Dietary pulses, including dry beans, lentils, chickpeas, and dry peas, have the highest proportion of fiber among different legume cultivars and are inexpensive, easily accessible, and have a long shelf-life. The inclusion of pulses in regular dietary patterns is an easy and effective solution for achieving recommended fiber intake and maintaining a healthier gut and overall health. Dietary pulses-derived resistant starch (RS) is a relatively less explored prebiotic ingredient. Several in vitro and preclinical studies have elucidated the crucial role of RS in fostering and shaping the gut microbiota composition towards homeostasis thereby improving host metabolic health. However, in humans and aged animal models, the effect of only the cereals and tubers derived RS has been studied. In this context, this review collates literature pertaining to the beneficial effects of dietary pulses and their RS on gut microbiome-metabolome signatures in preclinical and clinical studies while contemplating their potential and prospects for better aging-associated gut health. In a nutshell, the incorporation of dietary pulses and their RS in diet fosters the growth of beneficial gut bacteria and significantly enhances the production of short-chain fatty acids in the colon. Pulses are valuable dry grains from leguminous crops. Domesticated around 10,000 years ago, pulses have been consumed as a key staple food crop, especially in developing nations, thus providing a primary means of protein and energy . Based oLens culinaris L.), beans (Paseolus vulgaris L.), chickpeas (Cicer arietinum L.), and peas (Pisum sativum L.) are the most frequently consumed pulses worldwide [Although there are numerous pulse varieties available worldwide, Food and Agriculture Organization (FAO) has listed 11 main types, namely beans, broad beans, Bambara beans, chickpeas, lentils, cowpeas, peas, pigeon peas, vetches, lupins, and other “minor” pulses . Among torldwide . Pulses orldwide . The higorldwide . The daiorldwide . Still, orldwide . To addrorldwide .Starch is the major carbohydrate in pulses accounting for nearly 50% portion of carbohydrates . Certain14 microorganisms belonging to over 1000 species and has a bacterial genomic content of approximately 100 times over compared to the human genome [Clostridium coccoides group (or Clostridium cluster XIVa), Clostridium leptum group (or Clostridium cluster IV), and Bacteroides [Nowadays, it has been widely popularized that the human gastrointestinal (GI) tract is a frontline mediator system wherein the intestinal bacteria aid in the digestion of dietary constituents of consumed foods and synthesizes low molecular weight bioactive molecules, which ultimately exerts a crucial role on human health and well-being . The humn genome . About 9n genome . Severalteroides . The gutGut microbiota is dynamic in nature and changes continuously during the lifespan of an individual . During Over 50% of ‘baby boomers’ are considered at nutritional risk, and this statistic could grow to over 30% by 2050, indicating the economic burden on the American health care system while underscoring the significance of nutrient-dense, health-promoting food sources as a preventive strategy. To this end, pulses could be a ‘perfect’ food choice for older adults as they have a higher fiber and protein contents, and low glycemic index (and low saturated fat) and are easy to buy, prepare, and consume, thereby offering an inexpensive way to specifically promote gut health and overall health of all age groups including older subjects . Gut heaStarch is a dietary carbohydrate that is commonly found in everyday food. It is the second most abundant chemical compound in the plants after cellulose. Chemically, starch is composed of two monosaccharide molecules that are amylose (linear chain) and amylopectin (branched chain). These molecules are linked together with alpha 1-4 and/or alpha 1-6 glycosidic bonds. Based on physical and physiological properties, starch can be classified into three categories, namely rapidly digestible starch, slowly digestible starch, and resistant starch (RS) . EnglystResistant starch is further subdivided into five types depending upon its structural features. RS type 1 (RS1) is physically inaccessible starch and has the most complex structures as it is frequently found entrapped within protein matrix or non-starch components of the plant cell wall  . CompareResistant starch possesses many desirable functional and health-promoting properties . An overBifidobacterium adolescentis and Ruminococcus bromii. These species unmask the resistant starch with their complex enzymatic action. Ruminococcus bromii is an important part of the gut community, present to the tune of 3% of gut microbiota [R. bromii contain 17 GH-13 amylases, which catalyzes the hydrolysis of alpha 1,4 and alpha 1,6 glycosidic linkage [Ruminococcus species FMB-CY1 was identified, having a close resemblance to Ruminococcus bromii [Bifidobacteria adolescentis. After fermenting RS, B. adolescentis liberates acetate, lactate, and formate and is able to utilize more starch by-products when compared with R. bromii . A diverse community of microbes present in the gut degrades the dietary fiber, including resistant starch in the colon. The human genome encodes only 17 enzymes to metabolize food glycans viz., starch, sucrose, and lactose , while acrobiota . Five st linkage . It form linkage . Recentls bromii . This sps bromii  demonstrEubacterium rectale, Roseburia, Butyrivibrio, Bacteroides thetaiotaomicron, and Bifidobacteria. E. rectale is a key member of the bacterial community , which generates butyrate and helps in maintaining homeostasis of the gut. The amylopectin hydrolysis capacity of this bacteria is twice compared to amylose . Butyrogenic species such as Roseburia faecis utilize amylopectin more readily but amylose poorly [Bacteroides thetaiotaomicron is capable of degrading different types of glycans (at least 32) effectively [Bifidobacteria, e.g., B. infantis, B. longum, B. bifidum, and B. breve, could act as secondary degraders. In addition, improved growth of B. cuniculi and B. magnum on starch have been found when they are co-cultured [Secondary degraders can degrade regular starch due to the presence of amylases but poorly utilize resistant starch, or in some cases, they are unable to degrade RS. They can grow on RS in monoculture similar to primary degraders. However, they readily utilize starch by-products (oligosaccharides) that are generated by other degraders. For efficient working, they require primary degraders to act on the smooth surface of the resistant starch granule. The eroded surface is suitable for their attachment. Typically, secondary degraders align themselves near to the primary degraders and utilize their excess by-products . Secondary degraders consist of e poorly . Bacteroectively . Furtherectively . Severalcultured . R. hominis cannot utilize starch, but when co-cultured with B. adolescentis, they grow well on by-products (lactate and acetate), which are generated by B. adolescentis. In addition, R. hominis only utilize malto-oligosaccharide and cannot degrade amylose and amylopectin [Cross feeders cannot directly metabolize starch and are unable to grow in monoculture. These microbes play an important role in the conversion of upstream by-products and metabolites . Starch lopectin . Bacteroidetes and Actinobacteria, while decreasing the prevalence of Firmicutes as compared to RS2 [Bifidobacterium adolescentis and Parabacteroides distasonis was promoted after RS4 consumption, while RS2 promoted the abundance of Eubacterium rectale and Ruminococcus bromii [Faecalibacterium prausnitzii was increased while the number of Escherichia coli and Pseudomonas spp. decreased [Bifidobacterium spp., whereas RS3 with A-type polymorphic form enriched the prevalence of Atopobium spp. [Further, the structural difference in the RS can affect the metabolizing capacity of gut bacteria. It has also been observed that the growth of some gut bacteria is differentially upregulated based on RS types. In a human study, at the phyla level, RS4 consumption increased the abundance of d to RS2 . At the s bromii . In a piecreased . Furtherium spp. . Hence, Prebiotics are non-viable food ingredient which are selectively metabolized by beneficial gastrointestinal microbiota thereby inducing specific changes in the microbiota composition and/or activity, thus conferring benefit(s) upon host health . Dietary(i) Resistance to digestive enzymes and gastric acidity: Due to complex physico-chemical properties and structural characteristics of resistant starch, a specific value of resistance cannot be developed. Naturally occurring resistant starches, i.e., RS2 and RS3, are inaccessible to digestive enzymes present in the gut mainly due to their structure. Starch modification (RS3 and RS4) affects the resistance against gastric acidity. Resistance of the starch also depends on the amylose to amylopectin ratio. These molecules are arranged in semi-crystalline form and provide integrity and stability to the starch granules. Li et al.  reported(ii) Fermentable by the gut microbiota: Starch, after resisting the harsh condition of the digestive tract, finally enters the colon part of the human body, where it is utilized by a wide array of gut microbes. Bacteria responsible for starch fermentation can be characterized into two types, namely proteolytic and saccharolytic bacteria. Proteolytic bacteria act on the protein structures, and saccharolytic bacteria break down the carbohydrate molecules . (iii) Foster the growth of health-promoting bacteria: Dietary fibers are fermented by a wide range of gut microbiota, such as Bifidobacteria, Clostridia, Bacteroides, and Lactobacilli. Complex microbiological techniques are used to quantify the increased abundance of gut microbes. Selective quantification of target bacteria using molecular techniques  and assessment of change in entire gut bacterial composition relative to baseline via metagenomics approach are considered reliable tools for estimating the effects of RS treatment. Further, measuring the increased production of organic acids and gas can be indirectly associated with the significant growth of the bacteria community in the gut .The recent advances linking the role of dietary fibers in ameliorating different disease states have led to increased interest in pulse-based foods. Various types of fibers present in pulses include long-chain soluble and insoluble polysaccharides, resistant starch, and galactooligosaccharides. In addition, these components can act as prebiotic precursors, which are digested by beneficial microorganisms in the gut. The consumption of pulses in the diet has been linked to the reduction in serum cholesterol, increased satiety, and low post-prandial blood glucose levels, thus mitigating the risk of different metabolic diseases like cardiovascular diseases, obesity, diabetes, etc. ,60. In fBifidobacterium, Faecalibacterium, Clostridium, Eubacterium, and Roseburia along with enhanced butyrate and acetate production [Prevotella, Dorea, and Ruminococcus flavefaciens, and decreased abundance of Ruminococcus gnavus in mice models [Prevotella is a genus possessing a large spectrum of glycoside hydrolases and is known for its ability to produce SCFAs following the carbohydrates fermentation [Ruminococcus flavefaciens had been found to decrease in overweight (BMI: 25.0–29.9) and obese (BMI: >30.0) subjects [Ruminococcus gnavus, a mucolytic species, has been linked to an increase in gut-barrier pathologies in subjects with obesity and inflammatory bowel disease [Akkermansia muciniphila in the gut, which is often categorized as next-generation probiotics [R. gnavus [Muribaculaceae (S24-7), Rikenellaceae and B. acidifaciens [Roseburia in mouse feces [Roseburia is involved in butyrate production and has negative correlation with several diseases such as colitis and Crohn’s disease [In the last few years, more research has been directed towards pulses which could be a sustainable source of plant protein compared to animal protein to feed the growing population and to simultaneously address the food insecurity problems . Additiooduction . Severale models ,64,65,66entation . The spesubjects . The abuobiotics ,68. Inte. gnavus . Majorit. gnavus . Among tifaciens . Lentil  disease . AlthougBacteroidetes and Faecalibacterium prausnitzii; decreased total serum cholesterol, LDL- and HDL-cholesterol; boost in microbial richness, and significant change in metabolite profile  in colorectal cancer survivors.Common beans, chickpea, and lentils have been shown to exert positive effects in the modulation of the colonic microenvironment in animal models ,29,64,66The concentration of SCFAs in the lower GI tract normally reduces from the proximal to the distal colon. The amount of SCFAs production is majorly dependent upon the amount of fiber reaching the distal colon. Therefore, one way of increasing the SCFAs in the distal gut is the selection of dietary fibers, which are minimally digested prior to reaching the distal colon. Increasing the consumption of resistant starches in the diet is a promising strategy to modulate gut health and benefit the host. Lens culinaris, Medikus), RS content was further increased by the addition of lipids, resulting in the formation of amylose-lipid complexes (RS5 type) [Native RS is present in varying proportions in cereals, tubers, and legumes. In addition, the RS content can be altered using cooking and cooling operations. Interestingly, the comparison of RS content among cooked cereals, legumes, and tubers samples showed legumes with the highest RS content . BrummerS5 type) .Fermentation of resistant starch by the intestinal microbes in the distal gut brings about changes in the gut microbiota and metabolic profile. Although ample clinical studies have been conducted in relation to changes in gut microbial community structure post-consumption of RS from cereal or tuber sources , so far,Anaerotruncus and Bacteroides in mice fed with autoclaved retrograded lentil starch (RS3 type) relative to control and high-fat diet groups. Anaerotruncus is associated with the production of butyrate and/or propionate, while Bacteroides contribute to increased propionate production via the succinite pathway [C. leptum (cluster IV), a group known to increase in obese individuals [A recent study conducted by Xu, Ma, Li, Liu and Hu  exhibite pathway . They al pathway . Anotherividuals .Bacteroides fragilis to pH drop after fermentation could be one reason for reduced diversity [Blautia and Roseburia genera were found to increase post-fermentation of human fecal samples with RS derived from pinto beans and peas [Blautia and Roseburia are members of the Lachnospiracea family associated with high butyrate production.Zhou, Ma and Hu  reportediversity . Anotheriversity . Blautiaand peas ,87,88. BBacteroides occupy a large portion of Bacteroidetes, which have the inherent ability to ferment complex carbohydrates such as polysaccharides or RS. The production of SCFAs from RS fermentation is largely influenced by host health and diet, colonic environment, microbiota, and fiber’s structural characteristics . RecentlSenescence is an inevitable and irreversible growth process dictated by the cascade of complex natural phenomena. Advances in research point towards a close connection between the ecology of intestinal flora and aging, and the intestinal ecological disorders could cause accelerated aging and shortening of lifespan . AbnormaProteobacteria and its genus Desulfovibrio, the species of which are involved in LPS associated pathogenicity and hydrogen sulfide (H2S) production. H2S impairs mitochondrial respiration in colonocytes as well as butyrate oxidation that provides energy to cells, thereby promoting inflammation [Oscillibacter, Lachnoclostridium, Tyzzerella, Ruminiclostridum 9, and Helicobacteria are also reduced in this study. Alistipes, an aging-associated genus, was decreased in some studies post RS2 supplementation [Parabacteroides and Rikenella, which are usually linked with IBD [Bifidobacterium and Akkermansia spp. has been reported as person ages . RS2 is shown to increase these two taxa in older mice models [Allobaculum, a genus involved in butyrate production [Dietary modulation of gut microbiota composition and metabolites through the supplementation of resistant starch has the potential to extend the health span of the older population by improved gut barrier function and increased expression of gut peptides signaling glucose homeostasis together with lipid metabolism . The benammation . Other oentation ,116. RS with IBD ,117. TheAmong the SCFAs, butyrate is a key metabolite involved in intestinal homeostasis, enhancement of intestinal barrier functions, and promotion of gut peptides (proglucagon and PYY) involved in satiety . TherefoIn many regions of the world, dietary pulses fall in the category of neglected staple crops. However, pulses are a good source of high-quality protein and dietary fibers and could offer a cheaper and more sustainable alternative to animal-based protein to address food insecurity concerns. Besides, from a health point of view, several studies discussed herein have highlighted the potential of pulses to positively modulate gut health and to mitigate the risk of various metabolic diseases by beneficially modulating the gut microbiota and strengthening the colonic mucosal environment. The increase or decrease in specific gut microbial signatures after pulse consumption signifies improvement in various diseased states such as obesity, IBD, hypercholesterolemia, and colorectal cancer. However, resistant starches derived from dietary pulses remain relatively less explored functional ingredient. Although no clinical studies have been done to date, some of the pre-clinical studies and in-vitro fecal fermentation studies have revealed their potential in modulating the gut microbiome-metabolome arrays and ameliorating several non-communicable gut and metabolic diseases. Likewise, there is a paucity of studies examining the effect of resistant starch on aging-associated diseases except for a few existing studies examining the effect of maize, potato, acorn, and sago-derived RS on gut-associated healthy aging and well-being. Nevertheless, there is ample evidence suggesting that the incorporation of these ‘prebiotic’ food components in a healthy dietary pattern fosters the growth of beneficial gut bacteria and significantly enhances the production of SCFAs in the colon. However, the positive impact of RS on gut health from animal and in-vitro studies may not be directly translated or extrapolated to humans, largely due to considerable differences in the human gut microbial composition among individuals, and disparities in study design, habitual intake, dose and type of RS used during different intervention studies. This presents an exciting opportunity for future research involving rational and stringent design of longitudinal multi-omics clinical nutrition studies addressing the above variables for comprehensively deciphering the detailed mechanisms underlying the effects of different types of pulse-derived RS on the gut microbial ecology, metabolome, intestinal function, and host health. Studies to date have focused mostly on cereal- and tuber-derived RS2, whereas concomitant exploration of the effect of different pulses-derived RS types on human microbiome, which remains relatively limited, will certainly advance the knowledge in this field. Manipulation of RS naturally occurring in foods is quite complex owing to numerous intrinsic and external processing factors; therefore, more efforts should be directed to purify the RS from these complex matrices. However, more efforts are also imperative to develop standardized and systemic methods to appropriately characterize chemical composition of purified pulse RS, particular given the substantial structural heterogeneity based on the type of cultivars, method of isolation, interaction with other nutrients, and physical and chemical methods employed for its modifications. Moving forward, the use of purified RS could also further facilitate in drawing more robust conclusions for its use as functional ingredient particularly in the absence of interfering/overlapping effects of other food components such as proteins, polyphenols, other fibers, etc. on the health outcomes. Such studies will also be helpful in formulation and standardization of recommended daily intake dose of RS for various food and nutraceutical applications. Finally, further comprehensive understanding of the detailed structure of different RS types and their selective influence on the diverse gut microbiome structure and functions could further facilitate in the development of microbiome-specific or microbiome-targeted functional foods containing RS within the milieu of healthy dietary patterns.


C. esculentus porous starch (PS) and C. esculentus cross-linked porous starch (CPS) were prepared by enzymatic modification and sodium phytate cross-linking, and their physicochemical and structural properties were determined. The results showed that the adsorption and emulsification capacities of PS were 1.3606 g/g and 22.6 mL/g, respectively, which were significantly higher than 0.5419 g/g and 4.2 mL/g of C. esculentus starch (NS). The retrogradation curves of starch paste showed that the stability of PS was inferior to that of NS. In addition, the results of texture analysis showed that the gel strength of PS was also significantly reduced relative to NS. The PS exhibited a rough surface with pores and low molecular order and crystallinity according to scanning electron microscope (SEM), fourier infrared spectroscopy (FTIR), and X ray diffractometer (XRD) analyses. As compared to PS, CPS still presented a high adsorption capacity of 1.2744 g/g and the steadiness of starch paste was significantly better. XPS demonstrated the occurrence of the cross-linking reaction. Our results show that enzyme modification and dual modification by combining enzymatic treatment with sodium phytate cross-linking can impart different structures and functions to starch, creating reference material for the application of modified starch from C. esculentus.In this study,  Cyperus esculentus is an annual herb in the sedge family, which is also known as tiger nut and iron water chestnut, is widely cultivated in tropical and subtropical regions because of its high resistance to adversity and low soil requirements. Many studies have demonstrated that C. esculentus is rich in nutritional value and is a crop with a high yield and comprehensive use-value of its oil and grain; it has thus gained the reputation of an “underground walnut”. It is one of the most promising healthcare-related genera in the family Salviaceae, which is rich in bioactive substances and has been studied for pharmacological benefits [benefits ,2,3. In benefits .C. esculentus can be 20% or more. It has been shown that the surface of starch granules is smooth. They are mostly spherical or oval with particle diameters ranging from 3.41 to 12.12 μm and an average particle size of 8.60 μm. They have a typical A-type crystal structure with high thermal stability. In addition, as compared to potato starch, C. esculentus starch paste has lower viscosity and transparency and better freeze–thaw stability [Starch is a low-cost, readily available, biodegradable, and non-toxic natural polymer material. However, its inherent properties, such as high retrogradation tendency, water insolubility, and poor thermal stability limit its application in food, medicine, and chemical industries . The statability .C. esculentus by enzyme modification and to investigate the changes in these properties.Porous starch (PS), as a type of modified starch, has been receiving increasing attention because of its valuable functions and potential applications. At present, the methods of preparing PS include physical, chemical, enzymatic, and synergistic methods. The enzymatic method is more acceptable because of its mild reaction conditions, high efficiency, and specificity . The proC. esculentus starch.Being an important means of starch modification, cross-linking changes the overall structure and surface chemistry of starch by introducing chemical bonds at random locations in the starch granules. It can stabilize the starch granule structure and can enhance acid, heat, and shear resistance. Thus, it is often used in conjunction with the porous modification of starch to improve its properties ,11. TricC. esculentus starch must be > 90%, which was extracted from C. esculentus meal  by the alkaline extraction method [Aspergillus niger, and sodium phytate were purchased from Sinopharm Chemical Reagent Co., Ltd., Beijing, China. Potato amylose was purchased from Shanghai Yuanye Biotechnology Co., Ltd., Shanghai, China. All reagents were analytically pure, unless indicated otherwise.The purity of n method . The α-aC. esculentus starch in the ratio of 1:8, and the enzyme complex  was added. The reaction was then carried out on a magnetic stirrer  for 10 h ; it was then terminated by adding 0.1 M sodium hydroxide solution to adjust the pH of the starch suspension to 10 according to previous studies [A buffer solution containing 0.1 M citric acid and 0.2 M disodium hydrogen phosphate at pH 5 was prepared in advance and preheated. It was mixed with  studies . The staC. esculentus porous starch with sodium phytate was carried out by using the method of Sun et al., with slight modification [C. esculentus porous starch was used to prepare a 20% starch suspension in distilled water. The sodium phytate solution was then added slowly while stirring at 1% (w:w) as the cross-linking agent. The pH of the starch suspension was adjusted to 7, and the reaction temperature was kept at 50 °C. After 6 h of reaction, the contents were centrifuged at 4200 rpm for 10 min , and the precipitate was washed several times with distilled water until the supernatant did not produce a white turbid substance with 0.1 M silver nitrate solution, to ensure that free sodium phytate was not contained in CPS. Finally, the precipitate was dried in a blast drying oven at 50 °C to obtain the cross-linked porous starch (CPS) of C. esculentus.Cross-linking of fication . BrieflyM0) was placed in a 50 mL centrifuge tube (M1) of known mass. A certain amount of soybean oil was added and mixed well using a vortex mixer. The contents were allowed to stand for 30 min until the adsorption equilibrium was achieved. The contents were centrifuged at 4200 rpm for 20 min, and the oil layer was poured off until no more oil drops fell. The total mass of the centrifuge tube and solids (M2) was determined. The oil absorption of porous starch was calculated using Equation (1).The oil absorption capacity was evaluated by using the method described by Li et al. . An accuA gram of starch was configured into a starch suspension with a mass fraction of 1%. The contents were placed in a boiling water bath with stirring for 20 min to allow the starch to disperse. An equal volume of soybean oil was homogenized for 3 min and then dispensed into 10 mL centrifuge tubes. Centrifugation at 3000 rpm for 20 min caused visible stratification, and the amount of floating oil was measured. The volume of oil emulsified per gram of starch sample was calculated .The starch suspension was prepared according to the material–liquid ratio of 1:100. It was heated and stirred in a boiling water bath for 30 min, while adding boiling water continuously to keep the volume of starch suspension constant. After cooling to room temperature, the light transmittance was measured at a wavelength of 650 nm, with distilled water as a reference [A starch suspension with a mass concentration of 1% was prepared. It was heated in a boiling water bath and stirred for 30 min to obtain a uniform paste, which was poured into a 50-mL graduated test tube to cool. After that, the suspension was stored in a refrigerator at 4 °C, and the change in supernatant volume was recorded within 24 h .The determination of amylose content was done by the method of Castro et al. . The samw/w, dry basis) were prepared and transferred to an aluminum canister. The viscosity was measured using a rapid viscosity analyzer , whose procedure has been detailed by Sun et al. [The starch samples were ground in a mortar after moisture equilibration and then passed through a 100-mesh sieve. Twenty-five milliliters of 10% starch dispersion  starch suspension was heated in a water bath for 30 min with constant stirring. After cooling for 24 h (at 4 °C) to form a stable gel, the contents were taken out from the beaker (diameter = 40 mm) and were transferred into a cylinder with a bottom diameter of 40 mm and a height of 15 mm. The surface of starch was uniform and flat. The hardness, springiness, glueyness, and chewiness of the starch gels were determined using a texture analyzer . The TPA test model was used to determine the starch gel properties using a P/75 disc probe with pre-, mid-, and post-measurement rates set to 0, 30, and 60 mm/min. The time interval between two compressions was set to 2S, the compression ratio was set to 20%, and the trigger force was set to 0.05 N .The morphology of starch particles was observed under SEM . First, a small amount of starch sample was evenly spread on the conductive gel on the carrier table. Excess starch was then blown off with an ear wash ball, and the stage was put into the gold plating apparatus for a minute. Finally, the sample was put under the SEM for observation. A clear image was obtained at 2500× magnification.A starch sample (3 mg) was placed in a stainless-steel dish, and 7 mg of distilled water was added to it. The dish was then sealed and equilibrated at room temperature for 24 h. The thermal properties of starch were analyzed by DSC . DSC was performed as follows: temperature increase rate of 10 °C/min for a temperature range of 30–120 °C. Changes in the onset pasting temperature (To), peak temperature (Tp), conclusion temperature (Tc), and enthalpy (ΔH) were measured separately .X-ray diffraction patterns of the three types of starch were obtained using the Bruker D8 Advance diffractometer  with Cu-Ka as the characteristic ray. Data was acquired with a scan range of 5–40° (2θ) at a scan rate of 5°/min (40 kV and 40 mA) .−1 with a resolution of 4 cm−1 and 64 scans using a thin slice of KBr as a background.The FT-IR spectra of the starch samples were obtained by an FT-IR spectrophotometer . The starch sample was mixed with KBr in a certain ratio (1:100), and then poured into an agate mortar, ground to a powder, and transferred to a tablet press to be pressed into transparent flakes . KBr-pelPowdered starch samples were spread on double-sided tape on a sample table to analyze the structure and surface elemental composition of starch samples by in situ XPS  at a passage energy of 23.5 eV and a resolution of 0.05 eV .2-sorption/desorption isotherms of starch samples. The specific surface area and pore size distribution of the starch samples were calculated by multi-point BET (Brunner–Emmet–Teller) and BJH  methods, respectively [The starch samples were degassed under vacuum at 120 °C for 8 h to remove adsorbed gas molecules and residual solvent from the sample surface. A rapid specific surface area and pore size distribution tester  were used to determine the Nectively .p < 0.05).All experiments were repeated at least three times. Results were expressed as mean ± standard deviation (SD) and analyzed using the software, Origin 2018 . Statistical analysis was performed using SPSS 22 . One-way analysis of variance (ANOVA) and Duncan’s multiple range test was used to determine significant differences (C. esculentus starch and its modified starch were determined (In the present study, some physical properties of termined . The oilThe transparency of starch paste, which is usually expressed in terms of transmittance, directly affects the sensory acceptability of starchy foods and reflects the strength of the starch to bind with water. The transmittance of the three starch pastes is presented in C. esculentus starch and its modified form are shown in When gelatinized starch is placed at a low temperature, starch molecules rearrange and form insoluble precipitates through hydrogen bonding, in a process called retrogradation. The retrogradation stability of starch is usually measured by the percentage of liquid supernatant during the placement of starch paste. Since the volume of starch paste in all samples in this experiment is the same, it is directly expressed by the volume of liquid supernatant. The retrogradation curves of C. esculentus starch and its modified form are summarized in The pasting viscosity parameters of As shown in According to the study by Dura et al. , the setThe thermal characteristic parameters of starch are summarized in C. esculentus starch. The hardness, springiness, glueyness, and chewiness of porous starch were significantly reduced. Among them, the decrease of hardness may be due to the damage of the rigid and ordered structure of the starch particles in the PS processing process by the complex enzyme, which leads to the break of the starch molecular chain, thus affecting the arrangement behavior of starch molecules in the forming process of gel, resulting in the decrease of hardness. The springiness of starch gel is related to water holding capacity. It has been confirmed in The textural parameters of starch gel are shown in C. esculentus starch and its modified starches visualized under SEM are displayed in C. esculentus starch was dominated by spherical starch granules with uneven sizes and relatively smooth surfaces (The morphologies of surfaces A. FigureC. esculentus starch and its modified form are exhibited in A. niger on starch that broke down the starch chain and led to molecular recombination. However, a further decrease in the crystallinity of CPS relative to PS indicated that the reaction process with sodium phytate as a PS cross-linker was chemically modified, which was consistent with the results previously reported by Sun et al. [Starch granules are complex polycrystalline systems, and according to the X-ray diffraction patterns, starch can be classified into A-, B-, C-, and V-types . The X-rn et al. .C. esculentus starch and its modified form are shown in −1 is attributed to the stretching vibration of the hydroxyl group, where the intensity is positively correlated with the number of intermolecular hydrogen bonds of starch. As shown in The Fourier infrared spectra of −1 is one of the characteristic indexes of starch hydrophilicity. The order of strength of the absorption peak in this region was as follows: NS < PS < CPS, which indicated that both enzymatic modification and cross-linking increased the hydrophilicity of starch [The absorption peak near 930 cmf starch ,34. Howe−1. The ratio, R1047/1022, of the absorption peak intensities at 1047 cm−1 and 1022 cm−1 reflects the orderliness of starch structure in the short-range; the larger the ratio, the higher the crystallinity. The ratio, R995/1022, of the absorption peak intensities at 995 cm−1 and 1022 cm−1 reflects the internal changes of the starch double helix structure. Generally, the larger the ratio, the more double-helical structures are present [1047/1022 and R995/1022 of the PS were significantly lower than that of NS, which indicated that the enzymatic modification destroyed the orderliness of starch structure in the short-range and reduced the crystallinity, which was consistent with the results of X-ray diffraction analysis and also the reason for the lower △H of PS than NS. However, the R1047/1022 of CPS was higher than that of PS, while R995/1022 was smaller than that of the PS, because not all double-helical structures can form crystals [The infrared spectrum of starch typically has three absorption peaks corresponding to wave numbers of 995, 1022, and 1047 cm present . The rescrystals .The elemental composition at the surface of the starch is usually characterized by XPS. The binding energies and chemical states of C1s in PS and CPS are shown in The specific surface area and pore size of starch samples were determined by the nitrogen adsorption and desorption methods. The pore size distribution of the three starches is shown in C. esculentus starch with α-amylase and amyloglucosidase from A. niger, and then cross-linked with sodium phytate to obtain CPS. These two modification methods can significantly change many properties of C. esculentus starch and have great significance for expanding the application radius of C. esculentus starch. Finally, the study of these structural and property changes can provide a reference for the development of the starch industry.PS can be obtained by modifying


Bread has a high glycemic index (GI) and rich contents of quickly digestible carbohydrates, which is associated with insulin resistance and the risk of chronic diseases. (-)-Epigallocatechin Gallate (EGCG) is the primary catechin component that inhibits starch hydrolases, while the low release and absorption rates limit its utilization. In this study, EGCG was added to the bread matrix for fortification to reduce its glycemic index compared to white bread. EGCG fortification at 4% decreased the starch digestion rate of baked bread by 24.43% compared to unfortified bread and by 14.31% compared to white bread, with an identical amount of EGCG outside the matrix. Moreover, the predicted GI (pGI) was reduced by 13.17% compared to white bread. Further, 4% EGCG-matched bread enhanced the bioaccessibility and bioavailability of EGCG by 40.38% and 47.11%, respectively, compared to the control. The results of molecular docking demonstrated that EGCG had a higher binding affinity with α-amylase than with α-glucosidase, indicating that EGCG may effectively inhibit the accumulation of carbs during starch digestion. Thus, EGCG can be used as a functional ingredient in bread to reduce its glycemic potential, and the bread matrix can be used as a carrier for EGCG delivery to enhance its bioaccessibility and bioavailability. Bread is a typical staple food consumed in many nations that is high in carbohydrates and contains a lot of quickly digestible starch, which frequently results in a high glycemic index (GI). The GI value describes the contribution of food products to blood glucose response in the body based on the carbohydrate quality, but not the quantity of the food. To better evaluate the overall glycemic effect of foods, a concept, namely glycemic load (GL), was introduced as the product of GI and the carbohydrate content in a typical serving. Epidemiologic evidence strongly supports the excessive consumption of high dietary GI and GL food was associated with insulin resistance and the risk of some chronic diseases, such as cardiovascular diseases and type 2 diabetes mellitus . TherefoThe dietary intervention of phytochemicals, such as flavonoids, terpenoids, and polysaccharides, has been proven to be an effective treatment to reduce the activities of digestive starch enzymes, therefore, causing a reduction in GI and GL and improving the phytochemical function through synergistic interaction with the food matrix . Bread iAmong numerous natural phytochemicals, (-)-epigallocatechin gallate (EGCG), the principal component in green tea catechins, has the most significant capacity to inhibit α-glucosidase and α-amylase ,8. In reThere has been lots of work reporting on tea-catechin-fortified bread, assessing its quality attributes, sensory evaluation, catechin stability, antioxidant properties, and health benefits, indicating that EGCG-fortified bread is a potential functional food for anti-diabetes ,12,13. HThis study aimed to investigate the starch digestive profile and matrix effects of EGCG-fortified bread compared with the matched one . Moreover, the potential of utilizing the bread matrix to enhance the bioavailability and bioaccessibility of EGCG was evaluated through a simulated digestion model. The effects of EGCG on bread starch digestion, GI, and GL values were investigated. In addition, the potential molecular interactions of EGCG with starch hydrolase were analyzed to explore the mechanism of starch inhibition.Saccharomyces cerevisiae, S.I.Lesaffre, Maisons-Alfort Cedex, France), refined salt, pure cane sugar, and shortening. For the chemistry assay, chemicals were purchased from Sigma-Aldrich Co., Ltd. : Methanol, HCl, NaOH, 3,5-Dinitrosalicylic acid (DNS).EGCG  was obtained from . The ingredients were obtained from the local Fair Price supermarket: high-protein wheat bread flour , instant active dry yeast . In a mg  for 5 min. Five-times the solid fraction was re-extracted using the process described previously. For High-Performance Liquid Chromatography (HPLC) analysis, the mixed liquid extract was diluted to 50 mL with methanol.To measure the retention rate of EGCG in the bread before digestion, 5 g lyophilized bread-crumb powders was blended with 25 mL of methanol in 50 mL beakers using an orbital shaker  at 600 rpm for 2 h in a 60 °C water bath. Following this, liquid extract and solid fraction were obtained by centrifugation at 3500× The quantification of EGCG utilized a HPLC equipped with a diode array detector (DAD)  and a C18 reserved-phase column . The detection protocols were adapted from Wang’s research: Mobile phase A (1% acetic acid in DI water) and B (100% methanol) were applied according to the gradient elution with 1 mL/min flow rate, and 30 °C column oven temperature at 275 nm wavelength detection. Quantitative detection of EGCG calibration was accomplished by the external calibration ranging from 0.025 to 0.250 mg/mL . The cal2, 75 U/mL α-amylase, pH 7.0). After vortexing the suspension for 20 s, it was then incubated for 2 min at 37 °C in circulating water baths  with a magnetic stirring rod on a magnetic stirrer . In the gastric phase, oral bolus was mixed with 8 mL SGF buffer . After that, the mixture was blended using a stirring rod at 37 °C for 2 h. In the intestinal phase, gastric chyme was blended with SIF buffer . The resultant mixture was then transferred into a 14 kDa cut-off-size dialysis tube, submerged in 200 mL of phosphate-buffered saline , and dialyzed for 6 h at 37 °C [The simulated digestion in vitro protocols were adopted from the latest standardized static INFOGEST 2.0 . The schat 37 °C . For digIn the intestinal phase, a 0.5 mL aliquot of dialysate was collected from 0 to 180 min at 5 min intervals. The released reducing sugar in the dialysate at each time point was determined by using the DNS approach .g for 10 min, and the supernatant was collected and brought to 50 mL in a flask with DI water. In the interim, the dialysate was first concentrated to less than 50 mL at 40 °C using a vacuum evaporator  and then brought to 50 mL in the volumetric flask using DI water. The digesta were transferred into a 50 mL centrifuge tube and centrifuged. After that, the EGCG quantification by HPLC was held to measure its amount in the digesta and dialysate [After the intestinal phase, the digesta in the dialysis tube were centrifuged at 18,000× The bioaccessibility and bioavailability were calculated by using Equations (1) and (2), respectively.digA is the contents of EGCG detected in the digesta, diaA is the contents of EGCG in the dialysate, and iA is the contents of EGCG in the bread after baking. ‘Dialysate’ refers to the solution outside the dialysis tube and ‘digesta’ refers to the solution in the dialysis tube [sis tube .recR is the recovery rate. iniA is the total quantity of EGCG in bread aliquots after baking and before digestion, whereas digA is the total contents of EGCG detectable in the digesta.For fortified and matched samples, the recovery of EGCG from the digesta was determined using Equation (3)tC is the concentration of released reducing sugar (mg/mL) over time t (min) and k is the rate constant of starch digestion (min−1). Non-linear regression was performed to achieve the k and 2R, the root mean square error (RMSE) was employed to evaluate the quality of the established model [The starch digestion kinetics of bread aliquots followed a thermodynamic first-order reaction, which can be modeled using Equation (4) to determine the digestive profile of the bread aliquots.ed model .The total accessible carbohydrate contents in the bread aliquots were determined using an assay kit from Megazyme  . The brepGI) and predicted Glycemic Load (pGL) based on Equations (5)–(8) developed by Goñi [AUC) up to 180 min was integrated. By dividing the AUC of each bread sample by the reference, the control white baked bread, the hydrolysis index (HI) was computed (Equation (7)). White bread was chosen as the reference food for GI  (Equation (5)), while glucose served as the reference for estimating GI glucose  (Equation (6)). Each sample’s generated GI bread was then multiplied by 0.7. Based on a 50 g portion of bread and taking into account the TAC content of each sample, the predicted GL was determined (Equation (8)) [Assessment of the in vitro simulated digestion was conducted to calculate the predicted Glycemic Index ( by Goñi . The curion (8)) .(5)pGIbComputational molecular docking was employed to explore the potential binding of EGCG with α-amylase and α-glucosidase using AutoDock Vina 1.2.3 to illustrate the mechanism that EGCG inhibits the enzyme activities . The α-aAll the experiments were conducted in triplicates. Results were presented as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) with Duncan was used to determine the significant differences in the results. The non-linear curve fitting, regression, and integration were performed using OriginPro software  and all statistical analysis was conducted using SPSS 27.0  [To examine the influence of EGCG in the bread matrix on starch digestion and absorption, control bread aliquots were supplemented with the same quantity of EGCG as remained in the fortified bread samples after digestion . The curves continually rise from 0 to 100 min and level off afterward. At the end of dialysis, the released RS concentration was significantly decreased due to the formation of phenolic-starch complexes. It has been reported that the released reducing sugar of bread enriched with EGCG significantly decreased during pancreatic digestion . In thisWith increasing EGCG content in the bread matrix, the inhibitory effects of bread starch digestion increased, according to these findings. The inhibitory impact of EGCG on α-amylase was one probable explanation. As one of the principal phenolics, EGCG inhibits starch digestion by pancreatin, α-amylase, α-glucosidase, and other digestive enzymes to a greater extent . It is l∞C of RS and the digestion rate constant k value were estimated (Equation (1)). As demonstrated in k value decreased from 0% to 4% EGCG concentration in both fortified and unfortified bread. A similar pattern applied to the value of ∞C. The slower the rate of in vitro starch digestion, the lower the k value would be. Sui et al., showed a 20.5% decrease in the regression digestion coefficient of the 4% black rice extract anthocyanin-enhanced bread compared to the control bread in their investigation [Based on the mathematical modeling of the digestion profile, the equilibrium concentration k was 21.76%, 20.42%, and 24.43% higher than control bread with significance. It could be attributed to the fact that EGCG could interact with starch digestive and hydrolyze enzymes through hydrogen bonding and hydrophobic interactions, reducing the contents of rapidly digested starch in bread and inhibiting the starch enzyme digestion activities [The RS concentrations of EGCG-fortified bread aliquots were significantly lower than the control, and the parameters of the fitting curves could quantitatively discriminate the digestion profiles further. Compared to the control bread samples, the contents of released reducing sugar at the end of starch digestion were 1.27%, 8.50%, and 15.70% lower in the EGCG-fortified bread aliquots at 1%, 2%, and 4%; meanwhile, the starch digestion rate pGI and pGL for bread aliquots was listed in AUC, HI, pGIbread, pGIglucose, and pGL was observed for all the EGCG-fortified bread aliquots compared to the control. There were significant differences in pGI and pGL in the fortified with matched bread at a relatively higher EGCG dosage (4% and 2%), while no significant differences were observed at the lower dosage (1%). The maximum reduction in pGIbread in the fortified/matched bread samples was observed at 13.17% and 8.38%, respectively. Meanwhile, the maximum reduction in pGL value in the fortified/matched bread was 20.69% and 16.35%, respectively. Both the pGIbread and pGL value of fortified bread were significantly lower than that of the matched bread. It might be attributed to the formation of resistant starch. In the fortified bread, EGCG could interact with starch in the noncovalent binding, contributing to the generation of resistant starch, which was able to reduce the pGI, while in the matched bread, EGCG interacts with bread starch loosely out of the bread delivery system, forming a relatively unstable force with starch.The summarization of Moreover, starch digestion was hindered by the glutelin, gliadin, and lipids in the bread matrix. EGCG–protein complexes were proven to be formed in wheat bread delivery systems with the addition of black tea , which mThe terms ‘bioaccessibility’ and ‘bioavailability’ are used to evaluate the utilization efficacy of nutrients. ‘bioavailability’ refers to the nutrient release rate from the food matrix, while ‘bioavailability’ refers to the nutrient absorbed rate by the small intestine epithelium .The percentage of EGCG preserved in bread crumbs after baking is presented in On the other hand, the degradation and epimerization of EGCG would also be contributing factors. As reported by Mizukami et al., the number of cis-catechins decreased continually under the roasting at 180 °C, due to the degradation and epimerization . CompareBoth bioaccessibility and bioavailability are essential indexes for the evaluation of available components from specific food matrices. In vitro bioaccessibility refers to the contents of EGCG released after digestion in the liquid fraction of the digesta and dialysis as a fraction of the EGCG that was initially in the bread after baking. As passive diffusion was one of the main methods of EGCG absorption, the in vivo bioavailability refers to the EGCG released after digestion in the dialysate passing through the dialysis tube as a fraction of the EGCG that was initially retained in the bread after baking. Significant differences are observed between the fortified and the matched bread at the different dosages of EGCG. There was lower bioaccessibility of EGCG (32.34%) in the 4% matched bread compared with the fortified one (45.40%) . A similIt was reported that EGCG could strongly inhibit the activity of both α-amylase and α-glucosidase, but the molecular mechanism has still not been fully elucidated, especially under the circumstance of food matrix . Both α-The binding pocket of α- amylase with EGCG included Trp 59, Tyr 62, Gln 63, His 101, Gly 104, Leu 162, Thr 163, Leu 165, Arg 195, Asp 197, Ala 198, Lys 200, His 201, Glu 233, Val 234, Ile 235, Asp 300, and Gly 306 with 18 amino acid residues. The binding pocket of α-glucosidase with EGCG included Phe 307, Ala 427, Ala 429, Asp 456, Arg 459, Phe 461, Thr 462, Pro 465, Thr 466, Arg 467, Pro 469, Val 495, Asp 496, Gly 498, Tyr 499, Arg 500, Val 501, Trp 523, Cys 524, Trp 525, Met 546, Met 565, and Phe 571 with 23 amino acid residues. The binding pocket is relatively more minor in the α-glucosidase than α- amylase, resulting in the more flexible binding of EGCG with α-amylase, which meant EGCG could more easily enter the binding site and form a tight complex.pGI and pGL value of bread with EGCG incorporation, as well as the bioaccessibility and bioavailability of EGCG. Compared to the control bread samples, the content of reducing sugar released at the end of starch digestion was 1.27%, 8.50%, and 15.70% lower in the EGCG-fortified bread samples at 1%, 2%, and 4%, while the starch digestion rate was 21.76%, 20.42%, and 24.43% higher than control bread with significance. Further, 4% EGCG-fortified bread significantly reduced the pGIglucose and pGL by 13.17% and 20.69%, respectively, compared to the control. The resultant pGIglucose of the 4% EGCG-fortified bread at 60.78 ± 1.29 was lower than most high-carbohydrate foods, proven to be a healthy food with low glycemic potential for consumers. The matrix effect of bread on the starch digestive enzyme inhibition activity of EGCG was investigated. The 4% EGCG-fortified bread possessed a significantly lower starch digestion rate than that of the 4% matched EGCG bread. One possible mechanism is that the fortification of EGCG into bread before baking allowed EGCG to interact with starch via hydrophobic interactions and hydrogen bonding to change the starch structure of bread samples, therefore, hindering the digestion of starch, which is more efficient compared with the interaction in the solution or outside the bread matrix. Furthermore, incorporating 4% EGCG into the bread matrix could increase the bioaccessibility and bioavailability by 45.39% and 55.60%, respectively. One possible explanation is that EGCG could conjugate with gluten and form the disulfide bond through the SH-SS interchange reaction during the dough mixing and avoid being unstable throughout the digestion process and, therefore, increase the small intestinal absorption. The result reveals that the incorporation of EGCG into bread systems could significantly reduce the starch digestion rate of the bread samples and lower their pGI and pGL value compared with the EGCG out of the bread-delivery system. The molecular docking results effectively explain the binding affinity of EGCG with α-amylase and α-glucosidase, the disrupted structure of α-amylase and α-glucosidase with the insertion of EGCG in the active binding sites avoiding the entrance of substrate and decrease the enzyme activity ultimately. Therefore, bread was proven to be a promising food carrier for delivering EGCG more efficiently. The results of this study highlighted the feasibility of EGCG being used as a functional ingredient in the reformulation of bread for its glycemic reduction.In this study, simulated starch digestion in vitro was performed to assess the bread starch digestion kinetics, the


Plant Physiology initiated the Assistant Features Editor (AFE) program five years ago to help disseminate discoveries published in the journal and to train the next generation of editors and reviewers. Our AFEs are promising early-career scientists and they bring their passion for science to our journal, communicating to our readers each month some of the most exciting advances in research.News and Views articles, blog posts, and related material highlighting content of special interest in Plant Physiology. They have grown professionally and will build on their experience with the journal. Many of our previous AFEs have moved onto independent academic positions after completing their terms at Plant Physiology. We hope to call the AFEs to serve as regular members of the editorial board when they have become more established in their careers.The AFEs have added substantially to the plant science community and to the journal. Their contributions have expanded our content through https://plantae.org/2023-plant-physiology-assistant-features-editors/) who started last month and will work alongside our seasoned crew.The program is designed to rotate off a number of AFEs each year and to add new members at the beginning of each year. Sixteen of our AFEs stepped down from the editorial board this past month, and we would like to take this opportunity to thank them all for their contributions. We would also like to welcome the 19 new AFEs Jiawen Chen Joke De Jaeger-Braet Manuel Gonzalez-Fuente Henning Kirst Yee-Shan Ku Aida Maric Hannah McMillan Sebastian Moreno Dyoni Oliveira Lara Pereira Moona Rahikainen Janlo Robil Alaeddine Safi Maria-Angelica Sanclemente Henryk Straube Jiaqi Sun Kyle Swentowsky Ryo Yokoyama The new AFEs are joining those who started in January of 2022, listed below, who will stay on for one more year to continue writing News and Views, help with the transition, and mentor the new AFEs.Alexandra Burgess Amanda Cardoso Jian Chen Gustaf E. Degen Marieke Dubois Guadalupe Lucía Fernández-Milmanda Yana Kazachova Amy Lanctot Divya Mishra Míriam Osés-Ruiz .Yadukrishnan Premachandran José Manuel Ugalde Yajin Ye


The opalescence gradient was higher in Katana STML than in the e.max MT Multi. In both zirconia types, translucency increased from the dentin to enamel layer based on the AT, TP, and CR results, while OP decreased (p < 0.05). The higher translucency from the dentin to enamel layer in Katana STML was caused by the pigmentation gradient, while in e.max MT Multi, it was caused by the difference in phase fraction and the pigmentation gradient.The differences in the optical properties of multi-layered zirconia with and without yttria-gradient are not fully understood. This study aimed to evaluate and compare the optical properties, related microstructures, and phase fractions of multi-layered zirconia with and without yttria-gradient. For this, multi-layered zirconia of 5 mol% yttria (5Y) stabilized (Katana STML) and 4Y/5Y stabilized (e.max MT Multi) were cut layerwise, sintered, and analyzed using the opalescence parameter (OP), average transmittance (AT%), translucency parameter (TP), and contrast ratio (CR). The average grain size and phase fractions were obtained from field-emission scanning electron micrographs and X-ray diffraction patterns, respectively. Although the TP values of Katana STML and e.max MT Multi did not show a significant difference (except for transition layer 1), the results of AT and CR showed that the translucency of e.max MT Multi was slightly higher than that of Katana STML ( Recently, multi-layered zirconia with improved translucency has been widely used in dental prosthetics ,2. This Early dental zirconia (3Y-TZP) exhibited optically opaque properties that required feldspathic porcelain veneers with high translucency; however, chipping of veneered ceramics was common ,6,7. To Studies on the optical properties of zirconia, particularly those related to translucency, have been reported ,17,18,192O3 showed lower AT and TP and higher OP compared to 3Y-TZP with a single-phase structure [2O3 [In addition to translucency, natural tooth enamel exhibits opalescent optical properties ,26. Opaltructure . This efure [2O3 . There iTwo types of multi-layered zirconia (shade A2) were used . One hadn = 4/group). The specimen dimension for optical properties was 10.0 mm (width) × 10.0 mm (length) × 1.01 mm . The total light transmittance was obtained by placing the specimen in the inlet hole of the integrating sphere in transmission mode. This was divided by the transmittance measured with no specimen to obtain a percentage of light transmittance between 0% (opaque) and 100% (transparent). The average transmittance (AT) was obtained by dividing the sum of transmittance (%) at each wavelength by the number of data points [The spectral transmittance and spectral reflectance were measured three times at 10 nm intervals from 360 to 740 nm using a spectrophotometer , the Spectra-Magic software , a CIE standard D65 light source, and a 2° standard observer  and black  backgrounds. A software  was used to determine the color coordinates as follows: L * (brightness), a * (red–green chromaticity index), b * (yellow–blue chromaticity index), and Y . The TP was calculated using the following equation:ectively ,30,31.To obtain the CR of the specimen, the spectral reflectance (Y) of the specimen obtained on the black (Yb) and white (Yw) backgrounds were put in the equation CR = Yb/Yw . The conThe OP was determined according to the following equation ,35: OP =n = 1/group). The line interception method was used to measure the grain size [C is the length of the test line, M is the magnification of the micrograph, and n is the number of intercepts. In this equation, 1.56 was used as a correction factor [To observe the specimen’s microstructure, it was sputter-coated with platinum for 60 s and then subjected to FE-SEM analysis  at an acceleration voltage of 10 kV (ain size . More thn factor .n = 1/group). The measurements were performed at a tube voltage of 40 kV, tube current of 30 mA, and step size of 0.013° in the range of 20–80° (2θ). In addition, Cu-Kα radiation filtered with Ni was used. Topas Academic software V 7.20  was used to perform Rietveld analysis of the obtained X-ray diffraction patterns. Relative phase fraction and lattice parameters were analyzed for the monoclinic (space group: P21/c), tetragonal (space group: P42/nmc), and cubic (space group: Fm3m) phases. The Y2O3 content (mol%) in the tetragonal phase was calculated based on the lattice parameters  using Equations (4) and (5) [For the specimen’s XRD analysis, high-resolution XRD  was used ( and (5) ,38: (4)Yt-test. Grain-size data were analyzed using a generalized linear model, as the data did not satisfy normality.The experimental results were analyzed at a significance level of 0.05 using the statistical program SPSS 25.0 . The Shapiro–Wilk test was performed to analyze the normality of the data. Data for the AT, TP, CR, and OP were analyzed using two-way ANOVA, post-hoc Tukey’s HSD test, and Student’s p < 0.001), layer (p < 0.001), and the interaction between zirconia type and layer (p = 0.002) influenced AT. As a result of the post-hoc analysis (p < 0.05). The e.max MT Multi showed a statistically higher AT (p < 0.05) than Katana STML in all layers with the exception of transition layer (TL) 1. The total transmittance of the two types of zirconia at each wavelength  showed tanalysis , both zip < 0.001) and layer (p < 0.001) influenced the TP. As a result of the post-hoc analysis (p < 0.05). There was no statistically significant difference in the TP values between the two zirconia layers .The two-way ANOVA revealed that the zirconia type , layer (p < 0.001), and the interaction between zirconia type and layer (p < 0.001) influenced the CR. The post-hoc analysis (p < 0.05). The CR of Katana STML was higher than that of e.max MT Multi in all layers (p < 0.05).The two-way ANOVA revealed that zirconia type , layer (p < 0.001), and the interaction between zirconia type and layer (p = 0.002) influenced the OP. The OP decreased from the DL to the EL in both types of zirconia (p < 0.05). There was no significant difference in the OP values for the EL between Katana STML and e.max MT Multi; however, for the DL, Katana STML had a higher OP than that of e.max MT Multi (p < 0.05). The difference in the OP values between the EL and the DL was more significant for Katana STML than for e.max MT Multi.The two-way ANOVA revealed that zirconia type (p < 0.001), layer (p < 0.001), and the interaction between zirconia type and layer (p < 0.001) affected the average grain size. As shown in p < 0.05). For Katana STML, there was no significant difference in the average grain size in each layer (p > 0.05). Katana STML had a larger average grain size than e.max MT Multi in all layers (p < 0.05).Both types of zirconia showed an equiaxed crystal structure in the FE-SEM image . All layRietveld analysis of the XRD patterns , Table 5c/a√2, tetragonality) and yttria content of each tetragonal phase  were similar, regardless of the zirconia type and layer. The axial ratios (c/a√2) were ~1.016 for the T-phase and ~1.005 for the T′-phase. The yttria content was ~2.5 mol% in the T-phase and ~6.9 mol% in the T′-phase.The axial ratio . These results indicate that translucency increases from the DL to the EL. This tendency was also reported for multi-layered zirconia with lower yttria content [p < 0.05). The CR was lower in e.max MT Multi than in Katana STML (p < 0.05). Because translucency and the CR are inversely proportional, the results of the AT and CR showed that the translucency of e.max MT Multi was slightly higher than that of Katana STML. However, with the exception of TL1, the TP values of Katana STML and e.max MT Multi were not significantly different. In this study, the ELs of the two zirconia exhibited a TP value of ~13, which was slightly higher than the TP value reported without dividing the zirconia sample by layer  [Measuring the light transmittance at each wavelength showed a reduction in the peaks at 520 and 650 nm only in e.max MT Multi but not in Katana STML. This difference was owing to the difference in the type and content of the added pigments . In both content . The e.mT Multi) . This map < 0.05) for the DL. Visual inspection of the two types of zirconia (A2 shade) revealed that the color gradient of Kanata STML was more prominent than that of e.max MT Multi. This may be related to the higher opalescence gradient in Katana STML than in e.max MT Multi. In this study, translucency increased from the DL to the EL based on the AT, TP, and CR results, while the OP decreased (p < 0.05) for both types of zirconia. This trend was also reported in a study on the changes in translucency and the OP according to the thicknesses of 3Y- and 4Y-zirconia [Human tooth enamel is opalescent, appearing bluish in reflected color and orange/brown in transmitted color . In thiszirconia ,28. Simizirconia . The cubic phase of zirconia has an isotropic refractive index in all crystallographic directions, whereas it is anisotropic in the tetragonal phase, resulting in birefringence . Owing tc/a√2, tetragonality) obtained from the lattice constants of the two types of tetragonal phases shows that the T′-phase has a smaller axial ratio than the T-phase owing to the higher yttria content [p > 0.05). Moreover, because the phase fraction was similar in all layers, the translucency gradient in Katana STML must be caused simply by the gradient of pigmentation [The phase diagram presented by Scott  shows th content ,37,46. T content . In thisp < 0.05). This was considered to be due to an increase in the T-phase fraction from the EL to the DL. In this study, e.max MT Multi had a smaller average grain size than Katana STML in all the layers (p < 0.05). In addition, e.max MT Multi had a lower fraction of the C-phase, which contributes to translucency, than Katana STML in all layers. Nevertheless, the AT and CR results showed that the translucency of e.max MT Multi is slightly higher, possibly due to more pigmentation in Katana STML than in e.max MT Multi. Unlike Katana STML, e.max MT Multi has different yttria contents in each layer according to the manufacturer’s information. Rietveld analysis showed that each layer had a different phase fraction in e.max MT Multi. From the EL to the DL, the fraction of the T-phase increased with a simultaneous decrease in the T′- and C-phase fractions. In the EL with the highest yttria content, the fraction of each phase was ~50 wt% for the C-phase, ~37 wt% for the T’-phase, and ~13 wt% for the T-phase. The phase fraction of the TL was closer to that of the EL than to that of the DL. For the DL, which is the bottom layer, the fraction of the C-phase (~47 wt%) was not apparently different from that of the EL, but the fraction of the T′-phase was reduced to ~29 wt%, and that of the T-phase was increased to ~24 wt%. Such a change in the fraction of the T′- and T-phases increases the tetragonality in zirconia, and consequently, the translucency decreases. A study of 3-5Y-zirconia showed that with decreasing yttria content, the T-phase fraction increased as the T′-phase fraction decreased, showing the lowest translucency in 3Y-zirconia . SimilarBy investigating the optical properties of dental multi-layered zirconia, it is possible to develop esthetic zirconia materials that can mimic the optical properties of natural human teeth without the requirement of veneering porcelain. In this study, multi-layered zirconia of A2 shade, which is a widely used shade, was tested. The average TP values for the tested zirconia were lower than the reported values of 1 mm-thick human enamel and dentin . The obtIn conclusion, the translucency of e.max MT Multi was slightly higher than that of Katana STML, as assessed using the AT and CR, but was similar in the TP results. The opalescence gradient was higher in Katana STML than in e.max MT Multi. The translucency gradient in Katana STML was caused simply by the gradient of pigmentation. However, in e.max MT Multi, it was caused by the difference in phase fraction and the gradient of pigmentation.


Shear wave elastography (SWE) is widely used in clinical work. But there is no standard protocol and operation specification for SWE acquisition methods, which impacts the diagnosis and clinical staging. This study aimed to investigate the influence factors of diameter, depth, and stiffness on SWE using different probes at superficial depths and discuss SWE differences with two machines at superficial depths. We performed SWE on two elastic phantoms that each phantom contained six subjects with two stiffness (41.06 ± 4.62 kpa and 57.30 ± 4.31 kpa), three diameters , and two depths (15 and 25 mm). A total of 240 measurements were obtained by using two ultrasound machines (SuperSonic Imagine Aixplorer and Mindray Resona 7) and 4 probes . The measurements were compared among 4 probes, 3 diameters, and 2 depths. There was no significant difference in SWE measurements among the probes from the same machine. The SWE measurements were affected by diameter, and the degree of influence was related to the stiffness. The SWE measurements were unaffected at a 15–25 mm depth range. Shear wave elastography (SWE) is a novel technique and is widely used in clinical practice due to its non-invasive procedure and visualization property . The priIn recent years, the application of SWE in the breast, thyroid, and other superficial organs has gradually increased –11. ClinThe purpose of this study was to investigate the influence factors of diameter, depth, and stiffness on SWE using different probes from different machines at superficial depths.Two custom-made elastic tissue phantoms (Phantom 1 and 2) were fabricated by gelatin with tumor-mimicking subjects which were provided by the Department of Engineering Mechanics, the Institute of Biomechanics and Medical Engineering, Tsinghua University. The production process was consistent with that in the study by Zhang et al. .As shown in E = 2 × (1 + ν) × ρ × (SWV)2, the E is Young’s modulus, ν is the Poisson ratio (0.5), and ρ is the density (estimated as 1.0 g/cm3)  with transducers of SL15-4 and SL10-2 and Resona 7  with transducers of L11-3 and L14-5. Both systems were equipped with an SWE mode. The Young’s modulus can be calculated according to the equation: 0 g/cm3) .Routine thyroidal settings were applied while taking the SWE measurements. The maximal cross-section of the phantom subjects was observed by the B-mode imaging. Then, the instrument was switched to the SWE mode and the sampling frame was placed in the center of the image within the subject.mean) in the ROI was measured and repeated 5 times. The mean value of each subject (Ei) was calculated by averaging these values. In this experiment, each subject (n = 12) was measured by each of the probes (n = 4) in both systems. Thus, a total of 240 Emean values and 48 Ei values were obtained.The SWE imaging was frozen and measurements of the stiffness were performed after 5 s of real-time imaging for stabilization. The region of interest (ROI) of SWE was placed in the center of the phantom subject and the diameter of the ROI was adjusted according to the size of the target. The mean Young’s modulus E . All the data obtained from the experiments passed the normality tests. The Friedman test was used to compare the Ei| /actual stiffness values × 100%. When the percentage error was close to zero, the measured value could accurately reflect the actual stiffness values of phantom subjects.The coefficient of variation (CV) was used to evaluate the precision and repeatability of the measurements The smaller the CV, the better the repeatability of the measurement. The percentage error was calculated as follows: | actual stiffness values – EThe results of the SWE measurement value in two ultrasound machines with four probes are summarized in The Bland–Altman plots show the agreement between two machines. p = 0.000). For stiffness 2, the difference between probes from different machines is statistically significant (p = 0.000), on the contrary, the difference between probes of the same machine is not statistically significant (p > 0.05).p = 0.045), and the difference between SL15-4 and SL10-2 was statistically significant when the phantom subjects with 18 mm diameter and stiffness 1  or at 15 mm depth with 10 mm diameter and stiffness 1 (p = 0.023). In other cases, the difference between the two probes under the same ultrasound machine was not statistically significant (p > 0.05).The comparison of SWE measurement values of four probes for phantom subjects with different diameters, stiffness, and depth are summarized in p = 0.011, depth 25 mm p = 0.020) The difference between the measurement values of 15 and 18 mm phantom was statistically significant when the phantom subjects with stiffness 1 at depth 25 mm or with stiffness 2 at depth 15 mm . There was no significant difference in the measurement values of 10 and 18 mm phantoms in all conditions (p > 0.05)  .p = 0.261, stiffness 2 p = 0.950)  .The CVs show that the SWE measurement of four probes have a good reproducibility . For stiThe measurements on both probes were lower than the theoretical values of two kinds of stiffness . For theIn this study, we found that SWE measurements can be significantly different according to the machine, probe, and diameter. While the depth has no significant effect on SWE within superficial depth.mean, i.e., Young’s modulus. Our results show that the difference in the SWE measurement values between the two machines is significant under some conditions. And with the increase in the stiffness of the phantom, the difference between machines becomes significant. In this study, we found that the measurement values between the two probes under the same machine have good consistency, and the difference is not statistically significant. Therefore, we think that we should keep the consistency of the machine when follow-up lesions. In addition, the influence of different vendors should be considered when analyzing the change in elasticity value of lesions. Our results showed that the SWE measurements with all probes were lower than the theoretical values of the phantom which is in agreement with some previous studies (The Quantitative Imaging Biomarkers Alliance (QIBA) of the Radiological Society of North America (RSNA) concluded that there were significant differences in SWE measurements between four machines which including FibroScan, Philips, ACUSON S2000, and Aixplorer . However studies . We thinE = 3ρ.SWV2 (To our knowledge, few studies have assessed the effect of the diameter of the subject on SWE. Studies  have com 3ρ.SWV2 , 29, the 3ρ.SWV2 , 28. The 3ρ.SWV2  is consi 3ρ.SWV2  found thWith respect to the effect of depth on SWE, previous studies , 29, 30 The study has several limitations. We used the phantom for experiments; however, the human tissue is more complex, and patient age, fat content, respiration, and other factors can influence the measurements. Therefore, this experiment cannot mimic clinical cases. In addition, the purpose of this study was to discuss the influence of factors, such as diameter, depth, and stiffness, on SWE under superficial conditions, Therefore, the depth ranges chosen were not wide enough. Furthermore, stiffnesses 1 and 2 were medium, and SWE under other softer or harder conditions was not explored. Finally, the sample size of this experiment was relatively inadequate; thus, to obtain more reliable results, a number of tests should be conducted in future experiments.This study demonstrated that the SWE measurements could have a good consistency when the probes were under the same machine. Caution should be used when the diameter of the lesion is smaller than 15 mm and the degree of influence is related to the stiffness. Finally, the SWE is affected by depth and can be ignored when the lesion is located superficially.The original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.QC wrote the first draft of the manuscript. BS collected and collated the data. YZ made the phantoms. XH edited and revised the manuscript. All authors reviewed the manuscript and approved the submitted version.


Dear colleagues,I was greatly honored to be invited to be the Editor of the Brazilian Association of Otolaryngology – Cervico-Facial Surgery Journal. In any medical journal, such office requires great responsibility. Specially for me this demand will be even greater, because the teams that preceded me were outstanding, they were even able to index our journal internationally, in both Scielo and Medline, being considered Qualis national A and Qualis international C by CAPES.The quality achieved by our publication was only possible thanks to the effort of the colleagues who have submitted their scientific research all these years. My main goal is to keep our journal on its current track, and try to raise the standard we have reached so far. This will only be possible if we keep our scientific level, because only then our papers and journal will be quoted in other authors’ references. The more quotes we have in other publications, the more important our journal becomes. Thus, we may aim at achieving Qualis international B or class A. This objective will not be met overnight, requiring time and work, from the authors, editors, associates, editorial body and the whole journal team.As the Editor, and being part of a team coordinated by Prof. Sílvio Caldas Neto (Publications Director) and Prof. Regina H. G. Martins (Assistant Director of Publications), some points regarding our work have to be touched upon.Many colleagues seek us out to know why it takes so long to publish some of their papers, specially “case reports”. We have received a number of these papers, besides those already reviewed and awaiting publication. Unfortunately we can not publish as many of those as we wished, because in order to keep the classification we’ve reached we have to give priority to original papers. The more original work published, the greater is the chance of being quoted.In an attempt to mitigate the problem, soon the authors of this type of articles, already submitted to the journal will be questioned as to the feasibility of changing their current format. Our major goal is to make them more objective and clear for the readers. Those who choose to re-format them will have their papers published faster, because they will be printed in special issues. Those who wish to keep their case reports in the original format will have to be more patient because the number of such papers per journal issue is limited. The new format guidelines are defined and available in the files receiving system - through the Internet. Besides, in a temporary basis, new case reports are not being accepted, however, as soon as we can accepted again, they have to follow the new guidelines. At the journal discretion, special cases may follow other formats.Still, about the delay in reviewing and publishing papers, some colleagues that have submitted their papers now for some while, have contacted us to enquire the whereabouts of their work. We are now implementing changes in the review process in an attempt to expedite the whole process.Another important issue is the re-registration of our reviewers. Otolaryngology is a broad medical field, encompassing allergy, audiology, head and neck surgery, skull-maxilla-facial, facial plastic surgery, swallowing, stomatology, pharyngology, laryngology, voice, otoneurology, pediatric ENT, otology, rhinology, sleep disorders, etc. We have recently asked the reviewers to chose the areas they would most like to work with, so as to have the most qualified personnel to review the papers.As to maintaining the scientific quality of our journal, we reinforce to those colleagues who wish to publish their work involving human beings or animals, that these papers should be submitted together with the approval protocol number from the Ethics Committee where the investigation was carried out.Our journal is available to all those who wish to express their opinions about the papers published. Thus, following the suggestion of some colleagues, we are opening a new session called “Letter to the Editor”, where readers may express their opinions about some paper or theme published. However, in order to guarantee a right of response, this letter will be referred to the author of the commented upon paper, before it is published. If the author wishes to, his/her response will be published together with the letter. The guidelines for these texts are available at the journal internet site.And finally, these first lines I write as the Editor are part of a very special journal issue. In this issue we have published some of the award winning scientific work presented at the last Triological Society Meeting. Everyone will be able to appreciate the scientific quality of our colleagues, encompassing different fields of otolaryngology.I thank all of you who trusted me with this office.Best regards


H-style after precipitation. The chemical bonding of differentSNPs after the nanoprecipitation is analyzed by Fourier transforminfrared spectroscopy (FT-IR). Thermogravimetric analysis (TGA) demonstratesthe decomposition of starch nanoparticles and the starch matrix thatis related to the depolymerization of carbon chains in the range of260 to 350 °C. The mechanical properties of the SNP films versusthe temperature changing are discovered by dynamic mechanical analysis(DMA). The water contact angles of SNP films are measured using agoniometer, and the results showed the hydrophobic surfaces of theprepared films. Our study indicates that SNPs have a promising impacton the properties of corn starch films, which would be useful in biodegradablepackaging material.Starch nanoparticles (SNPs) are synthesized by differentprecipitationtechniques using corn starch, and SNP films are prepared by the evaporationcasting method. The morphological study is investigated by scanningelectron microscopy (SEM) and atomic force microscopy (AFM). The distributionand sizes of precipitated SNPs after synthesizing are discovered bythese methods as well. The crystallinity of the SNPs is studied bythe X-ray diffractometry (XRD) method that demonstrates reductioncompared to neat starch granules, and it is changed from A-style toV It has been suggested that acid hydrolysiswill produce starch nanocrystals when amorphous domains of semicrystallinegranules are disrupted, while gelatinization may produce SNPs withamorphous matrixes.3 Other authors conveythat it is almost impossible to distinguish between starch nanoparticlesand starch nanocrystals since both phrases have been applied to thecrystalline parts of starch after hydrolysis or other physicochemicaltreatments. They indicate that SNPs are used to represent elementswith a nanoscale dimension.4A significant advantageof nanoparticles (NPs) is their capabilityto be used in a broad range of applications in a variety of industries,including food, cosmetics, and healthcare. Natural starch is a nonallergic,plentiful polysaccharide that is renewable and biodegradable, makingit an ideal candidate to be used in eco-friendly bioformulations.5 In addition, a top-down process may be classifieddue to the number of stages it takes to prepare the final SNPs involvedin simple or hybrid processes. Acid hydrolysis and enzymatic hydrolysishave been described as popular top-down simple methods for generatingSNPs. In contrast to amorphous regions, crystalline regions are moreresistant to acid hydrolysis after long periods of time and low yieldsof hydrolysis.6 An X-ray diffraction studywas carried out by LeCorre et al. to see if the botany of starch affectedcrystallinity after acid hydrolysis. During this study, the amyloseamount in starches was found to be the most crucial factor determiningthe crystallinity of SNPs, when starches of different botanical originsbut with similar amylose contents were compared, there were no differencesfound.7Thepreparation of SNPs can be categorized into two classificationsbased on the precursor material utilized for synthesis, bottom-upand top-down. Nanoparticles can be synthesized from a top-down processwhere larger volumes of materials or microparticles are broken downwhile nanoparticles can be generated by a bottom-up process made upof small primary cores made from building blocks of atoms or moleculesthat is controlled by thermodynamic standards such as self-assembly.8 ultrasonification,9 and extrusion,10 requireless time andproduce higher yields but can be difficult to control when it comesto crystallization. Combining enzymatic and acid hydrolysis for thepreparation of SNPs has also been done with satisfactory results usingthe hybrid top-down method, since the procedure was able to be accomplishedin a shorter time frame.11 Furthermore,hydrolysis has been combined with ultrasonication as a final refinementmethod in several studies. It is possible to reduce crystallinityfor longer periods of time by ultrasonication, but this may changethe starch’s X-ray diffraction pattern.12However, physical treatment,such as homogenization,14Microemulsions and nanoprecipitation are the twomost common methodsof preparing SNPs from bottom-up processes. During nanoprecipitation,dilute polymer solutions are successively added to a solvent, whichthen results in the deposition of polymers at the surfaces after theremoval of a semipolar solvent that is soluble with water. The microemulsionprocess includes the preparation of water-in-oil microemulsions containingaqueous parts dispersed in a steady oil phase reinstated by the interfacialfilm of the surfactant molecules functioning as nanoreactors wherethe synthesis of the preferred SNPs happens. Both approaches are gentlechemical techniques that are gaining increasing interest due to theireffective control of shape, size, composition, and monodispersityof SNPs gained, instead of using toxic solvents or external energysources.In this study, we attempted to producestarch films with differentprecipitation techniques of starch nanoparticles and analyze theirphysical properties such as morphology, crystallinity, and mechanicalproperties (stress/strain as a function of temperature). Furthermore,chemical and thermal properties of the prepared specimens are investigatedby Fourier transform infrared spectroscopy (FT-IR) and thermogravimetricanalysis (TGA), respectively. The outstanding results of hydrophobicityare obtained by using water contact angle measurements.22.1First of all, 10.7 g of starch (Amido de Milho AMIDEX 3001) is addedto the reactor containing 200 mL of distilled water for all typesof prepared SNP films in this study. Then, 6.2 g of glycerol is addedto all of the solutions except for SNP1. The solutions are kept mixingby the stirrer at a speed of 425 rpm. The temperature of the solutionsis controlled by an oil bath which is located under the glass reactor,and the reactor is sunk into the oil for the heat exchange. The processof gelatinization starts when the temperature of the solution approaches90 °C. After reaching 90 °C, the stirrer mixes the solutionof SNP3 for 30 min and other samples for 60 min. Then, 200 mL of absoluteethanol are added dropwise to the gelatinized starch solutions exceptfor SNP4 under active mechanical stirring. The gelatinized starchsolutions are normally cooled down to room temperature. Afterward,200 mL of absolute ethanol are added dropwise to all the solutionsat room temperature. Thereafter, the solutions are kept under stirringfor more than 5 min. Finally, 30 mL of each solution are put in aPetri dish, and they need to be on the furnace with a temperatureof 50 °C for 24 h. The difference between the four types of theprepared solutions is summarized in Furthermore, the mean size, size distribution, andrelative standarddeviation (RSD) of the SNPs are calculated through a dynamic lightscattering . Two millilitersof obtained solutions are added to cuvettes and measured.2.215A scanning electron microscope  with an acceleration voltage of 15 kV is used to considerthe microstructure of the starch nanoparticles on the surface of thesamples. All of the analyzed samples are coated by gold with 3 nmas the thickness of the coating using a LEICA MICROSYSTEMS EMQSG100,USA. Furthermore, the morphology and the size of the nanoparticlesare analyzed by scanning electron microscopy .For the analysis, one droplet of the film solution is deposited ona (110) oriented silicon wafer (1 × 1 mm) and dried on a heatingplate at 50 °C. This procedure is done a total of four times.The samples are fixed on a stub by a carbon tape. The nanoparticlessize is calculated using a digital imaging analysis program.2.316An atomic force microscope is used to image the samples. The surface topography of the preparedfilms is obtained to exhibit the nanostructure of the starch nanoparticleson the surface. Furthermore, the dimension of the particles is measuredby using ImageJ software. Images are obtained in dynamic mode usinga Micromasch NSC 14/AIBS cantilever with a nominal spring constantof 5 N/m.2.417Dynamic mechanical analyses are accomplished in a TA Instrument(Q800 DMA) with prepared SNP film samples. The dimension of the specimenis approximately 35 mm × 10 mm × 2 mm. The analysis is carriedout at room temperature and 1 Hz in 0.01 N force-controlled deformationmode, from −80 to 150 °C, at a heating rate of 3 °C/min.The samples are kept at 21 °C and 50% relative humidity beforethe test for a period of 48 h.2.5–1 in the range of 800–4000cm–1 using ATR mode. The chemical structure of starchnanoparticles on the samples might be proven by this method basedon the formed peaks corresponding with specific chemical bonds.18FTIR spectra are recorded to study the effectof the different conditions of nanoparticle formation and the functionalgroups created on the SNP films using a TA Instruments SDT-Q600 spectrometer,with a resolution of 5 cm2.619The crystallinity of SNP films is gained by a Bruker D8 DISCOVER(Germany) diffractometer in the diffraction angles range 2θ= 10° and 40°, using a wavelength of 1.540A° with CuKα radiation and 90 s of time per step and a step size of 0.025°.The samples are placed in the sample holder, and the experiment isachieved in a static mode.2.720Thermogravimetric analysis is accomplished to appraise the degradationcharacteristics of the SNP films. The thermal stability of three specimensfor each kind of samples is distinguished by using a thermogravimetricanalyzer (THERMO NICOLET 6700 spectrometer), with the nitrogen flowrate of 20 mL/min as the atmosphere and a heating rate of 10 °C/min.The weight of each sample is 15 mg, and they are subjected to a temperaturerange of 25 to 600 °C.2.821The surface contact angle of the prepared samples is measured byoptical contact angle goniometer to investigate the hydrophobicity of the obtained films. A three-axishorizontal tilt stage is utilized to make the surface of the samplesperpendicular to the pipet. A droplet of water of about 10 μLis pipetted on the center of the surface at 25 °C. The watercontact angles outcome of each sample is listed immediately and 100s after the release when the droplet status stabilized.33.1During the process of SNPs precipitation, native granularcorn starch was gelatinized in water and formed a starch paste. Thepreparation of SNP in water by the dropwise addition of ethanol resultsin the nanoparticles 1a. Most A nonsolvent must be added to the polymeric solutionin order toobtain starch nanoparticles. Nanoparticle precipitation is the resultof the diffusion between a nonsolvent and solvent caused by the additionof a nonsolvent. In accordance with the parameters, the particle shapesand sizes can differ. The nanoparticles formed when ethanol was addedto the hot gelatinized starch and cooled down again to room temperature2.a. In cOn the other hand, the starch molecular agitationis low whilethe nonsolvent is added to the gelatinized starch only when it isalready cooled down to room temperature. Therefore, smaller sizesof particles will be formed, and less agglomeration will occur asillustrated in 3.222 and Zhou et al.23 Díaz-Visurraga et al.22 have stated that the random dispersion of starch nanoparticles inthe starch film matrix led to enhancement of the film surface roughness.Furthermore, based on the results of the present study, it seems thatthe low hydrophilicity of starch in comparison to the film componentscould be another probable reason for the tendency of starch nanoparticlesin the external part of the starch film, giving rise to the surfaceroughness.The qualitative(morphology) and quantitative (roughness) parameters of the filmsare analyzed by AFM. It is used to examine the dispersion of SNPsin the starch matrix. As can be seen in The mean size, RSD, and particle size distributionof the SNPsare presented in 3.325 According to 26 Theloss modulus is calculated based on the energy loss during heat orcycle deformation and is regarded as a viscous reproduction of thematerial. Having a variety of starch nanoparticles in prepared films,the loss modulus exhibits comparable trends with the storage modulus,as demonstrated in 27The purpose of this experiment is to understand the mechanicalperformance of polymeric films when they are subjected to varioustemperatures instead of room temperature.28 A positive displacement in the tan δ apexfor the SNP film might be the result of the strong chemical interactionbetween the TPS matrix and the nanoparticles, which limited the mobilityof the polymer chains in the proximity of the nanoparticles.3.4–1, whichis considered to be the firmly bound structural water existing inthe starch, associated with the angular deformation of hydroxyl groups.The absorption peaks in the range of 1000 and 1200 cm–1 are representative of the −C–O– stretchingon the polysaccharide structure. The peak at 2920 cm–1 is attributed to the asymmetric C–H stretching. The spectradisplay a resemblance to the investigation explained by Akrami etal.,29 which represents a wide peak inthe range of 3000 to 4000 cm–1 attributed to thevibration of the O–H functional groups’ deformation.The peak 1338 cm–1 relates to the angular distortionof the C–H bond. The set of peaks in the range of 900 to 1200cm–1 is assigned to the stretching C–O–Cglycosidic bonds.The existence of the different elements in theprovided specimens may be estimated by checking the translocationsof the absorption infrared peaks related to the wavenumber along withthe interactions among the functional groups. –1 peak, demonstratingalterations in the hydrogen bond model of the starch and glycerolcombination. The absorption bands at 3276 cm–1 areallocated to O–H stretching vibrations associated with freeand inter- and intramolecularly bound hydroxyl groups. In comparisonwith the SNP1 film, an increase in intensity observed for the filmsobtained after SNP formation indicates an increase in intermolecularhydrogen bonding mostly because of glycerol addition between the matrixand starch nanoparticles.30 On the otherhand, the intensity of the peak at 990 cm–1 is differentamong the prepared samples, associated with the angular deformationof the C–O–C glycosidic bonds.Furthermore, accretion in the intensity of thepeaks by the additionof glycerol is perceived veritably, which is associated with the glycerolinteractions and the matrix of starch. The glycerol as the plasticizeris a low molecular weight compound and gets into the spiral structureof starch by destroying the intermolecular bonds. The principal variationsof the peaks among plasticized starch matrixes with disparate glycerolcontents are in the 3276 cm3.5H-style crystallinity, which is different fromA-style crystallinity in corn starch, might have originated from aunique helical formation (inclusion complex) fabricated by amyloseand glycerol as the plasticizer. The plasticized corn starch alsoexhibits a similar VH-style crystallinity in its structure.During delivering the ethanol into the starch solution, the gelatinizedSNPs are precipitated slowly. Consequently, the gelatinization annihilatesA-style crystallinity of the starch, and starch nanoparticles demonstratethe VH-style crystallinity.As can be seen in 3.63.731 A high contact angle indicates better hydrophobicity of the surfaceand vice versa. The CA value of SNP films is presented in 33 Additionally, the figure shows that increasing the gelatinizationtime and glycerol content of the films results in a greater contactangle . These results suggest that there isa strong interaction between starch and nanoparticles.The method which normally has been used to compare the hydrophobicityof starch film surfaces is the determination of the contact angle(CA) of water droplets on the surfaces.4H-style after precipitation.Moreover, thermal stability of nanocomposites is improved by addingthe nanoparticles into the nanocomposites. Finally, surface hydrophobicityis increased on the samples by adding SNPs and leads to more stabilizationcompared to the samples without the nanoparticles.We have successfully producedSNP-containing starch films withhot and cold gelatinized starch solutions retaining ethanol in a singlestep. As a result of adding starch nanoparticles to the starch film,its wettability is reduced, and the existence of the plasticizer resultedin more homogeneous and more hydrophobic film. Furthermore, all samplescontaining SNPs displayed improved mechanical properties, which isconfirmed by DMA results. Compared with starch films that lack glycerol,nanocomposites with SNPs and glycerol show more flexibility. Accordingto the results of FTIR, the accretion in the intensity of the peaksby the addition of glycerol is perceived veritably, which is associatedwith the glycerol and SNP interactions and the matrix of starch. Thehomogeneous dispersion of SNPs and their interaction with the starchnanocomposites are confirmed by SEM. On the other hand, AFM showsthe exact topography of distributed SNPs and different zones enrichedwith starch and SNPs after the nanoprecipitation process. XRD patternsdemonstrate reduction crystallinity compared to neat starch granules,and it is changed from A-style to V