Nutritional characterization of Vasconcellea quercifolia A.St-Hil.: potential for the development of functional food

Brazil houses 15 to 20% of all species of global terrestrial biodiversity, but is also responsible for the deforestation of large forest extensions, which might be causing the extinction of many species even before they are known to Science, thus hampering the discovery of new products or substances that might be beneficial for human kind (Brasil, 2000). On addition, agricultural activities generally exploit few species. This paradox brings forth the need for increasing research to broaden knowledge and lead to a better use of Brazilian biodiversity (Garcia, 1995). Additionally, human kind is heading towards reduced food diversity, with consequent cessation of certain foods that were once consumed, and adopting calorie-rich diets with low contents of essential micronutrients which are crucial to a good quality of life (Moratoya et al., 2013).


Introduction
Brazil houses 15 to 20% of all species of global terrestrial biodiversity, but is also responsible for the deforestation of large forest extensions, which might be causing the extinction of many species even before they are known to Science, thus hampering the discovery of new products or substances that might be beneficial for human kind (Brasil, 2000).On addition, agricultural activities generally exploit few species.This paradox brings forth the need for increasing research to broaden knowledge and lead to a better use of Brazilian biodiversity (Garcia, 1995).Additionally, human kind is heading towards reduced food diversity, with consequent cessation of certain foods that were once consumed, and adopting calorie-rich diets with low contents of essential micronutrients which are crucial to a good quality of life (Moratoya et al., 2013).
Vasconcellea quercifolia A.St-Hil. (Caricaceae), popularly known as papaya of the woods or jacaratiá, occurs from the south of Bahia to Rio Grande do Sul (RS) and is considered a Non-Conventional Food Plant (PANC) (Kinupp et al., 2011;Kinupp & Lorenzi, 2014), however, its nutritional characteristics are poorly known.On addition, it is included in the list of the Brazilian flora, considered part of its sociobiodiversity, with feeding value that justifies its marketing in natura or that of its derivatives (Brasil, 2016).
Grated medullary parenchyma was used instead of coconut in the preparation of sweets in the past.However, its popular use had been gradually forgotten (Kinupp et al., 2011;Kinupp & Lorenzi, 2014) and was recently resumed with the broad dissemination of PANC's (Kinupp & Lorenzi, 2014) which describe the potentialities of this species (Kinupp & Lorenzi, 2014;Caetano et al., 2008).On light of that, the aim of the present study was to investigate the physical, chemical, and nutritional properties of V. quercifolia to encourage its use in the development of new food products.

Physical and chemical analyses
Percentage of total proteins (TP) was estimated by determining the total nitrogen with the Kjeldahl method using Digestion Blocks (Marconi, Brazil) and a Kjeldahl Distiller (Tecnal, Brazil).On order to determine ash (Ash), samples were weighed on porcelain crucibles and incinerated in a muffle furnace (Marconi, Brazil), with temperature programmed to reach 550 °C.For humidity (H), fresh samples were placed in an oven at 105 °C and the difference in dry mass was calculated.Carbohydrates were calculated using the difference in all parameters = 100 − % (humidity + protein + fat + ash +fibre).Titratable acidity (TA) was determined by titration with 0.1 N NaDH solution and expressed as percentage.The pH values of the samples were determined using a digital pHmeter (Digimed, Brazil) through direct measurement.All parameters were determined in triplicates for each sample of green fruit, ripe fruit, and medullary parenchyma, following the methodology described by Onstituto Adolf Lutz (2008).TA and pH were not defined for stem samples due to their difficult homogenization.Total contents of dietary fibre and amino acids were determined at the laboratory of the Eurofins Group using the ADAC 991.43 method (Association of Dfficial Analytical Chemists, 1991) and high-efficiency chromatography (EU 152/2009(F), Standards EN OSD 13904, OSD 13903:2005), respectively (European Union, 2009;Onternational Drganization for Standardization, 2005a, b).The ascorbic acid in the fruit was determined by 2,6-dichloroindophenol following the methodology proposed by the Onstituto de Tecnologia de Alimentos (1990).
Carotenoids, β-carotene, and lycopene were determined following the methodology proposed by Rodriguez-Amaya & Kimura (2004), aliquots of fruit (2.5 g) were ground in a mixer with 10 mL of extracting solvent (acetone:hexane 4:6) under constant agitation and protected from light for 30 min.The supernatant was centrifuged at 10,000 rpm for 5 min.The reading was performed at wavelengths of 453, 505, 645, and 663.The results were expressed in mg of β-carotene and lycopene per 100 g of medullary parenchyma and green fruits.Semi-mature and ripe fruits from sample 1 were not analysed due to the lack of material.All data were submitted to an analysis of variance (ANDVA) and Tukey's test using the BioEstat 5.3 program.

Results
Fruits (green and ripe) differed from each other in all parameters (humidity, proteins, carbohydrates, and fibres) except for ashes.Green fruits had higher protein and fibre contents, and ripe fruits had higher ash and carbohydrate contents (Table 1).Dn the other hand, the medulla had higher ash content and humidity.There was no significant difference between the physical and chemical parameters of the samples of the three individuals, showing uniformity in the contents of each nutritional component in the species, regardless of the site collected.
Protein contents significantly differed (p < 0.01) among the three structures evaluated, with the lowest value in the medullary parenchyma.Dn the other hand, medullary parenchyma was the structure with the highest ash content, significantly differing only from green fruit samples (p < 0.05).Ripe fruits showed intermediate values and did not differ from green fruits.
Table 1.Mean values and standard deviation (SD) of total proteins (TP), ashes (Ash), humidity (H), titratable acidity (TA), pH, carbohydrates (CH), and fibres (F) of green fruits (GF), ripe fruits (RF), and medullary parenchyma (MV), considering three analytical replicates, in g per 100 g of each sample (1, 2, and 3).Medullary parenchyma and green fruits had the lowest humidity contents, significantly differing (p < 0.01) from ripe fruits, whereas the opposite occurred with carbohydrates, which were higher in ripe fruits, significantly differing (p < 0.01) from the other two structures evaluated.Fibre determination showed that fibres do not follow carbohydrate proportion.Fibre content recorded in green fruits (3.67 g for every 100 g) corresponded to 47% of total carbohydrates while the lowest value recorded for medullary parenchyma (2.17 g) corresponded to 30.43% of carbohydrates, with significant differences among the three parts of the studied plants (p < 0.01).Ripe fruits had the lowest proportion of fibres in relation to total carbohydrates (28%), showing reduced fibre content with fruit ripening.Titratable acidity, analysed only in fruits, had citric acid indices between 0.62% (green fruit) and 0.97% (ripe fruit).Dn the other hand, pH values were close to neutrality with a slight acidic flavour (5.20 and 5.46 for green and ripe fruits, respectively).
Aminogram analysis showed that green and ripe fruits and stem medulla of V. quercifolia had higher glutamic acid, aspartic acid, and lysine (essential amino acid) contents than other amino acids (Table 2).On a 100-gram sample, the highest concentrations of the three amino acids were observed in green fruits, followed by ripe fruits, and medullary parenchyma, which was similar to total protein values.
Ascorbic acid (vitamin C) content progressively increased from green to ripe fruits (Table 3).The same occurred with β-carotene and lycopene contents, with values three times higher in ripe fruits.For the three parameters evaluated (ascorbic acid, β-carotene, and lycopene), medullary parenchyma had much lower contents than fruits.

Discussion
The homogeneity of the parameters observed in each structure of different individuals was also observed by Kinupp & Barros (2008) who studied the same species.Additionally, the protein content observed in fruits by the same author was similar to that observed in the present study, indicating a pattern in protein content, which encourages its exploitation as it ensures the quality of the product to be produced.However, when considering the same parameters in different structures (green fruits, ripe fruits, and medullary parenchyma), there was variation in chemical composition between individuals.Green fruits had higher protein and fibre contents, ripe fruits had higher ash content, carbohydrate content, and titratable acidity, whereas medullary parenchyma had higher ash content.This indicates a second advantage to this species, as it allows for the plant to be exploited in several ways and for different purposes, including the development of new dietary products, which includes functional foods.
Medullary parenchyma, the most frequently used as food, especially as a replacement for coconut (Cocos nucifera L.) in the preparation of sweets, contains a much lower protein content than fruits and coconut, whose protein content is 0.83% (Santana, 2012).Their contents, however, are similar to those recorded for Amaranthus viridis L. (caruru) (0.6%) (Bredariol, 2015), also considered PANC by Kinupp & Lorenzi (2014).
As it is rich in proteins, V. quercifolia also constitutes an important source of amino acids.Among the ten amino acids with the highest contents found in V. quercifolia, three are essential amino acids.Df those, lysine was the third highest both in green and ripe fruits (0.26% and 0.21%, respectively).Lysine-rich foods comprise a healthy diet, as they reduce the recurrence, severity, and incidence time of lip infections caused by herpes simplex and promote higher calcium absorption and lower calcium excretion in diets supplemented with calcium, which suggests their potential to prevent and treat osteoporosis with a daily dose of 400-800 mg/day of lysine (Bruno, 2013).This daily amount indicated for people with osteoporosis, for instance, can be easily reached, considering that there is 0.262 g of lysine in 100 g of green fruit.
Green and ripe fruits of this species were rich in fibres, with a higher content than that recorded for papaya (1.0%) and 'Formosa' papaya (1.8%); this emphasizes its potential for developing new food products.Dn the other hand, it was similar to loquat (3.0 g), pear (Pyrus communis L.) (3.0 g), and pitanga (Eugenia uniflora L.) (3.2 g); the latter is considered PANC (Kinupp & Lorenzi, 2014).Despite the low fibre content observed in the medullary parenchyma, these values are similar to those recorded in other fruits (Annona cherimola Mill.(cherimóia), Annona squamosa L. (atemoia), Musa spp.(banana), Theobroma cacao L. (cocoa), and Mangifera indica (mango) (Universidade Estadual de Campinas, 2011).Cocos nucifera, for instance, had much higher contents than in the grated medullary parenchyma of the study species (10.15 g); however, the analysis was performed with freeze-dried pulp, whereas the analysis in the present study was performed with in natura medulla.
Regarding carbohydrates, the results for ripe fruits are in accordance with the values recorded for 'Papaya' papaya and 'Formosa' papaya, which are 10.4% and 11.6%, respectively.The same occurs with other fruits consumed worldwide, such as Mangifera indica L. (mango, 'Tommy Atkins cultivar') and Vitis vinifera L. (grapes, 'Rubi cultivar').Carbohydrate contents in stem and in green fruits, on the other hand, are lower and similar to those recorded in Cucumis melo L. (melon), Fragaria vesca L. (strawberry), and Ananas comosus L. Merril (pineapple) pulp (Universidade Estadual de Campinas, 2011).However, this carbohydrate content includes fibres, which favours its consumption by people who need to control sugar uptake.When considering fibre to total carbohydrate ratio, green fruits had a higher ratio (47%), which a sharp decrease in ripe fruits (28%).Medullary parenchyma, on the other hand, showed an intermediate percentage of fibres (30.43%).
The data recorded also show that green fruits and medullary parenchyma of Vasconcellea quercifolia A.St-Hil. (Caricaceae) promote a fibre-rich diet, with daily indication of 20 to 35 g/d (Pilch, 1987).Therefore, they might constitute functional foods and meet the expectation of researchers who have investigated new sources of fibres (Du et al., 2002), as they improve intestinal functioning and decrease absorption of carbohydrates and fats (Schneeman & Tietyen, 1994).As they control the absorption of some diet components, they reduce obesity and risks of cardiovascular diseases; especially stroke (Bazzano et al., 2003).
Aside from representing an important source of fibres, medullary parenchyma is also rich in mineral salts, corroborating the findings by Kinupp & Barros (2008) for this species.Ot also confirms that the medullary parenchyma of Vasconcellea quercifolia A.St-Hil. (Caricaceae) is richer in minerals than other foods, such as Cocos nucifera pulp, which, according to Santana (2012), has only 0.75 g of ashes when it is grated, and this indicates the advantage of replacing it with grated medullary parenchyma of V. quercifolia.Ripe fruits, and even green fruits, which had the lowest mineral salt content among the three structures evaluated in this study, are also rich in minerals and have twice the value recorded by Universidade Estadual de Campinas (2011) for both papaya varieties.
Values of pH were similar to those recorded for 'Papaya' papaya (5.71) (Fagundes & Yamanishi, 2003;Albertini et al., 2016), which was expected, but with lower acidity level compared to ripe papaya fruits of 'Formosa' cultivar (4.1) (Souza et al., 2005).Acidity values influence the decision as to how the fruit shall be used since acidity helps reduce the development of pathogenic micro-organisms (Hansen et al., 2008); low acidic fruits are more indicated for consumption in natura and those with higher acidity, for the food industry (Botrel, 1994).Therefore, V. quercifolia fruits might be indicated for consumption in natura and for the preparation of other foods.Titratable acidity (TA) was higher in ripe fruits, indicating a relationship between TA and ripening stage; according to Caldas et al. (2010) citric acid content decreases with fruit ripening.Moura et al. (2003) also observed reduced TA in ripe acerola fruits and indicated as potential cause the decrease in organic acids that occurs during ripening.
Ascorbic acid and carotenoids (β-carotene and lycopene) increase with decreased CA in ripe V. quercifolia fruits, as much as three times the amount recorded for green fruits, which in turn, was a little lower than that recorded for semi-mature fruits.These results corroborate the findings of Leong & Shui (2002), according to whom the content of these phytochemicals with antioxidant action changes according to the type of fruit, variety, ripening degree, and edaphic and climatic conditions of the cultivation site.Despite the sharp increase in ascorbic acid in ripe fruits, this species falls in the category Low (50 mg 100g -1 ) according to the classification by Ramful et al. (2011), whereas the papaya varieties ('Formosa' and 'Papaya') studied by Reis et al. (2015), who evaluated ascorbic acid content in 'Formosa' papaya fruits (70.09 mg 100g -1 ) and 'Papaya' papaya fruits (114.52 mg 100g -1 ), fall into category High (> 50 mg 100g -1 ).Dn the other hand, V. quercifolia fruits have more ascorbic acid than that recorded by Universidade Estadual de Campinas (2011) in other fruits such as avocado (8.7 mg 100g -1 ), watermelon (6.37 mg 100g -1 ), and pineapple (5.77 mg 100g -1 ), and with a similar amount to that found for Valencia orange (47.8 mg 100g -1 ) and tangerine (48.8 mg 100g -1 ).The opposite occurs with β-carotene and lycopene contents; both were higher in ripe fruits, more than three times the values recorded for carrot (23.8 mg 100g -1 ), for instance.
Lycopene contents recorded for green and semi-mature fruits were similar to those recorded for guava (53.00 µg g -1 ) and watermelon (36.00 µg g -1 ).Dn the other hand, ripe fruits had a higher value compared to the samples analyzed, reaching 134.8 µg g -1 , which is similar to that of tomato; 174 µg g -1 (Rodrigues- Amaya et al., 2008).B-carotene is the most important precursor of vitamin-A (retinol), which plays a role in several physiological processes, such as improved sight, growth, teeth formation, collagen formation, and is related to the functioning of the immune system (Rodrigues-Amaya et al., 2008;Queiroz et al., 2013).B-carotene content in RF was, on average, 238 µg g -1 ; this corresponds to 23,800 µg 100 g -1 .This value is much higher than that found by Reis et al. (2015) for the two most consumed papaya varieties worldwide ('Formosa' papaya and 'Papaya' papaya), which have mean b-carotene values of 41.11 µg g -1 and 42.84 µg g -1 , respectively.
Considering that the conversion rate from b-carotene to vitamin-A is 12:1 (Campos & Rosado, 2005), 23,800 µg of b-carotene contained in 100 grams of ripe fruit shall comprise 1,983 µg of vitamin-A or retinol (ER).This value is higher than that found in boiled carrot, which generates 1,326 µg of ER, and in raw pumpkin, which is 1,108 µg of ER (Universidade Estadual de Campinas, 2011).
On addition to the previously explained nutritional characteristics of V. quercifolia, its carotenoid content emphasizes its potential as food.Several carotenoids, especially β-carotene, work as sequesters of free radicals and prevent singlet oxygen from being formed (Podsedek, 2007).Among natural carotenoids, lycopene is one of the most powerful absorbents of singlet oxygen and have an antioxidant activity, capable of preventing DNA lesions and chromosomal changes (break and loss), having a chemopreventive activity in case of mutations (Scolastici et al., 2007).For these reasons, carotenoids are antioxidants that help delay aging and reduce risks of diseases such as cancer, cataract, and atherosclerosis (Damodaran et al. 2017).According to Kruger & Mann (2003), carotenoids are among the compounds that provide benefits to health and that comprise functional ingredients.
According to Resolution nº 19, of April 30, 1999, of the Agência Nacional de Vigilância Sanitária (Brasil, 1999), food with functional properties has metabolic or physiological activity, promoting growth, development, and maintenance of the organism.For Hasler (1998), these food items have potential to mitigate diseases, promote health, and reduce costs to the healthcare system.Thus, the high fibre content associated to high contents of proteins, amino acids (including essential amino acids), ashes, and carotenoids place V. quercifolia in the category of functional food.These food items, as part of a diet, provide basic nutrients and benefits to metabolic and physiological functioning of the organisms, thus contributing to physical and mental health, as well as preventing chronic degenerative diseases (Angelis, 2001).
The search for new food options with functional properties is of great importance.For Kinupp & Barros (2008) low income populations strive to keep a balanced diet regarding nutrients, and therefore, the discovery of species with easy access and cultivation, and with high nutritious value is quite important.Dther food options indicated by the same authors are ripe fruits of Ananas bracteatus (Lindl.)Schult.& Schult (pineapple), Psidium cattleianum (araça-rosa) and Vitex megapotamica (Spreng.)Moldenke (tarumã), or even the medullar parenchyma of Jacaratia spinosa (Aubl.)DC. (jacaratiá), all of which are native of Brazil and have high protein content.These species are considered non-conventional food species, very similar to our study species, and their nutritious content is still poorly known and exploited (Kinupp & Barros, 2008).These species, as is the case of V. quercifolia, might be exploited as food, for safety, nutrition, agricultural development, economic improvement, and also as rotation crops.V. quercifolia can also contribute with the global production of foods due to its adaptation to several environmental conditions, high resistance to diseases, pests and rapid growth (Sridhar & Seena, 2006) and to its potential to produce functional food.

Conclusions
V. quercifolia has high dietary potential.Ots fruits, either green or ripe, and its medullary parenchyma have high contents of ashes, proteins, carbohydrates, fibres, and carotenoids, sometimes similar to or higher than those recorded in several plants consumed worldwide.Therefore, edible structures of the plant might be used for the development of new foods that have functional properties, meeting the demand for new food items which are more nutritious, combined with the possibility of contributing with good health.

Table 2 .
Mean amino acid values (g in 100 g of sample) present in green fruits, ripe fruits, and medullary parenchyma of Vasconcellea quercifolia.
< = lower than the quantification limit (DL) of the technique; *essential amino acids.