PROCESSING - Handbook of Fruits and Fruit Processing

Washing and Sanitizing Raw Materials for Fresh-cut Fruit Products

Washing and sanitizing of raw fruits is required to remove pesticide residues, plant debris, and other possible contamination 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, alco- hol, and CO2. Fermentative species of yeasts such as KloeckeraandHanseniasporaoccur 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; Gorny et al., 2000; Dong et al., 2000, Bett et al., 2001; Soliva- Fortuny et al., 2002b). In melon and watermelon, the 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 mi- crobicidal activity is related to the concentration of the sanitizer 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, 2000).

New sanitizing agents have been introduced in the past few years because of concern about the products obtained when chlorine is decomposed by or- ganic matter, resulting in the formation of potentially harmful substances.

Other sanitizers such as hydrogen peroxide (H2O2), chlorine dioxide, peroxyacetic acid, and or- ganic acids have been used for washing produce.

Hydrogen peroxide (H2O2) demonstrates a broad- spectrum efﬁcacy against virus, bacteria, yeasts, and

bacterial spores, although it is less active against fungi than bacteria (Block, 1991). Its bacteriocidal effect is based on the production of hydroxyl free rad- icals (OH), which attack essential cell components, including lipids, proteins, and DNA (McDonnell and Russell, 1999). Thus, this treatment was recommended for fresh-cut melon and analogous fruits because it extended the shelf life by 4–5 days in comparison with the chlorine treatment (Sapers and Simmons, 1998). 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 in combination with commercial sanitizing agents achieved more effectiveness in decontaminating apples, which contained non- pathogenic strains ofEscherichia coli, than by using chlorine or other commercial sanitizing agents for fruits or vegetables (Sapers et al., 1999). However, exposure to H2O2vapor caused bleaching of antho- cyanins in strawberries 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).

Several published studies have assessed the efﬁ- cacy of different sanitizers againstE. coliO157:H7 on inoculated apples. Apples washed with 80 ppm of peroxyacetic acid reduced 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, 80␮g/ml of chlorine dioxide, 16 times the recommended concentration, was needed to reduce the population of E. coli O157:H7 by 2.5 logs (Wisniewsky et al., 2000).

Ozone and UV light could be other alternatives to traditional sanitizing agents as these sanitizing pro- cesses are not only effective in destroying microorganisms 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,Botry- tis cinerea, Mucor piriformis, and Penicillium ex- pansum and stored for 4 weeks at 5^◦C and 90%

RH. Under 0.3 ppm ozone, gray mold, caused by B. cinerea, spread from the decayed fruit to adjacent healthy fruit among table grapes was also com- pletely inhibited, when fruits were stored for 7 weeks

8 Fresh-Cut Fruits 133 at 5^◦C (Palou et al., 2002). In citrus fruit, the expo-

sure to ozone did not reduce the ﬁnal incidence of postharvest green mold, caused byPenicillium dig- itatum, and postharvest blue mold, caused byPeni- cillium 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 also be effective as a minimal processing alternative for extending the shelf life of fresh-cut fruits. The effect of UV light (UVC,

␭=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 con- sequently, by inducing resistance mechanisms in different 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 re- sulted 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 light at a wavelength of 253.7 nm (UVC) was applied to apples inoculated withE. coliO157:H7, achieving a log reduction of approximately 3.3 logs at 24 mW/cm² (Yaun et al., 2004).

Mechanical Operations

Mechanical operations during minimal processing damages fruit tissues, which in turn limits the shelf life of products. Operations including peeling, cor- ing, 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 asSac- charomyces cerevisiaeandSaccharomyces exiguous (Heard, 2002).

Damage on plant tissues may make them more susceptible to attack by pathogenic microorganisms and contamination with human pathogens. Cross contamination may occur during cutting and shredding operations because sanitation in raw fruits may not have been carried out properly (Garg et al., 1990). The whole fresh fruits with bacterial soft rot and fungal

rot were shown to have a high incidence of contamination withSalmonellaspp. (Wells and Butterﬁeld, 1997, 1999).

Although safety is the most important attribute to be taken care in food, color, texture, ﬂavor, and nu- tritional value are also the primary limiting factors in determining the product’s acceptability by the consumer. Therefore, the inﬂuence of cutting operations on quality should be taken into account. It is clear that turgor pressure has a great effect on the textural response, as it has been reported for minimally processed melon by Rojas et al. (2001). In bananas, less ethylene production and lowest respiration rates were observed when a 1-cm thick transverse cutting sec- tion was chosen (Abe et al., 1998). In apples or pears, the core and adjacent tissues should be removed during cutting operations because these parts are susceptible to browning (Soliva-Fortuny et al., 2001).

Enzymatic browning is regarded as one of the most important problems related to color deterioration in fresh-cut fruit produce. Such phenomenon is caused by the discoloration of fruit by the action of a group of enzymes called polyphenol oxidases (PPOs). This enzymatic reaction consists of the oxidation of phenolic substrates, found naturally in many fruits, to o-quinones, which is highly reactive and will react with (Whitaker and Lee, 1995):

rother quinone molecules rother phenolic compounds

rthe amino group of proteins, peptides, and amino acids

raromatic 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 compartmentalization of enzymes and substrates (Nicoli et al., 1994; Rocha, 1998). Ac- cording to Soliva-Fortuny (2002b), in mature apples, the chloroplast begins to disintegrate, causing a sol- ubilization of PPOs, which would increase the over- sensitivity 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 conditions (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 signiﬁcant de- crease in the phenolic content occurred with delayed harvest times (Amiot et al., 1995). Reduced rates of enzymatic browning in pears may be related to low levels of PPO (Soliva-Fortuny et al., 2002b).

It has been shown that the pectinolytic and pro- teolytic enzymes may be responsible for softening when they are exuding from bruised cells during slicing operations. Not only do these enzymatic mechanisms play a signiﬁcant role in the softening process but also affect the morphology, cell wall–middle lamella structure, cell turgor, water content, and bio- chemical components (Harker et al., 1997). Peeling and cutting also result in high rates of moisture loss from cut surfaces as 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 (Toivonene and DeEll, 2002).

Low temperatures minimize the effects of mechanical injuries because they are able to reduce enzymatic activity, metabolic reactions, and microbial growth. Processing is performed 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 exudates released during mechanical operations.

Dipping Treatments

Dipping treatments after peeling and/or cutting reduce microbial loads and rinse tissue ﬂuids, thus reducing enzymatic oxidation during storage and the growth of microorganisms.

Due to the low pH values of most fruits, the main typical ﬂora consists of moulds and yeasts.

Both fungi and yeasts are responsible for the production of a wide range of enzymes. Among these, pectic enzymes should be taken into account because of their role in the degradation process of plant polymers. B. cinerea and Aspergillus niger were found to be important fungi on fruits as well as yeasts such asCanidia,Cryptococcus,Fa- bospora, Kluyveromyces, Pichia, Saccharomyces, andZygosaccharomyces(Chen, 2002). Also, the ability of lactic acid bacteria to alter the food ﬂavor might contribute 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 pseudomon- ads are believed to degrade the fruit tissues by the production of pectic enzymes.

Consumption of fresh-cut fruits is associated with foodborne disease due to some pathogenic bacteria such asCyclospora cayetanensisin raspberries, Salmonellaspp. in precut watermelons, andShigella spp. 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, including watermelon and raw papaya (Escart´ın et al., 1989). A recent study suggests that, after contamination,Campylobacter jejuni, a common cause of foodborne bacterial gastroenteritis in developed countries worldwide, may continue to survive on cantaloupe pieces and strawberries (K¨arenlampi and H¨anninen, 2004).Escherichia coli O157:H7 can grow within damaged or wounded apple tissues (Dingman, 2000).

The ability ofE. coliO157:H7 to grow in the moder- ate 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 in comparison to surface contamination of whole apples.

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 consumer demand for more natural food where the use of chemical additives is reduced or eliminated.

Hence, the use of antimicrobial agents from plants and plant products can represent a natural alternative to food additives. These substances, generally regarded as safe (GRAS), are able to inhibit microorganisms and determine ﬂavor and quality because of the presence of some volatile compounds (Utama et al., 2002). Some natural constituents, such as hexanal, hexanol, 2-(E)-hexenal, and 3-(Z)-hexenol, responsible for the aroma of some vegetables and fruits, provide protective action against microbial prolifer- ation in wounded areas (Gardini et al., 2002). The effectiveness of hexanal in improving the quality of minimally processed apples is based on its antimicrobial activity, its ability to delay color deterioration of slices, and its interconversion to volatile compounds

8 Fresh-Cut Fruits 135 giving an enhancement of aromatic properties. The

formation of volatile compounds such as hexanol and hexyl acetate may be beneficial as they are regarded as inhibitors of the polyphenol oxidase (Valero et al., 1990). Hexanal totally inhibited mesophilic bacteria at 4^◦C and considerably prolonged the lag phase of psychrotrophic bacteria. Its presence also signifi- cantly inhibited, at abuse temperatures, the growth of moulds, yeasts, mesophilic, and psychrotrophic bacteria (Lanciotti et al., 1999). Hexanal, 2-(E)-hexenal, as well as hexyl acetate are also capable of inhibiting some pathogenic bacteria. 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. monocytogenes, and caused a significant extension of lag phase ofE. coli andS. enteritidisinoculated at levels of 10⁴–10⁵cfu/g (Lanciotti et al., 2003) In addition, the antimicrobial activities of hexanal, 2-(E)-hexenal, and hexyl acetate are positively affected by a rise in temperature, since their action is dependent on vapor pressure (Lanciotti et al., 1999). The antimicrobial action of essential oil constituents seems to be related to their solubility in the microbial membrane (Karatzas et al., 2000), their partition in the cytoplasmatic microbial membranes (Juven et al., 1994) or the perturbation of membrane permeability (Tassou et al., 2000). Fruit essential oils may either reduce the growth ofS. cerevisiaeinocu- lated at levels of 10²cfu/ml or increase the death rate ofE. coliinoculated at levels of 10⁶cfu/ml, under temperature abuse conditions. Citrus essential oils may be compatible with the organoleptic characteristics of minimally processed fruit. Thus, some research carried out by Lanciotti et al. (1999) suggested the addition of citrus, mandarin, cider, lemon, and lime essential oils to fresh sliced fruit mixtures (apple, pear, grape, peach, and kiwifruit) to inhibit the prolif- eration of naturally occurring microbial populations.

In fact, citrus oxygenated monoterpenes have been reported as molecules with the highest antifungal activity, and citral as the most active compound against P. digitatumandP. italicum(Caccioni et al., 1995).

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 an antibrowning agent. This reducing agent indirectly inactivates the PPO enzyme by degrading the free radical of the histidine molecule at the active site and by reducing the cofactor Cu²⁺ to Cu⁺, thereby causing the cuprous ion to dissociate more readily from the enzyme (Osuga and Whitaker, 1995). AA is able to prevent the browning caused

by PPO reducing quinones back to phenolic compounds before they undergo further reaction to form brown-colored pigments. The antibrowning effects of AA have been widely demonstrated in several fresh- cut fruits under a wide range of conditions (Agar et al., 1999; Gorny et al., 1999; Rocha et al., 1998;

Dorantes et al., 1998; Soliva-Fortuny et al., 2002b;

Senesi et al., 1999; Buta et al., 1999; Soliva-Fortuny et al., 2001). Cysteine as a reducing agent is also capable of inhibiting enzymatic browning, although the amount required is often incompatible with product taste (Richard-Forget et al., 1992). However, among a wide variety of antibrowning compounds, Dorantes et al. (1998) chose cysteine as the best inhibitor of browning in minimally processed avocado slices.

Acidulants, such as citric acid, are effective in pre- venting 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). Opti- mum PPO activity is observed at pH 6.0–6.5, while little activity is detected below pH 4.5 (Whitaker, 1994). Sodium chloride, as potassium chloride, is known to control browning when they are used at pH<3.5 (Rouet-Mayer and Philippon, 1986). Cal- cium chloride may confer undesirable bitterness to the product when it is used at concentrations in excess of 1% (Perera and Baldwin, 2001). However, acidulants are not often used alone because it is difficult to achieve efficient browning inhibition. Neverthe- less, the acid combination with a chemical reductant may show a major effect. According to Pizzocaro et al. (1993), above 90% inhibition of PPO in apples 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 an improvement in color was shown by adding 1% CaCl2and storage under 2.5^◦C for 1 week, rather than water-treated control slices (Rosen and Kader, 1989). Sapers and Miller (1992) suggested that PPO inhibition could be due to the firming action of cal- cium, which reduces the leakage of PPO and its substrates at the exposed cut surfaces. Gorny et al.

(1998a) also reported the effectiveness of a dip for 1 min in 1.0% CaCl2 +2% AA, in reducing pear slice surface browning.

4-Hexylresorcinol (4-HR) inhibitory action is based on its interaction with PPO, which compro- mises the ability of the enzyme to catalyze the reaction. Luo and Barbosa-C´anovas (1996) showed a synergistic effect in browning inhibition using 0.01%

4-HR and 0.5% AA in combination. Thus, not only

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