Sodium chloride (NaCl), commonly known as salt, is a substance that is utilized in a variety of businesses, including the tourism and construction industries. Therefore, the main purpose of this article is to accommodate a salt-based building material called NaCl-binder for tourist and industrial applications. By utilizing salt mortar with varying grain sizes, food-grade corn starch as an exclusive binder agent (without using any non-starch binder), and water under microwave-cured conditions, environmentally friendly hydrophobic hybrid NaCl-binder samples with low bulk density were successfully produced. The fabrication of these samples involved an inventive utilization of small quantities of starch. This study evaluated the impact of microwave exposure time on the strength of salt samples, particle interconnectivity and chemical composition using SEM, XRD, and XRF analyses. The compressive strength of the samples showed a remarkable increase, with a 600% improvement when using 0 to 1% corn starch, and a 137% increment when using 1 to 10% corn starch, indicating a lower rate of increment with higher starch consumption. A key aspect of this research is the significant reduction in starch consumption compared to other corn starch-based materials during the manufacturing process of the incorporated materials, highlighting its novelty and importance. Because of this, research indicates that salt therapy significantly boosts tourism growth3.Salt therapy is the use of small particles of salt aerosols and mineral compounds to cure respiratory disorders, skin discomforts, and joint-bone problems, which has been employed in European salt mines since the nineteenth century. This therapy approach is non-invasive and physical, does not require medicines, and is safe. This method has a positive impact on therapy and drug reduction4. As the Egyptians did in the twelfth century, they used blocks of salt from the Salt Lake to construct buildings, although such constructions are still carried out by old masons5. The world is full of examples of these endeavors. It can be mentioned as an example of a salt restaurant that was built in Shiraz by Emtiaz Architectural Group using salt materials and a kind of natural resin, and its salt is provided from a salt lake near the city6. Palacio de Sal, a unique salt hotel in Bolivia, utilizes blocks mined from the salt lake, annually attracts foreign tourists and generates substantial income for the hotel investors7. In Qatar, the plan of using salt obtained from the desalination of seawater using solar energy has been studied for different constructions8. Technological projects like 3D printing using salt materials have also been studied by researchers11. A type of sustainable and innovative building material for use in hot, arid climates called SaltBlock has been introduced, which uses raw materials such as ethylene–vinyl acetate polymer, salt powder, and fine sand to produce lightweight blocks11.Nowadays, the construction of tourist complexes and villages has a high potential to attract domestic and foreign tourists and it is possible to use the potential of the salt deposited on the beaches to build complexes, parks  respecting scientific and ecological principles17. In the production of bricks and natural salt materials, the grinding of salt stones, which are extracted from the bottom of salt lakes or salt mines, could be used. Due to the brittleness of salt rock, the production of salt materials with a regular geometric shape is very expensive. Thus, by using salt particles as a mortar component it can be converted into salt building materials by in situ molding, pressing, or 3D printing18. In the phase of laboratory studies related to the Qatar salt project plan, salt powder, maltodextrin, isopropyl alcohol, water, and microwave processing were used to prepare salt samples19. The current research aims to find cost-effective alternatives for producing natural salt samples. This involves substituting high-cost maltodextrin with low-cost corn starch (starch gel) and eliminating the use of isopropyl alcohol. The goal is to make the production process more affordable and environmentally friendly. In the past decades, the production of biopolymers and the development of biodegradable and bioplastic materials from renewable sources such as starch and cellulose products have been the focus of research, and the use of these materials instead of synthetic polymers from non-renewable sources such as oil has been of great importance to reduce the environmental impact24.Nowadays, caves and salt-therapy complexes, as well as mud-therapy complexes, attract tourists from all parts of the world and even some modern swimming pools are equipped with salt rooms for halotherapy25. The adhesion mechanism of sand particles in this building material is caused by the gelatinization of starch particles, which binds coarse and fine sand particles together, forming a composite structure with the ability to harden quickly. Also, in further complementary research, the construction of sand-starch materials by microwave curing was further investigated and the use of conventional and natural coating materials for waterproofing the samples was also studied in detail26. Based on the results of these investigations, various factors influence the compressive strength of starch-based building products. Among these factors, the water content, the size distribution and composition of the grains, the temperature and duration of the hardening, the starch gelatinization process, and the binding of the starch matrix with sand grains can be mentioned26.In recent years, researchers at Delft University of Technology have introduced a new biodegradable building material called CoRncrete, made by curing corn starch mortar with sand and water in a microwave. The production of these innovative and environmentally friendly materials is economically viable and has acceptable mechanical strength compared to ordinary bricks25 and starch-sandstone26 is the use of corn starch to produce environmentally friendly building materials without using non-ecofriendly types of cement such as Portland cement. Starch is a suitable natural polymer for the production of biodegradable materials and building materials due to its advantages such as renewability, abundance, low cost, and biodegradability. The wide use of these materials as adhesives to improve the mechanical, rheological, thermal, insulating, and other physical properties of various materials such as cement, concrete, asphalt, mortar, etc. has been of interest to researchers31. For example, starch is used as an adhesive in thermal insulation composites36, as an additive to modify the viscosity of concrete43, as an asphalt modifier44, and as a retarder in cement45. It has also been reported that the use of 1% corn starch in the concrete composition increases the compressive strength of the samples46.The common point of the starchy NaCl-binder material presented in the current study with the investigations carried out at CoRncreteThe current research investigated the fabrication of NaCl-binder samples for the first time and in an innovative way using salt mortar with salt particles, and a small amount of corn starch and water under microwave curing condition. In which compared to CoRncrete, salt particles were used instead of sand particles in the manufacture of the samples. In general, corn starch at 0, 1, 5, and 10% by weight was used to prepare the samples. Corn starch in an amount of 1% (wt.) was mainly used in the form of a gel due to the proper distribution of the starch between the particles. Various parameters such as the granulation of the salt particles, curing time, and the type of starch were examined in relation to the mechanical strength , microstructure, and hydrophobic characteristics of NaCl-binder samples. Additionally, these samples were the first to successfully address the issue of samples' water solubility by hybrid paraffin wax and beeswax coating. The difference in the behavior of salt and sand particles, which leads to the possibility of significantly reducing starch consumption in the manufacture of introduced material compared to CoRncrete, is one of the important points of this research. Subsequently, the hydrophobicity of the samples was also examined with beeswax and a paraffin coating. In this study, the introduced NaCl-binder materials composed of salt particles (powder or aggregates) and corn starch (gel containing starch and water) can be used to produce lightweight salt materials without the need for pressure in the preparation stage (only by impact or vibration to remove excess air from the mortar).In the sample preparation, the waste salt as one of the raw materials was passed through the standard sieves, and the salt particles with four different sizes were obtained  at a temperature of 100 °C. Then, by increasing the speed of the stirrer to 150 rpm, the contents inside the Erlenmeyer flask were stirred for a further 10 min until the color of the resulting gel changed from white to milky. To increase the viscosity, gelatinized starch was stirred for an additional 5 min at 150–350 rpm at 150 °C, resulting in a whiter and more viscous gel. In the end, to cool the gel, the heat source of the stirrer (hot plate) was turned off, and the contents of the flask were stirred for a further 3 min at a speed of 150 rpm at room temperature. In the end, after measuring the weight, it was found that with the evaporation of water, the weight of the produced gel decreased by 300 g (280 g water & 20 g corn starch).In continue, Glucosan edible corn starch was used to prepare NaCl-binder samples, in which corn starch has been used in three forms: starch powder, starch suspension, and gelatinized starch to make salt mortar. First, the starch powder was mixed with salt particles, and then water was added. Secondly, the starch was added to the salt particles after stirring for 5 min at laboratory temperature. In this phase, the starch particles are insoluble in cold water (laboratory temperature), and not only a gelatinous state does not form, but also the starch particles become suspended in water. Thirdly, starch was added to water and converted to gelatinous starch by heating on a magnetic hot plate stirrer, and then salt particles were added. In all phases, tap water was used. When starch granules are heated in the presence of water, they swell and a phase transition occurs. With enough water, this transformation, known as gelatinization, causes the starch particles to burst and dissolve in water25. Therefore, the treatment time of the samples in the microwave is of considerable importance. As will be mentioned in the results, during the tests, it was observed that the curing time has a significant impact on the mechanical strength of the samples. Therefore, the effect of changing this parameter in the samples prepared with S2 salt was studied in detail. For all samples made from gelatinized starch, the weight of starch and water in the starch gel is reported separately.The significance of the current research lies in the use of starch in a very low weight (1% wt.) to produce NaCl-binder samples with good mechanical strength. Low starch consumption in the production of cost-effective salt-building materials is very important from an economic point of view. In addition, samples (without starch) were prepared as controls to evaluate the effect of not using 1% starch. Samples containing 5 and 10% starch were also prepared to compare the effect of changing the weight of starch used in the materials. The mixture design of cubic and prismatic NaCl-binder samples (without hydrophobic coating) for compressive strength and flexural strength evaluation is according to Table According to lines 1 to 10 of Table 25. It should be noted that due to the importance of the color of the samples, any oiling of the molds was avoided in this study. To prevent the salt mortar from sticking to the mold, to keep the samples intact, and to remove them safely from the mold, a binding cover was installed into the mold, allowing the NaCl-binder samples to be easily pulled out of the mold.To prepare NaCl-binder samples, a uniform salt mortar was prepared by mixing salt particles with starch gel or starch powder and water for 5 min at 50 rpm using an electric mixer (Sepehr Arya Azma-LM3). The weight ratio of starch to salt in all samples containing starch (powder or gel) equaled 1%. To produce NaCl-binder cube samples, three layers of the prepared salt mortar were poured into 5 × 5 × 5 cube molds  and cured in the microwave. At this stage, after pouring each layer of salt, each layer was tamped 10 times with a silicone rubber-capped steel rod3 and the volume of one prismatic sample is 256 cm3, to make similar samples in terms of energy absorption in the microwave; a 125 cm3 cubic sample was placed next to each prismatic sample to make the volume reach 381 cm3 (only slightly more than the 375 cm3 volume of the three cubic samples). Due to the lower weight of the starch used in the samples, the samples did not swell after microwave curing and therefore, overhead loading of the samples was not required. After removing the samples from the microwave and lowering their temperature to laboratory temperature (25 °C), the dimensions and mass of the samples were measured to calculate the bulk density, and then their compressive and flexural strengths were also measured.A 16 × 4 × 4 aluminum mold was used to make the prismatic samples, and a cellophane layer was used inside the mold to prevent the salt mortar from sticking to the aluminum mold. Salt mortar was poured into a prismatic mold in three layers and pounded. Then, using a cellophane layer, the samples were removed from the mold and transferred to the microwave . Three cubic samples or one prismatic sample from each test design were placed in a microwave with a power level of 900 W and processed for a specified time according to Table An overview of the process of fabricating and curing NaCl-binder samples with and without starch and the evaluation of their mechanical strength are shown in Fig. 26. As a result, in this study, samples of 1% starchy NaCl-binder were made using S2 salt, microwave cured for 7 min, and coated with paraffin wax and beeswax to assess their hydrophobic qualities for the first time. Then, the hydrophobic properties of the surface of the samples were qualitatively analyzed by measuring the contact angle of the samples with distilled water using the Jikan CAG-20 analyzer as a static contact angle analysis tool.The use of different materials to coat sand-starch samples was reported by Mansour et al., and according to the results, it was found that the use of paraffin wax and beeswax  leads to the formation of the highest contact angles50. A flexure testing machine with a 3-point loading method by ASTM C348-2151 was used to measure flexural strength. The X-ray diffraction (XRD) analysis of the corn starch powder, 0% (wt.) starchy NaCl-binder powder and 1% (wt.) starchy NaCl-binder samples in the angle range (2θ) from 10–80° was also recorded with an Xpert Pro diffractometer  with a Cu anode at room temperature with a step size of 0.05°, 40 kV and 30 mA. To evaluate the chemical composition of the samples, in continue, salt (Type1 and Type2), corn starch powder, and 1% starchy NaCl-binder samples cured for 4 and 7 min in the microwave was determined by an X-ray fluorescence (XRF) spectrometer . The target samples are exposed to X-rays during the XRF test, and secondary X-rays are generated as a result of the excitation of atoms. Afterward, the necessary element or elements can be found using Energy-dispersive X-ray spectroscopy (EDS). The microscopic morphology of the rock salt and 1% starchy NaCl-binder samples cured for 7 min were investigated at room temperature and 15 kV using MIRA3-FESEM .To measure the mechanical strength of NaCl-binder samples, a concrete compression testing machine was used to measure the compressive strength of the samples by ASTM C39In this study, the production of NaCl-binder samples was done using extremely small amounts of starch (1 wt.%), especially in gel form, which is considered innovative. The issue of the samples' solubility in water is also resolved by coating the samples with paraffin wax and beeswax. In addition, desirable compressive strength was attained despite small corn starch consumption.20. For the NaCl-binder samples with 1% starch, by increasing the curing time from 10 min, the starch is gradually burned, and its compressive strength tends towards the samples without starch, and the binding effect of the starch is lost. The color of samples without starch is white for different curing durations, while the color change of samples containing 1% starch with an increase in curing time, which leads to the burning of starch, is evident as shown in Fig. In this part, the possibility of the economical production of NaCl-binder with low corn starch consumption (1 wt.%) with the lowest energy was evaluated. The histogram of the compressive strength of samples without starch and with 1% starch, cured in the microwave  is presented according to the diagram in Fig. −3, which is due to the complete evaporation of the physical water in around 7 min. By increasing the curing time, there is no physical water in the sample to evaporate and therefore the bulk density of the samples remains almost constant during the curing time of 7 to 16 min.Figure The compressive strength test results and Fig. Figure Table 8. In which, salt, maltodextrin, water, and alcohol were used, and the weight ratio of maltodextrin to salt was 12.5%, and the compressive strength and bulk density of the manufactured cubic samples were reported 2.99–7.57 MPa and 1.48–10.3 g cm−3, respectively. In the current research, by using S2 salt (300–600 μm), while removing alcohol from raw materials and substituting cheaper food-grade corn starch instead of maltodextrin, it is possible to make salt samples with a very low weight ratio of starch to salt of 1%. The average compressive strength and bulk density of the produced cubic samples were 8.13 MPa and 13.13 kN m−3 (1.34 g cm−3), respectively. Whereas, with increasing starch consumption, the compressive strength was also reached (discussed in the next parts). Another study presented a type of sustainable and new building material for use in hot arid climates called SaltBlock, which used raw materials such as ethylene–vinyl acetate polymer, salt powder, and fine sand to produce lightweight salt blocks11. In salt construction materials such as salt-rich mud mortar and salt mixed with natural resin, the mechanical strength and physical characteristics of the materials have not been evaluated7.Furthermore, The results of this research differ significantly from those of other studies on salt materials, which supports the superior outcomes and innovation of the present study so that in the salt project related to Qatar, where the feasibility of making salty materials using salt extracted from seawater desalination using solar energy has been studied and mechanical strength and physical properties of the materials have been investigatedAggregate grading is an essential key parameter of the compressive strength of different mortar samples. In the following section, samples with and without starch (0 and 1% starchy NaCl-binder) were prepared and tested with an optimal curing time of 7 min to investigate the impact of variations in salt grain size  on compressive strength and flexural strength. According to Fig. As a result, S3 is the most suitable salt size out of the various salt sizes, and samples from S1, S2, and S4 all fall into the same strength range. Furthermore, according to Fig. 26. Additionally, unlike sand particles, salt particles of NaCl-binder can bind to one another in the presence of water and provide a favorable strength.In addition to the above, studies on CoRncrete and starch-sandstone have confirmed the same issue. Because the use of 1% starch is insufficient to bind the sand particles together, much higher amounts of starch were used in the studies to achieve the desired mechanical strength−3, respectively. In the current research, by removing alcohol from raw materials and substituting cheaper food-grade corn starch instead of maltodextrin, and using a very low weight ratio of starch to salt of 1%, NaCl-binder material is introduced. In the following, the effect of using higher amounts of starch was evaluated.As mentioned, in the salt project related to Qatar, salt, maltodextrin, water, and alcohol were used, and the weight ratio of maltodextrin to salt was 12.5%, and the compressive strength and bulk density of the manufactured cubic samples were reported 2.99–7.57 MPa and 1.48–10.3 g cmThe compressive strength of samples made with different amounts of starch  is shown in Fig. 52. The NaCl-binder samples with 1% and 5% corn starch conform to Grade NW and samples with 10% corn starch conform to Grade SW according to ASTM C62-17 in terms of load capacity53.The compressive strength of the NaCl-binder samples can be addressed in relation to the findings of the clay brick (unfired) test and the corresponding standardsAn experiment using three different mix designs were conducted to investigate the impact of the production methods on the compressive strength of the NaCl-binder Obtaining the optimal curing time of 7 min in the microwave to produce 1% starchy NaCl-binder samples with maximum compressive strength and white color without burning.Achieving the optimal curing time of 14 min in the microwave to produce 0% starchy NaCl-binder samples with the maximum compressive strength possible.Manufacturing 10% starchy NaCl-binder materials with salt matrix and co-binder of corn starch with a higher compressive strength of 19.52 MPa and hence higher cost by using more starchSuccessful hydrophobization of samples made with hybrid ecofriendly-based material, a mixture of paraffin wax and beeswax.The important results of this research include the following:These new materials can be a candidate for the materials of salt buildings and other structures along with increasing tourism. By using the salt obtained from the desalination of seawater using solar energy, it can be used to produce salt materials, which have dual environmental benefits. On the one hand, eco-compatible salt materials can be used for the construction of salt buildings and tourism development without the use of polluting materials and types of cement, and on the other hand, by preventing the discharge of separated salt water to the sea, related environmental problems are solved.


The aim of this study is to describe and validate a simple and reliable method to pre-operatively predict the size of the ACL graft in the double strand technique with autologous semitendinosus–gracilis tendons on the same MRI used for ACL rupture diagnosis.The study included 92 patients, with a median age of 31 years (IQR 26–41 years), 73/92 (79%) of whom were males. All patients that underwent an ACL reconstruction with doubled ST + GT between 2017 and 2022 were counted in the study.κ = 0.797 which corresponds to a level of agreement defined as “Strong”. Tendon sizes calculated on pre-operative MRI were evaluated both with intra-observer and inter-observer reliability demonstrating a statistically reproducible method. The predicted graft was then compared to the reported one with a statistically significant reliability found.Overall, the median predicted graft diameter from MR imaging was similar to the actual graft diameter with no significant differences (n.s.). Regarding the comparison between predicted and actual graft size, concordance was 78/92 , with This study can help the surgeons to perform a fast pre-operative planning of an ACL reconstruction for graft selection. If the planned graft with ST and GT is smaller than 8 mm, the clinician can decide to switch to a different type of graft or plan a different graft preparing technique and, therefore, reduce the risk of post-operative ligament re-rupture. The method proposed is reliable and reproducible. The major strength of the planning technique proposed is that it relies on data that are already available for the clinician before surgery, without the need of further analysis.IV. ACL reconstruction (ACLR) is, therefore, one of the most common procedures performed in orthopaedic surgery .Different grafts are used for the ACLR, mainly autologous tissues such as Hamstrings tendons (semitendinosus and/or gracilis), bone–patellar tendon–bone or quadriceps tendon , 16, 22.The standard procedure for preparing a hamstrings tendon (HT) graft require to flip both tendons in half obtaining a four-bundle graft. This technique, although easy and fast, has one major downside: the size of the graft cannot be decided by the surgeon because ST and GT are harvested as one single structure , 34.The graft diameter is patient dependent and may vary due to the individual biometric characteristics of the patient  , 18, 23.The overall ACLR re-rupture rate in literature ranges from 3% up to 25% and is around 8% at 2 years post-op , 33, 34.One of the risk factors for ACL re-rupture is an insufficient graft diameter, leading to a weaker neo-ligament. Different studies in literature have shown that an increased risk of re-rupture is statistically related to an ACL reconstructed with a graft with a diameter of 7 mm or less , 32, 33.The necessity to have a graft large enough led to the development of an alternative technique for graft preparation or pre-operative planning.Knowing pre-operatively the size of the planned graft could help the surgeon to different graft choices or different graft preparation techniques to maximise the outcome of the reconstruction.Most known procedures at the present time for pre-operative planning require techniques that, even if accurate, are impractical and difficult to apply in a clinical field , 20, 25.The aim of the study is to propose and validate a simple and reliable method to pre-operatively predict the size of the ACL graft in the double strand technique with autologous ST–GT tendons.The technique proposed is meant to be performed on the same MRI used for ACL rupture diagnosis, therefore making it available in most clinical settings.The hypothesis of this study is that the propose method for ACL graft diameter prediction is reliable and reproducible.The Institutional Review Board approved the study protocol .The study included 92 patients, with a median age of 31 years (IQR 26–41 years), most of the patients was males (73/92 or 79%).All patients that underwent an ACL reconstruction with doubled ST + GT between 2017 and 2022 were included in the study.Only patients who had known final diameter of the graft were selected. For all those patients, the pre-operative MRI was researched. Amongst those patients, only those that had an MRI performed during the investigation centre were considered eligible for the study. All MRI analysed were performed with the same 1.5 T machine and the same imaging reconstruction protocol. The measurements were performed by two independent researchers at time 0 and then repeated after 1 month to assess the inter- and intra-observer reliability of the proposed pre-operative planning method. All measures were performed using the same programme (Impax Client).The exclusion criteria for the study were as follows: neoplastic disease around the knee; radiological evidence of surgical procedures in the affected limb; any synthetic material in the affected limb; age less than 18 years old; open physis and past fractures around the knee.The collected data for the study were: initials; age at the moment of the MRI; gender; affected side; date of the surgical procedure; graft diameter as reported on the surgical report; major diameter of ST; minor diameter of ST; major diameter of GT; minor diameter of GT.From the collected data, for each patient, the mean diameters of both the ST and GT and pre-operative graft diameter predicted were calculated.The pre-operative predicted graft size and the intraoperative graft size reported were then confronted.The intra- and inter-observer reliability was then calculated between the major and minor diameter of the tendon measured by the two researchers at time 0 and 1 month after.The predicted graft size was then compared with the researchers to assess if, even if there were differences in the measurements of the single tendons diameters, the overall result of the predicted graft size was affected or not.The measures were performed in the pre-operative MRI. Transverse cuts in T2 fat sat, PD or STIR protocols were evaluated. One slice for each patient was used for the measurement. The image slice where the epicondyles were more represented was selected. After this first selection, the slices above and below were also evaluated to select the one where the ST and GT tendons were more round shaped Fig. .Fig. 1TrD) and minor (d) diameters were measured in mm . The measurement of tendons was carried out with one decimal place taken into account due to limitations in the measurement programme. However, the mathematical formula used later was adjusted to include two decimal places, enabling a more precise calculation of the mean tendon diameter (D + d/2) and consequently leading to a more accurate prediction of graft size /2). Then the mean diameter of ST and GT was added, to obtain the predicted MRI graft diameter. The final measure obtained was then rounded up by excess to the next full millimetre. The rounding was performed to the higher full diameter because, intraoperatively, the graft can fit through a bigger tunnel and not in a smaller one.For each tendon, the mean diameter was calculated  when quantitative, and as counts and percentages when qualitative. Differences between quantitative data distributions were evaluated with the Wilcoxon test. For both ST, GT and overall graft size, intra- and inter-reader reproducibility were appraised via Bland–Altman analyses and reported as bias and coefficient of repeatability (CoR). The agreement between predicted and actual graft size was assessed with raw concordance, 95% confidence intervals (CI) and linearly weighted Cohen’s ordingly .p values < 0.05 were chosen as a threshold for statistical significance.Statistical analyses were conducted using Python 3.7.6, and No sample size was calculated for the study, but rather all consecutive patients referred for pre-operative magnetic resonance imaging (MRI) at the research centre before anterior cruciate ligament reconstruction between 2017 and 2022 was retrospectively retrieved.Considering the entire patient group, in 48/92 (52%) patients, the left ACL was scheduled for reconstruction, whereas in 44/92 (48%) of patients, the right ACL was considered.Overall, the median predicted graft diameter from MR imaging was 7.5 mm (IQR 6.89–7.90 mm), whilst the actual graft diameter was 8.0 mm (IQR 7.00–8.00 mm), with no significant differences (n.s.).Concerning intra-reader reproducibility, ST diameter displayed a bias of 0.00 mm and a CoR of 0.07 mm, whilst GT diameter displayed a bias of 0.00 mm and a CoR of 0.06 mm. Bland–Altman plots for intra-reader reproducibility are reported in Fig. Concerning inter-reader reproducibility, ST diameter displayed a bias of 0.00 mm and a CoR of 0.09 mm, whilst GT diameter displayed a bias of − 0.01 mm and a CoR of 0.11 mm. Bland–Altman plots for intra-reader reproducibility are reported in Fig. κ = 0.797 which corresponds to a level of agreement defined as “Strong”. In 8/92 cases , the predicted graft size was smaller than the actual graft size, whilst in the remaining 6/92 cases , the predicted graft size was greater than the actual graft size. The distributions of predicted and actual graft sizes are reported in Fig. Regarding the comparison between predicted and actual graft size, concordance was 78/92 , with Concerning cases with a predicted graft size smaller than the actual graft size, the difference was 1 mm in all instances, with 5/8 (63%) cases with a predicted diameter (after rounding) of 7.00 mm versus an actual diameter of 8.00 mm, and 3/8 (37%) cases with a predicted diameter (after rounding) of 8.00 mm versus an actual diameter of 9.00 mm. In all these cases, patients were males with a median age of 27 years (IQR 24–30 years), whilst the ACL lesion was on the left side in 5/8 (63%) cases and on the right side in 3/8 (37%) cases, with no significant differences (n.s.).The main finding of this study is that the proposed method for pre-operative evaluation of expected graft diameter in ACL reconstruction with ST–GT is reliable and reproducible.In ACL reconstruction surgery, the importance of surgical planning is supported by the evidence that up to 8.2% of ACLR end in graft failure , 29, 34.Multiple causes could lead to an ACL re-rupture including non-compliance to the rehabilitation protocol, traumas, patients’ selection, or surgical error.One of the risk factors reported in literature for neo-ACL rupture is the reconstruction with a graft of insufficient diameter. Eight millimetre is reported as the threshold under which the risk of ACL re-rupture is statistically higher , 17, 29.Amongst the graft that the surgeon can decide to use, ST and GT have one major disadvantage over the others: the size of the graft is patient dependent and cannot be selected by the surgeon.Different studies are present in the literature showing the possibility of successful graft size prediction using analysis of MRI images of the knee both in children , 31 and Nearly every one of them succeeded, supporting the theory that a pre-operative planning is possible.The previously reported techniques, even if accurate, are complex to perform, requesting elaborated mathematical calculations, and therefore fail to present a clinically feasible planning method.Furthermore, most of the proposed systems have not been validated by the inter- and intra-observer reliability.Other studies tried different imaging techniques or planning performed on anthropometric parameters with poorer results , 24, 31.These studies require a pre-operative planning based on exams that are not necessary for the diagnosis nor the treatment of a patient with an ACL rupture, therefore representing a waste of time and resources either for the patient or the national health system.The authors’ idea was that all patients with a diagnosis of ACL rupture must have performed at least one MRI; therefore, this was the imaging method that the authors choose for the planning.The evaluation method proposed has been found to be reliable and reproducible.This method, although reliable, presents two main limitations.The first limitation is that the study has been performed with strict inclusion criteria that allowed only the selection of patients that performed the knee MRI at the research centre, with the same machine, the same image reconstruction protocol and the same measuring programme (Impax Client). More studies performed in different centres with different programmes should be performed to confirm the effectiveness of the proposed pre-operative planning method.The authors selected for this study a measuring programme that allowed for millimetre decimals (e.g. 0.1 mm). The measures of the tendons calculated are small, and a programme that automatically rounds one decimal, instead all mathematical calculations rounded to two decimals.The second limitation is that the study was retrospectively designed; therefore, it was not possible to directly evaluate the graft size reported in the surgical report. There is one patient in the study database that has a 2-mm difference between the predicted size (9 mm) and the reported size (7 mm).The main strength of this study is that the proposed method is highly reproducible, as confirmed by the statistically significant inter- and intra-observer reliability.The results of this study could help the clinicians to perform a fast pre-operative planning of an ACL reconstruction regarding graft selection. If the planned graft with ST and GT is smaller than 8 mm, the clinician could decide to switch to a different type of graft or plan a different graft preparing technique and, therefore, reduce the risk of post-operative ligament re-rupture.The method proposed is reliable and reproducible. The major strength of the planning technique proposed is that it relies on data that are already available for the clinician before surgery, without the need for further analysis or complex mathematical formulas. This method will help the clinician to select the graft or the graft preparing technique before surgery.


Panicum miliaceum L.) starch. Thus, this study examined the physicochemical characteristics of starch in two types of broomcorn millet (waxy and non-waxy) under four different conditions, including a control group (N0), nitrogen fertilizer treatment alone (N150), biochar treatment alone (N0+B), and a combination of biochar and nitrogen fertilizer treatments (N150+B). The results showed that, in comparison to the control, all the treatments, particularly N150+B, decreased the content of amylose and gelatinization temperature and enhanced the starch transparency gel consistency and swelling power. In addition, biochar can improve the water solubility of starch and the gelatinization enthalpy. Importantly, the combination of biochar and nitrogen fertilizer increased the proportion of A-granules, final viscosity, starch content, and the average degree of amylopectin in polymerization. Thus, this research indicates that the combinations of biochar and nitrogen fertilizer result in the most significant improvement in the quality of starch produced from broomcorn millet.The overuse of nitrogen fertilizers has led to environmental pollution, which has prompted the widespread adoption of biochar as a soil conditioner in agricultural production. To date, there has been a lack of research on the effects of biochar and its combination with nitrogen fertilizer on the quality of broomcorn millet ( Panicum miliaceum L.), commonly known as yellow rice, is the oldest crop in the world  Lam.). N150+B did not have any notable impact on SM1, whereas it significantly elevated the overall starch content in SM2 compared with N0, which resulted in a 3.19% increase. An ANOVA suggested that the treatments and varieties had a significant influence on the total starch. The amylose contents of waxy and non-waxy broomcorn millet ranged from 2.97% to 4.00% and 28.65% to 33.18%, respectively. However, there were no apparent significant differences among the various N and biochar treatments of SM1 (s of SM1 . The fins of SM1 . HoweverThe distribution of amylopectin (AP) chain lengths significantly influenced the pasting properties, gel properties, and crystalline structure of the starches . The dis−1 region. The degree of order was quantified by the ratio of 1045/1022 cm−1, and the ratio of amorphous to ordered carbohydrate structure in the starch was 1022/995 cm−1 [−1 ranged from 0.687 to 0.761, and N150+B of SM1 had the highest peak  of both the individual and combined impacts of biochar and N fertilizer, as well as the influence of different varieties, were observed. The SV indicates the performance of the starch paste after cooling. A higher value indicates a greater tendency to recede. In SM1, the SV was the highest under the N150 treatment and was significantly different from that of the other treatments. In SM2, the SV ranged from 394 to 648 mPa s and was the highest in the N150+B treatment. This is consistent with our retrogradation results. Compared with the non-waxy broomcorn millet, the waxy variety has a lower SV, which could explain the difference between varieties. The highest PT was observed for N0 in SM2 across all the treatments, and it consistently showed higher values in SM2 compared with those of SM1. The application of N fertilizer and biochar in SM1 resulted in decreased PT values compared with N0, although no significant variation was observed among the different treatments. The parameter denoted as FV refers to the viscosity of starch paste following a 50 °C holding period at the conclusion of the RVA test [N fertilizer and biochar significantly affected the gelatinization properties of broomcorn millet starch . The PV RVA test . A statiThe thermal properties of broomcorn millet starch samples under different treatments were measured by differential scanning calorimetry (DSC) . In SM1,p < 0.01), but there was no detectable significant interaction between the two factors. Some studies have shown that the distribution of AP chain length can influence its pasting properties. We found that fertilizer reduced the proportion of short chains in AP and increased the To, Tp, and ∆H. This may be because a decrease in short chains in AP increases the packaging efficacy within the crystal structure of starch, which leads to a higher To, Tp, and ∆H [In this study, most starch grains exhibited irregular polygonal shapes, and the N150+B treatment resulted in smoother grains compared with those of the other treatments. Starch grain morphology is influenced by a variety of factors, including amylose content, and starch with a higher amylose content has irregular grains . Previou, and ∆H .The water solubility of starch can be inhibited by its amylose content , which iFagopyrum esculentum Moench) [The retrogradation percentages of the starch paste of waxy broomcorn millet were significantly lower than those of non-waxy millet, which may be related to the differences in genotypes between these types of broomcorn millet . The app Moench) . The ret Moench) . Because Moench) . N150+B  Moench) . Cao et  Moench) . Furtherp < 0.05). This increases the ease of gelatinizing the treated starch. Interestingly, various varieties of rice behaved differently when treated with biochar (10 t hm−2) [p < 0.05). Wang et al. [The starch pasting temperature was reduced for both varieties of broomcorn millet whether biochar was applied alone or in combination with biochar and N fertilizer, and these results were statistically significant ( t hm−2) . Treatme t hm−2) . The BV  t hm−2) . In this t hm−2)  studied  t hm−2) . We foun t hm−2) . In SM2, t hm−2) . The PT  t hm−2) . The disg et al.  showed tA Pearson correlation analysis was utilized to analyze the physicochemical properties on broomcorn millet starch, and the results are shown in These results showed that biochar and N fertilizer increase the smoothness of the surface of starch particles and change their particle size distribution. For both varieties, the proportion of A-granules increased significantly in the N150+B treatment, while the proportion of B-granules and C-granules decreased. Importantly, N150+B has the lowest amylose content and the best light transmittance. The starch after N treatment has the lowest trend of retrogradation in SM2. Biochar can increase the water solubility of millet and needs more energy for the gelatinization of broomcorn millet. The heat resistance of SM1 was enhanced by N150+B. The addition of biochar can increase the thermal stability of broomcorn millet starch and improve its gelatinization characteristics. In summary, the utilization of biochar has the potential to augment the efficacy of N fertilizer, elevate the levels of seed starch, and enhance the characteristics of starch.


This study was conducted to evaluate the effect of cooking and storage temperature on the formation of resistant starch in Indian wheat products and its effect on blood glucose levels in humans and rats.The health benefits provided by resistant starch have been well documented; however, few studies are available on the resistant starch content of wheat products in India. Moreover, few studies have examined the Chapati), boiling , Shallow frying (Paratha), and Deep frying (Poori). They were then stored at different temperatures including freshly prepared within 1 h (T1), stored for 24 h at room temperature (20-22°C) (T2), kept at 4°C for 24 h (T3) and reheated after storing at 4°C for 24 h (T4). The products were then analyzed for proximate composition . The effect of different cooking methods and storage temperatures on Resistant, non-resistant and total starch, total dietary fibre (soluble and insoluble), in vitro starch digestion rate (rapidly and slowly digestible starch), amylose and amylopectin content were analysed using standard operating procedures. The effect of products found to have higher resistant starch was studied on the post prandial blood glucose response of 10 healthy individuals using change in by analysing their glycemic index and glycemic load of wheat products. Further, the effect of resistant starch rich chapati on the blood glucose level of rats was also studied. Tukey’s test in factorial CRD was used to assess the effect of cooking and temperature on various parameters.Wheat products were prepared by common cooking methods including roasting , followed by parantha , chapati  and poori (deep frying 2.47%). Under different storage temperatures, it was found high in products stored at 4°C (T3), followed by products stored at room temperature (T2), reheated products (T4) and lesser in freshly prepared products (T1). The glycemic index and glycemic load were found low in chapati  and dalia  stored at 4°C (T3) compared to others. The resistant starch content found in chapati stored at T3 was found to be more effective at reducing blood glucose levels in rats from 291.0 mg/100 mL to 225.2 mg/100 mL in 28 days of study compared to freshly prepared chapati (T1) and stored at room temperature (T2).The amount of resistant starch was found to be high in In vivo research showed that fibre and resistant starch in the digestive system of rats acts as the main factors in slowing glucose absorption and reducing a rise in blood glucose levels by promoting glycogen synthesis and inhibition of gluconeogenesis.Cooking methods including boiling, roasting and shallow frying increased the amount of resistant starch in foods, but cooking methods such as deep frying decreased the amount of resistant starch in food. Products stored at 4°C and at room temperature for 24 h increased the amount of resistant starch whereas the products that were freshly cooked and reheated decreased the amount of resistant starch in foods. At 4°C the stored products have a high amount of insoluble dietary fibre, slowly digestible starch, high amylose and low glycemic index. They take time to digest, meaning that they slowly increase blood glucose levels. The effect of insoluble dietary fibre and resistant starch in the inhibition of glucose diffusion in the small intestine is suggested to be due to the absorption or inclusion of the smaller sugar molecules.  The starch present in the food is indigestible and the duration of cooking improves the digestibility of starch. Starch is made up of amylose and amylopectin chains. Amylose contains α--linked gResistant starch skips digestion in the small intestine and enters directly into the large intestine. It is then fermented into short chain fatty acids (SCFAs) , like acThere are five forms of resistant starch present in foods. Resistant starch 1 (RS1) is starch that is not accessible to digestion owing to the presence of complete, undamaged cell walls in the grains, tubers and seeds . ResistaThe third type of starch (RS3), retrograded starch, is affected by the cooking technique used. Retrogradation is the process that causes the recrystallization of the starch chains after the gelatinized paste cools. Starch structures that are molecular or crystalline are affected by storage conditions like duration, temperature and water content, which determine the retrogradation rate and its extent. Wheat starches show higher retrogradation rates due to longer amylopectin chains and high amylose (21.7%) content compared to rice (17.55%) . A high The process of starch degradation leads to the formation of starch and its by-products, which are not assimilated and absorbed in the small intestine of healthy individuals . Both thThe ability of any food to raise the blood glucose level after being consumed depends on its glycemic response or glycemic index. Food that has a high glycemic index raises blood glucose levels quickly compared to low glycemic food, which slowly increases the blood glucose level. Hence, low glycemic foods are beneficial for controlling glycemic responses. Wheat, rice and maize food products have a high glycemic index and can easily raise blood glucose levels. The overconsumption of high glycemic food for longer periods can cause several metabolic disorders such as obesity and type-2 diabetes. Insulin resistance and insulin insensitivity in muscles leads to hyperinsulinemia caused by obesity .Some studies have suggested that retrograded starch may reduce serum cholesterol concentration through numerous mechanisms, along with an increase in faecal bile acid excretion . It has in vivo efficacy of resistant starch in wheat products to improve blood glucose levels.The main challenge of using resistant starch in the food industry is the process of manufacturing consumer-friendly foods that contain enough resistant starch to result in the significant enhancement of public health. In response to the potential health benefits of resistant starch, the present study was undertaken to determine the effect of cooking and storage temperature on the resistant starch of wheat products that are part of the staple diet of North Indian people. We also studied the 22.1dalia , dalia , parantha , and poori  (1) stored for 24 h at room temperature (20-22°C and 45–50% RH) (T2), stored at 4°C for 24 h (T3), and finally, reheated after being stored at 4°C for 24 h (T4) (The most commonly consumed Indian wheat variety (HD3086) was procured from the Department of Plant Breeding and Genetics, Punjab Agricultural University, Ludhiana. The grains were cleaned and ground using a sample milled with 60 mesh size for making flour and 22 mesh size for making dalia . We chos frying) . These f4 h (T4) . For the2.2Nutritional analyses of raw and cooked samples were undertaken, examining crude protein, crude Fat and ash using standardized methods. The macro-Kjeldahl method was used for the determination of crude protein. Crude fat and ash content were also measured by using (AOAC 2000) method .2.2.1The total dietary fibre was determined using a megazyme total dietary fibre (K-TDFR-200A) kit. The soluble and insoluble dietary fibre contents were also analyzed using the standard protocol given by . The die1 = residue weight 1 from m1, R2 = residue weight 2 from m2, m1 = sample weight 1, m2 = sample weight 2, A = ash weight from R1, p = protein weight from R2 and B = blank Where: BR = blank residue, BP = blank protein from BR1, BA = blank ash from BR2.Where: R2.2.2The total starch and resistant starch were determined using a megazyme K-RSTAR assay, as outlined previosuly . Resista2.2.3in vitro starch digestion rate was determined using the procedure given in another study (The er study . In tota2.2.4The glucometer reading at 15 min was converted to the percentage of starch digested using the following equationGG = Reading of the glucometer (mM/L). V = Digest volume (mL), 180 = glucose’s molecular weight W = sample weight (g). S = sample’s starch content (g per 100 g dry sample). M = moisture percentage in the sample (g per 100 g sample). 0.9 = starch stoichiometric constant from glucose concentrations.RDS% = percentage of starch digested at 15 min. SDS% = percentage of starch digested at 120 min – percentage of starch digested at 15 min.2.2.5The Amylose Content was measured by colorimetric estimation of the amylose-iodine complex . The defAmylopectin = 100-amylose.2.3The glycemic index was calculated using the method given by Goni . Ten heaGlycemic load was calculated as:2.4Previous human supplementation research has indicated that cooked wheat products that had been subject to different treatments had a low glycemic index. We hypothesized that lower glycemic index foods have a positive effect on treating diabetes maybe through enhanced insulin secretion or by reducing its sensitivity. For this, we conducted a rat experiment to have authentic, real and unbiased data. Moreover, rats are very similar to humans genetically, and biologically and their behavioral characteristics closely resemble that of humans.2.4.1ad libitum.35 Wistar albino rats aged 2–3 months with weights 180-220 g were obtained from the animal house and breeding centre (AHBC) at Akal College of Pharmacy and Technical Education Mastuana Sahib, Sangrur (Registered breeder of CCSEA). The experiment was conducted as per the permission provided by the Institutional Animal Ethics Committee (IAEC no.:- GADVASU/2023/1AEC/68/12). The animals were housed in cages, fed with commercial pellets and had access to water 2.4.2The Wistar albino rats were given an intraperitoneal injection of freshly prepared 230 mg/kg Nicotinamide (NA) with buffer saline NaCl 0.9%. After 15 min, the rats were again given intraperitoneal injections of Streptozotocin (STZ) at about 60 mg/kg. Rats were provided with 5% of glucose water after injection to prevent hypoglycaemia. After 5 days of induction, their blood samples were taken and used to measure blood glucose and insulin levels. A blood glucose level of more than 200 mg/kg was an indicator of diabetic rats. Rats were treated for 28 days and blood glucose levels were checked first, third, and last week of the experiment.2.4.3Group-I:  consists of normal rats given a normal diet for 28 days.Group-II: (Diabetic control) after induction of diabetes were given a normal diet for 28 days.chapati, T1) orally for 28 days.Group-III: (Treatment group) Diabetic rats with supplement FWC  orally for 28 days.Group-IV: (Treatment group) Diabetic rats with supplement 24WC  orally for 28 days.Group-V: (Treatment group) Diabetic rats with the supplement RehWC , which was prepared using roasting followed by dalia , parantha , and poori  (chapatti (11.06%) parantha (11.72%) and dalia (9.22%) was found highest in T3, i.e., when stored at 4°C for 24 h. This was followed by T2, T1, and T4 in chapatti and T2, T4, and T1 in parantha and T4, T1, and T2. Poori stored for 24 h at room temperature (20-22°C and 45–50% RH) showed the highest protein content of 10.98% compared to other storage temperatures. All methods of cooking led to a decline in the protein content of the products as compared to the raw uncooked sample of wheat flour (11.3%).The crude protein content was found to be highest in , 9.16%) . A signipoori  followed by parantha  than the raw and other cooked products because of the addition of extra oil. Among different storage temperatures, crude fat content was high in T1 with 5.43%, followed by T2 (4.80%), T4 (4.18%), and T3 (4.07%). Raw wheat samples contained 0.83% ash, which was increased after cooking. The ash content was found to be high in parantha , followed by chapati , dalia , and poori . Keeping the products at 4°C for 24 h  resulted in higher ash content, followed by the products stored at room temperature (T2).Contrary to protein, the crude fat content was observed to be higher in 3.2dalia, which was prepared by boiling. Storing of the prepared food products at different temperatures, affected the dietary fibre content. The insoluble and total dietary fibre content increased with an increase in storage period at low temperatures and was observed to be highest in products stored at 4°C (T3)  for 24 h while the soluble fibre content was higher in the fresh food samples (T1) (2%) (The soluble (2.05%), insoluble (10.78%) and total dietary fibre (12.84%) were found to be maximum in T1) (2%) .3.3dalia  followed by parantha , chapati , and poori . Wheat products stored at T3 were found to have a higher amount of resistant starch content (4.47%) followed by T2 (3.32%), T4 (2.97%) and a lesser amount in freshly prepared products T1 (2.57%)  and poori , respectively and during storage it was found to be high during T1 (68.8%) compared to T3 (66.1%)  increased after cooking and the amount of resistant starch was found to be highest in  (2.57%) . On the  (66.1%) . No sign chapati .3.4in vitro starch digestion rate is important in determining the potential of a food product to raise the blood glucose levels of an individual. The in vitro starch digestion rate of wheat products  was affected by different storage temperatures. and was determined at 120 min after completion of the digestion of the food sample  and T4(33%). The starch digestion rate of wheat paratha was high in T1 (41%) followed by T4 (38%), T2(33.5%) and T3 (32%) at 60 min. In poori, the starch digestion rate was also found high in T1 (56%) and T4 (53%) compared to products stored at low temperatures for 24 h (T2 and T3).The d sample –5. The w3.5in vitro starch digestion rate, the amount of SDS in dalia  was highest followed parantha , chapati  and poori . Treatment 3(T3) increased the level of SDS to the maximum followed by T2, T4 and T1. Due to higher SDS content  was observed to have the lowest RDS followed by chapati , parantha , and poori  when stored with treatment 3.Similar to the  content , 7. Dali3.6dalia , followed by parantha , chapati  and poori . Treatment 3 resulted in increased amylose content of the products followed by T2, T4 and T1 , followed by parantha , chapati  and dalia  respectively and under storage conditions, it was found highest with T1 followed by T4, T2, and T3  with T3 followed by chapati (45.2%) and treatment 2 with a glycemic load of 28.6 and 35.5%, respectively. Treatments 2 and 3 were found to be the best storage conditions for lowering the glycemic index and may prove beneficial for diabetic individuals  after giving the treatment Diet. In G3, chapati stored at T1 was given and blood glucose level reduced from 275.8 ± 29 to 225.5 ± 20.0. In G4, chapati stored at T2 was given and found blood glucose level reduced from 291.0 ± 9.8 to 225.1 ± 15.0. In G5 chapati stored at T4 was given and found blood glucose levels reduced from (292.0 ± 12.1 to 256.3 ± 12.4), respectively  and the products were reheated after being stored at 4°C for 24 h (T4).The present study aimed to find the best storage conditions and cooking methods for increasing the resistant starch content of wheat products commonly consumed in India. The protein content was found to be higher in raw flour (without any treatment) compared to cooked examples, as cooking leads to denaturation of the protein structure. The milling method and the mesh size used in the current study might have also had an effect on the nutritional composition of the flour samples. The protein content of all wheat products was found to be low in freshly prepared products, i.e., within 1 h  and insoluble dietary (36 per cent) increased after cooking. This increase in the total dietary fibre of ragi may be due to the formation of resistant starch (In-vitro rapidly digestible starch was reduced during boiling and frying because of a significant increase in both the resistant starch (RS) and water-insoluble dietary fibre (IDF). The total dietary fibre content increased after cooking because of the increase in cellulose, lignin and pectin content during soaking and cooking  followed by stored at room temperature (T2) had higher RS content than their counterparts. The storage and reheating of products in treatment 1  caused a reduction of RS content due to the degeneration of the crystalline structure of starch granules as compared to T3, meaning it led to retrogradation and recrystallization of starch granules, resulting in higher RS. Compared to the food products prepared using different cooking methods, we observed that dalia prepared by boiling had higher RS content because, during boiling, food comes into contact with both heat and water, leading to the swelling of starch cells and its gelatinization, which results in the release of amylose in the solution  due to the formation of lipid-amylose complex. This form of RS5 led to there being a higher amount of resistant starch than Poori, which was prepared by deep-frying. More water was evaporated during deep-frying, which led to less formation of crystalline structure and, hence, lower resistant starch formation  and lowest in chapati (roasting). During roasting, when food came into contact with heat, the total starch was broken down into sugar. The total starch content of Pearl millet was found to be reduced after the action of the amylase enzyme when total starch was converted into sugar  was affected. Results showed that the total starch content in wheat products was between 70.46 to 71.5%. Freshly prepared products had the highest level of total starch content present in to sugar . On the to sugar , 49.Plant genotype can also affect the digestibility of starch, with the amylose/amylopectin ratio being a crucial determinant of the rate of starch hydrolysis in both peas and wheat . The stadalia prepared by boiling had the lowest starch digestion rate compared to parantha , chapati (roasting), and poori (deep frying). This variation in the digestibility of wheat products occurred due to the heat–moisture remedy proving that these treatments alter the structure of starch and increase the quantities of SDS and RS at the same time as decreasing the proportion of RDS. Differences in the physical and morphological properties of cereal starches cause lower digestibility.The frequency and quantity of starch digestion are also affected by the structure, composition, processing, and cooking of starch granules associated with other nutrients like lipids, protein, fibre, minerals, and antinutritional factors , 51. ComCooking cereal starches brings about modifications, for example in the physical and chemical disruption and gelatinization of starch granules. The extent of gelatinization in turn is dependent on the amount of water present, the cooking time and the temperature , which iDalia (boiling) had a high amount of amylose content compared to parantha , chapati (roasting), and poori(deep frying) and was found to be highest at storage temperature T3 (4°C for 24 h). Wheat starches stored at T3 showed higher retrogradation rates due to longer amylopectin chains and high amylose content  the formation of amylose increased, resulting in low amylopectin content and a high amount of resistant starch. The formation of amylopectin content was inversely proportional to the amylose content. The starch from grains such as maize, wheat, rice and low amylose maize, tubers such as potatoes and sweet potatoes, and legumes such as kidney beans, has the highest starch content (8.51%), while kidney bean starch has the higher amylose content (49.50%). Large-size starches  showed longer amylopectin chains high amylose content and resistant starch confirming that they had the most slowly digestible starch content .dalia compared to chapati when stored at 4°C (T3) compared to freshly prepared (T1). Because of the presence of retrograded starch at low temperatures. It takes time to digest and hence, slowly raises blood glucose levels. Low glycemic index diets improve both diabetic and healthy people’s risk of coronary heart disease. Low-glycemic index meals promote satiety and help limit food consumption in obese or overweight people. A healthy person’s post-prandial glucose and lipid metabolism is improved by choosing foods with a low glycemic index (The glycemic index determines the rate of rise of blood glucose by a specific food. It was found low in ic index . The GL ic index . Food whchapati stored at T2 (room temperature for 24 h) for 28 days, it was found to be more effective in reducing blood glucose levels than consumption of products stored at T1 and T4. This happens because wheat products stored at room temperature comprise excessive quantities of nutritional fibre and resistant starch. These products release sugar slowly in the blood and also diminish glucose absorption. The gradual digestion of resistant starch (RS) has implications for the application of managed glucose release  in a rat study was found to be more beneficial in controlling the rise of blood glucose levels. Thus, it leads to a slower rise in blood glucose levels, providing longer satiety. In India, people consume a large variety of starchy preparations, meaning that modifications to the food cooking methods and storage temperatures used for starchy foods may lead to several health benefits. Greater awareness should be fostered regarding the nutritional and health benefits of resistant starch consumption to maintain blood levels. People also need to be educated about the correct ways of cooking and storing food products to increase the amount of resistant starch in food at the domestic level.Cooking methods including boiling, roasting and shallow frying increased while deep frying decreased the amount of resistant starch in foods. Products stored at 4°C (T3) and at room temperature for 24 h (T2) showed an increased amount of resistant starch whereas freshly cooking (T1) and reheating (T4) decreased the amount of resistant starch of foods. Products stored at 4°C (T3) have a high amount of insoluble dietary fibre, slowly digestible starch and amylose content. The glycemic index and glycemic load were also found to be low in T3 wheat products. These contained a high amount of resistant starch, which reduced the blood glucose level by regulating the promotion of glycogen synthesis and inhibition of gluconeogenesis. Consumption of The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author/s.The studies involving humans were approved by the Institutional ethics committee, PAU. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required from the participants or the participants’ legal guardians/next of kin because there was no chance to harm the human. In the study only their blood glucose levels was measured. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.PK: Writing – original draft. HK: Methodology, Writing – review & editing. RA: Writing – review & editing. KB: Visualization, Writing – review & editing. AM: Data curation, Writing – review & editing. OG: Project administration, Writing – review & editing. LS: Visualization, Writing – review & editing. KS: Visualization, Writing – review & editing.


Correction to: International Journal of Colorectal Disease (2023) 38:64 https://doi.org/10.1007/s00384-023-04358-0The Funding information in the published version of the above article was incomplete. The complete information is presented as follows:Funding Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This work was supported in part by grants from the Instituto de Salud Carlos III and the European Regional Development Fund ; the Spanish Ministry of the Economy (PID2020-115738 GB-I00); the Departments of Health (2010111098) and Education, Language Policy and Culture  of the Basque Government; the Research Committee of Galdakao Hospital; the REDISSEC  thematic network of the Instituto de Salud Carlos III; and the Department of Education of the Basque Government through the Consolidated Research Group MATHMODE (IT1456-22) and the Basque Government through BMTF “Mathematical Modeling Applied to Health” Project. This study has been funded by Instituto de Salud Carlos III (ISCIII) through the projects "RD16/0001/0001" and RD21CIII/0003/0017 and co-funded by the European UnionThe original article has been corrected.


Induction of mutation through chemical mutagenesis is a novel approach for preparing diverse germplasm. Introduction of functional alleles in the starch biosynthetic genes help in the improvement of the quality and yield of cereals.PHO1, PUL) in the high starch mutant lines, indicating that other starch metabolic genes may also contribute to the starch biosynthesis. The overexpression of SSII, SSIII and SBEI in JE0173 may be due to presence of missense mutations in these genes and SSI also showed overexpression which may be due to 3-primer_UTR variant. These mutations can affect the other starch related genes and help to increase the starch content in this mutant line (JE0173).In the present study, a set of 350 stable mutant lines were used to evaluate dynamic variation of the total starch contents. A megazyme kits were used for measuring the total starch content, resistant starch, amylose, and amylopectin content. Analysis of variance showed significant variation (p < 0.05) in starch content within the population. Furthermore, two high starch mutants (JE0173 and JE0218) and two low starch mutants (JE0089 and JE0418) were selected for studying different traits. A multiple comparison test showed that significant variation in all physiological and morphological traits, with respect to the parent variety (J411) in 2019–2020 and 2020–2021. The quantitative expression of starch metabolic genes revealed that eleven genes of JE0173 and twelve genes of JE0218 had consistent expression in high starch mutant lines. Similarly, in low starch mutant lines, eleven genes of JE0089 and thirteen genes of JE0418 had consistent expression in all stages of seed development. An additional two candidate genes showed over-expression (This study screened a large scale of mutant population and identified mutants, could provide useful genetic resources for the study of starch biosynthesis and genetic improvement of wheat in the future. Further study will help to understand new genes which are responsible for the fluctuation of total starch.The online version contains supplementary material available at 10.1186/s12870-023-04389-3. GBSSI gene to produce partial waxy wheat to create less amylose content which help in production of good quality noodles [The quality of wheat flour evaluates based on endosperm starch contents and its composition. The endosperm starch that is approximately 70% dry weight of seed has significant effect on the yield and quality with showing capacity of sink tissues in wheat . The sta noodles . By usin noodles , 5. Simi noodles .TaAGPL1-A, while overexpression of TaGPL1 and TaGPS1 helped to increase total starch and thousand grain weight in wheat [GBSSs and all the starch mainly composed of amylopectin in the endosperm of wheat [SSI in wheat help to increase the concentration amylose content. Similarly, inactivation of SSII also increased the amylose content in wheat [TaSSIVb-D had no significant affect at the reserve starch but there was less granules in leaves [agp.L-B1/ssIVb-D), grain starch and amylopectin contents were less than other single mutants in wheat [Glucose-1-phosphate play important role in the amyloplast during starch biosynthesis. The initiation of starch biosynthesis by ADP-glucose pyrophosphorylases (AGPase) with help of other enzymes  in the seed . The forin wheat . A waxy of wheat . Throughin wheat  The mutan leaves . Similarin wheat . The forin wheat .GBSSI gene [SSIIa, SBEIIa and SBEIIb used to produce high and low starch mutants [Phenotypic variation has been produced in cereal crops through chemical agent. One of the most frequently use chemical agent is ethyl methane sulphonate (EMS) in cereal crops . EMS worSSI gene . Further mutants . To find mutants . So for  mutants .In the present study, a set of 350 stable mutant lines were screened for total starch content. The parent variety of this stable population is J411. Further, two high starch mutant lines and two low starch mutant lines were used to study quantitative gene expression patterns of 19 starch metabolic pathway genes during seed development. The exome sequence had been done to know the allelic variations in these selected lines. Overall workflow is given in the Fig. Through starch analysis, it’s indicated that the total starch contents in mutant lines were stable and consistent. Similarly, analysis of variance showed significant variation between the mutant lines. Most of lines showed significant variations (60%) for total starch contents from the parent variety. The lowest value for total starch content was 48.2% and the highest value in the mutant lines was 80.6%, while the value of WT (J411) was 67.1%. Gluten, Protein, and hardness of seed also showed variation within the population. The lowest seed hardness value was 92% and highest was 120% while the value of WT was 96%. The maximum mutant lines whose values were 39% for gluten while the minimum value for protein was 14%. The results of these 350 lines are given in Fig. p < 0.05) were recorded in the case of physiological and biochemical parameters. The results of two years selected lines have been given in the Table Two high starch (JE0173 and JE0218) and two low starch (JE0089 and JE0418) mutant lines had been selected from mutant population. The average low starch was 50% and 52% in two mutant lines JE0089 and JE0418 respectively for both year data while highest starch was 76% and 77% in mutant lines JE0173 and JE0218 respectively, and average value of J411 was 66% in 2020 and 2021. These lines showed significant difference from J411 in two years data. Further, amylose content of JE0089 was significantly lower in both year of data while amylose content of high starch mutant line JE0218 was higher as compared to WT but there was lower amylose content for JE0173 mutant line. Similarly, amylopectin content of all mutant lines showed significant difference from WT. The resistant starch of high starch mutant lines showed significant difference from WT and both lines had higher resistant starch contents than low starch mutant lines. Highest resistant starch was observed in the JE0218 line. There was significant difference in all mutant lines as compared to WT for digestible starch. Protein contents was also higher in the high starch mutant lines. Similarly, gluten was high in high starch lines and less in less starch lines. Clear and significant differences (p < 0.05) were recorded in the case of morphological parameters. The results showed that low starch mutants and high starch mutants had significant effects on the four yield-related traits in wheat, namely, TGW, grain width, grain length and grain area in two years data. The grain width of JE0218 was higher than all mutant lines, while the Grain length of JE0173 was higher than all mutants. The TGW was higher of both high starch mutants in which the average TGW of JE0173 was 45.4 g and JE0218 had 46.6 g, while WT had 40.2 g. The gluten content of JE0218 was also higher than all other lines in both year of data (Table By taking pictures of the WT(J411) and mutant plants of wheat at heading stage, the mature plants were investigated for different biological traits, and the mature grains were photographed Fig. . Grain lTM4000 scanning electron microscopy was used to find out the difference of grain morphology between WT (J411) and high starch mutants  and low starch mutants   showed reduced expression in all stages while, from 12 genes of JE0218, nine genes showed overexpression and three genes  had reduced expression  showed that eleven genes of JE0173 and twelve genes of JE0218 had consistent expression in all stages of seed development Fig. . While eion Fig. .GBSSI, SSI, SBEI, ISA2, AGPase S) of JE0089 showed reduced expression and six genes  had over expression from these eleven genes. Similarly, from the thirteen genes of JE0418, six genes  had reduced expression while seven genes  had over-expression in all seed development stages. The expression pattern of remaining starch synthesis genes in both mutant lines were inconsistent by showing high and reduced expression in different stages of seed development. In the present study, the low expression of TaRSR1 in the low starch mutant lines may be this gene had no effect in starch metabolism. This differential expression analysis in four mutants give the idea that there is involvement of other starch metabolic pathway genes such as phosphorylase, isoamylase etc., in the starch biosynthesis.The low starch mutant’s expression analysis had been conducted. The quantitative gene expression analysis showed that from nineteen genes of starch biosynthesis, eleven genes of JE0089 and thirteen genes of JE0418 had consistent expression in four stages of seed development Fig. . Five geExon sequencing had been done for selected high starch mutant lines (JE0173 and JE0218) and low starch mutant lines (JE0089 and JE0418). It was depicted that in the mutant lines, the number of SNPs were more in all four mutant lines than Indels. The number of SNPs in JE0418 was higher than other three mutants. But Indels were almost in equal amount in all mutant lines. The different types of SNPs between the mutants suggested that there were more transitions than transversions in the four mutants figure . There wSSIII, SBEI, GBSSI, SSII) and four were synonymous mutation  while remaining two were 3_prime_UTR variant (SSI) and splice region variant (SSII). JE0218 had two variants that consist of one synonymous (AMY) and other was missense (TaRSR1) mutations. Similarly, JE0089 and JE0418 had six and four variants respectively.  mutations in JE0089 and two  in the JE0418 had been observed, while two were synonymous in both lines.From four mutant lines, twenty-two variants were identified in nineteen starch biosynthesis genes. Ten variants were discovered in JE0173 in which four were missense mutation . Similarly, in JE0218, there was missense mutation in TaRSRI and its showed overexpression as compared to WT (J411). In the low starch mutant line JE0089, SSI and PUL had reduced expression and these genes had missense mutation. PUL also had missense mutation, but the expression of this gene was inconsistent. So, these genes which showed reduced expression and other starch related genes might be the responsible for low starch contents in this mutant line. Similarly, in JE0418, SBEIII had reduced expression due to missense mutation and this mutation might be responsible for low starch content in this mutant line.The overexpression of Modification of starch composition in wheat help to increase starch content and yield of the crop which ultimately effect on the public health due to good quality of flour. Different chemical mutagenesis have been used in different crop for studying the functional genomic and breeding . DiffereThe deposition of protein increased or decreased due to mutation and there will be significant reduction of amino acids in seed tissues exposed to EMS chemical . The depAGP-S1, SS1, SSIIa, SSIIIa, SBEIIa, SBEIIb, and GBSSIa expression level play significant role in starch biosynthesis and accumulation. Changing of expression pattern of these main genes may have positive affect on the grain weight of the wheat during grain filling stages [GBSSII, SBEII, AGPaseSS and SSs) as compared to low starch mutants during grain filling stages may lead to accumulation of high starch [GBSSI. Similarly, due to silent and null mutants produce waxy and partial waxy wheat with low amount of total starch and lacking amylose contents [ISA help to reduction in the amylopectin contents in wheat [SBEII also increased amylose content with reduced total starch contents [The key genes of starch biosynthesis such as g stages , 26. In h starch . Amylosecontents , 29. Witin wheat , while scontents . TherefoAMY and BMY) with phosphorylases (Pho1 and Pho2) play significant role in starch metabolism along with hydrolysis and degradation [AMY and BMY fluctuate the starch structure and baking quality in wheat [RSR1) had been observed during co-expression in the rice which negatively regulate the some starch biosynthesis genes and modulate the starch metabolism [Amylases  and other mutagens to the J411 variety. J411 is the hexaploidy modern cultivar in China. After selecting the lines based on different traits, stable lines were developed. After maturing, seed samples from each mutant lines were collected and crushed these seeds to make the flour for studying the total starch contents and other physiological traits table . FurtherFor measuring the total starch content of 350 samples, Megazyme kit that was based on AOAC Method 996.11, AACC Method 76-13.01 was used. We followed the instruction mentioned in the kit for measuring the total starch content .A commercial Megazyme kit  was used for measuring the resistant starch and digestible starch from the flour of the wheat. Instruction was followed according to the kit guidance .The amylose/amylopectin megazyme kit  was used for measuring the amylose content of wheat flour . The proGrain analyzer had been used to measure the protein, gluten, and seed hardness. FOSS InfratecTM 1241 Grain Analyzer  was used for scanning NIR spectra. WinISI II v1.50  software was used to find out the final reading . All theSamples of seed from each mutant lines and WT were collected with three replications for measuring the grain width, grain length, grain area and thousand grain weight (TGW). SmartGrain was used for evaluating these traits by following methodology as described in previous studies , 39. TGWSeeds of mutant samples were crushed for extracting pure starch. The methodology was followed for starch purification as describe in somewhere else , 40. GraSelected mutants were used to capture the genetic diversity. The 4 mutant pools were subjected to exon capture and sequencing to compare the polymorphisms between different pools. The process of exon sequencing was followed as describe in Irshad et al.,.AGPaseS), while four starch synthase isoforms , three were starch branching isoforms  and remaining consisted of granule bound starch synthase (GBSSI) with three starch debranching isoforms and pullunase . Further we selected four starch degrading genes . Two transcription factors were also added for expression analysis that play role in starch biosynthesis (SPA and TaRSR1).Nineteen genes had been selected for quantitative expression analysis during different grain filling stages. These genes consist of starch synthase, starch branching enzymes, debranching enzymes, amylase, phosphorylase, and transcription factors. These genes play important role in amylose and amylopectin biosynthesis as well as total starch biosynthesis. Thirteen was main starch biosynthesis genes in which one was small subunit of ADP-glucose pyrophosphorylases , two high starch mutant lines (JE0173 and JE0218), and the parent wheat variety ‘J411’. Samples of these mutants and parent variety were collected after 6, 12, 18, and 24 days anthesis (DAA). These samples were quickly put in the liquid nitrogen after taking from spike and then shifted to the − 80c refrigerator for RNA extraction. The detailed protocol of RNA extraction, cDNA formation and qRT-PCR followed by as described by Zhang et al., (2019). In addition, Actin (GenBank accession no: AAW78915), a housekeeping gene, was used as an internal control. Similarly, genes and their primer information was obtained from Singh et al., . Three bMicrosoft excel formulas had been used for calculating the mean and standard deviation. One-way analysis of variance (ANOVA) was used to study variation in total starch, in the replications for the mutant lines. To test the significant difference between mutants and parent variety for the above traits, Dunnett’s test had been performed. For the purpose of these analyses, three biological replications were used for studying all of these traits.In this study, 350 mutant lines were used and mutants with starch content variations were identified, this useful germplasm can be used for a genome-wide study and help to improve the starch-based nutritional quality in wheat. The gene expression of nineteen starch metabolic genes in two high starch mutants and two low starch mutants indicate that with starch main genes, there may be other genes  responsible in starch biosynthesis. Further exon sequencing of these diverse mutant lines gives the information for further studies. There is a need to find out about starch metabolic genes through the backcrossing of these mutant lines and a developed NILs population for mapping.Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2