Starch is added to meat analogues for binding and water holding. In this study, we investigate whether starch can have an additional role as a structuring agent. Therefore, different types of starch were combined with wheat gluten at various amounts and sheared in a High Temperature Shear Cell to determine how starch influences the structuring behavior of gluten–starch blends. The starches were chosen based on their diverse amylose contents, leading to different technological properties. Remarkable differences were found between the starches investigated. The addition of Amioca starch (containing 1% amylose) had a strong negative influence on the ability of gluten to form fibers. Maize starch (25% amylose) and Hylon VII (68% amylose) formed fibrous materials up to high starch additions. The pre-gelatinizing of maize starch further increased the ability of gluten–starch mixtures to form fibrous structures. The influence of the different types of starch on the hardness, deformability, and stiffness of the sheared samples was also assessed, revealing a spectrum of achievable properties through the addition of starch. Most remarkable was the formation of a material with anisotropy in Young’s modules. This anisotropy is also found in chicken meat, but not in protein-based fibrous materials. Furthermore, it was observed that the pre-gelatinization of starch facilitated fiber formation. A similar effect of pre-gelatinizing the starch was found when using faba bean meal with added wheat gluten, where fibrous structures could even be formed in a recipe that previously failed to produce such structures without pre-treatment. In a world with an ever-growing population, meat analogues are gaining more and more momentum to contribute to a more sustainable diet, and this momentum is not expected to stop ,2,3,4,5.Currently, intensive fractionation/separation processes are used to purify protein-rich crops for plant-based meat analogues. However, these processes take away (part of) the positive effects on the sustainability of replacing animal products with plant-based products, as processing requires significant amounts of water and energy. Therefore, a transition from purified ingredients to functional fractions will be necessary in the future to keep the environmental impact of plant-based meat replacers to a minimum . For staTo fill this gap, dedicated studies using starches with different properties are necessary to understand their influence on fiber formation. The  properties of starch are not exclusively, but significantly defined by its amylose/amylopection composition. In this study, we explore the role of starch in fiber formation in the HTSC. Special emphasis will be on the opportunities to utilize the unique gelling behavior of starch to modify product properties. We investigate the influence of three different types of starch on fiber formation, tensile strength, deformability, and the stiffness of gluten–starch blends. The starches used are chosen for their different amylose contents, leading to different technological properties: a low amylose starch , an intermediate amylose starch , and a high amylose starch . Their characteristics are also heavily studied and reasonably well reported in the literature. Using starches with different amylose content to vary the properties of starch enables us to exclude factors such as the crop source of the starch, as all three starches used are variants of maize starch. Furthermore, we pre-gelatinized the intermediate amylose starch and used it in the same way as the other starches. Finally we test our findings in a recipe with gluten and faba bean meal (FM), an exemplary unrefined, starch-rich crop. FM was pre-gelatinized and processed in the HTSC in its native and pre-treated form, in combination with gluten, to transfer the findings on isolated starch to a less refined ingredient. Faba bean was chosen as it is a starch-rich crop, the isolated protein of which is suitable for the formation of a fibrous structure in the HTSC when combined with gluten , whereasVital wheat gluten (WG) was obtained from Roquette  and had a protein content of 74.6% w.b.  and a dry matter content of 93.9%. Amioca starch (AS), maize starch (MS), and Hylon VII (HVII) were provided by Ingredion , and had an amylose content of 1%, 25%, and 68%, respectively. Faba bean meal (FM) was also provided by Ingredion  and had a protein content of 28.6% . The composition of the materials is listed in Pre-gelatinized maize starch (PMS) and pre-heated FM were obtained by creating 33.3% (w.b.) MS or FM slurries in milliQ water, placing it in a sealed plastic bottle, and heating it for 60 −1) at 140 °C. The HTSC was cooled down to 60 °C before opening and removing the sample. The samples were placed in plastic bags, sealed, and allowed to cool to room temperature before further analysis. The sample without starch was repeated on every experiment day to monitor the reproducibility of the results throughout the experiment.Doughs containing demineralized water, NaCl, gluten, and starch  or FM were prepared. The doughs were prepared following a protocol previously reported by Grabowska et al. , with soThe fibrous structure of the samples was assessed visually following a bending procedure for HTSC samples previously described by Cornet et al. , revealiThe stiffness (Young’s Modulus), maximum tensile strength (true fracture stress), and maximum deformation (fracture strain) in the parallel and perpendicular directions to the shear flow were obtained through tensile tests with a texture analyzer . The tests were performed closely following a protocol previously described by Schreuders et al. . In shorTo study the effect of starches on fiber formation, we tested different starches at different concentrations in the gluten–starch blends. Gluten doughs with increasing amounts of maize starch (MS), pre-gelatinized maize starch (PMS), Amioca starch (AS), or Hylon VII (HVII) were sheared in the HTSC. The sheared samples were bent in parallel to the shear flow direction, as described by Cornet et al. , to visuSamples without added starch exhibited a fibrous structure, meaning that the short and long fibers being oriented in the shear flow direction were visible upon bending the sample. This fibrous structure is used as a point of reference for all samples with added starch. The fibrous structure faded with increased amounts of MS, PMS, and HVII in the blend. The nature of the fibrous structure noticeably changed when ≥20% MS was added, as the number of fibers seemed to decrease and they became less distinguishable. At 40% MS, the fibrous structure was hardly noticeable anymore, while the sample containing 50% MS was too soft to break upon bending and therefore, no fibrous structure could be observed. Samples containing HVII also showed a noticeably different fibrous structure, which became less visible at 30% HVII and disappeared at higher HVII contents. Contrary to the sample containing 50% MS, the samples containing 50% HVII broke upon bending, but exhibited no fibrous structure. The addition of HVII also appeared to make the samples more stiff and brittle when bending. Samples containing any amount of PMS appeared to have smaller sized fibers, but they were still noticeable at higher added starch contents of up to 60% PMS. The maximum amount of starch that could be added without completely losing the fibrous structure  and one pre-gelatinized starch (PMS) were added to WG at different ratios and processed in the HTSC to determine the structuring behaviors of starch–gluten blends and their rheological properties. Potential factors influencing the effect of the added starches are pH and salt content, which both depend on the purification process of the commercial starches. The pH range (pH 3.5–7 in 20% aqueous suspension) and salt content  starts to play a role, decreasing the strength and deformability. At higher starch contents, the starch dominates the properties; in the case of HVII, this led to a low deformability, though an increased strength. This is why the effect of pre-gelatinization, and so the difference between MS and PMS, mainly manifested in the parallel direction. The small differences in perpendicular direction could stem from the different conditions during gelatinization of the starch: while the starch in the PMS was allowed to gel freely with only water being present, the starch in MS was competing with the gluten for water and was sterically hindered by the gluten network during swelling and gelatinization. The anisotropy of the Young’s modulus largely increased with pre-gelatinization. This made the samples containing PMS best match the Young’s modulus of chicken strips. Such a resemblance of a real meat texture has not been reported before. The use of pre-gelatinized starch could be a unique option to advance the development of meat analogues that actually resemble real meat.The sample containing 50% FM did not form a fibrous structure, likely due to a limited water uptake of FM at room temperature that diluted the gluten phase. FM had a starch content of 38.5%, resulting in an added starch content of 19.25% in the sheared samples. This falls in the range of the starch contents tested that could form fibrous products. The starch of FM (40% amylose) has a higher amylose content than MS (27%), but lower than HVII (68%), so on one hand, one would expect it to form a fibrous structure up to an addition of between 30% and 40%. On the other hand, the gluten content in the sheared FM samples was 50%, a recipe that also failed to produce fibrous structures with MS and HVII. FM also contains native protein, which is generally regarded to be less suitable for fiber formation in the HTSC . FurtherThe effect of the addition of starch on the structural formation of gluten in the HTSC was investigated. It is possible to add substantial amounts of native starch (up to 40%) to gluten and still obtain a fibrous structure. With the addition of starch, the textural properties of the samples change depending on the type and amount of starch, which can result in textural properties not previously seen for protein blends. The differences in the amylose/amylopectin contents of the starches lead to different technological properties such as viscosity and water uptake, thereby affecting the fiber formation and textural properties. Pre-gelatinization of the starch enhances the ability of the starch/gluten blends to form fibrous products, allowing for the addition of up to 60% of starch. It also leads to an increased anisotropy of the Young’s modulus, resembling chicken meat. This effect, and the higher maximum addable amount of PMS, are attributed to the higher water uptake of PMS at room temperature, which increases the density of the gluten network and therefore strengthens it. This concept was applied to FM, a unrefined material containing a considerable amount of starch. It was shown that not only can starch can be used to move the properties of plant-based meat analogues closer to those of real meat, but unrefined, starch-rich crops can be modified “in-situ” to become suitable for the production of fibrous meat analogues.


Thermoplastic starch is a material that has the potential to be environmentally friendly and biodegradable. However, it has certain drawbacks concerning its mechanical performance and is sensitive to the presence of moisture. The current study assessed agar-containing thermoplastic sago starch (TPSS) properties at various loadings. Variable proportions of agar  were used to produce TPSS by the hot-pressing method. Then, the samples were subjected to characterisation using scanning electron microscopy (SEM), mechanical analysis, differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), Fourier transform infrared spectroscopy (FT-IR), and moisture absorption tests. The results demonstrated that adding agar to starch-based thermoplastic blends significantly improved their tensile, flexural, and impact properties. The samples’ morphology showed that the fracture had become more erratic and uneven after adding agar. FT-IR revealed that intermolecular hydrogen bonds formed between TPSS and agar. Moreover, with an increase in agar content, TPSS’s thermal stability was also increased. However, the moisture absorption values among the samples increased slightly as the amount of agar increased. Overall, the proposed TPSS/agar blend has the potential to be employed as biodegradable material due to its improved mechanical characteristics.  Commodity plastics, including polyethylene (PE), polypropylene (PP), polystyrene (PS), and polyethylene terephthalate (PET), have been used extensively in industries for many years. However, it takes hundreds of years for these materials to break down into their constituent parts after being used. Alternative processes for producing biodegradable materials have been the subject of several investigations . Materiad-glucopyranose [Amylose and amylopectin are the two macromolecules that constitute starch, which has a linear and highly branching molecular structure that consists of repeating units of α -pyranose ,10. Starpyranose . CurrentMetroxylon sagu), indigenous to Southeast Asia, has been used to make sago starch and is known as the “starch crop of the 21st century” [The true sago palm  [Contrary to the majority of plastics now in use, thermoplastic starch has two significant drawbacks: a strong hydrophilic nature and poor mechanical characteristics ,27,28. Nphyceae) ,34.Rhodophyceae species . Due to its low cost, biodegradability, high degree of biocompatibility, and distinctive rheological qualities, it is frequently used as a gel-forming agent, thickener, and stabiliser in a wide range of applications [l-galactopyranose and d-galactopyranose are the chain’s primary components, and the connections between these two sugars alternate between  and . Agarose, an essential element of agar, is a neutral polymer that is only very slightly sulphated, whereas agaropectin is a sulphated polymer. Depending on the source, the value of the molecular weight might range anywhere from 80,000 to 140,000 g/mol [Metroxylon sagu), with a different biology and ecology from sugar palm starch (Arenga Pinnata) [Agar is a cell wall polysaccharide derived from specific ications ,36. 3,6-00 g/mol . Agar is00 g/mol ,39,40,4100 g/mol , as well00 g/mol  on the ePinnata)  even thoThus, the present paper aims to determine the effect of agar addition on the materials’ thermal, mechanical, and moisture absorption behaviour. Thermoplastic sago starch/agar blend moulded sheets were prepared in different concentrations by mixing with glycerol as a plasticiser. The use of a plasticiser improves the flexibility, extensibility, and ductility of the material by minimising intermolecular interactions between starch molecules . BecauseIn this experiment, there were two compositions prepared. First is the preparation of thermoplastic sago starch (TPSS), and secondly the preparation of thermoplastic sago starch/agar (TPSS/agar) blends. TPSS samples were produced by incorporating 30 wt% glycerol before pre-mixing in a Phillips HR2115/02 Dry Mixer  at 1200 rpm for 5 min at room temperature. The blend was hot pressed at 158 °C for 30 min using the compression moulding machine TECHNOPRESS-40HC-B , which produced a moulded sheet of 3 mm in thickness. A comparable method was also performed for the preparation of TPSS with the presence of agar. Various agar ratios were used to modify the matrix’s characteristics . The prepared specimens were stored in a silica gel desiccator at 23 °C and 50% RH until further study. −1. For each measurement, the ATR diamond crystal was cleaned with ethanol. The background spectra were then captured and automatically eliminated from the recorded spectrum. Beforehand, an anvil was utilised to crush the sample into the ATR diamond crystal, creating intimate closeness between the ATR diamond crystal and the sample. Because of the manual adjustment, the ATR-anvil FTIR’s pressure setting varies and is not reproducible across all samples. After selecting the correct band of spectrum, OMNIC software was used to control and correct the baseline of findings.Thermoplastics sago starch and agar were analysed using Fourier transform infrared (FT-IR) spectroscopy to determine functional groups. The JASCO FTIR-6100 Spectrometer  was equipped with an ATR Platinum diamond crystal and the manufacturer’s OMNIC software 9.2.106, version 9 was utilised to obtain the spectrum of each sample. The FT-IR spectra of the materials were measured between 4000 and 600 cmThe morphological properties of fractured tensile specimens were investigated using a scanning electron microscope (SEM), Zeiss Evo 18 Research , operating at 10 kV acceleration voltage. Before the testing, the fractured surface of the tensile test specimen was taken and cut into smaller pieces of 10 mm (L) × 10 mm (W) × 3 mm (T) in dimension, and afterwards the surface of the samples was coated using gold sputter. The tensile test specimens that had been prepared were stored in sealed bags and were characterised using SEM.−1), temperature (25 to 600 °C), and dynamic nitrogen atmosphere. The derivative form of TGA was produced via the differential of TGA values (DTG).The weight loss at a temperature increase served as an indicator of the thermal degradation of the composites during TGA analysis. TGA was performed using the TGA/DSC 3+  to examine the samples’ thermal stability in aluminium pans, constant heating rate  were performed on 5 mg of the sample material. The samples were first weighed before being placed in an aluminium specimen pan and immediately sealed. The point of reference was established by using an empty sample pan. All specimens were heated with a Universal V3-9ATA  Instrument at 10 °C/min. The inert atmosphere was kept constant by flowing nitrogen gas in the DSC cell at a flow rate of 20 mL/min. The transition temperatures were established using the thermogram results.Following ASTM D638, tensile tests were conducted at a relative humidity of 50.5% and a temperature of 23.1 °C. A 50 kN load-cell-equipped Universal Testing Machine INSTRON 5969  with a crosshead speed of 5 mm/min was used for five (5) repetitions of the tests. The data were calculated to establish the results for the tensile properties.According to ASTM D790, a flexural test was performed at a relative humidity level of 50 ± 5% and a temperature of 23 ± 1 °C. The specimens were prepared with dimensions of 130 mm (L) × 13 mm (W) × 3 mm (T). The testing was conducted using the Universal Testing Machine INSTRON 5969  with a 50 kN load cell and a crosshead speed of 2 mm/min.The Izod impact tests were performed using ASTM D256 at a temperature of 23 ± 1 °C and relative humidity of 50 ± 5%. Five (5) replications of each sample were employed in a digital pendulum impact tester made by Victor Equipment Resources Sdn. Bhd. . The samples, which had dimensions of 60 mm (L) × 13 mm (W) × 3 mm (T), were prepared. The impact strength was calculated by considering the specimens’ cross-sectional area and impact energy (Equation (1)). All samples were preconditioned for 2 days and then processed at 53% RH before testing.iW and fW, respectively, for a predetermined time until a constant weight was obtained. Equation (2) was used to calculate the samples’ moisture absorption.The samples were put in a closed humidity chamber at a temperature of 25 °C and relative humidity (RH) of 75.2% to conduct the moisture absorption test. Before the moisture absorption tests, five (5) samples with dimensions of 10 mm (L) × 10 mm (W) × 3 mm (T) were created and dried for 24 h at 105 °C. The specimens were weighed before and after absorption, represented by p < 0.05) was assessed using one-way analysis of variance (ANOVA), and Duncan’s test was performed to determine the significance of each mean property value (p < 0.05).The significance of each mean property value  was between 70 wt% to 72 wt%. The highest decomposition temperature was between 348 and 349 °C. The initial temperature of TPSS decomposition (Ton) dropped from 282 °C to 251 °C with the addition of agar (0–15 wt %). This could be attributed to native agar’s characteristics, since it decomposes at a lower temperature than sago starch [As in o starch . In the o starch . g, for native sago starch, native agar, and their combinations. It can be seen that the Tg transition of the TPSS was increased after the addition of agar. The Tg transition for the amorphous nature of the plasticised starch matrix that is indicated by the neat sago starch occurred at approximately 168.5 °C. The results were higher than those previously seen for sugar palm starch [g transition temperature increased from 168.5 °C to 180.1 °C (10 wt% agar content). This can be associated with the interaction between starch and agar in the solid filler matrix, which in consequence has accelerated the matrix-to-glass transition [g transition.Differential scanning calorimetry (DSC) was used to examine the thermal characteristics of native sago starch, agar, and blends. m starch . Moreoveansition ,57. Thesg value for the TPSS with 15% agar content. A similar trend was observed by Rudnik [g transition. However, the poor adhesion between sago starch and agar when adding a more significant filler percentage may account for the drop in the Ty Rudnik  for modiy Rudnik , and henp < 0.05), respectively. Nevertheless, the strength of the blends increased until they reached the maximum reinforcement loading and optimal tensile strength at 10 wt% of agar content. Due to their similar chemical structures, which promote the intermolecular hydrogen bonding between both elements instead of their native intermolecular bonds, the finding indicates that agar strengthens the TPSS matrix. A more significant interaction between the TPSS and agar may be reflected by the FT-IR peak in the FT-IR study. This agreement is further supported by Wan et al. [The tensile characteristics of TPSS/agar blends at various agar loadings are presented in n et al. , who invn et al. ,59. Thisp < 0.05) when agar was added to the TPSS matrix. It dropped from approximately 4.18% to 0.56% when agar was incorporated into the matrix. This result is expected, since the filler integration imparts rigidity and prevents the matrix from deforming, which certainly causes a reduction in the material’s measure of ductility [Additionally, it was found that agar loading reduced the elongation at the TPSS matrix break c. The eluctility . As agaructility .p < 0.05). The flexural modulus of TPSS revealed a significant rising trend after the integration of agar 15 wt%, which resulted in improvement by a factor of nearly 20 in the modulus (p < 0.05). Flexural strength and modulus increased from 4.31 MPa to 12.91 MPa and 101 MPa to 1962 MPa, respectively. Numerous reasons could be responsible for a rise in the flexural strength and modulus of TPSS. First, the comparable hydrophilic behaviour and polysaccharide chemical structures of sago starch and agar contribute to their excellent miscibility [The capacity of a rigid material to endure deformation when subjected to bending stress is referred to as the material’s flexural strength in the field of mechanical engineering. The flexural strength and modulus of TPSS blended with varying amounts of agar (ranging from 5 to 15 wt%) are displayed in cibility . In ordecibility  studied cibility ,66. SubsIdeally, the flexural strength would be the same as the tensile strength if the material were homogeneous. However, the findings indicate that this TPSS/agar blends exhibits the highest value at different ratios for both tests. Apparently, the highest value for the tensile strength was at agar loading 10 wt%, while for flexural strength was at agar loading 15 wt%. This was due to the brittleness properties of TPSS/agar composites, in which the composites become more brittle when the loading of the agar increased. Flexural strength is the ability of composites or materials to resist bending deflection when energy is applied to the structure. As the flexural fracture behaviour of the composites is also ductile, the flexural strength increased with agar loading .p < 0.05) in the impact strength with the agar addition (5 wt%–10 wt%). The impact strength was gradually rising from 0.62 to 1.30 J/cm2 as the agar content increased. This behaviour could be attributed to the efficient interfacial adhesion between the two phases, which reduces the potential of crack propagation due to the interface’s lower strength than that of the fibre or matrix [The Izod impact strength from notched injected moulded test bars was evaluated as a function of agar loading at 23 °C and 50% humidity. Data reported in r matrix ,67. Hower matrix . The decr matrix .p-value for the experiment was lower than 0.05, the test results showed a statistically significant difference between the mixtures in terms of the mean impact strength of the thermoplastic blends.One-way ANOVA was used to analyse the impact test data statistically. Since the Moisture absorption results of TPSS and the blends are illustrated in This work successfully developed a novel biopolymer from sago starch and agar. Experimental research using various agar contents examined the impact of agar on thermoplastic sago starch. The use of agar in thermoplastic sago starch blends improved their tensile, flexural, and impact properties, as seen from the results. The thermoplastic sago starch blends with 10 wt% agar showed a maximum tensile strength of 8.61 MPa and tensile modulus of 995 Mpa. As for flexural strength and modulus, the thermoplastic sago starch blends were increased at 15 wt% of agar. The thermoplastic sago starch blends with 10 wt% agar showed the highest impact strength due to good interfacial adhesion, with the matrix resulting in less developed crack propagation. These findings also demonstrated that the presence of agar in the TPSS matrix had caused a rougher surface at the fracture, as the ratio of agar loading was increased. The thermal stability of the thermoplastic sago starch blends shows an increasing trend with the presence of agar. Nevertheless, the moisture absorption was elevated at a higher amount of agar. Overall, the characteristics of thermoplastic sago starch were enhanced by adding agar, especially in tensile strength and modulus, flexural strength and modulus, impact strength, as well as their thermal properties. Thermoplastic sago starch with 10 wt% agar shows the optimum loading, which increased the biopolymer’s potential for use in creating environmentally friendly materials.


This paper contributes to a new design of the three-dimensional printable robotic ball joints capable of creating the controllable stiffness linkage between two robot links through pneumatic actuation. The variable stiffness ball joint consists of a soft pneumatic elastomer actuator, a support platform, an inner ball and a socket. The ball joint structure, including the inner ball and the socket, is three-dimensionally printed using polyamide−12 (PA12) by selective laser sintering (SLS) technology as an integral mechanism without the requirement of assembly. The SLS technology can make the ball joint have the advantages of low weight, simple structure, easy to miniaturize and good MRI compatibility. The support platform is designed as a friction-based braking component to increase the stiffness of the ball joint while withstanding the external loads. The soft pneumatic elastomer actuator is responsible for providing the pushing force for the support platform, thereby modulating the frictional force between the inner ball, the socket and the support platform. The most remarkable feature of the proposed variable stiffness design is that the ball joint has ‘zero’ stiffness when no pressurized air is supplied. In the natural state, the inner ball can be freely rotated and twist inside the socket. The proposed ball joint can be quickly stiffened to lock the current position and orientation of the inner ball relative to the socket when the pressurized air is supplied to the soft pneumatic elastomer actuator. The relationship between the stiffness of the ball joint and the input air pressure is investigated in both rotating and twisting directions. The finite element analysis is conducted to optimize the design of the support platform. The stiffness tests are conducted, demonstrating that a significant stiffness enhancement, up to approximately 508.11 N·mm reaction torque in the rotational direction and 571.93 N·mm reaction torque in the twisting direction at the pressure of 400 kPa, can be obtained. Multiple ball joints can be easily assembled to form a variable stiffness structure, in which each ball joint has a relative position and an independent stiffness. Additionally, the degrees of freedom (DOF) of the ball joint can be readily restricted to build the single-DOF or two-DOFs variable stiffness joints for different robotic applications. Controllable stiffness is a valued function in various robotic applications, including surgical robotics ,2,3, walControllable stiffness mechanisms have been widely applied to both conventional rigid-bodied robots and soft-bodied robots fabricated with soft elastomeric materials with low Young’s modulus. The working principles of the existing variable stiffness designs for conventional rigid-bodied robots can be divided into four groups, including equilibrium-controlled stiffness, structure-controlled stiffness, mechanically controlled stiffness and antagonistic-controlled stiffness . Soft roVariable stiffness joints are receiving great attention due to rising interest in the research field of hyper-redundant robotic arms in recent years. Redundant robotic arms are very useful in a highly constrained space or multi-obstacle environments . The redDifferent from the currently existing research achievements on the ball joints with controllable stiffness, a new design of variable stiffness ball joints (VSBJ) is proposed to realize the ability of stiffness modulation for ball joints based on pneumatic actuation in this research. A support platform is designed to connect the inner ball to its socket upon pressurization. When the pressurized air is supplied, the soft pneumatic elastomer actuator is inflated to push the support platform against the inner ball, thereby restricting the rotation of the inner ball inside its socket. Thus, the stiffness of the ball joint can be modulated by the input of pressurized air. The contributions of this research and the key features of the proposed VSBJ are summarized as follows: (1) Simple structure: The ball joint is manufactured as an integral mechanism without the requirements of redundant fastening components, which allows the miniaturization design owing to the SLS technology; (2) fast response: The stiffness of the VSBJ can be suddenly enhanced once the pressurized air is supplied to the soft pneumatic elastomer actuator; (3) ‘Zero’ stiffness: The VSBJ can be freely rotated with very little force in its natural state when no air pressure is supplied; (4) positive pressure actuation: Compared with the variable stiffness mechanisms actuated by the smart materials, the pneumatically actuated stiffness modulation proposed in this research is not affected by magnetic/electrical fields or temperature fluctuation. Compared to the variable stiffness mechanisms actuated by vacuum pressure , the VSBJ actuated by the positive pressure has a wide range of actuation pressure; (5) MRI (magnetic resonance imaging) compatibility: The materials for fabricating the components of the VSBJ  and the actuation mode for the variable stiffness mechanism possess the feature of MRI compatibility . Thus, tThe design of the proposed VSBJ is schematically illustrated in The socket of the VSBJ is designed with three channels, allowing the support platform to linearly move inside the socket, while limiting the rotation of the support platform  that is a very soft platinum cure silicone elastomer with a durometer hardness of Shore 00–30 and high elongation at break (approximately 900%) . The sofThe working principle of the proposed VSBJ is illustrated in The dimensions of the proposed VSBJ are illustrated in In addition to the input air pressure, the support platform also plays an important role in VSBJ stiffness when the ball joint structure is determined and manufactured. The design of the support platform was optimized based on finite element analysis by investigating the relationship between the VSBJ stiffness and the intersection angle (θ) .The stiffness of the VSBJ indicates the ability to maintain the position and orientation of the inner ball against the external force. The variable stiffness feature of the VSBJ is a result of the change of the frictional force that is dependent on the inflation of the soft pneumatic elastomer actuator. To investigate the stiffness variation of the VSBJ in both rotating and twisting directions at different input air pressures, including 100 kPa, 200 kPa, 300 kPa and 400 kPa, for the soft pneumatic elastomer actuator, a series of stiffness measurement experiments were conducted. A compressor was employed to supply the air pressure and a precision regulator  was used to modulate the supplied air pressure to the soft pneumatic elastomer actuator.The experimental setup for evaluating the rotating stiffness of the VSBJ was shown in During the experiments, the soft pneumatic elastomer actuator was inflated with different constant pressures . For each input air pressure, the inner ball of the VSBJ was rotated ranging from 0 to 20 degrees by the force gauge through the linear motorized module at 1.25 mm/s constant speed. Fr was the applied pulling force along the X-axis perpendicular to the rod of the inner ball in the initial state, as illustrated in As illustrated in The experimental setup for evaluating the twisting stiffness of the VSBJ was illustrated in During the experiments, the VSBJ was twisted ranging from 0 to 20 degrees by the force gauge through the linear motorized module at 1.25 mm/s constant speed. Ft is the applied pulling force along the Y-axis perpendicular to the short bolt inserted through the rod of the inner ball, as illustrated in The proposed VSBJ can be modularized by redesigning the connectors  technology, the ball joint structure can be 3D printed as an integral mechanism without the need of assembly, thereby avoiding fastening components. Therefore, the proposed VSBJ can be miniaturized. A support platform was designed and combined with the socket to form the variable stiffness mechanism for resisting the rotational movements of the inner ball. The stiffness of the VSBJ was dependent on the friction force between the socket, the inner ball and the support platform. The design of the support platform was optimized by using finite element analysis. A soft pneumatic elastomer actuator was used to enable the variable stiffness mechanism to be actuated by pneumatic actuation. The stiffness of the VSBJ was dependent on the air pressure supplied to the soft pneumatic elastomer actuator. Experimental results have demonstrated a significant variation in stiffness at different pressure values. The higher stiffness of the proposed VSBJ can be obtained by increasing the supplied air pressure. The proposed VSBJ possessed the key features, including simple structure, fast response, ‘zero’ stiffness, positive pressure actuation and MRI compatibility. The stiffness of the VSBJ was independent on the position of the inner ball relative to its socket. Additionally, the proposed VSBJ can be used as a module for building the variable stiffness arm. It can also be customized to form the variable stiffness revolute joints with one or two degrees of freedom.The limitation of the proposed VSBJ is the abrasion between the inner ball and the socket due to friction. As for such friction-based stiffness variable mechanisms, abrasion is inevitable. The proposed VSBJ can be connected with links to form a positioning arm as described above. A future extension of this research will be adding the actuation mechanism based on pneumatic actuation for this passive robotic arm.


An experimental database was built with seven starchy food formulations for manufacturing dense and expanded foods made from starches, starch blends, breakfast cereals, pulse crop ingredients such as pea flour, fava bean flour, and fava bean starch concentrated, and wheat flour enriched with wheat bran. This database includes the thermal and physical properties of the formulations at solid and molten states, melt viscosity model, extruder configurations and operating parameters, and extruded foods properties. Using extrusion and viscosity models, melt temperature (T) and specific mechanical energy (SME) were satisfactorily predicted. A sensitivity analysis of variables at die exit was performed on formulation, extruder configuration, and operating parameters, generating the extruder operating charts. Results allowed the establishment of relationships between predicted variables  and product features such as starch and protein structural change, density and cellular structure, and functional properties. The extrusion operating conditions leading to targeted food properties can be assessed from these relationships and also the relationship between extrusion operating parameters and variables provided by simulation.A 1D global twin-screw extrusion model, implemented in numerical software, Ludovic Many starch-based foods, such as texturized ingredients, breakfast cereals, snacks, crackers, pasta, noodles, pet food, and many others, are produced by extrusion processes . StarcheUnder mechanical stresses and heat developed between screw and barrel during the extrusion process, the biopolymers undergo structural modifications: disruption of starch granules, melting of starch crystals, depolymerization of starch macromolecules, denaturation and aggregation of proteins, depolymerization of dietary fibers, and product browning due to Maillard reactions ,4,5. TheDue to an abrupt pressure drop at the die exit, the flash evaporation of superheated moisture forms a porous and expanded melt structure that is set after crossing its glass transition temperature. The structural features of the expanded foods govern their texture, functional and nutritional properties, and consumer acceptability ,10,11,12The development of mechanistic models of extrusion, based on the continuum mechanics approach, provides many benefits: a better understanding of the extrusion process and the development of computer-aided tools in simulation, optimization, and scale-up. In twin-screw extrusion modeling, two main approaches can be considered. In the first approach, local flow can be described with accuracy in a limited portion of the extruder by sophisticated numerical 2D or 3D models based on the finite element method (FEM) ,20,21,22® v7.0.0 Classic Edition . It is quite straightforward to provide predictions of flow variables with simple computing resources and time: about a few seconds per simulation using a laptop configured with Intel® Core™ i7-7600U processor . A fairly good agreement was obtained between simulation results from this 1D global model and a full 3D FEM model   led to a drop in intrinsic viscosity ΔVis between 20 to 80%, which can be interpreted by starch depolymerization a. Converh origin a. Their 0 kJ/kg] a. This tSince it is possible to predict macromolecular degradation from a computed extrusion variable, it is interesting to predict the conditions for processing maize starch, e.g., having an amylose content of 47%, with a targeted intrinsic viscosity drop ΔVis = 50 ± 5%. According to 2 (Ng), and (2) molecular one, revealed by the residual molar mass  that is obtained by normalization to native starch property.Here, we deal with specific products, namely partially melted  dense extruded starches, i.e., Potato 2 and pea (PS). This kind of product was obtained through extrusion at the moderate moisture content (30%) and at the low barrel and die temperatures, i.e., 75–95 °C . The sta2R = 0.56) and the fitting parameters depended on the raw material; this is likely due to the underestimation of SME prediction of pea starch extrusion. This inaccurate prediction comes from approaching the viscous behavior of pea starch melt with pea flour as an input of the 1D global extrusion model. Indeed, a previous study revealed that the shear viscosity of these feeds, determined by a pre-shearing capillary rheometer (Rheoplast®) , operated at SME < 250 kJ/kg, was very close [2R = 0.73).For both starches, the Ng, reflecting the extent of starch granule breakdown or starch crystallite melting, diminished with an increase in SME_com following exponential functions a. This pry close . Predict2R = 0.7–0.89). The general tendency is in accordance with experimental results; however it is obviously seen experimentally that potato starch is more sensitive to molecular degradation than pea starch , the remnant starch granules of noodles uptake water and swell, and cooking loss occurs through the diffusion (leaching) of depolymerized amylose and amylopectin outward the ghost starch granules, from the core to the surface of noodles. Cooking loss can be minimized by allowing starch swelling without excessive breakdown/leaching of its macromolecules. In other words, a compromise between two properties, Ng and RM, shall be sought, based on the assumption that the higher the RM, the more enhanced the leaching.As an example, we want to manufacture partially gelatinized potato starch with the following features: (1) highly amount of undisrupted starch granule: 150 < Ng < 300; (2) moderate level of RM (RM = 50 ± 10%). According to the relationship described in Here, the features of protein-rich snacks, extruded from pea and fava bean ingredients, were represented by protein solubility, in terms of insoluble proteins and density. While the latter reveals the macrostructure, the first can reflect the structure at supramolecular level. The gain in insoluble proteins indicates a higher amount of proteins crosslinked by covalent bonds other than S-S ones, i.e., isopeptide bonds. The higher is the density and the less expanded is the snack.2R ≈ 80, 3, in other words, expansion was magnified by a factor of eight, when T_com increased from 125 to 175 °C, regardless of formulation .The rising of melt temperature, at die exit, observed in the pea flour extrusion simulation promoted the protein cross-linking by non S-S bond R2 ≈ 80, a, in acc3), resulting from high-temperature extrusion (155 < T_com °C < 175 °C) at low MC (18% and 21%), is indispensable for obtaining crispy snacks. However, extrusion at these temperatures will be accompanied by a higher amount of insoluble proteins (from 5 to 25%). In addition, according to some authors, the protein cross-linking can deteriorate nutritional properties, such as, i.e., protein digestibility [A low density : 155 < T_com °C < 165 , macrostructure described by sectional  expansion index (SEI), and cellular structure expressed as cell density per cm3 (NC). The higher the WSIstarch, the more soluble are the starch molecules. This variable can thus indicate the state of starch depolymerization induced by extrusion. The higher the NC, the more porous the expanded structure.The features of wheat-based snacks are represented by hydro-solubility of starch (WSIRegardless of fiber (bran) content, all wheat flour-based snacks exhibited a unique linear and positive relationship between WSI of starch and SME_com a, in agrstarch from both computed and measured SMEs is poor (The prediction of WSI is poor f. If the2R = 0.7–0.8) were found between T_com and structure, such as SEI   b and celrection) c. Howevestarch < 60%). From the correlations between extrusion variables  and structure  moderately expanded (SEI > 4.5), (2) moderately porous, i.e., medium level of cell density (1000 < NC < 2000), and (3) moderate hydrosolubility of starch ), and melt rheological model as input. A large data set has been built, including results for extruded products’ properties and extruder variables , taken as output variables. Satisfactory correlations between process parameters predicted extrusion variables and features of extruded foods, such as structural and functional ones, justify the use of the extrusion model as a computer-aided tool for designing starchy food by extrusion. Moreover, the capacity for predicting these features is extremely vital to reducing time and labor costs in industrial foods R&D.Twin-screw extrusion processing of starchy products was simulated using the 1D global extrusion model (LudovicFlow variables at the die exit, such as melt temperature, specific mechanical energy and melt viscosity, can be computed and used, in turn, as the input variables for a phenomenological model of expansion, allowing predicting expansion indices and cellular structure of expanded starchy foods. The possibility to compute melt viscosity is particularly interesting since it is difficult to measure, either in-line or off-line and because it governs the expansion phenomenon, at least for low hydrated starchy foods. In addition, the knowledge of temperature profile and processing conditions resulting from the simulation is useful in setting extruder configuration and operating parameters for extrusion of thermo-sensitive foods.As a future prospect, extrusion simulation will be applied to the design of a wide range of starch-protein blends and expanded snacks from pulse crops. Further possible extension includes the processing of plant proteins for meat analogues. Besides classical structural and functional properties, the textural and nutritional features, such as starch and protein digestibility, inactivation of anti-nutritional factors, and destruction of pathogenic microbes and vitamins, will be tackled. The rheology database will be updated with melt viscosity models encompassing a wide range of moisture content, temperatures, and SME, taking into account protein and fiber content, as well as protein structural changes and starch-protein morphology.


Tropomyosin in shellfish is considered a major cross-reactive allergen in house dust mites and cockroaches; however, the specific epitopes have not been elucidated. Therefore, this study aimed to identify the consensus antigenic determinant among shrimp, house dust mites, and cockroaches using in silico methods. The protein sequences of tropomyosin, including Der f 10, Mac r 1, Pen a 1, Pen m 1, Per a 7, and Bla g 7, were retrieved from the UniProt database. The 3D structures were derived from the AlphaFold or modeled using the Robetta. The determination of linear epitopes was performed by AlgPRED and BepiPRED for B cell epitope, and NetMHCIIpan and NetMHCII for T cell epitope, while Ellipro was used to evaluate conformational epitopes. Fourteen peptides were discovered as the consensus linear B cell epitopes, while seventeen peptides were identified as linear T cell epitopes specific to high-frequency HLA-DR and HLA-DQ alleles. The conformational determination of B cell epitopes provided nine peptides, in which residues 209, 212, 255–256, and 258–259 were found in both linear B cell and linear T cell epitope analysis. This data could be utilized for further in vitro study and may contribute to immunotherapy for allergic diseases associated with tropomyosin. Seafood is considered one of the “big eight” food groups responsible for 90% of all food allergies and anaphylaxis worldwide . NowadayAlthough many natural proteins found in shellfish have been identified as allergens, the muscle protein, namely tropomyosin, seems to play the dominant role since IgE antibodies from 60–80% of shellfish allergic patients recognized this protein . MoreoveInterestingly, allergy to shellfish is associated with other arthropods, such as house dust mites and cockroaches . A studySome researchers highlighted that tropomyosin plays a critical role in cross-reactivity between house dust mites and shellfish. Boquete et al.  discoverAlthough the protein sequence and sensitization capacity of tropomyosin has been well studied, little information about the immunological mechanism of this protein, which may be related to hypersensitivity mediated by IgE response and cross-reactivity between shrimp, house dust mites, and cockroaches, exists. Therefore, this study aimed to elucidate cross-reactive epitope among these arthropods related to allergy, especially from high seafood consumption countries, using an in silico approach.Amino acid sequences of tropomyosin were selected from three shrimp species, two cockroaches, and American house dust mites that have been reported for their cross-reactivity ,17,18. TMost allergens contain epitopes that are specific to IgE antibodies, as this is crucial to determine the allergic reactions. In order to identify the possibly cross-reactive IgE epitopes of tropomyosin from animals studied, the AlgPRED tool was used to predict the possibility of the protein being allergenic, and IgE epitope motifs were extracted from the sequence based on known allergens. In addition, the candidate IgE epitope was analyzed using the BepiPRED server for cross-validation. When the AlgPRED tool was applied for all tropomyosin sequences of all species in the present study, more than fifty peptides were identified to be IgE epitopes. However, only fifteen peptides were discovered as the consensus sequences for all species . All pepHelper T cell plays a major role in contributing to the physiological changes during food allergy. This scenario needs the presence of a food epitope mediated by antigen-presenting cells (APCs) via MHC class II molecules  for alleNo tropomyosin in this study was resolved experimentally, and there are three types of tropomyosin  from three species that have been found in the AlphaFold database, the deep machine learning algorithm that demonstrated the highest accuracy of protein structure prediction among other servers . The strThe refined models were subjected to quality validation using a Ramachandran plot analysis derived from PROCHECK . The resAfter validation and quality evaluation of the tropomyosin structures, the Ellipro web tool was further employed to predict conformational epitopes of the refined tropomyosin structures. It was noted that brown shrimp and giant freshwater prawn were excluded from this analysis according to the QMEANDisCo score. In addition, the results were categorized as cross-reactive discontinuous B cell epitopes when the consensus of residue numbers was discovered without considering residues sequences. As shown in Shellfish sensitization is a common reaction found in 1–2% of the U.S. population, while 5% of South-East Asian individuals are affected ,28,29. TCross-reactive epitopes of tropomyosin from shellfish (shrimps), house dust mites, and cockroaches were identified. These arthropod species have been reported as the main cross-allergic reaction in shellfish sensitized patients ,18,19,31Currently, the possible linear B cell epitopes responsible for the allergic cross-reactivity between Der f 10, Mac r 1, Pen a 1, Pen m 1, Per a 7, and Bla g 7 were identified via in silico prediction based on the protein sequences. These peptides were not only possibly antigenic for B cells but also specific to IgE antibodies, suggesting that all identified sequences were associated with allergies. Interestingly, most predicted tropomyosin epitopes (11/14) had been previously proven for their antigenicity in other crustacean species by using inhibitory dot-blot analysis and patient serum ,37,38. IThe presentation of allergens was considered the first step of allergic reaction. Naive CD4 T cells recognize epitope via HLA class II-mediated antigen binding and transform into CD4 memory T cell that subsequently stimulates the differentiation of B cell to plasma cell, leading to the production of allergen-specific IgE antibodies. Therefore, the identification of T cell antigenic determinants is also essential for the treatment of allergy. For shellfish T cell epitope, Ravkov et al.  experimeAs described above, no crystal structure has been experimentally resolved for all tropomyosin in this study, although this allergen has been realized for its allergenicity for a long time. Fortunately, three tropomyosin from three species, including Der f 1 (American house dust mite), Pen m 1 (black tiger shrimp), and Per a 7 (American cockroach), were predicted for their structures in the AlphaFold database, the server that showed the rank 1 of accuracy testing for protein structure modeling . For theAn attempt to use the outperform online tools according to the standard testing benchmark was made; however, the tropomyosin structures of brown shrimp and giant freshwater prawn revealed the uncommon conformation with the low quality according to the QMEANDisCo score and Ramachandran plot. Although the bending form of tropomyosin had been suggested in crystallized analysis ,47, the This work mostly explored the possible cross-reactive tropomyosin epitopes between shellfish, house dust mite, and cockroaches based on experimental evidence from the literature on allergic patients. This study and other findings demonstrated a strong allergic reaction between shellfish and insects, which may raise an issue of concern about the promotion of eating edible insects as an alternative source of nutrients ,50,51. Ihttp://allergen.org/; accession date: 12 January 2022). The amino acid sequences of tropomyosin from 6 animals  were acquired from The Universal Protein Resource (UniProt) according to the accession number /International Union of Immunological Societies (IUIS) Allergen Nomenclature Sub-committee database  and BepiPRED-2.0 server  were used to predicate the cross-reactive epitopes. AlgPred employs a wide range of information and techniques, including machine learning techniques, BLAST, MEME (Multiple Em for Motif Elicitation)/MAST (Motif Alignment and Search Tool), and IgE epitope mapping to determine IgE-specific epitopes and allergen motifs [Epitopes of the allergens were predicted based on the selected parameters, including the mapping of IgE epitopes and linear B-cell epitope mapping of motifs. Two immunoinformatics tools, including AlgPRED 2.0  was used to predict HLA-DR-based T cell epitope in the regions of HLA-DR DRB101, HLA-DR DRB103, HLA-DRB301, HLA-DRB401, and HLA-DRB501. NetMHCII-2.3 was also applied to predict the HLA-DQ binding epitope. Those included HLA-DQA10101-DQB10501, HLA-DQA10102-DQB10602, HLA-DQA10301-DQB10302, HLA-DQA10401-DQB10402, HLA-DQA10501-DQB10201, and HLA-DQA10501-DQB10301. Both methods predicted peptide binding to any MHC class II of a known sequence using Artificial Neural Networks (ANNs), and the candidates were grouped as either a strong binder (≤2 percentile range), weak binder (percentile rank ≤ 10), or no binder (>10 percentile rank) [The identification of T cell epitopes is principally based on the exploration of peptide fragments that bind to the MHC class II. NetMHCIIpan-4.0 (le rank) . As a rehttps://alphafold.ebi.ac.uk; accession date: 15 February 2022) were predicted using a deep-learning-based artificial intelligence program that combines both physical and biological information about protein structure and multisequence alignments to predict protein conformation, which has been ranked as the number one protein prediction server in CASP14 [https://robetta.bakerlab.org; accession date: 15 February 2022) was also used to model tropomyosin from species that cannot be found in the AlphaFold database. This server uses both physics- and energy-based force field types and Monte Carlo, a filtering protocol to choose the suitable ab initio models [Since the crystal structure of tropomyosin of all species in this study has not yet been established, all structures were either retrieved from the AlphaFold database or predicted by the ROBETTA server . The 3D o models .https://galaxy.seoklab.org/cgi-bin/submit.cgi?type=REFINE2; accession date: 17 February 2022) was utilized to refine the 3D model obtained from the AlphaFold database and Robetta prediction tool. The selection of this refinement tool was based on QMEANDisCo score testing after structure refinement using several methods, involving GalaxyRefine server, DeepRefine, and PREFMD (data not shown). Initial structures were subjected to quality assessment by using PROCHECK [https://prosa.services.came.sbg.ac.at/prosa.php; accession date: 25 February 2022) was also applied to validate the tertiary structure. This server employs the data of the inputted structure to calculate the overall stereochemical quality of the protein structure [The GalaxyRefine2 server  for structure-based prediction of antibody epitopes with the threshold score > 0.5. This software is a most comprehensive protocol containing three algorithms based on three-dimensional structure data to predict discontinuous epitopes as protrusion index (P.I.) values. Ellipro was shown to demonstrate the best performance and provided an AUC value of 0.732 [Most of the B cell epitopes were determined to be discontinuous. For this reason, the refined model base in the PDB coordinate was subjected to ElliPro, an online server (of 0.732 .


The degree of order (DO) and degree of double helix (DD) of the modified starches were also not significantly different from the native sample, except for AHT and OPT, respectively. Physical modification decreased the swelling volume, while chemical modification increased its value and is inversely proportional to solubility. AHT and OPT starches have the best freeze–thaw stability among others, indicating that both starches have the potential to be applied in frozen food.Sago starch has weaknesses such as low thermal stability and high syneresis. Modifications were made to improve the characteristics of native sago starch. In this study, sago starch was modified by autoclave-heating treatment (AHT), osmotic-pressure treatment (OPT), octenyl-succinic anhydride modification (OSA), and citric acid cross-linking (CA). This study aimed to examine the changes in chemical composition, crystallinity, and functional properties of the native sago starch after physical and chemical modifications. The results show that physical modification caused greater granule damage than chemical modification. All modification treatments did not alter the type of crystallinity but decreased the relative crystallinity of native starch. New functional groups were formed in chemically modified starches at a wavelength of 1700–1725 cm Sago is one of the potential local commodities originating from the eastern part, which is abundant in Indonesia. This commodity has a high potential as an alternative to local starch because it has high starch productivity, which is more than 400 kg of dry starch in each sago tree trunk. Sago flour production reaches 25 MT/ha, several times more than potato or cassava flour . The DirThe native starch produced has some limitations in its physical, chemical, and functional properties, such as being unstable to mechanical treatment, easy to retrograde, hard, sticky, and unclear pasta formed, and low thermal and acid stability. Furthermore, Nordin et al.  have repSeveral modifications were performed to improve the weaknesses of sago starch. In this study, physical and chemical modifications of starch were carried out. Several methods in physical modification are commonly involved hydrothermal treatment or pre-gelatinization processes to improve the native starch properties, such as annealing, heat-moisture treatment, pre-gelatinization, etc. Heat-moisture treatment (HMT) has been widely used and produces starch characteristics that are more resistant to heat and mechanical processes. HMT is a hydrothermal treatment, where the starch is treated at high temperatures (80–120 °C) and low moisture levels (less than 30%) for a certain period . HoweverThe chemical modification consists of several methods, including cross-linking, esterification, etherification, oxidation, etc. In contrast to physical modification, chemical modification involves the introduction of functional groups into the starch molecule, resulting in changed physicochemical and functional properties . In thisMetroxylon sp.) . All chemicals used for modification, such as 2-octen-1-ylsuccinic anhydride (OSA) , acetic acid, hydrochloric acid, and sodium hydroxide, were of analytical grade.The raw material used in this research is sago starch . Then, it was milled and sieved to a particle size of ≥100-mesh.The osmotic pressure-treated starch was prepared using the previous method . Starch The OSA modification method refers to the method by Anwar et al. . In thisThe citric acid cross-linked starch method refers to a previous study , with a Moisture content was determined by referring to the AOAC  method, Granular morphology was determined using scanning electron microscopy (SEM)  . Starch The particle size distribution of starch was determined using a Particle Size Analyzer (PSA)  . Starch −1. Relative crystallinity was determined using a sigma plot program by calculating the area under the curve of the crystalline region and the amorphous region in the obtained diffractogram [The crystallinity of sago starch samples was analyzed using PANalytical, Malvern, UK, X’Pert PRO series PW3040/x0. Before analysis, the moisture content of samples was adjusted to 6% ± 0.5%. This instrument was used to collect the X-ray diffraction pattern with a copper tube operating at 40 kV and 30 mA. This diffractometer produces the Cu-K α-radiation with the wavelength of 1.5406 Å. The diffractogram of the starch samples was obtained by scanning the sample from 5° to 50° (2θ) at room temperature, with an increase of 0.02° every second, with a rotating speed of the holder of 30 minactogram . Relativw/w) and pressed into pellets and then subjected to attenuated total reflectance (ATR). The wavelength range and spectral resolution used were 4000–500 cm−1 and 8 cm−1 by 32 scans, respectively. Degree of order (DO) and degree of the double helix (DD) were also measured by the absorbance ratios of 1047/1022 cm−1 and 995/1022 cm−1, respectively [FTIR was used to analyze functional groups of sago starch samples. Samples were mixed with KBr . All data were analyzed using Statistical Software Program (SPSS) version 26.0.Data are presented as the mean ± SD of triplicate experiments. All data were analyzed by one-way analysis of variance (ANOVA) with a further Duncan’s test to compare the sample mean at a significance level of 5% , and corresponding images are presented in AHT starch showed an irregular-shaped granule surface, cracks, and a rough surface. Most of the granule particles had an incomplete shape, and were broken and fragmented, which might be due to the partial gelatinization of AHT starch granules caused by the presence of limited water and heat energy in the starch system . SimilarChemical modification of sago starch did not significantly change the shape of the granule surface compared to native starch. OSA starch mostly had a smooth texture, and there was minor damage in a small portion of the starch granules. Small amounts of the granules showed small holes and cavities and a slight depression at the ends of the granules due to the esterification treatment. This small change was triggered by the esterification reaction and hydrolysis due to the addition of NaOH at the beginning of the treatment . OSA canAHT- and OPT-modified starch increased the granule size of native starch . The incThe native sago starch has an A-type crystallinity pattern with firm peaks at 15° and 23° and unresolved peaks at 17° and 18°. All modification methods did not alter the crystallinity pattern of native sago starch .Although it did not change the type of crystallinity pattern, the relative crystallinity (RC) of all modified sago starches decreased compared to the native starch. The RC of physically modified sago starch tends to be lower than that of chemical modification. Following a study by Marta et al. , the RC −1 indicated the formation of an ester carbonyl group in the starch [FTIR spectra of native and modified sago starches are shown in e starch . The fore starch . The fore starch .−1 and the ratio of the absorbance band at 995 and 1022 cm−1, respectively. From FTIR, we could also measure the degree of order and internal changes in the degree of the double helix of starch granules, which was calculated by the ratio of the absorbance band at 1047 and 1022 cmOxalis tuberosa (oca) tubers. In this study, the DD of OSA starch also tended to decrease, but statistically, it was not significantly different from the DD of native starch.The degree of the double helix (DD) of all modified starches was not significantly different from native starch, except OPT starch. Increased DD in OPT was due to the presence of saturated sodium sulfate solution. Although the modification involves heat, the sulfate can prevent excessive gelatinization. Compared with both physical modification treatments, it was seen that DD between AHT and OPT was not significantly different. Similarly, between both chemical modification treatments, OSA and CA. Velásquez-Barreto et al.  have rep42− ions in the OPT modification can inhibit the absorption of water by starch granules. Furthermore, Bhosale and Singhal [ Singhal  have repThe solubility (SOL) of all modified sago starches was significantly different from native sago starch . SOL hasIn contrast to physically modified starch, the SOL of OSA and CA starches decreased compared to their native counterpart. The decrease of SOL on CA starch was due to the higher molecular weight, making it difficult to dissolve . FurtherThe water absorption capacity (WAC) of AHT and OPT starches increased significantly compared to other treatments . The incWAC between both chemical modifications, OSA and CA, was not significantly different, and was higher than native starch. The octenyl group penetrates the starch structure, which causes a change in the structural system of the starch by breaking the hydrogen bonds between the starch chains so that water penetration into the starch granules increases at low temperatures . This reOil absorption capacity (OAC) in AHT increased compared to the native starch . The higThe decrease in syneresis also occurred in taro starch modified using an autoclave . The dec−1. The degree of order (DO) and degree of double helix (DD) of the four modified starches were also not significantly different from the native sago starch, except for AHT starch which decreased the DO value, and osmotic-pressure treatment (OPT), which increased the DD value. The functional properties of sago starch underwent different changes after the modification process. Freeze–thaw stability or the percentage of syneresis of sago starch decreased in all modified sago starch. Physical modification decreased the swelling volume value, while chemical modification increased its value, and this was inversely proportional to the solubility of all physically modified starch, which increased while chemical modification decreased. All modifications improved the water absorption capacity (WAC), while oil absorption capacity (OAC) with the physical modification increased and with the chemical modification decreased. All modified starches had high freeze–thaw stability (low syneresis), indicating that this starch has the potential to be applied in frozen food.Physical (autoclave-heating treatment and osmotic-pressure treatment) and chemical (octenyl-succinic anhydride and cross-linking citric acid) modifications had different and significant effects on the chemical composition, granule morphology, crystallinity, and functional properties of native sago starch. For chemical composition, modifications significantly affected the water content, but only autoclave-heating treatment (AHT) significantly affected amylose content. Whereas AHT and octenyl-succinic anhydride (OSA) modifications significantly affected starch content. Physical modifications damaged the starch granules, while chemical modifications only slightly damaged the granules. All modifications did not change the type of crystallinity through the crystallinity pattern but decreased the relative crystallinity. In addition, there was no change in functional groups in physically modified sago starch, but new functional groups were formed in chemically modified sago starch at a wavelength of 1700–1725 cm


In addition, the field is predicted to have significant impact in biomedicine—for example, in vaccine development and the way in which vaccines are manufactured, with the contribution of Ginkgo Bioworks to the development of the Moderna vaccine for COVID‐19 a timely demonstration of this. There are also many other areas where engineering biology is being applied to replace existing industrial processes and products and we are delighted to have published some of this work in the journal over the last year including the development of synthetic spider silk at The University of Manchester, which is already having applications in a number of areas, and the application of machine learning‐driven protein engineering by LabGenius for computational drug discovery. At the end of the year, we also published the popular opinion piece ‘Synthetic biology, engineering biology, market expectation’ by Lionel Clarke, Co‐Chair of the UK Engineering Biology Leadership Council and Director of BionerG, in which he discusses the differences between the terms ‘engineering biology’ and ‘synthetic biology’ and their role in the market ‘ecosystem’. In this first issue of our fifth volume, we are pleased to present a review of the optimisation of protein synthesis in cell‐free systems—an area that has a number of applications; and the case study ‘Responsible Innovation: its role in an era of technological and regulatory transformation’.We start the fifth volume of We would like to take this opportunity to thank all of the contributors to the journal: the authors from academia and industry for sharing their interesting developments and insights; the reviewers for the time they have taken to assess and provide valuable feedback on the submissions, and the Associate Editors for their work to uphold the high standards of the journal. We are very grateful for the efforts of all involved and we are continuing our work with our international editorial board to build the journal into a key source of information for all aspects of engineering biology.Engineering Biology has a deliberately wide scope so that it can capture not only original research, but also industrial developments ; opinion pieces on the grand challenges, policies, new projects and developments in the field; and invited mini reviews of progress in important areas. This year the publisher of Engineering Biology, The Institution of Engineering and Technology (IET), has partnered with Wiley to publish its journals, and we believe that this will give Engineering Biology an even greater outreach and range of readership. Please do get in touch with us at theiet_enb.office@wiley.com if you are interested in submitting an article or have an idea for a special issue. You can also find more information about the journal and how to submit at http://wileyonlinelibrary.com/iet‐enb. We are looking forward to working with the engineering biology community over the coming year to publish more of the exciting developments in this area.


Metroxylon sagu). The starch was subjected to gamma irradiation  prior to ANN at 5 °C (To-5) and 10 °C (To-10) below the gelatinization temperature. Determination of amylose content, pH, carboxyl content, FTIR (Fourier Transform Infrared) intensity ratio (R1047/1022), swelling power and solubility, thermal behavior, pasting properties, and morphology were carried out. Annealing irradiated starch at To-5 promoted more crystalline perfection as compared to To-10, particularly when combined with 25 and 50 kGy, whereby a synergistic effect was observed. Dual-modified sago starch exhibited lower swelling power, improved gel firmness, and thermal stability with an intact granular structure. Results suggested the potential of gamma irradiation and annealing to induce some novel characteristics in sago starch for extended applications.The increase in health and safety concerns regarding chemical modification in recent years has caused a growing research interest in the modification of starch by physical techniques. There has been a growing trend toward using a combination of treatments in starch modification in producing desirable functional properties to widen the application of a specific starch. In this study, a novel combination of gamma irradiation and annealing (ANN) was used to modify sago starch ( Apart from being the most abundant carbohydrate source for human staple food, the low cost, easy tailoring, biocompatibility, renewability, and extensive sources of starch have made it a most promising biodegradable natural polymer for food and nonfood industries . In its The ionizing radiation processing is a progressive, fast, and environmentally friendly way to produce changes in the structural and functional properties of starch . The irrw/w) at a temperature that is above the glass transition temperature (Tg) but lower than the onset temperature (To) for gelatinization [In recent years, there has been a growing trend toward treating starch with hydrothermal methods, including annealing. Annealing (ANN) is carried out by incubating starch samples with moisture content higher than 65% . It is considered an underutilized starch in food applications due to its poor functional properties [Malaysia is one of the main producers of sago starch, which is extracted from the pith of a palm plant  and gradually decreased as the dose of the irradiation was increased  and showed higher amylose content than the ANN(To-5) (p ≤ 0.05). A similar observation was reported by Babu et al. [p ≤ 0.05) than their irradiated counterparts, but insignificant differences when compared to the annealed starch samples (p > 0.05). Depolymerization by gamma radiation may most probably facilitate the reorganization of the starch molecules during annealing to acquire more 3-D helicoidal structures that are suitable to fix in the iodine. More amylose-like molecules were formed by the reorganization of the dual-modified starch molecules.On the contrary, the annealed samples had significantly higher amylose content than the native starch with the ANN and 5kGyAnn(To-10) were lower than 5kGy (p ≤ 0.05); insignificant differences were found between 10kGy, 10kGyANN(To-5) and 10kGyAnn(To-10) (p ≤ 0.05); whereas 25kGyANN(To-5), 25kGyAnn(To-10), 50kGyANN(To-5), and 50kGyAnn(To-10) were reported higher than 25kGy and 50kGy, respectively. The incubation temperature had no influence in this case (p ≤ 0.05). When compared to 25kGy and 50kGy, the dual-modified counterparts (25kGyANN(To-5), 25kGyAnn(To-10), 50kGyANN(To-5), and 50kGyAnn(To-10)) turned out to be less acidic, indicating the potential involvement of the acid groups in the structural reconfiguration of the starch macromolecules during ANN.A substantial drop in pH was observed with an increase in the irradiation dose , suggesto-10) exhibited a slightly higher pH than the native counterpart (p ≤ 0.05). As the carboxyl content was mainly attributed to the starch degradation by irradiation, ANN did not remarkably alter the carboxyl content of the dual-modified starch, regardless of the ANN temperature. Since no hydrolysis of the glycosidic bonds was expected, no carboxyl content was detectable in the native and annealed samples, even though the ANN. In contrast, the R1047/1022 increased after ANN because the treatment provided conditions enabling the rearrangement of the starch molecules for crystalline perfection. Compared to the ANN(To-10), the higher annealing temperature of ANN(To-5) accelerated the rate of hydration and increased the kinetic energy of the glucan chains to take part in the molecular rearrangement. Therefore, the more pronounced effect of annealing on the structural change of the starch was obtained at a temperature closer to its gelatinization temperature. The recrystallisation-promoting effect of the higher annealing temperature of ANN(To-5) was also notable when combined with gamma irradiation. In the dual-modified samples, a synergistic effect in promoting crystalline perfection was observed, in which 25kGyANN(To-5) and 50kGyANN(To-5) achieved the highest R1047/1022 (p ≤ 0.05), indicating the higher molecular order of the double helix short-range in the starch granules [Starch samples were analyzed by FTIR spectroscopy to confirm the breakdown of the glycosidic bonds and the changes to the short-range crystalline order (double helices) in the crystalline region and the amorphous region near the granule surface . The deggranules  as the sgranules . p ≤ 0.05). In corroboration to the results of the FTIR, the ANN(To-5), which experienced more extensive reorganization of the structure, had lower swelling power and solubility than the ANN(To-10) (p < 0.05). Compared to gamma radiation, the suppression of solubility by ANN was more apparent than the suppression of the swelling power. According to Zavareze and Dias [p > 0.05), except for 50kGyANN(To-5), which displayed the lowest swelling ability (p ≤ 0.05), approximately 40% lower than 50 kGy. The ANN caused a solubility suppression effect on the irradiated sago starch with a more profound outcome by ANN at (To-5) than (To-10). The reduction in the solubility was in proportion with the increase in the irradiation dose, where a reduction from 59% to 89% was observed for the dual-modified samples as compared to the irradiated counterparts. In brief, the dual-modified samples behaved like irradiated samples in terms of the swelling power, but the solubility was lower than the irradiated samples due to the suppression effect by ANN.Upon irradiation, the swelling power of the sago starch was noticeably reduced with the increasing irradiation dose . A reducand Dias , the intp ≤ 0.05). The formation of carboxyl groups by the radiolytic degradation of the starch molecules caused the electrostatic repulsion of the molecular association during gel formation by retrogradation [p > 0.05). Depending on the starch origin and the radiation dose, the irradiated starch gel firmness/strength was found to be either improved [o-5) exhibited the highest gel firmness among all tested samples (p ≤ 0.05). The results obtained show that ANN may improve the gel firmness of irradiated starch without being affected by the incubation temperature (p > 0.05). The beneficial effect was, however, only reported at lower radiation doses of 5 kGy, 10 kGy, and 25 kGy. At 50 kGy, all the irradiated samples and dual-modified samples showed insignificant differences (p < 0.05).radation . As the improved  or reducimproved . Polesi improved  attributimproved  due to to (onset temperature), Tp (peak temperature), and Tc (conclusion temperature) at 50 kGy (p ≤ 0.05). This observation agrees with the results of the swelling power and solubility ((∆H) of irradiated maize starch were not statistically significant from 0 to 20 kGy, but a significant decrease in To, Tp, and ∆H was observed from 20 to 50 kGy. Chung et al. [H . The ∆H for irradiated wheat starch was also reported to be insignificantly different from that of the native starch until 50 kGy, which was ascribed to the unchanged crystallinity [The gelatinization temperature of the irradiated sago starch was gradually decreased with the increasing dose of gamma radiation , and a slubility , wherebyg et al.  reportedg et al.  revealedg et al. , gelatinallinity . It is to-5) and (To-10) brought about an increase in the To and Tp of the sago starch (p < 0.05). The increase in the gelatinization temperature by ANN has been shown to be most pronounced for To and least for Tc [o and Tp, a decrease in the gelatinization range, and either no change or an increase in the gelatinization enthalpy [H of the annealed sago starch implies an improved starch reinforcement within the granules and the attainment of a higher configuration stability [p ≤ 0.05). The narrowing of the gelatinization temperature range, which may imply the higher crystallite homogeneity  was also observed, particularly for ANN(To-5) and the corresponding dual-modified samples. Even though the values of Tc for the two annealed samples are high, they are statistically insignificant from the native and irradiated samples (o-5), 25kGyANN(To-5), and 50kGyANN(To-10). The high standard deviations indicate that the Tc values (the ending point of gelatinization) are spread out over a wider range, suggesting that these three samples contained more heterogeneous crystallites (induced by irradiation) as compared to the annealed counterparts.Annealing at  in the dual-modified samples, with a more substantial change in the ANN(To-5)-corresponding samples, whereby these samples appeared to have the highest gelatinization temperatures. This observation agreed well with the results obtained for the R1047/1022, swelling power, and solubility. By comparing the effect of ANN on high-amylose wheat (HAWS) and maize starch (HAMS) with a similar apparent amylose content, Li et al. [o-5 provided the required chain mobility for successive structural rearrangement in the samples. In the present study, an inconclusive change in ∆H was reported for all the dual-modified starches.Owing to the starch reinforcement by ANN, a notable increase in Ti et al.  concludeo-5)-corresponding samples.Investigation of the pasting profile plays a vital role to elucidate the technological functionalities that encompasses the gelatinization, pasting, and retrogradation characteristics of starch. The results obtained  show thap ≤ 0.05); moreover, in parallel with the effects of irradiation and ANN on the swelling capacity of the starch (o-10)-corresponding samples were reported higher than the ANN(To-5)-corresponding samples (p < 0.05). The alteration in viscosity was in corroboration to the swelling power and solubility behaviors of these samples (o-5), more extensive molecular interactions took place to restrict the starch swelling and solubility, and subsequently a lower peak viscosity.Starch pasting involves the process of viscosity development that occurs after heating starch above the gelatinization temperature . Native e starch , a reduc samples . It is ip ≤ 0.05) because a higher irradiation dose brought about more severe molecular destruction and weaker starch granules, and hence a lower ability to withstand shearing at a high temperature. The final viscosity and setback also decreased with the increasing radiation dose. The setback and final viscosity are attributed to a reordering or polymerization of the leached amylose and long linear amylopectin [p ≤ 0.05) without affecting the setback (p > 0.05). The starch bond strengthening by ANN mentioned in the earlier session had improved the granular stability against collapse by continuous shearing at the maximum swelling volume. In general, all the dual-modified samples were found to exhibit a relatively high breakdown and low final viscosities and setback, indicating the irradiation exerted a more pronounced effect than ANN in the pasting properties of the dual-modified samples. As expected, the breakdown decreased in proportion to the irradiation dose applied (lopectin  upon thelopectin . The reslopectin  pointed o-5) and the ANN(To-10), as depicted in Dent surfaces and the development of pores on the starch granules (indicated by the arrows) are found on the ANN(To-5) is closer to the onset gelatinization temperature of the sago starch, more extensive agglomerations are observed compared to those annealed at (To-10) (o), no agglomeration of the starch granules was observed by the researchers. Partial gelatinization had taken place in the annealed and dual-modified starches, as shown by the agglomeration of the starch granules . No morp (To-10) . DifferiMetroxylon sagu) starch  was bought from Nee Seng Ngeng and Sons Sago Industries Sdn. Bhd. . Sago starch was packed in a polyethylene bag and irradiated with a 60Co gamma source at the Malaysian Nuclear Agency . The starch samples were subjected to four doses of irradiation  as described by Chung et al. [Food-grade sago  and mixed in a 25 mL volumetric flask. The content was heated for 15 min with continuous swirling at 85 °C until the starch sample was fully dissolved, and the content was topped up to 25 mL using distilled water. After that, 1 mL of the starch solution was added with 40 mL of distilled water and 10 mL of iodine solution, and the solution was incubated for 15 min before the measurement. The absorbance was measured  at 625 nm against a reagent blank as the reference. A standard curve for mixtures of pure potato amylose and amylopectin was plotted and the regression equation of the standard curve was used to calculate the total amylose content of the samples .The amylose content was determined using the iodine-binding spectrophotometry method modified from Williams et al. . An amouAn amount of 4 g of starch was suspended in 50 mL of distilled water and the pH value was recorded using a pH meter . The method of Chattopadhyay et al.  was used−1 was calculated [The infrared spectra of the starch samples were obtained on KBr pellets  of the samples using a Spectrum 100 Spectrometer (Perkin Elmer), and the intensity ratio from 1047 to 1022 cmlculated  to evaluThe swelling power and solubility were determined as described by Schoch , with slTM Diamond, Perkin Elmer) with slight modification from Chung et al. [Thermal analysis was performed using a Differential Scanning Colorimeter . An amount of 3.0 g of starch (dry basis) was adjusted with distilled water to achieve constant weight of 28.0 g in the aluminum canister. The heating and cooling profile used was according to Ng et al. . The temw/v d.b.) was prepared according to Adawiyah et al. [Gel firmness was measured using a texture analyzer  with a load cell of 25 N. The cylindrical (23 mm diameter × 20 mm height) starch gel  with 500× and 2000× magnification. Starch sample was suspended in a carbon tape and coated with a fin layer of gold–palladium using a sputter coater .Data reported are the average of at least triplicate determinations (from three batches of samples prepared independently). SPSS  version 20 was used for statistical analysis. One-way analysis of variance (ANOVA) with Tukey’s HSD test was used to compare the means (95% level of significance).o-5) enhanced the crystalline perfection by increasing the short-range orders of the irradiated sago starch as compared to a lower ANN temperature (To-10). Synergism in molecular reorganization was observed when the modification was carried out by combining a high radiation dose (25 kGy and 50 kGy) with the ANN temperature of To-5. Among the properties investigated, the annealing temperature significantly influenced the swelling power, the gelatinization behavior, and the pasting profile of the irradiated sago starch. The dual-modified sago starch recorded a lower swelling power and a higher thermal stability by exhibiting higher gelatinization temperatures as well as a narrower melting temperature range when compared to the native starch or the single-modified starch. The gel firmness was also significantly improved. The physical modification methods employed also offer a sago starch with new properties without destroying the granular structure, which might be important in extending its applications. Future work investigating in-depth structural changes and starch digestibility are needed to provide a better insight of the modification techniques employed.Results obtained support the hypothesis that a higher annealing (ANN) temperature (T