Firmicutes. At the genus level, the USM and NM were dominated by Lactobacillus, Leuconostoc, and Weissella, belonging to lactic acid bacteria. However, as alpha diversity is higher in the USM than in the NM, the genus Akkermansia, belonging to the phylum Verrucomicrobia, was detected only in the USM. Compared to the NM, the USM showed a relatively higher ratio of transcripts related to “protein metabolism,” and the USM was suspected to be involved with the viable-but-nonculturable (VBNC) state. When comparing the sub-transcripts related to the “cell wall and capsule” of USM and NM, USM showed a proportion of transcripts suspected of being VBNC. In addition, the RNA virome was also identified, and both the USM and NM were confirmed to be dominated by pepper mild mottle virus (PMMoV). Additionally, the correlation between metataxonome and metatranscriptome identified USM and NM was estimated, however, only limited correlations between metataxonome and metatranscriptome were estimated. This study provided insights into the relationship between the potential metabolic activities of the USM of kimchi and the NM.Presently, pertinent information on the ultra-small microbiome (USM) in fermented vegetables is still lacking. This study analyzed the metatranscriptome and metataxonome for the USM of kimchi. Tangential flow filtration was used to obtain a USM with a size of 0.2 μm or less from kimchi. The microbial diversity in the USM was compared with that of the normal microbiome (NM). Alpha diversity was higher in the USM than in NM, and the diversity of bacterial members of the NM was higher than that of the USM. At the phylum level, both USM and NM were dominated by  Brassica rapa subsp. pekinensis), mixing it with condiments such as red pepper powder, onion, garlic, and ginger, and then allowing it to ferment. Kimchi is, therefore, considered unique in its manufacturing method compared to other fermented-salted vegetables that are primarily pickled in salt or vinegar. Further, kimchi has been reported to be a good source of probiotics containing lactic acid bacteria  were first defined by In our previous study , althougOur group recently confirmed the presence of communities of UMB of size 0.2 μm or less in fermented cabbages such as kimchi and sauerkraut using tangential flow filtration (TFF) and 16S rRNA gene amplicon sequencing . It was Brassica rapa subsp. pekinensis), and 10 packets of kimchi of 400 g each (to make handling easier) were purchased. After purchase, the pH of kimchi was measured with an automatic pH/mV titrator . All the kimchi samples were merged in a double-structured basket sterilized with a built-in strainer, and broths from merged kimchi samples were obtained and roughly filtered by the strainer. Then, 500 ml of Dulbecco’s phosphate buffered saline  was added to the basket to facilitate filtration. The work on sample preparation was done inside a clean bench.For this study, kimchi made in South Korea was purchased from an online market in October 2021. The main ingredient of kimchi is kimchi cabbage , and TFF was performed in two phases. In the first phase, a 0.22 μm pore size TFF cartridge  was used to confirm a normal microbiome (NM) larger than 0.2 um in size. Then the NM was concentrated to 20 ml, and after dividing by each 10 ml, 25 ml of RNAlater  for metatranscriptomic analysis was separately added to 10 ml of concentrate for stabilization and protection of RNA. The remaining 10 ml was used for metataxonomic analysis. The prepared NM samples were stored in a −20°C freezer until analysis. A 100 kDa molecular weight cut-off (MWCO) TFF cartridge  was used in the second phase to confirm a USM smaller than 0.2 μm in size. Then the USM was concentrated to 20 ml, and after dividing by each 10 ml, 25 ml of RNAlater  for metatranscriptomic analysis was separately added to 10 ml of concentrate for stabilization and protection of RNA. The remaining 10 ml was used for metataxonomic analysis. The prepared USM samples were stored in a −20°C freezer until analysis.For metataxonomic analysis based on the 16S rRNA gene, DNAs were extracted using a DNeasy PowerSoil kit  for each sample solution of NM and USM. The extracted DNAs were quantified using the Quant-IT PicoGreen assay kit  following the manufacturer’s instructions. The quantified nucleic acids were amplified for the V3 and V4 regions of the 16S rRNA gene by a universal primer pair with Illumina adapters . The polymerase chain reaction (PCR) conditions were as follows: initial denaturation at 94°C for 3 min, followed by 25 cycles of denaturation at 95°C for 30 s, then annealing at 55°C for 30 s, then extension at 72°C for 30 s, and a final extension at 72°C for 5 min. The PCR products were purified using AMPure beads . The purified PCR products were re-amplified to 15 cycles, including initial denaturation and final extension steps, as described with the Nextera XT indexed primer pair. The re-amplified PCR products were re-purified using AMPure beads and were quantified using the KAPA Library Quantification kits for Illumine sequencing platforms . The qualification was carried out using the TapeStation D1000 ScreenTape . The paired-end sequencing (2 × 300 bp) was performed using the MiSeq platform .The paired-end sequence reads gained from the MiSeq platform were merged by the FLASH software , and theFor metatranscriptomic analysis, RNAs were extracted using a Maxwell 16 LEV simplyRNA tissue kit  for each sample solution of NM and USM. The extracted RNAs were quantified using the Quant-IT RiboGreen assay kit  following the manufacturer’s instructions. RNAs for each sample were independently prepared using the Illumina Stranded Total RNA Library Prep . The rRNAs in the total RNAs were removed by Ribo-Zero Plus . The remaining mRNA fragments were converted to cDNA using SuperScript II reverse transcriptase  with random primers. The second strand cDNA synthesis was carried out using DNA polymerase I with RNase H and dUTP. When the second strand cDNA was synthesized, the fragments underwent end repair, adapter ligation, and adenine bases were added. The products were then purified and enriched with PCR to produce the final cDNA library. The cDNA libraries were quantified using the KAPA library quantification kits for Illumina sequencing platforms , and qualification was then carried out using the TapeStation D1000 ScreenTape . The paired-end (2 × 150 bp) sequencing was performed using the NovaSeq platform .The annotation for the metatranscriptome of USM and NM was carried out by the MG-RAST server, and the paired-end sequence read files were also merged and trimmed by the server. Viromic analysis was performed using the RefSeq database , and theStatistical analyses for the metataxonome and metatranscriptome of USM and NM were done using MicrobiomeAnalyst . The datThe kimchi samples were isolated to USM and NM by TFF for the metataxonomic diversity of USM and comparison with NM. The metataxonomic sequencing data for NM and USM were obtained . The seqMetatranscriptomic sequencing data for NM and USM were obtained for identification of the potential functional transcriptome of USM and comparison with NM . The seqThe diversity of the USM in the kimchi sample was compared with NM  by metatFirmicutes , Bacteriodetes (1.42%), and Actinobacteria (1.28%) were not so dominant (Firmicutes (99.72%), just like the USM (Lactobacillus (64.52%), while Leuconostoc (12.99%), Weissella (5.63%), and Clostridium (2.21%) were not so dominant (Lactobacillus (78.82%) was dominant in NM, while Weissella (14.07%) and Leuconostoc (6.30%) were not so dominant  were dominant, and this was presumed to be the ripening state with the most Lactobacillus spp. in LAB because of the pH of 4.1 in the kimchi sample known for having the high LAB content , it was determined that there was a possibility that only USM could be purified and used as a new probiotic since it has just been described as a beneficial bacteria  state in which the cell size decreases , aggregaIn the USM, the level 2 transcriptome associated with the “cell wall and capsule” was occupied by RNA transcripts associated with “capsular and extracellular polysaccharides” (39.10%), “Gram-positive cell wall components” (34.97%), “Gram-negative cell wall components” (2.95%), and “cell wall of mycobacteria” 2.35%; . In the In the USM, the level 2 transcriptome associated with the “stress response” was occupied by transcripts associated with “oxidative stress” (44.7%), “osmotic stress” (22.9%), “heat shock” (13.0%), and “cold shock” 4.3%; . In the In the USM, the level 2 transcriptome associated with “virulence, disease and defense” was occupied by transcripts associated with “resistance to antibiotics and toxic compounds” (57.9%), “invasion and intracellular resistance” (10.8%), “bacteriocins ” (6.6%), and “adhesion” 5.4%; . In the The transcripts at the functional level of the USM and NM overlapped (48.2%), and for the USM, only 3.2% had unique transcripts, while 37.9% of the NM had unique transcripts. However, the abundance of transcripts was not considered .Since the formation of USM is thought to be related to survival, the lower transcripts were identified in the functional classification categories of level 1 “cell wall and capsule,” “stress response,” and “virulence and defense” and compared with the NM. In the “cell wall and capsule,” the transcripts related to “Gram-negative cell wall components” and “Gram-positive cell wall components” had a relatively higher proportion in the USM than in the NM. It was possible that as the cell size became small enough to belong to the USM, the ratio of the transcripts related to the cell wall components became relatively high. The transcripts related to “oxidative stress” in “stress response” had a relatively higher proportion in the USM than in the NM. Some studies  have shoTobamovirus (82.09%) and Allexivirus (9.35%). Carlavirus (2.63%) and Nepovirus (1.93%) were minorly dominant in the USM (Carlavirus (33.98%), Allexivirus (26.01%), and Tobamovirus (25.63%). Those with minor dominance in the NM were Tospovirus (2.99%), Fabavirus (2.84%), Foveavirus (2.58%), and Potyvirus  was dominant in both the USM (80.10%) and NM  at the genus level. Transcripts related to “stress response” were negatively correlated with Butyricimonas (r = −0.52) and Delftia (r = −0.57). Both positive and negative correlation coefficients did not exceed 0.5 large in transcripts related to “cell wall and capsule,” so it was considered that the correlation with bacteria at the genus level was not.The correlation analysis between metataxonome and metatranscriptome was expressed as a heatmap . TranscrBacteroides negatively correlated (r = −0.51) with “virulence, disease and defense” was found only in USM (Butyricimonas and Delftia negatively correlated (r = −0.52 and r = −0.57) with “stress response” were found only in USM  and Hespellia (r = −0.57). Transcripts related to “secondary metabolism” were positively correlated with Lactococcus (r = 0.52) and Terrimonas (r = 0.55), were negatively correlated with Halomonas (r = −0.53) and Staphylococcus (r = −0.51). Transcripts related to “respiration” were positively correlated with Leuconostoc (r = 0.53). Transcripts related to “regulation and cell signaling” were positively correlated with Cobetia (r = 0.54), Fusobacterium (r = 0.58) and Legionella (r = 0.59), were negatively correlated with Butyrivibrio (r = −0.52). Transcripts related to “protein metabolism” were negatively correlated with Bacillus (r = −0.52). Transcripts related to “potassium metabolism” were positively correlated with Lactobacillus (r = 0.54), and were negatively correlated with Megamonas (r = −0.57). Transcripts related to “photosynthesis” were positively correlated with Enterobacter (r = 0.67), Pectobacterium (r = 0.57) and Weissella (r = 0.57). Transcripts related to “phosphorus metabolism” were positively correlated with Abiotrophia (r = 0.65) and Coenonia (r = 0.57), were negatively correlated with Salinivibrio (r = −0.57). Transcripts related to “phages, prophages, transposable elements, plasmids” were positively correlated with Bacillus (r = 0.57) and Faecalibacterium (r = 0.52), were negatively correlated with Pedobacter (r = −0.59). Transcripts related to “nucleosides and nucleotides” were negatively correlated with Ruminococcus (r = −0.66). Transcripts related to “nitrogen metabolism” were negatively correlated with Cronobacter (r = −0.54) and Weissella (r = −0.51). Transcripts related to “motility and chemotaxis” were positively correlated with Eubacterium (r = 0.52), and were negatively correlated with Capnocytophaga (r = −0.55). Transcripts related to “metabolism of aromatic compounds” were positively correlated with Sarcina (r = 0.51). Transcripts related to “Membrane Transport” were positively correlated with Ethanoligenens (r = 0.60), and were negatively correlated with Terrimonas (r = −0.57). Transcripts related to “iron acquisition and metabolism” were positively correlated with Catenibacterium (r = 0.50) and Chryseobacterium (r = 0.54), and were negatively correlated with Agrobacterium (r = −0.55). Transcripts related to “fatty acids, lipids, and isoprenoids” were positively correlated with Acidobacterium (r = 0.53), and were negatively correlated with Planktothrix (r = −0.51), Staphylococcus (r = −0.55) and Stenotrophomonas (r = −0.64). Transcripts related to “dormancy and sporulation” were negatively correlated with Bradyrhizobium (r = −0.57), Sarcina (r = −0.56) and Streptomyces (r = −0.56). Transcripts related to “DNA metabolism” were positively correlated with Dyella (r = 0.53), and were negatively correlated with Catenibacterium (r = −0.57) and Legionella (r = −0.60). Transcripts related to “clustering-based subsystems” were positively correlated with Robinsoniella (r = 0.55), Salinivibrio (r = 0.54) and Staphylococcus (r = 0.56). Transcripts related to “cell division and cell cycle” were negatively correlated with Stenotrophomonas (r = −0.60). Transcripts related to “amino acids and derivatives” were negatively correlated with Robinsoniella (r = −0.58). In addition, both positive and negative correlation coefficients did not exceed 0.5 in transcripts related to “RNA metabolism,” “cofactors, vitamins, prosthetic groups, pigments” and “carbohydrates,” so it was considered that the correlation with bacteria at genus level was not.Transcripts related to “sulfur metabolism” were negatively correlated with Acidobacterium and Hespellia for “sulfur metabolism”; genera Bradyrhizobium, Sarcina, and Streptomyces for “dormancy and sporulation”) belonging to USM, there is a possibility that the expression level of A-related transcripts is low in USM. On the other hand, “membrane transport” showed a positive correlation only with bacteria (genera Ethanoligenens and Terrimonas) belonging to USM, and it is possible that the expression level of transcripts related to “membrane transport” in USM is relatively higher than that of NM. Also, there was no relationship between metataxonome and metatranscriptome with positive or negative correlation coefficients greater than 0.8, but there were relationships with positive or negative correlation coefficients greater than 0.5. Therefore, it is thought that the metataxonome and metatranscriptome influence each other to some extent. However, only limited samples were obtained due to the difficulty of pretreatment. To overcome this, since the correlation was analyzed with samples obtained by bootstrapping, more research is needed to obtain clearer results.Since “sulfur metabolism” and “dormancy and sporulation” only showed a negative correlation with bacteria  can be found at: H-WL: writing—original draft, investigation, data curation, conceptualization, software, methodology, and funding acquisition. S-RY: writing—review and editing, and investigation. Y-MD: investigation and data curation. J-HY: investigation, methodology, and software. HJ and K-NK: methodology and data curation. J-HH: validation, resources, writing—review and editing, funding acquisition, and supervision. J-WB: writing—review and editing, data curation, and supervision. All authors contributed to the article and approved the submitted version.This work was supported by the National Research Foundation of Korea (NRF-2019R1F1A1061368) and the World Institute of Kimchi (KE2202-2).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.


The miscibility between hydrophobic and hydrophilic biopolymers has been of significant challenge. This study used a novel simplified chitin modification method to produce phthalic chitin using phthalic anhydride in a substitution reaction. The FT-IR functional group analysis was used to confirm the substitution reaction. The modified chitin was used as compatibilizer in polylactic acid (PLA)/starch biocomposite to enhance its properties. The biocomposite was prepared using melt extrusion and compression moulding technique. The biocomposite's morphological, thermomechanical and water absorption properties were characterized using scanning electron microscope, tensile test, dynamic mechanical analysis, thermogravimetry analysis, differential scanning calorimetry, thickness swelling and water absorption test. The FT-IR study shows a successful substitution reaction of the amine hydrogen ion present in the chitin as opposed to substituting the hydrogen ion in the hydroxide group. The tensile and impact properties of biocomposite incorporated with modified chitin showed better results compared with other samples. The SEM images showed uniform miscibility of the modified biocomposite. The dynamic mechanical analysis showed improved modulus value with the incorporation of modified chitin. The thermal properties showed improved thermal stability of the modified biocomposite. Furthermore, the percentage of water absorbed by biocomposite with modified chitin is reduced compared with the PLA/starch biocomposite. The produced biodegradable ternary blend can be used as a substitute for plastics in industrial applications. PLA becomes an environmentally beneficial biomaterial as a result.Biodegradable polymers, or polymers derived from biological sources, are a viable substitute for many petrochemical polymers. They are, however, challenging to work with because property variance is always source dependent ,2. PolylPLA undergoes a lengthy hydrolysis process that can take years to complete. It is extremely brittle, resulting in poor strength and a breaking point of less than 10% elongation. PLA is also hydrophobic and has poor gas barrier characteristics ,8. PLA iStarch is the most cost-effective biodegradable substance used in various non-food applications such as papermaking, cardboard, textile sizing, thickening, adhesives agents and kitchenware and film raw materials. Furthermore, starch has the advantage of having quick-dissolving qualities in polymers, and starch-based polymers are often used in food packaging, where the plastic can deteriorate with the food. However, a peculiar physical and chemical reaction occurs . BecausePLA has been blended with starch to achieve higher strength because of the semi-crystalline nature of starch. However, the agglomeration challenge between PLA and starch has resulted in limited application of the blend. Several techniques have been applied to improve the PLA/starch blend , classifChitin is a white, nitrogenous, inelastic polymer found in various places, including crustacean exoskeletons, fungal cell walls, diatoms, mushrooms, worms, arthropods, insects, crawfish, shrimp and crabs, excrement being the most common sources . Chitin,. 2. 2.1−1, a specific gravity of 1.24, and a melt flow rate (MFR) of 0.78 g min−1 . Lifeline Nutrition, Chicago, IL, USA, provided commercially available chitin deacetylated to 90%. Sigma Aldrich provided commercial high-amylose corn starch .The materials are polylactic acid, starch and chitin. The PLA was used as the matrix of the polymer blend biocomposite. Nature Works  supplied commercial quality poly(lactic acid) (4032D) in pellet form. PLA (4032D) was used because it possesses outstanding oil and grease resistance, excellent twist and machinability properties. It has a glass transition temperature of 60–65°C, a melting point of 155–170°C, a tensile strength of 53.5 MPa, and a modulus of 3500 MPa. PLA (4032D) has a molecular weight of 100 000 g mole. 2.23 for 1 h with continuous stirring to form a semi-solid gel. The semi-solid gel was removed and rinsed in distilled water until it reached a pH of 7. The gel precipitate was then dried by freezing and ground to powder. For testing and biocomposite manufacture, the modified chitin powder was stored in an air-tight bag. FT-IR functional group analysis of chitin and modified chitin was performed with FT-IR EFTEM Libra, Carl Zeiss, UK. The samples were mixed with potassium bromide (KBr) in ratio 1 : 10 and pressed into pellet.The chitin was mixed with phthalic anhydride in dried dimethylformamide (DMF) and heated to obtain phthayl chitin (PHCS), used to make modified chitin. It came in the form of a yellow powdered substance. In 100 ml of acetic acid, the chitin powder was dissolved. Phthalic anhydride in methanol was poured into the chitin solution and heated at 50°C with stirring for 8 h. The obtained colloid mixture was precipitated with NaHCODry air generator Luxor 50  was used to dry the PLA pellets at 60°C for 4 h. Using a Thermo electronic rheomixer 03, powdered chitin, Mchitin, and starch with various compositions were combined with a constant percentage of PLA fluctuation . A Process 11 twin-screw extruder was used to mix and extrude the mixture . The extruding temperature ranges from 120°C to 200°C, and the product is subsequently quenched in room temperature in water. The sample filaments were pelletized in a Thermo Scientific pelletizer  and pressed into characterization test forms in a Carver press compression moulding machine  at 170°C for 15 min at 10 MPa pressure. The obtained test samples were air-cooled in atmospheric air and stored in lock bags.. 2.3−1 from room temperature to 600°C in an air bath. The neat PLA and biocomposite differential scanning calorimetry (DSC) was analysed with DSC model 6 PerkinElmer  using ASTM 3418 to observe the heat flow properties change with temperature. The transition temperatures were obtained for a sample of 6 to 7 mg.The neat PLA and the biocomposite were characterized with dynamic mechanical analysis (DMA), thermogravimetry analysis (TGA), derivative thermogravimetry (DTG), tensile test and water absorption analysis at ASTM standard. The morphological properties were studied using scanning electron microscopy (SEM). This work employed dynamic mechanical analysis to assess the miscibility of polymeric blend systems and analyse polymers' mechanical and structural properties. Furthermore, the dynamic mechanical analysis depicts the composites' mechanical properties at various temperatures. A PerkinElmer dynamic mechanical analyser (DMA 8000) was used . At a frequency of 1 Hz and a temperature range of 50°C to 150°C, samples were manufactured according to the ASTM D4065 standard for polymer composites. The thermogravimetric study was used to investigate the composite's thermal deterioration behaviour. T.A. Instruments  provided the PerkinElmer TG-IR-GCMS Interface Q500. T.A. universal analysis programme was used to analyse the results . The thermogravimetric test was carried out according to ASTM E1131, with sample volumes ranging from 10 to 21 mg being heated at a rate of 10°C min−1 with a 30 kN load. The composites specimen was created and submitted to the American standard for a material test with cross-sectional dimensions of 5 by 10 mm and a gauge length of 30 mm. The ASTM D3039 tensile test technique was used with standard dimensions. The impact strength was obtained using Ceast Resil Impactor 7181 , a hammer. The purpose of the test is to determine how much energy is absorbed throughout the breaking process. Scaffolds are subjected to impact loading. Hence this is required. The ASTM D256 standard specimen with dimensions of 10 × 10 mm by 50 mm was used. Before being placed in the impactor, the samples were notched in the middle. The microstructure of the various samples (parts) of the experiments was thoroughly investigated using scanning electron microscopy (SEM). SEM with both secondary electron (SE) and backscattered electron (BSE) signals were used to examine the microstructure of carbon-coated materials. The SEM was operated at an accelerating voltage of 2.00 kV secondary electron imaging (SEI). The material samples were immersed in the distilled water for 24 h. The initial and final mass of the samples were used to calculate the absorption rate. The ASTM test method D570-81 was used to determine the water absorption.An Instron universal testing machine (model 5966)  was used to perform the tensile test, run at a constant crosshead speed of 2 mm s. 3−1, indicating presence of water, -OH bond and NH stretches, respectively. On the chitin graph, a minor peak was found between 2810 and 2920 cm−1, indicating aldehyde -C-H stretches which were not found in modified chitin, probably because of the substitution reaction. Both modified chitin and unmodified chitin showed C = C alkene stretch, C = O at 1670 cm−1, and sp2 C-H bends at 1380 and 1400 cm−1. However, C = C aromatic stretch at 1500 cm−1 in the modified chitin indicates phthalic aromatic alkyl substitution, which is not present in the unmodified chitin. Also, 1100 and 1154 cm−1 C-O-C bridge was present in both. The presence of 800 cm−1 bands in the modified chitin indicated an alkene C-H bend [−1 bands indicated a C-H bond, which indicated an alkyl group. A classic sign of alkyl group attachment (substitution) to the chitin chain is the appearance or alteration of new peaks bands 1500 and 800 cm−1 [2 were replaced.800 cm−1 . The alk800 cm−1 . That isa) increased more than the neat PLA (53.5 MPa). The highest tensile strength for the biocomposite was observed with PLA/Mchitin/starch with 87.5 MPa, followed by PLA/chitin/starch with chitin and starch filler. The result showed that the biocomposite's tensile strength is enhanced with chitin and starch, but both filler's effectiveness justifies blending PLA with chitin and starch. The increase in tensile strength is probably due to the interaction between the three polymeric materials [The tensile properties of the biocomposites are shown in figure 2aterials . The tenaterials . The preaterials ,25,26. Hb. The tensile modulus value of neat PLA is 3500 MPa. Generally, the modulus of the biocomposite is significantly higher than those of the neat PLA. Similar to the tensile strength results, the modulus of PLA/Mchitin/starch shows the highest strength while that of PLA/chitin/starch is next to it. The lowest among the biocomposites was obtained with PLA/starch, which has the highest starch percentage, due to the difference in nature of PLA and starch. The tensile modulus of biocomposites increased by more than 20% for PLA/Mchitin/starch compared with the neat PLA. This is quite a significant increase which showed the compatibility between PLA, Mchitin and starch.The tensile modulus of neat PLA and biocomposite is presented in figure 2c. The elongation of the neat PLA shows the lowest due to its brittleness. The elongation value of the biocomposite showed significant improvement compared with the neat PLA. Also, the elongation values were observed to increase with both starch and chitin. The highest elongation value was obtained for PLA/starch due to its film-forming ability. The elongation is seen to reduce with the inclusion of chitin in the biocomposite but is still higher than that of neat PLA. This showed that the addition of chitin and starch enhanced the elongation properties of PLA, which means the brittle nature of PLA was reduced. This is probably due to the film-forming property of chitin and starch [The elongation properties of the neat PLA and biocomposite are presented in figure 2d starch . This shd starch ,28.d. The impact strength of a material is a measure of its resistance to a sudden force. The impact strength follows a trend similar to tensile strength and modulus, with starch and chitin percentages increasing more than neat PLA. The low impact strength of neat PLA probably means that it has a brittle nature. Notably, the graph shows PLA/Mchitin/starch having the highest impact strength, which shows a good toughening percentage. The graphs show a steady increase in the impact of energy, having the highest impact value at 14 200 J m−2. The effect of chitin and starch on the impact strength of the samples is more significant, as shown with PLA/chitin/starch compared with PLA/starch. This results from the brittle nature of the starch-filled biocomposite, which results in its low value, as reported previously [The impact strength of the material was also carried out to show its resilience property. The graph of energy and resilience against samples is shown in figure 2eviously ,30.a) revealed a single deterioration curve for neat PLA and the biocomposites. The single curve deterioration is most likely a favourable indicator of the polymeric blends' good miscibility. The onset temperatures are 303.11°C, 290.8°C, 289.75°C and 291.45°C for plain PLA, PLA/starch, PLA/chitin/starch and PLA/Mchitin/starch, respectively. The onset temperature of the biocomposite is generally reduced with starch content due to its biodegradable nature. The onset temperature is further reduced with chitin and Mchitin, enhancing degradation [figure 3radation ,32. The radation .Figure b) peak temperature values for neat PLA, PLA/starch, PLA/chitin/starch, and PLA/Mchitin/starch are 366.67°C, 367.10°C, 358.58°C and 370.21°C respectively. These results show that the addition of starch has a minor effect on the DTG peak temperature compared with the plain PLA. However, combining starch and chitin (PLA/chitin/starch) resulted in a considerable reduction in DTG peak temperature, which improves the biocomposite's thermal degrading capabilities. Also, Mchitin loading increased the DTG, which is probably due to miscibility enhancement. The reduction in the DTG peak temperatures is corroborated with the residue percentage. The percentage weight losses for neat PLA, PLA/starch, PLA/chitin/starch and PLA/Mchitin/starch are 87.08%, 92.04%, 90.40% and 95.04%, respectively. The percentage of weight loss is increased due to the natural biopolymer filler degradation enhancement of neat PLA. The values of the residue for neat PLA, PLA/starch, PLA/chitin/starch and PLA/Mchitin/starch residue are 10.54%, 6.24%, 7.78% and 4.92%, respectively. The difference in weight loss and residue is due to the volatile content present in the biocomposite [Furthermore, the DTG curve b peak teomposite .The differential scanning calorimetry (DSC) result is shown in E′), loss modulus (E") and loss factor (tan δ) of the biocomposites with temperature under 1 Hz are shown in E′) value of the neat PLA and biocomposite is presented in figure 5a. The storage modulus graph shows the thermomechanical behaviour of the biocomposite from the glassy region to the rubbery region [E′ value of the biocomposites. The E′ reduced with the addition of starch, as can be seen in figure 5a. This meant that the damping properties of composites reduced, and they became more brittle with the increase in starch content. Compared with PLA/starch, the storage modulus increased with chitin (PLA/chitin/starch) but was slightly lower than neat PLA. However, the highest storage modulus was observed with PLA/Mchitin/starch, probably enhanced miscibility. The storage modulus shows the stiffness properties of the material with temperature change [The variation of storage modulus (y region . The adde change . The grab) shows the ability of the biocomposite to store energy from the glassy region to the rubbery region. The loss modulus lowest value was observed with PLA/starch followed with PLA/chitin/starch and neat PLA. The loss modulus value of PLA/Mchitin/starch showed the highest value, similar to the storage modulus graph. The peak of the loss modulus was observed at 72°C, above which the modulus value decreases. The loss modulus value E" increased up to 3600 GPa with a Mchitin sample, while the modulus value was observed to reduce with chitin content. This shows the modification process's significance, which enhances miscibility in PLA/Mchitin/starch biocomposites. Generally, the loss modulus is high because of the viscoelastic properties of biopolymer. The loss modulus increased in value until 72°C, which signifies the glass transition into the rubbery region. The modulus begins to reduce at a higher rate until it turns into a viscous liquid. Above this temperature, the material possesses low internal bonding due to viscosity. This is similar to the storage modulus reduction, which shows a uniform line that signifies that the polymer blend is homogeneously mixed [The loss (viscous) modulus graph b shows tly mixed . The PLAly mixed .c presents the corresponding loss factor (tan δ) curves of the biocomposites. This shows the damping properties of the biocomposite. The chitin-based (PLA/chitin/starch) biocomposites (PLA/starch) showed the lowest tan δ peak and temperature, attributed to poor interactions between the reinforcement and the matrix [Figure 5e matrix . There we matrix . This she matrix .a–d shows the SEM images of neat PLA and biocomposites. Homogeneous surface morphologies were observed for the samples with neat PLA. However, the surface characteristic of the image changed with the addition of starch (b) with characteristics of white starch patches. The PLA/starch SEM images show flakes of starch. This image shows that the starch was not miscible with the PLA matrix due to differences in their nature. This is expected due to the difference in the nature of the two polymers. Polylactic acid is hydrophobic, while starch is hydrophilic. This means that starch immiscibility is dependent on the presence of hydroxyl ions. Starch has been reported to mix with water-loving polymers such as seaweed. However, PLA is highly repellent to water, resulting in immiscibility and probably the features experienced in the SEM images [c) had a rougher surface than the PLA/starch.The material samples were examined under scanning electron microscopy to determine the influence of the composition variation on the morphological properties. Figure 6f starch b with chM images . The lacM images . These iM images . The samd). The image showed a rough surface, but the uniformity in its colour shows the uniform mix.However, distinct white patches observed with PLA/starch had disappeared. This can be attributed to the inclusion of chitin in the blend with mixes with the starch filler. Chitin has been reported to have miscibility with starch due to the hydroxyl group in its structure. However, chitin is still hydrophilic, which also makes it possible for mixing with PLA. The image was observed to have wedges of the filler in the matrix. The surface of the samples with the Mchitin and starch showed crater-like dips on its surface with a uniform colour mix and network of lines d. The imThe nature of the polymer blends can be used to explain the miscibility as shown in Water absorption is a critical feature of a biodegradable polymer composite that assesses the material's resistance to water. Polylactic acid and chitin are hydrophobic, while starch is a hydrophilic material ,41. As aet al. [The capacity of water molecules to enter through the starch network causes water absorption. The nature of the polymer, which has high interaction with water, is blamed for water diffusion in starch. The low water absorption of PLA and chitin has also been reported by Nasrin et al.  in theiret al. . BecauseThe thickness swelling of neat PLA, PLA/starch, PLA/chitin/starch and PLA/Mchitin/starch biocomposites after immersion in water for 24 h is plotted in The trend of the thickness is similar to that of water absorption. The thickness of the swelling of the neat PLA did not significantly change due to its hydrophobic nature. The thickness of the PLA's swelling increased with starch and chitin's addition, as observed in the biocomposite. Generally, this study's swelling thickness depends on the polymer mix's nature and the porosity (voids or space) between the polymer mix molecules . The valThe properties of the biocomposite were further analysed using contact angle analysis as shown in . 4The biocomposites were successfully developed. The brittleness of neat PLA was reduced with the addition of the blend for all samples. The composites' DMA revealed a strong storage modulus for rigidity and a low loss factor for good miscibility. It also displayed a single peak, indicating that the polymer blend was compatible. The glass temperature from the loss factor peak, on the other hand, indicated that the material was appropriate for industrial use. The thermogravimetry research reveals that the composites have a single deterioration curve and glass transition temperature, indicating stability thermally. The composite moulded with a Carver press demonstrated a homogeneous dispersion of the natural polymer blend in terms of physical appearance. The network distribution of the chitin/starch blend in PLA was seen in SEM images. The morphological features of the blend with no void reveal good compatibility and finely structured network distribution. The photos revealed a uniform combination of modified chitin/starch and PLA compared with other samples.


Maize accumulates large amounts of starch in seeds which have been used as food for human and animals. Maize starch is an importantly industrial raw material for bioethanol production. One critical step in bioethanol production is degrading starch to oligosaccharides and glucose by α-amylase and glucoamylase. This step usually requires high temperature and additional equipment, leading to an increased production cost. Currently, there remains a lack of specially designed maize cultivars with optimized starch (amylose and amylopectin) compositions for bioethanol production. We discussed the features of starch granules suitable for efficient enzymatic digestion. Thus far, great advances have been made in molecular characterization of the key proteins involved in starch metabolism in maize seeds. The review explores how these proteins affect starch metabolism pathway, especially in controlling the composition, size and features of starch. We highlight the roles of key enzymes in controlling amylose/amylopectin ratio and granules architecture. Based on current technological process of bioethanol production using maize starch, we propose that several key enzymes can be modified in abundance or activities via genetic engineering to synthesize easily degraded starch granules in maize seeds. The review provides a clue for developing special maize cultivars as raw material in the bioethanol industry. For example, the modified 1/pUFLA) , can pro1/pUFLA) . Moreovedrolysis . By overimproved . By geneimproved .Though the application of these engineering enzymes and yeast strains have successfully reduced energy consumption of fermentation, the starch hydrolysis process remains to be improved, especially on starch properties, engineering enzyme sources and reaction conditions, to convert starch into ethanol economically and efficiently.As discussed above, the easily degraded starchy substrates in bioethanol production would reduce energy costs and generate more economic benefits . AccordiThe amylose content affects the enzymatic hydrolysis rate of starch. Previously, maize starch hydrolysis experiment in vitro by human salivary α-amylase showed that the rate of digestion of 100% maize amylose < hybrid high-amylose (64–66% amylose) < waxy maize starch (99–100% amylopectin) . Similarsbe1a mutant was more resistant to digestion during germination due to the altered branching pattern of amylopectin and amylose [The rate of starch enzymatic hydrolysis is controlled by starch multi-scale structures such as chain length distribution , crystal amylose . In duck amylose . Moreove amylose . The efficiency and yield of bioethanol production were also affected by starch granules sizes. Small granules of barley starch were more suitable for ethanol production than large ones, because it can produce more dextrins during enzymatic hydrolysis . In addiIn addition, endosperm types , starch content and other components  in maize and sorghum seeds affect final ethanol yield and rate of fermentation ,74,75,76The analysis of the starch degradation pathway during seed germination can provide ideas for the optimization of starch degradation in the process of industrial fermentation. Thus, we compared the difference in starch degradation between maize seeds (in vivo) and industrial fermentation (in vitro).Maize seeds mainly consist of endosperm and embryo, which account for 90% and 10%, respectively, of the whole dry seed weight . Maize eStarch is the primary carbon and energy sources for crop seed germination and seedling early growth ,81. AfteStarch granules in maize endosperm are generally spherical or, rarely, polygonal, surrounded by many other structures, such as protein bodies, residual walls, and amyloplast membranes B. The siNotably, the degradation of starch granules needs several amylolytic enzymes ,90 to deIn summary, the degradation process of starch in vivo during seed germination is a series of complicated enzymatic reactions, occurring slowly in moderate growth conditions . By comparison, starch degradation in bioethanol production usually requires high temperature and violently mechanical shear to break apart starch granules from seeds and to break down starch (amylose and amylopectin) structure for easily enzyme attacks.Starch synthesis of cereal crops is the primary determinant of grain filling . The curBased on the relevant studies ,99,100, brittle2 (bt2) encodes the small subunit, and the genes shrunken2 (sh2) and AGP2 encodes the large subunits. In maize endosperms, active AGPase formation requires SHRUNKEN2 and BRITTLE2 subunits [sh1 (deficient in sucrose synthase), starch contents significantly decreased and soluble sugars dramatically increased [As the substrate for primer synthesis, ADPglucose (ADPG) determines the yield of starch synthesis to a great extent. The ADPG synthesis from sucrose needs ADPglucose pyrophosphorylase (AGPase)  Figure . AGPase subunits ,103. Thesubunits ,103. Witsubunits . In the ncreased . The movMoreover, ADPG can be originated from the oxidative pentose phosphate pathway . Malate Wx)  (encoded by Wx) . SeveralWx) . Compareamylose-extender, ae) of maize and rice, amylopectin structure was found to be shaped by BEIIb [For amylopectin synthesis in amyloplast, transglycosylating branching enzyme (BE) is the only enzyme that forms 1,6-α-branch points in amylopectin molecules. Two classes of BE exists in cereal crops: BEI and BEII (two isoforms: BEIIa and BEIIb). In the mutants , the structure of amylopectin was changed with the increased short glucans [In the mutant of ndosperm . The debndosperm ,120. In  glucans . Moreove glucans . Maize P glucans . SBEIIb  glucans . opaque2 in rice can change the amylopectin branching patterns [In plants, there are five classes of SS. These SSs are similar at the C-terminal region, but obviously differed at the N-terminal region, which provide unique functions. SSI, SSIIa and SSIIIa are also required for the synthesis of amylopectin in maize. They elongate amylopectin chains with DP 6–7 to DP 8–12, DP 6–12 to DP 13–24, and long chains connecting amylopectin clusters, respectively ,124. SSIpatterns . Proteome is the entire set of proteins expressed by a genome under particular conditions, and it represents the key enzymes and proteins in biochemical processes . Proteompul and isa mutants [In maize endosperms, certain SSs and SBEs exist in multisubunit complexes  ,99,133.  mutants ,121. Thu mutants . SSIIa, sbeIIb contains sucrose response elements (SURE), which can regulate seed starch synthesis based on sugar availability. The details in the regulatory network of starch synthesis needs to be clarified in the future. Some non-enzyme proteins are involved in starch synthesis. 14-3-3 protein, one of regulatory factors family, was associated with BEII, SSI, and SSII in the developing barely endosperm . ProteinMoreover, transcription factors are involved in controlling starch biosynthesis, starch content and amylose/amylopectin ratio, such as ZmMADS1a, ZmCBM48-1, ZmbZIP22 and ZmMYB14 ,140,141.In summary, the knowledge on starch biosynthesis in maize endosperm, especially the identification of key enzymes, proteins and regulatory factors, can provide the clue to change the properties of starch and to redesign special starch for bioethanol production ,145,146.After long term domestication and cultivation, modern maize has a large number of phenotypic and genotype diversities. Common maize is classified as flint, pop, flour, dent, sweet maize, etc., mainly based on the morphological, rheological, functional and thermal properties of starch in endosperms . For exaA combinatorial approach through genomics, proteomics, and other associated-omics branches of biotechnology is proving to be an effective way for accelerating genes or enzymes discovery and the crop improvement programs. Compared with traditional plant breeding methods , genetic engineering techniques can precisely target DNA sequence to obtain desired agronomic traits  of plants ,20,21,22SBEIIa or SBEIIb, with better applications in the areas of films, foods, medical treatments and textiles [Bt2, Sh2, Sh1 and GBSSIIa and suppression of SBEI and SBEIIb by RNAi, the starch content increased 2.8–7.7% and amylose content increased 37.8–43.7% in maize endosperm. Additionally, the 100-grain weight increased 20.1–34.7% [Wx1, which encodes the GBSS gene that is responsible for making amylose. In the absence of GBSS expression in the endosperm, amylose was not synthesized, and this created a high amylopectin (waxy) maize with improved digestibility and the potential for bio-industrial applications [Wx mutant allele acquisition and breeding efficiency by conversion of parental lines from normal to waxy maize were overcame [Below, we have summarized recent studies on modulation of starch composition, structure and properties in cereal crops through genetic engineering techniques  23,24,1,157,158.overcame .TaSBEIIa and TaSBEIIb of wheat were silenced simultaneously by RNAi [RNAi construct may silence multiple genes if these genes have high similarity. For example,  by RNAi . Many en by RNAi . TherefoWx gene from two elite cultivated rice lines (XS134 and 9522) with CRISPR/Cas9 technology, glutinous (sticky) rice varieties with reduced amylose content (0–5%) were developed, resulting in better brewing performance [SBEI and SBEIIb in rice were obtained using CRISPR/Cas9 technology. The content of amylose and resistant starch significantly increased in the sbeIIb mutant, but no difference in starch features was observed between the sbeI mutant and its wild type [IbGBSSI (encoding granule-bound starch synthase I) and IbSBEII (encoding starch branching enzyme II), the amylose percentage in sweet potato (ipomoea batatas) starch reached 5.5% and 40.3%, respectively. The two potatoes with different starch compositions are excellent parental lines in genetic crossing to produce novel properties for food and industrial applications [Wx1 has been targeted in the potato to produce waxy potatoes, with improved cultivars aimed predominantly at the industrial starch market [CRISPR/Cas9 system can be used to target the genes/enzymes related to starch biosynthesis pathways ,160. It formance . A similild type . After kications . The genh market . TherefoBrassica napus, the genomic study of the maize is relatively simple. Similar results may be obtained by manipulating homologous target genes in maize by analyzing the role of some target genes in improving starch in other crops. Therefore, the design of transgenic maize by genetic engineering strategies will produce unique starches as raw materials for bioethanol production [Compared to many allopolyploid crops, such as wheat, potato, and oduction . It is fWe are still facing many challenges in creating easily degraded maize starch as feedstock in bioethanol industry. Although the pathway of starch metabolism is largely clear, the network and molecular mechanism of regulation involved in starch biosynthesis remains unknown, especially the regulation of the ratio of amylose/amylopectin and their accumulation. Many genes and regulatory factors have been found to contribute to starch biosynthesis, but the key genes are still unclear. Thus, we here summarized proteomic and molecular studies on maize starch metabolism and indicated the key genes of amylose and amylopectin. Alterations in starch structure can be achieved by modifying genes encoding the enzymes responsible for starch synthesis. Based on the discussion above, it is proposed that GBSS, BE and DBE among others  are the At present, maize kernels are the main raw material used for bioethanol production. However, the composition of common maize endosperm starch is far from optimum to serve as feedstocks for yeast fermentation in bioethanol production. In particular, the amylose/amylopectin ratio, amylopectin architecture and the number of small starch granules need to be modified by genome editing technology for efficient enzymatic digestion during bioethanol production. The discovery and isolation of target genes is the premise of genetic engineering. Proteomic analysis and transgenic studies have identified the key enzymes and regulatory factors involved in starch biosynthesis. The knowledge on controlling the synthesis of amylose to amylopectin and the formation of starch structures provides a clue for designing easily degraded maize starch as feedstock in the bioethanol industry.


GROUP 3 (n = 10) Zirconia copings with self-adhesive resin cement without pretreatment. GROUP 4 (n = 10) Zirconia copings with self-adhesive resin cement pre-treated with Visio.link adhesive. Universal testing machine was used to evaluate the tensile bond strength of these copings. The results were analyzed using SPSS software Version 25.0 . One-way ANOVA and independent t-test were used to compare the mean scores. Statistically significant increase was observed in Tensile Bond Strength of samples when Visio.link adhesive was used. Tensile Bond Strength of PEEK copings and Zirconia copings with Visio.link adhesive is considerably greater than PEEK copings and Zirconia copings without adhesive. The mean Tensile Bond Strength of Zirconia (with or without adhesive) is less as compared to Tensile Bond Strength of PEEK (with or without adhesive), but the difference is not statistically significant.This in vitro research aimed to evaluate the Tensile Bond Strength of Poly Ether Ether Ketone and Zirconia copings using resin cement with or without Visio.link adhesive. From commercially available Zirconia and PEEK, blocks were machined milled using (CAD)/(CAM) to obtain 20 Zirconia and 20 PEEK copings. These specimens were sandblasted using 110 μm of alumina. The two main groups (20 Zirconia and 20 PEEK copings) were divided further into 4 subgroups, GROUP 1 ( PEEK (Poly Ether Ether Ketone), regarded as methacrylate-free semicrystalline thermoplastic material, is a crucial prototypical member of the polyacryletherketone (PAEK) family . It compPEEK is advantageous over other materials, however, its whitish color, low translucency, and absence of durable bond limits its use as a restorative material . Hence, Adhesives improve the bonding by complex exchange between the substructure and resin cement . Higher Zirconia, on the other hand, has proven chemical and dimensional stability. It has opened new horizons for metal-free dentistry in the past few years. It has superior wear resistance in comparison with other ceramic materials . The mosVisio.link is a light curing bonding agent (primer) for veneers and artificial teeth based on polymethylmethacrylate and composites. It ensures firm bonding of highly cross-linked PMMA teeth. Researchers have shown that visio.link has the highest bond strength with PEEK restorations when used following ideal protocol and alumina blasting. Visio.link is a bonding agent that incorporates methyl methacrylate monomer. As a result, it is possible that the visio.link dissolved the tested PEEK material at its surface, and the free carbon double bond polymerized with carbon compounds from the bonding agent and the resin composite cement. MMA and PETIA (pentaerythritol triacrylate) are the two primary components of Visio.link. Visio.link gave higher binding strength values to PEEK restorations because of PETIA’s high capacity to change the PEEK surface .Tensile Bond Strength helps us to determine the load at which the adhesive bond of resin cement with coping and tooth fails and dislodgement of coping takes place.The studies done before were on PEEK material to determine the best adhesive and pre-treatment required to increase the bond strength and thus the durability of the restoration. However, the most commonly used aesthetic material, along with PEEK, is Zirconia. Previous studies have shown that applying a 10-Methacryloyloxydecyl dihydrogen phosphate (MDP)-containing bonding or silane-bonding agent mixture to ceramic zirconia yields superior shear bond strength. In a study done by Hasan Sarfaraj et al. in 2020, 6-methacryloyloxyhexyl phosphonoacetate (6-MHPA) was used as an adhesive agent for zirconia and composite veneer. The good chemical interaction between the 6-MHPA or 6-MHPP (-P(=O)(OH)2) phosphonic acid group and metal oxides on the zirconia surface was assumed to be responsible for the effective zirconia bonding . PreviouThe null hypothesis tested was that there are no differences in the Tensile Bond Strength of Poly Ether Ether Ketone and Zirconia using resin cement with or without Visio.link adhesive.This experimental in vitro study aims to evaluate the Tensile Bond Strength of PEEK and Zirconia copings on freshly extracted maxillary central incisor under universal testing machine before the commencement of study IEC approval was obtained with reference number 2019/IEC/300/3.The inclusion criteria for the study was freshly extracted maxillary central incisor, and exclusion criteria were posterior tooth, carious tooth, restored tooth, or fractured tooth.A freshly extracted Maxillary Central Incisor was collected from Oral and Maxillofacial Surgery department. Patient consent was taken, patient was a known case of aggressive periodontitis, and extraction of mobile tooth was done. The calculus deposits and soft tissue debris were washed off using 0.5% sodium hypochlorite and tap water. The natural teeth were used in the study because we needed to gauge the bond strength of peek and zirconia adhesively luted to human dentin. The tooth was then entrenched into a self-cure acrylic resin block. The tooth was prepared under standard dimension for all ceramic crown, and a shoulder gingival finish line of 1 mm width was uniformly made. An impression of prepared tooth was made using addition polyvinylsiloxane material. The impression was then sent to a commercial lab  for fabrication of PEEK and Zirconia copings using CAD/CAM software  . The labThe specimens were surface treated using sandblasting of 110 μm of aluminum oxide.The sample consisted of 20 Zirconia and 20 PEEK copings. This sample size was selected because it was the minimum required number to obtain statistically significant result. These groups were further divided depending upon the use of adhesive. The groups are as follows:Groups surface Treatmentsn = 10): PEEK substructure cemented with self-adhesive resin cement using no pre-treatment.GROUP 1 (n = 10): PEEK substructure cemented with self-adhesive resin cement pre-treated using visio.link adhesiveGROUP 2 (n = 10): Zirconia copings cemented with self-adhesive resin cement using no pre-treatment.GROUP 3 (n = 10): Zirconia copings cemented with self-adhesive resin cement pre- treated using visio.link adhesive.GROUP 4 (n = 10):Group 1  was mixed and manipulated following manufacturer’s instruction and the PEEK copings were light cured for 5 s and cemented.n = 10):Group 2 . The polymerization time was chosen according to the manufacturer recommendation. After the application of adhesive, the resin cement  was mixed and manipulated according to the manufacturer’s instruction and the PEEK copings were cemented. Cement was light cured for 5 s per side.n = 10):Group 3  was mixed and manipulated according to the manufacturer’s instruction and the zirconia copings were cemented and light cured for 5 s per side.n = 10):Group 4  was mixed and manipulated according to the manufacturer’s instruction and the Zirconia copings were cemented. Cement was light cured for 5 s per side.Universal Testing Machine (UTM) was employed to evaluate the tensile bond strength of the samples. A 23-gauge wire was attached through the hole for testing the specimens as shown in The universal testing machine (INSTRON 3382 series operating on Bluehill software) with a crosshead speed of 0.5 mm/min was used to dislodge the cemented copings and the force required for the same was measured .2) of the prepared tooth. The below-mentioned formula was used to calculate the surface area of tooth.The readings at which copings were debonded were noted . The disThe dimension of the tooth was measured using Vernier Calliper. The following are the specification:d1 = 2 mmd2 = 4.7 mmh = 7.5 mmd3 = 5.7 mm2. The following formula was used to measure the tensile bond strength:Thus, the surface area calculated was 90.232 mmt-test were used to obtain the results.Data was calculated in Microsoft Excel spreadsheet and descriptive data was evaluated using SPSS software Version 25.0 . The statistical test one-way ANOVA and independent The dependent variables used in the study were zirconia, PEEK (a high-performance polymer), resin cement, and Visio.link adhesive.p-0.000) (The outcome of the result depicted that the mean tensile bond strength of Poly Ether Ether Ketone (PEEK) without visio.link adhesive group was 0.607 + 0.0211 (MPa) and for PEEK with visio.link adhesive group it was more 1.292 + 0.0282 (MPa), thus the result shows a significant difference between these groups (p-0.000)  Table 1p-0.000 (p-0.000) (The mean tensile bond strength of Zirconia without visio.link adhesive group was 0.549 + 0.0375 (MPa) and for Zirconia with visio.link adhesive group it was more 1.225 + 0.0317 (MPa) and the difference between the groups was also highly significant (p-0.000)  Table 2p-0.000 (p-0.000) (The comparative difference in Tensile Bond Strength between Zirconia and PEEKgroup was Zirconia without visio.link adhesive showed 0.549 + 0.0375 (MPa) and mean tensile bond strength of Poly Ether Ether Ketone (PEEK) without visio.link adhesive was 0.607 + 0.0211 (MPa). For PEEK with visio.link adhesive was 1.292 + 0.0282 (MPa), and for Zirconia with visio.link adhesive was 1.225 + 0.0317 (MPa). Both the groups with visio.link adhesive showed more mean tensile bond strength compared to those without visio.link adhesive group, and the result showed a significant difference between these groups (p-0.000)  Table 3p-0.000 (p-0.576) (When we compare the PEEK (with and without visio.link adhesive) and zirconia (with and without visio.link adhesive) groups, the mean tensile bond strength of Zirconia was less 0. 887 + 0.3484 compared to PEEK group 0.949 + 0.3522, but the result shows no statistically significant difference (p-0.576)  Table 4p-0.576 .The obtained results showed that mean Tensile bond strength of Poly Ether Ether Ketone (PEEK) and Zirconia without visio.link adhesive group was 0.607 + 0.0211 and 0.549 + 0.0375 (MPa), respectively, and for PEEK and Zirconia with visio.link adhesive group was 1.292 + 0.0282 and 1.225 + 0.0317 (MPa), respectively. Both the study groups which used visio.link showed statistically highly significant bond strength .When we compare the PEEK (with and without visio.link adhesive) and zirconia (with and without visio.link adhesive) groups, though the mean tensile bond strength of Zirconia was less 0.887 + 0.3484 compared to PEEK group 0.949 + 03522, the results were not statistically significant (In this article, the tensile bond strength was measured in vitro without thermocycling, and studies should be done after thermocycling for better evaluation of bond strength, although the appropriate and adequate measures were used to control the technicality in the specimen fabrication and to minimize errors while testing.Future studies should be done to incorporate the composition of Visio.link adhesive to resin cement for better clinical results.Adhesive helps to build a durable bond of copings with resin cement. The use of resin cement alone is not recommended. In clinical practice we have to include the use of adhesive, and the results will be favorable.Bonding of PEEK and Zirconia copings almost doubles after the use of Visio.link adhesive. Adhesives increase the surface area and thus the bonding of copings with natural tooth. Tensile bond strength of PEEK is more than Zirconia, but the difference is not significant.


G″/G′ > 0.1).The aim of the study was to assess the effect of soil type and the application of fertilizer composed of ashes from biomass combustion to potatoes on selected physicochemical, rheological, and thermal properties of potato starches isolated by using the laboratory method. Potatoes were grown in Haplic Luvisol (HL) and Gleyic Chernozem (GC) soil and fertilized with different doses of biomass combustion ash (D1–D6) with different mineral contents. The thermodynamic characteristics of gelatinization and retrogradation were identified by DSC. The analyses of rheological properties included the determination of the gelatinization characteristics by using the RVA method, flow curves, and assessment of the viscoelastic properties of starch gels. It was found that the starches tested contained from 24.7 to 29.7 g/100 g d.m. amylose, and the clarity of 1% starch pastes ranged from 59% to 68%. The gelatinization characteristics that were determined showed statistically significant differences between the starches analyzed in terms of the tested factors. The value of maximum viscosity and final viscosity varied, respectively, in the range of 2017–2404 mPa·s and 2811–3112 mPa·s, respectively. The samples of the potato starches studied showed a non-Newtonian flow, shear thinning, and the phenomenon of thixotropy. After cooling, the starch gels showed different viscoelastic properties, all of which were weak gels (tan δ =  Solanum tuberosum L.) are one of the most important consumer crops in the world. The annual global production of potato tubers exceeds 380 million tons [Potatoes  cv. Sagitta . The potatoes came from a field experiment involving the study of the effect of fertilizing with ashes from biomass combustion on the cultivation of potatoes. The details regarding the field trial are provided in The research material was provided by starches isolated from potatoes , and then grounded and sieved through a sieve with a mesh opening of 0.125 mm .2). The research factors were as follows. (I) Type of soil: Haplic Luvisol and Gleyic Chernozem. (II) Varied fertilizer application to the potato: Control—only N and P fertilizer; D1—NPK mineral fertilizer; and D2–D6—N and P mineral fertilizer + ash from biomass with different doses, i.e., 0.5, 1.0, 1.5, 2.0, and 2.5 t·ha−1, respectively.A field experiment located in Southeastern Poland (50°3′ N 22°47′ E) was carried out in 2020. The experiment had a two-factor design with four replications and 56 plots ), monoammonium phosphate (MAP) NH4H2PO4 (12% N-NH4 52% P2O5 (22.7% P)), and phosphorus (in the form of Monoammonium phosphate (MAP)) were constant. In variants D2 to D6, ashes from forest biomass combustion (70%) and agricultural biomass (30%) were used as fertilizer [Mineral nitrogen fertilizer (in the form of RSM−1); a very high availability of Mg (36.4 mg·100 g−1); and an average availability of K (12.85 mg·100 g−1), Fe (845 mg·kg−1), Mn (147 mg·kg−1), Zn (5.49 mg·kg−1), and Cu (2.20 mg·kg−1). In turn, the availability in Gleyic Chernozem soil of P (2.56 mg·100 g−1) was low; of K (7.23 mg·100g−1) was very low; of Mg (13.51 mg·100 g−1) was high; and of Fe (1439 mg·kg−1), Mn (216 mg·kg−1), Zn (11.39 mg·kg−1), and Cu (5.28 mg·kg−1) was average [The experiment was carried out on two types of soils: Haplic Luvisol with a Silt (Si) particle size composition and Gleyic Chernozem with a Silty Loam (SiL) particle size composition . The HapThe isolated potato starches were analyzed for selected physicochemical parameters. The dry-matter content was determined by measuring weight loss during drying at 130 °C . The appL* coordinates, describing the brightness; a*, expressing the red–green color balance; and b*, expressing the blue–yellow color balance. The analysis was performed in triplicate in the 400–700 nm spectral range. On the basis of the results obtained, the whiteness index (WI) [The starch color analysis was also performed by spectrophotometric method, using a Color i5 spectrophotometer . The illuminant D65 was used with the measurement geometry d/8. Measurement results were recorded in the X-Rite Color Master software. The value of was determined for the dex (WI)  was calc3, according to method described by Szpunar-Krok et al. [The contents of basic macro- and microelements  were determined by means of the atomic absorption spectrometry technique  after wet mineralization in 60% HNOk et al. . Phosphok et al. .O), peak (TP), and end set (TE) gelatinization temperature and enthalpy (∆HG) of gelatinization were calculated. After cooling, the samples were stored in the refrigerator at 4 ± 1 °C for 7 days. Retrogradation was measured by reheating the sample pans containing the starches investigated in the same conditions as for gelatinization. The onset (TO), peak (TP), and endset (TE) temperatures and enthalpy (∆HR) of retrogradation were calculated. The index of retrogradation (%R) was calculated from the ratio of ∆H retrogradation to ∆H of gelatinization [Thermodynamic gelatinization characteristics were obtained by using a DSC F204 Phoenix  differential scanning calorimeter. A water–starch dispersion (3:1) was heated in the DSC aluminum pan in the temperature range of 25–110 °C, with a heating rate of 10 °C/min. The empty aluminum pan was used as a reference. On the basis of the thermograms obtained, onset . Samples  were kept at 50 °C for 1 min, then heated to 95 °C at 12 °C/min, kept at 95 °C for 3 min, cooled to 50 °C at a rate of 12 °C/min, and finally kept at 50 °C for 2 min. On the basis of the viscograms, the following values were read: gelatinization temperature (PT); maximum viscosity during heating (PV); viscosity at 95 °C (HPV); final viscosity at 50 °C (FV); decrease in viscosity during heating, value = PV-HPV (BD); and viscosity increase during cooling, value = FV-HPV (SB) [−1. The experimental curves were described by the power law equation:apη is the apparent viscosity (Pa·s), K is the consistency coefficient (Pa·sn), −1), and n is the flow index [The starch pastes prepared in the RVA analyzer were used in these experiments. The rotary viscometer Rheolab QC  with a system of coaxial cylinders  was used for the determination of viscosity curves at temperature of 50 °C in the shear rate range from 1 to 300 sow index .G′ is the storage modulus (Pa); G″ is the loss modulus (Pa); ω is the angular frequency (rad/s); and K′, K″, n′, and n″ are the constants.The starch pastes prepared in the RVA analyzer were used for these experiments. The sweep frequency test at 25 °C was carried out by using Mars II rheometer  with cone/plate geometry  [All analyses were performed in triplicate. In order to check the normality of the distribution at α = 0.05, the Shapiro–Wilk test was performed. The homogeneity of variance was also checked. For the statistical evaluation of the results, the two-way ANOVA was used. The significance of differences between the means was determined by using Tukey’s post hoc test at a significance level of 0.05. Pearson’s linear correlation coefficients were also calculated between the study parameters characterizing potato starches, and their significance was assessed at the level of 0.05. All statistical calculations were performed in TIBCO Statistica 13.3 .G, n′, and K″ .A high clarity of starch pastes is an important feature affecting the use of starch in the production of fruit gel, jellies, and cake fillings. The results of statistical analysis indicate that the clarity of potato-starch pastes statistically significantly depended on the type of soil, type of fertilizer used, and the interaction of these factors. The clarity of 1% starch pastes ranged from 52.5% (D3GC) to 68.0% (D5HL). The mean clarity value of starch-based pastes from potatoes grown in HL soil was 3.4% higher when compared with starch-based pastes from potatoes grown in GC soil. The fertilizer use was also an important factor in this regard, as, in general, the clarity of starch pastes depended on the chemical composition of ash from biomass combustion. Therefore, fertilizing with ash, which has the highest content of potassium (D6), resulted in an increase in clarity in tandem with an increasing share of this element. Pycia et al. . MoreoveL* parameter, which is the brightness of the sample. The value of this parameter ranged from 90.2 (Control HL) to 92.9 (GCD4). The values obtained are similar to those presented by other authors [a* (in the range from green to red) and b* (in the range from blue to yellow). The b* parameter reached positive values for all the starches analyzed, which proves the color shift toward yellow. On the other hand, the values of the a* parameter in the case of HL had positive values, and in the case of GC, it had negative values, thus indicating a shift toward the green color. The mean value of the a* parameter of GC starch was 0.164, and it was five times higher than HL. Higher mean values of the b* parameter was achieved by starches from potatoes grown on HL and fertilized with D3. Whiter starch was obtained from potatoes grown in HL soil, as evidenced by the average values of the WI* parameter.Color is a parameter that provides information about the quality of raw materials and the products based on them. The color of raw materials is related to their chemical composition. Changes in color parameters usually take place under the influence of storage conditions or the processing process . The two authors ,21. The −1year−1 (D1) to 469 kg ha−1year−1 (D6). However, potassium was not present in the control fertilizer [−1year−1 of phosphorus. The type of soil, fertilizer applied, and interactions between soil type and fertilizer used had a statistically significant influence on the level of potassium content in the studied starches . On the other hand, the value of TP was influenced by fertilizer applied and the interaction of both factors, and the TE value was only influenced by the interaction of factors. The values obtained were higher than those presented by Pycia et al. [G, of potato starch. In turn, potato starch with low water absorption and water solubility has a higher value of To, TP, and TE of the gelatinization process [P and TE. Low temperature during the growing season of potatoes, sweet potatoes, and rice increased the gelatinization temperature and the gelatinization enthalpy value determined by the DSC method [G, in terms of quantity and quality, provides information about the crystallinity of the starch grain and is an indicator of the loss of the granular structure of starch [E) for starches from potatoes grown on GC soil was 2.6 °C higher than on HL. The retrograde enthalpy value ranged from 5.10 (GCD2) to 8.57 J/g (HLD4). The change in the thermal properties of starch during gelatinization and during refrigerated storage depends on the ratio of amylose to amylopectin, the size and shape of the granules, and the presence of lipids. The amylose content is considered to be the main factor influencing the starch retrogradation process [R and ΔHG, the retrogradation index (R%) was calculated. The value of this parameter ranged from 29.3% (GCD3) to 44.2% (HLD4). The calculated R values are slightly lower than those presented by Pycia et al. [Differential scanning calorimetry (DSC) makes it possible to determine the temperatures characteristic for starch gelatinization and the enthalpy of this transition. These parameters make it possible to indicate the differences between the starches isolated from potato tubers that are obtained from crops with different fertilizers applied . The para et al. ,29. Morea et al. , the tem process , the dif process  also obsf starch ,31. The f starch , the gelf starch ,14. The f starch ,14. The  process . Based oWhen starch is heated under hydrothermal conditions, the suspension is transformed into a dispersion system, the viscosity of which changes with time and temperature. Initially, starch granules absorb water, swell, and increase in volume until they break and release amylose. The temperature at which this occurs is called the pasting temperature, and the product obtained is starch paste. The viscosity of the paste depends largely on the concentration of starch. Thus, the starch paste is a two-phase system in which entangled amylose chains are the continuous phase, and the swollen starch granules are the dispersed phase. During the cooling of such a system, an increase in viscosity is observed as a result of amylose and amylopectin chains joining hydrogen bonds . Among sp ≤ 0.05).The parameters of the pasting characteristics of the study starches are summarized in Under hydrothermal conditions, the starch suspension is transformed into a colloidal system, in which the continuous phase is formed by amylose released from starch granules, and the dispersed phase is formed by fragments of starch granules containing amylopectin. The starch paste exhibits the properties of a non-Newtonian shear-thinning fluid. The rheological properties of starch paste strongly depend on, for example, time and speed of shear, starch concentration, measurement temperature, and botanical origin of the starch ,10. The 2. The parameters of the power model describing the experimental curves are the coefficient of consistency (K) and the flow index (n). It was shown that all the parameters analyzed had a statistically significant influence on the value of the K and n parameter. The mean value of the consistency coefficient, which proves the initial viscosity, for starches cultivated on HL soil was about 12% higher than those obtained from cultivation on GC soil. It was shown that fertilizer application had a different effect on the K value. The highest K value was found for starch from potatoes fertilized with D1. The melt index ranged from 0.397 (HLD6) to 0.506 (GCD5). The melt index indicates the rate of viscosity decrease with the increase of the shear rate [K and n, and the content of Mg in the starch , as well as between the values of the parameter n and K′ and n″ .To describe the flow curves, a power model was used, and it described the experimental data that were obtained well. The parameters of the model used to describe the experimental curves are summarized in 0.397 HLD to 0.506G′) and loss (G″) of the starch gel, depending on the angular frequency (ω). The conservative modulus (G′) characterizes the share of elastic properties in the tested material and is responsible for the part of the energy that is stored. On the other hand, the loss module (G″) gives information about the energy lost or dispersed during deformation. The ratio of lost-to-stored energy in each cycle is described as the tangent of the phase shift angle (tan δ), which gives information about the physical behavior of the system [G′) and the loss modulus (G″) on the angular velocity, are shown in G″/G′) greater than 0.1 (tan > 0.1)  b. MoreovG′ modulus depends on the amylose content in starch grains, the swelling capacity of starch grains, the degree of crystallinity, the ratio of amylose to amylopectin, the length and distribution of amylopectin chains, and the degree of amylopectin polymerization [K′, while the value of the constant K″ did not depend only on the type of soil. The value of the constant K′ and the constant K″ reflect, respectively, the modules G′ and G″ at an angular velocity equal to 1 rad/s. It was found that the value of the constant K′ representing the initial magnitude of the modulus G′ ranged from 92.7 (HLD5) to 131.5 (GCD2). On the other hand, the value of the constant K″ corresponding to the initial value of the modulus G″ ranged from 14.5 (GCD4) to 22.5 (GCD2). It was shown that the average highest values of the constants K′ and K″ were recorded for the cultivation of potatoes on HL soil and fertilized at the D2 level. The values of the K′ and K″ constants indicate that, in all starch gels analyzed, there was an advantage of viscous properties over elastic properties. The values of the n′ and n″ constants depended statistically significantly on the fertilizer applied and the interaction between the fertilizer applied and soil. The values of the n′ and n″ constants reflect the sensitivity of the modules to changes in angular velocity. The type of fertilizer applied and the interaction between the type of soil and fertilizer applied significantly influenced the values of these parameters. The GCD4 sample , as well as the parameters n′, K″, n″, and FV, respectively .The value of the rization ,34. The 4 sample  showed tAsh from biomass combustion can be an ecological and valuable mineral fertilizer used for fertilizing food and starch potatoes. The research conducted proved that the minerals present in this fertilizer, to some extent, affect the physicochemical, thermal, and rheological properties of starch. Moreover, a statistically significant influence of soil type and interactions between soil and fertilizer applied on the values of most of these parameters was demonstrated. Nevertheless, the direction of these changes was not clear cut. The amylose content generally decreased as the mineral content of the starches increased. A similar tendency was observed in the case of the clarity of starch pastes, which is an important feature in terms of their use in the development of food concentrates. The highest viscosity value was characteristic for paste based on starch isolated from potatoes grown in Haplic Luvisol soil and fertilized at the D6 level. The opposite trend was observed in the case of starch derived from potatoes from Gleyic Chernozem soil. The pastes based on all tested starches showed a non-Newtonian shear-diluted flow. On the other hand, after cooling, the starch paste had the character of weak gels.


Starch is a common biopolymer that can be used for removing heavy metal ions from aqueous solutions. A valuable property of starch is its functional diversity, which can be enhanced by chemical modification. Hydroxyl groups enclosed in the starch and formed during hydrolysis act as reducing agents of Cr(VI). The sorption properties of native starch depend mainly on the presence of carboxyl groups formed during redox processes and basic centers created during acid hydrolysis, while the superiority of phosphorylated starch is related to the presence of phosphate groups binding Cr(III) ions. The effectiveness of starch depends on a series of equilibria established in its aqueous suspension and chromate ions solution, where the pH is the driving force for these processes. In this article, a systematic discussion of pH changes being the consequence of chemical reactions unraveling the extraordinary functionalities of starch was given. It also explained the influence of establishing equilibria and chemical modifications of starch on the efficiency of chromium ion removal. This allowed for the development of a comprehensive mechanism for the interaction of Cr(VI) and Cr(III) ions with native and phosphorylated starch. Potato starch, of which Poland is one of the world’s largest producers, is an attractive sorbent because of its low cost and ease of modification. The main advantage of starch is its availability: corn, potato, rice, tapioca, wheat legume, and green fruit are common sources of commercially provided starch ,2. The mChemical modification of native starch is achieved through etherification, esterification, cross-linking, grafting of starch, or decomposition by hydrolysis and oxidization ,15,16,17One of the potential applications is the use of this native and modified biopolymer for removing transition metal ions from wastewater ,22,23,242O72− anions to Cr3+ and Cr5+ ions, which were subsequently adsorbed on starch. In native starch, the positive zeta potential on the surface of the starch grains facilitated the interaction of anionic Cr2O72− species with reducing -OH groups of the starch. Simultaneously with metal reduction, carboxyl groups of the starch were created, which served as adsorption centers for reduced chromium ions. The activation energy, calculated from kinetic data, indicated that the starch–chromium interaction was controlled by a chemical reaction formulated by D. Park as an adsorption-coupled reduction process [in statu nascendi by reduction of Cr(VI), are much more active in coordinating to starch functional groups than those ions delivered by dissolving of the chromium(III) salts in water. In the paper [43− phosphorus groups introduced into the starch structure. Therefore, the reduced Cr(III) ions were more rapidly depleted from the suspension of phosphorylated starch than from the suspension of native starch. In this way, the phosphorylated starch extorted further reduction of Cr(VI). However, it must be pointed out that during starch chemical treatment, in addition to the main reaction, hydrolysis processes take place, modifying the biopolymer properties. The mechanism of starch hydrolysis strongly depends on the pH of the solution. In the alkaline environment, the final products of hydrolysis are lower fatty acids [In two recently published works ,27, we d process . It was he paper , leads tty acids , while iThe current work aims to provide additional data, shedding light on the mechanism of interaction of chromium ions with starch in an aqueous medium of different pH values. The effect of the environment on the hydrolysis processes was examined in native and phosphorylated potato starch by following the changes in pH values over time. The chemical changes occurring in starch biopolymer during this process were correlated with the effectiveness of chromium anion sorption and reduction. The role additional reagents play, i.e., chromium ions on various oxidation states, was also considered. It was shown that simple experiments such as pH value measurements as a function of time allow for following reaction mechanisms. Additionally, it was documented that the well-known and described process of starch hydrolysis depends not only on pH but also on the presence of other components and starch modification. The formation of basic centers in starch in an acidic medium was proved, whereas in the basic medium, hydronium ions liberated during hydrolysis of starch participate in the reduction of Cr(VI).3O+ ions decreases almost 100 times. After 24 h. the pH of the solution stabilizes with a value equal to 6.1. Changes in pH are a consequence of starch hydrolysis [3O+ ions from the water, which leads to a lowering of the acidity of the solution and creates positive potential ζ of the surface of native starch grains, as can be seen in In an acidic medium (pH = 4), the pH rapidly increases to the value of 5.8, which means that the concentration of Hdrolysis , which, Significant changes in pH value occur also in the suspension with an initial pH value equal to pH = 11. During the first hour of contact time, the value of pH decreases to pH = 7.6, which indicates a significant increase in the number of hydronium ions in the solution.3O+ ions [In basic solutions, hydrolysis leads to the breaking of glucosidic rings of starch and the creation of lower organic acids, which dissociate with the production of H3O+ ions , resultiIn It should be seen that the results of measurements of ζ potential in an acidic medium for the phosphorylated starch differ from those obtained for native starch . The surChemical modification of the starch by phosphorylation increases the effectiveness of the hydrolysis in the basic medium, which results in a little change in initial pH value (∆pH = 3.5) as compared with native starch (∆pH = 3.4). During basic hydrolysis, more hydronium ions are created in phosphorylated starch occurring under the same conditions as for native starch.2Cr2O7 as a function of time are presented in The results of the pH measurements in an aqueous solution of KThe concentration of Cr(VI) = 1 g/L, room temperature.2Cr2O7, hydrolysis occurs according to the reaction described by the Equation (1):In a water solution of K42− as the dominating form of Cr(VI) concentration.In a basic medium (pH = 11), the equilibrium is established with CrO2Cr2O7 solution. Hydronium ions liberated by hydrolysis of potassium dichromate in a basic medium cause a lowering of the initial pH value of the K2Cr2O7 in the presence of native starch changes, which is associated with the hydrolysis of the starch and processes induced by the third component—Cr(VI) ions.As can be seen in 3O+ ions from the solution. This is responsible for the positive potential of the starch surface and facilitates the approaching of Cr2O72− anions to starch [In an acidic medium (pH = 4), basic centers with a negative charge, created during hydrolysis, interact with positively charged Ho starch . The Cr(VI) species became reduced mostly to Cr(III) ions according to the reaction described by Equation (2): − groups of the starch are oxidized to very active sorption centers -COO−. Freshly reduced Cr(III) cations are bonded to starch by these new adsorption sites. Additionally, a certain number of reduced Cr(III) ions are blocking the basic centers of the starch created by acidic hydrolysis, because they compete efficiently in the occupation of these negative sites with the hydronium ions due to their higher charge than the +1 positive charge of H3O+. As a result of the blockade of basic centers, a certain number of H3O+ ions persist in the solution such that the value of ∆pH = 1.9 in the acidic solution of potassium dichromate is smaller than that of native starch suspended in water  ions in smaller amounts were also created . Simulta2Cr2O7, the pH value decreases to 8.4. This change (∆pH = 2.6) is smaller than that observed for suspension of native starch in pure water (∆pH = 3.4) . The presence of Cr(III) ions in starch was confirmed by EPR data (In the basic medium of native starch in a water solution of KH = 3.4) . The difEPR data . HydroniIn a basic medium the following reaction is also possible:However, due to the significant difference in E° values, reaction (2) is privileged.The third component (Cr(VI)) influences pH values in the water-starch system.(1)Freshly reduced Cr(III) ions blockade the basic centers formed during acidic hydrolysis. As a result, a certain amount of hydronium ions remains free in the solution and may participate in the reduction of Cr(VI) in an acidic medium. Consumption of these ions in reaction (2) is a reason for a smaller ∆pH value (∆pH = 1.9) than that for native starch in water (∆pH = 2.1). Diversity of sorption centers increases because basic centers efficiently play the role of sorbents and quickly remove the reduction products (Cr(III)), stimulating in this way the further reduction of Cr(VI).(2)Changes in pH in a basic medium are smaller (∆pH = 2.6) than in pure water (∆pH = 3.4) because a part of hydronium ions formed during the basic hydrolysis of starch is consumed in the reduction of a certain amount of Cr(VI) ions.3O+ ions remain in solution. Comparing this effect (∆pH = 1.4) with that found for native starch  ions, pH value increases less (∆pH = 1.4 in vide infra). In a basic medium, in the presence of Cr(VI) ions, the decrease in ∆pH value of phosphorylated starch is also smaller 3O+ ions remain in the solution.The basic centers created by acidic hydrolysis play a role in especially active sorption centers of the reduction product (Cr(III)) in phosphorylated starch. In an acidic medium, blockade of basic centers by Cr(III) ions occurs and H(2)The ratio of reduction products (Cr(V)/Cr(III) ions) increases in phosphorylated starch much more significantly than in native starch with an increasing value of pH.The effect of the third component (Cr(VI) ions) in the phosphorylated starch–water system consists of:Chromium(III) in water solutions exists in the form of aqua complexes (Equation (4)) and aqua hydroxo complexes (Equation (5)) formed by hydrolysis. Hydrolysis leads to an acidic medium of the solutions:3)3, presented in Comparing the results obtained for native starch in water  with pH 3O+ ions and making changes in pH values less noticeable.These results can be rationalized by the assumption that for higher concentrations of Cr(III), products of hydrolysis (reactions 4 and 5) undergo sorption on basic centers of the starch, thereby blocking sorption of H2O7)2− ions and (2) provided from Cr(NO3)3 ·9H2O solution. In the first case the role of starch was double: to reduce Cr(VI) and adsorb the reduced ions on the polymer, whereas in the second case the starch plays only the role of adsorbent.In 2O72− ions are higher than those for the ions provided by Cr(NO3)3·9H2O compound. In the salt solution, the Cr(III) ions form hexaaqua complexes in which central Cr(III) cation is surrounded by H2O ligands shielding it from the sorption centers of the starch. As a consequence, the sorption is impeded. On the other hand, Cr(III) ions produced by in situ reductions of Cr2O72− ions exhibit an unoccupied coordination sphere and therefore may easily approach the sorption centers. The values concerning native starch and starch after pretreatment in acidic conditions are similar. In both cases, the results obtained for Cr(III) ions produced by freshly reduced CrHydrolysis in an acidic medium is a very rapid process and progresses to some extent, therefore the content of Cr(III) adsorbed on native starch at the applied conditions (pH = 1) is practically equal to that on starch pretreated for 24 h in an acid solution. Starch hydrolyzed in a basic environment reveals higher effectiveness of sorption than both native starch and starch hydrolyzed in an acidic medium. In the basic medium, opening of the glucosidic rings takes place , which lPretreatment of the starch in a basic medium increases its effectiveness in further contact with Cr(VI). Sorption properties are especially enhanced.2O72− ions, to Cr(III) ions, followed by adsorption of them on the starch. The aim of the experiment presented below was to investigate the interaction of starch with K2Cr2O7 in a range of pH values pH = 1–12 and to check how the pH value influences the reduction mechanism. After a certain time, the starch-containing reduced chromium was separated, and the content of paramagnetic chromium ions (Cr(III) and Cr(V)) in starch was determined by quantitative EPR spectroscopy.All the data reported in our previous papers were obtained in aqueous solutions with a pH value equal to 1, favorable for the reduction of Cr(VI), introduced into this system in the form of CrData presented in − sorption centers created proportionally to the quantity of reduced Cr(III) and/or Cr(V) ions and basic centers created by acidic hydrolysis are present. In phosphorylated starch, there are additionally -PO43− groups that act as very active sorption centers. In a basic medium, initial value of pH is more strongly reduced in phosphorylated (In native starch suspended in Cr(VI) solution, -COOorylated  than in orylated . The mecMaterials: Commercial food-grade potato starch was obtained from WPPZ SA, Poland. Analytical grade solutions 1 M of HCl and NaOH were supplied by Chempur, whereas K2Cr2O7 and chromium(III)nitrate (Cr(NO3)3·9H2O) were delivered by POCh, Poland. Starch Phosphorylation: The procedure of phosphorylation was based on papers Lim and Seib [w/w) and Na2SO4. After mixing this suspension for 1 h at room temperature, it was dried at 45–50 °C until the water content decreased below 20%. The solid residue was heated at 130 °C for 2 h, ground, and mixed with NaOH solution (pH = 6.5) in distilled water. This treatment was repeated three times, then the product was dried at 50 °C overnight.and Seib , Sang anand Seib , and Bidand Seib . In a tyAnalyses: Phosphorus content in the starch phosphate was determined according to Polish Norm [ish Norm . In the Starch hydrolysis in acidic medium: The suspension of 10 g of native starch in 45 cm3 water with pH = 1 (obtained with 1 M HCl) was shaken for 1 h. The pH value of the solution was measured, and the starch was washed on a Büchner funnel with distilled water until a negative reaction for Cl− ions was reached. The solid sample was dried for 24 h at 50 °C.Starch hydrolysis in basic medium: The pH of the 40% weight suspension of the starch in water was adjusted to the value equal to 10. The content was mixed for 50 min at room temperature. The pH was then lowered to 7, and the solid was filtered off and washed with distilled water. The sample was dried for 24 h at 50 °C.Quantitative EPR spectroscopy: To prepare samples for EPR measurements, 5 g of the starch pretreated in the above-mentioned way were contacted with 22.5 cm3 Cr(VI) or Cr(III) solutions with a concentration of 5 g Cr/L at pH = 1 and mixed for 24 h at room temperature. Then, the samples were filtered and dried for 24 h at 50 °C. EPR spectra were measured at room temperature by an Elexsys E-500 spectrometer  at X band (9.8 GHz) with a modulation amplitude of 0.1 mT and microwave power of 3 mW. The number of paramagnetic chromium ions was determined by comparison of the value of the double integral of the area of EPR signals of an appropriate sample with that of the EPR standard [standard .The procedure of pH measurements: The pH values, in the range 1–12, were obtained by adding the required amount of HCl and NaOH solutions to volumetric flasks filled with 22.5 cm3 of distilled water, K2Cr2O7, or Cr(NO3)3 solution containing 1 g of Cr/L. Then, either 5.00 g of native potato starch or 1.00 g of phosphorylated starch was added, and the suspensions were mixed at room temperature for 24 h to properly shake up the contents; apparatus from the firm Elpin was used. Every hour, the pH value of suspensions was measured by an Elmetron pH-meter equipped with a glass electrode from the firm Eurosensor. Measurements were performed at room temperature, with a precision of reading equal to 0.05.Zeta Potential Measurements: Zeta potentials were determined in the range of pH values from 1 to 10 by Zetasizer Nano S combined with the MPT-2 Autotitrator .Native starch in a neutral water suspension at ambient temperature is reasonably stable; however, it undergoes hydrolysis in contact with water of a pH value different than 7. The mechanism of starch hydrolysis in water suspension depends not only on pH but also on the presence of other components and starch modification. The mechanism can be followed by such simple experiments as suspension of starch in water or in solutions of chromium(VI) and chromium(III) with pH value measurements as a function of time.3O+ ions and the creation of the positive ζ potential of the native starch surface, whereas the ζ potential of phosphorylated starch is neutral. This difference indicates stronger interaction between hydronium ions and basic centers created by acidic hydrolysis in phosphorylated starch than with similar centers formed in native starch.In acidic solutions, the breaking of α-1,4- and α-1,6- bonds of native starch creates basic centers. The interaction of these negatively charged centers with hydronium ions present in the slurry leads to the attraction of H3O+ ions. The effect of hydrolysis in a basic medium results in an effective lowering of pH value. More hydronium ions are created under the same conditions in phosphorylated than in native starch.In basic solutions (pH = 11), hydrolysis leads to the breaking of glucosidic rings of starch and the creation of lower organic acids, which dissociate with the production of H− groups are formed. Both of them are negative and consist of attractive sorption centers for freshly reduced Cr(III) ions. In modified starch, additionally, the third sorption center exists, namely a very active sorption center incorporated into the structure of the -PO43− group, which not only immediately attracts Cr(III) ions, but also creates much more effective basic centers during acidic hydrolysis.The third component, namely Cr(VI) ions, influences changes in pH values of hydrolyzed starch by two mechanisms: BLOCADE in an acidic medium and REDUCTION in a basic medium. Negatively charged basic centers created in starch by acidic hydrolysis are blocked by Cr(III) ions, which are products of Cr(VI) reduction. Simultaneously, a suitable quantity of -COO− groups are formed. They have a negative charge; therefore, they are attractive sorption centers for freshly reduced chromium ions. The higher pH causes a change in the reduction mechanism comprised of lowering the content of Cr(III) and increasing the number of Cr(V) ions upon increasing the pH value of the solution.In the basic medium, part of hydronium ions formed during the basic hydrolysis of starch are consumed in the reduction of a certain amount of Cr(VI) ions. Simultaneously, an appropriate quantity of -COO