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Imaging the phase of starch–gelatin blends by confocal Raman microscopy
Food Hydrocolloids 60 (2016) 7e10
Contents lists available at ScienceDirect
Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd
Imaging the phase of starchegelatin blends by confocal Raman microscopy Xingxun Liu a, *, Nuozi Zhang b, Long Yu c, Sumei Zhou a, Robert Shanks b, Jinkai Zheng a a
Institute of Food Science and Technology (IFST), Chinese Academy of Agricultural Sciences (CAAS), Beijing, 100193, China School of Applied Sciences, RMIT University, Melbourne 3000, Australia c School of Light Industry and Food Science, CPFRR, South China University of Technology, Guangzhou 510640, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 July 2015 Received in revised form 28 January 2016 Accepted 1 March 2016 Available online 9 March 2016
The well recognized complex issue of compatibility between starch and gelatin was studied based on their interfacial and phase composition using Raman microscopy. Blending films with different ratios of gelatin and starch was used in this work. Raman beam focused on a 1.7 mm 1.5 mm detection region and the micro-spectroscope scanned across the gelatinestarch interface. The ratio of areas of saccharide bonds (1173-953 cm1) and amide I bands (1750-1550 cm1) was used to monitor the relative distributions of the two components of the blends. The Raman spectral maps confirmed that for all the blends investigated, gelatin formed a continuous matrix in which starch inclusions were dispersed. Intermediate phases consisted of amylose interacted with gelatin, which demonstrated this was an unique and effective method to investigate starch based blend and composite materials. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Gelatin Starch Raman spectroscopy Interface Polymer compatibility
1. Introduction Blends of gelatin with starch have attracted much attention since they are both renewable resources and they have filmforming ability. A blended film of polysaccharide and protein exhibited improved gas barrier (O2 and CO2) properties than either of the pure films (Arvanitoyannis, Nakayama, & Aiba, 1998; Baldwin, Nisperos-Carriedo, & Baker, 1995). Blends have been developed for use as medical capsule materials (L. Zhang et al., 2013; N. Zhang, H. Liu et al., 2013; N. Zhang, X. Liu et al., 2013). Previous research has shown that gelatin and starch are immiscible, their morphologies and compatibility are determined by several characteristics, such as processing time (Firoozmand, Murray, & Dickinson, 2009), temperature (Ioannis Arvanitoyannis et al., 1998), pH (Ong, Whitehouse, Abeysekera, Al-Ruqaie, & Kasapis, 1998) and volume fraction (Al-Hassan & Norziah, 2012). Mechanical properties of starchegelatin blends depend on their morphology, particularly the extent of homogeneity and the composition of their continuous and dispersed phases. Various techniques have been used to characterise phase composition and
* Corresponding author. IFST, Chinese Academy of Agricultural Science (CAAS), Beijing. E-mail address: [email protected] (X. Liu). http://dx.doi.org/10.1016/j.foodhyd.2016.03.005 0268-005X/© 2016 Elsevier Ltd. All rights reserved.
interface of this complex blended system by indirect methods such as differential scanning calorimetry (DSC) (Jagannath, Nanjappa, DasGupta, & Bawa, 2003), dynamic mechanical analysis (DMA) (Arvanitoyannis, Psomiadou, Nakayama, Aiba, & Yamamoto, 1997) and rheometry, as well as direct methods such as polarized optical microscopy and confocal laser scanning microscope (CLSM) (Firoozmand et al., 2009). In our previous paper (Liu et al., 2013), we have compared different microscopies such as polarized optical microscopy, scanning electron microscopy (SEM) and synchrotronFourier transform infrared spectroscopy microscopy (SynchroFTIRM) to study gelatinestarch blends. Of these methods, the Synchro-FTIRM provided capability to detect and measure composition and interface. Spatial resolution of FTIRM was limited to 10 10 mm, even application of synchrotron light technology only enabled achievement of a spatial of 5 5 mm (Wetzel, Shi, & Reffner, 2010). Corn-starch is an economic crop that is often used for the raw materials for edible or biodegradable materials. The average diameter of corn starch granules is from 8 to 12 mm (Chen, Yu, Chen, & Li, 2006), so high spatial resolution is needed to examine morphing of the granules into film by gelatinization. Raman microscopy is an effective method to study heterogeneous materials since it provides sub-micron spatial resolution with high sensitivity (Chrimes, Khoshmanesh, Stoddart, Mitchell, & Kalantar-zadeh,
8
X. Liu et al. / Food Hydrocolloids 60 (2016) 7e10
Fig. 1. Raman optical images, spectra and maps of G3S7 for excitation at 532 nm, (A) the selection zone chosen from the optical images; (B) one spectra of gelatinestarch blends of collecting data; (C) map obtained from the ratios between integrated intensities in the 1173-953 cm1and 1750-1550 cm1 range; (D) overlaid images shows that the same area was mapped.
2. Experimental 2.1. Materials and film preparation Starchegelatin blend film was prepared according to previous methods (N. Zhang, H. Liu et al., 2013; N. Zhang, X. Liu et al., 2013). Briefly, solutions were prepared with different ratios of starch (A1081, Penford, Australia):gelatin (GELITA UG719-N, Type A, 250 bloom, Sweden) equal 90:10, 70:30, 50:50, 30:70 and 10:90 including 1%$w/w sorbitol based on a total weight basis (2 g) in 100 mL distilled water. The mixed materials were dissolved in distilled water at 80 C for an initial 30 min at a slow stirring speed (100 min1), then stirred for a further 30 min at high speed (700 min1) until a clear solution was obtained. The solution (5 mL) was poured onto a poly(ethylene terephthalate) (PET) dish (diameter 5 cm) that was kept level to control film thickness. The cast film was dried overnight at 37 C. The dry films were peeled from the plate, placed in a desiccator containing saturated sodium bromide (NaBr) solution to control humidity, and stored at 56%$RH and 23 C until required for analysis. 2.2. Raman microscopy An XploRA plus Raman confocal microscope system (Horiba scientific) was used to analyze specimen surfaces. A532 nm diode laser (15 mW laser power) with an X100/0.90NA air objective was employed. Spatial resolution was obtained using 100 mm confocal pinholes. The Raman signal was acquired using a 1200 lines/mm grating centered between 200 and 2760 cm1. For each specimen, a 1.7 1.5 mm area of the surface was mapped at X and Y-axes. Data was analyzed using LabSpec 6. The integration time was 10 s for all
measurements. 3. Results and discussion Different ratios of starch:gelatin (90:10, 70:30, 50:50, 30:70 and 10:90, named G9S1, G7S3, G5S5, G3S7, G1S9) including 1%$w/w sorbitol plasticizer, were used as model materials, a Raman confocal microscope system with a 532 nm laser was used to study the gelatinestarch films. Fig. 1 shows the approach to acquisition of spectra, data analysis (image generation) and processing of the Raman images, Fig. 1(A) shows the heterogeneous nature of such blends and their phase distribution. Gelatin was a continuous phase while starch formed a separated phase distributed as spherulites of about 5 mm diameter. It should be noted that the diameters of starch particles depend on volume fraction of starch and drying temperature. The distribution of starch was more diffuse and the interfacial contrast became less distinct depending on volume fraction and water content. Fig. 1(B) and Fig. 2 show the Raman spectra of gelatinestarch
20000 Starch
Intensity(counts)
2013). For example, lateral and depth resolutions of approximately 0.25 and 1.7 mm, respectively, can be achieved when using a 633 nm laser source and an aperture of 50 mm in radius to give a 60 /1.2 numerical aperture (NA) objective. Therefore, the aim in this study was to study morphology and phase composition of gelatinestarch blends using Raman microscopy.
15000
10000
G3S7
5000 Gelatin 0 1700
1300
900
Raman Shift (cm-1) Fig. 2. Raman spectra of gelatin and gelatinestarch blend (G3S7).
500
X. Liu et al. / Food Hydrocolloids 60 (2016) 7e10
9
Fig. 3. Raman optical images, maps and overlaid maps of different gelatinestarch ratios.
and gelatin. A typical spectral feature of the pure gelatin film was the strong amide I band located at approximately 1654 cm1 which usually indicate the alpha helix dominated structure. The amide I absorption is primarily due to the stretching vibration of the C]O bond (Sun et al., 2011), and it is related to the protein conformation, such as a-helix or b-sheet. Another characteristic peak around 1250 cm1 is the amide III involving CeN stretching and NeH inplane bending vibrations of the peptide bond as well as contributions from Ca-C stretching and C]O in-plane bending (Sun et al., 2011). The typical starch spectra in the range 1300e800 cm1
was found in the gelatinestarch film, the peak 1150 (shoulder peak), 1120 and 1087 cm1 is due to the CeO, CeC stretching and CeOH bending. Overlapping peaks located in the region of 1173953 cm1 (labelled as band 1), were the most intense bands in the Raman spectra and they were chosen as the characteristic peaks for starch; while the integrated area of the amide I band (1750e1550 cm1), labelled as Band 2, was used to represent gelatin. The ratio of the peak area of the starch bands (band 1) to that of gelatin bands (band 2) was used to represent the distribution of starch or gelatin in the blends. The ratio of band areas rather
10
X. Liu et al. / Food Hydrocolloids 60 (2016) 7e10
than absolute values was used to enable normalization of the results, correcting for possible variations in film thickness, both within the same film and between different films. Raman intensity mapping of the band 1/band 2 under 532 nm laser excitation in Fig. 1(C) shows a 2D contour map that was established for each of the blends with different starchegelatin content. Fig. 1(C) gives a higher special resolution to observe the phase distribution compared with FTIR microscopy, where special solution could reach 1.7 mm 1.5 mm. The color code represents concentration of a component. The red in the scale denotes a high starch concentration while blue denotes a high gelatin concentration. The Raman maps confirmed that starch was a dispersed phase while the gelatin phase formed a continuous matrix in which starch inclusions were dispersed. The results demonstrated the heterogeneous nature of such blends with starch domains dispersed into a gelatin continuous phase, even with higher starch content blends. Fig. 1(D) shows overlay data of Raman spectral microscopy -optical microscopy images. The overlaid images show that exactly the same area was mapped by Raman microscopy, however Raman microscopy revealed the chemical composition. When the images were overlaid, the resultant image gave a replicate result for the distribution of starch and gelatin. Fig. 3 shows a chemical map of starch distribution acquired from a CCD (charge-coupled device) detector for various gelatinestarch blends. The size of starch domains decreased with increasing gelatin volume fraction. Raman concentration maps suggested that for all mixtures investigated gelatin formed a continuous matrix in which starch inclusions were dispersed. The results demonstrated the disperse and diffuse nature of such blends, with starch domains dispersed into a gelatin continuous phase, even with higher starch volume fraction blends. However, the interfacial contrast became less distinct with increasing gelatin volume fraction. During the film-forming process, the starch experiences gelatinization and gelation; it is well known that over short times retrogradation of amylose occurs, while over long times amylopectin retrogrades. Furthermore, while some amylose will be subject to retrogradation to V-type crystals thus enhancing separation of starch as it becomes spherical (Liao et al., 2014), so amylose may interact more with gelatin. The Raman results support the observation that starch and gelatin are compatible though not miscible, furthermore, it also provide some new results, in particular the inter-phase. More work of enlarging the mapping area will be studied in future. Compared with gelatinestarch blending observed by FTIR microscopy, large spatial resolution (1.7 mm 1.5 mm) resulted with Raman microscopy. There are some intermediate phases around the starch phase that display a green color in the gelatinestarch blends. This phenomenon may be due to the amylose leached from the starch phase during gelatinization during processing, and interacting with gelatin; some sorbitol plasticizer would assist by stabilizing the interface. Intermediate phases were observed using Raman microscopy, which further support the observation that starch and gelatin are compatible though not miscible. 4. Conclusion Raman microscopy mapping has been shown to be an efficient and effective method to characterize the phase composition and distribution of gelatinestarch blends. The ratio of the areas of starch and gelatin bands was used to determine the relative distributions of the two components in the blends. The Raman maps confirmed that gelatin formed a continuous matrix in which starch inclusions were dispersed, for all the blends investigated.
Intermediate phases, due to amylose interaction with gelatin, were observed using Raman microcopy. The gelatinestarch blends formed a compatible system with gelatin matrix and starch dispersed phase, with each component contributing to properties for application in capsules requiring enhanced gas barrier and facile dissolution to release contents. Conflict of interest The authors declare that there is no conflict of interests regarding the publication of this paper. Acknowledgments The authors from China would like to acknowledge the research funds NFSC (31301554, 21174043) and the Fundamental Research Funds for Chinese Academy of Agricultural Sciences (2015ZL048). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.foodhyd.2016.03.005. References Al-Hassan, A. A., & Norziah, M. H. (2012). Starchegelatin edible films: water vapor permeability and mechanical properties as affected by plasticizers. Food Hydrocolloids, 26(1), 108e117. Arvanitoyannis, I., Nakayama, A., & Aiba, S.-I. (1998). Edible films made from hydroxypropyl starch and gelatin and plasticized by polyols and water. Carbohydrate Polymers, 36(2e3), 105e119. Arvanitoyannis, I., Psomiadou, E., Nakayama, A., Aiba, S., & Yamamoto, N. (1997). Edible films made from gelatin, soluble starch and polyols, Part 3. Food Chemistry, 60(4), 593e604. Baldwin, E. A., Nisperos-Carriedo, M. O., & Baker, R. A. (1995). Use of edible coatings to preserve quality of lightly (and slightly) processed products. Critical Reviews in Food Science and Nutrition, 35(6), 509e524. Chen, P., Yu, L., Chen, L., & Li, X. X. (2006). Morphology and microstructure of maize starches with different amylose/amylopectin content. Starch-Starke, 58(12), 611e615. Chrimes, A. F., Khoshmanesh, K., Stoddart, P. R., Mitchell, A., & Kalantar-Zadeh, K. (2013). Microfluidics and Raman microscopy: current applications and future challenges. Chemical Society Reviews, 42(13), 5880e5906. Firoozmand, H., Murray, B. S., & Dickinson, E. (2009). Microstructure and rheology of phase-separated gels of gelatin þoxidized starch. Food Hydrocolloids, 23(4), 1081e1088. Jagannath, J. H., Nanjappa, C., Das Gupta, D. K., & Bawa, A. S. (2003). Mechanical and barrier properties of edible starcheprotein-based films. Journal of Applied Polymer Science, 88(1), 64e71. Liao, L.-s., Liu, H.-s., Liu, X.-x., Chen, L., Yu, L., & Chen, P. (2014). Development of microstructures and phase transitions of starch. Acta Polymerica Sinica, 2014(6), 761e773. Liu, X., Wang, Y., Yu, L., Zhang, N., Shanks, R. A., Liu, H., et al. (2013). Morphologies and phase composition of gelatinestarch blends. Chinese Journal of Polymer Science, 32(1), 108e114. Ong, M. H., Whitehouse, A. S., Abeysekera, R., Al-Ruqaie, I. M., & Kasapis, S. (1998). Glass transition-related or crystalline forms in the structural properties of gelatin/oxidised starch/glucose syrup mixtures. Food Hydrocolloids, 12(3), 273e281. Sun, W., Zhao, Q., Zhao, M., Yang, B., Cui, C., & Ren, J. (2011). Structural evaluation of myofibrillar proteins during processing of cantonese sausage by Raman spectroscopy. Journal of Agricultural and Food Chemistry, 59(20), 11070e11077. Wetzel, D. L., Shi, Y.-C., & Reffner, J. A. (2010). Synchrotron infrared confocal microspectroscopical detection of heterogeneity within chemically modified single starch granules. Applied Spectroscopy, 64(3), 282e285. Zhang, L., Wang, Y., Liu, H., Zhang, N., Liu, X., Chen, L., et al. (2013). Development of capsules from natural plant polymers. Acta Polymerica Sinica, 0(1), 1e10. Zhang, N., Liu, H., Yu, L., Liu, X., Zhang, L., Chen, L., et al. (2013). Developing gelatinestarch blends for use as capsule materials. Carbohydrate Polymers, 92(1), 455e461. Zhang, N., Liu, X., Yu, L., Shanks, R., Petinaks, E., & Liu, H. (2013). Phase composition and interface of starchegelatin blends studied by synchrotron FTIR microspectroscopy. Carbohydrate Polymers, 95(20), 649e653.
Contents lists available at ScienceDirect
Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd
Imaging the phase of starchegelatin blends by confocal Raman microscopy Xingxun Liu a, *, Nuozi Zhang b, Long Yu c, Sumei Zhou a, Robert Shanks b, Jinkai Zheng a a
Institute of Food Science and Technology (IFST), Chinese Academy of Agricultural Sciences (CAAS), Beijing, 100193, China School of Applied Sciences, RMIT University, Melbourne 3000, Australia c School of Light Industry and Food Science, CPFRR, South China University of Technology, Guangzhou 510640, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 July 2015 Received in revised form 28 January 2016 Accepted 1 March 2016 Available online 9 March 2016
The well recognized complex issue of compatibility between starch and gelatin was studied based on their interfacial and phase composition using Raman microscopy. Blending films with different ratios of gelatin and starch was used in this work. Raman beam focused on a 1.7 mm 1.5 mm detection region and the micro-spectroscope scanned across the gelatinestarch interface. The ratio of areas of saccharide bonds (1173-953 cm1) and amide I bands (1750-1550 cm1) was used to monitor the relative distributions of the two components of the blends. The Raman spectral maps confirmed that for all the blends investigated, gelatin formed a continuous matrix in which starch inclusions were dispersed. Intermediate phases consisted of amylose interacted with gelatin, which demonstrated this was an unique and effective method to investigate starch based blend and composite materials. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Gelatin Starch Raman spectroscopy Interface Polymer compatibility
1. Introduction Blends of gelatin with starch have attracted much attention since they are both renewable resources and they have filmforming ability. A blended film of polysaccharide and protein exhibited improved gas barrier (O2 and CO2) properties than either of the pure films (Arvanitoyannis, Nakayama, & Aiba, 1998; Baldwin, Nisperos-Carriedo, & Baker, 1995). Blends have been developed for use as medical capsule materials (L. Zhang et al., 2013; N. Zhang, H. Liu et al., 2013; N. Zhang, X. Liu et al., 2013). Previous research has shown that gelatin and starch are immiscible, their morphologies and compatibility are determined by several characteristics, such as processing time (Firoozmand, Murray, & Dickinson, 2009), temperature (Ioannis Arvanitoyannis et al., 1998), pH (Ong, Whitehouse, Abeysekera, Al-Ruqaie, & Kasapis, 1998) and volume fraction (Al-Hassan & Norziah, 2012). Mechanical properties of starchegelatin blends depend on their morphology, particularly the extent of homogeneity and the composition of their continuous and dispersed phases. Various techniques have been used to characterise phase composition and
* Corresponding author. IFST, Chinese Academy of Agricultural Science (CAAS), Beijing. E-mail address: [email protected] (X. Liu). http://dx.doi.org/10.1016/j.foodhyd.2016.03.005 0268-005X/© 2016 Elsevier Ltd. All rights reserved.
interface of this complex blended system by indirect methods such as differential scanning calorimetry (DSC) (Jagannath, Nanjappa, DasGupta, & Bawa, 2003), dynamic mechanical analysis (DMA) (Arvanitoyannis, Psomiadou, Nakayama, Aiba, & Yamamoto, 1997) and rheometry, as well as direct methods such as polarized optical microscopy and confocal laser scanning microscope (CLSM) (Firoozmand et al., 2009). In our previous paper (Liu et al., 2013), we have compared different microscopies such as polarized optical microscopy, scanning electron microscopy (SEM) and synchrotronFourier transform infrared spectroscopy microscopy (SynchroFTIRM) to study gelatinestarch blends. Of these methods, the Synchro-FTIRM provided capability to detect and measure composition and interface. Spatial resolution of FTIRM was limited to 10 10 mm, even application of synchrotron light technology only enabled achievement of a spatial of 5 5 mm (Wetzel, Shi, & Reffner, 2010). Corn-starch is an economic crop that is often used for the raw materials for edible or biodegradable materials. The average diameter of corn starch granules is from 8 to 12 mm (Chen, Yu, Chen, & Li, 2006), so high spatial resolution is needed to examine morphing of the granules into film by gelatinization. Raman microscopy is an effective method to study heterogeneous materials since it provides sub-micron spatial resolution with high sensitivity (Chrimes, Khoshmanesh, Stoddart, Mitchell, & Kalantar-zadeh,
8
X. Liu et al. / Food Hydrocolloids 60 (2016) 7e10
Fig. 1. Raman optical images, spectra and maps of G3S7 for excitation at 532 nm, (A) the selection zone chosen from the optical images; (B) one spectra of gelatinestarch blends of collecting data; (C) map obtained from the ratios between integrated intensities in the 1173-953 cm1and 1750-1550 cm1 range; (D) overlaid images shows that the same area was mapped.
2. Experimental 2.1. Materials and film preparation Starchegelatin blend film was prepared according to previous methods (N. Zhang, H. Liu et al., 2013; N. Zhang, X. Liu et al., 2013). Briefly, solutions were prepared with different ratios of starch (A1081, Penford, Australia):gelatin (GELITA UG719-N, Type A, 250 bloom, Sweden) equal 90:10, 70:30, 50:50, 30:70 and 10:90 including 1%$w/w sorbitol based on a total weight basis (2 g) in 100 mL distilled water. The mixed materials were dissolved in distilled water at 80 C for an initial 30 min at a slow stirring speed (100 min1), then stirred for a further 30 min at high speed (700 min1) until a clear solution was obtained. The solution (5 mL) was poured onto a poly(ethylene terephthalate) (PET) dish (diameter 5 cm) that was kept level to control film thickness. The cast film was dried overnight at 37 C. The dry films were peeled from the plate, placed in a desiccator containing saturated sodium bromide (NaBr) solution to control humidity, and stored at 56%$RH and 23 C until required for analysis. 2.2. Raman microscopy An XploRA plus Raman confocal microscope system (Horiba scientific) was used to analyze specimen surfaces. A532 nm diode laser (15 mW laser power) with an X100/0.90NA air objective was employed. Spatial resolution was obtained using 100 mm confocal pinholes. The Raman signal was acquired using a 1200 lines/mm grating centered between 200 and 2760 cm1. For each specimen, a 1.7 1.5 mm area of the surface was mapped at X and Y-axes. Data was analyzed using LabSpec 6. The integration time was 10 s for all
measurements. 3. Results and discussion Different ratios of starch:gelatin (90:10, 70:30, 50:50, 30:70 and 10:90, named G9S1, G7S3, G5S5, G3S7, G1S9) including 1%$w/w sorbitol plasticizer, were used as model materials, a Raman confocal microscope system with a 532 nm laser was used to study the gelatinestarch films. Fig. 1 shows the approach to acquisition of spectra, data analysis (image generation) and processing of the Raman images, Fig. 1(A) shows the heterogeneous nature of such blends and their phase distribution. Gelatin was a continuous phase while starch formed a separated phase distributed as spherulites of about 5 mm diameter. It should be noted that the diameters of starch particles depend on volume fraction of starch and drying temperature. The distribution of starch was more diffuse and the interfacial contrast became less distinct depending on volume fraction and water content. Fig. 1(B) and Fig. 2 show the Raman spectra of gelatinestarch
20000 Starch
Intensity(counts)
2013). For example, lateral and depth resolutions of approximately 0.25 and 1.7 mm, respectively, can be achieved when using a 633 nm laser source and an aperture of 50 mm in radius to give a 60 /1.2 numerical aperture (NA) objective. Therefore, the aim in this study was to study morphology and phase composition of gelatinestarch blends using Raman microscopy.
15000
10000
G3S7
5000 Gelatin 0 1700
1300
900
Raman Shift (cm-1) Fig. 2. Raman spectra of gelatin and gelatinestarch blend (G3S7).
500
X. Liu et al. / Food Hydrocolloids 60 (2016) 7e10
9
Fig. 3. Raman optical images, maps and overlaid maps of different gelatinestarch ratios.
and gelatin. A typical spectral feature of the pure gelatin film was the strong amide I band located at approximately 1654 cm1 which usually indicate the alpha helix dominated structure. The amide I absorption is primarily due to the stretching vibration of the C]O bond (Sun et al., 2011), and it is related to the protein conformation, such as a-helix or b-sheet. Another characteristic peak around 1250 cm1 is the amide III involving CeN stretching and NeH inplane bending vibrations of the peptide bond as well as contributions from Ca-C stretching and C]O in-plane bending (Sun et al., 2011). The typical starch spectra in the range 1300e800 cm1
was found in the gelatinestarch film, the peak 1150 (shoulder peak), 1120 and 1087 cm1 is due to the CeO, CeC stretching and CeOH bending. Overlapping peaks located in the region of 1173953 cm1 (labelled as band 1), were the most intense bands in the Raman spectra and they were chosen as the characteristic peaks for starch; while the integrated area of the amide I band (1750e1550 cm1), labelled as Band 2, was used to represent gelatin. The ratio of the peak area of the starch bands (band 1) to that of gelatin bands (band 2) was used to represent the distribution of starch or gelatin in the blends. The ratio of band areas rather
10
X. Liu et al. / Food Hydrocolloids 60 (2016) 7e10
than absolute values was used to enable normalization of the results, correcting for possible variations in film thickness, both within the same film and between different films. Raman intensity mapping of the band 1/band 2 under 532 nm laser excitation in Fig. 1(C) shows a 2D contour map that was established for each of the blends with different starchegelatin content. Fig. 1(C) gives a higher special resolution to observe the phase distribution compared with FTIR microscopy, where special solution could reach 1.7 mm 1.5 mm. The color code represents concentration of a component. The red in the scale denotes a high starch concentration while blue denotes a high gelatin concentration. The Raman maps confirmed that starch was a dispersed phase while the gelatin phase formed a continuous matrix in which starch inclusions were dispersed. The results demonstrated the heterogeneous nature of such blends with starch domains dispersed into a gelatin continuous phase, even with higher starch content blends. Fig. 1(D) shows overlay data of Raman spectral microscopy -optical microscopy images. The overlaid images show that exactly the same area was mapped by Raman microscopy, however Raman microscopy revealed the chemical composition. When the images were overlaid, the resultant image gave a replicate result for the distribution of starch and gelatin. Fig. 3 shows a chemical map of starch distribution acquired from a CCD (charge-coupled device) detector for various gelatinestarch blends. The size of starch domains decreased with increasing gelatin volume fraction. Raman concentration maps suggested that for all mixtures investigated gelatin formed a continuous matrix in which starch inclusions were dispersed. The results demonstrated the disperse and diffuse nature of such blends, with starch domains dispersed into a gelatin continuous phase, even with higher starch volume fraction blends. However, the interfacial contrast became less distinct with increasing gelatin volume fraction. During the film-forming process, the starch experiences gelatinization and gelation; it is well known that over short times retrogradation of amylose occurs, while over long times amylopectin retrogrades. Furthermore, while some amylose will be subject to retrogradation to V-type crystals thus enhancing separation of starch as it becomes spherical (Liao et al., 2014), so amylose may interact more with gelatin. The Raman results support the observation that starch and gelatin are compatible though not miscible, furthermore, it also provide some new results, in particular the inter-phase. More work of enlarging the mapping area will be studied in future. Compared with gelatinestarch blending observed by FTIR microscopy, large spatial resolution (1.7 mm 1.5 mm) resulted with Raman microscopy. There are some intermediate phases around the starch phase that display a green color in the gelatinestarch blends. This phenomenon may be due to the amylose leached from the starch phase during gelatinization during processing, and interacting with gelatin; some sorbitol plasticizer would assist by stabilizing the interface. Intermediate phases were observed using Raman microscopy, which further support the observation that starch and gelatin are compatible though not miscible. 4. Conclusion Raman microscopy mapping has been shown to be an efficient and effective method to characterize the phase composition and distribution of gelatinestarch blends. The ratio of the areas of starch and gelatin bands was used to determine the relative distributions of the two components in the blends. The Raman maps confirmed that gelatin formed a continuous matrix in which starch inclusions were dispersed, for all the blends investigated.
Intermediate phases, due to amylose interaction with gelatin, were observed using Raman microcopy. The gelatinestarch blends formed a compatible system with gelatin matrix and starch dispersed phase, with each component contributing to properties for application in capsules requiring enhanced gas barrier and facile dissolution to release contents. Conflict of interest The authors declare that there is no conflict of interests regarding the publication of this paper. Acknowledgments The authors from China would like to acknowledge the research funds NFSC (31301554, 21174043) and the Fundamental Research Funds for Chinese Academy of Agricultural Sciences (2015ZL048). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.foodhyd.2016.03.005. References Al-Hassan, A. A., & Norziah, M. H. (2012). Starchegelatin edible films: water vapor permeability and mechanical properties as affected by plasticizers. Food Hydrocolloids, 26(1), 108e117. Arvanitoyannis, I., Nakayama, A., & Aiba, S.-I. (1998). Edible films made from hydroxypropyl starch and gelatin and plasticized by polyols and water. Carbohydrate Polymers, 36(2e3), 105e119. Arvanitoyannis, I., Psomiadou, E., Nakayama, A., Aiba, S., & Yamamoto, N. (1997). Edible films made from gelatin, soluble starch and polyols, Part 3. Food Chemistry, 60(4), 593e604. Baldwin, E. A., Nisperos-Carriedo, M. O., & Baker, R. A. (1995). Use of edible coatings to preserve quality of lightly (and slightly) processed products. Critical Reviews in Food Science and Nutrition, 35(6), 509e524. Chen, P., Yu, L., Chen, L., & Li, X. X. (2006). Morphology and microstructure of maize starches with different amylose/amylopectin content. Starch-Starke, 58(12), 611e615. Chrimes, A. F., Khoshmanesh, K., Stoddart, P. R., Mitchell, A., & Kalantar-Zadeh, K. (2013). Microfluidics and Raman microscopy: current applications and future challenges. Chemical Society Reviews, 42(13), 5880e5906. Firoozmand, H., Murray, B. S., & Dickinson, E. (2009). Microstructure and rheology of phase-separated gels of gelatin þoxidized starch. Food Hydrocolloids, 23(4), 1081e1088. Jagannath, J. H., Nanjappa, C., Das Gupta, D. K., & Bawa, A. S. (2003). Mechanical and barrier properties of edible starcheprotein-based films. Journal of Applied Polymer Science, 88(1), 64e71. Liao, L.-s., Liu, H.-s., Liu, X.-x., Chen, L., Yu, L., & Chen, P. (2014). Development of microstructures and phase transitions of starch. Acta Polymerica Sinica, 2014(6), 761e773. Liu, X., Wang, Y., Yu, L., Zhang, N., Shanks, R. A., Liu, H., et al. (2013). Morphologies and phase composition of gelatinestarch blends. Chinese Journal of Polymer Science, 32(1), 108e114. Ong, M. H., Whitehouse, A. S., Abeysekera, R., Al-Ruqaie, I. M., & Kasapis, S. (1998). Glass transition-related or crystalline forms in the structural properties of gelatin/oxidised starch/glucose syrup mixtures. Food Hydrocolloids, 12(3), 273e281. Sun, W., Zhao, Q., Zhao, M., Yang, B., Cui, C., & Ren, J. (2011). Structural evaluation of myofibrillar proteins during processing of cantonese sausage by Raman spectroscopy. Journal of Agricultural and Food Chemistry, 59(20), 11070e11077. Wetzel, D. L., Shi, Y.-C., & Reffner, J. A. (2010). Synchrotron infrared confocal microspectroscopical detection of heterogeneity within chemically modified single starch granules. Applied Spectroscopy, 64(3), 282e285. Zhang, L., Wang, Y., Liu, H., Zhang, N., Liu, X., Chen, L., et al. (2013). Development of capsules from natural plant polymers. Acta Polymerica Sinica, 0(1), 1e10. Zhang, N., Liu, H., Yu, L., Liu, X., Zhang, L., Chen, L., et al. (2013). Developing gelatinestarch blends for use as capsule materials. Carbohydrate Polymers, 92(1), 455e461. Zhang, N., Liu, X., Yu, L., Shanks, R., Petinaks, E., & Liu, H. (2013). Phase composition and interface of starchegelatin blends studied by synchrotron FTIR microspectroscopy. Carbohydrate Polymers, 95(20), 649e653.