Washing and sanitizing of raw fruits is required to remove pesticide residues, plant debris, and other possible contam- ination as well as microorganisms responsible for quality loss and decay. Fruit products undergo fermentative spoilage by lactic acid bacteria or yeasts, resulting in the production of acids, alcohol, and CO2. Fermentative species of yeasts such asKloeckeraandHanseniasporaoccur naturally on the surfaces of fruits and are capable of causing fermentative spoilage (Barnett et al. 2000).
Raw material is generally immersed in tap water, whereas sanitizing agents are added to process water to effectively reduce the microbial loads on the fruit surface. The use of chlorine at a concentration no greater than 200 ppm has been widely reported as an effective sanitation treatment of both whole and fresh-cut fruits (Lanciotti et al. 1999, Dong et al.
2000, Gorny et al. 2000, Bett et al. 2001, Soliva-Fortuny et al.
2002b). In melon and watermelon, sanitation of the whole fruit is usually achieved by using dips ranging from 50 to 1000 ppm of sodium hypochlorite (NaOCl; Qi et al. 1999, Portela and Cantwell 2001). The effectiveness of NaOCl on microbicidal activity is related to the concentration of san- itizer as well as pH and temperature. On the other hand, chlorine efficacy may be influenced by the type of produce and diversity of microorganisms that fruits contain (Beuchat 2003).
New sanitizing agents have been introduced in the past few years because of concerns about the products obtained when chlorine is decomposed by organic matter, resulting in the formation of potentially harmful substances, such as chloroform or other trihalomethanes, which are known or suspected of being carcinogenic. Therefore, the use of chlo- rine in the fresh-cut industries has been forbidden in some European countries such as Germany, The Netherlands, Den- mark, Switzerland, and Belgium (Carlin and Nguyen-the 1999, Betts and Everis 2005). As a consequence, several innovative approaches have been explored for the decontam- ination of minimally processed fruits and vegetables.
Other sanitizers such as hydrogen peroxide (H2O2), chlo- rine dioxide, peroxyacetic acid, and organic acids have been used for washing produce. Hydrogen peroxide demonstrates a broad-spectrum efficacy against virus, bacteria, yeasts, and bacterial spores, although it is less active against fungi than against bacteria (Block 1991). Its bacteriocidal effect is based on the production of hydroxyl free radicals (ãOH), which attack essential cell components, including lipids, proteins, and DNA (McDonnell and Russell 1999). The efficacy of H2O2washing has been demonstrated to be similar to that of NaOCl for extending the shelf life and reducing the native microbial and pathogen populations, includingEscherichia coli, of whole grapes, prunes, apples, oranges, melons, toma-
toes, and fresh-cut melons (Art´es et al. 2007). Sapers (1996) also showed that hydrogen peroxide vapor treatments were highly effective in reducing loads of microorganisms on whole prunes and table grapes. Hydrogen peroxide solu- tions used alone or combined with commercial sanitizing agents achieved more effectiveness in decontaminating ap- ples, which contained nonpathogenic strains ofE. coli, than by using chlorine or other commercial sanitizing agents for fruits or vegetables (Sapers et al. 1999). However, exposure to H2O2 vapor caused bleaching of anthocyanins in straw- berries and raspberries. This treatment could also be unfit for pome fruits due to the presence of residual contents in the product (Sapers and Simmons 1998). Although H2O2is permitted for other uses in food processing and packaging because it leaves no potentially harmful residues, it is not yet approved by the Food and Drug Administration (FDA) as a sanitizing agent for fresh produce (Art´es et al. 2007).
Chlorine dioxide (ClO2) is more effective than free chlo- rine against many classes of microorganisms at lower concen- trations. Its major advantages over NaOCl include reduced re- activity with organic matter and greater activity at neutral pH.
Chlorine dioxide has been shown to produce lower amounts of potentially carcinogenic chlorinated reaction products than chlorine (Tsai et al. 1995). There are very few reports about the use of ClO2in fresh-cut products. It has shown, though, that for apple, lettuce, strawberry, and cantaloupe, concen- trations of 3–5 mg/L are effective for inhibiting the main epiphytic microbiota as well as some inoculated foodborne bacteria such as E. coli and L. monocytogenes (Rodgers et al. 2004, L´opez-G´alvez et al. 2010). A main drawback is that it has to be generated on-site by reacting sodium chlorite and acid or chlorine. Currently, new technology al- lows for an easier production by systems where the reactants are packed together. ClO2is unstable and can be explosive when concentrations reach 10% or more in air (Betts and Everis 2005).
Several published studies have assessed the efficacy of different sanitizers againstE. coli O157:H7 on inoculated apples. Apples washed with 80 ppm of peroxyacetic acid re- duced the microbial loads by about 2 logs, and a 5% acetic acid wash reduced the load by about 3 logs when compared to water wash (Wright et al. 2000). On the other hand, 80g/mL of chlorine dioxide, 16 times the recommended concentra- tion, was needed to reduce the population ofE. coliO157:H7 by 2.5 logs (Wisniewsky et al. 2000). A dip containing 68 ppm of peroxyacetic acid reduced psychrotrophic counts on fresh-cut Galia melon by 2 log units and mesophilic counts by 1 log units in comparison with a 150 ppm NaOCl dip, which allowed to extend shelf life to 10 days at 5◦C (Silveira et al. 2007).
Ozone, UV light, and pulsed light could be other alter- natives to traditional sanitizing agents as these sanitizing processes are not only effective in destroying microorgan- isms, but they could also improve the safety of fruits because
of the lack of residues on produce. Fungal deterioration of blackberries and grapes was decreased by ozonation of the fruits (Beuchat 1992). Recent studies supported this work;
ozone exposure at 0.3 ppm inhibited the normal aerial growth of the mycelia and prevented sporulation on peach wounds inoculated withMonilinia fructicola,Botrytis cinerea,Mu- cor piriformis, andPenicillium expansumand stored for 4 weeks at 5◦C and 90% RH. Under 0.3 ppm ozone, gray mold, caused byB. cinerea, spread from the decayed fruit to ad- jacent healthy fruit among table grapes was also completely inhibited, when fruits were stored for 7 weeks at 5◦C (Palou et al. 2002). In citrus fruit, the exposure to ozone did not reduce final incidence of postharvest green mold, caused by Penicillium digitatum, and postharvest blue mold, caused by Penicillium italicum Wehmer, although infections developed more slowly on fruits stored in an ozonated atmosphere than on fruits stored in an ambient air atmosphere (Palou et al.
2001).
UV light could be effective as a minimal processing al- ternative for extending the shelf life of fresh-cut fruits. The effect of UV light (UV-C,=254 nm) may be based on its direct effect on pathogens because of DNA damage as well as its ability to simulate biological stress in plants and, consequently, by inducing resistance mechanisms in differ- ent fruits against pathogens. Actually, the exposure of melon slices to UV light decreased the concentrations of most of the aliphatic esters by over 60% of the amounts present in fresh-cut fruit and resulted in the production of terpenoid compounds in response to biological stress, particularly- ionone, which is capable of inhibiting the microbial growth in the fruit tissue (Lamikanra et al. 2002). UV at a wave- length of 253.7 nm was applied to apples inoculated withE.
coli O157:H7, achieving a log reduction of approximately 3.3 logs at 24 mW/cm2(Yaun et al. 2004).
Intense light pulses are an interesting decontamination method for food surfaces approved by the U.S. FDA that could be suitable for disinfecting fresh-cut fruit. Different microorganisms, includingAlternaria alternate,A. niger,B.
cinerea,Fusarium oxysporum,Fusarium roseum,Monilinia fructicola,Penicillium expansum,Penicillium digitatum, and Rhizopus stolonifer, were completely or partially killed af- ter exposure of fruit surfaces to PL (248 nm) treatments (Lagunas-Solar et al. 2006). These authors observed that the energy threshold that causes injury in fruits such as apples, oranges, lemons, peaches, raspberries, and table grapes was below 2 J/cm2. Maximum 4.3 and 2.9 log CFU/mL reduc- tions forSalmonellaandE. coliO157:H7, respectively, were achieved after treating blueberries with a PL treatment of 22.6 J/cm2 for 60 (Bialka and Demirci 2007). On raspber- ries and strawberries, maximum 3.9 and 2.1 log reductions ofE. coliO157:H7 were obtained after treatments of 72 and 25.7 J/cm2, respectively, while 3.4 and 2.8 log reductions of Salmonellawere observed at 59.2 and 34.2 J/cm2(Bialka and Demirci 2008).
Mechanical Operations
Mechanical operations during minimal processing damages fruit tissues, which in turn limits the shelf life of products.
Operations including peeling, coring, cutting, and/or slicing are responsible for such phenomena as microbial spoilage, desiccation, discoloration or browning, textural changes, and development of off-flavor or off-odor. During the preparatory steps of minimal processing, the natural protection of fruit (the peel) is generally removed, and hence, they become highly susceptible to microbial spoilage. During processing, the leakage of juices and sugars from damaged tissues allow the growth and fermentation of some species of yeasts such asS. cerevisiaeandS. exiguous(Heard 2002).
Damage on plant tissues may make them more susceptible to attack by pathogenic microorganisms and contamination with human pathogen. Cross-contamination may occur dur- ing cutting and shredding operations because sanitation in raw fruits may have not been carried out properly (Garg et al.
1990). The whole fresh fruits with bacterial soft rot and fun- gal rot were shown to have a high incidence of contamination withSalmonellaspp. (Wells and Butterfield 1997, 1999).
Although food safety is the most important consideration, color, texture, flavor, and nutritional values of the produce are equally important for acceptability by consumer. There- fore, the influence of cutting operations on quality should be taken into account. It is clear that turgor-pressure has a great incidence on the textural response, as it has been reported for minimal processed melon by Rojas et al. (2001). In ba- nanas, less ethylene production and lowest respiration rate were observed when 1-cm thick transverse cutting section was chosen (Abe et al. 1998). In apples or pears, the core and adjacent tissues should be removed during cutting operation because these parts of the fruit are susceptible to browning (Soliva-Fortuny et al. 2001).
Enzymatic browning is regarded as one of the most impor- tant problems related to color deterioration in fresh-cut fruit produce. Such phenomenon is caused by the discoloration of fruit by the action of enzyme polyphenol oxidase (PPO). This enzymatic reaction consists of the oxidation of phenolic sub- strates, found naturally in many fruits, too-quinones, which are highly reactive compounds and will react with (Whitaker and Lee 1995)
r Other quinone molecules r Other phenolic compounds
r Amino groups of proteins, peptides, and amino acids r Aromatic amines, thiol compounds, ascorbic acid (AA),
etc.
Browning phenomena are caused when, after mechanical operations during processing, enzymes, which are liberated from the tissues, come in contact with phenolic compounds.
However, several factors may contribute to the development of brown pigments due to enzymatic browning. The tendency
toward browning may be influenced by high concentration or types of phenolic compounds in fruits as well as high PPO activity (Garcia and Barrett 2002), ripeness stage, activity of oxidative enzymes, oxygen availability, and compartmental- ization of enzymes and substrates (Nicoli et al. 1994, Rocha et al. 1998). According to Soliva-Fortuny et al. (2002b), in mature apples, the chloroplast begins to disintegrate, causing a solubilization of PPO, which would increase the oversensi- tivity of browning. In pears, browning is related to phenolic and PPO compositions, whose contents may vary according to cultivar, stage of maturity, and postharvest storage con- ditions (Amiot et al. 1992). It was found that, in pear fruits of different varieties, the susceptibility to browning and the phenolic content were not greatly different, although a signif- icant decrease in the phenolic content occurred with delayed harvest times (Amiot et al. 1995). Reduced rates of enzy- matic browning in pears may be related to low levels of PPO (Soliva-Fortuny et al. 2002b).
It has been shown that pectinolytic and proteolytic en- zymes may be responsible for softening when they are exuding from bruised cells during slicing operations. These enzymatic mechanisms not only play a significant role in the softening process but also affect morphology, cell wall- middle lamella structure, cell turgor, water content, and biochemical components (Harker et al. 1997). Peeling and cutting also results in high rates of moisture loss from cut surfaces as it was reported in pears by Gorny et al. (2000).
Increased rates of water loss lead to wilting and/or shriveling, limiting factors of quality in fresh-cut produce (Toivonen and DeEllm 2002).
Low temperatures minimize the effects of mechanical in- juries because they are able to reduce enzymatic activity, metabolic reactions, and microbial growth. Processing is per- formed at around 10–15◦C, and washing water is generally refrigerated (Ahvenainen 1996). Rinsing the peeled and/or cut product in cold water is suggested to keep products in a suitable range of temperature or for removing cellular exu- dates released during mechanical operations.
Dipping Treatments
Dipping treatments after peeling and/or cutting reduce mi- crobial loads and rinse tissue fluids, thus reducing enzymatic oxidation during storage and the growth of microorganisms.
Because of low pH values of most fruits, the main typical flora consists of molds and yeasts. Both fungi and yeasts are responsible for the production of a wide range of enzymes.
Among these, pectic enzymes should to be taken into account because of their role in the degradation process of plant poly- mers. B. cinerea and Aspergillus niger were found to be important fungi on fruits as well as yeasts such asCanidia, Cryptococcus,Fabospora,Kluyveromyces,Pichia,Saccha- romyces, and Zygosaccharomyces (Chen 2002). Also, the ability of lactic acid bacteria to alter food flavor might con- tribute to the relatively rapid flavor loss in fresh-cut fruits.
In fact, the deterioration of fresh-cut cantaloupe stored at 20◦C was related to gram-positive bacteria and an increased production of lactic acid (Lamikanra et al. 2000). During the spoilage of fruits, gram-negative bacteria such as pseu- domonads are believed to degrade the fruit tissues through the production of pectic enzymes.
Consumption of fresh-cut fruits are associated with food- borne disease due to some pathogenic bacteria such as Cyclospora cayetanensisin raspberries,Salmonellaspp. in precut watermelons, andShigellaspp. in fruit salad, among others (Heard 2002). In general, pathogens may often be able to grow on some fruit surfaces such as melon, watermelon, papaya, or avocado because of the high pH value of the fruits.
For example,Shigellaspecies can survive on sliced fruits, in- cluding watermelon and raw papaya (Escart´ın et al. 1989). A recent study suggests that, after contamination,Campylobac- ter jejuni, a common cause of foodborne bacterial gastroen- teritis in developed countries worldwide, may continue to survive on cantaloupe pieces and strawberries (K¨arenlampi and H¨anninen 2004).E. coliO157:H7 can grow within dam- aged or wounded apple tissues (Dingman 2000). The ability ofE. coliO157:H7 to grow in the moderate pH of a bruise will likely predispose the bacterium for survival in a fresh-cut fruit. Therefore, the use of damaged fruits will increase the risk for contamination of fresh-cut products.
Proliferation of microorganisms on the surface of fresh-cut fruit is currently retarded or inhibited by using low storage temperature, MA packaging, and antimicrobial substances (Rojas-Gra¨u and Mart´ın-Belloso 2008). Either spraying of antimicrobial agents throughout the cut surface or dipping into antimicrobial solutions is widely practiced to prevent microbial growth (Oms-Oliu et al. 2010). Organic acids are usually applied as a dip. Citric acid has been widely used as an effective preservative because it is able to reduce the pH of cut fruits such as orange (Pao and Petracek 1997), apple (Rocha et al. 1998), peach, apricot, kiwifruit (Senesi and Pastine 1996), avocado (Dorantes et al. 1998), and bananas (Moline et al. 1999). However, there is a growing demand for natural food, where the use of chemical additives is reduced or elimi- nated. Hence, the use of antimicrobial agents from plants and plant products can represent a natural alternative to food addi- tives. These substances, generally regarded as safe (GRAS), are able to inhibit microorganisms (Utama et al. 2002). Some natural constituents, such as hexanal, hexanol, 2-(E)-hexenal, and 3-(Z)-hexenol, responsible for the aroma of some fruits and vegetables provide protective action towards microbial proliferation in wounded areas (Gardini et al. 2002). The ef- fectiveness of hexanal in improving quality of minimally pro- cessed apples is based on its antimicrobial activity, its ability to delay color deterioration of slices, and its interconversion to volatile compounds, giving an enhancement of aromatic properties. The formation of volatile compounds such as hex- anol and hexyl acetate may be beneficial as they are regarded as inhibitors of the PPO (Valero et al. 1990). Hexanal inhib- ited mesophilic bacteria at 4◦C and considerably prolonged
the lag phase of psychrotrophic bacteria. Its presence also significantly inhibited, at abuse temperatures, the growth of molds, yeasts, mesophilic, and psychrotrophic bacteria (Lan- ciotti et al. 1999). Hexanal, 2-(E)-hexenal, as well as hexyl acetate are also capable of inhibiting some pathogenic bacte- ria. In fresh apple slices, their addition at levels of 150, 150, and 20 ppm for hexanal, hexyl acetate, and 2-(E)-hexenal, respectively, may have a bactericidal effect onL. monocyto- genesand caused a significant extension of lag phases ofE.
coliandS. enteritidisinoculated at levels of 104–105 cfu/g (Lanciotti et al. 2003). In addition, the antimicrobial activities of hexanal, 2-(E)-hexenal, and hexyl acetate are positively af- fected by a rise in temperature, since their action is dependent on vapor pressure (Lanciotti et al. 1999). The antimicrobial action of essential oil (EO) constituents seems to be related to their solubility in the microbial membrane (Karatzas et al.
2000), their partition in the cytoplasmatic microbial mem- branes (Juven et al. 1994), or the perturbation of membrane permeability (Tassou et al. 2000). While in vitro antimicro- bial activity has been well demonstrated, there are a limited number of studies reporting the use of EOs to inhibit micro- bial growth on foods. The difficulties of the application to foods underlies in their limited solubility and the impact of these substances on the organoleptic food properties, variabil- ity of their composition, and their variable activity in foods due to interactions with food components (Gutierrez et al.
2008). Citrus EOs may be compatible with the organoleptic characteristics of minimally processed fruits. Lanciotti et al.
(2004) also reported that the addition of 0.02% (v/v) citrus, mandarin, cider, lemon, and lime EOs to a minimally pro- cessed fruit mix inhibited the proliferation of the naturally occurring microbiota and reduced the growth rate of inocu- latedS. cerevisiaepopulations, thus extending the shelf life of the fruit salad without affecting its sensory properties. In this way, Ngarmsak et al. (2006) applied a vanillin dip (0.12%
w/v) to delay the development of total aerobic bacteria and yeast and mold populations of fresh-cut mangoes stored at 5◦C and 10◦C for up to 14 and 7 days, respectively. A dip of vanillin (0.18% w/v) inhibited 37% and 66% of the microbial growth on “Empire” and “Crispin” apple slices, respectively, after 19 days of storage (Rupasinghe et al. 2006).
The addition of chemical agents is the most common way to control browning phenomenon. They can either affect the enzyme or their substrates. AA has been generally used as anti-browning agent. This reducing agent indirectly inacti- vates the PPO enzyme by degrading the free radical of the histidine molecule at the active site and by reducing the co- factor Cu++to Cu+, thereby causing the cuprous ion to dis- sociate more readily from the enzyme (Osuga and Whitaker 1995). AA is able to prevent the browning caused by PPO re- ducing quinones back to phenolic compounds before they un- dergo further reaction to form brown-colored pigments. The anti-browning effects of AA have been widely demonstrated in several fresh-cut fruits under a wide range of conditions (Dorantes et al. 1998, Rocha et al. 1998, Agar et al. 1999,
Buta et al. 1999, Gorny et al. 1999, Senesi et al. 1999, Soliva- Fortuny et al. 2001, Soliva-Fortuny et al. 2002a). However, this treatment may not be completely effective to control enzymatic browning of fresh-cut fruits, since once the AA is completely oxidized to dehydroascorbic acid,o-quinones are no longer reduced and darkening occurs (Nicolas et al.
1994). In addition, recent works have demonstrated that AA may cause important oxidative damage in fresh-cut “Fuji”
apples (Larrigaudi`ere et al. 2008).
Other anti-browning agents include thiol-containing com- pounds such as cysteine, glutathione, andN-acetylcysteine, which are thought to form colorless thiol-conjugated o- quinones (He and Luo 2007). Dips in aqueous solu- tions containing sulfur-containing amino acids such as N- acetylcysteine and/or glutathione at concentrations around 0.75% have been shown to inhibit browning of fresh-cut pears and apples (Oms-Oliu et al. 2006, Rojas-Gra¨u et al.
2006).
Acidulants, such as citric acid, are effective in prevent- ing the fresh-cut produce from browning due to its dual effect on PPO enzyme by chelating copper and its action as an acidulant (Sapers 1993). Optimum PPO activity is ob- served at pH 6.0–6.5, while little activity is detected below pH 4.5 (Whitaker 1994). However, acidulants are not often used alone because it is difficult to achieve efficient browning in- hibition. Nevertheless, the acid combination with a chemical reductant may show a major effect. According to Pizzocaro et al. (1993), above 90% inhibition of PPO in apple cubes was reported by using a mixture of 1%AA+0.2% citric acid or 1%AA+0.5% sodium chloride. Effects of citric acid and/or AA dips were not effective in controlling the browning of pear slices but there was an improvement in color by adding 1% CaCl2and storage at 2.5◦C for 1 week (Rosen and Kader 1989). Other carboxylic acids such as oxalic and oxalacetic acid showed higher anti-browning activity than citric acid on fresh-cut apples (Son et al. 2001). Immersion of banana and apple slices in oxalic acid solutions was effective against browning (Son et al. 2001, Yoruk et al. 2004).
Among several resorcinol derivatives, 4-hexylresorcinol (4-HR) has proved to be effective in controlling browning on fresh-cut apples and pears (Monsalve-Gonz´alez et al. 1993, Dong et al. 2000, Son et al. 2001, Rojas-Gra¨u et al. 2006).
These latter authors stated that 4-HR concentrations lower than 0.5% were effective in preventing browning of fresh-cut Fuji apples for 14 days at 4◦C. The inhibitory action of 4-HR is based on its interaction with PPO, which compromises the ability of the enzyme to catalyze the reaction. Its applicability on fresh-cut fruit has been proven, especially when used in combination with reducing agents (Monsalve-Gonz´alez et al.
1993, Luo and Barbosa-Canovas 1997, Dong et al. 2000, Arias et al. 2008).
Some blends of additives have proved to extend the storage life of fresh-cut produce. A mixture of 0.001 M 4-HR+0.5 M isoascorbic acid+0.05 M calcium propionate+0.025 M homocysteine maintained the freshness of apple slices