Sbe) in tetraploid potato to develop starch with an increased amylose ratio and elongated amylopectin chains. By using ribonucleoprotein (RNP) transfection of potato protoplasts, a mutation frequency up to 72% was achieved. The large variation of mutations was grouped as follows: Group 1 lines with all alleles of Sbe1 mutated, Group 2 lines with all alleles of Sbe1 as well as two to three alleles of Sbe2 mutated and Group 3 lines having all alleles of both genes mutated. Starch from lines in Group 3 was found to be essentially free of amylopectin with no detectable branching and a chain length (CL) distribution where not only the major amylopectin fraction but also the shortest amylose chains were lost. Surprisingly, the starch still formed granules in a low-ordered crystalline structure. Starch from lines of Group 2 had an increased CL with a higher proportion of intermediate-sized chains, an altered granule phenotype but a crystalline structure in the granules similar to wild-type starch. Minor changes in CL could also be detected for the Group 1 starches when studied at a higher resolution.DNA-free genome editing was used to induce mutations in one or two branching enzyme genes ( In addition, using transient approaches for genome editing of potato is possible, which is a big advantage when plants free of recombinant DNA are desired. A transient approach has a clear benefit for highly heterozygous clonally propagated crops like potato, since segregation through seed generations is more or less impossible2.Genome editing in plants has opened up new possibilities, both in terms of research and development of crops with novel traits beneficial for human health or the environment. The method can be used to create knockouts of enzymatic functions, which in many cases has been difficult to achieve with traditional genetic engineering methods, e.g. RNAi. Protocols for genome editing through CRISPR-Cas9 have been successfully established for inducing mutations in potato3. Starch is composed of glucose units linearly linked by α(1 → 4) glycosidic bonds and branched by α(1 → 6) bonds. Starch is generally deposited as highly ordered granules directed by the highly branched amylopectin molecule with the addition of the essentially linear molecule amylose to a ratio of approximately 4:14. In food products, the high amylose content and the long chains of amylopectin contribute to formation of resistant starch and relate to a low glycaemic index (GI) after intake6. By increasing the average chain length of potato starch, a starch with health benefits can be developed. The increased chain length can yield resistant starch (RS) leading to a low GI and concur health benefits by promoting the growth of healthy gut flora, and lowering both the caloric intake and cholesterol levels in the blood5. The long-chain quality of starch also creates beneficial properties as a raw material for producing bioplastic films7, which in the future might replace some of the fossil-based plastics produced today.Potato is an important, high-yielding, nutritious and starch-rich staple crop11. The development of potatoes with a high ratio of amylose starch and/or altered starch chain length distribution has been achieved by targeting two starch branching enzymes (SBEs) using traditional gene silencing technologies and recently genome editing14. A very high amylose starch content was found synthesised at the expense of total starch content and plant development15. Based on those results and the fact that, so far, no potato studies have resulted in pure amylose starch, it could be speculated that the presence of a fraction of amylopectin in potato starch is essential for plant development. In contrast, barley with suppressed activity of three SBEs  was found to have an amylose-only starch in the endosperm16. In that study, the high amylose content only had a minor impact on grain yield and starch content.The research and development of potatoes with novel starch qualities has been ongoing for a long time. Potatoes that synthesise solely amylopectin starch have been developed by eliminating granule-bound starch synthase (GBSS) activity through traditional mutagenesis, antisense, RNAi and most recently CRISPR-Cas9.14, CRISPR-Cas9 was used to target the two SBEs in potato. Lines with mutations in Sbe1 or Sbe2 alone or in combination were developed using either traditional Agrobacterium-mediated transformation or PEG-mediated protoplast transfection with vector DNA. In that study, lines mutated in Sbe1 were not found affected in their starch structure, while tuber cells from Sbe2 mutated lines displayed an increased number of granules. One line had a strong reduction in both SBEs, resulting in starch with an altered granule phenotype, longer amylopectin chains and a degree of branching that was reduced by half.In a recent study by Tuncel et alSbe genes through CRISPR-Cas9 and Agrobacterium-mediated transformation17. In that study, mutations in the Sbe1 gene did not result in any major changes in the starch compared to the parental variety, while mutations in Sbe2 led to a starch with increased amylose content from ca. 15 to 25% and an amylopectin chain-length distribution which shifted towards longer chains17. Similar results were obtained on sweet potato mutated in Sbe2, with an increase in amylose content from ca. 27 to 40%18.Other crops have also been targeted for developing high-amylose starch genotypes using genome editing, such as rice and sweet potato. Rice was subjected to mutagenesis in the respective Sbe1 individually and Sbe1–Sbe2 simultaneously in potato. By using genome editing to induce mutations in all eight Sbe alleles, we were able to develop, for the first time, a unique potato starch essentially lacking branching. We further investigated the effects of this starch as well as starches with altered amylopectin structure on plant development and starch granular structure and phenotype.In this study, we used a previously established CRISPR-Cas9 RNP-method to induce mutations in Sbe1 was targeted alone or in combination with Sbe2 in the potato variety Desiree  and BE2T3 and BE2T4 (Sbe2) , six lines had four-allele mutations in Sbe1 combined with two to three alleles mutated in Sbe2, 104001, 104005, 104006, 104016, 104018 and 104034 (Group 2) and two lines, 104010 and 104023, had all eight alleles mutated (Group 3). Both lines in Group 3 contained in-frame indels in the Sbe1 and/or Sbe2 mutated alleles  and high-performance anion exchange chromatography (HPAEC) were used to investigate the chain-length distribution of debranched starches. In the HPSEC analysis, the debranched starch samples of Group 1 lines showed a similar chain-length distribution pattern as the parental variety Fig.  but withSubstantial changes to the chain-length distribution pattern, both in the amylose and amylopectin fractions, could be observed for the lines of Group 2 and Group 3 compared to the parental variety  was analysed using NMR spectroscopy on starch from lines in Group 3 and the parental variety Desiree. At the branching point, the anomeric proton at the α(1 → 6)-linkage has a different chemical shift compared to other anomeric protons and can thus be used for quantification of the DB. Neither starch from line 104010 nor 104023 showed any detectable branching (< 0.1%), whereas the starch from Desiree showed a DB of 3.0% , 17° (strong) and a doublet at 22–24° 2θ, which is the common pattern for the tuber starches. However, the X-ray diffraction pattern and the diffraction intensities of the Group 3 lines differed from the other lines. A decrease in the diffraction intensity for most of the peaks was observed in the Group 3 lines when compared to the other lines. However, it was still possible to observe all the major diffraction peaks that correspond to B type starches , 104006 and 104018 (Group 2), 104010 and 104023 (Group 3) and T-2012 (RNAi) Fig. . After fLeaf tissue from the top shoots harvested at ten weeks after planting was stained with iodine after a light and dark period. As can be seen in Fig. Sbe1 and Sbe2 of potato had a major impact on the starch quality. To develop a starch with a significant increase in chain lengths corresponding to the amylose fraction, mutations in all alleles of both genes were needed (Group 3). The lines from Group 3 were found to have starch with a reduced degree of branching from 3.0% in the control to below the detection limit of 0.1% and no amylopectin could be detected when analysed by HPAEC. Notably, the lines from Group 3 had at least one allele with an in-frame mutation in one or both of Sbe1 and Sbe2. In a study by Tuncel et al.14, the quantity of the SBEs was reduced but not absent in lines with in-frame deletions in the Sbe genes. These proteins could, even with a potential loss of activity, be of importance for starch synthesis to occur. The situation has not been thoroughly investigated in potato, but several studies in cereals have shown that enzymes involved in the synthesis of starch molecules and organisation into granules are active as complexes23. The remaining fraction of SBEI and SBEII in our lines from Group 3 might be crucial to maintaining protein–protein interactions and keeping other enzymes, such as soluble starch synthases, active.The number of alleles mutated, and the genomic structure of mutations induced in 15. In our study, lines with full knockout of SBEI together with two to three mutated Sbe2 alleles (Group 2) only had a minor effect on the amylose content. None of the Group 2 lines were found to have the same increased amylose content or the very large fraction of intermediate sized chains like the starch structure found in the RNAi line T-2012 included in this study, which seem to be common among high-amylose potato starches achieved by reduced transcript levels. In a previous study, using antisense to inhibit both SBEs, a reduction of branching enzyme activity to less than 1% was found in tubers with starch containing more than 50% amylose12. Hence, the presence of one or two Sbe2 wild type alleles is enough for the tubers to produce starch close to wild type levels of amylopectin.The chain length distribution of debranched starches suggests that the targeted mutagenesis of Sbe1 with or without Sbe2 had different effects on starch structure, which has also been shown in other studies28. This is believed to be due to different activities of the potato SBE isoforms25 affecting the amylopectin structure29. Simultaneous suppression of SBEs in potato has been shown to induce drastic effects on starch structure15. Consistent with these findings, our HPSEC results for debranched starch from Groups 2 and 3 showed a loss of the short-chain amylopectin fraction and elevated fractions of amylose. Accordingly, a decreased number of short chains of DP ≤ 13 and an increased proportion of intermediate-sized chains (DP14–33) were observed for starch from Group 2 by HPAEC analysis.Using the applied genome-editing method, we were not able to mimic the starch qualities of high-amylose potatoes that were previously developed by transgenic gene inhibition30. Also previous studies in potato, antisense suppression of SBEI did not affect the amylose content or the chain length distribution of the amylopectin30. This is in line with our HPSEC results for Group 1 starches, which showed a similar chain length distribution of amylopectin fraction in relation to the parental variety. One exception is line 104032, which can be found similar in structure to the Group 2 starches. It can be speculated that this line might be a chimera or having one Sbe2 allele with a very large deletion that was not amplified during the genomic analyses. Even though several attempts were made to elucidate that, only wild type alleles were found using HRFA and Sanger sequencing. When studying the starches from lines in Group 1 with higher resolution using HPAEC, an altered chain-length distribution was found, indicating that a complete knockout of SBEI alone influenced the starch structure somewhat. Moreover, light microscopy of stained starch granules from Group 1 lines revealed a minor truncation in the core of the granules.Many studies on cereals have reported that suppression of SBEI alone has either no detectable effect or only a minor effect on starch structure20. Thus, the significant loss of the amylopectin fraction in the Group 3 starches might be the reason for the considerably decreased X-ray diffraction pattern of the starches from Group 3. Moreover, the level of crystallinity has an inverse relationship to the amylose content31. Hence, the high-amylose feature of the potatoes from Group 3 might also contribute to the lower degree of crystallinity in the potato lines. However, the high-amylose potato starch in line T-2012, produced by RNAi, did not show a difference in the X-ray diffraction pattern compared to the parental variety.X-ray diffraction of starch correlates with crystalline organisation of double helixes in a starch granule, while the crystallinity of granular starches is mostly attributed to the amylopectin fraction32. It is known that the apparent intensity of the birefringence is not only affected by the degree of crystallinity and granule thickness but also by the orientation of the crystallites33. Therefore, the complete loss of Maltese crosses in the starch from Group 3 could be attributed to a deviation in the orientation of the crystallites from its radial arrangement. The results from X-ray diffraction, polarised light microscopy and NMR spectroscopy revealed a type of starch that has the ability to organise into granules with a poorly ordered amylose crystalline arrangement, even with the considerable loss of the amylopectin fraction. These results are also in agreement with the study of amylose-only barley starch16.Maltese crosses in starch granules generally indicate a higher degree of ordered structure in granules with molecules arranged in a radial pattern15. It was speculated that the gene inhibitions could also have an effect on diurnal starch, which might have led to a depletion in the energy turnover, or that an increase in sugars in the plant might disturb other metabolic processes. In this study, the development of the Group 3 lines was supressed during the first period after planting but those lines developed a higher green biomass than lines from Groups 1 and 2 up until senescence; despite that, the transitory starch was clearly decreased after a light period. One hypothesis could be that once tuber initiation occurs and throughout the tuber development phase, the low efficiency of channelling sugars into long-term storage as tuber starch is instead redistributed to produce green biomass.Lines from Group 3 had a significantly decreased tuber yield, tuber size and tuber dry matter content. In a previous study of high-amylose potato lines grown in the field for several years, plants were found to be stunted in growthTo our knowledge, this is the first time potato starch with no detectable branching has been developed and studied. It highlights the expanded knowledge we can gain from using genome editing to study enzymatic functions and consequences of the loss of enzymatic activities on biosynthesis and plant products. This study has also raised new questions, for example, how can the self-organisation of starch into granules occur even with complete absence of branching, or are they simple molecular aggregates?Sbe1 (GenBank accession no. NW_006238958.1:c2098376-2090439) and in exon 3 in Sbe2 (GenBank accession no. NW_006238947.1: c2592132-2611729) using CRISPR RGEN Tools (http://www.rgenome.net/cas-designer) and a previously available guide design tool (http://crispr.mit.edu/). Double sgRNA targets were selected for each gene and named BE1T3 and BE1T4 as well as BE2T3 and BE2T4  and sequenced through the Illumina TruSeq PCR-free library preparation (average fragment size 350 bp) and Illumina HiSeqX (PE 2 × 150 bp) . Targets were selected in regions of exon 5 in 2T4 Fig. . All sgRSolanum tuberosum L. cultivar Desiree through PEG-mediated protoplast transfection of RNPs. In vitro propagation, protoplast isolation, regeneration and mutation screening and characterisation were performed as described in Andersson et al.11. Transfection of RNPs to isolated protoplasts was done essentially as described in Andersson et al.35 using 5 ug in vitro transcribed RNA preassembled with 5 ug Cas9 per target  for lines named 82- or 0.1 nmol synthetically produced crRNA preassembled with 5 ug Cas9 per target  for lines named 104-.Mutations were induced in 11 by multiplexing primers SBE1f.-HEX and SBE1_r with SBE2_3f.-FAM and stbe2exonr and screening for mutations in both genes simultaneously. Sanger sequencing  was performed using unlabelled primers of SBE1f.-HEX and SBE1_r, SBE2_3f.-FAM and stbe2exonr as well as stbe2exonr and Stbe2exonf  was performed as previously described7, were grown in a greenhouse. In vitro cuttings of the lines were planted in soil  in three to five biological replicates in 7.5 L pots and cultivated under controlled greenhouse conditions: 16-h day length, 18/15 °C day/night temperature, supplementary light intensity up to approximately 200 µmol s−1 m−2 photons, 60% relative humidity. The lines were grown for 5 months between 13 December and 21 May and were regularly fertilised with SW Bouyant RikaS 7-1-5 + mikro .Thirteen genome edited lines, the parental variety Desiree and one RNAi line, T-2012 from the parental variety Dinamo developed in a previous study34 with a minor modification where the time for sedimentation after extraction and each buffer washing steps were performed overnight to secure that small granules were retained. Dry matter content was measured on freshly harvested tubers as previously described35. Amylose content was measured on isolated starch using both an enzymatic assay and a colorimetric assay. The enzymatic assay was made using an amylose/amylopectin kit  according to the supplier’s instructions. The colorimetric method was based on iodine complex formation stabilized with trichloroacetic acid. The amylose content was analyzed according to Chrastil36 with minor modification. After solubilisation of starch with Urea-dimethylsulphoxide (UDMSO) according to Morrison and Laignelet37, 25 µL of sample was transferred to each of two test tubes. 5 mL 0.5% trichloroacetic acid and 50 µL 0.01 N KI-I2 solution were directly added for 30 min incubation at 25 °C before the absorbance measurement. A standard curve (R2 = 0.999) was developed using waxy potato starch  and potato amylose standard  with varying amylose contents of 0%, 25%, 50%, 75% and 100%. All samples were analyzed in duplicate and results are reported as average values with standard deviation (SD) based on total starch content.Starch was extracted from tubers harvested after senescence according to Larsson et al.40. The starch samples were debranched with isoamylase from Pseudomonas sp  and pullulanase M1 from Klebsiella planticola  before both analyses. The enzymes were desalted and ten-fold diluted using acetate buffer  before applied for debranching.Starch samples solubilised in UDMSO (0.6 M urea in 90% DMSO) were used for chain-length distribution analysis by high-performance size exclusion chromatography (HPSEC) and high-performance anion exchange chromatography (HPAEC) as previously describedFor HPSEC, 300 μL each enzyme and 400 μL distilled water was mixed with 500 μL solubilised starch sample for debranching overnight at 40 °C. For HPAEC, 60 μL each enzyme and 880 μL distilled water and 400 μL sodium-acetate buffer  was mixed with 100 μL solubilised starch sample for debranching overnight at 40 °C. For both HPSEC and HPAEC, the samples were boiled for 10 min after debranching to terminate the enzyme reaction and then filtered through a 0.45 μm nylon filter before analysis.41 with minor modifications. The HPSEC system has two serially connected OHpak SB-802.5 HQ columns with a guard column  kept at 35 °C. The eluent was 0.1 M NaNO3, containing 0.02% NaN3 with a flow rate of 0.5 mL/min. The HPSEC is equipped with refractive index (RI) detector  and multiple-angle laser light scattering detector . Data for molecular-weight determinations were analysed using ASTRA software version 4.70.07  based on a dn/dc of 0.14741. The angular fit was based on the Debye procedure41. The HPSEC columns were calibrated with Dextran T2000, Dextran T500, maltoheptaose, maltopentaose, maltotriose, and glucose  coupled with a BioLC gradient pump and a pulsed amperometric detector (PAD) was used in this study. Separation was performed on a CarboPac PA-100 (4 × 250 mm) analytical column  equipped with a guard column. The elution was performed at 25 °C with a flow rate of 1 mL/min and injection volume of 25 μL using 0.15 M NaOH (A) and 0.50 M NaOAc + 0.15 M NaOH (B) with the following gradient: 0–15 min, 15–28% eluent B; 15–45 min, 28–55% B; 45–75 min, 55–70% B; and 75–80 min 70–15% B (return to the start mixture). The column was equilibrated with 15% eluent B for 15 min between runs. The PAD response was converted to molar percentage and normalised42. Starch samples (10 mg) were dissolved by heating in deuterated dimethyl sulfoxide  with the addition of deuterated trifluoroacetic acid  prior to NMR analysis to avoid spectral interference with hydroxyl protons. 1H NMR spectra were recorded on a Bruker Avance III 600 MHz spectrometer using a 5 mm broadband observe detection SmartProbe. Spectra were acquired at 50 °C using 128 scans and a relaxation delay of 12 s and were processed with TopSpin 3.6. The degree of branching was measured as the ratio I1−6/(I1−6 + I1−4), where I1−6 is the integrated signal at 4.77 ppm and I1−4 is the integrated signal at 5.12 ppm, corresponding to H1 of glucose at the α(1 → 6) and α(1 → 4)-linkages, respectively.Starch from the parental variety and the Group 3 lines were analysed by NMR for degree of branching according to Tizzotti, et al.5. Starch X-ray patterns were identified using a PANalytical X’Pert alpha1 powder X-ray diffractometer in Theta-2Theta geometry, and coupled with a focusing Johansson Ge monochromator producing pure Cu-Kα1 radiation (λ = 1.54060 Å). The starches were spread onto 1.5^2 cm2 Si wafers. The scanning region of the diffraction angle (2θ) was from 5° to 40°.The method for X-ray diffraction analysis has been previously described5 using a light microscope  equipped with an infinity X-32 digital camera . The images of starch granules were captured at a 20 × objective under polarised light to compare the birefringence of starch granules from lines from Group 3, 104016 (from Group 2) and the parental variety.Starch dispersions  in distilled water were freshly prepared. One drop (25 μL) of each starch dispersion was taken for microstructural analysisStarch granule distribution and phenotype was studied by staining thin tuber discs and purified starch granules with Lugol’s solution. The results were visualised with a light microscope  and documented with an assembled camera .Green biomass of five of the mutated lines, 82079, 104006, 104018, 104010, 104023 as well as Desiree and T-2012, were studied in a greenhouse. The plants were grown between 7 October and 22 January under the same growth conditions as described above.35. The plants were placed on a rotating disc and images were taken as side views with a standard camera  tethered to the software digiCamControl v2,1,2 (http://www.digicamcontrol.com)43. Four photos were taken of each plant and three biological replicates covering 360º of the plants. The images were processed using Easy Leaf Area44.Green biomass was measured at four, ten and fifteen weeks after planting using a low-cost RGB imaging phenotyping lab published by Armoniene et al.Top leaves were harvested at week 10, decoloured by boiling in 80% EtOH and stained with 50% Lugol’s solution.Harvested greenhouse grown potatoes were stored in the dark at 6 ℃ for five months before being incubated at 22 ℃ for 3 months. Sprout development was studied through naked eye observation once a week.Supplementary Information


The success of free vascularized fibular bone graft (FVFBG) has accelerated the osteo reconstruction which results from trauma, resection of a tumor or an infectious bone segment, or correction of congenital deformity. But the complication behind should not be overlooked. The failure could necessitate a second surgery, which prolong the rehabilitation period and produce further health cost. Worst, the patients may suffer a permanent impaired ankle function, or a sustained morpho-functional loss on reconstructive area which are hard to save. To provide an overview of the complication related to reconstruction by FVFBG, a narrative review is conducted to identify the complications including their types and rates, the contributing factors, the approaches to measure and the techniques to avoid. Methodologically, by quick research on Pubmed and abstract reading of reviews, we characterize five reconstructive areas where FVFBG were most frequently applied: extremities, mandible, spine, osteonecrosis of femoral head, and penile. Following, the complications on different reconstructive areas are retrieved, studied and presented in five  separate sections. By the way, meaningful difference between FVFBG and other bone flap was presented in a few words if necessary. Donor-site morbidities were studied and summarized as a whole. In these literatures, the evidences documented on limb and mandibular reconstruction have the fullest detail, followed by the spine and lastly the penile. In conclusion, FVFBG, though a mature technique, needs further deep and comprehensive study and maybe device-based assistance to achieve better reconstructive effect and minimize donor-site damage. Since 1986, the free vascularized fibular bone graft (FVFBG) has been increasingly applied in restoration of bone defect because of trauma, tumors, infection or congenital anomalies. Due to its integration potential and abundant blood supply, it also has a varied utilization in spinal fusion and osteonecrosis of femoral head. What is more, its advantages in rigidity extend its application in phalloplasty. Owing to the advancements in microsurgery, vascular compromise and flap failure have largely been avoided and many researches declare a low incidence rate of complication both on recipient site and donor site. However, complication like stress fracture and non-union could fail the rebuilding process, and donor-site morbidities like valgus ankle deformity or great-toe flexing contracture could lead to impaired walking ability and low-quality life. This review is aimed to summarize complication in types, incidence rates, and contributing factors of complication on donor and recipient site, and meanwhile update the measurement to quantize complication, technical refinement to avoid the complication. To the best of our knowledge, this is the first review focusing on complication both of donor and recipient area, covering all kinds of osteo reconstruction  by free vascularized fibular bone graft.Initially, a quick search was conducted by using term “(fibula [Title/Abstract]) AND (((flap [Title/Abstract]) OR (transfer [Title/Abstract])) OR (graft [Title/Abstract])) AND (review [Filter] OR systematic review [Filter])” on Pubmed to identify the complication types relevant to free vascularized fibular bone flap or fibular chimeric flap which has a bone component. In a generalized sense, the applied range of fibula bone flap may be roughly divided into four categories: extremities, spine, mandible and penis. However, according to the retrieved result-180 search results on fibular flap transfer, we found that the femoral head and the special condition of extremity reconstruction which necessitates a proximal fibular epiphyseal transfer are usually studied as isolated unit in published literatures. In fact, the purpose of hip-preserving treatment for femoral head necrosis is to introduce a new and stable blood supply, which is slightly different from that of repairing a segmental bone defect on extremities. In addition, the objective of proximal fibular epiphyseal transfer is to rebuild a near-nature growth potentiality for immature bone, or to reconstruct the spherical joint head (hip or shoulder joint) with the spherical fibular head. Both the reconstructive goal and the flap design have particularity. Therefore, we classified the applied ranges of FVFBG more detailedly into six categories: extremities, proximal fibular epiphyseal transfer, mandible, spine, osteonecrosis of femoral head, and penile. Accordingly, the complications on different reconstructive area were searched once again on Pubmed and studied in different separated section, such as “(extremit [Title/Abstract]) AND ((fibula [Title/Abstract]) AND (((flap [Title/Abstract]) OR (transfer [Title/Abstract])) OR (graft [Title/Abstract])))”. For the retrieval of donor site complications, we performed a separate search and excluded the fibular flap without bone component. Generally speaking, this is a Pubmed-based narrative review aimed to summarize all the potential risk involved in reconstruction by free vascularized fibular bone transfer. Evidences from systemic review, meta-analysis, and large-scale studies have been meticulously studied and updated if necessary. Certain emphasis is put on the highly-cited literatures. Sporadic cases are only adopted when uncommon complication is discussed, and would be presented as a whole after generalized search.FVFBG has been reported with higher success rate , superioWound healing problems in fibular flap donor areas, such as infection, bleeding, and delayed healing, are common early complications. But usually these do not affect the functional outcome, and will not cause long-term problems . As longSurgical detail is significantly related to the donor-site function. Approach to harvest is the first consideration: Medial approach refers to a surgical alternative by which the fibular graft was elevated from the medial side. Technically speaking, lateral approach is more convenient in clinical practice because the fibular bone lies on lateral side just beneath the skin. But a comparative study by Philip  showed tPreoperative angiography of both legs should be carried out routinely to specify the distribution of blood vessels in the donor area. Anatomic variations where peroneal artery is absent or is the dominant artery supplying the leg and foot  should be detected in advance, otherwise harvest failure or ischemia of the distal limb would happen . PreoperValgus ankle deformity happens particularly where there has been no fibular transfixion or where too little residual fibular bone has been left. In skeletally immature patients, the remaining distal fibula should be at least 6 cm or 10 cm  long andFlexion contracture of the great toe is caused by the contracture and a tenodesis effect of the long-toe flexor tendons due to loss of muscle origins after fibular graft removal. Biomechanical model  showed tp = 0.05) which are an important phase in a gait cycle [p < 0.05) decrease in the time-distance parameter and the peak plantarflexion, and dynamic range was seen. But at 3 months postoperatively only peak plantarflexion in swing was significantly (p < 0.05) decreased [Since 1988 , measureit cycle . It has it cycle  found thecreased . Completecreased . General. Ling [Incidence rate of function loss is confusing. In spite of these small-sized study in which evidence is not so convincing, and whether they are the different or the same reconstruction sites , the donor-site complication has widely-ranged incidence rates. In a prospective study with 157 Consecutive Patients, Adeyiza  found a . Ling  reported. Ling .Complication on reconstructed area can be classified into three types: one is about graft survival and usually happened at early stage. Another type is about incomplete reconstruction objective, for example, persistent malformation, repressed bone growth, delayed or unachieved bone union, stress fracture. The third type is recurrence of infection, tumor and radioactive ulcer which completely comes from problematic reconstructive area itself and is absent in reconstruction of congenital deformity. Vascular compromise is first noteworthy complication which could completely leads to transplantation failure. However, the common flap-monitoring methods like SPECT/CT , scintigThe vascularity of the graft, which depends directly on the vascular anastomosis, has been described as a major factor influencing the outcome after FVFBG . HoweverUnlike the common vascularized fibular graft, the blood supply of vascularized fibular epiphyseal (VFG) transfer does not always come from fibular artery. Actually, the pedicle harvest has been provided with five different choice in chronological order: peroneal vessel , bi-pediVFG has been used to reconstruct a lost growth plate in the skeletally immature subject and articular surfaces. David  reviewedThe common complications in the radius reconstruction are radial deviation and wrist subluxation, which often necessitate second surgery to correct or stabilize. Salah  reviewedThe cases of using vascularized fibular epiphysis to reconstruct the lower extremity is small and the effectiveness is dissatisfactory. There were overall only 4 cases about hip , 52, 3 aOverall speaking, this technique still leaves much to be desired for its high complication rate. However, the fibular head provides a good growth potential and its sphericityis close to the shape of humeral head and femoral head. At present, it is the most suitable choice for the repairing bone defects on immature skeleton, and the only autograft which could reshape into an articular head.Fibular graft is the workhorse in mandibular reconstruction , for itsRadiological change of non-union and bone absorption of fibular graft has been well documented and compared with those of other flap types . TransfeJust like the limbs, to assure a stable mechanical support to large defect, two-section or bilateral fibular grafts should be administrated in mandible reconstruction when necessary. In pediatric patients, impaired mandible growth after the reconstruction with fibular growth possible leads to malocclusion and facial asymmetry . LuckilyOld age does not increase the risk of donor site complication or flap failure , but it Complication relevant to plate system, which are used to stabilize the fibular graft, has also been debated. The evolution in type of metal, the size and malleability of the plates, and the locking mechanism has lower down the risk of complication like plate exposure, plate fracture, infection, screw loosening and worst surgical removal. Mandible plate, locking plate and reconstruction plate have been applied in mandibular reconstruction to fix the fibular graft, and mini-plate has gained an increasing use and currently become the dominant one, for its lower profiles and smaller screw diameters, which minimizing the risk of plate exposure , 71. HowVascular pedicle ossification refers to graft pedicle experiencing calcification which could be identified both on CT scanning and with naked eyes under operation. Though it has a low incidence (4.4%)  and onlyComputer-aided design and manufacturing (CAD/CAM) technology  in mandiFibular graft is indicated when there are complex primary or secondary defects and/or loss of structural integrity of one or more spinal segments that require adequate, long-term, mechanical stability. The application of fibular flap in spine reconstruction is based on the success of limb reconstruction. As a result, the developmental history is short and only sporadic cases are available, the documented complications of spine reconstruction are not as complete as the that in extremities.Non-union is the most-common complication and has been reported with a incidence rate of 4% , 6.3% 9 and 14% To ensure an adequate mechanical support to avoid fracture and collapse, the fibular should be double-, triple- or quadruple-barrel construct if necessary. But when multi-barrel fibular grafts are applied, it should be realized that there is a risk of pushing the most posterior fibular strut into the direction of the spinal cord . AnotherVascularized grafts can be used in the treatment of osteonecrosis at a variety of anatomical sites, but most commonly used in young patients with ONFH. In a systemic review of 1270 procedure at an average of 8.3 years  (32%) arThe incidence of conversion to total hip arthroplasty and radiographic necrosis progress are major parameter studied in literatures. Not included as complications though, they are definitely surgical failure to arrest necrosis progression. Risk factors for reconstructive failure or graft absorption has been routinely studied, like age, sex, different predisposition  and different disease stage. However, as the bone flap survivorship is monitored indirectly by radiography  and based on bone viability, there is an absence of key evidence for the vascular-anastomosis quality. Once this confounding viable-the quality of vascular anastomosis is not controlled. The true relationship between the risk factors and reconstructive failure will be deviated. Histopathological analysis should be the most convincing examination but is limited by small size , 102 . HCompared with non-vascularized fibular graft and core compression, the vascularized fibular graft is a superior choice for lower rate of conversion to THA or radiographic necrosis progress . But no Recipient vessel and surgical approach have also been studied. Different recipient vessels  (three kIn previous-published literature of 1196 patients under phalloplasty, only 141 patients were operated with fibula osteo-cutaneous flap including 5 without osteo component, which is secondary to the workhorse-the radial forearm flap . AdvantaIn summary, the complication of osteo reconstruction by vascularized fibular is acceptable and need further improvement. Technical tips on flap harvest and transplantation have been provided to lower down the risk of vascular compromise and other complications. Computer assistance is still under exploration and might be good option to avoid mismatch of graft, implant, and mandibular residual. Proximal fibular epiphyseal transfer is more challengeable than a diaphyseal transfer and further basic research is needed to ensure the growth of transplanted fibular proximal adjustable to the recipient site. Current monitoring devices on fibular flap survivorship is mainly rely on indirect approaches like SPECT/CT, scintigraphy, intraosseous microdialysis and so on. We are looking forward to more direct and real-time monitoring device on fibular flap perfusion. Dynamic measurement of functional loss on donor-site needs the large-scale research to quantize the complication and guide gradual weight-bearing.


To compare the effect of Er:YAG Laser and Air particle abrasion (APA) surface treatments on shear bond strength of Y-TZP to composite resin cuboids in the presence and absence of primer application and salivary contamination. Seventy-two cuboidal shaped specimens 7x7x3 were prepared from Y-TZP using CADCAM, cleaned and sintered. Specimens were divided into 2 main groups (n=36) according to surface treatment method; Air particle abrasion (A) and laser (L). Each group was subdivided into 2 subgroups (N = 18) according to surface modification using primer; each subgroup was further divided into 2 subdivisions (N=9) according to the presence of salivary contamination; APC , AP , AC , A (Air particle abrasion), LPC , LP , LC , L (Laser). Composite cuboids having dimensions of 6x6x3 were also fabricated using custom made plexi plates. Composite cuboids were cemented centrally to zirconia cuboids and light cured under 5 kg weight for 6 mins. Shear bond strength of specimens was measured utilizing universal testing machine at a crosshead speed of 0.5 mm/min. Failure loads were recorded in Newton. SBS was calculated according to equation: SBS (MPa) = load (N)/area(mm2).Viewing shear bond strength between studied groups, group APNC (484.02±85.02) showed higher mean value compared to ANPNC (122.09±55.80), also LNPNC (120.87±65.10) showed higher mean value in comparison to LPNC (170.78±53.22). APNC (484.02±85.02) and APC (592.22±189.65) showed higher mean values than LPNC (170.78±53.22) and LPC (3227.66±108.28) in sequence.APA showed higher SBS values than Er:YAG surface treatment. Primer showed better results than no primer coating. Artificial saliva contamination did not affect the SBS of zirconia compared with no contamination results. Key words:Shear bond strength, zirconia, air particle abrasion, Er:YAG laser, primer, contamination. Ceramics have received much praise and clinical success since their introduction, because of their ability to mimic color and translucency of natural teeth. This urges manufacturers to improve weak points inherited in ceramics, which included brittleness and low tensile strength. This led to marked increase in the use of metal free restorations.Despite its mechanical strength, zirconia was accused of having poor bond strength to cement and veneer material. Unfortunately, establishment of a durable chemical or mechanical bond to zirconia has been proven to be difficult because of its surface stability , which rIt goes without saying that; the clinical success of bonded ceramic restorations depends on the cementation process. There is a general agreement that air particle abrasion (APA) using 50–110 µm alumina particles at 0.25 PSI is effective in cleaning and roughening the bonding surface of zirconia ceramic , additioRecently, laser pretreatment is suggested to change surface of materials aiming to improve the bonding to dental structure. Some studies have suggested application of lasers to change zirconia surface in order to improve their bond to tooth structures. Whereas other studies, demonstrated that in comparison to conventional zirconia surface treatment methods; laser pretreatment was not efficient for increasing bond strength or even decreasing it .This raises another major issue, jeopardizing optimum ZrO2- resin bond related to potential contamination before cementation. After APA and clinical try-in procedures, contamination can’t be avoided by blood, saliva or silicone indicator. This may lead to compromised bonding protocol and questionable bond strength . To compSeveral methods were suggested to prevent contamination after try-in stage, the most commonly used, was application of primer to the APA surface prior to try-in step, which was found to be time saving method by eliminating the need of additional laboratory step to repeatAPA to the intaglio surface and cause further delay of cementation .-Specimen groupingTotal sample size was 72 cuboids divided into two main groups (N=36) according to surface treatment (Air particle abrasion (A) and laser (L)). Each group was further subdivided into two subgroups (N = 18) according to surface modification using primer. The two subgroups were further divided into two subdivisions (N=9), according to the presence of salivary contamination .-Fabrication of Zirconia ceramic specimensUsing AUTOCAD , a cuboidal shape of 7x7mm dimension and thickness of 3mm was drawn and imported as STL (Standard Triangulation Language) file to CAD/CAM software . A block of each material was mounted in the milling machine in dry mode using burs that are specially developed for milling dental zirconia Fig. . Zirconi-Fabrication of Resin blocks Transparent plexi-frames of 3mm thickness were prepared by laser cutting. In the middle of this frame, square mould of (6x6) was laser cut in order to standardize the size of resin cuboids obtained from plexi. Another transparent plexi-frame was cut to act as floor. The 2 plexis were placed above each other. SDR composite was then injected into cuboidal mold created in the middle of plexi. Histology glass slide was placed onto to ensure flat composite surface which was then light cured. After curing, 2 plexis were separated from each other and composite was pushed from cuboidal space.-Surface Treatment• Air Particle AbrasionJig was constructed to standardize 60 degrees angle and 1cm distance between application tip and zirconia surface for group (A) specimens Fig. . Zirconi•Laser surface treatment.Er:YAG laser was applied to group L with special handpiece. Zirconia holder was constructed to maintain laser tip perpendicular to ceramic surface at a distance of 1 mm, and restricted ceramic area was laser treated with water irrigation and air cooling for 15 sec. -Application of zirconia primer.Zirconia primer was applied to zirconia surface using clean disposable brush and was left for 20 sec and then thinned out by air.-Saliva contamination of specimens.Saliva substitute was prepared composed of  . Each su-Cementation of Zirconia Cuboids to Composite cuboids.Two transparent 3mm thick plexi frames of the same size were prepared by laser cut. One transparent plexi frame was laser cut with a space of (7.2x7.2) in middle to facilitate placement of zirconia into the plexi without touching it. Second one was also laser cut with (6.2x6.2) space in middle. Bond was applied on intaglio surface of composite cuboids then self-adhesive resin cement was auto-mixed and applied surface . Finally, zirconia specimens were placed over composite resin specimens under 5 kg weight for 6 min and light cured. Fig. -Specimen testingA framework to hold the specimens was fabricated. The plexi was laser cut with a dimension of 7x6x7 and cemented to another plexi to act as a floor. Chisel tip was applied vertically to the cement zirconia interface at cross head speed of 0.5mm/min until failure which was defined by sudden drop of load (in newton). SBS was calculated according to equation: SBS (MPa) = load (N)/area(mm2).-Statistical analysisKolmogorov-Smirnov normality tests was considered to evaluate normality data distributions. Independent T-student tests were conducted to analyze differences in shear bond strength according to surface treatment method, primer coating, and saliva contamination.All statistical analysis was conducted using SPSS v.17 . Charts were created using Microsoft Excel 2018. An alpha level of 0.05 was used as a decision point for statistical significance.Effect of saliva contamination on SBS values (MPa) according to primer coating and surface treatment:According to independent T student test results in A successful restoration is not only defined by its strength, but mainly by stability of the cement-restoration adhesion. Zirconia is widely applied in the dental field due to its strength, esthetics and biocompatibility. Unfortunately, debonding of zirconia restorations has been observed in clinic situations. Many studies were carried out to evaluate the best surface treatment protocol.et al., reported acceptable surface roughness by Er:YAG laser at 400 and 600 mJ pulse energy , they compared effect of different Er:YAG output power  with different lasers and particle abrasion on surface characteristics of zirconia ceramics, they concluded that treated surfaces by Er:YAG laser and air abrasion showed similar surface roughness  showed highest SBS, similar results were found by Özcan cle size . Skienheintering . These fondition . Su et aIn current study, results showed that primer coating after surface treatment, increases SBS, even in the presence of saliva contamination. In previous studies, it was concluded that primer application on zirconia treated surface will protect the surface treatment from any contamination, assure good strength. That’s why in current study, primer was used with and without contamination, to check its efficacy in protecting treated surface .Additionally, surface should be free from any contaminants to prevent decrease in bonding efficacy . Hence, in vitro study, results showed that APA effect on shear bond strength was higher than laser surface treatment. Demir and Ozturk et al. and Caglar et al., demonstrated that APA method provided rougher surfaces than Er:YAG laser radiation and this method can be used to obtain micromechanical retention  when associated with a primer gave the highest bond strength  and cemented with resin containing MDP which is considered best standard in enhancing and increasing bond strength (et al., evaluated effect of primer on bond strength and durability of zirconia and concluded that primer could improve the primary resin bond strengths of zirconia ceramics (In present study, it could be suggested that primer coating is effective in increasing bonding of zirconia with or without contamination, when used after APA surface treatment. Results of current study were in accordance with Cavalcanti strength . Moreovestrength . Moreoveceramics .The challenge in promoting a strong, reliable bond between the intaglio surface of zirconia and tooth structure, lies in achieving surface free of the contaminants that often result from intraoral try-in procedures. In this study, surprising results revealed that the contaminated surface of zirconia yielded better result than the contamination free surface when treated with APA and coated with primer. These results may be explained by the fact that some of the artificial saliva inorganic components left after water washing, could had bound to specific primer element that led to increase the SBS of Y-TZP. Furthermore, these results could be by the effect of primer on zirconia surface, by making it more hydrophobic, leading to decrease effect of contamination on treated surfaces.et al., they concluded that when primer is applied prior to salivary contamination, water spray is able to return bond strength to its original state, according to their results, application of primer made the zirconia surface slightly more hydrophobic, probably, reducing salivary wetting ability and deposition of organic residue (et al., studied effect of decontamination and cleaning on shear bond strength of Y-TZP, they concluded that, coating with primer containing 10-MDP prior to human saliva contamination and followed by water preserved bond strength, suggesting that the zirconia surface is somewhat saturated and that, interaction of phosphate compounds from saliva is impossible (et al., studied chemical interaction mechanism of 10-MDP with zirconia, according to their findings, presence of phosphate groups was confirmed originating from primer containing 10-MDP at zirconia surface, even after washing with acetone, thereby indicating that strong interaction of primer with zirconia resisted washing (et al., concluded that, cementation surface contamination of zirconia restoration and inadequate removal of contaminants increase risk of failure (Stated by Angkasith  residue . Krifka possible . Nagaoka washing . Neverth failure . Yoshida failure . Clearlyet al. stated that, abrasive process removes loose contaminated layers, increases surface area available for bonding and improves wettability of luting material. Yang et al., have suggested that an additional particle abrasion may give good bonding results after contamination, comparable to that seen in groups without contamination (et al., stated that use of a second particle abrasion could be controversial as a result of potentially deleterious effect on zirconia phase transformation that could possibly weaken the zirconia ceramic (Earlier studies, stated that, retreating the zirconia surface prior to cementation, will ensure good bonding results. Amaral mination . Moreove ceramic .Shear bond strength showed higher results when zirconia is treated with APA, laser surface treatment showed lower results. Treating zirconia surface with primer significantly increases shear bond strength of zirconia, it is recommended to coat zirconia surface using primer after treating with APA for better bonding. Artificial saliva significantly increased shear bond strength of resin to zirconia when used after APA or Laser surface treatment.


J Cell Mol Med. 2019; 23: 4991– 5005 (https://doi.org/10.1111/jcmm.14348)Retraction: microRNA‐374 inhibits proliferation and promotes apoptosis of mouse melanoma cells by inactivating the Wnt signalling pathway through its effect on tyrosinase. Xiao‐Jing Li, Zhi‐Feng Li, Yan‐Yan Xu, Zhao Han and Zhi‐Jun Liu. wileyonlinelibrary.com) in Volume 23, pp. 4991‐5005, has been retracted by agreement between the authors, the journal Editor‐in‐Chief and John Wiley & Sons Ltd. The journal was informed by the authors of improper images used in Figure 1 and Figure 2, where tumour tissue images have mistakenly been included instead of paracancerous tissue. More follow‐up experiments are needed to confirm the validity of the results.The above article from Journal of Cellular and Molecular Medicine, published online on 17 June 2019 on Wiley Online Library (


The complication of uretero-arterial fistula after prolong ureteral stenting is well recognized. The treatment is primarily endovascular stenting across the fistulous communication accepting the potential risk of stent graft infection. Herein we present a case of a 71-year-old female who developed an uretero-arterial fistula after prolong ureteral stenting and exchanges following ileal conduit obstruction. Initial treatment with left common iliac stenting controlled the hematuria, but only temporarily. Repeat angiography revealed a type 1b endoleak requiring stent extension. Unfortunately, persistent hematuria necessitating further angiography showed the development of a saccular pseudoaneurysm around the stent graft requiring proximal stent extension. A nuclear medicine indium 111-tagged white blood cell scan with single-photon emission CT (SPECT)/CT confirmed stent graft infection. Conservative therapy with antibiotics failed, causing graft failure that ultimately required bypass surgery. Uretero-arterial fistula (UAF) is a rare but potentially life-threatening complication that should be considered in patients presenting with intermittent gross hematuria and a history of pelvic surgery/radiation, ureteral manipulation and chronic cannulation ,2. UAF aA 71-year-old female with a significant history of endometrial and vaginal cancer ultimately underwent exenteration with ileal conduit reconstruction after radiation and chemotherapy. She developed a left ureteral-ileal anastomotic stricture, ultimately requiring a retrograde transileal nephroureteral stent (RNUS). During a routine exchange of the RNUS, profound arterial bleeding was noted in the ileal conduit. Immediate angiography confirmed a suspected UAF where the left ureter overlapped the left common iliac artery. Hemostasis was obtained following left common iliac artery stent graft placement across the fistula. RNUS stent exchange was then completed successfully /CT was performed, which showed focal, radiolabeled white blood cell localization involving the left common iliac artery stent, suspicious for indolent infection Figure . DespiteCurrently, there is no gold standard for the management of UAF, although endovascular repair has become the preferred treatment. A majority of published cases report no long-term procedural-associated complications, although data are scarce -11. OkadComplications of stent graft infections include aneurysms, sepsis, and graft failure. Conservative treatment of stent graft infections includes antibiotics, while surgical therapy usually involves stent removal with reconstruction and bypass ,15,17. REndoleaks are well documented and considered a risk factor for stent graft infection, so is having multiple reinterventions ,19. PrimThis case highlights a rare but dreaded sequela (stent graft infection) of an equally rare complication (UAF). Strategies to avoid stent graft infection include maintaining sterile technique, appropriate antibiotic coverage, and mitigating procedural complications associated with infection (e.g. endoleaks). As endovascular repair is quickly becoming the preferred method for treatment of UAFs, awareness of this morbid outcome and strategies to best manage the risks becomes more prudent.


During processing of cereal-based food products, starch undergoes dramatic changes. The objective of this work was to evaluate the impact of food processing on the starch digestibility profile of cereal-based foods using advanced imaging techniques, and to determine the effect of preserving starch in its native, slowly digestible form on its in vivo metabolic fate. Four different food products using different processing technologies were evaluated: extruded products, rusks, soft-baked cakes, and rotary-molded biscuits. Imaging techniques  were used to investigate changes in slowly digestible starch (SDS) structure that occurred during these different food processing technologies. For in vivo evaluation, International Standards for glycemic index (GI) methodology were applied on 12 healthy subjects. Rotary molding preserved starch in its intact form and resulted in the highest SDS content (28 g/100 g) and a significantly lower glycemic and insulinemic response, while the three other technologies resulted in SDS contents below 3 g/100 g. These low SDS values were due to greater disruption of the starch structure, which translated to a shift from a crystalline structure to an amorphous one. Modulation of postprandial glycemia, through starch digestibility modulation, is a meaningful target for the prevention of metabolic diseases. There has been increasing attention on food processing methods, as transforming food products can modify the physiological fate of their components. There is growing controversy regarding the potential risks of some food processing technologies on health ,2. For sStarch is a major daily source of carbohydrates, and represents approximately 50–70% of human energy intake. Food starch is derived from cereals , tubers  and legumes . It consists of a semi-crystalline material produced by plants that forms roughly spherical granules in plant tissues . The sizThe food processing technologies and cooking methods applied to cereal products result in these products exhibiting a wide range of available starch digestibility profiles ,11,12,13Complementary approaches can be used to observe structural modifications to starch during food processing. These effects can be observed and measured by X-ray diffraction, micro-X-ray-tomography, and scanning electron microscopy ,25,26. SFurthermore, postprandial glycemic excursions, as well as the related phenomena of insulinemia and lipemia, have been implicated in the etiology of chronic metabolic diseases such as type 2 diabetes ,30. In aAt a mechanistic level, comprehensive studies have described the potentially deleterious effects of increased carbohydrate intake on the glycemic response ,35,36. TThe innovative goal of this work was to determine the relationships between complementary parameters involved in starch digestibility, from its observable structure inside food products to its in vivo metabolic fate, to the physiological response to consumption of cereal-based food products. The findings from this study will improve our understanding of the impact of food processing on human health, which is currently a topic of considerable debate.Seven wheat flour–based products were prepared using four different technologies. “Technology” is used here to refer to the series of processes involved in making a finished product. These processes include the ingredients used, the mixing and cooking conditions, and the machines used to make the products. The resulting products differed greatly in aspect, taste, and texture. Four technologies were tested with and without wheat bran to evaluate the impact of bran on starch digestibility: two rotary-molded biscuits, with and without wheat bran ; two extruded products, with and without wheat bran ; two soft-baked cakes, with and without wheat bran ; and a rusk-type product without wheat bran (Rk) only. The rotary-molded products, soft-baked cakes, and rusk were made at the Mondelēz International R&D center in Saclay, France. The extruded products were made in collaboration with Welience, based at the University of Bourgogne, Dijon, France.All products were made using the same ingredients in similar quantities, although some adjustments were made to accommodate the specific requirements of certain technologies. Eggs and glycerin were added to the soft-baked cake recipes . Different amounts of water were added to the dough as needed (from 0% to 12%). Finally, 6% of yeast was added to the rusk recipe .The nutritional parameters were measured analytically . In vitro starch digestibility was assessed using the SDS method developed by Englyst et al. . This meThree different micro-imaging methods were carried out by Novitom , a company that specializes in microscopy analysis.X-ray diffraction enables analysis of molecular structures at the micrometer scale ,42. SampThe four products without bran  were analyzed by micro-X-ray tomography. Each sample was crushed prior to analysis. Measurements were taken by synchrotron radiation , in which the density of a material is reflected by different degrees of X-ray absorption. The resulting images are shown in different shades of gray: denser materials are shown in lighter gray, whereas air bubbles show up as black or very dark gray patches.The electronic microscope was used in Field Emission Gun mode, a technique which requires no prior sample preparation . The microscope used was a Zeiss Supra55VP at the Physics Laboratory of Solids . The imaging was performed with a voltage of 1.8 keV at magnifications of ×200 and ×600.2, respectively. The mean fasting blood glucose level and blood glucose level 2 h after ingestion of a 50-g glucose solution were 5.12 (SD 0.25) and 4.60 (SD 0.59) mM, respectively.Twelve healthy, non-smoking volunteers between 18 and 45 years old participated in a randomized, open trial with a cross over study design at the Human Nutrition Unit within the School of Life and Environmental Sciences at the University of Sydney, Australia. The ethical committee approval number was 2015/082. They consumed test portions of the cereal products containing 50 g of glycemic carbohydrates with 250 mL of Evian water over a 10–12-min period. Capillary blood samples were collected at the time of consumption (T0) and 15, 30, 45, 60, 90, and 120 min later to construct glycemic and insulinemic response curves for each test product. As recommended by the International Standards , the subEach volunteer consumed one portion of each test product, as well as three servings of a glucose solution (reference food), which corresponded to 10 test sessions in total, separated by a minimum of 1 day of wash out. Detailed descriptions of the portion sizes and macronutrient contents of the seven cereal products and the reference glucose solution are shown in The GI of a given food reflects how much its digestible carbohydrate content raises blood glucose levels. It is defined as the incremental area under the blood glucose response curve (iAUCg) after consumption of a portion of test food providing 50 g of available carbohydrates, and is expressed as a percentage of the average iAUCg for the same amount of carbohydrates from a reference food (glucose) ingested by the same subject on a separate occasion (iAUCg test food/average iAUCg reference food × 100). The iAUCg is the incremental area under the blood glucose response curve , ignoring the area beneath the fasting concentration . Other ® Statistics software (version 14). Repeated-measures analysis of variance (ANOVA) was used to determine whether there were any significant differences between the mean GI and II values of the test foods. If a statistically significant product-effect was found, a post hoc multiple comparisons test was performed in order to identify the specific significant differences. For normally distributed data, the Least Significant Difference (LSD) test was used as the post hoc test for multiple comparisons. For non-normally distributed data, a global comparison was performed using the non-parametric Friedman test, taking repeated measures into account.Descriptive statistics  of all measured and calculated (GI and II) variables related to each food were determined using JMPFor a portion size containing 50 g of available glucose, the protein and fat content of all products was similar, ranging from 4–6 g/portion and 10–12 g/portion, respectively . The sugThe starch digestibility analysis revealed major differences between the fractions of starch in the products. The conversion from SDS to RDS occurred to different extents depending on the different food processing techniques. The SDS fraction was much higher, and the RDS was correspondingly much lower in RB and RnB compared to the three other technologies, soft-baked cakes, rusk, and extruded product . The resThe diffraction pattern of the wheat flour, which was used as a control, displayed a typical A-Type crystallinity profile with peaks at 2-Theta values of 15°, 17°, 18°, and 20°. As shown in graph A in As shown in graph D, the diffraction patterns for EB and EnB were very similar to one another, but did not match any of the previous patterns. They were characterized by a multitude of punctual peaks that did not match any commonly known starch diffraction patterns.As shown in In both rotary-molded biscuits (Rb and RnB), starch granules were embedded in a rough matrix but remained visibly intact . The stap < 0.001), especially between T15 and T60 (p < 0.0001). Both rotary-molded biscuits (with or without bran) exhibited the lowest GI values, at 47 and 43, respectively, which were significantly lower than those for all the other products  (p = 0.026) and Rk (p = 0.013), at 77  . The sof), at 77 .p < 0.001) (p < 0.0001). The average II values for the rotary-molded biscuits (RB and RnB) were significantly lower than the II values for four of the other products . The average II value for Rk was significantly lower than the II values for EnB (p = 0.001), SB (p = 0.027), and SnB (p = 0.013)  b. Ingest< 0.001) . There w= 0.013) .The innovative design of this study, which investigated cereal-based food products from starch structure to in vivo metabolic fate, demonstrates how food processing intrinsically modifies starch organization in cereal products, leading to different postprandial metabolic responses. We showed that rotary molding better preserves starch in its native state and slows its digestion, which led to a significantly lower GI and II compared to the other three processing technologies studied. Each food product was examined in detail using very precise imaging techniques to characterize the starch in each product and better understand the different metabolic effects of the seven cereal products. As described previously, starch granules undergo drastic structural changes during processing, with a shift from a semi-crystalline structure to an ever more amorphous one ,46. The The addition of wheat bran (4% to 7%) did not modify the impact of processing on in vitro starch digestibility and starch crystallinity. Indeed, the products generated by all three food processing technologies  exhibited similar results for all studied parameters, whether or not they contained bran. There is some evidence that the addition of wheat bran to a cereal product can decrease the glycemic response in vivo, especially for extruded products, but this difference is not statistically significant ,52. AddiIn addition to measuring glycemic parameters , plasma insulin was also measured to better understand how ingestion of these products modified physiological factors ,29,36. NWhile considerable care was taken to limit differences in the ingredients and macronutrient contents of the seven cereal products, the recipes did vary somewhat. Glycerin and eggs were added to the soft-baked cake recipes to facilitate processing. These ingredients did not have a significant effect on the final macronutrient composition of the test portions. There were only minimal differences among the portions used for the in vivo study, which are unlikely to have affected the GI . Indeed,The two cereal products with intermediate GI values (SB and Rk) and EB, which had a significantly higher GI, had similarly low SDS values (<2 g/100 g). This discrepancy could potentially be explained by the nature of the structural changes that occurred in the starch during processing. The diffraction patterns of SB and Rk were close to the typical A-type crystallinity of wheat starch, but the diffraction peaks at a 2-Theta value of 19.5° were more rounded, indicating the presence of amorphous material ,42, as wIn summary, the results from this study linked the effect of food process on starch organization inside the food matrix, starch digestibility profile, and finally their metabolic effect. Some food processing technologies led to greater starch alteration which induced high glycemic and insulin excursions in postprandial period. By contrast, the preservation of starch structure as close as possible to its native form, i.e., intact starch granules with low porosity, as obtained when using a rotary molding approach, led to a lower glycemic response and insulin demand. Modulation of postprandial glycemia is a meaningful target in the prevention of metabolic diseases. This can be achieved through the modification of dietary factors such as starch digestibility.


Starch digestibility, measured by an in vitro digestion model, was applied to determine the effect on starch fractions. The bread glycemic index was calculated. Bread antioxidant capacity  and ferric-ion-reducing antioxidant power (FRAP) methods) and total phenolic compounds were assessed. Anti-inflammatory properties according to enzymatic matrix metalloproteinase (MMP)-9 inhibitory activity were also studied. Considering the higher level of both dairy products tested  and comparing with control bread results, a reduction of around 35% in the glycemic response of curd cheese bread was achieved, resulting in intermediate index level (glycemic index (GI) 55–69), with yogurt bread still showing a high glycemic index (GI > 70). In terms of bread bioactivity, curd cheese bread expressed better reducing power effects, whereas yogurt bread showed more effective radical-scavenging capacity. An increase in bread phenolic compounds by yogurt (55.3%) and curd cheese (73.0%) additions (at 20%) were also registered. MMP-9 inhibition activity was higher in the dairy bread than in control bread, suggesting an improvement in terms of anti-inflammatory properties. The supplementation of the gluten-free bread by yogurt or curd cheese was shown to be a promising strategy to reduce the glycemic response and to improve the bioactive properties of the bread, that which can contribute to preventive diets of celiac patients and irritable bowel syndrome individuals.The influence of flour replacement by yogurt or curd-cheese additions (from 10% to 20%,  Wheat and gluten-containing products have been associated with a wide range of gastrointestinal disorders. Celiac disease (CD) is the most studied form of gluten intolerance, characterized by a small gut inflammation via an immune response to specific peptides of gliadin, one of the gluten proteins . This inFurthermore, gluten is not the only triggering component in gastrointestinal disorders. Many other components coexist with gluten in wheat and gluten-related food, such as members of the short-chain carbohydrate group , collectively termed FODMAPs , which are associated with several gastrointestinal symptoms in non-celiac gluten sensitivity (NCGS). Irritable bowel syndrome is the common gastrointestinal inflammatory condition associated with non-celiac gluten sensitivity symptoms, characterized by abdominal pain, bloating symptoms, diarrhea, and irregular bowel microbiota , stronglAccordingly, there has been an increase in demand for gluten-free products, not only due to the greater prevalence of celiac disease but also due to the prevalence of irritable bowel syndrome diseases, since gluten-free grains and derived products tend to be lower in FODPAMs .The recent development of gluten-free foods has focused on research aimed at overcoming the technical challenge of gluten removal from bakery products ,6, whoseThe glycemic response depends on several intrinsic properties of flour, such as starch grain structure and molecular size, protein and/or lipid content, and the amylose–amylopectin ratio . PreviouAdditionally, recent works ,13 showeIn the last decade, the search for anti-inflammatory inhibitors in food has been an important branch of research since it can be a promising strategy for preventive diets.Matrix metalloproteinases (MMPs) are a zinc-dependent family of endopeptidases, highly involved in the biological human process of connective-tissue remodeling . SpecifiAccordingly, the search for new protein-rich sources that contribute to reducing the glycemic index and for bioactive ingredient sources capable of an inhibitory effect against MMP-9 is an important topic of research, since it can contribute to a preventive diet, not only for celiac patients but also for those suffering from irritable bowel disorders. Dairy products (DP) are promising protein sources, with recognized nutritional properties and functional benefits. Additionally, they present bioactive compounds associated with human physiological properties, such as antioxidant ,19, antiFurthermore, a few studies showed particular interest in these products, especially those deriving from lactic acid bacteria (LAB) fermentation , in which secondary metabolites may contribute to an anti-inflammatory activity toward human immune processes, due to the bioactive peptides generated by LAB fermentative activity ,22,23,24Yogurt (Yg) is one of the most nutritious dairy products, widely consumed around the world, due to its functional benefits to the human diet, highlighting it as an alternative ingredient for bakery product supplementation ,26. Curd cheese (Cc) is a derived dairy product obtained via the manufacturing of soluble whey proteins, characterized by a rich protein source, essential amino acids , and minerals , and arow/w) in terms of a reduction in glycemic index and an improvement in the bioactivity of gluten-free bread. The impact on starch performance (pasting properties), determined using heating–cooling microdoughLab assays, was first assessed. To mimic starch digestibility in the human body, an in vitro digestion model was applied. The glycemic index of the gluten-free bread was calculated. The antioxidant capacity of gluten-free bread was evaluated based on scavenging effects ) and ferric-ion-reducing antioxidant power (FRAP). Total phenolic content (TPC) was also determined. The gelatinolytic activity of MMP-9 inhibition was assessed using fluorometric quantification (dye-quenched (DQ) gelatin assay) to assess the anti-inflammatory potential of the gluten-free bread obtained. Linear correlations were tested among pasting properties, starch digestibility, glycemic index, and antioxidant capacity, to acquire additional information about the processes involved.This work aimed to study the influence of flour replacement with yogurt or curd-cheese additions  used is a product from LongaVida, Portugal; The yogurt dry extract  was determined as described earlier . The fresh curd cheese (from whey cow milk) used was a commercial product from Lacticínios do Paiva ; The dry extract of curd cheese  was determined as described earlier . The dry matter of both dairy products was determined to be considered in the optimization of the gluten-free bread formulations since the replacement was based gluten-free flour basis.Other ingredients used in bread formulations  were comGluten-free bread dough formulations were prepared in according to the procedure earlier described by Graça et al. : the yeaw/w. Replacements were based on gluten-free flour basis, i.e., substituting the dry matter coming from each percentage of yogurt or curd cheese added, on 100 g of flour, as earlier described [w/w), sugar—2.8 g , dry yeast—2.8 g , xanthan gum—0.5 g , and vegetable oil—5.5 g .Considering the water coming from each level of the dairy products tested, the water added (WAD) was determined by microdoughLab mixing curves, and the water absorption was calculated according to the procedure earlier described by Graça et al. . Doughs escribed ,29: IngrThe different gluten-free bread formulations tested are presented in Foods.The nutritional composition of the different bread formulations, presented in w/w) on the starch physical behavior of the gluten-free bread dough, in comparison to control dough (CD), was studied by MicrodoughLab measurements , according to the method earlier described [The effect of yogurt and Cc additions . The supernatant (digestible starch fraction) was used for glucose determination. Aliquots were treated with 3 mL of sodium acetate buffer , and 60 μL of amyloglucosidase (3300 U/mL) were added, followed by incubation for 45 min at 60 °C, under constant stirring; volumes were adjusted to 10 mL with distilled water and centrifuged  was employed to describe the starch hydrolysis kinetics: C is the concentration at t time, The hydrolysis index (HI) was obtained from Equation (2), dividing the estimated areas under the hydrolysis curve (AUC 0–180 min) obtained for gluten-free bread and reference food (white wheat bread) ,31,32:(2The estimation of glycemic indices (eGI) was calculated according to Equation (3) :eGI =  by preparing methanolic extracts (20 mL of methanol), followed by centrifugation (8000 g/4 °C/20 min) and filtration (0.2 μm filter). The extracts were stored at −4 °C.The scavenging effect of bread extracts was determined using the DPPH  methodology : Bread eBread extract reducing power was evaluated applying the ferric ion reducing antioxidant power (FRAP) method : The breResults of scavenging activity and reducing power were expressed as mg ascorbic acid equivalents (AAE) per gram of bread extract. w/v, 300 μL), followed by room temperature dark room incubation (2 h). Absorbance (760 nm) was measured, and TPC results were reported in mg of gallic acid equivalents (AGE) per gram of bread extract.The total phenolic content (TPC) of bread extracts was assessed following the method earlier described : The breTests were performed in triplicates for each antioxidant activity assay, in three bread, to ensure reproducibility of the results.w/v)), and it was kept stirring for 4 h at 4 °C. After centrifugation  (Beckman J2-21M/E), the supernatant was collected and stored (−20 °C). The MMP-9 inhibitory capacity of the yogurt and that of the curd cheese were also evaluated, applying the same procedure described above.To evaluate the MMP-9 inhibitory activity of different crumb bread, a buffer-solution extraction was performed: Bread samples (5 g) were added to Tris-HCl buffer solution . A black micro-assay plate  was used and each well was loaded with 0.1 mM  MMP-9 , and 80 µL of each bread extract were incubated (30 min at 37 °C). The dairy products and bread extract volume was kept constant (80 µL), equivalent to the following protein content (mg/5 g of dairy product or bread): 8.64 ± 0.40 mg to Yg and 83.0 ± 3.15 mg to Cc, 8.6 ± 1.0 mg to CB, 13.0 ± 0.05 mg to yogurt bread (YgB) and 17.0 ± 0.51 mg to curd cheese bread (CcB). MMP-inhibition activity was tested using the dye-quenched (DQ)-gelatin assay as described before . The fluSubsequently, remaining MMP-9 catalytic activity quantification, the DQ-gelatin , was added to each plate well followed by incubation, for 1 h. Since the gelatinolytic activity is present, the DQ-gelatin substrate is hydrolyzed and releases fluorescence (fluorescence measurement conditions: excitation 485 nm/emission 530 nm). To correct possible gelatinolytic activity in the bread extracts, positive (without bread extracts) and negative (without enzyme) controls were also included, and all experimental data were corrected by subtraction the respective negative controls. Tests were performed in triplicates and applied in three bread.Statistical analysis in the experimental data   using variance test in one factor (ANOVA), by Tukey test , were assessed. A Nonlinear Rheology Model, to fit the experimental rheology data, was applied . Starch behavior changes, induced by the addition of yogurt or Cc, were assessed by heating-cooling cycles, to simulate the mixing and baking steps of the breadmaking process.p > 0.05) differences from control dough.During the first seconds of mixing, at constant temperature (30 °C), the torque was very low for all the tested doughs. This result was expected since these doughs comprise mixtures of flours without gluten. Starting the heating stage, at about 60 °C, the systems tended to phase separation, resulting in a further decrease of dough consistency (measured as torque—C2). The values of C2 were too low, namely, for yogurt dough (YgD), expressing a difficulty for the equipment to measure such low torque values, compared to control dough (control dough = 4.3 mNm ± 0.6 mNm). These results suggest that the caseins and exopolysaccharides (EPS) coming from yogurt addition are acting as lubricants with destabilizing effects on the dough matrix , reducinAs the heating phase proceeds (up to 95 °C), starch granules start gelatinizing and take the dominance on torque values, whereas dough consistency increases as a result of starch swelling .Significant impact on starch gelatinization performance was obtained for higher levels of dairy products tested: YgD20%, 101.0 mNm ± 8.5 mNm, and CcD20%, 92.0 mNm ± 1.7 mNm, in which a reduction of around 13% and 21%, respectively, compared to control dough (CD: 116.0 mNm ± 5.0 mNm), was registered. The diminished starch gelatinization observed can probably be attributed to the interaction between starch and protein coming from dairy product additions, reducing the starch availability to swell and break, affecting considerably the dough consistency . p < 0.05) were obtained on C4 values by yogurt additions, presenting values close to control dough (98.0 mNm ± 2.1 mNm). However, for curd cheese incorporations, significant differences in C4 torque values were noticed, varying from 77.0 mNm ± 3.2 mNm for CcD10% to 65.0 mNm ± 0.6 mNm for Cc20%, compared to control dough, representing a reduction around of 22% and 34%, respectively. As a consequence of continuous starch granule physical breakdown, due to the mechanical shear stress, further reduction in torque units occurred , and at this phase of cooking, the amylase activity takes the dominance, followed by the amylose network stabilization . No signSubsequently, on cooling (from 95 °C to 50 °C), starch retrogrades and the torque values increase (C5). In terms of final consistency values (C5), slight differences were obtained for yogurt additions, varying from 230 mNm ± 15.1 mNm for Yg10% to 220 mNm ± 16.1 mNm for Yg20%, compared to control dough (250 mNm ± 5.6 mNm). Nevertheless, curd cheese additions promoted a significant impact on C5 values, ranging from 165.3 mNm ± 5.5 mNm for Cc10% to 150.0 mNm ± 1.5 mNm for Cc20%, representing a reduction of around 34% and 40%, compared to control dough values. This considerable reduction in final torque can be attributed to a sort of protective effect of curd cheese proteins on the starch granules hindering gelatinization, reducing the damage of starch granules, and lowering amylose concentrations on continuous phase. Besides, it suggests that the starch granules in the curd cheese dough should be less available to enzymatic hydrolysis than in yogurt doughs. Besides, curd cheese lipids may induce some lubricant effect, further reducing the final consistency.R2 = 0.922) were found between the final consistency and dairy products additions, meaning that the effect on reducing final consistency is proportional to the level of dairy products added (Linear correlations presented in Linear correlations ((i)Insufficient hydration of starch granules resulting from the competition for free water by denatured whey proteins (derived from curd cheese additions );(ii)Starch-protein interactions, reducing the accessibility of enzymatic attack to starch granules, thus limiting the degree of starch hydrolysis ;(iii)Amylose-lipid complexes formed during cooling, lowering the final peak consistency .The impact of curd cheese additions on starch behavior was remarkable compared to yogurt incorporations, and it could be attributed not only to the dilution effect on starch but also to the following scenarios:Results suggested that the different dairy proteins added by yogurt and curd cheese incorporations, promoted different effects on starch performance, mainly on starch gelatinization (C3) and on final dough consistency (C5), being higher for curd cheese incorporations, that probably can have an impact in reducing the glycemic response of bread.The influence of yogurt and curd cheese additions to gluten-free bread dough on digestible and resistant starch fractions was evaluated, and the results obtained are presented in Starch digestibility can be characterized into rapid digestion (RDS)—hydrolyzed within the first 30 min of human digestion; slowly digestion (SDS)—digested within 60–180 min; and resistant starch (RS)—not enzymatically hydrolyzed during human digestion (0–180 min) .p < 0.05) differences on the RDS and RS fractions were achieved, whereas no significant impact was registered in SDS fraction, compared to CB.From w/w) tested, where the SDS took the dominance, followed by the RDS and RS. The SDS fraction, being slowly digested, is more desirable than RDS since it promotes a gradual increase in plasma glucose and insulin levels [Starting from control bread, the dominant starch fraction was RDS followed by SDS and RS. However, upon the addition of yogurt and curd cheese to the dough, this pattern changed, essentially for higher levels (20% n levels .w/w), where a significant reduction of about 24% and 44% for yogurt bread and 40% and 67% for curd cheese bread were registered, compared to control bread. Moreover, considerably increases in RS values of around 20% and 80%, and 70% and 110%, respectively, were also obtained.Yogurt and curd cheese are known to be rich-protein sources (even richer in the latter’s case). Therefore, protein-starch interactions within bread structure, further boosted by the baking process, might affect the digestive enzymes’ accessibility to starch granules, hindering their hydrolytic performance.These significant decreases of RDS fraction and subsequent increase in RS levels can most probably be linked to the negative impact promoted on starch gelatinization performance (C3). Since the starch was less available to swell and break, probably due to the starch-protein interaction established, the accessibility of enzymatic attack to starch granules was diminished, thus limiting the degree of starch hydrolysis.R2 = 0.993) and RS (R2 = 0.933) results with the protein content of yogurt and curd cheese bread, at 10% and 20% addition  were found among dairy product additions to the dough, and RDS and RS fractions, as well as the relation between starch fractions and final consistency results , starch hydrolysis was estimated by the amount of glucose released. Hydrolysis curves and starch hydrolysis vs. time are plotted in All digested bread samples exhibited similar behavior, in which the extent of glucose release displayed an almost linear rise between 30 and 60 min of hydrolysis, followed by a gradually diminished as hydrolysis time proceeded (90–180 min).R2 > 0.915) to a nonlinear model (Equation (1)) [k), hydrolysis index (HI), and estimated glycemic index (eGI) and are summarized in The influence of yogurt and curd cheese supplementation, in vitro kinetics starch digestion of the bread, was also evaluated, based on primary and secondary parameters derived by fitting the experimental data (ion (1)) . These fp < 0.05) influence in C∞ reduction, varying from 28.0 g/100 g to 27.0 g/100 g for yogurt additions, and 20.0 g/100 g to 18.0 g/100 g for curd cheese incorporations, compared to 33.1 g/100 g obtained for CB. Considering the highest levels of yogurt and curd cheese tested , these variations represented a decrease of around 18% and 46% of the equilibrium concentration, respectively, compared to control bread. Incorporation of yogurt and curd cheese had a significant . Lower values of k suggest that the increase of dairy product in the dough is promoting a higher resistance to enzymatic starch hydrolysis, and this may be explained by a starch–protein interactions, limiting starch digestibility.The These results are in line with those obtained on digestible starch fractions, where a significant reduction in RDS and an increase in RS were noticed. Comparable findings were obtained recently by other researchers  by “Amalw/w). Despite the variations obtained for yogurt levels tested , the estimated values of the glycemic index are still showing that they maintain a high glycemic response (GI > 70).These results were reflected in estimated values for the glycemic index, resulting in an intermediate glycemic index for curd cheese bread (GI 55–69), a considerable reduction of about 35% for both dairy products levels tested , the incorporation of the higher level  of yoghurt  and of curd cheese  led to an increase in bread AC, representing an improvement of around 140% and 50% for yoghurt addition and about 71% and 70% for curd cheese incorporation, respectively. The RSC increment on yogurt bread can probably derive from α-tocopherol (vitamin E) since the bioactive properties of this compound contribute to oxidative stability properties [The antioxidant capacity (AC) of gluten-free bread enriched with yogurt or curd cheese additions was tested by DPPH and FRAP methods. From operties . Additiooperties  that prow/w) ranged from 13.2 mg·mg−1 AGE for YgB, and 15.0 mg·mg−1 AGE for CcB, compared to 8.50 mg·mg−1 in control bread, representing an increase of 55.3% and 73.0%, respectively. The increase of PC in bread, deriving from dairy products additions, probably contributed to enhancing the antioxidant activity of the bread, expressed as FRAP activity. The linear correlation (R2 = 0.855) calculated between TPC and FRAP activity supports this relation (Lycium ruthenicum addition to bread fortification and in vitro digestibility impact.Phenolic compounds (PC) are regarded as an important class of secondary metabolites, some of which exhibits health-promoting benefits, including antioxidant activity . The addrelation , Table 3The impact of yogurt or curd cheese supplementation on gluten-free bread was evaluated by its capacity to inhibit MMP-9 enzyme activity. The DQ-gelatin assay was used to evaluate the anti-inflammatory capacity of bread. The results obtained are illustrated in The MMP-9 inhibitory capacity of yogurt and curd cheese was assessed individually , in whicp < 0.05) on MMP-9 activity inhibition, compared to the control (C+). The highest inhibition of MMP-9 activity was obtained in CcB20% (40%), followed by YgB20% (30%), and finally by the control bread (17%). One can suggest that the supplementation of the control bread with each one of the two dairy products tested can be an interesting approach to improve its bioactivity in terms of anti-inflammatory properties, and these properties can probably be further enhanced by the breadmaking fermentation process.Several studies have been shown the anti-inflammatory properties of dairy products, especially those obtained by fermentation processes, associating them as anti-inflammatory agents on humans’ immune processes ,24.Limonium tetragonum extract and are also in line with those reported by other authors [These results agree with those obtained in a recent study , which s authors  based onIt can be stated that the MMP-9 inhibition activity exhibited by gluten-free bread with yogurt or curd cheese may constitute an interesting contribution to inflammatory bowel disease preventive diets. Besides, the incorporation of these nutritional dairy products creates an advantage since it can be consumed by the entire population, even by celiac patients, improving their daily diet.According to the results presented along with this research work, a considerable impact on glycemic index reduction by yogurt or curd cheese additions to gluten-free bread was obtained. The relationships among pasting properties, starch digestibility, antioxidant capacity, and glycemic index should be considered, to give additional support and consolidate these findings. R2 > 0.850) are presented in Linear correlations found between pasting properties, starch digestibility, antioxidant capacity, and glycemic index results (R2 = 0.922), RDS content (R2 = 0.910), RS fraction (R2 = 0.940), and the glycemic index (R2 = 0.922) of the breads were obtained. In addition, glycemic index results were also strongly correlated with all these parameters, FC (R2 = 0.900), RDS (R2 = 0.940) and RS (R2 = 0.950). These linear relations reflect a strong correlation between dairy product addition and starch performance changes that, in turn, led to a considerable impact on in vitro starch digestibility and, consequently, on the glycemic index of the gluten-free bread.Strong correlations between the dairy products addition and final consistency (FC) (R2 = 0.900) and glycemic index and FRAP (R2 = 0.920), suggest that the enrichment in PC and the increase of FRAP activity by the addition of dairy product probably promoted an additional effect on the reduction of the glycemic index of the gluten-free bread. These results agree with those published by other authors [According to previous studies ,12 diges authors ,12 reinfFrom this study, in which the influence of yogurt or curd cheese supplemented to gluten-free bread was evaluated, to reduce the glycemic response and to improve bioactivity, encouraging results were obtained.Considering the glycemic index results, the impact of curd cheese addition to dough was greater than those obtained by yogurt additions, significantly reducing the glycemic response to intermediate values of the glycemic index (glycemic index: 55–69).Improvements in the glycemic response can probably be associated not only to the dilution effect of starch granules and physical interactions between starch and proteins but also to the presence of some bioactive metabolites , derived from the dairy products additions, that we’re able to slow the enzyme’s hydrolysis activity performance while improving the bioactivity of gluten-free bread.It was possible to improve the bioactivity of bread with both dairy products additions, in terms of antioxidant capacity and phenolic compounds.The enrichment of gluten-free bread with yogurt or curd cheese resulted in effective inhibition of MMP-9 activity, suggesting that both can be interesting baker’s ingredients to improve the bioactivity of bread in terms of anti-inflammatory properties and possibly anticarcinogenic effects. Nevertheless, further in vitro and in vivo assays to consolidate these anti-inflammatory properties achieved would be tested in future surveys.The gluten-free bread obtained can give an important nutritional contribution to the celiac and irritable bowel syndrome patients’ daily diet as well as to an inflammation preventive diet strategy.These findings can be a new window of research to prove the positive impact of dairy product addition to reduce the glycemic index and increase the antioxidant and anti-inflammatory potential of foods.In summary, the incorporation of yogurt or curd cheese in gluten-free bread showed to be an interesting strategy to improve the glycemic response and anti-oxidative and anti-inflammatory activities, thus contributing to fulfill the needs of the celiac individual’s daily diet.