Antifungal Activity of Natural and Enzymatically-Modified Flavonoids

Food Chemistry 124 (2011) 1411–1415

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Food Chemistry

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Antifungal activity of natural and enzymatically-modiﬁed ﬂavonoids isolated from citrus species

Maria Paula Salas a,b,*, Gustavo Céliz c, Hugo Geronazzo c, Mirta Daz c, Silvia Liliana Resnik b,d a Agencia Nacional de Promoción Cientíﬁca y Tecnológica (ANCYPT), Argentina b Departamento de Industrias, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Intendente Güiraldes 2160, 1428, Ciudad Autónoma de Buenos Aires, Argentina c Instituto de Investigaciones para la Industria Química (CONICET) and Facultad de Ciencias Exactas, Universidad Nacional de Salta, Avenida Bolivia 5150, Salta, Argentina d Comisión Cientíﬁca de Investigaciones, Calle 526 entre 10 y 11, La Plata, Buenos Aires, Argentina article info abstract

Article history: The antifungal activity of isolated flavonoids from Citrus species, such as naringin, hesperidin and neohes- Received 2 November 2009 peridin, and enzymatically-modified derivatives of these compounds, was studied on four fungi often Received in revised form 17 June 2010 found as food contaminants: Aspergillus parasiticus, Aspergillus flavus, Fusarium semitectum and Penicillium Accepted 27 July 2010 expansum. Although all the flavonoids showed antifungal activity, the intensity of this activity depended on the type of fungus and compound used. The hesperetin glucoside laurate strongly inhibited the mycelial growth of P. expansum, while prunin decanoate was the most inhibiting flavonoid for A. flavus, A. par- Keywords: asiticus, and F. semitectum. Flavonoids The flavonoids naringin, hesperidin and neohesperidin, obtained as byproducts at low cost from the Mycelial growth Fungal growth residues of the citrus industries, present an interesting option for these industries. Citrus Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction protection against cardiovascular diseases and certain forms of cancer (Benavente-García et al., 1997; González-Molina, Domín- Flavonoids are widely distributed compounds that are found guez-Perles, Moreno, & García-Viguera, 2010; Salah et al., 1995; mainly in plants. The most common flavonoids found in Citrus sp. Tripoli, La Guardia, Giammanco, Di Majo, & Giammanco, 2007). can be classified into different groups: flavanones, flavones, flava- Several studies have assessed the antimicrobial activity of many nols and anthocyanins (Benavente-García, Castillo, Marin, Ortuño, essential oils obtained from a wide variety of plants (Demirci, Kosßar, & Del Río, 1997). In particular, flavanones are found in Citrus as Demirci, Dinç, & Basßer, 2007; Fraternale, Giamperi, & Ricci, 2003; glycosides. Rota, Herrera, Martínez, Sotomayor, & Jordán, 2007). Some articles Flavanone glycosides are abundant constituents of citrus leaves describe the antimicrobial activity and identify flavonoids, some of and fruits. The most common Citrus flavanone glycosides are hes- them found in Citrus. Among these works, we find that Arima, Ash- peridin or 30,5,7-trihydroxy-40-methoxyflavanone-7-a-L-rhamno- ida, and Danno (2002) studied the activity against Bacillus cereus syl(1?6)-b-D-glucoside, which is found in oranges, lemons and and Salmonella enteriditis, reporting that quercetin had antibacterial other citrus, naringin or 30,5,7-trihydroxy-40-methoxyflavanone- activity against these two bacteria. Another work showed that seven 7-a-L-rhamnosyl(1?6)-b-D-glucoside in grapefruits and sour pure flavonoids, including neohesperidioside, isolated from five oranges and neohesperidin or 30,5,7-trihydroxy-40-methoxyflava- moss species, proved to have antibacterial activity over some Gram none-7-a-L-rhamnosyl(1?2)-b-D-glucoside in sour oranges. This negative bacterial strains (Basile, Giordano, López-Sáez, & Cobianchi, variety of flavonoids has been obtained as a byproduct of the Citrus 1999). Rauha et al. (2000) found that the aglycone of a flavanone, industries, and it represents an interesting option for these compa- naringenin, exhibited activity against several bacteria and Proestos, nies (Ellenrieder, 2004). Boziaris, Nychas, and Komaitis (2006) showed that the extract of Flavonoids have aroused considerable interest recently because Astanea vulgaris, a plant with high concentrations of naringenin of their potential beneficial effects on human health such as antivi- and quercetin, had a high antimicrobial capacity against a strain of ral, anti-allergic, anti-inflammatory, antioxidant activities, and Listeria monocytogenes. Mandalari et al. (2007) worked with flavonoids isolated from bergamot peel, a byproduct of the Citrus fruit * Corresponding author at: Departamento de Industrias, Facultad de Ciencias processing industry, finding that eriodictyol strongly inhibited the Exactas y Naturales, Universidad de Buenos Aires, Intendente Güiraldes 2160, 1428, growth of seven bacteria and also Saccharomyces cervisiae. Neverthe- Ciudad Autónoma de Buenos Aires, Argentina. Tel.: +54 1145763389; fax: +54 1147920781. less, few studies have examined the antifungal activity of flavonoids. E-mail address: [email protected] (M.P. Salas). One of these studies, from Wächter, Hoffmann, Furbacher, Blake, and

Timmermann (1999), showed that the prenylated flavanone isolated liberates 1 lmol of p-nitrophenol per minute. The a-rhamnosidase from the shrub Eysenhardita texana showed activity against the specific activity was 18.6 U mg1, whereas the b-glucosidase was opportunistic pathogen Candida albicans. Two flavones from Artemi- very low (0.0074 U mg1). After that, the system was kept at 4 °C sia giraldii have been reported to exhibit activity against Aspergillus for 24 h: the solid was separated by filtration, dissolved to 10 % flavus (Zheng, Tan, Yang, & Liu, 1996). Galagin, a flavonol commonly at 96 °C, and hydrolysed again at 50 °C and pH 4.0 with a new por- found in propolis samples, proved to have inhibitory activity against tion of enzyme. After a new crystallization at 4 °C, the solid was fil- five moulds (Afaloyan & Meyer, 1997). Weidenbörner and Jha (1993) tered, washed with cold water, and dried at 50 °C. P was kept in a have focused on the study of the inhibitory effect of some flavonoids dry place. During the conversion of NAR and NEO to P and HG, against fungi commonly found in food, but so far there are not many respectively, there were no other substances that could be pro- studies about the possible antifungal activity of flavonoids, espe- duced by this hydrolysis, so the transformation to these two flava- cially flavanones, isolated from Citrus. Ortuño et al. (2006) showed nones was almost completed. that some modifications in Penicillium digitatum hyphae and the The syntheses of the other flavonoid glycoside esters were car- inhibition of the spore production were caused by neohesperidin ried out by enzymatic catalysis in organic medium, using the com- and naringin. mercial enzyme lipase B of Candida antartica, immobilized in an The increasing concern of the consumer for food safety has acrylic resin NovozymÒ 435 (donation of Novozymes Latin America pushed industries to the elimination of synthetic additives and Limited, Araucaria, Parana, Brazil) and different alkyl vinyl esters their replacement by natural additives. This change is seen as a which provided the acyl group (Sigma and Fluka). The method con- benefit in the quality and safety of food (Viuda-Martos, Ruiz-Nava- sisted in placing in a hermetic reactor acetone (150 ml) as a solvent jas, Fernández-López, & Pérez-Álvareza, 2008). and molecular sieves of 4 Å (Sigma). After that, the chosen flavo- The aim of this work is to study the activity of certain natural noid glycoside (30 mM) and the vinyl ester (300 mM) were added. and enzymatically-modified flavonoids from Citrus species against The system was heated up to 50 °C before the initiation of the reac- the growth of fungi commonly found in food. tion with the enzyme. Reaction times were variable, but were completed in 24–48 h, depending on the flavonoid synthesised. Each conversion was followed until the substrate was converted to al- 2. Materials and methods most 100%. All the reactions were followed by HPLC with auto injector (234 Gilson), with UV detector (118 Gilson) and using a 2.1. Flavonoid synthesis RP-8 LiChrospher 100 Merck column (25 cm length, 4 mm internal diameter and 5 lm particle size). The isocratic elution used aceto- The analysed flavonoids were naringin (NAR), prunin (P), prunin nitrile:water as mobile phase in different proportions, according to butyrate (PB), prunin decanoate (PD), prunin laurate (PL), prunin the ester analysed, and UV detection at 280 nm. In each case, a stearate (PS), hesperidin (HES), neohesperidin (NEO), hesperetin reaction product was observed that was only esterified at carbon glucoside (HG) and hesperetin glucoside laurate (HGL). Fig. 1 number 6 of the glucose. In order to purify the synthesised esters, shows the structures and known physicochemical properties of the solvent was evaporated from the reaction mixture with a ro- the different flavonoids tested. The flavonoids NAR, HES and NEO tary evaporator at 40 °C under vacuum (final pressure 14 mmHg). were obtained from immature aborted fruits of citric origin. The The residue mixture was washed several times with hexane in or- production processes consist of grinding the fruits to an average der to eliminate the unreacted vinyl ester, centrifuging after every size of 2 mm in diameter and then an extraction in a fixed bed col- wash at 200 rpm for 5 min. The remaining solid was the flavonoid umn. To obtain NAR, immature grapefruits are used (Ruby Red glycoside ester and insoluble material from the molecular sieve. variety) and the extraction is done with distilled water as solvent The ester was then dissolved in dimethylformamide (DMF) and fil- at 80 °C according to Geronazzo, Robin, Blanco, Cuevas, and Ellen- tered in order to discard the solid residue form the molecular sieve. rieder (2000). To obtain HES, immature oranges (Hamlin variety) This DMF solution was evaporated at 50 °C and solid crystals were are used with aqueous solution at pH 10.0–10.5 as extracting sol- obtained. Flavanone glucoside esters resulted in 99% of purity, vent at 70 °C according to Macoritto, Geronazzo, and Ellenrieder checked by HPLC. (2001). NEO is obtained from immature bitter orange (Citrus aurantium) with ethanol:water (25:50 v/v) at 25 °C according to Maco- 2.2. Antifungal activity ritto, Robin, Blanco, and Geronazzo (2004). In all three processes, the extract obtained is cooled, this leading to the crystallization 2.2.1. Fungal strains of the flavonoids. The precipitate is then filtered and washed. Final- The different moulds, Aspergillus parasiticus, A. flavus and Fusar- ly the solid is dried in an oven at 50 °C. The NAR obtained had 95% ium semitectum, were isolated from soy seeds and P. expansum from purity. HES was obtained with a purity of 87% and, by subsequent apples. All the above species were kept in the Type Culture Collec- recrystallization in water, reached 95% purity. NEO resulted in 99% tion of the Natural Science Faculty, University of Buenos Aires of purity. NAR, HES and NEO had most of their impurities arising (Buenos Aires). These moulds were previously cultivated in strains from water and reducing sugars, 0.08% from calcination residues containing Malt Extract Agar (MEA) during 7 days in order to as- and less than 20 ppm from heavy metals. sure fungus purity. Then, 10 ml of Tween 80 (Biokar) (0.02%) were Naringin glucoside, commonly known as prunin (P) and the added and the tubes were shaken for 1 min in a vortex to separate hesperetin glucoside (HG) were obtained by enzymatic hydrolysis the conidia from the rest of the medium. The concentration of con- of supersaturated solutions of naringin and neohesperidin accord- idia in suspension was determined, using a Neubauer counting ing to the methodology described by Ellenrieder, Blanco, and Daz chamber. The values obtained were: A. parasiticus, 1.5 105 coni- (1998) and Soria and Ellenrieder (2002). Briefly, the method is dia /ml (standard deviation SD = 0.2 105 conidia /ml); A. flavus, achieved by hydrolysing a 10% supersaturated NAR solution with 2.0 107 conidia /ml (SD = 0.3 107 conidia /ml); F. semitectum, 7Uml1 of an Aspergillus niger a-rhamnosidase (a commercial 8.2 107 conidia /ml (SD = 0.8 107 conidia /ml); P. expansum, naringinase preparation from TanabeÒ, Japan) at 50 °C and pH 4.0 1.6 108 conidia /ml (SD = 0.3 108 conidia /ml). for 6 h. The activity of a-rhamnosidase was determined using p- nitrophenyl-a-rhamnoside in the buffer (0.05 M citric acid/sodium 2.2.2. Agar dilution method citrate, pH 4.0), as described by Romero, Manjon, Bastida, and Ibor- The fungi were tested by the agar dilution method described by ra (1985). The unit of activity (U) was the amount of catalyst that Fraternale et al. (2003) with some modifications. Briefly, the M.P. Salas et al. / Food Chemistry 124 (2011) 1411–1415 1413

Product Characteristics Structure

Naringin - Formula weight: 580.5.

Cas Nº - Purity by HPLC ≥ 95%. OH H C 2 O 10236472 - High solubility in ethanol, acetone, DMF and boiling HO HO HO O OH water (100 mg/mL). HO O O

-Optical activity: -80±10º (1% in ethanol at 25ºC). HO O CH3 OH O - Appearance: off-white to light tan powder.

Neohesperidin - Formula weight: 610.6.

Cas Nº - Purity by HPLC ≥ 99%. OH H C 2 O HO OH 13241333 - High solubility in ethanol, acetone, DMF, not so HO HO O O CH3 much in boiling water (10mg/mL). HO O O

HO O - Optical activity: -103±5º (0.05% in ethanol at 25ºC) CH3 OH O - Appearance: yellow to green tan powder.

Hesperidin - Formula weight: 610.6.

Cas Nº - Purity by HPLC ≥ 95%.

520263 - Water solubility ≤ 20 mg/L (at room temp.). Soluble

CH3 in formamide and DMF at 60ºC. Slightly soluble in HO O

OH methanol, and hot glacial acetic acid. Almost insoluble OH O

H C 2 O OH in acetone, benzene and chloroform; readily soluble in HO HO OH O dilute base and pyridine. Slightly soluble in methanol. CH3 O O Practically soluble in dilute alkali aqueous solution.

OH O - Optical activity: -74±5º (2% in pyridine).

- Appearance: yellowish or almost white crystalline

powder.

Hesperetin - Formula weight: 464. OH OH H2C O HO O glucoside - Purity by HPLC ≥ 99%. HO CH3 OH O O - Water solubility 0.03 g/l. 1-Octanol solubility 0.01 g/l.

- Appearance: greenish white powder. OH O

Fig. 1. Main chemical characteristics and structures of all ﬂavonoids used in the experiment.

studied flavonoids were dissolved in DMF in order to prepare 100 peptone per litre of distilled water. Before moulds were inoculated, times more concentrated solutions to achieve a final concentration the dishes were left to dry out for two days. Twice a day, during of DMF of 1% (v/v) of the growth media and to have an uniform dis- 86 h, two measurements of two right-angled diameters of the col- persion of the flavonoids in the plates. The flavonoids were added onies were taken in order to evaluate the efficacy of treatment. For to the culture medium at a temperature of 42–45 °C and then those measurements, the plates were photographed with a Pana- 10 ml of this mixture were poured into disposable Petri dishes sonic Industrial Colour Camera (model GP-KR222). The diameters (90 15 mm, Massobact). The medium used was MEA (Biokar), were obtained from the photos by using the programme Corel which contained 30 g of malt extract, 15 g of agar and 3 g of Draw 11 Windows in the computer. The radius of the colony was 1414 M.P. Salas et al. / Food Chemistry 124 (2011) 1411–1415 plotted against time, and linear regression was applied in order to Table 2 obtain the growth rate constant (k) as the slope of the line to the x- Reduction percentage of growth rate constants against control. axis. The lag phase (lag) prior to growth was also calculated when % k reduction at 0.25 mM the colony diameter reached approximately 17 mm, the size that Flavonoids F. semitectum P. expansum A. parasiticus A. flavus duplicated the initial inoculum diameter. PB 36 22 49 49 In some published investigations, the concentrations most com- PD 54 44 54 49 monly used to prove the antifungal activity of flavonoids varied P29443841 from 0.2 to 0.8 mM (Carrillo, Gomez Molina, & Benitez Ahrendts, PS 7 11 36 39 1999; Weidenbörner, Hindorf, Jha, Tsotsonos, & Egge, 1990; PL 43 11 38 37 Weidenbörner & Jha, 1994a; Weidenbörner & Jha, 1994b; HG 43 33 38 41 HGL 46 56 54 41 Weidenbörner & Jha, 1997). In this study, we chose to work with HES 39 11 33 33 a concentration near the lower limit, 0.25 mM. The study was car- NEO 50 44 38 41 ried out in quintuplet, in disposable Petri dishes. In each case, the NAR 29 22 41 41 sowing volume was 10 ll (10 ± 0.2 ll). The dishes were incubated PB: prunin butyrate, PD: prunin decanoate, P: prunin, PS: prunin stearate, PL: at 25 °C±1°C in darkness for the different periods of time. prunin laurate, HG: hesperetin glucoside, HGL: hesperetin glucoside laurate, HES: hesperidin, NEO: neohesperidin, NAR: naringin.

2.2.3. Statistical analysis this concentration. However, Weidenbörner and Jha (1994b) Data analyses were performed by analysis of variance. Two-way showed that some flavonoids had antifungal effects against some ANOVA was applied to determine differences (p <0.05). To ascer- moulds but they stimulated the growths of others, depending, tain significant differences between levels of the main factor, Bon- not only on the fungus, but also on the concentration used. For ferroni´s test was applied between means. ANOVAs were used to example, at 0.2 mM, NAR and HES reduced the mycelial growth determine the influence of the different flavonoids among lag, k of Spicellum roseum, but NAR at the same concentration, stimulated and the interaction between lag and k. The analysis was conducted the growth of Alternaria tenuissima. Another study also showed using the programme R (Statistical Software). that NAR (at 1.72 mM) had no antifungal activity against A. niger or C. albicans (Rauha et al., 2000). In our study, NAR at 0.25 mM 3. Results and discussion inhibited the mycelial growth of all the studied moulds. Table 3 shows the effect of all ten flavonoids on lag (h) prior to All ten flavonoids, at 0.25 mM, showed the capacity to alter the growth for all the moulds. For F. semitectum, A. parasiticus and A. growth of A. parasiticus, A. flavus, F. semitectum and P. expansum. At flavus, PD was again the flavonoid with the strongest activity, this concentration total inhibition was not achieved for any of the showing the highest increment on lag. It is interesting to note that, moulds. For all fungi and flavonoid tested, the k and lag were cal- despite that all moulds having their lag phase affected, there was a culated. Table 1 shows the values of k obtained, calculated from similar tendency in the behaviour of the Aspergillus strains. P. linear regression in each case, and the range of quadratic regres- expansum was more dependent on the type of flavonoids than were sion coefficients (R2). Table 2 shows the values of percent growth the rest of the fungi. reductions at 86 h for all the moulds and flavonoids tested. The interaction between lag and k with the different flavonoids In the case of A. parasiticus, A. flavus and F. semitectun the PD was was examined by considering the variable flavonoid as a fixed the flavonoid that showed the strongest inhibition capacity. source for each mould and the results are shown in Tables 1 and Although PD showed antifungal activity against P. expansun,it 3 for k and lag, respectively. The interaction (mould–flavonoid) be- was HGL which provided the highest degree of inhibition in this tween k and lag was significant according to the ANOVA test (data mould. P. expasum was also sensitive to NEO and P. Besides PD, F. not shown). semitectun also showed a strong inhibition with NEO. For A. parasit- There is little information about the structure-antifungal activ- icus, HGL had the same effect in the inhibition as did PD. PB also ity relationships of flavonoids. Weidenbörner and Jha (1997) achieved an important percentage of growth reduction for A. para- showed, for some moulds, that in the case of flavones, fungicidal siticus, as well as A. flavus. Similar antifungal behaviour of the two activity is reduced if one or more hydroxyl or methoxy groups flavanones, at 0.8 mM against Penicillium digitatium, was also found are introduced; for flavanones, the unsubstituted molecule had a by Ortuño et al. (2006) who showed that HES and NAR reduced the higher effectivity than had hydroxylated or methoxylated flava- mycelial growth of these moulds by 38% and 25%, respectively, at nones. Another study (Silva, Weidenbörner, & Cavaleiro, 1998)

Table 1 Growth rate constants (mm/h) obtained for F. semitectun, P.expansum, A.parasiticus and A.ﬂavus with the different ﬂavonoids.

K (mean ± SD, n = 5) and range of R2 (quadratic correlation coefﬁcient) Flavonoids F. semitectum P. expansum A. parasiticus A. ﬂavus control 0.28 ± 0.04k (0.989–0.996) 0.09 ± 0.03i (0.983–0.980) 0.39 ± 0.01e (0.991–0.996) 0.49 ± 0.01a (0.980–0.984) PB 0.18 ± 0.06lm (0.970–0.996) 0.07 ± 0.02ij (0.965–0.983) 0.20 ± 0.01gh (0.990–0.967) 0.25 ± 0.01d (0.977–0.990) PD 0.13 ± 0.06m (0.975–0.999) 0.05 ± 0.01ij (0.969–0.987) 0.18 ± 0.01h (0.991–0.999) 0.25 ± 0.02d (0.986–0.989) P 0.20 ± 0.03klm (0.984–0.999) 0.05 ± 0.02ij (0.956–0.992) 0.24 ± 0.01f (0.992–0.997) 0.29 ± 0.03cd (0.981–0.989) PS 0.26 ± 0.02kl (0.984–0.998) 0.08 ± 0.01ij (0.960–0.988) 0.25 ± 0.01f (0.993–0.977) 0.30 ± 0.03bc (0.981–0.984) PL 0.16 ± 0.02m (0.983–0.996) 0.08 ± 0.02ij (0.981–0.999) 0.24 ± 0.01fg (0.986–0.992) 0.31 ± 0.03bc (0.981–0.989) HG 0.16 ± 0.03m (0.993–1.00) 0.06 ± 0.02ij (0.961–0.996) 0.24 ± 0.01fg (0.992–0.998) 0.29 ± 0.01cd (0.985–0.996) HGL 0.15 ± 0.05m(0.974–0.998) 0.04 ± 0.01j (0.856–0.983) 0.18 ± 0.04h (0.969–0.985) 0.29 ± 0.01bcd (0.980–0.986) HES 0.17 ± 0.04m (0.993–0.999) 0.08 ± 0.01ij (0.977–0.986) 0.26 ± 0.01f (0.991–0.946) 0.33 ± 0.01b (0.982–0.993) NEO 0.14 ± 0.03m (0.981–0.998) 0.05 ± 0.01j (0.975–0.995) 0.24 ± 0.01fg (0.992–0.996) 0.29 ± 0.01bcd (0.981–0.987) NAR 0.20 ± 0.03lm (0.995–0.998) 0.07 ± 0.01ij (0.980–0.984) 0.23 ± 0.01fg (0.994–0.996) 0.29 ± 0.03bcd (0.990–0.999)

PB: prunin butyrate, PD: prunin decanoate, P: prunin, PS: prunin stearate, PL: prunin laurate, HG: hesperetin glucoside, HGL: hesperetin glucoside laurate, HES: hesperidin, NEO: neohesperidin, NAR: naringin. Values followed by the same small letter within the same column are not signiﬁcantly different (p > 0.05) according to Bonferroni´s multiple range test. M.P. Salas et al. / Food Chemistry 124 (2011) 1411–1415 1415

Table 3 Benavente-García, O., Castillo, J., Marin, F. R., Ortuño, A., & Del Río, J. A. (1997). Uses Effects of the differents flavonoids on lag phase (h). and properties of citrus flavonoids. Journal of Agricultural and Food Chemistry, 45(12), 4505–4515. lag phase (h) (mean ± SD, n =5) Carrillo, L., Gomez Molina, S. E., & Benitez Ahrendts, M. (1999). La acción de la naringenina y la naringina sobre varios hongos contaminantes. Revista Científica Flavonoids F. semitectum P. expansum A. parasiticus A. flavus Agraria, 3, 11–14. Control 14 ± 7p 40 ± 7ijk 9±3g 25 ± 2c Demirci, B., Kosßar, M., Demirci, M., Dinç, M., & Basßer, K. H. C. (2007). Antimicrobial PB 11 ± 3p 57 ± 20hij 29 ± 2d 38 ± 7ab and antioxidant activities of the essential oil of Chaerophyllum libanoticum Boiss. PD 57 ± 8m 42 ± 12jki 29 ± 1d 40 ± 5a et Kotschy. Food Chemistry, 105(4), 1512–1517. P22±8nop 84 ± 13h 21 ± 2def 38 ± 3ab Ellenrieder, G., Blanco, S., & Daz, M. (1998). Hydrolysis of supersaturated naringin PS 37 ± 1mno 6±12kl 15 ± 1fg 32 ± 1abc solutions by free and immobilized naringinase. Biotechnology Techniques, 12, 63–65. PL 39 ± 2mn 2±12l 8±6g 30 ± 4bc Ellenrieder, G. (2004). Biotransformations of citrus flavanone glycosides. In A. HG 18 ± 2op 74 ± 25hi 18 ± 2fe 37 ± 2ab Pandey (Ed.), Concise encyclopaedia on bioresource technology (pp. 189–197). HGL 15 ± 12p 43 ± 15hijkl 7±7g 36 ± 2ab op jkl fg bc USA: Harworth Press Inc. HES 18 ± 3 25 ± 20 15 ± 15 31 ± 2 Fraternale, D., Giamperi, L., & Ricci, D. (2003). Chemical composition and antifungal p hi fe bc NEO 11 ± 3 73 ± 20 19 ± 4 30 ± 4 activity of essential oil obtained from in vitro plants of Thymus mastichina L. p hi de bc NAR 11 ± 11 69 ± 13 25 ± 3 29 ± 1 Journal of Essential Oil Research, 15, 278–281. Geronazzo, H., Robin, J., Blanco, S., Cuevas, C., & Ellenrieder, G. (2000). PB: prunin butyrate, PD: prunin decanoate, P: prunin, PS: prunin stearate, PL: Aprovechamiento integral de residuos de producción y procesamiento de prunin laurate, HG: hesperetin glucoside, HGL: hesperetin glucoside laurate, HES: pomelos. Un proyecto de innovación tecnológica. Anales del VIII Congreso hesperidin, NEO: neohesperidin, NAR: naringin. Values followed by the same small Argentino de Ciencia y Tecnología de los Alimentos. Libro de Resúmenes. 1900, letter within the same column are not significantly different (p > 0.05) according to (p.119). Available from www.sicytar.secyt.gov.ar/busqueda/prc_imp Bonferroni´s multiple range test. _cv_int?f_cod=0000534730. González-Molina, E., Domínguez-Perles, R., Moreno, D. A., & García-Viguera, C. (2010). Natural bioactive compounds of Citrus limon for food and health. Journal showed that the flavonoid mixture resulted in a synergistic effect, of Pharmaceutical and Biomedical Analysis, 51, 327–345. causing a greater inhibition than each substance alone at different Macoritto, A., Geronazzo, H., & Ellenrieder, G. (2001). Obtención de hesperidina a concentrations. Possible action mechanisms in which mycelial partir de naranjas de derrame. Información Tecnológica, 12(4), 3–8. Macoritto, A., Robin, J., Blanco, S., & Geronazzo, H. (2004). Obtencion de flavonoides growth could be reduced are not yet clear. Our results do not yet de frutas inmaduras de Citrus aurantium. Innovacion, 16, 39–42. indicate possible mechanisms by which fungi growth can be re- Mandalari, G., Bennett, R. N., Bisignano, G., Trombetta, D., Saija, A., Faulds, C. B., et al. duced, nor any structure–antioxidant capacity relationship. (2007). Antimicrobial activity of flavonoids extracted from bergamot (Citrus bergamia Risso) peel, byproduct of the essential oil industry. Journal of Applied Microbiology. ISSN 1364-5072. Ortuño, A., Bavidez, A., Gómez, P., Arcas, M. C., Porras, I., García-Lidón, A., et al. 4. Conclusions (2006). Citrus paradisi and Citrus sinensis flavonoids: Their influence in the defense mechanism against Penicillium digitatum. Food Chemistry, 98, 351–358. At the concentration tested, the flavonoids, naringin, prunin, Proestos, C., Boziaris, I., Nychas, J. E., & Komaitis, M. (2006). Analysis of flavonoids and phenolic acids in Greek aromatic plants: Investigation of their antioxidant prunin butyrate, prunin decanoate, prunin laurate, prunin stearate, capacity and antimicrobial activity. Food Chemistry, 95, 664–671. hesperidin, neohesperidin, hesperetin glucoside and hesperetin Rauha, J., Remes, S., Heinone, M., Hopia, A., Kähkönen, M., Kujala, T., et al. (2000). glucoside laurate reduced growth rate consistently. Nevertheless, Antimicrobial effects of Finnish plant extracts containing flavonoids and other phenolic compounds. International Journal of Food Microbiology, 56, 3–12. this activity depended on the type of mould and flavonoid used. Romero, C., Manjon, A., Bastida, J., & Iborra, J. L. (1985). A method for assaying the This investigation is, for some flavonoids, a first approach to the rhamnosidase activity of naringinase. Analytical Biochemistry, 149, 566–571. study of these compounds as potential natural fungicides. No gen- Rota, M. C., Herrera, A., Martínez, R. M., Sotomayor, J. A., & Jordán, M. J. (2007). Antimicrobial activity and Chemicals composition of Thymus vulgaris, Thymus eral rule can be proposed yet to explain the antifungal activity and zygis and Thymus hyemalis Essentials oils. Food Control, 19, 681–687. more studies about their activity and action mechanism should be Salah, N., Miller, N. J., Paganga, G., Tijburg, L., Bolwell, G. P., & Rice-Evans, C. (1995). carried out. Nonetheless, the possible use of these flavanones as Polyphenolic flavonols as scavenger of aqueous phase radicals and as chain– natural fungicidal agents opens an interesting option for the citrus breaking antioxidants. Archives of Biochemistry Biophysics, 2, 339–346. Silva, A. M. S., Weidenbörner, M., & Cavaleiro, J. A. S. (1998). Growth control of industries. different Fusarium species by selected flavones and flavonoid mixtures. At the concentration assayed, none of the flavonoids totally Mycological Research, 102(5), 638–640. inhibited the growth of the tested moulds, so further investigations Soria, F., & Ellenrieder, G. (2002). Thermal inactivation and product inhibition of Aspergillus terreus CECT 2663 a-l-rhamnosidase and their role on hydrolysis of of higher concentrations, mixtures of different flavonoids and other naringin solutions. Bioscience, Biotechnology and Biochemistry, 66, 1442–1449. dispersion solvents, should be tested in order to inhibit the mould Tripoli, E., La Guardia, M., Giammanco, S., Di Majo, D., & Giammanco, M. (2007). growth completely. Also, these flavonoids should be tested in dif- Citrus flavonoids: Molecular structure, biological activity and nutritional properties: A review. Food Chemistry, 104(2), 466–479. ferent food matrices. Viuda-Martos, M., Ruiz-Navajas, Y., Fernández-López, J., & Pérez-Álvareza, J. (2008). Antifungal activity of lemon (Citrus lemon L.), mandarin (Citrus reticulata L.), grapefruit (Citrus paradisi L.) and orange (Citrus sinensis L.) essential oils. Food Acknowledgments Control, 19, 1130–1138. Wächter, G. A., Hoffmann, J. J., Furbacher, T., Blake, M. E., & Timmermann, B. N. (1999). Antibacterial and antifungal flavanones from Esynhardita texana. 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