The main aim of this study is determination of the basic probabilistic characteristics of the effective stiffness for inelastic particulate composites with spherical reinforcement and an uncertain Gaussian volume fraction of the interphase defects. This is determined using a homogenization method with a cubic single-particle representative volume element (RVE) of such a composite and the finite element method solution. A reinforcing particle is spherical, located centrally in the RVE, surrounded by the thin interphase of constant thickness, and remains in an elastic reversible regime opposite to the matrix, which is hyper-elastic. The interphase defects are represented as semi-spherical voids, which are placed on the outer surface of this particle. The interphase is modeled as hyper-elastic and isotropic, whose effective stiffness is calculated by the spatial averaging of hyper-elastic parameters of the matrix and of the defects. A constitutive relation of the matrix is recovered experimentally by its uniaxial stretch. The 3D homogenization problem solution is based upon a numerical determination of strain energy density in the given RVE under specific uniaxial and biaxial stretches as well as under shear deformations. The analytical relation of the effective composite stiffness to the input uncertain parameter is recovered via the response function method, using a polynomial basis and an optimized order. Probabilistic calculations are completed using three concurrent approaches, namely the iterative stochastic finite element method (SFEM), Monte Carlo simulation and by the semi-analytical method. Previous papers consider the composite fully elastic, which limits the applicability of the resulting effective stiffness tensor computed therein. The current study voids this assumption and defines the composite as fully hyper-elastic, thus extending applicability of this tensor to strains up to 0.25. The most important research finding is that (1) the effective stiffness tensor is sensitive to random interface defects in its hyper-elastic range, (2) its resulting randomness is not close to Gaussian, (3) the semi-analytical method is not perfectly suited to stochastic calculations in this region of strains, as opposed to the linear elastic region, and (4) that the increase in random dispersion of defects volume fraction has a much higher effect on the stochastic characteristics of this stiffness tensor than fluctuation of the strain. Multiscale computational methods and simulations have attracted scientists and engineers for many years, which has been documented by the comprehensive review presented in . As is eSuch multiscale homogenization methods have been worked out in solid mechanics to predict the elastic effective properties of fiber-reinforced composites , metal–cNevertheless, the problem of stochastic micro or nanostructure and the realistic impact of the random spaces on the overall characteristics of the composite material (or system) is rather infrequent, and this is why it shall be discussed in this paper.The linear mechanical behavior of single phase isotropic and anisotropic materials is well understood and has been proved by a firm theoretical background. This is also true for composites with non-complex internal composition and geometry, for which analytical solutions apply, and when geometry of the microstructure represents a certain pattern and symmetries. This allows them to be included in certain class of behaviors, e.g., cubic or orthotropic, etc. When such symmetries are not recognizable straight away, various homogenization approaches are used that usually require multi-scale considerations; some modern ones include the FE2 scheme  or neuraMechanical behavior in the non-linear regime of materials is already complex in the isotropic case, where multiple constitutive laws may be utilized for the same material. This is true for the hyper-elastic regime for instance, where these laws are usually defined assuming material isotropy, so that the potential application area is strictly limited. There also exists some hyper-elastic theories accounting for the anisotropy of material microstructures , howeverIndependently from the composites’ response under the given loads, one of the most important aspects of both deterministic and stochastic numerical modelling is the connection of their constituents. A perfect bonding is not achieved in many cases because of the chemical, thermal, electrical or mechanical properties’ incompatibilities and also due to manufacturing processes. In such cases, an interphase is inserted in-between the original constituents of the composite , which fThe interface defects are considered in this work and the influence of uncertainty in their volume fraction in the given interphase on effective mechanical properties of some particulate composite is numerically studied using the generalized stochastic perturbation-based computational analysis. We verify the impact of this input uncertainty and, independently, of the strain level on the effective probabilistic stiffness tensor components, when particles are linear-elastic and the matrix is hyper-elastic. The iterative stochastic finite element method based on higher order Taylor expansions and polynomial bases is used to determine the first four probabilistic characteristics of the effective tensor components. Computational implementation has been completed using a hybrid application of the finite element method system Abaqus Standard and computer algebra system Maple 2017.Let us consider a periodic particulate composite  j=1 … r ,35. The Next, the effective composite stiffness Energy contributions to this effective stiffness come from the hyper-elastic matrix and the interphase as well as from the elastic particles60). It was selected as the closest approximation of the matrix response coming from laboratory tests in this specific case study. A constitutive relation of the interphase The interphase and the matrix work according to the Arruda–Boyce hyper-elastic potential . This po60) occupy 5% of the RVE, 5% of the interphase, and the last 90% of this composite is filled with a matrix. The mesh of this composite (The representative volume element (RVE) of the composite used in the finite element method (FEM) computations has a single centrally located particle, which is surrounded by an interphase of constant thickness, and they are both inserted into the matrix; this composition is shown in omposite a is madeA source of randomness in this work is the statistical dispersion of the volume fraction of interface defects echnique . In thislable in .Computations of the effective properties of the composite are made in the following order: firstly, a set of computations is completed in the FEM system Abaqus according to the boundary conditions shown schematically in on (MCS)  with 50,on (MCS) ,40. CharFinal diagrams of the effective stiffness tensor  Cijeff iThe expected values and the coefficients of variation for all the components and probabilistic methods are exclusively positive, skewness is predominantly negative and kurtosis is positive for the MCS and SAM, while negative for the ISFEM. All the coefficients, despite the expected values, increase in magnitude together with an increase in input uncertainty The expected values of the effective stiffness tensor  tension a and bia tension b are dec tension c slightlThe coefficients of variation of the effective stiffness tensor Skewness of the effective stiffness Kurtosis of all the effective stiffness tensor components is presented in 60 spherical fullerenes surrounded by the interphase defects. It is conducted in a hyper-elastic regime of this material, where the constitutive relation of the matrix has been adopted after initial laboratory tests. This work reports a successful determination of the random nonlinear cubic effective stiffness tensor The resulting effective stiffness tensor is sensitive to uncertainty in the volume fraction of the interface defects and it cannot have a Gaussian distribution regardless of the strain intensity applied. It has more than twice the lower uncertainty level than the input one and, unlike for the linear elastic case, its probabilistic characteristics highly depend upon the strain imposed on the RVE; this dependence has no evident pattern for the respective components and different probabilistic characteristics but it is basically monotonic. An influence of statistical scattering of the interface defects on the expected values of the effective stiffness tensor components is relatively small (limited to A coincidence of the three probabilistic methods is relatively high for the expectations There is no doubt that experimentally-based homogenization of particulate composites with stochastic interface defects brings new interesting results, so that further SFEM computational studies will concern numerical simulation of hysteresis of such a composite in the probabilistic context.


Metroxylon sagu) pith waste (SPW) with the assistance of ultrasound ensued by the transformation of extracted starch into a higher value-added bioplastic film. Sago starch with extraction yield of 71.4% was successfully obtained using the ultrasound-assisted extraction, with the following conditions: particle size <250 µm, solid loading of 10 wt.%, ultrasonic amplitude of 70% and duty cycle of 83% in 5 min. The rapid ultrasound approach was proven to be more effective than the conventional extraction with 60.9% extraction yield in 30 min. Ultrasound-extracted starch was found to exhibit higher starch purity than the control starch as indicated by the presence of lower protein and ash contents. The starch granules were found to have irregular and disrupted surfaces after ultrasonication. The disrupted starch granules reduced the particle size and increased the swelling power of starch which was beneficial in producing a film-forming solution. The ultrasound-extracted sago starch was subsequently used to prepare a bioplastic film via solution casting method. A brownish bioplastic film with tensile strength of 0.9 ± 0.1 MPa, Young’s modulus of 22 ± 0.8 MPa, elongation at break of 13.6 ± 2.0% and water vapour permeability (WVP) of 1.11 ± 0.1 × 10−8 g m−1 s−1 Pa−1 was obtained, suggesting its feasibility as bioplastic material. These findings provide a means of utilization for SPW which is in line with the contemporary trend towards greener and sustainable products and processes.The present study was conducted to optimize the extraction yield of starch from sago ( Generally, starch extraction methods can be categorized into chemical and physical techniques. Acid hydrolysis ,2 and enMetroxylon sagu) pith waste (SPW) can be extracted and further converted into higher value-added product. SPW represents an abundant source of agro-industrial waste which is derived from sago starch processing with starch content ranges from 60–80% [Similar to the starchy crops mentioned above, the residual starch present in the sago  towards the extraction yield of starch from SPW. The investigations of these influencing factors are essential to understand their relationship in obtaining the optimum extraction yield within the shortest duration and minimal use of renewable resources. The effectiveness of the ultrasound technique was subsequently compared with the conventional method in terms of extraction yield, extraction efficiency and chemical composition. The granule structure of the starch extracted from both methods was characterized in detailed by scanning electron microscopy (SEM), particle size distribution, X-ray diffraction (XRD), solubility and swelling power. Subsequently, the ultrasound-extracted starch was used to fabricate bioplastic film to validate its feasibility to be used as value-added product. The film properties were also compared with the bioplastic film fabricated using conventional method extracted starch to gauge its effectiveness.SPW was provided by Craun Research Sdn. Bhd., Kuching, Malaysia. Potato amylose (100% purity) was purchased from Sigma-Aldrich Co. . Ethanol (95% purity) from HmbG . Sodium hydroxide (99% purity), iodine (≥99.8%), potassium iodide (99.5% purity) and acetic acid (100% purity) were obtained from Merck . All the chemicals were used as received without further purification. Laboratory formulated distilled water was used throughout the experiments.Starch extraction from SPW was performed following the method of Pinyo et al.  with sliAmylose content of starch was determined according to the iodine colorimetric method of Babu and Parimalavalli . A beakeC is the sample concentration determined from the standard curve (mg/mL);DF is the dilution factor;V is the volume of the sample (mL);W is the weight of the sample (mg).Starch content was determined using the enzymatic colorimetric method according to AOAC 920.40 . A measuKjeldahl method was adopted to determine the nitrogen content of the sample which involved digestion, distillation and titration steps. One gram of the sample was digested with 30 mL of concentrated sulphuric acid in the presence of 2.0 g of catalyst mixture (potassium sulfate and selenium). The digested solution was subjected to acidic distillation in the presence of 25 mL of boric acid and 25 mL of distilled water. The distillate was neutralized with sodium hydroxide solution before titrating with sulphuric acid. The protein content was determined by multiplying the calculated nitrogen content with a factor of 6.25 .n-hexane as the extraction solvent for 6 h. The solvent was evaporated using a rotary vacuum evaporator after completion of the extraction. The fat content was determined from the weight of oil retained in the flask [One gram of the dried sample was extracted and refluxed in a Soxhlet extractor with he flask .Two grams of the sample was transferred to a pre-ashed crucible and was combusted over a low burner flame until no more flame was produced. It was then kept in a muffle furnace for overnight at 550 °C. The ash content was determined from the weight of the ash left in the crucible .Extraction yield was obtained by calculating the amount of dried starch recovered from SPW as expressed in Equation (2) .(2)ExtrAfter determined the optimum conditions, the weight of extracted pure starch was calculated by multiplying the starch content percentage with the weight of dried starch extracted from SPW. Extraction efficiency was calculated according to Equation (3) .(3)ExtrThe sago starch bioplastic film was prepared following the method of Agustin et al.  with sliThe surface morphology of the control and ultrasound-extracted starch samples was observed using a bench top scanning electron microscope . The samples were dried prior to imaging. A single layer of the starch samples was sprinkled evenly on a clean stub attached with double-sided tape without gold coating. The stubs were then placed in the SEM chamber and the images were captured at an accelerating voltage of 15 kV with magnification of 500× . The aveParticle size distribution and its cumulative curves of both control and ultrasound-extracted starch samples were determined using a laser particle size analyzer  equipped with a HydroMU sample dispersion unit  at 25 °C . Prior tThe diffraction patterns of the samples were recorded using an X-ray diffractometer . The diffractometer is equipped with a CuKα radiation operating at 45 kV and 30 mA. The diffracted intensity was measured from 3–60° as a function of Bragg angles (2θ) with the scanning speed of 1°/second. Starch crystallinity was calculated according to Equation (4) .(4)Crys0) and 20 mL distilled water were added into a 50 mL centrifuge tube. The tube was kept in a shaking water bath at 70 °C for 30 min. The suspension was then cooled to room temperature and centrifuged at 2000 rpm for 15 min. The supernatant was carefully decanted in a petri dish and dried in an oven to a constant weight (W1) at 103 °C for 2 h. The weight of the swollen granules (W2) was measured after decantation. The solubility and swelling power were calculated according to Equations (5) and (6), respectively.Solubility and swelling power of the control and ultrasound-extracted starch samples were determined according to the method of Sit et al.  with sliThe colour of the bioplastic film was determined using colour spectrophotometer  interfaced with computer operating Spectra Magic NX software . A white standard colour plate was used as the background for the colour measurements. The films were subjected to CIE-Lab system under illuminant D65 and observer 10° where L, a and b represent lightness, redness and yellowness. The total colour difference (tion (7) . The aveMechanical properties of bioplastic film such as tensile strength, Young’s Modulus and elongation at break were determined according to ASTM D882-02  using UnA bottle containing 2.0 g of fully dried calcium chloride (0% relative humidity) was sealed on top (11.5 mm in diameter) with the bioplastic film. It was then placed in a desiccator at room temperature of 25 °C with saturated sodium chloride solution (75% relative humidity). The changes in the weight of calcium chloride were recorded for every 1 h interval. The equilibrium state of saturation was reached after 24 h. The WVP of the film was calculated using Equation (8) . Three r2);p < 0.05) were accessed by one-way analysis of variance (ANOVA) with Tukey test using OriginPro 2018 software .The experiments were performed in triplicates and all the statistical analysis results were expressed as mean ± standard deviation. For results displayed on graph, standard deviation was indicated by an error bar. Significant differences between the mean values (p < 0.05). The highest extraction yield of 59.1 ± 3.0% was achieved at the smallest particle size of smaller than 250 µm. Shorter diffusion path and larger surface area possessed by smaller particle size would facilitate the mass transfer of the starch, thereby achieving higher extraction yield. Furthermore, reduction in the particle size would lead to an increase in the number of starch cells directly exposed to the solvent, thus improving the extraction yield [p < 0.05) in the extraction yield with the increasing particle size from smaller than 250 µm to larger than 1 mm. Particle size of more than 1 mm recorded the lowest extraction yield at 5.4 ± 0.1%. A similar trend was observed by Shirsath et al. [Curcuma amada using ultrasound by varying four different particle sizes . The highest (72%) and lowest (55%) extraction yields were achieved at the smallest (0.09 mm) and biggest (0.85 mm) particle sizes, respectively. In the present study, the weight distribution of SPW increased in the descending order of particle size. By grinding the SPW for 30 s and sieving them into different particle sizes, the weight distribution was as follows: 87.7% , 6.8% (250–500 µm), 4.6% (500 µm–1 mm) and 0.8% (>1 mm). Considering the extraction yield and the weight distribution for particle size smaller than 250 µm were the highest while the weight loss was not very significant which was only approximately 12%, particle size of smaller than 250 µm was selected for the subsequent parameter optimization.Particle size plays an important role in the ultrasound-assisted starch extraction from SPW. Reducing the particle size of the starch increases the number of cells directly exposed to the extraction solvent and the ultrasound cavitation, thus enhancing the extraction yield. This effect could be achieved by grinding the biomass before extraction . Energy on yield ,43. On ton yield . The resp > 0.05). Theoretically, particle collision frequency would increase with higher solid loading with the aid of ultrasound [p < 0.05). This can be explained by the saturation of the solvent beyond the optimum solid loading. The decreasing concentration gradient between the interior starch and the external solvent would lower the mass transfer rate from the solid matrix to the solvent and thus hindered the extraction yield. In addition, different solid loadings will also affect the concentration difference between the inner cells of sago starch and the solvent (water) which subsequently resulting in different viscosities of starch slurry [Curcuma longa. The extraction yield reduced from 86.7–31.3% when the solid loading was increased from 5–20% (w/v). At a solid loading of 12.5 wt.%, the amount of starch extracted was 1.2 times higher than that of 10 wt.% solid loading despite its extraction yield was slightly lower (57.9 ± 1.2% vs. 59.1 ± 3.0%). However, more water washing during slurry filtration and longer filtration time were required at solid loading of 12.5 wt.%. Similar extraction yields were also obtained at solid loadings of 5 and 10 wt.%. From the economical point of view, a solid loading of 10 wt.% was chosen for the subsequent parameter study since the final amount of starch produced was doubled compared to the 5 wt.% solid loading.The effect of solid loading on extraction yield was investigated over the range of 5–15 wt.% as depicted in trasound . This entrasound . The exth slurry . Increash slurry ,44,46. Th slurry  in the up > 0.05) and from 60.9 ± 1.1% to 66.1 ± 0.5% (p > 0.05) for ultrasonic amplitudes of 50 and 60%, respectively. These results could be related to the dominating effects of both cavitation and thermal effects with increment in sonication duration. Both the cavitation effect and thermal effect are crucial in ultrasound-assisted extraction. The cavitation effect will aid in the imploding of cavitation bubbles while thermal effect will help in the swelling and loosening of the cell structures. Their synergistic effects contribute to the augmented mass transfer of intracellular substances into the solvent [p > 0.05) at ultrasonic amplitudes of 50 and 60%.Changes in the extraction yield of starch from the SPW at different sonication durations and ultrasonic amplitudes were investigated on particle size of less than 250 µm, solid loading of 10 wt.% and duty cycle of 71.4%. Increment of the sonication duration had enhanced the extraction yield at constant ultrasonic amplitude as shown in  solvent . The cav solvent ,45,48. I solvent ,49. Nonep < 0.05). The highest extraction yield of 68.2 ± 0.7% was attained at an ultrasonic amplitude of 70% with sonication duration of 5 min. When the sonication duration was further increased to 15 min, the extraction yield dropped significantly to 41.7 ± 3.3% (p < 0.05). The decrease in the extraction yield could be related to the negative thermal effect with prolong sonication duration. Heat generated from the frequent asymmetric collapse of cavitation bubbles could increase the temperature to 66 °C at an ultrasonic amplitude of 70% with sonication time of 15 min. This temperature had exceeded the starch gelatinization temperature of 64 °C, causing some starch in the suspension started to gelatinize. As a result, some starch granules were trapped in the high viscosity suspension after extraction and could not be separated easily [Conversely, prolonged sonication duration could undermine the extraction yield at high ultrasonic amplitude. This was reflected by the significant decrement in extraction yield at the ultrasonic amplitude of 70% when the sonication duration was increased from 5–15 min  and 60% (p < 0.05) which can be explained by the aforementioned cavitation and thermal effects. Sit et al. [On the other hand, extraction yield increased with an increase in the ultrasonic amplitude at constant ultrasonic duration. This was because more energy could be transmitted for the cavitation event to occur and thus, cavitation bubbles generated at higher ultrasonic amplitude could collapse at higher intensity, thereby enabling more cracks and fissures to be formed on the starch surface. Impingement by high-speed jets also caused surface erosion and particle fragmentation simultaneously, leading to more channels and spaces for solvent diffusion. When solvent diffused into the particles easily, it facilitated the starch to be released into the solvent ,43,52. Nt et al.  also repp < 0.05). Pulse inflection of the ultrasound suppressed the formation of degassing bubbles and aided in the clearance of the cavitation area and intensified the sonochemical reactions [p > 0.05) difference in the extraction yield between duty cycles of 83–100%. It had been observed that similar extraction yield of around 71% was achieved for duty cycles of 83 and 100% in pulse and continuous mode, respectively. This might be due to the volumetric oscillation in continuous mode which had restricted the cavitation formation. The transmission of ultrasound might be curtailed by the cloud of degassing bubbles through absorption and scattering of the sound waves. The bubbles would then undergo intensive compression and expansion and restrict the sonochemical reaction [Spirulina platensis and antioxidants from pomegranate peel, respectively. Continuous mode usually requires shorter sonication time than pulse mode. However, it could accelerate tip erosion and would not be as energy efficient as the pulse mode [Duty cycle is referred as the percentage of ultrasound effective working time to the total ultrasound working time and idling time . It deteeactions . Higher eactions . Howeverreaction ,57. The reaction  as well reaction  where silse mode . TherefoRadix Puerariae was reported by Li et al. [To clearly demonstrate the benefits of the ultrasound technique over the conventional method, their extraction yields were compared under the same optimized conditions of particle size smaller than 250 µm, solid loading of 10 wt.% and temperature of 46 °C (temperature auto-generated at ultrasonic amplitude of 70% with duty cycle of 83%). The extraction efficiency at 89.7% was determined based on the total starch present in the SPW. From i et al.  with an Radix Puerariae conducted by Li et al. [2 as compared to lower power intensity of 20.38 kW/m2 used in Li et al. [Contrary to the lower extraction yield and efficiency, the chemical composition of the extracted starch from the conventional approach recorded higher amylose content than the ultrasound technique (37.4 ± 0.0% vs. 32.1 ± 0.0%). The longer extraction time (30 min) required in the conventional approach may cause more starch granules to swell and thus more amylose would be leached out . In addii et al. , the stai et al. . The higp < 0.05) and ash contents (0.7 ± 0.1% vs. 0.9 ± 0.0%) (p < 0.05) than the control starch, while fat content was not detected in either starches. Lower protein content in the ultrasound-extracted starch could be due to the bond disruptions between protein and starch by ultrasound which eased the starch-protein separation [paration . Park etparation  and Zhanparation  also obtparation . In the The comparison of the optimum reaction conditions between this study and the reported literatures is presented in SEM images of the control starch extracted conventionally and the ultrasound-extracted starches are displayed in After ultrasound-assisted extraction, the starch granules exhibited uneven, disrupted and irregular surfaces with cracks and fissures as indicated by the orange square shapes in The particle size distribution and the cumulative particle size distribution curves of control and ultrasound-extracted starches that expressed in volume basis are presented in The XRD patterns of control and ultrasound-extracted starches are displayed in Amylose content is also associated with swelling power and solubility of starch in which lower amylose content would have higher swelling power and solubility . The ultThe colour, mechanical and barrier properties of the bioplastic films prepared using control and ultrasound-extracted starches were evaluated in L and b values than that using control starch (p < 0.05). These results indicated that the film fabricated using ultrasound-extracted starch was darker and less yellowish. This might be caused by the presence of impurities along with the extracted starch after ultrasonication [The photographs of bioplastic films fabricated using control and ultrasound-extracted starches are shown in nication .p > 0.05) as shown in Both films prepared using control and ultrasound-extracted starches had comparable mechanical properties (−8 g m−1 s−1 Pa−1 (p > 0.05), respectively. Slightly lower WVP of film fabricated using ultrasound-extracted starch could be attributed to smaller particle size of starch in producing a film with a uniform matrix. This would then reduce the interstitial spaces between the polymer structure and the diffusion rate of water molecules [−10 g m−1 s−1 Pa−1 which was lower than that of both films in the present study. This could be due to higher glycerol loading used in the present study (40 vs. 20 wt.%) as compared to their study. Addition of glycerol which is more hydrophilic would increase the number of free hydroxyl groups and enhance the interaction with water molecules, making the film more favourable to adsorption and desorption of water molecules, thus increasing the WVP of films [Both the bioplastic films fabricated using control and ultrasound-extracted starches had comparable WVP of 1.13 ± 0.4 and 1.11 ± 0.1 × 10olecules . In the olecules , the perof films .Ultrasound is a feasible method to extract starch from sago pith waste (SPW). It is not only capable of eliminating mass transfer limitations but also eradicating the needs of separate heating and agitation due to the localized temperature increment and the formation of micro jets. The extraction technique outshines conventional extraction with 17.2% higher extraction yield and much shorter sonication duration. Combination of ultrasound extraction and utilization of low-cost SPW rendered the process to be economically feasible and potentially sustainable for production of bioplastic film.−8 g m−1 s−1 Pa−1 were attained which had further confirmed that SPW could serve as an alternative starch source in the bioplastic film industries. Starch extracted from SPW would generate added-value product and close its life cycle and encourage circular economy.From the tensile and WVP tests of the bioplastic film fabricated using starch extracted from SPW, tensile strength of 0.9 MPa and WVP of 1.11 × 10Further improvement in the mechanical properties and WVP of bioplastic film could be achieved by incorporating different types of plasticizer or additives. Future studies could also be directed towards the investigation of factors such as ultrasonic tip placement, size and shape of the sono-reactor that would affect the efficiency of ultrasound, for instance, intensity and energy density. In addition, kinetic modelling of the ultrasound-assisted extraction process could be conducted to determine the proportion of acoustic energy transferred to the medium and thus designing a more efficient process. Furthermore, the possible starch gelatinization effect for large-scale ultrasonication extraction system also requires more studies in the future. This information would be beneficial for scaling the process to the industrial level.


FCGR3A-V/F158 was earlier suggested to determine the potential of donor-specific HLA antibodies to trigger microcirculation inflammation, a key lesion of antibody-mediated renal allograft rejection. Associations with long-term transplant outcomes, however, have not been evaluated to date. To clarify the impact of FCGR3A-V/F158 polymorphism on kidney transplant survival, we genotyped a cohort of 1,940 recipient/donor pairs. Analyzing 10-year death-censored allograft survival, we found no significant differences in relation to FCGR3A-V/F158. There was also no independent survival effect in a multivariable Cox model. Similarly, functional polymorphisms in two other activating FcγR, FCGR2A-H/R131 (FcγRIIA) and FCGR3B-NA1/NA2 (FcγRIIIB), were not associated with outcome. There were also no significant survival differences among patient subgroups at increased risk of rejection-related injury, such as pre-sensitized recipients  or recipients treated for rejection within the first year after transplantation (n = 229). Our study results suggest that the earlier shown association of FcγR polymorphism with microcirculation inflammation may not be strong enough to exert a meaningful effect on graft survival.The functional Fc gamma receptor (FcγR) IIIA polymorphism  The latter includes the binding of the antibody’s Fc domain to Fc receptors on a variety of different immune cells, such as natural killer (NK) cells, which are suspected to play a key role in rejection (via activating Fc gamma receptors (FcγR) may initiate a variety of effector mechanisms, such as immune cell recruitment and activation, production and secretion of reactive oxygen species, cytotoxins and cytokines, all of which may contribute to allograft injury (Antibody-mediated rejection (ABMR) is a major obstacle to long time transplant survival , 2. Alloantation –5. Donorejection , 7. Signt injury .FCGR3A-V/F158) and is associated with decreased immune cell activation and altered binding characteristics to IgG1 and IgG3 (FCGR2A-H/R131), which is associated with decreased IgG2 binding and Fc-mediated phagocytosis  in the coding sequence of FcγRIIIA, which is expressed on the surface of monocytes/macrophages and natural killer (NK) cells, encodes a valine (V) to phenylalanine (F) amino acid substitution at position 158 , a characteristic feature of ABMR. In contrast, no such associations were found for functional variants of FcγRIIA and FcγRIIIB. There were no associations with renal functional course or graft loss rate, however, the study was not powered to detect subtle differences in renal allograft outcomes . AnalyziConsidering the presumed critical role of FcγIIIA-triggered NK cell activation in ABMR and the recent observation of an association of FcγIIIA polymorphisms with the extent of DSA-triggered microcirculation inflammation, we hypothesized a potential impact of FcγRIIIA functionality on clinically relevant endpoints. Our present study was primarily designed to clarify the impact of functional polymorphisms of low affinity Fcγ receptor FcγRIIIA  on long-term graft survival in a large cohort study powered to detect meaningful outcome differences. The study included approximately 2,000 donor-recipient pairs derived from the international Collaborative Transplant Study (CTS). Included patients were transplanted during a period from 1988 to 2006, which allowed for a long-term clinical follow-up of 10 years.www.ctstransplant.org). FcγR genotyping  and statistical data analysis (University of Heidelberg) were carried out in a blinded fashion.To assess the impact of functional FcγR polymorphisms on long-term transplant survival we analyzed a large cohort derived from the Collaborative Transplant Study . The present retrospective genotyping study was approved under the application number 083/2005 of the ethics committee of the University of Heidelberg.The criteria for inclusion of recipient and donor pairs were: (1) deceased donor kidney transplantation, (2) transplantation between 1988 and 2006 in Europe or Northern America; (3) single-organ transplantation; (4) Caucasian recipient and donor; (5) complete database record of original disease, recipient and donor age, graft number, or HLA typing; and (6) known immunosuppression scheme (intention to treat). Initially, 2,070 recipient-donor pairs were randomly chosen from the DNA bank of the CTS study. For 3,880 of the selected samples, sufficient DNA was available, and complete Fc receptor genotyping was possible for both recipient and donor, resulting in a total of 1,940 transplants from 52 transplant centers in 13 countries, which were finally included. There was no pairing of one donor with two recipients. As shown in FCGR3A-V/F158 (denoted as rs396991) and FCGR2A-H/R131 (rs1801274) and one intron polymorphism associated with FCGR3B-NA1/NA2 (rs35139848) was carried out in 384-well optical plates on a 7900HT fast real-time PCR system  using TaqMan® SNP Genotyping Assay and TaqMan® Universal PCR Master Mix (Fisher Scientific Austria). According to a previously published protocol, two TaqMan® MGB probes were used for each genotyping assay. The first probe detecting allele 1  was labelled with VIC® dye, and the second with FAM™ labelled probe was detecting allele 2  , and categorical variables as absolute and relative frequencies. Chi-quadrat tests were used to compare categorical data. Kruskal-Wallis tests were applied for continuous variables. For all genotype-related statistical analyses patient numbers were high enough to divide homozygotes and heterozygotes into three groups; combining a low-numbered homozygous group with heterozygotes was not necessary. Kaplan-Meier analysis was used for calculation of 10-year graft survival. The Mantel-Cox log-rank test was applied for the comparison of survival between groups. Multivariable Cox regression analyses with the variables year of transplantation, geographical region (continent), first or re-transplant, recipient and donor sex and age, cold ischemia time, number of HLA A+B+DR mismatches, preformed panel-reactive antibodies, original disease leading to end stage renal failure, and immunosuppressive therapy (intention to treat) was performed to eliminate the influence of possible confounders. Hazard ratios (HR) are presented with 95% confidence intervals (CI). A two-sided P value less than 0.05 was considered statistically significant. For statistical analysis IBM SPSS Statistics  was used. The R package “hwde” was used for exact testing of Hardy-Weinberg equilibrium .FCGR3A-V/F158 (denoted as rs396991), FCGR2A-H/R131 (rs1801274) and FCGR3B-NA1/NA2 (rs35139848), respectively. Baseline characteristics of the overall cohort stratified by FcγR genotypes are provided in FCGR3A-V/F158 locus was 64.0%, that of the donor 64.3%, and frequencies of variant H131 of the FCGR2A-H/R131 polymorphism was 53.7% and 52.7% (donors). The allele frequency of the NA2 variant of the FCGR3B locus was 64.5% in recipients and 65.4% in donors. Genotype frequencies did not deviate from Hardy-Weinberg equilibrium , first or re-transplant, recipient and donor sex and age, cold ischemia time, number of HLA A+B+DR mismatches, pre-formed panel-reactive antibodies, original disease leading to end stage renal failure, and immunosuppressive therapy (intention to treat). The hazard ratio (HR) for death-censored graft loss during the first 10 years post-transplant was with 1.09  for heterozygous V/F158 recipients and 0.84  for homozygous V/V158 recipients not significantly different from the homozygous reference group of F/F158 recipients. Furthemore, the FCGR2A-H/R131 and FCGR3B-NA1/NA2 genotypes did not reveal any impact on death-censored graft survival  at the time of transplantation. Despite their undefined DSA status, such patients are known to have a higher risk of developing ABMR and death-censored graft survival . In KaplFurther sub-analyses focused on 229 patients who were treated for rejection during the first year. Again, there were no statistically significant associations of FcγRIIIA, FcγRIIA and FcγRIIIB polymorphisms with death-censored 10-year graft survival  had a greater extent of ptc than homozygous carriers of the low-affinity allele F158  upon incubation with anti-HLA antibody-spiked T cells than the same cell line transfected with the homozygous F158 variant  (but not ptc) and with inferior 3-year graft survival after diagnosis. Yuan et al. and Ozkayin et al. reported a significant shift towards a higher allele frequency of FcγRIIA R131 in acute rejectors  actually had DSA-triggered ABMR, and a majority of them may have had TCMR without any preformed or We have found previously an interrelation between high-affinity FcγRIIIA V158 and ptc in a cohort of 85 DSA-positive long-time transplanted patients , however, we were not able to demonstrate an impact of FcγRIIIA V/F158 polymorphism (or of any of the other function-defining polymorphisms in the other two receptors) on 2-year allograft survival in the same study .In summary of this present register study we confirmed this previous finding and found no impact of FcγRIIIA V/F158 polymorphism (or of any of the other function-defining polymorphisms in the other two receptors) on 10-year allograft survival, neither in the large cohort of 1,940 recipients nor in our two subanalysis cohorts of 438 pre-transplant anti-HLA antibody-positive patients and 229 patients with rejection treatment during the first post-transplant year.Based on our current results, we believe that FcγR genotyping may have limited relevance for unselected transplant cohorts. Future studies, however, will have to clarify its usefulness for risk stratification in selected cohorts of patients, e.g. pre- or post-transplant DSA-positive subjects who are at particular risk of ABMR.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Ethics Committee of the University of Heidelberg. The patients/participants provided their written informed consent to participate in this study.Conceptualization, MW, BD, GB and CS. Methodology, M-LA, HH. Formal analysis, MW, SH. Investigation, MW, KM. Resources, HH. Data curation, BD, SS, CS. Writing-original draft preparation, MW, GB. Writing-review and editing, BD, KM. Visualization, BD. Supervision, CS. Funding acquisition, GB. All authors contributed to the article and approved the submitted version.This research was funded by the Austrian National Bank (to GB), grant number 12471).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Corypha umbraculifera L.) was subjected to three different thermal treatments  prior to phosphorylation. Upon dual modification of starch with thermal treatments and phosphorylation, the phosphorous content and degree of crosslinking significantly increased (p ≤ 0.05) and was confirmed by the increased peak intensity of P=O and P–O–C stretching vibrations compared to phosphorylated talipot starch in the FT-IR spectrum. The highest degree of crosslinking (0.00418) was observed in the autoclave pretreated phosphorylated talipot starch sample. Thermal pretreatment remarkably changed the granule morphology by creating fissures and grooves. The amylose content and relative crystallinity of all phosphorylated talipot starches significantly decreased (p ≤ 0.05) due to crosslinking by the formation of phosphodiester bonds, reducing the swelling power of dual-modified starches. Among all modified starches, dry-heat pretreated phosphorylated starch gel showed an improved light transmittance value of 28.4%, indicating reduced retrogradation tendency. Pasting and rheological properties represented that the thermal pretreated phosphorylated starch formed stronger gels that improved thermal and shear resistance. Autoclave treatment before phosphorylation of talipot starch showed the highest resistant starch content of 48.08%.Talipot starch, a non-conventional starch source with a high yield (76%) from the stem pith of talipot palm ( Starch is a naturally occurring biopolymer with an extended application in food and non-food industries, owing to its technological properties, bioavailability, biodegradability, and safety. It is a well-favored, multifunctional and renewable material majorly sourced from cereals, seeds, roots, and tubers . Starch w/w at a temperature ranging from 80–140 °C for a duration of 1–24 h [Physical modification of starch is further classified into thermal and non-thermal modifications. Among thermal modifications, dry-heat treatment (DHT), heat-moisture treatment (HMT), annealing, and autoclave treatments are most commonly used for starch modification. DHT is a thermal treatment of starch at high temperatures 130–140 °C) for a duration of 1–20 h . DHT chaf 1–24 h . The sigf 1–24 h . HMT majf 1–24 h . Heat-mof 1–24 h . Autoclaf 1–24 h . The prof 1–24 h  and enhaf 1–24 h . Autocla0–140 °C Phosphorylation is an established food-grade chemical treatment employed for modifying starch of varied sources . PhosphoCorypha umbraculifera L.). Talipot palm is a large hapaxanthic palm from the family of Arecaceae that has the largest terminal paniculate inflorescence in the entire flora [From the literature thus far, it is evident that dual modification is observed to make remarkable alterations in starch characteristics compared to single modifications. For instance, dual modification of waxy maize starch with crosslinking and HMT is reported to change the physicochemical properties, in vitro digestibility and increase the RS content of modified starches . Besidesre flora . Talipotre flora . The obj3, >99% purity), potassium iodide, α-amylase from A. oryzae (∼30 U/mg), amyloglucosidase from A. niger (≥300 U/mL) were procured from Sigma Aldrich . Sodium hydroxide, hydrochloric acid, acetic acid, and iodine were purchased from Merck Ltd. . The chemicals availed for the study were of analytical grade, otherwise mentioned. The talipot flour was acquired from the stem pith of a matured talipot palm from Malappuram, South India, by following the method of Navaf et al.  and storTalipot starch was isolated by adhering to the procedure of Sudheesh et al.  with nec2SO4. The pH of the suspension was adjusted to 11.0 using 1 M NaOH and POCl3  was incorporated dropwise into the suspension. The starch was subjected to phosphorylation reaction for 1 h with continuous stirring at 200 rpm by maintaining the pH at 11.0. Later, the reaction was ceased by adjusting the pH of the slurry to 6.5 using 0.1 N HCl. The resultant mixture was centrifuged  thrice with DW and once with absolute ethanol. The obtained starch sediment was then dried overnight in a hot air oven at 40 °C and finely ground, sieved, and stored for analysis. The phosphorylated talipot starch was referred to as PTS.Talipot starch was phosphorylated by following the method of Hazarika and Sit  with necDHT of native talipot starch was performed by following the method of Maniglia et al. , where tHMT was performed by following the method of Marboh and Mahanta  with sliAutoclave treatment was performed according to the method described by Deka and Sit  with sliTalipot starch samples thermally treated by DHT, HMT, and autoclave treatment were then subjected to phosphorylation (as mentioned in 2.3). The resultant mixtures were centrifuged  thrice with DW and once with absolute ethanol. The obtained starch sediments were then dried overnight in a hot air oven at 40 °C and finely ground, sieved, and stored for further analysis. The thermal pretreated phosphorylated talipot starch by DHT, HMT, and autoclave treatment was referred to as DH-PTS, HM-PTS, and AC-PTS, respectively.The phosphorus content (P) of the native and modified starches were quantified according to the spectrophotometric method of Smith and Caruso , and theThe rapid colorimetric method of Williams et al.  was adopThe morphological attributes of native and modified talipot starch samples were determined using scanning electron microscopy (SEM) . Each sample was mounted over a stub using double-sided carbon conductivity tape and carbon-coated in an automated sputter coater . The coated talipot starch samples were viewed at 1500× magnification.The crystalline characteristics of native and modified talipot starch samples were analyzed under an X-ray diffractometer (XRD) . The samples were molded flat onto the cavity of a sample holder, and the data on scanning was obtained at an angle of diffraction (2θ) = 4° to 60°. The relative crystallinity (RC) of the starch granules was estimated with the formula:−1.FT-IR spectra of native and modified talipot starch samples were studied using an FT-IR spectrophotometer . Each starch sample was blended with optical grade KBr in a ratio of 1:100 and made into pellets using a pelletizer . The FT-IR analysis was performed in the scanning range, 400–4000 cmThe swelling power and solubility of starch samples were analyzed by following the procedure detailed elsewhere  and calcLight transmittance was found by employing the method described in Navaf et al.  at 640 nThe pasting properties were found with the help of Rapid Visco Analyzer . A thoroughly mixed 12% starch slurry of each sample was taken in a canister. The slurry was equilibrated for 1 min at 50 °C, heated for 3.5 min at 95 °C, and held for 2.5 min at 95 °C. The temperature was cooled down to 50 °C for 4 min and held at 1 min. The parameters of pasting temperature (PT), peak viscosity (PV), breakdown viscosity (BDV), final viscosity (FV), and setback viscosity (SBV) were analyzed.plusC, Stable Micro Systems, Surrey, UK). The gelatinized starch samples in canisters after RVA analysis was stored at 20 ± 2 °C for 24 h by covering with parafilm. The starch gels were punctured at 1.0 mm/s to 30 mm distance using a stainless-steel punch probe (p/5 probe of 5 mm diameter). Textural properties such as hardness, adhesiveness, springiness, cohesiveness, and gumminess were determined from the curve between force and time. Textural properties of native and modified talipot starch gels were obtained using texture analyzer (TA.HDThe storage (G′) and loss (G″) moduli and loss factor (tan δ) of native and modified talipot starch gels were analyzed with the method described in Sudheesh et al.  at 25 °CRapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS) were determined by following the method of Englyst et al. .p ≤ 0.05 of the starch samples, NTS, PTS, DHTS, HMTS, ACTS, DH-PTS, HM-PTS and AC-PTS. The data from each experiment were analyzed with one-way analysis of variance (ANOVA) and Duncan’s multiple range test (DMRT) employing the software, IBM SPSS Statistics 23 .The data reported were the average of triplicate observations and conveyed as mean ± standard deviation (SD), at a significant difference of p > 0.05) change the P of talipot starches. Upon phosphorylation with POCl3, the P content of talipot starch increased from 0.018% to 0.058%. The introduction of orthophosphate groups into starch structure upon crosslinking increased the P content. Besides, when talipot starches were subjected to thermal pretreatment prior to phosphorylation, the P content and DC significantly increased (p ≤ 0.05) compared to P content and DC of PTS sample, where the highest P content (0.080%) and DC (0.00418) was shown by AC-PTS sample (Phosphorus content (P) is determined for interpreting the degree of crosslinking (DC) upon phosphorylation with any crosslinking agent . The phoS sample . The reap ≤ 0.05) upon modifications, except for ACTS (3 exhibited a similar decrease in amylose content [p ≤ 0.05) amylose content than PTS, where DH-PTS showed the lowest amylose content (19.26%). High-temperature treatment followed by phosphorylation introduced phosphodiester bonds and enhanced interaction between amylose-amylopectin, making amylose more insoluble and inaccessible for binding with iodine during quantification [Amylose renders a prime role in governing starch gelling and pasting properties. NTS possessed a higher amylose content of 28.81%, and it significantly decreased  in RC upon phosphorylation treatment  exhibited a significant decrease (p ≤ 0.05) in RC. This might be ascribed by the substitution of some hydroxyl groups with phosphate group during phosphorylation [p ≥ 0.05) the RC of DHTS, HMTS and ACTS. The decrease in the RC of DHTS may be attributed to the degradation of crystalline regions of starch, partial gelatinization of starch granules, and double helical movement during DHT [p ≤ 0.05) compared to PTS, and the lowest RC (10.60%) was observed in the DH-PTS sample. This decrease is due to enhanced degradation of starch crystallites or alteration in the orientation of crystallites [The RC of NTS was observed to be 16.97%, and PTS showed an insignificant decrease , where the least swelling power was observed in the AC-PTS sample. In the PTS sample, the reduction in swelling power might be attributed to the strengthened intermolecular interaction by the introduction of phosphodiester bonds upon crosslinking. The addition of phosphate groups thus tightly held the starch granules and formed a gelatinized mass that inhibited swelling even at higher temperatures [The interaction of the amorphous and crystalline region of the starch chain with water molecules is indicated by swelling power and solubility. The swelling power of native and modified talipot starches increased with the increase in temperature from 60 to 90 °C a. The aueratures . Phosphoeratures ,20.p ≤ 0.05) than NTS. The reduced swelling power of DHTS was attributed to the extent association between starch chains, which caused further reduction in the diffusion of amylopectin molecules [w/w)) represented a similar decrease in swelling power [The swelling power of thermally modified samples was significantly lower , with an increase in temperature from 60 to 90 °C (p ≤ 0.05) upon phosphorylation owing to the restricted leach out of starch chains due to the formation of phosphodiester bonds. Such formed crosslinks protected the structure of starch granules and hindered the disintegration and leaching out of starch molecules [p ≤ 0.05) than PTS. The decrease was due to the formation of crosslinks between starch chains that helps in the stabilization of readily soluble amylopectin presented in the amorphous regions [3 and dual modified by crosslinking and organic acids [p ≤ 0.05) than PTS. The increase in DH-PTS solubility might be attributed to the damage in the amorphous region as well as in the granule surface that led to amylose leaching and increased starch solubility during DHT [Starch solubility is associated with the amylose leaching from the granules, followed by maximum swelling. The solubility of native and modified talipot starch samples showed a significant increase  and HMT and autoclave treatment significantly decreased (p ≤ 0.05) the light transmittance of talipot starches. The lower light transmittance value of hydrothermally modified starches was attributed to the increase in flexibility of starch chains within the amorphous region of granules [p ≤ 0.05) than NTS. Upon phosphorylation, crosslinks formed after gelatinization changed the granular structure and made them more intact, restricted the light transmittance, and resulted in lower paste clarity [p ≤ 0.05) than PTS, and at the end of 168 h of storage, the light transmittance of DH-PTS showed a significant increase (p ≤ 0.05) compared to NTS. The augmentation in the paste clarity by pretreatment with DHT can be ascribed to the reduction in the amount of swollen starch granules followed by the gelling and fragmentation of amylopectin fractions. The double-helical structure and interaction of starch molecules were weakened upon DHT compared to native starch granules [Higher starch paste clarity is an important characteristic in the preparation of certain products such as fruit pastes, jellies, and pie fillings that require transparency, and it is indicated by a higher light transmittance value . The liggranules . The pro clarity . Moreovegranules . This ingranules ,36.p ≤ 0.05) than NTS, except for the DH-PTS sample. Higher PT is due to the development of crosslinks, which provides strong intermolecular associative forces and increases the structural stability of PTS [RVA profile depicting the pasting properties of native and modified talipot starch samples are shown in y of PTS . The incy of PTS . Howevery of PTS .p ≤ 0.05), whereas the DHTS and DH-PTS showed a significant decrease (p ≤ 0.05) compared to NTS. The increase in PV of PTS is due to the higher integration of starch granules. Modification of starch by phosphorylation enhanced the resistance of granules against disintegration by increasing the intra- and inter-bonding between starch molecules, which resulted in higher viscosity [Peak viscosity (PV) is the maximum viscosity shown at the equilibrium point between swelling and polymer leaching due to increased temperature, and break down viscosity (BDV) represents the starch granules’ stability against shear force and temperature. The PV and BDV of NTS were observed to be 3646 and 1881 cP. The PV of PTS, HMTS, ACTS, HM-PTS and AC-PTS showed a significant increase (iscosity . The PV iscosity . Besidesiscosity . Lower Pp ≤ 0.05) versus NTS, except for DM-PTS  indicates the increase in starch viscosity because of aggregation of amylose molecules on cooling, and setback viscosity (SBV) determines the gelation or retrogradation capacity of starch . The FV r DM-PTS . The decr DM-PTS . High FVp ≤ 0.05) from NTS. Gel hardness has been reported to be dependent on the binding capacity of starch molecules to water by hydrogen bonding [The textural parameters of native and modified talipot starches are presented in  bonding . Adhesiv bonding . The har bonding .The improved gel properties in DHTS might be due to the molecular depolymerization promoted by DHT that enhances the reassociation and packing of starch chains, which helps in developing a stronger three-dimensional network structure . In thisThe rheological properties dictate the flow and deformation behavior of starch under stress. The rheological properties of the starch gels are crucial to determine the processability and eating quality of starch-based food products. The G′ and G″ of native and modified talipot starch samples over an angular frequency (ω) of 1–100 rad/s are depicted in Tan δ value is the ratio of G″ and G′, where tan δ value > 1 implies the viscous behavior of starch gels, and tan δ value < 1 implies the elastic behavior of starch gel. The tan δ values of NTS, crosslinked and thermally modified talipot starches, were below unity b. This ip ≤ 0.05) for talipot starch. The RS and SDS content increased to 44.37% and 35.01% upon crosslinking with POCl3, owing to the restriction of swelling by the starch granules and enhanced resistance to digestion by α-amylases. Crosslinked waxy maize starch with STMP/STPP, 99:1  showed a consistent increase in RS content [p ≤ 0.05) in RDS content versus PTS. This might be due to the partial disruption of organized chain structures by DHT, which could promote the attack of the enzymes into the interiors of starch granules [Based on gastrointestinal absorption, starch is classified into RDS, SDS, and RS. The NTS after hydrolysis showed the RDS, SDS, and RS as 29.44%, 33.85%, and 36.71%, respectively. RS possesses varied physiological benefits by acting as a dietary fiber and reduces the risk of many degenerative diseases. From  content . The DHTp ≤ 0.05) in the RS content of AC-PTS is due to the combined effect of thermal force and pressure given by autoclave treatment and phosphodiester bond crosslinks. Upon hydrothermal treatments, the tight structure in the amorphous regions formed by starch granule expansion might have reduced enzyme susceptibility in AC-PTS for in vitro digestion [HMTS and ACTS showed an increased percentage of RS  compared to NTS by partial hydrolysis. RS3 is a type of RS formed via retrogradation mechanism by intentional modification or processing. The effect of hydrothermal treatment on in vitro digestibility is governed by starch source, moisture content during treatment, treatment time and temperature, and interactions between amylose–amylose, amylose–amylopectin, amylopectin–amylopectin interaction, and amylose–lipid interactions . Howeverigestion . Hence, igestion .p ≤ 0.05) than PTS. This increase in P content and DC is in agreement with the increased peak intensity of 1160 and 1010 cm−1 representing P=O and P–O–C stretching vibrations upon phosphorylation. The swelling power of PTS, DH-PTS, HM-PTS and AC-PTS was significantly lower than (p ≤ 0.05) NTS, due to reduced amylose content and relative crystallinity by the introduction of phosphodiester bonds upon crosslinking. The increased light transmittance of NTS and DH-PTS suggested its application in the preparation of soups, jellies, and pie fillings that requires higher paste clarity. The increased PT, PV and FV of HM-PTS and AC-PTS indicate the development of intermolecular crosslinks and reduced space between the molecules by fortified associative forces in starch chains. Such starches are suitable for application, which uses high-temperature processing or requires higher viscosity in the final products. The increased G′ and G″ values of DH-PTS, HM-PTS and AC-PTS depict that stronger gels are formed upon dual modification that is stable to shearing and heating. In vitro digestibility significantly increased (p ≤ 0.05) with DH-PTS sample and significantly decreased (p ≤ 0.05) with PTS, HM-PTS and AC-PTS. The improved RS content of HM-PTS and AC-PTS suggests its application in the preparation of low glycemic food products. Talipot starch crosslinked with phosphorylation with three different thermal modifications as pretreatments distinctly changed starch properties and indicated their functionality in varied food applications.Talipot starch modified by phosphorylation alone and phosphorylation and thermal pretreatments remarkably changed the starch granule characteristics and starch paste functionality compared to NTS. The phosphorylation modification in talipot starch was confirmed by the increased P content and DC of PTS versus NTS, and the P content and DC of thermal pretreated phosphorylated starches was significantly greater (


Small and medium enterprises (SMEs) in the midstream  segments of transforming value chains have proliferated rapidly over the past several decades in Africa, Asia, and Latin America. Their spread has been most rapid in the long transitional stage between the traditional and modern stages, when value chains grow long and developed with urbanization but are still fragmented, before consolidation. Most of Sub-Saharan Africa and South Asia, and parts of the other regions, are in that stage. The midstream SMEs in output and input value chains are important to overall food security (moving about 65% of food consumed in Africa and South Asia), and to employment, farmers, poor consumers, and the environment. The midstream of value chains is neglected in the national and international debates as the “missing middle.” We found that it is indeed not missing but rather hidden from the debate, hence “the hidden middle.” The midstream SMEs grow quickly and succeed where enabling conditions are present. Our main policy recommendations are to support the SMEs further growth through a focus on infrastructure investment, in particular on wholesale markets and roads, a reduction of policy-related constraints such as excessive red tape, and regulation for food safety and good commercial practices. Agrifood value chains (AVCs) have been growing and transforming rapidly over the past three decades in developing regions. The transformation has been “pulled” by urbanization, liberalization, privatization, and income growth, and facilitated by investments of millions of small and medium enterprises (SMEs)The midstream SMEs are crucial to food security, defined by FAO  as incluFrom observation of the national and international policy debates and the research literature we believe that food security policy debates neglect midstream SMEs despite their importance to food security. We focus on these SMEs to help redress that neglect. When midstream SMEs are evoked in international and national policy debates, they are often referred to as stagnant, traditional, severely constrained, part of a “missing middle.” Instead, we find evidence of rapid proliferation and dynamism of midstream SMEs and counter the term “missing middle” with our term the “hidden middle.” The latter indicates that the vibrant midstream segments of SMEs exist but have been relatively neglected in or hidden from the debate. We also contend that midstream SMEs do not need inventing, as that would be “reinventing the wheel;” rather, their further development should be encouraged and their constraints diminished so that they can grow faster and further yet.In this paper, in Sect. We draw on two sources of information: 1) we review other authors’ survey-based studies of the midstream SME sector; (2) we summarize findings from our own extensive primary surveys on midstream SMEs (and farm households and retailers interacting with them) in Asia and Africa in the past 15 years. We call these “stacked surveys.” The latter comprised a set or “stack” of surveys of representative samples of actors in each segment of the AVC of a product. Table  we revieThere has been a lot of variation in the timing of take-off and speed of transformation of AVCs across products, regions, countries within regions, and zones within countries. In general, the transformation is over three stages of change in structure and conduct. We summarize here from Reardon et al.  and RearThe “traditional” stage of AVC transformation is no longer common in Africa, Asia, and Latin America, and persists mainly in hinterland areas. AVCs in this stage tend to: (1) be spatially short ; (2) be fragmented in structure ; (3) use technologies intensive in labor but with little capital; (4) have exchange arrangements characterized by “spot markets,” that is, without contracts; (5) not have formal standards.The “transitional” stage is now strongly dominant in most of SSA and South Asia, but has reduced to half or less of the food economies of Southeast and East Asia and Latin America. The AVCs in this stage tend to: (1) be spatially long ; (2) be fragmented in structure, highly dominated by SMEs with only the bare emergence of LEs (large enterprises); (3) feature emerging disintermediation such as by urban wholesalers supplanting use of rural brokers with third-party logistics (3PLS) SMEs; (4) use mainly labor-intensive but increasingly capital-using technologies; (5) still mainly use “spot markets;” (6) have emerging public standards of quality (such as milk or fruit grades) in the emerging formal sector.The “modern” stage is now perhaps roughly at least half of the food economies of Southeast and East Asia and Latin America, and just emerging with perhaps about a fifth to a quarter of the food economies of Africa and South Asia. The AVCs in this stage tend to: (1) be spatially long; (2) be consolidating in various segments  and multi-nationalizing via FDI (foreign direct investment); (3) see a concomitant trend in the reduction of the share of SMEs in many segments as LEs outcompete them mainly due to economies of scale; (4) feature some “dis-intermediation” such as supermarkets buying directly from processors and processors from farmers; (5) feature capital intensification as the modern stage which tends to coincide with higher labor costs; (6) see the emergence of private standards and some use of contracts.A key driver of the diffusion and transformation of wholesale markets and wholesale SMEs has been urbanization of developing regions over the past five decades,The wholesale sector expanded rapidly over the past 50 years along with urbanization. Wholesaling expanded in and between rural areas and cities as AVCs lengthened geographically. It also expanded in volume to supply the rapidly growing food volumes demanded by cities  and rural brokers were dominant because the great majority of the population was rural. Emerging cities were supplied grain, animal products, and fruits and vegetables by peri-urban farmers and by rural brokers gathering in small “truck markets” .In the transitional stage, as cities started to grow rapidly, municipal and national governments worried about the ability of small, scattered truck markets to feed cities. They worried about urban food security’s reliance on informal grain traders, especially as then, governments and researchers saw traders as unreliable (given to speculation), exploitative, and having high marketing margins  markets4On the other hand, governments, especially in Asia, invested extensively in public wholesale markets in rural towns coupled with asphalting rural roads in the 1990s and 2000s. In China, Huang et al.  illustraWhere there were similar road and wholesale market investments in secondary and tertiary cities and rural towns in SSA, such as in Ethiopia, there was induced proliferation of wholesale and logistics SMEs , large aggregate investments by these SMEs in for example truck size increases, and subsequent reduction of marketing margins and transport costs despite the removal of fuel subsidies. Similar to China and Bangladesh, the development of these spontaneous clusters of wholesale and logistics SMEs induced farmers to upgrade to better varieties of teff, use more fertilizer, and market more  despite consolidation over wholesale markets  to fetch the produce from the farms. Evidence of this shift has been found in situations as diverse as maize trading in Nigeria  segment takes off as firms in the other segments outsource logistics rather than acquire and manage their own large trucks and warehouses. In the later modern stage, often LEs in the food industry invest in in-house capacity in logistics.3PLS SMEs have played two sets of roles supporting wholesale. On the one hand, 3PLS replaced the need for SME wholesalers to undertake the threshold investments of transport and storage. In Nigeria, maize wholesale markets were built in consumption zones in the South and production zones in the North and consumption cities in the North, creating a network of wholesale markets stretching 1000 km from North to South. In this network, thousands of wholesale SMEs move 75% of their maize via 3PLS trucking firms and obtain most of their storage by renting 3PLS warehouses  to multi-season availability and 65% of consumption from cold-storage potatoes, mainly from Agra. Rural brokers were sidelined as the cold storages themselves became the main locus of intermediation, with urban wholesalers coming  to buy potatoes from farmers at the storages.In the early modern stage, the share of wholesale markets in the overall wholesale sector gradually drops. For example, in Mexico there is evidence of a fall in wholesale market volumes in the first half of the 2000s. The estimates include a 25–30% drop in produce sales from the Mexico City wholesale market, and 40 and 50% reductions in the Guadalajara and Monterrey markets over 2002–2005  in developed countries where the share of the wholesale market has become minimal in retail and food service sourcing. The food industry firms thus reduce their transaction costs and outsource the enforcement of their private standards of quality and sometimes safety.Often one observes in food security debates a sharp distinction between “traditional” and “modern” enterprises. But abundant evidence is emerging that there is a category between these poles, an intermediate or transitional type of SME that has transformed and innovated beyond being traditional, but is not fully modern, and supplies local domestic markets; this point echoes this contention for SMEs in Reardon et al.  for riceIn the traditional setting, farmers had little rural nonfarm employment (RNFE) or other cash sources and so relied on wholesalers for advances. These “tied credit-output market” Bardhan,  arrangemHowever, there is substantial recent evidence that wholesalers’ provision of advances to farmers has declined substantially. Adjognon et al.  used farThe reasons given in these studies for the demise of the arrangement were that: (1) rural households had other sources of cash, especially RNFE ; (2) the improvement in roads and the proliferation of traders meant competition was keen and farmers “side sold” regardless of the tying. It is interesting that the exceptions to the demise include traders giving advances to large farmers, such as in Myanmar in aquaculture  or where they must reward farmers for practices that are costly in labor or capital, that the traders would have to do if they could not induce the farmers to do for them. An example is maize wholesalers in Nigeria paying farmers differential prices for dried (ready for milling) versus humid maize . The services fit the needs of the seller or buyer in the face of "idiosyncratic market constraints" that impede their clients from getting those services otherwise.Wholesale SMEs help small farmers indirectly by establishing resource-provision relations with farms that bring farmers services such as logistics and inputs and credit. These complementary services are usually associated with large companies in contract relations with farmers but in a scoping review of the literature, Liverpool-Tasie et al.  found th5In output value chains, wholesale and logistics SMEs help small farmers directly with resource-provision arrangements. This was illustrated in the cases discussed above of traders in Asia and Nigeria, as well as cold storages in India.In input and agricultural services value chains, wholesale and retail SMEs help small farmers as well. This is partly by solving the input access problem of farmers by bringing the fertilizer and seeds to farmers who do not have the time and means to go to towns to buy the inputs, such as in Nigeria . Its vertical axis is the degree of concentration in the processing sector. The traditional stage featured micro enterprises in a broad informal sector and large parastatals in a limited formal, urban sector. The middle, transitional stage  featured an expansion of the processing sector with a proliferation of SMEs. This led to a de-concentration of the processing sector and thus a dip in concentration relative to the traditional  stage. The modern stage (the right-most part of the J curve) featured competitive investment by foreign firms after FDI liberalization in the 1990s as well as competitive domestic investment which increased the share of food processing LEs, often at the expense of SMEs especially in categories like grains and edible oils where there are strong economies of scale in processing Reardon, .Overlaying the 50-year trend of de-concentration then re-concentration phases of the J curve, the processing segment transformed with respect to its technology and its degree of processing. This involved first the rise of first-stage processing, or minimal processing such as grain flour and milk powder and edible oils, and then the rise of second-stage processing such as breads and noodles and packaged sauces, and ultra-processed foods such as cookies and other confection and sweetened beverages. We explore the stages in more detail below.In the traditional stage, little processed food was purchased. The great majority of food was processed and prepared at home. Emerging late in the traditional stage are hammer mills (as occurred in Africa in the 1970s/1980s) ushering in the spread of custom milling of grain and edible oil. There was strong demand by women to use these custom mills to avoid the onerous hand pounding of grain and tubers  and on the supply side by the diffusion of processing equipment and technologies  FDI bought the South African FDI firm Blue Ribbon Foods to lead the handful of bakery firms dominating that sector in the 2010s . Our hypothesis runs contrary to the great attention paid in policy debates to setting up managed clusters; we contend that at least equal emphasis should be placed on supporting the “endogenous and indigenous” emergence of spontaneous clusters.There are a limited number of systematic surveys of wholesale and processing SMEs and few among those studies trace impacts on employment, farm incomes, the environment, and consumption. We use what evidence we found and illustrate pathways of impacts.To explore impacts on employment we draw on Dolislager et al.  which usFirst, what they refer to as "food systems employment"  is important in overall rural employment. It averages 20% of rural FTEs . That is only modestly below the share of own-farming in total rural FTEs, averaging 29% , and above the share of employment in farm wage labor, which averages only 9% of FTEs .Second, the food system employment impacts suggest inclusion of youth and women. (1) There is youth inclusion: in SSA, 24% of adults FTEs and 21% of youths' FTEs are in this employment; in Asia, 26% versus 32%; and in Latin America, 21% versus 23%. (2) There is female inclusion: over regions, the share of females’ total FTEs is 30%, versus only 19% for males. These findings coincide with SME enterprise studies for example in agro-processing in Ghana where Ampadu-Ameyaw and Omari  find thaHowever, there is mixed inclusion of hinterland or lagging zones. Overall, rural peri-urban, intermediate, and hinterland zone households show similar shares for food system employment in total FTEs: 26%, 25%, and 22% respectively. However, in Asia and Latin America, food system employment was negatively correlated with distance from towns: in Asia, 20%, 12%, and 11% from peri-urban to hinterland; in Latin America, 26%, 13%, and 15% respectively. This suggests agglomeration effects near towns spurring wholesale and processing.Third, they also found "food system employment" important in urban areas, averaging 25% of urban FTEs . Note that the share in rural Asia was but 18% so this shows that a lot of the processing and wholesale is agglomerated in urban areas in Asia but less so in SSA. As in rural areas, in cities the reliance on food system employment is similar between urban youths and adults.Most of this urban employment is in SMEs, at least in SSA and South Asia where the penetration of large-scale processing firms as well as large wholesale and logistics firms is still slight. For example, in Ghana, in a survey of 282 agro-processing firms, Afful-Koomson et al.  cited b found thFirst, technological change in SMEs can increase the employment they induce. An example is the shift in enjera processing from traditional wooden plates to new electric plates (mitads) which led to a higher capacity of the SMEs and better quality of the product, and a marked increase in employment in the sector . Above we noted that African females used to spend 4 h a day pounding staples, time that was saved by their being able to buy flour from SMEs. Liverpool-Tasie et al.  show thaFirst, a common finding in the limited number studies comparing midstream SMEs versus large enterprises is that that SMEs are less capital-intensive. Examples of this include the positive correlation of the capital/labor ratio shown for small versus large enjera enterprises in Minten et al. , and a pSecond, the corollary is that it appears that usually SMEs are more labor intensive (in terms of labor/output ratios). For example, for China, Reardon et al.  show thaHowever, larger firms often have a higher capacity  utilization rate and its correlate, the profit rate. This was shown for example in the rice mill survey results presented in Reardon et al.  for ChinThird, another factor that reduces the share of smaller SMEs (and thus by corollary reduces the SME labor impact over time) is the exit of smaller SMEs that cannot compete in product differentiation. For example, Reardon et al.  show in First, SMEs in the midstream of output value chains are the actors closest upstream and, with input retailers, downstream from farmers. SMEs have been crucial in creating a “vent-for-surplus” and value addition which facilitates farmers’ commercialization. SMEs signaling quality requirements and opening markets and services for high-value products help small farmers to shift from low-value basic food grains; an example is the rise of cold storages in the poor state of Bihar, India  by rural households . This income is a major source of cash for farm investments and technology adoption often much more important than credit , the efficiency of the SMEs, and whether they engage in non-competitive behavior. The impact can be positive, such as we illustrated above for Ethiopia where wholesale and transport SMEs reduced wholesale and retail margins and transport costs cheapening teff products for consumers.Second, SMEs' investments can reduce waste in value chains, increasing food access for consumers. For example, cold storage SMEs in Bihar, India, displaced traditional on-farm storage and thus reduced wastage rates  and permit quality differentiation. However, the labeling does not always guarantee safety or even improved quality as there are usually poor regulatory systems in developing countries such as lax or missing labeling and food safety regulations. For example, Minten et al. , b note Third, as expected, there is uneven adoption of food safety measures by SMEs in the midstream. Strict public standards as well as even more demanding private standards of large food manufacturers and supermarket chains can result in exclusion of food processing SMEs that cannot meet the standards, such as in Brazil for the cases of milk and shredded coconut  of the food output and farm inputs value chains in developing regions. We situated that development in the context of the stages of transformation of AVCs from traditional to transitional to modern. We focused on the growth and importance of SMEs in the long transitional stage, in which most of SSA and South Asia now are, as well as parts of Southeast Asia and Latin America. We reviewed evidence for and noted impact pathways of those SMEs on employment, farmers, consumers, and the environment. We relied on a broad review of the findings from our own surveys of thousands of SMEs mainly in SSA and Asia, and the findings of studies by others. Due to space constraints, we did not attempt to review the literature on the constraints SMEs face or policies and programs aimed at regulating and assisting SMEs. Following are the main findings and implications.First, there has been a rapid expansion of SMEs in the midstream segments of AVCs in the developing regions, paralleling the rapid growth of the volume and length of AVCs. The latter has been spurred mainly by the "demand pull" of urbanization accompanied by the facilitating influence of development of public infrastructure, in particular wholesale markets and roads.Second, the midstream SMEs taken as a whole handle roughly two-third of the food consumed in the developing regions; the rest is handled by modern firms and a minor share is subsistence consumption. Thus, midstream SMEs are crucial to the food security of billions of persons in these regions. Their share in the food economy is roughly similar to farms themselves, yet midstream SMEs are often "hidden" from the food security policy debates. They are often considered a "missing middle", but we argue that they are already a dynamic and massive presence. They should be termed the "hidden middle," as they are not missing but are merely usually hidden from the debate.Third, we found that midstream SMEs condition employment, farm incomes, the environment, and the food prices, food safety, and food quality facing consumers. It appears that often the effect is important and positive. The employment by SMEs forms roughly a fifth to a quarter of rural and urban employment in these regions and is disproportionately important as jobs for women and youths.Fourth, and crucial to the policy implications here, is that we found that where the enabling conditions  are present, SMEs emerge and grow rapidly, often in "spontaneous clusters" rather than in the "managed clusters" that dominate the policy debate. Emblematic are the cases of: (1) the wholesale, logistics, and processing SMEs involved in the teff value chains feeding Addis Ababa, Ethiopia, its leading staple food; over just a decade these firms tripled in number , they made investments in their own capacity that drove down transport costs and supply chain margins; (2) the potato cold storage SMEs in Agra, India, feeding Delhi its leading staple vegetable; over a decade these firms also tripled, making investments that reduced potato supply seasonality to cities and increased farmer incomes; (3) the wholesalers and input firms in the aquaculture supply chain in Bangladesh; over just a decade they tripled in numbers and more than tripled in volumes, facilitating a boom in fish supply to cities and creating substantial employment. These three cases represent many others we identified, and are important not only for the dynamism they portray, but because in none of the cases did any donor, NGO project, subsidy or direct government action play a significant role. In all three cases however the governments' provision of wholesale markets and roads and electrification were crucial. When the conditions were present, the SMEs spread and grew to meet the rapidly rising urban demand, creating rural and urban livelihoods.The policy implications of the above four findings are that multilateral agencies, donors, governments, and NGOs do not need to “kickstart” SMEs in the midstream and downstream of food supply chains. There is no need to put in place services that these firms are already providing and thus “reinvent the wheel.” Rather, support should focus on leveraging the rapid growth of SMEs already in place or emerging, and addressing their constraints to faster growth and better impacts. Donors and governments should not waste resources trying to “organize” SMEs into cooperatives or managed clusters, force them into formal patterns, or try to “create them” with programs that set up firms. Extrapolating from our findings that infrastructure coupled with urban demand was usually the key enabler, we recommend a focus on infrastructure provision.While we did not analyze the constraints to SMEs, our prior work has pointed to SMEs' being constrained by poor wholesale markets, bad roads, spotty electrification, and bribes on the roads. We believe that addressing these should be the first and main priority.Moreover, there is a need to address bad policies and regulations that constrain SMEs. Bribes are a kind of informal regulation or informal tax that the government at various levels imposes on SMEs and are a key regulation problem. Other bad regulation includes needless red tape for redundant certifications, confusion and ambiguity in zoning and premises, and in times of crisis like COVID-19, deleterious limitation on mobility (such as shown in Nigeria, Liverpool-Tasie et al., Finally, there are missing or poorly implemented food safety and hygiene regulations that make SMEs with unsafe practices a danger to consumers and a constraint to themselves. This also extends to the need for truth in labeling regulations. There is a role for education of SMEs in food safety standards to complement regulation.