Chemical, physico-chemical, technological, antibacterial and antioxidant properties of dietary fiber powder obtained from yellow passion fruit (Passiflora edulis var. flavicarpa) co-products

Food Research International 51 (2013) 756–763

Contents lists available at SciVerse ScienceDirect

Food Research International journal homepage: www.elsevier.com/locate/foodres

Chemical, physico-chemical, technological, antibacterial and antioxidant properties of dietary fiber powder obtained from yellow passion fruit (Passiflora edulis var. flavicarpa) co-products Jairo H. López-Vargas a, Juana Fernández-López b, José A. Pérez-Álvarez b, Manuel Viuda-Martos b,⁎ a

Instituto de Ciencia y Tecnología de Alimentos ICTA, Universidad Nacional de Colombia, Sede Bogotá 3465000 ext. 19225, Bogotá, Colombia Industrialización de Productos de Origen Animal (IPOA) Research Group (UMH-1 and REVIV-Generalitat Valenciana), AgroFood Technology Department, Escuela Politécnica Superior de Orihuela, Miguel Hernández University, Crta. Beniel km. 3,2. E-03312 Orihuela, Alicante, Spain

b

a r t i c l e

i n f o

Article history: Received 18 December 2012 Accepted 23 January 2013 Keywords: Passion fruits Co-products Fiber Antioxidant Antibacterial

a b s t r a c t The aims of this study were to ascertain (i) the chemical composition (ii) the physico-chemical properties, (iii) the technological properties, (iv) the total phenol and flavonoid content, (v) the antioxidant properties and (vi) the antibacterial properties of dietary fiber powder obtained from yellow passion fruit (Passiflora edulis var. flavicarpa) co-products (pulp and seeds or albedo) to determine its suitability for use as a natural food ingredient. The proximate composition and the total, insoluble and soluble fiber content were determined. The water holding (WHC), oil holding (OHC) and swelling (SWC) capacities were also determined, as well as the total phenolic and total flavonoid content. For the antioxidant activity, three different test systems were used (DPPH, FIC and FRAP) while the antibacterial activity was determined using the microdilution method. The dietary fiber content of passion fruit seeds and pulp (PFSP) was 53.51 g/100 g, while the fiber content in passion fruit albedo (PFA) was 71.79 g/100 g. Both types of fiber showed good technological properties. Phenol and flavonoid recovery was dependent on the fiber type and the solvent system used, DMSO being a more efficient solvent in this respect than methanol or water. All the samples analyzed showed good antioxidant and antibacterial capacities. © 2013 Elsevier Ltd. All rights reserved.

1. Introduction Colombia is one of the world's main producers and exporters of fresh tropical fruit, due, among other reasons, to the great variety of fruit species available, which are new to international markets and which exhibit excellent sensory, nutritional and/or nutraceutical qualities (Jiménez et al., 2011). One of these fruits is Passiflora edulis a tropical fruit belonging to the family of Passifloraceae. There are several types of this fruit: purple passion fruit (Passiflora edulis Sims), granadilla (Passiflora ligularis), gulupa (Passiflora edulis Sims. fo edulis) and yellow passion fruit (Passiflora edulis var. flavicarpa Degenerer) (Chen & Lu, 1994). The P. edulis var. flavicarpa fruits are round in shape, with a diameter of between 8 and 10 cm, and green peel at maturity. They contain many seeds (as do the other Passifloraceae species) surrounded by a gelatinous yellow pulp that has an intense aroma and sweet-acid taste. In Colombia, the area dedicated to passion fruit cultivation in 2011 was 5321 ha, with a production of 79,458 t, an average yield of 14.9 t/ha (Agronet, 2012). Most of the fruit is intended for the ⁎ Corresponding author at: AgroFood Technology Department, Orihuela 03312, Spain. Tel.: +34 966749737; fax: +34 966749677. E-mail address: [email protected] (M. Viuda-Martos). 0963-9969/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodres.2013.01.055

production of juices and soft drinks. However, the juice industry alone generates several thousand tons of seeds, pulp and peels as agricultural co-products. Depending on the availability of an adequate technology, these co-products can be converted into commercial products either as raw materials for secondary processes (intermediate foods ingredients), as operating supplies or as ingredients for new products (SánchezZapata et al., 2011). The co-products of tropical fruits contain high levels of bioactive compounds (vitamins, minerals, polyphenolic antioxidants and dietary fiber), which may have positive health effects and contribute to preventing some diseases such as, cancer, cardiovascular diseases and diabetes, among others (Ayala-Zavala et al., 2011; Viuda-Martos et al., 2010). Dietary fiber from different agro-industrial co-products has been added to a variety of foods, including meat products (ViudaMartos, Ruiz-Navajas, Fernández-López, & Pérez-Álvarez, 2010), breakfast cereals and bakery products (Vergara-Valencia et al., 2007) and dairy products (Sendra et al., 2008). There are some studies where the antioxidant and antibacterial activity of yellow passion fruit were determined. Similarly, there are some research which identifies some properties of the co-products of passion fruit, but only the pulp and seeds, or just taking the peel or the pulp, seeds and peel as a whole. This is the first study which determines the chemical, physico-chemical, technological, antibacterial and

J.H. López-Vargas et al. / Food Research International 51 (2013) 756–763

antioxidant properties of the seeds and the pulp of yellow passion fruit co-product on the one hand and the albedo present in the peel on the other side. Thus, the aims of this study were to analyze (i) the chemical composition (ii) the physico-chemical properties, (iii) the technological properties, (iv) the total phenol and flavonoid content, (v) the antioxidant properties and (vi) the antibacterial properties of dietary fiber powder obtained from the seeds and the pulp of yellow passion fruit (Passiflora edulis var. flavicarpa) co-products on the one hand and the albedo present in the peel of this fruits, on the other side, to determine whether it could be used as a natural food ingredient. 2. Material and methods 2.1. Plant material Five kilograms of mature yellow Passiflora edulis var. flavicarpa, with no visible external cuts or spoilage, were purchased from a local market. The identification of the plant material was made by Prof Dr. Francisca Hernandez Garcia, Plant Production Department of Miguel Hernandez University (Spain). To obtain the passion fruit seeds and pulp fiber (PFSP) the fruits were cut in half and squeezed in a vitapress 600 juicer (Moulinex, Barcelona, Spain), thus obtaining passion fruit juice and a co-product formed of pulp and seeds. To obtain passion fruit albedo (PFA) fiber, the squeezed fruit halves were peeled manually to remove the external peel, thus providing the albedo co-product. Both co-products were triturated for 60 s in a vertical cutter (Tecator 1094 Homogeneizer, Tekator, Hoganas, Sweden) to obtain uniformly sized pieces and to increase the contact time during washing (1 L of water per kg of product for PFSP and 5 L of water per kg of product for PFA). The mixture was stirred constantly and the water temperature was kept at 45 º C during the 8 min that the washing process lasted. The whole product was pressed to drain liquid waste and then dried at 60 °C for 24 h to improve the product shelf-life without the addition of chemical preservative. A grinder and sieves were used to obtain a powder particle size of less than 0.417 mm. These processes were carried out in triplicate. 2.2. Chemical analysis The proximate composition, including, protein, lipid and ash contents, of the samples was determined using the appropriate AOAC (2000). Protein content was determined by estimating the nitrogen content using the Kjedahl method (AOAC Method 920.152). Ash content was determined by incineration at 525 °C (AOAC Method 940.26) while fat was determined by the Soxhlet method (AOAC Method 963.15). Total dietary fiber (TDF) (g TDF/100 g d.m.) and insoluble dietary fiber (IDF) were determined following 991.43 AOAC methods (AOAC, 1997). Soluble dietary fiber (SDF) was calculated by subtracting the IDF proportion from the TDF. Each assay was carried out in triplicate. 2.3. Physic-chemical analysis The pH was measured in a suspension resulting from blending 10 g sample with 10 mL of deionized water for 2 min, using a pH meter (model pH/Ion 510, Eutech Instruments Pte Ltd., Singapore). The water activity (aw) was determined in a TH-500 Sprint Novasina Thermo-constanter (Pfäffikon, Switzerland) at 25 °C. The color was studied in the CIELAB color space using a Minolta CM-2600d (Minolta Camera Co., Osaka, Japan), with D65 as illuminant and an observer angle of 10°. Low reflectance glass (Minolta CR-A51/1829-752) was placed between the samples and the equipment. The CIELAB coordinates studied were lightness (L*), red/green (a*), yellow/blue (b*) and the psychophysical parameters, Chroma (C*) and hue angle (h*).

757

2.4. Technological properties The water-holding capacity (WHC), oil holding capacity (OHC) and swelling capacity (SWC) were determined according to Robertson et al. (2000) with some modifications. Twenty-five milliliters of buffer phosphate (1 M, pH 6.3) or commercial olive oil were added to 250 mg of dry sample, stirred and left at room temperature for 1 h. After centrifugation (3000 g; 5 min), the residue was weighed. For SWC determination, 0.1 g of sample with varying ranges of particle size was hydrated in 10 mL of distilled water in a calibrated cylinder (15 cm diameter) at room temperature. After equilibration for 18 h, the bed volume was recorded. The WHC was expressed as g of water held per g of sample; the OHC was expressed as g of oil held per g of sample, while the SWC was expressed as mL/g of sample. Each assay was carried out in triplicate. 2.5. Total phenol content The total phenol content (TPC) was determined using the Folin– Ciocalteu's reagent (Singleton & Rossi, 1965). The results were expressed as mg gallic acid equivalents (GAE)/g sample, as mean of three replicates. 2.6. Total flavonoid content For the total flavonoid content (TFC), the method based on Blasa et al. (2006), with some modifications, was used. One milliliter of PFA or PFSP (10 g/L) was mixed with 0.3 mL NaNO2 (5%), and 0.3 mL AlCl3 (10%) were added after 5 min. The samples were mixed in a Vortex for 2 min and after 6 min, were neutralized with 2 mL NaOH solution (1 M). The absorbance was read at 510 nm and the quantification was carried out using a calibration curve. Different concentrations of rutin (8.5–170 μg/mL) were used for calibration, giving a linearity of 0.997 (R2). The results were expressed in mg rutin equivalents (RE)/g of sample as mean of three replicates. 2.7. Determination of polyphenolic compounds 2.7.1. Extraction of polyphenolic compounds Samples (1 g PFA or PFSP) were weighed into a tube test and 5 mL of water, methanol or DMSO were added. The mixture was vigorously shaken for 2 min and left for 2 h in a Selecta ultrasonic water bath (Selecta S.A. Barcelona, Spain) without temperature control. Then, the extracts were centrifuged at 5000 g for 15 min at 4 °C. The supernatants were filtered through a 0.45 μm Millipore filter (Millipore Corporation, Bedford, USA). The collected fractions were maintained at −4 °C before HPLC analysis. The five fractions obtained were: PFA extracted with methanol (PFAM), and extracted with DMSO (PFAD) and PFSP extracted with methanol (PFSPM) extracted with water (PFSPw) and extracted with DMSO (PFSPD). 2.7.2. HPLC analysis Phenolic acids and flavonoids were analyzed by high performance liquid chromatography coupled with a diode array detector (HPLCDAD) as previously described by Benavente-García, Castillo, Lorente, Ortuño, and Del Río (1999). Twenty microliters were injected into a Hewlett–Packard HPLC series 1100 instrument (Woldbronn, Germany) equipped with a C18 Teknokroma column (Mediterranea sea18, 25× 0.4 cm, Teknokroma, Barcelona, Spain) and detected by absorbance at 280 or 320 nm. UV spectra of individual peaks were recorded in the range of 200–400 nm. The separation columns were controlled thermostatically at 40 °C. Phenolic compounds were analyzed in standard and sample solutions using a gradient elution at 1 mL/min with the following gradient program (0–20 min 95–75% A, 20–40 min 75–50% A, 40–50 min 50–20% A, 50–60 min 20% A) with 1% acetic acid in water as solvent A and acetonitrile as solvent B. All solvents were HPLC grade. Peaks were

758

J.H. López-Vargas et al. / Food Research International 51 (2013) 756–763

identified using authentic standards (phenolic acid standards: caffeic acid, ferulic acid, synapic acid, p-coumaric acid, gallic acid, chlorogenic acid; flavonoids standards: rutin, quercetin, luteolin, apigenin, isoorientin, isovitexin and luteolin-7-O-glucoside) (Extrasynthese, Genay, France) by comparing the retention times, peak spectral analysis and from the literature. The compounds were quantified through calibration curves of standard compounds as mean of three replicates. 2.8. Antioxidant activity assays 2.8.1. Sample preparation to determined the antioxidant activity To obtain the extracts for antioxidant activity the following procedure was used. Different weights of PFA or PFSP (3.9, 7.81, 15.62, 31.25, 62.5, 125, 250 and 500 mg) were extracted with 5 mL of three different solvents such as methanol, water and dimethylsulfoxide (DMSO), using an ultrasonic water bath (Selecta S.A. Barcelona, Spain) without temperature control, during 2 h. Then, the mixtures were centrifuged at 5000 g for 15 min at 4 °C. After centrifugation supernatants were filtered through a 0.45 μm Millipore filter (Millipore Corporation, Bedford, USA) and used to determine extractable antioxidant capacity content. The five fractions obtained were: PFA extracted with methanol (PFAM), and extracted with DMSO (PFAD) and PFSP extracted with methanol (PFSPM) extracted with water (PFSPw) and extracted with DMSO (PFSPD). 2.8.2. 2,2′-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging ability assay The antioxidant activity of different concentrations of PFSP and PFA dissolved in different solvents was measured in terms of radical scavenging ability, using the stable radical DPPH (Brand-Williams, Cuvelier, & Berset, 1995). The results were expressed in % of inhibition as mean of three replicates. 2.8.3. Ferric reducing antioxidant power The ferric reducing antioxidant power (FRAP) of different concentrations of PFSP and PFA dissolved in different solvents was determined by the potassium ferricyanide-ferric chloride method (Oyaizu, 1986). The FRAP of the samples was estimated in terms of Trolox equivalent antioxidant capacity (TEAC) in mM Trolox/g (mean of three replicates). 2.8.4. Ferrous ion-chelating ability assay Ferrous ion (Fe2+) chelating activity of different concentrations of PFSP and PFA dissolved in different solvents was measured by inhibiting the formation of Fe2+-ferrozine complex after treatment of the test material with Fe2+, following the method described by Carter (1971). The results are expressed in % of chelating effect (mean of three replicates). 2.9. Antimicrobial activity 2.9.1. Microbial strains The PFA and PFSP were individually tested against several bacteria: Listeria innocua CECT 910, Serratia marcescens CECT 854, Pseudomonas fragi CEPT 446, Pseudomonas fluorescens CECT 844, Aeromonas hydrophila CECT 5734, Shewanella putrefaciens CECT 5346, Achromobacter denitrificans CECT 449, Enterobacter amnigenus CECT 4078, Enterobacter gergoviae CECT 587, and Alcaligenes faecalis CECT 145. These microorganisms were chosen because they are commonly associated with refrigerated foods: as indicator of pathogenic microorganism or as spoilage microorganism. All species were supplied by the Spanish Type Culture Collection (CECT) of the University of Valencia (Spain). 2.9.2. Microdilution assay Antimicrobial activity was determined based on the colorimetric broth microdilution method proposed by Abate, Mshana, and Miörner (1998). The bacterial strains A. hydrophila, A. denitrificans, S. marcescens

and S. putrefaciens were cultured for 24 h at 26 °C, A. faecalis, E. amnigenus and E. gergoviae for 24 h at 37 °C in Nutrient Broth No. 2 (NB No2) (Oxoid Ltd, England) and L. innocua was cultured for 24 h at 37 °C in Brain Heart Infusion Broth (BHI) (Scharlau S.L., Spain), all adjusted to a final density of 106 CFU/mL before being used as inocula. PFSP or PFA was dissolved in DMSO to reach a final concentration of 100 mg/mL. Serial two fold dilutions were made in a concentration range from 0.1 to 100 mg/mL in sterile test tubes containing Muller Hinton broth. Nisin was used as positive control whilst DMSO was used as negative control. The procedure was repeated three times for each microorganism. 2.10. Statistical assay Statistical analysis and comparisons among means were carried out using the statistical package SPSS 19.0 (SPSS Inc., Chicago, IL.). The data collected for the chemical and technological properties were analyzed by one-way analysis of variance with one factor (fiber source). All the data collected for the antioxidant activity were analyzed by two-way analysis of variance to test the effects of two fixed factors: fiber source (levels: PFSP, PFA) and extracting agent (levels: Methanol, DMSO and Water). Tukey's post hoc test was applied for comparisons of means; differences were considered significant at p b 0.05. 3. Results and discussion 3.1. Chemical analysis Table 1 summarizes the chemical compositions of PFSP and PFA. The proximate composition analysis showed higher protein and fat contents in PFSP samples (pb 0.05) than in PFA, while PFA showed higher (pb 0.05) ash and total dietary fiber contents than PFPS. The PFA and PFSP had a protein content of 0.35 and 1.49 g/100 g d.m. respectively, values lower than those reported by Martínez et al. (2012a) in passion fruit extracts obtained from pulp, peel and seeds. As regards the lipid content, PFA had a lipid content of 1.00 g/100 g. On the other hand, PFSP had a very high fat content (29.54 g/100 g), which is similar to the value reported by Chau and Huang (2004) in passion fruit seed fibers (24.50 g/100 g). This high lipid content could be a limiting factor in its potential application as ingredient in food. The PFA and PFSP showed a very high ash content. Such high ash content could be a problem for the potential application of these co-products in food, since the amounts of metal ions would increase considerably and might facilitate the oxidation of the product in which they are incorporated (Martínez et al., 2012b). The total dietary fiber (TDF) insoluble dietary fiber (IDF) and soluble dietary fiber (SDF) contents of PFA and PFSP, along with the ratio between IDF and SDF, are presented in Table 1. The TDF, IDF and SDF contents were higher in PFA (71.79, 52.34 and 19.45 g/100 g d.m. respectively) (pb 0.05) than in PFSP (53.51, 48.25 and 5.26 g/100 g d.m. respectively). The ratio between IDF and SDF in PFA was 2.69 while in PFSP was 9.17. The TDF content of PFA was similar to that of dietary fiber preparations from other tropical fruits, such as Indian mango peels (78 g/100 g d.m.) (Ajila, Bhat, & Prasada Rao, 2007) or lime co-products (70.76 g/100 g d.m.) (Peerajit, Chiewchan, & Devahastin, 2012). In the case of PFPS, the TDF content was lower than previous reports for seed Table 1 Chemical composition of pulp and seeds (PFSP) and albedo (PFA) obtained from passion fruit co-products. Values expressed as g/100 g dry matter (Mean values). Sample PFA PFSP Ψ

Protein bЖ

0.35 1.49a

Ash

TDFΨ

Fat a

8.08 5.77b

b

1.00 29.54a

IDFΨ a

71.79 53.51b

SDFΨ a

52.34 48.25b

Ratio IDF/SDF a

19.45 5.26b

2.69 9.17

TDF: Total dietary fiber; IDF Insoluble dietary fiber; SDF Soluble dietary fiber. Values followed by the same letter (a–b) within the same column are not significantly different (p > 0.05) according to Tukey's Multiple Range Test. Ж

J.H. López-Vargas et al. / Food Research International 51 (2013) 756–763

fibers obtained from passion fruit (Chau & Huang, 2004) but similar to that obtained for pomegranate fruit bagasse (50.3 g/100 g) (ViudaMartos et al., 2012). As mentioned above, the SDF content in PFA was higher than in PFSP. This large amount of SDF indicated that PFA can be regarded as an interesting ingredient for inclusion in several foods due to the capacity of SDF to retain water and increase post-prandial satisfaction, as well as its ability to increase the time needed for nutrient absorption. In addition, this co-product would act technologically as important thickening agents, gelling and stabilizing foams and emulsions and as film-forming and fat-mimetic agents (Martínez et al., 2012b). Additionally, epidemiological studies have suggested that diets high in SDF may protect against CVD risk factors (Theuwissen & Mensink, 2008). In contrast, the IDF content in PFSP was higher than in PFA. A high proportion of IDF in dietary fibers could have beneficial health effects related with increased satiety and the volume and weight of fecal mass, thus promoting improved functioning of the digestive system (Ku & Mun, 2008). 3.2. Physic-chemical analysis The physic-chemical properties of PFA and PFSP were given in Table 2. PFA showed a pH of 4.36 while PFSP showed a pH of 3.75, the differences between them being statistically significant (p b 0.05). The PFA pH value was similar to that of other fruit fiber products such as pomegranate bagasse (4.40) (Viuda-Martos et al., 2012). However, the pH value of PFSP was lower than other co-product fibers such as lemon albedo (3.96) (Lario et al., 2004). The water activity of PFA and PFSP (pb 0.05) was 0.164 and 0.213, respectively. The low water activity and pH values (both parameters highly related to product deterioration) of PFA and PFSP indicate that the risk of deterioration (by microorganisms, enzymes or non-enzymatic reactions) is minimal. Table 2 also shows the color parameters (L*, a*, b*, C* and h*) of PFA and PFSP. The difference in lightness (L*) between PFA and PFSP (79.91 and 43.89 respectively) was statistically significant (pb 0.05). As regards the red-green coordinate, (a*), statistically significant differences (pb 0.05) existed between PFA and PFSP (1.53 and 2.96, respectively). The PFA yellow–blue coordinate, (b*) had a value of 14.79, while PFSP had a value of 8.93, again with statistically significant differences (pb 0.05) between them. In the same way, the differences in Chroma (C*) values and hue angle (h*) were statistically significant (pb 0.05) between PFA and PFSP (14.86 and 84.10, compared with 9.41 and 71.23, respectively). The incorporation of fiber rich products within a food system may affect the product's organoleptic characteristics and color. They are the most important quality parameters in this respect when dietary fiber products are used as food ingredients. In fact, one of the challenges that industries face when increasing the dietary fiber and whole grain content in food is the change in color and texture that these might produce (Scott-Thomas, 2011). 3.3. Technological properties The hydration properties of dietary fibers are related with the chemical structure of the polysaccharide component, and other factors such as porosity, particle size, ionic form, extraction condition,

Table 2 Physic-chemical properties of pulp and seeds (PFSP) and albedo (PFA) obtained from passion fruit co-products (Mean values). Sample

pH

aw

PFA PFSP

4.36a¥ 3.75b

0.164b 0.213a

¥

Color coordinates L*

a*

b*

C*

h*

79.91a 43.89b

1.53b 2.96a

14.79a 8.93b

14.86a 9.41b

84.10a 71.23b

Values followed by the same letter (a–b) within the same column are not significantly different (p > 0.05) according to Tukey's Multiple Range Test.

759

pH, the IDF/SDF ratio, temperature, ionic strength, type of ions in solution, vegetable source and stresses upon fibers (Elleuch et al., 2011). The results obtained for water holding capacity (WHC), oil holding capacity (OHC) and swelling capacity (SWC) are presented in Table 3. WHC is the ability of a moist material to retain water when subjected to an external centrifugal gravity force or compression. It consists of the sum of linked water, hydrodynamic water and physically trapped water, the latter of which contributes most to this capacity (Lan, Chen, Chen, & Tian, 2012). Additionally, WHC is an important property of dietary fiber from both physiological and technological points of view. As shown in Table 3, PFA and PFSP had a WHC of 13.00 and 1.80 g water/g sample, respectively, with statistically significant differences (p b 0.05) between them. PFA had a WHC similar to the results reported by Martínez et al. (2012a) for passion fruit extracts obtained from pulp, peel and seeds, or by Vergara-Valencia et al. (2007) for fiber concentrate from mango fruit. This value of WHC indicated that PFA has potential applications in products requiring hydration, viscosity development, and freshness preservation, such as baked foods or cooked meat products. Other co-products have been seen to have lower values than those mentioned above and similar to those found for PFSP e.g. date paste obtained from date co-products (Sánchez-Zapata et al., 2011) or ripe banana pulp flour (Al-karkhi, Ramli, Yong, & Easa, 2011). OHC is a technological property related with the chemical structure of plant polysaccharides and depends on surface properties, overall charge density, thickness, and the hydrophobic nature of the fiber particle (Fernández-López et al., 2009). PFA and PFSP had an OHC of 2.03 and 1.43 g oil/g sample, respectively, with statistically significant differences (pb 0.05) between them. In general, PFA and PFSP showed a significantly lower OHC than other fruit and vegetable-derived fibers, such as pomegranate bagasse, 5.9 g oil/g sample (Viuda-Martos et al., 2012), or ripe kiwi 6.00 g oil/g sample (Femenia et al., 2009). Because of its low OHC, PFA and PFSP are potential ingredients for fried products since it would not provide a greasy sensation. SWC indicates how much the fiber matrix swells when water is absorbed. PFA and PFSP had SWC values of 37.00 and 5.00 mL/g sample, respectively, the difference between them was statistically significant (pb 0.05). These results obtained for PFA were higher than those obtained for other fruit fibers, including those from passion fruit pulp, peel and seeds (7.2 mL/g) (Martínez et al., 2012a) or cocoa pod husks (5.81 mL/g) (Martínez et al., 2012b). A high SWC is related to the amount SDF, especially pectin. It is known that the structural characteristics and chemical composition of the fiber play an important role in the kinetics of water uptake (Figuerola, Hurtado, Estévez, Chiffelle, & Asenjo, 2005). 3.4. Total phenol and total flavonoid content The total phenol (TPC) and total flavonoid (TFC) contents of PFA and PFSP extracted with different solvents (methanol, water and DMSO) are presented in Table 4. The TPC of PFSP samples, expressed as gallic acid equivalent, ranged from 0.98 to 4.31 mg/g sample, with statistically significant differences (p b 0.05) between all the samples. PFSPD showed the highest TPC followed by PFPSM. PFPSW had the lowest TPC. As regards PFA, the samples extracted with DMSO had a TPC of 1.86 mg GAE/g sample whilst the PFA extracted with methanol Table 3 Technological properties of pulp and seeds (PFSP) and albedo (PFA) obtained from passion fruit co-products (Mean values). Sample

WHCΨ (g/g)

OHCΨ (g/g)

SWCΨ (mL/g)

PFA PFSP

13.00a¥ 1.80b

2.03a 1.43b

37.00a 5.00b

Ψ

WHC: Water holding capacity; OHC: Oil holding capacity; SWC: Swelling capacity. Values followed by the same letter (a–b) within the same column are not significantly different (p > 0.05) according to Tukey's Multiple Range Test. ¥

760

J.H. López-Vargas et al. / Food Research International 51 (2013) 756–763

showed a TPC value of 0.64 mg GAE/g sample with statistically significant differences (p b 0.05) between them. Comparing the results obtained for PFSP with those of the literature, similar values of TPC have been reported for strawberry (2.6–3.68 mg GAE/g), passion fruit (7.2 mg GAE/g), or guava (1.70–3.45 mg GAE/g) (Thaipong, Boonprakob, Crosby, Cisneros-Zevallos, & Hawkins, 2006; Wu et al., 2004). With reference to the total flavonoid content in the PFSP samples, the highest (p b 0.05) TFC was found in PFSPD (13.63 mg RE/g), followed by PFSPM (6.83 mg RE/g) and PFSPw (2.07 mg RE/g). In the case of PFA the samples extracted with DMSO had a TFC content of 5.12 mg RE/g sample (p b 0.05), while the PFAM showed a TFC value of 0.64 mg RE/g sample. Total phenolic and total flavonoid concentrations could be influenced by geographical origin, cultivar, harvesting, storage time and drying (Babbar, Oberoi, Uppal, & Patil, 2011). In addition, Naczk and Shahidi (2006) mentioned that the recovery of polyphenols from plant materials is influenced by the solubility of the phenolic compounds in the solvent used for the extraction process. Furthermore, solvent polarity plays a key role in increasing phenolic solubility.

3.5. Determination of polyphenolic compounds The flavonoid peaks were identified by their UV/DAD spectra due to their characteristic UV spectral pattern. This UV pattern allows for the selection of flavonoid peaks for quantitative analysis; hence, UV/DAD is an important alternative in the absence of a mass detector (Zeraik & Yariwake, 2010). The HPLC analysis of the PFSP and PFA, extracted with methanol, DMSO or water, by comparison with UV-spectra of authentic standards showed two main peaks, identified as C-glycosylflavones, in particular, isoorientin and isovitexin. PFSP showed higher content in polyphenolic compounds than PFA. PFSPD had the highest (pb 0.05) values for isoorientin (73.60 mg/100 g) and isovitexin (42.35 mg/100 g) followed by PFSPM with values, for isoorientin of 69.90 mg/100 g while for isovitexin of 18.70 mg/100 g. In PFSPw isoorientin or isovitexin were not found. As occurs with PFSP samples, PFAD showed the highest (pb 0.05) content in isoorientin (7.1 mg/100 g) and isovitexin (4.5 mg/100 g) whilst in PFAM the values for isoorientin and isovitexin were 1.10 and 0.53 mg/100 g, respectively. These results were in agreement with Zeraik and Yariwake (2010) or Dhawan, Dhawan, and Sharma (2004) who reported that the main flavonoid present in the pulp and rind of yellow passion fruit was isoorientin. Other flavonoids such as schaftoside, isoschaftoside, orientin, vitexin, luteolin-6-C-chinovoside and luteolin-6-C-fucoside had been identified in the fruit by Mareck, Galensa, and Herrmann (1990). However, in this work, these flavonoids were not found, probably, due to the heat treatment to obtain the extracts. In addition, another problem in identifying the polyphenolic profile is that only a few of passiflora C-glycosylflavones are commercially available as analytical standards.

Table 4 Total phenol (TPC) and total flavonoid (TFC) content of pulp and seeds (PFSP) and albedo (PFA), extracted with different solvents, obtained from passion fruit co-products (Mean values). Sample PFSPD PFSPM PFSPW PFAD PFAM Ж

TPC (mg GAE/g)Ж a§

4.31 2.98b 0.98d 1.86c 0.64e

TFC (mg RE/g)Ψ 13.63a 6.83b 2.07e 5.12c 3.18d

GAE: Gallic acid equivalent. RE: Rutin equivalent. § Values followed by the same letter (a–e) within the same column are not significantly different (p > 0.05) according to Tukey's Multiple Range Test. Ψ

3.6. Antioxidant activity DPPH radical scavenging, the ferric reducing antioxidant power (FRAP) and ferrous ion-chelating assays were performed to determine in vitro antioxidant activities of PFA and PFSP. Since the antioxidant capacity of food is determined by a mixture of different antioxidants with different action mechanisms, among them are synergistic interactions, it is necessary to combine more than one method in order to determine in vitro, the antioxidant capacity of foodstuffs (Pérez-Jiménez et al., 2008). Additionally, solubility of antioxidant compounds in solvent was proven to have strong influence on the recovery of those compounds during the extraction processes. In other words, polarity of solvents indirectly played a vital role in extraction process since it would increase the solubility of antioxidant compounds (Alothman, Bhat, & Karim, 2009). It was impossible to develop a standard solvent that was suitable for the all kinds of antioxidant compounds extracted from plants or fruits. Thus, screening processes are important to justify the best solvent in the extraction of antioxidant compounds. Fig. 1 shows the DPPH radical-scavenging activity of PFSP and PFA. The PFSPD and PFSPM samples showed a higher (p b 0.05) ability to inhibit DPPH radical than the PFAD and PFAM samples. With all solvents assayed, a high increase in radical scavenging activity was observed when the concentration of both PFSP and PFA was increased. At all concentrations, PFSPD had the highest (p b 0.05) DPPH radical inhibition percentage, with values of between 6.76 and 91.79%; followed by PFSPM with values ranging from 1.47 to 87.79%. As regards PFA, the samples extracted with DMSO showed higher (p b 0.05) radical scavenging activity at all concentrations than the samples extracted with methanol. BHT showed the highest (p b 0.05) radical scavenging activity of all the samples assayed. The IC50 (concentration that inhibits 50% of the DPPH radical) values were in the order BHT b PSPF−7 , 12.73, 25.16, D b PSPFM b PFAD b PSPFW b PFAM (values of 6.32 × 10 89.06, 252.68 and 289.74 mg/mL, respectively). The Fe2+ chelating capacity of different PFSP and PFA samples is shown in Fig. 2. All samples were capable of chelating Fe2+ with all solvents studied in a concentration-dependent manner. As regards the PFSP samples, PFSPD at all concentrations produced the highest (pb 0.05) inhibition percentage of ferrozine-Fe+2 complex formation, with values ranging between 68.79 and 95.40%; followed by PFSPM at higher concentrations (25, 50 and 100 mg/mL). At lower concentrations (0.78–12.5 mg/mL) PFSPw showed higher (pb 0.05) chelating activity than PFSPM. In PFA samples, as occurred with PFSP, the samples extracted with DMSO showed higher (pb 0.05) chelating activity at all concentrations than the samples extracted with methanol. At highest concentration (100 mg/mL), no statistically significant differences (p> 0.05) were found between the PFSPD and PFAD. In general terms, it should be noted that PFA and PFSP exhibited higher (pb 0.05) ion-chelating capacity than BHT, which was used as a positive control. The IC50 (concentration that inhibits 50% of ferrozine-Fe+2 complex formation) values were in the order PSPFD b PFAD b PSPFW b PSPFM b PFAM b BHT (0.01, 0.09, 1.23, 7.03, 8.63 and 217.90 mg/mL, respectively). Fig. 3 shows the FRAP analysis of PFSP or PFA extracted with different solvents. As occurred with the DPPH or FIC assays, a concentrationdependent reducing power was observed for all the samples assayed. BHT, at all concentrations showed the highest (pb 0.05) ferric reducing capacity in terms of mM trolox equivalents (mM TE). As occurred with the DPPH assay, the PFSPD and PFSPM samples showed a higher (pb 0.05) ferric reducing capacity than the PFAD and PFAM samples. In PFSP, no statistical differences were found (p> 0.05) at a high concentration (100 mg/mL) between the PFSPD and PFSPM samples. As regards PFA, the PFAD samples showed a higher (pb 0.05) ferric reducing capacity at all concentrations than PFAM samples. There are few studies in which the antioxidant activity of the co-products obtained from exotic fruits in general and passion fruits in particular has been determined. Martínez et al. (2012a) reported that the antioxidant activity of passion fruit fiber concentrate,

J.H. López-Vargas et al. / Food Research International 51 (2013) 756–763

761

Fig. 1. Determination of the antioxidant activity of pulp and seeds (PFSP) and albedo (PFA) obtained from passion fruit co-products extracted with different solvents and at different concentrations (0.78–100 mg/mL) using the DPPH assay.

measured using DPPH and FRAP assays, was 5.12 and 6.93 μM TE/g, respectively. However there are numerous studies in which the antioxidant activity of fresh exotic fruits has been determined. For example, Vasco, Ruales, and Kamal-Eldin (2008) reported that the antioxidant activity of passion fruit measured with the DPPH assay was 0.5 μM TE/g (fresh weight). Contreras-Calderón, Calderón-Jaimes, Guerra-Hernández, and García-Villanova (2011) reported that the antioxidant activity of Passiflora mollissima, measured with the ABTS and FRAP assays, was 34.4 and 42.2 μM TE/g (fresh weight), respectively, while the antioxidant activity of Passiflora tarminiana was 36.7 and 48.9 μM TE/g (fresh weight), respectively. Several studies have revealed that most antioxidant activities arise from flavonoids, isoflavones, flavones, anthocyanins, catechins and other phenolics (Isabelle et al., 2010; Mhatre, Tilak-Jain, De, & Devasagayam, 2009). Polyphenolic compounds are very important fruit constituents, by virtue of their antioxidant activity, chelating

redox-active metal ions, inactivating lipid free radical chains and preventing hydroperoxide conversion into reactive oxyradicals. However, Babbar et al. (2011) reported that antioxidant activity is not the result of phenolic compounds alone. Other constituents, such as ascorbates, reducing carbohydrates, tocopherols, carotenoids, terpenes, and pigments might contribute to the total antioxidant activity. Indeed, although individual phenolics may have substantial antioxidant potential; there may be synergistic or antagonistic interactions between phenolic and non-phenolic compounds. 3.7. Antibacterial activity The broth microdilution method was used to determine the antimicrobial activity of PFSP, PFA and nisin against Gram-positive and Gram-negative bacteria (Table 5). PFSP and PFA showed antimicrobial activity against all the bacteria analyzed. The MIC values of

Fig. 2. Determination of the chelating activity of pulp and seeds (PFSP) and albedo (PFA) obtained from passion fruit co-products extracted with different solvents and at different concentrations (0.78–100 mg/mL) using the FIC assay.

762

J.H. López-Vargas et al. / Food Research International 51 (2013) 756–763

Fig. 3. Determination of the reducing power activity of pulp and seeds (PFSP) and albedo (PFA) obtained from passion fruit co-products extracted with different solvents and at different concentrations (0.78–100 mg/mL) using the FRAP assay.

PFSP were ranged between 6.25 mg/mL for A. hydrophila and 50 mg/mL for P. fragi and P. fluorescens. The rest of the bacteria tested had an MIC of 12.50 mg/mL. As regards PFA, the MIC values ranged between 3.125 mg/mL for A. denitrificans and 50 mg/mL for P. fragi and P. fluorescens. Other bacteria assayed had an MIC of 25 mg/mL. All the bacteria tested in this assay had higher resistance to PFSP and PFA than to nisin, which is the only bacteriocin widely accepted as a natural food preserver. The antimicrobial activities observed in this study might be due to the presence, as occurred with the antioxidant activity, of phenolic acid and/or flavonoid compounds. Extracts of various fruits and fruits co-products containing phenolic and flavonoids have previously been reported to possess antimicrobial activity (Al-Zoreky, 2009; Engels, Gänzle, & Schieber, 2012; Yao et al., 2011). However, although the extracts of various fruits and fruits co-products show antimicrobial activity, the reason behind this capacity is not well documented. Several authors have suggested that the antibacterial activity of phenolic acids and flavonoids may be attributable to the cytoplasmic membrane damage caused by perforation and/or a reduction in membrane fluidity (Tsuchiya & Iinuma, 2000), the inhibition of energy metabolism (Haraguchi, Tanimoto, Tamura, Mizutani, & Kinoshita, 1998) or the inhibition of nucleic acid synthesis (Plaper et al., 2003). In addition, Chinnam et al. (2010) reported that several flavonoids (flavonol, flavan-3-ol and flavone) inhibit energy metabolism through ATP synthase inhibition, while Wu et al. (2008) indicated that the mechanisms of antibacterial activity of phenolic acids and flavonoids include the inhibition of cell wall synthesis and the inhibition of cell membrane synthesis.

4. Conclusions Since fiber-rich passion fruit co-products obtained from pulp and seed or albedo are available in large quantities as a co-product of juice production, it could be used as an intermediate food ingredient (IFI) in the development of functional foods because of its interesting antioxidant and antibacterial activities. In addition, these fiber-rich co-products have potential applications as ingredients in products requiring hydration, viscosity development and freshness preservation, such as baked foods or cooked meat products, due to their high total dietary fiber content and good technological properties, especially their water holding and swelling capacities.

Acknowledgment The authors are grateful to the project CYTED-IBEROFUN Codec: 110AC0386 and CajaMurcia for supporting the post-doctoral grant of one of the authors.

References Abate, G., Mshana, R. N., & Miörner, H. (1998). Evaluation of a colorimetric assay based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) for rapid detection of rimampicin resistance in Mycobacterium tuberculosis. The International Journal of Tuberculosis and Lung Disease, 2, 1011–1016. Agronet (2012). http://www.agronet.gov.co (Accesed: October 8th 2012) Ajila, C. M., Bhat, S. G., & Prasada Rao, U. J. S. (2007). Valuable components of raw and ripe peels from two Indian mango varieties. Food Chemistry, 102, 1006–1011.

Table 5 Minimum inhibitory concentration (MIC) values of pulp and seeds (PFSP) and albedo (PFA) obtained from passion fruit co-products against several bacterial strains. Sample

PFSP PFA Nisin

Minimum inhibitory concentration (mg/mL) L. innocua

A. hydrophila

A. denitrificans

A. faecalis

E. amnigenus

E. gergoviae

S. marcescens

S. putrefaciens

P. fragi

P. fluorescens

12.5 25 1.25

6.25 25 1.25

12.5 3.12 1.25

12.5 25 1.25

12.5 25 0.625

12.5 25 0.625

12.5 25 0.625

12.5 25 1.25

50 50 0.625

50 50 0.312

J.H. López-Vargas et al. / Food Research International 51 (2013) 756–763 Al-karkhi, A. F. M., Ramli, S. B., Yong, Y. S., & Easa, A. M. (2011). Comparing physicochemical properties of banana pulp and peel flours prepared from green and ripe fruits. Food Chemistry, 129, 312–318. Alothman, M., Bhat, R., & Karim, A. A. (2009). Antioxidant capacity and phenolic content of selected tropical fruits from Malaysia, extracted with different solvents. Food Chemistry, 115, 785–788. Al-Zoreky, N. S. (2009). Antimicrobial activity of pomegranate (Punica granatum L.) fruit peels. International Journal of Food Microbiology, 134, 244–248. AOAC (1997). Official methods of analysis of AOAC International (16th ed.). Washington, DC: Association of Official Analytical Chemists. AOAC (2000). Official methods of analysis of AOAC International (17th ed.). Gaithersburg, MD: Association of Official Analytical Chemists. Ayala-Zavala, J. F., Vega-Vega, V., Rosas-Domínguez, C., Palafox-Carlos, H., Villa-Rodriguez, J. A., Siddiqui, W., et al. (2011). Agro-industrial potential of exotic fruit byproducts as a source of food additives. Food Research International, 44(7), 1866–1874. Babbar, N., Oberoi, H. S., Uppal, D. S., & Patil, R. T. (2011). Total phenolic content and antioxidant capacity of extracts obtained from six important fruit residues. Food Research International, 44, 391–396. Benavente-García, O., Castillo, J., Lorente, J., Ortuño, A., & Del Río, J. A. (1999). Antioxidant activity of phenolics extracted from Olea europaea L. Leaves. Food Chemistry, 68, 457–462. Blasa, M., Candiracci, M., Accorsi, A., Piacentini, M. P., Albertini, M. C., & Piatti, E. (2006). Raw Millefiori honey is packed full of antioxidants. Food Chemistry, 97, 217–222. Brand-Williams, W., Cuvelier, M. E., & Berset, C. (1995). Use of free radical method to evaluate antioxidant activity. Lebensmittel-Wissenschaft und Technologie, 28, 25–30. Carter, P. (1971). Spectrophotometric determination of serum iron at the submicrogram level with a new reagent (ferrozine). Analytical Biochemistry, 40, 450–458. Chau, C. F., & Huang, Y. L. (2004). Characterization of passion fruit seed fibres—A potential fibre source. Food Chemistry, 85, 189–194. Chen, X. J., & Lu, L. X. (1994). Karyotype analysis of two major cultivated forms of Passiflora L. Journal of Fujian Agricultural University, 23, 24–25. Chinnam, N., Dadi, P. K., Sabri, S. A., Ahmad, M., Kabir, M. A., & Ahmad, Z. (2010). Dietary bio-flavonoids inhibit Escherichia coli ATP synthase in a differential manner. International Journal of Biological Macromolecules, 46, 478–486. Contreras-Calderón, J., Calderón-Jaimes, L., Guerra-Hernández, E., & García-Villanova, B. (2011). Antioxidant capacity, phenolic content and vitamin C in pulp, peel and seed from 24 exotic fruits from Colombia. Food Research International, 44, 2047–2053. Dhawan, K., Dhawan, S., & Sharma, A. (2004). Passiflora: A review update. Journal of Ethnopharmacology, 94(1), 1–23. Elleuch, M., Bedigian, D., Roiseux, O., Besbes, S., Blecker, C., & Attia, H. (2011). Dietary fibre and fibre-rich by-products of food processing: Characterisation, technological, functionality and commercial applications: A review. Food Chemistry, 124, 411–421. Engels, C., Gänzle, M. G., & Schieber, A. (2012). Fast LC–MS analysis of gallotannins from mango (Mangifera indica L.) kernels and effects of methanolysis on their antibacterial activity and iron binding capacity. Food Research International, 45, 422–426. Femenia, A., Sastre-Serrano, G., Simal, S., Garau, M. C., Eim, V., & Rosselló, C. (2009). Effects of air-drying temperature on the cell walls of kiwifruit processed at different stages of ripening. LWT—Food Science and Technology, 42, 106–112. Fernández-López, J., Sendra-Nadal, E., Navarro, C., Sayas, E., Viuda-Martos, M., & Pérez-Alvarez, J. A. (2009). Storage stability of a high dietary fibre powder from orange by-products. International Journal of Food Science and Technology, 44, 748–756. Figuerola, F., Hurtado, M. L., Estévez, A. M., Chiffelle, I., & Asenjo, F. (2005). Fibre concentrates from apple pomace and citrus peel as potential fibre sources for food enrichment. Food Chemistry, 91, 395–401. Haraguchi, H., Tanimoto, K., Tamura, Y., Mizutani, K., & Kinoshita, T. (1998). Mode of antibacterial action of retrochalcones from Glycyrrhiza inflata. Phytochemistry, 48, 125–129. Isabelle, M., Lee, B. L., Lim, M. T., Koh, M. T., Huang, D., & Nam, C. (2010). Antioxidant activity and profiles of common fruits in Singapore. Food Chemistry, 123, 77–84. Jiménez, A. M., Sierra, C. A., Rodríguez-Pulido, F. J., González-Miret, M. L., Heredia, F., & Osorio, C. (2011). Physicochemical characterisation of gulupa (Passiflora edulis Sims. Fo edulis) fruit from Colombia during the ripening. Food Research International, 44(7), 1912–1918. Ku, C. S., & Mun, S. P. (2008). Optimization of the extraction of anthocyanin from Bokbunja (Rubus coreanus Miq.) marc produced during traditional wine processing and characterization of the extracts. Bioresource Technology, 99, 8325–8330. Lan, G., Chen, H., Chen, S., & Tian, J. (2012). Chemical composition and physicochemical properties of dietary fiber from Polygonatum odoratum as affected by different processing methods. Food Research International, 49, 406–410. Lario, Y., Sendra, E., García-Pérez, J., Fuentes, C., Sayas-Barberá, E., Fernández-López, J., et al. (2004). Preparation of high dietary fibre powder from lemon juice by-products. Innovative Food Science and Emerging Technology, 5, 113–117. Mareck, U., Galensa, R., & Herrmann, K. (1990). Identizierung von passionsfruchtsaft in fruchtprodukten mitels HPLC. Zeitschrift für Le0bensmittel-Untersuchung und -Forschung, 191, 269–274.

763

Martínez, R., Torres, P., Meneses, M. A., Figueroa, J. G., Pérez-Álvarez, J. A., & ViudaMartos, M. (2012a). Chemical, technological and in vitro antioxidant properties of mango, guava, pineapple and passion fruit dietary fibreconcentrate. Food Chemistry, 135, 1520–1526. Martínez, R., Torres, P., Meneses, M. A., Figueroa, J. G., Pérez-Álvarez, J. A., & Viuda-Martos, M. (2012b). Chemical, technological and in vitro antioxidant properties of cocoa (Theobroma cacao L.) co-products. Food Research International, 49, 39–45. Mhatre, M., Tilak-Jain, J., De, S., & Devasagayam, T. P. A. (2009). Evaluation of the antioxidant activity of non-transformed and transformed pineapple: A comparative study. Food and Chemical Toxicology, 47, 2696–2702. Naczk, M., & Shahidi, F. (2006). Phenolics in cereals, fruits and vegetables: Occurrence, extraction and analysis. Journal of Pharmaceutical and Biomedical Analysis, 41, 1523–1542. Oyaizu, M. (1986). Studies on products of browning reaction: Antioxidative activity of products of browning reaction prepared from glucosamine. Japan Journal of Nutrition, 44, 307–315. Peerajit, P., Chiewchan, N., & Devahastin, S. (2012). Effects of pretreatment methods on health-related functional properties of high dietary fibre powder from lime residues. Food Chemistry, 132, 1891–1898. Pérez-Jiménez, J., Arranz, S., Tabernero, M., Díaz-Rubio, E., Serrano, J., Goñi, I., et al. (2008). Updated methodology to determine antioxidant capacity in plant foods, oils and beverages: Extraction, measurement and expression of results. Food Research International, 41, 274–285. Plaper, A., Golob, M., Hafner, I., Oblak, M., Solmajer, T., & Jerala, R. (2003). Characterization of quercetin binding site on DNA gyrase. Biochemical and Biophysical Research Communications, 306, 530–536. Robertson, J. A., Monredon, F. D., Dysseler, P., Guillon, F., Amadó, R., & Thibault, J. F. (2000). Hydratation properties of dietary fibre and resistant starch: A European collaborative study. Lebensmittel-Wissenschaft und Technologie, 33, 72–79. Sánchez-Zapata, E., Fernández-López, J., Peñaranda, M., Fuentes-Zaragoza, E., Sendra, E., Sayas, E., et al. (2011). Technological properties of date paste obtained from date by-products and its effect on the quality of a cooked meat product. Food Research International, 44, 2401–2407. Scott-Thomas, C. (2011). How the 2010 guidelines affect food technologists. Available from: http://www.foodnavigator-usa.com/content/view/print/356612 (Accesed: October 8th 2012) Sendra, E., Fayos, P., Lario, Y., Fernandez-López, J., Sayas-Barberá, E., & Perez-Alvarez, J. A. (2008). Incorporation of citrus fibers in fermented milk containing probiotic bacteria. Food Microbiology, 25(1), 13–21. Singleton, V. L., & Rossi, J. A. (1965). Colorimetry of total phenolics with phosphomolybdicphosphotungstic acid reagents. American Journal of Enology and Viticulture, 16, 144–158. Thaipong, K., Boonprakob, U., Crosby, K., Cisneros-Zevallos, L., & Hawkins, D. (2006). Comparison of ABTS, DPHH, FRAP, and ORAC assays for estimating antioxidant activity from guava fruit extracts. Journal of Food Composition and Analysis, 19, 669–675. Theuwissen, E., & Mensink, R. P. (2008). Water-soluble dietary fibers and cardiovascular disease. Review, Physiology and Behavior, 94, 285–292. Tsuchiya, H., & Iinuma, M. (2000). Reduction of membrane fluidity by antibacterial sophoraflavanone G isolated from Sophora exigua. Phytomedicine, 7, 161–165. Vasco, C., Ruales, J., & Kamal-Eldin, A. (2008). Total phenolic compounds and antioxidant capacities of major fruits from Ecuador. Food Chemistry, 111, 816–823. Vergara-Valencia, N., Granados-Pérez, E., Agama-Acevedo, E., Tovar, J., Ruales, J., & Bello-Pérez, L. A. (2007). Fibre concentrate from mango fruit: Characterization, associated antioxidant capacity and application as a bakery product ingredient. LWT —Food Science and Technology, 40, 722–729. Viuda-Martos, M., López-Marcos, M. C., Fernández-López, J., Sendra, E., López-Vargas, J. H., & Pérez-Alvarez, J. A. (2010a). Role of fibre in cardiovascular diseases: A review. Comprehensive Reviews in Food Science and Food Safety, 9, 240–258. Viuda-Martos, M., Ruiz-Navajas, Y., Fernández-López, J., & Pérez-Álvarez, J. A. (2010b). Effect of added citrus fibre and spice essential oils on quality characteristics and shelf-life of mortadella. Meat Science, 85, 568–576. Viuda-Martos, M., Ruiz-Navajas, Y., Martin-Sánchez, A., Sánchez-Zapata, E., FernándezLópez, J., Sendra, E., et al. (2012). Chemical, physico-chemical and functional properties of pomegranate (Punica granatum L.) bagasses powder co-product. Journal of Food Engineering, 110, 220–224. Wu, X., Beecher, G. R., Holden, J. M., Haytowitz, D. B., Gebhardt, S. E., & Prior, R. L. (2004). Lipophilic and hydrophilic antioxidant capacities of common foods in the United States. Journal of Agricultural and Food Chemistry, 52, 4026–4037. Wu, D., Kong, Y., Han, C., Chen, J., Hu, L., Jiang, H., et al. (2008). d-Alanine:d-alanine ligase as a new target for the flavonoids quercetin and apigenin. International Journal of Antimicrobial Agents, 32, 421–426. Yao, W. R., Wang, H. Y., Wang, S. T., Sun, S. L., Zhou, J., & Luan, Y. Y. (2011). Assessment of the antibacterial activity and the antidiarrheal function of flavonoids from bayberry fruit. Journal of Agricultural and Food Chemistry, 59, 5312–5317. Zeraik, M. L., & Yariwake, J. H. (2010). Quantification of isoorientin and total flavonoids in Passiflora edulis fruit pulp by HPLC-UV/DAD. Microchemical Journal, 96, 86–91.