Zinc has wide industrial applications; consequently, its extraction procedures have been extensively studied. Hydrometallurgy is one of the most common methods employed for zinc recovery. However, the electrooxidation of sphalerite and the effect of the pyrite content in the concentrate have not been investigated; thus, in this work, zinc recovery from low-iron sphalerite mineral with a relatively high pyrite content (EBHSS), in a sulfate medium was further explored. The reaction mechanism of the anodic dissolution of the EBHSS mineral was established by microelectrolysis using mineral carbon paste electrodes; these results were used to determine adequate conditions for the macroelectrolysis of the sample. The macroelectrolysis indicated that EBHSS has a low electrodissolution rate; additionally, different analyses of the species produced in the macroelectrolysis showed that the ohmic drop registered in the collector had no influence in the passivation of the EBHSS surface. It was also determined that the dissolution of EBHSS was driven by the charge transfer of the sphalerite particles, which are not very efficient for electronic conductivity. Experiments using doped EBHSS led to an increase of the electrodissolution rate, which consequently increased the recovered zinc. Zinc is an important base metal required for various industrial applications . It is mThe hydrometallurgical processes, on the other hand, are more environmentally friendly and rely on the use of aqueous solutions to decompose the sphalerite, forming an aqueous zinc sulfate electrolyte which is electrowon as metallic zinc at the cathode.2SO4 for zinc extraction is also a well-known method [A significant consideration during the hydrometallurgical process is the reaction between sphalerite and ferric ion; consequently, the leaching reactions in ferric sulfate media ,13 and fn method ,19,20,21n method ,23,24.Therefore, it is necessary to investigate other alternatives that allow overcoming the limitations of hydrometallurgical processes in terms of passivation caused by the formation of sulfur species, during the initial stages. In this work an electrochemical strategy is proposed to study the electrooxidation of a sphalerite concentrate (zinc sulfide), using an applied electric potential as the oxidant. To determine the optimal pH and electrical potential, microelectrolysis assays were carried out; subsequently, the massive electrodissolution (macroelectrolysis) of the mineral was tested using a tridimensional electrochemical reactor for experimentation.The two zinc sulfide concentrates  with an average particle size of 37 µm, from the Bismarck  and Rey de Plata  mining units, were provided by Industrias Peñoles, S.A. de C.V. for the micro- and macroelectrolysis experiments. The preliminary mineralogical composition  identifi2 injection. The working electrode consisted of a plastic tube  with a piston, into which was introduced a mineral carbon paste prepared using a homogeneous mixture of 0.7 g of graphite and 0.3 g of either low-iron or high-iron sphalerite, agglomerated with oil silicone (electrode CPE-ZnS). The active area of the working electrode (3.14 × 10−2 cm2) was polished using a Carbimet Piper disc . The electrical contact was a silver-welded copper-platinum connection.To determine the electrochemical response of the sphalerite samples, a 100 mL Pyrex cell was used to perform the voltametric analyses. The lid of the glass cell had four orifices to place a three-electrode system  and a N2 flow. The auxiliary electrode was a graphite bar of 9 cm length, 0.6 cm diameter, and purity of 99.9995% . The reference electrode was a saturated mercury sulfate electrode , connected to the glass cell by a Luggin capillary.After each voltametric measurement, the piston re-established the effective area of the working electrode. Subsequently, its active area was levelled again, and the dissolved oxygen was removed from the working solution using a N2SO4 , and H2SO4 , with no electrolyte agitation, over a range of −2 ≤ E ≤1 and at a scan speed of 100 mV each for 1 s, using CPE-ZnS samples (70:30 wt.%). The solutions were prepared using deionized water with a specific resistance of 18.2 mΩ−1 cm−1 and analytic-grade reagents. Scanning electron microscope analyses  were conducted to study the mineral passivation.An AUTOLAB potentiostat was used for experimental control and data acquisition of the high-iron (EAHSS) and low-iron (EBHSS) sphalerites. To determine the oxidation stages of the low-iron sphalerite (EBHSS), voltamperometric and chronoamperometric analyses were performed using the AUTOLAB software GPES , delivered with the AUTOLAB potentiostat. The voltammograms were obtained in solutions of NaBased on the oxidation stages detected during microelectrolysis of the EBHSS sphalerite, a parallel flow tri-dimensional nylamide electrochemical reactor (1 L) was used for the macroelectrolysis experiments. The working electrode was a graphite cloth containing 10 g of homogeneously distributed EBHSS sphalerite. The auxiliary electrode was prepared using five stainless steel meshes . The reference electrode was a saturated sulfate electrode  connected to the electrochemical reactor by a Luggin capillary.λ+) range was 0.4 ≤ Eλ+ ≤ 1.0 V, with pulse of 180 s. The experimental solutions of Na2SO4  and H2SO4  were prepared using analytical-grade reagents.The chronopotentiometric data were collected using an EG&G potentiostat . For measurement of the voltametric and chronoamperometric data, an AUTOLAB potentiostat was used. For the chronoamperometric study, the applied potential . The surface morphology of EBHSS after electroleaching was analyzed using a scanning electron microscope .The cyclic voltammetry analyses for comparing the electrochemical response to the oxidation of the EAHSS and EBHSS concentrates indicated that EAHSS and EBHSS presents an oxidation process, associated with the oxidation of sphalerite . However2SO4 (1.7 M), the reductions of EBHSS and EAHSS were different only in the value of their respective cathodic current peaks ):For an electrolytic medium of Hnt peaks c. It wasr et al.  showed tAnalyzing the relative iron-content influence in the redox behavior of the sphalerite samples (EBHSS and EAHSS), it was found that there is a more important oxidation process in EBHSS than in EAHSS. The electrodissolution of two zinc concentrates with different content of iron in solution (0.8% and 12.3%) was studied by Ahlberg et al. . The resHowever, the iron content in both zinc concentrates is present as solid solution and as pyrite, which can act as a galvanic couple of sphalerite. Mehta and Murr  observed2SO4 , λ+) increases, the current associated to the oxidation-reduction processes also increases.Regarding the effect of inversion anodic potential  at the working electrode-electrolyte interface. At potentials above 0.7 V, the passivation process disappears due to the transformation of elemental sulfur to more elevated oxidation states.The oxidation of EBHSS for different electrooxidation potentials is shown in λ+ < 0.7 V. A significant increase of transformed species  during the anodic dissolution was also observed at Eλ+ > 0.7 V.The evaluated charge densities from the voltammetries  and chroAccording λ+). The oxidation reactions related to such processes are:On the other hand, the voltametric and chronoamperometric analyses showed that the oxidation stages of EBHSS are dependent on the potential  is less positive than the value required in Equation (2).When an elevated overpotential (1 V) is applied to the EBHSS oxidation process, it is significantly modified because the sulfides are oxidized to higher oxidation states such as thiosulfates or sulfates. The elevated overpotential allows the oxidation of SE* = −0.547 V vs. SSEE* = −0.9215 V vs. SSEOxidation reactions of EBHSS at pH = 2The oxidation stages of EBHSS are practically the same at pH = 2 as at pH = 7; however, the conditional potential will change when elevated overpotentials are applied and in systems where pH has a remarkable influence.λ+ ≤ 0.7 V• Anodic potential EE* = −0.526 V vs. SSEλ+ ≥ 0.7 V• Anodic potential EE* = −0.3258 V vs. SSEE* = −0.556 V vs. SSE2SO4Oxidation reactions of EBHSS at 1.7 M H2SO4 electrolyte, the EBHSS redox processes dramatically change because the chemical species produced during the oxidation of EBHSS can be attached to the electrode surface. In addition, the oxidation stages of EBHSS cannot be clearly distinguished due to the passivation process occurring at Eλ+ = 0.7 and different pH.Using the 1.7 M HOxidation reactions of pyrite at pH = 2 and pH = 7Different experiments have suggested that sphalerite and pyrite oxidation can simultaneously occur. This study indicates that pyrite oxidation follows a reaction pathway where ferric ions or sulfates  are produced .FeS2 + The formation of intermediate species such as iron polysulfides has also been reported (reaction (9)), which turns the colorless electrolyte yellow . This re2+, and zinc, Zn2+ are released at the same time, iron may follow the reaction pathway shown in reaction (7) to produce iron hydroxides.In addition, when pyrite oxidizes (reaction (8)), the ferric ion can produce iron hydroxides (reaction (10)). On the other hand, when in solution iron, FeThe chronoamperometric study of the sphalerite EBHSS at 25 °C, pH = 7 and pH = 2 is shown in During the macroelectrolysis at pH = 7, intermediate iron species such as polysulfides could have been formed from the pyrite oxidation because the colorless electrolyte turned yellow . Conversλ+, occurs at pH = 2, which indicates that the amount of dissolved zinc via chemical dissolution is negligible compared to the amount of dissolved zinc obtained from EBHSS electroleaching.Changes in the electroleached zinc and iron at pH = 2 and 7 in function of electroleaching time and total initial concentration are shown in At 40 °C , the eleThe percentage of extracted zinc from the total content of EBHSS is shown in The scanning in the negative direction of both untreated EBHSS and EBHSS after electrolysis indicates that, at pH = 7 and pH = 2 , the ele0) attached to the EBHSS surface was not observed. The intensity changes of Zn, Fe, and S peaks related to the electrolytic recovering changes before and after electroleaching . In comparison to the macroelectrolysis I results, the current of the macroelectrolysis II increased by a ratio of 1:10 and 1:5 at pH = 7 and pH = 2, respectively. However, as the current increased, the fisiadsorption of the hydrogen bubbles on the auxiliary electrode also increased, causing electrical fluctuations .λ+ < 1 V vs. SSE can minimize the pyrite electrodissolution but cannot avoid the dissolution of the iron arranged in solid solution with sphalerite.Regarding the conversion of zinc and iron with respect to time, the iron concentration obtained in the macroelectrolysis II was higher than the zinc concentration, which suggests that when EBHSS is doped, the iron and zinc are simultaneously oxidized . AdditioIn this work, the electrochemical response of two zinc sulphide concentrates was initially studied, one with a high iron content in solid solution (EAHSS) and the other with a low iron content in solid solution (EBHSS), both concentrates with an amount of iron as pyrite. The oxidation process is more important for EBHSS, because in the range of potential of study, in addition to the oxidation of the sphalerite, the oxidation of the pyrite also occurs, a mineral that is present in appreciable quantities in this zinc concentrate. For this reason, this mineral was chosen to continue with the study.λ+ < 0.7 V vs. SSE, where sulfide is oxidized to elemental sulfur. The second stage of oxidation occurs at Eλ+ > 0.7 V vs. SSE where the elemental sulfur is converted to sulfates and thiosulfates, although iron can be electroleached at any oxidation stage when it is presented in dissolution or at Eλ+ > 0.6 V in the form of pyrite. However, when the reaction occurs at 1.7 M H2SO4, the oxidation stages are not clearly differentiated and require further study. The anodic dissolution of EBHSS is promoted as the pH of the experiments increase, probably caused by the proton concentration in the electrolyte. The chemical dissolution of sphalerite also creates more active surfaces, which favors its electrodissolution during the anodic polarization. The macroelectrolysis experiments do not show any passivation on the mineral according to the microelectrolysis results. However, the type of electrical contact occurring in the collector causes a slow kinetic electrodissolution rate. When the temperature increases, the electrodissolution improves due to an increase in the mineral conductivity. In addition, the electrodissolution speed also increases when the EBHSS is doped, due to the created metal-semiconductor contact. Therefore, EBHSS dissolution can be carried out through electroleaching, although it is not able to generate high electrodissolution kinetics using only an external potential. To increase the electrodissolution kinetics, the process must be externally stimulated.The electrochemical study allowed to establish the potential ranges where this zinc concentrate is oxidized. The microelectrolysis of EBHSS at pH = 2 and pH = 7 showed that oxidation occurs in two stages. The first stage was identified at E


Chestnut is popular worldwide for its unique flavor, high eating quality and nutrition. Here, we evaluated the influence of phosphorylation and acetylation on the structural, physicochemical and functional properties of chestnut starch. Scanning electron micrographs showed the agglomeration of starch granules and the appearance of numerous dents on the starch granule surface under phosphorylation and acetylation. X-ray diffractograms confirmed that the modification treatments did not affect the C-type crystal pattern, but reduced the relative crystallinity of the chestnut starch, particularly phosphorylation. Moreover, modification improved the paste transparency of the starch. Differential scanning calorimeter analysis revealed that the gelatinization temperature and enthalpy of the starch decreased with the increasing substitution degree, particularly in phosphorylated starch. The Rapid Visco Analyser analysis demonstrated that phosphorylation could greatly improve the pasting properties of chestnut starch. In addition, phosphorylated and acetylated starch had a smaller amount of slowly digested starch and a larger amount of resistant starch relative to native chestnut starch. In conclusion, the functional and physicochemical properties of chestnut starch can be significantly improved through phosphorylation and acetylation, demonstrating its great application potential as a food additive. Starch is the main carbohydrate reserve for many higher plants as well as the most abundant natural and renewable biopolymer next to cellulose. It has diverse applications in both food and industrial production. However, the application of native starch at the industrial level has been largely hindered by its low flowability and shear stress resistance, thermal decomposition during processing and high syneresis and retrogradation ,2. For vPhosphorylated starch is an abundantly produced and widely applied starch with chemical modifications. The introduction of phosphate groups into starch chains leads to repulsion between the phosphate groups on adjacent chains and elevates hydration. Therefore, increasing the phosphorylation degree can alter the physicochemical properties of starch, even when the degree of substitution (DS) is very low ,8. NeverAcetylation is another approach for starch modification through the substitution of hydroxyl groups of glucose with acetyl groups . The intCastanea mollissima Bl.), which belongs to the Fagaceae family, is a traditional nut and also a popular food around the world [The chestnut , one of the chestnut varieties with the largest yield and the best flavor in the Yanshan region, were collected from the Chestnut Industry Collaborative Innovation Center of Hebei province, China, in the 2020 season. The fruit were stored at 4 °C before analysis. Chestnut starch was obtained through the alkaline method following the procedures of our previous report [A glucose oxidase/peroxidase (GOPOD) kit and amyloglucosidase  were provided by the Megazyme International Ireland Ltd. . Amylose (A0512) and amylopectin (A8515) from potato starch were purchased from Sigma Chemical Co. .2PO4·2H2O) and urea were mixed at a ratio of 10:1 (w/w). The mixture was then dissolved in distilled water for preparing a 7% (w/v) NaH2PO4 salt solution for phosphorylation. Chestnut starch  was first suspended in an 80 mL NaH2PO4 solution, followed by gradual adjusting of the pH to 6.0 with 0.1 M solution of NaOH. The mixture was stirred for 30 min and then vacuum filtered. The starch was dried in an oven at 40 °C for 24 h, and then heated at 145 °C for 60, 120 or 180 min to obtain phosphorylated starch with different DS. After heat treatment, the resultant retentate was washed several times with distilled water to removed residual phosphorus salts. Finally, the sample was dried in an oven at 30 °C, ground and then sieved (80 μm mesh).Phosphorylated chestnut starch was prepared according to Hu et al. with minor modifications . Brieflyw/v) starch suspension. The pH of the suspension was adjusted to 9.0 by using a 3.0% (w/v) solution of NaOH. Acetic anhydride (6 g) was added dropwise to the stirred slurry, with the pH being maintained at 8.8–9.0 with the 3.0% NaOH solution. Then, the reaction was conducted at 40 °C for 30, 60 and 90 min. The pH of each starch slurry sample was adjusted to 6.5 by using 2.0% (w/v) HCl, and then the sample was centrifuged at 6000× g for 20 min. The obtained residue was washed repeatedly with distilled water until it had a constant pH of 6.5, and then oven dried at 30 °C for 48 h. Finally, the dried starch was ground and sieved (80 μm mesh), and acetylated starch was obtained.Chestnut starch acetylation was performed according to the method of Elijah et al. with some modifications . BrieflyThe starches modified with phosphorylation or acetylation are listed in p indicates the % phosphorus content .The phosphorus content in the phosphorylated chestnut starch was measured using the ammonium molybdate and vanadate spectrophotometric method . The DS The DS was determined according to Shubeena et al. with some modifications . A blankApparent amylose content in the starch was measured using the iodine binding method with a calibration curve of zero to 40% amylose mixed with amylopectin .The images of the chestnut starch were taken using the SU-8010 Scanning Electron Microscope . The accelerating voltage was 5 kV for imaging. Different samples were photographed under SEM at a magnification of 4000× and 6000×.X-ray diffraction analysis was performed with a D/max-2500vk/pc X-ray diffractometer . The equilibration of the starch was performed with a saturated potassium chloride (KCl) solution at room temperature for one week before analysis . The XRD−1. Starch samples were diluted with KBr  and pressed into tablets before measurement. The background value from pure KBr was acquired before scanning of the sample.FTIR spectroscopy was performed for all samples using a Tensor 27 FTIR Spectrometer  over a range between 4000 and 400 cmThe starch swelling power was determined in excess water at temperature from 40 °C to 90 °C following the descriptions of Konik Rose et al. .w/v) was prepared and heated in a water bath at 90 °C for 30 min with constant stirring. The paste clarity was determined as the percentage transmittance at 650 nm.The clarity of the chestnut starch gel was measured as described in our previous report . The aqu2O were mixed and sealed in an aluminum pan and allowed to stand still for 1 h before analysis. The samples were heated from 20 °C to 100 °C at a rate of 10 °C/min. The onset (To), peak (Tp) and conclusion (Tc) of the gelatinization temperature and gelatinization enthalpy (ΔH) were recorded.The thermal properties of the starch were measured using a differential scanning calorimeter  equipped with a thermal analysis data station. Starch  and 15 μL of HThe pasting profile of the starch was monitored using a Rapid Visco Analyser . Starch  was first dispersed in distilled water (25 mL), held at 50 °C for 1 min, heated to 95 °C (12 °C/min), held for 3.3 min, and then cooled (12 °C /min) to 50 °C and held for 2 min. The mixing paddle was set at a speed of 960 rpm for the first 10 s, and then at 160 rpm in the rest of the testing period.In vitro starch digestibility was determined by a procedure described in our previous report , which wp < 0.05) was used to examine significant differences among experimental mean values using the SPSS 19.0 Statistical Software Program .The results are presented as mean value ± standard deviation of at least triplicates, except for XRD, which was performed only once. A one-way analysis of variance (ANOVA) followed by a Duncan test  . The DS At the reaction times of 30, 60 and 90 min, the DS of the acetylated starch was 0.010, 0.020 and 0.024, respectively. Similarly, the starch acetylated for 90 min showed a higher DS than that acetylated for 30 and 60 min. The acetyl content ranged from 0.18 to 0.38% for the different reaction times, which is also below the maximum limit 2.5 g/100 g) for acetylated starch recommended by the United States Food and Drug Administration for food applications ,33. Gene g/100 g The SEM images of native and modified starches are presented in Acetylation led to the appearance of some concave pits on the starch granule surfaces and a slight aggregation of the starch granules, but the chestnut starch granules still maintained a relatively complete structure e–g. The The decrease in peak intensity after modification compared with that of native starch indicated that modification altered the relative crystallinity of chestnut starch. The relative crystallinity of starch followed the order of native starch > acetylated starch > phosphorylated starch. The increase in the DS of the modified starch was generally accompanied by decreases in relative crystallinity. Chemical modification can reduce the hydroxyl groups that may participate in the generation of intra/inter-molecular hydrogen bonds necessary for helical crystalline structure formation in starch. In the present study, phosphorylation decreased the formation of intra/inter-molecular hydrogen bonds more significantly than acetylation, making the crystalline structure of starch granules less ordered. Acetic anhydride shows a poor penetrating ability in starch granules. Acetylation occurs in all amorphous regions as well as at the outer lamellae of the crystalline regions, rather than throughout the crystalline regions of the whole starch granule. The decreasing degree of the crystalline order in starch after acetylation is dependent on the reaction time and DS as reported by Sindhu et al. .−1, there was a decrease in the intensity of the band, indicating a decline in the stretching vibration of O-H bonds, which were attributed to the substitution of hydroxyl groups by phosphate groups and acetyl groups in phosphorylated and acetylated starch. The spectra of the phosphorylated starch showed a characteristic absorption peak at 1016 cm−1 relative to that of native starch, which was related to the antisymmetric stretching vibration of the P-O-C bond. A new band appeared at 1417 cm−1, which has been reported to be the characteristic of the P = O stretching vibration [−1, which originates from the stretching vibration of C = O carbonyl ester and proves the occurrence of acetylation [−1 was assigned to the CH3 antisymmetric deformation vibration, which also confirms the increment of acetyl groups in acetylated starch molecules. Furthermore, there was a decrease in the intensity of the band at 1650 cm−1 in acetylated starch because of the incorporation of acetyl groups into the starch, which is due to the lower affinity of acetylated starch to water. Luo and Shi also reported a hydrophobic character of acetylated maize starch [Relative to those of native starch, the FTIR spectra of acetylated starch with a different DS exhibited a new band at 1720 cmtylation . A charae starch . Similare starch ,47.−1 and 1022 cm−1 have been reported to be sensitive to the ordered structure and the amorphous structure in starch, respectively. Hence, the ratio of absorbance at 1047 cm−1 and 1022 cm−1 was employed for characterizing the short-range molecular order of double helices [The absorbance bands at 1047 cm helices . As a reStarch paste clarity is a significant quality parameter closely associated with the botanical source of starch. Acetylation can also improve the clarity of chestnut starch, possibly because the introduction of acetyl groups hinders the formation of an ordered structure after gelatinization, and therefore suppresses retrogradation to result in a more fluid paste with higher clarity. Similar effects of acetylation on paste clarity have been reported for various types of starch ,48. HighClearly, the swelling power of chestnut starch phosphorylated and acetylated at 40 °C to 90 °C with a different DS was in the range of 12.30–24.89 g/g and 1.32–15.75 g/g, respectively . Native Acetylated chestnut starch showed a similar swelling power to native starch, which might be due to the low DS of acetylation. Moreover, the FTIR results implied that the starch with the incorporation of acetyl groups shows lower water affinity. Similar results were reported for rice starch . Howevero, Tp and Tc) and gelatinization enthalpy (ΔH) of the chestnut starch with the increase in the DS, indicating the accelerated destabilization of the starch structure, which requires less energy for gelatinization. The modification process, particularly phosphorylation, causes the collapse of the starch structure, which will facilitate internal molecular disorganization in the process of gelatinization. The decline in the gelatinization temperature is possibly due to the variations in starch granules, such as the early uncoiling of amylopectin double helices during phosphorylation, which is similar to the results reported in a previous study [us study . Howeverus study . The chao and Tp). The reduction of ΔH may be attributed to the disruption of double helices in both crystalline and non-crystalline regions of starch granules during acetylation, which can be confirmed by the XRD and FTIR results. Similar trends for the thermal parameters have been reported for acetylated starch from different sources [As shown in  sources ,37,45. TAccording to the RVA results, the acetylation process resulted in a slight decrease in the pasting temperature (from 74.2 °C to 71.9 °C), and did not significantly change the peak, trough and final viscosities, but decreased the breakdown and setback viscosity relative to those of native starch. The presence of acetyl groups hinders the formation of an ordered structure, thus delaying starch retrogradation. These results are inconsistent with the finding of some studies that acetylation may increase the viscosity of starch ,26,34. IMoreover, these properties may also be influenced by the size of the substituents. The phosphate group has a larger size than the acetyl group. The introduction of larger substituting groups to amylose or the outer branches of amylopectin starch can create more free space between starch macromolecules, which will hinder the establishment of intermolecular or intramolecular associations between the individual chain segments due to the effect of steric hindrance.Interestingly, the modified starch showed a higher proportion of RS than native starch in this study, and acetylated starch had a relatively higher RS proportion (81.4–84.0%) than phosphorylated starch (62.7–76.2%). These results may be explained by the introduction of phosphate and acetyl groups to the starch molecules, which may retard the formation of the enzyme-substrate complex. Some factors that may affect starch digestibility include the starch source, the amylose to amylopectin ratio, crystallinity, the granule size and the amylopectin molecular structure. The main reason for the higher RS content in chemically modified starches is the high specificity of digestive enzymes that do not recognize the chemically modified structure of amylose and amylopectin chains, which results in the high values of nonhydrolyzed starch fractions. The results of the digestibility test implied that a high RS can be achieved through modification, especially acetylation. RS can be added to some foods to reduce caloric content and the postprandial insulinemic response after consumption.Phosphorylation and acetylation were found to significantly influence the physico-functional and chemical properties of chestnut starch in this work. An increase in the reaction time can increase the DS of the modified starch. Phosphorylation and acetylation led to the appearance of dents on the surface and the aggregation of starch granules. Chemical modification does not influence the C-type crystalline pattern of chestnut starch but reduces the relative crystallinity, indicating that the crystalline structure is disrupted. Chemical modifications, especially phosphorylation, disrupt the long- and short-range molecular order of double helices in chestnut starch, which further decreases the gelatinization temperature and gelatinization enthalpy of the starch. Relative to native chestnut starch, phosphorylated chestnut starch has a higher swelling power, lower pasting and gelatinization temperature, higher paste clarity and better viscosity characteristics with increasing DS. Acetylated starch has a higher proportion of RS than native starch, suggesting a decrease in digestibility. Overall, our findings demonstrate that different technologies for starch modification may be employed to generate different characteristics in the final products, which will greatly improve the applicability of chestnut starch in the food industry.


Znk increases from 5.15 × 10−6 to −1.49 × 10−4 ms−1. For the experiments carried out in the furnace with a cold crucible (T = 953 K), a reduction in the operating pressure in the furnace from 1000 Pa to 10 Pa resulted in a decrease in zinc content in the alloy from 18 to 80%, compared with the initial value. For comparison, the experiments carried out in the crucible induction furnace at 953 K showed a reduction in zinc content in the alloy from 6 to 50%, which means that more intense zinc evaporation is seen in the furnace with a cold crucible. Comparison of ISM and VIM technologies in the removal efficiency of the Al-Zn alloy indicates a higher removal efficiency using the first technology, which, using the same conditions, achieves 80% of the removal efficiency of the component.Using a vacuum during the smelting and refining of alloys removes dissolved gasses, as well as impurities with high vapour pressure. When smelting is carried out in vacuum induction furnaces, the intensification of the discussed processes is achieved by intensive mixing of the bath, as well as an enhanced mass exchange surface  due to the formation of a meniscus. This is due to the electromagnetic field applied to the liquid metal. This study reports the removal of zinc from the Al-Zn alloy containing 6.3 wt.%. Zn. The experiments were carried out with the use of two types of metallurgical devices: the VIM and ISM furnaces. For the experiments carried out in the crucible induction furnace, reduction in the operating pressure in the furnace from 1000 Pa to 10 Pa, together with the increase in temperature from 953 K to 1103 K, is accompanied by a decrease in zinc content in the alloy from 6 to 96%, compared with the initial value. Simultaneously, the overall mass transfer coefficient  The dynamic development of the world economy requires the supply of more and more materials that meet high-quality requirements. Metals belong to the group of strategic construction materials, but the basic issue of their use in advanced solutions is their purity. Physicochemical properties of metals, such as chemical affinity for oxygen, reductions, surface tension, viscosity, and gas vapour pressure, constitute basic barriers that, in the currently used refining technologies, are limitations for the level of removal of impurities in metals and their alloys. The emerging solutions based on scientific studies and the increasing technical possibilities of building advanced metallurgical structures create new possibilities, especially in the area of selective metal preparation and purification.In the literature on the subject, one can find a well-documented database of results concerning metal refining processes with the use of melting aggregates based on inductive remelting under reduced pressure conditions, such as VIM. However, the emerging base of secondary raw materials and the exhausting existing resources of natural resources mean that the search for solutions that have the potential in the area of processing and selective recovery of materials with complex chemical compositions are particularly of interest. Such solutions include the induction skull melting (ISM) technology, which is based on induction melting using a phenomenon similar to levitation so that the melted metal does not come into contact with the crucible material, which can also often introduce contamination to the metal.Due to the very high intensity of the interaction of electromagnetic forces, the liquid metal is affected by large forces that significantly increase the interface between the liquid and gas phases. Taking into account the kinetic analysis of metal refining processes, a considerable role in this process is played by the mass transfer surface, which is the interface.The research results presented in this article constitute an innovative approach to the refining of metals, including zinc and aluminium. Thus far, due to high costs, ISM remelting solutions are used in the technologies of obtaining titanium, while the use of non-ferrous metals in this technology requires a number of tests to supplement the knowledge in this field. Comparison of ISM and VIM technologies in the removal efficiency of the Al-Zn alloy indicates a higher removal efficiency using the first technology, which, using the same conditions, achieves 80% of the removal efficiency of the component.The phenomenon of evaporation of volatile metal bath components can be observed in numerous processes of both extractive metallurgy and refining. In some of them, this phenomenon can be considered positive, while in others, however, to the contrary. In order to intensify the evaporation process or to restrain it, one must necessarily know the factors determining its rate. Among the most important of these factors are the pressure inside the system, the type of gas atmosphere, the temperature, and the hydrodynamic properties of the system in which the metallurgical process is carried out. Crucible-based vacuum induction furnaces (VIM technology), such as cold crucible induction furnaces (ISM technology), are melting machines used in modern refining or smelting processes. In these operations, due to the use of vacuum, it is possible to evaporate its components, characterised by high vapour pressure from the metallic bath.Induction furnace with a non-conductive crucible, e.g., ceramic crucible;Induction furnace with a conductive crucible, e.g., graphite crucible or copper crucible in case of the cold crucible furnace.The source of the electromagnetic field in induction furnaces is a coil made of copper. Taking into account the construction of crucible, we have two kinds of induction furnaces:Magnetic cores or magnetic field concentrators are applied to concentrate the electromagnetic field. The electromagnetic field generated by the alternating electric current flowing through the inductor, directly or indirectly, affects the workpiece placed inside the crucible. Eddy currents induced in the workpiece are responsible for heat generation in the metal. As a result of the interaction, the Lorentz force is generated ,3,4,5. IThe application of direct heating in the crucible may cause intense reactions of the crucible with the metal, e.g., titanium or zircon . It causAnother technology for induction smelting, one that partially eliminates the disadvantages of classical crucible smelting, is smelting in induction furnaces with a so-called cold crucible .The same phenomena occur in cold crucible furnaces (ISM technology). The water-cooled crucible is mostly made of copper ,9,10,11.When metals or their alloys are melted in induction furnaces (VIM or ISM), the surface area of the metal is strongly dependent on the electromagnetic field acting on the alloy and the properties of the liquid metal. This is evinced by the formation of a distinct meniscus on the surface of the bathtub. Increasing the surface of the liquid alloy can significantly intensify the discussed evaporation process. Approximating the surface of liquid Al-Zn alloy melted using VIM and ISM technologies;Laboratory smelting of these alloys in variable pressure and temperature;Estimation of the overall mass transfer coefficient of zinc on the basis of experimental data;Determination of the stages in finding the velocity of the analysed evaporation process.The aims of this work include the following:The selection of the alloy Al-Zn which the tests were conducted resulted from the fact that zinc as a component of this alloy is characterised by much higher vapour pressure, compared with aluminium. The paper presents the kinetic analysis of zinc evaporation from the Al-Zn alloy containing 6.3 wt.%. Zn. The experiments were carried out with the use of two types of metallurgical devices—the VIM and ISM furnaces, ranging from 953 K to 1103 K and a wide range of operating pressures, from 10 Pa to 1000 Pa.AP0, BP0—equilibrium vapour pressures over pure components A and B, respectively; Aγ, Bγ—coefficients of activity in the alloy for A and B, respectively.The potential for evaporation of the A element from a two-component A-B alloy is determined by the evaporation coefficient Ω which is described by the equation .(1)Ω=γAA-B alloy, with Ω ≈ 1, it is assumed that the chemical composition of the alloy does not change during the smelting process. When the coefficient value is Ω > 1, we can infer a loss (evaporation) of the A component from the alloy relative to the B component. When the following condition is met: Ω < 1, we can infer a loss (evaporation) of the B component from the alloy.For the two-component B—volatility ratio of the alloy B component; AM, BM—atomic (molar) mass of the alloy main component A and the evaporating component B.According to Oletta , the valIt is generally assumed that the process of ‘i’ component evaporation is possible if the following condition is met:BX—a molar fraction of the B component of the alloy; j—the number of atoms in the metal particle B in the gaseous phase.Metal can evaporate in both atomic and particulate forms. In the latter case, the volatility ratio equation takes the following form:Zn/Al and ΦZn ratios are >1. The values of the volatility and evaporation ratios presented in The data presented in The research experiments were performed on a multi-component Al-Zn5.5MgCu alloy; its composition is presented in The melting furnaces applied in the experiments were the VIM−20 vacuum induction melting furnace and the vacuum furnace with a cold crucible ISM 2–200 .All the experiments assumed in the research were conducted following a fixed scheme. In the first stage, when the sample was placed in the furnace, the pressure was lowered in the working chamber, and then the charge was heated and melted. Upon reaching the set temperature by the liquid metal, it was maintained in the furnace for 600 s. The temperature measurement was performed using a thermoelectric B-type sensor PtRh30-PtRh6 and an optical pyrometer . During the process, a sample of liquid metal was collected at defined intervals. When the experiment finished, the chemical composition of the metal was analysed. For this purpose, the atomic absorption spectrometry method with the ASA solar device was used.In the VIM unit, the process was performed within the temperature range of 953 K to 1103 K, while the ISM unit experiments were conducted at 953 K. The recorded changes in the furnace operating power ranged from 8 kW to 22 kW and from 70 kW to 130 kW for the VIM furnace and the ISM furnace, respectively. The operating pressure in both units was changed within the range of 10 Pa to 1000 Pa.ZnU) are shown. Graphic interpretations of these results are shown in Results obtained in all experiments are presented in As indicated by the data presented in For the experiments conducted at 935 K, the loss of zinc mass was as follows, depending on the unit applied: 6.19% to 49.84% for the experiments conducted in the crucible furnace; 18.57% to 79.20% for the experiments conducted in the furnace with a cold crucible.The elimination of impurities in vacuum induction melting is generally observed to follow first-order kinetics . As a reThis relationship can be written in the following integral form:ZnC0, ZntC—zinc concentrations in the alloy, initial and at t, respectively (wt.%); Znk—overall mass transfer coefficient (ms−1); F—evaporation surface area (m2); V—liquid metal volume (m3); (t − t0)—duration of the process (s). After integrating Equation (6), we have:Znk coefficient, it is necessary to know the volume of the melt and its surface. In the analysed experiments, within 953 K to 1103 K, the estimated value of the liquid metal volume fell within the range of 420 to 425 cm3. The values of liquid aluminium density within the analysed temperature range were determined based on the data in [−3.The form of Equation (7) shows that in order to determine the value of the  data in ,20. TheyWhen analysing the process of zinc evaporation from molten aluminium alloys during their smelting in a vacuum induction furnace, attention should be paid to the issue of increasing the liquid metal surface with the increase in power released in the charge. With greater power, we observe a significant increase in the bath surface due to the formation of a meniscus, which is an effect of the electromagnetic field acting on the liquid metal.Acquisition of images of the molten metal with the use of a high-speed camera;Determination of the resulting meniscus geometry on the basis of the obtained photos;Determination of the functions of the curves describing the meniscus for various process parameters;Estimation of the surface area using the programs Wolfram Mathematica (F1).To estimate the size of the surface, the following method of determining the area consisting of four stages was proposed :AcquisiAs part of the first stage, using a Phantom v5.1 camera and the FLIR thermal imaging camera, a series of photos of the liquid metal was taken during the process for the assumed operating parameters of the furnace. The use of the WebPlotDigitizer 4.1 program allowed us to place points on the photos describing the resulting geometry. Based on this, a line and a function describing the shape of the interface were determined. Znk, determined based on Equation (7), are presented in Znk values versus the operating pressure of the crucible induction furnace.The values of the zinc overall mass transfer coefficient Znk. This refers to the experiments carried out in both the crucible induction furnace and the induction furnace with a cold crucible. The values of the Znk coefficient obtained in the research are fairly consistent with the literature data reported by other authors studying the process of zinc evaporation from copper, iron, and aluminium alloys melted in the conditions of lower pressure [Znk due to an intensified process of liquid alloy stirring, with a higher furnace power.The data in pressure ,22,23,24lβ can be determined with the use of the recommended Machlin’s equation [mν—near-surface velocity of inductively stirred liquid metal; mr—radius of the liquid metal surface (most commonly assumed to be the crucible inner radius); ZnD—Zn diffusion coefficient in liquid aluminium.For a metal which is melted and stirred in the induction furnace, the arsenic mass transfer coefficient equation  as follolβ value is directly proportional to the near-surface velocity of the liquid metal. Most of the authors who study the evaporation kinetics of inductively stirred metal bath components assumed consistently with Machlin that this velocity is practically independent of the electrical parameters of furnace operation. For induction furnaces with a capacity of up to 1 Mg, the value of mν was assumed to be constant and equal to 0.1 ms−1. However, further studies showed that this velocity depends on parameters such as the furnace power, current frequency, crucible geometry, or the crucible location in the furnace relative to the inductor [lβ coefficient was determined using the near-surface velocity data from studies by [By analogy to penetration models of mass transfer, Machlin assumed that liquid metal components move along the tangent, i.e., liquid metal–gas or liquid metal–crucible, and the velocity gradient normal to the surface is near the zero value. The equation shows that the inductor ,27. In tudies by ,29, in wudies by .lβ coefficient determined based on Equation (8). They fell within the range of the analysed temperatures and ranged from 3.8 to 5.49 10−4 ms−1. The value of the zinc diffusion coefficient in liquid aluminium was estimated based on the data contained in the paper [he paper .The data in eZnk was assumed to be described by the following equation:α—evaporation constant; 0Znp—equilibrium vapour pressure of zinc over liquid metal; Znγ—activity coefficient of zinc in liquid aluminium; AlM, ZnM—the molar mass of aluminium and zinc, respectively; Alρ—aluminium density. In the present paper, the values of zinc vapour pressures p0Zn were determined based on the data from the HSC thermodynamic database [Znγ were determined based on the data reported in a study by [eZnk are summarised in When analysing the rate of zinc evaporation from the liquid alloy surface, the maximum value of rate constant database , and thestudy by . The esteR as a fraction of the overall process resistance, within the ranges of temperature and pressure applied in this research, showed that it changed from 1.5% to 28% for the experiments performed in the crucible induction furnace, and from 1.5% to 11% for the experiments carried out in the furnace with a cold crucible (only 953 K for this device).Estimation of the resistance βlR) and with the reaction on the liquid metal surface (eR) was up to 22% of the overall process resistance (βlR + eR) of up to 65%  was used according to the suggestion provided in . This paIt is assumed that transport in the gaseous phase does not determine the rate of the analysed metal evaporation process under reduced pressure when the following condition is met:To estimate the OPR coefficient for the analysed temperature range applied in the experiments, vapour pressures of all volatile components over liquid Al-Zn alloy were considered. The OPR values versus temperature are presented in Znk regarding the evaporating component;Determination of the values of the overall mass transfer coefficient lβ and the evaporation rate constant ek;Estimation of the values of mass transfer coefficients in the liquid phase Determination of the stages defining the investigated process.As part of the above research, a kinetic analysis of zinc evaporation from aluminium during its smelting in the crucible induction furnace and the induction furnace with a cold crucible was carried out. The analysis involved the following procedures:Znk increases from 5.15 × 10−6 to 1.49 × 10−4 ms−1.For the experiments carried out in the crucible induction furnace, reduction in the operating pressure in the furnace from 1000 Pa to 10 Pa, together with the increase in temperature from 953 K to 1103 K, is accompanied by a decrease in zinc content in the alloy from 6 to 96%, compared with the initial value. Simultaneously, the overall mass transfer coefficient Znk increases from 5.17 × 10−6 to 3.34 × 10−5 ms−1.For the experiments carried out in the furnace with a cold crucible (T = 953 K), a reduction in the operating pressure in the furnace from 1000 Pa to 10 Pa resulted in a decrease in zinc content in the alloy from 18 to 80%, compared with the initial value. Simultaneously, the overall mass transfer coefficient For comparison, the experiments carried out in the crucible induction furnace at 953 K showed a reduction in zinc content in the alloy from 6 to 50%, which means that more intense zinc evaporation is seen in the furnace with a cold crucible. This effect is caused by a far larger evaporation surface  for this device .βlR) and with the reaction on the liquid metal surface (eR) was up to 22% of the overall process resistance for this pressure range.The rate of analysed zinc evaporation within 100 Pa to1000 Pa is controlled by the mass transfer rate in the gaseous phase. The overall resistance associated with mass transfer in the liquid phase (βlR) and with the reaction on the liquid metal surface (eR) constitutes nearly 70% of the overall process resistance.For 10 Pa, the overall resistance associated with mass transfer in the liquid phase (The research findings and their analysis led to the following conclusions:


The extrusion process conditions were significantly affected by the starch type. In the extruded blends, the starch type had a significant impact on the protein solubility which decreased with increasing barrel temperature. Increasing barrel temperatures significantly increased the amount of soluble fiber fractions in the blends. At defined thermomechanical conditions, the starch type showed no significant impact on the protein solubility of the blends. Therefore, the observed effects of starch type on the protein solubility of extruded blends could be attributed to the indistinct process conditions due to differences in the rheological properties of the starches rather than to molecular interactions of the starches with the rapeseed proteins in the blends.For the valorization of oilseed press cakes into food products, extrusion can be used. A common way of applying the protein- and fiber-rich press cakes in directly expanded products is the combination thereof with starch, since starch gives a favourable texture, which correlates directly to expansion. To control product properties like expansion of protein and fiber-rich extruded products, the underlying physicochemical changes of proteins, fibers and starch due to thermomechanical input need to be comprehensively described. In this study, rapeseed press cake (RPC) was extruded and treated under defined thermomechanical conditions in a closed-cavity rheometer, pure and in combination with four starches. The impact of starch type  and temperature  on protein solubility, starch gelatinization (D The valorization of food production by-products into directly expanded food products using extrusion has been a well-established and well-studied technology in the past years ,6,7,8,9.High expansion properties of extruded snacks are mostly generated by extrusion processing of starch-based formulations, such as potato, rice, wheat or maize starch . When RPRPC exhibits protein content between 19–40 g/100 g ,23,24. IBesides proteins, RPC exhibits about 36 g/100 g of total dietary fiber content, whereof 88 g/100 g are soluble and 12 g/100 g are insoluble . A transIn order to increase the amount of RPC in starch-based extruded snacks and to control the quality of the extruded products, the underlying physicochemical changes of these biopolymers need to be investigated in relation to the applied process conditions.Therefore, in this study, the impact of extrusion processing on the physicochemical properties of rapeseed protein, rapeseed fiber, as well as of starch was evaluated. RPC as a pure component and combined in blends with four starch types was investigated, both untreated and after thermomechanical treatment.It was expected that the extruder response (SME and product temperature) during extrusion would be affected by the blend composition of the starch type and the resulting rheological properties, respectively, although the process conditions  were kept constant. Therefore, to overcome the hurdle of indistinct temperatures and shear rates applied to the blends in the extruder, a closed-cavity rheometer was used in order to execute thermomechanical treatments at defined temperatures and shear rates.TM 100) were kindly provided by Avebe . Maize starch (MS) and high-amylose maize starch (HAMS) (HylonTM VII PCR) were kindly provided by Ingredion . The amylose content of the starches, as reported in the specifications of the suppliers, were 25, 1, 26 and 67 g/100 g for PS, WPS, MS and HAMS. The RPC was milled to <500 µm before it was mixed in a 70:30 ratio wet basis (w.b.) with PS, WPS, MS or HAMS in a Spiral-Mixer SP 12  for 60 min, followed by an incubation at 20 °C for at least 8 h. Prior to extrusion, the dry matter content of the mixtures was analyzed  as described in the German Food Act [Cold-pressed 00-type RPC was kindly provided by Teutoburger Ölmühle . The temperature during pressing did not exceed 60 °C. The pH of RPC was 5.97 ± 0.25. Potato starch (PS) and waxy potato starch . The mass flow rate was kept at 10 kg/h, the moisture content of the melt was set to a 29 g/100 g dry matter basis (d.m.), the screws rotated with 300 rpm, and the temperature of the last barrel segment was set to 20, 80, 100, 120 or 140 °C. A detailed temperature and screw profile can be found in Defined thermomechanical conditions were applied using a closed-cavity rheometer . The cavity can be pressurized (4.5 MPa) and sealed and the device allows the analyses of low moisture samples at high temperatures without water vaporization or material slippage . Before −1 . The treatment time was 60 s to mimic the residence time in the used extruder. Since the temperature in the measuring chamber of the closed-cavity rheometer is cooled by air, the lowest possible treatment temperature was 25 °C. For each treatment, at least five samples were collected. After treatment, the samples were treated as the extruded samples  and stored at −20 °C until analysis.After the protein extraction step, 20 mL of the solution were placed in centrifuge tubes and centrifuged for 15 min at 15 °C and 20.000 rpm. The supernatant was filtered . V represents the initial volume of the sample (50 mL), PC refers to the protein content present in the supernatant as determined by the Dumas method, mgel and enthalpy ∆ H of RPC/starch blends were analyzed by using a differential scanning calorimeter (DSC) . Slurries were prepared with the unextruded and extruded samples to obtain a final moisture content of 60 g/100 g (w.b.) using a Thermomix . Slurry amounting to 15–20 g were placed into aluminum pans and sealed for analysis. A sealed empty pan was used as a reference. A heating ramp of 20–120 °C with a heating rate of 10 °C/min was applied for one scan. Analysis was carried out using the TA Universal Analysis software . Tgel was taken as the peak temperature of the gelatinization endotherm. The degree of gelatinization Dgel was calculated according to Equation (2), where ∆ H0 refers to the gelatinization enthalpy of the unextruded formulation and ∆Hgel indicates the gelatinization enthalpy of the extruded formulations. Analysis was carried out in triplicate.The gelatinization temperature TThe total hydrolyzed starch of RPC/starch blends was analyzed by using a Starch UV Test kit  according to Beutler et al. . Starch Soluble and insoluble fiber contents of non-extruded RPC and RPC/starch blends were analyzed by enzymatic-gravimetric analysis according to AOAC 991.43. Analyses were carried out in triplicate.p ≤ 0.05. When appropriate, means were compared using Tukey’s honest significance test.Data were analyzed using OriginPro  by means of two-way analysis of variance (ANOVA). Statistics were considered significant when The effect of starch type on the amounts of dietary fiber in untreated samples is likely to be an effect of the analysis method and the presence of resistant starch. Dietary fiber analysis in this study was initiated with an enzymatic starch digestion step (initiated by amylase) at the beginning of the extraction, and resistant starches, if present, remain unaffected by this treatment. It is known from previous studies that uncooked high-amylose starches are more resistant to enzymatic hydrolysis than high-amylopectin starches .B of 20 °C, the product temperature increased with starch addition irrespective of starch type. At a TB of 80 °C and 120 °C, RPC generated a similar product temperature as HAMS30/RPC70 and WPS30/RPC70. RPC as a single component could not be extruded at a TB of 140 °C due to severe extruder clogging. We assume that this was due to the high water absorption of fiber components in RPC.B had a significant impact on SME (p < 0.01). At all TB, RPC/WPS blends exhibited the highest SME values, and RPC/MS blends the lowest ones.A comparison of all RPC/starch blends exhibited that starch type and TB than RPC/MS and RPC/HAMS blends. With increasing TB, the SME significantly decreased in all RPC/starch blends, which was due to a temperature-induced decrease of melt viscosity. PS and WPS gelatinized at a lower temperature compared to maize starches  are the 11S globulin cruciferin (300 kDa ) and theSince cruciferin constitutes the major protein fraction in rapeseed, consequently, the amount of extractable protein at pH 4 is lower compared to pH 7 and 11, where cruciferin predominantly occurs in protein extracts. At high-alkaline conditions, protein hydrolysis (proteolysis) can appear, where cruciferin bands can be degraded, while napin bands are only slightly affected, which in turn increases the overall protein solubility in this pH range. The ratio of soluble cruciferin and napin can vary at neutral pH (2.1:1 to 2.6:1), wherefore pH 7 gives the most mediocre picture for the amount of extractable protein. .B of 100 °C, cruciferin is already denatured, whereas napin might still be native or partially native. When TB increased from 100 to 120 °C, it is likely that both main proteins were denatured and might have formed new protein bonds, which explains the significant decrease of extractable protein content and solubility at pH 7 and 11. The significant decrease in extractable protein at pH 4 with an increase of TB from 80 to 100 °C supports these assumptions, since napin, which denatures at temperatures > 80 °C, contributes majorly to the protein fractions extractable at acidic pH.A reduced solubilisation of proteins due to extrusion processing was described in a number of studies and was attributed to protein cross-linking induced by thermal denaturation and Maillard reactions ,51. NapiAdditionally to protein linkages that are formed due to protein unfolding and cross-linking, Maillard reactions products  glyoxal) can induce protein cross-linking through reactions with lysyl, agrinyl or tryptophanyl residues. We observed that the RPC samples changed from a light yellow colour to brown due to thermomechanical treatment, which strengthens this hypothesis.The amount of extractable RPC proteins at pH 7 and 11 a was sliOverall, the protein solubility of RPC was lower compared to rapeseed meal investigated in previous studies ,53. FetzB of 20 °C and extracted at pH 4. At a TT and TB of 25 and 20 °C, respectively, the effect of the mechanical energy input on protein extractability of RPC proteins dominates over the impact of temperature ; however, there was no significant interaction between starch type and TB (p < 0.05), and the amount of soluble protein was lower as at pH 7 and 11  and ∆Hgel (p < 0.01) of all starches, the starch types differed significantly in ∆Hgel (p < 0.01), but did not differ significantly in Tgel (p > 0.05). After extrusion, the thermal properties (∆Hgel) of the blends were significantly lower as in the unextruded samples.gel and the highest ∆ Hgel compared to PS and MS blends. Dgel increased with increasing TB and a Dgel of > 90% was found for WPS blends at a TB of 80 °C, for PS blends at 100 °C and for MS blends at 120 °C. PS30/RPC70 had a higher Tgel than WPS30/RPC70, but though lower than MS30/RPC70. This explains that for PS and MS blends, the stage of full gelatinization, indicated by the absence of a peak in the thermogram, was completed at 100 °C and 120 °C, respectively. The ∆ Hgel of PS, WPS and MS blends had already decreased by 58, 50 and 81% when the blends were extruded at 20 °C compared to unextruded blends. For HAMS, no thermal peak was observed at any TB.WPS blends exhibited the lowest TStarch gelatinization indicates the disorganization of the semi-crystalline structure into an amorphous state, and since HAMS exhibits >67% amorphous amylose and the blends only contained 30 g/100 g (w.b.) starch, the transformation of the remaining crystalline amylopectin into the amorphous state in the blends might not be detectable with the DSC. In a number of previous studies, there was also no distinct endothermic peak found for the gelatinization of high-amylose starches. Russel et al.  detectedgel of WPS30/RPC70, and the low Tgel compared to other starch types is also in accordance with the literature and can be attributed to a high water uptake and a high degree of transformation from crystalline to amorphous structures of amylopectin rich starches [The high ∆Hstarches . In contstarches ,64. Thisstarches ,66.gel with an increasing extrusion temperature was reported several times in the literature for sweet potato starch [An increase in Do starch  and maizo starch  as singlo starch .In the RPC/starch blends, severe competition for water likely takes place with both components requiring water for physicochemical transformations, especially since the starch and protein content in the blends was set to be relatively equal (27 and 30 g/100 g respectively). Furthermore, the fibers present in the RPC have a high capacity to hydrate, wherefore they restrict the availability of the plasticizer, increase the melt viscosity, and reduce the availability of water required for gelatinization . AlthougB had a significant impact on SH; furthermore, a significant interaction of starch type and TB was observed (p < 0.01). In the unextruded blends, almost the whole amount of PS, WPS and MS that was present in the blends (30 g/100 g), was hydrolysed, wherefore only 27 g/100 g HAMS could be hydrolyzed. MS in extruded blends at a TB of 140 °C could almost fully be hydrolyzed, whereas for PS, WPS and HAMS, SH decreased with increasing TB. A TB of 140 °C only marginally decreases SH for MS, WPS and HAMS blends, and slightly increases SH for the PS blend, compared to a TB of 100 °C.As illustrated in H can indicate the formation of non-covalent or covalent bonds between starch and other components in the blends, such as rapeseed proteins, lipids or fibers, during extrusion [A decrease of Sxtrusion . Extrusixtrusion ,70,71. TB increased from 100 to 140 °C, but the protein solubility decreased significantly in these samples, we assume, that rather than protein–starch bonds, protein–protein bonds between unfolded rapeseed proteins are formed during extrusion that cannot be solubilized in NaCl.Allen et al.  discusseThe lower degree of hydrolysis of HAMS-based blends compared to MS, PS and WPS blends, regardless of whether the samples were extruded or not, may be attributed to the low susceptibility to degradation of HAMS due to its high amylose content. Some studies report that high-amylose maize starch is less susceptible to various physicochemical treatments  than normal or waxy starches, due to its lower crystallinity, small particle size and high surface area ,74,75,76gel and the comparatively large decrease of protein solubility at TB 100 °C of PS30/RPC70, indicating that polymerization occurred from interactions between exposed binding sites from unfolded rapeseed proteins and gelatinized starch. However, since process conditions during extrusion processing were significantly influenced by the starch type, and this effect was not found in blends treated under defined thermomechanical conditions, we assume that rheological effects due to the starch type during the analysis of starch and protein solubility dominated over actual protein–starch bonds. To resolve this, future studies should systematically evaluate the impact of an isolated rapeseed protein addition to starch blends under defined thermomechanical conditions.In our study, the extrusion-induced reduction of hydrolyzed starch content in PS30/RPC70 may indicate that the starch has interacted with rapeseed components during extrusion, making the starch less accessible for the enzymes during analysis. This observation could be corroborated by the relatively low TB.B to 100 °C, the SDF/TDF of RPC increased, but an increase to 120 °C did not affect the SDF/TDF of RPC.RPC exhibited the least amount of SDF relative to TDF in an unextruded and extruded condition. With the addition of starch, SDF/TDF increased in the order of RPC/HAMS, RPC/MS, RPC/WPS and RPC/PS. With an increase of TB of 100 and 140 °C, the amount of SDF increased regardless of the starch type in the blends. There was only a slight increase of SDF in the samples when TB increased from 100 to 140 °C; however, SDF/TDF of RPC/HAMS increased to a larger extent.With a TThe increase of SDF/TDF with the addition of starch to RPC can be related to the methodology of dietary fiber analysis in our study, where the first step of sample treatment is an enzymatic digestion of amylose using amylase. The presence of antinutritional components in RPC, such as phytic acid, can inhibit the starch digestion by amylase, as shown in previous research ,77. FurtA number of studies based on rice bran or wheat bran described an increase of SDF/TDF due to extrusion ,34. An eB of 100 °C and 140 °C, but the increase of SDF/TDF was not larger than in MS or HAMS, which generated lower SMEs during extrusion. The slight increase of SDF when TB increased from 100 to 140 °C can be explained by the impact of thermal treatment on additional breaks of glycosidic bonds resulting in smaller and more soluble fractions of polysaccharides, as described in previous studies [An increase of SDF/TDF was accompanied by a high SME in the study of Naumann et al. . However studies .In this study, rapeseed press cake (RPC), both pure and in combination with four starch types, and varying in botanical origin and amylose content, was exposed to thermomechanical treatment at five temperatures. The impact of extrusion-processing on the solubility of rapeseed protein and fiber, hydrolyzed starch content and gelatinization properties was investigated using extraction, enzymatic and enzymatic-gravimetric analysis. Starch and protein content in the blends was equal in order to exclude an overage effect.Indistinct process conditions  were monitored during extrusion as a function of starch type. To compensate this effect, a closed-cavity rheometer was used to expose RPC and the blends to defined thermomechanical treatment at a constant shear rate and extrusion-like temperatures. The protein solubility of the thermomechanically treated blends was analyzed accordingly.Extrusion-processing of RPC significantly reduced the protein solubility with increasing barrel temperature, so did thermomechanical treatment at defined conditions with increasing treatment temperatures. At temperatures ≤100 °C, extruded RPC exhibited a lower protein solubility compared to thermomechanically treated RPC, whereas both treatments led to an equivalent protein solubility at 120 °C. Effects observed at temperatures ≤100 °C can be attributed to the higher shear forces and higher local temperatures in the extruder barrel promoting protein aggregation reactions. At ≥100 °C, it can be assumed that thermal energy input dominates over the influence of shear stress, wherefore extrusion and defined thermomechanical treatment resulted in equivalent protein solubilities.The protein solubility of extruded RPC/starch blends was significantly influenced by the starch type and decreased with increased barrel temperature. No effect of starch type was detected when samples were treated under defined thermomechanical conditions. This was considered as an indication that no protein–starch interactions were formed due to shear and heat under the given conditions. It can be assumed that the effect of starch type on the protein solubility in extruded blends was predominantly due to differences in the rheological properties of the starches, leading to indistinct process conditions during extrusion, such as shear rates or local temperatures.At temperatures of 100 and 140 °C, blends treated with defined thermomechanical conditions exhibited higher amounts of extractable proteins and a higher protein solubility compared to extruded blends, which was attributed to higher local shear rates in the extruder barrel compared to the shear rates applied in the rheometer.Increasing barrel temperatures decreased the degree of hydrolyzed starch, and increased the degree of gelatinization, as well as the amount of soluble dietary fibers of RPC; however, the latter was not affected by the starch type.Our findings emphasize that the closed-cavity rheometer is a suitable tool to analyze physicochemical transformations of biopolymers at defined thermomechanical conditions in order to overcome the hurdle of indistinct process conditions during extrusion, as they are significantly affected by the raw material.


Randomised Controlled Trial 1Randomized Trial of a Third Dose of mRNA-1273 Vaccine in Transplant Recipientsby Hall, V. G., et al. New England Journal of Medicine 2021; 385 (13): 1244–1246.Randomised Controlled Trial 2Conversion from Calcineurin Inhibitor to Belatacept-based Maintenance Immunosuppression in Renal Transplant Recipients: a Randomized Phase 3b Trialby Budde, K., et al. Journal of the American Society of Nephrology 2021 [record in progress].Centre for Evidence in Transplantation have developed the Transplant Trial Watch. The Transplant Trial Watch is a monthly overview of 10 new randomised controlled trials (RCTs) and systematic reviews. This page of Transplant International offers commentaries on methodological issues and clinical implications on two articles of particular interest from the CET Transplant Trial Watch monthly selection. For all high quality evidence in solid organ transplantation, visit the Transplant Library: www.transplantlibrary.com.To keep the transplantation community informed about recently published level 1 evidence in organ transplantation ESOT and the RANDOMISED CONTROLLED TRIAL 1Randomized Trial of a Third Dose of mRNA-1273 Vaccine in Transplant Recipientsby Hall, V. G., et al. New England Journal of Medicine 2021; 385 (13): 1244–1246.This study aimed to investigate the effect of a third dose of the mRNA1273 (Moderna) vaccine in solid organ transplant (SOT) recipients.Participants were randomised to receive either a third dose of mRNA1273 vaccine or saline placebo.120 adult solid organ transplant recipients.The primary endpoint was a serologic response. Secondary endpoints included the percent neutralization, and the polyfunctional T-cell response.4 months.Published as a correspondence letter in the NEJM, this study randomised transplant recipients to a third coronavirus vaccine dose (moderna) or to placebo. 120 transplant recipients were included, with median age 66 years and median time after transplant to third vaccine dose of 3 years. Making use of the supplemental appendix and protocol, we can see that this study was conducted in an adequately blinded and randomised fashion, with allocation concealment. Four months after the third injection, 55% in the study group and 18% in the placebo group had antibody levels over the threshold of 100 U/ml for anti-receptor-binding domain antibodies. There was also a significant improvement in virus neutralisation in the study group (71% vs 13%). The third moderna vaccine dose showed a significantly higher immunogenicity than placebo in this patient group, but the authors acknowledge that the study was not powered to, nor had long enough follow up, to assess clinical outcomes.5.RANDOMISED CONTROLLED TRIAL 2Conversion from Calcineurin Inhibitor to Belatacept-based Maintenance Immunosuppression in Renal Transplant Recipients: a Randomized Phase 3b Trialby Budde, K., et al. Journal of the American Society of Nephrology 2021 [record in progress].Per protocol analysis.Yes.ClinicalTrials.gov–NCT04885907.Non-industry funded.Participants were randomised to either the belatacept conversion group or the CNI continuation group.Participants were randomised into two groups: the intervention group, in which the patients participated in a personalised exercise rehabilitation program in addition to standard care, or the control group where the patients received standard care alone.446 stable adult kidney transplant recipients.The primary outcome was the percentage of patients surviving with a functioning graft. Secondary outcome included patient survival, graft survival, incidence and severity of biopsy-proven acute rejection (BPAR), renal function, mean changes in systolic and diastolic blood pressure, proportion of patients with preexisting donor-specific antibodies, and adverse events.24 months.de novo DSA, tempered by numerically higher acute rejection rates. It should be noted that the population recruited is relatively low risk, with no recent acute rejection, stable function and EBV seropositive due to risk of PTLD. In reality, the study is underpowered to demonstrate non-inferiority for the primary endpoint, although outcomes in both arms in this respect were excellent. Longer-term follow-up will be interesting to see, as it is quite possible that the improvements in graft function and reduction in dnDSA seen will translate to better long-term graft survival.This large multicentre phase 3b study randomised renal transplant recipients 6–60 months post-transplant to continue CNI, or to switch to Belatacept-based immunosuppression. The primary endpoint  did not differ between groups. There was, however, a clinically significant superior GFR in the Belatacept arm with lower rate of 3.Strict intention-to-treat analysis.Yes.ClinicalTrials.gov–NCT01820572.Industry funded.This is a large, well-conducted RCT that took place across multiple centres in several countries. The included patients were adult recipients of both live and deceased donor kidney transplants who were stable 6–60 months after surgery. Patients were also excluded if they had experienced: antibody-mediated rejection at any time; any form of rejection within 3 months of the study start; recurrent acute rejection; greater than or equal to Banff grade-IIA acute rejection in the current allograft; previous graft loss due to BPAR; or had a positive T cell crossmatch prior to the current transplant. Randomisation was adequate and included only one form of stratification; by GFR so that an equal distribution of low functioning kidneys was entered into each study arm. Renal biopsies were mandated in any suspected acute rejection episode, surveillance biopsies were not done.The primary endpoint was graft survival at 24 months and this was assessed in an intention to treat analysis to preserve randomisation. The withdrawals, dropouts and cross-overs are described and in any event were at tolerable levels for a study of this size. Due to the lack of prior data on graft function on which to base power calculations, the study was powered to exclude significant differences in graft survival instead.de novo DSA in the belatacept group than the CNI group, but there is no statistical analysis presented for this outcome.The results show that there was a similar patient and graft survival at 24 months after randomisation and there was no significant difference in acute rejection rates or overall adverse events. However, there was a significant and evolving improvement in eGFR in patients in the belatacept group from baseline, compared to a decline in eGFR in the CNI group. The paper demonstrates a lower proportion of patients that developed This study shows that low-risk, stable renal transplant recipients can be converted to belatacept from CNI-based immune suppression with comparable graft survival at 2 years. Whilst there were some post-conversion rejection events, they did not lead to any graft loss in this study and were successfully treated with steroids. Conversion to belatacept in this low-risk population is associated with an improvement renal function and provides a safe option in patients who are intolerant of CNI. If the follow up for the study could be extended, then this improvement in function might also be associated with improved graft survival.


NOTCH2NLC. NIID exhibits a wide range of clinical manifestations. However, patients presenting with recurrent migraine-like attacks and cerebral edema have only rarely been reported.Neuronal intranuclear inclusion disease (NIID) is a progressive neurodegenerative disease associated with the GGC repeats in the 5'-untranslated region (5'UTR) of NOTCH2NLC gene identified an abnormal expansion of 118 GGC repeats in the 5'UTR of NOTCH2NLC gene.A Chinese female suffered probable migraines with aura for 10 years. At age of 51, aggravating migraine-like attacks co-occurred with a sudden encephalopathy-like episode. Brain MRI showed right cerebral edema and cortical enhancement. Serum lactic acid level was elevated at rest and significantly increased after a simplified serum lactic acid exercise test. The initial diagnosis was MELAS, while NIID was suspected after faint DWI high-intensity signals in the corticomedullary junction was retrospectively recognized. Mitochondrial genome sequencing and gene panel analysis of nuclear genes related to mitochondrial diseases failed to find any causative variants. Repeat-primed PCR and fluorescence amplicon length PCR of NIID should be taken into account for differential diagnosis of migraines and MELAS-like episodes. Besides the corticomedullary hyperintensity on DWI, cortical enhancement in contrast-enhanced brain MRI may also be a brain image marker for the differential diagnosis between MELAS and NIID with MELAS-like episodes. NOTCH2NLC gene has been confirmed as the cause of familial and sporadic cases of NIID  is a progressive neurodegenerative disease characterized by the presence of eosinophilic hyaline intranuclear inclusions in the central and peripheral nervous systems and various organs . Recentl of NIID . NIID ex of NIID . Encepha of NIID , 5. HoweA 51-year-old female presented with recurrent headaches for 10 years. At the age of 41, she had probable migraines with aura. The headache attacks usually preceded by a paroxysmal blurred vision, which looked like a snowflake-like flashing dots in front of her eyes. These vision symptoms usually lasted for about 15 min, followed by numbness on the left fingers and the lip, which lasted for about 5 min. A severe throbbing headache in the right frontotemporal region occurred next, which sometimes accompanied by nausea and vomiting. Photophobia and phonophobia during attacks were also reported. The pain was often severe enough to hamper daily activities and may last from hours to half a day when painkillers were given. Initially, she had 3 to 4 attacks each year. Four years ago, she started to take flunarizine to prevent migraine when headaches increased. Two month before admission, she stopped taking flunarizine, and the headaches occurred twice a month.2-weighted images and fluid-attenuated inversion recovery (FLAIR) images along the cortex and partial subcortical area in the right occipito-parieto-temporal lobes and part of frontal lobe, with gadolinium enhancement selectively spreading along the surface of the cortex  showed abnormal hyperintensities on diffusion-weighted images (DWI), Te cortex . She wasRI image .NOTCH2NLC gene  and fluorescence amplicon length PCR (AL-PCR) was performed to screen the GGC expansion in the 5'UTR of the NLC gene . FluoresNLC gene . Her motDuring the follow-up, she was continued with coenzyme Q10 and riboflavin. Fatigue and poor appetite was complained, and no headache was reported. A follow-up brain MRI performed 3 months later after disease onset revealed the cortical enhancement was improved , while DIn this study, we report a patient with NIID presenting with a MELAS-like episode in long-standing history of migraine with aura. Our patient present with two rare situations occurred in NIID patients. First, migraine with aura and NIID occur concurrently in our patient, a condition that was only reported in a few patients with NIID. Our case gives further evidence that the two diseases could co-relate with each other. Second, extensive cerebral edema, with the cerebral cortex mainly involved, and elevated serum lactic acid level, strongly suggest a MELAS-like episode. This is a very uncommon presentation in NIID patients. Our case indicated again that differential diagnosis should be emphasized between adult-onset NIID and MELAS.NOTCH2NLC is also associated with essential tremor, Alzheimer's disease, frontotemporal dementia, Parkinson's disease, multiple system atrophy, leukoencephalopathy, amyotrophic lateral sclerosis, and oculopharyngodistal myopathy type 3  for the publication of any potentially identifiable images or data included in this article.FX designed the study and wrote the first draft of the manuscript. XH offered suggestions for the study and revised the manuscript. PL performed the genetic analysis and revised the manuscript. DZ organized the study, collected clinical data, and revised the manuscript. All authors reviewed and accepted the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


A 75-year-old man with an abdominal aortic aneurysm underwent endovascular aortic repair (EVAR) using an AFX2 endograft with no endoleaks. Nevertheless, the aneurysmal sac increased by 8 mm at 24 months after EVAR despite no detectable endoleaks. Open surgical treatment was performed because of the risk of rupture. Intraoperative findings of much viscous cloudy fluid with no blood flow in the sac suggested that perigraft seroma resulted in sac enlargement. The endografts were replaced by a Dacron graft. Perigraft seroma should be considered as a cause of sac growth after EVAR with AFX2 when there are no detectable endoleaks. To date, several reports have elucidated the possible etiology and pathogenesis of PGS after open AAA repair. Conversely, there have been few reports regarding PGS associated with endovascular aortic repair (EVAR).Perigraft seroma (PGS) complicates open abdominal aortic aneurysm (AAA) repair by using a vascular prosthesis with a relatively low incidence.Here, we report our experience of PGS after EVAR using an Endologix AFX2 endograft, which comprises extended polytetrafluoroethylene (ePTFE) graft.A 75-year-old man with a diagnosis of 65 mm AAA . Endoleaks can be diagnosed on a contrast-enhanced CT scan. By contrast, endotension can be diagnosed by no evidence of endoleaks despite increased aneurysmal sac. Endotension is associated both with transudate accumulation within the sac due to an endograft porosity and with pressure transmission through an endograft and intrasac thrombus. The former case is called “perigraft hygroma” or “PGS.” In our case, sac enlargement with a diameter increase of 8 mm per year was confirmed on a routine CT scan without any types of endoleaks after successful EVAR with AFX2. CT also showed homogeneous density in the sac, which seemed to be different from the findings of occult endoleaks. Much viscous cloudy fluid without no blood flow in the aneurysmal sac was recognized during surgery. Intrasac pressure was not measured, but fluid evacuation under tension implied increased intrasac pressure. On the basis of these clinical conditions, we eventually diagnosed PGS during the surgery.Aneurysm sac enlargement after EVAR is a crucial finding because it means treatment failure and can cause sac rupture. The causative factors have been reported to be a low-grade infection, an immunologic reaction to the graft material, and an extravasation of plasma fluid through the graft. PGS develops even after EVAR. Cho et al. reported late aneurysm enlargement after EVAR with an Excluder bifurcated endograft  made of ePTFE fabric. The authors showed that nine of 45 patients developed late sac enlargement with no active endoleak. Nano et al. reported a case of sac enlargement caused by seroma after EVAR with a PowerLink endograft  made of ePTFE fabric, which was a former product of AFX2. The difference between the PowerLink and AFX2 system is the graft processing methods. The AFX2 system characterizes tighter mechanical strength with helically wrapped ePTFE layers despite a reduced thickness of the graft wall, which is called the DURAPLY graft. The DURAPLY graft of AFX2 is a stronger and less permeable ePTFE graft than the first-generation graft of PowerLink. Although the exact pathogenesis of PGS with AFX2 remains unclear, direct pressure transmission through a strengthened graft material and serous fluid transudation through a thinner graft material can be the contributory mechanism of the PGS with AFX2 endograft. Risberg et al. have proposed a new mechanism of PGS. They described that thrombus degradation via stress-induced fibrinolysis and subsequent protein-rich perigraft fluid collection provoke the increased permeability of serum across the endograft. Regarding the association between the materials of the stent graft and PGS, there have been no reports on PGS after the implantation of stent grafts made of woven polyester  or Zenith Flex ). This suggests that woven polyester stent grafts seem to be rarely involved in PGS, unlike ePTFE stent grafts.PGS is well recognized after open AAA repair mainly using a PTFE graft. Symptomatic patients with increasing PGS need interventions. Interventions include open surgical procedures such as complete removal of an endograft followed by replacement and endovascular procedures such as relining. We preoperatively considered that sac enlargement was not caused by PGS but by indiscernible, very low flow endoleaks. We were also concerned that sac enlargement could threaten the proximal and distal sealing and cause type I endoleak, which could result in sac rupture. Fortunately, the patient was not frail without any comorbidities that made invasive open surgery difficult. We eventually decided to perform definitive graft replacement.In our case, we planned to perform open surgery for the asymptomatic patient with sac growth reaching 73 mm. Generally, close observation is recommended for asymptomatic patients with stable PGS. Graft replacement after EVAR includes demanding procedures such as suprarenal aortic clamp and dissection of the adhesion at the proximal and distal landing zone. Fortunately, these procedures were not so complicated and graft replacement was completed without any complications in our case. An alternative less invasive surgical option is a graft-preserved procedure such as a wrapping of the endografts with a Dacron graft and tight closure of the sac. This operation is time-saving in comparison with graft replacement, but careful observation of the recurrence is required after the operation.PGS was diagnosed during surgery in our case. We did not change the surgical strategy of replacing the whole endografts because it has been reported to be a curative approach with a high success rate.We experienced a rare case of PGS after EVAR by using the AFX2 endograft. In the case of sac enlargement with no detectable endoleaks on a routine contrast-enhanced CT scan, PGS should be considered as a cause of sac growth.


The mouse heterotopic cardiac transplant model has been extensively used to explore transplant immunity. Although the cuff technique facilitates the operation, the procedure remains difficult, and vessel eversion is the most difficult step. Cuff movement and everted vessel wall slippage are the main adverse factors in vessel eversion. Traditional strategies to prevent these factors focus on cuff fixation, while more steps or surgical instruments would be required.b) or BALB/c(H-2d) mice were transplanted into C57BL/6(H-2b) mice. The operating times of recent 90 operations, which were divided into 9 groups according to their sequence, were summarized and analyzed.According to the reported protocols and our experience, the vessel eversion skills were modified and used for transplantation. Cardiac grafts from C57BL/6(H-2The mouse cervical cardiac transplantation was successfully performed by using the modified vessel eversion skills. The cuff movement, which is the most important adverse factor to prevent vessel eversion, was effectively prevented. In the recent 90 operations, the total operating time was 47.3±7.9 min and the success rate was 98%.The modified surgical skills simplify the vessel eversion in mouse cervical cardiac transplantation with cuff technique, characterized by less cuff movement, fewer steps, and surgical instruments. Using these surgical skills, the transplant can be performed in a short time. In this nonfunctional transplant model, the cardiac graft aorta is anastomosed to the recipient artery, and the cardiac graft pulmonary artery is anastomosed to the recipient vein; the host blood flow perfuses the cardiac graft through the coronary circulation. Although the mouse heterotopic cardiac transplant model has been extensively used to explore transplant immunity,4, the procedure remains difficult. To simplify the operation, cuff technique was developed for mouse cervical cardiac transplantation in 1991.Corry first described the mouse heterotopic vascularized cardiac transplant model in 1973,6-8. Traditionally, strategies to prevent these problems have focused on cuff fixation, such as the use of a specially designed cuff that has an extension, or on modification of the manner of vessel anastomosis or the use of affiliating tools,6-8, which required more steps or surgical instruments. In this article, we present surgical skills to simplify vessel eversion that do not require specially designed cuffs or surgical instruments.Although the cuff technique facilitates the mouse cervical cardiac transplantation, the procedure is still difficult. During the surgery, vessel eversion is the most difficult step; cuff movement and everted vessel wall slippage are the main adverse factors in vessel eversionb) and BALB/c(H-2d) mice were purchased from Beijing HFK Bioscience Co., Ltd, or Beijing SPF Biotechnology Co., Ltd, or generated by the Institute of Zoology. All mice were kept in microisolator cages in a specific pathogen-free facility. All experiments were approved by the Animal Ethics Committee of the Institute of Zoology. All animals received human care in compliance with the Guide for the Care and Use of Laboratory Animals.The 6-12-week-old male C57BL/6 has a 0.8 mm external diameter and a 0.7 mm inner diameter. The cuff for the carotid artery (CA) has a 0.5 mm external diameter and a 0.4 mm inner diameter. The cuffs were cut in segments ranging from 1 to 1.3 mm . A set oThe operation was conducted using an operating microscope  with a magnification between 5x and 25x.,9,10, modified by our experience. In the step of recipient vessel eversion, the vessel was dragged into the cuff, and the vessel end was gently caught with two forceps, bilaterally, while the cuff was perpendicular to the clamp (The cardiac graft procurement and transplantation primarily followed the reported protocolshe clamp . One halhe clamp . To connhe clamp  and 2D. P <0.05 was considered statistically significant.All data were presented as mean±SEM. Graft survival was compared using the log-rank test. A The Mouse Cervical Cardiac Transplantation Model with Cuff Technique Was Successfully Established,9,10 and our modification during training, the mouse cervical cardiac transplantation was established successively (According to the reported protocolsessively  and 3B. essively . In the ,6-8. Following the surgical skills previously described, vessel eversion can be successfully performed (Vessel eversion is the most difficult step of mouse cervical cardiac transplantation using the cuff technique. Cuff movement and everted vessel wall slippage are the main adverse factors of vessel eversionerformed . Vessel erformed . TherefoDue to the benefit of vessel eversion surgical skills, the transplant can be completed in a short time. In our recent 90 consecutive operations, the total time was 47.3±7.9 min from the beginning of the graft procurement to the incision suture of the recipient mice. Furtherly, these 90 operations were divided into 9 groups according to their time sequence. In the last 10 operations, the operating time was stably decreased and totaling 39.0±6.4 min. The recipient time, including vessel preparation, graft implantation, and recipient incision suture, also decreased . Therefo,4. Compared to the suture anastomosis, deploying a cuff technique may be easier and saves time,11. The most difficult step of mouse cervical cardiac transplantation using the cuff technique is vessel eversion, especially for the CA,10. Cuff movement and everted vessel wall slippage are the primary adverse factors in vessel eversion,6-8. Some work has been done to address these adverse factors, such as using a cuff with an extension that can be held with a clamp and using instruments to hold the cuffs. These improvements help make vessel eversion easier in situations requiring more steps or surgical instruments. In addition, improving surgical skills may be another way to facilitate vessel eversion.The mouse heterotopic cardiac transplant model has been extensively used to explore transplant immunityDuring our operations, we first placed the cuff perpendicular to the clamp and then everted the vessel wall half in half. To prevent vessel wall slippage during ligation after eversion, one forceps held the vessel wall while the other forceps caught one end of a preset circumferential ligature. The second forceps encircled the everted vessel wall with this ligature, then gently pulled the end of the ligature to fix the everted vessel wall. The surgeon released the first forceps to tie the ligature. In our experience, the adverse effects of cuff movement and everted vessel wall slippage can be prevented using these surgical skills.-12; the total operating time of our recent 90 consecutive operations was 47.3±7.9 min. In addition to the total time, the time of vessel preparation, defined as the process from performing the recipient skin incision to finishing the vessel eversion of both EJV and CA, was 21.0±4.8 min for these 90 operations. Our improvement in surgical skills was effective in reducing the time of operations.Traditionally, the average total operating time has been reported to be 45 to 60 minSeveral useful points and some troubleshooting are as follows:,12,13.Watching a video of the operation is helpful for mastering the operation. Some videos by Ratschiller and Oberhuber are available onlineSuper-fine-tip forceps, such as the forceps with a 0.15 mm tip, are more appropriate during blood vessel isolation, eversion, and anastomosis.An inner core, which is placed into the vessel lumen to facilitate vessel eversion, may be useful during the early training stage. Our experience, however, indicates that it should be avoided as much as possible because of the risk of vessel intima injury and thrombosis for some genetically modified mice.The site of the ligature on the recipient CA must be at the proximal site of its bifurcation, and some distance should be left from the ligature to the bifurcation to avoid the risk of cerebral blood flow disorder.The length of the cuffs should be appropriate for vessel eversion and donor and recipient vessel anastomosis. In our experience, 1.0-1.3 mm may be suitable.In conclusion, in addition to the strategies that facilitate vessel eversion by fixing the cuff in mouse cervical cardiac transplantation with cuff technique, the modified surgical skills simplify the vessel eversion, characterized by less cuff movement, fewer steps and surgical instruments. Using these surgical skills, the transplantation could be performed in a short time.