The Optimum Disinfection Treatment According To The Antibacterial Efficacy & Impact On Vegetable Quality

RESFOOD
RESOURCE EFFICIENT AND SAFE FOOD PRODUCTION AND PROCESSING

GA No. 308316
Deliverable No. D5.1

Deliverable Title The optimum disinfection treatment
according to the antibacterial efficacy &
impact on vegetable quality
Document ID RESFOOD-D5.1 FINAL v2.0
Dissemination level Public
Main Author Eva Petri - CNTA
Issue date 8-OCTOBER-2014
EUROPEAN COMMISSION DG Environment

SEVENTH FRAMEWORK PROGRAMME THEME ENVIRONMENT FP7-ENV-2012.6.3-1
RESFOOD-D5.1-FINAL v2.0
Disclaimer and acknowledgement
This project is co-funded by the 7th FP (Seventh

Framework Programme) of the EC - European
Commission DG Environment
Disclaimer
The FP7 project has been made possible by a financial contribution by the European
Commission under Framework Programme 7.
This document reflects the views of the author(s) and does not necessarily reflect the views
or policy of the European Commission. Whilst efforts have been made to ensure the
accuracy and completeness of this document, the RESFOOD consortium shall not be liable
for any errors or omissions, however caused.
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Document information
Additional author(s) and contributing partners
Name Organisation
Silvia García CNTA
Eric Lefebvre KRONEN
Hülya Ölmez TÜBITAK
Sinem Malkoc TÜBITAK
Document Change Log
Version Date Comments
v0.1 19/06/14 First draft of document
v0.2 22/09/14 Revised version based on the comments of WP5 partners: Silvia García
(CNTA), Eric Lefebvre (KRONEN), Hülya Ölmez (TÜBITAK)
v0.3 01/10/14 Final draft, approved by WP leader and partners, submitted to Executive
Board
v1.0 03/10/14 First final version, approved by Executive Board, (will be) submitted to
EC.
v1.1 First draft based upon first final version
v2.0 08/10/14 Second final version, approved by Executive Board, (will be) submitted
to EC.
Document Distribution Log
Version Date Distributed to
v0.1 19/09/14 WP5 partners: KRONEN, TÜBITAK, TNO, VEGA MAYOR
v0.2 22/09/14 WP5 partners: KRONEN, TÜBITAK, TNO, VEGA MAYOR
v0.3 01/10/14 EXECUTIVE BOARD, KRONEN, TÜBITAK, TNO, VEGA MAYOR
Verification and approval
Name Date
Verification Final Draft by WP leader Eva Petri 30/09/2014
Approval Final Deliverable by Willy van Tongeren 03/10/14

coordinator
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Executive summary
The goal of the RESFOOD project is to develop and test/demonstrate innovative “green”
solutions towards resource efficient and safe food production and processing, leading to
reduced resource use, maximized resource productivity, recovery of valuable compounds
and increased food safety.
The main objective of WP5 of the RESFOOD project is reducing health and safety risks by
mitigating foodborne outbreaks by introducing more effective and less water consuming
disinfection technologies.
The first task focuses on the study of the antimicrobial effectiveness on the proposed
vegetables (multi-hurdle approach) and the two other tasks on the evaluation of effluent
and product quality, respectively.
Initially, research was based on the multiple hurdles concept, where a variety of physical-
chemical hurdles are applied simultaneously in combined form. The principle of this concept
is that the use of combined preservative factors may have greater effectiveness at
reducing/eliminating microorganisms than the use of any single factor. In this work, hurdle
technology consisted on the use of positive / negative pressure to improve the contact
between the sanitizing agent and bacteria attached in inaccessible sites on the product
surface by removing gas or liquid barriers than block penetration of the sanitizing agent.
However, the studies performed showed that combination treatments were equally
effective in reducing levels of microorganism than were single treatments, with the
exception of some isolated cases, and in some cases, negative effect on the sensory
characteristics was observed.
Therefore, bearing in mind that sanitizing treatment typically achieve 1-2 log units’
reductions in microbial populations on fresh-cut vegetables and they are unlikely to greatly
increase efficacy in decontaminating produce, the incremental improvements of work was
addressed to:
1. select the most adequate sanitizing strategies at atmospheric pressure (efficiency
comparison of numerous sanitizing agents, minimum effective dose, improved contact
between product and water, etc), for the prevention of waterborne product cross-
contamination
2. improve the water quality during washing for water reuse. So, the disinfection capacity
and efficacy in preventing cross-contamination of product as well as their effect on
physicochemical quality of wash water will be evaluated
In the present work, the effectiveness of chlorine and other sanitizer agents was evaluated
on the microorganism’s reduction, both on produce and on washing water. Different
sanitizer concentrations (based on literature and on manufacturer’s recommendation) were
tested. The antimicrobial efficacy of treatments was evaluated, being all the tested washing
solutions more effective in reducing the microbial load that water alone. However, the use
of some of them, did not show a sufficient level of antimicrobial activity (e.g. Neutral
Electrolyzed Water, Hydrogen peroxide, ozone) compared to chlorine, or its use was
associated with a detrimental quality of produce (e.g. lactic acid) or it had an negative
impact on the wash water quality, characterized mainly by an increase on COD content.
On the basis of the experiments performed with different agents on minimally processed
lettuce and carrots and on the discussion regarding their specific strengths and weakness,
two strategies (peroxyacetic acid based sanitizer and chlorine dioxide) have been selected
for subsequent industrial upscaling. This implementation is needed because of the growing
need for having a survey of scientific data and practical experiences related to the various
sanitizing techniques, their efficacy and usefulness in being applied to control the microbial
load on the real conditions of fresh-cut processing. Therefore, WP8 will evaluate the
available knowledge regarding those sanitizers, also providing some information on their
industrial application. The purpose of this study intends to provide information, based on
primary investigation, to help the decision making process and the establishments of
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policies and strategies taken by relevant authorities regarding the use of alternative
sanitizers during the washing stage.
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Table of contents
Disclaimer and acknowledgement ............................................................................ 2
Document information............................................................................................ 3
Executive summary ............................................................................................... 4
Table of contents ................................................................................................... 6
List of Figures ....................................................................................................... 8
List of Tables ........................................................................................................ 9
1. Introduction ...................................................................................................10
1.1 Background RESFOOD ..............................................................................10
1.2 Purpose of the document...........................................................................12
1.3 Document Structure .................................................................................12
1.4 Deviations from original DoW .....................................................................12
1.4.1 Description of work related to deliverable as given in DoW ......................12
1.4.2 Time deviations from original DoW .......................................................14
1.4.3 Content deviations from original DoW ...................................................14
2. Material and methods .....................................................................................16
2.1 Bacterial strains .......................................................................................16
2.2 Preparation of inoculum ............................................................................16
2.3 Sample preparation ..................................................................................16
2.4 Inoculation procedure ...............................................................................16
2.5 Application of sanitizing treatments ............................................................19
2.6 Washing solutions ....................................................................................21
2.6.1 Chlorine ............................................................................................21
2.6.2 Organic acids .....................................................................................21
2.6.3 Chlorine dioxide .................................................................................21
2.6.4 Hydrogen peroxide .............................................................................21
2.6.5 Electrolyzed water ..............................................................................22
2.6.6 Ozone ...............................................................................................22
2.6.7 Peroxyacetic acid ...............................................................................23
2.7 Standardized process wash water in the COD content ...................................23
2.8 Physical-chemical analysis .........................................................................23
2.8.1 Measurement of pH ............................................................................23
2.8.2 Determination of trihalomethane compounds .........................................23
2.8.3 Measurement of COD ..........................................................................24
2.8.4 Measurement of aquatic toxicity of waste process water..........................24
2.9 Microbiological analysis .............................................................................24
2.10 Sensory analysis ...................................................................................24
2.11 Data analysis ........................................................................................24
3. Results and discussion ....................................................................................25
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3.1 Multi hurdle approach: antimicrobial effectiveness of combined treatments on

vegetables to mitigate the risk of food-borne illness and with minimum impact on
environment and sustainability (experiments from Dec’12 to Nov’13) ......................25
3.1.1 Initial characterization of samples ........................................................25
3.1.2 Efficiency of different combined treatments to prevent E.coli O157:H7
contamination of fresh-cut products ..................................................................25
3.1.3 Effect of different combined treatments in the quality of fresh-cut products
28
3.1.4 Efficiency of different combined treatments to extend the shelf life of fresh-
cut products ..................................................................................................31
3.2 Efficiency of different sanitizing treatments to prevent cross- contamination of
fresh-cut products (experiments from Jan’14 to Sept’14) .......................................33
3.2.1 Experiments with decreasing initial chlorine concentrations in the washing
water 33
3.2.2 Effect of NEW washing solutions on bacterial inactivation on lettuce and carrot
processing .....................................................................................................35
3.2.3 Effect of ozone (O3) washing solutions on bacterial inactivation on lettuce and
carrot processing ............................................................................................37
3.2.4 Effect of hydrogen peroxide (H2O2) washing solutions on bacterial inactivation
on lettuce and carrot processing .......................................................................38
3.2.5 Effect of peroxyacetic acid (PAA) and hydrogen peroxide (H 2O2) washing
solutions on bacterial inactivation on lettuce and carrot processing .......................40
3.2.6 Effect of lactic acid (LA) washing solutions on bacterial inactivation on lettuce
and carrot processing .....................................................................................44
3.2.7 Effect of chlorine dioxide (ClO2) washing solutions on bacterial inactivation on
the processing water of lettuce and carrot .........................................................45
4. Conclusions and recommendations ...................................................................46
5. References ....................................................................................................49
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List of Figures
Figure 2-1 Comparison of attachment strength of pathogenic bacteria to iceberg lettuce
leaf after 5-60 min of inoculation a) immediately after inoculation, b) after 48 h. of
incubation at 4ºC .................................................................................................17
Figure 2-2 Formation of biofilms and penetration into the stomata by E.coli O157:H7, L.
monocytogenes, S. enterica on iceberg lettuce leaf. ..................................................18
Figure 2-3 Flow diagram of minimal processing for lettuce samples (same flow diagram for
carrot samples was used) ......................................................................................19
Figure 2-4 Semi-industrial equipment used for achieving: a) positive pressures
(Formulador PC-20) and b) negative pressures /vacuum (GASTROVAC) ......................20
Figure 2-5 Neutral electrolyzed water generator (ANE, INNOBAK SYSTEMS) ................22
Figure 3-1 Lettuce appearance after applying vacuum treatment at 30 mbar ...............29
Figure 3-2 Lettuce and carrot appearance after pressure treatment at 3 bar ................29
Figure 3-3 Lettuce and carrot appearance after applying vacuum treatment at 30 mbar +
lactic acid ............................................................................................................30
Figure 3-4 Lettuce and carrot appearance after applying vacuum and pressure treatments
+ hydrogen peroxide ............................................................................................30
Figure 3-5 Lettuce and carrot appearance after applying vacuum and pressure treatments
+ neutral electrolyzed water (NEW) ........................................................................31
Figure 3-6 Effect of the sanitizing treatment on the microbial counts during the shelf-life
study of lettuce processing at 4ºC (HP: hydrogen peroxide; P: positive pressure; Patm:
atmospheric pressure; CW: chlorinated water) .........................................................32
Figure 3-7 Effect of the sanitizing treatment on the microbial counts during the shelf-life
study of carrot processing at 4ºC (HP: hydrogen peroxide; P: positive pressure; Patm:
atmospheric pressure; CW: chlorinated water) .........................................................32
Figure 3-8 Effect of decreasing free chlorine levels on the formation of THMs in process
wash water ..........................................................................................................35
Figure 3-9 Aerobic mesophilic load in the processing of fresh-cut lettuce and carrot (COD:
1000 mg/L) treated with NEW (50, 75 and 100 ppm) chlorine (100 ppm) and tap water at
4ºC. Red line indicate the initial load of product (fresh-cut lettuce) before submerging in
washing solution ..................................................................................................35
Figure 3-10 Schematic representation of the system used for ozone treatments ...........37
1000 mg/L) treated with H2O2 (0,25%, 0,5%, 0,75% and 1%), chlorine (100 ppm) and
tap water at 4ºC. Red line indicate the initial load of product (fresh-cut lettuce) before
submerging in washing solution ..............................................................................38
1000 mg/L) treated with PAA + H2O2 (0,250%, 0,375%, 0,500 %), chlorine (100 ppm)
and tap water at 4ºC. Red line indicate the initial load of product (fresh-cut lettuce) before
submerging in washing solution ..............................................................................40
Figure 3-13 Appearance of minimally processed lettuce along the storage period at 4ºC41
1000 mg/L) treated with lactic acid (0,25, 0,1%), chlorine (100 ppm) and tap water at
4ºC. Red line indicate the initial load of product (fresh-cut lettuce) before submerging in
washing solution ..................................................................................................44
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List of Tables
Table 1-1 Activities within WP5 of the RESFOOD project ............................................10
Table 2-1 Sanitizer treatments and concentration of active components (based on technical
specification sheet from manufacturers) ..................................................................23
Table 3-1 Initial microbial population on Iceberg lettuce and carrots ...........................25
Table 3-2 E.coli O157:H7 log reduction of washed inoculated fresh-cut lettuce using tap
water, chlorine (100 mg/l) and lactic acid (0,25%, 0,50%, 1%, 2%) at 4ºC. ..............26
water, chlorine (100 mg/l) and citric acid (0,25%, 0,50%, 1%) at 4ºC........................26
water, chlorine (100 mg/l) and acetic acid (0,25%, 0,50%, 1%) at 4ºC. ....................26
Table 3-5 Log reductions in E. coli O157:H7 inoculated onto fresh-cut lettuce ..............27
Table 3-6 Log reductions in E. coli O157:H7 inoculated onto shredded carrots ..............27
Table 3-7 Effect of different concentrations of chlorine, as sanitizing treatment, on E.coli
O157:H7 reduction in lettuce processing (washing water with an initial load of organic
matter of 500 mg O2/L COD) (Treatment time: 45 seconds) ......................................33
Table 3-8 Effect of different concentrations of chlorine on THM’s compounds in process
water (washing water with an initial load of organic matter of 500 mg O2/L COD) .........34
Table 3-9 Physicochemical characteristics (COD and pH) of process wash water (after and
before NEW treatments) of lettuce. .........................................................................36
Table 3-10 Individual and total THM’s (µg/L) in the process wash water of lettuce. .......36
Table 3-11 Effect of ozone bubbling on the microbial load of process wash water .........38
before H2O2 treatments) of lettuce. .........................................................................39
before TSUNAMI treatments) of lettuce. ..................................................................40
Table 3-14 E. coli O157:H7 (log CFU/mL) population before and after CITROCIDE PLUS
(250, 375 and 500 ppm) treatment on lettuce and carrots. ........................................42
Table 3-15 Physicochemical characteristics (COD and pH) of process wash water
(CITROCIDE PLUS treatments) of lettuce and carrots. ...............................................42
Table 3-16 E. coli O157:H7 (log CFU/mL) population before and after CITROCIDE PC
(0,5%, 0,6% and 0,7%) treatment on lettuce and carrots. ........................................42
(CITROCIDE PC treatments) of lettuce and carrots. ..................................................43
Table 3-18 Individual and total THM’s (µg/L) in the process wash water of lettuce
processing, treated with a peroxyacetic based sanitizer. ............................................43
Table 3-19 Physicochemical characteristics (COD and pH) of process wash water (LA
treatments) of lettuce. ..........................................................................................44
Table 3-20 E. coli O157:H7 (log CFU/mL) population before and after chlorine dioxide
(CLODOS PURO) treatment on lettuce and carrots. Two different levels of inoculum tested
(3 and 6 log CFU/mL) ...........................................................................................45
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1. Introduction
1.1 Background RESFOOD
RESFOOD addresses the most important topics in the food chain towards resource efficient
and safe food production and processing, leading to maximized resource productivity and
recycling and re-use of valuable materials by research and demonstration of the proposed
green solutions: Increased output with reduced input.
Many natural resources (e.g. minerals, water, soil, biomass, land and fuels (energy) are
used to grow and process food products, but in many cases their usage is highly inefficient,
due to the lack of technological solutions and knowledge in combination with uncertainties
about health and safety issues. Another important challenge in the food chain management
is the large amount of wasted food. RESFOOD will overcome the main
bottlenecks and barriers leading to an Resource Efficient Food Chain by:
 Developing innovative technologies for re-use of Nutrients, Energy, Water and Biomass,
reducing input, maximizing resource productivity and minimizing waste
 Develop new methods for improving the disinfection processes for vegetables ensuring
appropriate monitoring of health and safety risks.
 Validate the solutions in five on site pilot demonstrations, also including Life Cycle
Assessment
In the RESFOOD resource efficiency concept the focus is a cascade approach: Look first for
the most efficient solutions with the lowest effort, like direct re-use of warm and cold water
(and energy) nutrients and biomass, followed by more complex solutions like withdrawal
of useful products and energy from the water and the recovery of high valuable
components from food waste (biomass).
This could lead to 30 to 75 % reduction of water, energy and nutrients use, 25 to 80 %

less emissions to surface and ground water, 20-30 % reduction of the use of fertilizer
products and overall 20 to 30 % more crop per resource input, combined with better
controlling and reducing food health and safety risks.
An overview of different tasks/activities within this report described work package 5 of the
RESFOOD project is presented in table 1-1:
Table 1-1 Activities within WP5 of the RESFOOD project
Task Title Main activities
5.1 Study of the antimicrobial effectiveness o Study of the antimicrobial

reached by combination of chemical + effectiveness of different
physical agents on the proposed sanitation methods and impact
vegetables: multi-hurdle approach of treatments on vegetables
o Study of the influence of
organic matter on the efficacy
of decontamination
o Selection of the optimum
disinfection technologies for
prototype development
5.2 Development of a pilot scale prototype for o Design and building of a pilot
the application of disinfection methods to scale prototype
fresh-cut vegetables
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5.3 Evaluation of quality effluent o Microbiological analysis

o Physicochemical analysis
o Residual by-products
/potential contaminants
5.4 Product quality assessment o Sensory analysis (shelf life)

o Microbiological analysis
o Physicochemical analysis
o Residual by-products /potential
contaminants
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1.2 Purpose of the document

The purpose of this document is to describe and summarize results regarding the
antimicrobial effectiveness achieved by means of the application of combined methods
(chemical strategies under positive and negative pressure conditions) and by the
application of chemical sanitizers at atmospheric pressure. The impact of the sanitizing
treatments, on product quality and on water quality, will also be presented and evaluated.
1.3 Document Structure

The structure of the report is as follows:
 Section 1 – Introduction
 Section 2 – Material and methods
 Section 3 – Results and discussion
 Section 4 – Conclusions
1.4 Deviations from original DoW
1.4.1 Description of work related to deliverable as given in DoW

The scope of tasks 5.1, 5.3 and 5.4 as introduced in the Description of Work (DoW) is as
follows:
TASK 5.1. Study of the antimicrobial effectiveness reached by combination of chemical +
physical agents on the proposed vegetables: multi-hurdle approach (months 1-18) Leader:
CNTA, Partners: TÜBITAK, Vega Mayor, KRONEN
A selection of bactericidal solutions will be studied, in combination with positive pressures
(P) and vacuum (V), on two kinds of vegetables: lettuce (Lactuca sativa L.) and shredded
carrots (Daucus carota L.) (two of the most important vegetables processed at VEGA
MAYOR and representative of the fresh-cut vegetable industry). Also, they have been
chosen due to their different behavior during disinfection treatments: lettuce is a leafy
vegetable with a high surface contact with natural irregularities and pores in which bacteria
tend to locate in and shredded carrot is a root vegetable with a usually high microbial load
due to the minimal processing operation and handling.
Disinfectants used for decontamination treatments will be organic acids (such as lactic acid
in a range of 0.5 to 3%), hydrogen peroxide, ozone and electrolyzed water. Samples will
be submerged in washing systems with these chemicals agents added, simulating industrial
washings, and then drained. Ozone concentration will be measured before sample
immersion using an ozone concentration kit based in DPD reaction. TÜBITAK will support
CNTA with the evaluation of the efficacy of ozone gas, in the inactivation of pathogens in
the selected vegetable matrices. Considering disinfected water is necessary to minimize
the risk of cross-contamination and bearing in mind some sanitizing agents used in water
disinfection may be the same as those tested on the vegetable surfaces, results from Task
3.2.2 will be taken into account for the final selection of chemical solutions. This will
contribute to the integration of the system and therefore to industrial upscalability. On the
other hand, literature on the effect of pressure on the mass transfer rate and its effect on
the quality of porous foods such as leafy vegetables will also be taken into account for the
selection of the most suitable sanitizing agents.
The physical treatments employed will be the application of positive pressures (P), vacuum
(V) and a combination of both of them (P+V; V+P). Treatments will be applied to
vegetables samples obtained from VEGA MAYOR factory a) without being inoculated with
the selected pathogens (only with their own naturally occurring flora), b) inoculated with
the selected pathogens (Escherichia coli O157:H7, Salmonella spp. and Listeria
monocytogenes), c) on raw pieces of lettuce and carrots prepared in lab (in sterile
conditions) and inoculated with the pathogens and finally d) on these vegetable pieces
after growth of Biofilms on them. These 4 scenarios will allow studying, on the one hand,
the influence of the competitive bacteria on the effectiveness of the selected treatments
for the chosen pathogens and, on the other hand, the effectiveness of the treatments on
biofilms.
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The selected pathogens will be inoculated in the samples individually and, therefore,
antimicrobial effectiveness will be quantified for each of them also individually. The
resulting processing water after the inoculation of the vegetables will be the one used in
WP6 for the development of the DHPLC (Denaturing high performance liquid
chromatography technique).
After optimizing the treatments, results will be analyzed and the most suitable conditions
will be applied on vegetables inoculated with a combination (at equal ratio) of the three
strains. The effectiveness of the different chemical interventions will be compared with
chlorinated water at a standard chlorine concentration (100 ppm, adjusted with citric acid
to pH 6,5). All experiments will be performed in triplicate.
The determination of the efficacy of decontamination methods will be reflected in the
microbiological reduction obtained and, even more important, in the maintenance of this
reduction during storage. Samples will be homogenized after treatments by manual
crushing until getting a vegetable puree, and survival counts will be quantified from this
puree. The goal is a microbial reduction of 2-3 log cycles via the use of chemical agents.
In addition, the influence of the presence of organic matter will be evaluated by comparing
the treatment effectiveness in presence and in absence of a known concentration of TSB
(Tryptone Soy Broth) in the washing solutions.
These tests will be performed at CNTA’s pilot plant, in a sealed chamber with 20 L capacity.
This chamber enables the possibility of applying vacuum and/or positive pressure alone or
in combination with the sanitizing agents selected. KRONEN will supervise the process and
will support its integration and upscaling and will supervise the washing protocols used and
will propose the possibilities according to its equipment and industrial machinery.
Extensive laboratory trials will be carried out to validate the anti-microbial efficacy of the
treatments. Samples will be homogenized after treatments by manual crushing until
getting a vegetable puree, and survival counts will be quantified from this puree. Treatment
time, pressure intensity and conditions/composition of the chemical aqueous solutions will
be the main studied variables. Contact and dwell times of the chemical agent with the
product will be tested and adjusted for optimal disinfection results. Alternative parameters
(e.g. agitation) will be taken into account to guarantee the contact of the selected
chemicals with the vegetables, also ensuring sufficient contact time between vegetables
and pretreated water.
Criteria for proper selection of the appropriate treatments will included its ability for
removing undesirable microbiological components from vegetables, in supplying water of
the necessary microbiological and chemical quality and also, it will depend on the cost-
effectiveness criteria.
TASK 5.3. Evaluation of quality of effluent (months 1-20) Leader: Vega Mayor, Partners:
CNTA, Tübitak, Vega Mayor, KRONEN
In order to evaluate the quality of water, the disinfection efficiency of proposed methods
will be also tested on the process water. Two important issues regarding the environmental
impact of the disinfection techniques are first, the physicochemical quality of the waste
water effluents, and second, the amount of wastewater generated.
The disinfection technique employed actually determines the physicochemical quality of
the wastewater effluents that is the presence of disinfection residuals, the formation of
toxic disinfection by-products (DBP) and the modification of organic matter, which can
harm environment. The maintenance of the quality of the process water by means of
avoiding the formation of hazardous and carcinogenic DBP as a result of chlorine
disinfection will be the goal of this task.
For achieving this aim, different quality variables will be evaluated in process water (such
as pH, organic load, turbidity, analysis of residues, sanitizer concentration, microbiological
analysis, etc.) to ensure that the water disinfectant of choice is effective in reducing the
negative effect of discharge in the environment and in human health.
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All the tests will be carried out with real process water. Before and after passing the fresh-
cut vegetables through the wash tank, containing potable water at 4ºC, samples of wash
water will be taken and kept at 4 ºC in darkness prior to analyze. The pH of water samples
will be measured using a pH meter. The turbidity will be calculated by the nephelometric
method using turbiquant turbidimeter and expressed in nephelometric turbidity units
(NTU). Conductivity will be measured using a conductometer. Additionally, free and total
chlorine will be determined using a chlorine (free & total) test kit. TÜBITAK will support
CNTA with the laboratory tests for analysis of residues.
TASK 5.4. Product quality assessment (months 2-20) Leader: CNTA, Partners: Vega Mayor
o The impact of the treatments in the treated vegetables will be evaluated in CNTA to
ensure their safety and to categorize them in terms of product quality and shelf-life. The
shelf-life is defined as the length of time which the vegetable can maintain the appearance,
safety and nutritional value that appeals to the consumer. For these reason, sensorial,
microbial (mesophiles and pathogens) and nutritional analyses will be carried out
throughout the entire storage time. The most promising decontamination strategy will be
scaled up and tested full-scale at the Vega Mayor facility (WP8). Analytical descriptive and
preference test will be used to evaluate the sensory quality attributes of fresh-cut lettuce
and shredded carrots prepared by the optimized treatment method. The panel, formed by
12 judges with sensory evaluation experience, will score changes in these products along
their storage. Sensory evaluation will be used to determine the shelf-life of these products,
as scores below to 5 will be taken as unacceptable (evaluation scale from 1 to 10), and to
indicate the end of shelf-life. Data analysis will be collected using Fizz sensory software
(Biosystemes, France).
o Microbiological analyses will be carried out on the samples before and after the treatment
at regular intervals through the storage period. Samples of 30 g will be homogenized for
90 s in 0,1% sterile buffered peptone water (BPW), (1:10 dilution) using sterile filter
stomacher bags and a stomacher. Total aerobic mesophilic bacteria will be enumerated
using plate count agar (PCA) incubated at 30 ºC for 48 h. Colonies of selected pathogens
(Listeria monocytogenes, Salmonella spp. and E. Coli) will be enumerated on plates of their
corresponding selective agar incubated for 48 h at 37 ºC. Three replicates will be analyzed
in duplicate and microbiological counts will be expressed as log CFU/g of tissue.
o In addition, nutritional quality of fresh-cut vegetables will be determined. Analysis of
residues will be carried out in order to determine the chemical compound used for the
decontamination process. The analysis of residues will be adapted according to the different
chemical agents to be identified. TÜBITAK will support CNTA with the laboratory tests for
analysis of residues.
1.4.2 Time deviations from original DoW

D5.1 was planned to be finished in month 20, so there was a delay of 3 months, due to
various last minute setbacks such as the delay in the delivery of some of the chemical
agents that should be subsequently tested at CNTA facilities.
1.4.3 Content deviations from original DoW

The initial purpose of WP5 was to investigate the efficacy of the combined use of physical
(application of positive and negative pressures) and chemical methods in the
decontamination of fresh-cut products. It is known that foodborne pathogens can be
internalized through vegetable tissue, becoming out of reach of disinfectants, whose action
is exclusively superficial. The expected goal of the combined use of these techniques was
to improve the contact of the chemical agents while applying physical forces (imposing
positive and vacuum pressures) in order to promote the gas release from the product pore
and facilitating the impregnation of product surface with the chemical solution. However,
preliminary studies with combined treatments showed not significant
effectiveness in reducing levels of microorganism than single treatments, with the
exception of some isolated cases. Therefore, due to these findings along the investigation
and to the weak synergistic effect observed in laboratory testing scale when applying
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combined physical and chemical treatments, this hurdle technology has no significance for
practical food preservation at industrial scale. Thus emphasis must continue to be placed
on the prevention of cross-contaminations and on the importance of the
maintenance of water quality during washing but without leaving aside product
decontamination.
Thus, the new approach of WP5 is to define the optimum disinfection method (focusing
on the application of chemical methods at atmospheric pressure) that not compromise
sanitation or causing any adulteration of product and that allows using the same water
during longer periods or allows recycling.
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2. Material and methods

2.1 Bacterial strains
Escherichia coli O157:H7, Salmonella spp. and Listeria monocytogenes are the main
pathogens implicated in several foodborne outbreaks related to fresh produce and the ones
chose for this study.
These bacterial strains were obtained from the Spanish Type Culture Collection (CECT,
Valencia, Spain) with the exception of E. coli which is unique of DSM (German Collection
of Microorganism).
2.2 Preparation of inoculum

Stock cultures were maintained adhered onto porous beads at -80ºC. Working cultures
were streaked onto Tryptic Soy Agar (TSA), incubated at 37ºC for 24 h and stored at 4ºC.
Cultures were regenerated by transferring one bead into 10 ml of tryptic soy broth (TSB)
supplemented with 0,6 g/100g yeast extract (YE) and incubating, in agitation, at 37ºC for
8h. Afterwards, 0,1 mL of this culture is transferred to a volume of 50 ml of TSB (+0,6%YE)
and incubating at 37ºc for 12h. Mixed culture cocktails were prepared by blending together
equal volumes of each three strains.
A final cell number of 109 CFU/mL was achieved using this procedure.
(TSA+0.6 % YE (TSA+0.6 % YE
(TSA+0.6 % YE 37ºC/24h) 37ºC/24h)
37ºC/24h)
CRIOVIAL CRIOVIAL ≈5010 9 CFU/mL ≈ 10 9 CFU/mL
Freeze-dried50
cultureFreeze-dried 9 (-80ºC)
≈culture
10 10 CFU/mL
mL 50 10
mLmL mL
10 mL mL (-80ºC) TSB+0.6
TSB+0.6 % YE TSB+0.6 % YE
% YE TSB+0.6 % YE
TSB+0.6 % YE TSB+0.6 % YE 37ºC/ 8h 37ºC/ 8h
37ºC/12h 37ºC/12h
37ºC/ 8h 37ºC/12h
2.3 Sample preparation
Iceberg lettuce (Lactuca sativa) was supplied by Vega Mayor industry on the day before
the experiment. The outer leaves, which usually correspond to dirty and damage leaves,
and the core were removed from the lettuce and discarded. Inner leaves were cut into
pieces using a cutting machine (at Vega Mayor) in transversal long narrow strips of 1 cm.
Fresh carrots (Daucus carota L.) were also kindly provided by Vega Mayor. They were
sorted for any cracks or defects, their ends removed using a sharp knife, peeled and
shredded in sticks with a food processor.
2.4 Inoculation procedure

Vegetable samples were immersed in water with the inoculum solution (sample:inoculum
ratio = 1:10 w/v) and kept under agitation for 5 minutes. After dipping, the samples were
drained for 30 seconds. A level of inoculation of approximately 10 6 CFU/mL was achieved
using this procedure. The samples were then placed into plastic containers and maintained
for 30 minutes at 37 ºC temperature until treatment with the sanitizer solutions.
A study based on surface attachment and biofilm formation by major foodborne pathogens
on iceberg lettuce leaf surface was performed. Although the level of attached cells on
lettuce leaf surfaces, were almost the same after 5-60 minutes of inoculation period, it is
important to note that the attachment strength (Sr) of the bacterial cells increases with
the time of inoculation for E.coli O157:H7 and for Salmonella enterica (Figure 2-1a).
Moreover, incubation at 4ºC for 48 hours after inoculation resulted in a significant increase
in the Sr for all the three types of strains studied (Figure 2-1b). Salmonella enterica has
the highest Sr, both immediately after inoculation and after 48 h. of incubation.
Page | 16
a) Attachment strength (Sr) of bacteria on iceberg lettuce leaf

(immediately after inoculation)
1,000
0,900
0,800
Attachment strength (Sr)
5 min
0,700
10 min
0,600
0,500 20 min
0,400 30 min
0,300 60 min
0,200
0,100
0,000
E. Coli O157:H7 L. monocytogenes Salmonella enterica
b) Attachment strength (Sr) of bacteria on iceberg lettuce leaf

(after 48 hrs of incubation at 4°C)
1,000
0,900
0,800
Attachment strength (Sr)
0,700
5 min
0,600
0,500 30 min
0,400 60 min
0,300
0,200
0,100
0,000
E. Coli O157:H7 - 48 hrs incubation
L. monocytogenes - 48 hrs incubation
Salmonella enterica - 48 hrs incubation
Figure 2-1 Comparison of attachment strength of pathogenic bacteria to iceberg

lettuce leaf after 5-60 min of inoculation a) immediately after inoculation, b) after
48 h. of incubation at 4ºC
The formation of biofilms was demonstrated by the Scanning Electron Microscope (SEM)
images for the three strains studied (Figure 2-2).
Page | 17
Figure 2-2 Formation of biofilms and penetration into the stomata by E.coli
O157:H7, L. monocytogenes, S. enterica on iceberg lettuce leaf.
Page | 18
In this work it will be evaluated the efficacy of decontamination treatments on reducing

the population of E. coli O157:H7 and on the naturally occurring flora (aerobic mesophilic
counts).
2.5 Application of sanitizing treatments

Raw material was processed according to the flow diagram shown in Figure 2-3.
Reception of iceberg lettuce Reception from

(Tª<8,5ºC) VEGA MAYOR
Cut off the lettuce stem and remove

outer leaves
Microbiological
Cutting characterization
of raw material
Weighting (500 g)
Inoculation (dipping time: 5 min) Microbiological

characterization
Water volume 5 L. (room Tª) of raw material
Washing without chlorine Washing with chlorine

Water volume: 5 L (Tª: 4ºC) SANITIZING TREATMENT (100 ppm, pH: 6,5 citric acid)
Time: 5 min (3 doses/each chemical agent) Water volume: 5 L (Tª:4ºC)
Water volume: 5 L (Tª:4ºC) Time: 5 min
Time: 5 min
Rinse in destilated water (Tª: 4ºC)

Time: 30''
(50g/0,5L)
Microbiological Microbiological Microbiological

characterization characterization after characterization
after treatment treatment (3 doses) after treatment
Figure 2-3 Flow diagram of minimal processing for lettuce samples (same flow
diagram for carrot samples was used)
Page | 19
Decontamination processes were conducted on pilot-plant scale with semi-industrial

equipment (Figure 2-4), carefully sanitized prior to processing. Positive pressure (P) values
tested were +1, +2 and +3 bar and the maximum vacuum (V) intensity achieved was 30
mbar. Also combined pressures were performed (P+V, V+P). After pressuring, the
atmospheric pressure was restored for 5 minutes more.
(a) (b)
Figure 2-4 Semi-industrial equipment used for achieving: a) positive pressures

(Formulador PC-20) and b) negative pressures /vacuum (GASTROVAC)
For each experimental run, fresh cut vegetables were immersed in a tank containing the
respective sanitizing solutions (using a product:solution ratio of 1:10 w/v) at 4ºC, followed
by an atmospheric pressure restoration time (5 min) and a rinsing step in distilled water
for 30 seconds at 4ºC.
As described before, inoculated products were immersed in tap water and in chlorine water,
which were used as reference samples (controls). There are two different controls due to
existing restrictions on chlorine use in some Europeans countries, including Germany, The
Netherlands, Switzerland and Belgium, where its use for washing fresh cut products is
banned (Beltrán, Selma, Tudela, & Gil, 2005).
Chlorine efficiency is extremely dependent on the amount of free available chlorine (as
HClO) in the wash water but also on the pH, temperature, treatment duration, organic
matter present and vegetable tissue components (Beuchat, 1998). Chlorinated water
solutions were prepared by adding commercial sodium hypochlorite to cold tap water to
obtain a final solution containing 100 ppm of active chlorine. The pH of the sanitizing
solution was adjusted to 6,5 using citric acid in order to improve chlorine disinfection
efficacy.
Finally, samples were centrifuged to remove excess water with a manually-operated
enclosed spinner.
Changes in levels of pH and temperature (ºC) were measured in the washing solutions
during the disinfection of the vegetables.
Page | 20
2.6 Washing solutions

Several sanitizing agents have been tested with the intention of reducing the risk of
microbial contamination. It should be noted that washing and sanitizing are unlikely to
totally eliminate all pathogens after the s is contaminated.
The effectiveness of the washing solutions will be always compared with tap water (W) and
with chlorinated water (CW) at a standard chlorine concentration (100 ppm)
2.6.1 Chlorine
Chlorine is the most widely used disinfectant in fresh-cut industry, with concentrations
varying from 50 to 200 ppm and with typical contact times of less than 5 min (Artes &
Allende, 2005) (Rico, Martin-Diana, Barat, & Barry-Ryan, 2007). Although chlorine is the
most commonly used sanitizer, it is inactivated by organic material and during production
can also lead to the formation of chlorinated by-products (DBP) with potential adverse
health effects (Parish, Beuchat, Suslow, Harris, Garret, & Farber, 2003). However the
benefits of chlorine use for the fresh-cut industry outweigh the concerns of potential
formation of harmful by-products. Anyway, in some European countries, the use of chlorine
has been forbidden.
In our experiments, chlorinated water was prepared by adding sodium hypochlorite (El
Leon, commercial bleach, with 35 g of active chlorine per litre) in water to a concentration
of 100 mg/L free chlorine. The amount of free chlorine was determined using a free & total
chlorine test kit (model HI95711, HANNA Instruments).
The recommended pH for chlorine disinfection ranges of 6,5 to 7,5 (to prevent corrosion
and to maintain efficacy). Under these conditions, the active killing form of chlorine (HClO)
predominates in the water solution. Therefore, the pH of the solution was adjusted with
citric acid to 6,5 ±0,5 and measured with a pH meter (model GLP-21, Crison, Spain).
With the purpose of optimizing the chlorination process of wash water, it has been also
tested other concentrations: 10 ppm, 25 ppm and 50 ppm (as well as 100 ppm).
2.6.2 Organic acids

Organic acids have been extensively investigated because of their bactericidal activity. The
antimicrobial action is due to the pH reduction in the environment, and it changes widely
among the organic acids.
Lactic acid (80%), citric acid monohydrate (E-330) and acetic acid (80%) were used in this
work. For the combined treatments, inoculated samples were immersed in 0,25, 0,5 and
1, 2, 4 and 5% of organic acids at 4ºC for 5 minutes with mild agitation. After treatment,
the excess liquid was eliminated.
2.6.3 Chlorine dioxide

Chlorine dioxide is also a powerful oxidant, more stable and has a higher oxidizing power
tan chlorine. Unlike chlorine, the effectiveness of ClO2 remains largely constant in a wide
range of pH.
A commercially available brand of chlorine dioxide (CLODOS PURO, STC, Spain) was used
in this experiment. The stock solution (3000 ppm of ClO2) was diluted with tap water to
achieve the working concentrations of 20 ppm and 30 ppm. The chlorine dioxide
concentration was measured by means of Palintest® ChlordioXense instrument.
2.6.4 Hydrogen peroxide

Hydrogen peroxide (H2O2) has both a bacteriostatic and a bactericidal activity due to its
strong oxidizing power. One of the main advantages of using H 2O2 as a disinfecting agent
is that it produces no residue as it is decomposed into water and oxygen by the enzyme
catalase which is naturally found in plants. The main drawback is its phytotoxicity, inducing
extensive browning on some products like lettuce (McWatters, Hashim, Walker, Doyle, &
Rimal, 2002).
Page | 21
Solutions of 1%, 2% and 5% (v/v) were prepared from a solution of 30% (w/v) of hydrogen
peroxide (PANREAC, Spain) stabilized by dilution with water.
2.6.5 Electrolyzed water

Neutral electrolyzed water (NEW) will be generated using an electrolyzed water generator
(Innobak Systems, Eurozon, Spain) following the manufacturer’s instructions. A saturated
sodium chloride solution is pumped into the equipment with a current passing through the
electrolyzed water generator. NEW is collected from outlet in containers and stored at 4ºC
until use (no more than one day). Solutions of NEW at 50, 75 and 100 mg/l of free chlorine
will be prepared by diluting in distilled water immediately before treatment.
Figure 2-5 Neutral electrolyzed water generator (ANE, INNOBAK SYSTEMS)
In this work it will be evaluated the efficacy of neutral electrolyzed water on reducing
bacterial population. The properties of each solution were determined, including ORP, pH
and free chlorine concentration. All measurements were made at 4 ºC immediately before
treatments. ORP and pH were measured with the use of a pH-meter (model GLP-21, Crison,
Spain), using an ORP electrode (Crison cod. 52-61) and a pH electrode (Crison cod. 52-
23) respectively. Free chlorine concentrations were determined using a free and total
chlorine photometer (model HI95711, HANNA Instruments).
2.6.6 Ozone
Ozone is a strong antimicrobial agent with high reactivity, penetrability and spontaneous
decomposition to a non-toxic product. When compared to chlorine, ozone has a greater
effect against certain microorganisms and rapidly decomposes to oxygen, leaving no
residues (Rico, Martin-Diana, Barat, & Barry-Ryan, 2007).
Ozonated water was prepared by ozonating sterile distilled water through an H 2O Mini
Ozone Injection system (Ozone Solutions, Inc., Sioux Center, IA, USA) and used
immediately after the desired ozone concentration was achieved. The system was equipped
with a pump, a contact tank having a water inlet tube, pressure gauges and regulators,
safety release valve, liquid withdrawal tube and gas inlet tube fitted with a venturi-type
injection dispenser unit. Ozone gas was produced from extra dry oxygen by means of a
corona discharge generator with a capacity of 4,5 g ozone/h (Hess Machine Int.,USA).
Ozone concentration was measured by colorimeter (Spectroquant Colorimeter Picco,
Merck, Darmstad, Germany).
Page | 22
2.6.7 Peroxyacetic acid

Peroxyacetic acid (PAA), which is also referred to as peracetic acid, is an aqueous
quaternary equilibrium mixture of acetic acid and hydrogen peroxide (Dell'Erbaa,
Falsanisia, Libertia, Notarnicolaa, & Santoroa, 2007). It is a strong disinfectant with a wide
spectrum of antimicrobial activity.
PAA is gaining increased interest among chlorine-alternative chemical disinfectants
claiming that only harmless disinfection by-products have been identified so far as acetic
acid, water and oxygen are formed from its spontaneous decomposition (Dell'Erbaa,
Falsanisia, Libertia, Notarnicolaa, & Santoroa, 2007). Moreover is important to note that
the efficacy of PAA is not affected by the organic compounds present in the process water
(Ruiz-Cruz, Acedo-Felix, Diaz-Cinco, & Islas-Osuna, 2007).
During this research, two commercially available peroxyacetic acid based sanitizers were
used: 1) TSUNAMI from Ecolab (Barcelona, Spain) and 2) CITROCIDE PC and CITROCIDE
PLUS from Citrosol S.A. (Valencia, Spain). These products are a combination of
peroxyacetic acid (PAA), acetic acid (AA) and hydrogen peroxide (H2O2).
Table 2-1 Sanitizer treatments and concentration of active components (based

on technical specification sheet from manufacturers)
PAA AA H2O2
TSUNAMI 15,2% -- 11,2%
CITROCIDE PLUS 15% 17% 23%
CITROCIDE PC 4,5-5,4% 7-9% 21-24%
The solution with TSUNAMI and CITROCIDE PLUS was prepared by adding the mixture to
cold water to achieve a concentration of 250 mg/L, 375 mg/L and 500 mg/L, and the
solution with CITROCIDE PC was performed at a concentration of 0,5%, 0,6%, and 0,7%,
based on the manufacturers’ recommended doses.
2.7 Standardized process wash water in the COD content

Lettuce and carrots were supply by Vega Mayor industry in Navarra (Spain) and
transported to the lab for further handling. After discarding the outer leaves in the case of
lettuce and after peeling in the case of carrots, vegetables were put in a blender (ECRON,
model BH-3381) in order to obtain a lettuce and carrot juice respectively. Subsequently,
the vegetable juice was diluted with tap water to obtain standardized process water. This
process gives place to process water with very high chemical oxygen demand (COD)
(≈1000 mg/L).
2.8 Physical-chemical analysis
2.8.1 Measurement of pH
Surface pH was measured on samples from each treatments group prior to microbiological
analysis. After centrifugation, 50 g of the vegetable sample were transferred to a
stomacher bag containing 50 ml distilled water (1:1, w:v), homogenized by mixing for 100
s with a Bag-Mixer 400 ml stomacher, and the pH of the homogenate was measured at 23
°C using a pH meter with glass electrode (model GLP-21, Crison, Spain).
2.8.2 Determination of trihalomethane compounds

Samples of process water were taken from the washing tank after treatment. 10 mL of
water samples were used to fill in 20 mL glass vials. Equilibration was achieved by heating
the vial for 15 min at 75ºC. Headspace THMs were collected using a Agilent G1888
Headspace Sampler. The syringe assembly unit with the transfer line was lowered into the
vial, suspended in the headspace above the liquid layer of the samples. After 15 min of
extraction, the transfer line was injected into the gas chromatograph (GC). Separation was
Page | 23
carried out on an Agilent 6890N GC equipped with an Agilent 5975B MS detector. The THMs
were separated on a CP-select 624, 30m x 0,25mm capillary column with helium as a
carrier gas.
Identification of THMs was based on the retention time of standard compounds. The
compounds quantifies were: chloroform, bromodichlotomethane, dibromochloromethane
and bromoform. Estimation of THM quantity was based on the areas of the peaks detected
by MS.
2.8.3 Measurement of COD

Chemical Oxygen Demand (COD) was determined by the standard photometric method
using the Spectroquant PHARO 100 photometer (MERCK). This method is based on the
photometric determination of the concentration of Cr3+ ions resulting from the oxidation of
organic matter by a hot mixture of K 2Cr2O7 acidified by H2SO4 that contains Ag2SO4 as
catalyst, containing HgSO4 as chloride masker. Results were expressed as mg O2/L.
2.8.4 Measurement of aquatic toxicity of waste process water

Toxicity tests of waste process water were performed using BioTox Kit (Aboatox 1243-500)
according to ISO 11348-3. The luminescent bacterium, Vibrio fischeri, was used as the
target species.
2.9 Microbiological analysis

Following treatments, all samples (each of 10 g) were transferred into a sterile stomacher
bag containing 90 ml of 1% buffered peptone water and then homogenized for 100 seconds
in a stomacher (AES Chemunex). For enumeration of pathogens, 0,1 ml volume of
appropriate serial dilutions of the homogenate was spread on the surface of TSA media.
All plates were incubated at 37ºC for 24 h before counting. Colony counts were transformed
to log CFU/ml.
2.10 Sensory analysis

Sensory evaluation of vegetables was conducted. A panel of seven trained judges was
asked to distinguish samples in a triangle test according to the following sensory attributes:
appearance, flavor and texture.
Besides a descriptive sensory analysis was performed and the following sensory
parameters were evaluated:
 For lettuce minimally processed: overall visual quality, off-odour, flavor, leaf edge
browning, leaf surface browning, wiltness and dryness (those 2 attributes are
related to texture).
 For carrot minimally processed: overall visual quality, odour, flavor and texture.
Panelists were also asked to provide any relevant comments. Scales 1 to 5 were used to
avoid panel fatigue. A scale 1 to 9 was used to emphasize assessments of overall visual
quality because it is the most important parameter for MP vegetables quality.
2.11 Data analysis

For each group of experiments, three replicate experiments were conducted. Microbial
counts were transformed to logarithms before computing means and standard deviations;
population densities were reported as log CFU/ml for lettuce and carrot samples. The
General Linear Models (GLM) procedure of the Statistical Package for the Social Sciences
(SPSS) was applied. Significant differences between treatments with respect to bacterial
reduction were analyzed by Tukey test at a significance level of 0,05.
Page | 24
3. Results and discussion

3.1 Multi hurdle approach: antimicrobial effectiveness of combined
treatments on vegetables to mitigate the risk of food-borne
illness and with minimum impact on environment and
sustainability (experiments from Dec’12 to Nov’13)
Hurdle technology refers to a combination or the different existing preservation techniques
that supplement and enhance each other. The most important hurdles commonly used are
based on storage temperatures, water activity, pH, redox potential, modified atmosphere
and addition of preservatives (Rahman, Jin, & Oh, 2011) (Randazzo, Pitino, Scifo, & Caggia,
2009) (Trias, Badosa, Montesinos, & Bañeras, 2008). The hurdle technology consists on
the use of a sequence of mils treatments to inhibit or inactivate the factors responsible for
food spoilage. Its main goal is to use preservation techniques that prolong storage stability
and do not have detrimental effects on the quality attributes of the product (Parish,
Beuchat, Suslow, Harris, Garret, & Farber, 2003).
On this basis, the initial purpose of WP5 was to investigate the efficacy of the combined
use of physical (application of positive and negative pressures) and chemical methods in
the decontamination of fresh-cut products. It is known that food-borne pathogens can be
internalized through vegetable tissue, becoming out of reach for disinfectants, whose
action is exclusively superficial. The expected goal of the combined use of these techniques
was to improve the contact of the chemical agents while applying physical forces (imposing
positive and vacuum pressures) in order to promote the gas release from the product pore
and facilitating the impregnation of product surface with the chemical solution.
3.1.1 Initial characterization of samples

In our study, it was determined 5 log cycles of mesophilic bacteria, <10 CFU/g of E.coli
and absence (in 25 g) of Listeria monocytogenes and Salmonella (table 3-1) for fresh-cut
carrot and lettuce.
Table 3-1 Initial microbial population on Iceberg lettuce and carrots
LETTUCE CARROT
Total viable counts
(CFU/mL) 1,70E+05 1,60E+05
E. coli (CFU/g) <10 <10
Listeria Absence/25 g Absence/25 g
Salmonella Absence/25 g Absence/25 g
3.1.2 Efficiency of different combined treatments to prevent E.coli O157:H7

contamination of fresh-cut products
The first step, when studying novel decontamination techniques, is to test the feasibility to
decontaminate product from a microbiological point of view.
With that purpose, fresh-cut product was inoculated with E. coli O157:H7, achieving a load,
in product, of around 6,5 ± 0,5 log CFU/mL. A pressure process (V, P, V+P, P+V) was used
to force bacteria located inside the pores, which were then subjected to a five-minute water
wash and a five-minute chemical treatment (lactic, citric and acetic acid, including
chlorine). After treatments, vegetable products were crushed to recover any remaining
E.coli bacteria.
As can be observed in table 3-2, 3-3 and 3-4, washing in chemical treatments did little to
reduce levels of E.coli in lettuce when comparing to washing with plain water (the drag
force exerted by plain water is enough to eliminate around 1 log cycle from product
surface).
Page | 25
Table 3-2 E.coli O157:H7 log reduction of washed inoculated fresh-cut lettuce
using tap water, chlorine (100 mg/l) and lactic acid (0,25%, 0,50%, 1%, 2%)
at 4ºC.
Log reduction
Treatment Doses P V P+V V+P
Water wash 1,4 1,7 0,9 1,2
Chlorinated water 2,1 1,2 2,0 2,0
Lactic acid 0,25% 1,0 1,4 1,2 1,1
0,5% 1,4 1,3 1,2 1,2
1% 1,1
2% 1,5
P:positive pressure (+1bar); V: negative pressure (400 mbar)
Log reduction= (log CFU/mL before treatment)-(log CFU/mL after treatment)
Citric acid treatments produced results similar to those of the tap water washing (table 3-
3).
using tap water, chlorine (100 mg/l) and citric acid (0,25%, 0,50%, 1%) at 4ºC.
Log reduction
Water wash 1,3 1,1 0,9 1,3
Citric acid 0,25% 1,1 1,0 1,4 1,4
0,5% 1,1 1,2 1,4 1,2
1% 1,2 1,5 1,7 1,5
using tap water, chlorine (100 mg/l) and acetic acid (0,25%, 0,50%, 1%) at
4ºC.
Log reduction
Water wash 1,1 0,6 0,9 1,0
Acetic acid 0,25% 1,0 1,2 1,1 1,2
0,5% 1,3 1,1 0,8 0,8
1% 1,1 1,0 1,0 1,0
Overall, washing inoculated fresh-cut lettuce in each washing solution, including tap water
and chlorine as reference samples, confirmed that washing in pressure conditions had little
Page | 26
effect on microbial reduction on product surface. E.coli population present on inoculated

fresh-cut lettuce was reduced by about 1-2 log CFU/mL (table 3-2, 3-3 and 3-4).
Besides, the results suggested that binary combination of pressure (P+V; V+P) did not
significantly enhance the effectiveness of these decontamination technique compared with
individual application of pressure (P; V), thus those treatments will not be taken into
account any more. Moreover, in this study, we also found that acetic acid induces changes
in the sensory quality of fresh-cut lettuce, conferring off odours to the products (vinegary
odour), so that it will not be tested in future experiments.
According to these results, the next step in the experimental design was to increase the
pressure and vacuum level, in order to promote a higher gas/liquid release from the pore
structure that block penetration of the sanitizing agent. Application of various sanitizers
(lactic acid, hydrogen peroxide, neutral electrolyzed water, chlorine dioxide) under a
positive pressure higher than +1 (+2 and +3 bar) and under vacuum lower than 400 mbar
(200, 100 and 30 mbar) was performed (table 3-5 and 3-6) on fresh-cut produce.
Table 3-5 Log reductions in E. coli O157:H7 inoculated onto fresh-cut lettuce
Log reduction
Pressure Vacuum
+1 +2 +3 400 200 100 30
Treatment Doses Atm P bar bar bar mbar mbar mbar mbar
0,6 -
Water wash 0,9 1,4 0,9 - 1,3 1,7 1,0
1,7 -
Chlorine 1,1 2,1 1,6 - 1,7 1,2 1,9
Lactic acid 0,25% 1,0 1,4
0,50% 0,7 1,4 1,3
1% 1,1 1,7 2,1 2,2 2,8
2% 1,5
5% 1,4 2,1 2,2 3,0
H2O2 1% 1,5 1,3 1,3
2% 1,6 1,6 1,2
5% 1,9 1,7 1,6
NEW 50ppm 1,4 1,1
75ppm 1,6 1,6
100ppm 1,6 1,7
ClO2 1ppm 0,4 0,5
2ppm
Table 3-6 Log reductions in E. coli O157:H7 inoculated onto shredded carrots
Page | 27
Log reduction
Pressure Vacuum
+1 +2 +3 400 200 100 30

Treatment Doses Atm P bar bar bar mbar mbar mbar mbar
Water wash 0,4 - 0,8 0,2 - 0,9 0,4 - 0,7

Chlorine 1,6 - 1,8 1,4 - 1,8 1,3 - 1,9
Lactic acid 0,25% 1,2
0,50% 1,7
1% 1,1 2,2 1,7 2,1 2,2 2,0
2% 3,1 3,3 1,7
4% 3,8 3,3
5% 4,9 3,7
H2O2 1% 1,2 1,6 0,9
2% 1,2 1,9 1,1
5% 1,9 2,8 1,6
NEW 50ppm 1,0 1,4
75ppm 1,2 1,7
100ppm 1,6 1,4 1,9
ClO2 1ppm 0,2 0,1 0,6
2ppm 0,6 0,6
Overall conclusion of multi hurdle approach: these studies with combined treatments
(increasing the pressure conditions and the vacuum level) showed not significant
effectiveness in reducing levels of microorganism than single treatments, with the
exception of some isolated cases (see lactic acid at 4 and 5%).
Information obtained in the present study, in line with most of the scientific literature on
the topic, suggest that the use of decontamination techniques cannot guarantee microbial
safety of these products, due to the limited reductions that can be obtained in the numbers
of microorganisms from nature present in the product itself. Therefore, use of sanitizers
would be mainly justified by their efficacy for cross-contamination avoidance in the
production process of fresh-cut vegetables.
3.1.3 Effect of different combined treatments in the quality of fresh-cut

products
By applying the higher vacuum level a sponge effect was observed on lettuce tissue,
due to its porous structure (intercellular spaces). These changes are related to the removal
of substantial amounts of water from the product and its substitution by an external
solution (chemical agent), resulting in a translucent product (Figure 3-1). In the case of
carrot tissue, since it does not present intercellular spaces (densely packed cells), it shows
a better performance to vacuum treatment than lettuce.
Page | 28
Figure 3-1 Lettuce appearance after applying vacuum treatment at 30 mbar
The impact of the positive pressure by itself on product was also evaluated, concluding
that positive pressure treatments don’t contribute to changes in the product appearance
neither for lettuce nor for carrots (Figure 3-2).
Figure 3-2 Lettuce and carrot appearance after pressure treatment at 3 bar
When including a chemical agent in combination with vacuum levels, the effect of the
negative pressure on product quality is enhanced, as can be observed in Figure 3-3. The
use of organics acids adversely affects to the sensory quality of lettuce, mainly, in flavor,
due to the product acidification and in appearance, because lettuce could be differentiated
from reference samples in terms of browning and wilting (P<0,05). Also the sensory
properties of carrots were affected by the use of lactic acid (slight browning).
Page | 29
5%LA +V 4%LA +V 2%LA +V 1%LA +V
Water+V Chlorinated water+V
Figure 3-3 Lettuce and carrot appearance after applying vacuum treatment at 30
mbar + lactic acid
When the positive pressure is combined with organics acids, negative effects on the
sensory properties were also found regarding the acidification of the product and the
changes in appearance.
In the case of hydrogen peroxide and neutral electrolyzed water, no negative effects were
observed due only to the chemical agent (Figures 3-4 and 3-5).
1% P.H. 2% P.H. 5% P.H.
VACUUM
Water + Vacuum
PRESSURE
No negative effect
1% P.H. 2% P.H. 5% P.H.
VACUUM
No negative effect
PRESSURE
No negative effect
Figure 3-4 Lettuce and carrot appearance after applying vacuum and pressure
treatments + hydrogen peroxide
Page | 30
50 ppm 75 ppm 100 ppm
VACUUM
Negative effect (translucent aspect)
PRESSURE
No negative effect on product
50 ppm 75 ppm 100 ppm

VACUUM

PRESSURE
Figure 3-5 Lettuce and carrot appearance after applying vacuum and pressure
treatments + neutral electrolyzed water (NEW)
3.1.4 Efficiency of different combined treatments to extend the shelf life of

fresh-cut products
The use of combined treatments for reducing the native micro flora on minimally processed
vegetables during storage at 4ºC for 12 days was also investigated. Only hydrogen
peroxide as chemical agent was tested.
The initial population of mesophilic bacteria on lettuce was 5,4 log CFU/g (Figure 3-6).
After the decontamination treatment a reduction of up to 2 log cycles was achieved by
using hydrogen peroxide and positive pressure. A level of 7 log CFU/g for mesophilic
population was set in as the maximum limit to consider the product acceptable from a
microbiological point of view. In fresh-cut lettuce, microbial populations reached the
microbial limit in all treated samples (including reference sample) after 12 days of storage,
with the exception of the combined treatment hydrogen peroxide 5% + positive pressure.
Page | 31
10
LETTUCE
Aerobic mesophilic bacteria

9
8
(log 10 CFU/g)
7
6
5
4
3 HP 2% Patm HP 2% P+3
2
HP 5% Patm HP 5% P+3
1
CW Patm
0
0 2 4 6 8 10 12 14
Storage time (days)
Figure 3-6 Effect of the sanitizing treatment on the microbial counts during the
shelf-life study of lettuce processing at 4ºC (HP: hydrogen peroxide; P: positive
pressure; Patm: atmospheric pressure; CW: chlorinated water)
In carrots (Figure 3-7), there was an initial reduction of the mesophilic counts in a range
of 1-2 log cycles after the decontamination treatment. The aerobic mesophilic counts on
the control samples were initially higher to those samples treated with hydrogen peroxide
but they reached the microbial limit at the same time than the others. Then, no shelf-life
prolongation is observed from a microbial point of view.
10
Aerobic mesophilic bacteria
9 CARROT
8
(log 10 CFU/g)
7
6
5
4
3
2
1
CW Patm
0
0 2 4 6 8 10 12 14
Storage time (days)
Figure 3-7 Effect of the sanitizing treatment on the microbial counts during the
shelf-life study of carrot processing at 4ºC (HP: hydrogen peroxide; P: positive
pressure; Patm: atmospheric pressure; CW: chlorinated water)
Regarding the sensory quality, no interferences have been detected by the use of combined
treatments. All treated samples kept product quality acceptability during the 12 days of
storage at 4 ºC.
Conclusions of the shelf-life study: Due to these findings along the investigation and
to the weak synergistic effect observed in laboratory testing scale when applying combined
physical and chemical treatments, this hurdle technology has no significance for practical
food preservation at industrial scale.
Thus emphasis must continue to be placed on the prevention of cross-contaminations and
on the importance of the maintenance of water quality during washing but without leaving
aside product decontamination.
Page | 32
3.2 Efficiency of different sanitizing treatments to prevent cross-

contamination of fresh-cut products (experiments from Jan’14
to Sept’14)
Processing wash water, if not properly sanitized, can become a source of microbiological
contamination for every vegetable that passes through. There is strong evidence that the
use of sanitizing agents helps to minimize the potential cross-contamination and thus,
maintaining the quality of processing water (FDA, 2008). However the selection of the most
effective sanitizers should be made in conditions similar to those found in the industrial
process in order to determine which sanitizing agent tolerates commercial processing
conditions whilst maintaining the microbial safety of the process water to prevent any risk
of cross-contamination to the uncontaminated products. So, in the next step of our study,
the COD contribution has been taking into account for the selection of the most suitable
sanitizers. Below are presented results obtained by using NEW, hydrogen peroxide,
different commercial mixtures of PAA+H2O2, lactic acid and chlorine dioxide as sanitation
treatments.
3.2.1 Experiments with decreasing initial chlorine concentrations in the

washing water
Chlorine is the most used water disinfectant in general in the fresh-cut industry due to the
low cost, the availability and the good effectiveness (when used properly) and due to the
minimal impact on the nutritional and sensorial quality of the product (Nou & Luo, 2010)
(Parish, Beuchat, Suslow, Harris, Garret, & Farber, 2003) (Tomas-Callejas, López-Gálvez,
Sbodio, Artés, Artés-Hernández, & Suslow, 2012).
Since it is generally agreed that the main role of sanitizers in process water is to keep the
water free of microorganisms and not mainly to “disinfect” the product itself, the effect of
diminishing levels of chlorine on the process water microbial safety has been evaluated
(Table 3-7). Moreover, in order to be able to assess the potential re-use of water with
reduced chlorine levels, the effect of decreasing the initial chlorine concentrations on the
formation of trihalomethanes (THMs) and toxicity was also evaluated (Table 3-8).
Table 3-7 Effect of different concentrations of chlorine, as sanitizing treatment,
on E.coli O157:H7 reduction in lettuce processing (washing water with an initial
load of organic matter of 500 mg O2/L COD) (Treatment time: 45 seconds)
Log reduction
Microbial load of Residual free
(CFU/g) of E.coli
Treatment process water chlorine
O157:H7 on
(log CFU/ml) (ppm)
lettuce
Chlorine 100 ppm 2,11 ± 0,06 <1 86
Chlorine 50 ppm 2,03 ± 0,15 <1 40
Chlorine 25 ppm 1,45 ± 0,15 <1 13
Chlorine 10 ppm 1,46 ± 0,17 <1 2,8
Chlorine 5 ppm 1,57 ±0,02 <1 1,7
Chlorine 2 ppm 1,62 ± 0,02 <1 Not detected
Control (wash water

1,21 ± 0,28 6,23 ± 0,18 Not detected
with 500 mg O2/L COD)
Page | 33
Data depicted in table 3-7 indicate the bacterial reduction of E. coli O157:H7 on fresh-cut
lettuce and the microbial load of process wash water when free chlorine concentration is
reduced from 100 ppm to 2 ppm (with a constant COD level of 500 mg O 2/L in the washing
tank).
Results revealed that, in terms of both the microbial safety of process water and the
microbial reductions achieved on the lettuce itself, there is no added advantage of using
higher than 5 ppm free chlorine levels even in the presence of high organic load (500 mg
O2/L) in the process water. Maintaining a residual chlorine level of around 2 ppm seems
to be enough to keep the process water free of microorganisms and to prevent the risk of
cross-contamination.
The use of high chlorine concentrations causes the generation of harmful disinfection by-
products in the water. Partially due to these possible DBPs generation, the use of chlorine
in fresh-cut produce washing is prohibited altogether in some european countries such as
Germany, Switzerland, the Netherlands, Denmark and Belgium (Artés, Gómez, Aguayo,
Escalona, & Artés-Hernández, 2009) (Rico, Martin-Diana, Barat, & Barry-Ryan, 2007)
(Tirpanalan, Zunabovic, Domig, & Kneifel, 2011).
A comparison of the concentrations of the THMs (µg/L) formed after these sanitizing
treatments were presented in table 3-8.
Table 3-8 Effect of different concentrations of chlorine on THM’s compounds in

process water (washing water with an initial load of organic matter of 500 mg
O2/L COD)
CHCl3 CHBrCl2 CHBr2Cl CHBr3

Treatment
(µg/L) (µg/L) (µg/L) (µg/L)
Chlorine 100 ppm 664,10±57,4 7,15±0,1 1,55±0,1 <3
Chlorine 50 ppm 552,80±11,4 4,21±0,0 <1 <3
Chlorine 25 ppm 109,49±7,1 6,32±0,8 1,68±0,2 <3
Chlorine 10 ppm 61,17±20,7 4,86±2,3 <1 <3
Chlorine 5 ppm 10,0±2,8 3,40±2,3 1,40±0,8 <0,468
Chlorine 1-3 ppm 12,20±5,0 5,35±1,5 1,55±0,5 <0,468
Control (waste water

13,03±1,7 5,64±1,7 1,63±0,3 <3
with 500 ppm COD)
CHCl3: chloroform, CHBrCl2: bromodichlotomethane, CHBr2Cl: dibromochloromethane and

CHBr3: bromoform
As shown, a decrease in initial free chlorine levels from 100 ppm to 5 ppm resulted in a
remarkable reduction in the levels of THMs. More important to note is that, the level of
THMs formed at 5 ppm free chlorine is comparable to that formed in 1-3 ppm ozonated
water as well as to control treatment that is the tap water. Therefore, a reduction in free
chlorine levels, which will be enough to maintain a residual free chlorine level of 2 ppm in
the process water during washing, will help in eliminating the risks associated with the
formation of chlorinated DBPs in waste water to a great extent. Lower than 25 ppm initial
free chlorine levels resulted in less than 100 ppb THMs in the waste water which is the
maximum allowable level of THM for drinking water in EU (Figure 3-8).
Page | 34
800
700
Total THMs (µg/L)

600
500
400
300
200 EU limit for TTHMs in drinking water
100
0
CW 100 CW 50 CW 25 CW 10 CW 5 CW 1-3 Control
ppm ppm ppm ppm ppm ppm W
Figure 3-8 Effect of decreasing free chlorine levels on the formation of THMs in
process wash water
3.2.2 Effect of NEW washing solutions on bacterial inactivation on lettuce and

carrot processing
NEW was adjusted to maintain a stable FC concentration (50, 75 and 100 ppm), keeping
a constant COD of 1000 mg/L. Figure 3-9 shows the effect of those doses on reducing the
aerobic mesophilic load on fresh-cut lettuce and carrots. The initial population of mesophilic
load was 5,8 ±0,1 log CFU/mL on lettuce and 6,5±0,1 log CFU/mL on carrots.
7,0 7,0
LETTUCE 6,0
Aerobic mesophilic counts
6,0
CARROT
5,0 5,0
45''
(CFU/mL)
(CFU/mL)
4,0 1' 4,0

1'30''
3,0 3,0
2'
2,0
2,0
1,0
1,0
0,0
0,0 NEW50 NEW75 NEW100 W CW
NEW50 NEW75 NEW100 W CW
Figure 3-9 Aerobic mesophilic load in the processing of fresh-cut lettuce and
carrot (COD: 1000 mg/L) treated with NEW (50, 75 and 100 ppm) chlorine (100
ppm) and tap water at 4ºC. Red line indicate the initial load of product (fresh-cut
lettuce) before submerging in washing solution
Under the tested conditions, it can be observed on lettuce that, after 45 seconds of
treatments, the washing control “Water” (W) and NEW50 only reduced the mesophilic
counts by 1,3 log and 1,5 CFU/mL, respectively and there was no significant difference
Page | 35
between them (P>0,05). NEW 75, NEW100 and the washing control “Chlorinated Water”
(CW) resulted in significantly higher reductions of mesophilic counts (P<0,05).
In the carrots processing, washing solution (W) resulted in 1,3 log CFU/mL. Meanwhile,
significant higher reductions in the number of mesophilic counts were obtained for samples
treated with NEW solutions (75 and 100 ppm) and chlorinated water (CW) comparing to
the washing control W (P<0,05).
Our results showed that the effectiveness of NEW solutions at a concentration of 75 and
100 ppm is comparable to the washing solution CW (P>0,05).
Table 3-9 and table 3-10 summarizes some quality parameters (COD, pH and THM values)
of the sanitation treatments on processing water, which give an idea about the potential
environmental impacts of this sanitizer.
(after and before NEW treatments) of lettuce.
COD (mg O2/L) pH

Washing
treatment Before After Before After
treatment treatment treatment treatment
W 942±83 1085±23 7,23±1,03 7,78±0,21

CW** (100 ppm) 1235±191 1169±25 6,42±0,17 6,39±0,31
NEW50 1160±28 1325±290 8,25±0,08 7,93±0,22
NEW75 1160±127 1228±145 8,40±0,09 7,97±0,24
NEW100 1265±120 1338±169 8,43±0,10 7,80±0,31
*Values are the mean ± standard deviation of at least three replicates
**CW solution was adjusted to a pH of 6,5 by adding citric acid.
Mention that these experiments have been run by adding organic matter (approximately
1000 mg O2/L) in the wash water in order to search more severe conditions.
So, according to these results, the processing water from the NEW treatments did not
resulted in a higher COD values as compared to the CW treatment. With regard to microbial
quality of processed water, this technique eliminates completely microorganisms in
suspension. But, the reaction of chlorine with organic matter from process water led to the
generation of the disinfection by-products, mainly trihalomethanes (THMs) (Table 3-10).
Table 3-10 Individual and total THM’s (µg/L) in the process wash water of
lettuce.
CHLORINE NEW100
Total Trihalomethanes (TTHMs) 74 109
Chloroform (µg/L) 61,0 81,2
Bromodichloromethane (µg/L) 7,3 12,0
Dibromochloromethane (µg/L) 3,4 11,5
Bromoform (µg/L) 2,8 4,0
As expected, the concentration of THM’s in NEW100 solution was similar to the

concentration in the CW solution, due to the same FC concentration in both wash water
solutions. The European Legislation states that, any contamination from disinfection by-
products should be kept as low as possible in drinking water with a maximum acceptable
level for THM’s of 100 µg/L (EU, 1998). Therefore, based on this limit established for
Page | 36
potable water, the total THM value of NEW100 solution exceed the authorized limit set by
legislation.
In summary, the main effects of the use of NEW treatment are:
a. With regards to fresh-cut vegetables, NEW75 and NEW100 reported similar
sanitation efficacy than chlorinated water.
b. At doses tested (50, 75 and 100 ppm), this sanitizer eliminates completely
microorganisms in suspension in process water, but due to the free chlorine
concentration of this wash water solutions the THM’s values are similar to
chlorinated water solutions, exceeding in some cases the authorized limit set
by legislation for drinking water.
c. This sanitation technique does not compromise product quality.
3.2.3 Effect of ozone (O3) washing solutions on bacterial inactivation on
lettuce and carrot processing
Ozonization of E. coli O157:H7 inoculated lettuce leaves were conducted at 1 ppm, 2 ppm
and 3 ppm ozone level for 45 seconds in process water with high organic load (500 mgO2/L
COD). Due to the high organic load of the process water, ozone showed only a limited level
of antimicrobial efficacy on the microbial load of the fresh-cut product. E. coli populations
on produce decreased by 1,2, 1,4 and 1,4 log CFU/g, respectively. Regarding carrot
processing, the antimicrobial efficacy of ozone treatment was even lower (<0,5 log CFU/g)
in the 3 doses tested (1 ppm, 2 ppm and 3 ppm). This is due to the high amount of organic
load present in the washing water of fresh-cut products (coming from the exudates) which
affects the efficacy of ozone treatment.
Despite the scarce efficacy of ozone treatment on the microbial reduction on products, it
is worth noting that bubbling ozone in the process water for 1 min resulted in a washing
water with less than 10 CFU/mL.
Since the aquatic toxicity of process wash water associated with the chlorinated by-
products is a major concern in terms of the protection water resources and the aquatic life,
a study on aquatic toxicity was conducted using the Microtox method (a rapid toxicity test
with the marine bioluminescent bacterium, Vibrio Fischeri, as the test organism). Based on
the classification categories of EC50, it can be reported that the application of 3 ppm ozone
treatment in lettuce was class 1 (low aquatic toxicity) compared to the aquatic toxicity
levels of chlorinated water which was evaluated to be class 4 (highly toxic) for up to 10
ppm FC levels (the toxicity test results for the 5 ppm FC is not available yet).
In order to investigate the possibility to reuse the ozonated water during lettuce washing
process, the effect of recirculating the ozonated process wash water on the microbial load
of lettuce and the process water was studied.
O3 generator
Figure 3-10 Schematic representation of the system used for ozone treatments
As demonstrated in Figure 3-10, starting with 3 ppm ozonated fresh water with no organic
load, and reusing this water with continuous bubbling of ozone resulted in 1,69 log CFU/mL
of E.coli O157:H7 in the first waste water, and the microbial load of waste water continued
Page | 37
to increase in each wash cycle being 1,78 log CFU/mL at the end of the second cycle and
2,50 log CFU/ml at the end of the third cycle (Table 3-11). Ozone bubbling of 1 minute
was enough to disinfect the waste water after each cycle. However, due to the increase in
the organic load of water after each cycle, the efficacy of ozone in keeping the water free
of microorganisms gradually decreased. Moreover, due to the high organic load it was not
possible to maintain a residual ozone level that is enough to keep the water free of
microorganisms during the wash treatment as obvious from the residual ozone levels
reported in Table 3-11.
Table 3-11 Effect of ozone bubbling on the microbial load of process wash water
Log
Microbial load Bubbling
reduction Residual
of process wastewater
Treatment (cfu/g) of E. ozone
water (log for 1 min
Coli O157:H7 (ppm)
cfu/ml) with O3
on lettuce
3 ppm O3 1,37 1,69 ND 0,25
1st wastewater –
1,48 1,78 ND 0,08
bubbling
2nd wastewater -
1,28 2,50 ND 0,09
bubbling
3.2.4 Effect of hydrogen peroxide (H2O2) washing solutions on bacterial

inactivation on lettuce and carrot processing
The effect of hydrogen peroxide on inactivating microorganisms on lettuce and carrots is
shown in Figure 3-11. The initial population of mesophilic load was 5,9 ±0,3 log CFU/mL
on lettuce and 6,6±0,0 log CFU/mL on carrots.
7,0 7,0
CARROT
6,0 LETTUCE
6,0
45''
5,0 5,0
1'
(log CFU/mL)
(log CFU/mL)
4,0 1'30'' 4,0

2'
3,0 3,0
2,0 2,0
1,0 1,0
0,0 0,0
HP HP HP HP 1% W CW HP HP HP HP 1% W CW
0,25% 0,50% 0,75% 0,25% 0,50% 0,75%
carrot (COD: 1000 mg/L) treated with H2O2 (0,25%, 0,5%, 0,75% and 1%),
chlorine (100 ppm) and tap water at 4ºC. Red line indicate the initial load of
product (fresh-cut lettuce) before submerging in washing solution
Page | 38
In our study with fresh-cut lettuce, washing solution (W) resulted in 0,8 log CFU/mL.
Compared to this washing control (W), treatments with H2O2 (0,25%, 0,5%, 0,75% and
1%) did not significantly enhanced the effectiveness of these washing solutions (P>0,05),
although it can be observed a slight additional bactericidal effect when using hydrogen
peroxide rather than water alone. However, no further benefit was provided by using H2O2
(at any dose) instead of chlorine (CW).
In carrot processing, the mesophilic counts were reduced by 1,3 log CFU/mL. In these
experiments, H2O2 did not show any advantage over the CW control treatment, which
showed to be the most effective treatment solution (6,2 log CFU/mL).
With regard to microbial quality of process water, H2O2 (at doses tested) eliminates
completely microorganisms in suspension. Mean values for physicochemical characteristics
of washing water of lettuce processed under different sanitation solutions (W, CW and
H2O2) are presented in table 3-12.
(after and before H2O2 treatments) of lettuce.
COD (mg O2/L) pH

Washing
W 942±83 1085±23 7,23±1,03 7,78±0,21

CW** (100 ppm) 1235±191 1169±25 6,42±0,17 6,39±0,31
H2O2 0,25% 1081±182 1148±258 7,93±0,07 7,81±0,14
H2O2 0,50% 1060±-- 1065±148 7,93±0,16 7,94±0,01
H2O2 0,75% 1170±-- 1108±202 7,82±0,25 7,87±0,17
H2O2 1,0% 1290±-- 3025±391 7,94±0,27 7,74±0,19
*Values are the mean ± standard deviation of at least three replicates (with some exceptions
indicated as “--“)
There was not a significant increase of COD and pH values when using hydrogen peroxide,
as sanitation treatment, instead of the washing water control (not taking into account the
pH values of the CW, which was acidified by adding citric acid).
In summary, the main effects of the use of hydrogen peroxide are:

a. At doses tested (1%, 2% and 5%), no further benefit was provided by using
H2O2 instead of chlorine (CW) on produce. By other way, mention that the
required doses of application to obtain any benefit are not appropriate and
realistic for the industry, since they are too high.
b. Regarding, the washing water, there is absence of microbial load for all doses
tested. Also regarding the final quality of water it is good to take into
consideration that this higienization agent produces no residue as it is
decomposed into water and oxygen.
c. There is no negative impact on the overall appearance of product.
Page | 39
3.2.5 Effect of peroxyacetic acid (PAA) and hydrogen peroxide (H2O2) washing
solutions on bacterial inactivation on lettuce and carrot processing
The effect of the mixture PAA+H2O2 from the commercial brand of Ecolab (TSUNAMI) on
the populations of mesophilic bacteria on fresh-cut lettuce and carrots is shown in figure
3-12.
The initial microbial load of the unwashed carrots and lettuce was respectively 6,7 and 5,7
log CFU/mL.
7,0 7,0
Aerobic mesophilic counts (CFU/mL)
LETTUCE
Aerobic mesophilic counts (CFU/mL)

6,0 6,0
5,0 5,0 CARROT

45''
4,0 1' 4,0
1'30'' 3,0
3,0
2'
2,0 2,0
1,0 1,0
0,0
0,0 PAA 0,025%
PAA 0,0375%
PAA 0,050% W CW
PAA 0,025%
PAA 0,0375%
PAA 0,050% W CW
carrot (COD: 1000 mg/L) treated with PAA + H2O2 (0,250%, 0,375%, 0,500 %),
chlorine (100 ppm) and tap water at 4ºC. Red line indicate the initial load of
product (fresh-cut lettuce) before submerging in washing solution
Reductions in meshophilic bacteria after washing with water (W) were about 1,2 log
CFU/mL in lettuce processing and 1,0 log CFU/mL in carrot processing. The highest
mesophilic reduction was observed after washing lettuce and carrots with TSUNAMI, not
being significant differences with CW solution (P>0,05) in the lettuce processing. However,
in the processing of carrots, TSUNAMI was able to result in significantly higher reduction
of mesophilic populations, at doses 375 and 500 ppm, than CW solution.
Chemical composition of process water, when using the commercial brand TSUNAMI as
sanitizer, is shown in table 3-13.
(after and before TSUNAMI treatments) of lettuce.
COD (mg O2/L) pH

Washing
W 942± 83 1085± 23 7,23±1,03 7,78±0,21

CW** (100 ppm) 1235±191 1169± 25 6,42±0,17 6,39±0,31
Tsunami 0,025% -- 1352±145 -- 6,92±0,09
Tsunami 0,0375% -- 1359± 93 -- 6,64±0,10
Tsunami 0,050% -- 1459±145 -- 6,44±0,05
Page | 40
*Values are the mean ± standard deviation of at least three replicates (with some exceptions
indicated as “--“)
The main component of the tested preparation is peroxyacetic acid, which is easily
decomposed to acetic acid. This fact supports the increase of COD values when using
TSUNAMI solution, when compared to water controls. However, no statistical differences
in COD values were observed derived from the increase in the concentration of the sanitizer
(P>0,05).
The impact of TSUNAMI sanitizer on the product quality of fresh-cut lettuce was also
evaluated by packaging the processed lettuce in individual bags, under a passive modified
atmosphere. Samples were stored simulating commercial conditions at 4ºC. The visual
appearance of product was assessed along its shelf life, resulting in an acceptable product,
at least, until day 7 (figure 3-13).
Day 0 Day 4
Day 7 Day 11
Figure 3-13 Appearance of minimally processed lettuce along the storage period
at 4ºC
In the treatments solutions using CITROSOL products, the assay was performed with
bacteria (E.coli O157:H7) artificially inoculated, instead of working with the natural
occurring flora (mesophilic bacteria). In order to simulate washing conditions of fresh-cut
industry not allowed to use sanitizers, the level of contamination was 4 log cycles of
pathogen in the washing tank. Besides, the initial COD level in processing water was
approximately 1000 mg O2/L.
CITROCIDE PC and CITROCIDE PLUS were applied at the manufacturer’s recommended

dose and results obtained are indicated below.
Page | 41
Table 3-14 E. coli O157:H7 (log CFU/mL) population before and after CITROCIDE
PLUS (250, 375 and 500 ppm) treatment on lettuce and carrots.
E. coli (Log CFU/mL)

LETTUCE CARROT
Before After Before After
treat. treat. treat. treat.
CITROCIDE PLUS 0,025% 4,49±0,17 <1,00 4,44±0,17 <1,00
CITROCIDE PLUS 0,0375% 4,52±0,07 <1,00 4,54±0,12 <1,00
CITROCIDE PLUS 0,050% 4,51±0,12 <1,00 4,54±0,02 <1,00
W 4,51±0,11 4,76±0,34 4,47±0,11 4,62±0,18
CW 4,42±0,03 <1,00 4,47±0,07 <1,00
The initial average of E.coli population ranged between 4,42 and 4,54 log CFU/mL in all
experiments performed, both in lettuce and carrots. As can be observed in table 3-14, the
higher reductions were obtained by washing in CITROCIDE PLUS and chlorinated water
(CW) solutions, while the lower reductions were obtained by the water washing (W). Thus,
washing with CITROCIDE PLUS for 45 seconds was equally effective against E.coli O157:H7
than chlorinated water.
(CITROCIDE PLUS treatments) of lettuce and carrots.
COD (mg O2/L) pH

Washing treatment
LETTUCE CARROT LETTUCE CARROT
W 916±81ª 851±18ª 7,82±0,22 7,81±0,00

CW** (100 ppm) 1055±7ª 998±32b 6,04±0,12 6,42±0,40
CITROCIDE PLUS 0,025% 1010±71ª 913±3ab 7,04±0,11 6,83±0,00
CITROCIDE PLUS 0,0375% 1060±42ª 980±29b 6,84±0,16 6,69±0,07
CITROCIDE PLUS 0,050% 1100±28ª 1003±10b 6,67±0,14 6,51±0,04
*Values are the mean ± standard deviation of at least three replicates. Data in the same column
followed by the same lowercase letter are not significantly different (P>0,05)
At CITROCIDE PLUS doses tested, there are no statistical differences (P>0,05) in the COD
levels depending on the sanitation procedure selected (CW or the mixture PAA+H2O2).
Table 3-16 E. coli O157:H7 (log CFU/mL) population before and after CITROCIDE
PC (0,5%, 0,6% and 0,7%) treatment on lettuce and carrots.
E. coli (Log CFU/mL)

LETTUCE CARROT
Before After Before After
treat. treat. treat. treat.
CITROCIDE PC 0,50% 4,51±0,00 <1,00 4,52±0,06 <1,00
CITROCIDE PC 0,60% 4,52±0,03 <1,00 4,54±0,10 <1,00
CITROCIDE PC 0,70% 4,63±0,06 <1,00 4,62±0,05 <1,00
W 4,52±0,10 4,47±0,02 4,52±0,07 4,63±0,15
CW 4,49±0,12 <1,00 4,50±0,10 --
Page | 42
The initial average of E.coli population ranged between 4,47 and 4,63 log CFU/mL in all
experiments performed, both in lettuce and carrots. As can be observed in table 3-13, the
higher reductions were obtained by washing in CITROCIDE PC and chlorinated water (CW)
solutions, while the lower reductions were obtained by the water washing (W). Thus,
washing with CITROCIDE PC for 45 seconds was equally effective against E.coli O157:H7
than chlorinated water.
(CITROCIDE PC treatments) of lettuce and carrots.
COD (mg O2/L) pH

Washing treatment
LETTUCE CARROT LETTUCE CARROT
W 933±45a 786±10a 8,00±0,04 8,00±0,12

CW** (100 ppm) 1130±42a 964±22a 6,30±0,22 6,48±0,08
CITROCIDE PC 0,50% 2145±163 b
1945±49 b
4,86±0,11 4,80±0,04
CITROCIDE PC 0,60% 2245±21bc 2160±71c 4,71±0,10 4,66±0,04
CITROCIDE PC 0,70% 2580±141c 2390±42c 4,59±0,16 4,51±0,00
*Values are the mean ± standard deviation of at least three replicates. Data in the same column
followed by the same lowercase letter are not significantly different (P>0,05)
There are statistical differences in COD levels between the two washing controls and the
solutions with CITROCIDE PC at the tested concentrations (P<0,05).
Table 3-18 indicates the concentration of THM’s in a peroxyacetic based sanitizer solution
at different doses. As can be observed, the values of total trihalomethanes found in the
washing solutions are well below the reference value established by legislation for drinking
water.
Table 3-18 Individual and total THM’s (µg/L) in the process wash water of lettuce
processing, treated with a peroxyacetic based sanitizer.
PAA 0,025% PAA 0,0375% PAA 0,05%

Total Trihalomethanes
11,3 8,8 11
(TTHMs)
Chloroform (µg/L) 3,3 2,9 8,4
Bromodichloromethane (µg/L) 2,0 3,8 3,5
Dibromochloromethane (µg/L) 3,7 6,8 7,2
Bromoform (µg/L) 2,3 4,0 3,8
In summary, the main effects of the use of peroxiacetic based sanitizers are:
a. In the study of the antimicrobial effectiveness on product, it has been
demonstrated that peroxiacetic acid based sanitizer, at 375 and 500 ppm,
provide further benefit than chlorine on product.
tested. According to the quality parameters of the effluent, mention that
there is a significant increase on COD values when using CITROCIDE PC at
doses tested (0,5, 0,6 and 0,7 %), not for washing solutions with CITROCIDE
PLUS (0,025, 0,0375 and 0,05%). Besides, no THMs generation by using
this sanitation technique.
Page | 43
3.2.6 Effect of lactic acid (LA) washing solutions on bacterial inactivation on

lettuce and carrot processing
Figure 3-14 shows the effect of lactic acid treatments on reducing the mesophilic population
on the processed water of lettuce processing. The initial population of mesophilic bacteria
was 5,9±0,3 log CFU/mL.
7,0
6,0
5,0 45''
(log CFU/mL)
1'
4,0
1'30''
3,0 2'
2,0
1,0
0,0
LA 0,25% LA 0,1% WATER CW
carrot (COD: 1000 mg/L) treated with lactic acid (0,25, 0,1%), chlorine (100
ppm) and tap water at 4ºC. Red line indicate the initial load of product (fresh-cut
lettuce) before submerging in washing solution
Water washing (W) resulted in 0,8 log CFU/mL reduction of the mesophilic population.
Regarding to the lactic acid solutions (0,25% and 0,1%) a higher reduction is observed
(3,6 log CFU/mL and 2,8 log CFU/mL, respectively) but it did not bring a significant
reduction comparing with chlorinated water (4,6 log CFU/mL).
(LA treatments) of lettuce.
Washing COD
pH
treatment (mg O2/L)
W 1085±23 7,78±0,21
CW** (100 ppm) 1169±25 6,39±0,31
LA (0,1%)* 2503 3,89
LA (0,25%)* 4160 3,28
LA (2%)* 23500 2,23
LA (4%)* -- --
LA (5%)* 55500 2,03
*Data from one repetition (guidance values). More repetitions are needed.
Page | 44
As observed in table 3-19, the use of lactic acid for higienization purposes has an impact
on the processed water quality, by increasing the COD values even at the minimum dose
tested (LA 0,1%). It must also be taken into account the acidification of the washing
solution and therefore, the resulting acidification of produce, conferring negative aspects
on their sensory quality.
In summary, the main effects of the use of lactic acid are:

a. At doses tested (0,25% and 0,1%) no further benefit was provided by using
LA instead of chlorine (CW) on product.
b. The use of lactic acid for higienization purposes has an impact on the
processed water quality, by increasing the COD values even at the minimum
dose tested.
c. There is a sensorial impact on the product due to the acidification.
3.2.7 Effect of chlorine dioxide (ClO2) washing solutions on bacterial

inactivation on the processing water of lettuce and carrot
Populations of E.coli O157:H7 surviving after treatments with chlorine dioxide (CLODOS
PURO) at two different concentrations are shown in table 3-20.
Table 3-20 E. coli O157:H7 (log CFU/mL) population before and after chlorine
dioxide (CLODOS PURO) treatment on lettuce and carrots. Two different levels of
inoculum tested (3 and 6 log CFU/mL)
Before treat. After treat.

[ClO2] [ClO2]
E.coli O157:H7 E.coli O157:H7
initial pH residual pH
(log CFU/mL) (log CFU/mL)
(ppm) (ppm)
ClO2 20 ppm 6,17±0,05 21,1±1,8 7,59±0,1 <1,00 4,4±0,6 6,93±0,1
ClO2 30 ppm 6,06±0,08 32,0±0,0 6,93±0,0 <1,00 5,5±1,6 6,75±0,1
ClO2 30 ppm 3,33±0,10 34,0±0,0 -- <1,00 19,3±1,2 6,87±0,1
The initial population of pathogen was around 6 log CFU/mL or 3 log CFU/mL depending of
the inoculum level tested.
Sanitation treatment with chlorine dioxide, for 45 seconds, resulted in a significant
(P<0,05) reduction of E.coli O157:H7 at both doses tested (20 and 30 ppm). No detectable
E. coli O157:H7 was found on artificially inoculated processing water after treatment.
In summary, the main effects of the use of chlorine dioxide are:

demonstrated that chlorine dioxide, at 20 and 30 ppm, eliminates completely
microorganisms in suspension in process water.
b. It has a reduced cross-reactivity with organic matter. No DBPs formation by
using this sanitation technique.
Page | 45
4. Conclusions and recommendations

Findings reported on the basis of the experiments performed and information gathered
during these 18 months are:
 According to the multi-hurdle approach (combination of physical and chemical

agents):
1. The main conclusion reached was that the use of combined treatments with +/-
pressures, to improve the contact of the chemical agent with the bacteria, does not
show enough added benefit (weak synergistic effect) regarding the treatment
performed at atmospheric pressure. Due to the findings along the investigation,
this hurdle technology has no significance for practical food preservation at industrial
scale.
Information obtained in the present study, in line with most of the scientific literature
on the topic, suggest that the use of decontamination techniques cannot guarantee
microbial safety of these products, due to the limited reductions in the numbers of
microorganisms, from nature, present in the product itself. Therefore, use of
sanitizers would be mainly justified by their efficacy for cross-contamination
avoidance in the production process of fresh-cut vegetables.
 According to the individual testing of sanitizing agents, the main results are detailed
below.
2. Chlorine (sodium hypochlorite):

a. The use of sodium hypochlorite at a level that will maintain residual free
chlorine levels around 2 ppm in the wash water would be a short-term
alternative for the fresh-cut industry as an effective way of wash water
sanitization with little disinfection by-products formation.
3. Neutral electrolyzed water:
a. With regards to fresh-cut vegetables, NEW75 and NEW100 reported similar
sanitation efficacy than chlorinated water.
b. At doses tested (50, 75 and 100 ppm), this sanitizer completely eliminates
microorganisms in suspension in process water, but due to the free chlorine
concentration of this wash water solutions the THM’s values are similar to
chlorinated water solutions, exceeding in some cases the authorized limit set
by legislation for drinking water.
4. Hydrogen Peroxide:
a. At doses tested (1%, 2% and 5%), no further benefit was provided by using
H2O2 instead of chlorine (CW) on product. The required doses of application
to obtain any benefit are not appropriate and realistic for the industry, since
they are too high.
tested. Also regarding the final quality of water it is good to take into
consideration that hydrogen peroxide produces no residue as it is
decomposed into water and oxygen.
c. There is no negative impact on the overall appearance of product.
Page | 46
5. Ozone:
a. With regards to fresh-cut vegetables, ozone reported a reduced sanitation
efficacy.
b. Taking into account the aquatic toxicity test results, ozone still remains to
be the most environmentally friendly alternative for wash water sanitization.
c. Since the high organic load of the wash waters makes it impossible to
maintain a residual ozone level during washing that will help to keep water
free of microorganisms, other sanitizing agents that could be used in
combination with ozone should be investigated. Moreover the potential use
of ozone for disinfecting the waste wash water before recirculating it to the
system should not be overlooked.
6. Peroxyacetic acid based sanitizers:
Two commercially available brands have been tested: TSUNAMI (from ECOLAB) and
CITROCIDE (from CITROSOL).
demonstrated that peroxiacetic acid based sanitizer, at 375 and 500 ppm,
provide more benefit than chlorine on product.
tested. According to the quality parameters of the effluent, a significant
increase on COD values was observed when using CITROCIDE PC at doses
tested (0,5, 0,6 and 0,7 %). This was not observed for washing solutions
with CITROCIDE PLUS (0,025, 0,0375 and 0,05%). Besides, THMs were not
generated by using this sanitation technique.
d. This approach therefore could be used as an effective way to sanitize fresh
product by food processors (tuning with WP8_ Pilot testing).
7. Lactic acid:
a. At doses tested (0,25% and 0,1%) no further benefit was provided by using
LA instead of chlorine (CW) on product.
b. The use of lactic acid for sanitation purposes has an impact on the processed
water quality, by increasing the COD values even at the minimum dose
tested.
c. There is a sensorial impact on the product due to the acidification.
8. Chlorine dioxide (CLODOS PURO):
c. In the study of the antimicrobial effectiveness on product, it has been
demonstrated that chlorine dioxide, at 20 and 30 ppm, eliminates completely
microorganisms in suspension in process water.
d. It has a reduced cross-reactivity with organic matter. No DBPs formation by
using this sanitation technique.
e. This approach therefore could be used as an effective way to sanitize fresh
produce by food processors (tuning with WP8_ Pilot testing).
The overall conclusions for the sanitizing agents tested are:

Results revealed that PAA and ClO2 treatments have comparable microbial decontamination
properties as chlorine, maintaining the overall quality of washed vegetables. Additionally,
their potential health impact and environmental effects regarding the by-products
generated from the reaction with organic matter are scarce. In summary, these sanitation
agents demonstrate their potential as an alternative sanitizing method to maintain safety
on the process wash water of the fresh-cut industry, avoiding cross-contamination during
Page | 47
processing. Hence, more realistic evaluation of their potential to replace chlorine

under simulated industrial conditions is needed (WP8_ pilot testing).
Further investigation is required regarding operational considerations, residues (both in

processed water and product), processed water quality parameters (such as microbial load,
COD, BOD, TOC values), product quality parameters (sensorial characteristics, microbial
load and other contaminants) number of product loads without refreshment, etc. A final
note is that sanitizer’s efficiency may be dependent on the kind of fresh vegetable and
different approaches should be considered by the food industry.
Page | 48
5. References
Artes, F., & Allende, A. (2005). Minimal fresh processing of vegetables, fruits and juices.
En Emerging technologies for food proccesing (págs. 677-716). London: Academic Press.
Artés, F., Gómez, P., Aguayo, E., Escalona, V., & Artés-Hernández, F. (2009). Sustainable
sanitation techniques for keeping quality and safety of fresh-cut plant commodities.
Postharvest Biology and Technology 51 , 287-296.
Beltrán, D., Selma, M., Tudela, J., & Gil, M. (2005). Effect of different sanitizers on
microbial and sensroy quality of fresh-cut potato strips strored under modified atmosphere
or vacuum packaging. Postharvest Biology and Technology 37(1) , 776.
Beuchat, L. (1998). Surface decontamination of fruits and vegetables eaten raw: A review.
Food Safety Unit, World Health Organisation WHO/FSF/FOS/98.2, 42 .
Dell'Erbaa, A., Falsanisia, D., Libertia, L., Notarnicolaa, M., & Santoroa, D. (2007).
Disinfection by-products formation during wastewater disinfection with peracetic acid.
Desalination, 215 , 177-186.
McWatters, K., Hashim, I., Walker, S., Doyle, M., & Rimal, A. (2002). Acceptability of
lettuce treated with a lactic acid and hydrogen peroxide antibacterial solution. Journal of
Food Safety 25 , 223-242.
Nou, X., & Luo, Y. (2010). Whole-leaf wsh improves chlorine efficacy for microbial reduction
and prevents pathogen cross-contamination during fresh-cut lettuce processing. Journal of
Food Science 75 , 283-290.
Parish, M., Beuchat, L., Suslow, T., Harris, L., Garret, E., & Farber, J. (2003). Methods to
reduce/eliminate pathogens from fresh and fresh cut produce. Comprehensive reviews in
Food Science and Food Safety 2 , 161-173.
Rahman, S., Jin, Y., & Oh, D. (2011). Combination treat,emt pf añkaline electrolysed water
and citric acid with mild heat to ensure microbial safety, shelf-life and sensory quality of
shredded carrots. Food microbiology, 28(3) , 484-491.
Randazzo, C., Pitino, I., Scifo, G., & Caggia, C. (2009). Biopreservation of minimally
processed Iceberg lettuce using a bacteriocin produced by Lactococcus lactic wild strain .
Food Control 20(8) , 756-763.
Rico, D., Martin-Diana, A., Barat, J., & Barry-Ryan, C. (2007). Extending and measuring
the quality of fresh-cut fruit and vegetables: a review. Trends in food science and
technology 18(7) , 373-386.
Ruiz-Cruz, S., Acedo-Felix, E., Diaz-Cinco, M., & Islas-Osuna, M. (2007). Efficacy of
sanitizers in reducing Escherichia coli O157:H7, Salmonella spp. and Listeria
monocytogenes populations on fresh-cut carrots. Food Control, 18 , 138.
Tagmat, T. (31 de January de 2013). RESFOOD public website. (Minerva) Obtenido de
www.resfood.eu
Tirpanalan, O., Zunabovic, M., Domig, K., & Kneifel, W. (2011). Mini review: Antimicrobial
strategies in the production of fresh-cut lettuce products.
http://www.formatex.org/microbiology3/chapters1.html. A. Mendez-Vilas (ed.).
Tomas-Callejas, A., López-Gálvez, F., Sbodio, A., Artés, F., Artés-Hernández, F., & Suslow,
T. (2012). Chlorine dioxide and chlorine effectiveness to prevent Escherichia coli O157:H7
and Salmonella cross-contamination on fresh-cut red chard. Food Control 23 , 325-332.
Trias, R., Badosa, E., Montesinos, E., & Bañeras, L. (2008). Bioprotective Leuconostoc
strains against Listeria monocytogenes in fresh fruits and vegetables. International Journal
of Food Microbiology, 127 (1-2) , 91-98.
van Tongeren, W., & Verschragen, E. (2013). Example presentation for RESFOOD
references in project deliverables. 7th IWA Specialist Conference on Efficient Use &
Management of Water. 2, págs. 333-349. Paris: XP-Dite Publications.
Page | 49
Page | 50
Definitions & Abbreviations

atm atmosphere
cfu colony forming units
ClO2 chlorine dioxide
COD chemical oxygen demand
COT Committee on toxicity
DBP disinfection by-products
E. coli Escherichia coli
FC free chlorine
FDA Food and Drug Administration
h hours
HClO Hypochlorus acid
H2O2 hydrogen peroxide
Kg kilogram
log log10
mg milligram
min minute
ml milliliter
MP minimally processed
n samples number/repetitions
NEW neutral electrolyzed water
NTU nephelometric turbidity units
O3 ozone
P positive pressure
PAA peroxyacetic acid
PCA plate count agar
ppm parts per million (mg/l)
SEM Scanning Electron Microscope
THMs trihalomethanes
TSA Tryptone Soy Agar
TSB Tryptone Soy Broth
V negative pressure (vacuum)
v/v volume/volume
YE yeast extract
w/v weight/volume
µg micrograms
Page | 51

The Optimum Disinfection Treatment According To The Antibacterial Efficacy & Impact On Vegetable Quality

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RESFOOD

RESOURCE EFFICIENT AND SAFE FOOD PRODUCTION AND PROCESSING

Deliverable No. D5.1

Document ID RESFOOD-D5.1 FINAL v2.0

Dissemination level Public

Main Author Eva Petri - CNTA

Issue date 8-OCTOBER-2014

EUROPEAN COMMISSION DG Environment

Disclaimer and acknowledgement

This project is co-funded by the 7th FP (Seventh

Silvia García CNTA

Eric Lefebvre KRONEN

Hülya Ölmez TÜBITAK

Sinem Malkoc TÜBITAK

Document Change Log

Version Date Comments

v0.1 19/06/14 First draft of document

v1.1 First draft based upon first final version

Document Distribution Log

Version Date Distributed to

v0.1 19/09/14 WP5 partners: KRONEN, TÜBITAK, TNO, VEGA MAYOR

v0.2 22/09/14 WP5 partners: KRONEN, TÜBITAK, TNO, VEGA MAYOR

v0.3 01/10/14 EXECUTIVE BOARD, KRONEN, TÜBITAK, TNO, VEGA MAYOR

Verification and approval

Verification Final Draft by WP leader Eva Petri 30/09/2014

Approval Final Deliverable by Willy van Tongeren 03/10/14

3.1 Multi hurdle approach: antimicrobial effectiveness of combined treatments on

This could lead to 30 to 75 % reduction of water, energy and nutrients use, 25 to 80 %

Table 1-1 Activities within WP5 of the RESFOOD project

Task Title Main activities

5.1 Study of the antimicrobial effectiveness o Study of the antimicrobial

5.3 Evaluation of quality effluent o Microbiological analysis

5.4 Product quality assessment o Sensory analysis (shelf life)

1.2 Purpose of the document

1.3 Document Structure

1.4 Deviations from original DoW

1.4.1 Description of work related to deliverable as given in DoW

1.4.2 Time deviations from original DoW

1.4.3 Content deviations from original DoW

2. Material and methods

2.2 Preparation of inoculum

2.4 Inoculation procedure

a) Attachment strength (Sr) of bacteria on iceberg lettuce leaf

b) Attachment strength (Sr) of bacteria on iceberg lettuce leaf

Figure 2-1 Comparison of attachment strength of pathogenic bacteria to iceberg

In this work it will be evaluated the efficacy of decontamination treatments on reducing

2.5 Application of sanitizing treatments

Reception of iceberg lettuce Reception from

Cut off the lettuce stem and remove

Inoculation (dipping time: 5 min) Microbiological

Washing without chlorine Washing with chlorine

Rinse in destilated water (Tª: 4ºC)

Microbiological Microbiological Microbiological

Decontamination processes were conducted on pilot-plant scale with semi-industrial

Figure 2-4 Semi-industrial equipment used for achieving: a) positive pressures

2.6 Washing solutions

2.6.2 Organic acids

2.6.3 Chlorine dioxide

2.6.4 Hydrogen peroxide

2.6.5 Electrolyzed water

Figure 2-5 Neutral electrolyzed water generator (ANE, INNOBAK SYSTEMS)

2.6.7 Peroxyacetic acid

Table 2-1 Sanitizer treatments and concentration of active components (based