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Laser-Induced Breakdown Spectroscopy Based Protein Assay for Cereal Samples Banu Sezer, Gonca Bilge, and #smail Hakki Boyaci J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04828 • Publication Date (Web): 23 Nov 2016 Downloaded from http://pubs.acs.org on November 26, 2016

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Journal of Agricultural and Food Chemistry

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Laser-Induced Breakdown Spectroscopy Based Protein Assay for Cereal Samples

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Banu Sezer, Gonca Bilge, Ismail Hakki Boyaci*

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Department of Food Engineering, Hacettepe University, Beytepe 06800, Ankara, Turkey

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*Corresponding Author:

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Phone: +90 312 297 61 46

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Fax: +90 312 299 21 23

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e-mail: [email protected]

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1 ACS Paragon Plus Environment


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ABSTRACT

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Protein content is an important quality parameter in terms of price, nutritional value and

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labeling of various cereal samples. However, conventional analysis methods, namely Kjeldahl

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and Dumas have major drawbacks such as long analysis time, titration mistakes and carrier

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gas dependence with high purity. For this reason, there is an urgent need for rapid, reliable

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and environmentally friendly technologies for protein analysis. The present study aims to

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develop a new method for protein analysis in wheat flour and whole meal by using laser

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induced breakdown spectroscopy (LIBS), which is a multi-elemental, fast and simple

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spectroscopic method. Unlike the Kjeldahl and Dumas method, it has potential to analyze

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high number of samples in considerably short time. In the study, nitrogen peaks in LIBS

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spectra of wheat flour and whole meal samples with different protein contents were correlated

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with results of standard Dumas method with the aid of chemometric methods. Calibration

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graph showed good linearity with the protein content between 7.9-20.9%, and 0.992

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coefficient of determination (R2). Limit of detection was calculated as 0.26%. The results

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indicated that LIBS is a promising and reliable method with its high sensitivity for routine

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protein analysis in wheat flour and whole meal samples.

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Key words: LIBS; protein analysis, wheat flour, whole meal, nitrogen content.


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INTRODUCTION

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Proteins are the main functional components in various food products and have ability to

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support dietary needs. They have a significant role in determining the textural, sensory and

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nutritional properties of products. Protein content affects technological properties and

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determines nutritional quality of foods. Therefore, determination of protein is an important

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quantitative analysis in terms of quality control, accurate nutrition labeling, pricing, functional

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property investigation and biological activity determination,1 which makes it a subject of both

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economic and social interest. The market value of major agricultural products (cereal grains,

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legumes, flour, oil seed, milk, livestock feeds) is determined partly by their protein content. It

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is also important for wheat products. Protein content of wheat varies from 6% to 20%

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depending on geographical factors and soil properties. Also, it is a quality index for wheat

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flour in bread making industry.2 Overall, protein analysis has nutritional, legal, health and

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economic results for food industry.3

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The analytical techniques used for determining protein content of food products can be

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divided into several groups based on measured parameters such as determination of nitrogen,

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peptide bonds, aromatic amino acids, ultraviolet absorptivity, and light scattering properties.

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The most commonly used reference analytical methods are Dumas and Kjeldahl methods,1

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which were developed in 1831 and 1883, respectively. Different from other methods, these

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two methods analyze protein content directly by measuring the nitrogen and amino acid

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content. In the Kjeldahl method, the sample is first digested with strong sulphuric acid in the

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presence of catalyst, and amine nitrogen is converted to ammonium ions. Then, ammonium

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ions are converted to ammonia gas, heated and distillated, then ammonia gas is converted

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back to ammonium ions by trapping into a solution. Following this process, the trapped



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ammonium ions are determined by titration, and protein content is calculated with some

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conversion factors. Despite its widespread use, Kjeldahl method has serious drawbacks such

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as need for sample pretreatment, titration mistakes, use of corrosive and potentially toxic

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reagents and long analysis time. In addition, breakdown of all organic compounds by

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sulphuric acid and conversion of nitrogen to ammonium in sample preparation step should be

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performed properly, otherwise it causes erroneous results and poor precision. In Dumas

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method, on the other hand, sample is purged of any atmospheric gas and heated at about 1000

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°C and combusted in the presence of pure oxygen. Combustion products such as carbon

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dioxide, water and nitrogen as several oxides are passed on to hot copper to remove any

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oxygen and convert nitrogen oxides to molecular nitrogen. Then, the obtained signal from

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thermal conductivity detector is converted to total nitrogen content, and protein amount is

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calculated with conversion factors. However, high purity carrier gas and expensive equipment

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is required to complete the analysis.

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Principles of other protein analysis methods (Biuret, Nessler’s Reagent, Folin-Ciocalteau,

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Lowry, Bradford and bicinchoninic acid methods4) are based on interaction of protein and

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reactive or dye molecules. In these techniques, results are influenced by the presence of

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pigment molecules which absorb visible, UV or infrared light, thus some difficulties can be

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witnessed in food samples. Recently, spectroscopic methods have come to the fore because of

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their simplicity and short analysis time. Near Infrared Spectroscopy (NIR) is another method

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used for protein analysis, which is based on absorption of a wavelength of infrared radiation

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specific to peptide bond. NIR combined with chemometric methods has been developed due

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to the need for a rapid and simple protein analysis method. However, it is an indirect method

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to measure protein content of the sample. There are many studies which use NIR as a tool for

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determining protein content of food products.5-8 It is also preferred by food industry, and there

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are many commercial NIR analysis systems developed for determination of protein content. 4 ACS Paragon Plus Environment

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However, in NIR spectra, finger print spectrum cannot be seen as distinctly as in mid-infrared

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and far-infrared regions. The obtained overtones in NIR spectra is strongly affected by the

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matrix of sample. Therefore, regular calibration updates are necessary. Given all these

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disadvantages, it can be clearly seen that food industry urgently needs a new, rapid, in-situ,

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robust, accurate and sensitive method that will determine protein content of food products.

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Laser induced breakdown spectroscopy (LIBS) is a new, rapid and in-situ technique for

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elemental analysis.9, 10 LIBS is a laser based optical spectroscopy technique in which plasma

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is created by means of focusing the laser through a lens on the sample surface. Plasma

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contains free atoms, ions and electrons, and it emits plasma light which is characteristic of

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each element.11,

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spectrometer, and spectroscopic signals are produced. These signals are related to chemical

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compositions of the sample.11 LIBS does not require complex sample preparation step and is a

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rapid method which has been used for qualitative and quantitative measurements of the

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elemental composition of different matrices such as solid, liquid and gas.13 The application

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field of LIBS has been increasingly expanding, including studies on metallurgy, mining,

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environmental analysis and pharmacology.14-16 There are also several studies on food

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applications such as analysis of rice powder, unpolished-rice flour and starch17 and Ca in

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breakfast cereals,18 Na and NaCl analysis in bakery products,19 determination of whey

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adulteration in milk powder,20 identification of meat species,21 oil analysis,22 determination of

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biological contaminants on foods and food surfaces,10 effect of processing techniques on Mg

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content of green leafy vegetables23 and analysis of medicinal herbs rich in flavonoids.24

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The obtained light is collected with fiber optics and directed into the

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The aim of this study was to develop a new, rapid, in-situ and advanced method to

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determine protein content, related to nitrogen content, in the protein structure of cereal

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samples by using LIBS combined with multivariate data analysis technique such as partial

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least square (PLS). The study focuses on developing a relatively rapid, practical and 5 ACS Paragon Plus Environment


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economical method compared to the conventional ones. For this purpose, wheat flour and

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whole meal samples with different protein contents were analyzed with LIBS, and the spectra

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were correlated with the results of Dumas method by using chemometric methods.

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MATERIALS AND METHODS

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Sample Preparation. Totally 140 wheat flour and whole meal samples with different

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protein contents ranging between 7.9-20.9%, were obtained from Turkish Republic Ministry

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of Food, Agriculture and Livestock, General Directorate of Agricultural Research and Policies

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(Ankara, Turkey). Two mL deionized water were added into a 3 g sample and mixed to obtain

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a basic dough. After dough formation, the samples were placed into Glutork 2020 device

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(Perten Instruments, Hägersten, Sweden), also known as dry gluten tester, which provides

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cooking of dough as a sheet, then rested for 4 min at 150 °Ϲ. For LIBS analysis, three

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replicates were prepared from each sample. Ninety different flour samples and 50 different

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whole meal samples with different protein contents were analyzed to obtain data for

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calibration and validation graphs.

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Protein Analysis. Protein analysis was conducted using the Flash 4000

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Nitrogen/Protein Analyzer (Thermo Scientific, Cambridge, England). 500 mg of wheat flour

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and whole meal samples were weighed into tin copper capsule and introduced into a

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combustion reactor in automated equipment. The released nitrogen was carried by helium gas

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and quantitated by using thermal conductivity detector (TCD). Then, total nitrogen content

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was converted to protein content by using conversion factor.1 Protein content was calculated

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as in Equation 1.1

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% protein = % N × c

(Equation 1) 6 ACS Paragon Plus Environment

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Where c: conversion factor. In cereals, conversion factor may vary because of the 25

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composition difference.

Based on this differentiation, conversion factors were chosen as

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5.70 and 5.83 for wheat flour and whole meal analysis, respectively.

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LIBS Instrumentation. In the LIBS experiments Nano SG 150 mJ Nd:YAG laser

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(Litron Lasers, Warwickshire, England) which emits a laser pulse at 1064 nm and 5 channel

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Aurora LIBS spectrometer (Applied Spectra, Fremont, CA) which records the spectrum

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between 186-888 nm were used. Figure 1 shows the experimental setup of LIBS system.

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Laser was operated in Q-switched mode at a repetition rate of 8 Hz and 44.6 mJ/pulse, while

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spectrometer was operated at 650 ns gate delay and 1.05 ms integration time. Surfaces of the

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samples were scanned with laser at 50 different regions (three replicates for each sample) by

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using a rotary system which was operated at 1.33 rpm.

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Data Analysis. Determination of LIBS spectra is very difficult because of its high-

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dimensional, large and complex data, thus multivariate data analysis was performed to obtain

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quantitative data, which also provided elimination of laser fluctuations from shot-to-shot and

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physical/chemical matrix effect. Complexity of the study lies in the correlation of

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spectroscopic variables (wavelengths) and chemical differences between samples. PLS is

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frequently used for such type of analyses. It is a multivariate data analysis method used for

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data quantitation. To evaluate wide range spectrum and reduce matrix effect of the samples,

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samples with different protein contents were analyzed using PLS_Toolbox ver. 7.5.2

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(Eigenvector Research, Wenatchee, WA). In this study, 100 different flours with various

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protein contents were used for calibration data set; and 40 different samples with various

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protein contents were used for validation data set. Orthogonal Signal Correction (OSC) pre-

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processing method was used to obtain a global model for protein prediction in both of flour

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and whole meal samples. In order to evaluate measurement sensitivity and precision, relative

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standard deviation (RSD), relative error of prediction (REP) and relative accuracy (RA) 7 ACS Paragon Plus Environment


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values of PLS method were calculated using the following equations (Equation 2-4).26 Also,

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limit of detection (LOD) and limit of quantitation (LOQ) values were calculated based on a

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new approach in multivariate calibration, which takes into account IUPAC official

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recommendations and the latest development in error-in-variables for PLS.27

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ĉ

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REP% =

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Where Nv : number of validation spectra; ci : true concentration; and ĉi : predicted

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concentration.

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RSD% =

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Where Nconc : number of different concentrations in the validation set; ρ : number of spectra

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per concentration; and σ : Standard deviation.

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RA =

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Where d: difference between LIBS measurement and reference method; S.D.: standard

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deviation of LIBS measurement; n: the number of measurement ; t0.95: t value at 2.5% error of

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confidence; and M: reference method result.

∑

!"#!

!"#! ∑(

$%& '&

(Equation 2)

with

,

σ'& ) = ∑

ĉ&*! + &

,

|/|01.3.× 45.67 /√:

(Equation 3)

(Equation 4)

;

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RESULTS AND DISCUSSION

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In this study, wheat flour and whole meal samples were analyzed by using LIBS. For the

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analysis, high power laser source was focused on the surface of cereal samples and absorbed

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energy rapidly was converted into heat. When local temperature reached between 8000-20000

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K, the value at which small portion of material vaporizes and turns into ionized gas, plasma


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formation occurred. In plasma formation, organic structure burns and molecular structure

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turns into elemental forms which emits characteristic light. These emissions can be analyzed

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quantitatively according to the intensity of peaks. In this study, full spectrum and major

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nitrogen peaks of wheat flour and whole meal are presented in Figure 2. LIBS spectra were

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recorded through laser-scanning of 50 shots for each flour sample, and triplicate

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measurements were performed with LIBS. Then, 150 spectra from triplicate measurements

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were used to obtain the average spectrum for that sample. In the literature, nitrogen peaks

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were located between 340-870 nm, and three major peaks were reported at 742, 744 and 746

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nm. 28, 29

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LIBS spectra were normalized to eliminate fluctuations of LIBS experiments such as

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laser energy variations shot-to-shot. Normalization was carried out by averaging the spectra

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over the number of measurements.

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were carried out by averaging the fully integrated peak intensity and normalizing the

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integrated continuum emission intensity (Figure 2). The method has been applied as a reliable

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analytical method

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lines and nitrogen content of wheat flour and whole meal.

31, 32

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For each spectral line, peak to base (P/B) calculations

as it provides linear relation between intensities of atomic spectral

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LIBS can measure all elements in flour including nitrogen, which makes it applicable for

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measuring protein content representing the total nitrogen content. For this reason, the obtained

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LIBS spectra were correlated with the results of Dumas analysis by using PLS method. Figure

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3 demonstrates calibration and validation graphs of wheat flour and whole meal samples

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together. Low root mean square error of calibration (RMSEC) (0.33) and root mean square

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error of cross validation (RMSECV) (1.73) values were chosen to develop the calibration

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model, and high coefficient of determination value was observed (latent variable 3). In

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addition, root mean square error of prediction (RMSEP) value was observed as 0.93 for

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validation. 9 ACS Paragon Plus Environment


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In order to evaluate measurement sensitivity and precision, RSD, REP, LOD and LOQ

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values of PLS method were calculated.26, 27 The accuracy of LIBS compared to Dumas was

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evaluated with relative accuracy (RA) value. RA is calculated using Equation 4.26 RSD, REP,

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LOD and LOQ values were calculated as 4%, 4.5%, 0.26% and 0.77%, respectively; and

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these values indicate that LIBS is a reliable and sensitive method for protein analysis in wheat

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flour and whole meal samples and has good prediction ability. Also, Dumas results of the

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samples as a function of protein level showed low RA values at about 0.13, which means that

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LIBS is a quite acceptable technique for protein measurement in wheat flour and whole

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meal.33

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The present study is a novel study since it has made use of LIBS on food applications for

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protein analysis in wheat flour and whole meal samples. Besides, other experiments in the

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literature used only a standard calibration curve method which utilized only a single band. It

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was reported in a previous study that nitrogen calibration can be successfully accomplished

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following only 746 nm in the spectrum and it is not possible to use other lines of nitrogen in

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the calibration study.

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wavelength range between 340-888 nm (resolution: 0.07 nm) with the help of PLS. With this

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method, all lines of nitrogen in the sample made contribution to the calibration model.

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However, in this work, calibration study was carried out by using a

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In previous studies, emissions from nitrogen were observed when laser plasma was

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formed in air or on a solid surface in air, and it had an impact on the results. This influence

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can be eliminated by reducing atmospheric pressure, using vacuum conditions or using inert

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gases to remove the air influence. This difficulty is caused by occurrence of strong nitrogen

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emission lines after the first few microseconds following plasma formation.28, 29 However, in

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this study, data set was collected under the same atmospheric conditions which produced the

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same influence on the whole dataset. Nitrogen, and also oxygen and hydrogen interferences

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coming from the air have a background role, and PLS can eliminate this interference effect 10 ACS Paragon Plus Environment

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from all the collected data while obtaining a model. Therefore, atmospheric nitrogen does not

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cause negative effects on determination of protein content if calibration and validation data set

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were collected under the same atmospheric conditions. Protein analysis has a vital role for

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understanding the nutritional value and quality of the product to determine utilization field,

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which makes it indispensable for manufacturers. Therefore, protein analysis is a routine and

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important analysis in food industry. In this study, LIBS method combined with multivariate

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data analysis was used as a rapid method for protein analysis in food applications for the first

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time. The results of the study are very promising for food industry considering that

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conventional protein analysis methods are both expensive and time consuming, and have

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complex sample preparation steps. In contrast, the developed method is rapid, economical, in-

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situ and requires minimum sample preparation step, which makes it very appealing for food

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industry. Unlike conventional protein analysis methods, LIBS has potential to analyze wheat

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flour and whole meal samples only in a few seconds, thus much higher numbers of flour

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samples can be analyzed rapidly by using LIBS. Consequently, this study can be promising

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not only for flour and whole meal samples, but also for other food products.

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Therefore, this method seems to be a useful tool for quality control laboratories of flour mills and other food processing companies.

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Acknowledgement

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The authors would like to thank to Turgay Sanal, General Directorate of Agricultural

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Research and Policies, Turkish Republic Ministry of Food, Agriculture and Livestock

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(Ankara) for his help in the sample collection and Prof. Durmus Ozdemir, Department of

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Chemistry, Izmir Institute of Technology (Izmir) for his contributions.

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remediation process of chromium polluted soil using LIBS. J. Hazard. Mater. 2009, 163,

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1265-1271.

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FIGURE CAPTIONS

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Figure 1. LIBS experimental setup.

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Figure 2. Full spectra of: (A) whole meal; (B) three major nitrogen peaks of the whole meal;

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(C) full spectra of the wheat flour; and (D) three major nitrogen peaks of the wheat flour.

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Figure 3. (A) PLS calibration; and (B) validation models of wheat flour and whole meal.


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Figure 1 177x84mm (300 x 300 DPI)

ACS Paragon Plus Environment


Figure 2 304x203mm (300 x 300 DPI)


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Figure 3 177x93mm (300 x 300 DPI)



For Table of Contents Only 83x47mm (300 x 300 DPI)


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Laser-Induced Breakdown Spectroscopy Based Protein Assay for

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