molecules

Article
Improvement of Quality Properties and Shelf Life
Stability of New Formulated Muffins Based on
Black Rice
Constantin Croitoru 1 , Claudia Mures, an 2 , Mihaela Turturică 3 , Nicoleta Stănciuc 3 ,
Doina Georgeta Andronoiu 3 , Loredana Dumitras, cu 3 , Vasilica Barbu 3 , Elena Enachi (Ionit, ă) 3 ,
Georgiana Horincar (Parfene) 3 and Gabriela Râpeanu 3, *
1 Academy of Agricultural and Forestry Sciences, 61 Marasti Blvd, 011464 Bucharest, Romania;
c.croitoru@sodinal.com
2 Faculty of Food Engineering, Tourism and Environmental Protection, Aurel Vlaicu University of Arad,
2 Elena Dragoi Street, 310330 Arad, Romania; claudia.muresan@uav.ro
3 Integrated Center for Research, Expertise and Technological Transfer in Food Industry,
Faculty of Food Science and Engineering, Dunarea de Jos University of Galati, 111 Domnească Street,
800201 Galati, Romania; mihaela.turturica@ugal.ro (M.T.); nsava@ugal.ro (N.S.);
Georgeta.Andronoiu@ugal.ro (D.G.A.); ldumitrascu@ugal.ro (L.D.); vbarbu@ugal.ro (V.B.);
elena.ionita@ugal.ro (E.E.); gparfene@ugal.ro (G.H.)
* Correspondence: Gabriela.Rapeanu@ugal.ro or grapeanu@ugal.ro; Tel.: +4-0336-130177; Fax: +4-0236-460165




Received: 25 October 2018; Accepted: 19 November 2018; Published: 21 November 2018


Abstract: Effects of partial (50%) and total replacement of wheat flour with black rice flour on the
phytochemical, physico-chemical, sensorial, and textural properties of muffins were studied. Partial or
total replacement of wheat flour with black rice flour in muffins improved their nutritional and
antioxidative properties with a positive effect on microbiological and color stability during the storage
period in accelerated conditions. The low gluten muffins had an anthocyanin content of 27.54 ± 2.22 mg
cyanidin-3-glucoside (C3G)/100 g dry weight (DW), whereas the gluten free muffins had 46.11 ± 3.91
mg C3G/100 g DW, with significant antioxidant values. Retention of 60% and 64% for anthocyanins and
72% and 80% for antioxidant activity after baking was found. The fracturability and hardness scores
increased with the addition of black rice flour, whereas firmness and chewiness increased for gluten free
muffins. The confocal analysis revealed a tendency of glucidic components to aggregate, with gathers
of small bunches of black rice starch granules comprising anthocyanin. The results allowed designing
two new value added bakery products, low and free gluten muffins, with significant high amounts of
bioactive compounds, suggesting the functional potential of black rice flour.

Keywords: black rice flour; anthocyanins; antioxidant activity; low gluten muffins; added value products

1. Introduction
Muffins are sweet baked products highly appreciated by consumers due to their good taste and
soft texture, perfect for breakfast, brunch and snacks. Muffin composition is a fat in water emulsion
obtained from an egg-sugar-water-fat mixture as a continuous phase, and air bubbles represent a
discontinuous phase where the flour is dispersed. Muffins are generally associated with a high porous
spongy texture [1,2]. Traditionally, a muffin recipe is composed of wheat flour, vegetable oil, eggs and
milk [3]. For this reason, many people with celiac disease are unable to consume this type of product
since they are made with wheat flour.
The demand for low gluten and gluten-free products is increasing because it is well known that
celiac disease is a common lifelong disorder, affecting 1% of the world’s population [4–6]. The reaction

Molecules 2018, 23, 3047; doi:10.3390/molecules23113047 www.mdpi.com/journal/molecules

Molecules 2018, 23, 3047 2 of 15

to gluten ingestion for those who sufferer from celiac disease is the inflammation of the small intestine
leading to malabsorption of the nutrients [7,8]. However, a gluten free diet is characterized by low
daily energy intake combined, with an unbalanced macronutrient content, compared to a balanced
normal daily diet [9]. In avoiding the use of gluten in foods, significant technological and quality
problems will have to be solved. Their sensorial properties are still different from similar products
containing gluten. The main ingredients of gluten free cereal products are gluten-free flours, corn,
rice and potato starches and different hydrocolloids that slow down the gluten viscoelastic properties.
Recent studies were performed to improve the nutritional profile of gluten-free products by using
pseudo-cereals as functional gluten-free ingredients [10–12]. In recent years, there have been many
research projects for the development of gluten free sweet bakery products aimed to improve the
organoleptic properties of the finished products [13,14].
Rice is a suitable cereal for developing gluten free products because it has a low level of prolamine
and is hypoallergenic [15]. The main ingredient of the gluten free muffins, cake or cupcakes recipes is
the rice flour [13,14,16], or different starch sources, such as corn, potato and wheat [17].
Rice flour has a big potential to be a wheat flour substitute in muffins because it has been used
before to prepare gluten free bakery products, such as breads and cakes, which are traditionally
made with wheat flour [8]. However, less information is available on the use of rice flour for
gluten free products such as muffins. Several researchers have developed gluten free products using
starches, dairy products, probiotics, gums and hydrocolloids to improve the structure and taste of the
products [8,18].
Black rice was grown at a small scale in the early history of agriculture. In fact, black rice is
considered to have the highest nutritional profile of all the cereals. The interest in black rice is growing
because is gluten free, cholesterol-free, and low in sugar, salt and fat. Among these properties it
also contains anthocyanins, antioxidants, B and E vitamins, iron, thiamine, magnesium, niacin and
phosphorous and high fiber content. There are a lot of scientific studies showing that black rice powder
is one of nature’s most well-balanced foods [19]. Black rice anthocyanins are about 26.3% and the
most effective constituents in a percentage of 90% are represented by the cyanidin-3-O-glucoside and
peonidin-3-O-glucosid anthocyanin [20]. Anthocyanins represent the flavonoid pigments of the black
rice and they are a source of antioxidants that have the ability to inhibit the formation or to reduce the
concentrations of reactive cell damaging free radicals [21].
The aim of the present study was to obtain value added low gluten and gluten-free black rice
based muffins. In order to demonstrate the added-value of the products, the muffins were tested for
total phyto-chemical, physico-chemical and microbiological properties, textural and sensorial analysis.
An accelerated storage test for phytochemicals and microbiological stability was performed over
21 days at a temperature of 25 ◦ C.

2. Results and Discussion

2.1. Black Rice Flour, Batter and Muffin Characterization

The black rice flours were characterized in terms of anthocyanins content using the
chromatographic technique. Figure 1 shows a typical HPLC chromatogram, where the major
anthocyanin found was cyanidin-3-glucoside. Four compounds were found in the black rice flour
extract among which only three of them were identified as follows: Peak 1, cyanidin-3,5-diglucoside
(1.08 mg/100 g dry weight (DW)); peak 2, cyanidin-3-glucoside (176.83 mg/100 g DW); peak 3,
peonidin-3-glucoside (7.08 mg/100 g DW); and peak 4, unidentified. The total anthocyanin content
in black rice flour was 192.36 ± 1.14 mg (C3G)/100 g DW. The results are similar with ones
reported by Bordiga et al. [22] and Melini et al. [23] who studied the same variety of black rice.
Bolea et al. [24] reported significant lower quantities of 21.00 µg/g cyanidin-3-glucoside and 0.10 µg/g
peonidin-3-glucoside in the whole black rice flour.
Molecules 2018, 23, 3047 3 of 15
Molecules 2018, 23, x FOR PEER REVIEW 3 of 15

Figure 1. HPLC
Figure 1. HPLC chromatogram
chromatogram of
of anthocyanins
anthocyanins from
fromblack
blackrice
riceflour.
flour.

Cyanidin-3-glucoside and peonidin-3-glucoside have been previously identified as the

Cyanidin-3-glucoside and peonidin-3-glucoside have been previously identified as the main
main anthocyanins present in the black rice [25,26]. According to other studies reported by
anthocyanins present in the black rice [25,26]. According to other studies reported by Zhang et al.
Zhang
[27,28]etfive
al. [27,28] five anthocyanins
anthocyanins have been and
have been separated separated and in
identified identified
waxy and in waxy and non-waxy
non-waxy black rice.
black
Theserice.anthocyanins
These anthocyaninswere were malvidin,pelargonidin-3,5-diglucoside,
malvidin, pelargonidin-3,5-diglucoside, cyanidin-3-glucoside,
cyanidin-3-glucoside,
cyanidin-3,5-
cyanidin-3,5-diglucoside
diglucosideand andpeonidin-3-glucoside.
peonidin-3-glucoside.
The presence of gluten in
The presence of gluten in blackblack ricerice
flour has has
flour beenbeen
checked with with
checked a gluten ELISAELISA
a gluten assay and
assayfound
and
no gluten
found no presence in blackinrice
gluten presence flour.
black rice flour.
The
The phytochemical
phytochemicalcharacteristics
characteristicsof ofbatters
battersare
aredescribed
describedin inTable
Table1.1. The
The physico-chemical
physico-chemicaland and
phytochemical characteristics of muffins are presented in Table 2. The total anthocyanin
phytochemical characteristics of muffins are presented in Table 2. The total anthocyanin content content (TAC) in
S2 batter was 69.93 ± 2.34 mg cyanidin-3-glucoside (C3G)/100 g DW and 125.4
(TAC) in S2 batter was 69.93 ± 2.34 mg cyanidin-3-glucoside (C3G)/100 g DW and 125.4 ± 6.64 mg ± 6.64 mg C3G/100 g
DW for S3 batter. After baking, the TAC was 27.54 ± 2.22 mg C3G//100 g
C3G/100 g DW for S3 batter. After baking, the TAC was 27.54 ± 2.22 mg C3G//100 g DW for S2DW for S2 muffins and 46.11
± 3.91 mg
muffins C3G//100
and g DW
46.11 ± 3.91 mgfor S3 muffins,
C3G//100 g DW respectively. Therefore,
for S3 muffins, the retention
respectively. of TAC
Therefore, theinretention
the S2 and of
S3 after baking was approximately 60% and 64%. The total polyphenolic content
TAC in the S2 and S3 after baking was approximately 60% and 64%. The total polyphenolic content (TPC) in batters were
(TPC)±in
254.1 5.52 mg gallic
batters wereacid (GA)/100
254.1 ± 5.52 mg g DW and
gallic 307.3
acid ± 1.02 mg
(GA)/100 g DW GA/100 g DW
and 307.3 in S2mg
± 1.02 andGA/100
S3, respectively,
g DW in
whereas baking
S2 and S3, caused a whereas
respectively, decrease tobaking ± 4.55 mg
170.3 caused GA/100tog 170.3
a decrease DW and mg±GA/100
226.5
± 4.55 2.14 mggGA/100
DW andg226.5DW.
± 2.14 mg GA/100 g DW.
Table 1. Phytochemical characteristics of batters.

Table 1. Phytochemical characteristics of batters.

Samples
Phytochemical Properties
S1 S2 Samples S3
Phytochemical Properties
69.93 ± S1 S2 ± 6.64 bS3
Total anthocyanin content (TAC), mg
n.d. 2.34 a 125.4
cyanidin-3-glucoside (C3G)/100 g dry weight (DW)
Total anthocyanin content (TAC), mg cyanidin-3-glucoside 69.93 ± 125.4 ±
Total polyphenolic content (TPC), mg gallic acid n.d.
(C3G)/100 g dry weight (DW) 82.1 ± 1.06 a 254.1 ± 5.52 b,c 2.34
307.3a ± 1.026.64
b b
(GA)/100 g DW
82.1 ± 254.1 ± 307.3 ±
Total polyphenolic
Total flavonoid contentcontent (TPC),
(TFC), mg mgequivalents
catechin gallic acid (GA)/100 g aDW
(CE)/100 g DW
71.2 ± 1.44 1.06
149.4 ± 3.10a b 5.52 b,c± 5.041.02
187.1 c b

Total flavonoid content (TFC), mg catechin equivalents (CE)/100 g

Antioxidant activity, mM
71.2 ± 149.4 ± 187.1 ±
DW
6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic 152.8 ± 2.10 a 611.2 ±1.44
a
8.32 b,c 3.10
552.71b
± 5.065.04
c c

Antioxidant
acid (Trolox)/100 g DWactivity, mM
152.8 ± 611.2 ± 552.71 ±
6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic
* Values with different acid different (p <a0.05).
letters in the same raw are significantly
2.10 8.32 b,c 5.06 c
(Trolox)/100 g DW
Therefore,* Values with different
the retention of TPC letters
in thein the sameS3
S2 and raw are baking
after significantly
was different (p < 0.05).
approximately 67% and 74%,
respectively. Total flavonoid content (TFC) in batter were 149.4 ± 3.10 mg catechin equivalents
Therefore,
(CE)/100 g DW the
andretention of TPC
187.1 ± 5.04 mginCE/100
the S2 and S3 after
g DW, baking was
respectively. approximately
Baking 67% decrease
caused a slight and 74%,
respectively. Total flavonoid content (TFC) in batter were 149.4 ± 3.10
in TFC to 133.4 ± 1.88 mg CE/100 g DW and to 158.6 ± 1.02 mg CE/100 gDW, respectively, with a mg catechin equivalents
(CE)/100 g
retention ofDW
89%and 187.1
in S2 and± 85%
5.04 inmgS3. CE/100 g DW, respectively.
The antioxidant activitiesBaking caused
in batter werea 611.2
slight±decrease
8.32 mM in
TFC to 133.4 ± 1.88 mg CE/100 g DW and to 158.6 acid
6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic ± 1.02 mg CE/100 DW
(Trolox)/100g gDW, and respectively,
687.72 ± 4.11 with
mMa
retention of DW
Trolox/100g 89%forin S2
S2and
andS385% in S3. The
respectively, antioxidant
whereas activities
in muffins in batter werevalues
the corresponding 611.2were
± 8.32 mM
445.89
6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox)/100g DW and 687.72 ± 4.11 mM
Trolox/100g DW for S2 and S3 respectively, whereas in muffins the corresponding values were
Molecules 2018, 23, 3047 4 of 15

± 2.22 mM Trolox/100g DW and 552.71 ± 5.06 mM Trolox/100g DW. Coefficients of 72% and 80%
respectively were found for antioxidant retention in muffins after cooking.

Table 2. Physico-chemical and phytochemical characteristics of muffins.

Samples
Physico-Chemical and Phytochemical Properties
S1 S2 S3
a 12.71 ± 0.92 a
Proteins, g/100 g 11.69 ± 0.57 b 12.16 ± 1.16
Fats, g/100 g 20.17 ± 1.37 b 20.22 ± 0.45 c 18.37 ± 1.91 a
Carbohydrates, g/100 g 45.44 ± 2.60 a 42.91 ± 1.68 b,c 42.38 ± 2.51 c
Moisture, g/100 g 20.60 ± 0.11 b 22.53 ± 0.23 c 24.13 ± 0.15 d
Ash, g/100 g 2.10 ± 0.01 a 2.18 ± 0.01 a 2.41 ± 0.01 b
Energy value, %:
kcal 421.81 413.83 396.71 a
kJ 1763.18 1729.82 1658.24 a
TAC, mg C3G/100 g DW n.d. 27.54 ± 2.22 a 46.11 ± 3.91 b
TPC, mg GA/100 g DW 64.4 ± 3.16 a 170.3 ± 4.55 b 226.5 ± 2.14 v
TFC, mg CE/100 g DW 57.2 ± 0.94 a 133.4 ± 1.88 b 158.6 ± 1.02 c
Antioxidant activity, mM Trolox/100 g DW 124.6 ± 3.20 a 445.89 ± 2.22 b,c 552.71 ± 5.06 c
L* 80.41 ± 9.13 a 27.71 ± 0.15 b 19.6 ± 3.58 b,c
Colorimetric parameters a* 0.06 ± 0.001 a 8.47 ± 1.08 b 6.53 ± 0.95 c
b* 51.83 ± 1.15 a 7.31 ± 0.41 b 1.49 ± 0.14 c
* Values with different letters in the same raw are significantly different (p < 0.05).

However, it is difficult to estimate the effect of food matrices on the different phytochemicals, due to
the complexity and different processing parameters. For example, retention of malvidin in the bun and
biscuit after baking were 95.9% and 98.6%, respectively as reported by Karakaya et al. [29]. Similarly,
a significant decrease ranging from 37.5% to 70% in the TAC content was determined during snack
production by Nemś et al. [30], whereas Barti et al. [31] found a decrease in the anthocyanin content of
breads produced using purple and blue wheat flours during the baking process. As expected regarding
the colorimetric parameters, a lower L * value was observed for S3, whereas a * and b * values suggested a
red dark (S2) to red brown (S3) color, with a pleasant taste due to the presence of black rice.

2.2. Sensory Analysis

Table 3 shows the average scores of sensorial attributes evaluated by the panelists.

Table 3. Sensory characteristics of muffins.

Samples
Sensorial Attribute
S1 S2 S3
Color 1.82 ± 0.87 a 5.63 ± 1.2 6.27 ± 1.27
Surface humidity 3.27 ± 1.84 a 3.82 ± 0.98 a 4.72 ± 1.19
Cross section appereance 1.73 ± 1.10 1.64 ± 0.92 2.55 ± 1.7
Denseness 2.82 ± 1.47 2.82 ± 1.25 2.82 ± 1.94
Fracturability 2.46 ± 1.7 2.82 ± 1.33 3.64 ± 1.7
Hardness 2.64 ± 1.57 a 3.73 ± 1.35 a 4.36 ± 1.5
Cohesivity 5.46 ± 1.21 4.73 ± 1.00 4.55 ± 1.44
Moistness of mass 3.36 ± 1.75 3.46 ± 1.58 3.81 ± 2.27
Taste 6.00 ± 0.89 5.09 ± 1.22 4.90 ± 1.38
Sweetness 4.90 ± 1.51 4.27 ± 1.67 4.63 ± 1.7
Overall acceptability 5.90 ± 0.83 5.18 ± 0.98 5.18 ± 1.4
a Based on Dunnett multiple comparisons with a control, means on the same row that do not share a letter are
significantly different (p < 0.05).
Molecules 2018, 23, 3047 5 of 15

Control muffins (S1) as expected showed the lightest color, while S3, was the darkest (p < 0.001).
Surface humidity was perceived as being higher (p < 0.05) for S3 compared with S1 and S2. The scores
given for fracturability and hardness attribute increased with the addition of black rice flour, reaching
a maximum for S3.
The taste of all samples was appreciated; however the control sample was evaluated by the
panelists with the highest score. Some panelists perceived that samples with black rice flour contained
some crispy particles as compared with the control sample. Overall acceptability indicates that the
panelists liked the analyzed muffins, regardless of whether or not they contained black rice flour.
These results indicate that gluten free muffins obtained with black rice flour could be an alternative for
people suffering from gluten intolerance.

2.3. Texture Analysis

Texture parameters revealed by instrumental analysis are shown in Table 4. Firmness, defined
as the maximum force required to compress the samples in the first cycle, varied between 4.75 ±
0.16 N for S1 and 6.67 ± 0.02 N for S3. Similar values for firmness were reported by Demirkesen et
al. [32] and Wronkowska et al. [33] for bread formulated with rice, wheat, chestnut flour or buckwheat.
The smaller value of control firmness could be explained by the presence of glutenin and prolamin
(the major fractions of gluten) which are responsible for the porous network in muffins.

Table 4. Texture parameters of muffins.

Samples
Textural Parameters, Unit
S1 S2 S3
Firmness, N 4.75 ± 0.16 a 5.82 ± 0.26 6.67 ± 0.02
Cohesiveness, dimensionless 0.37 ± 0.01 0.35 ± 0.02 0.33 ± 0.02
Springiness, mm 6.95 ± 0.06 6.83 ± 0.08 6.57 ± 0.23
Chewiness, mJ 10.15 ± 0.23 12.21 ± 0.25 15.13 ± 0.17
a Mean of the five determinations ± standard deviation.

In the samples containing black rice flour, the reduced porosity led to a higher resistance during
compression. Cohesiveness, determined as the ratio between the resistance of the samples during
the second and the first compression, and springiness, defined as the deformation recovered between
the two compression cycles, showed the highest values for S1 sample. These values may be due
to the presence of glutenin, which is responsible for elastic and cohesive properties of dough [32].
Chewiness, described as the energy required to disintegrate the food during mastication, raised from
10.15 ± 0.23 mJ for S1 sample, to 12.21 ± 0.23 mJ for S2 sample and 15.13 ± 0.17 mJ for S3.

2.4. Confocal Microscopy Analysis

The wheat flour that was analyzed as the first control sample (control 1) contained starch granules
that can be grouped into three categories. The major category (about 60%) was displayed as large,
lenticular or disc shaped granules with a diameter > 10 µm. Approximately 30% of the wheat starch
granules were spherical, medium-sized (3–10 µm), while 10% were really small grains (under 3 µm)
with irregular forms (as it can be seen in Figure 2a). The heterogeneity of the wheat flour starch granules
could be attributed to the wheat variety (soft or hard wheat), the amylose content, and especially
the moment in which is formed during anthesis or their different times of formation during grain
development [34–38]. There are also many studies that confirm the presence of amylose in the
peripheral region of the starch granules as it was likewise assessed in our study. As such, in Figure 2a
it can be observed an interaction between the lipophilic dye molecules and some granules that
afterwards displayed a green border whereas in the central location (in the hilum) longer amylopectin
chains were noticed to form several inclusion complexes with the ligands as it was also suggested by
Manca et al. [39].
Molecules 2018, 23, x FOR PEER REVIEW 6 of 15

amylopectin chains
Molecules 2018, 23, x FORwere
PEER noticed
REVIEW to form several inclusion complexes with the ligands as it was6 also
of 15
suggested by Manca et al. [39].
amylopectin chains
Molecules 2018, 23, 3047 were noticed to form several inclusion complexes with the ligands as it was6also
of 15
suggested by Manca et al. [39].

(a) (b1) (b2)

Figure 2. Confocal laser scanning microscopy images of control 1—wheat flour (a), and control
(a) (b1) (b2)
2—black rice flour (b1 and b2).
Figure
Figure 2. Confocallaser
2. Confocal laserscanning
scanning microscopy
microscopy images
images of control
of control 1—wheat
1—wheat flour
flour (a), and(a), and 2—black
control control
The starch granule sizes of the black rice flower (control 2) were somewhere around 2–10 μm,
2—black
rice flourrice
(b1 flour
and (b1
b2). and b2).
similar to the results obtained by BeMiller & Whistler [40]. Rice starch granules were polygonal,
The
Thestarch
irregular starch
in shape granule
granule
[41] withsizes
sizes of
ofthe
sharp the black
blackrice
angles, riceflower
and flower
without (control
(control 2)2)were
any obvious were somewhere
somewhere
concentric around
around2–10
striations, 2–10μm,
hilum µm,
or
similar
similar to
to the
the results
results obtained
obtained by
by BeMiller
BeMiller &
& Whistler
Whistler [40].
[40]. Rice
cleft (Figure 2(b1)), most of them were grouped into large aggregates as can be seen in Figure 2(b2). Rice starch
starch granules
granules were
were polygonal,
polygonal,
irregular
same in
irregular
The in shape
shape [41]
characteristics [41] with
weresharp
with sharp angles,
angles, and
also reported and without
without any any obvious
by Leewatchararongjaroen obvious concentric
concentric
& Anuntagool striations,
[42]. hilum
striations, hilum or or
cleft The
cleft(Figure
(Figure 2(b1)),analysis
confocal2(b1)), most ofof
most of them
the were
them were
muffins grouped
grouped
samples into
into large aggregates
large
displayed aggregates
a much greateras can be
as can be seen in Figure
seen
complexity in Figure
due to 2(b2).
2(b2).
the
The
The same
same characteristics
characteristics were
were also
also reported
reported by
by Leewatchararongjaroen
Leewatchararongjaroen
different biochemical composition of the ingredients that were used for the recipe (butter, sugar, &
& Anuntagool
Anuntagool [42].
[42].
eggs The
andconfocal
The confocal
wheat flour analysis
analysis andof the
the muffins
ofblack muffins
rice floursamples
samples displayed
displayed
in variable aa much
much greater
proportions). greater
It wascomplexity
complexity due
dueto
more difficult to the
the
to
different
different biochemical
biochemical composition
composition ofofthe
the ingredients
ingredients that
that were
distinguish the components in the cooked samples, possibly as a result of the complex interactionswere used
used for
for the
the recipe
recipe (butter,
(butter, sugar,
sugar,
eggs
between andthe
eggs and wheat
wheat flour
flour
gelatinized and and
orblack black
rice rice
expanded flour flour in variable
in variable
starch, denatured proportions).
proportions).
proteins It was
and It was
more
lipids. more
difficult
When todifficult
only distinguish
the wheat to
distinguish
the
flourcomponents
was used the components
(S1 sample), in
in the cooked in the
the cooked
texture samples,
samples, possibly as
of the baked possibly
a result of
dough,as alarge
the resultwheat
complex of thestarch
complex
interactions intactinteractions
between
granules the
between
gelatinized the gelatinized
or expanded or expanded
starch, denatured starch, denatured
proteins and proteins
lipids.
were also observed, the granules being isolated or grouped into the complex protein matrix. The and
When lipids.
only the When
wheat only
flour the
was wheat
used
size
flour
of the was
(S1 sample),
isolatedusedparticles
in (S1 sample),
the texture of in the texture
the
was variable, baked dough,
from 9.56 of the
large bakedμm,
to 43.44 wheat dough,
starch
and the large
intact wheat
granules
largest starch intact exceeded
were
conglomerates also granules
observed,
were
100 μmalso(Figure
the granules observed,
being 3(S1)).the granules
isolatedBy or beinginto
grouped
increasing isolated
the the or grouped
complex
proportion protein
of intomatrix.
black the
ricecomplex
The size
flour, theprotein
of matrix.
the isolated
glucidic The size
particles
components
of the
was isolated
variable, particles
from 9.56 was
to variable,
43.44 µm, from
and 9.56
the to
largest43.44 μm, and
conglomerates
displayed a more obvious tendency towards aggregation so that around the large granules of the the largest
exceeded conglomerates
100 µm exceeded
(Figure 3(S1)).
100
wheatμmstarch
(Figure
By increasing the3(S1)).
gatheredproportionBy increasing
small of black rice
bunches the proportion
flour, rice
of black of black
the glucidic
starch rice flour,
components
granules the glucidic
that displayed
come witha morecomponents
the obvious
intake of
displayed
tendency a more
towards obvious
aggregation tendency
so that towards
around aggregation
the large so
granules
anthocyanins (in green) (Figure 3(S2)). Confocal images taken for the muffins prepared with simple that around
of the the
wheat large
starch granules
gathered of the
small
wheat
bunches
black starch
rice offlour gathered
black rice starch
frequently small bunches
granules
showed thatofclusters
huge comeblack withrice
(overthestarch
intake
200 μmgranulessize)that
ofinanthocyanins come
consisting with
(in green) the
of starch intake
(Figure of
3(S2)).
granules,
anthocyanins
Confocal
most images
of them having (in green)
taken (Figuredue
for
expanded the 3(S2)).
muffins Confocal
prepared
to the cooking images
with taken for
simple
temperature black the muffins
(aboutrice 80 μm inprepared
flour frequently
diameter) with
andsimple
showed huge
at the
black rice
clusters flour
(over frequently
200 µm in showed
size) huge
consisting clusters
of starch (over
granules,200
same time being strongly colored in green due to the presence of anthocyanins, as it can be seenμm
most in size)
of them consisting
having of starch
expanded granules,
due to the
in
most of
cooking them
Figure 3(S3). having
temperature expanded
(about 80 due
µm to
in the cooking
diameter) and temperature
at the same (about
time 80
being μm in diameter)
strongly colored and in at the
green
same
due totime
the being
presence strongly colored in green
of anthocyanins, as it can duebetoseen the in presence of anthocyanins, as it can be seen in
Figure 3(S3).
Figure 3(S3).

S1 S2 S3
Figure 3. Confocal laser scanning microscopy images of muffin samples: (S1) (muffins with wheat
S1 S2 S3
flour), (S2) (muffins with 1:1 wheat and black rice flour) and (S3) (muffins with black rice flour).
Molecules 2018, 23, x FOR PEER REVIEW 7 of 15

Figure 3. Confocal laser scanning microscopy images of muffin samples: (S1) (muffins with wheat
flour), (S2) (muffins with 1:1 wheat and black rice flour) and (S3) (muffins with black rice flour).
Molecules 2018, 23, 3047 7 of 15

Our results were similar to those obtained by Malik et al. [43]. It has been found that by
replacing the wheat
Our results wereflour with
similar rice flour
to those in pastries
obtained or baked
by Malik goods,
et al. [43]. thebeen
It has firmness
foundand
thatthe sensorial
by replacing
attributes of freeze-thawed cake are improved due to a low amylose content of rice
the wheat flour with rice flour in pastries or baked goods, the firmness and the sensorial attributes flour [44].
Furthermore,
of freeze-thawedblack rice
cake areflour also brings
improved due toadditional bioactive
a low amylose contentcompounds such
of rice flour as Furthermore,
[44]. anthocyanin
pigments that are valuable in improving the food functionality.
black rice flour also brings additional bioactive compounds such as anthocyanin pigments that are
valuable in improving the food functionality.
2.5. Anthocyanin in Vitro Digestibility
2.5. Anthocyanin
To evaluateinthe Vitroanthocyanins
Digestibility in vitro digestibity of new formulated muffins, simulated
digestions conditions
To evaluate were applied.
the anthocyanins in The
vitrodigestion pattern
digestibity of new offormulated
formulatedmuffins,
muffinssimulated
is given indigestions
Figure 4.
conditions were applied. The digestion pattern of formulated muffins is given in Figure 4. sample
As can be seen from Figure 4a, the maximum release of anthocyanins registered for the S3 As can
wasseen
be 14.23
from± 1.02%
Figureafter 120maximum
4a, the min of reaction.
release ofTheanthocyanins
digestion ofregistered
S3 samples forwas limited
the S3 sample with a
was
maximum
14.23 release
± 1.02% afterof1207.22min
± 0.69% after 120
of reaction. Themin of reaction.
digestion The
of S3 results was
samples presented inwith
limited Figure 4b during
a maximum
duodenal
release digestion
of 7.22 ± 0.69% revealed thatmin
after 120 the of
anthocyanin
reaction. Therelease was
results faster in in
presented theFigure
case of
4bS3 compared
during with
duodenal
S2. From revealed
digestion our results,that it
theseems that less
anthocyanin than was
release 26%faster
of thein anthocyanins in S2 andwith
the case of S3 compared 18%S2.inFrom
S3 were
our
retaineditinseems
results, the formulated muffins
that less than 26%during
of thein vitro digestion.
anthocyanins in S2 and 18% in S3 were retained in the
Therefore,
formulated it canduring
muffins be appreciated that anthocyanins were slowly released from the muffins under
in vitro digestion.
simulated digestion
Therefore, it can conditions.
be appreciated Ourthat
results are similar
anthocyanins werewith those
slowly reported
released frombytheSari et al.under
muffins [44],
suggesting that curcumin is released slowly from the nanoemulsion under simulated
simulated digestion conditions. Our results are similar with those reported by Sari et al. [45], suggesting digestion
conditions.
that curcuminOurisinreleased
vitro digestibility
slowly from results
the support a slowlyunder
nanoemulsion releasesimulated
of anthocyanins
digestionfrom the food
conditions.
matrices
Our during
in vitro simulated
digestibility gastric
results digestion
support and arelease
a slowly significant release of thefrom
of anthocyanins bioactive compounds
the food matrices
into thesimulated
during gut. gastric digestion and a significant release of the bioactive compounds into the gut.

(a) (b)
Figure 4. The
Figure 4. The patterns
patternsofofgastric
gastric(a)(a) and
and duodenal
duodenal (b)(b) digestion
digestion of formulated
of formulated muffins
muffins S2 (muffins
S2 (muffins with
with
1:1 1:1 wheat
wheat and black
and black rice flour)
rice flour) and S3 and S3 (muffins
(muffins with black
with black rice flour).
rice flour).

2.6. Shelf-Life Assessment

2.6. Shelf-Life Assessment
To evaluate the phytochemicals and antioxidant activity and color stability in the newly
◦ for 21 days. At every seven
To evaluate the
formulated matrix, thephytochemicals and antioxidant
samples were stored activity
at a temperature andCcolor
of 25 stability in the newly
formulated
days, matrix, the
the following samples were
parameters were measured:
stored at a Total
temperature of 25°Cflavonoids
polyphenolic, for 21 days.
andAtanthocyanins
every seven
days, the following parameters were measured: Total polyphenolic, flavonoids and anthocyanins
content, antioxidant activity, color parameters and molds and yeasts. Data from Figure 5 showed the
content, antioxidant activity, color parameters and molds and yeasts. Data from Figure 5 showed
total anthocyanin content, total polyphenols, total flavonoids content and antioxidant activity the
changes
total
duringanthocyanin content,The
the storage period. total polyphenols,
anthocyanin total
content flavonoidsdecreased,
significantly content and antioxidant
up to 50% in S2 andactivity
33%
changes during
in S3 in the first the storage
14 days period. The
of storage, anthocyanin
whereas degradationcontent significantly
continued up to decreased,
68% and 39%, up to 50% in S2
respectively
and 33% in S3 in the first 14 days of storage, whereas degradation continued up to 68% and 39%,
after 21 days, probably due to degradation reactions (Figure 5a).
respectively after 21 days, probably due to degradation reactions (Figure 5a).
Molecules 2018, 23, 3047 8 of 15

Figure 5. The retention in anthocyanis content (a), total polyphenols (b), total flavonoids (c) and
antioxidant activity (d) of muffins during storage at a temperature of 25 ◦ C for 21 days.
Molecules 2018, 23, 3047 9 of 15

It can be noticed that the anthocyanin’s degradation was more significant in S2 compared to S3,
probably due to the higher concentration of the polyphenolic compounds, which exhibited a protective
action. Regarding the total polyphenolic content in muffins during storage, the decreases were up
to 50% (S2) and 45% (S3), and for flavonoids, up to 42% (S2) and 22% (S3) (Figure 5b,c). However,
a slow decrease in antioxidant activity was found in all samples (Figure 5d). Therefore, after 14 days
of storage, antioxidant activity decreases by 11% and 9% in S2 and S3, respectively, and by 33% and
15% after 21 days of storage. As expected, the decrease in antioxidant activity was lower in S3 when
compared with S2, due to the higher concentration of polyphenolic compounds. However, it seems
that the anthocyanin degradation does not significantly affect the antioxidant activity in all tested
samples. Malvidin stability in the anthocyanin enriched bun after 21 days at room temperature was
significantly lower than those of buns stored for 7 days. However, Karakaya et al. [29] reported that
storage of 21 days at room temperature did not cause huge losses in anthocyanin contents of the bun
and biscuits.
Table 5 shows the variation of color parameters. A slight increase in L * values can be observed
with increasing storage time for all samples, likely due to anthocyanin degradation. Significant
differences in brightness (p < 0.05) can also noticed, both in terms of samples and period of storage.
The control sample (S1) had a * value close to 0 with no variation during storage time, while for S2 and
S3 samples the a * value increased. Our results are in line with ones reported by Ursache et al. [46].

Table 5. Colorimetric analysis of muffins.

S1 S2 S3
Storage Period,
Colorimetric Parameters
Days
L* a* b* L* a* b* L* a* b*
80.41 ± 0.06 ± 0.001 51.83 ± 27.71 ± 8.47 ± 1.08 7.31 ± 0.41 19.6 ± 3.58 6.53 ± 0.95 1.49 ± 0.14
0
9.13 c,d b,c 1.15 a,b 0.15 b,c,d a,b,c,d a,b,c c ab a,b,c

88.02 ± 0.10 ± 0.001 60.11 ± 29.42 ± 9.10 ± 1.63 8.40 ± 0.83 23.9 ± 1.96 7.30 ± 1.30 1.71 ± 0.26
7
0.83 a b 4.96 b,c 0.30 d a,b a b,c a,b,d a,b,c

110.41 ± 0.18 ± 0.011 73.25 ± 35.80 ± 10.30 ± 9.20 ± 0.94 28.8 ± 2.49 11.6 ± 0.22 2.25 ± 0.66
14
5.25 a,b,c,d b 2.49 d 73.74 a,b 1.84 a c d c a

119.63 0.21 ± 75.53 ± 4.31 38.51 ± 1.41 11.11 ± 9.62 ± 0.31 30.1 ± 3.63 13.2 ± 0.95 3.51 ± 0.51
21
±4.60 b,c,d 0.057 b a,b a,b,c 1.10 a,b,c,d b,c a,b a,b b,c,d

Values with different letters in the same column are significantly different (p < 0.05) (L *—lightness, a *—redness,
b *—yellowness).

The b * values which denote a yellow color of the samples, had higher values for S1 and lower S3
baked with only black rice flour.
From microbiological point of view the results suggested that value-added muffins are
microbiologically satisfactory during the accelerated storage test compared to control (Table 6).

Table 6. Yeasts and molds during storage (colony forming unit CFU/g).

Storage Period, Days

Samples
0 7 14 21
S1 <10 1.33·× 102 ± 0.13 2.59 × 103 ± 0.08 5.16 × 105 ± 1.10
S2 <10 <10 <100 <100
S3 <10 <10 <10 <100

3. Materials and Methods

3.1. Materials and Chemicals

2,2-Diphenyl-1-picrylhydrazyl (DPPH), 6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic
acid (Trolox), Folin-Ciocalteu reagent, sodium carbonate, sodium hydroxide, sodium acetate, sodium
nitrite, potassium chloride, aluminum chloride, gallic acid, catehine, potassium persulfate, formic
acid, ethanol and methanol (HPLC grade), cyanidin and peonidin standards were obtained from
Molecules 2018, 23, 3047 10 of 15

Sigma Aldrich Steinheim, Germany. For muffin preparation, coconut butter with 80% fat content,
brown sugar, hen eggs, wheat flour and black rice (Oryza sativa L. ssp. Japonica, Nerone variety, Italy)
were purchased from the local supermarket, Galati, Romania.

3.2. Batters and Muffins Preparation

Preparation of the muffin batter was performed with the following steps: The coconut butter
was mixed continuously with salt and brown sugar until the sugar is dissolved and a foam is formed;
then the eggs were added, alternately with wheat flour (S1 sample, considered as control), wheat and
black rice flour (1:1) (S2 sample), black rice flour (S3 sample), and finally the baking powder was
added. The batter was mixed for 10 min at 300 rpm in order to get a uniform composition. Finally,
the batter was filled in paper cups and baked at 185 ◦ C for 25 min in a convection oven with forced
air circulation. Products from each recipe were produced, baked and analyzed in two independent
batches. The muffins were packed in vacuum bags at 800 mbar and stored at a temperature of 25 ◦ C
for 21 days.

3.3. HPLC Technique

The chromatographic analysis of the anthocyanins from black rice flour was performed as
described earlier by Bolea et al. [24]. HPLC analysis was performed using a Surveyor HPLC system,
controlled by an Xcalibur software system (Finnigan Surveyor LC, Thermo Scientific, Waltham, MA,
USA). The anthocyanins detected in black rice flour were analyzed at a wavelength of 520 nm.
The column used for this analysis was a C18 BDS Hypersil (150 mm × 4.6 mm, 5 mm). The gradient
used for the elution of the anthocyanins was: 0–20 min: 9–35% (A), 20–30 min: 35% (A), 30–40 min:
35–50% (A), 40–45 min: 50–9% (A), with an injection volume of 10 µL, and the flow rate maintained at
1.000 mL/min.

3.4. Sensorial Analysis

A panel consisting of 11 different panelists aged between 29–50 years old performed the
sensorial analysis of gluten free and added value muffins according to seven point hedonic scale.
The panelists assessed the muffins samples for color (light to dark), surface humidity (none to very
high), cross section appearance (non uniform to uniform), denseness (dense to airy), fracturability
(low to very high), hardness (low to very hard), cohesiveness (none to tight mass), moistness of mass
(low to very high), taste (dislike very much to like extremely), and overall likeability of the product
(dislike to like extremely). Muffins samples were served in random order to panelists on white papers.
Water was used for mouth rising before and between samples.

3.5. Physico-Chemical, Phytochemical and Microbiological Analysis of Muffins

Standardized and validated laboratory methods were used to determine the physico-chemical
characteristics of muffins, in terms of moisture, fat, protein, carbohydrates, ash and energy value.
The phytochemical content of extract (total polyphenols, total flavonoids, total monomeric
anthocyanins) and antioxidant activity were determined as described by Turturică et al. [47]. In brief,
1 g of muffin was crushed and then mixed with 8 mL of 70% ethanol and 1 mL HCl 1N. The mixture
was stirred for 8 h at room temperature on an orbital shaker at 150 rpm. After centrifugation at
6000 rpm for 10 min, the supernatant was collected and concentrated at 40 ◦ C to dryness under
reduced pressure (AVC 2-18, Christ, UK). The extracts were redissolved in 2 mL of MiliQ water and
used for phytochemical analysis.

3.6. Microbiological Assessment

The microbiological shelf life of muffins was evaluated by monitoring fungal growth over 1, 7,
14 and 21 days of storage. The standard pour plate method described by Ursache et al. [46] was used to
Molecules 2018, 23, 3047 11 of 15

count the number of yeasts and molds. The results were expressed as CFU/g of sample. Each sample
was analysed in duplicate on each day of storage.

3.7. Textural Analysis of Muffins

The textural analysis was achieved by the Texture Profile Analysis (TPA) Method, using the
Brookfield CT3-1000 analyzer. The sample preparation consisted of removing the rind of the muffins
and cutting the core in cube shapes with the sight length of 15 mm. Then, a double compression
was applied at a distance of 10 mm, at a speed of 1 mm/s, with no holding time between the two
compression cycles. The trigger load was 0.02 N and the load cell was 1000 g. The compression was
performed using an acrylic cylinder (diameter ~ 24.5 mm, height ~ 35 mm) (TA11/1000). The data
were recorded and processed using the TexturePro CT V1.5 software. For each sample, five tests were
performed. The textural parameters determined by TPA were firmness, cohesiveness, springiness
and chewiness.

3.8. Confocal Microscopy Analysis

The comparative confocal analysis of the samples was performed in order to capture the structural,
textural and compositional changes of the experimental variants, while for the control samples simple
wheat flour and black rice flour were used. The confocal microscope that was used for the analysis
is a Zeiss Axio Observer Z1 inverted microscope model (LSM 710) equipped with a laser scanning
system: Diode laser (405 nm), Ar laser (458 nm, 488 nm and 514 nm), DPSS (561 nm pumped solid
state diodes), and HeNe-laser (633 nm). The strong anthocyanin absorption in the visible range was
registered between 465 nm and 550 nm [48] with an in vivo peak, between 537 nm and 542 nm [49].
The distribution of the pigments into the protein matrix was observed at the excitation wavelength
of 488 nm and by applying the FS38 filter, whereas the emission was collected between 500–600 nm.
The powder that was stained with two dyes, DAPI (1 µg/mL) and Red Congo (40 µM), in a ratio of
3:1:1, was observed using a 40x apochromatic objective (numerical aperture 1.4) and the FS49 and FS15
filters. The 3D images were rendered and analyzed with ZEN 2012 SP1 software (Black Edition).

3.9. In Vitro Digestibility

In vitro digestibility was performed by using a method described by Oancea et al. [50]. Briefly,
1 g of muffins (S2 and S3) was mixed with Tris-HCl buffer (10 mM, pH 7.7). The gastric digestion
was performed by the addition of a simulated gastric fluid (SGF), which consisted of porcine pepsin
(40 mg/mL in 0.1 M HCl) that was added to the initial mixtures in a ratio of 0.5 g of pepsin per
100 g of sample and the pH was adjusted to 2.0 with 6 M HCl. Regarding the enteric digestion
step, the simulated intestinal fluid (SIF) consisted of a mixture containing pancreatin (2 mg/mL)
and afterwards the resulting mixture was neutralized to pH 5.3 with 0.9 M sodium bicarbonate.
The pH of the system was adjusted to 7.0 with 0.1M NaOH, prior to the incubation of the samples
for 2 h. The incubation was performed in an SI—300R orbital shaking incubator (Medline Scientific,
Oxfordshire, UK), at 100 rpm and 37 ◦ C. The total anthocyanin’s content of the samples was measured
at every 30 min during the in vitro digestion.

3.10. Colorimetric Study

The color parameter values of the muffins were measured using the Minolta CR-410 Chroma
Meter (Konica Minolta, Osaka, Japan) as described by Ursache et al. [46]. The results were expressed as
L * (a lower value indicates a darker color, black: L * = 0 and white: L * = 100), a * (indicate the balance
between red (>0), green (0) and blue (<0) color), and b * (the balance between yellow (>0) and blue (<0)
color). All the measurements were performed in triplicates.
Molecules 2018, 23, 3047 12 of 15

3.11. Storage Stability

No preservatives were used in the recipe formulation of the gluten free and added value
muffins. Therefore, an accelerated storage stability test was performed during a period of 21 days at
temperature of 25 ◦ C. Duplicate samples were considered for determination of the molds and yeast,
total polyphenolic content, total flavonoids and anthocyanins content, antioxidant activity and color at
every 7 days.

3.12. Statistical Analysis of Data

Minitab 18 statistical processing software was employed to perform the statistical evaluation of
the sensorial data. First, the data were checked for normality and homoscedasticity using the Ryan
Joiner test and the Bartlett test. Then, one-way ANOVA was used to identify if panelists detect any
differences between samples considering a significance level of 0.05. Post-hoc analysis via Dunnett
multiple comparisons with a control were performed when appropriate. All data reported in this study
represent the averages of duplicate analyses and is reported as mean ± standard error of the mean.

4. Conclusions
The muffins baked with black rice flour presented a high anthocyanin content and antioxidant
activity compared with the control sample baked with wheat flour. The textural analysis suggested
that the addition of black rice caused the increase of firmness, springiness and chewiness, while the
cohesiveness was lower compared with the control sample and was related to a weaker binding
between the constituents. Confocal images taken for the muffins baked with black rice flour showed
huge clusters (over 200 µm in size) consisting of starch granules, most of them having expanded due
to the cooking temperature (about 80 µm in diameter) and at the same time being strongly colored in
green due to the presence of anthocyanins. Sensorial analysis showed that all samples were appreciated;
some panelists even perceived that samples with black rice flour contain pleasant crispy particles
compared with the control sample.
Storage stability of muffins revealed a decrease of anthocyanins, antioxidant activity and color
parameters. The added value products showed a microbiological stability during the accelerated
storage period, probably due to the presence of polyphenolic compounds. These results indicated that
value added muffins obtained with black rice flour could be an alternative for people suffering from
gluten intolerance, whereas proving a significant amount of polyphenolic content, with potentially
beneficial effects on human health.

Author Contributions: G.R. and N.S. conceived and designed the experiments and reviewed the final manuscript;
C.C.; C.M.; D.G.A.; L.D. and G.H. performed the experiments, analyzed the data and prepared the manuscript;
M.T. performed the HPLC analysis; E.E. and V.B. performed the confocal microscopy analysis; G.R. reviewed the
final manuscript.
Funding: This research received no external funding.
Acknowledgments: The Integrated Center for Research, Expertise and Technological Transfer in Food Industry
is acknowledged for providing technical support (www.bioaliment.ugal.ro). The authors are grateful for the
technical support offered by the Grant POSCCE ID 1815, cod SMIS 48745 (www.moras.ugal.ro).
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Matos, M.E.; Sanz, T.; Rosell, C.M. Establishing the function of proteins on the rheological and quality
properties of rice based gluten free muffins. Food Hydrocoll. 2014, 35, 150–158. [CrossRef]
2. Martínez-Cervera, S.; Sanz, T.; Salvador, A.; Fiszman, S.M. Rheological, textural and sensorial properties of
low-sucrose muffins reformulated with sucralose/polydextrose. LWT Food Sci. Technol. 2012, 45, 213–220.
[CrossRef]
Molecules 2018, 23, 3047 13 of 15

3. Sanz, T.; Salvador, A.; Baixauli, R.; Fiszman, S.M. Evaluation of four types of resistant starch in muffins. II.
Effects in texture, colour and consumer response. Eur. Food Res. Technol. 2009, 229, 197–204. [CrossRef]
4. Gujral, N.; Freeman, J.H.; Thomson, A. Celiac disease: Prevalence, diagnosis, pathogenesis and treatment.
World J. Gastroenterol. 2012, 18, 6036–6059. [CrossRef] [PubMed]
5. Rai, S.; Kaur, A.; Singh, B. Quality characteristics of gluten free cookies prepared from different flour
combinations. J. Food Sci. Technol. 2014, 51, 785–789. [CrossRef] [PubMed]
6. Shevkani, K.; Singh, N. Influence of kidney bean, field pea and amaranth protein isolates on the characteristics
of starch-based gluten-free muffins. Int. J. Food Sci. Technol. 2014, 49, 2237–2244. [CrossRef]
7. Feighery, C.F. Coeliac disease. Br. Med. J. 1999, 319, 236–239. [CrossRef]
8. Man, S.; Păucean, A.; Muste, S.; Pop, A. Studies on the formulation and quality characteristics of gluten free
muffins. J. Agroaliment. Process. Technol. 2014, 20, 122–127.
9. Bardella, M.T.; Fredella, C.; Prampolini, L.; Molteni, N.; Giunta, A.M.; Bianchi, P.A. Body composition and
dietary intakes in adult celiac disease patients consuming a strict gluten-free diet. Am. J. Clin. Nutr. 2000, 72,
937–939. [CrossRef] [PubMed]
10. Islas-Rubio, A.R.; De la Barca, A.C.; Cabrera-Chávez, F.; Cota-Gastélum, A.G.; Beta, T. Effect of semolina
replacement with a raw: Popped amaranth flour blend on cooking quality and texture of pasta. LWT Food
Sci. Technol. 2014, 57, 217–222. [CrossRef]
11. Sakač, M.; Torbica, A.; Sedej, I.; Hadnađev, M. Influence of breadmaking on antioxidant capacity of gluten
free breads based on rice and buckwheat flours. Food Res. Int. 2011, 44, 2806–2813. [CrossRef]
12. Sedej, I.; Sakač, M.; Mandić, A.; Mišan, A.; Pestorić, M.; Šimurina, O.; Čanadanović-Brunet, J. Quality
assessment of gluten-free crackers based on buckwheat flour. LWT Food Sci. Technol. 2011, 44, 694–699.
[CrossRef]
13. Gularte, M.A.; de la Hera, E.; Gómez, M.; Rosell, C.M. Effect of different fibers on the enrichment of
gluten-free layer cake. LWT Food Sci. Technol. 2012, 48, 209–214. [CrossRef]
14. Gularte, M.A.; Gómez, M.; Rosell, C.M. Impact of legume flours on quality and in vitro digestibility of starch
and protein from gluten-free cakes. Food Bioprocess Technol. Int. J. 2012, 5, 3142–3150. [CrossRef]
15. Omary, M.B.; Fong, C.; Rothschild, J.; Finney, P. Effects of germination on the nutritional profile of gluten-free
cereals and pseudocereals: A review. Cereal Chem. 2012, 89, 1–14. [CrossRef]
16. Park, S.J.; Ha, K.-Y.; Shin, M. Properties and qualities of rice flours and gluten-free cupcakes made with
higher-yield rice varieties in Korea. Food Sci. Biotechnol. 2012, 21, 365–372. [CrossRef]
17. Ronda, F.; Oliete, B.; Gómez, M.; Caballero, P.A.; Pando, V. Rheological study of layer cake batters made
with soybean protein isolate and different starch sources. J. Food Eng. 2011, 102, 272–277. [CrossRef]
18. Schamne, C.; Dutcosky, S.D.; Demiate, I.M. Obtention and characterization of gluten free baked products.
Ciênc. Tecnol. Aliment. 2010, 30, 741–750. [CrossRef]
19. Ujjawal, K.; Kushwaha, S. Black rice. In Black Rice Research, History and Development; Springer International
Publishing AG: Basel, Switzerland, 2016; pp. 21–47, ISBN 978-3-319-30153-2.
20. Chang, K.K.; Kikuchi, S.; Kim, Y.K.; Park, S.H.; Yoon, U.; Lee, G.S.; Choi, J.W.; Kim, Y.H.; Park, S.C.
Computational identification of seed specific transcription factors involved in anthocyanin production in
black rice. Biochip. J. 2010, 4, 247–255. [CrossRef]
21. Adom, K.K.; Liu, R.H. Antioxidant activity of grains. J. Agric. Food. Chem. 2002, 50, 6170–6182. [CrossRef]
22. Bordiga, M.; Gomez-Alonso, S.; Locatelli, M.; Travaglia, F.; Coïsson, J.D.; Hermosin-Gutierrez, I.; Arlorio, M.
Phenolics characterization and antioxidant activity of six different pigmented Oryza sativa L. cultivars
grown in Piedmont (Italy). Food Res. Int. 2014, 65, 282–290. [CrossRef]
23. Melinia, V.; Panfilib, G.; Fratiannib, A.; Acquistuccia, R. Bioactive compounds in rice on Italian market:
Pigmented varieties as a source of carotenoids, total phenolic compounds and anthocyanins, before and
after cooking. Food Chem. 2019, 277, 119–127. [CrossRef]
24. Bolea, C.; Turturica, M.; Stanciuc, N.; Vizireanu, C. Thermal degradation kinetics of bioactive compounds
from black rice flour (Oryza sativa L.) extracts. J. Cereal Sci. 2016, 71, 160–166. [CrossRef]
25. Yawadio, R.; Morita, N. Color enhancing effect of carboxylic acids on anthocyanins. Food Chem. 2007, 105,
421–427. [CrossRef]
26. Hou, Z.; Qin, P.; Zhang, Y.; Cui, S.; Ren, G. Identification of anthocyanins isolated from black rice
(Oryza sativa L.) and their degradation kinetics. Food Res. Int. 2013, 50, 691–697. [CrossRef]
Molecules 2018, 23, 3047 14 of 15

27. Zhang, M.W.; Guo, B.J.; Zhang, R.F.; Chi, J.W.; Wei, Z.C.; Xu, Z.H.; Zhang, Y.; Tang, X.J. Separation,
purification and identification of antioxidant compositions in black rice. Agr. Sci. China 2006, 39, 153–160.
[CrossRef]
28. Zhang, M.W.; Zhang, R.F.; Guo, B.J.; Chi, J.W.; Wei, Z.C.; Xu, Z.H. The hypolipidemic and antioxidative
effects of black rice pericarp anthocyanin in rats. Acta Nutrimenta Sin. 2006, 28, 404–408.
29. Karakaya, S.; Simsek, S.; Tolga Eker, A.; Pineda-Vadillo, C.; Dupont, D.; Perez, B.; Viadel, B.;
Sanz-Buenhombre, M.; Guadarrama Rodriguez, A.; Kertész, Z.; et al. Stability and bioaccessibility of
anthocyanins in bakery products enriched with anthocyanins. Food Funct. 2016, 7, 3488–3496. [CrossRef]
[PubMed]
30. Nemś, A.; Peksa, A.; Kucharska, A.Z.; Sokół-Ł˛etowska, A.; Kita, A.; Drożdż, W.; Hamouz, K. Anthocyanin
and antioxidant activity of snacks with coloured potato. Food Chem. 2015, 172, 175–182. [CrossRef] [PubMed]
31. Barti, P.; Albreht, A.; Skrt, M.; Tremlová, B.; Ošt’ádalová, M.; Šmejkal, K.; Vovk, I.; Ulrih Poklar, N.
Anthocyanins in purple and blue wheat grains and in resulting bread: Quantity, composition, and thermal
stability. Int. J. Food Sci. Nutr. 2015, 66, 514–519. [CrossRef] [PubMed]
32. Demirkesen, I.; Mert, B.; Sumnu, G.; Sahin, S. Rheological properties of gluten-free bread formulations.
J. Food Eng. 2010, 96, 295–303. [CrossRef]
33. Wronkowska, M.; Troszynska, A.; Soral-Smietana, M.; Wolejszo, A. Effects of buckwheat flour (Fagopyrum
esculentum moench) on the quality of gluten-free bread. Pol. J. Food Nutr. Sci. 2008, 58, 211–216.
34. Langeveld, S.M.J.; Van, W.R.; Stuurman, N.; Kijne, J.W.; de Pater, S. B-type granule containing protrusions and
interconnections between amyloplasts in developing wheat endosperm revealed by transmission electron
microscopy and GFP expression. J. Exp. Bot. 2000, 51, 1357–1361. [CrossRef] [PubMed]
35. Bechtel, D.B.; Wilson, J.D. Amyloplast formation and starch granule development in hard red winter wheat.
Cereal Chem. 2003, 80, 175–183. [CrossRef]
36. Wilson, J.D.; Bechtel, D.B.; Todd, T.C.; Seib, P.A. Measurement of wheat starch granule size distribution using
image analysis and laser diffraction technology. Cereal Chem. 2006, 83, 259–268. [CrossRef]
37. Xie, X.J.; Cui, S.W.; Li, W.; Tsao, R. Isolation and characterization of wheat bran starch. Food Res. Int. 2008, 41,
882–887. [CrossRef]
38. Kumar, R.; Kumar, A.; Sharma, N.K.; Kaur, N.; Chunduri, V.; Chawla, M.; Sharma, S.; Singh, K.; Garg, M.
Soft and hard textured wheat differ in starch properties as indicated by trimodal distribution, morphology,
thermal and crystalline properties. PLoS ONE 2016, 11, e0147622. [CrossRef] [PubMed]
39. Manca, M.; Woortman, A.J.J.; Mura, A.; Loos, K.; Loi, M.A. Localization and dynamics of amylose–lipophilic
molecules inclusion complex formation in starch granules. Phys. Chem. Chem. Phys. 2015, 17, 7864–7871.
[CrossRef] [PubMed]
40. BeMiller, J.N.; Whisler, R.L. Starch: Chemistry and Technology, 3rd ed.; Academic Press: New York, NY, USA,
2009; ISBN 9780080926551.
41. Li, W.H.; Bai, Y.F.; Mousaa-Saleh, A.S.; Zhang, Q.; Shen, Q. Effect of high hydrostatic pressure on
physicochemical and structural properties of rice starch. Food Bioproc. Technol. 2011, 5, 2233–2241. [CrossRef]
42. Leewatchararongjaroen, J.; Anuntagool, J. Effects of Dry-Milling and Wet-Milling on Chemical, Physical and
Gelatinization Properties of Rice Flour. Rice Sci. 2016, 23, 274–281. [CrossRef]
43. Malik, A.N.; Gaiani, C.; Fukai, S.; Bhandari, B. X-ray photoelectron spectroscopic analysis of rice kernels and
flours: Measurement of surface chemical composition. Food Chem. 2016, 212, 349–357. [CrossRef]
44. Jongsutjarittam, N.; Charoenrein, S. Influence of waxy rice flour substitution for wheat flour on characteristics
of batter and freeze-thawed cake. Carbohydr. Polym. 2013, 97, 306–314. [CrossRef] [PubMed]
45. Sari, T.P.; Mann, B.; Kumar, R.; Singh, R.R.B.; Sharma, R.; Bhardwaj, M.; Athira, S. Preparation and
characterization of nanoemulsion encapsulating curcumin. Food Hydrocoll. 2015, 43, 540–546. [CrossRef]
46. Ursache, F.M.; Andronoiu, D.G.; Ghinea, I.O.; Barbu, V.; Ionită, E.; Cotârlet, M.; Dumitrascu, L.; Botez, E.;
Râpeanu, G.; Stănciuc, N. Valorizations of carotenoids from sea buckthorn extract by microencapsulation
and formulation of value-added food products. J. Food Eng. 2018, 219, 16–24. [CrossRef]
47. Turturică, M.; Stănciuc, N.; Bahrim, G.; Râpeanu, R. Effect of thermal treatment on phenolic compounds
from plum (Prunus domestica) extracts—A kinetic study. J. Food Eng. 2016, 171, 200–207. [CrossRef]
48. Glover, B.J.; Martin, C. Anthocyanins. Curr. Biol. 2012, 22, 147–150. [CrossRef] [PubMed]
Molecules 2018, 23, 3047 15 of 15

49. Merzlyak, M.N.; Chivkunova, O.B.; Solovchenko, A.E.; Naqvi, K.R. Light absorption by anthocyanins in
juvenile, stressed, and senescing leaves. J. Exp. Bot. 2008, 59, 3903–3911. [CrossRef] [PubMed]
50. Oancea, A.M.; Aprodu, I.; Ghinea, I.O.; Barbu, V.; Ionită, E.; Bahrim, G.; Rapeanu, G.; Stanciuc, N.
A bottom-up approach for encapsulation of sour cherries anthocyanins by using β-lactoglobulin as matrices.
J. Food Eng. 2017, 210, 83–90. [CrossRef]

Sample Availability: Samples of the compounds are available from the authors.

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