Microalgal starch can be exploited for bioenergy, food, and bioplastics. Production of starch by green algae has been concerned for many years. Currently commonly used methods such as nutrient stress will affect cell growth, thereby inhibiting the production efficiency and quality of starch production. Simpler and more efficient control strategies need to be developed.Platymonas helgolandica. By adding exogenous glucose and controlling the appropriate circadian light and dark time, the highest dry weight accumulation 6.53 g L−1 (Light:Dark = 12:12) can be achieved, and the highest starch concentration could reach 3.88 g L−1 (Light:Dark = 6:18). The highest production rate was 0.40 g L−1 d−1 after 9 days of production. And this method helps to improve the ability to produce amylose, with the highest accumulation of 39.79% DW amylose. We also discussed the possible mechanism of this phenomenon through revealing changes in the mRNA levels of key genes.We proposed a novel regulation method to promote the growth and starch accumulation by a newly isolated Chlorophyta This study provides a new idea to regulate the production of amylose by green algae. For the first time, it is proposed to combine organic carbon source addition and circadian rhythm regulation to increase the starch production from marine green alga. A new starch-producing microalga has been isolated that can efficiently utilize organic matter and grow with or without photosynthesis.The online version contains supplementary material available at 10.1186/s13068-022-02174-2. Microalgae can use light and carbon dioxide for photosynthesis, release oxygen and synthesize organic substances at the same time. It is one of the biological groups with the highest photosynthetic efficiency in nature. Microalgae can provide many metabolites with commercial value, such as lipid, starch, polysaccharide and phycobiliprotein , 2. Starnt years , 7.Chlamydomonas reinhardtii culture results in increased dry weight and accumulation of multiple biomass components  compared with adding low-concentration acetate [The accumulation of starch in microalgae can be promoted by various means. In the past few years, the commonly used methods are mainly the regulation of nutrient elements, such as changes in nitrogen, phosphorus, and sulfur –10. Nitr acetate . Another acetate , 16, 17.Chlorella zofingiensis showed that light and non-light conditions can regulate cells to converse added glucose to starch or lipids [The production rate is very important in starch production. It can be achieved by increasing the growth rate of algae. Optimizing the culture medium and culture conditions is the most used method , 18, 19.r lipids . Up to nr lipids .Platymonas helgolandica var. tsingtaoensis (P. helgolandica) is a unique species of Chlorophyta that grows in the coastal waters of China  production capacity of P. helgolandica was greatly improved. The produced starch can be used as a potential substitute for food starch, and can also be used to produce clean energy such as bioethanol and biohydrogen.P. helgolandica, glucose was added to the culture to a final concentration of 0, 2, 5, and 10 g L−1, and the growth parameters were measured under mixotrophic culture (continuously illuminated) during the cultivation process , increasing by 60% (p < 0.05) compared to the control group. The concentration of starch of the 10 g L−1 glucose group is the highest (0.91 g L−1), increasing by 167% compared to the control group. After 12 days, the dry weight stopped increasing and starch accumulation reached its peak.In addition to promoting growth, the presence of glucose also promotes the accumulation of carbohydrates (mainly starch). We determined the changes in starch accumulation during the growth of ica Fig. D, E. DurTetraselmis spp. is considered as a dominant group of green microalgae to produce starch. Its allied species T. subcordiformis tends to accumulate starch when exposed to stress, compound stimulation, etc. [Tetraselmis can also use glucose for growth. In general, the increase in glucose concentration can better promote the dry weight accumulation of Tetraselmis. However, the promotion effect becomes insignificant after a certain concentration (≥ 10 g L−1) [P. helgolandica accumulated starch relatively uniquely.on, etc. . Accordi0 g L−1) . In othe0 g L−1)  and prot0 g L−1) . HoweverP. helgolandica, and can strongly promote the growth and biomass accumulation. In the subsequent cultivation, 10 g L−1 glucose was added to the KWF medium. Glucose can support the growth of P. helgolandica in different culture modes . The heterotrophic cultivation with continuous darkness can also promote the dry weight accumulation (2.67 g L−1). Compared with the continuous light autotrophic cultivation, the dry weight increased by 125% and 92%, respectively. Under heterotrophic conditions, the number of algae cells is relatively small, but the cell size and weight increased, resulting in an increase in dry weight accumulation  were used for the cultivation of P. helgolandica, and samples were taken over time to determine growth and related parameters  has the strongest promotion of cell population. The three rhythm patterns can promote the accumulation of dry weight, the highest of which is the 12:12 group. Under this rhythm the dry weight can reach 6.53 g L−1, which is 104% higher than the dry weight accumulated under continuous illumination. When the circadian rhythm is L:D = 6:18 (h), the algae can accumulate the most starch. It accounts for 61.09% of dry weight, which was 128% higher than 24:0 group  and 0.40 g L−1 d−1 (day 9), 351% and 460% improved separately  is usually close to 1:1 (amylose ratio ≈ 50% TS). After the addition of glucose, there was no significant difference in Am/Ap (p > 0.05) regardless of whether it is mixotrophic (continuous light) or heterotrophic (continuous darkness) cultured. When cultured under circadian rhythm with glucose added, Am/Ap increased significantly (p < 0.05)  can carry out 39.79% DW of amylose. The yield of amylose on glucose could reach 0.28 gAm·gGlc−1 under this cycle  and amylopectin (Ap) in the starch was determined. Starch is the main storage carbohydrate in plants and some algae. Usually amylose accounts for < 35% of most natural starches, and the rest is amylopectin . Amylose5) Table . In P. h of gbss .Table 2AChlorella sp. AE10, 60.30% dry weight accumulation can be achieved through a two-stage method of large amounts of CO2 [−1 d−1. Nevertheless, it is worth noting that adding NaHCO3 combined with CO2 supply can lead to a starch productivity of 0.5 g L−1 d−1 in T. subcordiformis, mainly due to the high-speed accumulation of dry weight [Scenedesmus sp. ASK22, the externally added glucose brought a dry weight accumulation of 4.88 g L−1, but starch only accounted for 39.93% of the dry weight, which also limited the yield [P. helgolandica grow rapidly while accumulating a large amount of starch, and finally obtain a yield of 0.4 g L−1 d−1. More importantly, the need for less light time helps to save resources under indoor production conditions.Compared with the commonly used methods of nutritional stress in starch production, adding glucose under circadian rhythm is a competitive method Table . In Chlos of CO2 . Howevery weight . In Scenhe yield . In thisL:D = 6:18 (h) will enable P. helgolandica to achieve the highest starch production rate. We further determined photosynthetic parameters and respiration efficiency of the autotrophic group , the mixotrophic group , the circadian group , and the heterotrophic group , trying to further reveal the differences in the physiological status of P. helgolandica under different culture modes. Figure Fv/Fm of different groups over time. Fv/Fm (PSII maximum electron transfer efficiency) reflects the efficiency of photosystem II (PSII). The overall change trend of mixotrophic group is the same as that of the autotrophic group. The Fv/Fm of the circadian group was relatively stable in the early stage of culture, and decreased rapidly in the later stage of culture. Due to the rapidly increasing number of cells, single cell received more limited light. The heterotrophic group consistently maintained very low PSII efficiency.According to previous experiments, we have determined that with the addition of glucose, applying a day–night cycle of c group 2:0, −Glc,Fv/Fm of the two groups were reduced by 9.71% and 20.67%, respectively, reflecting the PSII efficiency decreased with the decrease of the light time.Figure ABS can better reflect the overall performance of photosynthesis. Compared with the autotrophic group, the PIABS values of the mixotrophic group and the circadian group were lower. It is worth noting that PIABS of the mixotrophic group was lower than that of the circadian group, which may be due to the increased efficiency of trapped electrons moving to Qa− (ETo/TRo) in the circadian group [p < 0.05) compared with the mixotrophic group, which may be caused by the closure of reaction centers in the mixotrophic group due to long-term light exposure. The TRo/ABS and ETo/ABS values of the three groups added with glucose all decreased compared with autotrophic group, indicating that the presence of glucose reduced the capture and transmission of absorbed energy. In the heterotrophic group, the PIABS value was only 4.353, which decreased by 77.7% and 59.15%, respectively, compared with the autotrophic group and the mixotrophic group. TRo/ABS decreased by 15.98% (p < 0.05) compared with the mixotrophic group, DIo/RC increased by 76.52% (p < 0.05), indicating that continuous dark culture resulted in a significant decrease in energy capture and a significant increase in energy consumption efficiency. ABS/RC increased by 18.03% (p < 0.05) compared with the mixotrophic group, which may reflect that reaction centers in the cells of heterotrophic group were closed in large numbers.PIan group . The parRespiration rates were also tested Fig. D. ComparP. helgolandica are close to heterotrophic group, indicating that under long darkness, P. helgolandica tends to accumulate starch and reduce protein and lipid synthesis. The intracellular ATP content changed greatly with time , total lipids and intracellular ATP were further determined Fig. . In geneime Fig. C. The ATime Fig. B. At theime Fig. A, and ceP. helgolandica have tremendous changes. Both growth and starch synthesis are related to carbon metabolism. In order to explore the metabolic regulation mechanism of algal cells in different modes, the relative mRNA expression levels of 9 genes related to central carbon metabolism pathway, CO2 fixation, starch synthesis, and circadian rhythm regulation were determined . The photosynthetic carbon fixation pathway is promoted due to the increase of CO2 and the increase of ribulose. The oxygen produced by carbon fixation further promotes aerobic respiration and brought more energy production. In addition, the existence of the dark period contributes to the repair of the photosynthetic system [P. helgolandica.Integrating the determination of mRNA expression levels and physiological parameters Figs. , 6A, we c system , which mP. helgolandica. By adding exogenous glucose and controlling the appropriate circadian light and dark time, the highest dry weight accumulation 6.53 g L−1 (L:D = 12:12 h) can be achieved, and the highest starch concentration could reach 3.88 g L−1 (L:D = 6:18 h). The increased growth rate also helps to reduce the risk of contamination caused by long-term culture with glucose. The highest production rate was 0.40 g L−1 d−1 after 9 days of production. And this method helps to improve the ability to produce amylose, with the highest accumulation of 39.79% DW amylose. We also discussed the possible mechanism of this phenomenon through revealing changes in the mRNA levels of key genes. The results of this study will help to develop new strategies from microalgae carbon fixation to the production of biologically active substances.In this study, we proposed a novel regulation method to promote the growth and high amylose starch accumulation by a new isolated Chlorophyta Platymonas helgolandica var. tsingtaoensis HL-1, a marine green microalga, was isolated from the Donghai Sea near Yancheng, Jiangsu Province, PR China, streaking on the plate repeatedly to purify to sterility in the laboratory. Evolutionary tree of 18S rRNA sequence was drawn with MEGA 7. The 18S rRNA sequence of P. helgolandica was sequenced by BioMarker, China, sequences of other species were from published sequences in NCBI (https://www.ncbi.nlm.nih.gov/). The microalgae were previously cultivated in KWF medium, with the components as follow (per liter): 30 g sea salt, 0.875 g NaNO3, 0.812 g Tris, 0.033 g H3BO3, 0.0408 g NaH2PO3, 1.3 mg FeCl3, 0.0986 mg (NH4)6Mo7O24·4H2O, 0.3125 mg CuSO4·5H2O, 0.4417 mg ZnSO4·7H2O, 0.3665 mg CoCl2·6H2O, 0.567 mg MnCl2·4H2O, 52 mg Na2EDTA·2H2O, and adjust pH to 7.6 with hydrochloric acid. The culture medium and flasks used in the experiment are sterilized by high-temperature steam . The cells were first cultured in a 400-mL glass air bubble column photobioreactor (working volume 300 mL). Sterile air was continuously blown into the medium. The temperature was maintained at 28 ℃, and continuously illuminated from single side with cool white fluorescent lamps that provided an average irradiance of 100 μmol m−2 s−1. The algae cells were harvested during the late exponential phase and resuspended with fresh KWF ± glucose . The initial cell density was 1.5 × 106 cells mL−1 (OD680 = 0.3). The culture solutions were divided into 250-mL flasks and placed in a shaker that can start lighting at a fixed time, and the culture was continuously shaken at a speed of 120 rpm. In the experiments under different culture models, the light adjustment ability of the incubator shakers was used to maintain continuous illumination for mixotrophic culture, continuous darkness for heterotrophic culture, and to turn on/off the light at designed time for circadian culture. All experiments were in triplicates.−1). Glucose concentration detection kit  was used to measure the concentration of glucose in the cultures.The cell concentration was determined by measuring the absorbance of the algae cultures at 680 nm and counting cells with a hemocytometer and microscope. 5 mL of algae culture was suction filtered onto a piece of dried 0.45-μm filter paper, and dried to a constant weight in a 55 ℃ stove. The weight of the filter paper before and after is measured to calculate the dry weight  was calculated with the equation reported in reference [The cell pellet from 1 to 4 mL of culture was sonicated in 5 mL 95% ethanol on ice. The extracts were centrifuged at 12,000eference .2-KI solution was used to react with the supernatant, and the absorbance at 618 and 550 nm were measured to calculate the concentration of amylose and amylopectin. The concentration of amylose , amylopectin , and the ratio of amylose and amylopectin (Am/Ap) were calculated according to the standard curve prepared in advance, which was prepared using standard products of amylose and amylopectin. The growth rate , total starch (TS) concentration , amylose concentration , dry weight , starch productivity , amylose productivity , total starch content in dry weight , amylose content in dry weight , amylopectin content in dry weight  were calculated according to the equations in reference [The starch concentration and amylose/amylopectin ratio (Am/Ap) was measured by the methods described by Hovenkamp-hermelink et al. . Simply eference , 38.595 was measured to reflect the protein concentration. The total lipid was measured by sulpho-phospho-vanillin (SPV) method described by Mishra et al. [The total soluble protein was measured by the Bradford method. To describe simply, the soluble protein released by ultrasonicating the cells was dissolved with 0.5 M NaOH, the supernatant was mixed with Bradford reagent, and ODa et al. . The ATPCells from day 9 were collected for transmission electron microscopy (TEM). Cell pretreatment and section preparation were performed according to standard procedures . Cutall −1 Chl, and performed dark adaption for 20 min. A super pulse at 2100 µmol m−2 s−1, and a flash at 0.009 µmol m−2 s−1 were applied to run the OJIP program. Maximal PS II quantum yield was termed as Fv/Fm, where Fv represented the variation of chlorophyll fluorescence between maximal fluorescence (Fm) induced by saturating pulse and initial fluorescence (F0).PS II activity of algal cells was measured by a chlorophyll fluorometer . The concentration of algae cultures was adjusted to 20 mg L680 ≈ 3, and recovered in light for 30 min. The probe was inserted into the culture medium, and the consumption rate of dissolved oxygen is measured in the dark. The samples were measured every 1 s for 10 min, and the data of the last 5 min were taken to calculate the respiratory rate (μmol O2 L−1 s−2 OD−1).Respiration rate was measured by an oxygen meter . The concentration of algae cultures was adjusted to ODRNA was extracted on Day 0, Day 1, Day 4, Day 7, once in light period and once in dark period. Microalgae cultivated in KWF medium with continuous light were used as control. All the RNA samples were extracted by Trizol , and cDNA was synthesized using FastKing RT Kit . The cDNAs were used for quantitative PCR analysis using M5 HiPer Realtime PCR mix (SYBRgreen) . Target genes were obtained through genome and transcriptome annotation . The primers were designed with Oligo 7 , and a value of Additional file 1: Fig. S1. Evolutionary tree constructed using 18 s rDNA sequences. Evolutionary tree of 18S rRNA sequence were drawn with MEGA 7. The 18S rRNA sequence of P. helgolandica was sequenced by BioMarker, China, sequences of other species were from published sequences in NCBI (https://www.ncbi.nlm.nih.gov/). Table S1. Primers in this study for gene expression analysis. Table S2. Experimental expressions of the OJIP-test and their calculated values obtained for different cultured group on 6th day .


Furthermore, it has been shown that starch biosynthetic enzymes can mutually and synergistically increase their activities by forming protein complexes. This review focuses on the more recent discoveries made in the last several years. Generation of single and double mutants and/or high-level expression of specific starch synthases (SSs) allowed us to better understand how the starch granule morphology is determined; how the complete absence of SSIIa affects starch structure; why the rice endosperm stores insoluble starch rather than soluble phytoglycogen; how to elevate amylose and resistant starch (RS) content to improve health benefits; and how SS isozymes mutually complement their activities. The introduction of active-type SSIIa and/or high-expression type GBSSI into ss3a ss4b, isa1, be2b, and ss3a be2b japonica rice mutants, with unique starch properties, and analyses of their starch properties are summarized in this review. High-level accumulation of RS is often accompanied by a reduction in grain yield as a trade-off. Backcrossing rice mutants with a high-yielding elite rice cultivar enabled the improvement of agricultural traits, while maintaining high RS levels. Designing starch structures for additional values, breeding and cultivating to increase yield will enable the development of a new type of rice starch that can be used in a wide variety of applications, and that can contribute to food and agricultural industries in the near future.Rice endosperm accumulates large amounts of photosynthetic products as insoluble starch within amyloplasts by properly arranging structured, highly branched, large amylopectin molecules, thus avoiding osmotic imbalance. The amount and characteristics of starch directly influence the yield and quality of rice grains, which in turn influence their application and market value. Therefore, understanding how various allelic combinations of starch biosynthetic genes, with different expression levels, affect starch properties is important for the identification of targets for breeding new rice cultivars. Research over the past few decades has revealed the spatiotemporal expression patterns and allelic variants of starch biosynthetic genes, and enhanced our understanding of the specific roles and compensatory functions of individual isozymes of starch biosynthetic enzymes through biochemical analyses of purified enzymes and characterization of  Starch (α-glucan), which is synthesized mainly from photosynthetic products, is deposited in the storage tissues of plants and represents the most important source of carbohydrates in the human diet. Starch is a gigantic polymer of glucose molecules connected by α-1,4- and α-1,6-glucosidc bonds. The major component of starch is amylopectin, a highly branched structure, while that remaining is composed of amylose, which is essentially a linear polymer. The structure of starch, as described above, has been extensively studied at the molecular level; however, the higher order structure of starch has yet to be fully understood. To date, the higher order structure of starch has been postulated by two models and is currently under debate: the Cluster Model Nikuni  has beenArabidopsis and Chlamydomonas. Our research group revealed the functions of each starch biosynthetic enzyme by exhaustively isolating japonica rice mutants lacking these enzymes and by comparing the characteristics of these mutants with those of the wild-type in detail. The results of our studies served as the basis for the model of starch biosynthesis , are likely to be utilized as functional rice because the intake of RS is thought to promote human health. Greater variation in the properties of rice starch will likely expand its utilization in various food applications. In this review, the process of generating new rice lines, with unique and novel starch properties, by designing the starch structure, is explained based on accumulating evidence obtained through the analysis of single and double mutants, and by the introduction of high-expressing alleles of starch biosynthetic genes. Please refer to the book “Starch” on rice lines generated before 2015 -treated populations of japonica rice cultivars (Kinmaze and Taichung 65) or from the Tos17 insertional mutant panels of japonica rice cultivar, Nipponbare. Mutant lines were identified by western blot analysis of mature seed extracts to confirm the absence of specific proteins. The presence of single nucleotide polymorphisms (SNPs) was confirmed by PCR amplification of genomic DNA isolated from seedlings, followed by genotyping using cleaved amplified polymorphic sequence (CAPS) or derived cleaved amplified polymorphic sequence (dCAPS) markers. SSIIa and/or GBSSI genes derived from indica rice were selected by PCR using SNP-specific primers. Mutant lines lacking starch biosynthetic enzyme(s) were backcrossed with elite rice cultivars, ‘Akita 63’ or ‘Akita Komachi’, and homozygous BC3F2 plants were identified as described above. The selected homozygous plants were grown to maturity, and the seeds were used for extracting starch using the cold-alkaline method. The purified starch was gelatinized, debranched using Pseudomonas isoamylase, and separated by gel filtration chromatography using ToyoPearl HW55s connected in series to three ToyoPearl 50S columns to analyze the apparent amylose content. The amylopectin branch structure was analyzed by capillary electrophoresis, and starch granule morphology was observed by scanning electron microscopy.Mutant rice lines described in this article were isolated from the indica rice cultivars possess active-type SSIIa (wild-type), whereas typical japonica rice cultivars possess mutant SSIIa harboring three amino acid substitutions, thus exhibiting only 10% of SSIIa activity relative to indica rice  13–24, and an increase in short amylopectin chains with DP ≤ 12 , which is located close to the SSIIa gene on chromosome 6, carries a SNP at the exon1–intron1 boundary in japonica rice, which reduces the amount of GBSSI protein, thus decreasing the amylose content in japonica rice compared with indica rice  by threefold, compared with the be2b single mutant. Further analysis demonstrated that RS extracted from the cooked rice of be1 be2b consisted of partially degraded amylose and long amylopectin branches. Although the be1 be2b double mutant lacked two major BE isozymes, its plant growth and fertility rate were not affected, and it showed rather higher seed weight than the be2b single mutant  was similar to that of the ss1 SS2a SS3a line, containing active-type SSIIa in the absence of SSI. The absolute value of the subtraction curve of “ss1 SS2a SS3a (rice containing active-type SSIIa in the absence of SSI)—SS1 SS2a SS3a (wild-type indica rice)” was considerably lower than that of “ss1 ss2aL SS3a (japonica ss1 mutant)—SS1 ss2aL SS3a (wild-type japonica rice)”. These results indicated that the active-type SSIIa enzyme derived from indica rice compensated most of the functions of SSI. The increase in the amount of amylopectin chains with DP 6–7 and the reduction in that of chains with DP 8–12 in the absolute value of the subtraction curve “ss1 SS2a SS3a—SS1 SS2a SS3a” were minimized by 80%; however, the increase of DP 16–19 was minimized by only 50%. This implies that SSIIa derived from indica rice compensates the function of SSI in elongating the non-reducing ends of A-chains with DP 6–7 into those with DP 8–12, but does not perfectly compensate its role in the elongation of the non-reducing ends of B1-chains into those with DP 16–19. By contrast, the SS1 SS2a ss3a line generated by introducing indica rice-derived active-type SS2a allele into the ss3a japonica mutant did not increase the amount of long amylopectin chains with DP > 30. This clearly indicates that SSIIa derived from indica rice does not compensate for the function of SSIIIa. Similarly, SSI could not compensate for the function of SSIIa or SSIIIa, and SSIIIa could not compensate for the function of SSI or SSIIa  showed a remarkably different starch granule morphology  compared with the wild-type  , and SS2a ss3a ss4b gbss1L, ss2aL ss3a ss4b GBSS1, and SS2a ss3a ss4b GBSSI were generated  was high, whereas that of lines with the GBSS1 allele was low, perhaps because ADP-glucose was used for amylose synthesis  allele was identified by map-based cloning  and more amylose–lipid complex, which increased the RS content to 6% compared to that of the control line (approximately 1%) . Analyses of the recombinant inbred lines, which possess SS2a and GBSS1 but lack BEIIb, also showed high amylose content (31–34%) but had some short branches on amylose  compared with wild-type and be2b single mutant japonica rice, because of a concomitant increase in GBSSI and AGPase protein levels , presumably because the ADP-glucose content reached its upper limit and was insufficient to meet the demand of increased GBSSI levels. The RS content of rice lines lacking BEIIb was higher than that of lines lacking SSIIIa and BEIIb, regardless of the presence or absence of SS2a and/or GBSS1 alleles. This is because the loss of SSIIIa increases the expression levels of SS1, thus increasing the number of short amylopectin branches and decreasing the number of long amylopectin chains connecting amylopectin clusters  compared with typical japonica rice (20 mg per grain), indicating its potential for practical applications.Compared with mutants lacking SS isozymes such as SSI, SSIIIa  mutants  using the transgenic approach resulted in the accumulation of iodine-stainable insoluble α-glucans in the endosperm , is essential for formulating properly structured insoluble starch and for generating plump seeds. Loss of ISA1 leads to the accumulation of water-soluble phytoglycogen and the production of shriveled seeds in many plant species, including maize  by conventional crossing, the resultant genotypes (SS2a gbss1L isa1 and SS2a GBSS1 isa1) accumulated starch instead of phytoglycogen and produced plump seeds  was phenotypically similar to EM653 and accumulated phytoglycogen in the central region of the shriveled seeds  were elongated, and their chain-length distribution patterns (DP < 24) were shifted toward longer amylopectin chains, as in the severe-type isa1 japonica mutant transformed with the SS2a allele. The mild- and severe-type isa1 mutants carrying the SS2a allele differed from each other in the amounts of long amylopectin chains (DP 30–60), which were higher in mild-type than in severe-type isa1 mutant background, although the amounts of these amylopectin chains in SS2a gbss1L isa1 and SS2a GBSS1 isa1 lines were less than those in the wild-type. In addition, introduction of the SS2a allele into the mild-type japonica isa1 mutant resulted in displaying clear A-type crystallinity and polygonal starch granules, similar to the wild-type; however, this result was not obtained by the introduction of SS2a into the severe-type japonica isa1 mutant  and approximately 1 month earlier than that of the original ss3a mutant. The seed weight and yield of ‘Akita Sarari’ and ‘Akita Parari’ were also similar to those of ‘Akita 63’ and ‘Akita Komachi’, respectively. ‘Akita Sarari’ presented high yield and high amylose content, and its flour could be used for the production of noodles and bread. ‘Akita Parari’ exhibited a less sticky texture relative to the typical japonica rice cultivars and could be used for making pilaf, fried rice, and risotto. Both ‘Akita Sarari’ and ‘Akita Parari’ are now commercially available (https://starchtec.com/).The y Fujita . Cultivass3a be2b mutant  .High-level expression of GBSSI in japonica rice mutant lines lacking starch biosynthetic enzymes, analyzed their starch phenotypes, and revealed the function of individual enzymes  that affect starch properties in indica rice is expected, such as ALKd described in the present review article. In fact, six starch biosynthetic enzymes, SSI, SSIIa, GBSSI, BEI, BEIIa, and BEIIb, are known to have different alleles between japonica and indica rice cultivars. Their recombinant inbred lines showed that not only SSIIa but also SSI, EBI, and BEIIa gave a minor effect on starch structure (Luo et al. indica rice that are different from the japonica rice. Accumulating the knowledge of different alleles in indica rice variety and combining with the alleles from japonica rice will enable to expand the variation of starch further. If these allelic combinations inhibit starch biosynthesis and deteriorate certain agricultural traits such as seed size, then the lines carrying these allelic combinations could be backcrossed with high-yielding elite varieties. Designing starch structures, breeding and cultivating to suit their growth environment and increase yield, and popularizing them will enable the development of a new type of rice starch, which can be used in a wide range of applications, and can contribute to the food industry and agricultural sector in the near future.The generation of new allelic combinations of starch biosynthetic genes exhibiting no and/or high expression levels in rice, and analysis of their starch properties are starting to allow us to freely design the starch structure according to the application-based requirements. We have isolated numerous s Fujita . However


A PCL graft with an inner diameter of 1 mm is created using electrospinning (control). The PCL nanofibers are coated with PVA, resulting in a PVA–PCL graft. Mechanical property tests demonstrate that the PVA coating significantly increases the stiffness and resilience of the PCL graft. The PVA–PCL surface exhibits a much smaller sessile drop contact angle when compared with that of the control, indicating that the PVA coating has hydrophilic properties. Additionally, the PVA–PCL graft shows significantly less platelet adsorption than the control. The proposed PVA–PCL graft is implanted into the rat’s abdominal aorta, and its in vivo performance is tested at 8 weeks. The patency rate is 83.3% (10/12). The histological analysis demonstrates autologous cell engraftment on and inside the scaffold, as well as CD31/α-smooth muscle positive neointima regeneration on the graft lumen. Thus, the PVA–PCL grafts exhibit biocompatibility in the rat model, which suggests that the PVA coating is a promising approach for functionalizing PCL.The low patency of synthetic vascular grafts hinders their practical applicability. Polyvinyl alcohol (PVA) is a non-toxic, highly hydrophilic polymer; thus, we created a PVA-coated polycaprolactone (PCL) nanofiber vascular graft (PVA–PCL graft). In this study, we examine whether PVA could improve the hydrophilicity of PCL grafts and evaluate its  Ischemic heart disease has been a major cause of death for decades, causing over 8.9 million deaths globally in 2019 , as it can provide a scaffold for the host, allowing cell engrafting and autologous regeneration , 3. PolyPolyvinyl alcohol (PVA) contains a hydroxyl group in its structure and is a highly hydrophilic polymer, unlike many other synthetic polymers . BecauseTherefore, we developed a PCL nanofiber-based vascular graft functionalized with PVA. In this study, we aimed to (1) clarify whether the PVA coating can prevent platelet adhesion on PCL grafts and (2) characterize its patency and autologous tissue-like regeneration in a rat implantation model.N, N-dimethylformamide and tetrahydrofuran (3:7 w:w) in accordance with our previous study  dissolved in us study . The spiPVA (10 g)  was dissolved in water (10 g) at room temperature. Subsequently, 10 g of ethanol was added to the solution. One milliliter of the solution was sucked up with a 5 ml syringe connected to a tapered nozzle. The taper nozzle was inserted into the PCL graft, and the solution was slowly extruded. This procedure was repeated twice. Air was then blown using an empty syringe to remove the excess solution remaining in the graft. Finally, the grafts were dried overnight at room temperature (approximately 25°C).The grafts were cut using single-edge industrial blades . The cut samples were mounted on aluminum bases, coated with gold using an ion-sputter coater , and observed under a scanning electron microscope .2. Then, the compressor was set back to release the force at the same velocity. The stress–strain curve  from O (the point corresponding to the absence of compressive force). The curve then descends according to the removal of the compressive force and finally reaches B, which is the point corresponding to the complete removal of the compressive force. When the sample completely recovers its form, point B corresponds to point O. C represents the compressed thickness at the maximal compressive force. The area under the curve (AUC) of the upper line obtained under compression is denoted by “a,” and “b” denotes the AUC of the lower line obtained from releasing the compressive force. From these values, the compression work (WC), compression resiliency (RC), and T0–TM were obtained using the following formula: WC = a, RC = b/a, T0–TM = Length [O-C]. WC is an indicator of the compression susceptibility. A higher value indicates that the sample is more susceptible to compression and collapses easily. RC and T0–TM can be used to evaluate the resiliency or recoverability; the sample is observed to have enhanced elastic properties as the values of RC and T0–TM approach 100 and 0, respectively.The mechanical properties of the PCL graft were measured using a Kawabata evaluation system (KES)  Supplem with a cin curve  was obtaθ) was measured using the half-angle method (θ1) from the edge to the vertex of the drop on the contact surface was assumed to be half of the geometrical contact angle (q = 2θ1) because a small and light drop behaves as a sphere. From the values of the radius (r) and height (h) of the sphere measured using a contact angle meter , the contact angle was calculated as tan θ1 = h/r, θ = 2 arctan [h/r] . Written informed consent was obtained from all participants.Blood harvested from healthy volunteers was centrifuged at 200 × g for 5 min at 4°C to obtain the platelet-rich plasma. Two milliliters of the plasma were run into a PCL graft  three times, and the graft was flushed with 10 ml of phosphate-buffered saline  to wash out the platelet-rich plasma. The inside of the graft was observed using an electron microscope to evaluate the platelet adhesion. The grafts were placed in 2% glutaraldehyde solution (0.1 M phosphate buffer (PB), pH 7.4) at 4°C for 24 h. For conductive staining, they were immersed in 1% tannic acid (0.1 M PB) for 1 h, rinsed with a buffer solution for 1 h, and immersed in 1% osmium tetroxide (0.1 M PB) for 1 h. The specimens were subsequently dehydrated using a graded ethanol series . After dehydration, t-butyl alcohol was substituted with ethanol (10 mm × three times) and dried in a freeze dryer . The dried specimens were mounted onto aluminum bases, coated with platinum-palladium using an ion-sputter coater , and observed with a field-emission scanning electron microscope .All animal experiments were approved by the Institutional Animal Care and Use Committee of Asahikawa Medical University (reference number 20129-2).via the cardiac apex.Male Wistar rats  were purchased from the Charles River animal facility (Japan) and anesthetized with 2.0% isoflurane. The abdominal aorta was isolated through a midline laparotomy. After clamping the aorta below the renal arteries on the proximal side and above the aortoiliac bifurcation on the distal side, the aorta was transected and replaced with a PCL or PVA–PCL graft with an inner diameter of 1 mm and length of approximately 5–7 mm. Patency at implantation was judged by restoration of the graft form and lower-limb discoloration upon pinching the graft. After closing the abdominal cavity and skin, the rats were kept in individual cages and monitored for 8 weeks. The rats were provided normal food and water, and no anticoagulation or antiplatelet drugs were administered throughout this study. Finally, the grafts were harvested under general anesthesia. The patency of the graft was determined by pulsating the blood flow from a hemi-resection site in the distal aorta of the graft anastomosis. The rats were then euthanized, and the grafts were harvested after injecting 4% paraformaldehyde (PFA) The harvested grafts were fixed in 4% PFA overnight at 4°C. The grafts were then cut (i) axially into three sections: proximal, middle, and distal or (ii) longitudinally from the proximal to a distal anastomosis. They were then embedded in an optimal cutting temperature compound . The frozen grafts were sliced with a cryostat to 5 μm thickness at −20°C and used for hematoxylin and eosin (HE) staining and immunofluorescence staining (IF). For IF, heat-induced epitope retrieval was performed using tris-ethylenediaminetetraacetic acid (EDTA) buffer , and the specimens were blocked with 1% bovine serum albumin in PBS before the occurrence of antibody reactions. Hoechst 33342  was used for nuclear staining, and the following antibodies were used for IF in this study . The HE and IF images were captured using an all-in-one fluorescence microscope .The neointimal thickness was measured with Fiji . By usint-tests were used to compare the mean values between the two groups. One-way analysis of variance followed by a post-hoc test was performed to compare more than two groups. The statistical significance was set at p < 0.05.All statistical analyses were performed using GraphPad Prism9. The data were expressed as mean ± standard deviation (SD). Unpaired To overcome two issues: (i) technical difficulty in creating a tubular structure with uniform thickness and small diameter, and (ii) hydrophilicity of PCL, we fabricated PVA-coated PCL nanofiber grafts with 1 mm inner diameter as described in 0–TM indicates the stiffness ; it was significantly decreased in the PVA–PCL graft (0.18 ± 0.05 mm vs. 0.73 ± 0.10 mm in the PCL graft). Thus, consistent with the results of the handleability test, the KES method demonstrated that the PVA coating improved the resilience and mechanical strength of the PCL graft.We observed a change in the firmness of the PCL graft after the PVA coating. Therefore, we tested the handleability of the PVA–PCL graft using surgical forceps. After repeated pinches with the forceps a few times, the control PCL graft collapsed, and the inner cavity was closed easily . By contPVA is a highly hydrophilic polymer . We exam2) when compared with that in the control PCL graft (87 ± 17 platelet granules/100 μm2)  . Thus, tin vivo performance of the PVA–PCL graft as a small-diameter vascular graft. The PVA–PCL grafts were implanted into the rat’s abdominal aorta with interposition. The patency rate at 8 weeks was 83.3% (10 of 12)  . The har0 of 12) . HE stai0 of 12) . CellulaWe developed a PVA–PCL graft, where PCL nanofibers were coated with hydrophilic polymer PVA and demonstrated that (1) the PVA coating increased the mechanical properties and hydrophilicity of the PCL graft and (2) the PVA–PCL graft exhibited patency beyond 8 weeks with neointima regeneration.We demonstrated that the PVA coating added hydrophilicity to the PCL graft and significantly reduced the platelet adsorption . It has Mechanical property tests indicated that the PVA–PCL graft was stiffer and more elastic than the control PCL graft. In the PVA–PCL graft, the voids between the PCL nanofibers are filled with PVA, and the PCL nanofibers are bonded together, which increases the graft density and limits the deviation occurring between the fibers during deformation. This is presumably the reason the PVA–PCL graft demonstrates improved mechanical properties and requires greater force to deform.As a vascular graft, mechanical properties such as stiffness and elasticity are of paramount importance because the graft is anastomosed to the native vessels with a needle suture. In our preliminary study, the PCL graft without PVA coating was used for the replacement of the abdominal aorta in the same rat model as applied in the present study. However, the PCL graft was fragile, and anastomosis was extremely difficult because the graft was easily broken during the process, resulting in very poor early patency (approximately 30%), even though these experiments were performed by cardiac surgeons with adequate experience. Although there is no standardized method and it is difficult to quantify the physical experience of the anastomosis operation, from the viewpoint of a cardiac surgeon, the mechanical properties of the PVA–PCL graft used in this study appear to be sufficient and satisfactory for anastomosis.Nanofiber-based scaffolds are believed to be ideal for VTE because they can provide biomimetic microenvironments for cells to regenerate tissue , 11, 20.in vivo. In our future study, further investigations on how to sustain the function for longer periods will be undertaken. In this regard, making blended polymer is one of the options. However, to our knowledge, it is difficult to make blended polymer of PVA and PCL because solvents for PVA and PCL are aqueous and organic, respectively, and do not mix. In contrast, there is a report on fabricating copolymer of PVA and PCL  In this study, PVA solution was infused into the PCL graft for coating. Since PVA was not immobilized, we speculate that PVA disappears at some point due to its solubility and degradability, and we did not clarify the duration for which the effect of PVA is maintained  and PCL . In thisin vivo performance test showed that the PVA–PCL graft with an inner diameter of 1 mm was patent beyond 8 weeks and demonstrated autologous tissue regeneration, including the neointima layer. Our data suggest that functionalization of the PCL grafts with PVA is a promising approach for increasing hemocompatibility. Considering the development of a clinical application, further animal model tests are needed to understand and overcome the limiting factors of graft failure, such as graft length, long-term patency, performance under disease conditions, and manner of anastomosis.We developed a PCL graft functionalized with a hydrophilic PVA polymer and tested its graft performance in a rat aorta implantation model. The PVA coating added hydrophilic properties to the PCL graft, which suppressed platelet adsorption. An The original contributions presented in this study are included in the article/The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of Asahikawa Medical University.NW and TY performed data collection, analysis, and manuscript writing. MT and YK helped NW with data collection. KO performed data analysis, manuscript writing, and coordination of the study. TY and DN performed material preparation. DK performed scanning electron microscope imaging. HK conceived the study and reviewed the manuscript. All authors read and approved the final manuscript.


The breakdown values and the swelling power at 95°C of core-shell starch/zein microparticles significantly (p < 0.05) decreased by 52.83–85.66% and 0.11–0.28%, respectively. The in vitro digestibility test showed that the contents of slowly digestible starch and resistant starch in the core-shell starch/zein microparticles increased to ∼42.66 and ∼34.75%, respectively, compared to those of native CS . Our research supports the application of food-grade core-shell starch/zein microparticles to formulate low-digestibility food products.The purpose of this study was to slow down the digestibility of starch granules by encapsulating it in zein shells. Drop of the preformed swollen corn starch (CS) granule suspension into thermal-treated zein ethanolic solution enables antisolvent precipitation of thermal-treated zein on the surface of the preformed swollen CS granules, leading to the formation of core-shell starch/zein microparticles. Confocal laser scanning microscopy images showed that the preformed swollen CS granules were coated by thermal-treated zein shells with a thickness of 0.48–0.95 μm. The volume average particle diameter of core-shell starch/zein microparticles was 14.70 μm and reached 18.59–30.98 μm after crosslinking by transglutaminase. The results of X-ray diffraction and Fourier transform infrared spectroscopy demonstrated that an interaction occurred between the preformed swollen CS granules and the thermal-treated zein. The results for thermodynamic characteristics, pasting properties, and swelling power indicated that the compact network structure of core-shell starch/zein microparticles crosslinked by transglutaminase could improve starch granule thermal stability and resistance to shearing forces. Compared to native CS, the peak gelatinization temperatures of core-shell starch/zein microparticles increased significantly ( Starch is the major source of carbohydrates in the human diet and represents a staple food ingredient on people’s dining tables all over the world . HoweverStarch digestion depends on both internal and external factors. The internal factors include the type, size, crystalline structure, and amylose-to-amylopectin ratio of the starch granules , and theSeveral studies have found that the constituents and content of the food matrix encapsulating the starch granules might protect starch granules from hydrolysis by digestive enzymes, thereby reducing starch digestion . In factHowever, the current methods used to prepare starch-protein complexes mostly involve simple blending and result in complexes with low adsorption and poor stability , 10. In Core-shell microparticles have wide applications in a variety of industries, including foods , pharmacIn the present study, we aimed to develop a food-grade, biocompatible core-shell starch-protein microparticles that inhibit starch digestibility. The microparticles were fabricated using antisolvent exchange and protein crosslinking. Zein, an FDA-approved generally recognized as safe (GRAS) material, is thermally treated and used as the shell material due to its excellent film-forming and hydrophobic properties . The corin vitro simulated digestion procedure to compare the digestibility of native CS and a series of core-shell starch/zein microparticles formed using different crosslinking periods. We further explored the mechanisms by which the thermal-treated zein shells decreases the starch digestibility in the core-shell microparticles. The findings are expected to facilitate the development of health products containing low-digestible starch.In this study, transglutaminase (TGase) was used to strengthen the network structure of thermal-treated zein shells. A further study was conducted on the impact of the thermal-treated zein shells on the digestion of the starch in the core-shell microparticles. This was accomplished by performing an Aspergillus niger, 260 U/mL) were purchased from Sigma-Aldrich Chemical Co. . Calcium chloride  was ordered from Sinopharm Chemical Reagent Co., Ltd. . TGase (1000 U/g) was provided by Beijing Solarbio Science & Technology Co., Ltd. Nile blue was purchased from Macklin Reagent Co., Ltd. . Ethanol was obtained from Fuyu Fine Chemical Engineering Company . All other chemicals and reagents were of analytical grade. Milli-Q water was used in all experiments.Corn starch was kindly provided by Zhucheng Xingmao Corn Developing Co., Ltd. . Zein from corn (Z3625), rhodamine B, pancreatin , and α-glucosidase (from 2). The mixture was stirred at 46°C with a thermostatic magnetic stirrer  at 250–300 rpm for 1 h to swell the CS granules. The mixture was then combined with 200 g Milli-Q water, and the CS granules were left for 20 min at room temperature to absorb more water. The mixture was centrifuged at 3,600 × g for 5 min to obtain the preformed swollen CS granules. The preformed swollen CS granules were washed with Milli-Q water and centrifuged at 3,600 × g for 5 min at least three times until the CaCl2 was completely removed. Milli-Q water was then added to the preformed swollen CS granules until the total volume of the preformed swollen CS granule suspension was 30 mL.The preformed swollen CS granules were prepared using the method of Wang et al. , with mig for 5 min at least three times until ethanol and unadsorbed thermal-treated zein were completely removed.Thermal pre-treatment of zein consisted of dispersing 1% w/v zein in 100 mL 70% aqueous ethanol and heating at 95°C for 30 min. To stimulate the antisolvent exchange of thermal-treated zein, 30 mL of the preformed swollen CS granule suspension was dripped into 45 mL of the thermal-treated zein ethanolic solution. The mixture was stirred at 100 rpm, and the antisolvent exchange process was normally completed in 2–3 h. The obtained core-shell starch/zein microparticles were washed and centrifuged at 3,600 × g for 5 min in a centrifuge . The supernatant was decanted to remove weakly adsorbed thermal-treated zein. The core-shell starch/zein microparticles were then lyophilized and designated CS-zein 0h microparticles, CS-zein 1h microparticles, and CS-zein 2 h microparticles, for the microparticles formed after 0, 1, and 2 h of crosslinking. Unshelled swollen CS granules was set as one of the control groups, where CS went through all the steps in Crosslinking was performed by using the method already published in literature , 23, witAn aqueous suspension of native CS or unshelled swollen CS granules or core-shell starch/zein microparticles was placed on a clean glass slide and covered with a coverslip, avoiding air bubble formation. The morphological structures of the microparticles were viewed with an optical microscope  with a 40× objective.–3 parts of 1% fluorophore in aqueous solution) by combining 2 g core-shell starch/zein microparticles and 25 mL aqueous Nile blue solution. After reaction for 1 min, the core-shell starch/zein microparticles were washed and centrifuged at 3600 × g for 5 min at least three times until the unreacted Nile blue was completely removed. All samples were wrapped in foil and stored at 4°C in a refrigerator until used. Confocal laser scanning microscopy (CLSM) images of the microparticles were acquired using an inverted laser-scanning confocal microscope  with a 10× objective and excitation at 633 nm.The thermal-treated zein component was stained with Nile blue  and over a sufficient number of core-shell starch/zein microparticles (>100).The morphologies of native CS and core-shell starch/zein microparticles were observed by field-emission scanning electron microscopy (SEM) . The cooked core-shell starch/zein microparticles were digested for 0, 20, and 120 min and observed with the same SEM instrument. Before observation, the digested core-shell starch/zein microparticles were washed with copious amounts of Milli-Q water, vacuum filtered at least three times, and lyophilized to inhibit digestive enzyme activity. The lyophilized samples were mounted on an aluminum stub with double-sided tape and sputtered with gold for SEM imaging at 15 kV. Images were captured under 3,000× magnification.The particle size distributions of the native CS, unshelled swollen CS granules, and core-shell starch/zein microparticles were determined by dynamic light scattering (DLS) . The samples at a concentration of 0.1% were ultrasonically  dispersed and evaluated at room temperature.A Megazyme Total Starch kit was used to determine the total starch content of the samples . A nitroThe crystal structures of native CS, unshelled swollen CS granules, and core-shell starch/zein microparticles were determined by X-ray diffraction  using Cu Kα radiation (λ = 1.5405 nm) with a divergence slit of 0.38 mm, generator voltage of 40 kV, and tube current of 40 mA. The scanning range was 4–40°, the scanning rate was 2°/min, and the step size was 0.01. The relative crystallinity of native CS and core-shell microparticles was obtained using a computerized spectrum analyzer. The relative crystallinity (RC) was calculated by the ratio of the crystalline area (Ac) to the entire area (Ae) according to the following equation:–1 and a resolution of 4 cm–1 to record the Fourier transform infrared (FTIR) spectra of native CS, unshelled swollen CS granules, and core-shell starch/zein microparticles.Structural investigations were performed using a NEXUS-870 spectrometer  with the wavenumber range of 4,000–400 cmThe thermal characteristics of the native CS, unshelled swollen CS granules, and core-shell starch/zein microparticles were determined by differential scanning calorimetry . Hermetic aluminum pans were filled with about 4.00 mg of dried microparticles and 8 μL of Milli-Q water. The samples were equilibrated overnight at room temperature and then heated from 25 to 125°C at 10°C/min. The thermal transition parameters, including onset (To), peak (Tp), and conclusion (Tc) temperatures and enthalpy change (ΔH) were determined using the data recording software. ΔH was calculated using the dry starch weight.A Rapid Visco Analyzer  was used to determine the pasting performance of the native CS and core-shell starch/zein microparticles by adding 3 g  sample to 25 mL Milli-Q water , equilibrating the resulting slurry at 50°C for 1 min, heating from 50 to 95°C in 4 min 20 s, maintaining at 95°C for 2 min, cooling to 50°C in 4 min 20 s, and maintaining at 50°C for 1.5 min. The analyzer paddle was programmed to spin at 960 rpm for the first 10 s and 160 rpm for the rest of the test. The pasting parameters, including the peak viscosity, trough viscosity, final viscosity, breakdown, setback, and peak time were evaluated.g for 15 min , and the supernatant was carefully decanted onto a glass plate with a specified weight (precision: 0.1 mg), dried to a constant weight at 105°C, and weighed. The residue was weighed for SP determination. The following formulas were used to determine WS and SP:The water solubility (WS) and swelling power (SP) measurements were made at temperatures ranging from 50 to 90°C, with 10°C intervals using sample suspensions consisting of 200 mg  sample and 20 mL Milli-Q water The suspensions were maintained in a water bath at each temperature for 30 min with constant shaking. The suspensions were cooled to room temperature in an ice bath, centrifuged at 3,000 × where S is the weight of the dried microparticles, A is the weight of the dried supernatant, and B is the weight of the microparticles in the swollen state.in vitro digestion test was carried out according to Englyst et al. (g for 10 min. The quantity of glucose in 0.l mL of the supernatant was determined using a glucose oxidase/peroxidase (GOPOD) kit  and following the manufacturer’s instructions. Digestograms were created by graphing the percentage of digested starch as a function of hydrolysis time. The following equation was used to compute the content of rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS):The t et al. , with slwhere G20 and G120 were the quantities of glucose (mg) released after 20 and 120 min of enzymatic hydrolysis, respectively, and C was the starch content (mg) in the sample.p < 0.05).All data were statistically evaluated and presented as mean values ± standard deviations (SD) of three replicates for each sample using SPSS 22.0 . Analysis of variance (ANOVA) and Duncan’s multiple range tests were used to establish statistical significance at a level of 0.05  higher than those of the native CS (12.44 μm), which could be caused by water permeation during the proceeding of starch gelatinization. The range of particle size of unshelled swollen CS granules was 3.89–37.00 μm, which was wider than those of the native CS (3.89–31.11 μm) for the same reason. As shown in p < 0.05) higher than those of the native CS (12.44 μm) and unshelled swollen CS granules (13.60 μm) and increased with the increasing crosslinking time. When the crosslinking period was 2 h, the volume average particle diameter d of core-shell starch/zein microparticles increased to 30.98 μm. The range of particle size of of core-shell starch/zein microparticles before crosslinking began was 4.62–37.00 μm, which was wider than those of the native CS (3.89–31.11 μm) and broadened with the increasing crosslinking time. In addition, we discovered that the particle diameter of core-shell starch/zein microparticles at the peak shifted to a larger size with increasing crosslinking time. This was possibly a result of aggregation of several adjacent core-shell starch/zein microparticles in response to the crosslinking effect of TGase. This result is consistent with the evidence in the microscopy images.As shown in g for 5 min at least three times until ethanol and unadsorbed thermal-treated zein were completely removed. The supernatant after centrifugation approximately contained 0.06 g thermal-treated zein, which approximately accounted for 14.28% of the totality of the thermal-treated zein used in this experiment. The dry weight of the core-shell starch/zein microparticles before crosslinking began  was 1.68 ± 0.11 g, the protein content of which was 21.43 ± 0.19%. Thus, the core-shell starch/zein microparticles before crosslinking began approximately contained 0.36 g thermal-treated zein, which approximately accounted for 85.71% of the totality of the thermal-treated zein used in this experiment. Namely, before crosslinking began approximately 85.71% of the totality of the thermal-treated zein used in this experiment adsorbed on the surface of the preformed swollen CS granules.To stimulate the antisolvent exchange of thermal-treated zein, 30 mL of the preformed swollen CS granule suspension was dripped into 45 mL of the thermal-treated zein ethanolic solution, which contained 0.42 ± 0.01 g thermal-treated zein, which is the totality of the thermal-treated zein used in this experiment. However, not all of the thermal-treated zein adsorbed on the surface of the preformed swollen CS granules. After antisolvent exchange process, the obtained core-shell starch/zein microparticles were washed and centrifuged at 3,600 × p < 0.05), perhaps because TGase treatment crosslinked the weakly adsorbed zein to the strongly adsorbed zein on starch granule surface. Previous work showed that increases in added exogenous protein could reduce the rate and degree of starch digestion , corresponding to the zein α-helix and β-sheet structures, respectively . HoweverThe XRD pattern of the core-shell microparticles showed the characteristic peak at 10° (2θ) for thermal-treated zein, and its low might reflect the low protein content . The add–1 that were related to the stretching vibration of free hydroxyl groups and intermolecular and intramolecular bound hydroxyl groups . The stretching vibrations of intermolecular hydroxyl groups were responsible for the broad band at 3285 cm–1 , amide II (stretching vibration of C–N bonds and bending vibrations of N–H bonds), and amide III , respectively  bonds, indicating an interaction between the thermal-treated zein and swollen CS granules  decrease in the thermal transition temperatures  and ΔH of unshelled swollen CS granules. The loose structure of unshelled swollen CS granules was responsible for the decrease in energy required to unravel the helical structure; thus, the ΔH was lower than that of native CS.Compared with the native CS, unshelled swollen CS granules underwent easier gelatinization, as determined by the significant (p < 0.05) compared with that of the native CS, indicating that the formation of core-shell starch/zein microparticles improved the thermal stability of the native CS. Potato starch/soy protein complexes (p < 0.05) decreased compared with that of the native CS, and decreased with increasing crosslinking time. This probably reflected the increased compactness of thermal-treated zein shells with the increasing crosslinking time. Gelatinization of some core-shell starch/zein microparticles was difficult, and these core-shell starch/zein microparticles may not be involved in endothermic processes.The Tp of the core-shell starch/zein microparticles was significantly increased  higher for the core-shell starch/zein microparticles than for the native CS, and was further increased with increasing crosslinking time. The original buried hydrophilic groups (such as asparagine) inside the zein were gradually exposed after heating and came into contact with water molecules (p < 0.05) decreased, mainly because of the low starch content. In addition, thermal-treated zein might compete for water molecules with swollen CS granules, thereby inhibiting starch granule expansion and amylose leaching (The pasting profiles of native CS and core-shell starch/zein microparticles are shown in olecules . The comolecules . Compareleaching . The peap < 0.05) greater for the core-shell starch/zein microparticles than for the native CS and increased with increasing crosslinking time. Thermal-treated zein might aid in the formation of the gel network structure of swollen CS granules, thereby speeding up the short-term retrogradation of swollen CS granules  the thermal-treated zein shell limits the swelling of starch granules and (2) compact thermal-treated zein shell serves as barrier between digestive enzymes and starch granules. Further investigation is required to determine the accurate physicochemical mechanisms involved.in vitro hydrolysis profiles of the various cooked samples  are shown in The in vitro hydrolysis profiles.The changes in core-shell starch/zein microparticles and thermal-treated zein shells at different digestion times were investigated by SEM and CLSM. The RDS, SDS, and RS contents of native CS and core-shell starch/zein microparticles are summarized in The film formed by the thermal-treated zein around the swollen CS granules prevented starch hydrolysis by the digestive enzymes. As the crosslinking periods increased, the degree of compactness of the thermal-treated zein film was improved, thereby improving the protective effect of the thermal-treated zein film. Previous studies have demonstrated that the interaction of starch with proteins in foods can significantly decrease its digestibility . Our resp < 0.05) by 52.83–85.66% and 0.11–0.28%, respectively. During the digestion process, the thermal-treated zein shells acted as physical barriers that protected starch granules from hydrolysis by digestive enzymes. Compared to the cooked CS, the SDS and RS contents of cooked CS-zein 2h microparticles increased by about 5- and 14-fold, respectively. The research provides a new strategy for the slow digestion of starch by encapsulating starch granules in thermal-treated zein shells, which will contribute to develop low digestible health products.This research presented a facile method to fabricate natural food-grade core-shell starch/zein microparticles, based on antisolvent exchange and protein crosslinking. The volume average particle diameter of core-shell starch/zein microparticles was 14.70 μm, and increased to 18.59–30.98 μm with a shell thickness of 0.48–0.95 μm after crosslinking by TGase. The CLSM and SEM images showed that swollen CS granules were encapsulated by thermal-treated zein shells. The results of DSC, RVA, and SP indicated that the compact network structure of core-shell microparticles crosslinked by TGase improved the thermal stability of the starch granules and their resistance to shearing forces. The CS-zein 2h microparticles showed the highest Tp, which was 6.62°C higher than the Tp for native CS. The breakdown values and the SP at 95°C for the core-shell starch/zein microparticles significantly decreased (The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.CW: writing original draft–lead, writing review and editing—supporting, and conceptualization—supporting. KQ: conceptualization—supporting; writing original draft–supporting, and investigation—supporting. QS: resources–lead, conceptualization—lead, funding acquisition—lead, supervision—lead, and writing review and editing—lead. XQ: resources—lead, conceptualization—lead, funding acquisition—lead, supervision—lead, writing review and editing—lead. All authors contributed to this work and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Therefore, these parameters allow to analyze quantitatively the effect of different structural factors on the acoustic absorption performance. It has been found that the parameters under study have a remarkable influence on the normalized acoustic absorption coefficient at different frequency ranges. In particular, it has been demonstrated that increasing the surface of the holes and the percentage of holes in the cell walls allows increasing the acoustic absorption of these types of foams, a promising statement for developing highly efficient acoustic insulators. Additionally, we could determine that a suitable minimum value of hole surface to reach the highest sound dissipation for these samples exists.Polyurethane foams with a hybrid structure between closed cell and open cell were fabricated and fully characterized. Sound absorption measurements were carried out in order to assess their acoustic performance at different frequency ranges. The cellular structure of these systems was studied in detail by defining some novel structural parameters that characterize the cell wall openings such as the average surface of holes ( Currently, one major environmental problem that is causing an undesirable and negative impact on human health is noise pollution. The World Health Organization (WHO) states that one in five Europeans is regularly exposed to sound levels at night that could significantly damage health. Therefore, this concern has led to increasingly hardened regulations under the European Directive on the Assessment and Management of Environmental Noise . For thiPorous materials are among the most commonly used as noise absorbers since the sound waves are allowed to penetrate and dissipate inside their complex structure. Their capacity to dissipate sound energy is mainly based the inertial loss promoted by the friction of air with the pore walls and thermal damping by thermal effects in the proximity of the solid walls ,3,4. SpeSeveral studies have focused their efforts on determining the effect of different structural parameters on the noise absorption capacity of porous materials. Sedeq  evaluateWhile there is wide literature focused on the acoustic absorption of flexible PU foams ,19,20,21In order to carry out a systematic study, a new approach to characterizing some key parameters of the cellular structure of these materials is presented in this paper. Herein we analyzed the effect of the holes present in the cell walls and their main characteristics on the acoustic performance of rigid PU foams. Density, open cell content and cell size are kept almost constant and by variations in the catalyst concentration other structural parameters were modified to evaluate its influence on the acoustic performance.3) provided by BASF Poliuretanos Iberia S.A. . The polyol component includes a mixture of two polyols (VORANOL™ CP 450 (50% by weight) and VORANOL™ CP 1055 (50% by weight)) obtained from DOW . The mixture of polyols is considered as 100 parts by weight and the rest of the components are expressed in relation to this value. Distilled water was used as a chemical blowing agent (4.8 ppw). Additionally, an additive based on carbon black (TEGOCOLOR® BLACK HI) (0.3 ppw), TEGOSTAB® B 8522 (non-hydrolysable poly-ether-polydimethyl-siloxane) (1 ppw), employed as surfactant, and two amine-based catalysts (TEGOAMIN® DMCHA and TEGOAMIN® PMDETA) were supplied by Evonik . The different foams differed on the catalyst amount as follows: PU1 , PU2 , PU3 , PU4 , PU5 . The proportion between isocyanate and polyol blend was 1.7:1.0.The formulations of this work are bi-component rigid polyurethane foams. The isocyanate component is a polymeric diphenylmethane diisocyanate (pMDI) (IsoPMDI 92140)  provided with a 50 mm diameter Lenart disc. The polyurethane foams were prepared by mixing the isocyanate and polyol blend (isocyanate-polyol ratio of 1.7:1.0) at 2000 rpm for ten seconds in a plastic bucket The mixture was poured into a paperboard box and was allowed to react by free-rising. After 48 h of ageing at room temperature, samples were saw-cut, obtaining cylinders with 30 mm diameter and 10 mm thickness.r) (the ratio between density of the foam and density of solid PU) was calculated by using 1160 kg/m3 as the solid density of the polyurethane matrix.Geometrical density (ρ) was measured as described by ASTM D1622/D1622M 14 . One cylThe open cell content (OC%) was determined through nitrogen pycnometry with an Accupyc II 1340 from Micromeritics, Georgia, USA, according to ASTM D6226-10 . A cylinincident–Ireflected) to the incident energy (Iincident) on the sample surface. The absorption coefficient was calculated as the average value of six measurements (three for each of the cylinder bases) to obtain consistent results. The selected cylinder (30 mm diameter × 10 mm height) for the acoustic measurements was the same as that used for the density and open cell calculations to ensure the accuracy of the data. The incident wave was parallel to the foam rise direction.The sound absorption properties were obtained by using a two-microphone impedance measurement tube from Bruel & Kjaer, Nærum, Denmark, type 4206, UA-1630, with a frequency range of 500–6500 Hz at an exposure time of 4.3 s. The measurements were performed according to the standard procedure detailed in ASTM E1050-10 . The acoD3), number of cells (n) in the analyzed total cell area (tS), average surface of the cells (pS), number of holes (h) in the analyzed area and the average surface of the holes (hS), i.e., the average area of the holes detected in the analyzed area for each sample.The cellular structure micrographs were acquired by using scanning electron microscopy (SEM) with a FlexSEM 1000 Hitachi  microscope. The foams were saw-cut and the surface was examined by SEM after vacuum coating with a gold monolayer. The evaluated plane is the growth plane of the foams (z). The SEM images were analyzed with a software based on Image J/FIJI  obtaininn) greater than 100 was analyzed. The number of cells per unit area (aN) and the number of holes per unit area (hN) were calculated (see h) higher than 30 and estimating its average surface (hS). Through the calculation of the cell size, assuming a pentagonal dodecahedron geometry [wS). To obtain this value, first Equation (1) relates the volume of a sphere and the volume of a pentagonal dodecahedron to extract the pentagonal side (L)-cell size (ΦD3) relationship (Equation (2)).The samples selected for the structural analysis were the same as used for the acoustic absorption, density and open cell measurements. For each sample, a number of cells  can be estimated:Then, with the expression of the dodecahedron area (Equation (3)), the surface area of each wall  was calculated by using Equation (5):Finally, taking into account that hN/aN gives the number of holes per cell (NHP).The factor 1/24 takes into account that each cell is composed of 12 walls that are shared between two cells. The relationship 3 and 0.023, respectively. The effect of the open cell content on sound absorption for polyurethane foams was studied by Park et al. [The main characteristics of the PU foams are listed in k et al.  by theork et al. ,33.Some features of the cellular structure, such as cell size and distribution or cell holes, may cause a variation on the final sound absorption capacity. The structural parameters are described and analyzed in this section.Cell size distributions and cellular structure micrographs are displayed in −2 mm2 as average value, and the narrowest distribution. Samples PU2 and PU4 present bigger hole sizes (2.61·10−2 and 3.66·10−2 mm2 respectively), followed by PU3 (with 5.34·10−2 mm2). The system with the largest average hole size is PU5 (6.37·10−2 mm2), showing a distribution which is more heterogeneous than those for the rest of the formulations.An exhaustive analysis of the holes in the pore walls was carried out. A similar image area was selected for each sample and the number, size, and surface of holes were estimated see . The holhN) was calculated as described in −2 for PU1. Nevertheless, according to the open cell values, in the case of holes with a larger area, hN decreases, reaching 0.34 mm−2 for PU5. The parameter defining the percentage of holes per cell wall (% HCW) covers a range between 0.045 for PU1 and 0.167 for PU5, following the same trend as the surface of holes as a result of its influence on this parameter.The number of holes per unit area α=∫f1α) (500–6500 Hz) and the open cell content .As shown in α) for each sample. Different ranges covering 2000 Hz were selected to determine possible variations on the absorption trend depending on the frequency range. As already observed in A deeper analysis of the influence of the structural parameters and the acoustic performance of these foams can be found in the following section.hS (first row), the number of holes per unit area hN (second row) and the percentage of holes’ area on the cell walls %HCW (third row) with the normalized absorption coefficient for every range of frequencies selected. The obtained results indicate that the higher the surface of holes (hS), the higher the acoustic absorption coefficient (first row). This trend was observed for all the frequency ranges under study (columns a–c).The results discussed above indicate that density, open cell content and cell size are not the critical factors controlling the differences in the sound absorption capacity of the samples under study since there is a slight variation in their numerical values. Thus, there must be other structural parameters contributing to govern the sound properties of the polyurethane foams. Aiming to look for them, other parameters of the cellular structure were analyzed. Thus, a noticeable relationship between the acoustic behavior and both the number and the surface of holes which are present on the cell walls was found. These results confirm that the sound absorption capacity of porous materials mainly depends on the pore interconnection. It is well known that the efficiency of sound absorption is based on a low sound reflection and a high sound dissipation. The acoustic waves lead to the vibration of the cell walls through stretching and bending; thus, cell walls and the air inside pores contribute to dissipating the sound energy by vibration damping . The norhS value of 0.04 mm2. However, for lower frequencies (500–2500 Hz and 2500–4500 Hz), this limiting value of hS was not found in this study since the acoustic coefficient continued increasing with the surface area of holes. Regarding the general trend for the whole frequency range (d column), the acoustic coefficient is similar when hS varies between 0.025 and 0.055 mm2, showing a plateau region, and it has a clear improvement for surface holes that reach a value higher than 0.060 mm2, as is the case of PU5.For higher frequencies (4500–6500 Hz) the maximum absorption capacity is reached at a hN (second row), the same tendency is shown for all the frequency ranges; that is, more holes reduce the sound absorption efficiency. This behavior might be explained by means of the sample tortuosity. For a fixed value of surface of holes, when the number of holes is lower, tortuosity increases, leading to a high air flow resistance by means of friction of viscosity through the vibration of air [Regarding the number of holes n of air ,11. ThushN parameter has an inverse relationship with the sound coefficient, whereas the surface of holes hS presented a direct one since the sound absorption capacity is higher as this parameter increases.Therefore, the HCW) that takes into account the number and surface of holes , as well as the number of cells (aN) and the average area of cell walls (wS), as shown in Equation (5). This structural parameter follows the same trend as the surface of holes, confirming that the percentage of holes’ area per wall has to be increased in order to enhance the acoustic behavior of rigid polyurethane foams. The relevance of hN and hS contributions on the final value of %HCW can be analysed by plotting each of these two parameters versus the %HCW, as shown in Finally, the third row of hN) for all the samples under study differ in a narrow range; hence, its contribution to the final result has a lower impact.Only the average surface of holes shows a direct correlation with the final percentage of holes’ area present in the cell walls, even though both parameters multiply in Equation (5). However, the values of the number of holes per unit area  was successfully developed.3), and similar open cell contents (80–91%) were fabricated. These systems present an intermediate cellular structure between the conventional rigid closed cell PU foams and flexible open cell PU foams. Thus, their main peculiarities, apart from the high stiffness of the polymer matrix, are the holes present in the cell walls that are the cause of their sound absorption capacity.Several rigid PU foams with cell size values in a narrow range (1000–1250 µm), having similar densities (around 27 kg/mhS), the number of holes per unit area (hN) and the percentage of cell wall area occupied by these holes (%HCW). Since tortuosity will change depending on the number and surface of holes, an equilibrium between these two factors should be reached for an optimum sound absorption. The obtained results show that a noticeable connection exists between the area of the holes (hS) and the final acoustic performance, obtaining a minimum surface hole of 0.04 mm2 for reaching the highest absorption coefficient between 4500 and 6500 Hz. In addition, the percentage of cell wall area (%HCW) also shows a direct correlation with the absorption coefficient, being larger when these parameters increase. Therefore, these results indicate that a small number of large size holes in the cell walls is the ideal structure to maximize the acoustic absorption of these rigid PU foams.An exhaustive structural characterization was carried out by defining new parameters in order to understand the relationship between the cellular structure and the acoustic absorption properties. The novel parameters introduced in this work are the area of the holes (