Bacteroides spp., Parabacteroides spp., Lactococcus spp., and Enterococcus spp. The spontaneous fermentation of cricket powder allowed for the isolation and characterization of seven lactic acid bacteria strains belonging to six species: Latilactobacillus curvatus, Lactiplantibacillus plantarum, Latilactobacillus sakei, Lactococcus garvieae, Weissella confusa, and Enterococcus durans. The strains were characterized and selected according to different technological properties. L. plantarum CR L1 and L. curvatus CR L13 showed the best performance in terms of general aminopeptidase activity, acidification, and growth rate in MRS broth and in dough with cricket powder and wheat flour, as well as robustness during consecutive backslopping. Thus, they were used as starter-mixed to produce sourdough to manufacture bread fortified with 20% cricket powder. The addition of cricket powder led to a significant increase of protein (up to 94%) and lipid content, from 0.7 up to 6 g/100 g of bread. Spontaneous fermentation represents a source of microbial diversity that can be exploited in order to obtain potential starters for food with innovative ingredients. Edible insects powder can be successfully added in leavened baked goods to enhance their nutritional value.The bacterial community profile of cricket powder highlighted the presence of four main genera:  Acheta domesticus) are included by the European Food Safety Authority (EFSA) in the list of the insects with the biggest potential for food and feed  of 154, with and without NaCl addition (2% w/w) and incubated at 30 °C. At the end of every fermentation step, microorganism enumeration was carried out on MRS agar. In order to monitor the strain trends during propagation, at least 20 colonies of the beginning of the experiment and at the end of the 4th, 7th, and 10th backsloppings were randomly selected from the plates containing the two highest sample dilutions and then were analyzed by RAPD-PCR to determine the percentage distribution of the inoculated LAB.Initially, a sourdough comprising 80% wheat flour (type 00) and 20% cricket powder with a dough yield of 154 was prepared. LAB were counted by a Neubauer improved counting chamber  to obtain an initial cell density in sourdough of about 7.3 log CFU/g and were simultaneously inoculated into the dough. The sourdough was backslopped ten times (25% V2 − V0)/V0] × 100, where V2 was the volume after the 2 h fermentation and V0 was the initial volume.The pH values of the sourdoughs were determined by a pH meter . The total titratable acidity (TTA) was calculated by weighing 10 g of dough samples and homogenizing them with 90 mL of distilled water for 3 min; the TTA was then expressed as the amount (mL) of 0.1 N NaOH to achieve a pH of 8.5. The increase of volume was assessed immediately and after 2 h of fermentation at 30 °C by placing 100 g of dough in a graduated cylinder (1 L). The volume increase was calculated using the following formula: [  before the injection. Organic acid determination was carried out by HPLC analysis  following [For the organic acid determination, the sourdough samples were diluted ten times with distilled water and then filtered by Amiconollowing . Data weThree breads were prepared according to recipes reported in Triticum aestivum L.) flour , while CSDBread and CBYBread (bread produced by baker’s yeast and wheat flour and cricket powder) were incorporated with 20% of cricket powder. The CBYBread was fermented by using only baker’s yeast . The ingredients were added at the same time and mixed for 10 min in a model RS12 twin arms mixer . The doughs were placed on trays at 30 °C with 88–90% relative humidity in a Unipan proofing chamber  for 2 h. Samples were taken at the beginning and end of the leavening time. Finally, doughs were baked at 180 °C for 25 min in an oven .The sourdough was prepared by inoculating the strains that showed promising technological traits at an initial cell density of approximately 7 log CFU/g of dough. The dough was fermented at 30 °C for 18 h before its utilization as ingredient for SDBread (bread produced by sourdough and only wheat flour) and CSDBread (bread produced by sourdough and wheat flour and cricket powder). SDBread was prepared with type “00” wheat  (GraphPad Prism 6 software package). A p value of <0.05 was considered to be significant. The kinetics of microbial growth and acidification were modelled in agreement with the Gompertz equation using GraphPad Prism 6 The results are presented as the mean values ± standard deviation of two or three separate experiments for each test. The level of statistical significance was determined using one-way ANOVA (for multiple groups) followed by Tukey’s Test or Student’s The results of cricket powder microbial diversity, assessed by the sequencing of the V3–V4 region of the 16S rRNA gene, are shown in Parabacteroides spp. and Bacteroides spp. were the dominant genera at 35.6% and 15.5%, respectively. Three genera were identified among Firmicutes: The genus Lactococcus spp. was found at the highest relative abundance, accounting for 6.9%, followed by Enterococcus spp. (4.7%) and Weissella spp. (0.6%). Leminorella spp. and Acinetobacter spp., belonging to Proteobacteria, were found in low abundance at less than 1%.A total of 57,811 reads passed the filters applied in the QIIME split_library.py script. Most of the operational taxonomic units (OTU) were identified at the order level as 96.7%, while at the genus level, sequences were identified at 69.0%. Cricket powder was dominated by three bacterial phyla—Bacteroidetes , Firmicutes (30.0%), and Proteobacteria (8.2%). Tenericutes and Fusobacteria were also detected in low abundance at less than 1%. Within Bacteroidetes, Microbiological analyses indicated that cricket powder was characterized by cell densities of 3.71 ± 0.27 log CFU/g of LAB, of 3.51 ± 0.27 log CFU/g of yeasts, and of 4.12 ± 0.28 log CFU/g of the total mesophilic bacteria. Enterobacteriaceae were below the detection limit. In order to isolate indigenous LAB, a dough was prepared using cricket powder. The dough was backslopped daily ten times. At the end of each backslopping, LAB, yeasts, Enterobacteriaceae, and the total mesophilic bacteria were enumerated .At the beginning, the microbial populations of the dough included 3.40 log CFU/g of LAB, 3.74 log CFU/g of the total mesophilic bacteria, and 3.17 CFU/g of yeasts. Enterobacteriaceae were below the detection limit. During the consecutive backsloppings the yeast population increased, reaching the maximum concentration of 6.08 log CFU/g at the end of the second day; then, it decreased, and yeasts were not detected anymore after the fourth backslopping. Unexpectedly, starting from the fourth backslopping, Enterobacteriaceae were detectable, attaining a value of approximately 5 log CFU/g. After the seventh propagation step, the population started decreasing, reaching a concentration of 3.30 log CFU/g at the end of dough propagation. The total mesophilic bacteria rapidly increased during the propagation, and they were found in concentration of 9.48 log CFU/g after the first 24 h of fermentation. The cell densities remained basically stable until the end of propagation.The LAB cell density after the first day of propagation was 7.25 log CFU/g. Over the successive backsloppings, their number increased, reaching concentrations higher than 9 log CFU/g and then remaining stable until the end. The initial pH of the dough was 6.38, and it remained stable at about 6.5 during all the propagation steps despite the high LAB concentration.Lactiplantibacillus plantarum  CR L13, Lactococcus garvieae CR L14 and CR L31, Latilactobacillus sakei (formerly known as Lactobacillus sakei) CR L15, and Enterococcus durans CR L36.A total of 60 isolates of presumptive lactic acid bacteria, isolated on each day of propagation, were subjected to RAPD-PCR. The analysis allowed us to identify seven patterns named CR L1, CR L2, CR L13, CR L14, CR L15, CR L31, and CR L36 . The amplantarum ) CR L1, The isolated lactic acid bacteria LAB strains were evaluated for their technological features.W. confusa CR L2) to 2.17 ± 0.23 mMol/L of p-NA reached by L. garvieae CR L14, which was not statistically different from the enzymatic activities of L. curvatus CR L13 and L. garvieae CR L31. PepA and PepO activities were low in all the LAB strains when compared to PepNC activities, not exceeding 0.26 mMol/L of released p-NA. No significant differences were detected among the strains.The PepNC, PepA, and PepO activities of the strains were determined using Leu-p-NA, Glu-p-NA, and NCBZ-Gly-Gly-Leu-p-NA as substrates. The highest variability and values were found for PepNC activity . The valW. confusa CR L12 strain was the only LAB that showed a notable ability to produce EPS from sucrose after 48 h of incubation at 30 °C. No production was observed in the other six strains.The The LAB growth and acidification kinetics in the MRS broth at 30 °C were monitored over 24 h , and dat2R values higher than 0.90 (data not shown). L. plantarum CR L1 showed the best performance of growth; indeed, it displayed the highest maximum yield (C) of 1.56 ± 0.03 and the highest maximum rate (µ max) of 0.29 ± 0.02, along with a short lag phase. The lowest growth was observed for both L. garvieae strains (less than 0.6), while the lowest µ max was displayed by L. curvatus CR L13. L. garvieae CR L31 showed the longest lag phase. All the LAB reached the stationary phase after approximately 20 h. The LAB acidification ability was assessed by measuring the pH for 24 h. The kinetic parameters obtained through the Gompertz equation were used to describe the potential acidifying performances of the tested strains. L. plantarum CR L1 proved to be the most acidifying strain, reaching a final pH value of 3.78 after 24 h and hence having the highest maximum yield (C) value. W. confusa CR L2, L. curvatus CR L13, L. sakei CR L15, and E. durans CR L36 acidified the MRS broth to values below 4.5, while the two L. garvieae strains final pH was approximately 4.5. However, the highest µ max was displayed by L. garvieae CR L31 at higher than 0.3 dpHh−1, and this strain also showed the longest lag phase.The goodness of fit of this model was appropriate for all the tested strains, with w/w) addition. The scatterplot  were 0.94 without NaCl and 0.40 with NaCl. L. plantarum CR L1, W. confusa CR L2, L. curvatus CR L13, and E. durans CR L36 reached a concentration of 9 log CFU/g, while the other strains reached a concentration of around 8.9 log CFU/g. The supplement of NaCl negatively influenced the final cell concentrations. In the presence of salt, none of the strains reached 9 log CFU/g; the highest concentration in the dough with NaCl was achieved by L. plantarum CR L1 at 8.6 log CFU/g, followed by L. curvatus CR L13 and E. durans CR L36.In all the sourdoughs, the addition of salt reduced the acidification. The pH median value of the dough without salt was 5.10, while this value was 5.56 in doughs containing NaCl. L. plantarum CR L1, followed by L. sakei CR L15 (13%); L. garvieae CR L31 was not detected.The strain robustness (the ability to dominate and persist during consecutive backsloppings) was evaluated by co-inoculating all the strains at the initial concentration of 7.3 log CFU/g of dough. Soon after the first day of backslopping, the total LAB concentration was attained at 9.78 log CFU/g, which remained stable until the end. After the fourth, seventh, and tenth backslopping, 20 colonies were subjected to RAPD-PCR in order to monitor the strain percentages. As shown in L. plantarum CR L31 was present at the highest percentage (50% of the isolates), followed by L. sakei CR L15 (28%). At the end of the propagation, L. plantarum CR L1, represented the majority of the isolates, 75%, followed by L. curvatus CR L13 (12%), L. sakei CR L15 (10%), and, in the lowest concentration, W. confusa CR L2 (2.5%). The E. durans strain and both L. garvieae strains were not detected, thus showing the lowest robustness.Our results showed that the proportions among the strains remained similar between the fourth and seventh refreshments. Particularly, L. plantarum CR L1 and L. curvatus CR L13, which were selected for their technological traits, were inoculated for the production of sourdough for bread manufacturing. At the end of fermentation, the SD dough reached a pH below 4.5, while the final pH of the CSD dough was higher at 5.15. The initial pH of the CSD dough was higher due to the buffering capacity of the cricket powder; indeed, the pH decrease was not statistically different between the samples, remaining at around 0.47. The final total titratable acidity values were higher in the CSD and CBY doughs, while the SD dough displayed the lowest value. This difference depended on the incorporation of cricket powder into the dough since the control bread without sourdough inoculum also displayed a considerable TTA value of 7.6 mL. Cricket powder negatively affected the volume increase; indeed, SD dough showed the highest final volume, as seen The results of the acidification, volume increase, and microorganism concentration of the doughs prepared with the use of only wheat flour and sourdough (SD), the addition of cricket powder and the use of sourdough (CSD), and the addition of cricket powder and use of only baker’s yeast (CBY) are reported in Regarding organic acid production, no difference in lactic acid concentration was observed between the two SD doughs, while the highest content of acetic acid was displayed in the CSD dough, thus leading to a different fermentation quotient. The microbiological analyses did not show any significant differences in the LAB and yeast concentrations, with values of about 8.6 and 7.8 log CFU/g, respectively.The addition of cricket powder in the formulation significantly increased the protein content of the breads by +82% and +94% in the CSDBread and CBYBread, respectively, compared to the SDBread. Lipid content was also affected by cricket powder addition, as both the CBreads presented a significantly higher content than the SDBread at more than 5.5%. The amount of carbohydrates was higher in breads without cricket powder due to the higher content of carbohydrates in wheat flour compared to cricket powder. In terms of moisture, no significant differences were found among the samples.Clostridiales and Lactobacillales of the genera Lactococcus spp., Enterococcus spp., and Weissella spp., all of which are of interest in food fermentation. As reported by Cappelli et al. [Enterococcus durans, Lactococcus garvieae, and Weissella confusa. Enterococcus durans has been frequently isolated in several dairy products made with pasteurized and raw milk, and it can occur as natural starter cultures during cheese making [Weissella confusa has been frequently detected in the spontaneous fermentation of fish products and starchy- or cereal-based foods [Lactococcus garvieae is an emerging zoonotic agent isolated from fish and has been the most frequently species isolated from insect frass samples and cricket powder [Lactobacilli. Nevertheless, four species were previously isolated in cereal sourdoughs. Lactiplantibacillus plantarum is often isolated in different ecosystems due to its versatile metabolism [Latilactobacillus curvatus is frequently isolated in association with other Lactobacilli (particularly Latilactobacillus sakei) in fermented meat, such as sausage [In the first part of the work, a bacterial characterization of cricket powder was carried out through a next generation sequencing (NGS) analysis that allowed for an in-depth evaluation of the occurring bacteria. In accordance with the findings of other authors ,20,21, ti et al. , the mice making ,38. Weised foods , as welled foods . Lactocot powder . It has t powder , wheat gt powder . The spotabolism . Latilac sausage , and dur sausage . The dis sausage ,47,48,49L. garvieae strains displayed the highest values of PepNC, which were not statistically different from the enzymatic activities of L. curvatus CR L13. The EPS production was also tested, since microbial EPS can improve the rheology and texture of gluten-free or low-gluten dough [W. confusa CR L2 strain was able to produce EPS from sucrose, corroborating that the ability to produce these polymers is one of the distinctive phenotypic features of the genus Weissella. Acidification and growth capabilities are essential characteristics for a microbial starter to be used in sourdough fermentation. A short lag phase, a high acidification rate, and a high yield are of particular interest. In our experiment, the kinetic parameters obtained through the measurements of pH and growth over 24 h indicated that the L. plantarum CR L1 strain was the best performing, showing the highest and fastest acidification both in the MRS medium and the sourdough made of 20% cricket powder. The acidification in cricket-wheat sourdough was also evaluated with the addition of 2% of salt, which is a common ingredient in bread and cereal-based products. As expected, the addition of salt worsened the acidification and growth parameters. Together with L. plantarum CR L1, L. curvatus CR L13 showed good performance in the presence of salt. In this regard, homofermentative LAB and facultative heterofermentative LAB such as L. plantarum and L. curvatus are considerably more resistant to high NaCl levels than heterofermentative species [L. plantarum has been reported to grow at 8% NaCl [L. plantarum CR L1 was already observed at the fourth backslopping and was retained until the end of propagation. L. curvatus CR L13 microbial population represented the second-highest percentage of LAB at the end of propagation, showing an increase of its prevalence during the consecutive backsloppings. Based on the obtained results, L. plantarum CR L1 and L. curvatus CR L13 were selected as starters for the production of sourdough for bread manufacturing. Both the doughs with cricket powder were characterized by a higher TTA than the SD dough, as found by Osimani et al. [The direct selection of starter microorganisms from the substrate to be fermented is considered an important feature in guaranteeing the rapid adaptation, intense acidification, and positive influence on the nutritional and technological properties of a specific matrix ,51. The en dough . Only th species , and L.  8% NaCl ,59. Fina 8% NaCl . Usually 8% NaCl ,62. The i et al. . This fii et al. ,13,63. ALactococcus spp., Enterococcus spp., and Weissella spp. genera. The spontaneous fermentation of cricket powder, monitored through a culture-dependent method, allowed for the development of seven lactic acid bacteria species that are highly adapted to this ecosystem. The technological characterization of the indigenous LAB led to the selection of two suitable strains, L. plantarum CR L1 and L. curvatus CR L13, to be used for the manufacturing of cricket-wheat-based sourdough products. Despite the negative effect on bread volume, cricket powder replacement provided higher protein and fat contents than those of bread with only wheat flour. Overall, these results highlighted the good suitability of the microbial starter for the production of cricket-wheat flour bread with improved nutritional quality. Further studies should be carried out in order to improve consumer acceptance, test the selected LAB in other products, and optimize the final product formulations. Nevertheless, the combination of lactic acid bacteria fermentation and the use of edible insects as alternative flour can lead to the manufacturing of baked goods with improved nutritional traits that meet the demand of more sustainable food.The microbiota of cricket powder were characterized by several gut-associated bacteria and, among lactic acid bacteria, by the


Variable-stiffness artificial muscles are important in many applications including running and hopping robots, human–robot interaction, and active suspension systems. Previously used technologies include pneumatic muscles, layer and granular jamming, series elastic actuators, and shape memory polymers. All these are limited in terms of cost, complexity, the need for fluid power supplies, or controllability. In this article, we present a new concept for variable-stiffness artificial muscles  made from twisted rubber cord that overcomes these limitations. Rubber cord is inexpensive, readily available, and inherently compliant. When an extended piece of rubber cord is twisted, the tensile force it exerts is reduced and its stiffness is altered. This behavior makes twisted rubber ideal for use as an artificial muscle, because its output force and natural stiffness are both controllable by varying twist angle. We investigate the behavior of four types of rubber cord and evaluate which type of rubber allows for the greatest reversible reduction in average stiffness  and initial stiffness . Tensile force and stiffness can be further altered by increasing the twist angle of the artificial muscle beyond a threshold angle, which initiates nonlinear buckling behavior. This, however, can cause plastic deformation of the artificial muscle. Using a single TRAM, we show how the equilibrium position and natural frequency of a system can be simultaneously altered by controlling twist angle. We further demonstrate independent position and stiffness control of a functional robotic arm system using an antagonistic pair of TRAMs. TRAMs are ready for immediate inclusion in a wide range of robotic systems. Compliance can be beneficial to running and hopping systems, allowing energy to be stored and reused as the leg shortens and extends,6–11 Similarly, in granular jamming, a particulate media such as coffee grounds contained within an elastic bag is transitioned from a compliant and malleable state to a rigid state by application of a vacuum, which causes the granular material to lock or “jam” together.13 Other systems have used antagonistic arrangements and variable-reduction-ratio transmissions that allow traditional linear springs to deliver variable-stiffness behavior.14–16 Similarly, the Mechanically Adjustable Compliance and Controllable Equilibrium Position Actuator (MACCEPA) actuator combines a tension-adjustable traditional linear spring and an offset lever arm to deliver decoupled controllable stiffness and equilibrium position for rotary joints.18 Many polymers exhibit glass transition behavior, transitioning from a hard, brittle, “glassy” state to a soft, flexible, “rubbery” state when heated above their glass transition temperature. This behavior has been well studied as a mechanism to deliver controllable stiffness variation.19–24 More unusual variable-stiffness mechanisms include an elastic ring with anisotropic stiffness, which can be rotated to change its loading direction resulting in a change in effective stiffness,25 and flexible structures coated in or containing wax, which phase transitions to a liquid when heated.26Various physical phenomena and technologies have been proposed to deliver materials with controllable stiffness. In laminar or layer jamming, interleaved thin sheets form a variable-stiffness structure. The sheets are free to slide past one another, allowing extension and flexion, until a vacuum is applied to the device, drawing the sheets together and preventing deformation.27–35 whereby one or more strings are twisted to exert a linear contractile force. Here, we replace the comparatively inelastic strings in twisted string actuators with a single strand of rubber cord. Surprisingly, when a load is suspended from a rubber cord and the cord is twisted, the cord length increases, in contrast to the shortening-with-twist behavior exhibited by twisted string actuators  or helical (double string actuator) form, reducing its length . In contThe source of the twist-induced compressive stress that acts to squeeze the rubber cord is not immediately clear; the mechanism can be imagined more clearly by considering an analogous system, a single cord of elastic material, lying vertically on the surface of a cylinder . If the L is the initial length of elastic cord and the height of the rigid cylinder and r is the radius of the rigid cylinder. Because the cord is elastic and has been stretched, it exerts a tensile force T resisting its extension. For a Hookean material,where k is the material's stiffness. This force can be resolved into two components: axial force FA and tangential force FT , the axial force reduction Fr isThis phenomenon has been well studied in the field of materials.r is the radius of the cylinder, C10 is the Neo-Hookean material constant and ψ is the torsion defined as φ/l, where φ is the total twist angle and l is the rubber cylinder length.37 Alternatively, using a Mooney SEF, the axial force reduction iswhere λ is the stretch ratio along the axis of the cylinder, and C10 and C01 are the Mooney SEF coefficients.37 These models have been evaluated for twist angles up to 0.2 rad for a rubber sample of length 32 mm, implying a maximum ψ of 6.25 rad/m.37where 37 this phenomenon is yet to be investigated by the Soft Robotics community. Twisted rubber cord has extremely attractive qualities for use as artificial muscles: rubber cord is readily available, inexpensive, inherently compliant, and matches the typically desired artificial muscle form factor. It lacks the disadvantages associated with state-of-the-art artificial muscles such as delamination (layer separation) in dielectric elastomer actuator (DEA) stacks38 and does not require processing before use such as precoiling as in coiled polymer actuators.39 Twisted rubber cord can be included within a robotic system and its twist angle can be varied to control axial force. In addition, controlling the twist angle of rubber cord can deliver variable-stiffness behavior, which could improve the performance of locomotion robots or enhance safety in human–robot interaction.Although the variation in axial tensile force exhibited by rubber cord when twisted has been studied in detail from a materials perspective,The force-extension behavior of rubber cord was investigated using a standard material testing setup . A NEMA-In addition to reducing tensile force, increasing twist angle alters the force-extension behavior of the rubber cord: the gradient of the force-extension curve changes, implying altered compliance. This is especially clear at low extensions: the gradient of the force-extension curve is considerably reduced, and the cord is considerably less stiff in the tensile direction. reversible stiffness reduction (that which could be achieved without plastic deformation occurring).In general, for robotics applications, twisted rubber should be used within its elastic range, such that neither twisting or extension plastically deforms the artificial muscle. In cases where extension or torsion permanently altered the sample length or imparted some permanent twist , these results were discarded. Results in For some robotics applications, particularly those where the entire extension range of an artificial muscle is required, an important metric is the amount by which overall stiffness of the muscle can be altered. We define this “average stiffness” as the mean stiffness over the full extension range of a rubber cord sample, calculated by dividing maximum tensile force by maximum reversible extension. For other applications, for example, where a system oscillates about its equilibrium position, only a small amount of extension will be applied to the artificial muscle. If a TRAM only experiences such small extensions, a much greater reduction in stiffness can be achieved. We define “initial stiffness” as the stiffness at an extension of 1 mm (0.6% of the length of the rubber cord). 40–42Thus far, analysis has been restricted to twist angles and extensions that do not permanently alter the rubber cord and cause plastic deformation. If this requirement is discarded, more complex behavior can be captured. At high twist angles, elastic rods can exhibit unusual buckling behavior, transitioning from a quasi-static short-wave helix form to a quasi-static writhing form.One application of TRAMs is controlling the natural frequency of a system. independent control of equilibrium position and stiffness, and enabling many applications in robotics and wearable assistive devices.In this article, we introduced the new concept of TRAMs. These artificial muscles leverage the fundamental material behavior of twisted rubber, which exhibits a reduction in tensile force and change in stiffness when it is twisted. Twisting induces a compressive stress that reduces tensile force and lengthens the TRAM. The compressive stress also causes the stiffness of the TRAM to either increase or decrease (depending on loading) because of the nonlinear S-shaped stress–strain relationship of rubber. By controlling the twist angle of these artificial muscles, tensile force and stiffness can be controlled. Both average and initial stiffness can be reduced considerably . This allows TRAMs to be used to control natural frequency of a system. Antagonistic arrangements of TRAMs further increase their utility, allowing for TRAMs do have limitations: compared with traditional (constant stiffness) engineering springs, which tend to store energy extremely efficiently; rubber has more associated viscous losses. This could increase energy requirements in, for example, running and hopping robots, and the benefits of controllable stiffness should be weighed against this reduced efficiency.In future, we plan to test a wider range of elastic materials, to improve the range of available tensile force variation, stiffness control, and elastic range of TRAMs. We also plan to further investigate buckling and coiling behavior and how they may be exploited to deliver greater functionality in TRAMs. Finally, we plan to develop a proportional–integral–derivative (PID) controller that controls twist angle in a TRAM robotic system, characterize the performance of such a system, and demonstrate setpoint tracking and speed control.Data necessary to support conclusions are included in the article.


There is a heightened concern for exposure to infectious aerosols during the intubation of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) patients. We took our previously designed portable, inexpensive, and easily constructed device and modified it for the prehospital setting.Using polyvinyl chloride pipe and automobile collision wrap, a prehospital unit can be constructed in less than 30 minutes, and the cost of material is under 50 U.S. dollars. The box stores easily in an ambulance and can be assembled on the scene within two minutes.This easily built device likely provides some limited protection from aerosolization during video laryngoscopy and can be replicated easily. Some limited experimentation has suggested that aerosolized novel human coronavirus that is named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can remain viable in the air for up to three hours . SimulatSimilar to our previously reported hospital box, the following materials were used to construct the device for EMS use: polyvinyl chloride (PVC) ½-inch pipe, PVC joints, and plastic collision wrap Table .The PVC pipe can be purchased as a single 10-feet piece that can be cut up into thirteen pieces using a miter saw or hacksaw. Completed box dimensions for this device are 24 inches in height, 22 inches wide, and 27 inches in length. We reduced the width in this version from our hospital designed boxes in order to better accommodate the narrower EMS stretcher. For the top four corners, three-way fittings are used, and on the base corners two 90-degree fittings are used. On the posterior  surface, two "T" fittings are used which will support an additional cross-bar when assembled. Following construction of the frame's two sides, collision wrap is then used to cover each side. Collision wrap has a single adhesive side and is used in automotive industry to temporarily cover broken car windows. The adhesive side of the wrap faces into the interior of the box  can be easily assembled rapidly in the prehospital setting with materials that are readily available in most areas. Given the possible difficulties in acquiring automobile collision wrap, any clear plastic material that could adequately cover the frame could be used. Other suggestions include a clear painter’s drape. Direct visualization of the patient may be limited given the opacity of any alternative material used.Concern over the adhesive nature of the inner surface and its possible interference with performing airway management has not been experienced in the limited time this box has been used.Use of the unit during airway intubation is not the only potential use of this device. Other suggestions include using the box as a protective covering for patients during EMS transfer through a hospital in either the awake or intubated COVID-19 (coronavirus disease 2019) patient.Due to the time-sensitive nature of disseminating this model, a trial of this model in a controlled setting could not be performed. This model does not completely contain aerosolized viral particles, and appropriate personal protective equipment should still be worn when intubating any suspected SARS-CoV-2 patient.The prehospital PRPU is an inexpensive, quick, and easy-to-construct protective device for prehospital clinicians to using during intubation of high-risk patients such as those with SARS-CoV-2. The device is easy to reuse and has many additional applications in the prehospital setting.


Quercus ilex) fruits. The chemical composition of acorn flour showed its richness in carbohydrates (64.43%), proteins (8%), and fat (10%). The extraction yield of acorn starch was about 34.5%. Thus, the composition of extracted acorn starch and its physical and functional properties were studied. Acorn starch had high purity represented by low proportions of proteins (0.92%) and lipids (0.51%) with a pH of 5.3. The swelling power was 20.76 g/g, while the solubility was about 64.22% at 90°C which suggests that acorn starch has potential for use in food industries. The FT-IR spectra of isolated native starches have shown the main bands characterizing the starch. However, X-ray diffractograms exhibited an A- and B-type diffraction pattern. Furthermore, the effect of acorn starch incorporation at different levels  on the quality parameters of a fermented dairy product was investigated at the beginning of storage. The results demonstrated that the most suitable dose of acorn starch to be incorporated in the fermented dairy product was lower than 1%. This low concentration reduced syneresis, improved functional properties, and enhanced the viscosity of the fermented dairy product.In this study, a new starch has been isolated from acorn ( Quercus acorns belong to the family Fagaceae, which includes several species such as Quercus robur, Quercus petraea, Quercus ilex, and Quercus pubescens [Quercus acorns, the chemical composition of this fruit varies with species and origin [Genus ubescens . Due to d origin . Being md origin . Generald origin . Howeverd origin .On the other hand, generally, 31 to 55% of acorns mass were composed of starch [Additionally, today's consumers are demanding food products improving their health. Since acorns have always been an attractive food resource in human diet and are good sources of starch, these fruits show a large potential for commercial use, and thus, acorn flour may be an interesting component .Streptococcus salivarius ssp. thermophilus (S. thermophilus) and Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) [Yoghurt is one of the most popular fermented milk products widely consumed around the world as it provides nutritional and health benefits . Yoghurtgaricus) .Some studies focused on the improvement of yoghurt quality by reducing syneresis and enhancing viscosity and sensorial characteristics by adding whey proteins , fiber-rIn Tunisian dairy industry, modified starch has been commonly used as a stabilizer, especially for many fermented dairy products in order to achieve desired viscosity and prevent syneresis . BesidesIn this regard, in the present study, we evaluated the effect of acorn starch incorporation in milk in order to improve the physicochemical, organoleptic, and rheological quality as well as syneresis variation of a new produced fermented product after manufacture.Quercus ilex were manually collected from Ayn Darahim region of Jendouba in the northwest of Tunisia. Then, kernels were hand-peeled, dried at 40°C for 3 days, and then milled into flour. The acorn flour was stored in glass flasks at about 4°C for further starch extraction and analysis.Acorns fruits belonging to the species Acorn starch was extracted as reported in . Acorn fL∗  to 100 (white)), a∗ (ranging from −100 (greenness) to +100 (redness)), and b∗ (ranging from −100 (blueness) to +100 (yellowness)).Chemical composition of acorn flour and extracted starch was determined according to the AOAC methods for moisture, ash, protein, lipid, and carbohydrate contents . The pH Solubility, water absorption, and swelling power patterns at 60, 70, 80, and 90°C were determined according to the modified method described in . BrieflyTo evaluate the stability of acorn starch under refrigeration and freezing, 400 ml of 6% starch suspension was heated to 95°C at a rate of 1.5°C/min. Then, the suspension was held at this temperature for 15 min, cooled to 50°C at the same rate, and held at this second temperature for another 15 min. Portions of 50 ml were placed in centrifuge tubes, cooled down to room temperature, and stored at 4°C and −20°C. These tubes were centrifuged at 4000 rpm for 10 min. Measurements of water separation from the starch gels at 24, 48, 72, and 96 h were taken [−1 with a spectral resolution of 4 cm−1.All spectra were collected using a PerkinElmer Spectrum single-bounce ATR accessory with a diamond crystal. Dry starches were equilibrated at laboratory humidity (50% RH) and were dispersed in a matrix of KBr at room temperature, and the mixture was then pressed into pellets. The range of spectra was from 400 to 4000 cmα radiation (k = 1.5418 Å) operated at 40 kV and 40 mA was used to determine the degree of starch crystallinity using the method reported in [X-ray diffraction  in the Bragg–Brentano configuration using Ni-filtered Cu Korted in . DiffracStreptococcus salivarius subs. thermophilus and Lactobacillus delbrueckii subs. bulgaricus. Control (0% acorn starch) and enriched batches were incubated at 45°C until the gel structure was formed and pH reached 4.8. Then, fermented dairy products were stirred, distributed in flasks, and stored at 4°C. Finally, sampling of the resulting fermented dairy product was performed on the first day of storage at 4°C. All determinations of physicochemical, technological, and nutritional characteristics of produced products were done in triplicate in order to choose the best added concentration of acorn starch improving this fermented dairy product quality.The fermented dairy product was manufactured from raw milk in the Tunisian SLD industry (industry of dairy products). Milk  was homogenized, pasteurized at 95°C for 5 min, and cooled down to 45°C. After that, three batches of pasteurized milk were prepared and each batch was incorporated with one concentration of acorn starch  and inoculated with a freeze-dried starter culture, consisting of a combination of −1) determined by the potentiometric method according to the IDF standard [Total solids of fermented dairy samples were determined at 90°C using an infrared dryer. The pH of the prepared samples was measured with a pH meter. The titratable acidity was  using a cone-plate geometry (60 mm in diameter). Fermented dairy samples were carefully placed in the measuring system, left to rest for 30 min for structure recovery, and analyzed to determine viscosity .Streptococci were counted on M17  agar, and Lactobacilli were counted on MRS agar . Yeasts, molds, and coliforms were enumerated according to the methods described in [Microbiological analyses of control and enriched fermented dairy product were performed to determine the influence of acorn starch addition on the viability of starter and total bacterial count during the refrigerated storage period. The total mesophilic bacteria were enumerated by using Plate Count Agar Medium , ribed in . The samAll tests were possessed in three replications, and the average values were performed by variance analysis (ANOVA) using STATISTICA software. Duncan's multiple range tests were used at the significance level of 5% to highlight significant differences among the fermented dairy samples.Quercus ilex species is shown in Quercus lobata (4.25%) and Quercus shrubs (4.05%) [Quercus suber. L. In the literature, acorn fat content varies from lower than 2 to 30% [Quercus ilex acorn kernels.The chemical composition of acorn flour and acorn starch extracted from lobata 4.% and Que (4.05%) , on Quer2 to 30%  and thisQuercus ilex acorn flour contains a high amount of carbohydrates, it may constitute a valuable source of starch which can be used in human food. Concerning starch, we noted that the yield of the extraction was approximately 34.5%. This result was in disagreement with that (63%) found by Masmoudi et al. [Quercus suber. L species but slightly similar to that registered by Nand et al. [Manihot esculenta (cassava) (32.10%). However, the obtained yield was higher than those reported, respectively, from D. pyrifolia tubers (26.64%) [D. hispida (11.42%) [Since i et al.  on Quercd et al.  for Mani26.64%)  and D. h11.42%) . The ric1.42% . T.64%  and(11.42%) .D. pyrifolia (3.43) [D. hispida (4.48) [Solanum tuberosum (potato) (6.22).After extraction, we observed that the moisture content of acorn starch under study was about 10.17%. This low moisture content of the acorn starch, less than 20%, was acceptable for commercial starch as it was recommended for safe storage with moisture content less than 13% as reported by Pérez-Pachecoa et al. . Besidesa (3.43)  and D. ha (4.48)  but loweL∗ value (85.03) compared to the acorn flour (51.13). For acorn starch, the obtained values of a∗ (0.52) and b∗ (10.2) were different from those found in acorn flour . These results indicated that acorn starch presents yellow tones. In the present study, the results found for color parameters were similar to those reported on Ramon starch [Acorn starch exhibited a slightly yellow-white color while acorn flour was darker. Indeed, extracted acorn starch showed a high lightness ∗ value 8.03 compan starch . This pon starch .As shown in D. pyrifolia, Dioscorea opposite, D. alata, D. nipponica, D. bulbifera, and D. septemloba [The solubility of the extracted acorn starch had significantly increased at 80°C–90°C to reach a value of 62%. This solubility of the extracted acorn starch increased significantly from 12.95% at 60°C to a value of 64.22% at 90°C. These values were higher than those registered on starches from ptemloba . The higFor the water absorption capacity of starch, it corresponds to the hydrogen bonding between water molecules and hydroxyl groups in the starch molecules and starch chains as well as diversification of the starch granule structures .The stability variation of the extracted acorn starch under refrigeration and freezing conditions during time is shown in θ were classified as the A-type pattern, which characterized most cereal starches [θ were characterized as the B-type pattern. Our results were similar to that reported for starch from Quercus glandulifera Bl. [Dioscorea pyrifolia tubers [θ ≤ ±0.02, the observed peak shifts are not experimental error, which is in good agreement with the previously reported phase.The X-ray diffraction and the FT-IR spectra of acorn starch are shown in Figures starches . Howeverfera Bl.  and Diosa tubers . Since t−1 are shown in −1 which indicated the stretch vibrations of O–H groups of the starch molecules. Besides, two second bands were observed at 2925.84 and 2855.35 cm−1, probably resulting from the stretching of C–H bonds. The amide group of proteins band was found between 1645.87 and 1710.84 cm−1 reflecting the C–O stretching. The observed peaks at 1645.87 cm−1 were also assigned to water molecules absorbed in the amorphous region [−1 originated mainly from carbohydrate vibrations. In fact, the bands at 1150.18 and 1076.9 cm−1 observed in the spectra of acorn starch were attributed to the presence of β-glycoside and glucoside and the stretching of the C–O bonds. However, the observed band at 995.51 cm−1 was characterized by partially crystalline materials [The FT-IR spectra of acorn starch in the range of 400 to 4000 cms region . The regaterials .In order to choose the best concentration of extracted acorn starch to be incorporated later in a fermented dairy product at industrial scale, biochemical, physical, rheological, and color properties, variations were observed as a function of starch dose variation at the first day of storage. As shown in P < 0.05) initial Dornic acidity values observed on control and yoghurt enriched with 1% of acorn starch. For higher acorn starch doses, we registered a decrease of acidity. This result may be due to the fact that adding high concentrations of extracted starch could reduce the amount of water which makes it difficult for starter culture to metabolize lactose sugar, and thereby, the amount of produced lactic acid was reduced [The initial composition of all tested fermented dairy products is shown in  reduced . Besides reduced , suggest reduced .P > 0.05) were noted between these two enriched fermented dairy products and the control. However, total solids values decreased with higher doses to reach the lowest value for 2% added acorn starch fermented dairy product. Hence, the presence of acorn starch at high concentration decreased the total solids content of milk bases.Results showed that initial dry matter levels were between 19.41 ± 1.68% and 28.5 ± 0.70% for all tested fermented dairy products . The totn. This concentration was shown able to improve the rates of aggregation and curd firming reactions in the casein gels. Moreover, the results revealed that the high concentration (2%) of acorn starch gives rise to the decrease of consistency coefficients (0.69 ± 0.01 Pa.sn). This result can be explained by the fact that the higher polysaccharide content resulted in the formation of polysaccharide-protein interactions that are weaker than protein-protein bonds [For consumer products, texture is an important parameter of the quality that makes an impact on the pleasure of eating. In this study, as shown in in bonds  leading P < 0.05).Also, this finding can be explained by the breakdown of the fermented dairy product structure after manufacture and the decrease of the total solid content in enriched fermented dairy product, especially when acorn starch concentration exceeds 1% (P > 0.05) between control and these two enriched fermented dairy products. These findings could be related to total solids. In fact, when dry extracts increased (fermented dairy product samples with 0.5% and 1% acorn starch), syneresis decreased as reported in [In this study, the addition of acorn starch was associated with a decrease in viscosity and an increase in syneresis. At the beginning of storage, the use of 0.5% and 1% of acorn starch was associated with weak whey separation. Our result did not show a significant difference  between all samples in L∗ (lightness). This finding was in agreement with those reported in [P < 0.05) on the first day of storage between control and fortified samples for a∗ (red color) and b∗ (P > 0.05) (yellow color) between the different obtained fermented dairy products. This result was due to the yellow color of added starch which may affect the aspect and the final color of the product, during processing. In the light of these findings, we can conclude, as shown by Masmoudi et al. [When evaluating color parameters, orted in , suggesti et al.  on acornIn this study, the findings corresponding to the enumeration of yeasts, molds, and total and fecal coliforms obtained on control and enriched fermented dairy products showed the absence of this flora at the beginning of storage (data not shown). Hence, our fermented dairy product enriched with different acorn starch concentrations had a satisfactory hygienic quality.The biochemical quality of acorn flour, a nonconventional source, showed its richness in carbohydrates and proteins. Acorn produced a starch yield of 35.4%. Thus, the functional and chemical properties of extracted starch from acorn fruit demonstrated that it can be used as an ingredient in food industries requiring high processing temperatures. The results of this study showed that addition of acorn starch during manufacturing of a fermented dairy product showed a slight distinction compared to the control sample. In light of these reports, it can be concluded that acorn starch dose lower than 1% was able to improve or maintain the characteristics of conventional fermented dairy product, but this result has to be examined further during the refrigerated storage.


Aspergillus nidulans.Lytic polysaccharide monooxygenases (LPMOs) are often studied in simple models involving activity measurements of a single LPMO or a blend thereof with hydrolytic enzymes towards an insoluble substrate. However, the contribution of LPMOs to polysaccharide breakdown in complex cocktails of hydrolytic and oxidative enzymes, similar to fungal secretomes, remains elusive. Typically, two starch-specific AA13 LPMOs are encoded by mainly Ascomycota genomes. Here, we investigate the impact of LPMO loss on the growth and degradation of starches of varying resistance to amylolytic hydrolases by AnAA13A that possesses a CBM20 starch-binding module, AnAA13B (lacking a CBM20) or both AA13 genes resulted in reduced growth on solid media with resistant, but not soluble processed potato starch. Larger size and amount of residual starch granules were observed for the AA13-deficient strains as compared to the reference and the impairment of starch degradation was more severe for the strain lacking AnAA13A based on a microscopic analysis. After 5 days of growth on raw potato starch in liquid media, the mount of residual starch was about fivefold higher for the AA13 gene deletion strains compared to the reference, which underscores the importance of LPMOs for degradation of especially resistant starches. Proteomic analyses revealed substantial changes in the secretomes of the double AA13 gene deletion, followed by the AnAA13A-deficient strain, whereas only a single protein was significantly different in the proteome of the AnAA13B-deficient strain as compared to the reference.Deletion of the genes encoding AnAA13A, impairs degradation of resistant potato starch, but has limited impact on less-resistant wheat starch and no impact on processed solubilized starch. The effects of LPMO loss are more pronounced at the later stages of fungal growth, likely due to the accumulation of the less-accessible regions of the substrate. The striking impairment in granular starch degradation due to the loss of a single LPMO from the secretome offers insight into the crucial role played by AA13 in the breakdown of resistant starch and presents a methodological framework to analyse the contribution of distinct LPMOs towards semi-crystalline polysaccharides under in vivo conditions.This study shows that the loss of AA13, especially the starch-binding  Uniquelubstrate , 6. Notaubstrate , celluloubstrate  and starubstrate , 9. Currubstrate , 13.Starch is a composite of the mainly linear amylose, consisting of α--linked glucosyl units and the major component amylopectin, constituting up to 70–80% per weight in most plants. Amylopectin consists of α--linked linear glucosyl chains and 5–6% α-1,6-glucosidic branch points , 15. In AnAA13B was ranked among the top 10 upregulated proteins in the Aspergillus nidulans secretomes during growth on three different starches, while the modular AnAA13A that is appended to a CBM20 was the most abundant in the starch-binding secretome fraction [Currently, starch-active LPMOs are classified into auxiliary activity family 13 (AA13) in the CAZy database . To dateA. nidulans and analysed the impact of these gene deletions on the growth and starch utilization by A. nidulans. The loss of AA13s, especially the enzyme with the CBM20, had a major impact on the ability of the fungus to degrade resistant potato starch. By contrast, AA13s appeared less crucial for the degradation of the less-resistant wheat starch and soluble processed potato starch. Altogether, these findings underscore the instrumental role of LPMOs, especially those appended to CBMs, in fungal breakdown of granular resistant starch and establish a framework to dissect the contribution of LPMOs in breakdown of recalcitrant polysaccharides.In this study, we have generated two single and a double knockout (KO) of the AA13s genes in Aspergillus nidulans encodes two LPMOs of AA13 sharing 83.8% primary structure identity in their catalytic modules: AnAA13A , which is the modular enzyme harbouring a C-terminal starch-binding CBM20 and AnAA13B   [aasA and pU2000-aasB, which were digested to yield linear gene-targeting substrates (GTSs). The non-homologous end-joining-deficient A. nidulans NID1 strain [aasAΔ and aasBΔ deletion mutants were identified as homokaryotic deletions by diagnostic tissue-PCR, respectively, following the mutant validation scheme in Additional file aasBΔ::AfpyrG on 5-FOA supplemented medium to select for the loss of the pyrG marker hereby generating a uracil and uridine auxotrophic aasBΔ strain  . This pr1 strain , 19, use-editing . A singlA. nidulans reference and AA13 gene deletion strains were grown for up to 9 days on minimal media agar plates supplemented with glucose, soluble potato starch, or resistant potato starch, which has been thoroughly washed as previously described [AnAA13B strain displayed the least severe phenotype, whereas the growth was most drastically affected for (-)AnAA13A and the double KO (-)AnAA13A + B  raw granular potato starch, 2) wheat granular starch and (3) processed soluble potato starch. The raw potato starch comprises large (up to 100 µm in diameter) granules of the tightly packed B-type allomorph, considered as a resistant starch type, as compared to the A-type allomorph of the smaller cereal (including wheat) granules that are considered more susceptible to amylolytic hydrolases , 16. Theescribed  to minim wheat grAnAA13 strains, especially those lacking the starch-binding AnAA13A on.These results revealed a significant difference in the utilization of resistant starch by the (-)To provide a rationale for the observed phenotypes, we used confocal laser scanning microscopy (CSLM) to analyse growth on the same resistant potato starch. The analysis was performed in a thin agar medium layer supplemented with resistant starch on a microscope slide for 20 h. The relatively short incubation period was set to avoid artefacts related to the drying the thin solidified medium layer and extensive hyphal growth. This analysis revealed that the number and size of granules in the reference strain culture were greatly reduced consistent with efficient degradation of the resistant starch Fig. a, b.Fig.AnAA13B lowered the efficiency of resistant starch utilization as evident from the larger number and average size distribution of residual starch granules as compared to the reference strain AnAA13A + B strains AnAA13A + B strain, was performed on wheat starch, which has been shown to sustain excellent growth and abundant secretion of AA13s by A. nidulans in our previous analysis [AnAA13A + B strain as compared to the reference  also observed in the (-)AnAA13A + B, was statistically regulated relative to the reference .To bring insight into the impact of the AA13 gene deletion on AnAA13A and (-)AnAA13A + B revealed 14 and 204 (37 CAZymes) significantly regulated proteins , respectively AnAA13A + B compared to the reference.Interestingly, this enzyme was downregulated in this dataset, suggestive of a limited access to the oligomers that mediate the transcriptional upregulation. Hypothetical proteins, proteases and a considerable proportion of intracellular proteins were amongst the upregulated proteins, which suggests a distinctly different physiological state of the (-)A. nidulans on starches with varying degree of resistance to enzymatic hydrolysis.The action of LPMOs, their synergy with other hydrolases , 22 and A. nidulans in our previously reported study [The visual phenotypic difference observed for the AA13 gene deletion strains indicated a marked change in growth on resistant potato starch on solid media Fig. . By contAnAA13A in the degradation of resistant starch, which is supported by: (1) the growth phenotype on solid media AnAA13A and (-)AnAA13A + B as compared to the reference strain AnAA13A and (-)AnAA13A + B strains AnAA13B, which does not apply to the same extent for the (-)AnAA13A strain. Only about a fifth of the significantly regulated proteins in the secretomes of the (-)AnAA13A + B strain at day 5 compared to the reference were CAZymes. Thus, the majority were proteins with other functions including proteases and intracellular stress proteins .Our findings highlight a paramount role of the modular starch binding dia Fig. ; 2) the theAnAA1A. nidulans on wheat, maize and pea starches observed in a previous study [AnAA13A displayed moderate affinity to the starch mimic β-cyclodextrin (Kd ≈ 40 µM), which is mediated by the CBM20 starch-binding module [AnAA13B homologue lacking the CBM20 was not captured by the β-cyclodextrin affinity column [AnAA13B is that the activity is less localized due to the more dynamic lower affinity binding and smaller size as compared to AnAA13A, which may allow deeper penetration in the starch granule. This feature, maybe more important in cereal starches, especially maize which has a channel structure [AnAA13A is likely to be more important than the smaller size of AnAA13B. Taken altogether, this study demonstrates a crucial role of LPMOs in the degradation of resistant starch, and possibly other semi-crystalline substrates.A deleterious effect on the activity of a bacterial modular cellulose-specific LPMO as a result of deleting the appended CBM has been reported . In anotus study , demonstg module . This afy column , 28, albtructure . A modelThe study provides evidence that the deletion of one or both AA13s reduces the degradation efficiency of granular resistant starch, but not processed solubilized starch. This impact of the AA13 deficiency is less pronounced on wheat starch that is more susceptible to degradation by hydrolytic enzymes. This study offers a framework to assess the contribution of distinct LPMOs in fungal secretomes on the utilization of specific semi-crystalline polysaccharides.g. The residual ethanol was evaporated in a laminar flow bench and each starch substrate was subsequently resuspended in autoclaved growth media and added to make a homogenous suspension in agar plate before solidification. p-Nitrophenyl-α-d-glucopyranoside (PNPG) was from Sigma-Aldrich, and insoluble Blue Starch (iBS) was a custom preparation from Pharmacia .Commercial granular raw resistant potato starch was from Bauckhof , soluble potato starch (S2630) and granular unmodified wheat starch (S5127) were from Sigma-Aldrich . The granular starches for the liquid or solid media growth experiments were not autoclaved to minimize changes of the native starch granule structure, but the soluble starch comprising was autoclaved and used for the growth phenotype assay on plates. The granular starches were thoroughly washed four times with five consecutive volumes of Milli-Q water, 70% ethanol, Milli-Q water and 70% ethanol at 30 °C with intermittent centrifugation at 5000xAnAA13A (Gene locus tag: AN5463 (gene designated aasA in this study), UniProt ID: Q5B1W7) and AnAA13B (Gene locus tag: AN5463 (aasB in this study), UniProt ID: Q5B1W7) starch-specific LPMO encoding genes were deleted in the present work. A. nidulans strain NID1  [A. nidulans strains with regard to ability to utilize complex glycans as carbon source. The argB and pyrG encode ornithine carbamoyltransferase and orotidine-5′-phosphate decarboxylase activities, respectively. The argB2 mutant was corrected to functional argB in the final mutant strains using CRISPR/Cas9 and the oligonucleotide mediated gene-editing strategy as described recently [pyrG89 mutation was complemented by the use of the heterologous AfpyrG from Aspergillus fumigatus. This gene, which was extended by its native promoter and terminator and flanked by a direct repeat (DR) of 290 bp [Escherichia coli strain DH5α was used as host for plasmid propagations.The , nkuAΔ)  was usedrecently . The pyrAspergillus nidulans was cultivated on solid and liquid glucose based minimal medium (MM) containing (per litre): 1% glucose (w/v), 6 g NaNO3, 0.52 g KCl, 0.52 g MgSO4·7H2O, 1.52 g KH2PO4 [l-arginine (Arg). Solid plates containing 5-fluoroorotic acid  were made from MM + Arg + Uri + Ura for counter-selecting pyrG. The glucose was replaced by 1 M sucrose for transformation media (TM). For protoplasting, 100 mL of liquid MM + arg + Ura + Uri in unbaffled shake flasks were inoculated to a concentration of 1 × 106 conidia mL−1, and incubated at 37 °C with stirring at 150 rpm for 20 h.g KH2PO4 , 0.001% g KH2PO4 , 2% (w/v® HF Buffer (New England Biolabs (NEB), USA), 0.2 mm dNTPs, 0.4 µM primers , 1 U PfuX7, < 10 ng of gDNA, 3% DMSO. PCR products were purified from agarose gels using a GFX purification kit . All vectors were assembled by USER cloning as described previously [A. nidulans, respectively, as described before [All PCR products used for cloning were amplified by the proof-reading PfuX7 polymerase  on an Ageviously . The plad before , 34. TraA. nidulans and the gene deletion strains derived thereof were cultivated on solid MM at 30 °C for 5 days. Conidia were gently harvested using ice-cold sterilized 0.9% (w/v) NaCl solution to a final concentration of 1 × 108 conidia mL−1, which was used to inoculate plates with the same medium containing 1% (w/v) or Glc, soluble potato starch or resistant potato starch at three positions. To minimize bacterial contamination of the plates with the washed non-autoclaved resistant potato starch, ampicillin (100 µg mL−1) and chloramphenicol (34 µg mL−1) were added to the medium together with the starch before solidification. Growth was monitored for up to 9 days.The reference 5 mL−1 conidia suspension. Prior to mounting the cover glass onto the incubated slides (with and without inoculum), two drops of 3 µL tenfold diluted 0.1% (w/v) calcofluor white  in Milli-Q were added to the agar with 1 cm distance between the drops. The microscopy slides were placed in petri dishes stacked in micro-perforated plastic bags at 37 °C for 20 h. The slides were assembled by a 50 × 24 mm cover glass #1  and incubated at room temperature for an hour prior microscopy analysis. The assembled slide-samples were mounted up-side-down on a Leica Microsystems SP8 confocal microscope . Images were alternatively captured using a 10× HC Plan Fluotar objective, or a 63× HC N Plan-Apochromat oil immersion objective. Cells stained with CFW were imaged in confocal mode, using 405 nm excitation and 480–545 nm emission detection. Transmitted light images were acquired via a non-confocal configuration mode using a 552 nm laser as illumination. CFW has optimal excitation and emission wavelengths below what were used here, but due to technical limitations, the indicated wavelengths still captured clear images of the fungal cell components.For the CLSM analysis, 700 µL MM containing 1% agar and either potato or wheat starch (1% w/v) as a sole carbon source, and supplemented with antibiotics as described above, were added as 1.5 × 4 cm square to sterilized microscope glass slides. After 30 min solidification, the media were inoculated by adding 10 µL of 1 × 10Aspergillus nidulans and the gene-deletion mutants were grown in 250 mL baffled Erlenmeyer flasks containing 50 mL MM containing 1% (v/w) resistant potato starch as a carbon source. The MM was supplemented with ampicillin (100 µg mL−1) and chloramphenicol (34 µg mL−1) to minimize bacterial contamination. Each flask was inoculated to a final concentration of 1 × 106 spores mL−1 and cultivated on a rotary shaker (150 rpm) for 5 days at 30 °C. The growth was performed in four biological replicates.2SO4 and incubated at 30 °C, 1 h. The samples were immediately cooled down and stored at 5 °C until further analysis. The mannose content in the samples was quantified using an HPAEC-PAD system equipped with PA1 column and post-column NaOH addition as described previously [Filtration through a cloth, was used to separate the final fungal biomass (retentate) from the culture suspension. The wet weight was determined and the biomass was frozen at − 20 °C until further use. The mannose content of the final fungal biomass was determined by acid hydrolysis. The biomass (from 40 mL suspension) was thawed, transferred to Teflon-capped pressure tubes, freeze dried, suspended in 1.5 mL 72% (w/v) Heviously .g for 30 min. The pelleted starch was washed with twice 96% ethanol, re-centrifuged as above. The ethanol was removed and the starch dried prior to determining the weight.To separate the residual insoluble resistant potato starch, the culture suspension (filtrate from the above step) was centrifuged at 5000×®Ultra Centrifugal Filters .For enzyme activity measurements, 500 µL culture supernatant was concentrated 50-fold and buffer exchanged to assay buffer  using Amicon−1) suspended in assay buffer. The reaction mixture (180 μL iBS + 20 μL supernatant) was incubated for 20 h at 37 °C, stopped by 0.5 M NaOH (40 μL), centrifuged , and 200 μL supernatant was transferred to a 96 well microtiter plate and the absorbance read at 620 nm. α-Glucosidase activity was determined towards 2 mM p-nitrophenyl-α-d-glucopyranoside (PNPG) in assay buffer. The reaction mixture (40 μL PNPG and 10 μL supernatant) was incubated for 20 h at 37 °C, stopped by adding 1 M Na2CO3 (200 μL), centrifuged . Then, 200 μL supernatant was transferred to a 96 well microtiter plate and the absorbance read at 410 nm.α-Amylase activity of the fungal cultures was assayed toward insoluble Blue Starch  according to the manufacturer´s instruction using bovine serum albumin as standard. Amylolytic activities protein concentration were measured in the four biological replicates and presented as the means with standard deviations.g, 10 min, 4 °C) and filtered  to remove mycelia and residual insoluble starch. The filtrate was flash frozen in liquid nitrogen and stored at − 80 °C until further use. Proteins of the culture filtrates were precipitated by addition of acetone to a final concentration of 80% (v/v), incubation over night at − 20 °C, and centrifugation  to pellet the precipitated protein. The protein pellets were dissolved in 50 µL buffer phosphine hydrochloride, 40 mM 2-chloroacetamide, 50 mM HEPES pH 8.5), boiled (5 min 95 °C) and sonicated (5x60 s at 4 °C) . For digestion, 45 µL sample were diluted 1:3 in digestion buffer  acetonitrile (ACN), pH 8.5) and incubated with 200 ng LysC  at 37 °C for 3 h. Subsequently, the samples were diluted 1:10 in digestion buffer and further digested with 200 ng trypsin  at 37 °C for 18 h. The digestion was stopped by addition of 450 µL 2% trifluoroacetic acid (TFA). Samples were desalted using SOLAµ HRP plate (Thermo Fisher Scientific) by activation with 200 µL methanol and 200 µL MS buffer , equilibration 2 × 200 µL 3% ACN, 1% TFA, sample application, 2x wash with MS buffer A (0.1% FA) and elution with 2× 300 µL 40% ACN, 0.1% FA. Between each solvent application, the plate was centrifuged . Eluted peptides were dried in an Eppendorf Speedvac (3 h at 60 °C) and reconstituted in 2% (v/v) ACN, 1% (w/v) TFA acid prior to mass spectrometry (MS) analysis. The peptides were loaded on the mass spectrometer by reverse phase chromatography through an inline 15 cm C18 column (Thermo EasySpray ES804A) connected to a 2 cm long C18 trap column  using a Thermo Easy nLC 1200 HPLC system. Peptides were eluted with a mobile phase of constant 0.1% (w/v) FA at 250 nL min−1 over 100 min with a five step gradient of buffer B (80% (v/w) CAN, 0.1% FA): 0–68 min gradient of 6–23%, 68–85 min 23–38%, 85–90 min 38–60%, 90–93 min 60–95%, and 93–100 min 95%. Analysis was performed on a Q-Exactive HF-X instrument (Thermo Fisher Scientific) run in a data-dependent manner using a “Top 15” method. Full MS spectra were collected at 60,000 resolution, with an AGC target set to 3 × 106 ions or maximum injection time of 50 ms. Peptides were fragmented via higher-energy collision dissociation . The intensity threshold was set to 1 × 104, dynamic exclusion to 60 s and ions with a charge state < 2, > 6 or unknown species were excluded. MS/MS spectra were acquired at a resolution of 15,000, with an AGC target value of 1 × 105 ions or a maximum injection time of 60 ms. The scan range was limited from 350 to 1850 m/z.Samples (2 mL) from four biological replicate cultures were collected from day 3 to 5 under sterile conditions. The samples were centrifuged .The raw files were analysed using Proteome Discoverer 2.4. Label-free quantitation (LFQ) was enabled in the processing and consensus steps, and spectra were matched against the Dynamic modifications were set as Oxidation (M), Deamidation  and Acetyl on protein N-termini. Cysteine carbamidomethyl was set as a static modification. All results were filtered to a 1% FDR, and protein quantitation done using the built-in Minora Feature Detector.2 transformed and used for further hypothesis testing. A linear modelling approach (LIMMA v3.40.6) was applied using the R statistical computing environment (v3.6.1). After a principal component analysis of the protein abundances from day 3 and day 5 samples, the largest source of variability was observed to be related to the sampling day, and that day 3 samples showed little effects due to the strain background. For this reason, only day 5 samples were used in the statistical model. A linear model was applied where log2-abundance values were used to estimate the parameters for each strain background and comparisons (contrasts) were made for each KO strain relative to the reference strain. Empirical Bayes statistics were used to estimate false discovery rates (q) using the Benjamini–Hochberg method for each comparison. The difference in the group means is represented by the log2-fold change (logFC) from the reference. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [The normalized abundance values for each sample were then loghe PRIDE  repositoAdditional file 1: Table S1. List of strains used in the study. Table S2. Primers for generation and validation of the A. nidulans mutant strains. Fig. S1. Gene targeting and mutant-strain validation. Fig. S2. Growth of reference and AA13 gene deletion strains on glucose. Fig. S3. Growth of reference and AA13 gene deletion strains on soluble potato starch. Fig. S4. Growth of reference and AA13 gene deletion strains on resistant potato starch. Fig. S5. Biomass analysis of reference and AA13 gene deletion strains on resistant potato starch at day 5. Fig. S6. Principal component analysis of the proteomic data sets during growth on resistant starch. Fig. S7. Venn diagram of the significantly upregulated proteins during growth on resistant potato starch.Additional file 2: Label-free Proteomics.


This research also enables the determination of the temperature conditions required for becoming the desired form of material. Therefore, it is necessary to achieve the requested compact porous structure in an activation process, because in the native state, the polymer exhibits limited applications as a result of thermal decomposition, low shear stress, retrogradation, and syneresis, hence the low solubility in organic solvents. Thermodynamic parameters and reactive hydroxyl groups in this article review are innovative and have not yet been found in the literature.The main aim of this study is to estimate the kinetic and thermodynamic parameters of thermal decomposition of starches by the Coats–Redfern method. This procedure is a commonly used thermogravimetric analysis/difference thermal gravimetry/differental thermal analysis (TG/DTG-DTA) kinetic method for single rate form. The study also shows a proposed method for reactive hydroxyl groups content on the starch surface determination, and values were in range of 960.21–1078.76 mg OH per 1 g of starch. Thermal processing revealed the thermophysical properties of biomass for the kinetics of decomposition estimation. Activation energies reached the values in range of approximately 66.5–167 kJ·mol Thus, innovations are also involved with applications with the most natural energy transformations possible; that are environmentally friendly. Mostly, the adjustments are associated with global warming remission due to rising greenhouse gas COmosphere ,2,3. Themosphere . The lowmosphere . The amy−3. Both compounds have α-O-1,4-glucosidic bands, and the structure of amylopectin additionally contains α-O-1,6-glucosidic bands. Approximately 95% of biopolymer exhibits α linkages and only 5% α bands [6H10O5)n, where n is a value between 300 and 1000 [According to the literature , within 6) bands . Starch and 1000 . There aand 1000 . The traand 1000 . The pyrand 1000 , at abouAll starches were purchased from Sigma-Aldrich . In research, rice starch, wheat starch, corn starch, and potato starch were applied . Glicerol is the 1,2,3-Propanetriol.−1. The gas flow rate was 20 mL·min−1. The process was conducted in the inert gas atmosphere (nitrogen). To conduct the analysis, amounts of 11.8, 12.3, 13.4, and 15.1 mg of starch from rice, potato, wheat, and corn, respectively, were used, and then the changes in the mass of the heat-treated material were obtained. It should be assumed that the results of the thermal analysis for these selected samples are always repeatable and the maximum number of samples depends on the instrument’s requirements. The result of analysis was shown using appropriate graphs, considering the mass loss in every 10 K per minute in a precisely chosen temperature range. The data graph, meanwhile, describes the physical and chemical transformations, such as crystallization, melting, and sublimation, and thus the phase transitions.The main aim was to define changes in the structure of starches from different botanical origins. It was possible to also obtain the amount of hydroxyl groups of starch molecules using TG/DTG-DTA  data. To carry out the analysis, a Perkin Elmer thermogravimetric analyzer (TGA) 8000  was used. TG/DTG-DTA techniques were carried out in a simultaneous TG/DTG-DTA instrument. In the study, an alumina crucible without any lid was used. The TG/DTG-DTA analysis enables the accurate assessment of thermal stability, and thus the lifetime of the material, using the range of temperatures of 29.6–1062.1 °C, while the heating rate ß was 10 K·minTo determine the kinetics of starch decomposition, the TG/DTG-DTA data were used. The fundamental equation, used in all TG/DTG-DTA calculations, is described as follows:im is the initial mass; tm is the mass in time t, constant in given temperature: K; and fm is the final mass.The conversion rate α is given by the following equation:k, has the following formula:The generally occurring Arrhenius equation, and thus the rate constant The heating rate is given by the following formula:k(t) is the reaction rate constant—min−1, f(α) is a function describing the transition (reaction model), aE is the activation energy—J·mol−1·K−1, A is the pre-exponential factor—min−1, T is the temperature of heating—K, and R is the gas constant reaching the value of 8.314 J·mol−1·K−1.To obtain the reaction rate constant, the modified Arrhenius equation, and thus the combination of Equations (1), (3), and (4), is recommended:g(α), given by the equation below, was used [g(α) and f(α) functions for suitable mechanisms are presented in Assuming the non-isothermal conditions of pyrolysis, for the data analysis, the form of function with infinite limits was used .(6)g(α)bk is the Boltzmann constant, equal to approximately 1.3806 × 10−23 J·K−1; and h is determined as the Planck constant, equal to circa 6.6261 × 10−34 J·s.To designate the Gibbs free enthalpy of decomposition, the general Eyring–Polanyi equation was used.Further, to estimate enthalpy, the combination of Equations (3) and (7) was used, given by Formula (8).Finally, to determine entropy of degradation, the Gibbs Function (9) is used.K, using the isotherm of van’t Hoff, described as follows:Furthermore, it was possible to calculate the equilibrium constant aE and A parameters from the equation (Coats–Redfern (CR)):For the graph, the best fits, as well as the slope and intercept values, are used to determine Algebraic expression and plot recommended for the adopted kinetic model  with ploTo determine the kinetics of starch, the TGA/DTG-DTA graphs were used.The DTA and DTG data allowed to distinguish the dehydratation and dehydrogenation processes, and thus the first and second loss of mass during the process adopted. The TG analysis a presentg(α)T−2)) versus (T−1) for each g(α) were created—Equation (12). Next, to determine the parameters, the best fit (slope and intercept) using the most equal to 1 R2 parameter based on 2, A3, A4, D1, and F1. For every starch, the best correlation . The results enabled obtaining other thermodynamic parameters  for the reaction. To obtain those parameters, Equations (8)–(10) were used. Those parameters include the second step of the TG graph, which means when the hydroxyl groups are degraded (±280–360 °C). A2, A3, A4, D1, and F1 in To obtain the kinetics of starch degradation, graphs  has the value of 17.008 g·mol−1.To quantify the hydroxyl group content on the starch molecule area, Equation (13) is used.The total hydroxyl group content was calculated from the entire second mass loss, so after all physisorbed water was removed from the structure, and all values are shown in −1 on the starch surface, which confirms their belonging to carbohydrates, which contain circa 60–70% of water, including the hydroxyl groups, and 30–40% of black carbon. Those values depend on the starch origin. Comparing starch to disaccharides such as saccharose, starch contains more water in the range of 6.23–9.83% and, per 1 g of starch, those values are in the range of 62.30 up to 98.28 mg; the values are presented in It was observed that the fewer the hydroxyl groups that stayed on the structure after the second step, the more stable the starch was, which was confirmed by the literature . AccordiThis study made it clear that TGA/DTG-DTA was suitably used to estimate the kinetics and thermodynamic parameters of starch degradation, and thus the amount of reactive hydroxyl groups on the surface of molecules. Really large mass loss during the process depends on the structure of polymer, and thus the chain length and functional groups transitions. Thus, the higher activation energy for corn starch could be the result of the possible higher molecular weight and thus more α-1,6 linkages. It may be attributed to the more advanced amylopectin content. Additionally, DTA shows endothermic character of the reaction, which the calculated kinetics and thermodynamics confirm. The obtained results of the kinetics of thermal degradation are mathematically and physically not synonymous with typical first-order kinetic reactions, which allows obtaining the half-life of materials from classic equations, and so kinetic formulas for the research were modified.


Furthermore, the cationic sago starch was characterized using Fourier transform infrared spectroscopy, FTIR, X-ray diffraction, XRD, and field emission scanning electron microscopy, FESEM.Modification and characterizations of cationic sago starch with 3-chloro-2-hydroxypropyl trimethylammonium chloride (CHPTAC) prepared via etherification reaction was reported in this study. The optimization of cationic sago starch modification was performed by utilizing the combination of response surface methodology and central composite design (RSM/CCD). The effect of each variable and the interaction between the three variables, the concentration of CHPTAC, concentration of the catalyst NaOH, and the reaction times on the degree of substitution (DS) of the product were investigated and modeled. Moderate conditions were employed and a water-soluble cationic sago starch with high DS value was obtained. Based on RSM, the highest DS = 1.195 was obtained at optimum conditions: 0.615 mol of CHPTAC concentration (CHPTAC/SS = 5), 30%  Cationic starches are important industrial derivatives in which the starch is given positive ionic charge by introducing ammonium, amino, imino, or phosphonium groups . UtilizaIn previous studies, most of the researchers used starch from maize, waxy maize, and potato for cationic modification. Different approaches were done to obtain the high DS value cationic starch. Conventionally, the cationic starch can be prepared by the etherifying ,18, grafSago starch is another type of starches that are used in the food industry and selected as the raw material because it is relatively cheap and widely available in Southeast Asia especially Malaysia and Indonesia. Sago can compete economically in terms of yield and price compared with other starch crops ,28. EvenResponse surface methodology (RSM) is one of the popular designs of experiment techniques. This method implements statistical and mathematical techniques for the development, improvement and optimizing process, in which the response of interest is affected by several variables. This statistical process works by defining the effect of independent variables, individually or in combination, thus allows the determination of the significance of each parameter studied, along with the significant interaction between parameters. Central composite design (CCD), Doehlert design (DD) and Box–Behnken design (BBD) are among the typical designs in RSM . Currentw/v) activation monomer concentration, methanol (99.7%), isopropanol (99.7%), and ethanol (95%) were purchased from Sigma-Aldrich (M) Sdn. Bhd., Kuala Lumpur, Malaysia. 85% (v/v) methanol was prepared from methanol (99.7%). All other chemicals such as isopropanol (99.7%), and ethanol (95%) were of analytical grade and used as received without further purification.Sago starch (food grade) was obtained from C.L Nee Sago Industries Sdn Bhd, Sarawak, Malaysia. The cationic etherifying agent, 3-chloro-2-hydroxypropyl trimethylammonium chloride (CHPTAC) with 60% (w/v) was dissolved in 20 mL water and added in the starch suspension. Subsequently, a solution of CHPTAC (0.123–0.615 mol), with molar ratio CHPTAC/SS = 1 to 5 was added dropwise into the suspension. The mixture was kept at 60 °C (3–5 h). After cooling down to ambient temperature, the product was neutralized with 1 M hydrochloric acid, filtered off and finally washed with ethanol (95%).The method from Heinze et al.  was modiImportant variables on the cationization of sago starch with CHPTAC were optimized by using the response surface methodology/central composite design (RSM/CCD) approach. CHPTAC concentration (0.123–0.615 mol), concentration of NaOH (30–70%), and reaction time (3–5 h) were the independent variables used in this study. Three factors with a total of 16 experiment runs were suggested by RSM/CCD using the statistical package from Design Expert 6.0, Stat Ease Inc., Minneapolis, MN, USA.ith linear coefficient, ijth interaction coefficient, while ix and jx represents factors. Validation of the model and optimization of the degree of substitution response were conducted. The optimum values of variables were predicted by the response surface analysis of the combined variables. The constraints in this study were applied to predict the optimum variable conditions that resulting in the highest degree of substitutions value response by inserting the desired conditions in the Design Expert 6.0 software. The efficiency of the model was verified by performing the suggested experiments and comparing the output with the predicted results. Residual standard errors (RSE) were used to determine the reliability of the model. The numerical optimization of the response was predicted based on the second-order polynomial model.Response surface modeling, statistical analysis, and optimization were conducted with the assistance of Design Expert 6.0. Analysis of variance (ANOVA) was used in the analysis of the output data. The degree of substitution  data from the suggested experiments was expressed by polynomial regression equation to generate a model as shown in the equation below:N = percentage of nitrogen; and CA = molecular weight of cationic reagent.The degree of substitution (DS) is the number of hydroxyl groups substituted per glucose unit with anhydrous glucose unit, AGU. The DS was determined by elemental analysis of a TruSpec Micro CHNS-932 Analyzer, St. Joseph, Michigan, MI, USA. To determine the percentage of nitrogen present. Five replicates were analyzed with a low standard deviation (<0.5). The DS value can be calculated using the following equation :DS=162N, X-ray diffraction spectroscopy XRD-6000 , and field emission scanning electron microscopy JSM-7600F  −1 using attenuated total reflectance (UTAR) technique.Fourier transform infrared, FTIR measurements were carried out with 3 mg of sample on the diamond holder was irradiated with infrared in the range of 4000 to 400 cmX-ray Diffraction, XRD characterization was carried out using Shimadzu XRD-6000 diffractometer with Cu Kα (λ  =  1.5418 Å) radiation at room temperature operated at 30 kV and 30 mA. A sample was placed in an aluminum sample holder and a diffraction pattern plots intensity against the angle of the detector, 2θ and the scanning range 2° to 30° with the rate of 2°/min with continuous scan mode.Field emission scanning electron microscopy (FESEM) was used for surface morphology studies. Samples were mounted on the stub followed by coating with gold to make the samples conductive before the scanning process is done. The prepared samples were examined under FESEM  at a voltage of 5.0 kV and recorded at 200 and 1000× magnification.A = concentration of CHPTAC, B = concentration of NaOH, and C = reaction of time. The quality of the model was evaluated based on correlated coefficient and standard deviation. Model capability to predict the response was translated to a small deviation and correlated coefficient closer to the R2 [The complete design matrix for preparing cationic sago starch is given in o the R2 . The stao the R2 . These iThe ANOVA was determined by using Design Expert 6.0 software. The statistical significance of this model was determined by F-value and prob > F. F-value is a measurement of the variance of data about the mean, based on the ratio of the mean square of group variance due to error. The model is significant when it possesses high F-value and prob. > F less than 0.05. The ANOVA for the quadratic model of the DS value is presented in The cationization starch involving several steps of reactions simultaneously. First, the chlorohydrin form of the CHPTAC is converted to epoxy intermediate. Next, the epoxy agent will react with the starch itself in the presence of an alkaline medium. Then, the epoxy unit will be converted via hydrolysis to the non-reactive 2,3 dihydroxyl derivative . The illThe effect of CHPTAC and NaOH concentrations on the value of DS with a constant reaction time at 4 h is illustrated in w/w of concentration increased the DS rapidly. However, the DS then gradually decreased after 30% w/w NaOH due to hydrolysis. This phenomenon can be explained that under the basic condition the hydroxyl groups of the anhydroglucose units of starch may react in a nucleophilic reaction with the etherification agent, as in the well-known Williamson ether synthesis [In the formation of cationic sago starch, NaOH acts as a catalyst in both reactions which are the formation of intermediate epoxide along with cationization of the starch . From Fiynthesis . But at ynthesis  who studValidation of the model was carried out by performing three sets of experiments generated from Design Expert 6.0 software. The validation was obtained by comparing the predicted and experimental values. The optimization process was carried out by inserting the desirable criteria such as the maximum DS value response was set as the main goal, while the other factors were in the studied range as mention in methodology −1, C-H stretching vibrations of aliphatic groups at 2906 cm−1, absorbed water signals at 1644 cm−1, C-H bending at 1365 cm−1, C-O stretching for ether at 1000 cm−1. The band around 1644 cm−1 corresponded to the tightly bound water present in starch due to its hygroscopic nature. In general, starch granules have two different populations of water, one that is part of the hydrogen bonding network of the starch crystalline structure and another which is freer and considered hydration water [FTIR spectra of sago starch, CHPTAC, and cationic sago starch are depicted in −1 which is assigned to the O–H stretching vibration of water since the CHPTAC is 60% w/v in liquid form. A strong band was observed at 1649 cm−1 due to intermolecular H-bond in the CHPTAC. Another strong band at 1478 cm−1 corresponded to C–N stretching vibration. There are two strong absorption bands at 944 cm−1 and 1081 cm−1 corresponded to an alkyl halide, C–Cl and C–O stretching, respectively.IR spectrum for CHPTAC in −1 attributed to the C–N stretching vibration of amide, which similar to Xia et al. [a et al.  observeda et al. .Starch is a semicrystalline material which contains both the crystalline and amorphous phases, determined by its principal components, the amylose and the amylopectin. The amylose has a linear structure, which produces the crystalline region of the starch, whereas the amylopectin has a branched structure that is responsible for the amorphous phase of the starch ,40. The a et al.  for corna et al.  for sagoa et al.  also obsa et al. .w/v NaOH, 5 h reaction time, and 60 °C reaction temperature. The value of DS obtained from these optimum conditions was 1.195. The validation and optimization based on the software prediction have shown that this model can predict with less than 2% error as compared with the experiment, suggesting that this model was reliable and able to predict the DS values response accurately.In this work, the central composite design/response surface methodology is successfully utilized for the optimization of DS in the cationization of sago starch. The effect of three major factors in the cationization of sago starch was studied and optimized statistically. Based on RSM, the optimum conditions to prepare water-soluble cationic sago starch in moderate conditions were at 0.615 mol of CHPTAC concentration (CHPTAC/SS = 5), 30%


Risks for living-liver donors are lower in case of a left liver donation, however, due to lower graft volume, the risk for small-for-size situations in the recipients increases. This study aims to prevent small-for-size situations in recipients using an auxiliary two-staged partial resection liver transplantation (LTX) of living-donated left liver lobes.Two patients received a two-stage auxiliary LTX using living-donated left liver lobes after left lateral liver resection. The native extended right liver was removed in a second operation after sufficient hypertrophy of the left liver graft had occurred. Neither donor developed postoperative complications. In both recipients, the graft volume increased by an average of 105% (329 ml to 641 ml), from a graft-to-body-weight ratio of 0.54 to 1.08 within 11 days after LTX, so that the remnant native right liver could be removed. No recipient developed small-for-size syndrome; graft function and overall condition is good in both recipients after a follow-up time of 25 months.Auxiliary two-staged partial resection LTX using living-donor left lobes is technically feasible and can prevent small-for-size situation. This new technique can expand the potential living-donor pool and contributes to increase donor safety. For some patients living-donor liver transplantation (LTX) remains the only treatment option for end-stage liver disease . Donor sIn the donors, a standard left liver resection  using crush-clamp technique with bipolar sealing and clipping of larger structures was performed. The left hepatic artery, the portal vein and middle and left hepatic vein were sutured and cut. The graft was flushed with heparin immediately and perfused with HTK solution via the portal vein.In the recipients, after liver mobilization and cholecystectomy, the liver hilum was dissected, and the main portal vein and left liver artery were isolated; then, the left-lateral liver was resected. The left portal vein and bile duct were sutured and cut, and the left hepatic vein clamped. The vena cava was dissected around the left hepatic vein orifice, so that an incision could be made from the left vein into the vena cava after clamping to enable a wide venous anastomosis.In the graft, the left and middle donor veins were sutured together to receive a common ostium. The venous and subsequent portal anastomosis (E/E) was performed. After reperfusion, the donor artery was anastomosed onto the left recipient liver artery (E/E). The biliodigestive anastomosis was performed during the planned relaparotomy. During hepatectomy completion, the liver was mobilized from the vena cava, and the right hilar structures and right and middle hepatic veins were dissected and cut between sutures.99mTc-mebrofenin hepatobiliary scintigraphy (HBS) scans were performed at postoperative day 1 and 10 to monitor future graft function, and liver perfusion was assessed by duplex and contrast enhanced ultrasound [Donors and recipients were evaluated using CT and MRI scans with arterial and portovenous contrast phases and additionally MRCP scans for evaluation of bile duct anatomy. Preoperative volumes for left and right liver lobes and whole liver after hypertrophy at postoperative day 10–12 were calculated from CT scans. Furthermore, trasound –6.3 graft  during a 1-day hospital admission. Until now (2½ years post-LTX) liver function has been good  and the patient is back to work and taking care of her child.3 graft , with a mild cellular rejection episode 1 year after LTX. Duplex ultrasounds showed normal liver graft perfusion and no signs of fibrosis. Liver function is good and the patient currently attends school now 20 months post-LTX.Both donors had an uncomplicated intra- and postoperative course, were discharged in good condition at postoperative day 7–8, and had no medical problems following living donation.We describe a new treatment concept for patients with end-stage liver disease in whom a living-donor left liver lobe is used in an auxiliary two-stage LTX setting to avoid a potential small-for-size situation. Our approach uses a left liver lobe instead of a left-lateral lobe in an auxiliary setting in cirrhotic patients. Here, we show the method is feasible and can be performed safely and successfully, at least in the first two patients. This is in contrast to a study that also described a treatment concept for cirrhosis where two recipients after left-lateral liver resection then received auxillary LTX using left-lateral grafts . After gSimilar approaches were undertaken in the treatment of irresectable colorectal liver metastases but for non-cirrhotic livers. In this setting, after left hepatectomy, a right portal vein ligation was performed, followed by transplantation of a living-donated left-lateral liver lobe as auxiliary partial orthotopic LTX followed by completion hepatectomy 2 weeks later . In contGenerally, management of portal vein perfusion is important to prevent small-for-size-syndrome with rather small liver grafts , 11. HemMultiple complications occurred in these cases. We had to deal with arterial stenosis, portal vein thrombosis, biliary leakage, venous outflow obstruction due to graft hypertrophy and portal vein stenosis in the long-term follow-up. If complications do occur, “hypersmall” left-lateral liver grafts as used in one of the described studies are at high risk of liver failure due to lacking functional reserve . InsteadDecide on timing of completion hepatectomy is difficult. For this decision we used CT-volumetric measurements and hepatobiliary scintigraphy together with regular laboratory parameters. While CT volumetry is an established method to quantify liver hypertrophy, hepatobiliary scintigraphy adds a functional parameter to simple volume measurement, but has not been routinely used in the setting of LTX or liver hyperthrophy. We suggest that this method needs to be critically evaluated further. While, in principal, the longer it is possible to wait, the better the liver hypertrophy will be, this must be balanced against potential increasing surgical problems regarding adhesions and reduced intraabdominal space. After this two case experience, we consider 10–12 days after the initial operative step as the best timing for completing hepatectomy with sufficient time for graft hypertrophy .Taking our complications into consideration—after this two case experience—our “optimized” technical approach would first be to anastomose the portal vein to the recipients’ main portal trunk, since this reconstruction is more stable with regard to positioning of the liver and not overly long after removal of the native liver. Second, we suggest resecting liver segment I in the recipient during the first operative step, providing an easier approach during the completion operation resulting in less manipulation of the transplanted graft. Third, we suggest a second look operation at POD 1 and delay of the biliodigestive anastomosis to this time point; this approach is meant to increase patient and graft safety by directly evaluating the graft, with assurance of optimal blood in- and outflow. Moreover, this step provides an important opportunity to further position the graft, which can change dramatically after completion hepatectomy.In conclusion, these cases demonstrate that living-donor left lobe auxiliary two-stage LTX to avoid small-for-size situation in adults/adolescents is safe and technically feasible. This new technique could expand the potential living-donor pool, and in selected recipient/donor combinations enables a living liver donation and additionally contributes to increase donor safety.


International Journal of Environmental Research and Public Health (IJERPH) [We wish to update Section 2.3 (Data Collection) in our paper published in the (IJERPH) . In the