Physicochemical characteristics of tea seed starches from twenty-five cultivars.

The physicochemical features of starches separated from tea seeds of 25 cultivars were analyzed. The distinct characteristic of tea seed starches was that they had high apparent amylose content (AAC, 28.94-39.91 %) and resistant starch contents (4.64-8.24 %), suggesting that tea starch can be used for production of low glycemic index food. One cultivar (T12) had smallest breakdown (74.2 RVU) and highest gel hardness, indicating it performed stably during shear thinning, resulting in a firm texture. Another cultivar (T25) had a peak viscosity of 417.6 RVU, a large breakdown and small setback, suggesting a low tendency for retrogradation. There was a range of 61.6 °C to 77.5 °C for the peak gelatinization temperature and 0.163 to 0.390 for the flow behavior index values. These parameters could serve for selecting suitable starches with minor differences in physicochemical properties for food use. Correlation analysis indicated that AAC is a key factor determining starch retrogradation properties. The broad genetic diversity in the tea seed starch physicochemical features provided potentially versatile applications in the food industry. The results gained from the present study contribute to a better understanding of tea seed starch quality, and encourage its application in many value-added food products.

Effect of ultrasound/CaCl2 co-treatment on the microstructure, gelatinization, and film-forming properties of high amylose corn starch.

The effect of ultrasound/CaCl2 co-treatment on aggregation structure, thermal stability, rheological, and film properties of high amylose corn starch (HACS) was investigated. The scanning electron microscopy (SEM) images revealed the number of starch fragments and malformed starch granules increased after co-treatment. The differential scanning calorimetry (DSC) results showed the co-treated HACS got a lower gelatinization temperature (92.65 ± 0.495 °C) and enthalpy values (ΔH, 4.14 ± 0.192 J/g). The optical microscope images indicated that lesser Maltase crosses were observed in co-treated HACS. The results of X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) indicated ultrasound influenced the compactness of amorphous zone and CaCl2 damaged the crystalline region of HACS granules. Additionally, the rheology properties of HACS dispersion demonstrated the apparent viscosity of co-treated dispersion increased as the ultrasound time prolonged. The mechanical strength and structural compactness of HACS films were improved after ultrasound treatment. The mechanism of ultrasound/CaCl2 co-treatment improved the gelatinization and film-forming ability of HACS was that (i) ultrasound wave loosened the HACS granules shell, promoted the treatment of CaCl2 on HACS granules, and (ii) ultrasound wave improved the uniform distribution of HACS dispersion, increased the interaction between CaCl2 and starch chains during the process of film-forming.

Correlation Study between Multi-Scale Structure and In Vitro Digestibility of Starch Modified by Temperature Difference.

Physical techniques are widely applied in the food industry due to their positive impact on food quality and the environment. Temperature differences can effectively modify starch, but the resulting changes in starch structure and quality remain unclear. In this study, the corn starch was processed with high temperature, low temperature, and temperature difference (TD), including high temperature before low temperature (H-L) and low temperature before high temperature (L-H). The results showed that high temperature induced the umbilicus to concave inward shape and sharply decreased the amylose content, while low temperature increased the surface micropores and reduced the A-chain. TD reduced the fluorescence intensity and increased the clearness of the growth ring. TD elevated the relative crystallinity (RC), short-range order, A/B1 chains, hydrolysis parameters, and resistant starch (RS), and reduced amylose content, B2/B3 chains, and viscosity. Moreover, the corn starches treated by H-L had lower amylose content and higher RC, 1047/1022, A-chain, and RS than those treated by L-H. Overall, high temperature degraded the amylose and low temperature destroyed the amylopectin. During the TD, H-L can accelerate the starch molecular rearrangement more than the opposite temperature treatment order. These results will help produce novel starches for better food applications.

Boosting resistant starch in rice: Bacterial inulin as a metabolic and glucose uptake modulator.

Bacillus stercoris PSSR12 (B. stercoris PE), an isolate from rice field soils, was identified via 16s rRNA sequencing. The synthesis of the inulin and inulin producing enzyme (IPE) in B. stercoris PE was verified using SDS-PAGE and FTIR. This study aimed to assess the impact of B. stercoris PE treatment on in vitro inhibition of α-amylase and α-glucosidase from traditional and commercial rice varieties of South India. Additionally, the study investigated enzymatic inhibition and mRNA expression of starch synthesis genes (RAmy1a, GBSSIa, SBEIIa, and SBEIIb). Glucose transporter gene expression (GLUT1 and GLUT4) patterns were analyzed in 3T3-L1 adipocytes to evaluate glucose uptake in B. stercoris PE treated rice varieties. The application of B. stercoris PE enhanced grain quality by imparting starch ultra-structural rigidity, inhibiting starch metabolizing enzymes, and inducing molecular changes in starch synthesis genes. This approach holds promise for managing type II diabetes mellitus and potentially reducing insulin dependence.

Effects of crystallization temperature on structure and digestibility of spherulites formed from debranched high-amylose maize starch.

High-amylose maize starch (69.3 % amylose) was debranched to increase the level of linear molecules and enhance the formation of spherulites. Debranched high-amylose maize starch (25 %, w/w) was heated to 180 °C in a Parr reactor followed by crystallization at different temperatures between 25 and 150 °C. The objectives of this study were to investigate the effects of crystallization temperature on the yield, morphology, structure, crystallinity, and digestibility of the spherulites formed. When the crystallization temperature was 150 °C, spherulites with negative birefringent sign were formed. High crystallization temperature caused molecular degradation and the degree of degradation was severe at 150 °C, resulting in relatively short chain amylose (DP < 150). When crystallized at 25 to 120 °C, spherulites with strong positive birefringence were produced. The long chain amylose was attributed to the positive birefringence. All spherulites had a predominant B-type crystalline structure. The spherulites with negative birefringence showed a lower degree of crystallinity and lower resistance to enzyme digestion, but all the spherulites with positive birefringence had a high resistant starch content (89-94 %). α-Amylase was not able to penetrate inside the spherulites as revealed by the confocal laser scanning microscopic images.

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.

Key structural factors that determine the in vitro enzymatic digestibility of amylose-complexes.

The effects of complexing conditions on the formation of amylose-lipid-protein complexes and relationships between structure and digestion of amylose-lipid and amylose-lipid-protein complexes were poorly understood. The objective of this study was to investigate the effects of complexing time (0, 0.5, 2, 4 and 6 h) and temperature (60, 70, 80, 90 and 100 °C) on the structure and in vitro amylolysis of amylose-lauric acid (AM-LA) and amylose-lauric acid-ß-lactoglobulin (AM-LA-ßLG) complexes, and to understand the relationships between structure and in vitro digestiblity of these complexes. Longer complexing time and higher complexing temperature promoted the formation of greater amounts of the more stable type II crystallites than type I crystallites in both AM-LA and AM-LA-ßLG complexes, which in turn decreased the rate and extent of the complexes digestion to a greater extent. Correlation analyses between parameters for structure and digestion kinetics showed that both the quantity of AM-LA and AM-LA-ßLG complexes and the quality of their arrangement into V-type crystallites influenced their rate and extent of digestion. This study demonstrates that AM-LA and AM-LA-ßLG complexes can be prepared with designed structural and functional properties tailored for various applications.

Transcription factor OsbZIP10 modulates rice grain quality by regulating OsGIF1.

Understanding and optimizing the process of grain filling helps the quest to maximize rice (Oryza sativa L.) seed yield and quality, yet the intricate mechanisms at play remain fragmented. Transcription factors (TFs) are major players in the gene networks underlying the grain filling process. Here, we employed grain incomplete filling (OsGIF1)/cell wall invertase 2, a key gene involved in grain filling, to explore its upstream TFs and identified a bZIP family TF, OsbZIP10, to be a transcriptional activator of OsGIF1. Rice grains of the knockouts of OsbZIP10 showed increased white-core rates but lower amylose content (AC), leading to better eating and cooking qualities in all genetic backgrounds investigated, though the impact of mutations in OsbZIP10 on grain weight depended on genetic background. Multi-omics analyses suggested that, in addition to OsGIF1, multiple genes involved in different biological processes contributing to grain filling were targeted by OsbZIP10, including OsAGPS1, a gene encoding the ADP-Glc pyrophosphorylase (AGPase) small subunit, and genes contributing to homeostasis of reactive oxygen species. Distinct genetic make-up was observed in OsbZIP10 between japonica and indica rice varieties, with the majority varieties of each subspecies belonging to two different haplotypes that were closely associated with AC. Overexpressing the haplotype linked to high-AC in the low-AC genetic background increased AC. Overall, this study sheds crucial light on the significance of the OsbZIP10-OsGIF1 module in the determination of rice grain quality, offering a potential avenue for genetic engineering of rice to produce seeds with tailored attributes.

Tuber crops could be a potential food component for lowering starch digestibility and estimated glycemic index in rice.

BACKGROUND: Rice is considered a high estimated glycemic index (eGI) food because of its higher starch digestibility, which leads to type II diabetes and obesity as a result of a sedentary life style. Furthermore, the incresaing diabetes cases in rice-consuming populations worldwide need alternative methods to reduce the glycemic impact of rice, with dietary prescriptions based on the eGI value of food being an attractive and practical concept. Rice is often paired with vegetables, pulses, tubers and roots, a staple food group in Africa, Latin America and Asia, which are rich in fibre and health-promoting compounds. RESULTS: Rice from four categories (high protein, scented, general and pigmented) was analyzed for eGI and resistant starch (RS) content. Among the genotypes, Improved Lalat had the lowest eGI (53.12) with a relatively higher RS content (2.17%), whereas Hue showed the lowest RS (0.19%) with the highest eGI (76.3) value. The addition of tuber crops to rice caused a significant lowering of eGI where the maximum beneficial effect was shown by elephant foot yam (49.37) followed by yam bean (53.07) and taro (54.43). CONCLUSION: The present study suggests that combining rice with suitable tuber crops can significantly reduce its eGI value, potentially reducing the burden of diet-associated lifestyle diseases particularly diabetics. © 2024 Society of Chemical Industry.

Effect of Fenton reaction parameters on the structure and properties of oxidized wheat starch.

Wheat starch was oxidized through a Fenton reaction by hydrogen peroxide and Iron II sulfate as a catalyst at various concentrations and reaction duration. The formation of carbonyl and carboxyl groups confirmed the starch oxidation as determined with Fourier-transform infrared (FTIR) spectroscopy. The degree of oxidation was estimated by carbonyl and carboxyl titration. The various oxidized wheat starches presented considerable variations in their oxidation level as a function of the catalyst concentration and oxidative process duration. The effect of the Fenton reaction parameters on the starch macromolecular chains and microstructure was evaluated by X-ray diffraction and amylose content estimation. Significant depolymerization of the starch macromolecules was observed, mainly in the starch amorphous phase, followed by a degradation of the crystalline phase at a higher oxidation level. SEM observations revealed changes in starch structure, which ranged from minor degradation of the starch granules to a more crosslinked morphology.

High-amylose starch-based gel as green adhesive for plywood: Adhesive property, water-resistance, and flame-retardancy.

The escalating demand for environmentally sustainable and cost-effective adhesives in the wood processing and manufacturing sector has prompted exploration into innovative solutions. This study introduces a novel gel adhesive composed of chemically unmodified high-amylose starch (G70, with 68 % amylose content) with a minimal proportion of urea-formaldehyde (UF) (UF/starch = 1:10, w/w). This G70/UF gel demonstrates remarkable adhesive capabilities for wooden boards under both dry conditions (with a shear stress of 4.13 ± 0.12 MPa) and wet conditions (with a shear strength of 0.93 ± 0.07 MPa after 2 h of water soaking). The study unveils that the elevated amylose content in the starch, coupled with a meticulously controlled isothermal process during bonding, is crucial for these enhancements. Specifically, the robust cohesion of amylose chains expedites phase separation between starch and UF, while the isothermal process facilitates the migration and enrichment of UF molecules at the gel-board and gel-air interfaces. Lacking these mechanisms, conventional amylopectin-rich starch/UF gels (27 % amylose content) show minimal improvement. Moreover, the G70/UF gel showcases exceptional fire retardancy. In all, the G70/UF gel presents a promising alternative for plywood production, reducing reliance on unhealthy UF resin while offering satisfactory bonding resistance in diverse conditions and superior flame retardancy.

The importance of starch chain-length distribution for in vitro digestion of ungelatinized and retrograded foxtail millet starch.

The digestibility of ungelatinized, short-term retrograded and long-term retrograded starch from foxtail millet was investigated and correlated with starch chain length distributions (CLDs). Some variations in starch CLDs of different varieties were obtained. Huangjingu and Zhonggu 9 had higher average chain lengths of debranched starch and lower average chain length ratios of amylopectin and amylose than Dajinmiao and Jigu 168. Compared to ungelatinized starch, retrogradation significantly increased the estimated glycemic index (eGI), whereas significantly decreased the resistant starch (RS). In contrast, long-term retrograded starches have lower eGI (93.33-97.37) and higher RS (8.04-14.55%) than short-term retrograded starch. PCA and correlation analysis showed that amylopectin with higher amounts of long chains and longer long chains contributed to reduced digestibility in ungelatinized starch. Both amylose and amylopectin CLDs were important for the digestibility of retrograded starch. This study helps a better understanding of the interaction of starch CLDs and digestibility during retrogradation.

Starch chain-length distributions affect the processing and digestion characteristics of proso millet starch.

Starch chain-length distributions play a key role in regulating the processing and digestion characteristics of proso millet starch. Waxy proso millet starch has higher endothermic enthalpy (13.06-16.73 J/g) owing to its higher relative crystallinity (27.83%-32.04%), while nonwaxy proso millet starch has lower peak viscosity (1.0630-1.1930 Pa∙s) and stronger viscoelasticity owing to its higher amylose content (21.72%-24.34%). Non-waxy proso millet starch exhibited two different digestion phases and its resistant starch content (18.37%-20.80%) was higher than waxy proso millet starch. Correlation analysis showed proso millet starch with longer amylopectin B1 chains and more amylopectin B2 chains exhibited excellent thermal ability and retrograde resistance, whereas proso millet starch with shorter and more amylose medium/long-chains not only reduced the digestion rate and increased the resistant starch content but also exhibited stronger viscoelasticity and excellent retrogradation properties. These results could provide more insights into efficient utilization of proso millet starch.

Quantum Chemical Investigation into the Structural Analysis and Calculated Raman Spectra of Amylose Modeled with Linked Glucose Molecules.

This study presents a quantum chemical investigation into the structural analysis and calculated Raman spectra of modeled amylose with varying units of linked glucose molecules. We systematically examined the rotation of hydroxymethyl groups and intramolecular hydrogen bonds within these amylose models. Our study found that as the number of linked glucose units increases, the linear structure becomes more complex, resulting in curled, cyclic, or helical structures facilitated by establishing various intramolecular interactions. The hydroxymethyl groups were confirmed to form interactions with oxygen atoms and with hydroxymethyl and hydroxyl groups from adjacent rings in the molecular structures. We identified distinct peaks and selected specific bands applicable in various analytical contexts by comparing their calculated Raman spectra. Representative vibrational modes within selected regions were identified across the different lengths of amylose models, serving as characteristic signatures for linear and more coiled structural conformations. Our findings contribute to a deeper understanding of amylose structures and spectroscopic signatures, with implications for theoretical studies and potential applications. This work provides valuable reference points for the detailed assignment of Raman peaks of amylose structure, facilitating their application in broader research on carbohydrate structures and their associated spectroscopic properties.

Multifunctional and edible egg white/amylose-tannin bilayer film for perishable fruit preservation.

The substantial waste of perishable foods during transportation significantly contributes to greenhouse gas emissions, intensifying the climate crisis. To mitigate the rapid spoilage of fruits, an eco-friendly bilayer film was developed using natural egg white (EW), amylose (Am), and tannic acid (TA). The EW/Am-TA bilayer film features a primary layer of amphiphilic EW, ensuring a uniform coating on hydrophobic fruit surfaces, and a secondary layer composed of Am and TA, imparting notable tensile strength (5.3 ± 0.5 MPa) and elongation at break (28.5 ± 4.1 %). This bilayer film effectively shields fruits from UV-B and UV-C radiation (~0 % transmittance at 280 and 330 nm) and exhibits antioxidant and antibacterial properties due to the presence of TA. Fruits such as bananas, avocados, and cherry tomatoes, when dip-coated with the optimized EW/Am-TA bilayer, maintained their freshness, color, weight, and texture for up to seven days, demonstrating the effectiveness of this bilayer coating in food preservation. The natural materials in the coated film are edible and can be safely removed with tap water at room temperature in <10 s, posing no food safety risks. Thus, the proposed bilayer coating presents a significant solution to the global problem of food waste.

Optimization of Liquid Crystalline Mixtures Enantioseparation on Polysaccharide-Based Chiral Stationary Phases by Reversed-Phase Chiral Liquid Chromatography.

Enantioseparation of nineteen liquid crystalline racemic mixtures obtained based on (R,S)-2-octanol was studied in reversed-phase mode on an amylose tris(3-chloro-5-methylphenylcarbamate) (ReproSil Chiral-MIG) and a cellulose tris(3,5-dichlorophenylcarbamate) (ReproSil Chiral-MIC). These polysaccharide-based chiral stationary phase (CSP) columns for High-Performance Liquid Chromatography (HPLC) were highly effective in recognizing isomers of minor structural differences. The mobile phase (MP), which consists of acetonitrile (ACN)/water (H2O) at different volume ratios, was used. The mobile phases were pumped at a flow rate of 0.3, 0.5, or 1 mL·min-1 with a column temperature of 25 °C, using a UV detector at 254 nm. The order of the elution was also determined. The chromatographic parameters, such as resolution (Rs), selectivity (α), and the number of theoretical plates, i.e., column efficiency (N), were determined. The polysaccharide-based CSP columns have unique advantages in separation technology, and this study has shown the potential usefulness of the CSP columns in separating liquid crystalline racemic mixtures belonging to the same homologous series.

The Waxy Gene Has Pleiotropic Effects on Hot Water-Soluble and -Insoluble Amylose Contents in Rice (Oryza sativa) Grains.

Rice (Oryza sativa) is a cereal crop with a starchy endosperm. Starch is composed of amylose and amylopectin. Amylose content (AC) is the principal determinant of rice quality, but varieties with similar ACs can still vary substantially in their quality. In this study, we analyzed the total AC (TAC) and its constituent fractions, the hot water-soluble amylose content (SAC) and hot water-insoluble amylose content (IAC), in two sets of related chromosome segment substitution lines of rice with a common genetic background grown in two years. We searched for quantitative trait loci (QTLs) associated with SAC, IAC, and TAC and identified one common QTL (qSAC-6, qIAC-6, and qTAC-6) on chromosome 6. Map-based cloning revealed that the gene underlying the trait associated with this common QTL is Waxy (Wx). An analysis of the colors of soluble and insoluble starch-iodine complexes and their λmax values (wavelengths at the positions of their peak absorbance values) as well as gel permeation chromatography revealed that Wx is responsible for the biosynthesis of amylose, comprising a large proportion of the soluble fractions of the SAC. Wx is also involved in the biosynthesis of long chains of amylopectin, comprising the hot water-insoluble fractions of the IAC. These findings highlight the pleiotropic effects of Wx on the SAC and IAC. This pleiotropy indicates that these traits have a positive genetic correlation. Therefore, further studies of rice quality should use rice varieties with the same Wx genotype to eliminate the pleiotropic effects of this gene, allowing the independent relationship between the SAC or IAC and rice quality to be elucidated through a multiple correlation analysis. These findings are applicable to other valuable cereal crops as well.

Design and Characterization of a Novel Core-Shell Nano Delivery System Based on Zein and Carboxymethylated Short-Chain Amylose for Encapsulation of Curcumin.

Curcumin is a naturally occurring hydrophobic polyphenolic compound with a rapid metabolism, poor absorption, and low stability, which severely limits its bioavailability. Here, we employed a starch-protein-based nanoparticle approach to improve the curcumin bioavailability. This study focused on synthesizing nanoparticles with a zein "core" and a carboxymethylated short-chain amylose (CSA) "shell" through anti-solvent precipitation for delivering curcumin. The zein@CSA core-shell nanoparticles were extensively characterized for physicochemical properties, structural integrity, ionic stability, in vitro digestibility, and antioxidant activity. Fourier-transform infrared (FTIR) spectroscopy indicates nanoparticle formation through hydrogen-bonding, hydrophobic, and electrostatic interactions between zein and CSA. Zein@CSA core-shell nanoparticles exhibited enhanced stability in NaCl solution. At a zein-to-CSA ratio of 1:1.25, only 15.7% curcumin was released after 90 min of gastric digestion, and 66% was released in the intestine after 240 min, demonstrating a notable sustained release effect. Furthermore, these nanoparticles increased the scavenging capacity of the 1,1-diphenyl-2-picrylhydrazyl (DPPH•) free radical compared to those composed solely of zein and were essentially nontoxic to Caco-2 cells. This research offers valuable insights into curcumin encapsulation and delivery using zein@CSA core-shell nanoparticles.

Insight to starch retrogradation through fine structure models: A review.

The retrogradation of starch is crucial for the texture and nutritional value of starchy foods products. There is mounting evidence highlighting the significant impact of starch's fine structures on starch retrogradation. Because of the complexity of starch fine structure, it is a formidable challenge to study the structure-property relationship of starch retrogradation. Several models have been proposed over the years to facilitate understanding of starch structure. In this review, from the perspective of starch models, the intricate structure-property relationship is sorted into the correlation between different types of structural parameters and starch retrogradation performance. Amylopectin B chains with DP 24-36 and DP ≥36 exhibit a higher tendency to form ordered crystalline structures, which promotes starch retrogradation. The chains with DP 6-12 mainly inhibit starch retrogradation. Based on the building block backbone model, a longer inter-block chain length (IB-CL) enhances the realignment and reordering of starch. The mathematical parameterization model reveals a positive correlation between amylopectin medium chains, amylose short chains, and amylose long chains with starch retrogradation. The review is structured according to starch models; this contributes to a clear and comprehensive elucidation of the structure-property relationship, thereby providing valuable references for the selection and utilization of starch.

Complexation Mechanisms of Aqueous Amylose: Molecular Dynamics Study Using 3-Pentadecylphenol.

Molecular dynamics (MD) simulations of linear amylose fragments containing 10 to 40 glucose units were used to study the complexation of the prototypical compound, 3-pentadecylphenol (PDP)─a natural product with surfactant-like properties─in aqueous solution. The amylose-PDP binding leverages mainly hydrophobic interactions together with excluded volume effects. It was found that while the most stable complexes contained PDP inside the helical structure of the amylose in the expected guest-host (inclusion) complexation manner, at higher temperatures, the commonly observed PDP-amylose complexes often involved more nonspecific interactions than inclusion complexation. In the case where a stoichiometric excess of PDP was added to the simulation box, self-aggregation of the small molecule precluded its ability to enter the internal helical part of the oligosaccharide, and as a result, inclusion complexation became ineffective. MD simulation trajectories were analyzed preliminarily using cluster analysis (CA), followed by more rigorous solvent accessible surface area (SASA) determination over the temperature range spanning from 277 to 433 K. It was found that using the SASA of PDP corrected for its intrinsic conformational changes, together with a generic hidden Markov model (HMM), an adequate quantification of the different types of PDP-amylose aggregates was obtained to allow further analysis. The enthalpy change associated with the guest-host binding equilibrium constant (Kgh) in aqueous solution was estimated to be -75 kJ/mol, which is about twice as high as one might expect based on experimentally measured values of similar complexes in the solid state where the (unsolvated) helical structure of amylose remains rigid. On the other hand, the nonspecific binding (Kns) enthalpy change associated with PDP-amylose interactions in the same solution environment was found to be about half of the inclusion complexation value.