Evaluating the potential neurotoxicity of hexanedione isomers:an in vitro approach

2,5-hexanedione (2,5HD) is the neurotoxic metabolite of the aliphatic hydrocarbon n-Hexane. The isomers, 2,3-hexanedione (2,3HD) and 3,4-hexanedione (3,4HD) are used as food additives. Although the neurotoxicity of 2,5HD is well established, there are no human data of the possible toxicity of the 2,3- and 3,4- isomers. MTT and flow cytometry were utilised to determine the cytotoxicity of hexanedione isomers in neuroblastoma cells. The neuroblastoma cell lines SK-N-SH and SH-SY5Y are sufficiently neuron-like to provide preliminary assessment of the neurotoxic potential of these isomers, in comparison with toxicity towards human non-neuronal cells. Initial studies showed that 2,5HD was the least toxic in all cell lines at all times (4, 24 and 48h). Although considerably lower than for 2,5HD, in general the IC50s for the α isomers were not significantly different from each other and, besides 4h exposure, the SH-SY5Y cells were significantly more sensitive to 2,3HD and 3,4HD than the SK-N-SH cells. All three isomers caused varying degrees of apoptosis in the neuroblastoma lines, with 3,4HD more potent than 2,3HD. Flow cytometry highlighted cell cycle arrest indicative of DNA damage with 2,3- and 3,4HD. The toxicity of the isomers towards 3 non-neuronal cell lines (MCF7, HepG2 and CaCo-2) was assessed by MTT assay. All 3 hexanedione isomers proved to be cytotoxic in all non-neuronal cell lines at all time points. These data suggest cytotoxicity of 2,3- and 3,4HD (mM range), but it is difficult to define this as specific neurotoxicity in the absence of specific neurotoxic endpoints. However, the neuroblastomas were significantly more susceptible to the cytotoxic effects of the α hexanedione isomers at exposures of 4 and 24 hours, compared to non-neuronal lines. Finally, a mechanism of toxicity is suggested for the α HD isomers whereby inhibition of the oxoglutarate carrier (OGC) releases apoptosis inducing factor (AIF), causing apoptosis-like cell death.

Polarized secretion of leukemia inhibitory factor

Background: The direction of cytokine secretion from polarized cells determines the cytokine's cellular targets. Leukemia inhibitory factor LIF) belongs to the interleukin-6 IL-6) family of cytokines and signals through LIFR/gp130. Three factors which may regulate the direction of LIF secretion were studied: the site of stimulation, signal peptides, and expression levels. Stimulation with IL-1 beta is known to promote IL-6 secretion from the stimulated membrane apical or basolateral) in the human intestinal epithelial cell line Caco-2. Since LIF is related to IL-6, LIF secretion was also tested in Caco-2 following IL-1 beta stimulation. Signal peptides may influence the trafficking of LIF. Two isoforms of murine LIF, LIF-M and LIF-D, encode different signal peptides which have been associated with different locations of the mature protein in fibroblasts. To determine the effect of the signal peptides on LIF secretion, secretion levels were compared in Madin-Darby canine kidney MDCK) clones which expressed murine LIF-M or LIF-D or human LIF under the control of an inducible promoter. Low and high levels of LIF expression were also compared since saturation of the apical or basolateral route would reveal specific transporters for LIF. Results: When Caco-2 was grown on permeable supports, LIF was secreted constitutively with around 40% secreted into the apical chamber. Stimulation with IL-1 beta increased LIF production. After treating the apical surface with IL-1 beta, the percentage secreted apically remained similar to the untreated, whereas, when the cells were stimulated at the basolateral surface only 20% was secreted apically. In MDCK cells, an endogenous LIF-like protein was detected entirely in the apical compartment. The two mLIF isoforms showed no difference in their secretion patterns in MDCK. Interestingly, about 70% of murine and human LIF was secreted apically from MDCK over a 400-fold range of expression levels within clones and a 200,000-fold range across clones. Conclusion: The site of stimulation affected the polarity of LIF secretion, while, signal peptides and expression levels did not. Exogenous LIF is transported in MDCK without readily saturated steps.

Amino acids in oral drug delivery:salts, ion-pairs and transcriptomics

Oral drug delivery is considered the most popular route of delivery because of the ease of administration, availability of a wide range of dosage forms and the large surface area for drug absorption via the intestinal membrane. However, besides the unfavourable biopharmaceutical properties of the therapeutic agents, efflux transporters such as Pglycoprotein (P-gp) and multiple resistance proteins (MRP) decrease the overall drug uptake by extruding the drug from the cells. Although, prodrugs have been investigated to improve drug partitioning by masking the polar groups covalently with pre-moieties promoting increased uptake, they present significant challenges including reduced solubility and increased toxicity. The current work investigates the use of amino acids as ion-pairs for three model drugs: indomethacin (weak acid), trimethoprim (weak base) and ciprofloxacin (zwitter ion) in an attempt to improve both solubility and uptake. Solubility was studied by salt formation while creating new routes for uptake across the membranes via amino acids transporter proteins or dipeptidyl transporters was the rationale to enhance absorption. New salts were prepared for the model drugs and the oppositely charged amino acids by freeze drying and they were characterised using FTIR, 1HNMR, DSC, SEM, pH solubility profile, solubility and dissolution. Permeability profiles were assessed using an in vitro cell based method; Caco-2 cells and the genetic changes occurring across the transporter genes and various pathways involved in the cellular activities were studied using DNA microarrays. Solubility data showed a significant increase in drug solubility upon preparing the new salts with the oppositely charged counter ions (ciprofloxacin glutamate salt exhibiting 2.9x103 fold enhancement when compared to the free drug). Moreover, permeability studies showed a 3 fold increase in trimethoprim and indomethacin permeabilities upon ion-pairing with amino acids and more than 10 fold when the zwitter ionic drug was paired with glutamic acid. Microarray data revealed that trimethoprim was absorbed actively via OCTN1 transporters while MRP7 is the main transporter gene that mediates its efflux. The absorption of trimethoprim from trimethoprim glutamic acid ion-paired formulations was affected by the ratio of glutamic acid in the formulation which was inversely proportional to the degree of expression of OCTN1. Interestingly, ciprofloxacin glutamic acid ion-pairs were found to decrease the up-regulation of ciprofloxacin efflux proteins (P-gp and MRP4) and over-express two solute carrier transporters; (PEPT2 and SLCO1A2) suggesting that a high aqueous binding constant (K11aq) enables the ion-paired formulations to be absorbed as one entity. In conclusion, formation of ion-pairs with amino acids can influence in a positive way solubility, transfer and gene expression effects of drugs.

A comparison of the apoptotic and cytotoxic effects of hexanedione derivatives on human non-neuronal lines and the neuroblastoma line SH-SY5Y

The effects of the alpha-diketone derivatives 2,3- and 3,4-hexanediones were investigated in three non-neuronal cell lines (MCF7, HepG2 and CaCo-2) as well as in the neuroblastoma line, SH-SY5Y. The MTT reduction assay was employed to determine the necrotic effects of the alpha-diketones and the neurotoxin 2,5-hexanedione over 4, 24 and 48 hr exposures. Flow cytometry was also used to study the effects of the three isomers on the cell cycle of the SH-SY5Y line only. With 2,5-hexanedione, the mean MTT IC50 decreased more than 10-fold from 4 to 48 hr. The toxicities of both alpha-diketones were similar, with a more than 18-fold increase in sensitivity of the SH-SY5Y at 24 hr compared to that of 4 hr. With flow cytometry at 48 hr, SH-SY5Y apoptosis with 2,5-hexanedione rose throughout the concentration range evaluated (0-30 mM) while 2,3- and 3,4-hexanediones showed apoptosis over the concentration range 1-1.6 mM, with 3,4-hexanedione being the more potent compared to the 2,3-isomer. At 1.6 mM nearly all the cells had entered apoptosis in the presence of the 3,4-isomer, (94.9 ± 1.4%) but only 57.5 ±4.1% of the 2,3-isomer-treated cells had reached that stage. The 2,3-and 3,4-isomers in diets alone may not pose a serious threat to human health. Further studies may be necessary to evaluate the effects of other dietary components on their toxicity. These alpha-diketones also display a degree of toxic selectivity towards neuroblastoma cells, which may have therapeutic implications.

Paediatric drug development:reformulation, in vitro, genomic and in vivo evaluation

Angiotensin converting enzyme (ACE) inhibitors lisinopril and ramipril were selected from EMA/480197/2010 and the potassium-sparing diuretic spironolactone was selected from the NHS specials list for November 2011 drug tariff with the view to produce oral liquid formulations providing dosage forms targeting paediatrics. Lisinopril, ramipril and spironolactone were chosen for their interaction with transporter proteins in the small intestine. Formulation limitations such as poor solubility or pH sensitivity needed consideration. Lisinopril was formulated without extensive development as drug and excipients were water soluble. Ramipril and spironolactone are both insoluble in water and strategies combating this were employed. Ramipril was successfully solubilised using low concentrations of acetic acid in a co-solvent system and also via complexation with hydroxypropyl-β-cyclodextrin. A ramipril suspension was produced to take formulation development in a third direction. Spironolactone dosages were too high for solubilisation techniques to be effective so suspensions were developed. A buffer controlled pH for the sensitive drug whilst a precisely balanced surfactant and suspending agent mix provided excellent physical stability. Characterisation, stability profiling and permeability assessment were performed following formulation development. The formulation process highlighted current shortcomings in techniques for taste assessment of pharmaceutical preparations resulting in early stage research into a novel in vitro cell based assay. The formulations developed in the initial phase of the research were used as model formulations investigating microarray application in an in vitro-in vivo correlation for carrier mediated drug absorption. Caco-2 cells were assessed following transport studies for changes in genetic expression of the ATP-binding cassette and solute carrier transporter superfamilies. Findings of which were compared to in vitro and in vivo permeability findings. It was not possible to ascertain a correlation between in vivo drug absorption and the expression of individual genes or even gene families, however there was a correlation (R2 = 0.9934) between the total number of genes with significantly changed expression levels and the predicted human absorption.

An in vitro investigation on the cytotoxic and nuclear receptor transcriptional activity of the mycotoxins fumonisin B1 and beauvericin

Fumonisin B1 (FB1) and beauvericin (BEA) are secondary metabolites of filamentous fungi, which under appropriate temperature and humidity conditions may develop on various foods and feeds. To date few studies have been performed to evaluate the toxicological and endocrine disrupting effects of FB1 and BEA. The present study makes use of various in vitro bioassays including; oestrogen, androgen, progestagen and glucocorticoid reporter gene assays (RGAs) for the study of nuclear receptor transcriptional activity, the thiazolyl blue tetrazolium bromide (MTT) assay to monitor cytotoxicity and high content analysis (HCA) for the detection of pre-lethal toxicity in the RGA and Caco-2 human colon adenocarcinoma cells. At the receptor level, 0.001-10μM BEA or FB1 did not induce any agonist responses in the RGAs. However at non-cytotoxic concentrations, an antagonistic effect was exhibited by FB1 on the androgen nuclear receptor transcriptional activity at 10μM and BEA on the progestagen and glucocorticoid receptors at 1μM. MTT analysis showed no decrease in cell viability at any concentration of FB1, whereas BEA showed a significant decrease in viability at 10μM. HCA analysis confirmed that the reduction in the progestagen receptor transcriptional activity at 1μM BEA was not due to pre-lethal toxicity. In addition, BEA (10μM) induced significant toxicity in both the TM-Luc (progestagen responsive) and Caco-2 cells.

Nanostructured lipid carriers as vehicles for the delivery of poorly water-soluble drugs

186 p.

Déterminer le rôle de C1orf106, un gène associé aux maladies inflammatoires de l’intestin

Les maladies inflammatoires de l’intestin (MIIs, [MIM 266600]) sont caractérisées par une inflammation chronique au niveau du tube gastro-intestinal. Les deux principales formes sont la maladie de Crohn (MC) et la colite ulcéreuse (CU). Les MIIs résulteraient d’un défaut du système immunitaire et de l’épithélium intestinal. Ce dernier forme une barrière physique et biochimique qui sépare notre système immunitaire des microorganismes commensaux et pathogènes de la microflore intestinale. Un défaut dans la barrière épithéliale intestinale pourrait donc mener à une réponse immunitaire soutenue contre notre microflore intestinale. Les études d’association pangénomiques (GWAS) ont permis d’identifier 201 régions de susceptibilité aux MIIs. Parmi celles-ci, la région 1q32 associée à la MC (p<2x10-11) et à la CU (p<6x10-7) contient 4 gènes, dont C1orf106, un gène codant pour une protéine de fonction inconnue. Le re-séquençage de la région 1q32 a permis d’identifier une variante génétique rare de C1orf106 (MAF˂1%) associée aux MIIs (p=0,009), Y333F. Nous avons démontré que la substitution de la tyr333 par une phénylalanine semble avoir un effet sur la stabilité protéique de C1orf106 tel que démontré lors de l’inhibition de la synthèse protéique induite par le cycloheximide. Nous avons déterminé que C1orf106 est exprimé dans le côlon et l’intestin grêle. De plus, son expression est augmentée lors de la différenciation des cellules épithéliales Caco-2 en épithélium intestinal polarisé. Son profil d’expression correspond aux types cellulaires et tissulaires affectés dans les MIIs. De plus, C1orf106 est partiellement co-localisée avec le marqueur des jonctions serrées, ZO-1. Toutefois, son marquage reproduit parfaitement celui du marqueur des jonctions adhérentes, E-cadhérine. Les jonctions serrées et adhérentes sont localisées du côté apical de la jonction intercellulaire et sont toutes deux impliquées dans l’établissement de la barrière épithéliale. Nous avons donc testé l’impact de C1orf106 sur la perméabilité de l’épithélium intestinal. Nous avons observé une augmentation de la perméabilité épithéliale chez un épithélium intestinal formé par des cellules Caco-2 sous-exprimant C1orf106. Nos résultats suggèrent que C1orf106 pourrait être le gène causal de la région 1q32.

Déterminer le rôle de C1orf106, un gène associé aux maladies inflammatoires de l’intestin

Les maladies inflammatoires de l’intestin (MIIs, [MIM 266600]) sont caractérisées par une inflammation chronique au niveau du tube gastro-intestinal. Les deux principales formes sont la maladie de Crohn (MC) et la colite ulcéreuse (CU). Les MIIs résulteraient d’un défaut du système immunitaire et de l’épithélium intestinal. Ce dernier forme une barrière physique et biochimique qui sépare notre système immunitaire des microorganismes commensaux et pathogènes de la microflore intestinale. Un défaut dans la barrière épithéliale intestinale pourrait donc mener à une réponse immunitaire soutenue contre notre microflore intestinale. Les études d’association pangénomiques (GWAS) ont permis d’identifier 201 régions de susceptibilité aux MIIs. Parmi celles-ci, la région 1q32 associée à la MC (p<2x10-11) et à la CU (p<6x10-7) contient 4 gènes, dont C1orf106, un gène codant pour une protéine de fonction inconnue. Le re-séquençage de la région 1q32 a permis d’identifier une variante génétique rare de C1orf106 (MAF˂1%) associée aux MIIs (p=0,009), Y333F. Nous avons démontré que la substitution de la tyr333 par une phénylalanine semble avoir un effet sur la stabilité protéique de C1orf106 tel que démontré lors de l’inhibition de la synthèse protéique induite par le cycloheximide. Nous avons déterminé que C1orf106 est exprimé dans le côlon et l’intestin grêle. De plus, son expression est augmentée lors de la différenciation des cellules épithéliales Caco-2 en épithélium intestinal polarisé. Son profil d’expression correspond aux types cellulaires et tissulaires affectés dans les MIIs. De plus, C1orf106 est partiellement co-localisée avec le marqueur des jonctions serrées, ZO-1. Toutefois, son marquage reproduit parfaitement celui du marqueur des jonctions adhérentes, E-cadhérine. Les jonctions serrées et adhérentes sont localisées du côté apical de la jonction intercellulaire et sont toutes deux impliquées dans l’établissement de la barrière épithéliale. Nous avons donc testé l’impact de C1orf106 sur la perméabilité de l’épithélium intestinal. Nous avons observé une augmentation de la perméabilité épithéliale chez un épithélium intestinal formé par des cellules Caco-2 sous-exprimant C1orf106. Nos résultats suggèrent que C1orf106 pourrait être le gène causal de la région 1q32.

Large-scale screening of a targeted Enterococcus faecalis mutant library identifies envelope fitness factors

Spread of antibiotic resistance among bacteria responsible for nosocomial and community-acquired infections urges for novel therapeutic or prophylactic targets and for innovative pathogen-specific antibacterial compounds. Major challenges are posed by opportunistic pathogens belonging to the low GC% gram-positive bacteria. Among those, Enterococcus faecalis is a leading cause of hospital-acquired infections associated with life-threatening issues and increased hospital costs. To better understand the molecular properties of enterococci that may be required for virulence, and that may explain the emergence of these bacteria in nosocomial infections, we performed the first large-scale functional analysis of E. faecalis V583, the first vancomycin-resistant isolate from a human bloodstream infection. E. faecalis V583 is within the high-risk clonal complex 2 group, which comprises mostly isolates derived from hospital infections worldwide. We conducted broad-range screenings of candidate genes likely involved in host adaptation (e.g., colonization and/or virulence). For this purpose, a library was constructed of targeted insertion mutations in 177 genes encoding putative surface or stress-response factors. Individual mutants were subsequently tested for their i) resistance to oxidative stress, ii) antibiotic resistance, iii) resistance to opsonophagocytosis, iv) adherence to the human colon carcinoma Caco-2 epithelial cells and v) virulence in a surrogate insect model. Our results identified a number of factors that are involved in the interaction between enterococci and their host environments. Their predicted functions highlight the importance of cell envelope glycopolymers in E. faecalis host adaptation. This study provides a valuable genetic database for understanding the steps leading E. faecalis to opportunistic virulence.

Unconjugated bile acids influence expression of circadian genes: a potential mechanism for microbe-host crosstalk

Disruptions to circadian rhythm in mice and humans have been associated with an increased risk of obesity and metabolic syndrome. The gut microbiota is known to be essential for the maintenance of circadian rhythm in the host suggesting a role for microbe-host interactions in the regulation of the peripheral circadian clock. Previous work suggested a role for gut bacterial bile salt hydrolase (BSH) activity in the regulation of host circadian gene expression. Here we demonstrate that unconjugated bile acids, known to be generated through the BSH activity of the gut microbiota, are potentially chronobiological regulators of host circadian gene expression. We utilised a synchronised Caco-2 epithelial colorectal cell model and demonstrated that unconjugated bile acids, but not the equivalent tauro-conjugated bile salts, enhance the expression levels of genes involved in circadian rhythm. In addition oral administration of mice with unconjugated bile acids significantly altered expression levels of circadian clock genes in the ileum and colon as well as the liver with significant changes to expression of hepatic regulators of circadian rhythm (including Dbp) and associated genes (Per2, Per3 and Cry2). The data demonstrate a potential mechanism for microbe-host crosstalk that significantly impacts upon host circadian gene expression. Disruptions to circadian rhythm in mice and humans have been associated with an increased risk of obesity and metabolic syndrome. The gut microbiota is known to be essential for the maintenance of circadian rhythm in the host suggesting a role for microbe-host interactions in the regulation of the peripheral circadian clock. Previous work suggested a role for gut bacterial bile salt hydrolase (BSH) activity in the regulation of host circadian gene expression. Here we demonstrate that unconjugated bile acids, known to be generated through the BSH activity of the gut microbiota, are potentially chronobiological regulators of host circadian gene expression. We utilised a synchronised Caco-2 epithelial colorectal cell model and demonstrated that unconjugated bile acids, but not the equivalent tauro-conjugated bile salts, enhance the expression levels of genes involved in circadian rhythm. In addition oral administration of mice with unconjugated bile acids significantly altered expression levels of circadian clock genes in the ileum and colon as well as the liver with significant changes to expression of hepatic regulators of circadian rhythm (including Dbp) and associated genes (Per2, Per3 and Cry2). The data demonstrate a potential mechanism for microbe-host crosstalk that significantly impacts upon host circadian gene expression.

Immune cells and oxidative stress in the endotoxin tolerance mouse model

Sepsis is a systemic inflammatory response that can lead to tissue damage and death. In order to increase our understanding of sepsis, experimental models are needed that produce relevant immune and inflammatory responses during a septic event. We describe a lipopolysaccharide tolerance mouse model to characterize the cellular and molecular alterations of immune cells during sepsis. The model presents a typical lipopolysaccharide tolerance pattern in which tolerance is related to decreased production and secretion of cytokines after a subsequent exposure to a lethal dose of lipopolysaccharide. The initial lipopolysaccharide exposure also altered the expression patterns of cytokines and was followed by an 8- and a 1.5-fold increase in the T helper 1 and 2 cell subpopulations. Behavioral data indicate a decrease in spontaneous activity and an increase in body temperature following exposure to lipopolysaccharide. In contrast, tolerant animals maintained production of reactive oxygen species and nitric oxide when terminally challenged by cecal ligation and puncture (CLP). Survival study after CLP showed protection in tolerant compared to naive animals. Spleen mass increased in tolerant animals followed by increases of B lymphocytes and subpopulation Th1 cells. An increase in the number of stem cells was found in spleen and bone marrow. We also showed that administration of spleen or bone marrow cells from tolerant to naive animals transfers the acquired resistance status. In conclusion, lipopolysaccharide tolerance is a natural reprogramming of the immune system that increases the number of immune cells, particularly T helper 1 cells, and does not reduce oxidative stress.

Epigenetic Silencing of CRABP2 and MX1 in Head and Neck Tumors

Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous disease affecting the epithelium of the oral cavity, pharynx and larynx. Conditions of most patients are diagnosed at late stages of the disease, and no sensitive and specific predictors of aggressive behavior have been identified yet. Therefore, early detection and prognostic biomarkers are highly desirable for a more rational management of the disease. Hypermethylation of CpG islands is one of the most important epigenetic mechanisms that leads to gene silencing in tumors and has been extensively used for the identification of biomarkers. In this study, we combined rapid subtractive hybridization and microarray analysis in a hierarchical manner to select genes that are putatively reactivated by the demethylating agent 5-aza-2'-deoxycytidine (5Aza-dC) in HNSCC cell lines (FaDu, UM-SCC-14A, UM-SCC-17A, UM-SCC-38A). This combined analysis identified 78 genes, 35 of which were reactivated in at least 2 cell lines and harbored a CpG island at their 5' region. Reactivation of 3 of these 35 genes (CRABP2, MX1, and SLC15A3) was confirmed by quantitative real-time polymerase chain reaction (PCR; fold change, >= 3). Bisulfite sequencing of their CpG islands revealed that they are indeed differentially methylated in the HNSCC cell lines. Using methylation-specific PCR, we detected a higher frequency of CRABP2 (58.1% for region 1) and MX1 (46.3%) hypermethylation in primary HNSCC when compared with lymphocytes from healthy individuals. Finally, absence of the CRABP2 protein was associated with decreased disease-free survival rates, supporting a potential use of CRABP2 expression as a prognostic biomarker for HNSCC patients.

Etiology of childhood diarrhea in the northeast of Brazil: significant emergent diarrheal pathogens

In a study conducted in Joao Pessoa, northeast of Brazil, 2344 Escherichia coli isolated from 290 infants with diarrhea and 290 healthy matched controls were analyzed for virulence traits. Enteroaggregative E. coli (EAEC) was the most prevalent pathogen associated to acute diarrhea. Based on the results of colony blot hybridization, serotyping, and HEp-2 cell adherence assays, strains were separated in categories as typical enteropathogenic E. coli (EPEC) (1.7%), atypical EPEC (a-EPEC) (9.3%), EAEC (25%), enterotoxigenic E. coli (10%), and enteroinvasive E. coli (EIEC) (1.4%). No enterohemorrhagic E. coli strains were isolated. Other enteropathogens were found, including Salmonella (7.9%), Shigella spp. (4.1%), thermophilic Campylobacter spp. (2.4%), Giardia lamblia (9.3%), and Entamoeba histolytica (5.8%). All enteropathogens were associated with diarrhea (P < 0.01). However, the association was lower for EPEC and EIEC (P < 0.03). Different pathogens associated with diarrhea may have been changing in Brazil where EAEC and a-EPEC seem to be the most prevalent pathogens among them. (C) 2010 Elsevier Inc. All rights reserved.

Production and characterization of sponge-dough bread using scalded rye

In this work, flatbed scanning, instrumental texture analysis, spectrophotometric color determination (L*, a*, b*), moisture and specific volume measurements were used to evaluate the effects of the addition of rye flour or rye flakes, yeast and boiling water in different amounts in sponge-dough rye bread production. The treatments changed significantly (P < 0.05) the crumb cell area (mm(2)), cell diameter (mm), cell perimeter (mm), texture parameters and light reflectance (L*, a*, b*). Scalding process could be used to produce new textures and color of baked products.