Academic Editor: Marcello Iriti
Background: Biofortification is a method that improves the nutritional
value of food crops through conventional plant breeding. The aim of this study
was to evaluate the effects of intra-amniotic administration of soluble extracts
from zinc (Zn) biofortified and Zn standard cowpea (Vigna unguiculata L.
Walp.) flour on intestinal functionality and morphology, inflammation, and gut
microbiota, in vivo. Methods: Seven treatment groups were
utilized: (1) No Injection; (2) 18 M
Zinc (Zn) is essential for human health due to its key role as a required cofactor in numerous enzymatic reactions in the body. Zn holds a vital role during infants’ growth and development phase, and contributes to immune system maintenance [1, 2]. Zn deficiency has been correlated with stunted growth, immune system depletion, and adverse pregnancy outcomes [3, 4]. An estimative of World Health Organization (WHO) showed that one-third of the global population is at risk for Zn deficiency, data calculated considering those individuals with intake lower than the daily requirements of Zn [5], thus improving Zn status through an increase of dietary Zn absorption is considered a critical challenge to public health [6, 7]. Worldwide, Zn deficiency is the second most prevalent mineral deficiency, just behind iron (Fe) deficiency, and is estimated to affect 17% of the global population. This is mainly attributed to the low Zn bioavailability in food [4, 8].
Cowpea is a nutritious crop and widely consumed in West Africa and North and
Northeast Brazil [9], and its high tolerance to heat and drought makes it a
relevant target crop for Zn biofortification. Biofortified cowpea cultivars
present equal to or above 40 and 60 mg Kg
The promising chemical and polyphenolic composition [12] of the grain, combined with its undemanding agronomic characteristics, make cowpea favourable to low-income farmers, who have limited access to nutritionally-balanced diets and are highly susceptible to micronutrient malnutrition [13]. Polyphenols are a class of compounds naturally present in beans; some coloured beans have a higher content of phenolic compounds, which can potentially inhibit Zn bioavailability [10, 14, 15]. However, phenolic compounds have also been associated with beneficial health effects, such as anti-inflammatory and antioxidant properties [16, 17] and improvement of intestinal health [18, 19].
Cowpea flour also contains soluble compounds, such as soluble dietary fiber, which can act as prebiotics. Prebiotics are non-digestible complex carbohydrates that resist digestion in the gastrointestinal tract and are fermented in the colon [20]. Metabolites produced by gut microbiota fermentation of prebiotics can confer benefits to host health [21]. Gut microbiota fermentation of prebiotics can lead to the production of short-chain fatty acids (SCFA) and a decrease in intestinal lumen pH, beneficially affecting the gut microbiome and intestinal health [22, 23, 24].
Previous studies have shown the role of Zn to support
Studies evaluating the effects of food intake as part of biofortification programs on intestinal functionality, morphology and microbiota are limited. This is the first study with Zn biofortified cowpea in this line of investigation; as the effects of intra-amniotic administration of soluble extracts from Zn biofortified cowpea cultivars on intestinal health are unknown. Hence, the objectives of this study were to investigate the effects of the Zn biofortified and standard cowpea soluble extracts on Zn and Fe related BBM proteins and BBM functionality and inflammation, as well as to assess the effects of cowpea cultivars on the cecal microbiota and intestinal morphology in vivo (Gallus gallus). In addition, this study aimed to contribute to scientific advances and the utilization of Zn biofortified foods, and provide the basis for developing dietary strategies aimed to combat micronutrient deficiencies in vulnerable populations.
Grains of four cowpea cultivars were used to conduct this experiment: Zn
standard BRS Pajeú, and Zn biofortified BRS Aracê, BRS Imponente and BRS
Xiquexique. All cultivars were obtained from Embrapa Meio-Norte, Teresina, PI,
Brazil. The cultivars’ grains were shipped to the Department of Nutrition and
Health, Federal University of Viçosa, Viçosa, Brazil, and were cooked in
three replicates in a conventional pressure cooker for 25 min using a
bean/distilled H
As previously described [33, 39], the cowpeas flour samples were homogenized in
distilled H
The dietary fiber and protein content were determined according to the methodology proposed by the Association of Official Analytical Chemistry (AOAC) [40], in duplicate. For dietary fiber assessment, samples were enzymatically hydrolyzed using heat-resistant amylase, protease and amyloglucosidase enzymes from total dietary fiber assay (Kiyonaga, Sigma®, Kawasaki, Japan). Dietary phytic acid (phytate)/total phosphorous assay was used to determine phytate content following specific kit instructions (K-PHYT 12/12, Megazyme International, Bray, Ireland).
Determination of Fe and Zn concentration in cowpeas flour was performed as
previously described [33, 35]. For analysis, 500 mg samples of each respective
cowpea flour were pre-processed at room temperature for 16 h, in borosilicate
glass tubes added with 3 mL concentrated nitric acid and perchloric acid (60:40
v/v). After, samples were maintained for 4 h in a heated (120
1 g of each respective cowpea flour was added with 5 mL of methanol/H
Extracts and standards were assessed using an Agilent 1220 Infinity Liquid
Chromatograph (LC; Agilent Technologies, Inc., Santa Clara, CA, USA) combined
with an Advion expression LC mass spectrometer (CMS; Advion Inc., Ithaca, NY,
USA). 10
Cornish-cross fertile broiler eggs (n = 63), acquired from a commercial hatchery
(Moyer’s Chicks, Quakertown, PA, USA), were properly incubated [41] at Cornell
University Animal Science Poultry Farm incubator. Lyophilized soluble extracts
were separately diluted in deionized H
Immediately after hatch (21 days), chicks were weighed and then euthanized by
CO
30 mg of the liver tissue or proximal duodenal tissue (n = 5) were weighed for
the total RNA extraction. Qiagen RNeasy Mini Kit (RNeasy Mini Kit, Qiagen Inc.,
Valencia, CA, USA) was applied according to the kit manufacturer’s protocol. All
stages were executed under RNase-free conditions. Briefly, with a rotor–stator
homogenizer and containing
Each sample (700
RT-PCR was performed as previously published [39, 42, 43]. Briefly, 20
The primers used in the real-time PCR were designed. This procedure was based on gene sequences from the GenBank database, using Real-Time Primer Design Tool software (IDT DNA, Coralville, IA, USA), as previously described [39, 42, 43]. Primers sequences used in this study were summarized in Table 1. Through performing a BLAST search against the genomic National Center for Biotechnology Information (NCBI) database, the specificity of the primers was tested. The reference gene used was the 18S rRNA specific for the Gallus gallus model.
Analyte | Forward primer (5′-3′) | Reverse primer (5′-3′) | Base Pairs Length | GI identifier |
Zinc and iron metabolism | ||||
DMT-1 | TTGATTCAGAGCCTCCCATTAG | GCGAGGAGTAGGCTTGTATTT | 101 | 206597489 |
Ferroportin | CTCAGCAATCACTGGCATCA | ACTGGGCAACTCCAGAAATAAG | 98 | 61098365 |
DcytB | CATGTGCATTCTCTTCCAAAGTC | CTCCTTGGTGACCGCATTAT | 103 | 20380692 |
ZnT-1 | GGTAACAGAGCTGCCTTAACT | GGTAACAGAGCTGCCTTAACT | 105 | 54109718 |
ZnT-7 | GGAAGATGTCAGGATGGTTCA | CGAAGGACAAATTGAGGCAAAG | 87 | 56555152 |
ZIP-9 | CTAAGCAAGAGCAGCAAAGAAG | CATGAACTGTGGCAACGTAAAG | 100 | 237874618 |
Δ-6-desaturase* | GGCGAAAGTCAGCCTATTGA | AGGTGGGAAGATGAGGAAGA | 93 | 261865208 |
Δ-5-desaturase* | GTACTTCTTCATCATTGGTCCC | CCCAGGATACCCTTCACAC | 171 | 423120 |
BBM functionality | ||||
AP | CGTCAGCCAGTTTGACTATGTA | CTCTCAAAGAAGCTGAGGATGG | 138 | 45382360 |
SI | CCAGCAATGCCAGCATATTG | CGGTTTCTCCTTACCACTTCTT | 95 | 2246388 |
SGLT-1 | GCATCCTTACTCTGTGGTACTG | TATCCGCACATCACACATCC | 106 | 8346783 |
MUC-2 | CTGCTGCAAGGAAGTAGAA | GGAAGATCAGAGTGGTGCATAG | 272 | 423101 |
Inflammation | ||||
NF-κB1 | CACAGCTGGAGGGAAGTAAAT | TTGAGTAAGGAAGTGAGGTTGAG | 100 | 2130627 |
TNF- |
GACAGCCTATGCCAACAAGTA | TTACAGGAAGGGCAACTCATC | 109 | 53854909 |
IL-8 | TCATCCATCCCAAGTTCATTCA | GACACACTTCTCTGCCATCTT | 105 | 395872 |
18s rRNA | GCAAGACGAACTAAAGCGAAAG | TCGGAACTACGACGGTATCT | 100 | 7262899 |
DMT-1, Divalent metal transporter-1; DcytB, Duodenal cytochrome B; Znt and ZIP,
Zinc transporter proteins; BBM, Brush border membrane; AP, Aminopeptidase; SI,
Sucrose isomaltase; SGLT-1, Sodium-glucose transport protein 1; MUC-2,
Mucin-secreting intestinal protein-2; NF- * Liver analysis. |
For the RT-qPCR design, all procedures were conducted as previously described
[35, 39, 42, 43]. Each 10
Gene expressions were quantified as Cp values based on the “second derivative
maximum” (automated method) as computed by Bio-Rad CFX Maestro 1.1 (Version
4.1.2433.1219, Hercules, CA, USA). All tests were measured by including a
standard curve in the real-time qPCR analysis. The standard curve was prepared
using 1:10 serial dilution, in duplicate. Software generated a graph with the
concentrations of Cp vs. log10, and the efficiencies were calculated as
10[1/slope]. The specificity of the amplified real-time RT-PCR products was
verified by melting curve analysis (60–95
As was previously described, the cecum was sterilely removed and treated
[24, 34]. To collect microbial samples, the cecum content was placed into a
sterile 15 mL tube, containing 9 mL of sterile PBS, and homogenized with glass
beads (3 mm diameter) for 3 min. Through centrifugation, debris was removed, at
1000
As previously described, primers for Lactobacillus, Bifidobacterium, Clostridium and E. coli were utilized [33, 39]. To estimate the relative proportion of each studied bacteria, each product was expressed relative to the content of the universal primer product, and proportions of each bacterial group are presented. PCR products were separated using electrophoresis on 2% agarose gel, stained with ethidium bromide, and quantified using the Quantity One 1-D analysis software (Bio-Rad, Hercules, CA, USA).
Analysis of the intestinal morphology was conducted as previously described
[39, 42]. Briefly, samples from the duodenum were fixed in fresh 4% (v/v)
buffered formaldehyde, dehydrated, cleared and implanted in paraffin. Serial
sections were cut at 5
The data were expressed as means and standard deviation. Experimental groups for
the intra-amniotic administration procedure were arranged in a completely
randomized design. Determined parameters were noticed to have a normal
distribution and equal variance through a Shapiro-Wilk test and were, therefore,
acceptable for one-way analysis of variance (ANOVA). For significant
“p-value”, test groups were compared using Duncan post-hoc test, with
the significance level established at p
The total dietary fiber and insoluble dietary fiber concentrations were higher
(p
BRS Pajeú | BRS Aracê | BRS Imponente | BRS Xiquexique | |
TDF (g/100 g) | 19.02 |
13.82 |
11.65 |
15.10 |
SDF (g/100 g) | 1.59 |
1.07 |
1.16 |
0.91 |
IDF (g/100 g) | 17.43 |
12.75 |
10.50 |
14.19 |
Protein (g/100 g) | 22.28 |
26.08 |
25.03 |
23.04 |
Fe (µg/g) | 55.27 |
54.54 |
49.47 |
61.25 |
Zn (µg/g) | 31.09 |
36.34 |
40.91 |
37.19 |
Phytate (g/100 g) | 0.78 |
0.76 |
0.90 |
0.81 |
Phytate:Fe molar ratio | 11.91 |
11.81 |
15.49 |
11.18 |
Phytate:Zn molar ratio | 24.78 |
20.74 |
21.92 |
21.55 |
Values are means BRS Pajeú, Zn-standard; BRS Aracê, BRS Imponente and BRS Xiquexique, Zn-biofortified; TDF, Total dietary fiber; SDF, Soluble dietary fiber; IDF, Insoluble dietary fiber; Fe, Iron; Zn, Zinc. |
The concentration of the eight most prevalent polyphenolic compounds found in
the Zn biofortified and Zn standard cowpea flours is shown in Table 3. BRS
Aracê flour showed the highest (p
BRS Pajeú | BRS Aracê | BRS Imponente | BRS Xiquexique | |
Epicatechin | 37.23 |
39.00 |
- | 38.94 |
Kaempferol 3-sambubioside | 0.19 |
0.24 |
0.13 |
0.19 |
Myricetin | 22.45 |
22.95 |
22.51 |
22.68 |
Myricetin 3-glucoside | 5.08 |
2.47 |
1.87 |
2.33 |
Protocatechuic acid | 12.19 |
0.21 |
0.70 |
0.26 |
Quercetin | 1.68 |
1.51 |
- | - |
Quercetin 3-glucoside | 1.62 |
0.36 |
0.32 |
0.42 |
Quercetin 3-rutinoside | 0.24 |
0.35 |
0.25 |
0.30 |
Values are means BRS Pajeú, Zn-standard; BRS Aracê, BRS Imponente and BRS Xiquexique, Zn-biofortified. |
There was no significant difference (p
The gene expression of duodenal cytochrome b (DcytB), divalent metal transporter
1 (DMT1) and ferroportin in the three Zn biofortified cowpea soluble extracts
were similar (p
Effect of the intra-amniotic administration of cowpea soluble
extracts on gene expression of proteins involved with Zn and Fe metabolism.
Values are the means
The gene expression of sodium-glucose transport protein 1 (SGLT1), sucrose
isomaltase (SI), aminopeptidase (AP), and mucin-secreting intestinal protein 2
(MUC2) are commonly used as biomarkers of BBM digestive and absorptive functions.
In the present study, the treatment with soluble extracts of Zn biofortified
cowpea did not alter (p
Effect of the intra-amniotic administration of cowpea soluble
extracts on gene expression of proteins involved with the BBM functionality and
inflammation. Values are the means
The expression of markers related to inflammatory mechanisms is presented in
Fig. 2. The expression of NF-
There was no difference (p
Effect of the intra-amniotic administration of Zn biofortified
cowpea soluble extract on genera- and species-level bacterial populations from
cecal contents measured on the day of hatch. Values are the means
In addition, the standard BRS Pajeú (Zn standard) soluble extract increased
the (p
The villus surface area was higher (p
Treatment group | Villus surface area (mm |
Villi goblet cell number (Unit) | Villi goblet diameter (µm) | Villus goblet cell number (Unit) | ||
Acid | Neutral | Mixed | ||||
No Injection | 353.39 |
39.63 |
3.45 |
31.89 |
1.85 |
5.89 |
18 MΩ H |
261.12 |
28.94 |
3.43 |
16.27 |
1.42 |
5.29 |
Inulin | 384.57 |
25.50 |
3.20 |
23.96 |
0.08 |
1.46 |
BRS Pajeú | 326.62 |
40.56 |
3.28 |
33.82 |
0.88 |
5.86 |
BRS Aracê | 318.97 |
34.14 |
3.31 |
30.63 |
0.44 |
3.20 |
BRS Imponente | 327.45 |
36.47 |
3.30 |
31.58 |
0.65 |
4.30 |
BRS Xiquexique | 351.28 |
36.05 |
3.30 |
32.31 |
0.33 |
3.41 |
Values are the means |
In relation to the types of goblet cells in the crypt epithelium, we observed an
increase (p
In the crypt, we observed an increase (p
Treatment group | Crypt goblet diameter (µm) | Crypt goblet cell number (Unit) | Crypt depth (µm) | Paneth cell/crypt (Unit) | Paneth cell diameter (µm) | Crypt goblet cell number (Unit) | ||
Acid | Neutral | Mixed | ||||||
No Injection | 3.24 |
10.15 |
22.04 |
1.81 |
2.88 |
7.74 |
1.56 |
0.86 |
18 MΩ H |
2.74 |
11.14 |
17.8 |
2.32 |
1.70 |
7.66 |
2.62 |
0.86 |
Inulin | 2.18 |
10.78 |
21.49 |
2.29 |
1.64 |
8.45 |
0.63 |
1.70 |
BRS Pajeú | 2.80 |
8.35 |
22.31 |
1.81 |
1.58 |
7.40 |
0.29 |
0.66 |
BRS Aracê | 2.02 |
11.01 |
21.7 |
1.89 |
1.80 |
7.49 |
2.04 |
1.48 |
BRS Imponente | 2.22 |
9.87 |
20.66 |
1.81 |
1.81 |
6.89 |
1.60 |
1.38 |
BRS Xiquexique | 3.27 |
10.26 |
25.22 |
2.04 |
1.77 |
8.96 |
0.66 |
0.65 |
Values are the means |
In relation to the types of goblet cells in the crypt, the BRS Xiquexique
presented the highest and BRS Imponente presented the lowest (p
In the present study, four cowpea cultivars (Vigna unguiculata L. Walp.) were assessed following intra-amniotic administration (Gallus gallus) of its soluble extracts, with the aim to investigate the potential of standard (BRS Pajeú) and Zn biofortified cowpeas (BRS Aracê, BRS Imponente and BRS Xiquexique) in improving intestinal bacterial composition and morphology, brush border membrane (BBM) functionality and inflammation. Cowpeas are a nutritious crop and a widely consumed legume in West Africa and North and Northeast Brazil with a high tolerance to heat and drought, making cowpeas a great target crop for Zn biofortification [9, 11]. The cowpea flour used in this study showed a significant concentration of protein, dietary fiber, Zn and Fe, and polyphenols, specifically, epicatechin, myricetin and quercetin (Tables 2 and 3). Studies have shown the potential of soluble fiber, phenolic compounds and minerals from biofortified foods to improve mineral bioavailability and gut functionality [23, 33, 35, 37, 47, 48].
In this study, we observed that BRS Imponente and BRS Xiquexique soluble
extracts decreased the populations of Clostridium and E. coli
in comparison to all the other experimental groups. Further, despite the
reduction in Bifidobacterium, the BRS Xiquexique treatment group
demonstrated an increased relative abundance of Lactobacillus compared
to BRS Imponente (Fig. 3). These observations are also associated with improved
intestinal morphology, as indicated by increased crypt depth, crypt goblet
diameter, villi goblet number and villi acidic goblet cell number, in the BRS
Xiquexique group, compared to the inulin and 18 M
The BRS Xiquexique flour showed the lowest phytate: Fe molar ratio compared to the other tested Zn biofortified cowpea flour, and a lower phytate: Zn molar ratio than the standard BRS Pajeú. This may indicate higher mineral bioavailability in the intestinal lumen, where minerals could be utilized by bacteria that colonize the gastrointestinal tract [48, 51]. The chemical composition of the food matrix of a bean cultivar can determine its effects on intestinal functionality and health [33, 35, 47]. BRS Xiquexique showed improved results, compared to the other varieties with increased levels of Zn, possibly due to its higher content of dietary fiber the lower phytate: Fe molar ratio, which increases the mineral bioavailability and is associated with its polyphenolic profile. Further, BRS Xiquexique showed high levels of gallic and ferulic acids, supporting an antioxidant and functional potential demonstrated in the present study [52]. Bacteria that inhabit the gut lumen are mineral dependent, therefore, an increased supply of Zn and Fe can increase the abundance of beneficial phyla and genera [31, 43]. Several bacterial species have the ability to ferment dietary soluble fibers and produce short-chain fatty acids (SCFA), which is a valuable metabolite used by enterocytes as a source of energy and nutrition [53]. Lactobacillus is a probiotic genus generally regarded as safe (GRAS); this genus harbors SCFA producing species, where SCFA production has been associated with anti-inflammatory properties [54, 55]. Further, the reduction in potentially pathogenic Clostridium and E. coli is associated with two treatment groups, Zn biofortified BRS Imponente and BRS Xiquexique, and suggests an improvement in the gut health [24, 33].
Among treatment groups with Zn biofortified cowpea beans soluble extracts, BRS
Aracê showed an increase total goblet cell number and acidic type goblet cell
number per villi, compared to the inulin and 18 M
Paneth cells play a key role in intestinal immunity and host defense, secreting
anti-microbial compounds and other substances that contribute to maintaining the
intestinal barrier [57]. Paneth cell number was increased in the Zn
biofortified-BRS Xiquexique treatment group, compared to the standard Zn
standard-BRS Pajeú. Further, an increased Paneth cell diameter was measured
in the three Zn biofortified soluble extracts treated groups compared to the Zn
standard (Table 5). Paneth cells number and size can reflect the early stage of
intestinal inflammation since Paneth cell-produced lysozyme regulates intestinal
anti- and pro-inflammatory responses [58, 59]. Current data agrees with the gene
expression of NF-
BRS Xiquexique and BRS Aracê flour presented higher epicatechin contents
than BRS Pajeú, which may explain the improved barrier function in these
treatment groups. Compared with cultivars of Fe biofortified and Fe standard
common bean [35], the cowpea flour polyphenolic profile assessed in the present
study had a higher concentration of epicatechin and quercetin 3-glucoside. As
previously demonstrated [60], derivates of myricetin and quercetin constitute the
most abundant flavonoids in the cowpea, and this flavonoid profile has a major
impact on the bioactive properties of this legume. Flavonoids, such as
epicatechin, are metabolized by the gut microbiota, generating metabolites that
are more potent than the primary compound, such as
epicatechin-3
The current study also assessed the potential effects of Zn biofortified and Zn
standard cowpea soluble extracts on the gene expression of key Fe and Zn
metabolism associated BBM proteins BBM functional and inflammation proteins. In
general, there were no significant differences in the gene expression of proteins
related to Zn and Fe absorption (DcytB, DMT1, ferroportin, ZIP9, ZnT1, ZnT7, and
Studies with biofortified foods show that the increased amounts of Zn and Fe in
food matrices have the potential to improve the absorption of these minerals by
improving BBM functionality [33, 35, 48, 67]. In the present study, we did not
observe differences in the gene expression of proteins associated with BBM
functionality (SGLT1, SI, AP, and MUC2) in the Zn biofortified and standard
treatment groups in comparison to the inulin and 18 M
Thus, our results demonstrate the potential benefit of biofortified cowpea extracts to improve intestinal morphology, BBM functionality, inflammation, and gut microbiota. These observations were significant specifically in the BRS Xiquexique group, with clear improvements in intestinal bacterial populations and intestinal morphological biomarkers.
The intra-amniotic administration of Zn biofortified cowpea soluble extracts demonstrated potential nutritional benefit, as was demonstrated by the improved intestinal morphology, BBM functionality, and cecal microbial composition. The promising effects shown by BRS Xiquexique and BRS Imponente in improving Zn BBM transport and by BRS Xiquexique in improving intestinal morphology indicate these are the most promising cultivars to be considered by biofortification programs.
In addition, we underlined the need for continuous studies on the benefits of new Zn biofortified cowpea cultivars, and we emphasize that the consumption of these beans cultivars should be encouraged in other regions of the world besides West Africa and Northern Brazil. Based on the results of our preliminary study, the new cowpea cultivars have the potential to improve human health, although further studies are necessary to support these findings.
ET led the research, conceived and supervised the project; MJCG, HSDM and ET developed and designed the experiment; MJCG, NK and ET conducted the experiment; MJCG, NK, JC and NA analyzed the data; MJCG drafted the manuscript; NK, JC, NA and MMR participated in data analysis and manuscript editing.
All animal care and experimental procedures complied with the Cornell University Institutional Animal Care and Use Committee (approval number: IACUC #2020-0077).
The authors would like to thank Embrapa Mid-North (Brazil) for providing the Zn-biofortified cowpea samples. The first author is grateful to the Fulbright Commission Brazil for the grant (2020–2021) that supported her research in the United States, and she thanks the Department of Food Science at Cornell University for providing facilities during her split Ph.D. in the United States.
This research received no external funding.
The authors declare no conflict of interest.