**4. Sanitizers as a control measure**

Minimally processed vegetables are products ready for consumption and must be free of pathogenic microorganisms. Its washing step must be done with good quality water followed by the addition of sanitizer solution aiming to reduce the microbial counting and increasing microbial safety and the product preservation. Thus, the sanitation plays an important role in reducing decay and maintaining quality. Therefore, the types of sanitizers, the forms of application, generally a function of time and concentration, will depend on the accompanying microbiota and characteristics of raw material processing.

Chlorine, in its various forms, is the group of most commonly used compound sanitizers because of its efficiency and low cost. They are compounds of broad-spectrum germicidal action by reacting with membrane proteins of the microorganism. Sodium hypochlorite is the most widely used chemical sanitizer because of its complete dissociation in water, easy application and quick action being effective in reducing populations of bacteria, fungi, viruses and nematodes. In water, produces sodium hydroxide (NaOH) and hypochlorous acid (HOCl), the latter being the germicidal agent, which dissociates into H+ and OCl- ion according to the following reactions:

NaOCl + H2O NaOH + HOCl

#### HOCl → H+ + OCl-

It is proved that the hypochlorous acid (HOCl) has greater disinfecting action (about 80 times more) than the same concentration of hypochlorite ion (OCl-). The amount of HOCl formed depends on the pH of the solution and its concentration is considerably higher at pH 4.0 decreasing as pH increases. Thus, at pH above 5.0 occurs an increase of the hypochlorite ion (OCl-). The sanitizing step is usually performed at pH between 6.5 and 7.0 because in this range there is still considerable amount of hypochlorous acid. The greatest disinfecting power of the hypochlorous acid is explained by the fact that being a small, neutral molecule has a greater ease of penetration through the cell wall. In turn, the hypochlorite ion due to its negative charge is more difficult to cross the cell wall and reach the enzyme system. It is therefore possible that the greatest difficulty in the elimination of sporulated forms is related to the penetration of the disinfecting agent as this may be hampered by the protective mantle of the microorganism.

A study carried out by Berbari et al. (2001) showed that soaking for 15 minutes in a solution containing a chlorine 70mg.L-1 enables a shelf-life of up to 6 days for minimally processed lettuce stored at 2°C, increasing to 9 days if treated with a solution containing 100 to 130mg.L-1 of chlorine. On the other hand, a study by Nunes et al. (2010) with Peruvian carrot minimally processed, showed that soaking for 10 minutes in a solution containing 100mg.L-1 of chlorine

stands at room temperature for a long time, such as two hours, is also at risk. It is important to assent that vegetables, now identified as a source of *Salmonella* sp., must be thoroughly washed in treated or healthy running water before they are eaten, as basic operations of food borne disease. In food industry, internal systems of quality control are essential to prevent occurrence of foodborne illness to consumer. As an example, the HACCP (Hazards Analysis and Control of Critical Points) system, adopted by major international markets, basically ensures that the manufactured products are developed without risk to public

Minimally processed vegetables are products ready for consumption and must be free of pathogenic microorganisms. Its washing step must be done with good quality water followed by the addition of sanitizer solution aiming to reduce the microbial counting and increasing microbial safety and the product preservation. Thus, the sanitation plays an important role in reducing decay and maintaining quality. Therefore, the types of sanitizers, the forms of application, generally a function of time and concentration, will depend on the

Chlorine, in its various forms, is the group of most commonly used compound sanitizers because of its efficiency and low cost. They are compounds of broad-spectrum germicidal action by reacting with membrane proteins of the microorganism. Sodium hypochlorite is the most widely used chemical sanitizer because of its complete dissociation in water, easy application and quick action being effective in reducing populations of bacteria, fungi, viruses and nematodes. In water, produces sodium hydroxide (NaOH) and hypochlorous acid (HOCl), the latter being the germicidal agent, which dissociates into H+ and OCl- ion

NaOCl + H2O NaOH + HOCl

HOCl → H+ + OCl-It is proved that the hypochlorous acid (HOCl) has greater disinfecting action (about 80 times more) than the same concentration of hypochlorite ion (OCl-). The amount of HOCl formed depends on the pH of the solution and its concentration is considerably higher at pH 4.0 decreasing as pH increases. Thus, at pH above 5.0 occurs an increase of the hypochlorite ion (OCl-). The sanitizing step is usually performed at pH between 6.5 and 7.0 because in this range there is still considerable amount of hypochlorous acid. The greatest disinfecting power of the hypochlorous acid is explained by the fact that being a small, neutral molecule has a greater ease of penetration through the cell wall. In turn, the hypochlorite ion due to its negative charge is more difficult to cross the cell wall and reach the enzyme system. It is therefore possible that the greatest difficulty in the elimination of sporulated forms is related to the penetration of the disinfecting agent as this may be hampered by the protective

A study carried out by Berbari et al. (2001) showed that soaking for 15 minutes in a solution containing a chlorine 70mg.L-1 enables a shelf-life of up to 6 days for minimally processed lettuce stored at 2°C, increasing to 9 days if treated with a solution containing 100 to 130mg.L-1 of chlorine. On the other hand, a study by Nunes et al. (2010) with Peruvian carrot minimally processed, showed that soaking for 10 minutes in a solution containing 100mg.L-1 of chlorine

health, and also have uniform standards of identity and quality (SILVA, 1999).

accompanying microbiota and characteristics of raw material processing.

**4. Sanitizers as a control measure** 

according to the following reactions:

mantle of the microorganism.

allowed a shelf-life of 6 days when stored at a temperature of 5°C ± 1°C. Nascimento (2002) showed that vegetables washed with a solution containing 50 ppm of free chlorine showed a significant reduction in the total count of aerobic and that fecal coliforms were even more sensitive to chlorine, not being more detected in vegetables after washing. Therefore, chlorine and its salts, especially hypochlorite, are effective and of low cost, and widely applied as a spray for bacteriological control in industries working with vegetables (KIM et al., 1999). However, in recent years there has been some concern in the use of chlorine due to the inconvenience of toxic compounds that can be formed and leave residual taste in food (OLIVEIRA & VALLE, 2000). Among these compounds, there are the trihalomethanes (THM), aldehydes, halocetonas and chloramines, which when hydrolyzed proved related to some types of cancer according to epidemiological studies of Meyer (1994). Depending on the toxicity of these compounds, there is a recognized need to find alternative sanitizers for hygiene and sanitization procedures for vegetables. Thus, chlorine dioxide (ClO2) has received special attention (ARENSTEIN, 2003) for, although it is a derivative of chlorine, generates negligible amount of by-products (trihalomethanes), characterized as a product of low carcinogenic potential (ANDRADE & MACEDO, 1999). In addition, chlorine dioxide is a strong oxidizing agent that reacts mostly through a mechanism of electron transfer by attacking the cell membrane, penetrating, dehydrating, and lastly, oxidizing the internal components of the microbial cell without however causing toxic effects, as most of the chlorine compounds do. It also has the advantage of being effective against gram negative and positive. Still, by the fact that hydrolyzes the phenolic compounds it reduces the possibility of formation of tastes and odors.

Another important aspect of chlorine dioxide is its sharp and sporicidal disinfectant action in lower concentrations than that of chlorine. The explanation of its high bactericidal action is due to the fact that it is soluble in oils, greases and substances of mixed composition, such as cells of virus and bacteria, whose membranes easily penetrates in, as opposed to other disinfectants of polar nature.

Chlorine dioxide is stable under a wide pH range (6-10) and its decomposition are first formed chlorite (ClO3) and then chlorate (ClO2) which can be seen in the equations:

> 2 NaClO2 + Cl2 2 ClO2 + 2 NaCl 2 ClO2 + H2O ClO-2 + ClO-3 + 2 H+ 4 ClO2- + H2+ 2 ClO-2 + ClO-<sup>3</sup> + 2 H2O

However, the major disadvantages of chlorine dioxide are its cost and its sensitivity to high temperatures.

Currently, several studies are being conducted with chlorine dioxide in different countries. Felkey et al. (2003) and Rash (2003) showed in their studies the efficiency of chlorine dioxide in reducing *Salmonella* on the surface of tomato and melon, respectively. Another sanitizing agent that has been used quite successfully is peracetic acid, also known as peroxide of acetic acid or peroxyacetic acid. It is obtained by the reaction of acetic acid or acetic anhydride with hydrogen peroxide in the presence of sulfuric acid, which has the function of catalyst. The decomposition products are acetic acid, hydrogen peroxide and water.

The peroxyacetic acid has currently one of the largest application as disinfectants in the food industry and its efficiency is similar or superior to sodium hypochlorite (NASCIMENTO, 2002), but more potent than hydrogen peroxide. It is an excellent sanitizer for the great

Occurrence of *Salmonella* in Minimally Processed Vegetables 117

The microbial counts on fresh materials rocket and chicory after washing followed by immersion in water for 15 min. showed high contamination of molds and yeasts (5.90 and 5.62 log CFU.g-1), total coliforms (6.22 and 5.59 log CFU.g-1) and *Escherichia coli* (2.61 and

It has also been seen that the samples of rocket showed initial contamination superior to the chicory for the same tests, which may be a consequence of the type of rocket leaf that by being rough ends up retaining contaminants on its surface, unlike the chicory which has the

Data regarding to the effects of chlorine dioxide and peracetic acid in the population of yeasts and molds in minimally processed chicory (Table 1) showed that the variables concentration and contact time influenced significantly (at 5%), and both concentrations as the contact times studied was inversely proportional to the population of yeasts and molds

(Min) 10ppm 25ppm 50ppm MSD1 2 3.312 0.212 a, A 2.871 0.157 b, A 2.436 0.120 c, A 0.419 5 3.026 0.266 a, A, B 2.598 0.182 a, b, A 2.242 0.084 b, A 0.482 10 2.541 0.278 a, B 2.026 0.046 b, B 2.000 0.000 b, B 0.407 DMS2 0.635 0.353 0.212 ----

(Min) 50ppm 75ppm 100ppm MSD1 4 3.445 0.279 a, A 3.247 0.185 a, A 2.716 0.119 b, A 0.514 7 3.131 0.174 a, A, B 2.785 0.094 b, B 2.452 0.119 b, B 0.334 10 2.902 0.139 a, B 2.308 0.166 b, C 2.000 0.000 b, C 0.313 MSD2 0.517 0.384 0.243 ---- Blank (washing and immersion in tap water for 15 minutes) \*\* .(log CFU.g-1) 5.616

hypochlorite: 120ppm/15min) \*\* ……………………………….…(log CFU.g-1) <2.000

MSD1 = for the data on the lines; MSD2 = for the data on the columns; small letter compares averages on the same line, capital letters compare means in the same column, different letters indicate that the data differ significantly at 5% probability; \* Time of contact with the sanitizer product; \*\* reference

Table 1. Yeast and mold count (log CFU.g-1) observed in samples of minimally processed

In the case of chlorine dioxide, the treatments performed with 25ppm/10min and 50ppm/10min were statistically superior to the others and there wasn't, however, significant differences between the two. Both treatments showed a reduction equivalent to 3 logarithmic cycles in the population of yeasts and molds when compared with the treatment by washing followed by immersion in water for 15 minutes. On the other hand, regarding the effect of peracetic acid in the population of yeasts and molds, the treatments carried out

2.37 log CFU.g-1).

naturally present in chicory minimally processed.

Time\* Treatment with chlorine dioxide (ClO2)

Time\* Treatment with peracetic acid (CH3-COOOH)

Standard (washing with water and immersion in a solution of sodium

smooth leaf.

treatments.

chicory.

oxidation capacity of the cellular components of microorganisms having a rapid action at low concentrations and still effective in the presence of organic material and therefore being an effective biocide.

Its biocide action is influenced by the concentration, shape and type of microorganism. It degrades rapidly in biodegradable and harmless substances such as acetic acid and active oxygen, which pose no risk of toxicity and does not affect the taste and odor of food. Do not have mutagenic or carcinogenic effects (COSTA, 2007). However peroxyacetic acid has low stability during storage and handling must be done carefully. A study performed by Hilgren & Salverda (2000) showed a significant reduction in the total count of bacteria and fungi in vegetables treated with peroxyacetic acid. Alvarenga et al. (1991) found that after 1, 3 and 5 minutes of contact with peracetic acid at a concentration of 300mg.L-1 reached respectively 0.43, 1.2 and 2.8 decimal reductions in the population of spores of *Bacillus subtilis*.

Also according to Nascimento (2002), there was no significant difference to the performances of the peracetic acid compared to sodium hypochlorite. Similar results were reported by Farrell et al. (1998), Sapers et al. (1999) and Wisniewsky et al. (2000). However other authors have demonstrated the superiority of peracetic acid when compared to the sodium hypochlorite in the presence of organic matter. Jones et al. (1992) got a reduction of 3 log cycle for *Vibrio cholerae* and *E. coli* using peracetic acid (25ppm) when compared to sodium hypochlorite (25 ppm). Thus, although there are a number of studies reported in the international literature, most of the time these were carried out under different conditions not allowing comparisons. Therefore, further studies are needed to know the effectiveness of sanitizers in the real conditions of use, working with vegetables available in the local market, with its natural contaminant microbiota unchanged. It is also interesting the implementation in the food sector, of a rotation between different sanitizers thereby preventing the development of resistance by microorganisms to the active principles of the same.
