agronomy

Article
Physicochemical Characterization and Functional Potential of
Phaseolus vulgaris L. and Phaseolus coccineus L. Landrace
Green Beans
Elia Nora Aquino-Bolaños 1 , Alma Karina Garzón-García 1 , Jimena Esther Alba-Jiménez 2, * ,
José Luis Chávez-Servia 3 , Araceli Minerva Vera-Guzmán 3 , José Cruz Carrillo-Rodríguez 4
and Manuel Alberto Santos-Basurto 5

1 Centro de Investigación y Desarrollo en Alimentos, Universidad Veracruzana, Xalapa 91190, Mexico;

[email protected] (E.N.A.-B.); [email protected] (A.K.G.-G.)
2 Centro de Investigación y Desarrollo en Alimentos, CONACyT-Universidad Veracruzana,
Xalapa 91190, Mexico
3 Instituto Politécnico Nacional, CIIDIR-Oaxaca, Oaxaca 71230, Mexico; [email protected] (J.L.C.-S.);
[email protected] (A.M.V.-G.)
4 Instituto Tecnológico del Valle de Oaxaca, Oaxaca 71230, Mexico; [email protected]
5 Campus Queretaro, Arkansas State University, Querétaro 76270, Mexico; [email protected]



* Correspondence: [email protected]; Tel.: +52-228-303-5426



Citation: Aquino-Bolaños, E.N.; Abstract: The green bean is an important crop worldwide, because it is rich in protein, dietary fiber,
Garzón-García, A.K.; Alba-Jiménez, vitamins, and minerals, as well as bioactive compounds that provide it with important functional
J.E.; Chávez-Servia, J.L.; properties; however, the composition of many landraces is still unknown. The purpose of this project
Vera-Guzmán, A.M.;
was to characterize Phaselus vulgaris and coccineus L. landrace green beans on pH, titratable acidity,
Carrillo-Rodríguez, J.C.;
total soluble solids, total sugars, color parameters, total phenols, monomeric anthocyanins, and
Santos-Basurto, M.A.
in vitro antioxidant activity (DPPH and FRAP). Regarding the content of total sugars, differences
Physicochemical Characterization
were registered between both species, as opposed to results observed in total soluble solids. Color
and Functional Potential of Phaseolus
vulgaris L. and Phaseolus coccineus L.
parameters showed higher reddish tones for P. vulgaris landraces, though P. coccineus had a higher total
Landrace Green Beans. Agronomy phenolic content, especially the reddish landraces, which correlated directly to a higher antioxidant
2021, 11, 803. https://doi.org/ activity by DPPH and FRAP. In the protein content, the species P. vulgaris registered the highest
10.3390/agronomy11040803 content. These results could contribute to a greater use and even promote the genetic improvement of
the outstanding pods that serve as one of the main food products in rural regions for higher benefits.
Academic Editors: Laura Siracusa
and Rosa Palmeri Keywords: total phenols; monomeric anthocyanins; antioxidant activity; protein

Received: 25 February 2021

Accepted: 14 April 2021
Published: 19 April 2021
1. Introduction
Legumes are among the most important crops in the world, since they represent one
Publisher’s Note: MDPI stays neutral
of the main sources of food in developing countries, in addition to being rich sources of
with regard to jurisdictional claims in
vegetable protein and can be used to supplement the deficiency of different cereals, due to
published maps and institutional affil-
iations.
their content of essential amino acids, fiber, vitamins, and minerals such as iron, potassium,
magnesium, and zinc, among others [1].
Among the cataloged legumes by the FAO is the genus Phaseolus, which includes up
to 117 species, considering the Mesoamerican region as the main center of both origin
and diversity [2]. Many of them, such as P. vulgaris and P. coccineus L., seem to have been
Copyright: © 2021 by the authors.
initially domesticated in western Mexico and are among the five most cultivated and
Licensee MDPI, Basel, Switzerland.
consumed species in Mexico by the number of commercial varieties and landraces [3], in
This article is an open access article
addition to being two of the three most relevant species worldwide [4].
distributed under the terms and
conditions of the Creative Commons
In addition to the seed, which would be the bean, the pod, known in Mexico as green
Attribution (CC BY) license (https://
beans, is also consumed. Prior to the senescence and dehiscence of the pods, they grow and
creativecommons.org/licenses/by/ reach their commercial maturity, in which they can develop shades from yellow and green
4.0/). to reddish and purple, depending on the variety to which they belong [5]. Regarding its

Agronomy 2021, 11, 803. https://doi.org/10.3390/agronomy11040803 https://www.mdpi.com/journal/agronomy

Agronomy 2021, 11, 803 2 of 12

nutritional composition, it has a high-water content, which contributes to its characteristic

turgor, in addition to minerals and vitamin A. To a lesser extent, it provides protein and
fiber [6]. Green bean is classified as a vegetable and is part of the daily diet in many coun-
tries, even being considered one of the main sources of phenolic acids (hydroxybenzoic
and hydroxycinnamic acids), flavonoids (flavonols, flavones, dihydroflavonols, flavanones,
isoflavones, and isoflavanones), lignans, and other polar compounds (Kutkoside) with po-
tential health benefits such as slowing the development of cancerous tumors and providing
antibacterial, antiviral, antispasmodic, and anti-inflammatory properties [7–9].
It has been shown that the anticancer activity of green beans is mainly related to the
presence of high amounts of chlorophyll, resistant starch, soluble and insoluble dietary
fiber, and phenolic compound, which suggests that its regular consumption may reduce
the risk of breast, colon, and prostate cancer. Likewise, it has been reported that due to
its high fiber content, it could help control diabetes by reducing insulin generation and
glucose levels that enter the bloodstream; besides, patients suffering from non-insulin
dependent diabetes can prevent the need for insulin by up to 40% by eating green beans in
their regular diet [10–13].
In addition to the nutritional aspect, endemic varieties represent a potential source of
income for households in rural communities, since small farmers in Latin America and the
Caribbean, Asia, and Africa contribute up to 77% of the world production of P. vulgaris [2].
However, a current trend has been observed in which endemic crops are being replaced
by commercial outdoor varieties that tend to present a higher demand, harvest yield, and,
therefore, commercial value. This leads to a gene pool loss since the landraces are limited
to be cultivated in small plots or farmers backyards for self-consumption of the edible
parts (flowers, leaves, fruits, and grains) as well as maintaining the germplasm for the
next planting [14]. In addition to the biodiversity loss, there is a dependence on external
resources that can compromise the economic and food security of these regions.
Part of the reason for the displacement of local varieties may be due to ignorance of
their properties, so their study contributes to a better understanding and, in turn, to promot-
ing their consumption. For this reason, the present work focuses on the physicochemical
characterization and functional potential, measured through the phenolic compounds
quantification and in vitro antioxidant activity, of various P. vulgaris and P. coccineus native
varieties from rural regions of Oaxaca, Mexico, in order to know its nutritional and func-
tional composition. This would contribute to promote both cultivation and consumption,
with direct benefits in the food and economic security of rural populations, in addition to
avoid the loss of unexplored germplasm with potential health benefits and maintaining the
local gene pool.

2. Materials and Methods

2.1. Plant Material
A total of 18 bean landraces were evaluated; nine from Phaseolus vulgaris L. (common
bean) and nine from Phaseolus coccineus (scarlet runner bean), which were collected in
different regions of the state of Oaxaca, Mexico where each seed lots correspond to a
farmers variety. This means that each farmer preserves one or more native varieties
because Oaxaca is part of the Mesoamerican region where P. vulgaris and P. coccineus have
their origin center. Seed lots of each landrace were sown and cultivated in an open field plot
in the Training Unit for Rural Development (UNCADER2) located in Coatepec, Veracruz,
carrying out a basic fertilization (60:60:60 N:P:K). Green bean was harvested at stage 4 of
maturity, when the color change was already observed in the dorsal suture and pedicel
(Figure 1), since it has been reported that at this stage of development there is a higher
phenolic compounds content and in vitro antioxidant activity [15]. The whole fruits in fresh
state were used to perform analysis of the physicochemical parameters and compounds
with in vitro antioxidant activity.
Agronomy 2021, 11, 803 3 of 12

Figure 1. Landrace green bean samples evaluated of P. vulgaris (a) and P. coccineus (b).

2.2. Physicochemical Parameters

pH was measured according to the AOAC (Association of Official Analytical Chemists) [16]
using a digital potentiometer (OAKTON, 510, Vernon Hills, IL, USA) and the titratable
acidity (TA) was determined using the AOAC methodology [17] by acid-base titration,
with some modifications. Using NaOH 0.1 N as a titrant agent and phenolphthalein as an
endpoint indicator (pH 8.2), results were expressed as grams of citric acid per 100 g of dry
weight (dw). Total sugar content (TSC) was determined by the phenol-sulfuric method [18].
Total soluble solids (TSS) were measured in a digital refractometer and expressed in ◦ Brix
(ATAGO, PR-32, Tokyo, Japan). Color parameters were determined by the CIEL* a* b*
system with a spectrophotometer for solids (Konica Minolta, CM-2600d, Osaka, Japan) and
the values of L*, a*, b*, chroma C* = (a*2 + b*2) 1/2 and hue angle h◦ = tan − 1 (b*/a*)
indexes were calculated according to McGuire [19]. Protein content was obtained of the
homogeneous sample from each bean sample to measure the protein content according
to the method described by Bradford [20]. The quantification was based on a standard
curve obtained using samples of bovine albumin (Sigma-Aldrich, Saint Louis, MO, USA)
at concentrations ranging from 0.006 to 0.018 mg mL−1 .

2.3. Ethanolic Extracts Preparation

An ethanolic extract was obtained with a 5 g sample and 20 mL of ethanol 80% (v/v),
which was homogenized for 30 s at 1 g × s (DAIHAN-brand HG-15-A, Gangneung, Korea).
Subsequently, it was centrifuged at 35,000 g × s for 20 min at 10 ◦ C (Hettich zentrifuge,
Universal 32R, Tuttlingen, Germany) and the supernatant was brought to a volume of
25 mL with ethanol 80% (v/v).

2.4. Total Polyphenols

They were determined by the method described by Singleton and Rossi [21], and the
reaction absorbance was measured at 750 nm in a UV-visible spectrophotometer (Jenway
6305, Bibby Scientific Ltd., Dunmow, Essex, UK). The quantification was performed based
on a standard curve of gallic acid (0.021 to 0.165 mg mL−1 ), and the results were expressed
in mg equivalents of gallic acid per gram of dry weight (mg GAE g −1 dw).

2.5. Monomeric Anthocyanins

Anthocyanin content was determined by the differential pH method [22]. Two di-
lutions of the extract were made, one with potassium chloride buffer at pH 1.0 and the
second with sodium acetate buffer at pH 4.5, diluting each by the previously determined
dilution factor. Subsequently, a spectrophotometer was used to generate an absorption
spectrum in the range of 460–710 nm to determine the maximum absorbance. The con-
Agronomy 2021, 11, 803 4 of 12

centration of monomeric anthocyanins (MA) was calculated according to the following

equation: MA = (A*PM*FD*1000)/(ε*I), where the absorbance of sample A corresponds
to (Aλ510–Aλ700) pH 1.0 − (Aλ510–Aλ700) pH 4.5; MW = 449.2 is the molecular weight
of cyanidin-3-glucoside; ε = 26,900 g/mol is the molar absorptivity of the cyanidin-3-
glucoside; FD is the dilution factor used; and I is the cell length (1 cm). The results were
expressed as mg of cyanidin-3-glucoside per gram of dry weight (mg C3G g−1 dw).

2.6. Antioxidant Assay Procedures

Antioxidant activity was evaluated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH)
method [23]. A 100 µL sample of the extract was reacted with 2.9 mL of DPPH reagent
and allowed to stand for 30 min at room temperature. The absorbance was measured
using a UV-vis spectrophotometer (Shimadzu UV-1800, Kyoto, Japan) at 517 nm using 80%
(v/v) methanol as the target. To quantify the in vitro antioxidant activity, it was performed
based on the inhibition percentage of a standard 6-hydroxy-2,5,7,8-tetramethylchroman-
2-carboxylic acid (Trolox) curve in a concentration range of 0.13–0.79 µmol equivalents
of Trolox (TE) per mL. The results were expressed in micromoles of Trolox equivalents
per gram of dry weight (µmol TE g−1 dw). The reducing power of the samples was also
evaluated through the FRAP method [24]. The FRAP reagent was prepared by acetate
buffer mixture (0.3 M pH 3.6), with a 10 mM TPTZ solution (2, 4, 6-tripyridyl-s-triazine)
in 40 mM HCl and a solution of 20 mM FeCl3-6H2O, in a ratio 10:1:1. A 100 µL sample
of the extract was reacted with 3 mL of FRAP and incubated at 37 ◦ C during 30 min. The
absorbance was measured using a UV-vis spectrophotometer (JENWAY 6305, Sttafordshire,
UK) at 593 nm. The in vitro antioxidant activity quantification was performed using
a standard 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) curve in a
concentration range of 0.1 to 1 µmol equivalents of Trolox (TE) per mL. The results were
expressed in micromoles of Trolox equivalents per gram of dry weight (µmol TE g−1 dw).

2.7. Statistical Analysis

All samples were analyzed by quadruplicate in each determination, reporting the
averages for the samples harvested at two different seasons on the same maturity stage.
A database with all results was integrated, and an analysis of variance was performed
using a completely random design with the nesting of landraces within species. Compar-
isons among species and landraces were made by the Tukey method (p ≤ 0.05). Pearson
analysis was used to determine the correlation between bioactive compounds, in vitro
antioxidant activity, and physicochemical parameters. All statistical analyses were per-
formed using SAS software version 9.0 [25], and the values are reported as the mean of
replicates ± standard deviation.

3. Results
In the variance analysis, significant differences were recorded in the genotype/species
interaction in the physicochemical parameters, protein, and bioactive components, such as
total phenolic compounds (TPC), total anthocyanin content (TAC), and in vitro antioxidant
activity by the DPPH and FRAP method, evaluated with a coefficient of variation less
than 26.5% (p < 0.01; Table 1). With the exception of pH and titratable acidity, considering
the mean squares value, it was estimated that the variance due to the species was greater
than in the genotype/species interaction (Table 1). This indicates that, in general, the
variation between species is greater than between landraces within each species and
reflects part of the evolutionary differences between P. vulgaris and P. Coccineus [26,27]. In
addition, statistical differences (p < 0.05; p < 0.01) were observed in the pH parameters,
total sugars, L*, protein and in vitro antioxidant activity evaluated by DPPH and FRAP
between repetitions of the genotypes sown in the experimental plot (Table 1). This could
be attributed to the heterogeneity between same genotype individuals, due to factors
such as agronomic practices, sampling randomness, and typical variation of the physical
characteristics (color, size, shape) between grains of each accession.
Agronomy 2021, 11, 803 5 of 12

Table 1. Mean square values of the variance analysis of the physicochemical parameters in
green beans.

Variation Sources
Variable
Repetition Species Genotype/Species Error CV (%) 4
pH 0.1 ** 0.0 ns 0.2 ** 0.0 1.8
TA 0.0 ns,1 0.1 ns 1.0 ** 0.1 23.8
TSC 5736.1 **,3 21802.8 * 7394.1 ** 301.3 25.8
TSS 0.0 ns 84.7 ** 59.4 ** 1.8 16.6
L* 249.4 *,2 83.1 * 42.0 41.99 11.8
a* 28.8 ** 1.4 ** 0.1 0.14 20.1
Color
b* 15.5 ** 2.1 ** 0.3 0.34 19.5
parameters
C 12.1 ** 1.8 ** 0.3 0.33 19.0
h◦ 23,406.2 ** 1392.6 ** 92.5 92.47 11.1
Protein 48.9 ** 15.3 ** 1.9 1.94 17.4
TPC 2918.3 ** 422.9 ** 7.2 7.19 26.5
TAC 10.3 ** 7.2 ** 0.1 0.14 15.4
DPPH 288.7 ** 33.2 ** 0.6 ** 0.61 ** 11.7
FRAP 458.5 ** 33.3 ** 0.8 0.76 14.0
1,ns not significant at p = 0.05, 2, * (p < 0.05), 3, ** (p < 0.01), 4 CV% (Coefficient of variation).

3.1. Physicochemical Parameters

Regarding total sugars content, the P. coccineus species was 1.3 times higher (76.2 mg
Glu g−1 dw) compared to the P. vulgaris species (59.3 mg Glu g−1 dw); on the contrary,
P. vulgaris presented the highest content of TSS (Table 2). The interspecific variation for total
sugars ranged from 47.9–125.5 mg Glu g−1 dw in P. coccineus green beans, and for P. vulgaris
from 36.8–81.8 mg Glu g−1 dw, with this latter species being the one that presented the
lowest variability (42%).
Among genotypes within each species, the SJA and P-69 landraces of P. coccineus
green beans obtained the highest total sugars content (125.5 and 107.1 mg Glu g−1 dw,
respectively), while in the case of P. vulgaris green beans, P-19, P-71, P-68, and P-34 landraces
showed the highest concentration, not presenting significant differences between them
(Table 2). In the TSS content, the FCA-04 green beans landrace of P. vulgaris obtained
the highest value (13.2 ◦ Brix) followed by P-58 (10.4 ◦ Brix) and SLMV (10.8 ◦ Brix) of the
P. coccineus species.
In the luminosity parameter, significant differences were observed between species
and landraces within each species, with P. coccineus being the one that presented the highest
value with respect to P. vulgaris (p < 0.05). Furthermore, between landraces of both species,
the P-65 green beans (P. vulgaris) registered a lower average value (57.8) in contrast to SJA
(P. coccineus) (59.2) (Table 2). In a* and b* color parameters, between species, P. coccineus
green beans showed average values with a tendency to yellow-green, while P. vulgaris
observed a tendency to red-purple, with variation between landraces of each species,
since CAA green beans of P. coccineus obtained a negative value for a* and positive for b*
corresponding to the yellow color, on the other hand, in P. vulgaris, beans of P-40 and P-71
presented positive values for a* and b* corresponding to the red color, without statistical
differences between them (p < 0.05). For chromaticity parameter (C *), it was observed
that P. coccineus species presented a higher average value (10.9) compared to P. vulgaris
(8.5), with the landraces P-69 and FCA-04 being those that presented the highest values,
respectively (Table 2). Regarding the hue angle parameter (h◦ ), a wide interval (87.5–100.1◦ )
was observed for P. coccineus green beans, where CAA obtained the highest value with a
tendency to green; while, for P. vulgaris green beans, hue angle ranged from 57.9–96.5◦ ,
with P-71 (57.9◦ ) being the one that presented a value with a tendency to red and P-65
(69.2◦ ) and P-58 (72.5◦ ) with a tendency to yellow (p < 0.05).
Agronomy 2021, 11, 803 6 of 12

Table 2. Physicochemical, color parameters, and protein content in green beans from bean landraces.

Species and Genotypes pH TA TS TSS L* a* b* C* h◦ P

P. coccineus L. 6.4 ± 0.1A 1 0.9 ± 0.3 A 76.2 ± 27.1 A 7.4 ± 1.9 B 55.9 ± 1.9 A −1.4 ± 0.7 B 10.8 ± 1.7 A 10.9 ± 1.7 A 96.3 ± 4.2 A 5.6 ± 2.3 B
Fat bean 6.4 ± 0.1 a–f 1 0.8 ± 0.1 e–g 48.6 ± 6.4 f–i 6.8 ± 0.6 g–i 55.0 ± 7.1 ab −1.7 ± 0.9 e–g 11.4 ± 3.5 a–d 11.6 ± 3.6 a–d 98.1 ± 2.1 abc 9.6 ± 0.8 c–e
SLMV 6.5 ± 0.2 a–e 0.9 ± 0.1 c–e 56.4 ± 11.3 e–i 10.8 ± 2.3 b 57.7 ± 4.2 ab −0.8 ± 0.6 d–g 7.9 ± 2.2 b–g 7.9 ± 2.3 b–f 95.2 ± 2.8 a–d 6.2 ± 1.1 gh
SJA 6.3 ± 0.2 gh 1.3 ± 0.4 ab 125.5 ± 39.7 a 5.3 ± 0.5 ij 59.2 ± 8.0 a −0.2 ± 1.6 d–f 9.6 ± 6.1 a–f 9.7 ± 6.1 a–f 87.5 ± 10.9 b–e 6.5 ± 1.0 gh
CAA 6.3 ± 0.1 f–h 1.2 ± 0.1 bc 93.5 ± 14.5 bc 5.0± 0.0 j 57.4 ± 5.7 ab −2.2 ± 0.9 g 11.9 ± 4.0 ab 12.1 ± 4.1 ab 100.1 ± 1.8 a 3.0 ± 1.6 j
P-69 6.5 ± 0.2 a–d 1.4 ± 0.4 a 107.1 ± 11.6 ab 8.6 ± 2.0 d–f 53.9 ± 5.9 ab −2.4 ± 0.7 g 13.6 ± 3.3 a 13.8 ± 3.3 a 99.8 ± 0.9 a 3.1 ± 1.7 j
P-26 6.3 ± 0.1 e–h 0.8 ± 0.1 d–f 66.8 ± 12.9 d–g 6.3 ± 1.0 h–j 55.6 ± 5.5 ab −1.7 ± 0.9 e–g 10.4 ± 3.7 a–f 10.5 ± 3.8 a–e 98.8 ± 2.0 ab 8.1 ± 0.5 e–g
P-96C2 6.5 ± 0.1 a–d 0.5 ± 0.2 g 47.9 ± 7.8 f–i 9.1 ± 3.3 cd 56.3 ± 7.7 ab −0.8 ± 1.5 de–g 9.4 ± 4.3 a–f 9.5 ± 4.4 a–f 92.0 ± 9.2 a–d 5.3 ± 0.3 hi
P-91C2 6.5 ± 0.0 a–f 0.7 ± 0.2 e–g 76.5 ± 14.2 c–e 8.0 ± 1.8 d–g 53.5 ± 6.9 ab −1.3 ± 1.2 e–g 11.0 ± 3.3 a–e 11.1 ± 3.4 a–e 96.2 ± 4.9 a–d 5.4 ± 0.7 h
P-102 6.5 ± 0.1 a–e 0.8 ± 0.2 ef 63.6 ± 11.5 d–g 7.1 ± 0.1 f–h 54.5 ± 5.6 ab −1.9 ± 0.7 fg 11.8 ± 3.2 abc 11.9 ± 3.3 a–c 98.8 ± 1.4 ab 3.3 ± 0.6 ij
P. vulgaris L. 6.4 ± 0.1 A 1.0 ± 0.2 A 59.3 ± 14.8 B 8.5 ± 2.0 A 54.1 ± 2.6 B 1.0 ± 1.4 A 8.2 ± 2.4 B 8.5 ± 2.2 B 78.7 ± 12.2 B 10.1 ± 1.8 A
P-34 6.4 ± 0.1 b–g 1.1 ± 0.2 b–d 69.8 ± 12.4 d–f 7.3 ± 0.9 e–h 55.4 ± 8.1 ab 0.0 ± 2.4 c–e 9.9 ± 3.7 a–f 10.1 ± 3.7 a–e 87.0 ± 14.0 b–e 10.1 ± 0.6 b–e
P-68 6.6 ± 0.1 a 1.1 ± 0.1 b–d 71.7 ± 23.2 c–e 8.2 ± 1.0 d–g 57.3 ± 6.4 ab −1.5 ± 1.2 e–g 11.8 ± 3.2 a–c 11.9 ± 3.3 a–c 96.5 ± 5.1 a–c 8.7 ± 1.3 d–f
P-76 6.4 ± 0.1 b–g 1.1 ± 0.4 bc 59.3 ± 21.1 e–h 8.3 ± 1.1 d–g 56.8 ± 7.5 ab −0.2 ± 1.0 d–f 7.5 ± 3.7 c–g 7.6 ± 3.7 c–f 86.6 ± 11.1 c–e 12.2 ± 2.5 ab
P-19 6.5 ± 0.1 ab 1.2 ± 0.1 ab 81.8 ± 25.1 cd 6.8 ± 0.4 g–i 52.6 ± 6.0 ab 0.4 ± 1.6 b–d 8.1 ± 2.4 b–g 8.3 ± 2.3 b–f 84.5 ± 14.1 d–f 9.1± 1.2 c–f
P-71 6.5 ± 0.0 a–c 0.6 ± 0.1 fg 72.2 ± 18.5 c–e 7.1 ± 1.0 f–h 53.9 ± 7.6 ab 2.6 ± 0.9 a 4.8 ± 2.4 g 5.6 ± 2.3 f 58.0 ± 13.4 h 10.7 ± 3.5 a–d
P-58 6.5 ± 0.1 a–e 0.9 ± 0.2 c–e 45.2 ± 3.8 g–i 10.4 ± 0.3 bc 52.5 ± 7.5 ab 1.9 ± 0.9 ab 6.5 ± 2.3 fg 6.8 ± 2.3 ef 72.6 ± 7.6 fg 12.3 ± 1.7 a
P-65 6.2 ± 0.1 h 0.8 ± 0.1 ef 36.8 ± 8.6 i 7.6 ± 0.4 d–h 57.8 ± 8.1 ab 2.0 ± 2.8 ab 7.4 ± 4.3 d–g 8.1 ± 4.3 b–f 69.2 ± 20.0 gh 11.8 ± 0.9 ab
P-16 6.4 ± 0.0 d–g 0.8 ± 0.2 d–f 41.5 ± 14.7 hi 7.1 ± 1.4 f–h 52.0 ± 6.0 ab 1.7 ± 0.7 a–c 6.9 ± 2.0 e–g 7.1 ± 2.0 ef 75.7 ± 5.7 eg 10.9 ± 1.1 a–c
P-40 6.4 ± 0.1 c–g 1.2 ± 0.5 ab 57.2 ± 13.9 e–i 8.8 ± 0.8 c–e 51.7 ± 5.7 ab 2.8 ± 2.4 a 6.5 ± 2.7 fg 7.3 ± 2.9 d–f 66.4 ± 16.1 gh 8.7 ± 0.4 d–f
FCA-04 6.5 ± 0.1 ab 0.9 ± 0.1 c–e 57.1 ± 23.1 e–i 13.2 ± 1.3 a 50.8 ± 4.5 b −0.2 ± 1.0 d–f 12.1 ± 2.8 ab 12.2 ± 2.8 ab 90.6 ± 4.1 a–d 7.0 ± 0.9 f–h
TA = titratable acidity (g Citric acid 100 g−1 dw), TS = Total sugars (mg Glucose g−1 dw), TSS = Total soluble solids (◦ Brix), L*: Luminosity, a*: Red/green Coordinates, b*: Yellow/blue Coordinates, C*:
Chromaticity, h◦ : Hue angle, P: Total protein (g 100 g−1 dw). SLMV: Santa Lucia Monte Verde, SJA: San Juan Atepec, CAA: Cerro Amole Ayutla. 1 Between species (capital letter) and among landraces (lowercase
letter), means with same letter indicate non-significant differences (Tukey’s test, p < 0.05).
Agronomy 2021, 11, 803 7 of 12

Protein content differs significantly between species and landraces within each species
(p < 0.05). The species P. vulgaris showed higher protein content (10.1 g protein 100 g−1 dw)
compared to P. coccineus (5.6 g protein 100 g−1 dw) (p < 0.05; Table 2). The protein content
variation ranged 7.0–12.2 g protein 100 g−1 dw for beans of the species P. vulgaris, and for
P. coccineus 3.0–9.6 g protein 100 g−1 dw, the latter being the one that presented the highest
variability with 68.8%. Among genotypes, the landraces P-76, P-71, P-58, P-65, and P-16
of the species P. vulgaris, presented the highest protein contents, with respect to the other
landraces and the P. coccineus species landraces, likewise, of the fat beans (p < 0.05; Table 2).

3.2. Total Phenols, Anthocyanins, and In Vitro Antioxidant Activity

Total polyphenols content (TPC), total anthocyanins content (TAC), and antioxidant
activity by DPPH and FRAP is shown in Table 3. TPC for P. coccineus species was signifi-
cantly (p < 0.05) higher than P. vulgaris with a value average of 13.4 mg GAE g−1 dw and
7.2 mg GAE g−1 dw, respectively, observing a high variability between results, with a
range of 6.0 to 26.5 mg GAE g−1 dw for P. coccineus and 4.2 to 10.1 mg GAE g−1 dw for
P. vulgaris, with the landraces with reddish tones being those that presented higher TPC
concentrations, probably due to anthocyanins presence.

Table 3. Content of total phenols, anthocyanins, and in vitro antioxidant activity in green beans from
bean landraces.

Species and Landraces TPC TAC DPPH FRAP

P. coccineus L. 13.4 ± 8.0 A 1 0.7 ± 0.0 B 62.9 ± 32.5 A 60.2 ± 32.3 A
Fat bean 7.8 ± 1.2 de–g 1 - 37.9 ± 12.6 f–j 54.3 ± 11.4 b–d
SLMV 26.5 ± 8.5 a 0.7 ± 0.1 c 58.6 ± 9.9 b–e 53.9 ± 17.7 b–d
SJA 12.9 ± 3.4 bc - 71.0 ± 19.5 b 65.6 ± 33.3 b
CAA 13.6 ± 1.9 b - 67.5 ± 9.5 bc 57.4 ± 5.1 bc
P-69 23.7 ± 4.9 a - 141.4 ± 50.9 a 141.0 ± 43.8 a
P-26 9.3 ± 0.8 de - 51.6 ± 6.9 c–f 42.4 ± 6.7 c–e
P-96C2 6.0 ± 0.6 f–h - 34.1 ± 7.3 g–j 34.3 ± 8.6 e
P-91C2 7.7 ± 1.2 d–g - 39.3 ± 7.8 f–j 33.8 ± 3.9 e
P-102 12.9 ± 3.0 bc - 65.2 ± 18.5 b–d 58.8 ± 10.5 bc
P. vulgaris L. 7.2 ± 1.9 B 2.2 ± 1.0A 34.3 ± 11.3 B 27.0 ± 10.9 B
P-34 6.5 ± 0.3 e–h - 31.5 ± 10.8 g–k 30.2 ± 8.9 ef
P-68 7.9 ± 0.4 d–g - 40.1 ± 2.7 f–j 28.9 ± 4.1 ef
P-76 6.5 ± 0.9 e–h - 45.6 ± 7.5 e–g 31.0 ± 10.7 ef
P-19 10.1 ± 1.0 cd - 45.2 ± 6.3 e–h 44.8 ± 7.4 c–e
P-71 4.2 ± 0.9 h - 14.3 ± 1.9 k 14.2 ± 5.1 f
P-58 9.0 ± 4.0 d–f - 28.1 ± 9.5 h–k 15.2 ± 3.7 f
P-65 5.7 ± 0.8 gh - 26.2 ± 2.0 i–k 16.1 ± 3.0 f
P-16 7.9 ± 1.1 d–g 2.9 ± 1.6 a 41.0 ± 7.0 f–i 34.9 ± 7.0 e
P-40 9.1 ± 4.1 d–f 1.2 ± 0.5 b 48.0 ± 15.6 d–g 37.8 ± 13.7 de
FCA-04 4.9 ± 0.6 gh - 23.4 ± 3.0 jk 16.4 ± 3.7 f
TPC: Total polyphenol content (mg GAE g−1 dw), TAC: Total anthocyanin content (mg C3G g−1 dw), DPPH:
Antioxidant activity (µmol TE g−1 dw), FRAP: Antioxidant capacity (µmol TE g−1 dw). SLMV: Santa Lucia Monte
Verde, SJA: San Juan Atepec, CAA: Cerro Amole Ayutla. 1 Between species (capital letter) and among landraces
(lowercase letter), means with same letter indicate non-significant differences (Tukey’s test, p < 0.05).

Regarding TAC, only green beans of three genotypes presented monomeric antho-
cyanins detectable values, following an order from higher to lower concentration, purple
beans of P-16 (2.9 mg C3G g−1 dw) and red beans of P-40 (1.2 mg C3G g−1 dw) from
P. vulgaris species, and green beans from SLMV (0.7 mg C3G g−1 dw) from P. coccineus
(Table 3).
The close relationship between TPC and antioxidant activity has been documented in
various fruits and vegetables, as observed in this study (Table 4). Most of the evaluated
green bean landraces showed a strong correlation (p < 0.05) between the phenolic com-
pounds content and the antioxidant activity determined by the DPPH free radical reduction.
Agronomy 2021, 11, 803 8 of 12

The P-69 landrace of the P. coccineus species that exhibited a high concentration of phenolic
compounds also presented high antioxidant activity (141.4 µmol TE g−1 dw), as well as its
ability to reduce iron (141.0 µmol TE g−1 dw). In contrast, P-71 which showed the lowest
phenolic compounds concentration (4.2 mg GAE g−1 dw) also showed the lowest antioxi-
dant activity by DPPH and FRAP method (14.3 µmol TE g−1 dw and 14.2 µmol TE g−1 dw,
respectively). However, this relationship was not observed in all landraces, since SLMV
presented the highest TPC average (26.5 mg GAE g−1 dw), reporting an activity against
DPPH of 58.6% lower compared to P-69, probably due to the variability in the individual
polyphenols profile and that each compound can exert a different reducing activity.

Table 4. Correlation coefficients between bioactive compounds, in vitro antioxidant activity, and
physicochemical parameters in green beans from bean landraces.

Variable TPC TA TS FRAP DPPH h◦ C*

TPC 1.0
TA 0.4 **,2 1.0
TS 0.3 ** 0.5 ** 1.0
FRAP 0.7 ** 0.5 ** 0.5 ** 1.0
DPPH 0.8 ** 0.5 ** 0.5 ** 0.9 ** 1.0
hº 0.3 ** 0.1 ** 0.2 ** 0.4 ** 0.4 ** 1.0
C* 0.1 ns,1 0.1 ns 0.1 ns 0.2 ** 0.2 ** 0.6 ** 1.0
1,ns not significant at p < 0.05, 2, **significant (Student’s test, p < 0.001).

A similar effect was observed for the antioxidant activity determined by the FRAP
technique. Most of the landraces that presented a high DPPH radical reducing activity
also showed an ability to reduce the ferric ion, although some landraces, mainly those of
the P. vulgaris genus, showed an activity between 15% (P-16) and 46 % (P-58) lower in the
FRAP technique compared to DPPH.

4. Discussion
In general, regarding the physicochemical parameters of pH and titratable acidity,
there were no differences between P. vulgaris and P. coccineus. Organic acids concentration
determines the physiological and commercial fruits maturity such as green beans [28]. In
this sense, the state of maturity chosen for this study shows pH values similar to those
reported before [29] with a pH value of 6.2 and 6.4 for the Strike and Bina green bean
varieties (P. vulgaris L.), respectively, and 5.8–6.4 for green beans (Phaseolus vulgaris L. var.
Helda) in an immature state [30]. In titratable acidity, Garzón-García et al. [15] reported
a higher value (1.6 g citric acid 100 g−1 dw) in green beans (Phaseolus vulgaris L.) in the
same state of maturity as that used in this study. Regarding the sugars content, and
color parameters L*, b*, C*, and h◦ , the P. coccineus species presented the highest average
values. It has been reported that the main sugars in green beans are glucose, fructose, and
sucrose [31], which could constitute the total sugar content in this study, presenting a lower
average value (32.7 mg Glu g−1 dw) compared to green beans (Phaseolus vulgaris L.) from a
native bean common accession from Oaxaca [32].
Regarding the luminosity parameter, an interval of 36.5–50.3 lower than that registered
in the two species evaluated in this study has been reported [29] in P. vulgaris L. green
beans in an advanced stage of development. Regarding the TSS content, P. vulgaris species
was higher than P. coccineus, whose values has been reported before [29] on a range of
9.5–20.1 ◦ Brix, in the Strike and Bina green bean varieties (P. vulgaris L.) in different
development stages, similar to those obtained in this study. Green beans (Phaseolus vulgaris
L. cv. Paulista) irrigated by drip and furrows presented values of 4.37 and 4.60 ◦ Brix,
respectively [33], similar to Phaseolus vulgaris ssp. Volubilis grown in soils rich in organic
matter and nitrogen fertilization with values of 4.9 to 5.7 ◦ Brix [34], being much lower
than that obtained in the two green beans species analyzed. Thus, the total soluble solids
content variation, constituted by organic acids, but also total sugars, can be influenced by
Agronomy 2021, 11, 803 9 of 12

agricultural practices, its post-harvest handling, as well as the state of maturity in which it
is harvested [35,36].
Regarding protein content, Martínez et al. [37] reported a range of 16.9–17.2 g protein
100 g−1 dw in the Cleo, Strike, and Sentry green bean varieties, while for beans of the
species P. vulgaris, P. coccineus, Black Horse, Michigan, and Peruvian, a range of 12.6–21.3 g
protein 100 g−1 dw is reported [38]. For their part, Bhagya et al. [39], recorded a protein
content in tender pods beans of Canavalia cathartica of 21.7 g protein 100 g−1 dw. In
contrast, in pea pod (Pisum sativum L.) and broad bean pod (Vicia faba L.), values of 10.8 and
13.6 g protein 100 g−1 dw were obtained [40], respectively, similar to what was observed in
this study in the species P. vulgaris.
Phenolic compounds concentration in the evaluated samples registered a high variabil-
ity, showing the highest values those P. coccineus species reddish landraces, which coincides
with that reported in Oaxaca landrace green beans of the P. vulgaris species [15]. Similar
results have been observed before [41,42], where the reddish bean landraces presented
higher phenolic compounds concentrations, compared to the green landraces. However,
in this study, no correlation was found between the reddish landraces of the P. vulgaris
species and the content of phenolic compounds, even though these species showed the
highest anthocyanin content. Still, the TPC average results in both species were higher than
the values reported [43] where concentrations of 3.6 mg GAE g−1 dw were observed for
varieties of green beans marketed in Turkey, as well as those recorded of 1.1–2.0 mg GAE
g−1 dw [44,45] for other Phaseolus vulgaris varieties, including Emerit, which showed a TPC
of 2251 mg GAE g−1 dw [46]. On the other hand, the evaluation of the phenolic compounds
content and antioxidant activity for three varieties of green beans has shown values of 14.6
for the green variety and 40.8 µmol TE g−1 dw for the reddish variety [41], which agrees
with values reported in the present study. This great variability observed in the phenolic
compounds content, anthocyanins, and antioxidant activity in the species P. coccineus and
P. vulgaris green beans could be directly related to the phenolic compounds profile, which
can vary widely due to the state of maturity, the accession, species, geographic location,
and cultural practices within that region [47]. This not only promotes phenotypic differ-
entiation, but also at the genetic level, in the identified phenolic compounds expression
and synthesis (gallic acid, chlorogenic, epicatechin, rutin, and more than 72 polyphenols
including 10 phenolic acids, 59 flavonoids, 2 lignans, and 1 iridoid) in different green
beans landraces (P. vulgaris L. var Perona, Helda and Strike), as well as in different parts
of this crop such as flowers [48], leaves and stems [49], and seeds [50,51]. Furthermore,
the aqueous extract of Phaseous vulgaris species green beans has been reported to have
anti-obesogenic and hypoglycemic activity [52–56], which could help combat the obesity
pandemic currently present in Mexico [57] and the world [58], as well as reduce the factors
associated with metabolic syndrome, such as cardiovascular diseases, which are the main
cause of mortality in the world [59].

5. Conclusions
Based on the two green beans species evaluated, we can conclude that there are
significant differences between P. coccineus and P. vulgaris in the content of TS, TSS, and color,
as well as in their content of polyphenols, flavonoids, anthocyanins, and antioxidant activity.
P. coccineus was higher in phenolic compounds and antioxidant activity, while P. vulgaris
was higher in anthocyanins. There was a high variability between the landraces evaluated,
and, specifically, P. vulgaris landraces had more than double the anthocyanins compared to
those of P. coccineus. Both species are a source of complementary polyphenols, flavonoids,
and anthocyanins, and this suggests that their use can be considered a complementary food
to improve the rural community’s nutrition or implement a genetic improvement program
towards a higher nutritional quality of the prominent pods where their food base is in
conjunction with other native crops such as corn, squash, and chili. However, other studies
are necessary to determine their potential in preventing some chronic degenerative diseases.
Agronomy 2021, 11, 803 10 of 12

Author Contributions: J.E.A.-J., E.N.A.-B., J.L.C.-S., J.C.C.-R., and M.A.S.-B. performed the research,
data analysis, and wrote the paper; A.K.G.-G., A.M.V.-G., and E.N.A.-B. contributed with laboratory
analysis. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by Consejo Nacional de Ciencia y Tecnología CONACYT Problemas
Nacionales (Project no. 2015-1119) and Instituto Politécnico Nacional (Project 20201270). A.K.G.-G.
received a scholarship from the CONACyT (No. 478457) to study a Master’s in Food Sciences.
Acknowledgments: Biological material were sown and cultivated by Dionisio Ruíz-Landa in the
Training Unit for Rural Development (UNCADER 2) located in Coatepec, Veracruz, Mexico.
Conflicts of Interest: No potential conflict of interest was reported by the authors.

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