Hyperbaric Storage of Fruit Juice and Impact on Composition

CHAPTER

HYPERBARIC STORAGE OF FRUIT JUICE AND IMPACT ON COMPOSITION

30

´ 1, Ricardo V. Duarte1, Ivonne Delgadillo1, Mauro D. Santos1, Liliana G. Fidalgo1, Rita S. Inacio 2 3 Shahin Roohinejad , Mohamed Koubaa , Francisco J. Barba4,5 and Jorge A. Saraiva1 1

University of Aveiro, Aveiro, Portugal 2Shiraz University of Medical Sciences, Shiraz, Iran 3 University of Technology of Compiegne, Compie`gne, France 4 University of Copenhagen, Frederiksberg, Denmark 5University of Valencia, Valencia, Spain

30.1 INTRODUCTION Refrigeration storage (RS) is one of the most energy-intensive technologies applied in the food supply chain, involving a number of sustainability-related challenges, since it accounts for  50% of electricity consumption in the food industry (James and James, 2010). At the moment, there is a growing demand from food industries to find new/improved processes, that are more efficient, economic, and also more environmentally friendly. Hyperbaric storage (HS) is a novel food preservation methodology using a moderate pressure (up to 200 MPa), involving very low energy consumption. Therefore, it can provide an interesting opportunity to reduce energy costs for food storage when compared to other preservation methodologies, such as RS. In 1969, the sunken research submarine Alvin was recovered from a depth of 1540 m ( 15 MPa). Surprisingly, after 10 months at this pressure and  4 C, wellpreserved foods (bouillon, sandwiches, and apples) were found (Jannasch et al., 1971). In general the taste, appearance, smell, and consistency of these food products were maintained, a clear improvement being observed relative to the expected microbial spoilage and putrefactive odor of these foods. Furthermore, an equal pH-value, and a half-activity value of tyrosinase were found in these samples when compared to fresh apples (Jannasch et al., 1971). The authors suggested that the use of pressure and low temperature during food storage led to microbial growth and biochemical activity inhibition, which resulted in an extended shelf-life, similar to RS at atmospheric pressure (AP). One year later, rice, wheat, and soybeans were stored under water at a depth of 30 m (  0.3 MPa) for 1 year; fewer changes were observed at the end in their composition (seed moisture, fatty acids, vitamin B12, and reducing sugars), compared to the conventional storage of these products (Mitsuda et al., 1972). Afterwards, storage under pressure (24.1 MPa/1 C) of fresh fish (pollock and cod) during 12 and 21 days confirmed the possibility to store food products and other biomaterials above AP and refrigerated temperatures as a possible enhancement of conventional RS, increasing their shelf-life (Charm et al., 1977). Fruit Juices. DOI: https://doi.org/10.1016/B978-0-12-802230-6.00030-8 © 2018 Elsevier Inc. All rights reserved.

607

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Two patents regarding the HS concept have already been published: (1) “Method of pressure preservation of food products” (US5593714) (Hirsch, 1997), and (2) “Hydraulic pressure sterilization and preservation of foodstuff and feedstuff” (US6033701) (Hirsch, 2000). In these patents, it is claimed that different food products can be preserved under pressure up to 250 MPa at room temperature (RT), from few hours to more than a month. However these patents have expired. More recently, some studies have been conducted to evaluate the HS feasibility at variable (uncontrolled) RTs. When used at RT, HS confers exceptional advantages compared to RS, since energy is not required to control the temperature over the storage period, the energetic costs only being associated with the compression/decompression phases (Moreira et al., 2015). Several studies evaluated the use of HS on solid food products, such as tilapia fish fillets (Ko and Hsu, 2001), dairy whey cheese (Duarte et al., 2015), carrot soup (Moreira et al., 2015), and sliced cooked ham (Fernandes et al., 2015). In these studies, very promising results regarding the inhibition of the microbial growth/microorganisms inactivation, and maintenance of physicochemical parameters (e.g., pH, titratable acidity, reducing sugars, color, among others) were observed.

30.2 STRAWBERRY JUICE Recently, several studies evaluated the effect of HS at RT on the physicochemical properties and natural microbiota of strawberry juice (Segovia-Bravo et al., 2012; Bermejo-Prada et al., 2015a,b,c; Bermejo-Prada and Otero, 2015). Strawberry juice was stored for 15 days at 20 C (RT), under different pressure levels (25 220 MPa). Those samples were then compared to raw juices, and in some cases with thermal-pasteurized juices (at 85 C for 90 s) stored at AP and RS during the same time period.

30.2.1 MICROBIAL STABILITY The HS effects on the natural microbiota, total aerobic mesophiles (TAM), lactic acid bacteria (LAB), and yeasts and molds (YM) of strawberry juice were studied by Segovia-Bravo et al. (2012) and Bermejo-Prada et al. (2015a) in frozen thawed and freshly squeezed (nonfrozen) strawberry juices, respectively (Table 30.1). In frozen thawed strawberry juice, the initial microbial loads were 2.9 and 2.6 log10 CFU/mL for TAM and YM, respectively (Segovia-Bravo et al., 2012). The frozen thawed strawberry juice stored at AP/RT for 15 days showed an increase above 3 log10 units for TAM and YM counts. Off-flavors, unpleasant odors, and gas production were detected in these samples. However, in nonfrozen juice, the initial microbial counts varied (several fresh batches were used) from 3.1 to 6.3, from 2.6 to 6.2, and from 3.6 to 6.1 log10 CFU/mL for TAM, LAB, and YM, respectively (Bermejo-Prada et al., 2015a). In fresh/nonfrozen strawberry juice, Bermejo-Prada et al. (2015a) observed a microbial growth of  1 log10 units for all studied microorganisms after 1 day of storage at AP. After 10 and 15 days under the same conditions, signs of spoilage were observed in all samples and the packages were swollen (one of them burst on the 15th day), revealing microbial counts above 6 log10 units in both storage periods. The samples stored under RS (AP) for 15 days showed a slower microbial growth than expected. TAM counts increased more than 2 log10 units and YM maintained the initial counts (Segovia-Bravo et al., 2012). These results highlighted that RS was not enough to completely

30.2 STRAWBERRY JUICE

609

Table 30.1 Microbial Effect of Pressure Level Used on Hyperbaric Storage of Strawberry Juice Microbial Effect Pressure (MPa)

Temperature  ( C)

Time (days)

0.1

20

1, 10, 15

25

5 20

15 15 1 10 15

50 100

20 20

200 220

20 20

15 1, 10, 15 1, 10, 15 15 1, 10, 15 15

Mesophiles No effect on growth No effect on growth Growth inhibition Inactivation Growth inhibition Inactivation Inactivation

Lactic Acid Bacteria

Yeasts and Molds

Reference (1)

NA

NA

No effect on growth

(2)

Inactivation

(1)

Inactivation

(2) (1) (2) (1) (2)

NA, not applicable. Source: Adapted from (1) Bermejo-Prada, A., Lo´pez-Caballero, M.E., Otero, L., 2015a. Hyperbaric storage at room temperature: effect of pressure level and storage time on the natural microbiota of strawberry juice. Innov. Food Sci. Emerg. Technol. 33 154 161; Bermejo-Prada, A., Segovia-Bravo, K.A., Guignon, B., Otero, L., 2015b. Effect of hyperbaric storage at room temperature on pectin methylesterase activity and serum viscosity of strawberry juice. Innov. Food Sci. Emerg. Technol. 30, ´ 170 176; Bermejo-Prada, A., Vega, E., Perez-Mateos, M., Otero, L., 2015c. Effect of hyperbaric storage at room temperature on the volatile profile of strawberry juice. LWT Food Sci. Technol. 62 (1), 906 914 and (2) Segovia-Bravo, K.A., Guignon, B., Bermejo-Prada, A., Sanz, P.D., Otero, L., 2012. Hyperbaric storage at room temperature for food preservation: a study in strawberry juice. Innov. Food Sci. Emerg. Technol. 15, 14 22.

avoid microbial growth, the application of an additional thermal treatment (i.e., pasteurization) to obtain a stable strawberry juice over storage time (15 days at 5 C) being required. On the other hand, when stored at low pressure (25 MPa) at RT, strawberry juice showed a growth inhibition in TAM and LAB, and a microbial reduction of YM, after 1 day of storage. Under 50 MPa, the growth of all studied microorganisms slightly decreased, and this reduction was more pronounced in samples stored under higher pressure levels (100 and 200 MPa). TAM, LAB, and YM counts were reduced by 1.4, 1.6, and 1.0 log10 units under 100 MPa, respectively, and by 3.6, 3.6, and 3.1 log10 units under 200 MPa, respectively (Bermejo-Prada et al., 2015a). Thus, the inhibition effect was found to be clear when lower pressure levels (25 MPa) were applied during storage, higher pressures (50 200 MPa) being capable of reducing the initial microbial loads (inactivation effect) after 1 day of storage. Probably, the moderate pressure used led to changes in bacterial components; e.g., activity modifications of key enzymes, reversible protein denaturation, alterations in cell morphology and cell membrane that may cause the leakage of cell contents (Abe, 2015). Bermejo-Prada et al. (2015a) also observed that longer storage times (10 15 days) produced

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CHAPTER 30 HYPERBARIC STORAGE OF FRUIT JUICE

a slight reduction in the initial microbial loads. In general, no growth was observed for all microorganisms after 10 or 15 days at pressure levels higher than 50 MPa, while HS at 25 MPa for 15 days revealed a growth inhibition for all studied microorganisms. However, these results were affected by the initial microbial load (depending on the fresh juice batch used), promoting differences in the microbial reduction after 15 days of storage (Bermejo-Prada et al., 2015a). A previous study reported by Segovia-Bravo et al. (2012) in frozen thawed strawberry juice, showed that the use of HS (25, 100, and 220 MPa), at 20 C for 15 days, revealed significant reductions of TAM and YM counts to levels below the detection limits. The authors suggested that these results could be enhanced by the stress of the previous freeze thaw treatment (Bermejo-Prada et al., 2015a).

30.2.2 MICROBIAL STABILITY DURING POST-HYPERBARIC STORAGE The time period between an HS period and the moment of food product consumption is relevant, since it allows us to understand the behavior of the product after the application of HS. This period at AP has been called post-hyperbaric storage (post-HS). After HS (25 220 MPa)/RT, strawberry juice samples were stored at AP/RS (Segovia-Bravo et al., 2012) or at AP/RT (Bermejo-Prada et al., 2015a). After HS of fresh juice (25 and 50 MPa for 1 day), an increase in the microbial load was observed in the post-HS period (AP/RT over 3 days), thus indicating a recovery of cell proliferation capacity. However, in fresh juice samples HS at 100 and 200 MPa, the microorganisms showed lower microbial recovery. A longer period of HS (15 days at 50 MPa) led to difficulties in cell recovery at AP (Bermejo-Prada et al., 2015a). On the other hand, strawberry juice samples previously frozen thawed stored at RS for 2 weeks after HS, kept the microbial load below the detection limits (Segovia-Bravo et al., 2012). These results showed that the reduction effect could be enhanced by the stress of a previous freeze thaw treatment (Bermejo-Prada et al., 2015a).

30.2.3 PHYSICOCHEMICAL PARAMETERS 30.2.3.1 Viscosity Viscosity is a relevant parameter reflecting the fruit juice quality, which affects its mouth-feel and the ability to hold its solid portion in suspension over the product shelf life (Segovia-Bravo et al., 2012). Fresh strawberry juice revealed a value of 35.32 6 1.80 cSt (Bermejo-Prada et al., 2015b), whereas, frozen thawed strawberry juice showed an initial value of 5.0 6 0.3 cSt (Segovia-Bravo et al., 2012). Generally, serum viscosity decreased in all strawberry juice samples during storage experiments (Bermejo-Prada et al., 2015b; Segovia-Bravo et al., 2012). A greater viscosity decay was measured in the first day of storage, being higher in fresh HS/RT juice samples (50 MPa: 55.5%, 200 MPa: 74.5%) than in samples stored at AP/RT (42.5%) (Bermejo-Prada et al., 2015b). Minor differences among juices were verified after 5 and 15 days of storage, being lower at the end of storage, closer to that of pure water (Bermejo-Prada et al., 2015b). Thus, samples stored under pressure showed an increase in the viscosity decay. Meanwhile, these results were different from those previously obtained for frozen thawed strawberry juice stored for 15 days (Segovia-Bravo et al., 2012). In the latter, the sharpest serum

30.2 STRAWBERRY JUICE

611

viscosity decay was detected in samples stored at AP/RT with phases separation (,1 cSt), whereas HS strawberry juice samples revealed smaller decays (25 MPa: 79.2%, 100 MPa: 71.1%, 200 MPa: 63.7%), compared to juice kept at AP/RT (83.6%). The loss of viscosity can be related to the depolymerization of pectin due to endogenous pectinase action (pectin methylesterase (PME) and polygalacturonase (PG)) during storage (Duvetter et al., 2009). Even further, RS was revealed to be more efficient in strawberry juice preservation presenting a lower viscosity decay (49.7%), possibly due to the reduction of pectin-hydrolyzing enzymes activity at low temperatures (Imsabai et al., 2002) or, probably, in the case of HS, due to the inhibitory effect of pressure on microbial growth and/or in other mechanisms associated with viscosity degradation (Bermejo-Prada et al., 2015b).

30.2.3.2 Color Color is another important quality parameter in strawberry juices, with the bright red color being preferred by consumers, which is easily degraded during processing and storage. The instrumental color parameters: lightness (L ), redness (a ), and yellowness (b ), were measured, which allowed calculating the total color change (ΔE ), hue angle (Ho) and chroma (C ). The initial frozen thawed strawberry juice presented values of 27.43 6 0.05, 8.23 6 0.22, and 3.68 6 0.16 for L , a , and b parameters, respectively (Segovia-Bravo et al., 2012). Bermejo-Prada and Otero (2015) obtained values of 33.87 6 0.10 for L , 15.75 6 0.09 for Ho, and 13.98 6 0.07 for C in strawberry juice, at day 0. HS/RT of strawberry juice samples over 15 days at 50 and 200 MPa showed a slight increase until day 5 in the L parameter and then a decrease was observed, with no differences at the end of storage compared to samples at day 0. These results were in agreement with another study performed by Segovia-Bravo et al. (2012). Assuming 1 as the basis for a color-perceptible difference for human eyes (Rein and Heinonen, 2004), the total color changes (ΔE ) were very small in all juices stored under the different conditions, ranging between 0.24 and 1.59 (BermejoPrada and Otero, 2015). Significant differences were only noted for samples stored over 10 days, with the HS samples being the ones that revealed minor ΔE values (0.24 0.63) compared to samples stored at AP (1.59). HS/RT samples at 200 MPa for 15 days showed two separated layers, a top clear layer and a cloudy bottom layer. However, Segovia-Bravo et al. (2012) verified that pressure levels between 25 and 200 MPa did not have a significant effect on color decay of strawberry juice. Regarding other color parameters, Bermejo-Prada and Otero (2015) reported that Ho value decreased until the 5th day in all samples, with an increase in this value being verified for HS/RT samples, while AP/RT-stored samples revealed lower values until the 10th day and then increased to values similar to that in HS samples. During storage, a slight decrease in C values was observed in the samples stored under higher pressure (mainly at 200 MPa), while samples stored at AP presented lower values. Segovia-Bravo et al. (2012) recorded slight decreases in Ho and C values during all storage period for all samples. Color losses were observed in frozen thawed strawberry juices stored at 20 C for 15 days, which were higher than samples stored under AP (Segovia-Bravo et al., 2012). Nevertheless, HS/RT (25 220 MPa, 20 C) attenuated color degradation, most probably due to polyphenoloxidase (PPO) and peroxidase (POD) activity, which could be responsible for the slowing down of anthocyanin degradation under pressure. However, RS was shown to be more efficient at reducing color decay.

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30.2.3.3 Bioactive compounds Strawberry juice is an important source of antioxidant compounds, including phenolic compounds, especially anthocyanins. Bermejo-Prada and Otero (2015) studied the effect of HS on the total phenolic and anthocyanin contents in strawberry juice at two pressure levels (50 and 200 MPa) and RT for 15 days. Initial total phenolics and total monomeric anthocyanin contents in strawberry juice were 781.30 6 28.06 mg GAE/L and 195.07 6 7.30 mg Pg-3-glu/L, respectively. At the 7th day of storage, the total content in phenolic compounds remained stable. However, a significant decrease of 11.6%, 23.2%, and 18.3% was observed between the 10th and the 15th days of storage for AP/ RT, 50 MPa/RT, and 200 MPa/RT, respectively. Similar behavior was recorded for total monomeric anthocyanin contents with a decrease by about 32%, 27%, and 28%, under the same respective storage conditions after 15 days of storage. The HS effect on total phenolics and total monomeric anthocyanin degradation should be further studied to elucidate the mechanisms involved in these effects.

30.2.3.4 Volatile compounds Bermejo-Prada et al. (2015c) also studied the effect of HS/RT (50 and 200 MPa) on the volatile compounds composition of strawberry juice stored over 15 days. The volatile profile was analyzed by gas-chromatography mass-spectroscopy, and compared with control samples stored under AP/ RT and RS during the same period. Thirty-one volatile compounds, including esters, aldehydes, alcohols, terpenoids, aromatic compounds, furanone, and ketone, were identified in the analyzed samples. After 15 days of storage, samples maintained at AP/RT exhibited clear signs of deterioration, with a characteristic musty aroma, while samples previously stored under HS/RT did not show any visible evidence of deterioration. Juices preserved under RS produced substantial drops in the trans-2-hexenyl acetate and nerolidol abundances after 15 days. HS prevented the spoilage of the samples, with no decreases in trans-2-hexenyl acetate, methyl hexanoate, and nerolidol. Compared to the control, juices stored under pressure retained most of the volatile compounds, while furan-2-methyl acetate, trans-2-hexenal, and 2,4-hexadienal contents decreased. Overall, HS/RT was found to be more efficient than RS in maintaining the volatile profile of strawberry juices, with no changes in any key aroma compounds.

30.2.3.5 Enzymatic activity As stated previously, viscosity decay is generally attributed to the action of endogenous enzymes (PME and PG), together with microbial growth, which implies an associated enzymatic activity. PME is particularly interesting because it affects not only serum viscosity but also the stability of the suspended particles in the juice cloudiness (Croak and Corredig, 2006). Bermejo-Prada et al. (2015b) reported that PME activity in strawberry juices decreased over storage in all samples stored under different conditions. Bermejo-Prada et al. (2015b) observed a significant decrease in residual PME activity in all juices stored for 15 days, which was 56%, 52%, and 57% for the samples stored at AP/RT, 50 MPa/RT, and 200 MPa/RT, respectively. The catalytic activity of PME could be evaluated by monitoring the methanol formation during incubation time. Results showed that the initial PME activity (2.49 6 1.79 μg/mL) increased in all samples during the storage period. After 7 days of storage, no significant differences in methanol content were found between the samples stored at AP/RT and those stored by HS/RT at 25 MPa. On the other

30.3 WATERMELON JUICE

613

hand, juice samples stored by HS/RT at 200 MPa revealed higher methanol content, most probably due to the pressure-induced structural changes in pectin that made it more susceptible to the action of PME. The presence of other enzymes, such as PPO and POD, may cause enzymatic browning and consequently degradation of fruit juice color. Bermejo-Prada and Otero (2015) studied the behavior of these enzymes during HS/RT for 15 days. These authors found significantly higher residual PPO activity, which was increased by 59% and 52% in the samples stored at 50 and 200 MPa, respectively, compared to 41% in the samples stored at AP/20 C. On the other hand, POD activity was unchanged in the samples stored at AP/20 C and at 50 MPa, whereas 15% reduction was observed in juice samples stored under 200 MPa. However, HS at a lower pressure level (50 MPa) resulted in an increase in PPO activity and no variations for POD activity were detected, which was similar to control samples. When the pressure level was increased up to 200 MPa, an increase in PPO activity and a decrease in POD activity were verified.

30.3 WATERMELON JUICE Due to its low acidity (pH . 4.6) and high water activity (aW . 0.85), untreated watermelon juice is highly perishable (as a result of microbial growth) with a very short shelf life (few hours). The feasibility of its preservation under pressure was evaluated using watermelon juice as a highly perishable food. For instance, HS conditions of 100 MPa over 60 h at variable/uncontrolled RT (Fidalgo et al., 2014) and at pressures between 25 and 150 MPa at controlled RT ranging from 20 C to 37 C (Santos et al., 2015) were studied.

30.3.1 MICROBIAL STABILITY The microbial stability of watermelon juice preserved by HS was evaluated through the analyses of several endogenous microorganisms, such as TAM, Enterobacteriaceae and YM, during 60 h, using a pressure level of 100 MPa and at variable/uncontrolled RT (  20 C) (Fidalgo et al., 2014). In this experiment, the temperature was not controlled, varying naturally with the environment (day/ night cycle) from 18 C to 21 C. Results demonstrated that after HS, the microbial load decreased in the first 8 h, and was then maintained up to 60 h of storage. TAM counts decreased from an initial value of 4.28 6 0.13 to 3.31 6 0.08 log10 CFU/mL after 8 h of storage. For Enterobacteriaceae and YM counts, the reductions were significant, and decreased from 2.86 6 0.18 and 2.48 6 0.13 log10 CFU/mL to values below the detection limit (,1.0 log10 CFU/mL), respectively. These juice samples showed the characteristic odor of fresh raw watermelon juice with no signs of off-flavors after 60 h of storage. Furthermore, no changes in the microbial loads of the samples stored under RS (5 C) were observed after 60 h of storage. Sample storage at AP/RT for 24 h, led to microbial loads close to or higher than 6 log10 CFU/mL. At 24 h, clear signs of spoilage, uncharacteristic and unpleasant odors, strong off-flavors, and high microbial load were observed in the juice. This study showed the possibility of food storage under pressure at variable RT with no temperature control and with no energetic costs. To clearly verify such a possibility, the same author carried out a test at 100 MPa and 30 C over 8 h, and observed microbial loads similar to those

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CHAPTER 30 HYPERBARIC STORAGE OF FRUIT JUICE

obtained in samples at 100 MPa and variable RT (18 21 C). These authors demonstrated that HS, at and above RT (regardless of temperature), is a promising food preservation method, which can be used to preserve perishable foods such as watermelon juice. In another study, the microbial stability of watermelon juice under different HS conditions was also evaluated by Santos et al. (2015). A time period of 8 h, pressures as low as 25 MPa, and different temperatures between 20 C and 37 C were studied, and a better microbial stability than in conventional RS was found (Table 30.2). For all microorganisms studied, it was observed that 25 MPa was insufficient to affect the microbial growth and higher pressure levels (50 or 75 MPa) to obtain an inhibition effect, or 100 and 150 MPa for an inactivation of the microorganisms was reported to be required. As shown in Table 30.2, for TAM counts, a minimum pressure of 50 MPa (at 25 C and 30 C) was necessary to obtain an inhibitory effect on the microbial growth. HS at 100 and 150 MPa showed additional microbial inactivation effects compared to those observed at lower pressures. Similar inactivation effects to RS were observed in samples stored at 75 MPa/20 37 C. Furthermore, the initial TAM load was reduced by about 1.12, 1.24, and 1.70 log10 units under 100 MPa at 20 C, 30 C, and 37 C, respectively, and by 1.44 and 1.95 log10 units under 150 MPa at 25 C and 30 C, respectively. Regarding Enterobacteriaceae counts, besides the growth inhibition observed under 50 MPa/ 30 C, it was found that a pressure level of 75 MPa was necessary to obtain a microbial inhibition, regardless of the temperature used. Increasing the pressure level up to 100 and 150 MPa resulted in a decrease in the initial microbial load (inactivation effect), causing the decrease in Enterobacteriaceae

Table 30.2 Microbial Effect of Pressure Level Used on Hyperbaric Storage of Watermelon Juice During 8 h Microbial Effect Pressure (MPa)

Temperature  ( C)

0.1

Growth inhibition No effect on growth

25 50

4 20, 25, 30, and 37 30 25 and 30

75

25 and 30

100 150

20, 30, and 37 25 and 30

25 C—no effect on growth 30 C—inactivation Inactivation

Mesophiles

Growth inhibition

Enterobacteriaceae

25 C—no effect on growth 30 C—growth inhibition Growth inhibition

Yeasts and Molds

Growth inhibition Inactivation

´ Source: Adapted from Santos, M.D., Queiro´s, R.P., Fidalgo, L.G., Inacio, R.S., Lopes, R.P., Mota, M.J., et al. 2015. Preservation of a highly perishable food, watermelon juice, at and above room temperature under mild pressure (hyperbaric storage) as an alternative to refrigeration. LWT Food Sci. Technol. 62(1), 901 905.

30.3 WATERMELON JUICE

615

to values below the detection limit (,1.0 log10 CFU/mL), which corresponded to a reduction higher than 1.68 log10 units. In the case of YM, the microbial effect was similar to TAM. As observed for Enterobacteriaceae counts, increasing the storage pressure up to 100 or 150 MPa was effective in YM count reduction to below the detection limit. Thus, the results obtained from this study clearly showed the possibility of food storage under pressure at variable (uncontrolled) RT. At all temperatures (20 37 C) studied, using pressure levels of 75 150 MPa during 8 h, a significant microbial growth inhibition was verified, with an additional microbial inactivation effect for pressure levels of 100 and 150 MPa (Santos et al., 2015).

30.3.2 MICROBIAL STABILITY DURING POST-HYPERBARIC STORAGE Fidalgo et al. (2014) studied the post-HS microbial stability of watermelon juice, initially at HS/RT (100 MPa for 60 h) and then stored at RS for 7 and 14 days. After 7 days of storage, under these refrigerated conditions, the microbial load was similar to the values observed immediately after HS, for TAM and Enterobacteriaceae, and an increase for YM (  2.5 log10 units) was detected. However, after 14 days under RS, the microbial loads were above 5.5 log10 CFU/mL for all studied microorganisms.

30.3.3 PHYSICOCHEMICAL PARAMETERS In both studies conducted by Fidalgo et al. (2014) and Santos et al. (2015), a similar initial pH value (5.91 6 0.04 and 5.78 6 0.19, respectively) was observed. After HS (100 MPa at uncontrolled RT,  20 C), a lower pH value than the one verified under RS over 60 h of storage was detected (Fidalgo et al., 2014). When the storage temperature was increased to 30 C, the initial pH value of watermelon juice was maintained at lower values than that in the other tested conditions (RS and AP/RT). Furthermore, a storage pressure between 50 and 150 MPa, at controlled RT of 20 30 C during 8 h, had no effect on the initial pH value of watermelon juice, while under RS, an increase was observed (Santos et al., 2015). Titratable acidity showed a pattern variation similar to pH, because HS/RT was clearly effective in substantially attenuating the observed titratable acidity increase at AP (Fidalgo et al., 2014; Santos et al., 2015). Total soluble solids are highly correlated with the concentration of sugars present in fruits and juices (Arocho et al., 2012). HS/RT of watermelon juice at 100 MPa, during 60 h did not influence the total soluble solids (Fidalgo et al., 2014), which was similar to the effect observed in watermelon juice stored at pressure levels between 25 and 150 MPa and controlled temperature of 20 37 C. However, during storage at AP/37 C, an increase in total soluble solids was reported (Santos et al., 2015). HS at 100 MPa and at uncontrolled RT (  20 C) for 60 h affected the browning degree of watermelon juice, causing a decrease in the initial value to values similar to those found when juice was stored at AP/RT, while under RS the browning degree showed no variations (Fidalgo et al., 2014). The pressure level decrease to 25, 50, and 75 MPa caused different variations in browning degree (Santos et al., 2015), contrasting with storage under RS, which showed similar values throughout the storage period. Color and cloudiness changes in fruit juices are usually associated with the catalytic action of enzymes, such as PME, PG, and PPO, causing sedimentation, loss of cloudiness, and browning

616

CHAPTER 30 HYPERBARIC STORAGE OF FRUIT JUICE

degree (Chisari et al., 2007). Color parameters (L , a , and b ) were measured in watermelon juice under a pressure level of 100 MPa for 60 h, at uncontrolled RT (  20 C) and compared with storage without pressure. An increase in color differences over storage under pressure was observed due to luminosity increase (L value) (Fidalgo et al., 2014). When the storage temperature of watermelon juice at 100 MPa (8 h) was increased to 30 C, the color was maintained at similar values to those under HS during the same period. During storage, anthocyanins may degrade due to several factors, such as light, temperature, pH, presence of oxygen, certain metal ions or L-ascorbic acid, causing the condensation of anthocyanins (self-association) and copigmentation phenomena (complex formation between anthocyanins and other polyphenols), resulting in color changes (Lo´pezSerrano and Ros Barcelo´, 2002).

30.3.4 BIOACTIVE COMPOSITION Fidalgo et al. (2014) evaluated the effect of HS and RS on the total content of phenolic compounds in watermelon juice during 60 h. Throughout storage, juice showed a general tendency to show a decrease in phenolic content under RS, which was similar to that observed in the HS/RT samples at 100 MPa during the same period. On the other hand, AP/RT storage revealed a tendency to present higher values, which increased over storage time.

30.4 MELON JUICE Recently, HS (8 h) of melon juice at different temperatures (25 C, 30 C, and 37 C) and pressures (25 150 MPa) was investigated and compared with AP storage at the same temperatures and under refrigeration (4 C) (Queiro´s et al., 2014). The characteristics of this juice, low acidity and high water activity, present a real challenge for the feasibility of HS methodology when applied to this food product.

30.4.1 MICROBIAL STABILITY The initial microbial profile of melon juice showed values of 4.24 6 0.06, 2.34 6 0.02, and 3.07 6 0.03 log10 CFU/mL for TAM, Enterobacteriaceae, and YM, respectively. As expected, when melon juice samples were stored under RS, the microbial load remained almost unchanged (4.26 6 0.01, 2.56 6 0.05, and 3.23 6 0.05 log10 CFU/mL, for TAM, Enterobacteriaceae, and YM, respectively) over 8 h. Melon juice stored at 25 C, 30 C, and 37 C during 8 h at AP revealed an accelerated microbial growth tendency in all analyzed microorganisms, with TAM counts reaching values of above 6 log10 CFU/mL at the three studied temperatures. Enterobacteriaceae and YM counts showed values higher than 5.65 6 0.10 and 3.64 6 0.31 log10 CFU/mL at 25 C, 30 C, and 37 C, respectively. The undeniable presence of spoilage signs was observed, with the existence of an unpleasant odor when these samples were stored at AP, and under the same temperatures. HS of melon juice demonstrated the capability of HS with regard to microbial growth inhibition when compared to samples stored at AP, and under the same temperatures. With the exception of HS/30 C at 25 MPa (8 h) conditions, all microbial counts were statistically lower (P , .05) than the

30.5 CONCLUSION

617

respective control samples (AP/30 C). Moreover, the authors pointed out that all microbial results were equal to or lower than the ones found in the samples stored over the same time period under RS (exception for Enterobacteriaceae when applying a pressure level of 50 MPa). This provided evidence that, regardless of temperature (25 C, 30 C, and 37 C), HS of melon juice using a pressure level higher than 50 MPa allows us to obtain a similar to better microbial result than for samples stored over the same time period under RS. For this food product, it was found that HS at 50 and 75 MPa had a microbial growth inhibition effect, with microbial counts similar to or lower than the ones initially present in the melon juice. Increasing the pressures up to 100 and 150 MPa showed additional inactivation effects of the microorganisms present in the samples. In fact, after 8 h under these conditions, the microbial load was lower than the initial one. Thus, it was concluded that, regardless of the temperature, the pressure level increase has a positive effect on the microbial load reduction, in which slopes of 20.011 and 20.020 log10 CFU/mL per MPa were found for TAM and YM, respectively (Queiro´s et al., 2014).

30.4.2 PHYSICOCHEMICAL PARAMETERS HS of melon juice over 8 h was also subjected to the following physicochemical analyses: pH, titratable acidity, total soluble solids, browning degree, and cloudiness (Queiro´s et al., 2014). In this work, the authors stated that the pH was not considerably affected by HS conditions. In fact, besides AP storage at 30 C and 37 C, where slight pH changes were observed (5.62 6 0.01 and 5.65 6 0.01, respectively), the other measured pH values were similar to the initial one. The titratable acidity values presented a linear increase tendency with the rise of temperature, when melon juice was stored at AP. This tendency was also noticed when increasing the storage pressure, but to a lower extent (lower levels). Regarding the total soluble solids in melon juice samples, no significant differences between the different HS conditions (with the exception of 150 MPa/30 C and 100 MPa/37 C) were observed. However, storage at AP at the different temperatures increased the total soluble solids to values of around 10 Brix. HS of melon juice for over 8 h showed higher browning degree values with the increase in pressure level, being more similar to the initial one, when compared to samples stored at AP and the same temperatures. It was also verified that cloudiness values presented an increased tendency with the increase in temperature (at AP) and with the increase in pressure (at HS), being observed, in the latter, cloudiness values similar to the initial one. As mentioned previously, these changes in cloudiness parameters are, most of the time, related to the catalytic actions of PG, PME, and PPO, and the authors did not expect the inactivation of these enzymes under these HS conditions (taking into consideration the level of pressure applied), which means that these enzymes preserved their catalytic action under this new food preservation methodology (Chisari et al., 2007).

30.5 CONCLUSION Several studies evaluated the possibility of preserving fruit juices for longer periods under pressure (HS) at room or refrigeration temperatures. This preservation method not only can reduce the energetic costs compared to RS (since energy is only applied to reach the desired pressure, with no

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CHAPTER 30 HYPERBARIC STORAGE OF FRUIT JUICE

energy requirements for temperature control), but also prevents the losses during storage, associated with temperature fluctuations. Research developments regarding HS/RT of food products are still very scarce because of economic and technological obstacles due to the need for new and more adequate equipment for these types of storage studies. Nevertheless, the few published studies regarding HS feasibility are very promising and demonstrate the potential of this new food preservation methodology applied to fruit juices, as well as to other food products.

ACKNOWLEDGMENTS Thanks are due to FCT/MEC for the financial support to the QOPNA research Unit (FCT UID/QUI/00062/ 2013), through national founds, and where applicable cofinanced by the FEDER, within the PT2020 Partnership Agreement. F.J. Barba was supported from the Union by a postdoctoral Marie Curie IntraEuropean Fellowship (Marie Curie IEF) within the 7th European Community Framework Programme (http:// cordis.europa.eu/fp7/mariecurieactions/ief_en.html) (project number 626524 -HPBIOACTIVE—Mechanistic modeling of the formation of bioactive compounds in high pressure processed seedlings of Brussels sprouts for effective solution to preserve healthy compounds in vegetables).

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