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Effect of fatty acid addition on properties of amylose nanoparticles prepared via complexing and precipitation
Industrial Crops & Products 145 (2020) 112097
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Effect of fatty acid addition on properties of amylose nanoparticles prepared via complexing and precipitation
T
Xiaoxia Yan, Lvheng Kou, Hongyuan Wei, Lili Ren, Jiang Zhou* Key Laboratory of Bionic Engineering (Ministry of Education), College of Biological and Agricultural Engineering, Jilin University, Changchun 130022, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Amylose Fatty acids Complexing Precipitation Nanoparticles
Amylose nanoparticles were prepared by complexing with fatty acids (palmitic acid, lauric acid and n-caproic acid) and then ethanol precipitation. The effects of the fatty acid addition and fatty acid carbon chain length on the size, crystallinity, morphology and fatty acid content of the complex nanoparticles were studied. Analyses of FT-IR, complexing index and 13C CP/MAS NMR suggested that amylose complexed with the fatty acids. The determined complexing indexes were 7.8 %, 36.2 % and 44.5 % for the n-caproic acid, lauric acid, and palmitic acid at the 10 % addition. The complexing index increased from 37.1%–44.7% for the palmitic acid when the addition was increased from 5 % to 20 %. XRD results showed that the complex nanoparticles possessed V-type crystalline structure, but the crystallinity decreased from 54.22%–36.38 % when the palmitic acid addition changed from 0 to 20 %. At the 10 % addition, the crystallinity decreased from 51.07%–42.15 % as fatty acid chain length increased from 6 carbon atoms to 16 carbon atoms. The result of 13C CP/MAS NMR analysis indicated that the fatty acid content in the complex nanoparticles increased with increasing of fatty acid chain length at 10 % addition. The findings of this study provide a guideline to prepare nanoparticles of amylose-fatty acid complexes with desired properties.
1. Introduction Starch nanoparticles have drawn much attention due to their physiochemical properties, renewability from natural resources and potential applications in several fields. Nanoprecipitation is a simple and fast method to produce starch nanoparticles. Amylose (a linear molecule of glucose units linked by (1–4) α-D-glycoside bonds) and amylopectin are the two main components in starch. Amylose nanoparticles prepared via ethanol precipitation possessed sizes around 200 nm and the V-type crystalline structure, and it was believed that the formation of this V-type crystalline structure was due to associations of amyloseethanol single helices (complexed during precipitation) in the drying stage (Yan et al., 2017, 2018). It is well known that amylose has a unique feature to form complexes with some ligands, such as alcohols and lipids, especially fatty acids (Godet et al., 1995; Helbert and Chanzy, 1994; Karkalas et al., 1995; Le Bail et al., 2005; Lebail et al., 2000). The formation of amylose complexes makes the conformation change of amylose into a single, left-handed helix with a hydrophobic cavity which can entrap the ligands (Lesmes et al., 2009; Putseys et al., 2010). The helix diameter and dimensions are dependent on the size of complexing agent (Gelders et al., 2004; Putseys et al., 2010). For the amylose-fatty acid complexes, ⁎
it is generally believed that sections of amylose chains with at least 18 glucose units form left-handed helices with three turns (at least 6 glucose units per helix turn) and the fatty acid locates in the helices with the polar head group outside the cavity (Lesmes et al., 2009; Putseys et al., 2010; Seo et al., 2015). These complex segments are interrupted by the section of uncomplexed amylose that permit arrangement of the complex helices and formation of the V-type crystalline structure with type I and type II polymorphs (Lalush et al., 2005; Lesmes et al., 2009). Chain length and unsaturation degree of fatty acids as well as amylose chain length affect the complexation degree (Oyeyinka et al., 2016; Putseys et al., 2010). The longer the fatty acid or the amylose chains, the more heat-stable the formed complexes (Cao et al., 2015; Putseys et al., 2010). Amylose complexes have many important functions, such as, inhibit retrogradation of products containing amylose, preserve and deliver functional substances (Lesmes et al., 2009; Putseys et al., 2010). Nano-sized starch particles were prepared by complex formation with n-butanol (Kim and Lim, 2009), the size of the obtained starch particles was 10–20 nm, but the yield was low and the production process took 7 days. Fanta et al. (2015) reported nanoparticle formation from amylose-oleic acid complexes by steam jet cooking, the obtained nanoparticles had the diameters of 63−375 nm and showed V-type crystalline structure.
Corresponding author. E-mail address: [email protected] (J. Zhou).
https://doi.org/10.1016/j.indcrop.2020.112097 Received 8 October 2019; Received in revised form 22 November 2019; Accepted 4 January 2020 0926-6690/ © 2020 Elsevier B.V. All rights reserved.
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with 25 mL distilled water at 50℃ in a 50 mL capped tube and vortexed for 2 min. Then, 500 μL of the resulting solution was mixed with 15 mL distilled water, followed by the addition of 2 mL iodine solution. The absorbance at 690 nm was measured by using a UV spectrophotometer (Ruili Analytical Instrument Company, Beijing, China). Amylose paste without adding fatty acid was used as a reference, the measurement was carried out three times for each sample and mean values were reported. Complexing index was calculated using the following equation:
As amylose can complex with fatty acids as well as ethanol and form single helices, it is interested in to investigate the effects of fatty acid addition on amylose nanoparticles formation via ethanol precipitation, i.e., how the complexation with fatty acids influences the ethanol precipitation of amylose. In this study, amylose and saturated fatty acids were used to form amylose-fatty acid complexes first, then the nanoparticles of amylose-fatty acid complexes were obtained via ethanol precipitation. The main objective of this study was to investigate the effects of fatty acid addition and the carbon chain length of fatty acid on the properties of the complex nanoparticles, including size, crystallinity, morphology and fatty acid content.
CI =
(AbsRe ference − AbsAmylose − fattyacid ) AbsRe ference
× 100
(1)
Where AbsReference is the absorbance of the amylose paste without adding fatty acid, and AbsAmylose-fatty acid is the absorbance of the amylose paste containing fatty acid.
2. Materials and methods 2.1. Materials Amylose with a content of 99.5 % was obtained from Shanxi Tianwei Biological Production Co. Ltd. (Xi’an, China). The amylose is isolated from corn starch, its molar mass (determined by size-exclusion chromatography) ranges 2.4 × 106 - 4.5 × 108 g/mol and the viscosity average molecular weight (calculated from measured intrinsic viscosity) is 3.36 × 105 (Chang et al., 2017). Absolute ethanol, iodine and potassium iodide were purchased from Beijing Chemical Works (Beijing, China). Palmitic acid (C16), lauric acid (C12) and n-caproic acid (C6) were purchased from Heng Ye Jingxi Chemical Co. Ltd. (Gu’an, China), Tianjin Guangfu Jingxi Chemical Institute (Tianjin, China) and Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China), respectively. All the used chemicals were the grade of analytical reagent.
2.5. Size analysis of nanoparticles Z average-size (d. nm) and polydispersity index (PDI) of the nanoparticles of amylose-fatty acid complexes were measured by dynamic light scattering (DLS) using a Malvern Zetasizer Nano-ZS90 (Malvern Instruments Ltd., UK).The samples were prepared by dispersing the nanoparticles (without drying) in distilled water at a concentration of about 0.1 %. The measurement was carried out three times for each sample, and then the mean values were reported. 2.6. X-ray diffraction (XRD) analysis The crystalline structure of the nanoparticles of amylose-fatty acid complexes was analyzed using a XRD-6100 Shimadzu X-ray Diffractometer (Shimadzu Corporation, Japan) with Cu-Kα radiation (λ =1.542 Å) at a voltage of 40 kV and electric current of 30 mA. The XRD patterns of the nanoparticles of amylose-fatty acid complexes were recorded over the 2θ range of 4-35° at a speed of 2°/min.
2.2. Preparation of amylose-fatty acid complex nanoparticles Amylose suspension with a concentration of 3 % was prepared by mixing amylose and distilled water, and then heated at 100 °C in a water bath for 1 h with stirring. When temperature of the obtained amylose paste reached 90 °C, certain amount fatty acid dissolved in 5 mL hot ethanol (70 °C) was added into 50 mL amylose paste, the paste was continuously agitated with a magnetic stirrer at 600 rpm for 30 min. Next, ethanol (three times of the amylose paste in volume) was added dropwise into the amylose paste which was continuously agitated with a magnetic stirrer at 600 rpm. The resulting mixture was cooled to room temperature (25 °C) and centrifuged at 6000 rpm for 5 min. The obtained sediments, nanoparticles of amylose-fatty acid complexes, were rinsed twice with ethanol by centrifugation, dried under 11 % relative humidity at 4 °C for 12 h and then at 40 °C for another 12 h (using a small desiccator containing saturated lithium chloride aqueous solution and putting the desiccator in a refrigerator at 4 °C and then an oven at 40 °C), and sealed in plastic bags for further tests.
2.7. Morphological characteristics Morphology of the nanoparticles of amylose-fatty acid complexes was observed by a XL-30 ESEM FEG scanning electron microscopy (FEI company, USA). Dried complex nanoparticles were dispersed in ethanol by using ultrasonic agitation to obtain a 0.2 % suspension, and 3 μL of the suspension was dropped onto a silicon wafer and freeze-dried at -70 ℃. The samples were mounted on specimen stubs with carbon black tape and sputter-coated with gold before observation. 2.8. 13C CP/MAS (cross-polarization/magic angle spinning) NMR spectroscopy The solid-state 13C CP/MAS NMR spectra were recorded on a Bruker AVANCE III 400 WB spectrometer equipped with a 4 mm standard bore CP/MAS probe head whose X channel was tuned to 100.62 MHz for 13C and the other channel was tuned to 400.18 MHz for broad band 1H decoupling, using a magnetic field of 9.39 T at 297 K. The nanoparticles were packed in a ZrO2 rotor closed with Kel-F cap which was spun at 8 kHz rate. The measurements were conducted at a contact time of 2 ms. Total of 2000 scans were recorded with 6 s recycle delay for each sample. All 13C CP/MAS chemical shifts were referenced to the resonances of adamantane (C10H16) standard (δCH2 = 38.4).
2.3. Fourier transform infrared spectroscopy (FT-IR) analysis The FT-IR spectra of amylose-fatty acid complex nanoparticles were obtained using a FT-IR (IRAffinity-1 spectrophotometer, Shimadzu, Japan). The specimens were prepared by grinding the nanoparticles of amylose-fatty acid complexes (1∼2 mg) together with KBr (about 200 mg) and then pressed into a disc. The spectra were scanned in the range of 4000–400 cm−1. The resolution was 4 cm−1and the total number of scans was 32. 2.4. Complexing index (CI)
2.9. Statistical analysis The complexing index (CI) of amylose was measured by the method of Tang and Copeland (2007) with some modifications. Iodine solution was prepared by dissolving iodine (1.3 %) and potassium iodide (2.0 %) in distilled water. Amylose paste containing fatty acid (5 g) was mixed
Analysis of variance (one-way ANOVA) was used to compare differences of the samples. Comparisons were performed using Duncan’s Multiple Range Test (DMRT) in SPSS 23.0 software (IBM). 2
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Fig. 1. FT-IR spectra of fatty acids (A) and the complex nanoparticles of amylose-fatty acids with different chain lengths (B).
3. Results and discussion 3.1. FT-IR analysis The FT-IR spectra of lauric acid (C12), palmitic acid (C16) (n-caproic acid (C6) is liquid, FT-IR analysis was not carried out) and the amylose nanoparticles containing fatty acid are presented in Fig. 1. As shown in Fig. 1A, the characteristic absorption peak of carbonyl band (C]O) stretch for the fatty acids located at 1705 cm−1 (Boeriu et al., 2004). The absorbance at 2850 cm−1 is CHe stretch of aliphatic compounds (Lammers et al., 2009). It can be seen in Fig. 1B that, there was a peak at 1650 cm−1 in the spectrum of amylose nanoparticles which was assigned to water molecules absorbed in the amorphous region and the stretching vibration of the C]O band (amide I) (Dankar et al., 2018; Shi et al., 2018). This peak shifted to 1645 cm−1 after the fatty acids were added. Compared with the FT-IR spectrum of the fatty acids, the C]O absorption peak at 1705 cm-1 disappeared for the amylose nanoparticles containing fatty acid. Since the absorption peak of C]O at 1705 cm-1 was absent in the FT-IR spectra of the nanoparticles, it could be concluded that either the fatty acids were not present in the nanoparticles or they complexed with amylose rather than physically mixed. In order to futher confirm the formation of the amylose-fatty acid complex in the nanoparticles, analyses of complexing index (CI) and 13C CP/MAS NMR were performed in the subsequent studies (Sections 3.2 and 3.6).
Fig. 2. Complexing index (CI) for the complexes of amylose-fatty acids. Values are given as mean ± standard deviation. Different letters indicate significantly different (p < 0.05) when analyzed by Duncan’s Multiple Range Test.
formation, as they are too soluble in the aqueous environment so that they can not be entrapped properly in the hydrophobic helix cavity (Putseys et al., 2010). It is also believed that the low solubility of the longer chain fatty acids is one reason of complex formation (Tufvesson et al., 2003). Fatty acids with longer carbon chains are more hydrophobic and therefore more likely form single-helix structure complexes with amylose and stay in the hydrophobic helix cavity, which gives rise to the increase of CI.
3.2. CI of amylose-fatty acid complex The value of CI can be used to characterize complexing degree between amylose and fatty acids (Kaur and Singh, 2000; Wang et al., 2016). Fig. 2 shows the CIs for the complexes of amylose and the fatty acids. With increasing of the amount of palmitic acid addition, the CI increased, but when the addition of palmitic acid beyond 10 %, the CI tended to remain stable. This is understandable, because when the amount of palmitic acid was small, even the palmitic acid was completely complexed, the CI was still low. As the palmitic acid addition increased, more palmitic acid was involved in complexation, which gave rise to higher CI. However, when the palmitic acid addition reached 10 %, there were no enough available sections of amylose chains to form the complexes with palmitic acid. Therefore, further increasing palmitic acid addition would not increase CI. In other words, for the amylose paste with concentration of 3 %, the 10 % addition of palmitic acid gave maximum complexation. Besides amount of fatty acid addition, carbon chain length of fatty acids could also affect the CI of the complexes. Fig. 2 also presents CIs of the complexes formed by amylose and fatty acids with different carbon chain lengths at 10 % addition. It was found that the CI increased with the increasing of fatty acid carbon chain length. This is because lipids with chain lengths less 10 carbon atoms seem not to induce complex
3.3. Size analysis of the complex nanoparticles Fig. 3 shows the mean size of the nanoparticles of amylose- fatty acid complexes. It was found that the more the palmitic acid was added, the larger the nanoparticles of amylose- palmitic acid complex were formed, and the longer the carbon chain length of the fatty acid, the larger the complex nanoparticles of amylose-fatty acid were formed. During the preparation of the complex nanoparticles, some sections of amylose chains complexed with fatty acid first, i.e. coiled into single helices with fatty acid molecule in the internal cavity (Lesmes et al., 2009; Putseys et al., 2010; Seo et al., 2015). Since these complex segments are interrupted by the section of uncomplexed amylose (Lalush et al., 2005), it can be imagined that, at the following ethanol precipitation stage, these formed amylose-fatty acid helices will be incorporated into the nanoparticles as the amylose chains contract and the nanoparticles take shape in the process of ethanol precipitation. As discussed in Section 3.2, the more the fatty acid was added and the longer the carbon chain length of fatty acid, the more the amylose-fatty 3
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inversely proportional to the crystallite size. The crystallinity of the complex nanoparticles decreased from 54.22%–36.38 % when the palmitic acid addition changed from 0 to 20 %. Author's previous works (Yan et al., 2017, 2018) showed that the Vtype crystalline structure in the amylose nanoparticles (precipitated using ethanol) formed in the drying process instead of the precipitation process. Amylose chains complexed with ethanol and formed single helices in the precipitation stage, and these single helices associated in an ordered crystalline structure in the drying stage. In other words, the V-type crystalline structure of the amylose nanoparticles without fatty acid addition was due to an ordered arrangement of the amyloseethanol helices. For the complex nanoparticles of amylose-fatty acid in this study, some sections of amylose chains complexed with the fatty acid first, and ethanol complexed with the uncomplexed sections of amylose chains during the precipitation stage, thus, there could be two kinds of single helices, amylose-fatty acid helices and amylose-ethanol helices in the system. Moreover, the more the palmitic acid was added, the more the amylose-palmitic acid single helices were formed, and at the same time, the less the amylose-ethanol single helices could be formed due to less of available uncomlexed sections of amylose chains. Since each ligand imposes its own specific helix dimensions when complexing with amylose (Putseys et al., 2010), the size of the amylosepalmitic acid helices should be larger than that of the amylose-ethanol helices. The larger size of the amylose-palmitic acid helices decreased their mobility and made them difficult to re-associate in an ordered crystalline structure at drying stage, which caused decrease in crystallite size and crystallinity of the complex nanoparticles. In addition, it can be imagined that the difference in the size of the amylose-palmitic acid helix and amylose-ethanol helix could reduced integrity of the ordered structure piled up by these two helices, which also led to a decrease in the crystallite size and crystallinity of the complex nanoparticles. Fig. 5 presents the X-ray diffraction patterns of the nanoparticles of amylose-fatty acids (10 % addition) with different chain lengths. Compared to the amylose nanoparticles without fatty acid addition, there were no obvious change in intensity and width of the diffraction peaks at 2θ of 7.5°, 13.5° and 20.8° for the amylose-C6 nanopaetricles and the amylose-C12 nanoparticles, but these peaks were slightly shorter and broader for the amylose-C16 nanoparticles. The crystallinity of the complex nanoparticles were 51.07 %, 49.24 % and 42.15 %, respectively, when the chain length of fatty acid had 6, 12 and 16 carbon atoms. As presented in Section 3.2, when fatty acid with shorter chain length was used, CI was low, suggesting that less the amylosefatty acid helices were formed. Thus, more amylose-ethanol helices
Fig. 3. Mean size of complex nanoparticles of amylose-fatty acids. Values are given as mean ± standard deviation. Different letters indicate significantly different (p < 0.05) when analyzed by Duncan’s Multiple Range Test.
acid complex helices were formed. Thus, when increasing fatty acid addition or using fatty acid with longer chain length, there were more amylose-fatty acid complex helices which could be incorporated into the nanoparticles during precipitation. This led to the size increase of the complex nanoparticles. It was noted that the amylose - n-caproic acid (C6) nanoparticles were lightly larger than the amylose nanoparticles without fatty acid addition. This is because lipids with chain lengths less 10 carbon atoms hardly induce complex formation (Putseys et al., 2010) as mentioned in Section 3.2. In other words, too few amylose- n-caproic acid (C6) complex helices in the system would not affect the size of precipitated nanoparticles. 3.4. X-ray diffraction analysis Fig. 4 presents X-ray diffraction patterns of the nanoparticles of amylose-palmitic acid complex with different palmitic acid additions. The complex nanoparticles possessed V-type crystalline structure (Seo et al., 2016; Zaba et al., 2009), and the addition of palmitic acid had no effect on the crystalline structure. The V-type crystalline structure was used to verify the formation of the amylose-lipid complexes (Wang et al., 2019). Compared with amylose nanoparticles without fatty acid addition, the diffraction peaks at 2θ of 7.5°, 13.5° and 20.8° for the nanoparticles of amylose-palmitic acid complex became wide with increasing amount of fatty acid addition, suggesting that the crystallite size became small as the width at half height of a diffraction peak is
Fig. 4. X-ray diffraction patterns of the nanoparticles of amylose-palmitic acid complex with different additions.
Fig. 5. X-ray diffraction patterns of the complex nanoparticles of amylose-fatty acids (10 % addition). 4
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Fig. 6. SEM micrographs of the complex nanoparticles of amylose-fatty acids (10 % addition), amylose-n-caproic acid (A) amylose-lauric acid (B) amylose-palmitic acid complex (C).
shifts were attributed to the formation of amylose complexes (Zhang et al., 2016). Fig. 7B shows enlarged view of the range from 40 ppm to 20 ppm. The resonance in the 31–32 ppm range is the characteristic of lipid complex (Lebail et al., 2000). As can be seen in Fig. 7B, the complex nanopartticles of the amylose-fatty acids had resonance at 31.38 ppm, while the amylose nanoparticles without fatty acid addition did not show any resonance at this chemical shift, suggesting that the fatty acids were complexed in the nanoparticles. The order of the resonance intensity and the peak area at this chemical shift was amylosen-caproic acid < amylose-lauric acid < amylose-palmitate. This result indicated that, at the 10 % fatty acid addition, the content of fatty acid in the complex nanoparticles increased with increasing of fatty acid carbon chain length, which is consistent with the complexing index.
would be formed during the precipitation stage. The higher mobility of the amylose-ethanol helices tends to associate to a higher order crystalline structure during drying. On the other hand, if less amylose-fatty acid helices were formed in the stage of complexation with fatty acids, there would be more sections of amylose chains available to form the amylose-ethanol helices in the stage of ethanol precipitation. Reduction of the amylose-fatty acid helices and increase of amylose-ethanol helices could improve integrity of the structure mainly piled up by the amylose-ethanol helices. All of these could give rise to higher crystallinity and larger crystallite size in the complex nanoparticles reflected by intensive and narrower diffraction peaks in XRD patterns. 3.5. Morphological characteristics Fig. 6 shows the SEM micrographs of the nanoparticles of amylosefatty acid (10 % addition) complexes. The nanoparticles of amylose-ncaproic acid complex (Fig. 6A) had smaller size. The size of the nanoparticles of the amylose-lauric acid complex (Fig. 6B) was larger than the nanoparticles of the amylose-n-caproic acid complex, and there were some aggregates. The nanoparticles of the amylose-palmitic acid complex (Fig. 6C) were spheres and the size was larger than the nanoparticles of the amylose-n-caproic acid complex and the amyloselauric acid complex. The results of SEM observations showed that the size of nanoparticles of the amylose-fatty acid complexes were consistent with that obtained by DLS in Fig. 3. 3.6. Analysis of solid state
4. Conclusions Nanoparticles of amylose-fatty acid (palmitic acid, lauric acid and ncaproic acid) complexes were prepared via complexation and ethanol precipitation. The size of the complex nanoparticles increased with increasing of fatty acid addition and fatty acid chain length. The complex nanoparticles possessed V-type crystalline structure, but the crystallite size and crystallinity decreased with increasing of fatty acid addition and fatty acid chain length. The above results can be explained by the fact that there are two kinds of single helices, amylose-fatty acid helices and amylose-ethanol helices, in the complex nanoparticles. The larger size and quantity of the amylose-fatty acid single helices gave rise to size increase and crystallinity decrease of the complex nanoparticles due to their lower mobility. The difference in size of the amylose-fatty acid helices and amylose-ethanol helices was also a reason to cause decrease in crystallinity of the complex nanoparticles. At the level of 10 % addition, fatty acid content in the complex nanoparticles increased with increasing of fatty acid chain length. The findings of this study provide a guideline to prepare nanoparticles of amylose-fatty acid complexes with desired properties via ethanol precipitation.
13
C NMR spectroscopy
Fig. 7 shows the 13C CP/MAS NMR spectra of nanoparticles of amylose and amylose-fatty acid (10 % addition) complexes. As shown in Fig. 7A, the resonances at chemical shifts of 94−105 ppm, 68−78 ppm, 80−84 ppm, and 58−65 ppm are associated with C1, C2,3,5, C4 and C6 of starch, respectively (Gidley and Bociek, 1985; Guo et al., 2015). Compared with the spectrum of amylose nanoparticles (a), the peaks of C1, C2,3,5, C4 and C6 shifted to a lower field in the spectra of the amylose-fatty acid complexes (b, c and d), respectively. These peak 5
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Fig. 7. 13C CP/MAS NMR of the nanoparticles (A) and enlarged view of the range from 40 ppm to 20 ppm (B): amylose nanoparticles (a), the complex nanoparticles of amylose-fatty acids (10 % addition), amylose-n-caproic acid (b), amylose-lauric acid (c), amylose-palmitic acid (d). Kaur, K., Singh, N., 2000. Amylose-lipid complex formation during cooking of rice flour. Food Chem. 71, 511–517. Kim, J.-Y., Lim, S.-T., 2009. Preparation of nano-sized starch particles by complex formation with n-butanol. Carbohydr. Polym. 76, 110–116. Lalush, I., Bar, H., Zakaria, I., Eichler, S., Shimoni, E., 2005. Utilization of amylose-lipid complexes as molecular nanocapsules for conjugated linoleic acid. Biomacromolecules 6, 121–130. Lammers, K., Arbuckle-Keil, G., Dighton, J., 2009. FT-IR study of the changes in carbohydrate chemistry of three New Jersey pine barrens leaf litters during simulated control burning. Soil Biol. Biochem. 41, 340–347. Lebail, P., Buleon, A., Shiftan, D., Marchessault, R.H., 2000. Mobility of lipid in complexes of amylose-fatty acids by deuterium and 13C solid state NMR. Carbohydr. Polym. 43, 317–326. Le Bail, P., Rondeau, C., Buleon, A., 2005. Structural investigation of amylose complexes with small ligands: helical conformation, crystalline structure and thermostability. Int. J. Biol. Macromol. 35, 1–7. Lesmes, U., Cohen, S.H., Shener, Y., Shimoni, E., 2009. Effects of long chain fatty acid unsaturation on the structure and controlled release properties of amylose complexes. Food Hydrocoll. 23, 667–675. Oyeyinka, S.A., Singh, S., Venter, S.L., Amonsou, E.O., 2016. Effect of lipid types on complexation and some physicochemical properties of bambara groundnut starch. StarchStrke 68, 1–10. Putseys, J.A., Lamberts, L., Delcour, J.A., 2010. Amylose-inclusion complexes: formation, identity and physico-chemical properties. J. Cereal Sci. 51, 238–247. Seo, T.-R., Kim, H.-Y., Lim, S.-T., 2016. Preparation and characterization of aqueous dispersions of high amylose starch and conjugated linoleic acid complex. Food Chem. 211, 530–537. Seo, T.R., Kim, J.-Y., Lim, S.-T., 2015. Preparation and characterization of crystalline complexes between amylose and C18 fatty acids. Lwt - Food Sci. Technol. 64, 889–897. Shi, M.M., Liang, X.W., Yan, Y.Z., Pan, H.H., Liu, Y.Q., 2018. Influence of ethanol-water solvent and ultra-high pressure on the stability of amylose-n-octanol complex. Food Hydrocoll. 74, 315–323. Tang, M.C., Copeland, L., 2007. Analysis of complexes between lipids and wheat starch. Carbohydr. Polym. 67, 80–85. Tufvesson, F., Wahlgren, M., Eliasson, A.-C., 2003. Formation of amylose-lipid complexes and effects of temperature treatment. Part 2. Fatty acids. StarchStrke 55, 138–149. Wang, R., Liu, P., Cui, B., Kang, X.M., Yu, B., 2019. Effects of different treatment methods on properties of potato starch-lauric acid complex and potato starch-based films. Int. J. Biol. Macromol. 124, 34–40. Wang, S.J., Wang, J.R., Yu, J.L., Wang, S., 2016. Effect of fatty acids on functional properties of normal wheat and waxy wheat starches: a structural basis. Food Chem. 190, 285–292. Yan, X.X., Chang, Y.J., Wang, Q., Fu, Y.J., Ren, L.L., Zhou, J., 2018. Influence of precipitation conditions on crystallinity of amylose nanoparticles. StarchStrke 70, 1700213. Yan, X.X., Chang, Y.J., Wang, Q., Fu, Y.J., Zhou, J., 2017. Effect of drying conditions on crystallinity of amylose nanoparticles prepared by nanoprecipitation. Int. J. Biol. Macromol. 97, 481–488. Zabar, S., Lesmes, U., Katz, I., Shimoni, E., Bianco-Peled, H., 2009. Studying different dimensions of amylose–long chain fatty acid complexes: molecular, nano and micro level characteristics. Food Hydrocoll. 23, 1918–1925. Zhang, S., Zhou, Y.B., Jin, S.S., Meng, X., Yang, L.P., Wang, H.S., 2016. Preparation and structural characterization of corn starch–aroma compound inclusion complexes. J. Sci. Food Agric. 97, 182–190.
CRediT authorship contribution statement Xiaoxia Yan: Data curation, Investigation, Writing - original draft. Lvheng Kou: Resources, Writing - review & editing. Hongyuan Wei: Formal analysis, Visualization. Lili Ren: Supervision, Validation. Jiang Zhou: Conceptualization, Funding acquisition, Project administration, Writing - review & editing. Declaration of Competing Interest All authors (Xiaoxia Yan, Lvheng Kou, Hongyuan Wei, Lili Ren, and Jiang Zhou) of the submission entitled “Effect of fatty acid addition on properties of amylose nanoparticles prepared via complexing and precipitation” state that there are no competing interests to declare. Acknowledgements This work was supported by the National Natural Science Foundation of China (grant number 51273083). References Boeriu, C.G., Bravo, D., Gosselink, R.J.A., van Dam, J.E.G., 2004. Characterisation of structure-dependent functional properties of lignin with infrared spectroscopy. Ind. Crops Prod. 20, 205–218. Chang, Y.J., Yan, X.X., Wang, Q., Ren, L.L., Tong, J., Zhou, J., 2017. Influence of ultrasonic treatment on formation of amylose nanoparticles prepared by nanoprecipitation. Carbohydrate Polymer 157, 1413–1418. Cao, Z., Woortman, A.J.J., Rudolf, P., Loos, K., 2015. Facile synthesis and structural characterization of amylose-fatty acid inclusion complexes. Macromol. Biosci. 15, 691–697. Dankar, I., Haddarah, A., Omar, F.E.L., Pujolà, M., Sepulcre, F., 2018. Characterization of food additive-potato starch complexes by FTIR and X-ray diffraction. Food Chem. 260, 7–12. Fanta, G.F., Kenar, J.A., Felker, F.C., 2015. Nanoparticle formation from amylose-fatty acid inclusion complexes prepared by steam jet cooking. Ind. Crops Prod. 74, 36–44. Gelders, G.G., Vanderstukken, T.C., Goesaert, H., Delcour, J.A., 2004. Amylose-lipid complexation: a new fractionation method. Carbohydr. Polym. 56, 447–458. Gidley, M.J., Bociek, S.M., 1985. Molecular organization in starches: a 13C CP/MAS NMR study. J. Am. Chem. Soc. 107, 7040–7044. Godet, M.C., Bizot, H., Buleon, A., 1995. Crystallization of amylose-fatty acid complexes prepared with different amylose chain lengths. Carbohydr. Polym. 27, 47–52. Guo, Z., Zeng, S.X., Zhang, Y., Lu, X., Tian, Y.T., Zheng, B.D., 2015. The effects of ultrahigh pressure on the structural, rheological and retrogradation properties of lotus seed starch. Food Hydrocoll. 44, 285–291. Helbert, W., Chanzy, H., 1994. Single crystals of V amylose complexed with n-butanol or n-pentanol: structural features and properties. Int. J. Biol. Macromol. 16, 207–213. Karkalas, J., Ma, S., Morrison, W.R., Pethrick, R.A., 1995. Some factors determining the thermal properties of amylose inclusion complexes with fatty acids. Carbohydr. Res. 268, 233–247.
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Effect of fatty acid addition on properties of amylose nanoparticles prepared via complexing and precipitation
T
Xiaoxia Yan, Lvheng Kou, Hongyuan Wei, Lili Ren, Jiang Zhou* Key Laboratory of Bionic Engineering (Ministry of Education), College of Biological and Agricultural Engineering, Jilin University, Changchun 130022, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Amylose Fatty acids Complexing Precipitation Nanoparticles
Amylose nanoparticles were prepared by complexing with fatty acids (palmitic acid, lauric acid and n-caproic acid) and then ethanol precipitation. The effects of the fatty acid addition and fatty acid carbon chain length on the size, crystallinity, morphology and fatty acid content of the complex nanoparticles were studied. Analyses of FT-IR, complexing index and 13C CP/MAS NMR suggested that amylose complexed with the fatty acids. The determined complexing indexes were 7.8 %, 36.2 % and 44.5 % for the n-caproic acid, lauric acid, and palmitic acid at the 10 % addition. The complexing index increased from 37.1%–44.7% for the palmitic acid when the addition was increased from 5 % to 20 %. XRD results showed that the complex nanoparticles possessed V-type crystalline structure, but the crystallinity decreased from 54.22%–36.38 % when the palmitic acid addition changed from 0 to 20 %. At the 10 % addition, the crystallinity decreased from 51.07%–42.15 % as fatty acid chain length increased from 6 carbon atoms to 16 carbon atoms. The result of 13C CP/MAS NMR analysis indicated that the fatty acid content in the complex nanoparticles increased with increasing of fatty acid chain length at 10 % addition. The findings of this study provide a guideline to prepare nanoparticles of amylose-fatty acid complexes with desired properties.
1. Introduction Starch nanoparticles have drawn much attention due to their physiochemical properties, renewability from natural resources and potential applications in several fields. Nanoprecipitation is a simple and fast method to produce starch nanoparticles. Amylose (a linear molecule of glucose units linked by (1–4) α-D-glycoside bonds) and amylopectin are the two main components in starch. Amylose nanoparticles prepared via ethanol precipitation possessed sizes around 200 nm and the V-type crystalline structure, and it was believed that the formation of this V-type crystalline structure was due to associations of amyloseethanol single helices (complexed during precipitation) in the drying stage (Yan et al., 2017, 2018). It is well known that amylose has a unique feature to form complexes with some ligands, such as alcohols and lipids, especially fatty acids (Godet et al., 1995; Helbert and Chanzy, 1994; Karkalas et al., 1995; Le Bail et al., 2005; Lebail et al., 2000). The formation of amylose complexes makes the conformation change of amylose into a single, left-handed helix with a hydrophobic cavity which can entrap the ligands (Lesmes et al., 2009; Putseys et al., 2010). The helix diameter and dimensions are dependent on the size of complexing agent (Gelders et al., 2004; Putseys et al., 2010). For the amylose-fatty acid complexes, ⁎
it is generally believed that sections of amylose chains with at least 18 glucose units form left-handed helices with three turns (at least 6 glucose units per helix turn) and the fatty acid locates in the helices with the polar head group outside the cavity (Lesmes et al., 2009; Putseys et al., 2010; Seo et al., 2015). These complex segments are interrupted by the section of uncomplexed amylose that permit arrangement of the complex helices and formation of the V-type crystalline structure with type I and type II polymorphs (Lalush et al., 2005; Lesmes et al., 2009). Chain length and unsaturation degree of fatty acids as well as amylose chain length affect the complexation degree (Oyeyinka et al., 2016; Putseys et al., 2010). The longer the fatty acid or the amylose chains, the more heat-stable the formed complexes (Cao et al., 2015; Putseys et al., 2010). Amylose complexes have many important functions, such as, inhibit retrogradation of products containing amylose, preserve and deliver functional substances (Lesmes et al., 2009; Putseys et al., 2010). Nano-sized starch particles were prepared by complex formation with n-butanol (Kim and Lim, 2009), the size of the obtained starch particles was 10–20 nm, but the yield was low and the production process took 7 days. Fanta et al. (2015) reported nanoparticle formation from amylose-oleic acid complexes by steam jet cooking, the obtained nanoparticles had the diameters of 63−375 nm and showed V-type crystalline structure.
Corresponding author. E-mail address: [email protected] (J. Zhou).
https://doi.org/10.1016/j.indcrop.2020.112097 Received 8 October 2019; Received in revised form 22 November 2019; Accepted 4 January 2020 0926-6690/ © 2020 Elsevier B.V. All rights reserved.
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with 25 mL distilled water at 50℃ in a 50 mL capped tube and vortexed for 2 min. Then, 500 μL of the resulting solution was mixed with 15 mL distilled water, followed by the addition of 2 mL iodine solution. The absorbance at 690 nm was measured by using a UV spectrophotometer (Ruili Analytical Instrument Company, Beijing, China). Amylose paste without adding fatty acid was used as a reference, the measurement was carried out three times for each sample and mean values were reported. Complexing index was calculated using the following equation:
As amylose can complex with fatty acids as well as ethanol and form single helices, it is interested in to investigate the effects of fatty acid addition on amylose nanoparticles formation via ethanol precipitation, i.e., how the complexation with fatty acids influences the ethanol precipitation of amylose. In this study, amylose and saturated fatty acids were used to form amylose-fatty acid complexes first, then the nanoparticles of amylose-fatty acid complexes were obtained via ethanol precipitation. The main objective of this study was to investigate the effects of fatty acid addition and the carbon chain length of fatty acid on the properties of the complex nanoparticles, including size, crystallinity, morphology and fatty acid content.
CI =
(AbsRe ference − AbsAmylose − fattyacid ) AbsRe ference
× 100
(1)
Where AbsReference is the absorbance of the amylose paste without adding fatty acid, and AbsAmylose-fatty acid is the absorbance of the amylose paste containing fatty acid.
2. Materials and methods 2.1. Materials Amylose with a content of 99.5 % was obtained from Shanxi Tianwei Biological Production Co. Ltd. (Xi’an, China). The amylose is isolated from corn starch, its molar mass (determined by size-exclusion chromatography) ranges 2.4 × 106 - 4.5 × 108 g/mol and the viscosity average molecular weight (calculated from measured intrinsic viscosity) is 3.36 × 105 (Chang et al., 2017). Absolute ethanol, iodine and potassium iodide were purchased from Beijing Chemical Works (Beijing, China). Palmitic acid (C16), lauric acid (C12) and n-caproic acid (C6) were purchased from Heng Ye Jingxi Chemical Co. Ltd. (Gu’an, China), Tianjin Guangfu Jingxi Chemical Institute (Tianjin, China) and Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China), respectively. All the used chemicals were the grade of analytical reagent.
2.5. Size analysis of nanoparticles Z average-size (d. nm) and polydispersity index (PDI) of the nanoparticles of amylose-fatty acid complexes were measured by dynamic light scattering (DLS) using a Malvern Zetasizer Nano-ZS90 (Malvern Instruments Ltd., UK).The samples were prepared by dispersing the nanoparticles (without drying) in distilled water at a concentration of about 0.1 %. The measurement was carried out three times for each sample, and then the mean values were reported. 2.6. X-ray diffraction (XRD) analysis The crystalline structure of the nanoparticles of amylose-fatty acid complexes was analyzed using a XRD-6100 Shimadzu X-ray Diffractometer (Shimadzu Corporation, Japan) with Cu-Kα radiation (λ =1.542 Å) at a voltage of 40 kV and electric current of 30 mA. The XRD patterns of the nanoparticles of amylose-fatty acid complexes were recorded over the 2θ range of 4-35° at a speed of 2°/min.
2.2. Preparation of amylose-fatty acid complex nanoparticles Amylose suspension with a concentration of 3 % was prepared by mixing amylose and distilled water, and then heated at 100 °C in a water bath for 1 h with stirring. When temperature of the obtained amylose paste reached 90 °C, certain amount fatty acid dissolved in 5 mL hot ethanol (70 °C) was added into 50 mL amylose paste, the paste was continuously agitated with a magnetic stirrer at 600 rpm for 30 min. Next, ethanol (three times of the amylose paste in volume) was added dropwise into the amylose paste which was continuously agitated with a magnetic stirrer at 600 rpm. The resulting mixture was cooled to room temperature (25 °C) and centrifuged at 6000 rpm for 5 min. The obtained sediments, nanoparticles of amylose-fatty acid complexes, were rinsed twice with ethanol by centrifugation, dried under 11 % relative humidity at 4 °C for 12 h and then at 40 °C for another 12 h (using a small desiccator containing saturated lithium chloride aqueous solution and putting the desiccator in a refrigerator at 4 °C and then an oven at 40 °C), and sealed in plastic bags for further tests.
2.7. Morphological characteristics Morphology of the nanoparticles of amylose-fatty acid complexes was observed by a XL-30 ESEM FEG scanning electron microscopy (FEI company, USA). Dried complex nanoparticles were dispersed in ethanol by using ultrasonic agitation to obtain a 0.2 % suspension, and 3 μL of the suspension was dropped onto a silicon wafer and freeze-dried at -70 ℃. The samples were mounted on specimen stubs with carbon black tape and sputter-coated with gold before observation. 2.8. 13C CP/MAS (cross-polarization/magic angle spinning) NMR spectroscopy The solid-state 13C CP/MAS NMR spectra were recorded on a Bruker AVANCE III 400 WB spectrometer equipped with a 4 mm standard bore CP/MAS probe head whose X channel was tuned to 100.62 MHz for 13C and the other channel was tuned to 400.18 MHz for broad band 1H decoupling, using a magnetic field of 9.39 T at 297 K. The nanoparticles were packed in a ZrO2 rotor closed with Kel-F cap which was spun at 8 kHz rate. The measurements were conducted at a contact time of 2 ms. Total of 2000 scans were recorded with 6 s recycle delay for each sample. All 13C CP/MAS chemical shifts were referenced to the resonances of adamantane (C10H16) standard (δCH2 = 38.4).
2.3. Fourier transform infrared spectroscopy (FT-IR) analysis The FT-IR spectra of amylose-fatty acid complex nanoparticles were obtained using a FT-IR (IRAffinity-1 spectrophotometer, Shimadzu, Japan). The specimens were prepared by grinding the nanoparticles of amylose-fatty acid complexes (1∼2 mg) together with KBr (about 200 mg) and then pressed into a disc. The spectra were scanned in the range of 4000–400 cm−1. The resolution was 4 cm−1and the total number of scans was 32. 2.4. Complexing index (CI)
2.9. Statistical analysis The complexing index (CI) of amylose was measured by the method of Tang and Copeland (2007) with some modifications. Iodine solution was prepared by dissolving iodine (1.3 %) and potassium iodide (2.0 %) in distilled water. Amylose paste containing fatty acid (5 g) was mixed
Analysis of variance (one-way ANOVA) was used to compare differences of the samples. Comparisons were performed using Duncan’s Multiple Range Test (DMRT) in SPSS 23.0 software (IBM). 2
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Fig. 1. FT-IR spectra of fatty acids (A) and the complex nanoparticles of amylose-fatty acids with different chain lengths (B).
3. Results and discussion 3.1. FT-IR analysis The FT-IR spectra of lauric acid (C12), palmitic acid (C16) (n-caproic acid (C6) is liquid, FT-IR analysis was not carried out) and the amylose nanoparticles containing fatty acid are presented in Fig. 1. As shown in Fig. 1A, the characteristic absorption peak of carbonyl band (C]O) stretch for the fatty acids located at 1705 cm−1 (Boeriu et al., 2004). The absorbance at 2850 cm−1 is CHe stretch of aliphatic compounds (Lammers et al., 2009). It can be seen in Fig. 1B that, there was a peak at 1650 cm−1 in the spectrum of amylose nanoparticles which was assigned to water molecules absorbed in the amorphous region and the stretching vibration of the C]O band (amide I) (Dankar et al., 2018; Shi et al., 2018). This peak shifted to 1645 cm−1 after the fatty acids were added. Compared with the FT-IR spectrum of the fatty acids, the C]O absorption peak at 1705 cm-1 disappeared for the amylose nanoparticles containing fatty acid. Since the absorption peak of C]O at 1705 cm-1 was absent in the FT-IR spectra of the nanoparticles, it could be concluded that either the fatty acids were not present in the nanoparticles or they complexed with amylose rather than physically mixed. In order to futher confirm the formation of the amylose-fatty acid complex in the nanoparticles, analyses of complexing index (CI) and 13C CP/MAS NMR were performed in the subsequent studies (Sections 3.2 and 3.6).
Fig. 2. Complexing index (CI) for the complexes of amylose-fatty acids. Values are given as mean ± standard deviation. Different letters indicate significantly different (p < 0.05) when analyzed by Duncan’s Multiple Range Test.
formation, as they are too soluble in the aqueous environment so that they can not be entrapped properly in the hydrophobic helix cavity (Putseys et al., 2010). It is also believed that the low solubility of the longer chain fatty acids is one reason of complex formation (Tufvesson et al., 2003). Fatty acids with longer carbon chains are more hydrophobic and therefore more likely form single-helix structure complexes with amylose and stay in the hydrophobic helix cavity, which gives rise to the increase of CI.
3.2. CI of amylose-fatty acid complex The value of CI can be used to characterize complexing degree between amylose and fatty acids (Kaur and Singh, 2000; Wang et al., 2016). Fig. 2 shows the CIs for the complexes of amylose and the fatty acids. With increasing of the amount of palmitic acid addition, the CI increased, but when the addition of palmitic acid beyond 10 %, the CI tended to remain stable. This is understandable, because when the amount of palmitic acid was small, even the palmitic acid was completely complexed, the CI was still low. As the palmitic acid addition increased, more palmitic acid was involved in complexation, which gave rise to higher CI. However, when the palmitic acid addition reached 10 %, there were no enough available sections of amylose chains to form the complexes with palmitic acid. Therefore, further increasing palmitic acid addition would not increase CI. In other words, for the amylose paste with concentration of 3 %, the 10 % addition of palmitic acid gave maximum complexation. Besides amount of fatty acid addition, carbon chain length of fatty acids could also affect the CI of the complexes. Fig. 2 also presents CIs of the complexes formed by amylose and fatty acids with different carbon chain lengths at 10 % addition. It was found that the CI increased with the increasing of fatty acid carbon chain length. This is because lipids with chain lengths less 10 carbon atoms seem not to induce complex
3.3. Size analysis of the complex nanoparticles Fig. 3 shows the mean size of the nanoparticles of amylose- fatty acid complexes. It was found that the more the palmitic acid was added, the larger the nanoparticles of amylose- palmitic acid complex were formed, and the longer the carbon chain length of the fatty acid, the larger the complex nanoparticles of amylose-fatty acid were formed. During the preparation of the complex nanoparticles, some sections of amylose chains complexed with fatty acid first, i.e. coiled into single helices with fatty acid molecule in the internal cavity (Lesmes et al., 2009; Putseys et al., 2010; Seo et al., 2015). Since these complex segments are interrupted by the section of uncomplexed amylose (Lalush et al., 2005), it can be imagined that, at the following ethanol precipitation stage, these formed amylose-fatty acid helices will be incorporated into the nanoparticles as the amylose chains contract and the nanoparticles take shape in the process of ethanol precipitation. As discussed in Section 3.2, the more the fatty acid was added and the longer the carbon chain length of fatty acid, the more the amylose-fatty 3
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inversely proportional to the crystallite size. The crystallinity of the complex nanoparticles decreased from 54.22%–36.38 % when the palmitic acid addition changed from 0 to 20 %. Author's previous works (Yan et al., 2017, 2018) showed that the Vtype crystalline structure in the amylose nanoparticles (precipitated using ethanol) formed in the drying process instead of the precipitation process. Amylose chains complexed with ethanol and formed single helices in the precipitation stage, and these single helices associated in an ordered crystalline structure in the drying stage. In other words, the V-type crystalline structure of the amylose nanoparticles without fatty acid addition was due to an ordered arrangement of the amyloseethanol helices. For the complex nanoparticles of amylose-fatty acid in this study, some sections of amylose chains complexed with the fatty acid first, and ethanol complexed with the uncomplexed sections of amylose chains during the precipitation stage, thus, there could be two kinds of single helices, amylose-fatty acid helices and amylose-ethanol helices in the system. Moreover, the more the palmitic acid was added, the more the amylose-palmitic acid single helices were formed, and at the same time, the less the amylose-ethanol single helices could be formed due to less of available uncomlexed sections of amylose chains. Since each ligand imposes its own specific helix dimensions when complexing with amylose (Putseys et al., 2010), the size of the amylosepalmitic acid helices should be larger than that of the amylose-ethanol helices. The larger size of the amylose-palmitic acid helices decreased their mobility and made them difficult to re-associate in an ordered crystalline structure at drying stage, which caused decrease in crystallite size and crystallinity of the complex nanoparticles. In addition, it can be imagined that the difference in the size of the amylose-palmitic acid helix and amylose-ethanol helix could reduced integrity of the ordered structure piled up by these two helices, which also led to a decrease in the crystallite size and crystallinity of the complex nanoparticles. Fig. 5 presents the X-ray diffraction patterns of the nanoparticles of amylose-fatty acids (10 % addition) with different chain lengths. Compared to the amylose nanoparticles without fatty acid addition, there were no obvious change in intensity and width of the diffraction peaks at 2θ of 7.5°, 13.5° and 20.8° for the amylose-C6 nanopaetricles and the amylose-C12 nanoparticles, but these peaks were slightly shorter and broader for the amylose-C16 nanoparticles. The crystallinity of the complex nanoparticles were 51.07 %, 49.24 % and 42.15 %, respectively, when the chain length of fatty acid had 6, 12 and 16 carbon atoms. As presented in Section 3.2, when fatty acid with shorter chain length was used, CI was low, suggesting that less the amylosefatty acid helices were formed. Thus, more amylose-ethanol helices
Fig. 3. Mean size of complex nanoparticles of amylose-fatty acids. Values are given as mean ± standard deviation. Different letters indicate significantly different (p < 0.05) when analyzed by Duncan’s Multiple Range Test.
acid complex helices were formed. Thus, when increasing fatty acid addition or using fatty acid with longer chain length, there were more amylose-fatty acid complex helices which could be incorporated into the nanoparticles during precipitation. This led to the size increase of the complex nanoparticles. It was noted that the amylose - n-caproic acid (C6) nanoparticles were lightly larger than the amylose nanoparticles without fatty acid addition. This is because lipids with chain lengths less 10 carbon atoms hardly induce complex formation (Putseys et al., 2010) as mentioned in Section 3.2. In other words, too few amylose- n-caproic acid (C6) complex helices in the system would not affect the size of precipitated nanoparticles. 3.4. X-ray diffraction analysis Fig. 4 presents X-ray diffraction patterns of the nanoparticles of amylose-palmitic acid complex with different palmitic acid additions. The complex nanoparticles possessed V-type crystalline structure (Seo et al., 2016; Zaba et al., 2009), and the addition of palmitic acid had no effect on the crystalline structure. The V-type crystalline structure was used to verify the formation of the amylose-lipid complexes (Wang et al., 2019). Compared with amylose nanoparticles without fatty acid addition, the diffraction peaks at 2θ of 7.5°, 13.5° and 20.8° for the nanoparticles of amylose-palmitic acid complex became wide with increasing amount of fatty acid addition, suggesting that the crystallite size became small as the width at half height of a diffraction peak is
Fig. 4. X-ray diffraction patterns of the nanoparticles of amylose-palmitic acid complex with different additions.
Fig. 5. X-ray diffraction patterns of the complex nanoparticles of amylose-fatty acids (10 % addition). 4
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Fig. 6. SEM micrographs of the complex nanoparticles of amylose-fatty acids (10 % addition), amylose-n-caproic acid (A) amylose-lauric acid (B) amylose-palmitic acid complex (C).
shifts were attributed to the formation of amylose complexes (Zhang et al., 2016). Fig. 7B shows enlarged view of the range from 40 ppm to 20 ppm. The resonance in the 31–32 ppm range is the characteristic of lipid complex (Lebail et al., 2000). As can be seen in Fig. 7B, the complex nanopartticles of the amylose-fatty acids had resonance at 31.38 ppm, while the amylose nanoparticles without fatty acid addition did not show any resonance at this chemical shift, suggesting that the fatty acids were complexed in the nanoparticles. The order of the resonance intensity and the peak area at this chemical shift was amylosen-caproic acid < amylose-lauric acid < amylose-palmitate. This result indicated that, at the 10 % fatty acid addition, the content of fatty acid in the complex nanoparticles increased with increasing of fatty acid carbon chain length, which is consistent with the complexing index.
would be formed during the precipitation stage. The higher mobility of the amylose-ethanol helices tends to associate to a higher order crystalline structure during drying. On the other hand, if less amylose-fatty acid helices were formed in the stage of complexation with fatty acids, there would be more sections of amylose chains available to form the amylose-ethanol helices in the stage of ethanol precipitation. Reduction of the amylose-fatty acid helices and increase of amylose-ethanol helices could improve integrity of the structure mainly piled up by the amylose-ethanol helices. All of these could give rise to higher crystallinity and larger crystallite size in the complex nanoparticles reflected by intensive and narrower diffraction peaks in XRD patterns. 3.5. Morphological characteristics Fig. 6 shows the SEM micrographs of the nanoparticles of amylosefatty acid (10 % addition) complexes. The nanoparticles of amylose-ncaproic acid complex (Fig. 6A) had smaller size. The size of the nanoparticles of the amylose-lauric acid complex (Fig. 6B) was larger than the nanoparticles of the amylose-n-caproic acid complex, and there were some aggregates. The nanoparticles of the amylose-palmitic acid complex (Fig. 6C) were spheres and the size was larger than the nanoparticles of the amylose-n-caproic acid complex and the amyloselauric acid complex. The results of SEM observations showed that the size of nanoparticles of the amylose-fatty acid complexes were consistent with that obtained by DLS in Fig. 3. 3.6. Analysis of solid state
4. Conclusions Nanoparticles of amylose-fatty acid (palmitic acid, lauric acid and ncaproic acid) complexes were prepared via complexation and ethanol precipitation. The size of the complex nanoparticles increased with increasing of fatty acid addition and fatty acid chain length. The complex nanoparticles possessed V-type crystalline structure, but the crystallite size and crystallinity decreased with increasing of fatty acid addition and fatty acid chain length. The above results can be explained by the fact that there are two kinds of single helices, amylose-fatty acid helices and amylose-ethanol helices, in the complex nanoparticles. The larger size and quantity of the amylose-fatty acid single helices gave rise to size increase and crystallinity decrease of the complex nanoparticles due to their lower mobility. The difference in size of the amylose-fatty acid helices and amylose-ethanol helices was also a reason to cause decrease in crystallinity of the complex nanoparticles. At the level of 10 % addition, fatty acid content in the complex nanoparticles increased with increasing of fatty acid chain length. The findings of this study provide a guideline to prepare nanoparticles of amylose-fatty acid complexes with desired properties via ethanol precipitation.
13
C NMR spectroscopy
Fig. 7 shows the 13C CP/MAS NMR spectra of nanoparticles of amylose and amylose-fatty acid (10 % addition) complexes. As shown in Fig. 7A, the resonances at chemical shifts of 94−105 ppm, 68−78 ppm, 80−84 ppm, and 58−65 ppm are associated with C1, C2,3,5, C4 and C6 of starch, respectively (Gidley and Bociek, 1985; Guo et al., 2015). Compared with the spectrum of amylose nanoparticles (a), the peaks of C1, C2,3,5, C4 and C6 shifted to a lower field in the spectra of the amylose-fatty acid complexes (b, c and d), respectively. These peak 5
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Fig. 7. 13C CP/MAS NMR of the nanoparticles (A) and enlarged view of the range from 40 ppm to 20 ppm (B): amylose nanoparticles (a), the complex nanoparticles of amylose-fatty acids (10 % addition), amylose-n-caproic acid (b), amylose-lauric acid (c), amylose-palmitic acid (d). Kaur, K., Singh, N., 2000. Amylose-lipid complex formation during cooking of rice flour. Food Chem. 71, 511–517. Kim, J.-Y., Lim, S.-T., 2009. Preparation of nano-sized starch particles by complex formation with n-butanol. Carbohydr. Polym. 76, 110–116. Lalush, I., Bar, H., Zakaria, I., Eichler, S., Shimoni, E., 2005. Utilization of amylose-lipid complexes as molecular nanocapsules for conjugated linoleic acid. Biomacromolecules 6, 121–130. Lammers, K., Arbuckle-Keil, G., Dighton, J., 2009. FT-IR study of the changes in carbohydrate chemistry of three New Jersey pine barrens leaf litters during simulated control burning. Soil Biol. Biochem. 41, 340–347. Lebail, P., Buleon, A., Shiftan, D., Marchessault, R.H., 2000. Mobility of lipid in complexes of amylose-fatty acids by deuterium and 13C solid state NMR. Carbohydr. Polym. 43, 317–326. Le Bail, P., Rondeau, C., Buleon, A., 2005. Structural investigation of amylose complexes with small ligands: helical conformation, crystalline structure and thermostability. Int. J. Biol. Macromol. 35, 1–7. Lesmes, U., Cohen, S.H., Shener, Y., Shimoni, E., 2009. Effects of long chain fatty acid unsaturation on the structure and controlled release properties of amylose complexes. Food Hydrocoll. 23, 667–675. Oyeyinka, S.A., Singh, S., Venter, S.L., Amonsou, E.O., 2016. Effect of lipid types on complexation and some physicochemical properties of bambara groundnut starch. StarchStrke 68, 1–10. Putseys, J.A., Lamberts, L., Delcour, J.A., 2010. Amylose-inclusion complexes: formation, identity and physico-chemical properties. J. Cereal Sci. 51, 238–247. Seo, T.-R., Kim, H.-Y., Lim, S.-T., 2016. Preparation and characterization of aqueous dispersions of high amylose starch and conjugated linoleic acid complex. Food Chem. 211, 530–537. Seo, T.R., Kim, J.-Y., Lim, S.-T., 2015. Preparation and characterization of crystalline complexes between amylose and C18 fatty acids. Lwt - Food Sci. Technol. 64, 889–897. Shi, M.M., Liang, X.W., Yan, Y.Z., Pan, H.H., Liu, Y.Q., 2018. Influence of ethanol-water solvent and ultra-high pressure on the stability of amylose-n-octanol complex. Food Hydrocoll. 74, 315–323. Tang, M.C., Copeland, L., 2007. Analysis of complexes between lipids and wheat starch. Carbohydr. Polym. 67, 80–85. Tufvesson, F., Wahlgren, M., Eliasson, A.-C., 2003. Formation of amylose-lipid complexes and effects of temperature treatment. Part 2. Fatty acids. StarchStrke 55, 138–149. Wang, R., Liu, P., Cui, B., Kang, X.M., Yu, B., 2019. Effects of different treatment methods on properties of potato starch-lauric acid complex and potato starch-based films. Int. J. Biol. Macromol. 124, 34–40. Wang, S.J., Wang, J.R., Yu, J.L., Wang, S., 2016. Effect of fatty acids on functional properties of normal wheat and waxy wheat starches: a structural basis. Food Chem. 190, 285–292. Yan, X.X., Chang, Y.J., Wang, Q., Fu, Y.J., Ren, L.L., Zhou, J., 2018. Influence of precipitation conditions on crystallinity of amylose nanoparticles. StarchStrke 70, 1700213. Yan, X.X., Chang, Y.J., Wang, Q., Fu, Y.J., Zhou, J., 2017. Effect of drying conditions on crystallinity of amylose nanoparticles prepared by nanoprecipitation. Int. J. Biol. Macromol. 97, 481–488. Zabar, S., Lesmes, U., Katz, I., Shimoni, E., Bianco-Peled, H., 2009. Studying different dimensions of amylose–long chain fatty acid complexes: molecular, nano and micro level characteristics. Food Hydrocoll. 23, 1918–1925. Zhang, S., Zhou, Y.B., Jin, S.S., Meng, X., Yang, L.P., Wang, H.S., 2016. Preparation and structural characterization of corn starch–aroma compound inclusion complexes. J. Sci. Food Agric. 97, 182–190.
CRediT authorship contribution statement Xiaoxia Yan: Data curation, Investigation, Writing - original draft. Lvheng Kou: Resources, Writing - review & editing. Hongyuan Wei: Formal analysis, Visualization. Lili Ren: Supervision, Validation. Jiang Zhou: Conceptualization, Funding acquisition, Project administration, Writing - review & editing. Declaration of Competing Interest All authors (Xiaoxia Yan, Lvheng Kou, Hongyuan Wei, Lili Ren, and Jiang Zhou) of the submission entitled “Effect of fatty acid addition on properties of amylose nanoparticles prepared via complexing and precipitation” state that there are no competing interests to declare. Acknowledgements This work was supported by the National Natural Science Foundation of China (grant number 51273083). References Boeriu, C.G., Bravo, D., Gosselink, R.J.A., van Dam, J.E.G., 2004. Characterisation of structure-dependent functional properties of lignin with infrared spectroscopy. Ind. Crops Prod. 20, 205–218. Chang, Y.J., Yan, X.X., Wang, Q., Ren, L.L., Tong, J., Zhou, J., 2017. Influence of ultrasonic treatment on formation of amylose nanoparticles prepared by nanoprecipitation. Carbohydrate Polymer 157, 1413–1418. Cao, Z., Woortman, A.J.J., Rudolf, P., Loos, K., 2015. Facile synthesis and structural characterization of amylose-fatty acid inclusion complexes. Macromol. Biosci. 15, 691–697. Dankar, I., Haddarah, A., Omar, F.E.L., Pujolà, M., Sepulcre, F., 2018. Characterization of food additive-potato starch complexes by FTIR and X-ray diffraction. Food Chem. 260, 7–12. Fanta, G.F., Kenar, J.A., Felker, F.C., 2015. Nanoparticle formation from amylose-fatty acid inclusion complexes prepared by steam jet cooking. Ind. Crops Prod. 74, 36–44. Gelders, G.G., Vanderstukken, T.C., Goesaert, H., Delcour, J.A., 2004. Amylose-lipid complexation: a new fractionation method. Carbohydr. Polym. 56, 447–458. Gidley, M.J., Bociek, S.M., 1985. Molecular organization in starches: a 13C CP/MAS NMR study. J. Am. Chem. Soc. 107, 7040–7044. Godet, M.C., Bizot, H., Buleon, A., 1995. Crystallization of amylose-fatty acid complexes prepared with different amylose chain lengths. Carbohydr. Polym. 27, 47–52. Guo, Z., Zeng, S.X., Zhang, Y., Lu, X., Tian, Y.T., Zheng, B.D., 2015. The effects of ultrahigh pressure on the structural, rheological and retrogradation properties of lotus seed starch. Food Hydrocoll. 44, 285–291. Helbert, W., Chanzy, H., 1994. Single crystals of V amylose complexed with n-butanol or n-pentanol: structural features and properties. Int. J. Biol. Macromol. 16, 207–213. Karkalas, J., Ma, S., Morrison, W.R., Pethrick, R.A., 1995. Some factors determining the thermal properties of amylose inclusion complexes with fatty acids. Carbohydr. Res. 268, 233–247.
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