Food Chemistry 170 (2015) 394–400

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Influence of technical processing units on chemical composition and antimicrobial activity of carrot (Daucus carrot L.) juice essential oil Tingting Ma a,1, Jiyang Luo b,1, Chengrui Tian a,⇑, Xiangyu Sun c, Meiping Quan a, Cuiping Zheng a, Lina Kang a, Jicheng Zhan c,⇑ a b c

College of Food Engineering and Nutritional Science, Shaanxi Normal University, Xi’an 710062, China Chinese Academy of Inspection and Quarantine, Beijing 100123, China College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China

a r t i c l e

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Article history: Received 13 June 2014 Received in revised form 3 August 2014 Accepted 5 August 2014 Available online 21 August 2014 Keywords: Essential oil Technical processing units Chemical composition Antimicrobial activity Carrot juice

a b s t r a c t The effect of three processing units (blanching, enzyme liquefaction, pasteurisation) on chemical composition and antimicrobial activity of carrot juice essential oil was investigated in this paper. A total of 36 compounds were identified by GC–MS from fresh carrot juice essential oil. The main constituents were carotol (20.20%), sabinene (12.80%), b-caryophyllene (8.04%) and a-pinene (6.05%). Compared with the oil of fresh juice, blanching and pasteurisation could significantly decrease the components of the juice essential oil, whereas enzyme liquefaction had no considerable effect on the composition of juice essential oil. With regard to the antimicrobial activity, carrot juice essential oil could cause physical damage and morphological alteration on microorganisms, while the three different processing units showed noticeable differences on the species of microorganisms, the minimum inhibitory concentration and minimum bactericidal concentration. Results revealed that the carrot juice essential oil has great potential for application as a natural antimicrobial applied in pharmaceutical and food industries. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Plants essential oils have great potential use as food flavours and preservatives. Thus, the antimicrobial effect of essential oils and their aromatic component analysis have longly been recognised (Boukhatem, Abdelkrim, & Fairouz, 2013). Recently, because of consumers’ growing concerns over the food safety issues, the substitution of synthetic antimicrobial chemicals by natural antimicrobial agents has attracted increasing interest. Many naturally occurring compounds in plants have shown to possess antimicrobial effect against food-borne pathogens (Boukhatem et al., 2013; Burt, 2004; Dorman & Deans, 2000). Meanwhile, synthetic antimicrobial chemicals are sometimes associated with adverse effects including hypersensitivity, toxicity, carcinogenicity and immunity suppression (Cakir, Kordali, Kilic, & Kaya, 2005; Wu & Zheng, 2007). As a result, use of essential oils as antimicrobial agents in food systems may be considered as additional intrinsic determinant to increase the safety and shelf life of foods (Sagdic, Karahan, Ozcan, & Ozkan, 2003; Salgueiro, Martins, & Correia, 2010). ⇑ Corresponding authors. Tel.: +86 15934891959; fax: +86 029 85310517 (C. Tian). Tel.: +86 010 62737535; fax: +86 010 6273755 (J. Zhan). E-mail addresses: [email protected] (C. Tian), [email protected] (J. Zhan). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.foodchem.2014.08.018 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.

Carrot (Daucus carrot L.) has been a favourite vegetable for a long time due to its nutritive value and culinary uses (Ma et al., 2013). Currently, carrot essential oil has been widely used in many food categories, and as a fragrance component in perfumes, cosmetics, and soaps (Lawless, 2002). It is a source of sesquiterpenic alcohols, carotol, daucol, and the sesquiterpene b-caryophyllene (Glisic et al., 2007). There have been several reports claimed properties of the essential oil include antibacterial (Giraud-Robert, 2005; Glisic et al., 2007; Staniszewska, Kula, Wieczorkiewicz, & Kusewicz, 2005), fungicidal (Batt, Solberg, & Ceponis, 1983; Dwivedi, Dwivedi, Pandey, & Dubey, 1991; Giraud-Robert, 2005), hepatocellular regenerator, general tonic and stimulant, lowering of high cholesterol and cicatrisant (Giraud-Robert, 2005). Traditional uses are for hepatic and renal insufficiency and skin disorders, e.g. burns and furuncles (Bergonzelli, Donnicola, Porta, & Corthesy-Theulaz, 2003; Giraud-Robert, 2005). Chemical composition as well as antimicrobial and fungicidal activities of carrot essential oil has been the subject of frequent researches. However, to the best of our knowledge, the researches of carrot essential oil are mostly limited to the carrot itself; there is no information available in the literatures on carrot processed products, particularly the carrot juice as its main processed products. As one of the main forms of people consumption of carrot, it would have important guiding significance to study the changes of chemical composition

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and antimicrobial effect of carrot juice essential oil during processing, especially the effect of the main processing units (blanching, enzyme liquefaction, and pasteurisation) on chemical composition and antimicrobial activity of carrot juice essential oil. Therefore, the aim of the present investigation was undertaken to identify the chemical composition of the essential oil obtained by hydrodistillation from carrot juice by gas chromatography-mass spectrometry (GC–MS), and to focus on characterising the changes in the essential oil of carrot juice during processing, including blanching, enzyme liquefaction and pasteurisation. In addition, its antibacterial effect on food spoilage bacteria and possible mechanism of action responsible for the antibacterial activity were discussed. Thus, it is expected to develop methods and provide some parameters which could facilitate the processing technology of carrot juice processing. 2. Materials and methods 2.1. Plant materials The carrots defined as No. 1 Orange-red variety grew in Gaoling (Shaanxi, China) Planting base in autumn season. After harvesting, the fresh carrots were transported to the laboratory by truck at about 15 °C, and stored in woven polypropylene bags in batches of approximately 10 kg each. Storage temperature was 1 °C and the relative humidity was 90–95%. The storage time was less than 15 days before processing. 2.2. Preparation of carrot juices with different treatments The raw material carrots were randomly separated into 4 batches. Washing process was conducted on a pilot plant scale with commonly used as industrial equipment. Then selected good ones, peeled and treated as follows (all treatments were selected for the best in preliminary experiments): (1) Fresh carrot juice: The carrots were sliced into 3 mm thick and pressed into juice by HR2864 juice extractor (Philips Electric Appliance Co., Holland) without any treatment used as control. (2) Blanching treatment: The carrots were placed in water bath (Model HH-S; Jiangsu Zhenjiang Instrument Co. Ltd, Jiangsu, China). Water blanching was carried out at 86 °C for 10 min. Then the carrots were sliced into pieces and pressed into juice as above. (3) Enzyme liquefaction treatment: The carrots were sliced into pieces and treated by 1.5% (w/w) pectase (12,475 U mL 1) and 1.5% (w/w) cellulase (18,200 U g 1). The optimal conditions of pectase and cellulase treatments were 45 °C, pH4.5, 120 min, and 50 °C, pH5.0, 60 min, respectively. After that, the pieces of carrots were pressed into juice. (4) Pasteurisation treatment: The carrots were sliced into pieces and pressed into juice, and then the juice was sterilized in water bath at 100 °C for 30 s. Each treatment was replicated thrice and all the carrot juices with different treatments were stored at 4 °C and the storage time was less than 15 days before analysis. 2.3. Extraction of the essential oil of carrot juice with different treatments The extraction of essential oil was carried out according to the method with slight modifications (Diao, Hu, Zhang, & Xu, 2014; Gao et al., 2011). The carrot juices with different treatments were

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subjected to hydro distill for 6 h in a Clevenger-type apparatus. The oily layer obtained on top of the aqueous distillate was separated and dried with anhydrous sodium sulphate, then stored in a tightly closed dark vial at 4 °C until chemical composition analysis and antimicrobial activity studies. 2.4. GC/MS analysis The carrot juice essential oil with different treatments were analysed by GC–MS, a gas chromatograph, Agilent Technologies 5973A using a HP 6890A Mass spectrometer with electron impact ionisation (70 eV). A SE-54 capillary column (30 m  0.25 mm; film thickness, 0.25 lm) was used. Oven temperature was programmed to rise from 40 to 280 °C at a flow rate of 4 °C/min, sample injection volume, 0.5 lL. The carrier gas was He with a flow rate of 1 ml/min and a split ratio of 30:1, scan speed and mass range were 0.5 s/dec and 45–450 m/z, respectively (Zaouali, Hnia, Rim, & Mohamed, 2013). The components were identified by comparing their mass spectra with the data from the library of essential oil constituents, U.S. National Bureau of Standards library, and Adams GC/MS libraries. Use INCOS data system for retrieval and mass spectrum analysis. Only the similarity of the components is greater than 80% would be recorded. 2.5. Microbial strains The antimicrobial activity of the carrot juice essential oil with different treatments was tested against 8 selected food-related microorganisms. Two gram-positive strains were Listeria monocytogenes ATCC 19115 and Staphylococcus aureus ATCC 6538 P. Four gram-negative bacteria were Salmonella typhimurium ATCC 19430, Salmonella enteritidis ATCC 13076, Shigella dysenteriae CMCC (B) 51252 and Escherichia coli ATCC 25922. Aspergillus niger ATCC 02512 and Yeast were the two fungal microorganisms studied. All the microorganisms were provided by the Microbiological Laboratory, College of Life Science, Shaanxi Normal University, China. 2.6. Determination of antimicrobial activity of the carrot juice essential oil with different treatments The antimicrobial activity of the essential oil with different treatments was evaluated using the standardised filter paper disk diffusion method as described previously (Gao et al., 2011). Briefly, 100 ml of a suspension containing approximately 107 colonyforming units (CFU)/ml of bacteria and 104 spore/ml of fungus were spread on nutrient agar (NA) and potato dextrose agar (PDA), respectively. Filter paper disks (6 mm in diameter) were impregnated with 10 lL (4 mg/ml) of dilutions in dimethyl sulfoxide (DMSO, Sigma) of the essential oil and placed onto the solid medium (20 ml) plates. The diameter of inhibition zones (DIZ) was measured after 24 h of incubation at 37 °C for bacteria, after 4–5 d of incubation at 28 °C for A. niger and after 24 h of incubation at 25 °C for Yeast. Ten microlitres of gentamicin (4 mg/ml) and DMSO were used as positive and negative controls respectively. Tests were performed in triplicate. 2.7. Determination of minimum inhibitory concentration (MIC) and minimum bactericide concentration (MBC) with different treatments MIC values and MBC values were determined for the microorganisms that were sensitive to the essential oil in the filter paper disk diffusion assay. The MIC values were determined by tube dilution method as described by Gao et al. (2011). Stock solution of essential oil with different treatments was prepared in DMSO. Two fold serial dilutions of essential oil were prepared in sterile

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Luria–Bertani (LB) medium ranging from 20 to 1280 mg/ml. To each tube, 100 ml of the inoculum containing approximately 107 CFU/ml microorganisms was added. A control test was also performed containing inoculated broth supplemented with only DMSO under identical conditions with gentamicin as reference. The tubes were then incubated at 37 °C for 24 h and examined for evidence of the growth. The first tube without turbidity was determined as the MIC value. All assays were performed in triplicate. The MBC was regarded as the lowest concentration of the samples that allowed less than 0.1% of the original inoculum treated with the extract or compound samples to survive and grow on the surface of the medium used. The MBC values were determined according to the method of Shan, Cai, John, and Cork (2008). In brief, the samples (50 ml) for the MBC assays were taken from the tubes of the MIC assays in which any visible turbidity was not observed. Then, they were spread on freshly prepared nutrient agar plates and were incubated at 37 °C for 24 h so as to determine the MBC values. After the incubation, the plates were observed for growth. Triplicate samples were performed for each test concentration. 2.8. Scanning electron microscope (SEM) observations To determine the efficacy of the essential oil and the morphological changes of bacteria, SEM observation was performed on the tested bacteria. The method of SEM was modified from Shan et al. (2008). The bacteria cells were incubated in nutrient broth at 37 °C for 10 h. The suspension was divided into two portions. One portion was added suitable concentrations (MIC) of the essential oil with enzyme liquefaction treatment, the other portion was left untreated as a control. The resuspension was incubated at 37 °C for 4 h, and then the cells from both tubes were harvested by centrifugation and were fixed with a 2.5% glutaraldehyde solution overnight at 4 °C. After this, a drop of each resuspension was filtered by 30%, 50%, 70%, 90%, and 100% ethanol, respectively. Then, cells were dried at ‘‘critical point’’ in liquid CO2 under 95 bar pressure. The samples were gold-covered by cathodic spraying. Finally, morphology of the bacterial cells was observed on a scanning electron microscope (SEM) (Quanta-200, Philips-FEI Co., Amsterdam, Netherlands). 2.9. Statistical analysis Experimental results were means ± SD of three parallel measurements. Statistical analyses were performed by Data Processing System (DPS, version 7.05) and Excel program. 3. Results and discussion 3.1. Influence of technical processing units on essential oil composition of carrot juice The chemical composition of essential oil of carrot juice with different treatments was analyzed by GC–MS, and the results were presented in Table 1. In total, 36 components were identified in fresh carrot juice. In previous literature, Glisic et al. (2007) reported that 46 components were identified from the essential oil of carrot fruit obtained by hydrodistillation. Thus it can be seen that the composition of carrot essential oil is differ from the carrot juice essential oil. This variation could be mainly caused by the variety diversity of the carrots and the technical processes. In addition, the pomace will be separated from carrot juice after juicing process, which inevitably resulted in the loss of essential oil. As can be seen from the Table 1, carotol (20.20%), sabinene (12.80%), b-caryophyllene (8.04%) and a-pinene (6.05%) were found to be the major compounds in fresh carrot juice, with minor

Table 1 Chemical composition (%) of essential oil from carrot juice with different treatments. No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37

Compound

EOF (%)

EOB (%)

EOE (%)

EOP (%)

a-Pinene

6.05 0.69 12.8 0.82 0.45 1.26

1.21 – 9.02 0.34 – 0.54

4.25 0.50 13.4 0.61 – 0.88

0.84 – 7.24 0.18 – 0.41

0.48 1.26 0.37 0.31 1.01 1.08 1.02 0.67 0.49 1.35 1.21 0.35 0.79 0.61 0.87 0.81 0.41 0.67 0.64 0.74

0.13 0.24

0.27 1.03 – 0.68 0.71 0.78 0.86 0.42 0.35 1.12 1.32 0.46 1.41 1.12 0.76 0.64 0.54 0.36 0.83 0.89 – 1.46 4.24 5.12 1.58 4.38 1.15 1.21 25.2 3.25 1.12 82.9

– – –

Camphene Sabinene b-Myrcene Carene 1-Isopropyl-2methylbenzene c-Terpinene Limonene Linalool Carene alcohol Pinocarveol Verbenol Sabinaketone Pinocarvone 4-Terpilenol Myrtenol Verbenone trans-Carveol Geraniol Bomyl acetate Humulene Borneol Bornyl acetate Terpinolene o-Xylene Curcumene Benzaldehyde Cumic alcohol Geraniolene b-Caryophyllene a-Caryophyllene b-Selinene a-Selinene b-Bisabolene Carotol Caryophyllene oxide Daucol Total identified (%)

– 0.86 4.04 8.04 0.53 4.22 1.08 1.11 20.20 2.18 1.05 80.52

– 0.14 0.41 0.32 – – 0.13 0.42 – 0.24 2.58 – 0.51 0.44 1.48 0.32 0.53 0.43 0.35 0.47 5.96 8.42 – 8.90 – 5.08 20.5 4.80 1.31 75.22

0.35 – – 0.46 0.43 – 0.86 0.51 – 0.57 – 0.34 0.54 0.87 – 0.56 0.54 0.56 0.36 4.48 10.02 – 4.24 0.87 4.12 24.4 6.85 2.54 73.14

The compounds were identified by comparison of retention indices and the mass spectrum with those of NIST library. –, Not detected.

amounts of b-selinene (4.22%) and myrcene (4.04%), and others were found in less quantity (1280 320

320 160

Influence of technical processing units on chemical composition and antimicrobial activity of carrot (Daucus carrot L.) juice essential oil.

The effect of three processing units (blanching, enzyme liquefaction, pasteurisation) on chemical composition and antimicrobial activity of carrot jui...
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