CHAPTER 2

LITERATURE REVIEW

2.1

Lettuce

Lettuce (Lactuca sativa L.) is a leafy vegetable from Asteraceae family. It is mainly cultivated in temperate region countries such as England, France, the Netherlands and Western Europe (Vries, 1997) and also in some tropical regions. Lettuce is the only member of the Lactuca genus grown commercially (Koike et al., 2007). The Food and Agriculture Organization of the United Nations (FAO, 2010) reported that world production of lettuce and chicory for year 2010 was 23,622,366 metric tons. These are primarily from China (53%), the United States (17%) and India (4%). Although China is the top world producer of lettuce, majority of the crop is consumed domestically. Jore L., (2012) revised that Spain is the world's largest exporter of lettuce with US ranked second.

In Malaysia, it was reported that lettuce production in 2009 to 2010 increased from 19, 662 to 38, 597 metric tons, while the acreage also increased from 1,182 to 2, 286 ha (DOA, 2010). In Tenth Malaysia Plan, (RMK-10), for years 2011 to 2015, the lettuce production is expected to increase from 10, 430 to 16, 362 metric tons and the planting area also increased from 883 ha to 1, 246 ha. Also, consumers demand for lettuce is on the increase in Malaysia.

There are several types of lettuce, but most common are the leaf, head and cos or romaine lettuce (Katz and Weaver, 2003). Butterhead lettuce, a head type lettuce, is one of the most popular varieties in Western Europe, where it accounts for about 80% of lettuce consumption. In North America, this lettuce is gaining popularity and also is known as Boston or Bibb lettuce (Davey et al., 2007).

Butterhead lettuce, unlike other types of lettuce such as Iceberg, Romaine, or leaf, forms open heads with softer leaves and has much smoother and delicate texture (Bradley et al., 2010). Plants have a height and spread of 6 to15 to 30 cm. Elzebroek and Wind

(2008) reported that lettuce contains 96% water, 2% protein, 1% carbohydrate and 0.5% fiber. Lettuce is a good source of vitamin A and potassium, with higher concentrations of vitamin A found in darker green lettuces.

Vegetable production and marketing have received increasing attention with regard to quality and safety of produce (Midmore and Jansen, 2003; Kader, 2005; Hewett, 2006). In plastic bags, wounded lettuce leaves produce ethylene, which may compromise the product quality (Hodges et al., 2008). Although temperatures close to 0 C are

recommended, fresh vegetables may be prepared, shipped and stored at 5 C and sometimes 10 C (Watada et al., 1996). The high water content (94.9%) of lettuce creates a problem when attempting to preserve the plant because they are highly susceptible to water loss and mechanical damage during storage and transportation. Butterhead lettuce damaged much easily due to its exceptionally tender leaves. Crushing and bruising caused about 28% postharvest losses of fresh harvested Butterhead lettuce (Boonyakiat, 1999).

2.2

Food Safety

In recent years, there had been an increased focus on food safety all over the world. Public food safety standards enforced through legislation and firms, at different levels of the supply chain, have developed various private standards (Hammoudi et al., 2009). Legislations adopted to improve food safety include standards regarding the characteristics of the final product, production practices in supply chain, traceability within the supply chain and legal liability of the supply chain.

Food safety is defined as the assurance that the food will not caused harm to the consumer when it is prepared and/or eaten according to its intended use (FAO/WHO, 1997). Food safety hazard is a biological, chemical, or physical agent that is reasonably likely to cause illness or injury in the absence of its control. A biological hazard can be defined as probability of step of food a biological if agent contaminating food during any ingesthe consumeronsumer can cause a

production, which

health disorder (Luning et al., 2006). Every living organism that can colonize foods and in some cases grow or survive in the food matrix, or any structure that needs the participation of a living being to reproduce itself or produce toxic metabolites, can be considered as a biological agent.

Biological hazards seem to have a more notorious impact on public opinion than physical or chemical risks, probably because biological risks are more frequently reported, and normally affect to a certain extent a large number of consumers (Luning et

al., 2006). In addition, biological hazards induce acute symptoms, which make them noticeable by the consumers. Bacteria is the most well-known biological hazards.

Microbial food safety is the way to prevent and manage the microbiological hazards that affect human being (Food, 2006). The factors that contaminate in the field are soil, insects, animals, irrigation water, and workers. During harvesting stage, the

contamination is due to workers, harvesting tools, crates and transport vehicles (Sinha et al., 2010). The microbial contamination problem also came from processing, The to

transportation, distribution, preparation and retail display (Gorny, 2006). governments all over the world are a concern about these problems and trying overcome the

increasing number of foodborne illnesses by improving food safety

management (Codex Alimentarius Commission, 2003).

Chemical hazard may cause adverse reactions to chemicals present in food and can be classified as food intoxications or food sensitiveness (Luning et al., 2006). Typically, every population is vulnerable to food intoxications, as long as the exposure is high enough. Chemicals provoking food intoxications can be classified as food additives, residues, contaminants and endogenous substances (De Meulenaer, 2006).

2.2.1

Nitrate and Nitrite

Nitrate is a chemical hazard, present naturally in soils, water and plant materials as a result of nitrogen fixation. More than 80-90% of the nitrogen absorbed by plants is in the form of nitrate. It is one of the consequences of the mechanisms by which plant absorb

nitrogen element from fertilizers or organic materials which are essential to the process of protein synthesis (Meah et al., 1994; Santamaria et al., 1999; Chung et al., 2003). Through the nitrogen cycle, plant received the nitrate from nitrogen fertilizer in two ways (Rutherford, 2010). Firstly, the nitrogen fertilizer will be converted into ammonium and then nitrified by bacteria to nitrate before uptake. Secondly, the plant absorbed the nitrate content after the nitrogen fertilizer converted it directly into nitrate.

Nitrate and nitrite occur widely in human food product both as intentional additives and undesirable contaminants (Fytianos and Zarogiannis, 1999). The presence of nitrates in vegetables, as in water and other food products, is a serious threat to human health, not so much due to their toxicity, which is low, but for the compounds they induced in the organism (Ikemoto et al., 2002; Ishiwata et al., 2002).

Fresh and processed vegetables, especially leafy vegetables, are major sources of dietary nitrate intake. Vegetables contribute up to 80-92% of the average daily intake (Penttil, 1995; Dich et al., 1996; Muramoto, 1999; Eichholzer et al., 2003). Acceptable daily intake of 0-3.7 mg kg-1 body weight for nitrate and 0-0.06 mg kg-1 body weight for nitrite has been established by the EU Scientific Committee for Food (EU Scientific Committee, 1995). Santamaria (2006) reported that increased use of synthetic nitrogen fertilizers and livestock manure in agriculture activities has led to high nitrate concentrations in vegetables and drinking water. Nitrite concentrations are usually low in fresh vegetables, but under adverse postharvest storage conditions, nitrite levels can

increase due to bacterial or endogenous nitrate reductase reducing the nitrate to nitrite (Hunt and Turner, 1994; Huarte-Mendicoa, 1997).

2.2.2

Factors Affecting Nitrate and Nitrite Accumulation

In general, the factors affecting the nitrate and nitrite content in the plant can be divided into three groups, which were physiological, environmental and nutritional (Anjana and Iqbal, 2007). The physiological factors include genotypic variability, plant parts and diurnal effects. The nitrate content varies markedly with plant species, cultivars of the same species, and even genotypes with different ploidy (Blom-Zandstra and Eenink, 1986; Siomos, 2000; Grzebelus and Baranski, 2001; Escobar-Gutierrez et al., 2002; Harada et al., 2003; Anjana et al., 2006). According to Harrison et al. (2004), the shoot nitrate content is genetically determined and likely to be controlled by several genes (QTLs).

In general, vegetables consumed with their roots, stems and leaves have a high nitrate accumulation, whereas those with fruits and melons as consumable parts have a low nitrate accumulation (Zhou et al., 2000). Nitrate content of various parts of a plant differs (Santamaria et al., 1999). Nitrate concentration in the petiole was more than double of those found in the lamina of rocket leaf (Elia et al., 2000) and as high as 6.6 folds in spinach (Anjana et al., 2006). Therefore, increasing the blade or petiole ratio of

spinach plants at harvest (Santamaria et al., 1999) and separation of those parts prior to processing or preparation may appreciably reduce the extent of nitrate consumption by humans. Vegetables that do not develop storage organs as foodstuff have a different pattern of nitrate accumulation where nitrate often continues accumulating with plant age (Maynard et al., 1976). Similarly, Anjana et al. (2006) showed that nitrate

concentration in spinach was high at later stages of plant growth. Therefore, optimum physiological age for harvesting needs to be standardized for different leafy vegetable crops. Since nitrate continues to accumulate in spinach plant till maturity, the extent of nitrate intake by humans through spinach leaves may be reduced by harvesting the crop during the vegetative stage (Anjana and Iqbal, 2007).

Nitrate accumulation in plants is affected by environmental factors such as light intensity, temperature, climate, and crop season (Zhou, 2000; Tosun and Ustun, 2004). Light intensity is correlated inversely to nitrate content of plants; therefore, diurnal changes in light intensity might cause a diurnal nitrate accumulation pattern. The

diurnal variation in nitrate content is kept low by selecting a proper harvest time. High light intensity reduces nitrate accumulation has been shown in several studies (BlomZandstra et al., 1988; van der Boon et al., 1990). Marked diurnal patterns showing a decrease of nitrate content during the day period followed by an increase during the night have also been demonstrated (Delhon et al., 1995a, 1995b; Cardenas-Navarro et al., 1998).

Effect of climate on nitrate accumulation was studied by Grzebelus and Baranski (2001) who found that nitrate content was lesser in the year that had a high rainfall. Plant

nitrate levels were influenced by weather conditions more significantly than by the form and application rates of fertilizers (Custic et al., 2003). Nitrate accumulation varies with season (Vieira et al., 1998), being higher in autumn-winter than in spring (Santamaria et al., 1999). Gruda (2005) has extensively reviewed the effect of different environmental factors on the nitrate content of greenhouse vegetables. The other factors affecting nitrate accumulation were harvesting period, amount and composition of fertilizers, (Zhou, 2000; Tuson and Ustun, 2004), soil texture and fertilizer type (Scaife et al., 1986; Gianquinto et al., 1992; Gunes et al., 1995). Fertilizing with nitrogen and light intensity has the greatest influence on the nitrate content in vegetables (Santamaria et al. 2001; Santamaria 2006).

The use of fertilizers based on ammonia or a mixture of nitrate and ammonium can reduce the nitrate content in plants (Inal and Tarakcioglu, 2001; Santamaria et al., 2001), It is possible to have a quality yield with low nitrate content by manipulating the nitrogen nutrition of plants (Izmailov, 2004). Proper application of nitrogenous,

phosphate, potassium fertilizers, and green and farmyard manures could materially reduce the nitrate accumulation in vegetables (Zhou et al., 2000). Nitrate concentration in plants can also be manipulated by stopping nitrogen supply for a few days before harvesting (Santamaria et al., 2001). Temme et al. (2010) stated that in general, the nitrate content is highest in early plant growth stages and decreases with maturity. Longer growth periods favor the reduction of nitrates in plant from time of harvesting.

2.2.3

Escherichia Coli O157:H7

E. coli strains are portions of the natural intestinal flora of human beings and other animals. Nevertheless, this species covers a range of virulent strains, characterized into several groups of pathogen, which may cause foodborne illnesses (Kaper et al.,

2004). The Institute of Food Technologists (2002) stated of all the E. coli strains, E. coli O157:H7 is possibly the most signify as an emerging pathogen. Cattle and other ruminants have been established as the major natural reservoirs for E. coli O157:H7 and play a significant role in the epidemiology of human infections (Rasmussen et al., 1993).

Clinical symptoms of the disease caused by E. coli O157:H7 range from haemorrhagic colitis, which is characterized by severe cramping (abdominal pain), and watery diarrhea (Peacock et al., 2001). In 2005, bagged salad containing romaine lettuce, red cabbage, and carrots, were linked to an outbreak of E. coli O157:H7 foodborne illness (Smith DeWaal and Bhuiya, 2007). They also found that spinach contaminated with E. coli O157:H7 was at the center of a large outbreak that resulted in 205 confirmed illnesses and three deaths in 2006.

2.2.4

Factors Affecting Escherichia Coli O157:H7 Contamination on Fresh

Produce

Demand for fresh fruit and vegetables had increased along with consumption of minimally processed foods (Bracket, 1992; DeRoever, 1998). Factors leading to the E. coli O157:H7 contaminations are numerous which include production field, transportation, packinghouses, storage, market outlets, and consumer abuse (Narciso and

Plotto, 2006). There were two main factors that influence bacterial contaminations, which were intrinsic and extrinsic factors.

Intrinsic factors include nutrients, microbial growth and moisture. All microorganisms require a source of nutrients for metabolism. Fermentative bacteria require carbohydrates, either simple sugars, such as glucose and fructose, or complex carbohydrates, such as starch or cellulose (Florkowski et al., 2009). Microbial growth is usually referred to as increase in the number of microbes, rather than a change in microbial mass. Recent studies have indicated that microbial flora associated with fresh produce exist as aggregates or biofilms (Fett, 2000; Rayner et al., 2004; Aruscavage et al., 2006; Brandl, 2006). Foodborne microbial Bacteria dominates in foods at high moisture content. pathogens tend to colonize leaf veins due to greater

wettability of the area and nutrient leaching (Brand and Mandrell, 2002). Plants have evolved barriers to limit microbial growth as part of their intrinsic defense. An important general defense mechanism is the production of phytoalexins (antimicrobial compounds) by many plants (Harborne, 1999).

The extrinsic factors were more environmental factors such as temperature and liability to contaminate. Storage at abusive temperature can facilitate rapid, quality decline as a result of proliferation of spoilage bacteria (Smyth et al., 1998; Kader 2002). There is evidence that the growth of E. coli O157:H7 is inhibited at temperatures of 4C or less (Francis and OBeirne 2001) whereas E. coli O157:H7 is capable of growing at 8 to 12C (Francis and OBeirne 1999, 2001; Li et al., 2001), the temperature range that

fresh-cut produce is frequently exposed to during marketing and distribution (Jol et al., 2006).

Liability to contamination had numerous sources. It was either at pre-t or postharvest levels. Potential sources of contamination at the preharvest level are soil, irrigation water, manure, wild and domestic animals, harvest workers, and harvest equipment (Oliveira et al., 2011). Soil is a natural environment for a variety of human pathogens and broadened considerably by the addition of animal wastes (Whipps et al., 2008). It has been reported that E. coli O157:H7 and Salmonella may survive in soil from 7 to 25 weeks depending on the soil type, moisture level, temperature and source of contamination (Guo et al., 2002; Lang and Smith, 2007; Zhang et al., 2009b; Erickson et al., 2010). Solomon et al. (2002) studied the effect of irrigation types on the presence of E. coli O157 and found that 90% of lettuce plants, which had been spray-irrigated with water containing 7 log CFU/ml of E. coli O157, were contaminated, while only 19% were contaminated when surface irrigation was used with the same concentration of E. coli O157.

Fields that contain animal manure are more likely to be contaminated with enteric pathogens because of their ability to survive in soils for months or years (Doyle and Erickson, 2008). Feces may naturally contain between 102 and 105 CFU/g E. coli and between 102 and 107 CFU/g Salmonella spp. (Himathongkham et al., 1999). Harvest maturity can also affect susceptibility of the produce to decay (Waller, 2002). The leaf age of Romaine lettuce was shown to be influenced on growth and population of Salmonella and E. coli O157:H7 (Brandl and Amundson, 2008).

At the postharvest level, washing water, workers, packing materials, process equipment, and transportation vehicles are potential sources of contamination (Beuchat and Ryu, 1997; Brackett, 1998; Dallaire et al., 2006). After processing, pathogens can survive and even grow on lettuce depending on temperature, water availability, and level of tissue damage, available nutrients, and native microbiota (Abdul-Raouf et al., 1993; Aruscavage et al., 2006; Brandl, 2006; Cooley et al., 2006).

2.3

Lettuce Production System

Production system is very important to determine the production yield, nutritional value, produce quality, and safety and profit. Commercial and subsistence farming are still relying on the use of inorganic fertilizers for growing crops (Lampkin, 1990). This is due to easy to use, quickly absorbed and utilized by crops. However, these fertilizers are believed to contribute substantially to the human, animal food intoxication and environmental instability or degradation. The chemical fertilizers used in conventional agriculture contain just a few minerals, which dissolve quickly in damp soil and give the plants large doses of minerals (Vernon, 1999). Restrictions on pesticides have the direct effect of reducing residual level of pesticides that are not so strict in conventional farming (Lairon, 2010). Increased consumer awareness of food safety issues and

environmental concerns have contributed to the development of organic farming over the last few years (Worthington, 1998; Worthington, 2001).

The studies comparing nutrient content of organic and conventional foods have been extensively reviewed (Woese et al., 1997; Heaton, 2001; Worthington, 2001; Bourn and Prescott, 2002; Gennaro and Quaglia, 2002; Williams, 2002; Magkos et al., 2003; Winter and Davis, 2006; Rembialkowska, 2007; Benbrook et al., 2008; Dangour et al., 2009; Lairon, 2010). Compared with conventional high-input production, organic plant tends to show higher levels of vitamin C, phytochemicals and essential amino acids, less nitrate and total protein, and lower contamination of mycotoxins and pesticide residues. The yields in organic systems are significantly lower than in conventional production, and it appears that the yield reduction and changes in composition caused by the restrictions in fertilizer use are directly linked (Brandt et al., 2011).

2.4

Reduction of Nitrate Content by Harvesting Stages

Numerous factors have been identified to cause nitrate accumulation in plant. Hence, many researchers were struggling to reduce the nitrate content in vegetables for safe human consumption. Most researchers tend to manipulate the fertilizer composition and application in order to reduce the nitrate level (Ahmed et al., 2000; Custic et al., 2003; Abu Rayyan et al., 2004; Chen et al., 2004; Konstantopoulou et al., 2012). According to Beis et al. (2002), they stressed on growth phase that affected the content of nitrates. These authors, who studied on spinach, claimed that the highest content of these compounds was in the youngest leaves.

The findings from Amr and Hadidi (2001) showed that harvest stage had a significant effect at P0.05 on the nitrite content of spinach, lettuce and cabbage but not on squash,

cauliflower and parsley. Late-harvested lettuce and cabbage had the highest nitrite levels while spinach had the lowest. However, different pattern of accumulations were observed on nitrate. The harvest stage was only significant at P0.05 on the nitrate content of spinach, cabbage and squash. Late harvested vegetables had the lowest nitrate content.

Ozone application could reduce the nitrate level after 30 minutes exposure (Vaccaro et al., 1962). Tingey et al. (2006) stated that ozone-induced alterations in metabolites, which are associated with nitrate reduction either as an energy source or an end product, suggested that nitrate reductase activity may also be altered by ozone. The ozone depression on nitrate reduction was not the result of direct ozone reaction with nitrate reductase protein but rather interference with reactions that supplied the nicotinamide adenine dinucleotide phosphate (NADPH) needed for nitrate reduction.

2.5

Ozone Treatment

Treating fruits and vegetables with ozone has been used to increase shelf-life (Norton et al., 1968; Rice et al., 1982). Treatment of apples with ozone resulted in lower weight loss and spoilage. An increase in the shelf-life of apples and oranges by ozone has been attributed to the oxidation of ethylene. Fungal deteriorations of blackberries and grapes were decreased by ozonation (Beuchat, 1992). Onions have been treated with ozone during storage had decreased mold and bacterial counts without any change in chemical composition and sensory quality (Song et al., 2000).

2.5.1

Aqueous Ozone

Aqueous ozone is partially soluble in water. Ozone in pure water is quickly degrades to oxygen and even more rapidly in impure solutions (Hill and Rice, 1982). Ozone

solubility in water is 13 times that of oxygen at 0-30 C and it is progressively more soluble in colder water (Rice, 1986). The most crucial factors that affect solubility of ozone in water are temperature and pH. Ozones solubility in water is only about 0.2 mg/L at 20 C, but increases as temperature, 0.54 mg/L at 0 C (Bablon et al., 1991).

Grimes et al. (1983) stated that, in aqueous ozone, ozone decomposes primarily to the hydroxyl radical and the presence of phenolic compounds enhances this decomposition. In fact, many different compounds act to initiate, promote, or inhibit free radical formation from ozone (Bablon et al., 1991). The half life of ozone at the room-

temperature water is only twenty minutes (Xu, 1991). In water with suspended soil and organic matter, the half-life of ozone is reduced to less than one minute (Suslow, 1998).

Applications of ozone could enhance the microbiological safety and quality of foods (Kim et al., 1999a; Khadre et al., 2001b). Aqueous ozone is a highly effective, broad spectrum antimicrobial agent, effective at low concentrations and short contact times (Wickramanayake, 1991; Restaino et al., 1995). Bialka and Demerci (2007) obtained large reductions in the populations of E. coli O157:H7 and Salmonella on inoculated raspberries and strawberries by treatment with aqueous ozone (1.7 to 8.9 mg/liter). Ozone at concentrations of 0.155.0 ppm has been shown to inhibit the growth of spoilage bacteria as well as yeasts (Jay et al., 2005).

The ozone treatment has also been effective in extending the shelf-life of oranges, raspberries, grapes, and pears and apples (Beuchat, 1998; Skog and Chu, 2001). However, aqueous ozone treatment was ineffective in reducing populations of E. coli O157:H7 in the stem and calyx regions of inoculated apples (Achen and Yousef, 2001) reducing postharvest fungal decay of pears (Spotts and Cervantes, 1992) and decontaminating alfalfa seeds inoculated with E. coli O157:H7 (Sharma et al., 2002) and L. monocytogenes (Wade et al., 2003), probably because of the difficulty in contacting and inactivating bacteria attached in inaccessible sites.

2.6

Postharvest Quality Related to Aqueous Ozone Treatment during Storage

Quality is an ambiguous concept, and many definitions have been suggested. Quality has been described as a degree of excellence, a high standard, or value (Barrett et al., 2010). In order to maximized the quality of food, ozone has been used to decontaminate catfish processing (Sopher, 2007), remove pesticides on vegetables (Wu et al., 2007) and as disinfection treatments on corn (Prudente and King, 2002), dried figs (Oztekin et al., 2005), fresh-cut celery (Zhang et al., 2005) and fresh-cut lettuce (Beltran et al., 2005). Ozone also can retard the ripening process of fruits and vegetables by oxidation of ethylene released during storage. Aguayo et al. (2006) indicated that the shelf life of strawberries increased when 0.35 ppm ozone was applied. Ozone, being highly reactive, causes many physiological changes in living plants, including harvested fresh fruits and vegetables. Ozone-induced physiological changes affect rates of decay, ripening, and overall quality.

According to Perez et al. (1999), ozone was ineffective in reducing fungal decay in strawberries and also, detrimental to aroma after four-day treatment with 0.35 ppm ozone. Ozone treatment may also affect quality parameters. Skog and Chu (2001) reported that ozone (0.4 L L-1) improved the quality of broccoli and cucumber stored at 3C, prolonging the storage life. Appearance of ozone treated berries were free from all damages (Gabler, et al., 2010).

2.6.1

Weight loss

Freshly harvested produce is sold based on weight. Therefore, any loss in weight has a great impact not only on the quality but also the economic value of the produce and its profitability. Maguire et al. (2001) in their review reported that excessive weight loss from fruits can lead to shriveled appearance; decreased turgidity that cause undesirable textural and flavor changes and make the produce unsalable.

Bialka (2007) found ozone at 1.5 ppm could reduce the weight loss of strawberries. Salvador et al. (2006) also found that ozone-treated persimmon fruit showed the highest weight loss, achieving values of 3.6% after 30 days of storage. Ozone treatment on fresh grapes decreased the weight loss noticeably (Geransayeh et al., 2011). They stated that this was due to water loss, and the reduction in stored materials in the process of respiration. Ozone leads to increase dehydration of samples only if it is used in high concentrations, which cause damage to the epidermis and cuticle tissues of the fruits (Palou, et al., 2002). Exposure to 0.7 and 1 L/L ozone caused increased weight loss

resulting in desiccation of broccoli florets (Forney et al., 2003) and tomatoes (Tzortzakis et al., 2007), respectively. Ozone induced membrane damaged, which caused increased membrane leakiness that could also contribute to enhance water loss (Tingey and Taylor, 1980).

It was in contrast with the findings of Liew and Prange (1994). They found ozone did not affect carrot weight loss. Similarly, no differences in the weight loss of rocket leaves were observed when tap, chlorinated or ozonated water were used (Martnez-Sa nchez et al., 2006). Furthermore, the exposition to gaseous O 3 did not affect the weight loss of tomatoes (Rodoni et al., 2010) and table grapes (Artes-Hernandez et al., 2004) treated before storage or stored in an ozone enriched atmosphere, respectively.

2.6.2

Firmness

Firmness is a critical quality attribute determining postharvest quality of fruit and vegetables (Gross et al., 2002). At the cellular level, firmness depends on cell size, cell wall thickness and strength, turgor pressure and the manner in which cells bind together (DeEll et al., 2001). In lettuce, texture refers to crispiness of the leaves and is affected negatively by moisture loss due to improper storage and handling procedures. Some researchers found that gaseous ozone improved firmness in tomatoes fruits (Aguayo et al., 2006). This was in agreement with Garcia et al. (1998) where in their studies, ozone improves firmness in citrus fruit skin. In previous studies, higher

firmness was found in citrus and cucumbers treated with ozone compared with control

(Nadas et al., 2000; Skog and Chu 2001). Ozone also induced a delay in fruit softening in strawberry during cold storage and storage at room temperature (Nadas et al., 2003). Peppers did not show significant differences in firmness after exposure to gaseous ozone (Horvitz and Cantalejo, 2012). In a similar way, ozone gas applied either continuously or in intermittent periods did not significantly affect the firmness of mushrooms (Escriche et al., 2001).

2.6.3

Colour

Color is a primary indicator of maturity and is the result of type and quantity of pigments in the product. Color is derived from natural pigments in fruits and vegetables, many of which change as the plant proceeds through maturation, ripening and senescence.

Nadas et al. (2000) observed that treatment with ozone delayed color evolution in citrus fruit. Ozone also affected carrot color (Liew and Prange 1994). Ozone treatment on grapes led to a lower hue angle and chroma compared with control (Geransayeh et al,. 2011). It appears that when the samples experience weight loss and dehydration during the storage period, the phenomenon of browning berries occurs and cause more color changes in them. Reduction in the amount of green pigment over time is the main index of berry color purity reduction (Artes-Hernandez and Aguayo, 2004). It seems that green pigments were reduced in ozone treated samples.

2.6.4

Titratable acidity

The titratable acidity (TA) is a measure of the amount of organic acid present and is used primarily to estimate consumption quality and hidden attributes. Acids can affect flavour directly and are also beneficial in processing since they affect the formation of off-flavours and the gelling properties of pectin (Manning, 1993).

Determination the acids in developing boysenberries also found that a decrease total titratable acidity as the fruit developed; malic acid decreased from 17.9% of the total acid in unripe fruit to 8.3% when the fruit was overripe (Manning, 1993). Wills et al. (2007) also stated that usually organic acids decline during ripening as they respired or converted to sugars. Perez et al. (1999) reported that sugar and organic acids

concentrations in strawberry were lower in ozone treatments than in control samples; however, these differences were not observed by Nadas et al. (2003).

2.6.5

Soluble solids concentration

Sugars are one of the main soluble components in fruit and provide energy for metabolic changes. Sugars in tomatoes which were treated with ozone with concentrations of 0.15 and 1.0 mol.mol-1 increased significantly compared with controls (Tzortzakis, 2007). Garcia et al. (1998) which studied on oranges and mandarins, stated that ozone exposure did not affect the quality of TA and SSC. Ozone also had no effect on the SSC of persimmon fruit (Salvador et al., 2006).

2.6.6

pH

The amount of TA indicated the amount of organic acids present while the pH value gives a measure of the acidity or alkalinity of the products. The pH is base on acid content, which was counted according to the free H ion (Wills et al., 2007). The free state of H ion is due to the dissociation of H ion from the carboxyl group (-COOH) of organic acid.

The increase in pH throughout maturation was due to metabolic processes resulted in the decrease of organic acid (Coseteng and Lee, 2006). This is because they are an

important source of respiratory energy in plant cells. Thus, decrease in organic acids will decrease free H ion and consequently increases the pH. During prolonged storage, the amount of pH declined because the acids are respired or converted to sugar (Wills et al, 2007). By day 14, a significant increase in the pH values occurred in control and both the chlorinated and the ozone-treated peppers (Horvitz and Cantalejo, 2012). The increase of pH also resulted from the breakdown of proteins with the release of basic compounds (Jacxsens et al., 2003). 2.6.7 Total chlorophyll

Chlorophylls are green pigments of ubiquitous occurrence in nature. Chlorophylls in photosynthesis allow the suns energy to be utilized to synthesized carbohydrates. Thus, causing change of light energy into chemical energy that is indispensable for the survival of all living organisms. In higher plants, chlorophylls a and b are imbedded in

the highly structured lamellae of chloroplasts. In green vegetables, retention of color is of paramount importance for consumer acceptance (Lamikanra et al., 2005). The

reduction of yellowing at 0.7 L/L ozone may be related to the inhibition of chlorophyll degrading enzymes. As a response to ozone, broccoli may increase levels of

antioxidant chemicals and enzymes which could slow down chlorophyll loss (Castillo and Greppin, 1988; Foyer et al., 2006). Diminution in the chlorophyll content of leaves after ozone exposure has been reported in many species such as Nicotiana tabacum L. (Saitanis et al., 2001), Spinacia oleracea L. cv. New Asia (Sakaki et al., 2006) and Triticum aestivum L (Pleijel et al., 2006), while no such reduction has been observed in Cucumis sativus L. (Agrawal et al., 1993) and Oryza sativa L. (Welfare et al., 1996).

2.6.8

Total phenolic compound

Total phenolic compound (TPC) can contribute to the astringency and bitter taste of some products. They are present in low concentrations, but in certain cases, such as in blueberry, they can reach levels of more than 0.1%. The general characteristic of compounds within this group is to have aromatic rings with variable degrees of hydroxylation (Mattila et al., 2006). TPC is oxidized easily to quinones. Polyphenolic compounds, widely distributed in higher plants, have potential health benefits believed to arise mainly from their antioxidant activity (Liu, 2003). When it added to foods, it can minimize rancidity, retard the formation of toxic oxidation products, maintain nutritional quality, and increase shelf life (Virgili et al., 2001).

Ozone treatment was not affected by total phenolic compounds in the fresh-cut of iceberg lettuce (Beltran et al., 2005). Zhang et al. (2005) reported an inhibition effect of ozonated water on the polyphenol oxidase activity of fresh-cut celery. Alothman et al. (2010) reported that 20 min of ozone treatment significantly increased TPC in both bananas and pineapples compared with 0 min exposure. For guava fruits, TPC

decreased with treatment time. This reaction to ozone treatment could be attributed to the activation of phenylalanine ammonia lyase (PAL; EC 4.3.1.5). PAL is one of the key enzymes used in the synthesis of phenolic compounds in plant tissues.

The increase in the phenolic and flavonoid contents of the fruits also caused by cell wall modification that occurred during ozone exposure; this modification may have increased the extractability and freed some of the conjugated phenolic compounds in the cell wall. Maier-Maercker and Koch (1992) suggested that the walls of guard and subsidiary cells of ozone treated spruce needles became de-lignified through the reaction with ozone (ozonolysis), which resulted in the accumulation of non-lignin phenolic compounds. According to Hoign and Bader (1983), byproducts of ozone decomposition such as hydroperoxyl (H2O), hydroxyl (OH), and superoxide (O 2 ) radicals were scavenged by phenolic compounds in the fruits, to different extents, might have led to the reduction of phenolic and flavonoid contents of the fruits after 30 min of exposure.

2.6.9

1,1-Diphenyl-2-picryl- hydrazyl (DPPH)

Antioxidants are considered essential nutraceuticals on account of many health benefits (Droge, 2002; Lee et al., 2004; Valko et al., 2007). The requirement of a standard assay

is very beneficial in order to compare the results of different laboratories and validation of the conclusions. DPPH is a stable free radical which has an unpaired valence electron at one atom of nitrogen bridge (Eklund et al., 2005). Scavenging of DPPH radical is the basis of the popular DPPH antioxidant assay (Alma et al., 2003; Karioti et al., 2004; Kordali et al., 2005). Ozone exposure up to 20 min increased the antioxidant activity in pineapple and banana, but the activity tended to decrease at 30 min of treatment (Alothman et al., 2010). Tzortzakis et al. (2007) reported that an ozone-enriched atmosphere (concentration up to 1.0 mol mol1 at 13C) did not change the antioxidant activity of tomatoes.

2.6.10 Vitamin C

Vitamin C is defined as the generic term for all compounds exhibiting the biological activity of L-ascorbic acid (AA) (Lee and Kader, 2000). Vitamin C is also known to have many biological functions in collagen formation, absorption of inorganic iron, reduction of plasma cholesterol level, inhibition of nitrosamine formation, enhancement of the immune system and reaction with single oxygen and other free radicals. Vitamin C, as an antioxidant, was reported to reduce the risk of arteriosclerosis and cardiovascular diseases (Harris, 1996).

Tiwari et al. (2008) found that ascorbic acid content of freshly squeezed orange juice decrease from 41.59 to 12.70 mg/100 ml after 10 min of ozonation treatment. Alothman et al. (2010) found that vitamin C content of the fruits decreased significantly when treated with gaseous ozone (P0.05). Vitamin C is a part of the anti-oxidation system in

the plant tissue was expected to prevent injuries of plant (Keutgen and Pawelzik, 2008). Ascorbic acid is thermo labile and highly sensitive to various processing and storage conditions (Tiwari et al., 2008). Therefore, vitamin C content decreased in the fruits due to its scavenging of the free radicals formed during decomposition of ozone. Degradation of ascorbic acid in ozone-treated fruit was due to activation of ascorbate oxidase (Lee and Kader, 2000).

The degradation of ascorbic acid by ozone is due likely to the free radical mechanism (Garcia-Viguera and Bridle, 1999). According to Geransayeh et al. (2011), ozone

treatment did not affect vitamin C content in grapes fruits. Similar findings were reported in tomato fruits (Shalluf et al., 2007; Tzortzakis, 2007) and strawberries (Kute et al. 1995).

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