Bioactive Compounds, Vitamin C and Antioxidant Capacities of Fresh and Industrialized Frozen Pulps of Guava (Psidium guajava L.)

196 The Natural Products Journal, 2012, 2, 196-204

Bioactive Compounds, Vitamin C and Antioxidant Capacities of Fresh and Industrialized Frozen Pulps of Guava (Psidium guajava L.)

Renata Polinati, Luciana Oliveira and Eliane Fialho*

Departamento de Nutrição Básica e Experimental, Instituto de Nutrição Josué de Castro, Universidade Federal do Rio de Janeiro, Avenida Carlos Chagas Filho, 393 RJ 21941-590, Brazil

Abstract: Consumption of bioactive compounds in fruits, vegetables and their derivatives is related to lower incidences of non-communicable diseases. This study determines the contents of bioactive compounds and vitamin C and verifies the antioxidant capacities of fresh (FG) and industrialized frozen pulps (IP1 and IP2) of guava. Results showed that the total soluble solids and pH for FG and IP were within the limits established by current legislation. IP and FG are excellent sources of phenolic compounds, especially proanthocyanidins, whose values were 38.45±0.21, 51.26±6.15, 38.03±2.48 mg catechin equivalent/100 g (fresh or wet weight) for FG and IP1 and IP2, respectively. There was only a significant difference between IP1 and IP2 in the amount of total flavonoids. The major carotenoid present in guava, lycopene, was significantly higher in FG (7.46±0.37 mg lycopene/100 g wet weight). The IP2 had the highest content of vitamin C (68.10 mg AA/100 g wet weight), while the FG (9.51 mg AA/100 g wet weight) had the smallest amount of this vitamin. After performing three different antioxidants methods, a significant difference was observed. FG had a lower antioxidant capacity measured by DPPH scavenging. For the other methods, no significant difference was found between samples. Finally, we conclude that fresh and frozen guava pulp have considerable levels of antioxidants. So frozen guava pulp may be an excellent choice for consumers who are looking for a convenient, healthy food.

Keywords: Bioactive compounds, vitamin C, antioxidant capacity, guava.

1. INTRODUCTION

Guava fruit (Psidium guajava L.) is native to tropical regions of the Americas and has spread to different regions of the world. Among tropical fruits grown in Brazil, guava occupies a special place, not only for its aroma and flavor but also for its nutritional value [1]. "Pedro Sato" is a variety that has a rough skin, pink pulp, is thick and firm and has few seeds [2]. It is considered to be the most preferred in the national market [3].

Guava is considered to be one of the best sources of vitamin C, but the ascorbic acid (AA) content can vary with cultivar, location and management [4] and is three to six times the amount found in oranges [5]. Additionally, guava has several bioactive compounds (BACs) such as phenolic compounds and carotenoids. These are mainly responsible for the high antioxidant potential of this fruit [1].

It can be consumed "in nature", and it is the primary raw material for the manufacture of guava paste, jams, soft drinks, juices and pulps, with significant market acceptance worldwide [3]. Recent data from the Consumer Expenditure Survey conducted in Brazil in 2008-2009 indicated that 4.2% to 4.6% of total monetary expenditures on fruits between

*Address correspondence to this author at the Departamento de Nutrição Básica e Experimental, Instituto de Nutrição Josué de Castro, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, UFRJ, Caixa Postal 68041, Cidade Universitária, Ilha do Fundão, Rio de Janeiro, CEP 21941-590, Brasil; Tel: + 55 21 2562 6599; Fax: + 55 21 2280 8343; E-mail: [email protected]

2003 and 2009 were due to guava. These values are close to the total expenditure of other foods such as sugars and derivatives (4.6%) and vegetables (3.3%) [6]. Although fruit consumption "in nature" is still small in Brazil, the use of frozen fruit pulp is expanding. It is put into dairy products, sherbet, pastries, juices and others. This is due to the ease of its use, which increases the interest of producers and consumers.

The major agroindustrial activity for guava fruit is processing it to obtain frozen pulps that add economic value to the fruit. This avoids waste, increases its shelf life and minimizes losses that may occur during the marketing of "in nature" products.

The pulps of tropical fruits marketed in Brazil can be frozen. They often contain high levels of phenolics and their antioxidant properties are preserved. Kuskoski et al. [7] found that frozen pulps can retain their properties and be excellent sources of phenolic compounds that have high antioxidant capacities.

Besides the nutritional aspect, consumers demand convenience. This is related to the ease of storage and preparation for domestic consumption. The awareness of the importance of fruit consumption, coupled with continuous improvements being made to the quality of food, indicate that frozen pulps of tropical fruits should continue to gain market share. However, consumers are setting new standards for convenience foods. They demand that the quality and nutritional value should be preserved [8].

2210-3163/12 $58.00+.00 © 2012 Bentham Science Publishers

Bioactive Compounds, Vitamin C and Antioxidant Capacities of Fresh The Natural Products Journal, 2012, Vol. 2, No. 3 197

In a previous study, it was found that storing orange and apple extracts frozen at -18 °C and -70 °C for 10 days, preserved the contents of soluble (SP) and hydrolyzable polyphenols (HP) as well as the amount of vitamin C and consequently the antioxidant capacities [9].

On the other hand, ascorbic acid losses during storage of other fruits, when they occurred, are minimal. Its stability in fruits "in nature" and in its products is attributed to its high levels and the protective effect of phenolic antioxidants [10]. Moreover, the stability of carotenoids in foods is very different, even when subjected to identical processing and storage conditions. The main cause of its destruction is oxidation (enzymatic and non enzymatic). Freezing process, especially the quick freezing and storage under freezing temperatures, generally favor the retention of carotenoids in foods [11].

Moreover, Pasteurization of frozen pulp may not initially change the nutritional content and antioxidant capacity of fruits. However, Pasteurization is a restrictive technique, which can influence the content of vitamin C and phenolic compounds, with losses of up to 30-60% in oranges [12].

Increasingly, consumers are concerned about the quality of products they consume and in adopting healthy dietary practices. Because of the importance of the higher consumption of fruits and vegetables, as recommended by World Health Organization, and the demand for food quality and easily accessibility, the purpose of this study was to determine the levels of bioactive compounds, vitamin C and the antioxidant capacities in red guava pulp ‘in nature” and industrialized frozen guava pulp, acquired in local markets of Rio de Janeiro, Brazil.

2. MATERIAL AND METHODS

2.1. Material and Chemicals

Mature samples of two brands of frozen pulps and fresh “Pedro sato” variety of red guava (Psidium guajava L.) were acquired at local markets in Rio de Janeiro, Brazil. Authentic standards of catechin, gallic acid, beta-carotene, ascorbic acid and the Folin-Ciocalteu’s reagent were obtained from Sigma–Aldrich (St. Louis, MO).

2.2. Sample Preparation

Guavas were washed in distilled water, dried on paper towels and peeled manually with the aid of a knife, in order to get only the red pulp. They were placed in a domestic processor (Vicini, model EPV – 80, Brazil) and blended to obtain a homogeneous extract. After the complete homo- genization, pulp was sieved in a screen to remove seeds. Immediately after extraction, the pulps were subdivided into polyethylene plastic bags and vacuum sealed. About 10g of each were stored at -22 ° C until analyzed. Two different brands of industrialized frozen pulps were used in this study. These were purchased in packs of 100g (integral pulp Pasteurized) and stored at -22 ° C until analyzed. The extracts of “in natura” guava and industrialized frozen pulps were acquired in three different times, thus obtaining three different lots of three samples each.

2.3. Total Soluble Solids and pH

The determination of total soluble solids (TSS) was performed using the Eppendorf 2763 refractometer and the results were expressed in °Brix and pH was obtained in a Micronal digital potentiometer according to the analytical standards of the Institute Adolfo Lutz [13].

2.4. Percent Composition

Moisture, ash, lipids and carbohydrates were measured in accordance with analytical standards of the Institute Adolfo Lutz [13]. The Kjeldahl method was used for protein analysis, using factor 6.25 for converting nitrogen in protein [14].

2.5. Soluble and Hydrolyzable Polyphenols

2.5.1. Polyphenol Extraction

The polyphenol extraction method was adapted from Vinson, Su, Zubik, and Bose [15]. Briefly, 100 µL of each sample was extracted using 500 µL of 50% methanol (methanol:water, 50:50, v/v) for the soluble polyphenols (SP), and 50% methanol (methanol:water, 50:50, v/v) acidified with 1.2 M HCl for the hydrolyzable polyphenols (HP), in screw capped Eppendorf vials. The samples were then placed in a water bath at 90°C for 180 min with constant shaking. After three hrs, the samples were removed from the water bath and cooled at room temperature (about 2 min). The volume was adjusted to 1 mL with 95% methanol, and the Eppendorf vials were centrifuged at 12,000g for 5 min. The supernatants, considered to be polyphenol extracts, were immediately used for the determination of polyphenol content.

2.5.2. Polyphenol Content

The polyphenol contents were determined using the Folin–Ciocalteu’s reagent as described by Karou, Dicko, Simpore, and Traore [16] in Eppendorfs tubes, 30 µL of either the soluble or hydrolyzable polyphenol extract was mixed with 75 µL of Folin–Ciocalteu’s reagent, diluted in distilled water (Folin:water, 50:50, v/v). The mixture was allowed to stand for 5 min before adding 75 µL of 20% sodium carbonate solution. After 30 min at room temperature, the absorbance at 750 nm was measured using a Beckman 6300 spectrophotometer. The contents of soluble and hydrolyzable polyphenols were calculated using a standard curve of gallic acid. The results were expressed as mg of gallic acid equivalents per 100 g, or GAE/100 g wet or fresh weight.

2.6. Total Flavonoids

Total flavonoids content was measured using the method described by Kim, Chun, Kim, Moon and Lee [17]. One mL of the diluted sample was added to 7 mL of distilled water and 0.3 mL of 5% sodium nitrite solution at time zero. Five minutes later, 0.3 mL of 10 % aluminum chloride solution were added; at six minutes, 2 mL of 1M sodium hydroxide were added. 2.4 mL of distilled water were immediately

198 The Natural Products Journal, 2012, Vol. 2, No. 3 Polinati et al.

added and the tubes were quickly agitated in vortex. The absorbance at 510 nm was read and the results were expressed in mg of catechin equivalents per 100 g, or CTE/100 g FW.

2.7. Condensed Tannins (Proanthocyanidins)

Condensed tannin content was determined based on the method described by Nakamura, Tsuji and Tonogai [18]. 100 µL of the samples were diluted in 900 µL of absolute methanol. After this extraction, 2.5mL of 1% vanillin was added, followed by addition of 2.5 mL of HCl 90%. The samples remained in a water bath at 30°C for 20 min and after this period were cooled at room temperature. The reading was performed in a spectrophotometer at 500 nm and results were expressed as mg catechin equivalents, CTE/100 g.

2.8. Total Carotenoids

One gram of sample was extracted with 10 mL of hexane/acetone/toluene/ethanol (10:7:7:6, v/v/v/v), 1 mL of KOH in 40% methanol and 10 mL of 10% Na2SO4. The standard curve was constructed using β-carotene in hexane (0 to 10 mg/mL). The absorbances of the samples at 450 nm of the upper phases were measured, using hexane as a blank. The results were expressed as mg β-carotene equivalents per 100 g, or BCE/100 g wet or fresh weight.

2.9. Lycopene

The method described by Javanmardi and Kubota [20] was used. Samples were extracted with acetone, ethanol and hexane (1:1:1, v/v/v). Then, 0.05% BHT in acetone was added to 1 mL of each sample. They were agitated for 15 mins in a vortex. After 15 mins, the absorbance at 503 nm was read. The content of lycopene was calculated using a molar extinction coefficient 3.12. Values were expressed as mg lycopene/100g wet weight.

2.10. HPLC Analyses of Ascorbic Acid

Ascorbic acid (AA) was extracted from 10 g of sample. The extract was diluted (1:2) and centrifuged at 5000 rpm for 10 mins. 200 µL of the supernatant was added to 800 µL of 2% potassium phosphate (KH2PO4) buffer, pH 2.32. The AA analysis was performed by HPLC using methods described by Silva et al. [21], with the following modifications. The chromatographic separations were performed using a Nucleosil 100 C18 pre-column (4.6 mm) and analytical column (250 mm) and an isocratic elution using 2% potassium phosphate (KH2PO4) buffer, pH 2.32. The absorbances at 243 nm and 254 nm were monitored for detection of AA and using dithiothreitol (DTT) for the conversion of dehydroascorbic acid (DHA) to AA in photodiode array UV detector SPD-M10A Shimadzu (Kyoto, Japan). The flow rate was 0.4 mL/min and the run time was 20 mins. Quantification of AA was performed using a calibration curve with ascorbic acid standards (2.7-21.8 mg/mL). Results were expressed as mg AA/100g PF. Standards of AA were diluted into 2% KH2PO4 buffer and samples were added to 100 µL of dithiothreitol (DTT) solution at 5 mg/mL.

2.11. Antioxidant Capacity

2.11.1. DPPH

Antioxidant capacity (AC) was measured by the DPPH radical method according to Kuskoski, et al. [7]. Briefly, a 100 mM DPPH solution was prepared with 80% methanol, giving an absorbance of 1.1 at 517 nm. 100 µL of each sample were placed in test tubes and 3.9 mL of the DPPH solution were added. The mixture was allowed to stand, in the absence of light, and the absorbance was measured at 15, 30, and 60 min. The DPPH solution alone was measured before the addition of the samples (A0) and 80% methanol was used as blank. The AC was represented as the percentage radical scavenging capacity (RSC) remaining after each time according to the equation below:

% RSC = (A0 – At) / A0 A0 represents the absorbance of the DPPH solution alone

measured at zero time, and At is the absorbance for each sample at 15, 30, and 60 min after the addition of the DPPH solution. The value of A0 is considered to be 100%.

2.11.2. Beta-carotene/Linoleic Acid Model System

Extracts of lycopene obtained as described in Section 2.9 were used. The method was performed as described by Melo, Maciel, Lima and Nascimento [22] with modifications. A solution of β-carotene (0.3 mg/mL) was placed in round-bottomed flask containing 20 µL of linoleic acid and 200 µL Tween20. After removal of chloroform in a rotary evaporator at 50°C, 50 mL of distilled water were added under vigorous stirring. Aliquots (250 µL) of this emulsion were transferred to an ELISA plate containing 25 µL of lycopene extract. Then plate was placed in a water bath at 50 °C for 120 mins and the absorbance at 470 nm was recorded every 30 mins. AC was expressed as percentage of inhibition of oxidation, calculated for 100% oxidation of control (no antioxidant), as described below:

% Inhibition = (rate degradation control – rate degradation sample x 100) / rate degradation control

2.11.3. Ferric Reduction Antioxidant Power Assay (FRAP)

Stock solutions, including 300 mM sodium acetate buffer, pH 3.6, 10 mM TPTZ (2,4,6-tripyridyl-s-triazine) in 40 mM HCl and 20 mM of FeCl3 were prepared. The FRAP solution was prepared in a mixture of 25 mL of acetate buffer, 2.5 mL of TPTZ and 2.5 mL of of FeCl3 and then heated to 37 ° C. Samples reacted with 285 µL of FRAP solution for 30 mins in the dark. The absorbance at 503 nm was read. Results were expressed in mM Trolox equivalent (TXE)/100 g fresh weight [5].

2.12. Statistical Analysis

The experiments were carried out in triplicate. The statistical analysis of the results was done using GraphPadPrism 5.0 for Windows (GraphPad Software, 2009) to apply one-way ANOVA followed by the Tukey test. Trends were considered significant when the means of compared sets differed at P < 0.05. The Pearson correlation was performed using Microsoft Office Excel 2007, where the


correlation coefficient was obtained from the average of the values found.

3. RESULTS AND DISCUSSION

3.1. Total Soluble Solids and pH

The content of TSS is usually used as an index of fruit ripening. The maturation stage in which fruits are harvested determines the quality of the product that will be offered to the consumer. There is no standard or consensus on the optimal maturity stage for harvesting guava, so little is known about the maturation index [23]. TSS levels and pH of fresh guava pulp (FG) and the industrialized frozen pulps (IPs) acquired in the same markets are shown in Table 1.

According to Azzolini, Jacomino and Bron [24], TSS does not indicate the maturity stages in the “Pedro Sato” variety. The total sugars (mostly fructose) represent about 50% to 91% of the soluble solids. Therefore, TSS content is under the influence of factors that affect the synthesis of fructose, which hinders the establishment of a range that represents a stage of maturity. This can be confirmed when we evaluated guava pulp, not considered mature because of the dark green peel of the fruit and the stiffness of the fruit. The immature fruit had a TSS content of 8.75± 0.25 °Brix, not considered statistically significant in relation to FG (data not shown). The results indicated that the FG has a content of TSS greater than IPs. The IP2 had the lowest TSS content of all the pulps analyzed. The Ministry of Agriculture and Supply, in its normative instruction N°. 01, dated January 7, 2000 [25], established the minimum content of TSS for the guava pulp (7°Brix), so the IPs and FG had the proper levels that were established by the legislation.

There was no significant difference in pH between samples. The Ministry of Agriculture and Supply, established the minimum pH (3.5) and maximum (4.2) for guava pulp. Thus, the pH found in all samples was within those established by legislation. According to Evangelista and Vieites [26], the quality of four different samples of industrialized frozen pulps acquired in São Paulo city, had pH values between 3.5 and 3.9. The TSS content ranged from 5.87 to 11.27 °Brix. These values were similar to those found in this study for the IPs.

3.2. Soluble and Hydrolyzable Polyphenols, Total Flavonoids and Proanthocyanidin

There was no significant difference in the amount of soluble and hydrolyzable phenolics between samples. The

soluble phenol contents were 9.3±2.0, 13±0.01, and 8.5±1.5 mg GAE/100 g for FG, IP1 and IP2, respectively as shown in Fig. (1A). For HP, the content was higher, being 17±2.3, 17.5±2.6, 14±1.7 mg GAE/100 g for FG, IP1 and IP2, respectively as shown in Fig. (1B). It was observed that IPs have similar amounts of polyphenols compared to the FG. In addition, the content of soluble polyphenols was less than total hydrolyzable polyphenols, indicating that some of these compounds need to be hydrolyzed. A similar result was found by Vinson et al. [15], who found that avocados, melons and oranges had higher amounts of soluble polyphenols than other fruits, a total of 16 mg GAE/100 g fresh weight, with a high percentage of polyphenols in the conjugated form, ranging from 31 to 94 %. Hassimotto, Genovese and Lajolo [27], studying fresh pulps and frozen pulps purchased at the central market of São Paulo, found that red guava pulp had a content of 124 mg/100 g GAE, while frozen guava pulp showed a similar content, 119 mg/100g GAE. Kuskoski et al. [7], in a study with 100% natural fruit pulps marketed in Florianópolis, Brazil, in packs of 100 g and stored under freezing (-15 ° C ± 0.1), found that guava pulp had 83.0 mg/100 g GAE, while other frozen fruit pulps had 20.0 mg GAE/100 g fresh weight (Passiflora edulis) and 897.6 mg/100g GAE (Taluma ovata).

There was a significant difference in the total flavonoid contents of IP1 and IP2 but there was no difference between FG and IPs. The values were: 0.3±0.005, 0.2±0.02 and 0.37±0.02 mg CTE/100 g wet weight for FG, IP1 and IP2, respectively, as shown in Fig. (1C). Luximon-Ramma, Bahorun and Crozier [28] found that the content of total flavonoids in the peel and pulp of red guavas was 11 mg CTE/100 g fresh weight, a value greater than that found in this study. Studies involving the content of total flavonoids in red guava, as well as its derivatives are still scarce. Franke, Custer, Arakaki and Murphy [29] found that the total flavonoid content of some fruits like apple, mango, pineapple, papaya and strawberries, can range from 1.1 mg/kg to 26 mg/kg, depending on variety of fruit. As for plums, Kim et al. [17] found even higher values - ranging from 64 mg to 257 mg CTE/100g fresh weight.

As for proanthocyanidins, there was no significant difference in the amount of proanthocyanidins between the samples. The amounts found for FG, IP1 and IP2 were 38.45±0.21, 51.26±6.15, 38.03±2.48 mg CTE/100 g fresh weight, respectively as shown in Fig. (1D). Few studies analyzed guava for proanthocyanidins in pulps and frozen derivatives. The IP1 had a higher content of condensed tannins in comparison to other pulps, but this was not statistically significant. The FG and IP2 had very similar values. Therefore, the IPs may show considerable concentrations of these compounds, since their values did not differ significantly from the FG. Pérez-Jiménez, Arranz and Saura-Calixto [30] determined the content of proanthocyanidins in apples, pears and nectarines. They found values of 28, 71 and 16 mg/100g, respectively, and these values are similar to the amounts found for guava pulp in this study. On the other hand, Luximon-Ramma et al. [28] found a content equivalent to 10.9 mg cyanidin/100 g fresh weight for the red guava. This is four times lower than that found for the red guava variety "Pedro Sato" in this study. Additionally, Melo et al. [22] quantified proanthocyanidins

Table 1. Total Soluble Solids and pH in Fresh Guava (FG) and Industrialized Pulps 1 (IP1) and 2 (IP2)

Sample oBrix (Total Solids) pH

FG 11.25±0.75 3.88±0.04

IP1 9.75±0.25 3.50±0.07

IP2 7.25±0.75 3.78±0.05

Values are averages ± standard deviations. The pH values for FG and IP2 were not significantly different when analyzed by the one-way ANOVA, followed by the Tukey Test (p<0.05).


in guava produced in Pernambuco-Brazil. They found a content that was four times higher than found for "Pedro Sato", 183 mg CTE/100 g fresh weight.

Freezing is one of the ways to preserve the bioactive compounds in food, such as polyphenols. During freezing, several changes may occur in the metabolism of polyphenols. One such change is the activation of the enzyme PAL (phenylalanine ammonium lyase). This activation may be related to the maintenance of phenolic compounds during storage at low temperatures [7, 31]. In addition, thermal treatments such as Pasteurization or bleaching, when applied before freezing, are of fundamental importance for the complete inactivation of enzymes such as PPO (polyphenol oxidase), thereby ensuring that no degradation of polyphenols occurs and there is no consequential enzymatic browning, which can cause the unacceptability of the product [11]. Thus, Pasteurization applied to the IPs may have been effective in the inactivation of this enzyme during freezing and therefore there was no degradation of polyphenols. The flavonoids, which are polyphenols, also showed some stability during freezing. As mentioned earlier, freezing can activate PAL and this can be related to the increase in phenolic compounds, including flavonoids. The stress caused by freezing induces the production of phenolic compounds, so the storage at low temperatures can be used as a means to increase the synthesis of phytochemicals with known

beneficial properties to health [31]. On the other hand, one must remember that the fruits used for the industrial production of frozen pulp can be from different cultivars. Often, this is not mentioned by the manufacturer. Also, the place of growth can affect the amount of phytochemicals present [32].

3.3. Total Carotenoids and Lycopene

The sample IP1 (0.072±0.009 mg BCE/100 g fresh weight) had a total carotenoid content that was significantly higher than that found in IP2 (0.023±0.0001 mg BCE/100 g fresh weight) and FG (0.024±0.007 mg BCE/100 g fresh weight) as shown in Fig. (2A). In addition, this content was also significantly higher in IP1 than IP2. Agostini-Costa et al. [8] found that acerola pulp retained its carotenoids content relatively well after freezing and storage for 11 months. Also, Lopes et al. [11] observed that there was a significant decrease in the total carotenoid content of cherry pulp in the first 30 days of storage at -18°C. However, on days 45, 60 and 90 no significant decrease was observed. There was a loss of 14% during the 90 days of freezing. Melo et al. [22] analyzed the content of total carotenoids in red guava pulp and found 4.3mg β-carotene/100 g fresh weight. Low storage temperatures generally favor the retention of carotenoids in foods. The carotenoids retention is also favored by protection from light, exclusion of oxygen

Fig. (1). Phenolic compounds in fresh guava (FG) and industrialized pulps 1 (IP1) and 2 (IP2). (A) Soluble polyphenols (mg GAE/100 g wet weight), (B). Hydrolyzable polyphenols (mg GAE/100 g wet weight), (C) Total flavonoids (mg TCE/100 g wet weight) and (D) Proanthocyanidin content (mg TCE/100 g wet weight). a,b,c Equal letters have no significant difference: One-Way ANOVA, followed by Tukey Test (p<0.05). GAE is gallic acid equivalents and TCE is total catechin equivalents.


(vacuum, filling the hot, modified atmosphere or packaging impermeable to oxygen). These are all conditions to which the pulps are commonly subjected. Also, heat treatment, like Pasteurization, can completely inactivate the enzymes present in fruit pulps, ensuring the stability of these pigments [11]. On the other hand, the carotenoid composition and content of food can vary depending on the cultivar or variety of plant, stage of maturity, climate, growing conditions, handling during harvesting, transport, storage and conservation post harvesting, processing and storage [33].

The sample FG (7.46±0.37 mg LYC/100 g wet weight) had a significantly higher content than IP2 (5.110.46 mg LYC/100 g wet weight) or IP1 (5.91±0.33 mg LYC/100 g wet weight) as shown in Fig. (2B). The differences in the amounts in IP1 and IP2 were not statistically significant. The frozen guava pulp samples may differ in their carotenoid contents. In a study of three different frozen pulps, the levels of β-carotene ranged from 0.27 to 0.45 mg/100 g fresh weight. The lycopene contents ranged from 3.7 to 7.3 mg/100 g fresh weight [33]. Also, there was no correlation between the contents of total carotenoids and lycopene. When the pulp had higher total carotenoids, the lycopene content was lower. This may be related to the degree of maturation in the fruits that are used for the preparation of industrial pulps. For instance, the ripening of fruit is related

to color. The change from pink to red is related to the content of β-carotene and lycopene. When the fruit is not fully mature, the β-carotene content is higher than that of lycopene. These changes are related to the activity of enzymes in the metabolism and biosynthesis of carotenoids, such as the enzyme lycopene β-cyclase. It is responsible for converting β-carotene into other carotenoids such as lutein, zeaxanthin and lycopene [34]. The stability of carotenoids in foods is very different, even when subjected to identical processing and storage conditions. The main cause of destruction is the oxidation of carotenoids (enzymatic or non enzymatic). Still, freezing and Pasteurization promote protection from oxidation, thus preserving antioxidant content and the functional quality of food [11].

3.4. Ascorbic Acid

The amount of ascorbic acid (AA) differed significantly for all samples. This confirms the variability of this vitamin in frozen pulp and also in the fruit consumed in the city of Rio de Janeiro. The sample IP2 had the highest content of ascorbic acid and FG demonstrated a significantly lower content as shown in Fig. (3). In this case, we must take into account that guava is usually used to make frozen pulp or sweets. The fruit is fully utilized, without wasting the peel. The results found were: 9.51 + 0.43 mg AA/100 g wet weight; 32.87±3.6 mg AA/100 g wet weight; 68.1±5.3 mg AA/100 g fresh weight in FG, IP1 and IP2, respectively. Analyzing the effect of freezing at -40°C, Gil-Izquierdo et al. [12] noted that this process does not affect the contents of AA in oranges. Nojavana, Khaliliana, Kiaiec, Rahimic, Arabanianc and Chalavia [35] showed that freezing oranges prevents oxidation and degradation of AA. Kalt et al. [10] reported that the stability of AA in fruits may be related to cellular compartmentalization of this vitamin with the phenolic compounds, which protects it from oxidation.

Fig. (3). Vitamin C content (mg AA/100g FW) by HPLC method in fresh guava (FG) and industrialized pulps 1 (IP1) and 2 (IP2). a,b,c

Equal letters have no significant difference: One-Way ANOVA, followed by Tukey Test (p<0,05).

Brunini et al. [4] reported that the levels of vitamin C in

guava pulp intact, frozen and stored at -20 °C decreased

Fig. (2). Carotenoid content in fresh guava (FG) and industrialized pulps 1 (IP1) and 2 (IP2). (A) Total carotenoids content (mg BCE/ 100 g wet weight) and (B) Lycopene content (mg lycopene/100 g wet weight). a,b,c Equal letters have no significant difference: One-Way ANOVA, followed by Tukey Test (p<0.05). BCE is β-carotene equivalents.

Ascorbic Acid

0 20 40 60 80 100

FG

IP1

IP2

mg AA/100g FW

a

b

c


from an initial value of 67.86 mg of AA/100g wet weight to 17.44 mg AA/100g wet weight after 18 weeks of being frozen. The level of ascorbic acid may range depending of how it is cultivated, its location and management. The manner that the fruit was processed, the storage conditions and type of packaging can increase or reduce the loss of ascorbic acid in guava pulps. So, this fruit continues to be an excellent source of this vitamin. Also the vitamin content can vary dramatically depending on the type of cultivar. Thus, the nutritional quality of frozen guava pulp purchased in markets can vary [26].

3.5. Antioxidant Capacity

There was a significant difference in the radical scavenging abilities of FG and IP samples, as measured by the DPPH method. The classification described by Melo et al. [22], suggests a strong antioxidant capacity (AC) when the percentage of free radical scavenging is > 70%. As shown in Table 2, all the samples had a strong antioxidant capacity. For the FRAP and β-carotene/linoleic acid methods, no significant difference between the samples was observed. Overall, the samples showed a similar profile of antioxidant capacity by three different methods. In descending order, IP2 had the highest antioxidant capacity, followed by IP1 and last FG.

All the samples contained the following antioxidants: AA, carotenoids and polyphenols. The number and amounts of these phytochemicals varied depending on the type, variety and ripeness of the fruit as well as the climatic conditions of cultivation. To evaluate the antioxidant capacity of a plant it is necessary to obtain the maximum extraction of bioactive compounds which have different polarities. Also, different foods have different matrix effects. Thus, the solubility in a particular solvent is characteristic of

each phytochemical and food, which explains the absence of a universal extraction procedure that is specific for each compound [22].

Kuskoski et al. [36] determined the antioxidant capacities by different methods in different frozen pulps from southern Brazil. They found that guava pulp had 100 mg AA/100g fresh weight. Of eleven pulps analyzed, guava appeared in fifth place behind only mango, cherry, grape and açaí. Mezadri, et al. [37] evaluated different forms of commercial acerola and found that frozen pulp have a wide range of antioxidant capacities, ranging from 38.31 to 85.11 mM Trolox equivalent/kg wet weight. But, when they analyzed the macerated fruit, they found that it could have a higher AC, with values up to 122.7 mM Trolox equivalent/kg wet weight. Still, pulps were found to have the greatest antioxidant potential. Kuskoski et al. [36] also analyzed different frozen pulps from Brazil and found the guava had an antioxidant capacity of 5.9 mM Trolox equivalent/g wet weight and concluded that the tropical fruits pulps contain high levels of total polyphenols and considerable antioxidant properties. Guava was considered to be the fruit with the sixth highest antioxidant potential. Melo et al. [22] analyzed the aqueous and acetone extracts of different Brazilian fruits and found that guava had 60 and 80% antioxidant capacity, respectively, similar to data found in this study. Thus, it can be concluded that acerola, cashew, papaya, guava, oranges, pears and pineapples stand out as sources of powerful antioxidants and can protect against oxidative damage. Also, they observed that inhibition of β-carotene oxidation for guava was 25 to 65%, respectively, lower values found in this study using extract containing lycopene. Above all, we observed that the extract containing lycopene had a high percentage of inhibition of β-carotene oxidation, and is therefore is considered to be an excellent antioxidant.

Table 2. Antioxidant Capacities in Fresh Guava (FG) and Industrialized Pulps 1 (IP1) and 2 (IP2), Based on Three Different Methods

Sample DPPH (% AC)

FRAP (mM TXE/100g Wet Weight)

β-carotene/Linoleic Acid (% Inhibition)

FG 85.70±1.30c 229.00±4.00a 81.70±0.05a

IP1 92.80±0.70a,b 230.00±19.80a 85.30±2.30a

IP2 93.70±0.50a 234.00±7.60a 87.80±0.30a

Values are averages ± standard deviations AC is antioxidant capacity and TXE is Trolox equivalents a,b,c Equal letters have no significant difference: One-Way ANOVA, followed by Tukey Test (p<0.05).

Table 3. Correlation Between the Pearson Coefficient Correlation, Bioactive Compounds, Vitamin C and Antioxidant Capacity

Method Soluble Phenols Hydrolyzable Phenols Proanthocyanidins Lycopene Vitamin C

DPPH 0.26 NC 0.38 NC 0.85

FRAP NC NC NC NC 0.97 NC No correlation. The Pearson coefficient correlation was calculated based on the values found for bioactive compounds and vitamin C and the values of antioxidant capacity by DPPH and FRAP between fresh guava (FG) and industrialized pulps 1 (IP1) and 2 (IP2).


3.6. Pearson Correlation

As shown in Table 3, there is not a good correlation between the amount of bioactive compounds, vitamin C and antioxidant capacity. However, vitamin C had the highest correlation to methods for measuring antioxidant capacities, with a correlation coefficient of 0.97 for the FRAP method. Thus, in the samples analyzed, vitamin C is primarily responsible for the antioxidant capacity. Kuskoski et al. [36] analyzed the correlations with the antioxidant capacities of frozen pulps and noted that they correlated positively with the average amounts of polyphenols and anthocyanins. They also asserted that phenolic compounds can contribute to the antioxidant capacities of fruits. Still, vitamin C is considered by many authors as the largest contributor to antioxidant capacity in fruits. Thaipong et al. [5] confirmed that a high correlation exists between antioxidant capacity and ascorbic acid content when analyzing fruits that contain high concentrations of this vitamin, such as oranges and guava. They also stated that both ascorbic acid and phenolic compounds have positive correlations with antioxidant levels, indicating that these compounds are important contributors to the total antioxidant capacities of guava extracts.

In conclusion, the differences found in the amounts of bioactive compounds in the FG and IP samples, indicate the variability that each plant can have in its composition. However, all of them had considerable amounts of bioactive compounds and high antioxidant capacities. IPs acquired in local retail markets in the city of Rio de Janeiro are excellent options for consumers who are looking for convenient foods which can easily preserve their functional properties.

ACKNOWLEDGEMENTS

The authors would like to thank the Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ) and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for support.

CONFLICT OF INTEREST

Declared none.

REFERENCES [1] Gutiérrez, R.M.P.; Mitchell, S.; Solis, R.V. Psidium guajava: A

review of its uses, phytochemistry and pharmacology. J. Ethnopharmacol., 2008, 117, 1-27.

[2] Pommer, C.; Murakami, K.; Watlington, F. Goiaba no mundo. O Agrônomo, 2006, 58, 22-26.

[3] Azzolini, M.; Jacomino, A.P.; Bron, I.U.; Kluge, R.A.; Schiavinato, M.A. Ripenning of “Pedro Sato”guava: study on its climacteric or non-climacteric nature. Braz. J. Plant Physiol., 2005, 17, 299-306.

[4] Brunini, M.A.; Oliveira, A.L.; Varanda, D.B. Avaliação da qualidade de polpa de goiaba ‘paluma’armazenada a –20ºC. Rev. Brasil. Fruticult., 2003, 25, 394-396.

[5] Thaipong, K.; Boonprakob, U.; Crosby, K.; Cisneros-Zevallos, L.; Byrne, D.H. Comparison of ABTS, DPPH, FRAP, and ORAC assays for estimating antioxidant activity from guava fruit extracts. J. Food Comp. Anal., 2006, 19, 669-675.

[6] IBGE – Instituto Brasileiro de Geografia e Estatística. Brasil. 2005, Available in: http://www.sidra.ibge.gov.br Acessed on Dec 14, 2010.

[7] Kuskoski, E.M.; Asuero, A.G.; Morales, M.T.; Fett, R. Frutos tropicais silvestres e polpas de frutas congeladas: atividade

antioxidante, polifenóis e antocianinas. Ciência Rural, 2006, 36, 1283-1287.

[8] Agostini-Costa, T.S.; Abreu, L.N.; Rossetti, A.G. Efeito do congelamento e do tempo de estocagem da polpa de acerola sobre o teor de carotenóides. Rev. Brasil. Fruticult., 2003, 25, 56-58.

[9] Polinati, R.M.; Faller, A.L.K.; Fialho, E. The effect of freezing at -18°C and -70°C with and without ascorbic acid on the stability of antioxidant in extracts of apple and orange fruits. Intl. J. Food Sci. Technol., 2010, 45, 1814-1820.

[10] Kalt, W.; Forney, C.F.; Martin, A.; Prior, R.L. Antioxidant capacity, vitamin C, phenolics, and anthocyanins after fresh storage of small fruits. J. Agric. Food Chem., 1999, 47, 4638-4644.

[11] Lopes, A.S.; Mattietto, R.A.; Menezes, H.C. Estabilidade da polpa de pitanga sob congelamento. Ciênc. Tecnol. Alimentos., 2005, 25, 553-559.

[12] Gil-Izquierdo, A.; Gil, M.I.; Ferreres, F. Effect of processing techniques at industrial scale on orange juice antioxidant and beneficial health compounds. J. Agric. Food Chem., 2002, 50, 5107-5114.

[13] IAL - Instituto Adolf Lutz. 2005. Normas Analíticas do Instituto Adolfo Lutz: métodos químicos e físicos para análise de alimentos. 4.ed., p.1018.

[14] AOAC - Association of Official Analytical Chemists. Official Methods of Analysis of the Association of Official Analytical Chemists, (method 934.06). Arlington: A.O.A.C., 1995. chapter 37. p. 4.

[15] Vinson, J.A.; Su, X.; Zubik, L.; Bose, P. Phenol antioxidant quantity and quality in foods: fruits. J. Agric. Food Chem., 2001, 49, 5315-5321.

[16] Karou, D.; Dicko, M.H.; Simpore, J.; Traore, S.F. Antioxidant and antibacterial activities of polyphenols from ethnomedicinal plants of Burkina Faso. Afr. J. Biotechnol., 2005, 4, 823-828.

[17] Kim, D.O.; Chun, O.K.; Kim, Y.J.; Moon, H.Y.; Lee, C.Y. Quantification of polyphenolics and their antioxidant capacity in fresh plums. J. Agric. Food Chem., 2003, 51, 6509-6515.

[18] Nakamura, Y.; Tsuji, S.; Tonogai, Y. Analysis of proanthocianidins in grape seeds extracts, health foods and grape seed oils. J. Health Sci., 2003, 40, 45-54.

[19] Corral-Aguayo, R.D.; Yahia, E.M.; Carrillo, L.A; González, A.G. Correlation between some nutritional components and the total antioxidant capacity measured with six different assays in eight horticultural crops. J. Agric. Food Chem., 2008, 56, 10498-10504.

[20] Javanmardi, J.; Kubota, C. Variation of lycopene, antioxidant activity, total soluble solids and weight loss of tomato during postharvest storage. Postharvest Biol. Technol., 2006, 41, 151-155.

[21] Silva, P.T.; Fialho, E.; Miguel, M.A.L.; Lopes, M.L.M.; Valente-Mesquita, V.L. Estabilidades química, físico-química e microbiológica de suco de laranja cv.“pera” submetido a diferentes condições de estocagem. B.Ceppa, 2007, 25, 235-246.

[22] Melo, E.A.; Maciel, M.I.S.; Lima, V.L.A.G.; Nascimento, R.J. Capacidade antioxidante de frutas. Braz. J. Pharm. Sci., 2008, 44, 193-201.

[23] Manica, I.; Icuma, I.M.; Junqueira, N.T.V.; Salvador, J.O.; Moreira, A; Mala Volta, E. Fruticultura tropical: goiaba. Porto Alegre: Cinco Continentes. 2000, cap. 6: 373.

[24] Azzolini, M.; Jacomino, A.P.; Bron, I.U. Índices para avaliar qualidade pós-colheita de goiabas em diferentes estádios de maturação. Pesquisa Agropecuária Bras., 2004, 39, 139-145.

[25] Brasil. Ministério da Agricultura e do Abastecimento. Instrução Normativa N.º 01, DE 7 DE JANEIRO DE 2000. Regulamento Técnico Geral para fixação dos Padrões de Identidade e Qualidade para Polpa de Fruta.Publicado no Diário Oficial da União de 10/01/2000, Seção 1, Página 54, 2000.

[26] Evangelista, R.M.; Vieites, R.L. Avaliação da qualidade de polpa de goiaba congelada, comercializada na cidade de São Paulo. Segurança Alimentar e Nutr., 2006, 13, 76-81.

[27] Hassimotto, N.M.A.; Genovese, M.I.; Lajolo, F.M. Antioxidant Activity of Dietary Fruits, Vegetables, and Commercial Frozen Fruit Pulps. J. Agric. Food Chem., 2005, 53, 2928-2935.

[28] Luximon-Ramma, A.; Bahorun, T.; Crozier, A. Antioxidant actions and phenolic and vitamin C contents of common Mauritian exotic fruits. J. Sci. Food Agric., 2003, 83, 496-502.

[29] Franke, A.A.; Custer, L.J.; Arakaki, C.; Murphy, S.P. Vitamin C and flavonoid levels of fruits and vegetables consumed in Hawaii. J. Food Comp. Anal., 2004, 17, 1-35.


[30] Pérez-Jiménez, J.; Arranz, S.; Saura-Calixto, F. Proanthocyanidin content in foods is largely underestimated in the literature data: An approach to quantification of the missing proanthocyanidins. Food Res. Intl., 2009, 42, 1381-1388.

[31] Padda, M.S.; Picha, D.H. Effect of low temperature storage on phenolic composition and antioxidant activity of sweet potatoes. Postharvest Biol. Technol., 2008, 47, 176-180.

[32] Khanizadeh, S.; Tsao, R.; Rekika, D.; Yang, R.; Charles, M.T.; Rupasinghe, H.P.V. Polyphenol composition and total antioxidant capacity of selected Apple genotypes for processing. J. Food Comp. Anal., 2008, 21, 396-401.

[33] Rodriguez-Amaya, D.B.; Kimura, M.; Godoy, H.T.; Amaya-Farfan, J. Updated Brazilian database on food carotenoids: Factors affecting carotenoid composition. J. Food Comp. Anal., 2008, 21, 445-463.

[34] Dalal, M.; Chinnusamy, V.; Bansal, K.C. Isolation and function characterization of Lycopene betacyclase (CYC-B) promoter from Solanum habrochaites. BMC Plant Biol., 2010, 10, 61-76.

[35] Nojavana, S.; Khaliliana, F.; Kiaiec, F.M.; Rahimic, A.; Arabanianc, A.; Chalavia, S. Extraction and quantitative determination of ascorbic acid during different maturity stages of Rosa canina L. fruit. J. Food Comp. Anal., 2008, 21, 300-305.

[36] Kuskoski, E.M.; Asuero, A.G.; Troncoso, A.M.; Mancini-Filho, J.; Fett, R. Aplicación de diversos métodos químicos para determinar actividad antioxidante en pulpa de frutos. Ciênc. Tecnol. Aliment., 2005, 25, 726-732.

[37] Mezadri, T.; Villaño, D.; Fernández-Pachón, M.S.; García-Parrilla, M.C.; Troncoso, A.M. Antioxidant compounds and antioxidant activity in acerola (Malpighia emarginata DC.) fruits and derivatives. J. Food Comp. Anal., 2008, 21, 282-290.

Received: December 11, 2011 Revised: February 26, 2012 Accepted: April 03, 2012

Documents

Bioactive Compounds, Vitamin C and Antioxidant Capacities of Fresh and Industrialized Frozen Pulps of Guava (Psidium guajava L.)