Proceedings of 2010 International Conference on Systems in Medicine and Biology 16-18 December 2010, liT Kharagpur, India

Characterization of Oil-in-water Gelatin Emulsion

Gels: Effect of Homogenization Time

Goutam Thakurt, Analava Mitral', Amit Basak2, Derick Rousseau3, Kunal Pal4

[School of Medical Science and Technology, Indian Institute of Technology Kharagpur, Kharagpur-721302, India 2Department of Chemistry, Indian Institute of Technology Kharagpur, Kharagpur-72 1302, India

3Department of Chemistry and Biology, Ryerson University, Toronto, Canada 4Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela, Orissa, India

*Correspondence: analavamitra@gmail.com

Abstract- Oil-in-water emulsion gels consisting sunflower oil as the internal phase and a chemically-crosslinked gelatin solution as the continuous aqueous phase were developed. The dispersion was homogenized at 60°C in a high pressure valve homogenizer at a pressure of 5000/500psi for different time periods (2, 5 or 10 min). The homogenized samples were formed into films at 5°C followed by crosslin king with genipin at room temperature. The microstructure of the gels was studied using confocal laser scanning microscopy. The results showed significant differences in the microstructure depending on homogenization time. Gel micrographs indicated a well-dispersed network of sunflower oil droplets in the gelatin matrix with a higher homogenization duration (10 min) while a less unorganized gel microstructure was evident at shorter homogenization times (2 and 5 min). Gels were also characterized using colourimetric analysis. Puncture tests of the gels were tested to establish their mechanical stability. The gels prepared with 10 min homogenization exhibited the highest puncture strength (0.23±0.20 MPa) (p<0.05). These results demonstrated that gelatin gels homogenized for longer periods were more stable, thus expanding their range of possible biomedical applications.

Keywords- gelatin; emulsion gel; homogenization; genipin; crosslin king; microstructure

I. INTRODUCTION

Oil-in-water emulsions consist of oil (e.g. vegetable or mineral oil) dispersed as droplets stabilized by a variety of emulsifiers (e.g., proteins or low molecular weight emulsifiers) and/or thickening agents. Emulsion gels are oil-in-water emulsions further stabilized by a highly-viscous continuous phase polymer matrix. Emulsion gels are now finding application in food and biomedical applications [1, 2]. Many biopolymers (e.g. milk protein, gelatin) have been used to prepare and help stabilize emulsion gels [3, 4].

High pressure homogenizers are used to emulsify, mix and process many food/pharmaceutical products. Proper homogenization can help improve a product's shelf life by effectively reducing the droplet size and by preventing phase

separation (e.g., sedimentation). The use of too Iow a pressure fails to lead to successful emulsification [5]. During high pressure homogenization, a coarse mixture is subjected to high turbulence which is the predominant mechanism that leads to the break-up of the dispersed phase into small droplets. There exists a dynamic equilibrium between breakage and coalescence of the droplets that is influenced by homogenization conditions [5].

Gelatin is a well-known biocompatible polymer that exhibits a sol-gel transition at ::; 30°C. The aqueous solubility of gelatin leads to its thermal and mechanical instability and limits its biomedical applications. As a result, chemical crosslinkers (e.g., glutaraldehyde, formaldehyde, genipin, etc.) are often used to form insoluble networks with improved mechanical and thermal stability. Given its negligible cytotoxicity, genipin has recently attracted considerable attention [6, 7].

There are several important factors influencing the quality of o/w emulsions produced by homogenization. Besides the pressure, the homogenization time plays an important role in determining the stability and quality of the emulsion gels. In the present study, we prepared gelatin-based emulsion gels (o/w) consisting of sunflower oil as the internal phase while the continuous aqueous phase consisted of a gelatin network crosslinked with genipin. Our objective was to investigate the effects of different homogenization times on emulsion gel properties, namely their microstructure and mechanical strength.

A. Materials

II. MATERIALS AND METHODS

Porcine gelatin (Type A; bloom strength 300), polysorbate 80 and glycine were procured from Sigma-Aldrich (USA). Sunflower oil was obtained from a local supermarket while genipin was purchased from Challenge Bioproducts (Taipei, Taiwan, PRC). Deionized water was used throughout the study.

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Proceedings of 2010 International Conference on Systems in Medicine and Biology 16-18 December 2010, liT Kharagpur, India

B. Methods

The aqueous phase consisted of a 10% (w/w) gelatin solution prepared by dissolving the gelatin powder in deionized water at 60°C for 10 min. A 1% (w/v) polysorbate 80 was added to help emulsify the sunflower oil within the emulsion gel continuous phase. Coarse emulsions were prepared by magnetic stirring by premixing all ingredients on a stirring hotplate at 60°C. The aqueous and oil phases were mixed at weight ratios of S: 1 and the coarse emulsions were homogenized at 60°C in a high pressure valve homogenizer (APV lab homogenizer) at a pressure of SOOO/SOOpsi for 2, S or 10 min. The gelatin emulsion gels (GEG) are hereafter referred to as GEG-2, GEG­S and GEG-1O. The schematic representation of the homogenization process is shown in Fig.I. Gelation of the homogenized samples was performed at SoC.

A genipin solution [10 mL of 0.4% (w/v)] was poured onto the GEGs and allowed to diffuse into the samples for 24 h. Afterwards, the genipin solution was replaced with glycine solution and was allowed to interact with the excess unreacted genipin for another 24 hr. Crosslinking was conducted at room temperature.

Colourimetric analysis of the gels was performed using a Hunterlab colourmeter (Hunter Associates Laboratory, Reston, Virginia, USA), which provided 'L *', 'a*' and 'b*' values, where L * is the lightness component ranging from 0 (black) to 100 (white), and parameters a* (from greenness to redness), and b* (blueness to yellowness) are the two chromatic components. Based on the L *a*b*, the Whiteness Index (WI) was calculated as follows [8]:

WI = 100-[(100-L*)2 +a*2 +b*2]OS

Vapourtnp

Heating element

Sunple retuming tube

OvedJead stirrer

/

PressureV1lves

(I)

Figure 1. Schematic representation of homogenization process and experimental set-up

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Figure 2. Experimental set up for the puncture test, where A and C are the sample holders and B represents an GEG sandwiched between plates A and C.

The morphology of the GEGs was analyzed at 100x using a confocal laser scanning microscope (Zeiss LSM-S1O, Zeiss Inc., Toronto, ON, Canada). A 488 nm laser (filter: HFT 488) was used for excitation while the emitted spectra was collected by using the LPS60 filter. Images were analyzed with the LSM-SIO software.

The puncture strength of the gels was measured using a Texture analyzer (TA-XTIi Texture Analyzer, Texture Technologies Corp, USA). A stainless steel probe (diameter: 0.9S mm) pierced the GEGs at a speed of O.l mm/sec. Sample holders were designed to prevent sample slippage whereby the GEGs were sandwiched between two plates (Fig. 2). A S kg load cell was used for calibration. The puncture strength was calculated as per the reported literature [9].

Results are reported in all experiments as arithmetic means ± standard deviation. Triplicate analyses were performed on all measurements. Analyses of variance (ANOVA) and Tukey's multiple comparison tests were analyzed using GrapbPad Prism S. Statistical differences were deemed significant at p<O.OS.

III. RESULTS AND DISCUSSION

Colour analysis of the genipin-crosslinked GEGs was performed to assess differences in cross linking as a function of homogenization time [10]. As cross linking of gelatin with genipin results in the formation of a blue pigment, it was surmised that the processing conditions employed may lead to differences in whiteness index (WI) (Eq. 1). However, Fig. 3

shows insignificant changes in the WI for all gel constructs (p>O.OS), suggesting that homogenization time no impact the extent of crosslinking with genipin.

The microstructure of the GEGs was evaluated with confocal microscopy (Fig. 4a-c). This figure illustrates the continuous gelatin gel phase, with the oil droplets visible as the dark regions. As the genipin-crosslinked structure autofluoresces, no dye was needed to analyze the gel microstructure [11].

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Proceedings of 2010 International Conference on Systems in Medicine and Biology 16-18 December 2010, liT Kharagpur, India

25

20

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en en Q) 10 c Q)

:t= ..c

S 5

o GEG-2 GEG-5 GEG-10

Gelatin emulsion gels

Figure 3. Whiteness index of the GEGs crosslinked for 2,5 or 10 min.

Fig. 4a shows that the oil droplets were larger, more polydispersed and aggregated with shorter homogenization (2 min). With an increase in homogenization time, the oil phase became more effectively dispersed, and any droplet flocs were broken up (Fig. 4b-c). Fig. 4c shows the microstructure of a GEG homogenized 10 min, where individual droplets �20-50 /--lm in diameter were evenly distributed within a closely-spaced gelatin network. During high pressure valve homogenization, composition and processing parameters play an important role. At a given pressure, homogenization duration is critical for the development of emulsions with small, narrowly-distributed dispersed droplets [5]. During our experiments, it was quite clear that shorter homogenization times (2 and 5 min) were insufficient to allow effective emulsification.

The mechanical properties of biopolymer gels provide an indication of gel integrity during operational conditions. To elucidate the effect of the homogenization time on the strength of the GEGs, the puncture strength of the GEGs was determined. Fig. 5 compares the puncture strength of the GEGs, showing the impact of homogenization time. The GEG-10 showed the highest puncture strength (�0.23 MPa) followed by GEG-5 (�0.21 MPa) and GEG-2 (�O.l9 MPa) (p < 0.05). If we presume that crosslinking was similar irrespective of homogenization duration (based on the WI results), then the droplet size and organization of the dispersed phase appears to have a played a significant role on GEG structural stability and mechanical strength. With GEG-IO, the dispersed was much more homogeneously distributed than in either GEG-2 or GEG-5.

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(a)

(b)

(c)

Figure 4. Confocal laser scanning micrographs showing changes in the microstructure of the crosslinked GEGs as a function of homogenization duration (2, 5 or 10 min). The size bars shown represent 50 /--lm.

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Proceedings of 2010 International Conference on Systems in Medicine and Biology 16-18 December 2010, liT Kharagpur, India

0.26

0.25

0.24

ro-a.. 0.23 ::::?: ........

..c. 0.22 OJ c

� 0.21 -

VI

� 0.20 ::J

t5 c 0.19 ::J

a.. 0.18

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GEG-2 GEG-5 GEG-10

Gelatin emulsion gels

Figure 5. Puncture strength of gelatin emulsion gels homogenized for 2, 5 or 10 minutes.

As emulsion gels may be thought of as particle-filled gels, the possible interaction between the dispersed and continuous phases likely played a role in the mechanical properties of the GEGs. Assuming an identical volume fraction for all GEGs, the more finely-dispersed network in GEG-lO consisted of the highest surface area per volume. Presumably, the 'filler' dispersed oil phase interacted more strongly in GEG-lO compared to GEG-5 or GEG-2 and was more effectively bound to the gel matrix [13]. Interactions between the dispersed oil droplets and surrounding gel matrix will strongly depend on the surface properties of the droplets, which depend on the nature and concentration of the surfactant(s) present at the oil­water interface [14]. It is thus possible that there were interactions (hydrogen bonding and/or electrostatic) between the hydrophilic moiety of the surfactant (polysorbate 80) present at the oil droplet surface and the gelatin polymer chains within the continuous network, leading to the strengthening of the polymer network. Thus the presence of an organized,network of small droplets in GEG-l 0 likely led to an increase in gel strength.

IV. CONCLUSION

The present work demonstrates that the extent of homogenization can significantly impact GEG mechanical strength. Future research efforts will further investigate the use of homogenization conditions to modulate the properties of emulsion gels for potential biomedical applications.

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ACKNOWLEDGMENT

Author Thakur gratefully acknowledges lIT Kharagpur for a PhD fellowship. Debasish Maji of lIT kharagpur is acknowledged for technical assistance. Funding from the Natural Sciences and Engineering Research Council of Canada (NSERC) is gratefully acknowledged.

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