Antioxidant Capacity , Mineral Content and Sensory Properties of Gluten-Free Rice and Buckwheat Cookies

A number of studies have indicated that the gluten-free diet is unbalanced in carbohydrates, proteins and fat and defi cient in certain essential nutrients (1). Due to the limitation of some nutrients, the fortifi cation of basic gluten-free formulations is recommended to develop value-added products. For instance, pseudocereal fl our such as buckwheat has been utilized to produce gluten-free bread (2–4) and pasta (5).


Introduction
Coeliac disease is one of the most common chronic autoimmune disorders.It is related to the intolerance of proteins of gluten complex present in many common cereals such as wheat, rye, barley and oat.The symptoms of coeliac disease can only be avoided by adhering to a strict lifelong gluten-free diet.
A number of studies have indicated that the gluten--free diet is unbalanced in carbohydrates, proteins and fat and defi cient in certain essential nutrients (1).Due to the limitation of some nutrients, the fortifi cation of basic gluten-free formulations is recommended to develop value--added products.For instance, pseudocereal fl our such as buckwheat has been utilized to produce gluten-free bread (2)(3)(4) and pasta (5).
Cookies are widely consumed bakery products due to their long shelf life and strong consumer preference.
Although the structure-forming ability of gluten infl uences the rheological properties of dough and aff ects overall appearance of bakery products, the development of a gluten network in biscuit and cookie dough is minimal and undesirable (6).Therefore, the eff orts in gluten--free cookie production are more frequently related to the fortifi cation of gluten-free formulations to achieve be er nutritional profi le of biscuits and cookies with acceptable sensory properties.There are several papers on the enrichment of gluten-free biscuits and cookies (7,8).
Rice and buckwheat fl our types are recommended as the safe ingredients for coeliac patients since they possess no gluten and can be used in the production of bread (9) and cookies (8).Rice fl our is known as the most suitable component for gluten-free formulations due to its mild taste, colourlessness, hypoallergenic properties and easily digestible carbohydrates (10).Buckwheat fl our is charac-terized as a gluten-free ingredient containing well-balanced amino acids and is rich in polyphenols (11,12).The dominant polyphenolic compound in buckwheat fl our is rutin (13), shown to be a potent antioxidant (14).Buckwheat fl our is abundant in minerals, especially magnesium and iron, which are lacking in gluten-free products (2)(3)(4).
Schönlechner et al. (15) incorporated pseudocereal fl our (amaranth, quinoa and buckwheat) in gluten-free biscuit formulations.Cookies based on rice and light buckwheat fl our were initially characterized by Torbica et al. (8) from the aspect of their physicochemical and sensory characteristics and compared to the control which was based on wheat fl our.According to the authors, the best quality of gluten-free rice cookies was achieved when they were enriched with 20 % of light buckwheat fl our.
Published papers in the available literature present results mainly focused on physicochemical or sensory characteristics of gluten-free cookies without any functional analysis such as antioxidant activity.Therefore, the objective of this paper is to investigate the antioxidant capacity, mineral content and sensory properties of the cookies made from rice and light buckwheat fl our in three diff erent ratios and to compare them with the control sample made from rice fl our.In addition, principal component analysis was used to study and visualize the correlation among all tested properties of gluten-free cookies.

Preparation of cookies
The formulation of gluten-free cookies with rice and buckwheat fl our was made according to Torbica et al. (8).Mixtures of the two types of fl our were prepared, with the ratio of rice to buckwheat fl our of 90:10, 80:20, and 70:30.Rice fl our was used for the preparation of control cookies.Dough mixing, processing and baking were performed on laboratory-scale equipment.The ingredients were weighed as follows (in g): fl our (rice fl our 300 for the control cookies or rice fl our and light buckwheat fl our, respectively: 270 and 30 for the cookies with 10 % fl our substitution, 240 and 60 for the cookies with 20 % fl our substitution, and 210 and 90 for the cookies with 30 % fl our substitution), deionized water 75, vegetable fat 85, granulated sugar 70, honey 45, NaHCO 3 9, diacetyl tartaric acid ester of monoglycerides 9, carboxymethyl cellulose 4.5, and salt 2.1.
Rice fl our or rice/buckwheat fl our mixtures were transferred into Farinograph mixing bowl (Brabender GmbH, Duisburg, Germany), which was previously tempered at 30 °C.A erwards, the rest of the dry ingredients and vegetable fat were added and mixed for 2 min.Finally, 45 g of honey which was previously dissolved in deionized water was poured into the mixer bowl and the dough mass was mixed for 25 min at 30 °C.The obtained cookie dough was le to rest at 8 °C for 24 h in order to allow the hydration of the added carboxymethyl cellulose.A er the resting period, the dough was tempered at ambient temperature for 30 min and then sheeted to a thickness of 4 mm using a pilot scale dough sheeter (Mignon, Mestrino, Italy).The dough was cut using a stainless mould (60 mm × 55 mm) and fi nally baked at 170 °C for 12 min in a laboratory oven (MIWE gusto ® , MIWE Michael Wenz GmbH, Arnstein, Germany).The obtained cookies were cooled for 2 h at ambient temperature and packed in sealed polypropylene bags.(16).Starch content was determined by hydrochloric acid dissolution according to the ICC Standard No. 123/1 (17).

Proximate composition and mineral content
Mineral content (Mg, K, Zn, Fe, Mn and Cu) of cookies was determined using a Varian Spectra AA 10 (Varian Techtron Pty Limited, Mulgrave, Victoria, Australia) atomic absorption spectrophotometer equipped with a background correction (D2 lamp).The samples were prepared according to the procedure described in Manuals of Food Quality Control (18).

Preparation of ethanolic extracts
Cookies were ground in a laboratory blender (Waring, Torrington, CT, USA) to obtain coarse powder which passed through an 800-mm sieve.Cookie powder (5 g) was mixed with 50 mL of 80 % ethanol.Extraction was carried out by shaking the mixture at ambient temperature ((23±1) °C) for 1 h.A er 1-hour shaking, the suspension was le overnight at ambient temperature.The procedure was repeated twice with 50 mL of solvent, and combined extracts were dried using a vacuum evaporator (Büchi, Flawil, Switzerland).The yield was calculated based on the wet mass of the samples.The dried extract was redissolved in 80 % ethanol to 10 mL volume and used for further investigation of antioxidant activity.

Total phenolic content
Total phenolic content of gluten-free rice and buckwheat cookie extracts was determined spectrophotomet-rically using Folin-Ciocalteu reagent (19).Gallic acid was used as a standard and results were expressed in mg of gallic acid equivalents (GAE) per g of sample on dry mass basis.The extract (0.1 mL) was diluted with pure water (7.9 mL, Millipore Elix 10 UV water purifi cation system).Folin-Ciocalteu reagent (0.5 mL) and sodium carbonate solution (1.5 mL; g=200 g/L) were added, and the reaction mixture was mixed thoroughly.The mixture was allowed to stand for 120 min with intermi ent shaking, and the absorbance was measured at 750 nm (6405 UV/VIS, Jenway, Stone, Staff ordshire, UK).

Antioxidant activity measured by b-carotene bleaching method
Oxidative loss of b-carotene in a b-carotene/linoleic acid emulsion was used to assess the antioxidant activity of the examined extracts (20).b-Carotene (2 mg) was dissolved in 10 mL of chloroform and 1 mL of b-carotene solution was mixed with 20 mg of purifi ed linoleic acid and 200 mg of Tween 40 in a round-bo om fl ask.Chloroform was removed by purging with nitrogen.Pure water (50 mL) was added into the b-carotene/linoleic acid emulsion and mixed using a vortex mixer V1 plus (BOECO, Hamburg, Germany).Cookie extracts (0.2 mL) at various concentrations (10.0-60.0mg/mL) and aliquots (5 mL) of the b-carotene/linoleic acid emulsion were placed in capped culture tubes and mixed thoroughly.The tubes were immediately placed in a water bath and incubated at 50 °C.Oxidation of b-carotene/linoleic acid emulsion was monitored spectrophotometrically by measuring the absorbance at 470 nm a er 120 min (6405 UV/VIS, Jenway).A control was prepared using 0.2 mL of 80 % ethanol instead of the extract.
Degradation rate (DR) of the extracts was calculated according to the fi rst order kinetics using the following equation ( 21): where A 0 is the initial absorbance (470 nm) at time zero, A is the absorbance (470 nm) at time 120 min and t is time (min).
The antioxidant activity (AA) was expressed as inhibition (in %) relative to the control using the following equation: The IC 50 value (mg/mL) was defi ned as the concentration of the extract at which the antioxidant activity was 50 % under the experimental conditions.

Reducing power
Reducing power of the cookie extracts was measured according to the method of Oyaizu (22).Various concentrations (15.0-60.00mg/mL) of the extracts (0.5 mL) were mixed with 2.5 mL of phosphate buff er (0.2 M, pH=6.6) and 2.5 mL of potassium ferricyanide (1 %).The mixtures were incubated at 50 °C for 20 min, and a er that trichloroacetic acid (10 %, 2.5 mL) was added.The mixtures were centrifuged at 650 × g for 10 min (LC-320, Tehtnica, Železniki, Slovenia).The supernatant (2.5 mL) was mixed with 2.5 mL of pure water and 0.5 mL of ferric chloride and the absorbance was measured at 700 nm with spectrophotometer (6405 UV/VIS, Jenway).Higher absorbance of the reaction mixture indicates greater reducing power.The IC 50 value (mg/mL) was defi ned as the concentration of the extract that causes a decrease in the absorbance of reaction mixture up to 0.5.

DPPH radical scavenging activity
Eff ect of the examined extracts on the content of 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH • ) was estimated according to the modifi ed method of Hatano et al. (23).The concentration of the DPPH • solution used in the assay was 90 mM, i.e. 22.5 mL of 0.4 mM DPPH • solution (0.01577 g of DPPH • in 100 mL of methanol) were diluted with 95 % methanol to 100 mL.An aliquot (1.0 mL) of the DPPH • solution (90 mM) was diluted in 2.9 mL of methanol, and 0.1 mL of the extracts at various concentrations (15.0-60.00mg/mL) was added.The mixture was shaken vigorously and le to set for 60 min in the dark, then the absorbance was measured at 517 nm (6405 UV/VIS, Jenway) against the blank (mixture without the extract).
The IC 50 value (mg/mL) was defi ned as the concentration of an antioxidant extract which was required to quench 50 % of the initial amount of DPPH • under the experimental conditions given.

Fe 2+ chelating activity
Fe 2+ chelating activity was measured according to the method of Decker and Welch (24).Aliquots of 1 mL of different concentrations of the extracts (5.0-60.00mg/mL) were mixed with 3.7 mL of pure water.The mixture was le to react with ferrous sulphate heptahydrate (2 mM, 0.1 mL) and ferrozine (5 mM, 0.2 mL) for 10 min at ambient temperature ((23±1) °C), and then the absorbance was measured at 562 nm (6405 UV/VIS, Jenway).A lower absorbance indicates a higher chelating power.Chelating activity (CA) was calculated according to the following equation: The IC 50 value (mg/mL) was defi ned as the concentration of an antioxidant extract which chelates 50 % of the present Fe 2+ under the experimental conditions.

HPLC determination of rutin
A mass of 5 g of cookie powder was extracted with 20 mL of 80 % boiling methanol for 10 min, ultrasonicated for 10 min and fi ltered through regenerated cellulose membrane fi lters (0.45 µm pore size; Agilent Technologies, Santa Clara, CA, USA) before injection into the HPLC system.HPLC analysis was performed using a liquid chromatograph (Agilent 1200 series), equipped with a diode array detector (DAD), on an Agilent Eclipse XDB-C18, 1.8 mm, 4.6 mm × 50 mm column, at a fl ow rate of 1 mL/min.A single rapid resolution HPLC method reported by Mišan et al. (25) was used.The solvent linear gradient mode was performed by varying the proportion of solvent A (methanol) to solvent B (1 mL of formic acid in 100 mL of water) as follows: initial 10 % A, then 0-10 min 10-25 % A, 10-20 min 25-60 % A, and 20-30 min 60-70 % A.
The run time and post-run time were 45 and 10 min, respectively.The column was operated at 30 °C.The injected volume of samples and standards was 5 mL and it was done automatically, using autosampler.The spectra were acquired in the range of 210-400 nm and chromatograms plo ed at 280, 330 and 350 nm with reference wavelength set at 550/100 nm.Rutin was identifi ed by matching its retention time and spectral characteristics against rutin standard.The external standard method was used for quantifi cation.
Stock solution of rutin was prepared in the concentration of 1 mg/mL in methanol.The solution was properly diluted with 1 % formic acid to obtain a series of dilutions in the range of 0.005-34 mg/mL in the mobile phase for external standard calibration.

Sensory evaluation
Sensory evaluation was conducted 24 h a er baking by eight experienced and trained panellists (7 females and 1 male, at the age of 30 to 43).Prior to sensory analysis, sensory profi le of gluten-free cookies was established by a multidimensional approach.The established sensory profi le included 10 descriptors with their defi nitions and evaluation techniques (26).To express the intensity of each perceived descriptor, the intensity scale was applied: from 0, i.e. the absence of perception to 5, i.e. strong perception/maximal intensity (27,28).The samples were evaluated on three separate occasions.Drinking water was provided for palate cleansing a er each sample.

Statistical analysis
Results were expressed as the mean values of three replications±standard deviation.ANOVA and Fisher's multiple range tests were used, and p<0.05 were regarded as signifi cant.Principal component analysis (PCA) was carried out to investigate the within-set data profi le and to study the correlation between the data.All analyses were made using the XLSTAT so ware v. 2012.2.02 (Addinso , New York, NY, USA).

Proximate composition of cookies
The main ingredients in gluten-free rice and buckwheat cookie formulation were rice and light buckwheat fl our whose mineral and phenolic content and antioxidant activities are presented in Table 1.
The produced gluten-free rice and buckwheat cookies had signifi cantly higher (p<0.05)protein content in comparison with rice cookies (Table 2).This fi nding is due to higher protein content of light buckwheat fl our (29) compared to rice fl our (9).Dietary fi bre content of the investigated cookies was found to be signifi cantly diff erent (p<0.05) in the descending order: cookies with 30 % light buckwheat fl our>cookies with 20 % light buckwheat fl our>cookies with 10 % light buckwheat fl our>control cookies.Similar increase in total dietary fi bre content was noticed in light and wholegrain buckwheat crackers when they were compared to their wheat counterparts, which corresponded with higher content of dietary fi bres in buckwheat fl our (30).The increased total dietary fi bre content in gluten-free rice and buckwheat cookies contributed to their overall functional properties for the consumption of both adults and children.Namely, children older than two are recommended to consume a minimal amount of dietary fi bre equivalent to their age plus 5 g per day (31).Regarding the highest content of total dietary fi bres in cookies containing 30 % light buckwheat fl our ((2.94±0.04)%) (Table 2), it can be concluded that 100 g of these cookies can satisfy 42 % of a daily dietary fi bre intake for 2-year-old children or a lower percentage depending on the age.Therefore, they cannot be considered as high-dietary fi bre cookies whose normal consumption Since the main ingredients in cookie formulation were rice fl our and light buckwheat fl our, both known as gluten-free ingredients, the produced cookies were considered to be gluten-free although the presence of gluten needs to be controlled before placing the product on the market.In the case of gluten-free products, their gluten content should be less than 20 mg/kg according to the Commission Regulation (EC) No 41/2009 of 20 January 2009 concerning the composition and labelling of foodstuff s suitable for people intolerant to gluten (32).This require ment could be satisfi ed only by purchasing the certifi ed gluten-free ingredients, and applying the strict standards in the production process to avoid cross-contamination.

Mineral content of cookies
The addition of light buckwheat fl our to control gluten-free cookie formulation contributed to the signifi cant increase (p<0.05) in their mineral content, especially magnesium, potassium, iron and copper (Table 3) due to their signifi cantly higher (p<0.05)amounts in light buckwheat fl our compared to rice fl our (Table 1).This fi nding is in agreement with previously obtained data published by Alvarez-Jubete et al. (2) and Peterson et al. (33).It is assumed that the consummation of gluten-free rice and buckwheat cookies with the improved mineral profi le can contribute to the reduction of mineral defi ciency in gluten-free diet.
Similar positive correlation between macroelement content and increased amount of buckwheat fl our in gluten-free bread was observed by Wronkowska et al. (4).Furthermore, the addition of buckwheat fl our signifi cantly increased potassium, magnesium and phosphorus contents of tarhana made with wheat fl our (34).

Total phenolic and rutin content
Due to higher total phenolic content of light buckwheat fl our than of rice fl our (Table 1), as it was previously determined by Sakač et al. (9), a signifi cant increase (p<0.05) in total phenolic content of gluten-free rice and buckwheat cookies was found in comparison with the rice ones (Table 4).The fortifi cation of gluten-free products using pseudocereals has been reported by Alvarez--Jubete et al. (3), while buckwheat was investigated as a component for gluten-free bread (9,35) and cookies (8).
Rutin, well known as a potent antioxidant (36), dominates in light buckwheat fl our (37), but is not present in rice fl our (Table 1), which contains ferulic and p-coumaric acids as the main polyphenols (38).Therefore, incorporation of light buckwheat fl our in gluten-free cookie formulation resulted in an increased concentration of rutin in the following order: cookies with 30 % light buckwheat fl our>cookies with 20 % light buckwheat fl our>cookies with 10 % light buckwheat fl our (Table 3).
Although offi cial dietary intake of polyphenols and rutin has not yet been established, the total dietary intake of polyphenols was recommended to be 1 g per day (39) or about 1.2 g per day (40 % of fl avonoids, 60 % of pheno-  (40), while the intake of 10-25 mg per day of rutin for adults and 5-10 mg per day for children are considered safe (41).In this view, 100 g of cookies with 30 % light buckwheat fl our, which contain the highest amount of polyphenols and rutin among the examined cookies (Table 4), could contribute to approx.12 % of daily polyphenol intake and less than 8 % of daily rutin intake for children.These fi ndings suggest that problems connected with the excessive polyphenol and rutin intake should not be expected at moderate consumption of gluten-free rice and buckwheat cookies.

Antioxidant properties
The antioxidant activity of buckwheat fl our is superior to that of rice fl our (Table 1), due to its higher total phenolic content (9), especially rutin (37).Therefore, the incorporation of light buckwheat fl our in gluten-free cookie formulation resulted in increased antioxidant activity of the enriched gluten-free cookies (Table 4), expressed as IC 50 .
The IC 50 values of antioxidant activity among all investigated buckwheat-enriched cookie extracts diff ered signifi cantly (p<0.05) from the IC 50 value of control cookies.Antioxidant activity negatively correlated with the total phenolic content (R= -0.951), suggesting that phenolic compounds in the produced cookies mainly contributed to their overall antioxidant properties.However, thermal processing of cereals, such as baking, also resulted in the formation of substances with antioxidant properties, namely Maillard reaction products (42) that contribute to the overall antioxidant activity of bakery products.Sensoy et al. (43) stated that the accumulation of these products in bread crust was responsible for signifi cant increase in its antioxidant activity.
Signifi cant diff erences (p<0.05) in reducing activity were found between the samples, except between the samples containing 10 and 20 % light buckwheat fl our (Table 4).The enrichment of cookie formulation with 30 % light buckwheat fl our resulted in superior reducing activity due to the fact that buckwheat presents one of the greatest sources of antioxidants amongst cereals and pseudocereals (37).Alvarez-Jubete et al. (11) reported that buckwheat seeds and sprouts as well as buckwheat bread were rich in reducing agents.
The buckwheat-enriched cookie extracts exhibited scavenging activity against DPPH • in the descending or-der: cookies containing 30 % buckwheat fl our>20 % buckwheat fl our>10 % buckwheat fl our~control cookies (Table 4).The increased amount of light buckwheat fl our in the formulation of cookies positively correlated with the scavenging activity against DPPH • due to the approx.20 times higher scavenging activity against DPPH • of light buckwheat fl our extract than of rice fl our extract (Table 1).The buckwheat fl our capacity to scavenge DPPH • is mainly a ributed to the presence of rutin, which was confi rmed as a potent DPPH • scavenger (14).
The addition of 10-30 % of light buckwheat fl our to the gluten-free cookie formulation improved the Fe 2+ chelating activity but without signifi cant diff erences (p<0.05)among diff erent samples (Table 4).It is assumed that the determined increase in Fe 2+ chelating activity of cookies enriched with light buckwheat fl our could be explained by the chelating properties of rutin, which was specifi ed as metal chelator and/or radical scavenger in the Fenton reaction (36).

Sensory properties
Most of the sensory properties were not signifi cantly infl uenced by the addition of light buckwheat fl our compared to the control sample (Table 5).However, the substitution with 20 % of light buckwheat fl our in control gluten-free cookie formulation resulted in signifi cantly distinct intensity (p<0.05) of colour and odour.These results are in agreement with previously published fi ndings of Luthar (44), who found that the presence of aromatic compounds in buckwheat fl our improved the pleasant odour and taste compared to the bland and neutral rice fl our (45).More intensive colour was found in the samples with 20 and 30 % of substituted fl our than in the other two samples, due to the diff erences in amino acid profi le (46) and the content of reducing sugars between buckwheat and rice fl our, which further caused the diff erences in nonenzymatic browning reaction during baking (47).
Regarding textural properties, supplementation of light buckwheat fl our negatively infl uenced the hardness of the cookies when compared to the control sample (Table 5).In a previous study (48) which focused on instrumental texture and dimensional measurements of these cookies, partial replacement of rice fl our with buckwheat fl our led to a decrease in cookie hardness, fracturability and percentage of contraction, and an increase in the per- centage of spread and eccentricity.Thus, lower scores for hardness of gluten-free rice and buckwheat cookies in our experiment could be a ributed to the diff erences in rheological properties between the rice dough and the dough made from rice fl our and light buckwheat fl our.Rice dough was stronger (higher elastic modulus and lower maximum creep compliance) and more elastic (lower tan δ, higher recovery) than the dough made with rice fl our and light buckwheat fl our mixtures (48).
Moreover, comparing the enriched cookies with the control, which was set as the reference/average value, cookies with 20 % light buckwheat fl our had be er sensory quality with higher scores or values closer to the average of the properties considered as positive for this type of product.On the contrary, as the crumbliness and adhesiveness are considered to be less positive properties, cookies with 20 % light buckwheat fl our showed acceptable sensory quality due to lower than average scores (Table 5).
Generally, the addition of light buckwheat fl our to the gluten-free cookie formulation did not have a negative infl uence on their sensory quality.In comparison with other investigated samples, the cookies containing 20 % light buckwheat fl our were found to have the most acceptable sensory properties.Our fi ndings are in accordance with the previous fi ndings of Torbica et al. (35), who applied a diff erent sensory method of evaluation.

Principal component analysis
The results of ANOVA revealed clear signifi cant differences among cookies for each determined parameter (Tables 2-5), while the principal component analysis (PCA) gave the insight into the relevant properties which provide a perceptual map of the gluten-free cookies.The data were standardized and submi ed to the correlation matrix.The fi rst two dimensions accounted for 90.97 % of the total variance, 78.22 % of which were explained by the fi rst dimension.F2 and F3 components carried out 12.75 and 9.03 % of the total information given by the gluten--free cookie profi les.The factor loadings (correlation coef-fi cients between variables and F-factors) are listed in Table 6.From this table it can be concluded which variables are well linked with an axis based on the squared cosine values (factor loadings).The higher the value of the factor loadings (≥0.5), the more important that variable is to the corresponding axis.Therefore, the properties with high positive or negative loadings summarized the meaning of the fi rst three components.
Sensory properties (colour, odour, fatness, crumbliness, fracturability, particle size and taste), chemical components (proteins, fat, ash and total dietary fi bre), all minerals except manganese, and total phenolic content and rutin exhibited positive factor loadings for F1, while the remaining properties exhibited negative factor loadings.Consequently, these properties were found in most of the samples at diff erent intensity levels and exhibited diff erences among the gluten-free cookies, because F1 had the highest eigenvalue (21.903).It should be pointed out that the second dimension of the PCA can be explained only by two sensory properties (odour and fracturability) and manganese.
According to Kallithraka et al. (49) and Bower (50), the samples that are close to each other in PCA score plot possess similar overall properties and samples that are far apart are very diff erent.In Fig. 1 the samples are positioned in diff erent areas except the sample with 10 % light buckwheat fl our, due to the diff erence in their composition.The main parameters characterizing the control sample are: starch, chelating activity, adhesiveness, hardness and sharpness.On the other hand, the PCA of all measurements and sensory evaluation resulted in eff ective classifi cation of the cookies with 20 and 30 % light buckwheat fl our into two groups of samples.The parameters which describe this grouping were primarily odour and fracturability of the cookies with 20 % light buckwheat fl our, and iron and protein content of the cookies with 30 % light buckwheat fl our.
Cookies with 10 % light buckwheat fl our could not be classifi ed in a separate group because they were not similar to the others (Fig. 1).Neither the sensory evaluation In general, it can be concluded that almost all antioxidant and positive sensory properties appeared to be much more eff ective in describing the gluten-free cookies with 20 and 30 % light buckwheat fl our than the other two samples.In Fig. 1 (F1 vs. F2) all mentioned parameters (variables) appeared to be very highly correlated (positively or negatively) with F1 factor.Therefore, the results derived using PCA showed a reasonable agreement with the number of variables relevant in discrimination among the cookies.

Conclusions
Light buckwheat fl our was used to enhance mineral content and antioxidant capacity of gluten-free rice and buckwheat cookies compared to the rice cookies used as the control.The substitution of rice fl our in gluten-free cookie formulation with 10 to 30 % light buckwheat fl our resulted in signifi cantly higher (p<0.05)mineral content, total phenolic content and rutin content, scavenging activity against DPPH • , antioxidant activity and reducing power than in the control cookies.Comparing all evaluated sensory properties, cookies enriched with 20 % light buckwheat fl our expressed the most acceptable sensory properties.The obtained results of PCA showed that almost all antioxidant and positive sensory properties were much more eff ective in describing cookies containing 20 and 30 % light buckwheat fl our than the other two samples.Therefore, the use of light buckwheat fl our in gluten-free cookies can be benefi cial due to the increased antioxidant activity and mineral content.The confi rmation of the mentioned benefi t of using light buckwheat fl our in the production of gluten-free cookies could be obtained in in vivo experiment, which might be part of our further investigations.

Fig. 1 .
Fig. 1.PCA score plot of the samples: CS=cookies made with rice fl our, samples 1, 2 and 3=rice fl our cookies containing 10, 20 and 30 % of light buckwheat fl our, respectively

Table 1 .
Mineral and phenolic content and antioxidant activities of rice fl our and light buckwheat fl our *Results are presented on dry mass basis.Values are means of three determinations±standard deviation.Values in the same column with the same le er in superscript are not statistically diff erent (p<0.05).

Table 2 .
Proximate composition of cookies Results are presented on dry mass basis.Values are means of three determinations±standard deviation.Values in the same row with the same le er in superscript are not statistically diff erent (p<0.05).Control=rice fl our cookies, sample 1=rice fl our cookies containing 10 % light buckwheat fl our, sample 2=rice fl our cookies containing 20 % light buckwheat fl our, sample 3=rice fl our cookies containing 30 % light buckwheat fl our *

Table 3 .
Mineral content in rice cookies and gluten-free rice and buckwheat cookies *Results are presented on dry mass basis.Values are means of three determinations±standard deviation.Values in the same column with the same le er in superscript are not statistically diff erent (p<0.05).Control=rice fl our cookies, sample 1=rice fl our cookies containing 10 % light buckwheat fl our, sample 2=rice fl our cookies containing 20 % light buckwheat fl our, sample 3=rice fl our cookies containing 30 % light buckwheat fl our lic acids)

Table 4 .
Phenolic content and antioxidant activities of rice cookies and gluten-free rice and buckwheat cookies Results are presented on dry mass basis.Values are means of three determinations±standard deviation.Values of the same column with the same le er in superscript are not statistically diff erent (p<0.05).Control=rice fl our cookies, sample 1=rice fl our cookies containing 10 % light buckwheat fl our, sample 2=rice fl our cookies containing 20 % light buckwheat fl our, sample 3=rice fl our cookies containing 30 % light buckwheat fl our *

Table 5 .
Sensory scores of cookies

Table 6 .
Principal component (PC) values, factor loadings and eingevalues for each PC