Sanitation and storage

Use of ozone in sanitation and storage of fresh fruits and vegetables

Abstract

The following study focuses on the efficiency of ozone sanitation in the food industry with specific reference to fresh fruits and vegetables. Recent research findings including mechanisms of action, artificial synthesis, sanitation food efficiency and effects, application with different preservation techniques, as well as pros and cons have been reported.

In particular, ozone reduces microbial spoilage and weight loss of apple.Onions treated with ozone showed that mould and bacterial counts were greatly reduced without any change in their chemical composition. Ozone treatments carried out on tomatoes did not affect their colour, sugar

content, acidity and antioxidant capacity while it reduced the amount of aflatoxins when applied to peanuts. Red peppers, strawberries and cress treated with ozone showed a reduction in the microbiological population. In addition to many other examples it is also reported that the phenolic

and flavonoid content of pineapples and bananas increased significantly when exposed to ozone for up to 20 minutes. While considering limitations and contraindication in ozone use, it has been pointed out that ozone is a highly instable and corrosive gas and due to its short life span, ozone must be generated on site as storage is not possible.

In conclusion ozone appears to be a more effective biocide than other substances due to its high reactivity and its strong oxidant power. However, it is necessary to determine the safety limits of exposure to ozone in order to prevent damage on food and human health.

Key words: Ozone, sanitation, fresh fruits and vegetables, foods, conservation techniques, IV range products, lightly processed foods.

Introduction

The increasing demand for sanitation as a means for controlling infection and disease in food and the need for reducing the emission of polluting substances have made researchers search for safe and new sanitizing methods. Ozone has proved to be suitable for this purpose.

The term “ozone” comes from the Greek word “ozein” which means “to give off odour”.

The molecule has a pungent and characteristic odour and athigh levels of concentration it is blue at ordinary temperatures 1. At the level of concentration at which it is normally used, the colour is not noticeable 2. Ozone was first discovered by the German researcher Christian Friedrich Schönbein in 1839, even if as early as 1783,Van Marum indicated the presence of a strange pungent smell of gas in proximity of equipment capable of conducting electrical discharge 3.

In 1902 Cologne used ozone to disinfect frozen beef in order to prolong its shelf life. In 1904, De la Coux noticed the ample opportunities of using ozone in gelatine, casein and albumin production plants 3. It was first used commercially in 1907 for disinfecting the municipal water supply in Nice and then in St. Petersburg in 1910 for the same purpose. In 1928 it was used for disinfecting eggshells and between 1953 and 1956 it was acknowledged as an effective method for sterilising food containers 3.

In 1995 and 1996, Japan, France and Australia made laws which permitted the use of ozone in the food industry. In 2001 the U.S. Food and Drug Administration (FDA) modified the regulation in such a way to allow the use of both aqueous and gaseous phases in food treatment, conservation and transformation 3.

The main physical and thermodynamic properties of pure ozone are reported in Table 1.

Table 1. Major physical properties of pure ozone 2.

Molecular weight 47.9982 g/mol

Density 0.001962 g/cm3

Dielectric constant 1.002

Boiling point -111.9 ± 0.3°C

Melting point -192.5 ± 0.4°C

Critical temperature -12.1°C

Critical pressure 54.6 atm

As it is an unstable gas, the average life of ozone is about 20 min, depending on the temperature 1. The molecule is generally more stable in the gas phase than in the aqueous phase 5.

In pure water, it degrades rather rapidly in oxygen. This degradation is faster in impure solutions 6. Rice 5 states that the solubility of ozone in water is 13 times higher than the solubility of oxygen in temperatures ranging from 0-30°C. The lower the temperature, the higher the level of solubility of ozone. In fact at high temperatures, the stability and effectiveness of ozone are reduced 7.

This study reports the effect of ozone treatments on the main fruits and vegetables and on some fresh-cut products.

Mechanisms Of Action

The bactericidal effect of ozone was studied and documented on a wide variety of organisms, including Gram positive and Gram negative bacteria as well as spores and vegetative cells. Ozone is efficient against Venezuelan equine encephalomyelitis virus, hepatitis A, influenza A, vesicular otitis virus, and infectious bovine rhino-tracheitis virus as well as several strains of bacteriophage 2.

In the case of virus and bacteria, as well as protozoa and insects, ozone acts through the catalytic oxidation of proteins and liposaccharides destroying their structure. Ozone oxidizes the organic matter of bacterial membranes, weakening cellular walls and causing cell damage and the consequent death of the cell. This distinguishes ozone from chlorine and other oxidant or nonoxidant

biocides which must be transferred through membranes in order to interfere with cellular enzymatic or non enzymatic activities and therefore perform biocide activity 8. Ozone treatments can cause death of cells due to the effect of nucleic acid destruction 9. Ozone has a short life span in water and in air, so it is safer than other food additives, as no residues remain in the food 9. Khadre and Yousef 10 compared the effects of ozone treatment to the effects caused by hydrogen peroxide treatment on Bacillus spores of food origin and ozone proved to be more efficient than hydrogen peroxide. When compared to chlorine and hypochlorous acid, the potential of ozone is from 1.5 to 3000 times greater than the other two respectively 11. Ikeura et al. 12 analyzed the efficiency of ozone efficacy in removing pesticides from vegetables by using gas in the form of micro-bubbles in rinsing water. The effectiveness varies according to method used for producing the micro-bubbles: production by means of decompression is more efficient than production by gas-water circulation. This could be due to a greater number of micro-bubbles which can infiltrate into the vegetables.

Ozone Generation

Oxygen molecules are ruptured, producing oxygen fragments which join other oxygen molecules to produce ozone, O3 13. In another process of ozone formation, oxygen floats upward into the atmosphere and in turn is converted into ozone through ultraviolet radiation 14.

Ozone can be generated artificially by means of the corona discharge; air or O2 passes through a high voltage electric field. In this case the stable molecule of oxygen is broken and splits into two oxygen radicals which react with each other to form ozone. The formation of Ozone produced by electrical discharges on a gas is based on the lack of homogeneity of the corona discharge in the air and in oxygen. There are many microdischarges distributed in space through which ozone is produced: every single micro-discharge only lasts a few nanoseconds and is greater (2.5-3 times) in the air than in pure oxygen. In order to create corona discharge an electrical potential of at least 5000V is required. The typical range within which the reactions are carried out is between 5000 V (with a frequency of 1000 Hz) and 16000 V (with a frequency of 50 Hz) 3.

The first ozone generator was developed in the United States in 1888 by Fewsn to deodorize gas pestilential. In Germany in 1902, Siemens and Halske built the first ozone generator for treating water.

Use Of Ozone In Food Hygiene

Ozone is a strong oxidant which is efficient for controlling bacteria, molds, protozoa and viruses 15. In 1997, ozone was declared a GRAS product (Generally Recognized As Safe) by a team of experts from FDA 17. This denomination soon caused an increase in research concerning the use of ozone in the food industry. Ozone can be used for various purposes such as cleaning surfaces or equipment and disinfecting water for recycling 8, 17. The effectiveness of disinfectants varies according to the type of product, the surface to be treated and the specific characteristics of the microorganisms 14. In addition, the susceptibility of microorganisms to ozone varies according to the physiology of the tissue, temperature, moisture, the pH level and the presence of additives such as acids, soaps and sugars 19. The effectiveness of the various types of treatment can be seen in Table 2. Other proven positive effects of the use of ozone concern the purification of micotoxins 19 and pesticide residuals 20 and for the control of classified microbes in the field of biological risk 21. Leesch and Tebbets 22 demonstrated that ozone is effective for controlling the presence of arthropods, like in the case of

grape fumigation with high doses of ozone to control spider populations belonging to the black widow spider species which are often found in boxes of grape exported from California.

Ozone can be used as a sanitizer in the form of a gas or dissolved in water. When ozone is utilized as a gas, the length of exposition is longer (1-4 h) than ozone dissolved in water (1-10 minutes) 3.

Ozonated water is a good alternative to traditional sanitizers because it is effective at a low concentration 13. Artes et al. 23 reported several studies in which the efficacy of ozonated water was successfully tested by inoculating targeted specific microorganisms on pure cell suspensions or on the food surface and treating these surfaces with O3. Singht et al. 24 and Kim et al. 8 demonstrated the controlling action of ozone on some pathogens: Staphylococcus aureus, Salmonella typhimurium, Bacillus cereus, Enterococcus faecalis, Pseudomonas aeruginosa, Pseudomonas fluorescens, Leuconostoc mesenteroides, Yersinia enterocolica, Listeria monocytogenes, Escherichia coli, Candida albicans, Zygosaccharomyces and Aspergillus niger spores.

Table 2. Treatments with ozone and relatives effects 4, modified.

Applications	Treatment	Micro-organism	Results
stainless steel surface	2 ppm ozone gas at atmospheric pressure 22°C and 77% HR for 4h	E.coli, S. liquefaciens, S.aureus, L.innocua Rhodorotura rubra	Reduction ranging  from 7.56 to 2.41 log  values
	2 ppm ozone gas in chamber at 20°C and 50% HR for 1 h	Micrococcus luteus	2-3 log reduction
Stainless steel in the presence of milk	2 ppm ozone gas at atmospheric pressure,    22°C and 77% H.R.     for 4 h	E.coli, S. liquefaciens       S. aureus, L. innocua, Rhodorotura rubra	Reduction ranging from 5.64 to 1.65 log
Equipment, walls, floors drains, tables and conveyors, previously well-cleaned	Ozonated water,             3.0-3.5 ppm	Trichophyton  mentagrophytes,S cholerasuis,S. aureus, Ps. aeruginosa Campylobacter jejuni,L.monocytogenes,  Aspergillus flavus  Brettanomyces bruxellensis, E. coli	4-6 log reduction

The Effect of Ozone Treatment On Fresh And Fresh-cut
Fruit And Vegetables

More recently, there has been a growing interest in the evaluation of ozone treatments during the processing and storage of fruits and vegetables 25, 26.

Microbial contamination of fruit and vegetables can occur at various stages from the farm to the table. The propagation of microorganisms occurs during growth in the field, harvesting, post-harvest handling and transportation, storage, processing and marketing for human consumption.

Beuchat 27 showed that treatment with ozone seems to have a beneficial effect in extending the storage life of fresh non-cut commodities such as broccoli, cucumber, apples, grapes, oranges, pears, raspberries and strawberries by reducing microbial populations and through ethylene oxidation. Treatments on apples with ozone resulted in a reduction of weight loss and spoilage.

Onions treated with ozone during storage showed a considerable decrease in mould and bacterial counts without causing any change in their chemical composition and sensory quality 28.

Continuous ozone exposure to 0.3 ppm (v/v) inhibited the growth of fungi and spores on ‘Elegant Lady’ peaches during storage 29. The authors also observed a reduction of grey mould in ‘Thompson Seedless’ table grapes.

Treatments on whole and fresh-cut Thomas tomatoes, stored for a long time at high concentrations (7μL L-1), show how ozone efficiently reduced bacterial and fungal population 30.

Gonzales-Barrio et al. 31 analyzed the induction of antioxidant accumulation in white table grapes (var. ‘Superior’) after ozone treatments at different concentrations (3.88 and 1.67 gh-1) for 1, 3, and 5 h during storage at 22°C.

Fresh-cut salad, washed with ozonated water and packed in ozone, showed an extension of shelf life 32.

Tzortzakis et al. 33 demonstrated that low-level ozone-enrichment (0.1 μmol mol-1) during storage at 13°C, reduces spore production in Botrydiscinerea and lesion development in tomatoes, strawberries, table grapes and prunes.

Tzortzakis et al. 34 report that ozone treatments between 0.005 and 5.0 μmol mol-1 reduce fungal lesion development due to Alternaria alternate and Colletotrichum coccodes.

Tiwari et al. 35 investigated the main effects and interaction of ozonation on orange juice colour degradation. The ozonation of organic dyes causes loss of colour because of the oxidative fission of the chromophores due to attack on conjugated double bonds. Similarly the chromophore of conjugated double bonds of carotenoids is responsible for the colour of orange juice. Carotenoid pigments which are responsible for the yellow, orange or red colour in orange juice contain one or more aromaticrings. The ozone and hydroxylradicals (OH-) generated in the aqueous solution may open these aromatic rings and lead to a partial oxidation of substances such as organic acids, aldehydes, and ketones.

Rodoni et al. 15 evaluated the effect of short-term gaseous ozone treatment (10 μL/L; 10 min) on the quality of tomato fruits. The treatments did not modify fruit colour, sugar content, acidity, or antioxidant capacity but reduced fruit damage by 27% after 9 days of storage at 20°C (Fig. 1). They also reduced weight loss and aided the accumulation of phenolic compounds (Fig. 2). The study

shows that short-term treatments with ozone are also effective in reducing the softening of tomato fruits.

Figure 1. Ozone treatment effects on tomatoes damage. The asterisk

indicates significant differences compared to control (P≤0.05) 15.

Figure 2. Ozone treatment effects on total phenols formation. The asterisk

indicates significant differences compared to control (P≤0.05) 15.

Alothman et al. 36 studied the effect of ozone treatments (8±0.2 ml/s for 0, 10, 20, and 30 min) on the total phenol, flavonoid, and vitamin C content of fresh-cut honey pineapple, ‘pisang mas’ bananas and guava demonstrating that the total phenol and flavonoid content of pineapple and banana increased significantly when exposed to ozone for more than 20 min. An opposite trend was observed for guava. The ozone treatment significantly decreased the vitamin C content of all three fruits.

Treatments carried out with ozonated water on red peppers, strawberries and cress showed a reduction in the microbiological population (0.5-1.0 log cycle-1). This reduction is even more evident when the treatment is combined with a scalding process 37. Ozone treatments on kiwi brought about an interruption in the production of ethylene and in the induction of the antioxidant

activity 38.

Alencar et al. 39 reported that peanuts treated with ozone showed a total aflatoxin reduction (about 3 log cycle-1) and a B1 aflanoxine and internal fungal population reduction.

Kechinsky et al. 40 observed that ozone treatments with wax and thermal-therapeutic treatments with water led to fungal absence and disinfection on papaya.

Limits And Contraindications Of Ozone Use

Ozone is an efficient sanitizer, but has some limitations when used on food. Moreover, due to its short life span, ozone must be generated on site as storage is not possible 14. Ozone is a highly instable and corrosive gas. The decomposition of ozone requires elaborate processes depending on the types of radicals formed in solution and on several types of organic matter in medium that induce, promote or inhibit the reaction chain 9. In addition, low doses of ozone, which can inactivate pure microbial cultures, can be inefficient against viruses, spores and cysts.

Perez et al. 25 reported the loss of aroma which is typical of strawberries treated with ozone during conservation, due to changes in the ratio between soluble solids and organic acids. The changes may be due to degradation of saccharose and glucose.

Kim et al. 9 observed modifications in the surface color of some fruits and vegetables such as peaches and carrots, a decrease in the amount of ascorbic acid in broccoli florets and thiamin in wheat flour, and lipid oxidation affecting the sensory quality of grains and crushed spices. Ozone in air, at concentrations higher than 0,1 ppm, has a strong odor which causes irritations of the nose, throat and eyes. Exposure to a high concentration of ozone may have mutagen effects and cause death due to acute toxicity. After 1-2 hours of exposure to ozone at a concentration of 0.65 ppm concentration, an acceleration in the respiration rate was observed in test dogs. Exposure to 0.02 ppm ozone concentrations for 4-6 weeks caused distension of the lung in young rats 41. Ozone concentrations higher than 0.02 ppm cause various levels of damage to the respiratory system according to the duration of exposure 42. It is important to establish safety limits for the treatment of food, find efficient systems for detecting and destroying ozone when there are high levels of concentration of the gas and carry out regular medical checkups on workers subject to chemical risk. In humans, ozone mainly effects and damages the respiratory system. Symptoms of intoxication are headache, weakness, loss of memory, increase in frequency of bronchitis and high muscle tension 43.

Use Of Combined Preservation Methods

Several studies analysed the use of combined preservation methods with ozone treatments.

Sharma 15 mentioned hydrostatic pressure, UV and H2O2. The application of pressure aids the penetration of the sanitizers into the inaccessible cracks and crevices of foods, thus enhancing microbial decontamination without compromising quality. The main advantage of applying hydrostatic pressure includes uniform transmission of pressure, regardless of the size and shape of sample

.

The use of ozone in combination with initiators such as UV or H2O2 can result in advanced oxidation processes that are highly effective against the most resistant microorganisms.

Conclusions

Ozone is a more efficient biocide than other chemical substances due to its high reactivity and strong oxidant power. Therefore it is widely used to decontaminate food, mainly fruits and vegetables despite its instability. The identification of the most efficient applications and their implementation at industrial level have been a slow process. This may be due to the fact that high capital investment and operational costs are required for ozone treatments 44. Further studies must be carried out on ozone treatment with the aim of finding a valid alternative to the chemical decontamination of fresh or cut-fresh fruits and vegetables. It is necessary to find food products for which ozone treatment is qualitatively effective and find synergies with other solutions like UV ray, hydrostatic pressure and sonication. In the future, ozone treatment will be considered a suitable post harvest operation for removing chemical and biological contaminants and improving the conservation as well as for recycling water.

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