The GEB5P bread showed the highest increase in extractable and bound trans-ferulic acid content, by 320% and 137%, respectively, compared to the control bread. Principal component analysis showed differences between the control bread and enriched breads in terms of their quality, sensory and nutritional properties. Breads with 2.5% and 5% “germinated + fermented” spelt flour had the most acceptable rheological, technological and sensory characteristics, in addition to a substantial improvement in their antioxidant content.In the present study, the partial substitution of common white wheat flour for a bread recipe with variously bioprocessed wholegrain spelt was investigated. The addition of 1% and pasteurised 5% “germinated + enzymatic treated” spelt flour to wheat flour significantly improved the specific volume of the bread, but their texture profile analysis and sensory evaluation were not satisfactory. A higher percentage of added bioprocessed spelt flour darkened the colour of the bread. Breads with the addition of more than 5% of bioprocessed spelt flour were unacceptable in terms of quality and sensory parameters. The highest extractable and bound individual phenolics were found in breads with 5% “germinated + fermented” spelt flour (GFB5) and 5% pasteurised “germinated + enzymatic treated” spelt flour (GEB5P). A strong positive correlation was determined between  Triticum aestivum L.) is an important crop in most of the world, accounting for between 20% and 50% of the total intake of calories. Furthermore, wheat flour is most commonly used for bread-making because of its gluten protein fraction, which permits the retention of CO2 in the viscoelastic dough matrix [Triticum spelta L.) [Wheat (h matrix . An attrelta L.) . In geneelta L.) ,4. To soTriticum seeds, most phenolics are covalently bound to structural components of the cell wall  [p-coumaric, caffeic and p-hydroxybenzoic acids [Saccharomyces cerevisiae was based on the optimisation experiments previously performed on raw, germinated, and enzymatically-treated spelt seeds [Lactobacillus plantarum), alcoholic (S. cerevisiae) and spontaneous fermentation individually and in combination and concluded that the highest extractable and bound individual phenolic contents and antioxidant activities of the extracts were obtained in germinated spelt seeds fermented with S. cerevisiae [Epidemiological studies have linked the consumption of wholegrain products with health benefits in relation to the development of chronic diseases . These eroteins) . Accordiic acids . Severalic acids . In our ic acids ,11,12. Ilt seeds . We testrevisiae . Previourevisiae ,14,15,16revisiae . Similarrevisiae  reportedrevisiae . Furtherrevisiae  showed trevisiae .The aims of this study were: (i) to investigate the potential of using bioprocessed spelt flour in a wheat bread recipe to improve the quality and sensory characteristics; and (ii) to evaluate the effects of added bioprocessed spelt flour on the total phenolic content (TPC), phenolic profile and antioxidant activity of enriched wheat bread.The farinographic parameters are presented in The extensiographic properties of the doughs are presented in The quality characteristics of bread samples were evaluated 3 h after baking and are presented in In terms of bread crust colour, the L* value (lightness) of the control was significantly higher than that of the bioprocessed breads. With a higher percentage of bioprocessed spelt flour in the bread recipe, the lightness of the bread’s crust decreased , Table 2The change in crumb firmness was measured only in three bread samples ; measurements of other samples could not be obtained because the crumbs were too sticky. The supplement of 5% wheat flour with “germinated + fermented” spelt resulted in a 111% increase in firmness, while flour replacement of 2.5% had no statistically significant impact on it. The wet sticky crumbs of other bread samples can be explained by the excessive hydrolysis of starch, proteins and arabinoxylan, resulting in the release of an excess of water previously bound to these polymers. The increase in enzyme activity was not only a consequence of the presence of microorganisms or the addition of exogenous enzymes but was also caused by the activation of endogenous enzymes during the germination of spelt seeds. Excessive darkening of the bread crust and stickiness of the dough were associated with increased activity of α-amylases and xylanases. It is known that the profound degradation of arabinoxylan by exogenous xylanases and the release of water previously bound to arabinoxylan can lead to very poor dough manageability . FurtherSensory analysis was carried out three hours after baking . OverallThe addition of 5% and 2.5% “germinated + fermented” spelt flour was characterised by a slightly more distinct smell and taste of spelt, as well as a crust taste of cheese and caramel. GFB2.5 and GFB5 had quite good porosity, uniform pores in the crumb, a fairly elastic-flexible crumb and a slightly sticky crumb, but this gives a feeling of freshness. The colour of the crumb was a little darker but still typical of bread. These two breads were pleasant in the mouth, not too sticky, and were preferred among all the bioprocessed breads. In general, the assessors preferred breads enriched with up to 5% “germinated + fermented” spelt flour.Bioprocessing did not improve the texture score of the breads. All sensory scores decreased when enzymatic treatment was combined with germination of spelt seeds, leading to the rejection of these breads in terms of appearance, flavour, texture, overall liking and, consequently, purchase intention. Although enzymes have become a promising strategy in baking positively to modify the rheological properties of dough without affecting sensory acceptance, in the present study, the addition of 5% of bioprocessed spelt flour already impacted the characterisation and acceptance of the bread samples, meanwhile breads with 30% substitution of white flour were completely inadequate for sensory assessment. On the other hand, the addition of 2.5% and 5% of “germinated + fermented” spelt flour resulted in bread with acceptable technological characteristics and sensory profile. All sensory properties decreased when the “germinated + enzymatic treated” spelt flour was added to the bread recipe. This is in agreement with the study of de Almeida et al. , who repFurthermore, de Almeida et al.  reportedIn our study, the TPC and antioxidant activity were determined in the five breads with the most promising characteristics. The results for TPC and antioxidant activity assays of extractable and bound phenolics are shown in The highest increase in bound TPC was also found in GFB5 (24%) and GEB5P (20%) breads compared to the control bread. The increase in bound TPC using “germinated + enzymatic treated” spelt flour was due to the fragmentation of arabinoxylans, which became more soluble and easily accessible during extraction. With respect to the increase of bound TPC, it is relevant to mention that both enzymatic and yeast treatment of germinated spelt flour affects the distribution of phenolics, which may have undergone polymerisation or conjugation reactions. The total (extractable + bound) TPC was thus significantly increased, from 15% in GEB1 to 27% in GFB5 bread compared to the control bread. A considerably higher proportion of bound TPC was observed for all bioprocessed and control bread (more than 90%). Interestingly, despite a 5-fold increase in the added “germinated + enzymatic treated” spelt flour (from 1% to 5%), no increase in bound TPC was achieved. In contrast, with a 2-fold increase to the added “germinated + fermented” spelt flour (from 2.5% to 5%), an increase was achieved, although it was minimal. It seems that the increase in extractable and bound TPCs did not depend on the type of bioprocessed spelt flour but rather on the percentage of bioprocessed flour added and, in the case of “germinated + enzymatic treated” spelt flour, on the pasteurisation.The increased TPC in bioprocessed breads was expected since bioprocessed flour contains significantly higher extractable and bound TPC than white wheat flour. Specifically, the “germinated + fermented” and “germinated + enzymatic treated” spelt flours contain 157% and 109% higher total (extractable + bound) TPC, respectively, compared to raw spelt flour . Cornejo• scavenging activity assay was at 0.054 mg TE/g DW for extractable TPC and at 0.209 mg TE/g DW for bound TPC (The obtained results indicate that the antioxidant activity of control bread determined by the DPPHound TPC . The higCoefficients of correlation (r) were calculated to explain the relationship between TPCs and the antioxidant activity of extractable and bound fractions. The extractable and bound fractions showed a positive and strong correlation between TPC and antioxidant activity . These results indicate that phenolics strongly contributed to the antioxidant activity of breads.The increase in antioxidant activity observed in our research in bioprocessed breads is consistent with research reports that indicate that the antioxidant activity (measured by various assays) increases with the addition of bioprocessed flour. Ktenioudaki et al.  reportedtrans-ferulic acid, by 320% and 137%, respectively, compared to the control bread. Interestingly, the total (extractable + bound) ferulic acid content in “germinated + fermented” spelt flour was higher than in “germinated + enzymatic treated” spelt flour by approx. 9% [trans-ferulic acid (extractable + bound) in breads was up to 5-fold, 34-fold, 142-fold and 15-fold higher than that of total cis-ferulic, p-coumaric, caffeic and p-hydroxybenzoic acids, respectively.The effect of adding bioprocessed spelt flour to breads on individual extractable and bound phenolics was evaluated with HPLC-MS . Five phprox. 9% , but whecis-Ferulic acid. Similar to trans-ferulic acid, the highest increase in extractable (250%) and bound (45%) cis-ferulic acid content was observed in GEB5P bread compared to the control bread. The type and percentage of added bioprocessed spelt flour had a greater influence on the extractable cis-ferulic acid content than on the bound content.The second most abundant phenolic in our analysed breads was p-coumaric acid content was observed in bread enriched with 5% “germinated + fermented” spelt flour, by 42% compared to the control bread. The difference between the content of extractable p-coumaric acid in breads enriched with 5% “germinated + fermented” spelt flour and 5% pasteurised “germinated + enzymatic treated” spelt flour was 13%, but the “germinated + fermented” spelt flour had a 24% higher total content of p-coumaric acid than “germinated + enzymatic treated” spelt flour [p-coumaric acid content was much higher, from 66% in GEB1 bread to 449% in GEB5P bread, compared to the control.The highest increase in extractable lt flour ,12. The p-hydroxybenzoic acids, the highest increase was observed in GFB5 bread, by 100% and 159%, respectively, compared to the control bread. This may be because of the content of total caffeic acid in the “germinated + fermented” spelt flour, which was 142% higher than in the “germinated + enzymatic treated” flour. Furthermore, the content of total p-hydroxybenzoic acid in “germinated + fermented” spelt flour was 27% higher than in “germinated + enzymatic treated” flour [p-hydroxybenzoic acid was observed in GEB5P bread compared to the control, while there was no statistical difference between GEB5P and GFB5 breads regarding the content of bound caffeic acid.For extractable caffeic and d” flour . The higWhen compared to the control, the highest relative increase in extractable apigenin hexoside pentoside I (89%), II (125%) and III (37%) was observed in GEB5P bread, while the preparation of GFB5 was equally relevant for the contents of bound apigenin hexoside pentosides I (165%), II (482%) and III (72%). On the other hand, the addition of “germinated + fermented” spelt flour to the bread recipe had a negative impact on the content of extractable unknown C-glycosyl derivative compared to the control bread, while the addition of 5% “germinated + fermented” spelt flour showed the highest increase (713%) in bound C-glycosyl derivative compared to the control.p-coumaric acid. They also reported that the free ferulic acid content increased by 69–93% in all bioprocessed bran breads compared to native bran bread. Furthermore, Anson et al. [p-coumaric and gallic acids. In the bound extracts of the bioprocessed breads, they found only ferulic and p-coumaric acids, and the concentrations averaged 387 and 95.5 μg/g DW bread, respectively. Koistinen et al. [p-coumaric acid.Interestingly, Amaya Villalva et al.  reportedn et al.  found thn et al.  found thn et al.  showed tIt is important that with the addition of bioprocessed flour, we increase the content of phenolics in the bread, especially in extractable form, because the bioaccessibility of phenolic acids is mainly associated with the amount of extractable phenolic acids present in bread, and if we want bread to have a positive effect on our health, the phenolics must be bioaccessible. The increase in extractable phenolic acids using yeast fermentation or enzymatic treatment was due to the action of hydrolytic enzymes.trans-ferulic, cis-ferulic and p-coumaric acids are the main contributors to the TPC and antioxidant activity of the extractable fraction. The main contributors for predicting bound TPC and antioxidant activity are trans-ferulic, p-coumaric and caffeic acid. Among phenolics analysed, trans-ferulic acid had the greatest influence on the extractable and bound TPC  and DPPH• radical scavenging activity . Bound cis-ferulic acid showed a lower correlation with bound TPC and DPPH• radical scavenging activity .We suggest that •, trans-ferulic acid, cis-ferulic acid, p-coumaric acid, caffeic acid, p-hydroxybenzoic acid, L* and a* values, crust appearance, sensory overall quality, crust and crumb colour. Then PC2, which explained 32.5% of the variance, separated the bread samples based on specific volume, b* value, crust crispiness, crumb porosity, elasticity, stickiness, odour and taste. Projecting the variables onto the plot showed that the control bread was defined particularly by its lightness (L*), crust appearance, sensory overall quality, elasticity and crumb porosity. On the other hand, the control bread had the lowest content of phenolic antioxidants. According to the PCA plot, there was a significant difference in total TPC and individual phenolic acids content between the control bread and bioprocessed breads, with the percentage of added bioprocessed flour having the greatest influence on the content of phenolics and their antioxidant activity against the DPPH• radical.We performed PCA for further analysis of the obtained data, based on a correlation matrix, using the bread quality and sensory parameters, as well as the results of the total (extractable + bound) TPC and individual phenolic acids and antioxidant activity. In the PCA, as shown in the two-dimensional plot in Plotting the data in a two-dimensional coordinate system  showed t• radicals and had a darker crust and crumb colour and higher redness.The PC1 separated breads enriched with 5% bioprocessed spelt flour from the control bread and breads enriched with a lower percentage (1% and 2.5%) of bioprocessed spelt flour. Breads with 5% bioprocessed spelt flour had a higher total (extractable + bound) TPC and individual phenolic acids content and higher antioxidant activity against DPPHTriticum spelta L. cv. Ostro) seeds were purchased from a Slovenian producer and were stored in the dark at 1 °C. Absolute methanol, formic acid, sodium hydroxide and sodium carbonate were purchased from Merck . DPPH•  reagent, Folin–Ciocalteu reagent, Trolox, p-hydroxybenzoic acid, trans-ferulic acid, p-coumaric acid, caffeic acid, α-amylase (EC 232-560-9) from Bacillus amyloliquefaciens (enzyme activity 250 U/g) and protease (EC 232-752-2) from Aspergillus oryzae (enzyme activity 500 U/g) were from Sigma-Aldrich . Cellulase (EC 3.2.1.4) from Talaromyces emersonii (enzyme activity 70 U/mg), xylanase (EC 3.2.1.8) from Aspergillus niger (enzyme activity 79 U/mg) and feruloyl esterase (EC 3.1.1.73) from rumen microorganism (enzyme activity 30 U/mg) were from Megazyme . All the chemicals were of analytical quality. For the preparation of the solution, ultrapure water  was used. For bread production, white wheat flour type 500 was obtained from Žito d.o.o. Quality parameters of wheat flour: dry matter: 854.9 g/kg; water: 14.52%; ash: 0.535% on a dry basis; protein content: 13.6%; wet gluten: 31.4%; dry gluten: 10.6%; gluten index (GI): 89; enzyme activity : 366 s.Dehulled spelt (Triticum spelta L. cv. Ostro) occurred under abiotic stress specified as darkness, at 25 °C for 144 h, with the addition of 25 mM NaCl after 48 h and 50 mM sorbitol after 96 h of germination according to Mencin et al. [® Works, Staufen, Germany) (particle size < 0.25 mm). The lyophilized germinated spelt flour was then fermented for 72 h at 30 °C under static conditions in a Roto-Therm incubated rotator , a sample-to-saline ratio of 1:1.5 (10 g:15 mL) and the addition of 0.75 mL of Saccharomyces cerevisiae inoculum (3% (v/v) inoculum) at an initial optical density of 1.6, corresponding to 2 × 108 cfu/mL (hereinafter referred to as “germinated + fermented” flour) [Germination of spelt seeds  . The bioThe bread was made according to a white wheat bread recipe that served as a control bread and was made from 1000 g of wheat flour, 600 mL of water, 7.5 g of instant baker’s yeast, 19 g of salt and 25 mL of sunflower oil. The ingredients were mixed, and the dough was then fermented for 30 min. The dough was then divided and transferred to a proofing chamber, where the dough fermented again for 45 min at 30 °C and 85% relative humidity. Baking of the bread started with a dosage of 0.5 L of 180 °C hot steam, which was automatically dosed in the oven . The baking regime was as follows: 5 min at 230 °C, 20 min at 190 °C and 4 min at 200 °C. The bread was cooled for 3 h at room temperature. Depending on the type of bioprocessed bread to be produced, the commercial wheat flour was replaced by an appropriate proportion of bioprocessed spelt flour, taking into account a correction of the dry matter content. All other ingredients in the recipe and bread-making protocol were the same as for the control bread. Different bioprocessed breads were prepared as described in Bread quality characterisation and sensory evaluation were performed on all breads. With pasteurisation, we wanted to increase the percentage of added bioprocessed flour to the bread recipe while maintaining the quality of the bread. The selection for analysis of the phenolic content and antioxidant activity of breads included a control bread and four bioprocessed breads with the best quality and sensory properties .The effect of adding 2.5% and 5% “germinated + fermented” spelt flour to wheat flour compared to the control flour (wheat flour) was investigated since these two breads had acceptable sensory quality. Farinographic and extensiographic properties were examined using a Farinograph and Extensiograph  according to the AACC 54-21 and AACC 54-10.01 methods, respectively . The farBread quality characterisation was performed after 3 h of bread cooling. The bread weights were taken, together with bread volumes measured using the rapeseed displacement method (AACC method 10-05.01). Specific volume was calculated as the ratio between the bread volume (mL) and weight (g).The crust colour of the breads was measured with a Minolta Chroma Meter CR-400 . The instrumental colour parameters (five replicates on the same bread) of lightness (L*), redness (a*), and yellowness (b*) were determined. The total colour change (ΔE) between the control and breads enriched with bioprocessed spelt flour was calculated by the following formula: +Δa2+Δb2 .Crumb firmness was determined by a texture analyser  with a 36 mm diameter compression plate. For the texture profile analysis, the samples were compressed to 40% (10 mm) of their original thickness at a crosshead speed of 10 mm/s. Two slices taken from the centre of every bread sample, each 2.5 cm thick, were placed under an aluminium compression plate, and the force required for penetration was measured . Three mThe sensory evaluation was carried out on bread samples with the different percentages of bioprocessed wholegrain spelt flour 3 h after baking. The slice of each type of bread (2 cm thick) was numbered and served to each panellist under normal (daylight) illumination, and water was provided for rinsing between samples. The panel consisted of five trained assessors (aged between 31 and 55 years) who evaluated the bread’s overall acceptability . In the sensory analysis of the bread, the external appearance of the bread was evaluated first . A slice of bread was then cut and the appearance of the crumb , elasticity and stickiness of the crumb, crispness of the crust, odour and taste were evaluated. The individual parameters were scored as the intensity of the respective parameter compared to the reference sample (control bread). The score of all parameters for the reference sample was 0. Scores ranged from −3 to +3 points, and a positive score did not necessarily mean a positive description of a single parameter, just a more intensively expressed characteristic. If necessary, the panel supplemented the numerical score with a description of the specific properties.g for 10 min at 10 °C and filtered (pore size 0.45 µm). The methanol was evaporated off in a vacuum evaporator  at 40 °C, and the residues were resuspended in water. These filtered supernatants contained extractable phenolics. After methanol extraction, the remaining solid residues were then hydrolysed with 20 mL of 2 M sodium hydroxide and shaken in the dark for 4 h at room temperature. The hydrolysed samples were acidified to pH 3 with the addition of concentrated formic acid. These filtrated hydrolysates contained bound phenolics.The extraction of extractable and bound phenolic compounds was carried out as previously described by Mencin et al. . ShortlyIn addition, solid phase extraction (SPE) was used to purify the extracts and concentrate the phenolics and was performed according to Mencin et al. . Firstlyw/v) was added. After 1 h, the absorbance was measured in a 1 cm cuvette at 765 nm on a UV–visible spectrophotometer . Final data were expressed as mg Trolox equivalents per g dry weight (mg TE/g DW).The TPCs in bread were evaluated using the Folin–Ciocalteu assay as described by Mencin et al. . Brieflyp-coumaric, trans-ferulic, caffeic and p-hydroxybenzoic acids and gallocatechin were calculated according to peak areas, as compared to calibration curves with the corresponding standard. Concentrations were expressed as µg per g bread DW (µg/g DW). One peak was tentatively identified as the cis-isomer of ferulic acid because data from HPLC-MS analysis showed mass spectra that were nearly identical to those of trans-ferulic acid, so it was quantified using the calibration curve of trans-ferulic acid. For compounds identified without standards, quantification was performed using similar compounds as standards. Apigenin hexoside pentoside  and an unknown C-glycosyl derivative were quantified according to p-coumaric acid.Reversed-phase HPLC-MS/MS analysis was used to quantify the individual phenolics (extractable and bound) in the bread samples. The HPLC system  equipped with an UV detector set at 280 nm and 310 nm was used. Chromatographic separation was carried out using a C18 column . All of the individual phenolic compounds were identified using a mass spectrometer , with electrospray ionisation, operating in negative ionisation mode. Identification of phenolics was confirmed by comparisons of their UV-VIS spectra and MS spectra and retention times with external standards, followed by fragmentation, as fully described in the study of Mencin et al. . Concent• radical was determined as described in our previous publication [• solution in 99.9% methanol was added to the appropriate diluted bread extracts. After 1 h, the decrease in absorbance was measured at 520 nm using a UV–visible spectrophotometer . Methanol (99%) was used as a blank. The reference solution consisted of 0.2 mM DPPH• solution and 99% methanol at a ratio of 1:1 (v:v). The DPPH• radical scavenging activity  was expressed as mg TE/g DW.Antioxidant activity against the DPPHlication . Brieflyp < 0.05. Principal component analysis (PCA) was applied using OriginPro 2015 to analyse the contribution of all studied parameters to the overall quality of the different bread samples.All of the analyses were carried out in two parallel runs on two separate extractions. The results are presented as the mean ± standard deviation (SD). Statistical assessment was performed with the SPSS programme for Windows (version 22). The results were analysed using one-way ANOVA. The significance of the differences between the means was determined using Duncan’s post-hoc test with the significant difference at In conclusion, breads with up to 5% of “germinated + fermented” spelt flour showed potential for further research due to their improved nutritional value with still acceptable technological and sensory characteristics. Breads GFB30, GEB30 and GEB10P were unacceptable in terms of quality parameters due to the excessive activity of enzymes present in the bioprocessed flour. In GEB1 and GEB5P breads, there was a significant improvement in specific volume. A higher percentage of added bioprocessed spelt flour darkened the colour of the bread.• radicals was also observed in GEB5P and GFB5 breads for extractable and bound fractions. The control bread had the lowest content of extractable and bound TPCs and, consequently, showed the lowest antioxidant activity. The predominant phenolic acid identified in the breads was trans-Ferulic acid, and more than 98% of total trans-ferulic acid was present in bound form. The GEB5P bread showed the highest increase in the content of extractable and bound trans-ferulic acid. We can assume that trans-ferulic and p-coumaric acids are the main contributors to the TPC and antioxidant activity of extractable and bound fractions.The highest content of extractable and bound TPCs was observed in GEB5P and GFB5 breads, and the greatest ability to scavenge DPPHThe incorporation of bioprocessed spelt into wheat flour affected the rheological behaviour of the blends. Flour replacement (from 2.5 to 5%) by “germinated + fermented” spelt slightly lowered the water absorption capability and worsened the dough handling properties.Evaluation of the bread-making process in terms of quality, sensory and especially nutritional properties confirmed that the addition of 2.5% or 5% “germinated + fermented” spelt flour is a promising and interesting ingredient for the formulation of bakery products, avoiding the use of enzymatic improvers and having a positive impact on consumer acceptance and facilitating the adoption of a “clean label”. It is crucial that consumers are informed about the presence of health-promoting substances in bioprocessed breads since this may improve the bread’s acceptability and, consequently, market potential. Our results indicate that the addition of bioprocessed flour significantly increases the phenolic content and antioxidant activity of bread. However, it is also important to determine whether these antioxidants are truly bioaccessible in the bioprocessed breads and, consequently, bioavailable for utilisation so that they can have beneficial health effects.


Talin , is a cytoskeletal adapter protein that binds integrin receptors and F-actin, and is a key factor in the formation and regulation of integrin-dependent cell–matrix adhesions. Talin forms the mechanical link between the cytoplasmic domain of integrins and the actin cytoskeleton. Through this linkage, talin is at the origin of mechanosignaling occurring at the plasma membrane–cytoskeleton interface. Despite its central position, talin is not able to fulfill its tasks alone, but requires help from kindlin and paxillin to detect and transform the mechanical tension along the integrin–talin–F-actin axis into intracellular signaling. The talin head forms a classical FERM domain, which is required to bind and regulate the conformation of the integrin receptor, as well as to induce intracellular force sensing. The FERM domain allows the strategic positioning of protein–protein and protein–lipid interfaces, including the membrane-binding and integrin affinity-regulating F1 loop, as well as the interaction with lipid-anchored Rap1  GTPase. Here, we summarize the structural and regulatory features of talin and explain how it regulates cell adhesion and force transmission, as well as intracellular signaling at integrin-containing cell–matrix attachment sites. Summary: Talin is a key protein controlling integrin-dependent cell–matrix adhesions. Here, we summarize its function in linking mechanical coupling to the actin cytoskeleton and adhesion signaling. Talin , was first reported as P235, and identified in human platelets as a dimeric, globular and highly abundant protein (3–8%) with a molecular mass of 235 kDa . PurifieAnother means by which talin regulates integrins was found to be through their α- and β-subunit association. Integrin mutations at the juxtamembrane region led to spontaneous activation of the αIIbβ3 integrin (platelet receptor) . This prTherefore, new biochemical assays were developed to study the direct interaction between β-integrin tails and different cytoplasmic adapters .Overall, it became clear that integrins are regulated by an interplay of competition and interaction between many cytoplasmic adapters; among these, talin stood out because of its integrin-activating function .2+-induced integrin clustering in cells plated on immobilized integrin ligands allowed the definition of a role for acidic phospholipids, such as phosphatidylinositolP2 (PIP2) in talin-mediated integrin activation, clustering and cell adhesion   . With thas PTK2) . On the as PTK2) . On the as PTK2) . Howeveras PTK2) .6VGU) (3IVF) . Recently, we presented the first talin head structure that demonstrated the ability of the talin head to fold into this FERM cloverleaf, together with a possible explanation for why earlier structures folded into an extended, linear structure instead. In the remaining part of the review, we will name the FERM-folded talin head ‘compact’ (PDB 6VGU) , in cont) (3IVF) . We willTalin exists as two highly similar isoforms , with differences in expression pattern and functions in specific adhesion types that are not yet fully elucidated . In thisThus, talin is a bona fide FERM domain protein sharing multiple features with other FERM family proteins, including binding sites for lipids, peptides and F-actin. The latter function is shared with ezrin, radixin and moesin, which exhibit a C-terminal F-actin-binding domain that can be activated from the autoinhibited FERM-bound configuration by a phosphorylation switch B,C.FERM-domain-containing proteins often bind to juxtamembrane domains of single-pass cell surface receptors, such as CD44, ICAM-2  or β-intLipid-binding orients FERM domain proteins on the plasma membrane C. These In addition to the major peptide-binding site in the F3 subdomain, an additional binding interface is present in the second β-strand in the F1 subdomain (see for example radixin in 6VGU), not all the FERM–bundle interfaces in talin might have been identified  in the context of the isolated F1 subdomain, the loop is entirely flexible, with a tendency to form a membrane-interacting helix . HoweverThis lysine-rich turn in the talin F3 domain is relevant for integrin activation. In comparison, the integrin-binding FERM domain of myosin-X lacks such a lysine-rich turn in F3, and thus can position integrins at filopodia, but fails to activate them, probably due to missing lipid and membrane-proximal integrin interactions .1MK7; 6VGU; Significant efforts have been made to determine the structure of talin and how it binds to integrins through its FERM-containing head domain . The C-t1MK7, 3G9W and 4F7G; 1MK7, 3IVF and 6U4K) , or the F1 subdomain (PDB 3G9W), the F3 subdomain twists towards the F2 subdomain and forms an atypical interactions from Glu269 and Tyr270 to Lys345 (6VGU), it is so far not clear whether the F3 subdomain can twist even more, as for example seen in the kindlin structure  , reveal K) 6VGU,  (Fig. 4Eo Lys345 . Owing ttructure . The exiThe C-terminal tail of integrins interact with the FERM domain proteins talin and kindlin, but also with adapter proteins exhibiting a classical PTB domain, such as Dok1, Dab-2 or tensin family proteins . SwitchiThe close association (clasp; in On the one hand, the basic motif at the C-terminal end of the F3 helix in talin is critical for the proper folding of its NPXY ligand-binding pocket. On the other hand, the basic motif-mediated link to the F1 domain prevents the movement of the C-terminal helix towards F3-bound integrin peptides, thereby avoiding an ‘induced-fit’ conformation, seen with classical PTB-domain-containing proteins . In analA hallmark of integrin-dependent formation of adhesions and subsequent signaling involves the recruitment of paxillin to nascent cell–matrix adhesions at the cell periphery. Paxilin has been shown to be recruited to focal adhesions upon tension and to have a role in mechanosignaling , and is Further research is required to dissect tension-dependent paxillin recruitment to talin-containing focal adhesions and to distinguish the mechanosensitive recruitment mediated by LIM domains from regulatory interactions between N-terminal paxillin LD motifs and focal adhesion proteins, such as talin, vinculin and FAK .The talin–integrin link is found in almost all vertebrate cells and thus covers a large tensional spectrum, ranging from very soft brain tissue to highly contractile myofibroblasts. Under tension, the talin rod bundles unfold, making formerly cryptic binding sites available for vinculin recruitment in a stress-dependent manner . When coDespite its critical role in the dynamic force adaptation of the talin rod bundles, the direct interaction between F-actin and talin is not that well understood. Recent studies suggest that the talin–F-actin interaction behaves like a catch-bond with a distinct directional preference . F-actinIn contrast to the R13 bundle, the second F-actin-binding site in talin R4 is not accessible under low tension. Apparently, this F-actin-binding site is exposed upon deletion of R2 and R3, mechanical destabilization of R3 or tension-mediated vinculin binding to exposed R3 helices, which all result in an enlargement of focal adhesions . Thus, tHow the creation of a second, force-bearing F-actin linkage in R4 affects adapter binding of the distal segment of the talin tail needs to be further explored.Talin is a highly complex integrin adapter protein that is modulated by membrane interactions, post-translational modifications and tension-mediated mechanical responses. Given that conformational activation of talin is highly regulated, the sequence of events leading to binding of integrin receptors, association with co-adapters such as kindlin  and its


Implementation researchers often combine the Theoretical Domain Framework (TDF) and Consolidated Framework for Implementation Research (CFIR) in their studies. However there is some debate on the merits of using multiple frameworks—whether they contribute to results or provide superfluous analysis. Our recent research combined the TDF and CFIR to identify determinants to widespread incorporation of patient held medication lists (PHML) in healthcare practice. The aim of this report is to provide guidance on the use of the TDF and CFIR; by assessing the degree of overlap between the two frameworks in their application to interviews about PHML.Semi-structured telephone interviews were conducted with healthcare professionals (HCPs) and non HCPs (people taking multiple medicines and caregivers).Interview data were transcribed and analysed using the TDF and CFIR. Within paired domains substantial intersection/overlap across constructs and domains within the two frameworks was classified as > 75% of coding references, consistent intersection/overlap was defined as > 50% and ≤ 75%, average intersection/overlap was defined as ≤ 50% and > 25% and non-substantial intersection/overlap was classified as ≤ 25% of coding references.Interview data were collected from 39 participants – 21 HCPs and 18 non HCPs.Environmental context & resources, Beliefs about capabilities, Beliefs about consequences, Social influences, Behavioural regulation and Social/professional role & identity; and five CFIR domains: Intervention Characteristics, Outer Setting, Inner Setting, Characteristics of Individual and Process. A pattern of substantial intersection/overlap in coding emerged with broad TDF domains such as Environmental context & resources often linked to well-defined CFIR domains and constructs . Broad CFIR constructs such as knowledge & beliefs about intervention within Characteristics of Individuals also linked to more descriptive TDF domains like Beliefs about capabilities. In addition there was some unexpected non-substantial intersection/overlap in coding with the TDF domain Social influences less frequently linked to the CFIR Inner Setting domain and constructs such as networks and communications.Mapping of TDF domains to CFIR domains/constructs identified key determinants in six TDF domains: Identifying intersections/overlaps in coding between CFIR and TDF can assist interpretation of findings in implementation research. The strengths of each framework were exploited in a reciprocal process which provided more information to broad/poorly defined domains and enabled identification of implementation determinants and innovation determinants.The online version contains supplementary material available at 10.1186/s43058-023-00466-8. Contributions to the literatureIt is unclear for many researchers if using multiple frameworks has advantages beyond the application of a single framework.Our findings contribute to the goal of data collection within implementation science—to predict outcomes based on setting-level and recipient-level characteristics.Combining the TDF and CFIR facilitated identification of implementation and innovation determinants.Numerous frameworks have been developed by implementation scientists to guide the implementation of new practices or changing exiting practices in real-world settings. Using theoretical frameworks can generate information on mechanisms of change that can be targeted in interventions . The TheIt has been suggested that combining the TDF and CFIR can assist studies by addressing distinct and multiple conceptual levels – system and individual – and process factors , 5. A reThe Irish Health Service Executive (HSE) National Quality Improvement (NQI) team are devising a national medication safety campaign – the ‘Know Check Ask’ . The purMethods are described in detail elsewhere . In brieDetails of the thematic analysis conducted based on the Framework approach, with TDF 12 domain) and CFIR informing the analysis framework have been described previously . In summ domain a. The frequency and proportion of coding references that intersected/overlapped was established for each individual CFIR construct and domain and each TDF domain using cross-tabulation. Substantial intersection/overlap across constructs and domains within the two frameworks was classified as > 75% of coding references, consistent intersection/overlap was defined as > 50% and ≤ 75%, average intersection/overlap was defined as ≤ 50% and > 25% and non-substantial intersection/overlap was classified as ≤ 25%. The Standards for reporting qualitative research (SRQR) guidelines were adhered to throughout this study [The TDF and CFIR constructs and domains were examined to establish common patterns in coding across all the constructs and domains. Interview text that was coded to both a TDF domain and a CFIR construct and domain was identified and classified as an intersection/overlapis study .The interview data about views on PHML included 39 participants – 21 HCPs and 18 non HCPs (patients and carers) – 74% (29/39) were females, median age was 45 years for HCPs (IQR = 37–48), 55 years (IQR = 49–61) for non-HCPs and average number of years of professional practice for HCPs was 18.37 years (SD ± 10.59). ; Outer Setting (OS); Inner Setting (IS); Characteristics of Individual (CI); and Process (P). All 39 interviews contained instances of intersection/overlaps; where the same interview text was linked to multiple TDF and CFIR constructs and domains. See Figs. Overall key determinants likely to influence use of PHML were previously identified in six key TDF domains: In total 382 (81%) coding references within the CFIR domain IC substantially intersected/overlapped with the six key TDF domains.  and seven associated constructs – ‘Intervention Source’, ‘Cost’, External policy and Incentives’; ‘Compatibility’, ‘Tension for Change’, ‘Relative Priority’ and ‘Individual Stage of Change’. These constructs each contributed ≤ 5% of total coding coverage within the paired TDF and CFIR domains and constructs  coding references within the TDF domain Beliefs about capabilities, 201 (61%) of total coding references consistently intersected/overlapped with the five CFIR domains. This TDF domain substantially intersected/overlapped with ‘Patient Needs and Resources’ within the CFIR OS domain and ‘Planning’ within the P domain. It also consistently intersected/overlapped with ‘Knowledge and Beliefs’ in the CFIR CI domain. Beliefs about capabilities did not code with six individual constructs within the IS domain and there was also non-substantial intersection/overlap between this TDF domain and twelve associated constructs. Three CFIR constructs—‘Identification with Organisation’, ‘Self-efficacy’ and ‘Other Personal Attributes’—in the CI domain and ‘Intervention Source’ within the IC domain each contributed ≤ 7% of total coding coverage.For the TDF domain Beliefs about consequences 422 (80%) of total coding references substantially intersected/overlapped with the five CFIR domains. There was substantial intersections/overlaps between this TDF domain and ‘Patient Needs and Resources’ in the CFIR OS domain. It also consistently intersected/overlapped with two individual CFIR constructs—‘Knowledge and Beliefs’ and ‘Planning’ in the CI and P domains respectively. There were non-substantial intersections/overlaps in coding with twenty associated constructs with six of these constructs each contributing ≤ 3% of total coding coverage.For the TDF domain Social influences consistently intersected/overlapped with the five CFIR domains. This TDF domain substantially intersected/overlapped with ‘Patient Needs and Resources’ within the OS domain and ‘Engaging’ within the P domain. It also consistently intersected/overlapped with ‘Knowledge and Beliefs’ in the CI domain. Social influences did not code with seven individual constructs within the CFIR domains and there was also non-substantial intersection/overlap between this TDF domain and ten associated constructs.In total 80 (61%) coding references within the TDF domain Behavioural regulation 250 (61%) coding references consistently intersected/overlapped with the five CFIR domains. There was substantial intersections/overlaps with ‘Patient Needs and Resources’ in the OS domain and consistent intersections/overlaps with ‘Knowledge and Beliefs’ and ‘Planning’ in the CI and P domains respectively. Behavioural regulation did not code with three constructs—‘Cost’ (IC domain), ‘Tension for Change’ (IS domain) or ‘Identification with Organisation’ (CI domain).Within the TDF domain Social/professional role and identity 266 (73%), consistently intersected/overlapped with the five CFIR domains. There was substantial intersections/overlaps in coding with ‘Patient Needs and Resources’ in the OS domain and consistent intersections/overlaps with ‘Engaging’ in the P domain. Social/professional role and identity did not code to ‘Intervention Source’ or ‘Relative Priority’ in the IC and IS domains respectively. There was non-substantial intersection/overlapping with eighteen CFIR constructs with three of these constructs each contributing ≤ 9% of total coding coverage within the paired TDF and CFIR domains and constructs .  were linked to more elaborate/descriptive TDF domains such as Beliefs about capabilities. Our findings suggest that the frameworks were often complimentary; combining the TDF and CFIR can lead to additional information on key determinants such as individual characteristics, context, resources and result in increased targets for implementation efforts [This study found it beneficial to combine the TDF and CFIR to address the limitations of the two frameworks identified by previous research such as broad or poorly defined domains, variation in which domains/constructs are used, gaps between determinants and outcomes , 5, 14.  efforts , 15.Social/professional role & identity and some CFIR constructs associated with organisational receptivity to intervention. This pattern of non-substantial intersection/overlapping in coding may indicate that organisational factors could exist in a hierarchy which mediates perceptions and influences HCPs to varying degrees. Prioritising constructs with substantial intersections/overlaps in coding, such as ‘Implementation climate’ when selecting targets for interventions could prove to be beneficial [Implementation research has suggested that overarching frameworks like the TDF can be augmented with broader frameworks such as the CFIR which can address contextual issues/barriers e.g. practical access to resources . In thisneficial .In general the frameworks were complementary however some non-substantial intersections/overlaps between TDF and CFIR emerged in the area of social support. It could be anticipated that TDF domains related to social support/norms would frequently map to equivalent CFIR domains and constructs associated with structured support at an organisational level such as ‘Networks and Communications’, ‘Culture’, ‘Peer Pressure’ and ‘Champions’. However non-substantial coding between the frameworks in this area could indicate that TDF provides more precise coding of social support than CFIR and that CFIR may identify structured social influences but possibly capture less information about social networks. Using the TDF ensures that the impact of social relationships at the individual level; which may vary across cultures and/or ethnicities; are thoroughly investigated , 18.Combining the TDF and CFIR had numerous benefits; increasing the depth and breadth of relevant information through complementary processes that provide useful information to broad/poorly defined domains. However, it should be noted that applying both frameworks to interview data had implications for project management and timelines. The iterative process of double-coding required considerable expenditure of research time and resources.A limitation of this study was the possibility of bias – selection and/or researcher. However attempts were made to address these issues with purposive sampling of participants; to achieve a range of clinical experience and health conditions. Methodological strategies were also employed to reduce researcher bias; mapping decisions were independently made and reviewed to ensure robust alignment of mappings . SequentRecent CFIR research has added outcomes to the CFIR framework, assessing implementation (setting-level) and innovation (recipient-level) determinants to explain/predict outcomes . ImplemeOur findings should be viewed as an initial attempt to systematically record intersections/overlaps in coding with the CFIR and TDF and clarify the value of their combined use in identifying key determinants. Identifying patterns can increase interpretation of findings and create a more robust picture of the implementation environment. This mapping will need continued refinement and validation to establish the benefits of combining these frameworks in implementation research. Future research could examine a greater number of cases to verify coding patterns across the TDF and CFIR.Additional file 1. CFIR & TDF analysis: subthemes, themes & illustrative quotes.Additional file 2. Total coding references & classification within key TDF domains & CFIR domains.Additional file 3. Frequency mapping of key TDF domains to key CFIR domains & constructs.


The activation energy of the deformation which decreased from 465 kJ mol−1 to 315 kJ mol−1 by the electric current indicated that the lattice diffusion of Zr cation during the deformation was enhanced. And the deformation rate of 3Y-TZP with the electric current may be controlled by the grain boundary diffusion of Zr cation.The aim of this paper is to investigate the mechanism of an electric current-assisted superplastic deformation on 3Y-TZP in an oxygen-lean atmosphere. The experiments were performed with different electric currents in the range of 0~5 A. The results show that the flow stress of 3Y-TZP during the deformation was significantly decreased by the combination of Joule heating and the applied current effect. The microstructures of the deformed specimens were all equiaxed grains without an obvious preferential grain growth. The stress exponent  Yttria-stabilized tetragonal zirconia 3Y-TZP) is generally considered a suitable candidate material in the aircraft engine and aerospace fields as a result of their excellent properties ,2,3. WhaY-TZP is ng (GBS) ,5,6,7. H−4 s−1, which would make it difficult to achieve the industrial production of structural ceramics.Recently, the electric field, which could affect the diffusion transport in ceramics in a significant way, was used for improving the superplastic deformation of the structural ceramics as a result of the low strain rate during deformation ,13,14,15−1) at low furnace temperatures (1000 °C). They believed that the applied electric field on yttria-stabilized zirconia may accelerate the diffusional transport during the sintering. Yoshida et al. [2 under flash sintering conditions and discovered that the superplasticity in ZrO2 can only be improved by the electric current within a certain range. They believed that the decreased flow stress by the electric current was attributed to a combination of the current-induced increase in diffusion and Joule heating. Meanwhile, Luo and coworkers [2-Al2O3-spinel composite ceramics can be achieved by using spark plasma sintering (SPS) equipment at a furnace temperature of 1100 °C. They believed that this high-speed superplastic forming was made possible by taking advantage of the enhanced diffusional transport in the SPS chamber environment. Furthermore, Dong and Chen [2 + 5%H2 atmosphere by an electric current. They pointed out that this remarkable grain growth was related to the enhancement of cation diffusion in an oxygen-lean atmosphere. Therefore, an oxygen-lean atmosphere will inevitably affect the deformation behavior and mechanism of the electric field/current-assisted superplastic deformation on ZrO2.More recently, Raj and coworkers ,16,17 rea et al. ,18,19 stoworkers  discoveroworkers  also disand Chen ,23 reporA is a constant, n is termed the stress exponent, the parameter p is the grain size exponents, and the Q, R, and T are the activation energy, gas constant, and absolute temperature, respectively.It is well-known that the deformation behavior and the corresponding mechanism for structure ceramics can be identified by the following constitutive equation:2 polycrystals and suggested that the mechanism for superplastic deformation was grain boundary sliding, which was characterized by a stress exponent of 2. In addition, Jimenen-Melendo et al. [Wakai, F. et al. ,24,25 ino et al. ,27 studio et al. , and theo et al. . They foo et al. ,9,10 ind2O3 stabilized tetragonal ZrO2 ceramics (3Y-TZP) in an oxygen-lean atmosphere. For this purpose, the flow behavior of 3Y-TZP was examined at different initial strain rates and temperatures.Therefore, the present work aims to investigate the mechanisms of the electric current-assisted plastic deformation on 3 mol% YThe starting materials used in this paper were powders of TZ-3Y, with a particle size of 27 nm . The specimens for the deformation were sintered by using these powders at 1200 °C, a 30 MPa applied pressure, and at a 1 h holding time in a Hot Press Sintering Furnace. The relative density of the as-sintered specimen was 99.3%, which was measured by the Archimedes method. The geometry of the as-sintered 3Y-TZP for the compression deformation was cylindrical, with 10 mm in diameter and 10 mm in height. The SEM micrographs of the as-sintered 3Y-TZP specimen are shown in −1 until it reached the set temperature. In order to guarantee a uniform temperature, the specimens were kept at the deformation temperature for 10 min before the deformation. The experiment was performed at different electric currents: 1 A, 3 A, and 5 A. The corresponding initial current densities through the specimen were 12.7 mA/mm2, 38.2 mA/mm2, and 63.7 mA/mm2. The temperature of the furnace and specimen were both measured and recorded by the deformation device. For each applied electric current, the immediate height xH and the load P were recorded by adjusting the punch rates at 0.1, 0.2, and 0.4 mm·min−1, respectively. The initial end face area A0 and the initial height H0 of the cylindrical specimen were directly measured. The true stress The deformation experiment was operated by self-designed equipment. In order to ensure the electric current only passed though the specimen, a special graphite with an alumina tube wan was designed. The upper/lower graphite indenter (electrode) was connected with a voltage-stabilized DC power supply device by using electric wires. The schematic diagram of the apparatus used for the experiment is shown in The specimens were polished by a diamond paste of 1.5 −4 s−1 at a furnace temperature of 1200 °C. And the flow stress for the specimen at 1300 °C and 1400 °C were 105.44 MPa and 48.52 MPa, respectively. By contrast, the flow stress of deformation decreased sharply as the electric current was applied. The flow stress of the specimen with the electric current of 5 A at 1200 °C, 1300 °C, and 1400 °C were 63.59 MPa, 30.72 MPa, and 18.25 MPa, respectively. The flow stress decreased from approximately 75 MPa to 30 MPa. In addition, with the electric current increased, the flow stress decreased significantly and the apparent strain hardening was eliminated  ,32,33. Aiffusion ,35. Furtn = 2 indicates that the high-temperature deformation of ceramic material was usually controlled by grain boundary sliding and the microstructure of the deformation sample was equiaxed grains. In the present study, the flow stress–strain rate relationship of n = 2~3 was consistent with the reported plastic flow relationship of fine-grained 3Y-TZP materials at high temperatures [The deformation mechanism is usually characterized by the stress exponent n and its microstructure. And the stress exponents (n) are obtained by the deformation experiments with the as-sintered grain size at the various initial strain rates. The flow stress as a function of the strain rate under different electric currents in log–log plots are shown in eratures ,26,27,37eratures , which weratures  have demn (from 2.61 to 2.05) suggests that the limitation of grain boundary sliding during the deformation was decreased. Recently, Charalambous et al. [2O3-stabilized ZrO2. Some recent reports [It is known that stress concentration will be inevitably created at the boundary ledges or multiple grain junctions during grain boundary sliding, which will cause a significant increase in the flow stress with the strain during the deformation of 3Y-TZP (strain hardening phenomenon) . As shows et al.  found th reports ,40 have Q was calculated from the slope lines. The apparent activation energies of 0 A, 1 A, 3 A, and 5 A were 465, 362, 339, and 315 kJ mol−1, respectively. It can be seen clearly that there was a significant decrease in the activation energy as the electric current was applied. And the apparent activation energy decreased as the electric current increased. Generally, the apparent activation energy is interpreted as the energy for the Zr cation diffusion of the high-temperature plastic flow in 3Y-TZP [2O3-stabilized ZrO2, worked on by Chen et al. [2O3-stabilized ZrO2 showed that an electric field of 500~1000 V·cm−1 could induce the formation of cation vacancy and increase the cation vacancy concentrate at the grain boundaries, which enhanced the diffusivities of cations and anions [Q during the deformation was attributed to the point defects generated by the electric current.The relationship between the temperatures and strain rates are shown in n 3Y-TZP . In thisn 3Y-TZP ,33,38,42n et al. , indicatd anions . Therefo−1 and 500 kJ mol−1, respectively. The activation energy for the deformation of 3Y-TZP without an electric current (Q = 465 kJ mol−1), which was close to the activation energy for the lattice diffusion of Zr cation, indicated that the deformation rate of 3Y-TZP was controlled by the lattice diffusion of Zr cation. The decrease in the apparent activation energy by the applied electric current indicated that the lattice diffusion of Zr cation was enhanced. And the deformation rate of 3Y-TZP with the electric current may be controlled by the grain boundary diffusion of Zr cation.In addition, cation diffusion is usually regarded as the rate-controlling step for the superplastic deformation of Y-TZP . Some stThe superplastic deformation of 3Y-TZP was improved by the electric current in an oxygen-lean atmosphere. With the electric current increased, the flow stress during deformation had a significant decrease. And this decreased flow stress resulted from a combined effect of Joule heating and the enhancement of the Zr cation diffusion due to the electric current. The relative contribution of Joule heating indicated that Joule heat had a more significant effect on decreasing the flow stress in a deformation with 5 A.n = 2.05~2.61, which suggested that the dominant deformation was grain boundary sliding.Significant grain growth was observed in the deformed specimen with the electric current. And the microstructures of the deformed specimens were all equiaxed grains without an obvious preferential grain growth. The stress exponents n of the deformation with different electric currents at 1400 °C were determined to be −1, which was significantly lower than without the electric current (465 kJ·mol−1). This decreased apparent activation energy indicated that the cation diffusion was accelerated during the deformation by the electric current, which may result in a transition of the rate-controlling step for the deformation from lattice diffusion to grain boundary diffusion.The apparent activation energies for the deformation with different electric currents were evaluated to be about 315~365 kJ·molThe compression deformation of 3Y-TZP ceramics with different electric currents in an oxygen-lean atmosphere was investigated. The results are summarized as follows:


Theobroma grandiflorum) pulp and flour on fermented milk beverages’ physicochemical, microbial, and sensory properties during refrigerated storage . Twelve formulations were realized with different percentages of cupuassu pulp  and flour . The treatments with 3% cupuassu flour presented the highest percentages of protein, fat, fiber, and carbohydrates, compared with the samples containing pulp. On the other hand, the addition of pulp increased water retention capacity and color parameters  and decreased pH and syneresis on day 0 of storage. During storage, the samples with pulp showed increases in pH values, consistency index, and apparent viscosity. In comparison, cupuassu flour addition decreased syneresis values and increased L* and b* during storage, as did pulp. In addition, sample HPHF (10% pulp and 3% cupuassu flour), based on just-about-right, penalty, and check-all-that-apply analyses, improved some sensory attributes of the fermented milk beverage, such as brown color, acid taste, bitter taste, cupuassu flavor, and firm texture. It can be concluded that cupuassu pulp and flour addition improves the physicochemical and sensory quality of fermented milk beverages and can provide nutritional value to the product.The use of fruits and their by-products in food has dramatically impacted the food industry due to the nutritional benefits and the technological and sensory effects of food matrices. Therefore, this research aimed to evaluate the effects of adding cupuassu ( Some ofv/v . In thisv/v ,5,6,7. TTheobroma grandiflorum) has been used in recent years for this purpose [Moreover, fruit preparations, starches, pectins, xylooligosaccharides, and flours are reported to improve milk beverages’ sensory acceptability and physicochemical and rheological properties ,9,10. Fo purpose ,12,13. I purpose ,15,16. L purpose ,13 due t purpose  and anti purpose . TherefoGAE/gDM), flavonoids (5.92 mgCA/gDM), epicatechin (20.74 mg/100 gDM), and antioxidant activity in ABTS and DPPH  [Cupuassu flour is a by-product of the fruit obtained from the fermentation, roasting, and grinding of seeds (up to 17% of the fruit) . This flctively) ,19. ThusThe use of cupuassu pulp in different food products has been employed to improve the sensory properties of some dairy products, such as goat milk yogurt ,20, and Besides their potential nutritional, sensory, and techno-functional benefits, cupuassu pulp and flour are not widely used in developing new products. The primary hypothesis of this work is that cupuassu pulp and flour addition enhances the nutritional, sensory, and techno-functional quality of fermented milk beverages. Therefore, this work aimed to evaluate the effect of cupuassu pulp and flour on the physicochemical, microbial, and sensory properties of a fermented milk beverage prepared with milk and whey during 28 days at 4 °C.3 and a freezing point of −0.44 ± 0.02 °C. The cupuassu pulp used was 100% natural, containing no water, sugar, nor preservatives, and was purchased at the Oliva Distribuidora store in Salvador de Bahia. The flour was packaged and distributed by C2 Alimentos and was purchased at local markets in São Paulo (Brazil).Raw cow milk was obtained from the Experimental Farm of Entre Rios of Federal University of Bahia (UFBA). The sweet whey (pH 6.29 ± 0.02) obtained from the processing of fresh cheese and used in this study had the following proximal composition : dry mat®, Fego Alimentos LTDA, Goiânia, Brazil) composed of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus were added. The mixture of ingredients was fermented in a water bath  at a constant temperature (42 ± 1 °C for 5 h) until it reached a pH of 4.6. After interrupting the fermentation process, cupuassu pulp (water 30% and pulp 70%) and/or flour were added to the fermented milk beverages at different concentrations according to Costa et al. [Artisanal fermented milk beverages were prepared as described by Costa et al.  with moda et al.  and Jovaa et al. , dependiThe proximate composition of the fermented milk beverages was analyzed on day 0. Moisture content was evaluated using the oven drying method; ash content, by ashing at 550 °C; protein content, with the Kjeldahl method, using 6.38 as a factor; and fat content, using soxhlet AOAC , where tpH, syneresis, water-holding capacity, and instrumental color were evaluated every seven days during storage .pH was determined with a bench pH meter for aqueous solutions . Before use, the electrode was calibrated with buffer solutions with pH 4.0, 7.0, and 10.0.g for 30 min at 10 °C, and the whey was separated. Syneresis (%) was calculated as the weight of generated supernatant (whey) per weight of fermented milk beverage multiplied by 100. In contrast, WHC was given as the percent weight of drained gel (precipitate) relative to the original weight of fermented milk beverages [Syneresis and water-holding capacity were evaluated using an Eppendorf laboratory centrifuge, model 5702R , according to Ladjvardi  with slieverages .L*; 100 = white and 0 = black), redness , and yellowness  of the fermented milk beverages were recorded with a Chroma Meter CR-5 colorimeter  according to Costa et al. [The values of lightness , hue angle (h°), and total color difference (ΔE*) were calculated based on the analyzed color coordinates (Equations (1)–(3)). In addition, ΔE was calculated by matching the spectrum of the freshly prepared fermented milk beverage (day 0) and its relative spectrum on subsequent storage days according to Lucas et al. [Chroma , γ is the shear rate (s−1), and n is the flow behavior index (dimensionless). This model was used to evaluate the pseudoplastic behavior of each dairy beverage sample.Rheological measurements were performed using a rheometer  with concentric cylinders coupled to a water bath for temperature control (25 °C). The flow curves of samples were determined at speeds between 0.56 and 243 rpm; their corresponding shear rates (γ) and shear stresses (σ) were computed from relations given by the instrument’s manufacturer and then recorded. The experimental data were fitted to the Ostwald–de Waele model  as in Eq−1, as in Equation (5).The rheological data were adapted to the Ostwald–de Waele model  to obtaiStaphylococcus, molds and yeasts, Salmonella spp., total and thermotolerant coliforms, and Escherichia coli on days 1 and 28 of storage according to the American Public Health Association [Staphylococcus was performed on Baird Parker agar  after incubation at 35–37 ± 1 °C for 48 h. Mold and yeast counts were determined according to growth on Dichloran Rose Bengal Chloramphenicol Base (DRBC) agar and incubated aerobically at 25 ± 1 °C for 3–5 days. The colonies were expressed as log colony-forming units (CFU) per mL. For the analysis of Salmonella spp., samples were pre-enriched with 1% buffered peptone water  and incubated at 35 ± 2 °C for 24 h, followed by selective enrichment in Tetrathionate broth  and Rappaport Vassiliadis broth (RV-Micromed Isofar)  at 42 ± 2 °C for 24 h. Afterwards, aliquots were streaked on Xylose Lysine Deoxycholate agar , Hektoen enteric agar , and Bright Green agar  and incubated at 35 ± 2 °C for 24 h.All samples were analyzed for coagulase-positive ociation . BrieflyEscherichia coli were performed following the most probable number (NMP) technique with Lauryl Sulfate Tryptose (LST) broth , followed by streaking on Eosine Methylene Blue Agar (EMB) for E. coli identification.Finally, the analyses of total and thermotolerant coliforms and For the sensory analysis, six treatments  were used. These artisanal treatments were selected aiming to measure the sensory effect generated by cupuassu flour in combination with pulp on fermented beverages, based on previous studies reported by Costa et al. ,20, who Sensory analysis was conducted by the ethical norms for research on humans  and after approval by the Ethics Committee of School of Nursing of Federal University of Bahia . The adherence of individuals to work was ensured with the signing of the Term of Free and Informed Consent form. The sensory panel consisted of 61 untrained participants (38 women and 23 men), aged 18 to 56 years, who were regular consumers of dairy products. Persons with allergy or lactose intolerance were not recruited. All participants signed the informed consent form. All participants were recruited from Escola de Medicina Veterinária e Zootecnia . They evaluated the product with a quantitative acceptance test, using a 9-point hedonic scale, where 1 meant “I dislike it very much” and 9 meant “I like it very much”. The session was conducted in individual booths. Fermented milk beverages were evaluated on day 0 of storage, presented in three-digit blind codes, and 20 mL samples were served one at a time at 7 °C, simultaneously with a glass of water for mouth rinsing among samples. The sensory attributes were appearance, color, aroma, flavor, consistency, firmness, and overall impression .The just-about-right (JAR) scale was used to identify the optimal intensity of each attribute . The scParticipants also performed CATA, using terms referenced in other studies ,39,40 tom = 1, …, M), o = optimism of the original model; M = number of bootstrap datasets.The performance of a predictive model is overestimated (optimism) when only determined on the sample used to construct the model. Therefore, internal validation methods aim to accurately estimate model performance in new samples . The estEquation (6) was used to calculate the coefficient of determination of the original model after validation.p < 0.05). In addition, Pearson’s correlation test with a 0.05 significance level was performed to evaluate the correlation between variables. The internal validation of the regression models was performed with the bootstrap method  [p < 0.05). When significant values were found, Sheskin’s multiple pairwise comparison test was employed at a 0.05 significance level to identify significant differences in each sensory term between samples. Statistical analyses were performed with a commercially available statistical package, XLSTAT, version 2022.3.2.1353 .All analyses were performed in analytical and experimental triplicate, and the data obtained were presented as means ± standard deviations (SDs). The comparison of multiple samples was performed using one-way analysis of variance (ANOVA) for centesimal results and two-way ANOVA for physicochemical  and microbial results, using GraphPad Prism 8 . ANOVA was followed by Tukey’s multiple comparison tests  . In CATAp < 0.05), indicating that cupuassu pulp and flour addition directly interfered with this parameter. This fact can be attributed to the high moisture content in pulp [The moisture, ash, protein, fat, fiber, and carbohydrate contents in the artisanal fermented beverages are presented in  in pulp  and the  in pulp , which ip < 0.05) of protein, fat, fiber, and carbohydrates were found in cupuassu flour samples. This pattern happened due to the concentration of nutrients resulting from the technological transformation to which the cupuassu seeds were subjected [The highest values , which was evident when comparing HPNF (10% pulp and 0% flour) and NPNF (control sample). In this case, pulp addition was a predictor of pH values  with the percentage of added flour, and the model had poor predictive power (Rv = 0.599), given that the higher the percentage of cupuassu flour was, the higher the pH value was, with NPHF (0% pulp and 3% flour) being the treatment with the highest value (p < 0.05). This behavior was also present in the samples with a combination of pulp and flour, whose pH values were intermediate, compared with the samples that contained only pulp or flour. According to Yadav et al. [In addition, it was observed that the pH of cupuassu flour (5.70 ± 0.02) presented a correlation  and day 28 . This reduction could be attributed to the post-acidification generated in the dairy beverages by lactic acid bacteria (LAB) transforming lactose into lactic acid, allowing to maintain metabolic activity and acidification during storage under refrigeration  and Kaurp < 0.05), except in the HPLF and MPLF treatments. On the other hand, it was observed that NPNF (control sample) had a decrease in pH values on days 0 and 21 of storage. While this is true, the treatments with a combination of cupuassu pulp and flour only showed this decrease on day 21, which indicates that the combination of pulp and flour in the beverage generated a slower post-acidification process, which would help to prolong the shelf life of the product [Pichia genus), which metabolize acid, causing the increase in pH [Furthermore, when comparing storage days 0 and 28, adding cupuassu pulp increased pH values ( product  and whicse in pH . Despitese in pH .p < 0.05). The contraction of the casein network formed during fermentation justifies this behavior [p < 0.05). Since syneresis is an indicator of quality, using these ingredients can mitigate this technological defect, which commonly affects the sensory parameters of this type of product. The reduced syneresis could be attributed to the pectin polysaccharides contained in cupuassu, which strengthen the casein network formed by interacting with the positive charges on the surface of micelles [The results of syneresis presented in behavior ,49. Cupumicelles ,50.p > 0.05). However, the addition of HPNF pulp produced a slight increase, contrary to the addition of 3% flour alone, which decreased syneresis values (p < 0.05), presenting a predictor , a* (greenness–redness), b* (blueness–yellowness), C* (chroma), and h° (hue angle) of artisanal fermented milk drinks during the 28 days of storage are presented in L* parameter (p < 0.05), except on day 0, where there was no significant difference from the control. This fact may be attributed to the pigments present in pulp, which make the beverage darker [L* values decreased more sharply in treatments with flour than in those with pulp. Thus, flour intensified the darkening when combined with pulp, as evidenced in LPHF (5% pulp and 3% flour), which exhibited the lowest value of this parameter (p < 0.05). Therefore, flour content was a strong predictor of L* . The reduction in the size of the casein micelles due to proteolysis during storage can increase L* values due to increased scattering light [During storage, the ng light . Howeverng light .a* compared with the control. However, the values obtained with pulp were higher than those obtained with flour due to the reddish color developed by cupuassu catechins due to oxidation, a product of fermentation [a* and pulp concentration, while a negative correlation between a* and flour was observed in the beverages , which could be associated with the pH behavior of the samples during storage being a low predictor of the a* parameter (p < 0.0001). On the other hand, in samples containing cupuassu, the values of a* remained unchanged, increased, or decreased. This could be attributed to the influence of pH, as well as the stability of catechin, on each treatment.The addition of cupuassu increased the values of entation ,24. Coheeverages . Thus, tb* values were significantly higher in the control (p < 0.05) and in the treatments with pulp alone, regardless of storage day. It was attributed to the yellow–green color of whey due to the presence of riboflavin and the yellowish white of cupuassu pulp [b* values. An increase in b* values is typical of non-enzymatic browning (Maillard) reactions [b* values. It is possible that the carbohydrate and protein contents increased with the two ingredients  results indicated that treatments with pulp alone presented c* values similar to or slightly lower than those in the control. In contrast, samples with flour alone sharply decreased chroma, including when combined with pulp , as reported in b*, which can be explained by the significant correlation between them (p < 0.001), as shown in The chroma (ith pulp a. Therefh°) values identify the relative orientation of the color with respect to the origin as 0° (red-purple), 90° (yellow), 180° (blue-green), and 270° (blue) to specify the color. The h° values obtained indicated that the values of pulp-only samples were significantly lower than those of the control, presenting a yellow color perception (p < 0.05). On the other hand, the samples containing flour showed an increase in the value of h°  than the control. The same behavior was found in the combined samples of pulp and flour, where MPLF and LPLF showed significantly lower values (p < 0.05), indicating that higher percentages of pulp and flour resulted in a more stable color during storage, which is excellent from the consumer’s point of view. During the remaining days of storage , ΔE values increased in all samples except NPHF and NPNF, which showed no significant difference during storage (p > 0.05).Δ2 ≥ 0.910), which indicates that the power law model provided suitable adjustment parameters. The consistency index (K) gives an idea of the fluid viscosity; the K values were generally unaffected by flour alone compared with the control, regardless of the added concentration (p > 0.05). On the other hand, the medium and high pulp concentrations elevated the K values, while the low concentration was insignificant. Similarly, 10% and 7.5% pulp in combined treatments provided higher consistency indexes than the control, regardless of flour concentration. In contrast, 5% pulp generally did not affect K, regardless of the combined flour concentration. The addition of pulp presented a positive correlation with K. Pulp acidified the pH and reduced syneresis, mainly when at a higher concentration. Accordingly, these factors contributed to increased K, as reported by the inverse correlation between them , while the medium and high concentrations of pulp decreased the values of n (p < 0.05). On the other hand, the addition of flour did not generally affect the values of n, regardless of the added concentration (p > 0.05). Differently combined samples with the high pulp concentration provided lower consistency indices than the control, regardless of flour concentration, while samples with the medium pulp concentration showed no significant difference, regardless of flour concentration, as did LPHF (containing 5% pulp and 3% flour) in contrast to LPLF (containing the same concentration of pulp but less flour), with which the index was significantly higher (p < 0.05).The n index indicates the degree of deviation from the Newtonian flow (n = 1). As n values were ≤ 0.534 , all samp > 0.05); in contrast to the samples with only flour and only pulp, LPNF and MPNF decreased n values, while HPNF increased with time, indicating that storage time, pulp addition, and pH favored n , while flour did not generate a significant effect, regardless of concentration (p > 0.05). On the other hand, the combination of flour and pulp ingredients in the LPLF, MPHF, and LPHF samples did not show significant changes compared with the control (p > 0.05), in contrast to HPLF, MPLF, and HPHF, which showed a significant increase in apparent viscosity values (p > 0.05).Apparent viscosity describes the flow behavior, an important parameter that influences sensory attributes and quality in dairy beverages, since the addition of whey generates low viscosity in this type of product . Table 5p < 0.05). Samples with only-pulp addition increased viscosity over time, which could be attributed to the reduction in pH due to the addition of pulp favoring higher apparent viscosity . However, LPLF showed no significant difference from the control on day 28 (p > 0.05). Concerning the samples combined with the medium pulp concentration (MPHF and MPLF), they showed a decrease on day 7, where MPHF subsequently increased until day 28 (p < 0.05), showing a higher apparent viscosity value than the control. In general, HPLF and HPHF samples had the highest apparent viscosity at the beginning of storage (p < 0.05). However, these had the lowest values on day 7, which can be attributed to the decrease in n during storage and the increase in K caused by the addition of pulp, which positively affected the viscosity . On day 28, samples MPNF, LPHF, HPLF, NPHF, and NPLF obtained the highest values (between 6.11 ± 0.12 and 6.20 ± 0.32), while NPNF had the lowest value, with 5.57 ± 0.13 (p > 0.05). Therefore, treatments without or with lower cupuassu content had less fungal growth than treatments with higher content. This could be attributed to the yeast (Pichia and Hanseniaspora) content in the cupuassu fruit microbiota and organic compounds in cupuassu pulp and seeds [p < 0.05), except those in HPHF and LPLF, which remained stable. The relatively high pH and water activity of artisanal fermented milk beverages favor the growth of this type of microorganism.In the count of molds and yeasts on day 0 of storage, the lowest values were found in the LPLF and NPNF samples, with counts ≤ 4 log CFU/mL, while HPLF had the highest value, with 5.83 ± 0.08 log CFU/mL  of the artisanal fermented milk beverages, which was attributed to the decrease in syneresis caused by the increase in WHC, which increased the scores in appearance and color attributes  among the samples. However, pulp concentration was a strong predictor of an alcoholic aroma , that is, the higher the perception of b* was, the lower the alcoholic aroma was .p < 0.05). On the other hand, flour concentration contributed to a less sweet taste but a more acidic and bitter taste. Indeed, the acidic score of MPHF was higher than those of HPLF and LPLH. In terms of bitter taste, the scores of HPHF and MPHF were superior to that of HPLF. In contrast, MPHF and LPHF had sweet taste scores that were lower than those of LPLF and HPLF. This pattern was attributed to the correlation between flour concentration and bitter taste, where flour addition was a strong predictor of bitter taste , unlike the pulp percentages, which generated increases in L* and C* color parameters, favoring cupuassu flavor  but no correlation concerning pulp and flour concentration  of the cupuassu microbiota, which are involved in alcoholic fermentation . AccordiHowever, it is important to note that a higher flour concentration increased acidity and bitterness, which were penalized attributes. Thus, an ideal flour concentration to harmonize these effects must be established. LPHF, MPHF, and MPLF presented excessive sandiness. Despite the penalty results, most evaluated attributes were close to “moderately more than ideal” (JAR between 2.49 and 3.85). The exceptions were sweetness and caramel flavor, which were less than “ideal” (JAR between 1.74 and 2.28) in all treatments. This can be attributed to the low percentage of sugar in the beverage (3%). The combination of the JAR profile data and the penalty analysis made it possible to establish an ideal product with an adjustment of the percentages of cupuassu pulp and flour, increasing the percentage of sweetener in the product, which would also reduce the acid aroma, acid taste, and bitter taste.p < 0.05). These terms were homogeneous appearance, dark brown color, light brown, beige color, white color, bitter taste, and cocoa aroma. Samples containing 3% cupuassu flour  were described as having bitter taste, dark brown color, and homogeneous appearance. MPHF and LPHF were also associated with a cocoa aroma, which could be attributed to the aromatic compounds in the fermented cupuassu and cocoa seeds, which impart an identical aroma [al aroma .On the other hand, samples containing 1.5% cupuassu flour  were described as white, light brown, and beige. These results showed similar behavior in the evaluators’ scores, for both CATA and JAR, where the samples with the highest concentration of cupuassu flour (3%) were perceived as having the most bitter taste and brown color. Likewise, the white and beige colors were associated with the samples with the lowest concentration of cupuassu flour (1.5%).L*, b*, and C*, and pulp increased the values of a* due to the pigmentation that each one presented as a result of the technological process. Therefore, the combination of samples in the sensory analysis allowed us to observe the effect that flour generated on the product, compared with the concentrations of cupuassu pulp studied by other researchers, where the pigmentation of flour allowed us to improve the color parameter in sensory acceptability. This study established that a higher flour concentration improves sensory appearance, consistency, and firmness in combined formulations, especially when containing 10% pulp. Pulp provided an alcoholic aroma and a cupuassu flavor. At the same time, flour contributed to an excessively acidic and bitter taste. Despite improving the rheological parameters by adding cupuassu, the beverages were still sensorially penalized regarding suboptimal firmness, consistency, and viscosity. Therefore, elaborating an artisanal fermented milk beverage with functional potential using cupuassu pulp or seed flour as ingredients is promising due to physicochemical, nutritional, and microbial quality. However, it is recommended, for future research, to optimize beverage formulations, mainly regarding the concentrations of cupuassu ingredients , and even the forms of addition, to improve the characteristics and achieve sensory fullness.Cupuassu pulp and flour had positive effects on some physicochemical parameters of artisanal fermented milk beverages by reducing syneresis and increasing water retention capacity. The high pulp concentration (10%) improved the rheological parameters, especially the consistency index and apparent viscosity. In addition, flour increased protein and fiber contents in the beverages. Likewise, these two ingredients affected the instrumental color parameters, where flour decreased the values of