Aseptically packaged UHPH-treated apple juice: Safety and quality parameters during storage

Journal of Food Engineering 109 (2012) 291–300

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Aseptically packaged UHPH-treated apple juice: Safety and quality parameters during storage Ángela Suárez-Jacobo a, Jordi Saldo b,⇑, Corinna E. Rüfer c, Buenaventura Guamis b, Artur X. Roig-Sagués b, Ramón Gervilla b a

Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco, A.C. Unidad Noreste, Parque PIIT, Via de Innovación 404, 66629 Apodaca, Nuevo León, Mexico Centre Especial de Recerca Planta de Tecnologia dels Aliments, Departament de Ciència Animal i dels Aliments, XaRTA, TECNIO, MALTA-Consolider, Facultat de Veterinària, edifici V, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain c Department of Safety and Quality of Fruit and Vegetables, Federal Research Centre for Nutrition and Food, Max Rubner-Institut, Haid-und-Neu-Strasse 9, 76131 Karlsruhe, Germany b

a r t i c l e

i n f o

a b s t r a c t

Article history: Received 20 May 2011 Received in revised form 6 August 2011 Accepted 5 September 2011 Available online 24 September 2011

Ultra-high pressure homogenization (UHPH) at 300 MPa and 4 °C inlet temperature were used to preserve apple juice, and shelf-life evaluation of aseptically packaged juice was investigated. After processing Tetra Brik containers were stored at temperatures of 4, 10, 20 and 30 °C during 60 days. In this article, the effect of processing on the spoilage inactivation was evaluated after processing and during the storage trial. Non-germinated and germinated spores were found in the UHPH-treated juice, being an inactive population during storage. Patulin content was also not modified by UHPH processing, but a significant decrease was observed during storage at 30 °C (P < 0.05). Polyphenoloxidase (PPO) and pectinmethylesterase (PME) activity was not found after UHPH-processing and during storage. A kinetic study of post-processing quality loss was conducted. Vitamin C, chlorogenic acid, total polyphenols and color change were measured during storage study and were used to model the UHPH-treated apple juice shelf-life. Loss of vitamin C was correlated with the hydroxymethylfurfural (HMF) accumulation (0.59, P < 0.05). A limiting quality parameter was polyphenolic content. UHPH-treated apple juice stored at 4 °C was found to show a shelf-life for about 21 months by preserving the color characteristics of the juice with low HMF accumulation. From 15 °C changes in quality parameters were more evident. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: UHPH-treated apple juice Aseptic package Storage Vitamin C Chlorogenic acid Total poyphenols Color HMF

1. Introduction Food processing by UHPH has been recently introduced as a non-thermal process alternative to conventional pasteurization process for the production of shelf-stable milk and soymilk and their by-products. Recently, fruit juices have been considered for application of UHPH treatments in order to produce safer products. Pressures above 200 MPa are able to inactivate food-spoilage and pathogenic microorganisms in juices with only minimal affects on sensory and nutritional characteristics (Briñez et al., 2006a,b; Campos and Cristianini, 2007; Kheadr et al., 2002; Tahiri et al., 2006; Vachon et al., 2002). UHPH treatment in apple juice was able to inactivate the endogenous microbiota and to extend the shelflife under refrigeration conditions to several weeks while preserving the original quality of fresh juice (Donsì et al., 2009; Suárez-Jacobo et al., 2010). Juices are highly sensitive products. They can easily change their composition and physicochemical properties during ⇑ Corresponding author. Tel.: +34 93581 4731; fax: +34 935812006. E-mail addresses: (R. Gervilla).

[email protected]

(J.

Saldo),

[email protected]

0260-8774/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2011.09.007

shelf-life. The main quality characteristics of fruit juice products are color, presence or absence of cloudiness and nutritional value. Thus, the choice of the packaging material for juices is a crucial point regarding the shelf-life study. The aseptic packaging technology has been widely accepted by the fruit juice industry for the production of shelf-stable fruit juices. Tetra Brik containers have been widely used as containers for fruit juices and nectars to avoid the degradation of biocompounds. Apple juice in Tetra Brik cartons is one of the most popular juice drinks on the Spanish market. The loss of some nutrients such as ascorbic acid (AA) might be a critical factor for the shelf-life of some juice products. AA is considered to be highly sensitive to oxidation and thermal degradation during thermal processing and storage, and is often used as a marker for product quality deterioration (Kennedy et al., 1992; Tiwari et al., 2009; Zerdin et al., 2003). Furthermore, apple juice is an important source of polyphenolic compounds, which are also currently considered as a measure for product quality due to them being the most important bioactive apple juice components (Gliszczynska-Swiglo and Tyrakowska, 2003). It has been reported that several reactive products are formed via degradation of ascorbic acid and these compounds may combine with amino acids, resulting in the formation of brown

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pigments. HMF is one of the decomposition products (Burdurlu et al., 2006). There are several causes of browning in processed and/or stored fruit and vegetables: ascorbic acid oxidation, enzymatic browning of phenols, caramelization, formation of browning polymers by oxidized lipids and Maillard reactions (Dogan et al., 2005; Vaikousi et al., 2008). Enzymatic browning, which is caused by the action of PPO, is the major problem for apple juice (Gui et al., 2007). PME is another enzyme responsible for the loss of quality characteristics due to the loss of cloudiness in the orange juice caused when promotes the precipitation of pectin. UHPH shows a good potential in the inactivation of PME and thus, in the stabilization of the juice (Lacroix et al., 2005; Welti-Chanes et al., 2009). The effect of UHPH processing on some quality characteristics in apple juice was studied by Suárez-Jacobo et al. (2011) concluding that UHPH processing does not disturb the original AA and dehydroascorbic acid (DHA) contents in apple juice, being highly decreased by pasteurization treatment. Additionally, the antioxidant capacity and the polyphenolic compounds were well preserved after UHPH-processing at 300 MPa and 4 and 20 °C inlet temperatures. Betoret et al. (2009) observed a better preservation in flavonoid content after homogenization of orange juice in comparison to thermal treatment. However, more knowledge of the storage stability of UHPHproducts is essential with respect to their microbial shelf-life, physical appearance and retention of nutritional value. Therefore, the objective of this study was to evaluate the stability of safety and quality parameters of UHPH-treated apple juice during different storage conditions. The optimal conditions to preserve UHPHtreated apple juice with minimal quality deterioration should be identified. It is important to note that this study is part of the first reported experiment of UHPH-product treated coupled to aseptic packaging technology. 2. Materials and methods 2.1. Apple juice supply Apple juice was supplied by Cal Valls (Vilanova de Bellpuig, Spain), the juice was obtained from Golden Delicious apples and details are given in previous research work (Suárez-Jacobo et al., 2010). No vitamin C was added to the juice in order to preserve the original characteristics of natural fresh apple juice. 2.2. UHPH treatment Apple juice was UHPH-treated at 300 MPa (single-stage) with inlet temperature of 4 °C by using an ultra-high pressure homogenizer (Model/DRG No. FPG 11300:400 Hygienic Homogenizer, Stansted Fluid Power Ltd., Essex, UK) at a flow rate of 100 L h 1. The UHPH system was described by Suárez-Jacobo et al. (2010). The UHPH-treated apple juice was aseptically transferred into a sterile holding tank and maintained at 4 °C until the aseptic packaging process. The full experiment was conducted independently twice. 2.3. Aseptic packaging and storage conditions The UHPH-treated apple juice was packaged in coated paperboard cartons (200 mL Tetra Brick containers) by using a Tetra Pak aseptic (TBA9 slim line, Lausanne, Switzerland). The Tetra Brick containers were stored in four controlled chambers at 4, 10, 20 and 30 °C. Samples were analyzed in triplicate immediately after processing (day 0) and after 15, 30, 45 and 60 days of storage. Samples below the microbial detection level of 1 cfu mL 1 were incubated at 37 °C for 9 days to check the lack of growing.

2.4. Microbial analysis The microbial analyses were performed as in Suárez-Jacobo et al. (2011). The presence of Listeria monocytogenes was detected using a 2-stage enrichment procedure. Twenty-five milliliters of juice was preenriched in half-Fraser broth (bioMérieux S.A., Marcy L’Etoile, France) and incubated at 37 °C for 24 h. One milliliter of the preenriched sample was then incubated in Fraser broth (bioMérieux S.A.) at 37 °C for 24 h. The enriched sample was then streaked onto Palcam agar medium (Oxoid) and incubated at 37 °C for 24 h. The presence of Salmonella spp. was detected using a 2-stage enrichment procedure. Twenty-five milliliters of juice was preenriched in buffered peptone water (Oxoid) and incubated at 37 °C for 24 h. One milliliter of the preenriched sample was then incubated in Muller–Kauffman broth (bioMérieux S.A.) and 0.1 mL in Rappaport–Vassiliadis broth (bioMérieux S.A.) at 37 °C and 42 °C for 24 h, respectively. Enrichments were then streaked onto XLD (Oxoid) and SMID2 media (bioMérieux S.A.) and incubated at 37 °C for 24 h. The detection limit was 1 cfu mL 1 of apple juice for all microorganisms except for Salmonella and Listeria whose limit was 1 cfu 25 mL 1.

2.4.1. Isolation and spore forming bacterial identification by polymerase chain reaction (PCR) Briefly, from SP plate three different types of colonies were selected according their appearance and morphology. Selected colonies were cultivated on an axenic culture. The axenic cultures were spread-plated in triptone-soy-agar (TSA) and incubated at 37 °C 48 h. The DNA from axenic cultures was extracted by using the DNeasy Tissue and Blood (Qiagen Iberia, S.L., Madrid, Spain) kit following manufacturer’s instructions. The sequences of the 16S rRNA gene aligned in order to search for specific primer sites. The PCR was cycled once at 94 °C for 5 min, 40 repetitions at 94 °C for 1 min, 65 °C as indicated for 1 min, 72 °C for 1 min, and once at 72 °C for 10 min. On completion of cycling, amplicons were directly analyzed by electrophoresis in (Tris–Acetate–EDTA electrophoresis buffer) TAE 1% agarose gel. 16S rRNA gene amplification was performed with primers 27F and 1492R (Lane, 1991). Sequencing was carried out by DNASTAR (DNASTAR, Madison, WI, USA) and these sequences were then compared to known submitted sequences within the National Center for Biotechnology Information (NCBI) database to determine taxonomic status of the rDNA sequences.

2.5. Determination of patulin Patulin (PAT) was extracted and quantified following the AOAC method (Official Methods of Analysis, 1996).

2.6. Enzyme activity measurements 2.6.1. Polyphenoloxidase (PPO) and pectinmethylesterase (PME) activity The extracts for determination of polyphenoloxidase (PPO) enzyme were carried out according with Cano et al. (1997). Reaction rate was obtained by measuring the change in A420 at 20 °C for 20 min and corresponds to PPO activity. The PME assay was carried out using the Rouse and Atkins (1955) method in automatic titrator (model Titrando 842, Metrohm, Herisau, Switzerland). PME was evaluated by titration of free carboxyl groups released for 30 min at pH 7.5 at 25 °C. The PME activity was expressed as milliequivalents of ester hydrolyzed per milliliter of juice sample at pH 7.5 by minute (unit).

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2.7. Vitamin C and total polyphenol content

3. Results and discussion

Ascorbic acid and total vitamin C (ascorbic plus dehydroascorbic acid) were determined by HPLC according to Sánchez-Moreno et al. (2003). High-performance liquid chromatographic analytical conditions and chromatographic data were performed as described by Suárez-Jacobo et al. (2011). Total polyphenol and chlorogenic acid were determined by HPLC in order to evaluate the effect of the temperature on this individual polyphenol and the total polyphenol content (Stracke et al., 2009).

UHPH conditions (300 MPa and 4 °C) were selected mainly based on microbial (Suárez-Jacobo et al., 2010), and enzymatic inactivation as well as on the better nutritional preservation in apple juice (Suárez-Jacobo et al., 2011). Quality parameters of the juice are listed in Table 1.

2.8. Hydroxymethylfurfural quantification by HPLC

3.1.1. Microbial analysis In the present study, the microbial groups that most typically comprise the natural apple juice microbiota were evaluated at day 0 (Table 2). The data obtained was in agreement to those obtained in a previous study (Suárez-Jacobo et al., 2010) with lethality values ranging from 4 to 6 log units by microorganism. A significant microbial growth was observed in raw apple juice stored at 4 °C after 15 days (reaching 107 cfu mL 1) demonstrating a microbial instability of the fresh product. Aerobic mesophilic (AM) and spores (SP) survivors were detected after processing and during storage. It is important to note that these microorganisms were not able to grow throughout the storage trial at any of the different conditions (Fig. 1). Neither L. monocytogenes nor Salmonella spp. were detected in either raw or UHPH-treated apple juice. Thus, enterobacteriaceae (EB) and fecal coliforms (FC) were used as indicators of undesirable microorganisms, and moulds and yeasts (MY), psychrotrophic bacteria (PS) and lactobacilli (LB) as spoilage microorganisms. At least 6.3, 5.6, 5.1 and 6.9 log unit reductions for EB, FC, MY and LB were obtained, respectively. According to the EC Regulation no. 2073/ 2007 on microbiological criteria for foodstuffs, the limit for a satisfactory microbial acceptance of a fruit juice is a total microbial contamination of 100 cfu g 1 (considering that coliform species can be

5-Hydroxymethylfurfural (HMF) was measured by using the IFFJP method. (IFFJP, International Federation of Fruit Juice Producers Methods, 1996). 2.9. Sugar content Sucrose, glucose and fructose were determined by HPLC using the amino-column Phenomenex Luna (250 mm  4.6 mm i.d., 5 lm, particle size) (Phenomenex, Inc., CA, USA.) and refraction index detector according to Truong et al. (1986). 2.10. Color and browning index measurements Apple juice color was measured using a Hunter Lab colorimeter (MiniScan XE) as described by Saldo et al. (2009). Browning index was measured as the absorbance at 420 nm (Cortés et al., 2008). 2.11. Determination of antimutagenic effect The antimutagenic potential was tested according to Butz et al. (1997) who modified a method by Maron and Ames (1983). The mutant strain of Salmonella typhimurium (ampicillin resistant and His ) TA 100 was used as an indicator for testing mutagenic activity. The mutagen used was 2-amino-3-methylimidazol [4,5-f]quinoline (IQ) (Toronto Chemical Research Chemicals Inc., Ontario, Canada). The S9 microsomal fraction from rat liver induced by Aroclor 1254 (S9) was provided by LMP (Laboratorium für Mutagenitätsprüfung, Darmstat, Germany). Antimutagenic assay of UHPH-treated apple juice against IQ was performed as follows: 500 lL of isotonic 0.15 M KCl (in 0.01 M sodium phosphate buffer, pH 7.4), 15 lL of IQ solution in bi-distilled water, 500 lL S9 mix and 100 lL bacterial suspension were mixed with 250 lL of juice filtered through 0.45 lm filters. Then, 2.7 mL of Top Agar were added and the mixture was poured onto plate with Difco BactoNutrient agar (Difco-BD, Heidelberg, Germany). Plates were incubated at 37 °C, and after 48 h Salmonella revertants were counted. Control without sample and mutagen added, as well as ampicillin resistance and spontaneous mutations were tested. Results were expressed as revertants (cfu) per plate.

3.1. Safety parameters

Table 1 Apple juice characterization.

2.13. Statistical analysis The experimental data were statistically analyzed performing the ANOVA procedure by using SAS statistical software (2004). Tukey test pairwise comparison was used to determine significance differences (P < 0.05). Pearson’s correlation coefficients were used to examine relationships between variables.

Values ± SD

pH TSS# (°Brix) TA# (g MA# L

3.88 ± 0.02a 11.98 ± 0.01a 3.60 ± 0.01a

1

)

Values are means of triplicate analysis from two different productions (n = 6). Values in the same row with different superscripts differ significantly (P < 0.05). Total soluble solids (TSS), titrable acidity (TA), malic acid (MA).

Table 2 Microbial population of raw apple juice and after UHPH-processing (day 0). Microbial populationA

Log (cfu mL 1) ± SD (6 2.0 log cfu/mL detection limit) Raw juice UHPH (day 0)

AM PS MY LB EB FC SP E. coli

5.6 ± 0.1a 2.4 ± 0.1a 3.1 ± 0.2a 4.9 ± 0.2a 4.3 ± 0.2a 3.6 ± 0.1a 1.4 ± 0.01a N.D.

2.12. Physicochemical determinations pH, titrable acidity (TA) and total soluble solids (TSS) were determined according to the IFFJP methods (International Federation of Fruit Juice Producers, 1996).

Parameters

1.8 ± 0.1b N.D.b N.D.b N.D.b N.D.b N.D.b 1.4 ± 0.1a N.D.

Values are means of triplicate analysis from two different productions (n = 6). Values in the same row with different superscripts differ significantly (P < 0.05). N.D., not detected. A Aerobic mesophilic (AM), psychrotrophic bacteria (PS), moulds and yeast (MY), lactobacilli (LB), enterobacteriaceae (EB), feacal coliforms (FC) and spores (SP).

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(η g g-1)

Log (cfu mL -1)

60

A

3

2

40

20

0 1

0

15

30

45

60

Storage time (days) 0 0

7

15

30

45

60

Fig. 2. Evolution of patulin content (gg g 1) on UHPH-treated apple juice during storage at 4 °C (N), 10 °C (j), 20 °C (), 30 °C (d).

Storage time (days)

Log (cfu mL -1)

3

B

2

1

0 0

7

15

30

45

60

Storage time (days) Fig. 1. Microbial population (log cfu mL 1) of UHPH-treated apple juice during storage at 4 °C (N), 10 °C (j), 20 °C (), 30 °C (d). (A) Aerobic mesophilics – AM, and (B) spores – SP.

excluded). In this research study the contamination was close but below this value. Additionally, samples were subjected to 37 °C over 9 days resulting in stable AM and SP counts (1.5 and 1.4 log cfu mL 1, respectively). In order to obtain information on the microorganisms resistant to UHPH processing, isolation of the different colonies and identification by taxonomic groups were carried out. Lysinibacillus sphaericus (99% 16S rRNA gene sequence similarity), Paenibacillus mendelii/Paenibacillus spp. (95% similarity) and Bacillus spp./Paenibacillus spp. (99% similarity) were identified, confirming that the population highly resistant to UHPH processing is constituted by non-germinated spores. Buffa et al. (2006) evaluated the microbial inactivation in almond milk and observed survivors at 300 MPa in AM counts, all the surviving microorganisms corresponded to non-germinated spores. Feijoo et al. (1997), studying the effect of high-pressure homogenization (microfluidization) on spores on ice cream at pressures from 50 to 200 MPa, obtained percentages of spore reduction from 6% to 68%. The combinations of pressure and temperature used were far from being sufficient to eliminate spores. Cruz et al. (2007) and Pereda et al. (2007) also reported that bacterial spores were not completely eliminated by UHPH treatments of soymilk and cow’s milk, respectively. Bevilacqua et al. (2007) have investigated the effectiveness of high-pressure homogenization against Alicyclobacillus acidoterrestris. The treatment (50–170 MPa) caused a significant reduction of the initial cell number (1–2 log units) at the highest pressures applied. The authors conclude that the application of several successive rounds of high-pressure homogenization could have an additive effect on the reduction of viability and increase of susceptibility of A. acidoterrestris spores. 3.1.2. Patulin content PAT content was detected in all samples, and the change in its amount during storage is shown in Fig. 2. In raw apple juice, the

mean value of PAT was found to be 43.26 ± 0.62 gg g 1 and after UHPH processing the concentration was 41.92 ± 2.48 gg g 1, showing no significant differences between them. This means that UHPH does not modify the original PAT content in the samples. The content was close to the limit of 50 gg g 1 which is the maximum permitted level in fruit juices, nectars, apple juices and other beverages marketed in Europe (European Commission, 2003). No differences were observed between samples stored at 4, 10 and 20 °C. However, a statistically significant decrease was observed after 30 days at 30 °C (34.33 ± 0.20 gg g 1) (Fig. 2), making evident that temperature is PAT degradation factor. Koca and Eksßi (2005) studied apple juice stored at 22 and 33 °C, and observed a PAT reduction between 45–64% and 66–86%, respectively. Finally, after 2 months of storage, PAT reduction was 75–86% and 78– 100% at 22 and 30 °C, respectively. In this study, 26–40% of reduction was observed after 30 and 60 days at 30 °C (34.44 ± 0.14 and 27.95 ± 0.14 gg g 1), respectively. According to Stinson et al. (1978) the PAT content of apple juice stored for 2 weeks at a temperature of 22 and 25 °C decreased by 10%. The decrease during storage could be attributed to the presence of sulfhydryl groups present in the juices (Scott and Somers, 1968). Scott and Somers (1968) reported on a 50% reduction of PAT (200 lg initial concentration) in canned apple juice, after heating at 80 °C for 10 min. In addition, heating apple juice for nearly 3 h at 100 °C resulted in a PAT reduction of 33% (Kryger, 2001). According to Drusch et al. (2007), AA content can also reduce the stability of PAT at acidic pH. The PAT concentration was decreased to 70% in the presence of AA compared to 32–29% in samples without AA. 3.2. Quality parameters 3.2.1. PPO and PME activity In this study PPO and PME activity were not detectable after processing and during the storage. 3.2.2. Vitamin C, chlorogenic acid and polyphenol degradation kinetics during storage In order to establish the shelf-life of UHPH-treated apple juice, the degradation kinetics of AA, chlorogenic acid, and total polyphenols were determined. Vitamin C is often used as a marker for fruit juice quality deterioration (Kennedy et al., 1992). Polyphenol compounds were subject to evaluation, because they have received much attention since many epidemiological studies suggest that their consumption is associated with disease prevention (Cao et al., 1995; Kaur and Kapoor, 2001). There is no information currently concerning the changes in the content of polyphenols during the shelf-life. Of the polyphenol content, chlorogenic acid has been found to be the dominant constituent in apple juice (Suárez-Jacobo et al., 2011). The initial vitamin C, chlorogenic acid and total polyphenol concentrations were 13.59 ± 0.31, 3.64 ± 0.22, 20.68 ± 0.60 mg L 1, respectively. After the UHPH processing, the vitamin C, chlorogenic

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A

1.0

Ln (C/C0)

0.0 -1.0

Table 3 Rate, kinetics constants and times of half destruction of some compounds as a function of temperature. Halflife (days)

k4°C = 2.9  10 2 (r = 0.93) k10°C = 4.6  10 2 (r = 0.87) k20°C = 9.3  10 2 (r = 0.98) k30°C = N.D.

50.58 (r = 0.99)

26 14 7 N.D.

Loss in chlorogenic acid

k4°C = 0.8  10 3 (r = 0.70) k10°C = 1.6  10 3 (r = 0.84) k20°C = 2.8  10 3 (r = 0.99) k30°C = 6.6  10 3 (r = 0.99)

55.18 (r = 0.99)

852 440 250 105

Loss in total polyphenols

k4°C = 1.1  10 3 (r = 0.90) k10°C = 1.6  10 3 (r = 0.95) k20°C = 2.9  10 3 (r = 0.97) k30°C = 4.7  10 3 (r = 0.90)

39.02 (r = 0.99)

630 330 239 147

Rate constants (days

Loss in vitamin C

)

-2.0 -3.0 -4.0 -5.0 0

15

30

45

60

Storage time (days)

B

0.2

-0.1

Ln (C/C0)

1

Ea values determined by Arrhenius eq. (kJ/mol)

Parameter

-0.3

N.D., not detected.

-0.6

-0.8 0

15

30

45

60

Storage time (days)

C

0.2

Ln (C/C0)

-0.1

-0.3 -0.6

-0.8 0

15

30

45

60

Storage time (days) Fig. 3. Vitamin C (A) chlorogenic acid (B) and total polyphenols (C) degradation kinetics on UHPH-treated apple juice throughout storage at 4 °C (N), 10 °C (j), 20 °C (), 30 °C (d).

acid and total polyphenol concentrations made up to 13.18 ± 0.33, 4.01 ± 0.16 mg and 22.75 ± 1.40 mg L 1, respectively. UHPH processing did not modify the original AA content, but a significant change was observed for chlorogenic acid and total polyphenol concentrations. Several works report higher polyphenol concentration after HHP or PEF as a consequence of their improved extraction from pulp particles or depolymerization from complexes induced by the treatment (Cao et al., 2011; Grimi et al., 2011). It is considered that degradation of AA follows a first-order kinetic: C = C0 exp ( k t) where C0 is the initial content (mg L 1), k is the first-order kinetic constant (day 1), C is the biological materials content at time t (mg L 1), and t is the storage time (days) (Burdurlu et al., 2006; Polydera et al., 2003; Zanoni et al., 2005). In order to study the degradation of vitamin C, chlorogenic acid and total polyphenols Ln(C/C0) of each compound were plotted against storage time for each temperature condition Fig. 3. Table 3 lists the vitamin C and chlorogenic acid degradation rates as well as the correlation coefficients, obtained for storage at 4, 10, 20 and 30 °C. At a storage temperature of 30 °C for 15 days, the vitamin C concentration in the juice was below detection limits. On the other hand, at 4 °C the degradation rates of these compounds were lowest. Nagy and Smoot (1977) concluded that storage temperature above the critical temperature (22–27 °C) caused a significant

increase in the rate of AA degradation. The AA degradation rates values in orange–carrot juice treated by high-pulsed electric field (PEF) during storage at 10 °C was similar to the data obtained in this study (0.04 days 1). However, the pasteurized juice showed a higher degradation rate (0.09 days 1) than in both PEF and UHPH studies. On the other hand, Tiwari et al. (2009) reported on the same degradation rate value for AA (0.04 days 1) in thermally processed juice (98 °C, 21 s) stored at 10 °C. The activation energies (Ea) were also calculated by using the Arrhenius equation (Table 3). Highest Ea values of 57.74 kJ mol 1 were reported for AA degradation in thermally treated (65 °C, 30 s) lemon juice after storage (Al-Zubaidy and Khalil, 2007). Kaanane et al. (1988) observed Ea values of 56 kJ mol 1 at 4–45 °C in orange juice as well as 53.1 kJ mol 1 for thermally pasteurized orange juice (Polydera et al., 2003). Moreover, Nagy and Smoot, 1977 found Ea values of 60 kJ mol 1 at 4.4–24 °C. These values suggest a great temperature dependence of the AA degradation rate for the thermally treated juice. The differences in Ea values between different fruit juices can be attributed to the polyphenols content. Flavonols in orange juice may protect the AA from degradation (Özkan et al., 2004) due to their antioxidant capacity. In this study the Ea for chlorogenic acid and total polyphenols were higher than AA, showing a greater stability which is in agreement with the study of Zheng and Lu (2011). However, in our study they did not protect AA degradation causing a decrease in the Ea value due to the low polyphenol concentration. The shelf-life of juice treated by UHPH stored at 4, 10, 20 and 30 °C was calculated as the time taken for vitamin C, chlorogenic acid and total polyphenol concentrations to be reduced to 50% of their original value (half-life). Table 3 lists the half-lives of these compounds. In general, lower degradation rates were found for chlorogenic acid and total polyphenols in comparison to vitamin C, which obtained a half-life greater than that of the vitamin C reference. There was a great increase in the shelf-life of the juice when the preservation temperature was 4 °C, meaning that this temperature is recommendable to ensure the nutritive value of UHPH-treated apple juice. Donsì et al. (2009) concluded that shelf-life of UHPH-treated apple juice can be prolonged for many weeks by storage at 4 °C which is in agreement with the results presented in this paper. In comparison with our results, Choi et al. (2002) found that more than 50% of the AA was lost within 3 week of storage at 4.5 °C (after thermal treatment 90 °C, 90 s), and it was completely degraded after 5 weeks of storage. In contrast, Gliszczynska-Swiglo and Tyrakowska (2003) reported similar ascorbic acid content.

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It is important to consider that the polyphenolic compounds are the main components of apple juice (Gardner et al., 2000; Gliszczynska-Swiglo and Tyrakowska, 2003), more relevant than vitamin C in pure apple juices, meaning that juice shelf-life life must be calculated according to the polyphenol content. 3.2.3. HMF degradation kinetics and evolution of sugars During the UHPH-treatment, the temperature of apple juice rose up to 88 °C at 4 °C of inlet temperature (data not shown). This increase in temperature was a result of the pressure increase together with the friction when passing through the valve, and can be estimated to be 2.2 °C per 10 MPa in this experiment. These data are in agreement with those reported by Popper and Knorr (1990). Afterwards, the juice was cooled down to 19 °C in just 0.7 s, thus the thermal effect only lasted for that short time (Saldo et al., 2009). Nevertheless, a significant (P < 0.05) accumulation of HMF content in UHPH-treated apple juice was observed as function of time and storage temperature. The HMF content after processing was 1.2  10 2 lg mL 1 and at the end of the storage trial (60 days) 1.0  10 2, 3.0  10 2, 8.0  10 2, and 17  10 2 lg mL 1 at 4, 10, 20 and 30 °C, respectively. This accumulation was clearly lower that the amount achieved due to thermal treatment (Saldo et al., 2009). Although that thermal damage can be evident after long storage at high temperature, the value on final product rely more on initial conditions, and UHPH causes lower changes than thermal treatments capable to produce shelf stable products. When HFM values of UHPH-treated juice are plotted versus storage time the best-fit model for HMF accumulation was a first-order kinetic (Fig. 4) and the determination coefficients in relation to this reaction are listed in Table 4. HMF formation is mainly attributed to decomposition of AA (Burdurlu et al., 2006). A significant correlation (0.58) was obtained between AA loss and HMF formation (P < 0.05) during storage. Sugar degradation might also contribute to HMF formation, since it is known that this reaction occurs in acidic media (Burdurlu et al., 2006). Table 5 lists the changes of fructose, glucose and sucrose of UHPH-treated apple juice during the storage trial. A significant decrease in the sucrose content (P < 0.05) can be observed. However, no significant changes were found for the monosaccharides. This could be because the increase of temperature favored the hydrolysis of sucrose producing more monosaccharides, which could have reacted in the acidic medium to form HMF. The Ea of HMF formation is also given in Table 4 and the value can be compared with the HMF accumulation in an apple juice model solution reported as 118–166 kJ mol 1 by Resnik and Chirife (1979). Burdurlu et al. (2006) found Ea values ranging from 181 to 334 kJ mol 1 in concentrated citrus juice during storage at 28, 37 and 45 °C. In conclusion, as the vitamin C initial content was low,

6 5

Ln (C/C0)

4 3 2 1 0 -1 0

15

30

45

60

Storage time (days) Fig. 4. HMF accumulation in a UHPH-treated apple juice throughout storage at 4 °C (N), 10 °C (j), 20 °C (), 30 °C (d).

Table 4 Kinetics constants of HMF, chroma and absorbance at 420 nm as a function of temperature. 1

Parameter

Rate constants (days

)

Ea values determined by Arrhenius eq. (kJ/mol)

HMF formation

k4°C = 0.1  10 2 (r = 0.80) k10°C = 1.5  10 2 (r = 0.93) k20°C = 3.4  10 2 (r = 0.98) k30°C = 9.0  10 2 (r = 0.97)

92.23 (r = 0.93)

Chroma value

k4°C = 0.5  10 3 (r = 0.98) k10°C = 2.4  10 3 (r = 0.96) k20°C = 4.4  10 3 (r = 0.84) k30°C = 13.3  10 3 (r = 0.89)

80.86 (r = 0.94)

A (420 nm)

k4°C = 0.6  10 3 (r = 0.80) k10°C = 3.1  10 3 (r = 0.97) k20°C = 4.9  10 3 (r = 0.84) k30°C = 12.1  10 3 (r = 0.81)

77.69 (r = 0.97)

Table 5 Changes in fructose, glucose and sucrose content (mg L 1) on UHPH-treated apple juice during storage at 4, 10, 20 and 30 °C throughout 60 days in Tetra Brik containers.

UHPH 0 day 4 °C 60 days 10 °C 60 days 20 °C 60 days 30 °C 60 days

Fructose

SD

Glucose

SD

Sucrose

SD

71.12 71.08 69.72 70.04 69.98

0.18a 1.18a 1.07a 1.52a 0.13a

30.92 33.05 32.81 33.18 33.17

1.00a 2.11a 0.82a 0.60a 2.81a

10.38 9.30 9.85 9.44 8.27

0.05a 1.47ab 0.40a 0.32ab 1.86b

Values are means of triplicate analysis from two different productions (n = 6), Values in the same column with different superscripts differ significantly (P < 0.05).

the HMF formation could depend on sucrose hydrolysis. The observed k and Ea could reflect such dependency since they are far from the reported values. 3.2.4. Color degradation kinetics During storage, thermal effects can cause changes in color characteristic which can be used as a quality parameter of the food product. Fig. 5 shows the changes in L⁄, a⁄ and b⁄ color parameters during storage of UHPH-treated apple juice over 60 days. Original L⁄ a⁄ and b⁄ values were found to be 54.99 ± 0.04, 1.63 ± 0.04, 56.35 ± 0.12, respectively. After UHPH processing L⁄a⁄b⁄ were 55.65 ± 0.12, 1.67 ± 0.06, 57.43 ± 0.13. A significant increase (P < 0.05) was found in L⁄ (lightness) value during storage (Fig. 5A) ranging from 57.04, 57.48, 56.7, 56.4 at 4, 10, 20 and 30 °C (day 60). L⁄ values indicated brightness and light increase with the storage time. The juice changed slightly its color from the initial yellow to whitish-yellow (bleaching effect). Discoloration and browning due to thermal effects have been explained as a result of several reactions. These include Maillard reaction condensation between reducing groups and amino acids, caramelization, AA browning processes (Cornwell and Wrolstad, 1981) and pigment destruction (Beveridge et al., 1986). The discoloration effect observed in this study may be related to a xylanase, a thermo stable enzyme from Paenibacillus spp. (one of the microorganisms found in UHPH-treated apple juice). This enzyme might have been found in the raw sample before the UHPH-processing producing a biobleaching in the juice (Ko et al., 2011). The a⁄ (red-green) value increased with storage temperature and decreased with time, but the b⁄ (blue-yellow) value was influenced by both time and storage temperature (Fig. 5B and C). Thus, color parameters were estimated in function of storage time at 4– 30 °C using the chroma value. In order to evaluate the color degradation the chroma value was compared with the absorbance at

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297

Fig. 5. Changes in L⁄ (A), a⁄ (B) and b⁄ (C) color parameters during storage of UHPH-treated apple juice over 60 days in Tetra Brik containers.

420 nm. Similarly to AA, the change of color was found to follow first order kinetics for both parameters (Fig. 6). Chroma and absorbance at 420 nm showed similar degradation rates and Ea values (80.86 and 77.69 kJ mol 1, respectively) (Table 4). In agreement with our results, Polydera et al. (2003) found similar degradation rates in high pressure processed orange juice. In the same study, high-pressure treatment led to lower rates of color change compared to thermal pasteurization. As can be observed, the results did not show a good correlation in some k values (20 and 30 °C), meaning that the degradation of color may not be a first order kinetic reaction. Ibarz et al. (1999) explained that it is not always possible to apply kinetics as simple as first or zero order to describe color changes since these changes cannot be only due to Maillard reaction (color formation), but also due to thermal destruction of pigment present in the samples (color destruction). Color changes occurring during apple juice storage

are more relevant that the changes produced by a simple thermal treatment and much larger than those produced by any of the UHPH treatments previously assayed by the authors Saldo et al. (2009)) capable to microbiologically stabilize apple juice (Suárez-Jacobo et al., 2010). Thermal- or UHPH-treatment effects are mainly related with Maillard reactions, while long term storage in TetraBrik allowed for pigment destruction. 3.2.5. Antimutagenic activity Fruits and vegetables contain inhibitors of carcinogenesis such as flavonoids and coumarins, but the identity of many others is still unknown. UHPH-treated samples were tested by the Salmonella/ reversion assay in order to evaluate the presence or effectiveness of protective antimutagenic action against IQ (Fig. 7). UHPH processing did not change antimutagenic activity of apple juice, and it was not changed during storage at 4, 10, 20 and 30 °C (day 60).

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A

-0.2

10000

log Δ 50 % (days)

Ln (A/A0)

0.1

-0.4

-0.7

100

-0.9 0

15

30

45

60

Storage time (days)

Ln (A/A0)

0.1

B

1 1

6

11

16

21

26

31

36

Temperature (ºC)

-0.2 Chlorogenic acid

-0.4

TPP

VIT C

HMF

Chroma

Fig. 8. Temperature–time plot showing the degradation/accumulation of some quality parameters in UHPH-treated apple juice. Log D 50% is the time (days) to reach fifty percent change on concentration of the quality parameters.

-0.7

-0.9 0

15

30

45

60

Storage time (days) Fig. 6. Chroma (A) and absorbance at 420 nm (B) degradation kinetics of UHPHtreated apple juice throughout storage at 4 °C (N), 10 °C (j), 20 °C (), 30 °C (d).

be optimal by also keeping the quality attribute of a desired color. At 15 °C the change in color properties was shown to be more obvious. When the UHPH-treated juice was stored at 4 °C it showed a shelf-life of about 21 months (calculated based on the polyphenol concentration as major components), while at room temperature (20 °C) the juice exhibited a shelf-life of 8 months. 4. Conclusions

600 4ºC

Revertants (cfu)

500

10ºC

20ºC

30ºC

400 300 200 100 0 Blank

R

UHPH

Sample amount (μL) Fig. 7. Revertants (cfu) of Salmonella typhimurium TA 100 at 60 days of storage on UHPH-treated apple juice (300 MPa and 4 °C inlet temperature), compared with raw apple juice.

In agreement, Butz et al. (2003) did not find a change in the antimutagenic effect in orange, lemon and carrot juice as well as in peach fruit before and after ultra high pressure treatment. 3.3. Selection of the optimal storage conditions A plot was constructed with the aid of the reaction kinetics obtained in this study (Fig. 8). This plot shows at a glance which time–temperature combination leads to a desired effect. HMF was more temperature dependent than the other quality parameters. This implies that reducing temperature will reduce the HMF accumulation. Due to its low AA content in the UHPH-treated samples, this parameter was not relevant to determine the optimal storage condition. On the other hand, the total polyphenol content seems to be the most important quality parameter in these samples. Thus, to obtain the optimal storage conditions, referred to 50% of change in the desired quality parameters, it is necessary to be below the total polyphenol line. Hence, storage at lower temperature for the preservation of the total polyphenol content can

The current study focused on defining the stability of apple juice after UHPH processing and aseptic packaging. For that purpose, UHPH processing treatment of 300 MPa at 4 °C (inlet temperature) was used to preserve apple juice and the samples were subjected to storage at different temperatures (4–30 °C) for 60 days. UHPH was not able to modify the original PAT content; but storage at the highest temperature caused a decrease in PAT content. Addition of AA before the UHPH-treatment could contribute to the reduction of PAT content. The weak aspects of UHPH regarding reduction of the PAT contents can be overcome by selecting good quality fruit. Furthermore, washing and sorting are the most important factor in the reduction of fungal contamination during the juice production. UHPH-treated apple juice was microbiologically stable at any storage temperature for up to 60 days, despite it not being able to destroy non-germinated spores. In the aseptically packaged UHPH-treated apple juice, the presence of enzyme activity was not observed after processing and during storage. The time–temperature effect of storage caused degradation of nutritional components (vitamin C, chlorogenic acid and total polyphenols) as well as color changes. This effect was confirmed by the HMF accumulation. Thus, storage temperature of 4 °C was the best condition to preserve the overall characteristics of the aseptically packaged UHPH-treated juice, by obtaining a shelf-life similar to a commercial juice. Acceptable commercial shelf-life was obtained when the juice was stored at room temperature. However, a decrease in their quality parameters was observed. Changes during storage are only dependent on the storage conditions and the initial characteristics of apple juice. As UHPH causes smaller changes to the original product, the final characteristics would be closer to those of the raw product that when a thermally treated product were stored on the same conditions. As a final remark, 300 MPa and 4 °C inlet temperature were applied satisfactorily to preserve apple juice. However, it is important to highlight that to obtain similar results in other liquid food prod-

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ucts might be necessary the combination of higher pressure and temperature conditions. These conditions will be applied, taking into account equilibrium between the safe and quality parameters by extending the shelf-life in a UHPH-treated product. Acknowledgement The authors ackwnoledge the financial support given by the Spanish Ministery of Education and Science. The author Suárez-Jacobo gratefully acknowledges the financial support given by the CONACyT (Mexico) Fellowship program. We also thank to Joan Miquel Quevedo for his valuable technical assistance. References Al-Zubaidy, M.M.I., Khalil, R.A., 2007. Kinetic and prediction studies of ascorbic acid degradation in normal and concentrate local lemon juice during storage. Food Chemistry 101 (1), 254–259. Betoret, E., Betoret, N., Carbonell, J.V., Fito, P., 2009. Effects of pressure homogenization on particle size and the functional properties of citrus juices. Journal of Food Engineering 92 (1), 18–23. Beveridge, T., Franz, K., Harrison, J.E., 1986. Clarified natural apple juice: production and storage stability of juice and concentrate. Journal of Food Science 51 (2), 411–414. Bevilacqua, A., Cibelli, F., Corbo, M., Sinigaglia, M., 2007. Effects of high-pressure homogenization on the survival of Alicyclobacillus acidoterrestris in a laboratory medium. Letters in Applied Microbiology 45 (4), 382–386. Briñez, W.J., Roig-Sagués, A.X., Hernández Herrero, M.M., Guamis, B., 2006a. Inactivation by ultrahigh-pressure homogenization of Escherichia coli strains inoculated into orange juice. Journal of Food Protection 69 (5), 984–989. Briñez, W.J., Roig-Sagués, A.X., Hernández Herrero, M.M., Guamis, B., 2006b. Inactivation of Listeria innocua in milk and orange juice by ultrahigh-pressure homogenization. Journal of Food Protection 69 (1), 86–92. Buffa, M., Herranz, R., Quevedo, J.M., Saldo, J., 2006. Treatment of almond milk by ultra high pressure homogenisation. Alimentaria 375, 116. Burdurlu, H.S., Koca, N., Karadeniz, F., 2006. Degradation of vitamin C in citrus juice concentrates during storage. Journal of Food Engineering 74 (2), 211–216. Butz, P., Edenharder, R., Fister, H., Tauscher, B., 1997. The influence of high pressure processing on antimutagenic activities of fruit and vegetable juices. Food Research International 30 (3–4), 287–291. Butz, P., Fernández Garcı´a, A., Lindauer, R., Dieterich, S., Bognár, A., Tauscher, B., 2003. Influence of ultra high pressure processing on fruit and vegetable products. Journal of Food Engineering 56 (2–3), 233–236. Campos, F.P., Cristianini, M., 2007. Inactivation of Saccharomyces cerevisiae and Lactobacillus plantarum in orange juice using ultra high-pressure homogenisation. Innovative Food Science and Emerging Technologies 8 (2), 226–229. Cano, M.P., Hernández, A., Ancos, B., 1997. High pressure and temperature effects on enzyme inactivation in strawberry and orange products. Journal of Food Science 62, 85–88. Cao, G., Verdon, C.P., Wu, A.H., Wang, H., Prior, R.L., 1995. Automated assay of oxygen radical absorbance capacity with the COBAS FARA II. Clinical Chemistry 41 (12), 1738–1744. Cao, X., Zhang, Y., Zhang, F., Wang, Y., Yi, J., Liao, X., 2011. Effects of high hydrostatic pressure on enzymes, phenolic compounds, anthocyanins, polymeric color and color of strawberry pulps. Journal of Science of Food and Agriculture 91, 877– 885. Choi, M.H., Kim, G.H., Lee, H.S., 2002. Effects of ascorbic acid retention on juice color and pigment stability in blood orange (Citrus sinensis) juice during refrigerated storage. Food Research International 35 (8), 753–759. Cornwell, C.J., Wrolstad, R.E., 1981. Causes of browning in pear juice concentrate during storage. Journal of Food Science 46 (2), 515–518. Cortés, C., Esteve, M.J., Frígola, A., 2008. Color of orange juice treated by high intensity pulsed electric fields during refrigerated storage and comparison with pasteurized juice. Food Control 19 (2), 151–158. Cruz, N., Capellas, M., Hernández, M., Trujillo, A.J., Guamis, B., Ferragut, V., 2007. Ultra high pressure homogenization of soymilk: microbiological, physicochemical and microstructural characteristics. Food Research International 40 (6), 725–732. Dogan, S., Turan, Y., Erturk, H., Arslan, O., 2005. Characterization and purification of polyphenol oxidase from artichoke (Cynara scolymus L.). Journal of Agricultural and Food Chemistry 53 (3), 776–785. Donsì, F., Esposito, L., Lenza, E., Senatore, B., Ferrari, G., 2009. Production of shelfstable annurca apple juice with pulp by high pressure homogenization. International Journal of Food Engineering 5, Article 12. Drusch, S., Kopka, S., Kaeding, J., 2007. Stability of patulin in a juice-like aqueous model system in the presence of ascorbic acid. Food Chemistry 100 (1), 192– 197. European Commission, 2003. Regulation N. 1425/2003 amending Regulation N. 466/2001 as regards patulin. Official Journal of the European Union 1425/ 2003(12.08.03), L 203/3.

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