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

Fat and fibre interfere with the dramatic effect that nanoemulsified d-limonene has on the heat resistance of Listeria monocytogenes Academic research paper on "Biological sciences"

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{Nanoemulsions / " d -limonene" / "Heat resistance" / " Listeria monocytogenes " / "Antimicrobial effect" / "Synergistic effect"}

Abstract of research paper on Biological sciences, author of scientific article — Javier Maté, Paula M. Periago, María Ros-Chumillas, Coralin Grullón, Juan Pablo Huertas, et al.

Abstract The application of d -limonene in form of nanoemulsion has been proved to reduce dramatically the thermal resistance of Listeria monocytogenes in culture media. The present research shows very promising results on the application in food products. The thermal resistance of L. monocytogenes was reduced 90 times when 0.5 mM nanoemulsified d -limonene was added to apple juice. This is the biggest reduction in the heat resistance of a microorganism caused by an antimicrobial described ever. However, no effect was found in carrot juice. A carrot juice system was prepared in an attempt to unravel which juice constituents were responsible for the lack of effect. When fat and fibre were not included in the carrot juice system formulation, the thermal resistance of L. monocytogenes was, again, dramatically reduced in presence of nanoemulsified d -limonene, so these components were shown to interfere with the effect. Once this interaction with food constituents becomes solved, the addition of nanoemulsified antimicrobials would allow to reduce greatly the intensity of the thermal treatments currently applied in the food processing industry.

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

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Food Microbiology

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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: DOI:

Reference: To appear in:

S0740-0020(16)30216-7 10.1016/j.fm.2016.10.031 YFMIC 2656

Food Microbiology

Received Date: Revised Date: Accepted Date:

18 March 2016 11 July 2016 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

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Highlights

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

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

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

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

Javier Maté1, Paula M. Periago1,2, María Ros-Chumillas1, Coralin Grullón1, J'an Pablo Huertas1 and Alfredo Palop12*

1 Dpto. Ingeniería de Alimentos y del Equipamiento Agrícola, Campxs de Excelencia Internacional Regional "Campus Mare Nostrum". Escuela féc-:ca Superior de Ingeniería Agronómica. Universidad Politécnica de CartagNna, Paseo Alfonso XIII 48, 30203 Cartagena, Spain

2 Instituto de Biotecnología Vegetal, Campus de Excelencia Internacional Regional "Campus Mare Nostrum". Universidad Politécnica de Cartagena, Spain

Corresponding author:

Alfredo Palop, Dpto. Ingenie/a je Alimentos y del Equipamiento Agrícola, Universidad Politécnica de Cu~tagena, Paseo Alfonso XIII 48, 30203 Cartagena, Spain.

Email: alfredc.palop@apct.es; Phone: ++34 968 32 5762; Fax: ++34 968 32 5433

1 Abstract

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

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

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

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

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

7 the heat resistance of a microorganism caused by an antimicrobia' described ever.

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

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

10 When fat and fibre were not included in the carrot juice sys.em formulation, the

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

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

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

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

15 thermal treatments currently applied in tie food processing industry.

16 Keywords:

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

18 effect, synergistic effect.

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 current society is to consume fresh-like products that keep their nutritional and quality properties next to the raw products. Hence, food industry needs to reduce the intensity of the thermal treatments and to seek strategies that permit tc keeh the same food safety levels without significantly increasing the costs of prciuc*.on (Devlieghere et al., 2004). However, mild heat treatments can result in the survival of pathogenic microorganisms.

Past occurrences of illnesses due to consumption oi contaminated unpasteurized fruit juices have led the United States Food and Drug Administration (USFDA) to enact the federal Juice Hazard Analy^s and Critical Control Point (HACCP) and ensure the safety of juice products (FDA, 2001). This program compels manufacturers to subject juice products to a processing step or combinations of processes capable of reducing J^e population of the target pathogen by 5 log10 cycles (Goodrich, Schneider, & Parish, 2005).

Bacterial pathogens pe.+'nent to juice safety have been identified as Escherichia coli O15/.H/, Salmonella enterica and Listeria monocytogenes. The identification of these pathogens was based on their historical association with juice products as wJl as he possibility of the pathogens to be involved in future outbreaks (FDA, 2001).

Oi° of the strategies followed in recent years to reduce the thermal treatments has been the application of antimicrobials. When they have been applied combined with thermal treatments, a reduction of treatment temperatures and times can be achieved, improving the nutritional and sensory properties of the product while

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

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

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

48 2008). Essential oils have flavoring, antioxidant and antimicrobial properties

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

50 their terpene or terpenoid nature and their interaction with microbial cJl membranes.

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

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

53 2013). The interest of incorporating essential oils in foods as preservatives is related

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

55 produce foods free of synthetic additives. Tn recent years, only a few

56 researches have evaluated the combined effec of heat treatments and essential oils

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

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

59 treatments against E. coli in a cuu-re medium. These authors found a reduction of

60 the initial population in 5 logl0 cycles when 200 |iL of citral and a temperature of 53

61 °C were applied to the heating medium. Later, Espina et al. (2012), investigated the

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

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

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

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

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

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

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

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

The incorporation of antimicrobial essential oils to foods still presents several drawbacks due to their poor water solubility and their strong flavor (Burt, 2004). Hence, there is a need to develop technologies that allow to increase the so'-'bility of the essential oils in water and to reduce the concentration of active ingredients. Nanoemulsions allow solving the non-solubility of the essential o:s in water (Donsi et al., 2012). These properties of nanoemulsions are determined by üeir droplet size and size distribution. Nanometric range emulsion droplets fure with the cell membrane of bacteria and destabilize it, resulting in leakage of intracellular constituents (Ghosh et al., 2014).

Recent researches have investigated the an+;microbial effect of essential oil nanoemulsions, but no thermal treatment was applied in combination. Ghosh et al. (2014) evaluated the antimicrobial effect of a nanoemulsion of eugenol in orange juice against Staphylococcus aureus. Donsi et al. (2014) evaluated the antimicrobial effect of a nanoemulsion of Ou"^acro in solid foods (zucchini and meat sausages) against E. coli. Severino et al. (2014) evaluated the antimicrobial effect of four nanoemulsions of essential oils, incorporated in chitosan based coatings over broccoli florets agaiist L. monocytogenes. Maté et al. (2016a) also evaluated the antimicrobial ffect of a nanoemulsion of D-limonene combined with nisin on different culture media and foods against L. monocytogenes. All these researches have p-°/ided 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

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

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

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

97 components of fruit juices interact with the nanoemulsion interfering with its

98 antimicrobial effect.

100 2. Materials and methods

101 2.1. Bacterial strains

102 Listeria monocytogenes CECT 4032 was used in this ^search and it was

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

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

105 inoculating a loop of the cryopreserved cultui- in +ryptic soy broth (TSB; Scharlau

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

107 growth phase was reached.

109 2.2. Antimicrobials andprenar^ ion of nanoemulsions

110 D-limonene was obtained from Sigma Aldrich Chemie (Steinheim,

111 Germany). The namen/isions of D-limonene were prepared following the

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

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

114 mixing 55 mL of sterile distilled water and 27.5 mL of propylene glycol (Panreac,

115 Barcelona, Spain). The oily phase was prepared mixing 6 mL of Tween 80 (Panreac)

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

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

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

stirring. A water-in-oil emulsion with a high oil-to-water ratio was formed, and then increasing amounts of water were added to the system, until a phase inversion occurred and an oil-in-water emulsion was formed, with continuous stirring for 6 h. Final concentration of D-limonene in the nanoemulsion was 1 M. All the ingredients of the nanoemulsion (D-limonene, propylene glycol and Tween 80) are considered as GRAS substances and permitted as food additives in the European Union.

Nanoemulsions were aliquoted in pre-sterilized test tubes rad :„..d in refrigeration until use. Droplet size was determinated at the beginning and at the end of the experiment. Size distribution of the oil droplets weie d^ermined by the laser light scattering method using Mastersizer 2000 (Malvern Instruments, Worcestershire, UK), as already described (Maté et al., 2016a).

2.3. Fruit juices andjuice 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 Thermomix ® (Vorwerk, Germany) for 20 minutes at 10000 rpm speed. The juice obtained was dispensed in boiJes and autoclaved for 20 min at 120° C.

A carrot juice sycem was also prepared by adding the majoritarian compounds described by the Agricultural Research Service of the USDA (2015) about nutritional composition of carrot juice. The composition per 100 g. was the 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 of cellulose fibre (Sigma Aldrich) and 86.5 g of distilled water. Then the carrot juice system was dispensed in bottles and sterilized in autoclave for 20 min at 120 °C. To explore the interaction of the food compounds with the nanoemulsion, some of these

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

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

146 distilled water.

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

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

149 neither fibre was present in the apple juice.

151 2.4. Heat treatments

152 Thermal inactivation kinetics were determined at constant temperature in a

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

154 the thermoresistometer was filled with 400 mL of pre-s^erilized fruit juice

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

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

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

158 had attained stability (±0.05 °C), / was • noculated with 0.2 mL of the cell culture

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

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

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

162 37 °C. Each treatment was assayed in triplicate in independent experiments

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

164 nanoemu-sion components (tween 80 and 1, 2- propanediol. Panreac, Spain) without

165 D-limonjne were developed at the same temperature (52.5 °C) to evaluate the

166 antimicrobial effect of these components and no antimicrobial effect was observed

167 for both compounds (data not shown).

2.5. Data analysis

Decimal reduction times (D-values) were calculated as the inverse negative of the slope of the regression line of the survival curves, drawn plotting the logarithm of the survivors in front of the corresponding heating times. Survival curves inclded all the counts obtained in the different repetitions.

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

3. Results and discussion

3.1. Droplet size distribution

Figure 1 shows the droplet size distribution oc the nanoemulsion of D-limonene, at the beginning of the experiment. The mean droplet size was of 0.399 |im, with a Sauter mean diameter (D (3,2)) of 0.262 |im, which falls into the consideration of nanoemulsion (Solans et al., 2005). No differences were found in size distribution along the time the present research was performed (data not shown). Phase separation was nei'her observed along this time. Data about size particles distribution showed similar results to these obtained previously (Maté et al., 2016a; 2016b).

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

Figure 2 shows the survival curves of L. monocytogenes in apple juice (A) and carrot juice (B), as affected by the presence of 0.5 mM nanoemulsified D-limonene. The results were completely different in one and other juice. While the thermal resistance was reduced 90 times in apple juice with 0.5 mM nanoemulsified

194 D-limonene added (from a D52.s °c = 149 min without D-limonene to a D525 °c =

195 0.0166 min with 0.5 mM D-limonene; fig. 2A and Table 1), this effect was

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

197 heat resistance of L. monocytogenes at 52.5 °C in apple juice with 0.5 mM

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

215 addition of nanoemulsified D-limonene (Table 1).

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

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

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

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

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 on the heat resistance of bacteria (Ocio et al., 1994; Palop et al., 1999; Est^an et al., 2013). Actually, in this research, the thermal resistance was greatly reduced from carrot juice, with a pH of 7.1 (D525 °c = 22.6 min), to apple juice, with a pH of 3.5 (D52.5 °C = 149 min), when no antimicrobials were added, so the pH seemed to have an effect on the thermal resistance of L. monocytogenes. Hence, in the same way, it could be expected that the pH would also be involved in the interaction with the essential oil nanoemulsion. However, the addition of lunoemulsified D-limonene led to a further decrease of 90 times in the D525 °c in the acidic apple juice, but to no significant decrease in the D value in the ne^tH carrot juice. If any, neutral pH was inhibiting the effect of nanoemulsified D-limonene while acidic pH was favoring this effect. However, the dramatic decease on the heat resistance of this same strain of L. monocytogenes was also shown in brain heart infusion broth at neutral pH when nanoemulsified D-limonene was present (Maté et al., 2016b), and no effect of the pH of the brain heart infusi'n broth was even observed. Therefore, the pH of the food did not seem to hav a relevant role on the lack of effect of the nanoemulsion.

3.3. Effec1 of fo>d 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 could be responsible for this drop of the antimicrobial effect. For this purpose, a carrot juice system, composed of its majoritarian components (USDA, 2015) was

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

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

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

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

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

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

250 its majoritarian components, at least in which microbial thermal rsist,— regards

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

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

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

254 (fig. 2A).

255 Different constituents were removed fom carrot juice system. The effect

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

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

258 proteins and fructose did not affec^ the thermal resistance of L. monocytogenes in the

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

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

261 increase on the thermal *esistance when fibre was removed from the formulation of

262 the carrot juice system (from a D52 5 °C = 24.76 min with fibre to a D525 °C = 68.49

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

264 times, wc'ld involve an important role of fibre on reducing the thermal resistance of

265 bacteria. When both fibre and fat were removed, a similar increase was also observed

266 (Table 1).

267 When nanoemulsified D-limonene was present in the heating medium,

268 proteins and fructose neither have an effect, since the removal of these compounds

from the carrot juice system led to no reduction of the thermal resistance of L. monocytogenes (data not shown). Fat had some effect, since the removal of fat from carrot juice system led to a reduction of the thermal resistance to about one half when adding nanoemulsified D-limonene (from a D525 °c = 18.42 min without D-limonene to a D525 °c = 8.31 min with nanoemulsified D-limonene; Table 1). Fat presence in foods has been linked to the lack of antimicrobial effect of essential oils, since they are supposed to move to the oily phase, turning absent from the watery phase, where the bacteria develop (Burt, 2004; Weiss et al., 2014; Perricone et al., 2015).

Also fibre had some effect, since the removal of fibre led to a reduction of the thermal resistance of L. monocytogenes in carrot juic^ system to about one half when adding nanoemulsified D-limonene (from a D525 °C = 68.49 min without D-limonene to a D525 °C = 38.16 min with nanoemulsified D-limonene; Table 1).

But the most impressive decrease ol me thermal resistance of L. monocytogenes in presence of nanoemulshmd D-limonene was evidenced when both fat and fibre were removed from the formulation of the carrot juice system. In this fat and fibre free carrot juice system a reduction of almost 70 times in the D value was observed (from D52 5 °c = 65.43 min without nanoemulsified D-limonene to D52 5 °c = 0.952 min with nanoeimisified D-limonene; figure 3 and table 1). This decrease in the thermal resistance was comparable to that found in apple juice (90 times; fig. 2A and Table 1) o" laboratory media (Maté et al., 2016a), in which no fat neither fibre are present. As already observed in apple juice, or even in laboratory media (Maté et al., 20^), an instantaneous inactivation of more than 90% of the initial population of L. monocytogenes was also shown in the fat and fibre free carrot juice system. It is hypothesised that this high percentage could correspond to an extremely sensitive portion of the population, and the less than 10% of the population remaining

corresponding to a fraction more resistant to the combination of heat plus nanoemulsified D-limonene. To our knowledge, this is the first report of such a dramatic effect of a combined preservation process using an antimicrobial in a food or a food system. Food constituents are known to interact with antimicrobials (Burt, 2004; Hyldgaard et al., 2012; Weiss et al., 2014; Perricone et al., 2015) an^ so, w^en antimicrobials are applied in foods to be heat treated, much of their armmimobial effect is lost (Espina et al., 2012; Luis-Villarroya et al., 2015). Hcwev„, the application of the antimicrobials in form of nanoemulsion helps to maintain the effect.

It is noteworthy that fat and fibre separately interfered in a small amount with the effect of nanoemulsified D-limonene on the heat resistance of L. monocytogenes, so when only one of these compounds were removed from the formulation, the reduction of the thermal resistance was scarce, but when both compounds were absent, then the reduction of the thermal resistance caused by the presence of nanoemulsified D-limonene was very la ge. Then, some kind of interference with the effect of D-limonene has been shown, and this interference is only avoided when both compounds are removed °rom the food.

This research smws that nanoemulsions of essential oils can be applied in a wide variety of foods w'th very satisfactory results, largely solving the problems raised by the mod industry for the application of these compounds. But, this research also shows that certain nutritional components of foods could influence the effect of nanoemulsions of essential oils. The mechanisms of interaction of the nanoemulsion with these food constituents should be elucidated, so improvements in their effect on the thermal resistance (and other properties of microorganisms) could be achieved.

This fact would expand the variety of foods in which nanoemulsions of essential oils can be applied.

Acknowledgements

This research was financially supported by the Ministry of Economy and Competitiveness of the Spanish Government and European Regional De-eloment Fund (ERDF) through project AGL2013-48993-C2-1-R

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Maté, J., Periago, P.M., Palop, A., 2016a. Combined effect of a nanoemulsion of D-limonene and nisin on Listeria monocytogenes growth and viability in culture media and foods. Food Science and Technology International 22, 146-152. Maté, J., Periago, P.M., Palop, A., 2016b. When nanoemulsified, D-limonene reduces Listeria monoyugenes heat resistance about one hundred times. Food Control 59, 824-828.

Ocio, M.J., Sánchez, T., Fernández, P.S., Rodrigo, M., Martínez, A., 1994. Thermal resistance characteristics of PA3679 in the temperature range of 110-121 °C as affected by pH, type of acidulant and substrate. International Journal of Food Microbiology 22, 239-247.

Palop, A., Raso, J., Pagán, R., Condón, S., Sala, F.J., 1999. Influence of pH on heat resistance of Bacillus coagulans in buffer and homogenized foods. International Journal of Food Microbiology 46, 243-249.

392 Perricone, M., Arace, E., Corbo, M., Sinigaglia, M., Bevilacqua, A., 2015.

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

394 Frontiers in Microbiology DOI: 10.3389/fmicb.2015.00076.

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

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

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

398 Antimicrobial effects of different combined non-thermal treatments against Listeria

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

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

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

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

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

404 Tajkarimi M.M., Ibrahim S.A., Cliver D.O., 2?1l. Antimicrobial herb and spice

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

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

407 National Nutrient Database for rcan.ard Reference, Release 28.

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

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

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

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

412 307-312.

Figure captions.

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

Figure 2. Survival curves of Listeria monocytogenes at 52.5 °C in apple juic~ (A) and carrot juice (B). Apple juice: ♦; apple juice with 0.5 mM D-limonene nanoemu'sified: 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. Carrot juice system: •; carrot juice system fat and f>re free: A; carrot juice system with 0.5 mM D-limonene nanoemulsified: o; caii^t juice system fat and fibre free with 0.5 mM D-limonene nanoemulsified: A. Dashed line represents the detection level of survival counts. Error bars stand for me staidard deviation.

Part-irk» Si7P DKt rihuHon

1 10 Particle Size (jim)

1000 3000

Figure 1

Time (min)

0 10 20 30 40 50 60 70

Time (min)

Figure 2

Figure 3

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

Javier Maté1, Paula M. Periago1'2, María Ros-Chumillas1, Coralin Grullón1, Jua' Pablo Huertas1 and Alfredo Palop12*

1 Dpto. Ingeniería de Alimentos y del Equipamiento Agrícola, Ca npus de Excelencia Internacional Regional "Campus Mare Nostrum". Escuela Técnica Superior de Ingeniería Agronómica. Universidad Politécnica de Cartagena, Paseo Alfonso XIII 48, 30203 Cartagena, Spain

2 Instituto de Biotecnología Vegetal, Campus de Excelencia Internacional Regional "Campus Mare Nostrum". Universidad Po,;técmca de Cartagena, Spain

Corresponding author:

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

Email: alfredo.palop@upct.es; Phone: ++34 968 32 5762; Fax: ++34 968 32 5433

1 Abstract

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

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

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

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

6 nanoemulsified D-limonene was added to apple juice. This is the biggest "edu'+ion in

7 the heat resistance of a microorganism caused by an antimicrobial de>cri'_d ever.

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

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

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

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

12 nanoemulsified D-limonene, so these component shown to interfere with the

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

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

15 treatments currently applied in the fo M processing industry.

16 Keywords:

17 Nanoemulsions, D-limonen,, he^t resistance, Listeria monocytogenes, antimicrobial

18 effect, synergistic effect.

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 current society is to consume fresh-like products that keep their nutritional and quality properties next to the raw products. Hence, food industry needs to reduce the ^ensuy of the thermal treatments and to seek strategies that permit to keep the same mod safety levels without significantly increasing the costs of production (Devli ghm, al., 2004). However, mild heat treatments can result in the survival of pathogenic microorganisms.

Past occurrences of illnesses due to consumption of contaminated unpasteurized fruit juices have led the United States Food and Drug Administration (USFDA) to enact the federal Juice Hazard Analysis and Critical Control Pomt (HACCP) and ensure the safety of juice products (FDA, 2001). This program compels manufacturers to subject juice products to a processing step or combinations of processes capable of reducing the population of the target pathogen by 5 log10 cycles (Goodrich, Schneider, & Parish, 2005).

Bacterial pathogens pertinent to juice safety have been identified as Escherichia coli O157:H7, Salmonella enter^a and Listeria monocytogenes. The identification of these pathogens was based on their historical association with juice products as well as the possibility of the pathogens to be involved in future outbreaks.

One of the strategies followed in recent years to reduce the thermal treatments has been the applic ation of antimicrobials. When they have been applied combined with thermal tmatments, a reduction of treatment temperatures and times can be achieved, improving the nutritional and sensory properties of the product while keeping its safety. Among natural antimicrobials, essential oils have been widely used. Essential oils contain a complex mixture of non-volatile and volatile compounds produced by

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

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

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

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

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

50 thus leading to cellular breakdown (Espina et al., 2013). The interest of in "oiporating

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

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

53 In recent years, only a few researches have evaluated the co mbined effect of heat

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

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

56 some citric fruit essential oils, and mild heat trea me^+s against E. coli in a culture

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

58 when 200 |iL of citral and a temperature of 53 °C were applied to the heating medium.

59 Later, in the year 2012, Espina et al., invest igated the combination of mild heat

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

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

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

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

64 of 75% of the tin. e to reduce 5 log10 cycles when carvacrol was added to the heating

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

66 essential o il and mild heat treatment in liquid whole egg against Salmonella Seftenberg

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

68 The incorporation of antimicrobial essential oils to foods still presents several

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

there is a need to develop technologies that allow to increase the solubility of the essential oils in water and to reduce the concentration of active ingredients. Nanoemulsions allow solving the non-solubility of the essential oils in water (Donsi et al., 2012). These properties of nanoemulsions are determined by their droplet size and size distribution. Nanometric range emulsion droplets fuse with the cell membrane of bacteria and destabilize the microorganism's lipid envelope resulting in leakage of intracellular constituents (Ghosh et al., 2014).

Recent researches have investigated the antimicrobial effect of essential oil nanoemulsions, but no thermal treatment was applied in com bimavn. Ghosh et al. (2014) evaluated the antimicrobial effect of a nanoemulsion of eugenol in orange juice against Staphylococcus aureus. Donsi et al. (2014) evaluated the antimicrobial effect of a nanoemulsion of carvacrol in solid foods (zuce úJ and meat sausages) against E. coli. Severino et al. (2014) evaluated the antimicrob'al eifect of four nanoemulsions of essential oils, incorporated in chitosan based coatings over broccoli florets against L. monocytogenes. Maté et al. (2016a) a'so evaluated the antimicrobial effect of a nanoemulsion of D-limonene combined with nisin on different culture media and foods against L. monocytogenes. All these researches have provided satisfactory results regarding the application ^ essential oils in form of nanoemulsions.

Maté et al. (2016b) showed the synergistic effect of mild heat treatment and a D-limonene nanoem ulsioi on L. monocytogenes in culture medium. In this investigation, the thermal msistaice of L. monocytogenes was reduced about one hundred times. The aim this research was to explore the effect of this same combined treatment on fruit juices and to unravel which components of fruit juices interact with the nanoemulsion inhibiting its antimicrobial effect.

95 2. Materials and methods

96 2.1. Bacterial strains

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

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

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

100 inoculating a loop of the cryopreserved culture in tryptic soy broth (TSB; Sa^lau

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

102 growth phase was reached.

104 2.2. Antimicrobials and preparation of nanoemulsions

105 D-limonene was obtained from Sigma Aldrich Chemie (Steinheim, Germany).

106 The nanoemulsions of D-limonene were prepare' flowing the procedure described by

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

108 2014). Final concentration of D-limonene in urn nanoemulsion was 1 M.

109 Nanoemulsions were aliquot^ in pe-sterilized test tubes and stored in

110 refrigeration until use. Droplet size war determinated at the beginning and at the end of

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

112 scattering method using M-stersizer 2000 (Malvern Instruments, Worcestershire, UK),

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

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

116 2.3. Fruit j ices and juice systems

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

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

Thermomix ® (Vorwerk, Germany) for 20 minutes at 10000 rpm speed. The juice obtained was dispensed in bottles and autoclaved for 20 min at 120° C.

A carrot juice system was also prepared by adding the majoritarian compounds described by the Agricultural Research Service of the USDA (2015) about nutritional composition of carrot juice. The composition per 100 g. was the 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 of cellu'ose fib e (Sigma Aldrich) and 86.5 g of distilled water. Then the carrot juice syste a was dispensed in bottles and sterilized in autoclave for 20 min at 120 °C. To explme the interaction of the food compounds with the nanoemulsion, some of these compounds were removed from the carrot juice system. When one (or more) compounds, was (were) removed, its (their) corresponding weight was replaced by distilled water.

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 present in the apple juice.

2.4. Heat treatments

Thermal inactivation kinetics were determined at constant temperature in a thermoresistometer M, stia as described by Conesa et al. (2009). Briefly, the vessel of the thermoresistometer was filled with 400 mL of pre-sterilized fruit juice supplemented (or not) with 0.5 mM D-limonene nanoemulsified. This concentration was chosen as it did not change sensory properties of fruit juices (data not shown). Heat treatments were conducted at 52.5 °C. Once the heating medium temperature had attained stability (±0.05 °C), it was inoculated with 0.2 mL of the cell culture (approx. 108 cells mL-1). At preset intervals, 1 mL samples were collected into sterile test tubes, which were kept

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

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

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

147 distilled water and foods with nanoemulsion components (tween 80 and 1, 2148 propanediol. Panreac, Spain) without D-limonene were developed at the same

149 temperature (52.5 °C) to evaluate the antimicrobial effect of these components and no

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

152 2.5. Data analysis

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

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

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

156 the counts obtained in the different repetitions.

157 Correlation coefficients (r0) o the i agression lines of survival curves and 95%

158 confidence limits (CL) were calculated by an appropriate statistical package.

160 3. Results and discus'ion

161 3.1. Effect of nanoemhlsifi d D-limonene on the heat resistance of Listeria

162 monocytogenes m foods

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

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

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

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

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

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

The dramatic decrease of the thermal resistance of L. monocytogenes found in apple juice when adding nanoemulsified D-limonene was similar to that previously reported for the same crop of this microorganism in TSB and other laboratory media (Maté et al., 2016b), and all them can be regarded as among the biggest rMuc/ons in thermal resistance caused by a combined process with an antimicrobial. Only Luis-Villarroya et al. (2015) have found similar (although smaller, i.e. about 40 times) decreases of the thermal resistance of E. coli O157:H7 when adding propolis to pH 4 buffer. However, this effect was reduce to about 6.25 times wi.en the thermal treatment was applied in apple juice (Luis-Villarroya et al., 2015). Opposite to this impressive decrease of the thermal resistance, the behavior of this same microorganism in carrot juice was completely different (fig. 1B and Table 1). A D52.5°c = 22.6 min was obtained in natural carrot juice and no significant decrece 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 it has been shown that the food matrix components may interact with the essential oils, impairing their antimicrou:J effects (Gutierrez et al., 2009; Hyldgaard et al., 2012; Weiss et al., 2014; Luis-Villarroya et al., 2015; Perricone et al., 2015; Rivera-Calo et al., 2015).

The food matrix also has an important effect on the thermal resistance of bacteria. A mong food characteristics, the pH is one of the most influencing factors on the heat resistance of bacteria (Ocio et al., 1994; Palop et al., 1999; Esteban et al., 2013). Actually, in this research, the thermal resistance was greatly reduced from carrot juice, with a pH of 7.1 (D52.5C = 22.6 min), to apple juice, with a pH of 3.5 (D52.5C =

193 1.49 min), when no antimicrobials were added, so the pH seemed to have an effect on

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

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

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

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

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

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

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

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

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

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

204 lack of effect of the nanoemulsion.

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

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

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

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

209 these foods.

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

211 could be responsible f r th's drop of the antimicrobial effect. For this purpose, a carrot

212 juice system, com pose* of its majoritarian components (USDA, 2015) was prepared.

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

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

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

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

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

These results point out, first that the composition of a food can be mimicked with its majoritarian components, at least in which microbial thermal resistance regards and, second, that the lack of effect of nanoemulsified D-limonene was still present in the carrot juice system: although in this artificial juice some significant effect was shown, the effect was very low, at least in comparison to that found in apple juice (fig. 1A).

Different constituents were removed from the carrot juice system. Promins and fructose did not have any effect, since the removal of these compouris f tm the carrot juice system led to no reduction of the thermal resistance of L. monocytogenes when nanoemulsified D-limonene was present in the heating medim ^aa'a not shown). Fat had some effect, since the removal of fat from carrot juice system led to a reduction of the thermal resistance to about one half when adding nanoemulsified D-limonene (from a D52.5°c = 18.42 min without D-limonene to a D52.5°c = 8.31 min with nanoemulsified D-limonene; Table 1). Fat presence in foods has been linked to the lack of antimicrobial effect of essential oils, since they are supposed +o move to the oily phase, turning absent from the watery phase, where the ba^ena d evelop (Burt, 2004; Weiss et al., 2014; Perricone et al., 2015).

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 when adding nanoemulsified D-limonene (from a D52.5°c = 68.49 min without D-limonene to a D52.5°c = 38.16 min with nanoemulsified D-limonene; Table 1). It is noteworthy to point out the increase on the thermal resistance of L. monocytogenes when fibre was removed from the formulation of the carrot juice system (from a D52.5°c = 24.76 min with fibre to a D52.5°c = 68.49 min without fibre; Table 1), regardless of the addition of nanoemulsified D-limonene. This increase on the thermal resistance, of

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

243 resistance of bacteria.

244 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 f-ee carrot

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

248 65.43 min without nanoemulsified D-limonene to D52.5C = 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 foo< sg+em. Food constituents are known

254 to interact with antimicrobials (Burt, 2004; Hyldg^ard 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 ffect i, 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.

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

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

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

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

263 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.

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 by the food industry for the application of these compounds. But, this research also shows that certain nutritional components of foods could influence the effect of nanoemulsions of essential oils. The mechanisms of interaction of the nanoem-'lsion with these food constituents should be elucidated, so improvements in their effect on the thermal resistance (and other properties of microorganisms) could be achieved. This fact would expand the variety of foods in which nanoemulsions of es:ential oils can be applied.

Acknowledgements

This research was financially supported by urn Ministry of Economy and Competitiveness of the Spanish Government and European Regional Development Fund (ERDF) through project AGL2013-4°993-C2-1-R

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

Heating medium D valup (min) 95% - CL 95% + CL r0

Apple juice 1.49c 1.19 1.99 0.991

Apple juice with 0.5 mM D-limonene nanoemulsified 0.0166a 0.0124 0.0251 0.984

Natural carrot juice 22.59g 20.30 25.47 0.998

Natural carrot juice with 0.5 mM D-limonene nanoemulsified 20.45fg 14.90 32.60 0.981

Carrot juice system 24.76fgh 17.89 40.22 0.979

Carrot juice system with 0.5 mM D-limonene nanoemulsified 12.94e 11.72 14.44 0.998

Carrot juice system without fat 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 nanoemulsfied 38.16h 32.77 45.53 0.950

Carrot juice system without fibre and fat 65.43i 50.37 93.33 0.968

Carrot juice system without fibre and fat with 0.5 mM D-l:monene nanoemulsified 0.952b 0.805 1.165 0.995

a-f: same letters indicate that there are no significant deferences.