Combining the ANN and the genetic algorithm by using the multiple objective and nonlinear constraint functions, the optimum point was determined based on the experimental and estimated data, respectively. The characterization analysis, performed by FTIR and XRD, was used to describe the effect of the inputs on the output. The results indicated that the statistics obtained show excellent MOR predictions by the feed-forward neural network using Levenberg–Marquardt algorithms. The comparison of the optimal output of the actual values obtained by the genetic algorithm resulting from the multi-objective function and the optimal output of the values estimated by the nonlinear constraint function indicates a minimum difference between both functions.The purpose of the present article is to study the bending strength of glulam prepared by plane tree  wood layers adhered by UF resin with different formaldehyde to urea molar ratios containing the modified starch adhesive with different NaOCl concentrations. Artificial neural network (ANN) as a modern tool was used to predict this response, too. The multilayer perceptron (MLP) models were used to predict the modulus of rapture (MOR) and the statistics, including the determination coefficient (R Starch is a polysaccharide found in many plants, such as cereals, and is very important in industrial consumptions due to its cheapness, biodegradability, renewability, and safety. However, when applied as an adhesive, it has constraints, such as low shear and thermal resistance, thermal decomposition, and weak stability in solidification-melting. Hence, it must be treated, which can be mainly based on three main hydrothermal, chemical, and enzymic methods to improve its quality of application. The modified starch can be a suitable substitute for synthetic resins, such as UF from which formaldehyde gas can be emitted. Polycondensation of formaldehyde-based resins is the main method of making these resins due to their high reactivity, chemical adaptability, and economic competition . The rea2O2 or NaOCl under certain temporal, temperature, and pH conditions [When applying modified starch as an adhesive to produce wood-based composites, the process of the chemical treatment and oxidation of the starch polymer chains is very important. Modified starch is usually prepared in the reaction with oxidizing agents such as Hnditions . These onditions . During nditions . Oxidatinditions ,5. A hignditions .Due to the decrease in the crystallinity index and lower thermal stability, adding starch could improve the properties of wood panels adhered by the UF resin. Adding starch also results in a uniform distribution of the adhesive in the wood panels . Luo et The connections made between two substrates by an adhesive layer are used normally in many consumptions. The efficiency of the connections from these adhesives is evaluated normally by common traditional experimental, analytical, or numerical methods. Due to the absence of valid conventional methods to present a certain phenomenon correctly, machine learning methods can be used to release the data-driven models ,12. In rDue to these advantages, ANNs have been used in different fields of wood science. Using the experimental test data obtained by the studies on many properties of wood and wood products, such as the veneer defects classification , the dryHowever, using the ANN methods has some disadvantages. During the modeling by ANN and its accuracy, two factors are very important: weight connecting the structure of ANN and modified quantities. This weight can have negative, positive, or zero values. With respect to the type of used ANN, these values, which are produced stochastically, may be real or an integer number . The estIn the present research, the effect of the F to U molar ratio at three levels , the weight ratio of the modified starch to UF resin at three levels , and the NaOCl solution concentration percent, as the starch treater to produce the starch adhesive used to make the UF-MS adhesive at three levels , as the independent input variables was evaluated on the bending strength of the three-layer glulam produced during the loading perpendicular on the glue line as the dependent output variable using the ANN technique. Using the genetic algorithm (GA) coupled with ANN and the multi-objective and nonlinear constraint functions, the optimum limit of the application of the independent variables to achieve the highest MOR was determined.First, 100 gr corn starch powder was loaded in a flask containing 200 mL distilled water. While the mixture was mixed on a magnetic stirrer-heater, the NaOCl solution with 10%, 15%, or 20% concentration  was added to the mixture, according to the test design used, and it was mixed for 30 min at the temperature 30 °C (with pH = 9.5). After being mixed well, the mixture was neutralized using sulfuric acid 20% and was put on the stirrer-heater for 10 min at the temperature 30 °C. After being put in the falcons, the resulting solution was centrifuged for 20 min at 2000 rpm to separate its water. Then, the starch deposited in the falcons was washed by distilled water for five times. To remove more moisture from the mixture, a Buchner funnel equipped with a vacuum pump was used and the obtained starch was dried on the filter paper at room temperature. This process was repeated with the different sodium hypochlorite percentages used in the chemical treatment of starch.The UF-MS adhesive synthesis was in two stages. According to the test design used, 42 g urea consumed at the first stage together with 120 cc, 150 cc, or 200 cc formalin  and 18 g, 24 g, or 30 g starch consumed to be added in the second stage were weighed. After installing the heater and setting the temperature in the oil bath and stabilizing the three-necked flask equipped with the condenser, thermometer, and pH-meter (blocked by cork) in the bath, formalin and the first part of urea were added to the flask while the mixture was mixed constantly by the magnetic stirrer. The mixture’s pH was increased to 8–8.5 by adding few drops of NaOH 0.5 mol with the concentration 20% and it was mixed for 15 min at the temperature 90–95 °C. To decrease the time of the reaction more, the mixture’s pH was decreased to 4–4.5 by adding sulfuric acid 1% (0.5–3 mL). The approximate period was 15 min at the temperature 90–95 °C. This time was enough to achieve a homogeneous single-phase mixture. After removing the condenser from the flask and opening two other necks, the extra water evaporated at the temperature 90–95 °C for 10–20 min. The formed acidic medium was neutralized by adding a few drops of NaOH. Decreasing the reaction medium to 50–60 °C, the starch which replaced urea at the second stage was added to the mixture at the three levels according to the test design. At this temperature, the mixture was mixed for 30 min until the starch dissolved completely. After cooling at room temperature, the formaldehyde urea adhesive was prepared.Platanus Orientalis-L) and slicing it, radial slats were cut by the band saw with the dimensions 400 × 70 × 7 mm and then dried to a moisture content of 8%. According to the test design used (2) put on two lower and upper surfaces of the middle board were pressed (at 15 kg/cm2) inside an experimental hydraulic press with the press plate temperature of 160 °C for 20 min. After keeping the boards in the laboratory conditions for 2 weeks, they were trimmed by a circular saw to prepare the bending test specimens. The direction of pressure during the bending test was perpendicular to the glue line surface. The bending test was performed at the loading speed 5 mm/min using a universal tester  according to the EN 302-1 standard [After cutting the plane tree  patterns of the samples were recorded by a STOE-STADV (Germany) wide angle X-ray diffractometer using a CuKα radiation source with a wavelength of λ = 0.154 nm. The chemical compound of natural starch, modified starch, pure UF resin, and the mixture of the UF resin with the modified starch treated in alkali medium with the concentration 10% and 20% (after complete curing at the temperature 160) (as the index adhesives) were analyzed by FT-IR (Fourier transform infrared) spectroscopy using the pelletized samples. For this purpose, after mixing 100 mg potassium bromide (KBr) with 2 mg adhesive sample that was chopped into flour, the prepared samples were scanned using a Thermo Scientific Nicolet 6700 FT-IR Spectrometer  in the wave number range 600–4000 cmiX is the i-th input value of the independent variable, ijw is the weight of connection between the i-th input neuron and the j-th hidden neuron, jβ is the bias of the j-th hidden neuron, jν is the weight of connection between the j-th hidden neuron and the output neuron, θ is the bias of the output neuron, and g(.) and f(.) are the active functions of the output and hidden neurons, respectively. The widest type of ANN used for prediction is the multi-layer perceptron (MLP). The typical example of the MLP structure is shown in In order to avoid underfitting and statistical bias on the one hand, and overfitting, high variance, and increasing time for training the network, the correct number of neurons in the hidden layer needs to be determined. For this porpoise, the best neuron numbers in the hidden layer can be calculated by different methods, such as the following: the number of hidden neurons should be less than twice the size of the input layer; the number of hidden neurons should be 2/3 the size of the input layer, plus the size of the output layer. According to obtained statistics, there was no difference between the mentioned methods and the trial-and-error process, which is described as the best way to obtain the hidden layer . Training a MLP using a backpropagation learning algorithm as the commonest neural network algorithm to model various engineering applications  means to2, RMSE and MAPE showing the best performance of each model, so that the three-layer ANN architecture, including one input layer, one hidden layer, and one output layer was chosen , scaled conjugate gradient (trainscg), and Bayesian regularization (trainbr) were selected as feed-forward neural network models for training data sets. As a result, the ANN model producing the closest values to the measured values of MOR was chosen as the prediction model. This was performed based on some statistical criteria, such as Rs chosen . 2, Equation (4)) for a configuration with six neurons according to the it is the measured value, itd is the predicted value, n is the total number of data, and The formaldehyde to urea molar ratio (MR), the weight ratio of starch to urea added in second stage (WR), and the NaOCl concentration treating starch (AC) were chosen as the network input in the chemical treatment of the starch used while the variable MOR was used as the network output in the prediction model. Thus, there are three input neurons and one output neuron in the ANN model developed to predict the MOR. The best performance of the ANN model was obtained by statistical indicators showing the performance, such as the mean absolute percentage error ), the root mean square error ) and the determination coefficient  in the hidden layer and the pure line transfer function (purelin) in the output layer were used as the activation functions. The Levenberg–Marquardt back-propagation algorithm was preferred as the training algorithm. The data in the range from −1 to 1 were normalized. Then, the normalized data were changed into the original values using an inverse normalization. The ANN programs were performed using MATLAB software, version R2015a, The MathWorks, Inc, Natick, MA, USA.FTIR and XRD analyses were conducted to investigate the possible interactions between oxidized starch and UF resin. FTIR spectra of native starch, modified starch, and synthesized adhesives are presented in −1 in all samples is attributed to OH stretching vibrations. For native starch, the characteristic bands were mainly observed at 2922 cm−1, 1650 cm−1, 1364 cm−1, 1149 cm−1, and 1075 cm−1. The bands at 2922 cm−1 and 1364 cm−1 originated from C-H stretching vibrations of methylene (−CH2) groups [−1 is assigned to C-O bending vibrations associated with hydroxyl groups. The stretching vibrations of asymmetric C-O-C and C-O functional groups would cause the absorption bands at 1149 cm−1 and 1075 cm−1, respectively [−1 range [−1 became stronger and it was also shifted to a lower wavenumber, confirming the formation of more aldehyde groups during the oxidation process. The symmetric stretching band of carboxyl groups (−COO) arising from oxidized starch might overlap with the band belonging to C-O bending vibrations of native starch, which resulted in band broadening. The band at ~3260 cm−1 was strengthened and widened, which was assigned to the formation of free hydroxyl groups as a result of chain scission in the oxidative process. The band’s broadening at 2922 cm−1 after oxidation is related to chain scission, which further evidenced the successful oxidation of starch [−1 and 1150 cm−1 are attributed to the stretching vibration of the N-H group and the deformation vibration of the C-H group, respectively. This finding verified the transformation of oxidized starch and UF into adhesive resin via a polycondensation reaction [−1 and 2920 cm−1 belonging to O-H and C-H stretching vibrations were weakened, which confirmed the generation of polycondensation and partial intermolecular hydrogen bonds [−1. Indeed, the intra-molecular O–H of oxidized starch at 1640–1650 cm−1 overlapped with the C = O bond of the amide group in the UF resins [The presence of a band at 3200–3300 cmectively ,32. Basef starch . For thereaction . Furtheren bonds . It was An XRD analysis was performed to investigate the phase structure of the samples during the synthesis and curing, too. The diffractograms of native starch and modified starch are shown in 2), WR (x22), and AC (x32) on the bending strength were significantly and statistically different with p < 0.05%. Moreover, the non-significance of the lack of fit also shows the fitness of the quadratic model.The effect of MR, WR, and AC was examined on the bending strength and was modeled by ANN. 2) had the highest effect on the response with the highest value (1110) and the quadratic effect of MR (x12), the interaction effects of MR×WR (x1x2) and WR×AC (x2x3) had the lowest effect on the response with the lowest value. However, when applying an average level of MR, the low level WR, and an average level AC, the maximum MOR (140 MPa) was obtained. Based on The predicted values of the experimental MOR and their error percentage in 2, RMSE, and MAPE were used to evaluate the performance of each ANN model with three different training algorithms for MOR , scaled conjugate gradient (trainscg), and Bayesian regularization (trainbr) algorithms. Meanwhile, the backpropagation learning algorithm was used in the feed-forward neural network with one hidden layer. In the produced model, the tansig activation and purelin functions were used between the input-hidden layer and hidden-output layer, respectively. The statistics R for MOR . The staDue to the high importance of MAPE as a statistic showing the performance of a model to make decisions, it had low values for predicting the MOR for the training, testing, and validation data sets . The results show that the ANN approach has enough precision to predict the MOR. Hence, according to 2 values to predict the MOR for testing, validation, training, and all data sets are 0.9639, 0.9817, 0.9937, and 0.9903, respectively. R2 indicates that the obtained network describes at least 96% of the actual data of MOR. These values confirm the applicability of ANNs to predict glulam’s MOR.When evaluating the validity and precision of a neural network, the regression analysis often connects the actual and predicted values  for MOR 2 have been diverse. However, in almost all cases, it is confirmed that different ANN prediction models could offer an acceptable estimate. Tiryaki and Hamzacebi (2014) showed that using ANN, the strength of the thermally treated wood can be predicted with an R2 of more than 0.99 [2 (0.98) when applying ANN to predict the mechanical properties of bamboo-wood composite [2 = 0.92 [Limited studies are conducted on the prediction of the bending strength of laminated wood products connected by bio-based adhesives. However, several studies are conducted on some strength properties of wood and wood-based products. The results obtained on the prediction of the strength behavior of these products based on Rhan 0.99 . You et omposite . Nguyen 2 = 0.92 . The comparison of the predicted and actual values of the test and all data sets for MOR is presented in An ideal ANN model can identify and apply complex nonlinear relationships between the inputs and outputs of any process with a high trainability . Hence, The interactive effects of the independent variables on the MOR are given as a 3D plot in The interactive effect of MR and AC on MOR is given in The interactive effect of WR and AC on MOR is shown in 2 and RMSE. At the same time and under the effect of the variables, MOR was affected by the variables differently. As MR increases from 1.5 to 2, MOR increases. However, as it increases more and reaches 2.5, MOR decreases. As the UF resin to MS weight ratio increases to 80%, the increase in MOR is slow. However, applying the minimum starch (10%), the intensity of the increase in MOR has increased and becomes maximum. It is observed that, as the treatment concentration increases from 10% to 15%, the strength increases, and when it increases beyond it to 20%, the bending strength decreases again. Based on the ANOVA table, the F-value of every variable indicates that the direct effect of the factors has been high on the MOR. In In the bending test, due to the difference in the deformation direction of the layers under pressure compared to the layers under tension, the shear stress can result in the failure of the samples. It was observed that the samples with the highest or lowest formaldehyde and also with the highest starch to UF weight ratio have a failure at the larger area in the intraphase or interphase region of the adhesive due to the dominance of the shear stress over the bending stress. However, as the F to U molar ratio approaches the middle level (2) and as the ratio of starch approaches 20%, there is a failure under the tensile stress so that it begins from the wood and moves along the wood layer fibers. Starch oxidation can decrease the adhesive viscosity due to the failure of the inter- and intra-molecular connections  and resuAs the solid substance and viscosity increase on average, the solid structures of the modified adhesive tend to get bigger through polycondensation and hence, the molecular weight of the resin system increases. Meanwhile, due to the formation of more hydroxymethyl in the modified starch, the formation of a hydrogen bond is more possible with the UF resin. However, the curing time decreases, which is the criterion of resin activity. As the modified starch increases more, or a more intense treatment of starch is applied, the effect becomes inverse and the curing and complete copolymerization in the modified starch are limited due to the thermoplastic nature of starch . The cheGenerally, the starch molecules in gel and paste forms are related to retrogradation. The chemical treatment of starch decreases the molecular weight of starch and the steric hinderance, providing a good opportunity for grafting between starch molecules and resin. Hence, the quality of adhesives increases when the gelatinization of starch molecules is hindered in the system . HoweverIn addition, based on the SEM images, it is evident that, during the oxidation, the spherical shape of starch can disappear completely and the surface structure becomes rough or bar-shaped when it is added to urea and the OS-U adhesive is formed, showing the polycondensation reaction between urea and the modified starch . However2) of resin in the presence of a catalyst, such as ammonium chloride, that develops a stronger cohesion together with the branched structure of amylopectin so that it was observed that the failure of the samples containing a suitable level of starch occurred mainly in the wood layers, not on the glue line. In addition, the chemical reaction between the active aldehyde and carboxyl groups of the starch molecules and the hydroxyl groups in wood can improve the bonding strength more, which is very effective for bearing the horizontal shear force in the bending test and the stress can be transferred from the surface under pressure to the surface under tension. It seems that under optimal ideal conditions, where the adhesive molecule is limited to the surface layers to some extent and the gel nail can form after hardening and more penetration of resin is prevented, rupture occurs completely in the wood region. When applying the modified starch, carbonyl and carboxyl groups react with amino groups (−NH2−O−CH2−) ether bridges that are more likely to occur in the more moderate conditions of oxidation treatment, (−CH2−) methylene bridges form at high pressure temperature [It is proved that the strength properties of wood composites decrease as the crystallinity index of resins increases. This index decreases using the UF resins known as a crystalline substance during the curing  because perature  that canThe problem of the optimization of the strength properties of wood composites must be defined as mathematical model equations or the fitness function. These equations are given as the dependent functions of measured value and construction parameters. Due to the complexity of the models, more accurate mathematical models include linear and nonlinear components. However, in practice, second-order polynomials are enough to develop a mathematical model to describe the production and construction process. For this purpose, based on the independent variables x1, x2, and x3, the multi-objective functions and nonlinear constraint functions were used respectively to determine the actual effect  and estimated effect  of each independent variable as a direct, interactive, and quadratic form on each source, including f, f(x1x3), and f(x2x3) using the genetic algorithm . The minUF resins were successfully modified by adding OS prepared by NaOCl oxidation. FTIR and XRD analyses detected both aldehyde and carboxyl groups in the OS. The OS was cross-linked with UF resin by forming the ester groups.2 = 0.9937, RMSE = 0.72, and MAPE = 0.55.The ANN-GA model with three neurons in the input layer and six neurons in a hidden layer showed the best performance of optimization with RThe proposed optimal network predicted a MOR value of more than 126 MPa, validated by experiments.Based on the optimization equations of the genetic algorithm, the difference between the optimal values given by the multi-objective functions based on the actual response and the values given by the nonlinear constraint function based on the estimated response (outputs) was minimum.According to the statistics and the coefficients of the optimization equations, the concentration of the chemical solution treating the starch has been the main factor affecting the changes in the response level.The present study has predicted the effect of the application of the starch modified at different concentrations of NaOCl in the UF resin synthesis with different F to U molar ratios as the inputs on the bending strength of glulam as the outputs using the ANN and has optimized it using the ANN-GA. The results showed that:


Incorporation of graft weight in a composite multivariate model allowed for better prediction of patients who presented an aminotransferase peak > 2000 IU/L after LT . The GRWR is important in determining early graft loss after adult LT, and the mEAD model is a useful predictive tool in this perspective, which may assist in improving the graft allocation process.The role of the graft-to-recipient weight ratio (GRWR) in adult liver transplantation (LT) has been poorly investigated so far. The aim is to evaluate the contribution of the GRWR to the well-recognized early allograft dysfunction (EAD) model  for the prediction of 90-day graft loss after LT in adults. Three hundred thirty-one consecutive adult patients undergoing LT between 2009 and 2018 at Tor Vergata and Sapienza University in Rome, Italy, served as the Training-Set. The Validation-Set included 123 LTs performed at the Erasmus Medical Center, Rotterdam, the Netherlands. The mEAD model for 90-day graft loss included the following variables: GRWR  Liver transplantation (LT) is the only cure for many acute and chronic end-stage liver diseases and selected liver tumors. Early allograft dysfunction (EAD) is a commonly used tool for evaluating the initial graft function after LT. The most cited and internationally accepted definition of EAD, as proposed by Olthoff et al. is based on the presence of one or more the following factors: an increase in aspartate aminotransferase (AST) or alanine aminotransferase (ALT) above 2000 IU/L within the first week after LT, serum bilirubin ≥ 10 mg/dL and/or an INR ≥ 1.6 on day seven after LT [We retrospectively analyzed the data collected prospectively from three European liver transplant centers. Three hundred thirty-one consecutive patients who underwent LT between January 2009 and December 2018 in two inter-consortium Roman transplant centers (Tor Vergata and Sapienza University of Rome) were analyzed to produce the model derivation cohort (Training-Set). One hundred twenty-three patients from the Liver Transplant Unit of the Erasmus MC, University Medical Center, Rotterdam, The Netherlands, who underwent LT between July 2011 and October 2016, were used to validate the mEAD . The exclusion criteria were patients who received livers from donors after cardiac death; patients subjected to a multi-organ transplant, split liver transplantation, or re-transplantation; patients who received livers after the machine perfusions; living donor liver transplantation and pediatric recipients.The manuscript was approved by the local Ethical Committee of the Fondazione Policlinico Tor Vergata (reference 253/19).The grafts were perfused with Celsior solution during organ procurement, being procured during the “cold phase” within 30 min . The orgLTs were performed with the modified piggyback technique , 12. TheRecipients’ data included age at LT, biochemical MELD score at the listing, indication for LT, ICU stay (days), duration of LT (minutes), total bilirubin (mg/dL), INR, AST (IU/L), ALT (IU/L) and incidence of EAD and graft loss. Donors’ data included: cause of death (trauma or other), age, gender, height, weight, body mass index (BMI), severe hypotension, regional share, warm and cold ischemic time, body surface area (BSA), liver weight and GRWR, graft loss (within 90 days) and mortality (within 90 days), biochemical parameters , biopsy-proven macrovesicular steatosis and EAD .Recipients’ data included age at LT, biochemical MELD score at the listing, indication for LT, ICU stay (days), duration of LT (minutes), total bilirubin (mg/dL), post-liver transplant INR, AST (IU/L), ALT (IU/L), graft loss (within 90 days) and mortality (within 90 days). Donors’ data included age, gender, height, weight, BSA, warm and cold ischemic time, biopsy-proven macrovesicular steatosis, and EAD.According to the primary and secondary end-points, the study follow-up was cut at 3 months. Serum aminotransferases were analyzed daily within the first postoperative week. The highest level of AST or ALT was considered the T-peak. Graft loss was defined as patient death or re-transplantation within the first 90 postoperative days.U test was used to compare groups. Categorical variables were reported as numbers and percentages; comparisons were performed using Fisher’s exact test.Continuous variables were reported as medians and interquartile ranges (IQRs); the Mann–Whitney n = 81); intermediate GRWR ; and high GRWR . The thresholds corresponded to the first quartile (1.57) and the third quartile (2.13).In the absence of internationally well-recognized threshold values, we identified two different GRWR cut-off values in the Training-Set able to divide the whole cohort into three groups: low GRWR  of the Training-Set and the end-points of the study, the univariate selection data were not computed as suggested by Heinze et al. [https://www.omnicalculator.com/statistics/relative-risk.Due to the sample size  and then analyzed using the SPSS statistical package version 23.0 .The 90-day patient and graft survival probabilities were estimated using the Kaplan–Meier method. Differences were considered statistically significant at a n = 331), 245 patients (74%) were males, the median age at LT was 57 years (IQR: 50–62), and the biochemical MELD score was 15 (IQR:11–20).In the Training-Set cohort (n = 46), T-peak > 2000 IU/L (n = 7), bilirubin ≥ 10 mg/dL (n = 44), and 78 (38.4%) with two features, namely aminotransferase and bilirubin (n = 7), aminotransferase and INR (n = 21), and bilirubin and INR (n = 50). Fifty-three (16%) patients experienced graft loss during the follow-up.A total of 203 (61.3%) patients experienced EAD after LT; of these, 28 (13.8%) simultaneously presented with all three EAD features, while 97 (47.8%) presented with only one feature, namely INR ≥ 1.6  IU/L and 673 (IQR: 469–1270) IU/L, respectively. The median INR and serum bilirubin on day 7 were 1.5 (IQR: 1.26–1.96) and 8.8 (IQR: 5.2–14) mg/dL, respectively.The median GW and GRWR were 1350 g (IQR: 1191–1648) and 1.83 (IQR: 1.56–2.13), respectively. Eighty-one (24.4%) recipients showed a GRWR < 1.57, 167 (50.4%) between 1.57 and 2.13, and 83 (25.1%) > 2.13. The abdomen was primarily closed in all patients.n = 4) or hepatic artery thrombosis (n = 1). Two (0.6%) patients died after re-transplantation, 24 due to infection/sepsis (7.1%), 20 (6.0%) for multiorgan failure and 7 (2.1%) due to other causes. The patients’ characteristics are summarized in Table The 90-day patient and graft survival rates were 85% and 84%, respectively. Five 1.5%) patients underwent re-transplantation due to primary non function (.5% patien = 123), 62 recipients  were male; the median age at LT was 55 years (IQR: 44–61), and the biochemical MELD score was 15 (IQR: 11–21). The indications for LT were cirrhosis in 77 (62.6%) recipients and hepatocellular carcinoma in 45 (36.5%). Remarkably, 40 (32.6%) LT recipients had an underlying cholestatic disease.In the Validation-Set (Forty-six patients (37.4%) experienced EAD after LT. In 25 cases, a T-peak > 2000 IU/L was reported, followed by 10 cases with bilirubin ≥ 10 mg/dL and 7 cases with INR ≥ 1.6 at day seven after LT. In three patients, bilirubin ≥ 10 mg/dL and aminotransferase > 2000 IU/L were reported, while one recipient had all three EAD risk factors. Six (4.9%) patients experienced graft loss within 90 days.The median values of the AST and ALT peaks after LT were 997 (IQR: 636–1718) IU/L and 706 (IQR: 447–1215) IU/L, respectively. The median values of INR and bilirubin on day 7 were 1.2 (IQR: 1.3–2.0) and 7.7 (IQR 1.5–4.3) mg/dL, respectively.The 90-day patient and graft survival rates were 96.7% and 95.1%, respectively.The median estGW (23) and estimated GRWR were 1359 (IQR: 1240–1442) g and 1.73 (IQR: 1.52–1.98), respectively. Thirty-seven (30.0%) recipients showed an estimated GRWR < 1.57; 67 (54.5%) between 1.57 and 2.13, and 19 (15.5%) > 2.13.WIT and CIT were significantly lower in the Validation-Set cohort. All demographic and clinical characteristics of both the Training-Set and Validation-Set are described in Table p = 0.03) an INR ≥ 1.6 on day 7 , and transaminase levels > 2000 IU/L within the first week after LT . In addition, GRWR was also identified, either at high values , or at low values , as reported in Table In multivariable logistic regression, four variables were identified as independent risk factors for 90-day graft loss. These included, all three components of EAD: a serum bilirubin ≥ 10 mg/dL on day 7  and in the Validation-Set  . Although the mEAD was superior in terms of diagnostic ability (z = 1.54) in the Validation Set, the test only merged statistical significance (p = 0.12).In the Training-Set, the mEAD model showed an NRI of 0.93 vs. the EAD, resulting from a correct reclassification of 0.4% of events (graft loss) and 8.9% of non-events (no-graft loss). Similarly, in the Validation Set, we observed a positive NRI value of 0.23 due to a correct reclassification of 0.5% of events and 1.8% of non-events. In the Training Set, the adopted The relative risk (RR) for 90-day graft loss according to the GRWR and mEAD are depicted in Figs. In the case of mEAD classes of risk, patients with a mEAD value of 0–1 (RR = 0.3) and 2–3 (RR = 0.5) had low relative risks for 90-day graft loss when compared with patients with a mEAD value of 4–5 . On the opposite, increased values corresponded to an increased RR .. those with a T-peak < 2000 IU/L were 1500 g (IQR: 1365–1748) and 1306 g (IQR: 1156–1540) (p < 0.0001), respectively. Using multivariate analysis, we investigated the effect of GW on the risk of presenting with a T-peak p = 0.0005). Among the other considered potential risk factors, the steatosis grade was the only one that showed a significant risk, with an estimated OR of 1.43 for every 10% increase in steatosis.The median T-peak observed within the first week after LT was 974 (IQR: 530–1830) IU/L. Sixty-eight (20.5%) LT recipients experienced a T-peak > 2000 IU/L. The median GW in recipients presenting with a T-peak Historically, in adult patients who have undergone LT, the EAD model proposed by Olthoff et al. in 2010  and the In Italy, the organ allocation system is based on the concept of urgency, utility, and “transplant benefit” , as reguGRWR variability” on 90-day graft survival. Since LT early outcome models taking into account preoperative donor/recipients variables as well as intraoperative features  have been reported so far [perioperative donor-recipient size match  in association with the well-known early postoperative liver functions variables .The GRWR represents a highly sensitive and critical parameter in pediatric patients . Even thoutcomes . In contd so far , 24, in z = 1.54), the test only merged statistical significance when compared with the Olthoff EAD (p = 0.12). This datum underlines the relative impact of mEAD when compared with the original EAD in the external validation. However, the fact that the p-value was 0.12 does not mean that no effect exists. The AUC of mEAD was superior (0.68 vs. 0.52), consenting to reclassify a relevant number of patients correctly. In detail, the mEAD model correctly reclassified 0.4% of graft loss and 8.9% of no-graft loss (NRI = 0.93) in the Training-Set and 0.5% of graft loss and 1.8% of no-graft loss in the Validation-Set (NRI = 0.23). The small number of cases (n = 123) and events of interest (only 4.9% of 90-day graft losses) reported in training set strongly conditioned the results of c-statistics. The smaller the number of events of interest, the greater the AUC confidence intervals (ranging 0.35–0.69), the greater the overlapping of the AUC values between mEAD and EAD, and inferior the statistical significance observed comparing the tests.The newly proposed mEAD model, which is an adjustment of the conventional Olthoff EAD model, showed that both high   and L-GrAs shown in Fig. Our study also shows that GW allowed estimation of the level of the post-transplant T-peak. This finding may be a consequence of the increased difficulty in liver perfusion of large and heavy grafts during procurement and slower reperfusion at LT. Undoubtedly, the homogeneous intrahepatic distribution of the perfusion solution during organ procurement or blood during reperfusion is influenced by the organ's size. Furthermore, GW may be influenced by the extent of macrosteatosis, leading to partial or complete sinusoid obstruction , the Kupp < 0.0001) (due to the difference in the surface area and transplant logistics in the two countries) (iii) an extended WIT could also justify the higher EAD incidence in the Training-Set cohort. Since a high incidence of the EAD can hamper the outcome of the primary end-point of the study, the mEAD model was externally validated. The study has other potential limitations that cannot be ruled out: (i) like other models mentioned above [c-statistics in the Validation-Set is close to 0.50; (iii) it is multicentred and retrospective, with a limited number of transplanted patients, and (iv) since the GW in the Validation-Set cohort was not available at backbench, the GRWR were calculated using the estimation standard liver weight formula; hence we cannot exclude the possibility of minor inaccuracies in estimating GRWR. Finally, GRWR values ranged between 1.0 and 2.13 in 75% of LTs; thus, we cannot make firm conclusions in the case of a higher GRWR commonly used in other Western countries [We are aware that the high incidence of the EAD , 2 obsered above , 21, theountries , 33.In conclusion, this study suggests that, in the setting of whole adult LT, surgeons should pay greater attention to the measurement of GW, which should always be obtained at the backbench or estimated using the appropriate formulas , 28. The


Oryza sativa) from Qujialing was already domesticated, and millet  had also been spread into the site since the Youziling Culture period (5800-5100 BP). Nevertheless, no direct evidence has been provided regarding the daily consumed plant foods, especially plant foods obtained by gathering, throughout the site occupation. This paper thus examines pottery sherds (n=41) associated with culinary practices from Qujialing with starch grain analysis. Apart from starch grains from rice and millet, the results indicate that job’s tears (Coix lacryma-jobi), lotus roots (Nelumbo nucifera), tubers possibly from Chinese yam (Dioscorea panthainca), acorns (Quercus sp.), and beans (Vigna sp. or/and Vicia sp.) were consumed by the ancient Qujialing people, within job’s tears and lotus roots were not discovered before in the macrofossil remains and phytoliths. Combining the starch data and multiple lines of evidence from macrofossil remains and phytoliths, it is suggested that rice was among the most frequently consumed plant foods since the first occupation phase at Qujialing, while acorns could have been gradually replaced by other agricultural products  and became less important food ingredients, especially when agriculture was more developed in the last occupation phase at Qujialing. These novel findings not only complement our previous research by providing the first line of evidence of paleodiet in the Jianghan Plain from the perspective of starch grain analysis but also delivers a better understanding of the characterized dietary trends and agricultural development in the research region.The site of Qujialing experienced a long, sustained process of the development of Neolithic culture in the Jianghan Plain, with a period of some1600 years. Our previous studies based on macrofossil remains and phytoliths revealed that rice ( Oryza sativa) and millet  cultivation accordingly  is located in the east of the lower catchment of the Han River in Jingmen city, where is about 130 kilometers from Wuhan, the capital city of Hubei Province Figure 1Our research team has been closely involved in the latest excavation at the site of Qujialing since the beginning, allowing us to conduct a more holistic archaeobotanical study at the site. In two of our previously published papers, macrofossil remains retrieved from flotation and phytolith remains discovered in the sedimentary soil samples from Qujialing have been studied , revealiThis paper studies 41 pottery sherds recovered from the latest excavation at the site of Qujialing between 2015 and 2017 Figure 33)6 to deflocculate clay minerals and other minor components, and then centrifuged in the presence of heavy liquid (CsCl with a density of 1.9 g/cm3) before being mounted on glass slides in a solution of 50% glycerine and 50% distilled water.The chosen pottery sherds were sealed in separate Ziplock bags in the field laboratory and then transported to the Archaeobotany Laboratory at the University of Science and Technology of China (USTC) for further analysis. First of all, each of the pottery sherds was briefly rinsed with a wash bottle with ultra-purified water. Then, liquid samples from the internal surfaces of the pottery sherds were collected using an ultrasonic toothbrush. These liquid samples were gathered in test tubes for subsequent extraction. The liquid samples were treated with 10% HCl and 5%  were taken of each grain using a Spot Flex Mono 15.0 digital camera and Zeiss Axiovision software. The size of the starch grains was measured using AxioVision Rel. 4.7 software.The identifications of starch grains were based on modern starch references at the Archaeobotany Laboratory of USTC e.g., Figure 4Setaria italica or/and Panicum miliaceum), job’s tears (Coix lacryma-jobi), acorns (Quercus sp.), beans (Fabaceae), lotus root (Nelumbo nucifera), and other types of underground storage organs (USO) possibly from Chinese yam (Dioscorea panthainca).A total of 1521 starch grains were extracted from 41 pottery samples from the Qujialing site Table 1.Starch grains from type I (n = 256) are polyhedral or round polyhedral in shape with the centric hilum closed, and they are common for the multigrain aggregation form  are comparable with the subfamily Panicoideae in the Poaceae grass family. They have a polyhedral shape with angular or round edges, the hilum is centric, and fissures are fairly common, appearing as stars with a radiating. The extinction cross is generally “+” in shape, and very few of the crosses show a twisted pattern at the end of the arm. Lamellae are always invisible. The size range is 6.38–23.23 µm. They are similar in morphology to foxtail millet (11.21–16.75 μm) and broomcorn millet (5.92–13 µm) in our reference collection. Meanwhile, it has been noticed that starch grains from foxtail millet and broomcorn millet cannot be fully separated because of their similar morphological features (Starch grains of this type (n = 281)  are polyhedral or oval-spherical in shape, exhibit Y-, V-, or linear-shaped fissures, centric or/and eccentric hilum, invisible lamellae, and have extinction crosses with straight or Zig-Zag arms ’s tears . The siz). Fissures and lamellae are not visible. The size range is 9.74–24.93 µm. These starch grains resemble the starch grains from seeds of Quercus in the Fagaceae family, according to our previously documented work (Starch grains of type VI (n = 47) are triangle ovate or water-drop shaped, with a centric hilum with an “×” shaped slightly bent extinction cross that appears under polarized light  are relatively large, showing subspherical or elongated oval shapes Figure 4yam (Dioscorea polystachya). However, some starch grains from Type VI do not fully match the existing modern plant starch references, so we cannot exclude the possibility that they may come from other plant species.starch grains of type III (n = 217) are round quadrilateral or elongated oval in shape, with extremely eccentric hila, visible lamellae in most cases, and extinction crosses with bent arms (Vigna sp. or/and Vicia sp.Starch grains of type VII (n = 9) are elliptical or nearly kidney-shaped starch grains that have radiating fissures with clear-cut lamellae Figure 4Glycine max), other types of beans (Leguminosae), acorns (Quercus sp.), jujube (Ziziphus sp.), persimmon (Diospyros sp.), plum (Armeniaca mume Sieb.), water chestnut (Trapa sp.), and Gorgon fruit  were found  dated to the Eastern Zhou Dynasty (770-256BC) were identified at Qujialing  among other types of cereals . During In terms of millet, the earliest remains of this plant have been recovered on pottery sherds at grinding tools at the sites of Nanzhuangtou (ca. 9.500-9.000 BC) and Donghulin (ca. 9000-7500 BC) in the upper Yellow River valley, as attested by starch grain analysis . Then, mThe yielded starch data shows that millet had been consumed since the Youziling Culture period and its ubiquity was even close to rice starches on the pottery vessels, although never exceeded Figure 7Triticum aestivum) had also been introduced into this region since at least the Warring States Period  distributed in the area 5400-4200BP  were detected on the Qujialing pottery vessels. The starch data thus consolidates the previous archaeobotanical work based on macrofossil and phytolith analysis at Qujialing, and put more new insights into the plant foods that had been consumed by the local people during the Neolithic period. Further quantitative analysis of the yielded data from different analytic methods, namely starch grain, phytolith, and macrofossil analysis, indicate that although millet had spread into the Jianghan plain since the earliest occupation period at Qujialing, rice had always been the predominate cultivated crop and probably more frequently consumed. Specific dietary choices and trends were also detected at Qujialing, including persistent exploitation of lotus roots throughout the site occupation and the abandonment of acorns in Phase III, which may be related to the local environment that was surrounded by water and developed agriculture accordingly. Yet, apart from the present research, starch grain analysis has rarely been applied to investigate the uses of Neolithic pottery vessels in the Jianghan Plain, more future work thus is still needed to deliver a more comprehensive understanding of the past human diet in the whole research region.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.YY, WL and JZ conceived and designed the study. YT, DZ, and YL provided the archaeological samples. MK and LY collected the study samples and analyzed the data. WL, YY, and MK wrote the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the International Partnership Program of the Chinese Academy of Sciences(132311KYSB20190008), the National Natural Science Foundation of China (41772172), the Philosophy and Social Science Planning Project of Anhui Province (Grant No. AHSKQ2021D52), the USTC Funding for Featured Liberal Arts (YD2110002016), and the USTC Youth Innovation Fund (WK2110000020). We would like to thank three reviewers for their insightful comments.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


To write about the beginnings of the Brazilian Journal of Otorhinolaryngology, back in 1933 through 1990, feels like recounting the very history of our specialty in Brazil. In the capacity of Editor, between 1989 and 1996, I strengthened my ties with the publication and became interested in all it represented to Otorhinolaryngology. When I was the Editor, my first job was to prevent the journal from not being published, so that it could grow and achieve relevance, together with national and international exposure. Some years before, in 1989, when I took on the challenge, the journal had three papers, usually review papers or case reports, published in each one of the three annual issues. There were no advertisements and the medical journal - which is among the five oldest papers in Brazil - was all paid for by the late Brazilian Society of Otorhinolaryngology (SBORL). It was no easy task to make the few Brazilians who published at the time to trust us and send us their papers. In 1996 we had four annual issues, with an average of ten papers per issue and eight advertisers, which amassed profits of R$ 80 thousand per year. Among the main indexing agencies at the time, not only the journal represented a surplus, but it was also the main source of income for the SBORL.This challenge motivated us to look for the origins and the people - like us, who were also associated with this enterprise and struggled for its survival. In 1990, I published the Memories of the Brazilian Journal of Otorhinolaryngology, in a special issue, and its table of contents and index through the date, then. We managed to collect all the journal issues, since the very first. The special edition is available at the current Brazilian Association of Otorhinolaryngology and Facial and Neck Surgery (ABORL-CCF) and its table of content and index served as a basis to recover all the papers present today in the official website of our journal.Founded in 1933 with the name of OTO-LARYN-GOLOGICAL JOURNAL OF SÃO PAULO, the publication was headed by Drs. Mário Otoni de Rezende and Homero Cordeiro, who remained at its front until 1963. At that time there was no national body representing the specialty, and most of the specialists in the field practiced Otorhinolaryngology and Ophthalmology. Notwithstanding, the journal published only papers in the field of Otorhinolaryngology, which already hinted at a separation between ENT and Ophthalmology as two independent specialties.In 1939, the journal became the BRAZILIAN JOURNAL OF OTORHINOLARYNGOLOGY, without disregard for the previous issues, continuing with the numbering started at the Oto-laryngological Journal of São Paulo. In 1964, Dr. José Eugênio Rezende Barbosa became the Journal's Editor, being succeeded by Drs. Rudolf Lang and Nicanor Letti, from 1970 through 1975. Lang remained in that capacity until 1985. At that time, the production was in the hands of the Director of Publications of the Brazilian Society of Otorhinolaryngology, Dr. Arthur Octavio Kós, between 1985-1986, and Dr. Fernando Sérgio Portinho, between 1987 and 1988.In a quantitative assessment of papers, we found 1,461 papers published between 1933 and 1990. In We have to stress our difficulties in publishing scientific papers. Brazilian publications are hammered by international papers, especially those from the United States. In order to correct this shortcoming, in recent years the Journal has been published also in English, aiming at achieving more indexing and exposure, with citations at the ISI. National authors have much difficulty in finding their space in high-impact journals because of prejudice against our publications in developing countries, such as China, Russia and India.The low government and private stimulus for state-of-the-art scientific research, often times prevents papers from reaching high-impact journals, such as Nature, Science and the New England Journal of Medicine. There is a worsening factor in our specialty: otorhinolaryngology Journals have the lowest impact rates when compared to others. The major publications in our field, the Laryngoscope; Otology and Neurotology; Otolaryngology and Head and Neck Surgery; Rhinology, all have a mean impact rate of 2.0.University and government research and graduate program agencies, such as CAPES and CNPq, do not respond to the argument that the impact rate in the specific field is most important, and there is a need to match other ENT authors in the world. These committees insist on associating us with other clinical and basic fields which publish in higher impact journals, inherent to their specialties. As the saying goes, they want to “compare apples with oranges”. This is something that has already happened in the USA, Canada and European countries, and in university institutions each field is rated vis-à -vis its international counterparts.The so-called Speech-and-Hearing papers, , involviGraphs 4, 5 and 6, dealing with nose and paranasal sinuses, oralpharyngology and head and neck surgery and miscellaneous (papers which do not fit the previous categories) papers had a constant drop between 1930 and 1990, with a mild recovery for nose and paranasal sinuses' papers as of 1970.Here, I stress some author-related points: During this period, some colleagues had a constant flow of publications, while others published a high number of papers for some time and, then, suddenly stopped, which may coincide with passing in some university program or peaking in their careers. Other colleagues, very productive in terms of attending meetings and conventions, and even famous specialists, are not frequent authors. We could also notice that very productive authors are not necessarily associated with medical schools. Independent specialists or some from private clinics also contribute to our scientific publications.The goal of these remarks is to stimulate scientific production from lab researchers, academics, graduate students, resident physicians, interns or even independent physicians. Everyone must help with their share, publishing clinical cases, sharing their experience and communicating their techniques. It is only with such procedures that we will grow our field of work.The Brazilian Journal of Otorhinolaryngology is a true asset of our specialty, and I am proud to be part of its steering team. The work of all the editors who came after me helped increase its credibility and strengthen it among its international counterparts. Today we have the highest possible index mark at ISI, and I continue to believe and invest in the future of our Journal, which success portrays the scientific development of Brazilian Otorhinolaryngology.


Gryllus bimaculatus), called the Mediterranean cricket, is increasingly being adapted for more commercial purposes. Adapting the mass cricket-rearing conditions for field cricket production, we found crickets had a typical growth rate and capacity for conversion of ingested feed into body mass. The efficiency of the deposition of major nutrients  in the field crickets from the ingested feed is possible to be measured under mass cricket-rearing conditions. The feed intake and mortality rate for cricket rearing should be considered for calculating major nutrient conversion efficiency as increased mortality rate resulted in higher conversion efficiency.Farming edible insects such as field crickets (Gryllus bimaculatus), due to their market value and sustainable development. The current study aimed to measure the production performance of field crickets and to quantify the major nutrient deposition rate using a new approach for a nutrient conversion efficiency calculation for the field crickets under mass-rearing conditions. The field crickets were reared under mass-rearing conditions in the rearing crates and fed with a commercial cricket feed. Measurements for daily feed offered, final body weight, and dead cricket quantity were carried out during the feeding trial period. There were three production rounds with the same procedure for farmed cricket management. The samples of diet, adult crickets, and dead crickets were collected and then analyzed for chemical analysis of macronutrients. The production performance and nutrient conversion efficiency were calculated and then compared with applicable earlier reports for both field and house (Acheta domesticus) crickets. The production performance for the studied field crickets under mass-rearing conditions had final a body weight, an average daily gain (ADG), a feed conversion ratio (FCR), and a survival rate of 0.95 g, 23.20 mg/day, 2.94 and 88.51%, respectively. The field crickets had nutrient conversion efficiency for dry matter (DM), ash, crude protein (CP), crude fat (EE), crude fiber (CF), and nitrogen-free extract (NFE) of 13.26, 8.03, 28.95, 88.94, 34.87, and 1.85, respectively, with an adjusted nutrient conversion efficiency of 14.85, 8.99, 32.37, 99.17, 38.95, and 2.10, respectively. Thus, the production of field crickets could be performed under mass-rearing conditions, and the nutrient conversion efficiency for both adjusted and non-adjusted values could be measured.Currently, there is an increased interest in mass producing edible insects, e.g., field crickets ( The global population is expected to reach 9.7 billion by 2050. Alternative food sources for humans and feed for animals are currently being explored to overcome the planet’s finite resources with a sense of sustainable production for food security . ConsumiAmong the species known to be edible, the insects in the orders Coleoptera, Lepidoptera, Hymenoptera, Orthoptera, and Hemiptera, are commonly consumed . Within Gryllus bimaculatus) and house (Acheta domesticus) crickets due to their short production cycle (45–60 days) among edible cricket production and abundant returns. The key difference between these two species of crickets is that G. bimaculatus stands out in terms of economic factors ), while A. domesticus has a more acceptable taste  [G. bimaculatus with A. domesticus [G. bimaculatus production performance and related points of view, although more important for the economic impact scale [From the informative report of Kemsawasd et al. , Thai faon cycle 5–60 daysmesticus . This woct scale . From thct scale , studiesct scale ,16. Manyct scale . House cct scale ,18. In tct scale ,19,20,21ct scale , and alsct scale , includiG. bimaculatus, the crickets were reared at a farm facility belonging to the Animal Science Division, Department of Animal Production Technology and Fisheries, School of Agricultural Technology at King Mongkut’s Institute of Technology Ladkrabang, Thailand. Eight rearing cricket crates with a size of 60 cm (width) × 120 cm (length) × 60 cm (height) were made with polycarbonate walls and flooring with a nylon-wire mesh cover, which aimed to allow for ventilation, protection from flies, and prevention of natural predators (such as geckos). Egg board cartons (29.5 cm × 29.5 cm × 4.7 cm) were continuously arranged in a zigzag direction and then arranged into each rearing crate by making a platform for the cartons at around 10 cm above the crate floor. This was to allow floor cleaning, collecting of dead crickets, and prevention of moisture accumulation. Approximately 10–15% of the crate floor area was free from egg cartons, allowing ventilation on the floor of the rearing crates and working convenience. All crates were placed on a shelf with their feet placed in vegetable oil traps to prevent ants and other predators from accessing the cricket eggs and their crickets. Other facilities and equipment required for mass rearing were also prepared, such as plastic bags, a small sweeper, record books, etc. Cricket eggs of the field crickets were purchased from a commercial cricket breeder in Thailand . Concerning the mass-rearing system, Parajulee et al. [ad libitum by adding several feeding trays and an amount of feed proportional to the larger size of crickets. The amount of feed offered to the experimental crickets was recorded and a new feed of approximately 600–800 milliliters of water was provided once, twice, and three times a day for the 1st–3rd, 4th–5th, and 6th-week old field crickets, respectively, through approximately 100 cm length and 3.81 cm diameter of PVC pipe (rectangle shape) with 22 holes on its rough surface filled with thread to allow the crickets to sip water through the thread.To measure the production performance and nutrient conversion efficiency of the field crickets, e et al.  mentione®; PP feed, the TFMS (Saraburi) Co. Ltd., Saraburi province, Thailand) containing 20.24% crude protein (CP) and 3.22% crude fat was used, which was also used as a dietary treatment in the study of Bawa et al. [To investigate the nutrient conversion efficiency, a commercial cricket feed , ash, crude protein (CP), crude fat (EE), crude fiber (CF), and nitrogen-free extract (NFE). The proximate compositions were estimated by following the standard methods recommended by the Association of Official Analytical Chemists . The moisture percentage was calculated by drying the sample in an oven at 60 °C for 3 days. The dried sample was put into a desiccator, allowed to cool and reweighed. The process was repeated until a constant weight was obtained. Crude protein was determined by the Kjeldahl method and the total protein content was calculated as the amount of total N determined multiplied by a nitrogen-to-protein conversion factor of 6.25 for the diet sample. For the samples of live and dead crickets, the conversion factor of 5.0 was used to multiply N contents instead of the conversion factor of 6.25 as an overestimate of protein for field and house crickets . The fatG. bimaculatus [The number of crickets per rearing crate was estimated by dividing the weight of biomass by an average adult body weight. The reported value of 1.36 mg for the first weight after the hatching of aculatus  was usedaculatus , demonstaculatus  used theaculatus . This ECaculatus  and Bawaaculatus , respectt-test.All data were analyzed using IBM SPSS Statistics 28  to look for differences in the results from the current experiment and the reference. Mean ± standard deviation was expressed where results were appropriate. Differences were tested by Student’s The results of production performance are illustrated in p < 0.05) of conversion for DM after correction by dead crickets and CF both before and after the correction. The statistical analysis (not shown) illustrated no difference (p > 0.05) between nutrient conversion efficiency and adjusted nutrient conversion efficiency for both DM and each nutrient. The calculations for the amounts of feed intake and deposition per 1 g of body weight cricket for DM and nutrients help us to make a comparison between the field and the house crickets (p < 0.05) CF than house crickets but deposited more of it (p < 0.05). Dead Mediterranean crickets deposited significantly less CP (p < 0.05) than house crickets did.The components of calculation for both ECI and adjusted ECI are shown in crickets .. From tThe adjusted ECI had a higher value compared to the ECI for the current study and the referral report, including other reports about ECI for crickets available at this moment . From thFor comparisons in the current study, it is likely improper to use results from other studies to compare with the results of the current study. A meta-analysis would be applicable for this kind of comparison. However, it is not the main objective of the current study. Instead, an approach for quantifying the DM and macro-nutrient conversion efficiency applied for the mass rearing of crickets was focused on for this study.For the production performance, a comparison between different experiments might be improper because production performance is mainly influenced by animal genetics, feed offered, rearing management, and environmental conditions. Among these factors for the comparison in the current study, animal genetics might be the main factor influencing the results of production performance. This might be the fact that other factors had small differences between both studies and the values of the production performance obtained from this study were in accordance with the earlier reports. All relevant values of production performance were used in calculating the DM and macro-nutrient conversion efficiency. Thus, it is implied as important background information for further calculations of the nutrient conversion efficiency. Obtained production performance of the field crickets under mass-rearing conditions was comparable with that of earlier reports performed in both mass-rearing and small-scale experiments, which indicates validation for the production performance values.There is important data of the field cricket production, which is available for calculation at the present. However, the environmental conditions and the overall management of the crickets, especially the CP content in the diet were comparable, although slight differences in CF contents did occur .Apolygus lucorum [The main productive differences between the field and house crickets are the bigger size, shorter duration of rearing, and faster growth rate for field crickets. These are advantages for the field crickets when compared with the house crickets. However, the field crickets had higher FCR and lower survival rates. The latter was also described in the study of Sorjonen et al. , who fou lucorum , but sta lucorum . From th lucorum , housing lucorum  in centr lucorum ,38 requi lucorum ,19,20,30 lucorum , and othThe results in However, when nutrient conversion efficiency (as a dry basis) was applied, the difference in values of nutrient conversion efficiency between the field and the house crickets tended to disappear, except for 1.4 times and around 4.0 times deposition of DM and CF, respectively, in the field crickets. This was in agreement with the results of higher ECI (DM conversion efficiency)  and highConcerning the calculation of adjusted ECI and nutrient conversion efficiency , the adjG. bimaculatus) could be performed under mass-rearing conditions without major adverse effects on the production performance. Recording feed intake and collecting dead crickets during the period of cricket rearing are key activities to monitor cricket performance, allowing possibilities to calculate the value of nutrient conversion efficiency for both adjusted and non-adjusted values under mass cricket rearing. This probably helps to get more insight into nutrient metabolism and utilization of crickets, at least by allowing comparable values across studies as the higher adjusted conversion efficiency related to a higher mortality rate of crickets.The production of field crickets (


Heart transplantation is the definitive treatment for many cardiovascular diseases. However, no ideal approach is established to evaluate heart grafts and it mostly relies on qualitative interpretation of surgeon based on the organ aspect including anatomy, color and manual palpation. In this study we propose to assess quantitatively the Shear Wave Velocity (SWV) using ultrasound as a biomarker of cardiac viability on a porcine model.The SWV was assessed quantitatively using a clinical ultrasound elastography device  linked to a robotic motorized arm  and the elastic anisotropy was obtained using a custom ultrasound research system.N = 8) with the heart removed and arrested by cold cardioplegia and immerged in a preservation solution. One ischemic group (N = 6) with the organ harvested after 30 min of in situ warm ischemia, to mimic a donation after cardiac death. Hearts graft were revived at two preservation times, at 4 h (N = 11) and 20 h (N = 10) and the parameters of the cardiac function evaluated.SWV was evaluated as function of time in two porcine heart model during 20h at controlled temperature (4°C). One control group (p = 0.007) and 20h  of preservation with significant differences. A good correlation between SWV and cardiac function index was found (r2=0.88) and manual palpation score (r2=0.81).On control hearts, SWV remained unchanged during the 4h of preservation. SWV increased significantly between 4 and 20h. For the ischemic group, SWV was found higher after 4h (3.04 +/- 0.69 vs 1.69+/-0.19 m/s, Myocardial stiffness increase was quantified as a function of preservation time and harvesting conditions. The correlation between SWV and cardiac function index suggests that SWV could be used as a marker of graft viability. This technique may be transposed to clinical transplantation for assessing the graft viability during transplantation process.FRM PME20170637799, Agence Biomédecine AOR Greffe 2017, ANR-18-CE18-0015. Non-invasive assessment of explanted cardiac grafts remains challenging as there is no ideal approach established. Ex situ assessment mostly relies on qualitative interpretation of surgeon based on the organ aspect including anatomy, color and manual palpation. Quantification of myocardial properties could be crucial in order to optimize preservation strategies, harvesting technique and finally to expand the number of grafts available for cardiac transplantation.We demonstrated the capacity of ultrasound elastography to monitor grafts stiffness after heart harvesting, during the hypothermic preservation duration, and after warm ischemia. We show that SWV is a reliable tissue marker for monitoring the organ preservation as a function of time.This could become a powerful tool to optimize preservation strategies and also for graft viability estimation, especially those from DCD. Possibility of real time non-invasive measurements associated with easy implementation, strongly suggests that this technique may be considered for a future using during the transplantation process. SWE has the potential to provide a tissue biomarker of the heart viability and could offer a new accessible, portable and predictive tool before heart transplant.Alt-text: Unlabelled box,,Heart transplantation remains the definitive therapy for end-stage heart failure patients but the number of cardiac grafts available for transplantation is still largely insufficient.Non-invasive assessment of explanted cardiac grafts remains challenging as there is no ideal approach established. Ex situ assessment mostly relies on qualitative interpretation of surgeon based on the organ aspect including anatomy, color and manual palpation.,,Ultrasound Shear Wave Elastography (SWE) has emerged during the last decade as a quantitative and non-invasive imaging modality to evaluate the viscoelastic properties of soft tissues.In this study, we propose to use SWE as a tool to assess cardiac grafts viability between heart harvesting and transplantation time. We developed a device to image an entire isolated porcine heart in different views to evaluate myocardial stiffness at several anatomical locations. A second ultrasonic system was also used to perform a multidirectional evaluation of the SWE.Experiments were performed in explanted hearts of a porcine model. The animals used are healthy, young adults, non-genetically modified farm pigs of both genders, weighing 30-35 kg, supplied by breeders approved by the veterinary authorities.at libitum, enrichments and controlled environmental conditions), the animals were anesthetized as follows: induction of anesthesia was performed by an IM injection of a mixture of Ketamine (6.5 mg/kg) and Xylazine (1.5 mg/kg). After introduction of cathlon (24G 11/10) into a marginal vein of the ear, a bolus of propofol  and Fentadon (0.035μg/kg) is injected by IV, in order to complete the anesthesia. The installation of a 7G tracheal probe, connected to an Alpha100 type respirator  ensures assisted ventilation and maintenance of the volatile anesthesia (with 2.5% Sevoflurane).To limit the stress of the animal and accustom it to human presence, daily care has been provided . After housing and acclimatization period of 72h in an approved animal facility (water and food N = 8), the heart was arrested by cold (4°C) cardioplegia and rapidly immerged in a preservation solution. In the second group (N = 6) heart harvesting was preceded by 30 min of in situ warm ischemia, to imitate a Maastricht 3 protocol . Due to the differences between the 2 types of surgical preparation, all participants were aware of the attribution of the groups.After stabilization of the hemodynamic parameters , a left thoracotomy was performed and the hearts were randomly allocated to one of the 2 following groups, at the end of the surgical preparation to limit confounding factors related to the animal. In the control group (+ (110 mmol/L), K+ (20 mmol/L), Ca2+ (1 mmol/L), Cl– (150 mmol/L), Mg2+ (4 mmol/L) and metabolic agents. More details can be found in Michel et al.After harvesting and cooling, each heart was suspended by the aorta and immerged on a preservation solution. This preservative technique was chosen due to simplicity and reproducibility without risk for the graft. The preservation solution used called LYPS was developed by Unité de recherche INSERM-UMR1060, Lyon, France. This preservation solution is mainly composed of NaEach graft was preserved during 20h at controlled temperature and their stiffness was measured three times. At first Shear Wave Velocity (SWV) measurement, which is the velocity of the traveling shear wave, were performed right after graft immersion as a reference point of a fresh harvested organ. A second set of measurements was obtained after 4h of preservation which corresponds to the time limit for organ transplantation. A last set of measurements was finally performed 20h after the harvesting in order to evaluate long preservation time impact on graft stiffness.Of all the experiments, approximately 10% of the animals were excluded for the following reasons: (i) surgical problem , (ii) problem related to a pathology of the animal .The measurements and analyzes were not carried out on the excluded animals.This study received the agreement by the French national ethics committee: APAFIS N°16758-201809171143620. All animals received humane care in compliance with the European Union Directive of 2010 (2010/63/EU), and the study was approved by the institutional and regional committees for animal care and followed the ARRIVE guidelines.At the end of the cold storage period, 13 control hearts and 8 ischemic hearts were prepared for the reperfusion step and assessment of cardiac function. 7 control and 4 ischemic hearts were preserved during 4h and 6 control and 4 ischemic hearts were preserved during 20h at 4°C. It should be noted that the heart revived were preserved and harvested in the same conditions than the hearts previously scanned. The grafts were next revived using a perfusion solution at 37.5°C, in practice, a latex balloon was introduced into the left ventricle and secured in the mitral annulus. The balloon was then connected to a pressure transducer and inflated with water to a diastolic left ventricular pressure of 5 mmHg. Hearts were submitted to aortic reperfusion with 600 ml of conserved heparinized whole blood at 38°C. The initial pH of the solution was 7.2-7.4 after equilibrium with a mixture of O2/CO2 (95%-5%) and 20 ml of Na2CO3 at 4.2%. Perfusion pressure was gradually increased from 2.9 KPa (30 cm H2O) at the beginning of reperfusion to 4.9 KPa (50 cm H2O) at 20 min to 30 min. Calcium was added after 20 min of reperfusion to obtain a final mean calcium level of 2.5 ± 0.19 mmol/l. At baseline and after 15, 30, 45 and 60 min of reperfusion, the functional state of the heart was evaluated by measuring Left Ventricular Developed Pressure (LVDP), heart rate (HR), RPP (= LVDP.HR), myocardial contractility and relaxation rate, and end diastolic pressure (EDP).To perform ultrasound evaluation of the cardiac graft a dedicated preservation box minimizing the absorption and the reflection of ultrasound energy was manufactured. This preservation container has a hexagonal shape. Each facet enables longitudinal and transversal scans of the heart allowing a full exploration. The whole box was manufactured by Goodfellow  using a TPX polymer, material used for his low acoustic attenuation and low acoustic impedance mismatch.We first performed a scan of the whole heart, B-mode images from short axis and long axis views were acquired on each side of the preservation box. On each of the faces 91 slices were obtained along long axis view at steps of 1mm and 95 slices along the short axis with the same step. The acquisition is automatic and each face of the box can be entirely scanned in 4 min. Cardiac structures and segments were labelled by a trained cardiologist and 10 segments were defined for SWE assessment. The right ventricle (RV) was imaged using three short axis slices, at basal, middle and the apical levels, and one long axis slice. Left ventricle (LV) was also explored using short axis slices at basal, middle, and apical levels and one long axis slice. Finally, the septum was imaged in short axis and long axis views.SWE maps obtained with Aixplorer system and the linear probe relies on two steps: firstly, a shear wave is remotely generated in the myocardium using acoustic radiation force, and secondly, plane wave images are transmitted at ultrafast frame rate to track the propagation of the shear wave and quantify shear wave velocity (SWV). The relationship between the stiffness measured by the shear modulus and the shear wave velocity can be found bellow:2] the density of the medium and 2 was defined using B-mode images to compute the mean and the standard deviation of the SWV estimates of each segment. We evaluated the myocardial stiffness of the 10 individual segments and defined also a global stiffness as the mean of all the individual values.With µ the shear modulus expressed in Pa, Because the myocardium is composed of fibers, the myocardial elastic properties are expected to be anisotropic. In clinical implementation of shear wave elastography, the medium is assumed to be isotropic. Therefore, to assess shear wave anisotropy, we used a dedicated research system, which was developed in our lab to assess myocardial stiffness in transverse isotropic medium such as skeletal muscle or the myocardium. The approach relies on a dedicated transducer developed in a previous studyIn this study we aimed at evaluating the stiffness of two heart models  and the evolution of myocardial stiffness as a function of time during preservation. Three measurement time points were defined, each heart was preserved during a total time of 20h and the initial stiffness was measured right after the collect of the graft. A second measurement was performed after 4h of preservation. Each time the stiffness was evaluated on each segment of the graft as previously described. A last measurement was performed at 20h of preservation.p < 0.05. We also investigated the correlations between markers of the heart function in post reanimation and SWV values. We used a Pearson correlation assuming that values are sampled from populations that follow a Gaussian distribution and choosing a two-tailed P value. The p-value style was chosen as follow * p < 0.05, ** p < 0.01, *** p < 0.001To estimates differences, between the two studied group, the statistical analysis was performed using GraphPad Prism software . To analyze the stiffness evolution as a function of time, we used an ordinary one-way ANOVA with a Šidák multiple comparison the assumption of normality was verified using a Chi-square normality test due to the small number of samples. The comparison of stiffness between the two groups we performed using a two-way ANOVA design with a Šidák multiple comparison assuming independence between tests. Significance was assumed for n = 6.The sample size of the experiments was decided after performing a priori power test. The analysis was performed using nQuery  a clinical trial design software. The power analysis was performed using a t-test after the first sample of each model to estimate properly the two groups mean value (1.5 m/s / 2.0 m/s) and the common standard deviation (0.2 m/s) was estimated from literature and previous studies. The sample size predicted for a 95% power was found of The funders did not have any role in study design, data collection, data analyzes, interpretation, or writing of report.In order to identify the cardiac structures, we first acquired ultrasound slices of an immerged heart graft. Whole heart images were reconstructed by combining the different slices acquired. N=8) as a function of preservation time. As shown on 0 (SWV = 1.87+/-0.35 m/s), after 4h (SWV = 1.86+/-0.23 m/s) and after 20h (SWV = 3.38+/-0.92 m/s). In yellow the same analysis was performed on the RV at T0 (1.52 +/- 0.37 m/s), after 4h (SWV = 1.50 +/- 0.24 m/s) and after 20h (SWV = 3.32 +/- 0.66 m/s). And finally, in orange, the septum was explored at T0 (SWV = 1.85+/-0.33 m/s), after 4h (SWV = 1.82+/-0.32 m/s) and after 20h (SWV = 3.38+/-0.88 m/s).First, we evaluated the evolution of myocardial stiffness in control hearts (0 defined as the harvesting time (one-way ANOVA with a Šidák multiple comparison). On each segment, no significant differences were observed on the LV (p = 0.392), the RV (p = 0.282) and septal wall (p = 0.334). The myocardial stiffness remained unchanged after 4h of preservation. Nevertheless, a strong stiffness increase was found between 0 and 20h and significant differences were found on the LV (p = 0.020), the RV (p = 0.002) and the septal wall (p = 0.018)The fourth hour of preservation was compared with the SWE value at T0 (SWV = 1.78+/-0.3 m/s), 4h (1.69+/-0.19 m/s) and 20h (3.40+/- 0.75 m/s) representative of a global SWV value of the organ.Measurements performed on each segment have been merged on the right of the figure to have a global evaluation of graft stiffness. A mean SWV value was computed for the three preservation times T0 and after 20h of preservation versus an ischemic graft on the In the same way, we evaluated the stiffness evolution of the ischemic heart model as a function of time. 0, 4h, and 20h of preservation. (two-way ANOVA design with a Šidák multiple comparison) We then compared the SWV between the two models and results are shown with a box plot.The analysis was performed on the 6 ischemic hearts , it alsop=0.211) at baseline, at 4h  and at 20h .In light blue, the SWV of the LV of control group was compared to ischemic group, in dark blue. Ischemic hearts were stiffer  and after four hours of preservation . No significant differences were found after 20h but we noticed a stiffer RV in the ischemic case . Regarding the septal wall, represented in orange on the figure, a difference was found between the two models at each preservation time, after harvest , at 4h  and after 20h The same observations can be done on the two other segments. The right ventricle, in yellow, show differences at T0 , 4h  and 20h  with significant differences at 4h and 20h of preservation time compared to control.With a global evaluation of graft stiffness, mean SWV values were computed at TTo analyze the stiffness alterations more finely, we performed experiments with a custom research system dedicated to the quantification of myocardial stiffness anisotropy. This new probe provides an estimation of the whole shear wave velocity using a multi-directional approach , the myocardial hardening from palpation performed by two operators (r2=0.81) and the relaxation rate (r2=0.81). The other parameters also showed a correlation with SWV values, the Rate Pressure Product (RPP), the product between heart rate and systolic blood pressure used to determinate the graft workload and the End Diastolic Pressure (EDP) have R squared scored of 0.77 and 0.67, respectively.The SWV values correlate well with all the cardiac function parameters as shown in In this study, we assessed the graft stiffness using elastography technique to predict its viability on preclinical situation close to human setting. A dedicated setup for ultrasound imaging and SWE was developed to image whole explanted hearts and assess quantitatively the myocardial stiffness over time.N=14) were assessed in two groups: a control group harvested in normal conditions and an ischemic group with 30 min of in situ warm ischemia before harvesting to mimic a DCD situation. Each model was studied during 20h to evaluate the stiffness alterations due to ischemia and to preservation time. The study was performed at a preservation temperature of 4°C. Finally, we found a strong correlation of the SWV values with cardiac function parameters measured when the hearts were revived. The anatomy and physiology of the porcine heart is close to the human heart. The surgical procedures used were very similar to those used in the clinical setting.Pig heart ((1)an entry of Na+ into the cells (following the reduced activity of cold sensitive Na/K-ATPases). This entry of Na+ is accompanied by a concomitant entry of water, which induces edema and therefore hardening of the tissues.(2)Ca++ entry due to the dysfunction of calcium regulatory pathways. This calcium invasion induces a progressive contracture of the cardiac myofibrils causing an increase in the rigidity of the myocardium. This phenomenon can be activated by the decrease of energy stores , that gradually keep the myofibrils in a contraction state.Myocardial stiffness is a biomarker which reflects the tissue architecture and its content at macroscopic and microscopic levels. However, the precise mechanism of graft deterioration and stiffness alterations during cold ischemic time is not yet elucidated. In deep hypothermia (4°C), heart is non-beating and cardiac metabolism is reduced by more than 95%. The oxygen demand is therefore identically reduced. Essentially an ion imbalance occurs between the intra and extracellular environments, with:These phenomena are intricated and occur globally throughout the myocardium, perhaps more marked in denser cellular areas such as the septum and the left ventricle. We can therefore expect to observe a progressive hardening of the graft with the time of ischemia, correlated with the suffering of the organ. This is precisely what clinicians try to assess qualitatively by manual palpation and what we measure quantitatively by elastography.31in vivo by SWV was significantly altered by age and pathologies such as hypertrophic cardiomyopathy,,Previous studies have shown that myocardial stiffness measured ,Because, the myocardium is composed of fibers locally oriented in a principal direction, the myocardial elastic properties are anisotropic.SWV was found to correlate strongly with the parameters of the cardiac function after reviving the hearts suggesting that SWV could be used as a marker of myocardial viability and a predictive marker of the cardiac function. The normal SWV was around 1.7 m/s, and a myocardial stiffness of about 3.0 m/s and higher was associated with a significant decrease of the cardiac function. Above 4.5m/s the hearts were definitively damaged and could not be restarted.,Various strategies have been proposed to expand the donor poolOne limitation of this study is the small sample size, to determine precise cutoff values, further validations will be required on animal and human hearts including measurements at other preservation times in particular before the 4h of preservation and after the reperfusion of the organ. One should note that shear wave velocity and myocardial stiffness are slightly dependent on preservation temperature (see supplementary data). The acquisition of the entire heart required 20 min of scanning, this duration could be significantly reduced by an optimization of the scan parameters  or by reducing the scan to 2 or 3 box facets.Before considering SWV as a marker of graft viability for clinical transplantation, the study needs to be translated to human grafts to validate the use of SWV as biomarker on real human hearts. SWE measurement needs to become widely available in dedicated devices for transplantation applications. The development of algorithms and precise SWV cut off values will be also required to propose clear guidelines on the use of SWV. Quantification of myocardial properties could be crucial in order to optimize preservation strategies, harvesting technique and finally to expand the number of grafts available for cardiac transplantation. The methodology developed in this work is relatively affordable compared to MRI systems, and can provide noninvasive whole heart scans in a very short time (few minutes), compatible with urgency encountered on clinical practice. Clinical ultrasound devices with shear wave elastography modality are widely available for several manufacturers and therefore, this approach, could be directly translated to the clinics.O.P., C.P., R.F. and M.P. did the concept and study design. L.A., J.L., M.L.G. and R.F. acquired data. O.P., C.P. and M.P. performed data processing. Writing, review, and/or revision of the manuscript: O.P., C.P., R.F. and M.P. All authors reviewed and commented on the manuscript, and approved its final submission. Underlying data have been verified by C.P and M.P.Data that support the findings of this study will be available from the corresponding author of the study.All authors have no conflicts of interest to disclose.


The objective of the present study is to enhance the resistant starch (RS) content of high amylose rice starch with heat–moisture treatment (HMT) for industrial application. The optimized HMT condition for achieving the highest RS content established using response surface methodology (RSM) was a temperature of 100 °C, moisture content of 24.2%, and a time of 11.5 h. Upon HMT, the RS content increased from 32.1% for native starch to 46.4% in HMT starch with optimized condition. HMT of the starches reduced the solubility and swelling power. The surface of HMT starch granules was more irregular than native starch. The X-ray diffraction (XRD) peak intensity at 2θ = 5° was greatly reduced by HMT, and the peaks at 22.7° and 24.2° were merged. HMT increased the gelatinization temperature and reduced the gelatinization enthalpy. HMT provides a method for the production of high-yield RS2 with high amylose rice starch in industrial application. With the interest in healthy eating, consumers are focused on increasing intake of plant-based food products and demanding foods which are natural, organic, less processed, and free from additives. Ingredient manufacturers and processed food companies are striving to meet these trends by offering clean label foods to the market . The cleNative starches are usually chemically modified to improve their functionality before use in processed food formulations because of the limited functionality and application of the native form. However, chemical modification has become less favorable among consumers because by-products of chemical reagents are present in the modified starch. By contrast, the physical treatments of starch have been gaining increased acceptance. The primary interest in physical modification is related to the clean label starches; they can be classified as natural products even though they are processed . In addiDuring HMT of starches, structural changes occur in both the crystalline and amorphous regions. The X-ray diffraction (XRD) pattern of starches with A-type crystallinity is not altered by HMT, while starches with B-type patterns exhibit a change to C-type (A + B) or A-type with an accompanying decrease in the XRD peak intensities ,5. HMT iStarch in foods can be classified as rapidly digestible starch, slowly digestible starch, and resistant starch (RS), according to its degree and speed of digestion. The RS is a fraction of the starch that is not digested in the small intestine of healthy individuals and enters the colon unchanged, where fermentation by resident microorganisms occurs, resulting in the formation of short-chain fatty acids . RS is cMost studies on high amylose rice starch have involved the structural and physicochemical properties of rice starch ,15 and eA series of experiments were statistically designed using RSM with a Box–Behnken design to establish the stable operating point of the HMT conditions that increased the RS level of high-amylose rice starch. A regression equation was used to describe the correlations between input parameters  and target parameter (RS). Based on the experimental design, three levels of each parameter  were studied. The RS values generated under the experimental conditions are shown in The RS content increased as the moisture content increased under the same temperature and time . PreviouA), moisture content (B), and time (C), as represented by Equation (1):The variables and responses had a certain relationship. One variable was fixed to visualize these relationships, and the changes in RS level according to the alteration of the other two variables were described by the contour plots . The con2) of the model. The R2 value was 0.7929, indicating that the fit between the model and the experimental data was adequate.The calculated optimal RS value was 46.4% and best potential processing condition was temperature of 100 °C, moisture content of 24.2%, and reaction time of 11 h 33 min. However, the predicted RS level of 46.4% was lower than experimental data. Analysis of variance (ANOVA) was used to evaluate the regression coefficient (RThe solubility and swelling power of heat–moisture treated starches are presented in SEM images of native and treated (HMT) rice starch granules are presented in Following HMT, the surface of the granules was more irregular, and the granules were more aggregated compared with the native starches. It is assumed that the loss of the physical integrity of the starch granule with distension of the granular surface was caused by a partial gelatinization .The XRD patterns of native and HMT rice starches are displayed in oT), peak (pT), and conclusion (cT) temperatures of GS was 59.6, 66.5 and 74.1 °C, respectively (cTo–T) of native starch was 14.6 °C, which was expanded for heat–moisture treated starches, except for HGS100-21-16. HMT decreased the gelatinization enthalpy (ΔH) of high-amylose rice starches. The enthalpy of native starch was 12.5 J/g and those of heat–moisture treated starches were 4.2–9.9 J/g. Previous studies also reported that the gelatinization temperature of heat–moisture treated starch was much higher than that of native starch, and ΔH was decreased during HMT [The gelatinization properties of the HMT rice starches were determined by differential scanning calorimetry (DSC). The onset  was provided by Rural Development Administration . The rice starch was isolated from the grain using an alkaline (0.2%) steeping method [The high amylose rice variety .The RSM was used to acquire the highest RS value by optimizing the HMT process, and the Box–Behnken design was used to understand the effects of reaction temperature, moisture content, and time on the RS level of rice starch. The HMT temperature, moisture content, and time were selected as independent variable, and RS level of high amylose rice starch was the dependent variable during HMT. Three levels of each factor were selected from the maximum and minimum values of the preliminary experiment conducted within the generally known HMT conditions. The HMT temperature range is above the gelatinization temperature, and low physical processing temperature are expected to reduce the effect of HMT due to small variations in physicochemical properties, so the temperature of the experiments was set at 100 °C or more. The three different levels for each factor were as follows: temperature, 100, 110 and 120 °C; moisture content, 15, 21, and 27%; time, 0, 8, and 16 h. The design consisted of 14 treatments in which the central point was replicated twice to calculate the experimental error. The results were calculated from nine experimental groups because there was no real data for the 0 h-treated group. A quadratic model with 10 coefficients was used to investigate the response of the RS value by Equation (2):After measuring the moisture content of raw rice starch, starch samples (20 g) were weighed into the screw cap glass container (100 mL). The starch was adjusted to 15, 21, and 27% moisture content by adding the appropriate amount of distilled water. The samples were mixed thoroughly to breakdown clumps. The containers were sealed, kept at ambient temperature for 24 h to equilibrate the moisture in starch, then placed in a forced air oven  at 100, 110, and 120 °C. The samples were thermally treated for 8 and 16 h at each temperature. Afterwards the containers were opened, and the starch samples were air-dried until moisture content reached around 10%. The samples were ground and passed through a 120 mesh (<125 mm) sieve, and stored in a desiccator until they were used.g for 10 min. The supernatant (2.7 mL) was transferred to a beaker and mixed with 0.3 mL of diluted amyloglucosidase  and 2 mg of invertase . This enzyme solution was freshly prepared for each digestion. Starch (100 mg) and 14 glass beads  were added into glass test tubes. The HCl/guar solution (2 mL) was added to each tube and mixed immediately on a vortex mixer. Then, 4 mL of 0.5 M sodium acetate buffer (pH 5.2) and 1 mL of enzyme solution was added to each test tube and incubated in a shaking water bath . Aliquots (100 μL) were taken and mixed with 1.5 mL of 50% ethanol. To measure the hydrolyzed glucose content, the Megazyme GOPOD assay kit  was used. RS was determined as the undigested fraction after 120 min of hydrolysis.The RS content was determined using the method of Englyst et al.  with ming for 30 min. The supernatant liquid was removed, and starch sediment in the tube was weighed. The supernatant was dried to constant weight in a drying oven  at 105 °C. The swelling power and solubility were calculated by the equations described by Schoch [Swelling power and solubility were determined at 80 °C by a modified method of Schoch . Starch y Schoch . The sweThe morphology of starches was characterized using a scanning electron microscope . The accelerating voltage was 15 kV for 85 s, and the magnification was 2000×. The starch samples dried in a desiccator were placed on an SEM stub with double-sided cellophane tape and coated with gold/platinum to increase the conductivity.A high-resolution three-dimensional X-ray diffractometer  was used to investigate the crystalline structure of the native and HMT starches. The diffractometer was operated at a 40 kV target voltage and a 34 mA target current with a scanning range of 5–40° (2θ) and a scanning rate of 2.0°/min.oT), peak (pT), conclusion (cT) gelatinization temperatures, and gelatinization enthalpy (ΔH) were determined from the DSC thermograms.The thermal properties of native and HMT starches were determined using a differential scanning calorimeter . A starch sample  and 14 mg of distilled water were loaded into aluminum pan and hermetically sealed. The pans were kept overnight at room temperature. For the analysis, the pans were heated from 5 to 180 °C at a heating rate of 10 °C/min. An empty pan was used as a reference. The onset (p < 0.05) using SPSS Statistics .All data were reported as mean values and standard deviations. Differences between factors were evaluated using one-way analysis of variance (ANOVA) and statistical analyses were carried out with Duncan’s multiple range test (To improve the industrial applicability of high amylose rice starch, RS content was increased through HMT and the physicochemical properties of HMT starches were investigated. This study clearly showed that optimal HMT condition for enhancing RS content was established using RSM. HMT leads to an increase in crystalline perfection due to strengthening the bonds between amylose molecules and between amylose and amylopectin of starch granules because the solubility and swelling power of the starch granules decreased, and the gelatinization temperature increased during HMT. Our results could provide useful information for establishing the HMT conditions of high amylose rice starch for the production of high-RS food in industrial application of rice materials.