Lamellar Repeat Distance Research Articles

Dynamic development of changes in multi-scale structure during grain filling affect gelatinization properties of rice starch

Rice was collected over the entire grain filling period (about 40 days) to explore the multi-structure evolution and gelatinization behavior changes of starch. During the early stage (DAA 6–14), the significant reduction in lamellar repeat distance (10.04 to 9.68 nm) and relative crystallinity (26.6 % to 22.7 %) was due to initial rapid accumulation of amylose (from 9.38 % to 14.05 %) and short amylopectin chains. Meanwhile, the decreased proportion of aggregation structure resulted in a decrease in the gelatinization temperature and a narrowed range of gelatinization temperature also indicated an increase in homogeneity as starch matured. Gelatinization enthalpy was mainly controlled by aggregation structure, which was negatively and positively related to the amylose content and the degree of order respectively. Peak viscosity of starch pasting increased and reached a maximum (924 cP) at DAA-21 due to larger granule size. Amylose and short amylopectin chains with degree of polymerization 6–12 showed positive and negative correlation with short-term retrogradation ability (setback value) respectively. The dynamics of different scale structure during grain filling had varying degrees of impact on gelatinization properties.

Combined effects of SSII-2RNAi and different Wx alleles on rice grain transparency and physicochemical properties

Near-isogenic lines Nip(Wxb/SSII-2), Nip(Wxb/ss2-2), Nip(Wxmw/SSII-2), Nip(Wxmw/ss2-2), Nip(Wxmp/SSII-2) and Nip(Wxmp/ss2-2) in the Nipponbare (Nip) background containing the SSII-2RNAi cassette combined with different Waxy (Wx) alleles were investigated in terms of rice grain transparency and quality profiles. Rice lines carrying the SSII-2RNAi cassette displayed downregulation of SSII-2, SSII-3 and Wx genes. Introduction of the SSII-2RNAi cassette decreased apparent amylose content (AAC) in all transgenic lines, but grain transparency differed between low AAC rice lines. Grains from Nip(Wxb/SSII-2) and Nip(Wxb/ss2-2) were transparent, while those of rice were increasingly translucent with decreasing moisture due to cavities within starch granules. Rice grain transparency was positively correlated with grain moisture and AAC, but negatively correlated with cavity area within starch granules. Starch fine structure analysis revealed a marked increase in short amylopectin chains with DP 6–12, but a decrease in intermediate chains with DP 13–24, resulting in decreased gelatinisation temperature. Starch crystalline structure analysis showed that the transgenic rice starches have lower crystallinity and lamellar repeat distance than controls due to differences in starch fine structure. The results highlight the molecular basis underpinning rice grain transparency, and provide strategies for improving rice grain transparency.

Investigating the influence of excipient batch variation on the structure, consistency and physical stability of polysorbate 60-based topical vehicles.

Fatty alcohol-polysorbate 60-water ternary systems were used as models to represent the continuous phases of the respective semisolid oil-in-water emulsions for topical delivery of cosmetic and medicinal agents. The influence of batch variation of polysorbate 60 and fatty alcohol on structure and consistency of these systems was investigated using microscopy, rheology, differential scanning calorimetry and X-ray scattering techniques. The polysorbate 60 : cetostearyl alcohol mixed emulsifying wax showed swelling in water, that is, the lamellar repeat distance continually augmented from 93 to 125Å with water percentage 20-90%. Cetostearyl alcohol ternary systems were thicker than cetyl alcohol ones independently of polysorbate 60 batches used. All the ternary systems showed an initial increase in consistency over the first 2weeks of storage, which was followed by slight changes in consistency (cetostearyl alcohol systems) due to the re-allocation of polysorbate 60 molecules in the gel network or significant breakdown of structure (cetyl alcohol systems) due to the transformation of swollen α-lamellar gel phase into β, γ crystals on 25°C storage. With all fatty alcohols, the consistency of polysorbate 60 ternary system was directly dependent upon interlamellar water thickness as governed by the length and distribution of polyoxyethylene groups within polysorbate 60molecules. In relation with the composition of polysorbate 60 batches used, the consistency of ternary systems was higher when prepared with the polysorbate 60 batch containing a greater amount of sorbitan polyoxyethylene monoesters. It was proposed that the swollen α-crystalline gel phase could be better formed by sorbitan polyoxyethylene monoesters rather than sorbitan polyoxyethylene diesters.

Insights into the mechanism of formation of non-conventional cochleates and its impact on their functional properties

The consequences of design of drug delivery system such as lipid cochleates are crucial on the functional outcome. This study describes the design, mechanism of formation and functional properties of Amikacin-bridged cochleates with and without the presence of sub-threshold concentration of Ca2+ in comparison to conventional Ca2+-bridged cochleates. The DOPS cochleates were successfully prepared with Ca2+, Amikacin, and Amikacin + sub-threshold Ca2+ as bridging agents. Synergistic bridging of the DOPS headgroups occurred in the presence of Amikacin + sub-threshold Ca2+ resulting in rigidly packed multiple bilayers with uniform lamellar repeat distance of 51 Å. The rigidity of the internal structure of the cochleates increased with increasing concentration of Ca2+. As a consequence, an increase in Amikacin encapsulation (71.63 ± 8.98%) and loading (266.70 ± 12.01 mg Amikacin/g cochleates) was obtained. Additionally, the in-vitro Amikacin release rate decreased as the concentration of Ca2+ was increased reflecting the tightly packed structures and suggesting sustained release. Drug release kinetics studied using the Higuchi model suggested tuneable Amikacin release by simply varying the Ca2+ concentrations used for cochleation. The use of higher Ca2+ concentration positively affected the long-term stability of the cochleates and showed increased half-lives during 12 weeks of storage. The proposed mechanistic model of the formation of non-conventional cochleates is supported well by the analytical data and promotes the synergistic effect of the bridging agents. This study reveals the importance of the type and concentration of the bridging agent to achieve a specific functional outcome and has the benefit to guide future design of cochleates with specific functions in drug delivery and formulation.

Ionic Liquid-Induced Phase-Separated Domains in Lipid Multilayers Probed by X-ray Scattering Studies.

A cellular membrane, primarily a lipid bilayer, surrounds the internal components of a biological cell from the external components. This self-assembled bilayer is known to be perturbed by ionic liquids (ILs) causing malfunctioning of a cellular organism. In the present study, surface-sensitive X-ray scattering techniques have been employed to understand this structural perturbation in a lipid multilayer system formed by a zwitterionic phospholipid, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine. The ammonium and phosphonium-based ILs with methanesulfonate anions are observed to induce phase-separated domains in the plane of a bilayer. The lamellar X-ray diffraction peaks suggest these domains to correlate across the bilayers in a smectic liquid crystalline phase. This induced IL-rich lamellar phase has a very low lamellar repeat distance, suggesting the formation of an interdigitated bilayer. The IL-poor phase closely related to the pristine lipid phase shows a decrement in the in-plane chain lattice parameters with a reduced tilt angle. The ammonium and phosphonium-based ILs with a relatively bulky anion, p-toluenemethanesulfonate, have shown a similar effect.

Relationship between molecular structure and lamellar and crystalline structure of rice starch

The relationship between molecular structure and crystalline and lamellar structures of fifteen types of rice starches was studied. GPC and HPAEC were used for the molecular chain analysis and WAXS, SAXS, and CP/MAS 13C NMR were employed for aggregation structural analysis. The amylopectin content and the average lengths of fb1-chains (the degree of polymerization (DP) 13–24) were positively correlated with the amount of double helices (r2 = 0.92 and 0.57, respectively). In contrast, amylose content was positively correlated with the amounts of amorphous materials in starch (r2 = 0.77). The amount of double helices, which constitute a major part of the crystalline matrix, was positively correlated with the lamellar ordering (r2 = 0.81), and negatively correlated with the thickness of crystalline lamellae (r2 = 0.90) and lamellar repeat distance (r2 = 0.84). Conversely, the amount of the amorphous matrix was correlated with these parameters in the opposite way (r2 = 0.50, 0.75, and 0.75, respectively).

Amylose content and specific fine structures affect lamellar structure and digestibility of maize starches

The molecular and lamellar structures and in vitro digestibility of high amylose maize starch granules were investigated. Size-exclusion chromatography (SEC) analysis of hydrodynamic radius (Rh) and relative contents (RC) of different amylose and amylopectin pools revealed that increased amylose content resulted in the decrease of RC of short amylopectin chains (AP1, DP < 36), and the increase of Rh and RC of long amylopectin chains (AP2, DP > 36). Fluorophore-assisted carbohydrate electrophoresis (FACE) analysis showed the average chain lengths of defined structures of fa, fb1, and fb3, and the content of fb3 were increased with the amylose content and at AC content of 43%, the chain lengths decreased with the amylose content. The relative contents of fa and fb1and the degree of branching had the converse trends. Hence, HA-2, having an AC content of 43%, was the tipping point for these structural changes. Small angle X-ray scattering (SAXS) and its normalized 1D correlation function and SAXS scattering curve parametric fitting showed that the crystalline lamellar thickness was negatively correlated with proportion of short amylose chains (AM1, 100 <DP ≤ 150). The Bragg lamellar repeat distance was positively correlated to Rh of the intermediate amylose chains (AM2, 150< DP ≤ 800). The amorphous lamellae thickness was positively correlated with the Rh of long amylose chains (AM3, 800 <DP ≤ 40,000). The positive correlation between Rh of AM2 on the Gaussian peak area and the scattering invariant suggests AM2 chains stabilize the lamellar structure. Both the digestion rate of the digestible starch as well as the resistant starch content were positively correlated with amylose content. Pearson correlation analysis demonstrated that longer amylopectin chains and lower content of AP1 chains were positively correlated with the digestion rate.

Comparison of physicochemical properties of very small granule starches from endosperms of dicotyledon plants

In this study, 6 very small granule starches (VSGSs) were isolated from endosperms of dicotyledon Amaranthus cruentus, Agriophyllum squarrosum, Chenopodium quinoa, Euryale ferox, Mirabilis jalapa, and Vaccaria hispanica. Their morphologies and physicochemical properties were investigated. Most VSGSs with granule size <3 μm were spherical or polygonal, and their apparent amylose contents ranged from 19.4 to 33.1% with A. cruentus starch the lowest and E. ferox starch the highest. All VSGSs had the same A-type crystalline structure with relative crystallinities from 23.3 to 29.6%. Though 6 VSGSs had slight differences in short-range ordered structure and lamellar repeat distance, their lamellar peak intensities exhibited significant differences. The gelatinization temperatures showed significant differences among 6 VSGSs with C. quinoa starch the lowest and M. jalapa starch the highest. The 6 VSGSs had significantly different pasting viscosities with peak viscosities from 1887 to 4579 mPa s, hot viscosities from 1704 to 3479 mPa s, breakdown viscosities from 56 to 1170 mPa s, final viscosities from 2419 to 4811 mPa s, and setback viscosities from 715 to 1821 mPa s. The digestion properties of starches were significantly different among 6 VSGSs. The above results could provide some references for applications of these VSGSs.

Peptide Kinetics in Symmetric and Asymmetric Membrane Mimics of Gram-Negative Bacteria

Antimicrobial peptides (AMPs) are well-studied compounds with high potential for application against multi-resistant bacterial strains. The AMPs L18W-PGLa and MG2a from the magainin family, exhibit a tenfold increase in killing efficacy when applied as a cocktail. Using Large Unilamellar Vesicles (LUVs) as simple lipid-only mimics of bacterial cytoplasmic membranes we were recently able to correlate this synergistic effect to the formation of a sponge phase with a co-existing lamellar phase. Here we used time-resolved small angle X-ray scattering and dye-leakage experiments to investigate the kinetics of the single peptides and their equimolar mixture, including a chemically linked L18W-PGLa/MG2a heterodimer (hybrid). For the hybrid peptide as well as the equimolar mixture, the sponge phase was already present after the experimental deadtime (i.e. 1 min after mixing). All peptides induced a transition from LUVs to multilamellar vesicles with a collapsed interbilayer spacing, apart from MG2a which just lead to an adhesion of the LUVs. The positional correlations of lamellae within the multibilayers increased with time for all systems but for the mixture, fastest for the hybrid, slowest for MG2a. For the peptide mixture, the multibilayers showed an increasing disorder after their initial formation indicating additional ongoing processes during the 30 minute time span of the experiment. Further, also the lamellar repeat distance exhibited non-monotonic behavior during the multibilayer formation process, showing first a decrease, followed by a swelling that started several minutes after the addition of the peptides. Finally, we report on the significance of cardiolipin and membrane asymmetry for the interaction kinetics with the AMPs.

Synergistic structures in lyotropic lamellar gels.

In this work we present a systematic study on the microstructure of soft materials which combine the anisotropy of lyotropic liquid crystals with the mechanical stability of a physical gel. Systematic small-angle neutron (SANS) and X-ray (SAXS) scattering experiments were successfully used to characterize the lyotropic lamellar phase (Lα) of the system D2O -n-decanol - SDS which was gelled by two low molecular weight organogelators, 1,3:2,4-dibenzylidene-d-sorbitol (DBS) and 12-hydroxyoctadecanoic acid (12-HOA). Surprisingly, a pronounced shoulder appeared in the scattering curves of the lamellar phase gelled with 12-HOA, whereas the curves of the DBS-gelled Lα phase remained almost unchanged compared to the ones of the gelator-free Lα phase. The appearance of this additional shoulder strongly indicates the formation of a synergistic structure, which neither exists in the gelator-free Lα phase nor in the isotropic binary gel. By comparing the thicknesses of the 12-HOA (25-30 nm) and DBS (4-8 nm) gel fibers with the lamellar repeat distance (7.5 nm), we suggest that the synergistic structure originates from the minimization of the elastic free energy of the lamellar phase. In the case of 12-HOA, where the fiber diameter is significantly larger than the lamellar repeat distance, energetically unfavored layer ends can be prevented, when the layers cylindrically enclose the gel fibers. Interestingly, such structures mimic similar schemes found in neural cells, where axons are surrounded by lamellar myelin sheets.

Structure and Interactions between Charged Lipid Membranes in the Presence of Multivalent Ions.

When aqueous salt solutions contain multivalent ions (like Ca2+ or Mg2+), strong correlation effects may lead to ion-bridging, net attraction, and tight-coupling between like-charged interfaces. To examine the effects of surface charge density, temperature, salt type, and salt concentration on the structures of tightly coupled charged interfaces, we have used mixed lipid membranes, containing either saturated or unsaturated tails in the presence of multivalent ions. We discovered that tightly coupled membrane lamellar phases, dominated by attractive interactions, coexisted with weakly coupled lamellar phases, dominated by repulsive interactions. To control the membrane charge density, we mixed lipids with negatively charged headgroups, DLPS and DOPS, with their zwitterionic analogue having the same tails, DLPC and DOPC, respectively. Using solution X-ray scattering we measured the lamellar repeat distance, D, at different ion concentrations, temperatures, and membrane charge densities. The multivalent ions tightly coupled the mixed lipid bilayers whose charged lipid molar fraction was between 0.1 and 1. The repeat distance of the tightly coupled phase was about 4 nm for the DLPS/DLPC mixtures and about 5 nm for the DOPS/DOPC mixtures. In this phase, the repeat distance slightly increased with increasing temperature and decreased with increasing charge density. When the molar fraction of charged lipid was 0.1 or 0.25, a less tightly coupled phase coexisted with the tightly coupled phase. The weakly coupled lamellar phase had significantly larger D values, although they were consistently shorter than the D values in monovalent salt solutions with similar screening lengths.

Comparison of the structural characterization and physicochemical properties of starches from seven purple sweet potato varieties cultivated in China

In this study, the structural characterization and physicochemical properties of starches from seven purple sweet potato varieties (Fuzi No. 1, Guang No. 161, Jihei No. 1, Ningzi No. 1, Ningzi No. 2, Xuzi No. 6 and Xuzi No. 8) cultivated in China were compared. Starch granules of purple sweet potatoes all exhibited round, polygonal and hemispherical shapes with granule sizes ranging from 4.3 to 23.6 μm. X-ray powder diffraction patterns and 13C nuclear magnetic resonance spectra revealed purple sweet potato starches were CA-type with relative crystallinity varying from 34.0% to 37.3%. Small-angle X-ray scattering spectra indicated the lamellar repeat distances of starch granules were in the range of 9.962–10.137 nm. Ratios of 1045/1022 cm−1 and 1022/995 cm−1 of Fourier transform infrared spectra varied in the range of 0.689–0.887 and 0.850–0.974, respectively. Amylose contents of purple sweet potato starches differed from 18.2 to 27.2%. Purple sweet potato starches exhibited different gelatinization properties but similar thermal stability. Moreover, resistant starch contents varied from 29.25% to 43.50%. Our study indicated the granule size, relative crystallinity, the degree of short-range order, amylose content, gelatinization property and in vitro digestibility of purple sweet potato starches were greatly influenced by the variety of purple sweet potato.

Physicochemical properties and in vitro digestibility of high hydrostatic pressure treated waxy rice starch

Waxy rice starch slurry (10%, w/w) was treated with high hydrostatic pressure (HHP) (100–600 MPa) for 20 min and morphology, gelatinization property, X-ray diffraction pattern, semi-crystalline structure, and in vitro digestibility was determined. The morphology analysis showed that HHP treatment significantly affected the shape of starch granules. The thermal properties indicated an increase in gelatinization temperatures and decreased enthalpy with increasing pressure. However, compared with lower pressure, a complete gelatinization of starch was induced by 600 MPa. The lamellar repeat distance increased with increased pressure, while scattering peak disappeared at 600 MPa as a result of starch gelatinization. Based on the in vitro digestibility results of slowly digestible starch, HHP treatment at 400 MPa could be beneficial for improving the functionality of the waxy rice starch.

Multi-scale structures of cassava and potato starch fractions varying in granule size

Large-, medium-, and small-sized granules were separated from cassava and potato starches. Fourier transform infrared spectroscopy, wide angle X-ray diffraction, small angle X-ray scattering, nuclear magnetic resonance and scanning electron microscope were used to investigate the supramolecular structures of cassava and potato starch fractions. The crystallinity of small-sized potato starch (SPS) was lower than that of its counterparts, while crystallinities of all cassava fractions were similar. The contents of lamellar structure of small-sized granules were the smallest, while those of their counterparts were similar. The lamellar repeat distance was similar for the starch fractions. Self-similar structure of SPS showed mass fractal with the lowest compactness, while that of small-sized corn starch showed surface fractal with the largest compactness. The NMR test revealed that SPS had the highest total double helix content, while its counterparts showed similar values. Moreover, a new 13C peak at 64 ppm was observed for SPS.

The long periodicity phase (LPP) controversy part I: The influence of a natural-like ratio of the CER[EOS] analogue [EOS]-br in a CER[NP]/[AP] based stratum corneum modelling system: A neutron diffraction study

This study used neutron diffraction to investigate a ceramide-[NP] C24/[AP] C24 /[EOS]-br C30/cholesterol/lignoceric acid (0.6: 0.3: 0.1: 0.7: 1) based stratum corneum modelling system. By adding specifically deuterated ceramides-[NP]-D3, [AP]-D3, and [EOS]-br-D3, detailed information on the lamellar and the nanostructure of the system was obtained. For the short periodicity phase a natural-like lamellar repeat distance of 5.47 ± 0.02 nm was observed, similar to the [NP]/[AP] base system without the [EOS]-br. Unlike in this system the ceramides here were slightly tilted, hinting towards a slightly less natural arrangement. Due to the deuteration it was possible to observe that the long ceramide chains were overlapping in the lamellar mid-plane. This is considered to be an important feature for the natural stratum corneum. Despite the presence of a ceramide [EOS] analogue – able to form a long phase arrangement – no distinct long periodicity phase was formed, despite a slightly higher than natural ω-acyl ceramide ratio of 10 mol%. The deuterated variant of this ceramide determined that the very long ceramide was integrated into the short periodicity phase, spanning multiple layers instead. The – compared to the base system – unchanged repeat distance highlights the stability of this structure. Furthermore, the localisation of the very long ceramide in the short periodicity phase indicates the possibility of a crosslinking effect and thus a multilayer stabilizing role for the ceramide [EOS]. It can be concluded, that additionally to the mere presence of ceramide-[EOS] more complex conditions have to be met in order to form this long phase. This has to be further investigated in the future.

Impact of the ceramide subspecies on the nanostructure of stratum corneum lipids using neutron scattering and molecular dynamics simulations. Part I: impact of CER[NS

For this study mixtures based on the ceramides [NS] (NS = non-hydroxy-sphingosine) and [AP] (AP = α-hydroxy-phytosphingosine) in a 2:1 and 1:2 ratio, together with cholesterol and lignoceric acid, were investigated. These mixtures are modelling the uppermost skin layer, the stratum corneum. Neutron diffraction, utilizing specifically deuterated ceramide molecules, was used to obtain a maximum amount of experimental detail. Highly detailed molecular dynamics simulations were used to generate even more information from the experimental data.It was possible to observe a single lamellar phase for both systems. They had a lamellar repeat distance of 5.43 ± 0.05 nm for the [NS]/[AP] 2:1 and a slightly shorter one of 5.34 ± 0.05 nm for the 1:2 system. The structure and water content was uninfluenced by excess humidity. Both the experimental and simulation data indicated slightly tilted ceramides, with their C24 chains overlapping in the lamellar mid-plane. This arrangement is well comparable to systems investigated before. The structure of both systems, except for the differing repeat distance, looks similar at first. However, on a smaller scale there were various distinct differences, demonstrating only low redundancy between the different ceramide species, despite only minor chemical differences. The mainly ceramide [AP] determined 1:2 system has a slightly smaller repeat distance. This is a result of a tighter arrangement of the lipids chain along the bilayer normal and increased overlapping of the long chains in the lamellar middle. For the CER[NS] some novel features could be shown, despite it being the overall most investigated ceramide. These include the low adaptability to changed lateral interactions, leading to an increased chain opening. This effect could explain its low miscibility with other lipids.The investigated model systems allows it to directly compare results from the literature which have used ceramide [NS] to the most recent studies using the phytosphingosine ceramides such as ceramide [AP].

Effect of alkan-1-ols on the structure of dopc model membrane

Abstract The effect of general anaesthetics alkan-1-ols (CnOH, where n = 10, 12, 14, 16 and 18 is the number of carbon atoms in the molecule) on the structure of dioleoylphosphatidylcholine (DOPC) model membrane was studied by small-angle neutron scattering (SANS) and small-angle neutron diffraction (SAND). Fluid bilayers were prepared at CnOH:DOPC = 0.3 molar ratio. The results of both the experiments show that bilayer thickness - a thickness parameter dg in the case of SANS and lamellar repeat distance D in the case of SAND - increases with increasing n. A coexistence of two lamellar phases with different D was detected by measuring the C18OH+DOPC oriented sample.

Electrostatic double-layer interaction between stacked charged bilayers.

The inapplicability of the DLVO theory to multilayered anionic bilayers is found in terms of the co-ion-valence dependence of the lamellar repeat distance. Most of the added salt is expelled from the interlamellar space to the bulk due to the Gibbs-Donnan effect on multiple bilayers with the bulk. The electrostatic double-layer interaction is well expressed by the formula recently proposed by Trefalt. The osmotic pressure due to the expelled ions, rather than the van der Waals interaction, is the main origin of the attractive force between the bilayers.

Low-pH induced reversible reorganizations of chloroplast thylakoid membranes — As revealed by small-angle neutron scattering

Energization of thylakoid membranes brings about the acidification of the lumenal aqueous phase, which activates important regulatory mechanisms. Earlier Jajoo and coworkers (2014 FEBS Lett. 588:970) have shown that low pH in isolated plant thylakoid membranes induces changes in the excitation energy distribution between the two photosystems. In order to elucidate the structural background of these changes, we used small-angle neutron scattering on thylakoid membranes exposed to low p2H (pD) and show that gradually lowering the p2H from 8.0 to 5.0 causes small but well discernible reversible diminishment of the periodic order and the lamellar repeat distance and an increased mosaicity — similar to the effects elicited by light-induced acidification of the lumen. Our data strongly suggest that thylakoids dynamically respond to the membrane energization and actively participate in different regulatory mechanisms.

Salt Effects on Lamellar Structure of Nonionic Surfactants

Effects of monovalent and monatomic salts on lamellar repeat distances d of nonionic surfactants (monomyristolein and $${\mathrm {C_{12}E_{2}}}$$ ) are investigated using small-angle X-ray diffraction. The lamellar repeat distances (sum of thicknesses of a bilayer and a sandwiched water layer) increase with increasing salt concentration with a strong anion dependence (Br $$^->$$ Cl $$^- $$ ). The increase of the thickness of the water layer is found to dominate the increase in d. Since the anion dependence is inconsistent with the ion dependence of the strength of the primary hydration, we reported previously (Hishida et al. J Chem Phys 142:171101, 2015), the hydration force classically considered is not the origin of the increase in d. This means the increase in d cannot be explained by the existing model of the forces between neutrally charged bilayers. The temperature dependence of d also supports the necessity for a new mechanism of the effect of ions. The new mechanism seems to be related to the water structure beyond the primary hydration water, i.e., the secondary hydration water, which depends on the ion species.