The purpose of this study is to determine if there is any correlation between the characteristicsof the user’s eye movements (EMs) and the preference of the user when wearingdifferent Progressive power lenses (PPLs) distributions. An eye-tracker system with asample rate of 120Hz and temporal resolution of 8.3ms (Tobii-X3-120) was used to registerEMs of 38 PPL users when reading in a computer screen with 2 types of PPLs (PPLsoftand PPL-hard). Number of fixations, complete fixation time, fixation duration mean,saccade duration mean, saccade distance mean, and number of regressions were analyzedfor 6 different regions of the computer screen. A statistically significant difference wasobserved between the characteristics of the user’s EMs and the user’s PPL subjectivepreference (p < 0.05*). Subjects that preferred the PPL-hard presented significantly lowercomplete fixation time, lower fixation duration mean and lower number of regressionsthan those subjects indicating a preference for the PPL-soft. Results of this study suggestthat eye-tracking systems can be used as PPL design recommendation systems accordingto the user EMs performance. Presbyopia is the last stage of a gradual loss of accommodativeamplitude due to the loss of elasticity of the crystalline lens.Accommodation amplitude starts to decrease from childhood, but it isaround the age of forty when difficulties performing tasks at nearvision start. At this age, the accommodative amplitude is between 3-4D,which is not enough to maintain clear and comfortable near vision. Thisdecline continues into the mid-fifties when accommodation reaches itsminimum value .Progressive power ophthalmic lenses (PPL) are one of the most commonsolutions for presbyopia. A PPL is a multifocal lens that provides acontinuously smooth increase in spherical power from the upper part ofthe lens to the lower portion, allowing the users to see clearly at alldistances by changing the gaze position .NeverthRecent advances in the manufacturing processes of PPLs usingFree-Form technology have provided a wide variety of PPL designs to theophthalmic industry . This teAlthough it is possible to characterize a PPL by means of powerdistribution maps, the selection of the lens that best fits the userneeds is complicated. Undistorted viewing areas calculated theoreticallythrough the power distribution maps, do not represent the visualperception while wearing a PPL, that varies for every subject. So, inorder to better understand the visual perception provided by a PPL,previous works have tried to characterize the performance of PPLs usingvisual tests as visual acuity ; 7,contAs indicated earlier, the optical properties of PPLs have someimplications in terms of ergonomics, mainly at mid-range and nearvision. The use of PPLs and the position of visual displays such asmonitor screens or laptops, can have musculoskeletal or visualimplications . In thosVideo-based eye trackers monitor eye position by video recording thereflection of an infra-red light projected in the subject’s eye; 21.EyeIn the beforementioned studies by Y. Han et al. and Hutchings et al.significant differences in some statistical descriptors of the EMs werefound when comparing PPLs and single-vision lenses, but no differenceswere detected when comparing different PPL designs ; 18; 23.A prospective observational longitudinal double-blind study followingthe tenets of the Declaration of Helsinki was carried out. Full approvalfor the study was obtained from the Hospital Clínico San Carlos EthicsCommittee’s (CEIC) Review Board. All participants provided their writteninformed consent and at the end of the trial participants werecompensated with two pair of spectacles.The study sample comprised presbyopic subjects of both genders agedover 42 years with the following inclusion criteria: 1) Refractive errorbetween -6.00D and +4.00D with an astigmatism lower or equal to 2.50Dand addition between +1.00D and +3.00D. 2) Best corrected VA better than0.05logMAR binocularly and 0.10logMAR monocularly. 3) Difference inrefractive error between both eyes lower than 1.50D. Subjects wereexcluded if they had significant binocular vision anomalies, ocularpathologies  or if they had been in anypharmacological treatment that could affect the visual function.The study required 4 visits. The recruitment visit (visit 1)consisted in a screening visit where refraction, selection of frames andsigning of the consent form were done. In this visit, all subjectsunderwent a complete visual exam to check whether they meet theinclusion/exclusion criteria. The visual exam was composed by VAtesting, binocular refraction, stereoacuity using Titmus test Stereo flytest, Stereoptical CO, USA), Worth test, cover test and binocularmotility. Once the optometrist confirmed the participant fulfilled theinclusion criteria, the subject selected a frame model that was adaptedprior to the measurements of the fitting and position-of-wearparameters. The pupillary distance was measured using an automatedpupillometer, segment height was measured manually and the additionalfitting parameters, which included pantoscopic tilt, back vertexdistance and frame wrap angle, were measured using a special ruler. At visit 2, optometrists gaveinstructions to participants about how to use the new progressivelenses. During this visit, the first pair of lenses (according to therandomization assignment) was dispensed. After using the lenses for 7days through the whole day, a register of EMs using eye-tracking wascarried out in visit 3. Also, assessment questionnaires were collected,and the subjects were provided with a second pair of spectacles. Atvisit 4, the same sequence of evaluation was conducted for the secondpair of spectacles after 7 days of wear.Two free-form PPL designs, soft and hard, were developed by IOT for this research. Cylinder maps and progression powerprofiles (as perceived by the user) are shown in Binocular eye position was registered when participants were readinga text in a computer screen when using the two different PPLs. An eyetracker Tobii-Pro-X3-120  was used to record thehorizontal and vertical EMs with a sample rate of 120 Hz and temporalresolution of 8.3ms, then data were processed using an open-sourcesoftware designed for the analysis of EMs called OGAMA . RelativParticipants were asked to read 2 texts composed by 10 lines withVA=0.5 logMAR located 67cm away from the participant’s eye, thereforesubtending a horizontal viewing angle of 41.5º. Different reading testswith similar difficulty level were used for the evaluation of PPL-softand PPL-hard in a randomized order. For statistical analysis, it was selected thelowest 4 reading rows recorded by the eye-tracker device, except for thefirst and last rows that were removed for all subjects to avoid EMserrors when subject reach the start and end of the reading test. Toanalyze differences in EMs between reading areas, the text was dividedin three vertical regions  withthe same width and two horizontal regions (upper and lower) formed bytwo text rows each as shown in Subjects were asked to use both pair of spectacles for 7 days each intheir daily activities. After using the lenses, a double-blindcomparison was carried out for mid-range vision. For this purpose,subjects were asked to change the spectacles when they were looking atext on a computer screen and choose the PPL which provides them abetter visual quality and comfort. According to their preference,subjects were divided into 2 groups: Group 1 including those subjectsthat preferred the design PPL-Soft and group 2 comprised by thosesubjects that preferred the lens PPL-hard.All statistical tests were done using Statgraphics Cen-turion XVI.IISoftware. The level of significance was set at 0.05. Statistical Powerwas set at 0.8. Multifactorial ANOVA was used to determine differencesin EM pattern depending on the user’s PPL preference. Design ofrandomized complete block test was used to determine the relationshipbetween the reading region and the EMs for each PPL. Finally, regardingthe comparison between naïve subjects and experienced subjects a designof randomized block test was used.Recordings of 49 subjects were collected for both pair of spectacles,11 of them were discarded due to data quality was not within the dataquality criteria. The final sample was comprised of 38 subjects of meanage 54±5 (44-64 years), 22 of them were experienced PPL subjects and 16were naïve. Experienced users are those subjects who had been using PPLat least 6 months before the study was carried out while naïve users arethose subjects who have never used PPL. According to the subjectiveevaluation, there were 21 subjects that preferred the PPL-soft (Group 1)and 17 participants whose preferred lens design was PPL-hard (Group 2).No statistical differences between EMs and user PPL previousexperience were found. Results of the analysis are shown in For each PPL design (PPL-soft and PPL-hard) an analysis was carriedout to compare the ocular movement performance between group 1 and group2. In both cases, using PPL-soft or PPL-hard, group 1 showed differentreading pattern than group 2. When participants were using the PPL-hard,complete fixation time, fixation duration mean and number of regressionswere statistically significantly lower for group 2 than for group 1,meaning that the time spent on the stops has less duration and lessgo-back movements influencing reading performance .The results of this study show that there is a relationship betweenthe EMs of the user and the preference for a specific power distribution of a PPL design. Subjects with lowercomplete fixation time, lower fixation duration mean and lower number ofregressions when reading at a computer prefer a PPL with widerundistorted viewing areas and faster growth of the unwanted astigmatismwhilst the participants with higher complete fixation time, higherfixation duration mean, and higher number of regressions prefer a PPLwith smoother transitions and a more limited undistorted viewing area.The results also showed how PPL optical performance is affected by thepower distribution of the PPL and how the lateral astigmatism and thepower of the lens influences the effectiveness of reading.Eye tracking techniques have been used to evaluate the performance ofthe lenses while tested by the subjects, and to determine the eye andhead movements when using the lenses. In this sense lateral aberrationsof PPLs have an important role in the visual behavior and satisfactionof the wearers. Y. Han et al.  comparedThe fact of being an experienced or naïve wearer of PPLs could be akey factor on the characteristics of the EMs. It could be expected thatan experienced user might have learnt to restrict head lateralmovements, while a naïve subject might use more EMs than head movementswhich would lead to the use of the distorted peripheral regions. Thus,an analysis between experienced and naïve users was carried out in thisstudy. The results of this analysis suggest that there is norelationship on EMs between experienced and naïve users. Therefore, thepreference results obtained in this research do not depend on thesubjects’ previous experience using PPLs in the same way than the studydone by Y. Han et al. where thThe main limitation in this study came from the type of device usedfor the eye-tracking. Tobii-Pro-X3-120 is a screen-based eye-tracker, itmeans, it is a stationary device capable of accurately recording EMsregardless of head movements. To use the PPLs in a more realistic way inour study, the subjects were allowed to use them without any headrestriction but with the eye tracking system used, the head movementcannot be recorded and compensated. Another limitation of this system isthe position of the lens. The lens is located between the eye and theeye tracker cameras, and therefore, the image of the eye recorded by theeye tracker was affected by a prismatic effect. This doesn’t allow tocorrectly determine the eye position and it could influence thedetermination of the saccade length and the determination of theposition on the text. Also, another limitation of the system is theobject distance used, in this case it was only used for intermediatevision but PPLs could have different performances at different objectdistances. To avoid these limitations, the idea of using wearable eyetrackers become important. This kind of eye-trackers incorporate agyroscope that provides pitch and yaw movements. In this way, EMs can becompensated with head movements obtaining a more realistic vision gazedata. In addition, wearable eye tracker can directly record the positionof the eye without prismatic effect as the eye tracker cameras arebetween the lens and the eyes. It also allows to record different objectdistances allowing the evaluation of the PPL for all working distancesin a dynamic and realistic environment. Another limitation of the studyis that the prior worn design was not collected, this could determinethe subjects’ EM behavior and preference. Although is difficult to knowexactly the power distribution of PPLs on the market it could beinteresting in future studies to consider the user’s prior powerdistribution.As a conclusion, the results from this study showed how theperipheral ocular motility is affected by the PPL power distribution andthe unwanted astigmatism. When a wearer is using the lateral zones ofthe lens, EM pattern is different than in the central part of the lensdue to the unwanted astigmatism of these areas of the PPL. As expected,the EMs improve when the object distance is well matched to the localaddition of the lens region the user is looking through. The main resultof this study is that while different subjects have different EM patternand the EMs are not significantly affected by the PPL used, thoseparticipants with lower complete fixation time, lower fixation durationmean and lower number of regressions have a stronger preference forharder lens designs compare to those with higher complete fixation time,higher fixation duration mean and higher number of regressions that tendto prefer a softer power distribution. The results of the study can beapplied by optometrists and ECPs to determine with an objective test thepower distribution that can be best suited for their customers,according to their EMs, and this can be combined with other opticalmetrics leading to a lens selection that can provide a betteradaptation, ergonomics, and performance.http://biblio.unibe.ch/portale/elibrary/BOP/jemr/ethics.htmland that there is no conflict of interest regarding the publication ofthis paper.The author(s) declare(s) that the contents of the article are inagreement with the ethics described inThis research was supported by IOT. We also thank the anonymousparticipant and the optometrists in charge of the measurements.


Breeding for less digestible starch in wheat can improve the health impact of bread and other wheat foods. The application of forward genetic approaches has lately opened opportunities for the discovery of new genes that influence the digestibility of starch, without the burden of detrimental effects on yield or on pasta and bread-making quality. In this study we developed a high-throughput in vitro starch digestibility assay (HTA) for use in forward genetic approaches to screen wheat germplasm. The HTA was validated using standard maize and wheat starches. Using the HTA we measured starch digestibility in hydrothermally processed flour samples and found wide variation among 118 wheat landraces from the A. E. Watkins collection and among eight elite UK varieties . We further investigated starch digestibility in fractions of sieved wholemeal flour and purified starch in a subset of the Watkins lines and elite varieties and found that the matrix properties of flour rather than the intrinsic properties of starch granules conferred lower starch digestibility. Triticum aestivum L.) is one of the most widely consumed crops worldwide and provides up to 50% of the calories in the human diet, mainly in the form of starch, so improving its nutritional quality could deliver health benefits to a large number of people [Starchy foods are a main source of carbohydrate in our diet and an important source of energy. Reducing the rate and extent of starch digestibility in foods can help to maintain healthy blood glucose levels, which is important for the prevention and management of obesity and chronic diseases like type II diabetes . Furtherf people . Thus, bw/w), which is the greatest contributor to calories, and also 10–15% (w/w) protein and 11–15% (w/w) dietary fiber [A mature wheat grain contains predominately starch, 60–70%  (accessed on 16 November 2022). Thus, a key aim of this study was to determine the extent of natural variation in starch digestibility of the c.Watkins lines and in modern elite UK varieties .To date, reverse genetic studies in wheat have demonstrated potential for increasing resistant starch levels using induced mutations in starch biosynthesis genes ,19,20,21Despite the availability of diverse wheat germplasm resources like the c.Watkins collection, forward screening approaches for starch digestibility have been limited due to the lack of informative, accurate, and efficient phenotyping methods. Screening based on amylose content, which has a positive association with resistant starch content, can identify lines with high levels of resistant starch but cannot identify factors beyond amylose content that may cause resistance to digestion ,28. OnlyTo facilitate rapid assessment of starch digestibility in the c.Watkins wheat landraces, an in vitro single-enzyme system was utilized , 4-Hydroxybenzhydrazide (PAHBAH) (5351-23-5), TRIS (77-86-1), EDTA (60-00-4), SDS (151-21-3), DTT (3483-12-3), Phosphate buffered saline (PBS) (P4417-100), Sodium Carbonate (497-19-8), DMSO (67-68-5), maltose (6363-53-7), sodium hydroxide (1310-73-2) and α-amylase  were purchased from Sigma-Aldrich Company Ltd., Poole, UK.3). Digestion was started by adding pancreatic α-amylase suspended in PBS targeting 2 U/mL activity into the samples. Enzyme activity was determined by applying the starch digestibility assay on gelatinized potato starch and obtaining the linear rate of maltose release every 3 min (mg/mL). Aliquots (50 μL) were then taken after 6, 12, 18, 24, 40, and 90 min from the onset of digestion and transferred to the stop solution.A graphical scheme of the HTA assay is presented in g for 5 min to avoid transferring any starch remnants, and 50 μL of the supernatant was transferred to a new deep well plate; 50 μL of maltose standards (5–1000 μM) were also added to the plate. The PAHBAH reducing end assay was used to quantify the reducing ends released [The stopped reactions were then centrifuged at 4000× released . BrieflyStarch digestion profiles from the HTA and the single-enzyme system were compared using standards of purified starch from standard maize, waxy maize, and high-amylose maize , standard wheat starch , and two high-amylose starches: sbeII and ssIIIa, previously characterized, respectively, by Corrado et al. and Fahy et al. ,21. Starhttps://www.seedstor.ac.uk/, (accessed on 16 November 2022); permission to use the materials for research purposes was obtained. The 118 c.Watkins lines were grown in Autumn 2018 in 1 m2 plots  at Church Farm, Norfolk UK (52°37′49.2″ N 1°10′40.2″ E) using standard agronomic practices (ahdb.org.uk) (accessed on 16 November 2022). Elite varieties were grown in 2013 in plots (one per genotype) of 1.5 m2 at Morley Farm, Norfolk, UK (52°33′15.57″ N 1°10′58.956″ E).Grains for the c.Watkins lines were ordered from the Germplasm Resources Unit  using the publicly accessible SeedStor system ractices . Based oractices . Seeds fGrains from the c.Watkins and elite varieties were coarsely milled in a cyclone mill fitted with a 0.5 mm screen (UDY Corporation). Milled samples were passed through a 0.3 mm sieve  to produce ‘wholemeal’ flour samples, and selected lines were passed through a 0.053 mm sieve to produce ‘sieved’ flour samples. The flour samples were kept in a vacuum desiccator for five days before analysis.g for 5 min, and the pellets were resuspended in 2 mL of water. The starch suspensions were then overlayed into a Percoll solution (90% v/v) and centrifuged at 2500× g for 15 min to remove cell walls and proteins. The recovered starch pellets were washed with 1 mL buffer , transferred into a 2 mL tube and left to incubate for 5 min. The starch suspension was then centrifuged at 4000× g for 1 min. The pellets were recovered, and the washing procedure was repeated once more. The pellets were then washed three times with 1 mL of water, then once with 100% ethanol. Samples were then kept one day in the fume hood, followed by five days in a desiccator containing silicon dioxide prior to analysis.Starch was isolated using an adapted method reported in Hawkins, et al. . Wheat fv/v) in 100 mM sodium acetate buffer, pH 5.0 . The deep well plate was sealed and secured with a Cap-mat . Samples were heated at 90 °C for 10 min at 1600 rpm using a thermal mixer with a 96 SmartBlock™ DWP 1000 n attachment . Total starch content was determined using a Total Starch HK kit  following manufacturer instructions, except volumes of reagents were scaled down by a factor of 10.Wholemeal flour samples were weighed (~8 mg) and transferred into a deep well plate , each well containing 20 μL of DMSO and a 3 mm glass ball to improve mixing. The plate was mixed for 5 min at 1600 rpm to disperse the samples before adding 500 μL of a thermostable α-amylase to each sample. The thermostable α-amylase was solubilized at 1:30  (accessed on 16 November 2022).During optimization of the HTA, a low-cost 3D-printed pipetting tool was developed, which allowed for manageable weighing and transferring of samples into 96-sample deep well plates and improved speed and control of pipette aspiration (Plate Z). The PLZ 3D design is available to download and print for free . Datasets of the validation of the in vitro starch digestibility HTA were analysed using the packages lme4 (v 1.1-30) and lmerTest (v 3.1-3) for a mixed model fit ,40,41. PFor validation of the HTA, the methods were compared by plotting the estimated starch digestion profiles from the HTA and the single-enzyme system  and by A linear mixed model including line as a fixed effect and experimental run as a random effect was used for analysis of in vitro starch digestibility of the c.Watkins lines and elite varieties. Marginal means with standard errors  are plotted for each line.The correlation between starch digestibility and total starch was estimated using linear regression. All values reported represent the mean, and the number of replicates and variance metrics are specified in the description of the corresponding figure and For complete details of analyses, all data and analysis code is available as The aim of this experiment was to determine the extent of natural variation in starch digestibility of the c.Watkins collection and compare it with elite UK varieties representing each commercial group .Results of the HTA revealed a wide range of variation in starch digestibility among c.Watkins landraces; starch digestibility profiles formed a gradient of low- to high- digestibility rather than two distinct groups, as expected from complex traits B. The reElite UK varieties showed less variation and greater starch digestibility compared to most c.Watkins lines, although a direct comparison is confounded by the different growing conditions between the two groups A. For exOur results demonstrate that natural phenotypic variation for starch digestibility exists in the c.Watkins collection  as well as in elite varieties representing commercial groups of bread, biscuit and animal feed groups, and so there is potential for identifying underlying genetic diversity. The variation observed among the c.Watkins lines was greater than among the elite varieties screened, which is consistent with the higher levels of genetic diversity previously reported for the c.Watkins collection compared to modern European bread wheat varieties, as well as increased phenotypic variation for agronomically desirable traits, such as stripe, leaf, and stem rust resistance, as well as grain surface area and grain width ,24,25,26p ≤ 0.001), and the elite varieties . The elite variety Diego had the highest TS content , and the c.Watkins line 651 had the lowest . Most of the samples had a TS content between 47 to 57 g/100 flour. Linear regression analysis showed that total starch content only weakly correlated (R2 = 0.0108) with starch digested at 90 min which suggests that the differences in total starch content do not explain the variation observed for starch digestibility  content of wholemeal flour varied significantly for c.Watkins landraces  to measure starch digestibility in sieved flour  and purified starch.Three low-digestibility lines: 777 (WATDE0111), 216 (WATDE0025), and 639 (WATDE0083); and five high-digestibility lines: including two c.Watkins landraces 816 (WATDE0117), 308 (WATDE0042) and the three UK elite varieties Myriad, Dickens, and Paragon were selected for further analysis. The selection was based on the starch digestibility HTA results, specifically the ranking (low to high), to represent the variation observed in the screen.p < 0.05) compared to the corresponding wholemeal samples, and while the third low-line (777) remained low  and flour properties . These results are described below. In summary, while differences between the lines themselves were evident for many properties, none of these factors significantly correlated with starch digestibility in this sample .p < 0.001) (p < 0.001), whereas minor differences were observed for the overall volume of A and B granules  and amylose (AM) (< 0.001) . Howevergranules . Low-digp < 0.003) in the particle size of wholemeal flour; line 639 had more particles in the range of 14–20 μm whereas Paragon showed a slightly smaller number of particles in that range. Micrographs of wholemeal flour (produced using scanning electron microscopy) showed aggregate formations ≥ 120μm in all lines apart from the low-digestibility line 639, which only had particles smaller than 120μm , , , howeverp < 0.001)  ; low-digThe different digestibility profiles observed across purification steps  suggest that multiple mechanisms in the selected lines could be affecting starch digestibility and that different factors in each line could have a distinct effect. This is consistent with prior studies which have shown effects of flour characteristics and starch structure on starch digestibility in cooked wheat samples including particle size of wholemeal flour, protein content, and chain-length distribution of starch polymers ,48,49. AThe aim of this experiment was to validate and assess the reliability of the 96-sample format starch digestibility assay using established starch standards of maize and wheat by comparing the HTA with the single-enzyme system protocol presented in Edwards et al., 2019 .Digestion profiles of maize and wheat starch standards were comparable to profiles generated using the single-enzyme system protocol in Edwards et al., 2019  Figure A. For exhackster.io, (accessed on 16 November 2022). A graphical scheme is presented in the The HTA (detailed in the There were no significant differences in the percent of starch digested at all the time points measured between the single-enzyme system protocol and the HTA. Thus, the HTA provided comparisons between samples that were accurate and reproducible, and the reliability of the assay was sufficient for use as a screening tool to aid in the selection of low- and high-digestibility samples.The variance using wholemeal flour (in the c.Watkins screen, Recent studies have reported improved methods to increase the throughput of starch digestibility assays. Wang et al.  developeIn this study we identified wide variation in starch digestibility among the c.Watkins lines which could be used to recover valuable alleles that were lost during modern breeding with the aim of improving wheat nutritional quality. Starch digestibility profiles of sieved flour and purified starch from selected lines suggested that the differences observed in starch digestibility are likely due to multiple factors in the flour matrix and are not limited to starch structural properties. Previously, strategies to reduce starch digestibility in wheat have mainly focused on reverse genetic approaches, but with new tools like the HTA there is potential to discover other useful sources of variation to support development of wheat varieties with improved health benefits. Furthermore, future work to determine starch digestibility in white flour samples will be useful for identifying sources of reduced starch digestibility for applications in foods made with refined flour.


Climate warming affects rice growth at different phenological stages, thereby increasing rice chalkiness and protein content and reducing eating and cooking quality (ECQ). The structural and physicochemical properties of rice starch played important roles in determining rice quality. However, differences in their response to high temperature during the reproductive stage have been rarely studied. In the present study, they were evaluated and compared between two contrasting natural temperature field conditions, namely, high seasonal temperature (HST) and low seasonal temperature (LST), during the reproductive stage of rice in 2017 and 2018. Compared with LST, HST significantly deteriorated rice quality, including increased grain chalkiness, setback, consistence, and pasting temperature and reduced taste values. HST considerably reduced the total starch and increased the protein content. Likewise, HST significantly reduced the short amylopectin chains [degree of polymerization (DP) <12] and increased the long amylopectin chains (DP > 12) and relative crystallinity. The starch structure, total starch content, and protein content explained 91.4%, 90.4%, and 89.2% of the total variations in pasting properties, taste value, and grain chalkiness degree, respectively. In conclusion, we suggested that rice quality variations were closely associated with the changes in chemical composition content  and starch structure in response to HST. These results indicated that we should improve the resistance of rice to high temperature during the reproductive stage to improve the fine structure of rice starch in further breeding and practice. TherefoVariations in growth temperature might be an important reason for differences in rice quality . NumerouIn addition to the flowering and grain filling stages, the reproductive stage (from panicle initiation to the heading stage) which is involved in pre-anthesis assimilate accumulation at early panicle development also plays a key role in determining the rice starch physicochemical properties . It has Furthermore, previous studies were usually conducted under controlled temperature chambers. This approach is limited in that it does not necessarily reproduce field conditions . In the 22.1−1 of organic matter content, 1.58 g kg−1 of total nitrogen, 8.75 mg kg−1 of Olsen-P, and 56.6 mg kg−1 of exchangeable K. The experiments were arranged in a randomized complete block design with three replications. Two japonica rice varieties with excellent sensory properties, i.e., Nanjing-9108 (NJ9108) and Jing-565 (J565), were used in the study. These cultivars were susceptible to high temperatures. The field management practices were similar in 2017 and 2018. The temperature conditions are shown in In 2017 and 2018, field experiments were carried out at the farm of Xiaowan Village, Zaoyang, Hubei Province . The soil was alluvial sandy loam with 34.3 g kg2.22 plants were harvested at the maturity stage. After that, the rice was threshed and air-dried. Seeds were stored for at least 3 months in a room temperature condition, and then they were hulled and milled by using a rice hulling machine (BLH-3250) and a rice polishing machine . Finally, the appearance quality  of the milled rice was measured by using a grain appearance analysis system  and an image analysis software.In the center of the plot, 5-m2.34 and 1 ml of 15% potassium ferrocyanide and then was filtrated to obtain a clear liquid. The filtrate was put into a polarimeter  and the total optical rotation was measured. The starch content was calculated by its total optical rotation according to the method of The total starch content was determined by the optical rotation method of The apparent amylose content (AAC) was determined by the iodine blue colorimetric method . The 1002SO4 was added overnight. The suspension was added with three to four drops of H2O2 and then heated at 320°C for 10-15 min by using a digestion oven . After cooling, two to three drops of H2O2 were added into the suspension and heated at 200°C for 5 min again. This step was repeated two to three times until the liquid is transparent. The protein content was determined according to the Kjeldahl method, and a nitrogen conversion factor of 5.95 was used to calculate the crude protein content of the milled rice  was used to determine the taste value. The 30-g milled rice sample was washed with water for 20 s. Then, it was transferred into a pot and added with water, following the ratio of rice to water of 1:1.35. It was soaked for 30 min and steamed for 40 min. After that, it was cooled at room temperature for 1.5 h to examine the taste value, including appearance, hardness, stickiness, and balance degree of the cooked rice. High taste value often indicates better taste quality . The vis2.5The rice starch sample was isolated following the method of 2.6First, the purified starch was debranched by using isoamylase (EC 3.2.1.68 Sigma). After that, high-performance anion-exchange chromatography (HPAEC) and amperometric detection were used to determine the length distributions of the starch branch chains similar to the method proposed by ; 150 mM 2.7θ range from 5° to 40° (1° min−1 rate and 0.02° step size). Relative crystallinity (%) was determined as the ratio of total crystalline peak areas to that of the total diffractogram by using the Origin 2018 software.The X-ray diffractometer  was used to analyze X-ray powder diffraction (XRD). The sample was canned over the 22.8P < 0.05), and Pearson correlation analysis. The principal component analysis was performed using Canoco 5. The figures were displayed by SigmaPlot 10.0 .The data in all of the tables and figures represented the average values of triplicate experiments. The SPSS 25.0 Statistical Software Program performed ANOVA, Tukey’s test , short chains (r = 0.64*), and S/L (DP > 12) (r = 0.65*). However, it was negatively correlated with protein content, relative crystallinity, and long chains. For chalkiness degree, it was negatively correlated with total starch content, short chains, and S/L, but it was positively correlated with protein content, relative crystallinity, and long chains. Furthermore, the principal components analysis demonstrated that PC1 and PC2 explained 91.4%, 90.4%, and 89.2% of the total variations in pasting properties, taste value, and grain chalkiness degree, respectively , increased long chains (DP > 12), and relative crystallinity. These results suggested that changes in starch structure also played an important role in determining rice quality under high natural temperature during the reproductive stage. These findings highlighted the importance of considering the effect of high natural temperature during the reproductive stage on the structure and physicochemical properties of rice starch in further breeding and practice.The original contributions presented in the study are included in the article/CC, WW, and DT initiated and designed the research, analyzed the data, and wrote the manuscript. DT, XS, and BC performed the experiments. YJ, AS, WW, MX and MC revised and edited the manuscript and also provided advice on the experiments. All authors contributed to the article and approved the submitted version.


The modification of flours by ultrasound (US) treatments requires excess water to suspend the sample to be treated, which must be removed after treatment to recover the ultrasonicated flour. The aim of this study was to determine the influence that the water removal method has on the final characteristics of US-treated gluten-free flours . US treatment parameters were constant, and two water removal methods were studied: freeze-drying and centrifugation + drying. The elimination of water by centrifugation resulted in the loss of solubilized compounds from the treated flours, which led to important differences between the final characteristics of US-treated flours. Ultrasonication resulted in the reduction of flours’ particle size and modification of their color parameters. Techno-functional properties were modified by US treatment, where the water removal method was more influential in whole grain samples (brown tef and quinoa). Few differences were found in thermal properties among pairs of US-treated samples, indicative that the effect caused to starch was mainly attributed to ultrasonication conditions than to the drying method. The water removal method markedly influenced the pasting properties of US-treated flours, resulting in lower profiles when freeze-drying was applied and higher profiles when flours were retrieved by centrifugation. Gels made with tef, corn and quinoa presented reduced tan(δ)₁ values after sonication, while gels made with rice did not show any modification. The water removal method is a decisive step in US treatments, defining the final characteristics of the treated matter, and having a great influence in the modification attributed to ultrasonication. The growing gluten-free (GF) market is constantly demanding improvement in their products, by the incorporation of novel ingredients and by the application of new technologies to expand the range of applicability of the gluten-free sources. Physical modifications of starches and flours are of great potential because they involve less byproducts and no chemical residues, and for being sustainable and environmentally friendly . DiffereUltrasound (US) treatment has shown many advantages in starch modification because of their higher selectivity and quality, reduced use of chemicals and processing time . UltrasoMany factors have been suggested to influence the modification caused by US treatments on starches, such as sonication power and frequency, time and temperature of the treatment and characteristics of the treated dispersion  ,10,11. OOryza sativa L.) is a major cereal crop, representing the staple food source for half of the world’s population. Rice flour is considered the most suitable cereal flour in GF formulations due to its soft taste, colorless, hypoallergenic properties, low levels of sodium and easy digestible carbohydrates [Eragostis tef (Zucc.) Trotter] grain, originated from Ethiopia, is always consumed as a whole meal given its extremely small size, resulting in higher iron, calcium and zinc than other cereal grains, and also presents proteins with an excellent balance among the essential amino acids  were used as dispersion parameters according to Abebe et al. (2015) [A Mastersizer 2000  equipped with a dry dispersion unit was used to determine the granulometry of the studied flours. The median diameter (D. (2015) . Samples2 + (Δa*)2 + (Δb*)2]1/2. Samples were measured five times.The color parameters of the flours were determined using a PCE-CSM 2 colorimeter , controlled with the 3nh Color Quality Controller System (CQCS3) software Version 3.2 . Results were obtained in the CIE L* a* b* and CIE L*C* h coordinates using the D65 standard illuminant and 10° standard observer. L* indicates lightness from black (0) to white (100), a* refers to tones from green (−) to red (+), and b* from blue (−) to yellow (+). The color difference (ΔE) of the US-modified flours with respect to their control flour was calculated using the equation: ΔE =  of the studied flours are presented in 50, and a consequent increase of the particles’ dispersion  in all cases, except for CO-C and QU-C. This behavior results from particle fragmentation caused by progressive erosion due to mechanical collision and shear forces from cavitation [50 values in centrifuged samples. In the case of the two exceptions, QU-C was not significantly different from QU-N, while CO-C was significantly higher than CO-N. The botanical origin of the sample is a factor that influences their susceptibility to fragmentation by US treatments, and by comparing the size reduction that the freeze-dried samples of these flours showed (QU-F and CO-F) with respect to their native counterparts, it can be concluded that their susceptibility was much lower than rice and tef flours. In the case of CO-C, the size increase after ultrasonication has been previously reported to happen due to agglomeration of granules in ultrasonicated starches [The determined values of median particle size . The increased amylose contents after ultrasonication could be explained due to molecular scission of chains by cavitation, contributing to the increase of linear fragments that are quantified as amylose . The deg−1 for starch analysis, and in the range of 1700–1600 cm−1 for protein secondary structure analysis. The absorption bands at 1047 cm−1, 1022 cm−1 and 995 cm−1 have been indicated to be particularly sensitive to modifications caused by starches, and are associated with the crystalline structure, the amorphous structures and C-OH bending vibrations, respectively [FTIR spectra were studied in the range of 1200–900 cmectively ,6. The aectively , while tectively . The valectively ,25. Thesectively . Linear ectively ,17.−1), β-turns (1690–1665 cm−1), random structure and α-helix (1665–1640 cm−1), and low frequency (LF) β-sheet (1640–1615 cm−1) [Changes in the protein secondary structure were studied in amide I see , the mos15 cm−1) ; the per15 cm−1) . The she15 cm−1) . The dryThe hydration properties , emulsion properties (EA and ES) and foaming properties (FC and CS) of the studied flours are presented in Proteins in flours are the main components influencing their emulsion activity and foaming capacity. Proteins, being surface active agents, can form and stabilize emulsions (EA and ES) by creating electrostatic repulsion on oil droplet surfaces , and cangel) after US treatment, where only those determined for TE-F, TE-C and QU-C were statistically lower than their corresponding native flour. A reduction in ΔHgel has been attributed to the disintegration of double helices present in the crystalline and non-crystalline regions of starch by cavitation [gel values could be due to the soft treatment conditions (short time and high concentration) and lower susceptibility of those flours to be modified by ultrasonication. ΔHgel results showed no significant differences between freeze-dried and centrifuged samples of the same flour (except for QU-F and QU-C), reflecting that the modification was mainly caused by US treatment and was not highly influenced by the drying method, as results are referred to the starch content in each flour. A significant reduction of ΔHgel has been reported in different flours (rice [O-gelT) and conclusion (C-gelT) gelatinization temperatures reflect the melting temperature of the weakest and high-perfection crystallites, respectively, in the starch granules [P-gelT) indicates the temperature when the highest energy absorption is recorded. Ultrasonication led to a reduction of all O-gelT, P-gelT and C-gelT values. The few differences observed between freeze-dried and centrifuged samples indicate that starch in the studied flours is not greatly affected by the drying method, and that the observed modified values were mainly attributed to ultrasonication. Lower gelatinization temperature values indicate that cavitation led to the structural weakening of starch configuration, allowing for greater mobility of starch polymers and promoting water accessibility to the crystalline regions [gel. Similar results have been reported for ultrasonicated quinoa flour [am-lip) was significantly increased by US treatments in RI-F, RI-C, TE-C and QU-C. The depolymerization of long chain amylose and amylopectin molecules caused by cavitation would generate very short amylose-like starch fragments that would increase the formation of amylose-lipid complex in US-treated samples [The thermal properties determined for the studied flours are presented in vitation ,35,36, avitation , so sonirs  and isors  was significantly increased in rice flour, reduced in quinoa flour and was unchanged in tef and corn flours. Ultrasonication led to a uniform effect on ΔHret regardless of the drying method applied (except in tef flour), varying depending on the nature of the sample. US treatment led to a significant increase of ΔHret in rice flours and a reduction in quinoa flours, while no significant modification was detected in tef and corn flours. The modified ΔHret values in rice and quinoa samples indicate that ultrasonication led to higher and lower, respectively, amylopectin retrogradation capacity. These results illustrate how influential the botanical origin of the studied sample is in determining the effect of ultrasonication over certain properties. The values determined for the dissociation of amylose-lipid complex in the second scan (ΔHam-lip-ret) showed no significant differences among flours, with all showing higher values than those determined in the first scan. The increased values in the second scan have been determined to be due to better conditions for complex formation after the first heating, because the leaking of amylose from starch occurs at temperatures above the starch gelatinization temperature range [O-retT of RI-F, TE-F, QU-F and QU-C, and P-am-lip-retT of QU-F and QU-C, with respect to their corresponding native flour. A second scan was performed after 7 days of sample storage at 4 °C to evaluate the thermal transitions of retrograded starch. After ultrasonication, the enthalpy determined for the melting of recrystallized amylopectin (ΔHre range . The traThe determined pasting properties of the studied flours are presented in max) and the cross over, or stress where the values of the elastic (G′) and viscous (G″) moduli are equal. Frequency sweeps were performed at a constant strain of 1%, within the LVR, and the data obtained were adjusted to potential equations. The coefficients G′1, G″1 and tan(δ)1 represent the elastic and viscous moduli and the loss tangent at a frequency of 1 Hz, respectively, while the exponents a, b and c quantify their dependence to the oscillation frequency (ω). The determined values for the strain and frequency sweeps are presented in max and cross over values determined after US treatment showed a different behavior depending on the nature of the studied flours. In rice, no significant differences were observed, while in tef, corn and quinoa, reduced values were observed after sonication (except for CO-C), presenting lower values in centrifuged samples than in freeze-dried samples. These results reflect the fragmentation of long-chain starch molecules by ultrasound treatments, leading to the formation of weaker gels (compared to those formed by native flours) [1 and G″1, but the proportion of change of both parameters remained unchanged, thus tan(δ)1 was not significantly modified. In the other flours , even though the values determined for G′1 and G″1 did not present a uniform trend, it could be seen that freeze-drying after ultrasonication mainly resulted in lower values, while centrifugation led to higher values. It has been indicated that the reduction of the viscoelastic moduli values by US treatments results from a combined effect of starch granule disruption and breakdown of the linear amylose molecules, since the rupture of polymeric chains leads to the straightening out of amylose molecules, and results in shorter linear amylose chains that are unable to form a consolidated viscoelastic network during gelatinization [1 were significantly reduced after ultrasonication, except in rice flour. Lower tan(δ)1 values are indicative of gels with higher strength [1 values after US treatments of starches [The rheological properties of the gels made with the studied flours were characterized by dynamic oscillatory assays, in which strain sweeps at a constant frequency of 1 Hz determined the end of the LVR (τ flours) . The bot flours) . The resnization ,9,15. Thstrength . A reducstarches ,9,32 andstarches ,31 has bThe natural characteristics and botanical origin of the flours influenced their susceptibility to be altered by US treatments. The water removal method greatly influenced the effect of ultrasonication on the modified flours, mainly resulting from different compositions due to the loss of soluble compounds  when flour samples were retrieved by centrifugation, resulting in flours with higher starch content. Ultrasonication caused a significant particle size reduction, which resulted in increased L* values, and a greater interaction with water, improving WAI and SP. Signs of partial depolymerization of starch macromolecules and a general weakening of starch structural arrangement were determined by increased values of amylose content after ultrasonication, the higher proportion of amorphous to ordered structure zones indicated by FTIR and the reduction of gelatinization transition temperatures showed by DSC. The pasting properties were markedly influenced by the water removal method, derived from the composition of the US-treated flours, where the higher starch content in centrifuged samples increased the viscometric profiles, while freeze-drying reflected the real effect of ultrasonication, mainly leading to reduced viscometric profiles. The gels’ rheological properties indicated that ultrasonication led to weaker gels that resisted lower stress before the rupture of their structure, probably related to fragmentation of starch macromolecules.


Starch can be found in the stems, roots, fruits, and seeds of plants such as sweet potato, cassava, corn, potato, and many more. In addition to its original form, starch can be modified by reducing its size. Starch nanoparticles have a small size and large active surface area, making them suitable for use as fillers or as a reinforcing material in bioplastics. The aim of reinforcing material is to improve the characteristics of bioplastics. This literature study aims to provide in-depth information on the potential use of starch nanoparticles as a reinforcing material in bioplastic packaging. This study also reviews starch size reduction methods including acid hydrolysis, nanoprecipitation, milling, and others; characteristics of the nano-starch particle; and methods to produce bioplastic and its characteristics. The use of starch nanoparticles as a reinforcing material can increase tensile strength, reduce water vapor and oxygen permeability, and increase the biodegradability of bioplastics. However, the use of starch nanoparticles as a reinforcing material for bioplastic packaging still encounters obstacles in its commercialization efforts, due to high production costs and ineffectiveness. BThe basic material commonly used for the manufacture of reinforcing materials in bioplastics is starch. Starch is a polysaccharide derivative compound in the form of granules and is used by plants as an energy source. Starch granules have a semi-crystalline structure and are composed of amylose and amylopectin, which are complex carbohydrate polymers ,21,22. AOne starch modification method is to reduce the size of starch particles to nanosize, which can be referred to as starch nanoparticles. Starch nanoparticles have at least 1 particle with a size of less than 300 nm and have a high active surface area . Starch Nowadays, the interest in research on bioplastics and reinforcing materials to produce bioplastics with better characteristics has increased. Starch is a promising resource because it is cheap, biodegradable, and abundant; thus, starch is promising in bioplastics and in reinforcing material production. This literature study is expected to provide in-depth information on the potential use of starch nanoparticles as reinforcing materials in bioplastics.There are two types of starch nanoparticle manufacturing methods, namely, top-down and bottom-up ,37. The Examples of treatments that include physical methods are milling ,47, highThe physical method tends to result in starch nanoparticles with low crystallinity because the use of energy is very large, so the crystalline structure becomes weak . This doAmong the physical method for starch nanoparticle production, milling produces a larger size of starch nanoparticle. During milling, the reduction in starch granules to nanosize is obtained from the weight and speed of the grinding ball that continues to move and rotate. This movement will produce kinetic energy. This energy will be channeled to the sample, resulting in a reduction in the size sample . It can The ultrasonication method is considered effective in reducing the size of starch. During ultrasonication, energy is transferred to the starch through a cavitation process, which is a condition where the microbubbles burst and spread in the solution suspension in the form of sound waves. This process involves high speed and generates shear forces that can break the covalent bonds of starch; thus, the starch particle size decreases . SeveralExamples of chemical production methods for starch nanoparticles are acid hydrolysis ,63,64 an2SO4) or chloride acid (HCl) [Chemical and enzymatic methods mostly result in starch nanoparticles with high crystallinity ,75. The id (HCl) , while eid (HCl) . Althougid (HCl) ,63,78, wid (HCl) ,80,81,82id (HCl) .Enzymatic hydrolysis tends to produce a higher yield of starch nanoparticle. A study showed potato starch nanoparticles with a manufacturing method using the pullulanase enzyme on elephant foot yam tuber starch, where the extraction yield obtained was 61.33% . AnotherRegarding size, acid hydrolysis can produce starch nanoparticles with smaller sizes compared to nanoprecipitation. Putro et al.  have repA combined method can be performed using two or more methods at one time of production. It is possible to combine chemical methods and chemical methods, physical methods, and physical methods, as well as chemical methods and physical methods. Examples of combined methods are the use of acid hydrolysis and ultrasonication, acid hydrolysis and precipitation, enzymatic hydrolysis and acid hydrolysis, and many more ,90,91,92The use of the acid hydrolysis method with other methods, such as enzymatic hydrolysis, ultrasonication, and milling as a pre-treatment, can overcome the shortcomings of the acid hydrolysis method, namely, the long treatment time . Such prThe use of ultrasonication before acid hydrolysis treatment can help acid molecules to reach the starch surface more quickly so that the required hydrolysis time is shorter . The cavAmylose content in starch can be analyzed using the iodine-binding method. When amylose meets iodine, it produces a blue color, while amylopectin reacts with iodine to produce a red–purple color that is not too bright . MinakawPotato starch nanoparticles obtained through the nanoprecipitation method had the highest amylose content (21.39%) compared to the acid hydrolysis method (4.79%) and the combined acid hydrolysis method with ultrasonication (3.08%) . In starFurthermore, LeCorre et al.  have repGranule morphology and the size of starch nanoparticles were analyzed using SEM (scanning electron microscopy), TEM (transmission electron microscopy), or FE-SEM (field-emission scanning electron microscopy) . GranuleFurthermore, Minakawa et al.  reportedCrystallinity can be interpreted as a comparison between the mass of the crystalline region with the mass of starch nanoparticles as a whole . Starch Starch crystallinity has a relationship with the double-helix structure of the amylopectin chain, so starches with high amylopectin content tend to have high crystallinity compared to starches with high amylose content . Starch o (onset temperature), Tc (endset temperature), Tp (peak temperature), Tc−To (melting temperature range), and ∆H  [o, Tp, Tc, and ∆H values of 57.07 °C, 66.33 °C, 73.73 °C, and 9.84 J/g, respectively. The nanoprecipitation + ultrasonication process reduces To, Tp, Tc, and ∆H by 40.77 °C, 50.39 °C, 63.8 °C, and 1.99 J/g, respectively [Thermal characteristics can be analyzed using DSC  and TGA . The parameters observed include T change) . Starch  change) . The ultectively . A lowerectively . The theBioplastic refers to plastics that are made from biomass or are bio-based, plastics that have the characteristics of being biodegradable, or plastics made from biomass that can be degraded naturally ,110. ExaThe methods commonly used for the manufacture of bioplastic packaging are solution casting ,119,120,The injection molding method produces packaging with good characteristics with effective production costs, including in large-scale production. Before printing, the dough must first be made which is a mixture of matrix and plasticizer. The injection process is carried out using a piston injection molding machine . The extThis paper will discuss the mechanical properties, water vapor permeability, thermal properties, and biodegradability of bioplastic with various starch nanoparticles for reinforcement. All the literature used in discussing the characteristics below use casting as a bioplastic production method. A lot of researchers use the casting method because the casting method is the simplest and most inexpensive method, compared to other methods. The characteristics of bioplastic with starch nanoparticle reinforcement will be influenced by filler concentration, filler–matrix interaction, and filler characteristics ,130.Parameters tested include flexibility, strength, and stiffness of the bioplastic film. Mechanical characteristics can be analyzed using a TA   or usingPiyada et al.  have repThe interaction of the reinforcing material and the matrix in bioplastic packaging forms a strong bond so that the water and air entry paths in the matrix molecules become torturous. This makes it difficult for air and water vapor to penetrate through the packaging film . With a The use of reinforcing material is effective in reducing water vapor and oxygen permeability. Bioplastic with a polyurethane matrix (PU) and a reinforcing material of corn starch nanoparticles has a water vapor permeability (WVP) of 3.17% without the use of reinforcing material, which decreases to 0.92% with the use of 30% reinforcing material. Oxygen permeability also decreased from 27.5% with 0% reinforcing material to 6.7% with 30% reinforcing material . A previg), onset temperature (To), melting point (Tm), and enthalpy change (∆H) [Analysis of the glass transition temperature can be carried out by using DSC  , DMA (dynge (∆H) . The mecnge (∆H) .According to Hakke et al. , there iThe biodegradability test was carried out to determine the time required for bioplastic packaging to decompose in the soil. Biodegradability causes a decrease in molecular weight, due to the activity of microorganisms, such as bacteria, microalgae, and fungi, resulting in molecular reduction . This te2, whereas without reinforcing material, the observed weight loss was 7 mg/cm2. Then, the observed weight loss was more than 2 times [The use of starch nanoparticles as a reinforcing material is effective in increasing the biodegradability of bioplastic packaging. A previous study has reported that within 1 week, bioplastic with a starch mung bean matrix and glycerol plasticizer experienced a weight loss of 28.67%. When adding 1% mung bean starch nanoparticle reinforcing material, in the same period, there was a decrease in packaging weight of 83.46%, and when 2–10% reinforcing material was added, the packaging was 100% degraded . The use 2 times . An incr 2 times . Accordi 2 times , the use 2 times . The bio 2 times ,145. TheImproving the properties of bioplastic polymers is a primary challenge of achieving sustainability in the food and packaging industry ,147,148.The use of starch nanoparticles as a reinforcing material as well as in the packaging field in general still finds many obstacles in its commercialization business. This is because production costs have not been effective. After all, the number of adaptations of this technology is still very low. On the other hand, reinforcing materials from inorganic materials have been widely applied, so that the production costs are cheaper and can produce bioplastic packaging with good characteristics. This causes the price of bioplastic packaging with reinforcing materials from starch nanoparticles to be expensive, and the option of using reinforcing materials from inorganic materials is preferred .Research related to bioplastics as an alternative to conventional plastics is increasingly being carried out. This is based on the environmentally friendly nature of bioplastics. However, bioplastics still have limited characteristics, such as low mechanical strength and high water vapor permeability; hence, reinforcing the material’s role becomes important. The use of starch nanoparticle as a reinforcing material in bioplastic decreases the elongation at break and water vapor permeability, and increases tensile strength and biodegradability. In general, starch nanoparticles also increase the thermal stability of bioplastics. The concentration of reinforcing material that exceeds the maximum threshold has an impact on increasing water vapor permeability and decreasing the mechanical and thermal characteristics of bioplastic packaging because starch nanoparticles at excessive concentrations tend to form aggregates.Starch nanoparticles have a great opportunity to be developed in the packaging field, mainly because of environmental interest. Although starch nanoparticles have many advantages as reinforcing materials, the utilization of starch nanoparticles on an industrial scale is still limited because adaptation to the use of starch nanoparticles as reinforcing materials in packaging is still low, causing production costs to be high and ineffective. Hence, continuous research about nano-reinforced bioplastic is needed.In bioplastic processing, the dispersion and homogeneity of mixing bioplastic with starch nanoparticles as a reinforcing material are also interesting to study to produce high-quality bioplastic. The modification of starch nanoparticles can also be a promising option to make better bioplastic characteristics. There have been several studies on starch nanoparticle modification, but the number is still very limited, and thus further research needs to be developed.


The specimens were characterized by density and translucency measurements, a three-point bending test, X-ray diffraction (XRD), scanning electron microscopy (SEM), and shrinkage measurement. The addition of Ga2O3 improved both translucency and flexural strength of 5Y zirconia high-speed sintered bodies. XRD and SEM observations revealed that this improvement in properties was due to the change in the crystal phase composition and the decrease in the amount and size of pores due to the addition of Ga2O3. Shrinkage measurements also revealed that the addition of Ga2O3 changed the sintering behavior of 5Y zirconia, suggesting that this change led to a reduction in porosity. From the above results, it was concluded that Ga2O3 addition is effective in improving the properties of 5Y zirconia high-speed sintered bodies.The high-speed sintering of zirconia has become essential for ceramic dental prosthesis treatment in a single visit. Previous studies have shown that 5 mol% yttria-stabilized zirconia (5Y zirconia), with the exception of some types, loses strength and translucency with high-speed sintering. In this study, 0.15–0.92 wt% Ga Of these stabilizers, Y2O3 is most commonly used for dental zirconia. The concentration of the stabilizer is an important factor for dental zirconia because the properties of zirconia are greatly affected by the concentration of the stabilizer: the mechanical properties improve and translucency decreases with increasing stabilizer concentrations [2O3 stabilized zirconia (3Y zirconia), 4 mol% Y2O3 stabilized zirconia (4Y zirconia), and 5 mol% Y2O3 stabilized zirconia (5Y zirconia) are mainly used. These materials are selected according to the clinical cases. For example, 5Y zirconia is used in cases with high esthetic demands such as anterior teeth due to its high translucency, and 3Y zirconia is used in the presence of heavy loads such as the case of long span bridges [Zirconia has become a major dental prosthetic material due to its excellent mechanical properties, chemical stability, high aesthetics, radiopacity and biocompatibility ,2. Zircotrations ,5. In th bridges ,7.Zirconia dental prostheses are made with a computer-aided design and manufacturing (CAD/CAM) approach. Since zirconia sintered bodies are too hard and strong to be machined, semi-sintered zirconia is first processed into a desired shape and then further sintered to full density. Since this sintering usually takes 6 to 10 h , the proIn recent years, several sintering furnaces capable of high speed sintering have been released. Among them, the most distinctive example is CEREC Speedfire (Dentsply Sirona). Zirconia can be densified in 30 min with employing this sintering furnace, so dentists can complete zirconia prosthetic treatment in one day (One Visit Treatment) ,10. GeneIt is well known that the various properties of ceramic sintered bodies are affected by sintering conditions such as heating rate and holding time. A number of reports have been published on the various properties of high-speed sintered dental zirconia ,14,15,162O3. Ga3+  is a trivalent cation that is smaller than Zr4+ . Furthermore, the colorlessness of Ga2O3 is a favorable factor because it does not affect the color tone of zirconia. Since Ga is not a dominant metal in dentistry, the biological effects of using Ga2O3 in dental materials are unknown. However, Cochis et al. [2O3 is also not cytotoxic.The formation of residual pores in zirconia sintered bodies is affected by their sintering behavior. The addition of metal oxides is known as a means of changing the sintering behavior of ceramics. The effect of oxide additives on the sintering behavior of zirconia was reported in a report by Flegler et al. . Their ss et al.  reported2O3 to yttria-stabilized zirconia. The present study aims to investigate the effect of the addition of Ga2O3 on the properties of high-speed sintered 5Y zirconia and to clarify the causes from the changes in crystal structure, microstructure, and sintering behavior.Although the effect of adding a number of different metal oxides has been investigated, to the best of our knowledge, there are no reports with regard to the adding of GaCommercially available 5Y zirconia powder containing 0.05 wt% of alumina  was used for a starting powder. The powder was pressed uniaxially by a press molding machine  to obtain zirconia blocks having a diameter of 19 mm × thickness of 19 mm and discs having a diameter of 100 mm × thickness of 17 mm. Next, the blocks and discs were isostatically pressed at 200 MPa using a cold hydrostatic isotropic press .The pressed zirconia blocks/discs were degreased at 500 °C in an inert gas oven  and then fired in a firing furnace  at 970 °C to obtain a pre-sintered zirconia block/disk (φ18 mm × 18 mm/φ98.5 mm × 16 mm).The pre-sintered zirconia blocks and disks were cut with cutting equipment , and surface polished with SiC water resistant polishing paper (# 1000) to reach the dimension required by each test.2O3 doping of each specimen was performed by impregnation with a Ga solution. 2O3 through the urea hydrolysis process and a firing process at 500 °C.The GaSamples produced as described above were finally subjected to high-speed sintering at 1510 °C according to the sintering schedule shown in Each sintered specimen was processed to the dimensions required for each test  by a sur2O3 content in each sample was evaluated by Wavelength dispersion X-ray fluorescence spectroscopy . The Ga2O3 concentration was semi-quantified by the fundamental parameter method (FP method) from the fluorescence X-ray data obtained by measuring the surface of each specimen .The GaThe sintering behavior of each sample was evaluated by measuring the shrinkage during sintering with a thermal dilatometer . The measurement was performed using the temperature range: room temperature to 1600 °C, heating rate: 5 °C/min, atmosphere: air.The microstructure of the specimen was evaluated by means of scanning electron microscopy . An acceleration voltage of 15 kV was used. The specimens were Au-coated to prevent charge-up.50) of each sample were determined by the image analysis of SEM images. Two hundred grains were randomly selected from the SEM images of each specimen and analyzed using image analysis software .The particle size distribution and median diameter  according to JIS R 1634: 1998. Relative density was calculated by dividing the measured density by the theoretical density (Equation (1)).A: content of alumina (wt%)G: content of Ga2O3 (wt%)ρ: measured density (g/cm3)ρ0: theoretical density (g/cm3)ρA: theoretical density of aluminaρG: theoretical density of Ga2O3ρx: theoretical density of zirconia containing 5.33 mol% of yttria (6.028 g/cm3 [28 g/cm3 ).The crystal structure of each specimen was characterized by X-ray diffraction  using Cukα radiation as the X-ray source. The specimens used for XRD measurement were the same as those used for XRF measurement. The measurement conditions are as follows: tube current; 40 mA, tube voltage; 40 kV, 2θ range; 20–120 °, scan step; 0.02 °. Rietveld refinement was carried out from the obtained diffraction pattern using a Rietan-FP . Two tetc/a) was calculated using the lattice constant refined by Rietveld refinement.The tetragonality  from chromaticity data measured on a white and black background (Equation (2)):L*, a*, b*: CIE L* a* b* valueSubscript w: measured on the white backgroundSubscript b: measured on the black background.The chromaticity value (L* a* b*) of each specimen was measured by a spectroscopic colorimeter . The specimens were measured on a black and white background (n = 2).σ (MPa) was calculated according to the following equation in accordance with ISO 6872:The flexural strength was measured by a three-point flexural test in accordance with ISO 6872  (n = 10). The test was performed using a universal testing machine . The test conditions are as follows: span, 12 mm; crosshead speed; 1.0 mm/min. Three-point flexural strength P: breaking load (N)l: test span (mm)w: width of the specimen (mm)b: thickness of the specimen (mm).A Tukey-Kramer multiple comparison test was used to compare each sample with regard to relative density, TP and flexural strength.2O3 content of each sample. The Ga2O3 content in the samples increased with increasing Ga concentration in solution , which is very small compared to Y3+ . Therefore, the increase in grain size by the addition of Ga2O3 is due to the enhancement of grain boundary diffusion due to the segregated Ga3+ at the grain boundaries. As the amount of Ga2O3 added increases, the amount of Ga3+ segregated to the grain boundaries also increases, and the grain boundary diffusion enhancement effect becomes greater. Therefore, the grain size increased as the amount of Ga2O3 added increased.The grain size increased as the amount of Gai et al. ,29 repori et al. ,31 suggea et al.  and Matsa et al.  reportedIn all samples, the grain size increased as the thickness of the pre-sintered specimen increased a. This i2O3 content and relative density for a fixed sintering schedule. Ga2O3-doped samples showed higher relative densities than Ga0. This indicates that Ga2O3 doping promoted densification. The relative density was highest for Ga1.5 and decreased slightly with increasing Ga2O3 content. However, as described in 2O3 content, is likely related to the calculation method of theoretical density. In this study, the theoretical density was calculated by Equation (1). This formula provides the density of a mixture of alumina, Ga2O3 and 5Y zirconia. However, the results of an X-ray diffraction analysis  may be calculated to be larger than the actual theoretical density, and this difference is expected to increase with increasing Ga2O3 concentration. We believe that this is the reason for the decrease in relative density in the Ga1.5-Ga5.0 range.analysis  suggest l et al.  reported2O3 does not affect the crystallinity of the zirconia sintered bodies. The X-ray diffraction peaks of all samples were assigned to only two tetragonal phases: a high yttrium tetragonal phase and a low yttrium tetragonal phase. No diffraction peaks from Ga compounds  were detected. An additional feature was that the diffraction peak of high yttrium tetragonal shifted towards higher angles upon the addition of Ga2O3. This suggests a decrease in lattice volume of tetragonal zirconia with the addition of Ga2O3. Accordingly, it is believed that the added Ga2O3 is dissolved in the zirconia crystal lattice. 2O3 content.2O3 content, while showing the opposite trend for the low yttrium tetragonal phase. In addition, the content of the high yttrium tetragonal phase increased with the addition of Ga2O3. These results suggest that phase separation into a high yttrium tetragonal and low yttrium tetragonal phase may be promoted with the addition of Ga2O3. It is known that yttria-stabilized zirconia begins to separate into yttria-rich and poor regions above 1300 °C, which eventually become high yttrium and low yttrium tetragonal phases [2O3 promotes phase separation is not clear, it is expected to be due to the enhancement of grain boundary diffusion due to the segregation of Ga3+ to the grain boundaries. As discussed in 3+ is expected to segregate at the grain boundaries. In addition, small cations segregated at the grain boundaries of zirconia improve grain boundary diffusion [3+ segregated at the grain boundaries promoted the diffusion of Y3+, resulting in enhanced phase separation.The tetragonality of high yttrium and low yttrium tetragonal phases displayed a contrasting behavior, with the tetragonality of the high yttrium tetragonal phase decreasing with increasing Gal phases ,28,29. Aiffusion ,32. TherAs mentioned above, the tetragonality of the two tetragonal phases showed an opposing behavior, so the stabilization behavior of the system as a whole is unclear. Therefore, we calculated the mean tetragonality as the tetragonality of the entire system using Equation (4).t: tetragonalityc: phase contenth: high yttrium tetragonal phaseSubscript l: low yttrium tetragonal phaseSubscript 2O3 content . Since the tetragonality was reduced in the high yttrium tetragonal phase, it is considered that Ga3+ replaced Zr4+.Lattice volume decreased with increasing Gantration , and thethan Y3+ . Thus, i2O3, or both, which cannot be distinguished in the results of this study.On the other hand, in the low yttrium tetragonal phase, the tetragonality increased, indicating a decrease in yttrium concentration. Therefore, the decrease in lattice volume in the low yttrium tetragonal phase could be due to either the decrease in yttrium concentration or the solid solution of Ga2O3 are summarized below.The crystallographic effects of the addition of Ga2O3 dissolves in 5Y zirconia and reduces the lattice volume;Ga2O3 functions as a stabilizer;Dissolved Ga2O3 promotes the phase separation into a high yttrium tetragonal and low yttrium tetragonal phase.GaA more detailed crystallographic investigation is necessary to clarify the relationship between the gallium addition and the crystalline phase composition of zirconia.2O3 concentration of a sample with a pre-sintered thickness of 1.9 mm. This result proves that the addition of Ga2O3 improved the translucency. This effect is considered to be a consequence of both microstructural change and crystal phase composition. As mentioned above, the addition of Ga2O3 decreased both the number and diameter of residual pores. The translucency of zirconia is sensitive to pore size and concentration, and even a small pore reduces translucency [2O3, which reduced both the number and diameter of pores, improved translucency. In addition, a decrease in grain boundary scattering due to the larger grain size might also contribute to the observed improvement in translucency.slucency . Therefo2O3 led to the improvement of translucency. In general, ceramic polycrystals have higher translucency, as the optical anisotropy of the crystal is smaller. XRD results show that all samples consist of optically anisotropic tetragonal phases , the higher flexural strength and fracture toughness [2O3 decreased the tetragonality and the content of the low yttrium tetragonal phase. This, in turn, makes the stress-induced phase transformation less likely and lowers the flexural strength.From a crystallographic viewpoint, the observed change in crystal phase composition upon the addition of Gaoughness . XRD res2O3 addition affects the flexural strength in both a positive way by decreasing the number and size of pores, and in a negative way by increasing grain size and decreasing the amount of stress-induced phase transition. In this study, Ga2.5 showed the highest value flexural strength because of the balance of these two contrasting effects.In summary, Ga2O3-added samples was 555~724 MPa. This strength level complies with ISO6872 Class 4 . Since ‘One Visit Treatment’ does not treat cases larger than 3-unit-prostheses, Ga2O3-added samples have clinically suitable flexural strength. Furthermore, the Ga2O3-added samples show less of an increase in pore number and diameter, even in thick specimens, and high reliability is obtained, even in thick prostheses. Therefore, Ga2O3-added 5Y zirconia is a promising material for high-speed sintering.The flexural strength of the Ga2O3 to 5Y zirconia improves translucency and flexural strength during high-speed sintering. The crystallographic characterization by XRD indicated that the added Ga2O3 dissolved in 5Y zirconia, leading to a decrease in tetragonality and an increase in the content of high yttrium tetragonal phases. Microstructural evaluation by SEM showed that the addition of Ga2O3 reduced the amount and size of pores in the sintered body, especially at the center of thick specimens. It was concluded that changes in the composition of these crystal phases and microstructure led to appreciable improvements in translucency and flexural strength.This study revealed that the addition of Ga2O3 increased the sintering rate from the early to middle stages of sintering, and decreased it beyond the middle stage. It was concluded that this change in sintering behavior was key to reducing both the number and size of pores in the sintered body.The measurement of the shrinkage rate with a dilatometer showed that the addition of Ga2O3-added 5Y zirconia exhibits high translucency and flexural strength, even when a thick specimen is sintered at high speed, making it a suitable material for one-visit treatment.Ga


No differences were found in the GRF characteristics among the shoe conditions. These findings suggest that an increase in midsole stiffness only in the midfoot region can reduce intersegmental foot medial-lateral movements during the stance phase of running. This may further decrease the tension of the foot muscles and tendons during prolonged exercises.We investigated how midfoot stiffness of running shoes influences foot segment kinematics and ground reaction force (GRF) during heel-toe running. Nineteen male rearfoot strike runners performed overground heel-toe running at 3.3 m/s when wearing shoes with different midfoot bending stiffnesses  in a randomized order. A synchronized motion capture system (200 Hz) and force plate (1000 Hz) were used to collect the foot-marker trajectories and GRF data. Foot kinematics, including rearfoot-lab, midfoot-rearfoot, forefoot-rearfoot, and forefoot-midfoot interactions, and kinetics, including GRF characteristics, were analyzed. Our results indicated that high midfoot stiffness shoes reduced the forefoot-rearfoot range of motion (mean ± SD; high stiffness, 7.8 ± 2.0°, low stiffness, 8.7 ± 2.1°;  Running is one of the most popular activities, and footwear is commonly used during running exercises, except for barefoot running. Footwear constructions and features such as midsole hardness, midsole thickness, heel-toe drop, heel counter, longitudinal bending stiffness, and torsional stiffness would significantly alter foot position and joint loading, which is aimed at improving running performance and preventing running injuries ,2. FootwThe longitudinal bending stiffness is described as shoe rotation around the medial-lateral axis and is considered to be one of the key features in running shoe development. This can be optimized by incorporating higher-density/thicker midsoles, carbon fiber/thermoplastic polyurethane (TPU) plates, or flex grooves . The fooPrevious studies examined the effect of shoe bending stiffness on arch movement. Cigoja et al.  suggesteMoreover, Renan et al.  found thStiff shoes with carbon fiber plates have been reported to systematically reduce the metatarsophalangeal (MTP) maximum dorsiflexion and plantar flexion velocities  and peakShoe bending stiffness also influences the ground reaction force (GRF) data. Shoes with carbon fiber insoles decreased the peak GRF in the anterior-posterior direction. They increased the stance duration while the anterior-posterior GRF impulse and peak vertical GRF remained unchanged . Flores The effect and underlying mechanism of shoe midfoot stiffness on intersegmental foot movement and GRF during running are still questionable. Hence, the objective of this study was to examine the effect of shoe midfoot bending stiffness on intersegmental foot kinematics using a multi-segment foot model and GRF characteristics. By gradually increasing midfoot stiffness, we hypothesized that stiffer midsoles would exhibit smaller intersegmental motion in the sagittal and frontal planes, slower propulsion velocity, and smaller GRF during running.p2 = 0.14, effect size f = 0.403) to examine the differences between the three tested conditions at 0.05 when the correlation among repeated measures was set at 0.5 using G*Power version 3.1.9.7 [A sample size of 12 was calculated based on an analysis of variance  to provide sufficient statistical power (0.8) to detect a large effect size  were built from a neutral running shoe model with a midsole ethylene-vinyl acetate copolymer (EVA) hardness of Asker C 45 . The medium stiffness shoe was identical to the original shoe model. The low stiffness shoe was modified by cutting with three 1 cm depth and 0.9–1.8 cm width transverse grooves at the midfoot region. In contrast, the high stiffness shoe was modified by replacing the original EVA midfoot plate with a stiffer TPU midfoot plate (Shore D 70). All three shoe conditions had the same size (9.5 cm × 2.5 cm × 0.2 cm) and location of the midfoot shank plate as per the manufacturer’s specification A. The loThere are different approaches to marker attachment for foot kinematics measurement: shoe surface markers, foot skin markers, and bone-mounted markers. Arnold and Bishop  reviewedAll reflective markers were placed at marker locations on the skin by the same experienced researcher across all tested conditions and participants . Eight mAccording to previous studies ,30, the A six-camera motion capture system  was synchronized with a force plate  to collect lower extremity kinematics and GRFs during running. Running trials were performed over a 16 m laboratory runway with a force plate mounted 8 m from the start line. The target running speed was set at 3.3 ± 5% m/s  and was Fourth-order Butterworth low-pass filters with cut-off frequencies of 150 and 30 Hz were applied to the GRF and kinematic data ,32, respz-axis pointed upward and along the line connecting the origin with the midpoint of MM and LM, and the x-axis was orthogonal to an anatomical plane, which was defined among four markers  using the least squares fit, and the y-axis was orthogonal to the x–z plane and followed the right-hand rule. A foot coordinate reference frame (x’y’z’) was built in our study to calculate joint angles, including midfoot-rearfoot (midfoot with respect to the rearfoot), forefoot-rearfoot  and forefoot-midfoot (forefoot with respect to the midfoot)  was built to calculate the segmental rotation  and joint rotation  kinematics A. The ormidfoot) B. The or segment , and thex-axis in the sagittal plane, eversion/inversion about the y-axis in the frontal plane, and internal/external rotation about the z-axis in the transverse plane. During the stance phase, the rearfoot, midfoot, and forefoot kinematics were investigated in the sagittal and frontal planes. Each participant performed a static calibration trial at the neutral standing position before dynamic running trials for each shoe condition, joint angles were defined to be 0° at this position. Dynamic joint angles were calculated relevant to the static calibration trial during the dynamic test. Kinematic data were normalized to 0–100% of the stance phase.Segmental and joint rotations were calculated using Euler angles  according to the International Society of Biomechanics recommendations ,37, thatThe vertical GRF variables included ground contact time, peak vertical force 1, maximum vertical loading rate 1, peak vertical force 2, and maximum vertical loading rate 2 . Ground p-value when Mauchly’s test of sphericity assumption was rejected. ηp2 was calculated to show the effect size of ANOVA and was categorized as small (0.01 ≤ ηp2 < 0.06), medium (0.06 ≤ ηp2 < 0.14), and large (ηp2 ≥ 0.14) [p < 0.05. All statistical analyses were performed using SPSS 26.0 .Kolmogorov–Smirnov tests were applied to verify that all data were normally distributed. One-way repeated-measures ANOVAs were performed to detect significant differences in shoe midfoot stiffness on the analyzed parameters. When a main effect of the shoe was present, post hoc tests with Bonferroni correction were applied to detect significant differences between every two conditions. Greenhouse–Geisser’s was chosen as an adjustment of the  ≥ 0.14) . StatistThe multi-segment foot kinematics, including rearfoot-lab, rearfoot-shank, midfoot-rearfoot, forefoot-rearfoot, and forefoot-midfoot interactions of the shoe conditions are displayed in p = 0.050, ηp2 = 0.154) did not show any significant difference. A significant main effect of the shoe was observed for peak plantar flexion angular velocity during the braking phase  = 3.697, p = 0.035, ηp2= 0.170). However, Bonferroni post hoc comparisons did not find any difference between shoe conditions. No significant difference was found in rearfoot-shank peak eversion angle and frontal plane range of motion between shoe conditions (p > 0.05).For rearfoot-lab interactions in the sagittal plane, the dorsiflexion angle at initial contact  = 3.270, p > 0.05).None of the midfoot-rearfoot variables, including the peak dorsiflexion angle, frontal plane range of motion, and peak plantar flexion angular velocities, showed any differences in sagittal and frontal plane movements between shoes during braking and propulsion phases  was observed in the frontal range of motion of the forefoot-rearfoot joint. The magnitude was greater in the low stiffness shoe (8.7 ± 2.1°) than that in the high stiffness shoe (7.8 ± 2.0°) during the stance phase.A significant main effect  = 6.247, p = 0.020, ηp2 = 0.196) in the medium stiffness shoe (4.6 ± 0.9°) than in the high stiffness shoe (4.2 ± 1.1°). A significant main effect  = 4.009, p = 0.043, ηp2 = 0.182) of the shoe condition was found when comparing the peak plantar flexion velocity during the propulsion phase, and no significant difference was observed for post hoc comparisons.The forefoot-midfoot variable indicated a significantly greater frontal plane range of motion  = 4.399, p > 0.05; The averaged vertical and anterior-posterior GRF curves are displayed in This study examined the influence of shoe midfoot stiffness on multi-segment foot movements and GRF during heel-toe running. The results of this study provide insights into the underlying mechanism of footwear midfoot stiffness on foot joint kinematics and GRF characteristics. Our study showed similar patterns of foot segmental motion across shoe conditions during the stance phase of running . HoweverOur study’s initial rearfoot-lab dorsiflexion angle >8.0° confirmed that all participants performed rearfoot strike patterns . The infDuring the propulsion phase, the participants displayed peak midfoot-rearfoot, forefoot-rearfoot, and forefoot-midfoot plantar flexion velocities. It is assumed that the increased midfoot bending stiffness could increase the resistance in the midfoot region, which could decrease the plantar flexion speed during the propulsion phase. Previous studies also found that an increase in shoe stiffness decreases the maximum MTP plantar flexion  and peakIncreased shoe forefoot  and fullParticipants wearing shoes with higher midfoot bending stiffness demonstrated smaller forefoot-midfoot and forefoot–rearfoot frontal plane range of motion during the running stance phase. Still, no significant differences were found for the midfoot-rearfoot joint. Renan et al.  found thPrevious studies indicated that increased foot torsional stiffness restricts the forefoot-rearfoot motion in the frontal plane, and thus leads to an excessive motion of the ankle pronation ,14, whicNo significant differences were found in the peak vertical forces and instantaneous loading rates between the shoe conditions. Oh and Park  also fouSome limitations of this study must be acknowledged when interpreting the results. First, the shoe upper was mostly modified, which may have sacrificed the original function of the shoe support . However, the elastic sock upper is a good alternative to bone-pin markers for the non-invasive measurement of actual foot multi-segment. Second, only laboratory ground running was measured in the present study and studying midfoot stiffness during long-distance running should be considered. Third, although our primary scope of study aimed to analyze the actual multi-segment foot kinematics, running shoe midfoot stiffness might influence the other proximal joint kinematics  according to the kinetic chain theory. Fourth, this study did not analyze the multi-segment foot kinetic influences of running shoe midfoot stiffness as no reliable anthropometric data are available, such as the center of mass, for accurate foot segment calculation. Fifth, only males were recruited in our study as the tested shoes were built in US size 9, and the current results may not be generalized to females. Finally, we just tested three shoe conditions whose stiffness are 0.069, 0.091, and 0.163 Nm/°, and future research should consider a larger range of midfoot stiffness conditions to optimize the midfoot stiffness in the running community.The midfoot stiffness of running shoes can influence multi-segment foot movements during heel-toe running. Shoes with increased midfoot bending stiffness can reduce foot medial-lateral movements during the stance phase of running, indicating that the length variation of intersegmental muscles and tendons should also be smaller, which may decrease the tension of the foot muscles and tendons connected between the metatarsals and tarsus. However, we found no difference between shoe conditions in ankle kinematics, thus the effect of shoe midfoot stiffness related to pronation injury was not evident.