Food Chemistry 211 (2016) 281–284

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Short communication

Effect on in vitro starch digestibility of Mexican blue maize anthocyanins

Gustavo A. Camelo-Méndez, Edith Agama-Acevedo, Mirna M. Sanchez-Rivera, Luis A. Bello-Pérez ⇑
Instituto Politécnico Nacional, CEPROBI, Yautepec, Morelos, Mexico

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

Article history: The purpose of this study was to evaluate the effect of blue maize extracts obtained by acid-methanol
Received 21 January 2016 treatment on the nutritional in vitro starch fractions such as: rapidly digestive starch (RDS), slowly diges-
Received in revised form 3 May 2016 tive starch (SDS) and resistant starch (RS) of native and gelatinized commercial maize starch.
Accepted 4 May 2016
Chromatographic analysis (HPLC-DAD/ESI-MS) of blue maize extracts showed the presence of seven
Available online 5 May 2016
anthocyanins, where cyanidin-3-(600 -malonylglucoside) was the main. Blue maize extracts modified
nutritional in vitro starch fractions (decrease of RDS) while RS content increased (1.17 and 2.02 times
Chemical compounds studied in this article:
for native and gelatinized commercial maize starch, respectively) when anthocyanins extracts were
Cyanidin-3-glucoside (PubChem CID:
92131208)
added to starch up to 75% (starch weight). This preliminary observation provides the basis for further
Peonidin-3-glucoside (PubChem CID: suitability evaluation of blue maize extract as natural starch-modifier by the possible anthocyanins-
443654) starch interaction. Anthocyanin extracts can be a suitable to produce functional foods with higher RS con-
Cyanidin 3-(600 -malonylglucoside) tent with potential human health benefits.
(PubChem CID: 44256740) Ó 2016 Elsevier Ltd. All rights reserved.
Cyanidin 3-(300 ,600 -dimalonylglucoside)
(PubChem CID: 44256743)

Keywords:
Blue maize
Anthocyanins
In vitro starch digestibility
Resistant starch

1. Introduction studied. Specifically, interactions between phenolics and carbohy-

In recent years, the development of functional foods has pre-
sented significant trends towards the development of gastroin-
testinal health food. In this respect, the indigestible
carbohydrates play an important role in the colon and which act
as prebiotics, increasing intestinal transit and production of
short-chain fatty acid (Englyst & Englyst, 2005). In food containing
carbohydrates, starch represents the main carbohydrate, providing
physical and chemical characteristics to food and energy after con-
sumption. Starch can be classified in nutritional fractions according
to their hydrolysis rates as: digestible starch (rapid and slowly
digestible starch) and resistant that is not digested in the upper
gastrointestinal tract (Englyst, Kingman, & Cummings, 1992).
Also, there has been a renewed interest in the study of food phe-
nolics due to their health-promoting properties. The biological
activity of dietary phenolics and the possibilities of enhancing
the nutritional properties of plant-derived food have recently been
⇑ Corresponding author.
E-mail address: labellop@ipn.mx (L.A. Bello-Pérez).
drates are mainly related to enhance and delay the absorption
(Bordenave, Hamaker, & Ferruzzi, 2014; Zhang et al., 2014). In
the literature, limited research is available on in vitro bioavailabil-
ity studies that investigate the effect of food matrices on phenolic
and the effect of phenolics in food model systems.
In studies it has been found that phenolic compounds mainly
affect starch digestibility because they inhibit digestive enzymes
(Lo Piparo et al., 2008; Miao et al., 2014). Furthermore, some
authors have reported in foods containing phenolic compounds
and carbohydrates that resistant starch content was increased in
presence of tannins (Mkandawire et al., 2013) and sorghum proan-
thocyanidins (Barros, Awika, & Rooney, 2012).
Currently, the pigmented maize (mainly blue) has received the
most interest for the development of functional foods such as:
tortillas, chips and cereal bars (Bello-Pérez, Flores-Silva, Camelo-
Méndez, Paredes-López, & Figueroa-Cárdenas, 2015; Del Pozo-
Insfran, Brenes, Serna-Saldivar, & Talcott, 2006; Utrilla-Coello,
Agama-Acevedo, Osorio-Díaz, Tovar, & Bello-Pérez, 2011) due to
the presence of phenolic compounds (principally anthocyanins).
In addition to the antioxidant properties of anthocyanins

http://dx.doi.org/10.1016/j.foodchem.2016.05.024
0308-8146/Ó 2016 Elsevier Ltd. All rights reserved.
282 G.A. Camelo-Méndez et al. / Food Chemistry 211 (2016) 281–284

(Aguayo-Rojas et al., 2012) and anti-proliferative activity (Urias-
Lugo et al., 2015) also they could be been associated beneficial
properties of these human consumption and benefits in colon
(Toydemir et al., 2013). However, scarce information exists of the
effect of anthocyanins present in pigmented maize on starch diges-
tion. In the current investigation, we evaluated the effect of Mexi-
can blue maize extracts on the modification of nutritional in vitro
starch fractions in food matrix systems (native and gelatinized
commercial maize starch) to simulate the effect in foods as
tortillas, snacks, etc., elaborated with pigmented maize.
2. Material and methods
2.1. Plant material
and amyloglucosidase (A7095, Sigma Aldrich, St Louis, MO, USA)
enzymes at 37 °C for 120 min. Starch classification was based on
the rate of hydrolysis, RDS (digested within 20 min), SDS (digested
between 20 and 120 min) and RS (undigested after 120 min).
2.5. Statistical analysis
For the statistical treatment of the data the Statgraphics Plus
version 5.1Ò (Manugistics, Inc., Rockville, MA, USA) was used.
One-way analysis of variance (ANOVA) was employed to establish
significant differences between: (a) type of extracts used (from
blue and white maize flours) (b) starch model system (raw and
cooked). In all cases, statistically significant level was considered
at p < 0.05.

Pigmented blue maize was obtained from the experimental 3. Results and discussion
field of INIFAP Texcoco, Edo. México, México during 2012–2013
season. The flour was obtained as reported previously (Camelo- 3.1. Polyphenol identification
Méndez & Bello-Pérez, 2014). Commercial white maize flour
(NatureloÒ, Queretaro, Mexico) was used as control. For in vitro The anthocyanins identified in blue maize are showed in Table 1.
starch digestibility essays normal maize starch (Ingredion México Seven peaks were monitored by an HPLC chromatogram at 520 nm
S.A, México) was used. (Abdel-Aal, Young, & Rabalski, 2006; Lopez-Martínez et al., 2009).
These pigments were identified by HPLC-DAD/ESI-MS, based on
2.2. Preparation of extracts the comparison of UV–vis absorption maxima, their retention
times, elution order and mass spectra (Abdel-Aal et al., 2006;
Samples (5 g of maize flour) were homogenized in 15 mL of an Montilla, Hillebrand, Antezana, & Winterhalter, 2011). The antho-
organic mixture is methanol-water (75% methanol acidified with cyanins identified in this study are similar than those reported in
5% of HCl [1 N]), kept under shaking for 1 h and further centrifuged other blue maize genotypes (Abdel-Aal et al., 2006; Urias-Lugo
at 3000g for 10 min at 4 °C. The resulting alcoholic extract was et al., 2015), base on majority peak area (46.5%), cyanidin-3-(600 -
filtered and the solvent was evaporated under nitrogen malonylglucoside) was the most abundant pigment in blue maize.
atmosphere. Maize extracts were prepared fresh prior to each It is noteworthy that chemical differences between blue and
experiment to prevent compounds degradation; extracts were white maize are mainly related to pigment presence (Montilla
rota-evaporated and re-suspended in 15 mL distilled water to et al., 2011). We found higher total phenolic content (p < 0.05) in
avoid interference from solvent on in vitro essays. blue maize than in white maize (unpublished data), similar results
have been reported in food products elaborated with blue maize
(Aguayo-Rojas et al., 2012; Bello-Pérez et al., 2015). The blue maize
2.3. HPLC-DAD/ESI-MS analysis of blue maize anthocyanins
used in this study presented total anthocyanins content of 0.2 mg/
g (Camelo-Méndez & Bello-Pérez, 2014). This is important issue
Analyses were carried out in an HPLC-MS 2020 chromatograph
because blue maize anthocyanins presented antioxidant
(Shimadzu LC–MS, Tokyo, Japan) Agilent Technologies, Palo Alto,
(Aguayo-Rojas et al., 2012; Camelo-Méndez & Bello-Pérez, 2014;
CA, USA) equipped with a diode-array detector, which was set to
Lopez-Martínez et al., 2009) and anti-proliferative properties
scan from 200 to 800 nm, and a C-18 column (Lichrospher 100,
against cancer cells (Urias-Lugo et al., 2015), which are important
250  4 mm, RP 18, 5 lm, Merck, Darmstadt, Germany) using an
features in search of functional ingredients for the food industry.
injection volume of 20 lL. The solvents were 5% formic acid in
deionized water (solvent A) and methanol (solvent B) at the fol-
lowing gradient: 0–2.5 min, 6% B, 2.5–6 min 14% B, 6–15 min 3.2. In vitro digestion of starch in the presence of maize polyphenols
15.8% B, 15–21.5 min 17% B, 21.5–30 min 20% B, 30–39 min 23%
B, 39–40 min 39% B, 40–44 min 42% B, 44–46 min 45% B, 46– Polyphenols were added in different levels (0, 25, 50, 75 and
56 min 100% B, 56–60 min 0% B. The flow-rate was 1 mL/min and 100% of starch content used in the enzymatic assay) and the differ-
the temperature of the column was set at 35 °C. Detection was also ent starch fractions (RDS, SDS and RS) were evaluated (Fig. 1). Is
performed in an UV–vis SPD-M20A equipped with an ESI source important to highlight that the ratio phenolic/starch content,
and a single quadrupole-ion trap mass analyser, which was con- remained in the range reported in blue maize-based foods, and that
nected to the HPLC equipment via the DAD cell outlet. Antho-
cyanins were identified by their retention time, UV–vis spectra Table 1
and mass spectra, as well as by comparison with our data library Anthocyanins determined by HPLC-DAD/ESI-MS analysis.
and standards when were available.
Peak kmax Major Relative Anthocyanins structures with
(nm) ionz (m/z) area (%) closely matching MS profiles
2.4. In vitro starch digestion in the presence of maize polyphenols 1 515 447 16.7 Cyanidin-3-glucoside
2 523 633 1.6 Peonidin-3-(dimalonyl glucoside)
The in vitro digestion of normal maize starch (raw and gela- 3 523 461 3.4 Peonidin-3-glucoside
tinised) was performed using the Englyst́s method (Englyst et al., 4 523 621 5.3 Cyanidin-3-glucoside-2-
malonylglucoside
1992) with modifications (Hoyos-Leyva, Bello-Pérez, Agama- 5 522 533 46.5 Cyanidin-3-(600 -malonylglucoside)
Acevedo, & Alvarez-Ramirez, 2015). Briefly, starch with different 6 523 575 10.6 Peonidin-3-(600 -
levels of polyphenols (0, 25, 50, 75, and 100 as a percentage of ethylmalonylglucoside)
the starch) (Mkandawire et al., 2013) were incubated with porcine 7 522 621 15.9 Cyanidin 3-(300 ,600 -
pancreatic a-amylase (A3176, Sigma Aldrich, St Louis, MO, USA) dimalonylglucoside)
 G.A. Camelo-Méndez et al. / Food Chemistry 211 (2016) 281–284 283

Uncooked Cooked
100 100
* aa a a *a *a
*
80 80 b b
bc c
RDS (%)

RDS (%)
60 60

40 40
aa a a
b b a b a b
20 20

0 0
0 25 50 75 100 0 25 50 75 100
Extract (%) Extract (%)

100 100

80 80
SDS (%)

SDS (%)
60 60

40 40

20 20

0 0
0 25 50 75 100 0 25 50 75 100
Extract (%) Extract (%)

100 100
* * * *
80 80

60 60
RS (%)

RS (%)

b cd d
40 ab ab ab 40
a a a a a a bc
aa ab
20 20 a a a a
0 0
0 25 50 75 100 0 25 50 75 100
Extract (%) Extract (%)

Fig. 1. Starch digestibility in the presence of extractable polyphenols of blue and white maize. Data represented by mean ± SEM (standard error of mean) of three replicates.
Different letters indicate significant differences (p < 0.05) between amounts of extract used of each sample. Significant differences between the effect of polyphenols from
blue and white maize in starch digestibility are denoted by lines where ⁄ p < 0.05. Polyphenols from blue maize are represented with a black bar and polyphenols from white
maize are represented with a white bar.

it was previously established that 100% of the extract (164 ± 14 mg
of gallic acid/g of DM) (unpublished data), inhibited over 90% of
alpha-amylase activity. This suggests that polyphenols hamper
starch hydrolysis, which would explain the decrease of RDS. There
were no significant differences (p < 0.05) between the SDS content
of the samples (starch with or without the addition of polyphenol
extract).
In contrast, RS content was importantly increased in relation to
the amount of extract added. The highest extract addition rates (75
and 100%) promoted significant differences in RDS and RS content
in comparison with the control sample (p < 0.05) on both uncooked
and cooked samples. RS content increased (1.17 and 2.02 times for
native and gelatinized commercial starch, respectively) when blue
maize extract were added (>75% of starch weight). Similar pattern
was reported with sorghum catechins on starch digestion, showing
decreased of RDS values with concomitant increase of RS content
(Mkandawire et al., 2013). So far, anthocyanins can affect starch
digestibility by two possible mechanisms, as inhibitors of digestive
enzymes (Worsztynowicz, Napierała, Białas, Grajek, & Olkowicz,
2014) and by interacting with starch components restricting the
amylolytic attack (Takahama, Yamauchi, & Hirota, 2013).
Also, the previous mentioned addition of blue maize flour phe-
nolics resulted in significant reductions of RDS and increased RS
values compared with the polyphenol preparation obtained from
commercial white maize flour (Fig. 1). Different polyphenol-
starch molecular interaction and/or polyphenols-enzyme interac-
tion may be reflected in the RS and total starch content. Non-
covalent interactions between polyphenols and polysaccharides
have been reported and one of the interactions is due to the forma-
tion hydrogen bonding between polyphenols and starch (Wu, Lin,
Chen, & Xiao, 2011).
The presence of polyphenols during starch gelatinization
resulted in higher RS content (p < 0.05). This increase in RS content
agrees with the observations by Barros et al. (2012), who showed
that tannins bind amylose, possibly through hydrophobic interac-
tions, an association that leads increase of the RS content.
Takahama et al. (2013) indicated that vignacyanidin can bind to
both amylose and amylopectin to inhibit their digestion by
a-amylase. We suggest that cyanidin pigment (the major polyphe-
nol in blue maize extracts) decreased in vitro starch hydrolysis by a
combination of the two proposed mechanisms previously men-
tioned, blocking the enzyme for starch hydrolysis (Dunn, Yang,
Girard, Bean, & Awika, 2015; Zhu, 2015) and likely form cross-
linked networks with starch during cooking, rendering structures
of limited availability for amylolytic attack, by physical shielding
and slowing the process (Zhu, 2015).
It was observed that blue maize extract (rich in anthocyanins)
could modify nutritional starch fraction, with an increase of RS
content in the maize starch. Therefore, the interaction between
anthocyanins and starch modified starch structure, which limits
284 G.A. Camelo-Méndez et al. / Food Chemistry 211 (2016) 281–284
the amylolytic enzymatic hydrolysis in the small intestine
(Takahama et al., 2013), so that anthocyanins can be released dur-
ing colonic fermentation (González-Barrio, Edwards, & Crozier,
2015) generating an antioxidant environment (Aura et al., 2005).
Thus, the fermentation of resistant starch and anthocyanins should
generate a high level of reactive species for colon cancer preven-
tion (Jing et al., 2008). The use of plant extracts for modification
of starch and modify starch digestibility must be done in future
studies.
4. Conclusions
This study showed that cyanidin-3-(600 -malonylglucoside) is the
major anthocyanin-pigment in blue maize extract. The extract rich
in anthocyanins modified the relation of starch fractions of
commercial maize starch (using above of 75% in relation to starch
weight) and contributed to RS formation due to the possible
anthocyanins-starch interactions. Further work must be per-
formed to evaluate the specific binding regions between polyphe-
nols and starch, to develop processes for designing novel functional
foods with higher RS content and to elucidate their potential rela-
tion to health benefits.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
The authors thank the support from CONACYT, México, SIP-IPN,
COFAA-IPN, and EDI-IPN. One of the authors (GACM) also acknowl-
edges the scholarship from CONACYT, Mexico.
References
Abdel-Aal, E.-S. M., Young, J. C., & Rabalski, I. (2006). Anthocyanin composition in
black, blue, pink, purple, and red cereal grains. Journal of Agriculture and Food
Chemistry, 54(13), 4696–4704.
Aguayo-Rojas, J., Mora-Rochín, S., Cuevas-Rodríguez, E. O., Serna-Saldivar, S. O.,
Gutierrez-Uribe, J. A., Reyes-Moreno, C., & Milán-Carrillo, J. (2012).
Phytochemicals and antioxidant capacity of tortillas obtained after lime-
cooking extrusion process of whole pigmented Mexican maize. Plant Foods for
Human Nutrition, 67(2), 178–185.
Aura, A. M., Martin-Lopez, P., O’Leary, K. A., Williamson, G., Oksman-Caldentey, K.
M., Poutanen, K., & Santos-Buelga, C. (2005). In vitro metabolism of
anthocyanins by human gut microflora. European Journal of Nutrition, 44,
133–142.
Barros, F., Awika, J. M., & Rooney, L. W. (2012). Interaction of tannins and other
sorghum phenolic compounds with starch and effects on in vitro starch
digestibility. Journal of Agricultural and Food Chemistry, 60(46), 11609–11617.
Bello-Pérez, L. A., Flores-Silva, P. C., Camelo-Méndez, G. A., Paredes-López, O., &
Figueroa-Cárdenas, J. (2015). Effect of nixtamalization process on dietary fiber
content, starch digestibility, and antioxidant capacity of blue maize tortilla.
Cereal Chemistry, 92(3), 265–270.
Bordenave, N., Hamaker, B. R., & Ferruzzi, M. G. (2014). Nature and consequences of
non-covalent interactions between flavonoids and macronutrients in foods.
Food & Function, 5, 18–34.
Camelo-Méndez, G. A., & Bello-Pérez, L. A. (2014). Antioxidant capacity of
extractable and non-extractable polyphenols of pigmented maize. Journal of
Chemical, Biological and Physical Sciences, 4(5), 6–13.
Del Pozo-Insfran, D., Brenes, C. H., Serna-Saldivar, S. O., & Talcott, S. T. (2006).
Polyphenolic and antioxidant content of white and blue corn (Zea mays L.)
products. Food Research International, 39, 696–703.
Dunn, K. L., Yang, L., Girard, A., Bean, S., & Awika, J. M. (2015). Interaction of
sorghum tannins with wheat proteins and effect on in vitro starch and protein
digestibility in a baked product matrix. Journal of Agriculture and Food Chemistry,
63(4), 1234–1241.
Englyst, K. N., & Englyst, H. N. (2005). Carbohydrate bioavailability. British Journal of
Nutrition, 94, 1–11.
Englyst, H. N., Kingman, S. M., & Cummings, J. H. (1992). Classification and
measurement of nutritionally important starch fractions. European Journal of
Clinical Nutrition, 46, 30S–50S.
González-Barrio, R., Edwards, C. A., & Crozier, A. (2015). Colonic catabolism of
ellagitannins, ellagic acid, and raspberry anthocyanins: in vivo and in vitro
studies. Drug Metabolism and Disposition, 39(9), 1680–1688.
Hoyos-Leyva, J. D., Bello-Pérez, L. A., Agama-Acevedo, E., & Alvarez-Ramirez, J.
(2015). Optimising the heat moisture treatment of Morado banana starch by
response surface analysis. Starch – Stärke, 11(12), 1026–1034.
Jing, P., Bomser, J. A., Schwartz, S. J., He, J., Magnuson, B. A., & Giusti, M. M. (2008).
Structure-function relationships of anthocyanins from various anthocyanin-rich
extracts on the inhibition of colon cancer cell growth. Journal of Agricultural and
Food Chemistry, 56(20), 9391–9398.
Lo Piparo, E., Scheib, H., Frei, N., Williamson, G., Grigorov, M., & Chou, C. J. (2008).
Flavonoids for controlling starch digestion: structural requirements for
inhibiting human a-amylase. Journal of Medicinal Chemistry, 51(12), 3555–3561.
Lopez-Martinez, L. X., Oliart-Ros, R. M., Valerio-Alfaro, G., Lee, C. H., Parkin, K. L., &
Garcia, H. S. (2009). Antioxidant activity, phenolic compounds and
anthocyanins content of eighteen strains of Mexican maize. LWT – Food
Science and Technology, 42(6), 1187–1192.
Miao, M., Jiang, H., Jiang, B., Zhang, T., Cui, S. W., & Jin, Z. (2014). Phytonutrients for
controlling starch digestion: Evaluation of grape skin extract. Food Chemistry,
145, 205–211.
Mkandawire, N. L., Kaufman, R. C., Bean, S. R., Weller, C. L., Jackson, D. S., & Rose, D. J.
(2013). Effects of sorghum (Sorghum bicolor (L.) Moench) tannins on a-amylase
activity and in vitro digestibility of starch in raw and processed flours. Journal of
Agricultural and Food Chemistry, 61(18), 4448–4454.
Montilla, C. E., Hillebrand, S., Antezana, A., & Winterhalter, P. (2011). Soluble and
bound phenolic compounds in different Bolivian purple corn (Zea mays L.)
cultivars. Journal of Agricultural and Food Chemistry, 59(13), 7068–7074.
Takahama, U., Yamauchi, R., & Hirota, S. (2013). Interactions of starch with a
cyanidin-catechin pigment (vignacyanidin) isolated from Vigna angularis bean.
Food Chemistry, 141(3), 2600–2605.
Toydemir, G., Boyacioglu, D., Capanoglu, E., Van Der Meer, I. M., Tomassen, M. M. M.,
Hall, R. D., & Beekwilder, J. (2013). Investigating the transport dynamics of
anthocyanins from unprocessed fruit and processed fruit juice from sour cherry
(Prunus cerasus L.) across intestinal epithelial cells. Journal of Agricultural and
Food Chemistry, 61(47), 11434–11441.
Urias-Lugo, D. A., Heredia, J. B., Muy-Rangel, M. D., Valdez-Torres, J. B., Serna-
Saldívar, S. O., & Gutiérrez-Uribe, J. A. (2015). Anthocyanins and phenolic acids
of hybrid and native blue maize (Zea mays L.) extracts and their antiproliferative
activity in Mammary (MCF7), Liver (HepG2), Colon (Caco2 and HT29) and
Prostate (PC3) Cancer Cells. Plant Foods for Human Nutrition, 70(2), 193–199.
Utrilla-Coello, R. G., Agama-Acevedo, E., Osorio-Díaz, P., Tovar, J., & Bello-Pérez, L. A.
(2011). Composition and starch digestibility of whole grain bars containing
maize or unripe banana flours. Starch/Stärke, 63, 416–423.
Worsztynowicz, P., Napierała, M., Białas, W., Grajek, W., & Olkowicz, M. (2014).
Pancreatic a-amylase and lipase inhibitory activity of polyphenolic compounds
present in the extract of black chokeberry (Aronia melanocarpa L.). Process
Biochemistry, 49, 1457–1463.
Wu, Y., Lin, Q., Chen, Z., & Xiao, H.-X. (2011). The interaction between tea
polyphenols and rice starch during gelatinization. Food Science and Technology
International, 17, 569–577.
Zhang, H., Yu, D., Sun, J., Liu, X., Jiang, L., Guo, H., & Ren, F. (2014). Interaction of
plant phenols with food macronutrients: Characterisation and nutritional-
physiological consequences. Nutrition Research Reviews, 27(1), 1–15.
Zhu, F. (2015). Interactions between starch and phenolic compound. Trends in Food
Science & Technology, 43(2), 129–143.