Chemical composition and antioxidant capacity of a fibre-rich flour prepared from Passiflora edulis peel
Lima, G.C., Vuolo, M. M., Batista, A.G., Maróstica, M.R.
Artigo preparado para submissão ao periódico “Food Science and Thecnology”
Abstract
In this work, chemical composition and antioxidant capacity of flour prepared from Passiflora edulis peel (PEPF) were evaluated. PEPF is a source of dietary fiber (DF) (60.08% ± 0.03) soluble (20.13% ± 0.9) and insoluble (39.96% ± 0.93), including pectin (17.27%). PEPF contains calcium, iron, magnesium, manganese and zinc. Total carotenoids (1.26 ± 0.12 mg 100g-1), tannin (308.21 ± 1.95 mg 100g-1) and phytic acid (37.0 ±0.04 mg 100g-1) content were also determined. A higher total phenolic content was extracted with methanol:acetone (9.19 ± 0.33 mg GAE g-1) than hydroalcoholic (3.26 ± 0.12 mg GAE g-1) extract. The antioxidant capacity was evaluated in both extracts through different tests. ORAC test showed higher antioxidant activity (99.75 ± 5.58 μM TE g-1
) in the hydroalcoholic extract when compared with DPPH (11.22 ± 0.184 μM TE g-1
) and FRAP (47.42 ± 0.529 μM TE g-1) tests. Value for DPPH test was higher (139.00 ± 15.93 μM TE g-1) in methanolic extract when compared with other tests. Hydrogen cyanide and heavy metals were not detected in PEPF. This work contributes for more information about this byproduct, which could be added in food as a source of health bioactive compounds, especially dietary fibers, such as pectin, and phenolic compounds.
Introduction
Yellow passion fruit (Passiflora edulis var. Flavicarpa) is native from Brazil, and one of the most popular and well known tropical fruits suited for processing throughout the world (Hernández-Santos et al., 2015). These fruits are round in shape, bigger than the other Passifloracea fruits, with a diameter ranging from 8 to 10 cm. Their many seeds are surrounded by a gelatinous yellow pulp that has an intense aroma and sweet-acid taste (López-Vargas et al., 2013). Brazil is the largest passion fruit producer in the world, having produced 838 thousand tons in 2013 (IBGE, 2013).
Although the consumption of the fresh fruit has increased, the juice industry is still the major consumer of the passion fruit (nearly 40%) (Silva, 2014), especially due to its acid taste and higher yield, resistance to pests and high productivity per hectare (Deng et al., 2010; Zeraik et al., 2010). Byproducts (peel and seeds) produced during this process correspond to approximately 65% of the fruit weight. The use of these products in food has been studied by many researchers that, besides intending to decrease their waste in the environment (Zeraik et al., 2010), evaluate their conversion into value-added food ingredients, due their bioactive compounds, such as dietary fiber (DF) and polyphenols (Martinés et al., 2012).
Extracts rich in dietary fiber and phytochemicals obtained from plants could be incorporated to food as a functional ingredient due their related-benefits (Viuda-Martos et. al, 2010). The consumption of food rich in DF and phytochemicals have been related to the prevention of type 2 diabetes, glucose intolerance (Nugent, 2005), obesity (Slavin, 2005), cardiovascular diseases and cancer (Viuda-Martos et. al, 2010). In addition, phytochemicals have a high antioxidant capacity, and this propriety is also inversely associated to obesity and other chronic diseases (Terra et al., 2009).
That said the aim of this research was to report some characteristics of fiber-rich flour prepared from Passiflora edulis peel that can influence in its suitability as a potential ingredient for functional foods.
Material and methods
Passiflora edulis peel flour (PEPF)
A commercial dried and milled Passion Fruit peel flour produced by M.W.A. Com. De Produtos Alimentícios Ltda (São José do Rio Preto – SP – Brazil) was obtained from a local market in Campinas-SP, Brazil.
Analytical reagents
Enzymes α-amylase, protease, amyloglucosidase and pectinase were purchased from Sigma-Aldrich (St. Louis, MO, USA). Galacturonic acid used for pectin determination was purchased from Sigma-Aldrich (St. Louis, MO, USA). Glass wolle used in fiber analysis was obtained from Merck (Damstadt, Germany). Standard solutions of cadmium, lead, manganese, potassium, selenium, sodium, and zinc were obtained from Ultrasonic Scientific (North Kingstown, USA). Calcium, copper, iron and magnesium standard solutions were obtained from Specsol (Jundiaí, Brazil). For antioxidants analyses β-nicotinamide adenine dinucleotide 2’-phosphate reduced tetrasodium salt hydrate (NADPH), (±) -6-hydroxy-2,5,7,8-tetramethylchromane-2- carboxylic acid (TROLOX); 2,2′-azobis (2-methylpropionamidine) dihydrochloride (APPH), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), FRAP reagent, catechin and gallic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Composition of PEPF Proximate composition
The PEPF proximate composition was determined evaluating protein (Kjeldahl method - AOAC, 2000), total lipids (Bligh & Dyer, 1959), ash (met 942.05) and moisture (met 934.01) (AOAC, 2000). DF (soluble and insoluble) was determined through enzymatic-gravimetric method described by Prosky et al. (1992). The crude carbohydrate content was calculated using the percentile difference from all the other constituents.
Total and soluble Pectin
Sugar were extracted from the PEPF using 95% ethanol and the residue was washed with 75% ethanol for two times. Total pectin was extracted using Versene solution, and 1.0 N NaOH solution until pH 11.5 for hydrolysis, followed by pH adjustment (5-5.5) and reaction with pectinase (Pectinase from Aspergillus niger, 1 U/mg; Sigma-Aldrich). The sugar-free PEPF obtained using the ethanol extraction was used for soluble pectin determination after agitation and solubilization in water. After the carbazole reaction method (Bitter and Muir, 1962), total and soluble pectin were measured at 530 nm using a microplate reader Synergy HT, Biotek (Winooski, USA); with Gen5™2.0 data analysis software. Galacturonic acid (Sigma-Aldrich) was used as standard.
Minerals determination
PEPF sample was submitted to an acid digestion using a microwave oven. The digested solution was used for cadmium, calcium, lead, copper, iron, manganese, magnesium, potassium, selenium, sodium and zinc determination. The analysis was
performed using an Inductively Coupled Plasma Optical Emission Spectrometer (Perkin Elmer- Optima 8300). The analytical curves were constructed using single standard solutions of the minerals (1000 µg mL-1).
Antinutritional factors
Phytic acid was determined through colorimetric method according to Latta and Eskin (1980). Tannin (met 952.03) and Hydrogen Cyanide (HCN) (met 915.03) were analysed using AOAC (2000) official methods.
Total Carotenoids content
According method described by Higby (1962), 10g of PEPF was added to 100mL of acetone and incubated for 1 h in a shaker at room temperature. The solution was filtered and 100mL of acetone was added to the residue. This process was performed three times and the combined extracted was evaporated and filled up to 25 mL. In an Erlenmeyer flask, the samples were mixed with isopropyl alcohol and hexane, and this solution was washed 3 times. The hexane layer was drained through a filter containing sodium sulfate and 5mL acetone was added. The volume was completed with hexane up to 25 mL. The absorbance was read at 450 nm in a spectrophotometer (Coleman 33 D) and total carotenoids content was calculated as follows:
Carotenoids = (mg 100g– 1) = (A. 100) /(250. L. W)
Where C= concentration, A= absorbance, L= wells pathlength (cm), W= weight of sample per mL of the final solution measured.
PEPF (1g) was weighed in a plastic tube and 15 mL of 60% ethanol were added. The extraction was done at 70 ° C for 1 h, stirring every 15 min. The samples were filtered and the filtrate was transferred to a volumetric flask (25 mL). To the residue was added an additional volume of 10 mL of the alcohol solution, and another extraction process was performed as above. The filtrates from the two extractions were homogenized and the final volume (25 mL) of the flask was completed with distilled water (Spagolla et al., 2009). The extract was stored in amber flasks at 4 ° C until analysis. This extract was used for the analysis of total phenolic and antioxidant potential.
Methanol: Acetone extract
The mathanolic extract was based in method described by LARRAURI et al. (1997). Briefely, PEPF (1g) was weighed in a plastic tube and 10 mL of 50% methanol were added. The extraction was done at 25 ° C for 1 h. The sample was centrifugated for 20 minutes at 4000 rpm. The supernatant was transferred to a volumetric flask (25 mL). To the residue was added an additional volume of 10 mL of 70% acetone solution, and another extraction process was performed as above. The filtrates from the two extractions were homogenized and the final volume (25 mL) of the flask was completed with distilled water. The extract was stored in amber flasks at 4 ° C until analysis. This extract was used for the analysis of total phenolic and antioxidant potential.
Total phenolic compounds content
The total phenolic compounds content of the PEPF hydroalcoholic and methanol: acetone extracts was determined according to Folin–Ciocalteu's method (Swain & Hillis, 1959), with modifications. In a vial, 50 μL of extract, 800 μL distilled water and 25 μL (0.25 N) of Folin–Ciocalteu's reagent were mixed and incubated at room
temperature for 3 min. Then, 100 μL of Na2CO3 (1 N) was added and further incubated
for 2 h at room temperature. The absorbance was read at 725 nm in a microplate reader Synergy HT, Biotek (Winooski, USA); using Gen5™2.0 data analysis software spectrophotometer. Gallic acid was used as standard, and the results were expressed in terms of gallic acid equivalent (GAE g−1).
Determination of antioxidant potential in Passiflora edulis peel flour extracts
For determination of antioxidant potential in Passiflora edulis peel hydroalcoholic and methanol: acetone extracts, the readings of absorbance and fluorescence were performed in a microplate reader Synergy HT, Biotek (Winooski, USA); with Gen5™ 2.0 data analysis software spectrophotometer.
DPPH assay
The free radical scavenging activity of the extracts was determined based on the DPPH (2,2-diphenyl-1-picrylhydrazyl) method (Roesler et. al, 2006; Rufino et al., 2010). 33 μL of the PEPF extract was added in a 1.3 mL DPPH solution diluted in methanol (0.024 mg/mL). This mixture was shaken in the dark for 30 min, and the absorbance was measured at 515 nm. A trolox calibration curve was also made in order to express the results as trolox equivalents (µM TE g -1 of sample).
Ferric Reducing Antioxidant Power (FRAP) assay
The ferric reducing ability of Passion fruit peel extracts was determined by FRAP method (Benzie & Strain, 1996), with adaptations. In the dark, FRAP reagent
was made using 300 mmol L−1 acetate buffer (pH 3.6), 10 mmol TPTZ (2,4,6-tris(2- pyridyl)-s-triazine) in a 40 mmol L−1 HCl and 20 mmol L−1 FeCl3 solution. Sample or
standard solutions, ultrapure water and FRAP reagent were mixed and kept in a water bath for 30 min at 37 °C. After cooling to room temperature, samples and standard were read at 595 nm. The Trolox standard curve was made with concentrations ranging from 10 to 800 μmol Trolox equivalent (TE) mL-1. Results were expressed as μM TE g−1
.
Hydrophilic Oxygen Radical Absorbance Capacity (ORAC) assay
In the dark, 20 μL of sample (prepared as described above), 120 μL of fluorescein in phosphate buffer — PB (pH 7.4), and 60 μL of AAPH (2,2′-azobis(2- methylpropionamidine) dihydrochloride) were added to black microplates. The microplate reader was adjusted as described elsewhere (fluorescent filters, excitation wavelength, 485 nm; emission wavelength, 520 nm) (Prior et al., 2003). ORAC values were expressed as μM TE g-1
extract by using the standard curves (2.5–50.0 μM TE L-1) (Prior et al., 2003).
Results
Proximate composition and other compounds in PEPF
The proximate composition of PEPF is presented in table 1. Moisture content of the PEPF was under 8%, and the analysis showed that its protein and lipids content are higher than passion fruit pulp (TACO, 2011). PEPF has a large amount insoluble (IDF) and soluble (SDF) dietary fiber, including pectin (Table 1). The ratio of IDF/SDF in PEPF is 2:1.
Regarding antinutritional factors, high Tannin and Phytic acid concentrations were found in PEPF, while Hydrogen Cyanide was not detected in the sample, as can be seen in Table 1.
Minerals determination
The concentrations for different elements present in PEPF are listed in Table 2. Metals that could be potentially toxic, such as cadmium, selenium and lead, were found to be below the method limit of detection in the PEPF sample. The other inorganic minerals investigated –calcium, iron, magnesium, manganese and zinc – were found in PEPF sample and their values were summarized in table 2.
Content (%) Moisture 7.42 ± 0.36 Ash 6.00 ± 0.23 Lipids 3.39 ± 0.26 Protein 8.87 ± 0.14 Carbohydrate† 14.24 ± 0.42 Total fiber 60.08 ± 0.03 -Insoluble fiber 39.96 ± 0.93 -Soluble fiber 20.13 ± 0.90 Soluble pectin 4.85 ± 0.13 Total pectin 17.27 ± 0.22 Other compounds Phytic acid (mg/100g) 37.0 ±0.04 Hydrogen Cyanid (mg/Kg) ND < 0.30 Tannin (mg/100g) 308.21 ± 1.95
* Dried and milled Passion Fruit peel produced by M.W.A. Com. De Produtos Alimentícios Ltda (São José do Rio Preto – SP – Brazil). Data are expressed as means ± standard deviation. All assays are carried out on triplicate; † Calculated by difference. excludes the dietary fiber fraction; ND= Not determinated
Table 2 Minerals determination in Passion fruit peel flour and their Recommended
Dietary Allowances (RDAs) *
Mineral PEPF (mg/100g) RDA (mg/day)†
%RDA in 30g portion of PEPF Female Male Cadmium < DL - - - Lead < DL - - - Copper < DL 0.9 - - Calcium 266 (± 0.3) 1000 8.0 8.0
Iron 20.0 (± 2.0) 18 (female); 8 (male) 33.3 75.0 Magnesium 111 (± 0.5) 310(female); 400 (male) 10.7 8.3
Sodium 164 (± 0.6) 1500 3.3 3.3
Potassium 2.200 (± 20) 4700 14.0 14.0
Zinc 1.48 (± 0.02) 8 (female); 11 (male) 5.6 4.0 Manganese 1.81 (± 0.05) 2.3 (female); 1.8 (male) 23.6 30.2
Selenium < DL 55 - -
PEPF, Passiflora edulis peel flour; RDA, Recommended Dietary Allowances,
LOD,limit of detection. * The results are expressed as means and ± standard deviation (n=3).
Total phenolic compounds, total carotenoids and antioxidant potential in Passiflora edulis peel flour extracts
Total carotenoids, total phenolics content, DPPH assay, FRAP assay and ORAC assays in the different extracts of PEPF are shown in Table 3.
Table 3 Determination of total carotenoids, total phenolic content and antioxidant activity in
different extracts of Passiflora edulis peel flour
Methanolic extract Hydroalcoholic extract
Total phenolic compounds 9.19 ± 0.33 μM GAE g−1 a 3.26 ± 0.12 mg GAE g−1 b DPPH anti-radical activity 139.00 ± 15.93 μM TE g−1 a 11.22 ± 0.184 μM TE g−1 b ORAC anti-radical activity 89.03 ± 10.41 μM TE g−1 ns 99.75 ± 5.58 μM TE g−1 ns FRAP anti-radical activity 33.52 ± 2.32 μM TE g−1 b 47.42 ± 0.529 μM TE g−1a
Acetone extract
Total carotenoids 1.26 ± 0.12 mg carotenoids 100 g-1
GAE, gallic acid equivalents; TE, Trolox equivalents; ns= not significative. Data are mean ± standard deviation (n=3). a, b
Mean values within a line with unlike superscript letters were significantly different. Data were analyzed using ANOVA followed by Student’s t-test (P < 0.05).
Discussion
Proximate composition and Dietary fiber in PEPF
The values found for proximate composition of PEPF were similar to the results reported by Cazarin et. al (2014). However, protein and fat contents (table 1) in PEPF sample of this study were higher than previous findings on PEPF from Mexico (4.62 g/100g and 0.64g/100g, respectively) (Hérnandes-Santos et al., 2015). Data also show that PEPF contains higher levels of protein than pulp (2.0g/100g) (TACO, 2011) or other byproducts from Passiflora edulis flavicarpa, such as albedo (0.35g/100g) and a pulp and seed mixture (1.49g/100g) (López-Vargas et. al, 2013). Regarding the determination of ash and moisture, previous studies using passion fruit peel showed similar results (Cazarin et. al, 2014; Hérnandes-Santos et al., 2015).
As expected, PEFP has a large amount of dietary fiber (Table 1), and could be source both insoluble and soluble fiber, especially pectin. The results for total dietary fibers found in this research are within the wide range of values (57.93 - 71.79%) reported in different studies using passion fruit peel (Hérnandez-Santos et al., 2015; Cazarin et al., 2014; López-Vargas et al., 2013). The IDF/SDF ratio in PEPF was 2:1, what is considered suitable to be used as a food ingredient, being an important factor related to structural and also sensorial properties (Jaime et.al, 2002). The IDF/SDF ratio close to 2:1 was also found in other studies with passion fruit peel (Cazarin et al., 2014; López-Vargas et al., 2013). Other byproducts have shown higher values for IDF/SDF (4.5:1- 12.9:1), such as those from pineapple, guava (Martínez et al., 2012), jaboticaba (Leite-Legatti et. al, 2012), grapefruit, lemon, orange and apple (Figuerola et. al, 2005). The lower IDF/SDF ratio in PEPF compared to other byproducts is due the high amount of soluble fiber in PEPF.
Recommended Dietary Reference Intakes (DRIs, Institute of Medicine, 1997) for dietary fibers are 25g for women and 38g for men in adulthood. The incorporation of passion fruit flour in the daily diet could be one way for increasing fiber intake and achieve these recommendations. In addition, several health benefits are attributed to a regular DF consumption, including improvement of glucose control, prevention of fatty liver (Brockman et. al., 2012), cardiovascular disease and some types of cancer, especially colon cancer (Viuda-Martos et. al, 2010).
Some of the effects of DF on health could be related to their fermentation capacity by colonic microbiota. Colonic fermentation of DF and other indigestible results in the formation of colonic metabolites, mainly short-chain fatty acids –acetate, propionate and butyrate (Vinolo et. al, 2011). In addition, they act as fuel for intestinal epithelial cells, have systemic effects, such as protection against diet-induced obesity and insulin resistance (Lin et. al, 2012), stimulate the release of anorectic and antidiabetic gut peptides (Tolhurst et. al, 2012) and regulate the inflammatory process (Vinolo et.al, 2011). In fact, the hability of passion fruit peel on increasing butyrate and acetate concentration in cecal content of Wistar rats has been reported recently (Silva et al, 2014).
In this study, total pectin in PEPF percentage was 17.27 % (Table 1). Similar value (19.1%) was reported by Yapo & Koffi (2008). Pectin intake has been related to cholesterol-lowering properties (Brouns et al., 2012). Ramos et al. (2007) evaluated the effect of PEPF on cholesterol levels in women, aged 30-60 years, presenting hypercholesterolemia. Reduction on total cholesterol and LDL-cholesterol levels was observed after daily doses of 30 g of PEPF for 60 days. Silva et. al (2011) evaluated the effects of the pectin isolated from P. edulis fruit peels on alloxan-induced diabetes in model rats. The findings revealed that pectin decreased blood glucose and triglyceride
levels in animals. Furthermore, pectin showed anti-inflammatory properties in the same study.
Other compounds of interest
Tannins and phytic acid were determined in PEPF and the results are presented in table 1. Tannins and phytate are present in many plant foods, and can lower nutritional of food, decreasing the digestibility or bioavailability of nutrients, especially minerals. On the other hand, tannin and phytate are also recognized by their functional effects (Chung et al, 1998; Campos-Veja et. al, 2010).
Studies have shown beneficial effects of tannins on health, such as antimutagenic, anticarcinogenic and antimicrobial activity (Chung et al, 1998). However, chromatography analysis is necessary to identify the category of the tannins presented in PEPF, since the beneficial effects are category-dependent (Chung et al, 1998). High amount of tannin results in astringent taste in food and decreased palatability, which coulb be a limiting factor for passion fruit flour addition in other foods.
Studies have also indicated antioxidant, anticarcinogenic and antidiabetic effects attributed to phytic acid. In addition, a recent study proved that phytic acid up-regulates cecal organic acids, especially butyrate and decreases serum levels of proinflammatory cytokines attributed in rats fed on high-fat (HF) diet. (Okazaki & Katayama, 2014).
As members of the Passifloraceae have long been recognized as cyanogenic (Spencer, 1983), Hydrogen Cyanide was evaluated in the PEPF, but it was not detected (table 1). Hydrogen cyanide in plants is generally released from cyanogenic glycosides, glycosidic derivatives of R- hydroxynitriles (Chassagne et. al, 1996). The investigation of these compounds is important, since they could be toxic depending of their levels
(Spencer, 1983). The cyanide content decreases during maturation of the fruit. Therefore, the practice of harvesting of the fruits in adequate ripeness could reduce the amount of cyanogen to safe consumption. (Spencer, 1983).
Mineral analysis
The determination of minerals and trace elements in foodstuffs is an important part of nutritional and toxicological analysis. Minerals are essential in the diet of humans due their several roles in human biochemistry and physiology. Many of them are co-factors for enzymes in different metabolic process (Meyer, 1997). PEPF can be considered a good source of calcium, iron, magnesium, manganese, potassium and zinc, when compared to RDA (Recommended Dietary Allowance) for adults (19-50 years old) (Food and Nutrition Board, Institute of Medicine, 1997). The values of minerals content in 100 g of PEPF are summarized in table 2. The percentage of each mineral was calculated in a portion of 30 g of Passion fruit according their daily recommendation (table 2). Considering the values of %RDA, it was evaluated that PEPF is a great source of these inorganic compounds, especially Iron, Manganese, Potassium and Magnesium, since their values reach over 10% of RDA. Iron amount is more expressive among minerals evaluated in PEPF, considering its % RDA (75% for male and 33% for female). Thus, PEPF could be used as a complement in the feeding, contributing to improve the nutritional profile of diet. In addition, potentially toxic elements were not detected in samples (table 2), indicating that PEPF is safe for consumption regarding this parameter.
Total phenolic and carotenoids content and antioxidant activity in the PEPF
Phenolic compounds are considered natural antioxidants. Their antioxidant action resides mainly in their ability to chelate metal ions, neutralize free radicals by
donating hydrogen ions, inhibiting lipid peroxidation (Carocho & Ferreira, 2013). Carotenoids are also antioxidants compounds scavenging singlet molecular oxygen and peroxyl radicals. Further, they can disable molecules involved in the generation of radicals and singlet oxygen (Ramel et al., 2012). Carotenoids content in PEPF (1.26 ± 0.12 mg carotenoids/100g) was 20-fold higher than the value found in P edulis pulp (0.215 mg carotenoids /g) (Oliveira et al, 2004). Nevertheless, other tropical fruits peel, such as mango (436.0 ± 0.22 µg carotenoids/g) (Ajila et. al, 2007) and orange (3.5 mg carotenoids/100g) (Zafiris et. al, 1992) revealed higher amounts of carotenoids than PEPF in previous studies.
The solvents applied to the extraction affected significantly (P < 0.001) the phenolic compounds content as well as antioxidant activity in PEPF. Methanol:acetone extract was found to be more efficient in the extraction of phenolic compounds compared to hydroalcoholic extract. The extraction of the phenolic compounds depends of their solubility in the applied solvent. Methanol has been generally found to be more efficient in extraction of lower molecular weight polyphenols (Dai &Mumper, 2010).
Regarding the antioxidant activity, there was no difference between the extracts for the ORAC values. However, the compounds extracted by the methanol:acetone solvent have more antioxidant activity when measured by DPPH methods than those