Morphologies and gelatinization behaviours of high-amylose maize starches during heat treatment

Carbohydrate Polymers 157 (2017) 637–642

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Morphologies and gelatinization behaviours of high-amylose maize starches during heat treatment Xu Chen a,1 , Xianfeng Du a,c,∗ , Peirong Chen b,∗ , Li Guo a , Yang Xu c , Xiuhong Zhou c a b c

School of Tea & Food Science and Technology, Anhui Agricultural University, Hefei 230036, China Department of Applied Chemistry, School of Science, Anhui Agricultural University, Hefei 230036, China Biotechnology Center of Anhui Agricultural University, Anhui Agricultural University, Hefei 230036, China

a r t i c l e

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Article history: Received 4 July 2016 Received in revised form 3 October 2016 Accepted 9 October 2016 Available online 13 October 2016 Keywords: High-amylose maize starches Gelatinization behaviours Heating treatment Maltese crosses

a b s t r a c t The granule morphologies and gelatinization behaviours of high-amylose maize starches during heating treatment were investigated by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Maltese crosses demonstrated that the high-amylose maize starches maintained a granular structure even at 120 ◦ C. The granules of high-amylose maize starches swelled slightly at 100 ◦ C and swelled remarkably at approximately 120 ◦ C. The destruction of the starch structure began at the centre and expanded rapidly to the periphery. The intense fluorescence of high-amylose maize starch granules gradually became feeble, and the darker region spread outward during heating at 130 ◦ C for 30 min, indicating that the amylose component may have been damaged and shifted. The starch granules treated at 140 ◦ C were substantially destroyed, and the CLSM, normal light microscopy (NL) and SEM images displayed no discernible granules, which indicated that the original starch granules formed a continuous integrated matrix. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction Starch is stored as discrete semi-crystalline granules and consists of two main biopolymers: linear amylose (20–30%) and highly branched amylopectin (70–80%) (Zhou et al., 2015). The amylose content and amylopectin structure of starch strongly influence its physicochemical properties and applications (Chung, Jeong, & Lim, 2003; Lin et al., 2016). Starch with a high amylose content is widely applied in the areas of support films, foods, paper making, medical treatments, and electronic chips because it has a high level of resistance to gelatinization and hydrolysis (Lin et al., 2016; Tan et al., 2015). Furthermore, high-amylose starch is especially suitable for producing thermoplastic materials because amylose can easily form crystallites and entanglements (Koch et al., 2010). However, high-amylose maize starches’ resistance to processing or treatment creates a substantial problem when they are used as raw materials because complete destruction of the original starch supramolecular struc-

∗ Corresponding authors at: NO.130, Western Changjiang Road, Anhui Agricultural University, Hefei City, Anhui Province 230036, China. E-mail addresses: [email protected] (X. Du), [email protected] (P. Chen). 1 Present address: NO. 130, Western Changjiang Road, Anhui Agricultural University, Hefei City, Anhui Province 230036, China. http://dx.doi.org/10.1016/j.carbpol.2016.10.024 0144-8617/© 2016 Elsevier Ltd. All rights reserved.

tures is required to form a continuous integrated phase (Yang et al., 2016a, 2016b). That is, because of its compact granule structure, native high-amylose starch exhibits strong resistance to being hydrolysed or disintegrated by small molecules such as water and enzymes, which limits its applications. For normal starches with excess water, the gelatinization endotherm can usually be observed during differential scanning calorimetry (DSC) temperature scanning in the low temperature range (54–73 ◦ C) (Liu et al., 2011). High-amylose maize starches are well known to exhibit higher gelatinization temperatures, a wider gelatinization range and lower gelatinization enthalpy than normal starches (Qiu et al., 2016). However, although much work has been reported on the thermal properties of various starches, few reports have addressed the gelatinization behaviours of highamylose maize starches under temperatures greater than 100 ◦ C (Cai et al., 2014a, 2014b; Mira, Villwock, & Persson, 2007). In particular, Chen et al. (2011) studied the internal structures and phase transitions of high-amylose starches below the gelatinization temperature, which was instructive in the exploration of the gelatinization mechanism to some extent (Chen et al., 2011). In this paper, the morphology, structure and gelatinization characteristics of high-amylose maize starches were investigated, with the objective of elucidating the granule morphologies and gelatinization behaviours of high-amylose maize starches during

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heating treatment, especially in the temperature range from 100 ◦ C to 140 ◦ C. 2. Materials and methods 2.1. Materials High-amylose maize starches with amylose contents of 70% were kindly supplied by the National Starch and Chemical Co. (Bridgewater, NJ, USA). HPLC-grade fluorescein 5-isothiocyanate (FITC) was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). 2.2. Starch paste preparation To achieve temperatures greater than 100 ◦ C, an autoclave was used. Starch paste was prepared for all the experiments, as previously described by Savary, Handschin, Conde-Petit, Cayot, and Doublier (2008) with some modifications. Four hundred grams of 8.0% (w/w) starch suspensions were stirred at a paddle speed of 300 rpm. The starch suspension was heated from room temperature to a preset temperature (100 ◦ C, 110 ◦ C, 120 ◦ C, 130 ◦ C or 140 ◦ C) at a constant rate of 2 ◦ C/min. The suspension was maintained at the preset temperature for 30 min, after this duration, the starch paste was decreased to 100 ◦ C. Subsequently, the starch paste was poured into a beaker immediately with a water bath maintained at 80 ◦ C. 2.3. Confocal laser scanning microscopy and light microscopy An Olympus FV10 (Tokyo, Japan) confocal laser scanning microscope equipped with an inverted microscope was used for observing the changing trends of starch granules during gelatinization. A stock solution of fluorescein 5-isothiocyanate (FITC) was prepared by dissolving 0.2 g of FITC in 100 mL of distilled water. Starch paste (100 ␮L) was stained by mixing with 20 ␮L of FITC stock solution (Qiu et al., 2016; Zhou et al., 2015). A drop of stained starch paste (approximately 10 ␮L) was deposited onto a concave slide, cooled to room temperature and observed within 15 min. The excitation wavelength was 488 nm, and the emission maxima were within 500–525 nm (Nagano, Tamaki, & Funami, 2008; Zhou et al., 2014). Each line was scanned four times and averaged to reduce noise. Light micrographs were obtained using an Olympus FV10 confocal scanning laser microscope (Tokyo, Japan) with normal light microscopy and polarized light microscopy to investigate the morphology of the unstained paste. A drop of starch paste was placed on the microscope slide with a glass coverslip. The specimen was viewed first under normal light microscopy. The same field was then viewed under polarized light microscopy. The observations were carried out in triplicate. The diameters of starch granules were measured and analyzed using Nano Measurer1.2.0 statistically software (Fudan Univ., Shanghai, China). 2.4. Scanning electron microscopy (SEM) The starch paste was stored in an electro- thermostatic blast oven at 25 ◦ C to be dried into a ‘solid state’, followed by drying at 105 ◦ C for 10 h. Evaporating the water slowly and slightly was key to ensuring that the samples had nearly identical moisture contents and to minimizing the influence of water loss on the intrinsic structure of the starch paste. The dried starch paste was affixed to a specimen holder using an aluminium plate, and was subsequently coating it with gold in a vacuum evaporator. The cross-sections were then observed by SEM on an electron microscope (S-4800, Hitachi, Japan) operated at an accelerating voltage of 3.0 kV.

2.5. Differential scanning calorimetry A PerkinElmer DSC Diamond-8000 equipped with a refrigerated cooling system was used to conduct DSC experiments, and nitrogen was used as the purge gas. High-pressure stainless steel pans (PE No. JYL0073) with a heat-resistant rubber ring were used because of the higher temperatures required. High-amylose maize starches and distilled water were mixed at the specific percentage of 8.0% (w/w) in a glass vial, then sealed and stored for 24 h for the water to reach equilibrium. Thereafter, the components (approximately 8 mg) were transferred to the stainless steel pan using a syringe. After the pan was sealed, it was frequently shaken to ensure that the starch granules were well immersed. The mixture was equilibrated at room temperature for 2 h before measurements. The DSC samples were heated from 40 ◦ C to 200 ◦ C at a rate of 10 ◦ C/min. 2.6. X-ray diffraction (XRD) X-ray analytical instrumentation (TTR-III, Rigaku, Japan) was used for detecting the crystalline structure of high-amylose maize starches and starch paste. The starch pastes were firstly dried at 25 ◦ C for almost two days; they were then milled into powder before absolute drying. The powder of the starch paste was sieved through 200 mesh and exposed to the X-ray beam at 200 mA and 40 kV. The scanning region of the diffraction angle (2) was from 3◦ to 50◦ with a step size of 0.02◦ . 3. Results and discussion 3.1. Morphological structure of starches The native high-amylose maize starch granules with typical Maltese crosses under polarized light microscopy are shown in Fig. 1A; granular birefringence of the small granules appeared somewhat weaker than that of the large granules when compared under the same background. The large granules with bright Maltese crosses disappeared when the samples were heated to 100 ◦ C, indicating that large granules exhibited less resistance to being gelatinized. This higher resistance stems from the amylose double helices in the small granules requiring a higher temperature and energy input to dissociate completely than the shorter double helices in the large granules (Cai et al., 2014a, 2014b; Lin et al., 2016; Naguleswaran, Vasanthan, Hoover, & Bressler, 2016). Despite the starch granules being remarkably gelatinized, a few Maltese crosses were still maintained, especially in the small granules, after the loss of birefringence when the granules were heated to 120 ◦ C. The starch granules tended to be smaller when the temperature was further increased to 130 ◦ C, where Maltese crosses could barely be discerned in the field of polarized light microscopy. The aforementioned phenomena reveal that high-amylose maize starches require temperatures greater than 130 ◦ C to be completely gelatinized (Ocloo, Minnaar, & Emmambux, 2016; Lin et al., 2016). Of particular interest, partial fragmentation with polarized light occurred via the rupture of high-amylose maize starches during gelatinization. As the temperature was increased, high-amylose maize starch granules were gradually broken into small fragments whose surface and internal structures were effectively destroyed, accompanied by the absence of Maltese crosses. Notably, the partial fragmentation that resulted from the fusion of high-amylose maize starch granules created particles that were highly bright, which may result from re-aggregation of small fragments. CLSM was used to study the effects of heating treatment on granule morphologies and gelatinization behaviours of high-amylose maize starches (Fig. 2). Native high-amylose maize starches showed greater fluorescence intensity in the centre of the granules. The

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Fig. 1. Polarized light microscopy images of high-amylose maize starches after heating to specific temperatures and native high-amylose maize starches: (A) Native starch; (B)-(F) 100 ◦ C, 110 ◦ C, 120 ◦ C, 130 ◦ C, and 140 ◦ C, respectively.

authors of previous studies used 8-amino-1, 3, 6-pyrenetrisulfonic acid (APTS) to label starch molecules, demonstrating that a higher concentration of amylose molecules was distributed in the hilum region (Chen et al., 2011; Naguleswaran et al., 2013). Chen et al. (2011) proposed that, because amylose is a much smaller molecule and contains a much higher molar ratio of reducing ends per anhydrous glucose residue than the amylopectin molecules, more extensive labelling of amylose by weight occurred (Chen et al., 2011). After the high-amylose maize starches were treated at 100 ◦ C, a bright fluorescence ring emerged and intense fluorescence in the interior of the granules disappeared, indicating that high concentrations of amylose and/or amylopectin were densely packed at the periphery of the granules. This phenomenon suggests that the central area of the granule was the most incompact region because this region was disrupted originally (Chen et al., 2009; Yang et al., 2016a, 2016b; Zhao et al., 2015). Similarly, at an elevated temperature, the morphology of the starch granules stabilized as a result of the integrity of swollen granules and the lipid-complexed amylose chains restricting both granular swelling and amylose leaching (BeMiller & Whistler 2009; Qiu et al., 2016; Zhao et al., 2015; Zhou et al., 2014). However, the intense fluorescence became feeble gradually and the darker region spread outward after the samples were heated at 130 ◦ C for 30 min, implying that the amylose component may have been damaged and gradually removed. The consistent changes of high-amylose maize starches during heating was observed using normal light microscopy, under which the starch granules were observed to swell slightly at 100 ◦ C and to swell remarkably at approximately 120 ◦ C. The phenomena were supported by the statistics analysis, the mean diameter of starch granules in 2A2 -E2 were 9.0, 9.9, 10.7, 13.5, 11.3 ␮m, respectively. A significant increase in mean diameter of starch granules when high-amylose maize starches were heated to 120 ◦ C, which was consistent with starch granules swelled remarkably. Finally, after treatment at 140 ◦ C, no distinct granules were observed, whereas

a homogeneous structure was clearly observed under normal light microscopy. SEM images of native high-amylose maize starch granules display substantially heterogeneous shapes consisting of spherical granules, irregular granules and elongated granules with smooth surfaces. The hollowed granules observed for large granules after heat treatments at 100 ◦ C, 110 ◦ C, and 120 ◦ C for 30 min may have resulted from inner-structure collapse; no pitting was observed on the surface of small granules. This phenomenon is attributed to the small granules of high-amylose starch having substantially higher contents of amylose and amylopectin long branch-chains, lower contents of amylopectin short branch-chains and smaller branching degrees than the large granules. Combined with the normal light microscopy images, the SEM images of the starch paste clearly show that the mean diameter of starch granules decreased with increasing temperature; these results seemingly contradict the observations under normal light microscopy. The reason for this discrepancy could be the different methods used to characterize the starch granules. In addition, the large granules gelatinized into gels, accompanied by the loss of the granules’ shapes, which led to them being not easily observed by SEM. No granules were observed in the starch paste treated at 140 ◦ C, in good agreement with the CLSM observations, which revealed greater destruction of the granules of high-amylose maize starches. On the basis of the aforementioned phenomena, the large granules exhibited less resistance to being gelatinized, possibly because large granules have lower amylose contents, which lead to a weak structure that is readily hydrolysed. The destruction of the starch structure begins at the centre and expands to the periphery rapidly. The change in granular morphology indicates that the heating treatment triggered substantial swelling or a gelatinization process. This process was accompanied by a series of variations, including the dissociation of amylopectin double helices and the amylose component leaching out. The starch granules treated at 140 ◦ C were substantially destroyed, and the images displayed no discernible

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Fig. 2. CLSM and NL and SEM images of high-amylose maize starches after heating to specific temperatures and native high-amylose starch: (A) Native starch; (B)-(F) 100 ◦ C, 110 ◦ C, 120 ◦ C, 130 ◦ C, 140 ◦ C, respectively.

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Temperature ( C) Fig. 3. DSC thermogram of native high-amylose maize starches. Fig. 4. X-ray diffractograms of high-amylose maize starches treated at different temperatures from 100 ◦ C to 140 ◦ C and native high-amylose maize starch (NS).

granules in the observation fields of CLSM, normal light microscopy, or SEM, indicating that the original starch granules formed a continuous integrated matrix.

3.2. Thermal properties of starches The transition temperatures corresponding to the onset (To ), peak (Tp ), and final gelatinization (Tc ), as determined by DSC, are presented in Fig. 3. Gelatinization of the native high-amylose maize starches started began at 115.3 ◦ C (To ), peaked at 125.7 ◦ C (Tp ), and completed at 130.7 ◦ C (Tc ), effectively confirming the previous microscopic observations that the starch granules swelled remarkably at approximately 120 ◦ C and broke down at 130 ◦ C. Fig. 3 clearly demonstrates that the unwinding temperatures of amylopectin double helices, lipid-complexed amylose and amylose double helices were approximately 115–130 ◦ C for this starch. According to the previous literatures, a broad endotherm of the gelatinization temperature for the high amylose starch (70%) was obtained in the temperature range between 65 and 125 ◦ C (Colonna & Mercier 1985; Haralampu, 2000). The reported results are incompletely consistent with our study because of the complexity of thermal behaviours of starches, differing starch sources and water content and measurement conditions (Liu, Yu, Xie, & Chen, 2006).

4. Conclusion The gelatinization behaviours of high-amylose maize starch granules exhibited slight swelling at 100 ◦ C and remarkable swelling at approximately 120 ◦ C. A few Maltese crosses were still observed after treatment at 120 ◦ C, especially in small granules, indicating that the high-amylose maize starch granules exhibited high resistance to gelatinization. Our results demonstrate that the destruction of the starch structure began at the centre of the granule and expanded rapidly to the periphery. The intense fluorescence of high-amylose maize starch granules gradually became feeble, and the darker region spread outward after the starch was heated at 130 ◦ C for 30 min, implying that the amylose component may have been damaged and removed. The starch granules treated at 140 ◦ C were substantially destroyed, and the images obtained by various microscopic techniques displayed no discernible granules in the field of view, which indicated that the original starch granules formed a continuous integrated matrix. Acknowledgement The authors would like to thank the National Natural Science Foundation of China (grant no. 31471700 and 31371735) for financial support.

3.3. Crystalline structures of starches Fig. 4 shows the XRD curves of native starch and high-amylose maize starches heated at different temperatures from 100 ◦ C to 140 ◦ C. Native high-amylose maize starches displayed similar evolutions of the XRD patterns compared with the other specimens. The XRD patterns of the native high-amylose maize starches exhibited the strongest diffraction peaks at a 2 angle of approximately 17.3◦ , with a few smaller peaks at 2 angles of approximately 20.0◦ and 22.0◦ . After the samples were treated at different temperatures for 0.5 h, the intensities of the peaks at 17.3◦ slightly decreased. In particular, the peak became flat at 140 ◦ C, indicating breakdown of the well-organized crystalline lamellae or a phase transition from an ordered granular structure into a disordered state. Combined with the results in Fig. 1, Fig. 2 and Fig. 3, the depression of the diffraction peaks suggest that the heating treatment disrupted the granule inner region. The crystalline structure was detected after gelatinization at 140 ◦ C, which we attributed to the conformational ordering of amylose solubilized within the continuous matrix (Huang et al., 2015; Wang et al., 2014).

References BeMiller, J. N., & Whistler, R. L. (2009). Starch-chemistry and technology (3rd ed.). New York: Academic Press. Cai, C. H., Lin, L. S., Man, J. M., Zhao, L. X., Wang, Z. F., & Wei, C. X. (2014). Different structural properties of high-amylose maize starch fractions varying in granule size. Journal of Agricultural and Food Chemistry, 62, 11711–11721. Cai, C. H., Zhao, L. X., Huang, J., Chen, Y. F., & Wei, C. X. (2014). Morphology: Structure and gelatinization properties of heterogeneous starch granules from high-amylose maize. Carbohydrate Polymers, 102, 606–614. Chen, P., Yu, L., Simon, G. P., Petinakis, E., Dean, K., & Chen, L. (2009). Morphologies and microstructures of corn starches with different amylase/amylopectin ratios studied by confocal laser scanning microscope. Journal of Cereal Science, 50, 241–247. Chen, P., Yu, L., Simon, G. P., Liu, X., Dean, K., & Chen, L. (2011). Internal structures and phase-transitions of starch granules during gelatinization. Carbohydrate Polymers, 83, 1975–1983. Chung, H. J., Jeong, H. Y., & Lim, S. T. (2003). Effects of hydrolysis and defatting on crystallinity and pasting properties of freeze-thawed high amylose corn starch. Carbohydrate Polymers, 54, 449–455. Colonna, P., & Mercier, C. (1985). Gelatinization and melting of maize normal and high-amylose and pea starches with genotypes. Phytochemistry, 24, 1667–1674. Haralampu, S. G. (2000). Resistant starch—a review of the physical properties and biological impact of RS3. Carbohydrate Polymers, 41, 285–292.

642

X. Chen et al. / Carbohydrate Polymers 157 (2017) 637–642

Huang, J., Shang, Z. Q., Man, J. M., Liu, Q. Q., Zhu, C. J., & Wei, C. X. (2015). Comparison of molecular structures and functional properties of high-amylose starches from rice transgenic line and commercial maize. Food Hydrocolloids, 46, 172–179. Koch, K., Gillgren, T., Stading, M., & Andersson, R. (2010). Mechanical and structure properties of solution-cast high-amylose maize starch films. International Journal of Biological Macromolecules, 46, 13–19. Lin, L. S., Guo, D. W., Zhao, L. X., Zhang, X. D., Wang, J., Zhang, F. M., et al. (2016). Comparative structure of starches from high-amylose maize inbred lines and their hybrids. Food Hydrocolloids, 52, 19–28. Liu, H. S., Yu, L., Xie, F. W., & Chen, L. (2006). Gelatinization of cornstarch with different amylose/amylopectin content. Carbohydrate Polymers, 65, 357–363. Liu, P., Xie, F., Li, M., Liu, X., Yu, L., Halley, P. J., et al. (2011). Phase transitions of maize starches with different amylose contents in glycerol-water systems. Carbohydrate Polymers, 85, 180–187. Mira, I., Villwock, V. K., & Persson, K. (2007). On the effect of surface active agents and their structure on the temperature-induced changes of normal and waxy wheat starch in aqueous suspension. Part II: A confocal laser scanning microscopy study. Carbohydrate Polymers, 68, 637–646. Nagano, T., Tamaki, E., & Funami, T. (2008). Influence of guar gum on granule morphologies and rheological properties of maize starch. Carbohydrate Polymers, 72, 95–101. Naguleswaran, S., Vasanthan, T., Hoover, P., & Bressler, D. (2013). The susceptibility of large and small granules of waxy, normal and high-amylose genotypes of barley and corn starches toward amylolysis at sub-gelatinization temperatures. Food Research International, 51, 771–782. Ocloo, F. C. K., Minnaar, A., & Emmambux, N. M. (2016). Effects of stearic acid and gamma irradiation, alone and in combination: On pasting properties of high amylose maize starch. Food Chemistry, 190, 12–19. Qiu, S., Yadav, M. P., Liu, Y., Chen, H., Tatsumi, E., & Yin, L. J. (2016). Effects of corn fiber gum with different molecular weights on the gelatinization behaviours of corn and wheat starch. Food Hydrocolloids, 53, 180–186.

Savary, G., Handschin, S., Conde-Petit, B., Cayot, N., & Doublier, J. L. (2008). Structure of polysaccharide-starch composite gels by rheology and confocal laser scanning microscopy: Effect of the composition and of the preparation procedure. Food Hydrocolloids, 22, 520–530. Tan, X. Y., Zhang, B. J., Chen, L., Li, X. X., Li, L., & Xie, F. W. (2015). Effect of planetary ball-milling on multi-scale structures and pasting properties of waxy and high-amylose cornstarches. Innovative Food Science and Emerging Technologies, 30, 198–207. Wang, S. J., Wang, J. R., Yu, J. L., & Wang, S. (2014). A comparative study of annealing of waxy: Normal and high-amylose maize starches: The role of amylose molecules. Food Chemistry, 164, 332–338. Yang, J. N., Xie, F. W., Wen, W. Q., Chen, L., Shang, X. Q., & Liu, P. (2016). Understanding the structural features of high-amylose maize starch through hydrothermal treatment. International Journal of Biological Macromolecules, 84, 268–274. Yang, Z., Swedlund, P., Hemar, Y., Mo, G., Wei, Y., Li, Z., & Wu, Z. (2016). Effect of high hydrostatic pressure on the supramolecular of corn starch with different amylose contents. International Journal of Biological Macromolecules, 85, 604–614. Zhao, D. D., Palaparthi, A. D., Huang, Q. R., Fu, X., Liu, H. S., & Yu, L. (2015). Effects of ionic liquid 1-allyl-3-methylimidazolium chloride treatment on the microstructure and phase transition of cornstarch. Industrial Crops and Products, 77, 139–145. Zhou, Y., Winkworth-Smith, C. G., Wang, Y., Liang, J. F., Foster, T. J., & Cheng, Y. Q. (2014). Effect of a small amount of sodium carbonate on konjac glucomannan-induced changes in thermal behavior of wheat starch. Carbohydrate Polymers, 114, 357–364. Zhou, Y., Zhao, D., Winkworth-Smith, C. G., Foster, T. J., Nirasawa, S., Tatsumi, E., et al. (2015). Effect of a small amount of sodium carbonate on konjac glucomannan-induced changes in wheat starch gel. Carbohydrate Polymers, 116, 182–188.