BIOACTIVE COMPOUNDS IN INDUSTRIAL TOMATO SAUCE AFTER PROCESSING AND STORAGE

Abstract

The effects of Industrial processing, product parameters (lethality F^sup 10^^sub 100^ and °Brix) and storage (up to 24 months at T^sub room^ on the content and antioxidant capacity (AC) of the bioactive compounds in industrial tomato sauce were studied. The total phenolic content (TPC) of both the hydrophilic and lipophilic fractions (using the Folin-Ciocalteu assay and a direct absorbance reading at 280 nm), in addition to the lycopene content and AC (using the ABTS+ assay), were analysed. Statistical analysis showed that the content and activity of the studied bioactive constituents were positively influenced by the °Brix, but not influenced by the thermal treatment severity (up to 40 min as F^sup 10^^sub 100^), that the TPC and its AC did not vary during storage, and that the lycopene AC decreased by 30% in the first year and then remained constant. The lycopene fraction and the hydrophific fractions contributed 83% and 27% respectively, of the sauce AC.

Headnote

ABSTRACT

The effects of industrial processing, product parameters (lethality F^sup 10^^sub 100^ and °Brix) and storage (up to 24 months at T^sub room^) on the content and antioxidant capacity (AC) of the bioactive compounds in industrial tomato sauce were studied. The total phenolic content (TPC) of both the hydrophilic and lipophilic fractions (using the Folin-Ciocalteu assay and a direct absorbance reading at 280 nm), in addition to the lycopene content and AC (using the ABTS^sup +^ assay), were analysed. Statistical analysis showed that the content and activity of the studied bioactive constituents were positively influenced by the °Brix but not influenced by the thermal treatment severity (up to 40 min as F^sup 10^^sub 100^), that the TPC and its AC did not vary during storage, and that the lycopene AC decreased by 30% in the first year and then remained constant. The lycopene fraction and the hydrophilic fractions contributed 83% and 27%, respectively, of the sauce AC.

Keywords: antioxidant activity, bioactive compounds, phenolics, shelf life, thermal treatment, tomato sauce

(ProQuest: ... denotes formulae omitted.)

INTRODUCTION

Tomatoes, the second most consumed vegetable in the world after potatoes, are the main component of the Mediterranean diet. The world production of tomatoes for processing in 2012 was estimated at 33.44 million tons, with California, Italy and China being the largest producers (11.46, 4.50 and 3.23 million tons, respectively) (WPTC, 2013). Tomatoes are a very important dietary source of carotenoids, especially lycopene, which represents approximately 80-90% of the total carotenoid content in tomatoes and confers their characteristic red colour; ß-carotene is the second most abundant carotenoid in tomatoes (3-5% total tomato carotenoid content) (SHI and LE MAGUER, 2000). Tomatoes, either fresh or as a processed product, contain different types of micronutrients with a high nutritional value, such as vitamins (C and E), folates, carotenoids and phenolic compounds (PERIAGO and GARCIA-ALONSO, 2009; SÁNCHEZ-RODRÍGUEZ et cd., 2012). In addition to being potent antioxidants, these compounds also have anti-inflammatory, antimutagenic and anticarcinogenic properties as well as the capacity to modulate key cellular enzyme functions (GIOVANNUCCI, 2005; VALLVERDÚ-QUERALT et aL, 201 la). It is known that thermal treatment and storage can cause a decrease in the nutritional value and a change in the colour of tomato products (CAPANOGLU et cd., 2010).

Numerous studies have investigated the micronutrient content of fresh and processed tomatoes, with the majority studying the loss of one or two types of micronutrients (SHARMA and LE MAGUER, 1996; RE et cd., 2002; GAHLER et al, 2003; GRAZIANI et al, 2003; LAVELLI and TORRESANI, 2011). However, to date, only a few studies have been published regarding the effect of technological processes or storage on antioxidants in tomatoes and tomato products (ABUSHITA et al, 2000; GIOVANELU and LAVELLI, 2002; LAVELLI and GIOVANELLI, 2003; CAPANOGLU etal, 2008; MURCIA et al, 2009; PÉREZ-CONESA et cd., 2009; CAPANOGLU étal, 2010; COLLE et aL, 2010; VALU VERDÚ-QUERALT et al, 2011a, 2011b, 2012). Furthermore, only a few of the cited authors have investigated changes in bioactive compounds due to processing at the industrial level, and none have investigated storage periods of longer than 12 months. The majority of the published studies involved laboratory-scale or pilot-scale experiments, and significant deviations have been found when comparisons were made with the end products of industrial-scale processes (CAPANOGLU et aL, 2010). Therefore, it is essential to examine actual commercial processes in order to obtain reliable data regarding the quality of the product that actually reaches the consumer.

In this context, the objectives of this work were to study the effects of various processes and product parameters, in particular the equivalent thermal effect or lethality (expressed as F1^, corresponding to equivalent minutes at 100°C, taking as reference a microorganism with a thermal resistance (z value) of 10°C), the °Brix, and prolonged storage periods (up to 24 months at room temperature, compared to an expected shelf-life of 3 years), on the content and activity of the bioactive compounds in industrial tomato sauce, applying simple and rapid analytical methods suitable for application in industrial quality control laboratories. The bioactive substances studied include lycopene and the total phenolics compounds.

MATERIALS AND METHODS

Reagents

Gallic acid, potassium persulfate, ascorbic acid and sodium carbonate were purchased from Carlo Erba (Milan, Italy), and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) was purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). Folin-Ciocalteu reagent was purchased from Merck (Darmstadt, Germany), and butylated hydroxytoluene (BHT) was purchased from Sigma Aldrich (St. Louis, MO, USA).

Acetone, ethanol (95%), methanol and hexane, all solvents of analytical reagent grade, were obtained from Carlo Erba Reagents (Milan, Italy).

Samples

Cans of tomato sauce (5 kg) were kindly provided by Steriltom Sri (Piacenza, Italy). This company uses both hydrostatic and continuous atmospheric retorts (AR). The products used in the present study were sterilised in two hydrostatic retorts (HRa and HRb). The temperature at the cold point of the product was measured using a Datatrace® Micropack III Temperature Data Logger for each sterilisation cycle. Industrial processing was carried out in order to achieve a minimum sterilising effect of 12 min as (F1^), which is required for the inactivation of spores of Clostridium pasteurianum and Bacillus coagulaos, the most important spoilage-causing microorganisms in tomato products with a pH of 4.5 or less.

The total soluble solids were determined using a digital refrac tome ter (RFM712, Bellingham + Stanley Ltd), and the results are reported as °Brix. The equivalent thermal effect obtained for each process was evaluated using Datatrace® equipment placed inside the can. For the specific aim of this study, the company provided samples of non-sterilised tomato (NS), for which the F1^ value was measured. Because preliminaiy studies had shown the influence of the position in the retort on the process lethality, for this investigation samples were collected from the hottest plant locations. Because the cans used to evaluate the F values could not be used for product analysis, the samples for experimentation were collected from positions in the retort close to the can with the temperature data logger.

For the analyses, each can was opened, the content was manually homogenised with a glass rod, and the hydrophilic fraction (HF), lipophilic fraction (LF) and lycopene fraction (LYF) were obtained. The HF and LF were tested for total phenolic content (TPC) and antioxidant capacity (AC). The LYF was tested for lycopene content and AC. All data in this work were obtained from the analysis of three cans in triplicate.

Experimental protocol

Because the first aim of this study was to assess the influence of process lethality on the content and activity of bioactive compounds, products were analysed with the same °Brix and storage age. To achieve different equivalent thermal effect values, five series of samples with different Tw (temperature of the product cold point at the start of heating, measured by the thermocouple) were collected (Table 1). For each series, the samples from HRa and HRb were collected at the same day and time (to ensure the same 0Brix). For the non-sterilised samples, the cans were not processed in the retorts but instead allowed to cool at room temperature, and a lethality value of 1.9 was assumed as the average of the F1^ values experimentally evaluated for the three different Thl values from the process Thl range; that is, F1^ wilues of 2.5, 1.8 and 1.4 min for Tw values of 84.9°, 83.6°C and 82°C, respectively.

The sampling of the cans was planned and carried out throughout the processing season in order to permit analysis of all the samples exactly 6 months after their collection and to sample the entire production from a single harvest. NS samples were unavailable for one of the series because the cans exploded before analysis; fortunately, this did not occur for the other series, demonstrating the high hygienic quality of the raw material. Furthermore, it was not possible to obtain exactly the same °Brix for all the samples (Table 1), and therefore the data were also elaborated to assess any possible influence of variations in the °Brix.

The second aim of the study was to assess the influence of long-term storage on the content and AC of bioactive compounds, analysing products with the same received lethality (21.3 and 1.8 min for sterilised and non-sterilised samples, respectively) and °Brix (6.15). All the samples were stored in a non-air-conditioned room and periodically analysed (after 0, 30, 64, 100, 133, 330, 407, 575 and 735 days). The non-sterilised samples were monitored for only 4 months.

Separation of hydrophilic and lipophilic fractions

The method was adapted from LARROSA et cd. (2003) and DJURIC and POWELL (2001). The tomato sauce (35 g) was centrifuged in a Varifuge 20 RS (Heraeus Sepatech GmbH, Hanau, Germany) at 10,000 g and 4°C for 10 min. Two fractions were obtained: a nearly colourless supernatant and a red pellet. The pellet was washed three times with 20 mL of distilled water. The four aqueous supernatants were pooled, and the total volume was measured (VHF) and filtered through filter paper (Whatman 595 V2) to give the hydrophilic fraction (HF). The red pellet was washed three times with 15 mL of acetone/methanol (70:30 v:v), shaken, and further centrifuged under the same conditions as specified for the HF. The reddish supernatants obtained were pooled, and the total volume was measured (VLF) and filtered through filter paper (Whatman 595 V2) to give the hydrophobic or lipophilic fraction (LF). The resulting colourless pellet was discarded. The LF was concentrated using a rotavapour BÜCHI B - R114 (BÜCHI, Flawil, Switzerland) to eliminate the acetone that could have interfered in the spectrophotometric analysis. After removing the acetone, the extract was brought to 50 mL with methanol and homogenised through agitation.

Recovery of Lycopene fraction

The lycopene fraction (LYF) was recovered according to the method proposed by FISH et cd. (2002). The tomato sauce (600 mg) was placed in an amber screw-top vial (40 mL) with 5 mL of 0.05% (w/v) BHT in acetone, 5 mL of ethanol, and 10 mL of hexane. The samples were extracted on an orbital shaker (HT INFORS AG CH-4103, Bottmingen, Switzerland) at 180 rpm for 15 min in an ice bath. After shaking, 3 mL of distilled water was added to each vial, and the samples were then shaken for 5 min on ice. The vials were left at room temperature for 5 min to allow the separation of the reddish upper hexane phase (LYF). For the estimation of AC, a BHT blank was obtained with the same extraction procedure but without tomato sauce, even though it is known from the literature that the ABTS response of BHT is negligible compared to that of lycopene (MÜLLER et cd., 2011).

The lycopene content was estimated by measuring the absorbance at 503 nm (Lambda Bio 40 UVVis spectrophotometer, Perkin-Elmer, Norwalk, CT) in a 1 cm path length quartz cuvette using hexane solvent as a reference. The total lycopene content in each sample was estimated from Eq. ( 1) :

... (1)

where: A^ is the absorbance at 530 nm; e is the molar extinction coefficient (17.2 x 104 M 'cm1) for lycopene in hexane, as reported by ZECHMEISTER et cd. (1943); b is the path length of the quartz cuvette (1 cm); Mw is the molecular weight of lycopene (536.9 g mol'1 cm1); and volume extract is the LYF or hexane phase in 10 2 L.

Although it is not the lycopene maximum absorption wavelength, the absorbance at 503 nm was used to minimise interference from other carotenoids because it has been reported that their contribution to the absorbance at 503 nm is less than 4% in fresh red tomatoes (FISH et al, 2002).

Total phenolic content

The total phenolics were determined using two different methods:

(1) Folin-Ciocalteu assay: this method was used because it is widely employed to study natural antioxidants and is considered the best method to determine the content of total phenolics (including tannins) (ENGELHARDT, 2001). Samples of HF and LF were analysed without dilution according to the method reported in AMENDOLA et al. (2010). Solid phase extraction to eliminate interference from, for example, ascorbic acid, amino acids and reductant sugars (VALLVERDÚ-QUERALT et al., 2012), was not carried out in order to simplify the procedure for potential routine industrial product control. Regarding the possible interference from reducing sugars, considering the maximum °Brix of the samples, that in tomatoes °Brix is not given only by reducing sugars and sample dilution in the experimental procedure, it was estimated that the minimum 2% sugar concentration necessary for the correction of Folin's results (WATERHOUSE, 2002) was not reached. To address possible interference from ascorbic acid (which was not measured in the samples), blank trials were carried out. Based on the literature values for the ascorbic acid content in fresh tomatoes and processed products (SINGH et cd., 2008; CHANFORAN et ad., 2012; KOH et ad., 2012), a content of 125 and 250 mg/kg ascorbic acid in the product was assumed. Assuming that all the ascorbic acid was extracted in the HF, blanks of standard ascorbic acid were prepared at concentrations of 50 and 100 mg/L and analysed with the Folin assay. The results demonstrated absorbance values of 0.050±0.003 and 0.101±0.005 for the 50 and 100 mg/L concentrations, respectively, while all of our samples had absorbance values above 0.2.

(2) Direct reading of the absorbance of the sample at 280 nm: this is a more rapid procedure based on the absorbance of the aromatic ring (AMENDOLA et cd., 2010) and the wavelength used to detect phenolic compounds in tomato products (CHANFORAN et cd., 2012). Samples of HF and LF were analysed after 50 dilutions.

In both cases, the total phenolics were expressed as gallic acid equivalents (GAE-Folin and GAE-280) by means of calibration curves with standard gallic acid in the concentration range of 100-750 and 2.5-30 mg/L for methods 1 and 2, respectively. In particular, the results were reported as mg/kg content of tomato sauce considering the VHF or the VLF and the amount of tomato sauce used for extraction, according to Eq. (2).

... (2)

Antioxidant capacity

The antioxidant capacity was assessed using the ABTS assay (AMENDOLA et cd., 2010), which is based on the ability of antioxidants to interact with the ABTS radical, decreasing its absorbance at 734 nm. A radical solution (7 mM ABTS and 2.45 mM potassium persulfate) was prepared and kept in the dark at room temper - ature for 12-16 h before use. This solution was then diluted with ethanol to obtain an absorbance of 0.70 (±0.02) at 734 nm and equilibrated at 30 °C. For the analysis, cuvettes for the samples and blanks were prepared, kept in the dark for 6 minutes and then read on the spectrophotometer (absorbance at 734 nm against ethanol). For the samples, 2 mL of the diluted radical solution was mixed with 20 pL of sample (HF, LF or LYF), and for each sample, three replicates were analysed. Two different blanks were prepared; one for the solvent, consisting of 2 mL of diluted ABTS mixed with 20 pL of solvent extraction (water for HF, methanol for LF, and hexane for LYF), and one for the ABTS, consisting of 2 mL of diluted ABTS. For each trial, two replicate blank samples were included. The antioxidant power (AOP) was calculated as the percentage inhibition of the absorbance at 734 nm according to Eq. (3):

... (3)

For the analysis of LYF, the AOP values were corrected with the AOP of the blank of BHT, giving values in the 20-40% range.

For the analysis of the HF, blanks of ascorbic acid were prepared as described for the total phenolic content and analysed for the AOP, giving values of 12.01±.13% and 23.70±0.84% for the 50 and 100 mg/L concentrations, respectively. The AOP in the analysed HF samples was always in the range 35-60%, while in the LF it was < 10%.

For the samples with an AOP < 30-40% it was not possible to obtain a proper dose-response curve (in terms of the change in AOP as a function of the sample concentration). Therefore, for a better comparison of all the samples, the specific antioxidant power (AOPj was also determined either in reference to the concentration of bioactive compounds (total phenolics or lycopene) in the sample according to Eq. (4) or as the specific power (AOP'j referred to in tomato sauce according to Eq. (5) (AMENDOLA et cd., 2010; AMENDOLA et cd., 2012):

... (4)

... (5)

Statistical analysis

The results reported in this paper are the average of the replicates ± SD. The statistical software SPSS (version 19.0, SPSS Inc., Chicago, IL, USA) was used to assess the influence of the F^q, the °Brix and the storage time on the content and activity of the bioactive compounds using univariate analysis of variance (ANOVA) at a confidence level of over 99%. When significance was found, a post-hoc test was carried out for mean discrimination. When variance homogeneity (according to Levene's test) was found, Tukey's post-hoc test was applied, while in the presence of non-homogeneous variances, a BrowneForsythe ANOVA and the Games Howell post-hoc test were applied.

RESULTS AND DISCUSSION

Analytical and methodological approach

The spectral analysis at 280 nm of the total phenolic compounds can be subject to influences from aromatic compounds other than phenolics, and each class of phenolic substances has a different absorptivity. However, this method is not influenced by the oxidative status of the molecules (AMENDOLA et cd., 2010), and it is a very rapid method, suitable for monitoring the influence of process parameters on the product.

On the other hand, the Folin assay, although traditionally used to estimate the total phenolic content, can be considered an antioxidant capacity method based on a single electron transfer (SETT) mechanism (PRIOR et cd. 2005); the results are thus influenced by the oxidative status of the molecules. The absolute values of the total phenolic content, although expressed in both cases as GAE, cannot be compared due to the different analytical principles. Folin analysis of the LF consistently gave absorbance values below 0.1, so the results are not reported.

Although seemingly redundant with the Folin assay, the ABTS test is usually classified as an SETT reaction, but the radical can be neutralised either by direct reduction via electron transfer or by radical quenching via H atom transfer (HAT) (PRIOR et cd., 2005). Therefore, even though currently a minimum of two assays are recommended in the evaluation of AC, we carried out only the Folin assay and the ABTSptest due to the screening nature of the present sturdy.

Ascorbic acid is a recognised bioactive compound in tomatoes and related products (LAVELLI and GIOVANNELLI, 2003; SÁNCHEZ-MORENO et cd., 2006) and it can interfere with both the Folin and the ABTS assay. Analysis of blanks (prepared based on the ascorbic acid content of fresh tomatoes and of processed products, as reported in the literature) revealed that ascorbic acid could contribute up to 50% of the Folin reducing power or of the ABTS AOP. On one hand, this means that in the evaluation of the total AC of a tomato product, ascorbic acid should not be removed from the HF in the purification steps, as was done in the present work. On the other hand, this also means that evaluation of the ascorbic acid content in the HF is advised in order to properly reference the Folin and ABTS values only to the phenolic content.

Effect of industrial process parameters

In the industrial process, the temperature of the product at filling is a pre-set value but inevitably undergoes some variations during operation. Similarly, the °Brix of the product varies slightly depending on the raw material and the harvest phase. Preliminary investigations had shown a great influence of Thl on the achieved lethality (Fig. la), independent of both the retort type and of °Brix, at least in the very narrow measured °Brix range (Figure lb). Based on these assumptions, in the experimental protocol samples with different Thl were collected in order to obtain different lethality values. For statistical analysis, the samples were grouped according to different F1^ ranges (1.9 min; 1721.5 min; 26.6-32 min; 37-39 min) and different °Brix (6.4-6.7; 6.9-7.3; 74-7.5). Considering the very limited °Brix range and that there should be no interaction between the °Brix and the process lethality for the solid contents (as also evidenced in Figure lb), statistical analysis was carried out separately on the two factors. The ANOVA results are summarised in Table 2.

As was expected given its very limited variation, the °Brix was found to have a significant influence only on the total content of phenolic compounds and on the tomato sauce specific AOP" based on the HF, with a slight tendency toward higher values at higher °Brix degrees (Table 3).

The content and activity of the studied bioac- tive constituents of tomato sauce were separated according to the F\° 0 range only in some cases. Lethality was observed to influence the total phenolic content of HF according to both analytical methods, but the means discrimination (Table 4) did not allow for a clear distinction between the samples. Overall, it was observed that an increase in the severity of thermal treatment within the observed range (2-40) did not cause any variation in the phenolic content, as also reported by PEREZ-CONESA et al. (2009). Our GAE-Folin results are consistent with the phenolic content of tomato and tomato products (between 100 and 500 mg kg'1) described in the literature (VALLVERDÚ - QUERALT et aL, 201 la). Considering the GAE-280 results and Eq. (2), the contribution of the HF to the total phenol content of the tomato sauce was calculated as the ratio between the mgoAE-HF/kgsauce ^ S(TM) ^GAE-HF/kg^e + H^AE-Lp/kgsauJ' confirming that almost all of the polyphenols (83-91%) were contained in the hydrophilic fraction (TOOR and SAVAGE, 2005).

Confirming the Folin results, lethality did not influence either the AOP' or the AOP", and a comparison among the hydrophilic, lipophilic and lycopene fractions showed that the greatest contribution to the AC of tomato sauce came from lycopene (Fig. 2). In fact, the LF should have given values close to those of LYF. This means that the procedure used to separate the LF was inadequate, while that used for lycopene extraction (in an ice bath and with the addition of an antioxidant) permitted the conservation of the bioactivity of the extracted compounds.

Likewise, for the lycopene content, despite the significant influence of lethality, the means could not be properly separated in the post-hoc test (Table 4), even though with increasing treatment severity an apparent trend of decreasing content and increasing activity was found. In the literature, it has been reported that heat treatment can exert a positive effect on carotenoid efficiency and absorption (ANESE et al., 2002) and that carotenoids can undergo isomerisation and oxidation, depending on the conditions, but generally remain quite stable during processing (GIOVANELLI and LAVELLI, 2002). The values we obtained were consistent with data reported by other authors for tomato and tomato juices (GARCÍA-ALONSO et al, 2009; PÉREZ-CONES A et al., 2009; COLLE étal, 2010).

Effect of storage on bioactive compounds and antioxidant capacity

Based on the aforementioned conclusions about the inadequacy of the procedure used to separate the LF, the LF was not analysed for the storage samples.

The unsterilised samples did not significantly differ from their sterilised counterparts for any of the investigated parameters at any time, confirming the previous finding. Only a few exceptions were found for the total phenolic content (as GAE-280) of the HF (with lower values for the NS samples) and for the related AOP' (with higher values for the NS samples).

The significance of the influence of storage time on the analysed parameters and the results of the means discrimination are reported in Tables 2 and 5, respectively. For the unsterilised samples (monitored for 4 months), only the AOP" based on the LYF was influenced by storage time, even though Tukey's post-hoc test could not discriminate the means. Regarding the processed samples, it can be concluded that the total phenolic content of the HF remained unvaried during the storage time, consistent with previous studies in which shorter periods were investigated (GIOVANELLI and PARADISO, 2002; GARCÍA-ALONSO et al, 2009; VALLVERDÚ-QUERALT et al, 2011a). However, a decrease in the specific antioxidant capacity of the tomato sauce (AOP") occurred after one year of storage.

As for the LYF, the lycopene content remained almost significantly unchanged. In fact, only two homogenous groups could be discriminated by Tukey's test, one from 0 to 407 days, and a second from 30 to 735 days. This is consistent with the findings of GARCÍA-ALONSO et cd. (2009), although other authors have reported final losses of approximately 60-70% (MIN et cd., 2003; LIN and CHEN, 2005; ODRIOZOLA-SERRANO et cd., 2008). However, comparisons between literature data are difficult due to differing tomato products and processing conditions. The specific antioxidant power of lycopene, and as a consequence the specific tomato sauce activity, decreased appreciably (30%) between 5 and 11 months of storage, and then remained constant thereafter up to 24 months.

CONCLUSIONS

The present study showed that the sterilisation step of tomato sauce processing, in a severity of up to almost 40 min (as influenced neither the content nor the AC of the hydrophilic (related to phenolics content) or the lycopene fractions. However, the extraction method can greatly influence the results because the addition of an antioxidant compound (BHT) and low temperature conditions are both required to prevent lycopene degradation.

Industrial tomato sauce has been confirmed as a potential rich dietary source of bioactive compounds, although the lycopene-associated nutritional antioxidant profile showed a 30% reduction between 5 and 11 months of storage at room temperature and then remained stable until 24 months. Furthermore, the effects of further processing during home cooking on antioxidant activity must be taken into account, given that tomato sauce is generally further cooked [Le., for pasta or pizza dressing) before consumption.

ACKNOWLEDGEMENTS

This research and publication have been partly funded by Università Cattolica under one of its programs for promotion and dissemination of scientific research (Linea D.3.I., year 2014)

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Paper received June 17, 2013 Accepted December 27,2013