Progression-free survival (PFS) and overall survival (OS) of these patients are favorably comparable with standard therapies. The protagonist in this type of therapy is a somatostatin-modified peptide fragment ([Tyr3] octreotide), equipped with a specific chelating system (DOTA) capable of creating a stable bond with β-emitting radionuclides, such as yttrium-90 and lutetium-177. In this review, covering twenty five years of literature, we describe the characteristics and performances of the two most used therapeutic radiopharmaceuticals for the NETs radio-treatment: [ There are several combinations of molecular targeting vectors and radionuclides suitable for theranostic use. Multi-element radiopharmaceuticals, consisting of two radioisotopes possessing similar chemical properties but having different physical emission properties, for example, Tc/188Re ,3,4, 68G68Ga/90Y , used foThe nuclear medicine research, in this particular therapeutic field, is constantly evolving thanks to the strong multidisciplinary synergy. In particular, the close collaboration of specialists from different disciplines such as physics, chemistry, radiochemistry, biochemistry, pharmacology, and nuclear medicine has determined the possibility to offer targeted therapies against solid neoplasms, such as NETs.NETs include a heterogeneous group of neoplasms, exhibiting a variable biological behavior, that can originate from various organs with an estimated incidence of about 5 new cases per 100,000 individuals per year .Overall, the highest incidence of these neoplasms is affecting the organs of the digestive system, in particular ileum and pancreas and, less frequently, stomach, duodenum, colon and appendix, constituting the gastro-entero-pancreatic NET (GEP) and representing 60–70% of all NETs. In addition to GEP NETs, other histotypes, affecting, in particular, the respiratory system and bronchi (20–30%) or other organs (10%) such as skin, thyroid, parathyroid, thymus, paraganglia and adrenal glands, can be classified as non-GEP NETs ,9.111In]In-pentetreotide or PET-CT with [68Ga]Ga-SST-As) Y-DOTATOC, [90Y]Y-DOTATATE, [177Lu]Lu-DOTATATE, [177Lu]Lu-DOTATOC. However, the greatest clinical experience (and the most abundant scientific literature) refers to the use of [90Y]Y-DOTATOC, which represented the first generation radiopharmaceutical for NETs. More recently, the [177Lu]Lu-DOTATATE was used in the phase 3 Netter-1 study Y-DOTATATE while the interest and use of [177Lu]Lu-DOTATOC is still growing Ga-DOTATOC), we proposed both [90Y]Y-DOTATOC and [177Lu]Lu-DOTATOC as therapeutic radiopharmaceuticals octreotide (TOC), octreotide (TATE) and [1-Nal3] octreotide (NOC), with peculiar SST receptors affinity. Focusing the attention on the chemical characteristics of these ligands, it was found that the replacement of Phe3 in octreotide by Tyr3 leads to an improved SST-R2 affinity, but the SST-R3 and SST-R5 affinity is reduced; the C-terminal introduction of Thr for Thr(ol) in TATE results in a SST-2- selective ligand with a 7-fold improvement of SST affinity. The NOC derivative  leads to a peptide with affinity to SST2,3,5. -octreotide or SMT487 or Edotreotide), somatostatin analogue, functionalized with an amide bonded to a chelating bifunctional macrocycle DOTA -Tyr3-octreotide -Tyr3-octreotide , representing the “VECTOR” component, is responsible for the pharmacokinetics and pharmacodynamics of the final radiopharmaceuticals Y-DOTATOC and [177Lu]Lu-DOTATOC radiopharmaceuticals respectively.The 90Y and 177Lu are responsible for the selective irradiation of the target and collateral irradiation of critical organs . It follows that the pharmacological characteristics of [111In] In-DOTATOC, [68Ga-]Ga-DOTATOC, [86Y]Y-DOTATOC, [90Y] Y-DOTATOC and [177Lu]Lu-DOTATOC can be considered almost superimposable, especially for the last two radiopharmaceuticals Y- or [111In]In-DOTATOC were used to predict the in vivo behavior of radiopharmaceuticals based on the use of DOTATOC radiolabelled with β- isotopes with higher energy emitters such as 90Y and 177Lu Y-DOTATOC was the first of the two radiopharmaceuticals to be produced as described in the review by Otte and collegues (1998) showing that, when administered systemically, [90Y]YDOTATOC stably binds to neoplastic lesions expressing SST-Rs  Lu-DOTATATE was the first radiopharmaceutical of 177Lu used in PRRT and subjected, starting from the 2000s, to phase II clinical studies. Consequently, the greatest pharmacological knowledge refers to this radiopharmaceutical, so much so that today it is the only radiopharmaceutical approved by the competent authorities for [177Lu] Lu-DOTATATE. DOTATATE, as mentioned above, has a different affinity (compared to DOTATOC) for SST-R2 Y-DOTATOC or [177Lu]Lu-DOTATOC is still mainly performed using manual procedures although the radioactivity managed during the synthesis and fractionation of these radiopharmaceuticals is very high. Normally, the radiopharmaceutical product is a 0.9% NaCl/buffer solution  containing an activity between 16.65 and 24.05 GBq in a total volume approximately 18 to 26 mL. The radioactive concentration of the solution is on average about 0.92 GBq/mL. However, regardless of the production method adopted, the synthesis and quality control procedure is the same and is described hereafter in accordance with European Pharmacopoeia. According to the revised procedure [177Lu]Lu-DOTA-TOC and [90Y]Y-DOTATOC were obtained conjugating the [177Lu]LuCl3 or [90Y]YCl3 with the DOTATOC peptide in the acetate form. The radiopharmaceutical synthesis must be carried out in a classified isolator. Both radiopharmaceuticals can be prepared by adding into the reactor, containing the radioisotope [177Lu]LuCl3 (or [90Y]YCl3), the solubilized peptide in ascorbate buffer , sterilized (0.2 mm sterile filter)  b and fin filter) c. The ad177Lu makes the radiopharmaceutical more suitable for the treatment of small lesions (≤20 mm) while the 90Y makes it more suitable for the treatment of large lesions (>20 mm) Lu-DOTATOC and [90Y]Y-DOTATOC.Since many researchers have produced and collected pharmacokinetic and pharmacodynamic data, comparing the different radiopharmaceuticals belonging to this radiopeptides category, these data will be presented in the following paragraphs with particular reference to MK-678 to evaluate the expression of surface membrane receptors and found that the binding of somatostatin peptides with high SSTR2 affinity and antiproliferative properties were powerful inhibitors of [125I]MK-678 binding to various tumor species, indicating that they can practice antitumor effects via the SSTR2 receptor.In a study conducted in 1994 by Taylor et al.  the pres111In]In-DTPA] octreotide, OctreoScan®; London, UK), was the ideal chelator for the radiosynthesis of radiolabeled somatostatin analogues. Moreover, a new modified octreotide Tyr3-octreotide (TOC), conjugated with the DOTA chelator (DOTATOC) and radiolabeled with indium-111 or yttrium-90 showed greater affinity for tumor cells expressing receptors for somatostatin with respect to [111In-DTPA0]-octreotide] and, therefore, a high diagnostic and therapeutic potential, respectively.De Jong et al. , in one 90Y]Y-DOTATOC has decreased by 65% intravenous administrated D-lysine, while the radioactivity of the blood, the pancreas and the adrenal glands were not affected. The D-lysine may be preferred to the L-lysine for reducing renal absorption of radioactivity during scintigraphy and PRRT due to lower toxicity and its disturbing with the metabolic equilibrium of natural amino acids.The potential nephrotoxicity following therapeutic use of radiolabeled somatostatin analogues was already known after the first pharmacological and scintigraphic studies with radiopeptides. In an animal study conducted by Bernard et al. , it was In a study published the following year, de Jong et al.  confirme90Y]Y-DOTATOC binds rapidly to the tumor of rats; (2) 24 h after administration, the ratio of radiopharmaceutical concentration in the tumor to blood is equal to 49.14; (3) a single administration of 10 mCi/kg of [90Y]Y-DOTATOC is able to determine a complete response in 5/7 experimental animals, with no signs of recurrence disease in the following eight months.In the same year, Stolz et al.  publishe0-(D) Phe1-octreotide (DOTAOc), DOTA0-(D) Phe1 Tyr3-octreotide (DOTATOC), DOTA0-(D)®Nal1-lanreotide (DOTALan) and DOTA0-(D) Phe1-vapreotide (DOTAVap) were tested, compared and better defined from a pharmacological point of view radiolabeled with indium-111, iodine-125 and yttrium-90 and tested in mouse models carriers of AR4-2J tumor with high SST-R2 receptor expression. For all DOTA-peptides included in the study, regardless of the incorporated isotope type, favorable biodistribution profiles were observed in animals with: (1) high receptor affinity for SST-R2 membrane receptors located on AR4-2J tumor cells; (2) a rapid clearance of radiopeptides from all tissues not expressing SST-R2 with the exception of the renal parenchyma and a specific uptake of radiopeptides in tissues expressing SST-R2 and in particular in tumor lesions; (3) excellent intra-tumor penetration of DOTA-peptides, and of DOTATOC in particular, which correlates with a high diagnostic and therapeutic efficacy.In a fundamental study by Froidevaux et al.  in 1999,50) of TOC, DOTATOC and Y-DOTATOC  for SSR-T2 has the highest values compared to the other analogs and that the bond with DOTA influences the binding affinity between the TOC peptide and the SST-R2, while the further incorporation of the metal inside the DOTA does not involve changes in receptor affinity.To get these results, chelator-free peptides [Oc and TyrOc (or TOC)], DOTA-chelated peptides (DOTATOC) and peptides conjugated with non-radioactive yttrium were tested, taking into account that the incorporation of the metal, radioactive or not, could influence the receptor affinity of the complex. The results obtained showed that the affinity  and, therefore, the highest tumor retention value in the 48-h observation period. Renal distribution was maximal at 4 h after administration and for DOTATOC was relatively low compared to the other peptides.A valid indicator of efficacy and safety for the therapeutic use of radiopeptides has been identified in the ratio between the surface area (under the distribution curve in the period 4–48 h after administration) relative to the tumor and the surface of the tumor area (under the distribution curve in the period 4–48 h after administration) relative to the renal parenchyma. The tumor/kidney ratio of DOTATOC was, once again, more advantageous than the other radioligands .This study has therefore shown that, among the DOTA-peptides examined in animals, DOTATOC has the best pharmacological profile, and it is probably the most suitable and promising radioligand for nuclear medical oncological clinical applications.90Y]Y-DOTATOC in CA20948 pancreatic tumors, of various sizes, transplanted into Lewis rats. The study showed the ability of ([90Y]Y-DOTA(0),Tyr(3))octreotide to control tumor growth, especially in medium-sized tumors and included in a volumetric range ≥1 cm2 and ≤14 cm2. The effect of radionuclide therapy appeared to be dependent on tumor size at the onset of therapy. They confirmed the therapeutic effect of [90Y]Y-DOTATOC previously demonstrated by studies Stoltz et al. Y-DOTATOC and [177Lu]Lu-DOTATATE in rats with tumor lesions (CA20948 or AR42J) expressing somatostatin receptors ranging in size from 0.1 to 15 cm2. The animals were treated with 1–2 intravenous administrations (one week apart) of 277.5–555 MBq of [177Lu]Lu-DOTATATE or 379 MBq of [90Y]Y-DOTATOC and followed for at least 150 days after therapy. They observed that the antitumor response obtained depended on the size of the lesions and, in particular, that 177Lu was more suitable for small lesions while 90Y showed greater efficacy on large lesions. These studies subsequently paved the way for clinical trials reaching conclusions still valid and applied in the most modern clinical protocols of PRRT Y-DOTATOC at doses of 120, 240, 360 mCi/m2 have documented a no-observed-effect-level (NOEL) value in monkeys at 120 mCi/m2. The NOEL values in rats treated daily for 4 weeks were: 1 mg/kg/day (6 mg/m2/day) and 0.3 mg/kg/day (1.8 mg/m2/day) in males and females, respectively. The NOEL values in monkeys, treated intravenously daily, for 4 weeks, were: 0.3 mg/kg/day (3.6 mg/m2/day) and 1 mg/kg/day (12 mg/m2/day) in males and females, respectively [The toxicity profile of DOTATOC was assessed—as such and after radiolabeling with ectively .86Y [111In [90Y and 177Lu allowed one to obtain accurate predictions in terms of absorbed dose to tumor lesions and healthy/critical organs and efficacy and safety profile. The evaluation of the kinetics of the system is carried out using a multi-compartmental developed by Cremonesi et al. [111In]In-DOTATOC [111In]In-DOTATOC from the blood compartment is very rapid and the circulating rate decreases to less than 9 ± 5% within the first hour and to less than 0.9 ± 0.4% within 10–12 h of intravenous administration.The DOTATOC pharmacokinetics and biodistribution have been studied in humans after labeling with 86Y  and withY Y-DOTATOC, obtained superimposable pharmacokinetic data and demonstrated that: (i) there is a rapid disappearance of the radiopeptide from the bloodstream with a plasma residue of the injected activity of approximately 5% after 5 h and ≤1% at 24 h; (ii) the circulating activity is entirely confined to plasma with a share linked to red blood cells of less than 1%; (iii) the fraction of stable radiopeptide circulating in the plasma is high (95.3 ± 4.7%) and that this value remains constant over time (with observations up to 3 h after administration) confirming the effectiveness of the chelating molecule DOTA; (iv) intravenous co-infusion of amino acids (L-Lysine and or L-Arginine) for the protection of the renal parenchyma did not significantly modify the plasma clearance curve of [89Y]Y-DOTATOC.Jamar et al.  with [89177Lu]Lu-DOTATATE is comparable to that of [111In]In-DTPA-octreotide, with rapid scintigraphic visualization of the renal parenchyma after administration and of the liver, spleen, kidneys, (and in some patients pituitary and thyroid) and tumor lesions 4 h after administration. The authors also reported in their study that the distribution patterns of the two radiopeptides at 24 h after administration are similar, with corresponding uptake on the liver, kidney and spleen; on the other hand, tumor lesions are more intensely (up to 3–4 times) and longer uplifting after administration of [177Lu]Lu-DOTATATE. From the percentage biostribution data obtained, the absorbed dose values for critical organs and tumor lesions were extrapolated. For the latter, it was demonstrated, by theoretical model, that the absorbed dose was 4 times higher for [177Lu]Lu-DOTATATE compared to [111In]In-DTPA-octreotide and 2 times higher for [177Lu]Lu-DOTATATE compared to [90Y]Y-DOTATOC [Kwekkeboom et al.  document-DOTATOC .177Lu]Lu-DOTATATE with [177Lu]Lu-DOTATOC. For both, the clearance in the blood is very rapid and the plasma concentration is less than 10% of the activity administered after 3 h. During the first 24 h the clearance of [177Lu]Lu-DOTATOC is slightly faster than that of [177Lu]Lu-DOTATATE but this difference is not statically significant and, therefore, the clearance is evaluated as overlapping for the two radiopeptides.Esser et al.  compared177Lu]Lu-DOTATATE, with a mean ratio of 2.1:1 compared to [177Lu]Lu-DOTATOC. Similarly, the value of τ is in the ratio of 1.4:1 and 1.5:1 for the renal and splenic parenchyma, respectively. The authors conclude that, based on residence times, a higher dose absorbed to the tumor but also a higher absorbed dose to the renal parenchyma is foreseeable in the case of [177Lu]Lu-DOTATOC, in the case of [177Lu]Lu-DOTATOC a lower absorbed dose to the tumor, but also a lower dose absorbed to the kidney which, however, remains the critical and limiting organ in PRRT.As regards the residence time τ in tumor lesions and in healthy/critical organs, however, a significant difference has been documented which, with the same clearance times and in the presence of the same isotope and the same chelating molecule, is imputable—as expected—to the different receptor affinity that the two distinct somatostatin analogues (TOC and TATE) have for SST-R2. In particular, the tumor τ value is significantly longer for patients treated with [177Lu]Lu-DOTATOC delivers the lowest doses to healthy organs and has a more advantageous ratio—in terms of absorbed dose—between tumor and kidney, compared to [177Lu]Lu-DOTATATE and [177Lu]Lu-DOTANOC  which deserves extensive consideration in the dosimetric evaluation, in order to limit renal damage. The deposit of radioactive urine at the level of the bladder cavity is a source of irradiation of the wall of this organ : this situation must also be taken into consideration, especially for when it concerns the aids to stimulate and facilitate diuresis.The elimination of the unbound portion of radiolabeled peptide occurs mainly through the renal emunctorium and, to a lesser extent, through the intestinal emuntorium. The amount of radiopetide present in the pre-urine undergoes reabsorption (mediated by an active transport mechanism) at the level of the proximal convoluted tubule cells. This reabsorption—if a somatostatin analogue radiolabelled with 111In]In-DOTATOC it was estimated that the cumulative activity of radiopeptide eliminated in urine was 52% ± 12% of the amount administered 4 h after administration and 73 ± 11% after 24 h from administration. Although the radioexposure of the renal parenchyma in the case of [177Lu]Lu-DOTA-SST-As is lower than the values reported in PRRT with [90Y]Y-DOTA-SST-As, it is still essential to consider the kidney as a critical organ and apply all available aids  in order to limit the damage.From the studies carried out, it has been shown that the amount of radiopetide eliminated through the intestinal emuntorium is reduced to the limits of significance and therefore determines a negligible dosimetric load. With [86Y]Y-DOTATOC Lu-DOTATOC compared to [177Lu]Lu-DOTATATE with percentages equal to 81% and 71%, respectively.Esser et al.  showed tIn two decades of follow-up, relating to numerous clinical studies, a lot of information was extracted about the safety and efficacy of PRRT. In terms of safety, PRRT is a well-tolerated treatment, with absent or mild-moderate toxicity in most cases, if performed with the correct prescribing methods and adopting all measures aimed at limiting the absorbed dose to the renal parenchyma and to the hematopoietic marrow, which represent the critical dose-limiting organs. In terms of efficacy, PRRT is able to determine an objective response with a reduction in size of lesions and tumor-specific markers, a reduction in symptoms and therefore an improvement in the quality of life and, overall, an increase in survival times.177Lu in PRRT was introduced in 2000, with the somatostatin analogue DOTATATE peptide. The use of [177Lu]Lu-DOTATATE has spread rapidly and—especially due to its potential better profile of renal toxicity due to the physical characteristics of the isotope used—has relegated the use of 90Y radiolabeled peptides to the background. The first studies with [177Lu]Lu-DOTATATE were carried out at the Erasmus University of Rotterdam. The key work regarding PRRT with [177Lu]Lu-DOTATATE is that of Kweekeboom et al. [177Lu]Lu-DOTATATE, for 4 consecutive administrations, with intervals of 8 weeks. As regards the objective response—measured with the SWOG criteria—the following results were obtained: CR: 2%, PR: 28%, MR: 16%, SD: 35%. In the same population, the median OS was found to be greater than 48 months and the PFS equal to 33 months. Direct comparison with a series of published data from a similar population estimated that patients undergoing PRRT with [177Lu]Lu-DOTATATE achieved an estimated survival benefit of 40–72 months. Although these data are not derived from a randomized study, the difference in survival reflects the real therapeutic impact that PRRT can have in the care of NET patients. The authors state that the data relating to the response obtainable from PRRT can compare well with those relating to other treatments, such as chemotherapy. In this study, it was also observed that the best therapeutic responses were obtained in those patients who showed high lesional uptake at baseline scintigraph with OctreoScan® and limited liver disease. In contrast, disease progression was more frequent in patients with low performance status and extensive liver disease. An attempt to categorize the objective response on the basis of histology allowed us to understand that NETs of pancreatic origin had a tendency to respond better than other GEP NETs and that functioning forms (pancreatic gastronomy) had a tendency to relapse earlier than to the remaining histotypes included in the study. In this study, the authors evaluated toxicity in 504 patients. Acute side effects that appeared within 24 h of radiopharmaceutical administration included nausea, vomiting and abdominal pain (10–25% of administrations). Grade 3–4 hematological toxicity (WHO), although transient, affected 9.5% of patients. Episodes of severe late toxicity  involved 13 patients (2%).The use of m et al. , which i177Lu]Lu-DOTATATE: these studies  of bronchial NETs (B -NETs) and other histotypes (some not strictly NETs but still expressing SST-R2). In particular, the following are summarized: the sample of treated patients, the histological variant, the objective response, the survival times, the toxicity profile and the most relevant clinical conclusions.On the experience of the Dutch group, other European Centers have carried out PRRT studies with [ studies  althoughR1 study  is the f177Lu]Lu-DOTATOC is another radiopeptide used in PRRT [1LuLu-DOTA in PRRT .177Lu]Lu-DOTATOC used in patients with advanced NETs. 56 patients with metastatic and progressive disease were studied  and treated with various cycles (1-4) of [177Lu]Lu-DOTATOC, with a median activity per cycle of 7.0 GBq and with intervals of 3 months between one cycle and the next. The results of this study confirmed that [177Lu]Lu-DOTATOC determines an objective response and survival times comparable to those obtained using [177Lu]Lu-DOTATATE. The most interesting thing that emerges from the results is the absence of significant side effects, especially affecting the renal parenchyma. 20% of the patients included in the study and treated presented (even before the PRRT) moderate renal insufficiency: in these patients, an objective lesion response was obtained, and no worsening related to the pre-existing renal deficit was manifested. 177Lu]Lu-DOTATOC was used, as the only radiopharmaceutical or in association with [90Y]Y-DOTATOC, for the treatment of NETs and other histotypes (some not strictly NETs but still expressing SST-R2). In particular, the following are summarized: the sample of treated patients, the histological variant, the objective response, the survival times, the toxicity profile and the most relevant clinical conclusions.Among the various studies in the literature, the phase 2 study by Baum et al.  is relev177Lu]Lu-DOTATOC , shows a high percentage of objective responses and high survival times. Furthermore, the safety profile is also favorable, with bone marrow toxicity percentages comparable (and in some cases lower) to those reported in the PRRT studies with [177Lu]Lu-DOTATATE.The analysis of the reported studies concerning the use of Y-DOTATOC and 3.8% per year after [177Lu]Lu-DOTATATE In-pentetreotide and mostly [68Ga]Ga-SS-As guarantee high accuracy to obtain this type of information. Numerous recent studies have confirmed that the lesion level uptake index  is potentially correlated with the magnitude of the objective response (OR) and the efficacy of the treatment in terms of overall survival (OS) and progression-free survival (PFS), reductions in symptoms and, therefore, improved quality of life (QoL).(a2)Histology positive for NET.In fact, a high receptor expression is essential to ensure adequate accumulation of radiopharmaceuticals and a consequent adequate radiation dose to tumor lesions. Positive imaging with [(a3)Well-differentiated low graded shapes (WHO) .NETs include the neoplasms that most frequently and most abundantly express SST-R2. Among these, the histological variants that statistically best respond to PRRT include those of the gastro-entero-pancreatic tract and the forms of broncho-pulmonary origin. NETs that originate in the remaining organs of the respiratory system and in other locations , as well as other histotypes with neuroendocrine phenotypes, generally have less responsiveness and efficacy to the treatment.(a4)Limited spread of disease.The histotypes with a high degree of differentiation, i.e., G1 (Ki67 ≤ 3%) and G2 (Ki67 ≤ 10%), provide the best profiles of objective response and efficacy in terms of survival at PRRT. Some histotypes with G2 (Ki67 > 10%) and especially the well-differentiated G3 (Ki67 > 20%) forms may respond to treatment but have lower PFS and OS values.(a5)Hepatic and pancreatic localization.The extent of disease spread is inversely proportional to the degree of objective response and therapeutic efficacy in terms of PFS and OS. Very often the NETs are indolent and slowly progressive, and their diagnosis occurs frequently when the disease is already systemic due to the presence of diffuse metastases or in any case not surgically attacked. Where technically possible and the patient’s general condition permits, surgical or interventional procedures aimed at eradicating or reducing the disease are recommended. Adjuvant post-surgical therapies, including PRRT, may be more successful after tumor debulking.(a6)Good performance status.Secondary hepatic lesions from NET are those that most frequently respond to PRRT, but primary NETs of the pancreas also show good responses to treatment. More resistant to treatment are secondary lymph node lesions and, above all, skeletal ones. PRRT with intra-arterial administration of the radiopharmaceutical through the hepatic artery represents—in patients with localized liver disease—an alternative modality (compared to the classic systemic intravenous administration) capable of expanding the therapeutic response and outcome of patients.(a7)Favorable genotype.A good general clinical status of the patient, a good life expectancy and the absence of comorbidities are potential factors directly related to the success of the treatment. In particular, the absence of risk factors for bone marrow toxicity  and for renal toxicity  allows, in a context of greater tolerability by of these two critical organs, the administration of the highest levels of administrable radiopharmaceutical activity, which results in a higher absorbed dose to the tumor lesions.It is known, in clinical practice, how similar clinical presentations of NETs can respond differently or even opposite to the various types of treatment, including PRRT. The evaluation of the cellular genome, which can be performed with specific tests still in the experimental phase (NeTest), will, in the near future, describe the state of the disease and predict its prognosis and possible response to therapeutic treatments ,106.(b)(b1)Multidisciplinary evaluation.Factors related to the therapeutic management of the patient to be treated:(b2)Adherence to PRRT protocols (and guidelines).The preliminary discussion of each single clinical case, the collegial choice of treatment, the timing and sequencing between the different treatments that make up the patient’s therapeutic plan, as well as the participatory evaluation of the follow-up, represent the ideal prerequisites for an efficient and effective management of the patient affected by NETs. Various studies correlate patient outcomes with how they are managed within structured specialized clinical paths and/or specialized and highly equipped Centers to respond to this type of patient.PRRT benefit predictors are ,62,71:Reduced bone marrow function.PRRT risk predictors are ,62,71:, especially if performed in the weeks preceding the PRRT, increase the risk of bone marrow toxicity after PRRT. In cases of suspected haematological compromise, it could be useful to perform a biopsy of the haematopoietic marrow to verify the pre-PRRT situation, in order to evaluate the possible risks that the PRRT itself could bring and therefore implement the precautions aimed at reducing potential risks . In any case, the overall bone marrow dose should always be ≤2 Gy. In relation to the activities (90Y or 177Lu) administered, the persistence of low platelet values, after the first courses of treatment, could affect the ability to recycle within the scheduled time and administer the planned activities at the start of treatment. Severe acute toxicity, albeit reversible, was reported in less than 10–13% of patients treated with 90Y and in 2–3% of those treated with 177Lu. In addition, sporadic cases of acute myelodysplasia and leukemia have been described.(b)Reduced kidney function.Adequate bone marrow reserve should be present in PRRT candidates. Reference values recommend: WBC > 3000/μL, with absolute neutrophil value >1000/μL, PLT > 75,000/μL for [90Y is recommended in situations in which renal function  is preserved. Treatments with 177Lu can also be admitted to patients with mild renal function impairment but, in any case, with creatinemia values ≤1.7 mg/dL. Renal parenchyma protection based on the administration of amino acids (L-Lysine and/or L-Arginine) before, during and after PRRT treatment is always recommended in all patients, regardless of starting renal function. For this concomitant treatment, attention should be paid to the possible hyperkalemia [The kidney represents the dose-limiting organ for radiopharmaceutical activities normally used in PRRT. Therapy with rkalemia ,108.90Y labeled peptides are used compared to those based on the use of analogues conjugated with 177Lu.Some chronic diseases such as uncompensated diabetes, uncontrolled hypertension, obstructive nephropathies, as well as any previous nephrotoxic chemotherapeutic treatments (based on platinum) represent risk factors for the development of a potential toxicity induced by PRRT. On the basis of these observations, the absorbed dose to the kidney, in terms of BED , was set at a threshold value of ≤40 Gy for the standard population and ≤28 Gy for subjects with a positive history of aforementioned pathologies favoring the onset of renal toxicity. Although the clinical experience accumulated in the last twenty years by the main PRRT centers has considerably reduced the occurrence of side effects, the most recent summary data confirm the possibility of a deterioration of renal function, more accentuated in the PRRT protocols in where Absolute contraindications to PRRT : pregnanAccording to what emerges from the literature and clinical experience, it can be said that PRRT constitutes an essential tool for the treatment of numerous patients with neuroendocrine neoplasia. In particular, taking into consideration the data concerning the potential toxicity and the documented therapeutic efficacy, the balance between the risks and the benefits clearly leans in favor of PRRT.


Adjuvant temozolomide (TMZ) chemotherapy with standard regimen remarkably improves survival in patients with high-grade glioma (HGG). However, the influence of long-term TMZ chemotherapy on serum ions concentration is unclear.One hundred and thirty-eight patients with HGG were included. Their blood samples were collected for blood biochemistry and routine test. The alteration in serum ions concentration, total protein, albumin, globin, and blood cells counts were used to identify the impact of long-term TMZ chemotherapy.p < 0.001; 1st vs. 12th, p < 0.001). Additionally, the correlation analysis showed that platelets was negatively correlated with chemotherapy cycles . The hematological adverse events mainly centered on grade 1 to 2.Through the comparation of quantitative value of diverse parameters among different chemotherapy cycles, we identified that serum potassium concentration had a downward trend after TMZ administration (1st vs. 6th, Long-term administration of TMZ may lead to serum ions disturbance. Besides the myelosuppression, we should pay attention to the alteration in serum ions concentration, and give patients proper symptomatic treatment when necessary.The online version contains supplementary material available at 10.1186/s41016-022-00271-7. As the most common intracranial primary malignant tumor in adult, glioma is characterized by high invasiveness and recurrence rate, especially for high-grade glioma  , 2. MediPostoperative radiotherapy with concurrent and adjuvant TMZ chemotherapy is universally recognized in clinical practice . In receBesides the effect on survival, there were studies assessing the influence of cisplatin on serum ion concentration and trace elements , 22. Howhttp://www.cgga.org.cn).There were two cohorts in this study. The first cohort included 73 recurrent HGG samples with postoperative chemotherapy information and RNA sequencing data available (48 with postoperative chemotherapy and 25 without) Table . The dat2/day for days 1–5 every 28 days) in our hospital. Seventy-three patients received at least 12 cycles of TMZ chemotherapy while 28 patients received at least 18 cycles. The range of chemotherapy cycles was from two to thirty-seven. After 6-cycle standard adjuvant chemotherapy, extended chemotherapy was performed according to the tumor residual and patients’ tolerance. The blood samples were collected before the next cycle of chemotherapy. Testing items included blood biochemistry  and blood routine   grade III and IV primary gliomas from Beijing Tiantan Hospital during May 2011 to March 2018 were enrolled in this study. Considering the large variation of time interval between two chemotherapy cycles in patients, patients with interval time more than 56 days were excluded. One hundred and thirty-eight postoperative patients were included finally. All patients received adjuvant TMZ  . The chaThe Common Terminology Criteria for Adverse Events (CTCAE) version 5.0 established by the National Cancer Institute was applied to assess the severity of the hematologic toxicity for patients in the second cohort. In CTCAE system, grade 1 to 5 indicates mild adverse effects, moderate adverse effects, severe or medically significant adverse effects, life-threatening adverse effects and death related to adverse effects, respectively. Here, the degree of thrombocytopenia, leukopenia, anemia, and erythropenia were evaluated by using CTCAE criteria .https://www.r-project.org/). Public packages “ggplot2,” “fgsea,” “qusage,” and “pheatmap” were used to perform statistical computation. Gene set enrichment analysis (GSEA) was applied to annotate the function of genes that were highly expressed in the chemotherapy group in the first cohort. In the second cohort, Spearman rank correlation analysis was applied to detect the correlation between chemotherapy cycles and the change of parameters. In patients with the same chemotherapeutic cycles, analysis of paired t test or paired Wilcoxon symbolic rank test was used to compare the change of ions concentration between different periods of chemotherapy. Chi-square test was used to compare the difference between hematological adverse events and chemotherapy cycles. In all statistical analysis, a p < 0.05 was considered significant.R language was used for statistical analysis and generating figures  > 1) were enriched in ion channels and ion transmembrane transport, including calcium, potassium, sodium, and chloride channels .To investigate the effect of TMZ chemotherapy on the expression profiles of HGG samples, we downloaded the RNA sequencing data of 73 recurrent HGGs with postoperative chemotherapy information available from CGGA database Table . Patientr = − 0.762, p = 0.004; Fig. r = 0.747, p = 0.005; p > 0.05). To detect the difference more directly, we compared the quantitative value of the ions in different chemotherapy cycles . The serum sodium concentration diminished slightly between the sixth cycles and the twelfth cycle (p = 0.029). While, an elevated serum concentration was observed in certain ions, such as iron  and chlorine . Therefore, we observed definite correlations between TMZ chemotherapy and ion concentrations in HGG patients.To validate the effect of TMZ chemotherapy on the concentration of serum ions, we collected the blood test results (blood biochemistry and blood routine test) from 138 patients in the second cohort before every cycle of TMZ chemotherapy. To reduce the variation of the baseline levels in each individual, we used their first blood test value as a baseline, and focused on the relative level  of the ions. With the increase of TMZ chemotherapy cycles, serum ions concentration showed slight fluctuations Fig. A–F. The p = 0.043) and potassium (p = 0.049) significantly declined after 18 cycles chemotherapy    Fig. . The num= 0.047) . With inThe toxicity of TMZ has been widely investigated. Fatigue, gastrointestinal side effects, leukopenia and thrombocytopenia were recognized as common adverse events , 23. To T test and Wilcoxon signed-rank test were applied for analyzing the differences among the first cycle, the sixth cycle and the eighteenth cycle chemotherapy. The results showed that the disorder of serum ions concentration was associated with long-term chemotherapy. However, except for some extremums, most indicators could maintain within normal range. Benefiting from the oral administration, patients did not need to stay in the hospital for a long time, which, on the other hand, affected the results to a certain extent. There were great differences in their dietary habit, digestive side effect as well as taking Chinese traditional medicine, which led to the inevitable biases. We attempted to enlarge the sample size to minimize such biases. Whereas, individual differences among patients could not be eliminated. Therefore, we performed data analysis to remind the clinicians paying attention to the risk of serum ions metabolism disorder after prolonged cycles of TMZ chemotherapy.According to the result of GSEA analysis, the function of differentially expressed genes was mainly involved in ion channels, such as potassium ion, calcium ion and sodium ion, etc. Compared with those without chemotherapy, patients received TMZ chemotherapy presented an up-regulation of genes associated with potassium, calcium and sodium ion transport, which reminded us that TMZ chemotherapy might change the serum ions concentration via regulating certain ion channels. To verify our findings, we included eligible patients with HGG and collected their blood samples to further analysis. For those patients who received at least 12 cycles chemotherapy, their serum ions concentration and the hematological parameters tended to fluctuate. The correlation analysis revealed that serum potassium concentration and PLT were decreased visibly with the increase of chemotherapy cycles. It has been reported that TMZ-induced cell death and apoptosis was mediated by O6-methylguanine (O6-meG) production and its reactive oxygen species production. TMZ-induced O6-meG production, involved in AMP-activated protein kinase (AMPK) activation, and contributed to cell apoptosis by promoting p53 activation and inhibiting mTORC1 signaling , 27. BasIn addition, the interaction among ions metabolism should be concerned with. Some studies suggested that the maintenance of serum potassium concentration resulted in sustained improvement in calcium balance . ApproxiLong-term TMZ chemotherapy may lead to the disturbance of ions concentration by influencing the specific ion channels. In addition to the focus on myelosuppression, we should pay attention to the alteration in serum ions concentration, and give patients proper symptomatic treatment when necessary. However, the dietary supplement, treatment before chemotherapy, anti-emetics and individual difference are unavoidable factors that may influence the results. Thus, well-designed large clinical trials and rigorous experiments are needed in the future.Additional file 1: Figure S1. The correlation analysis between serum parameters and chemotherapy cycles (with iron).Additional file 2: Figure S2. Chi-square analysis between hematological adverse events and chemotherapy cycles. (A) Leukopenia; (B) Thrombocytopenia.Additional file 3: Table S1. The normal ranges of blood routine and blood biochemistry test.Additional file 4: Table S2. Hematological adverse event assessment in TMZ administration according to the CTCAE criteria.Additional file 5: Table S3. The grade of adverse events according to CTCAE criteria.


Accumulating evidence also suggests that ER stress may play an important part in the pathogenesis of inflammatory myopathies and genetic muscle disorders. Among the different known proteins regulating ER structure and function, we focused on RTN-1C, a member of the reticulon proteins family localized on the ER membrane. We previously demonstrated that RTN-1C expression modulates cytosolic calcium concentration and ER stress pathway. Moreover, we recently reported a role for the reticulon protein in autophagy regulation. In this study, we found that muscle differentiation process positively correlates with RTN-1C expression and UPR pathway up-regulation during myogenesis. To better characterize the role of the reticulon protein alongside myogenic and muscle regenerative processes, we performed in vivo experiments using either a model of muscle injury or a photogenic model for Duchenne muscular dystrophy. The obtained results revealed RTN-1C up-regulation in mice undergoing active regeneration and localization in the injured myofibers. The presented results strongly suggested that RTN-1C, as a protein involved in key aspects of muscle metabolism, may represent a new target to promote muscle regeneration and repair upon injury.Skeletal muscle is a very dynamic and plastic tissue, being essential for posture, locomotion and respiratory movement. Muscle atrophy or genetic muscle disorders, such as muscular dystrophies, are characterized by myofiber degeneration and replacement with fibrotic tissue. Recent studies suggest that changes in muscle metabolism such as mitochondrial dysfunction and dysregulation of intracellular Ca Myogenic differentiation is an essential process of muscle development depending on the activity of different specific cells and environmental signals ,2. WhileLoss of skeletal muscle mass is associated with a wide array of disease states such as aging, cancer, HIV, chronic heart failure, burn injury, etc. . In addiER stress and the UPR pathways may regulate not only skeletal muscle adaptation in response to physiological stimuli, but also their formation during embryonic development and regeneration upon damage. Indeed, recent studies suggest that a physiological mild ER stress, which occurs during muscle cell differentiation, improves myogenic differentiation efficiency ,9. On thExisting evidence also links muscular dystrophies with mitochondrial and metabolic dysfunction. Indeed, it is now clear that energetics and mitochondrial dynamics are severely affected in muscle disorders and multiple metabolic adaptations are induced to maintain energy homeostasis in dystrophic muscle .2+ homeostasis, thus providing a possible mechanism by which this structural ER protein modulates the cellular metabolism. Recently, we also demonstrated that RTN-1C is involved in the regulation of autophagy [In this regard, the reticulon 1 protein (RTN-1C) has been demonstrated to be a key component of the ER compartment and a functional molecule in the induction of ER stress. RTN-1C is a member of the reticulon family, ER-associated proteins that play important role in bending and shaping the ER membrane, in trafficking of material from the ER to the Golgi apparatus, and in apoptosis . Our preutophagy . Specifiutophagy ,18,19. IBased on these findings, we here studied the role of RTN-1C in myogenic and muscle regenerative processes. We provided evidence that RTN-1C expression correlates with UPR pathway up-regulation and myogenic differentiation. Moreover, by using an in vivo model of Duchenne muscular dystrophy, we observed that RTN-1C modulation is also associated with myogenic and muscle regenerative processes in a pathogenic context.To investigate RTN-1C’s role in skeletal muscle differentiation, we used C2C12 myoblasts, a well-established cellular model, to study myogenesis. C2C12 can be cultivated in proliferative conditions when cultured in growth medium (GM) and undergo myogenic differentiation once shifted in differentiation medium (DM), forming multinucleated differentiated myotubes as shown in To study the effect of RTN-1C modulation along the differentiation process, we either over-expressed or silenced the protein in C2C12 cells and analyzed by western blotting the expression of different myogenic markers. Considering skeletal muscle’s ability to regenerate in response to injury, we investigated on the role of RTN-1C during the regenerative processes in vivo. Muscle regeneration was induced by muscle freezing of anterior tibialis in wild type (WT) mice and analysed for the expression of RTN-1C after 3 and 7 days post injury (d.p.i.). RTN-1C expression in injured anterior tibialis significantly increased at transcriptional A and traPrompted by this evidence, we investigated whether RTN-1C was involved not only in physiological regeneration, but also in disease-associated regenerative processes occurring in Duchenne Muscular Dystrophy (DMD). To this aim, we used mdx mice, the most common mouse model for DMD, characterized by the presence of a premature stop codon in exon 23 of the dystrophin gene. In mdx mice, muscle fibers became necrotic, initiated inflammatory processes, and progressed through cycles of degeneration and regeneration, starting at 2 weeks of age, peaking at 12 months, and continuing for the remaining lifetime of the mouse. Remarkably, the analysis of RTN-1C levels underlined a higher amount of the protein in both tibialis anterior and diaphragms of mdx mice with respect to the age-matched WT mice C,D. AccoDuring muscle regeneration, different cell types take part of the repair process. Among those, we can include muscle satellite cells, macrophages, and fibro-adipogenic precursors . In partTo trace RTC-1C expression in satellite cells in vivo, we analyzed its expression in freshly-isolated cells at different time points after muscle injury and analyzed right after isolation. Upon muscle injury, satellite cells exited from the quiescent state and became activated. After a proliferative expansion, they eventually underwent myogenic differentiation to repair damaged muscle ,22,23. WWe previously demonstrated that RTN-1C plays an important role in endoplasmic reticulum (ER) stress induction and the UPR pathway ,14; inteIn line with these findings, we observed an increase of ER stress-related genes such as GRP78 and the phosphorylated form of eIF2α that parallels RTN-1C upregulation in C2C12 D. These According to this evidence, we investigated whether RTN-1C could affect ER stress response during DMD pathogenesis. We unveiled a positive correlation among RTN-1C-increased expression in dystrophic tibialis and diaphragm and enhanced GRP78 and phosphorylated eIF2α protein levels E.In this study, we investigated the involvement of the reticulon protein 1C (RTN-1C) in skeletal muscle differentiation. We previously demonstrated that RTN-1C has a key role in different cellular processes associated to changes in ER homeostasis and function . SkeletaAnother important aspect of skeletal muscle metabolism and physiology is mitochondrial dynamics, which affect the signaling pathways regulating muscle mass. In this regard, we demonstrated that RTN-1C induces morphological and functional changes of mitochondria through the modulation of ER–mitochondria cross-talk .Moreover, we recently highlighted a new role for RTN-1C protein as a novel emerging regulator of autophagic processes . Taken tTo further support this notion, we decided to explore the involvement of the reticulon protein in myogenic and muscle regenerative processes in vivo. We investigated RTN-1C modulation after muscle freeze injury, a widely used model system to study the events involved in muscle regeneration. The increased expression of RTN-1C in the damaged tissue possibly suggests that the induction of the protein after muscle injury advances the muscle regeneration processes.To determine the contribution of RTN-1C, we analyzed RTN-1C levels in a mouse model of Duchenne muscular dystrophy (mdx), which is characterized by degeneration and regeneration of myofibers upon satellite cells activation. We demonstrated that mRNA and protein levels of the reticulon protein are up-regulated in mdx mice with respect to wild-type mice.The skeletal muscle has an intrinsic capacity to regenerate tissue damage deriving from physiological or pathological injuries. This ability is widely accomplished by undifferentiated skeletal muscle precursor cells, namely satellite cells, located in the niche between the muscle fiber plasmalemma and the basement membrane . SatelliThus, we investigated the expression of RTN-1C in primary satellite cells, isolated from mouse hind limbs muscles, and which underwent muscle differentiation. Here, again, we observed the up-regulation of the reticulon protein along with the early and late myogenic markers, while the specific silencing of RTN-1C showed an opposite effect, further supporting the involvement of the protein in the muscle differentiation process.The results obtained so far are not surprising considering the fact that among the different roles of reticulon proteins they have been originally indicated as markers of different cellular differentiations . MoreoveAs mentioned above, the RTN-1C protein is able to induce ER stress and UPR pathways ,14 that Taken together, these results suggested that the RTN-1C induction could be functional to generate ER stress during myogenesis favoring skeletal muscle differentiation.As previously mentioned, the preservation of a normal structure and function of skeletal muscle fibers depends on several factors such as cell-cell communication, vesicles-based delivery system, and the maintaining of calcium homeostasis. Considering the involvement of reticulons in ER- vesicle trafficking and the ability of RTN-1C to modulate intracellular calcium concentration and mitochondrial dymanics ,15, we r2 in a humidified atmosphere (GM). To induce differentiation, cells were cultured in Dulbecco’s modified Eagle’s medium (Lonza) supplemented with 2% horse serum (DM).C2C12 cells (less than 10 passage) were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum, 100 μg/mL streptomycin, and 100 units/mL penicillin, at 37 °C and 5% COTo silence RTN-1C, cells were transfected with 10-nM siRNA specific for mouse RTN-1C  for 72 h. Scramble siRNA, which does not target any known genes from any species, was used as negative control.To over-express RTN-1C, human RTN-1C cloned in pCDNA 3.1 Zeo(þ) vector was used .Transfections were performed using Lipofectamine 2000  according to the manufacturer’s instructions.Three-month-old male wild-type C57BL/6 (WT) mice or C57BL/6ScSn-Dmdmdx/J (mdx) mice purchased from Jackson Laboratories were used. Mice were maintained under regular housing conditions with standard access to food and drink in a pathogen-free facility. All the procedures in mice were approved by the local Ethics Committee for Animal Welfare  and complied with the NIH Guide for the Care and Use of Laboratory Animals.Muscle acute injury was performed in WT C57BL/6 mice, anesthetized by 1–4% L/min O2 isofluorane  inhalation, by inserting a 30-gauge 5/16” needle previously cooled in liquid nitrogen into tibialis muscle for 10 min.Tibialis anterior of mice were subjected to homogenization and enzymatic dissociation according to Marinkovic et al., 2019 ,29. The ® Western .Tissues were lysed in 50 mM Tris HCl pH 7.5, 50 mM NaCl, 320 mM sucrose, 1% Triton X-100, 10% glycerol supplemented with protease, and a phosphatase inhibitor cocktail. Cells were rinsed in ice-cold PBS and collected in lysis buffer containing 20 mM Tris–HCl pH 7.4, 150 mM NaCl, and 1% Triton X-100 with a protease inhibitor cocktail. Nuclear and cytosolic extracts were obtained using the NE-PER Nuclear and Cytoplasmic Extraction Kit . Protein concentrations were determined by the Bradford assay, using bovine serum albumin as a standard. Aliquots of total protein extracts from cells after different treatments were resolved on SDS–polyacrylamide gel and transferred to a nitrocellulose membrane. Blots were blocked in 5% non-fat dry milk in T-PBS (PBS + 0.05% Tween-20) for 1 h at room temperature and then incubated overnight with the described antibodies. The membranes were incubated with HRP-conjugated secondary antibody for 1 h at room temperature, and the signal was detected by ImmobilonAnti-tubulin , anti actin , anti GAPDH , anti RTN-1C , anti MyHC (DSHB), anti myogenin (DSHB), anti GRP78 , and anti p-eIF2a  were used.w/v (permeabilization solution) for one hour at RT and then incubated with a blocking solution composed of PBS plus Triton™ X-100 0.1% w/v, glycine 1% w/v and goat serum NGS  10% w/v for one hour at RT. Subsequently, the sections were incubated with a primary antibody appropriately diluted in blocking solution for one hour at RT. Two washes were performed, respectively at 15 and 5 min each, with PBS plus Triton™ X-100 0.2% w/v BSA (Bovine Serum Albumin) 1% w/v (washing solution), and then the sections were incubated for one hour at RT with a secondary antibody conjugated with a fluorophore appropriately diluted in blocking solution. After a 10 min rinse with the washing solution, DAPI was added , 0.1 mg/mL, in PBS for 10 min at RT. Finally, the sections were mounted with Aqua Poly/Mount .C2C12 cells were fixed in 2% PFA over night at 4 °C, and then were permeabilized with PBS plus Triton™ X-100  0.3% Mouse anti-MyHC (produced in our laboratory by hybridoma cells) diluted 1:2.Primary antibody used:Anti-mouse Alexa555 (Molecular Probes) diluted 1:100.The secondary antibody used:® software .The sections were photographed with the microscope Nikon Eclipse TE 2000, supported by MetaMorphTissues were lysed in Trizol reagent  and total RNA was extracted using Direct-Zol™ RNA MiniPrep Plus according to the manufacturer’s instructions. One μg of RNA was retro-transcribed using SensiFAST™ cDNA Synthesis Kit  and used in the quantitative RT-PCR (qPCR) experiment, using SensiFAST™ SYBR Hi-ROX Kit  following the manufacturer’s instructions. Thermocycling consisted of an initial polymerase activation step at 98 °C for 5 min, and amplification was performed with 35 cycles of 95 °C for 15 s, 68 °C for 10 s, and 72 °C for 20 s with data acquisition at this stage, and the reaction finished by the built-in melt curve. The relative amounts of mRNA were calculated by using the comparative Ct method.RTN-1C: 5′-TACCCGTCTCCAGGGTAGC-3′, 5′-TTGGTCAATCTGTGCCTGGT -3′GAPDH: 5′-AACTTTGGCATTGTGGAAGG-3′, 5′-CACATTGGGGGTAGGAACAC-3′t-test or one-way ANOVA test. p-value smaller than 0.05 (p < 0.05) was considered to be significant.Graph Pad was used for statistical analysis. ImageJ64 software was used for densitometric analysis. Statistical significance was determined using the Student’s


This study investigated the effects of transglutaminase (TG) concentrations  on the physicochemical properties of whole wheat dough (WWD) and noodles (WWN) during refrigerated storage . The yield, ferulic acid (FA) content, molecular weight (Mw), and apparent viscosity (AV) of water extractable arabinoxylan (WEAX) from refrigerated WWDs were analysed. The WEAX yield and FA tended to increase with refrigerated storage, while the Mw decreased. WEAX FA of from WWD with TG tended to be smaller than the control during refrigeration. The AV for all WEAXs gradually decreased during refrigeration. The TG concentration effects on WWD resistance to extension and extensibility and the WWN cooking properties and texture profile analysis (TPA) were studied. The water absorption and swelling index tended to decrease in WWNs with TG depending on refrigeration time compared to the control samples. The TPA results showed that WWNs with TG were significantly harder than the control after two days of refrigeration. This study demonstrated that TG affected not only WWD composition but also WWN physical properties during refrigerated storage. Triticum aestivum L.) is one of the major food sources consumed globally. Whole wheat grain includes endosperm, germ, and bran. Bran is removed when refining flour, while whole wheat grain provides many dietary fibres and phytochemicals [Wheat  is known to form isopeptide bonds within the gluten network. Since one of the problems in refrigerated refined flour dough is the loss of dough strength , informaThe wheat samples used in this study were the Keumkang cultivar , which is a typical medium wheat in Korea. Whole wheat flour was obtained by grinding with an Ultra Fine Particle Mill DSCH 550S . The moisture content and mean (d50) particle size of the WWF were 6.17% and 44.78 μm, respectively. TG (ACTIVA TG-B) was purchased from Ajinomoto . Alpha-amylase (Termamyl 120 L) and amyloglucosidase (AMG 300 L) were purchased from Novozymes . All reagents were analytical grade.w/w flour weight basis (fwb), distilled water—37.5% (based on 14% flour moisture content), and TG—0.1%/1% w/w (fwb). The TG concentrations used in this study were determined based on the minimum and maximum concentrations for foods recommended by the supplier. The solution (water + enzyme + NaCl) was mixed for 1 min at 60 rpm using a mixer . Then, flour was added to the bowl in solution by mixing it at 135 rpm for 3 min. The resulting dough was rounded and wrapped in plastic film. WWD samples were used for WWN preparation initially and after 1, 2, and 3 days of storage at 3 + 1 °C.The dough preparation procedure followed the AACC method 66-60.01 . EnzymesWWN samples were made by method AACC 66-60.01  with somg for 10 min. The supernatant pH was set to 6.5, and 0.3 mL amyloglucosidase was added. Then, the supernatant was kept at 55 °C for 16 h for degradation of liquefied starch by amyloglucosidase. After the reaction ended, the enzymes were inactivated by boiling in a water bath for 30 min. After passing the resulting solution through filter paper , 10% (v/v) bentonite solution (0.2% mass) was added to remove proteins and stirred for 30 min at room temperature (RT). The pellet was removed again by centrifugation .The water extractable arabinoxylan (WEAX) was extracted according to the methods described by Mansberger et al.  with somv/v), and the solution was stirred for 30 min at RT. The isolated pellet was washed with 200 mL of 65% (v/v) ethanol and twice with 50 mL of acetone. The final material was air-dried for 16 h. The dried pellet was resolved in 200 mL of deionized water, and the supernatant induced by centrifugation was freeze dried.To isolate arabinoxylan from the supernatant, ethanol  was added until the solution alcohol concentration reached 65% (3 and filtered with a 0.45 μm nylon filter. The solutions (200 μL) obtained were separated on TSKgel GMPWxl and TSKgel G2500PWxl  columns by isocratic elution with 0.1 M NaNO3 monitored with an RI detector. The flow rate was 1 mL/min, and the temperature was set at 40 °C. Pullulans (P50 to P800) were used for estimating Mw as a standard calibration.The molecular weight (Mw) of WEAX was determined with a Tosoh EcoSEC HLC-8320GPC  using EcoSEC software. WEAX sample aliquots were solubilized in 0.1 M NaNOw/v) was measured using an MCR 102 rheometer  at 25 °C according to Simsek et al. [−1.The apparent viscosity (AV) of WEAX aqueous solution  and acidified to pH 2 using 6 M HCl. The FA was extracted three times with 3 mL ethyl acetate. The combined extractions were dried under nitrogen followed by dissolution in 500 μL of H2O/MeOH solution at a 1:1 ratio. Twenty microliters of sample was injected for HPLC  with a Capcell Pak C18 UG120 column . The solvent system was composed of solvent A—0.1% (v/v) trifluoracetic acid (TFA) in water and solvent B—0.1% (v/v) TFA in methanol. The flow rate was 0.8 mL/min, and detection was performed on the absorbance at 320 nm. Elution was started isocratically at 35% solvent B, and then a linear gradient from 35% to 95% solvent B for 25 min was applied.The content of ferulic acid (FA) in WEAX was measured with a modified method by Wang et al. . WEAX (5w/v) CHAPS, 1% (w/v) DTTT, 2% pharmalyte, and 1 mM benzamidine). Protein was extracted for 1 h with vortexing followed by centrifugation . The upper layer was used for two-dimensional electric motion, and the protein concentration was determined by the Bradford method [Two DE WWD protein analyses were performed by the methods from Kim et al. . WWD prod method . The sold method , but theDough resistance to extension and extensibility were measured using a texture analyser  by the method from Barros et al. . The resDynamic rheological properties were measured using a MCR 102 rheometer  according to the method from Kaur et al. . The plaEach 30 g of noodles was cooked for 15 min in 500 mL of distilled water. After cooking, the noodles were removed from the water and drained for 15 s using noodle baskets. Cooking parameters were calculated by the method from Tudorica et al. . The remThe texture profile analysis (TPA) of cooked noodles was performed using a TA-XT plus texture analyser  using the method described by Niu et al.  with somp < 0.05. Two-way analysis of variance (ANOVA) was performed to investigate the effect of enzyme concentrations and refrigerated storage time.Three replications of Kieffer extensibility, dynamic rheological measurement, cooking parameters, and TPA as well as duplicate WEAX physicochemical property measurements were performed. Resulting data were represented as the mean ± standard deviation. Analysis of variance (ANOVA) was carried out using XLSTAT  to determine the differences among samples. When significant differences were detected, the Student–Newman–Keuls (SNK) multiple comparison test was performed to separate the means at The WEAX yield (%) from WWDs is represented in The content of ferulic acid (FA) in WEAX from WWD depended on TG concentration, ranging from 636 to 3007 μg/kg, and storage time at low temperature, as shown in The WWD WEAX Mw is presented in w/v) depending on the shear rate. The AV of the WEAX solution  in WWD showed Newtonian flow characteristics at a high shear rate (600–1200/s) after 3 days of refrigerated storage. Regardless of storage time, shear thinning behavior was observed as the shear rate was increased. At the state of rest, the un-crosslinked molecule polysaccharide solution contracts to shape balls, causing high viscosity. However, when shear is applied to a polymer solution, the ball unfolds and becomes an ellipsoid [llipsoid . The challipsoid . Therefollipsoid , which sProtein extractions of each of the groups with 0 and 3 day WWDs were separated by 2-DE, as shown in p < 0.001) in WWD resistance to extension and extensibility were observed by refrigerated storage time  and TG concentration , as shown in r = −0.809) between the WWD elastic modulus at 1 rad/s and extensibility in this study, TG, which prevents an elastic modulus decrease and gluten degradation during refrigeration, might prevent a dough strength decrease. However, to validate the TG function, which inhibits the gluten degradation induced by refrigeration storage, further study on the interaction between TG behavior and gluten is needed.p < 0.001) depending on the WWD TG concentration. The tan δ ranges for the control, 0.1% TG, and 1% TG groups were as follows: 0.37–0.40, 0.36, and 0.31–0.32, respectively. A decreasing trend in loss factor was shown by TG. These study results imply that TG could influence gluten degradation, affecting dough strength during refrigerated storage. The average loss factor (tan δ) at all frequency points, equal to G″/G′, was in the range of 0.31–0.40 in TG addition influenced both the WWD physicochemical properties and its product during refrigerated storage. During refrigeration, TG played an important role in modifying the WEAX structural properties by affecting its yield and Mw and prevented FA release from the gluten-WEAX structure. The WEAX yield (%) from WWD with 0.1% TG or the control was affected by storage time, while that with 1% TG was not affected by storage time. WEAX Mw ranges on day 0 and after 3 days of refrigerated storage were 105,406 to 109,692 and 93,209 to 96,971, respectively, which implied WWD WEAX decomposition during refrigeration. However, after 3 days of refrigerated storage, a significantly higher Mw was found in WEAX from WWD with TG than in the control implying inhibition of WWD WEAX decomposition by TG during refrigeration. The content of FA in WEAX from WWD depended on TG concentration, ranging from 636 to 3007 μg/kg, and storage time at low temperature. Overall, WEAX FA increased with time when stored at low temperatures. TG influenced FA in WEAX, which is positioned on O-5 of the arabinose residue, and the WEAX AV was altered, implying the modified WEAX branching degree. During refrigeration, the viscosity of the shear rate dependence for the control group continued to decline gradually, while the difference between the overall apparent viscosities depending on storage time was reduced when TG was incorporated in the WWD. Thus, WWD property improvement might be expected by the use of TG due to inhibited WEAX decomposition during refrigeration. However, when 1% TG was applied to the WWD, both resistance to extension and extensibility simultaneously decreased during refrigerated storage. This could be explained by the stiff WWD when 1% TG was added. The elastic modulus (G′) of WWD was decreased with refrigerated storage, while that of WWD with TG 1% was increased. Considering that gluten structure degradation reduces elastic modulus (G′), the reason for the increased G′ could be explained by TG which formulates disulfide bonds within gluten. In addition, after two days of refrigerated storage, 1% TG showed significantly higher cooking loss than the control and 0.1% TG. Consequently, even if TG ameliorates the WWD gluten structure or diminishes WEAX decomposition during refrigeration, the modified properties induced by TG do not always improve product quality. Significant decrease in hardness and chewiness was found in control WWN during refrigerated storage, while hardness and chewiness of WWN with TG 0.1% or 1% were not changed. Even though WWD with TG significantly maintained the WWN hardness during refrigerated storage, the TG concentration should be adjusted according to the target WWN properties since the WWN adhesiveness or cohesiveness differs with TG concentrations. This is the first report on the TG behaviour in refrigerated WWDs. However, further research on TG reaction in low-temperature storage is needed to confirm these findings.


Brewer’s spent grain (BSG) is the main side-stream of brewing. BSG is a potential source for nutritionally enriched cereal products due to its high content of fibre and protein. Two novel ingredients originating from BSG, EverVita FIBRA (EVF) and EverVita PRO (EVP), were incorporated into bread in two addition levels to achieve a ‘source of fibre’ (3 g/100 g) and a ‘high in fibre’ (6 g/100 g) nutrition claim for the breads. The impact of those two ingredients on dough and bread quality as well as on nutritional value was investigated and compared to baker’s flour (C1) and wholemeal flour (C2) breads. The addition of EVF performed outstandingly well in the bread system achieving high specific volumes (3.72–4.66 mL/g), a soft crumb texture (4.77–9.03 N) and a crumb structure comparable with C1. Furthermore, EVF barely restricted gluten network development and did not influence dough rheology. EVP increased the dough resistance (+150%) compared to C1 which led to a lower specific volume (2.17–4.38 mL/g) and a harder crumb (6.25–36.36 N). However, EVP increased the nutritional value of the breads by increasing protein content (+36%) and protein quality by elevating the amount of indispensable amino acids. Furthermore, a decrease in predicted glycaemic index by 26% was achieved and microbial shelf life was extended by up to 3 days. Although both ingredients originated from the same BSG, their impact on bread characteristics and nutritional value varied. EVF and EVP can be considered as game-changers in the development of bread fortified with BSG, increasing nutritional value, and promoting sustainability. Due to increased environmental awareness, the sustainable use of side-streams generated within the food industry has become increasingly important, focused on eliminating waste and the continual use of resources. Various studies have been conducted to valorise food waste by recycling these materials into biofuels, enzymes and bioactive compounds . From a w/w) and protein (19–30% w/w). The fibre is constituted of hemicellulose including arabinoxylans, cellulose and lignin [BSG has huge potential to enhance the nutritional value of food due to its high content of fibre  processed BSG and launched two ingredients, EverVita FIBRA and EverVita PRO, which differ in their protein and fibre content and particle size. A recent study characterised those ingredients and investigated their effect on pasta, resulting in outstanding product quality and increased nutritional value . The curBaker’s flour and stoneground wholemeal flour supplied by Odlums , sunflower oil , salt , sugar , baker’s instant active dried yeast  and tap water were used in the bread production. The two ingredients, EverVita FIBRA (EVF) and EverVita PRO (EVP), rejuvenated brewer’s spent grain, were supplied by EverGrain, LLC . Chemicals used for analysis were purchased from Sigma-Aldrich , unless stated otherwise.The compositional analysis of EVF and EVP were previously reported by Sahin et al., (2021) . Baker’sAmino acid analysis was conducted by Mérieux NutriScience CHELAB S.r.l., Resana, Italy. After protein extraction and hydrolysis , amino acids were quantified using ionic chromatography with post-column ninhydrin derivatisation coupled with a fluorescence and an ultraviolet (UV) detector. The concentration of the amino acids is given as % based on protein.Bread doughs were produced using the ingredients according to the recipes illustrated in Dry ingredients were pre-mixed for 1 min at minimum speed using a Kenwood Titanium Major KM020 mixer equipped with a hook attachment  to ensure a homogeneous distribution, and instant active dried yeast was activated by its addition to water (25 °C) for 10 min. Yeast solution and sunflower oil were added to the dry ingredients and mixed at speed 1 for 1 min first, followed by a second mixing step at speed 2 for 7 min. This procedure was used for the baking process as well as for the evaluation of the fermentation quality of the bread doughs.EverVita ingredients originate from BSG, which has been reported to impact bread dough quality negatively. Hence the effect of reinvented BSG-based ingredients on gluten network development, starch pasting properties as well as on dough rheology, extensibility and fermentability was investigated. Therefore, blends of baker’s flour and EVF/EVP according to the ratios given in The impact of EverVita ingredients on both gluten network and starch pasting was determined by the GlutoPeak and the Rapid Visco Analyser (RVA). For both measurements the solid part represents a mixing of baker’s flour and EverVita ingredient (EVF or EVP) in the proportion used for the bread baking process .®  was used to measure the gluten aggregation properties represented by the time of full aggregation (peak maximum time (PMT) in seconds (s)) and gluten network strength (torque maximum (TM) in Brabender units (BU)). 9 g of solids, based on 14% moisture, was added to 9 g of distilled water (36 °C) and the test was started using a rotation speed of 2750 rpm of the paddle.The GlutoPeakStarch pasting properties during heating and cooking were determined using a Rapid Visco Analyser ; 3 g of solids, based on 14% moisture, was added to 25 g distilled water in the metal sample cup and premixed briefly with the paddle to remove lumps. A constant shear rate of 160 rpm was applied during the measurement. The temperature profile used was equilibration at 50 °C for 1 min, followed by an increase to 95 °C with a heating rate of 0.2 °C/s, held at 95 °C for 162 s, cooled to 50 °C with a cooling rate of 0.2 °C/s, and held at 50 °C for 60 s. During the measurement the viscosity in centipoise (cP) was monitored resulting in a viscosity curve over time and peak viscosity (cP), breakdown viscosity (cP), trough viscosity (cP) and final viscosity (cP) were evaluated.® equipped with an automatic water dosing unit . Therefore, flour and EverVita ingredients were used in the ratio illustrated in The water content of the different formulations was adjusted using Farinograph-TS® . The dough was prepared following the bread dough preparation process omitting yeast. 150 ± 0.5 g of dough (without addition of yeast) was moulded and placed into the proofing chamber. Analysis was performed after 90 min of proofing (time used for proofing during the baking process).The dough extensibility (E) in mm, resistance to extension (RE) in extensograph units (EU) and the ratio of resistance over extensibility (RE/E) in (EU/mm) were determined using ExtensographA rheofermentometer  was used to measure gaseous release and dough rise during the proofing process. We prepared 300 g dough following the bread dough preparation process procedure mentioned before and transferred into the fermentation chamber (30 °C). A cylindrical weight of 1500 g was placed on the dough and the dough was fermented for 180 min. The maximum height of the dough (Hm) in mm, the time required to achieve maximum height (T1) in minutes, the total volume of carbon dioxide released by the dough (Vtot) in mL and the height of maximum gas formation (H’m) in mm were evaluated.Dough was separated into 450 g pieces, moulded, transferred into a bread pan (dimensions: 15 × 9.5 × 9.7 cm) and placed into the proofing chamber  for 90 min set to 30 °C and 85% relative humidity. After proofing, the tins with the leavened doughs were transferred into a deck oven  (220 °C top temperature and 230 °C bottom temperature). 400 mL of steam was injected prior to loading. The breads were baked for 35 min, removed from the baking tins and left to cool for 120 min on a rack before analysis. Two breads per batch were analysed after baking and two loaves were packed in plastic bags and stored at 20 °C for five days to determine the staling rate. Three batches per bread type were evaluated.The specific volume was measured with a 3D laser scan using a Volscan Profiler 300  and expressed as mL/g.The bread crumb texture was analysed using a TA-XT2i texture analyser  equipped with a 25 kg load cell. For analysis, the breads were sliced with a thickness of 25 mm. To imitate chewing activity, a two-compression test was carried out using a cylindric probe with a diameter of 35 mm, a test speed of 5 mm/s, a trigger force of 0.05 N and a compression of 40%. The crumb hardness in Newton (N) and chewiness (N) were evaluated 120 min after baking. Gumminess (N) showed the same values as chewiness and was not further considered in the manuscript. In addition, the hardness (N) was measured 120 h after baking to evaluate bread staling. Staling was expressed as staling rate as defined by Sahin, Axel, Zannini, and Arendt (2018) .2), number of cells and average cell diameter (mm) were evaluated. The crumb area for the evaluation of the number of cells and the cell diameter was the slice area. Furthermore, the microstructure of the crumb was visualised using a scanning electron microscope (SEM). Therefore, bread crumb was freeze-dried, immobilised, and coated with a layer of 25 mm of sputtered palladium-gold. The microstructure was observed using a working distance of 8 mm and micrographs were taken at an accelerating voltage of 5 kV. SEM Control User Interface software, Version 5.21  was used. For the evaluation of crumb structure, a C-cell Imaging System  was used and slice area  was used to determine the bread colour. To evaluate the influence of EverVita ingredients on the colour and the differences in colour compared to both controls, baker’s flour and wholemeal flour, the ΔL*2 = lightness value of the bread including EverVita ingredients; L*1 = lightness value of the control .a*2 = redness value of the bread including EverVita ingredients; a*1 = redness value of the control .b*2 = yellowness value of the bread including EverVita ingredients; b*1 = yellowness value of the control .The water activity was determined using a water activity metre . The microbial shelf life analysis of the breads were performed using the environmental challenge method as reported by Dal Bello et al. (2007) . BrieflyStarch can be present in different forms, digestible and resistant towards digestion. The digestible starch and resistant starch content of freeze-dried bread samples were determined by an enzymatic method using the K-RAPRS kit . The digestible and resistance starch content in fresh bread was calculated considering the moisture content. The sum of both starch components represents the total starch content. The predicted protein and fibre content were calculated based on nutritional information of the ingredients considering the bake loss. The expected indispensable amino acid , aromatic amino acids (AAA), threonine, tryptophan, valine) in the breads were calculated and are presented relative to the requirement pattern bread on an average intake of 0.66 g protein per kg composition in breads . TherefoAn in vitro digestion method, specific to fibre-enriched food, was carried out to determine the predicted glycaemic index (pGI) of the breads. The procedure was performed as described by Brennan and Tudorica (2008)  and 4 g sample refers to the absorbance at 546 nm of the sample treated with enzymes; 500 refers to the total volume of buffer; 0.95 is the conversion factor of starch to maltose by amylase; Amaltose refers to the absorbance of a 1 mg/mL maltose standard; digestible carbohydrates in 4 g sample expressed in mg.Reducing sugars released (RSR) is calculated as:The reducing sugar release index (RSRI) at 150 min is defined as:The ‘control’ refers to the baker’s flour control.maltose is the diffusion of the maltose blank . The percentage of maltose able to diffuse through the tube in the presence of sample (DIFF):blank +maltose refers to the absorbance of the sample blank with maltose addition; Ablank refers to the absorbance of the sample blank; 500 is the total volume of buffer; Amaltose refers to the absorbance of the maltose blank; 200 is the weight of maltose in ‘blank + maltose’-sample in mg.Sugar diffusion index: All tests were carried out in triplicate, unless stated otherwise. A variance analysis  was performed using Minitab 17. In addition, a two-way ANOVA was conducted to evaluate the influence of the type of ingredients and the addition level. Correlation analysis was performed using Microsoft Excel.The nutritional composition of the raw ingredients is an influencing factor of the final nutritional profile of the food product. The differences between baker’s flour (C1), wholemeal flour (C2) and EverVita ingredients regarding moisture, protein, fat, total carbohydrates, ash and sodium are illustrated in The total carbohydrate concentration in the EverVita ingredients is 6–19% lower compared to the flours, yet their dietary fibre content, illustrated in Apart from the macronutrients, such as protein, fat, and carbohydrates, the protein profile plays a key role in the final nutritional value of food products. The total amino acid composition of the protein fraction after hydrolysis of the raw ingredients based on their protein content is displayed in p < 0.05) influenced the PMT (63.3 ± 2.3 s (SF); 29.0 ± 1.0 s (HF)).The effect of EverVita ingredients on gluten network development compared to the controls baker’s flour (C1) and wholemeal flour (C2) is illustrated in C1 showed the highest trough viscosity (740 ± 6 cP), followed by formulations including EVF (SF) (655 ± 2 cP) and EVP (SF) (645 ± 1 cP). The incorporation of EVP (HF) showed a significant lower trough viscosity (465 ± 4 cP). The trough viscosity of C2 (516 ± 15 cP) was lower compared to C1 and did not significantly differ from samples including EVF (HF) (530 ± 10 cP). During cooling amylose and amylopectin reassociate, which is called retrogradation, and leads to an increase in viscosity. The highest final viscosity was determined in the C1 (1758 ± 9 cP), followed by the formulations with EverVita ingredients at a SF level (EVF (SF): 1603 ± 8 cP; EVP (SF): 1606 ± 1 cP). The addition of EVF at a higher level (HF) led to a significant lower value (1315 ± 8 cP) which is comparable to the final viscosity detected in C2 (1318 ± 28 cP). The incorporation of EVP at HF level resulted in the lowest final viscosity (1222 ± 11 cP).Rheological characteristics of dough include consistency changes during mixing (Farinograph) as well as elasticity and resistance to extension during stretching of the dough (Extensograph).The farinograph was not only used to adjust the water content (WA) of each dough reaching a final consistency of 500 ± 20 FU, rheological parameters such as dough development time (DDT), arrival of dough stability (S1) and the mixing tolerance index (MTI) were investigated. p < 0.04; r = 0.95). C2 resulted in a longer DDT (10.12 ± 0.28 min) and a delay in S1 (6.93 ± 0.32 min) compared to C1 . Both EverVita ingredients caused a quicker DDT  and S1 is reached earlier when added in ‘source of fibre’ levels (EVF (SF): 1.33 ± 0.12 min; EVP (SF): 1.27 ± 0.12 min). While the addition level of EVF did not have any impact on the dough rheology during mixing, the longest overall DDT (17.84 ± 0.65 min) and the highest S1 (8.07 ± 0.98 min) occurred in doughs with EVP (HF). The MTI showed the highest value for C1 (34.33 ± 1.53) while all other doughs had a significantly lower MTI (between 13.00 to 19.67).C1 showed the lowest water absorption resulting in a water addition level of 60.47% to achieve 500 FU, while C2 required 63.30% water. The addition of EVF (SF) and EVP (SF) caused an increased water addition level by 1.80% and 3.50% compared to C1, respectively. While the incorporation of EVF (HF) only affected the water absorption of the system to a relatively small extent (+3.83%) compared to C1, EVP (HF) caused an increase by 10.26% and showed the highest water content overall. DDT and S1 revealed a strong positive correlation (The Farinograph measurement of EVP (HF) did not give any MTI value which indicates no changes in peak consistency five minutes after the peak is reached. Furthermore, the curve showed fluctuations in the first 7.5 min of mixing.Extensograph measurements revealed the extensibility (E) and the resistance to extension (RE) during stretching of the dough and the results are demonstrated in 2 production by yeast, while in dough including EVP (HF) the lowest CO2 formation occurred (2426 ± 48 mL).The rheofermentometer was used to measure dough rise and CO2 formation during 180 min of fermentation. The results for maximum height of the dough (Hm), time required to achieve maximum height (T1), the total volume of carbon dioxide released by the dough (Vtot) and the height of maximum gas formation (H’m) are displayed in The specific volume, crumb texture, crumb macro- and microstructure as well as the crust and crumb colour, water activity and microbial shelf life of the breads were determined to evaluate bread quality. The results are illustrated in The addition of EverVita ingredients in a concentration to achieve a ‘source of fibre’ claim did not significantly impact the specific volume (EVF (SF): 4.66 ± 0.23 mL/g; EVP (SF): 4.38 ± 0.31 mL/g) compared to C1 (4.46 ± 0.26 mL/g). However, a higher addition level decreased the specific volume of the breads significantly resulting in 3.72 ± 0.37 mL/g and 2.17 ± 0.05 mL/g for EVF (HF) and EVP (HF) breads, respectively. EVP (HF) showed the same specific volume as C2 (2.28 ± 0.07 mL/g). In The texture of the bread crumb was evaluated considering crumb hardness (N) and chewiness (N), and the degree of staling over time was determined and expressed as the staling rate. The softest crumb was determined in C1 (4.76 ± 1.20 N) and EVF (SF) (4.77 ± 0.65 N), followed by EVP (SF) (6.25 ± 1.49 N) and EVF (HF) (9.03 ± 1.23 N). C2 showed a significant harder crumb (24.54 ± 3.68 N) and the highest crumb hardness was measured in bread containing EVP (HF) (36.36 ± 1.99 N). EVF (SF) resulted in the least chewy crumb (3.25 ± 0.56 N), while EVP (HF) had the highest chewiness value (20.32 ± 1.20 N). The staling rate was the highest in EVF (SF), EVP (SF) and EVF (HF) with values between 2.55 and 2.70. The lowest staling rates were detected in C2 (1.32 ± 0.45) and EVP (HF) (1.24 ± 0.15). 2), number of cells and average cell diameter (mm). A strong positive correlation between slice area and specific volume  occurred and thus the biggest slice area was measured in C1  and EVF (SF)  breads, while the smallest slice area was determined in C2 (4990 ± 388 mm2) and EVP (HF) (5701 ± 369 mm2). The highest number of cells in the bread crumb was determined in EVP (SF) (5997 ± 268). These cells were relatively small (diameter of 1.88 ± 0.10 mm) compared to those of the other samples. C2 showed the lowest number of cells (2794 ± 144) and also the smallest average cell diameter (1.25 ± 0.20 mm). C1 and EVF (SF) had the biggest cell diameter among all samples with 2.43 ± 0.20 mm and 2.32 ± 0.12 mm for C1 and EVF (SF), respectively. Differences in crumb structure were visualised using a SEM and are illustrated in Changes in crumb structure were evaluated considering slice area  and crumb colour (17.66 ± 2.86). The most similar crust colour to C1 was determined in breads including EVP (HF) (8.21 ± 4.47), while EVF (SF) (5.28 ± 3.39) and EVP (SF) (8.92 ± 2.80) showed the most similar crumb colour to C1. Compared to C2, breads containing EverVita ingredients with source of fibre addition level (EVF (SF): 15.08 ± 2.42; EVP (SF): 16.54 ± 2.55) had the highest ΔE-value, while EVP (HF) showed the most similar crust colour (8.93 ± 2.30). Regarding the colour difference in crumbs, EVP (HF) had the most similar crumb colour to C2 (5.69 ± 2.20), whereas C1 showed the highest difference. The microbial shelf life of the breads revealed the shortest shelf life in C1 and EVF (SF) breads. Compared to C1 which showed the first microbial growth after 6.00 ± 1.00 days, the growth started after 9.00 ± 1.00 days and 9.33 ± 0.58 days in breads including EVP (SF) and EVP (HF), respectively. Hence, an extension of microbial shelf life occurred, even though no significant differences in water activity was determined.The nutritional value of breads was evaluated considering the composition of the breads  as well as the amino acids composition and the predicted glycaemic index (pGI) and predicted glycaemic load (pGL).The predicted composition of the breads based on ingredient characteristics and the addition level of EverVita ingredients is illustrated in The main compound of the breads is moisture, which ranges between 42.04 ± 0.04% in EVF (SF) breads to 46.68 ± 0.26% in EVP (HF) breads. The second main compound in the breads is starch. The highest total starch content was determined in C1 (40.56 ± 0.42 g/100 g), whereas high fibre breads and C2 showed the lowest total starch concentrations. The same trend occurred in the digestible starch content. C1 had the lowest dietary fibre content (2.1 g/100 g) and C2 included 4.8 g/100 g dietary fibre based on calculation. EverVita ingredients were added in amounts needed to achieve either 3 g/100 g of dietary fibre (‘source of fibre’ claim) or 6 g/100 g (‘high in fibre’ claim). The inclusion of EverVita ingredients increased the protein content in the breads, which ranged between 8.4 g/100 g (C2) and 11.5 g/100 g (EVP (HF)). No major differences occurred in the fat content of the breads. C1 had the lowest fat content (3.18 g/100 g) and EVP (HF) breads included the highest fat content (3.47 g/100 g).The amount of indispensable amino acids was calculated and expressed relative to the requirement pattern established by the World Health Organisation (WHO) . Since tBy contrast, the fortification of wheat bread with EVP increased the concentration of some indispensable amino acids, in particular lysine (+24.5%) an amino acid that is known to be limiting in cereal based products. Moreover, the incorporation of EVP increased the concentration of aromatic amino acids (AAA) by 4% compared to C1, and valine (+1%). A significant difference occurred in the predicted tryptophan content in both high in fibre breads (EVF and EVP), which made 0.215% and 0.561% based on the total protein content in the breads, respectively, while no tryptophan was expected in both controls.The pGI and pGL values represent the in vitro starch digestibility of the breads during digestion. The pGI and pGL of the different bread samples are illustrated in The impact of BSG on bread dough characteristics and final bread quality has been extensively studied. Rejuvenated BSG in the form of two ingredients, EverVita FIBRA and EverVita PRO, was used to fortify bread in two inclusion levels, 3 g/100 g and 6 g/100 g bread. The type of EverVita ingredient significantly impacted on dough and bread quality and final bread characteristics.p < 0.001), the type of EverVita ingredient also influenced the network strength (p < 0.005) and development time (p < 0.001). As previously reported, the EverVita ingredients contain low molecular weight peptides, which promote intramolecular connections, such as disulphide-, hydrogen- and ionic bonds resulting in a faster network development [EVF contains 23.4% protein and 67.6% dietary fibre, mainly insoluble fibre, while EVP has a protein content of 36.8% and a dietary fibre content of 46.8% [elopment . Since Eelopment ,36. Secoelopment . Also, aelopment ,39. p < 0.036; r = 0.84) and dough rise during proving . This indicates the primary role of the gluten network on dough quality and the weakening effect caused by fibre ingredients [The gluten network strength influenced the dough rheology resulting in a positive correlation with extensibility  and the type of EverVita ingredient (p < 0.0001) and showed a strong positive correlation with the gluten network strength . Baker’s flour is rich in intact gluten proteins and starch, which participate in dough structure formation [p < 0.04; r = −0.84). Furthermore, the curves of the gluten network development (The dough rise during fermentation (Hm) is influenced by both, addition level .Vtot is the total volume of CO% starch , resultep < 0.003; r = 0.96), crumb hardness , chewiness  and slice area . The specific volume correlated negatively with the dough development time during mixing . To achieve the highest specific volume the formulation can reach, the dough needs to be fully developed, meaning all compounds need to be fully hydrated to form a network [p < 0.01; r = 0.92).It is known that dough extensibility and dough expansion during proofing influence the final bread quality. Hm and RE/E correlated with several bread quality parameters, such as specific volume , meaning a high degree of starch swelling during heating results in a high viscosity which enhances the stability of the system during the baking process [Besides the dough rheology, starch pasting during baking and the related changes in viscosity of the system impacts the final product quality. In general, the replacement of baker’s flour caused lower viscosity values which may be related to the reduced amount of total starch. The peak viscosity showed a strong positive correlation with the specific volume of the bread  caused a significant decrease in the specific volume of the breads, which affected the crumb structure. Food structure affects the starch digestibility of products making starch less accessible for digestive enzymes to bind on the substrate, in this case, starch . In part3 ,63. In a3 . Even thIn addition, the structural differences could explain the differences in microbial shelf life. The protein matrix, putatively, also acts as a barrier for microbial attack on starch, prolonging the shelf life by two to three days. Apart from the structural influence, the increased water binding capacity of fibre and especially proteins restrict the accessibility to water and hinder the growth of mould . BSG is a nutritious side-stream of the brewing industry that is used primarily for animal feed. Previously, using BSG as a food ingredient to enrich products, particularly cereal products, with highly nutritious dietary fibre and protein has been very challenging, more often than not leading to a significant decrease in final bread quality. The present study used two unique BSG-derived ingredients, EverVita FIBRA (EVF) and EverVita PRO (EVP), which differ in protein and dietary fibre content as well as particle size. These two ingredients performed very similarly in small inclusion levels but differently in bread systems with a ‘high in fibre’ addition level. The inclusion of EVF in high amounts resulted in outstanding bread characteristics such as high specific volume, soft crumb texture and a crumb structure comparable to a baker’s flour control. While the impact of EVP on the techno-functional properties was less desired, it significantly increased the nutritional value of the bread by increasing protein content by 36% and fibre content by 3-fold, lowering pGI-values by up to 25%, elevating the amount of indispensable amino acids and hence the protein quality, and prolonging microbial shelf life. Notwithstanding this, correlation analysis revealed the influence of dough characteristics on bread quality which can help make controlled changes in the baking process or even the formulation for further optimisation using functional ingredients. In the past, several studies have addressed the impact of the most abundant dietary fibre in BSG, arabinoxylans, on bread quality and the interaction between fibre and gluten network. However, the impact of the protein fraction present in BSG has been neglected in the literature. This study provided a deep insight into the impact of several BSG-derived fractions, including protein, on bread quality. This work demonstrated the potential game-changing impact of brewing side-streams to sustainably enhance the nutritional value of food products.