Wild pineapple (Ananas bracteatus (Lindl.), var. Albus) harvested in forest patches in rural area of Viçosa, minas Gerias, Brazil: Excellent source of minerals and good source of proteins and vitamin C

Abstract

ABSTRACT In the rural region of the municipality of Viçosa, Minas Gerais, forest patches are encountered and they present a great diversity of wild and edible fruit, where wild pineapple (Ananas bracteatus (Lindl.), var. albus) is one of those of greatest occurrence. Given that, little is known about the nutritional characteristics of this fruit, the present study aimed to investigate the physical and physic-chemical characteristics, chemical composition (titratable acidity, total soluble solids, pH, moisture content, ash, protein, lipids and dietary fiber), carotenoids (a-carotene, ß-carotene, ß-cryptoxanthin and lycopene), vitamin C (AA and DHA) by HPLC-DAD, vitamin E (a-, ß-, ?-, d-, tocopherols and tocotrienols) by HPLC-fluorescence, and minerals (P, K, Ca, Mg, Cu, Fe, Zn, Mn, Na, Cr, Se and Mo) by ICP-AES. Fruits showed a moisture content average of 78.5 g 100g-1, 16.3 ° Brix of soluble solids, titratable acidity equal to 1.71 g 100g-1 of citric acid, 1.66 g 100g-1 of dietary fiber, 1.41 g 100 g-1 of lipids and caloric density of 83.15 kcal 100g-1. We considered it a source of carbohydrates (12.82 g 100g-1), proteins (4.79 g 100g-1) and Zn (0.98 mg 100g-1); a good source of vitamin C (18.70 mg 100g-1); and an excellent source of Cu (0.48 mg 100g-1), Fe (2.05 mg 100g-1), Mn (8.87 mg 100g-1) and Mo (0.15 mg 100g-1).

Full text

Rev. Bras. Frutic., v. 38, n. 3: (e-526) DOI 10.1590/0100-29452016526 May/Jun 2016 Jaboticabal - SP
ISSN 0100-2945 http://dx.doi.org/10.1590/0100-29452016526
Rev. Bras. Frutic., Jaboticabal - SP, v. 38, n. 3 : e-526 1/8
WILD PINEAPPLE (Ananas bracteatus (LINDL.),
VAR. albus) HARVESTED IN FOREST PATCHES IN RURAL
AREA OF VIÇOSA, MINAS GERIAS, BRAZIL:
EXCELLENT SOURCE OF MINERALS AND GOOD SOURCE
OF PROTEINS AND VITAMIN C1
GALDINO XAVIER DE PAULA FILHO2, TIBÉRIO FONTENELLE BARREIRA3,
GILBERTO BERNARDO DE FREITAS4, HÉRCIA STAMPINI DUARTE MARTINO5,
HELENA MARIA PINHEIRO SANT’ANA6
ABSTRACT - In the rural region of the municipality of Viçosa, Minas Gerais, forest patches are encountered
and they present a great diversity of wild and edible fruit, where wild pineapple (Ananas bracteatus (Lindl.),
var. albus) is one of those of greatest occurrence. Given that, little is known about the nutritional characteristics
of this fruit, the present study aimed to investigate the physical and physic-chemical characteristics, chemical
composition (titratable acidity, total soluble solids, pH, moisture content, ash, protein, lipids and dietary
ber), carotenoids (α-carotene, β-carotene, β-cryptoxanthin and lycopene), vitamin C (AA and DHA) by
HPLC-DAD, vitamin E (α-, β-, γ-, δ-, tocopherols and tocotrienols) by HPLC-uorescence, and minerals
(P, K, Ca, Mg, Cu, Fe, Zn, Mn, Na, Cr, Se and Mo) by ICP-AES. Fruits showed a moisture content average
of 78.5 g 100g-1, 16.3 ° Brix of soluble solids, titratable acidity equal to 1.71 g 100g-1 of citric acid, 1.66 g
100g-1 of dietary ber, 1.41 g 100 g-1 of lipids and caloric density of 83.15 kcal 100g-1. We considered it a
source of carbohydrates (12.82 g 100g-1), proteins (4.79 g 100g-1) and Zn (0.98 mg 100g-1); a good source
of vitamin C (18.70 mg 100g-1); and an excellent source of Cu (0.48 mg 100g-1), Fe (2.05 mg 100g-1), Mn
(8.87 mg 100g-1) and Mo (0.15 mg 100g-1).
Index terms: Wild fruit, nutritional value, vitamins, nutrients.
ABACAXI DO MATO (Ananas bracteatus (Lindl.), var. albus) COLHIDO EM FRAGMENTOS
FLORESTAIS NA ZONA RURAL DE VIÇOSA, MINAS GERAIS: EXCELENTE FONTE DE
MINERAIS E BOA FONTE DE PROTEÍNAS E VITAMINA C
RESUMO-Na zona rural do município de Viçosa, Minas Gerais, encontram-se fragmentos orestais que
apresentam grande diversidade de frutas silvestres e alimentícias, sendo o abacaxi do mato (Ananas bracteatus
(Lindl.), var. albus) uma das frutas de maior ocorrência. Tendo em vista que pouco se conhece a respeito
das características nutricionais dessa fruta, este trabalho teve por objetivo investigar a caracterização física,
composição físico-química e química (acidez titulável, sólidos solúveis totais; pH; umidade, cinzas, proteínas,
lipídios e bra alimentar); carotenoides (α-caroteno, β-caroteno, β-criptoxantina e licopeno) e vitamina C
(AA e ADA) por CLAE-DAD; vitamina E (α-, β-, γ-, δ-, tocoferóis e tocotrienóis) por CLAE-uorescência;
e minerais (P, K, Ca, Mg, Cu, Fe, Zn, Mn, Na, Cr, Se e Mo) por ICP-AES. Os frutos apresentaram em
média 78,5 g 100 g-1 de umidade; 16,3 ºBrix de sólidos solúveis; acidez titulável igual a 1,71 g 100 g-1 de
ácido cítrico; 1,66 g 100 g-1 de bra alimentar; 1,41 g 100 g-1 de lipídios e 83,15 kcal 100 g-1 de densidade
calórica, sendo considerados fonte de carboidratos (12,82 g 100 g-1), proteínas (4,79 g 100 g-1) e Zn (0,98
mg 100 g-1); boa fonte de vitamina C (18,70 mg 100 g-1); e excelente fonte de Cu (0,48 mg 100 g-1), Fe (2,05
mg 100 g-1), Mn (8,87 mg 100 g-1) e Mo (0,15 mg 100 g-1).
Termos para indexação: Fruta silvestre, valor nutricional, vitaminas, nutrientes.
1(Trabalho 031-15). Recebido em: 09-01-2015. Aceito para publicação em: 18-09-2015.
2Professor Assistente, Dep. de Educação, Campus Mazagão, UNIFAP, Macapá-AP. E-mail: galdino.lho@unifap.br
3Doutorando em Fitotecnia, Dep. de Fitotecnia, Laboratório de Silvicultura, UENF, Campos dos Goytacazes-RJ. E-mail:
tiberiofbarreira@gmail.com
4Professor Associado, Dep. de Fitotecnia, Setor de Fruticultura, UFV, Viçosa-MG. E-mail: bernardo@ufv.br
5Profª Associada, Dep. de Nutrição e Saúde, Lab. de Nutrição Experimental, UFV, Viçosa-MG. E-mail: hercia@ufv.br
6Profª Associada, Dep. de Nutrição e Saúde, Lab. de Análise de Vitaminas, UFV, Viçosa-MG. E-mail: helena.santana@ufv.br

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Rev. Bras. Frutic., v. 38, n. 3: (e-526) DOI 10.1590/0100-29452016526 May/Jun 2016 Jaboticabal - SP
G. X. de PAULA FILHO et al.
INTRODUCTION
The fruit consumption has an important
role in the healthy eating because it increases life
expectancy, prevents chronic no infectious disease,
due to the presence of minerals, vitamins and dietary
bre (ARA et al. 2014; COSTA et al. 2014). The
protective effect that these foods have is attributed to
the presence of compounds capable of capturing free
radicals (antioxidants), highlighting the vitamins,
phenolic compounds and carotenoids (KAUR et al.
2015).
Despite of this importance, in Brazil, the fruit
consumption is still insufcient and inappropriate,
and the daily intake is below the recommendation
and this is lower in low-income families. The
recommended daily intake per capita is 400g of fruits
(LI et al. 2012). However, the fruits have a high cost
in the food groups and people in social vulnerability
less consume them (HERSEY et al. 2015). This
reality reinforces the need to appreciate wild fruits
and the potential that they have.
The Atlantic forest is rich in fruits with
nutritional potential that may be an alternative
to improve the supply of nutrients to the general
population (SILVA et al. 2014), extending this benet
to the rural population. However, non-conventional
food groups, like fruits and vegetables, are still
underutilized due to the lack of knowledge of their
nutritional value (NANDAL; BHARDWAJ, 2014).
In the rural area of the municipality of
Viçosa, there are forest patches of Atlantic Forest
with high diversity of wild fruits. Although theses
fruits are part of the local population eating habits,
information about their nutritional value are scarce
in the literature.
This study aimed to analyse the physical
and physic-chemical characteristics, centesimal
composition, occurrence and concentration of
carotenoids, vitamin C, vitamin E and mineral in
wild pineapple fruits (Ananas bracteatus (Lindl.) var.
albus) and to evaluate the nutritional contribution
potential of this fruit for adults between 19 and 30
years old.
MATERIAL AND METHODS
Collection area, raw material, sampling
and preparation of samples – The total area where
the fruit harvest of wild pineapple was carried out
is approximately 1.500 m of extension, in the wild
environment of the rural area of Viçosa, Minas
Gerais (south latitude 20º45’14’’ and west longitude
42º50’40”). The fruit harvest was carried out in
January 2013. The harvest points were spaced from
200 to 400 m. The fruit maturation was determined
according to Donadio (2007) and dened by the
red-orange peel colour and the characteristic smell.
After the harvest, the fruits were immediately
transported to the Vitamins Analysis Laboratory,
Department of Nutrition and Health of UFV,
protected from light (wrapped in plastic bags and
inside of cardboard boxes). The fruits were washed
in water and dried with paper tower. Then, the fruits
were peeled and the edible parts were homogenised
in food processor, freeze-dried and stored in plastic
bags in freezer at -18±1°C until the analyses that
happened in a maximum of 72 hours.
Physical characterization – The following
measures were carried out in 30 wild pineapple fruits:
length, diameter; total mass (TM), mass of the edible
part or pulp mass (PM) and mass of the inedible part
(peel mass). Then, the yield of the edible part was
calculated using the equation: (TM/PM) x 100.
Physic-chemical analysis – They were
determined according to the rules of Adolfo
Lutz Institute (2008). The total acidity (TA) was
obtained by the neutralization volumetry, using
standard solution of sodium hydroxide 0.1 mol/L
in the presence of phenolphthalein 1% solution
(C20H14O4) in ethanol, as an indicator. The soluble
solids content (SS) was measured by the refractive
index, using portable refractometer (Instrutherm,
RTD-45), calibrated with distilled water, making
the Brix correction to room temperature. The pH
was measured using pHmetro (Ultra Basic, UB-10),
calibrated with buffer solutions of 4 and 7.
Analysis of the centesimal composition –
moisture, ash, proteins, lipids and total dietary bre
were determined in triplicate (AOAC, 2010). The
moisture was determined in the oven (SP Labor®,
SP 200) at 65 ± 1°C, for 72 hours and ash in mufe
(Quimis®, Q318M) at 550 °C, for 6 hours. Proteins
were determined by the micro-Kjeldhal method,
and the crude protein was calculated multiplying
the nitrogen content (N) by 6.25 (AOAC< 2010).
The total dietary bre was determined by the non-
enzymatic gravimetric methods with changes.
The total carbohydrates were calculated by the
difference between 100 and the sum of protein,
lipid, moisture, ash and bre fractions (IBGE, 1999).
The caloric density was estimated considering
the conversion factors of 4, 9 and 4 kcal per g for
carbohydrates, lipids and proteins, respectively
(FRARY; JOHNSON, 2005).
Carotenoids analysis – the occurrence and
concentration of α-carotene, β-carotene, lycopene
and β-cryptoxanthin were explored. The extraction

Rev. Bras. Frutic., v. 38, n. 3: (e-526) DOI 10.1590/0100-29452016526 May/Jun 2016 Jaboticabal - SP
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WILD PINEAPPLE (Ananas bracteatus (LINDL.), VAR. albus) HARVESTED IN FOREST...
was carried out according to the method proposed
by Rodriguez-Amaya et al. (1976) and the analyses
were carried out by high performance liquid
chromatography (HPLC) with diode-array detection
(DAD). The vitamin A concentration was calculated
according to the recommendations of U. S. Institute
of Medicine (2001).
Vitamin C analyses – The occurrence
and concentration of ascorbic acid (AA) and
dehydroascorbic acid (DHA) were explored. The
vitamin C extraction and analyses were carried out by
(HPLC-DAD), according to the conditions proposed
by Campos et al. (2009), with changes. The vitamin
C total concentration was calculated by the sum of
the AA and DHA concentrations.
Vitamin E analyses – The occurrence and
concentration of α-, β-, γ- and δ- tocopherols and
tocotrienols were investigated, as proposed by
Pinheiro-Sant’Ana et al. (2011), in HPLC system
with uorescence detector. The vitamin E total
concentration was calculated by the sum of vitamin
E compounds in the fruits.
Mineral Analyses – Inductively Coupled
Plasma Atomic Emission Spectrometry (ICP-AES)
determined the concentration of P, K, Ca, Mg, Zn,
Mn, Fe, Cu, Na, Cr, Se and Mo, according to the
method proposed by Gomes and Oliveira (2011).
Determination of nutritional contribution
– The nutritional contribution potential was
estimated based on the Recommended Dietary
Allowance (RDA), for adults with age between 19
and 30, according to the recommendations of U. S.
Institute of Medicine (2001). The fruit portions were
calculated according to the ‘Guia Alimentar para a
População Brasileira’ (Food Guide for the Brazilian
Population) (BRAZIL, 2008), considering the caloric
density and the fruit portion equivalent to 70 kcal.
RESULTS AND DISCUSSION
Physic, physic-chemical characterization
and centesimal composition
The wild pineapple fruit (Figure 01) has a
slight resemblance to the conventional pineapple
(Ananas comosus L.), var. ‘Pérola’.
In table 01, there are the results obtained for
the physical characterization of the wild pineapple
fruits, which showed similar length, diameter and
weight to the conventional pineapple, var. ‘Pérola’.
The wild pineapple showed average mass of 687.84g
(with crown) and pulp yield (edible mass) of 52.4%.
The values found for SS were equivalent
to 16.30 ºBrix (Table 02), we highlight that is
difcult to compare the values found here with other
studies because the SS values are inuenced by the
environment, however due to the fact that this study
was not carried out under controlled experimental
conditions nor compared more than one specie in
that environment, the presented result is an estimate
obtained in the analysed sample (30 fruits).
The high acidity (1.71 g of citric acid 100g-1
of pulp) was higher than the values found by Pinheiro
et al. (2006) in pineapple juices, var. ‘Pérola’, ranging
between 0.68 and 0.98 g of citric acid 100g-1 of pulp.
The SS/TA relation in the wild pineapple fruits was
relatively high (9.53). This relation is one of the
best ways to evaluate the avour of a fruit, since it
is an indicator of fruit quality in terms of maturation,
avour and industrial value (PIMENTA et al. 2014).
The information regarding the chemical
composition and caloric density of the wild
pineapple, presented in Table 02, show a moisture
value of 78.51 g 100 g-1. This value is lower than
the pineapple moisture, var. ‘Pérola’ (TACO/NEPA/
UNICAMP, 2011), but it is a good indicator because
it can represent a lower perishability of this fruit and
a higher percentage of total dietary bre, which in
the fruits analysed in this study was 1.66 g 100 g-1
a concentration greater than the one found for the
conventional pineapple, var. ‘Pérola’ (TACO/NEPA/
UNICAMP, 2011).
The caloric density of wild pineapple was
higher than the banana (Musa paradisíaca L.)(78
Kcal 100 g-1) and orange (Citrus sinensis) (46 Kcal
100 g-1), which are between the ten most consumed
fruits in Brazil (TACO/NEPA/UNICAMP, 2011;
BUENO; BACCARIN, 2012). These results show
that the wild pineapple can be a good option of daily
intake for people who need to gain weight. However,
for obese people, this intake should be controlled.
The carbohydrates concentration found in
this study (12.8%) is similar to the pineapple, var.
‘Pérola’ (12.3%) and to other fruits available in the
Brazilian Table of Food Composition (TACO/NEPA/
UNICAMP, 2011), such as the ‘abiu’ (Pouteria
caimito Ruiz & Pav.) (14.9%), ‘cajá’ (Spondias lutea
L.) (11.4%) and cashew (Anacardium occidentale
L.) (10.3%)
Composition of carotenoids and vitamins
The wild pineapple fruits showed total
carotenoids concentrations of 0.24 mg 100g-1, referring
to the β-carotene that was the only compound found
in this fruit, resulting in 20.0 µg 100g1 of vitamin A in
RAE (Table 03). This value corresponds to 20 times
more than the one mentioned to the frozen pulp of
pineapple, var. ‘Pérola’ (NEPA/UNICAMP, 2011).
As for the vitamin C concentration, the results
found for the wild pineapple (18.70 mg 100 g-1)

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G. X. de PAULA FILHO et al.
are intermediated to the ones found by Berilli et al.
(2014) for the conventional pineapple, cv ‘Pérola’
and ‘Gold’ (13.94 and mg 100g-1, and 24,23 and mg
100g-1, respectively).
There are no data available in the specialized
literature about the vitamin E concentration in any
variety of the pineapple fruits, which reinforces
even more the importance of this study, where the
fruits analysed showed β-tocopherol, α-tocotrienol
and β-tocotrienol, resulting in 28.05 µg 100-1 of total
vitamin E.
Mineral Composition
Among the analysed minerals, we highlight
the K, Ca, Mg and Fe (Table 04). However, the
mineral concentration is not a safe indicator of
nutritional value because some minerals like calcium,
iron, zinc, copper and magnesium can form insoluble
complexes with antinutritional factors (phytate,
oxalate), decreasing its bioavailability (LEAL et
al. 2010).
Wild pineapple potential as a source of
nutrients
According to Philip (2008), food can be
classied as “source” of a nutrient when they supply
from 5 to 10% of the Dietary Reference Intake (DRI),
as “good source” when they supply from 10 to 20%
of the DRI and “excellent source” when they supply
more than 20% of the DRI.
The fruits analysed in this study were
divided in portion of 70 kcal, according to the ‘Guia
Alimentar para a População Brasileira’ (Food Guide
for the Brazilian Population) (BRAZIL, 2008). Thus,
84g of wild pineapple are necessary to provide a
portion of 70 kcal and it can be considered a source
of carbohydrates and proteins because they showed
8.28% and 7.20% of these nutrients, respectively. The
fruit can be considered a good source of vitamin C as
it can supply 17.45% of the DRIs for adults.
In relation to the minerals, this fruit can be
considered an excellent source of Cu, Fe, Mn and
Mo; and Zn source (Table 05). .
The proof in this study that the wild pineapple
show a signicant concentration of nutrients suggest
that strategies to introduce this fruit on the menu of
children and adults can be developed when there
is fruit availability, aiming at the possibility of
supplying the deciencies of some of these nutrients
identied in other studies that were carried out in the
same region, which identied risk factors associated
with inadequate nutrition (SOUZA et al. 2012;
FARIA et al. 2014).
TABLE 1 – Physical characteristics of wild pineapple fruits (Ananas bracteatus (Lindl.), var. albus) found
in the rural area of Viçosa, Minas Gerais, Brazil (2013).
Characteristic Average ± Sd
Diameter (cm) 9.46 ± 0.65
Length (cm) 13.64 ± 0.69*
Fruit mass (g) 687.84 ± 90.16
Peel mass (g) 329.17 ± 68.44
Pulp mass 358.66 ± 63.82
Pulp yield (%) 52.36 ± 6.69
* Without the crown. Sd: standard deviation
TABLE 2 – Physic-chemical and centesimal composition of wild pineapple fruits (Ananas bracteatus
(Lindl.), var. albus) found in the rural area of Viçosa, Minas Gerais, Brazil (2013).
Characteristic Average ± Sd
Soluble solids (SS) (oBrix) 16.30 ± 0.02
Total acidity (TA) (g of citric acid 100g-1) 1.71 ± 0.23
SS/TA relation 9.53 ± 0.08
pH 3.50 ± 0.05
Moisture (g 100-1) 78.51 ± 1.19
Total dietary bre (g 100-1) 1.66 ± 0.03
Lipids (g 100-1) 1.41 ± 0.12
Proteins (g 100-1) 4.79 ± 1.79
Ash (g 100-1) 0.80 ± 0.13
Carbohydrates (g 100-1) 12.82 ± 0.98
Caloric density (Kcal 100-1) 83.15 ± 3.14
Sd: standard deviation. 3 repetitions pattern.

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WILD PINEAPPLE (Ananas bracteatus (LINDL.), VAR. albus) HARVESTED IN FOREST...
TABLE 3 – Vitamin C, carotenoid and Vitamin E concentration of wild pineapple fruits (Ananas bracteatus
(Lindl.), var. albus) found in the rural area of Viçosa, Minas Gerais, Brazil (2013).
Characteristic Average ± Sd
Vitamin C mg 100-1
AA 15.06±1.81
DHA 3.64±2.57
Total (18.70±2.99)
Carotenoid mg 100-1
α-carotene ud
β-carotene 0.24±0.08
β-cryptoxanthin ud
Lycopene ud
Total (0.24±0.08)
Vitamin E (µg 100-1)
α-tocopherol ud
α-tocotrienol 8.55±0.22
β-tocopherol 15.98±6.25
β-tocotrienol 3.52±1.69
γ-tocopherol ud
γ-tocotrienol ud
δ-tocopherol ud
δ-tocotrienol ud
Total (28.05±6.94)
Vitamin A (RAE µg 100g-1)
Total (20.00±6.67)
Values expressed in fresh weight standard of 5 repetitions; ud: undetected. Sd: standard deviation
TABLE 4 – Minerals concentration in wild pineapple fruits (Ananas bracteatus (Lindl.), var. albus) found
in the rural area of Viçosa, Minas Gerais, Brazil (2013).
Nutrient (in mg/100-1) Average ± Sd
P0.23 ± 0.05
K10.58 ± 0.65
Ca 1.91 ± 0.05
Mg 1.76 ± 0.05
Cu 0.48 ± 0.04
Fe 2.05 ± 0.32
Zn 0.98 ± 0.11
Mn 8.87 ± 1.54
Na 0.07 ± 0.01
Cr ud
Se ud
Mo 0.15 ± 0.03
Values expressed in fresh weight; average of 3 repetitions; ud: undetected.

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G. X. de PAULA FILHO et al.
TABLE 5 – Potential of nutrient contribution of the wild pineapple (Ananas bracteatus (Lindl.) var. albus)
found in the rural area of Viçosa, Minas Gerais, Brazil (2013) for adults (19 to 30 years old).
Nutrient Wild pineapple (1 portion = 84g)*
Concentration/portion %**
Carbohydrates 10.77 g 8.28
Proteins 4.03 g 7.20
Fibres 1.39 g 3.67
Vitamin A 16.8 g 1.87
Vitamin C 15.71 g 17.45
Vitamin E (α-tocopherol) -- --
P 0.19 g 0.03
K 8.89 g 0.19
Ca 1.60 g 0.16
Mg 1.48 g 0.37
Cu 0.40 g 44.8
Fe 1.72 g 21.5
Zn 0.82 g 7.48
Mn 0.74 g 32.2
Na 0.06 g 0.00
Cr ud ud
Se ud ud
Mo 0.13 g 280
* Based on the fruit portions that supply 70 Kcal (BRAZIL, 2008); ** % of calculated contribution based on the Recommended Dietary
Allowance for adults between 19 and 30 years old, (U.S INSTITUTE OF MEDICINE, 2011), ud: undetected.
FIGURE 1-Whole fruit (A1) and longitudinal section (A2) of the wild pineapple (Ananas bracteatus (Lindl.),
var. albus) found in the rural area of Viçosa, Minas Gerais, Brazil (A2).

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WILD PINEAPPLE (Ananas bracteatus (LINDL.), VAR. albus) HARVESTED IN FOREST...
CONCLUSIONS
Wild pineapple fruits are source of
carbohydrates, proteins and zinc; good source of
vitamin C; and excellent source of copper, iron,
manganese and molybdenum.
Wild pineapple fruits can be an alternative
to the nutritional supply of people living in the
countryside, especially in the city of Viçosa, MG,
Brazil, where they are found.
ACKNOWLEDGEMENTS
We thank to FUNARBE, FAPEMIG, CNPq
and CAPES for the scholarships and nancial
support to carry out this research.
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