**5. Chlorine dioxide and peracetic acid as sanitizers to control microorganisms presents in minimally processed chicory (***cichorium endivia* **l.) and rocket (***eruca vesicaria sativa***)**

#### **Combined effect of type, concentration and action time of sanitizer in the microbial control of minimally processed chicory and rocket. An observation.**

Sodium hypochloride has been the sanitizer usually used to reduce the microbial counting in minimally processed vegetables, although its use is questioned due to be precursor in the formation of organic chloramines, compounds of high carcinogenic potential. As a consequence of this fact, other sanitizers have been proposed to replace it, among them chlorine dioxide and peracetic acid. Therefore in this work chlorine dioxide (10, 25 and 50ppm/2, 5 and 10min) and peracetic acid (50, 75 and 100ppm/4, 7 and 10min) were compared with sodium hypochloride (120ppm/15min) in the control of natural microbiota of minimally processed rocket and chicory.

In green leafy vegetables, the physical form of the vegetable being processed is very important because certain types of leaves are difficult to be washed and sanitized requiring greater care. The leafy vegetables, rocket and chicory, present this kind of difficulty, which by being consumed as salad, so fresh, are potentially risk factors, that's why they were chosen for the work associated with their high consumption.

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

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

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

**5. Chlorine dioxide and peracetic acid as sanitizers to control microorganisms presents in minimally processed chicory (***cichorium endivia* **l.) and rocket** 

Sodium hypochloride has been the sanitizer usually used to reduce the microbial counting in minimally processed vegetables, although its use is questioned due to be precursor in the formation of organic chloramines, compounds of high carcinogenic potential. As a consequence of this fact, other sanitizers have been proposed to replace it, among them chlorine dioxide and peracetic acid. Therefore in this work chlorine dioxide (10, 25 and 50ppm/2, 5 and 10min) and peracetic acid (50, 75 and 100ppm/4, 7 and 10min) were compared with sodium hypochloride (120ppm/15min) in the control of natural microbiota of

In green leafy vegetables, the physical form of the vegetable being processed is very important because certain types of leaves are difficult to be washed and sanitized requiring greater care. The leafy vegetables, rocket and chicory, present this kind of difficulty, which by being consumed as salad, so fresh, are potentially risk factors, that's why they were

**Combined effect of type, concentration and action time of sanitizer in the microbial** 

**control of minimally processed chicory and rocket. An observation.** 

chosen for the work associated with their high consumption.

0.43, 1.2 and 2.8 decimal reductions in the population of spores of *Bacillus subtilis*.

an effective biocide.

active principles of the same.

**(***eruca vesicaria sativa***)** 

minimally processed rocket and chicory.

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 2.37 log CFU.g-1).

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 smooth leaf.

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 naturally present in chicory minimally processed.


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 treatments.

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

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

Occurrence of *Salmonella* in Minimally Processed Vegetables 119

(1.48 log CFU.g-1) and statistically different from the others. In the case of peracetic acid, 2 treatments were better: 100ppm/10min (1.10 log CFU.g-1) and 100ppm/7min (1.44 log CFU.g-1) and they were statistically different from the others, however not different from each other. Therefore, as far as the control of total coliform in minimally processed chicory under the conditions of the treatments performed peracetic acid was more effective than

In the case of the action of chlorine dioxide on the total coliform in minimally processed rocket only one treatment (50ppm/10min) provided results (3.85 log CFU.g-1) in the same logarithmic cycle of the standard treatment (3.52 log CFU.g-1) being statistically different from the others. When peracetic acid was used as sanitizer, only one treatment (100ppm/10min) was able to reduce the count of total coliforms to below the standard, respectively 2.87 x 3.52 log CFU.g-1. Other 3 treatments (100ppm/4min, and 100ppm/7min 75ppm/10min) provided counts (3.65 log CFU.g-1, 3.33 log CFU.g-1 and 3.45 log CFU.g-1)

(Min) 10ppm 25ppm 50ppm MSD1 2 3.088 0.647 a, A 2.944 0.613 a, A 2.302 0.424 a, A 1.428 5 2.820 0.535 a, A 2.578 0.561 a, A 2.014 0.399 a, A, B 1.213 10 2.544 0.561 a, A 2.423 0.515 a,b, A 1.339 0.308 b, B 1.883 MSD2 1.460 1.415 0.953 ----

(Min) 50ppm 75ppm 100ppm MSD1 4 3.446 0.143 a, A 3.256 0.194 a, A 2.344 0.292 b, A 0.547 7 2.806 0.412 a, A, B 2.681 0.397 a, A, B 1.440 0.095 b, B 0.839 10 2.310 0.544 a, B 2.170 0.492 a,b, B 1.100 0.174 b, B 1.090 MSD2 1.008 0.957 0.510 ----

Blank (washing and immersion in tap water for 15 minutes) \*\* .(log CFU.g-1) 5.587

hypochlorite: 120ppm/15min) \*\* ……………………………….…(log CFU.g-1) 1.480

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

All samples of minimally processed chicory and rocket, treated with chlorine dioxide and peracetic acid were reduced by two logarithmic cycles for *Escherichia coli*, ie, an initial count of 2.86 log CFU.g-1 in the treatment by washing and immersion in water to less than 1.00 log CFU.g-1. However, in the sample of standard treatment there was a total control,

Table 3. Total coliform count (log CFU.g-1) observed in samples of minimally processed

similar to the standard (3.52 log CFU.g-1) being in the same log cycle.

Time\* Treatment with chlorine dioxide (ClO2)

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

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

chlorine dioxide.

treatments.

chicory.

that is, no growth.

at concentrations of 75ppm/10min and 100ppm/10min proved to be statically superior to others, but without showing any significant difference between them. Just as in the treatments with chlorine dioxide, peracetic acid treatments had reduced to the equivalent of 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 (blank). Treatment with chlorine dioxide and peracetic acid, described above as having showed the best results in terms of population control of yeasts and molds in chicory, showed the same level of standard treatment (2 log CFU.g-1). When the same treatments were performed using minimally processed rocket (Table 2), the counts were higher and showed no significant differences between them, as much for the treatments with chlorine dioxide as for treatment with peracetic acid. However, even with no significant difference between them, the greatest reductions in populations of yeasts and molds were obtained in the case of peracetic acid treatments, with 100ppm/10min and in the case of chlorine dioxide with 50ppm/10min.


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 treatments.

Table 2. Yeast and mold count (log CFU.g-1) observed in samples of minimally processed rocket.

As for the action of these sanitizers in counts of total coliform in chicory (Table 3) and rocket (Table 4), minimally processed, the response was almost linear and inversely proportional, that is, when the concentration of sanitizers or their periods of contact were increased, the population of total coliforms also decreased.

Referring to the action of chlorine dioxide on the total coliform in chicory only the treatment with 50ppm/10min showed the same log cycle (1.34 log CFU.g-1) of the standard treatment

at concentrations of 75ppm/10min and 100ppm/10min proved to be statically superior to others, but without showing any significant difference between them. Just as in the treatments with chlorine dioxide, peracetic acid treatments had reduced to the equivalent of 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 (blank). Treatment with chlorine dioxide and peracetic acid, described above as having showed the best results in terms of population control of yeasts and molds in chicory, showed the same level of standard treatment (2 log CFU.g-1). When the same treatments were performed using minimally processed rocket (Table 2), the counts were higher and showed no significant differences between them, as much for the treatments with chlorine dioxide as for treatment with peracetic acid. However, even with no significant difference between them, the greatest reductions in populations of yeasts and molds were obtained in the case of peracetic acid treatments, with 100ppm/10min and in the case of chlorine dioxide with 50ppm/10min.

(Min) 10ppm 25ppm 50ppm MSD1 2 5.149 0.544 a, A 4.433 0.538 a, A 4.078 0.479 a, A 1.305 5 4.839 0.504 a, A 4.127 0.463 a, A 3.709 0.387 a, A 1.138 10 4.327 0.375 a, A 3.797 0.439 a, A 3.371 0.370 a, A 0.992 MSD2 1.202 1.207 1.039 ----

(Min) 50ppm 75ppm 100ppm MSD1 4 4.314 0.425 a, A 3.869 0.577 a, A 3.400 0.593 a, A 1.345 7 3.998 0.472 a, A 3.563 0.640 a, A 3.020 0.692 a, A 1.525 10 3.594 0.468 a, A 3.160 0.690 a, A 2.644 0.673 a, A 1.549 MSD2 1.141 1.596 1.638 ---- Blank (washing and immersion in tap water for 15 minutes) \*\* (log CFU.g-1) 5.896

hypochlorite: 120ppm/15min) \*\* …………………………………(log CFU.g-1) 2.400

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

As for the action of these sanitizers in counts of total coliform in chicory (Table 3) and rocket (Table 4), minimally processed, the response was almost linear and inversely proportional, that is, when the concentration of sanitizers or their periods of contact were increased, the

Referring to the action of chlorine dioxide on the total coliform in chicory only the treatment with 50ppm/10min showed the same log cycle (1.34 log CFU.g-1) of the standard treatment

Time\* Treatment with chlorine dioxide (ClO2)

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

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

population of total coliforms also decreased.

treatments.

rocket.

(1.48 log CFU.g-1) and statistically different from the others. In the case of peracetic acid, 2 treatments were better: 100ppm/10min (1.10 log CFU.g-1) and 100ppm/7min (1.44 log CFU.g-1) and they were statistically different from the others, however not different from each other. Therefore, as far as the control of total coliform in minimally processed chicory under the conditions of the treatments performed peracetic acid was more effective than chlorine dioxide.

In the case of the action of chlorine dioxide on the total coliform in minimally processed rocket only one treatment (50ppm/10min) provided results (3.85 log CFU.g-1) in the same logarithmic cycle of the standard treatment (3.52 log CFU.g-1) being statistically different from the others. When peracetic acid was used as sanitizer, only one treatment (100ppm/10min) was able to reduce the count of total coliforms to below the standard, respectively 2.87 x 3.52 log CFU.g-1. Other 3 treatments (100ppm/4min, and 100ppm/7min 75ppm/10min) provided counts (3.65 log CFU.g-1, 3.33 log CFU.g-1 and 3.45 log CFU.g-1) similar to the standard (3.52 log CFU.g-1) being in the same log cycle.


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 treatments.

Table 3. Total coliform count (log CFU.g-1) observed in samples of minimally processed chicory.

All samples of minimally processed chicory and rocket, treated with chlorine dioxide and peracetic acid were reduced by two logarithmic cycles for *Escherichia coli*, ie, an initial count of 2.86 log CFU.g-1 in the treatment by washing and immersion in water to less than 1.00 log CFU.g-1. However, in the sample of standard treatment there was a total control, that is, no growth.

Occurrence of *Salmonella* in Minimally Processed Vegetables 121

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There was no *Salmonella* sp./25g in all samples analyzed.

Therefore, when the results of the best treatments were considered, the two sanitizers tested proved to be as effective as treatment with sodium hypochlorite. Thus, both chlorine dioxide and peracetic acid are able to replace the sodium hypochlorite in concentrations and times considered (50ppm/10min chlorine dioxide, peracetic acid and the 100ppm/10min 120ppm/15min sodium hypochlorite). On the other hand, none of the sanitizers caused any kind of physical or unfavorable organoleptic product changes (wilting, darkening, strong odor, color change etc.) at the concentration levels studied.


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 treatments.

Table 4. Total coliform count (log CFU.g-1) observed in samples of minimally processed rocket.
