Effects of Preheating and Storage Temperatures on Aroma Profile and

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The effects of preheating and storage temperature on aroma profile and physical properties of citrus-oil emulsions Ying Yang, Chengying Zhao, Guifang Tian, Chang Lu, Shaojie Zhao, Yuming Bao, David Julian McClements, Hang Xiao, and Jinkai Zheng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03270 • Publication Date (Web): 18 Aug 2017 Downloaded from http://pubs.acs.org on August 20, 2017

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Journal of Agricultural and Food Chemistry

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The effects of preheating and storage temperature on aroma

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profile and physical properties of citrus-oil emulsions

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Ying Yang1,#, Chengying Zhao1,#, Guifang Tian1, Chang Lu1, Shaojie Zhao1,Yuming

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Bao1, David Julian McClements2, Hang Xiao2,*, Jinkai Zheng1,2,*

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1

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Sciences, Beijing 100193, China

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2

8

United States

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Correspondence to: Jinkai Zheng; Hang Xiao.

Institute of Food Science and Technology, Chinese Academy of Agricultural

Department of Food Science, University of Massachusetts, Amherst, MA 01003,

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E-mail: [email protected] (J. Zheng); [email protected] (H. Xiao).

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#

Both authors have contributed equally to this work.

1

ACS Paragon Plus Environment


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Abstract

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Citrus oils are used as good carrier oil for emulsion fabrication due to their special

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flavor and various health-promoting functions. In this study, the effects of preheating

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temperature (30, 40, 50, 60, and 70 oC) and storage temperature (4, 25 and 37 oC) on

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aroma profiles and physical properties of three citrus-oil (i.e. mandarin, sweet orange

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and bergamot oils) emulsions were systematically investigated for the first time. The

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results demonstrated the significant impact of temperature on aroma profile and

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physical properties. The abundance of D-limonene was found to be the main factor

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determining the aroma of the three citrus-oil emulsions at different preheating and

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storage temperatures, while β-linalool and linalyl acetate were important for the

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aroma of bergamot oil emulsion. Preheating temperature showed profound impact on

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the aroma of citrus-oil emulsions, and the aroma of different citrus oil emulsions

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showed different sensitivity to preheating temperature. Storage temperature was also

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able to alter the properties of citrus oil emulsions. The higher the storage temperature,

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the more alteration of aroma and more instability of the emulsions, which could be

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attributed to the alteration of the oil components and the properties of emulsions.

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Among all three emulsions, bergamot-oil emulsion was the most stable and exhibited

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the most potent ability to preserve the aroma against high temperature. Our results

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would facilitate the application of citrus-oil emulsions in functional foods and

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beverages.

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Key words: citrus-oils, emulsions, temperature, aroma, physical properties 2


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Introduction

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A variety of lipophilic food components, such as carotenoids, capsaicin, resveratrol

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and some flavor components (e.g., D-limonene), show potent nutritional and health

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functions. However, the widespread applications in nutraceutical and pharmaceutical

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industries are dramatically limited by the low water-solubility and poor

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bioavailability.1-3 Lipid-based emulsion delivery systems are widely used as effective

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tools for encapsulating, protecting, and delivering of lipophilic components.4-6 On the

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one hand, these systems may inhibit degradation and improve solubility of

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nutraceuticals during delivery to increase their bioavailability as well as bioactivity.

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On the other hand, the lipid-based emulsion systems could protect aromatic scents

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from volatilization to ensure the food flavor quality.7-8 Lipid-based delivery systems

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with different compositions and structures have different physical and functional

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attributes.9 Based on this, appropriate emulsion systems could be fabricated by

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altering the type of carrier oil to delivery multiple nutraceuticals with different

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characteristics. Triacylglycerol oils, such as corn oil, sunflower oil, rapeseed oil, palm

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oil, fish oil and MCT, are commonly used to explore the influence of lipids on

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formation, stability and performance of emulsions.1,10-11

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It has been reported that citrus oils can also be used to encapsulate

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nutraceuticals.1,12 Citrus oils are widely utilized in beverages, confectionaries, pastries,

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cosmetics and other industries as natural aromatic and flavored agents.13-14 The

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aromatic compositions significantly depend on citrus genus and extraction method.15

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In addition, other multiple functions endow citrus oils with more wide applications in 3



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commercial productions, such as antibacterial, antioxidant, anti-inflammatory,

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anti-carcinogenic

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antimicrobial function made it a natural preservative to extend the shelf-life of

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commercial products.19 Therefore, the applications of citrus oils in food and other

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industries has attracted increasing interest. At present, citrus oil is mainly used as

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carrier oil to build emulsions in food and beverage industry. In our previous studies,

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orange oil-based emulsions exhibited various advantages over emulsions with other

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types of carrier oils (e.g. corn oil and MCT). The advantages include higher physical

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stability, smaller droplet size and higher loading capacity.1,12 More recently, we

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successfully used citrus oils to encapsulate polymethoxyflavones (PMFs). The results

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also showed that different types of citrus oils had different dissolving and loading

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capacity for PMFs under different temperatures.20 Until now, some studies have

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explored the major factors affecting the properties and stability of emulsions

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containing citrus oils, such as preparation method, oil fold, emulsifier types, addition

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of cosolvents and so on.21-26 The formation of citrus-oil emulsions had dramatically

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effects on their droplets characteristics and interfacial properties associated with their

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applications in different commercial products.

and

anti-acetylcholinesterase

potential.16-18

Especially,

the

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It was well-known that temperature is one of the most important parameters in the

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processing, transportation and storage of commercial foods. Temperature could alter

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the characteristics of emulsion droplets (e.g., droplet size, concentration and so on)

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and their capacity against environmental stress.26-29 Notably, differing from other

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common carrier oils, the main compositions of citrus oils were volatile, which could 4


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be as high as 90%. Therefore, the effects of temperature would be particularly obvious

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for flavor oils.25 Nevertheless, there is a lack of study on the effects of temperature,

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especially during processing and storage temperature on various citrus-oil emulsions

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properties. Therefore, in order to make a better application of citrus oil-emulsions in

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food industry, it is indeed necessary to investigate the influence of processing and

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storage temperature on various citrus oil-emulsions.

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In this study, three kinds of citrus (mandarin, sweet orange and bergamot) oils were

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used as oil phase to fabricate emulsions to systematically explore the effects of

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different preheating (30, 40, 50, 60, 70 oC) and storage (4, 25, 37 oC) temperature on

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the alteration of aroma and physical properties for the first time, as well as the

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potential mechanisms. Combination of GC-MS and electronic nose was used to

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elucidate the odor quality and determine the corresponding alteration of the

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components. The results obtained from this study provided better understanding on

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the relationship between temperature and flavor-oil emulsions. And at the same time,

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it would also provide scientific guidance for the application of citrus oils in food

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commercial products, which will be also beneficial to citrus by-products utilization

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and exploitation.

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Materials and methods

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Materials. Three typical kinds of citrus (mandarin, sweet orange and bergamot) oils

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were extracted by cold-press method, and data of the components, density, viscosity

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and refraction index had been reported in our previous study.20 Food-grade non-ionic

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surfactant (Tween 80) was provided by the Guoxin Co. Ltd. (Shenzhen, China). 5



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HPLC-grade methylene chloride and cyclohexanone were purchased from Fisher

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Scientific (Beijing, China). Other analytical reagents were obtained from Sinopharm

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Chemical Reagent Co., Ltd. (Beijing, China). Deionized water was obtained from our

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laboratory (Beijing, China).

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Preparation of citrus oil-in-water emulsions. The fresh emulsions were formed by

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mixing 10% oil phase and 90% aqueous phase (w/w). The aqueous phase was

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prepared by adding 1% (w/v) Tween 80 into deionized water. Coarse citrus

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oil-in-water emulsions were fabricated by mixing 10% oil phase and 90% aqueous

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phase using high-speed homogenization (ULTRA-TURRAX T25 digital, IKA,

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Germany) at 9000 rpm for 3 minutes. Then the mixture was passed through a

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high-press homogenizer (APV-2000, SPX, Germany) at 600 bar for 3 times to

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produce fresh emulsions samples.

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Effects of temperature on properties of citrus oil emulsions. In order to examine

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the impact of temperature on physical properties and aroma of the citrus-oil emulsions,

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before mixed, the oil phase and aqueous phase were preheated at 30, 40, 50, 60 and 70

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o

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were stored for 15 days at 4, 25 and 37 oC, respectively, and during which the particle

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size, turbidity, viscosity and the aroma alteration of emulsion samples were measured

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every day.

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Environmental stresses effects investigation. The three kinds of fresh emulsions

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were prepared at 30 oC, 600 bar for 3 times. 0.1 M HCl or 0.1 M NaOH was to

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modulate pH to 2, 7 and 9, respectively. 0.5 M NaCl, CaCl2 and AlCl3 solutions were

C for 0.5 h, respectively. The freshly prepared emulsions (30 oC, 600 bar / 3 times)

6


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separately mixed with fresh emulsions to make the final concentration of each salt

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0.25 M. Samples were kept at 25 °C for 12 h. The droplet size, zeta-potential and

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physical stability were measured.

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Electronic-nose analysis. Electronic nose (Aisense Analysitcs, GmBH, Schwerin,

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Germany) was used to analyze aroma alteration during encapsulation and storage at

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different temperature. Samples (2 ml) were put in head-space vials. Two needles, one

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connected with air, and the other connected with the detector and sample, were then

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inserted. Each measurement was repeated for 3 times. The results were analyzed by

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principle component analysis (PCA). Before measurement, the machine was

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preheated for 30 min and adjusted with air for three times to ensure the accuracy and

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stability.

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GC/MS analysis. The compositions of citrus oils-emulsions were analyzed by gas

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chromatograph coupled with a mass spectrometer (GCMS-QP 2010 plus, Shimadzu,

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Japan). A Shimadzu GC was equipped with DB-WAX fused silica capillary column

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(30 m × 0.25 mm i.d., film thickness, 0.25 µm), and a flame ionization detector (FID)

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was used. The column oven temperature was programmed from 40 °C for 4 minutes,

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to 220 °C in 3 °C/minute and held for 5 minutes, then rising to 240 °C in

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20 °C/minute. The injector and detector temperature was maintained at 245 °C.

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Helium was used as carrier gas at a flow rate of 1.0 ml/min. The citrus oils-emulsion

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samples were diluted 100-fold with chloroform and added cyclohexanone (1:500)

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prior to detection, and then injected by splitting and the split ratio was 1:30. The MS

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was set in electron impact mode at the ionization energy of 70 eV and scanned from 7



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m/z 40 to 600 at 2 scan/s. The ionization source and interface temperatures were 245

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and 250 °C, respectively. Components were preliminarily identified by comparison of

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their retention time (RT) and mass spectra with those in NIST and Willey database.

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Before analysis, the emulsion samples were extracted by methylene chloride and then

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2 µl cyclohexanone was added as internal standard into 1 ml extraction to realize the

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quantitative analysis of the principal component in citrus oil.

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Appearance of citrus oil emulsions. An image of the sample was acquired using

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digital image processing software (Canon SX610 HS) and stored on a personal

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computer.

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Particle size measurement. The mean particle size of each emulsion samples was

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determined by a commercial dynamic light scattering and micro-electrophoresis

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device (Zetasizer Nano ZS, Malvern Instruments, Malvern, UK). To prevent the effect

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of the multiple scattering and ensure the accuracy, each sample was diluted 1000

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times with same deionized water at room temperature prior to measurement. The

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angle of scattered light was 173o and the detection temperature was maintained at 25

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o

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cumulate mean diameter (size, nm) for particle size and volume-size for particle

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distribution.

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Turbidity and viscosity measurements of citrus-oils emulsion systems. The

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influence of thermal treatment on the turbidity (absorbance at 600 nm) of each sample

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was determined with a UV–visible spectrophotometer (Evolution Array, Thermo

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Scientific). Each sample was diluted 30 times by solutions at room temperature prior

C. All measurements were performed in triplicate and results were reported as

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to measurement. The solution without citrus oil was set as blank. Every sample was

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measured for three times. The shear viscosity of each citrus oil emulsion sample was

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determined using a glass capillary viscometer according to McClements.24 In brief,

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the time taken for citrus oil emulsion (τEO) to flow through a U-shaped glass tube was

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compared to the time taken for deionized water (τW) (a fluid of known viscosity, η) to

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flow through the same device (25 oC). The viscosity of the citrus oil emulsion was

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calculated as following: ηEO = η ˣ (τEO /τW).

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Statistical analysis. All measurements were performed on freshly prepared samples

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in triplicate at least. The results are reported as means and standard deviations.

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Statistical analysis was performed by ANOVA using SPSS Statistics Software (IBM).

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A P-value of < 0.05 was considered statistically significant.

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Results and discussion

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The impact of preheating temperature on the aroma of citrus oil emulsions.

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Aroma is one important index to evaluate the quality of commercial products. In order

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to determine the impact of preheating temperature on aroma, electronic nose and

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GC-MS were collectively used here to analyze the aroma alterations among different

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preheating temperature. As shown in Fig. 1, the aroma of three citrus oil emulsions at

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30 oC was similar to the original oils, especially for mandarin oil emulsion and

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bergamot oil emulsion. The results demonstrated that emulsion systems could protect

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their original flavor feature from losing at a relative low temperature. When

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temperature was 40 oC or higher (50, 60 and 70 oC), the aroma was apparently

9



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differed from the original oils. Although there were differences between 40, 50, 60

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and 70 oC, differences in mandarin oil and bergamot oil emulsions were not

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significant compared to sweet orange oil emulsions, especially for bergamot oil

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emulsions. According to previous data, mandarin oil was mainly consisted of

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D-limonene (72.4%) and some other minor components, such as camphene,

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(-)-β-pinene, γ-terpinene, β-linalool and so on. In sweet orange oil, D-limonene

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(97.4%) was almost the exclusive component. As for bergamot oil, the three main

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components were D-limonene (25.3%), linalyl acetate (34.6%) and β-linalool (15.4%).

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It has been verified that D-limonene (lemon, orange), linalool (flower, lavender) and

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linalyl acetate (flower, fruity) made the most contribution to the aroma of the citrus

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oils, especially D-limonene. Thus, the characteristic flavor of citrus oil emulsions was

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tightly associated with the concentration of these components.15,30,31

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As shown in Fig. 2, the volatilization of the main components at 30 oC was less

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significant than other higher temperature. Taking mandarin oil emulsions as an

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example, only 3.8% of D-limonene lost at 30 oC, while at 40 oC, the content of

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D-limonene had a dramatic decrease by about 30% compared to the original oil. The

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higher the temperature, the more decrease of D-limonene. However, although the

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content of the main components (D-limonene) were significantly different between

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emulsions at different temperature (43.2%, 32.1%, 22.6% and 7.9% for 40, 50, 60 and

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70 oC, respectively), the aroma was similar to each other. This was due to that along

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with the volatilization of D-limonene, the relative amount of D-limonene decreased

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dramatically and made the relative amount of the original minor components 10


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increased consequently. These changes might cause the flavor of lemon of

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mandarin-oil reduced significantly. In bergamot oil emulsions, the components almost

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had no significant alteration at all temperature, which might due to the high boiling

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point of the components, especially for the main components linalyl acetate and

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β-linalool. Even for D-limonene, only about 5% decreased for each 10 oC increased

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from 40 to 70 oC, indicating the interactions between the components could also

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decrease the volatilization. However, compared to the result of electronic nose, the

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aroma at 40-70 oC were different from that of at 30 oC, which might be attributed to

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that there were some components during other minor constituents (about 24.7% in

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total in the original oil) with higher boiling points, which could significant influence

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the aroma. Along with the volatilization of D-limonene and other aromatic

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components, the relative amount of the two main components (β-linalool and linalyl

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acetate) increased, which endowed more fragrance of flower of bergamot oil

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emulsion.

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The results of electronic nose and GC-MS demonstrated the significant impact on

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the aroma and components of citrus oil emulsions, which depended on the

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components of original citrus oils. The sensitivity of the aroma to preheating

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temperature was diverse in different citrus oil emulsions. The aroma of mandarin oil

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and sweet orange oil emulsions under different preheating temperature was

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dominantly affected by the concentration of D-limonene. The fragrance of lemon of

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these two kinds of emulsions decreased, while fragrance of flower of bergamot oil

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emulsion increased along with preheating temperature increased. More importantly, 11



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the results also indicated the aroma of citrus oil emulsions could not only depend on

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the genus selection, but also be modulated by the preheating temperature.

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Effects of preheating temperature on the physical properties of different citrus

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oil emulsions. Alteration of the citrus oil components caused by temperature might

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affect the density, viscosity and refractivity of the oil phase which played a vital role

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in the physical properties of the emulsions, including particle size, distribution, visual

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appearance and viscosity. As it’s known, these physical properties have significant

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effects on the quality of commercial products.32 As shown in Fig. 3, in general, the

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particle size of all the three kinds of citrus oil emulsions enhanced along with the

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temperature increasing from 30 to 60 oC. While the particle size of all the emulsions

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were decreased at 70 oC, which was almost similar to 30 oC. In different conditions,

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the dominant factors influencing the physical properties of citrus oil emulsions might

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be different. When preheating temperature ranged from 30 to 60 oC, high temperature

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accelerated the Brown action of the particles which made them easier to close and

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contact with each other to form larger particle, namely coalescence or flocculation.33

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When temperature increased to 70 oC, volatilization of components in citrus oil

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resulted in significant escape from oil phases. Thus, the amount of the oil in the

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emulsions decreased, which made the Tween 80 enough to encapsulate the oil phase

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to form smaller emulsion particle size.24 As for particle size distribution, sweet orange

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oil emulsions and bergamot oil emulsions were still maintained unimodal at different

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temperature, while mandarin oil emulsions were multimodal except 30 oC. In theory,

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similar viscosities of the two phases and low interfacial tension facilitate to produce 12


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fine oil droplets with small and uniform droplets during homogenization.32 Although

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the viscosity of mandarin oil (0.947 mPs) was similar to that of aqueous phase (1.152

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mPs), high temperature might lead to much loss of D-limonene and cause the

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reduction of the viscosity of oil phase, which led to the enlargement of the ratio of

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aqueous and oil phases. Therefore, it was not easy for the two phases to combine to

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form small and uniform particle droplets in emulsions. Moreover, the droplet growth

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by Ostwald ripening was accelerated by higher temperature to produce multimodal

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peaks. Generally, temperature had a significant influence on the viscosity of citrus oil

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emulsion and the viscosities of all the three emulsions decreased along with

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temperature increasing (Fig. 3). The decreased viscosity was mainly related to the

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volatilization of components which caused the reduction of the dispersion phase.4

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However, preheating temperature almost had no effect on turbidity (Fig. S1 in

264

Supporting Information).

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The aroma alterations at different temperature during storage. Citrus oils were

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composed of various volatile components which can be significantly affected by

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temperature and have substantial effects on the physical properties of the citrus oil

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emulsions, especially for the stability. As mentioned above, electronic nose and

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GC-MS were used in this study to investigate the aroma and composition alterations

270

of the emulsions during storage at 4, 25 and 37 oC which represented the refrigerated

271

storage temperature, ambient temperature and human body temperature, respectively.

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The data was analyzed each day, but only representative data on 1, 3, 7 and 15 d were

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shown. Electronic nose data (Fig. 4) showed that temperature had significant effects 13



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on the rate of aroma alteration of the citrus oil emulsions. In general, the higher of the

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temperature, the faster of the aroma alteration. The alteration at 4 oC was relatively

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slow, in which only minor alteration occurred stored for 7 days and significant

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alteration for 15 days for mandarin oil and sweet orange oil emulsion. While there

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was no significant alteration for bergamot oil emulsions for 7 days and only minor

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change after 15 days. However, for high temperature, aroma alteration dramatically

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speeded up. Especially at 37 oC, aroma could change significantly after 1 day. Among

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different citrus oil-emulsions, aroma of bergamot oil emulsion had minimum

282

alteration under the same condition, and followed by mandarin oil emulsion and sweet

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orange oil emulsion. Only minor variety occurred in the aroma of bergamot oil

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emulsions when stored for 7 days, and significant variety was exhibited after 15 days

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at 25 oC. Even at 37 oC, no significant change exhibited before 3 days. However, for

286

mandarin oil and sweet orange oil emulsions, aroma changed significantly after 1 day,

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and the former were more significant than the later. The results showed that emulsions

288

could preserve the aroma of citrus oil emulsions under certain conditions and the

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lower the storage temperature, the better for flavor preservation. This might be

290

attributed to the physical properties of the emulsions. For example, according to

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previous reports, smaller droplets have larger interfacial area, and the travel path from

292

the droplet center to the interface is shorter, both of which can facilitate mass transfer

293

of flavor molecules.34 In particular, at 4 oC, the aroma of mandarin oil emulsion and

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sweet orange oil emulsion could be preserved for 7 days, while for bergamot oil

295

emulsion, it could be 15 days. For higher temperature, bergamot oil emulsion could be 14


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stored for 7 days and 1 day at 25 and 37 oC, respectively. In addition, it was

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recommended that mandarin oil emulsion and sweet orange oil emulsion should be

298

stored at low temperature. In order to further investigate the alteration of the

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components at different temperature, GC-MS analysis was carried out, which verified

300

the relationship between aroma alteration and volatile components. Taking bergamot

301

oil emulsions as examples, most of the components had no significant alteration

302

during storage at 4 oC (Fig. 5). However, after 15 days, D-limonene and linalyl

303

acetate decreased by 11.0% and 5.4%, respectively. At 25 oC, D-limonene decreased

304

significantly from 7 days. D-limonene was decreased from to 23.2% to 14.7% on 7 d

305

and then to 6.17% on 15 d. The concentration of linalyl acetate decreased from 32.2%

306

to 26.2% after 15 days. For 37 oC, the main components D-limonene and linalyl

307

acetate had relatively significant alteration from 3 days, which decreased by 6.8% and

308

5.6%, respectively. And the other main component, β-linalool, also decreased.

309

Compared with 4 oC, the speed for loss of these three compounds was significantly

310

accelerated at high temperature and the tendency was fast at first and then slow. This

311

was might because that with the storage time prolonged, the concentration difference

312

between emulsions and air decreased and reduced the escape of the components. In

313

contrast to the results of electronic nose, the alteration of the aroma was coincident

314

with the components, which further verified the effects of storage temperature on

315

aroma. The GC-MS analysis data for mandarin oil emulsions and sweet orange oil

316

emulsions were shown in Fig. S2.

317

The changes of the physical properties of citrus oil emulsions at different storage 15



318

temperature. The visual appearance, turbidity, particle size and viscosity were

319

analyzed every day during storage. As shown in Fig. 6, when stored at 4 oC, creaming

320

ring began to appear on 1, 3 and 15 d for mandarin oil, sweet orange oil and bergamot

321

oil emulsions, respectively. However, three kinds of citrus oil emulsions still

322

maintained as emulsion systems under 4 oC, even stored for 15 days. When stored at

323

25 oC and 37 oC after 1 day, the appearances of the emulsions were almost no

324

difference with that of at 4 oC. With the extension of the storage time, the top of

325

mandarin oil emulsion and sweet orange oil emulsion both formed creaming ring on 3

326

d which were more significant than that of at 4 oC. At 25 oC, delamination occurred at

327

the bottom of mandarin oil and sweet orange emulsions, and the former was

328

significantly more apparent. Thin oil-layer on the top of all three kinds of emulsions

329

could be observed on 7 d. On 15 d, the creaming ring and phase separation of

330

mandarin oil emulsion were more obvious, while sweet orange oil emulsion became

331

slight clarified and bergamot oil-emulsion formed narrowed creaming ring. As for 37

332

o

333

be observed from 3 d. The bottom of mandarin oil emulsion was nearly transparent

334

and creaming ring appeared on the top of bergamot oil emulsion on 7 d. On 15 d, the

335

mandarin oil emulsion and sweet orange oil emulsion became single-phase rather than

336

emulsion systems, while for the bergamot oil emulsion, although creaming ring

337

expanded, it still maintained in emulsion condition. Correspondingly, the turbidities of

338

the emulsions reduced along with storage time and the speed at 37 oC was the fastest.

339

As for mandarin oil emulsion, the turbidity was near to 0 after stored for 15 days, as

C, thin oil-layer appeared in all the three emulsions, and apparent delamination could

16


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well as for sweet orange oil emulsions for 7 days at 37 oC (Fig. 7A). Considering the

341

visual appearance, it was abnormal that the turbidity of mandarin oil emulsion on 7

342

days was relative high at 37 oC, while for sweet orange oil, it was almost 0. That

343

might due to that before tested, the samples were mixed and the creaming ring on the

344

top of mandarin oil emulsion would affect the turbidity. In contrast, bergamot oil

345

emulsion still maintained with certain turbidity. The alterations of the particle size and

346

viscosity of all the emulsions were also investigated, which could also provide clues

347

for analysis of the stability. As shown in Fig. 7B, the particle size of all the three kinds

348

of citrus oil emulsion increased after 1 day and dramatically then decreased along

349

with storage time, especially for mandarin oil emulsions and sweet orange oil

350

emulsions at 37 oC. The growth of particle size of emulsions after 1 day might

351

because the unencapsulated oil droplet adhered to emulsion droplet surface.35

352

Moreover, Ostwald ripening might also facilitate the increase of the particle size.36

353

After that, large particle droplet might float upward and some of the components in

354

the oils escaped, resulting in the decrease of diameter of the emulsion systems.

355

Viscosity reflects the emulsions rheological behavior and the alteration can indicate

356

the stability of the emulsions in some extent.37,38 As showed in Fig.7 C, minor change

357

occurred for bergamot oil-emulsions during storage at different temperature. However,

358

for mandarin oil emulsions and sweet orange oil emulsions, the viscosity were in

359

downward trend at 4 oC and dramatically decreased after stored at 25 and 37 oC for 7

360

days, which was universal with the alteration of the appearance, turbidity and particle

361

size, reflecting the stability of the emulsions. Considering the alteration of all the 17



362

physical properties, the most stable emulsion was bergamot oil emulsion, which could

363

be stored for 15 days, even at 37 oC. However, for the other two emulsions, low

364

temperature was recommended, even at 4 oC, it should be stored no more than 7 days.

365

As discussed above, the aroma alteration should also be considered during storage.

366

All the results showed that on the one hand, the higher the storage temperature, the

367

more unstable of the citrus oil emulsions were. It was well known that the instability

368

of food emulsions was related to various physical mechanisms. Creaming,

369

flocculation coalescence and Ostwald ripening were the most common.33 The

370

formation of creaming ring could be attributed to the particle droplet increase and

371

floating upward. Higher storage temperature provided more energy for the Brown

372

motion of emulsion particles which assisted them stick together to form larger particle

373

and accelerated the volatile components to escape from aqueous phase to cause

374

instability. Importantly, higher temperature facilitated the volatilization of the

375

components which also dramatically influence the stability of the emulsions. On the

376

other hand, the results also demonstrated that bergamot oil emulsions were the most

377

stable ones, and followed by sweet orange oil emulsions and mandarin oil emulsions.

378

The difference of the stability between different citrus oil emulsions could be

379

attributed to the different physical properties of the citrus oils as shown in our

380

previous study.20 The larger of the ratio between two phases, the more easily of the

381

creaming ring to form due to the gravity effect. The density of mandarin oil (0.8470

382

g/cm3) had the biggest difference with aqueous (1.132 g/cm3), and therefore mandarin

383

oil emulsion was particularly unstable. In addition, the high water-soluble components 18


Page 18 of 34

Page 19 of 34


384

in bergamot oil, such as β-linalool (15.41%) and linalyl acetate (34.64%), benefited to

385

forming low size particle size (d≈120 nm), which could improve the stability by

386

inhibiting gravitational separation and droplet aggregation. The different components

387

in different type and amount made mandarin oil, sweet orange oil and bergamot oil

388

with different molecular weight which caused their critical emulsifier demands. Hence,

389

the micelle formed by 1% Tween 80 might be nearly enough for bergamot oil droplets

390

so that could maintain relative stability during storage at different temperature.26 The

391

oil phase that was not encapsulated floated upward and formed the oil-layer on the

392

surface of emulsions due to the lower density. In addition, the demulsification during

393

storage might also cause the oil-layer.

394

As it is known, other environmental stresses, such as pH and salt ion, are also

395

important for the physical properties of emulsions. Hence, the effect of different pH (2,

396

7 and 9) and salt ion (NaCl, CaCl2, and AlCl3) on the citrus oil emulsions were also

397

investigated. However, as shown in Fig. S3, there was almost no effect on the

398

emulsions. That might due to the non-ionic emulsifier used in this study and as a

399

result, there was almost no electric charge in the surface of the emulsion particles.

400

From this point of view, the results further demonstrated the importance of

401

temperature for citrus oil emulsions.

402

In summary, different citrus oil emulsions were successfully fabricated with

403

mandarin oil, sweet orange oil and bergamot oil. The impact of temperature

404

(preheating and storage) on their physical properties and aroma profile, as well as the

405

potential mechanisms were systematically studied in this paper for the first time. 19



406

Alteration of aroma was comprehensively analyzed by combination of electronic nose

407

and GC-MS. As for the effects of preheating temperature, 30 oC was optimal, at which

408

the citrus oil emulsions were all with relative small and uniform droplets. More

409

importantly, preheating temperature had significant effects on aroma, and it was the

410

first time to show the potential for the modulation of aroma of citrus oil emulsions. As

411

for storage, the temperature had significant influence on the aroma, appearance,

412

stability, particle size, turbidity and viscosity. Low temperature was recommended.

413

The higher the temperature, the more dramatic alteration of aroma and unstable of the

414

emulsions. This could be attributed to the properties of the emulsion itself and the

415

alteration of the components. In terms of different citrus oil emulsions, bergamot oil

416

emulsion was the most stable and most capable for preservation of the aroma,

417

followed by sweet oil and mandarin oil emulsions. And the shelf life for each

418

emulsion was also investigated. The results of this study provided scientific guidance

419

for rational design and storage of citrus oil based emulsion delivery systems in food

420

industry, which could also be used for other emulsion with volatile components.

421

Conflicts of interest

422

423 424

The authors declare no competing financial interests.

Acknowledgements The authors would like to acknowledge the financial support of the National

425

Natural Science Foundation of China (No. 31401581 and 31428017).

426

Supporting Information 20


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427

The effects of preheating temperature on turbidity, GC-MS analysis of alteration of

428

components at different storage temperature, and the effects of environmental stresses

429

(pH and salt ion) on the particle distributions of the citrus-oil emulsions were shown

430

in the Supporting Information file.

431

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548

Figure Captions

549

Fig. 1 The electronic nose profiles of mandarin-oil (A), sweet orange-oil (B), and

550

bergamot-oil emulsions (C) at the processing temperatures of 30, 40, 50, 60 and

551

70 oC for 0.5 h.

552

Fig. 2 The results of GC-MS analysis for alteration of main volatile components in

553

mandarin-oil (A), sweet orange-oil (B), and bergamot-oil emulsions (C) at

554

preheating temperatures 30, 40, 50, 60 and 70 oC for 0.5 h (*, 0.01 < p < 0.05;

555

**, p < 0.01).

556

Fig. 3 The particle size distributions (A) and viscosities (B) of three citrus-oil

557

emulsions at different preheating temperature (30, 40, 50, 60 and 70 oC).

558

Fig. 4 Electronic nose profiles of three citrus-oil emulsions at different storage

559

temperatures 4, 25 and 37 oC for 1, 3, 7 and 15 d.

560

Fig. 5 The GC-MS analysis of alteration of main volatile components in bergamot-oil

561

emulsions at different storage temperatures 4 oC (A), 25 oC (B) and 37 oC (C)

562

for 1, 3, 7 and 15 d (*, 0.01 < p < 0.05; **, p < 0.01).

563

Fig. 6 Appearances of mandarin-oil, sweet orange-oil, and bergamot-oil emulsions at

564

different storage temperature 4, 25 and 37 oC for 1, 3, 7 and 15 d (ME,

565

mandarin oil emulsion; SE, sweet orange oil emulsion; BE, bergamot oil

566

emulsion. Red line, creaming ring; red circle, oil-layer).

567

Fig. 7 The turbidity (A), main particle size (B) and viscosities (C) of mandarin-oil,

568

sweet orange-oil, and bergamot-oil emulsions at different storage temperature 4,

569

25 and 37 oC for 1, 3, 7 and 15 d. 26


Page 26 of 34

Page 27 of 34


Fig. 1 The electronic nose profiles of mandarin-oil (A), sweet orange-oil (B), and bergamot-oil emulsions (C) at the processing temperatures of 30, 40, 50, 60 and 70 oC for 0.5 h.

27



Fig. 2 The results of GC-MS analysis for alteration of main volatile components in mandarin-oil (A), sweet orange-oil (B) and bergamot-oil emulsions (C) at preheating temperatures 30, 40, 50, 60 and 70 oC for 0.5 h (*, 0.01 < p < 0.05; **, p < 0.01).

28


Page 28 of 34

Page 29 of 34


Fig. 3 The particle size distributions (A) and viscosities (B) of three citrus-oil emulsions at different preheating temperature (30, 40, 50, 60 and 70 oC) for 0.5 h.

29



Fig. 4 Electronic nose profiles of three citrus-oil emulsions at different storage temperatures 4, 25 and 37 oC for 1, 3, 7 and 15 d.

30


Page 30 of 34

Page 31 of 34


Fig. 5 The GC-MS analysis of alteration of main volatile components of bergamot-oil emulsions at different storage temperatures 4 oC (A), 25 oC (B) and 37 oC (C) for 1, 3, 7 and 15 d (*, 0.01 < p < 0.05; **, p < 0.01).

31



Fig. 6 Appearances of mandarin-oil, sweet orange-oil, and bergamot-oil emulsions at different storage temperature 4, 25 and 37 oC for 1, 3, 7 and 15 d (ME, mandarin oil emulsion; SE, sweet orange oil emulsion; BE, bergamot oil emulsion. Red line, creaming ring; red circle, oil-layer).

32


Page 32 of 34

Page 33 of 34


Fig. 7 The turbidity (A), main particle size (B) and viscosities (C) of mandarin-oil, sweet orange-oil, and bergamot-oil emulsions at different storage temperature 4, 25 and 37 oC for 1, 3, 7 and 15 d.

33



Graphical for table of contents

34


Page 34 of 34

Effects of Preheating and Storage Temperatures on Aroma Profile and

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