Pleurotus eryngii) flour on the rheological properties of the cookie dough and the final cookies was therefore determined. The rheological properties of wheat dough supplemented with 0%–25% (w/w) of mushroom flour were analyzed using a Mixolab instrument and a shear rheometer. Increasing the ratio of mushroom‐to‐wheat flour in the doughs increased the peak and final viscosities, but decreased dough stability and elastic modulus. Sensory evaluation using an orthogonal test showed that 15% mushroom flour, 10% maltodextrin, and 1.5% sodium bicarbonate were the optimal composition for producing cookies with the best sensory score. In conclusion, our results showed that cookies with acceptable textures and appearances could be produced by replacing up to 15% of wheat flour with mushroom flour.The objective of this study was to create a healthier version of a commonly consumed baked food (cookies) by replacing some of the wheat flour with a nutraceutical‐rich mushroom flour. The impact of incorporating different levels of powdered mushroom ( Pleurotus eryngii) flour would be achievable and valuable to provide people with nutraceutical‐rich product in their daily diet. It would be important that his study indicated that incorporation less than 15% P. eryngii flour had acceptable formation, texture, and appearance of cookies.Edible mushrooms are widely appreciated for their nutritional, bioactive activities and therapeutic potential. The enrichment of cookies with mushroom ( Cookies contain three major calorific components  and a small amount of water. Flour, sugar, fat, and water are mixed together with some minor components to form dough  flour on cookie manufacture, as well as on the quality of the dough and cookies produced. In particular, the study aimed to evaluate the effects of adding different levels of milled mushroom flour  on the rheological properties of doughs, as well as to optimize the levels of mushroom flour, maltodextrin, and sodium bicarbonate required to produce cookies with desirable sensory attributes.A few researchers have previously examined the impact of incorporating mushroom flours into breads and cookies on their physicochemical and sensory properties  were bought from a supermarket. They were then frozen at −20°C and freeze‐dried using a lab‐scale freeze dryer (Labconco Equipment Co.) at 40°C for 8 hr with chamber pressure of 100 Pa and condenser temperature of −84°C as previously reported  analysis was carried out to determine the pasting properties of the flour suspensions  as described previously. Suspensions were prepared by mixing deionized water (25 ml) with flour (3.5 g) and then manually homogenizing using a plastic paddle. Tests were conducted using a programmed heating and cooling method: holding at 50°C for 1 min; heat at 12°C/min to 95°C; holding at 95°C for 2.5 min; cool at 12°C/min to 50°C (3.7 min); hold at 50°C for 1.5 min. The plastic paddle was kept for 10 s at a speed of 1,000 2.4minimum torque produced by the dough related to protein weakening ; the maximum torque during the heating stage ; the minimum torque during the heating stage ; the torque after cooling to 50°C ; dough development time (DT); and dough stability time (ST). The control group refers to dough samples without mushroom flour added.A Mixolab instrument (Chopin Technologies) was used to determine the thermo‐mechanical properties. This device measures dough consistency during the mixing procedure of controlled heating and cooling . A convection Model TRTF32A oven  was used for the cookies bake. A T‐type thermocouple was used in the oven to monitor the temperature during the baking period. Then, the dough was kneaded and sheeted on a dough sheeter to obtain a uniform thickness of 0.5 cm and then cut into round shapes of 5 cm in diameter. The cookie was placed and baked at the temperature of 205°C for 10 min in the center of the oven. After that, the weights of the six cookies and cookie sheet were recorded. Then, the cookies were cooled at room temperature for further analysis. Overall, the baking cookies were replicated six times  equipped with a HDP/3PB three‐point bending rig according to the established method (AACC‐10‐50D). Setting procedure on texture analyzer of freshly cooled cookies was as follows: pretest speed of 1.0 mm/s, a test speed of 3 mm/s, and a post‐test speed of 10.0 mm/s with a 10‐mm distance. Six measurements were conducted for each sample.2.82.8.1The formulas of cookie samples preparation were 40% distilled water, 5% sugar, 20% maltodextrin, 0%, 5%, 10%, 15%, or 20% mushroom flour, with the remainder being wheat flour (wt% on 100 g basis).2.8.2The formulas of cookie samples preparation were 40% distilled water, 5% sugar, and 0%, 5%, 10%, 15%, 20%, 25%, or 30% maltodextrin, with the remainder being wheat/mushroom flour (wt% on 100 g basis).2.8.3The formulas of cookie samples preparation were 40% distilled water, 5% sugar, and 20% maltodextrin, 0%, 0.5%, 1.0%, 1.5%, 2.0%, 2.5% or 3.0% sodium bicarbonate, with the remainder being wheat/mushroom flour (wt% on 100 g basis).2.8.43 (C) were the three main factors affecting the quality of the product. Consequently, we carried out an L9 (3)4 orthogonal experiment.Based on single‐factor experiments, an orthogonal experiment was designed. Addition of mushroom flour (A), addition of maltodextrin (B), and addition of NaHCO2.8.5The sensory evaluation of the cookies was conducted in the sensory evaluation room in the College of Food Science and Technology in Nanjing Agricultural University. The sensory scores were evaluated by a panel of 20 semitrained people  of at least 3 repeated experiments. We used one‐way Analysis of Variance and Tukey's test  to evaluate the significant differences between different groups using statistical software .All results are presented as means ± standard deviation  mushroom flour were significantly higher than those for wheat flour alone (p < .05).The pasting properties of flours are particularly important in the cookie‐making process, and therefore, the impact of blending the wheat flour with different levels of mushroom flour was examined. The paste viscosity then increased progressively as the samples were cooled, which can be attributed to a coil‐to‐helix transition and hydrogen bond formation between amylose molecules. The paste viscosity profile of samples containing different levels of mushroom flour was analyzed and various parameters determined Table . The peap < .01; Table As the proportion of mushroom flour increased, the peak and holding viscosities of the blended flours decreased. The presence of high levels of mushroom flour has a dilution effect on the content of wheat gluten in the blended flours, which leads to a decrease in dough stability. The final viscosity decreased from around 2,930 cP for wheat flour alone to 2,149 cP for the blended flour containing 15% (w/w) mushroom flour (3.3Lentinus edode flour or up to 7.5% (w/w) Auricularia auricular flour had little impact on dough development time  is highly related to water absorption capacity of dough and the gluten matrix strength .The C2 values provide information about the extent of gluten network weakening during dough kneading: the higher the C2 value, the lower the degree of protein weakening  increased, the storage modulus (G′) and loss modulus (G″) of dough increased, with G′ > G″ at all ω values  on the hardness, brittleness, and sensory evaluation scores of cookies was investigated  was smaller than the D‐factor (Blank) suggested that maltodextrin addition had little influence on the hardness of the cookies. According to the R values, the factors influencing the brittleness were as follows: A > B > C.The effect of three factors . The maximum sensory evaluation scores of the cookies were obtained when mushroom flour addition, maltodextrin addition, and NaHCO3 addition were A2B2C2  according to the sensory evaluation scores. There were no significant differences in the sensory evaluation scores of A2B2C2 with the best combination on the orthogonal test table (A2B3C1) through validation tests. However, taking into account economic factors of factory production and the trend toward low‐carbohydrate foods, A2B2C2  was selected as the best cookie formulation because of the lower maltodextrin content and the cookies formulation was confirmed by addition of experiment cookies with the best formulation.In summary, considering the influence of all three properties of the cookies , the most significant factor influencing the quality of cookies was A (mushroom flour), followed by B , and then C (NaHCO4P. eryngii) flour influenced the mixing and rheological properties of the blended dough. The water absorption capacity and peak viscosity of mixed flours significantly increased when more than 15% mushroom flour was incorporated. The dough stability time decreased when the ratio of mushroom flour increased. The supplementation of mushroom flour lowered the gelatinization temperature and the shear modulus values. Analyses of cookie quality properties indicated that fortification with 15% mushroom flour was the maximum level of addition recommended for a better dough functionality and acceptable cookie quality. An orthogonal test showed that A2B2C2  was the best combination for producing healthier cookies with good taste attributes.The addition of wheat flour with mushroom (The authors declare no conflict of interest.This study does not involve any human or animal testing.


The arrangement of helices in the HI(CC) domain is reminiscent of the structure of the H1 domain of the first AAA+ module of ClpB chaperones. It has been hypothesized that LonA proteases with a single AAA+ module may also contain a part of another AAA+ module, the full version of which is present in ClpB. Here, we established and tested the structural basis of this hypothesis using the known crystal structures of various fragments of LonA proteases and ClpB chaperones, as well as the newly determined structure of the Escherichia coli LonA fragment (235–584). The similarities and differences in the corresponding domains of LonA proteases and ClpB chaperones were examined in structural terms. The results of our analysis, complemented by the finding of a singular match in the location of the most conserved axial pore‐1 loop between the LonA NB domain and the NB2 domain of ClpB, support our hypothesis that there is a structural and functional relationship between two coiled–coil fragments and implies a similar mechanism of engagement of the pore‐1 loops in the AAA+ modules of LonAs and ClpBs.LonA proteases and ClpB chaperones are key components of the protein quality control system in bacterial cells. LonA proteases form a unique family of ATPases associated with diverse cellular activities (AAA These proteins play a key role in the maintenance of cellular proteome in all natural kingdoms + proteins  Most chaperones and all proteases of the PQC system are heat‐shock proteins (Hsp). The PQC proteases are bifunctional oligomeric enzymes. Their proteolytic domains belong to different classes of peptide hydrolases, whereas the ATPase parts are related to the single Hsp100 superfamily denoted AAA+ proteins of the PQC system is shown in Fig. + protein is a two‐domain AAA+ module comprising a large nucleotide‐binding (NB or α/β) domain as well as a small α‐helical (H or α) domain. These modules contain a variety of conserved elements consisting of peptide fragments or, in some instances, single amino acid residues + proteins include nonhomologous ‘extra’ domains ; see Fig. The domain organization of AAA+ proteins are divided into two classes depending on the number of AAA+ modules. The ATPase components of most AAA+ proteases and proteolytic complexes (such as proteasomes) that contain only one such module  belong to class II of AAA+ proteins, whereas components ClpA, ClpC, and ClpE of other proteases, as well as chaperones‐disaggregases ClpB, have two AAA+ modules (D1 and D2) and are assigned to class I  domain, followed by an inserted domain which is located between the N domain and the AAA+ module https://embnet.vital-it.ch/software/COILS_form.html). It was suggested that the HI(CC) domain resembles the H1 domain of ClpB chaperone, which also contains a fragment of similar size with CC conformation . The interdomain interactions within monomers and oligomers of EcLon are also analyzed. As part of this study, we determined the structure of the fragment EcLon (235–584) that comprises a part of the HI(CC) domain and the AAA+ module, since no such structure of EcLon has been available to date.With the availability of extensive structural data for the N domain http://www.uniprot.org/) and the MEROPS database (www.merops.sanger.ac.uk) https://embnet.vital-it.ch/software/ClustalW.html; https://www.ebi.ac.uk/Tools/msa/clustalo/; https://embnet.vital-it.ch/software/COILS_form.html; http://distill.ucd.ie/porter/) was used to evaluate the degree of conservation in sequences, to identify the CC regions in proteins, and for the subsequent topology analysis.The amino acid sequences of the chaperones and proteases analyzed in this study were obtained from the UniProt Knowledgebase (EcLon (235–584) comprising the C‐terminal part of HI(CC) domain and the full‐length AAA+ module was produced by limited proteolysis from purified full‐length EcLonA, as previously described −1. The buffer consisted of 20 mm Tris/HCl pH 7.5, 0.2 m NaCl and 1 mm ADP. For crystallization, the protein was concentrated to 8 mg·mL−1 on a Microcon concentrator with 10 kDa MW cutoff .The fragment of m Li2SO4, 0.1 m Na cacodylate pH 6.5 from the Wizard II screen . Crystals were harvested and flash‐frozen in liquid nitrogen.Large needle‐shaped crystals were grown by the sitting drop vapor diffusion method with a HydraII crystallization robot  at 21 °C. The crystals grew in 7–10 days in 30% (w/v) PEG 400, 0.2 1 or its enantiomorph. Diffraction data were processed with HKL2000 X‐ray diffraction data were collected at the SER‐CAT 22‐ID beamline of the Advanced Photon Source at Argonne National Laboratory  with a MAR225 detector. The best crystal diffracted to ~ 3.0 Å and belonged to space group P6EcLon (235–584) was solved by molecular replacement using search models derived from the structures of fragments of LonA proteases from Meiothermus taiwanensis , might correspond to the C1–C4 helices of the H1 domain of the chaperone, combined with the helices L1–L4 of the inserted M domain  domain of LonA to the H1(M) fragment of ClpB has been previously suggested ain Fig. . The fraEcLon (1–245) fragment , as well as the Walker A motifs located in the following nucleotide‐binding domains  helix, consisting of 55 residues, is a distinctive feature of EcLon 1–25 fragmenent Fig.  between lpB Fig.  is > 45%+ module of ClpB (D1), since it includes the inserted CC region formed by four helices (α6–α9 or CC‐1–CC‐4), equivalent to the arrangement of the helices L1–L4 in the M domain of ClpB. In order to clarify this proposition in structural terms, we marked with identical colors the corresponding helices in the structure of the fragment (150–854) of TtClpB  and EcLon  and EcLon (247–299), highlighted by a gray background in Fig. We postulate that the first three helices (α3–α5), combined with the eighth helix (α10) of the HI(CC) domain, form a putative α‐helical domain in the N‐terminal part of the LonA proteases . Such a domain would topologically resemble the H1 domain of the first AAA EcLon 24–299, higEcLon and BsLon might be affected by truncations introduced into the constructs that have been used for crystallization. In the absence of a LonA structure with the intact HI(CC) domain, the existing structural fragments containing HI(CC) helices cannot be properly compared between themselves and to ClpB. Therefore, in order to test this hypothesis using the available structural data, we analyzed the resemblance between the two proteins on the level of both the secondary and tertiary structures, comparing either the individual helices, or the whole domains.As mentioned earlier, the mutual arrangements of the helices in HI(CC) in respect to the other domains in the partial structures of EcLon with their counterparts in the H1 domain of the first AAA+ module of ClpB EcLon  domain of EcLon (235–584) correspond to helices L3, L4, and C4 of ClpB  and BsLon (240–774), these helices are oriented toward the central pore  domain would be intact.Furthermore, helices α8 CC‐3), α9 (CC‐4), and α10 at the N terminus of , α9  with a similar three‐helix bundle at the N terminus of the molecule also indicate that formation of a closed hexameric ring does not tolerate the presence of all three‐helix bundles in the central pore, but requires to move three bundles out of six to the periphery of the ring  and (207–492) of  and  + proteins, in particular of the ATPase components of ClpXP (PDB ID 3HTE) ID 1KSF) IDs 1QVR, 1JBK) Structural data on the NB domains of LonA proteases include our structure of ere Fig. A, as wel+ ATPases, LonA proteases are expected to function as hexamers with a central pore that is lined with axial loops, required for protein unfolding and translocation. Three different pore loops, called ‘GYVG’ (or pore‐1), pore‐2 and ‘RKH’, are usually found in almost all AAA+ unfoldases Similarly to other AAA+ proteases and chaperones of the ClpB/Hsp104 family + modules of chaperones ClpA and ClpB revealed a distinct difference in the location of the functional motif within the individual loops in their NB1 and NB2 domains. Structure‐based sequence alignment of the NB domains of LonA proteases with the NB1 and NB2 domains of ClpA and ClpB chaperones  loops are found in all AAAnes Fig.  indicateEcLon, BsLon, and MtLon with those in the NB1 and NB2 domains of ClpA and ClpB chaperones reveals a striking similarity in the location of GYVG motif with a conserved tyrosine residue within the helical fragment of these loops between the LonA NB and ClpB NB2 domains  that do not contain a CC insertion have their GYVG signature located on the top of the loop, as shown in Fig. Our analysis indicates that there is a correlation between the presence of the CC fragment, embedded in the α‐helical domain that precedes either the single or second AAALocation of two other axial loops (pore‐2 and RKH) is highly conserved in the structures of LonA proteases and of both chaperones, ClpA and ClpB Fig. A,B. Pore+ molecular machine, stabilizing the contacts during initial complex formation RKH loops are located at the upper entry to the central pore and are expected to interact with the substrate at early stages of its approach to the AAA+ module, may also contain a part of the second AAA+ module, which is present in full in the Clp enzymes of class I AAA+ proteins. A comparative analysis of the corresponding domains in the available structures of LonA proteases and ClpB chaperones revealed similarities on all four levels of structural organization of these proteins (from primary to quaternary). There is a strong indication that the architecture of the unique HI(CC) domain of LonA proteases resembles the structure of the H domain of the D1 AAA+ module of ClpB chaperones. The number of helices, their topology, and the presence of the long helix in the HI(CC) domain, the structural equivalent of which in the M domain of D1 is engaged in regulation of the necessary dynamic rearrangement of the subunits in a hexamer of ClpB The principal aim of this study was to provide structural support for the hypothesis that LonA proteases, which bear a single classical AAAE. coli LonA mutant with deleted HI(CC) domain (residues 124–304) EcLon, binding of nucleotides and their complexes with magnesium ions did not affect the efficiency of peptide hydrolysis. It was concluded that the inserted HI(CC) domain is needed for formation of a functionally active enzyme, and the absence of this domain alters coupling between ATP hydrolysis and substrate proteolysis, as well as it affects interactions between LonA and a protein substrate.The other argument in favor of this hypothesis comes from studies evaluating functional characteristics of an + module of LonA with the corresponding ones in the NB1 and NB2 domains of two AAA+ modules of ClpA and ClpB chaperones, respectively. Significant results of that analysis are provided for the most conserved axial pore‐1 loops (GYVG) that are engaged in the interactions with substrate during all stages of protein function. A comparison of the fragment comprising this loop in LonA with the corresponding fragments of both the NB1 and NB2 domains of ClpA and ClpB chaperones unambiguously reveals a singular match in the location of the GYVG loop between the LonA NB domain and the NB2 domain of ClpB. This finding suggests that the mechanism of engagement of pore‐1 loops in these AAA+ modules of LonA and ClpB might be indeed similar. Our observations, combined with the data derived from the structure of ClpB with bound substrate +‐2 module as being the main motor of ClpB, give a new perspective to a functional meaning of the resemblance between the sole ATPase module of class II and the AAA+‐2 module of class I of the AAA+ proteins, and also correlate with the matching topology between the HI(CC) domain of LonAs and H1 domain of ClpBs.This idea is also supported by comparison of the functionally important axial loops in the NB domain of the single AAA+ proteins, may represent a novel subclass of AAA+ proteins, distinctive from the established class I and class II.In the absence of a structure of full‐length LonA protease, the structural elements that carry the functional or regulatory properties for these molecular machines cannot be accurately matched up between different partial structures. However, available structural data strongly support the hypothesis of their revised structural and functional relationship, thus suggesting that LonA proteases, exhibiting the structural features of both classes of AAAThe authors declare no conflict of interest.TVR and AG conceived this study and, together with AW, supervised its conduct. IB and ML performed the crystallographic experiments. AGA and AMK performed sequence alignments and generated the illustrations. All authors analyzed and discussed the data and contributed to writing the manuscript.


The optimization of legume incorporation , considering the influence of different maize flours , on consumer liking and sensory profiling of ‘broa’ was studied. A panel of 60 naïve tasters evaluated twenty different breads, divided in four sets for each legume flour fortification, each set including four breads with varying maize flour and a control (no legume). Tasters evaluated overall liking and the sensory profile through a check-all-that-apply ballot. Crude protein and water content were also analyzed. There were no significant differences in overall liking between the different types of legumes and maize. The incorporation of chickpea flour yields a sensory profile that most closely resembles the control. The protein content increased, on average, 21% in ‘broa’, with legume flours having the highest value obtained with faba bean incorporation (29% increase). Thus, incorporation of legume flours appears to be an interesting strategy to increase bread protein content, with no significant impact on consumer liking and the ‘broa’ bread sensory profile.The fortification of maize bread with legume flour was explored in order to increase the protein content of the traditional Portuguese bread ‘ Wheat is the most frequently used cereal for bread making in many parts of the world, but other common ingredients of bread are maize and rye . An intebroa’ . Broa’ is a Portuguese bread comprised of more than 50% maize flour, mixed with either wheat and/or rye flours, and highly consumed in the northern and central regions of Portugal [broa’ can be produced, depending on the type of maize variety and blending of the flours used, with regional maize landraces  being considered more suitable than hybrid varieties for bread production [broa’ bread, based on eleven regional OPV maize landraces, were evaluated [broa’ bread among specialty landraces of maize flours and the lowest scores for ‘broa’ bread from commercial (hybrid variety) maize flour. In that study, commercial flour presented the highest mean diameter and a larger flour particle distribution range of all the tested maize varieties. ‘Portugal . Severaloduction ,5. Maizevaluated . The stubroa’ consists of mixing maize flour , wheat and/or rye flour, hot water, yeast, salt, and leavened dough from a previous bread (acting as the sourdough) [broa’ bread and the microbial phenomena of dough fermentation and storage for ‘broa’ bread was studied in [The traditional bread making process used to prepare ‘urdough) . After murdough) ,3,7. Theudied in ,9.broa’ bread, with modification of the traditional composite maize/rye wheat flour, was tested and considered satisfactory for its sensory quality and bread making technology ability [A gluten-free ‘ ability . Maize d ability .Legumes are generally rich in protein and fiber and low in fat, and are considered to have a high nutritional value and key role in preventing metabolic diseases, such as diabetes mellitus and coronary heart diseases ,12. ThusThe objectives of this work were to select the optimal maize bread formulation with legume fortification (part of maize flour replaced by legume flour) accessed through overall consumer liking and a check-all-that-apply (CATA) profiling evaluation, in order to obtain bread claiming to have a high protein content that is also nutritiously enriched and well accepted by consumers.broa’ bread included 700 g·kg−1 maize, 200 g·kg−1 rye, 100 g·kg−1 wheat flour, 28 g·kg−1 sugar (wt/wt flour basis), 17.6 g·kg−1 salt (wt/wt flour basis), 10 g·kg−1 dry yeast (wt/wt flour basis), 100 g·kg−1 sourdough (wt/wt flour basis), and 100% (vol/wt flour basis) water, as described previously by Brites et al. [The base formulation of the ‘Cicer arietinum), faba bean—FB (Vicia faba), lentil—LC (Lens culinaris), pea—PS (Pisum sativum), and control—C (without legume flour) with four different maize flours: hybrid white—IW, hybrid yellow—IY, regional white—RW, regional yellow—RY. The regional whole maize flours were obtained after milling the grain in an artisanal water mill with millstones , and the hybrid flours correspond to commercial maize flour . Each of the four blocks corresponds to the replacement of 10% of the maize flour by one of the legume flours  and a control (C) sample with no replacement.Twenty breads were produced following a 4-block design combining four legume flours: chickpea—CH  consumer database. A sociodemographic characterization of consumers was performed. The company ensures data protection and confidentiality through the authorization 2063/2009 awarded by the National Data Protection Commission and an accomplished internal code of conduct.Sensory evaluation was carried out at Sense Test in a special room equipped with individual booths in accordance with ISO standard 8589:2007 , with peBroa’ breads were cut halfway and 2 cm-thick slices were cut from the central portion. A serving of one slice (≈100 g) was presented to each taster on white disposable plastic plates, identified by a three-digit random number, at the individual booths under normal white lighting  white regional maize with legume flour; (ii) white hybrid maize with legume flour; (iii) yellow regional maize with legume flour; (iv) yellow hybrid maize with legume flour; and a control sample made with yellow hybrid maize with no legume flour b. ‘Broa’lighting c. PaneliWithin a session, each participant received all the samples following a monadic sequential (one at a time) presentation, with a balanced sample serving order to compensate for possible carryover effects . OverallParticipants were invited to profile each sample over a CATA ballot, structured according to 6 sensory dimensions (Whole bread appearance (WBA), Slice appearance (SA), Texture at touch (TT), Mouth texture (MT), Aroma (A), and Flavor (F)). The bread samples were prepared according to the AACC 62-05.01 method , water c® system software . To synthesize the results of the overall liking test, descriptive statistics with mean and standard error (SE) for each session, corresponding to the different legume flour breads and the control sample, were computed. A two-way (type of maize and type of legume) ANOVA with blocks (tasters), and the Fisher-LSD test for multiple comparison (differences in the liking of each of the enriched breads), was applied at a 95% confidence level. A two-way (type of maize and type of legume) ANOVA, and Fisher-LSD test for multiple comparison, was applied to evaluate differences between both the protein and water content of the different ‘broa’ breads (with interaction as the error) at a 95% confidence level.Data analyses were performed using the XL-STAT 2019For CATA evaluations, the Cochran test was applied to identify which descriptive attributes were discriminating among samples . Subsequp > 0.05). Despite no significant effect, the bread incorporating chickpea flour with hybrid white maize was the only one presenting an average overall liking (6.97 ± 0.16) above the corresponding control sample (6.77 ± 0.17)  .Results from the CATA profiling by Cochran test yielded both discriminating and non-discriminating attributes. The non-discriminating attributes identified across all blocks are presented in broa’ bread samples, with the significant descriptive attributes, resulting from MDA analysis. Angles below 45° indicate a significant positive correlation between the projection of the sample and the projection of the attribute into the CA space, while angles above 135° indicate a significant negative correlation between the projection of the sample and the projection of the attribute [broa’ bread produced, combining the 4 legume flours and the control (without legume flour) with the 4 maize flours. Legume fortification significantly increased the ‘broa’ bread protein content, as expected. The mean protein content of the bread without legume incorporation was 56.2 (±1.3) g·kg−1 and increased, on average, by 21% in ‘broa’ with the incorporation of legume flours. The highest increase in protein content was obtained with faba bean (29%) incorporation rising to 72.3 (±2.4) g·kg−1, compared to the control without legume incorporation. In terms of protein content per dry weight, the differences were even higher between legume fortification and the control bread. The faba bean incorporation increased the protein content to 32% (118.3 (±1.8) g·kg−1 dry basis), followed by lentil incorporation of 22% (108.8 (±0.8) g·kg−1 dry basis), and chickpea and pea incorporation . The lowest water content was obtained from the bread with pea incorporation (362.5 (±5.6) g·kg−1) and the highest from faba bean incorporation (389.5 (±12.4) g·kg−1). A significantly lower water content was obtained from the hybrid varieties of bread in comparison with the regional varieties of bread.broa’ sensory profile appear related to apparent humidity (perceived moisture at touch and in mouth) and bread crumb cohesiveness (sticky texture at touch and in mouth). Incorporation of chickpea flour lead to liking scores closer to the control formulation. The incorporation of chickpea flour yielded the sensory profile that most closely resembled the control. Faba flour incorporation lead to ‘broa’ breads with the highest protein content. Incorporation of legume flours appears to be an interesting strategy to increase bread protein content without decreasing consumer liking. However, further research should also consider the impact of legume incorporation on the glycemic index of maize bread. Regarding the sensory profile method, one can observe that the CATA was an appropriate method to describe the maize bread formulations with legume fortification. The breads were produced in the bakery chain of Patrimvs S.A, with the intended positive effect of studying a real-life market production situation, but the industrial scale imposed limitations, such as the restricted choice of commercially available legumes flours. Major changes in the ‘These results can be seen as an opportunity for the bakery industry to develop new products that will respond to the growing consumer demand for high-protein food.


Cicer arietinum L.) was examined as a potential vehicle for Fe fortification. Fortificants (FeSO4·7H2O (ferrous sulfate hepta-hydrate), FeSO4·H2O (ferrous sulfate mono-hydrate) and NaFeEDTA (ethylenediaminetetraacetic acid iron (iii) sodium salt)) were applied by a spraying and drying method. At 2000 µg g−1 iron fortificant, the fortified split desi seeds , desi flour and kabuli flour supplied 18–19 mg, 16–20 mg and 11–19 mg Fe per 100 g, respectively. The overall consumer acceptability using a nine-point hedonic scale for sensory evaluation demonstrated that NaFeEDTA-fortified cooked chickpea (soup and chapatti) scored the highest among the three fortificants. Lightness (L*), redness (a*) and yellowness (b*) of Fe-fortified products changed over time. However, no organoleptic changes occurred. Fe bioavailability was increased by 5.8–10.5, 15.3–25.0 and 4.8–9.0 ng ferritin mg−1 protein for cooked split desi seeds (soup), desi chapatti and kabuli chapatti, respectively, when prepared using Fe-fortified chickpea. Desi chapatti showed significantly higher Fe bioavailability than the other two. The increase in Fe concentration and bioavailability in fortified chickpea products demonstrated that these products could provide a significant proportion of the recommended daily Fe requirement.Iron (Fe) deficiency is one of the most common nutritional disorders, and is mainly due to insufficient intake of bioavailable Fe. Chickpea ( Iron (Fe) deficiency, mainly due to low intake of bioavailable Fe, is one of the most common and widespread nutritional deficiencies in the world ,2,3. OveIron fortification of food is a key approach to addressing Fe deficiency . Fe defiIron bioavailability is generally considered to be the fraction of dietary Fe that is absorbed and used for normal body functions. For subjects with no Fe stores and who are non-anemic, the ranges of Fe bioavailability for mixed and vegetarian diets were 14%–18% and 5%–12%, respectively .−1) among commonly consumed pulses [Sensory evaluation provides important and useful information to product developers and food scientists regarding the sensory characteristics of their products ,17,18. Id pulses ,22.Phaseolus vulgaris L.) [−1 or 3.6 g day−1. South Asia is the largest consumer of chickpea (averaging 4.25 kg year −1 or 11.6 g day−1) followed by the Middle East and Northern Africa (averaging 2.11 kg year −1 or 5.9 g day−1) in 2006–2008 [Globally, chickpea is the second most consumed pulse after common bean (aris L.) . The ave006–2008 . Chickpe006–2008 . Chickpe006–2008 . It is a006–2008 ,29. More006–2008 . The ave006–2008 . BecauseTo date, no information is available on Fe fortification of chickpea as a potential solution to the Fe deficiency problem. The main objectives of this study were (1) to identify suitable Fe fortificants for the fortification of chickpea seeds and flour; (2) to determine consumer acceptance and preference for Fe-fortified raw and cooked chickpea products through sensory evaluation; and (3) to determine the relative amount of bioavailable Fe in Fe-fortified chickpea products.−1) and CDC Frontier  were used in this study. The former is a “desi” type chickpea with a dark tan seed coat color, and the latter is a “kabuli” type with a beige seed color. The seeds were provided by the Crop Development Centre, University of Saskatchewan. The kabuli chickpea seeds were used with the seed coats. Desi chickpea seeds were dehulled abrasively followed by sieving over 7.5 mm and 11 mm screens. Materials (cotyledons) retained on the 7.5 mm screen were passed through a column blower . The dehulled seeds were collected and stored at room temperature. The moisture content for both varieties was measured by drying samples in a convection oven at 80 °C for 12 h.Two chickpea varieties, CDC 551-1 , ferrous sulfate monohydrate (FeSO4·H2O) and sodium Fe EDTA (NaFeEDTA). All the three Fe fortificants were water soluble and relatively cost effective as well as had high relative bioavailability (~100%) compared to ferrous sulfate heptahydrate (FeSO4·7H2O) [−1 intervals ranging from 100 to 3200 µg g−1. Ten milliliters of each of the Fe fortificant solutions was sprayed onto 100 g of dehulled desi and whole kabuli chickpea seed samples. The samples were then dried at approximately 75 °C by placing them under a 250 watt light bulb with continuous shaking  and manual stirring for 15–20 min at 400 rpm. Half of the Fe-fortified desi seed sample and the entire Fe-fortified kabuli seed sample were then ground into flour with a maximum particle size of 106 µm using a cyclone sample mill .Three WHO-approved Fe fortificants were used in this study, namely ferrous sulfate heptahydrate (FeSOO4·7H2O) . Two typO4·7H2O) . The forThe color of all samples (control and Fe-fortified) from split desi seeds , desi flour and kabuli flour was evaluated using a HunterLab Colorimeter . Iron-fortified samples were compared to unfortified controls. The color values  for each sample were recorded at three-month intervals over a one-year period .The fortified desi and kabuli flours along with controls were assessed for rancidity. The samples were stored individually for 6 and 12 months in plastic bags at room temperature (19–22 °C) and a relative humidity of 45%–55%. The moisture contents ranged from 8.3%–9.3%. Five-gram samples were weighed and transferred into test tubes, followed by the addition of 50 mL of methylene chloride to each tube. The flour and methylene chloride mixture was pressed in a syringe and enough oil–solvent mixture collected to recover approximately 1g of oil. The solvent was removed using a rotary evaporator  and the oil was transferred to a 5 mm diameter NMR tube . The sam−1 of FeSO4·7H2O, FeSO4·H2O or NaFeEDTA, along with controls (sprayed with similar amount of deionized water with no fortificants), were soaked for 3 h in 3 L of deionized water. Each sample with the same 3 L deionized water that was used for soaking was cooked over medium heat for 40 min. Stainless steel kitchen utensils were used to cook the samples. This resulted in a semi-thick soup, a south Asian traditional chickpea dish called “dal”, to which the following ingredients were added after 20 min of cooking: 20 g of table salt, 10 g of turmeric powder, 30 mL of canola oil and 100 g of chopped onion [−1 of FeSO4·7H2O, FeSO4·H2O or NaFeEDTA, along with controls. Chapattis were made from 125 g of the fortified chickpea flour (“besan”) and 125 g of all-purpose refined wheat flour mixed with 2.5 mg of table salt, 38 mL of canola oil and 60 mL of warm water in a stainless-steel bowl. Another 30 g of all-purpose refined wheat flour was used during rolling of the chapattis using a wooden rolling pin and board. The chapattis were then fried on a cooking device of 250 mm diameter and 2100 W power for 3–5 min, followed by cooling at room temperature for approximately 2 h before transferring them into small, foil containers. The samples were kept at −80 °C for 24 h and then freeze-dried using a FreeZone 12 L Console Freeze Dry System  for 72 h followed by storage at room temperature [Dehulled desi seeds (500 g), fortified using fortificants containing 2000 µg ged onion . Chapattperature .−1) in the split desi seeds  and both desi and kabuli flours, before and after fortification, were quantified by flame atomic absorption spectrometry  at the analytical laboratory at the Department of Plant Sciences, University of Saskatchewan. Before analysis, the samples were digested using a Vulcan digester . The digestion was done with two repeats and three technical replications per repeat. The protocols used for digestion and the analysis of Fe concentration were described previously [The Fe concentrations  were quantified at the USDA-ARS Robert Holley Center for Agriculture and Health  using ICP-AES  . One-hal−1 of each of FeSO4·7H2O, FeSO4·H2O and NaFeEDTA) was assessed at the USDA–ARS Robert Holley Center for Agriculture and Health  using the in vitro digestion/Caco-2 cell model, where ferritin formation is used as an indicator of Fe uptake [The bioavailability of Fe in soup and chapatti samples prepared from fortified chickpea  and visually assessed 12 corresponding uncooked samples (split seeds and flour).For the screening of cooked (soup and chapattis) and uncooked chickpea samples (split seeds and flour), respondents provided verbal consent and completed consent forms prior to testing. After reading the consent form, participants were asked if they understood the purpose and objectives of the study, procedures for cooked food samples, potential risks by participating in this research, confidentiality of respondents and data and the right to withdraw and follow up, and were informed that any questions from any participants regarding this research could be addressed to the researchers through the Ethical Research Board, University of Saskatchewan. Signed consent forms were collected from the participants for record keeping purposes.A “sensory evaluation form” was provided to all participants. Participants were requested to evaluate cooked (soup and chapattis) and uncooked samples (split seeds and flour), along with filling in their participant code, age and sex, as well as the date and sample code. Twelve cooked and uncooked samples, along with controls, were presented in small, white plastic containers, each marked with a three-digit number. Each participant was given a spoon, 30 mL of chickpea soup and 60 g of chickpea chapatti, as well as 50 g of split desi seeds and 30 g of flour prepared from cooked and uncooked samples, respectively. Participants were asked to rinse their mouths before and after testing each of the dishes. For the cooked samples, participants evaluated five attributes: Appearance, odor, taste, texture and overall acceptability, whereas for the uncooked samples, attributes included appearance, odor and overall acceptability only. Participants used a nine-point hedonic scale . In addition, participants were allowed to record any opinions regarding this study, which were kept for record keeping purposes. Of the initial 45 participants, five did not attend the second session. Therefore, data from only 40 participants were analyzed.p < 0.05.The Fe concentrations in fortified uncooked  and cooked  chickpea samples and controls were averaged over three replications with two repeats. Values for the Fe bioavailability of cooked chickpea dishes were also averaged over three replications. Sensory data for cooked samples were averaged across two repeats. Descriptive statistics were used to calculate means ± standard deviations (SDs). The PROC GLM of SAS version 9.4  was used to compare means of Fe concentration, Fe bioavailability and sensory data. The correlation between Fe concentration and Fe bioavailability was calculated using Pearson’s correlation coefficient. Statistical significance was assumed at 4·7H2O, FeSO4·H2O and NaFeEDTA) at nine Fe concentrations ranging from 100–3200 µg g−1, with 400 µg g−1 intervals on Fe concentration in split desi seeds, desi flour and kabuli flour are presented in The effects of three food-grade Fe fortificants (FeSOp < 0.001) the Fe concentration relative to the control of split desi seeds sprayed with deionized water. The Fe concentration in unfortified split desi seeds was 43.8 µg g−1, whereas the Fe concentration in unfortified desi flour and kabuli flour was 44.8 µg g−1 and 54.3 µg g−1, respectively. The Fe concentrations in split desi seeds fortified with FeSO4·7H2O increased in parallel with the Fe concentrations in the fortificant, and ranged from 64.7 to 384.1 µg g−1. The Fe concentration in split desi seeds fortified with FeSO4·H2O and NaFeEDTA ranged from 59.7 to 333.2 µg g−1 and from 58.2 to 361.2 µg g−1, respectively. The Fe concentration in desi flour and kabuli flour fortified with FeSO4·7H2O also increased in parallel with the Fe concentrations in the fortificant. The values ranged from 62.4 to 316.5 µg g−1 in desi flour and from 80.9 to 373.2 µg g−1 in kabuli flour. The Fe concentrations in desi flour fortified with FeSO4·H2O and NaFeEDTA ranged from 63.7 to 364.5 µg g−1 and from 64.1 to 325.2 µg g−1, respectively, and the Fe concentrations in kabuli flour fortified with FeSO4·H2O and NaFeEDTA ranged from 62.7 to 278.5 µg g−1 and from 66.1 to 237.6 µg g−1, respectively.Fortification of split desi seeds significantly increased  also demonstrated the Fe concentrations in the product increased following the Fe concentrations in fortificants [A recent study on dehulled lentil using the same three food-grade Fe fortificants (FeSOificants . The maiificants . Similarificants . Our fin−1) and bioavailability (ng ferritin mg−1 protein) for three cooked chickpea samples (cooked split desi seeds and chapattis prepared from desi flour or kabuli flour) fortified with 2000 µg g−1 Fe concentration from FeSO4·7H2O, FeSO4·H2O and NaFeEDTA fortificants are shown in The iron concentration (µg gp < 0.0001). Moreover, for cooked split desi seeds (soup), the relationship between Fe concentration and Fe bioavailability was highly significant, with a correlation coefficient of 0.78 (p < 0.05)  A. The chp < 0.0001). The relationship between Fe concentration and Fe bioavailability was also highly significant, with a correlation coefficient of 0.99 (p < 0.05) . Since the chapattis were prepared using flour from dehulled desi seeds and refined wheat flour, the anticipated superior absorption of Fe from NaFeEDTA was not observed. The phytate content of refined wheat flour is 2–4 mg g−1, compared to 6–10 mg g−1 in whole wheat flour [Fe concentrations and bioavailability for chapattis prepared from desi flour treated with different Fe fortificants are shown in  < 0.05) B. As wasat flour . Currentat flour ,44.p < 0.0001). The choice of fortificant had no effect on either the Fe content or the Fe bioavailability of kabuli chapattis. Chapattis prepared from kabuli flour exhibited a moderate correlation between Fe concentration and Fe bioavailability . Chapattis prepared from kabuli flour included the seed coat. Research has shown that colored chickpea seeds (desi) contain more polyphenolic compounds than cream or beige colored seeds (kabuli). However, light colored seed is not completely free of bioactive substances [Iron concentrations and bioavailability for chapattis prepared from fortified kabuli flour are shown in bstances . Furtherbstances . In the The relationship between Fe concentration and Fe bioavailability has been reported in previous studies, where an elevated Fe concentration in lentil increased Fe bioavailability ,47. Our 4·7H2O, FeSO4·H2O and NaFeEDTA) on Fe bioavailability are presented in p < 0.05) on Fe bioavailability. In contrast, the effect of the fortificant was not significant.The effects of three different food matrices (cooked split desi seeds (soup), desi chapatti and kabuli chapatti) and three food-grade Fe fortificants  on Fe bioavailability are presented in The interaction effects between food matrices (cooked split desi seeds (soup), desi chapatti and kabuli chapatti)) and fortificants  had no effect on Fe bioavailability. Apparently, none the food matrices contained sufficient phytate for the reported superiority of NaFeEDTA as a fortificant to be observed [Across the three food matrices, the choice of fortificant  on split desi seed, desi flour and kabuli flour included color changes, as reflected in L*, a* and b* values over four time intervals  are presented in 4·7H2O, FeSO4·H2O and NaFeEDTA). For all three attributes, the control and the FeSO4·H2O-fortified sample had the lowest and highest scores, respectively, for split desi seed, desi flour and kabuli flour. The mean values for uncooked split desi seeds were 6.9 for appearance, 6.7 for odor and 6.8 for overall acceptability. The mean scores for NaFeEDTA-fortified samples were higher than those of FeSO4·7H2O- and FeSO4·H2O-fortified samples for all attributes. The mean scores for Fe-fortified uncooked split desi seeds were significantly lower (p < 0.05) than those of the control for all three attributes. For uncooked desi flour, mean scores were 7.5 for appearance, 7.0 for odor and 7.4 for overall acceptability. For the three attributes, the highest scores were for the control and NaFeEDTA-fortified samples. The lowest scores were obtained for FeSO4·H2O-fortified samples. However, there was no significant variation for any of the attributes across all fortificants, except for uncooked split seeds and overall acceptability in desi flour. For uncooked kabuli flour, mean scores were 6.7 for appearance, 6.5 for odor and 6.6 for overall acceptability. As for uncooked desi flour, NaFeEDTA-fortified samples scored higher than or equal to the control for all attributes. There was no significant variation for the control, NaFeEDTA-, FeSO4·7H2O- and FeSO4·H2O-fortified samples for all three sensory attributes. Among the three samples, the mean value for chapattis prepared from desi flour was higher than for cooked split desi seeds and chapatti prepared from kabuli flour for all five sensory attributes, except for taste in cooked split desi seeds. The reason behind this finding is that in sensory evaluation, Fe-fortified and unfortified cooked products from desi flour could not be differentiated from the control due to seed coat, cooking product and method.The uncooked samples were fortified with three Fe compounds  are presented in Results for five sensory attributes  for three cooked products  fortified using a fortificant Fe concentration of 2000 µg g4·7H2O-fortified samples for all five attributes. The lowest score was obtained for the FeSO4·H2O-fortified product. Mean scores for cooked split desi seeds were 7.1 for appearance, 6.9 for odor, 7.2 for taste, 6.9 for texture and 7.0 for overall acceptability. For all five attributes, the mean values for Fe-fortified cooked split desi seeds were significantly lower (p < 0.05) than for the control. The mean scores for NaFeEDTA-fortified samples were higher than for FeSO4·7H2O- and FeSO4·H2O-fortified samples for all attributes. These findings are similar to those for NaFeEDTA where fortified cooked samples scored higher than did FeSO4·7H2O- and FeSO4·H2O-fortified samples. For chapattis prepared from desi flour, the mean scores were 7.4 for appearance, 7.2 for odor, 7.1 for taste, 7.0 for texture and 7.1 for overall acceptability. For chapattis prepared from kabuli flour, the mean scores were 7.0 for appearance, 6.8 for odor, 6.9 for taste, 6.9 for texture and 6.9 for overall acceptability. As for cooked split desi seeds, chapattis prepared from desi flour and kabuli flour exhibited the highest scores for the control and the lowest scores for FeSO4·H2O-fortified samples. However, in contrast to cooked split desi seeds, FeSO4·7H2O-fortified desi chapattis and kabuli chapattis exhibited scores higher than or similar to those of NaFeEDTA-fortified desi chapattis. As for cooked split desi seeds, the average scores for Fe-fortified desi chapattis were significantly lower (p < 0.05) than those of the control for all five attributes. The attributes of Fe-fortified desi chapattis were not significantly different (p > 0.05) from that of the control. Similarly, for kabuli chapattis, Fe-fortified products did not exhibit significant differences (p > 0.05) for any of the five attributes. Some studies have found no significant differences between unfortified and fortified rice [For cooked split desi seeds (soup), the maximum average score was observed for unfortified samples, followed by NaFeEDTA- and FeSOied rice ,52. Our −1. It was found that fortificant with this Fe concentration provided approximately 180–190 µg g−1, 160–200 µg g−1 and 110–190 µg g−1 of additional Fe per 100 g in the cooked split desi seeds, desi chapatti and kabuli chapatti, respectively. Therefore, per serving (50 g), Fe-fortified split desi seeds or desi chapatti could supply more than 10 mg of Fe . A wide range (9–13 mg) in Fe content (6–10 mg from fortified kabuli chapatti plus 3 mg from the base chickpea) was observed for fortified kabuli products. These results indicated that fortified chickpea products could provide a major part of the estimated average requirements (EAR) for Fe. A previous study with lentil showed that Fe fortification using a fortificant Fe concentration of 1600 µg g−1 could provide an extra 10 mg of Fe [In the current study, panelists evaluated Fe-fortified cooked products which had been fortified using a fortificant with Fe concentration of 2000 µg gmg of Fe .4·7H2O, compared to FeSO4·H2O and NaFeEDTA, and higher consumer preference. A major finding from this study was that Fe bioavailability increased after fortification with a fortificant Fe concentration of 2000 µg g−1 for the three Fe fortificants. Approximately 11–12 mg of additional dietary Fe could be provided by a 50 g serving of cooked fortified chickpea products. Therefore, fortified chickpea could provide a major portion of the recommended daily allowance for Fe, which could help to reduce Fe deficiency in target populations.Chickpea is an economically valuable pulse grown in 59 countries around the world, of which approximately 96% is grown in developing countries. Moreover, chickpea is consumed as a daily staple in many developing countries where the prevalence of Fe deficiency is severe. Chickpea occupies an important role in the protein and mineral micronutrient supply, including Fe, for people who depend on plant-based diets for their total calories, especially in South Asia. However, Fe bioavailability from chickpea is relatively low. Therefore, iron fortification of chickpea is worth considering as a means of providing more Fe to mitigate Fe deficiency. This study demonstrates that it is possible to increase both Fe concentration and the amount of deliverable Fe in foods made from chickpea. In terms of Fe bioavailability, NaFeEDTA did not show any superiority over the two ferrous salts in cooked split desi seeds (soup), which is inconsistent with previous reports and fortification guidelines , and mayWe now have baseline information for fortifying desi chickpea dal and flour of both desi and kabuli chickpea. To our knowledge, this is the first report on Fe fortification of chickpea seed and flour. This information is important for future commercial Fe fortification of chickpea. Although this laboratory-based study suggested feasibility, effective scaling up of Fe fortification at the industrial level needs to be considered. Given the strong influence of Fe fortification on Fe deficiency, it will be important to effectively market Fe-fortified chickpea dal and flour to consumers. As for other pulses, chickpea dal is processed by washing, soaking and cooking of the dal prior to consumption. This may reduce the nutritional value (Fe content and availability) of fortified split chickpea seed. However, one of the positive aspects of Fe fortification of chickpea flour is that people do not wash flour prior to consumption. Therefore, it is recommended that Fe fortified chickpea products be labelled “ready to cook” and “ready to eat”. Moreover, large-scale human trials in Fe-deficient populations need to be conducted to determine the efficacy of Fe fortification of chickpea in mixed diets. Overall, it is recommended that effective systems for Fe fortification of chickpea dal and flour be established to alleviate Fe deficiency in affected populations.


Raloxifene (RXF) is a hormone-like medication used for treating postmenopausal osteoporosis and estrogen-dependent breast cancer, yet associated with bad low bioavailability due to poor solubility. This study was intended to develop cyclodextrin/chitosan nanoparticles (ccNPs) for oral delivery of RXF in order to enhance the oral bioavailability. RXF-loaded ccNPs (RXF-ccNPs) were prepared by cyclodextrin inclusion followed by complexation with chitosan. RXF-ccNPs were fully characterized by particle size, morphology and in vitro drug release. The oral delivery efficacy and transepithelial transport potential were evaluated by pharmacokinetics, in situ single-pass intestinal perfusion, cellular uptake and ex vivo imaging. The resulting RXF-ccNPs were around 165 nm in particle size with a narrow distribution. The oral bioavailability of RXF was enhanced by 2.6 folds through ccNPs compared to RXF suspensions in rats. It was shown that RXF-ccNPs could improve the intestinal permeability of RXF, increase the cellular uptake of RXF and facilitate its transport across the absorptive epithelia. The results indicate that our developed ccNPs based on sulfobutylether-β-cyclodextrin and oligochitosan are a promising vehicle to orally deliver poorly water-soluble drugs over and above RXF. Raloxifene (RXF) is clinically used as a selective estrogen receptor modulator for the treatment of postmenopausal osteoporosis and estrogen-dependent breast cancer ,2. The aPoor bioavailability tends to result in an unsatisfactory therapeutic effect, especially in the treatment of breast cancer. In order to break through the bottleneck of absorption, a variety of formulation strategies have been explored for oral delivery of RXF, including solid dispersions , lipid nIn this study, we utilized anionic sulfobutylether-β-cyclodextrin (SBE-β-CD) and cationic oligochitosan to fabricate composite nanoparticles in an attempt to achieve RXF loading and bioavailability enhancement. RXF-loaded cyclodextrin/chitosan nanoparticles (RXF-ccNPs) were prepared via inclusion/electrostatic complexation and characterized with particle size, morphology, in vitro release and gastrointestinal stability. The oral delivery efficacy of nanoparticles for RXF was evaluated by in situ single-pass intestinal perfusion, cellular uptake, and oral pharmacokinetics.Raloxifene hydrochloride (RXF) was purchased from Guangzhou Yiqing Biotechnology Co., Ltd. . Sulfobutylether-β-cyclodextrin (SBE-β-CD) and water-soluble oligochitosan (MW < 2000) were obtained from Meilun Biotechnology Co., Ltd. . CY3-labeled chitosan (CY3-chitosan) was from Ruixi Biological Technology Co., Ltd. . HPLC-grade methanol was provided by Merck . Deionized water was manufactured by a water purifying system . All other chemicals or reagents were of analytical grade.Kf) was calculated based on the equation: Kf = S/S0(1 − S), where S and S0 denote the slope of linear equation (if applicable) and drug solubility in the absence of SBE-β-CD, respectively.Phase solubility diagram of RXF as a function of SBE-β-CD was plotted according to the reported procedure . Brieflyv/v) and then evaporated to dry under reduced pressure at 35 °C. The residues were subsequently dissolved with an appropriate amount of deionized water to result in a solution equivalent to 10 mg/mL of SBE-β-CD. Finally, the obtained RXF-SICs solution was stepwise dropped into a 5 mg/mL of chitosan solution that was previously prepared with oligochitosan in water. To gain a preferred formulation, particle size and polydispersity (PDI) of RXF-ccNPs as indices were adopted to optimize the mass ratio of SBE-β-CD to oligochitosan upon complexation.RXF-ccNPs were prepared through two steps. The first step was to prepare RXF/SBE-β-CD inclusion complexes (RXF-SICs) by the solvent evaporation method . In the The particle size and ζ potential of RXF-ccNPs were measured at 25 °C by Zetasizer Nano ZS . The sample was properly diluted with deionized water and then subjected to laser diffraction or Doppler velocimetry. The data were collected with the built-in software for output of particle size ζ potential. The morphology of RXF-ccNPs was inspected by transmission electron microscopy . The sample was diluted and fixed on a carbon-coated copper grid by drying under the lamp light. The micrographs were taken at an accelerated voltage of 100 kv.H-indazol-1-yl)-2--4-hydroxycyclohexylamino)benzamide (SNX-2112) was designated as an internal standard substance for accurate quantification. The HPLC system was equipped with a degasser, a quaternary pump, an auto sampler and a VWD detector. The samples were eluted against a Kromasil C18 column  at 40 °C using a gradient of formic acid (0.1%) in water (mobile phase A) vs. formic acid (0.1%) in acetonitrile (mobile phase B) at a flow rate of 0.45 mL/min and detected at 288 nm [Raloxifene in all samples was determined by Agilent 1100 HPLC  coupled with an API 4000 triple quadrupole mass spectrometry . The compound of 4--4,5,6,7-tetrahydro-1® Ultra 0.5 mL, Merck, Darmstadt, Germany). RXF in the supernatants was quantified by HPLC described above. All experiments were performed in triplicate.The release of RXF from RXF-ccNPs was investigated in vitro in water, 0.1 N HCl and pH 6.8 PBS. Briefly, 5 mL of RXF-ccNPs were put in into 900 mL of release medium and maintained at 37 °C under stirring (100 rpm). At predetermined intervals, 2 mL of medium were withdrawn and immediately centrifuged at 10,000 rpm using a centrifugal filter with a molecular weight cut-off (MWCO) of 10,000 (Amiconn = 6). They were administered with RXF suspensions, RXF-SICs solution and RXF-ccNPs by gavage at the dose of 50 mg/kg, respectively. Here, the suspensions formulation was prepared by dispersing RXF in 0.5% CMC-Na solution. At predetermined time points, approximately 0.25 mL of blood were collected into heparinized tubes from the tail vein of rats. The blood was immediately centrifuged at 5000 rpm for 5 min to prepare the plasma. A deproteinization procedure was utilized to extract RXF from the plasma [Sprague Dawley (SD) rats  were used to appreciate the oral pharmacokinetics. The animals were treated as per the Guidelines on the Care and Use of Animals for Scientific Purposes (2004) and the experimental protocols were approved by the Institutional Review Board of Guocaoxiafang Biotechnology (GB20171203). The rats were fasted overnight but ad lib accessible to water. Rats were randomly divided into 3 groups  was calculated with the following equation:Q represents the flow rate (0.2 mL/min), r and L denote the radius and length of the perfused intestinal segment (cm), Cin and Cout are the inlet and outlet concentration of RXF, respectively.In situ single-pass intestinal perfusion was adopted to evaluate the intestinal permeability of free RXF and RXF-ccNPs as described by Li et al. . Briefly5 cells/well and cultured for two days under a 37 °C/5% CO2 atmosphere. Afterwards, the culture medium was removed and replaced with DMEM-diluted RXF or RXF-ccNPs (25 μg/mL). The cells were treated for 2 h and 4 h at 37 °C respectively, then washed twice with cold PBS, and followed by lysis with RIPA buffer. The lysates were then centrifuged at 10,000 rpm for 5 min under 4 °C to obtain the supernatant. RXF in the supernatant was determined by HPLC/MS established above. The cell concentration was gauged by protein content using a BCA protein assay kit.Caco-2 cells from ATCC were cultured in DMEM, in which fetal bovine serum  and penicillin-streptomycin solution (100 IU/mL) were supplemented. For cellular uptake study, Caco-2 cells were seeded in 12-well plate at a density of 5 × 10Atri/Acon) × 100%, where Atri and Acon representing the absorbance of living cell treated with drug and blank culture medium, respectively.The cytotoxicity of nanoparticles was evaluated by MTT assay. The well-cultured Caco-2 cells were washed thrice with PBS. Then, blank ccNPs with various levels were added into the cells and incubated for 24 h at 37 °C. After that, MTT solution  was added and incubated for another 4 h. At last, DMSO was used to dissolve the resultant formazan. The absorbance in the microwells was measured at 570 nm with a microplate reader . The cell viability was calculated using the equation: Cell viability (%) = . This type of plot indicated the formation of soluble cyclodextrin inclusion complexes at a stoichiometric rate of 1:1. The intrinsic solubility of RXF was approximately 0.465 mg/mL (9.83 × 10−4 mol/L) determined in our laboratory, close to that reported by Tran TH et al. [Kf) was 106.91 L/mol. The Kf value was considerably large for an inclusion compound, demonstrating a stable complexation. Phase solubility study indicates that it is practicable to use SBE-β-CD to include RXF.The phase solubility diagram of RXF vs. SBE-β-CD in water is shown in H et al. . Thus, tThe electrostatic complexation technique is a commonly used approach to fabricate polyelectrolyte nanoparticles ,20. In tThe particle size of RXF-ccNPs prepared by the formulation above established was 165.8 nm, showing a narrow distribution (PDI = 0.137) A. Due tor2) were 0.7862 for Zero-order, 0.9243 for First-order, 0.8351 for Higuchi model, 0.8654 for Hixson–Crowell model, and 0.8718 for Korsmeyer–Peppas model, respectively. It could be inferred that RXF release from RXF-ccNPs was in agreement with the first-order kinetic process, which could be ascribed to passive diffusion. The accumulative release was less than 33% at 4 h and approximately 70% within 24 h. In 0.1 N HCl, RXF-ccNPs presented a somewhat fast RXF release relative to in water and in pH 6.8 PBS. However, the similarity factors (f2) between three release curves were larger than 70, indicating no significant difference among them [The in vitro release profiles of RXF from RXF-ccNPs with RXF suspensions as control are shown in ong them . Low druCmax) of 108.46 ng/mL. Different from RXF suspensions, RXF-SICs solution gave rise to an elevated Cmax, which was almost two times as high as that of RXF suspensions. However, RXF-ccNPs resulted in a higher Cmax, up to 367.48 ng/mL. The time to Cmax (tmax) and the terminal half-life (t1/2) among three formulations appeared to be a little distinct. The formulation with good absorption possesses smaller tmax and shorter t1/2, which is related to the fast drug disposition, including absorption and elimination. The area under plasma drug concentration vs. time curve (AUC0-t) of RXF-ccNPs was up to 2400.78 ng/mL·h that was notably larger than that of RXF suspensions and RXF-SICs solution. The oral bioavailability of RXF-ccNPs relative to RXF suspensions was calculated to be 360.76%, whereas it was just 249.38% for RXF-SICs solution. RXF-ccNPs demonstrated an excellent absorption-promoting effect on RXF.The pharmacokinetic profiles of RXF concentration vs. time in rats after oral administration of RXF suspensions, RXF-SICs solution and RXF-ccNPs are displayed in Cyclodextrin inclusion has been shown able to promote the oral absorption of poorly water-soluble drug by improving the dissolution ,24. In oPeff values of free RXF and RXF-ccNPs in various intestinal segments are listed in Peff smaller than 5 × 10−6 cm/s. It indicates that the intestinal permeability of free RXF is poor, since a Peff of ca. 10−6 cm/s is predicted to be difficultly absorbed by the intestine [Peff notably increased for ccNPs-encapsulated RXF where the Peff values came to the order of 10−5 cm/s. In terms of different intestinal segments, a more significant improvement in the permeability took place in the ilium. The Peff value of RXF-ccNPs was up to 2 × 10−4 cm/s, suggesting a high absorption in this site. It can be attributed to the facilitating effect from chitosan. Depending on polycationic property, chitosan-based nanoparticles can protect labile drugs against rapid degradation and interact with the enterocytes to overcome the transport barriers by opening the tight junctions and positive cytosis [The ntestine . The Pef cytosis . Peff did not exclude the effect of intestinal metabolism on drug elimination. If the intestinal metabolism being taken into account, the Peff of free RXF may be lower. Thus it follows that RXF-ccNPs greatly boost the intestinal permeability of RXF that is fairly favorable for RXF absorption after oral administration.RXF available on the market is mostly the hydrochloride form. Low permeability of free RXF can be explained by the dissociation of the drug in the intestinal lumen. In addition, the determination of High cellular uptake guarantees good absorption and bioavailability. The safety of biomaterials is a great concern for oral drug delivery. The histomorphology of absorptive epithelia as well as fluorescence distribution on them after oral administration of fluorescent RXF-ccNPs are shown in We have successfully developed an oral nano-delivery system of RXF based on cyclodextrin and chitosan. It found that RXF could be well included into SBE-β-CD and further form nanoparticles with oligochitosan via electrostatic interaction. The resulting nanoparticles (cc-NPs) possessed a small particle size and exhibited a sustained drug release. The oral bioavailability of RXF was significantly enhanced through cc-NPs, up to 3.6 folds relative to RXF suspensions. RXF-ccNPs were provided with high intestinal permeability, excellent cellular uptake and biocompatibility as well as good transepithelial aptness. These findings support the assumption of ccNPs as a safe and efficient vehicle for oral delivery of RXF. The reported system provides a formulation strategy to tackle amphiphobic drugs that may serve the settlement of low bioavailability thereof.


Stabilization of covalent organic frameworks (COFs) by post-synthetic locking strategies is a powerful tool to push the limits of COF utilization, which are imposed by the reversible COF linkage. Here we introduce a sulfur-assisted chemical conversion of a two-dimensional imine-linked COF into a thiazole-linked COF, with full retention of crystallinity and porosity. This post-synthetic modification entails significantly enhanced chemical and electron beam stability, enabling investigation of the real framework structure at a high level of detail. An in-depth study by electron diffraction and transmission electron microscopy reveals a myriad of previously unknown or unverified structural features such as grain boundaries and edge dislocations, which are likely generic to the in-plane structure of 2D COFs. The visualization of such real structural features is key to understand, design and control structure–property relationships in COFs, which can have major implications for adsorption, catalytic, and transport properties of such crystalline porous polymers. Stabilization of covalent organic frameworks (COFs) by post-synthetic locking is a powerful tool to push the limits of COF utilization. Here the authors demonstrate a sulfur-assisted conversion of an imine-linked COF into a thiazole-linked COF, with retention of crystallinity and porosity, allowing for direct imaging of defects in COFs. Reversibility of the COF linkage is key to obtain ordered materials by error correction and defect healing6, but at the same time makes COFs inherently unstable and rich in defects. This dilemma can be circumvented by performing a reversible order-inducing step under thermodynamic control and subsequently arresting this order via a post-synthetic treatment, a concept that has been explored for the synthesis of crystalline, “unfeasible” zeolites7 as well as to stabilize the molecular cages formed by dynamic covalent chemistry8. In order to arrest COFs in their crystalline state (arrested linkage COFs: ALCOFs) the chemical linkage of the COF needs to be converted2 from a reversible to an irreversible type of bond in a topochemical fashion.Covalent organic frameworks (COFs) distinguish themselves from conventional polymers by three defining features: covalent connectivity, porosity, and crystallinity. This generation of two- (2D) and three-dimensional (3D) polymers is synthesized under solvothermal conditions by reversible covalent bond forming reactions9. In spite of these challenges, analysis of the ideal and real structure of COFs has been in the focus since the very beginning of the field, and it continues to attract attention as the field12.The competition between reversibility and stability, which is dominated by subtle changes in the reaction conditions, often leads to COFs with only moderate chemical stability and low crystallinity. As a result, nanocrystalline COFs with ubiquitous structural disorder are obtained. Such structural disorder is difficult to probe or even quantify by conventional diffraction methods such as X-ray powder diffraction (XRPD), while imaging with transmission electron microscopy (TEM) is difficult due to the sensitivity of COFs – similar to most soft matter systems – to the electron beam in comparison to inorganic crystalline materials14. In-plane defects, which also impact the electronic transport properties of COFs, have been invoked to explain the low crystallinity of many COFs15. In closely related systems such as on-surface grown, monolayer 2D polymers, rings with five or seven edges in an otherwise hexagonal system have been observed as defects by scanning tunneling microscopy17, very similar to the formation of defects in graphene18. While often detrimental, such defects can be highly beneficial, for example in (electro)catalysis where they act as high-energy binding sites for the adsorption of reaction intermediates19. In addition, the typically limited size of the crystallites of COFs leads to grain boundaries and domain intergrowth, which can drastically influence the long-range charge carrier percolation in COFs20 as well as influence ion conduction22. These examples show that disorder can have a significant influence on the chemical, structural and (opto-)electronic properties of COFs. However, such defects were never directly imaged. Hence, it is of paramount importance to understand the types of defects and disorder present in COFs and to elucidate and control their influence on the properties of the material.In 2D COFs, various kinds of out-of-plane disorder are prevalent, such as random or poorly defined stacking sequences23 with elemental sulfur into a thiazole-linked COF through a post-synthetic locking strategy, thereby establishing a class of thiazole-based COFs. This type of post-synthetic modification is fundamentally different from previous examples, such as the introduction of functionalities by tethering side groups24 or heterogeneous linker exchange:25 These approaches either do not change the reversible bond of the starting COFs, or they even utilize the reversibility to introduce functionality. The post-synthetic oxidation of an imine COF linkage to an amide, as reported recently, provides a direct transformation of the reversible bond of the COF;2 however it was recently shown that even amides are, in principle, reversible enough to be used for the synthesis of COFs26. Modification of the imine linkage of the TTI-COF leads to excellent contrast and high electron beam stability of the sulfur-modified TTI-COF , which enables imaging and analysis of in-plane defects with TEM, thus revealing details of real structure effects that have not been amenable to direct observation in any COF so far.In this work, we investigate the topochemical conversion of the triphenyl triazine imine COF (TTI-COF)2 the promise of COFs lies in the fact that COFs, though being extended solids, are amenable to the versatile toolbox of molecular synthesis. While this concept is particularly useful at the precursor level and hence formation of COFs, strategies for modifying the backbone of COFs once they are formed are extremely scarce2. We thus explored the post-synthetic reaction of COFs with elemental sulfur. At high temperatures, elemental sulfur reacts with aromatic imines to first oxidize the imine to a thioamide, and subsequently oxidatively cyclizes the thioamide group to form a thiazole ring  and as a nucleophile, attaching first to the imine carbon and afterwards to the phenyl ring on the nitrogen side of the imine.As pointed out by Yaghi et al.,ing Fig. 27. Thus,23, we synthesized TTT-COF in two successive steps: First, TTI-COF was infiltrated with molten sulfur at 155 °C. At this temperature, sulfur has minimum viscosity, enabling mixing with the COF material. Subsequently, by using a thermal treatment at higher temperature (350 °C), the conversion of the TTI-COF to the TTT-COF took place. After removal of the excess sulfur by Soxhlet extraction and under high vacuum, the obtained material was investigated by 13C and 15N solid state NMR (ssNMR) to probe the imine to thiazole conversion and retention of the framework structure  in the 13C ssNMR spectra, together with the appearance of the 3′ signal at 156 ppm for the TTT-COF, indicate the conversion of the nitrogen bearing phenyl ring to the thiazole in TTT-COF. Small residual intensity at 151 ppm might indicate some unreacted TTI-COF. Furthermore, the characteristic imine carbon 2 is shifted in the thiazole 2′ as a shoulder to the triazine carbon 1′29. Also, the absence of a 13C signal between 210 and 180 ppm30 hints at the conversion of the imine to the thiazole and the absence of thioamide. 15N ssNMR shows the triazine nitrogen, 6 and 6′, at the same position for TTT-COF as for TTI-COF and a shift of the imine nitrogen 5 from −55 ppm to the thiazole 5′ at −71 ppm. As additional confirmation of the determined thiazole structure of the TTT-COF, the calculated NMR chemical shifts of excised fragments  on the B97-2/3/pcS-2//PBE-D3/def2-TZVP level of theory  spectra further confirm the conversion of TTI-COF to TTT-COF, as evident by the disappearance of the characteristic imine (N = CH) vibration at 1627 cmm−1 Fig. . In addim−1 Fig.  of TTT-C2 g−1 for the TTT-COF  being close to the BET surface area of 1362 m2 g−1 for the precursor TTI-COF   and a higher symmetry than derived for the precursor TTI-COF (P1)   Å vs 25.244(8) Å), and a larger interlayer stacking distance (c = 7.002(5) Å) than rs-TTI-COF (c = 6.905(7) Å) as is evident from Rietveld refinement  Fig. , which i32, which was transformed to the Pyrene tetra(phenyl) biphenyl thiazole-COF  biphenyl imine-COF ; likewise, the crystallinity seen already for the TTI-COF is clearly retained as wellk0] Fig. . Both rek0] Fig.  are in ak0] Fig. , 23, 24.c-axes but are rotated against each other in the ab-plane  synthesis of the precursor TTI-COF and thus can be considered as lasting fingerprints of the COF formation process. In particular, we find prevalent one-dimensional defects such as edge dislocations as well as co-aligned COF grains with grain boundaries that are likely covalently connected. Unraveling the nature of defects in COFs is not only key to better understand their impact on the optical, electronic and catalytic properties of COFs, but also to control and design new COFs by targeting properties imbued by such defects.−1 heating rate) and maintained there for 3 h. Subsequently, the temperature was raised to 350 °C (100 K h−1 heating rate) and kept for 3 h. After cooling down, the resulting material was washed via Soxhlet extraction with toluene and THF for 24 h, respectively. The samples were dried at 70 °C in an oven and then at 150 °C under high vacuum.Triazine triphenyl thiazole COF (TTT-COF) and Pyrene tetra(phenyl) biphenyl thiazole (PBT-COF): The respective imine COF was activated under high vacuum at 150 °C and subsequently mixed with the 15-fold amount (by weight) of sulfur in a ball mill. The resulting homogeneous mixture was transferred to a quartz boat in a horizontal tubular furnace and purged at 60 °C under flowing argon. The temperature was increased to 155 °C : TT-CHO , TT-NH2 , di(n-octly)phthalate (1 ml), triphenyl phosphate (1 ml), aqueous acetic acid  were added successively to a Biotage2NNH2) and 1 M sodium borohydride (NaBH4) for 16 h at room temperature unless denoted otherwise. Afterwards, the sample was filtered off and washed thoroughly with water, ethanol, THF, chloroform and DCM. After drying at ambient conditions, the crystallinity was assessed by XRPD.Chemical stability of the TTI- and TTT-COF was assessed by immersing ~20 mg of the COF in an aqueous solution of each 12.5 M hydrochloric acid (HCl) (50 °C), 12 M potassium hydroxide (KOH), 1 M hydrazine . Molecular connectivity was based on geometric considerations and the obtained evidence from FT-IR and ssNMR. The structures and the unit cell was relaxed using force fields . These models were then used to refine the unit cell parameters by Rietveld refinement.The structural models were built successively and based on each other starting from the well defined TTI-COF model6 cathode). The samples were suspended in n-butanol and drop-cast onto a lacey carbon film (Plano). Processing of TEM and SAED images was performed with the help of ImageJ 1.47 v.TEM was performed with a Philips CM30 ST .Stability measurements were performed by taking images of the sample after defined time intervals in-between pictures, relative to the first image ) in Debye-Scherrer geometry. The sample was measured inside a sealed glass capillary (1.0 mm) that was spun for improved particle statistics.XRPD patterns were collected on a Stoe Stadi P diffractometer  at the frequencies of 400.1, 100.6 and 40.8 MHz, for 1H, 13C and 15N, respectively. The ssNMR experiments were performed on a Bruker double resonance 4 mm MAS probe with the COF samples packed in ZrO2 rotors. The 1H-13C and 1H-15N cross-polarization (CP) MAS spectra were recorded with a rotation frequency of 10–12 kHz using a ramped-amplitude (RAMP) spin-locking pulse on the proton channel. The contact time for both nuclei was set to 5 ms, which was found to be optimal. The recycle delay in the CP-experiments was 2 s, defined primarily by the spin-lattice relaxation of protons. All solid-state experiments were carried out using SPINAL64 composite-pulse proton decoupling with radio frequency power between 70 and 80 kHz. The reported 1H and 13C chemical shifts were referenced to tetramethylsilane (TMS), while the 15N shifts were referenced to nitromethane.The ssNMR spectra were recorded on a Bruker Avance III 400 MHz spectrometer (B47 level of theory using an acceleration scheme based on the resolution of the identity (RI) technique and the continuous fast multipole method (CFMM)48 in a developer version of Turbomole49.Atom positions and lattices of periodic structures were optimized on PBE-D3/def2‑TZVP-9 a.u. Grid 7 was used for the numerical integration of the exchange-correlation term. The norm of the gradient was converged to 10-4 a.u. and the total energy is converged to 10-8 Hartree within the structure optimization using an equidistant 5 × 5 k-point grid.The CFMM uses multipole moments of maximum order 20 together with a well-separateness value 3 and a basis function extent threshold of 1051 using the Turbomole program package in version 7.0.2 for geometries and the FermiONs++53 program package for the calculation of NMR chemical shifts performed on excised sections (Supplementary Figs. NMR chemical shifts were obtained on B97-2/pcS-2//PBE-D3/def2-TZVP level of theoryAll relevant data are available from the authors upon reasonable request.Supplementary Information


The effects of extruded corn flour (ECF) on the rheological properties of the wheat‐based composite dough and quality of the bread were investigated. The RVA results of the composite flour with ECF showed weak thermal viscosity and resistance to starch retrogradation. Mixolab tests revealed that the water absorption capacity increased with the increasing amount of ECF, while dough development time (DT) and dough stability (ST) showed a downward trend, and the composite dough became more resistant to retrogradation. The microstructure of the composite dough showed that the presence of both ECF and unextruded corn flour (UECF) resulted in a more broken gluten matrix. The breads made from the composite flour with ECF had significantly softer texture, lower hardening percentage with storage time, darker crust color, larger specific volume, and higher sensory scores than the UECF ones. It is concluded that the extrusion of corn flour is an effective way to improve the quality of the composite bread and retard staling during storage. The moisture was adjusted to reach final moisture content of 21% prior to extrusion. The extruded temperature and the screw speed were set at 100°C and 180 rpm, respectively. The circular matrix with 8 mm × 3 diameter was used. Both extruded and unextruded corn grits were ground with a universal grinder  for 2 min and screened through a sieve (80 mesh) to make extruded corn flour (ECF) and unextruded corn flour (UECF).The premixed flour of WF and ECF or UECF with substitution of 10%, 20%, 30%, and 40% was thoroughly blended by rotating and shaking in a resealable plastic bag.2.22.2.1Pasting properties of WF, ECF, UECF, and mixed flour were determined according to the method of LS/T 6101‐2002  was used to study the agitation and batter characteristics of WF, ECF, UECF, and mixed flour using chopin + protocol according to ICC No. 173  according to the method of Ma et al. . Small p2.2.42 in top, 15 × 6 cm2 in bottom, and 6.5 cm in depth). After proofing for 40 min at 30°C and RH of 85%, the dough was baked in an oven for 20 min (upper temperature 190°C and bottom temperature 200°C). The breads were removed from molds and cooled for 2 hr at 20°C before further analyses. The specific volume of bread was determined by the rapeseed displacement method and calculated in ml/g.A straight dough method  according to the method of 74‐09 AACC, . The bre2.2.6L*a* b*. Every measurement was duplicated for seven times.The color of bread was measured using colorimeter  with measurement area ø = 10 mm and Illuminant D65. The color values of the central point on each quarter of crust or crumb were measured and recorded according to color space of CIE 2.2.7Sensory evaluation of breads was performed with a group of twenty trained panelists using nine‐point hedonic scale according to the method of Patil et al. . The bre2.2.8p < .05). All statistical analyses were performed using DPS version 7.05 for windows.Measurements were performed at least in duplicate and analyzed using analysis of variance (ANOVA); then, the results were expressed as mean value ± standard deviation. A Tukey's multiple range test was conducted to establish the significant differences among experimental mean values (33.1p < .05) decrease in PV, FV, BD, and SB, indicating a severe breakage of the amylose chain, a loss of retrograde ability (Martínez, Rosell, & Gómez, The pasting parameters of WF, ECF, UECF, and composite flours are shown in Table 3.2The parameters WA, DT, ST, C2, C3, and C5‐C4 are shown in Table 3.3SEM is presented in Figure The morphology of composite dough observed by 3.4The effects of substitution of corn flour on the hardness of crumb are shown in Table 3.5The effects of ECF and UECF on the specific volume and bread color are presented in Table With increase in substitutive level, the value L* and b* of both ECB and UECB crust increased, respectively, while the value a* of UECB crust decreased. Both the values L* and b* of ECB crust were lower than those of UECB at all levels of substitution. The value L* of WB crust was higher than that of 10% ECB, but lower than those of the 40% ECB and all samples of the UECB group, and had no difference to the 20% ECB and 30% ECB. This was mainly due to the higher loaf height Figure . The lowWith increase in substitutive level, the values a* and b* of both ECB and UECB crumb increased, respectively, while the value L* decreased. The change in value a* was slight, especially for the ECB group. Both the values a* and b* of ECB crumb were lower than that of UECB crumb at all levels of substitution, while the L* values were all higher. The difference of both value L* and a* between crumb of ECB and UECB at all levels of substitution was slight. The main change in color of crumb was in yellowness. The color of crumb is mainly determined by materials (Turkut, Cakmak, Kumcuoglu, & Tavman, 3.6The sensory scores of composite breads were shown in stacked column graph Figure . Texture4The composite flour with ECF showed higher pasting temperature; but lower values of peak time, peak viscosity, final viscosity, and breakdown from the results of RVA tests; higher water absorption capacity; shorter dough development time; and lower dough stability from the results of Mixolab tests. The microstructure morphology of the composite dough structure showed that the presence of both ECF and UECF resulted in a more broken gluten matrix, and more entire starch granules were retained for the UECF one. The above facts indicated that the composite flour with ECF would result in softer dough with lower proofing resistance but weak ability of gas retaining during fermentation. The interactive balance of the above factors may be the reason why ECB had larger specific volume than the UECB ones but lower than WB.Both the results from the RVA and Mixolab tests showed the resistance to starch retrogradation of ECF. The ECB with 20%, 30%, and 40% addition had significantly softer texture than that of UECB, and the breads with 20% and 30% substitution of ECF were significantly softer than both WB and UECB during storage. The above facts sufficiently approved the antistaling ability of ECF.The bread from composite flour with ECF had lower hardening percentage with storage time, darker crust color, larger specific volume, and higher sensory scores than the UECB. Consequently, it can be concluded that the extrusion of corn flour is an effective way to improve the quality of composite bread.The authors declare no conflict of interest.This study has nothing to do with human and animal testing.