Physicochemical characterization of black seed oil-milk emulsions through ultrasonication

Physicochemical characterization of black seed oil-milk emulsions through ultrasonication

Accepted Manuscript Physicochemical Characterization of Black Seed Oil-Milk emulsions through Ultrasonication S. Anandan, M. Keerthiga, S. Vijaya, A.M...

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Accepted Manuscript Physicochemical Characterization of Black Seed Oil-Milk emulsions through Ultrasonication S. Anandan, M. Keerthiga, S. Vijaya, A.M. Asiri, V. Bogush, O. Krasulyaa PII: DOI: Reference:

S1350-4177(16)30374-1 http://dx.doi.org/10.1016/j.ultsonch.2016.11.005 ULTSON 3419

To appear in:

Ultrasonics Sonochemistry

Received Date: Revised Date: Accepted Date:

8 August 2016 26 October 2016 3 November 2016

Please cite this article as: S. Anandan, M. Keerthiga, S. Vijaya, A.M. Asiri, V. Bogush, O. Krasulyaa, Physicochemical Characterization of Black Seed Oil-Milk emulsions through Ultrasonication, Ultrasonics Sonochemistry (2016), doi: http://dx.doi.org/10.1016/j.ultsonch.2016.11.005

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Physicochemical Characterization of Black Seed OilMilk emulsions through Ultrasonication S. Anandan†,*, M. Keerthiga†, S. Vijaya†, A.M. Asiri€, V. Bogush#, O. Krasulyaa#,*, †

Nanomaterials and Solar Energy Conversion Lab, Department of Chemistry, National Institute of Technology, Trichy 620 015, India. #

Moscow State University of Technology and Management, Moscow, Russia



The Center of Excellence for Advanced Materials Research, King Abdulaziz University, Jeddah 21413, P.O. Box 80203, Saudi Arabia

AUTHOR EMAIL ADDRESS ([email protected]) *

To whom correspondence should be addressed: [email protected] Tel.: +91-431-2503639. Fax: +91431-2500133.

ABSTRACT The ultrasonic formation of stable emulsions of a bioactive material, black seed oil, in skim milk was investigated. The incorporation of 7% of black seed oil in pasteurized homogenized skim milk (PHSM) using 20 kHz high intensity ultrasound was successfully achieved. The effect of sonication time and acoustic power on the emulsion stability was studied. A minimum process time of 8 min at an applied acoustic power of 100 W was sufficient to produce emulsion droplets stable for at least 8 days upon storage at 4 ± 2̊ C, which was confirmed through creaming stability, particle size, rheology and color analysis. Partially denatured whey proteins may provide stability to the emulsion droplets and in addition to the cavitation effects of ultrasound are responsible for the production of smaller sized emulsion droplets.

KEYWORDS black seed oil; milk emulsion; ultrasound; creaming stability; rheological properties

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INTRODUCTION In recent years, increasing awareness of the relationship between nanoscopic, microscopic and macroscopic features of food and its physiological performance has made a significant shift in the scientific approach among researchers working with food materials [1-4]. The creation of rationally designed structural features within foods may provide specific functional performances. Further, the addition of nutraceutical and functional food components (such as vitamins, bioactive peptides, antimicrobials, antioxidants, flavors, colors, minerals and preservatives) leads to differences in physicochemical properties (solubility, partitioning, physical state, interactions, optical characteristics and stability) and hence different delivery systems are usually needed to address such issues [4-7]. Traditionally homogenizaton is employed in food industry even though such process is highly energy intense [8] and later microfluidizer has gained interest which caused particle size reduction due to particle-particle collision [9,10]. Ultimately, a new ultrasonic technique has been developed to avoid damage to the food constituents during processing [11,12]. Many researchers have used ultrasound (US) with additional external pressure and heat treatment (thermosonication & manothermosonication) to achieve benefits like microbial deactivation or fat globule homogenization, which are the basic prerequisite for preservation and processing [13-16]. The delivery of bioactives in a complex/real food matrix (health beverage) using US remains as a vast area to be explored. Tornberg and Hermansson [17] produced soybean oil emulsions in water using sonifier cell disruptor model B-12 at a residence time of 1 min. Later through ultrasound batch processes at 20 kHz and flow-through process at 24 kHz, Kentish et al. [18] prepared nanoemulsions of flaxseed oil and Tween 40 surfactant because it possess good stability against creaming, sedimentation, flocculation and coalescence because of the small droplet size. Following this, a handful of studies have demonstrated the usage of ultrasound in dispersing bioactives such as wheat bran oil, fish oil, lemon grass oil, etc. in aqueous surfactant solutions to produce coarse emulsions. [19-21] Like-wise Ashokkumar and Shanmugam [22] incorporated flax seed oil in pasteurized 2

homogenized skim milk using high intensity ultrasound to produce emulsion droplets of 0.64 µm size which was stable for at least 9 days. In this context, here we choose black seed oil (extracts of Nigella Sativa seeds) for preparation of a milk emulsion using ultrasonication to analyze its storage stability (creaming stability) and properties (colour, particle size, and rheology). The reason for choosing Nigella Sativa seed since it has lots of medicinal values (suppress cough, retard carcinogenic processes, treat diarrhea, promote menstruation and milk production, etc) and it is called "the Blessed seed" because it is identified as the curative black cumin in the Holy Bible. [23] In addition, it contains lots of nutritional ingredients such as vitamins and proteins including amino acids such as Thymoquinone, Thymol, Thymohydroquinone, Nigellicine, Nigellimine and α-Hederin, which are responsible towards growing number of phytochemical and pharmacological investigations. This study is aimed at proving the versatility of the ultrasonic technology for delivering bioactive materials in complex food matrices. EXPERIMENTAL DETAILS Pasteurised homogenized skim milk (PHSM) was purchased from a local retailer in India and its composition was 3.5% fat, and 8.0% solid not fat (SNF) as labeled by the manufacturer. Unrefined black seed oil purchased from Prano Flax India Pvt. Ltd, Jaipur, India. The hydrophobicity of milk proteins were measured on aqueous portion in the presence of a fluorescent molecular probe, Sudan III dye, purchased from Sigma-Aldrich. All other chemicals used here of the highest purity available and were used as received without further purification. Unless otherwise specified, all the reagents used were of analytical grade and the solutions were prepared using millipore DDI water (18.2 MΩ). Emulsification of Pasteurised homogenized skim milk by Ultrasound approach Pasteurised homogenized skim milk (PHSM) samples was placed in a Sonicator vessel jacketed with ice and treated by a 13 mm horn type ultrasonic probe placed at the centre and to a depth of 3 cm. Ultrasound was performed at a frequency of 20 kHz and a power of 100 W/cm2 for treatment times 2-12 min. For black seed oil milk emulsions, the composition was 7% black seed oil (v/v) in 93% PHSM. During sonication, thermostated water was circulated continuously through a jacket surrounding the 3

sonication cell and the water temperature was maintained at 25±2°C. The sonicated or emulsified samples were stored in a refrigerator for about 8 days at 4±2°C. The analysis and storage studies were performed on both fresh and stored samples. The black seed oil/milk is referred to as oil-milk emulsion (OM). Cream stability refers to physical stability of emulsions against separation into individual phases, viz., oil and aqueous. Initially, visual inspection of samples were carried out to check the phase separation and oiling-off or creaming. Next quantitatively, the amount of creaming was measured by storing them in sealed graduated tubes at 4 ± 2°C for 8 days. It was monitored by measuring the volume of the lipid-rich layer on top (VL) and the volume of total emulsion (VE) in the tube. It is represented as creaming index (%) using Equation 1. Creaming Index (%) = (VE - VL / VE) × 100

(1)

As an example, if the creaming index is 100%, there is no phase separation in the emulsions.

Size Analysis of Emulsions A laser diffraction method was used to measure the size distribution of emulsion droplets using Zetasizer 2000 (Malvern Instruments Ltd., Worcestershire, UK) in both fresh and stored samples. Laser diffraction measures droplets size distributions by measuring the angular variation in intensity of light scattered as a laser beam passes through a dispersed particulate sample. Large particles scatter light at small angles relative to the laser beam and small particles scatter light at large angles. The angular scattering intensity data was then analyzed to calculate the size of droplets responsible for creating the scattering pattern, using the Mie theory. In a typical measurement, a few droplets of the emulsion were suspended in re-circulating water (1250 rpm) and the sizes were recorded at the refractive index of black seed oil (1.4697). The distribution of the particles provided in this manuscript is the average of 3 runs.

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Rheological Properties of Emulsions The viscosity of the black seed oil-milk emulsion was measured using Brookfield cone and plate viscometer (LVDV-I PRIME C/P) equipped with a 2.4 cm 0.8ο cone supplied by Brookfield engineering laboratories, USA. The cone was connected to the spindle drive while the plate was mounted on the sample cup. Spindle used was CPE-40, suitable for samples in the viscosity range of 0.3–1028 cP. Using electronic gap adjusting feature provided with the viscometer, a gap of 0.013 mm between the cone and the plate was maintained within which the test fluid was placed. As the spindle was rotated, the viscous drag of the fluid against the spindle measured by the deflection of the calibrated spring. Cone and plate geometry required a sample volume of only 0.5–2 ml and hence the temperature equilibrium was achieved rapidly within a minute. The spindle type and speed combination produced satisfactory results when the applied torque was between 10% and 100% of the maximum permissible torque. Hence, during measurements, the readings were discarded if the applied torque did not fall within this range. The spindle speeds available with this viscometer fell in the range of 0–100 rpm and the shear rate range was 0–750 s-1. The viscometer was benchmarked with distilled water, glycerin and ethylene glycol at room temperature. The measured values of viscosity for distilled water, glycerin and ethylene glycol were 0.82, 10.9 and 360.5 cP, respectively, which agree well with the literature values of 0.79, 10.7 and 352 cP, respectively, with ±5% accuracy. Hydrophobicity of Emulsions The hydrophobicity of milk proteins were measured on aqueous portion in the presence of a fluorescent molecular probe, Sudan III dye. About 1 x 10-4 M Sudan III dye was dissolved in black seed oil (7%) by stirring for 3 hours. Then it was added to 93% PHSM solution and an emulsion was created. 2 mL of OM emulsion was mixed with 2 mL of chloroform and methanol in the composition ratio 2:1, shaken vigorously the mixture for 5 minutes and the organic layer was discarded. The obtained solid part of the milk was mixed with 6 mL water and 6 mL chloroform at a single time, 5

shaken well and once again, the chloroform layer was discarded. The aqueous layer left behind was analyzed by absorbance and fluorescence techniques (Specord S 600 diode-array spectrophotometer and Shimadzu RF5301PC spectrophotometer). RESULTS AND DISCUSSION The research presented here is focused on ultrasonic formulation of stable food emulsions for the delivery of nutrients in general. The work explores the emulsification capability of ultrasound to deliver stable emulsions of black seed oil in milk, particularly in the absence of external food additives and pre-emulsification procedures. The study is further extended to investigate the ultrasound induced changes to the inherent properties of milk and emulsion following sono-emulsification. The size of emulsion droplets is vital to the whole emulsion system as it influences the color, texture, and stability of emulsions. Good stability is usually associated with fine, uniform droplet size [24]. Figures 1A-C show the droplet size measurement of the black seed oil:milk (OM) emulsions sonicated for 2, 8 and 12 minutes. After storage at 4±2ºC, droplet sizes were once again measured on the eighth day for comparison (Figures 1D-F).The droplet size distribution curve of 2 min emulsions was bimodal on the first and eighth days (Figure 1A & D) suggesting that droplets coalesced during sonication [25]. The droplet size distributions of 8 and 12 min emulsions are bimodal on the first day (Figures 1B & C) and monomodal on the eighth day (Figures 1E & F). Two types of emulsion particles were detected, the one with sizes ranged from 30-110 nm and the other considerably larger particles of 400-2810 nm. The larger size particles could be generate by coalescence of fat globules or aggregation of proteins onto fat droplets. The droplet size of first and eighth day ranges between 408-448 nm (2 min emulsion), 404-435 nm (6 min emulsion), 400-415 nm (8 min emulsion), 416-450 nm (10 min emulsion), and 462-502 nm (12 min emulsion), suggesting that the average droplet size was not affected much, however, the droplet size distribution is narrower for 8 min emulsion [26,27]. However, the droplet size increased upon increasing the sonication time for 12 min, which might be due to coalescence (Fig 1G). 6

Creaming stability refers to physical stability of emulsions against separation into individual phases, viz., oil and water. 7% black seed oil (v/v) in 93% PHSM was chosen as a model system based on optimized conditions found in literature for oil milk emulsion standards [22]. Figures 2A -B show the creaming stability of 7% OM emulsions sonicated at 100 W (40% percentage amplitude) for 2-12 min and upon storage at 4±2ºC for 8 days. Initially, visual inspection of samples were carried out to check the phase separation and oiling-off or creaming. Such stored liquid emulsions did not show any distinctive change that could be observed by visual inspections (Fig 2 A). The emulsions processed using ultrasonication for 6 to 8 min showed 100% stability against creaming until eight days of storage, while those processed for 2-4 min and 10-12 min showed stability for only one or two days (Fig 2 B). Figures 2 C-D show the creaming stability of 7% OM emulsions sonicated at different power amplitudes (20, 30 and 40%) for 8 min. It can be seen that the prepared emulsions using 40% amplitude are 100% stable for 8 days whereas the other two samples (20 and 30% amplitudes) gave rise to a slight increase in yellowness. This is because of partial coalescence and clustering between the fat droplets. [28,29] That is, the first two samples were performed with 20% and 30% amplitude, and the third sample was performed with 40% amplitude which resulted incomplete emulsion formation whereas the first two resulted in phase separation. Complete emulsion formation with 40% amplitude is the consequence of the high intense collapse of the cavitation bubble at higher amplitude (or ultrasound power). Due to high intense collapse and very high velocity of shock waves at higher amplitude, the mixture undergoes complete mixing resulting in uniform emulsion formation without phase separation. At lower amplitude, phase separation happens due to low collision of molecules because of less intense collapse and lower velocity of shock waves. The rheological properties of OM emulsion are dependent on the composition and properties of the emulsion. [8] Fig. 3A shows the rheological properties of the OM emulsion sonicated after 2, 4, 6, 8, 10 and 12 minutes on the day of experiment performed and on the eighth day of the sample after 7

storage at 4±2ºC. No significant change in viscosity (2-6 min samples) illustrates all emulsions showed flow Newtonian behaviour (i.e., low particle - particle interaction). [30] A minor increase in viscosity is noticed for 8 min sample compared to 2-6 min samples, which might be due to the presence of finely emulsified fat globules generated by the intense shear forces of ultrasound. [31] Viscosity gets decreased after 8 min sample may be due to coalescence. The oil or fat content influences the color and opacity of emulsions. Hence oil soluble dye Sudan III was added to the emulsion prepared after 2-12 min sonication,

which shows pink colored

continuous phase illustrating the homogeneous nature of the emulsion (Fig 3B). The samples were extracted and studied by absorbance and fluorescence spectral measurements. The high absorbance and fluorescence intensity for eight minute sonicated emulsion may be due to a considerable increase in interaction between proteins adsorbed at the interface, which resulted in creating more hydrophobic sites [32]. In addition, the optical microscopic images of pure milk (Fig 4A), black seed OM emulsion sonicated after 2, 8 and 12 minutes samples (Fig 4 B-D) and upon storage at 4±2ºC for 8 days (Fig 4 EG) indicate that there is less change in the emulsion microstructure even after 8 days, when compared to initial emulsions. The reason for such stability due to the adsorption of partially denatured whey proteins. In addition, the optical microscopic images of pure milk (Fig 4A) and 2 min emulsions after the eighth day (Fig 4E) looks like an agglomeration of particles may be due to droplets get coalesced during sonication. In summary, stable emulsions of 7% black seed oil and PHSM were obtained using 20 kHz US (100 W). The cavitation effects of US are responsible for producing stable dairy emulsions. The whey proteins may contribute to the stability of the emulsions. A minimum process time of 8 min is recommended to produce stable emulsions of 7% black seed oil and milk. The data suggest that black seed oil can be effectively incorporated into a complex food matrix in the form of an emulsion by employing US technique which may useful to various medical applications.

ACKNOWLEDGMENT 8

The author SA and OK thank DST, India for the sanction of India–Russia collaborative research grant (INT/RUS/RFBR/P-209 dated 15-6-15) and NSC (Russia) for the financial support through (research grant No. 15-58-45028/15 dated 19-6-2015).

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Figure Captions

Fig 1 The particle size measurement of the black seed oil:Milk (OM) emulsion sonicated after 2, 8 and 12 minutes samples (A-C) and upon storage at 4±2ºC for 8 days (D-F). Particle size vs sonication time plot (G).

Fig 2 (A-B) Creaming stability of 7% OM emulsions sonicated at 100 W (40% percentage amplitude) for 2-12 min and upon storage at 4±2ºC for 8 days. (C-D) Creaming stability of 7% OM emulsions sonicated at power density (100 W) and at different percentage amplitude (20, 30 and 40%) for 8 min..

Fig 3 Viscosity vs sonication time plot (A). Color of 7% OM emulsions sonicated at 100 W (40% percentage amplitude) for 2-12 min in the presence of Sudan III dye (B). Absorbance (C) and Fluorescence spectra of 7% OM emulsions sonicated at 100 W (40% percentage amplitude) for 2-12 min in the presence of Sudan III dye.

Fig 4 Optical microscopic image at 100x magnification of pure milk (A), black seed oil:Milk (OM) emulsion sonicated after 2, 8 and 12 minutes samples (B-D) and upon storage at 4±2ºC for 8 days (EG).

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G

Day 1 Day 8

500

Particle Size (nm)

480

460

440

420

400 2 min

6 min

8 min

10 min

12 min

Sonication Time (min)

Figure 1

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Figure 2

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Figure 3

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Figure 4

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Highlights •

Ultrasonic formation of stable emulsions of a bioactive material, black seed oil, in skim milk was investigated



The effect of sonication time and acoustic power on the emulsion stability was studied



Partially denatured whey proteins may provide stability to the emulsion droplets

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