Starch Molecules Research Articles

Characterization of chestnut starch acetate with different degrees of substitution

Abstract Chestnut starch acetates (CSA) with different degrees of substitution (DS) were prepared. The structure and physicochemical properties of CSA were then determined, with scanning electron microscopy showing that most of the CSA granules were damaged and dented, and adhered with increased in DS. X-ray diffraction results indicated that the crystal form of CS and CSA was type C. Chemical structure analysis showed that the starch molecule was grafted with acetyl groups. The transparency, freeze–thaw stability, solubility and swelling power of CSA improved with an increase in DS. The viscosity and stability of CSA were significantly improved and pasting temperatures reduced compared with native CS. The cohesion, hardness, gumminess, chewiness and springiness of CSA decreased with an increase in DS, whereas adhesiveness increased. By comparing the properties of CSA with different DS, a new option was provided for the application of renewable natural polymer CSA in food fields.

Pho1 cooperates with DPE1 to control short maltooligosaccharide mobilization during starch synthesis initiation in rice endosperm.

Plastidial α-glucan phosphorylase is a key factor that cooperates with plastidial disproportionating enzyme to control short maltooligosaccharide mobilization during the initiation process of starch molecule synthesis in developing rice endosperm. Storage starch synthesis is essential for grain filling. However, little is known about how cereal endosperm controls starch synthesis initiation. One of core events for starch synthesis initiation is short maltooligosaccharide (MOS) mobilization consisting of long MOS primer production and excess MOS breakdown. By mutant analyses and biochemical investigations, we present here functional identifications of plastidial α-glucan phosphorylase (Pho1) and disproportionating enzyme (DPE1) during starch synthesis initiation in rice (Oryza sativa) endosperm. Pho1 deficiency impaired MOS mobilization, triggering short MOS accumulation and starch synthesis reduction during early seed development. The mutant seeds differed significantly in MOS level and starch content at 15days after flowering and exhibited diverse endosperm phenotypes during mid-late seed development: ranging from pseudonormal to shrunken (Shr), severely or excessively Shr. The level of DPE1 was almost normal in the PN seeds but significantly reduced in the Shr seeds. Overexpression of DPE1 in pho1 resulted in plump seeds only. DPE1 deficiency had no obvious effects on MOS mobilization. Knockout of DPE1 in pho1 completely blocked MOS mobilization, resulting in severely and excessively Shr seeds only. These findings show that Pho1 cooperates with DPE1 to control short MOS mobilization during starch synthesis initiation in rice endosperm.

Arabinoxylan of varied structural features distinctively affects the functional and in vitro digestibility of wheat starch

This study explored the effects of wheat bran arabinoxylan (AX) of various molecular structures on the functional and in vitro digestibility of wheat starch. AX was separated with sequential ethanol precipitation into different fractions (L-Fra and H-Fra), followed by acidic treatment at varied time to convert L-Fra into different subfractions (H-Sub, M-Sub and L-Sub), and the structural features of AX and its subsequent fractions were compared. The Mw and branching degree of the fractions were decreased in the order of H-Fra (545 kDa, 1.15), L-Fra (86 kDa, 0.35), H-Sub (38 kDa, 0.16), M-Sub (21 kDa, 0.10) and L-Sub (7 kDa, 0.08). The functional properties, such as pasting properties, rheological properties, thermal properties, and their effect on the in vitro digestibility of wheat starch-AX combinations, were closely related to the molecular structures of AX. AX with higher Mw, higher branching degree and phenolic content could better inhibit the starch gelatinization and retrogradation, thus reducing the apparent viscosity and gelling properties of wheat starch. The possible mechanisms can be illustrated as 1) AX could compete for water with starch to inhibit granule expansion; 2) AX could not only adsorb on the surface of starch granules through hydrogen bonds but also entangle with the leached amylose and wrap on the surface of granules; 3) AX contained phenols, leading them to inhibit granule expansion through non-covalent interactions with starch molecules. Overall, this study provides a theoretical basis to improve the quality of wheat starch foods and extend the shelf life of wheat foods.

Production of highly branched α-limit dextrins with enhanced slow digestibility by various glycogen-branching enzymes

α-Limit dextrins (α-LDx) are slowly digestible carbohydrates that attenuate postprandial glycemic response and trigger the secretion of satiety-related hormones. In this study, more highly branched α-LDx were enzymatically synthesized to enhance the slowly digestible property by various origins of glycogen branching enzyme (GBE), which catalyzes the transglycosylation to form α-1,6 branching points after cleaving α-1,4 linkages. Results showed that the proportion of branched α-LDx in starch molecules increased around 2.2–8.1 % compared to α-LDx from starch without GBE treatment as the ratio of α-1,6 linkages increased after different types of GBE treatments. Furthermore, the enzymatic increment of branching points enhanced the slowly digestible properties of α-LDx at the mammalian α-glucosidase level by 17.3–28.5 %, although the rates of glucose generation were different depending on the source of GBE treatment. Thus, the highly branched α-LDx with a higher amount of α-1,6 linkages and a higher molecular weight can be applied as a functional ingredient to deliver glucose throughout the entire small intestine without a glycemic spike which has the potential to control metabolic diseases such as obesity and type 2 diabetes.

Insight in changes in starch and proteins molecular structure of non-wheat cereal flours influenced by roasting and extrusion treatments

Barley, rye, triticale, oat, sorghum and millet flours, subjected to roasting or extrusion treatment, were analyzed by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), Fourier-transform infrared (FTIR) and Raman spectroscopy with aim to determine structural changes within starch and protein molecules that caused specific rheological properties of heat treated flours. Mixolab was used to determine how changes in starch and protein structure are reflected in rheological properties of dough. Extrusion treatment caused more extensive changes both in starch and protein structure than roasting treatment. Changes of protein secondary structure from α-helix to β-turn aggregated by hydrogen bonds, formed mainly between tyrosyl groups, as well as change in disulphide bond conformation, were more pronounced in extruded flour samples. Changes in the starch structure were also more extensive in extruded flours samples, including complete gelatinization and formation of amylose-lipid complexes. The results obtained by Mixolab showed that all extruded doughs showed resistance at C2 point, which was shifted to approximately 30 min, indicating higher mechanical resistance. It was supposed that this change in dough behaviour is related to protein polymerization during extrusion treatment and starch aggregation during roasting treatment.

Complexation between rice starch and cellulose nanocrystal from black tea residues: Gelatinization properties and digestibility in vitro

In this work, cellulose nanocrystal (CNC) was extracted from black tea waste and its effects on the physicochemical properties of rice starch were explored. It was revealed that CNC improved the viscosity of starch during pasting and inhibited its short-term retrogradation. The addition of CNC changed the gelatinization enthalpy and improved the shear resistance, viscoelasticity, and short-range ordering of starch paste, which meant that CNC made the starch paste system more stable. The interaction of CNC with starch was analyzed using quantum chemistry methods, and it was demonstrated that the hydrogen bonds were formed between starch molecules and the hydroxyl groups of CNC. In addition, the digestibility of starch gels containing CNC was significantly decreased because CNC could dissociate and act as an inhibitor of amylase. This study further expanded the understanding of the interactions between CNC and starch during processing, which could provide a reference for the application of CNC in starch-based foods and the development of functional foods with a low glycemic index.

Preparation and Physico-Chemical Characterization of OSA-Modified Starches from Different Botanical Origins and Study on the Properties of Pickering Emulsions Stabilized by These Starches.

Native starch (NS) from different botanical origins (native rice/tapioca/oat starch, NRS/NTS/NOS) were hydrophobically modified by octenyl succinic anhydride (OSA), and the octenyl succinic (OS) groups were successfully introduced in the starch molecules which obtained OS-starch (OSRS, OSTS and OSOS) with different levels of modification (0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%) and degree of substitution (DS). The structural properties of the OS-starch, such as granule size, crystal, wettability and morphology were studied, and the OS-starch was used as particulate stabilizers to produce oil-in-water (O/W) Pickering emulsions. The emulsion index, droplet size distribution and microstructures of Pickering emulsions produced by different OS-starches were compared. OSA modification had almost no effect on the morphology or crystal structure types of three kinds of NS and OS-starch but markedly increased the contact angle and particle size distribution of OSRS, OSTS and OSOS. Esterification reaction of OSA and starch mainly occurred in amorphous regions of starch, and the OSA significantly improved the emulsifying capacity of OSRS, OSTS and OSOS granules and thus stabilized emulsions formed at higher levels (2.5% and 3.0%) of modification of OS-Starch exhibited better stability; the ability of OS-starch to stabilize Pickering emulsion was 3.0% OSRS > 3.0% OSOS > 3.0% OSTS, respectively. Observation and structural properties analysis of OS-starch granules and Pickering emulsion droplets showed that the number and thickness of the starch granules on the oil-water interface of the emulsion droplets increased with improvement of the OSA modification level, and an aggregation state was formed between the OS-starch granules, which was also enhanced with the OSA modification levels. These were all necessary for the Pickering emulsion stabilized by starch granules to remain in a steady state.

Shape and size-controlled starch nanoparticles prepared by self-assembly in natural deep eutectic solvents: Effect and mechanism

Regulating particle size and shape of starch nanoparticles (SNPs) is imperative to producing natural nanomaterials with controllable physicochemical properties enabling novel applications. In this study, natural deep eutectic solvents (NADES) based on lactic acid and glucose were obtained and used for the self-assembly of SNPs with different shapes and sizes. The size of fabricated SNPs ranged from 406.7 to 104.6 nm, and the shape of SNPs changed from spherical to rice-like with increasing proportion of NADES. X-ray diffraction was shown that SNPs were in B + V crystalline structure, and the relative crystallinity was decreased with the increase of NADES content, indicating that the NADES disrupted the recrystallization of starch molecules. DSC and TGA analysis demonstrated that the longer starch chains self-assembled in NADES system to form SNPs with less ordered crystallization structure. Fourier transform infrared spectra exhibited a shift of O–H stretching band at 3500-3300 cm−1 towards higher wavenumber values due to the weakened hydrogen bond strength of SNPs. In addition, turbidity analysis indicated that the formation of rice-like and smaller size SNPs was related to the inhibition of nucleation and growth of starch by NADES, which was confirmed with the results of yield results. This study has significant implications for the preparation of SNPs with various size and shape in a completely new and highly effective approaches.

Insight into property, function, and digestion of potato starch modified by phenolic compounds with varying structures.

Influences of structural characteristics of phenolic compounds on the properties of potato starch were investigated, and their effects on the quality, function, and digestion of potato starch noodles were further determined. All testing phenolic compounds (including protocatechuic acid [PA], naringin [NA], and tannic acid [TA]) exhibited the significant capability to modify the thermal properties, rheological properties, and enzymatic hydrolysis of potato starch. The high amount of hydroxyl groups, the presence of glycoside structure, appropriate molecular size, and steric hindrance were beneficial to enhance their effects on potato starch. In addition, by changing the microstructure of starch hydrocolloids, PA, NA, and TA could affect the color, texture properties, and cooking properties of potato starch noodles. Meanwhile, PA and TA could endow potato starch noodles with remarkable antioxidant activity. Furthermore, the in vitro digestion of potato starch noodles was obviously inhibited by phenolic compounds, especially for TA. All present results suggested that structural characteristics of phenolic compounds affected their interaction affinity and combination degree with potato starch molecules, so as to modify the properties of starch and affect the quality, function, and digestion of starchy foods, which showed the valuable applications in food and chemical industries. PRACTICAL APPLICATION: The property, function, and digestion characteristics of potato starch show the significant effects on the quality of potato starch products in food industry. In present study, the thermal properties, rheological properties, and enzymatic hydrolysis of potato starch were modified by different phenolic compounds (including protocatechuic acid, naringin, and tannic acid) in varying degrees, whereas the quality, antioxidant activity, and digestion characteristic of potato starch noodles were modified by phenolic compounds with different structures. All results showed the potential application and interaction regularities of phenolic compounds as natural additives for potato starch processing in food industry.

Effects of rice protein, soy isolate protein, and whey concentrate protein on the digestibility and physicochemical properties of extruded rice starch.

Protein, as the second major component in starchy foods, is crucial for its influence on the physicochemical properties and digestibility of starch. However, the effect of different sources of protein on starch digestibility is still unclear. In this paper, the effects of different sources of proteins (rice protein: RP, soybean isolate protein: SPI, and whey concentrate protein: WPC) on structural features, digestibility, and enzyme activity of extruded rice starch were investigated. The addition of all three proteins reduced the starch digestibility of extrudates. Native SPI and WPC suppressed amyloglucosidase activity, and all three proteins exhibited stronger amyloglucosidase inhibition when hydrolyzed. The rheological properties and Fourier transform infrared spectroscopy results revealed the exogenous proteins and starch interacted through non-covalent bonds and improved the ordered structures in the extrudates. The extrusion process also facilitated the formation of a V-type structure. The sum of SDS and RS content of extrudates was negatively correlated with the content of leached amylose and positively correlated with the ratio of 1047/1022cm-1 . These findings suggest that the inclusion of exogenous proteins during extrusion can affect starch digestibility through mechanisms such as the interaction with starch molecules, as well as the inhibition of amylase activity. PRACTICAL APPLICATION: This result indicated that the addition of protein during extrusion not only increased the nutritional value of the extrudate, but also decreased the starch digestibility. Extrusion technology can efficiently produce extruded products with protein, expanding further applications of protein in food and providing new healthy staple food options for special populations, such as diabetic and overweight people.

Multi-scale structure characterization of ozone oxidized waxy rice starch

The elucidation of multi-scale structural variation and oxidation reaction mechanism of ozone oxidized waxy rice starch molecules remains a big challenge, limiting its development of intensive processing. In the present work, the changes in the structure of waxy rice starch after ozone treatment were systematically researched by various characterization methods. The study has shown that with the increase in ozone oxidation time, the granules of oxidized starch were polygons with multiple face depressions. It was also observed that ozone first attacked the amorphous zone of the starch granules and then penetrated the crystalline zone. Combining 1D and 2D NMR (1H NMR, 13C NMR, HSQC and HMBC) and other methods, it was proved that ozone oxidation led to ring splitting between C2 and C3 of the glucose unit. The resulting hemiacetal groups showed different types of structures. Among them, the main structures were intramolecular acetals and intermolecular hemiacetals. This research offered theoretical guidance for the utilization of ozone oxidation technology for starch modification and the development of waxy rice new foods.

Enhancing I0/I− Conversion Efficiency by Starch Confinement in Zinc–Iodine Battery

The redox couple of I0/I− in aqueous rechargeable iodine–zinc (I2‐Zn) batteries is a promising energy storage resource since it is safe and cost‐effective, and provides steady output voltage. However, the cycle life and efficiency of these batteries remain unsatisfactory due to the uncontrolled shuttling of polyiodide (I3− and I5−) and side reactions on the Zn anode. Starch is a very low‐cost and widely sourced food used daily around the world. “Starch turns blue when it encounters iodine” is a classic chemical reaction, which results from the unique structure of the helix starch molecule–iodine complex. Inspired by this, we employ starch to confine the shuttling of polyiodide, and thus, the I0/I− conversion efficiency of an I2‐Zn battery is clearly enhanced. According to the detailed characterizations and theoretical DFT calculation results, the enhancement of I0/I− conversion efficiency is mainly originated from the strong bonding between the charged products of I3− and I5− and the rich hydroxyl groups in starch. This work provides inspiration for the rational design of high‐performance and low‐cost I2‐Zn in AZIBs.

Impact of oxalate on the physicochemical properties of potato starch: An insight closely associated with Indian cuisine

Potato is one of the widely used vegetables in Indian cuisine. The starch of potato could undergo modification in presence of oxalic acid of tomato during cooking in Indian cuisine leading to the increased cooking time of potato. Present study analyses the effect of oxalic acid on the physicochemical properties of potato starch and investigate the reason behind the increased cooking time of potatoes in Indian food dishes. Water binding capacity, swelling power, solubility and paste clarity were determined for potato starch in the reaction mixture containing a varied concentration of oxalic acid. A significant increase in the water binding capacity and swelling power was observed on increasing the incubation temperature and concentration of oxalic acid. There was a sharp decline in the solubility of starch at higher temperatures and at 1 mg/mL oxalic acid concentration. The unexpected behaviour of solubility at a higher concentration of oxalic acid indicates that oxalic acid at 1 mg/mL alter the solubility of starch molecules in the bulk water. Treated starch showed high transmittance suggesting lesser opaqueness. Scanning Electron microscope results exhibited non-uniformity in starch granules with size varying from 5 μm to 50 μm. Most granules under Scanning Electron Microscope were smooth surfaces with few pores and defects.

Structure, thermal stability, and in vitro digestibility of rice starch–protein hydrolysate complexes prepared using different hydrothermal treatments

In this study, rice starch-protein hydrolysate (WPH-S) complexes with high resistant starch (RS) content were prepared by heat-moisture treatment (HMT) and annealing (ANN). The effects of different hydrothermal treatments on the structure and thermal stability of the WPH-S complexes and their relationship with starch digestibility were further discussed. The results showed that RS contents of ANN-WPH-S complexes (35.09–40.26 g/100 g) were higher than that of HMT-WPH-S complexes (24.15–38.74 g/100 g). Under hydrothermal treatments, WPH decreased the hydrolysis kinetic constant (k) of starch form 4.07 × 10−2–4.63 × 10−2 min−1 to 3.29 × 10−2–3.67 × 10−2 min−1. HMT and ANN promoted hydrogen bonding between WPH and starch molecules, thus increasing the molecular size of starch. In addition, the shear stability of WPH-S mixture was improved with the hysteresis loop area decreased after HMT/ANN treatments, resulting in a more stable structure. Most importantly, the hydrothermal treatment made the scatterers of WPH-S complexes denser and the surface smoother. Especially after ANN treatment, the WPH60-S complex formed a denser aggregate structure, which hindered the in vitro digestion of starch to a certain extent. These results enrich our understanding of the regulation of starch digestion by protein hydrolysates under different hydrothermal treatments and have guiding significance for the development of foods with a low glycemic index.

Effects of storage temperatures on the starch digestibility of whole rice with distinct starch fine molecular structure.

Although the retrogradation of rice starch has been extensively investigated, there remain questions as to how storage temperature affects starch inter- and intramolecular interactions in cooked white rice, and the relationship of these interactions with the digestion rate. To this end, micromorphology, crystallinity polymorphisms, molecular interaction patterns and in vitro starch digestibility of 3 rice varieties kept under 5 different storage temperature programs (room temperature (RT), 4 °C, -18 °C, 4 °C/RT (4 °C for 24 h and then RT for 24 h), -18 °C/RT (-18 °C for 24 h and then RT for 24 h)) were investigated. As expected, a significant variance in starch digestibility was observed for samples after storage at different temperatures. Overall, storage at 4 °C could most effectively decrease the starch digestibility of retrograded rice. The digestion rate constant was for the first time found to be determined by short-range amylopectin intermolecular interactions rather than long-range starch molecular interactions, for all different storage conditions. Furthermore, the digestion extent was determined by both inter- and intramolecular interactions among starch molecules as well as by the long-range order of the retrograded double helices. These results could prove useful to devise storage regimes which use retrogradation to produce cooked rice with lower glycemic index.

A new method for preparing the instant quinoa by piecewise gelatinization

In recent years, the market has great demand for the instant quinoa. However, the high content of protein and fat in quinoa grain inhibited the gelatinization and extension of starch molecules. The instant quinoa prepared by conventional gelatinization method has poor rehydration. In order to solve this problem, the sectioned heating method was used according to the properties of quinoa starch and protein. The purpose of the first heating stage was to gelatinize the starch with sufficient water, and the technological conditions were: the mass ratio of water to quinoa 4:1, the temperature of water bath 70 °C, the time of water bath 60 min. The purpose of the second heating stage was to denature the protein with less water, and the technological condition was that the quinoa was heated by atmospheric steam for 5 min. The instant quinoa prepared by above method had excellent rehydration. Compared to the raw quinoa, the content of protein, fat and reducing sugar in instant quinoa increased slightly, while the content of starch and polyphenols decreased slightly. The mass loss percentage of instant quinoa was 11.5% and the score of sensory evaluation for the sample prepared under the optimum conditions was 87.

Effects of acidity, number of hydroxyl group, and carbon chain length of carboxylic acids on starch cross-linking

Corn starch film samples cross-linked using malic, malonic, and succinic acids, and thermoplastic starch were characterized and tested for their water absorption, surface morphology, structural change, and thermomechanical properties. The acids vary in acidity, number of hydroxyl groups, and carbon chain length. The presence of an additional hydroxyl group has helped malic acid form more hydrogen bonding between starch molecules. The relatively higher acidity in malic acid compared to succinic acid is found to be another factor for its better cross-linking potential. The additional carbon chain in succinic acid, which reduces its solubility and acidity; has negatively affected its cross-linking potential. Among the three variables studied, number of hydroxyl group has highly influenced the cross-linking potential, followed by acidity and carbon chain length, respectively. Consequently, the elongation at break and water absorption resistance of thermoplastic starch were improved from 108.63 ​MPa to 175.72 ​MPa and from 140% to 80%, respectively, cross-linking corn starch with malic acid.

Effect of sonochemical pretreatment of slurry depressors on sylvin flotation performance

The main source of potassium fertilizers is sylvinite ores consisting primarily of halite (NaCl), silicate and clay-carbonate slurries (clay-salt slurries). Processing of natural potash ores is mainly carried out by the flotation method, which separates KCl, NaCl, and clay-salt slurry. The research is aimed at revealing the effect of sonochemical pretreatment of the depressor reagents, CMC and starch, on dynamic viscosity, aggregate size, electrokinetic potential of these reagent solutions and sylvin flotation performance. It has been established that sonochemical treatment of depressor solutions decreases the size of aggregates of starch molecules by more than 133 times and that of aggregates of CMC molecules from 6 to 4 nm. It has been revealed that sonochemical treatment of anionic CMC solution shifts the electrokinetic potential towards the area of negative values with an increase in acoustic power, while sonochemical treatment of any acoustic power has no effect on the zeta potential of nonionic starch. It has been found that the sonochemical treatment lowers the dynamic viscosity of CMC and starch solutions: the viscosity of CMC solution at a maximum acoustic power of 420 W decreases by 44 % and the viscosity of starch solution at the same acoustic (ultrasonic) power decreases by 70 %. Furthermore, sonochemical pretreatment of sylvin flotation depressors contributes to an increase in KCl recovery and a decrease in the slurry content in the flotation concentrate. The possibility of reducing the consumption of ultrasonic treated depressor is also demonstrated. It is expedient to test the obtained findings in pilot-plant conditions.

Advances in heterologous expression, structural elucidation and molecular modification of pullulanase

Starch is composed of glucose units linked by α-1, 4-glucoside bond and α-1, 6-glucoside bond. It is the main component of foods and the primary raw material for starch processing industry. Pullulanase can effectively hydrolyze the α-1, 6-glucoside bond in starch molecules. Combined with other starch processing enzymes, it can effectively improve the starch utilization rate. Therefore, it has been widely used in the starch processing industry. This paper summarized the screening of pullulanase-producing strain and its encoding genes. In addition, the effects of expression elements and fermentation conditions on the production of pullulanase were summarized. Moreover, the progress in crystal structure elucidation and molecular modification of pullulanase was discussed. Lastly, future perspectives on pullulanase research were proposed.

Effects of Heat-Moisture Treatment on the Digestibility and Physicochemical Properties of Waxy and Normal Potato Starches.

Heat-moisture treatment (HMT) is a safe, environmentally friendly starch modification method that reduces the digestibility of starch and changes its physicochemical properties while maintaining its granular state. Normal potato starch (NPS) and waxy potato starch (WPS) were subjected to HMT at different temperatures. Due to erosion by high-temperature water vapor, both starches developed indentations and cracks after HMT. Changes were not evident in the amylose content since the interaction between the starch molecules affected the complexation of amylose and iodine. HMT increased pasting temperature of NPS from 64.37 °C to 91.25 °C and WPS from 68.06 °C to 74.44 °C. The peak viscosity of NPS decreased from 504 BU to 105 BU and WPS decreased from 384 BU to 334 BU. The crystallinity of NPS decreased from 33.0% to 24.6% and WPS decreased from 35.4% to 29.5%. While the enthalpy values of the NPS declined from 15.74 (J/g) to 6.75 (J/g) and WPS declined from 14.68 (J/g) to 8.31 (J/g) at 120 °C. The solubility and swelling power of NPS decreased while that of WPS increased at 95 °C. Due to the lack of amylose in WPS, at the same HMT processing temperature, the reduction in peak viscosity of treated WPS compared to that of native starch was smaller than that of NPS. The resistant starch (RS) content of NPS after HMT at 120 °C was 73.0%. The slowly digestible starch (SDS) content of WPS after HMT at 110 °C was 37.6%.