Open access peer-reviewed chapter

Antioxidant Capacity and Food Pathogenic Bacteria Inhibition of Citrus limetta and Citrus reticulata

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Andrés Alejandro Damian-Reyna, Juan Carlos González-Hernández, Jesús Fernando Ayala-Zavala, Consuelo de Jesús Cortes Penagos, Rafael Maya-Yescas and Ma del C. Chávez-Parga

Submitted: April 7th, 2018 Reviewed: June 1st, 2018 Published: November 5th, 2018

DOI: 10.5772/intechopen.79253

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Abstract

In this study, phenolic compounds in the juice, seed and bagasse of C. limetta and C. reticulata cultivated in Mexico at two ripening stages were determined, and their antioxidant capacities were evaluated using 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH), 2,2′-azin-bis-(3-etilbenzotiazolin-6-sulfonic acid) (ABTS) and oxygen radical absorption capacity test (ORAC) methods, as well as their antibacterial growth inhibition. We found that bagasse had the highest total phenol content and the highest total flavonoid content. The dominant flavonoid, hesperidin, was observed to be the highest in bagasse. Ascorbic acid was analyzed and C. limetta juice and C. reticulata bagasse had the highest contents. Antioxidant capacity showed variations in both, C. limetta and C. reticulata, juices which had the highest ABTS value; C. limetta juice and C. reticulata bagasse had the highest DPPH value; C. limetta juice and C. reticulata bagasse had the highest ORAC value. C. limetta and C. reticulata extracts showed the bactericidal effect at the range of 4–40 mg/mL, assayed against Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella enterica and Staphylococcus aureus. Overall, ripeness increased total phenol content (TPC), total flavonoid content (TFC), hesperidin content, antioxidant capacity and bactericidal effect. These results may provide useful information for future utilization of C. limetta and C. reticulata.

Keywords

  • citrus
  • phenolic compounds
  • antioxidant
  • bactericidal
  • ripeness

1. Introduction

At present, there is a growing interest in the effective, economic and innocuous obtaining of extracts with antioxidant and antibiotic properties from natural matrices [1], which opens a field of investigation in the extraction fields, purification and characterization of plant extracts obtained from secondary products [2]. Given a large number of bioactive compounds present in plant extracts, several mechanisms of action may act simultaneously. The two main antibacterial effects that have been identified are (i) the opening of pores in the bacterial membrane at concentrations below the minimum inhibitory concentration (MIC), which leads to the leakage of intracellular components in the period initial contact, without causing a decrease in the viability of the bacteria. This effect can be reversed inappropriate cultivation conditions only when there have been short periods of exposure to the bactericidal agent. When the bacteria are exposed to concentrations above the MIC, the second effect occurs, (ii) causing irreversible damage to the membrane, due to the modification of the carboxyl groups of the fatty acids of the membrane or by reaction with the polysaccharides of the cell wall. This effect can also be seen when bacteria are exposed to MIC for an extended period, affecting cell viability [3]. Citrus species such as C. aurantifolia, C. sinensis, C. paradisi, C. reticulataand C. limettaare widely cultivated in Mexico and other parts of the world; some secondary metabolites of these fruits have shown bactericidal activity, helping to combat some infections in humans [4, 5, 6]; they are also useful as antivirals (herpes, influenza, etc.) [6], antifungal [7] and antibacterial [8, 9]. Similarly, these bioactive molecules can form the structural basis for developing new antibiotics, more effective, less expensive and with fewer collateral effects [6, 10]. A group of polyphenols’ with microbicide properties present in citrus fruits are flavonoids [6]. In particular, phenolic and flavonoid compounds [11] have attracted the attention of the scientific community due to a well-established connection between the consumption of flavonoids and the prevention of sufferings [12]. These compounds have shown the antibacterial effect at diluted concentrations, with the rupture of the membrane show the mechanism of action [13]. Altering the macromolecular structure of bacterial membranes affected the carboxylic groups of the fatty acid in the membrane [3]. The almost daily obtaining of new evidence of these compounds interfering with several virulence factors in pathogenic bacteria, including enzymes, toxins and signal receptors opens the possibility of using them in anti-infection therapies or even the development of new drugs [5]. Several recent publications report the regular presence of antibacterial activity of the isolated flavonoids [14]; with MIC for heperidine against E. coliCCTCC AB94014 de 800 μg/mL and against S. aureusCCTCC AB9105 of 200 μg/mL [15]; for naringine against E. coliATCC 35218 of 4 μg/mL, against P. aeruginosaATCC 10145 de 2 μg/mL and against S. aureusATCC 25923 of 16 μg/mL; for nobiletine against E. coliCCTCC AB94014 of 1600 μg/mL and against S. aureusCCTCC AB9105 of 1600 μg/mL [15]; for quercitine against E. coliATCC 35218 de 4 μg/mL, against P. aeruginosaATCC 10145 de 4 μg/mL and against S. aureusATCC 25923 de 2 μg/mL; for tangeretine against E. coliCCTCC AB94014 of 1600 μg/mL and against S. aureusCCTCC AB9105 of 1600 μg/mL [15]; of gallic acid against E. coliATCC 35218 of 4 μg/mL, against P. aeruginosaATCC 10145 of 2 μg/mL, against S. aureusATCC 25923 of 16 μg/mL [6], which are similar to bacterial inhibition of grape seed extracts with MIC against S. aureusATCC 25923 of 1.25 mg/mL, against L. monocytogenesZM58 > 10 mg/mL, against E. coliO157: H7 of 2.5 mg/mL [16].

IMDiameter (cm)High (cm)Weight (g)Bagasse (%)Juice (%)Seed (%)
C. limetta17.75.83 ± 0.31a5.73 ± 0.32a92.33 ± 9.06a53.76 ± 3.50a,b39.51 ± 6.15a,b1.93 ± 0.37a
9.95.03 ± 0.46a4.97 ± 0.35a64.79 ± 10.81a60.49 ± 6.15b35.02 ± 0.24a1.18 ± 0.64a
C. reticulata9.56.23 ± 0.21a5.13 ± 0.21a109.54 ± 17.22a26.76 ± 7.26a52.57 ± 0.26b2.83 ± 0.75a
6.36.07 ± 0.40a5.00 ± 0.36a107.32 ± 7.81a31.83 ± 2.53a,b49.46 ± 0.23a,b2.06 ± 0.49a

Table 1.

Characterization of the used fruits.

Each value is the average of three replications ± deviation standard. The different superscripts indicate significant differences between samples (p < 0.05).

This has boosted the development of commercial citrus extracts, with a good bactericidal performance and MIC of 20–80 ppm against S. entericaCECT 4300 [3]. Still, some citrus compounds do not inhibit certain bacteria such as L. monocytogenes01/155 and 99/287, P. aeruginosaATCC27853, S. aureusATCC29213, and E. coliO157:H7, such as those who showed no inhibition at concentrations up to 0.25 mM of Naringin [17]. Currently, research on citrus-derived antioxidant compounds has neglected the study of low-crop species, such as C. limettaand C. reticulata, which also have compounds with potential bactericidal effect [11]. The extracts of C. limettaand C. reticulatahave shown good antioxidant capacity in tests with the 2,2-diphenyl-1-picrylhydrazyl-hydrate (DPPH), with values of 261 ± 9 μM Trolox equivalent (TE) in juice of C. limetta[11], 210 ± 37.0 mg/mL in seed of C. reticulata[18], 48.8 ± 0.91 mg vitamin C equivalent (VCE)/100 ml in C. reticulatajuice [19], de 9.10 ± 0.68 a 19.75 ± 0.87 μM TE/g, in pulp of C. reticulata[20] and 29.04 ± 1.20 to 50.46 ± 3.57 μM TE/g, in shell of C. reticulata[21]. By the method with acid 2,2′-azin-bis-(3-etilbenzotiazolin-6-sulfonic acid) (ABTS) have been found 1446 ± 30 μM TE in C. limettajuice [11], 59.3 ± 0.23 mg VCE/100 ml in C. reticulatajuice [19], of 22.92 ± 0.32 to 34.28 ± 1.12 μM TE /g in C. reticulatapulp [20]; and of 65.62 ± 1.43 to 108.60 ± 0.24 μM TE/g in C. reticulatashell [21]. Currently, research on citrus-derived antioxidant compounds has neglected the study of low-crop species, such as C. limettaand C. reticulatawhich also have compounds with potential bactericidal effect [11]. The extracts of C. limettaand C. reticulatahave shown good antioxidant capacity in test with the 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH), with values of 261 ± 9 μM Trolox equivalent (TE) in juice of C. limetta[11], 210 ± 37.0 mg/mL C. reticulataseed [18], 48.8 ± 0.91 mg vitamin C equivalent (VCE)/100 ml in juice of C. reticulata[19], de 9.10 ± 0.68 to 19.75 ± 0.87 μM TE/g, in pulp of C. reticulata[20] and 29.04 ± 1.20 to 50.46 ± 3.57 μM TE/g, in C. reticulatashell [21]. With the method of 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), it has been found 1446 ± 30 μM TE in C. limetta juice [11], 59.3 ± 0.23 mg VCE/100 ml in C. reticulata juice [19], de 22.92 ± 0.32 a 34.28 ± 1.12 μM TE/g in C. reticulata pulp [20] and de 65.62 ± 1.43 a 108.60 ± 0.24 μM TE/g in C. reticulata shell [21]. Moreover, these extracts have shown antioxidant capacity of oxygen radicals in the oxygen radical absorption capacity test (ORAC), with values of 126.90 ± 1.96–13.22 ± 231.29 μM TE/g in C. reticulatapulp [20] and of 395.66 ± 14.42 to 834.37 ± 16.98 μM TE/g in C. reticulatashell [21].

Consequently, citrus fruits are of great importance due to the interest of finding new sources of antibacterial and antioxidant compounds. However, no work has been done in bacterial inhibition and antioxidant capacity of citrus fruits to different stages of maturation [22]. Therefore, in this research, it was determined the bactericidal activity and antioxidant capacity of bagasse, juice and seeds of two citrus species cultivated in Mexico, C. limettaand C. reticulata, in two stages of maturity (Table 1). The results were compared with those found in the literature, in order to provide innovative information for future applications of these citrus species.

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2. Methodology

2.1. Obtaining the extracts

The fruits of C. limetta and C. reticulatawere acquired in the city of Morelia, Michoacán, from different regions of the same state. To obtain the extract, the juice, the shells and the seeds were separately dried in the oven at a temperature of 40°C for 48 h and then ground. The materials were washed with acetone (Meyer, México) in proportion 1:1 (g of material: mL of acetone) in a flask. The mixture was stirred for 30 min at 200 rpm, at room temperature and then filtered to vacuum. The operation was repeated with hexane (J.T. Baker, México) in proportion 1:4 (g of material: mL of hexane). For the extraction of phenolic and flavonoid compounds, 4 mL of methanol (J.T. Baker, México) was used for each gram of material, stirring the mixture for 30 min at 200 rpm [23] at room temperature. The mixture is vacuum-filtered and the extracts obtained are stored in refrigeration at 4°C until posters test.

Total phenol content (TPC) was tested using the reagent of Folin-Ciocalteau (Hycel, Mexico) with a standard of Gallic acid (Golden Bell, Mexico), and absorbance was measured at 750 nm [24]. An aliquot of 0.1 mL of the methanol extract is diluted with 0.4 ml of distilled water; then, the solution was mixed with 2.25 mL of Folin-Ciocalteau reagent to 10% and 2.25 mL of solution (Golden Bell, Mexico) (20 g/mL of distilled water) of sodium carbonate.

Absorbance was measured after 2 h of incubation at 750 nm compared to a methanol target (J.T. Baker, Mexico), using a UV/Vis spectrophotometer (Jenway, Model 7305). The TPC was expressed in mg equivalents of Gallic acid (GAE) by g of dry matter (DM) of bagasse and seeds and in mg GAE/mL for the juices. The total flavonoid content (TFC) was analyzed with a modification of the spectrophotometric method described by Abeysinghe et al. [12]. A 1.5 mL of methanol was added to 0.1 ml of the diluted extract, to then add 0.1 mL of solution 6.8 g/50 mL of distilled water of aluminum chloride (Golden Bell, Mexico), and the resulting solution was diluted with distilled water to a final volume of 5 mL. The mixture was stirred, it was left to rest for 30 min and its absorbance was measured at 510 nm. The TFC was calculated using a calibration curve of quercetin (Aldrich, USA) and the results were expressed in mg equivalent of quercetin (QE) per grams of dry matter (DM) of bagasse and seeds and in mg QE/mL for the juices. The quantitative analysis of hesperidin and ascorbic acid was performed in a high-performance liquid chromatography (HPLC) Varian LC920 equipped with a column C18 Varian, 25 cm × 4.6 mm I.D. and a diode array detector. The TFC was calculated using a calibration curve of quercetin (Aldrich, USA) and the results were expressed in mg equivalent of quercetin (QE) per grams of dry matter (DM) of bagasse and seeds and in mg QE/mL for juices. The quantitative analysis of hesperidin and ascorbic acid was performed in a high-performance liquid chromatography (HPLC) Varian LC920 equipped with a column C18 Varian, 25 cm × 4.6 mm I.D. and a diode array detector. Mobile phases of KH2PO4 20 mM, phosphoric acid at 0.1%v, and 1%v methanol for the determination of ascorbic acid were used; 33%v methanol and 67%v water to determine hesperidin. Absorbances were measured at 215 nm and 283 nm, respectively. The samples were analyzed in duplicate, and the calibration curves were constructed from the average areas of the peaks. The contents of hesperidin (HD) and ascorbic acid (AA) expressed in mg/g DM for bagasse and seeds and in mg/mL for juices.

2.2. Determination of minimum inhibitory and bactericidal concentrations

The evaluation of the minimum inhibitory concentration (MIC) and minimum concentration bactericidal (MBC) It was carried out by microdilution of the extract in culture medium [25], in concentrations of 2–40 mg/μL. A 5 mL of bacterial suspension was placed in culture medium (cetrimide broth for P. aeruginosa, Muller-Hinton broth for the rest of the bacteria) in a sterile microplate of 96 wells. The volume was completed with the dilutions of the extract in the culture broth to obtain the test concentrations. Control wells were prepared with culture broth, diluted extract and bacterial suspension separately. The microplate was incubated for 24 h at 37°C. The MIC was the lowest concentration where no viability was observed in the well after 24 h [16]. To determine the MBC, 20 μL were taken from wells where no growth was observed and transferred to a plate with solid agar (Pseudomonasagar for P. aeruginosa, Muller-Hinton agar for the other bacteria) and incubated for 24 h at 37°C. MBC was the lowest concentration of colony-forming units (CFU) growth after the incubation period. Positive controls were wells with bacterial suspension in breeding stock. The negative controls were wells with culture broth and with the dilution of the extract. All the determinations of MIC and MBC were repeated in triplicate.

2.3. Test organisms

Escherichia coliATCC 15597, Listeria monocytogenesATCC 7644, Pseudomonas aeruginosaATCC 10145, Salmonella entericaATCC 14028 and Staphylococcus aureusATCC 6538 were used as test organisms.

2.4. Antioxidant capacity

The antioxidant capacity was determined by testing the oxidation inhibition of the acid radicals 2,2′-Azino-bis-(3-etilbenzotiazolin-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH), as well as the absorption capacity of oxygen radicals (ORAC).

The capacity of the extracts was measured to inhibit the DPPH radical by placing in a microplate 10 μL of the extract solution to the given concentration and added 140 μL of the DPPH radical adjusted to an absorbance of 0.70. The reaction was allowed to elapse for 30 min and the absorbance of the solution was read at 518 nm. The percentage of inhibition was calculated and the results were expressed in μmol equivalents of Trolox per milligram of a sample (μmol TE/mg), using a standard curve of this antioxidant. The tests were carried out in triplicate [26].

The determination of the inhibition of the radical ABTS was carried out by adding in a well of microplate 5 μL of extract to the appropriate dilution of test and 245 μL of radical ABTS solution adjusted to an absorbance of 0.70. It was allowed to react for 5 min and the absorbance was measured at 754 nm. The percentage of inhibition was calculated and the results were expressed in μmol TE/mg using a standard curve of Trolox [1]. Tests were carried out in triplicate.

The reaction mix for the ORAC test was prepared with 150 μL de fluorescein 10 μM, 25 μL of Trolox standard (standard curve of 6.25–200 μM), 25 μL of phosphate buffers (75 μM, pH 7.4) like control and 25 μL of extract; the reaction begin with the addition of AAPH (2,2′-Azobis(2-amidinopropane) dihydrochloride, 240 mM). The fluorescein drop was evaluated every 90 s during an hour and a half, at an excitation wavelength of 485 nm and a wavelength of 520 nm with a microplate reader FLUOstar Omega (BMG Labtech Inc., USA). The ORAC values were calculated using a linear regression equation of a standard Trolox curve [27]. The results were expressed as equivalent μmol of Trolox per milligram (μmol TE/mg).

2.5. Analysis of statistics

There were three replicas and at least three independent experiments. The data were presented as mean ± deviation standard (SD). STATGRAPHICS Centurion XVI.I version was used for the statistical analysis. (Statpoint Technologies, Inc., Warrenton, VA, USA). Differences between groups were detected by ANOVA and Tukey multiple comparison tests; pvalues less than 0.05 were considered as statistically significant.

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3. Results and discussion

3.1. Effect of maturity on the contents of hesperidin, ascorbic acid, TPC and TFC

The highest values of TPC were found in bagasse, followed by juice and finally in the seed, for both fruits regardless of the level of maturity (Figure 1). Changes in the maturation in the TPC showed similar trends in pulp, juice and seed. It was determined an increase of 13.3% 14.4% at the TPC of bagasse for the maturity of 20.38 ± 0.97 to 23.09 2.57 ± and 34.95 ± 0.11 to 39.97 ± 1.25 mg GAE/g DW) for C. limettaand C. reticulata, respectively. The TPC juice content increased 16.62% and 0.90% during maturation (of 10.98 ± 0.14 to 12.80 ± 1.20 and 17.15 ± 0.42 to 17.30 ± 1.14 mg GAE/g DW). The content TPC in seed increased 2.59 ± 0.95 to 5.63 ± 0.19 and of 6.85 ± 0.95 to 7.46 ± 0.95 mg GAE/g DW, which did not represent a significant difference to p > 0.05. It is possible that the variations are due to differences in culture, origin, growth conditions and the same extraction process. Phenolic compounds are secondary metabolites that have mainly been correlated with antioxidant activity in several fruits, vegetables and grains [12]. The presence of a high TPC in pulp and juice C. limettaand C. reticulataconfirms the nutritional value of these fruits and the presence of phenols in the seeds makes them an alternative source for later uses.

Figure 1.

Content of total phenols (TPC) enC. limettayC. reticulata.

The highest values of TFC were found in bagasse, followed by juice and finally in the seed, for both fruits regardless of the level of maturity (Figure 2). Changes in the maturation in the TFC showed similar trends in pulp, juice and seed. The higher content of TFC was found in bagasse for both fruits. It measured an increase of 7.11% and 2.23% in the TFC of bagasse during ripening of 1579.03 ± 12.85 to 1691.33 ± 22.83 and of 1892.63 ± 6.89 to 1934.89 ± 7.58 μg QE/g DW) for C. limettaand C. reticulata, respectively. The content of total flavonoids in juice increased 48.33% and 84.62% during maturation (of 694.70 ± 15.19 to 1030.44 ± 59.58 and from 1709.78 ± 6.23 to 1892.63 ± 6.89 μg QE/ g DW). The TFC in seed increased from 230.74 ± 14.56 to 344.76 ± 15.59 and from 499.65 ± 18.61 to 535.15 ± 40.96 μg QE/g DW, which did not represent a significant difference to p > 0.05. It is possible that the variations are due to differences in culture, origin, growth conditions and the same extraction process. Phenolic compounds show flavonoids may inhibit radical free and catch reactive oxygen species (ROS) and therefore provide an effective means to prevent and treat ailments promoted by free radicals [28]. The presence of high TFC in C. limettaand C. reticulatadefines them as a significant source of antioxidants with potential prophylactic applications and in the development of functional foods.

Figure 2.

Content of total flavonoids (TFC) inC. limettayC. reticulata.

Hesperidin content was determined by an analysis of HPLC-DAD. The presence of hesperidin was obviously different between bagasse, juice and seed and was influenced by maturation (Figure 3). In general, the highest value of hesperidin was registered in bagasse, followed by juice and seed. However, the variation was larger in C. limettaduring maturation. Large amounts of hesperidin were identified not only in bagasse but also in juice and seed, which is consistent with levels reported previously for C. reticulata[28, 29, 30]. The concentration of hesperidin in bagasse C. limettaand C. reticulataincreasing during ripening of 397.37 ± 20.01 to 617.21 ± 70.73 and 966.49 ± 14.68 to 978.89 ± 43.46 μg/g DW, respectively. Similarly, it increased the amount of hesperidin in the juice of 129.45 ± 24.81 to 444.97 ± 109.57 and 200.69 ± 22.01 to 255.54 ± 37.21 μg/g DW, respectively. Hesperidin is accumulated in seeds of C. limettaand C. reticulataduring maturation, and their quantities increased 90.25 and 14.46%, respectively.

Figure 3.

Contents of hesperidin inC. limettayC. reticulata.

The highest content of Ascorbic acid was found in bagasse, followed by juice and finally the seeds regardless of maturity for both fruits (Figure 4). The content of ascorbic acid in the pulp of C. limettaand C. reticulatadecreased slightly during the maturation of 3.13 ± 0.41 to 3.02 ± 0.22 and 3.83 ± 0.37 to 3.46 ± 0.20 mg/g DW for C. limettaand C. reticulata, respectively. During maturation, the content of ascorbic acid in juice decreased 1.96 ± 0.20 to 1.39 ± 0.10 and 3.47 ± 0.31 to 1.30 ± 0.08 mg/g DW. Ascorbic acid decreased from the seeds of C. limettaand C. reticulatawith maturation, and their number was reduced by a 20.80 and 27.31%, respectively. How the ascorbic acid is an essential nutrient to prevent scurvy, cancer, cardiovascular and nervous system diseases [31], they seem to be a beneficial and important source of this compound. The concentrations of metabolites found in the juices of C. reticulataare consistent with those reported previously [19, 32, 33]; also the amounts found in seed [18].

Figure 4.

Ascorbic acid content inC. limettayC. reticulata.

3.2. Antioxidant capacity

The results of this study showed, in general, similar trends in the inhibition of radical capacity for pulp, juice and seed and increased with maturation, as shown in Table 2. In the ABTS test, the highest values were found in juice, of 72.71 ± 1.72 to 82.19 ± 6.39 μmol TE/g DW from C. limettaand of 61.48 ± 1.40 to 62.00 ± 5.47 μmol TE/g DW of C. reticulata, being higher than the data reported in the literature [11, 19]; the inhibitory activity of bagasse with ABTS for C. limettaincreased during maturation, while it came down to the bagasse of C. reticulata, and their values agreed with previously reported data [20, 21]. ABTS of seed value did not change significantly with maturation and ranged from 6.06 ± 0.21 to 13.30 ± 1.07 μmol TE/g DW for C. limettaand 16.73 ± 1.39 to 42.82 ± 1.67 μmol TE/g DW for C. reticulata. The DPPH of juice C. limettais consistent with that reported by Barreca et al. [11]. The seeds of C. reticulatashowed good DPPH [18], but below that obtained for juice, as expected [19]; the highest DPPH antioxidant capability found in this study were for bagasse of C. reticulata(7.56 ± 0.88 μmol TE/g DW), which is higher than the values reported in previous studies [20, 21]. Although the DPPH values of seeds were lower than those of juice and pulp, this inhibiting activity DPPH implies a potential application as a valuable source of antioxidants. In general, values of DPPH showed similar trends for pulp, juice and seeds during maturation, with the exception of C. limettaand of C. reticulatajuice.

IMABTSDPPHORAC
C. limetta
Bagasse17.747.34 ± 2.84c,d,e1.64 ± 0.27a,b14.82 ± 0.33d
9.925.26 ± 2.21a,b,c3.31 ± 0.11a,b12.51 ± 0.17c
Juice17.782.19 ± 6.39f5.61 ± 0.62a,b11.97 ± 0.33c
9.972.71 ± 1.72e,f3.78 ± 1.06a,b11.33 ± 0.04c
Seed17.713.30 ± 1.07a,b0.61 ± 0.24a1.69 ± 0.01a
9.96.06 ± 0.21a0.13 ± 0.02a0.79 ± 0.04a
C. reticulata
Bagasse9.536.04 ± 2.27b,c,d7.56 ± 0.88b22.56 ± 0.28f
6.359.67 ± 5.31d,e,f3.65 ± 0.37a,b16.97 ± 0.13e
Juice9.562.00 ± 5.47d,e,f2.72 ± 0.86a,b22.56 ± 0.36f
6.361.48 ± 1.40d,e,f3.50 ± 1.27a,b22.07 ± 0.29f
Seed9.542.82 ± 1.67b,c,d,e2.08 ± 0.13a,b3.73 ± 0.12b
6.316.73 ± 1.39a,b1.60 ± 0.60a,b2.26 ± 0.05a,b

Table 2.

Antioxidant capacity of C. limettay C. reticulata, μmol TE/g DW ± SD.

Each value is the average of three replications ± standard deviation. The different superscripts indicate significant differences between samples (p < 0.05).

ORAC testing revealed that the pulp and juice of C. reticulatahad ORAC values significantly higher than pulp and juice of C. limetta, probably due to the higher levels of phenolic compounds and flavonoids in C. reticulata[11, 20, 21] which can lead to further investigation of this fruit. The ORAC values of seeds did not change significantly during maturation and were 0.79 ± 0.04 to 1.69 ± 0.01 μmol TE/g DW for C. limettaand 2.26 ± 0.05 to 3.73 ± 0.12 μmol TE/g DW for C. reticulata. A comparative evaluation indicates that the seeds of C. limettaand C. reticulatamay be an attractive source of antioxidants for future applications. The results suggest that both C. limettaas C. reticulatapossess a remarkable inhibitory activity of radical and therefore a significant antioxidant capacity.

3.3. Bacterial inhibition

Table 3 shows testing of antibacterial susceptibility of extracts against some Gram-positive and Gram-negative bacterial strains. Observed growth inhibition varied from one organism to another and a summary to another. Gram-positive strains showed values higher than the Gram-negative strains MIC and MBC. Extracts that showed greater bactericide effect were those obtained from bagasse for both fruits, with up to 3 mg/mL and MIC MBC 4 mg/mL (Table 3), which is consistent with the reported values of for essential oil of C. sinensis[34], this effect may be due to the increase in phenolic compounds and flavonoids during the ripening of fruit. S. aureuswas the microorganism tested that showed greater resistance to the extracts, with MIC above 31 mg/mL and MBC greater than 40 mg/mL for the majority of tests. L. monocytogeneshad MIC from 18 mg/mL and MBC from 13 mg/mL. E. colishowed MIC from 13 mg/mL and MBC from 22 mg/mL. P. aeruginosarecorded MIC from 3 mg/mL and MBC from 4 mg/mL. S. entericarecorded MIC from 9 mg/mL and MBC from 13 mg/mL. Extracts of C. reticulatashowed one inhibitory effect greater than the C. limetta, but still below that reported for extracts of green tea [16]. Lower bacterial inhibition was in seed for both fruits extracts, with MIC values higher to 36 mg/mL, but still below those obtained with extracts from grape seed [16]. Even so, extracts showed better antibacterial compounds isolated citruses such as quercetin, gallic acid [6] and naringin [17]; still better than the performance shown by some commercial fruit extracts [3].

Gram positiveGram negative
Listeria monocytogenesStaphylococcus aureusEscherichia coliPseudomonas aeruginosaSalmonella enterica
IMMICMBCMICMBCMICMBCMICMBCMICMBC
C. limetta
Bagasse17.7182231401322341322
9.99133140132234913
Juice17.740>40>40>40364031403140
9.940>40>40>40>40>4036403640
Seed17.7>40>40>40>40>40>4036403640
9.9>40>40>40>40>40>40364040>40
C. reticulata
Bagasse9.5223140>403140341822
6.3223140>4031409131822
Juice9.5273140>40314013222731
6.3314040>40364031403140
Seed9.5>40>40>40>40>40>40364040>40
>40>40>40>40>40>40>40>40>40>40

Table 3.

Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the extracts, mg/mL.

MIC, Minimum inhibitory concentration; MBC, minimum bactericidal concentration. Each value is the mean of three replicates with standard deviation <10%.

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4. Conclusions

We investigated the content and antioxidant activity of the phenolic compounds and flavonoids from two Mexican varieties of C. reticulataand C. limetta. Bagasse, juice and seeds of C. reticulataand C. limettacontain phenolic compounds and flavonoids. Its content increases as the maturity increases. The extracts show microbicide effect on microorganisms of study under in vitro conditions. The effect increases when rising the ripening of the fruit. Extracts of C. reticulatahave a higher bactericide effect than those obtained C. limetta, test microorganisms. As a result, the inhibition is directly related to the content of phenolic compounds and flavonoids. The extracts show antioxidative effect in vitro tests. The effect increases when rising the ripening of the fruit C. reticulatahas antioxidant capacity and content of secondary metabolites greater than C. limetta. Inhibition of oxidation is directly related to the content of phenolic compounds and flavonoids. Our findings suggest that C. reticulataand C. limetta, especially its bagasse, are good sources of antioxidant and antibacterial compounds. The results of citrus species analyzed in this study can motivate more widely used them in the pharmaceutical and food products.

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Acknowledgments

Authors express their sincere gratitude to the coordination of food of vegetable origin of the Center for Research in Nutrition and Development (Hermosillo, Sonora, Mexico) by providing microorganisms test and for their invaluable assistance in the determination of antioxidant activity. The work was partially supported by the National Council for Science and Technology, Mexico - Conacyt [Scholarship 220045/206495].

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Conflict of interest

I confirm there are no conflicts of interest.

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Nomenclature

AAPH2,2′-azo-bis-(2-aminopropane)-dihydrochloride)
ABTS2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)
ANOVAanalysis of variance
ATCCAmerican Type Culture Collection
CCTCCChina Center for Type Culture Collection
DPPH2,2-diphenyl-1-picrylhydrazyl-hydrate
DWdry weight
GAEgallic acid equivalents
HPLChigh performance liquid chromatography
MBCminimum bactericidal concentration, mg mL−1
MICminimum inhibitory concentration, mg mL−1
ORACoxygen radical absorption capacity, μmol TE/g DW
QEquercetin equivalents
SDstandard deviation
TETrolox equivalents
TFCtotal flavonoid content, mg GAE g DW−1
TPCtotal phenol content, mg QE g DW−1
Trolox6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid
UFCcolony forming units

References

  1. 1. Papetti A. Isolation and characterization of antimicrobial food components. Current Opinion in Biotechnology. 2012;23(2):168-173. DOI: 10.1016/j.copbio.2011.09.001
  2. 2. Damián-Reyna AA, González-Hernández JC, Chávez-Parga MDC. Procedimientos actuales para la extracción y purificación de flavonoides cítricos. Revista Colombiana de Biotecnología. 2016;18(1):135-147. DOI: 10.15446/rev.colomb.biote.v18n1.57724
  3. 3. Álvarez-Ordóñez A, Carvajal A, Arguello H, Martínez-Lobo FJ, Naharro G, Rubio P. Antibacterial activity and mode of action of a commercial citrus fruit extract. Journal of Applied Microbiology. 2013;115(1):50-60. DOI: 10.1111/jam.12216
  4. 4. Vikram A, Jayaprakasha GK, Jesudhasan PR, Pillai SD, Patil BS. Suppression of bacterial cell-cell signalling, biofilm formation and type III secretion system by citrus flavonoids. Journal of Applied Microbiology.2010;09(2):515-527. DOI: 10.1111/j.1365-2672.2010.04677.x
  5. 5. Cushnie TPT, Lamb AJ. Recent advances in understanding the antibacterial properties of flavonoids. International Journal of Antimicrobial Agents. 2011;38(2):99-107. DOI: 10.1016/j.ijantimicag.2011.02.01
  6. 6. Özçelik B, Kartal M, Orhan I. Cytotoxicity, antiviral and antimicrobial activities of alkaloids, flavonoids, and phenolic acids. Pharmaceutical Biology. 2011;49(4):396-402. DOI: 10.3109/13880209.2010.519390
  7. 7. Wu T, Cheng D, He M, Pan S, Yao X, Xu X. Antifungal action and inhibitory mechanism of polymethoxylated flavones fromCitrus reticulataBlanco peel against Aspergillus niger. Food Control. 2014;35(1):354-359. DOI: 10.1016/j.foodcont.2013.07.027
  8. 8. Martínez-Flórez S, González-Gallego J, Culebras JM, et al. Los flavonoides: propiedades y acciones antioxidantes. Nutrición Hospitalaria. 2002;17(06):271-278. Available from:http://www.aulamedica.es/gdcr/index.php/nh/article/download/3338/3338
  9. 9. Vollmerhausen TL, Ramos NL, Dzung DTN, Brauner A. Decoctions from Citrus reticulata Blanco seeds protect the uroepithelium againstEscherichia coliinvasion. Journal of Ethnopharmacology. 2013;150(2):770-774. DOI: 10.1016/j.jep.2013.09.050
  10. 10. Palacios-Espinosa F, Escobedo-Hinojosa W, Romero I. Panorama actual del Estudio de las plantas con actividad anti-Helicobacter pylori. Tip. Revista Especializada en Ciencias Químico-Biológicas. 2013;14(1):51-61. Available from:http://www.revistas.unam.mx/index.php/tip/article/view/43063
  11. 11. Barreca D, Bellocco E, Caristi C, Leuzzi U, Gattuso G. Flavonoid profile and radical-scavenging activity of Mediterranean sweet lemon (Citrus limetta Risso) juice. Food Chemistry. 2011;129(2):417-422. DOI: 10.1016/j.foodchem.2011.04.093
  12. 12. Abeysinghe D, Li X, Sun C, Zhang W, Zhou C, Chen K. Bioactive compounds and antioxidant capacities in different edible tissues of citrus fruit of four species. Food Chemistry. 2007;104(4):1338-1344. DOI: 10.1016/j.foodchem.2007.01.047
  13. 13. Heggers JP, Cottingham J, Gusman J, Reagor L, McCoy L, Carino E, Zhao J-G. The effectiveness of processed grapefruit-seed extract as an antibacterial agent: II. Mechanism of action and in vitro toxicity. The Journal of Alternative and Complementary Medicine. 2002;8(3):333-340. DOI: 10.1089/10755530260128023
  14. 14. Harborne JB, Williams CA. Advances in flavonoid research since 1992. Phytochemistry. 2000;55(6):481-504. DOI: 10.1016/S0031-9422(00)00235-1
  15. 15. ZhiBiao Y, Yan Y, YiZeng L, Bao Z. In vitro antioxidant and antimicrobial activities of the extract of Pericarpium Citri Reticulatae of a new Citrus cultivar and its main flavonoids. Food Science and Technology, LWT. 2008;41(4):597-603. DOI: 10.1016/j.lwt.2007.04.008
  16. 16. Klančnik A, Piskernik S, Jeršek B, Možina SS. Evaluation of diffusion and dilution methods to determine the antibacterial activity of plant extracts. Journal of Microbiological Methods. 2010;81(2):121-126
  17. 17. Céliz G, Daz M, Audisio MC. Antibacterial activity of naringin derivatives against pathogenic strains: Naringin derivatives as antibacterial agents. Journal of Applied Microbiology. 2011;111(3):731-738. DOI: 10.1111/j.1365-2672.2011.05070.x
  18. 18. Moulehi I, Bourgou S, Ourghemmi I, Tounsi MS. Variety and ripening impact on phenolic composition and antioxidant activity of mandarin (Citrus reticulate Blanco) and bitter orange (Citrus aurantiumL.) seeds extracts. Industrial Crops and Products. 2012;39:74-80. DOI: 10.1016/j.indcrop.2012.02.013
  19. 19. Kelebek H. Sugars, organic acids, phenolic compositions and antioxidant activity of grapefruit (Citrus paradisi) cultivars grown in Turkey. Industrial Crops and Products. 2010;32(3):269-274. DOI: 10.1016/j.indcrop.2010.04.023
  20. 20. Xi W, Zhang Y, Sun Y, Shen Y, Ye X, Zhou Z. Phenolic composition of Chinese wild mandarin (Citrus reticulata Blanco.) pulps and their antioxidant properties. Industrial Crops and Products. 2014;52:466-474. DOI: 10.1016/j.indcrop.2013.11.016
  21. 21. Zhang Y, Sun Y, Xi W, Shen Y, Qiao L, Zhong L, Zhou Z. Phenolic compositions and antioxidant capacities of Chinese wild mandarin (Citrus reticulata Blanco) fruits. Food Chemistry. 2014;145:674-680. DOI: 10.1016/j.foodchem.2013.08.012
  22. 22. Tounsi MS, Moulehi I, Ouerghemmi I, Mejri H, Wannes WA, Hamdaoui G, Marzouk B. Changes in lipid composition and antioxidant capacity of bitter orange (Citrus aurantium. L) and mandarin (Citrus reticulata. Blanco) oilseeds on different stages of maturity. Journal of the American Oil Chemists’ Society. 2011;88(7):961-966. DOI: 10.1007/s11746-010-1751-2
  23. 23. de Rijke E, Out P, Niessen WMA, Ariese F, Gooijer C, Brinkman UAT. Analytical separation and detection methods for flavonoids. Journal of Chromatography A. 2006;1112(1-2):31-63. DOI: 10.1016/j.chroma.2006.01.019
  24. 24. Vinson JA, Su X, Zubik L, Bose P. Phenol antioxidant quantity and quality in foods: Fruits. Journal of Agricultural and Food Chemistry. 2001;49(11):5315-5321. DOI: 10.1021/jf0009293
  25. 25. Andrews JM. Determination of minimum inhibitory concentrations. Journal of Antimicrobial Chemotherapy. 2001;48(Suppl 1):5-16. DOI: 10.1093/jac/48.suppl_1.5
  26. 26. Molyneux P. The use of the stable free radical diphenylpicrylhydrazyl (DPPH) for estimating antioxidant activity. Songklanakarin Journal of Science and Technology. 2004;26(2):211-219
  27. 27. Ou B, Hampsch-Woodill M, Prior RL. Development and validation of an improved oxygen radical absorbance capacity assay using fluorescein as the fluorescent probe. Journal of Agricultural and Food Chemistry. 2001;49(10):4619-4626. DOI: 10.1021/jf010586o
  28. 28. Ramful D, Bahorun T, Bourdon E, Tarnus E, Aruoma OI. Bioactive phenolics and antioxidant propensity of flavedo extracts of Mauritian citrus fruits: Potential prophylactic ingredients for functional foods application. Toxicology. 2010;278(1):75-87. DOI: 10.1016/j.tox.2010.01.012
  29. 29. Sun Y, Wang J, Gu S, Liu Z, Zhang Y, Zhang X. Simultaneous determination of flavonoids in different parts ofCitrus reticulata‘Chachi’ fruit by high-performance liquid chromatography-photodiode Array detection. Molecules. 2010;15(8):5378-5388. DOI: 10.3390/molecules15085378
  30. 30. Wang H, Chen G, Guo X, Abbasi AM, Liu RH. Influence of the stage of ripeness on the phytochemical profiles, antioxidant and antiproliferative activities in different parts of Citrus reticulata Blanco cv. Chachiensis. LWT – Food Science and Technology. 2016;69:67-75. DOI: 10.1016/j.lwt.2016.01.021
  31. 31. Ramful D, Tarnus E, Aruoma OI, Bourdon E, Bahorun T. Polyphenol composition, vitamin C content and antioxidant capacity of Mauritian citrus fruit pulps. Food Research International. 2011;44(7):2088-2099. DOI: 10.1016/j.foodres.2011.03.056
  32. 32. Abad-García B, Garmón-Lobato S, Sánchez-Ilárduya MB, Berrueta LA, Gallo B, Vicente F, Alonso-Salces RM. Polyphenolic contents in Citrus fruit juices: Authenticity assessment. European Food Research and Technology. 2014;238(5):803-818. DOI: 10.1007/s00217-014-2160-9
  33. 33. Kelebek H, Selli S. Identification of phenolic compositions and the antioxidant capacity of mandarin juices and wines. Journal of Food Science and Technology. 2014;51(6):1094-1101. DOI: 10.1007/s13197-011-0606-7
  34. 34. Debbarma J, Kishore P, Nayak BB, Kannuchamy N, Gudipati V. Antibacterial activity of ginger, eucalyptus and sweet orange peel essential oils on fish-borne bacteria. Journal of Food Processing and Preservation. 2013;37(5):1022-1030. DOI: 10.1111/j.1745-4549.2012.00753.x

Written By

Andrés Alejandro Damian-Reyna, Juan Carlos González-Hernández, Jesús Fernando Ayala-Zavala, Consuelo de Jesús Cortes Penagos, Rafael Maya-Yescas and Ma del C. Chávez-Parga

Submitted: April 7th, 2018 Reviewed: June 1st, 2018 Published: November 5th, 2018