The influence of the addition of four legume flours, chickpea, broad bean, common bean and red lentil , in ratios of 50:45:5; 50:40:10; 50:35:15) was studied by analyzing the rheological properties of dough in order to further exploit the functionality of legume flours in bakery products. The rheological properties of dough were monitored using a Mixolab 2. A Rheofermentometer F4 was used to check the dough fermentation, and a Volscan was used for evaluating the baking trials. The addition of different legume flours in the mixtures resulted in different viscoelastic properties of the dough. The results showed a weakening of the protein network depending on the amount of legume flour added and on the specific legume flour. On the contrary, all samples with a higher proportion of legume flour showed an increased resistance to starch retrogradation. All flours had the ability to produce a sufficient volume of fermenting gases, with the exception of flours with a higher addition of broad bean flour, and the baking test confirmed a lower bread volume for bread with this addition. The results of the sensory evaluation indicated that legume flour additions resulted in breads with an acceptable sensory quality, in the case of additions of 5% at the same level as the bread controls, or even better. The aromas and flavors of the added non-cereal ingredients improved the sensory profile of wheat-rye bread. Breads with additions of chickpea, common bean and broad bean had a considerable proportion of darker colors in comparison to the control bread and bread with red lentil. Bread has been part of the human diet for thousands of years and it remains the most regularly consumed food in the world, due to its convenience, portability, nutrition, and taste ,2. BreadEfforts have been made to improve the nutritional quality of bread. One of the possibilities is partial replacement of wheat with non-bakery raw materials, which are valued thanks to the content of ingredients that do not appear in traditional breads. This creates diverse products with beneficial health effects ,9,10. AnRegardless of the type of legumes, the substitution of wheat flour with legume flour considerably improves the nutritional value by increasing the protein, mineral and fiber content of bread, and decreasing the glycemic index ,21,22,23Saccharomyces cerevisiae) and the gluten network. The rheofermentometer analysis is useful in predicting the real proofing performance of bread dough. This is used to estimate the dough properties during fermentation by measuring the released CO2 or produced pressure, considering that the produced CO2 serves to expand the dough and achieve the final bread loaf volume [The suitability of alternative crops for the production of quality bread is examined in particular by measuring the properties of their mixtures with wheat or some other flour. The dough components  and their interactions play an important role in the conformational structure as well as in the rheological properties ,20,25. Tf volume ,30. The f volume . The finThe main aim of this research was to model some experimental doughs containing different levels of wheat flour, rye flour and legume flour , in order to study the influence of the different ingredients on the rheological properties of the dough, its ability to retain fermentation gases, and the objective properties of the final product.In this study, commercial flours , were used namely WF (wheat flour), RF (rye flour), ChF (chickpea flour), BBF (broad bean flour), CBF (common bean flour), and RLF (red lentil flour). These commercially available flours have defined quality parameters . AccordiThe selected tested legumes represent the widely used varieties found to be nutritionally enriching according to recent publications.Saccharomyces cerevisiae  were purchased from the local market.All other ingredients such as salt , sucrose (Slovenské cukrovary s.r.o.), and dry yeast of the species Three types of mixture of wheat flour and rye flour and different proportions of four legume flours, as well as a control flour without legume flour, were prepared in the following ratios: 50:45:5, 50:40:10, 50:35:15, and 50:50:0, respectively. The proportion of wheat flour in composite flours did not change (50% in each sample), but the mutual ratios of rye flour and legume flours varied as indicated in The mixing and pasting behavior of the composite flours were determined in a Mixolab 2  according to the ICC Standard Method 173  using th2), volume of CO2 lost (carbon dioxide volume in mL that the dough has lost during proofing), and retention volume (carbon dioxide volume in mL still retained in the dough at the end of the test). The method conforms to the AACC 89-01 [A Rheofermentometer F4  was applied to detect the gas release kinetics depending on the varied quantities of legume flours. Immediately after kneading, 315 g of a biologically leavened dough sample was placed into the fermentation basket and was covered with the optical sensor. The proofing chamber was closed hermetically to begin the measuring series at 28.5 °C for 180 min. The following dough parameters were measured: Hm (maximum dough height (mm); Hm′ (maximum height of the gas release curve (mm); Tx (time of porosity (hours); total volume  two hours after baking. The following parameters were evaluated: weight of the bread (g), bread volume (mL), specific volume (mL/g), volume yield (mL/100g flour), aspect ratio of a middle slice. The laser step was set to 1 mm and the rotation speed was set to 1 rps. The specific volume was calculated on the bases of loaf volume and mass.The organoleptic characteristics of the breads  were evaBread products were analyzed by an E-eye analysis using an IRIS VA400 visual analyzer (Alpha M.O.S.) with a charge-coupled device camera. The bread samples were placed in the measurement chamber. The collected color data were represented by IRIS color codes, which encompass 4096 colors. The analyzed data (color spectrum) was obtained as numerical values of signal intensities.An analysis of variance (ANOVA) and a Fisher’s multiple range test were applied at a significance level of 5% to describe the significance of the differences between the reference and the samples with different levels of legume flours incorporated. All analyses were performed in triplicate and average values were calculated. XLSTAT 2020.5.1 by Addinsoft was used as the statistical analysis software. Linear regression  was used to display the trend line and the calculated correlation coefficient was evaluated at the significance level α = 0.05 (Microsoft Excel 2010).Bread dough is a viscoelastic material that exhibits an intermediate rheological behavior between that of a viscous liquid and an elastic solid. The viscoelastic network plays a predominant role in both dough machinability and the textural characteristics of the finished bread.Water absorption (WA) is an important parameter showing the ability of flour compounds to absorb water and form dough of optimal consistency ,36. WhenBlending the wheat flour with legume flour affects the viscoelastic and mixing properties of the dough. Turfani et al.  reportedp < 0.05) increased with the addition of common bean flour (CBF), directly depending on the amount added, so the ability of the beans to increase the absorption capacity of composite flours was confirmed. The significant increase of the WA was also recorded in composite flours with added broad bean flour (BBF), but in this case the amount of the addition was not important. Composite flours with an addition of CBF and BBF therefore needed the highest amount of water to reach the optimal dough consistency. An addition of RLF and ChF slightly reduced the binding of the composite flour, significantly in the case of WF(50) + RF(35) + RLF(15)  + RF(50), the water absorption in our samples significantly ( RLF(15) . Experim heating ,37.Regarding the dough development time (DDT) and dough stability, the additions of legume flour did not considerably affect them. DDT ranged in very close values from 1.5 min in flour WF(50) + RF(35) + RLF(15) to 3.5 min in WF(50) + RF(50). Compared to WF(50) + RF(50), the time required to form dough with the needed consistency was shorter in composite flours with ChF and RLF. In other flours with a proportion of legume flour, DDT remained unchanged. Turfani et al. , howeverThe stability of our doughs was relatively short (1.5 to 3.5 min), including the control flour, because we worked with wheat-rye flour, which has a lower stability due to the content of RF than it would have with pure WF. During dough development, pentosans disturb the protein network formation and influence the dough properties. In wheat flours, dough formation is mainly achieved by hydration of glutenin and gliadin proteins and the disulfide bond formation between them. In case of rye types, the lower protein content requires less time for hydration in spite of the presence of dietary fiber. The rye flours are unable to form a continuous gluten network, therefore rye flour and whole grain rye flour have low DDT and stability values . Voicu eA positive and expected benefit of replacing a certain proportion of rye flour with legume flour would be to improve DDT and the dough stability. The results, however, showed that none of the additives (legume flours), replacing the proportion of RF in composite flours, had a potentiating effect on the stability of the dough, nor did they improve the pasting properties of the dough by the input of protein structures.By means of the Mixolab “Chopin+” protocol it is possible to record the mechanical changes due to mixing and heating, simulating the mechanical work, as well as the heat conditions that might be expected during the baking process. The typical Mixolab curve can distinguish different pertaining stages of the dough changes due to both the mixing force and the temperature. During initial mixing, the distribution of the material, the disruption of the initially spherical protein particles and the hydration of the flour compounds occur together with the stretching and alignment of the proteins, leading to the formation of a three-dimensional viscoelastic structure with gas-retaining properties, where the polymeric proteins play the main role . During The torque of all evaluated samples started at the optimal value of the dough consistency (1.100 ± 0.095 Nm) and in individual indicative parameters  it changed depending on the composition of the composite flour. At the point C1, the dough is able to resist the deformation for a certain time, which determines the dough stability . The parp < 0.05), and the additions of ChF and CBF were significantly less stable. Weakening based on mechanical processing at 30 °C was the lowest in flours with the addition of CBF and the highest in flours with RLF, especially at higher additions  of ChF and CBF. In the samples WF(50) + RF(35) + RLF(15) and WF(50) + RF(35) + CBF(15), it was reduced by up to 31.4%, and 27.6% respectively, in comparison to the control sample, which is a considerable reduction. However, if we take into account the calculated value (C1.2 (torque at the end of the holding time at 30 °C)—C2), the effect of legumes on the thermal stability of the protein would be shown more precisely, as the weakening based on mechanical processing at 30 °C would be considered. The higher the value of C1.2–C2, the lower the thermal stability, and in this respect it would be possible to evaluate the additions of BBF and RLF as the most suitable in terms of thermal stability of the protein network in composite flour with an addition of legumes. Statistical differences were confirmed in these samples compared to the control  decrease in case of the addition of 15% of CBF.During the heating stage, doughs containing legume flours showed different pasting properties  in comparison to the wheat-rye dough, but we cannot generalize their influence . During A decrease in the temperature resulted in an increase of the torque due to the augmentation of the dough resistance. That increase corresponds to the starch gelation process, in which the amylose chains, which leached outside the starch granules during the heating, are prompted to re-crystalize. The re-association between the starch molecules, especially amylose, leads to the formation of a gel structure. This stage is related to the retrogradation and reordering of the starch molecules. In the cereal slurries, low setback values indicate a low rate of starch retrogradation and low syneresis . The firp < 0.05) increase in resistance to starch retrogradation  in the values of alpha slope, beta slope and gamma slope depending on the added legume flour and the amount of its addition. Alpha slope had significantly lower values than the control flour in samples with an addition of 15% of CBF and BBF and higher values with the RLF addition. Samples with added ChF and BBF (5% and 10%) had higher beta slope values than in the control flour (significantly in BBF with an addition of 10%). Compared to the control flour the gamma slope showed significantly lower values with higher additions of ChF, RLF and CBF, and significantly higher values with additions of BBF. It was BBF flour that most significantly affected the observed properties.The presented results are also supported by the data given in The alpha slope shows the protein weakening speed under heating effect, and its values correlate negatively with the rheofermentogram parameters  . Beta slThe dough behavior during mixing, pasting and gelling was influenced by the composition of the composite flour, i.e., the specific legume flour and its amount. The authors also list other possible influences, for example granules of starch having lost their integrity during milling ,47. SuchAccording to the results obtained by Mixolab measurements, flours from different raw materials exhibited mixolab profiles which greatly differ from the wheat flour profile . Our floDetermining the ability to form fermenting gases is crucial in order to produce bread with a good volume. Rheofermentometer analysis of flour and dough enables accurate simulation of processing conditions during production of baked goods containing yeast. The instrument records two curves during the dough fermentation and rising, one describing the development of the dough and another depicting the production and retention of gas.2 production during the fermentation of doughs with an addition of legume flour. Rheofermentometer curves very similar to those of the control wheat-rye dough were observed with doughs with an addition of chickpea flour and there were no significant differences between these composite flours. The doughs with an addition of broad bean flour had clearly the worst course of the rheofermentometer curves. This was confirmed by a low gas production followed by a low total volume. These flours had a low fermentation capacity, and there were significant differences (p < 0.05) between the individual samples caused by the amount of addition. The higher the addition of BBF applied, the lower the total volume, but also the other evaluated parameters.2 volume produced during the analysis, but this gas leaked and the retention volume remained without a significant change in comparison to control.On the other hand, the addition of 5% and 10% of RLF significantly increased the total CO2 produced, the gas retention capacity of dough is also important (p < 0.05) retention volume for doughs with higher additions (10 and 15%) of CBF and BBF. It is evident that three out of the four examined flour mixtures tested by the Rheofermentometer showed similar properties and overall the additives, with the exception of BBF, can be assessed based on the results of this analysis as potentially suitable and applicable in bakery technology.In addition to the amount of COmportant . In gene2 is produced during fermentation. While a critical CO2 volume is noted, the additional gas likely renders the dough matrix unstable due to disproportionation and coalescence. By progressive coalescence and the mechanical destabilization of the gluten network, CO2 as well as evaporating water and ethanol escape during baking and result in a reduction of the specific bread volume [The bread volume tends to increase when more COd volume . These fd volume ,53.The main problem in applying non-wheat flour to dough is a disturbance of the starch–gluten matrix. This is likely to worsen the gas retention (due to the diluting effect), leading to the observed reduction in the specific bread volume and porosity, and to an increase in the hardness and chewability of the crumbs . Another2 volume, which induces disruption of the membranes between individual gas bubbles. Such a mechanism could have been responsible for the results related to the volume of CO2 loss. In the case of our samples the amount of losses was positively correlated with gas production (r = 0.947*), i.e., at a higher gas production there was also a higher volume of CO2 lost. Such a dependence was also found in the case of total volume gas production and retention volume (r = 0.972*).Babin et al.  conclude2 produced. The impact of various gas production rates on the time of dough porosity Tx and the corresponding CO2 volume were studied by Verheyen et al. [The rheofermentometer curve and measured parameters indicated that, for the doughs with an addition of legume flours, the fermentation completely retained on average 78.1% of the total COn et al. . They evr = 0.846) and a negative correlation with the fructose content after 120 min of fermentation (r = −0.993). Dough density after kneading depends on the type of yeast and the number of colony forming units were studied by Verheyen et al. [The main yeast substrates in wheat flour dough are fructose, glucose, sucrose, and maltose. The content of these fermentable sugars is continuously modified throughout the technological process due to both their amylolytic production and yeast consumption during the fermentation process. Codina et al.  assessedn et al. , who fouThe results of the evaluation using a Rheofermentometer indicated that the weakest flours in terms of the ability to create and maintain the largest possible volume of gases in the dough were samples with the addition of BBF, depending on the amount of the addition. This was also confirmed by a bakery experiment in which it was mainly these flours which produced low-volume bread.Basically, the process involved in the production of bread is a highly complex one that entails a range of parameters which have to be regulated . The rolOur results of the evaluation of dough prepared from wheat–rye flour and from composite flours with an addition of legume flour, which to some extent replace rye flour in the recipe, give indications that these composite flours are suitable for bread production. It is clear from p < 0.05). The smallest reduction was in breads with the addition of RLF and ChF. When compared with the prediction of bread volume based on data from a Rheofermentometer, we can conclude that the bread volume correlated (α = 0.05) with the retention volume measured by a Rheofermentometer (r = 0.643*). This correlation was higher than the correlation with the total volume measured by a Rheofermentometer and volume CO2 lost .Turfani et al.  confirmeOther bread parameters were also affected by the addition of legume flour, e.g., the specific bread volume and the aspect ratio of a middle slice, which expresses cambering, and for which the same trend was found as for bread volume.Based on a comprehensive evaluation of the physical properties of the trial breads, we can conclude that, although differences between the samples were found, all breads have properties that are acceptable.In addition to technological properties, sensory properties are also important, which ultimately determine the final quality and acceptability of the product. The trial breads from wheat–rye flour, or with legume flour added in a proportion of 5%, 10% and 15% instead of rye flour, were subjected to a sensory evaluation. Only based on this parameter it is possible to present the final conclusions.The sensory evaluation showed that the breads were characterized by a regular shape, a slight surface roughness and a crust color ranging from light yellow to light brown. The results of the evaluation by means of a 100–point system are shown in The extent to which bread will receive a positive sensory evaluation is largely determined by its smell and taste, and it is for these criteria that the ratings of breads with legume flour were the best. The aromas and flavors of the added non-cereal ingredients have improved the sensory profile of wheat-rye bread, which we clearly consider a benefit. In the case of ChF and CBF this was up to an addition of 10%, and in the case of RLF and BBF up to an addition of 5%. The other variants had an aroma and taste that was too pronounced or foreign, and were rated as worse compared to the control bread. The worst rated bread was with the addition of 15% of BBF .These findings were not surprising, because the addition of non-cereal raw materials, apart from nutritional benefits, also brings interesting sensory properties. Lower additions in general keep the quality attributes of conventional bread. Sabanis and Tzia  indicateIn terms of the intensity of the crust and crumb color and compared to the control bread , the addp < 0.05) in colors were found, which occurred in all samples between their signal intensity in the control bread and bread with the addition of legume flours. Of the darker bread colors, colors with codes 3529 and 3530 were present in the control bread, but in breads with BBF and CBF, the intensity of these colors was significantly higher. Another group of lighter colors (3082–3804) was already significantly lower in breads with the addition of BBF and CBF compared to the control bread, and such a trend in the spectrum of lighter colors typical of ordinary bread was also found in other cases. In particular, bread with CBF had a considerably lower proportion of lighter bread colors. In the case of bread with RLF, the light bread color 3785 was significantly more important compared to the control bread, but for most colors there were no considerable differences between the control bread and bread with RLF.The color spectrum by E-eye was also measured and converted to numerical values of signal intensities. The spectra of breads with the addition of individual legumes and control bread are shown in Although it is a very challenging task to mimic the unique bread making properties of wheat flour, it is possible to create products having a similar behavior to wheat flour dough and bread, but with improved functional properties . The nut-water absorption significantly increased with the addition of common bean flour (CBF) and broad bean flour (BBF), and an addition of red lentil flour (RLF) and chickpea flour (ChF) slightly reduced the binding of the composite flour; none of the additives (legume flours), replacing the proportion of rye flour (RF) in composite flours, had a potentiating effect on the stability of the dough;-the mixolab curve can distinguish different stages pertaining to the dough changes due to both the mixing force and the temperature, and the results showed a weakening of the protein network depending on the amount of legume flour added and on the specific legume flour. In terms of the interaction of mechanical processing and thermal procedure, the most stable (at the level of the control wheat–rye flour) was flour with the addition of CBF, in all applied proportions of this addition;-the addition of legume flours mainly influenced the dough behavior during the cooling phase, and in all samples with a higher proportion of legume flour there was an increase in resistance to starch retrogradation. From the evaluated samples, the additions of CBF had the biggest impact on increasing the resistance to retrogradation;-in comparison with the control flour, significant differences were found in the values of the slopes alpha, beta and gamma depending on the added legume flour and the amount of its addition. BBF addition most significantly affected the observed properties;-determining the ability to form fermenting gases is crucial in order to produce bread with a good volume, and the doughs with an addition of BBF clearly had the worst course of the rheofermentometer curves. The other three examined flour mixtures showed similar properties and overall the additives, with the exception of BBF, can be assessed as potentially suitable and applicable in bakery technology on the basis of the results of this analysis;-the bread volume depends on the dough expansion during fermentation and baking, and the ability of the matrix to stabilize the retained gas. In all our samples with additions of legume flour, the bread volume was significantly lower compared to wheat–rye breads. RLF proved to be the most suitable legume flour in terms of bread volume, even with the highest selected addition of 15%. The least suitable legume flour was BBF, in all additions;-the sensory evaluation confirmed that the addition of legume flour of 5% gives breads rated approximately the same or even better than the control bread; higher additions (15%) add extra typical legume flavors and aromas to breads, which have been assessed less positively, especially in the case of BBF;-the effect of individual additions on crust and crumb color was significant, which was also confirmed by the E-eye color spectrum; breads with the addition of ChF, CBF and BBF had a considerable proportion of darker colors compared to the control bread and bread with RLF.The use of non-bakery raw materials as an addition to wheat flour is becoming increasingly popular, mainly due to the nutritional benefits of different plant resources. On the other hand, these non-bakery raw materials change the technological parameters of the flours, doughs, and final products, mostly in a negative way. The results of the present study suggest that the incorporation of legume flours influences considerably the evaluated properties, and those flours with the addition of legumes behaved differently than wheat–rye flour. The properties of the final products (breads) also changed, both objectively and from a sensory point of view. It could be concluded that:In terms of dough formation and its mechanical properties, based on a comprehensive evaluation, it is possible to consider as the best flours those with the addition of CBF. Regarding the formation and retention of fermentation gases, flour with the addition of RLF was determined to be the most suitable, which was also confirmed by a baking experiment and the volume of bread. From the point of view of sensory evaluation, even with higher additions, breads with the addition of CBF were evaluated positively, but the additions of other legume flours at lower proportions were also approximately at the level of a control. In general, the least suitable addition was BBF.


We determined crystal structures of the truncated T–C bidomain of AmbB in three forms, the apo enzyme with disordered T domain, the holo form with serine linked phosphopantetheine (Ppant) and a holo form with substrate  loaded onto Ppant. The two holo forms feature the T domain in a substrate-donation conformation. Mutagenesis combined with functional assays identified residues essential for the attachment of Ppant, anchoring the Ppant-l-Ala in the donor catalytic channel and the role of the conserved His953 in condensation activity. Altogether, these results provide structural insights into the condensation reaction at the donor site with a substrate-bound C domain of AmbB and lay the foundation for understanding the molecular mechanism of condensation which is crucial for AMB synthesis.Non-ribosomal peptide synthetases (NRPS) are multi-modular/domain enzymes that catalyze the synthesis of bioactive peptides. A crucial step in the process is peptide elongation accomplished by the condensation (C) domain with the aid of a peptidyl carrier or thiolation (T) domain. Here, we examined condensation reaction carried out by NRPS AmbB involved in biosynthesis of  Pseudomonas aeruginosa is a ubiquitous opportunistic Gram-negative pathogen that infects animals, plants and humans. It produces several virulence factors and secondary metabolites that contribute to its pathogenicity and overcome competition1. Among these secondary metabolites, an oxyvinylglycine, l-2-amino-methoxy-trans-3-butenoic acid (AMB), interferes with amino acid metabolism by inhibiting a wide array of pyridoxal phosphate-dependent enzymes2. In addition, AMB has been shown to prevent the growth of E. coli K12, Bacillus sp. and the plant pathogen Erwinia amylovora5.P. aeruginosa PAO1 by a five-gene cluster, ambABCDE that encodes two non-ribosomal peptide synthetases (NRPS), AmbB and AmbE; two iron containing (II)/α-ketoglutarate-dependent oxygenases, AmbC and AmbD; and a putative transporter, AmbA6 . AmbB and AmbE are large multi-domain enzymes made up of three or more conserved domains, each having a defined function in the biosynthesis of AMB . In NRPS, three essential domains are required for peptide assembly namely adenylation (A), peptidyl carrier or thiolation (T) and condensation (C) domain. The A domain is responsible for the specificity and activation of an amino acid using ATP as co-substrate, and the subsequent transfer of the activated monomer to the phosphopantetheine (Ppant) arm of a T domain. The tethered Ppant, which spans 20 Å, plays a central part in transporting covalently attached substrates to domains in the NRPS assembly line. Peptide elongation is achieved by the C domain, through amide bond formation between substrates bound to an upstream and a downstream T domain. Some NRPS incorporate modifications in the growing peptide through tailoring enzymes resulting in structural and functional diversity of non-ribosomal peptides7. In most bacteria, a thioesterase (TE) domain finally cleaves the final product from the NRPS, although in some cases, this step can be catalyzed by a reductase (R) domain9.AMB is synthesized in l-Ala and l-Glu, respectively10. AmbB loads l-Ala on its own T domain and the T2 domain of AmbE, whereas the A domain of AmbE aminoacylates l-Glu on its T1 domain. Peptide elongation between l-Ala on AmbB and l-Glu on T1 of AmbE by the C domain of AmbB would then proceed. The resulting l-Ala-l-Glu would undergo condensation with l-Ala on T2 domain of AmbE forming the tripeptide l-Ala-l-Glu-l-Ala. Subsequently L-Glu would be modified by AmbC, AmbD and C domain of AmbE to give rise to l-Ala-AMB-l-Ala10. In a more recent study using deuterium labeling, it was shown that l-Glu gets sequentially hydroxylated at the C3 and C4 by AmbC and AmbD respectively, followed by methylation by MT domain of AmbE11. These modifications occur while l-Glu is still tethered to the T1 domain of AmbE prior to condensation with l-Ala from AmbB. It was also suggested that the resulting intermediate would undergo further modifications including dehydration (probably by the C domain of AmbE) and decarboxylation to generate the methoxy enol ether moiety11. The C domain of AmbE is atypical as phylogenetic analyses do not group this enzyme with other C domains , epimerization (E), dual C/E)11. Finally, hydrolysis by thioesterase domain of AmbE would yield the end-product, Ala-AMB, instead of the tripeptide l-Ala-AMB-l-Ala that was formerly proposed11. Although these studies provide detailed information on AMB biosynthesis pathway, open questions remain, such as the precise role of the non-canonical C domain of AmbE, how are AmbC and AmbD recruited, and what enzyme is involved in processing the AMB precursor.During AMB biosynthesis, the A domain of AmbB and AmbE activates 20. The C domain, which has binding sites for one donor and one acceptor T domain, displays specificity towards both donor and acceptor substrates23. To date, existing structures of C domain include excised C domain, bidomains of C domain with either acceptor or/and donor T domain, or as part of an entire module of NRPS27. This gate-keeping mechanism, along with the unidirectional condensation reaction ensure correct elongation of product28. However, only few of the characterized structures of C domain contain Ppant tethered substrate in the donor or acceptor catalytic channel27.Many studies have focused on elucidating the function of individual domain of NRPS and domain–domain interactions. Structural data, combined with extensive biochemical and mutagenesis studies have provided some insights into the catalytic mechanism of condensation in NRPSl-Ala loaded Ppant. Cell based bioassays and in vitro condensation assays coupled with mutagenesis, reinforce the indispensable role of active site histidine in the conserved HHxxxD motif of the C domain of AmbB. This work provides structural insights into a donor substrate-trapped state of a C domain, relevant for condensation reaction in NRPS.In this work, we report the crystal structures of T–C bidomain of AmbB (AmbB T–C) in apo form, AmbB T–C with bound Ppant and AmbB T–C in complex with 24 from Bacillus brevis for 351 α-C atoms. A detergent molecule from the crystallization condition, MEGA-9 is bound to the peripheral side of the C terminal subdomain between helix α8 and helix α11  was determined at a resolution of 2.4 Å , was used to trans-aminoacylate l-Ala onto the Ppant of the T domain of AmbB T–C. MS analysis showed a mass shift (+71.04) of the alkylated Ppant arm as compared to holo AmbB T–C, which corresponds to the molecular weight of l-Ala .In order to understand the role of T domain and the Ppant arm in the condensation reaction catalyzed by the C domain of AmbB, we determined the crystal structures of holo AmbB T–C and AmbB T–C with ely Fig. b, c. Thel-Ala on apo AmbB: 317 α-C atoms). Likewise, the T domains between the two holo structures are very similar (RMSD: 0.26 Å for 58 α-C atoms) . The T domain occupies the donor binding site of the C domain, which supports its role as an aminoacyl donor for C domain of AmbB during the first elongation step of AMB biosynthesis. T domain is a four-helix bundle (Ser736–Gly812) that adopts an A/H configuration previously observed in apo and holo T domains of NRPS29 . The interactions of Ppant moiety with C domain are essentially identical to those observed in the structure of holo AmbB T–C tethered with Ppant . The thiol moiety of Ppant is covalently attached to the carboxyl group of l-Ala via a thioester bond. The positioning of the Ppant places l-Ala in close vicinity to the active site, with the reactive carbonyl group facing the donor side of the C domain. l-Ala is surrounded by two residues namely Phe1181 and Leu846 -CoA was chemically synthesised and subsequently covalently tethered to the T1 domain of LgrA in vitro. The structure showed that delivery of the substrate as well as simultaneous binding of donor T1 and acceptor T2 domains have no influence on the conformation of the C2 domain26. Ppant-l-Ala of AmbB follows the same trajectory in the donor side as Ppant-fVal, placing the substrate l-Ala and fVal, at similar distances to the catalytic His953 and His908, respectively , the side chain of l-Ala is oriented towards the hydrophobic residues Leu846 and Phe1181 while the free amino group is in close proximity to His953, poised for condensation reaction  of a dimodular NRPS LgrA from gramicidin synthetase was reportedl-Ala  surrounding the spot inoculation whereas the mutant Ser768A did not display any zone of inhibition , implying significant impaired AMB biosynthesis pathway. The mutants Asp958A, Ser1058A and Phe1060S did not show any significant difference in inhibitory activity in comparison to wild type P. aeruginosa. We also mutated two other residues Tyr762 (between helix α1 and α2) and His773 (in helix α2) on the T domain to assess the interaction at the T–C bidomain interface. Mutating Tyr762 to aspartate did not cause any disruption in AMB biosynthesis. The mutant His773A displayed a slight, but significant decrease in inhibitory activity with mean inhibition zone of 21.87 mm ± 0.66.Based on the residues involved in the interaction with Ppant-Ala Fig. b, we conion Fig. a, b. Mutl-[14C]-Ala as indicator for formation of the dipeptide l-Ala-l-Glu. Aminoacylation of wild type or mutants AmbB was monitored for 20 min (Stage I), after which an equimolar amount of full-length AmbE Ser1819A that was priorly incubated with l-Glu, was added to initiate product formation (Stage II). Due to the mutation of the conserved Ser1819A on the T2 domain of AmbE, the dipeptide formed is not released from the protein and is stalled on the T1 domain of AmbE. Upon addition of AmbE Ser1819A to wild type AmbB, an increase equivalent to 867 CPM in l-[14C]-Ala labeling was detected. This almost doubling in l-[14C]-Ala labeling is attributed to the simultaneous translocation of l-[14C]-Ala to the T1 domain of AmbE as part of l-Ala-l-Glu dipeptide and rapid reloading of l-[14C]-Ala on the freed T domain of wild type AmbB  , this increase is attributed to the loading of l-[14C]-Ala in trans on the T2 domain of wild type AmbE, as previously reported10.We next examined the condensation activity of full-length AmbB mutants in vitro, by using mbB Fig. a. The syl-1C-Ala as 20. In this model, the C domain of AmbB is sandwiched between the T domain of AmbB loaded with Ppant-l-Ala and T1 domain of AmbE loaded with Ppant-l-Glu* (hydroxylated and O-methylated l-Glu). Such an arrangement of the domains positions the two amino acid substrates in close proximity around the active site residue His953 so as to mimic the beginning step of the condensation reaction between the l-Ala and l-Glu* on the donor and acceptor sides of the C domain, respectively. The model places the ε N atom of the active His953 of AmbB at a reasonable distance of ~ 3.1 Å away from the amino group of the acceptor substrate, which would allow deprotonation of this α-amino group by His953  (PDB:7KW0) of fuscachelin synthetase27. In this model, following deprotonation, the amino group of the acceptor substrate, which is positioned ~ 3.3 Å away from the Ppant-l-Ala thioester carbonyl, would then proceed with the nucleophile attack of the carbonyl group in order to release the l-Ala-l-Glu* dipeptide, tethered to the Ppant of the downstream acceptor T1 domain of AmbE.To gain a detailed insight into the mechanism of condensation reaction in AmbB, we modelled a hypothetical condensation complex using the C–T domains reported in the structure of holo-AB3403 (PDB: 4ZXH)953 Fig. . This dil-Ala, bound to the C domain. The importance of several residues in the donor solvent channel as well as in the active site of the C domain were assessed via site-specific mutagenesis, followed by in vitro and in vivo assays.The biosynthesis of NRPs requires the interplay of different domains and modules of NRPS. For peptide elongation, the C domain depends on the coordinated docking of two T domains for the delivery of substrates into the active site. The molecular mechanism of condensation is elusive due to limited number of structures with bound substrates. In this work, three crystal structures of T–C bidomain of AmbB were solved; apo form in which the T domain is disordered, and two holo forms with Ppant, or Ppant loaded with the native substrate, 31. The presence of the T domain in the holo and AmbB T–C with Ppant-l-Ala bidomain structures showed that the Ppant arm locks the condensation-carrier domains in a stable conformation, and in turn stabilises the highly flexible carrier protein. It is also possible that AmbB may adopt a more compact fold in its holo state than apo form, as suggested by the shift in gel filtration peaks . Further biophysical analyses would be required to interrogate this possibility.During the various stages of an NRPS synthesis cycle, the different domains form a dynamic catalytic platform while the mobile T domain handles the intermediates, by adopting different orientations relative to respective domains18. Comparison to other homologue structures shows that the C domain of AmbB adopts a conformation quite similar to that in EntF from enterobactin synthetase (PDB: 5T3D) . The C domain of apo and holo AmbB T–C show a quasi-identical conformation suggesting that it does not undergo obvious conformation change upon Ppant binding, at least by its upstream T domain. In addition, the delivery of the substrate l-Ala, did not cause any discernible conformation change in the C domain.The C domains of NRPS exhibit different relative arrangement of the N-terminal lobe with respect to the C-terminal sub domain. This structural variability has previously led to suggest that the enzyme may adopt an ‘open’ and ‘closed’ state during various functional stages of NRPS biosynthesis27. In addition to C domain of LgrA which features the synthesized donor CoA-fVal26, two other structures show mimics of acceptor substrate loaded Ppant in the C domain of CDA synthethase22, and stabilised-Glycine acceptor substrate in C domain of fuscachelin synthethase, respectively27. It is plausible that a mixture of Ppant and Ppant-l-Ala states may exist in the crystal structure of holo AmbB T–C with l-Ala due to the thioester linkage undergoing cleavage over the course of crystallisation. The Ppant arm which is intrinsically flexible as observed by the fragmented electron density of Ppant and Ppant-l-Ala in our structures, may also suggest that l-Ala may adopt different conformations in the active site of C domain of AmbB.Three structures of substrate-bound C domains of NRPS have been solvedl-Ala and Ppant-l-Glu* tethered on the donor T domain of AmbB and acceptor T1 domain of AmbE respectively11. Mutating the second Histidine (His953) to Ala in the consensus HHxxxDG motif of C domain was sufficient to abolish condensation activity of AmbB, thus reiterating its essential importance for catalytic activity. The dependence of C domain on the second His has also been demonstrated in EntF, TycB1 and SrfA-A, where a mutation to Ala/Val led to complete abolition of condensation activity32. However, little to moderate effect was reported for VibH and VibF15. Since the role of the second His seems not to be universally conserved in all C domains, it was recently proposed to be involved in positioning the acceptor substrate for nucleophilic attack, rather than acting as a general base in catalysis22.During AMB biosynthesis, the C domain of AmbB catalyses peptide bond formation between Ppant-17. In EntF, an Asp to Met mutation not only abolished condensation activity, but also disrupted the global fold of the protein as confirmed by CD. This residue was assigned to be structurally important owing to its ionic interaction with a highly conserved Arg, rather than playing a direct role in catalysis16. In our study, the Asp957A mutant could not be purified as it underwent severe degradation, suggesting a crucial structural role in the C domain of AmbB. A 300-fold and 1500-fold decrease in catalysis was observed for VibH and CAT respectively, upon mutating the equivalent Asp residue to Ala33. In VibH, the decreased activity of the mutant is due to local active site perturbations14.In AmbB, an in vivo mutation of the well-conserved Asp957 has a detrimental effect on AMB biosynthesis as judged by the plate bioassay. Similarly, mutation of the equivalent Asp in TycB and Srf A–B was proven deleterious34. In our work, Leu769S mutant was able to auto-aminoacylate its own T domain with radiolabeled l-[14C]-Ala at the same rate as wild type AmbB, which showed that the decrease in dipeptide synthesis is probably due to its impaired interaction with the phosphopantetheine arm, rather than a defect in phosphopantetheinylation.Functional analyses have also shown that the conserved Leu769 on the T domain of AmbB plays an important role in anchoring the Ppant in the donor site, as judged by its decreased AMB biosynthesis and condensation activity. This interaction, may in turn contribute to further stabilise the interaction between the T and C domain of AmbB, thus maintaining the T domain in a productive conformation during substrate delivery. Interestingly, the enterobactin synthethase EntF, bearing a mutation of the equivalent Leu (Leu1007A), could not be phosphopantetheinylated by EntD and Sfp, which the authors attributed to a defect in recognition of Leu1007 by Ppant transferase enzymesl-Ala, in the active site of the C domain of AmbB. Using functional assays, we demonstrated that S768 is essential for the attachment of Ppant, while L769 is important for anchoring the Ppant-l-Ala in the donor site of the C domain of AmbB. Additionally, we showed that the second Histidine in the conserved HHxxxDG motif of C domain plays a crucial role in condensation activity. Further structural investigations of C domain with donor and/or acceptor substrates are needed in order to advance our knowledge on the mechanism of peptide synthesis in NRPS. This understanding is vital for successful and efficient NRPS bioengineering, which at present, still represents a challenge.This study provides structural insights into the donor substrate, E. coli and P. aeruginosa were routinely cultivated at 37 °C in lysogeny broth (LB) and agar. Purified stocks of bacteria strains were maintained at − 80 °C in LB containing 15% glycerol. When necessary, antibiotics were added to the media of E. coli strains at the following concentrations: ampicillin 100 μg ml−1, chloramphenicol 34 μg ml−1 and gentamycin 5 μg ml−1, For conjugation, P. aeruginosa was grown in LB medium at 42 °C overnight statically. Gentamycin at concentration of 60 μg ml−1 was used to select mero-diploids of P. aeruginosa following conjugation.P. aeruginosa PAO1 using appropriate primers  which harbours a cleavable N-terminal SUMO tag. The amplified product of ambE was digested with NdeI and HindIII and cloned into pET21b vector with a C-terminal 6xHIS tag (pET21b-AmbE).The gene encoding AmbB T–C bidomain was amplified using the High-Fidelity Q5 DNA polymerase (NEB) from genomic DNA of E. coli DH5α. Positive transformants were confirmed by sequencing and the plasmid transformed into E. coli BL21 Codon Plus for overexpression.To generate full length AmbB mutants and AmbE S1819A, the plasmid pET28SUMO-AmbB and pET21b-AmbE was used respectively as template and amplified by inverse PCR using back-to-back primers. The amplified PCR product was incubated with DpnI, T4 PNK and T4 ligase for two hours at room temperature and 2 μl of the reaction mixture was transformed into chemically competent P. aeruginosa’s chromosome, the amplified 1000 bp gene fragment was digested from pET28aSUMO vector using EcoRI and HindIII restriction enzymes and ligated into the suicide vector pk18-Gm-mobsacB between the same sites. Using S17-1 λpir E. coli as donor cells, the suicide plasmid was mobilized from E. coli DH5α into P. aeruginosa.The plasmid pET28SUMO-AmbB was used as template to amplify a 1000 bp-gene carrying respective single amino acid substitution (Supplementary Table ambB to confirm the right insertion of the homologous mutator fragment into the chromosome. Single amino acid substitution was confirmed by sequencing.Single homologous recombination event between ambB gene on the chromosome and the mutator fragment on pk18-Gm-mobsacB was confirmed by selection for gentamycin resistance. Excision of the vector resulting from the second recombination event was achieved on LB plates without sodium chloride (NaCl), supplemented with 10% sucrose. Single colonies were screened using primers upstream and downstream of E. coli Codon Plus carrying the relevant plasmid. The cultures were grown at 37 °C until OD600 reached 0.5–0.7 and induced with a final concentration of 0.1 mM isopropyl-β-d-thiogalactopyranoside (IPTG), after which growth was continued for another 16 h at 18 °C.Cultures were set up in two flasks each containing 2 l of LB and each flask was inoculated with 20 ml of an overnight culture of E. coli Codon Plus (AmbB WT and AmbB mutants) and E. coli pLysS (AmbE WT and AmbE S1819A). At OD600 of 0.5–0.7, IPTG at a final concentration of 0.5 mM was used for induction and the cultures were grown for another 4 h at 24 °C.2 L of LB were inoculated with an overnight culture of −1), 1 mM phenylmethylsulfonyl fluroride (PMSF) and 2 mM benzamidine hydrochloride. The solution was incubated on ice for 30 min prior to sonication. A total of 5 min  was used to lyse the resuspended pellet. The soluble and insoluble fractions were separated by centrifugation at 18,000 RCF at 4 °C for 30 min. The supernatant was filter sterilized using 0.22 μM filters and incubated with 5 ml of gluthatione sepharose beads for 30 min at 4 °C. The mixture was loaded onto a polypropylene column, after which the beads were washed with 50 ml buffer A to remove unbound contaminants. GST-tagged AmbB T–C protein was then eluted with 50 ml of 20 mM reduced glutathione prepared in buffer A. To cleave the N-terminal GST tag from AmbB T–C protein, the evaluate was incubated with precision protease in a ratio of 50:1 for 2 h at 4 °C. The mixture was passed through a desalting column and then bound again to GST beads to remove GST tag. The flow-through containing AmbB T–C protein was collected and was purified further on an anion exchange chromatography column  using 20 mM Tris pH 8.0, 50 mM NaCl and 20 mM Tris pH 8.0, 1 M NaCl as running and eluting buffer respectively. AmbB T–C protein was lastly purified on a Superdex 75 column  using buffer B consisting of 20 mM Tris pH 8.0, 150 mM NaCl and 2 mM DTT. Pure AmbB T–C was subsequently concentrated with Amicon Ultra centrifugal filter with molecular weight cut–off of 10,000. Concentration of AmbB T–C was determined based on the calculated extinction coefficient and the protein (55 mg ml−1) was flash frozen in liquid nitrogen and stored in aliquots at − 80 °C.Following heterologous expression, cultures were centrifuged at 4000 RCF at 4 °C for 30 min. Supernatant was discarded and pellet was resuspended in 100 ml buffer A containing 50 mM Tris pH 8.0, 500 mM NaCl, 12% v/v glycerol and 2 mM dithiothreitol supplemented with 2 mM magnesium chloride (MgCl2), 500 μl lysozyme  that had been equilibrated with buffer B. After purification, fractions of pure proteins were concentrated with Amicon Ultra centrifugal filter with molecular weight cut-off of 30,000. Concentration of AmbB WT, AmbB mutants, AmbE and AmbE S1819A were determined based on the calculated extinction coefficient and respective protein was flash frozen in liquid nitrogen and stored in aliquots at − 80 °C.The conversion of apo to holo form of proteins was done prior to size exclusion chromatography step. In a 500 μl reaction, concentrated protein was incubated with 5 mM coenzymeA, 1 μM sfp enzyme (NEB) and 10 mM MgCl2. The mixture was incubated at 28 °C for 2 h.l-Ala and 10 mM MgCl2. The reaction mixture was incubated at 28 °C for 3 h followed by overnight incubation at room temperature. The mixture was run on a mono Q column to separate full length AmbB from truncated AmbB T–C. The samples corresponding to AmbB T–C was pooled, concentrated and further purified on a Superdex 75 size exclusion chromatography column.Apo full length AmbB was used to load L-alanine onto the T domain of the holo truncated AmbB T–C. 100 μM of full length AmbB was mixed with 500 μM AmbB T–C in an assay buffer containing 20 mM Tris pH 8.0, 150 mM NaCl, 5 mM ATP, 5 mM 35. For holo AmbB T-C, a total of 480 conditions were screened using commercially available kits (Hampton Reasearch and Qiagen). 0.2 μl of holo AmbB T–C protein at concentration of 10 mg ml−1 was mixed with 0.2 μl of precipitant solution and equilibrated against 56 μl of reservoir condition. After one day at 18 °C, crystals were observed in several conditions. Optimization of the initial hits using the hanging drop method led to single plate-like crystals that grew in a condition containing 0.1 M MES pH 6.6, 0.2 M ammonium nitrate, 15% PEG 3350 and 5% glycerol. A typical drop consisted of 2 μl protein with 2 μl precipitant against 120 μl of reservoir in 48-well crystallization plates (Hampton Research). Crystals were soaked briefly in a cryoprotectant solution made up of mother liquor supplemented with 25% (v/v) 2-methyl-2,4-pentanediol (MPD) and harvested with cryoloops . Crystals were flash- frozen in liquid nitrogen and sent for X-ray analysis at the European Synchroton Radiation Facility in Grenoble, France. Crystals of holo AmbB T–C with l-alanine grew in the same original condition as AmbB T–C. The crystals were harvested in mother liquor containing 0.2 M ammonium nitrate, 15% PEG3350 and 25% MPD and flash frozen in liquid nitrogen.Crystallization of apo AmbB T-C was reported previously522, unit-cell parameters a = b = 87.8 Å, c = 286.3 Å, α = β = 90°, γ = 120°, and contained one molecule per asymmetric unit. A diffraction dataset was collected at SSRF (BL17U1) and was processed using CCP436. The AmbB-T–C domain has only two methionine sites in 500 amino acids. We introduced three additional methionines in place of leucines and prepared cement crystals suitable for SAD phasing. These crystals diffracted poorly after initial 30–40 frames of data collection. So, we merged datasets from four different crystals to obtain initial SAD phases using HySS program of phenix suite37. Five sites were identified with a figure of merit of 0.2 and correlation of local RMS density of 0.68 suggesting a possible correct solution. Subsequent refinement of heavy atom positions, phasing and density modification using RESOLVE allowed identification of the correct handedness of the map. Initial model was built with 391 amino acids in nine different fragments corresponding to one chain of AmbB-T-C domain with a map-model correlation of 0.834.Crystals structure of apo T–C AmbB were determined using Set-Met labeled protein which diffracted to 2.40 Å, with space group P6l-Ala diffracted to 2.5 Å, with the same space group and cell parameters as holo AmbB T–C. The structure was auto built using Phenix and using holo AmbB T-C as model. Statistics of the structure determination and refinement are summarized in Supplementary Table Crystals of holo T-C AmbB was solved at a resolution of 2.2 Å, with space group C121, unit-cell parameters a = 198.6 Å, b = 71.4 Å and c = 172 Å, α = γ = 90°, β = 109. 9°, and contained four polypeptides per asymmetric unit. The structure was determined by molecular replacement method using Phaser, with the structure of C domain of apo AmbB T–C as search model. The T domain of AmbB was manually built based on available density map using COOT, and crystallographic refinement was performed using REFMAC. Crystals of holo T–C AmbB with Ppant-P. aeruginosa strains to inhibit E. coli K126. Wildtype and mutant P. aeruginosa strains were grown in GYP medium  plates supplemented with 0.5% glucose. The plates were incubated at 37 °C for 16 h and then exposed to ultra violet (UV) light for 5 min. 4 ml of soft agar (0.5% w/v) inoculated with 0.4 ml of E. coli K12, grown overnight in MME medium supplemented with 0.5% glucose and 1 mM threonine, and brought to an OD600 of 0.1 with 0.9% NaCl, was overlaid on the MME plates. Plates were incubated at 37 °C for 24 h. The zones of clearance around P. aeruginosa spots were visually assessed and measured using Image J software. The assay was done in triplicates (n = 3 independent samples) for each mutant. Unpaired t-test in GraphPad Prism was used to analyze any significant difference in the diameter of zones of clearance between wild type and mutants. Data was considered significant at a p value of ≤ 0.05.AMB production was assessed by the ability of 38. Briefly, 100 μg ml−1 of AmbB or AmbE was added to a tube containing 50 mM Tris pH 8.0, 5 mM ATP, 5 mM MgCl2, 32 mM hydroxylamine (pH 7.2), 2 mM l-Ala or l-Glu and the reaction mixture was topped up with water to 100 μl. The mixture was incubated at 30 °C for 60 min. 50 μl of the reaction was then transferred to an Eppendorf tube containing 500 μl of disodium molybdate (VI) dihydrate solution and incubated at room temperature for 3 min. 10 μl of bis (triphenylphosphoranylidene) ammonium chloride (BTPPACl) in acetonitrile was added, and the reaction mixture was further incubated for 5 min. The sample was centrifuged at 13,000 RPM for 10 min and the supernatant was discarded. The precipitant was dissolved in 100 μl of acetonitrile and mixed thoroughly. 10 μl of the solution was transferred to a 96-well flat bottom plate, to which 10 μl of ascorbic acid solution  was added. The mixture was pipetted a few times and absorbance of the well was read at 620 nm. For negative control, amino acid solution was substituted with water. The readings were compared to a standard curve, which was performed with sodium tetrasodium pyrophosphate using the same above described method.A colorimetric detection of pyrophosphate was used as indicator to assess the substrate specificity for the A domain of AmbB and AmbEl-alanine to assess dipeptide formation. A mutant AmbE_S1819A was generated so as to abolish loading of l-alanine in trans by AmbB onto the T2 domain of AmbE, that could interfere with the 14C-l-ala-l-glu reading. 1 μM of AmbES1819A was pre-incubated with 5 mM non-radiolabeled l-glutamic acid, 5 mM ATP and 5 mM MgCl2 for 1 h at room temperature. The aminoacylation reaction of wildtype or mutant AmbB in assay buffer containing 5 mM ATP and 5 mM MgCl2 was started by adding 4 μM radiolabeled 14C l-alanine. At 20 min time point, product formation was initiated by mixing equal volumes of pre-incubated AmbE_S1819A with wildtype or mutant AmbB. At various time points, 25 μl aliquots were taken and quenched immediately with ice-cold 10% w/v TCA containing 100 μl of 100 mg ml−1 BSA. The samples were centrifuged at 12,000 RCF for 5 min and washed twice with 10% TCA. The final pellet was resuspended in 150 μl formic acid and added to 3 ml of LSC fluid (Ultima Gold). 14C-radioactivity in the samples were quantified by LSC.To assay the in vitro condensation activity of wildtype and mutant full length AmbB, we used radiolabeled 14C-l-Ala on AmbB T–C, 60 μg of protein in 60 μl 20 mM Tris (pH 8.0) solution containing 150 mM sodium chloride and 2 mM DTT (Sigma-Aldrich) was reduced by the addition of 6 μl 55 mM DTT and incubation at room temperature for 30 min. This was followed by addition of 6 μl of 120 mM iodoacetamide (Sigma-Aldrich) and incubation at room temperature in the dark for 30 min. 6 μl of 0.2 μg μl−1 trypsin (Promega) was then added to the sample and incubated further at 37 °C overnight. The digested sample was treated with 100 μl 0.1% formic acid to stop trypsin digestion and the sample was cleaned up using stage tip39. The sample was stored in the stage tip at 4 °C until it is ready for mass spectrometry analysis. The sample eluted from the stage tip was vacuum-dried, reconstituted in 30 μl 0.1% formic acid and diluted to 0.5 μg μl−1 for liquid chromatography mass spectrometry analysis.To confirm the covalent attachment of Ppant or Ppant-m/z 310–2000) were acquired with a resolution of 7e5 at an AGC target of 3e6, and a maximum injection time of 10 ms. Top 20 most intense peptide ions in each MS scan were sequentially isolated to an ACG target value of 5e4 with resolution of 17,500 and fragmented using a normalized collision energy of 25 at MS2 level with fixed first mass at 110 and isolation window of 2.0. The mass spectrometry data was processed manually by performing “Extracted Ion Chromatogram” on the product ion and the MS/MS spectra was manually interpreted.2 μg of peptides was analyzed on Thermo Easy nLC 1000 that was connected to Q Exactive Plus Mass Spectrometer (Thermo Scientific). The trap column used was C18 Acclaim PepMap 100  and the analytical column was PepMap RSLC C18 . Mobile phase A was 2% acetonitrile/0.1% formic acid while mobile phase B was 95% acetonitrile/0.1% formic acid. The separation of the peptides was carried out with a flow rate of 200 nl/min at 50 °C using a 95 min gradient: 0–34% mobile phase B for the first 70 min, followed by a 10 min gradient ranging from 34 to 100% mobile phase B and maintained at 100% mobile phase B for 15 min. The mass spectrometer was set in the data dependent acquisition mode. Full scan MS spectra 20. Next, we superposed the C domains of 4ZXH and AmbB-Ppant-l-Ala to obtain the condensation complex of AmbB–AmbE. As expected, the modeled T1 domain occupied a position on the acceptor side of the C domain of AmbB. l-Glu* was manually connected to the Ppant bound to the acceptor site of the C domain of AmbB and the whole condensation complex was subjected to rigid body and energy minimization refinement to optimize the interactions between the modeled T1 domain of AmbE and the C domain of AmbB. In the resultant condensation complex, both the acceptor and donor sites on the C domain of AmbB are occupied by Ppant-l-amino acid loaded T domain.Our structure of TC bidomain of AmbB with Ppant-Supplementary Information.


In the performing arts, such as music and dance performances, people actively interact with each other and show their exciting performances. Some studies have proposed that this interaction is a social origin of the performing arts. Some have further investigated this phenomenon based on the synchronization and coordination theory. Though the majority of these studies have focused on the collaborative context, several genres of the performing arts, such as jazz sessions and breakdance battles, have a competitive context. Several studies have suggested that, in this competitive context, performers actively interact with each other and construct some correspondence. Moreover, a few recent studies focusing on competitive conversations, such as debates, have shown that, compared to people's interactions in collaborative conversations, people in competitive contexts frequently coordinate their behaviors in complicated ways. However, the interaction and coordination among performers in these competitive contexts have not been sufficiently investigated. Therefore, we investigated the coordination of expert breakdancers in battle scenes and measured their rhythmic movements using a motion capture system. We calculated the relative phase of the rhythmic movements between two dancers to investigate their coordination. The results showed that the dancers' rhythmic movements tended to synchronize in an anti-phase fashion, which means that there were similarities as well as differences between the two dancers' rhythmic movements. Furthermore, this pattern of coordination changed dynamically as time elapsed, from an in-phase synchronization or leader-follower relationships to an anti-phase synchronization and then leader-follower relationships. In the domain of the performing arts, which includes dance, theater, and music performances, performers actively interact with each other while showing interesting performances Bailey, . Some peAdditionally, some theories have proposed that this interaction plays a vital role in human society Fitch,  because In recent years, several researchers have attempted to investigate this complicated interaction by applying the synchronization and coordination theory  seen among insects . Several studies have suggested that rhythmic movement is the foundation of dance expression . All experts had the experience of winning first or second prizes in breakdancing competitions held in Japan. The experts were divided into two groups , and each group participated in the experiment separately.Four expert dancers in each group paired up and conducted the battles in a round robin tournament (six pairs in each group), meaning that 12 battles were conducted. Each battle allowed each dancer to perform three times in turn see . We exclWe set the experimental situations as close to real battle scenes as possible OHJI, . TherefoWe measured the movement data of each dancer using an infrared motion capture system . For the movement measurements, we put seven markers on each dancer in locations where the markers did not interrupt their performances based on a pilot study (five around their necks and two on their trunks). We used, in the analysis, the markers' z-axis movement data around their necks . The necThe experimental procedures were conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the University of Tokyo. All dancers provided a written informed consent and were paid for their participation.A is the vector of the rhythmic movements of one dancer and RB is that of another dancer.We calculated the relative phase of the rhythmic movements between two dancers using the z-axis data of the above markers. We conducted these analyses by developing a method based on several studies that investigated the coordination of movements in the space between two players in Kendo matches and a game of tag  Second, we used the Hilbert transformation to calculate a phase of each dancer's rhythmic movement vector.(3) Then, we calculated the relative phase between the two dancers' rhythmic movements using the phase data of each dancer.This relative phase of the rhythmic movement indicates the relationships of the upward and downward neck movements between two dancers. The relative phase at 0 degrees shows that the dancers moved in the same z-axis direction . The relative phase at −180/180 degrees shows that they moved in opposite directions . Furthermore, we can investigate the leader-follower relationships between two dancers' movements by checking this relative phase. If the relative phases' frequencies at slightly over 0 (like 0–20 and 20–40 degrees) are high, the dancer who performs first leads their movements. If those slightly below 0 (like −20–0 and −40-−20) are high, the dancer who performs second leads their movements. .This study also generated the Virtual pair condition and compared the results of coordination with those of the Real pair condition  = 2.36, p < 0.01, η2 = 0.07]. Post-hoc analysis (simple main effect and multiple comparisons with Holm's correction) showed that in the Real pair condition, the frequencies of the relative phases at −180–−160 and 160–180 degrees were higher than those at other degrees . Significant differences were observed between the relative phase at−180-−160 degrees and each of −60–−40 , −40–−20 , −20–0 , and 0–20 degrees . They were also seen between 160–180 degrees and each of −60–−40 , −40–−20 , −20–0 , and 0–20 degrees . However, in the Virtual pair condition, the frequency of the relative phase at −180–−160 and 160–180 degrees was not higher than that of the other phases . Furthermore, we assessed the differences of frequency between the two conditions. The frequency of the relative phase at 160–180 degrees in the Real pair condition was higher than that in the Virtual pair condition , although at −180–−160 degrees, there was no significant difference between the conditions .We also investigated the dynamic change in the patterns of this synchronization/coordination. F = 5.97, p < 0.001, η2 = 0.44]. Post-hoc analysis showed that in Turn 1, the frequency of the relative phase at 20–40 degrees was higher than that of the other phases . Significant differences were observed between the relative phase at 20–40 degrees and those at −180–0 and 100–180 degrees . However, no significant differences were indicated between the phase at 0–20 degrees and other phases . Additionally, in Turn 4, there were no significant differences among the phases . In Turns 2 and 3, the frequencies at −180–−160 and 160–180 degrees were higher than those at other phases . In Turn 2, significant differences were observed between the relative phase at −180–−160 degrees and −140–20 degrees , and at 160–180 and −140–0 degrees . In Turn 3, significant differences were observed between the relative phases at −180–−160 and −20–0 degrees , and at 160–180 and −20–0 degrees . These results suggest that in Turn 1, the first dancer frequently led their rhythmic movements, and in both Turns 2 and 3, the dancers took their rhythms in an anti-phase synchronization manner.The outcomes of the statistical testing support these results. The results of the two-way ANOVA showed a significant interaction between the turn and the relative phase in the Real pair condition . For 0–20 degrees, a significant difference was observed between Turns 1 and 2 . The difference between Turns 1 and 3 did not reach a significance, but it did have a large effect size . For 20–40 degrees, a significant difference was observed between Turn 1 and Turns 2 and 3 . Furthermore, the frequencies at −180–−160 and 160–180 degrees in Turns 2 and 3 were higher than those in other turns . For −180–−160 degrees, a significant difference was observed between Turn 1 and Turns 2, 3, and 4  and between Turns 2 and 3 and Turn 4 . For 160–180 degrees, a significant difference was observed between Turn 1 and Turns 2 and 3  and between Turns 2 and 3 and Turn 4 .When we checked the differences among turns, we found that the frequencies of the relative phases at 0–20 and 20–40 degrees in Turn 1 were higher than those in other Turns [0–20: These results support the observations made in This study showed that in the breakdance battle scene where two dancers competed against each other, they coordinated their rhythmic movements in an anti-phase synchronization. This result suggests the importance of focusing on the collaborative/competitive contexts when we investigate people's coordination in the performing arts. Studies investigating the performers' coordination are limited. Most of them have focused on the coordination in the collaborative context  Performer coordination in the competitive context is an exciting subject that requires much more scientific investigation.The raw data supporting the conclusions of this article will be made available by the corresponding author.The studies involving human participants were reviewed and approved by The University of Tokyo. The patients/participants provided their written informed consent to participate in this study.DS performed the experiment, analysis, and interpretation. DS drafted the manuscript and TO provided critical revisions. All authors contributed to the study design and approved the final version of the manuscript for submission.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.


The objective of this study was the production of innovative functional cookies enriched with two different sizes (100 and 28 mesh) xiquexique flour by substitution ratio 50% of wheat flour and monitoring the impact of these enrichments on the nutritional, physicochemical, texture characteristics and consumer acceptance. The physicochemical characteristics and sensorial properties of the xiquexique cookies were evaluated in a pursuit to identify an innovative bakery ingredient with high nutritional value and potential function that could be exploited by the food industry. The water activity and moisture values were low, which can provide greater stability during storage of food matrices, such as cookies. The xiquexique cookies had greater ash (2.47–2.74%), protein (0.94–1.36%), fiber (4.41–8.10%), and resistant starch (3.65–2.10%) contents than their respective controls with 100% wheat flour. The functional cookies were rich in minerals: mainly calcium, iron, potassium, magnesium and manganese and can be consumed by all individuals to help meet daily needs, especially those of people who have increased needs for these essential nutrients. In addition to the darker color of the xiquexique cookies, the hardness of these was higher than that of the control cookies, while the expansion index was smaller. The data from the Check All That Apply sensory method, which consists of a test used mainly for recipe adjustments and the development of ideal food products, confirmed that xiquexique flour have the potential for the development of bakery products such as cookies. Unconventional plant species have gained global attention for use in the food industry because they are nutritive and particularly rich in bioactive compounds. Several studies have observed the potential of these species for the development of functional food products –3. UnconPilosocereus gounellei which is a cactus in the Cactaceae family that grows in the semiarid region of Brazil and multiplies regularly, covering extensive areas of the Caatinga; moreover, this plant is popularly known as xiquexique [As an example of UFP we have quexique . The claquexique –8 and jaquexique .in natura cooked or dry) can be an alternative crop with multiple uses and benefits, providing human beings with excellent nutritional characteristics, as they are rich in minerals, carbohydrates, and fibers and low in fat and sugars [This UFP  are suitable for the production of flour, while the pulp is more appropriate for the preparation of products such as jam and candies , 10. TheNew technologies and ingredients are being introduced worldwide to fulfill nutritional needs , 16. FunCookies occupy an important position in the snack food industry due to their variety in taste, crispiness and digestibility, consequently, they represent a valuable vehicle of supplementation with nutrients because of their popularity, relatively low cost, varied taste, ease of availability, high nutrient density and long shelf life . CookiesTo date, few studies have focused on the physicochemical parameters, mineral content and antioxidant potential of natural xiquexique . HoweverP. gounellei plants were collected in Boa Vista (PB), Brazil, in November 2016. Botanical identification was conducted by Prof. Dr. Leonardo Felix Person (DF/CCA/UFPB), and a voucher specimen was deposited in the Professor Jaime Coelho de Morais—Center for Agricultural Sciences Herbarium, Federal University of Paraíba (CCA/UFPB) under code 17.562.Cladodes of ®, Piracicaba, São Paulo) and screened with a 100- and 28-mesh sieve on a sieve shaker, resulting in two samples, flour 1 and flour 2 (F1 and F2), which are shown in To obtain the xiquexique flour (tamized at 100 and 28 mesh), the collected material (cladodes) was carefully sanitized with running water and sodium hypochlorite (100 ppm/15 min) for soil removal and decontamination. The central stem was stripped, and the pulp and peel were removed. Then, the central stems were cut into 1 cm slices, and their pieces were autoclaved (121 ±1°C/20 min). Subsequently, samples were cooled at room temperature, followed by drying in an air circulation oven (40 ±1°C) until reaching approximately 4% moisture. After drying, the xiquexique was ground in a knife mill  at medium speed and equipment level 4 (144 rpm) with a flat beater attachment. The sugars  were added and mixed for 3 min at medium speed. Then, the remaining dry ingredients were added and homogenized for 1.5 min at low speed, level 2 (96 rpm), until complete homogenization.Cookies processing was performed using a methodology adapted from Kaur et al. . The ing®, Curitiba, Paraná) preheated to 180°C. The cookies were packed in paper packages and then in laminate bags, sealed under vacuum and stored at room temperature (25°C) until analysis (1 day).The dough was rolled out on a table with a Teflon rolling pin to a thickness of 8 mm by use of a guide. Cookies were cut with a 3.5 mm (diameter) aluminum mold, placed on a perforated metal tray, and baked for 9 min in a Vipinho 0448 oven (Perfectaw) and moisture, ash, lipid, protein, soluble, insoluble e total fiber, starch and resistant starch contents were determined. The aw at 25°C was determined using an Aqualab® apparatus  in accordance with the manufacturer’s instructions. The soluble, insoluble, and total dietary fiber contents were determined using Megazyme K-TDFR kit  and following the instructions of the kit which based on an integrated enzymatic-gravimetric analysis procedure [Physicochemical and nutritional characteristics analyses were conducted in triplicate according to standard procedures , 25: Watrocedure . The moirocedure . The detrocedure . Resista®, Tokyo, Japan) instrument. The analytical conditions were as follows: radiofrequency power, 1.2 kW; plasma flow, 12 L/min; auxiliary flow rate, 1.0 L/min; nebulization flow, 0.7 L/min; plasma vision, axial to Fe, Mn, Se and Zn and radial to Ca, Mg, P, and Na; Fe, 259.553 nm; Mn, 257.610 nm; Se, 196.026 nm; Zn, 206.2200 nm; Ca, 317.933 nm; Mg, 279.553 nm; P, 213.618 nm; and Na, 589.592 nm.Mineral content analysis was performed in triplicate on an inductively coupled plasma atomic emission spectrometry . The CIE Lab color scale (L*a*b*) was used with a D65 illuminant (standard daylight) and a 10° observer angle. The L*, a* and b* parameters were determined by values reported for the lightness, L* , chroma, a* , and hue, b*  parameters of the CIELAB system were the mean of three measurements at three different locations on each cookies sample.The texture was determined using a texture analyzer  to measure the hardness (as fracture force) of cookies via a 3-point bending test using a 3-point bending rig, a trigger force of 25 g, and a load cell of 50 kg. The textural studies were conducted at a pretest speed of 1.5 mm/s, a test speed of 2.0 mm/sec, a posttest speed of 10 mm/sec, and a distance of 10 mm, and the distance between the two bottom supports was adjusted to 50 mm. The peak value was measured as fracture force was recorded at a point when the cookies were broken into two major pieces and reported as hardness . The peaThe spread ratio was determined according to AACC  as the rSalmonella/25g, Presumptive B. cereus/g, Escherichia coli/g, Molds and yeasts/g) to determine the security for consumption followed the methodology recommended by APHA [Evaluation of microbiological parameters .The tests were performed in individual booths using white light, away from noises and odors, and equipped with Compusense Five version 5.6 for data collection and analysis.The data were submitted to analysis of variance (ANOVA) followed by Student’s t-test to verify significant difference between the samples, considering p < 0.05, and using XLSTAT software version 2019.1.2  .w values and moisture contents, and the inclusion of xiquexique flour in xiquexique cookies (C2 and C4) contributed to a reduction in these values when compared to their control cookies , which indicates that these flours (F1 and F2) will be stable during storage.Physicochemical and nutritional characteristics analyses were conducted to examine the composition and nutritional value of the xiquexique cookies, and the results are summarized in Opuntia ficus-indica L.) cookies.The ash content of both xiquexique cookies (C2 and C4) was greater than that of their respective controls (C1 and C3), probably due to the xiquexique flour (tamized at 100 and 28 mesh) being a rich source of minerals . The proConcerning the fiber content, the substitution of wheat flour with the xiquexique flour with two different particle sizes (100 and 28 mesh), contributed to an increase in the total and insoluble fiber contents, in addition to reducing the content of soluble fibers in the xiquexique cookies C2 (p < 0.05). This could have been because of the granulometry difference; the 100-mesh flour had lower soluble fiber content than that of the 28-mesh flour .Opuntia ficus-indica L.) in the preparation of cookies. Dick et al. [Opuntia monacantha) as alternative ingredients in gluten-free cookies observed that the addition of 10 and 15% of this flour contributed to the increase in the total and insoluble dietary fiber content. Equal behavior about fibers was also observed in our study with the replacement of up to 50% of wheat flour by xiquexique flour sifted in 100 or 20 mesh. However, the results in our study for total and insoluble fibers exceeded the values detected in the above-mentioned surveys.In the literature it was seen that the addition of cactaceous flours or substitution of wheat flour for cacti flours contributed to the increase of total and insoluble dietary fibers in cookies. This was the case in the study by Bouazizi et al. , who work et al.  working Fibers can be considered key compounds that preserve the gut microbiota because they can be fermented by the microbiota, producing metabolites that work to improve consumer health . In thisConsidering a 50 g portion (3 cookies with approximately 16.6 g each), according to recommendations of the dietary reference intake (DRI), C2 and C4 would be responsible for supplying 5.80 and 10.66% of the total fiber DRI, respectively, while control cookies (C1 and C3) supplied just 2.80 and 4.45% of the DRI for men (19 to 50 years old).The recommendations for women (19 to 50 years old) and children (1 to 3 years old), these values were higher, providing 8.82 (C2) and 16.20% (C4) of the DRI for women and 11.61 (C2) and 21.32% (C4) of the DRI for children, while control samples provided just 4.26 (C2) and 6.76% (C4) of the DRI for women and 5.61 (C2) and 8.89% (C4) of the DRI for children. These control cookies values corresponded to less than 50% of fiber supplied by the xiquexique cookies.The starch content of xiquexique cookies was lower than that of control cookies, while the resistant starch content was higher (p < 0.05). This could be justified by the low starch content and high resistant starch content determined in the xiquexique flour (F1 and F2) used, respectively, in the processing of C2 and C4 cookies .Principal carbohydrate of all cereals is starch, and wheat cookies present an average of 42% starch . Starch Agave tequilana ingredients, it was found that the replacement of whole wheat flour by agave dietary fiber contributed to the reduction of starch content and increase in the concentration of total and insoluble dietary fibers in the experimental bars. Similar behavior was also observed in our research, in which it was found that the substitution of 50% of wheat flour (refined or whole) by xiquexique flour with granulometry of 100 or 28 mesh resulted in cookies with lower starch contents and larger quantities of total and insoluble dietary fibers in their constitution.In a study by Zamora-Gasga et al.  with graThe mineral profiles of xiquexique cookies are presented in The results presented relevant amounts of minerals for C2 and C4, including, respectively, the following notable macrominerals: potassium (508.08 and 555.77 mg/100 g), calcium (201.45 and 124.88 mg/100 g), magnesium (151.08 and 132.88 mg/100 g) and sodium (222.94 and 218.36 mg/100 g). Among the microminerals, the highest contents obtained were of iron and manganese in C2  and manganese (5.36 mg/100 g of cookies) in C4. Corroborating the results mentioned above, Msaddak et al.  noticed Minerals are nutrients essential to metabolism and homeostasis in the human body, and deficiency of these bioactive constituents can result in common disorders and diseases . SimilarBased on the consumption of a 50 g portion, equivalent to 3 cookies, and according to the DRIs, the calcium content in the cookies would account for 10.86 and 6.24% of the DRI when consuming C2 and C4 cookies, respectively, while control cookies C1 and C3 contained 4.52 and 4.93% of the DRI of calcium, respectively. Calcium is one of the most abundant minerals in the body and plays an important role in the structure and conservation of bones and teeth, muscle development, and regulation of the heart rate and blood pressure . ConcernThe iron content of C2 was 17.50% of the DRI of adults, while C1 provides only 15.88% of the DRI, despite wheat flour being supplemented with iron by law. Deficiency of this nutrient is associated with the development of iron deficiency anemia, decreased cognitive ability and behavioral changes in children , 44.For magnesium, both xiquexique cookies (C2 and C4) presented high contents (p < 0.05), supplying 17.99 and 15.82% of the DRI, respectively, while their controls supplied only 12.33 (C1) and 7.75% (C3) of the DRI; therefore, we can say that xiquexique cookies are a source of magnesium and can be consumed for the purpose of supplying this nutrient. This mineral is related to several important metabolic reactions in the human body, as it is a cofactor in more than 300 enzymatic reactions .The xiquexique cookies presented potassium contents that were two times higher than those of their respective controls (p < 0.05); 50 g of C2 and C4 supplied 5.41 and 5.91% of the DRI of potassium, while C1 and C3 supplied only 2.26 and 2.81% of the DRI, respectively. The addition of xiquexique flour in cookies can contribute to achieving the DRI of this mineral. Potassium is important for fluid balance, nerve transmission, muscle contraction, the maintenance of blood pressure and waste elimination [Opuntia spp.The observed manganese values were particularly notable; the cookies supplied 157.50 (C2) and 116.52% (C4) of the adult RDI of manganese; therefore, xiquexique cookies contain more than enough manganese to meet the daily needs of adults. Manganese is an important trace element that is responsible for activating various enzymes in charge of scavenging free radicals, regulating glucose homeostasis, mobilizing calcium , increasThe consumption of 50 g of xiquexique cookies (3 units with approximately 16.6 g each) according to the DRI supplies 7.43 (C2) and 7.28% (C4) for sodium, 10.86 (C2) and 16.99% (C4) for phosphorus, 4.09 (C2) and 6.73% (C4) for zinc.The values of minerals contained in the xiquexique cookies suggest that these cookies are a potential supply of these minerals. Even minerals that presented relatively low values, if combined with a balanced diet, can contribute to the provision of these nutrients. Therefore, it is verified that the xiquexique cookies present relevant contents of minerals important for human health and therefore could be additions to a daily diet.The cookies are pictorially represented in C2 and C4 exhibited higher fracture force values than C1 and C3 (p < 0.05), however when the types of cookies were compared through the PF difference (ΔE), it was greater for conventional cookies. Giuberti et al.  observedThe spread ratio was lower in the cookies with xiquexique flour (C2 and C4) than in the control cookies (p < 0.05), as well as in bamboo cookies , chestnuThe sensorial tests were carried out using 123 voluntary individuals (aged 18–60 years). The results of the acceptance and preference of the cookies are shown in The general perception for C3 and C4 was the same (p ≥ 0.05), suggesting that the addition of whole xiquexique flour did not influence this parameter. Moro et al.  observedThe percentages of acceptance , indifference  and rejection  are presented in In It was observed that 34.1% of the consumers judged the crunchiness of C2 to be less intense than ideal, which caused a significant reduction in general perception. In addition, for 22.7% of the consumers, C2 was evaluated as harder than ideal but had no impact on the average of general perception . LikewisThe differences in the consumers’ perception of cookies can be observed by the CATA test in C2 was described as having a characteristic flavor (30.1%), having a characteristic taste (28.5%), having a mild taste (26.8%), being artisanal (27.6%), having a mild flavor (38.2%), being natural (22.0%), having a strong taste (29.3%), having an uncharacteristic taste (23.6%) and being artificial (26.0%) by consumers, with an average positive impact on the overall acceptability of 0.7 to 1.5 points, in addition to being described as artificial and noncharacteristic and as having strong flavor by 23.6 and 30.1% of consumers, with a reduction of 0.9 to 1.2 points in overall acceptability.Concerning C3 was described as being natural (29.8%) and having a characteristic whole cookies aroma (45.2%), with an increase in the overall acceptability average between 0.9 and 0.5 points. C4 was described as being natural (31.4%), being artisanal (33.1%), having a mild taste (31.4%), being homemade (46.8%) and having a mild flavor (41.9%) by consumers, which had a positive impact on the overall acceptability average of 0.5 to 0.9 points. C3 and C4 samples were described by more than 20% of consumers as having attributes that caused a reduction in the overall acceptability average.The consumer intent results are presented in Wheat is a food matrix for global consumption, but its cultivation in the quantities necessary to reach consumer demand is not viable due to the damage that its cultivation brings to the soil as impoverishment and desertification. In addition, the food industry is constantly developing strategies to reduce manufacturing increase profitability and improve the nutritional quality of products. In this matter, the incorporation of alternative ingredients in wheat-based food products represents a promising strategy to meet the demand for nutritious, sustainable and agro-ecological products, meeting consumer demands .Within this context, the replacement of wheat flour by alternative flours of cacti , 36, speIn this study, the initial hypothesis of the viability of using xiquexique flour as a partial gluten substitute was verified by the acceptability of the cookies and by the verified textural properties. In addition, there is a greater richness in fibers and minerals in cookies made with partial replacement of wheat flour by xiquexique flour. It is noteworthy that this substitution did not compromise technological properties, such as the kneading ability, nor the acceptance of most of the evaluated sensory attributes, reinforcing the importance of this study. So, the partial substitution of wheat flour with xiquexique flour to produce cookies is a promising strategy in the manufacturing of bakery products. The possibility of testing the xiquexique flour for cake preparation could be another interesting field of application.This is the first evidence of the use of xiquexique flour in the processing of cookies and suggests it as an excellent ingredient for production this type of product. The cookies with added xiquexique flour obtained the highest values of darkness, hardness, ash, protein and resistant starch contents, as well as low water activity values, moisture contents and spread ratios when compared to their respective controls. Xiquexique cookies can be considered as an innovative product for the functional food industry, especially for its bioactive and nutritional characteristics, being excellent sources of fibers and minerals, essential nutrients for the general population. Our study shows that cookies added from finely xiquexique flour (tamized at 100 mesh) and the xiquexique flour (tamized at 28 mesh) were well accepted by consumers. The CATA method added another dimension  to describe products. The results of this method can be used for the adjustment of cookies recipes and the development of optimal cookies and other baked goods with partial replacement of wheat flour.The authors declare the Impact of the Novelty, considering all the aspects already reported and reinforcing that the present invention (cookies enriched with xiquexique flour) has unique characteristics, being a potential alternative for nutritionally rich, tasty, safe and practical food; and thus represents a new form of economic use of this plant species, especially in relation to sustainability.S1 Tablea, bMedia ± standard deviation with different letters on the same line differed by Student’s t-test (p < 0.05), between treatments.F1—Xiquexique flour tamized at 100 mesh; F2—Xiquexique flour tamized at 28 mesh—Flours chosen to process cookies C2 and C4, respectively. (DOCX)Click here for additional data file.S2 Tablea, bMedia ± standard deviation with different letters on the same line differed by Student’s t-test (p < 0.05), between treatments. *Based in Institute of Medicine. Dietary Reference Intakes, Washington D. C., National Academy Press; 2003 (1997–2005). Based on a 70 kg man, 31–50 years old [F1—Xiquexique flour tamized at 100 mesh; F2—Xiquexique flour tamized at 28 mesh—Flours chosen to process cookies C2 and C4, respectively. ears old . (1) Ade(DOCX)Click here for additional data file.