Carvacrol-loaded chitosan nanoparticles maintain quality of fresh-cut carrots

Innovative Food Science and Emerging Technologies 41 (2017) 56–63

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Carvacrol-loaded chitosan nanoparticles maintain quality of fresh-cut carrots Ginés Benito Martínez-Hernández, Maria Luisa Amodio, Giancarlo Colelli ⁎ Dipto. SAFE, Università degli Studi di Foggia, Via Napoli 25, 71122 Foggia, Italy

a r t i c l e

i n f o

Article history: Received 8 July 2016 Received in revised form 1 February 2017 Accepted 13 February 2017 Available online 14 February 2017 Keywords: Essential oil Antimicrobial activity Chitosan Encapsulation Fresh-cut Whitening

a b s t r a c t The effects of carvacrol-loaded chitosan-tripolyphosphate nanoparticles (Np-EO) on the physicochemical, sensory and microbial quality of fresh-cut (FC) carrot slices stored up to 13 days at 5 °C were studied. Np-EO was compared to samples treated by NaOCl (100 mg L−1), Np (chitosan-tripolyphosphate nanoparticles without carvacrol) or individual chitosan (0.5%) and carvacrol (0.5%) solutions. Np-EO achieved the best sensory scores also avoiding carvacrol-related off-flavours found with the carvacrol solution. Furthermore, whitening of FC carrot slices was highly reduced in Np-EO samples. Np-EO reduced microbial levels in FC carrot slices by 0.6–3.0 log units on processing day compared to untreated (control) samples. Np-EO allowed to reduce the microbial growth in FC carrot slices during the first 9 days of storage similarly to carvacrol solution. Furthermore, Np-EO highly controlled microbial loads at the end of storage showing 2.3 (lactic acid bacteria), 6.1 (yeasts and moulds) and 5.1– 5.4 (mesophiles, psychrophiles and Enterobacteriaceae) lower log CFU g−1 units compared to control samples. Conclusively, Np-EO highly maintained microbial (2–6 lower log CFU g−1 units compared to control), sensory (up to 2.5 better scores than control) and physicochemical quality of FC carrot slices than control for 13 days at 5 °C. Industrial relevance: Natural essential oils industrially extracted from plants are potential alternative substances with high antimicrobial properties when tested in vitro. However, their microbicidal efficacy is greatly reduced due to their low solubility in washing solutions of fresh-cut products. Accordingly, chitosan-tripolyphosphate nanoencapsulation of essential oils such as carvacrol is a great opportunity to increase the antimicrobial properties of carvacrol to be used in fresh-cut fruit and vegetables alternatively to conventional NaOCl sanitation. © 2017 Elsevier Ltd. All rights reserved.

1. Introduction Carrot is a vegetable that occupies an important segment in the fresh-cut (FC) industry due to their versatility of use, pleasant flavour and nutritional and health-promoting benefits. However, minimal processing of FC carrots such as slicing induces tissue damage, which triggers non-microbial and microbial spoilage during storage and a subsequent negative impact on sensory quality (Martínez-Hernández, Amodio, & Colelli, 2016). Washing with 50–150 mg L− 1 chlorinated water (NaOCl) is a method widely used in the FC fruits and vegetables industry, thus reducing their initial microbial loads and ensure the food safety of these products (Martínez-Hernández et al., 2015). However, NaOCl may be potentially harmful for humans and the environment (Hrudey, 2009). Thus, several natural antimicrobials such as plant essential oils (EOs) are being studied as alternatives to NaOCl (Sivakumar & Bautista-Baños, 2014).

⁎ Corresponding author. E-mail address: [email protected] (G. Colelli).

http://dx.doi.org/10.1016/j.ifset.2017.02.005 1466-8564/© 2017 Elsevier Ltd. All rights reserved.

Generally, EOs possessing the strongest antibacterial properties are those that contain phenolic compounds such as carvacrol, eugenol, and thymol (Hirasa & Takemasa, 1998; Rota, Carraminana, Burillo, & Herrera, 2004). Carvacrol is a major component of the EOs derived from oregano, thyme, marjoram and summer savoury, and is generally recognized as a safe food-grade additive. The mechanism of action of carvacrol and EOs in general against microorganisms involves the interaction of phenolic compounds with the proteins (porins) in the cytoplasmic membrane that can precipitate and lead to leakage of ions and other cell contents causing cell lysis (Nychas, Skandamis, & Tassou, 2000). Carvacrol is reported to have strong bactericidal action (Lambert, Skandamis, Coote, & Nychas, 2001). Accordingly, good antimicrobial effects have been reported in different FC fruits and vegetables, such as lettuce, kiwifruit, apples and melons, treated with carvacrol-containing washing solutions (Bagamboula, Uyttendaele, & Debevere, 2004; Onursal et al., 2014; Roller & Seedhar, 2002). EOs have a strong antibacterial activity according to in vitro studies although it has generally been found that a greater concentration of EOs is needed to achieve the same effect in foods (Burt, 2004). However, high concentrations of EOs may transfer strong off-flavours perceived as non-

G.B. Martínez-Hernández et al. / Innovative Food Science and Emerging Technologies 41 (2017) 56–63

characteristic of the food product to FC fruits and vegetables. Furthermore, due to their lipophilic nature, it is difficult to dissolve EOs in aqueous solutions used as sanitising washing treatments for FC fruits and vegetables. Moreover, carvacrol is a volatile compound which easily evaporates and/or decomposes (oxidation) during food processing owing to direct exposure to light or oxygen. Chitosan has received great interest in the encapsulation of bioactive compounds due to its biocompatibility, low toxicity and biodegradability (Muzzarelli, 2010). The chitosan–tripolyphosphate (TPP) nanoparticles, composed of food-safe ingredients, have shown their capacity for the encapsulation and delivery of carvacrol and oregano oil allowing to retain the functional properties of these EOs (Hosseini, Zandi, Rezaei, & Farahmandghavi, 2013; Keawchaoon & Yoksan, 2011). Furthermore, encapsulation of carvacrol may reduce the characteristic off-flavours occurred in carvacrol crude solutions (occurring for the high concentrations needed for a good sanitising effect) due to the controlled release of the encapsulated EO. In this way, sanitising solutions containing encapsulated carvacrol may provide an excellent sanitising washing treatment with similar or higher antimicrobial activity compared to NaOCl without the problems related to dissolution in water of carvacrol and their characteristics off-flavours. To our knowledge there has been no prior work regarding the use of carvacrol nanoparticles in FC products so the study of its potential as a substitute of NaOCl is relevant for this sector. Among the variety of methods developed to prepare chitosan nanoparticles, ionic gelation technique has attracted considerable attention as this process is non-toxic, organic, solvent-free, convenient and controllable (Agnihotri, Mallikarjuna, & Aminabhavi, 2004). The ionic gelation technique is based on the electrostatic interaction between the positively charged primary amino groups of chitosan and the negatively charged groups of polyanions such as TPP. Present safety and risk assessment methods are based on knowledge gathered for conventional chemicals. Accordingly, knowledge on the toxicity of nanoparticles has been limited although it is rapidly growing (Bouwmeester et al., 2009). The aim of this study was to investigate the effects of a sanitising solution containing chitosan–TPP nanoparticles loaded with carvacrol (Np-EO) on the microbial and physiochemical quality of FC carrots throughout storage. Such treatment may be a potential substitute of NaOCl in the FC industry. 2. Material and methods 2.1. Plant material Carrots (Daucus carota L.) were obtained from a local market (Foggia, Italy) and stored at 5 °C and 90–95% RH until the next day, when they were processed. Minimal processing was accomplished in a disinfected cold room at 10 °C. Plant material was inspected; carrots were free from defects and with similar visual appearance. The carrots were washed (2 min) with NaOCl (100 mg L− 1; 5 °C; pH 6.5 ± 0.1) with a ratio of 300 g of plant material to 5 L of disinfected water (w/ v), rinsed (1 min) with tap water (5 °C) and drained in a perforated basket. The carrots were peeled using a manual peeler and cut into slices of 8-mm thickness using a manual slicer. The peeler and slicer were regularly disinfected with 70% ethanol during preparation. Slices corresponding to the central part of the carrots ranging from 14 to 18 mm in diameter were selected for the treatments. 2.2. Preparation of washing solution containing carvacrol-loaded Ch-TPP nanoparticles Np-EO were prepared according to Keawchaoon and Yoksan (2011). Chitosan solution (1% w/v) was prepared by dissolving chitosan flakes in aqueous acetic acid solution (1% v/v) at ambient temperature overnight. Tween 80 (HLB 15.9, 2.25 g) was then added to the solution and stirred at 60 °C for 2 h to obtain a homogeneous mixture. Carvacrol (2.5 g) was dissolved separately in ethanol (20 mL) and then this oil

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phase was gradually added drop-by-drop into the aqueous chitosan solution (200 mL) during homogenization (Ultra-Turrax T25 basic, IKA, Germany) at a speed of 13,500 × g for 10 min under a cold water bath (containing ice) to obtain an oil-in-water emulsion. Subsequently, a TPP solution (0.5% w/v, 200 mL) was slowly added into the oil-inwater emulsion drop-by-drop with stirring. Agitation was continuously done for 30 min at 13,500 × g. The particles were collected by centrifugation at 10,500 × g for 10 min at 25 °C and washed with aqueous Tween 80 solution (1% v/v) and distilled water several times to remove free carvacrol. The obtained wet particles were dispersed in aqueous acetic acid solution (0.48% v/v; 420 mL; pH = 4.3). A weight ratio of chitosan to carvacrol 1:1.25 was used for the present study. The carvacrol was successfully loaded into chitosan-TPP nanoparticles as confirmed by UV–vis spectrophotometry (Shimadzu UV1700, Kyoto, Japan) using a standard curve with different carvacrol concentrations (diluted with ethanol). Np-EO were immersed in ethanol for 1 h and maximum absorption peak at 275 nm, corresponding to carvacrol, was registered while it was not observed Np samples (Fig. 1). 2.3. Washing treatments Plant material slices were submerged in the washing treatments (5 °C) for 4 min with a ratio of 300 g of plant material to 5 L of sanitising washing (w/v). The washing treatments are detailed below. • Control: water. • NaOCl: 100 mg L−1 (acidified with citric acid to pH 6.5 ± 0.1). The product was rinsed after washing treatments for 1 min with cold tap water. • Chitosan solution: prepared at 0.5% as described in the previous section. pH of final solution was 4.36 ± 0.02. • Carvacrol solution: carvacrol was previously dissolved in ethanol (98%) and subsequently in water to final concentration of 0.5%. pH of final solution was 4.35 ± 0.03. • Solution with Ch-TPP nanoparticles (Np). Nanoparticles were prepared as previously described using 2.5 mL of distilled water instead of carvacrol. pH of final solution was 4.44 ± 0.02. • Solution containing carvacrol-loaded Ch-TPP nanoparticles (Np-EO). Nanoparticles were prepared as previously described pH of final solution was 4.33 ± 0.02. After treatments, product was drained in a perforated basket. Samples (150 g) were placed in 1-L rigid polypropylene (PP) clamshells (12 × 17 × 5 cm). Three clamshells (analytical replicates) per treatment were prepared for this experiment (one experiment replicate). Subsequently, the samples were stored at 5 °C (90–95% RH) up to 13 days. A synergism between reduced oxygen atmospheres and the antimicrobial effects of EOs may occur (Burt, 2004; Galvez, Abriouel, Lopez, & Ben Omar, 2007). Accordingly, samples were stored under aerobic conditions, using individual polyethylene bags to prevent water loss, to better understand the simple effect of the treatment during storage independently of the atmosphere conditions. Analyses were conducted on the processing day and after 3, 6, 9 and 13 days of storage. 2.4. Microbial analysis Standard enumeration methods were used to determine the microbial growth according to Rinaldi et al. (2013) but with slight modifications. A 10-g sample of carrots was mixed with 90 mL of sterile saline solution (8.5 g NaCl L− 1; Sigma Aldrich, Germany) for 1 min with a stomacher (Colwort Stomacher 400 Lab, Seward Medical, London, UK). One millilitre of the appropriate sample dilution was pour-plated on (1) plate count agar (PCA, Oxoid, Basingstoke, United Kingdom) incubated at 30 °C/24–48 h and 5 °C/7 days for total aerobic mesophilic and psychrophilic bacteria, respectively; (2) violet red bile dextrose agar (VRBD, Oxoid, Basingstoke, United Kingdom) incubated at 37 °C/

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G.B. Martínez-Hernández et al. / Innovative Food Science and Emerging Technologies 41 (2017) 56–63

Fig. 1. UV–vis absorption spectra from 250 to 400 nm of chitosan-TPP nanoparticles (dotted line) and carvacrol-loaded chitosan-TPP nanoparticles (solid line).

48 h for Enterobacteriaceae; and (3) de Man-Rogosa-Sharpe agar (MRS, Sigma Chemical Co., St. Louis, MO, USA) overlaid with the same medium and incubated aerobically at 37 °C/48 h for lactic acid bacteria (LAB). For yeast and moulds (Y + M), 100 μL of the appropriate sample dilution was spread-plated on potato dextrose agar base (PDA, Oxoid, Basingstoke, United Kingdom) with 100 mg L−1 chloramphenicol (Sigma Chemical Co., St. Louis, MO, USA) at 25 °C/4–6 days. All microbial counts were reported as log colony-forming units per gram (log CFU g−1). Each of the three replicates per treatment was analysed in duplicate.

system were recorded using on three equidistant points of each replicate. Three colour readings were recorded for each piece, and the mean measurement of five pieces for each replicate was recorded. The whiteness index (%) was calculated according to previously reported equation (Pushkala, Parvathy, & Srividya, 2012): rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi h iffi 2 ð100−LÞ2 þ a 2 þ b

WI ð%Þ ¼ 100−

2.5. pH, titratable acidity and soluble solid content The samples were analysed for soluble solid content (SSC), pH and titratable acidity (TA). Sample juice was prepared from five carrot slices by grinding with an Ultra-Turrax (T25 B, IKA, Germany) instrument and filtering through four layers of cheesecloth. A pH meter was used to determine the pH. The SSC of the juice was determined by a digital handheld refractometer (Atago N1, Tokyo, Japan) at 20 °C and expressed as °Brix. TA was determined by the titration of 7 mL of juice plus 33 mL of distilled water with 0.1 mol L−1 NaOH to pH 8.1 (T50, Metter Toledo, Milan, Italy) and expressed as % (g malic acid 100 mL−1). Three replicates per treatment were analysed. 2.6. Colour analysis Colour was determined using a colourimeter (Minolta CM-2600d, Japan). The standard tristimulus parameters (L*, a*, b*) of the CIE Lab

2.7. Sensory evaluation Sensory analyses were performed at the end of storage according to international standards (ASTM, 1986). The panel consisted of eight assessors (four women/four men, aged 24–40 years) screened for sensory ability (colour, odour detection, firmness and basic taste). A sample consisting of four carrot slices was prepared for every treatment and provided to each of the assessors in a covered white plastic plate with a random numeric code. Samples were allowed to temper 10 min at room temperature prior to sensory analysis. A 5-point scale of damage incidence and severity was scored for off-odours, off-flavours and whitening (5: extreme; 4: severe; 3: moderate, limit of usability; 2: slight; 1: none). Flavour, aroma and fresh-like firmness were assessed using another 5-point hedonic scale of acceptation (5: excellent, 4: good, 3: fair, limit of usability, 2: poor; 1: extremely bad).

G.B. Martínez-Hernández et al. / Innovative Food Science and Emerging Technologies 41 (2017) 56–63

2.8. Statistical analysis The experiment was subjected to analysis of variance using Statgraphics Plus (version 5.1) software. When treatment effect was compared with the control samples a one-way ANOVA for the treatment effect was run at each time of storage. Statistical significance was assessed at the level P = 0.05, and Tukey's multiple range test was used to separate means. 3. Results 3.1. Microbiological analysis The initial mesophilic, psychrophilic, Enterobacteriaceae, LAB and Y + M counts of unwashed carrots (data not shown) were 4.8, 3.4,

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3.5, 3.0 and 4.8 log CFU g−1, respectively (data not shown). Water washing, established as control treatment, did not induce significant microbial reductions. 3.1.1. Mesophiles The initial mesophilic level of control samples (4.5 log CFU g−1) were reduced by 1.6–2.1 log units after Np-EO, Ch and carvacrol solution treatments, without significant differences among them (Fig. 2A). Contrary, Np and NaOCl only reduced mesophilic levels by 1.1–1.3 log units without significant differences among them. During the first 9 days of storage, control and NaOCl showed the highest mesophilic growth rates with increments of 2.2 and 2.7 log units, respectively. Np and Ch registered intermediate increases of 1.8 and 1.3 log units after 9 days although a high increase was observed after 13 days reaching similar values to NaOCl and control. Np-EO and carvacrol solution

Fig. 2. Mesophilic (A), psychrophilic (B), Enterobacteriaceae (C), lactic acid bacteria (D) and yeasts and moulds loads (E) (log CFU g−1) of fresh-cut carrot slices subjected to different sanitising treatments and stored for up to 13 days at 5 °C. Np, chitosan-tripolyphosphate nanoparticles; Np-EO, carvacrol-loaded chitosan-tripolyphosphate nanoparticles. Different capital letters denote significant differences (P ≤ 0.05) among treatments for the same sampling day. Different lowercase letters denote significant differences (P ≤ 0.05) among sampling days for the same treatment.

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registered the lowest increments with 0.7–0.8 log units after 9 days comparing to their respective initial levels. While control and NaOCl registered low mesophilic increases (b0.6 log units) from day 9 to day 13, Ch and Np registered a high mesophilic increment of 1.9 and 2.1 log units, respectively. However, microbial levels of Np-EO samples decreased by 1.3 log units from day 9 to day 13. Accordingly, Np-EO was the only treatment that registered at the end of storage similar mesophilic levels to its initial values (3.1 log CFU g−1) while the mesophilic loads for the rest of treatments were between 6 and 7 log CFU g−1. 3.1.2. Psychrophiles The initial psychrophilic level of control samples (2.9 log CFU g−1) decreased by 0.4 log units after Np-EO treatment (Fig. 2B). The rest of treatments did not achieve significant psychrophilic reductions except chitosan and carvacrol solutions which showed an increase of 0.4–0.5 log units. Similar to mesophiles, control and NaOCl registered the highest psychrophilic increases with 3.9 and 3.3 log units increments after 9 days followed by Np and Ch. High microbial increases of 2.1 and 1.1 log units for NaOCl and Np, respectively, were registered from day 9 to day 13. Accordingly, control, NaOCl, Np and chitosan registered the highest psychrophilic levels ranging from 6.4 to 7.3 log CFU g− 1 after 13 days of storage. Similar to mesophiles, Np-EO showed the lowest psychrophilic level of 2.4 log CFU g−1 after 13 days which was similar to its initial level. 3.1.3. Enterobacteriaceae The initial Enterobacteriaceae counts of control samples (3.5 log CFU g−1) were not significantly reduced after washing treatments except carvacrol solution which achieved a reduction of 0.9 log units (Fig. 2C). Similar to mesophilic and psychrophilic levels, control and NaOCl registered the highest Enterobacteriaceae levels for the first 9 days with 3.3–3.4 log units remaining without great changes (b 0.4 log units) until the end of storage. Np and Ch Enterobacteriaceae levels remained stable for the first 3 days although a great and constant growth of approximately 4 log units was registered from day 3 to the end of storage. Np-EO and carvacrol solution registered the lowest increases of 0.5 and 1.1 log units, respectively, after 9 days. However, while levels of samples treated with carvacrol solution remained unchanged from day 9 to day 13, Np-EO decreased in 1.1 log units. According to mesophiles and psychrophiles, Np-EO was the only treatment that registered similar mesophilic levels at the end of storage compared to its initial levels (2.9 log CFU g−1). 3.1.4. Lactic acid bacteria Initial lactic acid bacteria (LAB) levels of control samples (2.7 log CFU g−1) were reduced by 1.2 log units after NaOCl (Fig. 2D) followed by a low microbial growth throughout storage with an increment of 0.6 log units after 13 days of storage. Similarly, control samples only registered an increment of 0.4 log units after 13 days of storage. The rest of treatments reduced LAB levels below the detection limit (1 log CFU g−1). Chitosan and Np showed the highest microbial levels with LAB of approximately 5 log CFU g−1 after 13 days. Samples treated with carvacrol solution increased through storage registering a microbial value of 3.2 log CFU g−1 at the end of storage. Np-EO showed the same LAB growth pattern compared to chitosan until day 6. However, from day 6 to day 9 LAB loads of Np-EO remaining stable followed by a decrease of 0.8 log units from day 9 to day 13. According to previous microbial groups, Np-EO showed the lowest LAB levels of 2.2 log CFU g−1 after 13 days of storage. 3.1.5. Yeasts and moulds The initial Y + M loads of control samples (4.7 log CFU g−1) were only reduced after carvacrol solution and Np-EO by 1.3 and 1.7 log units, respectively, without significant differences among them (Fig. 2E). Control registered the highest Y + M increase of 3.8 log units

after 13 days of storage. NaOCl showed a similar growth trend compared to control until day 6 with an increase of 1.5 log units and then showing only slight changes (b 0.5 log units) from day 6 to the end of storage. The Y + M loads of the rest of treatments (chitosan, carvacrol, Np and Np-EO) did not highly increase for the first 3 days showing levels of approximately 3.5 log CFU g−1. From day 3 to day 6 an increment of 1.4–2.4 log units was observed in the latter treatments without significant differences among them. However, while loads of chitosan and Np increased from day 6 reaching similar values to NaOCl on day 13, samples treated with Np-EO and carvacrol solution decreased showing Y + M levels of 2.2 and 3.0 log CFU g−1 on day 13, respectively. According to all microbial groups, Np-EO showed the lowest Y + M levels after 13 days of storage with 0.9 log units lower compared to its respective initial load. 3.2. pH, TA and SSC Control samples registered an initial pH of 6.10 (Table 1). While NaOCl induced a pH increment of 0.12 pH units, pH decreases of 0.22 for Np and 0.5–0.6 for the rest of treatments were observed. Throughout storage, pH of chitosan and Np increased reaching on day 6 similar levels to NaOCl and control, remaining all of those treatments without high changes (b 0.19 pH units) until the end of storage. Samples treated with Np-EO and carvacrol showed a slight pH decrement of 0.22 pH units after 9 days. However, while pH of Np-EO remained unchanged from day 9 to day 13, pH of samples treated with carvacrol highly increased in 2.16 pH units in those 4 last days of storage. The initial TA of controls samples was 0.081% (Table 1). According to pH data, TA of control samples decreased in 0.007 TA units after NaOCl treatment and increased in the remaining treatments of about 0.018– 0.053 TA units. An opposite but correspondent behaviour to pH was observed for TA throughout storage. Accordingly, at the end of storage NpEO registered the highest TA (0.188%) and samples treated with carvacrol the lowest one (0.010%) while the rest of treatments showed intermediate values. SSC of control samples (7.0 °Brix) decreased 0.15/0.30 °Brix after chitosan and carvacrol treatments but increased 0.20/0.35 after Np/ Np-EO treatments (Table 1) and did not change after NaOCl. SSC increased by 0.3–0.8 °Brix through storage in all conditions reaching maximum values generally after 6 days, although carvacrol and Np showed this maximum values earlier on day 3. Nevertheless, Np-EO remained unchanged for the first 6 days of storage. After those maximum levels, SSC decreased reaching in NaOCl and chitosan/Np samples the highest (7.1 °Brix) and the lowest (6.0–6.1 °Brix) values, respectively, with intermediate levels of 6.6–6.8 °Brix in the remaining treatments. 3.3. Colour analysis Control samples showed initial colour L* (Table 1), a* and b* values of 48.3, 21.7 and 29.99, respectively (data not shown), which corresponded to a WI of 36.1% (Table 1). Initial WI and L* of control samples decreased to 30.8–32.6% and 43.2–44.7, respectively, after treatments without significant differences among them. WI and L* increased throughout storage showing NaOCl/control the greatest increments of 13.6/11.7 and 12.1/13.6 units. On the other side, Np-EO registered the lowest WI increments of 5.0 units after 13 days and it was the only treatment where L* value remained unchanged after 13 days. 3.4. Sensory analysis Sensory scores of treated FC carrots at the end of storage are shown in Fig. 3. Samples treated with carvacrol showed higher off-odours and off-flavours scores than Np-EO which had absence of these carvacrol-related off-odours and off-flavours. Furthermore, chitosan showed a similar off-flavour score of 2 as carvacrol. The best aroma, flavour and freshlike firmness scores were reported for Np-EO treatment. Carvacrol

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Table 1 Soluble solids content (SSC), pH, titratable acidity (TA), L* and whitening index (WI) of fresh-cut carrot slices subjected to different sanitising treatments and stored for up to 13 days at 5 °C. Np, chitosan-tripolyphosphate nanoparticles; Np-EO, carvacrol-loaded chitosan-tripolyphosphate nanoparticles. Different capital letters denote significant differences (P ≤ 0.05) among treatments for the same sampling day. Different lowercase letters denote significant differences (P ≤ 0.05) among sampling days for the same treatment.

SSC

a

pH

TAb

L*

WI

a b

Processing day

Day 3

Day 6

Day 9

Day 13

Control NaOCl Chitosan Carvacrol Np Np-EO Control NaOCl Chitosan Carvacrol Np Np-EO Control NaOCl Chitosan Carvacrol Np Np-EO

7.0 ± 0.2Cb 7.0 ± 0.1Cb 6.9 ± 0.1D c 6.7 ± 0.1Ec 7.2 ± 0.2Bb 7.4 ± 0.1Aa

7.2 ± 0.1Cb 7.2 ± 0.2Cb 6.9 ± 0.1D b 7.5 ± 0.1Aa 7.5 ± 0.1AB a 7.4 ± 0.3Ba

7.6 ± 0.2Aa 7.5 ± 0.1AB a 7.1 ± 0.2Ca 7.1 ± 0.1Cb 7.1 ± 0.1Cb 7.4 ± 0.1Ba

7.1 ± 0.1Bb 7.2 ± 0.1Ab 6.1 ± 0.2Ed 6.6 ± 0.1D d 6.1 ± 0.1Ed 6.7 ± 0.1Cb

6.10 ± 0.01Bb 6.21 ± 0.20Aa 5.57 ± 0.15D d 5.52 ± 0.01Eb 5.88 ± 0.01Cb 5.57 ± 0.02D a 0.081 ± 0.015aD 0.074 ± 0.009D b 0.133 ± 0.022Ba 0.118 ± 0.018Aa 0.099 ± 0.012Ca 0.116 ± 0.014AB c

6.06 ± 0.01Bc 6.01 ± 0.16BC c 5.78 ± 0.18BCD c 5.50 ± 0.20CD b 6.64 ± 0.73Aa 5.33 ± 0.03D b 0.084 ± 0.018aB 0.092 ± 0.019Bb 0.124 ± 0.011Aab 0.133 ± 0.020Aa 0.106 ± 0.026AB a 0.147 ± 0.024Abc

6.04 ± 0.01Bd 6.01 ± 0.06Cc 6.01 ± 0.002Cb 5.27 ± 0.01Ec 6.05 ± 0.15Aab 5.30 ± 0.11D bc 0.090 ± 0.011aB 0.080 ± 0.016Bb 0.099 ± 0.021Bb 0.144 ± 0.019Aa 0.099 ± 0.011Ba 0.152 ± 0.017Aabc

6.10 ± 0.02Ab 5.98 ± 0.03Bd 6.00 ± 0.16Bb 5.30 ± 0.01Cc 6.11 ± 0.02Aab 5.35 ± 0.12Cb 0.088 ± 0.012aB 0.109 ± 0.008Ba 0.105 ± 0.017Bb 0.155 ± 0.026Aa 0.091 ± 0.013Ba 0.174 ± 0.031Aab

6.7 ± 0.1Cc 7.1 ± 0.2Ab 6.0 ± 0.1D e 6.7 ± 0.1Cc 6.1 ± 0.3Bc 6.6 ± 0.1bC 6.15 ± 0.01Ba 6.05 ± 0.03Cb 6.20 ± 0.05Ba 7.46 ± 0.04Aa 6.18 ± 0.02Bab 5.21 ± 0.03D c 0.090 ± 0.012aB 0.096 ± 0.016Bb 0.109 ± 0.019Bb 0.010 ± 0.002Cb 0.102 ± 0.014Ba 0.188 ± 0.027Aa

Control NaOCl Chitosan Carvacrol Np Np-EO Control NaOCl Chitosan Carvacrol Np Np-EO

48.3 ± 4.2bA 43.4 ± 4.9Ac 44.0 ± 3.6Ab 43.2 ± 3.6Ab 43.9 ± 4.6Ab 44.7 ± 4.1Aab 36.1 ± 3.4Ab 30.9 ± 2.6cC 32.6 ± 3.0BC c 32.4 ± 2.1Bb 31.6 ± 1.6BC c 30.8 ± 1.1Cc

49.4 ± 4.1bA 44.5 ± 3.0ABC bc 47.2 ± 1.9ABC ab 49.1 ± 2.1AB a 45.6 ± 2.8Cb 45.2 ± 1.7BC ab 38.9 ± 2.5Ab A 33.7 ± 2.1bc 38.8 ± 1.3Aa 38.6 ± 1.9Aa 36.5 ± 2.3Ab 38.3 ± 1.1Aa

60.5 ± 1.1aA 54.8 ± 5.4Bab 44.2 ± 2.4Cb 46.3 ± 4.0BC a 46.0 ± 3.1BCA ab 47.2 ± 1.9BC ab 48.2 ± 3.5Aa B 43.3 ± 4.5ab 35.1 ± 1.9Cbc 35.0 ± 2.3Cab 35.0 ± 1.6Cbc 32.5 ± 1.6Cbc

55.0 ± 2.5bA 55.8 ± 3.3Aa 49.1 ± 2.8Ba 49.6 ± 2.5Ba 51.8 ± 3.3Ba 49.6 ± 1.3Ba 46.8 ± 2.1Aa 48.5 ± 4.2aA 38.7 ± 1.9Ba 37.3 ± 1.8Ba 37.6 ± 1.3Bb 36.8 ± 2.3Bab

61.9 ± 3.7aA 55.6 ± 1.5Ba 47.7 ± 4.0D ab 47.6 ± 1.5CDA a 50.2 ± 4.9BC a 44.4 ± 0.1D b 47.8 ± 0.7Aa 44.6 ± 0.9aAB 39.1 ± 1.7CA ab 37.8 ± 1.3Ca 40.2 ± 3.2BC a 35.9 ± 2.0BC a

Expressed in °Brix. Expressed in % (mg malic acid 100 g−1).

showed the lowest aroma score related to the mentioned high offodours related to carvacrol EO. Control and NaOCl samples showed the highest whitening. However, carvacrol containing treatments (carvacrol and Np-EO) showed reduced whitening score, being equal to 1.5. 4. Discussion Sanitising dipping solutions containing nanoparticles of carvacrol can be an effective technique to avoid carvacrol-related off-flavour and off-odours keeping its excellent antimicrobial properties. Encapsulation of carvacrol into chitosan-TPP nanoparticles by ionic gelation

Fig. 3. Sensory scores of fresh-cut carrot slices subjected to different sanitising treatments after 13 days at 5 °C. Ch, chitosan solution at 0.5%; Np, chitosan-tripolyphosphate nanoparticles; Np-EO, carvacrol-loaded chitosan-tripolyphosphate nanoparticles.

nanoencapsulation by a two-step process (i.e., droplet formation and droplet solidification) has been successfully conducted by Keawchaoon and Yoksan (2011). The formation of carvacrol droplets in chitosan solution was achieved by an oil-in-water emulsion technique. Each droplet was solidified by ionic cross-linking of protonated amino groups (NH+ 3 ) along chitosan molecules surrounding the carvacrol droplet and polyphosphate groups (P3O5− 10 ) of TPP molecules. The carvacrol was successfully loaded into chitosan-TPP nanoparticles as confirmed by UV–vis spectrophotometry. Accordingly to Keawchaoon and Yoksan (2011), the carvacrol-loaded chitosan-TPP nanoparticles showed a maximum absorption peak at 275 nm after immersion in ethanol for 1 h. The achieved chitosan-TPP nanoparticle formulation is expected to enhance the solubility or dispersibility of the poorly watersoluble carvacrol in an aqueous system, and also improve its stability. Carvacrol release from chitosan-TPP nanoparticles takes place by several mechanisms including surface erosion, disintegration, diffusion and desorption (Hariharan et al., 2006). The in vitro release profile of carvacrol from chitosan-TPP nanoparticles can be described as a twostep biphasic process, i.e., an initial burst release followed by subsequent slower release (Hosseini et al., 2013). The initial burst release was attributed to the carvacrol molecules adsorbed on the surface of the nanoparticles and carvacrol entrapped near the surface, as the dissolution rate of the polymer near the surface is high, the amount of carvacrol released will be also high (Anitha et al., 2011). In order to obtain a gradual carvacrol release from the chitosan-TPP nanoparticles and consequently extend the shelf-life of FC carrots, the burst effect should be alleviated. Accordingly, the chitosan:carvacrol ratio of 1:1.25 was selected in this study since Hosseini et al. (2013) reported that the burst effect, which occurred within the first 3 h, was greatly alleviated from 82 to 12% at chitosan:carvacrol ratios of 1:0.1 and 1:0.8, respectively. When Keawchaoon and Yoksan (2011) studied the in vitro release of carvacrol from chitosan-TPP nanoparticles for 2 months, they reported that the

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low cumulative release of carvacrol of approximately 28% after 13 days at pH = 7 was increased to approximately 42% at pH = 3. Latter authors attributed the greater release of carvacrol in acidic medium to the swelling and partial dissolution of nanoparticles caused by ionic repulsion of protonated free amino groups on one chitosan chain with its neighbouring chains. Due to the short shelf-life of FC products, the carvacrol release from chitosan-TPP nanoparticles needs to be incremented. Such low pH of 3 may induce structural changes in plant cells and related acidic off-flavours. Accordingly, the carvacrol-loaded chitosan-TPP nanoparticles were re-suspended in slightly acidified (pH = 4) water solution to achieve an appropriate carvacrol release during the studied 13 days of storage. In addition to an improved carvacrol release in acidic medium, at low pH the hydrophobicity of EOs increases, enabling it to more easily dissolve in the lipids of the cell membrane of target bacteria (Juven, Kanner, Schved, & Weisslowicz, 1994). Samples treated with Np-EO showed the best sensory scores with best flavour, aroma, fresh-like firmness and absence of off-flavours and off-odours related to EOs. Contrary, carvacrol solution showed higher off-flavours and off-odours, and lower aroma scores. Carvacrol containing treatments greatly reduced whitening of FC carrots. Accordingly, this antioxidant compound may have induced partial inhibition of enzymes related to colour discolouration of FC carrots (Howard, Griffin, & Lee, 1994). Furthermore, the physicochemical quality of Np-EO-treated samples was well maintained during storage showing even a reduced whitening in these samples after 13 days of storage. In general, microbial levels of samples were below 7 log CFU g− 1 after 13 days being this threshold limit slightly exceeded (approximately 0.5 log units) by control and NP samples. The 7 log units can be considered as the microbiological threshold limit to define FC products shelf-life (Gilbert et al., 2000) being safe for consumption. The mesophilic reductions after Np-EO was almost double of that reported by Gutierrez, Bourke, Lonchamp, and Barry-Ryan (2009) in FC carrot discs treated with oregano EO crude solution (0.25 mL L−1 for 2 min) compared to distilled water treatment. Similarly, Singh, Singh, Bhunia, and Stroshine (2002) found a 1.3 log CFU g−1 Escherichia coli O157:H7 reduction in baby carrots after thyme EO treatment (1.0 mL L−1 for 5 min). Generally, the carvacrol-loaded chitosan-TPP nanoparticles allowed to reduce the growth of the hereby studied microbial groups in the FC carrot slices similarly to carvacrol solution during the first 9 days of storage. However, from day 9 to day 13 the microbial growth from Np-EO samples was reduced in a greater extend compare to carvacrol solution. The latter effect may be due to an enhanced carvacrol release from nanoparticles. Guo, Jin, Wang, Scullen, and Sommers (2014) reported similar unchanged Listeria innocua loads in ready-toeat meat for 28 days at 10 °C treated either with an edible film containing microemulsified allyl isothiocyanate or a film with the non-emulsified isothiocyanate. Additionally, Guo, Jin, Yadav, and Yang (2015) reported a reduction of L. innocua loads from day 28 to day 35 in the microemulsified film whereas it was not observed in the other film. These authors attributed this behaviour to the smaller emulsion sizes and micro-particles. Keawchaoon and Yoksan (2011) reported that Mueller Hinton broth mixture containing carvacrol-loaded chitosanTPP nanoparticles and Staphylococcus aureus, Bacillus cereus and E. coli showed a minimum inhibitory concentration of 0.257 mg mL−1 against those bacteria and minimal bactericidal concentrations (MBC) of 4.113, 2.056 and 8.225 mg mL−1, respectively. The action mode of EOs has not been completely elucidated although the antimicrobial activity of the EOs seems to be related to their composition, to the structural configuration of the constituents and to their functional groups, as well as to the possible synergistic interactions among the components. Accordingly, it is known that the antimicrobial activity of each EO is due to their phenolic compounds and it largely depends on their concentration as well as on their chemical structure (Dorman & Deans, 2000). In general, the mechanism of action of EOs against microorganisms involves the interaction of phenolic compounds with the proteins (porins) in the

cytoplasmic membrane that can precipitate and lead to leakage of ions and other cell content causing the cell breakdown (Nychas et al., 2000). As reviewed Burt (2004), Gram-negative bacteria appear more resistant to EOs. This might be due to the lower antibacterial susceptibility of hydrophobic compounds against Gram-negative microorganisms, owing to the restricted diffusion of these compounds through the lipopolysaccharide covering on the outer membrane. Accordingly, the great LAB (Gram-positive bacteria) reductions hereby observed in carvacrol containing treatments may be explained by latter hypothesis. Similarly, the previous MBC reported by Keawchaoon and Yoksan (2011) corroborates the greater growth inhibition by carvacrol-loaded chitosan-TPP nanoparticles against Gram-positive bacteria compared to Gram-negative microorganisms. Similarly to our results, fruit salads (kiwifruit and pineapple) packaged (50 g) with fructose syrup (70 mL) containing silver-montmorillonite nanoparticles (20 mg) highly extended the shelf-life of the product with stabilized microbial loads from day 9 to the end of shelf-life (21 days) (Costa, Conte, Buonocore, & Del Nobile, 2011). The EU safety regulation limits the silver ions amount in food matrices to 0.05 mg Ag kg−1 (Fernández et al., 2009). The much larger surface area of nanoparticles allows a greater contact with cell membranes, as well as greater capacity for absorption and migration (de Azeredo, 2013). Hence, toxicity aspects related to silver ions profiles cannot be extrapolated from those of their non-nanosized counterparts. Moreover, nanostructures can have more free movement through the body when compared to their higher scale counterparts (Brayner, 2008). However, EOs are considered recognized as safe (GRAS) products (Burt, 2004) so the carvacrol-loaded chitosan-TPP may have no healthrisks. 5. Conclusions The effect of sanitising solutions containing carvacrol-loaded chitosan-tripolyphosphate nanoparticles (Np-EO) to maintain the microbial, sensory and physicochemical quality of FC carrot slices during cold storage was studied. The carrot slices treated with Np-EO solution did not show carvacrol-related off-flavours compared to those samples treated with a carvacrol solution at the same concentration. Physicochemical quality of carrot slices was also well maintained during storage showing even a reduced whitening in these samples after 13 days of storage. The controlled carvacrol release from Np-EO during storage highly allowed to control microbial growth showing similar microbial levels to processing day after 13 days at 5 °C while chlorine-treated samples registered 2–3 log units higher levels for the same storage period. Accordingly, this study shows the potential application of sanitising solutions containing Np-EO in the FC industry to highly maintain the quality of FC products such as carrot slices. Further research should be conducted to study the use of different nanoencapsulated EOs. References Agnihotri, S. A., Mallikarjuna, N. N., & Aminabhavi, T. M. (2004). Recent advances on chitosan-based micro- and nanoparticles in drug delivery. Journal of Controlled Release, 100(1), 5–28. Anitha, A., Deepagan, V. G., Divya Rani, V. V., Menon, D., Nair, S. V., & Jayakumar, R. (2011). Preparation, characterization, in vitro drug release and biological studies of curcumin loaded dextran sulphate–chitosan nanoparticles. Carbohydrate Polymers, 84(3), 1158–1164. ASTM (1986). Physical requirements guidelines for sensory evaluation laboratories. Vol. 913. Philadelphia, USA: American Society for Testing Materials. Bagamboula, C. F., Uyttendaele, M., & Debevere, J. (2004). Inhibitory effect of thyme and basil essential oils, carvacrol, thymol, estragol, linalool and p-cymene towards Shigella sonnei and S. flexneri. Food Microbiology, 21(1), 33–42. Bouwmeester, H., Dekkers, S., Noordam, M. Y., Hagens, W. I., Bulder, A. S., de Heer, C., ... Sips, A. J. A. M. (2009). Review of health safety aspects of nanotechnologies in food production. Regulatory Toxicology and Pharmacology, 53(1), 52–62. Brayner, R. (2008). The toxicological impact of nanoparticles. Nano Today, 3(1–2), 48–55. Burt, S. (2004). Essential oils: Their antibacterial properties and potential applications in foods–A review. International Journal of Food Microbiology, 94(3), 223–253. Costa, C., Conte, A., Buonocore, G. G., & Del Nobile, M. A. (2011). Antimicrobial silvermontmorillonite nanoparticles to prolong the shelf life of ESSEN fruit salad. International Journal of Food Microbiology, 148(3), 164–167.

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