F) in the e"ternal validation were, respectively, as follows/ 0.12 and 3.(4 (5 oil)6 0.4( and (.24 7mol g#2 dw6 0.43 and (.80 (5 dw). 'he results o tained in this work indicate that N$%! is a le to predict ANFs in the Brassica seed with sufficient accuracy for screening purposes. 1e!2ords. Brassicas, 3IRS, erucic acid, ADF, glucosinolates

Introduction
'he germination capacity of Brassica seeds to germinate and plant resistance to low temperatures, have made Brassica oilseeds one of the few edi le oil crops that can e cultivated in the temperate agricultural &ones of the world, at high elevations and, as winter crops, under relatively cool growing conditions (9im er : ;c-regor, 211<). !pecies such as ,thiopian mustard (Brassica carinata A. =raun) and $ndian mustard (Brassica "uncea .. >&ern. : >oss.) are a le to grow under environmental stress conditions in high temperature climates, and can e cultivated as oilseed crops in ;editerranean conditions (Fereres et al, 2143). 'he seed of Brassica oilseeds is mainly used for the high oil and meal contents. Among the different uses of the oil are the edi le oils and margarines for human consumption, and the industrial use, mainly as engine lu ricants and additives, as well as its use in the plastic and nylon industries. On the other hand, their meal is a good source of protein in human and animal nutrition. However, Brassica oil and meal contain antinutritive factors (ANFs) that make them unaccepta le for these purposes. 'he main ANFs in the Brassica seeds are the erucic acid (,A) of the oil, and the glucosinolates (-!.) and fi re of the meal. 'he standard methods of analysis used for determining these ANFs are e"pensive, time#consuming and re+uires specialised personnel. 'his makes the analysis of large num ers of seed samples difficult for monitoring ANFs, as is re+uired in plant reeding programs. $n the last 30 years, Near $nfrared %eflectance !pectroscopy (N$%!) has een widely used as a fast and accurate method for +ualitative and +uantitative analysis in many fields (?illiams : Norris, 214@). 'he *epartment of Agronomy and )lant =reeding (*A)=) of the $nstitute for !ustaina le Agriculture ($A!, >!$>, >Ardo a), has een using N$%! for the last

fifteen years to determine the seed +uality components in different plant species (*e Haro et al, 21416 Belasco et al, 211(6 Font et al, 2114, (00(). 'he most attractive features of analysis using N$%! are its speed, minimal sample preparation and its non#destructive nature thus making it possi le to analyse large num ers of samples +uickly. 'he o Cective of this work was to test the potential of N$%! for determining the ,A, -!. and acid detergent fi re (A*F) contents of intact Brassicas seeds.

Material and Methods

Plant material )lant material used in this work comprises the oilseed species Brassica "uncea, B. carinata and B. napus, forming part of the germplasm collection at the *A)=. 'his collection is composed of accessions from different geographical origins and comprises most of the total genetic varia ility of these species. 'he plants used to conduct this work were grown over different years in >Ardo a (!pain), eing harvested individually and their seeds stored for N$%! analysis. Reference Methods of Analysis 'he ,A content of the seed was determined y gas li+uid chromatography (-arcDs : ;ancha, 2113). A*F analysis was performed y following the AOA> methods (AOA>, 2110 method 141.03). -!. content was analysed according to Euinsac : %i aillier, (214<). NIRS analysis 'he N$%! analysis consisted in the following steps/ 2. !eed samples elonging to each plant species were scanned in an N$%! spectrometer model F<00 (Foss#N$%!ystems, $nc., !ilver !pring, ;*, G!A) in reflectance mode, e+uipped with a transport module, and their N$% spectra recorder as log (2H%eflectance) in the range from 800 to (<00 nm, as independent files. (. ,A was analysed in B. carinata samples, while the -!. content was determined in B. "uncea samples. For A*F analysis the seed sample set was comprised y B. "uncea, B. carinata and B. napus samples. For ,A and -!. analysis, representative samples of B. carinata and B. "uncea files, respectively, containing the whole spectral varia ility e"isting in the germplasm collection, were selected for cali ration ((H3 of the whole set), and the rest for validation. 'his spectral selection was done y determining the ;ahalano is (H) distance of each spectrum to the mean spectrum of the population. 'he selected samples were then analysed y the reference methods for these parameters. >oncerning A*F analysis, the spectra files of the three Brassica species were considered together and the cali ration and validation groups were made, following the same procedure descri ed for ,A and -!.. 3. >ali ration e+uations for the different components were developed on the cali ration sets y using ;odified )artial .east !+uare (;).!) regression (-.O=A. v. 2.<0 program, ?$N$!$ $$, $nfrasoft $nternational, ..>, )ort ;atilda, )A, G!A), with different mathematical pretreatments I0,0,2,2 (derivative, gap, first smooth, second smooth)6 2,8,8,26 (,<,<,(J of the original spectra. 'he e+uations o tained in the cali ration process were then validated on the validation sets, to test the prediction a ility of each e+uation over samples of the same characteristics. 'he prediction

a ility of the different e+uations o tained for each parameter was evaluated attending their coefficients of determination in the e"ternal validation (r () and ratio of the standard deviation (!*) to standard error of prediction (!,)), which is known as %)* (?illiams : !o ering, 211F).

Results and iscussion

!econd derivative transformation of the raw optical data ((, <, <, () gave the est com ination of r( and %)* for ,A and A*F6 while the first derivative transformation (2, 8, 8, 2) of the original spectra, showed the high r( and %)* for -!.. 'he selected cali ration e+uations for ,A, -!. and A*F resulted in standard errors of cali ration (!,>s) of 3.22 (5 oil, dw), 2@.00 (7mol g#2, dw) and 0.F0 (5 dw), and coefficients of determination in the cali ration (%() of 0.1F, 0.40 and 0.4(, respectively ('a le 2), which indicate e+uations from good (-!.) to e"cellent (,A and A*F) precision (!henk : ?esterhaus, 211F). $n the e"ternal validation, the three components were predicted with sufficient accuracy for screening purposes, as their %)* values were over 3 or close to it (?illiams : !o ering, 211F). 'he level of precision shown y the e+uations in the cali ration was then validated in the e"ternal validation, confirming the prediction a ility shown y the three components in the cali ration. ;any authors have een e"tensively used N$%! as an alternative method for the analysis of ANFs in Brassica (=iston et al, 21446 *avis et al, 21126 ;ichalski et al, 211(6 Font et al, 2111, (003). 'he prediction a ilities of the reported e+uations y these authors for the ANFs, varied widely depending on factors such as species analysed, sample pre#treatment of the sample (intact or ground seed) or the spectroscopic techni+ue used (reflectance or transmittance), and also y the ranges and !*s of the sample groups used in these studies. 4able (. ,alibration and e5ternal 6alidation statistics 7or -A 8n9 :;<, #S= 8n9 >*+<, and ADF 8n9 (?*< 7or t/e selected e@uations.
AN F range 0.02#<0.F8 2F.22# 21F.F1 <.33#2F.32 mean 33.1@ 2(3.F < 22.00 !* 2<.( 4 3@.F 3 (.24 !,> 3.22 2@.0 0 0.F0 %( 0.1 F 0.4 0 0.1 ( range 0.03#81.@8 (2.41# 24@.(1 F.F<#2<.8@ mean 33.@0 2(@.F ( 22.0( !* 2<.0 F 38.2 < 2.1@ %) * 3.( 4 (.2 4 (.8 0 r( 0.1 2 0.4 ( 0.4 3 >ali ration ,"ternal validation

,A2 -! .( A* F3
2 (

B. carinata B. "uncea 3 B. carinata, B. "uncea and B. napus

!onclusions
%esults reported in this work show that N$%! is a le to determine the ,A, -!. and A*F contents on intact seed samples of Brassica with sufficient accuracy for screening and plant reeding purposes. 'he use of this non destructive techni+ue represents an important reduction of the analysis time at a low cost and without the use of ha&ardous chemicals.

References
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