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2nd Mercosur Congress on Chemical Engineering
4th Mercosur Congress on Process Systems Engineering
1
Comparison of the Decontamination Treatments Used For Reducing the
Initial Levels of Microorganisms From Fresh-cut Carrot
Abreu, M.1*; Alves, A.2.; Gonçalves, E. M.1.; Alegria, C.1; Peito, A.1.; Fernandes,.I.1; Moldão-Martins, M.2
1DTIA, Instituto Nacional de Engenharia, Tecnologia e Inovação. Portugal;
2 Centro de Estudos Agro-Alimentares, Instituto Superior de Agronomia. Portugal
Abstract. Shredded carrot represents an important component of the fresh-cut vegetable production, due to
their convenience and freshness. Like other minimally fresh-cut vegetables, shredded carrots may have high
levels of microorganisms. In commercial production, mesophilic counts of about 106 to 107 have been
reported. The decontamination of fresh produce can reduce the numbers of spoilage microorganisms and
consequently help to achieve a longer shelf life for the product. Commercial processes for preparing fresh-cut
carrots usually use chlorinated water to control the microbial population. Nevertheless, several drawbacks
have been identified, including the formation of potentially hazardous chlorinated organic compounds upon
treatment of vegetables. Therefore new and innovative environmentally friendly treatments need to be
developed. The objective of the present study was to compare the potential of decontamination treatments for
inactivation and/or removal of microorganisms on the surfaces of shredded carrot. The treatments tested
were: chlorinated water (200 ppm / 1 minute), ultrasound with water (40 and 47 kHz, for 1 / 2 minutes),
ultrasound (at the same frequencies and times) with chlorinated water (200 ppm), and ozonated water (0.5
ppm/15 minutes). Mesophilic aerobic flora, and yeasts & moulds were enumerated as Log 10 colony-forming
units (CFU) g-1 in the shredded carrot samples, commercially prepared, before and after each
decontamination treatment (laboratory scale). Sonication treatments of shredded carrot in water had only a
marginal effect on microbial reduction, regardless the frequencies and times applied. Reductions in
mesophilic flora yeast and moulds, obtained by the combined ultrasonic in chlorinated water attained a
similar efficacy compared to populations detected on shredded carrot treated with chlorine, as single
treatment. The ozonation treatment containing an initial concentration of 0.5 ppm ozone, failed to cause any
reduction in microbial populations on shredded carrot.
Keywords: Fresh-cut products, carrot, anti-microbial treatments, chlorine, ultrasound, ozone.
1. Introduction
Minimally processed (or fresh-cut) fruits and vegetables are fresh-like products, raw foods, which are
usually processed and sold to consumers in a ready-to-eat form (Wiley, 1994). However, the physical effects of
minimal processing induce or accelerate many physiological/biochemical aspects on the vegetables. Minimal
processing also accelerates microbial proliferation on vegetables by breaking protective surface structures and
increasing the availability of nutrients. The presence of spoilage bacteria, yeasts & moulds and occasional
pathogen on fresh produce has been recognized for many years (Beuchat, 1998). In commercial shredded
carrots, an important component of the fresh-cut vegetable production due to their convenience, mesophilic
counts of ca 106 to 107 have been reported (Nguyen and Carlin, 1994). The decontamination of fresh produce
can reduce the numbers of spoilage microorganisms and consequently help to achieve a longer shelf life.
However, in general, the usual decontamination techniques cannot guarantee the microbiological safety of
fresh-cut products without compromising their quality (Seymour, 1999).
* Address: DTIA - INETI, Estrada do Paço do Lumiar, 22, 1649-038 Lisboa - Portugal
E-mail: marta.abreu@mail2.ineti.pt
2nd Mercosur Congress on Chemical Engineering
4th Mercosur Congress on Process Systems Engineering
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Anti-microbial agent usually used to reduce initial contamination of minimally processed vegetables is
chlorinated water (ranging from 50 to 200 ppm with a contact time of 1 to 2 min). Nevertheless, several
drawbacks have been identified, including the real capacity of free chlorine in destroy or eliminate bacteria
present in vegetables surfaces (Cena, 1998; Liangji Xu, 1999) and the production of chlorinated organic
compounds, such as trihalomethanes that impact on human and environmental (Liangji Xu, 1999; Wei et al.,
1999). Therefore new and innovative environmentally friendly methodologies need to be developed.
Power ultrasound at the frequencies range between 20-100 kHz, has a potential application to fresh produce
decontamination, namely combined with other treatments, such as chorine. Ultrasound fields origin cavitation,
which is, the formation of bubbles in agitation. The bubble oscillation generates mechanical energy, which has
an effective action during decontamination of vegetables surfaces. To ensure the inactivation of the most
resistant microorganisms high-intensity ultrasound insonation is required as single treatment but the impact on
food quality may be negative (Seymour et al., 2002). Ultrasound in combination with chlorine shows higher
potentialities in decontamination because it helps the release of microorganisms from difficult access locations
in the vegetables surfaces, becoming more vulnerable to the lethal action of the chemical (Seymour et al.,
2002).
Ozone presents the capacity of destruct bacteria in a faster way than chlorine solutions and other
decontaminants without produce any secondary compounds. The potential application of ozone in the food
industry is based on ozone power oxidant capacity, even in lower concentrations (<3 ppm) and presents an
effective action over a higher number of microorganisms (Liangji Xu, 1999).
The objective of the present study was to assess the anti-microbial effect of different methodologies
(chlorine, ultrasound and ozonated water) on shredded carrot.
2. Materials and Methods
2.1 Carrot
Carrot (Daucus carrota L.) cv Nante, was used in the experiments. Shredded carrot was minimal processed
(not subjected to any anti-microbial agent) in a fresh-cut industry near Lisbon (Campotec), and immediately
transported (refrigerated) to the laboratory in bags. At the laboratory the shredded carrot was immediately
submitted to the decontamination treatments (laboratory scale). Aseptic conditions were maintained throughout
all the experiments to ensure accurate assessment of decontamination.
2.2 Anti-microbial methods
For each decontamination treatments shredded carrot directly from the bags were used as control samples.
Chlorinated water. The decontaminant treatment involved washing for 1 min in a chlorine solution (200
ppm free chlorine) at 5ºC (identified as HIPO samples).
Shredded carrot decontaminated was allowed to drip dry (manual centrifuge) for 1 min to remove excess
moisture and packed in aseptic bags (100 g). The bags were sealed and analysed for mesophilic aerobic flora
and yeasts & moulds groups. Three replicates were used performing the treatments independently.
2nd Mercosur Congress on Chemical Engineering
4th Mercosur Congress on Process Systems Engineering
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Ultrasound. The ultrasound decontamination treatments (single and combined with chlorine) were
established combining 3 variables: operating frequency (0, 40 and 47 kHz), treatment time (1 and 2 minutes)
and immersion solution (water and chlorine water with 200 ppm free chlorine). The total of 10 different
condition treatments (single and combined) taken in triplicates (100 g each) were identified as: HIPO_1min;
HIPO_2min; 40 kHz_1min; 40 kHz_2 min; 47 kHz_1min; 47 kHz_2 min; HIPO x 40 kHz_1min; HIPO x 40
kHz_2min; HIPO x 47 kHz_1min; HIPO x 47 kHz_2min.
An ultrasound tank (Branson 3510 for 40 kHz or Branson 2200 for 47 kHz) was filled with distilled water,
with / without 200 ppm free chlorine and degassed at the operating frequency for 5 min before treatments. The
decontamination processes were conducted by dipping the shredded carrot into the tank at the different
conditions. After dipping treatments the shredded carrots were processed as described above.
Ozonated water. Ozone gas bubbled into a tank containing water (120 L) was produced using an SPO3
model OZ5 equipment. For preparation of aqueous ozone, concentration was monitored continuously by
measuring the redox potential (Redox probe pH 7685, calibrated with Redox Buffer solutions of 220 mV and
468 mV from Mettler Toledo). Each sample treatment (identified as O3_samples) was comprised of 100 g of
shredded carrot dipped into ozonated water during 5 minutes that had been previously ozonated to achieve an
aqueous ozone concentration of 0.5 ppm. After dipping treatments the shredded carrots samples (triplicates)
were processed as described above.
2.3 Microbial analysis
Mesophilic aerobic flora was enumerated according to EN ISO 4833. Yeasts & moulds were enumerated
using Rose Bengal Chloramphenicol Agar, surface inoculation, incubated at 25°C during 5 days.
Microbiological counts were expressed as Log10 CFU per gram. The mean of the results for each of the
treatments was subtracted from the mean of the respective control to give an average log reduction in the
attached microorganisms groups.
3. Results and Discussion
Reductions in mesophilic aerobic flora and yeasts & moulds populations as affected by decontaminated
treatments with chlorinated water (200 ppm free chlorine), ultrasound (40 and 47 kHz; 1 and 2 min) and
ozonated water (0.5 ppm) are shown in fig. 1.
In what concerns single decontamination treatments “per si” (fig. 1) the chlorinated treatment, was the only
one that effectively reduced the initial microbial population present in shredded carrot, being more effective
against mesophilic flora (around 2 Log10 CFU/g) than yeasts & moulds group (around 1 Log10 CFU/g).
Regardless frequency and time treatments applied, the anti-microbial effects exerted by sonication as single
treatment, was diminutive, never exceeding 0.3 and 0.1 Log10 reduction for mesophilic flora and yeasts &
moulds group, respectively. Again yeasts & moulds group showed to be more resistant to the treatments than
mesophilic group, except for 47 kHz / 1 minute condition that attained 0.7 Log10 reduction. In fact this result
2nd Mercosur Congress on Chemical Engineering
4th Mercosur Congress on Process Systems Engineering
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appears to be misleading because when the treatment time increased (2 min) the microbial reduction was similar
to the other sonication conditions.
Ozonated water (0.5 ppm during 5 min) showed to be the least effective treatment (up to 0.1 Log10 reduction
maximum) for the mesophilic group, being ineffective against yeasts & moulds group. The reduced efficacy of
ozonated water bath might be due to more ozone demand of organic material in the medium competing with
microorganisms for the available ozone (Achen & Yousef, 2001). This effect can be critical in shredded carrot
due to the great surface area of internal tissue exposed.
Fig. 1. Average microbial reductions (Log 10) attained with chlorine, ultrasound and ozone decontamination as single
treatments, in shredded carrot industrial processed. Bars indicate one-sided standard deviations.
Fig. 2. Average microbial reductions (Log 10) attained with chlorine and ultrasound combined decontamination at two
frequencies (40 and 47 kHz) and two treatment times (1 and 2 min) in shredded carrot industrial processed. Bars indicate
one-sided standard deviations.
2nd Mercosur Congress on Chemical Engineering
4th Mercosur Congress on Process Systems Engineering
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The results obtained (fig. 2) indicate that chlorine and ultrasound in combination (throughout frequencies
and times assayed in chlorinated water with 200 ppm) were less effective at decontaminating shredded carrot
than chlorine alone for both microbial groups enumerated. While the effectiveness of 47 kHz x HIPO (1 and 2
min) was similar to that of chlorine alone for the mesophilic group (almost 2 Log10 reduction), nearly half
efficacy was attained for the yeasts & mould group. Worst microbial reductions were obtained with the
combined treatment at 40 kHz (1 and 2 min), around 1 Log10 and 0.5 Log10 reductions for mesophilic and yeasts
& mould group, respectively, with one exception. That one exception was 1.5 Log10 reduction obtained for the
yeast & mould group with the combined treatment 47 kHz x HIPO_2.
The decontamination efficacy of shredded carrot was not improved by sonication in chlorinated water (at
both frequencies and times tested) as observed on iceberg lettuce (Seymour et al, 2002). The great surface area
associated to shredded carrot for microbial attachment and the infiltration of microorganisms into points below
the surfaces providing some level of physical protection for bacterial against cavitation might explain these
failure results.
4. Conclusion
Among all treatments assayed the washing with chlorinated water at 200 ppm, was the most effective
methodology to reduced microbial load of shredded carrot, being the reduction levels of 2 and 1 Log10 cycles
for mesophilic and yeasts & moulds groups, respectively. In order to achieve an anti-microbial effect
comparable to chlorine, a more efficient ozone and ultrasound technologies are required.
5. References
Achen, M. & Yousef, A.E. (2001) Efficacy of Ozone Against Escherichia coli O157:H7 on apples. Journal of Food Science
66 (9): 1380-1384.
Beuchat, L.R. (1998). Surface decontamination of fruits and vegetables eaten raw: a review. Food Safety Issues. Food Safety
Unit. World Health Organization.
Cena, A., (1998). Ozone: Keep it fresh for food processing. Water Conditioning Purification. Sept: 112-115.
Liangji Xu (1999). Use of ozone to improve the safety of fresh fruits and vegetables. Food Technology 53(10): 58-63.
Nguyen-The, C., Carlin, F. (1994). The microbiology of minimally processed fresh fruits and vegetables. Critical Reviews In
Food Science and Nutrition 34:371-401.
Seymour I.J. (1999). Review of current industry practice on fruit and vegetable descontamination. Campden and
Chorleywood Food Research Association, Glos, UK: CCFRA Review nº14.
Seymour, I.J., Burfoot, D., Smith, R.L., Cox, L.A., Lockwood, A. (2002). Ultrasound decontamination of minimally
processed fruits and vegetables. International Journal of Food Science and Technology, 37, 547-557pp.
Wei, C.I., Huang, T.S., Kim, J.M., Lin, W.F., Tamplin, M.L., Bartz, J.A. (1999). Growth and survival of Salmonella
montevideo on tomatoes and disinfection with clorinated water. Journal of Food Protection 58: 829-836.
Willey, R.C. (1994). Minimally Processed Refrigerated Fruits and Vegetables. London, UK: Chapman & Hall.
Acknowledgments
Financial support provided by POCTI Med. 2.3 – CLEANTECH Project.
