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Journal of Food Research; Vol. 9, No. 1; 2020
ISSN 1927-0887 E-ISSN 1927-0895
Published by Canadian Center of Science and Education
EDITORIAL TEAM
EDITOR-IN-CHIEF
Antonello Santini, University of Napoli "Federico II", Italy
ASSOCIATE EDITORS
Anna Iwaniak, Warmia and Mazury University, Poland
Bülent Ergönül, Celal Bayar University, Turkey
Corina-aurelia Zugravu, University of Medicine and Pharmacy Carol Davila, Romania
Zafar Iqbal, Carleton University, Canada
EDITORIAL ASSISTANT
Bella Dong, Canadian Center of Science and Education, Canada
EDITORIAL BOARD MEMBERS
Adele Papetti, Italy
Afef Janen, United States
Akhila Vasan, USA
Akshay Kumar anugu, United States
Alex Augusto Gonçalves, Brazil
Alexandre Navarro Silva, Brazil
Alexandrina Sirbu, Romania
Alicia Guadalupe Marroquin-Cardona, Mexico
Aly R Abdel-Moemin, Egypt
Amin Mousavi Khaneghah, Brazil
Amira Mohamed Elkholy, Egypt
Ammar Eltayeb Ali Hassan, Norway
Ana Silva, Portugal
Ancuta Elena Prisacaru, Romania
Andreea Stanila, Romania
Anna Iwaniak, Poland
Anna Maria Pappalardo, Italy
Antonella Santillo, Italy
Asima Asi Begic-Akagic, Bosnian
Aytunga Bagdatli, Turkey
Beatriz Sevilla-Moran, Spain
Belay Berza Beyene, Ethiopia
Bernardo Pace, Italy
Bojana Filipcev, Serbia
Bruno Alejandro Irigaray, Uruguay
Bülent Ergönül, Turkey
Cemil Kurekci, Turkey
Cheryl Rosita Rock, United States
Claudia Alejandra Narvaez, Canada
Codina Georgiana Gabriela, Romania
Coman Gigi, Romania
Constantina Nasopoulou, Greece
Corina-aurelia Zugravu, Romania
Cristiano Ragagnin de Menezes, Brazil
Devinder Dhingra, India
Diego A. Moreno-Fernández, Spain
Djilani Abdelouaheb, Algeria
Domitila Augusta Huber, Brazil
Eduardo Esteves, Portugal
Efstathios S Giotis, United Kingdom
Eganathan Palanisami, India
Elke Rauscher-Gabernig , Austria
Elsa M Goncalves, Portugal
Emma Chiavaro, Italy
Essence Jeanne Picones Logan, Philippines
Estela de Rezende Queiroz, Brazil
Fernanda Papa Spada, Brazil
Fernando Sanches Lima, Brazil
Francesco Vizzarri, Italy
Fu Chen, United States
Gisele Fátima Morais Nunes, Brazil
Greta Faccio, Switzerland
Guojie Cao, United States
Haihan Chen, United States
Hamid El Bilali, Italy
Ilija Djekic, Serbia
Ilkin Yucel Sengun, Turkey
Ionel D. Bondoc, Romania
Isabela Mateus Martins, Brazil
J. Basilio Heredia, Mexico
Jasdeep Saini, United States
Jelena Dragisic Maksimovic, Serbia
http://jfr.ccsenet.org Journal of Food Research Vol. 9, No. 1; 2020
Jelena Vulic, Serbia
Jintana Wiboonsirikul, Thailand
Jose M. Camina, Argentina
Jose Maria Zubeldia, Spain
Juan José Villaverde, Spain
Juliana Vinholes, Portugal
Juliano De Dea Lindner, Brazil
Kamila Goderska , Poland
Kelly Mulder, Brazil
Koo Bok Chin, Republic of
Lenka Kourimska, Czech Republic
Leonardo Martín Pérez, Argentina
Liana Claudia Salanta, Romania
Lilia Calheiros De Oliveira Barretto, Brazil
Liyan Chen, United States
Lucia Martinelli, Italy
Lucy Mlipano Chove, Tanzania
Luis Patarata, Portugal
Lupu Mirabela Ioana, Romania
Ma Lourdes Vazquez-Odériz, Spain
Magdalena Polak-Berecka, Poland
Magdalena Surma, Poland
Mahantappa Halimani, United States
Mamdouh El-Bakry, Egypt
Marcel Bassil, Lebanon
Marco Iammarino, Italy
Maria Fernanda Pessoa, Portugal
Mariana de Lourdes Almeida Vieira, Brazil
Marta Mesias, Spain
Marwa Ibrahim Abd El Hamid, Egypt
Massimiliano Renna, Italy
Meena Somanchi, United States
Miguel Elias, Portugal
Milla Santos, Brazil
Mudita Verma, India
Muhammed Yüceer, Turkey
Mulunda Mwanza Mulunda, South Africa
Na-Hyung Kim, Korea
Nicola Caporaso, Italy
Ningning Zhao, United States
Palak Gupta, United States
Palmiro Poltronieri, Italy
Paolo Polidori, Italy
Paulo Henrique Gonçalves Dias Diniz, Brazil
Philippa Chinyere Ojimelukwe, Nigeria
Poonam Singha, USA
Poorna CR Yalagala, USA
Qinlu Lin, China
Rafael Porto Ineu, Brazil
Raza Hussain, Canada
Razieh Sadraei, Italy
Renata Dobrucka, Poland
Renuka Nayar, India
Reyhan Irkin, Turkey
Richard Nyanzi, South Africa
Rigane Ghayth, Tunisia
Roberta Melo, Brazil
Romdhane Karoui, France
Ruchi Singh, USA
Sachin Kumar Samuchiwal, United States
Sasha C Marine, United States
Sefat E Khuda, United States
Shalini A. Neeliah, Mauritius
Shao Quan Liu, Singapore
Slavica Grujic, Bosnia Herzegovina
Sonchieu Jean, Cameroon
Stine Ronholt, Denmark
Stuart Munson-McGee, United States
Suresh Kumar, Korea
Sushil Kumar Singh, USA
Suzana Rimac Brncic, Croatia
Tanima Bhattacharya, India
Teodora Emilia Coldea, Romania
Tinna Austen Ng'ong'ola-Manani, Malawi
Tzortzis Nomikos, Greece
Vasudha Bansal, India
Violeta Ivanova-Petropulos, Republic of Macedonia
Wesam Al-Jeddawi, USA
Winny Routray, Canada
Won Choi, United States
Xingjun Li, China
Xinyin Jiang, United States
Xuefeng Yang, China
Yong Yang, USA
Zelalem Yilma, Ethiopia
Zhilong Yu, USA
Journal of Food Research; Vol. 9, No. 1; 2020
ISSN 1927-0887 E-ISSN 1927-0895
Published by Canadian Center of Science and Education
I
CONTENTS
Incorporation of Multi-Strain Probiotic Preparation in a Traditional Brazilian Cheese: Effects on
Microbiological Safety and Bacterial Community
Moysés Estevão de S. F. Pehrson, Viviane Lívia C. Souza, Ismael M. Mancilha
1
Reasons Why Vegetable Cultivation Increases or Does not Increase Vegetable Intake among Adult
Vegetable Growers Living in a City in Gunma Prefecture, Japan: a Qualitative Study
Daisuke Machida, Tohru Yoshida
11
Effect of Boiling and Wet Frying on Nutritional and Antinutrients Content of Traditional Vegetables
Commonly Consumed in Malawi
Joseph Y. Issa, Arnold Onyango, Anselimo O. Makokha, Judith Okoth
19
Some Nutritional and Physical Properties of Different Zambian Market Classes of Bambara Groundnut
(Vigna subterranea)
Vincent Nyau, Lukonde M
welwa-Zgambo, Taonga Chirwa-
Moonga, Dorothy Nthani, Shiv Prakash, Jerry
Rodrigues, Jill Farrant
34
Corn Flour Formulation and Fortification Tests: Evaluation of Acceptability of Local Derived Product
Called “Kabato” Case of Napalakaha, Nibolikaha and Tiangakah
a of Region of Korhogo
Kouassi Amenan Elodie, Gbogouri Grodji Albarin, Ndri Yao Denis, Niaba Koffi Pierre Valery, Amoakon
Léonce, Clemens Korboi Vanessa, Menzan Guy-Roland
41
A Hospital Based Cross Sectional Study on Dietary Status and Associated Factors among People Living
with HIV/AIDS in Kigali, Rwanda
Tafadzwa Dzinamarira, Gashema Pierre, Elyse Jeanne Umuhire, Michael Habtu, Rosemary Okova
50
GC-MS and HPLC-ESI-MS-MS Characterization of Sanchezia oblonga (Acanthaceae) Extracts
Juliana Mourão Ravasi, Giuseppina Negri, Antonio Salatino, Maria Luiza Faria Salatino, Marco Aurelio
Sivero Mayworm
57
Reviewer Acknowledgements for Journal of Food Research, Vol. 9 No. 1
Bella Dong
72
Journal of Food Research; Vol. 9, No. 1; 2020
ISSN 1927-0887 E-ISSN 1927-0895
Published by Canadian Center of Science and Education
1
Incorporation of Multi-Strain Probiotic Preparation in a Traditional
Brazilian Cheese: Effects on Microbiological Safety and Bacterial
Community
Moysés Estevão de S. F. Pehrson1, Viviane Lívia C. Souza1 & Ismael M. Mancilha1
1Escola de Engenharia de Lorena, University of São Paulo, Lorena, SP, Brazil
Correspondence: Moysés Estevão S. F. Pehrson, Escola de Engenharia de Lorena, Department of industrial
biotechnology, Universidade de São Paulo São Paulo, Lorena, CEP 12600-970, Brazil. Tel: 55-249-8824-6079.
E-mail: moyses.freitas@gmail.com
Received: September 3, 2019 Accepted: October 29, 2019 Online Published: November 6, 2019
doi:10.5539/jfr.v9n1p1 URL: https://doi.org/10.5539/jfr.v9n1p1
Abstract
Consumer preference for raw milk cheeses has increased in the past few years. This occurred partly due to their
more diverse, enjoyable characteristics, but also due to claims that certain members of the autochthonous
microbiota of milk can be beneficial to human health. These microorganisms can inhibit the growth of
undesirable microorganisms and may also be used to establish a biogeographic identity for these products. The
aim of this study was to assess the effect of a multi-strain probiotic preparation on the microbiological safety and
composition of bacterial community of a traditional Brazilian raw milk cheese by means of culture dependent
and methods and pyrosequencing. Probiotic enriched cheeses presented an average of 50% less sequence reads
belonging to Enterobacteriaceae than control cheeses. Total and thermotolerant coliforms cell viability
decreased throughout ripening in two seasons (summer and autumn), while in the winter the presence of these
microorganisms was negligible since the beginning of ripening. Results obtained through culture dependent
method did not correlate with culture independent method, which pointed to a relatively constant number of
Enterobacteriaceae reads during ripening. Viable cells of coagulase positive Staphylococcus aureus stayed
within legal limits in both groups of cheeses since the first day and decreased to zero at the 15th day in probiotic
enriched cheeses. Salmonella sp. and Listeria sp. were absent in both control and probiotic groups. Our results
support that enriching raw milk cheeses with probiotic bacteria or other bioprotective bacteria may help mitigate
off flavors produced by Enterobacteriaceae and result in safer products by inhibiting the growth of these
microorganisms, while maintaining the microbial diversity that may be beneficial to sensory profiles and
health-promoting characteristics. We also showed that this traditional cheese, if made under right the conditions,
can meet legal parameters in much less than 60 days of ripening.
Keywords: artisanal cheese, microbiota, probiotics, raw milk cheese
1. Introduction
Global cheese market is expected to reach $ 164,338 million dollars in 2023. In 2016, North America and Europe
occupied nearly 80% of this market, with cheeses like mozzarella and cheddar dominating the exportation
market (www.alliedmarketresearch.com). Brazil is a minor player in the global market of cheeses, however, the
recent slow food movements contributed in increasing both the consumption and the production of traditional
products such as artisanal cheeses and other naturally fermented and minimally processed foods.
Raw milk cheeses are becoming preferred over pasteurized milk cheeses by an increasing percentage of
consumers for having stronger and/or more pleasant sensory characteristics. These characteristics have been
linked to various members of milk’s autochthonous microbiota, like Lactococcus spp., Lactobacillus spp.,
Leuconostoc spp and Enterococcus spp (Casalta, Sorba, Aigle, & Ogier, 2009; Masoud et al., 2012). These
fermented products can harbor a large variety of bacteria which are capable of inhibiting both spoilage as well as
pathogenic microorganisms, thus extending shelf-life and increasing safety of these products (O’Sullivan &
Cotter, 2017). Additionally, raw milk cheeses present a big potential as reservoirs of health-promoting bacteria
known as probiotics. This can be verified by the abundance studies like the ones from Elkenany, Mona, Eltaysh,
Zakaria & El-Baz (2018), Eid et al. (2016) and Valente et al. (2019) which evaluated probiotic characteristics in
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2
microorganisms originally isolated from raw milk or raw milk cheeses.
According to the slight redefinition of the term by Hill et al. (2014), probiotics are “live microorganisms that,
when administered in adequate amounts, confer a health benefit on the host”. One of the benefits offered by
probiotics is the protection against colonization by potentially pathogenic bacteria and in some cases, active
inhibition of already established detrimental intestinal bacteria. This inhibition can be attributed to the
production of antimicrobial substances, in special organic acids, bacteriocins and other inhibitory metabolites,
which can be applied to other fields such as food quality and safety. The main representatives of probiotic
bacteria belong to a phylogenetically diverse group known as Lactic Acid Bacteria (LAB).
Lactic acid bacteria are naturally occurring in raw milk and are capable of inhibiting a wide range of bacterial
contaminants, such as Salmonella sp. Staphylococcus aureus, Listeria monocytogenes and members of the
Enterobacteriaceae family. During the processing and ripening of raw milk cheeses LAB produce organic acids
and other antimicrobial compounds, which inhibit the growth of undesirable microorganisms, acting as
bioprotectants and rendering these products safer for consumption after a period of time that depends on ripening
conditions, type and abundance of beneficial LAB. However, microbial composition may vary greatly between
different batches of cheese, therefore a consistent percentage of bioprotective microorganisms must be ensured
in order to guarantee the safety of raw milk cheeses (Yoon, Lee, & Choi, 2016).
According to Chambers, Esteve & Retiveau (2010), pasteurization directly interferes in sensory profiles of
ripened cheeses by stripping them of their regional and seasonal characteristics. This effect can be attributed to
the elimination of milk’s autochthonous microbiota during pasteurization, which would then alter the proportion
of fermentation end products during ripening, thus altering flavor and aroma profiles. However, food safety is
also linked to microbial composition of raw milk and there are inherent risks of utilizing raw milk for cheese
making (Law & Tamime, 2010; Quigley et al., 2013). In this context, elimination of autochthonous microbiota
can also strip these products of most of their microbiota-associated benefits like the presence of probiotic lactic
acid bacteria and microbial digestive enzymes.
Moreover, studying the composition of microbial communities in fermented foods can help to establish a link
between microbial diversity or specific community compositions to their practical consequences, such as
desirable/undesirable flavor profiles, increased protection against spoilage and pathogenic bacteria and in
establishing a product identity (Dutton & Wolfe, 2015).
Our hypothesis was that enriching the milk’s autochthonous microbiota with probiotic microorganisms prior to
cheese making would result in safer products through the inhibition of potentially dangerous microorganisms,
reducing the ripening time required for raw milk cheeses to be considered safe for consumption.
In order to test this hypothesis, a traditional Brazilian raw milk cheese, known as “Queijo da Mantiqueira” was
studied in three different seasons, with and without the addition of probiotics. Effects of probiotics on
microbiological quality indicators such as total and thermotolerant coliforms, Listeria sp, Salmonella sp. and
coagulase positive Staphylococcus aureus were evaluated by culture-dependent methods, as well as on the
composition of the bacterial community, which was assessed by means of 454-pyrosequencing.
2. Materials and Methods
2.1 Probiotic Cultures
The probiotic preparation consisted of a mixture of Lactobacillus acidophilus (CUL-60), Lactobacillus
acidophilus (CUL-21), Bifidobacterium bifidum (CUL-20), Bifidobacterium animalis subsp. lactis (CUL-34),
Lactobacillus salivarius (CUL-61), Lactobacillus paracasei (CUL-08), Lactobacillus plantarum (CUL-66),
Lactobacillus casei (CUL-06), Lactobacillus fermentum (CUL-67), Lactobacillus gasseri (CUL-09),
Bifidobacterium animalis subsp. lactis (CUL-62), Bifidobacterium breve (CUL-74), Streptococcus salivarius
subsp. thermophilus (CUL-68), Lactobacillus acidophilus (NCFM™), Bifidobacterium animalis subsp. lactis
(HN019) and Lactobacillus rhamnosus (HN001) in equal proportions.
2.2 Production of Raw Milk Cheeses, Ripening and Sampling
Two groups of cheeses (probiotics and control) were made in three different seasons (winter, summer and
autumn) in order to account for seasonal changes in cheese microbial communities. Cheese production was
undertaken in artisanal scale in a local dairy manufacturer of a region known as “Serra da Mantiqueira”, starting
from 50L of raw cow’s milk for each group. Both groups of cheeses were inoculated with a natural whey starter,
obtained from the whey collected after cheese making in the previous day. The collected whey was stored in a
sealed polypropylene container and left to ferment at room temperature overnight.
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3
Cheeses belonging to the probiotic group were inoculated with a sufficient amount of probiotic preparation to
obtain a final concentration of 106 CFU/ml of probiotic bacteria and 1% of natural whey starter, while the control
group received only 1 % of the natural starter. The milk in the vats belonging to each group was ripened for 30
minutes at 35-37 oC after addition of whey starter and adjunct cultures, and then rennet (Ha-La™ – Christian
Hansen) was added according to the manufacturer’s instructions. After 30 minutes, the curd was cut, stirred for
20 minutes, then heated to 48oC and stirred until the consistency was adequate for molding. Afterwards, the
curds were transferred into 1kg molds and pressed for 30 minutes on each side. Following, the cheeses were
transferred to a brine (20o Baumé) for 48 hours at 4 oC, air dried, vacuum packed and then ripened at 10-15 oC.
Samples were collected at day 1, 15, 30 and 45 of ripening and tested for the presence of total and thermotolerant
coliforms, Salmonella sp., coagulase-positive Staphylococcus aureus, Listeria sp. and also stored for
culture-independent analyses.
2.3 Culture Dependent Analyses
Microbiological analyses were carried out using FDA’s Bacteriological Analytical Manual (BAM) preferred
procedures. Total and thermotolerant coliform populations were estimated according to Kornacki & Johnson
(2001). Coagulase-positive Staphylococcus aureus populations were determined according to Bennet & Lancette
(2001). Salmonella sp. quantification was performed according to Wallace, Andrews, Jacobson, & Hammack
(2016). Listeria sp. populations were determined according to Hitchins (2016).
2.4 Bacterial Community Identification: Total DNA Extraction and PCR Amplification
Bacterial community in the cheese samples was identified to the genus level by means of 454 pyrosequencing of
the 16S rRNA genes. All genera representing less than 0.01% of the total bacterial community were omitted from
the results.
Total DNA of cheese samples was extracted using MoBio Powerfood Microbial DNA Isolation Kit (Mobio
Laboratories), following manufacturer’s instructions using 0.25 g of homogenized cheese samples. The resulting
extracts were used as templates for PCR amplification and were stored at -80 oC until used. The 16S rRNA genes
were amplified using primers 968F (5'-AACGCGAAGAACCTTAC-3') and 1378R
(5´-CGGTGTGTACAAGGCCCGGGAACG-3´). For this, 5 μL of total DNA extracted from the respective
samples were added to microtubes containing 10mM dNTPs, 0,1 μM of the respective primers, 500 mM KCl,
100 mM Tris-HCl pH 8.3, and 2U of Taq polymerase. Distilled water was used to bring the final volume of the
reaction to 50 μL. Amplification program consisted of an initial denaturation step at 95oC for 5 min, followed by
35 cycles of denaturation for 30 s at 94oC, annealing at 62oC for 30 s, extension at 72oC for 40 s, followed by a
final elongation step at 72oC for 10 min.
2.5 Purification of Amplicons and Pyrosequencing
Samples resulting from PCR amplification were purified using Illustra GFX PCR DNA and gel Band
Purification Kit (GE Healthcare), followed by sequencing in Roche/454 (Roche Applied Science) using a
GS-FLX Titanium sequencing kit. Sequence classification was carried out using MOTHUR software and RDP
(Ribossomal Database Project – Michigan State) database.
2.6 Statistical Analysis
Results were analyzed by means of one-way ANOVA, followed by Tukey’s post hoc test for paired comparisons
with 95% confidence interval.
3. Results and Discussion
3.1 Bacterial Communities of Raw Milk Cheeses with and Without the Addition of Probiotic Bacteria
Raw milk cheeses inherently harbor a diverse microbial community, which accounts for their diversified sensory
profiles. Compared to commercially available starters used in cheese making, indigenous lactic acid bacteria
isolated from raw milk cheeses, bacteria found on surfaces of ripening racks and in traditional brines have been
associated with higher scores for sensory characteristics. Also, the low presence of pathogens in most traditional
ripened cheeses is attributed to the antimicrobial activities of several species of bacteria that form the
autochthonous milk microbiota (Montel et al., 2014).
Results obtained through pyrosequencing (Table 1 and Figure 1) showed that microbial communities were very
distinct in the three seasons. However, the genera Lactobacillus, Streptococcus and Lactococcus were the
dominant taxa in all of them. Lactobacillus was a major constituent of the bacterial community of cheeses in the
winter (≈ 95-97%) and autumn (≈ 70%), while in the summer, Streptococcus was the dominant genus (≈
51-54%). Masoud et al. (2012) found similar results while evaluating Danish raw milk cheeses by means of
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4
pyrosequencing, however, in their study, Lactococcus was the predominant genus.
Table 1. Percentages of total sequence reads belonging to different bacterial genera in artisanal cheeses,
produced in different seasons
Family
Genus
Control
Probiotic-enriched
Winter
Summer
Autumn
Winter
Summer
Autumn
Bifidobacteriaceae
Bifidobacterium
0.00% a
0.00% a
0.00% a
0.1% a
0.24% a
0.12% a
Flavobacteriaceae
Riemerella
0.04% a
0.03% a
0.07% a
0.1% a
0.02% a
0.08% a
Soonwooa
0.21% a
0.29% a
0.50% a
0.2% a
0.18% a
0.62% a
Staphylococcaceae
Staphylococcus
0.11% a
0.18% a
0.01% a
0.1% a
0.10% a
0.01% a
Carnobacteriaceae
Isobaculum
0.05% a
0.19% a
0.18% a
0.1% a
0.17% a
0.25% a
Jeotgalibaca
0.12% a
0.07% a
0.18% a
0.1% a
0.06% a
0.16% a
Lacticigenium
0.04% a
0.14% a
0.27% a
0.0% a
0.16% a
0.22% a
Enterococcaceae
Bavariicoccus
0.07% a
0.56% a
0.09% a
0.1% a
0.42% a
0.10% a
Catellicoccus
0.33% a
4.28% a
4.17% a
0.8% a
4.22% a
5.34% a
Enterococcus
0.02% a
0.05% a
0.06% a
0.0% a
0.04% a
0.02% a
Melissococcus
0.01% a
1.92% a
0.20% a
0.0% a
1.42% a
0.29% a
Pilibacter
0.01% a
3.99% a
0.12% a
0.1% a
3.75% a
0.16% a
Lactobacillaceae
Lactobacillus
97.39%a
24.07% a
69.79% a
95.7%b
32.07% b
69.39% a
Streptococcaceae
Lactococcus
0.13% a
5.37% a
8.87% a
0.2% a
2.26% b
14.23% b
Streptococcus
0.11% a
53.96% a
2.14% a
1.0% a
51.02% b
1.31% b
Lactovum
0.01% a
0.87% a
0.22% a
0.0% a
1.02% a
0.25% a
Enterobacteriaceae
Citrobacter
0.01% a
0.03% a
1.39% a
0.0% a
0.01% a
0.61% b
Enterobacter
0.01% a
0.10% a
4.93% a
0.0% a
0.02% b
3.20% b
Klebsiella
0.00% a
0.01% a
2.44% a
0.0% a
0.01% a
0.69% b
Kluyvera
0.01% a
0.02% a
0.66% a
0.0% a
0.01% a
0.25% b
Leclercia
0.01% a
0.14% a
1.55% a
0.0% a
0.05% b
0.95% b
Salmonella
0.00% a
0.00% a
0.44% a
0.0% a
0.00% a
0.22% b
Morganella
0.00% a
0.01% a
0.17% a
0.0% a
0.01% a
0.16% a
Obesumbacterium
0.00% a
0.11% a
0.52% a
0.0% a
0.06% a
0.34% b
Pluralibacter
0.00% a
0.00% a
0.28% a
0.0% a
0.00% a
0.23% a
Moraxellaceae
Acinetobacter
0.78% a
3.06% a
0.31% a
0.7% a
2.33% a
0.12% a
Enhydrobacter
0.51% a
0.56% a
0.40% a
0.6% a
0.34% a
0.62% a
Values of control and probiotic-enriched cheeses made in the same season with different superscripted letters are
significantly different (p<0.05).
Considering that all cheeses were subjected to the same conditions during processing and ripening, the main
factors that could have interfered in the composition of cheese bacterial communities in this study is the natural
whey starter and the raw milk used for production. In this context, the bacterial community in the whey starter
that is left to ferment overnight at room temperature could be influenced by the different temperatures in each
season, as well as by the different species of bacteria introduced with the milk that was used in the production of
the previous batch of cheeses. In the region that this study took place (Serra da Mantiqueira), which is situated at
1200 meters of altitude, the temperature usually ranges from 20-33 ºC in the summer and from 8-22 in the winter
and autumn.
Several species of bacteria are able to produce antimicrobial substances that act as biopreservatives. There are
many types of antimicrobial compounds found in foods produced with raw milk, such as organic acids,
bacteriocins, antifungals, diacetyl, hydrogen peroxide and others (O’Sullivan & Cotter, 2017). These
antimicrobial compounds, which are produced mainly by lactic acid bacteria, alter the intrinsic properties of
fermented foods to the extent that they actually kill and inhibit the growth of spoilage and pathogenic
microorganisms, rendering the product safer to consume and more shelf-stable. Perhaps the more complex of
these substances are bacteriocins, which are antimicrobial peptides, produced by bacteria, which kill other
competing bacteria in the environment (Deegan, Cotter, Hill, & Ross, 2006).
On the other hand, the antimicrobial properties of bacteriocins and other antimicrobial compounds is not directed
exclusively towards pathogenic or spoilage bacteria and can they also inhibit other beneficial or innocuous
starter and non-starter lactic acid bacteria that may be sharing the environment and competing for the same
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5
resources. Corroborating this affirmation, Tymoszewska, Diep, Wirtek & Alesandrzak-Piekaczyk (2017)
demonstrated that Garvicin Q, a bacteriocin produced by Lactococcus garvieae, has a very broad spectrum of
action, being capable of inhibiting Listeria monocytogenes and also several lactic acid bacteria species belonging
to the genera Leuconostoc, Lactobacillus, Carnobacterium, Enterococcus and Pediococcus.
Our results showed that the incorporation of the multi-strain probiotic preparation exerted different effects on the
predominant genera (Lactococcus, Streptococcus and Lactobacillus) in each season. In the winter, the addition of
the preparation promoted a slight, but significant (p<0.05) reduction in Lactobacillus percentage, even though
the preparation itself included 10 Lactobacillus species. This could indicate that some of the bacterial
components of the preparation may have exerted inhibitory effects on autochthonous Lactobacillus species,
which was not observed in the other seasons. On the other hand, in the summer, the percentage of Lactobacillus
was higher in probiotic cheeses.
The effect of the preparation on the genus Lactococcus was also different in the three seasons. Probiotic-enriched
cheeses presented lower Lactococcus sequence reads on summer (p<0.05) and a higher number of sequence
reads (p<0.05) on autumn relative to the control group while in the winter, no effect was observed and
Lactococcus was a minor member of the microbiota. Regarding the genus Streptococcus, we observed no
significant effect in cheeses made in the winter and a significant (p<0.05) negative effect in both summer and
autumn cheeses, regardless of the fact that Streptococcus thermophilus was present in the probiotic preparation.
Members of the genus Bifidobacterium were absent in the control cheeses and apparently did not adapt well to
cheese conditions, as the percentages for this genus were no higher than 0.25% in probiotic-enriched cheeses.
Figure 1. Main bacterial genera identified in the microbial community of traditional Brazilian semi-hard cheese
We observed that the cheeses produced in the winter had very low percentages of members of the
Enterobacteriaceae family, relative to the percentages observed in summer and autumn. In Brazil this is a
common observation because, in these months, the quality of water used in rural areas, which is often not treated,
tends to worsen as well as the hygienic conditions, due to the abundance of rainfall. In summer and autumn, the
probiotic-enriched cheeses presented significantly (p<0.05) lower percentages of Enterobacteriaceae when
compared to the control group (Figure 2). In both mentioned seasons, total number of sequence reads of
Enterobacteriaceae on probiotic group was approximately half of that observed in the control group, however, in
the winter, the control and probiotic groups did not differ regarding Enterobacteriaceae concentrations. To
establish a sense of proportion, it is important to point out that Enterobacteriaceae were present at greater
quantities on autumn cheeses (up to 5,000 sequence reads), to a lesser extent on summer cheeses (up to 170
sequence reads) and much lesser in winter cheeses (up to 20 sequence reads). This seasonality can cause a great
impact on raw milk cheeses quality because Enterobacteriaceae are known for causing off-flavors in raw milk
cheeses. The fact that just adding probiotic cultures along with the starter reduced an average of 50% of
Enterobacteriaceae sequence reads (Figure 2) on the seasons in which they were most prevalent may indicate
0 5000 10000 15000 20000 25000 30000 35000
40000
Winter
Summer
Autumn
Winter
Summer
Autumn
Control Probiotics
Number of Sequence Reads
Bifidobacterium Riemerella Soonwooa Staphylococcus Catellicoccus
Isobaculum Jeotgalibaca Lacticigenium Bavariicoccus Enterococcus
Melissococcus Pilibacter Lactobacillus Lactococcus Streptococcus
Lactovum Citrobacter Enterobacter Klebsiella Kluyvera
Leclercia Salmonella Moellerella Morganella Obesumbacterium
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6
that selected bioprotective cultures can be used as adjuncts on artisanal cheeses to mitigate or avoid off-flavors
and defects such as early blowing, which is also caused by Enterobacteriaceae.
Westling et al. (2016) conducted a study on the contribution of this family to the sensory characteristics of soft
raw milk cheeses and observed that the presence of Enterobacteriaceae could be predicted through the
perception of taste and odor alone. Moderate correlation was found between the sensory characteristics identified
as “manure”, “ammonia”, “pungent”, “bitter” and “metallic” and high levels of Enterobacteriaceae cultivable at
37 ºC. In this context, reducing the occurrence of members of this family can be a positive consequence of
employing bioprotective strains in artisanal cheeses, providing they do not alter their peculiar sensory
characteristics or biogeographic identity of cheeses.
Figure 2. Comparison of sequence reads belonging to Enterobacteriaceae in Brazilian artisanal cheeses made
with and without probiotics in different seasons
3.2 Microbiological Safety Parameters of Brazilian “Queijo da Mantiqueira” with or without Added Probiotics
With regards to parameters that indicate microbiological quality, it was observed that all but one set of cheese
samples exceeded legal limits for thermotolerant coliforms (Table 2), which belonged to the control group.
Samples from the first day after manufacturing belonging to this group had excessive numbers of thermotolerant
coliforms, however, after 15 days of ripening this number decreased, fell below the limit accepted by Brazilian
law and decreased even more with further ripening. It is important to remark that most of the raw milk cheese
related laws require cheeses to be ripened for a minimum of 60 days to ensure safety, including the
corresponding Brazilian law (Brasil, 2013). However, in this study both control and probiotic enriched cheeses
met legal parameters since the 15th of ripening (probiotic-enriched cheeses since day 1), which points out that
raw material quality and good processing practices are major factors that influence these indicators.
Despite presenting a lower number of sequence reads belonging to Enterobacteriaceae than cheeses made on
autumn, cheeses produced in the summer presented higher counts of total and thermotolerant coliform per gram
of product. This could indicate that the Enterobacteriaceae present in autumn cheeses were mostly inactive or in
a non-cultivable state, a concept previously explored by authors like Alegría, Gonzáles, Diaz, & Mayo (2011). In
both cases, the number of sequence reads was approximately 50% lower in the probiotic enriched cheeses,
despite the fact that the same relation was not found in the culture-dependent method.
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7
Table 2. MPN (Most Probable Number) of total and thermotolerant coliforms in cheese manufactured in different
seasons, subjected to different treatments
Group
Ripening (days)
Total coliforms (MPN/g)
Thermotolerant coliforms (MPN/g)
Winter
Summer
Autumn
Winter
Summer
Autumn
Control
1
< 3,0
>1.100
14
< 3,0
>1.100
< 3,0
15
< 3,0
1.100
< 3,0
< 3,0
240
< 3,0
30
< 3,0
1.100
43,0
< 3,0
460
9,2
45
< 3,0
43
23
< 3,0
43
< 3,0
Probiotic-enriched
1
< 3,0
460
1.100
< 3,0
460
< 3,0
15
< 3,0
>1.100
23
< 3,0
240
< 3,0
30
< 3,0
1.100
460
< 3,0
210
3,6
45
< 3,0
93
93
< 3,0
93
< 3,0
Limits allowed by specific legislation
Not specified
Max. 1.000 MPN/g
Numbers in bold: Exceeded legislation upper limit.
Staphylococcal Foodborne Disease (SFD) is one of the most common causes of diseases associated with food
consumption and is a major public health concern. Although S. aureus can be completely eliminated from foods
by heat treatment prior to consumption, staphylococcal enterotoxins produced during growth are resistant to heat,
gastrointestinal proteases and can cause serious food poisoning. Symptoms include profuse vomiting, nausea,
abdominal cramps and diarrhea (Kadariya, Smith, & Thapaliva, 2014). Salmonella and Listeria can be found
naturally in the environment and are the causative agents of several and severe foodborne disease outbreaks
worldwide. Dairy can be a common source of infections caused by these bacteria because the animals which are
the natural reservoirs of these microorganisms (fowl, sheep, goats, pigs) often share the same environment with
milk producing animals (Heredia & Garcia, 2018), especially in properties that adopt a free range farming
system and produce cheeses in an artisanal manner. Brazilian law admits a limit of 1000 CFU/g of S. aureus in
finished cheeses (Brasil, 2001), while Salmonella and Listeria must be absent in 25 g of finished product.
Overall contamination with coagulase positive Staphylococcus aureus was low and only was detected in five sets
of cheese samples by culture-dependent method (Table 3), three of them belonging to the control group.
Sequence reads belonging to Listeria sp. and Salmonella sp. were detected inconsistently by pyrosequencing, at
very low numbers (lower than 250 sequence reads) and only in cheeses manufactured on autumn, but were not
detected in any of the samples by culture-dependent methods.
Table 3. Results of viable counts of Coagulase + Staphylococcus aureus, Salmonella sp. and Listeria sp. in
cheese manufactured in different seasons, subjected to different treatments
Group
Ripening (days)
Coagulase + Staphylococcus
aureus (CFU/g)
Salmonella sp. and Listeria sp.
(Presence in 25 g)
Winter
Summer
Autumn
Winter
Summer
Autumn
Control
1
200
600
-
-
-
-
15
-
700
-
-
-
-
30
-
-
-
-
-
-
45
-
-
-
-
-
-
Probiotics
1
200
700
-
-
-
-
15
-
-
-
-
-
-
30
-
-
-
-
-
-
45
-
-
-
-
-
-
Limits allowed by Brazilian law
1000 CFU/g.
Absent in 25 g of product.
Although not negligible, the incidence of Listeria monocytogenes in Brazil is relatively low. Oxaran et al. (2018)
studied the incidence of Listeria monocytogenes in cheese, milk and brines of five dairies and retail stores in two
states of Brazil over the course of 7 months. The authors analyzed a total of 437 samples and found that only 4
samples were contaminated with this microorganism.
The factors that govern the incidence and survival of Listeria sp. in cheeses are well established. Some
categories of cheese, mainly non-acidic, semisoft, soft and smear-ripened cheeses are the most susceptible to the
growth of Listeria sp. There seems to be no evidence-based difference between pasteurized and raw milk cheeses
http://jfr.ccsenet.org Journal of Food Research Vol. 9, No. 1; 2020
8
when it comes to Listeria incidence because most of the contaminations are not linked to the quality of the raw
milk, but to the lack of hygiene during post pasteurization or post processing steps (Gérard, El-Hajjaji,
Nivonzima, Daube, & Sindic, 2018). Like Listeria monocytogenes, Salmonella sp. is also more prone to be
found at low acidity, high moisture cheeses that were manufactured under unsanitary conditions or were packed
or stored improperly. Soft ripened cheeses like Camembert and red smear cheeses in general present a higher risk
of Salmonella incidence than hard cheeses like parmesan and cheddar (Choi, Lee, Lee, Kim, & Yoon 2016).
4. Conclusions
Our results showed that incorporating probiotic microorganisms in raw milk cheeses prior to processing can
significantly reduce the numbers of Enterobacteriaceae since the beginning of ripening, improving overall
quality. This can result in safer products and lead to less accumulation of fermentation end products produced by
members of this family, thus reducing the occurrence of off-flavors. Overall microbial diversity was preserved in
probiotic-enriched cheeses, indicating that no negative effect was exerted upon the potentially health promoting
characteristics of the microbiota or the ability to generate positive sensory characteristics. Also, it was
demonstrated that this type of cheese can be made in such a way that it is safe for consumption in a much shorter
time than that which is established by Brazilian legislation.
The bacterial composition of “Queijo da Mantiqueira” varied significantly when produced in different seasons,
however three genera of bacteria were always dominant, namely Lactobacillus, Streptococcus and Lactococcus.
Major factors that seemed to interfere with this composition were the microbial composition of the raw milk
used for the production of cheeses and the microbial composition of the natural whey starter, as well as the
temperature in which the starter is left to ferment.
Acknowledgements
This study was partly funded by FAPESP and CAPES and was executed at the Biotechnology Department of
Lorena School of Engineering, University of São Paulo, Lorena, SP, Brazil.
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Copyright for this article is retained by the author(s), with first publication rights granted to the journal.
This is an open-access article distributed under the terms and conditions of the Creative Commons Attribution
license (http://creativecommons.org/licenses/by/4.0/).
Journal of Food Research; Vol. 9, No. 1; 2020
ISSN 1927-0887 E-ISSN 1927-0895
Published by Canadian Center of Science and Education
11
Reasons Why Vegetable Cultivation Increases or Does not Increase
Vegetable Intake among Adult Vegetable Growers Living in a City in
Gunma Prefecture, Japan: a Qualitative Study
Daisuke Machida1 & Tohru Yoshida2
1Takasaki University of Health and Welfare, Gunma, Japan
2Jumonji University, Saitama, Japan
Correspondence: Daisuke Machida, Takasaki University of Health and Welfare, 370-0033, 37-1, Nakaoorui,
Takasaki, Gunma, Japan. Tel: 81-27-352-1290. E-mail: machida-d@takasaki-u.ac.jp
Received: October 11, 2019 Accepted: November 2, 2019 Online Published: November 6, 2019
doi:10.5539/jfr.v9n1p11 URL: https://doi.org/10.5539/jfr.v9n1p11
Abstract
We examined the reasons why vegetable cultivation increases or does not increase vegetable intake among adult
Japanese vegetable growers. A qualitative cross-sectional study using a self-completed anonymous questionnaire
was sent to participants (aged 20–74 years residing in three areas of a city in Gunma Prefecture, Japan) in
September 2016. The questionnaire addressed perceptions of whether vegetable cultivation would increase
vegetable intake, with four possible answers: strongly disagree, disagree, agree, and strongly agree. Respondents
were then asked reasons for their view, with free-text responses. We also asked about participants’ characteristics
and whether they found that growing vegetables had changed their vegetable intake and access to vegetables. We
categorized the free-text answers by content. We analyzed 442 answers, and reasons for vegetable growing
increasing vegetable intake were grouped into five categories: “availability,” “purpose of cultivation,” “quality,”
“increased positive emotions toward vegetables,” and “unconsciousness”; for it not increasing intake were also
grouped into five categories: “limited quantities,” “negative emotions toward vegetables,” “cultivation for a
purpose other than eating vegetables,” “access to vegetables from other sources,” and “limits associated with
self-cultivation.”
Keywords: vegetable intake, vegetable growing, qualitative study, Japanese, adults
1. Introduction
In recent years, a positive relationship has frequently been reported between vegetable intake and vegetable
growing among adults. In previous studies, positive influence of having a vegetable garden (Devine, Wolfe,
Frongillo, & Bisogni, 1999), home-grown vegetable intake (Billson, Pryer, & Nichols, 1999; Nanney, Johnson,
Elliott, & Haire-Joshu, 2007; Umezawa et al., 2012), gardening or cultivation (Carney et al., 2012; Sommerfeld,
McFarland, Waliczek, & Zajicek, 2010; Machida, Onoe, & Yoshida, 2016; Machida & Yoshida, 2017; Machida
& Yoshida, 2018a; Machida & Yoshida, 2018b; Machida, 2019), and participation in community gardening
(Machida, 2019; Alaimo, Packnett, Miles, & Kruger, 2008; Blair, Giesecke, & Sherman, 1991, Johnson & Smith,
2006; Barnidge et al., 2013; Litt et al., 2011; Soga et al., 2017; Machida & Yoshida, 2017) on vegetable intake
has been indicated. Increased vegetable intake reduces the risk of certain chronic diseases and overall mortality
(Boeing et al., 2012; Wang et al., 2017). It is therefore expected that increasing vegetable intake by promoting
the growing of vegetables will have a positive impact on health.
In the previous studies, the reasons why vegetable cultivation increases vegetable intake are discussed as follows:
improvement of the accessibility of vegetables and fruits owing to the existence of the community garden
(Barnidge et al., 2013), improvement of the availability of vegetables due to having a vegetable garden (Devine
et al., 1999), and using vegetables naturally for daily meals due to the participation of community garden
(Alaimo et al., 2008). However, empirical studies addressing the reasons why vegetable cultivation increases
vegetable intake among adults remains scarce. In addition, there may be scenarios where vegetable intake is not
increased by vegetable cultivation, e.g., in circumstances where only a few vegetables are grown, or if they are
grown with the intention of being sold. By clarifying these, the understanding of the mechanism of the influence
of vegetable growing on increased vegetable intake will be deepened. Thus, it may contribute to further effective
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12
practices that encourage increased vegetable intake by vegetable growing. It is therefore necessary to clarify the
reasons for vegetable intake increasing (or not) as a result of vegetable cultivation.
This study aimed to examine the reasons why vegetable cultivation increases or does not increase vegetable
intake among adult Japanese vegetable growers. This study addresses the question from a qualitative perspective.
2. Method
2.1 Study Participants and Methods
We conducted a qualitative study using responses to an open-ended question in a self-administered questionnaire,
drawing on data collected in a previous study (Machida & Yoshida, 2018c). Participants were residents aged 20–
74 years from three areas of a city in Gunma Prefecture, Japan. We identified three geographic regions of the city:
rural, suburban, and urban. Rural area B is located northwest of the city and vegetable cultivation flourishes
there (607 households). Suburban area C is located in the middle of the city; vegetable cultivation flourished
there once but now the amount of agriculture has reduced owing to land conversion for residential or commercial
use (743 households). Urban area D, on the east side of the city, is the most urbanized region of the city (910
households).The questionnaire was sent to a total of 2,260 households, reaching about 3,000 residents aged 20–
74 years, according to the most recent Japanese national census (E-stat, 2015). We collected survey data in
September and October 2016 using a self-administered questionnaire. We mailed two sets of the questionnaire to
all households in the three areas on September 12, using TOWNPLUS by Japan Post Co., Ltd.; this service
helped us to send mail to all households in the selected areas, even without knowing the specific addresses. An
explanatory letter and a stamped, self-addressed return envelope were enclosed with the questionnaire. We
explained that (1) there was no need to reply if there were no residents aged 20–74 years in the household; (2) if
there were three or more residents aged 20–74 years in the household, then two residents should reply; and (3)
the survey was anonymous, and submitting a response would constitute consent to participate. In addition, we
numbered each questionnaire so that we could identify from which of the three geographic areas each response
came.
2.2 Survey Instruments
The questionnaire aimed to establish why vegetable cultivation increased or did not increase vegetable intake.
First, participants were asked: “Do you think vegetable intake will be increased by growing vegetables?” A
choice of four possible responses was given (strongly disagree, disagree, agree, and strongly agree). Participants
were then asked to respond in their own words to the question: “Why do you think this?” Moroever, we asked
participants’ sex, age, cultivation style, and perceptions of changes in vegetable intake and the availability of
vegetables as a result of vegetable cultivation. It is worthy to note that there were many farmers among the
participants. However, a previous study has shown that 96% of farmers cultivate vegetables for home
consumption (Machida & Yoshida, 2018b), so we deemed their inclusion appropriate.
2.3 Analysis
We divided participants into two groups according to their answer to the question: “Do you think vegetable
intake will be increased by growing vegetables?” (“agree or strongly agree” vs. “disagree or strongly disagree”).
Responses to the above open-ended question were categorized using the grounded theory approach (Flick, 1995).
We segmented the answers and identified reasons why respondents thought that vegetable intake would increase,
or not, as a result of growing vegetables. We then coded the segmented answers and generated abstracted
categories using these codes. These steps were repeated multiple times to ensure the validity of the results. We
also calculated the perceptions of changes in vegetable intake and vegetable availability as a result of vegetable
cultivation. Answers from those who said that there was no time when they had not grown vegetables were
excluded.
2.4 Ethical Approval
This study was approved by the Gunma University Ethical Review Board for Medical Research Involving
Human Subjects (Submission No.160074, approved on August 16, 2016).
3. Results
A total of 873 residents from 586 households responded (25.9% household response rate). Of these, 421 were
excluded because they did not grow vegetables and 10 because they did not respond to the question: “Do you
think vegetable intake will be increased by growing vegetables?” The study hence included responses from 442
people.
The distribution of respondents' characteristics is shown in Table 1. In total, 55 people (12%) thought that
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13
vegetable growing would not increase vegetable consumption (disagree), and 387 (88%) thought that it would
(agree). By characteristics, those who responded “agree” in rural and suburban had high percentages, and those
in urban were low (rural: 89%, suburban: 88%, urban 80%). The percentage of “agree” was higher among
women compared with men (women: 89%, men: 85%). There was no difference in the percentage of “agree” by
age (<60: 88, ؤ 60: 87). The percentage of those who answered “agree” by cultivation style was as follows:
farmer = 88%, home gardener = 89%, community gardener = 33%, and others = 67%.
Table 1. Distribution of characteristics
㻌
㻌
disagree*
n = 55 (12%)
agree*
n = 387 (88%)
㻌
㻌
n
%
n
%
Area
㻌
㻌
㻌
㻌
㻌
rural
19
11
154
89
㻌
suburban
26
12
192
88
㻌
urban
10
20
41
80
Sex
㻌
㻌
㻌
㻌
female
26
11
218
89
㻌
male
29
15
166
85
㻌
(no response)
0
0
3
100
Age
㻌
㻌
㻌
㻌
<60
22
12
168
88
㻌
≥60
33
13
217
87
㻌
(no response)
0
0
2
100
Cultivation style (multiple answers permitted)
㻌
㻌
㻌
㻌
farmer
15
12
109
88
㻌
home gardener
35
11
279
89
㻌
community gardener
2
67
1
33
㻌
others
4
33
8
67
㻌
(no response)
0
0
10
100
* “Disagree” denotes those who responded “disagree” or “strongly disagree” and “agree” denotes those who
responded “agree” or “strongly agree” to the question “Do you think vegetable intake will increase by growing
vegetables?” The total number of responses included in the analysis was 442.
The subjective changes in vegetable intake and vegetable availability as a result of vegetable cultivation are
shown in Table 2. Of the 442 participants who grew vegetables, 145 (33%) said that there had been no time when
they did not grow vegetables, and were hence excluded. Of the remaining 297 participants, 172 (58%; in total of
“increased” (77 [26%]) and “somewhat increased” (95 [32%]) said that their vegetable intake had increased
since they began growing vegetables. There were 105 (35%) participants who answered no change, and few
respondents answered “decreased” (2 [1%]) and “somewhat decreased” (13 [4%]). The participants of 217 (73%)
said that the availability of vegetables had increased (in total of “agree” (118 [40%]) and “strongly agree” (99
[33%]). The participants of “Disagree” and “strongly disagree” were 63 (21%) and 10 (3%), respectively.
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14
Table 2. Subjective changes in vegetable intake and vegetable availability as a result of growing vegetables
㻌
㻌
n
%
How has your vegetable intake changed since starting vegetable cultivation?㻌
㻌
decreased
2
1
㻌
somewhat decreased
13
4
㻌
no change
105
35
㻌
somewhat increased
95
32
㻌
increased
77
26
㻌
(no response)
5
2
Do you have more access to vegetables than you did before you started growing vegetables?㻌
㻌
strongly disagree
10
3
㻌
disagree
63
21
㻌
agree
118
40
㻌
strongly agree
99
33
㻌
(no response)
7
2
Number and percentage of responses excludes 145 participants (33%) who said there was no time when they did
not grow vegetables.
The reasons for respondents thinking that vegetable cultivated increased vegetable intake (or not) are shown in
Table 3. Reasons for this increase were placed into five categories, across 18 codes: “availability,” “purpose of
cultivation,” “quality,” “increased positive emotions toward vegetables,” and “unconsciousness.” Similarly, the
reasons why vegetable cultivation did not increase vegetable intake were placed into five categories, across 13
codes: “limited quantities,” “negative emotions toward vegetables,” ”cultivation for a purpose other than eating
vegetables,” “access to vegetables from other sources,” and “limits associated with self-cultivation.”
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Table 3. Reasons why vegetable growing increases or does not increase vegetable intake
Reasons why vegetable growing increases vegetable intake
category
code
availability
large amounts
no need to purchase
immediate access
large variety
continuous availability
ease of access
purpose of cultivation
cultivating what one wants to eat
cultivating vegetables to eat
quality
safe
worry-free
tasty
fresh
increased positive emotions toward vegetables
interest in vegetables
desire to avoid waste
㻌
attachment
㻌
fun
unconsciousness
shifting menus to include what is grown
㻌
eating vegetables without thinking about it
㻌
㻌
Reasons why vegetable growing does not increase vegetable intake
category
code
limited quantities
already eat enough vegetables
ability to eat only small meals
negative emotions towards vegetables
preference (do not like vegetables)
no sense of waste
cultivation for a purpose other than eating vegetables
cultivation for fun
cultivation for sale
cultivation with no intention of eating produce
access to vegetables from other sources
receive a lot of vegetables
buy enough vegetables
limits associated with self-cultivation
limited kinds
limited amount
limited cultivation skill
limited harvest period
4. Discussion
This study attempted to clarify the reasons why vegetable cultivation did or did not increase vegetable intake
among adults. The topic has not been widely studied; nevertheless, it is an important issue for public health.
Results will help to identify more effective intervention strategies to increase vegetable intake through vegetable
cultivation. Of those who had not grown vegetables over a period of time, 58% answered that their vegetable
intake had increased since they began to grow vegetables, and 73% observed that the availability of vegetables
had increased. This suggests that vegetable growing improves both vegetable intake and the availability of
vegetables. Perhaps, as discussed in previous studies, increased availability increases vegetable intake (Barnidge
et al., 2013; Devine et al., 1999).
As to why vegetable cultivation increases vegetable intake, “availability” may be one of the key reasons.
“purpose of cultivation” may be a factor linked to availability. In other words, growing vegetables with the aim
of eating them increases their availability and vegetable intake therefore also increases. The category “quality”
may be related to safety, whether actual or perceived. Soil contamination can be a problem in community
gardens (Alaimo, Beavers, Crawford, Snyder & Litt, 2016). However, participants seemed to feel “safe” and
“worry-free” when managing the growing space themselves. The code “tasty” may be biased because of both the
fact that participants equated taste with freshness and cognitive dissonance (Festinger, 1962). The code
“increased positive emotions toward vegetables” suggests that participants may have developed greater
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16
attachment toward vegetables as a result of exposure (Zajonc, 1968). As a result, they may seek to avoid waste.
Knowledge about vegetables develops through cultivation. Participants may thus become more interested in
vegetables and find growing them fun as their knowledge increases, which in turn may encourage them to eat
more vegetables. The code “unconsciousness” suggests that vegetable cultivation may steer participants toward
increasing their vegetable intake at an unconscious level (Alaimo et al., 2008; Thaler & Sunstein, 2008).
Growing vegetables may therefore encourage increased vegetable intake without the requirement for a conscious
choice on the part of the grower.
Regarding the reasons why vegetable cultivation does not increase vegetable intake, the category “limited
quantities” was used to refer to the amount that people can physically eat. For example, older people may eat less
because they find it harder to chew and swallow. Some may be unable to eat, even if the supply of food is
unlimited. Different approaches would be required to encourage these people to increase their vegetable intake.
The category “negative emotions toward vegetables” was divided into reasons related to preference and those
related to an absent sense of waste. People who do not like vegetables are unlikely to increase their intake
because they have grown their own produce. Encouraged or forced to do so, they would likely waste
considerable amounts. However, encouraging vegetable cultivation may help those who like vegetables or who
are indifferent to increases in their vegetable intake. A number of respondents felt that “cultivation for a purpose
other than eating vegetables” may not result in an increased vegetable intake. However, as previously noted,
greater exposure to vegetables may have a subconscious effect on intake (Alaimo et al., 2006; Thaler & Sunstein,
2008). It is therefore possible that those growing vegetables for other purposes may also increase their intake,
possibly without being aware of it. It is hence unclear whether this category is a genuine reason for vegetable
intake not increasing or simply an unfounded perception held by some of the respondents. Vegetable cultivation
may have no effect among those who are already getting enough vegetables from other sources, hence the
category “access to vegetables from other sources.” For example, a previous study in a rural area of Japan
reported an interactive effect on vegetable intake between vegetable cultivation and participants being given
vegetables (Machida & Yoshida, 2018a). In an area where vegetable cultivation flourished, many people grew
their own vegetables; moreover, they were also given vegetables or able to purchase them from farmers' markets
(Machida & Yoshida, 2018c). This area therefore had large numbers of people with high vegetable intakes
(Machida & Yoshida, 2018c). Interventions to encourage vegetable cultivation may therefore be more effective
in areas with little vegetable growing and where access to other sources or vegetables (e.g., supermarkets) is
relatively poor. Some respondents commented on the “limits associated with self-cultivation.” It may therefore
be helpful to provide guidance to improve the effectiveness of interventions encouraging people to grow
vegetables. This may require cooperation with professional growers and experts.
This study had some limitations. First, the methodology was only qualitative. Quantitative studies are therefore
also needed to explore the findings over a larger sample. It also cannot be said that the data was theoretically
saturated, as we did not use gradual theoretical sampling. However, a strength of the study was that we were able
to incorporate data from a range of people, including those with different cultivation styles and living in three
types of geographic area.
5. Conclusion
We clarified the reasons for vegetable intake increasing (or not) due to vegetable growing. As a result, there were
five main reasons why vegetable growing both may and may not increase vegetable intake. In the future,
quantitative studies are needed to explore the findings.
Authors’ contributions
DM designed the study and analysis and wrote the initial draft of the manuscript. TY provided assistance in
designing the study, analysis, and interpretation in addition to critically reviewing the manuscript. Both authors
approved the final version of the manuscript and agreed to be hold accountability for all aspects of the work in
ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated
and resolved.
Acknowledgments
We would like to thank the participants in this study. This study was funded by the Nakatani Suzuyo Memorial
Fund for Nutrition and Dietetics and JSPS KAKENHI (JP18K13037). The authors would like to thank Enago
(www.enago.jp) for the English language review.
Confrict of Interest
The authors have no conflict of interest to report.
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17
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This is an open-access article distributed under the terms and conditions of the Creative Commons Attribution
license (http://creativecommons.org/licenses/by/3.0/).
Journal of Food Research; Vol. 9, No. 1; 2020
ISSN 1927-0887 E-ISSN 1927-0895
Published by Canadian Center of Science and Education
19
Effect of Boiling and Wet Frying on Nutritional and Antinutrients
Content of Traditional Vegetables Commonly Consumed in Malawi
Joseph Y. Issa1, 2, Arnold Onyango1, Anselimo O. Makokha1 & Judith Okoth1
1Department of Human Nutrition Sciences, School of Food and Nutrition Sciences, Jomo Kenyatta University of
Agriculture and Technology, P.O. Box 62000 0020 Nairobi, Kenya
2Centre for Innovation and Industrial Research, Malawi University of Science and Technology, P.O.Box 5196,
Limbe, Malawi
Correspondence: Centre for Innovation and Industrial Research, Malawi University of Science and Technology,
P.O.Box 5196, Limbe, Malawi. E-mail: jissa@must.ac.mw
Received: August 9, 2019 Accepted: October 29, 2019 Online Published: November 7, 2019
doi:10.5539/jfr.v9n1p19 URL: https://doi.org/10.5539/jfr.v9n1p19
Abstract
This study was carried out to evaluate the effects of boiling and wet frying on nutritional and antinutrients
content of Amaranth hybridus, Moringa oleifera, Bidens pilosa (black jack), Corchorus olitorius (Jute mallow)
and Ipomea batatas (sweet potato) leaves. The edible portions of the vegetables were either boiled or wet fried
for ten minutes then dried alongside the raw vegetables under the shade. Crude fats, minerals, vitamins and
antinutrients were determined in the dried materials. Wet frying increased the oil content of the vegetables by a
range of 15.49% to 28.40 % and was hence associated with lower % ash and mineral contents. Wet frying
significantly reduced (P≤0.05) beta-carotene in all the vegetables except in jute mallow. Boiling had no
significant effect on beta-carotene in most of the vegetables. Boiling significantly reduced (P≤0.05) ascorbic acid
in all the vegetables while wet frying preserved ascorbic acid in all the vegetables. Both boiling and wet frying
significantly reduced (P≤0.05) oxalates in all the vegetables except in black jack. Both boiling and wet frying
significantly (P≤0.05) reduced the concentration of phytates in most of the vegetables. However, boiling was
more effective in reducing the amount of phytates. Boiling reduced higher concentrations of tannins in all the
vegetables as compared to wet frying. Boiling was associated with better retention of minerals and beta-carotene,
and greater reduction of antinutrients in most of the vegetables. Wet frying was more advantageous in retaining
vitamin C. The different species showed differences in retention of various minerals and vitamins.
Keywords: Vitamins, minerals, anti-nutrients, boiling, wet frying, traditional vegetables, Malawi
1. Introduction
Vegetables are the most affordable source of minerals and vitamins for families in most developing countries
(Kenya Demographic Health Survey, 2014). In Malawi, most households especially from the rural and
peri-urban areas depend on plant based food and vegetables as source of minerals and vitamins ( Cathoric Relief
Services [CRS], 2017). Traditional leafy vegetables are widely utilized in Malawi and thus play a big role in
contributing to nutrition. Leafy vegetables are rich sources of iron, pro-vitamin A (beta-carotene), vitamin C and
zinc among other vitamins and minerals (Maundu, 2014).
According to the assessment survey conducted by CRS, (2017), the following traditional vegetables are widely
consumed and preferred within the southern region of Malawi: Amaranth, Corchorus olitorius (jute mallow)
leaves, Moringa oleifera , Ipomea batatas (sweet potato) leaves, Bidens pilosa (black jack) leaves , pumpkins
leaves, Cleome gynandra leaves and cowpea leaves. Among these, Amaranth, Jute mallow, Black jack and
Cleome gynandra plants grow wildly in the bush or as weeds in the fields.
Farmers harvest these traditional and wild vegetables in the bush and the gardens for consumption and sale.
Sweet potato leaves, pumpkins leaves and cowpea leaves are harvested from the cultivated fields and are
consumed or sold as vegetables. These commonly consumed vegetables in Malawi play a big role in contributing
to health and nutrition through the vitamins and minerals that they possess. According to CRS, (2017), Amaranth
leaves are nutritionally rich in calcium, iron, beta carotene and ascorbic acid; sweet potato leaves leaves are rich
sources of iron, ascorbic acid and beta carotene; black jack leaves are rich in beta carotene, iron, Zinc and
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20
ascorbic acid; Cleome gynandra leaves are rich sources of ascorbic acid, beta carotene, folic acid and calcium;
Moringa oleifera leaves are rich sources of beta carotene, vitamin E, iron, folic acid and calcium; and Jute
mallow leaves are rich in beta carotene, iron, folic acid ascorbic acid, calcium and protein.
The nutrients being supplied by these indigenous and traditional vegetables are very important to human health
and nutrition. Most of the vitamins and minerals found in vegetables such as beta carotene, ascorbic acid, iron,
zinc and magnesium contributes to improvement of the immune function ( Ebrahimzadeh, M. A., Pourmorad, F.,
& Bekhradnia, A. R. (2008). Beta carotene which is a precursor of vitamin A is essential for growth,
development, immunity and good vision. Beta carotene and ascorbic acid also play a role of antioxidants and
helps to reduce the risk of diseases related to oxidative stress such as diabetes mellitus, cardiovascular diseases
and some cancers (Yang & Keding, 2009 ; Uusiku, N. P., Oelofse, A., Duodu, K. G., Bester, M. J., & Faber, M.
(2010). Iron is essential for prevention of iron deficiency anemia which is the main causes of anemia in the
world. The main risk factors of iron deficiency anemia are low intake of iron, poor bioavailability from iron food
containing phytates and polyphenols (Passone, M. A., Resnik, S. L., & Etcheverry, M. G. (2005). Zinc is also a
micronutrient which is responsible for normal growth function and immune function; its biological roles are
structural, as catalyst and as a regulatory ion ( Etcheverry, P., Grusak, M. A., & Fleige, L. E. (2012).
Vegetable containing diets however, may lead to reduced availability of minerals from the diets due to presence
of antinutrients (Etcheverry et al., 2012). Traditional leafy vegetables are plenty in Sub-Saharan Africa including
Malawi and they are known to contain anti-nutrients such as phytates, tannins, glycosides, oxalates, alkaloids
and hydrocyanic acid. Tannins are identified as plant polyphenols that are capable of forming complexes with
metal ions and macro-molecules like proteins and polysaccharides (Olawoye & Gbadamosi, 2017). Tannins
affect the nutritional value of food products by chelating minerals like iron and zinc and reducing the absorption
of these minerals as well as forming complexes with protein thereby inhibiting their digestion and absorption
(Olawoye et al., 2017). Tannins are responsible for a decrease in growth rate as well as a non-palatable taste as
they contribute to protein inhibition by forming complexes (Etcheverry et al., 2012).
Phytic acid (phytates) is the major phosphorous storage compound in traditional leafy vegetables. Phytic acid
has been reported to inhibit the absorption of minerals such as calcium, iron and zinc; and reduce the
bioavailability of these minerals in food (Etcheverry et al., 2012). Too much of a phytate rich diet is associated
with nutritional diseases such as rickets and osteomalacia or osteoporosis in children and adults (Otunola &
Afolayan, 2017) respectively.
Oxalic acid (Oxalates) exists in many leafy vegetables and plant foods. Depending on species, oxalic acid can
occur as soluble salts of potassium and sodium or as insoluble salts of calcium, magnesium or iron or it can
occur as a combination of soluble and insoluble salts (Essack, Odhav, & Mellem, 2017) . This forms strong
chelates with dietary calcium thereby inhibiting its absorption (Akwaowo, Ndon, & Etuk, 2000) . It is well
known that oxalic acid and its salts can have a deleterious effect on human nutrition and health, mostly by
decreasing calcium absorption and aiding the formation of kidney stones (Olawoye et al., 2017).
Food processing techniques are often utilized in order to reduce antinutrients in food. Cooking is a common form
of processing of plants that are consumed as a food source at household level. Some studies have suggested that
different cooking methods may have different effects on different nutrients and antinutrients. Thus, the
micronutrients and antinutrients are affected differently by processing, depending on the type of processing, as
well as the type of vegetable species. Cooking causes changes in the phytochemistry of the leafy vegetable
affecting its nutrients bioavailability and health benefit properties. The degree of these changes depends largely
on the cooking methods as well as the type of the vegetable ( Odhav, B., Beekrum, S., Akula, U., & Baijnath, H.,
2007).
This study was therefore conducted to investigate the effect of short time boiling and sautéing (wet frying) on
nutrients retention and antinutrients content in five traditional vegetables commonly consumed in Malawi.
Boiling and wet frying are the common methods of cooking vegetables in Malawi.
2. Materials and Methods
2.1 Sample Collection and Preparation
Five traditional leafy vegetables that are commonly consumed in Malawi (Amaranth hybridus, Moringa oleifera,
Bidens pilosa (black jack), Corchorus olitorius (Jute mallow) and Ipomea batatas (sweet potato leaves) were
used in this study. Vegetable samples were randomly collected from the gardens in the southern region of Malawi.
Moringa oleifera leaves were sampled from the Moringa trees in the district of Chikwawa, Malawi. Edible
portions of the fresh vegetable leaves in this study were harvested and put in the sealed plastic bags. The plastic
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21
bags containing the vegetables were placed in portable coolers and transferred to a laboratory at Malawi
university of Science and Technology (MUST) for processing. Vegetable leaf samples were washed with tap
water followed by distilled water before further processing. The vegetables were then cooked using the methods
that are commonly used in Malawi, thus boiling and wet frying (sautéing). A portion of each of the vegetable
samples (about 1 kilogram) was boiled in 800ml of distilled water for 10 minutes with frequent turning to allow
even cooking in stainless steel pots. Similar portions (1 kilogram) of the samples were wet fried in 200ml of
vegetable oil for 10 minutes with frequent turning to allow even cooking in stainless steel pots. Other similar
portions (1kilogram) of the raw samples were air dried in a shade until crispy dry without cooking. The boiled
and wet fried samples were also dried under shade as was done with the raw samples until crispy dry. The
temperature during the drying period ranged from 29-35 degrees Celsius. Dried samples were wrapped in
alluminium foils and kept in tightly closed plastic containers. All the samples were then transferred to Jomo
Kenyatta University of Agriculture and Technology (JKUAT), Food Biochemistry laboratory for chemical
analysis.
2.2 Nutritional Analysis
2.2.1 Determination of Crude Fats
Crude fat content was determined using Soxhlet method according to AOAC. To achieve this, 5g of ground dry
sample was placed in the thimble then the thimble was placed in the soxhlet extractor. Then 150mL round
bottom flask was cleaned and filled with 90 ml petroleum ether. The whole setting was placed on a heating
mantle to allow the petroleum ether to boil. The extraction was done for almost 6 hours. The condensing unit
was then removed from extraction unit to allow the sample to cool down as it had finally removed all the lipids.
The flasks containing extracted fat were then placed in the oven at 102oC to dry to constant weight then were
placed in a desiccator before they were weighed. The % Crude fat was calculated using the following method:
% Crude fat = ( W2-W1) × 100 / S (1)
Where W1 = weight of empty flask, W2=weight of flask and extracted fat, and S= weight of sample.
2.2.2 Determination of Ash and Minerals
Minerals (zinc, iron, calcium, magnesium and copper) were determined according to AOAC (1995, Method 970:
12). To achieve this, about 2 grams of each sample was weighed and delivered into the crucibles. The crucibles
were placed on a hot plate under a fume hood and the temperature was increased slowly until smoking ceased to
char the samples. Samples were then put in muffle furnace and temperature was increased gradually to 2500C
and heated for 1 hour. The temperature was then increased to 550 0C and incinerated to complete ashing.
Thereafter, the temperature was decreased to 300 0C, and the crucibles were removed and cooled to room
temperature. The ash was measured using an analytical balance to determine percentage of ash in the samples.
The ash was then transferred quantitatively to 100 mL beakers containing 20 mL of 1N HCL, then heated at
about 80-90 0C on a hot plate for 5 minutes. These were then transferred to 100 mL volumetric flask and were
filled to the mark with 1N HCL. Insoluble matter was filtered and the filtrate was kept in a labeled polyethylene
bottles. The absorbances of the solutions were read by Atomic Absorption Spectrophotometer (AAS) Shimadzu
Japan. Minerals standards were prepared at varying concentrations to make the calibration curves. Iron was
measured at a wavelength of 284.3nm, zinc was measured at 213.9nm, calcium was measured at 422.7nm,
magnesium was measured at 285.5nm and copper was measured at 324.8nm.
2.2.3 Determination of Beta Carotene
Beta carotene content in the vegetable samples was analyzed using column chromatography and UV
Spectrophotometer. Extraction of the carotenoids was done using acetone and petroleum ether as described by
Rodriguez-Amaya, D. & Kimura, M., (2004) . Briefly, approximately 2 grams of each sample was weighed using
analytical balance and placed in a mortar with about 10 mL of acetone. This was followed by thorough grinding
with a pestle then the acetone extracts were transferred into 100 mL volumetric flasks. The residues were again
extracted with 10 mL acetone and the extracts were added to the contents of the volumetric flasks. The extraction
with acetone was continued until the residues no longer gave color. The combined extracts were then made to a
volume of 100 mL with acetone. About 50mL of each extract was evaporated to dryness using rotary evaporator.
The residue was dissolved with 10 mL petroleum ether and the solution was introduced into a chromatographic
column to elute beta-carotene. The eluted beta-carotene was collected in 50ml volumetric flasks and made to a
volume of 25 mL with petroleum ether. The absorbances of the solutions were read at 440 nm using a UV-Vis
spectrophotometer. Beta carotene standards were prepared and read together with the samples at 440nm.
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2.2.4 Determination of Ascorbic Acid
Ascorbic acid content of the samples was determined by High Performance Liquid Chromatography (HPLC)
method (Vikram, V. B., Ramesh, M. N., & Prapulla, S. G. (2005) with some modifications. To achieve this, about
2 g of each sample was weighed and extracted with 30mL 0.8% metaphosphoric acid. The liquid extract was
centrifuged at 10,000 rpm. The supernatant was filtered and diluted with 10 mL of 0.8% metaphosphoric acid
using 0.45 µL filter and 20 µL of the filtrate was injected in HPLC for analysis. Various concentrations of
ascorbic acid standards were prepared to make a calibration curve. HPLC analysis was done using Shimadzu
UV-VIS detector at a wavelength of 266.0nm. The mobile phase was 0.8% metaphosphoric acid with flow rate
of 1.2 mL/minute.
2.3 Antinutrients Determination
2.3.1 Determination of Phytates
Phytates content was determined using HPLC as described by (Camire, & Clydesdale. (2006). To achieve this,
about 0.5 g of each sample was extracted with 10 mL of 3% H2SO4 and shaken at the automatic shaker for
45minutes. The contents were filtered and the filtrate was transferred to a boiling water bath to heat for 5
minutes followed by addition of 3 mL of FeCl 3 solution (6 mg ferric iron per mL in 3% H2SO4). The contents
were then heated for 45 minutes to complete precipitation of the ferric phytate complex. Samples were then
centrifuged at 2500 rpm for 10 minutes and the supernatant was discarded. The precipitate was washed with 30
mL of distilled water, centrifuged and the supernatant was discarded. A 3 mL of 1.5 N NaOH was added to the
residues and the volumes were brought to 30 mL with distilled water. The contents were then heated for 30
minutes in a boiling water bath to precipitate the ferric hydroxide. Samples were then cooled and centrifuged and
the supernatant was transferred into a 50 mL volumetric flask. The precipitate was rinsed with 10 mL distilled
water, centrifuged and the supernatant was added to the contents of the volumetric flask. This was filtered using
0.45µL filter before HPLC analysis. The mobile phase was 0.005 N sodium acetate in distilled water, at a flow
rate of 0.5 µL/minute.
2.3.2 Determination of Oxalates
Oxalic acid contents of the vegetable samples was determined by HPLC method as described by ( Chong, Y., Liu,
Y., & Yanping, F. (2002). To achieve this, about 0.3g of each sample was homogenized in 10 mL of 0.5N HCL.
The homogenate was heated at 80 0C for 10 minutes with intermittent shaking on the water bath. To the
homogenate, distilled water was added up to a volume of 25 mL. About 3 mL of the solution was withdrawn and
centrifuged at 12,000 rpm for 10 minutes. About 1 ml of supernatant was passed through a micro filter (0.45µL)
before HPLC analysis. Phytic acid standards were prepared at varying concentrations for quantification. HPLC
analysis was done by UV-VIS detector using a solution of 0.01 N H2SO4 as mobile phase. The flow rate was 0.6
mL/ minute and was detected at a wavelength length of 221 nm.
2.3.3 Determination of Tannins
Tannins were estimated by Vanillin-HCl method as described by Millet P, (2013). Approximately 0.1g of ground
dry leaf vegetable samples were accurately weighed into Erlenmeyer flasks. 10 ml of 4% HCL in methanol was
pipetted into each flask, sealed with parafilm and shaken (KS 250 basic, Germany) for 20 minutes, centrifuged
(HPLC-CTO-10AVP Shimadzu; detector used- Shimadzu RID6A Refractive Index Detector) for 10 minutes at
4500 rpm and supernatants were transferred to 25 ml volumetric flasks. The residues were extracted for the
second time using 5 ml of 1% HCL in methanol for 10 minutes. The aliquots of the first and the second extracts
were combined and made up to 25 ml volume using methanol. A set of catechin (Sigma) standards solutions
were prepared ranging from 10 to 100 ppm using methanol as a solvent. One (1 ml) of suitably diluted extracts
were taken in a test tube and 5ml of freshly prepared vanillin-HCl reagent was added to each test tube. To correct
for interference of natural pigments in the dry vegetables, a blank sample was prepared by subjecting the original
extract to the reaction conditions without the vanillin reagent. These were prepared by adding 5 ml of 4% HCL
in methanol to 1 ml of the aliquots of the extracts that were pipetted into the test tubes. The absorbances of the
standard solutions, sample extracts and blanks were read using a UV-VIS Spectrophotometer (Shimadzu, UV
mini 1240, Japan) at 500 nm exactly 20 minutes after adding Vanillin-HCL reagent to the samples and standards.
A standard curve was prepared from the readings of the catechin standard solutions. Tannin content was
expressed in mg of catechin equivalent (CE) per 100g of sample (mg CE/100g).
2.4 Data Analysis
Data was analysed using STATA software (STATA 14.0, 2015, Texas, USA). The mean differences for nutrients
and antinutrients within the sample groups as affected by treatments was tested for its significance differences
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using one way analysis of variance (ANOVA) by Bonferroni test. A p-value of ≤ 0.05 was used for the test of
significance. All samples were analyzed in triplicates.
3. Results and Discussion
3.1 Effects of Wet Frying and Boiling on Nutrients Content
3.1.1 Effects of Wet Frying and Boiling on Crude Fats
The results for Crude fats in the five traditional vegetables and its respective wet fried and boiled counterparts
for each vegetable are presented in Table 1. The percentages of crude fats in the dry uncooked vegetables ranged
from 1.07% to 2.28%. The mean amount of crude fats in the five vegetables were 2.28%, 2.18%, 1.91%, 1.32%
and 1.07% in Moringa oleifera, black jack, sweet potato leaves, jute mallow and Amaranth hybridus
respectively.
Table 1. Crude fat content (%) in traditional vegetables as influenced by cooking methods
Vegetable leaves
Dried (n=3)
Wet fried (n=3)
Boiled (n=3)
Sweet potato
1.91±0.06b
30.31±0.46a
2.82±0.13b
Black jack
2.18±0.45b
31.61±1.47a
2.70±0.35b
Moringa oleifera
2.28±0.48b
19.00±0.26a
1.81±0.17b
Jute mallow
1.32±0.62b
16.81.1.31a
2.42±0.33b
Amaranth hybridus
1.07±0.40b
28.07±2.53a
1.68±1.06b
Entries are Mean ±SD
Values in the same row followed by the same superscripts are not significantly different at α=0.05 using
Bonferroni ANOVA test.
There was no significant difference in the percentage of fats between the boiled vegetables and the raw
vegetables. Wet frying significantly increased (P≥0.05) the amount of fats in all the vegetables. The increase of
fats in the wet fried samples is due to absorption of fats by the vegetables during the process of wet frying. The
percentages of fats in the wet fried vegetables ranged from 16.81% to 30.31%. The differences in the percentage
of absorbed fats in the wet fried vegetables might be due to different matrix of the vegetable leaves.
3.1.2 Effect of Boiling and Wet Frying on Ash Content
Figure 1. Mean % of ash in traditional vegetables as influenced by cooking methods. SP=Sweet potato; BJ=
Black jack; MO= Moringa oleifera; JM= Jute mallow and AH=Amaranthus hybridus. Mean ±SD values (n=3) in
the same group followed by the same superscripts are not significantly different at (p≤0.05) using Bonferroni
ANOVA test
a
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b
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c
b
a
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b
a
b
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c
b
6 8 10 12 14 16
SP BJ MO JM AH
Dried Wet fried
Boiled
a
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The ash content in the dry uncooked vegetables ranged from 10.33% to 16.61%. The ash content in these dry
uncooked vegetables were 16.61%, 11.67%, 11.62%, 10.60%, 10.33% and in the leaves of Amaranth hybridus,
black jack, sweet potato, Moringa oleifera and jute mallow respectively. Figure 1 indicate that there were
significant differences in the percentages of ash as a result of wet frying and boiling in all the vegetables. Wet
frying resulted to higher losses of ash in all the vegetables compared to boiling except in jute mallow vegetables
where the ash reduction was statistically similar (P≥0.05) for wet fried and boiled jute mallow. The higher
reduction of ash percentages in wet fried as compared to boiled vegetables is as a result of high absorption of oils
in the wet fried vegetables that led to apparent decrease in percentages of ash. The loss of ash due to boiling
could be as a result of leaching of minerals in the boiling water. Thus most minerals were leached into the
boiling water and this led to reduced ash content because the ash contains minerals. These results tally with the
work done by Traoré, K., Parkouda, C., Savadogo, A., Ba, F., Regine, H., & Yves, K., (2017) where it was found
that the amount of ash reduced after blanching and boiling of vegetables that included jute mallow and Amaranth
cruentus L. The authors found that the amount of ash reduced in jute mallow and Amaranth from 12.40% and
16.33% to 9.37% and 14.27% respectively after boiling for 30 minutes due to leaching of minerals in the boiling
water.
In this study, the percentage of ash in jute mallow and Amaranthus hybridus reduced from 10.60% and 16.61%
to 8.06% and 9.83% after wet frying; and to 8.99% and 13.78% after boiling them respectively. The apparent
reduction of percentages of ash in the wet fried vegetables is due to increased oil content. Gunathilake, K. D. P.
P., Ranaweera, K. K. D. S., & Rupasinghe, H. P. V., (2018) reported that during deep-fat frying, oils undergo
physicochemical changes; the food dehydrates, and oils penetrates the food.
3.1.3 Effect of Boiling and Wet Frying on Iron Content
Figure 2 presents the iron content of the five traditional leafy vegetables; sweet potato, black jack, Moringa
oleifera, jute mallow and Amaranthus hybridus. It also presents the amount of iron in the vegetables as
influenced by wet frying and boiling of each of the traditional vegetables. The amount of iron in the dry uncooked
vegetables ranged from 37.22mg/100g to 48.21mg/100g on dry wet basis. The iron content in the dry uncooked
vegetables were 48.21mg/100g, 40.43mg/100g, 37.22mg/100g, 37.30mg/100g and 44.89mg/100g in the leaves of
sweet potato, black jack, Moringa oleifera, jute mallow and Amaranth hybridus respectively. Boiling and wet
frying for 10 minutes did not significantly reduce (P≥0.05) iron content in black jack leaves although there was
some slight losses of iron due to these processes. There were significant losses (P≤0.05) of iron in sweet potato
leaves, Moringa oleifera leaves and jute mallow leaves as a result of wet frying and boiling. Wet frying
significantly (P≤0.05) reduced iron content in Moringa oleifera and jute mallow vegetables as compared to
boiling the same vegetables for 10 minutes. The losses of iron due to boiling is as a result of leaching of the
mineral iron into the boiling water. These results are similar to work done by Lewu, M. N., Adebola, P. O., &
Afolayan, A. J. (2009) where the authors found that there were losses of iron due to boiling in most of the
vegetables. Habwe, F. O., Walingo, M. K., Abukutsa-onyango, M. O., & Oluoch, M. O. (2009) also found a
high reduction of iron content as a result of wet frying as compared to boiling for 5 minutes in Amaranthus
hybridus. However, Ojo, O. O., Taiwo, K. A., Scalon, M., Oyedele, D. J., & Akinremi, O. O. (2016) reported
slight increase of iron in Solanum marcrocarpon after boiling for 5 minutes in seven cultivars.
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Figure 2. Mean amount of iron in traditional vegetables as influenced by cooking methods. SP=sweet potato,
BJ= Black jack, MO=Moringa oleifera, JM=Jute mallow and AH=Amaranthus hybridus. Mean ±SD values in
the same group followed by the same superscripts are not significantly different at (p≤0.05) using Bonferroni
ANOVA test
3.1.4 Effect of Boiling and Wet Frying on Zinc Content
The results for Zinc content of the five traditional vegetables and its respective wet fried and boiled counterparts
for each vegetable are presented in Table 2. The amount of zinc in the dry uncooked vegetables ranged from
4.42mg/100g to 10.59mg/100g on dry weight basis. The mean amount of zinc in these vegetables were
5.47mg/100g, 7.27mg/100g, 10.59mg/100g, 4.42mg/100g and 5.70mg/100g in the leaves of sweet potato, black
jack, Moringa oleifera, jute mallow and Amaranth hybridus respectively. There was a significant reduction
(P≤0.05) of zinc in sweet potato leaves, Moringa oleifera leaves, and Jute mallow due to wet frying and boiling
for 10 minutes. Boiling of vegetables was also reported to reduce amount of zinc in different cultivars of
Colocasia esculenta vegetables (Lewu et al., 2009). Boiling did not significantly reduce the amount of zinc in
black jack leaves, however, wet frying reduced the zinc concentration significantly (P≤0.05). Wet frying also
decreased the amount of zinc in Jute mallow significantly (P≤0.05) compared to boiling the same vegetables for
10 minutes due to apparent increase in fats. There was also a significant reduction of zinc in Amaranthus
hybridus due to wet frying as compared to boiling.
Table 2. Zinc content (mg/100g DM) in traditional vegetables as influenced by cooking methods
Vegetable leaves
Dried (n=3)
Wet fried (n=3)
Boiled (n=3)
Sweet potato
5.47±2.38a
2.43±0.70b
6.15±0.87b
Black jack
7.27±0.98a
4.82±0.64b
6.49±0.16a
Moringa oleifera
10.59±2.32a
1.78±0.04b
2.48±0.03b
Jute mallow
4.42±0.12a
2.37±0.05c
3.01±0.07b
Amaranth hybridus
5.70±0.20b
4.73±0.12c
9.03±0.20a
Entries are mean ± SD
Values in the same row followed by the same superscripts are not significantly different at α=0.05 using
Bonferroni ANOVA test.
3.1.5 Effect of Boiling and Wet Frying on Copper Content
Figure 3 presents the results for amount of Copper in the raw dried vegetables and their respective wet fried and
boiled vegetables. The amount of Copper in the raw dried vegetables ranged from 1.29mg/100g to 3.90mg/100g.
The mean amount of Copper in the vegetables were 1.29mg/100g, 1.55mg/100g, 1.98mg/100g, 3.20mg/100g and
3.90mg/100g in Moringa oleifera, Amaranth hybridus, jute mallow, sweet potato leaves and black jack
respectively. The results indicate that there was no significant loss (P≤0.05) of copper due to boiling of sweet
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b
a
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a
c
b
a
c
b
bb
a
20 30 40 50 60
SP BJ MO JM AH
Dried Wet fried
Boiled
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potato leaves, black jack leaves and jute mallow leaves. There was however significant decrease (P≤0.05) of
copper due to wet frying in sweet potato leaves, black jack leaves, and jute mallow leaves because these
vegetables absorbed higher oils. There was no significant loss of copper due to wet frying in Moringa oleifera
leaves because Moringa oleifera absorbed relatively less amount of oils that resulted to apparent less effect on
copper content. Lewu et al., 2009 reported good retention of copper after boiling in several cultivars of Colocasia
esculenta with little or no losses of the mineral in other cultivars. Ojo et al., (2016) also reported slight reduction
of copper in Solanum marcrocarpon after boiling for 5 minutes.
3.1.6 Effect of Boiling and Wet Frying on Calcium Content
The results for amount of Calcium in the five vegetables and its respective wet fried and boiled counterparts for
each vegetable are presented in Figure 4. The amount of Calcium in the uncooked dried vegetables ranged from
150.86mg/100g to 559.75mg/100g. The mean amount of Calcium in the leafy vegetables were 150.86mg/100g,
176.54mg/100g, 242.95mg/100g, 396.10mg/100g and 559.75mg/100g in black jack , jute mallow, sweet potato
leaves, Amaranth hybridus and Moringa oleifera respectively. There were significant losses (P≤0.05) of calcium
due to wet frying in the vegetable leaves of sweet potato, black jack, Moringa oleifera and Amaranthus hybridus
due to apparent increase in crude fats. There was no significant loss of calcium as a result of boiling in black jack,
Moringa oleifera, jute mallow and Amaranthus hybridus leafy vegetables due to possible insolubility of calcium
in these vegetables. In a study by Ojo et al., 2016, it was found that boiling retained the amount of Calcium after
boiling Solanum marcrocarpon for five minutes. Wet frying did not significantly reduce calcium content in jute
mallow leafy vegetables.
Figure 3. Mean amount of Copper in traditional vegetables as influenced by cooking methods. SP=sweet potato,
BJ=Black jack, MO=Moringa oleifera, JM=Jute mallow and AH= Amaranthus hybridus. Mean ±SD values in
the same group followed by the same superscripts are not significantly different at α=0.05 using Bonferroni
ANOVA test
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a
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b
a
a
b
a
bb
a
1 2 3 4
SP BJ MO JM AH
Dried Wet fried
Boiled
a
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Figure 4. Mean amount of Calcium in traditional vegetables as influenced by cooking methods. SP=sweet potato,
BJ=Black jack, MO=Moringa oleifera, JM=Jute mallow and AH= Amaranthus hybridus. Mean ±SD values in
the same group followed by the same superscripts are not significantly different at α=0.05 using Bonferroni
ANOVA test
3.1.7 Effect of Boiling and Wet Frying on Magnesium Content
Magnesium content of the five traditional leafy vegetables and their respective boiled and wet fried counterparts
are presented in table 3. The amount of Magnesium in the uncooked dry vegetables ranged from 101.19mg/100g
to 196.22mg/100g. The mean amount of Magnesium in the leafy vegetables were 101.19mg/100g,
142.23mg/100g, 154.67mg/100g, 156.07mg/100g and 196.22mg/100g in jute mallow, black jack, Moringa
oleifera, sweet potato leaves and Amaranth hybridus respectively. There were significant losses (P≤0.05) of
Magnesium due to wet frying in leafy vegetables of sweet potato, black jack, Moringa oleifera, and Amaranth
hybridus. Boiling significantly (P≤0.05) reduced the amount of Magnesium in sweet potato leaves but not as
much as wet frying. There was no significant loss of Magnesium as a result of boiling in the vegetable leaves of
black jack, Moringa oleifera, jute mallow and Amaranthus hybridus. Lewu et al., 2009 however reported slight
reduction of Magnesium in some cultivars of Colocasia esculenta after boiling for five minutes. The reduction of
Magnesium in the vegetables as a result of boiling is due to leaching of the mineral in the boiling water.
Table 3. Magnesium content (mg/100g) in traditional vegetables as influenced by cooking methods
Vegetable leaves
Dried (n=3)
Wet fried (n=3)
Boiled (n=3)
Sweet potato
156.07±2.49a
93.47±2.34c
134.83±5.45b
Black jack
142.23±2.09a
101.37±5.08b
146.89±5.01a
Moringa oleifera
154.67±4.40b
148.25±4.91b
179.61±5.85a
Jute mallow
101.19±5.77a
93.99±1.33a
106.47±1.17a
Amaranth hybridus
196.22±0.60a
189.45±1.59b
196.96±3.18a
Entries are Mean ±SD
Values in the same row followed by the same superscripts are not significantly different at α=0.05 using
Bonferroni ANOVA test.
3.1.8 Effect of Boiling and Wet Frying on Beta-carotene Content
Figure 5 presents the results for mean amount of beta-carotene in the five traditional vegetables and the influence
of 10 minutes wet frying and boiling for each of the vegetables on beta-carotene. The amount of beta-carotene in
the uncooked dried vegetables ranged from 829.68µg/100g to 5791.64µg/100g on dry weight basis. The mean
amount of beta-carotene in the leafy vegetables were 5791.64µg/100g, 2153.29µg/100g, 1242.21µg/100g,
1055µg/100g and 829.68µg/100g in Moringa oleifera, jute mallow, black jack, sweet potato leaves and
Amaranth hybridus respectively. Boiling did not significantly reduce (P≤0.05) beta-carotene content in the
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0
200 400 600 800
SP BJ MO JM AH
Dried Wet fried
Boiled
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vegetables leaves of sweet potato, black jack and Moringa oleifera. There was however better retention of
beta-carotene in sweet potato and black jack leaves as a result of boiling. Wet frying significantly reduced
beta-carotene content in black jack leafy vegetables. There was no significant loss (P≤0.05) of beta-carotene in
Amaranthus hybridus due to wet frying, however, boiling retained the vitamin significantly in the same
vegetable. There was also a significant retention of (P≥0.05) beta-carotene due to boiling for 10 minutes in jute
mallow vegetables. These results agrees with the work by Ogliano, V. I. F., & Ellegrini, N. I. P. (2008) where
they found that in most vegetables frying reduced the amount of beta-carotene and boiling for a short period
increased beta-carotene in broccoli and courgettes vegetables. Cooking of green fresh vegetables has been
reported to promote release of carotenoids from the matrix because of the disruption of carotenoids-protein
complexes, leading to better extractability and higher concentrations in cooked samples (Ogliano et al., 2008).
Beta-carotene losses due to frying has been attributed to loss of the initial carotenoids concentration because of
leaching into oil at higher processing temperature. Traoré et al., 2017 also reported a reduction of beta-carotene
in jute mallow and amaranth due to shade drying alone. The results for shade dried samples of jute mallow and
amaranth in this study agrees with those reported by Traoré et al., 2017. Results from the study by Putra, U.
(2009) showed that both boiling and stir-frying increased the beta-carotene (2 to 4.2 times) of pumpkin. It was
found that boiling for 4 minutes resulted in the highest (4.2 times) increase of beta-carotene to 8.0 mg/100g from
2.0mg/100g. Six minutes boiling, on the other hand, resulted in 270% (3.7 times) increase in beta-carotene
content. In this study all the vegetables were either wet fried or boiled for 10 minutes. The 10 minutes cooking in
this study may attribute to more losses of beta-carotene as compared to when they would be cooked at a shorter
period. Long time cooking is attributed to longer exposure to oxidation and heat thereby leading to more losses
of beta-carotene. The higher losses of beta-carotene due to wet frying compared to boiling at 10 minutes in this
study might be attributed to the fact that carotenoids are fat-soluble compounds and solubilized readily in oil
during stir-frying as explained by Putra, 2009.
Figure 5. Mean amount of beta-carotene in traditional vegetables as influenced by cooking methods. SP=sweet
potato, BJ=Black jack, MO=Moringa oleifera, JM= Jute mallow and AH= Amaranthus hybridus. Mean ±SD
values in the same group followed by the same superscripts are not significantly different at α=0.05 using
Bonferroni ANOVA test
3.1.9 Effect of Boiling and Wet Frying on Vitamin C (Ascorbic acid) Content
The results for Vitamin C content of the five traditional vegetables and its respective wet fried and boiled
counterparts for each vegetable are presented in Figure 6. The amount of ascorbic acid in the uncooked dry
vegetables ranged from 7.93mg/100g to 35.81mg/100g on dry weight basis. The mean amount of beta-carotene
in the leafy vegetables were 35.81mg/100g, 28.78mg/100g, 25.48mg/100g, 22.85mg/100g and 7.93mg/100g in
the leaves of sweet potatoes, black jack, Moringa oleifera, jute mallow and Amaranth hybridus respectively.
Boiling significantly reduced (P≤0.05) vitamin C content in all the vegetables except in Amaranthus hybridus
where the amount of vitamin C was slightly reduced. Wet frying significantly retained (P≥0.05) vitamin C in all
the five traditional vegetables. The vitamin C losses in the vegetables due to boiling might be attributed to
a
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2000 4000 6000 8000
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Dried Wet fried
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leaching of the vitamin in the boiling water because Vitamin C is water soluble. Singh & Harshal, 2016 reported
losses of Vitamin C content in several vegetables after boiling for 10 minutes and after blanching and
microwaving. In their study, microwaving the vegetables significantly reduced the amount of Vitamin C than
blanching and boiling. The high retention of vitamin C due to wet frying in the wet fried samples in this study
might be due to its preservation in the cooking oil because vitamin C is slightly soluble in oil. There were slight
higher vitamin C content in almost all the wet fried vegetables compared to uncooked dried vegetables because
the wet fried vegetables were dried alongside the uncooked vegetables and more losses of vitamin C in uncooked
vegetables might occur during drying. The results for wet frying in this study, agrees with Masrizal, M., &
Giraud, D. (1996) where it was found that stir frying in cooking oil had the highest retention (by 1.31-1.83 fold)
of vitamin C in several vegetables than boiling. Armesto, J., Gómez-limia, L., Carballo, J., Martínez, S., Armesto,
J., Gómez-limia, L., … Armesto, J. (2019) also reported significant losses of vitamin C in Brassica oleracea after
boiling for 20 and 30 minutes.
Figure 6. Mean amount of ascorbic acid in traditional vegetables as influenced by cooking methods. SP= sweet
potato, BJ=Black jack, MO=Moringa oleifera, JM=Jute mallow and AH=Amaranthus hybridus. Mean ±SD
values in the same group followed by the same superscripts are not significantly different at α=0.05 using
Bonferroni ANOVA test
3.2 Effects of Wet Frying and Boiling on Anti-nutrients
3.2.1 Effects of Wet Frying and Boiling on Oxalates
The results for oxalates in the five traditional vegetables and its respective wet fried and boiled counterparts for
each vegetable are presented in figure 7. The amount of oxalates in the uncooked dry vegetables ranged from
77.06mg/100g to 771.95mg/100g on dry weight basis. The mean amount of oxalates in these uncooked
vegetables were 77.06mg/100g, 276.07mg/100g, 286.73mg/100g, 306.95mg/100g and 771.95mg/100g in black
jack, jute mallow, sweet potato leaves, Moringa oleifera and Amaranth hybridus respectively. Both boiling and
wet frying significantly reduced (P≤0.05) amount of oxalates in vegetable leaves of sweet potato, Moringa
oleifera, jute mallow and Amaranthus hybridus. Ojo et al., (2016) also reported a reduction of oxalates in
Solanum marcrocarpon vegetables after boiling for five minutes. In this study, there was no significant reduction
(P≥0.05) of oxalates in black jack leaves. The loss of oxalates in both wet fried and boiled black lack leaves was
17%. Also, Mcewan, R., Shangase, F. N., Djarova, T., & Opoku, A. R.,(2014) reported a significant reduction
of Oxalates in Colocasia esculenta after boiling as compared to stir frying.
3.2.2 Effects of Wet Frying and Boiling on Phytates Content
The results for phytates content in the five traditional vegetables and its respective wet fried and boiled
counterparts for each vegetable are presented in Table 4. Phytates content in the uncooked dry vegetables ranged
from 72.17mg/100g to 129.15mg/100g on dry weight basis. The mean amount of phytates in these uncooked
a
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a
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020 40 60 80
SP BJ MO JM AH
Dried Wet fried
Boiled
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vegetables were 72.17mg/100g, 86.64mg/100g, 99.80mg/100g, 124.95mg/100g and 129.15mg/100g in
Amaranth hybridus, jute mallow, Moringa oleifera, black jack and sweet potato leaves respectively. Boiling
significantly reduced (P≤0.05) the amount of phytates in jute mallow and Amaranthus hybridus vegetables.
There were significant losses (P≤0.05) of phytates due to wet frying in Moringa oleifera and jute mallow
vegetables. There were slight losses of phytates in both wet fried and boiled sweet potato and black jack leafy
vegetables though not significant (P≥0.05). Wet frying reduced the amount of phytates in Amaranthus hybridus
and black jack vegetables by 47% and 32% respectively. Results by Mcewan et al., (2014) revealed a significant
reduction of phytates after boiling and stir frying, where stir frying reduced the phytates more than boiling for
the same period in the tubers of Colocasia esculenta. The losses of phytates due to wet frying and boiling might
be as a result of thermal degradation and dissolution in water.
Figure 7. Mean amount of Oxalates in traditional vegetables as influenced by cooking methods. SP=sweet potato,
BJ=Black jack, MO=Moringa oleifera, JM=Jute mallow and AH=Amaranthus hybridus. Mean ±SD values in
the same group followed by the same superscripts are not significantly different at α=0.05 using Bonferroni
ANOVA test
Table 4. Phytate content (mg/100g) in traditional vegetables as influenced by cooking methods
Vegetable leaves
Dried (n=3)
Wet fried (n=3)
Boiled (n=3)
Sweet potato
129.15±42.47a
123.91±13.55a
118.48±11.41a
Black jack
124.95±52.25a
84.98±13.57a
99.35±40.76a
Moringa oleifera
99.80±10.29a
69.28±5.46b
87.11±0.36a
Jute mallow
86.64±12.51a
43.95±12.51b
44.75±8.14b
Amaranth hybridus
72.17±18.19a
37.90±14.11a
12.42±9.63b
Entries are Mean ±SD
Values in the same row followed by the same superscripts are not significantly different at α=0.05 using
Bonferroni ANOVA test.
3.2.3 Effects of Wet Frying and Boiling on Tannins Content
The results for tannins content in the five traditional vegetables and its respective wet fried and boiled
counterparts for each vegetable are presented in Table 5. The amount of tannins in the uncooked dry vegetables
ranged from 42.94mg/100g to 77.16mg/100g on dry weight basis. The mean amount of tannins in these dry
uncooked vegetables were 42.94mg/100g, 47.91mg/100g, 49.53mg/100g, 61.18mg/100g and 77.16mg/100g in
jute mallow, Amaranth hybridus, Moringa oleifera, sweet potato leaves and black jack leaves respectively.
Boiling significantly reduced (P≤0.05) the amount of tannins in jute mallow and sweet potato leafy vegetables.
There was no significant loss of tannins due to wet frying in all the vegetables. Boiling reduced the amount of
a
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0
200 400 600 800 1000
SP BJ MO JM AH
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tannins in sweet potato leaves and Amaranthus hybridus by 42.69% and 36% respectively. Wet frying reduced
the amount of tannins in black jack leafy vegetables by 31%. Significant losses of tannins were reported by
Jugran, A. K., & Chaudhary, W. Y.,2016 in Peeonia emodi leaves after boiling the leaves from different stages of
growth. The losses of tannins due to boiling and frying are attributed to thermal degradation of tannins during
cooking as reported by Gunathilake et al., 2018. In this study, the amount of tannins in wet fried jute mallow
increased by 8.09% compared to the dry uncooked jute mallow. The amount of tannins in Moringa oleifera leafy
vegetables increased by 13.79% and 30.67% as a result of wet frying and boiling the vegetables for ten minutes
respectively. These results agrees with Gunathilake et al.,2018 where it was found that in some vegetables,
tannins content increase due to breakdown of the complex tannins compounds present in the vegetables during
heat processing to simple tannins. Also, increase in total polyphenols such as tannins during thermal processing
might be due to the liberation of polyphenols from the intracellular protein complexes, changes in plant cell
structure, matrix modifications, or the inactivation of the polyphenol oxidases (Gunathilake et al., 2018).
Table 5. Tannins content (mg/100g DM) in traditional vegetables as influenced by cooking methods
Vegetable leaves
Dried (n=3)
Wet fried (n=3)
Boiled (n=3)
Sweet potato
61.18±15.48a
52.73±25.96a
35.06±8.84b
Black jack
77.16±23.27a
53.42±14.448a
70.56±4.51a
Moringa oleifera
49.53±7.10a
56.36±13.52a
64.72±8.65a
Jute mallow
42.94±17.48a
46.37±12.46a
32.98±19.58b
Amaranth hybridus
47.91±3.53a
33.87±11.19a
30.50±6.01a
Entries are mean ±SD
Values in the same row followed by the same superscripts are not significantly different at α=0.05 using
Bonferroni ANOVA test.
4. Conclusions
The findings from the present study indicate that minerals, crude fats, beta-carotene, vitamin C, oxalates,
phytates and tannins of selected five traditional vegetables are altered during common cooking practices; boiling
and wet frying. Wet frying reduced the amount of nutrients such as iron, zinc, copper, calcium, magnesium and
beta-carotene in most of the vegetables as compared to boiling. On the other hand, wet frying retained and
preserved the amount of Vitamin C in all the vegetables while boiling reduced vitamin C content in all the
vegetables. Wet frying significantly increased the amount of crude fats in all the vegetables while boiling slightly
reduced crude fats in the vegetables. Boiling has shown to have varying effects on iron, zinc, calcium,
magnesium, and beta-carotene depending on the type of leafy vegetable. Boiling retained significant amounts of
beta-carotene in all the vegetables. Both boiling and wet frying reduced the amount of antinutrients (oxalates,
phytates and tannins) in most of the vegetables. Boiling had the highest effect to reduce tannins as compared to
wet frying. The results of the study can be used for making recommendations on food processing methods to
preserve the health benefits of the studied vegetables and reduce the antinutrients of the vegetables.
Acknowledgements
Support for this research was made possible through a capacity building competitive grant “ Training the next
generation of Scientists” provided by Carnegie Cooperation of New York through the Regional Universities
Forum for Capacity Building in Agriculture (RUFORUM).
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Copyrights
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This is an open-access article distributed under the terms and conditions of the Creative Commons Attribution
license (http://creativecommons.org/licenses/by/4.0/).
Journal of Food Research; Vol. 9, No. 1; 2020
ISSN 1927-0887 E-ISSN 1927-0895
Published by Canadian Center of Science and Education
34
Some Nutritional and Physical Properties of Different Zambian
Market Classes of Bambara Groundnut (Vigna subterranea)
Vincent Nyau1, Lukonde Mwelwa-Zgambo1, Taonga Chirwa-Moonga1, Dorothy Nthani1, Shiv Prakash2, Jerry
Rodrigues3 & Jill Farrant3
1Department of Food Science and Nutrition, University of Zambia, Lusaka, Zambia
2Department of Chemistry, University of Zambia, Lusaka, Zambia
3Department of Molecular and Cell Biology, University of Cape Town, 7701 Rondebosch, South Africa
Correspondence: Vincent Nyau, Department of Food Science and Nutrition, University of Zambia, Lusaka,
Zambia. E-mail: vincentnyau@yahoo.co.uk
Received: October 19, 2019 Accepted: November 28, 2019 Online Published: December 7, 2019
doi:10.5539/jfr.v9n1p34 URL: https://doi.org/10.5539/jfr.v9n1p34
Abstract
Selected nutritional and physical properties of the two commonly cultivated Zambian market classes of Bambara
groundnut (red and brown) were investigated to establish their end-user traits. Nutritional (essential amino acids,
proximate and mineral compositions) and physical (hydration capacity, hydration index, swelling capacity,
swelling index and 100 seed mass) properties were investigated using established standard methods. All the
essential amino acids were present in both the red and brown Bambara groundnuts. The highest amount was
recorded for leucine (9.7 g/100g protein) in the brown Bambara groundnut and least for methionine (1.2 g/100g
protein) in the red market class. All the essential amino acids except methionine in the brown Bambara
groundnut market class were above the recommended WHO/FAO provisional requirements for children (2 – 5
years) and adults. The crude protein values ranged from 14.62-18.55 g/100g, total ash (4.21 – 4.29 g/100g),
crude fibre (2.79 – 5.33 g/100g), fat (6.28 – 6.54 g/100g), moisture (8.95 – 9.13 g/100g) and carbohydrate (59.23
– 60.34 g/100g). Potassium was found to be the most concentrated mineral in both market classes, followed by
phosphorous, magnesium and calcium, while lead, mercury and arsenic were not detected. The swelling capacity,
swelling index, hydration capacity, and hydration index of the two market classes of Bambara groundnuts were
not significantly different, whereas their 100 seed mass was significantly different, with the brown recording the
highest.
Keywords: Bambara groundnut, amino acid composition, proximate and mineral composition, physical
properties
1. Introduction
Bambara groundnut (Vigna subterranean) is traditionally one of the most important food resources among
peasant farmers in Zambia and other countries in the Sub-Sahara region. The crop has however not been
embraced by the commercial farmer and its annual production levels are generally low. Inadequate supplies and
high shortages of food in the world, particularly in developing countries necessitate the search for new sources
(Mahala & Mohammed, 2010). According to Mkandawire (2007), most food requirements are provided by fewer
than 20 crop species, and there remains a vast repository of many hundreds of underutilised species that have
been grown locally for centuries. These have the potential to contribute to the food and nutritional security of the
world’s poorest people.
Previous studies on Bambara groundnuts have reported that the crop has several advantages over other species in
terms of nutritional quality and can withstand adverse environmental conditions. The crop has a number of
production advantages in that it can yield highly on poor soils with little rainfall, as well as produce substantial
yields under better conditions (Mkandawire, 2007). Linnemann (1990) described Bambara groundnut as
nutritionally superior to other legumes and a preferred food crop for many local people. A study by Nyau,
Prakash, Rodriques and Farrant (2017) further demonstrated that Bambara groundnuts exhibit enhanced
nutraceutical profiles in cooked form.
This study was carried out to generate scientific baseline data on physical and nutritional end-user traits of the
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35
red and brown market classes of Bambara groundnuts commonly grown in Zambia. This baseline data is
important as it could possibly facilitate utilization of this neglected crop as a food source. The nutrition related
parameters studied included essential amino acids, mineral content, moisture, ash, crude protein, crude lipid,
crude fibre and total carbohydrate compositions. Physical properties that were investigated included 100 seed
mass, water hydration capacity, swelling capacity, hydration index, and swelling index respectively. These data
are valuable when it comes to the utilization and exploitation of orphaned crops such as Bambara groundnut by
breeders, food processors, nutritionists, farmers and policy makers. Water hydration capacity, swelling capacity,
hydration index, and swelling index give valuable tips in grain processing as they are correlated with cooking
time (Tazazu & Emire, 2010). The 100 seed mass is also an important characteristic that is well correlated with
grain yield and therefore useful in selection for yield (Fageria, Baligar, Moreira, & Aquino Portes, 2010)
2. Materials and Methods
2.1 Sample Collection
The Bambara groundnut seed samples were sourced from farmers in the Eastern region of Zambia at harvest.
The seeds were cleaned by winnowing to remove all external physical materials. Each market class of Bambara
groundnut was collected in batches of 0.5 kg from 15 farmers to ensure that the samples were representative.
2.2 HPLC Amino Acid Analysis
Quantitative analysis of essential amino acids was performed on Waters 2707 Module HPLC system attached to
a PDA using the procedure described by Dhillon et al. (2014) with slight modifications. After acid hydrolysis,
the samples were derivatised with o-phthalaldehyde (OPA) and transferred to maximum recovery vials. The vials
were heated for 10 min in a water bath at 55ć before separation of amino acids using the HPLC. About 10 µL
sample was injected into a cation-exchange resin column C18 (3.9 mm X 150 mm) using auto sampler (Waters
2707). The Waters AccQ Tag Eluent A Concentrate (WAT052890) was diluted to 10% in Milli-Q water and used
as eluent A, and 60% acetonitrile as eluent B in a separation gradient with a flow rate of 1 mL/min. The
separation gradient used was 0-2 min (100% A), and 2 min (98% A), 15 min (93% A), 19 min (90% A), 32 min
(67% A), 38 min (0% A) and 56 min (100% A). The amino acids were detected using PDA at 254 nm with the
column conditions set at 37 ć. The amino acid peaks were acquired using Empower Pro software ® by Waters
Corporation (2005-08) and were calculated based on amino acid standards. The concentrations of individual
essential amino acids were obtained from linear regression equations of the standard curves and expressed as
g/100 g crude protein.
2.3 Proximate Composition
The grounded Bambara seed samples were analysed for crude moisture, crude fat, crude protein, total ash and
crude fiber using AOAC official methods of 934.01, 920.39 (A), 984 (A – D), 942.05 and 978.10 respectively
(AOAC, 2006). Total carbohydrate content was calculated by difference as described in equation 1:
Carbohydrate content (%) = 100 – [crude protein content (%) + crude fat content (%) + total ash (%) + crude
fibre (%) + crude moisture content (%)] (1)
2.4 Mineral Analysis
The mineral content of Bambara groundnut was determined using three methods. The GBC Atomic Absorption
Spectrophotometer was used to measure the contents of calcium and magnesium while potassium and sodium
were assayed using a Corning 410 flame photometer (Sena et al., 1998). Technicon Auto-analyzer methodology
was used to measure the phosphorus content (Lockett, Calvet, & Grivetti, 2000). The Perkin-Elmer 2001 Model
Inductively Coupled Plasma Mass Spectrometry (ICP-MS) was used for assaying iron, zinc, copper, manganese,
lead, selenium and arsenic. About 2 g of the grounded seed was used for each determination and the experiment
was performed in triplicate.
2.5 Hydration, Swelling Capacity and Indices, and 100 Seed Mass
Hydration and swelling capacities were established following the method described by Bishnoi and Khetarpaul
(1993); Tazazu and Emire (2010). One hundred seeds were counted, their weight noted, followed by transferring
them into a measuring cylinder to which 100 ml of water was added. The cylinder was left at room temperature
for 24 hours with its open end covered with aluminium foil. After 24 hours, all the water was drained out from
the cylinder content, and the filter paper was used to remove excess water from the seeds. Hydrated seeds were
reweighed and their weight noted. Hydration capacity per seed was calculated by dividing the mass gained by
the seeds after hydration, by the number of seeds, while the hydration index was computed as the ratio of
average hydration capacity per seed and the mass of one seed. For swelling capacity, 100 seeds were counted,
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36
put in the measuring cylinder containing 100 ml of water and their initial volume noted. The seeds were then left
to hydrate for 24 hours as described above. The volume of the soaked seeds was noted in the graduated cylinder
after 24 hours. Swelling capacity per seed was calculated as the volume gained by the seeds divided by the
number of the seeds, while the swelling index was computed as the ratio of the swelling capacity per seed to the
volume of one seed. The 100 seed mass was determined by counting 100 seeds and noting their mass.
2.6 Statistical Analysis
Data were analysed using S-PLUS 6 Windows Professional 2001. The results were presented as mean values ±
standard deviation. The two-sample t-test was used to compare the means for the various Bambara attributes
investigated, and values at p < 0.05 were considered statistically significant.
3. Results and Discussion
3.1 Essential Amino Acid Composition
Essential amino acids composition (g/100 g crude protein) of the red and brown market classes of Bambara
groundnuts are presented in Table 1. Generally, all essential amino acids were present in both the red and brown
Bambara groundnuts. The highest amount was recorded for leucine (9.7 g/100g protein) in the brown Bambara
groundnut and least for methionine (1.2 g/100g protein) in the red market class. The total essential amino acids
were found to be 55 and 57 g/100g protein for the brown and red Bambara groundnuts, respectively. These
values are above the total amino acid content (46 g/100g protein) reported by Abdualrahman, Ali, Elkhalifa,
and Ma (2015) for Sudanese Bambara groundnut. All the essential amino acids, except methionine in the brown
Bambara groundnut market class, were above the recommended WHO/FAO, (2007) essential amino acid
requirement for children (2 – 5 years) and adults (Table 1). This observation suggests that only methionine would
require supplementation in the Bambara groundnut based diet. Leucine was found to be the most concentrated
essential amino acid in both market classes and this has been reported previously for Bambara groundnuts by
Aremu, Olaleke, Akintayo and Emmanuel (2006). Methionine was found to be the least concentrated essential
amino acid in both market classes as reported previously by Aremu, Olaleke, Akintayo and Emmanuel (2006).
The presence of all essential amino acids in both the brown and red market classes of Bambara groundnuts
suggests that this underutilized legume could be an alternative source of protein in local dishes especially in rural
and peri-urban areas where child malnutrition is highly prevalent. According to Masiye, Chama, Chitah and
Jonsson (2010), malnutrition is wide spread among children in Zambia and falls disproportionately on rural
children. Increased utilization of Bambara groundnut could enhance dietary diversity and help to combat most
nutritional disorders given the variety of products that can be prepared from it. Mumbaiwa, Fogliano, Chidewe
and Linnemann (2016) reported a number of preparation methods for Bambara groundnuts including cooking
with alkaline salts, milling, roasting, fermentation, and malting.
Table 1. Essential amino acids (g/100 g crude protein) of Bambara groundnuts and the recommended FAO/WHO
essential amino acids Provisional Pattern
EAA
FAO/WHO*
Brown Bambara
groundnut
Red Bambara
groundnut
Child
Adult
Threonine
3.4
0.9
3.9a
4.5b
Valine
3.5
1.5
6.4a
5.6b
Methionine
2.7
1.7
1.2a
2.8b
Isoleucine
2.8
1.3
4.9a
4.8a
Leucine
6.6
1.9
8.8a
9.7b
Tryptophan
1.1
0.5
ND
ND
Phenylanine
6.3
1.9
6.4a
6.6a
Histidine
1.9
1.6
8.4a
6.0b
Lysine
5.8
1.6
7.8a
8.5b
Arginine
-
-
7.2a
8.4b
EAA = Essential amino acid. ND = not determined. *Source: FAO/WHO (2007)
Values in the same row for the market classes of Bambara groundnuts with different superscripts were
significantly (p < 0.05) different, and n =1.
3.2 Proximate Composition of the Seeds
The results on proximate composition of the Bambara seeds are presented in Table 2. The protein content of the
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37
red market class ranged between 16 – 21 %, and this was similar to that reported by Brough and Azam-Ali
(1992); Brough, Taylor and Azam-Ali (1993); Purseglove, (1992), but much higher than that reported by Aremu,
Olaleke, Akintayo and Emmanuel (2006). Contrary to this, the brown market class had a lower protein content of
14.62 % compared to the red, which was also lower than other reported literature. This lower protein value could
be attributed to the genotype and possibly the environmental conditions in which they were grown (Salunkhe,
Kadon, & Charan, 1985).
The carbohydrate content of both the red and brown market classes was similar to that reported by Purseglove
(1992) in the range of 50 – 60 %, but slightly lower than that reported by Aremu, Olaleke, Akintayo and
Emmanuel (2006). However, when compared to Azam-Ali (1992), the carbohydrate contents of the red and
brown market classes were higher. The ash content for both market classes was higher than that reported by
Mahala and Mohammed (2010) and this difference could be attributed to differences in soil types (Mesquita,
Corrêa, Abreu, & Lima, 2007). The fat concentrations in the current study were similar to those reported by
Purseglove, (1992); Ijarotimi and Esho (2009) and were in the range of 4.5 – 6.5 %.
Table 2. Proximate composition (g / 100 g DW a) of the whole seed sample
Property
Market classes of Bambara groundnuts
Red
Brown
Moisture
9.05 ± 1.26a
9.13 ± 0.71a
Crude protein
18.55 ± 1.55a
14.62 ± 0.01b
Total ash
4.21 ± 0.68a
4.29 ± 0.51a
Fat
6.54 ± 0.38a
6.28 ± 0.77a
Crude fibre
2.79 ± 0.01a
5.33 ± 0.37b
Carbohydrates
59.23 ± 3.11a
60.34 ± 0.81a
aValues except moisture content are expressed in dry weight basis, DW = Dry weight
Means ± standard deviation, n = 3, means in the same row with different superscripts were significantly (p
< 0.05) different.
3.3 Mineral Composition of the Seeds
The results for various mineral contents of the two market classes of Bambara groundnuts are presented in Table
3. Potassium was the most abundant mineral in both the red and brown market classes and ranged from 16282 –
16670 mg / kg DW. This was followed by phosphorous (3221 –3398 mg / kg DW), magnesium (2024 –2054 mg
/ kg DW) and calcium (516 –593 mg / kg DW), respectively. Other minerals such as iron, copper and manganese
were found in very low quantities, while mercury, lead and arsenic were undetected in both samples. The
potassium concentrations were within the range of 15780 – 17420 mg / kg DW, reported by Amarteifio, Karikari
and Modise (2002) for Bambara groundnut landraces from Botswana. When the other minerals were compared
with previous literature, several differences in concentration were observed from those reported by Amarteifio,
Tibe and Njogu (2006); Amarteifio et al. (2002); Kemo (2000); Ijarotimi and Esho (2009). Mineral content of
agricultural products varies with geographical location and agricultural practices and therefore, the differences
seen in mineral content could possibly be attributed to these factors (Amarteifio et al., 2006). Information
gathered from the local people where sampling of the seeds was done revealed that the Bambara groundnut
landraces were normally intercropped with maize and no chemicals or fertilizers are used. Further, most growers
in Zambia prefer growing the Bambara groundnuts in sandy loam soil that is well drained. Thus, the differences
in agricultural practices that are seen from one growing region of Africa to another could possibly be one of the
key factors affecting mineral content of Bambara nuts and other crops in general.
The ratios of sodium to potassium (Na/K) and calcium to phosphorous (Ca/P) were also computed and are
presented in Table 3. The Na/K ratio was 0.0015 for the red market class and 0.0023 for the brown market class,
whereas the Ca/P ratio was 0.15 for the red market class and 0.18 for the brown market class of Bambara
groundnuts respectively. According to the report by Aremu, Olaleke, Akintayo and Emmanuel (2006), the Na/K
ratio in the body is of great significance for the prevention of high blood pressure and it is recommended to be
less than one. The findings on the Na/K ratio in the current study suggest that both the red and the brown market
classes of Bambara groundnuts would fit in the category of foods for high blood pressure prevention, given that
both their Na/K ratios are less than one. The Ca/p ratios for both the red and brown market classes of Bambara
groundnuts were lower than what was previously reported by Aremu, Olaleke, Akintayo and Emmanuel (2006),
but suffice to say that the two legumes would still serve as alternative sources of calcium and phosphorous for
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the body.
Table 3. Mineral composition (mg / kg DW) of the whole seed sample
Minerals
Market classes of Bambara groundnuts
Red
Brown
Calcium
516.6 ± 0.59a
593.7 ± 0.67b
Magnesium
2024.4 ± 0.20a
2054 ± 0.21b
Potassium
16670.1 ± 1.08a
16282.2 ± 1.56b
Phosphorous
3398.5 ± 0.03a
3221.1 ± 0.02b
Sodium
26.2 ± 1.97a
38.4 ± 2.89b
Iron
27.25 ± 1.3a
20.97 ± 1.9b
Arsenic
ND
ND
Zinc
19.02 ± 1.4a
18.45 ± 1.0a
Lead
ND
ND
Copper
5.51 ± 2.4a
5.12 ± 1.0a
Manganese
13.87 ± 2.3a
19.62 ± 1.9b
Mercury
ND
ND
Ca/P
0.15
0.18
Na/K
0.0015
0.0023
Means ± standard deviation, n = 3, means in the same row with different superscripts were significantly (p <
0.05) different.
Ca/P = Calcium to Phosphorus, Na/K = Sodium to Potassium ratio.
ND = Not detected
DW = Dry Weight
3.4 Physicochemical Properties of the Seeds
Table 4 summarizes the physicochemical properties of the red and brown Bambara groundnuts. There were no
significant differences (p > 0.05) observed in the swelling capacity, swelling index, hydration capacity, and
hydration index of the two market classes. Previous studies by Tazazu and Emire (2010); Bishnoi and Khetarpaul
(1993) reported an inverse relationship between these parameters and the cooking time. Lower values for these
parameters suggest longer cooking time. Hence, the red and brown Bambara groundnuts would most likely have
comparable cooking times. The brown Bambara groundnut market class had a higher 100 seed mass compared to
the red and the difference between the two was significant (p < 0.05). This suggests that in terms of yield and
marketed weight, the brown market class would be a better choice.
Table 4. Physicochemical properties of the seeds
Property
Market classes of Bambara groundnuts
Red
Brown
100 seed mass (g / 100 seeds)
72.922 ± 2.969a
88.812 ± 1.979b
Hydration capacity (g / seed)
0.173 ± 0.014a
0.176 ± 0.006a
Hydration index
0.125 ± 0.004a
0.120 ± 0.008a
Swelling capacity (ml / seed)
0.180 ± 0.036a
0.184 ± 0.031a
Swelling index
0.120 ± 0.005a
0.122 ± 0.002a
Means ± standard deviation, n = 3, means in the same row with different superscripts were significantly
(p<0.05) different.
4. Conclusion
The study has shown that Bambara groundnuts have considerable amino acid, macronutrients and mineral
profiles that could be exploited for use in local dishes especially in rural areas of developing countries where
child malnutrition is highly prevalent. The hydration and swelling behaviours for the two market classes are
similar, suggesting comparable cooking times. The brown is of higher quality than the red with regards to seed
mass.
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39
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Copyrights
Copyright for this article is retained by the author(s), with first publication rights granted to the journal.
This is an open-access article distributed under the terms and conditions of the Creative Commons Attribution
license (http://creativecommons.org/licenses/by/4.0/).
Journal of Food Research; Vol. 9, No. 1; 2020
ISSN 1927-0887 E-ISSN 1927-0895
Published by Canadian Center of Science and Education
41
Corn Flour Formulation and Fortification Tests: Evaluation of
Acceptability of Local Derived Product Called “Kabato” Case of
Napalakaha, Nibolikaha and Tiangakaha of Region of Korhogo
Kouassi Amenan Elodie1, 2, Gbogouri Grodji Albarin1, Ndri Yao Denis1, Niaba Koffi Pierre Valery2, Amoakon
Léonce3, Clemens Korboi Vanessa4 & Menzan Guy-Roland5
1UFR des Sciences et Technologies des Aliments, Université Nangui Abrogoua, 02 BP 801 Abidjan 02, Côte
d’Ivoire
2UFR Agroforesterie, Université Jean Lorougnon Guédé de Daloa, Bp 150 Daloa, Côte d’Ivoire
3Helen Keller International, Responsable de Programmes, 17 BP 1334 Abidjan 17, Côte d’Ivoire
4American Urogynecoologic Society, Memebership and Events Assistant, 1100 Wayne Ave, Sliver spring, MD,
20910, USA
5Ministère de l’Economie et des Finances, Direction Générale de l’Economie de la Côte d’Ivoire, BPV 163
Abidjan, Côte d’Ivoire
Correspondence: Gbogouri Grodji Albarin, UFR des Sciences et Technologies des Aliments, Université Nangui
Abrogoua, 02 BP 801 Abidjan 02, Côte d’Ivoire. E-mail: albringrodji@yahoo.fr
Received: October 3, 2019 Accepted: October 28, 2019 Online Published: January 7, 2020
doi:10.5539/jfr.v9n1p41 URL: https://doi.org/10.5539/jfr.v9n1p41
Abstract
The good use of food is one of the fundamental points of the food security of the populations especially in the
developing countries. Therefore, for convincing results, the methods of strengthening nutritional knowledge by
improving the culinary practices of vulnerable populations must take into account the dietary habits of the
targets.
The objective of the present study was to contribute to the consumption of the project crops to develop food
formulations. In practice, eight (8) cornmeal formulas using soybeans and orange-fleshed sweet potatoes have
been proposed and submitted to the grantees. The different proportions of ingredient to be mixed were obtained
by the Pearson's Square method. Analysis of the sensory evaluation data was possible to the Statistical Package
for Social Sciences (SPSS) software version 21 and the different results were presented in the form of radar
graphs.
The results showed that simultaneously flours and “kabato” accepted by the populations of the study area were
formulations of:
- E: 72.26 percent of maize flour and 27.74 percent of sweet potato flour
- F: 53.76 percent of corn flour and 46.24 percent of sweet potato flour
- G: 89.3 percent of composite flour (maize and sweet potato) and 10.7 percent of soya flour
- H: 78.09 percent of composite flour (maize and sweet potato) and 21.91 percent of soya flour
So, it can be envisaged to implement a strategy for a better vulgarization of these methods.
Keywords: corn, orange-fleshed sweet potato, Pearson's Square, Sensory analysis, soy
1. Introduction
Healthy eating is a fundamental component of overall health as it reduces the risk of chronic nutrition-related
illness (Santé Canada, 2013). However, according to FAO estimates in 2018, Africa remains the continent most
affected by the prevalence of undernourishment, with nearly 21% of the population or more than 56 million
people (Food and Agriculture Organization of the United Nations [FAO], The International Fund for Agricultural
Development [IFAD], World Health Organization [WHO], World Food Program [WFP] & United Nations
Children's Fund [UNICEF], 2018).
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In Côte d'Ivoire, nearly 12.6% of rural households are food insecure, ie 1.2 million people, 232,602 of whom are
reported to be severely food insecure. Moreover, the results of this same study show that several regions of the
country, particularly the North, have a level of global acute malnutrition exceeding the 5% threshold (FAO,
2010).
All these elements justify a contribution to food security in the northern regions of Côte d'Ivoire. To this end,
food supplementation would be a palliative to nutritional problems recurring in developing countries. However,
the choice of foods offered to the target group must take into account several criteria, one of the most important
of which is acceptance (Lateur, Planchon & Moons, 2001)
Thus, this current work, with the objective of helping the consumption of crops of nutritional interest (soybean,
sweet potato with orange flesh) of the project has proposed formulations based on corn flour. The study
consisted specifically to:
- Elaborate soy flour and sweet potato with orange flesh;
- Determine using Pearson's square the different proportions to be mixed;
- Evaluate the acceptability of flours and “kabato” by the beneficiaries.
2. Materials and Methods
2.1 Materials
The formulations used in this study consist of yellow corn powder, orange-fleshed sweet potato flour and / or soy
flour. These flours were obtained from field raw materials (sweet potato tubers and soya beans) and households
from villages concerned (yellow corn flour).
2.2 Flour Making
The details for this section have been summarized in the diagrams below:
Figure 1. Flour production diagram from sweet (A) potato and soybean (B)
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2.3 Preparation of Formulations
Eight (8) formulations were made using the Pearson's Square method (Figure 2). This method was used to
determine the quantities of corn powder, soybean and orange-fleshed sweet potato flour to be mixed (Table 1)
(Lee, 2009).
Figure 2. Representation of Pearson's Square
X: is the nutritional need in protein or provitamin A to be satisfied in this study
TM and TS / TP: represent protein or provitamin A levels of corn and soybean / sweet potato to meet the protein
or provitamin A requirement
PC: is the part of corn in the mixture; it is the difference between TS / TP and X without taking into account the
sign
Ps: is the proportion of soya or sweet potato in the mixture; it is the difference between TM and X without taking
into account the sign.
The proportion of Maize P1 (%) is determined as follows:
ܲ
ଵൌܲ
ܲ
ܲ
௦
ൈ ͳͲͲ
The proportion of soybean or sweet potato P2 (%) is determined as follows:
ܲ
ଶൌܲ
௦
ܲ
ܲ
௦
ൈ ͳͲͲ
Table 1. Different proportions of the mixture
Formulations
T
M
(%)
T
S
(%)
TP30+M70
TMixture
T
P
(μg/100
g)
X
(%)
P
C
P
s
P1
(g/100g
of the
mixture)
P2
(g/100g
of the
mixture)
A
4.61
21.94
5.95
6
15.94
1.39
91.98
8.02
B
4.61
21.94
7.08
8
13.94
3.39
80.44
19.56
C
4.61
21.94
12.82
12
9.94
7.39
57.36
42.64
E
0
14.37
54.07
15
39.07
15
72.26
27.74
F
0
22.53
54.07
25
29.07
25
53.76
46.24
G
21.94
4.091
12.03
6
15.94
1.91
89.3
10.7
H
21.94
4.091
11.41
8
13.94
3.91
78.09
21.91
I
21.94
4.091
7.87
12
9.94
7.91
55.69
44.31
Note
Corn and soy formulations
A: 91.98 % of maize flour and 8.02 % of soy flour; B: 80.44 % of maize flour and 19.56 % of soy flour; C:
57.36 % of maize flour and 42.64 % of soy flour.
Corn and sweet potato formulations
E: 72.26 % of maize flour and 27.74 % of sweet potato flour, F: 53.76 % of corn flour and 46.24 % of sweet
potato flour.
Corn, sweet potato and soy formulations
G: 89.3 % of composite flour (maize and sweet potato) and 10.7 % of soya flour, H: 78.09 % of composite flour
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(maize and sweet potato) and 21.91 % of soya flour, I: 55.69 % of composite flour (maize and sweet potato) and
44.31 % of soya flour.
TMixture which is protein or beta carotene content of the mixture.
TP30+M70 which is protein content of the mixture of corn (70 percent) and sweet potato (30 percent).
2.4 Sensory Evaluation
These tests were carried out according to the modified method of MEITE and collaborators (Meite, Kouame,
Coulibaly, & Offoumou 2008). In practice, they were carried out on samples coded by letters (flours, kaboto)
after an interview in local language with the panelists on the meaning descriptors. The hedonic test was
performed on flours with thirty naive panelists. During the evaluation, flours were presented simultaneously and
randomly to each panelist who was to give the degree of appreciation of the color and smell of the flours.
The descriptive test was used for the assessment of kabato. It consisted of giving each trained taster (twenty)
monadically sequential according to a rating criterion ranging from 1 to 10. The various criteria were related to
color, odor, taste and consistency.
However, a control sample (maize flour and derived kabato) was introduced to better assess the panelist's ability
to assess.
2.5 Data Analysis
The collected data was captured using the Census and Survey Processing System (CSPro) 7.0 software and
exported to the Statistical Package for Social Sciences (SPSS) version 21 software for calculation of average
scores and tabulation. The tables were then exported to the Excel MS for making radar graphs.
3. Results
3.1 Hedonic Test
The hedonic test was carried out on formulated flours. The results obtained are summarized in the following
tables 2 and figure 3 (i, ii, iii, iv). It’s showed that the panelists of all the localities gave scores higher than 2 to
the different formulations. However, formulations with scores below 2 are I (1.8 for both sensory attributes), B
(1.6 for odor), F (1.7 for color) in Napalakaha, and C, I formulations (1.9 for color) in Nibolikaha.
Table 2. Hedonic test of the formulated flours
Flours
Napalakaha
Nibolikaha
Tiangakaha
All the localities
Color
Odour
Color
Odour
Color
Odour
Color
Odour
A
2,4
2
2,9
2,7
2,8
2,9
2,7
2,5
B
2,2
1,6
2,9
2,9
2,9
2,8
2,7
2,4
C
2,1
2,9
1,9
2,2
2,4
2,2
2,1
2,4
D
2,6
2,6
3
2,9
2,9
2,9
2,8
2,8
E
2
2,7
2,8
2,8
2,9
2,9
2,6
2,8
F
1,7
2,3
2,6
2,7
2,8
2,7
2,4
2,6
G
2
2,2
2,6
2,7
2,4
2,3
2,3
2,4
H
2,4
2,3
2,5
2,8
2,6
2,8
2,5
2,6
I
1,8
1,8
1,9
2,2
2,3
2,2
2,0
2,1
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Figure 3. Average value of sensory attributes for (i) Napalakaha, (ii) Nibolikaha, (iii) Tiangakaha, (iv) all
localities
3.2 Descriptive Test
The descriptive test was carried out on the kabato resulting from the different formulations.
The results obtained are summarized in the following table 3 and figure 4 (a, b, c, d).
The analysis showed that for all localities, 3/8 kabato in particular A, B and C received ratings of less than 5 for
all sensory attributes (color, smell, taste and consistency). However, in Tiangakaha all kabatos evaluated by the
panelists obtained an average between 5.77 and 7.4.
Table 3. Average sensory attributes by kabato type
Kabato
Napalakaha
Nibolikaha
Tiangakaha
All the localities
Co
O
T
Cn
Co
O
T
Cn
Co
O
T
Cn
Co
O
T
Cn
A
4,2
3,5
3,0
4,5
5,1
4,7
4,1
3,3
5,4
5,5
6,1
6,6,
4,9
4,6
4,4
4,8
B
3,1
3,8
3,4
4,6
5,4
4,8
5
4,2
6,3
5,8
6,9
5,3
4,9
4,8
5,1
4,7
C
4,4
4,2
4,2
5
5,2
6,1
6
6,3
5,2
5
5,2
5,7
4,9
5,1
5,1
5,7
D
4,2
4,7
5,2
4,7
5,6
5
4,5
5,2
6,4
6,2
7,4
7,5
5,4
5,3
5,7
5,8
E
5,4
5,5
6,4
7,8
7,3
7,1
7,7
7,4
6,6
7
8,6
7
6,4
6,5
7,6
7,4
F
4,6
5,9
6,1
6,5
7,4
6
6,7
6,8
6,5
7,5
8,5
7,1
6,2
6,5
7,1
6,8
G
5,2
4,7
6,2
5,9
6,5
7
7,5
7,4
5,4
6,2
7
6,8
5,7
6,0
6,9
6,7
H
5,4
5
5,9
6,1
6,3
7,3
7,3
7
5,9
5,5
8
6,6
5,9
5,9
7,1
6,6
I
5,1
6,5
6,8
7,1
6,4
6,4
7,5
6,7
4
5,7
6,6
6,8
5,2
6,2
7,0
6,9
Note. Co: Color, O: Odor, T: Taste, Cn: Consistency
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Figure 4. Average value of sensory attributes for (a) Napalakaha, (b) Nibolikaha, (c) Tiangakaha, (d) all
localities
4. Discussion
This study was carried out with the aim of improving the protein content (6%, 8%, 12%) and adding value to the
maize flour used in the study area, including beta carotene (15%, 25%). Thus, three types of formulations were
developed, namely corn-based formulations containing soy (A, B, C), corn-based formulations containing sweet
potatoes with orange flesh (E, F) and those containing both soy and sweet potato (G, H, I). Formulations A, B, C
with soy incorporation levels of 8.02%, 19.56% and 42.64%, respectively, improved corn protein content from
4.61% to 5.95% for A, 7.08% for B and 12.82% for C. These rates are satisfactory for a Recommended Dietary
Nutrient Intake of 0.83 g/kg/day (Agence Française de Sécurité Sanitaire des Aliments [AFSSA], 2007). In
addition, according to Endres and collaborators. (Endres, Welch, Ashraf, Banz, & Gower, 2000); the soy proteins
are of good quality because of their biological value similar to those of animal proteins. As a result, the addition
of soybeans to corn flour would be beneficial to rural communities. Indeed, the value of a protein is justified by
the quality of the amino acids which constitute it and their bioavailability (that is to say the proportion of amino
acids which after digestion and absorption becomes accessible to the metabolic phenomena) (Agence Nationale
de sécurité sanitaire, alimentation, Environnement et travail [ANSES], 2016). As a result, the essential amino
acids specifically lysine would be higher in soybean than in many other plant sources. In addition, regular
consumption of soy products would reduce the risk of prostate cancer; breast cancer; kidney disease and heart
disease. This beneficial effect would be possible by bioactive compounds such as isoflavones that act as
estrogens, antioxidants and / or enzyme regulators (Hubert, 2006). Formulations E and F, by adding 27.74
percent and 46.24 percent of orange-fleshed sweet potato flour, were obtained; 14.37 μg / 100 g (E) and 22.53 μg
/ 100 g (F) of beta carotene in a corn meal which was free of it. Formulations G, H, I were obtained by
incorporation of 10.7 percent, 21.91 percent, 44.31 percent of soybeans into a composite flour (corn, sweet
potato) containing 30% sweet potato flour. orange flesh. Thus, they contain simultaneously proteins and
beta-carotene levels respectively of 5.27 percent, 6.58 percent and 8.89 percent (proteins) and 12.03 μg/100 g
(G). 11.41 μg/100g (H), 9.87 μg/100 g (I) (béta-carotène). So these formulations may be important for vision,
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47
growth, reproduction, resistance to bacterial or fungal infections (normal development of skin and mucous
membranes) (Bulvestre, 2007). In addition to these properties, they would protect cells against oxidative stress and
thus against chronic diseases such as cancer, cardiovascular diseases, osteoporosis and diabetes (De carbon, 2015).
So, the proposed formulations would be of great interest to the populations of the North, particularly those in the
Poro region, where the prevalence of vitamin A deficiency is estimated at 24%, with a higher rate in rural areas
(28%) than in the middle urban area (20%) (Sangare, Koffi, Akamou. & Fall, 2009).
The organoleptic tests of this study were carried out in order to highlight through the choice of target populations
the appropriate formulations for an extension. The radar diagrams obtained for the different sensory characters
analyzed are presented in the different figures. The evaluation of sensory attributes of flours focused on color
and odor. The score showed that the panelists from the three localities appreciated flours A, D, E, F, G, and H
(Figure 7). However, the flours retained by locality are H, D for Napalakaha; D, E, F, G, B, A for Nibolikaha and
A, B, D, E, F, G, H for Tiangakaha.
As for the test carried out on “kabatos”, the sensory attributes were essentially based on color, odor, taste and
consistency. The radar analysis shows that the subjects of Napalakaha did not have homogeneous responses.
However, three “kabatos” received at least a score of 5 for the 4 sensory attributes especially: E, H, I.
Nibolikaha's panelists made their choice on the “kabatos” of the formulations E, C, F, G, H, I. However, samples
enriched in soy (A, B) obtained the lowest average justified by the depreciation of taste, odor and consistency.
This observation could be due to the method used for the preparation of the flour. Indeed, according to the work
of Tshite and collaborators (Tshite, Mulamba, & Ndianabo, 2015); soybean meal obtained by the dry method as
in this study would have a brown color and a very pronounced soy aroma. In addition, the poverty of
polysaccharide soy flour could justify the depreciation of the consistency of kabatos that contain it. Indeed,
according to the work of Kadji (Kadji, 2018) the richness of a polysaccharide meal would be at the origin of a
high water absorption capacity. This characteristic would improve with the heat treatment giving this type of
flour a better viscosity and therefore a satisfactory consistency. Finally, the analysis of Tiangakaha subjects'
responses reveals that most formulations obtained a minimum score of 5/10 for all sensory attributes except the
formulation I whose color received a rating of 4/10. This could be attributed to the high proportion of soybeans,
44, 31 percent in the mix. Indeed, the soybeans having been subjected to roasting, slightly brown colored flours
were obtained. As a result, these flours in large quantities in the mix could affect the color of kabato. However,
from their choice spring out formulations D, E, F. Thus, at the end of the descriptive test; the kabatos appreciated
by all the localities are: E, F, G, and H, I (Figure. 11) with a maximum average of 6, 97 for the formulation E.
5. Conclusion
It should be noted that this study was carried out with the aim of proposing a maize flour of improved nutritional
value which would be accepted by the population of the study area. Thus, the results show that eight
formulations could be developed during this study, which are: Corn and soy formulations (A, B and C), Corn and
sweet potato formulations (E and F), Corn, sweet potato and soy formulations (G, H and I).
The sensory evaluation of these formulations made it possible to highlight in each locality the formulation of
good sensory quality. Thus, the following formulations were retained:
- Napalakaha: Flours H, D and kabato E, H and I
- Nibolikaha: flours D, E, F, G, B, A and kabato E, C, F, G, H and I
- Tiangakaha: flours A, B, D, E, F, G, H and kabato D, E and F
However, the selection criterion for this study is based on the degree of appreciation of the quality of flours and
kabatos derived from them; the formulas selected for all the localities are:
- E: 72.26 percent of maize flour and 27.74 percent of sweet potato flour
- F: 53.76 percent of corn flour and 46.24 percent of sweet potato flour
- G: 89.3 percent of composite flour (maize and sweet potato) and 10.7 percent of soya flour
- H: 78.09 percent of composite flour (maize and sweet potato) and 21.91 percent of soya flour
So, it can be envisaged to implement a strategy for a better vulgarization of these methods.
Acknowledgments
This study is part of the project "Operational Strategy for Improving the Productivity of Nutrient Crops for
Vulnerable Rural Populations in Côte d'Ivoire".
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48
We would like to thank the ECOWAS Commission, the Spanish Cooperation (Agence Espagnole de Coopération
Internationale pour le Développement: AECID) for their financial support in the context of the regional
agricultural policy of the ECOWAS, ECOWAP / CAADP. We would also thank Helen Keller International for
the financial and technical support in carrying out this study.
Conflict of Interest
The authors declare that there is no conflict of interest.
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Copyrights
Copyright for this article is retained by the author(s), with first publication rights granted to the journal.
This is an open-access article distributed under the terms and conditions of the Creative Commons Attribution
license (http://creativecommons.org/licenses/by/4.0/).
Journal of Food Research; Vol. 9, No. 1; 2020
ISSN 1927-0887 E-ISSN 1927-0895
Published by Canadian Center of Science and Education
50
A Hospital Based Cross Sectional Study on Dietary Status and
Associated Factors among People Living with HIV/AIDS in Kigali,
Rwanda
Tafadzwa Dzinamarira1, 3, Gashema Pierre2, Elyse Jeanne Umuhire3, Michael Habtu1 & Rosemary Okova1
1Department of Public Health, Mount Kenya University Rwanda, Kigali, Rwanda
2College of Medicine and Health Sciences, University of Rwanda, Kigali, Rwanda
3ICAP, Mailman School of Public Health, Kigali, Rwanda
Correspondence: Tafadzwa Dzinamarira, Department of Public Health, Mount Kenya University Rwanda, Kigali,
Rwanda. E-mail: anthonydzina@gmail.com
Received: November 20, 2019 Accepted: December 30, 2019 Online Published: January 9, 2020
doi:10.5539/jfr.v9n1p50 URL: https://doi.org/10.5539/jfr.v9n1p50
Abstract
Background
Good nutrition empowers PLWH with the ability to fight against infection ultimately slowing down disease
progression. Consequently, nutrition management is a crucial component of HIV treatment, care, and support.
This study aimed at assessing dietary status and associated factors among PLWH in Kigali, Rwanda.
Methods
We conducted a cross sectional study in three selected hospitals in Kigali from over a six-week period in July –
August, 2019 to collect data from 204 HIV positive adults enrolled using systematic random sampling. Data was
collected using an adapted, validated and pre-tested food frequency questionnaire (FFQ). Descriptive and
multiple logistic regression analyses were performed using SPSS version 25 for windows.
Results
The proportion of participants with poor dietary status was 15% based on FFQ responses. The study found only
three factors to be independently associated with dietary status. There was an association between dietary status
and HIV status disclosure (AOR 2.5; CI 1.25 - 4.83; p=0.014). There was an association between dietary status
and travel time to place of collection of ARVs (AOR 3.2; CI 1.7 - 5.8; p=0.006). There was an association
between dietary status and BMI (AOR 10.2; CI 8.30 – 16.0; p<0.001).
Conclusions
Poor dietary status among PLWH remains a concern. The strong association between dietary status and BMI
underlines the need for interventions that target PLWH to improve dietary status and ultimately nutrition status
Keywords: dietary status, people living with HIV/AIDS, Rwanda
1. Introduction
As of 2016, HIV accounted for more than 1.8 million incidents yearly, with most occurring in resource poor
countries (UNAIDS, 2017). Earlier data showed that in 2015, 36.7 million people globally were infected with
HIV and AIDS, 1.8 million being children under 15 years (Organization, 2016). In 2015, 1.1 million deaths were
reported from HIV with 2.1 million new infections, including 150,000 children (WHO, 2018). It has been
reported that close to 70 percent of the burden is in Africa (WHO, 2018), a region with the highest rates of food
insecurity. Available evidence shows that PLWH who are undernourished when they start ART are 2–6 times
more likely to die within 6 months of ART initiation compared to their normal body mass index counterparts
(Munthali, Jacobs, Sitali, Dambe, & Michelo, 2015). Even on ART, there is a continuous need for PLWH to
consume a nutritious diet to maintain weight and prevent micronutrient deficiencies (Audain, Zotor, Amuna, &
Ellahi, 2015). There is also a growing recognition of the role nutritional support within clinical and community
services plays in engagement, adherence and retention in care and treatment (Berhe, Tegabu, & Alemayehu,
2013; Kendall et al., 2014; Tang, Jacobson, Spiegelman, Knox, & Wanke, 2005). Proper nutrition complements
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51
the properly adhered ART. Closer to Rwanda, randomized control trials conducted in Kenya and Uganda showed
nutritional support to significantly decrease mortality among PLWH initiating ART (PrayGod, Friis, & Filteau,
2018).
As of a decade ago, more than 800 million people were chronically undernourished (Ivers et al., 2009). The
highest burden of both undernutrition and HIV/AIDS has been reported to be in SSA (UNAIDS, 2016). A study
conducted in Brazil by Andrade et al. revealed high levels on undernutrition among PLWH at hospitalization
with a reported prevalence of 43% (Andrade et al., 2012). Similar findings were reported in Asia by Hu et al,
2011 with a reported prevalence of malnutrition among PLWH of 37.2% (Hu et al., 2011). In Senegal, slightly
lower prevalence was reported. The prevalence of malnutrition among PLWH, defined by BMI, was 19.2% in
Dakar and 26.3% in Ziguinchor (Benzekri et al., 2015).
In East Africa, an institution based cross sectional study conducted by Gedle et al., 2015 in Southern Ethiopia
reported an overall prevalence of malnutrition was 25.2% of which 49, 19, and 9 patients were mildly,
moderately, and severely malnourished, respectively (Gedle, Gelaw, Muluye, & Mesele, 2015). Similarly, a
multi-center study in Central Ethiopia reported a prevalence of 23.6% (Gebremichael, Hadush, Kebede, &
Zegeye, 2018). These figures raise concerns as lower dietary diversity has been associated with greater mortality
and poor clinical outcomes among PLWH (Palermo, Rawat, Weiser, & Kadiyala, 2013; Rawat, McCoy, &
Kadiyala, 2013). There is no published literature for dietary status and associated factors among PLWH in
Rwanda. The current study aimed to assess dietary status and associated factors among PLWH in Kigali,
Rwanda.
2. Materials and Methods
2.1 Study Design
The study was a cross sectional survey. We adopted this study design given the nature of the research question.
To obtain a snapshot of the current proportion of dietary status and associated factors among PLWH in Kigali,
Rwanda, such a study design was best to survey the study population and answer the research questions.
2.2 Study Setting
Kigali City Province is the Capital City of Rwanda. The province has a total of 42 health facilities spread over
the three districts; 21 in Gasabo, 10 in Kicukiro and 11 in Nyarugenge (NISR & Rwanda, 2014) , it has a surface
area of 730 km2. The population of Kigali was 1,132,686 as of the 2012 national census (NISR & Rwanda, 2014).
Among adults 15 – 64 years old, HIV prevalence in City of Kigali is 4.3% (ICAP, 2019). One health facility in
each district in Kigali Province was purposively selected based on researcher convenience and volumes of ARV
clinic attendees.
2.3 Sampling and Data Collection Instrument
At each study site, participants were enrolled into the study by using simple random sampling. The questionnaire
addressed: socio-demographic characteristics of the interviewee (sex, age, education, religion, marital status,
occupation, duration on ART); outcome variable (dietary status). The food frequency questionnaire (FFQ) was
adapted from a validation study conducted in the Rwanda context in 2016 (Yanagisawa et al., 2016).
2.4 Data Quality
We conducted a pretest of the survey questionnaire on 15 respondents in a non-sampled hospital. Consistency,
understandability, and flow of questions was tested and revised accordingly. To ensure high data quality, the
interviewers (GP and EJU) received a survey specific training for one-week. TD provided close supervision of
the interviewers during data collection and the questionnaires were thoroughly edited to make sure that relevant
questions have been responded to and coded according to the code designed for the study.
2.5 Data Entry and Statistical Analysis
Data coding and verification of responses were made on the same day and any missing information corrected.
The cleaned data were entered into the computer using SPSS version 25 platform. Quantitative data was
analyzed using descriptive statistics. The FFQ assessed consumption of energy, protein, iron and vitamin A.
Based on the participant responses a mean food frequency assessment score of 49.5% was calculated.
Participants that scored less than the mean were classified as poor dietary status while mean and over were
classified as good dietary status. This allowed for the dependent variable to be dichotomous with subsequent
analysis. The student t-test or chi-square test and logistic regression were used to determine study variables
associated with dietary status. All statistical tests were concluded at 5% level of significance.
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2.6 Ethical Considerations
This study has been ethically reviewed and approved by the University Teaching Hospital of Kigali Ethics
Committee (Approval number: EC/CHUK/0129/2019).
3. Results
3.1 Socio-demographic Characteristics
A total of 204 participants were enrolled with average age of 30.3 and majority (55%) being female. Of these, 88%
reported to have ever attended school while only 5% were currently enrolled in school. 61% were educated up to
primary level only while 38% reported to have worked in the past 12 months. 18% were married and of these
only 3% reported to have more than one wife/husband. Participant demographic characteristics are presented in
Table 1.
Table 1. Demographic characteristics of the PLWH enrolled.
Variable
Value
Age in years, mean (SD)
30.3 (5.3)
Sex, n (%)
Male
92 (45)
Education level, n (%)
Nursery
4 (2)
Primary
121 (59)
Secondary
65 (32)
Higher
14 (7)
Work in the last 12 months for which you received cash or goods as payment n (%)
Yes
78 (38)
Work in the last seven days for which you received cash or goods as payment n (%)
Yes
49 (24)
Ever been married or lived together with a [man/woman] as if married n (%)
Yes
169 (83)
Marital status, n (%)
Married
35 (18)
Living together
45 (22)
Widowed
50 (24)
Divorced
6 (3)
Separated
35 (17)
Single
38 (16)
Number of live-in partners n (%)
0
124 (63)
1
68 (35)
2
3 (2)
3
2 (1)
Number of live-in partners who live elsewhere? n (%)
0
130 (66)
1
64 (32)
2
3 (2)
3.2 Dietary Status
Of the 204 participants, 15% had poor dietary status based on their FFQ responses.
3.3 Factors Associated with Dietary Status
The study found only three factors to be independently associated with dietary status. There was an association
between dietary status and HIV status disclosure (AOR 2.5; CI 1.25 - 4.83; p=0.014). There was an association
between dietary status and travel time to place of collection of ARVs (AOR 3.2; CI 1.7 - 5.8; p=0.006). There
was an association between dietary status and BMI (AOR 10.2; CI 8.30 – 16.0; p<0.001). More information is
presented on Figure 1.
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53
Figure 1. Factors associated with poor dietary status among PLWH in Kigali, Rwanda
4. Discussion
The current study revealed 15% of PLWH in Kigali, Rwanda to have poor dietary status. The findings of the
current study mainly noted that some of the main factors, which are associated with poor dietary status include
HIV status disclosure, travel time to clinic and BMI. Globally, multi-organizational efforts have been launched
and recommendations made by The U.S. President’s Emergency Plan for AIDS Relief (PEPFAR), and endorsed
by WHO, UNAIDS, and the World Food Program. The nutrition assessment, counseling and support model is
known as NACS (Tang, Quick, Chung, & Wanke, 2015). As of 2017, South Africa, Mozambique and Nigeria
were in early planning phases. Cote d’Ivoire, Ghana, Ethiopia, Tanzania, Namibia and Zambia were at program
expansion stage. Only Kenya and Malawi were in full implementation at national scale. Findings of the current
study underscore the need for nutritional management of PLWH to be prioritized in Rwanda.
In the current study, 15% of the participants had poor dietary status based on their FFQ responses. A slightly
higher prevalence of 25.2% was reported by Gedle et al. 2015 in Ethiopia (Gedle et al., 2015). However, a study
by Hailemariam et al. in Ethiopia established that 12.3% of PLWH had poor dietary status (Hailemariam, Bune,
& Ayele, 2013). The findings of the study that was done by Gebremichael et al. noted that the prevalence of poor
dietary status was 23.6% (Gebremichael et al., 2018). In Senegal, Benzekri et al. established that 19.2% in Dakar
and 26.3% in Ziguinchor had poor dietary status (Benzekri et al., 2015). It is worth noting that the prevalence the
current study reports is comparable to what has been reported by Argemi et al. who established that 11.2% adults
initiating ART were malnourished (Argemi et al., 2012). The difference between these findings and the current
study findings could be attributed to the lower BMI lower limit set by Argemi et al of 16 (Argemi et al., 2012).
However, much higher prevalence have been reported elsewhere. Mulu et al. noted a much higher prevalence
[46.8%] in comparison to the findings of the current study (Mulu, Hamza, & Alemseged, 2016). Hadgu et al.
reported a prevalence of 42.3% (Hadgu, Worku, Tetemke, & Berhe, 2013). This is way higher than the findings
of the current study.
Most of the studies have tried to explore some of the main factors that are associated with poor dietary status
among PLWH. For instance, the findings of Gedle et al. revealed that living within the rural areas, anemia, as
well as intestinal parasitic co-infection was significantly linked to poor dietary status (Gedle et al., 2015). The
study also made the conclusion that the prevalence of malnutrition among PLWH getting ART in Butajira was
very high. Similarly, the study, which was carried out by Hailemariam et al. indicated that some of the factors,
which were closely linked to poor dietary status among PLWH include unemployment, WHO clinical stage four,
gastrointestinal symptoms, as well as past opportunistic infections (Hailemariam et al., 2013). Some of the main
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54
factors, which have been linked to poor dietary status among the HIV/AIDS patients included unemployment,
clinical stages of AIDS progression, CD4 count less than 350 cells/μl, tuberculosis, the duration on antiretroviral
therapy as well as household food insecurity (Gebremichael et al., 2018). The study by Benzekri et al. noted that
severe food insecurity was linked to the missing of clinic appointments and also to the failure of the PLWH to
take antiretroviral therapy because of hunger (Benzekri et al., 2015). The findings of the study that was done by
Hadgu et al. noted that household food insecurity, inadequate dietary diversity, anemia, as well as the general
lack of nutritional support were the major independent predictors of poor dietary status (Hadgu et al., 2013). It
can be noted that most of the previous studies have given different factors to be associated with dietary status.
5. Conclusion
Based on the findings of the current study, poor dietary status among PLWH remains a concern. The strong
association between dietary status and BMI underlines the need for interventions that target PLWH to improve
dietary status and ultimately nutrition status. Larger studies with designs of more scientific rigour evaluating
dietary status and associated factors may help shed more light into the research problem. This study has
presented baseline findings which may be used to guide future research.
Limitations
Cross sectional studies by nature are not able to demonstrate causality. Further, the findings may not be
generalized for the entire population. Additionally, the data collection tool to be used for dietary assessment
collected descriptive qualitative information only. However, it is anticipated the tool yielded useful findings as
the tool used was adapted from a Rwanda validated food FFQ with guidelines from WHO. Finally, the research
did not include biomarkers, the gold standard for any nutritional assessment to validate the self-reported dietary
status.
Declarations
Acknowledgements
We thank all the participants who took time to complete the questionnaires for this study.
Competing Interests
The authors declare that they have no competing interests, which may have inappropriately influenced them in
writing this article.
Funding
N/A
Availability of Data and Materials
All data generated or analysed in this study is available from the corresponding author following a written
request with permission from University Teaching Hospital of Kigali Ethics Committee.
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Copyrights
Copyright for this article is retained by the author(s), with first publication rights granted to the journal.
This is an open-access article distributed under the terms and conditions of the Creative Commons Attribution
license (http://creativecommons.org/licenses/by/4.0/).
Journal of Food Research; Vol. 9, No. 1; 2020
ISSN 1927-0887 E-ISSN 1927-0895
Published by Canadian Center of Science and Education
57
GC-MS and HPLC-ESI-MS-MS Characterization of Sanchezia
oblonga (Acanthaceae) Extracts
Juliana Mourão Ravasi1, Giuseppina Negri2, Antonio Salatino2, Maria Luiza Faria Salatino2 & Marco Aurelio
Sivero Mayworm1
1Curso de Ciências Biológicas, Universidade Santo Amaro (UNISA), 04829-300, São Paulo, SP, Brazil
2Instituto de Biociências, Universidade de São Paulo, 05508-090, São Paulo, SP, Brazil
Correspondence: Giuseppina Negri, Laboratory of Phytochemistry, Department of Botany, Institute of
Biosciences, University of São Paulo, Rua do Matão 277 sala 154, CEP 05508-090, São Paulo SP, Brazil. E-mail:
gnegri@terra.com.br
Received: November 13, 2019 Accepted: December 15, 2019 Online Published: January 22, 2020
doi:10.5539/jfr.v9n1p57 URL: https://doi.org/10.5539/jfr.v9n1p57
Abstract
The genus Sanchezia (Acanthaceae) comprises neotropical herbs and shrubs with showy flowers. Sanchezia
oblonga (syn. S. nobilis) is a shrub of the rainforests of central and south America. The ethanolic extracts of
leaves and stems from S. oblonga were analyzed by GC-EI-MS and RPHPLC-DAD-ESI-MS/MS. Fatty acids
(free and esterified) and phytosterols were detected by the former method. Benzyl alcohol glycosides (21 and 25),
sinapic acid glycoside esters (29 and 31), ethyl rosmarinate (24), sinapic acid-O-glucoside (28), dihydrosinapic
acid-O-glucoside (26), catechin-O-arabinoside (36), in addition to flavonols glycosides (23, 32, 33 and 35) and
rosmarinic acid-3’-O-glucoside (34) were detected by RPHPLC-DAD-ESI-MS/MS. Three new compounds,
detected only in leaves, were tentatively identified as phenylpropane glyceride derivatives
1-O-coumaroyl-2-hydroxy propanal (20) and 1-O-coumaroyl-2-O-glycosyl propanal (22, 30). Compounds 20, 22
and 30 from S. oblonga are similar with phenylpropane glycerides present in red sorghum (Sorghum bicolor L.
(Moench) and Lilium longiflorum Thunb. It is noteworthy that S. oblonga could be used in cooking as a
complement after more detailed studies. Sorghum grain foods exhibit potential health benefits against chronic
diseases related to over-nutrition. Lilium longiflorum possess flower buds and bulbs that are used for both
culinary and medicinal purposes in many parts of the world. Studies on chemical composition and biological
activity of the genus Sanchezia are scarce. The presence of phytosterols and flavonol glycosides were recently
reported in leaves from this species. However, the chemical profile of the extracts analyzed in this work differs
from that previously reported for aerial parts of S. nobilis (sin. S. oblonga). Further studies, including statistical
methods, such as principal component analysis and hierarchical cluster analysis will be needed to evaluate
chemical markers for this species.
Keywords: Sanchezia oblonga, Acanthaceae, sinapic acid ester glycosides, phenylpropane glycerides,
phytosterols
1. Introduction
Species of Sanchezia Ruiz & Pav. (Acanthaceae) are neotropical herbs and shrubs. Several species of Sanchezia,
such as S. parvibracteata, S. nobilis and S. speciosa, are cultivated as ornamentals in tropical areas and botanical
gardens due to their showy leaves and conspicuous bright and colorful flowers. Recently, this genus was revised
by Leonard and Smith (1964); among 58 species, over half were newly described (Tripp and Koenemann, 2015).
There are few reports on chemical constituents and pharmacological activity of Sanchezia species. Therefore,
there is no exhaustive identification of the basic chemical constituents and comprehensive quality control of this
genus. S. speciosa contains cardiac and flavonoid glycosides and their extracts exhibited antioxidant and
anti-inflammatory activities (Bui Thanh et al., 2017). Extracts of the same species exerted cytotoxicity in human
epithelial cervical cancer cell lines (Shaheen et al., 2017, Parvin et al., 2015).
Sanchezia oblonga Ruiz & Pav. (syn. Sanchezia nobilis Hook. f.) is a perennial evergreen shrub from the
rainforests of Central and South America (Ellah et al. 2013, 2014). An earlier study reported that S. nobilis
extracts contain syringin, flavone glycosides, cinnamyl and benzyl alcohol glycosides, neolignan glucoside,
besides matsutake alcohol glycosides (Ellah et al. 2013, 2014). The presence of the phytosterols daucosterol
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58
and stigmasterol, flavonol glycosides, scopoletin and 3’-O-methyl-3,4- methylenedioxy ellagic acid in
ethylacetate extract of the leaves of S. nobilis was reported recently (Xuan et al., 2019a, Loi et al., 2019a). On
the other hand, mangiferin, β-sitosterol, in addition to margaric, ursolic and oleanolic acids were isolated from
n-hexane extracts (Xuan et al., 2019b), while 4',5,7-trihydroxy-3',5'-dimethoxyflavone and kaempferol
glycosides were isolated from aqueous extracts of leaves (Loi et al., 2019b).
In this study, the ethanolic extracts of S. oblonga leaves and stems were analyzed by GC-EI-MS and
RPHPLC-ESI-MS / MS. Phenylpropane glycerides similar with thoses found in red sorghum (Sorghum bicolor L.
(Moench) and and Lilium longiflorum Thunb. were detected only in ethanolic extracts of leaves, while flavonol
glycosides and phytosterols were detected only in ethanolic extracts of stems.
2. Material and Methods
2.1 Plants Material
S. oblonga leaves and stems were collected from plants growing on the campus I of Santo Amaro University
(UNISA), São Paulo, State of São Paulo (southeast Brazil). A voucher sample was deposited at the Santo Amaro
University Herbarium (UNISA 4121). The leaves and stems were washed and cleaned prior to air drying at room
temperature for one week. The dried and powdered leaf and stem material (2 g each) was macerated separately
for 30 days in a 500 ml conical flask containing 200 mL of high quality ethanol (Sigma Chemical Co). The
solvent was replaced weekly during extraction at room temperature and protected from light. The extracts were
filtered first through cotton and then through Whatman paper # 1 and then concentrated in rotary evaporator
(Büchi, Switzerland). Leaves and stems provided 130 mg and 90 mg, respectively of dried extracts, which were
stored in amber flasks at 5 ° C.
2.2 Analyses of the Leac Extract by Fourier Transform Infrared Spectrometry (FT-IR)
Only the ethanolic extract from leaves was analyzed by FT-IR. Several drops of the extract from leaves were
placed onto a KBr aperture plate and sandwiched under another aperture plate such that no gas bubbles were
trapped. The FT-IR spectrum was obtained using a Frontier PerkinElmer spectrometer equipped with a KBr
beam splitter. The spectra were recorded at room temperature from an average of 32 scans in the range 4000-400
cm−1, with 5 cm−1 spectrum resolution, normalization at 1030 cm−1 and aperture of 5.0 mm. The influences of
H2O and CO2 were subtracted automatically by the software operating the spectrometer.
2.3 GC/EIMS Analyses of Ethanolic Extracts from Leaves and Stems
Crude extracts from leaves and stems (5 mg) were dissolved in 1 mL of high grade ethanol supplied (Sigma
Chemical Co). An aliquot of 1 µL of each extract was analyzed using a Shimadzu GCMS-QP505A gas
chromatograph equipped with a ZB-5ms fused silica capillary column BPX5 (5% phenyl arylene/95%
dimethylpolysiloxane, 30 m x 0.25 mm internal diameter x 0.25 µm film thickness) coupled to an ion-trap mass
detector. Mass spectra were acquired in the electron-impact (EI) mode with an ionization voltage of 70 eV. The
GC conditions were set as follows: the oven was programmed with an initial temperature of 100 °C, maintained
for 5 min and then increased to 320 °C at a rate of 6 °C/min. The final temperature was maintained for 10 min.
The air and hydrogen flow rates were 400 mL/min and 29.5 mL/min, respectively. Helium was used as the
carrier gas at a flow rate of 2.1 mL/min, linear velocity of 53.8 cm/sec, column pressure of 150.0 KPa and total
flow of 29.5 mL/min. The MS conditions were set as follows: filament current 0.3 mA; detector voltage -0.7 kV,
ion source temperature 300 °C; interface temperature, 300 °C; split ratio of 11 and scan speed of 2 scans s-1. The
mass range was 120-700 Da over 52 min (full scan mode) (Negri et al., 2018).
Fragmentation patterns of the EI mass spectra of compounds were compared via spectrum matching with
reference mass spectra of libraries NIST 21, Wiley 275 and Wiley 229 (Hewlett Packard, Wiley/NBS) (Koo et al.
2013). The identification of constituents was based on the best mass spectrum (MS) matching score (NIST Mass
Spectrum number was added in Table 1) and MS data published in literature. Relative content was estimated by
integration of the areas under the corresponding chromatogram peaks.
2.4 RPHPLC-SPD-ESI-MS/MS Analyses of Ethanolic Extracts from Leaves and Stems
HPLC grade methanol was purchased from Merck (Darmstadt, Germany). HPLC grade water was prepared with
distilled water using a Milli-Q system (Millipore, Waters, Milford, MA, USA). The ethanolic extracts of leaves
and stems (5 mg dissolved in 2 ml of methanol – 2.5 mg/mL) were filtered through 0.45 μm
polytetrafluoroethylene (PTFE) membrane filters, before a 30 μL aliquot was injected into the HPLC
system (Coelho et al., 2018). The RPHPLC-SPD-ESI-MS/MS analysis were conducted using an SPD-M10AVP
Shimadzu system equipped with a photodiode array detector coupled to an Esquire 3000 Plus Mass Spectrometry
system (Bruke Daltonics, Billerica, MA, USA), coupled with two LC-20AD pumps, SPD-20A diode array
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59
detector, CTO-20A column oven and SIL 20AC autosampler (Shimadzu Corporation Kyoto, Japan). All the
operations, acquisitions and data analyses were controlled by the Shimadzu CBM-20A software. Separations
were carried out using a C18 RP Luna Phenomenex reverse phase column (4.6 x 250 mm i.d., 5 µm of particle
size) protected with a security guard cartridge (Gemini C18, 4.0 × 2.0 mm i.d.). The mass spectrometer was an
ion trap with atmospheric pressure ionization method through electrospray ionization interface (ESI) operating in
the scan MS mode from m/z 100 to 1500. The mobile phase was composed of eluent A (0.1% aq. formic acid)
and eluent B (methanol) at the constant flow rate 1.0 mL min−1 and constant temperature of the column oven at
40 °C. The following elution program, based on concentrations of the B solvent, was used: 0 min, 20%; 10 min,
40%; 20 min, 60%; 30 min, 80%; 40 min, 100%; 50 min, 20%. The time for the whole run was 60 min, 10
minutes were needed for equilibration of the column. Helium was used as the collision and nitrogen as the
nebulizing gas, respectively. Nebulization was aided with a coaxial nitrogen sheath gas provided at pressure of
27 psi. Mass spectra were acquired in negative mode with ion spray voltage at 3.0 kV, capillary temperature at
300qC, capillary voltage at 45 V and drying gas flow 6 L/min. Collision induced dissociation spectra were
obtained in the ion trap using helium as the collision gas, with voltage ramping cycles from 0.5 to 1.3 V. Data
were analyzed by an HP Chemistation System and Bruker Daltonics Data Analysis.
The analytes were characterized by their respective ultraviolet and mass spectra, which were compared with
literature data and the mass spectra database Phenol-Explorer (www.phenol-explorer.eu), ChemSpider (http:
//www.chemspider. com), Metlin (http://metlin.scripps.edu) and HMDB (www.hmdb.ca).
3. Results
3.1 Analyses of the Leaf Extract by Fourier Transform Infrared Spectrometry (FT-IR)
Fourier transform infrared spectrometry is used for analysis, because it represents a non-invasive analytical tool
allowing a fast and simultaneous qualitative and quantitative characterization of natural products and their
constituents. The infrared (IR) spectrum of the ethanolic extract of leaves exhibited bands at 3369, 3012, 2925,
2854, 1737, 1640, 1454, 1409, 1378, 1346, 1240, 1162 and 1073 cm-1. The maximum absorptions were
attributed to the following functional groups: at 3369 - 3200 cm–1 (stretching of free OH, as sharp peak), 2925 -
2854 cm–1 (C-H aliphatic stretching), 1737 cm–1 (C=O ester bond) and 1640 cm–1 (C=C aromatic). The C–O
stretching characteristic from esters appeared in the region 1346 – 1240 cm-1. The peak at 1737 cm-1 assigned to
the C=O stretching vibration means that some carbonyl compounds exist in the leaves of S. Oblonga. Bands
from 1240 to 1162 were attributed to C-O stretching of phenolics and asymmetric C-C-O stretching of esters.
The bands between 1454- 1409 cm-1 are attributed to the asymmetric in-plane bending of – CH3, which is also
the same spectral region reflecting to the phenyl (C=C bonds). The absorption bands that were attributed to C=C
stretching vibration at 1640 cm-1 and the band at 1378 cm-1 referred to C-O stretching vibration of phenyl
groups were observed indicating the presence of phenolic compounds. FT-IR analysis results revealed the
presence of fatty acids and its respective ethyl esters and phenolic compounds, such as phenylpropane
glycerides.
3.2 Compounds Detected In Leaves and Stems by GC-EI-MS Analysis
The extraction of phenolic compounds depends mainly on the nature of the sample matrix and its chemical
properties including molecular structure, polarity of solvent, concentration, number of aromatic rings and
hydroxyl groups. Identification of compounds 1-18 (Table 1) was performed by mass spectra through molecular
ions M+ ∙) and fragmentation patterns, which were compared with NIST 21 and Wiley-275 libraries. For
compounds 1-18, the similarity percentages obtained are equal to or greater than 95% with the NIST Match
Factor - 850.6. Mass spectral data from the literature has also been used to identify fatty acids (4, 7, 10 and 11)
(Hong and Kim, 2013) and their ethyl esters (5, 9 and 12) (Monção et al., 2015, Sudha et al., 2013), phytosterols
(14-18) (Chen et al., 2015, Suttiarporn et al., 2015), squalene (13), butanoic acid butyl ester (1) (Oliveira-Garcia
et al., 2015), methyl salicylate (2) (Jayasekara et al., 2002), 5-methyl-4-hepten-3-one (3), 9,12-octadecadienal (8)
(Hong et al., 2013) and phytol (6) (Sudha et al., 2013). High content of 5-methyl-4-hepten-3-one (3) was detected
in both extracts. Phytosterols 14-18 were not detected in the ethanolic extract of the leaves. Figure 1 shows the
GC-EI-MS mass spectrum of ethyl palmitate (5), stigmasterol (15) and sitosterol (16).
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Table 1. Compounds 1- 18 detected in the analysis of ethanol extract of stems and leaves of Sanchezia oblonga
by GC-IE-MS, identified through NIST Mass Spectra and MS data from literature (see text)
Cp.N*
Rt**(m
in)
EIMS[M+] m/z
(%)
EIMS – Fragments m/z (%)
Identification and
(NIST Mass Spectrum number)
Stems
(%)
Leaves
(%)
1
4.6
144 (34)
101 (40), 89 (70), 71 (100)
butanoic acid butyl ester (228835)
2.8
1.5
2
5.6
152 (50)
121 (25), 120(100), 92 (60)
methyl salicylate (291552)
1.1
-
3
6.8
126 (50)
97 (100), 69 (50)
5-methyl-4-hepten-3-one (63310)
31.1
21.4
4
24.3
256 (11)
213 (13), 157 (14), 129 (32),
115 (14), 83 (40), 73 (100)
palmitic acid (151973)
5.4
7.3
5
24.8
284 (5)
241 (7), 157 (15), 101 (60), 88
(100)
ethyl palmitate (233204)
3.7
4.7
6
27.0
296 (1)
123 (40), 111 (40), 95 (50), 81
(60), 71 (100)
phytol (375015)
1.9
41.5
7
27.4
280 (4)
124 (10), 110 (17), 95 (57), 81
(75), 67 (100)
linoleic acid (229327)
8.2
1.5
8
27.8
264 (4)
165 (2), 108 (30), 95 (50), 79
(60), 67 (70)
9,12-octadecadienal (35820)
7.0
-
9
28.0
308 (3)
262 (6), 153 (15), 135 (14),
109
(23), 107 (10), 95
(59), 81
(81), 67 (100)
ethyl linoleate (155747)
5.5
2.7
10
28.4
282 (1)
264 (7), 222 (20), 180 (30),
135
(12), 121 (16), 108
(24),
101 (27), 95 (53), 69 (100)
oleic acid (134027)
4.5
8.5
11
28.7
284 (10)
241 (30), 185 (40), 129 (60),
73 (100).
stearic acid (290961)
1.0
-
12
30.2
312 (8)
157 (13), 101 (50), 88 (100)
ethyl octadecanoate (23294)
0.6
-
13
38.7
410 (1)
149 (30), 136 (30), 121 (30),
109
(30), 95 (30), 81
(63), 69
(100)
squalene (227620)
0.5
3.1
14
44.3
400 (21)
382 (30), 367 (10), 315 (22),
289 (22), 255 (15), 213 (57),
145 (50), 107 (60), 95 (70)
campesterol (151556)
1.3
-
15
44.6
412 (24)
397 (3), 394 (3), 369 (5), 351
(20), 300 (30), 271 (19), 255
(20), 159 (40), 145 (30), 133
(60)
stigmasterol (352610)
5.8
-
16
45.4
414 (35)
399 (18), 396 (19), 381 (21),
329 (33), 303 (17), 273 (13),
255 (22), 213 (39), 207 (41),
173 (21),161 (36), 147 (28),
133 (32)
sitosterol (251915)
4.8
-
17
46.2
410 (12)
367 (33), 298 (23), 271 (20),
245 (20), 191 (17), 147 (23),
133 (27), 119 (15)
stigmasta-4,22-dien-3-one (255385)
0.6
-
18
47.0
412 (30)
398 (20), 370 (30), 289 (50),
229 (40), 149 (40), 135 (25),
124 (100), 107 (22)
stigmast-4-en-3-one (17165)
0.9
-
*Cp.N: compound number. **Rt: retention time.
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GC-EI-MS mass spectrum of ethyl palmitate (5)
GC-EI-MS mass spectrum of stigmasterol (15)
GC-EI-MS mass spectrum of sitosterol (16)
Figure 1. GC-EI-MS mass spectra of compounds 5, 15, 16
3.3 Compounds Detected in Leaves and Stems by RPHPLC-ESI-MS/MS Analyses
The identification of compounds 19-36 (Table 2) was based on the MS data in negative ionization mode
conjugated with the UV-DAD, which were compared with literature data. In negative ion mode, high abundances
of quasi-molecular ions [M-H]- and [M-H + HCOOH]- were exhibited by compounds 19-36. (Falção et al., 2013).
The glycosyl and alkyl residues are weak chromophores, so these substituents did not change significantly, the
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62
maximum absorption coefficients and absorption wavelengths of compounds 19, 21-23 and 25-36 (Table 2),
indicating that the loss of 132, 146, 162 and 176 Da, in these compounds corresponds to arabinose, rhamnose,
glucose and glucuronide, respectively.
3.4 Compounds Detected by RPHPLC-ESI-MS/MS Analyses of Leaf Ethanolic Extract
Compounds 20, 22, 29, 30, 31, 35 and 36 were detected only in ethanolic extract from leaves (Table 2).
Compounds 20, 22 and 30 are new and their identification was based on UV and mass spectra, which were
compared with that phenylpropane glycerides isolated from sorghum grain (Kang et al., 2016, Kadam et al.,
2018; Nguyen et al., 2015, and Svensson et al., 2010) and Lilium longiflorum Thunb. (Munafo Jr and Gianfagna,
2015). Sorghum is the fifth most-produced cereal in the world and the major producers are USA, Mexico,
Nigeria, Sudan and India. Sorghum grain foods is used in many parts of Africa, Asia and the semi-arid tropics
world-wide and exhibited potential health benefits against chronic diseases related to over-nutrition. Compound
20 (Rt - 3.6 min) exhibited UV maximum absorption at 310 nm, characteristic of coumaric acid derivatives.
Their mass spectra exhibited [M - H]- ion at m/z 235, base peak at m/z 217 and fragment ion at m/z 199, both
resulting from water loss (18 Da). The fragment ion at m/z 155 was formed by the loss of carbon dioxide (44 Da)
from fragment ion at m/z 199. Compound 20 was tentatively identified as 1-O-coumaroyl-2-hydroxy propanal.
Compounds 22 (Rt - 8.3 min) and 30 (Rt - 13.3 min) displayed the same fragmentation pattern with a base peak
at m/z 235 and were characterized as 1-O-coumaroyl-2-hydroxy propanal derivatives. Compound 22 exhibited
[M − H + HCOOH]− adduct ion at m/z 413, [M - H]- ion at m/z 367 and the base peak at m/z 235 [M – H -
132]-, was produced by loss of arabinose (132 Da). Based on literature data (Kang et al., 2016, Kadam et al.,
2018; Nguyen et al., 2015, Munafo Jr and Gianfagna, 2015; and Svensson et al., 2010), compound 22 was
tentatively identified as 1-O-coumaroyl-2-O-arabinosyl propanal. Compound 30 exhibited [M − H + HCOOH]−
adduct ion at m/z 427, [M - H]- at m/z 381 and the base peak at m/z 235 ([M – H - 146]-, was produced by loss
of rhamnose (146 Da). Based on literature data, (Kang et al., 2016; Kadam et al., 2018; Nguyen et al., 2015;
Munafo Jr and Gianfagna, 2015; and Svensson et al., 2010), compound 30 was tentatively identified as
1-O-coumaroyl-2-O-rhamnosyl propanal.
p-Coumaroylglycolic acid was reported for Lepidium sativum (Kadam et al., 2018). Caffeoylglycolic acid methyl
ester was isolated from the grains of Sorghum bicolor (L.) Moench var. hwanggeumchal Kang et al., 2016;
Nguyen et al., 2015). 2-O-coumaroylglycerol and 1-O-coumaroyl-2-O-glucosylglycerol were detected in
hydromethanolic extracts (50:50) of brown, red and white sorghum whole grains (Kang et al., 2016; and
Svensson et al., 2010). Feruloyl-caffeoylglycerol was found in Ananas comosus L. leaves and sorghum grain
(Wu et al., 2016). Sorghum grains in the diet promoted cardiovascular health and exhibited beneficial effects for
weight control (Kang et al., 2016). Caffeoylglycolic acid methyl ester and 1-O-caffeoylglycerol isolated from
Sorghum bicolor, showed inhibitory potential on nitric oxide production (Salazar-López et al., 2018). Lilium
longiflorum, an attractive ornamental plant, possesses flower buds and bulbs that are used for both culinary and
medicinal purposes in many parts of the world. This species contains significant amounts of phenylpropanoid
glycerol glucosides, which may contribute to plant pathogen defense, ultraviolet/high-intensity visible light
(UV/high light) protection, and use in traditional medicine (Munafo Jr and Gianfagna, 2015).
Compound 29 (Rt - 12.8 min) exhibited UV maximum absorption at 318 nm, characteristic of cinnamic acid
derivatives. Its mass spectrum exhibited [M − H + HCOOH]− adduct ion at m/z 491, [M - H]- at m/z 411 and
base peak at m/z 279 (deprotonated butyl sinapate), produced by the loss of arabinose (132 Da). Based on
literature data (Teixeira et al., 2013; Nićiforović et al., 2017; Martinović et al., 2019), compound 29 was
tentatively identified as 4-O-arabinosyl butyl sinapate. For compound 31 (Rt - 13.5 min) the mass spectrum
exhibited [M − H + HCOOH]− adduct ion at m/z 461 and [M - H]- at m/z 415. Based on literature data (Teixeira
et al., 2013; Nićiforović et al., 2017; Martinović et al., 2019), compound 31 was tentatively identified as
4-O-glucosyl ethyl dihydrosinapate.
Compound 35 (25.1 min) exhibited UV maximum absorption at 266 and 355 nm, characteristic of flavonol.
Band I (330 – 380 nm) is attributed to the B-ring cinnamoyl system, and Band II (240 – 280 nm) is attributed to
the A-ring benzoyl system. Mass spectrum of 35 exhibited [M − H + HCOOH]− adduct ion at m/z 493 and [M -
H]- ion at m/z 447. Based on UV and MS data compared with literature (Soares et al., 2019; Vukics et al., 2010;
Pitura and Arntfield, 2019, Loi et al., 2019), compound 35 was identified as kaempferol-3-O-glucoside.
Compound 36 (Rt - 26.5) exhibited UV maximum absorption at 280 nm and the mass spectrum gave [M - H]- at
m/z 421. Its MS/MS fragmentation yielded base peak at m/z 289 (deprotonated catechin), produced by the loss of
arabinose (132 Da). Based on literature data (Kang et al., 2016; Zerbib et al., 2018; Karar and Kuhnert, 2015),
compound 36 was tentatively identified as catechin arabinoside.
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63
3.5 Compounds detected by RPHPLC-ESI-MS/MS Analyses of Stem Ethanolic Extract
Compounds 21, 23, 24, 26, 32, 33 and 34 were detected only in ethanolic extract from stems (Table 2).
Compound 21 (Rt - 6.1 min, Table 2) exhibited UV maximum absorption at 254 nm. Mass spectrum exhibited
[M - H]- at m/z 315 and base peak at m/z 153 ([M – H - 162]-, corresponding to the loss of glucose moiety.
Compound 21 was identified as 4-hydroxy-3-methoxybenzyl alcohol glucoside, based on literature data (Karar
and Kuhnert, 2015; Kumar, 2017; Veličković et al., 2012). The mass spectra of compounds 24 (Rt - 10.3 min)
and 26 (Rt - 11.0 min) displayed the same [M - H]- at m/z 387. The mass spectrum of compound 24 (Rt - 10.3
min) exhibited base peak at m/z 163, that resulted to the loss of 225 Da from the molecular ion at m/z 387, which
was formed by cleavage of the ester bond (Aziz et al., 2014; Shen et al., 2018; Wicha et al., 2015). Compound 24
was tentatively identified as ethyl rosmarinate. The mass spectrum of 26 exhibited base peak at m/z 207,
corresponding to dihydrosinapoyl moiety, which was formed by cleavage of the ester bond that resulted in loss
of glucose (180 Da) (Tian et al., 2013; El-Sayed et al., 2017). Compound 26 was tentatively assigned as
dihydrosinapic acid-O-glucoside.
Compound 34 (Rt - 16.6 min, Table 2) exhibited UV maximum absorption at 328 nm, characteristic of caffeic
acid derivatives. The mass spectrum exhibited [M - H]- at m/z 521 and base peak at m/z 179 (deprotonated
caffeic acid) (Liang et al., 2017; Yang et al., 2018, Borrás-Linares et al., 2014; Lee et al., 2017). This fragment
ion was not observed in mass spectra of rosmarinic acid-3-O-glucoside (Yang et al., 2018, Borrás-Linares et al.,
2014, Lee et al., 2017), but it was detected in rosmarinic acid, the second most common ester of caffeic acid in
the plant kingdom (Lee et al., 2017; Hashem et al., 2019). The base peak at m/z 179 was formed by cleavage of
the ester bond, which produced the loss of 342 Da, corresponding to the 3,4-dihydroxyphenyl-lactic moiety plus
a glucose, probably attached at the 3'-O position. Compound 34 was tentatively identified as rosmarinic
acid-3'-O-glucoside.
Compound 23 (Rt - 8.5 min) and 33 (Rt - 15.9 min) exhibited UV maximum absorption at 266 and 355 nm,
characteristic of flavonols. The mass spectrum of compound 23 exhibited [M − H + HCOOH]− adduct ion at m/z
523 and [M - H]- at m/z 477 and was identified as quercetin glucuronide (23) (Soares et al., 2019). The mass
spectrum of compound 33 exhibited [M - H]- at m/z 579 and base peak at m/z 417, produced by the loss of
glucose moiety (162 Da) (Soares et al., 2019, Vukics and Guttman, 2010) and was identified as
kaempferol-7-O-arabinosyl-3-O-glucoside. Compound 32 (Rt - 14.0 min) exhibited UV maximum absorption at
290 nm, characteristic of flavanonol. Its mass spectrum exhibited [M - H]- at m/z 581, a fragment ion at m/z 419,
resulting from loss of glucose moiety (162 Da) and base peak at m/z 389 (Pitura and Arntfield, 2019). Compound
32 was assigned as dihydrokaempferol-7-O-arabinosyl-3-O-glucoside.
3.6 Compounds Detected by RPHPLC-ESI-MS/MS Analyses of Leaf and Stem Ethanolic Extracts
Compounds 19, 25, 27 and 28 were detected in ethanolic extracts from leaves and stems (Table 2). The mass
spectrum of 19, (Rt - 2.7 min) exhibited [M - H]- at m/z 341, and base peak at m/z 179 (deprotonated caffeic
acid), produced by the loss of glucose moiety (162 Da) (Karar and Kuhnert, 2015; Liang et al., 2017). Compared
with its elution order and MS/MS fragmentation pattern, compound 19 was assigned as caffeic acid glucoside.
For compound 25 (Rt - 10.6 min), the mass spectrum exhibited [M - H]- at m/z 401 (Figure 2), and base peak at
m/z 269 (C13H17O6), produced by the loss of arabinose moiety (132 Da). Benzyl alcohol-7-O-arabinosyl
glucoside 25 was detected in Sanchezia nobilis (Ellah et al., 2014) and was identified based on literature data
(Karar and Kuhnert, 2015).
Compounds 27 (Rt - 11.4 min) and 28 (Rt - 11.8 min) exhibited maximum UV absorption at 310 nm, which is
characteristic of coumaric acid derivatives. Mass spectrum of 27 exhibited [M − H + HCOOH]− adduct ion at m/z
491, [M - H]- at m/z 445 and base peak at m/z 293, which correspond to sinapyl alcohol diacetate after the loss of
galloyl moiety (152 Da). Compound 27 was tentatively identified as 4-O-galloyl-sinapyl alcohol diacetate based
on literature data (Nićiforović et al. 2017, Tian et al., 2013). The mass spectrum of 28 exhibited [M − H +
HCOOH]− adduct ion at m/z 431 and [M - H]
- at m/z 385. Based on literature data (Nićiforović et al., 2017;
El-Sayed et al., 2017, Yang et al., 2018), compound 28 was identified as sinapic acid-O-glucoside. The mass
spectrum of compounds 20, 22, 24, 25 and 34 are shown on Figure 2. The proposed structures of compounds 20,
22, 24, 30 and 34 are shown on Figure 3. Further studies should be performed through the isolation of these
compounds in order to confirm the identification by NMR methods.
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64
A
B
C
D
E
Figure 2. HPLC-ESI-MS/MS spectra in negative ion mode of compounds 20 (1-O-coumaroyl-2-hydroxy
propanal; A), 22 (1-O-coumaroyl-2-O-arabinosyl propanal; B), 24 (ethyl rosmarinate; C), 25 (benzyl
alcohol-7-O-arabinosyl glucoside; D) and 34 (rosmarinic acid-3’-O-glucoside; E)
155.2
199.1
217.1
-MS2(235.1), 3.6min #322
0.0
0.2
0.4
0.6
0.8
1.0
5
x10
Intens.
100 200 300 400 500 600 700 m/z
125.5 161.3 211.1
235.2
321.0 366.1
-MS2(367.1), 8.3min #778
0
2
4
6
4
x10
Intens.
0200 400 600 800 1000 m/z
113.6
163.4
192.3
225.2 297.3 346.0
369.1
-MS2(387.2), 10.3min #1033
0.0
0.5
1.0
1.5
2.0
2.5
4
x10
Intens.
100 200 300 400 500 600 700 800 m/z
125.5
161.3
197.6
233.0
269.1
293.1 336.9357.0 401.1
-MS2(401.1), 10.6min #1020
0
2
4
6
4
x10
Intens.
100 200 300 400 500 600 m/z
179.2
255.1 297.0 341.3
364.8
387.0420.9
453.1
521.2
558.0
-MS2(567.4), 16.6min #1721
0
1
2
3
4
5
4
x10
Intens.
200 400 600 800 1000 m/z
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65
Table 2. Pseudomolecular ions and MS/MS fragment ions obtained for compounds 19-36 by
RPHPLC-SPD-ESI-MS/MS analyses in negative ionization modes
Cp.Na
Rtb
(min)
UV
(nm)
RPHPLC/(-)ESI-
MS/MS mz (%)
Proposed
Compound
c
Refer.d
Stemse
Leavesf
19
2.7
320
[M–H]-
:
341
MS/MS:
179
(100),
161 (40), 143 (40)
caffeic acid
glucoside
32,43
+
+
20
3.6
310
[M–H]-
:
235
MS/MS:
217
(100),
199 (50), 155 (20)
1-O-coumaroyl-
2
-hydroxy propanal
19-24
-
+
21
6.1
254
[M–H]-
:
315
MS/MS:
153
4-Hydroxy-
3
-methoxybenzyl
alcohol glucoside
34,35
+
-
22
8.3
310
[M–H+HCOOH]-:
413;
[M–H]-:
367
MS/MS: 235
1-O-coumaroyl-
2
-O-arabinosyl
propanal
19-24
-
+
23
8.5
260,
355
[M–H+HCOOH]-:
523; [M–H]
-
: 477
Quercetin
glucuronide
28
+
-
24
10.3
320
[M–H]-: 387
Ethyl rosmarinate
36-39
+
-
25
10.6
254
[M–H]-: 401
MS/MS: 269
benzyl alcohol-7-O-
arabinosyl glucoside
3, 32
+
+
26
11.0
-
[M–H]-: 387
MS/MS: 207 (100)
dihydrosinapic acid-
O-glucoside
41,42
+
-
27
11.4
325
[M–H+HCOOH]-:
491;
[M–H]-:
445
MS/MS: 293
4-O-galloyl-sinapyl
alcohol
diacetate
26,41
+
+
28
11.8
330
[M–H+HCOOH]-:
431; [M–H]
-
: 385
sinapic acid-O-
glucoside
26,42,44
+
+
29
12.8
318
[M–H+HCOOH]-:
457;
[M–H]-:
411
MS/MS: 279
4-O-arabinosyl
butyl
sinapate
25
-
27
-
+
30
13.3
315
[M–H+HCOOH]-:
427;
[M–H]-:
381
MS/MS: 235
1-O-coumaroyl-2-O-rhamnosyl
propanal
19
-
24
-
+
31
13.5
-
[M–H+HCOOH]-:
461; [M–H]
-
: 415
4-O-glucosyl-ethyl-
dihydrosinapate
25-27
-
+
32
14.0
290,
330 sh
[M–H]-: 581
MS/MS: 419 (50),
389 (100)
dihydrokaempferol-
7
-O-arabinosyl-3-O-
glucoside
30
+
-
33
15.9
266,
348
[M–H]-: 579
MS/MS: 417
kaempferol-7-O-
arabinosyl
-3-O-
glucoside
28,29
+
-
34
16.6
328
[M–H+HCOOH]-:
567; [M
–H]-
: 521
MS/MS: 179
rosmarinic acid-
3’
-O-glucoside
43-46
+
-
35
25.1
266,
355
[M–H+HCOOH]-:
493; [M–H]
-
: 447
kaempferol-7-O-glucoside
28-30
-
+
36
26.5
280
[M–H]-: 421
MS/MS: 289
catechin-O-arabinoside
21,31-33
-
+
aCp.N: compound number.
bRt: retention time.
cidentified based on MS/MS and UV data and their comparison with MS/MS and other data from reference sources.
dreferences used in identification (see text)
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66
Figure 3. Proposed structures for compounds 20, 22, 24, 29, 30 and 34
4. Discussion
Differences were observed comparing the distribution of constituents of leaves and stems extracts by GC-EI-MS
(Table 1) and RPHPLC-DAD-ESI-MS/MS analyses (Table 2). In the analyses by GC-EI-MS (Table 1), the
steroids 14-18 were found only in extract from stems. Other unique constituents of stem extracts were methyl
salicylate (2) and 9,12-octadecadienal (8). Stigmasterol (15) and sitosterol (16) were detected in higher amounts
(5.8% and 4.8%, respectively) than campesterol (14, 1.3%), stigmasta-4,22-dien-3-one (17, 0.6%) and
stigmasta-4-en-3-one (18, 0.9%). 5-Methyl-4-hepten-3-one (3) was the main constituent of the ethanolic extract
from stems (31.1%) and was also detected with high content in leaf ethanolic extract (21.4%, Table 1).
Regarding the RPHPLC-ESI-MS/MS analyses (Table 2), the new compounds, tentatively identified as
phenylpropane glycerides derivatives (20, 22 and 30), sinapic acid glycoside esters (29 and 31),
kaempferol-7-O-glucoside (35) and catechin-O-arabinoside (36) were detected only in ethanolic extract from
leaves. Caffeic acid glucoside (19), benzyl alcohol-7-O-arabinosyl glucoside (25), 4-O-galloyl-sinapyl alcohol
diacetate (27) and sinapic acid-O-glucoside (28) were detected in the two ethanolic extracts. On the other hand,
4-hydroxy-3-methoxybenzyl alcohol glucoside (21), ethyl rosmarinate (24), dihydrosinapic acid-O-glucoside
(26), rosmarinic acid-3’-O-glucoside (34) and the flavonols glycosides 23, 32 and 33 were detected only in
ethanolic extract from stems (Table 2). It is noteworthy that ethyl rosmarinate (24) and rosmarinic
acid-3'-O-glucoside (34) were unique in the ethanolic extract of stems. Differences in gene expression between
plant parts is well known. For example, plant polyphenol profile varied widely ontogenetically and among
organs of Geranium sylvaticum (Tuominem and Salminem, 2017).
There have been few chemical studies about genus Sanchezia. In this study a hyphenated chromatographic
analysis of ethanolic extracts of leaves and stems of S. oblonga was performed. Although the isolation of
constituents is necessary for their identification by NMR methods, it is possible to observe that the results are
different from those previously obtained for this species (Ellah et al., 2014). Table 2 lists various synapic acids
and their esters, phenylpropane glycerides derivatives and rosmarinic acid derivatives, which had not been
reported previously for this species. In this work, phytosterols and kaempferol glycosides were detected only in
stems. However, a recent work reported that phytosterols were isolated from n-hexane and ethylacetate extract of
the leaves (Xuan et al., 2019a,b, Loi et al., 2019a), while kaempferol glycosides were isolated from ethylacetate
and aqueous extracts of leaves of S. nobilis (syn. S. oblonga) (Xuan et al., 2019b, Loi et al., 2019b).
The differences in chemical composition between the extracts analyzed in the present work and the previously
published papers regarding S. nobilis can be explained by two possibilities: a) they may correspond to the same
species but to different varieties or chemotypes; b) differences may be due to climate, season or growing
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67
conditions (Liu et al., 2015).
S. oblonga may turn out an interesting source of bioactive substances. Phytosterols exhibited antibacterial and
antifungal activity (Aldini et al., 2014), are active against leukemic cell lines (Suttiarporn et al., 2015) and useful
in the treatment of gastrointestinal inflammatory diseases by association with systemic and local metabolic
anti-inflammatory drugs (Burčová et al., 2018). Stigmasterol inhibited tumor endothelial cells and
cholangiocarcinoma (Saeidnia et al., 2014, Kangsamaksin et al., 2017). Sterols inhibited cholesterol absorption
and exhibited trypanocidal and mosquito larvicidal activity (Ghosh et al., 2013). Hydroxycinnamic acids and
their derivatives promote a variety of health benefits, e.g. reducing obesity and adverse health complications
(Alam et al., 2016), as well as exhibiting antibacterial, antifungal (Guzman, 2014), antityrosinase, UV protection,
anti-aging and anti-inflammatory effects (Alam et al., 2016).
Synapic acid derivatives exhibited antioxidant, antimicrobial, anti-inflammatory, anti-cancer, anti-anxiety
(Nićiforović et al., 2017), anti-glycemic, neuroprotective, antibacterial (Chen, 2016) and analgesic activities
(Hameed et al., 2016). Rosmarinic acid esters exhibited a broad spectrum of biological effects (Shen et al., 2018;
Liang et al., 2017; Thammason et al., 2018). Glycerol phenylpropanoid glucosides act as hepatic glucose
production inhibitors; structure–activity relationships indicated that the hydroxylation pattern of the
hydroxycinnamic acid moiety and acetylation accounted for differences in activity (Murray et al., 2019).
5. Conclusion
Leaves and stems of Sanchezia oblonga contain a variety of biologically active compounds, such as phytosterols,
flavonol glycosides, benzyl alcohol glycosides, sinapic acid glycoside esters, phenylpropane glycerides and
rosmaric acid derivatives, which were detected using GC-EI-MS and RPHPLC-DAD-ESI-MS/MS. These
techniques revealed that ethanolic extracts of leaves and stems contain distinct chemical profiles. The leaves
contain phenylpropane glycerides and can be used in cooking as supplement. Comparison of the current results
with previous studies suggests the possibility of chemical polymorphisms within species. Further studies are
needed to explore this possibility. Compounds should be isolated and identified by NMR methods. Statistical
methods, principal component analysis and hierarchical cluster analysis can be applied to evaluate intrinsic
quality and identify chemical markers, which is useful for the chemical standardization of this species.
Acknowledgments
This investigation was partly funded by CNPq (Conselho Nacional do Desenvolvimento Científico e
Tecnológico) and FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo).
Conflict of Interests
We confirm that there is no conflict of interest associated with this work.
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