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The Effects of High Hydrostatic Pressure on the Polyphenols and Anthocyanins in Red Fruit Products
Available online at www.sciencedirect.com
Available online at www.sciencedirect.com
Procedia Food Science
Procedia – Food Science 00 (2011) 000–000
Procedia Food Science 1 (2011) 847 – 853 www.elsevier.com/locate/procedia
11th International Congress on Engineering and Food (ICEF11)
The effects of high hydrostatic pressure on the polyphenols and anthocyanins in red fruit products Giovanna Ferrari a,b, Paola Maresca b*a, Rossella Ciccarone b a
Department of Industrial Engineering, University of Salerno,via ponte don melillo, 84084 Fisciano (SA), Italy b ProdAl Scarl, University of Salerno,via ponte don melillo, 84084 Fisciano (SA), Italy
Abstract Bioactive compounds, such as polyphenols and anthocyanins, are rapidly affected by exogenic factors such as oxygen, light, as well as pH and temperature. The challenge to preserve polyphenols and anthocyanins suggests the application of high hydrostatic pressure for the sanitation of products rich in bioactive compounds, such as red-fruits derivatives.The aim of this paper is to study the effects of high pressure (HP) on the polyphenol and anthocyanin content of several red fruit-based products (strawberry and wild strawberry mousses, pomegranate juice). The processing conditions were set at 500 MPa, 50°C, 10 min for the mousse samples and 400 MPa, 25 °C and 5 min for the pomegranate juice samples, according to the results of the process cycle optimization obtained in a previous experimental campaign. The products, microbiologically stable, were stored under refrigerated conditions for 3 months, with the bioactive molecules being determined weekly.The results demonstrate the dependence of the anthocyanin and polyphenol content on the processing conditions, raw materials and storage conditions. The observed stability of these molecules could be transient, due to the residual activity of the enzymes, which along with a small concentration of dissolved oxygen could cause the degradation of the anthocyanins. Moreover, HP treatment at moderate temperatures promotes the extractability of coloured pigments and increases the polyphenol content. The results confirm the potentiality of the HP process for the treatment of products rich in thermolable and nutraceutical compounds. © 2011 Published by Elsevier B.V. Selection and/or peer-review under responsibility of 11th International Congress © 2011 Published by Elsevier Ltd. Selection and/or peer-review under responsibility of ICEF11 on Engineering and Food (ICEF 11) Executive Committee.
Executive Committee Members
Keywords: high pressure; functional foods; polyphenols; anthocyanins
1. Introduction The increasing amount of interest being given to attaining wellness through nutrition as well as the results of scientific research, which has demonstrated the potential health benefits of particular biologically active components (nutraceutical compounds), are the main factors supporting the demand
* Corresponding author. Tel.: +39-089-969438; fax: +39-089-964168 E-mail address: [email protected]
2211–601X © 2011 Published by Elsevier B.V. Selection and/or peer-review under responsibility of 11th International Congress on Engineering and Food (ICEF 11) Executive Committee. doi:10.1016/j.profoo.2011.09.128
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Giovanna et al. / Procedia Food Science 1 (2011) 847 – 853 Author Ferrari name / Procedia – Food Science 00 (2011) 000–000
for foods with evident effects on human health. These have been generally defined as “functional foods”. There has been a particular focus of attention on fresh red fruits and their derivatives (juice, nectars, mousses and purees), due to their possible health benefits as dietary antioxidant, anti-mutagenic, and chemo-preventive nutraceuticals that can possibly contribute to reducing chronic diseases. The functional properties of red fruit-based foodstuffs are mainly related to the concentration or presence of anthocyanins, polyphenols, tannins and vitamins, as well as bioactive molecules, which are capable of ensuring antioxidant properties, colour intensity, astringency and nutritional value. Unfavourably, bioactive compounds are quickly affected by exogenic factors such as oxygen, light, and, in particular, both pH as well as temperature. Despite of the optimization of the process conditions and the mild technological solution applied in the case of red-fruit derivatives, the thermal stabilization of these products induces the degradation of the bioactive compounds with an evident effect on both the quality perception (colour, taste, aroma) as well as the nutritional value. The natural pigments, mainly represented by the anthocyanidins, usually red or blue or variations, due to their high reactivity, readily degrade and form colourless or undesirable brown-coloured compounds. Many factors affect the stability of the anthocyanins, including temperature, pH, oxygen, enzymes, the presence of co-pigments and metallic ions, ascorbic acid, sulphur dioxide as well as sugars and their degradation products. During pasteurization and storage, several red fruit derivatives lose their bright red colour and become a dirtybrown one. Similarly, the behaviour of the polyphenol content is similar to that observed for the anthocyanin content in several liquid, semi-solid or solid foodstuffs. The challenge to preserve the polyphenols and anthocyanins suggests the application of the high hydrostatic pressure for the sanitation of the products rich in bioactive compounds, such as red-fruits derivatives. In current literature, the scientific results reporting the stability or preservation of these compounds are contradictory. Several authors have reported that the anthocyanins of different liquid foods (red-fruit juices) are stable to HHP treatment at moderate temperatures. For example, in pomegranate juice at room temperature, the nutraceutical and sensorial properties are strictly related to the anthocyanin and polyphenol content. The concentration of these molecules decreases with the intensity of the treatment in terms of pressure level and processing time [1]. Therefore, both the higher pressure levels as well as longer processing times cause a decrease of the anthocyanin content. In contrast, some authors have reported increased extractability of coloured pigments from the pulp suspended in the clear juices at extreme pressures. However, the observed stability of these molecules could be transient, due to the pressure and temperature levels applied during the experiments, which cannot induce an irreversible inactivation of the enzymes involved in the degradation of the natural pigments [1, 2, 3]. The aim of this paper is to investigate the stability of bioactive compounds and, in particular, of the total anthocyanin and polyphenol content of high pressure treated red fruit-based products. Liquid (pomegranate juice) and semi-solid foods (strawberry, wild strawberry mousses) were processed in high hydrostatic pressure cycles, which were optimized in previous experiments. The products, microbiologically stable, were stored under refrigerated conditions for 3 months, with the bioactive molecules being determined weekly. 2. Materials and Methods 2.1. Sample preparation Strawberries and wild strawberries, purchased from a local market, were washed twice, cut into small pieces, had citric acid and saccharose added, in order to correct the acidity and °Brix% content of the fresh fruits, and finely homogenized using an electric blender. The samples were stored under refrigerated conditions before the HP experiments. Pomegranates obtained from the local market were used for the extraction of the juice. The pomegranates were washed with water to remove any surface dirt and superficial microbial flora. The fruits were cut into halves, pressed with a pilot press in order to have a 40–45% yield. The extracted juice was sequentially flocculated with 300 mg/l of gelatine A (Sigma-
GiovannaAuthor Ferrari et al./ Procedia / Procedia FoodScience Science00 1 (2011) (2011) 000–000 847 – 853 name – Food
Aldrich Co., Bloom:90-100) and 300 mg/l of bentonite (Sigma-Aldrich Co.), at room temperature for 1h. Clear juice was obtained by filtration through cheese paper. 50 ml samples were stored frozen and thawed at room temperature before the HP experiments. 2.2 Experimental apparatus and protocols The experiments were carried out in a High Pressure (HP) pilot plant MINI FOODLAB FPG5620, realized by Stansted Fluid Power Ltd (UK). The pilot system FPG5620 is designed for the high pressure treatments of biological material (foodstuffs, microbial suspension, standard solutions of enzymes, proteins, starches) at temperatures ranging from -20°C to 90°C. In the high pressure treatments, the processing conditions were set at 500 MPa, 50°C, 10 min for the mousse samples, and 400 MPa, 25 °C and 5 min for the pomegranate juice samples, according to the results of the process cycle optimization published in a previous paper [1]. Several samples of the mousses and juice were prepared, sealed in flexible pouches made from a multilayer polymer/aluminium/polymer film (Polyethylene-Aluminium-Polypropylene) and processed under the chosen experimental conditions. At the end of the treatment, the pouches were stored at 4°C. 2.3 Total anthocyanins and polyphenols determinations The total anthocyanins were determined by a modified pH-differential method, as reported by Alper et al (1996) [4]. The spectrophotometric measurements were carried out using a UV-Vis spectrophotometer (Jasco Europe) and all the absorbance readings were made against distilled water as a blank. The Folin–Ciocalteu assay method was used for the determination of the total phenol content in the samples, colourimetrically measured and expressed as Gallic acid equivalents. The analytical determinations were carried out in triplicate. Analysis of variance (ANOVA) was used to evaluate the repeatability of the experimental data. Student tests were used as paired comparisons between sample means. The level of significance was set to 0.05. 3. Results and Discussion The red fruit-based samples were processed and analysed according to the previously described procedure. The results were separately reported for semi-solid and liquid samples. Figures 1 and 2 show the values of the estimated total polyphenol content, respectively, for the strawberry and wild strawberry samples, and for the pomegranate juice samples processed in the optimized high pressure cycles and stored under refrigerated conditions. On the left side of the diagram, the value of the polyphenol content estimated for the fresh samples before the processing (Reference) is reported. The semi-solid foods, i.e. the strawberry and wild strawberry mousses, show an increase of the polyphenol content immediately after the HP treatment, thus demonstrating that the high pressure allows for a better extractability of the polyphenol compounds, as already observed in current scientific literature. For example, the phenol levels in high pressure strawberry purees treated at pressure levels up to 600 MPa increased significantly (9.8%) when compared to unprocessed purees. A similar trend was observed for blackberry purees [3]. Corrales et al. (2008) reported the increase in the total phenolic content of grape by-products following high pressure processing [5]. The authors attribute the increase of the total phenolic content to the release of some antioxidant components from solid suspended particles following the high pressure processing [3]. Therefore, for semi-solid foods the HP treatment could potentially improve the total polyphenol content and prevent the degradation deriving from the application of high temperatures, as observed in traditional thermal treatments for food pasteurization/sterilization. The HP treatment of liquid foodstuffs, as shown in Figure 2, has a clear effect on the polyphenol content detected for the fresh juice, inducing a 22% reduction of the initial value. In this case, according to the experimental results, the high pressure process
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4
causes a decrease of the polyphenol content with respect to the natural content of the fresh juice. Moreover, as observed in a previous paper, the total polyphenol content in pomegranate juice depends on the treatment time, in relation to room temperature and the pressure level, thus decreasing the value of this parameter [1]. Therefore, the optimized process cycle was set-up to minimize the effect of the processing conditions on the functional compounds of the pomegranate juices. Similar results are reported for other juices rich in polyphenols and nutraceutical compounds. For example, the measured concentration of the total polyphenols in Annurca apple juice reached 40-60% of the initial content following HHP treatments at either room or a mild temperature and a fixed processing time (5 min) [6]. 8,00 Strawberry mousse
Polyphenol Content (g/L)
7,00
Wild Strawberry mousse
6,00 5,00 4,00 3,00 2,00 1,00 0,00 Reference
0
7
14
21
28
35
72
Time (days)
Fig. 1. Polyphenol content of High pressure treated samples of strawberry mousse and wild strawberry mousse evaluated at fixed storage times under refrigerated conditions (4 °C) 2,5 Pomegranate juice 2 Polyphenol Content (g/L)
850
1,5
1
0,5
0 Reference
0
7
14
21
28
35
42
49
56
Time (days)
Fig. 2. Polyphenol content of High pressure treated samples of pomegranate juice evaluated at fixed storage times under refrigerated conditions (4 °C)
During the storage under refrigerated conditions, a significant degradation of the total polyphenols of the processed strawberry and wild strawberry mousses occurs, as shown in Fig. 1. The decrease of the
GiovannaAuthor Ferrari et al./ Procedia / Procedia FoodScience Science00 1 (2011) (2011) 000–000 847 – 853 name – Food
post-processing value ranges from 30 to 50-60% upon increasing the storage time. Therefore, the benefits deriving from the application of HP process for food stabilization are definitively annulled during the preservation of the products. As shown in Figure 2, the polyphenol content of the pomegranate juice is quite stable up to 14 days of storage, while a slight decrease of this parameter (maximum 10%) is observed in the following period. At the end of the observation period, the trend of the polyphenol content reverses and a partial recovery of the post-processing value occurs. Therefore, for pomegranate juice, probably due to its different and homogeneous composition, the polyphenol content mainly depends on the HP processing conditions, while the variation of this parameter during storage can be considered negligible. The trend of polyphenol content during the preservation of the HP treated mousses can be explained by taking into account the residual enzymatic activity as well as the concentration of dissolved oxygen, which could both contribute to the pressure-induced degradation of the polyphenols. Similar results have been reported in current literature for blueberry products [7]. Polyphenolics in processed blueberry products are also susceptible to losses during storage at room temperature, decreasing markedly during the storage of berries canned in water or syrup [9, 10], and purées [9,10]. Although the mechanisms responsible for polyphenolic losses during storage of processed blueberry products have not been elucidated, refrigerated storage has been shown to improve polyphenolic retention in juices [8]. This observation confirms the result attained for the pomegranate juice. The values of the anthocyanin content are listed in Table 1 for the three analyzed samples and reported as a percentage of the initial value estimated for the reference fresh samples. The HP process causes a slight decrease of this parameter if evaluated for both the mousses, showing a 11% reduction. In general, anthocyanins (pelargonidin-3-glucoside and cyanidin-3-glucoside) were well retained at all pressure treatments, with this being reflected in a greater retention of the antioxidant activity of fruit purées. Moreover, a positive significant correlation was also found between the anthocyanin content of strawberry (pelargonidin-3-glucoside) and antiradical power [3]. A more relevant effect of the process is observed for the pomegranate juice, which retains only 63% of the initial anthocyanin content, notwithstanding the less drastic processing conditions. Table 1. Anthocyanin content (percentage of content estimated for the fresh raw material) of high pressure treated samples of strawberry mousse, wild strawberry mousse and pomegranate juice evaluated at fixed storage times under refrigerated conditions (4 °C). Anthocyanins content (%) Time (days) 0
Strawberry mousse
Wild Strawberry mousse
Pomegranate juice
89%
89%
63%
7
90%
71%
77%
14
74%
69%
69%
21
75%
72%
65%
28
77%
67%
69%
35
76%
65%
67%
72
65%
63%
63%
However, anthocyanin degradation in HHP processed red fruit-based products has been reported to occur as a result of indirect oxidation by the phenolic quinones generated by polyphenol oxidase and peroxidase [3]. Other authors have also reported that anthocyanins are stable to HHP treatment at moderate temperatures, while anthocyanin degradation occurs during the storage of the processed foodstuffs [11]. In contrast, several authors have also reported the increased extractability of coloured
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pigments in food components at extreme pressures, along with the polyphenol content increase [3, 11, 12, 13]. These results highlight that the effect of high pressure on the anthocyanin content of red-fruit derivatives cannot be generalized, while the composition of the product, the activity of the oxidative enzymes and the processing and operative conditions could compromise the efficiency of the HP treatment. Despite these contradictory observations on the effect of HP treatment on the anthocyanins, current scientific literature, as well as the results reported in Table 1, indicate a decrease of the total anthocyanin content during the storage of the red-fruit products. A degradation of the anthocyanin pigments was observed in several products after a HHP treatment, due to the presence of oxidase enzymes during or after processing as demonstrated in several fruit systems [3]. Other authors propose a different mechanism underlying the decrease of the detectable anthocyanin content during preservation. For example, Srivastava et al. (2007) [8], observing that the losses of anthocyanins and procyanidins during the storage of processed blueberry products were accompanied by increased polymeric colour values, supposed that the formation of anthocyanins−procyanidin polymers occurred during the storage of the products [8]. The same observation has been confirmed and reported in current literature for similar products [9, 10]. Anthocyanin−procyanidin polymers have been shown to form through direct condensation reactions [14] or reactions mediated by acetaldehyde and furfural [15]. This mechanism could be suitable in explaining the trend of the anthocyanin content in pomegranate juice. In this case, according to the experimental results, while the values of the anthocyanin content as well as the tannins are stable during the observation period, the total colour intensity and the polymeric content of the tannins of the high pressure treated juice increase (data not shown). Therefore, the high pressure treatment allows the anthocyanin content to be preserved, which attributes the intensity of the red colour and the antioxidant properties to pomegranate juice, as well as the typical astringency of the pomegranate taste. Simultaneously, several phenomena occur during storage, which contribute to increasing the colour intensity due to the formation of anthocyanin-tannin complexes. These changes may be mostly attributed to the anthocyanin condensation reactions, which involve the covalent association of anthocyanins with other flavanols through ethyl bridges or with other small molecules, such as pyruvic acid, vinylphenol and glyoxilic acid. They are also precursors of highly polymerized anthocyanins with different colour ranges, which may be of interest from an industrial point of view. 4. Conclusions The experimental campaign demonstrates that high pressure technology can be successfully applied to stabilize semi-solid and liquid products derived from red fruits, such as strawberry and wild strawberry mousses and pomegranate juice. The preservation of the nutraceutical quality of these products is the major challenge of the stabilization treatments. Their added value is mainly due to the high content of nutraceutical compounds, such as anthocyanins, polyphenols, and tannins, which are generally compromised by traditional thermal treatments. High processing temperature or longer processing times drastically reduce the nutraceutical compounds content and modify the natural colour of the red-fruit derivatives. As widely demonstrated by the discussed experimental data, the high pressure process allows the natural quality of the strawberry mousses and pomegranate juice to be preserved. According to the experimental data, the HP treatment improves the extractability of the polyphenols in mousses and slightly affects their content in the clear juices. During storage, due to the residual oxygen concentration and the activity of oxidase enzymes (PPO and PDO) a decreasing trend of this parameter is detected for both the semi-solid and liquid products tested in the experiments. However, the observed decrease of the polyphenol content can be considered negligible in the case of the clear juice. Similar observations hold if the total anthocyanin content is considered. However, the HP treatment does not allow the preservation of the anthocyanin content during storage of both the strawberry mousses. In fact, the degradation of these
GiovannaAuthor Ferrari et al./ Procedia / Procedia FoodScience Science00 1 (2011) (2011) 000–000 847 – 853 name – Food
compounds occurs due to the oxidative mechanism described above. The anthocyanin condensation reaction may explain the stability of these compounds during the preservation of the pomegranate juice. References [1] Ferrari G, Maresca P, Ciccarone R.. The application of high hydrostatic pressure for the stabilization of functional foods: pomegranate juice. J Food Eng 2010; 100 (2): 245-53. [2] Sanchez-Moreno C, Plaza L, Elez-Martinez P, De Ancos B, Martin-Belloso O, Cano M P. Impact of high pressure and pulsed electric fields on bioactive compounds and antioxidant activity of orange juice in comparison with traditional thermal processing. J Agric Food Chem 2005; 53(11):4403-9. [3] Patras A, Brunton NP, Da Pieve S, Butler F. Impact of high pressure processing on total antioxidant activity, phenolic, ascorbic acid, anthocyanin content and colour of strawberry and blackberry purees. Innov Food Sci Emerg Technol 2009; 10(3):30813. [4] Alper N, Savas KB, Acar J.. Influence of processing and pasteurization on color values and total phenolic compounds of pomegranate juice. J Food Proc Pres 2005; 29:357–68. [5] Corrales M, Toepfl S, Butz P, Knorr D, Tauscher B. Extraction of anthocyanins from grape by-products assisted by ultrasonics, high hydrostatic pressure or pulsed electric fields: a comparison. Innov Food Sci Emerg Technol 2008; 9(1):85-91. [6] Donsì F, Ferrari G, Maresca P, Pataro G. Effects of emerging technologies on food quality. In Food Quality: Control, Analysis and Consumer Concerns. Nova Science Publishers, Inc. 2011 ISBN: 978-1-61122-917-2. [7] Howard LR, Castrodale C, Brownmiller C, Mauromoustakos A. Jam processing and storage effects on blueberry polyphenolics and antioxidant capacity. J Agric Food Chem 2010; 58 (7), 4022–9. [8] Srivastava A, Akoh CC, Yi W, Fischer J, Krewer G. Effect of storage conditions on the biological activity of phenolic compounds of blueberry extract packed in glass bottles. J Agric Food Chem 2007; 55:2705– 13. [9] Brownmiller C, Howard LR., Prior RL. Processing and storage effects on monomeric anthocyanins, percent polymeric color, and antioxidant capacity of processed blueberry products J Food Sci 2008; 73:72–9. [10] Brownmiller C, Howard LR., Prior RL. Processing and storage effects on procyanidin composition and concentration of processed blueberry products. J Agric Food Chem 2009; 57, 1896– 1902. [11]Garcia-Palazon A, Suthanthangjai W, Kajda P, Zabetakis L.The effects of High Hydrostatic Pressure on Beta-Glucosidase, Peroxidase and Polyphenoloxidase in red raspberry (Rubus Idaeus) and strawberry (Fragaria X Ananassa). Food Chem 2004; 88 (1): 7-10. [12]Sanchez-Moreno C, Plaza L, De Ancos B, Cano MP. Impact of high-pressure and traditional thermal processing of tomato puree on carotenoids, vitamin c and antioxidant activity. J Sci Food Agric 2006; 86 (2): 171-9. [13] Plaza L, Munoz M, De Ancos B, Cano MP. Effect of combined treatments of high-pressure, citric acid and sodium chloride on quality parameters of tomato puree. Eur Food Res Technol 2003; 216 (6):514-9. [14] Remy, S., Fulcrand, H., Labarbe, B., Cheynier, V., Moutounet, M.. First confirmation in red wine of products resulting from direct anthocyanin−tannin reactions. J Sci Food Agric 2000; 80:745– 51. [15] Es-Safi N, Fulcrand H, Cheynier V, Moutounet M.. Studies on acetaldehyde induced condensation of (−)-epicatechin and malvidin-3-O-glucoside in a model solution system. Food Chem 1999; 47:2096– 2102. [16] Van der Geer J, Hanraads JAJ, Lupton RA. The art of writing a scientific article. J Sci Commun 2000;163:51–9.
Presented at ICEF11 (May 22-26, 2011 – Athens, Greece) as paper NFP067.
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Available online at www.sciencedirect.com
Procedia Food Science
Procedia – Food Science 00 (2011) 000–000
Procedia Food Science 1 (2011) 847 – 853 www.elsevier.com/locate/procedia
11th International Congress on Engineering and Food (ICEF11)
The effects of high hydrostatic pressure on the polyphenols and anthocyanins in red fruit products Giovanna Ferrari a,b, Paola Maresca b*a, Rossella Ciccarone b a
Department of Industrial Engineering, University of Salerno,via ponte don melillo, 84084 Fisciano (SA), Italy b ProdAl Scarl, University of Salerno,via ponte don melillo, 84084 Fisciano (SA), Italy
Abstract Bioactive compounds, such as polyphenols and anthocyanins, are rapidly affected by exogenic factors such as oxygen, light, as well as pH and temperature. The challenge to preserve polyphenols and anthocyanins suggests the application of high hydrostatic pressure for the sanitation of products rich in bioactive compounds, such as red-fruits derivatives.The aim of this paper is to study the effects of high pressure (HP) on the polyphenol and anthocyanin content of several red fruit-based products (strawberry and wild strawberry mousses, pomegranate juice). The processing conditions were set at 500 MPa, 50°C, 10 min for the mousse samples and 400 MPa, 25 °C and 5 min for the pomegranate juice samples, according to the results of the process cycle optimization obtained in a previous experimental campaign. The products, microbiologically stable, were stored under refrigerated conditions for 3 months, with the bioactive molecules being determined weekly.The results demonstrate the dependence of the anthocyanin and polyphenol content on the processing conditions, raw materials and storage conditions. The observed stability of these molecules could be transient, due to the residual activity of the enzymes, which along with a small concentration of dissolved oxygen could cause the degradation of the anthocyanins. Moreover, HP treatment at moderate temperatures promotes the extractability of coloured pigments and increases the polyphenol content. The results confirm the potentiality of the HP process for the treatment of products rich in thermolable and nutraceutical compounds. © 2011 Published by Elsevier B.V. Selection and/or peer-review under responsibility of 11th International Congress © 2011 Published by Elsevier Ltd. Selection and/or peer-review under responsibility of ICEF11 on Engineering and Food (ICEF 11) Executive Committee.
Executive Committee Members
Keywords: high pressure; functional foods; polyphenols; anthocyanins
1. Introduction The increasing amount of interest being given to attaining wellness through nutrition as well as the results of scientific research, which has demonstrated the potential health benefits of particular biologically active components (nutraceutical compounds), are the main factors supporting the demand
* Corresponding author. Tel.: +39-089-969438; fax: +39-089-964168 E-mail address: [email protected]
2211–601X © 2011 Published by Elsevier B.V. Selection and/or peer-review under responsibility of 11th International Congress on Engineering and Food (ICEF 11) Executive Committee. doi:10.1016/j.profoo.2011.09.128
848
2
Giovanna et al. / Procedia Food Science 1 (2011) 847 – 853 Author Ferrari name / Procedia – Food Science 00 (2011) 000–000
for foods with evident effects on human health. These have been generally defined as “functional foods”. There has been a particular focus of attention on fresh red fruits and their derivatives (juice, nectars, mousses and purees), due to their possible health benefits as dietary antioxidant, anti-mutagenic, and chemo-preventive nutraceuticals that can possibly contribute to reducing chronic diseases. The functional properties of red fruit-based foodstuffs are mainly related to the concentration or presence of anthocyanins, polyphenols, tannins and vitamins, as well as bioactive molecules, which are capable of ensuring antioxidant properties, colour intensity, astringency and nutritional value. Unfavourably, bioactive compounds are quickly affected by exogenic factors such as oxygen, light, and, in particular, both pH as well as temperature. Despite of the optimization of the process conditions and the mild technological solution applied in the case of red-fruit derivatives, the thermal stabilization of these products induces the degradation of the bioactive compounds with an evident effect on both the quality perception (colour, taste, aroma) as well as the nutritional value. The natural pigments, mainly represented by the anthocyanidins, usually red or blue or variations, due to their high reactivity, readily degrade and form colourless or undesirable brown-coloured compounds. Many factors affect the stability of the anthocyanins, including temperature, pH, oxygen, enzymes, the presence of co-pigments and metallic ions, ascorbic acid, sulphur dioxide as well as sugars and their degradation products. During pasteurization and storage, several red fruit derivatives lose their bright red colour and become a dirtybrown one. Similarly, the behaviour of the polyphenol content is similar to that observed for the anthocyanin content in several liquid, semi-solid or solid foodstuffs. The challenge to preserve the polyphenols and anthocyanins suggests the application of the high hydrostatic pressure for the sanitation of the products rich in bioactive compounds, such as red-fruits derivatives. In current literature, the scientific results reporting the stability or preservation of these compounds are contradictory. Several authors have reported that the anthocyanins of different liquid foods (red-fruit juices) are stable to HHP treatment at moderate temperatures. For example, in pomegranate juice at room temperature, the nutraceutical and sensorial properties are strictly related to the anthocyanin and polyphenol content. The concentration of these molecules decreases with the intensity of the treatment in terms of pressure level and processing time [1]. Therefore, both the higher pressure levels as well as longer processing times cause a decrease of the anthocyanin content. In contrast, some authors have reported increased extractability of coloured pigments from the pulp suspended in the clear juices at extreme pressures. However, the observed stability of these molecules could be transient, due to the pressure and temperature levels applied during the experiments, which cannot induce an irreversible inactivation of the enzymes involved in the degradation of the natural pigments [1, 2, 3]. The aim of this paper is to investigate the stability of bioactive compounds and, in particular, of the total anthocyanin and polyphenol content of high pressure treated red fruit-based products. Liquid (pomegranate juice) and semi-solid foods (strawberry, wild strawberry mousses) were processed in high hydrostatic pressure cycles, which were optimized in previous experiments. The products, microbiologically stable, were stored under refrigerated conditions for 3 months, with the bioactive molecules being determined weekly. 2. Materials and Methods 2.1. Sample preparation Strawberries and wild strawberries, purchased from a local market, were washed twice, cut into small pieces, had citric acid and saccharose added, in order to correct the acidity and °Brix% content of the fresh fruits, and finely homogenized using an electric blender. The samples were stored under refrigerated conditions before the HP experiments. Pomegranates obtained from the local market were used for the extraction of the juice. The pomegranates were washed with water to remove any surface dirt and superficial microbial flora. The fruits were cut into halves, pressed with a pilot press in order to have a 40–45% yield. The extracted juice was sequentially flocculated with 300 mg/l of gelatine A (Sigma-
GiovannaAuthor Ferrari et al./ Procedia / Procedia FoodScience Science00 1 (2011) (2011) 000–000 847 – 853 name – Food
Aldrich Co., Bloom:90-100) and 300 mg/l of bentonite (Sigma-Aldrich Co.), at room temperature for 1h. Clear juice was obtained by filtration through cheese paper. 50 ml samples were stored frozen and thawed at room temperature before the HP experiments. 2.2 Experimental apparatus and protocols The experiments were carried out in a High Pressure (HP) pilot plant MINI FOODLAB FPG5620, realized by Stansted Fluid Power Ltd (UK). The pilot system FPG5620 is designed for the high pressure treatments of biological material (foodstuffs, microbial suspension, standard solutions of enzymes, proteins, starches) at temperatures ranging from -20°C to 90°C. In the high pressure treatments, the processing conditions were set at 500 MPa, 50°C, 10 min for the mousse samples, and 400 MPa, 25 °C and 5 min for the pomegranate juice samples, according to the results of the process cycle optimization published in a previous paper [1]. Several samples of the mousses and juice were prepared, sealed in flexible pouches made from a multilayer polymer/aluminium/polymer film (Polyethylene-Aluminium-Polypropylene) and processed under the chosen experimental conditions. At the end of the treatment, the pouches were stored at 4°C. 2.3 Total anthocyanins and polyphenols determinations The total anthocyanins were determined by a modified pH-differential method, as reported by Alper et al (1996) [4]. The spectrophotometric measurements were carried out using a UV-Vis spectrophotometer (Jasco Europe) and all the absorbance readings were made against distilled water as a blank. The Folin–Ciocalteu assay method was used for the determination of the total phenol content in the samples, colourimetrically measured and expressed as Gallic acid equivalents. The analytical determinations were carried out in triplicate. Analysis of variance (ANOVA) was used to evaluate the repeatability of the experimental data. Student tests were used as paired comparisons between sample means. The level of significance was set to 0.05. 3. Results and Discussion The red fruit-based samples were processed and analysed according to the previously described procedure. The results were separately reported for semi-solid and liquid samples. Figures 1 and 2 show the values of the estimated total polyphenol content, respectively, for the strawberry and wild strawberry samples, and for the pomegranate juice samples processed in the optimized high pressure cycles and stored under refrigerated conditions. On the left side of the diagram, the value of the polyphenol content estimated for the fresh samples before the processing (Reference) is reported. The semi-solid foods, i.e. the strawberry and wild strawberry mousses, show an increase of the polyphenol content immediately after the HP treatment, thus demonstrating that the high pressure allows for a better extractability of the polyphenol compounds, as already observed in current scientific literature. For example, the phenol levels in high pressure strawberry purees treated at pressure levels up to 600 MPa increased significantly (9.8%) when compared to unprocessed purees. A similar trend was observed for blackberry purees [3]. Corrales et al. (2008) reported the increase in the total phenolic content of grape by-products following high pressure processing [5]. The authors attribute the increase of the total phenolic content to the release of some antioxidant components from solid suspended particles following the high pressure processing [3]. Therefore, for semi-solid foods the HP treatment could potentially improve the total polyphenol content and prevent the degradation deriving from the application of high temperatures, as observed in traditional thermal treatments for food pasteurization/sterilization. The HP treatment of liquid foodstuffs, as shown in Figure 2, has a clear effect on the polyphenol content detected for the fresh juice, inducing a 22% reduction of the initial value. In this case, according to the experimental results, the high pressure process
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causes a decrease of the polyphenol content with respect to the natural content of the fresh juice. Moreover, as observed in a previous paper, the total polyphenol content in pomegranate juice depends on the treatment time, in relation to room temperature and the pressure level, thus decreasing the value of this parameter [1]. Therefore, the optimized process cycle was set-up to minimize the effect of the processing conditions on the functional compounds of the pomegranate juices. Similar results are reported for other juices rich in polyphenols and nutraceutical compounds. For example, the measured concentration of the total polyphenols in Annurca apple juice reached 40-60% of the initial content following HHP treatments at either room or a mild temperature and a fixed processing time (5 min) [6]. 8,00 Strawberry mousse
Polyphenol Content (g/L)
7,00
Wild Strawberry mousse
6,00 5,00 4,00 3,00 2,00 1,00 0,00 Reference
0
7
14
21
28
35
72
Time (days)
Fig. 1. Polyphenol content of High pressure treated samples of strawberry mousse and wild strawberry mousse evaluated at fixed storage times under refrigerated conditions (4 °C) 2,5 Pomegranate juice 2 Polyphenol Content (g/L)
850
1,5
1
0,5
0 Reference
0
7
14
21
28
35
42
49
56
Time (days)
Fig. 2. Polyphenol content of High pressure treated samples of pomegranate juice evaluated at fixed storage times under refrigerated conditions (4 °C)
During the storage under refrigerated conditions, a significant degradation of the total polyphenols of the processed strawberry and wild strawberry mousses occurs, as shown in Fig. 1. The decrease of the
GiovannaAuthor Ferrari et al./ Procedia / Procedia FoodScience Science00 1 (2011) (2011) 000–000 847 – 853 name – Food
post-processing value ranges from 30 to 50-60% upon increasing the storage time. Therefore, the benefits deriving from the application of HP process for food stabilization are definitively annulled during the preservation of the products. As shown in Figure 2, the polyphenol content of the pomegranate juice is quite stable up to 14 days of storage, while a slight decrease of this parameter (maximum 10%) is observed in the following period. At the end of the observation period, the trend of the polyphenol content reverses and a partial recovery of the post-processing value occurs. Therefore, for pomegranate juice, probably due to its different and homogeneous composition, the polyphenol content mainly depends on the HP processing conditions, while the variation of this parameter during storage can be considered negligible. The trend of polyphenol content during the preservation of the HP treated mousses can be explained by taking into account the residual enzymatic activity as well as the concentration of dissolved oxygen, which could both contribute to the pressure-induced degradation of the polyphenols. Similar results have been reported in current literature for blueberry products [7]. Polyphenolics in processed blueberry products are also susceptible to losses during storage at room temperature, decreasing markedly during the storage of berries canned in water or syrup [9, 10], and purées [9,10]. Although the mechanisms responsible for polyphenolic losses during storage of processed blueberry products have not been elucidated, refrigerated storage has been shown to improve polyphenolic retention in juices [8]. This observation confirms the result attained for the pomegranate juice. The values of the anthocyanin content are listed in Table 1 for the three analyzed samples and reported as a percentage of the initial value estimated for the reference fresh samples. The HP process causes a slight decrease of this parameter if evaluated for both the mousses, showing a 11% reduction. In general, anthocyanins (pelargonidin-3-glucoside and cyanidin-3-glucoside) were well retained at all pressure treatments, with this being reflected in a greater retention of the antioxidant activity of fruit purées. Moreover, a positive significant correlation was also found between the anthocyanin content of strawberry (pelargonidin-3-glucoside) and antiradical power [3]. A more relevant effect of the process is observed for the pomegranate juice, which retains only 63% of the initial anthocyanin content, notwithstanding the less drastic processing conditions. Table 1. Anthocyanin content (percentage of content estimated for the fresh raw material) of high pressure treated samples of strawberry mousse, wild strawberry mousse and pomegranate juice evaluated at fixed storage times under refrigerated conditions (4 °C). Anthocyanins content (%) Time (days) 0
Strawberry mousse
Wild Strawberry mousse
Pomegranate juice
89%
89%
63%
7
90%
71%
77%
14
74%
69%
69%
21
75%
72%
65%
28
77%
67%
69%
35
76%
65%
67%
72
65%
63%
63%
However, anthocyanin degradation in HHP processed red fruit-based products has been reported to occur as a result of indirect oxidation by the phenolic quinones generated by polyphenol oxidase and peroxidase [3]. Other authors have also reported that anthocyanins are stable to HHP treatment at moderate temperatures, while anthocyanin degradation occurs during the storage of the processed foodstuffs [11]. In contrast, several authors have also reported the increased extractability of coloured
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pigments in food components at extreme pressures, along with the polyphenol content increase [3, 11, 12, 13]. These results highlight that the effect of high pressure on the anthocyanin content of red-fruit derivatives cannot be generalized, while the composition of the product, the activity of the oxidative enzymes and the processing and operative conditions could compromise the efficiency of the HP treatment. Despite these contradictory observations on the effect of HP treatment on the anthocyanins, current scientific literature, as well as the results reported in Table 1, indicate a decrease of the total anthocyanin content during the storage of the red-fruit products. A degradation of the anthocyanin pigments was observed in several products after a HHP treatment, due to the presence of oxidase enzymes during or after processing as demonstrated in several fruit systems [3]. Other authors propose a different mechanism underlying the decrease of the detectable anthocyanin content during preservation. For example, Srivastava et al. (2007) [8], observing that the losses of anthocyanins and procyanidins during the storage of processed blueberry products were accompanied by increased polymeric colour values, supposed that the formation of anthocyanins−procyanidin polymers occurred during the storage of the products [8]. The same observation has been confirmed and reported in current literature for similar products [9, 10]. Anthocyanin−procyanidin polymers have been shown to form through direct condensation reactions [14] or reactions mediated by acetaldehyde and furfural [15]. This mechanism could be suitable in explaining the trend of the anthocyanin content in pomegranate juice. In this case, according to the experimental results, while the values of the anthocyanin content as well as the tannins are stable during the observation period, the total colour intensity and the polymeric content of the tannins of the high pressure treated juice increase (data not shown). Therefore, the high pressure treatment allows the anthocyanin content to be preserved, which attributes the intensity of the red colour and the antioxidant properties to pomegranate juice, as well as the typical astringency of the pomegranate taste. Simultaneously, several phenomena occur during storage, which contribute to increasing the colour intensity due to the formation of anthocyanin-tannin complexes. These changes may be mostly attributed to the anthocyanin condensation reactions, which involve the covalent association of anthocyanins with other flavanols through ethyl bridges or with other small molecules, such as pyruvic acid, vinylphenol and glyoxilic acid. They are also precursors of highly polymerized anthocyanins with different colour ranges, which may be of interest from an industrial point of view. 4. Conclusions The experimental campaign demonstrates that high pressure technology can be successfully applied to stabilize semi-solid and liquid products derived from red fruits, such as strawberry and wild strawberry mousses and pomegranate juice. The preservation of the nutraceutical quality of these products is the major challenge of the stabilization treatments. Their added value is mainly due to the high content of nutraceutical compounds, such as anthocyanins, polyphenols, and tannins, which are generally compromised by traditional thermal treatments. High processing temperature or longer processing times drastically reduce the nutraceutical compounds content and modify the natural colour of the red-fruit derivatives. As widely demonstrated by the discussed experimental data, the high pressure process allows the natural quality of the strawberry mousses and pomegranate juice to be preserved. According to the experimental data, the HP treatment improves the extractability of the polyphenols in mousses and slightly affects their content in the clear juices. During storage, due to the residual oxygen concentration and the activity of oxidase enzymes (PPO and PDO) a decreasing trend of this parameter is detected for both the semi-solid and liquid products tested in the experiments. However, the observed decrease of the polyphenol content can be considered negligible in the case of the clear juice. Similar observations hold if the total anthocyanin content is considered. However, the HP treatment does not allow the preservation of the anthocyanin content during storage of both the strawberry mousses. In fact, the degradation of these
GiovannaAuthor Ferrari et al./ Procedia / Procedia FoodScience Science00 1 (2011) (2011) 000–000 847 – 853 name – Food
compounds occurs due to the oxidative mechanism described above. The anthocyanin condensation reaction may explain the stability of these compounds during the preservation of the pomegranate juice. References [1] Ferrari G, Maresca P, Ciccarone R.. The application of high hydrostatic pressure for the stabilization of functional foods: pomegranate juice. J Food Eng 2010; 100 (2): 245-53. [2] Sanchez-Moreno C, Plaza L, Elez-Martinez P, De Ancos B, Martin-Belloso O, Cano M P. Impact of high pressure and pulsed electric fields on bioactive compounds and antioxidant activity of orange juice in comparison with traditional thermal processing. J Agric Food Chem 2005; 53(11):4403-9. [3] Patras A, Brunton NP, Da Pieve S, Butler F. Impact of high pressure processing on total antioxidant activity, phenolic, ascorbic acid, anthocyanin content and colour of strawberry and blackberry purees. Innov Food Sci Emerg Technol 2009; 10(3):30813. [4] Alper N, Savas KB, Acar J.. Influence of processing and pasteurization on color values and total phenolic compounds of pomegranate juice. J Food Proc Pres 2005; 29:357–68. [5] Corrales M, Toepfl S, Butz P, Knorr D, Tauscher B. Extraction of anthocyanins from grape by-products assisted by ultrasonics, high hydrostatic pressure or pulsed electric fields: a comparison. Innov Food Sci Emerg Technol 2008; 9(1):85-91. [6] Donsì F, Ferrari G, Maresca P, Pataro G. Effects of emerging technologies on food quality. In Food Quality: Control, Analysis and Consumer Concerns. Nova Science Publishers, Inc. 2011 ISBN: 978-1-61122-917-2. [7] Howard LR, Castrodale C, Brownmiller C, Mauromoustakos A. Jam processing and storage effects on blueberry polyphenolics and antioxidant capacity. J Agric Food Chem 2010; 58 (7), 4022–9. [8] Srivastava A, Akoh CC, Yi W, Fischer J, Krewer G. Effect of storage conditions on the biological activity of phenolic compounds of blueberry extract packed in glass bottles. J Agric Food Chem 2007; 55:2705– 13. [9] Brownmiller C, Howard LR., Prior RL. Processing and storage effects on monomeric anthocyanins, percent polymeric color, and antioxidant capacity of processed blueberry products J Food Sci 2008; 73:72–9. [10] Brownmiller C, Howard LR., Prior RL. Processing and storage effects on procyanidin composition and concentration of processed blueberry products. J Agric Food Chem 2009; 57, 1896– 1902. [11]Garcia-Palazon A, Suthanthangjai W, Kajda P, Zabetakis L.The effects of High Hydrostatic Pressure on Beta-Glucosidase, Peroxidase and Polyphenoloxidase in red raspberry (Rubus Idaeus) and strawberry (Fragaria X Ananassa). Food Chem 2004; 88 (1): 7-10. [12]Sanchez-Moreno C, Plaza L, De Ancos B, Cano MP. Impact of high-pressure and traditional thermal processing of tomato puree on carotenoids, vitamin c and antioxidant activity. J Sci Food Agric 2006; 86 (2): 171-9. [13] Plaza L, Munoz M, De Ancos B, Cano MP. Effect of combined treatments of high-pressure, citric acid and sodium chloride on quality parameters of tomato puree. Eur Food Res Technol 2003; 216 (6):514-9. [14] Remy, S., Fulcrand, H., Labarbe, B., Cheynier, V., Moutounet, M.. First confirmation in red wine of products resulting from direct anthocyanin−tannin reactions. J Sci Food Agric 2000; 80:745– 51. [15] Es-Safi N, Fulcrand H, Cheynier V, Moutounet M.. Studies on acetaldehyde induced condensation of (−)-epicatechin and malvidin-3-O-glucoside in a model solution system. Food Chem 1999; 47:2096– 2102. [16] Van der Geer J, Hanraads JAJ, Lupton RA. The art of writing a scientific article. J Sci Commun 2000;163:51–9.
Presented at ICEF11 (May 22-26, 2011 – Athens, Greece) as paper NFP067.
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