Bacteria

The genus Listeria consists of small, non-spore forming, motile and Gram-positive rods. Listeria monocytogenes is the primary pathogenic species. L. monocytogenes is a soil reservoir, moderately heat-resistant microorganism that is able to grow under refrigeration conditions with or without the presence of oxygen in a wide range of ready-to-eat products with extended shelf-lives (Doğan and Erkmen, 2004). Although Listeria is not known to have caused outbreaks through the consumption of unpasteurized fruit juices, it has been isolated from unpasteurized apple juice (Sado et al., 1998). Table 3.1 shows the main results of the inactivation of Listeria species (L. monocytogenes and L. innocua) achieved by HHP technology in fruit juices. A high pressure treatment of 600 MPa for 5 min at room temperature is typically sufficient to obtain a 5 log reduction of L. monocytogenes in fruit juices. However, the media used to conduct the studies seems to influence the effectiveness of the technology. When comparing the baroresistance of L. monocytogenes inoculated in different substrates (buffer, whole milk, and fruit juices), HHP treatment in milk was not as effective as in other food systems. Fat and protein content in milk seems to protect the microorganism against pressure, whereas the low pH of fruit juices can be an additional inhibitory factor enhancing the effectiveness of HHP technology (Doğan and Erkmen, 2004).

Temperature plays an important role when combined with pressure enhancing the overall process effectiveness. In addition, refrigeration conditions will keep the survival fraction of microorganisms under control. However, due to the psychrotrophic nature of Listeria, the microorganism could survive in the product during the shelf-life period. Fortunately, in most fruit juices, the acidic environment will prevent recovery of injured cells and reduce the surviving Listeria cells to safe levels. This further reduction can be explained by the fact that most cells injured by the pressure become more sensitive to the acidic environment and are not able to survive during the storage period.

Escherichia coli belongs to the family Enterobacteriaceae (enteric bacteria), which are Gram-negative, rod-shaped, motile and facultative anaerobes that live in the intestinal tracts of animals (Ramaswamy et al., 2003). The baroresistance of E. coli in apple juice (AJ) and orange juice (OJ) has been studied by several authors (Table 3.1). Differences in pressure-resistance are known to occur depending on the media and strain used in the study. Ramaswamy et al. (2003) found that a single pressure pulse at 400 MPa and 25 °C was enough to inactivate initial counts of E. coli by 8 log cycles in AJ (pH 3.5). The combination of pressure and mild heat composes a good strategy to improve the overall effectiveness of the HHP technology without compromising food quality. Muñoz et al. (2007) found an optimal combination of moderate levels of high pressure (200–250 MPa) and mild temperature (57–60 °C) for E. coli inactivation (6 log cycles) in OJ and AJ. At these treatment conditions, all natural flora present in the juices were reduced to almost undetectable levels.

In a selective environment of a high-pressure fruit juice processing plant, the occurrence of naturally E. coli pressure-resistant strains cannot be ruled out. In this regard, Garcia-Graells et al. (1998) isolated several mutants that were pressure-resistant (800 MPa at 10–40 °C) from a pressure-sensitive E. coli. The non-treated E. coli mutants were able to survive in acidic substrates (AJ and mango juice pH 3.3–4.0) at refrigeration conditions (8 °C) for at least 30 days. The survival at refrigeration conditions even in acidic conditions could be explained by the fact of a reduced permeability of cell membrane to protons or reduced metabolic activity at reduced temperatures. However, after pressure treatment (300–500 MPa) and further storage (5 days of storage at 8 °C) the number of survivors was below the detection limit (high numbers of cells were injured during the treatment, resulting in a reduced resistance to the low pH during storage). This fact illustrates that sublethal injury is a crucial parameter which should be monitored after HHP treatment to detect any recovery of injured cells, particularly over prolonged storage periods.

It is known that E. coli O157:H7 strains have been implicated in several outbreaks related to unpasteurized fruit juices due to its high resistance to acidic environment and low infective dose. Several studies have been performed using this strain and HHP treatment in different fruit juices (Table 3.1). In a first study, Linton et al. (1999a, 1999b) studied the survival of E. coli O157:H7 in OJ at different pH levels (3.4 to 5.0). The survival of E. coli in OJ was pH dependent. After 20 and 25 days at 3 °C at pH 3.4 and 3.6, respectively, no cells were detected. After 25 d at higher pH levels (3.9, 4.5, and 5.0) the reduction was 4.5, 1.3, and 0.6 log units. This fact could risk the occurrence of food poisoning, if OJ became contaminated with E. coli O157:H7. This is particularly true since its survival is longer than the length of time required for juice spoilage to occur. The authors found an optimal treatment (6 log reduction) of 550 MPa for 5 min at 20 °C at pH levels of 3.4 to 4.5 and 550 MPa at 30 °C at pH 5.0. Other studies have also shown the higher pressure resistance of the E. coli O157:H7 strain. Jordan et al. (2001) used two E. coli strains (type-strain and O157:H7) in AJ and OJ substrates. Both strains were more resistant to the pressure treatment in OJ than in AJ. Slight pH differences (higher pH in OJ), viscosity or other physical-chemical characteristics seemed to affect the microorganism resistance. A reduction of 1 log in E. coli O157:H7 was achieved in OJ, whereas 5 log was reduced in AJ after a treatment of 500 MPa for 5 min. The type-strain of E. coli was much more pressure-sensitive and after 350 MPa, a 5 log reduction was achieved in both substrates. After storage at 4, 25, and 37 °C, a further 3.3 and 7 log reduction at 4 and 25–37 °C temperatures, respectively, was achieved in O157:H7 strains in OJ. This fact also corroborates that refrigeration temperatures seem to protect the pressurized E. coli cells against the acidic environment.

In some cases, more than one bacterial strain can be present in the fruit juice and it is interesting to study the pressure resistance of E. coli strains composed of a cocktail. In this regard, Teo et al. (2001) and Whitney et al. (2007) used a cocktail of several E. coli O157:H7 strains related to different outbreaks to inoculate different juices (OJ, AJ, grapefruit (GJ), and carrot (CJ)). Treatment at 615 MPa for 2 min at 15 °C was able to inactivate more than 5 log cycles in CJ and GJ samples but not in the rest of the substrates. After storage for 24 h, further inactivation was observed (3.2–5.6 log reductions). It seemed that acid-resistant strains were also more resistant to pressurization. These differences among strains could be due to differences in their membrane composition and ability to repair membrane damage in acid environments after pressurization. In addition, differences in gene expression related to stress response could also contribute to increased resistance to pressure.

Some strains of Salmonella are able to survive acidic conditions and therefore are present in the fruit-based products, if low hygienic conditions are present, raw material is highly contaminated, or the product is recontaminated after processing. Several studies have been conducted to assess the pressure sensivity of these acid-resistant strains (Table 3.1). Teo et al. (2001) and Whitney et al. (2007) have shown an optimal treatment of 2 min at 550–615 MPa and 15 °C in the inactivation of several strains of Salmonella (S. hartford, S. muenchen, S. agona, S. enteritidis and S. typhimurium) in several fruit juices (AJ, OJ, GJ and CJ) achieving more than 5 log cycles in all samples.

Several lactic acid bacteria species can spoil fruit juices in optimal conditions for their growth. Leuconostoc mesenteroides is one important species among them. L. mesenteroides inactivation was studied by Basak et al. (2002) in fresh and concentrated OJ (42°Brix) after high pressure treatment. The survivor curves showed a biphasic phenomenon. The pressure instantaneously reduced the counts of the microorganism (4.4 log cycles at 400 MPa and 20 °C), then the survivor curves followed first-order rate destruction. The study also showed that the effectiveness was significantly reduced in the concentrated OJ where both the lower a_w and higher soluble solids content protected the microorganism from pressure.

Most spore-forming pathogenic bacteria will not germinate or grow in an acidic environment. However, some spoilage spore-forming bacteria such as Alicyclobacillus acidoterrestris have been implicated in acidic beverage and fruit juice spoilage (Lee et al., 2006). A. acidoterrestris is a soilborne and thermoacidophilic microorganism, and can be present in the final product through soil adhering to the surface of fruits during harvest or by the water used during juice processing. Their spores can germinate, grow and cause spoilage in a pH range of 2.5 to 6.0, a level below the typical range for spore-forming bacteria. The germination and growth has been observed in OJ incubated at 44 °C for 24 h and can occur even at higher temperatures (80 °C) (Pettipher et al., 1997). Their spores are resistant to the normal pasteurization conditions normally applied to acidic fruit products and can germinate and grow during storage of retail products where spoilage can occur (Jensen, 1999). This exceptional fact is possibly attributable to its unique cellular membrane composition containing ring structures (cyclohexane fatty acids) closely packed leading to a high stabilization of the membrane and retaining more divalent cations than other bacterial spores, explaining their high resistance to demineralization (Lee et al., 2006). The typical spoilage effects are organoleptic taint due the production of guaiacol leading to a ‘medicinal’ or ‘phenolic’ off-flavor and light cloudiness (Lee et al., 2002).

Some studies have investigated the pressure resistance of A. acidoterrestris spores to high pressure (Table 3.1). It seems that the spores are also baro-resistant, primarily due to the dehydrated state of the core. The inactivation of bacterial spores seems to occur in two steps: high pressure germination and inactivation of germinated spores (350 MPa for 20 min at 50 °C) (Alphas et al., 2003; Lee et al., 2006). Again, the a_w of the fruit juice influence the degree of inactivation; HHP is less effective in concentrated juices. The contamination of concentrated fruit juices with A. acidoterrestris and the dilution to produce commercial juice will rapidly spread the microorganism.

Molds and yeasts

Yeasts such as fermentative Saccharomyces cerevisiae and Zygosaccharomyces bailii can spoil fruit juices. Both yeasts have the ability to produce ascospores. The ascospore protoplast has a structure similar to vegetative cells, but the wall consists of an outer and inner coat (Raso et al., 1998). Spore formation can be induced on fruit surfaces where low concentrations of sugars and ethanol are present. During juice extraction, ascospores may contaminate the juice, eventually causing spoilage. Ascospores are known to be more resistant than vegetative cells to different food processing and chemical and physical agents (Zook et al, 1999).

Vegetative cells are easily inactivated by pressure and usually a treatment of 350 MPa at room temperature is enough to instantaneously reduce the initial yeast load in a fruit juice (Table 3.2) (Bang and Swanson, 2008). When studying the microflora of OJ, Parish (1998) found that the inactivation kinetics parameters of juice microbial flora were similar to the ascospores, meaning that a high percentage of the yeasts present in the juice were in the form of spores. This fact points out the need for validating high pressure treatment in a juice with high populations of ascospores.

The fact than ascospores are more pressure-resistant than vegetative cells in fruit juices has been corroborated in several studies (Raso et al., 1998; Parish, 1998). In ascospore survivor curves, a shouldering effect, mainly at low pressure values (300 MPa), indicates that at lower pressures, a time threshold was required before substantial inactivation was observed. The pH of fruit juice (3.5–5) seems not to affect ascospores inactivation by HHP. In addition, no differences are observed among different juices and model systems indicating no protective effects (Zook et al., 1999). This is different than the situation with bacteria, where inactivation increases at pH lower than 4.5. This phenomenon corroborated the ability of yeasts to grow at low pH. Comparing the inactivation data of several studies on yeasts with those obtained by other authors on bacteria species confirmed that yeasts are more sensitive to pressure than non-sporulating bacteria. In addition, a lower pressure-resistance of yeast ascospores is observed in comparison with bacterial spores (Reyns et al., 2000). The ascospores are larger and higher in lipid and carbohydrate content, and do not have dipicolinic acid and a cortex, which is considered very important in the resistance of the spore due to its ability in maintaining the state of osmotic dehydration of the protoplast. However, when using a concentrated juice (42°Brix) the a_w can affect the S. cerevisiae inactivation. The inactivation rate significantly decreased in the concentrated OJ. The high content of soluble solids or low a_w seems to be the main reason for the low inactivation rate (Basak et al., 2002; Donsí et al., 2007).

Fruits used for juice processing can be contaminated with molds. Most fungi are heat sensitive and usual pasteurization processes in fruit juices are adequate to inactivate them. However, several molds, especially ascospores producing molds, have high heat-resistance even at elevated temperatures of 90 °C (Palou et al., 1998). Ascospores of several Ascomycetes such as Byssochlamys nivea have been found to contaminate commercial fruit juice concentrate due to its wide pH growth range (2.0–9.0). Other molds such as Talaromyces avellanus can also be found as natural contamination in fruit products (Voldøich et al., 2004). Regarding the effect of physico-chemical characteristics of substrates on efficacy of HHP technology, the water activity or soluble solids content plays an important role (Table 3.2). This is especially true when HHP treatment is considered for the pasteurization or decontamination of concentrated fruit juices where severe treatment conditions are necessary.

Enzymes

Enzymes are a special type of protein with enormous catalytic power and great specificity. Their biological activity arises from active sites brought together by a three-dimensional configuration. They have two important regions; one that recognizes the substrate and the other that catalyzes the reaction once the substrate has been bound (Hendrickx et al., 1998). These two are called the active site and take place in a small part of the enzyme total volume. Changes in the active site interfering in the enzyme-substrate union or protein denaturation can produce an activity loss or functionality variations (Tsou, 1986). In general, covalent bonds are not affected by HHP treatment because the primary structure of the enzyme will not be damaged. The hydrogen bonds are also relatively baroresistant and the secondary structure will not be affected up to pressure values around 700 MPa. However, HHP treatment affects electrostatic and hydrophobic interactions that maintain the tertiary and quaternary structures stability (Ludikhuyze et al., 2002).

Within the food quality related enzymes, the most important in fruit juices are the following:

In fruit juices, enzyme baroresistance is generally higher than the majority of naturally found microorganisms. For that reason, fruit juice preservation treatment is based on the inactivation of the enzymes responsible for its quality deterioration (PME in citrus juices, PPO in apple juice, among others). However, in some cases, no relation is observed between enzyme baroresistance and thermoresistance.

Enzyme baroresistance also depends greatly on:

A primary purpose of HHP processing is food preservation by maintaining the quality of fresh product and thus inactivation of quality deteriorative enzymes. Among these enzymes, PME has been extensively studied by HHP technology in order to find the optimal inactivation conditions. PME is a texture-related enzyme mainly associated with the pulp content of citrus juices. It destabilizes the suspension formed by pectin mycelia (cloud loss) leading to a clarified product with low commercial value. Different studies have demonstrated the baroresistance of the enzyme to elevated pressures in OJ. Treatments below 500 MPa at room temperature seem not to affect the native PME activity in OJ (Cano et al., 1997; Nienaber and Shellhammer, 2001). Processing conditions of 600–700 MPa for 1–3 min combined with mild temperatures (50–60 °C) seems to be effective in inactivating the native PME (Nienaber and Shell-hammer, 2001; Polydera, et al., 2004, Sampedro et al., 2008), which stabilizes the OJ between 90 d and 16 wks at 4 °C (Parish, 1998; Goodner et al, 1999). Low pH seems to enhance the PME inactivation while increasing soluble solids content (concentrated OJ) seems to decrease the effectiveness of HPP showing a protective effect (Basak and Ramaswamy, 1996). Regarding the application of HHP technology to smoothies, Sampedro et al. (2008) studied the influence of HHP processing in a beverage based on a mixture of orange juice and milk (50% of OJ, 20% of milk, 30% of water, 0.3% of pectin and 7.5% of sugar). The authors found optimum conditions for PME inactivation at 90 ⁰C for 1 min or 700 MPa at 55 °C for 2 min showing the protective effect of the orange-milk media. In addition, PME was more thermostable and baroresistant in the OJ-milk based beverage system than in OJ.

POD is an oxidoreductase related to the oxidation of a wide range of natural substances present in fruits, especially those containing aromatic groups. The mode of action involves the generation of free radicals able to abstract hydrogen from such substrates. Usually, hydrogen peroxide or oxygen act as oxidizing agents. POD contributes to phenolic oxidation leading to deteriorative changes in flavor, texture, color and nutrition. In OJ, POD is involved in the loss of flavor quality (Elez et al., 2006). Several studies have shown the high baroresistance of POD in different substrates. In one study, Cano et al. (1997) achieved only a 25 and 50% inactivation of POD in strawberry (230 MPa for 15 min at 43 °C) and OJ (400 MPa for 15 min at 32 °C). Below or above these conditions, higher enzyme activity was observed. It seemed that the activation was pH dependent and at higher pH, the activation was strong as in the case of strawberry. In this sense, Garcia-Palazon et al. (2004) only observed a 35% inactivation of POD in strawberry after 600 MPa for 15 min at 20 °C and no more inactivation was achieved with pressures increasing up to 800 MPa, whereas Fang et al. (2008) observed a residual activity of 30% after 600 MPa for 30 min at 50 °C.

PPO is an oxidative enzyme responsible for undesirable color changes, undesirable flavors and nutritional losses. It is mainly related to the browning reactions, catalyzing the hydroxylation of mono-phenols, leading to the formation of di-phenols and the following oxidation of di-phenols to form quinones in the presence of oxygen. Next, the condensation of quinones generates dark substances (melanines) which negatively influence the quality and marketability of commercial fruit juices (Giner et al., 2002). Cano et al. (1997) studied the effects of HHP processing on PPO in strawberry puree. A maximum of 60% of inactivation was achieved combining 285 MPa at room temperature. At higher pressures or temperature levels, higher enzyme activity was observed (enzyme activation). The low pressure and temperature conditions applied in this study could explain the low treatment effectiveness and the activation phenomenon. Palou et al. (1999) studied PPO inactivation in a banana purée. Pressure treatment alone (689 MPa for 10 min at room temperature) was able to reduce enzyme activity by only 20%. Only after a blanching treatment (saturated steam for 7 min) followed by pressure treatment (689 MPa) they were able to reduce the initial activity by more than 95%. In a later study, García-Palazón et al. (2004) showed a high baroresistance of PPO in red raspberries, resulting in a remaining activity of 70% after 800 MPa for 15 min. In contrast, PPO in strawberries was more sensitive to the pressure treatment and 600 MPa for 15 min or 800 MPa for 10 min was enough for a complete inactivation. The authors linked the enzyme stability in raspberries and strawberries with the stability of their anthocyanins and the consequent color loss. Higher PPO activity decreased the stability of anthocyanins. Because raspberries retain high levels of activity of PPO after HPP treatment, anthocyanins in raspberries were more susceptible to HHP treatment.

Color

The color observed by human beings is the perception of the wavelengths coming from the surface of the object on the retina of the eyes (Tijsken et al., 2001). Food appearance can change depending on the amount of light, the light source, the observer’s angle of view, size and background differences. However, standardized instrumental color measurements used in the food industry such as the Hunter color (L*, a* and b*). L* is a measure of brightness/whiteness that ranges from 0 to 100 (white if L* = 100, black if L* = 0), a* is an indicator of redness that varies from –a* to + a* (–a* = green, + a* = red) and b* is a measure of yellowness that varies from –b* to + b* (–b* = blue, + b* = yellow). The CIELAB system is used as a quality index in fruit juices to assess the conformity to specifications or measure the changes as a result of food processing or storage (Giese, 2000). Maintaining the natural color of fruit juices is a major challenge to the application of HHP technology, because color is the first characteristic that is noticed in food and predetermines consumer perceptions of freshness and expectations of both flavor and quality (Rodrigo et al., 2007). The changes in the natural color of fruit juices are based on the degradation of pigments by enzymatic and non-enzymatic reactions. Compounds such as anthocyanins are responsible for the color in some fruits. Rodrigo et al. (2007) studied the degradation kinetics of strawberry juice color after HHP processing and concluded that the combination of L*, a* and b* parameters in the form of L*x a*/b* was the most accurate way to describe the color degradation in strawberry juice. No differences were found between treated and control samples up to 700 MPa for 60 min at 65 ⁰C. However, the effect of pH was found to be significant in the strawberry samples. The a* parameter after HHP processing increased as the pH increased from 3.7 to 5. It seems that pelargonidin-3-glucoside anthocyanin is the main compound responsible for the red color in strawberries and it is not stable in a pH range of 5–7.

Sensory and consumer studies

Some sensory studies have been conducted in fruit juices in order to demonstrate the advantages of HHP processing versus thermal pasteurization on preserving the natural sensory properties. Some studies have used trained sensory panelists in order to compare the freshness and acceptability of samples treated by HHP and traditional thermal pasteurization. However, due to their training, sensory panelists may not be representative of the typical consumers of fruit juices. In these cases, consumer acceptance studies are necessary. In addition, the analytical profile of volatile compounds related to the fruit juices aroma is performed, to try to connect the unique flavor of fruit juices with specific chemical compounds.

Baxter et al. (2005) studied changes in the sensory properties and flavor compounds during a 12-wk shelf-life storage of OJ processed by HHP and thermal pasteurization at 4 and 10 °C. Ten trained sensory panel members were used for the descriptive sensory analysis of samples and 30–40 regular consumers of OJ participated in a consumer acceptability study. Regarding the color, the trained panel did not observe differences among the different samples during the overall period. Increasing the duration of the storage period led to a decrease in the sweetness and strength of orange odors and an increase of aged, artificial and fermented odors. At the end of the storage period, consumers did not differentiate between control, thermally and pressure-treated samples at 4 °C. However, scores were lower in the HHP for 10 °C samples and were unacceptable for the thermally processed samples at 10 °C. Twenty volatile compounds were analyzed in the storage of OJ at 4 and 10 °C. Considerable reductions were found for most compounds in both HHP and thermally processed juices compared with the control sample at –20 °C. The compounds showing the greatest reductions were octanal, citral, ethyl butanoate and limonene with the final concentration of compounds 6–38% lower than the initial level. The decrease in the volatile compounds concentration during the storage was produced by a combination of factors. PET bottles used for the OJ storage seemed to absorb some volatile compounds. In addition, oxidation, hydrolysis and acid-catalyzed reactions were responsible for the degradation of volatile compounds.

Working with an OJ-milk beverage, Sampedro et al. (2009a,b) studied the volatiles profile after HHP treatment. After HHP treatment (650 MPa for 15 min at 30 °C), some volatile compounds increased. The authors argued that in a complex matrix such as OJ, with the presence of suspended solids, a portion of analytes could be entrapped in the pulp. HHP treatment could increase the membrane permeabilization and facilitate the release of several compounds from the suspended solids to the liquid phase, facilitating its extraction into the headspace. The average loss of volatile compounds concentration was between – 14.2 and 7.5% at 30 °C and 22.9 and 42.3% at 50 °C.

As for tropical fruit juices, Laboissiere et al. (2007) conducted a study on the effects of HHP processing on the sensory characteristics of a Brazilian yellow passion fruit juice. They found very similar patterns for sensory properties between fresh passion fruit juice and HHP processed juice. The only parameter that differentiated both samples was color. In addition, a panel could distinguish between fresh and HHP treated samples and commercially pasteurized samples. The main sensory attributes that differentiated those samples were the presence of suspended particles, phase separation, natural aroma and flavor, artificial aroma and flavor, cooked aroma and flavor and fermented flavor. Most of these sensory attributes considered as sensory defects possibly resulted from the heat pasteurization, addition of artificial aromas, flavor compounds and stabilizers.

Consumers are becoming more conscious about the potentially negative impact of food processing on their health and the environment. Healthy and natural foods are the most important area of research by the majority of food companies (Katz, 2000). ‘Fresh’ remains the most desirable food label claim. Other aspects such as country-of-origin, organic food, local foods and environmental concerns, have continued to rank high in public attention (Deliza et al., 2005). A positive consumer attitude towards the use of HHP technology is necessary to guarantee the success of the product in today’s competitive global market, where new food product innovation is required for survival. That means that the role of the consumer in the technology validation process must be taken into account.

In this sense, Deliza et al. (2003 and 2005) conducted two studies concerning the Brazilian consumer attitudes toward to the use of high pressure processing. They chose four consumer groups consisting mainly of women and fruit juice consumers. The product chosen for the study was pineapple juice processed by HHP with three different information labels (low, medium and high information). The first statement, pointed out by the majority, was the price as an important attribute during their decision-making process. The study also revealed that most consumers inferred the product taste based on the label information which affected the product expectation and perception. When the information about the technology was presented, three of the four groups perceived the product as having higher quality. The information about the technology had a significant impact on the intention of purchase. However, information about the technology without further explanation led to a negative impact on the consumer intention of purchase. These results lead to important information for the food producers. The information about the technology in the product label is essential for the product to be perceived by consumers as higher quality. In addition, factors influencing fruit juice purchasing include convenience, taste and cost.

In a later study, Nielsen et al. (2009) conducted a consumer study using focus groups in six European countries (two from northern Europe and four from eastern Europe). A baby food and fruit juice were used as selected products for focus group discussions. Participants were positive towards HHP products for naturalness, improved taste, and high nutritional value (high vitamin content). Longer shelf-life in comparison to fresh squeezed juice products, high prices and lack of information were seen as negative toward the technology. Environmentally friendly and natural products (no preservatives) were positive towards the technology. Differences were observed among the different cultures. Participants from northern countries were more skeptical about the new technologies and were more worried about the impact on the environment, whereas eastern countries saw the higher price solely as negative. Differences were also seen among the products. Longer shelf-life and higher prices were seen negatively in fruit juices, since consumers are more accustomed to fresh squeezed juices. In contrast, participants saw higher price and longer shelf-life as positives for baby food; however, some participants were negative to HHP baby food, since it is not homemade. In a potential buying situation, quality and especially taste play a critical role in accepting and maintaining the commercial marketability of these novel products.

Bioactive compounds

Daily intake of fruits and vegetables has been related with the prevention of degenerative processes such as cardiovascular disease and certain cancers. This protective action has been attributed to their bioactive compounds, which have antioxidant properties. HHP treatment is expected to be less detrimental than thermal treatment to low molecular weight food compounds such as flavoring agents, pigments and vitamins as covalent bonds are generally not affected by pressure (Butz et al., 2004). Vitamin C is the most important water-soluble nutrient and is related to the antioxidant capacity of the OJ (Cortés et al., 2008).

Regarding the effects of HHP treatment on the stability of vitamin C, ascorbic acid (L-AA) and total antioxidant capacity in OJ were studied. Specifically, (Sánchez-Moreno et al. 2003b, 2005) and Plaza et al. (2006) studied the effects of different high pressure (100–400 MPa for 1–5 min at 30–60 ⁰C) and thermal treatments (70 °C, 30 s and 90 °C, 1 min) in an OJ stored at 4 °C for 40 d. Around 10% of the vitamin C content was lost after the combined treatment (400 MPa for 1 min at 40 °C or 100 MPa for 5 min at 60 °C) but no loss was found at 30 °C. They argued that the high contents after processing could be attributed to a partial elimination of enzymes (POD and ascorbate oxidase) responsible for L-AA and vitamin C oxidative degradation. At higher temperatures, greater decreases in vitamin C content were observed due to possible thermal degradation. During storage, the losses reached 24 and 32% after thermal and HHP treatments, respectively. The untreated sample only lost 10% of the initial content. These differences were attributed to the different levels of enzyme inactivation (POD and ascorbate oxidase) achieved by the treatments that could degrade the L-AA during storage by an oxidative process. The antioxidant capacity was unaffected by HHP and low pasteurization processes, whereas high pasteurization processing (90 °C for 1 min) reduced the antioxidant capacity by 6.5%. The authors found a correlation between L-AA, vitamin C and antioxidant capacity, thus the high stability of both compounds after the different treatments also stabilized the antioxidant capacity. On the other hand, no correlation was found between total carotenoids and flavonones and antioxidant capacity, indicating the lack of relevant effect of these compounds on the total OJ antioxidant capacity.

The type of packaging used in the storage of the fruit juice after processing can influence the stability of the antioxidant capacity of OJ. Polydera et al. (2003) studied the degradation kinetics of ascorbic acid after a HHP treatment (500 MPa at 35 °C for 5 min) and thermal treatment (80 °C for 20 s) using two different packaging materials, an intermediate oxygen barrier (polypropylene bottles) and a high oxygen barrier (polyethylene, aluminum and cellophane) during 1–2 months storage of an OJ at 0, 5, 10, and 15 °C. When polypropylene bottles were used, the degradation kinetics of L-AA during the storage period seemed to follow a first-order reaction. The rate parameter (k) was lower in the pressure-treated sample than in the thermally-treated sample, indicating HPP juice had a lower degradation rate in L-AA than thermally processed juice during storage. The degradation rate of L-AA increased in both samples as storage temperatures increased. When flexible pouches were used, the degradation rates seemed to have two stages. The first part followed first-order kinetics and the second part followed zero-order kinetics. The more rapid decrease of L-AA at the beginning of storage can be attributed to autoxidation, the reaction of L-AA with dissolved oxygen, and then the lower rates could be controlled by the low diffusion of oxygen of the material or by an anaerobic decomposition. Inactivation rates of anaerobic decomposition are usually 2–3 orders of magnitude lower than the oxidative degradation (Gregory, 1996). For these reasons, the inactivation rates were significantly higher in the propylene bottles at both treatments. The authors estimated the shelf-life of OJ based on the vitamin C content (> 20 mg/100 mL of vitamin C in the OJ at the expiration date) regulated by the Association of the Industry of Juices and Nectars from Fruits and Vegetables of the European Union. Using polypropylene bottles, the shelf-life at 5 °C was estimated to be 50 and 34 d for the HHP and thermal samples, respectively. When the storage temperature was increased to 15 °C, the shelf-life period decreased to 20 and 18 d, respectively. On the other hand, using the flexible pouches, the shelf-life was estimated to be 90 and 62 d after HHP and thermal treatment at 5 °C and 62 and 50 d at 15 °C, respectively. The lower degradation rates of vitamin C in the HHP-treated samples extended the shelf-life compared to thermal pasteurization. Owing to the high oxygen barrier of the flexible pouches, the shelf-life was increased with respect to the polypropylene bottles. The sensory evaluation during the shelf-life indicated higher scores for the pressure-treated samples when comparing with the thermal ones.

The degradation of water soluble vitamins (vitamin C, B1 and B6) after high pressure treatments were studied by Sancho et al. (1999) using a model system and strawberry smoothie to check the effects of HHP treatment as well as thermal pasteurization (76C for 20 s) and sterilization (120C for 20 min) on water soluble vitamins. In the model system, the vitamin C content varied from 10 to 12% after 200–600 MPa for 30 min at room temperature. Changes in B1 and B6 vitamins after the pressure treatment were insignificant. In the strawberry system, changes in vitamin C content were not significant after the HHP processing and thermal pasteurization. However, a 33% loss was observed after the sterilization process.

Carotenoids are among the most abundant bioactive compounds in fruits and have diverse biological functions and actions. Provitamin A activity carotenoids and xantophylls are known to provide protection against macular degeneration. They also are potent antioxidant and free-radical scavengers and modulate the pathogenesis of cancers and coronary heart disease (Torregrosa et al., 2005).

Studies conducted by de Ancos et al. (2002) and Sánchez-Moreno et al. (2003a) showed an increase in the total carotenoid content by 23 and 43% after a pressure treatment at 100 and 350 MPa, respectively. Regarding the individual carotenoids, β-carotene increased by 50%, a-carotene by 60%, α-cryptoxanthin by 42% and β-cryptoxanthin by 63% after 350 MPa for 5 min at 30 °C. Differences in the oxygen and hydrocarbon carotenoids in the intracellular locations of juice vesicles could lead to variability in the release of carotenoids after HHP treatment. The higher carotenoid content could be explained by the release of carotenoids from the food matrix (orange cloud) after denaturation of protein-carotenoid complexes induced by pressure. The carotenoids extraction was pressure-dependent and increased at higher pressure levels. These changes could increase the amount of antioxidant carotenoids available, improving the bioavailability and absorption in HHP treated juices. The effect of treatment time was checked at 350 MPa at 30 °C. The carotenoids content was increased by 43% after 5 min, but increasing the treatment time to 15 min did not show any further improvement. The effect of temperature (30 and 60 °C) was studied at 100 MPa. Results showed that an increase in the temperature did not exhibit any improvement in the carotenoid content. The carotenoid degradation is mainly due to oxidation and geometric isomerization. The isomerization of carotenoids goes through covalent bonding rupture and it seems that pressure does not significantly affect covalent bonding. During the refrigerated storage of OJ after different treatments, no losses were found after 15 d at 4 °C. At the end of storage (30 d) losses in the 50 MPa and control samples were 17 and 42%, respectively, whereas the 350 MPa sample had 72% higher content than the fresh one. Long treatment times increased carotenoid content after storage (30 d) with 68, 72 and 100% increasing after 2.5, 5 and 15 min, respectively. The sample at the higher treatment temperature (60 °C) showed higher losses during the storage (28%) with respect to the sample at 30 °C. Vit-A carotenoid content increased (52 and 45%) after treatments at 100 and 300 MPa. Neither increasing the time nor temperature increased the carotenoid content. During the storage, Vit-A carotenoid content increased in the 200 and 350 MPa samples (36 and 63%) but decreased in the control, 50 and 100 MPa samples (9, 42 and 24%). It seemed that at lower pressure, Vit-A carotenoid losses were higher possibly due to the residual POD activity that survived the lower pressure treatments.

Flavonoids belong to a group of natural substances with variable phenolic structures and are found in different quantities in fruit juices. They possess anti-inflamatory, antiallergic, anti-viral, hypocholesterolemic, and anticarcinogenic properties (Sánchez-Moreno et al., 2003a). Orange juice is a dietary source of flavonoids, mainly flavanones. (Sánchez-Moreno et al. 2003a, 2005) studied the effects of HHP treatment at different temperatures and thermal pasteurization (70 °C for 30 s and 90 °C for 1 min) on the main flavanones (hesperidin and naringenin) in OJ. Just after pressure treatments at 350 and 400 MPa, the naringenin content increased by 13 and 12%, respectively, and by 34 and 22%, respectively, for hesperidin content. They argued that some structural changes and permeabilization of cell walls of OJ sacs could release phenols from proteins and increase the extraction of flavanones. Pasteurization processes led to diminishing naringenin content (16.0%) but hesperidin content was unaffected. During the storage of pressure treated samples, the naringenin content increased around 11% for 350 and 400 MPa samples and no differences were observed between the 100 MPa samples and the control. Regarding hesperidin content, an increase was observed for 350 and 400 MPa samples (19 and 21%) with no differences among the 100 MPa sample and the control. The authors argued that the remaining activities of POD and PPO suggested for the degradation of polyphenols in the 100 MPa sample could explain the lower content of flavanones at that processing condition. Flavanones tend to precipitate at low pH from the soluble fraction to the cloud in OJ, leading to an increase in the proportion of flavanones in the cloud after processing. For that reason, a lower release of naringenin after processing during extraction could occur.

Folates are hematopoietic vitamins with special importance during pregnancy. Regarding the nutritional needs of humans, folate deficiency occurs frequently, probably due to poor diet selection and losses during food processing (Sauberlich et al., 1987). Fruits are an important source of folate. For example, a 200 g portion of fresh oranges contains nearly 50% of the recommended daily intake (Butz et al., 2004). Very little data has been published in relation to the effects of HHP treatment and folate stability. One study conducted by Butz et al. (2004) studied the stability of three main types of folates found in OJ (tetrahydrofolate, 5-methyltetrahydrofolate and 5-formyltetrahydrofolate) after HHP treatment (600 MPa at 25 and 80 °C). An orange juice model containing ascorbic acid was used to check its protective effect against pressure in the folates content. At 25 °C, the losses ranged from 10 to 40% and at 80 °C from 25 to 95% after a 24 min treatment. The pressure sensivity was as follows: 5-methylfolate > 5-formylfolate > tetrahydrofolate. To check the thermal effects on the folates content, the authors performed a thermal treatment at 80 °C, observing that heat alone decreases folates content by 20, 50 and 80% in 5-methylfolate, 5-formylfolate and tetrahydrofolate, respectively. Combinations of HHP and thermal treatment did not increase the losses in 5-methylfolate but increased the losses in 5-formylfolate and tetrahydrofolate by 30 and 20% respectively. This indicates that small molecules such as vitamins are stable to HHP treatment and do not undergo cleavage of covalent bonds, certain reactions are accelerated by pressure. This is the case with 5-formylfolate where the formation of a 5, 10-methenylfolate derivative is accelerated by pressure. When comparing the model juice with fresh squeezed orange juice, the authors observed that natural folates in OJ were more stable than in the model system after the high pressure treatment. As the model and fresh juice had the same L-AA, the authors suggested that the presence of other substances such as vitamin C and flavonoids in the natural juice protected folate.

Anthocyanins play an important role in the antioxidant and antiradical capacities of fruits. Twenty anthocyanins are known, but only six (pelargonidin, cyanidin, peonidin, delphinidin, petunidin and malvidin) are important in food (Zabetakis et al., 2000). They are known for their brilliant red and purple colors and products containing anthocyanins are more susceptible to color changes during processing and storage. The degradation of anthocyanins can be influenced by different parameters such as temperature, enzymes, oxygen, and sugar content.

Grape juice is known for its high content of polyphenolics and anthocyanins. Talcott et al. (2003) and del Pozo-Insfran et al. (2007) studied the effect of HHP (600 MPa for 15 min) and thermal (90 °C for 15 min) treatments on anthocyanin content of a grape juice. The fortification of L-AA is a common practice in the food industry to protect against oxidation, but its combination with anthocyanins may be mutually destructive in the presence of oxygen. Treatment at 400 MPa caused the highest loss (70%) of anthocyanin content due to the activation of PPO activity, whereas at 600 MPa or thermal treatment achieved low losses (3–5%). The presence of L-AA enhanced the losses (12.4 and 18.1%) after thermal and HHP treatments, respectively. In addition, the formation of hydrogen peroxides from the oxidation of L-AA may contribute to the degradation of anthocyanin. Furthermore, these peroxides may activate residual POD, which further degrades L-AA and anthocyanins. After 21 d of storage at 25 °C, the anthocyanins content were reduced by 28–34% for all the samples. By-products from the degradation of L-AA and/or monosaccharides, such as furfuraldehydes, could contribute to anthocyanin degradation during storage. The individual anthocyanins differed in their resistance to the HHP treatment. The anthocyanins containing ï-diphenolic groups were less stable to HHP processing, possibly because they were more susceptible to the enzymatic oxidation.

Strawberry juice is also a medium in which thermal treatment can damage the natural color by the degradation of anthocyanins, thus the use of HHP technology can be a challenge. Zabetakis et al. (2000) focused on the pelargonidin derivatives, 3-glucoside and 3-rutinoside, which are the main anthocyanins in strawberry. At 4 °C, the losses the of 3-glucoside derivative were similar for all pressure treatments (400–600 MPa at 22 °C) and the control sample lost 20% at the end of 9d of storage. However, at 400 MPa the losses were higher and reached 40% after 9 d. The behavior of 3-rutinoside was similar, with higher losses after 400 MPa (50% after 9d) and similar losses after 600 MPa and control sample (25%). However, at 200 and 800 MPa, the losses were lower (10%). At 20 °C, there were no differences in the losses of 3-glucoside of all samples at the end of storage (50%), whereas 3-rutinoside treated at 200 and 800 MPa had a higher anthocyanin content (25% losses). At 30 °C, there were no differences in 3-glucoside in all samples (70–80% losses after 9d), whereas no differences (60–80%) in 3-rutinoside were found except for 200 and 800 MPa with losses of 42%. The authors explained this behavior by the residual enzyme activity after the different pressure treatments. Three main enzymes are involved in the oxidation of the anthocyanins: POD, PPO, and β-glucosidase. These enzymes have lower activity at lower temperatures, thus the losses of antho-cyanins were lower at 4 °C in comparison with 20 and 30 °C. In addition, β-glucosidase seems to be activated at pressures around 400 MPa, which could explain the higher losses at that pressure. At higher pressures (800 MPa) the activity of these enzymes is irreversibly reduced, corresponding to lower losses in anthocyanin content. The differences in the degradation of both derivatives could be explained by the differences in substrate specificity of β-glucosidase which was higher for 3-glucoside than 3-rutinoside, and the breakdown would therefore be more extensive and losses higher for 3-glucoside.

Blackcurrants contain high levels of flavonoids with anthocyanins as the most important group. They are present in the skin of the berries and are responsible for the characteristic aroma and color. Kounaki et al. (2004) studied the effects of pressure (200–800 MPa for 15 min at room temperature) on two important anthocyanins in black currant, delphinidin-3-rutinoside and cyanidin-3-rutinoside during the storage at 5, 20 and 30 °C for 7 d. At 5 °C, the losses of anthocyanins were the lowest because they were mediated by enzymatic action (PPO activity) and the enzyme activity would be low at that temperature. Losses of delphinin-3-rutinoside and cyanidin-3-rutinoside contents reached 58 and 40%, respectively, after 7 d of storage at 4 °C. Treatment at 600 MPa seemed to retain higher levels of both anthocyanins. At 20 °C, the losses reached 58% for both anthocyanins. The sample treated at 200 MPa counted for the highest losses (55-60%). At 30 °C, the losses were around 70–75%, and the samples treated at 200 MPa had the lowest anthocyanin content with less than 20%. The authors found a relation between L-AA content and anthocyanin degradation, where the rapid loss of L-AA appeared to contribute to the lower rate of anthocyanin loss. Also, the higher loss after 200 MPa could respond to an oxidative enzyme activation.

Case study: HHP fruit juice processing in Australia

In the last few years, Australia has become a leader in the developing of HHP-treated juices and derivatives. The Australian Research organization (CSIRO) and their joint venture Food Research Group, Food Science Australia have been developing high pressure processing systems in Australia for over a decade. Several companies are using HHP technology in their processes.

A questionnaire was compiled and sent from the authors of the present chapter to the fruit juice manufacturing companies to obtain the relevant data about the industrial experience of using HHP technology. General trends were drawn from the questionnaire. Companies were small to medium in size (from 30 to 100 employees). Although most of companies have been in operation for over a decade, the introduction of HHP processing occurred more recently. In addition, thermal processing is still used in many of the companies for the production of vegetable and herb products, stabilized fruit preparations for yogurt, ice-creams (ripples) and fruit ices, diced chicken (cooked) and egg and mayonnaise salad for sandwiches.

The main reasons reported by companies for choosing HHP technology were their efforts to provide a product with a clearly perceptible improvement and the ability to achieve a premium price for a premium product. Companies added that HHP products are superior in taste, texture and color to current competitor products, and consumers have to be informed about these differences. In addition, the product must be value-added to be able to support the extra processing costs. Supply chains must be in place or developed for the delivery of high quality raw materials, to ensure that the products are microbiologically safe. Optimum market survival is improved by creating new products that have never been made before. Fresh processed juices, stabilized fruit preparations, chilled packaged salads, whole and value-added egg products and processed diced chicken products for sandwich bars, chilled cooking sauces and curry pastes and guacamole, pomegranate juices and fruit puraes are the main products processed by companies using the HHP technology.

When asked if sensory tests were performed, companies answered that small group sensory tests were conducted based on preference testing to confirm that the product flavor and color benefits could be translated to the consumer’s preference and purchase intent. In addition, changes in the product labeling were introduced after HHP processing, highlighting the benefits of HPP as a cold pasteurization process that produces the most natural, freshest tasting, and most nutritious juice on the market (Fig. 3.1).

The main clients of HHP fruit juice products were supermarket chains, and specialty and deli stores. No special legal approvals were required for the domestic products. Regarding consumer attitudes, no negative attitudes, apart from the higher price, were observed, and mostly there were difficulties explaining the technology and its benefits to the consumer without becoming too technical and either losing their interest or confusing them.

One of the problems mentioned by HHP fruit juice processors was to develop a PET bottle for the unit that was extremely space-efficient to allow for maximum utilization of the cylindrical processing cavity to maximize throughput. A clever bottle design with a local supplier solved this issue by creating an immediately differentiated and individual wedge design.

On average (due to differences in packaging size), the cost comparisons for the actual processing step was approximately 40% more expensive than traditional thermal pasteurization. The recovery of the investment was around 5–10 yr. The packaging materials used in the HHP process included: blow molded PET bottles, standup high barrier laminated spout pouches, high barrier laminated bag in box systems, and high barrier laminated preformed bags, which were thermally sealed.

Changes in the product formulation, such as the performance of hydrocolloid and polysaccharide stabilizers, post HPP treatment, meant that it was possible in some cases to actually reduce the amount of stabilizers required and still achieve the same viscosity, texture and mouthfeel.