Journal of Cereal Science 80 (2018) 37e43

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Journal of Cereal Science

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Tartary buckwheat malt as ingredient of gluten-free cookies

Romina Molinari a, Lara Costantini a, Anna Maria Timperio a, Veronica Lelli a,

Merendino a, *
Francesco Bonafaccia a, Giovanni Bonafaccia b, Nicolo
a
snc, 01100 Viterbo, Italy
Department of Ecological and Biological Sciences (DEB), Tuscia University, Largo dell’Universita
b
Food and Nutrition Research Center (CREA-NUT), Via Ardeatina 546, 00178 Roma, Italy

a r t i c l e i n f o a b s t r a c t

Article history: The impact of malting on the profile of the phenolic compounds and the antioxidant properties of tartary
Received 22 September 2017 buckwheat (Fagopyrum tataricum L. Gaertn.) was investigated. A considerable increase in total phenolic
Received in revised form compounds and higher antioxidant activity were observed after 88 h of germination. The highest relative
17 November 2017
increases in phenolic compounds were observed for quercetin, orientin, and vitexin, which are conse-
Accepted 21 November 2017
Available online 22 November 2017
quently major inducible phenolic compounds during malting. Only a minor relative increase was
observed for the most abundant phenolic compound, rutin. Formulations of gluten-free cookies based on
rice flour and buckwheat malt or flour in ratios 70:30, have been produced. In the raw material and
Keywords:
Tartary buckwheat malt
cookies the proximate composition, starch, resistant starch, total polyphenols, profile of polyphenols,
Antioxidant activity antioxidant activity and expected glycemic index were determined. Gluten-free cookies made with rice
Flavonoids flour and buckwheat malt exhibited significantly higher total phenolic and quercetin contents.
Gluten-free cookies Comparing to control cookies higher antioxidant activity and lower glycemic index (p < 0.05) was found.
© 2017 Elsevier Ltd. All rights reserved.

1. Introduction essentially the same as common buckwheat, but it is rarely

Buckwheat is a gluten-free pseudo-cereal that belongs to the
Polygonaceae family. The two major species with agricultural sig-
nificance are common buckwheat (Fagopyrum esculentum Moench)
(sweet buckwheat) and tartary buckwheat (Fagopyrum. tataricum
(L.) Gaertn.) (bitter buckwheat).
Buckwheat is rich in fiber, essential amino acids, vitamins,
minerals and polyphenols. The dominant phenolic compounds in
buckwheat are rutin and quercetin. The general composition of
tartary buckwheat in terms of crude protein, fiber, fat, and ash is
Abbreviation: TMC, (Tartary Malt Cookies); RC, (Rice Cookies); TFC, (Tartary
Flour Cookies); RB, (Raw Buckwheat); SB, (Soaked Buckwheat); GB, (Germinated
Buckwheat at 16,40,64, 88 h); WTM, (Whole Tartary Malt); RF, (Rice Flour); WTF,
(Whole Tartary Flour); RS, (Resistant Starch); eGI, (expected glycemic index); GAE,
(Gallic Acid Equivalents); RE, (Rutin Equivalents); DW, (Dry Weight); TE, (Trolox
Equivalents); RDS, (Rapidly Digestible Starch); SDS, (Slowly Digestible Starch); TDF,
(Total Dietary Fiber); IDF, (Insoluble Dietary Fiber); SDF, (Soluble Dietary Fiber).
* Corresponding author. Department of Ecological and Biological Sciences (DEB),
Laboratory of Cellular and Molecular Nutrition, Tuscia University, Largo
dell’Universita
snc, 01100 Viterbo, Italy.
E-mail addresses: rominamolinari@libero.it (R. Molinari), lara.cost@unitus.it
(L. Costantini), timperio@unitus.it (A.M. Timperio), veronicalelli@yahoo.it
(V. Lelli), francesco.bonafaccia29@gmail.com (F. Bonafaccia), giovanni.bonafaccia@
cra.gov.it (G. Bonafaccia), merendin@unitus.it (N. Merendino).
consumed because of its bitter taste, which is caused by the
enzymatic degradation of the high rutin content. Rutin, which is
particularly abundant, is 4 times higher in tartary buckwheat than
in common buckwheat (Li and Zhang, 2001). Buckwheat grain is
considered to have higher content of phenolic compounds and
higher antioxidant activity in comparison to other cereal grains and
for this reason it have healing effects in some chronic diseases
(Zhang et al., 2012).
Buckwheat contains a high level of starch similar to many cereal
grains, and it is known for its high levels of resistant starch.
(Skrabanja et al., 2001). Buckwheat does not contain gluten so it is
edible by celiac disease patients. The gluten-free diet is unbalanced
in carbohydrates, proteins and fat and deficient in certain essential
nutrients. Due to the limitation of some nutrients, the fortification
of basic gluten-free formulations is recommended to develop value
added products. In literature, it has been reported that gluten-free
based products have been enriched by buckwheat flour in order to
achieve better functionality in the final product (Costantini et al.,
2014).
Formulations of gluten-free cookies based on rice and buck-
wheat flour in three different ratios, 90:10, 80:20 and 70:30, were
developed, and characterized by Torbica et al. (2012). However, in
the production of several foods, common buckwheat is used

https://doi.org/10.1016/j.jcs.2017.11.011
0733-5210/© 2017 Elsevier Ltd. All rights reserved.
38 R. Molinari et al. / Journal of Cereal Science 80 (2018) 37e43
because of tartary buckwheat strong bitter taste due to the high
concentration of rutin. One of the possible ways to promote the use
of tartary buckwheat could be its transformation into a more
palatable product.
Malting the grains includes three steps: steeping, to ensure
adequate absorption of water by the grain; germination, to estab-
lish embryo growth, enzyme synthesis, and limited endosperm
breakdown; and kilning, to ensure the typical flavor characteristics
and induces phytochemical alterations (Nelson et al., 2013).
A number of studies have indicated that malting is a process that
transforms the grain into a more palatable and nutritionally richer
form (Donkor et al., 2012); for example the amount of phenolic
compounds, and phytosterols in oat increase during germination
and oat malt can be used in wheat products to improve nutritional
quality (Ma €kinen and Arendt, 2012).
Malting transforms common buckwheat into a more agreeable
product and a considerable increase in total phenolic compounds
and higher antioxidant activity were observed during this process
(Terpinc et al., 2016).
The present study investigated the effects of soaking, germina-
tion and kilning on the phenolic compound profile and antioxidant
properties in tartary buckwheat during malting and evaluate its
suitability for gluten free cookie production. Therefore, this study
was aimed at determining the effect of tartary buckwheat malt
addition on the antioxidant, nutritional and functional character-
istics of cookies made from rice and tartary buckwheat malt (TMC)
in 70:30 ratio compering them to the control samples made from
rice flour (RC), and rice and tartary buckwheat flour in 70:30 ratio
(TFC).
2. Materials and methods
2.1. Raw materials
Tartary buckwheat seeds were obtained from Rangus mill

(Vrhpolje pri Sentjerneju, Slovenia), commercially available rice
flour was used in the formulation of cookies, other baking in-
gredients were purchased from the local food store. All chemicals
used, unless stated otherwise, were of analytical grade and were
purchased from SigmaeAldrich (Milano, Italy). Stock standard so-
lutions of phenolic compounds were prepared by rigorous disso-
lution of the commercial reagent in methanol. High-purity water
from a Direct-Q 3 UV water purification system (Millipore) was
used for all analyses.
2.2. Malting performance
The grain samples (raw buckwheat, RB) were carefully cleaned
and freed from foreign material before they were soaked in water
for 8 h at 20
C. Every hour, these samples were aerated and the
water was changed.
Following the soaking process (soaked buckwheat, SB), the
water was drained and the grains were spread out in a thin layer on
a stainless-steel tray with a perforated bottom, and then left to
germinate for 16 h (germinated buckwheat 16 h, GB16), 40 h
(germinated buckwheat 40 h, GB40), 64 h (germinated buckwheat
64 h, GB64), and 88 h (germinated buckwheat 88 h, GB88). The
germination period was conducted in a germination chamber
(WK3-180/0 series, Weiss Umwelttechnik GmbH, Deutschland) at
20
C and relative humidity of 98%. After the soaking (8 h after the
start) and at each step of the germination (16, 40, 64 and 88 h)
samples were collected and placed on perforated metal trays in
ventilated oven, in which they were dried at 60
C for 22 h to
provide the whole tartary final malt (WTM).
2.3. Preparation of cookies
Mixtures of the flours were prepared, with the ratio of rice flour
(RF) to whole tartary malt (WTM) or whole tartary flour of 70:30
(WTF, RB). According to our preliminary study (Merendino et al.,
2014) and to Torbica et al. (2012), the best quality of gluten-free
rice cookies was achieved when they were enriched with 30% of
buckwheat flour. Rice and tartaric flours were used for the prepa-
ration of control cookies. The ingredients were weighed as follows
(in grams, g): RF 100 g for the control cookies or RF and WTF or
WTM, respectively 70 g and 30 g for the cookies with 30% flour
substitution, deionized water 30 g, butter 28 g, granulated sugar
20 g, baking powder 6 g, salt 2 g.
All ingredients were mixed in mixer for 10 min. All the made
doughs were wrapped in polyethylene bags and left to rest at room
temperature for an hour, and then they were manually sheeted to
5 mm thickness and cut by pressing molds onto dough sheet.
Baking was done in an oven at 170
C for 20 min. After cooling for
30 min to room temperature, the cookies were packed in sealed
polypropylene bags and kept at room temperature for further ex-
amination. All raw samples and cookies were frozen prior to being
freeze dried (Thermo Electron Corporation, Waltham, MA, USA)
and then the cookies were ground in a laboratory mill (Laboratory
mill 3100, 0.5 mm sieve, Perten PerkinElmer Company, Sweden).
Cookies powder was used for all the analyses.
2.4. Extracts preparation
To measure the antioxidant activity, total polyphenol and total
flavonoid contents, raw material and powdered cookies samples
were extracted with a solvent consisting of methanol:water (80:20,
v/v) in a ratio 1:25 (w/v). The 1:25 mixture was shaken at room
temperature for 8 h, and then centrifuged at 1000 g for 10 min, the
supernatant was used for all of the analyses listed above.
2.5. Determination of total phenols
Total phenols were determined using a modification of the
FolineCiocalteu standard method (Singleton and Rossi, 1965).
Briefly, the assay was conducted by mixing 4 mL of deionized water,
0.25 mL of extracts (see ‘Extracts preparations’ in this section),
0.25 mL of FolineCiocalteu reagent, and 0.5 mL of Na2CO3. After
30 min at room temperature, the absorbance of the mixture was
measured on Uvikon spectrophotometer at 725 nm (942, Kontron
Instruments, Zurich, Switzerland). A standard curve with gallic acid
was prepared. The final results were expressed as mg of gallic acid
equivalents (GAE) per g of dry weight (DW). All of the analyses were
conducted in triplicate.
2.6. Determination of total flavonoids
The total flavonoid contents in raw and cookies samples were
determined using the aluminium chloride colorimetric method
described by Qin et al. (2010).
Briefly, the appropriate dilutions of extract (0.5 mL) were mixed
with 1.5 mL of 95% ethanol, 0.1 mL of 10% aluminium chloride
hexahydrate (AlCl3 6 H2O), 0.1 mL of 1 M potassium acetate
(CH3COOK), and 2.8 mL of deionized water. After incubation at
room temperature for 30 min, the absorbance of the reaction
mixture was measured at 415 nm on Uvikon spectrophotometer
(942, Kontron Instruments, Zurich, Switzerland) against deionized
water blank. The total flavonoid contents were expressed as mg
rutin equivalents (RE)/g of DW. Samples were analyzed in triplicate.
 R. Molinari et al. / Journal of Cereal Science 80 (2018) 37e43
2.7. Analysis of phenolic compound composition by LCeMS
Reversed-phase HPLC separation was carried out using a
Kinetex C18 column (100  2.00 mm; 2.6 mm), protected by a guard
cartridge (4.0  2.0 mm; Gemini C18 Security Guard; Phenomenex,
USA). The mobile phase components were 0.1% formic acid (A) and
acetonitrile (B). The mobile phase gradient was: 0e2 min, 10% B;
2e20 min, 10%e60% B; 20e21 min, 60%e80% B; 21e25 min, 80% B;
25e26 min, 80%e10% B; 26e30 min, 10% B. The injection volume
was 10 mL and the column temperature was 25
C. The flow rate of
the mobile phase was 0.300 mL/min.
Mass analysis was carried out on an electrospray hybrid quad-
rupole time-of-flight instrument MicroTOF-Q (Bruker-Daltonik,
Bremen, Germany) equipped with an ESI ion source. Mass spectra
for metabolite-extracted samples were acquired in positive ESI,
capillary voltage was set at 4500 V () ion mode. The liquid
nebulizer was set at 27 psi, and the nitrogen drying gas was set to a
flow rate of 6 L/min. Dry gas temperature was maintained at 200
C.
Data were stored in centroid mode and acquired with a stored mass
range of 50e1200 m/z. Instrument calibration was performed
externally every day with 10 mM sodium hydroxide in 50% iso-
propanol: water, 0.1% formic acid. Automated internal mass scale
calibration was performed through direct automated injection of
the calibration solution at the beginning and at the end of each run
by a six-port divert valve.
The flavonols were identified on the basis of their MS spectra
and molecular-ion identification. Quantification of these com-
pounds was calculated relative to the corresponding external
standards from the calibration curves that covered the range from
0.1 mg/L to 10 mg/L. Replicates were exported as mzXML files and
processed through MAVEN.52; mass spectrometry chromatograms
were elaborated for peak alignment, matching and comparison of
parent and fragment ions, and tentative metabolite identification
(within a 10 ppm mass-deviation range between observed and
expected results against the imported KEGG database). MAVEN is
an open-source software that could be freely downloaded from the
official project websites (http://genomics-pubs.princeton.edu/
mzroll/index.php?show¼download).
2.8. Measurement of total antioxidant capacity using ORAC assay
Antioxidant activity was measured using oxygen radical absor-
bance capacity (ORAC), a widely used fluorescent method for
assessing antioxidant capacity. The ORAC was determined using the
hydroxyl radical antioxidant capacity (HORAC) assay kit according
to the manufacturer's instructions (Cell Biolabs Inc, USA). The final
ORAC values were expressed as gallic acid equivalents (mM GAE/g
DW) and determined according to the standard curve. All of the
analyses were conducted in triplicate.
2.9. Measurement of total antioxidant capacity using FRAP assay
A ferric reducing antioxidant power (FRAP) assay was per-
formed according to the method described by Benzie and Strain
(1999) which was adapted for 96-well plates and an automatic
reader (Infinite 2000, Tecan, Salzburg, Austria). The method is
based on the reduction of the Fe3þ-2,4,6-tripyridyl-s-triazine
(TPTZ) complex to its ferrous form at a low pH. Briefly, 160 mL of
FRAP assay solution (consisting of 20 mM ferric chloride solution,
10 mM TPTZ solution, and 0.3 M acetate buffer at pH 3.6) was
prepared daily, mixed with 10 mL of the sample, standard, or blank,
and dispensed into each well of a 96-well plate. The absorbance
was measured at 595 nm at 37
C after 30 min of incubation. All of
the analyses were conducted in triplicate. The final results are
expressed as mM Trolox equivalents per g of the DW samples (mM
39
TE/g DW).
2.10. In vitro starch digestibility
2.10.1. Resistant starch (RS) content
In the method of determining the RS using the Megazyme Kit
(Megazyme, Ireland), the samples were hydrolyzed using a-
amylase and amyloglucosidase (provided with the kit) for 16 h at
37
C. All methodologies quantified the free glucose from the
enzymatic hydrolysis by the colorimetric method with an oxidase-
peroxidase glucose reagent and the starch content was calculated
by multiplying the glucose content by 0.9. The kit allows the
measurement of resistant starch, solubilized starch and total starch
content of samples.
2.10.2. In vitro starch digestion rate and expected glycemic index
(eGI)
A classical method was used to determine the in vitro di-
gestibility of starch (Go~ ni et al., 1997) with some modification. In
brief, to 50 mg of samples (raw material and cookies) HCl-KCl buffer
(0.01 M, pH l.5, 10 mL) was added; subsequently, pepsin solution
(0.2 mL) prepared by dissolving pepsin (1 g) in 10 mL of HCl-KCl
buffer was added to each samples, and then incubated for 60 min
in a shaking water bath at 40
C. After pepsin digestion, the total
sample volume was adjusted to 25 mL with maleate buffer (0.2 M,
pH 6.9). Maleate buffer (0.2 M, 5 mL) containing a-amylase (48 UI/g
sample) was added to each samples, and then incubated for 3 h in a
shaking water bath at 37
C. Aliquots (1 mL) at 0, 30, 60, 90, and
120,180 min were obtained from each samples and incubated at
100
C for 5 min to inactivate the enzyme. Each test was cooled at
the end of the incubation time. After centrifugation (10,000 g at
4
C) 500 mL of each supernatant was taken to a volume of 2 mL
with sodium acetate buffer (0.2 M, pH 4.75). Then, 60 mL of amy-
loglucosidase, were added and incubated at 60
C for 45 min with
constant stirring. Glucose content was measured by the GOPOD kit
(Megazyme, Ireland). The starch amount was calculated by multi-
plying the glucose content by 0.9. The rate of starch digestion was
expressed as the percentage of total starch hydrolyzed at 0, 30, 60,
90, 120 and 180 min.
A non-linear model following the equation [C ¼ C1∞/(1 e e-kt)]
was applied to describe the kinetics of starch hydrolysis, where C,
C1, and k were the hydrolysis degree at each time, the maximum
hydrolysis extent and the kinetic constant, respectively. The hy-
drolysis index (HI) was calculated as the ratio between the areas
under the hydrolysis curve (0e180 min) of the experimental sam-
ples and the area of reference sample (white bread).
The eGI was calculated using the equation proposed by Go ~ ni
et al. (1997), that is:
eGI ¼ 39.71 þ 0.549  HI
2.11. Proximate composition
The proximate compositions of flour and cookies for moisture,
ash, soluble and insoluble fibers, protein and fats were respectively
determined on dry matter using the methods of the ICC number:
110/1, 104/1, 156, 105/2 and 136 (ICC, 2010). Protein content was
determined using 6.25 conversion factors (AACC, 2008). The car-
bohydrate content (%) was calculated by subtracting the contents of
ash, fat, fiber and protein from 100% dry matter. Calorie contents
were calculated using the following specific energy factors: 9 for
fats, 4 for carbohydrates and 4 for proteins, calculated based on
100 g of the edible portion (carbohydrate, fat, and protein content
40 R. Molinari et al. / Journal of Cereal Science 80 (2018) 37e43

in the edible portion: data not shown). Kjoule contents were
calculated using the 4.184 factor on the calorie obtained.
2.12. Statistical analysis
The mean and standard deviation (SD) of the three replicates
were calculated for all samples. Statistical analysis was performed
with the XLSTAT 2016 (Addinsoft SARL, New York, USA) software
using one-way ANOVA. Fisher's least significant differences test
was used to describe statistical differences between means at the
p < 0.05 significance level.
3. Results and discussion
3.1. Total phenols compounds content
The total phenols content at different malting stages of tartary
buckwheat are presented in Table 1. As shown, the phenols ranged
from 47.7 mg (RB) to 58.8 mg GAE/g DW after germination (GB88).
Here, 8 h of soaking of the buckwheat had no statistically sig-
nificant influence on total phenols (Table 1, SB). Analysis of the
literature revealed different data related to the influence of soaking
on phenols. In particular a negative impact has been reported by
several studies (Khandelwal et al., 2010), with reductions in the
phenolic compounds believed to be connected to their leaching
into the soaking water and/or to the formation of insoluble com-
plexes with proteins or polymerization of lower molecular weight
phenolic compounds. On the contrary, the soaking stage was shown
to be an active period of antioxidant synthesis for most plant
samples (Cevallos-Casals and Cisneros-Zevallos, 2010).
In our results, the non-significant changes in total phenols after
germination for 16 h (Table 1, GB16) can be attributed to the active
phenolic metabolism, or due to the formation of insoluble com-
plexes with other molecules that affect their extraction. After
germination for 64 h, there was a significant increase in phenols
(Table 1, GB64). An additional significant increase coincided with
the later stages of germination (Table 1, GB88), where the total
phenols in the extracts were increased by 23% when compared to
the raw buckwheat before germination (Table 1, RB). Our data are in
agreement with other studies in the literature, where germination
also increased the phenols of common buckwheat (Terpinc et al.,
2016); on the contrary, other studies have reported that germina-
tion can lead to lower total phenols (Donkor et al., 2012); however,
direct comparisons with our study cannot be completely relevant,
because in each study was applied different extraction processes.
The increase in total phenols upon germination could be attributed
to several factors: the de novo biosynthesis of phenolic compounds
(Cevallos-Casals and Cisneros-Zevallos, 2010), the release of the
bound phenolic compounds by the action of glycosidases, or the
hydrolysis of polymeric phenolic compounds.
Due to higher total phenolic content of buckwheat malt (WTM),
than of rice flour (RF) (Table 1) and buckwheat tartary flour (WTF),
a significant increase (p < 0.05) in total phenolic content of gluten-
free rice and malt tartary buckwheat cookies (70:30 ratio, TMC)
was found in comparison with the rice ones (Table 1, RC) and rice
and tartary buckwheat flour (70:30 ratio, TFC). The obtained results
indicated that an increase of total phenolic content in the supple-
mented cookies was much lower than expected e there was no
linear correlation between the percentage addition of the supple-
ment and the determined phenolic content.
3.2. Total contents and individual concentrations of flavonoid
compounds
The contents of total flavonoids of non-germinated/germinated
buckwheat seeds are given in Table 1. The total flavonoids increased
slightly with germination time. Soaking and germination increased
the amount of rutin, quercetin, orientin and vitexin in buckwheat
seeds, but the content had no regular changing trend. The rutin,
quercetin and total flavonoid contents in the RB (WTF) were 2.2, 1.9
and 15.3 mg/g respectively; the rutin, quercentin and total flavo-
noid contents in GB88 (WTM) were 3.7, 4.1 and 16.3 mg/g,
respectively. The content of total flavonoids was higher than the
sum of rutin, quercetin, orientin and vitexin content, indicating that
there were other flavonoids present in the seeds, which might be
synthesized or transformed from other compounds during germi-
nation (i.e. quercetrin, luteolin, apigenin, kaempferol, myricetin,
genistein etc.); studies have reported that the contents of total
flavonoids increased with germination time and leveled off after
the third germination day with the changing trend of rutin and

Table 1
Polyphenol, flavonoid and total antioxidant capacity contents of raw material and experimental cookies.

Total Phenols Total Flavonoids Rutin Quercetin Orientin Vitexin Total Antioxidant Capacity
mg GAEb/ga mg REc/ga mg/ga mg/ga mg/ga mg/ga
FRAPe ORACf
mM TEd/ga mM TEd/ga

Raw materials
RB (WTF) 47.7 ± 1.2d 15.3 ± 0.2b 2.2 ± 0.2b 1.9 ± 0.2c 81.1 ± 1.1c 15.0 ± 0.8e 6.9 ± 0.3c 146.7 ± 2.2c
SB 44.6 ± 1.7d 13.9 ± 0.3c 2.4 ± 0.1b 3.6 ± 0.1b 81.0 ± 1.0c 17.1 ± 0.2d 5.6 ± 0.4d 136.8 ± 1.0e
GB16 46.9 ± 2.2d 14.4 ± 0.5c 2.2 ± 0.1b 3.9 ± 0.2a 87.1 ± 1.2b 13.2 ± 0.5e 6.1 ± 0.2d 141.4 ± 1.1d
GB40 49.8 ± 1.2cd 14.6 ± 0.4c 2.1 ± 0.2b 3.8 ± 0.1a 80.2 ± 1.1c 19.0 ± 1.2c 6.8 ± 0.1c 138.1 ± 1.0e
GB64 53.7 ± 1.1b 14.8 ± 0.3c 2.3 ± 0.1b 3.7 ± 0.1 ab 95.4 ± 1.2a 111.2 ± 2.3b 8.3 ± 0.3b 159.8 ± 1.5b
GB88 (WTM) 58.8 ± 1.7a 16.3 ± 0.2a 3.7 ± 0.2a 4.1 ± 0.1a 85.2 ± 1.3b 194.2 ± 2.8a 10.2 ± 0.4a 171.4 ± 1.9a
RF 1.1 ± 0.4e 0.1 ± 0.01d nd nd nd nd 0.1 ± 0.01e 14.2 ± 1.0f
Experimental cookies
RC 0.9 ± 0.1c 0.2 ± 0.01c nd nd nd nd 0.14 ± 0.02c 10.8 ± 1.0c
TFC 7.9 ± 0.1b 4.3 ± 0.1b 0.2 ± 0.01b 3.2 ± 0.1b 13.1 ± 0.2b 18.1 ± 0.9b 0.21 ± 0.03b 22.9 ± 1.2b
TMC 8.9 ± 0.2a 5.2 ± 0.1a 0.1 ± 0.02a 3.9 ± 0.2a 31.2 ± 1.1a 29.3 ± 1.3a 0.42 ± 0.01a 32.4 ± 1.9a

Data are means ± SD (n ¼ 3). Means with different letters within a column of the same group (raw material samples and experimental cookies samples) are significantly
different (p < 0.05). RB raw tartary buckwheat, SB soaked tartary buckwheat, GB germinated tartary buckwheat at different hours, RF rice flour, WTF whole tartary flour, WTM
whole tartary malt, RC rice cookies, TFC tartary flour cookies, TMC tartary malt cookies. Nd: not detectable.
a
Of dry weight sample.
b
GAE: gallic acid equivalents.
c
RE: rutin equivalents.
d
TE: trolox equivalents.
e
FRAP: ferric reducing antioxidant power.
f
ORAC: oxygen radical absorption capacity.
 R. Molinari et al. / Journal of Cereal Science 80 (2018) 37e43
quercetin opposite to each other (Yiming et al., 2015).
Due to higher total flavonoids content of WTM than of WTF and
RF (Table 1), a significant increase (p < 0.05) in total flavonoid
content of TMC was found in comparison with the TFC and RC
(Table 1). In addition, our results showed that during the cookies-
making process there was an increase in quercetin. The trans-
formation of rutin to quercetin is expected due to the presence of
rutin-degrading enzymes in the tartary buckwheat grain, indeed it
has been proven that in pasta made with tartary buckwheat
sprouts, the rutin concentration decreased, whereas the quercetin
concentration increased and remained stable during processing
(Merendino et al., 2014).
The results indicated that germination is a good way for accu-
mulation of bioactive flavonoids, such as quercetin.
3.3. Total antioxidant capacity
In general, because the antioxidant compounds in plant extracts
are chemically different and structurally complex, no single assay is
suitable to accurately determine the antioxidant activity for all
compounds present in a sample, the use of multiple assays is
therefore preferable. In our study, antioxidant activity was deter-
mined using two different in vitro antioxidant assays: FRAP and
ORAC. The FRAP assay is based on electron transfer mechanism
without the involvement of free radicals, moreover FRAP does not
measure thiol-based antioxidants, such as glutathione. On the
other hand, in the ORAC assay the reaction between radicals and
antioxidants is achieved through hydrogen atom transfer mecha-
nisms. Both analyses were performed to make relative comparisons
among the total antioxidant capacity of the raw materials and of the
experimental cookies.
Analysis of the antioxidant activities of the tartary buckwheat
malt extracts revealed similar profiles to those determined in the
Folin Ciocalteu assays. The differences in the antioxidant activities
between the raw buckwheat and the germination stages 16e40 h
were not statistically significant (Table 1). GB64 and GB88 resulted
in large increases in the antioxidant activities determined by FRAP
and ORAC methods, which is in agreement with the results of the
Folin-Ciocalteu assay. After 88 h of germination, the antioxidant
activity measured with FRAP (GB88) was increased by 47.8% when
compared to those for the RB (RB 6.9 ± 0.3 vs GB88
10.2 ± 0.4 mM TE/g); whereas the increase in the antioxidant ac-
tivities in the ORAC method was 16.8% (RB 146.7 ± 2.2 vs GB88
171.4 ± 1.9 mM TE/g). Indeed, GB88 had the highest antioxidant
capacity. Antioxidant compounds can also be formed as a result of
the Maillard reaction and pyrolysis. On the other hand, during heat
treatments, the phenolic compounds might also be degraded and/
or oxidized. The data in the literature on the influence of germi-
nation and kilning on these antioxidant activities show large vari-
ations between studies, due to the different temperatures of
processing, types of grain, and way of expressing the results (e.g.,
normalization to dry or fresh weight). Some studies indicate that
the antioxidant activity of enriched foods may be masked by the
interactions of phenolic compounds with food matrix components
(Se˛ czyk et al., 2015).
As for the types of flour, the FRAP and ORAC methods suggested
similar differences among the cookies samples. The lowest anti-
oxidant capacity among all the cookies samples was in RC (FRAP
0.14 ± 0.02 mM TE/g and ORAC 10.8 ± 1.0 mM TE/g), whereas the
highest antioxidant capacity were in TMC (FRAP 0.42 ± 0.01 mM TE/
g and ORAC 32.4 ± 1.9 mM TE/g), and these antioxidant capacity
value is statistically significant also versus TFC.
The results suggest that phenolic compounds in the produced
cookies mainly contributed to their overall antioxidant properties.
However, thermal processing of cereals, such as baking, also
41
resulted in the formation of substances with antioxidant proper-
ties, namely Maillard reaction products (Lindenmeier and
Hofmann, 2004) that contribute to the overall antioxidant activity
of bakery products.
Maillard reactions and lipid oxidation contribute to non-
enzymatic browning of foods and these reactions occur during
malting (Coghe et al., 2006). Depending on the temperature applied
and production processes, these complex, interconnected in some
pathways, contribute to the development of various degrees of
color and flavor characteristics, and in antioxidant activity increase.
The fortification of gluten-free products using pseudo-cereals
has been reported by Alvarez-Jubete et al. (2010), while buck-
wheat was investigated as component for gluten-free bread and
cookies; indeed gluten-free cookies made with rice flour and
buckwheat flour exhibited significantly higher total phenolic con-
tent, antioxidant activity than the control rice cookies (p < 0.05),
(Torbica et al., 2012).
3.4. In vitro digestibility
Based on its hydrolysis rate, starch is classified into three frac-
tions: rapidly digestible starch (RDS, digested within 20 min),
slowly digestible starch (SDS, digested between 20 and 120 min),
and resistant starch (RS, indigestible) (Tuyishime et al., 2017). RDS
induces a sudden rise in blood glucose levels, whereas SDS results
in a gradual increase in blood glucose levels, which is important in
the prevention of diabetes and cardiovascular diseases; RS protects
against colorectal cancer, controls serum cholesterol levels, de-
creases the glycemic index, and reduces insulinemic responses
(Tuyishime et al., 2017).
The total, soluble and resistant starch in flour and cookies
samples were investigated (Table 2).
The RF contains the greatest amount of total starch, while the
minor quantity of total starch was obtained for the WTM (81.7 vs
45.8 g/100 g DW). WTM and WTF have the highest content of
resistant starch (2 g/100 g DW) compared to the RF (0.7 g/100 g
DW).
In cookies the addition of 30% of WTF does not increase signif-
icantly the content of resistant starch compared to cookies con-
taining only RF, while the addition of 30% of WTM leads to a
significant increase of the resistant starch content (5.9 g/100 g DW
vs 4.9 g/100 g DW of RC). Normally, RS formation depends on food
processing, temperature, starch gelatinization, storage conditions,
and starch retro-gradation (Tuyishime et al., 2017).
Fig. 1 shows the starch digestibility of RC, TFC and TMC. In
general, the curves plotted for all the studied cookies contained the
first part where the hydrolysis rates increased (0e60 min) and the
second one where a plateau was reached (60e180 min). The results
(Fig. 1) shown that less starch was digest after 30 min in TFC and
TMC (about 50%) compared to RC (about 67%); in vitro starch
digestion progressed much slowly for TMC than the TFC after
60 min.
Significant differences in starch hydrolysis rates were observed
between the studied cookies. In particular, the amount of the
released starch after 30 min from the TMC sample was statistically
lower than that of RC sample (45% vs 67% respectively), the hy-
drolysis rate of TMC was statistically lower than that of TFC after
120 min. This result could be due to the fact that the TMC had a
higher content of dietary fiber and resistant starch respect to the RC
and TFC samples. Addition of dietary fiber can further reduce the
in vitro glycemic response.
Table 3 shows significant differences in eGI between the RC and
supplemented cookies. The values for eGI among samples varied
from 100% (RC) to 62.8 and 57.6% for TFC and TMC respectively;
significant differences were found also in flour samples.
42 R. Molinari et al. / Journal of Cereal Science 80 (2018) 37e43

Table 2
Chemical composition (g/100 g) and energetic value of raw material and experimental cookies samples.

Carbohydratea Starcha Proteina Lipidsa Dietary fibera Asha Kcalb Kjoulesb

Soluble Resistant Total Soluble Insoluble Total

Raw materials
RF 89.4 81.0 ± 2.6a 0.7 ± 0.01b 81.7 6.5 ± 0.1b 1.0 ± 0.3b 0.2 ± 0.03c 1.4 ± 0.02c 1.6 1.5 ± 0.01c 333.7 1394.9
WTF 61.9 50.2 ± 1.2b 2.0 ± 0.50a 52.2 14.5 ± 0.2a 2.2 ± 0.1a 0.9 ± 0.01a 17.9 ± 0.10b 18.8 2.6 ± 0.02a 294.8 1232.3
WTM 56.7 43.7 ± 0. c 2.1 ± 0.40a 45.8 12.7 ± 0.3a 2.5 ± 0.1a 0.5 ± 0.02b 25.5 ± 0.20a 26.0 2.1 ± 0.01b 287.8 1202.9
Experimental cookies
RC 77.7 32.4 ± 0.1a 4.9 ± 0.1b 37.3 5.28 ± 0.1c 14.5 ± 0.1a 0.1 ± 0.02c 0.7 ± 0.01c 0.8 1.69 ± 0.04b 431.1 1801.9
TFC 65.2 32.2 ± 0.5a 4.4 ± 0.1c 36.6 5.71 ± 0.1b 14.5 ± 0.2a 0.5 ± 0.01a 12.2 ± 0.20b 12.7 1.85 ± 0.07a 377.9 1579.6
TMC 59.9 33.9 ± 0.2a 5.9 ± 0.5a 39.8 6.20 ± 0.2a 14.5 ± 0.2a 0.3 ± 0.01b 17.5 ± 0.10a 17.8 1.73 ± 0.03b 351.4 1469.1

Data are means ± SD (n ¼ 3). Means with different letters within a column of the same group (raw material samples and experimental cookies samples) are significantly
different (p < 0.05). RF rice flour, WTF whole tartary flour, WTM whole tartary malt, RC rice cookies, TFC tartary flour cookies, TMC tartary malt cookies.
a
Values of 100 g of dry weight.
b
Values of 100 g of edible portion.

degree of branching) and physical state. These two factors are

related to processing conditions as well as the presence of other
constituents of a food matrix (e.g. proteins, lipids and poly-
saccharides) (Singh et al., 2010). Processing of buckwheat may
affect the enzyme susceptibility of starch; for example roasting
decreased the enzyme susceptibility of starch (Christa et al., 2009).
Other factors physically and chemically affect enzyme susceptibility
of starch in the food matrix; for example lipids (e.g., fatty acids),
proteins, dietary fiber, rutin, phytic acid, protease inhibitors, and
tannins present in the buckwheat flour interact with starch and/or
a-amylase, and make the starch less available to the enzymatic
hydrolysis. The presence of protease inhibitors in buckwheat
lowers the enzyme susceptibility and digestion of starch (Wolter
et al., 2013).
Researchers classify high, medium, and low glycemic index
foods as good, better, and the best choices for nutrition. In relation
to this approach, foods are classified as: low glycemic index foods
(GI < 55), medium glycemic index foods (56 < GI < 69), and high
glycemic index foods (GI > 70) with respect to glucose as reference
(Foster-Powell et al., 2002). According to the classification of food
glycemic index, TMC should be considered medium glycemic food
because their GI values are 57.6 ± 1.0.

Fig. 1. In vitro starch hydrolysis rate of experimental cookies. Data are means ± SD 3.5. Proximate analysis
(n ¼ 3). R, reference sample (commercial white bread); RC, rice cookies; TFC, tartary
flour cookies; TMC, tartary malt cookies.
Proximate analysis of the flours and cookies is shown in Table 2.
RF had significantly lower amounts of protein, ash and fat in
Table 3
comparison with WTF and WTM; moreover RF had higher amounts
Hydrolysis index (HI) and expected glycemic index (eGI) of raw material and of carbohydrate. WTM was characterized by higher amounts of
experimental cookies samples. insoluble fiber as compared to WTF. The WTM had significantly
HI180 eGI
higher insoluble fiber content in comparison to RF and WTF.
Moreover, investigated WTM and WTF have higher ash content
Raw materials
than RF (Table 2) due to high mineral content found in buckwheat
RF 70.5 ± 1.3c 78.4 ± 1.1c
WTF 15.2 ± 1.8a 48.1 ± 1.7a seeds (Baljeet et al., 2010).
WTM 34.2 ± 1.2b 58.4 ± 1.2b The produced gluten-free TMC had significantly higher
Experimental cookies (p < 0.05) protein content in comparison to RC (Table 2). This
RC 110.0 ± 2.0c 100.2 ± 1.0c
finding is due to higher protein content of WTM compared RF.
TFC 42.1 ± 1.7b 62.8 ± 1.1b
TMC 32.6 ± 1.4a 57.6 ± 1.0a
RC had the higher calorie content (431.1 Kcal), which was
significantly different from the TFC (377.9 Kcal) and TMC
Data are means ± SD (n ¼ 3). Means with different letters within a column of the
(351.4 Kcal). The addition of buckwheat malt or flour to the cookies
same group (raw material samples and experimental cookies samples) are signifi-
cantly different (p < 0.05). RF rice flour; WTF whole tartary flour; WTM whole modified its caloric value; the decrease energy intake is due to
tartary malt; RC rice cookies, TFC tartary flour cookies; TMB tartary malt cookies; higher fiber content of tartary buckwheat malt or flour.
DW dry weight; HI180 hydrolysis index at 180 min. Concentrations of dietary fiber in its different fractions are
presented in Table 2. It was evaluated that the addition of buck-
wheat malt into cookies increased total dietary fiber content (TDF)
The kinetics of starch hydrolysis and starch digestion rate
in comparison to the rice cookies. The same results were observed
depend mainly on its structural characteristics (starch granule
for buckwheat flour. The major fraction was the insoluble dietary
morphology, amylose/amylopectin ratio, molecular structure,
fiber (IDF). TDF consisted of 12.5% soluble dietary fraction (SDF) in
 R. Molinari et al. / Journal of Cereal Science 80 (2018) 37e43
the rice cookies, and only 3.9% for buckwheat flour cookies and 1.7%
for malt buckwheat cookies. In terms of TDF, cookies may be or-
dered as follows: TMC > TFC > RC. Fiber exerts a significant influ-
ence on human health and wellbeing. The results of many studies
have shown its potential in the prevention of arteriosclerosis,
reducing the risk of coronary heart disease, protection against co-
lon cancer, and lowering glucose, cholesterol and triglycerides
contents in blood (Chawla and Patil, 2010).
Highest dietary fiber content in buckwheat malt enriched
cookies observed in this study indicates that buckwheat malt could
be a potential component of functional food.
4. Conclusions
In conclusion, the data obtained in the present study clearly
demonstrate that the malting process influences the phenolic
compound composition and antioxidant activity of buckwheat.
Changes in the phenol compound composition were confirmed in
several ways. Here, 88 h of germination with subsequent kilning at
60
C significantly increased the antioxidant properties of the
buckwheat seeds, as compared to the untreated seeds.
Tartary buckwheat malt (WTM) was used to enhance poly-
phenols and flavonoids content and antioxidant capacity of gluten-
free rice and buckwheat cookies (TMC) compared to the rice
cookies (RC) or rice and buckwheat flour (TFC) used as the control.
The substitution of rice flour in gluten-free cookie formulation with
30% tartary buckwheat malt resulted in significantly higher
(p < 0.05) total phenolic, quercetin and rutin content, and antiox-
idant activity than the control cookies. Cookies enriched with 30%
buckwheat malt expressed the high content in protein, fiber and
resistant starch and the lower caloric value compared to RC and
TFC. Therefore, the use of small concentrations of buckwheat malt
flour in gluten-free cookies can be beneficial due to the increased
antioxidant activity, phenols content and lower glycemic index. The
confirmation of the mentioned benefit on human health of using
tartary buckwheat malt in the production of gluten-free cookies
could be obtained through studies on healthy or pathological
subjects, which might be part of our further investigations. All of
these data can provide helpful information for a better use of
buckwheat in food industry.
Compliance with ethics requirements
This research does not contain any studies with human or ani-
mal subjects.
Conflicts of interest
The authors declare that they have no conflicts of interest.
Funding sources
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
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