Accepted Manuscript

Authentication of indigenous flours (Quinoa, Amaranth and kañiwa) from the Andean
region using a portable ATR-Infrared device in combination with pattern recognition
analysis

Mei-Ling Shotts, Marcal Plans Pujolras, Collen Rossell, Luis Rodriguez-Saona

PII: S0733-5210(18)30024-9
DOI: 10.1016/j.jcs.2018.04.005
Reference: YJCRS 2557

To appear in: Journal of Cereal Science

Received Date: 10 January 2018

Revised Date: 18 April 2018
Accepted Date: 25 April 2018

Please cite this article as: Shotts, M.-L., Plans Pujolras, M., Rossell, C., Rodriguez-Saona, L.,
Authentication of indigenous flours (Quinoa, Amaranth and kañiwa) from the Andean region using a
portable ATR-Infrared device in combination with pattern recognition analysis, Journal of Cereal Science
(2018), doi: 10.1016/j.jcs.2018.04.005.

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1 Authentication of indigenous flours (Quinoa, Amaranth and kañiwa) from the Andean region using

2 a Portable ATR-Infrared Device in Combination with Pattern Recognition Analysis

3 Mei-Ling Shotts, Marcal Plans Pujolras, Collen Rossell and Luis Rodriguez-Saona*

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6 The Ohio State University Department of Food Science and Technology

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7 Parker Food Science and Technology2015 Fyffe Road, Columbus, Ohio 43210

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8 *To whom correspondence should be addressed:
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9 Luis Rodriguez-Saona: rodriguez-saona.1@osu.edu
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20 ABSTRACT

21 Quinoa (Chenopodium quinoa Willd.), kañiwa (Chenopodium pallidicaule Aellen) and kiwicha

22 (amaranth, Chenopodium pallidicaule) are high nutritional value grains that are at risk for adulteration

23 with less expensive grains. Our objective was to develop a rapid untargeted approach to authenticate high-

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24 value Andean grain flours by combining infrared spectroscopy and pattern recognition analysis. Samples

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25 (n=106) were collected from Peruvian (n=89) and US (n=17) markets that included quinoa, amaranth,

26 kañiwa and other common flours from the Andean region. Flour composition was characterized by

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27 reference methods including fat (Soxhlet, AOAC#922.06), protein (Dumas, ICC Standard #167), and

28 fatty acid profile (AOAC #996.0). Spectra was collected by using a portable infrared system with a

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29 single-reflection diamond ATR crystal, and analyzed by Soft Independent Model of Class Analogies and
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30 Partial Least Square Regression. Authentic grain flours formed distinct clusters and commercial samples

31 from Peruvian markets showed a prevalence of adulteration. Spectral differences were associated with the
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32 fingerprint region (1100-900 cm-1) due to differences in starch structure. Regression models were

33 generated for rapid determination of fat (Rcv=0.98, SECV=0.3) and protein (Rcv=0.98, SECV=0.96)
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34 levels. New generation of portable infrared devices provided a viable tool for chemical profiling allowing
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35 for rapid, “in-field”, and reliable authentication of food ingredients.

36 Key Words: portable Mid-Infrared spectroscopy, Andean flour, authentication, quinoa, kañiwa, amaranth
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37 Highlights
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38 ► Mid-infrared spectroscopy combined with pattern recognition analysis discriminated different types of
39 commercial flours including selected high-value Andean grains (Quinoa, Amaranth and Cañihua) and
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40 allowed to predict dietary constituents (fat and protein) of these grains.

41 ► Spectral differences responsible for classification of flours were associated with the fingerprint region
42 (1100–900cm-1) due to differences in starch structure.

43 ► The validated classification algorithm showed the prevalence of adulteration of Andean grain flours
44 obtained from several Peruvian street markets.

45 ► New generation of portable infrared devices provide a viable tool for in-situ chemical profiling making
46 it an alternative to time-consuming traditional testing methods
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47 1. Introduction

48 The United States (US) and Western diets are mostly based on wheat-derived foods which are

49 less satiating than other more nutrient grains (FAO, 2012). Wheat based products have been gaining a lot

50 of attention due to diet based allergies, diet intolerances (i.e. Celiac’s disease) and other health concerns

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51 (FAO, 2012). The U.S. Department of Agriculture (USDA) dietary guidelines is recommending

52 consumers to make a shift from high calorie food to more nutrient dense food, for example refined grains

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53 to whole grains, snacks with fruit products with added sugars to fruit. Currently US companies are

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54 moving towards functional foods and foods providing more nutritional impacts; in particular, alternative

55 crops like buckwheat, oat, quinoa, and amaranth grain are of interest for developing nutritional and

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56 healthier foods (Alvarez-Jubete et al 2010).

57 Three grains from the Andean region that are of interest to the food industry as alternatives for
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58 potential healthy alternatives to gluten-containing grains in the gluten-free diet are quinoa (Chenopodium

59 quinoa), kañiwa (Chenopodium pallidicaule) and kiwicha (Amaranthus caudatus) (FAO, 2012). These
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60 pseudo cereals are nutrient dense, gluten free, provide unique flavor profiles, baking functionalities, have
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61 been associated with health benefits, containing a variety of minerals, vitamins, and a balanced amino
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62 acid profile have a capacity to be transformed into a large range of products and are often substituted for

63 sparse animal protein in the Andes region (Alvarez-Jubete et al., 2010; Rosell et al., 2010, FOA, 2012).
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64 The most recognizable grain coming from the Andean region is quinoa, which has seen

65 unprecedented success throughout the world. Quinoa was nominated by FAO as a crop most likely to
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66 significantly contribute to global food security in the 21st century (FAO, 2012). Quinoa, kañiwa and
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67 amaranth are rich in polyunsaturated fatty acids; these acids are beneficial against cardiovascular disease

68 (Repo-Carrasco-Valencia et al, 2009). The highest percentage of fatty acids present in these flours is

69 Omega 6 (linoleic acid), being 50% for quinoa and 43% for kañiwa and ranging from 33 to 44% for

70 amaranth (Repo-Carrasco-Valencia et al, 2009). All three of these grains are high in protein and provide

71 other nutritional benefits including high antioxidant activity, rich in total phenolics and anthocyanins

72 (Gorinstein et al., 2008). Amaranth, which belongs to the same family as quinoa, has been gaining much
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73 attention for containing high lysine content compared to the other grains (Gorinstein et al., 2008).

74 Kañiwa, has a balanced essential amino acid (protein) profile similar to that of casein and it contains γ-

75 tocopherol, calcium and phosphorous (Alvarez-Jubete et al., 2010).

76 The new resurgence of Andean grains have not been met due to supply and demand in the South

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77 American region of the Peruvian–Bolivian altiplano due to farm lands being in rural areas, high altitudes,

78 and harsh climate (Jacobsen, 2011). In 2016 the FDA Food Code 21 CFR 2.25 was revised to prevent

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79 adulteration of pseudo-cereal grain and grain seed; since these Andean flours are hard to cultivate and

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80 obtain there is a concern that they could be mixed with less expensive flours (Jacobsen et. al, 2011).

81 Adulteration in the food industry is a growing concern and economically motivated adulteration (EMA) is

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82 one of the most common types of adulteration, driven by the demand for higher cost products and the

83 ability for companies to make more money by adulterating with less expensive ingredients. According to
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84 a Grocery Manufacturing Association report, companies generating inflated profits made by fraudulent

85 means comprise up to 10% of food products in retail stores, costing the industry an estimated $10 to $15
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86 billion per year (Grocery Manufacturing Association, 2015) and affecting food-trade systems threatening
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87 the livelihoods of honest purveyors of food in the global marketplace and the integrity and viability of
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88 their brand names. A public health concern is the potential adulteration with less expensive flours

89 containing undeclared allergens that could cause serious harm to the consumer. In 2015, there were
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90 numerous recalls of cumin as a result of purposeful adulteration with undeclared peanuts and almonds

91 affecting 700 different products by more than 40 manufactures causing the FDA to report the cumin recall
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92 as the “largest allergen recall” (Agres, 2015).

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93 Authentication of Andean flours is typically performed using gas chromatography, nuclear

94 magnetic resonance spectroscopy (NMR), polymerase chain reaction (PCR), Kjeldahl, Dumas, and

95 Soxhlet analysis (Li-Chan et al., 2010). All of these analytical techniques are accurate but time-

96 consuming, ranging from one hour to one day for results. Thus, technologies that can provide real-time

97 screening and field-based assessment are under high demand to control the quality and safety of raw

98 materials (Rodriguez-Saona et al., 2016). Vibrational spectroscopy (NIR, mid-IR and Raman) are rapid
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99 methods to establish reliable authentication for raw materials, offering a simple, high throughput,

100 sensitive and robust approach for detection and identification of adulterants and unavoidable

101 contaminants based on specific infrared signature profiles in combination with supervised pattern

102 recognition techniques (Rodriguez-Saona et. al, 2016). Developments in components miniaturization such

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103 as sources, wavelength selectors, and detectors have overcome crucial bottleneck issues of laser tuning,

104 power consumption, portability and cost (Rodriguez-Saona et al., 2016; Ellis et al., 2015). Portable and

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105 handheld spectroscopy has demonstrated its potential to move from the confines of the relatively stable

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106 and controlled laboratory environment and out into the potentially more challenging and dynamic

107 environments that would aid in the tractability of the food supply chain (Ellis et al., 2015). IR offers

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108 companies a more efficient process control to aid in a quick, efficient, and reliable technology to monitor

109 food (Rodriguez-Saona et al., 2016).

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110 The objective of this study was to develop a simple and rapid method to authenticate high-value

111 Andean grains by combining portable infrared spectroscopy technology and supervised pattern
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112 recognition techniques. This portable analytical technique can be an ideal screening tool for
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113 discriminating between high value Andean grains and cheaper alternatives as well as assessing different
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114 quality parameters including protein, fat and fatty acid profile.

115
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116 2. Materials and Methods

117 2.1 Materials

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118 A total of 106 samples were obtained from Peru and the United States, including authentic and
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119 samples from vendors from different wholesale street markets. Authentic flours (56), flour mixtures (4)

120 and market flours (29) were kindly provided by Ms. Tarazona from the Departamento de Industrias

121 Alimentarias at Universidad Nacional Agraria (La Molina, Lima – Perú). In addition, seventeen Andean

122 flour samples were purchased from online US retail stores and specialty markets in Columbus, Ohio;

123 these samples were from various regions in North and South America to account for varietal and

124 geographical differences.

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125 We identified 17 authentic flours of quinoa, 12 authentic flours of amaranth and 16 authentic

126 flours of kañiwa. Additional authentic flour samples included: barley (10), corn (5), fava bean (12), soy

127 (8), wheat (13) and negative control mixtures (4). The 29 samples bought from a market in Lima (Peru)

128 were labeled as quinoa (12), amaranth (3), kañiwa (7), barley (2), fava beans (3) and soy (2).

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129 The 89 flours obtained from Peru were sieved through a 600 mm screen in order to standardize

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130 granule size. The 17 grains obtained in the US were milled with a NutriMill Classic 760200 High Speed

131 Grain Mill (Bosch Kitchen Center, Munich, Germany) and sieved (600 mm screen) to ensure

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132 homogeneity and uniformity between all 106 samples.

133 2.2. Reference Analysis

134
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In order to develop qualitative and quantitative models with infrared technology, all the flour
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135 samples were characterized by determining the protein and fat levels and their fatty acid profile using

136 approved reference techniques in order to confirm their authenticity.

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137 2.3 Fat Analysis

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138 Fat analysis was done by using a Soxhlet gravimetric method (AOAC #922.06). Five grams of
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139 homogenous flour was placed into a cellulose thimble (33 x 80mm), the thimble was then placed into a

140 Soxhlet extractor connected to a round bottom flask containing 200 ml of petroleum ether that was heated
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141 on an isomantle set at 125°C. The percolating process ran for a total of 6 hours allowing for 240

142 cycles/refluxes. The percent fat was calculated by the difference in weight from the thimble plus sample,
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143 before and after the Soxhelt run. The round bottom flasks were then placed on a rotovaporator to remove
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144 the petroleum ether and collect the oil from the bottom of the flask. The oil was stored at refrigeration

145 temperature (4°C) until used for gas chromatography analysis.

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148 2.4 Fatty Acid Composition

149 Determination of fatty acid composition by capillary gas chromatography (AOAC Method

150 996.0). The oil extracted from flour samples was trans-esterified to their FAMEs with potassium

151 hydroxide for determining the relative distribution of fatty acids in the flour sample. Oil (0.1g) was placed

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152 into a 2 mL centrifuge tube and 1 mL of hexane was added and vortexed for 30 seconds for homogeneity.

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153 Then 20 µL of a 2 N potassium hydroxide in methanol was added with a pinch of sodium sulfate

154 anhydrous. Samples were centrifuged in an Eppendorf Centrifuge (Eppendorf North America,

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155 Hauppauge, NY) for 10 minutes at 16080 G- force at 15ºC and the supernatant (1.5 mL) was carefully

156 transferred to a GC vial avoiding extracting the salt residue and analyzed in duplicate on a Hewlett

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157 Packard 6890 GC equipped with a flame ionization detector (FID) and a HP G1513A auto-sampler.
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158 Separation of the fatty acid methyl esters was done using a HP-88 column (60 m x 0.32 mm x 0.5 µm)

159 (Agilent technology, Santa Clara, CA, USA) using helium gas as the carrier gas. The injection volume
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160 was 1.0 µL in split ratio 120:1. The oven conditions were 120 °C hold for 1 minute, then increased to 175

161 °C (10 °C/min), hold for 10 minutes at 175 °C, increased to 210 °C (5 °C/min), hold for 5 minutes at 210
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162 °C, increased to 230 °C (5 °C/min) and hold for 2 minutes. The injector temperature was 250 °C and the
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163 detector temperature was set to 280 °C.

164 2.5 Protein Analysis

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165 Protein analysis was done by the Dumas method for determination of nitrogen (% protein) in the
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166 flour samples based on the ICC Standard No. 167 from the International Association for Cereal Science
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167 and Technology; a factor of 6.25 was multiplied to the % nitrogen in order to calculate total % protein in

168 the pseudo cereals. The Dumas method is based on combustion of the whole sample in an oxygen-

169 enriched atmosphere at high temperature (about 900°C) in order to ensure complete combustion. Samples

170 were analyzed by The Ohio Agricultural Research and Development Center Star Lab.

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172 2.6 FT-IR Spectroscopy

173 Infrared spectral data was collected on portable Cary 630 Fourier transform- infrared

174 spectrometer (Agilent Technologies Inc., Santa Clara, CA) equipped with a 1.3 mm diameter,

175 thermoelectrically-cooled dTGS, single-bounce attenuated total reflectance (ATR) diamond crystal

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176 interface. Milled and sieved flour powder samples (~0.1 g) were placed directly on the surface of the

177 diamond ATR crystal and spectrum was collected in the 4,000–700 cm−1 region at 4 cm−1 resolution by

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178 pressing the powder sample onto the crystal using a pressure clamp. A background was taken prior to

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179 each sample run and the data was collected by co-adding 64 scans. Two independent spectra were

180 collected per flour sample.

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181 2.7 Data Analysis

182 The target components from Andean flours were evaluated using a randomized ANOVA
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183 experimental design with generalized linear model (GZLM) because the data set was unbalanced by

184 unequal number of samples per flour type. GZLM generalizes the response variables that are not
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185 distributed normally and generalizes the error, allowing the model to be more powerful (Hilbe, 1994).
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186 All analyses were done in duplicate. Comparisons were carried out by Tukey-Kramer post hoc test with
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187 probability values lower than 0.05 were considered significantly different. Tukey-Kramer Post hoc

188 comparison was used due to unequal sample sizes of the flours; the method can estimate standard
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189 deviation for each pairwise comparison (Brillinger, 1984). The data was analyzed using IBM SPSS

190 Statistics, version 23 (IBM Corp., Armonk, NY, USA).

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191 The spectral data was analyzed using multivariate statistical analysis software (Pirouette version
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192 4.0, Infometrix Inc., Bothell, WA, USA). The spectra were imported into the software from the

193 instruments as GRAMS (.spc) files and normalized and smoothed (S-G polynomial fitting algorithm with

194 a 35-point window) prior to multivariate analysis.

195 2.9 Soft independent modeling of class analogy (SIMCA)

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196 Because the richness of information often results in high spectral complexity, it requires the use

197 of multivariate data analysis to extract meaningful information (Beebe, 1998). SIMCA was used to

198 classify authentic Andean grain flour samples by grouping samples that have common infrared spectral

199 features and distinguishing them from samples with different profiles. SIMCA is a supervised pattern

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200 recognition methods, whereby a training set with known classes (kañiwa (class 1), quinoa (class 2) and

201 amaranth (class 3)) are used to build a mathematical model which is then evaluated by an independent

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202 validation data set (Beebe et al., 1998). Each class is generated by a separate PCA model providing

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203 information about the relevance of variables and outlier detection. Unknown samples are projected into

204 each PCA model and the degree of the fit allows to determine whether a sample is assigned to a class, fits

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205 several classes or does not fit in any of the classes (Massart et al., 2001).

206 In addition, other authentic flours that included fava bean (class 4), wheat (class 5), barley (class
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207 6), soy (class 7), corn (class 8) and known mixtures of flour samples containing at least two of the

208 Andean flours were used as negative controls to determine the robustness of the classification model.
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209 Probability clouds (α ≥ 0.05) surrounding the clusters allow SIMCA to be used as a predictive modeling
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210 system. The validated model was used to predict wholesale market samples from Peru to determine if
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211 these flours match their “market” labels.

212 SIMCA performance was examined in terms of class projections, misclassifications (percentage
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213 of samples correctly allocated to their original groups) and interclass distance (ICD) describing

214 quantitatively the similarity or dissimilarity of the different classes, being generally accepted that samples
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215 can be differentiated when ICD > 3 (Massart et al., 2001).

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216 2.10 Partial Least Squares Regression (PLSR)

217 PLSR uses features adapted from both Principal Component Analysis and Multi-linear

218 Regression, allowing data reduction techniques from spectra to extract important variables that provide

219 chemical signatures for analysis (Moseholm, 1988). Partial least squares (PLS) was developed to handle

220 data sets with more variables than samples by replacing the original variables with a few latent variables
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221 (LVs). Known as a bilinear factor method, PLSR attempts to find multidimensional directions in the

222 spectral matrix (X) that explains the maximum variance in the column vector (Y) (Wu et al., 2014). Both

223 the spectra (response variables) and concentration (dependent variables) matrixes are decomposed

224 simultaneously to generate a set of orthogonal LVs with the first few most related to predict dependent

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225 variables selected for the model calibration. PLS regression takes into consideration that measured values

226 from reference analyses (i.e. protein and fat levels) may contain errors associated with sample preparation

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227 (Moseholm, 1988). However, PLS is a method that has high tendency of over-fitting by selecting too

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228 many LVs which not only describes the information of the data but also the noise, while choosing too few

229 LVs implies an under-fitted model which incorporates insufficient information of the data (Wu et al.,

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230 2014). Overfitting of models was prevented by using cross-validation (leave-one-out) approach selecting

231 the optimum number of PLS components according to the minimum RMSEV criteria. In addition,
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232 variable selection was used to lower the risk of over-fitting by reducing the dimensionality of the variable

233 space.
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234 The fitness of the model was evaluated using the standard error of cross validation, coefficient of
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235 determination (r) and outlier diagnostics. The standard error of cross validation (SECV) shows the
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236 magnitude of error expected when independent samples are introduced into the calibration models and the

237 compound concentrations in the unknown samples are predicted (Moseholm, 1988; Vinzi et al, 2010).
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238 During the model development, samples with large residuals, unusual pattern and high leverage are

239 considered outliers and removed from the models (Vinzi et al, 2010).
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240 3. Results and Discussion

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241 3.1 Characterization of Flours

242 Table 1 summarizes the sample diversity between the flours with regards to levels for protein, fat

243 and fatty acid composition that were used to develop calibration models based on mid-infrared spectra to

244 authenticate high value Andean flours. Protein and fat levels of Andean flours ranged from 13.8 - 16.4%

245 and 8.0 - 9.8%, respectively (Table 1). The range of protein and fat were comparable to literature values

246 (Table 2) for the three Andean flours of interest (quinoa, kañiwa and amaranth), confirming that the
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247 Andean flours were authentic and matched values reported for the genus Chenopodium. The other flours

248 showed a larger range in protein (9.7- 35.4%) and fat (4.4-23.4%), with soybean flours having the highest

249 protein and fat levels among all flours while barley (8.6%) and fava bean (4.2%) flours had the lowest

250 protein and fat levels, respectively. In general, the pseudo-cereals (quinoa, amaranth, kañiwa, barley,

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251 wheat, corn and soy) showed similar ratios of protein to fat (1.8:1) while fava beans (legume) showed a

252 higher ratio of 6:1. Our data is supported by similar protein to fat ratios reported in the literature (Repo-

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253 Carrasco-Valencia et al, 2009; Rosell et al, 2009; Aluwi et al, 2017). Proc GZLM analysis for the three

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254 Andean flours (quinoa, amaranth and kañiwa) showed differences in levels of fat (p-value=0.013), protein

255 (p-value = 0.0019) and some of the minor fatty acids that included steric acid (p-value = 0.005), linolenic

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256 acid (p-value = 0.0002) and arachidic acid (p-value = 0.001). Tukey-Kramer Post hoc comparisons

257 indicated that kañiwa and amaranth were similar in protein and fat but showed significant differences
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258 (p<0.01) for quinoa. Fatty acid composition was similar in kañiwa and quinoa (p>0.1) while both showed

259 a significant difference compared to amaranth (p-value ≤ 0.05) in steric, linolenic and arachidic acid
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260 levels. Interesting amaranth had the highest level of arachidic acid (10%) among all flours evaluated.
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261 Ryan and others (2007) reported similar levels in arachidic acid (10.5%) in amaranth. Although, the three
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262 Andean flours, belonging to the genus Chenopodium, shared similar composition profiles, there were

263 distinct compositional variations that allowed differentiation amongst the flours.
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264 Even though the Andean flour samples were collected from different locations, the coefficient of

265 variability (COV) within flour type was ≤10% indicating uniform compositional traits suggesting that
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266 related cultivars are used for commercial applications. Aluwi and others (2017) compared the proximal
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267 analysis of 28 different varieties of quinoa from 8 different origins (United States of America, South

268 America and Europe) and found similar protein and fat levels in all varieties.

269 3.2 Soft independent modeling of class analogy (SIMCA) of Andean flours

270 Figure 1 shows the characteristic spectrum of quinoa, amaranth, and kañiwa using a portable

271 mid-infrared single reflection ATR system. The most prevalent bands were associated to the starch
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272 structure (1100-900 cm-1), amide I (1645 cm−1), amide II (1550 cm-1), lipids (1750 cm-1; 2800-3000 cm-1)

273 and water (3300 and 1680 cm-1) (Cozzolino et al., 2015). The OH-stretching vibration at ~3300 cm-1

274 showed some variation among samples likely due the drying of the samples. The Andean flours (Figure

275 1) showed strong intensity of the lipid bands centered at 2925 and 2855 cm-1, attributed to C-H stretching

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276 vibrational modes of alkylic CH2 and CH3 groups. Amaranth showed a well-defined and intense band at

277 1745 cm-1, typical of the ester carbonyl stretching common of the esterification of fatty acids in the

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278 glycerol backbone (Li-Chan, 2010). The information-rich fingerprint region from 900-1500 cm-1 contains

signal from amylose-lipid complexes present in the whole grain, amide III (1330-1230 cm-1), or structural

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280 carbohydrates such as starch and cellulose (Cozzolino et al., 2015).

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281 Mid-infrared spectroscopy is a powerful analytical technique but the interpretation of a spectrum
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282 collected from foods can be very complex due to the large number of compounds in the mixture, and the

283 signal are often overlapping. Visual spectral inspection of such data is subjective, impractical,
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284 cumbersome and subtle changes are difficult to detect (Rodriguez-Saona et al., 2016). Thus, we paired the

285 infrared data with pattern recognition to simultaneously analyze the large number of spectral variables by
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286 reducing them to a few key features, providing an effective tool for profile discrimination. We used soft
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287 independent modeling of class analogy (SIMCA) classification and defined a priori the number of

288 categories (flour types) and the samples that belong to each category confirmed by the reference analysis.
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289 We used forty-five flour samples to train the classification models using reference material

belonging to the genus Chenopodium. Class projections using the first three principal components
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291 generated well-separated clusters for the three Andean flours (Figure 2A) based on their unique infrared
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292 spectral patterns. The three classes had an interclass distance (ICD) > 3, indicating large Euclidian

293 distances between centers of clusters to be considered significant for identification. The kañiwa class had

294 an ICD of 4.8 and 3.6 with regard to quinoa and amaranth classes, respectively. In addition, amaranth and

295 quinoa classes had an ICD of 5.6. Interestingly, the closest ICD was obtained between kañiwa and

296 amaranth that showed the greatest similarities in chemical composition among flours evaluated (Mujica,
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297 1994). The discriminating power plot (Figure 2B), defining the variables (wavenumbers) that have a

298 predominant effect on sample classification by minimizing the difference between samples within clusters

299 and maximizing those from different clusters (Massart et al., 2001), showed that bands centered at 960,

300 1000, 1040 and 1100 cm-1 associated with the crystalline/amorphous structures of starch were important

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301 in clustering the different Andean flours. The infrared band at 960 cm-1 is attributed to the glycosidic

302 linkages in starches (Deeyai et al., 2013). The band at 1000 cm-1 is related to C-O-C linkages in the

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303 glucopyranose ring and the D-glucosidic bindings and its intensity can be affected by changes in the

molecular environment (Li-Chan, 2010). The band at 1040 cm-1 has been related to the degree of

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305 crystallinity of the material while the C-OH stretching vibration shoulder band near 1020 cm-1 is

associated to amorphous compounds (Li-Chan, 2010). The band at 1110 cm-1 can be attributed to the C–O

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307 stretching mode of secondary alcohols in polysaccharides and cellulosic compounds (1290−1180 cm-1)
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308 show an intense band centered at 1240 cm-1. The simple C-H bending vibrations of methyl (CH2, 1470

309 cm-1) and methylene (CH3, 1380 cm-1) groups of lipids and proteins side chains occur between 1500 and
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310 1300 cm-1. Lastly, there is influence of the amide II (~1550 cm-1) band originating from peptide N-H
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311 bending modes, coupled with C-N stretching modes (Fabian and Mäntele, 2002). A second SIMCA
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312 model was developed with some less expensive flours commonly traded in the region that included white

313 corn, wheat, barley, fava beans and soybeans. Figure 2C shows the SIMCA class projection displaying
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314 that all classes were clustered in well-separated groups indicating marked compositional differences

315 among the Andean (quinoa, amaranth and kañiwa) and the other flours that are commonly used in
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316 mixtures to complement their nutrient make-up. The interclass distances between the Andean and other
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317 flours ranged from 3.9 (quinoa & wheat) to 18 (quinoa & soy). The discriminating power plot (Figure

318 2B) showed that the frequencies associated with starch structure (1500 - 950 cm-1) were the most

319 important in classifying among flour types. Besides the influence of starch, the inclusion of additional

320 flour samples resulted in extra bands that were important in explaining the variance in the model

321 including the carbonyl group (C=O) stretching at around 1745 cm-1, the broad and strong Amide I band at

322 1645 cm-1 due to C=O stretching coupled with an in- plane bending of the N-H and C-N stretching
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323 modes, and the amide II band centered at around 1540 cm-1 due to C=O stretching coupled with C-N

324 stretching and bending deformation of N-H in the protein backbones (Li-Chan, 2010). Also, the bands in

325 the range from 1500 to 1300 cm-1 are due to bending deformation modes of CH/CH2/CH3 groups

326 originating from the various amino acid side chains and lipids and the band centered at 1180 cm-1

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327 associated to the C-O groups of carbohydrates (Li-Chan, 2010) arising from the other flours.

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328 After confirming that the SIMCA models correctly classified the Andean flours from other less

329 expensive flours commonly traded in the region, we challenged the model by including mixtures of flours

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330 that are marketed as a nutritious breakfast meal. These mixtures contained seven grains (“7 semillas”) and

331 were purchased in various markets in Lima (Peru). All products included amaranth and quinoa with the

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332 other grains including kañiwa, wheat, barley, white corn, fava beans, flaxseed, maca or carob bean. An
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333 important advantage of SIMCA is the ability not only to determine whether a sample belongs to any of

334 the predefined categories, but also to determine if it does not belong to any class (Massart et al., 2001).
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335 By using the prediction algorithm generated by the SIMCA model, all 4 mixtures were correctly

336 identified as not belonging to any of the designated flour classes and were given a class zero (0)
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337 indicating no class could be assigned (Figure 3A).

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338 Lastly, twenty-nine flour samples purchased from various wholesale markets in Lima (Peru) were

339 authenticated by using the SIMCA prediction algorithm. Figure 3A shows the SIMCA class projection
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340 for the predictions of the wholesale market samples indicating some prevalence of adulteration of the

Andean grains likely with less expensive grains, such as wheat, fava beans, corn or barley flours. All
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342 barley, soy and fava bean flours were correctly predicted as authentic but only ~20% of the Andean
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343 samples were predicted as authentic. The SIMCA prediction results were corroborated with reference

344 analysis that included fat and protein content and fatty acid profile (Figure 3B), showing 100%

345 agreement between the reference and infrared analysis. Quinoa, amaranth and kañiwa are often described

346 as a "supergrain" and have become popular among the health conscious consumers because of its

347 exceptional nutritional content. The informal nature of wholesale markets in Lima (Peru) creates settings
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348 for fraud for economic gain, targeting high-value products for adulteration by deliberate substitution or

349 misrepresentation of food ingredients. Our results support the application of a portable infrared system for

350 rapid, cost-effective and in-field testing to detect potential adulteration of Andean grain flours enabling

351 simple surveillance of ingredients by regulatory agencies and food industry to prevent economically

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352 motivated food fraud.

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353 3.3 PLSR analysis for Determination of Protein and Fat Contents

354 The infrared spectra collected using the portable unit was utilized to develop quantitative models

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355 with partial least squares regression (PLSR) based on reference values for protein, fat and fatty acid

356 contents. A full cross-validation was used to generate the calibration dataset using the relevant spectral

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357 ranges associated with fat content (1800-1700 cm-1) representing the carbonyl v[C=O] stretching vibration
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358 associated to the degree of lipid esterification while for protein content (1700 to 1400 cm-1) targeting the

359 amide I and amide II regions. Figure 4 shows the PLS regression plot developed for the fat (Fig. 4A) and
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360 protein (Fig. 4B) content needed an optimal number of PCs of four, providing a correlation coefficient

361 (R2) of 0.98 and a standard error of cross validation (SECV) of 0.3% and 0.96%, respectively. We used
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362 all flour samples (n=106) that included calibration (n=56), seven grains mixtures (4) and wholesale
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363 market (n=29) that gave minimum–maximum range of 2-14% for fat and 5-37% w/w for protein. The fat

364 content of soybean flours was not included in the PLSR model because it was much higher than the levels
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365 in other flours (~23%) that exerted a high leverage for the model. Ferrão and Davanzo (2005) reported the

366 use of a horizontal attenuated total reflectance (HATR) accessory combined with PLSR to generate
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367 predictive models for the determination of protein content (range 8.9 to 13.2%) of wheat flours with root
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368 mean standard error of prediction (RMSEP) of 0.198 and R2 of 0.98, however, the models required 9

369 factors having the risk of overfitting the models causing the regression coefficients to adapt to

370 peculiarities of a specific dataset be misleading (random noise) results in poor generalizability (Massart et

371 al., 2001). Near infrared reflectance spectroscopy (NIRS) has been vastly reported for the determination

372 of protein content in flours such as rice (n=218) samples collected over 2 years (range of protein: 4.89–
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373 12.48%; SECV=0.23% with 4 factors), maize (n=276) samples (range of 7.8-15.1%; SECV=0.32 and R2

374 = 0.78), and wheat (n=96) samples (range of 6.43-14.94%; SEP = 0.425 and R2 = 0.91 using 10 factors)

375 (Rosales et al., 2011).

376 Using a comparable large range of protein levels (15-35%) to our study, Hacisalihoglu and others

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377 (2010) reported standard error of prediction (SEP) of 1.9% and R2 of 0.82 using 12 factors to determine

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378 individual common beans seed protein content from 267 bean seeds representing 91 diverse genotypes

379 collected by NIRS spectra. The results reported in the study represent lower accuracy and robustness

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380 compared to our protein models using a portable mid-infrared spectrometer utilizing a wide distribution of

381 pseudo-cereal types available in retail markets.

382
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Likewise, our PLSR models generated to estimate the fat levels in various flours performed better
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383 than models developed by NIR (1100-2498 nm) reflectance for prediction of fat in diverse cereal products

384 with fat content range of 0.1-30%, reporting prediction errors of ~0.9% and coefficient of determination
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385 (r2) of 0.98 (Kays et al., 1998). Using a wider range of total fat content from 0.5- 43.2% the performance

statistics gave a SEP of 1.07 and r2 of 0.99 using a dispersive NIR spectrometer (Vines et al., 2005).
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387 According to the authors, NIR spectroscopy has potential for quality monitoring of cereal food products
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388 and the models predicted within NLEA accuracy guidelines (Kays et al., 1998; Vines et al., 2005).
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389 Encina-Zelada and others (2017) described a Near-Infrared (850-1048 nm region) transmission

390 spectroscopy approach for the routine determination of dietary constituents, including fat (5.35-7.78% db)
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391 and protein (8.33-11.38% db), of quinoa (Chenopodium quinoa Willd.) grains from 77 cultivars. Fair
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392 prediction were reported by the authors, with best models for protein and fat content giving correlation

393 coefficients of <0.8 and SEP of 0.53% and 0.25%, respectively.

394 4. Conclusion

395 We have described a rapid (< 1 min) and robust approach for authentication of Andean grain

396 (kañiwa, amaranth and quinoa) flours by interfacing the mid-infrared spectra collected by a battery-
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397 powered and portable device to a pattern recognition algorithm achieving a full system automation for

398 screening high-value Andean grain flours and to discourage adulteration practices.

399 The calibration model was developed using commercial Andean grain flour samples to provide

400 diversity, making the model robust to variations in composition encountered in market products. All flour

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401 samples were characterized by monitoring the protein, fat and fatty acid profiles to confirm their

402 identification, providing excellent discrimination ability between flour types and correctly distinguishing

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403 the Andean grain flours from other prevalent flours common in the market and popular seven grain flour

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404 mixtures that contained varied proportions of these Andean grains. The discriminating power plot

405 indicated that the spectral features that were most important for the classification model were associated

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406 to variations in the starch structure. The classification model was effective in identifying the miss-

407 representation of Andean grain flour samples purchased from street markets in Lima, Peru.
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408 Furthermore, the same spectra was used to generate regression models (PLSR) to estimate the

409 protein (range 5-37% w/w) and fat (range 2-14% w/w) content of flours giving correlation coefficients
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410 >0.98 and SECV of 0.96% and 0.3%, respectively. The predictive models employed an optimal number
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411 of latent variables of four, preventing the risk of overfitting. When comparing the performances of
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412 published NIR predictive models for protein and fat in flours and cereal products using comparable

413 content ranges, our models outperformed the NIR technique giving lower SECV with fewer numbers of
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414 latent variables. The portable infrared device can be employed for routine screening at the receiving point

415 of flour trade companies and also has the potential to be used in supporting breeding programs for
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416 monitoring composition traits.

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417

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419

420

421
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422 References

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428 contribute to world food security. Regional Office for Latin America and the Caribbean. Vitacura,
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439 in vitro antioxidant activity of amaranth, quinoa buckwheat and wheat as affected by sprouting and
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443 8. Brillinger, J.A., 1984. The Collected Works of John W. Tukey: Time (Vol. 1). CRC Press.
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446 10. Deeyai, P., Suphantharika, M., Wongsagonsup, R. and Dangtip, S., 2013. Characterization of
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449 11. Ellis, DI; Muhamadali, H; Haughey, SA; Elliott, CT; Goodacre, R. 2015. Point-and-Shoot: Rapid
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456 13. Fabian, H. and Mäntele, W., 2002. Infrared spectroscopy of proteins. Handbook of vibrational
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464 16. Hacisalihoglu, G., Larbi, B. and Settles, A.M., 2009. Near-infrared reflectance spectroscopy predicts
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477 22. Moseholm, L., 1988. Analysis of air pollution plant exposure data: the soft independent modelling of
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480 23. Mujica, A., 1994. Andean grains and legumes. Neglected crops, 1492, pp.131-148.
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482 functional components in two varieties of crude and extruded kiwicha (Amaranthus caudatus).
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485 food authenticity testing. In Advances in Food Authenticity Testing (pp. 71-116).
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488 breeding programs. Journal of agricultural and food chemistry, 59(20), pp.10781-10786.
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490 kañiwa, kiwicha, and tarwi. Cereal chemistry, 86(4), pp.386-392..

491 28. Ryan, E., Galvin, K., O’connor, T.P., Maguire, A.R. and O’brien, N.M., 2007. Phytosterol, squalene,
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494 29. Vinzi, V.E., Chin, W.W., Henseler, J. and Wang, H. eds., 2010. Handbook of partial least squares:
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497 spectroscopy and nuclear magnetic resonance techniques in tandem with multivariable selection for
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499 technology, 7(6), pp.1555-1569.

500
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501 LIST OF FIGURES

502 Figure 1: Representative ATR-Infrared spectra of Kañiwa, Quinoa and Amaranth flours, indicating band
503 assignments for functional groups found in the spectrum.

504 Figure 2: Soft Independent Model of Class Analogies (SIMCA) class projection of (A) Andean (Kañiwa,
505 Quinoa and Amaranth) grain flours, (B) Andean grain flours and 5 other common pseudo-cereals and

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506 legume flours sold in these markets and (C) Discriminating power plot indicating spectral signatures that
507 allow for discrimination between classes.
508
509 Figure 3: SIMCA prediction plots for authentication of market samples. All flours were purchased in

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510 various street stands in wholesale markets in Lima, Peru.

511 Figure 4: Partial Least Squares Regression plots for (A) total fat based on Soxhlet reference method and

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512 (B) total protein based on Dumas method, including all flour samples (n= 106).

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513

N-H Bend (Amide I & II)

C-O Stretch
(glucose-starch)
514

O-H Stretch (water)

C-H Stretch (lipid)

N-H Bend (Amide

515

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C-H Bend (lipid)
C-O Bend (TAGS)
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Absorbance

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519 Amaranth

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520

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521 Quinoa

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523 Kañiwa

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524

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3070 2138 1206
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525 Wavenumber (cm )
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526

527 Figure 1
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(A) (C)
Corn Barley
Amaranth
PC2

PC2 Wheat

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Quinoa
Kañiwa Fava Beans

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PC1 PC3 PC1
PC3

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Amaranth
Quinoa

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Kañiwa
Soy

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(B)

1055

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1000
160
Discriminating Power

1552

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120 1180
1480

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1440
1718

1634 962
1390 1042
80 1296 1112
1215 B
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40 A
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1765 1392 1019

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529 Wavenumber (cm-1)

530

531 Figure 2
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532 (A)

Corn Barley 533

PC2
534

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Wheat
535

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Quinoa Mixtures
536
Fava Beans

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537
PC3 PC1
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Mixtures
Kañiwa 539

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Amaranth
7 grains Soy 540
(negative control)

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541 (B)

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Wholesale Label Samples (#n) Pure Mixtures
kaniwa 7 3 4

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barley 2 2 0
fava bean 3 3 0
quinoa 12 2 10
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soy 2 2 0
amaranth 3 0 3
Total 29 12 17
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GC-FAME Analysis of Predicted Mixture Samples

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Fatty Acid Composition

Sample Fat Protein C16:0 C18:0 C18:1 C18:2 C18:3 C20:0
Kañiwa 5.4 - 8.0 8.4 -12.9 12.5 - 39.1 4.7 - 22.7 32.8 - 47.8 2.5 - 44.3 0.5 - 3.7 0.5 -4.4
Amaranth 4.7 - 11.8 4.9 -14.2 19.3 - 24.5 2.5 - 3.9 28.1 - 55.7 14.1-45.6 0.9 - 2.9 0.3 -2.4
Quinoa 4.0 - 24.9 6.1 - 48.0 5.1 - 35.1 0.1 - 27.7 8.3 - 46.8 3.1 - 53.2 0.3 - 23 0.1 - 13
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543 Figure 3
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544
Factors: 4 12 Factors: 4
545 30 R= 0.99 R= 0.99

Predicted Protein (%)

SECV=0.96 10 SECV=0.33

Predicted Fat (%)

546

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547 20
6

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548
4
10

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2
10 20 30 2 4 6 8 10 12
550
Reference Fat Content (%)

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Reference Protein Content (%)
551

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553 Figure 4

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563 Table 1: Summary composition (mean ± standard deviation) of protein, crude fat, and fatty acid profile in authentic (n=89) flours

GC Composition
Sample Sample Size Fat Protein C16:0 C18:0 C18:1 C18:2 C18:3 C20:0
Kañiwa 14 9.41 ± 1.6 b 16.39 ± 1b 14.34 ± 0.9 1.64 ± 0.4 b 27.53 ± 1.3 49.43 ± 2.4 3.83 ± 0.8 b 3.24 ± 0.3 b

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Amaranth 10 9.8 ± 0.4b 14.43 ± 1 a 16.19 ± 1 4.2 ± 0.4a 24.15 ± 0.5 44.22 ± 0.6 0.83 ± 0 a 10.41 ± 0.3 a
Quinoa 13 8 ± 1a 13.78 ± 0.9 a 13.38 ± 1 2.82 ± 0.5 b 26.7 ± 0.6 48.33 ± 1 5.54 ± 0.5 b 3.6 ± 0.4 b

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Fava Beans 12 4.22 ± 0.7 25.48 ± 2 18.11 ± 0.8 3.03 ± 0.5 24.48 ± 1 50.54 ± 2 3.46 ± 0.1 0.38 ± 0
Wheat 13 4.61 ± 1 10.28 ± 1.4 30.26 ± 2 1.82 ± 0.4 24.95 ± 0.9 41 ± 2 1.57 ± 0.4 0.63 ± 0.2
Barley 10 4.4 ± 0.5 8.57 ± 0.6 37.63 ± 0.7 5.17 ± 0.8 26.18 ± 0.9 28.97 ± 1 1.68 ± 0.3 0.37 ± 0

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Soy 8 23.4 ± 1.5 35.36 ± 0.4 12.28 ± 0.2 4.38 ± 0.3 22.85 ± 1 54.36 ± 1.7 5.8 ± 0.6 0.33 ± 0
564 Corn 5 5.59 ± .7 9.71 ± 1.8 13.17 ± 0.5 1.36 ± 0.5 30.73 ± 1 50.22 ± 1.2 1.32 ± 0.4 3.2 ± 0.2

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565 In each column averages with different characters (a, b) have significant differences (p<0.01) according to Tukey test. The comparison test was done for the
566 Andean grains (Amaranth, Quinoa and Kañiwa).

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567

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568 Table 2: Literature values of chemical compositions for quinoa, kañiwa and amaranth.

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Fatty Acid Composition
570 Sample Fat Protein References (*)
C16:0 C18:0 C18:1 C18:2 C18:3

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571 Kañiwa 6.4 -10.0 12.8 -18.8 12.5 - 15.5 1.5 - 4.7 26.0 - 32.8 48.9 - 54.3 3.5 - 8.5 1, 2, 3,
Amaranth 6.4-10.5 14.5-14.7 16.2 - 19.3 2.5 – 5.0 22.8 - 25.7 49.2 - 56.3 0.9 - 7.9 1, 2, 3, 4
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572 Quinoa 5.0-12.4 11.7-14.4 13.1 - 15.6 1.8 – 3.0 24.4 - 28.0 47.0 - 55.8 0.3 -7.1 1, 2, 3, 4
573 * 1- Repo Carrasco, 1992; 2- Rosell et al, 2009; 3- Ryan et al, 2007;4 - Ahamed et al., 1998;
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