Physiochemical and Antibacterial Characterization of Fruits of Three Chilean Trees

Fruits (2), 87–96 | ISSN 0248-1294 print, 1625-967X online | https://doi.org/10.17660/th.2017/72.2.4 | © ISHS 2017 Original article

Citronella mucronata (Cardiopteridaceae), Pitavia punctata (Rutaceae)Physiochemical and Beilschmiediaand antibacterial berteroana characterization (Lauraceae), of fruits three of endemic and threatened Chilean trees

, G.F. Narváez2, M.F. Morales3 3 4 and C.R. Figueroa 1 5,a F.A.12 Sáez , H.M. Bello , C. Balbontín 3 Master Program in Forest Sciences, Faculty of Forest Sciences, University of Concepción, Concepción, Chile 4 Faculty of Forest Sciences, University of Concepción, Concepción, Chile Research Lab of Antibacterial Agents, Faculty of Biological Sciences, University of Concepción, Concepción, Chile

5 Small Fruits and Berry Crops Research, Institute for Agricultural Research (INIA)-Quilamapu, Chillán, Chile Phytohormone Research Laboratory, Institute of Biological Sciences, University of Talca, Talca, Chile Summary Significance of this study Introduction – Several native tree species are What is already known on this subject? scarcely studied in relation to fruit properties. In or- • Citronella mucronata, Pitavia punctata and Beilschmie- der to bring about information of these plant resourc- dia berteroana are threatened endemic trees of central es, the characterization of ripening-associated properties of the fruit of three endemic and threatened studied. Chilean trees (Citronella mucronata, Pitavia punctata Chile whose fruit properties have been scarcely and Beilschmiedia berteroana) was performed in the What are the new findings? present study. Materials and methods – The physio- • C. mucronata and P. punctata chemical characterization of two developmental fruit a high amount of pectin and bacteriostatic effect, stages in each species included the measurement of fruits extracts showed soluble solid content (SSC), titratable acidity (TA), pH, for both fruits. color and chlorophyll content; cell wall composition respectively. Harvest indexes were described and lignin content; the total phenolic content and What is the expected impact on horticulture? antioxidant capacity of the fruit extracts. Moreover, • the minimal inhibitory concentration (MIC) was car- used for food and pharmaceutical industries. ried out in fruit extracts against 11 bacterial strains. Under a sustainable management, these fruits can be Results and discussion – Pitavia punctata and B. berteroana fruits exhibited similar SSC and TA values showing a significant increase of the SSC:TA ratio during devel- Résumé opment. However, C. mucronata showed the highest SSC value at stage II with 8.1 °Brix, accompanied by riennes des fruits de Citronella mucronata a noticeable color change. The analysis of pectin frac- Caractéristiquespteridaceae), de Pitavia physico-chimiques punctata (Rutaceae) et anti-bacté et de- tions suggests that solubilisation of pectic polymers (Cardio- increase during fruit development in all species. The Beilschmiedia berteroana (Lauraceae), trois arbres fruits of C. mucronata presented the highest level of total uronic acids (for stage I and II, a 63% and 71% of Introduction – Nombreuses sont les espèces the cell wall material, respectively). The phenolic con- d’arbreschiliens endémiquesindigènes dont et menacés.les propriétés des fruits sont tent, antioxidant capacity and MIC were particularly peu étudiées. Afin d’obtenir des informations sur ces remarkable in P. punctata fruits at stage II, when fruit ressources végétales, l’étude présente a caractérisé les reaches 6.5 °Brix, with values of 924.4 mg GAE 100 g-1 propriétés associées au stade de maturité des fruits -1 FW, 2.51 mM FeSO4 g FW, and 6.25% v/v, respective- de trois arbres chiliens endémiques et menacés (Ci- ly. Conclusion – These results relate interesting prop- tronella mucronata, Pitavia punctata et Beilschmiedia erties of C. mucronata and P. punctata fruits for food berteroana). Matériel et méthodes – La caractérisation and pharmaceutical industries with physiological in- physicochimique de deux stades de développement dexes at stage II, mainly soluble solids and color. de chaque espèce comprenait les mesures suivantes: la teneur en matières solubles (SSC), l’acidité titrable Keywords (TA), le pH, la couleur et la teneur en chlorophylle des extraits de ces fruits, ainsi que la composition des parois cellulaires, les teneurs en lignine, en composés Chile, endemic species, non-timber forest products, phénoliques totaux et la capacité anti-oxydante. De underutilized species, fruit quality, cell wall composition, plus, la concentration inhibitrice minimale (CMI) des antioxidant compounds a Corresponding author: extraits de ces fruits a été effectuée contre 11 souches or

[email protected] [email protected].

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| Physiochemical and antibacterial characterization of fruits of three Chilean trees

bactériennes. Résultats et discussion – Les fruits de P. et al Pitavia punctata is a perennial tree belonging punctata et de B. berteroana ont présenté des valeurs to1.2 the cm Rutaceae diameter, dark purple at ripe stage (Hechenleitner semblables de SSC et de TA, montrant une augmenta- ., 2005). tion significative du rapport SSC: TA au cours de leur for antimicrobial family, properties and its (Esterhuizen potential could et al be., 2006).similar to développement. Cependant, C. mucronata a montré other species of the family which have been characterized la plus grande valeur de SSC au stade II avec 8,1 °Brix P. punctata Anet accompagné d’un changement notable de coloration. alearly characterizationP. ofpunctata extracts from stems and leaves of L’analyse des fractions de pectine suggère que la so- reported several flavonoid compounds (Silva lubilisation des polymères pectiques augmente au ., 1971). Fruits of are ovoid drupeset al (1.8–2.5 cours du développement des fruits chez toutes les es- Beilschmiediacm diameter) berteroana green at the is an first evergreen developmental tree that stages belongs and to pèces étudiées. Les fruits de C. mucronata ont présen- theyellowish Lauraceae green at ripe stage (Hechenleitner ., 2005). té le niveau le plus élevé d’acides uroniques totaux (2×2 cm) (Hechenleitner et al (pour les stades I et II, respectivement 63% et 71% de about the fruit family properties with globular of this species and greyish-yellow is scarce, however drupes la paroi cellulaire). Le contenu phénolique, la capaci- ., 2005). The information té anti-oxydante et la CMI ont été particulièrement re- et al marquables dans les fruits de P. punctata au stade II, the fruits could be interesting since they would present high quand les fruits atteignent 6,5 °Brix, avec des valeurs speciesamounts has of mucilagenot been reported.(Rodríguez Chilean ., 1983).native fruits have the -1 -1 de 924,4 mg GAE 100 g FW, 2,51 mM FeSO4 g FW et potentialThe characterization to become in appreciated of fruit properties supplies for in nutraceuticalthis group of 6,25% v/v, respectivement. Conclusion – Ces résultats rapportent des propriétés particulières des fruits de Aristotelia chilensis Ugni C. mucronata et P. punctata peuvent intéresser l’in- molinaeindustry. In this sense, extracts fromLuma the apiculata Chilean native berries dustrie alimentaire ou pharmaceutique avec des va- ‘maqui’ ( [Molina] Stuntz), ‘murtilla’ ( leurs de référence au stade II du développement des Turcz.) and ‘arrayan’ ( [DC.] Burret) fruits, surtout en matières solubles et coloration. duehave to shown the phenolic interesting composition results forof their their leaves antioxidant and fruits and antibacterial activitieset al and potential ethealth al benefits, mainlyet Mots-clés al (Genskowsky ., 2016; Suwalsky ., 2007; Fuentes pectin,., 2016). a main Nevertheless, component other of the interesting cell wall metabolitesfound in a wide and Chili, espèces endémiques, produits forestiers non-ligneux, polymers could be found in endemic fruits. For instance, espèces sous-utilisées, qualité du fruit, composition des and pharmaceutical industries (Willats et al., 2006). In parois cellulaires, composés anti-oxydants variety of fruits, is a valuable ingredient for food, cosmetic processes can be described in indigenous or underutilized Introduction fruitsaddition, that othercould help interesting understand and the particular ripening physiologicalphenomena, Fragaria chiloensis [L.] Mill.) (Cherian et al Chile iset considered al a ‘biogeographic island’, with diverse as in the case of the Chilean strawberry ( climates, ecosystems, and an extraordinary biodiversity potential of Citronella., 2014).mucronata, Pitavia punctata and (Armesto ., 1998). While the diversity of native plants Chile has a high percentage of endemism what makes it a BeilschmiediaThe objective berteroana of this fruits work as materials was to for determine the food and the may be low, considering with otheret Southal., 2000). American However, countries, there

of‘hotspot’ these endemic of biodiversity resources (Myers (Hechenleitner et al pharmaceutical industries. The characterization included is little information about the biology and usefulness of many the analysis of physiochemical and antimicrobial properties sustainable management of forests and their .,species 2005). withThis of the fruits. In a first step, fruit quality parameters such as conservationsituation constitutes problems. a major While economicforests have opportunity been studied for for the a color, solid soluble concentration (SSC) and titratable acidity long time under the approach of obtaining wood for industrial general(TA), and characterization. chemical attributes In a secondsuch as step,total tophenolic deepen content insight purposes, now its properties have gained renewed interest as (TPC) and antioxidant capacity (AOX) were evaluated as a

in certain fruit attributes, analyses of cell wall fractions and Somea source of ofthe many threatened non-timber endemic forest trees products of native (NTFP), forests which of antibacterial assays were carried out on fruit extracts. add value to forests (Nepstad and Schwartzman, 1992). Materials and methods Citronella mucronata [Ruiz & Pav.] Plant material central Chile whosePitavia fruits punctata can be classified as fleshy drupe- Fruits of the tree species were collected from three type, Beilschmiediaare ‘huillipatagua’ berteroana ( D. Don), ‘pitao’ ( Molina) and ‘belloto del sur’ ( [Gay] Kosterm.). These three C. mucronata, natural populations located in Biobío Region, Chile: ‘Hualpén’ species share a restricted distribution in wet ravines in south- et (36°47’40’’S, 73°09’42’’W, 54 m a.s.l.) for central Chile and according to their conservation status they P. punctata, al ‘Nonguén’ (36°49’55’’S, 72°58’08’W, 250 m a.s.l.) for are threatened to extremely threatened (Hechenleitner a.s.l.) for B. berteroana and ‘Bulnes’ (36°43’35’’S, 72°15’26’W, 102 m ., 2005). Although there are no reports that these fruits are for food, pharmaceutical or natural products industries (Figure 1). The harvested fruits were edible, they could be interesting as raw materials or additives under a sustainable management. immediately transported to the laboratory under refrigerated Citronella mucronata is a perennial tree belonging to the conditions. The fruits were classified in two developmental Cardiopteridaceae (II)stages includes according bigger to theand evolution pigmented of fruits.weight and/or color. The first stage (I) includes smaller fruits; and the second stage family whose species are used in some African countries as an effective molluscicide (Brackenbury, 1999). The fruits of this species are small drupes of about

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| Physiochemical and antibacterial characterization of fruits of three Chilean trees

Figure 1. FIGURE 1. Citronella mucronata Pitavia punctata Beilschmiedia berteroana, Fruits and locations of the Citronellathree tree mucronataspecies studied in the presentPitavia research.punctata (A) Representative photographs of Fruits and locations of the three tree species studied in the present research. (A) Representative theBeilschmiedia tree fruits of: berteroana1) , bar = 25 mm; 2) , bar = 25 mm and; 3) forphotographs C. mucronata of the tree fruits of: 1)P. punctata , bar = 25B. mm;berteroana 2) , bar = 25 mm and; bar = 20 mm. (B) Geographical locations of theC. naturalmucronata populations in Biobío RegionP. punctata (Chile) for the three species: ‘Hualpén’ each3) location. , bar = 20 mm. (B) Geographical locations of the natural populations in Biobı́o Region B. berteroana (1), ‘Nonguén’ for (2) and ‘Bulnes’ for (3). See text for geographic coordinates of (Chile) for the three species: ‘Hualpén’ for (1), ‘Nonguén’ for (2) and ‘Bulnes’ for Fruit quality assessments(3). See text for and geographic chlorophyll coordinates of each location. determination FW. using 20 mM NaOH with a digital-1 burette (Jencons, UK) and expressed as g citric acid 100 g For each species and stage 30 fruits without external Chlorophyll quantification was performed according to damage were analysed for weight, diameter and color. The the method described by Lichtenthaler and Wellburn (1983) fruits were weighed (g) and the equatorial diameter was with some modifications. Briefly, fruit skin (0.2 g) was skin color of the fruits was characterized using a colorimeter H2 measured (mm) using a digital caliper (Mitutoyo, USA). The ground with liquid nitrogen, homogenized in 2 mL acetone/ O (80/20, v/v) and centrifuged for 10 min at 12,000 rpm at L a b H2 (model CR-400, Konica Minolta, Japan) and expressed as 4 °C. The supernatant was then diluted (1/3, v/v) in acetone/ CIELAB scale ( *, *, *) along with the dimensions of color g O fruit (80/20, skin. v/v) and then measured at 663–646 nm. The chroma and hue angle (h°). Two measurements on each results-1 of three replicates were expressed as µg chlorophyll equatorial side were performed for each fruit. Firmness Cell wall analysis of stage II fruits was determined using a texture analyser (model CT3, Brookfield Engineering Labs., USA) expressing Cell wall isolation and lignin determination the results in Newton (N). The fruits were punctured at the andequatorial a bulk regiontissue onsample opposite was sides,prepared using in aeach 1-mm species diameter and et al. stagecylinder and probe. separated Thereafter, in three the replicates, fruits were which cut were into frozen pieces Cell wall material was extracted according to Figueroa (2012), with some modifications. Five g of frozen fruit tissue was ground with liquid nitrogen, homogenized in in liquidFor the nitrogen determination and stored of at solid -20 °Csoluble until usecontent for the (SSC), rest 40 mL of 95% ethanol and boiled for 45 min. The insoluble of the analyses. material was filtered through miracloth and sequentially washed with 15 mL boiling ethanol, 15 mL chloroform/ titratable acidity (TA) and pH, 2 g of each replicate were methanol (1/1, v/v) and 15 mL acetone. The alcohol insoluble FW. ground in liquid nitrogen, homogenized in 5 mL distilled residue (AIR) was dried overnight at 37 °C and weighed.-1 The water and filtered through miracloth obtaining three results of three replicates were expressed as mg AIR g different juice extractions. The SSC was determined on Lignin was extracted as described by Campbell and Ellis the juice of each replicate at 20 °C using a hand-held (1992). After cell wall preparation, the samples were diluted temperature compensated refractometer (Atago, Japan) and in 1 M NaOH (1/3, v/v) and hydrolysed. A colorimetric17 assay expressed as °Brix. The pH was measured in the juice using a was performed using thioglycolic acid (Sigma-Aldrich, FW. USA), pH-meter (Model pH 20, HANNA Instruments, USA). TA was and the absorbance was measured at 280 nm.-1 The results of determined by diluting the remaining juice in distilled water three replicates were expressed as µg lignin g (1/10, v/v), and then titrating an aliquot of 13 mL to pH 8.2

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| Physiochemical and antibacterial characterization of fruits of three Chilean trees Cell wall fractionation bacterial strains were obtained from the Research Lab of ATCC BAA-1706, ATCC BAA-1705 and ATCC 700603. All The fractionation of cell wall materialet al was performed using a sequential chemical treatment of each AIR replicate Antibacterial Agents, University of Concepción, Chile. The as previously described (Figueroa ., 2012). The water- strains were maintained at -80 °C in a 2:1 ratio of 50% 2 3 soluble (WSF), the trans-1,2-diaminocyclohexane-N,N,N’,N’- glycerol and broth. Cultures for antimicrobial activity tests tetraacetic acid (CDTA)-soluble (CSF), and Na CO -soluble were grown in 3 mL Tryptone Soy Broth at 37 °C overnight (NSF) fractions were obtained and considered mainly Determinationbefore the assay. of minimal inhibitory concentration (MIC) neutralpectin-enriched sugars (NS) fractions. concentrations Two independent in the different extractions pectin were obtained from each replicate. The uronic acid (UA) and The MIC was determined by broth microdilution assay fractions were determined colorimetrically as previously according to the Clinical and Laboratory Standards Institute described (Blumenkrantz and Asboe-Hansen, 1973; Yemm (CLSI, 2012). Briefly, five microliter of inoculum adjusted to and Willis, 1954). The results were calculated using standard 0.5 McFarland of each of the strains were pipetted into 96- curves of galacturonic acid and glucose for UA and NS, well microtiter plates with 100 µL Mueller-Hinton broth). orrespectively. glucose (NS) Measurements mg were performed in triplicate, (Oxoid Ltd., UK) followed by a two-fold serial dilution5 of fruit-1 and the results were-1 expressed as µg galacturonic acid (UA) extract samples (final concentration of 5×10 CFU mL Total phenolic content AIR. (TPC) and antioxidant capacity A positive control (not including extracts) and a negative (AOX) determination lowestcontrol concentration (without inoculum) at which no were growth also occurred included. (devoid After incubation at 37 °C for 18–24 h, MIC was determined as the

Two grams of stage II fruits were ground with liquid Determinationof turbidity). of minimal bactericide concentration nitrogen, homogenized in 10 mL of 100% methanol (MBC) and centrifuged for 20 min at 5,000 rpm. The samples were filtered through miracloth to obtain a clear extract. Total phenolic content (TPC) were determined using the The MBC was calculated by taking 100 µL of broth from colorimetric method described by Singleton and Rossi determinedeach well without as the growth lowest andconcentration transferred at to which 5 mL Trypticaseno growth (1965), using gallic acid as the standard. Briefly, 500 µL of occurred.soy broth. After incubation at 37 °C for 24–36 h, MBC were methanol extract was mixed with 3.5 mL of distilled water 2 3 wasand 250added µL Folin-Ciocalteuand samples were reagent, incubated and incubated in darkness at room for Statistical analysis temperature for 5 min. Then, 500 µL of 10% (w/v) Na CO randomized design, with the main factors being the three 1 h at room temperature. Finally, the absorbance was read speciesThe (entireC. mucronata experiment, P. punctata was conducted, and B. berteroanausing a completely) at one at 765 nm using an UV/Vis spectrophotometer FW. (Spectronic Genesys, USA). The results of three replicates-1 were expressed as mg gallic acid equivalents (GAE) 100 g or two developmental stages, as the case. The data were Measurement of antioxidant capacity (AOX) of the fruit Panalysed by ANOVA using INFOSTAT (version 2008) software, extracts was evaluated by the ferric reducing ability of and differences were considered statistically significant at plasma (FRAP) assay according to Benzie and Strain (1996). Results≤0.05 (LSD and test). discussion The FRAP reagent was prepared by mixing together 10 mM 2,4,6-tripyridyl triazine (TPTZ) and 20 mM ferric chloride Characterization of fruit quality attributes in 0.25 M acetate buffer, pH 3.6. One hundred microliter of methanol extract was added to 300 µL distilled water C. mucronata fruit grows during development, since an followed by 3 mL FRAP reagent. The absorbance was read at The physical analysis of the fruit revealed that only 593 nm after 5 min of incubation at room temperature against 4·7H2 FW. C. mucronata presented a blank. The results of three-1 replicates were expressed as mM theincrease smallest in fresh fruits weight among and the diameter three species was only (2.3 observed g FW and in AntibacterialFeSO Oequivalent assays g this species (Table 1). In addition, B. berteroana P. punctata at Fruit samples 13 mm diameter at stage II), and together with toexhibited B. berteroana lower levels of fruit firmness than diameterthe stage atII (datastage notII. shown). The biggest fruit corresponds usingFruit a foodsamples processor. of each Sevenspecies grams from stageof each II weredried oven- and species, reaching 9.5 g FW and 23.1 mm dried at 70 °C for 48 h. Dried samples were finely pulverized development in C. mucronata and P. punctata, and increased in B.Regarding berteroana pH with values, the they highest remained value constant compared during with fruit the pulverized sample were mixed with 105 mL of 100% methanol in an orbital shaker at 100 rpm for 24 h at room C. mucronata temperature. After extraction, each sample was filtered and Chileanother species native (TableA. chilensis 1). The SSC values reported here for Bacterialstored at 4 strains °C until use. differet markedlyal when compared with the Each fruit sample was tested against Gram positive berry, which showed a SSC close Staphylococcus aureus C.to mucronata19 °Brix (Fredes fruits. In this., 2012). sense, Nevertheless, it has been other reported Chilean for Enterococcus faecalis native berries showBerberis SSC valuesmicrophylla closer G. to Forst.) those aexhibited wide range by Acinetobacter baumannii ATCC 29213 Enterobacter and ATCC cloacae 25923, Escherichia coli ATCC 29212; Gram-negative location‘calafate’ (Mariangel berry ( et al Pseudomonas aeruginosa ATCC 19606,Klebsiella pneumoniae of SSC in ripe fruits from 9.3 to 22.9 °Brix, depending on ATCC E705, ATCC 25922 and ATCC 35218, ., 2013). In addition, it has recently ATCC 27853, been reported SSC values from 5.20 to 12.56 °Brix in green

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| Physiochemical and antibacterial characterization of fruits of three Chilean trees Table 1. Citronella mucronata, Pitavia punctata and Beilschmiedia berteroana fruits in two develop n Quality assessments of - Fresh weight Diameter SSC TA mentalSpecies stages. DataStages are means ± standard deviations ( =3). SSC:pH soluble solid content, TA: titratable acidity. SSC/TA (g) (mm) (°Brix) (g citric acid 100 g-1 FW) C. mucronata I 1.0 ± 0.3 by 11.8 ± 0.9 b 6.3 ± 0.2 a 8.0 ± 0.3 a 0.23 ± 0.03 b 34.4 ± 3.4 a II 2.3 ± 0.4 a 13.0 ± 1.1 a 6.1 ± 0.1 a 8.1 ± 0.9 a 0.33 ± 0.03 a 24.5 ± 1.1 b P. punctata I 5.0 ± 0.6 a 21.4 ± 1.5 a 6.6 ± 0.1 a 4.2 ± 0.4 b 0.07 ± 0.00 a 61.3 ± 6.6 b II 5.2 ± 0.9 a 20.9 ± 1.2 a 6.5 ± 0.1 a 6.5 ± 0.9 a 0.05 ± 0.01 b 108.4 ± 11.3 a B. berteroana I 8.7 ± 0.4 a 23.0 ± 1.3 a 7.0 ± 0.0 a 4.7 ± 0.1 a 0.04 ± 0.01 a 118.3 ± 13.9 b II 9.5 ± 0.5 a 23.1 ± 1.5 a 6.7 ± 0.1 b 6.3 ± 0.4 a 0.04 ± 0.01 a 164.8 ± 12.3 a y Different letters indicate significant differences between developmental stages in each species (P≤0.05). and ripe fruits of L. apiculata et al., suggesting that color change is not a good indicator for fruit C. mucronata fruits are close ripening in B. berteroana. In contrast, the results of a b to the lowest value for B. microphylla, respectively fruit (Fuentes(i.e. for C. mucronata and P. punctata and2016). with The those SSC valuesobserved for for cultivated berries such as * and * et al , 9.3 °Brix) a species showed significant et al P. punctata differencesin b between theC. developmental mucronata fruit stages at stage under II accord study. andcranberry B. bert eroana (9.3 °Brix) (Celik ., 2008) and raspberry The highest value in the * parameter and the lowest value (10.9than those °Brix) of (Wang C. mucronata ., 2009). In the case of * were observedPitavia in punctata - , their fruits exhibited lower SSCet alat stage II stageing to II the according purplish to color the b that it reaches at maturity (Table (Table 1), similar to those values 2; Figure 1A). fruit turns to yellow color in andreported the growing for ripe practices, fruits of amongblackberry other (Guedes factors. For instance,., 2013). * value, which is the highest valuea inDifferences B. microphylla in SSC ofhigh fruit SSC (juice) values depend are inon accordancethe environment with registeredand b (Table 2; Figure 1A). higher degrees of south latitude (Mariangel et al the Thecolor chroma vividness and and hue color angle appearance (h°) are combinationsof each fruits inof the * and in L. apiculata a variation between samples collected at * parameters and its behaviora can beb easily differentiate different harvest seasons was reported (Fuentes et al., 2013), C. mucronata and P. punctata showed changes in chroma and developmental stages. As well as * and * parameters, only ., 2016). ofIn these the present threatened study, plant it wasspecies. difficult to compare between h° between developmental stages. The fruits of all speciesPitavia different locations and growingP. punctata seasons and B. due berteroana to the scarcity fruits punctataat stage I and have B. similar berteroana h° and fruits chroma remained values in (greenthis range tones, at 117-120°,the stage II, with although middle P. punctata chroma values 36 to 39). I to OnII, suggestingthe other hand,that ripening process takes place between C. mucronata fruits theseshowed two a significant stages. Fruit increase at green of the stage SSC:TA has ratio higher from pectin stage exhibited a more vivid color content and starch, which decrease as the fruit ripens due to indicatingin stage II (chromaa change 43.9). of color On theand contrary, saturation in the fruit skin showed a severe reduction in chroma and h° values (Table 2), C. mucronata, P. punctata the action of enzymes that hydrolyse and produce an increase (Figureand B. 1A).berteroana propertiesin the concentration which affects of sugarsthe organoleptic (Osorio and properties, Fernie, 2014). since The analysis of chlorophylls of Moreover, the TA is considered as one of the physicochemical fruitsa for revealedB. berteroana a significant which remained decrease in the first two species during development, except in the the Fruit organic pigmentation acid:sugar is ratio a result defines of a qualitycoordinate parameters and devel at C. case mucr of onata chlorophyll fruits showed a dramatic decrease of both harvest time in fruit (Osorio and Fernie, 2014). without significant differences (Table 2). Remarkably, only turnover of green chloroplasts into colored chromoplasts- a b). opment-regulated process during ripening thatet involves al the chlorophylls duringP. punctata development and B. berteroana (almost 95%fruits reductionstill have for chlorophyll and a 85% reductionC. for mucronata chlorophyll fruits. in yellow, orange and red colored etfruits al (Pech ., 2014), At the stage II, and the accumulation of anthocyanins that pigmented fruits high levels of chlorophyll in relation to (ofL red, blue and black (Ageorges a., 2014).b The CIELAB However, the total content of chlorophyll becomes lower system represented in rectangularL coordinates the lightness during fruit ripening in all the studied fruits. This loss of *) and chromaticity (composedL by * and *). Lightness C.chlorophyll mucronata is related with the decrease in the hue value, nentsgoes froma 0 b to 100, where * = 100a representsa a perfectb from green to yellow-orange colors tones, as occurred with (+reflectingb diffuser,a and * = 0 represents black.b The compo- Analysis of cell fruits wall (Table material, 2). lignin and cell wall * and * have values from (- *) to (+ *)L and (- *) to composition *), where * goes from green to red, and * goes from stagesblue to inyellow C. mucronata (Yam and andPapadakis, P. punct 2004).ata For * parameter, P. punctata and B. berteroana a significant decrease and increase were observed between The alcohol insoluble residue (AIR) contentC. mucronata decreased observed in C. mucronata and P. punctata fruits, respectively during the development only in (TableIn the case2). This of B. confirms berteroana a greater fruits, darkeningno changes and in lightness occurringfruits, without during significant the fruit differences development between of both P. puncta- were observed, with values close to the middle fruits, of respectively. the L fruitsta and (Table B. berteroana 3). This suggestsspecies. Regardinga degradation the oflignin the cellcontent, wall C. mucronata and a * scaleb P. punctata fruits during development. In contrast, B. ber- (Table 2). Alongside this, the fruits of this species neither a significant decrease was recorded in exhibited significant changes in the parameters * and *,

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| Physiochemical and antibacterial characterization of fruits of three Chilean trees FW) NS -1 11.8 ± 1.9 a 11.8 17.0 ± 2.0 a 2.0 ± 0.4 b 4.8 ± 0.7 a 3.6 ± 0.8 a 2.7 ± 1.0 a (μg g Chlorophyll b 253.2 ±15.1 a 38.8 ± 22.2 b 278.6 ± 14.3 a 246.1 ± 2.4 b 277.0 ± 5.9 a b 245.5 ± 11.6 NSF -soluble (NSF) fractions 3 CO 2 UA FW) -1 116.6 ± 9.8 b 116.6 16.3 ± 9.0 a 15.5 ± 2.9 a 64.2 ± 0.8 b 157.1 ± 14.1 a 237.0 ± 15.0 a (μg g Chlorophyll a 468.5 ± 36.1 a 25.2 ± 14.3 b 170.9 ± 10.1 a 144.3 ± 5.7 b 170.5 ± 10.5 a 141.8 ± 17.8 a AIR) NS -1 3.1 ± 1.0 b 7.3 ± 0.4 a 1.4 ± 0.1 b 3.2 ± 0.4 a 32.2 ± 3.1 a 8.9 ±1.1 b (hº) CSF Hue angle 117.7 ± 0.8 a 117.7 119.9 ± 0.2 a 119.9 ± 1.0 a 118.3 ± 3.6 a 114.8 47.1 ± 3.9 b 101.2 ± 2.5 b =3). UA berteroana Beilschmiedia fruits in two developmental stages. Data are Cell wall fractions (μg mg 8.8 ± 3.2 b 19.7 ± 2.1 a 30.3 ± 2.9 b 81.9 ± 5.41 a 124.9 ± 7.2 b 319.1 ± 25.9 a Chroma 38.3 ± 2.3 a 36.7 ± 2.4 b 39.1 ± 3.1 a 38.4 ± 1.4 a 2.4 ± 0.8 b 43.9 ± 0.9 a NS and punctata Pitavia 0.5 ± 0.3 b 23.8 ± 1.7 a 3.9 ± 0.5 a 4.8 ± 0.9 a 3.4 ± 0.7 b 27.6 ± 1.8 a

WSF * b ≤0.05). ≤0.05). P ( P 33.8 ± 2.4 a 31.6 ± 0.9 b 34.6 ± 3.7 a 35.4 ± 5.0 a 1.8 ± 0.3 b 43.3 ± 0.5 a UA 7.2 ± 2.6 b 66.2 ± 2.9 a 62.4 ± 3.7 a 71.8 ± 4.7 a 383.3 ± 20.2 a 154.6 ± 11.6 b 154.6 ± 11.6 , mucronata Citronella

* a FW) -1 -9.4 ± 1.1 a 1.7 ± 0.3 a -17.9 ± 1.2 b -18.2 ± 0.4 b -18.2 ± 1.6 a -16.0 ± 2.6 a Lignin (μg g 24.0 ± 0.3 b 31.0 ± 0.1 a 53.0 ± 0.2 a 21.0 ± 0.4 b 45.0 ± 0.3 a 36.0 ± 0.5 b y y fruits in two developmental stages. Data are means ± standard deviations ( n deviations means ± standard stages. Data are developmental fruits in two FW) L * -1 (AIR) 52.5 ± 3.0 a 52.4 ± 2.5 b 49.8 ± 2.8 a 22.2 ± 1.6 b 62.7 ± 0.9 a 45.1 ± 2.1 a (mg g berteroana

207.9 ± 3.3 b 247.1 ± 3.5 a 167.8 ± 0.9 b 270.7 ± 7.7 a 171.6 ± 3.7 a 151.5 ± 9.5 a Cell wall material =3). B. and I I I I I I II II II II II II Stages Stages punctata punctata

Char Cell wall material (AIR), lignin content, and content of uronic acids (UA) and neutral sugars (NS) in water-soluble (WSF), CDTA-soluble (CSF) and Na Cell material (AIR), wall lignin content, and and sugars (NS) content neutral of (WSF), acids in uronic CDTA-soluble (UA) water-soluble acterization of color and composition of chlorophylls of

2. 3.

mucronata, P. mucronata,

C. B. berteroana P. punctata P. C. mucronata C. mucronata Species P. punctata P. B. berteroana Species Different letters indicate significant differences between developmental stages in each species letters indicate significant differences Different Different letters indicate significant differences between developmental stages in each species ( letters indicate significant differences Different means ± standard deviations ( n deviations means ± standard y Table y Table of

92 International Journal of Tropical and Subtropical Horticulture Sáez et al.

| Physiochemical and antibacterial characterization of fruits of three Chilean trees teroana Table 4. C. mucronata, P. punctata fruits exhibited an increase in lignin during fruit and B. berteroana Total phenolic content (TPC) and antioxidant development (Table 3). Generally, the unripe or green-stage capacity (AOX) by FRAP methodn=3). of fruits have higher fiber content, because the fruit has many fruits at developmental stage II. Data are TPC FRAP duringpolymeric the carbohydratesripening process (cellulose, (Waldron hemicellulose, ., 2003). In lignin the means ± standard deviations ( et al Species -1 -1 and pectin substances), which are enzymatically degraded (mg GAE 100 g FW) (mM FeSO4 g FW) y seems to occur during fruit development, either of cell wall C. mucronata 742.7 ± 56.0 b 0.710 ± 0.001 c inthree P. punctata species, someand B. extent berteroana of degradation, or of lignin of these in the polymers case of P. punctata 942.4 ± 64.2 a 2.510 ± 0.100 a C. mucronata B. berteroana 724.5 ± 60.7 b 1.660 ± 0.124 b In order to determine the proportion of the different y Different letters indicate significant differences between species (Table 3). (P≤0.05). 2 3 to obtain the WSF, pectic substances in the fruits, the AIR was sequentially extracted with water, CDTA and Na CO could accumulate bioactive compounds as most studied CSF and NSF fractions, respectively (Table 3). Cell wall compositionfractionation associated and quantification to WSF, CSF of uronic and NSF acids fractions. (UA) and It neutral sugars (NS) allowed the identification of pectic (Kanellis and Manganaris, 2014). The results ofP. TPCpunctata and AOX showed significant differences between species. The is well known that high methoxylated pectins can be easily ingreatest C. mucronata values of TPC and AOX were detected in extracted with water, although it can only partially solubilize fruits whereas that the lowest values of AOX were observed some of the pectic substances of the primary cell walls and fruits (Table 4). This result suggestsP. punctata that middle lamellae (Selvendran and O’Neill, 1987). On the other phytochemicals rather than anthocyanins can be other hand, chelating agents, such as CDTA ions solubilises related with the higher TPC and AOX founded in low methoxylated pectin fractions, and this procedure also fruit since this fruit has a light yellow color (chroma 43.9 and ofpreferentially neutral sugar solubilises content pecticcompared substances with the from CSF the fraction. middle P.h° punctata101.2) at stage II (Table 2). It has been reported that in lamellae. WSF fraction usually has a relatively higher degree Citrus crops, which belong to the same botanical family as 2 3 captured pectin fractions , severalet al biocompounds such as flavanones and Disruptive treatment with Na CO triterpenoid acids can contribute to aP. significant punctata antioxidantfruits are with lower molecular weight due to cleavage of glycosidic capacity (Yu ., 2005). bonds mainly from primary cell walls (Selvendran and The TPC values exhibited by theO’Neill, stage 1987). II of C. mucronata and P. punctata in CSF and NSF comparatively higher than those found in the worldwide fractions.In terms In ofthe UA case content, of B. higher berteroana values were observed at known berries grown-1 in Chile such as raspberry ‘Heritage’ FW)(300 (Fredesmg-1 GAE et100 al g FW), blueberry ‘Brigitta’ (610 mg GAE-1 C. mucronata , higher UA values 100 g FW) and strawberry FW) (Ruiz ‘Camarosa’ et al (630 mg GAE 100 g were observed at the stage II in WSF and CSF fractions. Only -1 ., 2014), and in the native FW) ‘calafate’ (Ruiz berryet al., proportion of fruitpectins showed is in differenta reduction fractions in UA contentdepending in theon (870 mg GAE g ., 2010),-1 being overcome WSF fraction during development. Interestingly, the greater foronly C. by mucronata the ‘maqui’ berry (1,460 mg GAE g in B. berteroana, C. mucronata and P. punctata 2010). On the other hand, the antioxidant2+ g capacity observed the species. Thus, WSF, CSF and NSF are the majority fraction fruit is equivalentet al -1 to those reported for the , respectively ‘maqui’ berry (0.152 mmol Fe FW) also measured by (Table 3). In terms of NS content, values are markedly lower Determinationmeans of FRAP assay of antibacterial (Ruiz ., 2010).activity of fruit extracts in all species and stages withB. respect berteroana to UA and values. C. mucronata Among different fractions, WSF exhibited the greater NS values: it anrepresents increase 70% in NS and content 50% for can be observed in P. punctata The results of antibacterial activity on various bacterial andat the B. berteroana stage II, respectively.in the CSF fraction, During and fruit in development,C. mucronata species are shown in Table 5. Additionally, the minimal and B. berteroana inhibitory concentration (MIC) for methanol was evaluated being this 50% v/v. The results indicated that most of increases during fruit in development the WSF fraction and differences (Table 3). in These the lowestmethanol MIC extracts was from obtained P. punctata from against fruits P. aeruginosa samples showed data suggested that the solubilisation of pectic polymers a MIC>12.5% (v/v). The only extract that presented the (6,25% pectin metabolismIn this sense, could C. mucronata be specific presented to each species, the highest both [v/v]). When minimal bactericide concentrationP. punctata against (MBC) events widely reported for many fruits (Ruiz-May and Rose, Pseudomonaswas determined aeruginosa for this extract,has a bacteriostatic a value >12.5% behavior. (v/v) was 2013). obtained, indicating that the extract fromPseudomonas aeru- tolevel the of observed total UA inin thisrelation species, to AIR P. punctata in the two and developmental B. berteroana ginosa to antibiotics and compounds from plants or fungal stages (for I and II, 63% and 71%, respectively). In contrast The high resistance presentedet al by et al., et al wereshowed observed lower UA between levels inspecies, stage Isuggesting and II: 16% that and P. 31%;punctata 3% producing antibiotics (Adwan ., 2010; Sharifi-Rad and 10%,B. berteroana respectively. might Thus, have differences a higher in pectinproportion contents of 2015; Mota ., 2015)et isal apparently resulting from a par- C. mucronata. ticular waterproofing membrane and the presence of efflux mechanisms (Mayaud ., 2008; Tyagi and Malik, 2011). Analysishemicellulosic of total polymers phenolic in comparisoncontent (TPC) to and antioxidant Furthermore, it has been reported that the low activity of capacity (AOX) lipophilic compounds from plants may be due to multidrug pumps expulsion, including ATP-binding cassette (ABC)et al., performed in fruits at stage II, since these fruits, when ripen, transporters type (active transport) that remove antibiotics The analysis of TPC, AOX and antimicrobial assays was and other harmful lipophilic substances (Mulyaningsih 2011). In this sense, it could be interesting to deepen into the

Volume 72 | Issue 2 | March/April 2017 93 Sáez et al.

| Physiochemical and antibacterial characterization of fruits of three Chilean trees Table 5. P. punctata, C. mucronata and B. berteroana fruits at developmental stage II Minimum inhibitory concentration (MIC, % v/v) of extracts of MICs (% v/v) Bacterial strains against American Type Culture Collection (ATCC) bacterial strain. P. punctata C. mucronata B. berteroana Gram negative Acinetobacter baumannii ATCC 19606 > 12.5 > 12.5 > 12.5 Enterobacter cloacae ATCC E705 > 12.5 > 12.5 > 12.5 Escherichia coli ATCC 25922 > 12.5 > 12.5 > 12.5 Escherichia coli ATCC 35218 > 12.5 > 12.5 > 12.5 Klebsiella pneumoniae ATCC BAA-1706 > 12.5 > 12.5 > 12.5 Klebsiella pneumoniae ATCC BAA-1705 > 12.5 > 12.5 > 12.5 Klebsiella pneumoniae ATCC 700603 > 12.5 > 12.5 > 12.5 Pseudomonas aeruginosa ATCC 27853 6.25 > 12.5 > 12.5 Gram positive Staphylococcus aureus ATCC 29213 > 12.5 > 12.5 > 12.5 Staphylococcus aureus ATCC 25923 > 12.5 > 12.5 > 12.5 Enterococcus faecalis ATCC 29212 > 12.5 > 12.5 > 12.5

mechanisms of the bacteriostatic effect of P. punctata fruit

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Received: Nov. 20, 2015 Accepted: Dec. 12, 2016

96 International Journal of Tropical and Subtropical Horticulture