Fat and fibre interfere with the dramatic effect that nanoemulsified d -limonene has on the heat resistance of Listeria monocytogenes

Accepted Manuscript Fat and fibre interfere with the dramatic effect that nanoemulsified D-limonene has on the heat resistance of Listeria monocytogenes

Javier Maté, Paula M. Periago, María Ros-Chumillas, Coralin Grullón, Juan Pablo Huertas, Alfredo Palop PII:

S0740-0020(16)30216-7

DOI:

10.1016/j.fm.2016.10.031

Reference:

YFMIC 2656

To appear in:

Food Microbiology

Received Date:

18 March 2016

Revised Date:

11 July 2016

Accepted Date:

20 October 2016

Please cite this article as: Javier Maté, Paula M. Periago, María Ros-Chumillas, Coralin Grullón, Juan Pablo Huertas, Alfredo Palop, Fat and fibre interfere with the dramatic effect that nanoemulsified D-limonene has on the heat resistance of Listeria monocytogenes, Food Microbiology (2016), doi: 10.1016/j.fm.2016.10.031

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Highlights

Nanoemulsified D-limonene reduced L. monocytogenes heat resistance 90 times in apple juice

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Nanoemulsified D-limonene also reduced L. monocytogenes heat resistance in a fat and fibre free carrot juice system

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Food fat and fibre interfere with the effect of nanoemulsified D-limonene

ACCEPTED MANUSCRIPT Fat and fibre interfere with the dramatic effect that nanoemulsified D-limonene has on the heat resistance of Listeria monocytogenes

Pablo Huertas1 and Alfredo Palop1,2*

1 Dpto.

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Javier Maté1, Paula M. Periago1,2, María Ros-Chumillas1, Coralin Grullón1, Juan

Ingeniería de Alimentos y del Equipamiento Agrícola, Campus de Excelencia

Internacional Regional "Campus Mare Nostrum". Escuela Técnica Superior de

Ingeniería Agronómica. Universidad Politécnica de Cartagena, Paseo Alfonso XIII 48, 30203 Cartagena, Spain

de Biotecnología Vegetal, Campus de Excelencia Internacional Regional

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2 Instituto

Corresponding author:

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"Campus Mare Nostrum". Universidad Politécnica de Cartagena, Spain

Alfredo Palop, Dpto. Ingeniería de Alimentos y del Equipamiento Agrícola,

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Spain.

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Universidad Politécnica de Cartagena, Paseo Alfonso XIII 48, 30203 Cartagena,

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Email: [email protected]; Phone: ++34 968 32 5762; Fax: ++34 968 32 5433

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ACCEPTED MANUSCRIPT Abstract

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The application of D-limonene in form of nanoemulsion has been proved to reduce

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dramatically the thermal resistance of Listeria monocytogenes in culture media. The

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present research shows very promising results on the application in food products.

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The thermal resistance of L. monocytogenes was reduced 90 times when 0.5 mM

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nanoemulsified D-limonene was added to apple juice. This is the biggest reduction in

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the heat resistance of a microorganism caused by an antimicrobial described ever.

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However, no effect was found in carrot juice. A carrot juice system was prepared in

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an attempt to unravel which juice constituents were responsible for the lack of effect.

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When fat and fibre were not included in the carrot juice system formulation, the

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thermal resistance of L. monocytogenes was, again, dramatically reduced in presence

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of nanoemulsified D-limonene, so these components were shown to interfere with

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the effect. Once this interaction with food constituents becomes solved, the addition

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of nanoemulsified antimicrobials would allow to reduce greatly the intensity of the

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thermal treatments currently applied in the food processing industry.

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Keywords:

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Nanoemulsions, D-limonene, heat resistance, Listeria monocytogenes, antimicrobial

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effect, synergistic effect.

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1. Introduction Fruit juices are healthy and nutritious drinks. Development of new packaging technologies has increased fruit juice consumption in the last decades. The trend of

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current society is to consume fresh-like products that keep their nutritional and

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quality properties next to the raw products. Hence, food industry needs to reduce the

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intensity of the thermal treatments and to seek strategies that permit to keep the same

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food safety levels without significantly increasing the costs of production

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(Devlieghere et al., 2004). However, mild heat treatments can result in the survival of

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pathogenic microorganisms.

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Past occurrences of illnesses due to consumption of contaminated

unpasteurized fruit juices have led the United States Food and Drug Administration

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(USFDA) to enact the federal Juice Hazard Analysis and Critical Control Point

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(HACCP) and ensure the safety of juice products (FDA, 2001). This program

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compels manufacturers to subject juice products to a processing step or combinations

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of processes capable of reducing the population of the target pathogen by 5 log10

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cycles (Goodrich, Schneider, & Parish, 2005).

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Bacterial pathogens pertinent to juice safety have been identified as

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Escherichia coli O157:H7, Salmonella enterica and Listeria monocytogenes. The

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identification of these pathogens was based on their historical association with juice

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products as well as the possibility of the pathogens to be involved in future outbreaks

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(FDA, 2001).

One of the strategies followed in recent years to reduce the thermal treatments

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has been the application of antimicrobials. When they have been applied combined

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with thermal treatments, a reduction of treatment temperatures and times can be

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achieved, improving the nutritional and sensory properties of the product while

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ACCEPTED MANUSCRIPT keeping its safety. Among natural antimicrobials, essential oils have been widely

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used. Essential oils contain a complex mixture of non-volatile and volatile

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compounds produced by aromatic plants as secondary metabolites (Bakkali et al.,

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2008). Essential oils have flavoring, antioxidant and antimicrobial properties

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(Tajkarimi et al., 2010). Antimicrobial action of essential oils has been attributed to

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their terpene or terpenoid nature and their interaction with microbial cell membranes.

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They are known to penetrate through the microbial membrane and cause the leakage

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of ions and cytoplasmic content, thus leading to cellular breakdown (Espina et al.,

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2013). The interest of incorporating essential oils in foods as preservatives is related

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to their recognition as safe natural compounds, being a potential alternative to

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produce foods free of synthetic additives.

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researches have evaluated the combined effect of heat treatments and essential oils

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applied directly in fruit juices. Somolinos et al. (2010) evaluated the combined effect

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of citral, a terpenoid that is present in some citric fruit essential oils, and mild heat

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treatments against E. coli in a culture medium. These authors found a reduction of

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the initial population in 5 log10 cycles when 200 µL of citral and a temperature of 53

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ºC were applied to the heating medium. Later, Espina et al. (2012), investigated the

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combination of mild heat treatments with citrus fruit essential oils against E. coli in

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apple juice finding that treatment times were about 6 times lower when lemon

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essential oil was added. Ait-Ouazzou et al. (2012) evaluated the bactericidal effect of

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In recent years, only a few

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carvacrol in combination with mild heat against E. coli O157:H7 in fruit juices,

reaching a reduction of 75% of the time to reduce 5 log10 cycles when carvacrol was

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added to the heating medium. Espina et al. (2014) evaluated the combined effect of

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lemon essential oil and mild heat treatment in liquid whole egg against Salmonella

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ACCEPTED MANUSCRIPT Seftenberg and L. monocytogenes, inactivating up to 4 log10 of initial population in

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both cases.

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The incorporation of antimicrobial essential oils to foods still presents several

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drawbacks due to their poor water solubility and their strong flavor (Burt, 2004).

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Hence, there is a need to develop technologies that allow to increase the solubility of

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the essential oils in water and to reduce the concentration of active ingredients.

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Nanoemulsions allow solving the non-solubility of the essential oils in water (Donsi

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et al., 2012). These properties of nanoemulsions are determined by their droplet size

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and size distribution. Nanometric range emulsion droplets fuse with the cell

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membrane of bacteria and destabilize it, resulting in leakage of intracellular

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constituents (Ghosh et al., 2014).

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Recent researches have investigated the antimicrobial effect of essential oil

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nanoemulsions, but no thermal treatment was applied in combination. Ghosh et al.

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(2014) evaluated the antimicrobial effect of a nanoemulsion of eugenol in orange

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juice against Staphylococcus aureus. Donsi et al. (2014) evaluated the antimicrobial

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effect of a nanoemulsion of carvacrol in solid foods (zucchini and meat sausages)

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against E. coli. Severino et al. (2014) evaluated the antimicrobial effect of four

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nanoemulsions of essential oils, incorporated in chitosan based coatings over

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broccoli florets against L. monocytogenes. Maté et al. (2016a) also evaluated the

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antimicrobial effect of a nanoemulsion of D-limonene combined with nisin on

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different culture media and foods against L. monocytogenes. All these researches

have provided satisfactory results regarding the application of essential oils in form

of nanoemulsions. Maté et al. (2016b) showed the synergistic effect of mild heat treatment and a D-limonene nanoemulsion on L. monocytogenes in culture medium. In this

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ACCEPTED MANUSCRIPT investigation, the thermal resistance of L. monocytogenes was reduced about one

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hundred times. The aim of this research was to explore the effect of this same

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combined treatment on L. monocytogenes in fruit juices and to unravel which

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components of fruit juices interact with the nanoemulsion interfering with its

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antimicrobial effect.

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2. Materials and methods

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2.1. Bacterial strains

Listeria monocytogenes CECT 4032 was used in this research and it was

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provided by the Spanish Type Culture Collection (CECT, Valencia, Spain). This

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strain was stored at -80 °C (30% glycerol) until use. Fresh cultures were prepared by

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inoculating a loop of the cryopreserved culture in tryptic soy broth (TSB; Scharlau

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Chemie S.A., Barcelona, Spain) and incubating overnight at 37 ºC until the stationary

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growth phase was reached.

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2.2. Antimicrobials and preparation of nanoemulsions D-limonene was obtained from Sigma Aldrich Chemie (Steinheim,

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Germany). The nanoemulsions of D-limonene were prepared following the

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procedure described by Maté et al. (2016a) based on catastrophic phase inversion

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(CPI) method (Zhang et al., 2014). Briefly, the aqueous phase was prepared by

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mixing 55 mL of sterile distilled water and 27.5 mL of propylene glycol (Panreac, Barcelona, Spain). The oily phase was prepared mixing 6 mL of Tween 80 (Panreac)

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and 11.5 mL of D-limonene. Nanoemulsions were prepared by slowly adding

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aqueous phase into the oily phase with gentle magnetic agitation. The addition rate of

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aqueous phase was kept constant at approximately 1.0 mL/min with continuous

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ACCEPTED MANUSCRIPT stirring. A water-in-oil emulsion with a high oil-to-water ratio was formed, and then

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increasing amounts of water were added to the system, until a phase inversion

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occurred and an oil-in-water emulsion was formed, with continuous stirring for 6 h.

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Final concentration of D-limonene in the nanoemulsion was 1 M. All the ingredients

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of the nanoemulsion (D-limonene, propylene glycol and Tween 80) are considered as

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GRAS substances and permitted as food additives in the European Union.

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Nanoemulsions were aliquoted in pre-sterilized test tubes and stored in

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refrigeration until use. Droplet size was determinated at the beginning and at the end

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of the experiment. Size distribution of the oil droplets were determined by the laser

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light scattering method using Mastersizer 2000 (Malvern Instruments,

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Worcestershire, UK), as already described (Maté et al., 2016a).

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2.3. Fruit juices and juice systems

The natural carrot juice was prepared in the laboratory. Carrots obtained from a

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local market were washed with distilled water. Then carrots were homogenized with

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Thermomix ® (Vorwerk, Germany) for 20 minutes at 10000 rpm speed. The juice

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obtained was dispensed in bottles and autoclaved for 20 min at 120° C.

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A carrot juice system was also prepared by adding the majoritarian

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compounds described by the Agricultural Research Service of the USDA (2015)

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about nutritional composition of carrot juice. The composition per 100 g. was the

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following: 0.9 g of protein hydrolysate from wheat gluten (Sigma Aldrich, Spain), 0.2 g of sunflower seed oil (Sigma Aldrich), 9.6 g of fructose (Sigma Aldrich), 2.8 g

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of cellulose fibre (Sigma Aldrich) and 86.5 g of distilled water. Then the carrot juice

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system was dispensed in bottles and sterilized in autoclave for 20 min at 120 ºC. To

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explore the interaction of the food compounds with the nanoemulsion, some of these

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compounds were removed from the carrot juice system. When one (or more)

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compound(s) was (were) removed, its (their) corresponding weight was replaced by

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distilled water. Pasteurized apple juice was acquired from a local market. The nutritional

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composition was 0.2 g of protein, 20.2 g of carbohydrates and 79.6 g of water. No fat

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neither fibre was present in the apple juice.

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2.4. Heat treatments

Thermal inactivation kinetics were determined at constant temperature in a

thermoresistometer Mastia as described by Conesa et al. (2009). Briefly, the vessel of

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the thermoresistometer was filled with 400 mL of pre-sterilized fruit juice

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supplemented (or not) with 0.5 mM D-limonene nanoemulsified. This concentration

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was chosen as it did not change sensory properties of fruit juices (data not shown).

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Heat treatments were conducted at 52.5 °C. Once the heating medium temperature

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had attained stability (±0.05 °C), it was inoculated with 0.2 mL of the cell culture

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(approx. 109 cells mL-1). At preset intervals, 1 mL samples were collected into sterile

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test tubes, which were kept in ice until decimal dilutions were performed. Surviving

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cells were pour plated in TSA (Scharlau Chemie). Plates were incubated for 24 h at

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37 °C. Each treatment was assayed in triplicate in independent experiments

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performed in different days. Controls in sterile distilled water and foods with

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nanoemulsion components (tween 80 and 1, 2- propanediol. Panreac, Spain) without

D-limonene were developed at the same temperature (52.5 ºC) to evaluate the

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antimicrobial effect of these components and no antimicrobial effect was observed

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for both compounds (data not shown).

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2.5. Data analysis Decimal reduction times (D-values) were calculated as the inverse negative of

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the slope of the regression line of the survival curves, drawn plotting the logarithm of

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the survivors in front of the corresponding heating times. Survival curves included all

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the counts obtained in the different repetitions.

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Correlation coefficients (r0) of the regression lines of survival curves and 95% confidence limits (CL) were calculated by an appropriate statistical package.

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3. Results and discussion

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3.1. Droplet size distribution

Figure 1 shows the droplet size distribution of the nanoemulsion of D-

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limonene, at the beginning of the experiment. The mean droplet size was of 0.399

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µm, with a Sauter mean diameter (D (3,2)) of 0.262 µm, which falls into the

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consideration of nanoemulsion (Solans et al., 2005). No differences were found in

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size distribution along the time the present research was performed (data not shown).

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Phase separation was neither observed along this time. Data about size particles

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distribution showed similar results to these obtained previously (Maté et al., 2016a;

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2016b).

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3.2. Effect of nanoemulsified D-limonene on the heat resistance of Listeria

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monocytogenes in foods Figure 2 shows the survival curves of L. monocytogenes in apple juice (A)

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and carrot juice (B), as affected by the presence of 0.5 mM nanoemulsified D-

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limonene. The results were completely different in one and other juice. While the

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thermal resistance was reduced 90 times in apple juice with 0.5 mM nanoemulsified 9

ACCEPTED MANUSCRIPT D-limonene added (from a D52.5 ºC = 1.49 min without D-limonene to a D52.5 ºC =

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0.0166 min with 0.5 mM D-limonene; fig. 2A and Table 1), this effect was

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completely absent in the carrot juice (fig. 2B and Table 1). It is worth to note that the

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heat resistance of L. monocytogenes at 52.5 ºC in apple juice with 0.5 mM

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nanoemulsified D-limonene was so low that it was necessary to use a fraction

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collector to take the samples, since the whole experiment lasted less than 3 s.

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Actually, more than 90% of the initial population was inactivated instantaneously,

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just before the first sample had been taken, i.e. in less than 0.5 s.

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The dramatic decrease of the thermal resistance of L. monocytogenes found in

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apple juice when adding nanoemulsified D-limonene was of similar magnitude to the

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decrease previously reported for the same strain of this microorganism in TSB and

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other laboratory media (Maté et al., 2016b). All these decreases can be regarded as

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among the biggest reductions in thermal resistance caused by a combined process

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with heat and an antimicrobial. Only Luis-Villarroya et al. (2015) have found similar

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(although smaller, i.e. about 40 times) decreases of the thermal resistance of E. coli

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O157:H7 when adding propolis to pH 4 buffer. However, this effect was reduced to

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about 6.25 times when the thermal treatment was applied in apple juice (Luis-

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Villarroya et al., 2015). Opposite to this impressive decrease of the thermal

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resistance, the behavior of this same microorganism in carrot juice was completely

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different (fig. 2B and Table 1). A D52.5 ºC = 22.6 min was obtained in natural carrot

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juice and no significant decrease of the thermal resistance was observed after the

addition of nanoemulsified D-limonene (Table 1). The reason for the lack of effect could be attributed to the food matrix, since

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it has been shown that the food matrix components may interact with the essential

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oils, impairing their antimicrobial effects (Gutierrez et al., 2009; Hyldgaard et al.,

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2012; Weiss et al., 2014; Luis-Villarroya et al., 2015; Perricone et al., 2015; Rivera-

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Calo et al., 2015).

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The food matrix also has an important effect on the thermal resistance of bacteria. Among food characteristics, the pH is one of the most influencing factors

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on the heat resistance of bacteria (Ocio et al., 1994; Palop et al., 1999; Esteban et al.,

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2013). Actually, in this research, the thermal resistance was greatly reduced from

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carrot juice, with a pH of 7.1 (D52.5 ºC = 22.6 min), to apple juice, with a pH of 3.5

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(D52.5 ºC = 1.49 min), when no antimicrobials were added, so the pH seemed to have

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an effect on the thermal resistance of L. monocytogenes. Hence, in the same way, it

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could be expected that the pH would also be involved in the interaction with the

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essential oil nanoemulsion. However, the addition of nanoemulsified D-limonene led

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to a further decrease of 90 times in the D52.5 ºC in the acidic apple juice, but to no

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significant decrease in the D value in the neutral carrot juice. If any, neutral pH was

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inhibiting the effect of nanoemulsified D-limonene while acidic pH was favoring this

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effect. However, the dramatic decrease on the heat resistance of this same strain of

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L. monocytogenes was also shown in brain heart infusion broth at neutral pH when

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nanoemulsified D-limonene was present (Maté et al., 2016b), and no effect of the pH

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of the brain heart infusion broth was even observed. Therefore, the pH of the food

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did not seem to have a relevant role on the lack of effect of the nanoemulsion.

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3.3. Effect of food constituents on the heat resistance of Listeria monocytogenes in presence of nanoemulsified D-limonene An attempt was made to unravel the compounds present in carrot juice, which

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could be responsible for this drop of the antimicrobial effect. For this purpose, a

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carrot juice system, composed of its majoritarian components (USDA, 2015) was

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ACCEPTED MANUSCRIPT prepared. The thermal resistance in this carrot juice system was similar to that shown

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in the natural carrot juice (figures 2b and 3 and table 1). No significant differences

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were found among the D52.5 ºC values obtained in both media, and only a slight, but

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significant, reduction of the thermal resistance, to about one half, was achieved when

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nanoemulsified D-limonene was present in the carrot juice system (fig. 3 and Table

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1). These results point out, first that the composition of a food can be mimicked with

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its majoritarian components, at least in which microbial thermal resistance regards

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and, second, that the lack of effect of nanoemulsified D-limonene was still present in

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the carrot juice system: although in this artificial juice some significant effect was

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shown, the effect was very low, at least in comparison to that found in apple juice

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(fig. 2A).

Different constituents were removed from the carrot juice system. The effect

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of this removal on the heat resistance of L. monocytogenes, when the nanoemulsified

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D-limonene was not present in the heating medium, was as follows. The removal of

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proteins and fructose did not affect the thermal resistance of L. monocytogenes in the

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carrot juice system (data not shown). Also, the removal of fat led to a non-significant

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variation in the heat resistance (Table 1). However, it is noteworthy to point out the

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increase on the thermal resistance when fibre was removed from the formulation of

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the carrot juice system (from a D52.5 ºC = 24.76 min with fibre to a D52.5 ºC = 68.49

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min without fibre; Table 1). This increase on the thermal resistance, of almost three

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times, would involve an important role of fibre on reducing the thermal resistance of bacteria. When both fibre and fat were removed, a similar increase was also observed (Table 1). When nanoemulsified D-limonene was present in the heating medium, proteins and fructose neither have an effect, since the removal of these compounds

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ACCEPTED MANUSCRIPT from the carrot juice system led to no reduction of the thermal resistance of L.

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monocytogenes (data not shown). Fat had some effect, since the removal of fat from

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carrot juice system led to a reduction of the thermal resistance to about one half when

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adding nanoemulsified D-limonene (from a D52.5 ºC = 18.42 min without D-limonene

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to a D52.5 ºC = 8.31 min with nanoemulsified D-limonene; Table 1). Fat presence in

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foods has been linked to the lack of antimicrobial effect of essential oils, since they

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are supposed to move to the oily phase, turning absent from the watery phase, where

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the bacteria develop (Burt, 2004; Weiss et al., 2014; Perricone et al., 2015).

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Also fibre had some effect, since the removal of fibre also led to a reduction of the thermal resistance of L. monocytogenes in carrot juice system to about one

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half when adding nanoemulsified D-limonene (from a D52.5 ºC = 68.49 min without

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D-limonene to a D52.5 ºC = 38.16 min with nanoemulsified D-limonene; Table 1).

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But the most impressive decrease of the thermal resistance of L. monocytogenes in presence of nanoemulsified D-limonene was evidenced when both

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fat and fibre were removed from the formulation of the carrot juice system. In this fat

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and fibre free carrot juice system a reduction of almost 70 times in the D value was

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observed (from D52.5 ºC = 65.43 min without nanoemulsified D-limonene to D52.5 ºC =

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0.952 min with nanoemulsified D-limonene; figure 3 and table 1). This decrease in

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the thermal resistance was comparable to that found in apple juice (90 times; fig. 2A

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and Table 1) or laboratory media (Maté et al., 2016a), in which no fat neither fibre

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are present. As already observed in apple juice, or even in laboratory media (Maté et al., 2016a), an instantaneous inactivation of more than 90% of the initial population

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of L. monocytogenes was also shown in the fat and fibre free carrot juice system. It is

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hypothesised that this high percentage could correspond to an extremely sensitive

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portion of the population, and the less than 10% of the population remaining

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ACCEPTED MANUSCRIPT corresponding to a fraction more resistant to the combination of heat plus

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nanoemulsified D-limonene. To our knowledge, this is the first report of such a

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dramatic effect of a combined preservation process using an antimicrobial in a food

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or a food system. Food constituents are known to interact with antimicrobials (Burt,

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2004; Hyldgaard et al., 2012; Weiss et al., 2014; Perricone et al., 2015) and so, when

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antimicrobials are applied in foods to be heat treated, much of their antimicrobial

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effect is lost (Espina et al., 2012; Luis-Villarroya et al., 2015). However, the

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application of the antimicrobials in form of nanoemulsion helps to maintain the

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effect.

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It is noteworthy that fat and fibre separately interfered in a small amount with

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the effect of nanoemulsified D-limonene on the heat resistance of L. monocytogenes,

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so when only one of these compounds were removed from the formulation, the

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reduction of the thermal resistance was scarce, but when both compounds were

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absent, then the reduction of the thermal resistance caused by the presence of

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nanoemulsified D-limonene was very large. Then, some kind of interference with the

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effect of D-limonene has been shown, and this interference is only avoided when

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both compounds are removed from the food.

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This research shows that nanoemulsions of essential oils can be applied in a

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wide variety of foods with very satisfactory results, largely solving the problems

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raised by the food industry for the application of these compounds. But, this research

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also shows that certain nutritional components of foods could influence the effect of

nanoemulsions of essential oils. The mechanisms of interaction of the nanoemulsion

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with these food constituents should be elucidated, so improvements in their effect on

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the thermal resistance (and other properties of microorganisms) could be achieved.

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This fact would expand the variety of foods in which nanoemulsions of essential oils

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can be applied.

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Acknowledgements This research was financially supported by the Ministry of Economy and

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Competitiveness of the Spanish Government and European Regional Development

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Fund (ERDF) through project AGL2013-48993-C2-1-R

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References

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Ait-Ouazzou, A., Espina, L., García-Gonzalo, D., Pagán, R., 2012. Synergistic

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combination of physical treatments and carvacrol for Escherichia coli O157:H7

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inactivation in apple, mango, orange, and tomato juices. Food Control 32, 159-167.

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Bakkali, F., Averbeck, S., Averbeck, D., Idaomar, M., 2008. Biological effects of

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essential oils. Food and Chemical Toxicology 46, 446–475.

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Burt, S., 2004. Essential oils: their antibacterial properties and potential applications

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in foods. International Journal of Food Microbiology 94, 223-253.

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Conesa, R., Andreu, S., Fernández, P.S., Esnoz, A., Palop, A., 2009. Nonisothermal

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heat resistance determinations with the thermoresistometer Mastia. Journal of

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Applied Microbiology 107, 506-513.

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Devlieghere, F., Vermeulen, A., Debevere, J., 2004. Chitosan: antimicrobial activity,

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interactions with food components and applicability as a coating on fruit and

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vegetables. Food Microbiology 21, 703-714.

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Donsì, F., Annunziata, M., Vincensi, M., Ferrari, G., 2012. Design of

nanoemulsionbased delivery systems of natural antimicrobials: effect of the

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emulsifier. Journal of Biotechnology 159, 342-350.

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Donsì, F., Cuomo, A., Marchese, E., Ferrari, G., 2014. Infusion of essential oils for

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food stabilization: unraveling the role of nanoemulsion-based delivery systems on 15

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Technologies 22, 212-220.

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Espina, L., Somolinos, M., Ait-Ouazzou, A., Condón, S., García-Gonzalo, D., Pagán,

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R., 2012. Inactivation of Escherichia coli O157:H7 in fruit juices by combined

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treatments of citrus fruits essential oils and heat. International Journal of Food

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Microbiology 159, 9-16.

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Espina, L., Gelaw, T.K., de Lamo-Castellvi, S., Pagán, R., García-Gonzalo, D., 2013.

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Mechanism of bacterial inactivation by (+)-limonene and its potential use in food

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preservation combined processes. PloS ONE 8 (2): e56769.

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Espina, L., Condón, S., Pagán, R., García-Gonzalo, D., 2014. Synergistic effect of

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orange essential oil or D-limonene with heat treatments to inactivate Escherichia coli

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O157:H7 in orange juice at lower intensities while maintaining hedonic acceptability.

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Food and Bioprocess Technology 7, 471-481.

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Esteban, M.D., Huertas, J.P., Fernández, P.S., Palop, A., 2013. Effect of the medium

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characteristics and the heating and cooling rates on the nonisothermal heat resistance

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of Bacillus sporothermodurans IC4 spores in buffers and food. Food Microbiology

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34, 158-163.

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FDA, 2001. Hazard Analysis and Critical Control Point (HACCP): Procedures for

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the Safe and Sanitary Processing and Importing of Juice: Final Rule (21 CFR Part

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120): Fed. Regist., vol. 66. U.S. Food and Drug Administration, Washington, D. C.,

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pp. 6137–6202.

Ghosh, V., Mukherjee, A., Chandrasekaran, N., 2014. Eugenol-loaded antimicrobial

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nanoemulsion preserves fruit juice against, microbial spoilage. Colloids and Surfaces

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Biointerfaces 114, 392-397.

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An overview. Food Safety and Toxicology Series 15, 1-4.

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Gutierrez, J., Barry-Ryan, C., Bourke, P., 2009. Antimicrobial activity of plant

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essential oils using food model media: efficacy, synergistic potential and interactions

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with food components. Food Microbiology 26, 142-150.

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Hyldgaard, M., Myding, T., Meyer, R.L., 2012. Essential oils in food preservation:

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mode of action, synergies, and interactions with food matrix components. Frontiers

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in Microbiology 3, article 12.

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Luis-Villarroya, A., Espina, L., García-Gonzalo, D., Bayarri, S., Pérez, C., Pagán, R.,

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2015. Bioactive properties of a propolis-based dietary supplement and its use in

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combination with mild heat for apple juice preservation. International Journal of

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Food Microbiology 205, 90-97.

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Maté, J., Periago, P.M., Palop, A., 2016a. Combined effect of a nanoemulsion of D-

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limonene and nisin on Listeria monocytogenes growth and viability in culture media

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and foods. Food Science and Technology International 22, 146-152.

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Maté, J., Periago, P.M., Palop, A., 2016b. When nanoemulsified, D-limonene

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reduces Listeria monocytogenes heat resistance about one hundred times. Food

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Control 59, 824-828.

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Ocio, M.J., Sánchez, T., Fernández, P.S., Rodrigo, M., Martínez, A., 1994. Thermal

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resistance characteristics of PA3679 in the temperature range of 110-121 ºC as

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affected by pH, type of acidulant and substrate. International Journal of Food Microbiology 22, 239-247.

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Palop, A., Raso, J., Pagán, R., Condón, S., Sala, F.J., 1999. Influence of pH on heat

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resistance of Bacillus coagulans in buffer and homogenized foods. International

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Bioactivity of essential oils: a review on their interaction with food components.

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Frontiers in Microbiology DOI: 10.3389/fmicb.2015.00076.

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Rivera-Calo, J., Crandall, P., O´Bryan, C., Ricke, S., 2015. Essential oils as

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antimicrobials in food systems - A review. Food Control 54, 111-119.

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Severino, R., Vu, K.D., Donsì, F., Salmieri, S., Ferrari, G., Lacroix, M., 2014.

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Antimicrobial effects of different combined non-thermal treatments against Listeria

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monocytogenes in broccoli florets. Journal of Food Engineering 124, 1-10.

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Solans, C., Izquierdo, P., Nolla, J., Azemar, N., Garcia-Celma, M., 2005.

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Nanoemulsions. Current Opinion in Colloid and Interface Science 10, 102-110.

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Somolinos M., García D., Condón S., Mackey B., Pagán R., 2010. Inactivation of

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Escherichia coli by citral. Journal of Applied Microbiology 108, 1928–1939.

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Tajkarimi M.M., Ibrahim S.A., Cliver D.O., 2010. Antimicrobial herb and spice

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compounds in food. Food Control 21, 1199-1218.

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United States Department of Agriculture. Agricultural Research Service, 2015.

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National Nutrient Database for Standard Reference, Release 28.

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Weiss, J., McClements, D. J., Davidson, P.M., 2014. Nanoscalar dispersion of

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antimicrobials: effect on food safety. The World of Food Science 16, 8-19.

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Zhang, Z., Vriesekoop, F., Yuan, Q., Liang, H., 2014. Effects of nisin on the

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antimicrobial activity of D-limonene and its nanoemulsion. Food Chemistry 150,

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307-312.

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ACCEPTED MANUSCRIPT Figure captions.

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Figure 1. Droplet size distribution of the nanoemulsion of D-limonene.

Figure 2. Survival curves of Listeria monocytogenes at 52.5 °C in apple juice (A) and

carrot juice (B). Apple juice: ♦; apple juice with 0.5 mM D-limonene nanoemulsified: ♦; carrot juice: ■; carrot juice with 0.5 mM D-limonene nanoemulsified: □. Dashed lines represent the detection level of survival counts. Error bars stand for the standard deviation.

Figure 3. Survival curves of Listeria monocytogenes at 52.5 °C in carrot juice system.

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Carrot juice system: ●; carrot juice system fat and fibre free: ▲; carrot juice system

with 0.5 mM D-limonene nanoemulsified: ○; carrot juice system fat and fibre free with

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0.5 mM D-limonene nanoemulsified: ▲. Dashed line represents the detection level of

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survival counts. Error bars stand for the standard deviation.

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Figure 1

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Figure 2

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Figure 3

ACCEPTED MANUSCRIPT Fat and fibre interfere with the dramatic effect that nanoemulsified D-limonene has on the heat resistance of Listeria monocytogenes

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Javier Maté1, Paula M. Periago1,2, María Ros-Chumillas1, Coralin Grullón1, Juan Pablo Huertas1 and Alfredo Palop1,2*

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Dpto. Ingeniería de Alimentos y del Equipamiento Agrícola, Campus de Excelencia

Internacional Regional "Campus Mare Nostrum". Escuela Técnica Superior de

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Ingeniería Agronómica. Universidad Politécnica de Cartagena, Paseo Alfonso XIII 48, 30203 Cartagena, Spain

Instituto de Biotecnología Vegetal, Campus de Excelencia Internacional Regional

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Corresponding author:

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"Campus Mare Nostrum". Universidad Politécnica de Cartagena, Spain

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Alfredo Palop, Dpto. Ingeniería de Alimentos y del Equipamiento Agrícola, Universidad Politécnica de Cartagena, Paseo Alfonso XIII 48, 30203 Cartagena, Spain.

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Email: [email protected]; Phone: ++34 968 32 5762; Fax: ++34 968 32 5433

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ACCEPTED MANUSCRIPT Abstract

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The application of D-limonene in form of nanoemulsion has been proved to reduce

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dramatically the thermal resistance of Listeria monocytogenes in culture media. The

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present research shows very promising results on the application in food products. The

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thermal resistance of L. monocytogenes was reduced 90 times when 0.5 mM

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nanoemulsified D-limonene was added to apple juice. This is the biggest reduction in

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the heat resistance of a microorganism caused by an antimicrobial described ever.

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However, no effect was found in carrot juice. A carrot juice system was prepared in an

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attempt to unravel which juice constituents were responsible for the lack of effect.

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When fat and fibre were not included in the carrot juice system formulation, the thermal

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resistance of L. monocytogenes was, again, dramatically reduced in presence of

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nanoemulsified D-limonene, so these components were shown to interfere with the

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effect. Once this interaction with food constituents becomes solved, the addition of

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nanoemulsified antimicrobials would allow to reduce greatly the intensity of the thermal

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treatments currently applied in the food processing industry.

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Keywords:

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Nanoemulsions, D-limonene, heat resistance, Listeria monocytogenes, antimicrobial

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effect, synergistic effect.

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1. Introduction

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Fruit juices are healthy and nutritious drinks. Development of new packaging technologies has increased fruit juice consumption in the last decades. The trend of

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current society is to consume fresh-like products that keep their nutritional and quality

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properties next to the raw products. Hence, food industry needs to reduce the intensity

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of the thermal treatments and to seek strategies that permit to keep the same food safety

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levels without significantly increasing the costs of production (Devlieghere et al., 2004).

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However, mild heat treatments can result in the survival of pathogenic microorganisms.

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Past occurrences of illnesses due to consumption of contaminated unpasteurized

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fruit juices have led the United States Food and Drug Administration (USFDA) to enact

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the federal Juice Hazard Analysis and Critical Control Point (HACCP) and ensure the

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safety of juice products (FDA, 2001). This program compels manufacturers to subject

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juice products to a processing step or combinations of processes capable of reducing the

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population of the target pathogen by 5 log10 cycles (Goodrich, Schneider, & Parish,

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2005).

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Bacterial pathogens pertinent to juice safety have been identified as Escherichia

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coli O157:H7, Salmonella enterica and Listeria monocytogenes. The identification of

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these pathogens was based on their historical association with juice products as well as

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the possibility of the pathogens to be involved in future outbreaks. One of the strategies followed in recent years to reduce the thermal treatments

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has been the application of antimicrobials. When they have been applied combined with

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thermal treatments, a reduction of treatment temperatures and times can be achieved,

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improving the nutritional and sensory properties of the product while keeping its safety.

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Among natural antimicrobials, essential oils have been widely used. Essential oils

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contain a complex mixture of non-volatile and volatile compounds produced by

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ACCEPTED MANUSCRIPT aromatic plants as secondary metabolites (Bakkali et al., 2008). Essential oils have

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flavoring, antioxidant and antimicrobial properties (Tajkarimi et al., 2010).

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Antimicrobial action of essential oils has been attributed to their phenolic compounds

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and their interaction with microbial cell membranes. They are known to penetrate

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through the microbial membrane and cause the leakage of ions and cytoplasmic content,

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thus leading to cellular breakdown (Espina et al., 2013). The interest of incorporating

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essential oils in foods as preservatives is related to their recognition as safe natural

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compounds, being a potential alternative to produce foods free of synthetic additives.

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In recent years, only a few researches have evaluated the combined effect of heat

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treatments and essential oils applied directly in fruit juices. In the year 2010, Somolinos

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et al. evaluated the combined effect of citral, a phenolic compound that is present in

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some citric fruit essential oils, and mild heat treatments against E. coli in a culture

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medium. These authors found a reduction of the initial population in 5 log10 cycles

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when 200 µL of citral and a temperature of 53 ºC were applied to the heating medium.

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Later, in the year 2012, Espina et al., investigated the combination of mild heat

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treatments with citrus fruit essential oils against E. coli in apple juice finding that

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treatment times were about 6 times lower when lemon essential oil was added. In the

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same year, Ait-Ouazzou et al., evaluated the bactericidal effect of carvacrol in

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combination with mild heat against E. coli O157:H7 in fruit juices, reaching a reduction

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of 75% of the time to reduce 5 log10 cycles when carvacrol was added to the heating

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medium. Espina et al., in the year 2014, evaluated the combined effect of lemon

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essential oil and mild heat treatment in liquid whole egg against Salmonella Seftenberg

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and L. monocytogenes, inactivating up to 4 log10 of initial population in both cases.

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The incorporation of antimicrobial essential oils to foods still presents several

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drawbacks due to their poor water solubility and their strong flavor (Burt, 2004). Hence,

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ACCEPTED MANUSCRIPT there is a need to develop technologies that allow to increase the solubility of the

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essential oils in water and to reduce the concentration of active ingredients.

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Nanoemulsions allow solving the non-solubility of the essential oils in water (Donsi et

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al., 2012). These properties of nanoemulsions are determined by their droplet size and

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size distribution. Nanometric range emulsion droplets fuse with the cell membrane of

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bacteria and destabilize the microorganism's lipid envelope resulting in leakage of

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intracellular constituents (Ghosh et al., 2014).

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Recent researches have investigated the antimicrobial effect of essential oil

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nanoemulsions, but no thermal treatment was applied in combination. Ghosh et al.

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(2014) evaluated the antimicrobial effect of a nanoemulsion of eugenol in orange juice

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against Staphylococcus aureus. Donsi et al. (2014) evaluated the antimicrobial effect of

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a nanoemulsion of carvacrol in solid foods (zucchini and meat sausages) against E. coli.

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Severino et al. (2014) evaluated the antimicrobial effect of four nanoemulsions of

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essential oils, incorporated in chitosan based coatings over broccoli florets against L.

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monocytogenes. Maté et al. (2016a) also evaluated the antimicrobial effect of a

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nanoemulsion of D-limonene combined with nisin on different culture media and foods

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against L. monocytogenes. All these researches have provided satisfactory results

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regarding the application of essential oils in form of nanoemulsions.

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Maté et al. (2016b) showed the synergistic effect of mild heat treatment and a Dlimonene nanoemulsion on L. monocytogenes in culture medium. In this investigation,

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the thermal resistance of L. monocytogenes was reduced about one hundred times. The

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aim this research was to explore the effect of this same combined treatment on fruit

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juices and to unravel which components of fruit juices interact with the nanoemulsion

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inhibiting its antimicrobial effect.

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2. Materials and methods

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2.1. Bacterial strains Listeria monocytogenes CECT 4032 was used in this research and it was

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provided by the Spanish Type Culture Collection (CECT, Valencia, Spain). This strain

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was stored at -80°C (30% glycerol) until use. Fresh cultures were prepared by

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inoculating a loop of the cryopreserved culture in tryptic soy broth (TSB; Scharlau

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Chemie S.A., Barcelona, Spain) and incubating overnight at 37ºC until the stationary

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growth phase was reached.

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2.2. Antimicrobials and preparation of nanoemulsions

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D-limonene was obtained from Sigma Aldrich Chemie (Steinheim, Germany). The nanoemulsions of D-limonene were prepared following the procedure described by

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Maté et al. (2016a) based on catastrophic phase inversion (CPI) method (Zhang et al.,

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2014). Final concentration of D-limonene in the nanoemulsion was 1 M.

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Nanoemulsions were aliquoted in pre-sterilized test tubes and stored in refrigeration until use. Droplet size was determinated at the beginning and at the end of

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the experiment. Size distribution of the oil droplets were determined by the laser light

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scattering method using Mastersizer 2000 (Malvern Instruments, Worcestershire, UK),

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as already described (Maté et al., 2016a). Data about size particles distribution showed

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similar results to these obtained previously (Maté et al., 2016a) (data not shown).

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2.3. Fruit juices and juice systems The natural carrot juice was prepared in the laboratory. Carrots obtained from a local market were washed with distilled water. Then carrots were homogenized with

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Thermomix ® (Vorwerk, Germany) for 20 minutes at 10000 rpm speed. The juice

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obtained was dispensed in bottles and autoclaved for 20 min at 120° C.

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A carrot juice system was also prepared by adding the majoritarian compounds described by the Agricultural Research Service of the USDA (2015) about nutritional

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composition of carrot juice. The composition per 100 g. was the following: 0.9 g of

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protein hydrolysate from wheat gluten (Sigma Aldrich, Spain), 0.2 g of sunflower seed

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oil (Sigma Aldrich), 9.6 g of fructose (Sigma Aldrich), 2.8 g of cellulose fibre (Sigma

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Aldrich) and 86.5 g of distilled water. Then the carrot juice system was dispensed in

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bottles and sterilized in autoclave for 20 min at 120 ºC. To explore the interaction of the

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food compounds with the nanoemulsion, some of these compounds were removed from

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the carrot juice system. When one (or more) compound(s) was (were) removed, its

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(their) corresponding weight was replaced by distilled water.

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Apple juice was acquired from a local market. The nutritional composition was 0.2 g of protein, 20.2 g of carbohydrates and 79.6 g of water. No fat neither fibre was

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present in the apple juice.

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2.4. Heat treatments

Thermal inactivation kinetics were determined at constant temperature in a

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thermoresistometer Mastia as described by Conesa et al. (2009). Briefly, the vessel of

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the thermoresistometer was filled with 400 mL of pre-sterilized fruit juice supplemented

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(or not) with 0.5 mM D-limonene nanoemulsified. This concentration was chosen as it

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did not change sensory properties of fruit juices (data not shown). Heat treatments were

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conducted at 52.5 °C. Once the heating medium temperature had attained stability

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(±0.05 °C), it was inoculated with 0.2 mL of the cell culture (approx. 108 cells mL−1).

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At preset intervals, 1 mL samples were collected into sterile test tubes, which were kept

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ACCEPTED MANUSCRIPT in ice until decimal dilutions were performed. Surviving cells were enumerated in TSA

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(Scharlau Chemie). Plates were incubated for 24 h at 37 °C. Each treatment was assayed

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in triplicate in independent experiments performed in different days. Controls in sterile

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distilled water and foods with nanoemulsion components (tween 80 and 1, 2-

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propanediol. Panreac, Spain) without D-limonene were developed at the same

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temperature (52.5 ºC) to evaluate the antimicrobial effect of these components and no

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antimicrobial effect was observed for both compounds (data not shown).

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2.5. Data analysis

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Decimal reduction times (D-values) were calculated as the inverse negative of

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the slope of the regression line of the survival curves, drawn plotting the logarithm of

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the survivors in front of the corresponding heating times. Survival curves included all

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the counts obtained in the different repetitions.

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Correlation coefficients (r0) of the regression lines of survival curves and 95% confidence limits (CL) were calculated by an appropriate statistical package.

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3. Results and discussion

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3.1. Effect of nanoemulsified D-limonene on the heat resistance of Listeria

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monocytogenes in foods

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Figure 1 shows the survival curves of L. monocytogenes in apple juice (A) and

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carrot juice (B), as affected by the presence of 0.5 mM nanoemulsified D-limonene. The

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results were completely different in one and other juice. While the thermal resistance

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was reduced 90 times in apple juice with 0.5 mM nanoemulsified D-limonene added

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(from a D52.5ºC = 1.49 min without D-limonene to a D52.5ºC = 0.0166 min with 0.5 mM

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D-limonene; fig. 1A and Table 1), this effect was completely absent in the carrot juice

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(fig. 1B and Table 1).

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The dramatic decrease of the thermal resistance of L. monocytogenes found in apple juice when adding nanoemulsified D-limonene was similar to that previously

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reported for the same crop of this microorganism in TSB and other laboratory media

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(Maté et al., 2016b), and all them can be regarded as among the biggest reductions in

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thermal resistance caused by a combined process with an antimicrobial. Only Luis-

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Villarroya et al. (2015) have found similar (although smaller, i.e. about 40 times)

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decreases of the thermal resistance of E. coli O157:H7 when adding propolis to pH 4

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buffer. However, this effect was reduce to about 6.25 times when the thermal treatment

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was applied in apple juice (Luis-Villarroya et al., 2015). Opposite to this impressive

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decrease of the thermal resistance, the behavior of this same microorganism in carrot

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juice was completely different (fig. 1B and Table 1). A D52.5ºC = 22.6 min was obtained

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in natural carrot juice and no significant decrease of the thermal resistance was observed

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after the addition of nanoemulsified D-limonene (Table 1).

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The reason for the lack of effect could be attributed to the food matrix, since it

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has been shown that the food matrix components may interact with the essential oils,

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impairing their antimicrobial effects (Gutierrez et al., 2009; Hyldgaard et al., 2012;

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Weiss et al., 2014; Luis-Villarroya et al., 2015; Perricone et al., 2015; Rivera-Calo et

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al., 2015).

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The food matrix also has an important effect on the thermal resistance of

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bacteria. Among food characteristics, the pH is one of the most influencing factors on

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the heat resistance of bacteria (Ocio et al., 1994; Palop et al., 1999; Esteban et al.,

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2013). Actually, in this research, the thermal resistance was greatly reduced from carrot

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juice, with a pH of 7.1 (D52.5ºC = 22.6 min), to apple juice, with a pH of 3.5 (D52.5ºC =

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ACCEPTED MANUSCRIPT 1.49 min), when no antimicrobials were added, so the pH seemed to have an effect on

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the thermal resistance of L. monocytogenes. Hence, in the same way, it could be

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expected that the pH would also be involved in the interaction with the essential oil

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nanoemulsion. However, the addition of nanoemulsified D-limonene led to a further

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decrease of 90 times in the D52.5ºC in the acidic apple juice, but to no significant decrease

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in the D value in the neutral carrot juice. If any, neutral pH was inhibiting the effect of

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nanoemulsified D-limonene while acidic pH was favoring this effect. However, the

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dramatic decrease on the heat resistance of this same crop of L. monocytogenes was also

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shown in brain heart infusion broth at neutral pH when nanoemulsified D-limonene was

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present (Maté et al., 2016b), and no effect of the pH of the brain heart infusion broth

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was even observed. Therefore, the pH of the food did not seem to be involved in this

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lack of effect of the nanoemulsion.

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Water activity is another of the most influencing factors on the thermal

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resistance of bacteria, but both juices have similarly high water activity values. Hence,

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the reason for this impressive inhibition of the antimicrobial effect of nanoemulsified D-

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limonene in carrot juice (and other liquid foods) should be linked to the composition of

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these foods.

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An attempt was made to unravel the compounds present in carrot juice, which

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could be responsible for this drop of the antimicrobial effect. For this purpose, a carrot

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juice system, composed of its majoritarian components (USDA, 2015) was prepared.

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The thermal resistance in this carrot juice system was similar to that shown in the

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natural carrot juice (figures 1b and 2 and table 1). No significant differences were found

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among the D52.5ºC values obtained in both media, and only a slight, but significant,

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reduction of the thermal resistance, to about one half, was achieved when

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nanoemulsified D-limonene was present in the carrot juice system (fig. 2 and Table 1).

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majoritarian components, at least in which microbial thermal resistance regards and,

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second, that the lack of effect of nanoemulsified D-limonene was still present in the

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carrot juice system: although in this artificial juice some significant effect was shown,

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the effect was very low, at least in comparison to that found in apple juice (fig. 1A).

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Different constituents were removed from the carrot juice system. Proteins and

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fructose did not have any effect, since the removal of these compounds from the carrot

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juice system led to no reduction of the thermal resistance of L. monocytogenes when

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nanoemulsified D-limonene was present in the heating medium (data not shown). Fat

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had some effect, since the removal of fat from carrot juice system led to a reduction of

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the thermal resistance to about one half when adding nanoemulsified D-limonene (from

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a D52.5ºC = 18.42 min without D-limonene to a D52.5ºC = 8.31 min with nanoemulsified

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D-limonene; Table 1). Fat presence in foods has been linked to the lack of antimicrobial

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effect of essential oils, since they are supposed to move to the oily phase, turning absent

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from the watery phase, where the bacteria develop (Burt, 2004; Weiss et al., 2014;

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Perricone et al., 2015).

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Also fibre had some effect, since the removal of fibre also led to a reduction of the thermal resistance of L. monocytogenes in carrot juice system to about one half

236

when adding nanoemulsified D-limonene (from a D52.5ºC = 68.49 min without D-

237

limonene to a D52.5ºC = 38.16 min with nanoemulsified D-limonene; Table 1). It is

238

noteworthy to point out the increase on the thermal resistance of L. monocytogenes

239

when fibre was removed from the formulation of the carrot juice system (from a D52.5ºC

240

= 24.76 min with fibre to a D52.5ºC = 68.49 min without fibre; Table 1), regardless of the

241

addition of nanoemulsified D-limonene. This increase on the thermal resistance, of

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almost three times, would involve an important role of fibre on reducing the thermal

243

resistance of bacteria. But the most impressive decrease of the thermal resistance of L. monocytogenes

245

in presence of nanoemulsified D-limonene was evidenced when both fat and fibre were

246

removed from the formulation of the carrot juice system. In this fat and fibre free carrot

247

juice system a reduction of almost 70 times in the D value was observed (from D52.5ºC =

248

65.43 min without nanoemulsified D-limonene to D52.5ºC = 0.952 min with

249

nanoemulsified D-limonene; figure 2 and table 1). This decrease in the thermal

250

resistance was comparable to that found in apple juice (90 times; fig. 1A and Table 1) or

251

laboratory media (Maté et al., 2016a), in which no fat neither fibre are present. To our

252

knowledge, this is the first report of such a dramatic effect of a combined preservation

253

process using an antimicrobial in a food or a food system. Food constituents are known

254

to interact with antimicrobials (Burt, 2004; Hyldgaard et al., 2012; Weiss et al., 2014;

255

Perricone et al., 2015) and so, when antimicrobials are applied in foods to be heat

256

treated, much of their antimicrobial effect is lost (Espina et al., 2012; Luis-Villarroya et

257

al., 2015). However, the application of the antimicrobials in form of nanoemulsion

258

helps to maintain the effect.

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It is noteworthy that fat and fibre separately interfered in a small amount with

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the effect of nanoemulsified D-limonene on the heat resistance of L. monocytogenes, so

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when only one of these compounds were removed from the formulation, the reduction

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of the thermal resistance was scarce, but when both compounds were absent, then the

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reduction of the thermal resistance caused by the presence of nanoemulsified D-

264

limonene was very large. Then, some kind of interference with the effect of D-limonene

265

has been shown, and this interference is only avoided when both compounds are

266

removed from the food.

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ACCEPTED MANUSCRIPT 267

This research shows that nanoemulsions of essential oils can be applied in a wide variety of foods with very satisfactory results, largely solving the problems raised

269

by the food industry for the application of these compounds. But, this research also

270

shows that certain nutritional components of foods could influence the effect of

271

nanoemulsions of essential oils. The mechanisms of interaction of the nanoemulsion

272

with these food constituents should be elucidated, so improvements in their effect on the

273

thermal resistance (and other properties of microorganisms) could be achieved. This fact

274

would expand the variety of foods in which nanoemulsions of essential oils can be

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applied.

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Acknowledgements

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This research was financially supported by the Ministry of Economy and

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Competitiveness of the Spanish Government and European Regional Development

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Fund (ERDF) through project AGL2013-48993-C2-1-R

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Table 1. D values, 95% confidence limits and r0 values of Listeria monocytogenes at 52.5 ºC in different heating media without or with 0.5 mM D-limonene nanoemulsified. Heating medium

95% – CL

95% + CL

r₀

1.49c

1.19

1.99

0.991

0.0166a

0.0124

0.0251

0.984

22.59g

20.30

25.47

0.998

20.45fg

14.90

32.60

0.981

24.76fgh

17.89

40.22

0.979

12.94e

11.72

14.44

0.998

18.42f

16.37

19.11

0.982

Carrot juice system without fat with 0.5 mM D-limonene nanoemulsified

8.31d

7.17

10.31

0.987

Carrot juice system without fibre

68.49i

53.95

93.25

0.930

Carrot juice system without fibre with 0.5 mM D-limonene nanoemulsified

38.16h

32.77

45.53

0.950

Carrot juice system without fibre and fat

65.43i

50.37

93.33

0.968

0.952b

0.805

1.165

0.995

SC

D value (min)

Apple juice Apple juice with 0.5 mM D-limonene nanoemulsified

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Natural carrot juice Natural carrot juice with 0.5 mM D-limonene nanoemulsified Carrot juice system with 0.5 mM D-limonene nanoemulsified

ED

Carrot juice system without fat

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Carrot juice system

same letters indicate that there are no significant differences.

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a-f:

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Carrot juice system without fibre and fat with 0.5 mM D-limonene nanoemulsified