The main objective of this study was the development of gluten-free cracker-type snacks with a balanced supply of essential amino acids, a lower glycemic index, and a lower caloric intake that were sensorially acceptable. For this purpose, chickpea flour was replaced by quinoa  and the fat was partially (75%) replaced by chicory inulin. The flours were characterized in terms of their proximate composition, individual mineral content, particle size distribution, and functional properties. The parameters analyzed for the crackers, once baked, were the water content, water activity, weight, dimensions, color, and texture. A sensory analysis was performed as well, using the formulations containing 50% chickpea flour and 50% quinoa flour (g/100 g flour), with and without inulin, as well as those made with 100% chickpea flour. From the analysis of the raw flours, it can be concluded that snack products developed from them could be a nutritive option for children, in terms of the protein, magnesium, and fiber content. The functional properties revealed that both flours are suitable for producing doughs and baked products. The obtained results indicate that snacks made with 50% quinoa flour (g/100 g flour) and 75% chicory inulin (g/100 g high oleic sunflower oil) could be an interesting alternative for children as, in addition to offering a very interesting nutritional contribution, the energy intake from fat is reduced by 57%. The World Health Organization has determined that in 2016, worldwide, more than 39% of the adult male population, and 40% of the female population were overweight, and that around 13% of the global population suffered from obesity ), was 273 μm for the QF, and 192 for the CPF . The obtUsing plant proteins in the food industry becomes a challenge, as several changes may occur in the technofunctional properties of the meat or animal proteins involved in the characteristics of products, such as the appearance, texture, and mouthfeel. Thus, understanding protein behavior, and changes through the process chain, is imperative to optimizing and reducing production hurdles when working with plant proteins, such as chickpeas or quinoa. p < 0.05) than the QF.The water retention capacity (WHC) allows the evaluation of the flour’s ability to retain water under a gravitational centrifugal force; macromolecules such as carbohydrates and proteins, by offering hydrophilic sidechains, increase this retention . Authorsp < 0.05), independent of the chickpea flour’s replacement with quinoa or inulin. The water activity was below 0.6, which generally inhibits bacterial, yeast, and mould growth, as well as chemical and enzymatic changes in food [The obtained values for the water content, water activity, final weight, and final thickness of the cracker-type snacks  did not  in food .w/w) level, but the energy coming from fat is considerably diminished, as the fat is reduced by 57%. As expected, there is also a remarkable increase in the fiber intake as a result of the inulin incorporation, reaching 27% of the average requirement of 16 g/day for children aged 7–10 years [w/w. Some evidence suggests that fiber intake may play a role in the prevention and treatment of childhood obesity and constipation [Based on a single serving size of 30 g , the 50C10 years , as valutipation . The proConcerning the mineral contribution, the magnesium, phosphorus, calcium, and iron intakes were found to be, respectively, approximately 6, 9, 1.5, and 6% of the adequate values (AI) from the European Food Safety Authority for children .w/w) by this fiber. Different behavior could be observed with the amount of quinoa flour, depending on the sunflower oil substitution with inulin. Contrary to the expected results, the firmness was only significantly increased (p < 0.05) when the chickpea flour was replaced by quinoa flour at the 50% (w/w) level (for the non-inulin crackers), and significantly decreased with the quinoa amount (for the inulin crackers). Previous studies on cookies [The obtained values for the snack firmness are shown in  cookies  and bisc cookies  found thMicrostructural observations  allowed ab) and the psychometric coordinates (C*ab and h*ab) of the assessed snacks are summarized in Color is one of the attributes that determines the acceptability of baked products . It is iw/w flour), reduces the snack lightness, chrome, and hue angle, causing it to become darker and browner. Similar results were obtained by [ab values) significantly increased (p < 0.05) independent of the flour mixture used in the formulation. Inulins are thermally stable in short food-processing steps, but some hydrolysis may occur; in fact, inulin undergoes the Maillard reaction and caramelization, meaning that bakery products made with inulin have a nice, brown color [As a global tendency, it seems that quinoa flour, when used above 20% , and the effect of using inulin instead of sunflower oil. The panelists were asked to evaluate the appearance, color, odor, texture, crispness, taste, and overall acceptability. The obtained data showed a clear preference for those crackers based only on chickpea flour (the control sample), with higher scores for all the assessed attributes, except the odor, which was slightly lower. The results for the empirical firmness (w/w) of sunflower oil with inulin (50CPF50QF+I) but, in this case, the color, appearance, and odor obtained better scores, close to those of the chickpea snacks (100 CPF).The scores for the sensory properties of the cracker-type snacks are summarized in firmness  and sensThe panelists also stated that they would regularly consume the cracker formulated only with chickpea flour and would add it to their shopping cart, and as for the formulations with quinoa, they were considered to be a very interesting healthy alternative both for children and adults, although the texture should be improved to achieve a crisp product. Regarding the taste, panelists did not detect differences between the sample with inulin and the one that did not have this fiber in its composition.w/w total flour used), and with 75% of their high oleic sunflower oil substituted by chicory inulin, could be an interesting alternative for children as, in addition to offering a very interesting nutritional contribution , its energy density decreases by 5.4% . More relevant is the reduction in the energy intake from fat (57%). However, it is necessary to continue with further research, to achieve a crunchier texture.Taking into account all of the assessed parameters, it may be concluded that cracker-type snacks made using 50% quinoa flour (


Populus. The first actor of this signalling pathway belongs to the histidine-aspartate kinase (HK) family, which also includes the yeast osmosensor Sln1, as well as the Arabidopsis putative osmosensor AHK1. In poplar, the homologous AHK1 protein corresponds to a pair of paralogous proteins, HK1a and HK1b, exhibiting an extracellular domain (ECD), as in Sln1 and AHK1. An ECD alignment of AHK1-like proteins, from different plant species, showed a particularly well conserved ECD and revealed the presence of a cache domain. This level of conservation suggested a functional role of this domain in osmosensing. Thus, we tested this possibility by modelling assisted mutational analysis of the cache domain of the Populus HK1 proteins. The mutants were assessed for their ability to respond to different osmotic stress and the results point to an involvement of this domain in HK1 functionality. Furthermore, since HK1b was shown to respond better to stress than HK1a, these two receptors constituted a good system to search for osmosensing determinants responsible for this difference in efficiency. With domain swapping experiments, we finally demonstrated that the cache domain, as well as the second transmembrane domain, are involved in the osmosensing efficiency of these receptors.Previous works have shown the existence of protein partnership, belonging to a MultiStep Phosphorelay (MSP), potentially involved in osmosensing in  Saccharomyces cerevisiae [Water deficit is one of the most important environmental stresses affecting plant growth. Therefore, understanding the mechanism of drought stress perception is a major challenge to select new drought-tolerant plants. At the cellular level, this abiotic stress is perceived as an osmotic stress, and the plant mechanism of osmosensing is still unresolved, thus constituting an ongoing research field of particular interest ,2. So farevisiae . This parevisiae ,5. This revisiae .In planta analysis of ahk1 mutants gave evidence that AHK1 (i) acts as a positive regulator of osmotic stress signalling, (ii) is involved in drought tolerance [In yeast, Sln1 is integrated into the membrane by two transmembrane domains, flanking an extracellular domain (ECD) in the N-terminal part . It has olerance ,11 and  channel subunits and prokaryotic chemotaxis (che) receptors [Recently, a large computational analysis on sensor receptors was performed and revealed that the so-called “extracellular PAS” (Per/Arnt/Sim) domain is indeed a cache domain . This steceptors . This doBased on these findings, we performed an alignment of the ECD of AHK1 homologous proteins from different plant species, revealing a well-conserved cache domain in sequence and structure. Since AHK1 and HK1a/b were characterized as osmosensors in yeast, this alignment result allowed us to hypothesize that this cache domain may be responsible for the osmosensing capacity of these receptors. We tested this possibility by a genetic approach with a mutational analysis of poplar HK1a and HK1b cache domains. A rational mutation design, assisted by modelling, was used to direct point mutations, and mutants were tested in a complementation assay, with an osmodeficient yeast strain challenged on different osmotic stress and strength. These assays allowed us to identify positions that could be involved in protein functionality. Moreover, according to previous results demonstrating that HK1b is able to respond to osmotic stress more efficiently than HK1a , we perfAccording to Upadhyay and colleagues (2016), a cache domain is present in the osmosensor Sln1 and the putative plant osmosensors AHK1 and HK1a A. This cSince a cache domain corresponds to small ligand-binding domain, we were interested to know whether the cache domain of our poplar HK1a/b could also bind small molecules, unknown so far, to trigger their osmosensing activity. As a first step, we tried to model the 3D structure of the full HK1a and HK1b ECD in order to define potential positions in interaction with a ligand within the cache domain. These 3D models were built using a method based on deep learning neural networks .Pseudomonas aeruginosa PAO1, with a bound histamine (PDB 6FU4) [These models were then submitted to the DALI server to identify structural homologues among a subset of representative Protein Data Bank (PDB) structures (database PDB25) . The cloDB 6FU4) . Both moS. cerevisiae deletion mutant strain (MH179) where the two proteins involved in osmosensing were deleted (sln1Δ sho1Δ). These deletions are lethal due to constitutive HOG pathway activation, but viability can be restored by expression of the phosphatase Ptp2, induced by galactose, which dephosphorylates Hog1 in the nucleus, thus inhibiting its transactivation of response genes.In order to test these mutants, we performed a complementation assay in a 6000) infused into the medium (This strain was transformed with plasmids carrying the wild type (WT) or mutated cDNAs of HK1a (T324A/S304A/K296A and triple mutant TSK) and HK1b (T331A/Q311A/K303A and triple mutant TQK), along with plasmids corresponding to positive (SLN1) and negative (pYX212) control. The complemented mutant yeasts were tested for their capacity to grow on normal or high-osmolarity mediums. The survival of all transformants is allowed on galactose medium . On glucose medium (−U + Glu), inhibition of HOG response by Ppt2 is released and lethal phenotype can be observed, unless the expressed osmosensor restores the yeast osmodeficiency. We applied osmotic stress with increasing concentration of NaCl, as well as polyethylene glycol 6000 , infused into the medium (PEG50). This osmotic agent, as a non-permeant molecule, allows osmotic stress without the ionic component of salt stress. The osmotic strength of each medium was measured to compare the PEG stress medium to the NaCl stress medium  differed from only one amino acid. Consequently, we performed the exchange of TM2 domain between HK1a and HK1b by a single point mutation (F470C mutation in HK1a and C478F mutation in HK1b). This domain swapping was also tested in combination with cache domain exchange .In the case of HK1a, the TM domain exchange mutant (TM1b) exhibited better growth compared to WT, even on 0.9 M NaCl medium, overpassing cache domain exchange mutant (cache1b), which allowed a better growth on 0.6 NaCl medium only. Furthermore, this positive effect was still present on PEG50 medium A. HoweveSince no effect of the cache and TM domain exchanges are observed for HK1b, we assume that this discrepancy with HK1a exchange mutants may be due to the strong growth of HK1b, even in a stress condition as high as 0.9 M NaCl medium. Thus, in order to observe a possible effect, we challenged these HK1b mutants with stronger stress conditions .With these high NaCl concentrations, we were able to observe a decreasing effect of TM domain exchange (TM1a) on HK1b. The negative effect was slightly visible on 1.2M NaCl but became more noticeable on 1.5 M NaCl medium . The douAs sessile organisms, plants must cope with abiotic stress, such as soil salinity, drought and extreme temperatures. Drought stress is one of the most serious abiotic stresses that have profound effects on plant growth and survival. Understanding the biochemical and molecular mechanisms for drought stress perception, transduction and tolerance is still a major challenge in sustaining crop yield . Faced wSaccharomyces cerevisiae, the osmosensing pathway employs Sln1 as an osmosensor HK that monitors changes in turgor pressures [Campylobacter jejuni possesses a cache domain, and structural studies have revealed a downward displacement of the TM helix 2 towards the cytoplasm upon ligand binding to its cache domain [In the yeast ressures . Furtherressures , is presressures . On the ressures . After uressures . In the ressures . Indeed,e domain . This moPopulus and identified determinants that could be involved in HK1s response to osmotic stress. Through a genetic analysis, we tested point mutations in the cache domain of both HK1s and domain exchange mutants by cache and/or TM2 domains swapping. These mutants were tested in our osmodeficient yeast strain in order to carry out functional complementation assays in the presence of NaCl and PEG6000 stresses.By referring to these bibliographic data, in this present study, we demonstrated, experimentally, the importance of cache and TM domains in HK1 receptors of 6000, the three mutated positions in HK1a and HK1b conferred an improved osmotic stress response compared to WT, supporting the implication of these amino acids in osmosensing function. These three positions correspond to homologous positions of three amino acids involved in ligand binding of the histamine chemoreceptor of Pseudomonas aeruginosa PAO1. Therefore, our results suggest that the amino acids in both HK1s may also be involved in the binding of a ligand, though this remains to be determined. Consistently, it has been demonstrated in S. cerevisiae that the cell wall protein Ccw12 can affect the activation of Sln1, allowing the authors to postulate that physical contact between Sln1 and some cell wall or periplasmic component may occur [S. cerevisiae, it has been shown that the integrity of the ECD is essential for Sln1 function [AHK1, AHK2, AHK3 and AHK4, it was shown in Arabidopsis that AHK1 acts as a positive regulator in osmotic stress response and AHK2-3-4 act as negative regulators [in planta involvement in drought response, it would be interesting to test these receptors in an Arabidopsis ahk1 mutant line by complementation assay in stress conditions. In such in planta assays, the use of the cache domain mutants characterized in this study would allow us to confirm the contribution of these domains to the osmotic stress response in a plant background. Furthermore, the study of these two receptors, directly into Populus lines, would definitely bring information in the genuine homologous background. Such analyses with poplar mutant lines (overexpressed or knock-out) are currently under investigation.Our results showed that, in the presence of stress NaCl or PEGay occur . Thus, ifunction . Interesfunction ,34. Sincfunction ,35, and function ,37, the gulators . These agulators . For ourIn order to identify the determining factors in the differential response to osmotic stress between HK1a and HK1b, we tested mutants by cache and/or TM2 domain swapping. We showed that HK1a, with cache or TM2 domains of HK1b, exhibited a better tolerance to osmotic stress than WT HK1a. This result implies that these domains are responsible, at least in part, for the better osmosensing efficiency of HK1b. These results were in agreement with those observed for Sln1 , AHK4 2 and AHK2In summary, this study provided clues about the involvement of cache domain in the functionality of HK1 receptors. We found three amino acids in this domain that may play a role in osmotic stress response. This result shows the functional importance of this domain in poplar HK1s and, given that a cache domain is present in a wide range of plant HK1-like proteins, we can hypothesize that the involvement of this domain may be a general feature in plant osmosensing. Furthermore, the TM2 domain appears to be one of the factors responsible for the differential response observed between both HK1s. These new results shed some light on the functionality of HK1 receptors in osmosensing.As a next step, it could be interesting to explore the role of the cytoplasmic part of the HK1b receptor in the differential response, alone or in combination with other domains (cache or TM2), so we can precisely define all the determining elements in its efficiency. Moreover, further experiments at the structural level could bring valuable information on cache domain function. Structural determination of such a domain in plants would definitively provide precious insights since no structural data exist for a plant cache domain in the PDB. This approach is currently under progress. Finally, since this cache domain is involved in osmosensing and corresponds to a small ligand-binding domain, the identification of such a ligand would provide unprecedented clues to understanding the osmosensing mechanism. We are currently exploring this question with the use of an original reporter system to screen for molecules able to bind poplar HK1s. The determination of the true ligand responsible for osmotic stress response will definitely lead to valuable applications in developing drought tolerant plants for sustainable agriculture.www.ebi.ac.uk/Tools/services/rest/clustalo, accessed on 13 February 2023) or Clustal W  with Arabidopsis , saltwater cress , papaya , cocoa , cotton , peach , apple , rose gum , grape vine , tomato , Madagascar periwinkle , yellow monkeyflower , purple willow , castor bean , cassava , flax , barrel clover , common bean  and soybean . A portion of this alignment is shown in According to Upadhyay et al. (2016), a cache domain is present in poplar HK1a from aa 154 to aa 364. Therefore, we aligned sequences of HK1a/b ECD (starting from the end of the first TM domain to the beginning of the second TM domain) with ECD sequences from 27 plant AHK1-like proteins. Amino acid sequences of HK1a (accession No AJ937747) and HK1b (accession No LT622839) were aligned by Clustal Omega .The determination of secondary structures was performed with the “Secondary structure consensus prediction” program of NPS@ web server from the Pôle Rhône-Alpes de BioInformatique (http://robetta.bakerlab.org), and the 3D structure prediction was based on RoseTTAFold deep learning methodology [https://www.pymol.org/pymol, accessed on 13 February 2023) was used to superimpose the HK1a and HK1b ECD models to the Pseudomonas aeruginosa PAO1 cache domain bound with histamine. Selection of the residues, located 5 Å from the histamine in HK1a and HK1b models, enabled the identification of the putative ligand binding regions that were targeted for site-directed mutagenesis.The ECD peptide sequences of HK1a (residues 100–451) and HK1b (residues 107–458)  were submitted to the ROBETTA server (hodology . The besEcoRI-HindIII) by homologous recombination in yeast. DNA sequencing confirmed the presence of the desired mutations and that no undesired mutations were present.Point mutations in HK1a and HK1b full-length CDS sequences were introduced by PCR with specific mutated primers  to creatEcoRI-HindIII) by homologous recombination. The correct mutated sequences were confirmed by sequencing.Domain exchange mutations in HK1a and HK1b sequences were introduced by PCR, with specific primers , to creaura3 leu2 his3 sln1::LEU2 sho1::TRP1 + pGP22, with pGP22: GALp-PTP2/pRS413 ) was used for transformation, according to [w/v) overnight. Three 10-fold dilution series, starting from an OD600 0.2, was realized and 5 µL of each were spotted onto medium.The pPD2133 plasmid, expressing the Sln1 osmosensor, was used as a positive control (pPD-Sln1) and the empty pYX212 vector was used as a negative control. The yeast strain MH179  on liquid solutions. The PEG gel medium was melted before measurement. Results are the mean of 3 repeats.


The amine-linkages enhance the binding ability of CO2 molecules, and the ionic frameworks enable to improve the electronic conductivity and the charge transfer along the frameworks. However, directly synthesis of covalent organic frameworks with amine-linkages and ionic frameworks is hardly achieved due to the electrostatic repulsion and predicament for the strength of the linkage. Herein, we demonstrate covalent organic frameworks for CO2 reduction reaction by modulating the linkers and linkages of the template covalent organic framework to build the correlation between the catalytic performance and the structures of covalent organic frameworks. Through the double modifications, the CO2 binding ability and the electronic states are well tuned, resulting in controllable activity and selectivity for CO2 reduction reaction. Notably, the dual-functional covalent organic framework achieves high selectivity with a maximum CO Faradaic efficiency of 97.32% and the turnover frequencies value of 9922.68 h−1, which are higher than those of the base covalent organic framework and the single-modified covalent organic frameworks. Moreover, the theoretical calculations further reveal that the higher activity is attributed to the easier formation of immediate *CO from COOH*. This study provides insights into developing covalent organic frameworks for CO2 reduction reaction.To achieve high-efficiency catalysts for CO 2 reduction.Covalent organic frameworks constructed with ionic skeletons and C-N linkages by multilevel post-synthetic modification. These frameworks achieved high activity and stability for electrocatalytic CO The diversity of their building units allows them to have a high degree of structural tenability in terms of skeletons, porosities and topologies, resulting in tailorable functions8. The functional COFs have been utilized in molecular absorptions, light emitters, photo conduction/catalysis, proton/lithium-ion conduction, and lithium-ion or lithium-sulfur batteries16. Notably, the stable frameworks, large surface areas, open porous channels, and predictable catalytic sites of COFs also allow them to be used as electrocatalysts in the electrochemical reactions, such as oxygen reduction reaction, oxygen evolution reaction, hydrogen evolution reaction, CO2 reduction reactions (CO2RR), and H2O2 synthesis22. Electrocatalytic CO2RR, which can produce high-value products from the greenhouse gas, is a promising strategy for addressing the CO2 crisis. Owing to the molecular designability, COFs are an ideal class of templates to construct catalysts for CO2RR26. The catalytic behavior of COFs for CO2RR is dependent on the structure and properties30. From the perspective of structure, the types of catalytic centers, electronic states of the linker molecules, and linkage diversities have been adopted to assemble different catalytic COFs34. By changing the building units or linkages, the corresponding properties of COFs, such as the binding ability of CO2 and the electron conductivity, can be adjusted37. Thus, a COF structure containing both amine bonds and ionizing skeletons, which enhance the binding ability of CO2 molecules and the framework conductivity, respectively, is desirable for highly efficient CO2RR.Covalent organic frameworks (COFs) are fully designable porous polymers, being comprised of organic knots and covalently connected linkers42. Challenged by the steric hindrance effect, the solubility difference and the microscopic reversibility, however, some functionalities cannot be introduced directly into COFs via the bottom-up syntheses47. For the pre-designed COF that contains amine bonds and ionizing skeletons, the existing covalent connection methods  do not support to obtain C–N linkage through the bottom-up synthesis directly, and the electrostatic repulsion effects of ionic building units impede the direct formation of ionizing skeletons. Alternatively, the post-synthetic modification strategy provides a promising chance to construct functional skeletons, pores, and linkages at the molecular level with controllable catalytic properties56. Several single-step post-modifications were proposed to endow the base COFs with special properties60. Deng et al. constructed imine-COFs by a post-reduction method to improve gas diffusion on electrode61. Guo et al. used Viologens (C5H4NR)2n+ to construct a cationic radical framework from the 2,2′-bipyridine-based COF, which showed high photothermal conversion efficiencies62. Thus, it is expectable to obtain the COF with reduced imine linkages and ionic skeletons if the single-step post-modification of reduction and ionization can be well integrated. However, the integrated multilevel post-modification is rarely reported, because that not only a robust base COF is required but also the interference of multistep should be well avoided. In the view of COF design, 4,4′,4″,4‴-)tetrabenzaldehyde (PATA) unit contains ammonium groups which are potential to transfer to ionic skeleton, and 5,10,15,20-tetrakis(4-aminophenyl)porphinato]-cobalt (TAPP(Co)) unit can achieve efficient charge transfer due to their conjugated macrocyclic structures. In addition, we previously proved that CoTAPP-PATA-COF, composed by PATA and TAPP(Co), possesses good crystallinity, high surface areas, and excellent chemical stability28. Thus, CoTAPP-PATA-COF is expected as a template for constructing multilevel post-synthetic modification COFs for CO2RR.The bottom-up synthetic approach is the most common method to directly construct functional COFs2RR. The N+-NH-COF , which was constructed from CoTAPP-PATA-COF by the multilevel post-synthetic modification, showed a maximum CO faradic efficiency of 97.32% at −0.8 V with a CO current density of −28.01 mA cm−2 and the TOF value of 9922.68 h−1 at −1.0 V vs. RHE. The excellent electrocatalytic properties can be attributed to the superior binding ability of CO2 molecules from C–N bonds and high conductivity from ionization skeletons in these COFs. The results showed that reduction of C=N linkages into the C–N bond and ionization of the linkers significantly improve the selectivity and activity.In this study, we demonstrate a multilevel post-synthetic modification strategy to construct catalytic COFs for CO17. The C=N linkages of the COF were reduced by adding NaBH4 in dimethylacetamide (DMAC) to yield NH-COF, and the PATA units were ionized through in situ ammonium groups via Menshutkin reactions to obtain the N+-COF  to obtain the N+-NH-COF  was synthesized from CoTAPP and PATA using the solvothermal method described in our previous studyCOF Fig. 63. The bCOF Fig. .Fig. 1Sc−1 were retained, while a peak raised at 1470 cm−1, being ascribed to -N+-(CH3)2−, in the FTIR spectrum of ionized N+-COF 2– was also detected for the FTIR spectrum of N+-NH-COF. Moreover, the Co–N bonds at 660 cm–1 were observed in all four COFs, indicating that the post-modifications were harmless to the Co-N coordinations.The successful functionalization on the linkage reduction and the skeleton ionization were elucidated by the Fourier transform infrared spectroscopy (FTIR). Compared with the base COF, the C=N linkages at 1622 cm13C cross-polarization/magic-angle spinning solid-state nuclear magnetic resonance  measurements. The PXRD pattern of base CoTAPP-PATA-COF showed the peaks of (011), (022), (031) and (001) facets at 5.15, 11.02, 12.23 and 21.84 °, respectively , 11.12° (022), and 21.97° (001) , (002), (202) and (001) at 5.15, 11.12, 12.32 and 22.17 °, respectively ,2,+-COF, 01) Fig. . Both ofely Fig. . The hig (022), 01 and (00 (022), 01 and (00BET) of 943.72 m2 g−1 . Specifically, NH-COF exhibited a higher CO2 desorption signal than that of base CoTAPP-PATA-COF, indicating the enhanced CO2 adsorption capacity on C–N bonds  surface area  and transmission electron microscopy (TEM). The post-modified COFs exhibited similar morphologies to that of base COF without significant morphological changes, suggesting that the structure is well preserved , which are in good agreement with the hydrophobic behaviours  measurements  measurements . Furthermore, the high-resolution Co 2p spectra of the four COFs exhibited Co-N coordination, which confirmed that the Co-N sites were well retained after the multilevel post-modifications  and N+-NH-COF (780.90 eV) showed a negative shift of ~0.45 eV, which is ascribed to the electron-withdrawing effect of methyl groups56. This proves that the skeletons in N+-COF and N+-NH-COF have been ionized by the CH3I modification. The high-resolution N 1 s spectrum of the CoTAPP-PATA-COF was deconvoluted into three peaks of pyrrole N (398.29 eV), C=N (399.62 eV) and C–N (401.03 eV) with relative contents of 28.14, 55.88 and 15.97 at.%, respectively . The XPS spectra showed peaks corresponding to C, N, O, and Co in the prepared COFs  and NH-COF (3.0 × 10−10 S m−1). It suggests that the ionized skeletons can promote the electron transfer along the frameworks, and thus improving the activity. The highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) were calculated to investigate the electron conduction properties using the Mott–Schottky method  spectroscopy was adopted to determine their band gaps Fig. . Accordiely Fig. , inset. OFs Fig. 58.2RR performance of the catalytic COFs was investigated in a KHCO3 aqueous solution  saturated with CO2 using a standard two-compartment electrochemical cell. The COFs were mixed with carbon black at a weight ratio of 5/8. Firstly, the linear sweep voltammetry (LSV) measurements were conducted at a scan rate of 10 mV s–1 from 0 to –1.0 V vs. RHE  of the CoTAPP-PATA-COF were 67.84%, 73.11%, 76.26%, 81.75%, 73.42%, and 67.54% at –0.5, –0.6, –0.7, –0.8, –0.9, and –1.0 V, respectively  of the catalyst. The CoTAPP-PATA-COF exhibited the highest jCO of 13.26 mA cm−2 at a potential of –1.0 V  were calculated using the cyclic voltammogram (CV) plots in a potential range of –0.16 to –0.36 V at the scan rates of 10–50 mV s–1  of the COFs were then calculated by ECSA = Cdl/Cs, where the Cs is 0.04 mF cm−2 65. N+-NH-COF offered the highest ECSA (719) relative to CoTAPP-PATA-COF (423), N+-COF (659), and NH-COF (609), which is in line with its CO2RR performance. Moreover, the ECSA of N+-COF was higher than the base COF and NH-COF, suggesting that the ionic frameworks provided more active sites65. The normalized current densities on the COFs, i.e., jCO per ECSA, were further evaluated  over N+-COF and N+-NH-COF were 36 and 24 Ω, respectively, being smaller than that of CoTAPP-PATA-COF (52 Ω) and NH-COF (42 Ω). This suggested that the ionization of skeletons enhanced the charge transfer capacity of COFs  was >94.5% within 25 h, indicating the excellent long-term stability of N+-NH-COF for CO2RR. To confirm the stability, the used N+-NH-COF was checked by the PXRD, FTIR and XPS measurements after the long-term stability test. The PXRD patterns showed that all peaks were fully retained, and peaks were well-retained after the long-term stability test  calculations were carried out at the theoretical level of CAM-B3LYP/6-311 G(d) (SDD for Co) for the cluster models of these four COFs. Reasonable geometrical, electronical and thermodynamic information could be obtained with cluster models for COFs and similar systems68. All structures along the potential energy surfaces were optimized without any restrictions. Frequencies were further performed to confirm that all optimized geometries are local minima and to obtain the Gibbs free energies. In the calculation, the four model molecules were labelled as M-CoTAPP-PATA-COF, M-I-N+-COF, M-NH-COF and M-I-N+-NH-COF, respectively, where the counter ion I− was considered for the N+-COF and N+-NH-COF , while the process from *COOH to the final product CO are largely exergonic. This indicates that the formation of *COOH is the rate control step. The lowest free energy changes of *COOH state for M-I-N+-NH-COF is in line with its best performance from a thermodynamic viewpoint. In addition, both M-I-N+-COF and M-I-N+-NH-COF have the lower free energy change values (∆G) of *COOH than those of M-CoTAPP-PATA-COF and M-NH-COF, which indicates a stronger promotion effect of ionization than the neutral ones. As shown in Fig. +-NH-COF catalysing CO2RR. The oxygen O1 atom in CO2 coordinates with Co atom in M-I-N+-NH-COF. The Co-O1 bond is 2.734 Å and the charge transfer from M-I-N+-NH-COF to CO2 is negligible because the total charge on CO2 is nearly zero , which can contribute to their tuneable catalytic performance for COA mixture of 1-butanol / 1,2-dichlorobenzene (0.5 mL/0.5 mL), , TAPP(Co) , and an aqueous acetic acid solution  was degassed in a Pyrex tube (10 mL) by three freeze-pump-thaw cycles. The tube was sealed and heated at 120 °C for 3 days. The precipitate was collected by centrifugation, washed with tetrahydrofuran, and dried at 120 °C under vacuum overnight to give CoTAPP-PATA-COF in a yield of 87.6%.4 (100 mg), and methanol (50 mL) was stirred in a 100 mL flask for 12 h at 0 °C. The mixture was then poured into a large amount of ice water. After filtration, the white solid was washed three times with water and dried.A mixture of CoTAPP-PATA-COF (50 mg), NaBH3I) liquid was continuously added into a solution of the CoTAPP-PATA-COF (50 mg) in ethanol (200 mL) to be magnetically stirred at ambient temperature. Afterward, the mixture was vibrated at 60 °C for 24 h. The obtained white precipitate was collected and washed with ether followed by drying in vacuum for 6 h.The cool stored 5 mL iodomethane (CH+-COF (50 mg), NaBH4 , and methanol (50 mL) was stirred in a 100 mL flask for 12 h at 0 °C. The mixture was then poured into a large amount of ice water. After filtration, the white solid was washed three times with water and dried.A mixture of NSupplementary InformationPeer Review File


STS-1 and STS-2 form a small family of proteins that are involved in the regulation of signal transduction by protein–tyrosine kinases. Both proteins are composed of a UBA domain, an esterase domain, an SH3 domain, and a PGM domain. They use their UBA and SH3 domains to modify or rearrange protein–protein interactions and their PGM domain to catalyze protein–tyrosine dephosphorylation. In this manuscript, we discuss the various proteins that have been found to interact with STS-1 or STS-2 and describe the experiments used to uncover their interactions. Scott and co-workers were characterizing RNA transcripts that originate in an area of chromosome 21 that is linked to a familial form of deafness. They isolated a full-length transcript that encodes a protein with a UBA (ubiquitin-associated) domain and an SH3 (Src-homology 3) domain and named it UBASH3A . Cbl is STS-1 and STS-2 form a small family of soluble proteins that are present in the cytoplasm of many cell types. They are composed of an amino-terminal UBA domain, followed by a phosphodiesterase domain, an SH3 domain, and a carboxy-terminal PGM (phosphoglycerate mutase) domain . UBA domJanus kinases are protein–tyrosine kinases that are associated with various cytokine receptors, including interleukin receptors, the GM-CSF receptor, and the EPO receptor . JAK2, aTo investigate whether tyrosine phosphorylation sites on JAK2 can also function as binding sites for signaling proteins, Ihle and coworkers linked a synthetic phosphopeptide identical to a major JAK2 autophosphorylation site, Tyr (tyrosine) 966, to Sepharose beads . These pZAP-70 is a cytoplasmic protein–tyrosine kinase that is composed of two amino-terminal SH2 domains and a carboxy-terminal protein–tyrosine kinase domain. The SH2 domains mediate phosphorylation-dependent protein–protein interactions by binding to phosphorylated tyrosine residues in its binding partners . ZAP-70 Gene knockout experiments carried out by Ihle and coworkers led to the observation that mice lacking functional genes for both STS-1 and STS-2 have hyperactive T cells ,21. T-ceIn contrast to what was observed in gene knockout studies, overexpression studies in Jurkat cells suggested that STS-2 increases T-cell receptor signaling . In addiThese observations prompted Tsygankov and coworkers to investigate whether STS-1 and STS-2 proteins also regulate the Syk protein–tyrosine kinase. To do so, they overexpressed STS-1 or STS-2, together with Syk and a chimeric receptor composed of the extracellular domain and transmembrane region of CD-8 and the cytoplasmic region of the T cell receptor ζ chain . Their eCIN85 was identified as an 85 kDa protein that associates with Cbl ,24. It iInterestingly, a T cell-specific CIN85 knock-out mouse has a phenotype that is very similar to the STS-1 and STS-2 double knockout . T cellsTo identify CIN85 bound proteins involved in inhibition of T cell receptor signaling wild type CIN85 and a mutant lacking the proline-rich region were isolated by immunoprecipitation. CIN85 and associated proteins were digested with trypsin and peptides were identified by HPLC and mass spectrometry. This led to the identification of STS-2 as a CIN85-associated protein . The intCbl proteins are E3 ubiquitin ligases that negatively regulate signaling by protein–tyrosine kinases ,6. Cbl pTo learn more about the biochemistry of Cbl, Tsygankov, and coworkers purified Cbl and associated proteins from a human T cell line. They took advantage of the fact that the human Cbl protein contains a string of seven histidine residues close to its amino-terminal end that can bind to a Ni-affinity column. Proteins eluted from the Ni-affinity column were further purified using an anti-Cbl affinity column. Bound proteins were resolved by SDS-PAGE, digested with trypsin, and identified by mass spectroscopy . This leCo-immunoprecipitation experiments in which both proteins were overexpressed showed that Cbl and STS-2 interact within the cell. Deletion of the proline-rich region in Cbl or mutation of the SH3 domain in STS-2 blocked co-immunoprecipitation ,8. PurifIt is well established that Cbl uses its TKB domain to bind to activated receptors . FollowiAfter the identification of STS-2 as a binding partner for Cbl and a negative regulator of EGF receptor downregulation, Dikic, and coworkers looked in detail at STS-1. They observed robust binding of STS-1 to the EGF receptor. Binding was dependent on receptor activation . STS-1 oThe binding of STS-1 to Cbl was independent of EGF receptor activation, while the binding of STS-1 to the receptor was stimulated by EGF . InteresBCR-ABL1 is an oncogene that is formed in a chromosomal translocation between chromosome 9 and chromosome 22. This translocation involves both the BCR and the ABL1 genes and results in the formation of the Philadelphia chromosome . FormatiBCR is a multidomain protein that is widely expressed. It is composed of an amino-terminal dimerization domain, followed by a serine/threonine-protein kinase domain, a pair of SH2 domains, a Rho guanine nucleotide exchange domain, a RAC-GTPase activating domain, and a carboxy-terminal PDZ  domain . The RhoBCR-ABL) or a 210 kDa protein (p210BCR-ABL). p210BCR-ABL includes the Rho-GEF domain that is missing in p190BCR-ABL. Chronic myelogenous leukemia is usually associated with the expression of p210BCR-ABL [BCR-ABL [BCR-ABL or p210BCR-ABL.BCR-ABL1 protein is a constitutively active protein–tyrosine kinase composed of the amino-terminal half of BCR fused to most of the ABL1 protein. Depending on the location of the breakpoint in the BCR gene, the BCR-ABL1 gene produces a 190 kDa protein  was discovered as a mitochondrial protein that can induce various aspects of apoptosis in nuclei . AIF is The STS-1 and STS-2 proteins are involved in the regulation of protein–tyrosine kinases. They use their SH3 domain to bind to proteins that are thought to be involved in stimulating endocytosis and degradation of activated protein–tyrosine kinases, including Cbl, CIN85, and dynamin . Most ofSTS-2, which lacks robust protein phosphatase activity, could use its SH3 domain to sequester negative regulators and prevent them from interacting with active protein–tyrosine kinases. It may even be possible that STS-2 targets some of these negative regulators for degradation. This is likely to result in prolonged protein–tyrosine kinase signaling. While receptor stabilization by STS-2 was shown to require a functional UBA domain in several studies, it remains unclear exactly how the UBA domain contributes to this process. It is possible that the STS-2 UBA domain binds to ubiquitinated receptors thereby preventing other ubiquitin-binding proteins from targeting those receptors for internalization or degradation.Exactly how STS-1 or STS-2 recognize activated protein–tyrosine kinase remains unresolved. This holds true for the interactions with ShcA and AIF as well. Interestingly, these interactions most often involve docking onto tyrosine phosphorylated binding sites. Neither STS-1 nor STS-2 contains a domain that is known to bind in a sequence-specific manner to phosphotyrosine. This suggests that the interaction with these tyrosine phosphorylated proteins may be indirect. Candidate proteins that could mediate these phosphotyrosine-dependent interactions are Cbl and ShcA.The interactions between STS-1 and STS-2 and Cbl are well documented. STS-2 has been shown to use its SH3 domain to bind to the proline-rich region in Cbl. This leaves the Cbl TKB domain to mediate the interaction with tyrosine phosphorylated receptors. However, this model is inconsistent with some of the data showing that the binding of STS-1 to the EGF receptor is independent of Cbl. It remains possible that protein–tyrosine kinase recognition is different for STS-1 as compared to STS-2.A second candidate protein that could mediate the interaction between STS-1 or STS-2 and active protein–tyrosine kinases is ShcA. ShcA has been identified in several studies as a binding partner for STS-1. ShcA also happens to be well-equipped to function as an intermediate between STS-1 or STS-2 and their targets, because it carries a PTB domain as well as an SH2 domain. Both domains mediate phosphotyrosine-dependent protein–protein interactions. Unfortunately, the interaction between ShcA and STS-1 remains poorly characterized.Currently, the function of the STS-1 PGM domain and the function of the STS-2 SH3 domain are reasonably well understood. In contrast, it remains unclear how the UBA domains, the esterase domains, and the STS-2 PGM domain contribute to the regulation of protein–tyrosine kinase signaling. Finally, finding out exactly how STS-1 and STS-2 engage active protein–tyrosine kinases is important and this warrants further investigation.


The physico-functional and proximate parameters were determined using standard protocols. The color analysis showed that germination significantly increased the flour samples’ lightness (L*)  by almost 1.5-fold. Germination resulted in almost 1.1 times higher oil absorption capacity than the control . The foaming capacity of the germinated samples significantly (p = 0.015) increased by 6.4%. Germination significantly reduced the loose bulk density  but significantly increased the tapped bulk density . The germinated samples recorded significantly  lower crude fat, about 1.2 times lower than the raw flour. Germination significantly increased the flour’s total ash  and crude protein  content. Germinated pigeon pea flour will perform better in formulating baked products, aerated foods and food extenders than non-germinated pigeon pea flour. Hence, the germination of pigeon peas should be encouraged because it harnesses the functional and proximate attributes measured.The study investigated the effect of germination on pigeon pea flour’s physico-functional (pH, color Malnutrition or hidden hunger negatively impacts physical and mental growth, the immune system, and general health, limiting one’s ability to achieve their full potential4. Malnutrition affects one out of every three people, and almost every nation in Sub-Saharan Africa (SSA) is dealing with a significant public health problem due to malnutrition5.Childhood malnutrition (undernutrition and overnutrition) is a global problem6. The attributable cause of this poor statistic in rural Ghana is the inability of poor households to afford diversified diets to meet their nutritional needs due to poverty. In addition, the over-dependence on cheap energy-dense, nutrient-poor staples with high levels of anti-nutritional factors also contribute to the burden of hidden hunger.Over the years, Ghana has made marginal gains in reducing malnutrition among pre-schoolers. Nevertheless, malnutrition continues to be a significant  public health concern in rural parts of Ghana. To illustrate, in the Northern region, 29% of children younger than 5 years are moderately or severely stunted (too short for their age.), and 11% are wasting (too thin for their height)7. Legumes, particularly pigeon pea, is rich in protein, minerals, and phytochemicals, making them an essential and inexpensive food staple in the Global South8.The utilization of legumes in food-to-food fortification has been recognized as a sustainable approach to enhance the traditionally nutrient-deficient cereal-based diets, effectively tackling the problem of malnutrition in developing nations9.Despite the nutritional potentials of pigeon pea in addressing hidden hunger, the key limitation for their utilization and consumption in our daily diet include poor digestibility and the presence of anti-nutrients, such as phytic acids, trypsin inhibitors, oligosaccharides and phenolic compounds10 without much effort. Traditional household food processing techniques such as fermentation, germination/malting, soaking and thermal processes have been previously employed to improve the nutritional quality of cereals, legumes/pulses, and root tubers or minimize the levels of anti-nutrients11. The application of such technological processes provokes changes in the physicochemical characteristics of the components9. In particular, germination triggers endogenous enzymes to modify the grain’s constituents13. Germination has been reported to be most promising in this regard, resulting in desirable changes in consistency and nutritional properties11. It is simple, less expensive, culturally acceptable and can be employed at the household level without much demand on time, labor or space15. Germination offers numerous nutritional and organoleptic advantages17 because it helps synthesize vitamin C and converts starch to simple sugar18. There has been some evidence of the enhancement in the availability of proteins20, carbohydrates20, vitamins21 and minerals22 with germination. The reduction of several anti-nutritional factors using germination has also been documented26, while some evidence also suggests a reduction in viscosity by germination27, a desirable attribute for complementary food ingredients.Nevertheless, adequate processing methods can be used to minimize the anti-nutrients and improve the digestibility and bioavailabilityTherefore, this study investigated the effect of germination on the physico-fucntional properties of pigeon pea flour as a functional food ingredient. Also, the nutritional qualities of the germinated pigeon pea flour and total polyphenols and antioxidant activity were considered. The study hypothesized that germination would improve the functional and nutritional properties and decrease the anti-nutrient content of pigeon peas. This will significantly improve their digestibility and nutrient bioavailability and consequently address the issue of malnutrition among pre-schoolers in Ghana and other developing countries. Besides, it will broaden the frontiers of pigeon pea utilization in Ghana.The physico-functional properties of germinated pigeon pea flour are presented in Fig. Germination significantly influenced all the color parameters evaluated. The germinated flour showed significantly (p = 0.009) higher luminosity compared to the raw samples. In contrast, the raw samples had significantly higher a* values denoting redness Table . The b* The germinated flour recorded significantly (p = 0.006) lower moisture content relative to the raw flour samples Fig. A. The crAlthough not significant (p = 0.192), germination resulted in reduced total polyphenols content Fig. . However29.Although the pH of the germinated pigeon pea samples was still acidic, the gradual increase in pH of the samples could be due to the initiation of lactic acid fermentation moving towards alkaline fermentation because of the protein-rich nature of the substrate. Generally, microbial growth and diversity are affected by a myriad of factors including pH, and temperature among others. Alkaline fermentation involves the fermentation of protein-rich raw materials such as legumes and tends to increase the pH of the substrates30 and chickpea protein isolate powder31. A lower bulk density is significant for flours meant for infant foods because samples could be prepared with a small amount of water yet provide the desired energy and nutrient densities and semi-solid consistency. Besides, the packaging requirements of the flour are determined by the bulk density, which is a function of the flour's particle size and moisture content.The loose bulk density, an important functional property of flours, measures the highest attainable compaction without compression. The decrease in bulk density with germination could be attributed to the lowering of heaviness and dispersibility of the flour particles due to the breakdown of complex compounds like starch and protein. Similar findings have been reported in germinated pigeon pea flour32. The enhancement in the foaming capacity of the germinated pigeon pea flour samples, as observed in this study, corresponds to the ability of the protein to provide an interfacial area for foam formation and to stabilize it against gravitational and mechanical stresses30. The findings agree with previous works that reported increased foaming capacity with germination33. The improved foaming capacity in flours could be attributed to the hydrolysis of large peptides into smaller peptides and amino acids by proteolytic enzymes released during germination. Good foam capacity is a desired property for flours intended for use in the production of various baked products such as cakes and cookies. Also, it performs as a functional agent in other food formulations34.The foaming capacity of flour is a measure of the amount of interfacial area created by whipping the flour32. Swelling capacity (index) is regarded as a quality measure in some food products such as bakery products. The high swelling capacity of flour is suitable for making dough with high elasticity as reported earlier35. Therefore, pigeon pea flours with high swelling capacity could be utilised effectively as functional ingredients for pastry products such as bread, pasta, and other viscous foods.The swelling capacity is the measure of the ability of starch to imbibe water and swell and also indicates the extent of associative forces in the starch granules32. The high WAC indicates the usefulness of flour especially in food formulations involving dough due to its ability to absorb water and swell for improved consistency in food. Thus, the higher WAC of the flour samples will enable bakers to add more water to doughs, improving handling and maintaining freshness in product development. One advantage of high-water absorption is its influence on easy softening, and easy, as well as increased digestibility. However, the disadvantage is that it also increases water activity, which is likely to cause food spoilage.Water absorption capacity (WAC) is the amount of water taken up by flour to achieve the desirable consistency and create quality food product36. The oil absorption capacity of flour has been reported to be influenced mainly by the amount of hydrophilic, and lipophilic amino acids and their spatial arrangements30. Thus, the oil absorption capacity is a function of fat, binding the non-polar side chains of protein, and has been affected by the number of hydrophobic sites and protein–lipid–carbohydrate interactions. Increment in the oil absorption capacity after germination has previously been reported in germinated cowpea33, germinated wheat, brown rice and triticale30. Oil absorption capacity is an essential functional property that enhances the flavor and mouthfeel of food since oil is reported to act as a flavor retainer37. Therefore, this property is required in most food applications, such as bakery products, which require flavor retention and improvement of palatability38. In contrast, this flour with high oil absorption capacities, such as germinated pigeon pea as found in this study, may not be ideal for foods that are meant to be fried as the high oil absorption may have some dire economic and storage stability consequences.The trend observed in the oil absorption capacity in this study could be ascribed to the protein quality, its surface hydrophobicity, and its ability to hold fat globules39. The variety of pigeon pea used could account for the discrepancies in the findings. However, the increase in yellowness and chroma with germination could be due to the concentration of carotenoids.Color is one of the most critical quality attributes for determining a food product’s overall quality since it considerably influences the consumer acceptability for their utilization of various products. As found in this study, the increased lightness and decrease in redness with germination disagree with earlier works that reported lightness and increased redness with pigeon pea germination40. In this regard, the proximate composition of germinated pigeon pea flour was compared with ungerminated (raw) pigeon pea flour. The relatively low moisture content of the germinated pigeon pea flour indicates superior keeping quality, as high moisture content predisposes samples to spoilage. This is because increased moisture content can enhance microorganisms’ activity, leading to flour deterioration.The proximate composition  is an essential criterion for determining the nutritional values and quality of food41. The germination process evokes lipolytic enzymes that hydrolyze fat into fatty acid and glycerol and its subsequent oxidation for energy production, hence the reduction in fat into fatty acid and glycerol and its subsequent oxidation for energy production41, reducing the crude fat content in the germinated samples. Besides, the reduction in crude fat content could also be ascribed to the reduction of stored fat due to the high catabolic activities in seeds during germination.The decline in crude fat content of the germinated flour agrees with earlier reports of about a 32% reduction in crude fat content of germinated pigeon pea flour41.The increase in total ash content of the germinated pigeon pea flour samples in this study corroborates earlier findings that showed an almost 6% increase in total ash content of pigeon pea due to germination41 chickpea42, mungbean43 and faba beans44. The rise in crude proteins may be attributed to the synthesis of proteases required for certain amino acids during protein synthesis39.The increase in crude protein content of the germinated pigeon pea flour samples in this study supports other previous studies. For example, an increased crude protein content due to germination was also reported in pigeon pea45. Another study also reported a 2.3% decrease in carbohydrate content after 24 h of germination in cowpea46. The decline in total carbohydrates could be due to the breakdown of complex carbohydrates by alpha-amylase into absorbable sugars that are further used by the growing seedling during the early stages of germination.The decline in total carbohydrates with fermentation is consistent with earlier studies that reported decreased carbohydrate content in mung bean, chickpea and cowpea39. Several studies have reported increased phenolic compounds with germination in grains47. However, in the current study, the solubilization of water-soluble polyphenols that may have leached into the soak water could plausibly explain the decline in total polyphenols content with germination.The slight decline in total polyphenols content in the germinated pigeon pea flour samples in this study could be due to the soaking of the grains in water before the germination process as soaking before germination was found to reduce the total polyphenols content in pigeon pea by nearly 4.6%48. Samples with high free radical scavenging activity refers to flour that contains compounds known as antioxidants, which can help counteract oxidative stress in the body49. Oxidative stress occurs when there is an imbalance between the production of harmful molecules called free radicals and the body’s ability to neutralize them with antioxidants50. Prolonged oxidative stress is associated with various health issues, including non-communicable diseases (NCDs) such as cardiovascular diseases, diabetes, cancer, and neurodegenerative disorders51. Moreover, it has also been reported that vitamins, carotenoids, and other bioactive compounds are also present in different concentration in germinated grains other than polyphenol compounds and phenolic acids, which may function as additional antioxidants and significantly enhances the antioxidant activity of the grains during germination52. This, therefore, could explain the reason for the observed higher antioxidant activity in the germinated samples, although the samples recorded lower total phenolic content.Many previous works have reported a linear relationship between total polyphenols content and antioxidant activity. Thus, samples with higher total polyphenol content possess higher antioxidant activity. However, despite the relatively low total polyphenols content in the germinated pigeon pea flour samples, they showed superior antioxidant activity, using ascorbic acid as an index. It is reported that during germination, the amount of phenolic compounds increases due to the presence of more hydroxyl groups that can provide the required component as free radical scavengers, which enhances the antioxidant activity of germination legumesGermination improved the biochemical composition of pigeon pea grains, which positively influenced the functional properties of flour, such as oil and water binding capacities, foaming capacity and bulk densities. Further, it enhanced the crude protein and total ash contents that contributed to the high nutritional value of the flour. The germinated pigeon pea flour possesses antioxidant potential and could be useful against free radical-induced disorders.Based on the enhanced functional properties, germinated flour may be suitable for preparing innovative bakery products. Besides, the germinated flours could be used in cereal-based diets to address protein deficiency in developing countries. This will help promote the utilization of underutilized pigeon pea by incorporating them for the development of functional food products with high nutritional value and health-promoting bioactive compounds. The current investigation did not involve the creation of tangible food products to assess the applicability of germinated flour in such contexts. This aspect is recognised as a constraint in our study. Nevertheless, though this limitation might influence the breadth and understanding of our results, it also opens avenues for forthcoming research to confront these restrictions and expand upon our study's foundation.The pigeon pea grains were purchased from the open market in the Tamale Metropolis in the Northern region of Ghana. The study of physico-functional and proximate properties was conducted at the Spanish Laboratory complex of the University for Development Studies, Nyankpala campus, Tamale, Ghana.26 as shown in Fig. The grains, 5 kg, were first sorted to make sure all foreign materials and broken grains were removed. The germination of the pigeon pea was done following previous method53 using the pH meter (JENWAY model: 3510).The pH of the pigeon pea flour samples was determined as described by the method of AOAC (2005)54. Briefly, 20 g of the flour samples were put into a 50-ml measuring cylinder. The cylinder was gently tapped on the benchtop 10 times from a height of 5 m from the ground. The bulk density was calculated as the weight per unit volume of the sample and computed as follows:The tapped bulk density was determined according to the method described by Abe-Inge et al.55. Approximately, 2 g of flour sample were weighed and added to 50 ml distilled water in a 100 ml measuring cylinder, The suspension was mixed and properly shaken to foam and the total volume after 30 s was recorded. The percentage increase in volume after 30 s is expressed as foaming capacityFoaming capacity was determined according to the method described elsewhere56 method. Two (2) grammes of the sample were weighed into a measuring cylinder, and the volume was recorded. Ten (10) milliliters of distilled water were then added to each sample and the solution was allowed to stand for 30 min at room temperature (25 °C). The volume of each sample was retaken and recorded. The index of the swelling ability of the sample was calculated using the equation belowThe swelling capacity of the flour samples was determined by a modification of Onwuka57. Two grammes of each flour sample was weighed into a pre-weighed centrifuge tube and 20 ml of distilled water were added. For oil binding, 20 ml sunflower oil was added. Samples were vortexed and allowed to stand for 30 min at room temperature before being centrifuged at 4000 rpm for 25 min. Excess water or oil was decanted by inverting the tubes over absorbent paper and samples were allowed to drain. The weights of water and bound oil samples were determined by difference.Water- and oil absorption capacity of samples were carried out as reported elsewhere by Elkhalifa et al.58 were used to determine the moisture (AOAC 925.10) with some slight modification by drying the samples at 105 °C overnight for approximately 12 h instead of 24 h, crude protein (AOAC 960.52), Ash (923.03) and crude fat (AOAC 922.06). Total carbohydrate was computed by difference.The methods described in the Official Methods of Analysis of the Association of Official Analytical Chemists (AOAC) International59. Briefly, the Chroma meter was first calibrated with a white tile. Each flour sample was poured to fill a petri dish and then covered. The lens of the Chroma meter was placed on the petri dish at three different parts and the color was the taken. L* denotes darkness/lightness , a* (− a = greenness and + a = redness), and b*  as earlier described60. In brief, 1 ml of each sample extract was transferred into a 25 ml volumetric flask containing 2.5 ml of 3.54 g/l Iron (III) chloridehexahydrate solution. The volumetric flask containing the sample solution was then placed in a water bath and maintained at 80 ˚C for 20 min. After this, 2.5 ml of acetate buffer solution pH (4.6), 5.0 ml of 3.28 g/l 1,10-phenanthrolinehydrate  and 2.5 ml of 3.72 g/l ethylene diaminetetraaceticaciddihydrate (EDTA) solutions were added, respectively. Finally, each flask was added. Finally, each flask was filled to the mark with distilled water, cooled and then the absorbance measurements were made at 511 nm.In this study 100% methanol was used as solvent to extract the polyphenols in the pigeon pea flour samples. The total polyphenols content of the fermented flour samples was determined using the method developed in a previous study61. Approximately, 4 ml (0.004% w/v) of DPPH solution was mixed with 1.0 ml of extract (in methanol). The reaction mixture was vortex mixed thoroughly and incubated at room temperature in the dark for 30 min. Reduction in the absorbance of the mixture was measured at 517 nm using ascorbic acid as a control. Scavenging of DPPH radicals by the extract was calculated using the following formula:The DPPH-free radical scavenging activity of the samples was determined according to previous workData on physico-functional, proximate and total phenolic content was analyzed using two-sample t-test in Minitab. The antioxidant activity of the samples was also analyzed using a one-way Analysis of Variance (ANOVA) to compare the means of the germinated flour, raw flours and the standard (ascorbic acid). The Fisher’s Least Significant Difference test was used to compare differences between means when the ANOVA result was significant (p < 0.05) .Supplementary Information.


This collection of traits changes predictably through time, regardless of flour type, highlighting patterns of bacterial constraints and dynamics that are conserved across systems and scales. Yet, while sourdough climax communities exhibit similar markers of maturity , Pediococcus (mean abundance 22.7%), and/or Gluconobacter (mean abundance 19.1%)), we also detected specific taxa and aromas associated with each type of flour. Our results address important ecological questions about the relationship between community structure and starter performance, and may enable bakers to deliberately select for specific sourdough starter and bread characteristics.The microbial fermentation behind sourdough bread is among our oldest technologies, yet there are many opportunities for sourdough science to learn from traditional bakers. We analyzed 16S rRNA sequences in R to assess the bacterial community structure and performance of 40 starters grown from 10 types of flour over 14 days, and identified six distinct stages of succession. At each stage, bacterial taxa correlate with determinants of bread quality including pH, rise, and aromatic profile. Day 1 starter cultures were dominated by microorganisms commonly associated with plants and flour, and by aromas similar to toasted grain/cereal. Bacterial diversity peaked from days 2–6 as taxa shifted from opportunistic/generalist bacteria associated with flour inputs, toward specialized climax bacterial communities (days 10–14) characterized by acid-tolerant taxa and fruity ( Bread has played a crucial role in human civilizations and cultures  for at lhttp://studentsdiscover.org/lesson/sourdough-for-science/). In ecology, ideal model systems tend to be relatively low in diversity , easily manipulated, and amenable to detailed experiments on particular species pairs  and acid-tolerant yeasts, which metabolize sugars to produce carbon dioxide and aromatic compounds , starter performance , and the number of days it took for starters to reach maturity .From the perspective of bakers, sourdough starters have reached a “mature” climax community when pH and rise have stabilized and the starter can be reliably used to make bread, in 7 to 14 days . If we ae.g., the ability to produce or tolerate acids). Finally, we predict that community differences throughout succession should manifest functional differences among the starters, specifically their aromas.https://www.kingarthurbaking.com/recipes/sourdough-starter-recipe), utilize locally available ingredients (flour and distilled water) and easily accessible equipment, and are scaled to generate minimal waste. The following activities were completed for evaluation purposes only and as a result, the North Carolina State University IRB determined that they were not regulated by 45 CFR 46 and IRB approval was not required. No personal information was used to contribute to generalizable knowledge.All sourdough production methods used in this research were developed to mimic artisanal and home bakers’ practices. As such, our protocols are based on online recipes (Triticum aestivum), red turkey wheat (Triticum aestivum), rye , emmer (Triticum dicoccon), and einkorn (Triticum monococcum)—and five gluten-free flours—teff (Eragostis tef), millet (Eleusine coracana), sorghum (Sorghum spp), buckwheat (Fagopyrum esculentum), and amaranth (Aramanthus caudatus). Cereal grains are small, hard seeds without a fruit layer, grown as crops and harvested to be dried and either fed to livestock; boiled and consumed as a porridge or pilaf; or milled into flour for baking. While most cereals are grasses , the seeds of other plant families  are also described as “whole grains”. The grains used in this project represent four glutenous and five gluten free cereal lineages that together span more than 527 million years across the angiosperm phylogeny. The all-purpose, red turkey wheat, emmer, einkorn, rye, and millet flours were milled at Boulted Bread Bakery , while the teff, sorghum, buckwheat, and amaranth flours were purchased from commercial vendors online.Five researchers grew a total of 40 starters from 10 different flour types: four replicates each of five flours containing gluten—unbleached all-purpose flour . This point of maximum height can be identified by the residue left on the side of the jar, which resembles a “high tide” mark in cases where an active starter has subsequently deflated by the time of measurement. The height measurements provide a proxy for quantifying carbon dioxide production by each starter community, and changes in community behavior  over time. While sourdough starters grown from different flours may have different gas holding capacities, measuring change in height over time offers insight to gas production for starters grown from the same flour type via baseline comparison.In studying sourdough starters and other traditional fermentations, there is a tradeoff between using approaches that best match standard laboratory protocol and those that best match actual baking practices. Because we aim to generate results that can be directly applied by bakers, we focused on an approach that is in line with standard baker/bakery practices. We have illustrated our protocol in i.e., earthy, fermented, fruit, grain/cereal, sour, toasted, and other). While sensory assessments may provide subjective interpretation of bread volatiles  of the starter using free association, without the aid of a standardized word bank. Each researcher recorded the aromas produced by every individual starter, each day (1–14). We performed text analysis to classify individual descriptors into super-categories . Next, we removed one tablespoon of sourdough (the discard). and measured the pH using short-range  Hydrion Brilliant Paper . Finally, we added one tablespoon fresh flour and one tablespoon water to the remaining starter and mixed thoroughly with the spoon. These measuring and refreshing steps were repeated once a day  for 14 days. Conventional wisdom holds that a sourdough starter is mature and ready for baking after 14 feedings, although some bakers and instructions for growing a starter from scratch suggest that the starter is mature after only 10 days. After completing data collection, we also baked breads from the starters we grew for this project for an informal tasting, both to celebrate the completion of the project and to verify that all starters successfully leavened bread . We used Kruskal-Wallis and Pairwise Wilcoxon rank-sum tests with Bonferroni correction to identify significant differences in pH and height associated with time  and flour type. We focus on days 1, 2, and 3 to determine the impact of flour type on early succession dynamics and the potential importance of priority effects on community membership and starter performance at maturity. We include day 6 to represent intermediate succession, and days 10 and 14 to acknowledge two common lengths of time by which bakers assume that starters to have reached maturity. We binned aromas by category  and quantified (presence/absence) whether each aroma category was detected during each sensory assessment).We graphed the pH and height of each starter using ggplot2 in R  to identn = 30) and three 1-mL samples of distilled water to represent the resource inputs to each starter on day zero. We also collected a daily 1-mL aliquot from each starter after measuring pH, as part of the discard. All aliquots were stored at −20 °C. After analyzing the pH and height data from all starters to identify functionally significant stages in succession, we elected to sequence starter samples collected on days 1, 2, 3, 6, 10, and 14 (n = 240). Together with the flour and water aliquots, a total of 273 samples were shipped on dry ice for DNA extraction, PCR amplification, and Illumina multiplexed sequencing of the bacterial 16S v4 region on the MiSeq platform, using the 515f (AATGATACGGCGACCACCGAGATCTACACGCT TATGGTAATT GT GTGCCAGCMGCCGCGGTAA) and 806r (CAAGCAGAAGACGGCATACGAGAT XXXXXXXXXXXX AGTCAGTCAG CC GGACTACHVGGGTWTCTAAT) primers for bacteria at the Fierer Microbial Community Sequencing Lab  as previously described , and the data and code are available in the Dryad database (https://doi.org/10.5061/dryad.bk3j9kdh3).We collected three 1-mL samples of each flour type (escribed . PCR neghttps://github.com/fiererlab/dada2_fiererlab) and performed previously by Lactobacillus species using the “lactotax” tool (http://lactotax.embl.de/wuyts/lactotax/) developed by Lactobacillus but without species designations, we used BLAST to determine the new genus designations. First we cross-referenced each of the hits that met the 99% threshold with the new taxonomy. If all of the hits for a specific ASV matched a single genus, we assigned that new genus. If the BLAST results did not agree on a single genus, the ASV was assigned taxonomy only at the family level. We then created two datasets for downstream analyses on alpha and beta diversity:We compared all water, flour, and day 1 starters to assess the influence of inputs on early starter communities. This data set was un-rarefied.We removed all water and flour samples to compare changes in starter community composition from day 1–14. This dataset was rarefied to 1,200 bacterial reads per sample and the ASV table was converted to percent relative abundances for downstream analysis. We retained 232 starter samples for downstream analysis.The Fierer Microbial Community Sequencing Lab demultiplexed and processed raw sequences to produce exact sequence variant (ASV) tables with the DADA2 pipeline, as described at (https://github.com/leffj/mctoolsr/) to visualize all bacterial genera present at ≥0.1% relative abundance for each time point or flour type, and aromas detected in at least 25% (10/40) of starter samples.We first plotted heatmaps and produced nonmetric multidimensional scaling (NMDS) plots with Bray-Curtis distance calculations using the metaMDS function to compare the bacterial contents of day 1 starters to flour and water inputs. We next turned our attention to focus on the rarefied starter dataset. We calculated alpha diversity as ASV richness and Simpson diversity. We used Kruskal-Wallis tests to measure the effects of days elapsed on alpha diversity and on Lactobacillaceae ASV abundance. As a non-parametric method, the Kruskal-Wallis test is appropriate for comparing microbial membership and other data that do not follow normal distribution . We calcThe raw dataset included 1,219,763 forward reads and 1,215,774 reverse reads, with 665,156 joined reads remaining after quality control.p < 2.2e−16) and starter height  (p < 3.8e−09) while height increased . pH continued to fall on day 3, , after which starters stabilized at a pH of 3.6; we detected no significant changes in pH throughout the following days of the experiment . By contrast, height continued to rise significantly through day 6  and day 10 . By day 14, both pH and height were maintained, suggesting that communities were functionally stable.We identified six functionally distinct succession stages across flour types based on starter pH  . Notably2.2e−16)  and simi2.2e−16)  across fPantoea and an unknown genus within Enterobacteriaceae dominated both flour  and day 1 starter samples ; and Pseudomonas was present at similar relative abundance in water (13%), flour (10.1%), and day 1 starters (12%)  . These srs (12%) , despiters (12%)  and S2D.rs (12%)  and 3B, rs (12%)  and pH srs (12%) . Bacterirs (12%) .p < 2.2e−16); Erwinia , Pseudomonas , Klebsiella , Pantoea , Enterobacteriaceae unkn , Weissella , Leuconostoc , Latilactobacillus , Levilactobacillus , Gluconobacter , and Pediococcus . Erwinia (11.5% on day 1), Pseudomonas (12.8–18.2%), Klebsiella (11.2–13.2%), Pantoea (21.3% on day 1), and Enterobacteriaceae unkn (16.3–28.5%) occurred at higher relative abundance in younger starters (≦3 days old) than in older starters (≧10 days old)  (The observed changes in sourdough starter performance correlated significantly with (and are likely driven by) predictable shifts in the composition of bacterial communities. For example, pH decreased significantly as Lactobacillaceae grew more abundant   and S5A.ays old)  and S5A.Pediococcus (79.2%) and also contained Leuconostoc, but lacked genera belonging to the family Lactobacillaceae; and amaranth starters were dominated by Latilactobacillus  in sourdough starters grown from 10 different flour types. Given that the generation times of  an hour , this pe an hour . As succe.g., fruit, sour, and fermented aromas).Community membership at each successional stage comprised bacteria with specific functional traits: generalists and plant endophytes gave way to lactic acid producing genera, which were outcompeted by a subset of acid producing genera that are also acid-tolerant. Our results are entirely in line with the three-phase model of succession elaborated through the study of wheat-based sourdough . In the http://robdunnlab.com/projects/science-of-sourdough/). For example, many starter-keepers note a dramatic increase in height on day 2 or 3, followed by a plateau or decrease in activity until roughly day 7. We verify this widely noted behavioral pattern across flour types  by day 10 across flour types . The retions in . Why migtions in , making Gluconobacter, Pediococcus, and other genera of Lactobacillaceae were most abundant in mature starters. The ubiquity of the genus Gluconobacter, which accounted for 12.6–45.8% of the microbial communities present in 7 of 10 flour types studied  that are present and the aromas associated with those communities (Pantoea is much more abundant in mature buckwheat starters (12.2%) than in other starters (≦3.5%). Acid-tolerant Leuconostoc is enriched in teff (18.1%), and an unknown genus of Enterobacteriaceae is enriched in rye starters (7%). Other starters were characterized by the absence of particular taxa: for example, Gluconobacter is present in all starters except teff and amaranth , suggesting that they may compete for resources. This is especially apparent in teff  and amaranth  starters. e.g., alcohols, esters, carbonyl compounds  and viawith LAB .i.e., the same cereal grown in different fields or years) could yield additional insights to the variation within vs among cereals.Many of the bacteria we detected are novel compared to previous studies of the same flour types . These de.g., sourdough starter, sauerkraut, and kimchi) take only 2 weeks to develop stable climax communities. Chinese sauerkraut  and disus casei .i.e., the “10-day rule”). Yet, while the general patterns of succession hold across starters, bacterial community composition and aromas in mature starters differed significantly by flour type and are likely driven by grain-specific bacterial and nutritional components, suggesting that bakers (like brewers) can manipulate inputs to produce specific bread characteristics. To this point, we call for more thorough investigations into the sensory qualities of sourdough starters grown from different flours, and of the breads baked from those starters. A comparison of the aromas detected by a trained sensory panel vs the general public  would be particularly helpful to validate citizen science and other broad applications.Together, our results confirm that bacterial communities in sourdough starters follow predictable patterns of succession driven by resource availability and relative niche breadth over time. Changes in community composition coincided with changes in pH as well as height and aromas, offering empirical support for common baking practices  Heat map comparing the average relative abundance (%) of bacterial taxa detected in each type of flour, compared to the day 1 starters grown from that flour type.(A) Bar chart comparing average relative abundance (%) of bacterial taxa detected in water, flour, and day 1 starters. (B) Bray-Curtis NMDS plot comparing all water, flour, and day 1 starter samples. Water samples cluster separately. Overlap between flour and Day 1 samples is minimal and driven by a few flour samples. (C) Bray-Curtis NMDS plot comparing all flour inputs Click here for additional data file.10.7717/peerj.16163/supp-6Supplemental Information 6Values are averaged by flour type on days 1, 2, 3, 6, 10, and 14.Click here for additional data file.10.7717/peerj.16163/supp-7Supplemental Information 7Starters were sampled at 1, 2, 3, 6, 10, and 14 days elapsed and grown from all purpose, amaranth, buckwheat, einkorn, emmer, millet, rye, sorghum, teff, and turkey wheat flour. Pearson correlation = −0.5321391.Click here for additional data file.10.7717/peerj.16163/supp-8Supplemental Information 8p < 0.05.(A) Bacterial genera and (B) aromas that were identified as significantly different at different time points, across flour types. Significance determined based on Kruskal-Wallis tests after Bonferroni correction, Click here for additional data file.10.7717/peerj.16163/supp-9Supplemental Information 9Each sample is labeled by flour type .Click here for additional data file.10.7717/peerj.16163/supp-10Supplemental Information 10p < 0.05.Significance determined based on Kruskal-Wallis tests after Bonferroni correction, Click here for additional data file.