Efficiency under conditions that more accurately mime the human body will provide encouraging evidence towards future clinical applications.Autologous vein and artery remains the first choice for vascular grafting procedures in small-diameter vessels such as coronary and lower limb districts. Unfortunately, these vessels are often found to be unsuitable in atherosclerotic patients due to the presence of calcifications or to insufficient size. Synthetic grafts composed of materials such as expanded polytetrafluoroethylene (ePTFE) are frequently employed as second choice, because of their widespread availability and success in the reconstruction of larger arteries. However, ePTFE grafts with small diameter are plagued by poor patency rates due to surface thrombogenicity and intimal hyperplasia, caused by the bioinertness of the synthetic material and aggravated by low flow conditions. Several bioresorbable and biodegradable polymers have been developed and tested to exploit such issues for their potential stimulation to endothelialization and cell infiltration. Among these, silk fibroin (SF) has shown promising pre-clinical results as material for small-diameter vascular grafts (SDVGs) because of its favorable mechanical and biological properties. A putative advantage in graft infection in comparison with synthetic materials is plausible, although it remains to be demonstrated. Our literature review will focus on the performance of SF-SDVGs  Therefore, knowledge of the behavior of surgical or endovascular grafts and their long-term impacts onto large and small vessels is vital to the cardiovascular specialist.This scientific approach improved the management of vascular pathologies through different techniques and materials. A constant dialogue between clinicians and bioengineers is essential for the development new effective graft materials, with desirable characteristics over the aforementioned issues.Cardiovascular diseases are projected to grow in the following years to an estimated overall mortality of 23.4 million in 2030 . MoreoveHowever, the use of these conduits is often limited because atherosclerosis is a systemic disease that can involve all arterial districts, including arterial conduits most harvested for revascularization. Another issue arises in patients who underwent a prior harvesting of an arterial or venous conduit, limiting the availability of a possible graft for further interventions.Synthetic grafts provide a viable solution for large vascular replacement, but their performance with low flow conditions in smaller vessels is disappointing. For this reason, vascular tissue engineering aims to find a solution to have a functional and infection-resistant graft to replace the diseased artery. We consider the need for endovascular but also open revascularization and look for available and safe alternative materials applicable to all the small vessels requiring intervention.Indeed, the most critical problem is to obtain suitable materials for small grafts assuring a long-term patency and infection resistance.About the first aspect, vascular and cardiac surgeons are skilled in the harvest of autologous vessels, proven to maintain patency with low thrombogenicity. The most employed vessels for open surgical revascularization include mammary or radial arteries, or different veins of the superficial system of the upper and lower limbs, . In surgery of peripheral arterial disease, revascularization by means of in-situ venous conduits is also used to bypass occlusions of the distal arteries of the limbs.Secondly, to prevent infection, autologous grafts should be preferred over synthetic ones, particularly in case of revascularization of wet gangrene of the lower limbs.in vivo in anima  models in a preclinical phase as an arterial substitute and its further development, in terms of patency, endotelialization, low restenosis, surrounding tissues involvement, and resistance to infections.Different authors are reporting experiences of using silk fibroin (SF) Silk is a natural, versatile protein biopolymer which is produced by various insects . This ancient textile material has long been used to prepare sutures in surgery thanks to its biocompatibility and remarkable tensile strength  of the extracellular matrix and to realize a range of biochemical, topographical, and mechanical properties conducive to improved cell interactions  Figures, 2.Our review aims to provide a comprehensive overview of the principal fields of preclinical use of silk fibroin as a vascular substitute in different animal models evaluating silk fibroin (SF) behaviour in term of endothelialization, biocompatibility and short and long term patency.2.in vivo by performing vascular anastomosis and interposition procedures within small and large animal models and different arterial districts.Although other reviews on tissue-engineered vascular grafts have been previously published , includiThe Preferred Reporting Items for Systematic Reviews and Meta-Analysis Protocols (PRISMA-P) statement was used to obtain a strict paper selection .The PubMTo perform the initial review process, based on paper and abstract title search, the combination of keywords used, connecting Boolean operators, was:in vivo)((silk fibroin) AND (vascular)) AND (https://rayyan.qcri.org/)] was chosen to manage and screen selected papers. After the collection of all search results, duplicates were automatically excluded before the screening process.The Rayyan QCRI [Qatar Computing Research, Doha, Qatar  were also excluded. Finally, remaining articles utilizing vascular grafts developed from materials not including SF were discarded.A series of exclusion criteria were considered to select the articles that best applied to our objectives. Articles and abstracts not containing the selected keywords in their title were excluded. Subsequently, the remaining articles were screened for relevance of topics according to the aims of our review, as well as pertinence of the experimental model in which SF was evaluated. Indeed, studies exclusively including in vivo assessment of SF-based vascular grafts were reviewed, with experimental models ranging from small to large animals: murine, leporine, canine, porcine and ovine. Henceforth, the included studies are presented according to the respective animal model. Because the results were not fit for a meta-analytic investigation due to the heterogeneity of the in vivo animal models and graft materials, they were discussed in a narrative way.As a result of this selection process, a total of 31 articles concerning The flow chart for the selection of studies is shown 3.in vivo was performed on murine and leporine models.Compared to larger animals, small animals are easier to handle and less expensive. However, the technical difficulty of their size makes the surgical insertion of vascular conduits difficult. Rats and rabbits are the most employed, and both can often receive conduits with very small (2 to 5 mm) and small (2 mm) diameters . Accordi3.1.Some authors discourage the use of murine models to test the patency of short graft portions, especially in the absence of a clinically relevant control . Althougin vitro demonstration of rapid endothelial cell attachment and excellent resistance to blood clot and fibrin network formation suggesting that silk fibroin might be a promising material to develop vascular prostheses for smaller arteries  introduced into the SF heavy chain to improve its properties showed greater enhancement of cellularization behavior compared to wild type (WT)-SF . VEGF-SFMoreover, VEGF-SF has been shown to support significantly higher and earlier endothelialization in the central part of aortic grafts in mice compared to WT-SF . HoweverIn vivo substitution of arterial segments with SF grafts has also been attempted with carotid arteries in murine models. In one of such models, a small-sized graft of 0.9 mm inner diameter was braided from a SF thread and interposed to carotid arteries in mice using a cuff technique  and allowed complete re-endothelialization on the SF patches at 2 weeks after implantation in rat carotid arteries.In a different model employing patches of SF with a gelatin hydrogel incorporating simvastatin micelles for sustained release of simvastatin was investigated for its promotional effects on endothelial progenitor cell mobilization from bone marrow and recruitment to sites of vascular injury, exhibiting acceleration of re-endothelialization . In vivoin vivo into small animal models do so by combining it with other synthetic graft materials to obtain different mechanical and physiological outcomes, especially by developing a SF coating of the inner lumen. Indeed, coating is an important factor to maintain the strength of the anastomotic region of vascular grafts, and to prevent the blood leak from the vascular grafts after implantation.A significant number of studies employing SF A study evaluating electrospun poly (L-lactic caprolactone) /SF small-diameter grafts, loaded with VEGF and heparin, found that such artificial blood vessels have good cytocompatibility and histocompatibility, but the patency and degradability need to be improved . Indeed,in vivo , vessel patency was maintained, and the inner lumen of the scaffold showed regenerated endothelial cells. Platelet adhesion was supposedly countered by the presence of Spirulina extract in the inner SF layer.In an alternative combination, a tri-layered nano-fiber scaffold of SF and poly-caprolactone (PCL), fabricated using a sequential electrospinning method and containing Spirulina extract in its inner SF layer, was implanted into rat carotid artery and evaluated for histological analysis after 3 weeks . BesidesIn vivo outcomes of SF(Glyc)-PET graft were compared with a widely used commercial gelatin-coated PET graft -coated PET graft.A different research group developed a SF-coated poly (PET) graft of diameter <6 mm. The tubular PET graft was produced through the double-raschel knitting technology and then coated with porous SF prepared using glycerin (Glyc) as porogen. ET graft . In impln = 6, respectively) and explanted 2 weeks or 3 months later. Two weeks after implantation, no significant differences were found among the kinds of grafts in biological reactions evaluated by histopathologic examination. However, a remarkable difference was observed after 3 months in terms of area of tissue infiltration into the graft wall, 2.5 times larger in SF/SF than that in PET/G. The endothelialization was achieved almost 100% in SF/SF, despite only 50% was achieved in PET/G. Therefore, SF delivered promising results both as base and as coating materials for small-diameter vascular prostheses.In a parallel study SF and PET tubular grafts of 1.5 mm diameter, produced with a double-raschel knitting machine, were coated with aqueous SF or gelatin (G) to make four types of vascular grafts  . The fouAnother study of the same group focused on the coating of 1.5 mm diameter SF grafts produced by double-raschel knitting with SF solutions at different concentrations  containing polyethylene glycol diglycidyl ether (PGDE) as cross-linker. The effect the different coatings on tissue infiltration and remodeling was investigated . The graIn the work reported in a distinct study, small-diameter vascular grafts 1.5 mm in diameter were prepared by coating a double-raschel knitted silk fiber graft with SF aqueous solution containing PGDE as a cross-linking agent . Eight wAs an alternative application, a blend of SF and thermoplastic polyurethane  was employed in order to produce by electrospinning a cardiovascular patch with appropriate elastomeric characteristics without sacrificing the excellent tissue affinity and biocompatibility of silk . Upon imin vivo into the abdominal aorta of 22 rats and fixed using methylmethacrylate. Such technique of stent anastomosis produced significantly shorter ischemia time during implantation compared to conventional sutures and, after 4 months, the anastomosis was shown to be functionally patent in all cases. However, thrombus formation, frequent and severe abdominal infections, and heavy host rejection remain critical issues.SF has also been applied as substrate for cylinder stents of small diameter . In a si3.2.Rabbits are the small animal of choice for conduits 1 to 4 mm in diameter, enabling bilateral implantation of longer conduits and having a greater similarity than rats to humans in coagulation, endothelialization, and patency. Carotid artery grafting is convenient in the rabbit due to technical feasibility, similar longitudinal tension to humans, as well as comparable endothelial response to prosthetic conduits. In fact, patency rate over time closely simulates the clinical response to chronic ePTFE grafts seen in humans.in vivo by implanting and replacing a common carotid artery in rabbits  (SF/PLCL) and an inner layer obtained by shell-core electrospinning, in which the shell was SF/PLCL or PLCL alone and the core was a 15% aqueous heparin (Hep) solution, yielding SF/PLCL/Hep or PLCL/Hep grafts . Compared to the graft with PLCL/Hep as inner membrane, the hydrophilicity of the graft including SF (SF/PLCL/Hep) was significantly improved, with optimal biocompatibility and maintained lumen patency for 3 months after carotid artery transplantation at ultrasound examination.For what concerns SF blends with other polymers as vascular grafts in leporine models, a study implemented electrospun bi-layered scaffolds made of an outer layer of SF-poly, manufactured by electrospinning, was employed to evaluate patency and short-term remodeling in a sheep model of arteriovenous shunt . Nine SiSome authors initiated pilot studies to assess whether the porcine or the ovine animal model would be better to use for future trials regarding the suitability of the surgical model and the long-term biological outcomes of interposed SF-SDVGs. In a single study, the carotid arteries of 1 minipig and 1 sheep were replaced with novel a tri-layered SF graft, monitored with Doppler sonography and angiography, and histopathology of explants was examined after four weeks  Figure . CarotidWhile in the mini-pig endothelial-like cells were found along the whole surface of the graft neointimal layer, endothelialization within the grafted sheep carotids was restricted to peri-anastomotic sites. This reflects a slower rate of endothelialization in the ovine model, closer to what occurs in humans. Microscopic evidence of an ongoing foreign body inflammatory reaction was observed in both animals, but only in the ovine model no concurring subendothelial hyperplasia and no damage to neighboring tissues had occurred. Therefore, in light of the biological outcomes observed, as well as several technical and practical advantages, the ovine model was deemed more suitable than the minipig for long-term studies aimed at assessing the performance of SF grafts. According to the findings of such pilot study in large animals, the use of a small caliber vascular graft entirely made of pure SF has been validated by favorable biologic outcomes coupled to its simplicity in handling and surgical feasibility and, as a result, can be designed as an “off-the-shelf” device meant for preliminary clinical trials on humans.5.While synthetic grafts (including PET or ePTFE) are a viable option in the replacement of large vessels, these have been shown to perform poorly when replacing small diameter vessels. Consequently, the need for an effective non-autologous vascular graft material for districts such as coronary and peripheral arteries appear to be still an open task for surgeons and bioengineers.The optimal vascular graft should have scaffolds that mold the graft with an adhesive matrix primarily made of fibrin and vascular cells. Such biomaterials should also be capable of replacing the host's tissue, have a composition that is strong enough to prevent rupture, and have a sufficient size for clinical use.The benefit of employing natural polymers is their built-in bioactivity, which results in favorable characteristics for graft regeneration.Silk shows some properties that make it suitable to be a substrate for the creation of vascular grafts or in general vascular scaffolds for the healing of endothelial cells. They could be considered in two different groups: mechanical property and on the other hand biological properties as immune response and its interaction with vascular cells, biodegradation and haemocompatibility.Multidisciplinary scientific efforts have converged to pinpoint the advantageous characteristics of vascular scaffolds in the quest to increase the clinical viability of vascular grafts. Indeed, the physical and chemical characteristics of the vascular scaffolds pragmatically dictate their therapeutic outcomes. Additionally, by adjusting the graft's characteristics, it is possible to calibrate the remodeling and formation of a neo-intimal layer in an implanted vessel. The ability to attract and recruit host cells while also providing them with a favorable microenvironment for growth, hemocompatibility, and minimal immunogenicity are just a few of the desirable features from an ideal vascular biomaterial.Recruiting and attracting host cells is the base of endothelialization and it is a critical point in such a research especiallyfacing potentially biologic scaffolds. Endothelialization might be defined as cells and smooth muscle cells migrated into the fibroin graft early after implantation becoming organized into an endothelium and a media-like smooth muscle layer14. More specifically a transanastomotic endothelialization consists in the growth of the host intimal layer across the anastomosis because of a migration of native endothelial cells .in situ biodegradation that facilitates constructive graft remodeling, mechanical resilience, and stability throughout the process of graft remodeling, the ability to attract and recruit host cells, and minimal immunogenicity  respect to a polystyrene surface .All the cell types, once cultured on either SilkGraft or polystyrene, exhibited similarly low basal levels of TIMP-2, a metalloprotease blocker which regulates extracellular matrix (ECM) remodeling processes and interactions between cells and ECM. It is interesting to note that no significant pro-inflammatory cytokines (Tumor Necrosis Factor-α (TNF-α), or of profibrotic cytokines (Transforming growth factor-b (TGF-b) were secreted by the three cell types grown on either substrate. The patterns of cytokines and chemokines secreted suggest a proliferative attitude while neatly excluding a pro-inflammatory and/or profibrogenic proclivity.The biological haemocompatibility of SF is corroborate by lack complement activation and hemolysis meanwhile the cell adhesion, intense metabolic activities and the expansion of the cell populations cultured with no secretion of pro-inflammatory or pro-fibrotic cytokines show the high level of compatibility of SF rather than a synthetic graft  and thisAn important tool of Silk fibroin is the possibility to seed it with different cells. It has been reported that silk fibroin is a good scaffold for the cells of the endothelial wall but also bone marrow seeding or other cells type useful for the different use of the graft to reduce the thrombogenicity . The difAlthough silk grafts have demonstrated extraordinary long-term regeneration capability in trials on small animals, their effectiveness in larger animals remains underdeveloped and field for future studies.The development of other silk variants is still in its infancy and requires further attention. A couple of silk-based products that have been validated for clinical use belong to the tissue regeneration . While FNatural materials offer a solid blueprint to reinterpret, reengineer, and simplify modern manufacturing while preserving sustainability.SF is minimally immunogenic and avoid fibrous capsule encapsulation by chronic immune response. The factors which are determining the bio response of SF-based tissue-engineered grafts include the format of the material, implantation site and degradation rate and time. Hard tissue implantation and shorter degradation time would result in minimal immunogenicity, while SF matrices having longer degradation time and implanted at soft tissue sites would elicit comparatively elevated immunogenic response . This rein vivo and become unrecognized at the site less or equal to 2 years , but considering the opportunities of both, in the next future an increase of the possibilities of revascularization could be really evaluated .in vivo in large animal models before trying the silk fibroin grafts in humans (In our review all preclinical studies were analyzed. If a new material for vascular grafts should be studied, the way to the clinical phase is surely longer than other districts especially if it is a scaffold for the cellular growing. Literature reports some good results of pre-clinical phase and good efficacy of the graft as vascular scaffold for arterial, venous and artero-venous reimplantation. Clinical phase requires a particular assurance of the strength and resistance of the graft especially in the anastomotic sites. Results from different experiences are encouraging but we need more data from n humans , 55.6.Our review showed how vascular grafts encompassing silk fibroin are promising for further biological development in animals and humans. Biodegradability and biocompatibility of this natural polymer will likely foster the development as biomaterial of choice for vascular reconstruction.The key point remains that future studies should investigate silk-based vascular grafts in preclinical large animal models, focusing on the optimization of graft design parameters. Since the exact mechanism of graft remodeling and neo-tissue formation is not clear considering the heterogeneity of studies involving SF vascular grafts, further insight into the endothelialization of the grafts must be provided, along with long term outcomes on thrombogenicity and patency rates. Inevitably, the coming of age of SF as predilected biomaterial for vascular reconstruction will be sanctioned by the first clinical trial in humans.


The dry heat treatment (DHT) of starch and hydrocolloid mixtures is gaining acknowledgement since hydrocolloids can enhance the efficiency of DHT. However, the DHT of a starch–protein mixture has been less investigated. In this study, the effects of different proteins including sodium caseinate (SC), gelatin, and whey protein isolate (WPI) added to high amylose and waxy corn starches  prepared by the dry mixing and wet method before and after DHT were studied. The DHT of both starches with WPI and SC prepared by the wet method increased the peak viscosity, but no change was observed when gelatin was added. Dry mixing of HACS with the proteins did not affect the peak viscosity before and after DHT. The gelatinization temperatures and enthalpy of both starches showed a slight decrease with the addition of all proteins and reduced further after DHT. The firmness, gumminess, and cohesiveness of the samples decreased upon DHT. The SEM results revealed that the granules were coated by proteins and formed clusters. Particle size analysis showed an increase in the particle size with the addition of proteins, which reduced after DHT. Under the conditions used, the wet method was more successful than dry mixing and the effects of WPI > SC > gelatin in enhancing the physicochemical properties of the tested starches after DHT. Starch is broadly used in industrial and food applications as a gelling agent, thickener, bulking agent, hygroscopic agent, colloidal stabilizer, and adhesive material. With the increase in industrial demand, there is a constant need to explore alternative and new sources of starch . For manHeat treatment is one of the physical methods of starch modification that is gaining popularity in the food and pharmaceutical industries as it is an affordable, easy, safe, and green  method of starch modification .Dry heat treatment (DHT) involves the heating of starches with low moisture content (less than 10%) at 120–130 °C for 2–4 h . DHT canIt has been reported that the presence of other hydrocolloids such as proteins and polysaccharides can enhance the cross-linking effects of DHT on starch ,13,14,15Whey protein isolate (WPI), sodium caseinate (SC), and bovine gelatin (G) are commonly used in the food industry due to their availability, cost-effectiveness, nutritional value, and diverse functional properties ,21. TherHigh amylose corn starch  and the unmodified waxy corn starch (WCS) of pure amylopectin with only a traceable amount of amylose were purchased from Sigma-Aldrich Pty. Ltd. . Gelatin with a bloom number of 125 and SC with a protein content of 91% was also obtained from Sigma-Aldrich Pty. Ltd. . WPI (protein content: 94%) was secured from Fonterra .To study the effects of starch type (waxy and high amylose), selected proteins , DHT, and preparation methods (wet or dry mixing of the starch and protein), the following samples were prepared according to Lim et al.  with somProtein solutions were prepared separately by adding 3.7 g (dry basis) of WPI, SC, or gelatin to approximately 95 mL MilliQ water  and stirred continuously on a magnetic stirrer at 65 °C for gelatin and at room temperature (21 °C) for SC and WPI until complete dissolution. The gelatin solution was left at room temperature to cool down. Then, 120 g (dry basis) of either HACS or WCS was dispersed into the prepared protein solutions, respectively, to achieve a starch:protein weight ratio of 97:3 while the starch + protein to water weight ratio was maintained at 4:3. The dispersions in a closed container were stirred for 60 min at room temperature using a magnetic stirrer. These were then transferred to Petri dishes and dried in a conventional oven at 45 °C until the moisture content was less than 10%. A drying temperature of 45 °C was selected to ensure that the starch granules and proteins retained their native structure. The drying step may cause some unintentional annealing of the starch samples. The samples were then cooled down to room temperature, ground using a coffee grinder, and sifted through a 200 μm sieve, packed in glass jars, and stored in a desiccator at room temperature for further experiments. The samples prepared with the wet method before DHT are referred to as BHT (before heat treatment), followed by the combination of protein and starch used .The powders obtained in the previous step were used for DHT by heating them in a conventional air-forced oven at 120 °C for 4 h . The samSamples of starch and proteins with the same concentrations above-mentioned were mixed in dry form (without solubilizing in water) as follows: Dry mix—(WPI/SC/G—HACS/WCS) BHT by the dry mixing of native starches and proteins (no heat treatment); and Dry mix—(WPI/SC/G—HACS/WCS)-AHT where the dry mixtures of starch and proteins were heated at 120 °C for 4 h.The pasting properties of the controls and the modified starches were determined in triplicate using a Rapid Visco Analyzer (RVA)  following the method described by Qiu et al.  with sliThe samples were mixed vigorously a” 960’rpm for the first 10 s to disperse the starch granules. After mixing, the paddle speed was lowered to 160 rpm and kept constant throughout the test. Samples were heated from 21 °C to 50 °C and equilibrated for 1 min before ramping up the temperature to 95 °C. The samples were held at 95 °C for 2 min 30 s before being cooled down to 50 °C, and finally held at 50 °C for 2 min. A ramp rate of 12 °C/min was used throughout. Parameters including pasting temperature, peak viscosity, trough viscosity, final viscosity, breakdown (peak viscosity—trough viscosity), and setback  were recorded.o), peak temperature (Tp), conclusion temperature (Tc), and enthalpy of gelatinization (ΔH) were calculated directly from the DSC curves.The thermal properties of all samples were determined following the method outlined by Majzoobi, Saberi, Farahnaky, and Tongdang  using a Starch gels were prepared by directly pouring the hot starch paste obtained at the end of the RVA run into a 10 mm deep plastic cylindrical mold with a 10 mm diameter. To avoid moisture loss, the molds were covered with parafilm and stored at 4 °C overnight. This ensured uniformity in the cooling and gelation conditions for all samples prior to further experimentation.Textural profile analysis of the prepared starch gels was carried out using a Texture Analyzer  equipped with a 25 mm diameter cylindrical aluminum probe. The samples were compressed to 30% of their original height using a double compression test under the following conditions: 3.0 g trigger force, compression speed of 0.25 mm/s, and an interval of 10 s between the two cycles. The TPA parameters measured were based on the compression force versus time curves. Gel firmness was determined as the maximum force during the first compression (height of the first peak), the cohesiveness was calculated as the ratio of the area under the second peak (A2) to the area under the first peak (A1), gumminess was calculated by multiplying the value of hardness and cohesiveness, and elasticity (springiness) was determined as the ratio between the recovered height after the first compression and the original gel height .The particle size distribution was determined by a Malvern Mastersizer 3000 Laser diffraction Aero system . The 100 mg starch samples were weighed and swiftly transferred into the sample cell of the particle size analyzer. The dispersion conditions including 2 bar air pressure and 60 % feed rate were maintained for all samples. The refractive index used was 1.5. Each sample was analyzed in triplicate .Suspensions of all samples were prepared using a 4:1 water–sample ratio. A drop of the suspension was added onto a glass slide and covered with a coverslip before observations under a polarized light microscope  at 50× magnification . The morphological characteristics of all samples were observed using a scanning electron microscope . A dry, finely ground sample was placed on 12.6 mm carbon-coated aluminum pans and coated with a thin film of iridium. Surface micrographs were captured in high vacuum mode with an accelerated electron beam of 25 kV, spot size of 4, and 1000× magnification .p < 0.05, 95% confidence). Statistical Package for Social Science  v 17.0 was used for conducting statistical analysis.All experiments were performed in triplicate. The experimental data presented in the tables were the average of the triplicate value ± standard deviation. Analysis of variance (ANOVA) was conducted while Tukey’s Honest Significant Difference tests were applied to determine the significant differences  had significant effects on the pasting properties of the samples. For example, the mixing of HACS with the tested proteins had no effect on the peak viscosity before and after heat treatment, however, it reduced the final viscosity of all samples. This may indicate that the low concentrations of the tested proteins could not enhance the limited water uptake and swelling characteristics of the high amylose. For dry mixtures of WCS with proteins, WPI caused an increase while SC and gelatin reduced the peak viscosity after DHT. WPI and gelatin had no considerable effect while SC caused a reduction in the final viscosity of WCS.For wet mixtures of HACS and proteins, the peak viscosity increased when WPI and SC were added while it remained unchanged when gelatin was included. Similar results were observed for the WCS and proteins. The final viscosity of the HACS remained unaffected by the DHT of the wet mixtures of WPI and SC, but it was reduced when gelatin was added. For wet mixtures of WCS and proteins, the final viscosity increased when WPI was added, but decreased when SC and gelatin were included. The observed changes could be due to the interactions between the starch and proteins, the denaturation or coagulation of proteins during DHT, the interactions with water as well as the molecular and structural changes of starch and the ratio of amylose to amylopectin. Comparing the values of peak viscosity for HACS and WCS, it seems that the interactions of WCS with WPI or SC were more significant compared with the interactions of HACS with the same proteins before dry heating. Similar results were observed by Zhu et al.  for DHT Protein structure and concentration have significant effects on the pasting properties of starch . Kumar, o), peak (Tp), and conclusion (Tc) gelatinization temperature and enthalpy of the two starches with and without added proteins, before and after DHT are reported in o’ showed a slight increase, which dropped after heat treatment. Ma, Zhu, and Wang [The onset . It was found that the wet preparation method was more successful than the dry mixing method in enhancing the functional properties of the treated starches through the addition of low concentrations of the proteins. Amongst the proteins, WPI was the most effective and gelatin was the least effective protein on the functional properties of starch before and after DHT.The positive outcomes of this study are encouraging since a small concentration of protein can boost the starch viscosity without using chemicals. With the further exploration of different protein–starch interactions, the results of this study can find widespread applications in the food and pharmaceutical industries. Further work is required to study the effects of dry heat treatment on the molecular structure of both starches and proteins.


This research investigated the impact of air plasma and high-pressure plasma treatments on corn starch. The resulting samples were characterized by particle morphology, molecular polymerization degree, molecular functional groups, and crystallinity. SEM analysis revealed that plasma treatment altered the surface morphology of corn starch, with variations observed depending on the duration of treatment. UV/Vis spectroscopy results indicated that longer plasma exposure times increased maximum absorbance values with less complete peak shapes. FTIR results demonstrated that plasma treatment disrupted the crystalline structure of starch, resulting in decreased molecular polymerization. Lastly, XRD results showed a proportional relationship between plasma treatment duration and the intensity of the diffuse peak, indicating that prolonged plasma exposure increased the amorphous nature of starch. Starch serves as the primary source of carbohydrates in the human diet, offering essential energy and playing roles as a bulking agent, thickener, gelling agent, and water retention enhancer . It holdPlasma technology is used in the field of food because of its simple operation, green, pollution-free nature, and ability to save time and effort. Current research on plasma starch mainly includes the following aspects: one is the use of plasma technology to improve starch’s solubility; the second is the use of plasma technology and starch grafting, the cross-linking or depolymerization reaction, which changes the structure and characteristics of starch; and third is the use of plasma-processing starch to destroy its chemical bond and crystal structure and change the structure and characteristics of natural starch so as to achieve the purpose of obtaining modified starch. Plasma, an ionized gas containing a wide variety of active particles, such as electrons, positive and negative ions, free radicals, atoms, and excited or non-excited molecules, possesses the energy required to disturb covalent bonds and initiate various reactions, resulting in the formation of volatile compounds ,7,8. In This study investigates the structural characteristics of corn starch using two distinct plasma treatments as follows: air plasma and low-temperature, low-pressure plasma, under various treatment durations. On the one hand, it guarantees microbial safety in the process of starch modification, and a green way is adopted to enhance the availability of starch and broaden the application scope of starch. On the other hand, it also expands the application scope of plasma technology and broadens its application prospects in the field of food. The molecular structure and functional properties of corn starch were elucidated under these two different plasma treatments, providing valuable insights into the potential application of plasma technology in starch modification.Crystalline iodine, potassium iodide, citric acid monohydrate, and disodium hydrogen phosphate dodecahydrate were all of the analytical grade and sourced from the Beijing Chemical Factory. Soluble starch of an analytical grade was obtained from Beijing Auboxin Biotechnology Co., Ltd. .Plasma generator type A , UV-visible spectrophotometer UV2550 , scanning electron microscope Model TESCAN VEGA II , Fourier transform infrared (FTIR) spectrometer Model Avatar 370 , X-ray Diffraction Spectrometer Model XRD-6000 , High-Speed Refrigerated Centrifuge Model Himac CR 22G , Digital Constant Temperature Water Bath Model HH-2 , and Electronic Analytical Balance Model JA5003 . A test tube, triangle bottle, beaker, volumetric flask, centrifuge tube, glass bar, timer, cuvette, and four layers of gauze were all the instruments used in this experiment.Air plasma action starch solid powder sample: weigh 0.100 g of starch in the ampere tube; use spark discharge excitation to produce air plasma action on starch; set the action time to 0 s, 30 s, 60 s, 90 s, 120 s, 150 s, 180 s, and 300 s; and perform three parallel experiments at each time point. Also, to reduce the thermal effect of plasma produced by excessive heat on starch using the 10 s interval discharge method, starch samples with different plasma action times were obtained. Put the samples after action into the dryer for analysis and testing.Low-temperature and low-pressure plasma starch solid powder sample: weigh 0.100 g of starch in amp tubes and plug skimmed cotton in the seal to prevent the starch powder from sucking during the vacuum; connect the ampere with starch to the vacuum system; spark discharge to generate plasma at 0 s, 30 s, 60 s, 90 s, 120 s, 150 s, 180 s, and 300 s; and perform three parallel experiments at each time point. To reduce the thermal effect of plasma, the effect of excessive heat on the 10 s interval was measured. The starch samples with different plasma action times were obtained. Put the samples after action into the dryer for analysis and testing.6H12O6. It consists of α-D-(+) dehydrated glucose basic units. Starch can be divided into two categories as follows: straight-chain starch and branched-chain starch. The former has a branchless helical structure, while the latter consists of 24–30 glucose residues linked head-to-tail by α-1,4-glycosidic bonds with α-1,6-glycosidic bonds at the branched chains. Spectroscopy was used to characterize the functional groups and structures of the glucose units of starch and to explore the structural changes in starch before and after plasma treatment.Starch is a polysaccharide polymer compound formed by the polymerization of glucose with the chemical formula CAs the starch under examination existed in a powdered form, the prepared samples were placed inside the scanning electron microscope’s sample chamber. Creating a vacuum environment is essential to prevent the powder particles from dispersing, contaminating the scanning electron microscope’s chamber, or compromising the quality of analysis. Therefore, the adhesive paper tape method was employed for sample preparation. The prepared sample was then inserted into the sample compartment of the ion-sputtering instrument, where they were subjected to a 90 s gold coating process under a current of 15 mA. Subsequently, the sample was removed and loaded into the observation chamber of the scanning electron microscope for analysis. The starch samples intended for electron microscopy observation were categorized into two groups as follows: solid powder samples subjected directly to air plasma treatment and samples that underwent low-pressure, low-temperature plasma treatment [The complex formed by starch and iodine was subjected to full-wavelength UV scanning to compare the changes in its maximum absorption wavelength and absorption intensity before and after the plasma treatment and to examine the effect of plasma on its molecular polymerization. Scanning was conducted over a wavelength range of 200 to 900 nm .−1 range, generating infrared spectra [A small quantity of the dried sample was mixed with KBr powder. Under the illumination of an infrared lamp, this mixture was ground in an agate mortar for 2 to 5 min to ensure thorough homogenization. Subsequently, a small portion of the ground mixture powder was pressed into pellets and placed in the sample slot inside a Fourier transform infrared spectrometer. Full-wavelength scanning was performed within the 500 to 4500 cm spectra .The plasma treatment was applied to the starch sample for various durations , and the treated sample powder was used to create an X-ray diffraction sample. The analysis conditions were as follows: Cu Kα characteristic X-rays, copper target, graphite monochromator, voltage set at 40 KV, the emission and anti-reflection slit set at 1°, the receiving slit at 0.3 mm, scanning speed at 1.5 °/min, scanning range at 2θ = 10–65°, and scanning speed at 2 °/min .The size and shape of starch particles can also be used to distinguish the starch species. The morphology and structure of starch are affected by a variety of physical and chemical factors, and the size and shape of natural starch particles after plasma treatment is changed, so the mechanism of plasma action on starch can be inferred by analyzing the change in the structure of starch. Corn starch samples treated with air plasma and low-temperature, low-pressure plasma were subjected to scanning electron microscopy (SEM) to observe and capture representative images of the starch particle morphology. The objective was to investigate the impact of plasma treatment on the starch particle morphology. The results are illustrated in Based on Starch and iodine complexate and form colored complexes, and different colors correspond to different maximum absorption wavelengths. The UV-visible absorption spectrum is the absorption spectrum generated by the ultraviolet–visible light region (200~800 nm) causing the energy level transition, which can reflect the maximum absorption wavelength and wavelength range of the complex. After the action of plasma, starch produces an unsaturated structure, and the absorption of ultraviolet light makes the starch and iodine complex react. Therefore, the UV-visible absorption spectrum reflects the structural characteristics of starch and, to some extent, some of the properties of starch. According to the method outlined in As can be seen from As a method of infrared spectroscopy to characterize and identify compounds, it produces energy level transitions according to the selective absorption degree of radiation in the infrared light region, according to the waveform, intensity, position, and the number of peaks of the spectrum, and is often used to analyze the structure and chemical bond properties of molecules. Infrared spectroscopy is highly specific. In this experiment, a Fourier Infrared Spectrometer was used to analyze the change in the infrared absorption of starch before and after the action of air and low-temperature plasma and to explore the influence of plasma on the molecular group and crystallinity of the sample. There are many methods for infrared spectrometers, and the method in this experiment was the KBr pressing method. Following the procedure outlined in −1, 2914 cm−1, 1655 cm−1, and 1163 cm−1, representing stretching vibrations of O-H bonds involved in hydrogen bonding, asymmetric stretching vibrations of C-CH2-C, bending vibrations of water molecules, and asymmetric stretching motions of C-O-C, respectively [ectively .−1 weakened or disappeared, indicating that plasma weakened the stretching vibration of O-H bonds involved in hydrogen bonding. It could also be due to a feedback inhibition effect, where hydroxyl groups generated by the plasma itself interact with the hydroxyl groups in starch molecules [−1 and 1163 cm−1 were lower than those in the control group, and the area of the C-O-C peak decreased, indicating starch depolymerization. At this point, the plasma disrupts the crystalline structure of starch, leading to increased starch reactivity [olecules . The intactivity .X-ray diffraction technology is mainly used to analyze the long-range ordered structure of starch particles. In the process of starch granule biosynthesis, circadian photosynthesis is very different, so the rate of glucose transfer to cells is also very different. The crystallization area and amorphous area appear alternately, making the internal density of starch different to produce a “growth ring”. X-ray diffraction, when applied to plasma starch, can analyze the crystallinity and type of crystallization of starch molecules. Following the procedure outlined in As can be seen from This study demonstrates that both air plasma and low-temperature, low-pressure plasma treatments have a certain impact on the structural properties of microcrystalline starch. The active substances in the plasma-induced depolymerization of starch chains, resulting in etching on the particle surface and a decrease in the degree of molecular polymerization, led to a reduction in relative crystallinity and an increase in the amorphousness of starch. Importantly, it should be noted that these plasma treatments do not induce any alterations in the crystalline polymorph of starch, nor do they facilitate the generation of novel substances. Instead, their principal effect lies in inducing the transformation of the internal structural configuration of starch molecules between crystalline and amorphous states.This study provides a theoretical foundation for applying air plasma, low-temperature, and low-pressure plasma technologies in the modification of starch. However, research on some starch properties remains incomplete, necessitating further investigative strides in the future. Therefore, plasma technology, as an innovative approach to starch modification, is anticipated to attract increased attention.


Electrospray (ESI) positive ion mode detection and multireaction monitoring (MRM) quantitative analysis were performed. A total of 36 rats were given tenacissoside G, tenacissoside H, and tenacissoside I, respectively, orally (5 mg/kg) and intravenously (1 mg/kg), with 6 rats in each group, to evaluate the pharmacokinetic difference of tenacissoside G, tenacissoside H, and tenacissoside I in rats. The calibration curves showed good linearity in the range of 5–2000 ng/mL, where r was greater than 0.99. The results of precision, accuracy, recovery, matrix effect, and stability met the requirements of biological sample detection methods. The established UPLC-MS/MS method was successfully applied to pharmacokinetic studies of tenacissoside G, tenacissoside H, and tenacissoside I, and the bioavailability was 22.9%, 89.8%, and 9.4%, respectively.An ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method was developed for the determination of tenacissoside G, tenacissoside H, and tenacissoside I in rat plasma. The rat plasma was treated with liquid-liquid extraction using ethyl acetate. The determination was performed on the UPLC HSS T3 column (50 mm × 2.1 mm, 1.8  Marsdenia tenacissima, also known as glaucescent fissistigma root, Rosa banksiae f. lutea (Lindl.) Rehd, etc., is the dry stem and rattan of Marsdenia tenacissima (Roxb) Wight et Am., a plant belonging to the family Asclepiadaceae [Marsdenia tenacissima tastes bitter and slightly cold. It has the effects of clearing heat and detoxifying, relieving cough, and asthma, dispersing knots and relieving pain, and fighting cancer. The series of preparations made from its single medicinal material are widely used in clinics [iadaceae –3, whichiadaceae , 5. It i clinics –8.Marsdenia tenacissima can be used as medicine. Its effective components are steroidal glycosides, alkaloids, and polysaccharides [Marsdenia tenacissima polysaccharides and some fat-soluble components also have antitumor effects [Marsdenia tenacissima is C21 steroidal glycosides [vivo pharmacokinetics of tenacissoside H and tenacissoside I have been reported in literature [The roots, stems, and leaves of charides –11. Its  effects . At presycosides . Polysacycosides , 9, of wterature –16, but High-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) technology has the advantages of high sensitivity, low detection limit, and small sample consumption and is widely used in drug analysis of chemical composition, drug metabolism, and impurity identification , 18. UPLTherefore, this study was to establish an UPLC-MS/MS for the determination of tenacissoside G, tenacissoside H, and tenacissoside I in rat plasma and study the pharmacokinetics and bioavailability to provide a scientific experimental basis for the basic research of clinical pharmacy.Tenacissoside G, tenacissoside H, tenacissoside I, and astragaloside IV   and column temperature set at 40°C. The mobile phase was acetonitrile-water (containing 0.1% formic acid) with gradient elution at a flow rate of 0.4 mL/min and elution time of 6 min. 0–0.2 min, acetonitrile 10%; 0.2–2.4 min, acetonitrile 10%–75%; 2.4–5.0 min, acetonitrile 75%–90%; 5.0–5.1 min, acetonitrile 90%–10%; and 5.1–6.0 min, acetonitrile 10%.Chromatographic conditions were as follows: UPLC HSS T3 column , m/z 817.4 ⟶ 757.5 for tenacissoside H , m/z 837.4 ⟶ 777.5 for tenacissoside I , and m/z 785.4 ⟶ 143.0 for astragaloside IV .Mass spectrometry conditions: nitrogen as conical gas (50 L/h) and desolvated gas (900 L/h); capillary voltage set at 2.5 kV; ion source temperature at 150°C; and desolvent temperature at 450°C. ESI positive ion mode detection and MRM were used for quantitative analysis: μg/mL) were prepared with methanol, respectively. Tenacissoside G, tenacissoside H, and tenacissoside I working solution was obtained by diluting the reserve solution with methanol. Both the reserve solution and working solution were stored at 4°C. Appropriate amount of tenacissoside G, tenacissoside H, and tenacissoside I working solution was added to the blank rat plasma, and then the tenacissoside G, tenacissoside H, and tenacissoside I in rat plasma were 5, 10, 20, 50, 100, 200, 500, 1000, and 2000 ng/mL. Three quality control (QC) samples with plasma concentrations  were prepared by the same method.Tenacissoside G, tenacissoside H, tenacissoside I, and astragaloside IV reserve solution  and 1.0 mL ethyl acetate were added, vortex was mixed for 1.0 min, and it was centrifuged . The organic phase was transferred into another tube and evaporated to dryness at 40°C under a gentle stream of nitrogen. The residue was reconstituted in 100 μL methanol and centrifuged at 13,000 rpm for 5 min. The supernatant was pipetted to an auto sampler vial, and 2 μL was injected into the UPLC-MS/MS for analysis.A plasma sample of 100 μL of the plasma was then transferred to a new 1.5 mL microcentrifuge tube and held at −80°C prior to analysis. Pharmacokinetic parameters were statistically calculated using the pharmacokinetic software (DAS 2.0 version).Tenacissoside G, tenacissoside H, and tenacissoside I were given sublingual intravenous administration (iv) of 1 mg/kg and oral administration (po) 5 mg/kg, respectively, with 6 rats in each group, for a total of 36 rats. All experimental procedures and protocols were approved by the Animal Care Committee of Wenzhou Medical University (xmsq 2023-0689). The 0.4 mL blood was collected from the caudal vein at 0.083 3, 0.5, 1, 2, 3, 4, 6, and 8 h for tenacissoside G; 0.083 3, 0.5, 1, 2, 3, 4, 6, 8 and 12 h for tenacissoside H; and 0.083 3, 0.5, 1, 2, 3, 4, and 6 h for tenacissoside I, collected in heparinized test tubes and centrifuged at 13000 r/min for 10 min. 100 The retention times of tenacissoside G, tenacissoside H, tenacissoside I, and astragaloside IV were 3.32, 3.42, 3.41, and 2.63 min, as shown in r greater than 0.99. The typical regression equation of the tenacissoside G in rat plasma was as follows: y1 = 0.0045x1 + 0.0102 (r = 0.9976), x1 was the concentration of tenacissoside G in plasma, and y1 was the ratio of tenacissoside G peak area to the internal standard. The typical regression equation for tenacissoside H in rat plasma was y2 = 0.0046x2 + 0.0022 (r = 0.9986), where x2 was the concentration of tenacissoside H in plasma, and y2 was the ratio of the tenacissoside H peak area to the internal standard. The typical regression equation for tenacissoside I in rat plasma was y3 = 0.0020x3 + 0.0097 (r = 0.9977), x3 was the concentration of tenacissoside I in plasma, and y3 was the ratio of tenacissoside I peak area to internal standard. The lower limit of quantitation of tenacissoside G, tenacissoside H, and tenacissoside I in rat plasma was 5 ng/mL, and the detection limit was 1.5 ng/mL.The calibration curves of tenacissoside G, tenacissoside H, and tenacissoside I in rat plasma showed good linearity in the range of 5–2000 ng/mL, with The intraday and interday precision of tenacissoside G was within 10%, the accuracy was 90% to 111%, the recovery was over 92%, and the matrix effect was in the range of 94% to 109% .The intraday and interday precision of te nacissoside H was within 13%, the accuracy was 88% to 115%, the recovery was above 88%, and the matrix effect was in the range of 101% to 108% .The intraday and interday precision of tenacissoside I was within 15%, the accuracy was 88% to 110%, the recovery was above 80%, and the matrix effect range was 91% to 99% .The plasma samples of rats were stored in an automatic injector for 2 h, pretreated, and placed at room temperature for 24 h. The plasma samples underwent three freeze-thawing cycles, and the stability test was conducted at −20°C for 30 days. The accuracy of tenacissoside G was 88%–112%; RSD was within 13%. The accuracy of tenacissoside H was 88%–111%; RSD was within 15%. The accuracy of tenacissoside I was 88%–111%; RSD was within 15% . The resThe concentration-time curves of tenacissoside G, tenacissoside H, and tenacissoside I in rat plasma are shown in In order to obtain the best mass spectrum conditions, the positive and negative ion modes were used for monitoring. The responses of tenacissoside G, tenacissoside H, and tenacissoside I were higher in the positive ion mode than the negative ion modes. After optimizing various parameters, the mass spectrum parameters can meet the requirements of accurate quantification of effective substances in biological samples. Through the standard sample, the capillary voltage and the collision energy were optimized.The commonly used biological sample pretreatment methods were liquid-liquid extraction (LLE) –24, soliDuring quantitative analysis, it is necessary to add an internal standard substance of known concentration as a quantitative reference for the compounds to be measured in the sample. The internal standard substance should have similar physical and chemical properties to the compound to be tested, and be stable in the sample, and be easy to detect and quantify. Commonly used internal standard substances include isotope-labeled compounds and structural analogues. Astragaloside IV has similar physical and chemical properties to tenacissoside, and it was selected as the internal standard.β-tenacigenin B, tenacigenine B, and tenacigenine A) and their corresponding glycosides (tenacissoside A and tenacissoside B) in rat plasma [Zhao et al. studied the plasma concentration and pharmacokinetic process of tenacissoside A in rats by the LC-MS/MS method . Medroxyt plasma . After dIn this study, the UPLC-MS/MS technique was established for the determination of tenacissoside G, tenacissoside H, and tenacissoside I in rat plasma in the range of 5–2000 ng/mL. The rat plasma was treated with liquid-liquid extraction using ethyl acetate, and astragaloside IV was used as internal standard. The selectivity, linearity, precision, accuracy, recovery, and stability of this method have been verified, and it has been applied to the pharmacokinetic study of tenacissoside G, tenacissoside H, and tenacissoside I in rats, and the bioavailability was calculated to be 22.9%, 89.8%, and 9.4%, respectively.


Texture analysis (TA) provides additional tissue heterogeneity data that may assist in differentiating peripheral zone (PZ) lesions in multiparametric magnetic resonance imaging (mpMRI). This study investigates the role of magnetic resonance imaging texture analysis (MRTA) in detecting clinically significant prostate cancer (csPCa) in the PZ.This retrospective study included 80 consecutive patients who had an mpMRI and a prostate biopsy for suspected prostate cancer. Two radiologists in consensus interpreted mpMRI and performed texture analysis based on their histopathology. The first-, second-, and higher-order texture parameters were extracted from mpMRI and were compared between groups. Univariate and multivariate logistic regression analyses were performed using the texture parameters to determine the independent predictors of csPCa. Receiver operating characteristic (ROC) curve analysis was conducted to assess the diagnostic performance of the texture parameters.In the periferal zone, 39 men had csPCa, while 41 had benign lesions or clinically insignificant prostate cancer (cisPCa). The majority of texture parameters showed statistically significant differences between the groups. Univariate ROC analysis showed that the ADC mean and ADC median were the best variables in differentiating csPCa (p < 0.001). The first-order logistic regression model (mean + entropy) based on the ADC maps had a higher AUC value  than other texture-based logistic regression models (p < 0.001).MRTA is useful in differentiating csPCa from other lesions in the PZ. Consequently, the first-order multivariate regression model based on ADC maps had the highest diagnostic performance in differentiating csPCa. Prostate cancer is the second most frequently diagnosed cancer in men and the fifth leading cause of death worldwide ,2. DiagnMultiparametric magnetic resonance imaging (mpMRI) of the prostate is useful in detecting csPCa –9. csPCaTexture analysis (TA) is a quantitative image analysis method that can evaluate the signal heterogeneity of the images by examining the distribution and connectivity of pixel intensities within normal and pathological tissues. In medical images, macroscopic heterogeneity can reflect histopathological heterogeneity –22. ReceTA provides additional tissue heterogeneity data that may assist in differentiating PZ lesions in mpMRI. This study investigates the role of MRTA in detecting csPCa in patients with suspicion of prostate cancer.Between January 2018 and January 2020, 127 patients who had a clinical suspicion of prostate cancer and underwent mpMRI were evaluated retrospectively. mpMRI scans were accessed via the picture archiving and communication system. The following inclusion criteria were queried from our electronic hospital medical records system: (1) mpMRI performed within 6 months before prostate biopsy; (2) csPCa in the PZ using targeted biopsy or radical prostatectomy; and (3) benign lesions or clinically insignificant prostate cancer (cisPCa) in the PZ using targeted or systematic biopsy. Patients with csPCa in the transition zone (n = 10), those who did not have mpMRI (n = 13), those who did not have pathological diagnosis (n = 6), those who had imaging artifacts (n = 6), those who had previous treatment (n = 7), and those who had biopsy before mpMRI (n = 5) were excluded from the study. Finally, 80 consecutive cases were included in the study. The study was approved by the institutional ethics committee of our hospital, and the requirement for written informed consent was waived.2), and dynamic contrast-enhanced MRI (DCE-MRI) with a T1-weighted volumetric interpolated breath-hold examination sequences . On dynamic series, after the first two phases, 0.1 mmol/kg of gadobutrol  was injected intravenously at a rate of 3 mL/s with an MR-compatible automatic injector, followed by 30 mL of saline flush.MRI examinations were performed using a 3T scanner  with a pelvic-phased array coil. The MRI protocols were consistent with PI-RADS v2.1. All patients received a spasmolytic to reduce bowel peristalsis. The MRI protocols included axial, coronal, and sagittal T2-weighted turbo spin-echo sequence , diffusion-weighted imaging (DWI) with a single-shot echo planar imaging sequence . These forms of textural analysis consider only pixel intensity and do not provide spatial information between pixels. The most common histogram features include mean, standard deviation (SD) or variance, skewness (asymmetry), kurtosis (pointedness or flatness), first-order entropy (irregularity), and mean of positive pixels ,22,27,28More complex computations such as second- or higher-order statistics, which explore the relationship between pixels within the ROI, provide spatial information between pixels. Second-order statistics, such as second-order entropy, energy, homogeneity, dissimilarity, and correlation, are based on gray-level cooccurrence matrix (GLCM) and compare the relationship between two pixels. GLCM is a two-dimensional histogram capturing the frequency of cooccurrence of pixel pairs of certain values in a given spatial range. Higher-order statistics are based on neighborhood gray-level difference matrix (NGLDM) and include contrast, coarseness, and busyness. NGLDM can demonstrate coarseness and complexity within an image by comparing the relationship between more than two pixels ,22,27,28® 3.0, Olea Medical, La Ciotat, France) for segmentation and TA. The ROI was drawn using the freehand technique to encompass the entire lesion on the ADC map, T2-weighted images (T2WIs), and early and late postcontrast T1-weighted images (T1WIs) in patients with csPCa. In patients with benign lesions or cisPCa, the ROI was determined to include the lesion if visible on mpMRI, and to include the entire peripheral zone if it is not visible or has indistinct margins. After the segmentation, the first-, second-, and higher-order texture parameters were extracted from the selected ROI , aware of all clinical, and histopathological findings, interpreted mpMRI in consensus. According to the histopathological findings, csPCa and benign lesions or cisPCa were identified at dedicated workstations. All images were imported to commercially available software  and MedCalc for Windows, version 19.2.6 . The normality of the distribution of continuous numerical variables was analyzed using the Shapiro–Wilk test. Descriptive statistics were presented as mean ± SD or median (range) for continuous numerical variables and count (percentage) for categorical variables.t-test was used to compare two independent groups. Univariate and multivariate logistic regression analyses were performed using texture parameters to define the independent predictors of csPCa. The variables highly associated with each other were excluded from the regression model due to a multicollinearity problem using the backward stepwise analysis method used in multivariate regression analysis. Odds ratios (ORs) were used to denote the effect size of the variables in the regression model. The diagnostic performance of univariate and multivariate texture parameters was assessed using receiver operating characteristic (ROC) curve analysis. During multivariate analysis, the area under curve (AUC) values were derived according to the predictive values of the regression model. Comparisons among AUC values were assessed using the Delong method [The Student g method . In all The study enrolled 80 patients  with a mean age of 68.05 ± 8.45 years . For all patients, the mean prostate volume was 61.85 ± 34.43 mL and the mean PSA level was 9.76 ± 8.70 ng/mL. Histopathological analysis showed that 39 patients (48.8%) had csPCa, while 41 patients (51.2%) had benign lesions or cisPCa.Except the kurtosis, all first-order texture parameters in csPCa in the PZ were significantly different. In csPCa; mean, median, and entropy were significantly lower, while skewness and uniformity were significantly higher (p < 0.01).Except the GLCM correlation, GLCM difference entropy, NGLDM coarseness, NGLDM complexity, and NGLDM strength, all second- and higher-order texture parameters were significantly different in csPCa in the PZ. Furthermore, csPCa had significantly lower GLCM joint entropy, GLCM inverse variance, and GLCM sum entropy while having significantly higher GLCM contrast, GLCM joint energy, NGLDM contrast, and NGLDM busyness (p < 0.05).All first-order texture parameters in csPCa in PZ showed significant differences; mean, median, and entropy were lower, while skewness, kurtosis, and uniformity were higher (p < 0.05).Except the GLCM difference entropy, NGLDM coarseness, NGLDM complexity, and NGLDM strength, all second- and higher-order texture parameters in csPCa in the PZ were significantly different. Furthermore, GLCM correlation, GLCM joint entropy, GLCM inverse variance, and GLCM sum entropy were significantly lower for csPCa, whereas GLCM contrast, GLCM joint energy, NGLDM contrast, and NGLDM busyness were significantly higher (p < 0.05).On early postcontrast T1WIs, entropy was significantly lower among the first-order texture parameters, whereas uniformity was significantly higher in csPCa in PZ (p < 0.05).On late postcontrast T1WIs, mean, median, and uniformity were significantly higher among the first-order texture parameters, whereas skewness and entropy were significantly lower in csPCa in PZ (p < 0.05).On early and late postcontrast T1WIs, GLCM difference entropy, GLCM joint entropy, and GLCM sum entropy were significantly lower in csPCa in PZ, whereas GLCM contrast, GLCM joint energy, and NGLDM busyness were significantly higher. Furthermore, GLCM correlation in csPCa in the PZ was significantly lower on late postcontrast T1WIs (p < 0.05).On early and late postcontrast T1WIs, there was no significant difference in other texture parameters in the PZ (p > 0.05).The results of the univariate and multivariate logistic regression analyses on texture parameters are shown in Among the first-order texture parameters, mean and entropy and, among the second- and higher-order texture parameters, GLCM contrast, GLCM difference entropy, GLCM joint energy, GLCM joint entropy, GLCM sum entropy, and NGLDM busyness were independent predictors of csPCa on the ADC maps.The ROC analysis results of multivariate logistic regression models for detecting csPCa in PZ are shown in Univariate ROC analysis showed that the ADC mean and ADC median had the highest AUC value  for differentiating csPCa in PZ (p < 0.001). In addition, the first-order entropy based on the ADC maps had a higher AUC value  than other texture features (p < 0.001).The first-order logistic regression model (mean + entropy) based on the ADC maps had a higher AUC value  than other texture-based logistic regression models (p < 0.001).Recently, radiomics has been used for cancer detection, staging, and treatment response assessment ,30,31. MTA assesses tumor heterogeneity by analyzing the distribution and relationship of pixel intensities in medical images and provides quantitative information about the lesion that cannot be distinguished by a radiologist –22,28.Studies have shown that TA could be useful in diagnosing prostate cancer ,28,31,32b values, which limit the role of the ADC mean values in the distinction between benign and malignant lesions and the cut-off value [The mean ADC values vary according to the cell membranes, nuclei, and extracellular space at the tissue level. The mean ADC values are low in high-cellular tumors . Studiesff value .In this study, various first-order , GLCM  and NGLDM (busyness) texture parameters provided good diagnostic performance for identifying csPCa. Among these texture parameters, first-order entropy obtained from the ADC maps had the excellent diagnostic performance and the higher AUC value. In addition, other entropy parameters obtained using second-order statistics showed significant diagnostic performance. Entropy indicates uncertainty/randomness within the image. High entropy values represent an increase in heterogeneity in the image ,28,31. WUniformity, which is inversely correlated with entropy, is a measure of the gray-level distribution of the image; lower values represent heterogeneity ,28,31. ISkewness measures the asymmetry of the histogram curve and can be negative or positive ,28,31. WContrast, also called variance or inertia, represents local signal intensity variation in an image; higher values indicate a greater difference in signal intensity between adjacent voxels. Correlation measures the linear dependency of signal intensity between voxel pairs. It can be positive or negative and takes values between 0 (uncorrelated) and 1 (perfectly correlated) ,28,31. WMultivariate regression analysis showed that models based on ADC maps had higher diagnostic performance in the assessment of PZ lesions than T2WI and DCE-MRI. Texture parameters derived from ADC maps have provided more diagnostic benefits in the diagnosis of prostate cancer . In the Some limitations in this study should be considered. First, the sample size is small. Second, the PI-RADS score of the lesions was not considered in the study. Prospective TA studies using the PI-RADS in a larger series will form the basis of machine learning techniques for prostate cancer. Finally, mpMRI-directed transrectal US-guided fusion biopsy was used as a reference standard instead of radical prostatectomy in most patients.In conclusion, MRTA is useful in differentiating csPCa from benign lesions or cisPCa. In addition, the first-order multivariate regression model obtained by adding entropy to the mean had the highest diagnostic performance in identifying csPCa.


Heterosigma akashiwo occur in the warm-temperate Sundays Estuary on the southeast coast of South Africa. The response in body condition and assemblage structure on larval estuarine roundherring (Gilchristella aestuaria) was measured in conjunction with bloom conditions, water quality and zooplanktonic prey and predators. Larvae and early juveniles were sampled during varying intensity levels, duration and frequency of hypereutrophic blooms. This study demonstrated that extensive HABs could significantly impact larval roundherring, G. aestuaria, by decreasing larval nutritional condition and limiting their growth, resulting in poor grow-out into the juvenile phase. Poor condition and growth may likely affect recruitment success to adult populations, and since G. aestuaria is an important forage fish and zooplanktivore, poor recruitment will hold consequences for estuarine food webs.Eutrophication-driven harmful algal blooms (HABs) can have secondary effects on larval fishes that rely on estuaries as nurseries. However, few studies worldwide have quantified these effects despite the global rise in eutrophication. This study presents a novel approach using biochemical body condition analyses to evaluate the impact of HABs on the growth and body condition of the larvae of an estuarine resident fish. Recurrent phytoplankton blooms of  Recruitment of juvenile fishes into adult populations is intricately linked to the success of early life history stages . TherefoThe growth-mortality hypothesis  posits tEnvironmental conditions are critical in larval growth and mortality in estuarine nursery areas. Physico-chemical conditions may directly affect growth rate by regulating biochemical processes (temperature)  (a·L−1) during the monospecific blooms of H. akashiwo (Raphidophyceae) . A local study in the Sundays Estuary has shown negative effects from HABs on the gills of juvenile Mugilidae , were selected as suitable candidates because it completes its entire life cycle within the estuary and are commonly found in high abundance in South African estuaries . The diets of juvenile R. terranatalis and adult M. wooldridgei overlap with G. aestuaria  and biochemical body condition  in The warm-temperate, permanently open Sundays Estuary  is located on the southeast coast of South Africa . The staH. akashiwo blooms are most prominent , a commonly used indicator in eutrophication assessment methodologies  bloom concentrations. These were defined as standard blooms ≥20 μg·L−1 (H. akashiwo ≥ 204 cells·mL−1) and hypereutrophic blooms ≥80 g·L−1 (≥ 2781 cells·mL−1).Water samples were collected at depth intervals of 0 (sub-surface), 0.5, 1 m and the bottom of the water column and preserved with 1 mL of 25% glutaraldehyde solution. Phytoplankton biomass  fitted with flowmeters . The plankton nets were lowered into the water on either side of the boat, one at the sub-surface and the other at the near-bottom (± 1.5 m) and towed for 3 min at one to two knots at an oblique towing course to sample across the channel     were enu3) of 32.7 . Length-frequency graphs were created for each of the sampling days. The developmental stage of each larva was recorded using descriptions by G. aestuaria for body condition analyses. These were preserved in individual vials containing RNAlater®. Only flexion and postflexion larvae between 7 and 16 mm were included in the biochemical body condition analyses since this was the most dominant size range represented in the samples.Larvae collected for biochemical analyses were measured separately from those collected for assemblage structure analyses. Additional plankton tows were performed each sampling day within the 5–10 salinity range to collect 20 individuals per tow of larval G. aestuaria followed techniques described in G. aestuaria larvae was measured in ImageJ v1.8.0  and (ii) TE buffer . Fluorescence was measured in steps to determine the concentration of RNA using self-fluorescence (pure samples), total fluorescence (after addition of EB) and the remaining DNA fluorescence post-incubation in RNase  for 30 min at 37°C. Sample fluorescence was measured at an excitation wavelength of 355 nm and an emission wavelength of 590 nm at 25°C. RNA fluorescence was obtained by subtracting the total fluorescence from the DNA fluorescence. The RNA amount (μg) of each G. aestuaria larva was calculated from the relative fluorescence values using calibration curves and dilution factors. The slope of the RNA calibration curve was multiplied by 2.2 to calculate the slope of the DNA calibration curve, which adjusted for the relative fluorescence intensity difference of RNA and DNA  to indicate the body condition and growth (Fluorescence measurements were determined using an Ascent Fluoroscan (Thermo Fisher). Two dispensers of the Ascent Fluoroscan were prepared with (i) ethidium bromide . Copepods, P. hessei and P. longipatella were included as the prey components of the fish larvae, and ovigerous P. hessei was also included due to the previous findings of the importance of these copepod eggs in larval G. aestuaria diet  was performed using a negative binomial distribution, a log-linked function and automatic model selection based on Bayesian Information Criterion. GAMs were generated using R statistical software  with MuMc levels . Chloropestuaria . GAMs weestuaria , a time-H. akashiwo bloom density recorded during 2018 was more than double that of 2016 . The contrasting conditions of 2016 and 2018 provided two scenarios, whereby 2016 represented short bloom duration and low persistence, whereas 2018 represented persistent, longer enduring HABs. For this reason, the years were used as a comparison of bloom conditions in addition to bloom density.Both 2016 and 2018 experienced hypereutrophic blooms in the Sundays Estuary. However, the years differed in bloom duration, persistence (blooms recorded consecutively) and maximum density. In 2016, standard blooms were absent for 55.6% of the study period, whereas a single hypereutrophic bloom, which lasted for two sampling events  between 2016 and 2018, with higher salinities in the mesohaline zone in 2016. Salinity ranged from 9.7 to 22.5 in the pilot study and 4.3–25.8 in 2018. Turbidity fluctuated throughout the study period in 2016 and 2018 with no significant difference . Even though there was no notable difference in dissolved oxygen between the years, 2018 reached a slightly lower minimum (3.3 mg·L−1) and higher maximum (13.8 mg·L−1) than 2016  . Dissolved oxygen (mg·L−1) was greater during hypereutrophic blooms , whereas periods of low bloom presence and absence were similar  in both sampling years. A Spearman correlation test indicated a positive association between dissolved oxygen and H. akashiwo density .The mean temperature in the study area ranged from 20.0 to 26.2°C during 2016 and 20.7 to 25.8°C in 2018 . Salinit mg·L−1) . FurtherP. hessei was greater in 2016 (2409 per m3) than 2018 , mean densities of ovigerous P. hessei were similar between the years  . Both mysid species occurred at greater densities during 2016 . In terms of bloom presence, only R. terranatalis differed notably and was lower during the hypereutrophic blooms .The density of dominant zooplankton species was greater during 2016 compared with 2018 . Althoug= 0.433) . The denP. hessei  and R. terranatalis  were positively correlated with H. akashiwo density, whereas M. wooldridgei  was negatively correlated. Furthermore, dissolved oxygen was positively correlated with P. hessei  and R. terranatalis  and negatively correlated with P. longipatella .Spearman rank correlation analyses revealed that ovigerous G. aestuaria density was higher on average during 2018 (1010.69 per m3) compared with 2016 , although the highest density in this study occurred during 2016 (P = 0.0012), whereas preflexion larvae contributed similarly to both years  (P = 0.040). Early juveniles constituted a higher proportion of the 2018 assemblage than 2016 .Mean larval ing 2016 . The 201= 0.507) . FlexionG. aestuaria assemblage  consistently contributed >50% to the 2018 semblage . Since psemblage . In contsemblage . During G. aestuaria larvae at different developmental stages was assessed to understand the impacts of the blooms on growth. Time-sequenced length-frequency graphs  compared with 2018 . During 2018, there were only slight changes in the length classes per day . However, no larvae were encountered between 15.9 and 21.0 mm. During 2016, mean length ranged between 6.0 and 12.5 mm . The length-frequency analysis also indicated that many small larvae were consistently present in samples (P < 0.001). However, differences in larval length were negligible (P > 0.1) when compared among the states of bloom presence. The GAM results on G. aestuaria length explained little deviance (18.8–25.6%) (The size of the y graphs  show the per day . Mean le samples . Preflex8–25.6%) .P = 0.002) despite similar lengths during the sampling years . Mean RNA/DW was greater during the pilot study in 2016 (13.83 per μg), compared with 2018  .The mean dry weight of larvae collected for body condition analyses was higher during 2016 (625.1 μg) compared with 2018  . MoreoveG. aestuaria RNA/DW correlated with dissolved oxygen, turbidity and density of M. wooldridgei and P. longipatella (−1 (P < 0.001). There was a linear relationship between turbidity and RNA/DW, whereby higher RNA/DW was related to lower turbidity (P < 0.001). Similarly, RNA/DW decreased with the density of P. longipatella (P < 0.01) while increasing with the density of M. wooldridgei (P < 0.001). The GAM explained 46.2% of the deviance in RNA/DW.A time-lagged GAM analysis (3–4 days prior) indicated that ipatella . The amoG. aestuaria body condition would fluctuate between bloom phases with larvae being in a worse condition during peak HAB bloom conditions. Poorer body condition of G. aestuaria larvae was related to hypereutrophic blooms, exacerbated by extended bloom periods. It was observed that larval length stage progression was poor during more persistent phytoplankton blooms compared with sporadic, short-lived HABs. Moreover, larval G. aestuaria assemblage consisted of fewer late-stage (flexion/postflexion/early juvenile) larvae when bloom conditions were persistent, and a low proportion of the larvae reached the early juvenile stage (maximum proportion = 1.3%). Despite the year-round spawning of G. aestuaria, with spring and summer peaks  would determine larval condition, especially P. hessei eggs, which are frequently consumed by the larvae , the physical environment and prey availability. Some studies have reported positive and negative relationships between larval condition and predator density  the ecological effects of shifts in diet and the nutritional value of prey of G. aestuaria larvae under bloom conditions. During the research in the Sundays Estuary on HABs, mucilage from H. akashiwo has been observed  the direct effects of plankton . It is, −1). It was demonstrated that extensive HABs could significantly impact larval roundherring, G. aestuaria, by decreasing larval nutritional condition and limiting growth into older developmental stages. Poor condition and growth may affect the recruitment success of future adult populations of fishes (H. akashiwo HAB-affected estuarine nurseries. Furthermore, the loss of key role players in the estuarine food chain such as abundant forage fishes and planktivores can also lower estuarine nursery quality for other early stage fishes. These findings motivate for future research into the mechanisms that drive the loss in condition and growth of larvae as well as the subsequent effects on other young fishes and nursery quality in estuaries.This study presents a novel approach using biochemical body condition analyses and size frequency distributions to evaluate the impact of HABs on larval fish growth and body condition. To the authors’ knowledge, no other published works exist using this approach worldwide. HABs were linked to supersaturated oxygen concentrations (max 13.8 mg·LSupplementary_Materials_Table_S1_fbad013Click here for additional data file.Supplementary_Materials_Table_S2_fbad013Click here for additional data file.


In addition, this review will highlight the implications of each starch property adjustment.Starch has been a convenient, economically important polymer with substantial applications in the food and processing industry. However, native starches present restricted applications, which hinder their industrial usage. Therefore, modification of starch is carried out to augment the positive characteristics and eliminate the limitations of the native starches. Modifications of starch can result in generating novel polymers with numerous functional and value-added properties that suit the needs of the industry. Here, we summarize the possible starch modifications  However, the ratio of amylose and amylopectin in any native starch varies with the plant genotype.Starch is an essential biopolymer found abundantly in nature. It is the main energy and nutrient source of plants to overcome periods where photosynthesis is not possible but also for heterotrophic species such as humans and animals. Furthermore, starch is not only a source of dietary carbohydrates but also an ubiquitous raw material commonly utilized in non-food industry. In principle two types of starch exist, storage starch and transitory starch. The latter is synthesized in chloroplasts of leaves and followed a diurnal cycle of synthesis and degradation. By contrast, storage starch is synthesized in non-photosynthetic active plastids, such as amyloplasts in seeds or storage organs and remains unmetabolized for a longer time period until it is degraded once to support regrowth/sprouting or germination. Typically, the amount of storage starch is much higher than that of transitory starch and therefore this type of starch is commercially used. Compared to other polymers, starch is a multifunctional biomaterial with great potential due to its enormous amount, low cost, and non-toxic properties. Starch is biosynthesized as semi-crystalline granules and is composed of two glucans amylose and amylopectin. Chemically, both homopolysaccharides consist of α-D-glucose units, whereby the linear chains formed by α-1,4-glycosidic linkages and the branching points are introduced by α-1,6 glycosidic bonds [Moreover, amylopectin is responsible for the semicrystalline structure of the starch granules. The branching points of the amylopectin molecules are clustered, so neighboring glucan chains form double helices, creating crystalline lamellae. The crystalline lamellae alternate with amorphous regions where the branching points are located . The diahttps://www.marketsandmarkets.com/Market-Reports/modified-starch-market-511.html, accessed on 15 August 2023). In addition, the demand for processed and convenience foods is lifting the need for modified starch. Therefore, the usage of modified starch across numerous sectors is expanding as innovative experiments and new technologies in many industrial sectors (food and non-food) progress. Furthermore, due to its high economic and social impact on human life, the starch modification area will be more appealing in the future. Generally, modifications may be divided into two groups, the in planta modifications, which can be achieved by genetic manipulation via breeding and molecular biology technologies. The second group includes all modifications outside the plant system that can be obtained by physical, chemical, and/or enzymatic methods. These modifications will be explained in more detail in the following sections.During the last decade, the modified starch market has constantly been growing. According to Markets data, the worldwide modified starch trade is expected to be valued at USD 13.7 billion in 2022 and set to gain USD 15.9 billion by 2027, recording a compound annual growth rate (CAGR) of 3.1% , phosphoryl chloride, adipic acid mixed with anhydride and sodium tripolyphosphate. Cross linking involves the molecular reaction between the reactive hydroxyl groups on starch with multifunctional reagents resulting in ether or ester linkages. Such interactions not only take place between single chains but also links side by side chains as well . As a reEsterification takes place on hydroxyl groups at the C3, C2 and C6 positions (order indicates increasing reactivity) , wherebyImproved properties regarding thermal stability, rheological behavior, and, with restrictions, ionic activity are observed for etherified starch. The etherification substitutes hydroxyl groups with anhydroglucose units using functional groups, which can be positively or negatively charged. Moreover, the generation of amphoteric or non-ionic starches are possible. One important example of a negatively charged starch ether is carboxymethyl starch, classified as a green polymer that covers a broad spectrum of applications in industry, due to its great hydrophilicity and therefore, cold-water solubility, as well as flocculation behavior, for example, in pharmaceutical approaches as a drug delivery system or in waste water purification as biosorbents to sequestrate heavy metals ,51.Another opportunity for modifying starch is oxidation, which can cause depolymerization of starch, whereby it enhances hydrophilicity, disrupts molecule linearity and crystallinity, and reduces retrogradation as well as enthalpy by the formation of carbonyl and carboxyl groups in native starch by the use of an oxidizing agent . There aDuring grafting, a polymer chain (grafting onto) or monomers such as vinyl units (grafting from) is introduced into the starch, resulting in copolymerization that combines the features of both polymers. The initiation of the copolymerization process occurs in particularly at the C1–C2 end groups and the C2–C3 position in the presence of a free radical initiator ,53. TherThe acid hydrolysis of starch is performed at temperatures below the gelatinization temperature and favors properties such as solubility, pasting viscosity, gelatinization enthalpy, and swelling power, whereas the effect on granular morphology differs among species, tissues, and degrees of hydrolysis. For instance, a slight hydrolysis reveals no significant morphological alteration, which presupposes that the starch has not been boiled . In geneGenerally, physical modifications can be used to interrupt or alter the granular size and/or packing arrangement. Thus, physical modifications can generate starch with similar characteristics as those obtained from chemical treatments, but without any agent which is toxic or must be removed thereafter. The methods are easy, cost-effective, and largely environmentally friendly . OverallPregelatinization is a very simple modification that includes the boiling of starch in an aqueous solution followed by drum or spray drying, which then results in reduced hydrogen bonding, fragmentation, and the molecular disintegration of starch granules. The spray dried gelatinized starch finds its application in the microcapsule industry for the purpose of drug release due to its low cost and availability . The preExtrusion is a thermomechanical method involving continuously forcing gelatinized starch under pressure out of a shaping aperture. This technique plays a role in the packaging and food industry . HoweverThe most practiced thermal methods are heat-moisture treatment (HMT) and annealing. The physicochemical properties of starch are greatly enhanced by both of these hydrothermal processes without affecting the granular structure. Both methods differ in the moisture content used. While annealing employs an excessive amount of water (>40%), a long processing time, and temperatures over the glass transition but below the gelatinization temperature, HMT requires a high temperature and a low moisture content range (10–35%). HMT alters the crystalline and amorphous layers of granules and thus reduces swelling property, solubility, and viscosity, simultaneously raising the pasting temperature. However, the efficiency of this technique greatly depends upon the process variables and the source of starch. The lower the moisture used, the greater is the alteration on the starch granule surface and hence increased susceptibility to other types of modifications. Additionally, starch derived from cereals shows lesser sensitivity to alterations than the tuber and root starches .Annealing improves the crystallinity by providing molecular rearrangement and lowering the free energy; thus, more organized structures can form . Since tMicrowaves are electromagnetic waves with a wavelength between 1 mm and 1 m corresponding to the frequency range between 300 MHz and 300 GHz . Polar mMicronization is a significant reduction of the average particle size. It can be a thermal treatment with infrared radiation in the range of 1800 to 3400 nm. It also heats starch in a short amount of time . Non-theAnother technology used for physical modification is the γ-ray irradiation treatment of starch. Plenty of research has been done in the past few decades to assess the impact of γ-radiation on starch granules. Radiation processing via γ-irradiation includes the utilization of a radioactive isotope, either in the form of cesium 137 or cobalt-60. These isotopes can emit high energy γ-rays or photons and are capable of invading great depth into the target product. The γ-irradiation causes a decrease in crystallinity, the swelling index, and pasting properties. In addition, starch granules become internally cracked, but surface cracking is not observed .Factors Furthermore, starch can be exposed to ultra-high pressures (100–1000 MPa). High pressure is a green and eco-friendly technique capable of altering non-covalent chemical linkages with very minor effects on the covalent linkages. Depending on duration, pressure intensity, temperature, and starch origin, the swelling power and solubility increase and retrogradation is delayed. Moreover, the thermal and pasting properties, as well as the amounts of resistant, fast, and slow digestible starch, are altered significantly . Ultra-hThe application of frequencies above 20 kHz to agitate particles is known as ultrasonication. The sonication results in acoustic cavitation which is characterized by the rapid development and dissolution of bubbles in a liquid exposed to heat and pressure. The effects of ultrasonic treatment on starch properties depend on the time, intensity, frequency, type, and structure of the starch. However, it has been demonstrated that ultra sonification generates cracks and cavities on the granule surface and thereby increases the water-solubility and rheological characteristics . This kiPulse electric field technology, wherein a field strength of around 10–80 kV/cm for a period of micro- or milliseconds is used, can result in split or deformed starch granules. With increasing electric field strength, pasting properties such as the peak, breakdown, and final viscosity drop, and the crystallinity decreases. However, product properties  and temperature conditions of the reaction system can affect the modification of starch by influencing the pulse electric field intensity. Furthermore, ohmic heating can lead to gelatinization ,97.Another innovative non-thermal technology for modifying starch is cold plasma treatment. Plasma is a mixture of ions, radicals, free electrons, and neutral atoms and molecules. Accordingly, the degree of ionization can vary between 1 and 100% and is generated via an energy input through electric or magnetic fields and, radio waves, microwaves, or heat. Among these, an electric field is the most frequently used energy source. Likewise, the effect on starch properties can significantly differ depending on the exposure time, power and volt supply, and the gas composition used, so several modifications are possible, such as cross-linking, depolymerization, and etching. Thus, plasma treatment leads to either an increase or decrease in molecular weight. The same is also valid for the gelatinization temperature. Furthermore, this physical method destroys starch granules’ crystallinity. The degree of moisture content must also be considered because the bombardment with plasma can produce hydroxyl radicals that favor further breakdown of crystalline regions. This can also cause cracks and holes in granules. The use of plasma is a promising technique with numerous applications, as it allows for several types of starch modification. Importantly, it is also fast, simple, and eco-friendly, leaving no chemical and/or toxic waste products. Starch-related plasma research is in its early stages and there is an enormous energy demand that accompanies the process of utilization, so, in the end, a cost-benefit calculation is crucial . This teEnzymatic modification is referred to as the utilization of enzymes that alter starch parameters. This kind of modification is primarily applied in the food industry. Traditionally, starch modifications by an enzymatic reaction are generally divided into four categories: endoamylase, exoamylase, debranching enzymes, and transferases  However,α-amylases are a well-known example of the endoamylase category. These enzymes randomly hydrolyze starch at any -linkage within the two starch polymers, amylose, and amylopectin. As a result, they reduce the chain length . DependiThe debranching enzyme category includes isoamylase  and pullThe transferases include amylomaltase ,133, cycThe hydrolysis rate values for each enzyme mentioned above significantly differ depending on the starch. For native starch, the differences mainly depend on its structural properties ,135.Sinc2, which is available as a quasi-inexhaustible resource, and on the other hand the use of cold plasma.Finally, there are numerous possibilities and a great need to modify starch to change the properties in the direction required for the respective application area see . Not allThe key to success, however, is most likely to be found in the use of interdisciplinary methods; in a combination of chemical/physical methods and the use of enzymes, the reactions can occur under very mild and efficient conditions. One such example is the starch esterification, where a very high degree of substitution of the hydroxyl groups can be achieved by using lipase in combination with microwave irradiation in an ionic solution .The impact of starch and its utilization has enormously increased in recent years and with it the development of novel technologies that pursue the aim of modifying starch in the best conceivable manner for the respective demands of the application. The precise modulation of starch enhances its industrial applicability. Several ways have been devised to adjust the starch characteristics which can be implemented either inside the plant or outside the plant system . However, each method comes with its own set of pros and cons. For example, the modification of starch by means of chemicals is a preferred method due to its ease of modification and low cost of operation. However, in the current scenario, it is not acceptable due to increasing consumer demand for safe, high-quality products as the use of acids, acetates, hypochlorites, phosphates, etc., employed for modification can lead to the generation of chemical wastes that may be detrimental to the ecosystem and requires recycling. Furthermore, the physical modifications on the one hand are mostly eco-friendly but on the other hand, sometimes the alterations in starch structure are not as severe or obvious as those created through chemical modifications. Though enzyme modification methods can be used to effectively control the specific properties of starch, its application is hindered by factors such as low thermostability and catalytic efficiency of the enzymes. Similarly, although the genetic modification of plants offers the potential to generate novel starches which can lessen or eradicate the use of environmentally perilous post-harvest chemical and enzymatic modification, its usage is subject to legislative approval, limiting its global application. Thus, there is an inherent need for the development of integrative methods that are efficient, politically acceptable, inexpensive, as well as environmentally friendly. One such strategy could be the multiple modifications of starch with the desired functional and nutritional properties which employ either combination of several similar methods (homogeneous) or several different methods (heterogeneous). However, the choice of modification types and treatment methods depends on the types of changes in functionality and reactivity of starch that are required for a specific application. Despite some remarkable progress that has been made in starch analytics, time, and spatial resolutions often remain limited. Consequently, establishing and developing new starch analytical techniques are also essential in the future.