Basic chemical composition and bioactive compounds content in selected cultivars of buckwheat whole seeds, dehulled seeds and hulls

Basic chemical composition and bioactive compounds content in selected cultivars of buckwheat whole seeds, dehulled seeds and hulls

Journal of Cereal Science 69 (2016) 1e8 Contents lists available at ScienceDirect Journal of Cereal Science journal homepage: www.elsevier.com/locat...

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Journal of Cereal Science 69 (2016) 1e8

Contents lists available at ScienceDirect

Journal of Cereal Science journal homepage: www.elsevier.com/locate/jcs

Basic chemical composition and bioactive compounds content in selected cultivars of buckwheat whole seeds, dehulled seeds and hulls Kinga Dziadek a, Aneta Kope c a, *, Edyta Pastucha a, Ewa Pia˛ tkowska a, _  ska a, Elzbieta Pisulewska b, Robert Witkowicz b, Renata Francik c, d Teresa Leszczyn a

Department of Human Nutrition, Faculty of Food Technology, University of Agriculture in Krakow, Balicka 122, 30-149 Krakow, Poland Department of Crop Production, Faculty of Agriculture and Economics, University of Agriculture in Krakow, al. Mickiewicza 21, 31-120 Krakow, Poland Department of Bioorganic Chemistry, Jagiellonian University Medical College, Pharmacy Faculty, Medyczna 9, 30-688 Krakow, Poland d Institute of Health, State Higher Vocational School, Staszica 1, 33-300 Nowy Sacz, Poland b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 26 June 2015 Received in revised form 24 January 2016 Accepted 7 February 2016 Available online 10 February 2016

The objective of this study was to determine the basic chemical composition and the content of selected bioactive components in hulls, dehulled seeds and whole seeds of the selected cultivars/strains of buckwheat. In the tested material the content of ash, protein, fat, fatty acids, total carbohydrates, starch, dietary fibre, resistant starch, total polyphenols, profile of polyphenols and antioxidant activity was determined. We have found that buckwheat seeds were a rich source of protein and total carbohydrates. Dehulled seeds were also a source of resistant starch. Palmitic, oleic and linoleic acids were the dominant fatty acids in all samples. Analysis of extracts from whole seeds, dehulled seeds and hulls showed that there were twenty polyphenolic compounds in the tested material. The highest content of dietary fibre and total polyphenols as well as the highest antioxidant activity were found in the hulls and the lowest one in the dehulled seeds. Because of the rich composition, buckwheat can be used in the development of new food products especially for people on gluten free diet. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Buckwheat Dietary fibre Fatty acids Polyphenols

1. Introduction Recent studies have shown increasing interest in buckwheat (Fagopyrum esculentum), because of its high nutritional value and  ska health beneficial properties (Bonafaccia et al., 2003; Stempin   ska and Soral-Smietana, et al., 2007; Stempin 2006; Verardo et al., 2010). In 2013 the buckwheat world production reached over 2.3 million tons and its cultivation area was over 2.2 million hectares (FAOSTAT, 2015). The main polysaccharide in buckwheat seeds is starch. Seeds are also a good source of dietary fibre, among other  ska et al., 2007; Stempin  ska and resistant starch (RS) (Stempin  Soral-Smietana, 2006). Other carbohydrates present in small amounts are dextrin as well as sucrose (Ga˛ siorowski, 2008). The content of protein in buckwheat seeds fluctuates from 8.5 to 19% and is dependent on the cultivar. Buckwheat protein has high biological value, which is corresponding to 92.3% of the biological value of egg protein (Ga˛ siorowski, 2008; Wronkowska and Haros, 2014). Buckwheat protein has balanced amino acid composition.

* Corresponding author. E-mail address: [email protected] (A. Kope c). http://dx.doi.org/10.1016/j.jcs.2016.02.004 0733-5210/© 2016 Elsevier Ltd. All rights reserved.

Lysine, which is recognized as the first limiting amino acid in most cereals, is present in the buckwheat in a relatively large quantities (Ga˛ siorowski, 2008). Moreover, buckwheat is a gluten-free product (Khan et al., 2012; Sedej et al., 2011). It allows to use it for patients on gluten free diet, especially for these suffering from celiac disease. The buckwheat lipids (1.8e3.9% of seed) have the similar composition to those present in other cereals (Mazza, 1988; Ga˛ siorowski, 2008). Beside of the basic components, buckwheat contains also several other heath beneficial components, for example polyphenolic compounds (Kiprovski et al., 2015; Verardo et al., 2010). Therefore buckwheat is a good source of nutrients and nonnutrient compounds and should be consumed more frequently. Based on above information it is reasonable to undertake research concerning the chemical composition of buckwheat, not only known cultivars, but especially the new strains too. The objective of this research was assessment of basic chemical composition as well as concentration of selected bioactive compounds in selected cultivars and the new strains of whole seeds, dehulled seeds and hulls of buckwheat.

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2. Materials and methods 2.1. Buckwheat materials The analyzed material were seeds of selected cultivars (cv.) (Kora and Panda) and strains (8/2008, 5/2006, 21/2002 and 9/2006) of buckwheat (F. esculentum). The seeds of buckwheat were obtained from the Department of Cultivation and Production in Palikije (Poland) harvested in 2011. The seeds were separated into following fractions: hulls and dehulled seeds. In order to obtain high precision of separation and analysis results, dehulled seeds and hulls have been separated manually. The whole seeds and fractions were crushed in an electric grinder (Tecator Foss, Hillerød, Sweden). The samples were stored at room temperature in the paper bags until analysis. The fractions of buckwheat were grinded directly before analysis of polyphenolic compounds and antioxidant activity. 2.2. Basic chemical composition In all of samples the chemical composition was determined. Total proteins (proced. no 950.36), raw fat (proced. no 935.38), total dietary fibre (proced. no 991.43) and ash (proced. no 930.05) were measured according to the AOAC (2006) methods. The total carbohydrates content was calculated by the equation: total carbohydrates ¼ 100-(protein þ crude fat þ ash) (Fortuna et al., 2003). The concentration of starch was determined by calcium chloride dissolution (ICC Standard, 1994). Resistant starch (RS) was measured by commercially available kit (cat no. K-RSTAR 09/14, Megazyme International Ireland, Wicklow, Ireland). 2.3. Composition of fatty acids The composition of fatty acids was analyzed by gas chromatography after extraction of lipids from the whole seeds, dehulled seeds and hulls using the Folch's method (Folch et al., 1957). The free fatty acids were transformed into their respective methylated derivatives (methyl esters) in 14% (v/v) BF3/MeeOH and extracted using hexane as fatty acids methyl esters and separated with the GC-17A-QP5050 GCeMS model (Shimadzu, Japan), using the capillary SP-2560 column (30 m x 0.25 mm x 0.25 mm; Supelco, Bellefonte, PA, USA). The carrier gas was helium with the flow rate of 5 ml/min. The temperature of column was kept at 60  C for 5 min and then increased up to 220  C (5  C/min). This temperature was maintained for 23 min. 2.4. Extracts preparation For the measurements of total polyphenols content and antioxidant activity, one gram of sample of buckwheat was weighed into an Erlenmeyer flask and then 40 ml of 0.08N hydrochloric acid in 80% methanol (POCH, Gliwice, Poland) was added. The mixtures were extracted at room temperature for 2 h by shaking (Elpan, water bath shaker type 357, Poland). Then the samples were centrifuged at 1500 rpm for 15 min (Centrifuge type MPW-340, Warsaw, Poland). The supernatants were decanted. The residues were re-extracted with 40 ml of 70% acetone (POCH, Gliwice, Poland) in the same conditions and centrifuged as mentioned above. Both supernatants were combined and stored at temperature 20  C until analysis. 2.5. Determination of total polyphenols The content of total polyphenols in extracts was estimated by the Folin-Ciocialteu reagent (SigmaeAldrich, st. Louis, MO, USA)

(Poli-Swain and Hillis, 1959). Extracts were diluted, where ratio of extracts to distilled water was 1:20. The reaction mixtures were prepared by mixing 5 ml of diluted extract, 0.5 ml of the FolineCiocalteu reagent and 0.25 ml of 25% sodium carbonate (POCH, Gliwice, Poland). The samples were left for 20 min. The absorbance was measured at 760 nm using the spectrophotometer (UV-1800, RayLeigh, Beijing Beifen-Ruili Analytical Instrument Co., Ltd., Beijing, China). Results were expressed as chlorogenic acid equivalents (CGA) in mg CGA per 100 g of sample.

2.6. High-performance liquid chromatography with electrochemical detection (HPLC/EC) To isolate the phenolic fraction, the protocol reported by Van Hung and Morita (2008) was used. The chromatographic separation was performed on an Agilent HPLC 1100 series system (Agilent, Waldbronn, Germany), which was equipped with a degasser, a binary pump, an auto-sampler and a thermo-stated column compartment. The extracts of whole seeds, dehulled seeds and hulls were separated on a LiChrospher 60 RP-select B column (125 mm  4.6 mm ID, 5 mm particle size) in combination with an appropriate guard column (4 mm  4 mm; 5 mm particle size) (Merck, Germany). The column was thermo stated at 25  C. The mobile phase and gradient program were used as previously described by Verardo et al. (2010) with some modifications. A gradient elution was carried out using the following solvent system: mobile phase A, water/formic acid (99:1, v/v); mobile phase B, mobile phase A/acetonitrile (60:40, v/v). The gradient program was as follows: from 2% B to 6% B in 5 min, from 6% to 10% in 10 min, from 10% to 17% in 5 min, from 17% to 36% in 8 min, from 36% to 38.5% in 6 min, from 38.5% to 60% in 6 min, from 60% to 100% in 5 min and from 100% to 2% in 2 min. The flow rate was 0.5 mL/min; the injection volume was 20 mL. Mass spectrometry analyses were accomplished on an Applied Biosystems MDS Sciex (Concord, Ontario, Canada) API 2000 triple quadrupole mass spectrometer equipped with an electrospray ionization (ESI) interface. ESI ionization was performed in the positive ion mode. High purity nitrogen used as a sheath gas, was produced by a nitrogen generator. All experiments were carried out in the negative ion mode. The ion source parameters were as follows: ion spray voltage (IS): 4400 V; nebulizer gas (gas 1): 30 psi; turbo gas (gas 2): 10 psi; temperature of the heated nebulizer (TEM): 250  C; curtain gas (CUR): 30 psi. Nitrogen (99.9%) from Peak NM20ZA was used as the curtain and collision gas. The ion path parameters for analyzed compounds were as follows: declustering potential (DP): 20 V; focusing potential (FP): 200 V; entrance potential (EP): 10 V; collision cell entrance potential (CEP): 0 V; collision cell exit potential (CXP): 2 V, respectively.

2.7. Determination of antioxidant activity Extracts were also used to determine (spectrometrically) antioxidant activity by identifying the samples ability to extinguish ABTSþ (2, 2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) free radical (Re et al., 1999). The method involved colorimetric determination of the amount of the colored solution of ABTSþ which was reduced by the antioxidants present in the analyzed product. The absorbance was measured at a wavelength 734 nm with a spectrometer (UV-1800, RayLeigh, Beijing Beifen-Ruili Analitical Instrument Co., Ltd., Beijing, China). Values obtained for each sample were compared to the concentrationeresponse curve of the standard Trolox solution and expressed as micromoles of Trolox equivalent per gram of fresh weight (TEAC).

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2.8. Statistical analysis Results were expressed as the means ± S.D. Differences between groups were analyzed using Statistica 10 statistical software (Tulsa, OK, USA). Duncan's multiple range test was used for testing the differences. P values <0.05 were regarded as significant. 3. Results Significantly the highest content of ash in the hulls was measured in strains 8/2008 and 9/2006 in comparison to other cultivars/strains (Table 1). The cv. Kora was characterized by the lowest content of ash in the hulls. Significantly the highest content of ash in dehulled seeds was found in strain 21/2002 as compared to other cultivars/strains. The richest in ash were whole seeds of the strains 8/2008 and 9/2006 in comparison to cultivars Kora and Panda. Statistically significant effect of tested fractions on the content of ash in particular cultivar/strain was found. The highest content of ash was in the whole seeds and the lowest one in the hulls in each cultivar/strain. The content of protein in hulls was not affected by cultivar or strain (Table 1). The highest amount of protein in the dehulled seeds was in strain 8/2008 in comparison to other cultivars/strains. The cv. Kora was characterized by the significantly highest content of protein in the whole seeds compared to strains 21/2002 and 5/ 2006. Hulls contained the smallest amount of protein in all examined cultivars/strains. The significantly highest content of fat in the hulls was reported in strain 21/2002 in comparison to other cultivars/strains (Table 1). In the dehulled seeds, the largest amount of the crude fat was determined in cv. Panda in comparison to cv. Kora and strains 21/ 2002 and 5/2006. The significantly highest content of this compound in the whole seeds was found in strain 8/2008 and cv. Panda in comparison to other cultivar/strains The highest content of crude fat was in the dehulled seeds and the lowest in the hulls of buckwheat. The hulls of cv. Panda were characterized by the highest content of palmitic acid. In the dehulled seeds, the largest amount of this acid was determined in strain 5/2006 in comparison to other samples. The significantly highest content of palmitic acid in the whole seeds was found in strain 21/2002 compared to other strains/cultivars (Table 2). The significantly highest amount of the

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stearic acid was observed in the hulls of strain 5/2006, in the dehulled seeds of strain 21/2002 as well as in the whole seeds of cv. Kora in comparison to other strains/cultivars. The strain 9/2006 was characterized by the highest content of oleic acid in the hulls and dehulled seeds compared to others. The significantly highest amount of this acid in the whole seeds was found in strain 5/2006 in comparison to others. The highest content of linoleic acid in the hulls was measured in strain 8/2008 in comparison to other strains/ cultivars. In the dehulled seeds, the richest in this acid was cv. Panda and amongst the whole seeds strain 9/2006 compared to others. In the hulls, the contents of arachidonic acid and behenic acid were not determined. In the dehulled seeds, the largest amount of arachidonic acid was measured in strain 9/2006 in comparison to others. The whole seeds of strain 21/2002 contained significantly highest amount of this acid compared to others. The highest content of behenic acid in the dehulled seeds was found in the cv. Kora, in comparison to the strains 8/2008 and 9/2006. The cv. Panda was characterized by the highest amount of this acid in the whole seeds, compared to others. Statistically significant differences in the content of fatty acids in each cultivar/strain between the whole seeds, dehulled seeds and hulls were found. The highest content of total carbohydrates in the dehulled seeds was measured in cv. Kora in comparison to strains 8/2008, 5/2006, 9/2006 and cv. Panda (Table 1). In the whole seeds, the richest in total carbohydrates were strains 5/2006 and 21/2002 compared to others. Statistically significant differences in the content of total carbohydrates between the whole seeds, dehulled seeds and hulls were found in the strains 5/2006, 9/2006 and cv. Kora. The hulls contained the significantly highest amount of total carbohydrates in all the cultivars/strains. The significantly highest content of starch in the hulls was reported in strains 8/2008 and 21/2002 in comparison to other cultivars/strains. In the dehulled seeds, the highest level of this compound was determined in strain 9/2006 in comparison to strains 8/2008, 5/2006 and 21/2002. The highest content of starch in the whole seeds was found in cv. Kora in comparison to strains 8/ 2008, 5/2006 and 21/2002. Additionally, we have found that significantly highest content of starch was measured in the dehulled seeds as compared to the hulls and whole seeds in each cultivars/strains (Table 1). The highest content of dietary fibre was measured in the hulls of cv. Panda and strain 9/2006 in comparison to the cv. Kora and

Table 1 Basic composition of buckwheat whole seeds, dehulled seeds and hulls of individual cultivar/strain. Cultivar/strain

Tested material

Ash [g$100g1]

8/2008

hull dehulled seed whole seed hull dehulled seed whole seed hull dehulled seed whole seed hull dehulled seed whole seed hull dehulled seed whole seed hull dehulled seed whole seed

2.06 2.45 4.95 1.94 2.48 4.62 1.96 2.78 4.73 2.03 2.28 4.98 1.88 2.30 4.16 1.97 2.47 4.42

5/2006

21/2002

9/2006

Kora

Panda

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.03 0.03 0.02 0.04 0.02 0.24 0.05 0.04 0.29 0.03 0.03 0.00 0.01 0.01 0.13 0.01 0.04 0.17

d.* a** a*** ab* a** ab*** ab* c** ab*** cd* b** a*** a* b** c*** bc* a** bc***

Protein [g$100g1]

Crude fat [g$100g1]

5.68 ± 0.24 a* 16.96 ± 0.02 c*** 15.45 ± 0.55 a** 5.30 ± 0.01 a* 16.21 ± 0.03 ab*** 12.28 ± 0.10 b** 5.13 ± 0.03 a* 15.29 ± 0.20 d*** 12.63 ± 0.42 b** 5.44 ± 0.09 a** 16.27 ± 0.37 ab* 15.53 ± 0.37 a* 5.40 ± 0.47 a** 15.82 ± 0.15 a* 15.61 ± 0.23 a* 5.57 ± 0.03 a* 16.67 ± 0.02 bc*** 15.39 ± 0.51 a**

0.54 2.41 2.24 0.62 2.25 1.72 0.81 2.36 1.74 0.50 2.41 1.96 0.52 2.19 1.80 0.55 2.48 2.17

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.03 0.15 0.04 0.00 0.06 0.01 0.03 0.06 0.02 0.01 0.04 0.03 0.04 0.06 0.01 0.01 0.06 0.11

a** ab* b* b* ac*** a** c* ac*** a** a* ab*** c** a* c*** a** a* b*** b**

Total carbohydrates [g$100g1] 91.72 78.18 77.36 92.15 79.06 81.38 92.11 79.56 80.90 92.02 79.04 77.53 92.19 79.68 78.43 91.92 78.39 78.01

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.30 0.16 0.52 0.04 0.07 0.34 0.05 0.29 0.69 0.05 0.36 0.39 0.44 0.22 0.11 0.05 0.09 0.46

a** b* a* a*** a** b* a** ac* b* a*** a** a* a*** c** a* a** b* a.

Starch [g$100g1] 2.25 ± 0.13 b* 48.01 ± 1.37 c*** 38.41 ± 0.28 a** 1.36 ± 0.00 d* 45.24 ± 1.62 d*** 38.50 ± 0.82 a** 2.26 ± 0.13 b* 48.63 ± 0.90 ac*** 39.13 ± 0.49 a** 0.31 ± 0.00 a* 52.12 ± 1.08 b*** 40.27 ± 2.06 ab** 0.15 ± 0.00 a* 51.01 ± 0.47 ab*** 43.11 ± 1.96 b** 0.85 ± 0.05 c* 51.11 ± 0.04 ab*** 39.94 ± 1.20 ab**

Results are expressed as mean ± SD. Statistically significant differences of variety/family on the content of compounds in the whole seeds, dehulled seeds and hulls of buckwheat are indicated by letter code aee. Statistically significant differences of tested material on the content of compounds in the individual varieties/families of buckwheat are indicated by asterisks code.

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Table 2 Fatty acid profile of buckwheat whole seeds, dehulled seeds and hulls of individual cultivar/strain. Cultivar/strain

Tested material

Fatty acid [% of total fatty acid] Palmitic (C16:0)

8/2008

5/2006

21/2002

9/2006

Kora

Panda

hull dehulled seed whole seed hull dehulled seed whole seed hull dehulled seed whole seed hull dehulled seed whole seed hull dehulled seed whole seed hull dehulled seed whole seed

20.17 16.17 15.51 21.26 16.55 14.57 27.62 15.83 15.82 22.83 15.69 14.84 22.33 15.24 14.69 27.87 14.74 13.99

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.03 0.06 0.04 0.06 0.04 0.03 0.01 0.01 0.06 0.03 0.08 0.06 0.04 0.04 0.14 0.10 0.04 0.11

a*** d** c* b*** e** a* e** a* d* d*** a** b* c*** c** ab* f*** ab** b*

Stearic (C18:0) 3.96 2.87 4.02 9.28 2.49 3.86 4.11 4.09 3.79 0.34 2.71 3.35 4.29 3.72 4.18 3.31 2.79 3.89

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.01 0.10 0.01 0.08 0.03 0.01 0.03 0.08 0.07 0.03 0.01 0.01 0.03 0.01 0.10 0.01 0.03 0.07

c* b** b* f*** c* a** d** e** a* a* a** c*** e* d** d* b** ab* ab***

Oleic (C18:1)

Linoleic (C18:2)

39.13 ± 0.04 d* 41.62 ± 0.03 a*** 40.68 ± 0.04 a** 35.37 ± 0.11 b* 40.95 ± 0.08 b** 41.26 ± 0.04 d*** 38.23 ± 0.06 c* 41.49 ± 0.04 a*** 39.44 ± 0.16 b** 43.32 ± 0.10 e*** 42.2 ± 0.07 d** 40.61 ± 0.10 a* 41.17 ± 0.10 a** 41.91 ± 0.10 c*** 40.29 ± 0.04 c* 41.28 ± 0.06 a** 41.48 ± 0.07 a*** 40.62 ± 0.04 a*

36.74 38.83 38.44 34.09 39.06 38.91 30.04 37.62 39.06 33.51 38.57 39.71 32.21 38.36 39.33 27.54 40.94 39.25

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.01 0.01 0.08 0.04 0.07 0.08 0.03 0.06 0.04 0.06 0.11 0.06 0.06 0.06 0.07 0.04 0.04 0.14

f* d*** d** e** e* a* b* a** ab*** d* c** e*** c* b** c*** a* f*** bc**

Arachidonic (C20:0)

Behenic (C22:0)

not determined 0.30 ± 0.03 d* 0.63 ± 0.08 a** not determined 0.47 ± 0.01 b* 0.59 ± 0.01 ab** not determined 0.41 ± 0.01 ab* 1.17 ± 0.03 d** not determined 0.54 ± 0.06 e* 0.96 ± 0.03 c** not determined 0.19 ± 0.01 c* 0.68 ± 0.04 a** not determined 0.38 ± 0.01 a* 0.52 ± 0.01 b**

not determined 0.21 ± 0.01 b* 0.72 ± 0.04 a** not determined 0.48 ± 0.03 a* 0.81 ± 0.06 ab** not determined 0.56 ± 0.13 a* 0.72 ± 0.01 a** not determined 0.29 ± 0.07 bc* 0.53 ± 0.01 c** not determined 0.58 ± 0.03 a* 0.83 ± 0.03 b** not determined 0.42 ± 0.01 ac* 0.98 ± 0.04 d**

Results are expressed as mean ± SD. Statistically significant differences of variety/family on the content of compounds in the whole seeds, dehulled seeds and hulls of buckwheat are indicated by letter code aee. Statistically significant differences of tested material on the content of compounds in the individual varieties/families of buckwheat are indicated by asterisks code.

strains 8/2008 as well as 5/2006. The highest content of dietary fibre in the dehulled seeds was found in the strain 8/2008, in comparison to the cv. Kora and Panda as well as strains 21/2002 and 9/2006. The strain 8/2008 was characterized by the highest amount of fibre in the whole seeds, compared to strains 5/2006, 21/2002 and 9/2006. The highest content of this component was in the hulls and the lowest in the dehulled seeds in each cultivar/strain (Table 3). RS was not found in the hulls. The significantly highest content of RS in the dehulled seeds was measured in strains 21/2002 in comparison to other cultivars/strains. In whole seeds statistically significant effect of cultivar/strain on RS content was not observed. Statistically significant differences in the content of this compound between the whole seeds, and dehulled seeds were not found (Table 3). In the hulls, the highest content of polyphenols was found in the strain 8/2008, compared to the strain 21/2002 as well as cultivars Kora and Panda (Table 3). The dehulled seeds of strain 9/2006 were

richer in polyphenols, compared to the cultivars Kora and Panda as well as strain 8/2008. The whole seeds of cv. Panda contained significantly the highest amount of polyphenols, in comparison to the strains 9/2006 and 21/2002. Statistically significant differences in the content of polyphenols between the hulls, dehulled seeds and whole seeds were demonstrated in the strain 8/2008 and cultivars Kora and Panda. The hulls contained the highest amount of polyphenols in all cultivars/strains. Analysis of methanolic extracts from whole seeds, dehulled seeds and hulls showed that there were many polyphenol compounds in the tested material. Fig. 1 shows the selected profile of polyphenolic compound in hulls (Fig. 1A), dehulled seeds (Fig. 1B) and in whole seeds (Fig. 1C) of cv. Panda. In all samples from hulls, the compounds shown in Table 4 were identified. In the hulls of the strain 21/2002 the 2-hydroxy-3-O-b-p-glucopyranosil-benzoic acid (C13H15O9,; m/z 315.0722), caffeic acid hexose (C15H17O9; m/z 341.0889), procyanidin B2 (C30H25O12; m/z 577.1351), (-)-epicatechin-3-(300 -O-methyl) gallate (C23H19O10; m/z 455.0962) and

Table 3 Bioactive compounds of buckwheat whole seeds, dehulled seeds and hulls of individual cultivar/strain. Cultivar/strain

Tested material

Dietary fibre [g$100g1]

Resistant starch [g$100g1]

Total polyphenols [mg$100g1]

ABTSþ [mmol Trolox$g1]

8/2008

hull dehulled seed whole seed hull dehulled seed whole seed hull dehulled seed whole seed hull dehulled seed whole seed hull dehulled seed whole seed hull dehulled seed whole seed

76.52 ± 0.66 c*** 6.69 ± 0.19 a* 25.45 ± 0.68 b** 78.45 ± 0.10 a*** 6.36 ± 0.19 a* 21.84 ± 0.16 a** 79.65 ± 0.77 ab*** 3.10 ± 0.14 c* 20.32 ± 0.46 a** 80.63 ± 0.49 b*** 1.16 ± 0.06 b* 21.55 ± 0.74 a** 78.70 ± 0.28 a*** 3.59 ± 0.10 d* 23.85 ± 0.62 b** 80.73 ± 0.40 b*** 5.69 ± 0.04 e* 25.14 ± 0.26 b**

not determined 6.13 ± 0.07 a* 5.64 ± 0.48 a* not determined 7.35 ± 0.18 a* 3.29 ± 0.33 a* not determined 11.04 ± 1.71 b* 4.95 ± 0.44 a* not determined 7.68 ± 0.15 a* 6.58 ± 0.70 a* not determined 6.17 ± 0.36 a* 5.18 ± 0.78 a* not determined 6.34 ± 0.84 a* 4.82 ± 0.80 a*

525.45 ± 16.84 c*** 280.00 ± 3.33 a* 352.96 ± 8.36 ab** 476.44 ± 0.00 abc** 305.30 ± 10.96 ab* 336.8 ± 8.30 ab* 463.73 ± 18.28 ab** 302.00 ± 10.95 ab* 311.98 ± 3.22 c* 513.18 ± 4.95 bc** 328.03 ± 9.55b* 326.80 ± 14.78 ac* 434.06 ± 13.25 a*** 284.19 ± 6.32 a* 350.35 ± 0.00 ab** 465.05 ± 18.34 ab*** 290.81 ± 6.19 a* 357.14 ± 4.98 b**

65.79 34.93 45.16 63.61 31.25 42.93 63.40 33.06 44.71 65.60 33.11 43.20 66.50 31.28 46.34 63.20 31.87 49.05

5/2006

21/2002

9/2006

Kora

Panda

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.58 0.45 0.92 0.19 0.01 0.96 0.57 0.81 0.69 0.19 0.03 0.90 0.57 0.72 0.58 0.96 0.32 0.81

b*** b* ab** a*** a* a** a*** ab* ab** b*** ab* a** b*** a* bc** a*** a* c**

Results are expressed as mean ± SD. Statistically significant differences of variety/family on the content of compounds in the whole seeds, dehulled seeds and hulls of buckwheat are indicated by letter code aee. Statistically significant differences of tested material on the content of compounds in the individual varieties/families of buckwheat are indicated by asterisks code.

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epicatechin gallate (C22H17O10; m/z 441.0827) were detected. Epicatechin gallate was also identified in the hulls of strains 8/2008, 5/ 2006 as well as cv. Kora and Panda. Epiafzelchin-epiafzelchinepicatechin (C45H37O16; m/z 833.2087) was present only in the sample of hulls Panda. There was no presence of compounds such as glucoside catechin, epicatechin, epiafzelchin-epicatechin-Omethyl gallate and procyanidin B2 dimethyl gallate in the extracts from the hulls of the tested cultivars. Epicatechin (C24H21O10; m/z 469.1136) appeared only in the seeds extracts of strain 21/2002 as well as cv. Kora and Panda. In extracts from the whole seeds, the same polyphenols as in the hull extracts were identified. In addition, the presence of substances listed in Table 5 was revealed. Compound 2-hydroxy-3-O-b-p-glucopyranosil-benzoic acid (C13H15O9,; m/z 315.0722) was identified in the whole seeds extract: 9/2006, 5/2006 and 21/2002. Procyanidin B2 was identified in the whole seeds of cv. Panda. In the extracts from dehulled seeds most of the compounds identified in the extracts of whole seeds were also detected. However, only the samples 5/2006, 21/2002 and Panda did not contain vitexin phenol. In the samples 8/2008, 5/ 2006, 21/2002, 9/2006, hiperin or/and isoquercitin were not detected. The highest antioxidant activity was found in the hulls of cv. Kora compared to the cv. Panda as well as strains 5/2006 and 21/ 2002 (Table 3). The highest antioxidant activity was determined in the dehulled seeds of strain 8/2008, compared to cv. Kora and Panda. In the whole seeds, significantly higher ability to scavenge free radical ABTSþ was measured in the cv. Panda, compared to strains 8/2008, 5/2006, 21/2002 and 9/2006. Statistically significant differences in the antioxidant activity in each cultivar/strain between the whole seeds, dehulled seeds and hulls were found. The highest ability to scavenge free radical was in the hulls and the lowest in the dehulled seeds. 4. Discussion The content of chemical compounds differed between hulls, dehulled seeds and whole seeds of buckwheat. The average content of ash, which was determined in buckwheat hulls, was 1.97 g$100 g1. Our results correspond to data published by Ga˛ siorowski (2008), which reported the content of ash at the level of 2.0 g$100 g1. We have found that the average content of ash in the dehulled seeds was 2.46 g$100 g1. Results of Ga˛ siorowski (2008) research indicated the similar content of ash (2.3e2.4 g$100 g1). Lower content of ash was reported in dehulled  ska et al. (2007) and Kowalewski et al. (2004) seeds by Stempin (2.0 g$100 g1, 1.72 g$100 g1 respectively). We found that the highest amount of ash was determined in whole seeds (on average zova  (2005) reported lower 4.64 g$100 g1). Krkoskov a and Mra content of ash in this fraction, which was 2.1 g$100 g1. Similar results were obtained by Bonafaccia et al. (2003) and Steadman et al. (2001). Among the examined cultivars of buckwheat, cv. Panda was characterized by a higher content of ash in whole seeds (4.42 g$100 g1), in comparison to the cv. Kora (4.16 g$100 g1).   ska and Soral-Smietana Similar results were obtained by Stempin

Fig. 1. Exemplary chromatogram of selected polyphenols of strain Panda in: A-hulls, Bdehulled seeds, C-whole seeds.

(2006). They carried out research on buckwheat seeds, which were obtained from the Department of Cultivation and Production in Palikije (Poland) and were harvested in 2005. The lowest content of protein was determined in buckwheat  et al. (2005) reported hulls (on average 5.42 g$100 g1). Barton lower content of protein in this fraction, e.g. 4.17 g$100 g1. Dehulled seeds were characterized by the highest protein content, which was 16.20 g$100 g1. Similar results were shown by  ska et al. (2007), which reported Ga˛ siorowski (2008), and Stempin the content of protein in dehulled seeds 13.05e20.3 g$100 g1 and 15.85 g$100 g1, respectively. Kowalewski et al. (2004) reported

6

K. Dziadek et al. / Journal of Cereal Science 69 (2016) 1e8

Table 4 Phenolic compounds determined in the extract of hull family. Phenolic compounds

Molecular formula

Selected ion

m/z m/z experimental calculated

MS fragment

Retention time (min)

Catechin 1-O-Caffeoyl-6-O-alpha-rhamnopyranosyl-beta-glycopyranoside (swertiamacroside) Procyanidin B2-3-O-gallate Orientin/isorientin

C15H13O6 C21H27O13

[MH][MH]-

289.0718 487.1457

289.0726 487.1463

e 451.1248, 179.0360

32.2 39.7

C37H29O16 C21H19O11

[MH][MH]-

35.2 35.5/36.5

C21H19O11

[MH]-

729.1461 447.0932 447.0901 447.0909

577.1196, 289.0719 357.0614, 327.0480

Quercetrin

729.1460 447.0933 447.0933 447.0933

43.4

Rutin Vitexin Hiperin Isoquercitrin Epiafzelchin-epicatechin-O-dimethyl gallate

C27H29O16 C21H19O10 C21H19O12 C21H19O12 C39H33O15

[MH][MH][MH][MH][MH]-

609.1461 431.0983 463.0882 463.0856 741.1825

609.1451 431.0972 463.0839 463.0856 741.1812

Epicatechin-O-3,4-dimethylgallate

C24H21O10

[MH]-

469.1136

469.1140

301.0299, 179.0264, 151.0448 301.0045 e 271.0618 e 469.1118, 319.0812, 271.0594 319.0818, 271.0601, 125.0276

38.8 38.9 40.3 39.7 46.5 38.8

Table 5 Phenolic compounds determined in the extract of whole seeds and dehulled seeds of family. Phenolic compounds

Molecular formula

Selected ion

m/z experimental

m/z calculated

MS fragment

Retention time (min)

Caffeic acid hexose Catechin glucoside Epiafzelchin-epiafzelchin-epicatechin Procyanidin B2

C15H17O9 C21H23O11 C45H37O16 C30H25O12

[MH][MH][MH][MH]-

341.0889 451.1222 833.2087 577.1351

341.0878 451.1246 833.2035 577.1349

543.1229, 289.0691 407.0793, 289.0728,

27.5 29.3 40.3 42.9

Epicatechin gallate Epiafzelchin-epicatechin-O-methyl gallate (-)-Epicatechin-3-(300 -O-methyl) gallate Procyanidin B2 dimethyl gallate

C22H17O10 C38H31O15

[MH][MH]-

441.0827 727.1668

441.0824 727.1669

169.0156 455.0968, 289.0722,

39.8 42.6

C23H19O10 C39H33O16

[MH][MH]-

455.0962 757.1774

455.0984 757.1782

251.1031 289.0715 561.1389, 425.0883, 271.0603 289.0702, 561.1356, 271.0607 289.0689, 605.1242,

183.0293 289.0735

43.5 44.3

lower content of protein in dehulled seeds (9.77 g$100 g1). In whole seeds, the average protein level was 14.48 g$100 g1. The other authors reported lower content of this compound in whole  and Mra zova , 2005; seeds (Bonafaccia et al., 2003; Krkoskova   ska and Soral-Smietana Steadman et al., 2001). Stempin (2006) as well as Klepacka et al. (2011), who studied cv. Kora and Panda, presented lower content of protein in whole seeds in these cultivars. Our results indicate higher protein content in the whole seeds of buckwheat, than the data presented by other authors. It is advantageous due to the high biological value of the buckwheat protein. The content of crude fat, in the hulls and dehulled seeds of buckwheat, was on average 0.59 g$100 g1 and 2.35 g$100 g1, respectively. Our results were similar to data published by Ga˛ siorowski (2008). However, Kowalewski et al. (2004) and Qin et al. (2013) reported lower level of fat in the dehulled seeds of buckwheat (1.73 g$100 g1, 0.67 g$100 g1, respectively). The content of fat in the whole seeds of buckwheat was 1.94 g$100 g1. Other authors determined fat quantity in whole seeds of buck and Mra zova  (2005) e wheat at a higher level: Krkoskova 2.3 g$100 g1, Bonafaccia et al. (2003) e 2.88 g$100 g1, and Rosell et al. (2014) e 3.4 g$100 g1. Steadman et al. (2001) reported that fat content in whole seeds of buckwheat was 3.8 g$100 g1. Among the analyzed cultivars we found that, cv. Panda was characterized by higher content of fat in whole seeds (2.17 g$100 g1) in comparison to the cv. Kora (1.80 g$100 g1). Our results do not corre  ska and Soral-Smietana spond to the results published by Stempin (2006). These authors reported higher content of fat in whole seeds of the cultivars Panda and Kora (2.46 g$100 g1, 2.71 g$100 g1, respectively).

Examined buckwheat seeds contained fatty acids from C16:0 to C22:0. In all cultivars/strains, palmitic acid (saturated), oleic acid and linoleic acid (unsaturated) were the dominant fatty acids.  ska and These results were similar to the data obtained by Stempin  Soral-Smietana (2006). The average content of palmitic acid in the examined whole seeds of buckwheat was 15.02%. This data correspond to the results presented by Bonafaccia et al. (2003), who studied the cv. Siva from Slovenia. Mazza (1988) reported similar amount of this acid for the cultivars Mankan and Tokyo, and lower content of palmitic acid for a cv. Manor. In buckwheat whole seeds, the content of oleic acid, obtained by other authors, was lower than in the tested cultivars (Gulpinar et al., 2012; Mazza, 1988). A similar amount of linoleic acid was reported by Bonafaccia et al. (2003) and Mazza (1988) for the cultivars Manor and Tokyo. Lower content of this acid was determined by Gulpinar et al. (2012). In the whole seeds of buckwheat the content of stearic acid was on average 3.84%. According to Bonafaccia et al. (2003), the amount of this acid was 2.0% and according to Gulpinar et al. (2012) was 1.62%. The contents of arachidonic and behenic acids in whole seeds of buckwheat, gave by Bonafaccia et al. (2003) and Mazza (1988) were higher than in the tested cultivars. In the available literature, there is no data about content of fatty acid in buckwheat hulls and dehulled seeds. The hulls of buckwheat were the richest source of total carbohydrates and had the lowest content of the starch (Table 1). In the available literature, there is no data about content of carbohydrates in buckwheat hulls. The average content of total carbohydrates and starch in the dehulled seeds was 78.99 g$100 g1 and 49.35 g$100 g1 respectively. In available literature usually the concentration of starch in various fractions of buckwheat has

K. Dziadek et al. / Journal of Cereal Science 69 (2016) 1e8

usually been reported. Results obtained in this study were similar to data reported by Bonafaccia et al. (2003), which showed that the content of starch in grains of buckwheat was at the level of 55.8%. Higher content of starch was reported by Qin et al. (2013). These authors have shown that starch levels in the whole and dehulled seeds were 62.46 g$100 g1 and 57.96 g$100 g1, respectively.  ska et al. (2007) reported higher concentration of the Stempin starch in dehulled grains (77.29 g$100 g1). The average level of carbohydrates in whole seeds was 78.94 g$100 g1 and was similar to their concentration in the dehulled seeds. Krkoskov a and  (2005) reported that the buckwheat grains contained Mr azova 73.3 g$100 g1of carbohydrates. Basic composition of examined buckwheat seeds differed from those described by other authors. These differences come from the fact that the content of chemical components in the seeds of buckwheat depends on its cultivar/ strain, climatic and soil conditions, and the methods of seeds storage. The average content of dietary fibre in the hulls was 79.11 g$100 g1. The amount, gave by Ga˛ siorowski (2008), was lower (49.4 g$100 g1). The average content of dietary fibre in the dehulled seeds of buckwheat was at the level of 4.43 g$100 g1. Among the analyzed cultivars, dehulled seeds of cv. Kora contained  ska et al. 3.59 g$100 g1 of fibre. Research carried out by Stempin (2007) on the dehulled seeds of cv. Kora, indicate higher amount of fibre in the dehulled seeds of this cv. (8.94 g$100 g1). Kowalewski et al. (2004) reported a lower content of fibre in the dehulled seeds of buckwheat (3.70 g$100 g1). The content of dietary fibre in whole seeds of buckwheat was similar to the data published by Bonafaccia et al. (2003). They showed that whole seeds of buckwheat cv. Siva, which came from Dolenjska (Slovenia) contained 27.38 g$100 g1 of fibre. Lower level of fibre was reported zova  (2005) as well as by Rosell et al. (2014). by Krkoskov a and Mra It can be suggested that the differences in content of dietary fibre, in whole seeds of individual cultivars/strains of buckwheat reported in our study, were caused by different proportions of milling fractions in the seeds. We have found that cv. Panda was characterized by higher content of dietary fibre in whole seeds (25.14 g$100 g1), in comparison to the cv. Kora (23.85 g$100 g1). Similar results   ska and Soral-Smietana were obtained by Stempin (2006). Buckwheat is also a source of resistant starch (RS). The concentration of this compound in the dehulled seeds and in whole seeds was in the range 6.13e11.04 g$100 g1and 3.29e6.58 g$100 g1 respectively. Higher concentration of RS in  ska et al. (2007). Also dehulled seeds was determined by Stempin   ska and Soral-Smietana Stempin (2006) reported higher concentration of RS in whole seeds of buckwheat in cultivars Kora, Luba and Panda. The average concentration of total polyphenols in hulls of buckwheat was 479.65 mg$100 g1. Sedej et al. (2012) reported lower content of polyphenols in hulls of buckwheat which came from Ukraine. In whole seeds of buckwheat the concentration of polyphenols was on average 339.34 mg$100 g1. Higher content of polyphenols in whole seeds was reported by Holasova et al. (2002). Sedej et al. (2012) found lower levels of these compounds in whole seeds. Klepacka et al. (2011) reported lower content of polyphenols in cv. Panda (245.4 mg$100 g1) and higher level of these compounds in cv. Kora (281.1 mg$100 g1) as compared to our results. We have found that among all examined cultivars/strains of buckwheat, the hulls of strain 8/2008 and the dehulled seeds of strain 9/ 2006 had the highest content of total polyphenols. Based on the results of our study, the hulls were the richest source of total polyphenols and the poorest source of these compounds was dehulled seeds. It can be suggested that hulls should be introduced to the consumers diet. They should be used for example in the form of bran or whole seeds for meals preparation, as the rich

7

source of polyphenolic compounds. Results obtained from identification of polyphenolic compounds in analyzed material are similar to the data published by Kiprovski et al. (2015) which assessed the polyphenolic compounds in whole seeds of buckwheat cultivars cultivated in Serbia. Additionally we determined Epiafzelchin-epicatechin-O-methyl gallate, (-)-Epicatechin-3-(300 O-methyl) gallate, Epiafzelchin-epicatechin-O-dimethyl gallate. These compounds were more specific for dehulled seeds. Epiafzelchin-epicatechin-O-methyl gallate, (-)-Epicatechin-3-(300 O-methyl) gallate, Epiafzelchin-epicatechin-O-dimethyl gallate were detected by Verardo et al. (2010) in flour of buckwheat. The highest antioxidant activity was measured in the dehulled  ska seeds of strain 8/2008. It was 34.93 mmol Trolox$g1. Stempin et al. (2007) reported lower antioxidant activity milling fraction. Antioxidant activity of buckwheat whole seeds were on average 45.23 mmol Trolox$g1. Karama c (2010) reported lower antioxidant activity for whole seeds of cv. Panda from Olsztyn (Poland). The largest quantity of polyphenolic compounds (525.45 mg$100 g1) and one of the highest antioxidant activity (65.79 mmol Trolox$g1) were determined in the hulls of strain 8/ 2008. The dehulled seeds of cv. Kora were characterized by the lowest antioxidant activity (31.28 mmol Trolox$g1), and the lowest content of total polyphenols (284.19 mg$100 g1). There were also results that did not confirm this relationship. The dehulled seeds of strain 8/2008 contained low levels of polyphenols (280.0 mg$100 g1) and strong antioxidant activity (34.09 mmol Trolox$g1). Similarly, the whole seeds of strain 21/2002, which were characterized by the lowest content of polyphenols (311.98 mg$100 g1) and one of the highest antioxidant activity (53.31 mmol Trolox$g1). It was probably caused by the fact that the whole seeds of buckwheat may contain other antioxidant compounds which affect the antioxidant activity. 5. Conclusions Buckwheat seeds are a source of protein and carbohydrates. Statistically significant effect of cultivar/strain on the content of individual components and differences in the content of bioactive compounds and antioxidant activity between the whole seeds, dehulled seeds and hulls was observed. The highest content of dietary fibre and total polyphenols as well as the highest antioxidant activity were found in the hulls and the lowest e in the dehulled seeds of buckwheat. The strains 8/2008 and 9/2006 were recognized as a good sources of nutrients and bioactive compounds. This knowledge will allow us to develop the new functional foods, which can help in the prevention and treatment of noncommunicable diseases as well as better using of the byproducts of production (for example the hull). Acknowledgment This research was financed by Ministry of Science and Higher Education of the Republic of Poland. References AOAC, 2006. Official Methods of Analysis, 18-th Edition. association of Offcial Analitical Chemists International, Gaintersburg.  , H.J., Fołta, M., Chłopicka, J., Zachwieja, Z., Gumul, D., 2005. Antioxidant Barton activity of five varieties of buckwheat seeds (Fagopyrum esculentum Moench). Bromat. Chem. Toksykol. (Suppl. 71e74) (in Polish). Bonafaccia, G., Marocchini, M., Kreft, I., 2003. Composition and technological properties of the flour and bran from common and tartary buckwheat. Food Chem. 80, 9e15. FAOSTAT database: http://faostat3.fao.org/home/index.html. Folch, J., Lees, M., Sloane-Stanley, G.H., 1957. A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 226, 497e509.

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antioxidant and a-glucosidase inhibitory activities during the processing of tartary buckwheat tea. Food Res. Int. 50, 562e567. Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., Rice-Evans, C., 1999. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic. Biol. Med. 26, 1231e1237. Rosell, C.M., Barro, F., Sousa, C., Mena, M.C., 2014. Cereals for developing gluten-free products and analytical tools for gluten detection. J. Cereal Sci. 59, 354e364.    Sedej, I., Saka c, M., Mandi c, A., Misan, A., Pestori c, M., Simurina, O., Canadanovi cBrunet, J., 2011. Quality assessment of gluten-free crackers based on buckwheat flour. LWT Food Sci. Technol. 44, 694e699.   Sedej, I., Saka c, M., Mandi c, A., Misan, A., Tumbas, V., Canadanovi c-Brunet, J., 2012. Buckwheat (Fagopyrum esculentum Moench) grain and fractions: antioxidant compounds and activities. J. Food Sci. 9, 954e959. Steadman, K.J., Burgoon, M.S., Lewis, B.A., Edwardson, S.E., Obendorf, R.L., 2001. Buckwheat seed milling fractions: description, macronutrient composition and dietary fibre. J. Cereal Sci. 33, 271e278.   ska, K., Soral-Smietana, Stempin M., 2006. Chemical compounds and physicochemical estimation of buckwheat grains e comparison of three polish vari_ s eties. Zywno c. Nauka. Technol. Jakos c 2 (Suppl. l.), 348e357 (in Polish).   ska, K., Soral-Smietana,  ski, H., Michalska, A., 2007. Effect of Stempin M., Zielin thermal treatment on chemical and antioxidant properties of buckwheat grains. _ s Zywno c. Nauka. Technol. Jakos c 5, 66e76 (in Polish). Van Hung, P., Morita, N., 2008. Distribution of phenolic compounds in the graded flours milled from whole buckwheat grains and their antioxidant capacities. Food Chem. 109, 325e331. ez-Roma n, D., Segura-Carretero, A., Marconi, E., Fern Verardo, V., Arra andezrrez, A., Fiorenza-Caboni, M., 2010. Identification of buckwheat phenolic Gutie compounds by reverse phase high performance liquid chromatography e electrospray ionization-time of flight-mass spectrometry (RP-HPLCeESI-TOFMS). J. Cereal Sci. 52, 170e176. Wronkowska, M., Haros, M., 2014. Wet-milling of buckwheat with hull and dehulled e the properties of the obtained starch fraction. J. Cereal Sci. 60, 477e483.